Cryopreservation of Equine Semen - Where Do We Go From Here?
Dickson D Varner
Department of Large Animal Medicine and Surgery, College of Veterinary Medicine
Texas A&M University, College Station, Texas USA

Cryopreserved spermatozoa have been used commercially in the dairy cattle for half a century, and this form of semen technology has virtually revolutionized this industry.  Such success has not been realized in horses, probably owing to biophysical and biochemical differences in the spermatozoa of these two species.  Per-cycle pregnancy rate in mares bred with frozen/thawed semen is reported to range from over 70% to 10% 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% range for many stallions.  One study (Tischner 1979) revealed that, in response to freezing and thawing, approximately 20% of stallions have semen which responds very favorably; approximately 60% of stallions have semen which is affected adversely, and approximately 20% have semen which is damaged severely.  Other investigators have reported similar findings.   Not only does freezability of semen vary greatly among individual stallions, but ejaculates from a given stallion can also respond differently to the freeze-thaw process. (Vidament et al.  1997).  The contribution of factors, such as genetics, nutrition, and environmental toxicants, to such variability among stallions is virtually 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 spermatozoal quality have been culled from freezing programs.

The intrinsic quality of spermatozoa 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 ³ 35%) to the total number of ejaculates frozen  (Vidament et al.  1997).  Seminal plasma may also play an important role in spermatozoal response to freezing.  Aurich et al. (1996) reported that the incorporation of seminal plasma from stallions with good post-thaw motility into semen from stallions with poor post-thaw motility will improve the post-thaw characteristics of the later.  The reverse is also the case, whereby seminal plasma from stallions with poor post-thaw motility will actually increase the cryo-susceptibility of spermatozoa from stallions with good post-thaw motility.

A wide variety of extender types have been used to process semen for freezing.  No single extender has been identified as superior for this purpose.  Most extenders contain a source of lipoproteins, through incorporation of milk products, egg yolk, or a combination of these two ingredients.  The lipoproteins are adsorbed to the plasma membrane of spermatozoa and are thought to aid stabilization of membranes during the freeze/thaw cycles.  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.  Monosaccharides also have osmotic properties.  In addition, they can be utilized by spermatozoa as a source of energy.  Glycerol has been used universally as a cryoprotectant, although investigators have recently reported that other cryoprotectants, such as dimethyl sulfoxide (Chenieret al. 2000), ethylene glycol, methyl formamide or dimethyl formamide (Graham 2000) may yield similar or superior results.   An assortment of electrolytes, and sometimes detergents, are also incorporated into some freezing extenders.  It appears that the composition of the freezing extender will impact the length of the cooling phase required prior to freezing, to optimize results (Heitland et al. 1996)

Many types of packaging systems are available for semen freezing.  Most commonly, 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 when the same packaging system is used.  Palmer (1984) reported that spermatozoal concentrations greater than 100 x 10⁶/ml were detrimental to frozen-thawed spermatozoa, whereas two more recent reports indicated that spermatozoal concentrations up to 400 x 10⁶/ml (Heitland et al. 1996) or 1600 x 10⁶/ml (Leipold et al. 1998) were satisfactory, when 0.5-ml straws were used.  The number of packages required for an insemination dose varies from one to ten 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 versus 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 150 x 10⁶ spermatozoa when post-thaw spermatozoal motility is ³ 35% (Vidament et al.1997) and semen is deposited in the uterine body.  Insemination of 800 million total (Palmer, 1984) or motile (Leipold et al. 1998) frozen-thawed spermatozoa does not appear to improve upon pregnancy rates established when half that sperm number is inseminated.  Insemination of fresh semen into the tip of the uterine horn ipsilateral to the ovary containing a pre-ovulatory follicle will increase the number and percentage of oviductal sperm in the ipsilateral oviduct, as compared to uterine body insemination (Rigby et al. 2000).  Therefore, this insemination protocol may increase sperm colonization of the oviduct and enhance fertility.  Morris et al. (2000) recently reported a one-cycle pregnancy rate of 79% (11/14) when mares were bred using a hysteroscopic method whereby only 5 million frozen-thawed motile spermatozoa were deposited on the uterotubal papilla ipsilateral to the ovary containing a preovulatory follicle.

Laboratory-based tests are commonly used to evaluate viability of frozen/thawed spermatozoa.  The percentage of progressively motile spermatozoa in samples is the most widely employed parameter of measurement.  While post-thaw motility of spermatozoa provides useful information regarding sperm viability, it is not an exact measure of fertilizing potential.  Some semen samples express a high percentage of progressively motile sperm, yet yield low pregnancy rates.  Likewise, some samples contain a lowered percentage of progressively motile spermatozoa but possess acceptable fertility.  Investigators are presently attempting to develop additional tests which would improve the predictive power of laboratory testing, and the use of fluorescent probes and flow cytometric methods to assess spermatozoal integrity is becoming more commonplace.

In conclusion, considerable variation exists among stallions with regard to susceptibility of spermatozoa to cryo-injury.  In order for spermatozoa to be capable of establishing a healthy pregnancy following cryopreservation, certain spermatozoal qualities must be maintained.  The freeze-thaw process generally results in lethal damage to a relatively high percentage of the sperm population, whereas a portion of the remaining cryo-susceptible spermatozoa may suffer sublethal damage that, nevertheless, hinders or neutralizes their fertilization potential.   Disruptions in function could be attributed to alterations in membrane lipids/proteins, cytoskeletal elements, nuclear components, or a combination, thereof (for review, see Watson, 2000).   In addition to many reports emphasizing the detrimental effects of cryopreservation on post-thaw sperm motility and membrane viability, recent studies are revealing that cryopreservation may accelerate the spermatozoal capacitation process, interfere with spermatozoal binding to the oviductal epithelium (thereby reducing the oviductal sperm reservoir), and reduce the life expectancy of the spermatozoa following thawing (for review, see Curry 2000).  Given these intrinsic 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 spermatozoal entry into the oviduct. 

Without question, we require a greater understanding of the injurious effects that cryopreservation exerts on spermatozoa, so that we can develop repeatable methods for obtaining a commercially valuable product. The intensified interest of the horse industry in frozen semen may be the impetus required for development of reliable and successful cryopreservation techniques. As investigators, our challenge will be to clarify the mechanisms of cryoinjury, and devise strategies to minimize these impairments, so that a uniformly-applicable cryopreservation technique will be effective for a large percentage of breeding stallions.

References

1.        Aurich JE, Kühne A, Hoppe H, Aurich C 1996.  Seminal plasma affects membrane integrity and motility of equine spermatozoa after cryopreservation. Theriogenology 46: 791-797.

2.        Chenier T, Merkies K, Liebo S, Plante C, Johnson W 1998. Evaluation of cryoprotective agents for use in the cryopreservation of equine spermatozoa.  Proceedings of the Annual Meeting of the American Association of Equine Practitioners: 5-6. 

3.        Curry MR 2000. Cryopreservation of semen from domestic livestock. Reviews of Reproduction 5: 46-52.

4.        Graham JK 2000. Evaluation of alternative cryoprotectants for preserving stallion spermatozoa.  Proceedings International Congress on Animal Reproduction: Abstract 307 Abstract.

5.        Heitland AV, Jasko DJ, Squires EL, Graham JK, Pickett BW, Hamilton C 1996. Factors affecting motion characteristics of frozen-thawed stallion spermatozoa.  Equine Veterinary Journal 28: 47-53.

6.        Leipold SD, Graham JK, Squires EL, McCue PM, Brinsko SP, Vanderwall DK 1998. Effect of spermatozoal concentration and number on fertility of frozen equine semen.  Theriogenology 49: 1537-1543.

7.        Morris LHA, Tiplady C, Cook B, Wilsher S, Li X, Allen WR 2000. Hysteroscopic insemination of mares with low numbers of frozen-thawed ejaculated and epididymal spermatozoa. Proceedings 5th International Symposium on Equine Embryo Transfer, Saari, Finland: 12.

8.        Palmer E 1984. Factors affecting stallion semen survival and fertility.  Proceedings International Congress on Animal Reproduction: 377.

9.        Rigby S, Derczo S, Brinsko S, Blanchard T, Taylor T, Forrest DW, Varner D in press. Oviductal sperm numbers following proximal uterine horn or uterine body insemination. Proceedings of the Annual Meeting of the American Association of Equine Practitioners.

10.     Tischner M 1979. Evaluation of deep-frozen semen in stallions.  Journal of Reproduction and Fertility (Suppl) 27: 53-59.

11.     Vidament M, Dupere AM, Julienne P, Evain A, Noue P, Palmer E 1997. Equine frozen semen: Freezability and fertility field results.  Theriogenology 48: 907-917.

12.     Watson, PF 2000. The causes of reduced fertility with cryopreserved semen. Animal Reproduction Science 60-61: 481-492.