from the Vet Corner Archives
Part of Horse Previews Magazine website. Posted on 02/07/2001; 2:00:00PM.
Veterinary Corner 02/01: Breeding Your Mare with Frozen/Thawed Semen
by Frosty Franklin, DVM
Edgecliff Equine Hospital
S. 1322 Park Road, Spokane, WA 99212 * 509/924-6069
In 1950 investigators discovered that stallion spermatozoa could be separated from seminal plasma and re-suspended with buffers, glucose and glycerol, frozen to -110 degrees F, thawed and about 25 % of the spermatozoa were motile. For economic reasons research has focused on bull semen. When applying the basic procedures of cryopreservation of bull semen to other species the researchers have not been as successful.
The first pregnancy from frozen stallion semen occurred in 1957. Investigators inseminated seven mares with frozen/thawed semen and one mare subsequently foaled. Frozen/thawed has not been widely accepted and used by the equine industry because of poor pregnancy rates with many stallions and restrictions imposed by breed registries.
However, that is not the future. Today over 35 different breed registries permit the use of frozen semen. The notable exceptions are the Quarter Horse and Paint Horse Registries. As we select our breeding stock and determine the pregnancy rate per cycle of stallions breeding with frozen/thawed, and researchers elucidate the most successful freezing and inseminating protocols, mare owners will begin to experience pregnancy rates in their mares very much like that of natural breeding.
In fact, the future can be now with careful selection of stallions with high pregnancy rates using frozen/thawed semen and a normal fertile mare less than 15 years of age.
Here is what you need to know:
Success in cryopreservation of stallion spermatozoa depends on a complex series of interactions among extender, cryoprotectants, cooling and warming rates to minimize damage from cold shock, formation of ice crystals, and dehydration.
For a spermatozoon to fertilize an oocyte, it must retain four attributes after freezing and thawing: (1) metabolism for production of energy, (2) progressive motility, (3) have intact enzymes essential for penetration on the oocyte (4) have a plasma membrane that will survive within the female reproductive tract. Destruction of one these components will render the spermatozoon infertile.
Spermatozoa from individual stallions differ in their ability to survive freezing and thawing. Extreme differences in pregnancy rate have been found using frozen/thawed semen from stallions selected for normality of their semen at initial evaluation. Even after rejecting 24% to 67% of all ejaculates frozen from a given stallion, because they contained less than 30% progressively motile spermatozoa after thawing, one cycle pregnancy rates ranged from 8% to 61%. It is clear the motility of thawed spermatozoa is the easiest attribute to observe, however, that quality alone does not necessarily indicate the spermatozoa is fertile. The dosage for optimal pregnancy rates is well established using fresh semen at 500 million progressively motile sperm. The optimal dosage for frozen/thawed semen is unknown. Dosages of 150 to 800 million progressive motile sperm have produced acceptable pregnancy rates. Therefore, in order to maximize the percent per cycle pregnancy rate of your mare one needs to breed to a stallion with a documented highly fertile frozen/thawed semen breeding history.
Pregnancy rates ranging from 31% to 73% have been reported, with the highest rates achieved when mares of known fertility were examined every 6 hours with transrectal ultrasound, administered HCG, and inseminated just before and within 6 hours after ovulation. Such wide variability in pregnancy rates is due to selection of the breeding animals as well as timing, frequency, and total numbers of progressively motile spermatozoa in the insemination.
Selection of the mare is as important as selection of the stallion in a frozen/thawed semen breeding program. Young (4-12 years), foaling mares have been reported to have the highest first cycle pregnancy rates, followed by maiden mares, and the least fertile are barren mares, especially mares that have been barren two years or more. It is best if mares going to be bred with frozen/thawed semen are fully examined by a veterinarian experienced in equine reproduction. Most experts agree that rectal palpation, transrectal ultrasound of the reproductive tract, vaginal exam, uterine culture, uterine cytology and in selected cases uterine biopsy are current standards of practice in evaluating a mare for breeding. Mares that have fluid accumulations within their uterus, mares that have inflammatory cells or uterine cytology, mares with multiple endometrial cysts, or mares with signs of chronic inflammatory changes indicated by the uterine biopsy are not candidates for breeding with frozen/thawed semen.
There is a tremendous amount of effort that goes into timing the insemination of thawed semen. The estrous cycle of the mare is about 22 days. During those 22 days mares usually display receptivity (heat) to the stallion for 4-8 days. Most mares ovulate 24 to 48 hours before the end heat. The mare needs to be examined with transrectal ultrasound and palpation at least daily during the heat until the mare develops an ovarian follicle >35mm with the presence of mucosal folds of endometrium. Then the mare needs examined every 6-8 hours transrectally. With the administration of the hormone HCG(human chorionic gonadotrophin) 75% of the mares will ovulated between 24 and 48 hours. This hormone helps in timing ovulation, but there is still a sizable percentage of mares (25%) that ovulate outside that 24 hour window. Once ovulation has been determined to be imminent then proper thawing procedures of the frozen semen can occur. The thawed semen then needs to be inseminated within 5 minutes. Assuming the mare ovulates within 12 hours of the insemination, we recommend she be inseminated again within 6 hours post-ovulation. The mare should be examined transrectally with ultrasound 12 hours after breeding to evaluate her reaction to the extenders, cryoprotectants, glycerol and semen. Mares that have over 25 mm diameter of fluid accumulation within the lumen of the uterus need treated with oxytocin, uterine lavage, or both.
Breeding mares with frozen semen will be common place in the future. As freezing techniques and media improve, as well as the expertise of the inseminator with improved protocols and procedures, pregnancy rates will continue to improve. However, in my opinion, horses will continue to be selected for many other attributes besides fertility. Therefore, horses will likely never be as reproductively efficient as other domestic species.
Breeding mares with frozen/thawed semen is an exciting challenge as it opens doors to gene pools all over the world. It can add fun and endless possibilities to the breeding program of your horses.