STALLION REPRODUCTION SERVICES
RECOMMENDATIONS FOR THE USE OF STALLION SEMEN FROZEN BY
STALLION REPRODUCTION SERVICES

1. Do NOT use frozen semen to inseminate a mare if there is any doubt about her fertility.

Timeline of insemination

2. To get the best conception rate, frozen semen should be inseminated during the period from 12 hrs before until 6 hrs after ovulation. However, the frozen/thawed semen from some stallions with proven fertility has been shown to achieve acceptable conception rates if mares are inseminated at 24 hr intervals. If insemination takes place more than 6 hrs after ovulation, the chance of conception decreases and the incidence of early foetal loss rises. As each insemination dose is costly to produce, it is im-portant to use as few as possible per cycle.

3. There are various ways a mare can be managed during the insemination period depending on the facilities and organisation at the inseminating stud. Any of the following procedures (all but the last of which depend on the use of ultrasound) can be adapted to fit in with local conditions.

a. 6 hourly examination Maies should be examined at least every 6 hours during the critical part of their cycle and inseminated immediately ovulation has been detected, In this way, one can ensure that the mare is inseminated at the optimum time whilst using only one insemination dose.

b. 12 or 24 hourly examination If 6 hourly monitoring is impractical, then a mare should be examined at least every 24 hrs (ideally every 12 hrs) and insemination delayed for as long as one dares, depending on the appearance of the follicle. Once a snare has been inseminated, she should be checked at 24 (or 12) hourly intervals and reinseminated at each inspection until ovulation has occurred. This way, one risks missing a cycle if insemination is delayed too long. It may also be more expensive in terms of the doses of semen used.

c. Use of hCG If hCG (eg. 1,500- 3,000 iu Chorulon: Intervet) is given i/V when the follicle is 3.0-3.5 cm, the majority of mares ovulate approximately 44 hrs (36hrs- 48hrs) later. Therefore, if the mare is inseminated 36-40 bows after the injection there is a good chance of being reasonably close to ovulation in. However, there is a risk of missing a snare if she ovulates early. The following regime should help to overcome this possibility; carry out a normal daily examination during early oestrus until the follicle has reached 3.0-3.5cm in diameter and then inject the hCG. Thereafter, the mare should be examined at bCG +24, +30 and +36 hours, If, at any of these times, she has ovulated, she should be inseminated immediately. If at hCG +36, the follicle is still present, she should be inseminated anyway. She should be re-examined 12 - 24 hours later and, if she still has not ovulated, re-inseminated. This regime limits the period of intensive monitoring and, with a maximum of two inseminations, should catch all but the most awkward mares. It should be followed flexibly and be modified as necessary to take account of local knowledge of the individual mare and the way the follicle is judged to be developing.

d. Timing of insemination without examining follicles If there is no scanner on the stud or the vet lacks the experience to assess follicle size and maturity manually, then the routines described in either b. or c. above can be adapted using oestrous behaviour as a guide. This will inevitably be less accurate than other methods and will use more insemination doses per pregnancy.

Equipment required for insemination

4. The insemination pipette (bovine Uterine irrigation catheter), suitable syringe (one piece plastic plunger without a rubber tip) and clean sterile receptacle (eg 50 ml glass beaker well rinsed with de-ionized water) in which to collect the semen once it has been thawed should all be kept at 3~C(it helps if an incubator and/or waterbath, set at 37~C, are/is available close to the insemination area). Clean scissors should also be available for cutting the straws after thawing.

Preparation of the mare for insemination

5. Before the semen is thawed, prepare the mare for insemination. If possible, she should be restrained in stocks with her tail bandaged and held out of the way. It is a sensible precaution to rake out her rectum manually before washing her vulva which should then be rinsed well with clean water and thoroughly dried. Residues of soap, disinfectant or detergent may be spermicidal.

Thawing procedure

6. Semen frozen by Stallion Reproduction Services is delivered, immersed in liquid nitrogen. It is important that the straws remain in the liquid nitrogen at all times until an insemination dose is removed just before use. The semen is packed in plastic goblets each containing 4- lOx 0.5 ml straws which is enough for a single insemination dose (i.e. 2 5 ml of semen when thawed - the number of straws per dose depends on the concentration and motility of the sperm in the batch of semen being used for the insemination). The date of collection and the name of the stallion that produced the semen are printed on each straw. (Semen frozen by other organizations may be delivered in dif-ferent containers - eg. large 4 ml straws, 15 ml aluminium tubes &c and should be accompanied by instructions as to how they are be used).

7. A water bath (or bucket of water with a great enough volume to prevent temperature fluctuations while straws are being thawed) should be set at 370C (± not more than 1CC). Once all is prepared, remove one goblet from the liquid nitrogen - if there is more than one dose in the canister, take care not to lift the canister above the neck of the flask. Immediately, tsp the 6- 12 straws Out of the goblet into the water bath and leave for 30 sees. (Gardening gloves and large forceps are useful accessories when handling anything that has been stored in liquid nitrogen). Remove the straws and dry well - it is very important to avoid any risk of contaminating semen with water from the waterbath. Quickly examine each straw to check that the details printed on it identify the semen as coming from the correct stallion. If, for any reason, semen is not used once it has been thawed, it must be discarded; any attempt to refreeze the thawed semen by re-immersing it in the liquid nitrogen will kill the sperm.

8. With clean scissors, cut the coloured tips from the straws and hold them, cut ends down, over the warmed receptacle. Cut the opposite end containing the "cotton" plug from the straws and allow the contents to rim into the receptacle. Draw the semen into the wanned syringe leaving 3 - 4 ml of air space above the semen. Alternatively, the straws can be emptied directly into the warmed insemination syringe provided its end is blocked to prevent the semen running out Check that all the straws have emptied (it is important to ensure that the 'cotton' plug has been completely removed otherwise semen may be retained in the straw). All this must be done quickly to prevent cooling of the semen once It has been removed from the water bath.

Insemination of the mare

10. Once frozen semen has been thawed, it is very fragile and will be irreparably damaged by any kind of abuse or unnecessary temperature fluctuations. Lubricate the mare's vulva lightly with KY gel (not obstetric lubricant, which may contain spermicidal preservatives), attach the syringe to the insemination pipette and inseminate without delay: the time from removing the semen from the liquid nitrogen to depositing it in the mare's uterus should no exceed 90 seconds.

11. Ideally, the pipette should be in place in the mare's uterus whilst the semen is thawed by an assistant. In this way, the pipette is kept warm and there is no risk of damage to the sperm as the small volume of semen passes through a cold pipette. During insemination, the catheter should be introduced well into the uterus over the pelvic brim otherwise there is a risk of the semen spilling back through the cervix into the vagina especially if the bladder is full. Recent research suggests that conception rates may he improved if sensed is deposited in the uterine horn ipsilateral to the ovulatory follicle. Use the air space in the syringe to ensure that all the semen remaining in the pipette is expel-led into the mare. Examine her 12 hrs later and, if she has not ovulated, repeat the process.

12. Immediately after insemination, a drop of semen remaining in the syringe or container should be examined under a microscope to cheek sperm viability (make sure the microscope slide has been pre-warmed). It helps to visualize the sperm through the egg yolk and glycerol in the freezing medium if the semen is further diluted with a few drops of warm milk glucose extender.

(NB Acceptable conception rates are possible using frozen stallion semen but, once thawed, the semen is fragile and short lived. Accurate timing of insemination and great care in handling the semen are essential. Contaminants, eg. tapwater, detergents, disinfectants & on any surface with which the semen comes in contact, or excessive temperature fluctuations and exposure to air or sunlight during handling, will all reduce fertility)

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