Effect of Number and Timing of Equine Frozen Semen Inseminations on Fertility

December 03, 2014

Further expansion in the use of frozen semen is dependent upon developing simplified strategies for insemination. SBS has developed a timed insemination protocol where mares are only examined once per day during estrus and inseminated at 24 and 40 hr after hCG or 30 and 40 hr after GnRH. This approach allows frozen semen mares to be managed similar to those bred with cooled shipped semen. A couple of arguments against this approach with frozen semen is that it takes too much semen when mares are bred twice in one cycle and that if the mare is inseminated more than once per cycle the fertility will be lowered because of post-breeding induced endometritis.

Review of Previous Research

There have been several studies that have reported equal or better fertility when mares are bred more than once in a cycle with frozen semen.

Metcalf (2000) reported no adverse effect on fertility for mares bred within 4-10 hr of a previous insemination.

Reger et al (2003) compared pregnancy rates of mares inseminated 24 and 40 hr after hcG (SBS timed AI protocol) to those inseminated once within 6 hr post ovulation.

  • Embryo recovery was similar for these two AI strategies (11/20 and 12/20).
  • Those bred twice were inseminated with a half dose (400 million) each time in order not to use more semen than the mares in the other group.
  • This study also reported the results of a Clinical trial in Italy where mares were either bred within 6 hr post- ovulation or inseminated at 24 and 40 hr after hcG. They also reported no difference in pregnancy rate per cycle or seasonal pregnancy rate between the two groups.
  • Uterine fluid 24 hours after the last breeding was not different between the two group.

Squires et al (2006) reported on a retrospective study of mares inseminated with fresh, cooled and frozen semen at 6 different SBS facilities in the USA and Europe.

  • Data were available from 407 cycles in 2002 and 554 cycles in 2003.
  • Not all semen was processed by SBS.
  • Mares were bred either once or multiple times during the cycle with over 90% of the mares in the multiple AI groups being bred only twice.
  • Mares bred with cooled semen more than once had higher pregnancy rates but fertility was similar for those bred once or twice in a cycle with frozen semen.
  • This indicates that there was no adverse effect of breeding a mare with frozen semen more than once in a cycle.

Barbacini et al (2006) reported higher pregnancy rates for mares inseminated twice (76%) vs once (57%) with 800 million sperm. An 80% pregnancy rate was obtained for those inseminated with 400 million twice in a cycle

Recent Retrospective Study

During 2010-2014, additional data were obtained from SBS Maryland and SBSW plus a total of 8 additional SBS affiliate labs. This data was used to 1.) further determine the fertility of mares bred more than once in a cycle with frozen semen and 2.) to determine when in the cycle it is best to breed with frozen semen. Several hundred mares were bred with frozen semen in the SBS network each year to provide data on a total of 1871 mare cycles.

The effect of the number of inseminations per cycle on pregnancy rates at 16-21 days is shown in the table below. Frequency of AI - Table 1 Fertility Article

  • As has been reported by SBS before, inseminating twice in the cycle resulted in a higher pregnancy rate (54%) compared to inseminating only once (46%).
  • The pregnancy rate was even higher for those mares bred three times in one cycle (68%) but the number of mare cycles for this group was quite low (28 cycles).

The reason for this increased fertility with more frequent insemination is not truly known but may be due to providing sperm to the mare closer to the time of ovulation when mares are inseminated twice per cycle.

The other possibility is that more frequent inseminations increase the number of sperm in the reservoir. It is thought that upon insemination sperm travel to the oviduct and attach to the oviductal lining for a period of time. When they are released from this reservoir they are capable of fertilizing the egg for only a short period of time and if the egg is not available then the sperm die. By inseminating a second time it is possible that the reservoir is replenished.

We recommend a mare be bred with frozen semen within the window of 12 hr before ovulation to up to 6 hr after ovulation. With the timed insemination protocol recommended by SBS, mares can ovulate anywhere from 18 to 52 hr after hcG and they will have semen that was inseminated in this window of optimal fertility. With frozen semen, if one is not using the timed protocol and inseminates a mare with frozen semen and she has not ovulated by 12 hr then the mare should be bred a second time.

If the semen inventory is limited the veterinarian will often palpate and ultrasound the mare every 6-12 hr once she has acquired a large follicle and has been given an ovulatory agent (hcG or deslorelin). Once it has been determined the mare has ovulated she is then inseminated within 6 hr of ovulation. Barbacini from SBS Italia has published that fertility with post-ovulation breeding was similar to mares bred with frozen semen within 12 hr prior to ovulation.

Based on the data collected by SBS and their affiliates during the 2010-2014 breeding seasons, pregnancy rates were highest when mares were bred twice, once before and once after ovulation (290/524, 55%) compared to a 47% pregnancy rate (593/1262) for those inseminated after ovulation. The lowest fertility was for mares bred only once prior to ovulation (33/85, 39%).


Thus, based on a large number of mares, it appears that for the best fertility with frozen semen and the least amount of mare management, mares need to be bred both before and after ovulation and that one breeding before ovulation is not sufficient. If semen is limited then mares will need to be bred once within 6 hr after ovulation.