STORAGEABILITY AND SURVIVAL  OF FRENCH ALPINE GOAT SPERMATOZOA AS AFFECTED BY TYPE OF EXTENDER AND SUGAR.

Abd Elhakeam, A. A¹ And Zenat B. Rabie²

¹Department of Animal Production, Faculty of Agriculture, Minia University, Egypt. ²Animal Production Research Institute, El-Doki, Egypt.

ABSTRACT

French Alpine goat semen was collected and diluted  at 1:10 (v/v) semen to extender. Three basic buffers (Tris-citric acid, Na-citrate and Na-chloride) and ten sugars (Fructose, Manose, Maltose, Lactose, Sucrose, Trehalose, Rafinose, Erythritol, Inositol and Sorbitol) were used to prepare 30 extenders (3 buffers x 10 sugar solutions) at 300 mOsm osmotic pressure and 7.0 pH. Each  extender contained 10% (v/v) sugar solutions, 10% (v/v) fresh egg yolk and 4% (v/v) glycerol . Diluted semen samples (30) were cooled slowly to 5ºC within 2 hr , then packaged into 0.25 cc plastic straws and left for another 2 hr before freezing. Straws were mounted horizontally in a freezing boat at 7 cm above liquid nitrogen, which were exposed to liquid nitrogen vapor for 10 min. before plunging directly into liquid nitrogen and stored for 24 hr before thawing in water bath at 37-40 ºC for 15 sec. for analysis.

   Type of extender significantly affected the percent progressive motility of both unfrozen and frozen-thawed French Alpine goat spermatozoa. This effect was more pronounced  with longer storage period (4 hr and above) of liquid semen and with freezing and thawing. Tris-citric acid was superior for unfrozen and frozen goat semen followed by Na-citrate, while Na-chloride was the worst. Although, the differences among sugars were not significant during liquid preservation and freezing, fructose and erythritol maintained the highest motility at 4 and 24 hr storage at 5C, while trehalose was the best for freezing, regardless to type of extender. Generally the highest survival rate (50%)  post-thawing was obtained when French Alpine goat semen was diluted in Tris-citric acid containing trehalose sugar.

INTRODUCTION

            Preservation of genetically superior male germ plasm is very important and useful for improvement of animal productivity resulted from improvement of genetic make up of individuals. Successful preservation of superior male germ plasm and by using artificial insemination  will give the chance for future recalling even in the absence of those males.

            Cryopreservation of genetically superior imported male germ plasm gives the potential for overcome  the problems of adaptation and susciptability to local diseases; at the same time it enhances the chance of long term programs for genetic improvement and subsequently increasing of the productive and reproductive  performance of local Egyptian goats. Meanwhile, it has been reported that type of extender used for semen dilution and  processing is considered one of  the important factors which affect  the successful preservation of goat spermatozoa (Memon and Ott ,1981; Salamon and Ritar, 1982;  Perez and Mateos, 1995; Muren et al., 1997). In addition, type of sugar added to the extender plays an important role in the survival rate of unfrozen and frozen goat spermatozoa (Salamon and Ritar, 1982; Muren et al., 1997).

            French Alpine goats were imported in order to improve productivity of local Egyptian goats, since they have the genetic potential of higher milk production, higher growth and twinning rates (Ensminger and Parker, 1986). Therefore, the objectives of this study  were to investigate the effects  of three different buffers and ten different sugars in an attempt  to develop an extender to be successfully used  for the preservation  and cryopreservation of  French Alpine goat spermatozoa,

MATERIALS AND METHODS

This is research was conducted at the animal research farm, Faculty of Agriculture, Minia University.

Semen collection:

            Two French Alpine goat bucks were used throughout the study. Semen was collected by using artificial vagina. Two to three ejaculates were collected from each buck to obtain certain volume (3 ml) of pooled fresh semen. Semen was collected in the same collecting tube and mixed thoroughly before dilution. Collected semen was evaluated for percent  progressive motility (to be more than 70% for using) and sperm concentration/ml (3 billion).

Extenders preparation:

            Three basic buffer solutions were prepared (tris-citric acid, sodium citrate and sodium chloride) at 300mOsm/kg osmotic pressure (measured by  vapor pressure osmometer) and pH 7.0. Each extender contained 4.0% (v/v) glycerol and 10% (v/v) fresh egg yolk. Ten sugar solutions were prepared at the same osmotic pressure (300 mOsm) and each sugar solution was added to each extender at 10% (v/v) resulting in 30 diluents (3 buffers x 10 sugars). Extenders were centrifuged at 5000 r.p.m. for 10 min. and the supernatants were decanted and used for semen dilution.

Semen dilution and processing:

            Pooled French Alpine goat semen was mixed thoroughly, then diluted at 1:10 (v/v, semen to extender) dilution rate. Dilution was made at room temperature soon after collection. Diluted semen samples (30) were cooled slowly to 5ºC within 2 hr, then packaged into 0.25 cc plastic straws (3 straws/treatment). Straws were sealed using polyvinyl ethylene powder and were transferred to a water bath at same  temperature and left for another 2 hr before freezing. Straws were frozen horizontally  in the freezing boat (Abd Elhakeam, 1988) at 7cm above liquid nitrogen surface (after 4 hr from transferring to the refrigerator) , where they were exposed to liquid nitrogen vapor for 10 min, then they were plunged directly into liquid nitrogen and stored for 24 hr before thawing and analysis.

Semen evaluation:

            A total of 270 straws were frozen (30 extenders x 3 straws x 3 replicates). Straws were thawed in water bath at 37-40 ºC for 15 sec. and evaluated for percentage progressive motility immediately (0.0 hr) post-thawing and after 1 hr at room temperature post-thawing. Also, percentages of progressive motility of unfrozen semen stored at 5 ºC were recorded for 0.0 hr (immediately post dilution), 4 hr (pre-freezing) and 24 hr (stored at 5 ºC). Motility was estimated by using a microscope (450x)  with closed circuit  T.V. camera and color monitor. The experiment was repeated three times. Data were subjected to analysis of variance to obtain the main effects using GLM in SAS (1994). Differences among means were tested using Duncan’s New Multiple Range test ( Duncan, 1955).

RESULTS AND DISCUSSION

I-Unfrozen semen:

            The results (Tables 1,2 and 3) indicated that type of extender significantly affected the progressive motility of French Alpine goat spermatozoa   stored at 5 ºC for 0.0 hr (immediately  after dilution), 4 hr (pre-freezing) and 24 hr . Both Tris-citric acid and Na-citrate extenders were superior to Na-chloride extender, especially with longer storage time (> 4 hr) as shown in Table (1).

It was observed that, although Na-chloride extender and Na-citrate maintained the highest sperm-cell motility immediately after dilution compared to Tris-citric acid extender, however the later was superior at longer storage period (24 hr). Na-chloride extender was not a good buffer for storageability of liquid French Alpine goat spermatozoa, especially with longer storage time (24.6% at 4 hr and 0.0% progressive motility at 24 hr storage period at 5 ºC).

Meanwhile, type of sugar did not significantly affect storageability of French Alpine goat spermatozoa either at 0.0 hr post-dilution or at 24 hr storage periods at 5 ºC. However fructose sugar maintained the highest (p<0.05) value (57.2%) of progressive motility after 4 hr at 5 ºC (pre-freezing) followed by sorbitol  (55.6%) compared with the other eight sugars studied (Table, 1).

Table (1): Overall mean of percentages of progressive motility of unfrozen and frozen-thawed French Alpine goat spermatozoa as affected by  type of extender and sugars.

a,b,c Means in the same column under the same factor followed by different letters are significantly different .

* p<0.05            ** p<0.01            NS= Not Significant.

There was significant interaction at 0.0 and 4 hr storage period at 5 ºC between type of  extender and sugar. It was observed that trehalose and rafinose maintained the highest  motility with Tris-citric acid, while manose was the best with Na-citrate and fructose was the best with Na-chloride extender (Table, 2).

Table (2): Progressive motility (%) of unfrozen French Alpine goat spermatozoa stored for 4 hr  (pre-freezing) at 5 ºC as affected by type of sugar and extender.

a-g Means followed by the same letters vertically or horizontally are not significantly different (p>0.05)..

Generally, the highest progressive motility (70.0%) of French Alpine goat spermatozoa at 4hr storage period at 5 C was obtained when semen was diluted in Tris-citric acid contained either trehalose or rafinose sugars or when diluted with Na-citrate extender containing manose sugar. At 24 hr storage time at 5 ºC the highest progressive motility (46.7%) was obtained with Tris-citric acid extender contained erythritol sugar (Table 3).

Table (3): Progressive motility (%) of unfrozen (liquid) French Alpine goat spermatozoa stored for 24hr at 5ºC as affected by type of sugars and extenders.

a-g Means followed by the same letters vertically or horizontally are not significantly different (p>0.05).

II-Frozen-thawed:

            The results (Tables, 1, 4 and 5) indicated that regardless to the type of sugar, post-thaw progressive motility of French Alpine goat spermatozoa was also significantly influenced by type of extender. Tris-citric acid extender was superior (41.9%) to Na-citrate (36.9%), while Na-chloride yielded the lowest progressive motility (7.4%) as shown in Table (1). The same trend was observed after 1 hr storage at room temperature post-thawing.

            Type of sugar did not significantly affect post-thaw progressive motility, however trehalose sugar yielded the highest motility (33.1%) compared to the other nine sugars studied, regardless  to the type of extender (Table, 1). At the same time the differences in progressive motility between this sugar (trehalose) and the common used sugar (fructose, 28.9%) was not significant . Meanwhile, with longer time post-thawing (1 hr) monosacharide  sugars (fructose and manose) maintained higher motility values (22.6, 22.9%) compared with disacharide sugars (maltose, lactose, sucrose and trehalose) which yielded 19.3 to 20.5% and trisacharide one (rafinose),

 which gave 20.9% as well as sugars alchohol (erythritol, inositol and sorbitol) which yielded 20.4 to 21.5% progressive motility (Table 1).

Table (4): Effect of type of sugars and extenders on the percentage progressive motility of frozen-thawed French Alpine goat spermatozoa (immediately post-thawing).

a-d Means followed by the same letters vertically or horizontally are not significantly different (p>0.05).

            The interaction  between type of extender and sugar was not significant. Generally, the highest post-thaw motility (50.0%) was obtained when French Alpine goat semen was extended in tris-citric acid extender containing trehalose or rafinose sugars, followed by inositol (47.2%) and lactose (45.0%) as shown in Table (4). The same trend was observed at 1 hr storage at room temperature post-thawing (Table, 5).

Table (5): Effect of type of sugars and extenders on the percentage progressive motility of frozen-thawed French Alpine goat spermatozoa (1.0 hr at room temp. post-thawing).

 a-i Means followed by the same letters vertically or horizontally are not significantly different (p>0.05).

            From the present results it was observed that type of buffer used in diluting French Alpine goat semen is an important factor for successful preservation and cryopreservation of buck spermatozoa either at 5 ºC in the refrigerator or frozen at –196 ºC in the liquid nitrogen. Tris-citric acid was superior compared with sodium citrate and  sodium chloride, but the later was the worst especially with longer storage period (> 4 hr at 5 ºC) or when freezing buck semen. Many investigators reported that type of extender plays an important role in the successful preservation of semen (Abd Elhakeam, 2000; Abd Elhakeam et al., 1991; Memon and Ott, 1981; Salamon and Ritar, 1982; Muren et al., 1997). Also, Deshpande and Mehta, (1991), Mareco and Arostequy (1995) and  Perez and Mateos (1995) reported that  the use of Tris-yolk  diluent resulted in significantly higher sperm motility 2-6 hr after thawing and a higher overall semen quality than the other two diluents (milk with 3-6% yolk or milk with 7% glycerol). Tuli and Holtz (1992) reported that goat semen extended in Tris had significantly higher percentages of progressive motility and live spermatozoa than those extended in TEST, TEX and BES buffers. Moreover, Duta et al. (1996) diluted goat semen with Tris or egg yolk-citrate, they found that the highest percentage of motile and live spermatozoa in pre-freezing and post-thawing conditions was in Tris diluent. Furthermore, Choudhury et al. (1987) reported that the non return rate was 78.95% and 88.46% in females inseminated with semen frozen in Tris-egg yolk-citric acid-fructose-glycerol or egg yolk-citrate-fructose-glycerol diluents, respectively, although the percentage of motile spermatozoa after freezing was 64 and 61.1% (p<0.05) in the two diluents, respectively. At the same time Mareco and Arosteguy (1995) found that goats inseminated with semen diluted in Tris-glucose-citric acid –glycerol with  2-5% egg yolk and packaged into 0.25 ml French straws and thawed at 37 C in water bath, gave healthy  offspring.

Factors known to influence the degree of cryoprotection when sugars are used include their molecular weight (Nagase et al., 19964). Generally, the cryoprotective effects of sugars increase as the molecular weight of sugar is increased. Unal et al. (1978) reported that fructose (monosaccharide) was not beneficial to post-thaw motility in the absence of glycerol compared with lactose (disccharide) and raffinose (trisaccharide). Also, Marinov et al. (1980) and Abd Elhakeam et al. (1991) reported that sucrose and maltose added to the extender cryoprotected acrosome integrity and the motile apparatus of frozen spermatozoa. Furthermore, Molina et al. (1994) examined the cryoprotective effects of monosaccharides (fructose, glucose) and disaccharides (lactose, sucrose or trehalose) in Tris-egg yolk-glycerol based diluents on the post-thawing motility, acrosome integrity and fertility of frozen semen. They found that the recovery of spermatozoa was better (P<0.01) in the presence of sucrose and trehalose than in the presence of glucose. Also, Muren et al. (1997) reported that the addition of sucrose to egg yolk glycerol-citrate diluent improved post-thawing survival of spermatozoa, but albumin and Zn had only a small improving effect. It seems likely that the cryoprotective ability of sugar depends also, upon the type of buffer system used (Abd Elhakeam, 1988) and the storage temperature of semen (Glover and Watson, 1987). It could be concluded that French Alpine goat semen could be diluted in Tris-citric acid extender containing 10% (v/v) trehalose sugar solution for successful cryopreservation.

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