Breeding your mare with frozen semen

Breeding your mare with frozen semen.

As frozen/thawed semen has a short half-life, insemination must take place close to ovulation. This necessitates frequent examination of the mare as often as may be required to accurately predict ovulation. This means that a veterinarian who is skilled in reproductive management of mares must be either able to visit your farm frequently or the mare is left in the care of a veterinary hospital. At the Goulburn Valley Equine Hospital (GVEH) we routinely breed many mares with frozen semen each year. People come from all over Australia for this service.

Housing
At the GVEH mares can be kept on pasture (with their foals) either separately or in small groups. It is no trouble to keep your mare by herself either totally isolated or just separate from other horses. Mares can be placed in yards or boxes.

Facilities
Should be available to breed the mare under cover with adequate restraint to avoid damage to the mare or personnel involved in with the procedure. Restraining devices such as stocks should be immediately adjacent to the semen handling area or laboratory and should be constructed to avoid damage to the semen by temperature fluctuation or contamination before insemination.

Semen Handling: Stallion spermatozoa are fragile, easily damaged and short lived. They are susceptible to cold-shock, direct sunlight, many antibiotics and low levels of residues that may be left on the liners of artificial vaginas, glassware and other receptacles with which the semen may come in contact. Care should be taken to assure that all contact surfaces are made of non-spermicidal material, are thoroughly cleaned and have been rinsed with de-ionised water.

Before insemination, all handling and preparation of semen should take place in a clean room close to the insemination area. This room should be heated to maintain a minimum temperature of at least 24⁰C and the following equipment must be available:

	a microscope (preferably with a heated stage) for evaluating viability of spermatozoa (progressively motile sperm) at 200 x magnification and preferably with phase contrast,
	an incubator maintaining temperature at 37⁰C , and
	a water bath for thawing spermatozoa which is capable of accurately maintaining the temperature specified for thawing different batches of semen.

Mare Insemination: The mare should be cleaned and prepared following normal procedures for artificial insemination. Prior to thawing semen, all equipment should be prepared and made ready. Care should be taken to avoid spermicidal preservatives in lubricants by using KY gel. If the thawed semen needs to be removed from its receptacle (ie. glass ampoule or 5 ml straws) prior to insemination, then care should be taken to avoid using syringes with rubber plungers (the lubricant in these syringes has been demonstrated to be spermicidal).

Management of mares inseminated artificially with frozen semen.

Try to assure that only mares of normal fertility are bred with frozen semen.

Pre-breeding Evaluation: The requirements for pre-breeding gynaecological examination will vary according to the veterinarian and status of the mare. Most mares should be cultured and demonstrated negative for venereal pathogens . Ultrasonographic examination should be used to determine that the mare has no signs of uterine inflammation (fluid). Other examination procedures such as uterine cytology, biopsy, endoscopic exam, digital manipulation of the cervix, etc. may be necessary depending upon the history of the mare to be bred, her age and recent foaling complications etc.

Timing of insemination with frozen semen: Although the frozen thawed spermatozoa from some stallions may survive 24 hours or more after insemination, best results are achieved if the semen is inseminated in a period from 12 hours before, until a maximum of 6 hours after ovulation. Insemination greater than 6 hours after ovulation results in a decreased chance of conception and an increased incidence of early embryonic death. Because each breeding unit of frozen semen is expensive to produce, it is recommended mares are bred a maximum of 2 times per cycle, and preferably once. There are various ways that a mare can be managed during the insemination period to optimise the time of insemination and vary according to economic considerations, facilities and veterinary experience:

	Ultrasonographic examination every 6 hours. Mares are inseminated immediately ovulation has been detected.
	12-24 hourly examination. Ultrasonography is used to determine when the follicle(s) is immediately pre-ovulatory. The mare is inseminated immediately and re-checked in 12-24 hours. If ovulation has not occurred 24 hours later, the mare is re-inseminated at each inspection until ovulation has occurred. This will result in the use of more semen.
	Ovulation induction. When ultrasonographic evaluation of a mare in early oestrus reveals the presence of 30-35 mm follicle and she is treated with either Ovuplant S/C or 3000 IU of hCG (i.v.), then most mares will have ovulated in 36 - 48 hours. Under these circumstances, the mare is examined at ovulation induction + 24 hours. The mare may be inseminated at this time and re-inseminated 24 hours later if she has not ovulated. An alternative method is to inseminate all mares once at ovulation induction + 36 hours. The advantages of using ovulation induction are that it limits the number of examinations, while still allowing flexibility in relation to time of insemination. If pregnancy is not established, then subsequent cycles may be best managed with the use the other ovulation-inducing agent.
	At our practice we most commonly use ovulation induction and examine the mare every few hours when they are close to ovulation. We aim to breed just once per cycle. Ideally we would aim to breed immediately post ovulation as this ensures that a normal ovulation has occurred.

Artificial insemination: The mare should be restrained and then prepared for breeding as hygienically as possible and clean water should remove all traces of spermicidal antiseptic soaps, etc. After the frozen semen has been thawed according to the suppliers recommendations, it should be inseminated immediately. When the breeding dose is contained in one 0.5 ml straw, it will be difficult to evaluate the motility of the spermatozoa from samples bred. In these cases, recommendations are to sacrifice 1-2 straws in every 10 processed to assess the motility from each ejaculate. On other occasions, when semen needs to be placed inside a syringe prior to insemination, it is possible to check the motility after breeding from residues left within the AI pipette or syringe. Accurate evaluation will only be possible after the semen has been warmed to 37^oC for 5 minutes (use a warmed slide and cover slip). If possible, insemination should be performed as far up the uterine horn ipsilateral to the ovulatory follicle as possible.

Post-artificial insemination examination: Mares should routinely be examined 24 hours after breeding to ensure ovulation has occurred and that there is no evidence of uterine fluid which may necessitate intra-uterine or systemic anti-bacterial treatment. Post-breeding surgery such as Caslick may be performed at this time. Ultrasonography should be used 14-15 days after breeding to determine if the mare has conceived, has twins, etc.

Pregnancy rates.

The only accurate indicator of fertility is pregnancy rate per cycle. Pregnancy rate at the end of the season is influenced by the number of cycles that mares are bred, the fertility per cycle and in many cases good or poor management practices. Table 5 shows the theoretical pregnancy rate for matings with a theoretical probability of between 10% and 70% pregnancy rate per cycle. Examination of table shows that a stallion a 20% pregnancy rate/cycle can have 80% of his mares pregnant after 7 cycles which would not be different from a stallion with an expected outcome of 40% per cycle after three cycles or two cycles at 50% pregnant per cycle. Similarly 88% of mares would be pregnant after three cycles at 50% per cycle, 4 cycles at 40% per cycle, 6 cycles at 30% per cycle and 9 cycles at 20% per cycle.

*Pregnancy rate per cycle*	*70%*	*60%*	*50%*	*40%*	*30%*	*20%*	*10%*
*Cycle 1*	70	60	50	40	30	20	10
2	91	84	75	64	51	36	19
3	97.3	93.6	87.5	78.4	65.7	48.8	27.1
4	99.1	97.4	93.7	87.0	75.9	59.0	34.3
5	99.7	98.9	96.8	92.2	83.1	67.2	40.9
6	99.9	99.5	98.4	95.3	88.2	73.7	46.8
7	99.9	99.8	99.2	97.2	91.7	79.0	52.1

The effect of multiple cycles on cumulative pregnancy rate for matings of various theoretical outcomes (pregnancy rate per cycle).

Many studies and many semen centres report the overall pregnancy rate only and from the above discussion it should be clear that the pregnancy rate per cycle is the important figure to ascertain to recognise the true fertility of the sample. This becomes even more important when we are advising clients on the purchase of a set number of straws. If the fertility is only 30% per cycle and the number of straws retailed is 3 then the probability of having a mare pregnant in this scenario is only 66% after three attempts (cycles).