Squires said the perception in the industry is that mares must be examined four to six times per day to use frozen semen successfully. He said the reasons for frequent examination are because limited semen is made available and the desire to breed as close to ovulation as possible. If multiple doses of semen are available, mares could be examined once a day and treated identically to mares bred with cooled semen.

Perceived negatives to using frozen semen are that it is usually sold by the dose, it can be difficult to get, and it is often expensive if imported, he noted. There is also concern about uterine inflammation with multiple insemination doses with frozen semen and reduced fertility if mares are not bred close to ovulation. Therefore, there is a need to have a better method for breeding with frozen semen.

When frozen semen was first introduced, pregnancies were achieved by  checking the mare frequently each day and breeding with a single dose of semen as close to ovulation as possible. Therefore, this was adopted as standard operating procedure, said Squires. Veterinarians checked a mare every six hours once she was in heat and had been given hCG (human chorionic gonadotropin) or Ovuplant (deslorelin).

The problems with this protocol, said Squires, are the time demands on the veterinarian, cost to the client, and lower conception rates than with cooled or fresh semen in spite of this tremendous effort. The end result was that lots of owners stayed away from frozen semen.

Squires said the emerging protocol for frozen semen is to inseminate the mare twice with a timed insemination technique. This helps balance the cost of semen and the cost of veterinary care.

This new protocol for breeding with frozen semen centers around breeding a mare twice each cycle after administration of hCG or Ovuplant.  If the mare ovulates within 18 and 52 hours of treatment, then viable sperm should be available within the window of six hours after or 12 hours prior to ovulation.

Squires said two studies compared pregnancy rates for mares inseminated only once within six hours post-ovulation with mares inseminated on a timed insemination protocol 24 and 40 hours after hCG administration.

In the first study at Colorado State, one group of mares was given hCG once they developed a follicle greater than 35 mm. The mares were scanned with ultrasound every six hours until ovulation was detected, then they were bred using 800 million frozen thawed sperm post ovulation.

In the second group, Squires developed a timed insemination program. Once these mares developed a follicle, they were only scanned once a day, administered hCG, then bred 24 and 40 hours after hCG administration using 400 million sperm at each breeding.

Pregnancy rates were similar between the two groups, and there was no difference in the occurrence of post-breeding fluid accumulation.

Squires said data from Italy showed that the pregnancy rates in the timed insemination group were higher than for mares bred after ovulation.

Data from Select Breeders Services laboratories in 2002 and 2003 showed that mares bred one time with frozen semen had similar pregnancy rates as those bred multiple times.

Therefore, said Squires, the optimum window for AI with cooled semen is 24 hours before ovulation to six to eight hours after. With frozen semen the optimum time is 12 hours before to six-to-eight hours after ovulation.

Conclusions
Squires said daily exams were sufficient when mares were inseminated at fixed times after hCG or GnRH (gonadatropin-releasing hormone) administration. Multiple inseminations did not adversely affect fertility; there were no detrimental effects to the mares bred more than once in the cycle with frozen semen.