The role of osmotic resistance on equine spermatozoal function

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1 Theriogenology 58 (2002) 1373±1384 The role of osmotic resistance on equine spermatozoal function Angela C. Pommer, Josep Rutllant, Stuart A. Meyers * Sperm Biology Laboratory, Department of Anatomy, Physiology, and Cell Biology, School of Veterinary Medicine, University of California, Davis, CA 95616, USA Received27 March 2001; accepted15 March 2002 Abstract Cryopreservation requires exposure of sperm to extreme variations in temperature andosmolality. The goal of this experiment was to determine the osmotic tolerance levels of equine sperm by analyzing motility, viability, mitochondrial membrane potential (MMP), and mean cell volume (MCV). Spermatozoa were incubatedat 22 8C for 10 min in isosmolal TALP (300 mosm/kg), or a range of anisosmolal TALP solutions (75±900 mosm/kg), for initial analysis, andthen returnedto isosmolal conditions for 10 min for further analysis. Total sperm motility was lower (P < 0:05) in anisosmolal conditions compared to sperm motility in control medium. When cells were returned to isosmolal conditions, only sperm previously incubated in 450 mosm/kg TALP were able to recover to control levels of motility. Sperm viability andmmp were lower (P < 0:05) when exposedto hypotonic solutions in comparison to control solutions. Sperm suspensions that were returnedto isosmolal conditions from 75, 150, and 900 mosm/kg had lower (P < 0:05) percentages of viable sperm than control suspensions (300 mosm/kg). MMP was lower (P < 0:05) in cells previously incubatedin 75 and900 mosm/kg when returnedto isosmolal, as comparedto control cells. MCV differed (P < 0:05) from control cell volume in all anisosmolal solutions. Cells in all treatments were able to recover initial volume when returnedto isosmolal medium. Although most spermatozoa are able to recover initial volume after osmotic stress, irreversible damage to cell membranes may render some sperm incapable of fertilizing an oocyte following cryopreservation. # 2002 Elsevier Science Inc. All rights reserved. Keywords: Osmotic tolerance; Stallion; Sperm function; Motility; Mitochondrial membrane potential 1. Introduction It has been estimatedthat only 24% of stallions produce ejaculates that are suitable for cryopreservation, andfertility of frozen semen is approximately 40% that of fresh, * Corresponding author. Tel.: ; fax: address: smeyers@ucdavis.edu (S.A. Meyers) X/02/$ ± see front matter # 2002 Elsevier Science Inc. All rights reserved. PII: S X(02)

2 1374 A.C. Pommer et al. / Theriogenology 58 (2002) 1373±1384 nonfrozen semen [1]. Cooledor frozen storage of equine spermatozoa has been reported to result in markedreductions in sperm fertility for a majority of stallions in commercial breeding programs [2]. Although the mechanisms responsible for decreased fertility of cryopreservedspermatozoa are unknown, disruption of certain cellular processes has been reported [3,4]. Cryopreservation requires exposure of spermatozoa to extreme variations in temperature andosmolality. Cells undergoing freezing are initially exposed to extracellular ice crystallization that results in hyperosmolal concentration of solutes in the unfrozen aqueous channels between ice crystals [3,5]. The cell responds to this insult by losing water andshrinking in volume so that the solute concentrations between intracellular andextracellular compartments can equilibrate. Conversely, as cells are exposedto a hypotonic extracellular environment, as is the case during thawing, cell volume is increasedby passive diffusion of water. It is not known whether this osmotic stress results in sublethal, irreversible damage to cell membranes, causing decreased fertilizing capacity. Determining osmotic resistance for equine spermatozoa is critical to understanding the biophysical capabilities of spermatozoa during the freeze±thaw cycle. However, there are few publishedstudies concerning osmotic resistance of equine spermatozoa [6,7]. Therefore, the goal of the present study was to determine the osmotic tolerance limits of equine spermatozoa by analyzing motility, viability, mitochondrial membrane potential (MMP), andmean cell volume (MCV) in anisosmolal Tyrode's medium containing albumin, lactate, andpyruvate (TALP). Once the osmotic resistance of equine spermatozoa is determined, this knowledge can be used to understand membrane behavior and to modify current cryopreservation methods that could increase the fertility of frozen±thawed stallion spermatozoa. 2. Materials and methods 2.1. Chemicals and reagents Propidium iodide and JC-1 were purchased from Molecular Probes (Eugene, OR). All other chemicals were obtainedfrom Sigma Chemical Company (St. Louis, MO) Animals and semen Semen was obtained from three stallions individually housed at the Veterinary Medicine Teaching Hospital andthe Animal Science Horse Barn locatedat the University of CA, Davis. Stallions were maintainedon a diet of mixedgrass hay andomolene 200, with fresh water ad libitum and daily exercise according to Institutional Animal Care and Use Committee protocols at the University of California. Semen was collectedusing an arti cial vagina anda phantom mare. A nylon mesh lter was usedto eliminate the gel fraction, and the gel-free semen was immediately diluted 1:1 (v/v) into TALP capacitation medium [8]. The diluted semen was transported to the laboratory within 5 min of collection. Upon arrival at the laboratory, the sperm concentration was determined using a hemacytometer anddilutedto ml 1.

3 A.C. Pommer et al. / Theriogenology 58 (2002) 1373± Media The media used were isosmolal (300 5 mosm/kg) andanisosmolal TALP. Anisosmolal solutions were preparedby diluting isosmolal TALP (hyposmolal solutions, 75 and 150 mosm/kg) andby diluting 10-strength TALP (hyperosmolal solutions, 450, 600, and 900 mosm/kg) with HPLC-puri ed water for experiments in which MCV was determined. To achieve the nal osmolalities of 75, 150, 450, 600, and900 mosm/kg in the experiments in which the sperm motility, MMP, and viability were assessed, an adjusted set of anisosmolal solutions was prepared(20, 115, 490, 675, and1050 mosm/kg, respectively) in order to avoid the dilution effect (1:5) of the sperm sample in the anisosmolal media. Final osmolalities were always con rmedusing a vapor pressure osmometer (Model 5100 C; Wescor Inc., Logan, UT) Sperm processing Evaluation of sperm motility For motility experiments, 50 ml of the initial sperm suspension ( ml 1 ) was placedin 200 ml isosmolal TALP (300 mosm/kg), or a range of anisosmolal TALP solutions to get a nal concentration of 75, 150, 450, 600, and900 mosm/kg, and incubatedat 22 8C for 10 min ( ml 1 ) prior to motility analysis. Sperm suspensions were then returned to near-isosmotic conditions by adding 1.25 ml of isosmolal TALP ( ml 1 ) as described by Gilmore et al. [9]. After 5 min incubation, motility was analyzedagain. At least 200 cells were evaluatedusing computer-assistedsperm analysis (CASA) for all experiments in which motility was determined (CEROS, Version 10.9i, Hamilton Thorne Biosciences, Inc., Beverly, MA). The settings usedwere: frame rate 60 Hz, frames acquired30, minimum contrast 80, minimum cell size 3, threshold straightness 80, medium VAP cut-off 25, low VAP cut-off 5, low VSL cut-off 11, nonmotile headsize 6, nonmotile headintensity 160, static size limits 1.0±2.9, static intensity limits 0.6±1.4, static elongation limits 0± Evaluation of mitochondrial function For MMP experiments, 200 ml of the sperm suspension ( ml 1 ) was added to 800 ml of isosmolal or anisosmolal TALP for a nal sperm concentration of ml 1, and incubated at room temperature for 10 min. Then, each 1 ml sample was divided into two 500 ml samples. To the rst set of tubes in each treatment, a nal concentration of 2 mm JC-1 was added for 10 min and the samples were evaluated using a ow cytometer (FACSCalibur, Becton-Dickinson Immunocytometry Systems, San Jose, CA). Using the appropriate gating parameters for equine spermatozoa determined during preliminary experiments, the cell population (10,000 events) was divided into either high membrane potential (red/green uorescence) or low membrane potential (green uorescence), according to natural partitioning. In the second set of tubes, sperm were diluted with 1.5 ml isosmolal TALP (7: ml 1 ) for 10 min, andthen incubatedanother 10 min with 2 mm JC-1 before being analyzedon the ow cytometer. This same protocol was used for the viability studies, the only difference being that propidium iodide (5 mm) was used insteadof JC-1.

4 1376 A.C. Pommer et al. / Theriogenology 58 (2002) 1373± Determination of sperm volume A Coulter counter (Z2 model; Coulter Corp., Miami, FL) with a standard 50-mm aperture tube was used. Sperm MCV was calibrated using spherical polystyrene latex beads of three different diameters (3, 5, and 10 mm, CC Size Standard reference: , , , respectively; Coulter Corp.) as suggestedby the manufacturer. The Coulter counter was interfacedto a microcomputer anddata were acquiredby means of speci c software (Accucomp TM, Coulter Corp.). For MCV analysis, 20 ml of the sperm suspension ( ml 1 ) was added to 20 ml of isosmolal or anisosmolal TALP ( ml 1 ) for 10 min. The MCV was then determined by evaluating 10,000 cells. For determination of MCVafter return to isomolality, in separate tubes, 20 ml of sperm were added to 2 ml of isosmolal or anisosmolal TALP andincubatedfor 10 min. Then, 18 ml of isosmolal TALP was added to each treatment tube and incubated an additional 10 min before MCV was determined. Since the instruments used to determine MMP and MCV count a preselected number of cells for analysis, dilution factors do not affect results. In order to determine the inactive cell volume and estimate whether equine sperm membranes behave as linear osmometers, sperm volumes recorded in isosmolal and anisosmolal conditions were tted to the following Boyle van't Hoff equation: V V iso ˆ Miso M 1 V b V iso V b V iso where V is the cell volume at the osmolality M, V iso is the cell volume at isosmolality (M iso ), and V b is the osmotically inactive cell volume Statistical analysis Data were analyzedusing one-way ANOVAwith Minitab 1 statistical software (Minitab, Inc., State College, PA). ANOVA was usedto evaluate treatment differences over a range of anisosmolality treatments. Endpoints included sperm total motility, progressive motility, viability, MMP, andmcv. 3. Results 3.1. Motility Normalizedtotal sperm motility was lower (P < 0:05) in anisosmolal conditions as comparedto controls (Fig. 1). When sperm were returnedto isosmolal conditions, only sperm previously incubatedin 450 mosm/kg TALP were able to recover to control levels of motility. In control samples (300 mosm/kg) a decrease in motility was observed following dilution into isosmolal TALP; however, signi cant differences were not observed (P ˆ 0:27) Viability and mitochondrial membrane potential Spermatozoal viability (Fig. 2A) andmmp (Fig. 3A) were lower (P < 0:05) when cells were exposedto hypotonic solutions as comparedto control cells andcells exposedto

5 A.C. Pommer et al. / Theriogenology 58 (2002) 1373± Fig. 1. Sperm samples were incubatedfor 10 min in isosmolal or anisosmolal TALP andmotility was analyzed using a CASA system. Samples were then diluted with isosmolal TALP, incubated 5 min, and motility was analyzed again. Letters (a±d) denotes signi cant difference (P < 0:05) from control (300 mosm/kg) within each treatment group, respectively; n ˆ 5 experiments, three stallions. hypertonic solutions. Sperm suspensions that were returnedto isosmolal conditions from 75, 150, and900 mosm/kg hadlower (P < 0:05) numbers of viable spermatozoa than control cells (Fig. 2B). MMP was lower in a majority of cells previously incubatedin 75 and900 mosm/kg when returnedto isosmolal medium, as comparedto control cells (P < 0:05) (Fig. 3B) Mean cell volume The isosmotic MCV (mean S:E:M:) of equine spermatozoa was determined to be 24:4 0:6 mm 3 at 22 8C. MCV was different than control volume in all anisosmolal solutions (P < 0:05). Spermatozoa in all treatments were able to recover initial volume when returnedto isosmolal medium (P > 0:05) (Fig. 4). MCV swelledto 1.60 times initial volume when exposedto 75 mosm/kg TALP anddecreasedto 0.81 times initial volume when incubatedin 900 mosm/kg TALP (Table 1), indicating an active osmoregulatory volume range. The osmotic response of equine spermatozoa was determined over the range 150± 900 mosm at 22 8C. Data are presentedas a Boyle van't Hoff plot (volume versus 1/osmolality) anddepicts a linear response (r 2 ˆ 0:999) anda V b of 70.7% (Fig. 5). 4. Discussion An understanding of the osmotic tolerance limits and cellular processes involved is essential to develop new methods for semen processing and storage that will be useful for a

6 1378 A.C. Pommer et al. / Theriogenology 58 (2002) 1373±1384 Fig. 2. Sperm samples were incubatedfor 10 min at room temperature in isosmolal or anisosmolal TALP. (A) A nal concentration of 5 mm propidium iodide was added to each treatment for 10 min, and the samples were read using a ow cytometer. (B) Treatments were diluted with 1.5 ml isosmolal TALP for 10 min, and then incubated another 10 min with 5 mm propidium iodide before being analyzed on the ow cytometer. Nonlabeled cells were considered live. Letter (a) denotes signi cance from control (300 mosm/kg) viability (P < 0:05); n ˆ 4 experiments, three stallions. large number of genetically valuable sires. The rate at which osmotic volume regulation, andhence, cooling or warming, may take place is highly dependent upon cell hydraulic conductivity (water permeability), L p. In spermatozoa, L p has been shown to be dependent on species [10], temperature, cryoprotective agent (CPA), andice crystal formation [9]. Thus, further progress in improving sperm survival wouldnot be achievedsimply by modifying established cryopreservation diluents. A more fundamental understanding of the biophysical andbiochemical processes that accompany sperm freezing andthawing will be essential to design new successful cryopreservation protocols [11] Osmotic behavior of equine spermatozoa The results of our study showed that the mean volume (average of multiple mean values) of isotonic equine spermatozoa measuredwith a Coulter counter was 24.4 mm 3. Equine sperm cell volume has not been previously reported, therefore, we cannot compare our

7 A.C. Pommer et al. / Theriogenology 58 (2002) 1373± Fig. 3. Sperm samples were incubatedfor 10 min at room temperature in isosmolal or anisosmolal TALP. (A) A nal concentration of 2 mm JC-1 was added to each treatment for 10 min, and the samples were read using a ow cytometer. (B) Treatments were diluted with 1.5 ml isosmolal TALP for 10 min, and then incubated another 10 min with 2 mm JC-1 before being analyzedon the ow cytometer. Redlabeledcells were consideredto have high MMP, while those labeled green were considered to have low MMP. Letter (a) denotes signi cance from control (300 mosm/kg) values (P < 0:05); n ˆ 3 experiments, three stallions. results to other studies. However, using measures of equine spermatozoa obtained by automatedsperm morphometric analysis andoptical microscopy [12,13], the estimated cell volume is approximately 37 mm 3. As has been described in mouse [14] andhuman [15±17] sperm, the estimates of cell volume obtainedfrom electronic cell sizers are smaller than estimates obtainedfrom microscopic measurements. The origin of these discrepancies is not clear. However, considering that the resolution limits of optical microscopy are close to some of the diameters measured in the spermatozoa, an over-estimation of as little as 10% is not unreasonable, andcouldexplain the difference between the two estimated volumes for equine spermatozoa.

8 1380 A.C. Pommer et al. / Theriogenology 58 (2002) 1373±1384 Fig. 4. Sperm samples were incubatedfor 10 min at room temperature in isosmolal or anisosmolal buffer. MCV was then determined using a Z2 Beckman Coulter Counter. In separate tubes, after the initial 10 min incubation, samples were diluted with isosmolal buffer and incubated for 10 min before volume was determined. Letter (a) denotes signi cant difference from control (300 mosm/kg) within each treatment group, respectively (P < 0:05); n ˆ 3 experiments, three stallions. In the present study, equine spermatozoa behaved as linear osmometers in the range of 150±900 mosm/kg (r 2 ˆ 0:999) with 70.7% of the total cell volume (V b ) (both solids and water) being osmotically inactive. When a cell behaves as an ideal osmometer, the volume of osmotically available water containedin the cell, in our case almost 30% of total sperm volume in isosmotic conditions, will be inversely related to the osmolality of nonpermeable solutes in the external medium [18]. In addition, the observed linear osmotic behavior Table 1 Normalizedcell volume, motility, viability, andmmp of equine spermatozoa in anisosmotic TALP Osmolality (mosm/kg) Normalized volume Normalized motility (%) Normalized viability (%) Normalized high MMP (%) Sperm samples were incubatedfor 10 min at room temperature in isosmolal or anisosmolal TALP prior to all analyses. MCV was then determined using a Z2 Beckman Coulter Counter and motility was analyzed using a CASA system. For viability determination, a nal concentration of 5 mm propidium iodide was added to each treatment for 10 min andthe samples were readusing a ow cytometer. Nonlabeledcells were consideredlive. For MMP determination, a nal concentration of 2 mm JC-1 was added to each treatment for 10 min and the samples were readusing a ow cytometer. Redlabeledcells were consideredto have high MMP, while those labeledgreen were consideredto have low MMP.

9 A.C. Pommer et al. / Theriogenology 58 (2002) 1373± Fig. 5. Boyle van't Hoff plot (volume vs. 1/osmolality) of equine spermatozoa at 22 8C. Sperm samples were exposedto different osmolalities: 900, 600, 450, 300, and150 mosm/kg TALP. The y-intercept indicates that V b, the osmotically inactive volume, is 70.7% of the isosmotic cell volume. included hypoosmolal and hyperosmolal conditions (150±900 mosm/kg), suggesting that the values for exosmotic and endosmotic hydraulic ows were similar in the tested osmolality range. A recent paper addressing optimal cooling rates for equine sperm [19] usedestimated sperm measurements (sperm isosmotic volume, osmotically inactive cell volume) basedon geometrical shape models since objective data was not previously available. The data reportedhere will be of help in determining water andcpa permeabilities in equine spermatozoa during freezing±thawing processes. The effect of four permeable CPAs on equine sperm function has been recently reported [7]. In that study, the use of glycerol resulted in the most marked decline in sperm function (viability, motility, and MMP) while propylene glycol, dimethylsulfoxide and particularly ethylene glycol caused the least osmotic damage. During CPA addition, cells shrink due to the increasedexternal hyperosmotic environment andthe reverse occurs during CPA removal. These volume excursions associatedwith CPA use may be the cause of cell function disorders. It has been demonstrated in other species [9,20] that an optimal CPA is one that has rapidpermeability to the cell minimizing cell volume excursions, low temperature dependence, and low toxicity to the cell. Estimates from regression analysis of our data indicate that equine spermatozoa can tolerate approximately 20% swelling and 11% shrinkage of their isosmotic cell volume andstill maintain more than 70% of control motility. These limits will be used in future studies to determine the optimal CPA type and concentration to cryopreserve equine sperm Osmotic tolerance of equine spermatozoa Osmotic tolerance limits are critical to understanding the capabilities of equine spermatozoa during freezing and thawing. Resistance to anisosmolality is essential to prevent cell lysis anddeath. The capacity for spermatozoa to respondwith cell volume adjustment is determined by several factors including membrane phospholipid composi-

10 1382 A.C. Pommer et al. / Theriogenology 58 (2002) 1373±1384 tion, water permeability, lipidphase transition temperature, Na /K ATPase activity, ion channels, and cytoskeletal elements. Semen from individual stallions and boars can have signi cant differences in the ability to withstand the freeze±thaw cycle suggesting differences in sperm membrane characteristics and biochemical composition between species [4,17]. Within the range of osmotic conditions evaluated in this study, our data demonstrate that equine spermatozoa are capable of signi cant and reversible adaptation of mean cellular volume (Fig. 4). MCV was maximal at 75 mosm/kg, the lowest osmolality tested, and sperm cells reached an increase in volume of 60% over that of isosmolality. Noiles et al. [6] reportedthat stallion spermatozoa are capable of swelling to volumes of 6.1 times that of isosmotic volume. In that study, equine spermatozoa were evaluated for cell volume changes using a wider range of hyposmotic conditions (3±215 mosm/kg) than in the present study. Although the number of ejaculates was low, that study also indicated a highly responsive osmoregulatory function of equine spermatozoa. The trendbetween the two studies was similar, but there was a large difference observed in cell volume excursions while subjectedto hyposmolal solutions (1.6 versus 6.1 times isosmotic sperm volume). There are two possible explanations for this discrepancy. First, the osmotic ranges tested were lower in the study by Noiles et al. (3 mosm/kg), and second, those authors used an indirect ow cytometric technique to estimate sperm volume in response to anisosmolal conditions. The present study demonstrates that equine spermatozoa are highly susceptible to osmotic stress since sperm motility decreased in anisosmolal solutions and spermatozoa from most treatments demonstrated poor return to normal motility following this brief osmotic stress period. Both motility and viability of equine spermatozoa decreased signi cantly in hyposmotic media demonstrating a behavior similar to boar spermatozoa [4], which appear to be more sensitive to osmolality changes andto have a limitedosmotic tolerance comparedto mouse [14] andhuman sperm [17,20]. A recent study [7] reporteda limitedosmotic tolerance of equine spermatozoa when subjectedto anisosmolal conditions, in agreement with our results. It was interesting that MCV returnedto control levels in light of the failure of sperm motility to return to control levels. It is likely that the 10 min anisosmotic stress induced sublethal damage to equine spermatozoa. Although the appearance of the cells was normal using light microscopy, andthe cell volume returnedto normal, there were signi cant detrimental changes in sperm motility. It is clear that for equine spermatozoa, the swelling process is more detrimental to sperm function than shrinking, and that the mechanisms that affect motility seem to be different in these situations. Spermatozoa subjected to a hyperosmolal environment appear to have intact membranes (viable cells) andare capable of preserving their mitochondria, as demonstrated by a high MMP. However, when spermatozoa were subjectedto hypotonic solutions, MMP, andviability markedly decreased. At 450 mosm/kg cell volume was reduced to 89% of initial volume, and retained70% of initial motility. However, at 150 mosm/kg volume increased28% from control andmotility decreasedto 57% of initial motility. Furthermore, when the cells were returned to isosmolal conditions, spermatozoa in 900 mosm/kg showed decreased MMP andviability as cell volume increased. This suggests that the sperm's ability to maintain electrolyte balance andmembrane potential is hinderedwhen cells swell. Thus, the thawing process may be more detrimental to spermatozoa than the freezing process.

11 A.C. Pommer et al. / Theriogenology 58 (2002) 1373± Future studies should involve determining the optimal cryoprotectant agent to minimize such uctuations in cell volume. In summary, this study has determined several important cryobiological characteristics of equine spermatozoa that can be appliedto improve current cryopreservation protocols. The results showedthat: (a) equine spermatozoa exhibit a linear osmotic response in the range of 150±900 mosm/kg; (b) equine spermatozoa have a cell volume of 24.4 mm 3, with 70.7% being osmotically inactive; (c) although most spermatozoa are able to recover initial volume after osmotic stress, they are not able to recover initial motility andmmp; and(d) the membrane changes resulting from cellular swelling or shrinkage causes fundamental irreversible damage to plasma membrane and cellular organelles that, in turn, cause perturbations in cell function. Acknowledgements The authors gratefully acknowledge Dr. Mary Delany, Department of Animal Science, College of Agriculture andenvironmental Sciences, for technical assistance with the Z2 Coulter Counter andabigail Spinner, California Regional Primate Research Center, for technical assistance with ow cytometry. This work was supportedby a grant from the USDA (no ). References [1] Vidament M, Dupere AM, Julienne P, Evain A, Noue P, Palmer E. Equine frozen semen: freezability and fertility eldresults. Theriogenology 1997;48:907±17. [2] Jasko DJ, Moran DM, Farlin ME, Squires EL, Amann RP, Pickett BW. Pregnancy rates utilizing fresh, cooled, and frozen±thawed stallion semen. Proc Annu Conv Am Assoc Eq Pract 1992;38:649±60. [3] Watson PF. The causes of reduced fertility with cryopreserved semen. Anim Reprod Sci 2000;60/61: 481±92. [4] Holt WV. Basic aspects of frozen storage of semen. Anim ReprodSci 2000;62:3±22. [5] Watson PF. Recent developments andconcepts in the cryopreservation of spermatozoa andthe assessment of their post-thawing function. ReprodFertil Dev 1995;7:871±91. [6] Noiles EE, Mazur P, Benker FW, Kleinhans FW, Amann RP, Critser JK. Critical osmolality, water, and glycerol permeability coef cient determination of equine spermatozoa. Biol Reprod 1992;46:95 [abstract]. [7] Ball BA, Vo A. Osmotic tolerance of equine spermatozoa andthe effect of soluble cryoprotectants on equine sperm motility, viability, and mitochondrial membrane potential. J Androl 2001;22:1061±9. [8] Meyers S, Overstreet J, Liu I, Drobnis E. Capacitation in vitro of stallion spermatozoaðcomparison of progesterone-induced acrosome reactions in fertile and subfertile males. J Androl 1995;16:47±54. [9] Gilmore JA, Liu J, Peter AT, Critser JK. Determination of plasma membrane characteristics of boar spermatozoa andtheir relevance to cryopreservation. Biol Reprod1998;58:28±36. [10] Curry MR, Kleinhans FW, Watson PF. Measurements of the water permeability of the membranes of boar, ram, and rabbit spermatozoa using concentration-dependent self-quenching of an entrapped uorophore. Cryobiology 2000;41:167±73. [11] Hammerstedt RH, Graham JK, Nolan JP. Cryopreservation of mammalian sperm: what we ask them to survive. J Androl 1990;11:73±88. [12] Amann R, Graham J. Spermatozoal function. In: McKinnon A, Voss JL, editors. Equine reproduction. Philadelphia: Lea and Febiger, p. 715±45 [chapter 80].

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