Elizabeth S. Metcalf, MS, DVM, DACT

Although artificial insemination in horses has been reported since the 18^th century, it was not until the 1980’s that the successful shipment of cooled stallion semen rapidly emerged as an integral part of most equine practices and breeding programs. In the beginning, there was tremendous variability in the fertility of semen shipped throughout the country and subsequent pregnancy rates. Fortunately, many advances have been made in the technology of shipped semen as well as improved training of personnel responsible for the collection, processing and shipment of cooled stallion semen.

Still, the basics for success have not changed. Pregnancy rates of mares bred with cooled shipped semen are dependent on three variables: 

1) the fertility of the mare.

2) the fertility of the semen with which they are inseminated. 

3) the expertise of the veterinarian and technicians coordinating the insemination.

Assuming good fertility of the mare, there are many ways to optimize the stallion’s contribution to a successful pregnancy. First, the experience of the veterinarian who not only coordinates the timing of insemination with ovulation, but also evaluates and processes the stallion semen for shipment, is paramount to success. Unlike many other countries, the US does not provide regulations for the selection of breeding stallions based on fertility, but instead stands stallions based on their show record. Therefore, the consultation and oversight from veterinarians is even more important in the education of all stakeholders involved in the equine breeding industry — stallion owners, mare owners, breeding managers and technicians. A stallion should be evaluated for breeding soundness at least on an annual basis, and more frequently if he has a heavy breeding schedule.

Standards for a adequate breeding dose have not changed since the early 1980’s when Householder et al reported that the minimum insemination dose for optimal pregnancy rate contained 500 million progressively motile spermatozoa (PMS) inseminated every other day while a mare was in heat. The group also reported the highest pregnancy rates were obtained when cool-shipped semen was diluted at least 3 parts extender to 1 part raw semen to a final concentration that ranged between 25-50 million PMS/ ml. The volume of the inseminate does not have any influence on pregnancy rate.

This recommendation remains the gold standard in the industry. However, it appears that this minimum insemination dose for maximal pregnancy rates is actually quite stallion-dependent. Many stallions show maximal pregnancy rates with a much lower dose (same dilution standards). It takes many inseminations and critical analysis of records to lower a stallion minimum dose from the accepted number of 500 million.

An early retrospective study (Metcalf) determined the pregnancy rate per cycle that can be expected with semen shipped from many different sources into a private equine practice. Fifty-three mares, of various breeds, were bred over 81 cycles with semen from 41 different stallions that was shipped by a commercial carrier. The semen arrived in either an Equitainer, a Bioflyte, an Expect-A-Foal, or a modified version of a shipping container. The mares were inseminated at least once within 48 hours preceding ovulation. Ovulation was documented by ultrasonographic examination. Pregnancy was confirmed by the same method 12-18 days following ovulation or by the attainment of an embryo seven to eight days following ovulation.

The criteria used for the subjective evaluation of shipped semen included progressive motility, morphology of the spermatozoa, total number of progressively motile spermatozoa, concentration of the semen, packaging of the semen, the container in which the semen was shipped, and accompanying paperwork that included a semen analysis, instructions for use, and adequate labeling of the semen. Out of 81 heat cycles in which mares were bred with shipped semen, 39 resulted in a pregnancy, thereby resulting in an overall 48% per cycle pregnancy rate. The heat cycles in which mares were inseminated with excellent versus good versus fair versus poor quality semen resulted in 87.5% (14/16), 62% (18/29), 33% (6/18), and 11% (2/18) pregnancy rates, respectively. In this study, pregnancy rates in mares inseminated with shipped semen in private practice reflect the quality of the semen received. Many of the insemination dosages received during this study failed to meet the recommended insemination dose for maximal pregnancy rates.

More recent studies have compared pregnancy rates between mares bred at the collection facility versus mares bred with cool-shipped semen (Brinkerhoff et al). In a facility where stallions were collected every other day, pregnancy rates were significantly higher in mares that were inseminated on the facility where the stallions were collected when compared to mares that were inseminated with cool-shipped semen. The authors cited mare management as a potential factor in the difference in pregnancy rates.

Following this report, our facility performed a retrospective analysis on pregnancy rates of mares bred with semen from stallions that were available to be collected on a daily basis. We compared the pregnancy rates of mares bred on the same farm as the semen collection to mares bred the following day with cool-shipped semen (unpublished). The results from both studies are presented in Table I. Similar to the results reported from the study performed by Brinkerhoff et al, we also found that there was a significantly lower pregnancy rate in mares that had been bred with cool-shipped semen versus on-farm insemination. Interestingly, we also found a significantly higher pregnancy rate in both groups of mares (on-farm versus shipped) when the stallion was available for collection every day versus every other day. We concluded that higher pregnancy rates were achieved when the stallion’s collection scheduled is based on the mare’s readiness to breed as opposed to a set schedule that accommodates the stallion farm. Again, it should be emphasized that fertility of semen in mares bred on-farm versus by shipped semen is likely stallion-dependent.

A study published in 2015 imparts important information on the potential reason that cool-shipped semen may be associated with lower pregnancy rates than on-farm breeding. This group (Love CC et al) compared the embryo recovery rates from mares that were inseminated with cool-shipped semen on the same day of collection versus mare that were inseminated with cool-shipped semen that had been stored at 40C for 24 hours. They found that embryo recovery rates were significantly lower in mares bred with semen that had been stored for 24 hours despite finding no significant drop in motility between same day and 24-hour sperm. There were also no differences found in other semen parameters such as progressive motility, DNA integrity, or morphology. These results suggest that there are factors responsible for fertility, other than these semen parameters or mare management that are necessary for maximal fertility when breeding with cool-shipped semen.

Lastly, the number of inseminations per cycle may also contribute favorably to increased pregnancy rates, for Squires et al. reported that the embryo recovery rate increases significantly as the number of inseminations on each cycle increases. This increase may be due to replenishment of sperm oviductal sperm reservoirs with each insemination.

The information from these studies is integral to a successful equine breeding operation. The expertise of veterinarians and skills of trained technicians, on both the stallion and mare side of a breeding operation, can optimize the chances of success. In summary, the following recommendations are made:

1) An insemination dose should contain at least 500 million PMS unless past pregnancy data shows acceptable rates with lower dose.

2) The concentration of cool=shipped semen should be 25-50 million PMS/ml.

3) The semen should be diluted at least 3:1 (extender:raw semen).

4) Mares should be bred on the same day as collection.

Elizabeth S. Metcalf, MS, DVM, Diplomate
American College of Theriogenologists

 

REFERENCED ARTICLES

1. Metcalf, ES. Pregnancy rates with cooled equine semen received in private practice. 44th Ann AAEP Conv Proc, p. 16-18, 1998.

2. Householder, DD, Pickett, B.W., Voss, J.L., and Olar, T.T. Effect of extender, number of spermatozoa and hCG on equine fertility. Eq Vet Sci Jan/Feb, p. 9-13, 1981.

3. Brinkerhoff et al. Influence of mare age, pre-breeding mare status, breeding method, and stallion on first cycle pregnancy rates on a large commercial breeding farm. An Repro Sci 121S: 159, 2010.

4. Love CC et al. The relationship between sperm quality in cool-shipped semen and embryo recovery rate in horses. Theriogenology 84(9): 1587-1583, 2015.

5. Squires, EL et al. Factors affecting embryo recovery: retrospective study based on commercial embryo transfer. J Eq Vet Sci 41: 61, 2016

 

Table I

	EOD collection	Daily collection
Number of mare cycles	1203	417
Number of Stallions	18	4
Number on farm Inseminations		73
Number shipped Inseminations		344
On farm per cycle pregnancy rate	68%A	75% C
Shipped per cycle pregnancy rate	50% B	63% D
On farm seasonal pregnancy rate		93
Shipped seasonal pregnancy rate		70

A is significantly different from B (P<0.0001)

C is significantly different from D (P<0.05)

A is significantly different from C (P<0.05)

 

Reviewed and updated by original author in 2016.