It is a common belief of many veterinarians and breeders that the lifespan of frozen semen within the mare’s reproductive tract is reduced compared to fresh semen. While no experimental fertility trials have been conducted to directly address this question the type of damage that can occur to sperm membranes during the freezing and thawing process theoretically could reduce the longevity of semen. Sperm binding to the lining of the oviduct can extend fertilizing capacity in the mare and there is in vitro evidence that frozen-thawed sperm do not bind as well to the oviductal epithelium. Therefore, it is generally accepted that the optimum time for insemination of frozen semen is in the period from 12 hours prior and up to 6 hours after ovulation. The timing of insemination is a good topic for debate and many mare owners and veterinarians have their preferred method of choice. Generally opinion is split between a one dose post-ovulatory insemination or a two dose timed insemination protocol. We review the pros and cons of both options here.

One Dose Protocol
The most reliable way to use one dose of frozen semen is to inseminate post-ovulation. Post-ovulation inseminations must be made within 6-8 hours of ovulation due to the lifespan of the ovum within the oviduct. Obviously, the closer to ovulation the insemination is made, the better the result, this is true for fresh and frozen semen. The one dose insemination protocol generally involves single daily ultrasound examination until a 35mm pre-ovulatory follicle is detected, then administration of an ovulation agent (hCG or Deslorelin). The mare would then be checked 12 – 24 hrs after administration of the ovulatory agent and every 6 hrs (range 4-8hr) thereafter until ovulation is detected, at which point insemination occurs. Insemination can be performed pre-ovulation, however it is difficult to accurately predict when a follicle will ovulate, therefore it is preferable to wait for ovulation to occur and inseminate within six hours rather than risk losing a dose on a premature pre-ovulatory insemination.

Two Dose Protocol
SBS has developed and tested a simple and effective two dose timed insemination protocol for managing mares with frozen semen. This protocol involves a single daily examination until a 35mm pre-ovulatory follicle is detected, administration of an ovulation agent (hCG or Deslorelin), and insemination with two doses of semen; one each at 24 and 40 hours after administering the ovulation inducing agent. Use of this protocol insures that viable sperm are available for fertilization in the mare’s reproductive tract during the time of 12 hours before to 6 hours after ovulation for mares ovulating 18 to 52 hours after administration of the ovulatory agent.

Is Fertility Equal?
When deciding between the 1 or 2 dose protocol, the first consideration should be whether there is a difference in fertility outcome between the two methods. In a clinical trial conducted in Italy (Reger et al., 2003), 26 out of 34 mares became pregnant (76%) after two timed inseminations versus 15 of 21 (71%) following a single insemination within 6 hours post-ovulation. In the same paper, the authors also reported the outcome of a controlled study carried out in Colorado where they found no difference in embryo recovery rates for mares inseminated once within 6 hours post-ovulation with 1 dose of 800 x 106 total frozen-thawed spermatozoa (60%) versus mares inseminated twice at 24 and 40 hours post-deslorelin with a half dose of 400 x 106 total spermatozoa per insemination (55%). Two data sets collected in the USA during the 2002 and 2003 breeding seasons were examined retrospectively and reported similar pregnancy rates for mares inseminated once or multiple times in a given cycle (51.5% versus 51.7% for 2002 and 47.1% versus 46.1% for 2003). Mares inseminated twice on a cycle, once before and once after ovulation, became pregnant at a rate similar to mares inseminated once within 6 hours post-ovulation (48.1% versus 47.3%). Furthermore, additional studies performed in France, Germany and Canada have reported that pregnancy rates with frozen semen were higher when mares were inseminated more than once per cycle.

This published data shows that comparable pregnancy rates can be achieved with one or two dose insemination protocols, in fact multiple inseminations on a cycle may result in higher pregnancy rates, presumably because of a decrease in the interval between insemination and ovulation.

Cost Comparison
Given comparable fertility the next consideration is usually financial. Obviously the cost of veterinary care across the US and more so around the world varies significantly, so it is difficult for us to make generalizations here. Nonetheless, it is fair to assume that the frequent ultrasound examinations required when inseminating with one dose of frozen semen results in greater costs for veterinary management per cycle than the once daily examination utilized for the two dose timed insemination protocol. For convenience to the veterinarian the one dose protocol may also necessitate the mare being boarded at a breeding farm or clinic so that late night checks can easily be performed to insure that insemination occurs within the critical 6 hour window after ovulation.

The two-dose timed insemination protocol allows the veterinarian the flexibility to examine mares once daily during normal hours without compromising fertility, concurrently reducing management costs for the mare owner.

However….The purchase price of the frozen semen also has a significant influence on the anticipated costs for each breeding cycle. Of course, as horse owners and breeders we place just as much value on the selection of the stallion as we do in the consideration of veterinary expenses for mare management, in some cases perhaps more bias towards stallion selection. By choosing that top international stallion, with limited frozen semen available at a significant cost per dose, we essentially limit our choice in insemination protocol. Semen sold on a per dose basis for hundreds or even thousands of dollars imparts a significant pressure on the mare owner and veterinarian to use only one dose of frozen semen per cycle, since the cost of the extra ultrasound examinations incurred when breeding with one dose is generally less than the cost of purchasing an additional dose of semen.

At SBS we recommend to our stallion owners that they sell frozen semen as a breeding and provide at least two doses per cycle, as opposed to selling frozen semen by the dose. This allows mare owners to choose the timed insemination protocol and benefit from the flexibility in scheduling and reduced costs in veterinary management.

For further discussion on purchasing one or two doses of frozen semen, see our recent blog article, Frozen Semen – Sell by the Dose or As a Breeding?

Does Breeding With Two Doses Result in More Post Breeding Fluid Accumulation?
The numerous reports on timed-insemination protocols demonstrate that multiple inseminations in normal mares are not harmful. Insemination with frozen-thawed semen twice per cycle does not increase the incidence of fluid accumulation and post-breeding endometritis. All mares experience a natural inflammatory response within the uterus, after the deposition of semen. The post-breeding inflammation initiated by the first insemination is cleared within 12 hours in reproductively healthy mares. Thus, a second insemination can safely be performed 16 hours later without negatively affecting fertility.

However, use of multiple inseminations per cycle may not be appropriate for all breeding situations. For example, mares that are susceptible to post breeding endometritis are less efficient at clearing the uterine lumen of semen and inflammatory products accumulated after breeding. Fertility is reduced in this group of mares and, unless properly addressed, the condition may aggravate with subsequent matings. These mares may require a more intense management scheme and be bred only once per cycle in order to minimize invasion of the susceptible uterus.

For more information on this subject, see the blog article, The Facts About Frozen Semen and Post Breeding Uterine Fluid.

Further Considerations
Remember, not all semen is created equally. There is a huge variation in the quality of frozen semen on the market, both in the technique and standard of quality control applied to the semen processing as well as in the semen quality itself. Furthermore there is inherent variability between stallions in the longevity and fertility of their frozen semen. The industry recommended minimum is 200 million progressively motile sperm per frozen semen dose. But to complicate matters further the evaluation of semen quality can vary depending upon the technician and technique used when performing the analysis. Mare owners should gather as much information about the quality and fertility of the frozen semen before purchase.

See the blog article, Questions Every Mare Owner Should Ask Before Breeding With Frozen Semen and the FAQ Can’t Post-Thaw Motility Vary With the Laboratory Performing The Analysis?

Both the selection of your mare and veterinarian could influence the outcome of your proposed breeding; we plan to review these variables in future blog posts.

In summary, when considering breeding your mare with frozen semen it is important to remember that you have choices and options available, both in how you purchase your frozen semen and in how you manage the insemination of these doses. Talk with your veterinarian to review all the options, work up cost comparisons considering the best and worse case scenarios. Weigh your anticipated costs in breeding against the potential value of the foal and make an informed decision.

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