cryopreservation of jacks semen and artificial insemination in equines

7/31/2019 Cryopreservation of Jacks Semen and Artificial Insemination in Equines

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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 aselected 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 processesof the spermatozoa, allowing indefinite storage without loss of fertility.


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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.


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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. Normalinsemination 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 5C. Hold for

10 minute at 5C.

Cooling @ 10C/ 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 waterbath 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.


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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 technologycan 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.

cryopreservation of jacks semen and artificial insemination in equines

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