Physiological Aspects of Stallion Semen Cryopreservation

Transcription

1 Physiological Aspects of Stallion Semen Cryopreservation Bo G. Crabo, DVM, PhD Author s address: Professor Emeritus, University of Minnesota, P.O. Box 4105, Cave Creek, AZ AAEP. 1. Introduction The history of artificial insemination (AI) begins with a story about some Arabs stealing stallion semen from a rival tribe and bringing it home for breeding in a sponge. When AI began its modern development in Russia during the early 20th century by Ivanov, the main object was not cattle but horses as documented by Milovanov. 1 In the late 1940s, Christopher Polge, working with chicken and the economically more important bull semen, pioneered development of frozen-thawed semen technology. This, however, was done by unknowingly using a bottle containing glycerol and allowing the semen to remain in glycerolated extender for 18 h ( glycerol equilibration time ). 2 Thus, frozen semen technology was developed without much knowledge of the physiology of spermatozoa or the events leading to fertilization. Sperm motility was then, and largely remains, the main criterion upon which success or failure of the freezing procedure is evaluated in the laboratory. Sperm motility, however, proved to be of very little value in predicting the success of freezing swine semen. Polge had already shown in 1956 that the glycerol concentration in liquid semen was inversely related to its fertility. Glycerol concentrations of less than 2% were necessary to achieve good fertility also after freezing of boar semen, although maximum sperm motility was seen at a concentration of 7% glycerol. 3 In 1957, Canadians Barker and Gandier reported the first foaling following insemination with frozen epididymal spermatozoa. 3 During the next decade a dozen more successful reports with ejaculated semen followed from Germany, Japan, Russia, and the U.S. Sugar-containing diluents were most commonly used. Per cycle pregnancy rates varied between 13% and 44%, and the number of mares inseminated per trial varied between 5 and 116. Greater numbers were, however, used in the American Breeders Service (ABS) study utilizing 342 mares, and in China, where He Wen Ye reported respectable fertility in over 40,000 mares a inseminated with semen diluted in sucrose-egg yolk and vapor frozen as pellets. All this happened before inseminations could be timed to ovulation by the use of ultrasonography. Moreover, wide individual variations in fertility of frozen semen among stallions may not yet have been recognized. 4 A successfully preserved spermatozoon must be able to produce a viable embryo following an in vivo insemination. Many widely different mechanisms involved must be maintained. The spermatozoa must survive the uterine environment, be transported to the oviduct, be maintained there until the oocyte arrives, and be prepared to penetrate the NOTES AAEP PROCEEDINGS Vol

2 oocyte. For most species it means that the spermatozoon must undergo capacitation. The equine spermatozoon must be able to attach to the oviductal epithelium while stored, be released when the oocyte arrives, be motile during penetration of the ovum, be able to undergo a timely acrosome reaction to release enzymes necessary for reaching the cell membrane of the oocyte, and finally fuse with the cell membrane of the oocyte before being engulfed. 5,6 After getting inside the oocyte, the sperm chromatin has to form the male pronucleus, which needs to fuse with the female pronucleus to complete fertilization. Fertility trials are the only way to assess the whole process but it is easy to see that many of the steps involved are membrane-related, and most likely dependent on the protein components of the membrane. In the following, the freezing and thawing process will be discussed in relation to aspects of membrane composition and integrity. Information on the horse is very limited in this respect and references on other species will be used where available. 2. Principles of Cryopreservation Freezing Rate In the absence of cryoprotective agents in the medium, there is one optimal freeze rate for every cell type. 7 This freeze rate is directly related to the water permeability of the cell membrane. Cells that have higher water permeability require a higher freeze rate for survival. Ideally, extracellular ice formed during freezing will lead to increased osmotic pressure in the unfrozen medium, which in turn will pull more water out of the cell. The concentration of salts will thus increase both intra- and extracellularly as water freezes, and the temperature needs to decrease to protect proteins from salting out. Thus, too rapid freezing rates cause structural damage by intracellular ice formation, and too slow freeze rates cause salt damage to proteins. Damage to most cells occurs during freezing and thawing although some cells, most notably boar sperm, are susceptible to above freezing chilling injuries. While in the frozen state at temperatures below 60 C, cells are dehydrated and may be stored unharmed for over 1,000 years. Thawing of semen generally should be done at very rapid rates to decrease the possibility of damage from extracellular ice crystal growth. Supercooling and Seeding The temperature of the semen will invariably cool below the freezing point before freezing occurs (supercooling). A high degree of supercooling is generally considered unwanted because it will cause a rapid upward and downward temperature fluctuation. Seeding at a minimum temperature below the freezing point will initiate ice crystal formation and avoid the temperature drop. Seeding may be initiated by vibration or touching of the straw with an instrument colder than the package as is done in Vol. 47 AAEP PROCEEDINGS embryo freezing. Seeding will otherwise be initiated in the straw where it touches the freezing rack. Most heat is conducted away from the straw through the metal rack if the rack is in contact with the liquid nitrogen and therefore colder than the vapor. Freezing racks offering multiple contact points have been used for maxistraws (2.5 5 ml) but there is no data indicating an advantage for such racks. Cryoprotectants Glycerol has remained the major penetrating cryoprotectant for freezing semen although others have been or are being investigated. 8 Its mode of action remains obscure even if it is known that it will modify ice crystal formation during freezing and thawing. One may perhaps also speculate that it replaces water in frozen macromolecules to prevent folding and denaturation. 2 Glycerol was initially used in concentrations of 7 10%. A detrimental effect of glycerol was unknown until Polge 10 found that a concentration of glycerol between 2 4% decreased fertility in swine. Subsequently, boar sperm cell membrane damage, as evidenced by increased cell membrane leakage of an intracellular enzyme, was shown to be caused by glycerol in concentrations over 2%. 10 Although the fertility of frozen stallion semen was similar with 7% and 2% glycerol, 3 glycerol addition to fresh stallion semen decreased fertility as had been shown for pig semen. 11 A final glycerol concentration of 2 2.5% is now most commonly used for freezing stallion semen. Osmotic rupture of cells containing 2% glycerol will occur if glycerolated cells are suspended in glycerolfree medium, 10 but this is likely not the only way that glycerol affects fertility in frozen semen. Egg yolk has been an ingredient of many extenders since its presence was shown to protect against cold shock. 12 Its contents of phospholipids may help to protect the integrity of phospholipid bilayer of the cell membrane. Many stallion extenders for freezing contain Equex STM (formerly known as Orvus Es Paste) which is an emulsifier believed to keep more of the egg yolk lipids in solution. 3 It is very toxic to sperm in the absence of egg yolk or in higher concentrations. Sugars have been known to possess cryprotective properties and are favored with stallion semen. 3 A system with hydrogen buffers (Good s buffers) and egg yolk was developed to enable freezing of bull and boar spermatozoa in the absence of glycerol. 3 The Good s buffers and Tris, however, appear to be harsh on erythrocyte membranes in the absence of phosphates, and also on stallion sperm. 13,14 Although they have been tested, they are probably no longer used. 3 Diluents and Dilution Freezing of undiluted semen with some success is possible only with human semen. 15 In early studies with bull semen, the expression glycerol equilibration time was coined. 2 It is possible that the

3 long time necessary between dilution and freezing in bull semen had very little to do with glycerol penetration of the spermatozoa, but rather reflected the time needed for a seminal protein envelope surrounding the sperm to be reestablished, as was shown for boar semen. 16 Also, the dilution effect may represent instability in the phospholipid bilayer of the sperm membrane, possibly caused by movement of the surface proteins. Stallion spermatozoa also possess a protein coat of seminal plasma origin, which is fairly firmly attached and may be removed during capacitation. 17 Some diluents containing skim milk proteins may be more beneficial to stallion sperm than other ones. It is practically impossible to freeze stallion semen without removing most of the bulky seminal plasma, as in boar semen by centrifugation. Damage to the spermatozoa during centrifugation is likely occurring when the spermatozoa are packed in the tube and is believed to be mechanical. Early studies with rabbit semen suggested a maximum centrifugal force of g, but slightly higher forces have been used for stallion semen. b Interactions Although there is an ideal freeze rate for stallion spermatozoa, it is virtually impossible to achieve it even in a very small package. 18 Packaging for stallion semen has varied from 5- or 2.5-ml to 0.5-ml plastic straws or flat bags to minimize variations in the freeze rate. There is, however, an interaction between the ideal freeze rate and the concentration of glycerol which has been illustrated with the socalled Fiser s tent structures. 7 At an optimal glycerol level (for ram semen, 4%), a wide range of freezes rate is acceptable (for ram semen, C/ min) for maximal sperm motility. At higher glycerol concentrations, optimal recovery occurs at a freeze rate of 5 C/min and decreases as the freeze rate increases, while at lesser glycerol concentrations, the greatest recovery occurs at the highest freeze rate tested (100 C/min). Thus, the presence of glycerol in the diluent will compensate for our inability to optimize freezing rate due to the shape and volume of the package. Interactions should be expected between salts and proteins, also including ph. The effect of such protein electrolyte interactions is not well known. However, the solubility of many proteins is dependent on the ionic strength of the solute, and this may have an effect on the amount of seminal proteins remaining on the sperm membrane after the freeze thaw cycle. The importance of this will be discussed later in this article relative to sperm elimination from the uterus. 19 Sodium citrate has sometimes been used in pre-freezing extenders for stallion semen and is commonly used for bull semen. It will keep all seminal proteins in solution whereas lactose, commonly used as freezing extender for stallion semen, has by itself an ionic strength of zero, which may lead to precipitation of certain proteins. A more detailed discussion of protein/electrolyte relationships as regards boar semen appears elsewhere Role of Seminal Proteins in Sperm Elimination from the Uterus For most species investigated, epididymal sperm maturation is essential for the spermatozoa to acquire fertilizing ability. It is very probable that this is dependent more on the protein composition of the sperm membranes than on the acquisition of ability for motility. 20 During ejaculation, a seminal protein of non-epididymal origin is layered over the spermatozoa. 17,20 As discussed previously, this protein layer may be modified by dilutors used for freezing of the semen as well as by the capacitation process. It is not known if the inclusion of EDTA in some extenders used for freeze preservation of stallion semen is done to bind the calcium necessary for capacitation. 21,22 Capacitated spermatozoa additionally have more rapid forward motility and greater lateral head displacement during motility. It is therefore possible that using maximal sperm motility for formulating media for freezing stallion semen may favor capacitated spermatozoa or those possessing minimal protein coats. It was shown that rabbit epididymal spermatozoa briefly incubated in seminal plasma remained longer in ligated uterine pockets before being phagocytosed than those that had not. 16 It is now accepted that equine spermatozoa trigger the inflammatory response that will lead to the elimination of semen and debris following insemination or mating. Seminal plasma has a modulating effect on this influx of neutrophilic leukocytes (PMN) following insemination. 23,24 When stallion semen is frozen, the seminal plasma is removed by centrifugation. A more pronounced influx of PMNs is often seen following insemination with frozen semen than with fresh. 25 One might therefore speculate that loss of a seminal coat could make spermatozoa more vulnerable to the attack of PMNs. The presence of seminal plasma has been shown to protect equine spermatozoa from being phagocytized by PMNs Sperm Surface Probes A semen assay originally developed by Graham and consisting of filtration of semen through a Sephadex gel supported by glass wool yielded results remarkably well correlated to the fertility of many species but particularly water buffalo and stallion. 4 Evidence subsequently obtained suggested that the trapping of stallion spermatozoa in Sephadex was caused by clusterin, a heterodimeric glycoprotein produced as different glycoforms in testis and epididymis. 17,24 Studies in domestic animals have shown that acrosome damaged spermatozoa are trapped in glass wool/sephadex filters but it is not known if such spermatozoa attach to the glass wool or Sephadex component of the filters. The acrosomes of stallion spermatozoa (like human sperma- AAEP PROCEEDINGS Vol

4 tozoa) are very difficult to evaluate without special staining, unlike those of other domestic animals. However, virtually all acrosome reacted stallion spermatozoa were eliminated in the filters only when they contained both glass wool and Sephadex. 26 Earlier, we had hypothesized that testicular or epididymal clusterin would be incorporated in the sperm membrane and then covered with coating proteins from the lower reproductive tract. 17 The Sephadex assay would then be an indication of the fraction of capacitated spermatozoa. However, we did not obtain much evidence for that hypothesis. Instead, in cattle and sheep the epididymal form of clusterin was present on abnormal or damaged spermatozoa only and the incidence of clusterinpositive spermatozoa very well correlated to bull fertility. 24,27,28 Clusterin is a potent inhibitor of complement-induced cytolysis. It is very possible that clusterin in the seminal plasma attaches to the sperm membrane of injured spermatozoa in an attempt to rescue them as was suggested for injured kidney cells. 29 The incidence of clusterin-positive cells in the semen would then be a powerful indicator of the quality of the semen. Stallion clusterin is sufficiently different from the clusterin of other species so that most antibodies against rabbit, sheep or cattle clusterin do not cross-react with equine clusterin. 30 It appears to me that the clusterin assay would be worthwhile to develop for stallion semen in future approaches towards improved frozen stallion technology. 5. Which is the Frozen Semen Technology of My Choice? Improvements in technology of freezing stallion semen from the early 1970s until now may be fewer than we think. The greatest progress has likely been made in timing of insemination thanks to the use of ultrasonography, understanding of uterine physiology, and the realization that there are wide differences in fertility among stallions, particularly following freezing of the semen. Before a reliable semen assay that truly reflects the fertilizing ability of the semen (compare above) is developed, there is not much chance that real improvements in the freezing technology will occur. Sperm motility is useful to monitor whether something went wrong during processing. Using the Sephadex filtration and sperm motility as assay, Minnesota research has confirmed that replacing the initial dilutor of Martin, Klug, and Guenzel (1979) with Kenney s extender but otherwise following that technique works well. 21,31 There may be a beneficial effect of keeping some seminal plasma after centrifugation. The second dilutor contains EDTA which chelates calcium; Equex STM which may keep more egg yolk lipids in solution; and has a final concentration of glycerol of 2 2.5% which should not cause osmotic or membrane changes. It is, however, in the light of the early findings with bull semen both as regards cold shock and equilibration time interesting that Vol. 47 AAEP PROCEEDINGS stallion semen does not seem to need slow cooling or an extended time before freezing. There are, however, freezing techniques for stallion semen that take those aspects into account. 32 The advantage of being able to freeze the semen in a relatively short time span is obvious. On the other hand, the use of a programmable freezing machine does not seem to be necessary since vapor freezing 2.5 cm over liquid nitrogen produces very similar results. Racks on which the straws rest on several wires may be advantageous since seeding is induced at multiple points, but there are no data to support this. I am indebted to my former collaborators M. H. T. Troedsson and Kathryn J. Loseth for valuable advice and assistance. References and Footnotes 1. Milovanov VK. Principles of artificial insemination [in Russian]. Moscow: State Publishing House, Polge C. Sperm freezing: Past, present and future. In Johnson LA, Larsson K, eds. Deep freezing of boar semen, Beltsville, MD: USDA, 1985; Graham EF, Crabo BG, Pace MM. Current status of semen preservation in the ram, boar and stallion. J Anim Sci 1978; 47(Suppl 2): Samper JC, Hellander JC, Crabo BG. Relationship between the fertility of fresh and frozen stallion semen and semen quality. J Reprod Fertil 1991;44(Suppl): O Rand MG. Changes in sperm surface properties correlated with capacitation, In: Fawcett DW, Bedford JM, eds.: The spermatozoon: maturation, motility, surface properties and comparative aspects. Baltimore: Urban & Schwartzenberg, Thomas PGA, Ball BA, Miller PG, Brinsko SP, Southwood LA. A subpopulation of morphologically normal, motile spermatozoa attach to equine oviductal epithelial cell monolayers. Biol Reprod 1996;57: Mazur P. Basic concepts in freezing cells. In Johnson LA, Larsson K, eds.: Deep freezing of boar semen. Beltsville, MD: USDA, 1985; Alvarenga MA, Graham JK, Keith SL, Landim-Alvarenga FC, Squires EL. Alternative cryoprotectors for freezing stallion spermatozoa, in Proceedings. 14th Int Cong Anim Reprod & AI, 2000;2: Polge C. Artificial insemination in pigs. Vet Rec 1956;68: Bower RE, Crabo BG, Pace MM, Graham EF. Effects of dilution and glycerol on the release of glutamic-oxaloacetic transaminase (GOT) from boar spermatozoa. J Anim Sci 1973;36: Picket BW, Voss JL, Demick DS. Stallion semen extenders, in Proceedings. 20th Annu Conv Am Assoc Equine Practnr Conv 1975; Phillips PH, Lardy HA. A yolk-buffer pablum for the preservation of bull semen. J Dairy Sci 1940;23: Picket BW, Burwash LD, Voss JL, Back DG. Effect of seminal extender on equine fertility. J Anim Sci 1975;40: Crabo BG, Graham EF, Brown KJ, Mäki-Laurila M. Effect of some zwitterionic buffers on the cell membrane of dog and cow erythrocytes. Cryobiology 1973;10: Sherman JK. Banks for frozen human semen: Current status and prospects. In The Integrity of Frozen Spermatozoa. Washington, DC: The National Research Council 1978; Pavelko MK, Crabo BG. Possible importance of some sperm coating proteins and their behavior during preservation of boar semen, in Proceedings. AI 1976; th Intl Cong Anim Reprod &

5 17. Samper JC, Hamilton DW, Pryor JL, et al. Mechanism of Sephadex trapping of capacitated stallion spermatozoa. Biol Reprod 1991; Larson EV, Graham EF. Measurements of cooling rates and cell survival at different points within a sphere. Cryobiology 1973;10: Troedsson MHT, Lee C-S, Franklin RD, Crabo BG. The role of seminal plasma in post-breeding uterine inflammation. J Reprod Fert 2000;56(Suppl): Crabo BG. Post-testicular sperm maturation and its importance to deep freezing of boar sperm. In Johnson LA, Larsson K, eds. Deep freezing of boar semen, Beltsville, MD: USDA, 1985; Merkt H, Klug E. Deep freezing of stallion spermatozoa, Hannover experience, in Proceedings. Symp Equine Reprod 1987; Squires EL, Amann RP, Picket BW. Preservation of stallion semen Colorado experience, in Proceedings. Symp Equine Reprod 1987; Troedsson MHT, Liu IKM, Crabo BG. Sperm transport and survival in the mare. Theriogenology 1998;49: Ibrahim NM, Troedsson MHT, Foster DN, et al. Reproductive tract secretions and bull spermatozoa contain different clusterin isoforms that cluster cells and inhibit complementinduced cytolysis. J Androl 1999;20: Kotilainen T, Huhtinen M, Katila T. Sperm-induced leukocytosis in the equine uterus. Theriogenology 1994;41: Esser AI. Assessment of glass wool and Sephadex filtration in trapping stallion spermatozoa with altered acrosomes. Science in Agriculture Thesis, University of Minnesota, Ibrahim NM, Gilbert GR, Loseth KJ, Crabo BG. Correlation between clusterin-positive spermatozoa determined by flow cytometry in bull semen and fertility. J Androl 2000;21: Ibrahim N, Loseth K, Romano J, et al. Effect of scrotal insulation on sperm and seminal clusterin in rams and its relation to semen quality, in Proceedings. 14th Intl Congr Anim Reprod AI 2000; Rosenberg ME, Silkensen J. Clusterin: Physiologic and pathophysiologic considerations. Int J Biochem Cell Biol 1995;27: Barber JA, Farris JA, Troedsson MHT, et al. Nucleotide sequence of the complimentary DNA encoding equine clusterin. Anim Biotech 1996;7: Alghamdi A, Troedsson MHT, Xue J-L, Crabo BG. Freezing of stallion semen: Improved technique for freezing semen from stallions with poor freezing ability. Submitted. 32. Magistrini M, Couty I, Palmer E. Interactions between sperm packaging, gas environment, temperature and diluent on fresh stallion sperm survival. Acta Vet Scand 1992; 88(Suppl): a He Wen Ye: (personal communication) b Orgebin-Cirst MC: (personal communication) AAEP PROCEEDINGS Vol