Cryopreservation of Equine Semen for Optimum Utilization of Stallions

R.A. Legha and Yash Pal

Equine production Campus, Bikaner

• AI in mares was initiated with• Liquid• Cooled• Frozen

• More number of mares can be inseminated but with in a very short duration.

• Cooled semen Technology- very successful for short term storage.

• Long term storage is not possible, viability maintained for only 48 hours.

• To realize potential advantages of AI- Long term storage is necessary.

Advantages of AI

• Facilitation and acceleration of genetic improvement in equines.

• Decreased stress and risk to jenny/mare. • Decreased transport costs.• Longer breeding season for the stallion. • Genetic insurance for the stallion. • Opens new international markets. • Ensuring routine semen evaluation and monitoring.• Allowing the covering of injured mares, precluded from

natural covering.• Storage of semen for posterity and preservation of

endangered breeds.• Increasing the numbers of jennies/mares covered per

jack/stallion.• Aiding in control of disease and reduction in the risk of

injury.• Permitting the use of injured jack/stallion.

Cryopreservation

It is a system that halts the metabolic processes of spermatozoa, allowing indefinite storage without loss of fertility.

• Discovery of cryoprotectant properties of glycerol was a boon in the development of cryopreservation technique.

• Some damage occur to spermatozoa during freezing process.

• Reasons for spermatozoa damage are• Internal ice crystal formation • Increase in solute concentration• Changes in plasma membrane permeability to

calcium.

Steps in semen freezing:

Collection of semen: • Semen is collected by means of A.V. over a

jenny/mare well in estrous or dummy. • Stallion should be kept at reasonable distance

for proper stimulus.• The temperature of A.V. should be maintained

near about 42C.• 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.

• The tail should be properly covered by bandage.

Gel removal and evaluation:

• Immediately after collection, remove gel by filtering through gauge or IMV gel filter .

• 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.

Table 1: Macroscopic and microscopic evaluation of Marwari stallions and exotic donkey stallions semen.

Sr. No Parameters Horse (n=42) Donkey (n=50)

1 Total Volume (ml) 76.90±5.52 (30-225) 70.6±4.07 (5-150)

2 Gel volume (ml) 23.21 ±6.48 (0-200) 19.0±3.45 (0-95)

3 Gel free volume (ml) 53.69 ±2.64 (25-90) 51.30±3.56 (15-125)

4 Consistency Thin to thick Thin to thick

5 Colour Milky white to creamy Off white to creamy

6 pH 7.20 ±0.02 (7.0-7.5) 7.17±0.02 (7.0-7.5)

7 Initial motility (%) 79.76 ±0.93 (70-90) 82.60±1.02 (60-90)

8 Progressive motility (%) 73.33 ±0.94 (60-90) 77.80±1.00 (60-90)

Dilution of gel free semen:

• Dilute gel free semen in 1:1 ratio with sodium citrate medium (primary extender) at 37C as soon as possible after collection.

• Centrifuge at 2000 rpm for 4-6 minutes in refrigerated centrifuge 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.

• Keep the freezing media mixed semen at 5oC.

Marking of Straws:• Mark the name of the stallion/Jack and

date of freezing.

Filling of straws:Manual fillingMachine filling

• Fill the straws with extended semen. Seal them with sealing powder or with automatic sealing machine.

Freezing of semen in programmable cryo-freezer:

• The straws should be 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 minutes.• Plunge into LN2 container for preservation.

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 daily till the mare is in estrus.

• Normal insemination dose is 4 ml means 8 straws.

Semen collection in field

AI in field for mule production

Foal produced at farmer’s door