- Email: [email protected]
Influence of thawing method on motility, plasma membrane integrity and morphology of frozen-thawed stallion spermatozoa
ELSEVIER
INFLUENCE OF THAWING METHOD ON MCYfILITY, PLASMA MEMBRANE
INTEGRITY
AND MORPHOLOGY K.Borgf
OF FROZEN-THAWED
STALLION
B.Colenbmnde&2 A.Faz.elif JParlevliet?
SPERMATOZOA
LMalmgrenia
1
2
Department of Obstetrics and Gynaecology Swedish University of Agricultural Sciences Uppsala, Sweden
Department of Herd Health and Reproduction University of Utrecht, Utrecht, The Netherlands Receivedforpublication: Accepted:
February 6, 1997 May 16, 1997
ABSTRACT Different thawing methods are used for stallion semen, however, it is unclear which method is the optimal one. To determine if the thawing temperature has an effect on semen quality, we compared 2 thawing temperatures, 75°C and 37°C. The following parameters were used to measure sperm quality: sperm motility, sperm viability, plasma membrane integrity and sperm morphology. Twenty-three ejaculates from 10 Dutch Warmblood stallions were thawed either at 37°C for 30 set or at 75°C for 7 sec. Sperm motility was evaluated by a Hamilton Thorn Motility Analyser. Plasma membrane integrity and sperm viability were evaluated by using a live/dead fluorescein stain containing a calcein AM probe and ethidium homodimer-1 probe. The eosinaniline blue staining method was used to evaluate the percentage of live and dead cells, as well as sperm morphology. There was no significant difference (P=O.S4) between sperm motility after thawing at 37°C and 7SC. There was also no sianificant difference (P=O.O53) between the percentage of live spermatozoa using the calcein &/ethidium homodimer stain after thawingat 37°C &d 7PC. There was.however, a significant difference (P=O.O32) between the percentage of live spermatozoa using the eosin-aniline blue stain after thawing at 37°C compared with that at 75°C. In conclusion, our laboratory results indicated that stud farms using frozen semen should thaw the straws at 37’C instead of 75°C. The lower temperature is easier to work with, as thawing at the higher temperature requires special equipment and has to be timed very carefully to avoid damage to the spermatozoa. 0 1997 by Elsewer Science Inc. Key words: stallion, frozen semen, thawing temperature, sperm motility, plasma membrane integrity Acknowledgements The authors thank Peter Ursem for helping with sperm morphology determination. a Correspondence.
Theriogenology 46:531-536, Q 1997 by Elsevier Science
1997 Inc.
0093-691X/97/%17.00 PII SOO93-691X(97)00269-0
532
Theriogenology
INTRODUCTION The integrity of the plasma membrane is of crucial importance for the functioning of the sperm cell. This membrane forms a semi-permeable barrier for molecules, and serves to maintain and modulate the intracellular composition. The membrane protects the cell from influences from the extracellular environment, both in the male or female genital tract, and also from such nonphysiological influences as extenders during sperm preservation. Adequate functioning of the plasma membranes is essential for survival of the sperm cell on its way to the oocyte at the site of fertilization (4). Processing of the semen subjects the spermatozoa to stress. Freezing and thawing is detrimental to the plasma membrane. There are different methods for freezing and thawing, some of which may be more or less harmful to the plasma membrane. It is important to ensure that an optimal thawing temperature and time arc used in order to minimize the damage to the plasma membranes of the spermatozoa. Graham (5) described how the freezing extenders consist of lactose-EDTA or a milk containing extender (FR5). The semen was packaged in 0.5.2.5 or Xl-ml straws. Martin et al. (9) froze stallion semen in large volume straws, containing 4 ml of diluted semen. These straws were thawed in a waterbath at 50°C for 45 sec. Cochran et al. (2) found that O.lml straws of equine semen which were thawed at 75°C for 7 set, followed immediately by immersion in 37°C water for at least 5 set, showed a higher percentage of progressive sperm motility than the semen thawed at 37°C for 30 sec. However, it is not clear which freezing and thawing method is the optimal one. Further basic research is needed to determine and adopt ideal freezing-thawing rates for stallion semen (4). Sperm motility is by far the most commonly used criterion for evaluating post-thaw semen quality (12). New methods that provide more information, such as different tests for investigating plasma membrane integrity are now being introduced. Harrison et al. (6) used carboxyfluorescein diacetate and propidium iodide as fluorescent stains to assess membrane integrity in sperm populations from the ram and boar. Althouse and Hopkins (1) used 2 fluorescent stains, calcein acetylmethyl ester (calcein AM) and ethidium homodimer, to assess plasma membrane integrity and sperm viability of ejaculated fresh boar spermatozoa The aims of this study were: 1) to compare equine semen quality, i.e. sperm motility, sperm viability, plasma membrane integrity and sperm morphology after thawing at 37°C and 75”C, 2) to determine if there is a correlation between sperm motility and the percentage of live spermatozoa using eosin-aniline blue staining, and between sperm motility and the percentage of live spermatozoa using calcein AMethidium homodimer stain; and 3) to determine the correlation between the percentage of live spermatozoa stained with calcein AM/ethidium homodimer and e&n-aniline blue. MATERIALS AND METHODS Twenty-three ejaculates from 10 Dutch warmblood stallions (1 to 3 ejaculates from each stallion) were used in the study. The stallions were all of proven fertility. The different ejaculates had been frozen between July and December 1994. The semen had been frozen according to the Utrecht method: The semen was diluted at 1:2 with 37°C Citrate-EDTA and then centrifugated at 900 to 1OOOGfor 15 min at 20°C. The supematant was removed, and the semen was resuspended (DV-II lactose-egg yolk-glycerole) at 20°C to a concentration of 375 x ld sperm/ml. The semen was packed in OS-ml straws, and frozen in the KRYO 10 freezing machine. The straws were cooled at a rate of -0.5Wmin from +20” to +rC, and frozen at a rate of -lOWmin from +5”C to -lfC,and at a rate of -25Wmin from -15°C to -150°C. The straws were then placed in liquid nitrogen.
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533
Straws for each ejaculate from the same sperm batch were thawed either at 37°C for 30 set or at 75°C for 7 sec. The straws were placed in waterbaths at 37°C and 75”C, respectively, immediately wiped off, cut open and emptied into a clean prewarmed vial (37°C). which was immediately placed in a heat chamber at a temperature of 37°C. Sperm motility and sperm concentration determinations were carried out using a Hamilton Thorn Motility Analyser, HTM 2000 (Hamilton Thorn Research, Dawers, MA, USA). The following settings were used: Frames acquired, 20; Frame rate, 30/s min; Contrast, 8 min; Size, 6; lo/hi size gates, 050.8; lo/hi intensity gates, 0X.8; Non-motile head size, 13; Nonmotile intensity, 25, Medium Path Velocity Value, 25umls; Low Path Velocity Value, 9um/s; Slow cells motile, no; Threshold Straightness, 80; Temperature, 37.3”C; Chamber, microcell 20um; Phase contrast. Fifty microliters of thawed semen were diluted at 15 in filtered (20 micron) prewarmed (37’C) egg yolk extender and incubated in a heat chamber at 37°C for 15 minutes (12). A prewarmed glass slide with a chamber of 20 pm depth was filled with diluted, mixed semen and put on a warming plate. Five fields were selected and motility was presented as the percentage of rapid, medium, slow and static cells. Fast and medium-fast moving cells were used to calculate the percentage of motility. Plasma membrane integrity and sperm viability were both evaluated using a live/dead fluorescein stain containing a calcein AM probe and ethidium homodimer-I probe, (Molecular Probes, Inc., Eugene, OR). Calcein AM probe is a fluorogenic substrate which is cleaved intracellularly by an estetase in viable cells. The green fluorescent product, calcein, is a polar derivative which is impermeable to cell membranes. The calcein product, however, is able to leak out of dead cells or cells with damaged plasma membranes. The ethidium homodimer-1 probe has a high affinity for DNA and is only able to pass through compromised plasma membranes of dead or damaged spermatozoa. These cells fluoresce red (7). The stain was prepared by mixing the 2 probes of 2yM with 1 ml of sterile, filtered 3PC PBS. Then20 til of semen were mixed with 20 ul of stamina medium and incubated in a dark heat chamber’at 37°C for 15 to 45 min. For the’evaluation,ilides were made and studied under a fluorescence microscope with a filter of 295 to 3 10 nm at a magnification of x 1OCO.The spermatozoa were divided into 4 groups: wholly green cells, green acrosomes with red heads, red cells with green midpieces and wholly red cells. For the evaluation of the results the green cells were considered to be live whereas the red, red/green midpieces cells and green acrosome/red head cells were considered to be dead or damaged. In total 200 sperm cells were counted per sample. In addition, the e&n-aniline blue staining method was used to evaluate the percentages of live and dead cells, respectively. Colenbrander et al. (3) described how the dye may diffuse passively into sperm cells with damaged plasma membranes. The viable cells exclude the dye, giving them a white color. Dead cells and cells with damaged membranes stain red. Sperm morphology was evaluated using an eosin-aniline blue stain. From each thawed straw, slide was prepared by mixing a drop of semen with a drop of stain. The slides were evaluated under a light microscope at x 1000 magnification. A muumum amount of 200 cells was scored of which at least 100 were unstained (i.e. considered to be live).The morphology of the sperm cells was categorized according to a standard of Brettschneider (3). Statistical analysis was carried out using a paired t-test and Pearson’s correlation test (JMP, statistical analysis program, SAS Institute Inc., Guy, NC, USA).
534
Theriogenology RESULTS
Progressive sperm motility was 40.9 f 14.6% (mean f SD) after thawing at 37’C and 41.2 f 14.8% (mean f SD) after thawing at 75’C. There was no significant difference (F=O.84) between the thawing temperatures (Figure 1). The percentage of live spermatozoa (cakein AMIethidium homodimer stain) was 46.6 f 15.2% (mean f SD) after thawing at 37°C and 52.7 f 13.6% (mean f SD) after thawing at 75’C. There was no significant difference (FW.053) between the thawing temperatures. The percentage of live spermatozoa (eosin-aniline blue stain) was 69.3 f 10.7% (mean f SD) after thawing at 37°C and 71.9 f 10.2% (mean f SD) after thawing at 7SC. There was a significant difference (kO.032) between the thawing temperatures (Figure 1).
90
1
0”
70 60 -
40 30 20 10 MOT 370
MOT 7s0
AB 37”
AB 75”
CE 37”
CE NORM NORM ACR 75O 370 7Y 37O
ACR 75”
Figure 1. The percentages of sperm motility (MOT), live cells measured by eosin-aniline blue stain (AB), and calcein AMIethidium homodimer (CE), morphologically normal spermatozoa (NORM) and acrosome defects (ACR) after thawing at 37°C and 75°C. respectively (Mean f SD). Percentage of morphologically normal spermatozoa (eosin-aniline blue stain) was 59.7 f 15.8% (mean +.SD) after thawing at 37Y and 61.6 f 13.9% (mean f SD) after thawing at 75°C. There was no significant difference (fW.36) between the thawing temperatures. The percentage of acrosome defects (eosine-aniline blue stain) was 8.2 f 8.4 (mean f SD) after thawing at 37°C and 7.4 f 5.2% (mean f SD) after thawing at 7YC. There was no significant difference (F=O.36) between the thawing temperatures (Figure 1). The correlation between motility and percentage of live spermatozoa (cakein AM/ ethidium homodimer stain) was 0.56 (kO.006) after thawing at 37°C and 0.70 (F=O.O002)after thawing at 75°C. The correlation between motility and percentage of live spermatozoa (eosinaniline blue stain) was 0.63 (F=O.O013)after thawing at 37°C and 0.81 (P=O.OOOO) after
Theriogenology
535
thawing at 75’C. The correlation between the percentage of live spermatozoa with the caicein AM/ethidium homodimer stain versus the eosin-aniline blue stain was 0.65 (P=O.OOCV) after thawing at 37°C and 0.78 (P=O.OOOO) after thawing at 75°C. DISCUSSION Successful artificial insemination in the equine species depends on the ability of spermatozoa to keep their fertilizing capacity during and after storage (11). Maintenance of membrane stabilitv and membrane-related functions is crucial for the oreservation of fertilitv durina freezmg and thawing (4). In our study we found no significant difference between spe& motility of semen thawed at 37’C and at 75°C. Cochmn et al. (2) found a higher, although not significant, sperm motility after thawing at 75V for 7 set than at 37°C for 30 sec. The discrepancy between the 2 studies may be.explained by slight difference in thawing techniques. Cochran et al. (2) immersed the straws which thev had thawed at 75°C immediately into 37°C water for at least 5 set, which we did not do. * Sperm motility is the most commonly used criterion for assessing post-thaw semen quality. However, Squires et al. (14) found that the percentage of progressively motile spermatozoa in frozen-thawed semen was a notoriously poor predictor of pregnancy rates. Samper (13) also stated that most laboratory assays employed for semen evaluation are poorly correlated with the fertility of spermatozoa. Membrane integrity was also extremely poorly correlated with the motility, particularly of preserved semen (13). In our study, however, there seemed to be positive correlation between the motility and plasma membrane integrity. Althouse and Hopkins ( 1) found good correlation between motility and green fluorescing spermatozoa in their calcein AUlethidium homodimer staining of fresh boar spermatozoa. The authors stated that the viability of a semen sample could be approximated by visual estimation of motile sperm cells, but that this method was not as accurate as fluorophore determination. New evaluation techniques have made it possible to get better information. The correlation between sperm motility and plasma membrane integrity, as measured by calcein AM/ethidium homodimer, appears to be more effective for fresh spermatozoa than for frozen-thawed spermatozoa (1). Using calcein AM/ethidium homodimer stain rather than motility may thus provide more accurate results when assessing the post-thaw quality of frozen-thawed stallion spermatozoa. There were individual differences between the stallions in our study. One stallion, for instance, showed very low sperm motility after freezing and thawing, while the percentage of live spermatozoa in calceinAM/ethidium homodimer staining was high. The fertility rate for this stallion was also acceptable. The difference between the number of live spermatozoa stained with calcein AM/ethidium homodimer and spermatozoa stained with eosin-aniline blue was greater than suspected. The number of live spermatozoa in the eosin-aniline blue stain may be unusually high. This discrepancy may depend on the fact that calcein AMlethidium homodimer is a more sensitive method for measuring damage to plasma membranes. Small defects or damage may not be visible in spermatozoa stained with eosin-aniline blue stain, because of the slower passage of this stain through plasma membranes. Damaged cells may therefor falsely look like live ones. In conclusion, our laboratory results indicate that stud farms using frozen semen should thaw the straws at 37°C instead of at 75’C. The lower temperature is easier to work with, as thawing at the higher temperature requires special equipment and needs to be carefully timed to avoid damage to the spermatozoa.
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REFERENCES
1. Althouse GC, Hopkins SM. Assessment of boar sperm viability using a combination of two fluoroohores. Therioeenoloev 1995: 43: 595603. 2. Cochran JD, Amann RP, PromcbP. Pickett BW. Effects of centrifugation. glycerol level, cooling to 5°C. freezing rate and thawing rate on the post-thaw motility. Theriogenology 1984; 22: 25-38. 3, Colenbrander B, Fazeli AR, van Buiten A, Parlevliet J, Gadella BM. Assessment of sperm cell membrane integrity in the horse. Acta Vet Stand 1992; 88: 49-58. 4. Einarsson S. Concluding remarks. Acta Vet Stand 1992; 88: 165166. 5. Graham JR. Current techniaues for freezine equine semen. In: Proc. Techniaues for handling and utilization of tmnsported coolgd &d frozen equine spermatozoa.~Fort Collins 1994; 89-99. 6. Harrison RAP, Vickers SE. Use of fluorescent probes to assess membrane integrity in mammalian spermatozoa J Reprod Fertill990; 88: 343-352. 7. HaugIand RP. Handbook of Fluorescent Probes and Research Chemicals. City: Eugene OR. Molecular Probes Inc. 1992: 172-175. 8. Klug E, Robbelen I, Kneissl S, Sieme H. Cryopreservation of stallion spermatozoa. Acta Vet Stand 1992; 88: 129-135. 9. Martin JC, Klug E, Gunzel A-R. Centrifugation of stallion semen and its storage in large volume straws. J Reprod Fert 1979; 27: 47-51. 10. Palmer E. Magistrini M. Automated analysis of stallion semen post-thaw motility. Acta Vet Stand 1992; 88: 137-152. 11. Parlevliet J, MaImgren L, Boyle M, Wiickener A, Bader H, Colenbrander B. Influence of conservation method on the motility and morphology of stallion semen (an international project). Acta Vet Stand 1992,88: 153-162. 12. Rasbeck NO. Instnmtental inseminering i hestavlen. Dansk Vet Tidskrift 1984,67: 116 13. Samper JC. Evaluation of cryopreserved semen: an alternative assay. Acta Vet Stand 1992; 88: 59-65. 14. Squires EL, Amann RP, Pickett BW. Preservation of stallion semen - Colorado experience. In: Symposium on equine reproduction. Intervet Scandinavia Skovlunde Denmark 1987; 51-56.
INFLUENCE OF THAWING METHOD ON MCYfILITY, PLASMA MEMBRANE
INTEGRITY
AND MORPHOLOGY K.Borgf
OF FROZEN-THAWED
STALLION
B.Colenbmnde&2 A.Faz.elif JParlevliet?
SPERMATOZOA
LMalmgrenia
1
2
Department of Obstetrics and Gynaecology Swedish University of Agricultural Sciences Uppsala, Sweden
Department of Herd Health and Reproduction University of Utrecht, Utrecht, The Netherlands Receivedforpublication: Accepted:
February 6, 1997 May 16, 1997
ABSTRACT Different thawing methods are used for stallion semen, however, it is unclear which method is the optimal one. To determine if the thawing temperature has an effect on semen quality, we compared 2 thawing temperatures, 75°C and 37°C. The following parameters were used to measure sperm quality: sperm motility, sperm viability, plasma membrane integrity and sperm morphology. Twenty-three ejaculates from 10 Dutch Warmblood stallions were thawed either at 37°C for 30 set or at 75°C for 7 sec. Sperm motility was evaluated by a Hamilton Thorn Motility Analyser. Plasma membrane integrity and sperm viability were evaluated by using a live/dead fluorescein stain containing a calcein AM probe and ethidium homodimer-1 probe. The eosinaniline blue staining method was used to evaluate the percentage of live and dead cells, as well as sperm morphology. There was no significant difference (P=O.S4) between sperm motility after thawing at 37°C and 7SC. There was also no sianificant difference (P=O.O53) between the percentage of live spermatozoa using the calcein &/ethidium homodimer stain after thawingat 37°C &d 7PC. There was.however, a significant difference (P=O.O32) between the percentage of live spermatozoa using the eosin-aniline blue stain after thawing at 37°C compared with that at 75°C. In conclusion, our laboratory results indicated that stud farms using frozen semen should thaw the straws at 37’C instead of 75°C. The lower temperature is easier to work with, as thawing at the higher temperature requires special equipment and has to be timed very carefully to avoid damage to the spermatozoa. 0 1997 by Elsewer Science Inc. Key words: stallion, frozen semen, thawing temperature, sperm motility, plasma membrane integrity Acknowledgements The authors thank Peter Ursem for helping with sperm morphology determination. a Correspondence.
Theriogenology 46:531-536, Q 1997 by Elsevier Science
1997 Inc.
0093-691X/97/%17.00 PII SOO93-691X(97)00269-0
532
Theriogenology
INTRODUCTION The integrity of the plasma membrane is of crucial importance for the functioning of the sperm cell. This membrane forms a semi-permeable barrier for molecules, and serves to maintain and modulate the intracellular composition. The membrane protects the cell from influences from the extracellular environment, both in the male or female genital tract, and also from such nonphysiological influences as extenders during sperm preservation. Adequate functioning of the plasma membranes is essential for survival of the sperm cell on its way to the oocyte at the site of fertilization (4). Processing of the semen subjects the spermatozoa to stress. Freezing and thawing is detrimental to the plasma membrane. There are different methods for freezing and thawing, some of which may be more or less harmful to the plasma membrane. It is important to ensure that an optimal thawing temperature and time arc used in order to minimize the damage to the plasma membranes of the spermatozoa. Graham (5) described how the freezing extenders consist of lactose-EDTA or a milk containing extender (FR5). The semen was packaged in 0.5.2.5 or Xl-ml straws. Martin et al. (9) froze stallion semen in large volume straws, containing 4 ml of diluted semen. These straws were thawed in a waterbath at 50°C for 45 sec. Cochran et al. (2) found that O.lml straws of equine semen which were thawed at 75°C for 7 set, followed immediately by immersion in 37°C water for at least 5 set, showed a higher percentage of progressive sperm motility than the semen thawed at 37°C for 30 sec. However, it is not clear which freezing and thawing method is the optimal one. Further basic research is needed to determine and adopt ideal freezing-thawing rates for stallion semen (4). Sperm motility is by far the most commonly used criterion for evaluating post-thaw semen quality (12). New methods that provide more information, such as different tests for investigating plasma membrane integrity are now being introduced. Harrison et al. (6) used carboxyfluorescein diacetate and propidium iodide as fluorescent stains to assess membrane integrity in sperm populations from the ram and boar. Althouse and Hopkins (1) used 2 fluorescent stains, calcein acetylmethyl ester (calcein AM) and ethidium homodimer, to assess plasma membrane integrity and sperm viability of ejaculated fresh boar spermatozoa The aims of this study were: 1) to compare equine semen quality, i.e. sperm motility, sperm viability, plasma membrane integrity and sperm morphology after thawing at 37°C and 75”C, 2) to determine if there is a correlation between sperm motility and the percentage of live spermatozoa using eosin-aniline blue staining, and between sperm motility and the percentage of live spermatozoa using calcein AMethidium homodimer stain; and 3) to determine the correlation between the percentage of live spermatozoa stained with calcein AM/ethidium homodimer and e&n-aniline blue. MATERIALS AND METHODS Twenty-three ejaculates from 10 Dutch warmblood stallions (1 to 3 ejaculates from each stallion) were used in the study. The stallions were all of proven fertility. The different ejaculates had been frozen between July and December 1994. The semen had been frozen according to the Utrecht method: The semen was diluted at 1:2 with 37°C Citrate-EDTA and then centrifugated at 900 to 1OOOGfor 15 min at 20°C. The supematant was removed, and the semen was resuspended (DV-II lactose-egg yolk-glycerole) at 20°C to a concentration of 375 x ld sperm/ml. The semen was packed in OS-ml straws, and frozen in the KRYO 10 freezing machine. The straws were cooled at a rate of -0.5Wmin from +20” to +rC, and frozen at a rate of -lOWmin from +5”C to -lfC,and at a rate of -25Wmin from -15°C to -150°C. The straws were then placed in liquid nitrogen.
Theriogenology
533
Straws for each ejaculate from the same sperm batch were thawed either at 37°C for 30 set or at 75°C for 7 sec. The straws were placed in waterbaths at 37°C and 75”C, respectively, immediately wiped off, cut open and emptied into a clean prewarmed vial (37°C). which was immediately placed in a heat chamber at a temperature of 37°C. Sperm motility and sperm concentration determinations were carried out using a Hamilton Thorn Motility Analyser, HTM 2000 (Hamilton Thorn Research, Dawers, MA, USA). The following settings were used: Frames acquired, 20; Frame rate, 30/s min; Contrast, 8 min; Size, 6; lo/hi size gates, 050.8; lo/hi intensity gates, 0X.8; Non-motile head size, 13; Nonmotile intensity, 25, Medium Path Velocity Value, 25umls; Low Path Velocity Value, 9um/s; Slow cells motile, no; Threshold Straightness, 80; Temperature, 37.3”C; Chamber, microcell 20um; Phase contrast. Fifty microliters of thawed semen were diluted at 15 in filtered (20 micron) prewarmed (37’C) egg yolk extender and incubated in a heat chamber at 37°C for 15 minutes (12). A prewarmed glass slide with a chamber of 20 pm depth was filled with diluted, mixed semen and put on a warming plate. Five fields were selected and motility was presented as the percentage of rapid, medium, slow and static cells. Fast and medium-fast moving cells were used to calculate the percentage of motility. Plasma membrane integrity and sperm viability were both evaluated using a live/dead fluorescein stain containing a calcein AM probe and ethidium homodimer-I probe, (Molecular Probes, Inc., Eugene, OR). Calcein AM probe is a fluorogenic substrate which is cleaved intracellularly by an estetase in viable cells. The green fluorescent product, calcein, is a polar derivative which is impermeable to cell membranes. The calcein product, however, is able to leak out of dead cells or cells with damaged plasma membranes. The ethidium homodimer-1 probe has a high affinity for DNA and is only able to pass through compromised plasma membranes of dead or damaged spermatozoa. These cells fluoresce red (7). The stain was prepared by mixing the 2 probes of 2yM with 1 ml of sterile, filtered 3PC PBS. Then20 til of semen were mixed with 20 ul of stamina medium and incubated in a dark heat chamber’at 37°C for 15 to 45 min. For the’evaluation,ilides were made and studied under a fluorescence microscope with a filter of 295 to 3 10 nm at a magnification of x 1OCO.The spermatozoa were divided into 4 groups: wholly green cells, green acrosomes with red heads, red cells with green midpieces and wholly red cells. For the evaluation of the results the green cells were considered to be live whereas the red, red/green midpieces cells and green acrosome/red head cells were considered to be dead or damaged. In total 200 sperm cells were counted per sample. In addition, the e&n-aniline blue staining method was used to evaluate the percentages of live and dead cells, respectively. Colenbrander et al. (3) described how the dye may diffuse passively into sperm cells with damaged plasma membranes. The viable cells exclude the dye, giving them a white color. Dead cells and cells with damaged membranes stain red. Sperm morphology was evaluated using an eosin-aniline blue stain. From each thawed straw, slide was prepared by mixing a drop of semen with a drop of stain. The slides were evaluated under a light microscope at x 1000 magnification. A muumum amount of 200 cells was scored of which at least 100 were unstained (i.e. considered to be live).The morphology of the sperm cells was categorized according to a standard of Brettschneider (3). Statistical analysis was carried out using a paired t-test and Pearson’s correlation test (JMP, statistical analysis program, SAS Institute Inc., Guy, NC, USA).
534
Theriogenology RESULTS
Progressive sperm motility was 40.9 f 14.6% (mean f SD) after thawing at 37’C and 41.2 f 14.8% (mean f SD) after thawing at 75’C. There was no significant difference (F=O.84) between the thawing temperatures (Figure 1). The percentage of live spermatozoa (cakein AMIethidium homodimer stain) was 46.6 f 15.2% (mean f SD) after thawing at 37°C and 52.7 f 13.6% (mean f SD) after thawing at 75’C. There was no significant difference (FW.053) between the thawing temperatures. The percentage of live spermatozoa (eosin-aniline blue stain) was 69.3 f 10.7% (mean f SD) after thawing at 37°C and 71.9 f 10.2% (mean f SD) after thawing at 7SC. There was a significant difference (kO.032) between the thawing temperatures (Figure 1).
90
1
0”
70 60 -
40 30 20 10 MOT 370
MOT 7s0
AB 37”
AB 75”
CE 37”
CE NORM NORM ACR 75O 370 7Y 37O
ACR 75”
Figure 1. The percentages of sperm motility (MOT), live cells measured by eosin-aniline blue stain (AB), and calcein AMIethidium homodimer (CE), morphologically normal spermatozoa (NORM) and acrosome defects (ACR) after thawing at 37°C and 75°C. respectively (Mean f SD). Percentage of morphologically normal spermatozoa (eosin-aniline blue stain) was 59.7 f 15.8% (mean +.SD) after thawing at 37Y and 61.6 f 13.9% (mean f SD) after thawing at 75°C. There was no significant difference (fW.36) between the thawing temperatures. The percentage of acrosome defects (eosine-aniline blue stain) was 8.2 f 8.4 (mean f SD) after thawing at 37°C and 7.4 f 5.2% (mean f SD) after thawing at 7YC. There was no significant difference (F=O.36) between the thawing temperatures (Figure 1). The correlation between motility and percentage of live spermatozoa (cakein AM/ ethidium homodimer stain) was 0.56 (kO.006) after thawing at 37°C and 0.70 (F=O.O002)after thawing at 75°C. The correlation between motility and percentage of live spermatozoa (eosinaniline blue stain) was 0.63 (F=O.O013)after thawing at 37°C and 0.81 (P=O.OOOO) after
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thawing at 75’C. The correlation between the percentage of live spermatozoa with the caicein AM/ethidium homodimer stain versus the eosin-aniline blue stain was 0.65 (P=O.OOCV) after thawing at 37°C and 0.78 (P=O.OOOO) after thawing at 75°C. DISCUSSION Successful artificial insemination in the equine species depends on the ability of spermatozoa to keep their fertilizing capacity during and after storage (11). Maintenance of membrane stabilitv and membrane-related functions is crucial for the oreservation of fertilitv durina freezmg and thawing (4). In our study we found no significant difference between spe& motility of semen thawed at 37’C and at 75°C. Cochmn et al. (2) found a higher, although not significant, sperm motility after thawing at 75V for 7 set than at 37°C for 30 sec. The discrepancy between the 2 studies may be.explained by slight difference in thawing techniques. Cochran et al. (2) immersed the straws which thev had thawed at 75°C immediately into 37°C water for at least 5 set, which we did not do. * Sperm motility is the most commonly used criterion for assessing post-thaw semen quality. However, Squires et al. (14) found that the percentage of progressively motile spermatozoa in frozen-thawed semen was a notoriously poor predictor of pregnancy rates. Samper (13) also stated that most laboratory assays employed for semen evaluation are poorly correlated with the fertility of spermatozoa. Membrane integrity was also extremely poorly correlated with the motility, particularly of preserved semen (13). In our study, however, there seemed to be positive correlation between the motility and plasma membrane integrity. Althouse and Hopkins ( 1) found good correlation between motility and green fluorescing spermatozoa in their calcein AUlethidium homodimer staining of fresh boar spermatozoa. The authors stated that the viability of a semen sample could be approximated by visual estimation of motile sperm cells, but that this method was not as accurate as fluorophore determination. New evaluation techniques have made it possible to get better information. The correlation between sperm motility and plasma membrane integrity, as measured by calcein AM/ethidium homodimer, appears to be more effective for fresh spermatozoa than for frozen-thawed spermatozoa (1). Using calcein AM/ethidium homodimer stain rather than motility may thus provide more accurate results when assessing the post-thaw quality of frozen-thawed stallion spermatozoa. There were individual differences between the stallions in our study. One stallion, for instance, showed very low sperm motility after freezing and thawing, while the percentage of live spermatozoa in calceinAM/ethidium homodimer staining was high. The fertility rate for this stallion was also acceptable. The difference between the number of live spermatozoa stained with calcein AM/ethidium homodimer and spermatozoa stained with eosin-aniline blue was greater than suspected. The number of live spermatozoa in the eosin-aniline blue stain may be unusually high. This discrepancy may depend on the fact that calcein AMlethidium homodimer is a more sensitive method for measuring damage to plasma membranes. Small defects or damage may not be visible in spermatozoa stained with eosin-aniline blue stain, because of the slower passage of this stain through plasma membranes. Damaged cells may therefor falsely look like live ones. In conclusion, our laboratory results indicate that stud farms using frozen semen should thaw the straws at 37°C instead of at 75’C. The lower temperature is easier to work with, as thawing at the higher temperature requires special equipment and needs to be carefully timed to avoid damage to the spermatozoa.
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REFERENCES
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