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Indian Veterinary Journal (2012). 89 (2): 54-55

Stallion semen freezing and thawing protocols.
Yash Pal*and R. A. Legha Equine Production Campus, National Research Centre on Equines, Post Bag. No. 60, Bikaner 334 001 Rajasthan

Abstract Glycerol is the first penetrating cryoprotectant identified which enter the cell within 1 min of addition to the surrounding medium (Polge et al., 1949) Concentration of glycerol and equilibration time affects the post-thaw motility (PTM) of frozen semen. Researchers have used wide range of glycerol concentration (2.5 to 10%) and equilibration time (1 to 5 hours) in various studies. Piao and Wang (1988) suggested that 6% glycerol was the most beneficial in freezing of stallion semen. While Pace and Sullivan (1975) demonstrated that reducing glycerol from 7 to 2% was advantageous. Thawing of frozen semen is also one of the important factors affecting the PTM of spermatozoa, which further plays a major role in fertility. The study was carried out to optimize glycerol concentration and equilibration time for freezing of stallion semen as well as thawing protocols.

Materials and Methods Semen was collected from four adult, apparently healthy Marwari stallions using Colorado model of artificial vagina (AV) equipped with a disposable liner as per the standard method at four different occasions. The stallions were kept at distance for visual stimuli to get proper erection before mounting for ejaculation in the AV. An estrus mare was used as dummy. Soon after the semen collection, the semen was passed through sterilized gauze filter to remove the gel. The microscopic and macroscopic analyses were performed. The semen having progressive motility of more than 60% was processed for cryopreservation. Gel free semen was mixed with modified Glucose-EDTA primary extender (Glucose

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Corresponding author email: yashpal@scientist.com

0.15g, Sodium citrate dehydrate 2.6g, Disodium EDTA 0.37g, Sodium bicarbonate 0.12g, Streptomycin 0.10g, Benzyl penicillin 0.10g, made up to 100ml with double distilled water) as per the method described by Cochran et al. (1984) in the ratio of 1:1. Each semen sample was divided in two parts after mixing with primary extender and centrifuged at 2000 rpm for 4-5 min at 8-10C. The supernatant was aspirated off and the sperm pellet was dissolved in the modified secondary extender (mixture of two solutions i.e. 25ml from Solution 1 + 50ml from Solution 2 with 20ml egg yolk centrifuged at 3000rpm at 10C for 30 minutes to collect clear supernatant fluid and added glycerol 3% and 5% of the total volume). Solution 1 contains Glucose EDTA (Glucose 6g, Sodium citrate dehydrate 0.37g, Disodium EDTA 0.37g, Sodium bicarbonate 0.12g, Streptomycin 0.08g, Benzyl pencillin 0.08g, made up to 100ml with double distilled water. Solution 2 contains lactose 11g, Streptomycin 0.08g, Benzyl pencillin 0.08g, made up to 100ml with double distilled water. After filling the diluted semen in 0.5 ml French straws containing glycerol 3% and 5%, half of the straws of each group were given equilibration time of 1hour and the other half for 3 hours. The semen-filled straws were frozen in the programmable Bio-med Planner and plunged into liquid nitrogen (LN2), and kept stored in LN2 as described by Pal et al. (2011). Frozen straws filled with freezing media containing 3% glycerol were thawed at 37°C for 30 seconds, 370C for one minute and 450C for 15 seconds in water bath and emptied into a clean vial maintained at 370C and evaluated for post-thaw motility. The data was analyzed by standard statistical methods (Snedecor and Cochran, 1989). Results and Discussion Stallion semen is very less tolerant to the freezing and thawing processes than bull semen. In order to get maximum reproductive efficiency from the use of frozen stallion semen, it is essential that the semen be frozen, stored and thawed correctly. In the study, PTM was 41.25±3.15 and 36.25±4.73 per cent for semen cryopreserved with freezing media containing 3% and 5% glycerol with 1hour equilibration time, respectively, whereas it was 40.00±2.04 and 33.75±2.39 per cent for semen cryopreserved with freezing media containing 3% and 5% glycerol with 3hour equilibration time, respectively. Freezing media

3 containing 3% glycerol was observed to be significantly (P<0.05) superior than that containing 5% glycerol based on PTM, whereas equilibration time had no significant effect on stallion semen freezability. Pace and Sullivan (1975) also demonstrated that reducing glycerol from 7% to 2% was advantageous for horse semen freezing. The results indicated that equillibration time had no significant effect on semen freezability and 1hour equillibration time is sufficient to get optimum PTM. In similar type of study, Nagase (1967) obtained optimum effect of glycerol with equilibration time of 3-5 hours. Thawing of frozen straws is also one of the important factors affecting the post-thaw motility of spermatozoa. The cryopreserved straws were thawed in water at 370C for 30 seconds, at 370C for one minute and at 450C for 15 seconds. The PTM was observed as 40.00±1.86, 34.44±1.54 and 46.67±2.35%, respectively during the above three thawing protocols. PTM of semen straws thawed in water at 450C for 15 seconds was significantly (P<0.05) higher than the other two methods of thawing. Hence, faster thawing rate (450C for 15 seconds) was observed to be superior for obtaining good PTM in frozen stallion spermatozoa. Cochran et al. (1984) also demonstrated that immersion of 0.5ml straws in a water bath at 750C for 7 seconds followed by immediate immersion in a water bath at 370C for longer than 5 seconds, resulted in better PTM than immersion in a 370C water bath for 30 seconds. Based on results obtained in the study, it is evident that PTM of semen frozen using media containing 3% glycerol was observed to be superior to the media containing 5% glycerol. The equilibration time had no significant effect on stallion semen freezability. It was further recorded that faster thawing rate (450C for 15 seconds) was found superior to obtain maximum PTM.

Summary The aim of the present study was to optimize glycerol concentration (3 or 5%) and equilibration time (1 or 3 h) for freezing of stallion semen in 0.5ml straws and to evaluate three thawing rates (37 °C for 30 s, 37 °C for 60 s and 45 °C for 15 s). Freezing media containing 3% glycerol was observed to be superior than 5% glycerol media based on post-thaw motility, whereas equilibration time had no significant effect on

4 stallion semen freezability. It was also recorded that faster thawing rate (45 0C for 15 seconds) was found superior for obtaining maximum post-thaw motility. References Cochran, J. D., Amann, R. P., Froman, D. P. and Pickett, B. W. (1984)…Theriogenology 22: 25. Nagase, H. (1967). ..Japenese J. Anim. Reprod. 12: 52. Pace, M. M. and Sullivan, J. J. (1975)…J.Reprod. Fert., Suppl 23: 115. Pal, Y., Arangasamy, A., Legha, R. A,, Singh, J., Bansal, R. S., Khurana, S. K. and Tandon, S. N. (2011)… Indian J. Anim. Sci.: 81 (5): 445. Piao, S. and Wang, Y. (1988)…In Proceedings of the International Congress for Animal Reproduction and Artificial Insemination, 3, pp 286a. Polge, C., Smith, A.U. and Parkes, A. S. (1949)…Nature 164: 666 Snedecor, G. W. and Cochran, W. G. (1989). Statistical Methods. 8th edn. Iowa State University Press, London.