|Thirteenth Annual Conference|
ADAPTATION OF THE CAMEL TO DESERT ENVIRONMENT
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 .
1- Anatomical adaptation
a- Head and neck
¨ 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
¨ ( selective feeding ) and avoid the thorny plants .
¨ The camel has a long arched neck helping him to manipulate the high tree plants and to explore the enemy from long distances .
b- Trunk and tail
¨ 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 .
2- Physiological adaptation
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)
2- Energy balance
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 .
3- Water balance
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.
3- Milk and milk quality .
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
· Ingestive behaviour
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 .
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 “
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. 1st edition . Bailliere Tindall , london .
Higgins ,A (1986): The camel in Health and Disease .1st Edition . Bailliere Tindall , London , Tokyo , Sydney.
Nielsen , K.S (1979): Desert Animals . physiological problems of heat and water . 2nd Edition Dover publications , Inc , Newyork , USA .
Wilson ,R.T (1984): The camel . 1st Edition , longman group limited . Burnt Mill , Harlow Essex ,UK .
Yagil , R. (1985): Compartive physiological adaptation . 1st 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 .
Yousri , R.M (1976): World Review of Animal Reproduction . XII (4) : 75
SEXUAL BEHVIOUR FROM FISH TO MAN
E.M. ABDEL GAWAD
Dept. of Hygiene and Husbandry. Faculty of Veterinary Medicine, Cairo University, Giza
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.
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
( Grzimek, 1972 ).
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».
The Functions Of Courtship
1- Advertisement : -
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.
2- Overcoming Of Aggression : -
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)
3- Physiological co–ordination of male and female :-
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) .
4- Reproductive isolation : -
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).
5- Prevent Pridation Risk
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.
1- Sexual behavior in Fish
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.
2. Sexual behavior in Fiddler–Crab :
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.
3- Sexual behavior in Frogs : -
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.
4- Sexual behavior in lizards : -
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
5- Sexual behavior in Queen butterfly
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).
6- Sexual behavior in birds : -
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.
-The marriage market of the mallard.
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.
6- Sexual behavior in mammals : -
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.
6-1 Primates : -
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
6-3 Rodents : -
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
1- Monogamy :-
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 .
2- polygamy :
polygamy is much more common among mammals. An individual generally has two or more mates either successfully or simultaneously.
3- polyandry: -
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.
4- Promiscuous : -
|There are no pair bends, and both males and females mate with more than one member of the opposite sex
Bliss. D.E (1990): Shrimps, lobsters and Crabs. Columbia University press. New York
Brower,L.p.,Brower,J.V.and Cranston,F.P.(1965): Courtship behaviour of the Queen butterfly Danaus gilippus berenice (Cramer). Zoological, 50, 1-40
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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 17th 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.
Role of veterinarians and recent technology in cattle improvements :
· 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.
· Tools for farm improving :
1. Recording system through computer-based data.
2. Sonar as a diagnostic procedure.
3. Commercial hormones and drugs used to shorten the calving intervals and improve production (GnRH, PGF 2alva , L.H.,FSH…etc.).
· Technology of breeding included the following:
1. A.I. ( Liquid then Frozen semen).
2. Embryo transfer (Superovulation, fertilization, synchronization, embryo collection, sexing,embryo evaluation and preservation).
3. Gene transplantation.
Economic value of gynecological care in dairy farm :
· 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)
Reference : UN and FAO Report (1999)
Population Rate Growth
Numbers of livestock in egypt and their rate of development
FAO Report (2000)
General Information : ( FA),1993) :
· The largest numbers of cattle and buffalo were in India (198.4 and 77.0 million respectively)
· The largest number of sheep was in Australia (162.8 million)
Japan has no sheep
Syria has 15.3 million
Egypt has 4.9 million
· The largest number of farm animals per human population unit was in Newzealand.
· Animals in the world (FAO 1999)
GENDER PRESELECTION BY SPERM SEPARATION
Head of AI & ET Research Department
Animal Reproduction Research Institute
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 in farm animals :
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.
Gender preselection in human:
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.
Basis for X and Y sperm separation:
All manipulation of sperm separation takes place prior to fertilization.
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.
Table – 1 : Flow cytometric analysis of X- and Y-bearing
sperm for DNA difference.
( Johnson and Clarke 1990 ) .
Separation methods of X- and Y-bearing sperm
A - Physical:
Many attempts were used to separate 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.
However, there has been no confirmation of these results by others.
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.
C – Sperm separation by using markers: Long arm of Y chromosome; DNA:
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
D – Sperm separation by “ Flow – Cytometer / Cell –Sorter:
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 %.
Validation of the proportions of X and Y sperm by DNA analysis :
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 percent separation of the two populations is calculated by the difference:
100 [ X-Y / 0.5 ] ( X + Y ) =
where X and Y is the respective channel means for the two peaks .
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.
Sperm sort validation in the laboratory
A – Reanalysis of sorted sperm for DNA content :
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 .
Fertility using Flow-sorted X and Y sperm
Factors affecting the fertility of sperm sorting ( Table 2 )
1 – Uniformity of staining ,
2 - Orientation of the sperm head to the laser beam , and
3 – maintaining conditions to ensure the viability of the sperm.
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 .
Litters pigs resulting females averaged 74 % while males averaged 68% .
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).
Flow cytometric sorting of Human sperm
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 .
Ali, J. L., Edridge, F. E., Koo, G. C. Schanbacher, B. D. (1990) – Enrichment of bovine X and Y chromosome –bearing sperm with monoclonal H-Y antibody-fluorescence-activated cell sorter. Arch. Androl. 24: 235 –245.
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Table – 2 :Fertility data . Progeny produced from insemination with sexed semen ; IVF with sexed semen to produce sexed embryos for transfer .
Treatment No. No % %
Insem ./ No. offspring Male Female
Sorted Y 16 / 5 21 81 19
Sorted X 14 / 3 16 6 94
Recombined 17 / 5 50 43 57
Sorted Y 9 / 5 45 75 25
Sorted X 10 / 5 32 20 80
Unsorted / stain 11 / 5 40 43 57
Unsorted / unstain 7 / 5 46 42 48
In Vitro Fertilized 2 10 0 100
Sorted for X 4 / 2 2 0 100
Sorted for Y 5 / 2 3 100 0
*Johnson et al . , 1989
**Johnson et al . , 1991
*** Cran et al . , 1993 , 1995
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Cran, D. G., Johnson, L. A., Polge, and C. (1995) – Sex preselection in cattle: A field trial. Vet. Rec. 136: 495 – 496.
Eichwald, E. J. and Silmer, C. R. (1955) – Untitled communication. Transplant Bull, 2: 148 – 149.
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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**
* Dept. of Theriogenology, Faculty of Vet. Med.,
** Dept. of Animal Production, Faculty of Agriculture, Assiut University
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 7th 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.
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
Animals and treatment:
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 7th 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 and pregnancy diagnosis:
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.
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.
§ 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.
-a-b (P < 0.05) , a-c-b (P < 0.01).
-Means in the same column with the same superscript number are not significantly different.
-Significance = 0.05.
* P < 0.01 versus control pregnant animals.
N.S non-significance versus control non-pregnant animals.
Table 2 : Subsequent fertility.
* Nine animals exhibited behavioural heat after insemination.
** Five animals exhibited behavioural heat after insemination.
§Six animals exhibited behavioural heat at 40 after insemination.
©Four animals exhibited behavioural heat at 35 and 40 days after insemination.
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
Z. SAMIA MESHREKY AND H. E. ABBAS*
Animal Production Research Institute, Agricultural Research Centre, Egypt
*Animal Health Research Institute, Agricultural Research Centre, Egypt
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.
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, Ł0.03% 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 SASŇ program (1988). The significance of differences among means was evaluated by Duncan’s New Multiple Range Test.
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.
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.
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).
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)
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 şC) 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).
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
* =(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).
Triiodothyronine concentration (T3 ):
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 T3 level (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)
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 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).
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
E. B. ABDALLA
Department of Animal Production, Faculty of Agriculture, Ain Shams University,
Shobra El-Kheima 11241, Cairo, Egypt
The objective of the present study was to investigate the influence of prostaglandin F2µ (PGF2µ) 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 PGF2µ / 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 PGF2µ 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 , PGF2µ 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-PGF2µ injection , compared with the pre-injection basal level . However, PGF2µ 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 PGF2µ 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 PGF2µ and PGE2. Prostaglandin F2µ 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 PGF2µ 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 PGF2µ 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 PGF2µ induced a hyperthermic effect in rabbits. The effect of PGF2µ on the thermoregulatory mechanisms in buffaloes has not been investigated. Therefore, the objective of this study was to determine the effect of exogenous PGF2µ on the body temperature of buffalo-cows. The possible effect of PGF2µ on the cortisol concentration was also evaluated.
MATERIALS AND METHODS
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 PGF2µ (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).
Average rectal (RT), vaginal (VT), and skin (ST) temperatures gradually increased in response to PGF2µ 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 PGF2µ - treated group than the control at 12 hours post-injection . Prostaglandin F2µ 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 PGF2µ - injected buffaloes had greater (P>0.05) PR than control animals at 15 and 30 minutes post-injection. Moreover, PGF2µ administration increased (P>0.01) respiration rate (RR) after one hour from injection as compared to pre-injection basal level.
On the other hand, PGF2µ 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 PGF2µ - injected group and remained lower until late evening.
Table (1): Effect of PGF2µ administration on rectal, vaginal and skin
temperatures (°C) in suckling buffalo-cows
0 = Time just before PGF2 µ injection.
* Significant at P>0.01 compared with the basal pre-injection level within the same group.
+ Significant at P>0.05 compared with the control group at the same time.
Table (2): Effect of PGF2µ administration on pulse rate (pulses / min),
respiration rate (breaths / min) and plasma cortisol
concentration (ng / ml) in suckling buffalo-cows
0 = Time just before PGF2 µ injection.
* Significant at P>0.01 compared with the basal pre-injection level within the same group.
+ Significant at P>0.05 compared with the control group at the same time.
Administration of PGF2µ 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 PGF2µ - injected mares. One of the most important side effects of exogenous PGF2µ 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 PGF2µ injection, probably to decrease body temperature through radiation and respiratory evaporative cooling mechanisms. However, the elevated PR and RR in response to PGF2µ 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 PGF2µ 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 PGF2µ 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 PGF2µ- induced hyperthermia than turkeys.
In the present study, PGF2µ 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 PGF2µ 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 PGF2µ were mediated through activation of the protein kinase C pathway (Niswender et al., 1994). Prostaglandin F2µ 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 PGF2µ and its metabolites on hypothalamic CRF and pituitary ACTH is not fully clear. In conclusion , PGF2µ administration influenced thermoregulatory responses and reduced plasma cortisol concentration of suckling buffaloes. The author speculates that the administration of PGF2µ 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
A. KH. ABDEL-RAZEK*, M. GESHI2 AND M. SHIMIZU**
*Dept. Theriogenology, Fac. of Vet. Med., Assiut University, Assiut, Egypt
**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 Collection and Evaluation:
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 106 sp./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 50x10 6 sperm /ml and stored in 4 oC up to 14 days.
Percoll Gradient Separation:
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 50x10 6 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 38.5oC 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.
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
Table (1): Motility Percentage of stored spermatozoa in EYC, Washed
(EFE-W) and Percoll (EFE-P)
-0* is the first day of examination
-Data represents the means±SEM of replicates
-ab Means within the same row with different superscripts are different (p< 0.05)
Table(2): Alive Sperm Percentage of stored spermatozoa in EYC, Washed
(EFE-W) and Percoll (EFE-P)
-0* is the first day of examination
-Data represents the means±SEM of replicates
ab Means within the same row with different superscripts are different (p< 0.05)
Table (3): Intact Acrosome Percentage of stored spermatozoa in EYC,
Washed (EFE-W) and Percoll (EFE-P)
-0* is the first day of examination
-Data represents the means±SEM of replicates
-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
-Numbers within the same row with different superscripts are different (p<0.05)
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.
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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.
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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. 5th world buffalo cong. Royal Palace, Italy p 833-35.
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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.
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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
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 /H 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 .