Veterinary Practitioner Vol. 13 No. 1 June 2012,88-89 A COMPARATIVE STUDY OF FREEZING TECHNIQUES OF JACK SEMEN R.A.

Legha and Yash Pal Equine Production Campus, National Research Centre on Equines P.B. No.80, Bikaner-334 001, Rajasthan ABSTRACT Only a few superior jacks are available in field for the production of superior mules. The study was planned to compare the two semen freezing techniques for Poitou donkey semen preservation. The semen was collected from four apparently healthy jacks of normal fertility. Four samples of each jack were processed using primary and secondary extenders. Semen filled and sealed straws were divided in four groups and cryopreserved in LN2 vapours for 8, 10 and 12 minutes and using Bio-med planner. The straws were thawed at 37C for 1 min in water bath and evaluated for motility. It was observed that the semen straws cryopreserved in LN2 vapours for a period of 12 minutes and using Bio-med planner had significantly (P<0.05) higher postthaw motility as compared to other two groups where as post-thaw motility was not significantly different in the semen straws cryopreserved by placing in LN2 vapours for a period of 12 minutes and frozen using Bio-med planner indicating that results of both techniques were comparable. The study revealed that the technique of semen freezing in LN2 vapours for a period of 12 minutes may be used successfully for freezing of jack semen under field as well as laboratory conditions. Key words: Semen freezing, jack, donkey Introduction Artificial insemination (AI) using cryopreserved semen is integral part of reproduction. However, under field conditions natural service is generally in practice for mule production. Only a few superior jacks are available in field for the production of superior mules. Due to limited good stock of jacks, they had over burden of service and loss of libido (Pal and Legha, 2008). Programmable bio-freezer is a costly instrument used in equine semen freezing. Hence, its use in field semen freezing is not feasible due to risk of damage during transportation. Simplified semen freezing technique in liquid nitrogen (LN2) vapours will help in introducing the facility to veterinary hospitals as it only requires LN2 which is available there. The present study was planned to compare the two semen freezing techniques in terms of post-thaw semen motility in Poitou donkeys and suggest suitable technique of jack semen freezing for laboratory and field conditions. Materials and Methods Semen was collected from four apparently healthy adult Poitou donkeys (four collections each) of normal fertility maintained at Equine Production Campus, Bikaner in the morning hours before feeding between 7 and 8 A.M., semen was collected by artificial vagina (Colorado model) equipped with a disposable liner as per the standard method. The jacks were kept at distance for visual stimuli to get proper erection before mounting for ejaculation in the AV. An oestrus jennet was used as a dummy. Soon after the semen collection, the semen was passed through sterilized gauze filter to remove the gel and microscopic and macroscopic analysis was 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 0.15 g, sodium citrate dehydrate 2.6 g, disodium EDTA 0.37 g, sodium bicarbonate 0.12 g, streptomycin 0.10 g, benzyl penicillin 0.10 g, made up to 100 ml with double distilled water) as per the method described by Cochran et al. (1984) in the ratio of 1:1 and centrifuged at 2000 rpm for 4-5 min

at 8-10C. The supernatant was aspirated off and the sperm pellet was dissolved in the modified secondary extender (mixture of two solutions i.e. 25 ml from solution 1 + 50 ml from solution 2 with 20 ml egg yolk centrifuged at 3000 rpm at 10C for 30 minutes to collect clear supernatant fluid and added glycerol 3% of the total volume). Solution 1 contains Glucose EDTA (Glucose 6 g, sodium citrate dehydrate 0.37 g, disodium EDTA 0.37 g, sodium bicarbonate 0.12 g, streptomycin 0.08 g, benzyl pencillin 0.08 g, made up to 100 ml with double distilled water. Solution 2 contains lactose 11 g, streptomycin 0.08 g, benzyl pencillin 0.08 g, made up to 100 ml with double distilled water. The diluted semen was kept in semen cooling cabinet at 5C for 2 hour as equilibration time. The equilibrated semen was filled in the polyvinyl chloride (PVC) straws of 0.5 ml capacity (IMV make) and sealed with filling-sealing machine. The semen filled straws were divided in four groups and put on LN2 vapours for 8, 10 and 12 minutes and plunged into liquid nitrogen. One group of filled straws was cryopreserved in programmable biofreezer. The straws in Bio-med planner were cooled at a rate of - 0.3C/min from 18C to 5C, and frozen at a rate of - 10C/min from +5C to -15C and at a rate of -19C/ min from -15C to -100C. After reaching -100C, the straws were finally plunged into LN2 (-196C) and kept stored in LN2 till further use for AI. The other three groups of straws were placed for 8, 10 and 12 minutes in LN2 vapours. In this technique, the straws were laid horizontally onto a freezing rack and lowered into a styrofoam box that contains at least one inch of liquid nitrogen. The freezing rack is designed to support the straws three cm above the liquid nitrogen. After being held in that position for 8- 12 minutes, the straws were then plunged into liquid N2 and stored at -196C (Leipold et al., 1998). During vapour freezing, the cooling rate at the bottom of the straw is generally much faster than at the top and to avoid variation in temperature the position of straws are turned around. After one day, two cryopreserved straws from each group were thawed at 37C for 1 min in water bath, immediately wiped off, cut open and emptied into a clean pre-warmed vial (370C), which was maintained at 370C and evaluated for post-thaw motility. The semen was also tested for the presence of pathogens before and after freezing using standard methods. The data was analyzed by standard statistical methods (Snedecor and Cochran, 1994). Results and Discussion The semen was analyzed for physical and morphological characteristics before processing for freezing. The colour of stallion semen was milky white to creamy. Consistency of the semen was observed as thick to thin. Pre-freezing motility of the semen was 78.0}2.5%. Post-thaw semen motility was observed as 22.5}1.21 (range 15- 30%), 40.31}2.16 (range 20-50%) and 53.73}2.26 (range 35-65%) for the straws placed for 8, 10 and 12 minutes in LN2 vapours, respectively, where as postthaw motility of the straws frozen using Bio-med planner was recorded as 54.38}1.28 (range 45- 60%). It was also observed that the semen straws cryopreserved in LN2 vapours for a period of 12 minutes and using Bio-med planner had significantly (P<0.05) higher post-thaw motility as compared to other two groups where as postthaw motility was not significantly different in the semen straws placed in LN2 vapours for a period of 12 minutes and frozen using programmable planner indicating that results of both techniques are comparable and the technique of semen freezing in LN2 vapours for a period of 12 minutes may be used successfully for freezing of stallion semen under field as well as laboratory conditions. However, wide range in post thaw motility was observed in LN2 group than programmable freezer. The study revealed that the technique of semen freezing in LN2 vapours for a period of 12 minutes may be used successfully for freezing of jack semen under field as well as laboratory conditions.

References Cochran, J. D. et al. (1984) Theriogenology. 22: 25-38. Leipold et al. (1998) Theriogenology. 49: 1537-43. Pal, Y. and Legha, R. A. (2008) Indian J. Anim. Sci. 78 (11):1281-1284. Snedecor, G.W. and Cochran, W.C. (1994) Statistical Methods. 8th ed. Oxford and IBH Publishing Co. New Delhi.