August 31, 2012
A look at the potential and pitfalls of using frozen semen for horse breeding.
By Dr. Dickson D. Varner
Cryopreservation implies subjecting cells or tissues to an extremely low temperature in an effort to slow or stop biological activity. The storage temperature is typically -196 C (-321 F), the temperature of liquid nitrogen. When sperm are stored at this temperature, they are literally “suspended in time” in hopes that, once thawed, the sperm will be capable of fertilization.
As metabolism is essentially nil, some project that frozen sperm may be stored effectively for centuries.
Cryopreserved semen has been used commercially in the dairy cattle for more than half a century, and this form of semen technology virtually revolutionized this industry. Such success has not been realized in horses, probably owing to biophysical and biochemical differences in the sperm of these two species.
Per-cycle pregnancy rate in mares bred with frozen and thawed semen can range from more than 70 percent to 10 percent or less. Although the upper level of this range would lead one to suspect that it might be commercially feasible, the per-cycle pregnancy rate is in the 25-40 percent range for many stallions. The tolerance of sperm to freezing varies greatly among individual stallions, and also among ejaculates from a given stallion.
The contribution of factors, such as genetics, nutrition and environmental toxicants, to such variability among stallions is largely unstudied. It has been postulated that the present-day success with frozen semen in the dairy industry may have arisen from a decades-long selection process, whereby bulls with unacceptable post-thaw sperm quality had been culled from freezing programs.
The initial quality of ejaculated sperm appears to impact their ability to withstand the freeze-thaw process. Fertility of freshly ejaculated spermatozoa is strongly correlated with the “freezability” of semen, where freezability is defined as the ratio of acceptable ejaculates (i.e.., those with a post-thaw motility greater than 35 percent) to the total number of ejaculates frozen. Seminal plasma (the liquid portion of semen) may also play an important role in sperm response to freezing.
A wide variety of extenders have been developed to process semen for freezing. No single extender has been identified as superior for this purpose. Most extenders contain a source of lipoproteins (fat-protein molecules), through incorporation of milk products, egg yolk, or a combination of these two ingredients. The lipoproteins attach to the plasma membrane of sperm and are thought to aid stabilization of membranes during the freeze-thaw cycle.
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Sugars, such as monosaccharides (e.g., glucose or fructose), disaccharides (e.g., sucrose or lactose) and trisaccharides (e.g., raffinose), are also commonly incorporated into semen extenders. Disaccharides and trisaccharides are nonpermeable sugars and are used, primarily because of their osmotic effects, to aid in partial dehydration of sperm prior to freezing. Monosaccharides also have osmotic properties. In addition, they can be utilized by sperm as a source of energy. Glycerol ha
s been used most commonly as the primary cryoprotectant in extender; however, other cryoprotectants, such as dimethyl sulfoxide, ethylene glycol, methylformamide or dimethyl formamide have also yielded good results. An assortment of electrolytes and sometimes detergents are also incorporated into some freezing extenders.
Many types of packaging systems are available for semen freezing. Most commonly, equine semen is packaged in polyvinyl chloride straws with a volume capacity ranging from 0.5 – 5 ml. Occasionally, polypropylene bags or flattened aluminum tubes are used as semen packages. These containers accommodate volumes of 10-25 ml of extended semen. The general consensus is that 0.5-ml capacity straws are the most suitable package for semen because they provide the most uniform freeze and thaw rates for the package contents. The optimal sperm number per package is controversial and tends to vary between laboratories, even when the same packaging system is used. Depending on the laboratory, sperm concentration can vary between 100 million and 1.6 billion sperm per milliliter. The number of packages required for an insemination dose can vary from one to 10 or more.
Information available to date suggests that frozen-thawed semen is not dramatically affected by extender type, packaging system or freezing method (i.e., freezing semen in static nitrogen vapor vs. a computerized nitrogen vapor freezer with specific pre-programmed freeze rates). Fertility can be affected by insemination dose. An insemination dose should probably contain a minimum of 200 million motile sperm when thawed semen is deposited in the uterine body. If pregnancy rate is low with this insemination dose, it is unlikely that increasing the dose substantially will improve pregnancy rate. Insemination of semen into the tip of the uterine horn adjacent to the ovary with a dominant follicle or recent ovulation (typically referred to as low-dose horn insemination) will probably result in a similar pregnancy rate with a reduced insemination dose.
Laboratory-based tests are commonly used to evaluate viability of frozen-thawed sperm. The percentage of progressively motile sperm in samples is the most widely used measure of sperm function. While post-thaw motility of sperm provides useful information, it is not an exact measure of sperm fertilizing potential. Some semen samples exhibit a high percentage of progressively motile sperm, yet yield low pregnancy rates. Likewise, some samples contain a low percentage of progressively motile spermatozoa but possess acceptable fertility. More studies are needed to identify a battery of laboratory tests that will have reliable predictive value in determining the fertility of frozen-thawed semen.
Frozen-thawed sperm has a shorter life span in the mare’s reproductive tract than that of fresh or cooled semen. As such, it is important to breed mares as close to ovulation as possible. Interestingly, sperm are thought to undergo changes when frozen and thawed (called cryocapacitation), such that they are more ready for fertilizing an egg immediately after insemination than fresh semen (a process called capacitation). Therefore, one can breed mares within a few hours after ovulation and expect the same pregnancy rate as when breeding prior to ovulation.
In summary, considerable variation exists among stallions with regard to susceptibility of spermatozoa to cryo-injury. Given these side effects of cryopreservation, it is advisable to breed mares close to the time of ovulation and to use an insemination method that will maximize sperm entry into the oviduct, where fertilization occurs. Without question, we require a greater understanding of the injurious effects that cryopreservation exerts on sperm, so that we can develop repeatable methods for obtaining a commercially valuable product from a higher percentage of stallions.
Dr. Dickson D. Varner is professor and Pin Oak Stud Chair of Stallion Reproductive Studies in the Department of Large Animal Clinical Studies at the College of Veterinary Medicine and Biomedical Sciences at Texas A&M University.