Commercial semen freezing: Individual male variation in cryosurvival and the response of stallion sperm to customized freezing protocols

Animal Reproduction Science 105 (2008) 119–128

Commercial semen freezing: Individual male variation in cryosurvival and the response of stallion sperm to customized freezing protocols夽 P.R. Loomis a,∗ , J.K. Graham b b

a Select Breeders Service, Inc., 1088 Nesbitt Road, Colora, MD 21917, USA Animal Reproduction and Biotechnology Laboratory, Department of Biomedical Sciences, Colorado State University, Fort Collins, CO 80523-1680, USA

Available online 26 November 2007

Abstract One of the challenges for those attempting to cryopreserve stallion spermatozoa is dealing with the stallion to stallion variability in the cryosurvival of their semen. In the dairy industry, each bull stud, essentially utilizes a single cryopreservation technique, and bulls that produce sperm that do not cryopreserve well using that technique are replaced by other bulls. However, replacing stallions is unlikely to prove acceptable to the equine industry, where specific genotypes are desired. Instead, to increase the number of stallions that can be effectively utilized for cryopreserved semen production, it is likely that more than one method for cryopreserving sperm will be necessary. This manuscript reviews some of the processes involved in cryopreservation, how individual sperm physiology affects the ability to survive freezing and thawing, and how cryopreservation protocols can be customized to maximize sperm cryosurvival on an individual stallion basis. © 2007 Elsevier B.V. All rights reserved. Keywords: Cryopreservation; Equine; Sperm; Male variation; Customizing protocols

1. Introduction A survey conducted in 2004, by the American Horse Council Foundation, indicated that there are approximately 9.2 million horses in the United States, and it is estimated that the U.S. horse industry contributes approximately $101.5 billion to the country’s GDP. Since its introduction over 30 years ago, artificial insemination within the horse breeding industry has grown steadily. 夽 This paper is part of the special issue entitled “Understanding and Exploiting Spermatozoa – A Festschrift for Rupert P. Amann” guest edited by George E. Seidel Jr, James K. Graham and D.N. Rao Veeramachaneni. ∗ Corresponding author. Tel.: +1 410 658 3328; fax: +1 410 658 3329. E-mail address: [email protected] (P.R. Loomis).

0378-4320/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.anireprosci.2007.11.010

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In 2004, approximately 440,000 registered mares were bred, of which 88% (387,200) were breeds that permit the use of artificial insemination. Following the development of commercial liquidcooled transported semen techniques about 20 years ago, breeders quickly realized the benefits of transporting semen to mares rather than mares to stallions. More recently, the logistical limitations (time, distance and transportation delays) on liquid-cooled semen and the added benefits of frozen semen have led breeders to look towards frozen semen as a practical and efficient method for transporting or storing stallion semen. Impediments to the widespread commercial application of frozen equine semen include: 1. Greater veterinary costs for reproductive management of mares inseminated with frozen compared to liquid-cooled semen. 2. The need for stallion owners to ship their stallions to centralized laboratories for access to experienced professionals able to efficiently cryopreserved semen. 3. Less fertility with frozen semen than with liquid-cooled semen for many stallions. 4. There is no selection of stallions for fertility with frozen semen. Therefore, customized protocols are required to maximize post-thaw semen quality. 5. Historically, a lack of quality standards has led to the distribution of semen with very variable post-thaw quality resulting in reluctance among breeders and veterinarians to use frozen semen. Recently, a simple, cost-effective timed insemination protocol for managing mares inseminated with frozen semen has been adopted by many practitioners as a way to decrease the costs associated with frozen semen AI. The protocol involves two inseminations at 24 and 40 h following administration of an ovulation inducing agent such as hCG or Deslorelin to a mare with a mature, pre-ovulatory follicle; the induced ovulation typically occurs between the two inseminations. This technique allows mares to be examined once daily during estrus until ovulation is confirmed, similar to the management of mares inseminated with liquid-cooled semen. Controlled studies (Reger et al., 2005) and clinical trials (Barbacini et al., 2000; Barbacini et al., 2005; Loomis and Squires, 2005) have confirmed that this protocol can be used for many stallions with no decrease in fertility compared to liquid-cooled semen. 2. Freezability of stallion sperm In dairy cattle, bulls have been selected by the AI industry for many years based on the ability of their sperm to withstand the stresses of standard cryopreservation protocols. This selection has led to an increasingly uniform and positive response of selected AI bulls to cryopreservation. No such selection has been applied to stallions and as a result there is a wide variation in semen freezability among individuals. A 1976 study conducted by Japanese workers cited by Amann and Pickett (1987) examined pre-freeze and post-thaw sperm motility of 336 ejaculates from 40 stallions. Using a single standard protocol, 38% of stallions produced spermatozoa with average post-thaw motility that was 80–100% of initial pre-freeze values. The remaining 62% produced sperm that had post-thaw motility <65% of the pre-freeze value. Similar results were reported by M¨uller in 1986 (personal communication) who evaluated 341 stallions in Czechoslovakia and classified 35% as “good” freezers based on >60% initial motility, >70% normal morphology and ≥30% post-thaw motility; 25% were considered “average” and 40% were “poor” freezers. Tischner (1979) evaluated the post-thaw quality of 200 ejaculates from 36 stallions and concluded that stallions could be grouped into three categories based on percent motility and longevity of motility after thawing. Approximately 20% of stallions examined produced semen that with-

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stood the stresses associated with cryopreservation well and were considered “good freezers,” 60% were considered “fair/sufficient freezers” and 20% of the stallions produced spermatozoa that did not withstand cryopreservation and were considered “poor/insufficient freezers.” Good freezers produced spermatozoa that had post-thaw motility >40%, fair freezers 20–40% and poor freezers <20%. Tischner concluded that “before frozen semen becomes widely used it will be necessary to select stallions whose semen is conducive to freezing and thawing.” In their 1987 review of stallion semen cryopreservation, Amann and Pickett estimated that around 25% of stallions from the general population could achieve “acceptable” pregnancy rates with frozen semen while 30% would yield extremely low pregnancy rates. Even when ejaculates were selected for post-thaw progressive motility ≥35%, pregnancy rates, after insemination of mares, ranged from 8 to 61% per estrous cycle. Vidament et al. (1997) reported on semen frozen from 161 stallions using a single standard cryopreservation protocol in France over a 10-year period (1985–1994). After thawing, sperm motility was evaluated and ejaculates possessing ≥35% progressive motility (measured subjectively) or those with >35% “rapid” sperm (path velocity >30 ␮m/s measured using CASA) were considered acceptable. They considered stallions that produced acceptable post-thaw motility for ≥33% of ejaculates to be acceptable for use in a frozen semen program. Fifty one percent of tested stallions met these criteria. 3. Male variation and its effect on sperm cryosurvival Inherent differences exist in the ability of sperm, from different males, to survive cryopreservation. Differences exist not only between species in the survival rates of sperm (Darin-Bennett and White, 1977), but also between individual males within a species. These differences are probably due to inherent differences between both species and individuals within a species in sperm biochemistry and metabolism. However, to understand how these differences affect cryosurvival, we need to understand what happens to cells during cryopreservation, what types of damage occur to the cells and when during the process that damage occurs. The principles of sperm cryopreservation have been reviewed previously (Amann and Pickett, 1987; Hammerstedt et al., 1990; Amann, 1999). Basic principles of cell cryopreservation include: 1. During cooling physical changes occur in the cell membrane. When the membrane is cooled below its phase transition temperature, the membrane changes from a liquid crystalline state to a gel state. Lipid and protein rearrangements that occur as the membrane undergoes this transition during either cooling or warming can lead to cold shock and cooling damage (Amann and Pickett, 1987; Hammerstedt et al., 1990; Amann, 1999). 2. Metabolism changes with temperature. The general rule of thumb is that metabolism is reduced by half with each drop of 10 ◦ C. Therefore, when stallion sperm are reduced from body temperature (39 ◦ C) to 5 ◦ C, sperm metabolism is reduced to about 10% of the original level. In addition, when the cells are frozen at sufficiently low temperatures, their metabolism essentially ceases (Hammerstedt et al., 1990; Amann, 1999). 3. Cryoprotectants are added to increase cell survival. In addition to protecting cells from cryodamage (these mechanisms are described elsewhere, Amann and Pickett, 1987; Hammerstedt et al., 1990; Amann, 1999) penetrating cryoprotectants, such as glycerol, also act as both a solvent and a solute in the medium; non-penetrating cryoprotectants act purely as a solute. However, when added or removed, both types of cryoprotectant induce cell volume changes that can damage the cells (Amann and Pickett, 1987; Hammerstedt et al., 1990; Amann, 1999).

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4. Cryopreservation affects the cells that are frozen. When external water freezes, the resulting ice is “pure water” interspersed with unfrozen channels of highly concentrated solutes. The cells remain in these unfrozen channels where they are exposed to hyperosmotic conditions which cause the loss of water such that the cells shrink. When the temperature decreases sufficiently, the unfrozen channels vitrify at which time most biochemical and biophysical processes cease. The rate at which the cells are cooled determines both the extent and duration of cellular dehydration and the hypertonicity to which the cells are exposed. Indeed, if cooling is sufficiently rapid, intracellular water can freeze into ice crystals before the water can leave the cell; this intracellular ice can damage the cells (Amann and Pickett, 1987; Hammerstedt et al., 1990; Amann, 1999). 5. Removing penetrating cryoprotectants. After the cells are thawed, the osmolality of the solution and then cell size returns to normal (Hammerstedt et al., 1990). However, because penetrating cryoprotectants possess lower membrane diffusion rates than water, when the cells are diluted in cryoprotectant-free fluid, either in medium prior to insemination or in female reproductive tract fluids at the time of insemination, the osmotic gradient across the cell plasma membrane caused by the cryoprotectant, induces an increase in cell volume (Amann and Pickett, 1987; Hammerstedt et al., 1990; Amann, 1999). The extent of the cell excursion and the duration is dependent upon the amount of cryoprotectant (higher amounts lead to greater cell volume increases) and the membrane diffusion rate of the cryoprotectant (cryoprotectants with lower diffusion rates will induce higher cell volume increases). Although it is beyond the scope of this paper to describe in detail how these principles might affect cryosurvival of sperm from different males, they are instrumental to understanding the probable reasons why sperm from different species or different males within a species survive cryopreservation so differently. For example, an understanding of sperm membrane composition and how the membranes are affected by cooling can shed light on why sperm from some species exhibit ’cold shock’ when cooled improperly, while sperm from other species do not. The causes and effects of cold shock have been thoroughly investigated by Watson (1981) and Amann (1999). Rapid cooling through the membrane transition temperature range, induces lipid loss from (DarinBennett and White, 1973; Watson, 1981) and lipid/protein rearrangements within (Hammerstedt et al., 1990; Amann, 1999) the sperm membranes. These in turn lead to compromised membranes, which become permeable to water and ions ultimately leading to the sperm to exhibit abnormal motility (circular motion and midpiece reflex defects) and die prematurely (Watson, 1981). Interestingly, while sperm from many species (including cattle, horses, sheep and pigs) are susceptible to cold shock damage, sperm from other species (humans and chickens) are not. At least part of the reason for this species-dependant difference in susceptibility to cold shock is likely due to the lipid composition of the sperm membranes (Darin-Bennett and White, 1977). The membrane cholesterol to phospholipid ratios differ markedly human sperm (0.99) and ram or bull sperm (0.38–0.44; Darin-Bennett and White, 1977), and membranes containing higher cholesterol:phospholipid ratios exhibit lower transition temperatures (Ladbrooke et al., 1968; Rottem et al., 1973). Indeed, if the cholesterol content is sufficiently high, the membrane will not undergo a phase transition into the gel state (Ladbrooke et al., 1968). Although greater cholesterol content may be the reason that human sperm are resistant to cold shock damage, rooster sperm have similar membrane cholesterol:phospholipid ratios (0.30) to ram, stallion, boar and bull sperm (Parks and Kynch, 1992). However, rooster sperm have a very different membrane glycolipid composition than mammalian sperm, and whereas the glycolipids in mammalian sperm undergo phase transition to the gel state between 33 and 43 ◦ C, rooster sperm glycolipids do not appear to

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Table 1 The cholesterol:phospholipid ratio of sperm membranes and the percentage of motile sperm after cryopreservation, for sperm from four mouse strains, when the cells were treated with 0 or 2 mg cholesterol-loaded cyclodextrin (CLC)/120 million sperm, prior to freezing (n = 3) Strain

ICR B6D2F1 B6C3F1 B6SJLF/J *

Cholesterol:phospholipid ratio

Percent motile sperm

Amount of CLC added (mg)

Amount of CLC added (mg)

0

2

0

2

S.E.M.

.885 .958 .493 .490

1.033 1.101 .939 .899

52 43 22 9

55 48 42* 38*

3 3 2 3

Row means, within a parameter, are different at P < 0.05.

undergo phase transition (Parks and Kynch, 1992). Regardless of whether a species has greater amounts of a sterol or a specific lipid class, the overall lipid composition of a cell appears to affect its ability to survive cooling damage. It follows that differences in membrane composition between sires within a species may similarly result in sire differences in sperm cryosurvival. In this respect, when sperm from four different mouse strains were cryopreserved, greater cryosurvival rates were achieved from strains with the greatest sperm membrane cholesterol:phospholipid ratios (Table 1). Moreover, to demonstrate that a cryopreservation protocol can be customized to an individual, when these sperm were treated with cholesterol-loaded cyclodextrins, both the cholesterol content of the sperm, as well as their cryosurvival rates increased (Table 1). Similar effects have been observed for stallion sperm (Moore et al., 2005). Interestingly, stallion sperm that did not cool or freeze well showed a greater increase in survival rates after treatment with cholesterol than sperm that did normally survive cooling well (Table 1; Moore et al., 2005). Although the cholesterol content of sperm was not determined in the stallion study (Moore et al., 2005), it is possible that membrane cholesterol content differences between stallions, as between mouse strains, affects cryosurvival. Cell volume changes when the cryoprotectants are added or removed can also be detrimental to sperm survival. Although cryoprotectant removal appears to be more damaging than addition, the extent of damage incurred (due to cell swelling) depends upon both the size and shape of the sperm and the permeability of the membrane to the cryoprotectant (Watson et al., 1992; Gilmore et al., 1998). For example, poultry and rodent sperm, due to respective cylindrical and hook-shapes, cannot swell extensively without rupturing (Watson et al., 1992), while boar sperm exhibit membrane permeability coefficients roughly 100 times less than bull sperm (Gilmore et al., 1998; Guthrie et al., 2002). These phenomena both contribute to the relatively low osmotic tolerances of rooster, mouse and boar sperm (Watson et al., 1992; Guthrie et al., 2002). Although not responsible for sperm shape and size, differences in sperm membrane composition between species and males within a species may be responsible for differences in membrane permeability to water and cryoprotectants. Increasing cryoprotectant permeability, either by altering membrane composition or by using alternative cryoprotectants may improve cryosurvival rates of sperm that normally survive freezing poorly. Indeed, increasing the cholesterol content of stallion (Moore, 2005) and rat sperm (Graham, unpublished) increases their osmotic tolerance. In addition, for stallion sperm that do not survive cryopreservation well using standard procedures, changing the cryoprotectant to a smaller more permeable cryoprotectant such as for-

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mamide or dimethyl formamide can improve cryosurvival (Squires et al., 2004; Alvarenga et al., 2005). 4. Customizing freezing protocols Amann and Pickett (1987) concluded “Perhaps it is unrealistic to assume that spermatozoa from all stallions should be frozen by the same procedure. It may be desirable to optimize extender(s), cooling rate and warming rate for each valuable stallion.” Many commercial semen freezing laboratories have now adopted a split-ejaculate “test-freeze” procedure to evaluate new stallions. During this procedure, semen from one or more ejaculates is divided and processed using protocols that differ with regard to centrifugation techniques, extender composition, cooling rates and sometimes package type to determine if the stallion can produce acceptable post-thaw quality with one or more of the techniques. Samper et al. (1994) evaluated the interaction between individual stallion and extender type for 10 stallions classified on the basis of post-thaw sperm motility as “good freezers” or “bad freezers.” Semen from ejaculates of good freezers was split and extended with three different extenders before freezing using a common technique. The authors found no significant interaction between stallion and extender type and concluded that there was no value in performing split-ejaculate test-freeze procedures on stallions presented for commercial semen freezing. In a series of experiments investigating interactions among cooling treatments, semen extenders and individual stallions, Ecot and co-workers from France (Ecot et al., 2000) found significant interactions between cooling treatment and stallion and between semen extender and individual stallion in one experiment, but no interaction between stallion and semen extender in another. The authors concluded that while changes in key processing steps could have a significant impact on post-thaw quality and that the benefits could be variable among individuals, they did not demonstrate a consistent interaction between extender used and individual stallion. Previously, we reported on the freezability of sperm from stallions presented to our commercial laboratory (Loomis, 1999). We used a split-ejaculate test-freeze procedure to evaluate various cryopreservation protocols on the basis post-thaw sperm motility. The protocols varied with respect to freezing extender, cooling rate, package size and thawing rate. Of 148 stallions whose semen had ≥45% initial progressive motility, 121 (82%) were found to be ‘acceptable freezers’ using at least one of the protocols. An acceptable stallion was one that yielded postthaw progressive motility ≥30% following 30 min incubation at 37 ◦ C for ≥25% of ejaculates frozen. The conclusion of over 20 years experience in our laboratory is that a greater percentage of stallions can produce semen with acceptable post-thaw quality than has historically been reported in the literature and sperm from some stallions often tolerate cryopreservation better when certain aspects of the protocol are customized. To demonstrate this, we examined semen production and quality records for stallions presented to our laboratory for commercial semen freezing from 2000 to 2006. Here we report on pass/fail rates for a large number of stallions presented for semen freezing and the results of split-ejaculate test-freeze procedures. 5. Test-freeze procedure Our standard procedure for assessing suitability of a stallion for commercial frozen semen production is to perform one or more split-ejaculate test-freezes following a series of col-

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lections to deplete extra-gonadal sperm reserves. Semen is evaluated for total sperm number, progressive motility, sperm morphology and bacteriology. Stallions whose initial semen quality was very poor (<35% progressive motility and/or <40% morphologically normal sperm or persistent shedding of known mare pathogens non-responsive to treatment with extender containing antibiotics) were generally excluded as candidates for commercial semen cryopreservation. Currently, our standard test-freeze procedure consists of diluting the semen in a skimmilk-glucose based extender, centrifuging to concentrate sperm and remove >90% seminal plasma and re-suspending the sperm into four different freezing extenders. Semen is then packaged in 0.5-ml straws and cooled and frozen in a controlled rate cell freezer. The straws are plunged into liquid nitrogen and stored for >24 h prior to thawing and evaluation as described below. The four standard protocols vary in the extender used and cooling rate employed. The four extenders are proprietary variations on published formulations whose compositions vary with respect to; type and amounts of sugars and salts, presence or absence of milk as a primary component and amount used, percentage of egg-yolk, types of buffers and percentage of cryoprotectant. For every ejaculate or every treatment for split-ejaculates, the following procedure is used to assess post-thaw semen quality. Two (0.5 ml) straws are thawed and the contents combined. The thawed semen is cultured to confirm the absence of bacterial pathogens and an aliquot is removed and diluted to approximately 30–40 million spermatozoa/ml in a standard skim milk-glucose extender and incubated for 30 min at 37 ◦ C. Following incubation, the motility of the extended semen is determined using computer assisted semen analysis (CASA, Hamilton Thorne CEROS Model). Minimums of 400 motile or 1000 total sperm from >5 fields are analyzed for motion characteristics at 60 frames/s for 40 frames. The stage of the microscope is maintained at 37 ◦ C throughout analysis. For this program, a sperm is considered progressively motile if it has an average path velocity (VAP) >50 ␮m/s and straightness (STR) value >75%. A sperm with VAP <20 ␮m/s is considered non-motile in the calculation of percent motile. 6. Data summary During the period 2000–2006, 332 different stallions were presented for semen cryopreservation. Of those 322 stallions, 281 (84.6%) produced spermatozoa that exhibited ≥30% post-thaw progressive motility in >25% of the ejaculates frozen using one of our standard procedures This is similar to the results for 148 stallions presented to our laboratory in 1997 and 1998 (Loomis, 1999). Even if more stringent criteria were applied to determine a stallion’s suitability for a commercial freezing program (≥30% post-thaw progressive motility in ≥50% of the ejaculates frozen), 74.7% of the 332 stallions presented would still be considered suitable. Split-ejaculate test-freeze data (1-4 ejaculates per stallion) were available from 224 stallions who were presented to our main facility for assessment and for whom computer assisted semen analysis (CASA) was employed for both pre-freeze and post-thaw motility analysis. Of the 224 stallions, 191 (85.3%) produced semen that yielded ≥30% post-thaw progressive sperm motility after freezing using one or more of the standard protocols. The procedure codes for the four standard protocols are C, D, E and F. For each stallion, one of these protocols was selected as the preferred protocol based on the highest value for post-thaw progressive motility. The protocol preference distribution was; F (58%), E (28%), D (11%) and C (3%).

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Of those stallions whose sperm were subjected to multiple test-freeze procedures, protocol preference from the initial test was confirmed in subsequent tests for 70% of stallions while 30% had conflicting results in subsequent tests. 7. Summary Our experience over the last 20 years of commercial semen freezing, reflected in the data presented here and in a previous report (Loomis, 1999) support the notion that a greater percentage of stallions than has traditionally been reported in the literature are capable of producing semen with commercially acceptable post-thaw semen quality. We believe that this is a result of our multiple protocol approach to testing new stallions. These results are in contrast to those reported by Samper et al. (1994) who found no interaction between stallion and extender in a study in which all other processing variables, such as cooling rate were constant. One reason for failure (Samper et al., 1994) to detect an interaction between extender and stallion may be that despite initial average pre-freeze motility of 70% for the tested stallions, the mean post-thaw motility for the three extenders was 19, 18 and 14%, well below the 30% considered as the minimal acceptable value by most commercial laboratories. Our data are in agreement with a recent report, summarizing 20 years of field results with frozen semen in France (Vidament, 2005). Vidament reported significant increases in post-thaw motility, efficiency of freezing, and fertility following modification of the standard freezing protocol and insemination strategy. Stallions and ejaculates were selected on the basis of objectively measured post-thaw motility; an ejaculate was considered acceptable if it possessed ≥35% rapid sperm (CASA), after thawing and dilution. A rapid sperm was defined as a motile cell with an average path velocity >30 or 40 ␮m/s (depending on the CASA instrument used). The major modification that improved results was changing the freezing protocol and, in particular, changing the temperature at which semen was centrifuged to remove seminal plasma and the temperature at which the glycerol containing extender was added. In the old protocol, semen was diluted at 37 ◦ C with an extender containing egg-yolk and cooled to 4 ◦ C over 4 h. Centrifugation, addition of the freezing extender containing glycerol and packaging were also performed at 4 ◦ C before rapid freezing to −140 ◦ C and storage in liquid nitrogen. In the new protocol, semen is diluted in a milk-based extender without egg-yolk at 37 ◦ C and cooled to 22 ◦ C over 10 min. Centrifugation and addition of the freezing extender containing glycerol is performed at 22 ◦ C prior to cooling to 4 ◦ C over 75 min. The semen is packaged at 4 ◦ C and then frozen rapidly to −140◦ C prior to storage. Using this new protocol, Vidament (2005) reported that 90% of 344 stallions presented during 2003–2005 were acceptable for commercial AI (>33% of ejaculates selected after thawing). Coincidentally, all of the standard freezing protocols employed in our laboratory involve centrifugation at 22–25 ◦ C and addition of freezing extender and cryoprotectant to sperm pellets at 22 ◦ C prior to slow or rapid cooling before freezing. A major drawback to the split-ejaculate test-freeze procedure in a commercial setting is the need to perform this procedure on multiple ejaculates prior to selecting the preferred protocol. Our results indicate that a single split-ejaculate test-freeze performed as described above may result in selection of a protocol other than the “best protocol” for as many as 30% of the stallions. Performing multiple test-freeze procedures reduces the production of straws during that period because some of the protocols may result in semen that does not meet acceptable standards. Some stallion owners are reluctant to agree to the time and cost associated with three or four test-freeze procedures, especially when the results of one or two tests indicate that acceptable post-thaw quality can be obtained using one of the protocols. Another drawback is the cost to the freezing

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laboratory associated with producing and maintaining multiple freezing extenders to allow testing of all new stallions presented for freezing. However, despite these limitations, we will continue to perform a split-ejaculate test-freeze procedure on all new stallions at our commercial freezing laboratory because, in order for frozen semen to become more widely accepted in the horse breeding industry, we must attempt to produce the highest quality semen from each individual stallion. References Alvarenga, M.A., Papa, F.O., Landim-Alvarenga, F.C., Medeiros, A.S.L., 2005. Amides as cryoprotectants for freezing stallion semen: a review. Anim. Reprod. Sci. 89, 105–113. Amann, R.P., 1999. Cryopreservation of sperm. Encyclopedia Reprod. 1, 773–783. Amann, R.P., Pickett, B.W., 1987. Principles of cryopreservation and a review of cryopreservation of stallion spermatozoa. J. Equine Vet. Sci. 7, 145–173. Barbacini, S., Zavaglia, G., Gulden, P., Marchi, V., Neechi, D., 2000. Retrospective study on the efficacy of hCG in an equine artificial insemination programme using frozen semen. Equine Vet. Educ. 2, 404–408. Barbacini, S., Loomis, P., Squires, E.L., 2005. The effect of sperm number and frequency of insemination on pregnancy rates of mares inseminated with frozen-thawed spermatozoa. Anim. Reprod. Sci. 89, 203–205. Darin-Bennett, A., White, I.G., 1973. The effect of cold shock and freeze-thawing on release of phospholipids by ram, bull and boar spermatozoa. Aust. J. Biol. Sci. 26, 1409–1420. Darin-Bennett, A., White, I.G., 1977. Influence of the cholesterol content of mammalian spermatozoa on susceptibility to cold-shock. Cryobiology 14, 466–470. Ecot, P., Vidament, M., deMornac, A., Perigault, K., Clement, F., Palmer, E., 2000. Freezing of stallion semen: interactions among cooling treatments, semen extenders and stallions. J. Reprod. Fertil. Suppl. 56, 141–150. Gilmore, J.A., Liu, J., Peter, A.T., Critser, J.K., 1998. Determination of plasma membrane characteristics of boar spermatozoa and their relevance to cryopreservation. Biol. Reprod. 58, 28–36. Guthrie, H.D., Liu, J., Critser, J.K., 2002. Osmotic tolerance limits and effects of cryoprotectants on motility of bovine spermatozoa. Biol. Reprod. 67, 1811–1816. Hammerstedt, R.H., Graham, J.K., Nolan, J.P., 1990. Cryopreservation of mammalian sperm: what we ask them to survive. J. Androl. 11, 73–88. Ladbrooke, D.C., Williams, R.M., Chapman, D., 1968. Studies on lecithin-cholesterol-water interaction by differential scanning calorimetry and X-ray diffraction. Biochim. Biohys. Acta 150, 333–340. Loomis, P.R., 1999. Artificial insemination of horses: where is it going? In: Proc. Ann. Conf. Soc. Theriogenol., Nashville, Tennessee, pp. 325–336. Loomis, P.R., Squires, E.L., 2005. Frozen semen management in equine breeding programs. Theriogenology 64, 480– 491. Moore, A.I., 2005. Effect of cholesterol supplementation on the cryosurvival of equine spermatozoa. Dissertation, Colorado State University. Moore, A.I., Squires, E.L., Graham, J.K., 2005. Adding cholesterol to the stallion sperm plasma membrane improves cryosurvival. Cryobiology 51, 241–249. Parks, J.E., Kynch, D.V., 1992. Lipid composition and thermotropic phase behavior of boar, bull, stallion, and rooster sperm membranes. Cryobiology 29, 255–266. Reger, H.P., Bruemmer, J.E., Squires, E.L., Maclellan, L.J., Barbacini, S., Necchi, D., et al., 2005. Effects of timing and placement of cryopreserved semen on fertility of mares. Equine Vet. Educ., 128–136. Rottem, S., Yashouv, J., Ne’eman, A., Razin, A., 1973. Composition, ultra-structure and biological properties of membrane from Mycoplasma mycoides var. capri cells adapted to grow with low cholesterol concentrations. Biochim. Biophys. Acta 323, 495–508. Samper, J.C., Hearn, P., Ganheim, A., Curtis, E., 1994. Pregnancy rates and effect of extender on motility and acrosome status of frozen-thawed stallion spermatozoa. In: Proc. 40th Ann. Conv. Am. Assoc., Equine Pract, pp. 41–42. Squires, E.L., Keith, S.L., Graham, J.K., 2004. Evaluation of alternative cryoprotectants for preserving stallion spermatozoa. Theriogenology 62, 1056–1065. Tischner, M., 1979. Evaluation of deep-frozen semen in stallions. J. Reprod. Fert. Suppl. 27, 53–59. Vidament, M., 2005. French field results (1985–2005) on factors affecting fertility of frozen stallion semen. Anim. Reprod. Sci. 89, 115–136.

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Vidament, M., Dupree, A.M., Julienne, P., Evian, A., Noue, P., Palmer, E., 1997. Equine frozen semen: freezability and fertility field results. Theriogenology 48, 907–917. Watson, P.F., 1981. The effects of cold shock on sperm cell membranes. In: Morris, G.J., Clarke, A. (Eds.), Effects of Low Temperatures on Biological Membranes. Academic Press, New York, NY, pp. 189–218. Watson, P.F., Kunze, E., Cramer, P., Hammerstedt, R.H., 1992. A comparison of critical osmolality and hydraulic conductivity and its activation energy in fowl and bull spermatozoa. J. Androl. 13, 131–138.