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Cryopreservation of Jacks Semen and Artificial Insemination in equines

R. A. Legha and, Yash Pal Equine Production Campus, National Research Centre on Equines, Post Bag. No. 60, Bikaner 334 001 Rajasthan Artificial insemination (AI) is one of most important technique derived for the genetic improvement of animals at faster pace because enough spermatozoa produced by selected males can be inseminated in thousands of females per year. It involves mainly collection of semen from a selected males, evaluation and ultimately deposition of semen into a sexually receptive female at the time of ovulation, in order to result in fertilization. Artificial insemination with liquid or frozen semen is being used world wide in bovines. However, AI in equines could not gain popularity for a long time till the horse breeding societies permitted the use of this technique in thorough bred horses. The other factors responsible for its non popular in equines are decline in population size, lack of research efforts for developing frozen semen technology etc. AI with frozen Jacks semen is an important aspect in production of superior mules and up-gradation of indigenous donkey. Natural breeding or AI with fresh liquid semen may not be much advantageous because of certain limitations such as transportation of animals, venereal diseases, lesser use of good stallion and other associated problems. To overcome the stated problems, NRCE first time started the research work on freezing the semen of exotic Jacks during the year 1997 and standardized the preservation technique of jacks semen. Usually, there is a shortage of superior Jacks in the field and it is one of the serious limitations in production of improved mules and upgraded native donkeys. Therefore, AI with frozen semen of jacks can solve this problem. Salient features of the technology Cooled semen is successful for short-term storage only but long-term storage of semen is not possible through cooling of semen because in general, viability is being maintained for 48 hrs only. In view of ex situ conservation for posterity and realizing the potential advantages of AI long term storage of semen is necessary. It has become possible by cryopreserving the semen i.e. a system that halts the metabolic processes of the spermatozoa, allowing indefinite storage without loss of fertility.

AI with frozen semen of good quality jack is mainly used for breed improvement, mule production as well as for controlling venereal disease in equids. This technique is safe and more number of foals can be produced in a year by judicious use of the valuable semen. It makes countrywide transport of semen easy and use of jack even after death. Towards developing a standard protocol for cryopreserving the jack semen, three different primary as well as secondary extender were tried from the year 1997. After conducting several experiments on freezing of semen with different extenders, the technique is standardized with following primary and secondary extenders which yielded encouraging results in terms of post- thaw sperm motility. Primary extender: Citrate- EDTA; Secondary extender: Lactose- Glucose -EDTA-egg yolk. The post thaw sperm motility with the said extender has been 45-55%. Pilot trials on AI with frozen semen of jacks were undertaken during the year 2002 and 2003 in cluster of villages of Panipat and Karnal districts in Haryana. A total of 154 animals were covered during the period and average conception rate has been 43%. Steps Collection of semen: Semen is collected by means of A.V. over a jenny/mare well in estrous or dummy. The temperature of A.V. should be maintained near about 42C and the penis of the stallion should be washed with Luke warm water before mounting. The female should be restrained properly during collection to avoid any type of injury to stallion/handler.

Removal of gel and evaluation: Immediately after collection, remove gel by filtering through gauge or IMV gel filter and a small sample of gel free semen is evaluated for its quality i.e. initial motility, progressive motility, pH, volume, sperm concentration, live-dead count and abnormality of sperms etc. Dilution of semen: Dilute gel free semen in 1:1 ratio with sodium citrate medium (primary extender) at 37C and should be completed within 2-3 min of collection. Centrifuge at 2000 RPM for 4-6 minutes to get the nice pellet of the sperm in the bottom. Remove supernatant carefully without disturbing the sperm pellet and resuspend the pellet in freezing media (secondary extender). Add secondary extender to an extent so that 250x106 progressively motile sperm per insemination dose should be maintained. Normal insemination dose is 4 ml. Filling of straws: Mark the name of the stallion/Jack and date of freezing. Fill the straws with extended semen. Seal them with sealing powder or with automatic sealing machine. 1. Freezing of semen in programmable cryofreezer: The straws were loaded in automatic cryo-bio freezer for cooling as per following protocol. Cooling of semen @ -0.3 C/ min from 18 C to 5 C. Hold for 10 minute at 5C. Cooling @ 10 C/ min from 5C to -15C. Cooling @ 19 C/min from 15C to 100 C. Hold for 10 minute. Plunge into Ln2 container for preservation.
2. Thawing and Artificial Insemination: Thaw at 37C for 1 min in water

bath and evaluate post thaw semen quality. Now the semen is ready for AI and motile sperm rich semen is deposited in the body of the uterus through vagina by means of disposable sterile, non spermicidal catheter or plastic pipette and syringe. The optimum time to inseminate the mare is just before ovulation. Rectal palpation of ovaries and ultrasound scanning are done daily during estrus period to predict the ovulation. If it is not possible to accurately predict the time of ovulation, it is advisable to inseminate the mare on day 4 of estrus and subsequently every alternate day till the mare is in estrus.

Scope for commercialization As the conception rate in the field was 43% and the response of the equine owners in regards to production of superior mules through frozen semen technology was also good, therefore this technology can be attempted at large scale. Risk involvement in adoption Poor quality of semen, mishandling and improper way of insemination may result in lower conception rate and spreading of infection in the animal. Cost involved The cost of per dose of cryopreserved Jacks semen would be Rs.100/per dose. Mode of technology transfer The technology can be transferred directly in nearby areas of the Institute by us. For distant locations, the cryopreserved semen can be supplied to KVK, NGO and Veterinary hospitals for production of superior mules at farmers door through AI by skilled personals. Benefits to the equine owners Equine owners will get improved mules in terms of greater strengths, size and work efficiency. Ultimately farmers will fetch more money from their superior mules.