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Research Article |
Department of Anatomy, Physiology and Cell Biology, School of Veterinary Medicine;3
Electron Microscopy Laboratory, Department of Pathology, School of Medicine;4
Veterinary Medical Teaching Hospital;5
Center for Biostabilization;6
Section of Molecular and Cellular Biology;7
Department of Population Health and Reproduction, School of Veterinary Medicine,8 University of California, Davis, California 95616
ABSTRACT
The plasma membrane of sperm can undergo lipid phase separation during freezing, resulting in irreversible damage to the cell. The objective of our study was to examine the membrane phase behavior of equine spermatozoa in the absence and presence of lipid-based cryoprotectants. Biophysical properties of sperm membranes were investigated with Fourier-transform infrared spectroscopy. Compared to fresh untreated sperm, postthaw untreated sperm showed extensive lipid phase separation and rearrangement. In contrast, postthaw sperm that were cryopreserved in egg phosphatidylcholine (egg PC)- or soy phosphatidylcholine (soy PC)-based diluents showed similar lipid phase behavior to that of fresh, untreated sperm. Studies with a deuterium-labeled PC lipid (POPCd-31) suggest that exogenous lipid from the diluents are strongly associated with the sperm membrane, and scanning electron microscopy images of treated sperm show the presence of lipid aggregates on the membrane surface. Thus, the exogenous lipid does not appear to be integrated into the sperm membrane after cryopreservation. When compared to a standard egg-yolk-based diluent (INRA 82), the soy and egg PC media preserved viability and motility equally well in postthaw sperm. A preliminary fertility study determined that sperm cryopreserved in the soy PC-based medium were capable of fertilization at the same rate as sperm frozen in the conventional INRA 82 medium. Our results show that pure lipid-based diluents can prevent membrane damage during cryopreservation and perform as well as a standard egg-yolk-based diluent in preserving sperm viability, motility, and fertility.
gamete biology, sperm, sperm motility and transport
INTRODUCTION
The process of cryopreserving semen has profound effects on spermatozoa, many of which result in sublethal damage to the cells, and subsequent reduction of fertility. The sperm plasma membrane serves as the main physical barrier to the outside environment and is a primary site of freeze-thaw damage. Such damage includes membrane destabilization due to lateral lipid rearrangement [1–3], loss of lipids from the membrane [4], and peroxidation of membrane lipids as a result of formation of reactive oxygen species (ROS) [5–8]. These events can affect sperm motility, response to osmotic stress, and signaling pathways; as a result, the ability to reach, bind, and react with the zona pellucida is compromised [9].
Effects of temperature on the structure and organization of sperm membranes [2, 3] and on the phase behavior of such membranes [10–14] have been studied in some detail. Freeze-fracture studies showed that cooling induces massive phase separation and rearrangement of membrane components; events that appear to be irreversible on warming [2, 3]. Cold shock damage has been directly linked to lipid phase transitions that cause the sperm membrane to become transiently leaky, thereby compromising membrane integrity [12, 13]. The phase transition studies used Fourier-transform infrared spectroscopy (FTIR), a technique that is extremely sensitive to lipid conformational order [15]. By measuring changes in the CH2 symmetric stretching frequency (which reflects lipid acyl chain conformation) over a range of temperatures, the phase behavior of cell membranes can be monitored. FTIR has been used to study the effects of chilling on other whole cell systems, such as oocytes [13, 14], erythrocytes [16], and platelets [17]. On cooling, lipid phase separation was detected in erythrocytes and platelets with FTIR [16, 17]. As mentioned previously, such events were visualized in cooled spermatozoa with freeze fracture [2, 3].
Defining causes of damage to sperm during cryopreservation is further complicated because the processing of semen for cryopreservation is not standardized and there is a wide variety of freezing diluents in use. Most, however, contain some form of lipid, the most common being egg yolk lipids [18, 19]. The role that lipids play in protecting spermatozoa during freeze-thaw is unclear. Proposed mechanisms range from reversible binding of exogenous lipid to fusion of liposomes with the sperm membrane (for a summary, see De Leeuw et al. [20]). One FTIR study showed that exogenous lipid changed the phase transition profile of bull sperm [14], suggesting that the added lipids affected packing of membrane lipids via interactions with the surface. The effects of different lipids correlated with their fluidity; a fluid lipid (egg phosphatidylcholine[PC]), containing highly mobile acyl chains, decreased the sperm lipid main transition temperature and concurrently decreased chilling sensitivity, while a more rigid lipid (dipalmitoyl PC), with relatively less mobile acyl chains, had the opposite effect [14].
A further understanding of the mechanisms through which freeze-thaw injury occurs in spermatozoa and those that govern the protective action of lipids in freezing diluents is necessary to improve preservation methods. In this study, we have examined the membrane phase behavior of equine spermatozoa, before and after freeze-thaw, and in the presence of various freezing diluents. We investigated the interaction between the sperm membrane and lipids in the diluents by using FTIR spectroscopy and scanning electron microscopy. We also present data concerning the diluents' effects on sperm motility and viability and results from a preliminary fertility study.
MATERIALS AND METHODS
Chemicals and Reagents
Soybean phosphatidylcholine (soy PC), egg yolk phosphatidylcholine (egg PC), and 1-palmitoyl(D31)-2-oleoyl-sn-glycero-3-phosphocholine (POPCd-31) were purchased from Avanti Polar Lipids (Alabaster, AL). Glutaraldehyde and sodium phosphate were purchased from Fisher Scientific (Fair Lawn, NJ). The LIVE/DEAD Sperm Viability Kit (SYBR-14 and propidium iodide [PI]) was purchased from Molecular Probes (Eugene, OR). Skim milk extender was purchased from Animal Reproduction Services (Chino, CA), and Dulbecco phosphate-buffered saline (DPBS) was purchased from Gibco (Grand Island, NY). One-day-old chicken egg yolk was obtained from laying chickens at the UC Davis Hopkins Avian Facility (Davis, CA).
Animals and Semen Collection
Semen was obtained from four fertile stallions individually housed either at the UC Davis School of Veterinary Medicine Teaching Hospital or Center for Equine Health, according to Institutional Animal Care and Use Committee protocols of the University of California. Two ejaculates from each of four stallions were collected using an artificial vagina and ovariectomized jump mare. The sperm concentration of the gel-free fraction was determined by densitometer and diluted in skim milk extender or DPBS to 50 x 106 cells/ml.
Sperm Freezing and Thawing
All ejaculates were handled in the same manner, according to an established protocol used by the Equine Reproduction Service at the Veterinary Medical Teaching Hospital, University of California, Davis. Sperm were washed by centrifugation for 15 min at 400 x g, and the pellets were resuspended to 200 x 106 cells/ml in one of four diluents: 1) INRA 82 [21], 2) soy PC-INRA, 3) egg PC-INRA, or 4) DPBS ("untreated"). The soy and egg PC-INRA formulations consisted of the INRA 82 medium with egg yolk replaced by the same volume of either soybean PC or egg yolk PC, respectively. Aliquots of the resuspended treatments were either taken for experiments or loaded into 0.5-ml straws and frozen in a controlled rate freezer (Planer Products, Ltd, Middlesex, UK) using the following freezing curve: –0.5°C/min from 20 to 5°C, –10°C/min from 5 to –15°C, and –25°C/min from –15 to –150°C. Frozen samples were then plunged into liquid nitrogen for storage. Following cryopreservation for 24–48 h, one straw per treatment was thawed for 30 sec in a 37°C water bath. The samples were washed twice by centrifugation for 3 min at 800 x g and resuspended in DPBS. Computer-assisted sperm analysis using a CEROS system (Hamilton Thorne Inc., Beverly, MA) was used to determine sperm motility characteristics. Postthaw membrane integrity was monitored with SYBR-14 and PI. Fluorescence labeling of cells was visualized (100 cells evaluated per treatment) at 1000x under oil, on an Olympus BX-60 microscope (ex. 480/30 nm, em. 535/40 nm). Prefreeze samples were evaluated for motility just before loading into freezing straws, and postthaw samples were evaluated at 5, 30, and 60 min for motility and membrane integrity.
Fourier-Transform Infrared Spectroscopy
Prefreeze and postthaw samples (processed as stated previously) were concentrated by centrifugation at 16000 x g for 1 min and the pellets spread onto CaF2 windows for FITR analysis. Data were obtained with a Spectrum 2000 FTIR spectrometer interfaced to a PC with Spectrum 3.1 software (Perkin Elmer, Norwalk, CT). The instrument was purged of water vapor with a dry air generator (Balston, Haverhill, MA). Sample temperature was controlled by a Peltier device and monitored with a thermocouple. Temperature was ramped at a rate of 2°C/min for all samples, and a total of 16 spectra were averaged for each temperature point. The CH2 stretching region, from 3000 to 2800 cm–1, was analyzed. Band positions were determined by taking second derivatives of the original spectra and averaging intercepts at 80% peak intensity. Phase transitions were determined by plotting the band positions as a function of temperature and taking the first derivative of the data. To determine if exogenous lipid was associating with the sperm membrane, we replaced the PC lipid in our experimental media with deuterium-labeled POPCd-31. The vibrational modes of POPCd-31 can be distinguished from endogenous sperm lipid in the IR spectra [15]. In these samples, the CD2 stretching region, from 2200 to 2000 cm–1, was also analyzed, in the same manner as described previously. We were unable to obtain profiles for INRA 82 samples because of strong interference from the egg yolk. All FTIR data shown are from a single ejaculate, and figures present typical plots.
Scanning Electron Microscopy
Lipid-free samples were made using INRA 82 medium with no egg yolk or lipid added. Cells were fixed for at least 24 h in 3% glutaraldehyde at 4°C. The samples were washed four times by centrifugation at 16000 x g for 5 sec and resuspended in 0.1 M sodium phosphate buffer (pH 7.2). This buffer was then replaced with buffered osmium tetroxide (1%) and left for 1 h, and samples were washed with sodium phosphate buffer twice by centrifugation as described. Samples were deposited onto 0.2-µm filters, dehydrated in a graded series of ethanol up to 100%, and then critical point dried (Samdri PVT-3; Tousimis, Rockville, MD). The dried samples were mounted on aluminum stubs and sputter coated with gold. Imaging was performed with an FEI XL30 scanning electron microscope (Hillsboro, OR) at 25 kV accelerating voltage.
Fertility Study
We conducted a fertility study to compare the performance of soy PC-INRA to that of the conventional INRA 82 extender containing egg yolk. Eleven light horse mares ranging in age from 3 to 11 yr were used in a single blind, crossover design. Breeding soundness was based on obtainable breeding history and transrectal palpation and ultrasound of the uterus and ovaries (5-MHz linear probe, Sonovet 2000; Medison, Cypress, CA). The estrous cycle of each mare was followed daily; during estrus, an ovulatory agent (BioRelease Deslorelin Injection, 1.5 mg; BETPharm, Lexington, KY) was given intramuscularly when a follicle 
30 mm in size was detected, along with at least one other sign of estrus (behavioral, uterine edema, or relaxed cervix). Mares were randomly assigned to one of two treatment groups: group 1 (five mares) was inseminated using semen cryopreserved in INRA 82 extender on the first cycle, followed by semen cryopreserved in soy PC-INRA on the second cycle; group 2 (six mares) was treated with extenders in reverse order from group 1. Each animal served as their own control. A single operator inseminated each mare at 24 h postinjection with approximately 150 x 106 progressively motile thawed sperm, using a 75-cm flexible deep intrauterine insemination pipette and stylet (Minitube, Verona, WI), with pipette placements verified by transrectal palpation. This technique allows for placement of semen near the uterotubal junction. Ovulation was confirmed via transrectal palpation and ultrasound, and all mares had ovulated by 52 h postinjection. Pregnancy diagnosis was performed 14–16 days postovulation by transrectal palpation and ultrasound. Pregnant mares were given 7.5 mg PGF2
(Lutalyse; Pharmacia & Upjohn, Kalamazoo, MI) intramuscularly to induce return to estrus. The insemination process was then repeated on the subsequent estrus cycle, using semen frozen in the alternate extender for each group, respectively.
Statistical Analysis
For viability and motility data, treatment differences were calculated using a general linear model analysis of variance with Minitab12 software (Minitab Inc., State College, PA). The data were normally distributed, and post hoc treatment comparisons were performed using the Tukey method. A significance level of P < 0.05 was used. Pregnancy data were analyzed using a one-sided exact chi-square test of homogeneity, and a significance level of P < 0.05 was used.
RESULTS
FTIR profiles of untreated prefreeze and postthaw equine spermatozoa are shown in Figure 1. A change in the slope of the profile over a defined temperature range indicates that a lipid phase transition is occurring, as shown by the solid line (first derivative). Fresh, untreated sperm showed lipid phase transitions at 10–15°C, 30–40°C, and 55–60°C (Fig. 1A). Slight differences in midpoints of the transitions were observed between prefreeze profiles from different animals but were always within the ranges stated. After freeze-thaw, unprotected sperm showed transitions at 14, 27, 47, and 59°C (Fig. 1B). As shown by the first derivative (solid line), postthaw transitions were both more numerous and more cooperative (sharper peaks) than for fresh cells.
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Figure 2 shows postthaw profiles of spermatozoa from the same ejaculate that were frozen in experimental cryopreservation media. Sperm treated with egg PC-INRA (A) and soy PC-INRA (B) showed similar lipid phase behavior after washing, with transitions at 15–20°C, 30–35°C, 45–50°C, and 55–60°C. In contrast to postthaw untreated sperm (Fig. 1B), the majority of lipid in treated sperm melted at lower temperatures, similar to that of untreated prefreeze samples (Fig. 1A). Treated sperm also showed smoother, less cooperative transitions than untreated sperm, similar to those of the fresh untreated samples.
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Figure 3 shows a comparison of FTIR spectra from postthaw untreated (dashed line) and treated (solid line) spermatozoa. Sperm exposed to POPCd-31 INRA showed the characteristic peaks of deuterated lipid, even after washing to remove excess media. The peaks were absent in the untreated samples. Postthaw phase behavior of the deuterated lipid in the presence and absence of sperm is shown in Figure 4. The POPCd-31 shows similar behavior in both conditions, at temperatures ranging from 5 to 68°C.
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Figure 5 shows an SEM image comparison of spermatozoa from various treatments. Fresh, untreated sperm (A) had much less visible damage than those frozen and thawed in the lipid-free medium (B), which showed considerable damage at the head-neck junction and extensive damage to the membrane surface (B, inset). Sperm cryopreserved in soy PC-INRA (C and D) were intact and showed very few damaged sites, if any. Lipid aggregates, with an average diameter of approximately 300 nm, were observed on the surface of the lipid-treated sperm (C and D). Similar effects were seen in INRA 82 and egg PC-INRA samples (not shown) as those observed for soy PC-INRA-treated sperm.
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A comparison of prefreeze motility data reveals that sperm showed similar percentages of total and progressive motility in each of the cryopreservation media studied, with average values of 87% for total and 31% for progressive motility (P > 0.05) (Fig. 6). The motility of (untreated) sperm in DPBS averaged 45% total and 13% progressive (data not shown). Postthaw sperm viability was also similar for the three treatment media studied, with an average of 62% viable cells. No significant difference was observed for postthaw motility values (P > 0.05), with average values of 59% total and 20% progressively motile sperm. Motility and viability values were not included for cells frozen in DPBS.
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Results from preliminary fertility studies with sperm cryopreserved in INRA 82 and soy PC-INRA are shown in Table 1. On the first cycle, pregnancy was detected in two mares inseminated with INRA 82 (40%) and in three mares inseminated with soy PC-INRA (50%). On the second cycle, four pregnancies were detected in mares inseminated with INRA 82 (67%), and one pregnancy was detected in mares inseminated with soy PC-INRA (20%). The total number of pregnancies obtained with the INRA 82 diluent was 6/11 (55%), and the total for the soy PC-INRA diluent was 4/11 (36%), with no significant difference between treatments (P > 0.05).
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DISCUSSION
Sperm Membrane Phase Behavior
Fresh untreated spermatozoa showed three distinct phase transitions that likely represent different lipid fractions in the sperm membrane (Fig. 1A), in agreement with differential scanning calorimetry results for stallion sperm lipid extracts, which showed a phospholipid transition at 20°C and a glycolipid transition at 33°C [11]. The composition of stallion sperm cell membranes includes a variety of lipid classes and acyl chain lengths/saturation levels [11, 22]. Multiple thermal events seen during heating in human platelets have previously been assigned to membrane domains that phase separate because of differences in acyl chain length and saturation [23, 24]. We suggest that the multiple transitions seen here are due to a similar formation of distinct domains in the sperm membrane. For instance, stallion spermatozoa contain significant amounts of saturated fatty acids (16:0 and 15:0 acyl chains) [11, 12], which could contribute to a high-melting lipid fraction. Sulfogalactosylglycerolipid (SGG), a major glycolipid present in mammalian sperm [25], contains predominantly fully saturated 16:0/16:0 chains [11, 26–28]. Extracts of SGG from ram sperm show a main lipid phase transition at 46°C [28].
Comparison of the FTIR profiles of untreated spermatozoa before and after freeze-thaw reveals marked differences between their membrane phase behavior (Fig. 1, A and B). In contrast to fresh, untreated sperm, postthaw sperm showed multiple, sharp phase transitions, an effect seen in human erythrocytes [16] and equine platelets [17] after cold shock. Coupled with the observed shift of phase transitions toward higher temperatures in postthaw profiles, these data suggest that large-scale lipid rearrangement has occurred in the membrane during the freeze-thaw process. Freeze fracture studies of cooled ram and blackbuck sperm [2] and bull and boar sperm [3] have shown such membrane phase separation events to be irreversible, thereby permanently compromising membrane integrity and function. Since the FTIR technique used to acquire these data averages the signal over an entire population, it is not possible to determine whether the observed changes are due to the presence of dead cells or damaged membranes. However, the comparison across all samples is made in the same way, including any dead/dying or damaged cells, which are present even in prefreeze samples. In addition, we have previously scanned spermatozoa selected for motility and viability via Percoll separation and found no difference in Tm values compared to nonseparated samples (data not shown). Therefore, we feel it is unlikely that death in the population could result in the extensive changes shown in the FTIR data, although this possibility cannot be dismissed.
Alterations in membrane composition may contribute to the observed changes in postthaw sperm phase behavior, such as loss of cholesterol from the membrane, which would increase the cooperativity of lipid phase transitions [16, 17, 29]. Generation of lysolipids and free fatty acids from peroxidation of lipids during cryopreservation [5–8] may also affect the thermal profile.
Effect of Cryopreservation Media on Membrane Phase Behavior
We have replaced the yolk in a conventional freezing medium (INRA 82 [21]) with purified PC lipids, which yield a more defined product. Because of strong interference arising from egg yolk lipids, we were not able to obtain CH2 profiles for INRA 82 samples. Postthaw IR profiles of spermatozoa treated with egg PC and soy PC showed similar patterns of lipid phase transitions, suggesting nearly equivalent effects on membrane organization and/or composition (Fig. 2). These two phospholipids have similar phase transitions and fatty acid contents, so it is not surprising that they behave similarly in this context. The preservative effect of the diluents is readily seen when comparing their profiles to that of untreated sperm before and after freeze-thaw (Fig. 1). Postthaw treated sperm show smooth phase transitions in temperature ranges similar to that of fresh samples, in contrast to the sharp transitions of postthaw untreated sperm, where the majority of lipid in the membrane melts at high temperatures (Fig. 1B). These data suggest that the experimental media are effective in preventing membrane damage due to lipid phase separation.
Mechanism of Sperm Membrane Stabilization
De Leeuw et al. [20] summarized several hypotheses concerning the mechanism of cryoprotection by exogenously added lipids: exogenous lipids can 1) associate with the surface of the sperm plasma membrane or 2) modify membrane composition. The former suggests that lipids from cryoprotectants reversibly bind to the cell surface, providing a physical barrier to freezing damage without changing the lipid content of the membrane [14, 18, 30–33]. The latter implies that exchange of exogenous lipids occurs between the media and the cell membrane, thereby replacing lost lipid and leading to stabilization against cold shock [4, 34].
As a test of whether the exogenously added lipid inserts into the plasma membrane, we replaced the PC in the experimental media with deuterated POPC (POPCd-31). The acyl chains of the synthetic lipid are uniformly deuterated, thus providing molecules with CD2 vibrations rather than CH2 found in the native membrane. The CD2 stretching vibrations occur at lower frequencies than those of CH2 groups, thus permitting one to monitor both the added lipid and the native membrane simultaneously in a single sample.
After freeze-thaw, treated spermatozoa showed the presence of the deuterated lipid as detected by the characteristic CD2 stretching vibrations, even after washing (Fig. 3), suggesting that the lipid strongly associates with the sperm membrane during the freeze-thaw process. Support for this idea is reflected in comparison of postthaw untreated and treated sperm (Figs. 1B and 2), where the majority of lipid melts at lower temperatures in the treated samples, in contrast to the untreated sample. We hypothesize that fluid lipids from the diluents may affect sperm lipid packing at the membrane surface, thereby decreasing lipid transition temperatures and maintaining membrane integrity and organization.
To test this hypothesis, we monitored the postthaw phase behavior of POPCd-31 in the presence and absence of spermatozoa (Fig. 4) to determine if the lipid had incorporated into the sperm membrane. Coincident phase transition(s) of the POPCd-31 and sperm lipid would indicate that the exogenously added lipid was incorporated into the sperm membrane. If this were the case, we would also observe a change in the phase profile of POPCd-31 in the sperm/lipid mixture relative to the profile of the lipid alone. Our data show that there are no changes in the POPCd-31 phase behavior when added to the sperm, with the sperm/lipid mixture profile almost identical to that of the lipid alone (Fig. 4). In addition, the profile of POPCd-31 in the sperm/lipid mixture (filled circles) showed no transitions in the temperature range over which multiple transitions were observed for sperm frozen in PC diluents (Fig. 2). Therefore, the exogenous lipid behavior is independent from the sperm lipid, suggesting that it does not incorporate into the sperm membrane.
Evidence of external association of lipid with the sperm membrane can be seen in scanning electron micoscopy (SEM) images of spermatozoa that were cryopreserved in soy PC-INRA (Fig. 5). The cracked appearance of samples is likely a result of the SEM processing, which includes dehydration steps. Lipid-treated sperm showed the presence of lipid aggregates on the membrane surface that were absent in samples that had been frozen and thawed in the same diluent lacking lipid. Thus, even after postthaw washing, exogenous lipid remains associated with the sperm membrane. In contrast to untreated samples, lipid-treated sperm were morphologically intact, with smooth, continuous membrane surfaces. Such samples also showed less surface cracking than untreated samples, suggesting a protective effect of the lipid during processing for imaging. Taken together, our FTIR and SEM results show that exogenous lipid from the cryoprotectants does not incorporate into the sperm membrane but strongly associates with the membrane surface. In doing so, the lipid may provide a physical barrier to freeze-thaw damage and prevent membrane phase separation by influencing lipid packing at the membrane surface.
Effect of Cryoprotectants on Sperm Motility, Viability, and Fertility
In addition to the biophysical studies, we have assessed sperm motility and viability in the presence of cryoprotectants (Fig. 5). No significant differences were observed in prefreeze motility or postthaw viability values. The spermatozoa showed higher apparent total and progressive motility postthaw in the soy PC- and egg PC-based media. However, these distinctions were not statistically significant (P > 0.05). Therefore, the pure lipid diluents perform comparably to the conventional egg-yolk-based INRA in preserving sperm viability and motility.
As a practical test of cryoprotectant efficacy, a preliminary fertility trial was performed using sperm frozen in the conventional INRA 82 diluent and the soy PC-INRA formulation. The number of mares used in the study was necessarily small because of the labor and resource intensity of such trials. However, attempts were made to minimize artifactual results due to factors such as variation in individual mare fertility and estrous cycle, heterogeneity of semen samples, sperm-mare interactions, and insemination timing and technique. The results show that spermatozoa cryopreserved in the experimental soy PC-INRA diluent are capable of fertilization. Within this study group, we did not observe any statistically significant difference in pregnancy rate between the two diluents (P > 0.05). Therefore, according to our data, the soy PC- and egg-yolk-based media perform equally well clinically. Because of the small number of mares used, it is difficult to make more specific conclusions about the fertility of the sperm frozen in each diluent. A larger study will be necessary to investigate further the differences between diluents and to determine whether the experimental media may improve fertility rates for stallions whose sperm do not freeze well in conventional diluents.
In conclusion, we observe that the freeze-thaw process induces large-scale rearrangement of the membrane lipids in stallion spermatozoa and that the presence of egg PC- and soy PC-based diluents can minimize such damage by associating with the membrane surface. The advantages of these formulations over egg yolk preparations include ease of storage, reduction of risk for microbial contamination, and elimination of the need for specific pathogen-free eggs used by many semen processors. Coupled with the diluents' ability to preserve sperm viability and motility, our data suggest that egg and soy PC INRA are viable alternatives to conventional egg-yolk-based freezing diluents for cryopreserving equine spermatozoa.
ACKNOWLEDGMENTS
The authors would like to thank Barbara L. Stewart and James A. Brown for their assistance in the fertility trials and Drs. Allen Enders and Nelly Tsvetkova for helpful discussions.
FOOTNOTES
1 Supported through the UC Davis Center for Equine Health, with funds provided by the Oak Tree Racing Association, the State of California Pari-Mutuel Fund, and contributions by private donors, as well as USDA Cooperative State Research, Education and Extension Service NRI grant 2002–35203–12260. 
2 Correspondence: S.A. Meyers, Department of Anatomy, Physiology and Cell Biology, School of Veterinary Medicine, UC Davis, One Shields Ave., Davis, CA 95616. FAX: 530 752 7690; smeyers{at}ucdavis.edu
Received: 28 July 2005.
First decision: 26 August 2005.
Accepted: 7 October 2005.
REFERENCES
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