Instructions for use of Umm Qarn Farm semen
frozen by Stallion Reproduction Services*
Do NOT use frozen semen to inseminate a mare if there is any doubt about her fertility.
To get the best conception rate, frozen semen should be inseminated during the period from 12 hours before until 6 hours after ovulation. However, frozen semen from some stallions with proven fertility has been shown to achieve acceptable conception rates if mares are inseminated once daily at 24 hour intervals. If insemination takes place more than 6 hours 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 important to use as few as possible per cycle.
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:
- 6 hourly examination. Mares should be examined at least every 6 hours during the critical part of their cycle and inseminated immediately ovulation has been detected.
- 12/24 hourly examination. If 6 hourly monitoring is impractical, a mare should be examined at least every 24 hours (ideally every 12 hours) and insemination delayed for as long as one dares, depending on appearance of the follicle. Once inseminated, a mare should be checked at 24 (or 12) hourly interval and re-inseminated at each inspection until ovulation has occurred. This way the conception rate may be slightly reduced and 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.
- Use of hCG. If hCG (eg 1500 - 3000 i.u. Chorulon:Intervet) is given i/v when the follicle is 3.0-3.5 cm, the majority of mares ovulate approximately 44 hours (36-48hours) later. Therefore, if the mare is inseminated 36 hours after the injection, there is a good chance of being reasonably close to ovulation. However, there is a risk of missing a mare 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 3.0-3.5 cm in diameter. Then inject the hCG.
- Thereafter, the mare should be examined at hCG+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, 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 insemination, 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.
- 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 2 or 3 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.
The insemination pipette (bovine uterine irrigation catheter) suitable syringe (eg 10ml with plastic plunger without a rubber tip) and clean sterile receptacle (eg 50ml glass beaker well rinsed with de-ionised water) in which to collect the semen once it has been thawed should all be kept at 37 degrees C (it helps if an incubator and/or water bath, set at 37 degrees C, are/is available close to the insemination area). Clean scissors should also be available for cutting the straws after thawing.
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.
Frozen/thawed semen is fragile and short lived. Great care should be taken in handling it. Contaminants e.g. tap water, detergents, disinfectants etc. on any surface with which the semen comes into contact, or temperature fluctuations and exposure to air or sunlight during handling, will all reduce the chance of conception.
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 frozen semen is packed in plastic "goblets" each containing 3 - 10 x 0.5ml straws which is enough for a single insemination dose (ie 2-5ml 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 the 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 different containers - eg large 4ml straws, 15 ml aluminium tubes etc. and should be accompanied by instructions on how they are to be used.)
PLEASE MAKE A NOTE OF THE STRAW DETAILS (eg straw and tip colour, label and number of straws used) FOR INCLUSION ON THE INSEMINATION DETAILS FORM THAT MUST BE RETURNED TO UMM QARN.
A water bath (or jug with a great enough volume to prevent temperature fluctuations during thawing) should be set at 37 degrees C (plus or minus not more than 1 degree C). Once all is prepared, remove one insemination dose 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, tip the straws separately into the water bath and leave for 40 seconds. (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 water bath. 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 re-freeze the thawed semen by re-immersing it in the liquid nitrogen will kill the sperm.
With clean scissors, cut off the coloured tips from all the straws and hold the straws, cut end down, over the warmed receptacle. Cut the opposite end containing the "cotton" plug from the straws and allow the contents to run into the receptacle. Check that all the straws have emptied (it is important to ensure that the "cotton" plug as 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.
Once frozen semen has been thawed, it will be irreparably damaged by any kind of abuse or unnecessary temperature fluctuations. Immediately after thawing, draw the semen into the warmed syringe allowing 3-4 ml of air space above the semen. Attach the syringe to the warmed pipette and run semen down into the pipette to expel air from its lumen. Lubricate the mare's vulva lightly with KY gel (not obstetric lubricant, which may contain spermicidal preservatives) and inseminate without delay; the time from removing the semen from the liquid nitrogen to depositing in the mare's uterus should be no more than 90 seconds.
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. Use the air space in the syringe to ensure that all semen remaining in the pipette is expelled into the mare. Examine the mare 12-24 hours after and if she has not ovulated, repeat the insemination process.
Immediately after insemination, a drop of semen remaining in the syringe or container should be examined under a microscope to check sperm viability ( make sure the microscope slide has been pre-warmed to 27 degrees C). It helps to visualise 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.
* Reproduced by king permission of Martin Boyle MA, VetMB, MRCVS, Stallion Reproduction Services