ESARF 11th Annual Conference (2001) Proceedings

Dept, of Vet. Hygiene and Management , Faculty of Veterinary Medicine, Cairo University

The camel attained its greatest importance to man in times of warfare and when warfare become mechanized, interest in the camel tapered off . Physiological research renewed the interest of camel first purely academically then the camel’s social ability to survive could be used for the benefit of man and therefore interest in the camel today in congresses dealing with the problem of food in drought areas .

Adaptation of animal to its environment in general is used often for the process of adjustment to the environmental changes . Adaptations of the camel to the desert environment encompass anatomical , behavioural and physiological changes . It is quite clear that , the camel does not have any special mechanism for survival but relies on mechanisms known to and utilized by another animals . All the desert species have fluctuating body temperature decline in metabolism and utilization of intestinal water. The camel, however is able to utilize these mechanisms more effectively when exposed to the direct rays of the sun and for extremely long periods without drinking water (Nielsen,1979).

Regarding the desert environment , there are several problems facing the desert animals, those are lack of food and water ( starvation ) , sandy or stony ground , thorny plants or trees , hot and windy climate and finally the presence of natural enemies .The camel is one of the most important desert animal and the following points discussing how the camel cope with the desert environment .

¨ The head of the camel is small in comparison to that of other domestic animals . It bears no horns and has small bluntly erected ears to hear the minimal sound vibration and hear for long distance in the desert . Also , the ear contains small hairs to filter and warm the air entered the ears in sandy environment .

¨ The eyes are large and prominent enable the camel to see in different directions and for long distances . The massive supraorbital fossa or processes give some protection with the long lashes against the sandy environment of the desert in windy day .

¨ Also , the nostrils of the camel are long slit- like appearance having wing , so the camel is the only animal who can close its nostril as protection against sand and winds .

¨ The upper lip is split and hairy , extensible and slightly prehensile , it is very sensitive . This modification help the camel to select its food

¨ The camel has a long arched neck helping him to manipulate the high tree plants and to explore the enemy from long distances .

¨ Most of the fatty tissues of camel is stored in the hump than being diffused throughout the body . The hump acts as food ( fat ) storage which will be converted to energy and water in case of starvation in the desert .

¨ Skin of camel is attached rather tightly to the underlying tissues and has short fine hairs ( weber) which help in thermoregulation .

¨ Prepuce of camel is normally directed posteriorally , it is possible that this elevated position keeps the organ from touching the hot sand when the camel squats on the ground and avoiding its contamination with sand .

¨ Placenta of she camel is simple diffuse smooth type as in mare with no cotyledons so the retention of placenta is rare.

The legs are relatively long and slender , an adaptation , perhaps to a long easy gait in sandy environment , and to adaptive cooling , and terminate in large disk -like feet .More than 65% of the camel’s total weight is supported by the front limbs . the chest is deep and narrow which allows the balance to be shifted easily , so that it is directly over the weight bearing foreleg during locomotion .

The foot of the camel is well designed to cops with the loose sandy soils of the desert . The bearing surface of the foot is like a large plate , this plate is able to maintain flat contact with the ground throughout the duration of the stride due to exception rotation at the first digital joint . The foot stays out on taking the weight of the camel and thus act as a firm base for levering the weight forward to the next stride . the camel foot is excellent for movements on sand . It is less suitable for traversing stony desert althought some hardening occurs in animals habituated to this kind of country .The presence of the peculiar horny pads on the elbows , stifle and chest prevent more injuries to camel from the stony desert .

Physiological adaptation defined by biologists as the physiological processes involved in adjustments by the individual to climatic changes and changes in food quality …… etc.The requirements for survival in hot arid areas are very important . Temperature must be maintained and water must be conserved . The camel losses body heat by sweating more efficiently than other mammals .Adaptation of camel to desert environment are listed below

¨ In most mammals fat is spread over the body surface just under the skin . In the camel , the fat is concentrated in the hump which enables sweat to be evaporated easily over the rest of the body surface and this is adaptation to heat transmission .

¨ The skin is supple , covered with short fine hairs ( waber ) , which act as insulating medium and may be longer in cooler climates or during the cool seasons in hot areas ( thermoregulation )

¨ The poll glands which are situated towards the top of the back of the neck behind ears and cover an area of about 6x4 cm in both sexes . It is more active under condition of heat and fatigue than that at any other time except when the male is in rut , so it act as modified sweat gland to help in the evapration .

¨ Also , the coat of the camel is fairly sparse which allows sweat to evaporate at the surface of the skin. In mammals with very thick coats evaporation occurs at the ends of the hair a less efficient process .

¨ The body temperature can vary over a wide range under condition of dehydration . The large mass of the camel acts as a heat buffer .

¨ The camel can lose 25% of its body weight over a period of time without losing its appetite for food and can then make up this amount in just 10 minutes by drinking . While in other animals , water lost is drawn from the body tissues and the blood plasma .As a result the blood becomes viscous and the heart can no longer pump and explosive heat death then occurs . In camels, very little water is drawn from the blood, which remains fluid and can thus cotinue its function of heat transfer (Dorman,1984)

The camel is able to save considerable amounts of energy by allowing its body temperature to rise during the day , thus absorbing heat would be dissipated by some form of cooling . The variations in the camel’s temperature were formerly thought to be an indication of poor thermoregulation . It is now realised that the rises in temperature indicate a sophisticated control mechanism rather than poor regulation .

Water is essential to life and the camel has often to survive on limited quantities for long periods of time . To do this , it has developed not only a very low rate of water use but mechanism for restricting water loss as soon as its intake is reduced

a- The hump is mainly comprised of fat and thus the metabolic water content is high , complete oxidation of fat in the hump results water ( 20 kg of fat , would release a total of just over 21 kg of water ) . Oxidation of an equivalent amount of starch yields less water .

b- The camel’s stomach contains a large amount of fluid secreted by the glandular sac areas which called “ water sacs “

c- Water is lost from the body by evaporative cooling , in the urine , and in the feces . The structure and function of the kidney are extreme importance in water conservation the long loops of Henle in the medulla have the function of urine concentration . Urea is reabsorbed from the intestine and transferred back to the stomach for reconservation to protein . The kidney controls water loss in two ways : either by the absolute concentration achieved or by reduction in flow of urine . Concentration of urine not only serves to conserve water but allow camels to drink water even more concentrated than sea water and to eat very salty plant that would otherwise be poisonous . A reduction in urine flow is also achieved by reducing th glomelular filtration rate from a norm of 55-65 ml/100kg body weight / minute to 15 ml / 100kg / min .

d- Fecal water loss is also small in camel . Final reabsorption of water occurs in the colon.

In the meaning of koran “ which explain what the prophet Mohammed preached “ it clearly states that , when men who lived in the desert turned to God for help in surviving in the inhospitable climate . God answered their pleas and gave them the she- camel to drink of their milk . It was the camel’s ability to convert the scent desert food sources into milk and meat for human consumption that was instrumental in the domestication of this animal. Only the camel can survive and continue producing milk in arid areas(Yagil,1985) . It is common practice for nomads crossing the desert to take milch camel with them because the long marches without water do not depress milk secretion . Young camels can derive their nutritional and water requirements entirely from milk in times of water restriction(Yagil,1985).When lactating camels were subjected to chronic dehydration ( 10 days of water restriction followed by1 h . water followed by another 10 days dehydration ) from spring to the end of summer ,milk secretion was not affected( Wilson,1984) . Full exploitation of the camel’s ability to survive and lactate in drought areas will only be possible when the reproductive performance is improved .

Camels milk contains an average of 70 kal/ 100g milk . It is calculated that 3 –4 kg of milk would cover the daily caloric requirements of an adult man as 1.8kg milk would covered a man’s daily protein requirements ,therefore camel milk is an ideal source of nutrition for man in the desert and is often the only source . Camel milk normally has a sweet sharp taste but some times it tastes salty. The taste is affected by the nutritional and environmental factors.The she-camel secretes a highly diluated milk with a low fat content whereas , the cow , ewe and goat all secretes concentrated milk when drinking water is scarce. Chronically dehydrated camels (drinking water for only 1 hr once a week ) secrete milk with over 90% water and with only 1% fat , this is superadaptation for a desert environment ( normal milk contains 84% water and 4.5% fat ). The physiological explanation of this phenomenon is as lactation is a water losing process for the mother, the hormone ADH and aldosterone are secreted in addition to milk secreting hormones ( prolactin and oxytocin ). ADH and oxytocin cause the release of ACTH aldestrone and prolactin , respectively . ADH , oxytocin and prolactin all have an antidiuretic effect while aldesterone , prolactin and ADH act on the intestines to absorb water and ADH cause the secretion of water into the milk as occurs in the sweat gland.

The extensive discussion about milk fat is appropriate as in times of water depletion it is the ability of camels to replace fat with water that will guarantee the survival of a species and the low –fat and high- water contents of camel’s milk decrease the calf-death rate in arid areas and it is appropriate for the need of children from milk ( normally severe dehydrated mammals give less water and more fat milk )(Yagil andEtzion,1980).

The increased lactose , with increased water , would explain the continuing lactation under adverse conditions . In general , camel milk is rich in chloride and vit.C ( varying from 5-7 % to 9.8 % ) . The vit C level is three times higher than those in cow milk . As lactation progresses so the vit C concentration in milk increased because fruit and vegetables are scarce in the arid zones , milk becomes an important source of vit C for the human diet in the desert .

III Behavioural adaptation

Under conditions of dehydration and intense heat the camel adopts behavioural mechanisms to conserve energy

1- The camel sits down in the early morning before the ground has warmed up . It tucks its legs underneath it’s body so that it absorbs little heat from the ground by conduction

2- The camel orientates itself towards the sun presenting the least possible body area for the absorption of radiant heat . Any heat absorbed from the ground or the sun would have to be dissipated later in the day

3- A group of animals may lie down together , thus presenting an even smaller target area for heat accummulation

4- The camel’s metabolic rate increases in the normal way as the temperature rises

Camels are selective feeder not only with regard to plants but also in respect of the parts of the plants they eat .The natural selective feeding habits of the camel are considered the morpho physiological adaptation of the camel’s digestive tract

The digestive anatomy and physiology of the camel are peculiar in the adaptation to a wide rang of food types and in particular , coarse forage , without the necessity of a specialized cellulose fermenting rumen . The camel does , however posses adaptive mechanisms to compensate for long periods of poor quality food and water deprivation , for example , there is a slow decease in weight through fluid loss during exposure to high temperature (41c ) allowing adjustments of fluid spaces and a stable metabolism(Yousri,1976).

Camels are browsing animals, they feed on thorny plants of the desert. Anatomical adaptation as the mobile and prehensive split upper lip enable them to avoid the injuries of the desert plants , the camel jaw and dental pad enable it to seize and tear branches off trees if required , and with slow lateral movements of the jaw , the thorns of these plants are destructed . Also a small but mobile tongue with numerous hard , dentigerous papillae protruding from the lining of the cheeks and lateral aspect of the tongue assist in the mastication and ingestion of food . Although camels can resist thorns to some extent they are not completely immune to them and on very thorny species feeding is a slow business(Higgins,1986) .

The oesophagus has a large potential diameter with many mucus –secreting glands . The areopharyngeal and oesophageal anatomy assist in the movement of hard materials without causing irritation to mucosa .

CONCLUSIONS

The camel is a triple-purpose animal producing milk, meat and transport. It also provides hair and hide in some areas . Its comparative advantages over other domestic animals within the camel’s optimal environmental limits as follows :

1- Its water economy resulting from the ability to reduce water loss to a considerable degree

2- Its ability to support a very high degree of water loss amounting to as much as 30 % of its initial body weight .

3- Its massive capacity for making up water loss in a very short time when water becomes available ( can drink 180 litre / 24 hr .) .

4- The three previous factors enable the camel to go voluntary without water for up 10 days.

5- The camel doesn’t lose its appetite for food as it dehydrates, unlike other food producing domestic animals .

6- The ability of camel to fluctuate its body temperature coupled with anatomical and behavioural adaptation enable considerable saving in energy to be made .

7- The camel is primarly a browsing animal enabling it to make use of fodder often not relised by other domestic animals .

8- The huge plate-like feet themselves are less damaging to soil structure than the smaller cloved hooves of the other common domestic animals .

By utilizing all the knowledge concerning the physiology and behaviour of the camel, this animal which is so capable of surviving the extreme conditions in the desert can return to be a provider of food for man, as it was in Biblical times when man turned to God for help and was given the camel “ to drink of its milk “

REFERENCES

Dorman , A.E . (1984): Aspects of the husbandry and management of the genus camelus . In : The camel in Health and Disease .Edited by Higgins ,A.J. 1^st edition . Bailliere Tindall , london .

Higgins ,A (1986): The camel in Health and Disease .1^st Edition . Bailliere Tindall , London , Tokyo , Sydney.

Nielsen , K.S (1979): Desert Animals . physiological problems of heat and water . 2^nd Edition Dover publications , Inc , Newyork , USA .

Wilson ,R.T (1984): The camel . 1^st Edition , longman group limited . Burnt Mill , Harlow Essex ,UK .

Yagil , R. (1985): Compartive physiological adaptation . 1^st Edition , KARGER , Basel , Munchen , london , Tokyo and sydney .

Yagil ,R and Etzion , Z. (1980): Effect of drought condition on the quality of camel milk .J. of Dairy Research , 47 :159-166 .

E.M. ABDEL GAWAD

Dept. of Hygiene and Husbandry. Faculty of Veterinary Medicine, Cairo University, Giza

ABSTRACT

Sexual behaviour involves not only the act of mating itself, but also all those elements of courtship, display, motor activities and postures which have a functional unity with spawing or coitus to ensure fertilization, pregnency and propagation of the species.

Animal Courtship is essentially similar to human courtship. Both animals and humans like to formalize the mating transaction with some form of ritual, whether it be a display or a wedding ceremony.

Throughout the animal Kingdom there is a bewildering variety of courtship behaviour patterns, ranging from those species that reproduce without any behavioural interaction at all, to species that show very complex courtship sequences lasting for several hours or even days.

In Sexual interaction, some behavioural elements will be characteristic of the female and others of the male. The Sexually mature animal must be capable of recognizing the species, sex, and exact status of sexual readiness of a prospective mate.

Many animals have an intromittent organ (The penis in mammals) by means of which the sperm transfer is effected ,. Some accomplish copulation without specialized intromittent organ as in birds except ducks and few other by a quick apposition of their cloacae (cloacal kiss)

The extrusion of the sex products into the surrounding medium (external fertilization),common in water-living forms, is of course impossible in land living animals, since the gametes would be quickly killed by drying in air, thus internal fertilization is necessary in land-form.

The mating system that is typical of a species provides a fundamental aspect of its social organization, monogamy, polygamy, polyandry and promiscuous are common

Sexual behaviour in fishs, birds, reptiles, insects and mammals has been illustrated and analyzed.

INTRODUCTION

Man’s interest in animal life has perhaps never been greater that it is today. The latest studies in comparative behaviour have shown that essential components of human behaviour can be understood genetically as something we inherited from our ancestors. Therefore, an understanding of animals become a prerequisite for an understanding of man

The time it takes for an animal to reach sexual maturity depends on two factors : the size of the creature and its span of life. Small animals rarely live long. They have opted for a strategy of rapid reproduction and hence, quick colonization of new habitates. The other extreme of this situation is to leave reproduction until late in life and become a large animal. Many Creatures fall between these two extremes ( Whitfield 1979)

The reproductive urge is released by chemical and physical changes in the body, As the breeding season approaches, the gonads secrete sex hormones into the blood stream. The overwhelming coercion that these hormones exert on the body leads to surprising changes in behaviour (Nicolai,1975).

General environment, recent events, and presently occurring stimuli ( or lack of such ) also influence its behavior. Level of nutrition, seasonal effects such as day length and temperature, presence or absence of good health, earlier experience, and learning can all influence the activity seen ( Criage, 1981 )

Biologists have shown beyond any reasonable doubt that, with a few exceptions, the performance of species–typical behaviour contributes to the biological fitness or more accurately the inclusive fitness (Hamilton, 1964) of the behaving animal ( Hind 1975 ). Behaviours which are neutral or confer a biological disadvantage will disappear from the population under the forces of mutation and natural selection (Hind, 1975)

COURTSHIP AND DISPLAY

Courtship is the term used by ethologists to embrace all behaviour that precedes and accompanies the sexual act that leads to the conception of the young ( Mcfarland, 1981 ) . The essential components of courtship are : attraction, pursuit and a period of time before mating occurs. For many animals the relationship, or pair bond, may continue while the young are brought up or even for life ( Whitfield, 1979 )

Displays are stereotyped motor patterns involved in animal communication., Displays are largely genetically determined and species-specific. Related species often haven similar displays. Display postures often show distinctive color patterns, weaons, or other physical characteristics. (Mcfarland, 1981)

Sexual display consists of discrete components of behaviour which usually occur in a fairly strict order to give a sequence of behaviour which will be species-specific a phenomenon known as «Behavioural chains».

Clearly one of the problems in mating is the recognition of appropriate mates not only as to sex but also to readiness to mate. Behavaiours that “ advertise “ the sex and readiness of the mate constitute one class of courtship. Whereas the male of many vertebrate species is continuously sexually active during the reproductive season, the female is suitable as a mate only during a limited estrus (Etkin,1969)

· Many male birds posture infront of the female in such away that their plumage patterns are shown off to best advantage,. the displays of peacocks (pavo cristatus) are particularly spectacular example (McFarland. 1981 )

· The male smooth newt performs a complex displays that includes posturing to show off his brilliant colouration, and a tail fanning movement that produce a water current directed toward the female which carries his smell to her the female newt is-thus stimulated through three sensory channels, visual, olfactory and tactile

· The male fiddler crab of the species Uca maracoani performs lateral waving of large claw as female approaches then descends into burrow. Female often respands by following male into burrow and mating there (Bliss, 1990)

· Secretions produced by the female in one fish were found to stimulate mating behaviour.in the male (Tavolga, 1956)

Since most mammals are primary nocturnel non-visual animals, they use the sense of smell widely. Many female mammals in heat can apparently be recognized by odors, sometimes form special anal gland like those of the cat. Male rates distinguish estrous from non-estrous females, even at some distance, by odor, and male dogs are attracted to the urine of the estrous bitch (Etkin, 1969).

· In female chimpanzees and some other female primates the estrus state includes swelling and reddening of the so called sexual skin in the perineal region. In addition, the estrous female in mammals generally shows behaviour that differs from the non-estrous condition, she stands still for the male and tolerates his sniffing and eventually his mounting behaviour. The advertisement display of the female in estrus are, however, not a particularly elaborate aspect of courtship in vertebrates, because the male generally maintians sexual receptivity continuously during the rut and actively seeks the female .

Advertising displays of the male are more conspicuous though perhaps not more common aspect of animal courtship. We appreciate this best in birds, lizards, and some fish which like the human are visual minded.

Another function which courtship serves is that of overcoming the aggressive responses of one animal to another. When a female baboon comes into heat she displays her genitals in the so-called persentation posture thus courtship behaviour in the female monkey consists principally of subordination, sexual receptivity and acceptance of punishment from the dominant male (Carpenter, 1942).

Solitary preadaceous mammals, particulary of the cat and weasel family, often do considerable fighting as part of their coutship. The mating of cats is accompanied by much scratching, biting, and appropriate vocalization. This behavior differs from than fighting. Behaviorally, it appears to be fighting behaviour normal to a predator but modified and controlled by stimuli emanating from the sexual situation (Etkin – 1969).

In many spiders the male ties the female down with silken threads before mating with her to suppress or circumvent her cannibalistic tendencies (Mcfarland 1981)

Successful reproduction requires as a minimum the co- ordination of male and female activity with respect to insemination. In many rodents, rats, guinea pig., queen and sow, etc, when stimulated by the male during estrus, the female show a special posture called Lordosis. In this, the back is arched downward in the center, bringing the genital region up and the tail is lordotic posture, of course, directly assist in permitting the male to achieve intromission.

In birds, cloacal kiss facilitats transfer of the sperm to the female during breeding as when the male mounts the back of the female momentarily so that their cloacas are closely appressed and the transfer effected quickly (Wallace and Mahan 1975) .

The function of many courtship patterns is to minimize the risk of hybrid matings, and to ensure that mating occurs only with an individual of the same species. This phenomenon is called reproductive isolation, and any behavior pettern that achieves it is called «isolating mechanism». To ensure the survival of its offspring it is of paramount importance that an animal mates with a member of its own species, since hybrid mating rarely produces any offspring at all, and those that are produced are unlikely to survive or to produce further offspring themselves. ( Etkin, 1969 Whitfield. 1979, and Mcfarlond. 1981).

Mated pairs of colonial birds often display within sight of their neighbours, and the mutually stimulating effect of this displays tends to bring the members of the colony into reproductive condition at the same time. This has the advantage that the eggs and young are produced in large numbers during short period of time, thus swamping the predators (Mcfarland, 1981). This is called the Fraser Darling effect.

Copulation is that part of sexual behavior which is most closely associated with fertilization of the egg by the sperm. The copulatory behavior may be seen as a sequence of chain responses in which one event leads to another in a sterotyped manner.

A recent Painstakingly–researched study is that of Myrberg (1972) on the bicolor damselfish ( Eupomacentrus partitus ) . This is a tropical marine fish – the male exhibits a number of displays to the female which, since they precede spawing, can be considered as courtship these are : -

1- Tilt :- the body is quickly tilted to one side some 20 – 40 and then returned to the vertical

2- Dip : - A rapid, eye–catching vertical dive from 1 – 2 metres above the bottom.

3- Nudge :- the male approach the female with his head lowered and pelvic fin expanded, and attempt to push his head and neck against the female’s cloaca

4- Lead : - the male move rapidly in front of the female and toward his nest conch. His tail movements are exaggerated

5- If the female follow the male, then a further series of elements are displayed

6- Quiver : - the male stands on his head near his nest conch and quiver his body rapidly.

7- Close swim : - If the female enters the nest area, the male swims quickly over to her and they move together in a circle around the nest.

8- Skim : - both sexes swim a short distance over the nest, vibrating slightly.

9- Spawing : - this is skimming over the nest during which the female deposits eggs and the male sperm (milt) the spawing period may last between 10 minutes and and 2 hours.

10- Flutter :- if, during these elements the female moves away from the nest area, the male perform fluttering to attract her back.

The Courtship displays of fiddler–Crabs consists of rhythmic waving of the enlarged claw accompanied by body movements, so that the display looks like a dance. The movement varies in different species, but it is always Stereotyped within each species (Crane, 1957). In the Philippine species Uca zamboagana a series of vertical waves of claws is accompanied by raising and lowering of the body. In South American species Uca pugnax. rapax the enlarged claw moves outwards and upwards in three jerks and descends smoothly.

In frogs and toads ( Anura ), the male clasps the female from above and maintains this position throughout the deposition of the eggs, fertilizing them as they emerge from the females cloaca. If the object clasped another male, a few rapid vocalizations from him serving as a signal, and the clasping grip is released (McFarland, 1981). Copulatory behavior may be seen as a sequence of chain responses. Male clasps object female forms lordosis position male forms pasket female discharges eggs male fertilizes eggs.

Ferguson (1970) in a study of side – blotched lizards (Uta stansburiana) which live in the desert and semi – desert regions of southern United States, mentioned that the basic pattern of copulation in this species has six stages.

Stage 1: - the male approaches the female, while maintaining aggressive display in which the back is arched, he gives a sene^,s of puch–ups and down. These threat postures relax as he approaches the female, and perform courtship nods of the head.

Stage 2 and 3 : - As the male nears the female he licks her tail, hind legs, and pelvic region, continuing to head nod. Sometimes he circles the female.

Stage 4 : - the neck in grasped, often with some preliminary nipping, and the female is swung about or dragged along by the male. The female during this stage tells the male that she is receptive.

Stage 5 : - the male puts on leg over the pelvic region of the female and moves it rhythmically back and forth, pushing the base of his tail against her

Stage 6 : - In copulation proper , the male thrusts his Cloaca forwards until the penis is inserted. There are then a series of thrusts, culminating in ejaculation

Brower, Brower and Cranston, (1965) studies the courtship behavior of the Queen butterfly Danaus gilippus and mentioned that the sequences of responses performed by the male and female are kept in phase with one another. This occurs because each act of one individual is released by the previous response of the partner and provides the stimuli for the Partner’s next act. The whole ritual consists of an elaborate chain of responses analogy with Courtship in some vertebrates, such as the three-Spined Stickleback fish (Evans, 1968).

Etholgists have given a great deal of attention to the specific variations in the behaviour of waterfowl. In fact, following Darwin it was Oskar Heinroth (1910) Who in his paper, on the biology and ethology of Anatidse drew exactly his conclusion from the analogies found in the behavior of birds and human. Heinroth used Courtship in water fowl as a mean of taxonomy to distinguish between species. Lorenz (1971) has fully confirmed Heinroth‘s assertion.

After the mallard drakes have assumed their significant breeding plumage following the autumn moult,they gather on fine days in October and November on their home waters and form small groups for communal display (Nicolai. 1975). This display is usually triggered off by a duck suddenly shooting out among the drakes, stretching out her neck along the water surface and then retracting and extending it in quick succession, this is called (Nod–Swimming) . All the drakes present react together to the ducks nod – swimming by a specific synchronous display movement, the ( grunt – whistle ), this is followed by further display movement: down-up , head-up-tail-up, turning the head towards the female, again carried out simultaneously by all the drakes. The group displays of the mallard drakes gives the marriage – hungry females on opportunity to compare the attractiveness of the assembled males.

The duck signifies her intention to the drake she has selected by a peculiar behavior pattern. She goes up to him, swim eagerly behind him and with grumbling sequences of sounds she threatens over her shoulder at another drake in the display group. This so called “ inciting “ is to indicate her intended that she in fond enough of him alone to keep other equally valid competitors at bay.

If a duck continues to propose to the some drake on several consecutive days, the bond between then gradually strengthens and they eventually become a pair. The drake accompanies the duck in the search for a suitable nesting site, but he does not participate in incubating the eggs.

Copulation in mammalian species is characterized by intromission, a term which refers to vaginal penetration by the penis. In some species ejaculation is achieved with a single intromission, while in others multiple intromissions are necessary.

The copulatory behavior of primates is varied, multiple intromisston occur in some monkeys such as the rhesus macaque, but a single intromission is also common, especially among the apes. Thrusting is universal, multiple ejaculation normal, with the exception of gorilles and chimpanzees foot clasp is found in some monkeys ( Mcfarland, 1981).

The human female is continuously sexually receptive the sustained sex interest of the partners thus favors the intgration of the male ( Chance , 1962). Furthermore, in the absence of a definite estrus period, the specific stimuli furnished by the usual mammalian female as a sign of estrus (i.e. odors, sexual skin color,etc) are–no langer functional and have dropped out. Sexual play in humans is greatly facilitated by the assumption of the upright posture, which makes ventral copulation possible, replacing the awkward posture found in other Primates (Etkin, 1969).

In some species there is a genital lock which results in the copulating partners being temporarily unable to separate once intromission is achieved. In dog (cans) for example, the end of the penis become enlarged, and a vaginal sphincter muscle closes arround this enlargement, in cats (Felidae) the neck grip occurs

Rodent Copulatory behavior is characterized by multiple intromissions and ejaculations. Evidence from the laboratory rat (Ratus norvegicus) suggests that this facilitate the transport of sperm within the female reproductive organ . A period of quiescence following ejaclation is necssary for successful implantation of the fertilized ova

The mating system is a species – specific provides the social nature of the animal in relation to their ecological niche

Over 90% of the birds of the world are monogamous, but monogamy is rare in other animals, including mammals each breeding adult mates with only one member of the opposite sex such relationships are found amongst Swans, Geese, Cranes and gibbons .

polygamy is much more common among mammals. An individual generally has two or more mates either successfully or simultaneously.

The female mates with too or more males. The American jacana is an example. Having successively counted, the female lays the eggs, which are incubated solely by the male, while the female attempts to gain more males to incubate successive clutches.

|There are no pair bends, and both males and females mate with more than one member of the opposite sex

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ROLE OF VETERINARIANS AND RECENT BREEDING TECHNOLOGY IN ANIMAL WEALTH DEVELOPMENT IN EGYPT

A. M. OSMAN

Department of Theriogenology , Faculty of Veterinary Medicine, Assiut University, Assiut.

During the last decade, there has been an increased concern about overpopulation problem among human being in developing countries including Egypt. Our population reached 61.5 million (1996) with a developing rate of 2.1% from 1986 to 1996. In the year 1999 our population reached 66 million.

Each person in Egypt has an average of 16 gm animal protein per day which is half the minimum requirement for man as announced by the United Nation (35 gm per day).

Cattle and buffaloes as well as their products play a major supply for animal protein in Egypt (more than 55% of the total protein which include poultry, eggs and fish). Moreover, dairy animal is an important segment in live stock production. The numbers of cattle and buffalo in Egypt were 3.15 and 3.18 million while in the world the numbers were 1335.8 and 165.02 million in both species respectively. (FAO,1999).

On the other hand, Egypt occupies the rank of 17^th for the total human population in the world and our population represent 1.0% of the world population (FAO,1999) .

It is worthy to mention that the average rate of development in the world human population was 75% (from 1950/1980) which exceed those reported for cattle (52%) and buffaloes (47%). It is suggested by demographics that by the year 2025, 83% of the world population will be Living in the developing countries compared with 74% at 1982 and 67% at 195.

This difficult situation in such dangerous poor sources of animal protein required great effort for our local live stock.

The objectives of this article are to discuss a philosophy that production is reproduction and gynecological care to dairy cows could improve significantly their production.

Role of reproduction in dairy herd production and evaluation : (General precaution and care)

· Every phase of breeding ,calving and nursing animals must be managed at maximum efficiency to avoid losses in the new borne and milk yield.

· Calving every 12-13 months brings maximum milk yield from the cow with maximum profit for the owner.

· Cycling animals should exceed 90% and conception rate should not be less than 70%.

· Postpartum conception should not exceed 85 day. Any day beyond this period will cost 5-7 E.P. loss to the farm in Egypt.

· Heifers that calved late always tend to calve late in the subsequent years with longer calving intervals than others.

· Heifers with delayed puberty usually suffered from a lot of physiological malfunction and ovarian inactivity.

· Cows with poor milk yield or short lactation period usually accompanied with infertility problems and hormonal imbalance.

· Milk yield, fat and protein percentages, feed efficiency, mammary system, milk flow per minute, lactation period, mature size of the animal and ovarian condition have a high heritability nature and should be considered for breeding through selection.

· The experience, knowledge, cleverness of veterinarians and their co-workers could raise satisfactorily the income of any farm. Specialists are required for all aspects of Theriogenology.

3. Commercial hormones and drugs used to shorten the calving intervals and improve production (GnRH, PGF 2alva , L.H.,FSH…etc.).

2. Embryo transfer (Superovulation, fertilization, synchronization, embryo collection, sexing,embryo evaluation and preservation).

· Routine gynecological examination in a local buffalo farm could improve the following paramaters in 3 years:

Human population and numbers of cattle and buffalo in some countries (million)

Countries

Rate of growth

Countries

Rate of growth

Gaza Strip

Syria

Iraq

West Bank

Saudi Arabia

Egypt

+4.56

+3.82

+3.66

+3.58

+3.45

+2.1

Spain

Greece

Denmark

+0.16

+0.05

+0.04

Vatican

0.00

Hungary

Germany

Bulgaria

-0.16

-0.06

-0.04

Animals	Number (Millions)		Rate of Development %
	1992	2000
Cattle	2.97	3.18	7.07
Buffaloes	3.16	3.20	1.27
Camels	0.16	0.116	-27.5
Horses	0.053	0.045	50
Mules	0.00085	0.0012	41.18
Asses	2.75	3.05	10.91
Sheep	3.38	4.45	31.67
Goats	2.75	3.30	20
Pigs	0.027	0.029	7.41

· The largest numbers of cattle and buffalo were in India (198.4 and 77.0 million respectively)

· The largest number of farm animals per human population unit was in Newzealand.

GENDER PRESELECTION BY SPERM SEPARATION

G. ABDEL-MALAK

Preselection for sex in man and animals has occupied the imagination of mankind ever since the beginning of modern civilization.

Many theories have originated with the Greek philosophers. For example, there were suggested that various body positions during intercourse could give a child of one sex or the other. Others believed that females developed on the left side of the uterus, males on the right.

Even as late as the eighteenth century some believed that sperm from the right testes produced more male young while sperm from the left testes produced more females.

In mammals X- chromosome bearing sperm produce female offspring and Y-chromosome bearing sperm produce male offspring always in nearly equal numbers (50: 50).

Gender preselection of livestock offspring would enable producers to increase their rate of genetic improvement and reduce production costs.

Gamete manipulation prior to fertilization is the most efficient process for gender predetermination. This approach has an obvious advantage of allowing one to choose the sex before fertilization.

In production settings, preselection for sex would have the advantage of factoring the sex of offspring into a particular management plan. This would lead to increased flexibility in the utilization of facilities.

Males of high genetic merit can be more efficiently selected in order to propagate a large pool of offspring with improved genetic merit. Furthermore, only dams of the very highest genetic merit would need to be used for herd replacement.

Further, sex preselection offers the producer an important economic advantage since better control of his or her product helps to maximize production efficiency.

In human there is interest in the development of sex selection in conjunction with genetic testing for couples who risk transmitting a sex - linked disease to their offspring.

There are more than 350 X - linked diseases known in the human ( Mc Kusick , 1992 ) . Common examples of X - linked recessives include hemophilia, Duchenne’s muscular dystrophy and X- linked hydrocephalus.

The Ethics Committee of the American Society of Reproductive Medicine (1999) recommended to employ the reproductive technologies for sex selection and Preimplantation Genetic Diagnosis (PGD) to avoid the birth of children suffering from X - linked genetic disorders.

However to use Preimplantation Genetic Diagnosis and Sex selection solely for non – medical reasons should be discouraged because it poses a risk of unwarranted gender bias, social harm, and results in the diversion of medical resources from genuine medical need (Savulescu and Dahl, 2000).

Sex selection should be available, at least within privately funded health care.

In mammals, the male is the heterogametic sex and, thereby, determines the sex of the progeny. Since sperm carry an X or a Y chromosome, union with the egg results in an XX (female) or X Y (male).

In birds, the situation is markedly different in that all the sperm contain similar chromosome complements and similar DNA content and the female is heterogametic and determines the sex of progeny.

The basis for the DNA difference between X and Y sperm is that the sex chromosomes and the DNA within them carry genetic information ( Guyer , 1910 ) . The X-chromosomes is larger and carries more DNA than the smaller Y chromosome in all mammals . The autosomes carried by X or Y sperm are identical in DNA content.

The DNA difference between X - and Y - bearing sperm varies from about 2.8 % in the human (Johnson et al., 1993) to 12.5 % in the creeping vole (Johnson and Clarke, 1990). The DNA difference between X and Y sperm of domestic livestock (Johnson, 1992 and 1994) ranges from 3.5% to 4.2 % (Table 1).

Since production of spermatozoa occurs in the seminiferous tubules , it follows that one would expect equality of X and Y sperm production . A division of the normal chromosome complement creates them .

No experimental data exist to demonstrate that sperm development may be different for X sperm than for Y sperm . It is generally assumed that survival and maturation rates of all sperm are the same .

The fact that sperm differ according to their chromosome constitution provides the basis for separation by DNA content .

However , intracellular sperm DNA does not lend itself to surface measurement . If one were to choose an ideal means of differentiating X from Y sperm , it would be a specific surface marker ( antigenic protein ) . Such a protein would lend itself to the production of an antibody and subsequent adaptation to large-scale preparation of sexed semen . However , no such marker exists at the present time . Thus, DNA is the only sex-specific marker known, measured, and validated.

Separation methods of X- and Y-bearing sperm

1 – Separation of the sperm based on density using “ Percool gradients “

( Kaneko et al . , 1983 ). However, validation of these results has not been forthcoming . Bull sperm fractions collected from “ Percool gradients “ were subjected to DNA flow cytometric analysis to determine proportions of X and Y sperm in the separate fractions. There was no change in the sperm sex ratio based on the Flow cytometric DNA evaluation (Upreti et al., 1988).

2 – According to Roberts (1972), there is a theoretical basis for difference in size of X -and Y - bearing sperm, since X sperm carry the larger chromosome. However, no experimental evidence has been presented to substantiate the hypothesis. Morphologically, there has never been any data to back up the claim by some (Shettles, 1961), that the X sperm head is morphologically any different than the Y sperm head.

3 – Theoretically, it is possible that one can separate sperm on the basis of dry weight, since DNA mass differs between the X and Y sperm (Johnson, 1992). However, the difference in mass between X and Y sperm would be no more than 1 %. Thus it would require exceptional measuring technology to accurately measure a 1- % difference in the weight of X and Y sperm.

4 – The technique of Free - flow electrophoresis (Johnson, 1994 has given some indication of being useful in differentiating between two populations of sperm based on a difference in sperm surface charge.

5 – Surface charge was the basis for others attempts to separate sperm. Bhattacharya et al., (1977) used the principal of thermal convection and counter – streaming sedimentation in combination with galvanization to attempt to move sperm at different velocities. Using their application the lighter Y sperm would lag and accumulate at some distance below the X sperm. The claim success for this process has not been confirmed by others workers .

6 – Separation of sperm by Sephadex gel filtration as described by Steeno et al., (1975) has received some attention, particularly with human sperm. The ability of the sephadex method to separate X and Y sperm has not been confirmed ( Lobel et al ., 1993).

7 – Albumin column separation has been used on many species ( Ericcson et al, 1973 ) , but especially in humans . Although it is used in numerous clinics world – wide , there is still a good deal of controversy concerning its efficacy . The method seeks to enrich the Y sperm population, which is based on the view that the Y sperm swim faster and thus can be collected off the column prior to the arrival of the X sperm at the bottom of the column.

Clinical data put forth by Beernink et al ., (1993) claim 72 % males in 1034 births (data collected over many years) after the use of the method.

A study designed to test the method through the isolated sperm for hamster sperm penetrating experiments and subsequent karyotyping of the fertilized embryo resulted in no confirmation of the Ericsson data (Brandriff et al ., 1986).

In fact, the results showed a tendency to greater number of females from the separated semen.

B – Sperm separation by Immunological Technique (H- Y antigen and Y- bearing sperm) :

Attempts to inactivate the Y sperm by immunological methods have focussed on the H - Y antigen (Goldberg et. 1971).

Others reports have suggested the usefulness of H - Y antibody as a means of selecting only the X - bearing rabbit sperm (Zavos, 1985).

The existence of a male - specific antigen, the H-Y antigen was described in the mid 1950 s by Eichwald and Silmer.

Studies to determine whether or not anti -Y antibodies bind preferentially to Y- chromosome - bearing sperm have until recently lacked a reliable method of estimating the percentages X and Y sperm in the fraction examined.

Hoppe and Koo (1984) suggested that X- and Y- bearing sperm probably share the same surface antigen due to their origin in the same testicular milieu, also suggesting that sex specific expression of H- antigen on sperm is questionable.

The data of Hendriksen et al., (1993) appear to confirm that the presence of the H-antigen on sperm may not be restricted to Y-bearing sperm. If specific surface antigens should be present on the two sperm types, it may be possible to use an appropriate immunological technique to separate sperm on a large scale. In this, it might be possible to kind a relevant antibody to a matrix within a column and then pass diluted bull semen through it. The X- bearing sperm would bind to the antibody – coated matrix and remain within the column. The Y-bearing sperm would be washed through and collected. It would then be possible to release sperm bound to the antibodies to give a second sorted population. Since such a technique would result in the production of large quantities of undamaged sperm.

In USA, Ali et al., (1990) attempted to separate bull sperm semen into X- and Y- bearing sperm using monoclonal H-Y antibodies.

New Zealand and Australian researches have also attempted to isolate H-Y positive and H-Y negative sperm populations (Bradley and Heslop, 1988; Bradley, 1989).

With the advent of cell sorting by flow cytometry, however, it has now been suggested by Hendriksen et al., (1993) that the presence of the H-antigen on sperm may not be restricted to Y – bearing sperm. Experiments were conducted to determine if H-Y antigen is preferentially expressed on Y – chromosome – bearing sperm that have been flow cytometrically sorted into X or Y populations based on DNA. No difference in binding between the sorted into X or Y sperm populations of the boar and bull was found. Flow cytometric evaluation of bull semen samples resulting from treatment with antibodies to H-Y antigen and subsequent removal of dead sperm showed no deviation from the expected 50: 50 sex ratio (Johnson, 1988).

In the USA, however, on the assumption that only Y – chromosome – bearing sperm process cell – surface H – Y antigen, a novel rapids immunomagnetic method has been reported for identifying sperm that contain these antigens (Peter et al., (1993). These workers claimed a 98 % purity of X – chromosome – bearing sperm.

Identification of Y sperm based on the long arm of chromosome (human) was reported by Barlow and Vosa ( 1970 ) . This resulted in the quinacrine hydrochloride fluorescent technique being used to monitor sperm preparation The validity and repeatability of the quinacrine technique has been questioned because of the variability of sperm preparation procedures and the difficulty of consistently reading the fluorescent signal .

Alternately , the application of flow cytometric analysis to measure individual sperm DNA content in order to determine the proportions of X-and Y-bearing sperm in a sample of semen has served as a reliable check on procedures purposed to show the sex ratio ( Pinkel et al ; 1985 ; Johnson , 1988 ; Upreti et al ; 1988 ) . In no case has there been evidence to support the claims made for various “ physical separation “ procedures or other non-DNA-based separation procedures mentioned in the previous section

Modern FCM /CELL –sorting technology was first developed by Fuleoyler (1965) and Kamenstsky (1967). Flow systems were commercialized in the 1970 and have developed rapidly in conjunction with the computer revolution in the 1980 and continues in the 1990 .

The primary application has been in medical research and diagnosis with respect to blood and tumor cells . FCM is an effective tool for use with many types of cell suspensions .

Since sperm are produced in suspension , they are readily adapted to FCM analysis and sorting in classic illustration of the usefulness of FCM for measuring the relative DNA content of individual sperm at a very rapid rate .

This technology is now standardized within research facilities to the degree that skewing the sex ratio to 85 – 90 % for either males or females is effective in cattle , pigs , sheep and rabbits . The effectiveness in humans is on average 75 – 80 % for skewness of the sex ratio in either the X or the Y direction . The lower efficacy rate for human is due to the smaller difference between the amount of DNA in the X- and Y – bearing chromosomes ( 2.8 % ) which is less than the difference in most domestic mammals .

Sperm from any species , including birds , can be analyzed by Flow –Cytometer. A bimodal distribution representing X – and Y – bearing sperm occurs with mammalian species , whereas with birds only a unimodal peak occurs because all sperm contain the same sex chromosome .

Successful sorting of sperm depends on the amount of DNA difference, morphology of the sperm , orientation of the sperm during Flow – cytometric analysis , and the number and viability of the sperm sample .

The technology of isolating sperm with the Flow – Cytometer has been available , at different levels of practical use , for more than 10 years . The practical uses of separated sperm are only now beginning to be established in commercial agriculture and in clinical medicine .

This technique is based on a difference DNA content in the sperm cell . The X-chromosome is larger than the Y – chromosome . For that reason, the total amount of DNA in an X-sperm cell is larger than in any sperm cell.

Flow cytometry permits such separation after measurement of the relative DNA content, based on the fluorescence emission intensity after staining with a DNA –specific- Fluorochrome such as “ Hoechst 33342 “ (fluorescent dye that binds to DNA ) .

The sperm are stained with Hoechst 33342, a bisbenzimide (Fluorochrome dye) which preferentially binds to the Adenine- Thymine regions of the DNA helix (Muller and Gautier, 1975; Johnson, 1984; Johnson et al., 1987). The more DNA is present in the cell , the more fluoresence .

In this machine several thousand of cells per second can be sorted purities of up to 90 %.

Flow cytometric analysis of mammalian sperm DNA content in a given sample of semen results in a bimodal distribution representing X and Y sperm population( Fig . 1 ) .

Sperm from birds gives a unimodal peak because all sperm contain the same sex chromosome.

Data collected from a single semen sample are fitted to a pair of Gaussian distributions whose means relative areas and coefficients of variation (CV) are adjusted to give the best least –squares fit to the data (Johnson et al; 1987).

The histograms show illustrate the standard proportions of X and Y sperm in a sample of semen from several mammals ( always 50 : 50 ) .

The species are characterized by differences in DNA content , the latter of which is related to the presence and size of the X or Y chromosome .

The wider the difference , the easier it is to conduct a sort of the two populations . On the other hand, the closer the two peaks; the more difficult it is to sort high purity samples of X or Y sperm.

Aliquot of sperm are removed from the sorting tube , sonicated to remove the tails , and Hoechst 33342 added to maintain staining uniformity and then flow cytometrically analyzed but not resorted . The proportion of X and Y sperm is determined based on the DNA difference and histograms are analyzed by computer fitting to double Gaussian peaks (Johnson et al; 1987).

B – Molecular Genetic Analysis of single sorted sperm (PCR : Polymerase Chain Reaction ) :

This technique can be used as a tool for molecular genetic analysis (Zhang et al; 1992; Arnheim, 1994). This would be important when testing the sperm for allelic disomy . The sperm sorting technique can also be used to search for sex-linked molecular markers .

The PCR is an excellent method for determining X or Y sperm purity and therefore can be used to validate the results obtained by flow-cytometric reanalysis ( Welch et al ; 1995 ) .

The sex chromosome-specific alleles , zfx and zfy ( zinc finger allele which resides on both the X and Y chromosome ) ) Page et al ; 1987 ; Schneider-Gadick et al ; 1989 ) were differentially amplified using allele-specific PCR ( Kirkpatrick and Manson , 1993 ) and detected by agarose gel electrophoresis . Since these molecular markers detected the chromosomal constituents .

The application of PCR to individual sperm is highly accurate in determining the presence of an X or Y sperm.

PCR is also effective for confirming the sex of the embryo after fertilization with sexed sperm under in vitro fertilization ( IVF ) conditions ( Cran et al ; 1993 ) .

Reanalysis can be completed within one hour , but PCR determination takes many hours to complete . Therefore, while lacking a specific marker and being dependent on the separation method itself, quantitative flow cytometric reanalysis is preferred for routine determination of the percentages of X- and Y-chromosome sperm in a given population.

C – Fluorescence In Situ Hybridization ( FISH ) to validate proportions of X and y sperm :

In the livestock semen sexing work they have always used the reanalysis of the sorted sperm populations to determine the proportions of final sorted X or Y sperm.

In the human however, the X and Y DNA difference (2.8 %) is so small, it is difficult to consistently reanalyze the sorted sperm with unquestioned accuracy.

The sorting can be test by using fluorescence in situ hybridization ( FISH ) technology in combination with microsatellite DNA probes ( Johnson et al ; 1993 ) . In this study they used both X and Y probes to eliminate the possibility of counting false positive .

The FISH technology has proved to be an effective method for assessing the proportions of X and Y sperm in a sample of sorted semen or the respective control samples .

Revalidation of a sorted X or Y sperm population before using it for producing embryos by IVF for transfer or by insemination intracervically or intratubaally or into the uterus is essential to knowing the outcome.

Using this monitoring technique one can avoid the expense and consequences of the undesired sex .

The rabbit was chosen as the first experimental animal in which to demonstrate the skewing of the sex ratio ( Johonson et al ; 1989 ) . This initial study involved the staining sperm DNA with Hoechst 33342 .The flow sorting stained X and Y sperm based on the fact that they differ in DNA content by about 3 % ( specific for the rabbit ) and the subsequent insemination of the sorted sperm into the tip of the uterus via surgical means .

litters resulting from insemination of X bearing sperm were 94 % female . Correspondingly the litters resulting from insemination of Y bearing sperm were 81 % male.

The validity of the DNA sexing method was further demonstrated with the production of litters of pigs that showed a skewing of the sex ratio (Johnson, (1988). Swine X and Y sperm differ in DNA content by about 3 % , which should have made the skewing of the sex ratio of the resulting litters easier to achieve than was the case in the rabbit .

Recently, the sorting of the swine sperm was improved so that 85 % skewing of the sex rate can easily be achieved (Johonson and Welch, unpublished data, Hendriksen et al; 1996) using standard protocols.

In the UK , workers at the Mill Hill Medical Research Center were the first to use sperm sorted into X- and Y- enriched populations to inseminate small numbers of cattle ( Morrell et al ; 1988 ) ; the offspring produced showed a statistically significant bias from the normal sex ratio ( 50:50 ) . No evidence of teratogenic effects arising from the use of the fluorochrome DNA-staining dye was evident in studies conducted with sheep , pigs and rabbits ( Morrell and Dresser , 1989 ) .

In the Netherlands, the fluorescence method was used for in vitro production of embryos by the R&D Team of Holland Genetics (Division of CR Delta) in the early 1990, using the flow –cytometer / cell -sorter of the Academical Medical Center in Amsterdam. Unfortunately , fertility sexed sperm was found to be lower and the development of embryos was not as good as with unsexed sperm . This is an indication that there is some negative influence of either the fluorescent-dye or the UV light .

In the USA, the usefulness of the DNA sexing method has been demonstrated by the Beltsville Sperm Sexing Technology for producing off-spring. The initial study resulted in production of six calves, all of the predicted sex (Cran et al; 1993). The second study, a field trial designed to produce male calves from sorted Y sperm, produced 41 calves from 106 embryo transfers. Of the 41 calves 37 was male’s equivalent to a 90 % skewing of the sex ratio (Cran et al; 1995).

In 1993, Johnson et al; (1993), reported the results of studies in which they were able to demonstrate the feasibility of using this technology for human sperm, particularly for those couple who were at risk for a genetic sex-linked disease. The Genetics and IVF Institute, Fairfax, Virginia directed by J.D.Schulman collaborated on this project.

By adapting the sorting technology to a somewhat higher level of detection, they were able to repeatably sort the X and Y human sperm into separate populations.

The human sperm is somewhat more difficult to sort to high purity because it carries only about 2.8 % more DNA in the sex sperm than in the Y sperm.

However, despite the fact that bullet-shaped sperm such as human sperm may be much less easily separated by flow cytomtry than the paddle-shaped sperm of farm ruminants and pigs.

Studies showed that could repeatably separate X sperm with 82 % purity and Y sperm to 76 % purity (Johnson et al; 1993).

Since the initial demonstration that human sperm could be separated into X and Y populations based on DNA (Johnson et al; 1993) difference and flow cytometrically sorting, Levinson et al; (1995) have demonstrated the effectiveness of the DNA sexing method in human clinical trials. This Landmark work has produced the first baby, a girl, born in June of 1995. The parents of the girl chose sex selection to avoid having a son born with the X-linked disease , hydrocephalus . Previous pregnancies and births to this family had been males that expressed the disease.

In 1999 CR Delta and the American XY , Inc in cooperation with the Unity of Amsterdam started the development of a new machine . Basically , this machine measures the size of every passing sperm cell , using a technique that does not require any dye or UV . The method is still under development, but it may become an interesting alternative .

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Ethics Committee of the American Society of Reproduction (1999) – Sex selection and preimplantation genetic diagnosis. Fertil. Steril. 72: 595 – 598.

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Goldberg, E. H., Boyse, E. A., Bennett, D., Scheid, M. and Carswell , E. A. (1971) – Serological demonstration of H-Y (male) antigen on mouse sperm. Nature, 232: 478 – 480.

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Johnson, L. A. (1992) – Gender preselection in domestic animals using flow cytometrically sorted sperm. J. Anim. Sci. 70 (suppl. 2): 8 – 18.

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Johnson, L. A., Aalbers, J. G, Grooten, H. J. G. (1988) – Artificial Insemination of Swine: Fecundity of boar semen stored in Beltsville (BTS), modified modema (MM), or MR-A and inseminated on one, three and four days after collection, Zuchthygiene, 23: 49- 55.

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INFLUENCE OF PRE-MATING ADMINISTRATION OF GNRH ON THE CORPUS LUTEUM ACTIVITY AND SUBSEQUENT FERTILITY IN BUFFALOES DURING SUMMER IN

G.A. MEGAHED* , A.KH. ABDEL-RAZEK* AND H.A. DAGHASH**

The present study was conducted during summer season for 2 consecution year (1998 and 1999) when temperatures were hot enough to cause a measurable stress response in buffaloes. The objective of this work was to determine the effect of GnRH administration at time of breeding on the secretory activity of corpus luteum (C.L) and subsequent fertility under summer stress. Fifty buffalo-cows aged 4 - 7 years were selected for this work. These animals were inseminated artificially using freshly prepared diluted semen. The animals were injected , before insemination, either with 5 ml of receptal (Hochest, each ml contains 0.004 mg buserelin, n =35) or 5 ml physiological saline (control, n = 15). Out of these buffaloes 35 (20 treated and 15 control) were used to monitor progesterone concentration. The blood samples were collected at 7^th day post-insemination then at each 5 days till 47^th day. Conception and pregnancy rates were calculated for all for animals.

The obtained results revealed that, serum progesterone concentrations were markedly increased significantly (P< 0.01) in the treated pregnant animals than in the pregnant control ones. In non-pregnant treated buffaloes, progesterone concentration decreased significantly (P< 0.05) on day 17 post-insemination and at the same time, these animals exhibited the extend signs of heat. The treated buffaloes had a significant increase in the conception and pregnancy rates (74.29% and 76.92% respectively) when compared with control animals (66.67% and 60.00% respectively).

It could be concluded that GnRH administration at the time of breeding before insemination enhanced the secretion of C.L progesterone and could be increased the conception and pregnancy rates overcoming the bad effect of heat stress ,expected during summer, upon the fertility in buffaloes.

INTRODUCTION

Summer conditions, especially heat stress, create a bad effect on reproduction in lactating cattle. Generally, when dairy cattle are moved from their origin to environments of high temperature and humidity, stress responses to summer conditions are severe with a reduction on the reproductive performance (Badinga et al., 1985 and Younas et al., 1993). Although elevated temperature is a factor that contributes to reproductive responses. The primary reproductive responses to heat stress include reduced intensity of estrus and lower fertility (Her et al., 1988; Imtiaz Hussain et al., 1992 and Younas et al., 1993).

Buffaloes are polyoestrous and breed throughout the year. Moreover, buffaloes that calve in summer or autumn resume oestrous cyclicity earlier than those calving in winter or spring (Gorden, 1996 a). High ambient temperature in the summer may depress the libido of the male buffalo and this way contribute to the seasonality pattern of reproduction in the female (Parmar and Mehta, 1992). Moreover, female buffaloes are not as sexually active during the hot summer months as during the winter (Pandey and Razada, 1979). Shah et al., (1989) concluded that winter and spring calving buffaloes need to be pregnant before enter the summer season, otherwise they may remain anoestrous until the autumn season due to the bad effect of summer conditions upon the function of the ovaries.

The concentration of progesterone in body fluid is closely related to growth and secretory activity of the corpus luteum during the different stage of reproduction in bovine. A major factor limiting efficient utilization of buffaloes is their low fertility. In order to offset this problem, systematic and accurate oestrus detections and early detection of mated animals that return to oestrus are necessary, especially in buffaloes since they undergo silent heat (Kamboj and Prakash, 1993).

Gonadotrophin releasing hormone (GnRH) is hypothalamic peptides hormone, which binds to specific high affinity gonadotrophin receptors, to stimulate the release and biosynthesis of LH and FSH. This hormone plays a crucial role in regulating ovarian activity during normal oestrous cycle (Sealfon and Millar, 1995 and Gordon, 1996 b). GnRH has been given at random stages of the oestrous cycle to induce ovulation of the dominant follicle and synchronize emergence of a new wave, but its effectiveness appears to be affected by the stage of follicular development at the time of treatment (Pursley, et al., 1995 and Twagiramungu, et al., 1995).

In an economical study, GnRH treatment was more likely to be beneficial for herds with low fertility than for herds with conception rates more than 60% (Weaver, et al., 1988). The present work aimed to study the influence of GnRH administration before insemination of buffaloes during summer on function of the corpus luteum through progesterone profile and subsequent fertility.

MATERIALS AND METHODS

Fifty multiparious buffalo-cows apparently healthy were included in the present experimental trail which was performed during summer season (from May to August of each 1998 and 1999) at the veterinary service station (VSS) of Mosha village, Assiut. Meterological data at the time of study air temperature and relative humidity (RH %) were 40.2 ±1.8^°C and 59.0 ± 3.0 respectively. Their age ranged from 4-7 years and their parity was 2-4. The animals were submitted to VSS for the purpose of insemination. All of them exhibited the external signs of heat. Gynaecological examination does not reveal any pathological affections. One of the ovaries contained a graffian follicle. These animals were housed under nearly simillar condition of feeding and management with free access to drinking water.

All animals were inseminated artificially using freshly prepared diluted buffalo-semen. A specialist carried out the inseminations using the recto-vaginal technique. The treated animals (n = 35) were injected, before insemination with either 5 ml Receptal (Hochest, each ml contains 0.004 mg buserelin,) while the rest, the control animals (n=15) were injected with physiological saline.

Out of 50 buffalo-cows, 35 animals (20 treated with GnRH and 15 Controls) were used to monitor progesterone concentration. The blood samples (each 10 ml) were collected by direct venipuncture at 7^th day post-insemination then at, 5 day intervals until day 47 . The blood samples were kept at ice box and immediately transported to the laboratory then kept at 4°C for 6-12 h. After centrifugation (at 3000 rpm for 20 min.), serum samples were transferred to labeled vials and maintained at -20 °C until hormonal analysis.

Serum progesterone concentrations were determined by using commercial ELISA Kits (BIOSOURCE, EUROPS S.A). The sensitivity of the assay, defined as the smallest concentration distinguishable from zero, was 0.1 ng/ml. The intra and interassay coefficient of variation were 6.8% and 8.2% respectively.

Conception was calculated for all animals, as the animals which not return to heat after 21 days from insemination. Pregnancy diagnosis was carried out on all animals at day 60-75 post-insemination through rectal palpation.

Data were expressed as the Mean ± S.D for all treatment. Analysis of variance (ANOVA) was done and differences between treatment were analyzed by least significant difference (LSD) using PC-stat computer programme. Results were considered significant at P < 0.05 or less.

RESULTS

The obtained results are presented in Table 1 and Figure 1. Serum progesterone concentrations were higher (P < 0.01) in buffalo-cows treated with GnRH than control. In pregnant treated buffaloes, progesterone showed a significant (P < 0.05) increase until day 27. A further non-significant increase was occurred at day 32 till 47. In non-pregnant treated buffaloes, progesterone concentrations increased significantly (P <0.05) till day 12, then decreased significantly (P < 0.05 ) till day 17 after insemination. The lower level of progesterone was 0.19 ± 0.08 ng/ml which observed at day 22. In addition these animals exhibited the extend signs of heat. Overall means of progesterone in both treated and control pregnant animals were 4.78 ± 1.84 and 4.23 ± 1.76 ng/ml respectively as well as there is a significant difference (P < 0.05) between them. There is non-significant difference between overall means of progesterone in both treated non-pregnant (2.35 ± 1.22) and control non-pregnant animals (2.24 ± 1.15)

The fertility of the studied buffaloes was presented in Fig. 2. Conception rates, as determined by the animals which not returned to heat after insemination by 21 day, were 74.29 and 66.67% for the treated and control buffaloes respectively. Pregnant treated buffaloes had a significant higher (P< 0.01) conception rate in comparison with conception rate of the control animals. Pregnancy rates were 76.92% for treated buffaloes and 60.00% for control animals. In addition, there was a significant (P < 0.01) increase in the pregnancy rates of the treated group.

Table 1: Concentrations (Mean ± S.D) of progesterone (ng/ml) in GnRH treated and control buffaloes.

Post-insemination	Treated Group^§		Control Group^©
Time (days)	Pregnant (n = 14)	Non-pregnant (n = 6 )	Pregnant (n = 10)	Non-pregnant (n = 5)
0	0.52±0.14^a,9	0.48±0.03^a,4	0.51±0.09^a,8	0.47±0.05^a,7
7	2.78±0.32^a,8	2.39±0.46^c,b,5	2.34±0.46^{a, b,7}	2.38±0.56^a,b,c,3,4
12	3.88±0.41^a,7	2.86±0.35^{c, b,2}	3.26±0.39^b,6	2.62±0.57^c,3,4
17	4.63±0.32^a,6	2.34±0.42^c,5	3.98±0.49^b,5	2.37±0.71^c,4
22	5.30±0.31^a,5	0.19±0.08^c,4	4.58±0.57^b,4	0.21±0.07^c,6
27	5.73±0.32^a,4	1.76±0.29^c,3	5.02±0.66^b,4	1.83±0.51^c,5
32	6.02±0.39^a,3,4	2.88±0.28^c,2	5.44±0.43^b,3	2.55±0.49^c,3,4
37	6.15±0.35^a,2,3	3.06±0.34^c,2	5.66±0.34^b,3	2.91±0.53^c,2,3
42	6.33±0.30^a,1,2	3.60±0.32^c,1	5.92±0.36^b,1,2	3.15±0.30^c,2
47	6.46±0.22^a,1	3.98±0.29^c,1	6.10±0.32^b,1	3.73±0.20^c,1
Mean±S.D	4.78±1.84*	2.35±1.22^N.S	4.23±1.76	2.24±1.15

^§ Number of treated animals (n = 20) used to monitor progesterone concentrations.

^© Number of control animals (n = 15) used to monitor progesterone concentrations.

-Means in the same row with the same superscript letters are not significantly different.

-Means in the same column with the same superscript number are not significantly different.

^©Four animals exhibited behavioural heat at 35 and 40 days after insemination.

DISCUSSION

The present results indicated that serum progesterone concentrations after GnRH injection increased significantly in treated pregnant buffaloes than that in pregnant control. These findings are in agreement with those reported by Ullah et al., (1996). They concluded that means progesterone concentrations were higher for the cows treated with GnRH than for control. Similar findings were recorded by Mee et al., (1993) who mentioned that injection of GnRH causes a significant increase in serum progesterone. However, Badr and Sadia (1997) reported that plasma progesterone concentration was significantly higher in both the control and treated pregnant buffaloes than in non-pregnant ones when injected GnRH on day 12 post-service. In contrast, Kirkwood et al. (1990) reported that, there was no significant effect for GnRH on subsequent cow progesterone concentrations either in cultured C.L or plasma.

The etiology of the higher progesterone concentrations is unclear as well as the mechanisms for this stimulatory effect in the corpus luteum is unknown (Kirkwood, et al., 1990 and Ullah, et al., 1996). However, the obtained result attributed to GnRH leads to increasing the proportion of large luteal cells in corpora lutea (Mee, et al., 1993). Indeed, it has been suggested that exogenous GnRH may be enhanced development and functioning of corpora lutea (Macmillan, et al., 1985). This is might be through the relationship between LH and LH receptors concentrations which expected to increase the sensitivity of the corpus luteum to GnRH stimutation (Nusser et al., 1989). Support for this suggestion is provided by the association between GnRH injection at breeding and subsequent higher circulating progesterone concentrations in cattle (Lee, et al., 1985). In addition, Archibong et al., (1987) demonstrated that GnRH injection at the onset of the estrus in gilts leads to an increased number of corpora lutea. Moreover, previous studies have concluded that GnRH may be induced the formation of accessory corpora lutea (Macmillan and Thatcher, 1988, Rettmer, et al., 1992 and Harevey, et al., 1994) or follicle luteinization (Thatcher, et al., 1989. Ryan, et al., 1994).

The treated pregnant buffaloes had a significant increasing concentrations of progesterone as well as increasing in conception and pregnancy rates when compared with pregnant control animals. These results are in agreement with that reported by Mee et al., (1993) and Morgan and Lean (1993). They concluded that, the use of GnRH at time of insemination increased significantly the pregnancy rate. In contrast, BonDurant et al., (1991) reported that no difference in pregnancy rate when cows were treated with GnRH or saline.

There is a clear evidence that GnRH given at oestrus can increase serum progesterone levels and the proportion of large luteal cells in the corpus luteum (Mee et al., 1993). In other studies, indicating improved pregnancy rates in cattle after GnRH injection, it is possible that, this increased LH binding capacity may be mediating the effect (Kirkwood, et al., 1990). It is obvious that the high incidence of early embryonic losses in bovine are associated with low concentrations of progesterone (Lamming, et al., 1989). The establishment of pregnancy depends on a balance between the timing of development of the luteolytic mechanism in the mother and the production of antiluteolytic trophoblastic interferon by the conceptus (Flint, et al., 1992). When this balance fails, embryo loss occurs and this loss continues to be a major cause of reproductive failure in cattle. Progesterone is the main ovarian hormone which influence the development of the luteolytic mechanism (Beard, et al., 1994) and the production of the endometrial secretions necessary for embryo development (Geisert, et al., 1992). Moreover, the latter authors reported that progesterone induces changes in the uterine environment conductive to conceptus growth and developments. They added that, conceptus development may be impaired, when an embryo does not receive sufficient progesterone-mediate stimulus. In addition, progesterone is involved in the control of the development of the luteolytic mechanism itself and inhibiting the development of endometrial oxytocin receptors (Lau, et al., 1992). In cattle with lower progesterone concentrations, the luteolytic mechanism develops earlier as well as stop the ability of the conceptus to produce sufficient trophoblastic interferon to provide an adequate block to luteolysis (Lamming and Mann, 1993).

In conclusion, the present results demonstrated that, the administration of GnRH to buffaloes at the time of breeding increased and improved significantly the conception and pregnancy rates. This might be due to increase in the secretory ability of C.L through increasing the proportion of large luteal cells which subsequently leads to increasing the progesterone production. Generally administration of GnRH reduce the loss of conception and/or pregnancy which would obviously be of great economic benefit to the dairy large or small farm.

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EFFECT OF VITAMIN E AND SELENIUM INJECTION ON ADAPTIVE AND REPRODUCTIVE PERFORMANCE OF MALE RABBITS UNDER HOT CLIMATIC CONDITIONS

This experiment was carried out at Seds Research Station, Bani-Suef Governorate (Middle Egypt), during April to September, 1999, to study the effect of vitamin E (Vit E) alone or with selenium (Se) injections on the adaptive and reproductive performance of New Zealand White (NZW) and Baladi Black (BB) male rabbits. Twelve NZW and 12 BB (aged 8-10 months) bucks were caged individually in cement pen under open shed and maintained under normal nutritional status. The bucks of each breed were randomly divided into 3 groups (each of 4 NZW & 4 BB). The first group was injected subcutaneously with 100 IU Vit E (dl-a tocopherol acetate)/ head/week. The second group was injected subcutaneously with 100 IU Vit E/head/week plus 0.1 mg Se/ kg body weight/ week intramuscularly as sodium selenite. The third group was kept without treatment as control. Semen was collected weekly using artificial vagina to assess semen characteristics. Rectal temperature and respiration rate were measured between 12.00 and 15.00 hr.

Temperature-humidity index from April to September was estimated as 87.2 to 103.9, indicating exposure of the bucks to severe heat stress. Severe hot months (July and August) increased (P<0.05) rectal temperature (°C) and (P<0.01) respiration rate (breaths/minute), decreased (P<0.01) hematocrit (%), hemoglobin (g/dl), plasma testosterone, T₃ , aldosterone levels and semen quality. Vitamin E alone or Vit E + Se injection increased (P<0.05) respiration rate, blood hematocrit and hemoglobin concentration. Moreover, Vit E in combination with Se improved (P<0.01) libido, reaction time, ejaculate volume, mass motility, progressive motility (%), live sperm (%), sperm-cell concentration (x10⁶/ml), total sperm output (x10⁶/ejaculate), and decreased (P<0.05) sperm abnormalities (%). Plasma level of Vit E and Se were higher (P<0.001) in the treated bucks than in the control ones. The NZW bucks were more responsive to the treatment than BB bucks. It could be concluded that injection of rabbit bucks with Vit E+Se during the hot months (April-September) could improve adaptive and reproductive performance.

Key words: Vitamin E, Se, rabbit bucks, semen characteristics, hot climate.

INTRODUCTION

The reproductive efficiency of males depends on their ability to produce large number of viable sperm per ejaculate having good fertilization capacity (El-Harairy, 1981). In middle Egypt, the climate is characterized by long hot (from April to September) period. In hot summer, rabbit bucks suffer from disorders in spermatogenesis, libido, semen quality, in addition to ejaculatory disturbances and reproductive failure (Abdel-Samee et al., 1997). Alleviation of heat-stress can be carried out with minerals, amino acids or vitamins supplementation (Marai et al., 1999b).

Vitamin E acts as a non specific biological antioxidant (Liu, 1988). Its deficiency causes reproductive failure in rabbit (Yamini and Stein, 1989) and as supplementation produces favorable adaptive and reproductive responses, either in thermoneuteral conditions or when exposed to heat stress (Hassanein et al, 1995). Also, Abdel-Samee et al. (1997) stated that vitamin E is important for normal reproduction of the male rabbit under the hot climate conditions. More specific action of vitamin E is associated with selenium, in which it protects vital phospholipids from peroxidative change. Combination of vitamin E and sodium selenate have a synergistic effect on enhancement of cell-mediated immunity in rabbit (Liu, 1988). Blood testosterone concentration and total sperm concentration of NZW rabbit increased significantly by Vit E+Se supplementation (El-Masry et al., 1994).

The aim of the present study was to investigate the efficiency of vitamin E alone or with selenium injection on the adaptive and reproductive responses of NZW and BB bucks under hot climate conditions of middle Egypt.

MATERIALS AND METHODS

The present study was conducted on 12 New Zealand White rabbit bucks (NZW) and 12 Baladi Black (BB) aged 8-10 months. The bucks were raised at Animal Production Research Station at Seds, Bani-Suef Governorate (middle Egypt), belonging to Animal Production Research Institute, Ministry of Agriculture, during the period from April to September, 1999. The bucks were individually caged in cement pen (85 x 60 x 60 cm) under open shed with 6 metres height. The bucks in each breed were randomly divided into three groups (each of 4 NZW and 4 BB bucks) and maintained under normal nutritional status. The first group was injected subcutaneously with 100 IU vitamin E (Vit E)/ head/ week as dl-a tocopherol acetate (Cairo Company for Medicine) dissolved in soybean oil. The second group was treated with the same dose of Vit E and injected intramuscularly with 0.1 mg selenium (Se)/ kg body weight/ week as sodium selenite. The third group was kept without treatment as a control. Semen was collected weekly from the bucks using artificial vagina to assess semen characteristics according to Salisbury et al. (1978). Libido was determined as the time elapsed between the introduction of the doe to the buck and the collection of ejaculate. Reaction time was recorded from the moment that the buck mounting the doe and the completion of ejaculation. Rectal temperature in°C (RT) and respiration rate in breaths/minute (RR) were weekly measured between 12.00 and 15.00 hr.

Blood samples were collected from marginal ear vein weekly in a heparinized tubes. Hematocrit (%) and hemoglobin concentration (g/dl) were determined in the fresh blood. Heparinized blood samples were centrifuged at 3000 rpm. for 15 minutes and plasma was separated and stored at -20 °C till analysis. Plasma vitamin E concentrations were determined using High Performance Liquid Chromatography (HPLC) according to the method adopted by Bieri et al. (1979) under the following conditions: Column: Bond clone x 18; Mobile phase: Methanol : water as 95 : 5; Flow rate: 1.5 ml/minute; Detector: Spectro colorimeter; Injection volume: 20 ug; Weave length: 365 nm and Temperature: Under room temperature. Plasma Se concentrations were determined using Unicam 929 Atomic Absorption Spectrometer under the following conditions: Instrument mode: Furnace; Spectrometer mode: Absorbance; Wave length : 196.0 nm; Bandpass: Full 0.5; Lamp current: 80%; Transient type: height.

Plasma testosterone level of the bucks was determined (RIA kits from Immunotech, A Coulter Co., France) according to the manufacturer information. The antiserum used had cross reaction values of 100% with testosterone, £0.03% with progesterone and 0.002% with corticosterone. The standard curve ranged between 0.0 and 20 ng/ml and sensitivity value was reported to be 0.025 ng/ml. The intra- and inter-assay coefficients of variation were found to be 7.2 and 15.0%, respectively. Plasma triiodothyronine (T₃) was determined (RIA kits from Diagnostic System Laboratories, Inc., USA) according to the manufacturer information. The antiserum used had cross reaction values of 100% with triiodo-L-thyronine, 2.76% with triiodothyroacetic acid and 0.001% with 3,5-diiodo-L-tyrosine. The standard curve ranged between 0.0 and 1000 ng/dl and sensitivity value was reported to be 4.3 ng/dl. The intra- and inter-assay coefficients of variation were found to be 5 and 6%. Plasma aldosterone was determined (RIA kits from Diagnostic System Laboratories, Inc., USA) according to the manufacturer information. The antiserum used had cross reaction values of 100% with aldosterone, 0.03% with corticosterone and 0.05% with deoxycorticosterone. The standard curve ranged between 0.0 and 1600 pg/ml and sensitivity value was reported to be 25 pg/ml. The intra- and inter-assay coefficients of variation were found to be 3.6 and 7.3%, respectively.

Maximum and minimum air temperature (°C), relative humidity (RH%) and temperature-humidity index (THI) under shed during the experimental period are shown in Table 1. THI was estimated according to Livestock and Poultry Heat Stress Indices, Agricultural Engineering Technology Guide, Clemson University, Clemson, Sc 29634, USA, using the following formula: THI= db°F -(0.55 - 0.55 RH)(db°F - 58.00), where db°F = dry bulb temperature in Fahrenheit and RH = RH% ¸100. The obtained values of THI were classified as follows: less than 82 = absence of heat stress, 82 to < 84 = moderate heat stress, 84 to <86 = severe heat stress and those over 86 = very severe heat stress.

The data were analyzed using the following model in GLM procedure in SAS^Ò program (1988). The significance of differences among means was evaluated by Duncan’s New Multiple Range Test.

Y_ijklm	=	m + T_i + C_j (T_i) +B_k + M_l+T_i * B_k + T_i * M_l + B_k * M_l + T_i * B_k * M_l + e_ijklm
Where:
Y_ijklm	=	An observation on the buck,
m	=	Common mean,
T_i	=	Fixed effect of the i^th treatment (i= 1, 2 & 3),
C_j (T_i)	=	Random effect of the j^th buck within i^th treatment,
B_k	=	Fixed effect of the k^th breed (k= 1 & 2),
M_l	=	Fixed effect of l^th month of injection (l= 1, 2,......,6),
T_i* B_k	=	Interaction between the i^th treatment and k^th breed,
T_i* M_l	=	Interaction between the i^th treatment and l^th month of injection,
B_k* M_l	=	Interaction between the k^th breed and l^th month of injection,
T_i* B_k* M_l	=	Interaction between the i^th treatment, k^th breed and l^th month of injection, and
e_ijklm	=	The random error.

Table (1): Maximum and minimum values of ambient temperature (°C), relative humidity (%) and temperature humidity index (THI) in the rabbitry during the experimental period under middle Egypt conditions.

Months	Ambient Temperature (°C)		Relative humidity (%)		Temperature humidity index (THI)
	Max	Min	Max	Min	Max	Min
April	31.5	14.3	91.0	23.0	87.2	57.9
May	36.5	20.0	87.0	23.0	94.9	63.8
June	38.4	22.8	90.0	26.0	98.8	66.9
July	41.2	24.1	90.0	28.0	103.5	68.5
August	41.3	24.7	91.0	30.0	103.9	69.4
Sept.	37.7	23.6	91.0	35.0	97.8	68.6

RESULTS AND DISCUSSION

The maximum THI values in Table 1 indicating exposure of the bucks to very severe heat stress. These findings are higher than those (86.0) reported by Marai et al. (1999a) during summer at east Delta region of Egypt.

Thermoregulatory Responses:

Increase of air temperature from month to month during the experimental period caused an increase (P<0.05) in RT (Table 2). These results agree with Amici and Merendino (1996). Also, the increase of air temperature increased (P<0.01) RR (Table 2). Lebas et al. (1986) explained that when ambient temperature increased, the animal pants to enhance heat loss through water evaporation.

Injected bucks with Vit E alone or Vit E + Se increased nonsignificantly RT and significantly (P<0.05) RR (Table 2). Amici and Merendino (1996) found a positive correlation between rectal temperature and plasma concentration of vitamin E. Also, Hassanein et al. (1995) found that Vit E supplementation increased respiration rate.

The NZW bucks injected with Vit E+Se had higher (P<0.05) RT and RR than BB bucks (Table 2). Similar trend was obtained by Hassanein et al. (1995). The bucks treated with Vit E+Se recorded the highest RT in July and RR in August (Figure 1).

Hematological Responses:

Increased ambient temperature and relative humidity by advancing month from April to August led to decrease the Ht and Hb values (Table 2 and Figure 1). Similar results were obtained by Habeeb et al. (1994).

Injected bucks with Vit E alone or Vit E+Se increased (P<0.05) Ht values by 7.6 and 8.6%, respectively, and increased Hb values by 9.2 and 10.7%, respectively, than control group (Table 2 and Figur 1). These results were higher than those reported by Hassanein et al. (1995) that Vit E supplementation improved Ht and Hb values by 7.2 and 7.0% respectively, than control.

Baladi Black bucks had higher (P<0.05) Ht value and nonsignificantly higher Hb value than NZW bucks (Table 2). However, Hassanein et al. (1995) found that NZW had higher Ht and Hb values than Baladi rabbit.

Table (2): Rectal temperature (RT), respiration rate (RR), hematocrit% (Ht) and hemoglobin (Hb) concentration of NZW and BB rabbit bucks as affected by month and treatment (Means ± SE)

Item	No. Obs¹	RT (°C)	RR (Breaths/min.)	Ht (%)	Hb (g/dl)
Overall mean	576	39.47±0.81	137.5±8.2	33.17±0.82	11.79±0.82
Month:		*	**	**	**
April	96	39.12±0.16^b	102.3±1.7^e	35.58±0.17^a	12.90±0.16^a
May	96	39.32±0.16^b	123.5±1.7^d	34.33±0.17^b	12.23±0.16^b
June	96	39.55±0.16^b	139.8±1.7^c	32.78±0.17^d	11.68±0.16^c
July	96	40.10±0.16^a	158.0±1.7^b	31.58±0.17^e	11.10±0.16^d
August	96	39.43±0.16^b	163.9±1.7^a	31.00±0.17^f	10.78±0.16^d
September	96	39.28±0.16^b	137.6±1.7^c	33.77±0.17^c	12.07±0.16^bc
Treatment:		NS	*	*	*
Control	192	39.35±0.12^a	132.6±1.2^b	31.47±0.12^c	11.06±0.11^b
Vit E	192	39.47±0.12^a	134.7±1.2^b	33.86±0.12^b	12.08±0.11^a
Vit E+Se	192	39.58±0.12^a	145.2±1.2^a	34.19±0.12^a	12.24±0.11^a
Breed:		*	*	*	NS
NZW	288	39.72±0.09^a	145.3±0.96^a	32.96±0.10^b	11.76±0.09^a
BB	288	39.21±0.09^b	129.7±0.96^b	33.39±0.10^a	11.83±0.09^a
Treatment*Breed:		NS	**	*	NS
Control * NZW	96	39.52±0.2^a	143.1±1.7^b	30.84±0.2^d	10.90±0.16^a
Control * BB	96	39.18±0.2^a	122.1±1.7^e	32.10±0.2^c	11.22±0.16^a
Vit E * NZW	96	39.73±0.2^a	140.0±1.7^bc	33.92±0.2^b	12.12±0.16^a
Vit E * BB	96	39.20±0.2^a	129.5±1.7^d	33.80±0.2^b	12.05±0.16^a
34.12±0.2^ab	96	39.92±0.2^a	152.8±1.7^a		12.25±0.16^a
Vit+Se * BB	96	39.25±0.2^a	137.7±1.7^c	34.27±0.2^a	12.23±0.16^a

¹ =Number of observation. * = (P<0.05). * * = (P<0.01). NS= Non Significant. NZW= New Zealand White. BB= Baladi Black.

^a,b,c,d,e,f Values with the same superscripts in the same column within item are not significantly different (P>0.05).

Figure (1): Rectal temperature (°C), respiration rate (breaths/minute), hematocrit (%) and hemoglobin concentration (g/dl) of rabbit bucks as affected by month and treatment.

Libido, Reaction Time and Semen Characteristics:

The lowest values for semen characteristics were obtained during July and August months (Table 3 and Figure 2). Semen quality was deteriorated as a result of the harmful effect of the severe hot weather prevailed. These findings agree with the findings of Marai et al. (1998). Chou et al. (1974) reported that increasing ambient temperature lead to the degeneration of the germinal epithelium and partial atrophy in the seminiferous tubules. Also, O’loufa et al. (1951) mentioned that the increased the percentage of dead spermatozoa in high ambient temperature (35 ºC) is due to sustained damage during the late stage of spermatogenesis.

Bucks injected with Vitamin E showed improved libido, reaction time and increased (P< 0.01) ejaculate volume by 32.14%, sperm cell concentration (x10⁶/ml) by 12.6%, total sperm output (x10⁶/ejaculate) by 42.2%, mass motility by 10.1%, progressive motility by 11.3%, live sperm by 5% and reduced (P< 0.01) sperm abnormality by 17.0% than control group (Table 3). Similar findings were obtained by Magda-Salem (1995). Manner and Mason (1975) mentioned that Vit E has been recognized as a requirement for normal testicular function. Abdel-Samee et al. (1997) concluded that Vit E is important for normal reproduction of the male rabbit under hot climatic conditions. The improvement of spermatozoan motility could be attributed to the effect of Vit E on epithelial cells of the reproductive tract of bucks that responsible for acquiring the progressive motility spermatozoa. Improving of live spermatozoa might be due to the effect of Vit E on maintaining the viability and permeability of cell membranes of the spermatozoa. However, injected bucks with Vit E+Se increased ejaculate volume by 8.1%, sperm-cell concentration by 6.3%, total sperm output by 17.2%, mass motility by 2.6%, progressive motility by 2.1% and live sperm by 1.34% than bucks injected with Vit E alone (Table 3 and Figure 2). Similarly, El-Masry et al. (1994) reported that semen quality was significantly increased due to Vit E and Se supplementation for heat stressed rabbit bucks. Close (1999) reported the important role of Se in improving semen quality of Sow.

The NZW bucks had higher (P<0.05) ejaculate volume, mass motility, live sperm % and abnormal spermatozoa % than BB bucks (Table 3 and Figure 2). These results are in agreement with Abo-Warda (1994) who mentioned that Baladi breed had a significantly lower percentage of abnormal sperm-cells than Bouscat, Flander and Pepion rabbits. On the contrary, El-Harairy (1981) reported that the percentage of abnormal spermatozoa was higher in Baladi than the Bouscat rabbit. Sperm-cell concentration was higher in BB bucks than NZW rabbit by 11.75% (Table 3). This finding is in accordance with El-Harairy (1981) who found that ejaculates of Baladi rabbit had higher sperm cell concentration than that of Bouscat rabbits.

The NZW bucks were more responsive to the injection of Vit E+ Se than the BB bucks for ejaculate volume, mass motility, progressive motility and live sperm% (Table 3 and Figure 2).

Plasma Vit E and Se Concentrations:

The bucks injected with Vit E alone or Vit E+Se had higher (P<0.001) plasma Vit E level than the control bucks (Table 4). These findings agree with Castellini et al. (2000). Plasma vitamin E level of BB was higher (P<0.05) than NZW by 15.4% (Table 4). The bucks injected with Vit E+Se had higher plasma Vit E level than those injected with Vit E alone and control groups after 2, 4 and 6 months of the initiation of the experiment (Figure 3). Similar results were reported by Walsh et al. (1993) in calves.

Plasma Se concentration increased (P<0.05) by advancing interval from 0.0162 mg/ml after two months to 0.0282 mg/ml after 6 months of the initiation of the treatment (Table 4). Similar trend was observed by Metry et al. (1998) in buffalo calves. Selenium with Vit E injection increased (P<0.01) plasma Se level than Vit E and control groups (Table 4). This is in accordance with that reported by Stowe and Herdt (1992) and El-Gaafrawy et al. (2000) who found that Se in combination with Vit E increased plasma Se level. Treatment with Vit E+Se increased (P<0.01) Se concentration in plasma of bucks at intervals of 2, 4 and 6 months after the onset of treatment (Figure 3).

Hormonal Responses:

Increased ambient temperature from April to August decreased (P<0.01) plasma testosterone hormone concentration (Table 5). Similar trend was reported by El-Masry et al. (1994). The decline in testosterone level during summer season causing disorders in accessory glands secretion and spermatogenesis (Katongola et al. 1974). This reduction resulted from reducing the ability of leyding and sertoli cells to respond to LH (Jegou et al., 1984).

Injection with Vit E+Se increased the testosterone level by 21.1% than the injection with Vit E alone (Table 5). These findings agree with El-Masry et al. (1994) in rabbit and Metry et al. (1999) inbuffalo. Behne et al. (1991) reported that selenium is necessary for the biosynthesis of testosterone

Table (4): Plasma Vit E (mg/ml) and Se (mg/ml) concentrations of NZW and BB rabbit bucks as affected by month and treatment (Mean±SE

		Intervals (Month)				Treatment			Breed
	Item	0	2	4	6	Control	Vit E	VitE+Se	NZW	BB
No. of observations		-	10	10	10	6	12	12	15	15
		**				***			*
Plasma VitE (mg/ml)		-	11.05^c ±0.03	14.28^b ±0.03	19.87^a ±0.03	1.68^b ±0.04	20.86^a ±0.04	22.65^a ±0.04	13.98^b ±0.02	16.14^a ±0.02
No. of observations		16	16	16	16	16	16	32	32	32
		*				**			NS
Plasma Se (mg/ml)		.0162^d ±0.003	.0199^c ±0.002	.0253^b ±0.002	.0282^a ±0.002	.0157^b ±0.002	.0174^b ±0.001	.0342^a ±0.001	.0221^a ±0.002	.0227^a ±0.002

* =(P<0.05). ** =(P<0.01). *** =(P<0.001). NS= Nonsignificant. NZW= New Zealand White. BB= Baladi Black.

^a,b,c,d Values with the same superscripts in the same row within intervals, treatment or breed are not significantly different (P>0.05).

Figure (3): Plasma Vit E (dl-a tocopherol) and selenium concentrations in rabbit bucks, during post-treatment period.

and the production and normal development of the spermatozoa in male rats. Also, Youssef et al. (1997) noticed a significant increase of serum testosterone level after Se injection in low fertile Egyptian buffalo-bulls. The authors suggested that Se may have an effect directly on the interstitial cells of testes or indirectly via its effect on the anterior pituitary hormone secretion.

The NZW bucks had nonsignificantly higher plasma testosterone concentration than BB bucks (Table 5). Although, testosterone levels were decreased linearly in all groups from April to August, bucks injected with Vit E+Se had the highest level of testosterone followed by those injected with Vit E alone and control groups, respectively, (Figure 4).

The increase in air temperature from April to August decreased (P<0.01) plasma T₃ levels (Table 5 and Figure 4). Similar trend was observed by Chiericato et al. (1995) and Habeeb et al. (1999). Vitamin E alone and Vit E+Se injection increased (P<0.05) T₃ level by 24.5 and 28.1%, respectively, than control group (Table 5 and Figure 4). Behne et al. (1990) found that Se was involved in the formation of the active form of thyroxin. Baladi Black bucks had higher (P<0.05) plasma T₃ concentration than NZW rabbits. The highest plasma T₃level (199.7 ng/dl) was obtained for BB bucks treated with Vit E+Se (Table 5). This finding could be explained by that BB bucks injected with Vit E+Se were more adaptive than NZW bucks for increasing ambient temperature.

There was a trend toward decreasing (P<0.01) plasma aldosterone level from May to August due to the increase of ambient temperature (Table 5 and Figure 4). These results are in agreement with the findings of Abdel-Samee et al. (1997) who reported that under hot climatic conditions and in attempt by the animal to decrease it’s endogenous heat production to tolerate heat, most of anabolic and thermogenic hormones such as T₃ and aldosterone decrease appreciably.

Table (5): Plasma testosterone (ng/ml), triiodothyronine (T₃, ng/dl) and aldosterone (pg/ml) levels of NZW and BB rabbit bucks as affected by month and treatment (Means±SE)

Item	No. of Obs¹	Testosterone (ng/ml)	Triiodothyro-nine (ng/dl)	Aldosterone (pg/ml)
Overall mean	144	4.92±0.81	181.25±8.16	544.4±8.01
Month:		**	**	**
April	24	5.89±1.6^a	202.7±16.7^a	530.0±16.4^c
May	24	5.52±1.6^ab	198.3±16.7^a	732.4±16.4^a
June	24	4.79±1.6^c	177.4±16.7^c	561.0±16.4^b
July	24	3.89±1.6^d	173.9±16.7^c	490.9±16.4^d
August	24	4.12±1.6^d	152.5±16.7^d	412.9±16.4^e
September	24	5.34±1.6^b	182.7±16.7^b	539.1±16.4^c
Treatment:		**	*	**
Control	48	3.12±1.2^c	154.2±11.8^b	405.6±11.6^c
Vit E	48	5.27±1.2^b	192.0±11.8^a	583.4±11.6^b
Vit E+Se	48	6.38±1.2^a	197.6±11.8^a	644.1±11.6^a
Breed:		NS	*	**
NZW	72	5.01±0.9^a	177.4±9.6^b	487.5±9.4^b
BB	72	4.84±1.0^a	185.1±9.6^a	601.3±9.4^a
*Treatment Breed:**		NS	*	*
Control * NZW	24	3.19±1.6^a	145.8±17.0^d	396.8±16.0^e
Control * BB	24	3.05±1.6^a	162.5±17.0^c	414.4±16.0^e
Vit E * NZW	24	5.36±1.6^a	190.8±17.0^b	507.4±16.0^d
Vit E * BB	24	5.19±1.6^a	193.2±17.0^ab	659.3±16.0^b
Vit E+Se * NZW	24	6.47±1.6^a	195.5±17.0^ab	558.3±1.60^c
Vit E+Se * BB	24	6.29±1.6^a	199.7±17.0^a	730.0±1.60^a

* = (P<0.05). * * = (P<0.01). NS= Non Significant. NZW= New Zealand White. BB= Baladi Black.

^a,b,c,d,e Values with the same superscripts in the same column within item are not significantly different (P>0.05).

Bucks injected with Vit E and Vit E+Se had higher (P<0.01) plasma aldosterone concentration than the control group (Table 5 and Figure 4). Dvorak (1977) mentioned that vitamin E has been found to increase resistance to stress by increasing the adrenocortical activity.

Baladi Black bucks had higher (P<0.01) plasma aldosterone concentration by 23.3% than NZW bucks. In addition the BB bucks injected with Vit E+Se had the highest plasma aldosterone level (Table 5).

CONCLUSIONS

Vitamin E and selenium could be used to overcome the temporary sterility, which commonly occurred for bucks during hot summer months. More investigations are needed to evaluate the effect of Se and higher doses of Vit E and Se.

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EFFECT OF PROSTAGLANDIN F₂_µ ADMINISTRATION ON BODY TEMPERATURE AND PLASMA CORTISOL CONCENTRATION IN EGYPTIAN BUFFALOES

Department of Animal Production, Faculty of Agriculture, Ain Shams University,

ABSTRACT

The objective of the present study was to investigate the influence of prostaglandin F₂_µ (PGF₂_µ) administration on rectal , vaginal and skin temperatures , respiration rate , pulse rate and plasma cortisol concentration in postpartum suckling Egyptian buffaloes. Eight healthy multiparous buffalo cows were randomly divided into two equal groups. The animals in the first group were injected intramuscularly with 5 ml of isotonic saline and served as control, while those in the second group were injected with 25 mg PGF₂_µ/ animal. Data were collected just before injection (0 time) , then at 15 and 30 minutes , 1 , 2 , 3 , 6 , 12 and 24 hours after injection. Administration of PGF₂_µ caused an increase in rectal , vaginal and skin temperatures at 12 hours post – injection , compared with the pre-injection (P>0.01) and control (P>0.05) values . Moreover , PGF₂_µ induced an earlier (15 and 30 minutes post-injection) increase in pulse rate , compared with the pre-injection (P>0.01) and control (P>0.05) values . Respiration rate also increased (P>0.01) at one hour post-PGF₂_µ injection , compared with the pre-injection basal level . However, PGF₂_µ administration resulted in a decline in plasma cortisol concentration at 3 and 6 hours post-injection as compared with the pre-injection (P> 0.01) or the control (P>0.05) values. It is concluded that PGF₂_µ administration may induce a hyperthermic effect associated with hypocortisolemia in buffalo cows.

Prostaglandin E series (PGE) and prostaglandin F series (PGF) are of great biological interest. The most closely compounds associated with reproduction are PGF₂_µ and PGE₂. Prostaglandin F₂_µ is widely used as a luteolytic agent to control oestrus in several animal species (Hackett et al., 1981 ; Diaz et al., 1994 ; Pankowski et al., 1995 ; Pursley et al., 1995). Quite apart from control of oestrus , prostaglandins have been also used in dairy reproductive herd health programmes (Wenzel et al., 1995). Moreover , it has been reported that prostaglandin E and F series are highly active hyperthermic agents in the brain (Hoffman et al. , 1986).

A change in maternal body temperature prior to parturition and ovulation has been reported in different animal species (Ruppenthal and Goodlin , 1982 ; Ammons et al., 1989 ; Mosher et al., 1990 ; Cross et al., 1992). The concentration of PGF₂_µ is greatly elevated shortly before parturition (Haluska and Currie , 1988) and this is temporally associated with the prepartum drop in maternal body temperature. Moreover, exogenous administration of PGF₂_µ was found to reduce body temperature in mares (Cross and Threlfall, 1995). However, contradictory results were obtained by Emtethal et al. (1994) and Fayed and Abdalla (1999) who revealed that administration of PGF₂_µ induced a hyperthermic effect in rabbits. The effect of PGF₂_µ on the thermoregulatory mechanisms in buffaloes has not been investigated. Therefore, the objective of this study was to determine the effect of exogenous PGF₂_µ on the body temperature of buffalo-cows. The possible effect of PGF₂_µ on the cortisol concentration was also evaluated.

Eight Egyptian buffalo-cows calved within about 10 days , weighing approximately 450 kg and raised at the Experimental Research Station, Faculty of Agriculture, Ain Shams University, Cairo were used in this study .All animals were kept under the same managerial conditions, tie–housed in semi–shed open yards. Animals were fed about 7 kg / head / day of pelleted concentrate mixture, while the rice straw and drinking water were allowed at all times. The buffalo cows were allowed to nurse their calves twice daily at 10:00 am and 2:00 pm, and kept with their calves in pens during night. This experiment was conducted in May when ambient temperature and relative humidity ranged from 22 to 31°C and 38 to 60 % , respectively.

At about 30 - 40 days postpartum , the animals were divided into two equal groups and the following measurements were taken in both groups just before treatment (basal or pre-injection values). Rectal , vaginal and skin temperatures were recorded using a tele-thermometer (model 43 TD, Yellow Springs Instrument Co., Ohio, USA). Pulse rate was measured manually , using the index finger from the ventral coccygeal artery for 15 seconds . Respiration rate was counted from the flank movement for one minute. Jugular blood samples were withdrawn by venipuncture , then plasma was separated by centrifugation (20 min at 1,500 X g) and stored at – 20 °C for cortisol determination by RIA kits (Immunotech, A Coulter Company , France).

At 9:00 am , buffaloes in the first group were injected intramuscularly with 5 ml isotonic saline (0.9% Na C1) to serve as control, while animals in the second group were injected on the same day with PGF₂_µ (Lutalyse , The Upjohn Company , Belgium) at a dose of 25 mg / animal . In the two groups , all the above mentioned measurements were also taken at 15 and 30 minutes , 1 , 2 , 3 , 6 , 12 and 24 hours post-injection. Data were analyzed by least squares analysis of variance using the General Linear Models procedure of the Statistical Analysis System (SAS, 1985).

RESULTS

Average rectal (RT), vaginal (VT), and skin (ST) temperatures gradually increased in response to PGF₂_µ injection , reaching a peak (P>0.01) at 12 hours post-injection (Table 1). Thereafter , they receded toward their basal levels at 24 hours post-injection. Moreover , RT, VT and ST were higher (P>0.05) in the PGF₂_µ - treated group than the control at 12 hours post-injection . Prostaglandin F₂_µ induced a marked (P>0.01) increase in the pulse rate (PR) at 15 and 30 minutes post-injection, then it receded toward its pre-injection basal level with advancing time (Table 2) . Also , the PGF₂_µ - injected buffaloes had greater (P>0.05) PR than control animals at 15 and 30 minutes post-injection. Moreover, PGF₂_µ administration increased (P>0.01) respiration rate (RR) after one hour from injection as compared to pre-injection basal level.

On the other hand, PGF₂_µ administration decreased plasma cortisol concentration at all times of the experiment , being significant (P>0.01) starting from 3 up to 12 hours after injection as compared with the pre-injection basal level (Table 2) . A marked decrease (P>0.05) in plasma cortisol levels was also detected in the treated group at 3 , 6 and 24 hours post-injection , compared with the corresponding values in the control group. Present results (Table 2) also indicated that the circadian rhythm of plasma cortisol concentration was nearly similar in both groups, being higher in the morning (at 0 time ; 9:00 am) and lower in the evening (at 12 hours ; 9:00 pm) . However, the decrease in the cortisol level occurred earlier ; at mid-day (at 3 hours ; 12:00 pm) in the PGF₂_µ - injected group and remained lower until late evening.

Time	Rectal temperature		Vaginal temperature		Skin temperature
Time	Control	PGF₂_µ	Control	PGF₂_µ	Control	PGF₂_µ
0	38.4	37.9	38.1	37.7	33.1	32.2
15 min	38.5	37.9	38.1	38.0	33.5	32.5
30 min	38.4	38.2	38.1	37.9	34.1	33.1
1 hr	38.5	38.3	38.1	38.1	34.0	33.3
2 hr	38.4	38.3	38.0	38.0	34.1	33.4
3 hr	38.5	38.4	38.2	38.1	34.4	33.6
6 hr	38.6	38.4	38.2	38.3^*	34.3	34.8^*
12 hr	38.3	38.9^*,⁺	38.1	38.6^*,+	29.8	34.9^*,+
24 hr	38.0	38.2	37.9	37.8	31.5^*	32.6
SE	0.22	0.22	0.20	0.20	0.48	0.48

^* Significant at P>0.01 compared with the basal pre-injection level within the same group.

Time	Pulse rate		Respiration rate		Cortisol level
Time	Control	PGF₂_µ	Control	PGF₂_µ	Control	PGF₂_µ
0	64	61	19	17	22.2	17.0
15 min	62	69^*,+	20	18	20.0	16.6
30 min	64	70^*,+	20	19	18.8	15.4
1 hr	64	65	21	22^*	19.6	17.3
2 hr	62	61	19	20	19.5	15.4
3 hr	63	62	20	19	18.9	12.2^*,+
6 hr	63	63	21	20	19.6	12.3^*,+
12 hr	60	62	17	16	15.7^*	11.9^*
24 hr	62	62	18	18	20.9	13.9⁺
SE	1.9	1.9	0.99	0.99	2.4	2.4

^* Significant at P>0.01 compared with the basal pre-injection level within the same group.

DISCUSSION

Administration of PGF₂_µ had a significant hyperthermic effect in the suckling buffaloes after 12 hours from its injection, as indicated by increasing RT, VT and ST. This finding is different from that reported by Cross and Threlfall (1995) who showed a decrease in body temperature of PGF₂_µ- injected mares. One of the most important side effects of exogenous PGF₂_µ treatment in mares is sweating and this may account for such decrease observed in the body temperature . However , heat loss through sweating is poorly displayed in buffaloes , but instead an elevation in PR and RR was developed by PGF₂_µ injection, probably to decrease body temperature through radiation and respiratory evaporative cooling mechanisms. However, the elevated PR and RR in response to PGF₂_µ injection were noted within 30 minutes and lasted approximately 60 minutes, while developing hyperthermia occurred at 12 hours post-injection.

The exact mechanism by which PGF2µ increased RT, VT and ST in buffaloes is not fully understood. Prostaglandin F2µ is known to have a vasoconstrictive effect (Knickerbocker et al.,1988) , but can increase the firing rate of warm sensitive neurons in the hypothalamus resulting in stimulation of neurons that control heat loss responses such as sweating (Boulant,1991). Moreover, the elevated body temperature following PGF₂_µ administration may be attributed to the release of some endogenous secretions which may induce hyperthermia. It has been reported that endogenous inflammatory substances such as some prostaglandins (PGE) were released and induced an increase in body temperature (Boulant, 1991). A similar hyperthermic effect caused by PGF₂_µ injection was also reported in rabbits (Emetethal et al., 1994 ; Fayed and Abdalla, 1999) and in hens and turkeys (Fayed, 2000). The latter concluded that hens were more sensetive to PGF₂_µ- induced hyperthermia than turkeys.

In the present study, PGF₂_µ treatment caused a significant decrease in plasma cortisol concentration after 3 hours from administration . This decrease was maintained till 24 hours post-injection. The exact mechanism by which PGF₂_µ reduced cortisol level is not well known . There are two possible mechanisms to reduce the cortisol level. First , a reduced biosynthesis of cortisol due to decrease cholesterol uptake by adrenal cortical cells. Second , a reduced cortisol release due to the inhibition of hypothalamic corticotrophin releasing factor (CRF) and / or inhibition of adrenocorticotropic hormone (ACTH) secretion.

In the corpus luteum, the antisteroidogenic actions of PGF₂_µ were mediated through activation of the protein kinase C pathway (Niswender et al., 1994). Prostaglandin F₂_µ decreased plasma membrane phospholipid fluidity, increased superoxide radical formation, increased intracellular calcium level and increased heat shock protein 70 which interrupted the translocation of cholesterol into the mitochondria and reduced progesterone biosynthesis in the corpus luteum (Sawada and Carlson, 1991; Pepperell et al., 1995 ; Khanna et al., 1995). These mechanisms may also occur in the adrenal cortex resulting in a decreased cortisol biosynthesis, but this hypothesis need investigations. Anyhow, the effect of PGF₂_µ and its metabolites on hypothalamic CRF and pituitary ACTH is not fully clear. In conclusion , PGF₂_µ administration influenced thermoregulatory responses and reduced plasma cortisol concentration of suckling buffaloes. The author speculates that the administration of PGF₂_µ under heat stress conditions could aggravate the hyperthermic reaction of animals.

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VIABILITY AND FUNCTION OF BOVINE SPERMATOZOA IN ELECTROLYTE - FREE COLD EXTENDER

^**Dept. Anim. Repd., National Institute of Animal Industry, Kukizaki, Ibaraki, 305- 0901,Japan

Bovine spermatozoa were preserved in electrolyte free extender (EFE) after being separated from seminal plasma by either simple washing procedure(EFE-W) or centrifugation in a discontinuous Percoll gradient(EFE-P). The respective effects on sperm motility, alive percentage and acrosome status were analysed in comparison with control sample extended by egg yolk citrate (EGC). The samples extended with EFE after Percoll separation (EFE-P) showed a significantly higher percentage of motile, alive and intact acrosome cells.

In vitro matured oocytes inseminated with EFE-P had lower penetration rate than that of EFE-W. The later showed a higher percentage rate of polyspermia after one day storage. After four days storage, EFE-P showed higher percentage of penetrated oocyte than EFE-W, but with higher number of polyspermia.

In conclusion, spermatozoa separated by Percoll and stored in EFE showed long durability for function and fertilizability.

Preservation of livestock semen requires either complete arrest of the sperm metabolism by cryopreservation, or just reduction of this metabolism by liquid refrigeration.

However cryopreservation of spermatozoa results in motility percentage between 36.43 and 67.5% (Kjaestad et al 1993 and Kloves, 1997) and conception rate ranged from 32% to 67% (Mortimer et al 1976 and Johnson et al 1995). Liquid semen stored at 4 ^oC in buffered media resulted in higher motility and higher fertility rate (Vishwanath,et al,1996).

At low temperature, the activity of Na-K dependent adenosine triphosphatase (ATP- ase) decreased. Subsequently extra cellular sodium ions enter the cells causing swelling and rupture of the cells. This explains short life span of cells stored at refrigerator temperature (Gruener and Avi-Dor, 1966).

Recently, Saito et al (1996a) and Kanno et al (1998) stated that when electrolyte surrounding the sperm was depleted by washing or separation with Percoll gradient and extended with electrolyte free extender, the motility rate reached 40% after two weeks preservation. In this case the activity of stored sperm in electrolyte free extender must be reiniated by addition of potassium ions and extracellular alkalization (Saito et al 1996b).

This work was planned to determine the effect of electrolyte- free extension of bull sperm on the viability, acrosome integrity and fertilizability during liquid storage. Parallel the differences between spermatozoa separated by washing (EFE-W) or Percoll (EFE-P) gradient methods were specially investigated.

MATERIAL AND METHODS

Semen was collected from 4 bulls (2 Japanese black and 2 Holstein) once a week and transported to the laboratory within 5 minutes. After evaluation of ejaculates macroscopically and microscopically, the samples which had a gross motility score (+++) and individual motility above 75% were pooled to avoid individual variations in the semen characteristics. The pooled sample was re-examined for motility, concentration and alive sperm percentage.

The pooled semen sample was divided to three aliquots, where one aliquot was extended by egg yolk citrate (EYC) to a concentration of 25x 10⁶sp./ml and stored in refrigerator as control. The other two aliquots were subjected to either washing or separation by Percoll gradient method.

Ten ml of electrolyte free extender (EFE) which consists of 5% glucose and 3% bovine serum albumin (BSA, Sigma Chemical Co., St. Louis, Mo) with adjusted pH and osmolarity to be around 7 and 320 mOs respectively, was added to 0.5 ml semen and centrifuged at 400xg for 10 minutes. The supernatant was aspirated gently by pasteur pipette. The pellted semen was then extended using EFE to a concentration 50x10⁶ sperm /ml and stored in 4 ^oC up to 14 days.

Three layers of the electrolyte free percoll gradients (22%, 44% and 66%) were prepared according to Saito et.al. (1996 a,b) in a sterile 15-ml centrifuge tube. One ml of semen sample was layered on the surface of the Percoll gradient and centrifuge at 400xg for 20 minutes. The above layers were gently aspirated and the concentration of the pelleted sperm at the bottom of the tube were determined and resuspended in EFE to a concentration 50x10⁶ sperm/ ml and stored at 4^oC for 14 days.

After 1,4,7 and 14 days storage, the stored samples were diluted with equal volume of 25mM HEPES-buffered TCM199 (Gibco BRL, Grand Island, NY, USA) and incubated for 1 hour in water bath (37^oC).

Motility percentage was evaluated using a phase contrast microscope with hot stage. Examination of alive and intact acrosome sperm were determined from film stained by eosin-nigrosin, giemsa triple stain (Tamuli and Watson,1994).

Bovine ovaries were obtained from slaughtered cows and were transported to the laboratory in 0.9% NaCl solution at 35 ^oC within 4 h of evisceration. Oocytes were aspirated from follicles of 3-8 mm in diameter with 18-gauge needle attached to a disposable syringe. The oocytes were collected in centrifuge tubes. After settling, the sediment was aspirated and diluted in a Petri dish with a suitable amount of dulbeccos phosphate buffer saline supplemented with 3 mg/ml BSA. Examination was done with inverted microscope and only oocytes with cumulus cells were selected for the study. The cumulus oocyt complexes (COC) were washed four times in 25 mM HEPES buffered TCM199 supplemented with 5% Fetal Calf Serum (FCS, Cansera International Inc Canada), 0.02 IU/ml FSH (pFSH; Denka Pharmaceutical, Kwawsaki, Japan), 22 µg/ml Sodium Pyruvate (Kanto Chemical CO. Inc.), 1 µg/ml Estradiol (Sigma, St. Louis, MO, USA) and antibiotic as 100 IU/ml penicillin G and 50 µg /ml streptomycin. After washing, the oocytes were placed in droplets (100 µL) of the same medium covered with liquid paraffin (Nacali Tesque, Kyoto, Japan) in 35mm Petri dishes and incubated in 38.5 ^oC with 5% CO₂ in air for 20- 24 hours for maturation.

Half ml of semen extended by each of EFE after separation by washing and Percoll and that extended by EYC were washed twice by centrifugation for 5 minutes at 600 x g using BO medium (Brackett and Oliphant, 1975) with 10 mM caffeine (Sigma). The pelleted sperm was resuspended using the same medium to 2x10⁶ sp./ml. For making fertilizing droplets, 50 µl of diluted sperm were added to other 50 µl droplet of BO medium supplemented with 20 mg/ml crystallized BSA (Sigma) and 10 IU/ml heparin (Hoechst Marion Roussel Ltd. Akasaka, Minatoku, Tokyo) and covered with liquid paraffin.

After maturation, oocytes were washed twice in BO medium with 5 U/ml heparin, 5mM caffeine and 10mg/ml BSA. Oocytes were placed in 100µl droplet of fertilization medium (5-10 oocytes/droplet) and incubated at 38.5 ^oC and 5% CO₂ in air. After 6 hours the oocytes were washed by passing it several times in culture medium (25 mM HEPES buffered TCM 199 supplemented with 3mg/ml BSA and 0.2 mM sodium pyruvate and antibiotics) then, transferred to 100 µl droplets of the same medium and incubated for another 6 hours in 38.5^oC and 5% CO₂ in air. The oocytes then denuded to remove their cumulus cells and the cumulus free oocytes were fixed with aceto-ethanol (1:3,v:v) for 24 hours. The fixed oocytes were stained with 1% aceto-orcein. Examination was done under phase contrast microscope. Oocytes were evaluated for penetration and normal fertilization. Normally fertilized oocytes were those with two pronuclei and a sperm tail.

Data of semen analysis are given as mean ± SE and were analyzed by one-way ANOVA followed by Tukeys post test after arcsin square root transformation using Graph Pad PRISM software. Difference were considered to be significant at p<0.05.

RESULTS

The recovery rate of sperm after separation by washing or Percoll was 86.20±1.06 and 65.66±1.43% (M±SE) respectively. Examination of electrolyte free extended and control (EYC) samples were done in 0, 1, 4, 7 and 14 days of storage.

The motility rate of sperm extended with EFE-P was higher than that of EFE-W and that of control samples. The difference between EFE-W and EFE-P separated samples was significant (table1).

Estimation of alive sperm percentage during 0, 1, 4, 7 and 14 days of storage revealed that, EFE-P was higher than EFE-W and EYC samples (table 2).

The effect of storage on the acrosome integrity in EFE-W and EFE-P separated samples is shown in table3. The treated samples (EFE-W and EFE-P) showed less intact acrosome percentage than the EYC. At the same time, EFE-W showed significant lower intact acrosome percentage.

The percentage of alive sperm with non-reacted acrosome is presented in figure1. The Percoll-separated samples are higher than the washed samples and both are less than the control. Moreover, in alive reacted acrosome sperm, the EFE-P had higher percentage than EFE-W sperm and both were higher than the control samples (fig.2).

Table (4) represents the fertility percentages of semen stored in EFE-W, EFE-P and EYC after 1 and 4 days. The difference of penetrated oocytes between semen extended by EFE-W and EYC after 1 day storage was low (83% vs 85%), but both were higher than that extended by EFE–P (67%). The washed samples also represented the highest percentage in oocytes showing polyspermia. After 4 days storage, the normally penetrated oocytes with EFE-W semen (15%) was significantly lower than that of EFE-P (50%) and EYC (45%). EFE-P had the highest percentage of polyspermic oocytes

-^ab Means within the same row with different superscripts are different (p< 0.05)

^ab Means within the same row with different superscripts are different (p< 0.05)

-Means within the same row with different superscripts are different (p< 0.05)

Table(4): Fertilizability Percentage of stored spermatozoa in EYC, Washed (EFE-W) and Percoll (EFE-P) Extender

Semen Examined	Total oocytes Examined n	Non-penetrated oocytes n (%)		Penetrated oocytes
Semen Examined	Total oocytes Examined n	Non-penetrated oocytes n (%)		Normal n (%)	Polyspermic n (%)	Undeveloped n (%)

1 day storage
EYC	39	6 (15.4)^a	18 (46.2)^a		7 (17.9)^a	8 (20.5)^a
EFE-P	43	14 (32.6)^b	9 (20.9)^b		8 (18.6)^a	12 (27.9)^a
EFE-W	29	5 (17.2)^a	9 (31.0)^b		11 (37.9)^b	4 (13.8)^b
4 days storage
EYC	35	19 (54.3)^a	4 (11.4)^a		2 (5.7)^a	10 (28.6)^a
EFE-P	32	16 (50.0)^a	2 (6.3)^a		12 (37.5)^b	2 (6.3)^b
EFE-W	39	33 (84.6)^b	2 (5.1)^a		1 (2.6)^a	3 (7.7)^b

-Numbers within the same row with different superscripts are different (p<0.05)

DISCUSSION

In the last few years few reports dealt with the role of electrolyte-free extender in preservation of human spermatozoa after separation of seminal plasma (Saito et al 1996a&b, Kanno et al, 1998). These reports depended on the fact that depletion of surrounding electrolyte prevents the transfer of sodium and potassium ions across the cell wall. Consequently, swelling and disruption of the cells will not occur ( Gruener and Avi-Dor, 1966, Macknight and Leaf, 1977).

Demonstration of the current study revealed that, recovery rate of bull sperm by washing technique was higher than that recovered by Percoll gradient method. The high sperm recovery was explained by Parrish et al (1995), who reported that active motile spermatozoa settled in the bottom of the tube, wherever the weak and dead sperm hanged in Percoll after centrifugation.

A significant difference was found between EFE-P and EFE-W samples with respect to motility and alive sperm percentage. The presence of dead and non-functional sperm within the non-fractionated (washed) samples causing damage of the sperm membrane (Aitken,1988, Aitken and Clarkson,1988) and influence sperm movement characteristics (Aitken et al.,1993). In the same time the EFE samples did not differ significantly than EYC extended sample, which is not in accord with the results of Saito et al (1996a) in human spermatozoa.

The samples stored in the EFE (both washed and Percoll separated) showed a high percentage of sperm with reacted acrosome in comparison with control samples. However, samples of washed sperm contained high percentage of the dead reacted sperm. Presence of dead spermatozoa has its toxic and lytic effects on companion cells (Linderman et al,1982 , Shannon and Curson, 1972). The Percoll separated spermatozoa represented the highest percentage of alive sperm with reacted acrosome than those of washed and control samples during storage. We can conclude that separation of seminal plasma leads to liability of acrosome to be reacted.

Although many studies dealt with fertility of separated spermatozoa from seminal plasma, no study was done on this sperm after storage in EFE. The Penetrated oocyte percentage with EFE-P semen after one day storage was significantly lower than those of EFE-W and EYC samples. This result was in agreement with that of Parrish et al (1995), who recorded that fewer oocytes were penetrated by Percoll separated spermatozoa.

The EFE-W spermatozoa showed highly percentage of penetrated oocytes, but with noticeable number of polyspermic oocytes. This result supports that of Grant et al (1994), who reported high polyspermic percentage of oocytes inseminated with washed semen. The authors denied that increase polyspermic oocytes to be the cause of underdevelopment.

The stored samples for four days has its distinct deleterious effect on the fertilizing ability of all samples, but these was clear for EFE-W semen than that of EFE-P and EYC. In the same time, EFE-P showed the highest percentage of polyspermic oocytes, this may be due to conservation of sperm activity to some extent in EFE-P more than EFE-W and control sample.

The general conclusion of this study is that, the motility and activity of the stored Percoll separated semen were improved, although the fertility was less than that of washed semen. However, Percoll separated semen kept its fertilizing ability after long storage.

REFERENCES

Aitken, R.J.(1988): Assessment of sperm function for IVF. Human. Reproduction, 3: 89-95.

Aitken, R.J. and Clarkson, J.S.(1988): Significance of reactive oxygen species and anti-oxidant in defining the efficacy of sperm preparation techniques. J. andrology, 9: 367-376.

Aitken, R.J. Harkiss, D. and Buckingham, D (1993): Relationship between ironcatalysed lipid peroxidation potential and human sperm function. J. Reprod. Fertil., 98: 257-265.

Grant, S. A. ; Long, S. E. and Parkinson, T. J. (1994): Fertilzability and structural properties of boar spermatozoa prepared by Percoll gradient centrifugation. J. Reprod. Fertil., 100: 477-483.

Gruener, N. and Avi-Dor,Y. (1966): Temperature-dependence of activation and ihibition of rat-brain adenosine triphosphatase activated by sodium and potassium ions. Biochem.J.,100:762-767.

Johnson, M.S.; Senger, P.L.; Allen, C.H.; Hancock, D.D.; Alexander, B.M. and Sasser, R.G.( 1995): Fertility of bull semen Packaged in 0.25 and 0.5 milliliter French straws. J. Anim. Sci., 73(7): 1914-1917.

Kanno, H.; Saito, K.; Ogawa, T.; Takeda, M.; Iwasaki, A. and Kinoshita, Y. (1998): Viability and function of human sperm in electrolyte-free cold preservation. Fertil. Steril., 69:127-131.

Kjaestad, H.; Ropstad, E. and Berg, K. A.(1993): Evaluation of spermatological parameters used to predict the fertility of frozen bull semen. Acta Vet. Scand., 34(3): 299-303.

Kloves,S.I.(1977): And acrosomal integrity of cryopreserved buffalo bulls spermatozoa. 5^th world buffalo cong. Royal Palace, Italy p 833-35.

Lindermann,C.B.; Fisher, M. and Lipton, M.(1982): A comparative study of the effect of freezing and frozen storage on intact and demembranated bull spermatozoa. Cryobiology, 19: 20-28.

Macknight,A.D.C. and Leaf, A.(1977): Regulation of cellular volume. Physiol. Rev., 57:510-573.

Mortimer,R. G.; Brendtson, W.E.; Pickett, B.W. and Ball, L.(1976): Fertility of frozen bovine spermatozoa packaged in continental straws or ampules. J. Dairy Sci.,59(9): 1595-1598.

Parrish,J.J.; Krogenaes, A. and Susko-Parrish, J.L.(1995): Effect of bovine sperm separation by either swim-up or percoll method on success of in vitro fertilization and early embryonic development. Theriogenology, 44:859-869.

Saito, K.; Kinoshita, Y.; Kanno, H. and Iwasaki, A.(1996b): The role of potassium ion and exracellular alkalization in reinitiation of human spermatozoa preserved in electrolyte-free solution at 4 °C. Fertil. Steril. 65:1214-1218.

Saito, K.; Kinoshita, Y.; Kanno, H.; Iwasaki, A. and Hosaka, M. (1996a): A new method of the electrolyte-free long term preservation of human sperm at 4 °C. Fertil. Steril., 65:1210-1213.

Shannon, P. and Curson, B. (1972): Toxic effect and action of dead sperm of diluted bovine semen. J. Dairy Sci., 55:615-620.

Tamuli,M.K. and Watson, P.F.(1994): Use of a simple staining technique to ditinguish acrosomal changes in the live sperm sub-population. Anim. Reprod. Sci., 35:247-254.

Vishwanath, R.; Pitt, P. and Shannon, P.( 1996): Sperm numbers, semen age and fertility in fresh and frozen bovine semen. Proc. N.Z. Soc. Anim. Prod., 56: 31-34.

REPRODUCTIVE TRAITS OF IL-DE-FRANCE AND FINNISH LANDRACE RAMS UNDER EGYPTIAN CONDITION WITH SPECIAL EMPHASIS TO BREED AND SEASONAL VARIATIONS

F.I. HELAL*, R.I. ELSHESHTAWY AND W.M. AHMED

Departments of Animal and Poultry Nutrition and Production*, and Animal Reproduction & AI, National Research Center, Cairo, Egypt

ABSTRACT

Ten-sexually mature rams (5 IL-de-France and 5 Finnish Landrace ) were evaluated for sex drive and semen characteristics, taking in consideration breed and seasonal variations. Two successive ejaculates were collected from each ram every two weeks for a period of one year. Semen characteristics and some biochemical constituents were analyzed.

Results indicated that the reaction time and semen characteristics, including volume, pH, sperm concentration, total sperm / ejaculate and percent of sperm abnormalities were better in IL-de-France than in Finnish Landrace breed. In both breeds,the shortest reaction time was recorded in summer while, semen triats were better in winter . Seminal biochemical constituents revealed no breed difference except for the total nitrogen, which was higher in IL de France. In both breeds, significant seasonal variations were recorded for initial fructose, inorganic phosphorus and non-protein- nitrogen .

It was concluded that IL-de-France and Finnish Landrace rams produced good quality semen especially during winter .

INTRODUCTION

Nowadays, sheep breeding has been spread in Egypt especially in the new reclaimed areas due to socioeconomic factors. Local breeds of sheep usually lamb once every year with a limited percentage of fecundity (El-Khadrawy, 1997). In the same time, as reproductive performance is an important criterion in animal production, a great attention should be paid to the prolificacy. This aim can be achieved by crossing the local breeds with some standard European breeds which can tolerate the Egyptian environment, promote the reproductive potential and improve the genetic constitution (Fahmy, 1997).

Semen quality of a ram is a good indicator for its reproductive potential. However, it was reported that semen quality of sheep significantly varied among different seasons of the year especially in animals existed at high or mid latitudes (Roca et al., 1992). There is a relatively little information on the effect of season of the year on semen quality of exotic breeds of sheep under the prevailing local Egyptian conditions, therefore, the current investigation was designed to evaluate the reproductive traits of two of the most commonly imported foreign breeds of rams(IL-de-France and Finnish Landrace)

The study was carried out during a period of one year on the Experimental Farm of the Faculty of agriculture, Al-Azhar University . Ten pure breed rams (3-4 years old) were used ( five IL-de-France and five Finnish Landrace). Rams were imported from England and raised under local Egyptian conditions in semi open sheds and kept under the routine managemental system. Each ram was fed on 0.9-1.0 Kg commercial concentrated ration and 250 of corn or 2 Kg Egyptian clover.

Sex derive and semen characteristics were evaluated during the different seasonal of the year taking in consideration the differences due to breed as well as some climatic data.

During the period of the study, ambient temperature and relative humidity were determined using a thermohygrometer, while data of the photoperiod were obtained from the Egyptian Meteorological Organization.

The sex derive of rams were evaluated by recording the reaction time( Ashurbekov 1990).

Rams were allowed to exercise twice daily and trained to mount anoestrous ewes before beginning of the experiment. Semen collection was performed indoors at 7-10 a.m. using A.V (Fernandez-Abella et al.,1992). Two successive ejaculates were obtained from each ram every two weeks with 30 minutes interval. False mounts were performed once for each ram before collection to improve the quality of the ejaculate.

Immediately after collection, ejaculates were maintained in a water bath at 37C°. Physical characteristics including, colour, consistency and pH ( Kalous and Samkova, 1992), microscopical examination for sperm concentration, motility , live dead ratio and abnormalities (Alexopoulos et al., 1991; Demirci, 1993). Chemical constituents including fructose, inorganic phosphorus and protein contents ( Markandeya and Pargaonkar, 1990) were estimated.

The obtained data were statistically analyze as outlined by Snedecor and Cochran(1980).

In the present study, the reproductive traits in terms of sex drive and semen picture of two exotic breeds of rams under local Egyptian conditions were evaluated.

Seasonal variations in the prevailing meteorological data at the area of the investigation were recorded in table(1). Temperature and photoperiod differed markedly during winter and summer seasons, while, relative humidity revealed little changes among the four seasons of the year.

Table (1): Seasonal variation in ambient temperature, relative humidity and photoperiod in Egypt (Mean ± SE)

The reaction time and semen characteristics including volume, pH, sperm concentration, total sperm / ejaculate and percent of sperm abnormalities (Table 2 ) revealed significant differences between the two breeds and were better in IL-de- France than in Finnish Landrace. In this respect, Hoak et al.(1989), Trejo et al. (1990), Ley et al. (1990) and Mehouachi 1995) recorded variations in sexual desire and semen characteristics among different breeds of rams, while, Fernandez-Abella et al.1992) found variations in the reaction time only. However, differences in sex derive and semen characteristics were attributed mainly to andrological variations ( Soylu et al., 1991) among different breeds of rams .

Tables (2 and 3) revealed significant seasonal variations in libido and semen characteristics for both breeds of rams. The shortest reaction time was recorded during summer, while the longest time was noted during winter. El-Gamal (1975) recorded similar results. In this respect, a significant positive correlation was recorded between ambient temperature and testosterone level (Grasselli et al., 1996). However, more or less short reaction time was recorded during autumn. The longest reaction time during winter may be due to the highest relative humidity. The ejaculate volume and sperm motility were higher in winter and sperm concentration was higher in winter for IL-de-France and in spring for Finnish Landrace . Moreover, the incidence of sperm abnormalities and dead sperms was higher in summer for both breeds. Such seasonal variations agreed with the findings of Al- Hakim et al.(1989), Trejo et al.(1990) and Mehouachi (1995). In the same time, the improvement in semen triats during winter as indicated by the significant negative correlation between each of volume, motility and sperm concentration in one hand and photoperiod or ambient temperature and both on the other hand was evident (Table 5). The high percent of sperm abnormalities and dead sperms met with during summer may be related to the drastic environmental conditions during this season as indicated by the significant positive correlation between these parameters and photoperiod or ambient temperature or both. In this respect, Besancon et al (1991) and Sarlos et al.(1996) recorded seasonal changes in testosterone levels.

However, the improvement of semen quality in winter may be due to the short day light exposure which has a beneficial effect on semen quality ( Mies-Filho et al.,1990) and improving of the nutritional status ( Bedo et al.,1990).

In the present study, little breed variations were observed in the studied biochemical seminal constituents ( Table 4) except of the total nitrogen, which was significantly higher in IL-de-France than Finnish Landrace. In the same time, significant seasonal fluctuations were noticed in initial fructose, inorganic phosphorus and non -protein nitrogen where the highest and lowest values were recorded during summer and winter ; winter and summer, and summer and autumn, respectively. In this respect, Girao and Mies-Filho (1989) recorded higher values for fructose in semen of rams during summer. Variations in seminal phosphorus and protein contents may be related to nutrition ( Pineda, 1989) as well as sperm concentration ( Graner and Hafez,1993) . In the same time, the higher levels of fructose during summer were positively correlated with ambient temperature (Table 5).

In conclusion, IL-de-France and Finnish Landrace breeds of rams produced semen of good quality especially during winter despite that their libido was optimum during summer and moderate during autumn and this phenomenon could be potentiated by the finding of Fernandez-Abella et al. (1992) who found no significant correlation between

semen picture and libido. Also, it was recorded that during summer, the increases in ambient temperature and day light were significantly correlated with hyperprolactinaemia which is responsible for decreased fertility during this season( Singh and Madan, 1993). Therefore, we recommended that great care should be directed to semen evaluation not for libido before usage of such foreign breeds in breeding purposes for improving the fecundity of native breeds of sheep.

Alexopoulos, K. ; Karagiannidis, A. and Tsakalof, P. (1991): Development of macroscopic and microscopic characteristics of ejaculates from Chio, Serres and Karaguniki lambs. Theriogenology, 36: 667 - 680.

Al-Hakim, M. K. ; Issa, H. H. and Juburi, S.A. (1989): Monthly and seasonal variations in some characters of Awassi rams. In “Proc. The 3^rd Egypt. British Conf. Anim. Fish and Poultry Prod.”, Alex., Egypt. ,2: 691- 696.

Ashurbekov,A.A.(1990): The effectiveness of year – round collection of semen from rams. Zootekhniya, 6: 66 - 68.

Bedo, S.; Mezes, M.; Baresk-Tolh,G.; Saiar, L. and Mikus,G.(1991): Effect of feeding on sperm production of rams of different genotype. Allattenyesztes es Takarmanyozas, 39: 225 - 235.

Besancon, J.; Demers, P.; Lemayl, P. and Tremblay, R.R.(1991): Opposite variations of two-epididymal components and blood plasma testosterone in two breeds of rams. Comp. Biochem. Physiol., 99:173 - 177.

Demirci, E. (1993): Studies on semen characters of Awassi rams, and correlation of semen volume with age and testis size. Vet. Fakult.Derg., Uludag Univ., 12: 98 -106.

El-Gamal,A.A. (1975): Genetic studies on sexual behaviour of rams. M.Sc. Thesis (Anim.Prod.), Ain Shams Univ.

El Khadrawy, H.H. (1997): Biochemical changes of hormone in relation to immunization against protein- linked steroid in sheep. Ph . D.Vet. Thesis ( Biochem.), Cairo Univ.

Fernandez-Abella, D.; Villegas, N.; Klappenbach, A. and Machado, A. (1992): Effect of age and breed on semen production and sexual activity. Bul. Tecn. Cienc. Biol., 2 :37 - 49.

Girao, R.N. and Mies-Filho, A. (1989): Concentration of fructose and citric acid in semen from Corriedable rams subjected to natural or artificial photoperiod and temperature. Revista Brasil. Reprod. Anim., 13: 137 - 142.

Graner, D.L. and Hafez, E.S. (1993): Spermatozoa an d seminal plasma. In: “Reproduction in Farm Animal” . 6^th ed., E.S. Hafez(Ed.), Lea and Febiger, Philadelphia, USA, pp : 165-187.

Grasselli, F.;Quaranta, A.; Saleri, R. and Tamanini, C. (1996): Seasonal variations of pituitary and gonadal activity and responsiveness to GnRH in the male goat. Zootec. Nutr. Anim., 22: 333 - 338.

Horak, F.; Mares, V. and Barina, V. (1989): The importance of Booroola rams imported to Czechoslovakia. Nas Chov., 49: 514 - 518.

Kalous, R. and Samkova, E. (1992): Ejaculate quality of rams in relation to semen collection and housing conditions. Ceske Budejovice Zootec. Roda, 9: 83-84.

Ley, W.B.; Sprecher, D.J.; Thaichjer, C.D.; Pelzer, K.D. and Umberger, S.H. (1990): Use of the point-score system for breeding soundness examination in yearling Dordset, Hampshire and Suffolk rams. Theriogenology, 34: 721 - 733.

Markandeya, N. and Paragaonkar, D.R. (1990): Studies on biochemistry of Osmanabadi buck semen . Indian J. Anim. Reprod., 11: 36 - 38.

Mehouachi, M. (1995): Reproductive traits in Barbary and Thibar rams. Cahiers Options Mediterraneennes, 6: 5 -41.

Mies-Filho, A. ; Girao, R.N.; Oberst, E.R. and Wald, V.B. (1990): Benficial effect of 8 hours of artificial light daily on spermatogenesis in rams in Brazil. Bresil Buul. Acad. Vet. De Freana, 63: 409 - 413.

Pineda, M.H. (1989): Male reproduction. In: “ Veterinary Endocrinology and Reproduction”. 4^th ed., E. McDonald (Ed.), Lea and Febiger, Philadelphia, USA, pp: 261-301.

Roca, J.; Martinez, E.; Vazquez, J.M. and Coy, P. (1992) : Characteristics and seasonal variations in the semen of Murciano-Granadina goats in the Mediterranean area. Anim. Reprod. Sci., 29: 255 - 262.

Sarlos, J.; Molnar, A.; Huszar, S. ; Ratky, J. and Brussow, K.P.(1996): Seasonal changes of andrological characteristics in British Milk ram. Archiv fur Tierzucht, 39: 265 - 275.

Sigh, J. and Madan, M.I. (1993): RIA of prolacrtin as related to circadian changes in buffalo. Buff. J., 9: 159 - 164.

Snedecor, G.W. and Cochran,W.G. (1980): Statistical Method. 7^th ed., Iwoa State Univ Press., USA.

Soylu, M.K.; Gokcen, H.; Tumen, H. and Dogan, I. (1991) : Studies on some andrological characters of imported rams of different breeds. Hayvanctlik Arastirma Dergis, 1: 15 - 18.

Trejo, G.A.; Gonzalez, P.E. and Vasquez, P.C. (1990): Seasonal effects on fertility in rams of five breeds on high plateau in Mexico. 2. Semen traits. Proc. The 3^rd Natn. Congr. Sheep Prod., Tlaxcala, Mexico, pp: 193 - 197.

Dept of Vet. Hygiene and Management , Fac. Vet. Med, Cairo University

ABSTRACT

Shearing has been reported to impinge on the productivity, health and welfare of sheep. The following study examined the behavioural, physiological and performance response of sheep to the shearing process. Fifty male Barki sheep of 12-15 months of age (wethers) were used in this work , they were divided equally into five groups. Group one (G1), control not-shorn, Ns group and the other four groups were shorn(s) as group two (G2), hand shorn in early morning time of the day , group three (G3) was hand shorn in afternoon time, group four (G4), mechanically shorn in early morning and group five (G5), mechanically shorn in afternoon . The assessed variables were behavioral of and physiological response as well as the productive aspect (body weight gain). The results showed that there was a significant effect of shearing on ingestive, ruminating frequencies as it reduced these behaviours and consequently reduces the body weight gain of shorn sheep . The sheep shorn mechanically in early morning time of the day were the less affected by shearing than other groups. At the same time the shearing reduced the resting time and increase the locomotor activities among the shorn sheep while there was no significant effect of method of shearing and time of the day on locomotor activities and resting behaviour.

Regarding physiological responses, there is a significant elevation in plasma cortisol level, Haematocrit % and glucose level due to shearing procedure, the elevation was higher in the hand shorn in afternoon time group than other groups of sheep. The body weight gain was affected significantly by shearing as the shorn sheep gained significantly less body weights than the not shorn control group of sheep. There was no significant effect of the method of shearing or the time of the day on the body weight gain in this study.

INTRODUCTION

Community concern about methods of farm animal production has mainly concentrated on the intensive animal industries. But recently attention has shifted to grazing industries, in particular the welfare aspects of sheep production. However, more information is needed before judgements or decisions concerning the welfare of sheep in extensive systems can be made (Townend, 1985 and whilloughby, 19988).

Shearing of sheep is a management practice performed for economic purposes, to protect the animal from skin parasites and diseases, and also for surgical reasons. In Egypt, usually shearing takes place twice per year, in March and September.

Shearing has been reported to impinge on the productivity (Ensminger, 1979, El hadi, 1988 and Morris and McCutcheon, 1997), health (Baker, et al, 1982, Sumner et al, 1992 and Newman et al, 1996), and welfare (Hill,1983, Hargreaves, 1988, Hargreaves and Huston, 1990b and Gross and Siegel, 1993) of sheep. Recently, welfare issues have gained greater prominence as awareness and attitudes of the community to the treatment of farm animal changes (Selye, 1976 and Hargreaves and Hutson, 1990 c and d).

The wool is removed by noisy, sharp machinery, which is capable of cutting both wool and skin and consequently the sheep become under stressful conditions. Also, wool removal is of a fundamental importance to wool harvesting, yet it is the prime contributor to the stress response to handling (shearing) (Hargreaves and Huston, 1990 d).

A number of studies reported that many routine sheep handling procedures are stressful in that they result in elevated levels of cortisol, glucose, haematocrit and heart rate (Marks, 1959 and Pierzchala et al, 1983).The most stressful of these procedures is shearing (wheeler et al 1963 and Hargreaves and Hutson,1990 a). Gross and Siegel, (1993 ) reported that responses to stress can include anatomical, physiological and/or behavioral changes. Plasma cortisol and /or corticosterone are frequently used as criteria for measuring response to stressors.

Another studies have described the response of sheep to shearing and transportation. these studies have had a variety of aims including assessment of the effects of shearing on ingestive behavior ( Webster and Lyneh, 1966 and Arnold, 1976 ) .

A-Response of sheep to one of he most important handling procedures i.e shearing. The response involved the following parameters which are thought to indicate different aspects of a response to stressors.

1- Behavioral response: ingestive behaviour, locomotion activities and resting behaviour

B-Effect of the method of shearing and day time on the above mentioned parameters.

Materials and Methods

Fifty male Barki sheep of about 12-15 months of age (wether) were used in this study. They were obtained from a sheep farm located at El – Nobaria region, Behaira Governorate and kept in a private sheep farm . The sheep were maintained under grazing (free range) conditions until one week prior to shearing process when each group of sheep was kept in covered pen and giving access to pasture with hay and water ad-libitum.

This work was carried out in a factorial design. The factors studied are the shearing itself, the time of the day (early morning or afternoon) and the method of shearing (hand or mechanical shearing).

The fifty male Barki sheep were randomly divided into five groups, each of ten animals, a control group (G1) including the not shorn sheep (NS) and four treated group (shorn sheep) as follow:

All five groups of sheep were washed 2-3 days before shearing. Shearing process was carried out in March in a covered yard with a wooden floor (shearing board), the entire fleece was removed using either very sharp, well greased hand shears (hand shearing) or using clipping machine (mechanical shearing) (Trevalyan, 1982 and Holmes et al,1992).

The shearing took place either in the early morning or in afternoon times of the day (Alexander et al, 1979, Lynch and Alexander, 1980 and Al –Jaryan, 1991).

Six blood samples were taken from each of the five treated groups of sheep, 15 minutes prior to shearing and five samples during the 90 minutes following shearing process (Just after shearing 0 time), 15, 30, 60 and 90 minutes post-shearing (Hargreaves and Hutson, 1990a and b)

10 ml samples were collected, transferred into two heparanized tubes and chilled, blood in one of these tubes (approximately 2 ml) was used for Haematocrit determination and blood the other tube was centrifugated and the plasma collected and stored frozen until assayed for total cortisol and glucose levels (Fulkerson and Jamieson, 1982)

Daily observation was done between 8.00 and 10.00 a.m. and between 13.00 and 15.00 p.m. for one week post-shearing. The following behavioural patterns were observed and recorded during the observation time for each group of sheep (Moberg, 1987 and Gross ad siegel 1993).

- Rumination :chewing regurgitated food while standing or laying and its frequency of occurrence (Arnold, 1976).

B- Locomotor activities :as walking, running, feeding and drinking (Friend, 1991)

C- Resting behaviour: the time spent resting or sleeping during observation time was recorded. (Friend, 1991).

Cortisol level, Hematocrit%, and glucose level were recommended as physiological parameter responses (indicators) to the stressful conditions ( Hargreaves and Hutson, 1990 b and d ) .

The previously frozen plasma was used for cortisol assay using a radioimmunoassay according to the method of Jephcott et al, 1986 in duplicate at two dilutions using a commercial antisera (st C100,Steranti Resead, St. Albans,UK) (Meyer et al, 1988).

A 2 ml of each 10 ml blood sample was chillled in heperanized tube and used for determination of haematocrit by centrifuging samples, in triplicate, in mirco – haematocrit centrifuge for 5 minutes and measuring the red cell materials as a proportion of the total height of blood in the tube (Dobson, 1986 and Hargreaves and Hutson, 1990b).

Glucose level wa determined by using the glucose oxidase/ O- tolidine method. Samples were analysed on UV. Spectophotometer (Shimadza UV–120-02) (Fulkerson and Jamieson, 1982 and Meyer et al, 1988).

The initial body weights of all groups of sheep were determined immediately before shearing and the final body weights were also recorded after 2 weeks of shearing to calculate the body weight gain for each of treated and control group of sheep (Al-Jaryan 1996 and Scobie, 1998).

The difference among populations were tested using ANOVA test according to the methods of Snedecor and Cochran, (1989) .

RESULTS AND DISCUSSION

The welfare of an individual is its state as regards its attempts to cope with its environment. There are more than one measure showed that there is a problem or the animal is severely affected by the treatment. The most obvious indicators are changes in behaviour which show that some aspects of the situation is aversive ( Hill, 1983 ). Physiological changes are associated with the behavioural responses ( Hargreaves and hutson 1990a,b) .

(1) Behavioural response to shearing

As shown in table (1), the shorn sheep in all four treated groups showed variation in the frequency of eating in comparison with the not shorn control group. The eating frequency was significantly (p < 0.05) reduced immediately and for one week after shearing specially among sheeps shorn in the afternoon time of the day of shearing. The recorded frequencies sere (18.32±3.9 , 11.31± 2.3 , 8.18 ±1.8,10.18±1.3 and 12.11±1.7) for control group (G1 ) and the four treated groups ( G2 , G3, G4 and G5 ), respectively. There was no significant effect of the method of shearing on the eating frequency.

Regarding ruminaition, animals in the treated groups showed a significant (p < 0.05 ) decline in frequency of rumination than those in not shorn control group and at the same time there was no significent effect of the method of shearing.

A number of studies have described the response of sheep to handling procedures , including shearing , transportation and restraint. These studies have had a variety of aims, including assessment of the effect of handling procedures on ingestive behaviour ( wodzicka – Tomaszewska 1963., Arnold, 1976 and penning et al; 1991).

Rushen and Cogdon, ( 1986 ) and Hargeaves , (1988) stated that , it was established that electroimmibolization due to shearing may exacerbate the behavioural responses to shearing . The observed results agreed with that recorded by Webster and 1ynch (1966) and Al – Joryan (1991).

Table (1) also showed that, there was a significant ( p < 0.0 5) effect of shearing procedure on the locomotor activities and resting behaviors while there was no significant effect of the method of shearing and the time of day on these parameters, as the shorn sheep in all treated groups spent more time walking , running and less time resting than those in cont rol group .The less time spent resting may be due to the pain caused by shearing process or may be due to the fear reaction as a result of shearing noise or accidental injury of skin as fear is a hypothetical state of the brain or neuroendocrine system arising under certain conditions certain forms of behaviour .This result coincides with that observed by Dyrmundsson ( 1991 ) and Friend ( 1991).

Table (1) Behavioural responses of shearing in sheep (Mean ± SE)

Treatment

Not shorn control G1

Hand shorn

Mechanically shorn

Parameter

Early morning G2

Afternoon

Early morning G4

After noon

Ingestive

Eating

frequency

abcde

18.32 +3.9

abc

11.31+2.3

abce

8.18 +1.8

acd

10.18+1.3

ace

12.11+1.7

Rumination frequency

abcde

12.22 +2.1

abc

8.31 + 1.9

abcd

5.71 +1.1

acd

8.20 + 1.9

ace

7.32 +1.4

Resting

behavior (min)

abcde

32.10+2.8

23.24+1.9

22.18+1.7

24.66+1.8

22.23+2.3

Locomotor activities (min)

abcde

24.70+2.1

34.12+3.2

33.11+2.8

32.18+2.7

34.10+3.0

^abcde values with the same superscripts within columns were significantly differ at p < 0.05

Table (2) showed that , there was a significant ( p <0.05) elevation in plasma cortisol level following shearing procedure in all shorn groups of sheep than that in not–shorn control group (G1). The recorded level of cortisol were (27.0±0.2 , 52.1 ± 3.6 , 68.2 ± 6.8 , 38.2±3.2, 48.2 ± 4.8 ng / ml-1 ) for control (G1) and the four treated groups (G2 , G3 , G4 and G5) , respectively.

Also as shown in table ( 2 ), it can be noticed that , there was a significant ( p <0.05) effect of both the method of shearing and the time of day, as the highest rise in cortisol ( 68.2 ± 6.8 ng /m-l) was recorded in sheep hand shorn in the afternoon time while the lowest level was in sheep mechanically shorn in the early morning time of the day (38.2 ± 3.2 ng / ml-1).

Generally the highest rise in cortisol level was at o time ( immediately after shearing ), then declined gradually in the 4 treated groups with time till up-ending at 9o minutes was at the basal level . On the other hand, there was no fluctuation of the cortisol level in the not shorn control group during the experimental period. These findings were agreed with those reported by (kilgour and de langen, 1970 ; Fulkerson and Jamieson, 1982; Pierzchala et al; 1983 , Hargreaves and Hutson, 1990b).

Haematocrit % rose significantly ( p <0.05) after shearing , it was 32.7 ± 2.3 , 38.1 ± 2.0 , 33.0 ± 1.9 , 32.1 ± 2.5 and 24.0 ± 1.7 for the four shorn groups ( G2 , G3, G4, G5 ) and G1 , respectively ) and the control not shorn group , respectively just after shearing , then declined for all treated groups to the pre- treatment levels within 9o minutes after shearing as it becomes 22.1 ± 1.7 , 24.1 ± 1.6 , 21.3 ± 1.5 and 22.8 ± 1.3 for the treated groups ( G2 , G3, G4, and G5) respectively .The effects of the method of shearing and time of the day were significant only just after shearing as the higher elevation in haematocrit was among sheep hand shorn specially in the afternoon time of the day. The recorded values were agree to a large extent with those reported by Hargreaves, ( 1988), Hargreavs and hutson , ( 1990b) and Holmes et al, . (1992).

The elevation of cortisol and haematocrit levels after shearing is may be due to the potency of the acute short- term stress response to shearing due to wool removal or may due to noise of shearing itself which elicit the stress response ( increasing haematocrit and cortisol level ) as it effectively confers auditory isolation on the sheep.Haematocrit and plasma cortisol concentration were chosen as parameters of stress, reflecting catecholamine and pituitary adrenal response respectively. These parameters are thought to indicate different aspects of non specific response to stressors ( Dantzer and Mormede 1983) or different stages of the general adaptation syndrome ( Hill, 1983).

Table (2) : plasma cortisol level (ng / ml-1) of sheep which shorn or not (Mean+ SE).

Treatment

Not shorn control G1

Hand shorn

Mechanially shorn

Time after shearing

Early

morning G2

Afternoon G3

Early

morning G4

Afternoon

- 15 min before

25.8 +1.9

28.1+2.1

26.5 +1.6

25.6+2.2

26.4+1.8

O time just after

Abcde

27.0+2.0

Adcd

52.1+3.6

Abcd

68.2+6.8

abcd

38.2+3.2

Acde

48.2+4.8

15 min

Abcde

24.8+1.6

Abcd

50.0+4.0

Abcde

64.1+5.4

abcd

32.4+3.0

Ace

41.1+3.6

30 min

Abcde

23.4+1.7

Abcd

42.7+3.2

abcde

54.8+4.6

abcd

29.7+2.7

Ace

38.2+2.9

60 min

Abc

25.0+2.0

Abc

34.2+2.6

Abcde

48.2+3.8

acde

23.5+1.6

Cde

30.5+1.9

90 min after shearing

23.0+1.9

27.1+2.5

31.0+1.9

22.4+1.2

25.2 +1.2

^abcde values with the same superscripts within columns were significantly differ at p < 0.05

Table (3) Haematocrit % ( mean + SE) of sheep as affect by shearing

Treatment

Not shorn control G1

Hand shorn

Mechanially shorn

Time after shearing

Early morning G2

Afternoon G3

Early morning G4

After noon G5

- 15 min before

22.1+1.9

22.4+2.1

23.2+1.8

22.4+2.0

24.5+1.9

O time just after a

Abcde

24.0+1.7

abc

32.7+2.3

abcde

38.1+2.5

Acd

33.0+1.9

ace

32.1+2.0

15 min

Abcde

23.7+2.0

abce

31.1+1.9

abce

35.2+1.7

32.1+1.7

ace

30.8+1.3

30 min

adcde

22.9+1.3

27.3+1.4

ace

29.4+1.5

26.8+1.2

ace

25.1+1.7

60 min

22.0+1.8

24.0+1.3

26.1+2.0

24.2+1.2

23.8+1.4

90 min after shearing

22.8+1.8

22.1+1.7

24.1+1.6

21.3+1.5

22.8+1.3

^abcde values with the same superscripts within columns were significantly differ at

Table (4) Glucose level ( mg/dl) of sheep which were shorn (s) or not shorn (ns)

Treatment

Not shorn control G1

Hand shorn

Mechanially shorn

Time after shearing

Early morning G2

Afternoon G3

Early morning G4

Afternoon G5

- 15 min before

50.0+4.3

50.8+4.1

49.8+4.5

51.8+3.9

53.0+4.0

O time just after

Abcde

52.1+4.1

abde

68.3+4.8

acd

66.5+4.0

Abd

59.8+3.7

abe

60.1+3.9

15 min

abcde

52.0+3.9

abde

62.1+3.3

acde

63.0+3.5

Abcd

56.1+3.2

Abce

56.2+3.5

30 min

abce

50.8+4.2

58.0+3.6

58.5+3.2

Bcd

54.4+2.9

55.3+3.0

60 min

abc

49.8+4.0

55.6+3.3

55.0+4.0

53.2+3.8

54.0+3.1

90 min after shearing

50.2+3.9

52.8+3.8

51.4+3.9

50.2+3.1

52.1+3.0

^abcde values with the same superscripts within columns were significantly differ at

Treatment	Not shorn Control G1	Hand shorn		Mechanially shorn
time after shearing		Early morning G2	Afternoon G3	Early morning G4	After noon G5
Initial body weight (KG)	32.1+2.1	32.8+1.9	32.8+2.3	30.3+3.0	32.6+1.7
Final body	34.9+2.8	34.1+2.3	33.0+2.9	31.6+2.7	33.8+1.9
Body weight gain (kg)	Abcde 2.8+0.6	Ab 1.3+0.7	ac 1.0+0.5	Ad 1.6+0.3	ae 1.2+0.8

^abcde values with the same superscripts within columns were significantly differ at p< 0.05

Table (4) showed that, there was a significant rise in the glucose level (p<0.05) immediately after shearing ( 0 time ). The mean values were ( 52.1 ± 4.1, 68.3 ± 4.8, 66.5 ± 4.0 , 59. 8 ± 3.7 and 60.1 ± 3.9 mg/dl for the control not shorn and the four shorn groups of sheep ( ( G2 , G3, G4, and G5), respectively. This rise in glucose level declined again till reaching the baseline level within 90 min after shearing . The effect of method of shearing and time of the day of shearing was significant only till 30 minutes after shearing . The result agree to some extent with that reported by Holmes et al (1992) while marks ( 1959) reported that the glucose level wasn,t affected by shearing as a handling procedure .

Wool removal is the most potent component in eliciting a sympathetic adrenal medullary or pituitary- adrenal response to shearing and consequently secretion of adrenalin and non adrenalin from adrenal medulla which cause elevation of haematocrit and glucose level in blood ( hyperglycaemia ). Also as stated by Holmes et al. (1992) the secretion of adrenalin and noradrenalin as a result of stress factor ( shearing procedure ) produce glucose from the non carbohydrate source and also causing breakdown of glycogen through glycogenlysis producing hyperglycaemia i.e increase glucose level in blood.

As shown in table (5) it can be noticed that, there were significant (p< 0.05) differences among sheep in control not shorn group and those in other four treated groups ( shorn sheep ) regarding their body weight gain as sheep in shorn groups attained signifcantly low body weight gain ( 1.3 ± 0.7 , 1.0 ± 0.5 , 1.6 ± 0.3 and 1.2 ± 0.8 kg.) in comparison with the corresponding non treated group ( 2.8 ± 0.6 kg.) . On the other hand there was no significant differences among the four treated groups of sheep and this indicate that there was no effect of both the method of shearing and time of the day on the body weight gain.

The low body weight gain due to shearing may be du to the reduced eating frequency and also may be due to the less time spent resting following shearing which may be lead to high energy expanditure. These results coincided with those reported by ( El hadi, 1988; Holmes et al, 1992) and Aksoy, 1996) while sumner et al , (1992) and Newman et al, (1996) stated that, there was no significant effect of shearing as handling procedure on the body weight gain of sheep.

It can be concluded that, sheep shearing is an important management practice to take in account when improving techniques of sheep husbandry in order to attain high productivity without neglecting animal welfare , so shearing could be made less stressful by minimizing physical manipulation and social disruption to sheep through automated (mechanical ) shearing in the early morning time of the day of shearing .

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EFFECT OF SOME PARASITIC INFESTATION ON THE BEHAVIOUR AND PERFORMANCE OF CATTLE AND BUFFALOES

E.M. ABDEL–GAWAD and O.K. ZAHRAN

Dept. of Hygiene, Husbandry. Faculty of veterinary Medicine Cairo University. Giza

This study has been conducted on a herd of 22 animals of cattle and buffaloes infested with one host ticks (Boophilus annulatus), at the department of Hygiene and husbandry. Faculty of vet. Med., Cairo University. The aim of the study was to illustrate the main behaviour patterns that have been drastically affected by ticks infestation in both cattle and buffaloes, to determine the level at which animals can tolerate and overcome ticks infestation, as well as to study the effect of Diazinone as an acaricide to alleviate and mitigate the stressful effects caused by ticks on the infested animals.

Animals were classified according to their age and species into 4 distinct groups (Gr.1) Adult cattle, (Gr.2) calf cattle, (Gr.3) Adult buffaloes and (Gr.4) buffaloes calves respectively.

Obtained results revealed a marked changes of some behaviour patterns of animals infested by tick as a defensive mechanism. Two levels of adjustment were obtained by the infested animals according to the severity of infestation. First level when ticks counted on the animal body reached 300 to 500 here the animal react by increased behaviour patterns related to skin hygiene significantly (P< 0.05), rubbing, Scratching, ear flick, tail wagging and Social grooming increased among infested animal, while behaviour patterns related to rest, sleep, feeding and rumination decreased in both cattle and buffaloes. Second level was obtained when ticks counted on the animal body reached 1000 or more here the animal failed to adapt (frustrated), all body care behaviour patterns were completely arrested, animal showed laziness, slugh movement, appeared as if being shocked, ingestion and rumination reached a minimum peak (p <0.01), sleeping and laying behaviour arrested, signs of anemia, emaciation and showing ribs were the sequali.

Treatment by spraying with Diazinone in a concentration of 0.01% in two successive dose with 15 ds in between has been alleviated and mitigated the stresses caused by ticks, all body care behaviour, ingestive and ruminating behaviour returned its normality rest and sleep exceeded than normal as if the animal tried to compensate periods of stress.

The role of the veterinarian in the field of animal production can be divided into tow main aspects. First, the control of reduced production due to disease, parasitism, inadequate or improper nutrition and, secondly, the more positive and general approach of deliberately trying by various means to increase production directly by egg transplantation, cloning, sex determination of offspring, and control of oestrus. (Wooldridge, 1974).

Parasitism is an interaction among members of different species, in which the parasite exploits the host, but generally does not kill it. Many parasites are fully dependent upon the host at some stage of their life cycle, and natural selection therefore exerts a strong pressure for them to become highly adapted to their nich. At the same time many host species have evolved behaviourl defensive mechanisms, so that there may be an evolutionary (arm race) between parasite and host (Mcfarland, 1981).

Ticks may harm their hosts by (1) injuries done by bites, (2) sucking blood, (3) transmitting various. Protozoal, rickettsial, and bacterial diseases, and (4) causing tick paralysis (Scott, 1988). In addition, economic losses through hide damage and diagnostic, therapeutic, or control programs may be sizable. In tropical and temperate areas, ticks are responsible for the loss of hundreds of millions of dollars annually.

Many workers in the field of animal behaviour are impressed by the fact that behaviour like so many life processes, is obviously directed to a certain end (goal, or purpose). Behaviour is purposive or directive. Behaviour helps the animal to maintain itself in a hostile world. Relations between the animal and its environment are always mutual (Hill, 1976). Each species, is continuously protecting itself against the destructive forces of the environment. Behaviour is an essential element in the equipment serving this end.

Animals take care of their body surface in an organized, deliberate way and become involved in various activities in which areas of the body surface are treated in apparently satisfying ways (Fraser and Broom, 1990). Grooming in ungulates has been shown to very effective in removing ectoparasites. Ectoparasites, especially ticks, may be costly to an animal’s resources in terms of blood removed and depression of appetite (Hart, et al 1992). Prevention of grooming in cattle, Bos taurus, increase ticks infestation. Other studies on grooming behaviour in african ungulates has been conducted by (Borchelt, 1980; Pantenaude and Bovet, 1983, and Hart, 1990, Hart et al, 1992, McKenzie, 1990, Seebeck et al., 1971, Little 1963, Turner and Short, 1972; Williams et al., 1978).

A herd of 22 animals of cattle and buffaloes was used in this study. Animals were classified according to their age and species into 4 groups. (Group 1) contained 10 adult cows, (Group 2) contained 3 calves cattle of 4 month, (Group 3) contained 6 adult buffaloe gows and, (Group 4) contained 3 buffaloe calves respectively.

Animals that were maintained by the hygiene and hushandry farm, were kept in loose housing paddocks with the feeding manger and standing area covered with asbestos sheets.

The animal house was infested by one host ticks “Boophilus spp) which has been identified by the Department of Parasitology. The maximum and minimum temperature and relative humidity during the studying period ranged from 23^oC to 15^oC and 70-79% respectively. The feeding and managemental conditions offered to all animal groups were the same.

Technique for studying and recording the behaviour patterns of infested animals before and treatment with Diazinone has been used according to the introductory guide of measuring behaviour by Martin and Bateson (1986).

Four-hours daily observations have be measured on the studied animals, All animal sampling method have been used, each animal in all groups have given 15’ minutes daily observation for recording and categorizing its behaviour during the 15’ minutes session.

Time spent feeding, drinking and rumination in minutes has bee recorded/15’ min.

Frequency of grooming, licking, scratching, ear flick, tail wagging, foot stamping, head toothing and vocalization have been recorded/ the observation cission, numbers of social grooming bouts received by animals were also recorded.

For each group studied collected data were pooled and analysied according to Snedecor & Cochran, 1980).

Comparisons between the behavioural traits before and after treatment by Diazinone have been carried out by using student –T- test (two – tailed).

From table (1) and Fig (1) it can be mentioned that cattle infested with ticks showed a marked increase in the behaviour patterns related to skin hygiene licking, rubbing, ear flick, tail wagging recorded 24.8, 2.8, 35,8 and 56 respectively. The number of social grooming bouts increased among infested animals, 4.29 bout. Resting behaviour decreased as a result of the irritation caused by ticks. Time spent feeding and rumination also decreased.

Treated animals showed a significant increase in the time spent loafing, laying and rumination, flicking the ear, tail wagging and grooming frequency reduced indicating relief from pain and stress.

Result obtained in table (2) and Fig (2) indicated that calf cattle is more sensitive than adult, laying and rumination time decreased to minimum (0.12 min and zero min.) respectively, calf cattle used another means as a defensive mechanism against tick infestation, foot stamping, exploration, ear flick and

Table (1) Behaviour patterns of adult cattle infested with ticks before and after treatment with Diazinone

	Behaviour patterns		Before treatment				After treatment				Statistics
		Mean		St.dev.		mean		St.dev.		F		P		t		*
Loafing		4.6		3.6		7.6		5.6		4.1		4.7.		2		*
	Laying		0.75		3		3		6		1.9		0.17		1.3
	Social grooming		4.29		6.5		0.2		0.56		5.94		2.13		2.34		*
	Rubbing		2.8		4.32		0.66		2.1		2.9		9.6		1.7
	Foot stamp		0.4		0.8		0.13		0.5		1.12		0.29		1.0
	Head- toothing		0.62		1.4		0.125		0.5		1.79		0.19		1.33
	Ear flick		25.8		18.5		2.4		2.4		23.5		4.1		4.8		*
	Tail wagging		56		28.5		8.6		6.9		38.9		1.0		6.2		*
	Rumination		0.53		1.99		2.8		5.4		2.49		0.12		1.51
	Exploration		0.75		0.35		3		2		2.2		0.2		1.4
	Licking		24.8		31.7		1.7		5.7		7.6		9.7		2.7		*
	Vocalization		3		6.6		0.0		0.0		3.2		8.0		1.8

Table (2) Behaviour patterns of calf cattle infested with ticks before and after treatment with Diazinone

	Behaviour patterns		Before treatment				After treatment				Statistics
		Mean		St.dev.		mean		St.dev.		F		P			t			*
Loafing		5.6		5.4		1.6		2.6		3.6			7.9			1.9
	Laying		0.0		0.0		7.8		7.5		5.2			4.1			2.2		*
	Social grooming		1.03		1.47		0.33		1		1.13			0.3			1
	Rubbing		0.2		0.4		0.2		0.6		4.3			0.9			6.6
	Foot stamp		1		1.4		0.0		0.0		4.8			4.8			2.1		*
	Ear flick		10		14.7		0.6		2		4.2			6.1			2
	Tail wagging		13.2		14.1		9.3		18		0.16			0.68			0.4
	Rumination		0.0		0.0		6.4		6.3		5			4.4			2.2		*
	Exploration		1.2		1.6		0.8		1.1		0.18			0.67			0.45
	Licking		0.7		1.19		0.05		0.16		2.9			0.11			1.7
	Vocalization		0.0		0.0		1.2		2.9		0.8			0.38			0.89

Table (3) Behaviour patterns of adult buffaloes infested with ticks before and after treatment with Diazinone

	Behaviour patterns		Before treatment				After treatment				Statistics
		Mean		St.dev.		Mean		St.dev.		F		P			t				*
Loafing		3.8		3.1		9.7		6.5		6.8			1.55			2.6		*
	Laying		0.127		0.4		1.8		4.7		1.2			0.28			1.1
	Social grooming		1.7		2.3		0.06		0.25		7.4			1.1			2.7			*
	Rubbing		0.846		0.76		0.06		0.25		13.6			1.2			3.6			*
	Foot stamp		1.72		2.3		0.06		0.25		7.4			1.19			2.8			*
	Head- toothing		0.3		0.94		0.0		0.0		1.6			0.2			1.2
	Ear flick		36.5		36.6		5.5		5.3		10.6			3.4			3.2			*
	Tail wagging		43.4		53.1		5		3.2		7.9			9.6			2.8			*
	Rumination		0.0		0.0		2.1		5.2		1.6			0.21			1.2
	Exploration		0.8		1.03		0.0		0.0		9.2			5.9			3			*
	Licking		6		12.6		0.0		0.0		3.45			0.07			1.8
	Vocalization		0.27		0.64		0.0		0.0		2.9			0.1			1.7

Table (4) Behaviour patterns of buffalo calves infested with ticks before and after treatment with Diazinone

	Behaviour patterns		Before treatment				After treatment				Statistics
		Mean		St.dev.		Mean		St.dev.		F		P			t			*
Loafing		4		3.2		6		7.3		0.2			0.59			0.54
	Laying		0.0		0.0		1.6		5		0.42			0.52			0.65
	Social grooming		3		2.9		0.33		0.7		7.2			2.1			2.6		*
	Rubbing		0.37		0.75		0.0		0.0		1			0.35			1
	Foot stamp		0.25		5		0.0		0.0		2.5			0.13			1.5
	Ear flick		16.3		15.7		6.4		5.76		2.9			0.11			1.7
	Tail wagging		11		9.6		5		3.		2.9			0.11			1.7
	Rumination		0.87		0.75		3.1		6.1		0.48			0.5			0.69
	Exploration		1		1.2		0.1		0.3		4.9			4.8			2.2		*
	Licking		0.87		1.75		0.33		0.7		0.67			0.42			0.82

respectively. Calves treated with diazinone showed more time spent laying, 4.7 min and ruminating 2.1 min. volcalizations increased as a signs of normal health.

Obtained results in table (3) and Fig (3) revealed an increased in the frequency of the body care behaviour in the form of ear flick, tail wagging, licking, foot stamping and social grooming of the infested animals 10, 13.2, 0.7, 1 and 1.03 respectively. Body contact are more common in buffaloe than in cattle. Time spent laying and rumination increased in treated animals.

The mean time spent, laying, and ruminating as mentioned in table (4) and fig (4) recorded 0,0 and 0.87 min respectively. Social grooming increased between calves and their dams. Other body care behaviour used by adult animals may be used by small calves but to a lesser degree.

The behaviour patterns of treated calves returned its normality with time spent laying and rumination exceed the normal as a compensatory behaviour.

Grooming is an activity of primary importance to the survival of an animal, and encompasses all forms of care and attention to the body surface, either by an animal or by conspecific. Grooming is also the active means by which the animal defends its integument against attack from biological factors, specially ectoparasites and other micro-organism (McFarland, 1981).

Grooming in ungulates has been shown to be very effective in removing ectoparasites, especially ticks (Hart et al, 1992). Studies by (Lewis et al, 1967) showed that prevention of grooming in cattle, allow lice to increase about six-fold, but louse load is reduced to baseline levels within 3 days after grooming is once again allowed. Cattle groom with their tongues, whereas antelope use mostly the lower incisors in an upward scraping motion. In gazellas and impala the lateral incisors and canine teeth comprise the lateral dental grooming apparatus (McKenzie, 1990). The behaviour of both cattle and buffaloes infested with ticks indicated that tick infestation represents a source of irritation to the animal which inquire an effort to git ride of such stress by increaseing the body care behaviour related to skin hygiene. Licking behaviour as a mean of grooming used much more in cattle than by buffalo adults (24.8 and 6 time/15’m) respectively. Social grooming significantly increased among infested animals indicating a social cooperation between infested member to clean the head, ears and other inaccessible parts that an animal can not reached. Social grooming is a bovine trait that presumably promotes maternal-offspring bonds and social bonds among herd members (Estes, 1992). Bovine social grooming is directed mainly to the head and neck. Studies of social licking in domestic cattle suggest that it is a basic drive, satisfaction of which is essential to well-being.

Scratching is the main response to sources of peripheral irritation caused by fleas, lice and ticks (McFarland, 1981). When cattle groom, they lick all parts of the body that they can reach and scratch themselves against trees or fence posts (Broom, 1981). The hind foot is most commonly used for scratching by observed cattle and buffalo members.

Tail wagging and ear flick reached a maximum peak in tick infested animals as showed in all tables and figures in this respect buffaloe showed more ear flicking than cattle do (36.5 and 25.8) respectively while cattle used more tail wagging than the buffaloes. Young calves used foot stamping as stress indicators to get rid of the irrtating ticks, they are more sensitive than adults. (Barnard and Behnke, 1990), mentioned that a variety of defense behaviours were seen on parasite infested animals and birds: shaking, rubbling and scratching the head, pecking, stamping, slapping, shaking and lifting the legs and feet. Animals clearly vary in their individual resonses to attack by ectoparasites.

Regarding the altration caused upon the ingestive behaviour, obtained results revealed a loss of appetite of the all infested animal groups, and rumination behaviour reduced as a behavioural response of stress. Several studies have documented that in growing calves the equivalent of one engorging tick produces a growth decrement of at least 0.6 kg on an annualized basis (Little 1963; Seebeck et al., 1971; Turner & Short 1972; Williams et al. 1978). This effect of ticks on body weight is a result of the removal of blood and the depression of appetite due to toxins in tick saliva (Seebeck et al., 1971).

Results obtained revealed a marked decrease in the time spent sleeping and laying in all studied animal groups, this confermed by (Ruckebusch, 1975) who reported that, each species of animal has a particular circadian sleep profil (hypnogram). Factors, such as difficult in foraging, availability of food, digestive processes, predator susceptibility and other social and environmental pressures affect this hypnogram.

From results obtained and from observation it has been found that two levels of adjustment have been obtained by infested animal at the first level when ticks count reached 300-500 tick/animal. The behaviour related to skin hygiene increased and feeding, rumination, sheep bouts decreased. At the second level when ticks count reached 1000 or more tick/animal, the behaviour pattern in the form of feeding, resting and sleeping and other comfort movement were arrested and signs of anaemia, showing ribs, wound from crustation and scratching were the sequali.

Treatment with diazinone 0.01% returned the animals to their normal mainteinance and comfort movements. The increasing in time spent laying and sleeping after treatment is confirmed by (Swenson, 1975) who mentioned that in most species, sleep deprivation is followed by a compensatory rebound increase in sleeping when the opportunity becomes available.

1- some behavioural traits (body care behaviour, locomotor activity as well as ingestive and sleeping behaviour) can be used as stress indicators of cattle and buffalo infested with ticks.

2- Different species of bovidae used different traits to defend itself against ticks (buffaloes use ear flicks, rubbing and social grooming more than cattle which use licking, tail wegg more.

3- Young cattle tolerates ticks by another position, they increased shuddering of legs, and stamping with a marked head shaking.

4- Successful treatment and controle regimens must focus on the animal before reaching the second state of adjustment or frustreturn and on the environment surrounding the animal.

Barnard, C.J. and Behnke, J.M. (1990): Parasitism and host behaviour (editors). Taylor & Francis. London, New York. Philadelphia.

Borchelt, P.L. (1980): Care of the body surface (COBS). In : Comparative psychology: in Evolutionary Analysis of Animal Behaviour (Ed. By M.R. Denny), PP. 363-384. New York: John Wiley.

Broom, D.M., (1981) : Biology of Behaviour. (ed.) Cambridge University Press, Cambridge.

Estes, R., (1992): The behaviour of African Mammals. First edition. The University of California Press.

Fraser, A.F. & Broom, D.M. (1990) : Farm Animal Beaviour and Welfare. Bailliere Tindall, London.

Hart, B.L. (1990) : Behavioural adaptations to pathogens and parasites. Neurosci. Biobehav. Rev., 1, 274-294.

Hart, B.L., Hart. L.A., Mooring, M.S., and Olubayo, R. (1992): Biological basis of grooming behaviour in antelope: the body. Size, Vigilance and habitate principles. Anim. Behav., 44, 615-631.

Hill. R.W., (1976): Comparative physiology of Animals: An Environmental approach. Harper & Row, Publisher, New York, Hagerstown, San Francisco, London.

Lewis, L.F., Christenson, D.M. & Eddy, G.W. (1967): Rearing the long-nosed cattle louse and cattle biting louse on host animals in Oregon. J. econ. Entomol., 60, 755-757.

Little, D.A., (1963): The effect of cattle tick infestation on the growth rate of cattle. Austral, Vet. J., 39, 6-10.

Martin, A. and Bateson, A, (1986): Measuring behaviour : an introductory guide, Cambridge University Press.

McFarland, D.J. (1981): The Oxford Companion to animal behaviour (ed). Oxford University Press. Oxford, New York, Toronto, Melbourne.

McKenzie, A.A., (1990): The ruminant dental grooming apparatus. Zool. J. Linn. Soc., 99, 117-128.

Patenaude, F. & Bovet, J. (1983): Self-grooming and social grooming in North American beaver (Caster Canadensis). Can. J. Zool., 62, 1872-1878.

Ruckebusch, Y., (1975): The hypogram as an index of adaptation of farm animals to change in their environment. App. Anim. Ethol: 2, 3-18.

Scott. D.W. (1988): Large Animal Dermatology (ed.). Parasitic diseases, part 2, pp.233-283.

Seebeck, R.M., Spingell, P.H. & O’Kelley, J.C. (1971): Alterations in host metabolism by the specific and anorectic effects of the cattle ticks (Boophilus microplus) I, Food intake and body weight growth. Austral. J. Biol. Sci., 24, 373-380.

Snedecor, G.W. & CocHran, W.G. (1980): Statistical Methods. Ames, IOWA State University Press.

Swenson. M.J. (1975): Dukes’ Physiology of Domestic Animals. (9^th. ed.). Comstock Publishing Associates. Ithaco and London.

Turner, H.C. & Short, A.J. (1972): Effects of field infestations of gastrointestinal helminths and the cattle ticks (Boophilus micropuls) on growth of three breeds of cattle. Austral. J. agric. Res., 23, 177-193.

Williams, R.E., Hair, J.A. & McNew, R.W. (1978) : Effects of Gulf Coast Ticks on blood composition and weight of pastured Hereford stress. J. Parasitol., 64, 336-342.

Wooldridge, (1974) : The role of research in animal reproduction. Vet. Record., 23, 276-280.

EFFECT OF TWO IN VITRO CULTURE TREATMENTS OF EMBRYOS ON PREGNANCY RATES IN COWS

Embryo Transfer Lab.,College Of Veterinary Medicine And Biomedical Sciences,Colorado State University,Fort Collins,Colorado 80523,USA.

ABSTRACT

Ovaries were collected from cull cattle after slaughter in a slaughter house then send to the laboratory. Oocytes were collected from ovaries. Oviduct epithelial cells were isolated from oviducts. Oocytes were matured in Hepes-buffered TCM-199.Frozen semen from two fertile bulls was thawed,capacitated using swim up technique. Capacitated sperm were added to matured oocytes for in vitro fertilization. Half of the embryos was cultured with oviduct cells and the other half was cultured with chemically defined medium. Thirty two recipients (16/treatment) were synchronized using PGF2 alpha, examined ultrasonographically to locate the ovaries containing CLs. Two of the best embryos were transferred to each recipient cow. Ultrasonographical scannings were applied for detection of pregnancy in cows. Pregnancy rate of recipients cows recieving in vitro embryos with cultured in oviduct epithelial cells was significantly higher (45%) than in chemically defined medium (37.5%).

INTRODUCTION

Reports of resulting live offsprings following embryo transfer of in vitro fertilized embryos were found in rabbits (Chang,1959) ,mice (Whittingham,1968) ,rats (Toyoda and Chang,1974) and human (Steptoe and Edwards,1978).

Although embryo transfer in cattle ,involving transfer of embryos from the uterus of a valuable donor cow into uteri of less valuable recipients for development has been widely practiced in veterinary medicine during the last decade (Seidel,1981),effects towards in vitro fertilization and embryo transfer in ungulates were termed notoriously unsuccessful in 1980 by Blandau, who cautioned against equally potential developmental risks of embryo transfer with those that remain largely unknown for in vitro fertilization and embryo transfer .

Investigations of morphological and physiological aspects of bovine fertilization and early development , along with earlier in vitro efforts (Brackett et al., 1977,1978,1980 and 1981).

The goals of this investigation were to compare the effect of two in vitro culture treatments for embryos (oviduct epithelial cells in 199 lite and chemically defined medium) on conception rates in cows.

MATERIALS AND METHODS

Ovaries of cull cattle were obtained after slaughter in a slaughter house and transported to embryo transfer laboratory at Fort Collins, Colorado, USA in a thermally insulated container of sterile 0.9% NaCl at 30^o C.Ovaries and oviducts were rinsed two times in sterile saline , 20 seconds in 70% ETHOH , and then rinsed another two times in sterile saline. Follicles 1-6 mm were aspirated within 8 hours of slaughter with an 18 ga needle attached to washed and sterilized syringes using modified Dulbecco's phosphate-buffered saline. Oocytes were selected for experiments if they had homogenous,evenly granulated cytoplasm and a compact cumuls mass and then placed into 35 x 10 mm plastic dishes (Falcon # 1008) containing PBS + 1% bovine serum albumin before being transferred into maturation treatment.

Oviducts were cleaned by the method described for ovaries, except oviducts were rinsed in ETHOH no longer than 10 seconds.Epithelial cells were isolated by scraping the outer surface of the oviduct with a sterile microscope slide while grasping the other end with sterile forceps. Cells were forced out the lumen of the opposite end of the oviduct into a plastic dish containing modified Dulbecco's PBS. Tissue was broken up by aspirating through progressively smaller needle, until it could pass through 26 ga needle. Cells were washed four times by centrifuging 5 seconds at 900 x g and replacing supernatent twice with fresh PBS and then twice with TCM-199 lite. Each culture drop of TCM-199 lite under oil was added with a volume of cells approximately equal to the volume of 10-20 oocytes with their attached cumuls cells. Epithelial cells were cultured in these drops and 15 oocytes were matured per 200 u drop.

Hepes-buffered TCM (Sigma cat.No.2520) was supplemented with 10% (v/v) heat-treated estrous cow serum, bicrbonate and antibiotics added and pH adjusted to 7.45 with 1 NaOH. Estradiol-17 B (Sigma cat.No. E-8875,lot 34 F-0540), FSH (NIH-FSH-5-17) and LH (USDA-bLH-8-5) were stored frozen as 100 x stock solutions and added to maturation medium no longer than 24 hours before oocytes were added. Forty excellent oocytes were added and matured (20 oocytes /drop ). Maturation culture was in 0.2 ml. drops of medium under sterile, washed paraffin oil in a high humidity , 5% Co2 in air, 39^o C incubator for 24 hours.

Frozen ejaculated semen from two fertile bulls was thawed (at 35 C water bath for 12 seconds ) and then mixed in a 12 x 75 mm plastic tube (Falcon # 2058). Sperm were separated from extender by a 1 hour swim-up into Sp-talp (Parrish ;Susko-Parrish;Winner and First,1988),a modified Tyrode medium ,containing 5 U/ml heparin (porcine intestinal mucosa heparin,Sigma cat.No.H-3125).

Following the swim up, Sp-talp containing sperm was aspirated from tubes that contained a layer of semen in extender beneath a 1-ml column of Sp-talp. Thus , the extender layer was discarded. The resulting sperm suspension was centrifuged for 5 minutes at 900 X g and the volume of Sp-talp adjusted to concentrate sperm to 2x10⁷ sperm /ml. Sperm were allowed to capacitate an additional 2 hours in Sp-talp heparin before being added to the oocytes

Oocytes were removed from maturation medium , washed 3 times in Fert-talp (Parrish et al., 1988) and placed into 0.2 ml droplet of Fert-talp (20 matured oocytes/droplet) under oil. Sperm suspension was added (0.05 ml) , which gave a final concentration at insemination of 2 X 10 sperm/ml.Gametes were coincubated 18 hours in a high humidity , 5% Co₂ in air , 39^o C incubator.

Half of the embryos were cultured with oviduct epithelial cells (collected at the same day of collection of ovaries) in TCM-199 lite; the other half was cultured in chemically defined medium + 0.12 U insulin/ml + amino acids (Seidel; Glass and Olson,1991).

Thirty two recipient cows were used in this study , 32 embryos per treatment. Prostaglandin F2 alpha (Lutalyse ; Pharmaicia and Upjon Inc., Kalmazoo, Ml) was used for estrus synchronization on day 15 of the estrous cycle. After 6-7.5 days from estrus detection, each recipient was examined ultrasonographically to locate the ovary containing corpus luteum. The best embryos of each treatment were selected and precise descriptions were recorded. A random set of two of the best half of embryos was transferred ipsilateral to the CL of each recipient. Recipients were examined ultrasonographically on day 35 of pregnancy.

RESULTS

Tables 1 and 2 show that pregnancy rate of recipient cows after transfer of in vitro cultured embryos using oviduct epithelial cells was significantly higher (45.0%) than that in using chemically defined medium(37.5%).

Table (1): Effect of using oviduct epithelial cells or chemically defined medium during transferring of bovine embryos to recipient cows on pregnancy rate.

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DISCUSSION

The pregnancy rate of recipient cows after transfer of in vitro cultured embryos using oviduct epithelial cells was higher (45.0%) than in using chemically defined medium (37.5%). These results are in accord with Gandofli and Moore (1987) who produced a higher pregnancy rate from sheep embryos co-cultured with oviduct epithelial cells than without co-culture. It appears that oviduct epithelial cells support embryonic growth and nidation via secretions into the medium more than conditioned media (Eyestone and First ;1989).

So ,we advice adding oviduct epithelial cells as a good choice for in vitro co-culture media of embryos which are transferring to recipient cows, since they play a very important role in sustaining embryos development and nidation.

REFERENCES

Blandau,R.J.(1980): In vitro fertilization and embryo transfer. Fertil.Steril. 33:3-11.

Brackett, B.G. ;Bousquet,D.; Boice,M.L.;Donawich,W.J. and Evans,J.F.(1981):Pregnancy following cow in vitro fertilization. Biol.Reprod.24(Suppl.)173:190.

Brackett,B.G.;Evans,J.F.;DonawicK,W.J.;Boice,M.L.and Cofone,M.A. (1980): In vitro penetration of cow oocytes by bull sperm. Arech.Anrol.5:69-71.

Brackett,B.G.;Oh,Y.K.;Evans,J.F.and Donawick,W.J.(1978): In vitro fertilization of cow ova .Theriogenology 9:89.

Brackett,B.G.;Evans,J.F.and Donwich,W.J.(1980): Fertilization and early development of cow ova. Biol.Reprod.23:189-205.

Brackett,B.G.;Oh,Y.K.;Evans,J.F.and Donawick,W.J.(1977): Bovine fertilization and early development in vivo and in vitro.10 th Ann.Mtg.Soc.Study Reprod.,Abstr.56-57.

Chang,M.C.(1959): Fertilization of rabbit ova in vitro .Nature(Lond.).184:466-467.Eyestone,W.H. and N.L.First(1989):Co-culture of early cattle embryos to the blastocyst stage with oviductal tissue or in conditioned medium. J.Reprod.Fertil.85:715-720.

Gandofli,F. and R.M. Moore(1987): Stimulation of early embryonic development in sheep by co-culture with oviduct epithelial cells.J.Reprod.Fertil.81:23-38.

Parrish,J.J.; J.L.Susko-Parrish; M.A.Winner and N.L.Frist(1988): Capacitation of bovine sperm by heparin. Biol.Reprod.38:1171-1180.

Seidel,G.E.,Jr.(1981): Superovulationand embryo transfer in cattle.Science 211:351-35

Seidel,G.E.,;Glass and S.E.Olson (1991): Culture of 1-cell bovine embryos to blastocysts in chemically defined media. Biol.Reprod.44:88(Abstr.)

Snedecor,G.W.and Cochran,W.(1989): Statistical methods . 8th ED.Iwa State Univ.Press.Ames,Iwa,USA.

Steptoe,P.C.and Edwards,R.G.(1978): Birth after the reimplantation of a human embryo.Lancet 2:366.

Toyoda,Y.and Chang,M.C.(1974): Fertilization of rat eggs in vitro by epididymal spermatozoa and the development of egg following transfer. J.Reprod.Fertil.31:9.

Whittingham,D.G.(1968): Fertilization of mouse eggs in vitro .Nature(Lond.) 220:592-593.

OVARIAN AND TESTICULAR HISTOLOGICAL CHANGES RELATED TO HUMAN CHORIONIC GONADOTROPHIN ADMINISTRATION IN CHICKENS

A.M.M. HAMDY, A.K.I ABD EL MOTY, A.H. EL BOGDADY, A.A. ABD EL HAKEAM AND H.H. M. HASSANEIN

Animal Production Department, Faculty of Agriculture, Minia University, Egypt

ABSTRACT

This study was conducted to examine the effect of Human chorionic gonadotrophin (HCG) on ovarian and testicular histological changes. A total number of 192 of Fayoumi chickens (96 females and 24 males) at 16 weeks of age were used. Birds were divided into two groups; each of 48 females and 12 males. Birds of group 1 were injected by saline solution at 18 weeks of age, while those of group 2 were daily injected subcutaneously by 50 IU of HCG for 7 successive days. The pregnyl trademark was used as a source of the HCG. All birds were reared under the standard managerial conditions. At 18 and 21 weeks of age, 4 females and 4 males from each group were slaughtered. The ovary and testis were removed and processed for obtained tissue sections. From the obtained results, it could be concluded that HCG injection improved the number, the size and the maturation of the ovarian follicles. In cocks, it improves integrity of the seminiferous tubules and precocious spermatogenesis.

INTRODUCTION

Human chorionic gonadotrophin (HCG) is synthesized by the syncytiotrophoplastic cells of the placenta of pregnant primates and is found in the blood and urine. It has been detected in the urine eight days after conception by sensitive radioimmunoassay (Jaffe, 1978). HCG has both LH and FSH-like biologic action. Both avian LH and FSH are glycoprotein hormones with molecular weight of approximately 300,00 (Burke et al., 1979 and Popkoff et al., 1982). Avian LH and FSH like their mammalian homologue, consist of two glycoprotein subunits with molecular weight 15,000 each (Sturkie, 1986). The subunit has 92 amino acids and two carbohydrate chains, while subunit has 145 amino acids and five carbohydrate chains (Hafez, 1985). The function of the ovary and tests, in birds, which are controlled by the hypothalamic and hypophysecal hormones has been studied by several investigators. It is well known that, in the females, FSH mainly causes follicular growth and maturation while LH induces ovulation. While in the males, FSH initiate the growth of seminiferous tubules and LH causes the development of the extratubular leydig cells. Plamar and Bahr (1992) reported that the injection by FSH for old hens (90 wks.) significantly increase the growth of ovarian follicles. Ismail (1994) concluded that daily injection by 50 or 75 IU of HCG led to increased growth and number of follicles and egg production from force molted hens treated during or after fast period. Hamdy et al., (1999) and Hassona, (1998) speculated that HCG injection improved the reproductive performance of Hubbard parent cockerels and prolonged the production period and the semen quality during the second year of production.

The objective of this study was to examine the ovary and testicular histological changes related to HCG administration.

MATERIALS AND METHODS

This work was carried out in the Poultry Research Farm of Animal Production Department, Faculty of Agriculture, Minia University. A total number of 120 (96 females and 24 males) of Fayoumi birds, aged 16 weeks were used. Birds were divided into two groups. Each group contained 48 females and 12 males. Birds in each group were divided into four replicates (12 females and 3 males). All birds were kept under the same managerial and environmental condition regime. Birds were exposed to naturally light regime during the experimental period. Birds of each replicate were housed in floor pen provided with wood sharing as a litter. All birds were fed laying hen ration ad libitum during the entire experimental period and water was available all time. This laying ration contained 16.53% crude protein, 2750 Kcal / kg ME, 3.41% calcium and 0.70% total phosphorus.

Pregnyl HCG (Nile Company, Cairo, Egypt) was used for individually subcutaneously injection of females and males. Birds of the two groups were treated as follows:

Group 2: Daily injection by 50 I.U / ml HCG in saline solution for successive of 7 days at 18 weeks (before sexual maturity.

At 18 and 21 weeks of age 4 females and 4 males were chosen randomly from each group at each time. Both ovary and testis were removed after slaughtering and preserved in Bouin’s solution for 24 hrs. then dehydrated in ascending grades of ethyl alcohol, cleaned, embedded, sectioned and stained using Haematoxyline and Eosin stain for microscopically identification of histological structure microscopy. The pictures were taken at magnification power of X100.

RESULTS AND DISCUSSION

Plates 1,2 and 3 show the histological sections of the ovary at 18 and at 21 (non-injected and injected), respectively. The sections showed that the injection of HCG clearly increased the number of mature follicles. At the same time the size of both follicles and oocytes increased. The possibility remains that each of FSH and LH has a certain role and strong relationship with the metabolic activity tools especially thyroid hormones. This may led to increased yolk volume deposition. It was observed that age has a positive effect on the development of ovarian follicles as it can be seen from the comparison between plates 1 and 2.The positive effect of HCG injection on the ovarian follicles development may explain the earlier sexual maturity, and the increase in egg production data, which was published elsewhere. The histological effects of HCG injection are in agreement with those stated by Sturkie (1986). Moreover, Plamar and Bahr (1992) and Ismail (1994) concluded similar observations.

Plates 4, 5 and 6 show the histological sections of the testes at 18 and 21. It seems logic that the reproductive organs grow by advantage of age, that could be observed clearly when comparing the plates 4 with plate 5. However, HCG injection has an analeptic effect on seminiferous tubules and precocious spermatogenesis. These differences are clear when plate 5 compared with plate 6. Furthermore, HCG injection caused significantly improvement the reproduction characters in males (Hamdy et al., 1999 and Hassona, 1998). Hocking (1991) reported that stimulating the sections of androgen from leydig cells through the hypothalamus-hypophysial gonadal axis could cause the histological differences in testis. Moreover, the physiological effects of HCG on increasing metabolic activity and semen quality were published elsewhere.

REFERENCES

Burke, W., P.L., Licht, H. Popkoff, and G.Bona, (1979): Action and characterization of Luteinizing hormone and Follicle stimulating hormone for pituitary glands of the turkey (Meleagris Gallopavo) Gen. Comp. Endocrinol., 37:521-532.

Hafez, E.S.E. (1985): Endocrinology of reproduction. In: Reproduction in Farm Animals. Ed. By E.S.E. Hafez, 5^th edition PP:57-113. Philadelphia.

Hamdy,A.M.M., A.A., Abd El Hakeam, Kh.A. Mohammed and M.A. Hassona (1999): Enhancing semen quality of Hubbard parent cockerels during the second year of production. Egyptian J.Anim. Prod. 36(1): 51-70.

Hassona. M.A.(1998): Reproductive performance of Hubbard parent cockerels as affected by force molting after the first year of production. M. SC. Thesis. Fac. Of Agric., Minia Univ., Egypt.

Hocking, P.M. (1991): Effect of controlling body weight on the semen production of large white turkey males. Br. Poultry Sci., 32:211-218.

Ismail, H.T.M. (1994): Studies on follicle stimulating hormone during molting of laying fowls. M.Sc. Thesis. Fac. of Agric. Zagazig Univ., Egypt.

Jaffe, R.B. (1978): The endocrinology of pregnancy. In: Reproduction Physiology. Ed. By S.S.C Yen and R.B. Jaffe, P:521-536. W.B. Sounders Co. Philadelohia, USA.

Palmar,S.S., and J.M. Bahr (1992): Follicle stimulating hormone increases serum oestradiol 17 β concentrations, number of growing follicles and yolk deposition in aging hens (Gallus Domesticus) with decreased egg production. Br. Poultry Sci., 33:403-414.

Popkoff, H., A. Plicht, B. Gallo, D.S. Mackenzine, W. Oelofsen and M.M. Oosthuizen (1982): Biochemical and immunological characterization of pituitary hormones from the ostriol (Stutbio Camelus). Gen. Comp. Endocrinol, 48:182-195.

Sturkie, P.D. (1986): Avian Physiology. Spriner-Verlag, New York, Berlin, Heidelberg, Tokoyo.

Plate 1. Cross-section of ovary at 18 weeks of age, showing normal follicular development.

Plate 2. Cross section of ovary at 21 weeks of age (non-injected), showing fast growing of follicles.

Plate 3. Cross-section of ovary at 21 weeks of age (injected by HCG at week 18), showing large and mature follicles.

Plate 4. Tests of cock at 18 weeks of age, showing stage of spermatogenesis and interstitial cells.

Plate 5. Testis of a cock at 21 weeks of age (non-injection), showing several seminiferous tubules and thick C.T septa between tubules.

Plate 6. Testis of a cock at 21 weeks of age ( injected by HCG at week 18), showing many large seminiferous tubules contain numerous sperms.

INFLEUNCE OF EGG INJECTION IN DIFFERENT PLACES BY VITAMIN E AND SODIUM SELENITE ON HATCHABILITY AND SUBSEQUENT PRODUCTIVE PERFORMANCE OF JAPANESE QUAIL CHICKS

A.M.M. HAMDY AND S.A. ABD-ELATIF

Animal Production Department, Faculty of Agriculture, Minia University, Egypt

ABSTRACT

One thousand fertile eggs of Japanese quail, collected from one flock, through sequence of five days were performed. Eggs were in average of 12.7± 0.7 gm. All eggs were randomly divided into equal five groups. Eggs in group 1, were uninjected, those of group 2 were sham injected. While those of group 3,4 and 5 were injected in large end, middle and narrow end , respectively. The injection applied was by 0.2 ml saline solution / egg. Saline solution contained of 300 µg of Vit.E; and 2.5 µg /0.2ml of Se.

All eggs were set in the same time in one incubator at 37-38 °C and 55% relative humidity. After hatching, chicks of each group were divided randomly into 4 replicates. All replicates were housed in one battery line and reared under standard husbandry practices. The experiment continued till three weeks of age.

Obtained results indicate that egg injection applied improved (P<0.05) the hatchability percentage. There were no significant differences on hatchability among injection places. However, there was no significantly (P>0.5) effect of injections applied on percentages of dead embryo in prehatching stages. Moreover, obtained data indicated that, there was no significant (P>0.5) effect of injection on body weight and feed intake. While few significances appeared on feed conversion ratio. Values of mortality rate in post hatch period indicate that birds hatched from eggs injected by Vit. E and Se recorded significant (P<0.5) lower mortality rate when compared with those hatched from noninjected or sham injected eggs.

It could be concluded that, egg injection by Vit. E and Se improved (P<0.05) hatchability, and livability during posthatch period. While there was no notify effect of injection place on the hatchability and livability during post hatch period.

Keywords: Egg injection, vitamin E, sodium selenite, hatchability, productive performance.

INTRODUCTION

It is now well accepted that soluble growth factors have critical roles to play in the control of growth and differentiation during embryonic development. In 1997, Sanders and Wride, studied some factors affecting the embryonic developments, and suggested that the possible functions in overall pattern formation and embryonic induction to controls at the cellular level of morphogenesis, including phenotypic transformation, cell adhesion, cell proliferation and cell death. It is also accepted that free radicals can cause metabolic disturbances and cell injury in many ways. A reactive free radical formed close to DNA may produce a change in the molecular structure resulting in a mutation or cytotoxicity (Collins et al., 1994). Reactive free radicals may cause, profound changes in enzyme activity, and damage cells by lipid peroxidation of polyunsaturated fatty acids. This is by far, the most important damage produced by free radicals in the animal cells.

Vitamin E (Vit. E), is a term used to describe two compounds: tocopherols and tocotrienols. Vit. E, is through its intra-membrane antioxidant properties, may protect tissue membranes from lipid peroxidation caused by free radical attack. It could, therefore, reduce the associated loss of integrity of function of cell membranes and associated increased cellular permeability and play a role in improving the livability (Lee et al., 1998).

Trace minerals such as zinc, copper, iron, manganese, selenium and iodine are essential nutrients required in small amount for normal growth and development of the avian embryo (Richards, 1997). Thus, it is crucial to the survival of the embryo that the requisite amount of each essential trace mineral be available at the appropriate time during its growth and development within the egg.

The objectives of the present study were to determine the influence of injecting eggs in different places by vitamin E (Vit.E) and sodium selenite (Na₂SeO₃) on hatchability, dead embryos and productive performance of Japanese quail chicks.

MATERIALS AND METHODS

One thousand fertile eggs of Japanese quail, collected from one flock, through sequence of five days were performed. Eggs were in average of 12.7± 0.7 gm. All eggs were stored for maximum of five days at –17 °C and 50-60% relative humidity till the start of the experiment.

At the beginning of the experiment, all eggs were randomly divided into equal five groups. Eggs in group 1, were uninjected, those of group 2 were sham injected. While those of group 3,4 and 5 were injected, in large end, middle and narrow end, respectivily; by 0.2 ml saline solution / egg. Saline solution contained of 300 µg / 0.2 ml of Vit.E; and 2.5 µg / 0.2 ml of Se. These doses were based on and calculated as recommended by NRC (1994). For the egg injections, 0.2 ml of solution was injected per egg at day 0 of incubation, just before incubation, using a separate syringe with needle for each individual egg.

Egg injection mechanism was done according to the method explained by Robel (1993 a,b). Egg injections were performed in a sanitized clean room with a particle-free environment using filtered, high efficiency particulate air, and personnel were aseptically equipped. A 2% iodine tincture was applied to the injection site (large, middle and narrow end). A very slight indentation was made in the swabbed shell area using a sharp sterile punch. The force used to make the indentation was of a minor degree to avoid the formation of hairline cracks in the shell. The punch site exactly accommodated the microfine Ш, 28-gauge sterile needle that was locked to a 1-ml sterile insulin syringe. The needle was carefully inserted through the indentation site to a depth of 6 to 8 mm, and 0.2 ml of solution was injected over the inner eggshell membrane. The puncture was sealed with a small drop of fast-drying wax.

Eggs of each group were divided into four replicates each of 50. Each tray loaded by 250 eggs. Each tray of the incubator included 50 eggs presenting each group to avoid place effect in the incubator. The four trays were set in the same time in one incubator at 37-38 °C and 55% relative humidity. The incubator was fumigated before setting the trays and eggs were fumigated after 8 hrs. of incubation. The eggs were turned after each 4 hrs. for the first 24 hrs. and then every two hrs. up to day 15^th of incubation. At day 15^th of incubation the four trays were transferred to one hatcher set at 37.3-37.5 °C and 80-90% relative humidity. At day 18^th, the trays were taken off from the hatcher. Within each group hatched chicks were counted, leg banded and weighed individually to the nearest 0.1 gm. Percentages of hatchability were then calculated. Dead embryos were counted and expressed as a percentage from the basic number within each egg replicate (50). Dead embryo was concerned as that the not complete developed and without feathers at day 18^th. Within each group, the rest of 100% of incubated eggs (completed but not hatched chicks) was also counted and analyzed.

After hatching, chicks of each group were divided randomly into 4 replicates. All replicates were housed in one battery line and reared under standard husbandry practices with ad lib feed of 24 % crude protein and 2950 Kcal ME/Kg. Water and light were provided continuously. The experiment continued till three weeks of age.

Body weight was recorded at hatch and weekly then after for each replicate. Weekly feed intake was determined and feed conversion was calculated. Dead birds were counted weekly and expressed as a percentage from the basic number within each replicate.

Obtained data other than mortality rate and hatchability were subjected to ANOVA (SAS, 1992). If probability (P values)<0.5 were obtained, significant differences among groups were analyzed using Duncan’s multiple range test (Duncan, 1955). Percentage data of mortality rate and hatchability were transformed to arcsine √ % for the analysis of variance.

RESULTS AND DISCUSSION

Data in Table 1 show that there were no significant differences among injection places on hatchability. Obtained data reveal that egg injection significantly (P<0.05) improved the hatchability percentage (Table 1). These results are in good agreements with those reported by Gore and Qureshi (1997), they speculated that Vit. E prevents oxidation of unsaturated lipids materials within cell, thus protection the cell membrane from oxidative damage. Moreover, obtained results are supporting the conclusion of Latshaw et al (1977). They concluded that, the addition of sodium selenite to laying diets, improved (P<0.5) egg production, fertility and hatchability. They also, added that the effectiveness of Se is dependent on the form of which supplement to the diet; is it in sodium selenite, selenonethionine or seleno-cystine form.

Obtained data on dead embryo and completed but not hatched chicks in prehatching stages are shown in Table 1.The analyzed values indicate that there was no significant (P>0.5) effect of injections applied of the dead embryos. However, in recent years intensive selection for rapid growth, body conformation, and feed efficiency may have augmented the safety level required to sustain hatchability. Selection of formal size of egg and controlling the incubation conditions may lead to chicks of good quality.

Table 1. Percentages of hatchability and dead embryo as resulted from egg injection by vitamin E and sodium selenite in large end, middle and narrow end.

Item	G1	G2	G3	G4	G5
Hatchability %	57.3±1.8^b	56.5±1.5^b	59.3±2.0^a	59.7±2.0^a	59.6±2.1^a
Dead embryo %	33.4±1.7^a	34.4±1.5^a	31.7±1.4^a	31.5±1.2^a	31.9±1.1^a
Completed but not hatched chicks %	9.3±2.2^a	9.1±2.9^a	9.0±2.8^a	8.8±2.8^a	8.5±2.9^a

Least squares means in the same row with no common superscript are differ significantly (P<0.5).

G1: Control non injected eggs. G2: Sham injected eggs G3: Eggs injected at large end

Data in Table 2 show the body weight, feed intake, feed conversion and mortality percentages during the first three weeks after hatch. Obtained data indicated that, there was no significant (P>0.5) effect of injection place on body weight and feed intake. While few significances appeared on feed conversion ratio. From these results, it seems that increasing body weight and decreasing feed intake were going in one direction, which lead to improving feed conversion.

Data on mortality percentages are shown in Table 2. Table 2 shows that there were no significant effect of injection place on the mortality percentages. Obtained values indicated that birds hatched from eggs injected by Vit. E and Se recorded significant (P<0.5) lower mortality rate when compared with those hatched from noninjected or sham injected eggs. Differences in this percentage values probably reflect the effect of injection applied Hereagain, the mechanisms of Vit. E and Se, protection of liver or other organs against oxidative damage and maintaining more normal function of cellular processes regulating growth and increase livability.

It could be concluded that, egg injection by Vit. E and Se improved (P<0.05) hatchability, and livability during post hatch period. While there was no notify effect of injection place on the hatchability and livability during post hatch period.

REFERENCE

Collins,A., S.Duthie, and M. Ross (1994): Micronutrientsd and oxidative stress in the aetiology of cancer. Proceedings of the Nutrition Society, 53:67-75.

Duncan, D.B.(1955): Multiple range and multiple F test. Biometrics, 11:1-42.

Gore, A.B., and M.A. Qureshi (1997): Enhancement of humoral and cellular immunity by vitamin E after embryonic exposure. Poultry Sci., 76:984-991.

Halliwell, B., and J.M.C. Gutteridge (1989): Free radicals in biology and medicine, 2^nd edn (Oxford, UK, Clarenton Press).

Latshaw, J.D., J.F. Ort and C.D. Diesem (1977): The selenium requirements of the hen and effects of a deficiency. Poultry Sci., 56:1876-1881

Lee, S.B., M.A. Mitchell, D.B. Utomo, P.E.V. Williams and C.C. Whitehead. (1998): Influence of high dietary vitamin E supplementation on egg production and plasma characteristics in hens subjected to heat stress. British Poultry Sci., 39:106-112.

Table 2. Least squares means (± SE) of hatch weight (H.W), body weight (B.W), feed intake (F.I) gm/ bird, feed conversion (F.C) gm feed/gm body gain and mortality rate of chicks hatched from injected eggs by vitamin E and sodium selenite in large end, middle and narrow end

Item	G1	G2	G3	G4	G5
H.W (gm)	8.2±0.2^a	8.4±0.2^a	8.4±0.1^a	8.3±0.2^a	8.5±0.1^a
B.W at wk 1	16.8±0.5^a	16.8±0.6^a	17.9±0.5^a	18.3±0.5^a	18.8±0.6^a
B. W at wk 2	31.6±1.8^a	32.4±1.8^a	32.7±1.7^a	34.6±1.8^a	35.4±1.6^a
B W at wk 3	75.8±3.2^a	78.5±3.7^a	82.2±3.0^a	83.8±5.4^a	86.6±5.2^a
F.I at wk. 1	38.5±1.5^a	39.8±1.7^a	37.6±1.4^a	37.6±2.0^a	36.2±1.5^a
F.I at wk. 2	62.6± 2.4^a	64.3± 2.8^a	61.5± 2.3^a	56.5± 2.0^a	54.9± 2.2^a
F.I at wk.3	114.3±4.8^a	118.5±5.6^a	117.8±5.9^a	104.9±6.2^a	106.5±5.5^a
F.C at wk.1	4.5±0.1^a	4.7±0.1^a	4.0±0.1^a	3.7±0.1^b	3.5±0.1^b
F.C at wk.2	4.2±0.1^a	4.1±0.2^a	4.1±0.2^a	3.5±0.2^b	3.3±0.2^b
F.C at wk.3	2.5±0.3^a	2.6±0.3^a	2.4±0.3^a	2.2±0.2^a	2.1±0.3^a
Mortality % at wk. 1	5.6±0.5^a	5.8±0.6^a	3.6±0.3^b	3.8±0.4^b	3.4±0.3^c
Mortality % at wk .2	2.1±0.2^a	2.3±0.3^a	1.4±0.2^b	1.5±0.2^b	1.2±0.2^b
Mortality % at wk. 3	0.5±0.01^a	0.6±0.01^a	0.5±0.01^a	0.5±0.01^a	0.4±0.01^a

Least squares means in the same row with no common superscript are differ significantly (P<0.5).

G1: Control non injected eggs. G2: Sham injected eggs G3: Eggs injected at large end

National Research Council (1994): Nutrient requirements of poultry. 9^th rev.ed. National Academy Press. Washington.

Richards, M.P.(1997): Trace mineral metabolism in the avian embryo. Poultry Sci., 76:152-164.

Robel, E.J. (1993a.): Evaluation of egg injection of folic acid and effect of supplemental folic acid on hatchability and poult weight. Poultry Sci., 72:546-553.

Robel.E.J. (1993b): Evaluation of egg injection method of pantothenic acid in turkey eggs and effect of Supplemental pantothenic acid on hatchability. Poultry Sci., 72:1740-1745.

Sanders, E.J., and M.A. Wride (1997): Roles for growth and differentiation factors in avian embryonic development. Poultry Sci., 76:111-117.

SAS Institute, (1992): Guide for personal computer. SAS Institute, Inc. Cary, NC.

STUDIES ON PRESERVATION OF GOAT SEMEN:

A.A. ABD ELHAKEAM

Animal Production Department, Faculty of Agriculture,University of Minia, Egypt.

ABSTRACT

A method of cold dilution at 5^oC was developed for improving storage-ability of both liquid and frozen-thawed goat semen and compared with the conventional method of warm dilution at 30-37^oC.

Diluting goat semen cold at 5^oC later after collection increased the storage-ability of both liquid and frozen-thawed compared to diluting warm at 30-37^oC immediately after collection, either in presence or absence of glycerol. Allowing time at 5^oC after dilution and before freezing improved freeze-ability of semen.

Presence of certain level (3.0%,v/v) of glycerol in the extender improved storage-ability of unfrozen and frozen-thawed goat semen.

The best storage-ability was obtained when fresh buck semen was cooled gradually for 3 hr at 5^oC then diluted and held for another 1 hr at 5^oC after dilution (3+1) before freezing. Post-thaw progressive motility of 42.3% was obtained for this cold dilution method, while diluting at 30-37^oC immediately after collection followed by 4 hr holding at 5^oC before freezing (0+4) yielded 35.2% in presence of 3.0% glycerol.

Also, the cold dilution method improved storage-ability of liquid semen for 24 hr at 5^oC by 40.7 to 250% increase compared with the conventional used warm dilution method, in presence or absence of glycerol, respectively.

It is suggested that holding and slow cooling of goat spermatozoa in its own seminal plasma for 2 to 3 hr at 5^oC before dilution allows coating of sperm cell with seminal plasma proteins, which may stabilize the plasma membrane to withstand the freezing and thawing process. Further studies are needed to study other factors that might affect freeze-ability of buck semen using the cold dilution method.

INTRODUCTION

Semen is routinely diluted immediately after collection and gradually cooled to 5^oC to maintain the viability of spermatozoa (Foote, 1974 and Graham, 1978). However, it has been shown that some factors in the seminal plasma might play an important role in protecting spermatozoa during freezing (Desjardins and Hafes, 1962; Weil and Rodenburg, 1962 and Abd Elhakeam, et al., 1991). Holding semen prior to dilution permits seminal plasma constituents to bind to sperm membrane (Quinn and White, 1968; Pursel et al., 1973; Jeyendran and Hunter, 1980 and Pavelko and Crabo, 1976).

Roberts and Houlaban (1961) reported higher pregnancy rate of ewes inseminated with undiluted stored ram semen (at 5^oC for 24 hrs) compared with that of diluted stored semen. Gibson and Graham (1969) demonstrated that there is a significant relationship between after thaw motility with non glycerolated semen and the 60-90 days non-return rate (correlation coefficient of 0.67). It would appear that with semen frozen without glycerol the after thaw motility would be a better indicator of fertilizing capacity than the before freeze motility.

Therefore, the objectives of this study were to investigate if holding and slow cooling of fresh goat semen to 5^oC prior to dilution is beneficial for liquid storage-ability and freeze-ability in absence or presence of glycerol. If, so, what is the optimal period of time at 5^oC required to attain maximum post-thaw recovery in presence or absence of glycerol compared with dilution immediately after collection at 30-37^oC?

MATERIALS AND METHODS

This study was carried out in the Experimental Farm of Animal Production Department, Faculty of Agriculture, El-Minia University.

Semen was collected from four mature bucks (2 French Alpine and 2 Zarabi) using artificial vagina. Before collection, each buck was sexually stimulated by allowing one or two false mounts and two to three minutes restraint. Two to three ejaculates were collected from each buck and pooled directly into the same collecting tube . Semen with good progressive motility (> 70%) was used.

Sodium-citrate (2.9410 g%) buffer at pH 7.0 and osmotic pressure of 300 mOsm/kg (measured by vapor pressure osmometer) was mixed at 1:1 with fructose (5.4048 g%) solution at same osmolarity. Fresh egg yolk at 10% (v/v) was added and two levels of glycerol (0.0 or 3.0%, v/v) were used.

After collection, semen was mixed thoroughly before dilution . Pooled semen was diluted one part semen to ten parts of extender (1:10, v/v). Basically, two methods of dilution were applied to the semen. The first one was the warm dilution at 30-37^oC immediately after collection, where warm diluted semen was cooled gradually and held at 5^oC for 2, 3 and 4 hr before freezing. The second one was the cold dilution at 5^oC, where the fresh semen was cooled gradually, and diluted later after 1,2 and 3 hr at 5^oC, then frozen according to Table (1). Table (1) shows the experimental design for time at 5^oC from collection to dilution and from dilution to freezing.

Semen samples were cooled gradually from 30-37^oC to 5^oC and the whole period at 5^oC was 4 hr from collection to freezing. The quarter cc plastic French straws (3 straws/ treatment) were mounted on a freezing boat (Abd Elhakeam,1988) at 7 cm above the surface of liquid nitrogen and were left for 10 minutes in liquid nitrogen vapor, then transferred directly to liquid nitrogen and stored for 24 hour until examined.

Straws were thawed individually in water bath at 37-40^oC for about 20 seconds. Pre-freeze and post-thaw percent of progressive motility were estimated for each treatment using a light microscope attached to closed circuit color television monitor. The experiment was repeated three times. Data were subjected to statistical analysis using the general linear model (GLM) procedure (Goodnight,1979). Significant differences between means were determined by Duncan’s multiple range test (Sall, 1979).

RESULTS AND DISCUSSION

Results in Tables 2, 4 and 5 show that both dilution methods and glycerol level significantly affected storage-ability of liquid goat semen as indicated by percent progressive motility after storage period of four or 24 hr at 5^oC. It was observed that, although the differences among the dilution methods were not large and obvious at shorter storage period (4 hr) at 5^oC, they became larger and more obvious at longer storage period (24 hr), and either in presence or absence of glycerol (Tables, 2 , 4 and 5). Indeed, it was noticed that the conventional warm dilution method immediately after collection (0+4) yielded lower progressive motility (75%) after 4 hr storage period at 5^oC compared with the cold dilution method later at 5^oC, which yielded 78.7% progressive motility. Meanwhile, the difference between the two methods of dilution became much larger after 24 hr storage time and the average value was 36.7% for warm dilution vs72.5% for the cold dilution method. Generally, holding goat spermatozoa in its own seminal plasma for more than one hour period (2-3 hr) at 5^oC improved the percent progressive motility compared to immediate dilution of semen , which is commonly used.

The cold dilution method (3+1) improved the storage-ability of goat spermatozoa for longer time (24 hr at 5^oC ) compared to the other methods used, as it was shown in Tables (2,4 and 5). The percentages of improvement ranged from 3.1-6.7% at 4 hr to 40.7-250% at 24 hr according to the absence (0.0%) or the presence (3.0%) of glycerol, respectively (Table, 5).

Results (Tables , 2,4 and 5) revealed that the presence of 3.0% glycerol significantly improved the storage-ability of goat spermatozoa either at 4 hr or 24 hr and for all dilution methods (Tables, 2 and 5). From the present results, it could be stated that dilution of goat semen later at 5^oC instead of immediately after collection improved its storage-ability. Meanwhile, the cold dilution method (3+1) was the best method as compared with the warm conventional method , either in presence or absence of glycerol. Also, presence of 3.0% glycerol increased the storage-ability of unfrozen goat spermatozoa.

Methods of dilution

Percentage Motility *

Time before dilution PLUS

4 hr at 5^oC

24 hr at 5^oC

Time after dilution and before freezing

0.0% glyc.

3.0% glyc.

0.0% glyc.

3.0% glyc.

0 + 2

0 + 3

0 + 4

1 + 1

1 + 2

1 + 3

2 + 1

2 + 2

3 + 1

82.3 ± 0.7 a

72.3± 0.7 c

75.0 ± 0.0 bc

80.0 ± 0.0 a

75.0± 0.0 bc

77.3 ± 0.7 b

81.7 ± 0.8 a

80.0 ± 0.0 a

75.0 ± 0.0 bc

81.7 ± 0.8 a

78.3 ± 0.0 a

78.3 ± 0.8 ab

76.7 ± 0.8 b

80.0 ± 0.0 a

20.0 ± 1.4 e

17.3 ± 0.7 e

20.0 ± 0.0 e

46.7 ± 2.2 c

32.3 ± 6.5 d

56.7 ± 2.2 c

65.0 ± 0.0 b

70.0 ± 1.1 a

48.3 ± 4.6 c

38.3 ± 2.2 d

53.3 ± 4.4 c

61.7 ± 3.6 b

66.7 ± 0.8 ab

51.7 ± 3.6 c

72.3 ± 0.7 a

75.3 ± 0.0 a

*Means under same storage period followed by the same letters vertically or horizontally are not significantly different (P>0.05)

Effect of dilution methods and glycerol level on post-thaw progressive motility (%) of goat spermatozoa as level is shown in Tables 3, 4 and 5. Both factors significantly affected the freeze-ability of goat spermatozoa. Post-thaw progressive motility improved by diluting goat semen later at 5^oC rather than by diluting immediately at 30-37^oC either in absence or presence of glycerol (Tables, 3 and 5). The same trend was also found at 1 hr post-thawing.

Allowing time at 5^oC after dilution and before freezing in both dilution methods was required for better recovery after thawing. Three to four hours was necessary for the warm dilution method and one to two hours for the cold method, as shown in Table (4).

Generally, the cold dilution method (3+1) significantly improved freeze-ability of goat spermatozoa compared to the conventional warm dilution method (o+4) and the other treatments of dilution used. Improvement in post-thaw motility ranged from 20.2-123.3% immediately post-thawing (0.0 hr) to 27.1-183.3% at 1 hr post-thawing according to presence or absence of glycerol, respectively (Table,5).

Methods of dilution	Percentage Motility *
Time before dilution PLUS	0.0 hr post-thaw		1.0 hr post-thaw
Time after dilution and before freezing	0.0% glyc.	3.0% glyc.	0.0% glyc.	3.0% glyc.
0 + 2 0 + 3 0 + 4 1 + 1 1 + 2 1 + 3 2 + 1 2 + 2 3 + 1	1.2 ± 0.1 g 2.7 ± 0.5 fg 3.0 ± 0.4 f 1.7 ± 0.3 g 2.3 ± 0.2 fg 5.0 ± 0.8 e 4.4 ± 0.5 ef 5.6 ± 0.7 e 6.8 ± 0.8 e	20.6 ± 1.7 d 26.7 ± 1.4 c 35.2 ± 2.3 b 19.1 ± 1.4 d 33.3 ± 1.2 b 36.9 ± 1.0 b 26.6 ± 1.8 c 40.0 ± 1.9 a 42.3 ± 0.7 a	1.0 ± 0.0 h 1.2 ± 0.1 h 2.4 ± 0.4 g 1.0 ± 0.0 h 1.3 ± 6.5 h 5.0 ± 0.8 f 3.7 ± 0.7 g 4.7 ± 0.2 fg 6.8 ± 0.8 f	19.2 ± 1.4 d 23.9 ± 1.6 c 30.6 ± 2.3 b 12.4 ± 0.8 e 26.3 ± 2.1 bc 33.9 ± 1.8 b 23.3 ± 1.2 c 37.2 ± 1.2 ab 38.9 ± 1.8 a

*Means under same stored period followed by the same letters vertically or horizontally are not significantly different (P>0.05).

From Table 5, it is important to realize that the percent improvement in freeze-ability of goat spermatozoa due to using cold dilution was much higher in absence of glycerol (123.3%-183.3%), which may indicate the importance of certain seminal plasma constituents that might protect spermatozoa during freezing and thawing process.

With regard to glycerol effects, the results (Tables,3,4 and 5) show the importance of glycerol for successful cryopreservation of goat spermatozoa. The presence of 3.0% (v/v) glycerol in the extender significantly improved the percent progressive motility for all dilution methods compared to its absence in the extender (Tables, 3 and 5).

It was obvious that cooling and holding fresh goat semen at 5^oC without dilution for 3 hr then diluting and holding 1 hr after dilution and before freezing significantly increased the after thaw recovery. These results indicated that the new method of cold dilution (3hr +1hr) was the best treatment for goat semen processing and freezing. The results of the present study showed that time and temperature at dilution as well as glycerol level are important for successful preservation of buck spermatozoa. Holding and cooling of fresh goat semen gradually then diluting at 5^oC gave much better storage-ability of liquid semen and higher freeze-ability with better progressive sperm movement and long survival.

This improvement in storage-ability of goat spermatozoa due to the cold dilution at 5^oC is in complete agreement with the previous work on ram semen (Abd Elhakeam, et al., 1991). It has been shown that some factors in seminal plasma play an important role in protecting spermatozoa during freezing (Desjardens and Hafs, 1962; Weil and Rodenburg, 1962).

Holding semen prior to dilution permits seminal plasma constituents to bind to the sperm membranes. This binding assists in protecting the sperm from cold shock in the freezing process (Quinn and White, 1968; Pursel et al., 1973; Pavelko and Crabo, 1976; Crabo et al., 1980; Jeyendran and hunter, 1980). The binding is more rapid in bull semen and at least one additional specific antigen is found after mixing with egg yolk extenders (Jeyendran and Hunter, 1980). However, bull semen held in its own seminal plasma for 20 min at 26^oC before dilution resulted in higher fertility than semen diluted 0.0 min at 26^oC (Rajamannan et al., 1971). Boar spermatozoa, which are highly susceptible to cooling damage, apparently acquire resistance if held above 15^oC for several hours (Pursel et al., 1973).

	Unfrozen		Frozen-thawed
Factors	4 hr	24 hr	0.0 hr	1.0 hr
	At 5^oC		At 30^oC
Dilution methods 0 + 2 0 + 3 0 + 4 1 + 1 1+ 2 1 + 3 2 +1 2 + 2 3 + 1 Glycerol level: 0.0 % 3.0 %	82.0 ± 0.7 a 76.2 ± 0.3 b 75.0 ± 0.0 c 80.9 ± 0.4 a 77.5 ± 0.0 ab 76.7 ± 0.4 b 77.0 ± 0.7 b 78.7 ± 0.3 ab 78.7 ± 0.3 ab 76.9 ± 0.4 b 79.3 ± 0.3 a	34.2 ± 3.0 e 27.8 ± 1.5 f 36.7 ± 2.2 e 54.2 ± 2.9 c 49.5 ± 3.7 cd 54.2 ± 2.9 c 65.0 ± 1.5 b 68.7 ± 0.3 ab 72.5 ± 0.7 a 42.7 ± 2.3 b 59.9 ± 1.6 a	10.9 ± 1.4 d 14.7 ± 0.9 c 19.1 ± 1.4 b 10.4 ± 0.9 d 17.8 ± 0.7 b 21.0 ± 0.9 a 15.0 ± 1.2 c 22.8 ± 1.3 a 24.6 ± 0.7 a 3.6 ± 0.3 b 31.2 ± 1.0 a	10.1 ± 0.7 c 12.6 ± 0.8 bc 16.5 ± 1.4 b 6.7 ± 0.4 d 13.8 ± 1.1 b 19.5 ± 1.3 a 13.5 ± 0.9 bc 21.0 ± 0.7 a 22.9 ± 1.3 a 3.0 ± 0.3 b 27.3 ± 1.1 a

Means in same column within the same factor followed by the same letters are not significantly different (P>0.05)

Meanwhile, several reports (Graham et al., 1971) showed that slow cooling (1-2 h) to 22^oC is important to maintain fertility in the use of frozen boar semen. Waston (1979) proposed that this effect was a membrane ‘stabilization’ brought about by allowing time for phase change to occur. Dilution or washing of semen during processing can eliminate these exogenous proteins (Pavelko and Crabo, 1976).

Quinn and White (1968) showed that electroejaculated ram semen became more resistant to cold shock upon standing like semen collected by artificial vagina. They reported that seminal plasma lipoproteins play an important role in sperm freezing in agreement with Desjardens and Hafs(1962) and Hammerstedt et al.(1984).

Furthermore, Ozar et al. (1988) reported that the conception rate in goats inseminated with washed and unwashed goat semen was 53.3 and 63.3%, respectively. Also, motility of washed and unwashed spermatozoa in milk extender was 50 and 60-65%, respectively, indicating the important of the seminal plasma and its constituents to goat spermatozoa. Ritar (1993) stated that there appears to be no improvement in the fertility of stored goat semen when seminal plasma is removed.

Type of semen Warm dilution Cold dilution %

(0+4) (3+1) improvement

Unfrozen 4 hr at 5^oC:

0.0% glycerol 75.0 ab 77.3 a + 3.1

3.0% glycerol 75.0 b 80.0 a + 6.7

Overall mean 75.0 b 78.7 a + 4.9

Unfrozen 24 hr at 5^oC:

0.0% glycerol 20.0 b 70.0 a + 250.0

3.0% glycerol 53.3 b 75.0 a + 40.7

Overall mean 36.7 b 72.5 a + 97.5

Frozen-thawed (0.0 hr post-thaw):

0.0% glycerol 3.0 b 6.7 a + 123.3

3.0% glycerol 35.2 b 42.3 a + 20.2

Overall mean 19.1 b 24.5 a + 28.3

Frozen-thawed (0.0 hr post-thaw):

0.0% glycerol 2.4 b 6.8 a + 183.3

3.0% glycerol 30.6 b 38.9.a + 27.1

Overall mean 16.5 b 22.9 a + 38.9

a,b Means in the same row followed by different letters are significantly different (P<0.05).

The progress in membrane biology research has demonstrated that dramatic alteration in the organization of membrane lipids can be induced as a result of temperature- related phase transitions from the gel to the liquid-crystalline state and vice versa. The occurrence of liquid phase-transition events in ram sperm plasma membrane has been demonstrated by Holt and North (1984) and showed that phase transitions had occurred once the cells had been cooled to 5^oC. Moreover, the cholesterol:phopholipids ratio of ram spermatozoa has been reported s 0.38 (Darin-Bennet and White, 1977) and 0.59 (Holt and North, 1985), increasing this value towards the comparable estimate of 0.99 for human spermatozoa (Darin-Bennet and White, 1977) must be expected to protect against cold-shock. .It is significant that spermatozoa with higher cholesterol level and lesser degree of unsaturation in the phospholipids and therefore presumably the more compact membrane structure should also be resistant to cold -shock (White and Wales, 1960). It is also, likely to speculate that holding and slow cooling of fresh buck semen from 37^oC to 5^oC may be prompt some changes in these ratios resulting in more stabilization of sperm plasma membrane to withstand freezing process. It has been reported that in bacteria (E. coli), the ratio between unsaturated to saturated fatty acids changes when the temperature of the medium decreases from 42 to 27^oC (Stryer, 1981). The changes are important for membrane fluidity and stability to maintain viability (Bruce et al., 1983).

Freezing after short time after dilution reduced the recovery after thawing compared with allowing time before freezing . Sinha et al. (1992) found that post-thawing progressive motility of goat semen was significantly affected by equilibration time. They concluded that a 4 hr equilibration period in 4% glycerol gave the best results of both progressive motility and intact acrosome. Also, Das and Rajkonwar (1995) found that the percentage of motile spermatozoa after thawing was higher for 3 hr equilibration than for shorter (1 hr) or longer (6 hr) equilibration periods. Time after dilution promote the interaction between the extender constituents, i.e egg yolk glycerol and sugars and the sperm plasma membrane for more glyco-and lipo protein coating for more protection (Waston,1975)

The present results show also, that the presence of glycerol in the extender is essential for improving storage-ability of both unfrozen and frozen goat spermatozoa compared to its absence in extender. This finding is in agreement with those reported in the literatures since its discovery by polge et al.(1949) due to its ability to reduce the production and the accumulation of lactic acid from the sperm which have direct effect on sperm motility. Also, it can be metabolized by sperm for the production of energy. In addition, due to its role and protecting action to sperm cells during freezing process (Abd Elhakeam et al., 1978; Graham, 1978; Deka and Rao, 1988; Sinha et al,1992 and El-Hassanein, 2000).

Fertility trial could confirm the importance of holding and slow cooling of fresh semen before dilution at 5^oC and the role of buck seminal plasma constituents such as proteins and lipo- glycoproteins and constituents that may increase the freeze-ability and fertility of frozen semen.

REFERENCES

Abd Elhakeam, A.A. (1988): studies on freezing ram semen in absence of glycerol. Ph. D. Thesis, dept. Anim. Sci., Univ. of Minnesota, U.S.A.

Abd Elhakeam, A.A.; graham, E. F. and Vazquez, I. A.(1991): Studies on the presence of glycerol in unfrozen and frozen ram semen: Fertility trails and the effect of dilution methods on freezing ram semen in the absence of glycerol. Cryobiology 28:36-42.

Abd Elhakeam, A. A.; Tony, S. M.; Yassen, A. M. and El-Alamy, M. A. (1978): Ram sperm motility aged in glucose and Tris buffered extenders. Alex. J. Agric. Res. 26 (2): 301-308.

Bruce, A., Dennis, B. Julian, L.; Martin, R., Keith, R., and James, D.W. (1983): “ Molecular biology of the cell” . pp. 259-260. New York: Garland

Crabo, B.G.; Larsen, R.E. and Zimmerman, K.J. (1980): Binding of seminal plasma proteins to boar spermatozoa. In Ixth International congress on Animal Reproduction and Artificial Insemination. Madrid, 2:269.

Darin-Bennett, A. and White, I.G. (1977): Influence of cholesterol content of mammalian spermatozoa on susceptibility to cold-shock. Cryobiology 14:466-470.

Das, K.K. and Rajknowar, C.K. (1995): Effects of equilibration periods on the motility of frozen buck semen in rafinose egg yolk glycerol extender. indian J. Anim. Res., 29(2): 141-144..

Deka, B.C. and Rao, A.R. (1986): Effect of glycerol level in Tris-based extender and equilibration period on quality of frozen goat semen. Theriogenology, 26(2):231-238.

Desjardens, C. and Hafz, H.D. (1962): Motility and fertility during post-thawing storage of bovine spermatozoa frozen concentrated, thawed and re-diluted . J.Dairy Sci. 45:1242-1247.

El-Hassanein, E. E. (2000): Effect of glycerol level, packaging method and thawing rate on post-thawing motility and acrosomal integrity of Barki buck semen. All Africa & Egypt. Soc. Anim. Prod. Conf. Of animal production, Alexandria, Egypt. Nov. 6-9.

Foote, R.H. (1974): in: Reproduction in Farm Animals . E.S.E. Hafez ed. Lea and Febiger, Phil. pp.409-431.

Gibson, C.D. and Graham, E.F. (1969): The relationship between fertility and post-freeze motility of bull spermatozoa (by pellet freezing) without glycerol. J. Reprod. Fert. 20:155-157.

Goodnight, J. H. (1979): pp. 121-130 and 237-264, SAS Institute, Statistical Analysis System.

Graham, E. F. (1978): Fundamentals of preservation of spermatozoa. In: The integrity of frozen spermatozoa. Nat. Acad. Of Sci., Was. D. C. pp 4-44.

Graham, E.F., Rajamannan, A. H. J.; Schmehl, M. K. L.; Maki-Laurila, M. and Bower, R. E. (1971): Fertility studies with frozen boar spermatozoa. Artif. Insemination Dig. 19: 6.

Hammerstedt, P. S.; Inskeep, P. I. And Magarqu, S. M. (1984): Alterations in Motility and metabolism associated with sperm interaction with accessory sex gland fluids (bull). Fed. Proc. 43: 1517 (Abstr. 584).

Holt, W. V. and North, R. D. (1984): Partially irreversible cold-induced lipid Phase transitions in mammalian sperm plasma membrane domains: Freeze-Fracture study. J. Exp. Zool. 230, 473-483.

Holt, W. V. and North, R. D. (1985): Determination of lipid composition and thermal phase transition temperature in an enriched plasma membrane fraction from ram spermatozoa. J. Reprod. Fert. 73, 285-294.

Jeyendran, R. S. and Hunter, A. C. (1980): Properties of the bovine seminal Plasma-egg yolk protein interaction complex formed during cryopreservation. In: Ixth Int. Cong. Anim. Reprod. & A. i., Madrid, 2:382.

Ozar, S.; Guven, B.; Ekici, A.; Kalkandclen, G. and Erkoc, F. U. (1988): Artificial insemination with frozen thawed semen in the Angora goat in Turkey. IIth Int. Cong. Anim. Reprod. & A. I., Dublin, Ireland, Vol. 3,paper no. 283.

Pavelko, M.K. and Crabo, B. G. (1976): Possible important of some sperm coating proteins and their behavior during preservation of boar spermatozoa. In: VIIIthe Int. cong. Anim. Reprod. & A.I., Cracow, 4:1045-1048.

Polge, C.; Smith, A. U. and Parkes, A.S. (1949): Revival of sperm after vertification and dehydration at low temperature. Nature (London) 164:666.

Pursel, V.G.; Johnson, L. A. and Schulman, L.L (1973): Effect of dilution ,seminal plasma and incubation period on cold shock susceptibility of boar spermatozoa. J. Anim. Sci. 37:528-531.

Quinn, P. J. and White, I.G. (1968): The effect of pH cations and protective agents on the susceptibility of ram spermatozoa to cold shock. Exp. Cell res. 49:31-32.

Rajamannan, A. H. J.; Graham, E.F and Schmehl, M. K. L. (1971): Effects of holding on bovine semen. Artif. Ins. Pig. 19:6.

Ritar, A. J. (1993): Control of ovulation , storage of semen and artificial Insemination of Fibre-producing goats in Australia: a review.Australian J. Exp. Agric., 33: 6, 807-80.

Roberts, E. M. and Houlaban, M. (1961): A comparison of the fertility of stored and fresh sheep semen. Aust. J. Exp. Agric. and Anim. Hus. 1:156-158.

Sall, J. P. (1979): In: SAS User’s Guide (J. T. Helwig and K. A. council, Eds) pp. 191-194, SAS Institute, Statistical Analysis System.

Sinha, S.; Deka, B. C.; Borgohain, B. N and Tamuli, M. K. (1992): study on freezing of goat semen in skim milk extender with different glycerol levels and equilibration periods. Indian j. Anim. Reprod., 13(1): 38-41.

Stryer, L. (1981): Biochemistry. 2nd ed., pp. 206-215. San Francisco: freeman.

Watson, P.F. (1975): The interaction of egg yolk and ram spermatozoa studied with a fluoroscent probe. j. Reprod. Fert. 42:105-111

Watson, P.F. (1979): The preservation of semen in mammals. Oxford Rev. Reprod, Biol. 1:288-350.

Weil, A. J. and Rodenburg, J. M. (1962): The seminal vesicle as the source of the sperm coating antigen of seminal plasma. Proc. Soc. Exp. Biol. Med. 119:567.

REPRODUCTIVE RESPONSE OF BUFFALO AND FRIESIAN MALE CALVES SUBJECTED TO SEXUAL BIOSTIMULANT (FERTIL)

Anim.Production.Department , Faculty. of Agriculture , University of Minia Egypt

REPRODUCTIVE RESPONSE OF BUFFALO AND FRIESIAN MALE CALVES SUBJECTED TO SEXUAL BIOSTIMULANT (FERTIL)

Anim.Production.Department , Faculty. of Agriculture , University of Minia Egypt

The natural biostimulant Fertil was used as a feed additive to improve reproductive performance of both buffalo and Friesian male calves . Twelve male calves of 6 buffalo ( 410 ± 14.0 days average age and 204 ± 5.8 kg average body weight) and 6 Friesian ( 350 ± 15.3 days average age and 190 ±5.8 kg average body weight ) were divided into two equal groups of six males (three each species) . One group was supplemented with Fertil in the ration ( 40g/head /day) and the other group served as control . Body weight , age , testes circumference , testosterone levels and semen characteristics were recorded at puberty , sexual maturity and 60 days post- sexual maturity . The experiment lasted for eight months. The results indicated that Fertil had no significant effect on the all studied parameters in both species at puberty. At sexual maturity, the difference became significant for testosterone level in both species , and Fertil groups had younger age. Body weight and testes circumference at 60 days post-sexual maturity were significantly higher in Fertil groups than the control in both species . Meanwhile, Fertil supplementation significantly improved semen quality at puberty , sexual maturity and 60 days post- sexual maturity . It could be concluded that Fertil could be used as a natural biostimulant feed additive to improve reproductive performance of both buffalo and Friesian male calves .

keywords: Reproductive performance - buffalo - Friesian - male - natural

INTRODUCTION

Wide spread of Artificial Insemination paid the attention of many investigators to coined several schedules for bull selection . The number of required bulls for such purposes will be reduced while their qualities will become a matter of vital importance .Accordingly , greater attention and care has been paid to health and genital soundness of such individuals in the herd . Hafez (1987) reported that the nutrition of bulls during weaning period and thereafter had significant effect on reproduction performance such as libido, spermatozoa production….etc.

The last few decades showed great attention on the use of some chemical products , antibiotics , enzymes and / or natural extractions that may serve as an aid factor in improving livestock performance .However , the use of chemical products and antibiotics might have some unfavorable side effects Therefore, most of animal producers tended to use the natural extractives as alternative growth promoters . Mohamed et al. (1996) studied that the effect of supplementation of ration with the herbal extraction Grow-vital, which has most of the same ingredient as Fertil, on semen quality of Friesian calves. They reported that different physical characteristics of semen improved in treated than control. Fertil has been recorded as a feed additive extracted from edible plants and is suggested to be used as a biostimulant activator of rabbits male fertility (Rashwan et al.1997). They reported that Fertil supplementation could be successfully used alone to improve reproductive performance of new zealand white bucks.

The aim of the present study was to test the reproductive traits of buffalo and Friesian bulls when using dietary herbs biostimulant ( Fertil ) as sexual activator.

MATERIALS AND METHODS

This study was carried out at Animal Production Department , Faculty of Agriculture , Minia University from February till December 1997 . A number of 12 males of buffalo and Friesian male calves were used. Buffalo males ( n = 6 ) , aged 410 ± 14.0 days and averaged 204 ± 5.8 kg of body weight . Friesian males ( n = 6 ), aged 350 ± 15.3 days and averaged 190 ± 5.8 kg of body weight at the beginning of the experiment . Animals were housed in semi open shed yard and assigned randomly into two equal groups (n=6 contains three of each species). The first group served as a control was fed diet consisted of berseem ( Trifolium alexnadrnium ) in winter or drawa (green maize) in summer with rice straw and concentrate mixture to fulfill their maintenance and growth requirements according to Tommi (1963 ) . Diets were offered twice daily at 8.00 a.m. and 4.00 p.m. . The second group of each species was fed the same control diet and supplemented with Fertil ( 40 gm / head / day ) from the starting of the experiment till animals were reached sexual maturity . The amount of Fertil (40 g/head/day) was mixed in one kilogram of concentrate mixture and was offered to each animal.

Fertil contains Lactuca sativa (20%), Cyperus esculentus (10%), Raphanus sativa (20%), Peganum harmala (15%), Palm pollen grains (15%) and male goat testicules (20%). It contains 20.8% crude protein, 22.8% ether extract,11.1% crude fiber and 8.3% ash. Water was available all day and minerals were supplied in salt licking stones .

Fatty acids of “fertil” were determined by using Gas - Liquid Chromatography (G L C) according to Stahle (1967) using PYE- Unicam Model P V 4550 gas - Liquid Chromatography with dual - flame ionization detector.

All male calves of both species were tested for semen collection and all of them failed to produce sperm cells during ejaculation. The sexual behavior was observed every week starting from the beginning of the experiment till males reached sexual maturity . Age at puberty was considered the age at which first sperm was collected according to Asdell (1955) and Hussam (1979) . Bulls were considered sexually matured when produced two successive ejaculates of one billion sperm / ml each (Hussam, 1979; Hafez, 1987) . Body weight, scrotal circumference and age as well as semen quality at puberty, sexual maturity and 60 day post sexual maturity were also recorded.

Males were sexually stimulated by allowing two minutes restraint and two false mounts prior to first ejaculation (Almquist and Hale, 1973) . Semen was collected once weekly between 8.00 and 10.00 a.m. using an artificial vagina .At each collection two successive ejaculates were separately obtained . Ejaculate volume (ml)was measured directly in milliliters to the nearest 0.1 ml using a transparent graduated glass tube .Progressive motility of spermatozoa was estimated on a percentage score under the low power of closed circuit TV microscope using a hot stage at 37^oc. Number of spermatozoa/ml of semen was determined using a heamocytometer and total motile sperm ml was calculated .

Blood samples (10 ml) were monthly collected from the jugular vein in the morning before excess to drinking and feeding. The collected serum was centrifuged at 3000 r.p.m. for 10 minutes for clearance and was kept in deep freezer at - 20^OC to be analyzed for testosterone concentration . Assessment of testosterone concentration was performed by Jaffe and Behrman ,(1974) using coat -A- count I¹²⁵ radioimmunoassay kits purchased from Diagnostic Products Corporation, Los Anglos, California, 90045, U.S.A. According to the manufacture information, the antiserum is highly specific for testosterone. The cross activity was 20% with 4 –Estern-17-01-3-one,16 % with 11-keto-testosterone,3.3 % with 5-dihydrotestosterone , 1.7 % with Methyl test,1.2 with 11-B-Hydroxtestosterone and less than 1% with the other steroids. The coat – A count, total testosterone assay has a detection limit of approximately 0.04 ng/ml. All samples were determined in one assay. The intra assay coefficient of variation was 4.8% according to the following equation : Intra assay coefficient of variation = (S.D /x ) x 100 Standard curve ranged between 0.0 and 6.0 ng/ml (0.0, 0.2, 1.0, 4.0, 8.0, and 16.0 ng/ml testosterone) was used to determine testosterone concentration.

Data were statistically analyzed according to the General Linear Model and the differences between means were detected by Duncan`s Mulitiple Range Test , S.A.S (1992 ) . The following statistical model was used to study the effect of treatment on body weight, scrotal circumference, age, semen physical characteristics and serum concentration of testosterone at puberty, sexual maturity and 60 days post sexual maturity:

RESULTS AND DISCUSSION

A- Fatty acids composition of Fertil

Type and percent of Fatty acids contents in Fertil were illustrated in Table(1). It could be noted that Fertil contains adequate levels of unsaturated fatty acids (29.50%, Table 1). Linoleic (C18₂), Linolenic (C18₃) and arachidonic (C20₄), which represent 30.5% from unsaturated fatty acids considered as a precursor in the biosynthesis of prostaglandin (Martin et al., 1981; Yves et al.,1983 and Devlin ,1986). This may increase circulating gonadotropin hormone and stimulate testosterone hormone secretion that is essential for normal reproductive function in males (Hafez,1987).

Table 1. Fatty acids of Fertil

Results in Table (2). showed that Fertil supplementation in the ration has no significant effect on age , body weight, testes circumference and testosterone concentration of both buffalo and Friesian at puberty. .From the present results , it could be observed that supplementation of Fertil in the ration resulted in a slight enhancement of puberty of both species , but the effect was more obvious in buffalo (479.0 ± 20.1 day vs. 492.0 ± 7.9 ) day than in Friesian (379.7 ± 7.36 vs. 385.3 ± 23.3) day as shown in Table ( 2).

Table 2 . Mean (± SE) for age , body weight , testes circumference and testosterone concentration of Buffalo and Friesian bulls at puberty as affected by Fertile.

Items	Buffalo bulls		Friesian bulls
Items	Control (0)	40gm / head / day	Control (0)	40gm / head / day
Age at puberty (day)	492.7±7.9 a	479.0±20.1a	385.3± 23.3 a	379.7±7.4 a
Body weight (kg )	233.0±9.0 a	251.0±23.3a	203.7± 03.2 a	206.7±8.8 a
Testes circumference (cm)	21.0±0.7 a	21.5 ±0.3 a	25.0± 01.0 a	25.67±0.3 a
Testosterone (ng /ml)	0.60±0.2 a	0.63±0.2 a	0.61± 0.1 a	0.68±0.1 a

a , b means in the same row followed by the same letter are not significantly different.

It may be worth, mentioning that the experiment was started one month pre-puberty , therefore the Fertil effect was not more pronounced at puberty as it is at sexual maturity , which will be discussed later. The effect of Fertil supplementation on semen quality of both species at puberty was significantly more pronounced (Table 3 ).

Table 3. Means ( ±SE) for semen characteristics of buffalo and Friesian bulls at puberty as affected by Fertil

Semen characteristics

Buffalo bulls

Friesian bulls

Control

40/gm/head

/day

Control

40/gm/head

/day

Ejaculate volume (ml)

1.1±

0.07 b

1.3±

0.06 a

2.03±

0.141a

2.2±

0.15 a

Concentration /ml (x10⁶)

28.0±

0.01 a

30.5±

0.01 a

27.50±

0.002a

28.5±

0.01 a

Sperm cell motility (%)

16.7±

0.83 b

21.7±

2.70 a

25.00 ±

2.191b

32.5 ±

2.20 a

Motile sperm /ml (x10⁶)

4.6±

0.03 b

6.5±

0.06 a

6.67 ±

0.036b

9.1±

0.04 a

Motile sperm /ejac (x10⁶)

4.9±

0.06 b

8.5±

0.07a

13.73±

0.121b

20.1±

0.11a

Most of physical semen characteristics of both species was significantly (p<0.05 & p<0.01 ) improved and affected by Fertil supplementation .This significant improvement in semen quality was more obvious in Buffalo . The amount of improvements due to Fertil supplementation in Buffalo was 74.9 % increase in motile sperm / ejac. compared to the control group while in case of Friesian it was 46.6% for the same parameter compared with the control . ( Table , 3 ).

The significant improvement in semen quality due to Fertil supplementation is mainly attributed to the improvement in body weight, testes circumference and testosterone concentration related to Fertil effect ( Table ,2). Testosterone plays the major role in the development of reproductive organs and their functions (Hafez ,1987). Yassen and Mahmoud (1972) found a positive and significant relationship between body weight and testicular size and their production of semen .

Results in Table, (4) showed a significant differences in age and testosterone at sexual maturity between Fertil and control groups of both Buffalo and Friesian bulls .Meanwhile , there were no significant differences in body weight and testes circumference due to Fertil supplementation in both Buffalo and Friesian. However , treated males of both species had positive effect on body weight and testes circumference compared to the control .

It was found that buffalo and Friesian treated groups reached sexual maturity earlier (p<0.05) than control group (590.3 day vs. 516.3 day for buffalo than 647.3 day vs. 572.7 day for Friesian). This significant positive effect of Fertil on age at sexual maturity was reflected also on a positive significant effect on serum testosterone levels ( Tables ,4), in which treated bulls had higher ( p<0.05 ) values for serum testosterone concentration compared to that of control bulls (0.880 ng/ml vs. 0.81 ng/ml for Buffalo and 0.92 ng/ml vs. 0.84 ng /ml for Friesian as it can be seen in Table ( 4) .

Generally the results indicated that , Fertil supplementation in the ration had a positive effect on sexual maturity of both species . It hastened the age of sexual maturity with heavier body weight, larger testicular size and higher testosterone level . These four traits are related to each other . Yassen and Mahmoud (1972 ) found a positive and significant relationship between body weight and testicular size and their production of semen . Also , Ahmed et al (1984) reported a prepubertal increase in testicular growth rate in Buffalo bulls but at considerably later stage of development ( 15- 25 months) based on measurement of scrotal circumference as an index of testicular size . An increase in testicular weight during the later part of prepubertal development has also been described in Holstein bulls (Curtis and Amann , 1981) and has been related to corresponding peak in levels of growth hormone (Joakimsen and Blom, 1976 ) and increased testosterone levels (Sundby and Velle , 1980) . Recently Abd El-Hakeam et al .(1998) reported a highly significant positive correlation coefficients between body weight , testes circumference and testosterone levels in both Buffalo and Friesian bulls .

Table 4. Means (± SE) for age, body weight, testes circumference and

testosterone concentrations of buffalo and Friesian bulls at

sexual maturity as affected by Fertil .

Items

buffalo bulls

Friesian bulls

Control

40gm / head

/ day

Control

40gm / head/ day

Age at sexual maturatiy (day)

647.3±

2.3 a

590.3±

19.8b

572.7±

19.2 a

516.3±

0.4 b

Body weight (kg )

318.3±

11.4 a

332.3±

18.5a

303.3±

1.9 a

311.7±

12.8 a

Testes circumference (cm)

23.2±

1.09 a

23.5 ±

0.3 a

28.00±

1.0 a

28.83±

0.4 a

Testosterone (ng /ml)

0.81±

0.01b

0.88±

0.01a

0.844±

0.02 b

0.921±

0.02a

Table 5. Means ( ±SE) for semen characteristics of buffalo and Friesian bulls at sexual maturity as affected by Fertil.

Semen characteristics

Buffalo bulls

Friesian bulls

Control

40/gm/head

/day

Control

40/gm/head

/day

Ejaculate volume (ml)

2.0±

0.06 b

2.5±

0.06 a

2.5 ±

0.05 b

2.9±0.05 a

Concentration /ml (x10⁹)

1.1±

0.01 a

1.1±

0.02 a

1.03±

0.01b

1.05±0.01 a

Sperm cells motility (%)

72.5±

1.9 b

82.5±

1.9 a

80.83±

0.6 b

85.0±0.6 a

Motile sperm /ml (x10⁹)

79.2±

1.5 b

90.9±

1.4 a

83.10±

1.1 b

89.5±1.1 a

Motile sperm /ejac (x10⁹)

156.8±

2.9 b

228.7±

2.9 a

206.51±

7.3 b

261.2±7.3 a

The results in table 5 indicated that physical semen characteristics are significantly ( p<0.01 ) affected by the presence of Fertil in the ration . Buffalo and friesian treated bulls showed a marked improvement in most physical semen characteristics studied . The same trend of positive significant effect and improvement in semen quality due to Fertil was also observed and found in Friesian bulls ( Table , 5) . The higher testosterone concentration of both species due to Fertil supplementation is behind the significant improvement in reproductive performance and testes functions , as it is mentioned above . The present results agree with those reported by El-Alamy (1973), Yassen et al .(1975) , Hussam (1979), Batosamnia et al.(1987), Darwish ( 1990 ) and El- Feel et al .(1992) . They reported that after puberty most semen characteristics of both buffalo and cattle bull calves increased and improved with advancing age .

The results in Table (6) show that body weight. and testes circumference at 60 days post-sexual maturity were significantly (p<0.05) affected by presence of Fertil in treated groups both buffalo and Friesian bulls. Both parameters had higher values in treated bulls than those of the control ones in both species.

Table 6. Means (± SE) body weight and testes circumference of Buffalo and Friesian bulls at 60 day post sexual maturity as affected by Fertil .

The positive effect of Fertil on body weight. and testes circumference was observed beginning at puberty and continued and became more pronounced and significant at sexual maturity and post sexual maturity (60 days). The same trend was also found on semen quality and testosterone levels during those stages of male reproduction . All physical semen characteristics of buffalo bulls at 60 days (2 months ) post sexual maturity were markedly improved (P<0.01) by Fertil supplementation (Table 7). The same trend was found on semen quality of Friesian , except on sperm - cell concentration /ml and motile sperm /ml in which both parameters were not statistically significant. Buffalo bulls usually have lower semen quality. According to this study , Fertil supplementation improved significantly the reproductive performance of buffalo bulls as it has been shown in (Tables 6 and 7). These findings of the present study are in agreement with those reported recently in the literature by Rashwan et al (1997). Generally, most feed additives especially those used as growth promoters to improve reproductive performance including Fertil (Rashwan et al 1997) , Grow- Vital ( Mohamed et al., 1996 ), Flavomycin (Abd El-Hakeam et al ,1998), resulted in a significant improvement in body weight, age at puberty and age at sexual maturity and the related measurements , especially testicular size and functions as well as semen production and quality and testosterone secretions

Further more Mohamed et al (1996) studied the effect of supplementation of ration with the natural herbal extraction growth promoter (grow -vital, which has most of the same ingredients as Fertil on semen quality of Friesian calves. They reported that the overall mean of the ejaculate volume , live sperm percentage , sperm concentration /ml and sperm out put /ejaculate were slightly higher , while the percentage of sperm motility and abnormalities were slightly lower in treated group than the control .

3- Effect of age and Fertil supplementation on serum testosterone concentration of Buffalo and Friesian bulls.

Data in Table (8) shows that Fertil supplementation have insignificant effect on testosterone concentration at one and two month post - supplementation and its effect began to be significant (p<0.05) and obvious at three months and above post - supplementation in both species . Treated bulls had higher values of testosterone concentration than control one (Table, 8) . This positive effect started at three months and continued till five months post - supplementation . Also , from Table (8) it is clear that with the advance in the experimental period and with the increase or advance in the age of males , testosterone increased significantly and this increase was gradually . Such rise was due to the continuous development of the testes as a result of age progress toward attainment of puberty and sexual maturity. Such effect is in agreement with that reported by several investigators (Miyamoto et al ., 1989 ; Abd El-Hakeam et al ,1998 ).

From the present study it could be concluded that supplementation of Fertil (40g/head /day ) in the rations of both buffalo and Friesian males enhanced reproductive performance , since it hastened age of puberty and sexual maturity and improved body weight and testicular size which were reflected in a significant increase in blood testosterone concentration high quality of semen production . This positive effect of Fertil was more obvious in case of buffalo bulls.

Semen characteristics

Buffalo bulls

Friesian bulls

Control

40/gm/head

/day

Control

40/gm/head

/day

Ejaculate volume

(ml)

2.1±

0.2 b

2.6±

0.1 a

2.7±

0.2 b

3.03±

0.2 a

Concentration /ml

(x10⁹)

1.18±

0.05 b

1.27±

0.10 a

1.23±

0.18 a

1.22±

0.5 a

Sperm cell motility

(%)

78.9±

3.8 b

84.7±

2.5 a

82.5±

0.01 b

86.7±

2.5 a

Motile sperm /ml

(x10⁹)

93.0±

8.0 b

107.6±

9.7 a

101.3±

15.0 a

105.7±

5.6 a

Motile sperm /ejac

(x10⁹)

197.1±

23.0 b

280.3±

36.9 a

268.2±

36.0b

323.8±

2.4 a

Table 8. Means (± SE) for testosterone (ng/ml) profile of Buffalo and Friesian bulls as affected by Fertil.

Periods	Buffalo bulls		Friesian bulls
	control	40 gm /head /day	control	40 gm /head /day
Pre-treatment	0.39±0.01 a	0.39±0.01 a	0.398±0.012 a	0.40±0.01 a
1-month post treatment	0.41±0.01 a	0.44±0.01 a	0.436±0.012 a	0.48±0.02 a
2-month post treatment	0.48±0.03 a	0.51±0.01 a	0.440±0.002 a	0.56±0.08 a
3-month post treatment	0.66±0.01 b	0.71±0.01 a	0.609±0.024 b	0.74±0.08 a
4-month post treatment	0.71±0.01 b	0.78±0.02 a	0.782±0.019 b	0.90±0.02 a
5-month post treatment	0.81±0.01 b	0.88±0.01 a	0.834±0.010 b	0.92±0.02 a

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Devlin,T.M.,(1986). Text book of biochemistry with clinical correlation pp 355-436. A wiley Medical publication, New York,U.S.A.

El-Alamy, M.A.,(1973). Studies on some physiological aspects in semen of buffalo , Jersey and Friesian cattle .Ph. D. Thesis , Assiut Univ., Fac. of Agric., Egypt.

El-Feel , F.M.R.;A.K.I. Abd El -Moty ;A.A. Abd El-Hakeam, (1992). Puberty and post - pubertal changes in semen characteristics of Buffalo and Friesian bulls as affected by weaning systems. Eygpt .J. Aim.Prod.29.nol, pp.1.

Hafez , E. S. E. , (1987) . Reproduction in farm animals . Lea and Febiger , Phladephia, U SA.

Hussam ,H. A., (1979). Sexual development and sperm output in Friesian and buffalo bulls . M. Sc. Thesis , Fac. of Agric. Alex. Univ. Egypt .

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Joakinsen, O. and A.K. Blom, (1976). Growth hormone concentration in Jugular blood plasma in relation to growth rate and age in young bulls . Acta Agric Scand ., 26:239.

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Stahle , E . ,(1967).Thinlayer chromatogrophy . A laboratory Hand book , ed . Springer verloag Berlin , Heidelpery , New P . 359.

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Yassen, A.M.; and M.N. Mahmoud, (1972). Relationship between body weight and testicular size in buffalo bulls. J. Agric. Sci., Camb 78:367.

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EFFECT OF ADDING SOME BIOLOGICAL FLUIDS ON QUALITY OF NEAT AND CHILLED GOAT SEMEN.

Agglutination of goat semen after short period of collection was found to redyce its quality. In a trial to investigate this agglutination phenomenon and improve semen quality of Zaraibi goat semen, the present study designed to clarify the effect of adding some biological fluids such as anti-snake venom (A-SV), bovine seminal plasma (BSP), goat blood plasma (GBP) and anti-goat seminal plasma (A-GSP) on the quality of neat and chilled goat semen before and after its dilution. Tow experiments were conducted; The first experiment revealed that addition of both GBP and BSP were effective in reducing sperm agglutination in goat semen (66.00 vs 43.50 and 67.00 vs 46.00 % for GBP and BSP respectively). GBP was found to improve sperm motility (54.17vs31.00 %) and live sperm percentages (57.47vs 33.80 %). Concerning BSP, it achieved sperm motility of 50.00 vs 34.00 % and percent of live sperm of 54.10 vs 36.60 %. The lower percentages of acrosomal aberrations (10.83 vs 13.00 %) and total sperm abnormalities (8.32 and 8.00 %) were calculated for samples incubated for 2 hr at 37°c with addition of GBP. The second experiment showed that semen diluted with Tris 20% egg yolk-fructose citric acid diluent with different concentrations of the above mentioned biological fluids, and kept at +5ْC for 24hrs. improved semen quality. The highest sperm motility was recorded with inclusion of 100ml/ml of both BSP and GBP (62.00 and 61.00 % respectively). Adding 100 ml/ml of BSP and 80 ml/ml of GBP achieved low percent of spermatozoa with defected acrosomes (9.18 and 8.82%, respectively). In conclusion, the results obtained from the current work raised the possibility that GBP and BSP may be extremely useful as additives to neat goat semen incubated at 37ْc or diluted goat semen chilled at +5ْc, since these biological fluids may enhance semen quality by inhibiting phospholipase A2 action

INTRODUCTION

One of the principle problems in the worldwide development of A.I. in goats is related to the use of frozen semen, since the freezing process reduces the viability of the sperm cells. Researchers believed that an important contribution to the choice of storage medium for goat sperm was the discovery of enzymes produced by the bulbourethral glands of the male; these enzymes are phospholipase A2 and lysophospholipase “egg yolk coagulating enzymes” (Roy, 1957; Iritani and Nishikawa, 1961). Phospholibase A2 (PLA2) are ubiquitous enzymes capable of hydrolyzing the phospholipids and are believed to be important regulatory enzymes in numerous physiological systems such as inflammation, membrane remodeling and cell signalization (Kudo, et al., 1993). In general, the effect of PLA2 enzyme on phospholipids was found to be inactivated by anti-snake (Naja naja) venom (Kim, and Bonventre, 1993) and bovine seminal plasma proteins (Manjunath, et al., 1994). Also, goat blood plasma and anti-goat seminal plasma contain higher concentration of the anti-sticking factor (ASF-1) that showed high affinity for inhibiting agglutination of corpus epididymal spermatozoa (Roy, and Majumder, 1989).

The present study was, therefore, undertaken to investigate the agglutination phenomenon of goat semen after short period of collection and the effect of adding different biological fluids on sperm agglutination and some semen parameters. The study was also done to select the potent biological fluid and its optimal concentration in semen diluted with Tris-egg yolk- fructose extender and kept at +5ْc for 1,2 and 24 hr.

MATERAL AND METHODS

Ten adult Zaraibi male goats (19-20 months and 20-28 Kg. Body weight) were chosen from a farm belonging to Anim. Reprod. Res. Institute. Semen samples were collected from each buck once a week using artificial vagina. Tow experiments were conducted in the current study using different concentrations of some biological fluids viz., Anti-snake venom (A-SV), Buffalo seminal plasma (BSP), Goat blood plasma (GBP) and Anti-goat seminal plasma (A-GSP). A-SV was obtained from Egyptian organization for biological products and vaccines, Egypt. BSP was separated from semen samples of mature buffalo bulls. GBP was separated from goat blood samples while A-GSP was prepared by immunization of two mature female goats with goat seminal plasma according to Baldo and Boettcher (1971). BSP, GBP and A-GSP were stored at -20ْc.

It was designed to study the agglutination phenomenon (according to Chandler et al. 1988) of buck semen and the effect of adding the above mentioned biological fluids (A-SV, BSP, GBP and A-GSP) on semen agglutination and some semen parameters. They were added to neat semen (collected from ten bucks and good ejaculates were pooled) at concentrations of 0, 20, 40, 60, 80, and 100 μl/ml semen and the semen was incubated at 37ْC for 2hr. Sperm agglutination was examined for incubated samples at 37ْC at o, 30, 60, 90 and 120 min., Sperm motility, live sperm percentages, percentages of acrosomal aberrations and total sperm abnormalities were calculated for incubated samples at 37ْC at o min. and after 120 min.

Egg yolk fructose-citric acid extender containing different concentrations of the aforementioned biological fluids. Soon after 10 min. of dilution, treated semen was evaluated for progressively motile spermatozoa, live sperm percent, percentages of acrosomal aberrations and total sperm abnormalities. Extended treated semen was cooled to 5ْC within 45 min., kept in refrigerator for 24hr.and then were evaluated for previously mentioned parameters at 1, 2 and 27hr.

Statistical analyses of the recorded data were done according to Snedecor and Cochran (1980).

Percentages of sperm agglutination through two hours of incubation at 37ْC after collection with or without addition of Anti-snake venom (A-SV), Bovine seminal plasma (BSP), Goat blood plasma (GBP) and Anti - goat seminal plasma (A-GSP) in different concentrations are shown in Tables (1). It is obvious that GBP and BSP were effective in reducing sperm agglutination in goat semen. GBP (100 μl/ml) was found to reduce agglutination of neat semen from 66.00 % in control samples to 32.00 % in treated samples after 120min.of semen incubation at 37ْC. Analysis of variance (Table, 2) showed that there was a significant effect for adding biological fluids to neat semen in reducing sperm agglutination percentage specially after 90 and120 min. of incubation at 37ْC.

Sperm motility was highly improved by solving sperm agglutination problem where some biological fluids were used (Table,3).Addition of 100 ml/ml of GBP was highly effective on sperm motility improvement (65.00 %) after 120 min. incubation at 37°c. Analysis of variance (Table 4) showed a significant difference in sperm motility where addition of biological fluids at different

concentrations revealed higher sperm motility than control samples, with different degrees.

Percentages of live sperm were of highly significant difference by using different concentrations of biological fluids. The highest percentage was found by using 60 ml A-SV/ml semen (48.60 %) after 120 min. of incubation at 37°c., compared with control samples (32.00). Meanwhile, GBP was found to affect percentage of live sperm as the highest value was obtained by adding 100 ml/ml (68.60 %). Generally all concentrations of GBP used revealed a significantly higher percent of live sperm than control samples. Analysis of variance (Table 4) showed a significant difference between live sperm percent in control samples than that with some biological fluids which maintains the live sperm percent at any concentrations.

Table (2): Analysis of variance for the effect of different concentrations of some biological fluids on sperm agglutination of Zaraibi male goats incubated at 37°c through 2 hours.

			F- value
S. V		d.f	0 min.	30 min	60 min.	90 min.	120 min.
	A-SV	5	0.59	1.47	0.97	7.00 ***	45.85***
Conc.	BSP	5	1.46	6.12***	2.21	3.86 *	9.75***
	GBP	5	0.42	2.59	5.61**	17.90 *******	11.80 ***
	A-GSP	5	2.87 *	2.45	2.45	2.45	3.63 *
Error		24

Percentages of deformed sperm were classified into percent of acrosomal defects and other sperm aberrations. Addition of A-SV was found to increase percent of sperm with acrosomal defects significantly (Table 3) but the differences in total sperm abnormalities in treated samples did not reach the level of significance (Table 4). Addition of 40 ml/ml and 60 ml/ml BSP insignificantly decreases the total sperm abnormalities (Table 3). GBP at concentrations of 60 ml/ml and 100 ml/ml were found to insignificantly decrease the total abnormal sperm counts. A-GSP was found to increase percentage of sperm with acrosomal defects at 0 min., but after 120 min. of incubation at 37°c the differences between control and other concentrations were insignificant (Table 4).

Table ( 4 ): Analysis of variance for the effect of different concentrations of some biological fluids on semen parameters of Zaraibi male goats incubated at 37°c through 2 hr.

				F-value
S.V		d.f		Mot.		Live		Acro.		Abn.
			0 min.		120 min.	0 min.	120 min.	0 min.	120 min.	0 min.	120min.
	A-SV	5	1.56		6.46 *******	1.68	3.52 *	1.88	7.60 *******	0.34	0.64
Conc.	BSP	5	0.83		7.95 *******	0.38	9.14 *******	1.26	0.64	1.10	2.02
	GBP	5	0.80		20.84 *******	0.53	16.24***	0.37	1.26	1.85	1.90
	A-GSP	5	2.00		3.62 *	1.22	3.74 *	3.52 *	0.37	1.59	1.10
Error		24

- Acro. = acrosome defects of spermatozoa (%); - Abn. = sperm abnormalities (%).

Experiment 2: Influence of some biological fluids on semen quality, diluted and stored at 5°c, of Zaraibi male goats.

Dilution of Zaraibi buck semen with Tris- 20 % egg yolk- fructose-citric acid diluent containing different concentrations of biological fluids and cooled to 5°c and examined through 24 hr showed insignificant differences in sperm motility percentages between different concentrations throughout incubation times (Tables 5 and 6). Irrespective to A-SV concentration, sperm motility decreased by time as it averaged 78.50 % at 37°c after 0 hr and 54.00 % at 5°c after 24 hours. Diluted semen samples containing 100 ml/ml BSP and kept at 5°c for 24 hour achieved the highest sperm motility (62.00 %) while sperm motility of control samples was 34.00 %. Treatment of diluted semen with different concentrations of goat blood plasma at 5°c for 24 hours was found to increase percentages of sperm motility. The highest sperm motility (61.00 %) was observed for diluted semen samples inoculated with 100 ml/ml of GBP while it was 41.80 % for control samples at the same incubation period. Generally, addition of 40 ml/ml A-GSP to diluted semen revealed the highest sperm motility which was 82.00 % at 0 hr of incubation followed by 69.00, 58.00 and 42.00 % at 1, 2 and 24 hr of incubation at 5°c respectively. Analysis of variance (Table 6) showed that GBP and BSP significantly maintain sperm motility to 24 hr than control samples. The highest values for alive sperm percent of diluted buck semen treated with 100 ml/ml of goat blood plasma and kept at 5°c for 2 and 24 hr were 74.20 and 65.00 % respectively (Table 7), where the lowest values were 65.00 and 51.80 % respectively for control samples for the same two periods. Analysis of variance (Table 8) showed that the differences in live spermatozoa were statistically significant between concentrations of GBP at 2 hr (p<0.05) and 24 hr (p<0.0001), while insignificant at 0 and 1 hr of incubation at the same temperature. Analysis of variance for the effect of different concentrations of A-GSP on live spermatozoa were also significant at 0 hr (p<0.001), 1 hr (p<0.05) and 2 hr (p<0.05) of incubation at 5°c, with no significant difference at 24 hr.

Percentages of spermatozoa with defected acrosomes due to treatment of diluted semen with different concentrations of ASV averaged 10.65 and 13.35% for 2 hr and 24 hr incubation at 5°c respectively. The minimum values of acrosomal defects (8.00and 9.10 %) were recorded for diluted samples treated with 100 ml/ml BSP after 2 and 24 hr of incubation respectively(Table 9).Analysis of variance (Table10)showed that biological fluids significantly decreased percentages of spermatozoa with defected acrosomes especially after 2 and 24 hr. of incubation at 5°c.

Total sperm abnormality percentages of diluted samples and incubated with different concentrations of biological fluids are recorded in Table 11. It showed that 80 ml/ml. and 100 ml/ml. GBP maintained the lowest percent of sperm abnormalities(8.00 and 9.80) after 24hr of incubation at 5°c. It was evident from Table (11), that treatment of diluted spermatozoa with different concentrations of BSP decreased total sperm abnormalities if compared with non treated samples. After incubation at 5°c abnormal sperm was found to be 7.10 % vs. 10.60 % after 2 hr , and 8.70 % vs. 15.20 % after 24 hr of incubation. Analysis of variance (Table 12) revealed that effects of different concentrations of bovine seminal plasma after different incubation times at 5°c were significantly different at 2 hr (p<0.0001) and 24 hr (p<0.0001), while were insignificant at 0 and 1 hr of incubation.

Table (6): Analysis of variance for the effect of different concentrations of some biological fluids on sperm motility of Zaraibi male goats’ semen diluted with Tris- 20 % egg yolk- fructose diluent.

			F- value
S. V.		d.f	0 hr	1 hr	2 hr	24 hr
	A-SV	5	0.98	0.14	0.83	0.60
Conc.	BSP	5	2.37	1.02	5.93**	18.40 ***
	GBP	5	0.59	0.85	11.62***	13.43 ***
	A-GSP	5	5.81**	4.04**	2.71*	1.15
Error		24

Table (8): Analysis of variance for the effect of different concentrations of some biological fluids on alive spermatozoa of Zaraibi male goats’ semen diluted with Tris- 20 % egg yolk- fructose diluent.

				F- value
S.V.		d.f	0 hr at 37°c		1 hr at 5°c	2 hr at 5°c	24 hr at 5°c
	A-SV	5	2.44		0.52	0.46	1.24
Conc.s	BSP	5	1.86		1.37	4.94 **	6.16 ***
	GBP	5	1.30		1.51	3.62 *	8.80 ***
	A-GSP	5	5.25 **		3.73 *	2.69 *	1.26
Error		24

Table (10): Analysis of variance for the effect of different concentrations of some biological fluids on sperm acrosomal defect of Zaraibi male goats’ semen diluted with Tris- 20 % egg yolk- fructose diluent.

			F- value
S.V.		d.f	0 hr at 37°c	1hr at 5°c	2 hr at 5°c	24 hr at 5°c
	A-SV	5	1.39	1.60	1.03	0.39
Conc.	BSP	5	1.36	0.83	7.63 ***	11.42***
	GBP	5	1.81	0.86	5.99 ***	17.47***
	A-GSP	5	4.12 **	2.78*	3.32 *	3.12 *
Error		24

Table (12): Analysis of variance for the effect of different concentrations of some biological fluids on total sperm abnormalities of Zaraibi male goats’ semen diluted with Tris- 20 % egg yolk- fructose diluent.

				F- value
S.V.		d.f	0 hr at37°c		1 hr at 5°c	2 hr at 5°c	24 hr at 5°c
	A-SV	5	0.93		1.45	1.14	1.03
Conc.s	BSP	5	0.60		0.31	6.52 *******	12.31 *******
	GBP	5	1.27		2.38	14.01 *******	5.81 **
	A-GSP	5	2.57		3.54 *	5.32 **	4.84 **
Error		24

DISCUSSION

The lack of knowledge available in the literature concerning addition of some biological fluids(A-SV, BSP, GBP and A-GSP ), on goat semen before, during and after extention limits the discussion of our findings. However, in relation to the neat semen that incubated at 37°c for 2 hr.; GBP and BSP were superior in hindering sperm agglutination than other fluids. These finding were obvious from data presented in Table (1). The highest sperm agglutination was observed in control samples (66.00 %), but the lowest value (32.00 %) was at 100 ml/ml goat blood plasma after 2 hr incubation at 37°c . Concerning BSP, the sperm agglutination percent was 67.00 % vs 46.00 % at 37°c for 2 hr. Consequently, sperm motility, alive sperm percentage, total sperm cell aberrations and sperm acrosomal defects were improved (Table 3). It was clear from the results that the motility percent of control samples through 120 min incubation at 37°c was 31 % and addition of 100 ml/ml of goat blood plasma improved motility to reach 65.00 %. These are true for live sperm percent (68.60 % vs 33.80 %); total sperm abnormalities (6.60 % vs 8.00 %) and acrosome defects (10.70 % vs 13.00 %). Also, addition of BSP (100 ml/ml) increases the progressively motile sperm percent (52.00 % vs 34.00 %) and percent of alive sperm (58.40 % vs 36.60 %) in goat semen incubated at 37°c for 2 hrs. Rapid decline in semen quality within two hours of in vitro incubation of goat (undiluted) semen at 37°c might be due to high concentration of potassium (233 mg %) and very low Sodium / Potassium ratio (0.5 mg %) (Hemeida, 1975). Low concentration of potassium is necessary for optimum metabolism of spermatozoa whereas high level is detrimental. It is thought that this could be vital in the formation of semen extenders suited for goat semen to use diluents free entirely of potassium salts. Also, killed spermatozoa during incubation of semen at 37°c had a toxic effect on the live cells, a phenomenon reported previously for dead bull spermatozoa due to their amino acid oxidase activity (Shannon and Curson, 1972).

In the current study, improvement of semen quality by addition of GBP and BSP after 2 hr of incubation at 37°c might be due to their effects on potassium i.e. increase the affinity of their proteins for K⁺. It is conceivable but unlikely that the differences in activities of goat blood plasma and bovine seminal plasma are due to a species difference in intrinsic specific activities between goat and bovine proteins. However, Roy and Majumder (1989) isolated an anti-sticking factor (ASF-1) from goat epididymal plasma (47 ~ KDa). Evidence has been presented to support the view that the outer surface of sperm possesses specific ASF-1 receptors. The ASF activity was found to be distributed in all tissues and its specific activity was markedly higher in blood plasma than in the other tissues. So, the good results obtained in this work could be due to suggestion that ASF in goat blood plasma may play an important biological role by serving as a specific inhibitor of cell- substratum and cell - cell adhesions (Roy, and Majumder, 1989). Moreover, several lines of studies have been demonstrated that highly motile mammalian sperm suspended in epididymal or seminal plasma lose motility upon dilution with an isotonic synthetic medium. This loss of flagellar motility from dilution can be reduced substantially by the addition of seminal plasma, epididymal plasma, blood plasma, or their fractions (Mann, 1964; Brown and Senger, 1980).

Dilution of goat semen with Tris- 20 % egg yolk- fructose citric acid, in the current work (experiment II), with addition of GBP or BSP to the extenders at a level of 80 to 100 ml/ml was favorable for semen quality chilled at +5°c for 24 hrs. GBP was found to increase percentages of sperm motility (61.00 % vs 41.80 %); alive sperm percent (65.00 % vs 51.80 %) and decreases sperm acrosome defects (11.30 % vs 19.70 %) and total sperm abnormalities (9.80 % vs 13.30 %). An improvement in sperm motility (62.00 % vs 34.00 %); live sperm percent (57.00 % vs 44.40 %) with a decrease in acrosomal abnormalities (9.10 % vs 18.30 %) and total sperm deformities (8.70 % vs 15.20 %) was also recorded with inclusion of BSP (Tables 5,7,9 and 11). In our study, we have found that the incubation temperature (+5°c) was low for activity of phospholipase A2 like enzyme in the buck seminal plasma. This is in agreement with Iritani and Nishikawa (1961) who reported that the incubation temperature of goat semen extended in diluent contained egg yolk was 37°c which was close to the temperature reported (Ritar and Salamon, 1982; Memon, et al., 1985) to be optimum for activity of PLA2 enzyme. Also, Jansson, et al. (1991) found that pre-incubation of human seminal plasma for 1 hr. at 37°c resulted in an even higher PLA activity. Nevertheless, no explanation can be offered for the positive effects of GBP in diluted goat semen with egg yolk containing medium and the possibility of deactivation of the PLA2 enzyme cannot be excluded.

It is generally agreed that bovine seminal fluid, the extracellular milieu PLA2; however using phosphatidyl-choline (PC) as substrate, the activity is almost undetectable. This is presumably because BSP proteins are inhibitors of PLA2 activity, are present at a very high concentration (20 - 45 mg /ml) in seminal fluid. BSP proteins showed different effects on PLA2 activity depending on whether substrate or enzyme was preincubated with the BSP proteins, the type of substrate (phosphatidylcholine or phosphatidylethanolamine) and enzyme sources (Manjunath, et al., 1994). BSP proteins may sequester phospholipids substrates by inhibitor proteins rather than direct interaction with PLA; by substrate depletion or via a substrate coating mechanism (Davidson, et al., 1987; Haigler, et al., 1987). The inhibition involved, also, a reduced accessibility to the lipidic substrate (Soubeyrand and Manjunath, 1997). The latter authors suggested that the reduced accessibility of BSP proteins appeared to proceed via two distinct interactions: binding to the substrate on one hand and to the enzyme on the other hand and it may act as spermatozoa stabilizing agents by preventing premature lipolysis of the sperm surface.

The results reported in the present work in respect to the involvement of GBP and BSP proteins in preventing the deleterious effects of egg yolk (20 %) on the survival of spermatozoa, open the possibility of inhibiting the activity of PLA2 with highly specific inhibitors and this would avoid the harmful step of washing goat sperm and would retain the factors from seminal plasma exerting favorable effect on sperm motility and survival. However, there is as yet no consensus about the precise mechanism of PLA2 inhibition. It is possible that the BSP proteins may exert different specificities. This specificity may have some significance in the regulation of the sperm enzyme, for example, the BSP proteins may limit sperm PLA2 acting on choline phospholipids and permit PLA2 to hydrolyse other phospholipids to provide energy required for motility and / or other activities (Therian, et al., 1997).

It is worthy to mention that specific phospholipases A2 have been reported in the seminal plasma and / or accessory secretions from the human, bull, ram and goat, whereas lipase activity has not been detected in these species (Kunze, et al., 1974). It is thus evident that the role of GBP and BSP proteins in PLA2 inhibition requires further definition. To our opinion, studies are in need to explore phospholipase A2 action from kinetic approaches; to purify and prepare a monoclonal antibodies against PLA2 of goat seminal plasma.

In conclusion, the data from this study (experiments I and II) raised the possibility that the GBP and BSP proteins may be extremely useful as additives to undiluted goat semen incubated at 37°c or goat semen diluted with Tris- 20 % egg yolk fructose citric acid and chilled at +5°c, since these biological fluids may enhance semen quality by inhibiting PLA2 actions.

REFERENCES

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Brown, D. V. and Senger, P. L.(1980): Influence of homologous blood serum on motility and head-to-head agglutination in nonmotile ejaculated bovine spermatozoa. Biol. Reprod. 23: 271.

Chandler, J., Painter, C. L., Adkison, R. M., Memon, M. A., and Hoyt, P. G.(1988): Semen quality characteristics of dairy goats. J. Dairy Sci., 71: 1638.

Davidson, F. F., Dennis, E. A., Powell, M., and Glenney, J. R. (1987): Inhibition of phospholipase A2 by “Lipocortins “ and calpactins. ; of binding to substrate phospholipids. J. Biol. Chem., 262: 1698.

Haigler, H. T., Schlaepfer, D. D., and Burgess, W. H.(1987): Characterization of lipocortin I and an immunologically unrelated 33-KDa protein as epidermal growth factor receptor/ kinase substrates and phospholipase A2 inhibitors. J. Biol. Chem., 262: 6921.

Hemeida, N. A. (1975): Dilution and preservation of goat semen. Ph. D. Thesis, Fac. Vet. Med., Cairo Univ. Egypt.

Iritani, A., and Nishikawa, Y.(1961): Studies on the egg yolk coagulating factor in goat semen. II. Properties of the coagulating factor and influential conditions for coagulation. Proc. Silver Jubilee Lab. Anim. Husb. Kyoto Univ., pp.97.

Jansson, A., Ronquist, G., and Frithz, G.(1991): Phospholipase A2 activity in seminal plasma of men attending an infertility clinic. Urol. Int., 46: 184.

Kim, Y. H., and Bonventre, R. O. (1993): Purification of a 100 kDa phospholipase A2 from spleen, lung and kidney: antiserum raised to pig spleen phospholipase A2 recognizes a similar form in bovine lung, kidney and platelets, and immunoprecipitates phospholipase A2 activity. Biochem. J., 294: 261.

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Kunze, H., Nahas, N., and Wurl, M.(1974): Phospholipases in human seminal plasma. Biochim. Biophys. Acta, 348: 35.

Mann, T.(1964): Biochemistry of semen and of the male reproductive tract. Barnes and Noble, Inc., New York.

Manjunath, P., Soubeyrand, S., Chandonnet, L., and Roberts, K. D.(1994): Major proteins of bovine seminal plasma inhibited phospholipase A2. Biochem. J., 303: 121.

Memon, M. A., Bretzlaff, K. N., and Ott, R. S.(1985): Effect of washing on motility and acrosome morphology of frozen-thawed goat spermatozoa. Am. J. Vet. Res., 46: 473.

Ritar, A. J., and Salamon, S.(1982): Effects of seminal plasma and of its removal and of egg yolk in the diluent on the survival of fresh and frozen-thawed spermatozoa of the Angora goat. Aust. J. Biol. Sci., 35: 305.

Roy, A.(1957): Egg-yolk coagulating enzyme in the semen and Cowpers gland of the goat. Nature(London), 179: 318.

Roy, N., and Majumder, G. C.(1989): Purification and characterization of an anti-sticking factor from goat epididymal plasma that inhibits sperm- glass and sperm- sperm adhesions. Biochim. Biophys. Acta, 991: 114

Shannon, P. and Curson, B.(1972): Toxic effect and action of dead sperm on diluted bovine semen. J. Dairy Sci., 55: 614.

Snedecor, G. W. and Cochran, W. G. (1980): Statistical Methods. 7^th ed. J. B. H. Publishing Comp., Oxford.

Soubeyrand, S., and Manjunath, P.(1997): Novel seminal plasma phospholipase A2 is inhibited by the major proteins of bovine seminal plasma. Biochim. Biophys. Acta, 1341: 183.

Therian, I., Soubeyrand, S., and Manjunath, P.(1997): Major proteins of bovine seminal plasma modulate sperm ca-pacitation by high density lipoprotein. Biol. Reprod., 57: 1080.

FREEZABILITY OF ZARAIBI GOAT SEMEN IN RELATION TO DIFFERENT DILUENTS, EGG YOLK CONCENTRATIONS AND GLYCEROL LEVELS

The present experiment was carried out to reveal the suitable extender, egg yolk concentration and glycerol level used for dilution and freezing of Zaraibi goat semen. Four extenders (Milk, Tris, Sod. Citrate and CEGLY), four egg yolk concentrations (5, 10, 15 and 20 %) and three levels of glycerol (5, 7 and 9 %) were used for this purpose. In general, Tris diluent revealed the highest post-thaw semen quality. The overall percentages of post-thaw sperm motility averaged 43.42, 41.42 and 40.17% for semen samples frozen in Tris, milk and sod. Citrate diluents, respectively while CEGLY extender showed the least value (29.25%). Also, viabilty of spermatozoa post thawing was higher in Tris (87.92) and Sod.Citrate (77.42) extender. The overall means of acrosomal damages and AST and LDH enzymes post- freezing and thawing in Tris diluent were decline to 9.30 %, 141.48 IU/ml and 205.57 U/L, respectively. A general analysis of the buffering ability of Tris diluent used in the present study revealed that the performance of Tris extender was superior followed by Sod. Citrate diluent over other diluents in respect of all semen characters studies during freezing of buck semen at -196°c.

Irrespective of type of extenders and glycerol levels, the differences in post- thawing spermatozoal survival between different concentrations of egg yolk were highly significant. Higher egg yolk concentrations (15 and 20 %) revealed better post-thaw motility (41.67 and 47.83%, respectively), viability (76.04 and 78.63, respectively) and lower acrosomal defects (17.97 and 17.58%, respectively).

Irrespective of type of diluents and egg yolk concentrations, the progressive motility percent after thawing was higher for 7 and 9 % glycerol (39.31 and 39.75 % respectively) than for 5 % (36.63 %). Percent of post-thaw acrosomal damage (18.26 %), AST (155.53 IU/ml) and LDH (225.24 U/L) were decreased in using 7%glycerol than other glycerol levels.

INTRODUCTION

The wide distribution of goats in the tropics and sub-tropics reflects their ability to adapt to a variety of environments. In the dry tropics they perform best and thrive in large numbers. The inherent characteristics of goats such as resistance to dehydration, preference for browse and wide ranging feeding habits enable them to thrive in regions that receive less than 750 mm of rainfall. The genetic variability within and between goat breeds is so great that selection for improving animal productivity is possible, and little attention has been paid to the feeding , management and health. For these reasons, increased investment in dairy goat schemes is necessary in the year ahead (Devendra and McLeroy, 1990). Zaraibi goats originates in upper Egypt and have features typical of the Nubian group. This breed may have ancestors from Damascus goat. It is valued for milk production and to a lesser extent for meat production. Productivity potential in goats could be augmented through use of sires which may have a direct effect on the production traits of the resultant progeny. The use of artificial insemination (A.I.) can greatly increase the number of offsprings produced per sire per year.

For storage goat spermatozoa at reduced temperature, it is necessary to extend the semen in special diluents. These generally, should have proper pH and buffering capacity, suitable osmolality and should protect the spermatozoa from cryogenic injury (Salamon, and Maxwell, 1995), to obtain high sperm quality after thawing (Berridge, 1975; Mann and Lutuak-Mann, 1981; Jeyendran et al 1984; Dhami, and Kodagali, 1990 and Murase et al 1990). The present study was, therefore, undertaken to compare the efficiency of different storage media with different egg yolk concentrations and glycerol levels to obtain high post- freezing and thawing semen quality.

The present investigation was undertaken to study the comparative efficiency of different diluents (Milk, Tris, Sod.Citrate and CEGLY), four concentrations of egg yolk (5, 10, 15 and 20%) and three levels of glycerol (5,7 and 9%) on semen quality after freezing and thawing. Tris-citric acid fructose, Goat milk, Sodium citrate diluent 2.9% and CEGLY extenders were prepared as described by Evans and Maxwell (1987), Pellicer-Rubio, et al. (1997), Polge and Rowson (1952) and El-Chahidi, (1976) respectively.

Semen samples were collected, pooled, divided into 48 portions and then diluted (1:10) with different aforementioned diluents. Each with different egg yolk and glycerol levels (4 x 4 x 3) and kept at 37ْC. Extended semen samples were cooled to 5ْC within 45 min. and then equilibrated for 2 hr. at 5ْC. Equilibrated samples were packaged in 0.25 ml minitubes and sealed. Minitubes were frozen on L.N. vapor (about 6 cm above the level of L.N.) for 15 min., then plunged and stored in L.N. container. After 3 days storage, the minitubes were thawed in water bath at 37ْC for 30 sec. Diluted and post-frozen thawed semen samples were evaluated for progressively motile spermatozoa, percentage of sperm with acrosomal defects. Aspartate aminotransferase activity (AST), Calcium concentration, Lactic dehydrogenase activity were estimated in seminal plasma of post-diluted and post-frozen thawed semen samples. Viability of post-thawed semen samples was estimated by recording of sperm motility percentage for three hours. after thawing to estimate the viability indices according to Milovanov (1962). Statistical analyses of the recorded data were done according to Snedecor and Cochran (1980).

The present work was carried out to reveal the suitable media for freezing Zaraibi buck semen. Sperm motility after extension of semen samples in different diluents with various egg yolk concentrations and glycerol levels is shown in Table (1). It is obvious that Tris is better diluent for maintaining sperm motility (77.08 %) after semen dilution than Milk, CEGLY and Sod.Citrate extenders (66.92, 66.00 or 64.00 % respectively). Post-thaw sperm motility was found to be significantly higher for semen samples frozen in Tris diluent (43.42 %) as well as milk (41.42 %) and Sod.Citrate extenders (40.17%). Semen frozen in CEGLY extender showed the least value of sperm motility (29.25 %). Higher egg yolk concentrations (15% and 20%) revealed better post-thaw motility (41.67 and 42.83 % respectively) than lower (5 and 10 %) concentrations (34.83 or 34.92% respectively). Glycerol level differences failed to affect significantly post-thaw sperm motility (39.75; 39.31 and 36.63 % for 9 %, 7 % and 5 % glycerol concentrations respectively) in different diluents used. Analysis of variance (Table, 7) showed highly significant effects (p<0.0001) of diluents as well as egg yolk concentrations on post-frozen -thawed sperm motility while glycerol level did not reach the level of significance. There was no significant interaction could be found between the three variables studied (Diluent, Egg yolk and Glycerol).

Variations in sperm viability of post-thawed semen were highly significant due to egg yolk concentrations (p<0.0001), and diluents (p<0.0001) used (Table 2). On the other hand, there were insignificant differences due to glycerol levels. The average sperm viability was highest for Tris, Sod. Citrate and Milk extenders (87.92, 77.42 and 67.46 respectively). A sharp decrease for CEGLY (42.63 ) extender was peculiar. Irrespective of diluents used, egg yolk with percentages of 15 % and 20 % showed the highest sperm viability value (76.04 and 78.63 respectively) while the lowest values were recorded (60.92 and 59.83) at 5 % and 10 % .

Pre-freezing acrosomal defected spermatozoa (Table 3) were found to be better for diluted samples with Sod. Citrate and Tris extenders (6.02 and 6.18 % respectively). Percentages of post- frozen- thawed spermatozoa with defected acrosomes were found to be of minimal value for samples frozen in Tris diluent (9.30 %).However, different egg yolk concentrations and glycerol levels did not varied significantly in their effects on post- thaw acrosomal integrity (Table 7).

Exposure of goat semen to freezing and thawing caused a noticeable increase in AST-activity in different diluents (Table 4), compared with that before freezing. The average values of AST-activity recorded in Milk, Tris, and Sod. Citrate and CEGLY extenders post-thawing were 142.02, 141.48, 160.40 and 178.23 IU/ ml, respectively compared with 83.33, 65.85, 74.75 and 93.37 IU /ml, respectively before freezing. Irrespective to diluents, both egg yolk concentrations and glycerol levels did not indicate any significant differences in AST-activity before freezing and after thawing (Table 7) as analysis of variance showed that differences in AST-activity was statistically significant between diluents (p<0.0001) before freezing and after thawing . However, no significant interactions between different treatments have been recorded.

The average calcium concentration (Table 5) in seminal plasma before freezing decreased from 5.33 mmol/L in milk diluent to reach 3.39; 2.94 and 2.89 mmol/L in Tris, Sod.Citrate and CEGLY diluents respectively. After thawing, it decreased from 4.49 mmol/L in milk diluent to 2.73, 2.64 and 2.67 mmol/L in Tris, Sod. Citrate and CEGLY extenders respectively. Analysis of variance (Table 7) indicated that the effects of diluents on calcium level in buck seminal plasma before freezing and after thawing was statistically highly significant (p<0.0001). On the other hand, all other treatments and interactions before freezing and after thawing were insignificant.

Lactic dehydrogenase activity in goat seminal plasma after freezing and thawing displayed a significant increase from 205.57 U/L in Tris diluent to reach maximum values (239.60 and 237.93 U/L) in milk and CEGLY diluents respectively (Table 6). Before freezing, it displayed a significant increase from 114.77 U/L in Tris diluent to reach maximum values of 139.64 and 138.24 U/L in CEGLY and Sod. Citrate diluents respectively. Analysis of variance (Table 7) revealed that the differences in lactic dehydrogenase activity were statistically significant between diluents (p<0.0001) pre- and post- freezing and thawing. Egg yolk concentrations were statistically significant (p<0.05) in their effect on lactic dehydrogenase activity only after thawing as the lowest average value was recorded (218.68 U/L) for 20 % egg yolk, and the highest (230.11 U/L) for 5% egg yolk. There was also an interaction between egg yolk and diluents (p<0.05) after freezing and thawing. On the other hand, no significant differences were observed due to egg yolk and glycerol concentrations pre-freezing.

The correlation coefficients studied in the current work showed that sperm motility pre- and post-freezing and thawing had significantly negative relationships with AST (r= -0.22 ; P<0.05; r= -0.47; p<0.001 respectively) and LDH (r= -0.30;p<0.05 and r= -0.67; p<0.001 respectively) leakage. The level of AST and LDH released extracellularly during dilution and freezing- thawing were positively and significantly interrelated with each other (r= +0.22; p<0.05 and r= +0.33; p<0.001 respectively).

In the current work, the factorial design (4 x 4 x 3) aimed at testing the effect of four diluents [ Milk, Tris, Sod. Citrate and CEGLY- based diluents]; four different concentrations of egg yolk (5, 10, 15 and 20 %) and three levels of glycerol (5, 7 and 9 %) on Zaraibi buck semen (pre- and Post- freezing and thawing). AST and LDH enzymes and calcium selected in this study were based on the previous observations (Yanagimachi and Usui, 1974; Tuli, et al., 1992; Dhami and Kodagali, 1988; 1990; Upreti, et al., 1996) that these enzymes leak during the cryopreservation of buffalo, bull and ram spermatozoa. AST is located primarily in the middle piece of the sperm cell (Mann and Lutuak-Mann, 1981) and the measurement of the release of this enzymes from the spermatozoa is considered to be a sensitive indicator of sperm damage occurring during freezing and thawing and has been used during recent past to evaluate the quality of frozen- thawed spermatozoa (Sahni, 1987). LDH is predominantly localized in cytosol of spermatozoa (Morton, 1973) and its leakage has been correlated with cell membrane damage and used as a marker of membrane damage both during pre-freezing and post-thaw incubations (Upreti, et al., 1996).The major site of calcium accumulation is the mitochondria (Babcock, et al., 1976). Calcium ions in spermatozoa play an important role e.g. they influence motility (Nelson, et al., 1982) and are involved in the acrosomal reaction (Yanagimachi and Usui, 1974). Alterations in intracalcium may interfer with normal sperm function.

Irrespective of egg yolk concentrations and glycerol levels, the four extenders employed differ statistically in their ability to protect sperm against freeze damage as revealed by overall motility; viability; acrosomal damage and enzyme leakage rates (Tables 1-6). Similar findings for freezability and leakage of AST and LDH enzymes during the deep freezing of goat semen were reported by Chauhan and Anand, 1990; Sinha, et al., 1996 and Singh and Purbey (1996). The differences in post-thawing spermatozoal motility and cell survival between different concentrations of egg yolk were highly significant (Tables 7). These results were in accord with Moussa (1987); Sahni (1987); Chauhan and Anand (1990) and Tuli, et al. (1991). The egg yolk concentrations did not play an important role determining the release of AST and LDH enzymes in extracellular medium. The glycerol levels used in the current work have no significant effects on some characteristics of spermatozoa and enzymes leakage. These results are in agreement with those of Deka and Rao (1986) and Sahni (1987). The two- and three- way interaction effects studied between sperm motility, diluents, egg yolk concentrations and glycerol levels (post- freezing and thawing) were statistically non significant. Similar results were obtained with viability (Table 7), AST leakage (Table 4) and calcium concentration (Table 5) in seminal plasma. On the contrary, Drobnis, et al. (1980) found interaction between diluent and glycerol level for motility of thawed semen.The correlation coefficients studied herein showed that sperm motility pre- and post- freezing and thawing had significantly negative relationships with AST (r= -0.23; p<0.05 and r= -0.47; p<0.001respectively) and LDH (r= -0.62; p<0.001 and r= -0.67; p<0.001 respectively) enzyme leakage. The level of enzymes (AST and LDH) released extracellularly during extension and freezing-thawing were positively and significantly interrelated with each other (r= 0.22; p<0.05 and r= 0.36; p<0.001. Similar correlations for sperm motility with enzyme leakage of AST and LDH have been reported in goats (Graham, et al., 1973), rams (RoyChoudhury, et al., 1975) and Bovine (Sidhu and Quraya, 1978; Dhami and Kodagali, 1988; 1990). These correlations indicate that lower freezability of semen may be due to initial as well as post-thaw semen damage and leakage of intracellular enzymes involved in the process of cellular metabolism and fertilization, especially in poor semen quality ejaculates (Dhami and Kodagali, 1990). Stallcup and Hayden (1960) reported a correlation coeficient between LDH activity of semen and 30- to 60- non return data to be -0.414 (p<0.001). On the same line, AST in the seminal plasma was found to be negatively correlated with fertility in bull (r= -0.67), which may suggested that the higher release of spermatozoa AST into plasma resulted in low fertility (Pangawkar, et al., 1988). Of neat semen, Dube, et al. (1982) found that the higher the concentration of LDH in the semen, the lower was the keeping quality and the activity of LDH in neat semen was directly associated with the metabolic activity of spermatozoa.

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FOLLICLE STIMULATING HORMONE MODULATES SOME BIOCHEMICAL CONSTITUENTS IN UTERINE MILIEU OF PRE- AND POSTIMPLANTED RABBIT EMBRYOS

M.M. ZEITOUN, A.Z. FATHELBAB , ZAHRAA R. ABO EL-EZZ AND Y.R.HASSANEIN

ABSTRACT

Twelve adult female rabbits (New Zealand White x California) were used. The study aimed to investigate the effects of FSH/LH regime (n=6) as compared to control (n=6) on the reproductive traits as well as biochemical constituents in uterine media pre- (2 days postcoitum, n=3) and post- (9 days postcoitum, n=3) implantation.

Rabbit females treated with FSH/LH have shown significant decrease in; sexual receptivity (60 vs 100%), conception rate (67 vs 100%), percent ova recovery (49 vs 84%), and survival at birth (51 vs 94%) for FSH vs control does. Moreover, none of kids born of FSH-treated does were alive (0% survival) at weaning as compared with 65.6% survival for control kids. In contrary, there were significant increases at day 2 (preimplantation) in ovulation rate (27 vs 13.7) and total ova recovered (15 vs 11.3) for FSH vs control does. Number of implanted embryos was highly significant in FSH (30±7.4) than control (9.0±0.6) does. However, percentage of implantation was higher in control (100%) than FSH (69.3%) does.

Treatment with FSH has no significant effect on protein synthesis which is secreted in uterine fluids, however as day of embryonic development progresses, protein levels increase significantly. FSH caused a significant increase in K⁺ concentration and a significant decrease of Cu⁺⁺ at both stages.

There are 6 small peptide subunits (M. Wt » 20-40 k Da) in fallopian fluids at day 2, which disappeared at day 9 postcoitum. Contrariwise, uterine fluids did not show a peptide subunit in the pattern at day 2 of pregnancy however, at day 9 there exist 7 peptides of large molecular weights in uterine fluids.

Rabbits are considered a main source of white meat among domestic animals. As polytocus animals, rabbit does occasionally face some reproductive inefficiencies (i.e. pseudopregnancy, low conception rate, small litters size, low sexual receptivity… etc.). Moreover, in commercial rabbit enterprises in Egypt there is about 20% of does in a herd suffer of such drawbacks (Radwan, 1986).

Ovarian stimulation and superovulation have been intensively applied in monotocus animals last three decades (Maurer and Echternkamp, 1985). One of the alternatives to avoid culling subfertile does, is the treatment with FSH/LH (Donaldson and Ward, 1986) or GnRH (El-Keraby et al., 1991).

Among the obstacles related with the superovulatory treatments is the lower quality of embryos. These embryos undergo early degeneration or abnormal embryonic development (Zeitoun, 1990). Roberts and Bazer (1980) found that uterine secretions comprise a complex nutrient-rich culture medium for the developing conceptus.

Therefore, the current study aimed at; 1) investigating effects of administering FSH/LH on the reproductive traits of rabbit does, and 2) determining some biochemical constituents in the fallopian and uterine milieu pre- and postimplantation.

MATERIALS AND METHODS

Twelve mature female New Zealand White x Californian does with average body weight of 3.0±0.3 kg and age 8.0±0.6 months, were used. The animals were individually fed commercial pellets of alfalfa hay, wheat bran, barley, soybean meal, vitamins and minerals, with 18.2% crude protein, 14% crude fiber and 2670 kcal D.E./ kg feedstuffs.

Females were slaughtered, the reproductive tracts were excised and separated. Five or two milliliters of flushing medium (0.9% NaCl, with 1000IU penicillin/ml and 1000g streptomycin/ml) were used to flush the uterine horn or fallopian tube, respectively. Dilution ratio was considered at the time of calculation of nutrient concentration.

Samples of 10l were centrifuged at 3000 rpm for 20 minutes in cooled centrifuge (Beckman Model TI-6). The supernatant was aspirated and protein was measured using the method of Lowry et al. (1951) by adding Folin-phenol reagent. The color intensity was measured at wavelength 750nm (Shimadzu UV, visible recording spectrophotometer).

These ions were determined by wet ashing and the extract (in 5N HCl) was applied to a clinical flame photometer (410C).

These ions were determined in the extract (5N HCl) of the wet ash using Perkin-Elmer 2380 atomic absorption spectrophotometer.

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was carried out using the discontinuous buffer system described by Laemmli (1970) and quoted by Hames and Rickwood (1990).

Standard protein markers were used according to the method described by Weber and Osborn (1969). After electrophoresis, both the length of the slab gels and the distance moved by the dye (bromophenol blue) were measured. Also, after staining and destaining, the length of the gels and the positions of the protein zones were recorded. Mobility of the protein fraction was determined using the following equation :

Mobility was plotted against known molecular weights expressed on a semi-logarithmic scale.

Data were statistically analyzed by least square analysis of variance using general linear models (GLM) procedure (SAS, 1995). Differences between means were detected using Duncan’s Multiple Range Test (DMRT). Simple correlation coefficients were tested between traits studied.

RESULTS AND DISCUSSION

As shown in Table (1) on day 2 postcoitum (preimplantation stage) FSH significantly (p<0.05) increased ovulation rate (number corpora lutea, CL) by about two folds (27±4.1) than in control does (13.7±0.9). This finding is in concordance with Fisher and Schumacher (1991) who obtained a higher number of CLs when rabbits were treated with FSH. There were no significant differences due to FSH treatment in number of different stages of embryos. Although, total ova/embryos collected per doe tended to be higher in FSH-treated (15±7.9) as compared to control (11.3±0.7) does. There existed no significant differences due to the high variability of the response of individual does to FSH treatment.

Parameters	Control	FSH
No. of CL/Doe	13.7 ± 0.9^a	27.0 ± 4.1^b
No. of UFO/ Doe	0.0 ± 0.0	1.0 ± 1.0
No. of Morulae/ Doe	10.7 ± 1.3	14.0 ± 8.7
No. of Blastocysts / Doe	0.3 ± 0.3	0.0 ± 0.0
No. of Degenerate / Doe	0.3 ± 0.3	0.0 ± 0.0
Total No. of ova / Doe	11.3 ± 0.7	15.0 ± 7.9
Total No. of fert. Ova / Doe	11.0 ± 1.0	14.0 ± 1.5
Fertilization (%)	97.0	93.0
Ova recovery (%)	84.3 ± 9.6^a	49.1 ± 21.8^b

Means in the same row with different superscripts are significantly different at P < 0.05.

Although, difference was not significant, treatment with FSH resulted in unfertilized oocytes which decreased percent fertilization in treated than control does. Zeitoun et al. (1991) reported that the superovulatory treatment might impede the normal fertilization process. Moreover, FSH significantly (p<0.05) decreased percentage of ova/embryo recovery (84.3±9.6 vs 49.1±21.8 for FSH vs control does). The lower recovery in superovulated does could be attributed to the increase in ovarian size which reduced the ability of the fimbriae to pick up the released oocytes (Wise et al., 1986).

Table (2) presents data collected from pregnant does slaughtered 9 days postcoitum (postimplantation stage). Likewise to the results of day 2, there is a considerable (P<0.05) increase in ovulation rate (#CLs) in FSH (43.7±11.5) than control (9.0±0.6) does. Moreover, number of implanted conceptuses was about 3 folds in FSH (30.0±7.4) as compared to that in control (9.0±0.6) does. The purity of the FSH preparation (SuperOv.^®) resulted in such big number of ovulations. In contrary, percentage of implantation was significantly (p<0.05) lower in FSH (69.3%) than in control (100%) does. Mongkol (1986) reported that superovulation with FSH/LH decreased rate of recovered and normal embryos. Moreover, Hafez (1963) indicated that superovulatory treatment may induce some early ovulations which degenerate in their early stages. More recently, Gurthrie et al. (1997) stated that co-administration of LH with high doses of FSH increased estradiol secretion which impede implantation process.

Means in the same row with different superscripts are significantly different at P < 0.05.

As shown in Table (3) there found a 3-4 folds increase of protein concentration in genital fluids in late stage (day 9) as compared to early stage (day 2) of embryonic development. Uterine secretions comprise a complex nutrient-rich culture medium for the developing conceptus (Roberts and Bazer, 1980). In the present study at day 2 postcoitum there are five times increase of uterine than oviductal proteins, however, this was 25 folds at day 9 of embryo age. This finding is in accordance with Bazer et al. (1991) in gilts. Vallet et al. (1998) attributed such increase of endometrial activity to secrete proteins with advancement of conceptus age to the action of increased progesterone level.

Parameters	Control			FSH
Parameters	Day 2		Day 9	Day 2	Day 9
Protein (mg/ml)	8.7 ± 2.1^a	35.1 ± 11.9^b		10.1 ± 3.1^a	30.1 ± 8.9^b
Sodium (Na⁺, mg/ml)	6.3 ± 0.9	4.7 ± 0.6		5.2 ± 0.6	5.2 ± 0.7
Potassium (K⁺, ppm)	160.4 ± 41.5^a	202.1 ± 43.1^a		554.2 ± 127.7^b	550 ± 107.7^b
Copper (Cu⁺⁺, ppm)	1.9 ± 0.7^a	3.9 ± 0.7^b		1.2 ± 0.3^c	0.1 ± 0.1^d
Zinc (Zn⁺⁺, ppm)	9.5 ± 1.7^a	8.1 ± 1.3^a		10.5 ± 2.4^a	6.3 ± 1.3^b

Means in the same row with different superscripts are significantly different at P < 0.05.

It was found that, no significant differences between day 2 and day 9 in concentrations of Na⁺, K⁺ or Zn⁺⁺, however, in control does Cu⁺⁺ concentration was higher at day 9 than at day 2. The treatment with FSH appreciably (P<0.05) decreased such a level at day 9. Cao and Chavez (1995) found a smaller litter size in gilts fed low-Cu⁺⁺ diet during pregnancy. Also, House and Bell (1993) in their study on late gestation in Holstein cows found that Cu⁺⁺ accumulated in the conceptuses at a rate of 1.6mg/day. The low concentration of such cation in fluids around embryo in the FSH-treated does still needs more investigations. Eventhough this was accompanied by a lower embryonic growth. Sodium ion was not affected either by treatment or age of embryo. On the other hand, potassium ion concentration increased by about 2 folds in FSH than control does. Jauniaux et al. (1998) studied the biochemical composition of the fluids surrounded embryos in early pregnancy in rabbits. They found higher sodium, potassium and total proteins in vesicular than in amniotic fluids. Additionally, Waelchli et al. (1997) showed an evidence of the presence of Na⁺ and K⁺-dependent adenosine triphosphatase (ATPase) ₁ and ₁ subunits isoforms, and their probable role in blastocyst expansion in the pre-attachment horse conceptus.

Plate (1) illustrates a comparisons between fluids of genital tract of control versus FSH-treated does at day 2. Fluids were separately collected of fallopian tubes as well as uterine horns. The gel shows that FSH treatment caused disappearance of 3 major peptides with high molecular weights (» 51-77 kDa) and disappearance 7 low molecular weight-subunits (» 20-40 kDa) in oviductal fluids. The disappearance of such small peptides of the oviductal fluids at this early stage (day 2) was accompanied by the presence of 6.5% unfertilized oocytes. This indicates the great importance of such small peptides (» 20-40 kDa) in the process of fertilization. The 3 major peptides probably have certain roles in postovulatory oocyte development in fallopian tube. Suzuki et al. (1982) reported that uterine proteins show daily changes in protein patterns of the secreta of fallopian tubes and uterine horns in superovulated as compared with normally mated female rabbits. Uterine secretions did not show obvious peptides at this early stage of embryo age.

On the other hand, plate (2) illustrates the peptide patterns of genital tract fluid proteins at day 9 of embryo age. At this later stage, there existed a disappearance of the 7 small peptides which appeared at day 2 in oviductal fluids. This could confirm the fact that these peptides are needless to the embryos at this stage. Rather, there appeared 2 small peptides (28 and 33.5kDa) in the uterine fluids, which might have important role(s) in growth, development and metabolism. This finding is partially in accordance with results of Buhi et al. (1982) who reported that iron bound to uteroferrin was transferred to transferrin in allantoic fluids and enters fetal circulation. The molecular weights of 28 and 33.5 kDa are close to the protein “uteroferrin” which is indispensable for the conceptus respiration.

In conclusion, such a study gave insights on some biochemical parameters in the media surrounding the rabbit embryos in pre- and postimplantation. Further studies are needed to investigate different roles of such compounds during course of pregnancy.

Acknowledegement : The donation of SuperOV ^®and Lutogen of the AUSA, Tyler, TX is highly considered.

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TOTAL MOTILE SPERM OUTPUT AS A COMBINING PARAMETER FOR FERTILITY PREDICTION IN RABBITS

*Department of Environmental Studies, Institute of Graduate Studies and Research, Alexandria University, Alexandria, Egypt

**Departments of Animal and Fish Production, 3Poultry Production, Faculty of Agriculture and.

ABSTRACT

This study aimed at using some semen characteristics as laboratory tools to predict fertility status of male Spanish rabbits. Twelve mature V. line Spanish bucks were utilized for mating 36 V. line Spanish does (n = 9-13 mating/buck). Semen samples were collected once a week for 10 consecutive weeks and semen parameters were determined (ejaculate volume, sperm concentration, percentage of motile sperm, total number of motile sperm per ejaculate, semen initial fructose, reaction time, and percentage of dead, live and abnormal spermatozoa). Data of semen characteristics were analysed against the productive data obtained from females (conception rate, litter size and body weight at weaning).

Results indicated that as percentage of progressive motility, number of motile sperm per ejaculate and total sperm output increase, conception rate increased linearly and there is positive correlation between these parameters. Contrariwise, there found no clear relationship between doe fertility traits and reaction time, ejaculate volume and semen initial fructose. Also, as litter size increases, percentage of young rabbits viability decreases. It is concluded of this study that number of motile sperm per ejaculate is an accurate predictor for male fertilizing ability as well as selection of best sires in rabbit breeding programs.

INTRODUCTION

Several methods have been used for routine analysis of mammalian semen, each describing specific quality criteria of the sperm. The predictive value of the standard semen analysis which is based on investigations in a microscope (morphology, motility and sperm concentration) is limited (Liu and Baker, 1992). More specific and objective in vitro methods have been elaborated to characterize male fertility, e.g. acrosome reaction (AR), hamster zona-free oocyte penetration , hypoosmotic swelling test (HOS) and supravital staining (Eosin-Y). Also, measurement of adenosine triphosphate (ATP) and detection of mitochondrial function using flow cytometry (Graham et al., 1990) were carried out.

It has been found in different species that sperm motility is one of the strongest criteria for semen evaluation. In human, the total count of motile cells and the velocity of their motility have been implicated as crucial parameters for fertility (Hinting et al., 1988). In bulls, it was found that evaluation of the quality of frozen semen on basis of post-thaw sperm motility and velocity revealed a positive correlation with non return rate in cows (Kjoestad et al., 1992).

Fertilization in rabbits requires several numbers of motile sperm to ensure fertilization of such multi-ovulated oocytes. Therefore, the objective of this study aimed at validating such phenomena in rabbits and to correlate, where it is the percent of sperm motility and/or number of motile sperm per ejaculate in males with conception rate and litter size in females is the definitive criterion.

Sexually mature V. line Spanish rabbits, imported from Spain, were acclimated for 4 weeks. Twelve mature bucks (mean age = 9 months, range = 8-10 months, and mean body weight = 2.6± 0.5 kg) were trained for 4-8 weeks to give semen. Virgin female rabbits were 6-7 months old (mean body weight = 2.4 ± 0.4 kg). The animals were individually housed in stainless steel cages (dimensions, 24 ´ 24 ´ 14.5 in). Feed and water were provided ad lib throughout the experimental period (10 weeks). The animals were fed on a ration pellets consisting of 30 % berseem hay (Trifolium alexandrinum), 25 % yellow maize, 26.2% wheat bran, 14 % soybean meal, 3 % molasses, 1 % calcium chloride, 0.4 % sodium chloride, 0.3 % mixture of minerals and vitamins, and 0.1 % methionine. Chemical analysis indicated that this ration contained 17.5 % crude protein, 14.0 % crude fibers and 2.7 % fat.

Semen Collection and Evaluation

Semen was collected once a week for 10 consecutive weeks during April-June, 1999. Ejaculates were collected using an artificial vagina and a teaser doe (Tesh and Tesh, 19971). Since sexual preparation enhances the quality of semen produced (MacMillan and Hafs, 1967), each male was allowed three false mounts on the female before collecting semen. Reaction time was recorded from the moment of subjecting a doe to the buck until a complete erection occurs using a stopwatch. The volume of each ejaculate was recorded using a graduated collection tube after removal of the gel mass. A weak eosin solution, described by Smith and Mayer (1955) was used for evaluation of sperm concentration by the improved Neubauer hemocytometer slide. Determination of seminal initial fructose was carried out immediately after collection according to Mann (1948). Assessment of live, dead and abnormal spermatozoa were performed using an eosin-nigrosine blue staining mixture (Blom, 1950). Two parameters of sperm motility were evaluated to calculate sperm motility index (SMI): percentage motile and quality of motility (motility grade). The percentages of motile sperm and motility grade were estimated by visual examination under a microscope (10x). Motility grade was classified as follows: 0 = no movement; grade 1 = twitching, no forward progressive movement (fpm); grade 2 = slow fpm; grade 3 = good fpm; and grade 4 = fast fpm. For calculation of the final test scores, the two motility parameters were combined to yield a sperm motility index: SMI = percentage motile x motility grade (Yousef et al., 1996). Total number of motile sperm per ejaculate was calculated by multiplying; percentage motile sperm ´ concentration/ml ´ ejaculate volume.

Female Reproductive Traits

Twelve mature V. line Spanish bucks were utilized for mating 36 V. line Spanish does (n = 9-13 mating/buck). Conception rate, survival at birth, litter size and mean individual body weight at day 21^st post parturition were all recorded for each breeding of each buck.

Statistical Analysis

Data were collected of 12 males over 10 weeks. Therefore, data were analyzed by the 2-way analysis of variance for weeks of semen collection and differences between bucks. As no significant differences were observed between weeks, and male differences were highly significant. Therefore, males were classified according to the results of their conception rate into: high (90 % and up), medium (65 % and up) and low (> 60 %) fertile bucks. Least Square analysis of variance and mean comparisons were carried out between the three cateogries (SAS, 1984) Simple correlates were also tested between semen characteristics and doe fertility traits.

Standard semen analysis including sperm concentration, motility, and morphology is widely used as a fundamental indicator of male fertility (Liu and Baker, 1992). Semen analysis is the initial and most important step in the evaluation of male reproductive performance (Mohamed et al., 1986). Magistrini (1998) reported that prediction of fertility in stallion selection programs is routinely based on the analysis of seminal characteristics (spermatozoal morphology, motilit and zona pellucida-sperm binding assay) in addition to behavioural parameters. As shown in Table (1), there were no significant differences between the three categories (high, medium and low) of bucks with regards to libido (reaction time), ejaculate volume, percentage progressive motility or fructose concentration in seminal plasma. Furthermore, no significant differences (Table 2) were found between different fertile bucks from the point of litter size, survival at birth or mean kid weight at weaning (day 21).

On the other hand, sperm concentration per ml (Figure 1), total sperm output (Figure 2) and total motile sperm per ejaculate (Figure 3) were all higher (p < 0.01) in high than in medium or low fertile bucks. The trend of these semen characteristics coincided with the trend of conception rate (Figure 4). There found positive correlation coefficient between conception rate and; percentage of progressive motility (r = 0.25, p<0.05); total sperm output (r = 0.32, p < 0.01) and total motile sperm per ejaculate (r = 0.31, p<0.01). None of these semen parameters have shown a significant correlation with litter size or survival at birth. Most recently, Saacke et al. (2000) in cattle and Castellini and Lattaioli (1999) in rabbits found that sperm number and number of motile sperms are positively associated with both fertilization rate and embryonic quality. Mohamed et al. (1986) reported that the percentage of motile spermatozoa and the quality of their forward progression have appeared to be the best predictors of human male fertility potential. In view of the significance of sperm motility, it is not surprising that this criterion of sperm function has assumed a central role in the routine clinical diagnosis of male fertility (Aitken, 1990). Furthermore, sperm motility was found to be one of the most important criteria of semen quality and a determinant in the success of fertilization (Ijaz et al., 1994). Since, motility is involved in defining the ability of the spermatozoa to migrate through female reproductive tract to the Table 1: Sexual activity and semen characteristics of different fertile rabbit bucks (mean + SE)

Table 2: Fertility traits of female rabbits mated with rabbit bucks with different semen quality (mean + SE)

site of fertilization. Moreover, motility helps sperm to penetrate the vestments surrounding the oocyte, including the cumulus oophorus and zona pellucida to accomplish fertilization. (Pousette et al., 1986). In cattel, Soderqist (1991) has found a positive correlation between non-return rate and post-thaw percentage of motile sperm. Additionally, motility was found to be a critical factor in process prerequisite to gamete-interaction (Kjaestad et al., 1993).

In conclusion, in rabbits it is possible to select bucks as sires for breeding programs depending on number of motile sperm per ejaculate, but not percentage of progressive motility per se. Moreover, number of motile sperm of 650 million per ejaculate is a good predictor for high conception rates in such a specie.

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Liu, D. Y. and Baker, H. W. (1992): Tests of human sperm function and fertilization in vitro. Fertil. Steril., 58: 465-483.

MacMillan, K. L. and Hafs, H. D. (1967): Semen output of rabbits ejaculated after varying sexual preparation. PSEBM., 125: 1276-1281.

Magistrini, M., Vidament, M., Clement, F. and Palmer, E. (1998): Fertility prediction in stallions. Anim. Reprod. Sci., 42 (1-4): 181-188.

Mann T. (1948): Fructose content and fructolysis in semen: Practical application in the evaluation of semen quality. J. Agric. Sci. Camb., 38: 323-331.

Mohamed, M. K., Lee, W. I., Mottet, N. K. and Burbacher, T. M. (1986): Laser light-scattering study of the toxic effects of methyl mercury on sperm motility. J. Androl., 7: 11-15.

Pousette, A., Akerlof, E., Lundin, A., Rosenborg, L. and Fredricsson, B. (1986): On the use of adenosine triphosphate for estimation of motility and energy status in human spermatozoa. Intern. J. Andro., 9: 331-340.

Saacke, R. G., Dalton, J. C., Nadir, S., Nebel, R. L. and Bame, J. H. (2000): Relationship of seminal traits and insemination time to fertilization rate and embryo quality. Anim. Reprod. Sci. 60-61 (1-4): 663-677.

Smith, J. T. and Mayer, D. T. (1955): Evaluation of sperm concentration by the hemocytometer method. Fertil. Steril., 6: 271-275.

Soderquist, L. (1991): Sperm characteristics and fertility in dairy A.I. bulls. Thesis. SLU/Repro, Uppsala.

Statistical Analysis System, SAS. (1984): Principles and Procedure of Statistics, 2nd ed. Carry, NC, Mc Graw-Hill Inc.

Tesh, S. A. and Tesh, J. M. (1971): Artificial insemination in the rabbit and its use in routine teratogenic studies. Excerpta medica (Amsterdam) Int. Congerss Series 220: 332-336.

Yousef, M. I., Bertheussen K, Ibrahim H. Z., Helmi, S., Seehy, M. A. and Salem, M. H. (1996): A sensitive sperm-motility test for the assessment of cytotoxic effect of pesticides. J. Environm. Sci. Health B31: 99-115.

Animal Health Research Institute, Dokki Giza

ABSTRACT

One thousand and eight hundred -twenty blood samples were collected from different Friesian dairy farms in Gharbia, Mansoura and Behera province for detecting of brucella antibodies using RBPT; BAPT as screening tests compared with ELISA test. The results indicated that the high sensitivity of the ELISA test which it will be economically if it used as screening and confirmatory test.

INTRODUCTION

In the recent years, attention has been directed to re-study the prevalence of brucellosis in Egypt due to continuous importation of large numbers of cattle and establishment of several dairy farms with extensive cattle population, among animals. (Adway, 1985)

Brucellosis still constitutes one of the major health problems in both man and animal in Egypt. It is the main causes of breeding failure and infertility of affected animal. The infection spreads incessantly from animal to animal and from herd to herd all over the year. The recent wide, importation of cattle and establishment of modern dairy farms leads to high prevalence of brucellosis among cattle and buffaloes (Arthur, 1975; Shalaby, 1986)

The aim of this investigation is to evaluate the efficacy of different serological screening tests in picking up infected cases compared with ELISA test.

MATERIALS AND METHODS

1-Animals: 1820 Friesian dairy cows were included in these study composed 14 dairy farms in Gharbia and Behera governorates.

2-Samples: about 10 ml of blood from the examined animals in the different farms were collected in sterile silicon coated vacuum tubes, identified, kept for separation of serum.

3-Antigens: Rose Bengal Plate Antigen and Buffered Acidifies Plate Antigen were supplied by Veterinary Sera and Vaccine Research Institute, Abasia, Cairo,Egypt.

A-Rose Bengal Test was carried out according to the method described by Alton et al (1988).

B-Buffered Acidified Plate Test was carried out according to (N.V.S.L., Ames, Lowa, 1964).

RESULTS

DISCUSSION

Control of the disease requires basic understanding of the pathogenesis as well as the prophylaxis and surveillance methods available for its control/eradication. Before 1986 in Egypt, attempts to control brucellosis in animals have been reported in different countries of the world without prospective results (FAO/WHO, 1964).

In this study, as shown in table (1) the highest percentage of reactors was recognized in Mansoura, followed by Behera province. In Mansoura province The screening tests revealed a 4.5% (n=14/310) for both RBPT and BAPT while ELISA test revealed a 7.09% (n=22/310) moreover in Behera the screening tests revealed 1.08% (n=2/185) for RBPT and BAPT while ELISA test revealed a 5.4%(n=10/185). Collectively the screening tests revealed 1.6%(n=29/1820) in Gharbia, Mansoura and Behera provinces while the ELISA revealed 3.29%(n=60/1820).

Rose Bengal plate test and Buffered acidified plate test can be considered more reliable tests (Gones et al 1981; and Salem et al 1984). Rose Bengal test may be helpful in detecting cases of recent infection (Soliman 1993).

In Egypt the schedule for brucellosis control and eradication depends upon using the screening tests (RBPT and BAPT) then confirm the positive samples with confirmatory tests such as Rivanol, Mercaptoethanol, complement fixation and ELISA tests. Considering the farm is negative for brucellosis when the screening tests (RBPT and BAPT) are negative.

Table (1) shows that in Gharbia, farm1and farm3, in Mansoura, farm3 revealed a negative results with the screening tests while the same samples revealed 5.8% (n=5/86), 0.5%(n=2/400) and 7.1%(n=1/14) respectively with ELISA test which means a false negative results with these screening tests giving a good chance for spreading the infection through the missed positive cases in these farms.

Our results disagree with Corbel (1973) and Stemshorn et al (1985) who reported that RBPT and BAPT have a higher sensitivity and (Soliman 1993) as it may be helpful in detecting recent infection.

Concerning the total samples examined The ELISA test revealed 3.29% (n=60/1820) while the screening tests revealed 15.9%(n=29/1820) which means the high sensitivity of the ELISA test.

From the previous results we can conclude that these results throw light on using of ELISA test to be the first step as an alternative test for screening and confirming in the same time will be more economically which provides a rapid, simple, accurate and inexpensive diagnostic assay for brucellosis. This assay will also enable us to assess diagnosis accurately and reduce the eradication time

REFERENCES

Adway,A.T. (1985): Incidence of brucellosis in Egypt. Conference of Rabies and Brucellosis in Mediterranean and Arabian Countries, Pensiula, Montpelier-France.

Alton, G.G.; Jones, L.M.; Angus, R.D. and Verger, J.M. (1988): Techniques for the brucellosis laboratory INRA, Paris ISBN, 1988.

Arthur, G.H. (1975): Veterinary Reproduction and Obstetrics. 4^th Edition. 422-426.

Corbel, M.J. (1973): Studies on the mechanisms of the rose Bengal plate test for bovine brucellosis. Br.Vet.J. 129,157.

FAO/WHO(1964): The joint FAO/WHO expert committee on brucellosis Report, 1964.

Gones,I.F.; Gruner,A.M. ad Arouyo , M.(1981): Plate agglutination test for bovine brucellosis. Comparison of rose bengal and other serological tests. Border line reactons in the serological diagnosis

Merck andSharp (1986): The Merck Veterinary Manual, 6th Edition. Fraser, C.M. (Ed.) Merck and Co.,

National Veterinary Services Lab.Ames, Lowa U.S.A. (1984): Instruction manual on conducting brucellosis serological tests. of bovine brucellosis. Vet.Bull.51(1).

Salem,S.F; El-Gibally,S.M.;Farag,Y.Y.A.;El-Molla,A.A. and El Molla,A.H.(1984): Evaluation of some of the commonly used serological methods for diagnosis of brucellosis..2^nd Conf..R.C.Giza. 9-11 April 1984 XII.

Shalaby, M.N.H. ((1986): A survey on brucellosis as a cause of reproduction disorders in farm animals in Egypt. Ph.D.Thesis (Obst.Gyaen. &A.I) Vet.Med.Cairo Univ.

Soliman, S.A.(1993): Efficacy of serological tests in recognetion of bovine brucellosis in Suez Canal and Sinia provinces. M.V.Sc. Fac.Vet.Med .Seuz Canal Univ.

Stemshorn, B.W.; Forbes, L.B.; Eaglesome, M.D.; Nielsen, K.H.; Robertson, F.J.; Samagh, B.S. (1985): A comparison of standard serological test for the diagnosis of bovine brucellosis in Canada. Canadian Journal of comparative medicine.49 (4), 391.

** Theriogenology Dept., Faculty of Veterinary Medicine, Cairo University, Egypt.

The present study aimed to study freezability of Zaraibi goat semen and effect of adding some biological fluids such as anti-snake venom (A-SV), bovine seminal plasma (BSP), goat blood plasma (GBP) and anti-goat seminal plasma (A-GSP) on post-frozen-thawed semen quality. Post-freezing and thawing, sperm motility percenage was slightly improved (49.00 vs 44.00 %) with addition of BSP to semen diluted with Sod. Citrate diluent. Also, spermatozoal viability was improved (110.00 vs 94.50) without any signficant changes in acrosome defects (18.20 vs 17.00 %). Concerning enzymes leakage (AST, LDH and acrosin), which were increased post- freezing in control samples, addition of GBP to semen diluted in Tris diluent (80 - 100 ml/ml diluent) decreased, to some extent, the release of these enzymes. With regard to hypo osmotic swelling test performed in this experiment, as sperm membrane integrity and functional activity, the results do show a significant decline in percent of sperm swelling post- freezing and thawing.The in vitro assay of mucus penetration test used in the present study, aside from slight differences between control and treated samples (with GBP and BSP), freeze preservation of buck semen reduces the mucus penetration ability of spermatozoa. The post- freezing sperm penetration distance was 21.80 mm compared to 43.70 mm for control samples. Adding GBP and BSP increased the penetration distance to reach 24.00 mm for GBP and 24.57 mm for BSP. The acrosin activity was decreased from pre-freezing to the frozen- thawed stage (29.48 vs 19.03 mg /ml) and also with inclusion of GBP or BSP in diluted semen at its optimum concentrations (80 - 100 ml/ml diluent).

In general, it can be concluded that the findings of the current study have clearly revealed that Zaraibi goat semen can be frozen efficiently in Tris- citric acid fructose egg yolk and Sod. Citrate egg yolk diluents with inclusion of 80-100 ml/ml of GBP or BSP. However, the effects of these developed extenders on the fertility of frozen Zaraibi goat semen need further investigation.

Goats are important animals in tropical and sub-tropical livestock production systems and can be exploited for the benefit of the people. In their subsistence sector, pastoralists and agriculturists often depend on goats for much of their livelihood and the economic importance of goats depends on the value of their production or services which include meat, milk, fibers and skin among others (Devendra, 1981). Some of the major reasons for promoting animal production include the human populations are growing very rapidly creating a significant and increasing demand for additional animal protein foods. This demand can be met most easily by rapidly increasing the ruminant livestock population and it is easier to increase the population of small ruminants (such as goats) than large ruminants.

Phospholipase A2 and lysophospholipase enzymes (produced by the bulbourethral glands of male goat) were found to be egg yolk coagulating enzymes (Roy, 1957 and Iritani and Nishikawa, 1961) and dilution of goat semen in extenders containing egg yolk maintained activity of phospholipases enzymes during dilution, cooling, glycerolization, equilibration and freezing and thawing(Chauhan, and Anand (1990)). In general, there are many biological fluids which have the ability to inactivate the effect of PLA2 enzyme such as bovine seminal plasma proteins (Manjunath, et al., 1994) and goat blood plasma which contains higher concentration of the anti- sticking factor (ASF-1) that showed high affinity for inhibiting agglutination of corpus epididymal spermatozoa (Roy, and Majumder, 1989 ).

So, the present study was aimed to evaluate the potent biological fluid and best diluent for dilution of goat semen in freezing program.

The objective of this study was to assess the use of best concentrations of the biological fluids tested on neat and diluted semen (Chehadeh et al 2001) in freezing goat semen.

Semen was harvested, pooled and then diluted with Tris-citric acid- fructose diluent and Sod. Citrate diluent (both with 20% egg yolk and 7% glycerol). Immediately after dilution, goat blood plasma and buffalo seminal plasma at two levels (80 and 100 μl/ml) were added. Extended semen samples were cooled to 5ْC withen 45 min. and then equilibrated for 2 hr. at 5ْC. Equilibrated samples were packaged in 0.25 ml minitubes and sealed. Minitubes were frozen on liquid nitrogen (L.N). vapor (about 6 cm above the level of L.N.) for 15 min., then plunged and stored in L.N. container. After 3 days storage, the minitubes were thawed in water bath at 37ْC for 30 sec. Progressively motile spermatozoa and acrosome defects were estimated pre- and post-freezing and thawing. Viability of post-thawed semen samples was estimated by recording of sperm motility percentage for three hr. after thawing to estimate the viability indices according to Milovanov (1962). Aspartate aminotransferase activity (AST), Calcium concentration, Lactic dehydrogenase activity were estimated in seminal plasma of post-diluted and post-frozen thawed semen samples. In addition, Acrosine activity, Hypo-osmotic swelling test (HOST) and cervical mucus penetration test (CMPT) were performed according to Kennedy, et al. (1989), Soderquist, et al. (1997) and EL-Sheltawy (1989) respectively. Statistical analyses of the recorded data were done according to Snedecor and Cochran (1980).

Sperm motility of treated goat semen with 80 and 100 ml/ml of GBP and BSP before and after freezing and thawing was presented in Table (1). Before freezing, the highest sperm motility was found for semen samples diluted in Tris with 100 ml/ml GBP (89.00 %) or 100 ml/ml BSP (86.00 %) but with statistically insignificant results. After freezing and thawing, Sod. Citrate diluent was found to maintain significantly higher post-thaw sperm motility (45.67 %) than Tris (40.33 % ) diluent.

Overall mean percentages of spermatozoa with acrosomal defects of treated semen samples with 80 ml/ml GBP or BSP in Tris or Sod. Citrate diluents revealed significantly lower acrosomal defects (7.55 %) than 100 ml/ml (9.25 %) or control samples ( 9.80 % ) as shown in Table (1). Post- freezing and thawing, control samples achieved insignificant lower acrosomal defects (17.80 %) than treated samples (20.05 and 20.65 % for 100 and 80 ml/ml, respectively).

Post-thaw sperm viability of semen samples frozen in Tris diluent increased from 85.50 (for control samples) to reach 91.50 and 90.50 for treated samples with 100 ml/ml of GBP and BSP respectively (Table 2).

Results revealinging the effect of different concentrations of GBP and BSP and different diluents (Tris and Sod.citrate) on AST- activity in seminal plasma of Zaraibi bucks pre- freezing and post- thawing were shown in Table (3). The total average of AST- activity in seminal plasma pre- freezing (63.28 IU/ml) was found to be increased to reach 139.82 IU/ml post-freezing and thawing.

Concentrations of calcium in goat seminal plasma of semen pre-and post- freezing and thawing are shown in Table (3). Irrespective of biological fluids and its concentrations and diluents, the calcium level in seminal plasma of Zaraibi buck semen averaged 2.04 ±0.13 mmol/L pre- freezing and decreased to reach 1.12 ±0.05 mmol/L post- freezing and thawing.

Lactic dehydrogenase (LDH) activity in goat seminal plasma of semen post-freezing and thawing in Tris diluent was found to be highly decreased from 203.70 U/L for control samples to 165.15 and 184.56 U/L for treated samples with 80 ml/ml of GBP and 100 ml/ml of BSP, respectively. Also, for samples frozen in Sod. Citrate, LDH- activity decreased from 207.66 U/L for control samples to 200.90 , but increased to 241.10 U/L for treated samples with 80 ml/ml of GBP and 100 ml/ml of BSP respectively (Table 3).

Hypo- osmotic swollen spermatozoa showed changes associated with different concentrations of some biological fluids and diluents pre- and post- freezing and thawing. (Table 4). The percentage of swollen spermatozoa post- thawing averaged 24.80 % for control samples, which decreased to reach minimum values of 16.20 ± and 13.20 % for treated samples with 100 ml/ml of GBP and BSP respectively in Tris extender. However, in Sod. Citrate extender percentage of swollen sperm cells in control samples averaged 14.60 % that decreased to 13.40 and 11.40 % for treated samples with 100 ml/ml of GBP and 80 ml/ml of BSP, respectively.

Mucus penetrating spermatozoa post-freezing and thawing (Table 4) were found to be higher (25.80 and 32.00 mm) for semen samples treated with 100 ml/ml of GBP and BSP, respectively in Tris diluent than control samples (22.00 mm). On the other hand, samples frozen in Sod. Citrate, mucus penetrating spermatozoa were found to be higher (26.80 and 24.60 mm) in samples treated with 80 ml/ml GBP and BSP, respectively than control samples (21.60 mm).

Acrosin activity of treated semen with some biological fluids of post-frozen and thawed goat semen varied between 10.19 mg acrosin/ml at concentration of 100 ml/ml of GBP and 22.15 mg acrosin/ml at 80 ml/ml of BSP for samples frozen in Tris diluent, while it ranged 14.61mg acrosin/ ml at 80 ml/ml of BSP and 20.04 mg acrosin/ ml in control for those frozen in Sod. Citrate diluent. Addition of BSP and GBP revealed insignificant decreases in acrosin activity (18.55 mg acrosin/ml and 17.85 mg acrosin/ml, respectively). Before freezing, the overall mean of control samples achieved significant higher acrosin activity in spermatozoa (29.48 mg acrosin/ml) than treated samples (23.09 and 22.64 mg acrosin/ml for 80 and 100 ml/ml, respectively) as observed in table (4).

The best diluents (Tris and Sod. Citrate) developed for goat semen (Chehadeh et al 2001) with selected biological fluids (GBP and BSP) at their optimal concentrations (80 and 100 ml/ml) were used for freezing of Zaraibi goat semen in the current work to reveal the success of adding such biological fluids on freezability of buck semen.

This is, to our knowledge, the first report concerning the effect of adding GBP and BSP at concentrations of 80 and 100 ml/ml on goat semen (extended and processed in frozen condition). The lack of information available in the literature regarding that effect will limit the discussion of our results.

In addition to sperm motility, acrosomal sperm defects and sperm viability, we select and perform other tests used as additional parameters for fertility assessment. Hypo- osmotic swelling (HOS) test was used as membrane integrity and functional activity (Correa and Zavos, 1994) of soermatozoa. Sperm penetration into homologus mucus that was collected from does in estrus was performed as a test of fertility. Also, acrosin activity which provides a sensitive indicator for the acrosomal damage sustained by the spermatozoa during the different process of semen handling (Church and Graves, 1976) was carried out.

Sperm motility post-freezing and thawing in the current study (Table 1) was slightly improved (49.00 % vs 44.00 %) with addition of BSP to Sod. Citrate diluent which was used for dilution and freezing of buck semen. Also, viability of spermatozoa (Table 2)was improved (97.25 vs 90.oo) without any significant changes in percent acrosome (Table 1) defects (18.20 % vs 17.00 %) with inclusion of such biological fluid (BSP) to Sod. Citrate diluent. Concerning enzymes release (AST, LDH and acrosin) which were increased post-freezing and thawing confirm that the process of freezing damages the spermatozoa membrane and leads to leakage of these enzymes into the extracellular media (Mack and Zaneveld, 1987). Meanwhile, the inclusion of GBP in the Tris diluent used for freezing of Zaraibi goat semen decreased, to some extent, the release of AST; LDH and acrosin enzymes (Table 3). There are not firm grounds for ascribing this effect to GBP but it can be stated that GBP might play a role with Tris diluent for protection of spermatozoa during cryopreservation and the reason for the differences between GBP and BSP and their effect is unclear. Sinha, et al. (1996) speculated that the sudden increase in oxygen utilization by spermatozoa during thawing, following the dormant metabolic stage, might be responsible for increased protection of free radicals, toxic oxygen metabolites, leading to increased lipid peroxidation and thus spermatozoal membrane damage.

The in vitro assay of mucus penetration test (MPT) used in the current work measures the ability of spermatozoa to migrate through cervical mucus. Aside from slight differences between control and treated samples (with GBP and BSP), freeze preservation of buck semen reduces the mucus- penetration ability of spermatozoa. The post freezing sperm penetration distance was 21.80 mm compared to 43.70 mm for control samples. Addition of biological fluids, the penetration distance was increased to reach 24.00 mm in GBP and 24.57 mm in BSP. Our results (for control samples) support some of those observations recorded for bull spermatozoa (Murase and Braun, 1990). Okuda, et al. (1988) reported that there was high correlation between mucus penetration results of both fresh and frozen bull semen and therefore the authors propose that the MPT, using fresh or frozen semen, may serve as an effective screening method for eliminating the bulls which will produce a lower than 60 % conception rate. Also, Fjallbrant and Ackerman (1969) reported that the reduction in mucus penetration ability of spermatozoa is limited to a degree that might be compatible with fertility, even for storage periods approaching 3 years in liquid nitrogen. So, this technique can be used to study sublethal cryo-damage to spermatozoa and to compare various cryopreservation methods for their ability to reduce this form of damage to the sperm acrosome (Drobnis, et al., 1993).

A decrease in acrosin activity from pre freezing to the frozen thawed stage (19.03 vs 17.85 mg /ml) was clear for control samples and also with inclusion of GBP (17.85 vs 25.83 mg/ml) or BSP (18.55 vs 24.31 mg/ml) in diluted semen at its optimal concentrations (Table 4). A similar tendancy for control samples was observed in ram (Nehring, 1988). Such decline in acrosin activity reported in the present study could be attributed primarily to increased percentages of damaged acrosome post- freezing and thawing.

A negative correlation between intracellular acrosin enzyme activity and acrosomal damage in bucks (Sinha, et al., 1996) were previously reported. Luthra and Georigiev (1997) found that the acrosin activity was higher in the ejaculates of bull more than 50 % motility and they concluded that the differences in acrosin contents may reflect the potential ability of the sperm to fertilize and may be related to various aspects of the underlying physiology of the sperm.

In general, it can be concluded that Zaraibi goat semen can be frozen successfully in Tris-citric acid- fructose -egg yolk extender with inclusion of GBP (80-100ml/ml) or in Sod. Citrate egg yolk glycerol with addition of BSP (80-100ml/ml).

Chauhan, M. S., and Anand, S. R.(1990): Effect of egg yolk lipids on the freezing of goat semen. Theriogenology, 34: 1003.

Chehadeh, R. Y., Ziada, M. S. Seida, A. A. M. and Ghallab, A. M. (2001): Effect of adding some biological fluids on quality of neat and chilled goat semen. Proc. 13^th Annual Congr. Egypt. Soc. Anim. Reorod. And Fert.

Church, K. E., and Graves, C. N.(1976): Loss of acrosin from bovine spermatozoa following cold shock : protective effect of seminal plasma. Cryobiology, 13: 341.

Correa, J. R., and Zavos, P. M.(1994): The hypo-osmotic swelling test: its employment as an assay to evaluate the functional integrity of the frozen-thawed bovine sperm membrane. Theriogenology, 42: 351.

Drobnis, E. Z., Clisham, P. R., Brazil, C. R., Wisner, L. W., Zhong, C. Q., and Overstreet, J. W.(1993): Detection of altered acrosomal physiology of cryopreserved human spermatozoa after sperm residence in the female reproductive tract. J. Reprod. Fertil., 99: 159.

EL- Sheltawy, M. A. (1989): Evaluation of locally processed frozen semen. Ph. D. Faculty Vet. Med., Cairo Univ.

Fjallbrant, B., and Ackerman, D. R.(1969): Cervical mucus penetration in vitro by fresh and frozen-preserved human semen specimens. J. Reprod. Fertil., 20: 515.

Kennedy, W. P., Kaminski, J. M., Van der Ven, H. H., Jeyendran, R. S., Reid, D. S., Blackwell, J., Bielfeld, P. and Zaneveld, L. J. (1989): A simple, clinical assay to evaluate the acrosin activity of human spermatozoa. J. Androl., 10: 221.

Luthra, R. A. and Georigiev, G. H.(1997): Acrosin activity in bull spermatozoa. Ind. Vet. J., 74: 570.

Mack, S. R. and Zaneveld, L. J. D.(1987): Acrosomal enzymes and ultrastructure of unfrozen and cryotreated human spermatozoa. Gamete Res., 18: 375.

Manjunath, P., Soubeyrand, S., Chandonnet, L., and Roberts, K. D.(1994): Major proteins of bovine seminal plasma inhibited phospholipase A2. Biochem. J., 303: 121.

Milovanov, V. K. (1962): Biology of reproduction and artificial insemination of animals (in Russian). Seljhozizdat, Moscow, pp. 696.

Murase, T., and Braun, J. W.(1990): Impact of methodological factors on sperm penetration into cervical mucus in cattle. Theriogenology, 34: 73.

Nehring, H. (1988): Evaluation of acrosome integrity during semen storage. Wiss. Zeitschrift der Humboldt University Zu Berlin. R. Med., 371: 109.

Okuda, K., Murase, T., Sato, K., Matsuzaki, S., Iwano, S., and Iwama, N.(1988): Penetration ability of bull spermatozoa into bovine cervical mucus. In: Proc. 11^th Inter. Cong. Anim. Reprod. A.I., vol. III, No. 279.

Roy, A.(1957): Egg-yolk coagulating enzyme in the semen and Cowper's gland of the goat. Nature(London), 179: 318.

Sinha, M. P., Sinha, A. K., Singh, B. K., and Prasad, R. L.(1996): Effect of cryoprotectants on release of various enzymes from buck spermatozoa during freezing. Theriogenology, 45: 405.

Snedecor, G. W. and Cochran, W. G. (1980): Statistical Methods. 7^th ed. J. B. H. Publishing Comp., Oxford.

Soderquist, L., Bury, N. M., and Martinez, H. R.(1997): Assessment of ram sperm membrane integrity following different thawing procedures. Theriogenology, 48: 1115.

Suttiyotin, P., Thwaites, C. J., Sanchez-Partida, L. G., and Setchell, B. P.(1995): Evaluation of a modified sperm penetration test in ram semen. Theriogenology,44:29.

R.H. FAYED

III Behavioural adaptation

CONCLUSIONS

REFERENCES

E.M. ABDEL GAWAD

ABSTRACT

INTRODUCTION

COURTSHIP AND DISPLAY

A. M. OSMAN

Rate of improvements

Cattle

Cattle

Pigs

Mules

Sheep

GENDER PRESELECTION BY SPERM SEPARATION

G. ABDEL-MALAK

Separation methods of X- and Y-bearing sperm

B – Sperm separation by Immunological Technique (H- Y antigen and Y- bearing sperm) :

Attempts to inactivate the Y sperm by immunological methods have focussed on the H - Y antigen (Goldberg et. 1971).

G.A. MEGAHED* , A.KH. ABDEL-RAZEK* AND H.A. DAGHASH**

INTRODUCTION

MATERIALS AND METHODS

RESULTS

DISCUSSION

REFERENCES

INTRODUCTION

MATERIALS AND METHODS

RESULTS AND DISCUSSION

Thermoregulatory Responses:

Hematological Responses:

Treatment*Breed:

Libido, Reaction Time and Semen Characteristics:

Plasma Vit E and Se Concentrations:

Hormonal Responses:

Figure (3): Plasma Vit E (dl-a tocopherol) and selenium concentrations in rabbit bucks, during post-treatment period.

CONCLUSIONS

REFERENCES

ABSTRACT

RESULTS

Rectal temperature

Control

Pulse rate

Control

SE

DISCUSSION

MATERIAL AND METHODS

RESULTS

__________________Extender__________________

DISCUSSION

REFERENCES

F.I. HELAL*, R.I. ELSHESHTAWY AND W.M. AHMED

ABSTRACT

INTRODUCTION

Season

Dept of Vet. Hygiene and Management , Fac. Vet. Med, Cairo University

ABSTRACT

INTRODUCTION

Materials and Methods

RESULTS AND DISCUSSION

(1) Behavioural response to shearing

Table (1) Behavioural responses of shearing in sheep (Mean ± SE)

Table (3) Haematocrit % ( mean + SE) of sheep as affect by shearing

E.M. ABDEL–GAWAD and O.K. ZAHRAN

Table (1) Behaviour patterns of adult cattle infested with ticks before and after treatment with Diazinone

Table (2) Behaviour patterns of calf cattle infested with ticks before and after treatment with Diazinone

Table (3) Behaviour patterns of adult buffaloes infested with ticks before and after treatment with Diazinone

Table (4) Behaviour patterns of buffalo calves infested with ticks before and after treatment with Diazinone

ABSTRACT

INTRODUCTION

MATERIALS AND METHODS

RESULTS

DISCUSSION

REFERENCES

Animal Production Department, Faculty of Agriculture, Minia University, Egypt

ABSTRACT

INTRODUCTION

MATERIALS AND METHODS

RESULTS AND DISCUSSION

REFERENCES

Extender

Total Unsaturated C16₁-C20₄