CRYOBIOLOGY
28, 454-459 (1991)
New Thermal
Stress Test to Assess the Viability Boar Sperm’
P. S. FISER, Animal
C. HANSEN, Research
Centre,
L. UNDERHILL, Agriculture
Canada,
of Cryopreserved
AND G. J. MARCUS Ottawa,
Ontario
KIA
005
A new, rapid, thermal stress test for assessing the viability of boar semen, requiring only 45 min of incubation at 42.X, was developed and compared with a widely used stress test of 180 min incubation at 37°C. The shorter procedure was found to have the same discriminatory ability as the standard test in assessing the effects of freezing conditions on the percentage of spermatozoa remaining motile. Neither test was able to show differences in the kinetic rating of motile sperm after freezing in relation to the glycerol concentration present during freezing. However, the new test had a greater ability to distinguish the effects of different concentrations of glycerol, over the range of 0 to 6%, and to reveal different degrees of acrosomal damage sustained during freezing. The longer procedure was unable to distinguish among glycerol concentrations from 0 to 4% with respect to acrosomal damage and produced an overall lower proportion of sperm having a normal apical ridge. The new thermal stress test thus has the advantages of greater sensitivity and more rapid execution over the test hitherto in widespread use.
Prediction of the fertilizing potential of fresh or frozen mammalian semen is usually based on in vitro assessment. However, there is no single in vitro test applicable to fresh and frozen mammalian semen in general and porcine semen in particular whose results correlate well with fertility. A fair assessment of semen requires evaluation by two or more different tests in vitro. Two tests commonly used with boar semen evaluate sperm motility and membrane integrity, usually expressed in terms of the proportions of spermatozoa with progressive forward motion and a normal apical acrosoma1 ridge. After processing and initial microscopic evaluation, semen is often subjected to a second test, termed a “thermal stress test,” involving in vitro incubation for several hours followed by reexamination, as an estimate of the capacity of sperm to survive in the female reproductive tract and retain their fertilizing capability (8, 9). Exposure of frozen-thawed sperm to the “thermal stress test” may reveal latent
Received July 30, 1990; accepted January 28, 1991. ’ ARC Contribution No. 16%. 454 001 l-2240/91 $3.00
damage, that is, damage which is not apparent immediately after ejaculation and processing. The test is thus an additional aid in assessing different processing procedures and their effect on sperm survival. Most thermal stress tests entail incubation at 3738°C (1, 4, 6, 10-12) for various times, usually 2 to 5 h. It has been reported (10) that the semen of boars of high fertility shows good motility after a 3-h stress test at 37°C. However, the acrosomal integrity of frozen-thawed sperm, evaluated as a percentage of sperm with a normal apical ridge (NAR), deteriorates substantially after incubation for 3 to 5 h, making discrimination among various treatments more difficult. Some investigators, therefore, have used an incubation of 2 h, which reveals fewer differences in motility, but distinguishes differences in the proportions of sperm with NAR more readily (1, 13). To shorten the test while preserving its discriminatory ability, we increased the incubation temperature to 42.5”C and reduced the incubation period to 45 min. We describe a comparison of the new test with a widely used, standard thermal stress test (incubation for 3 h at 37”C), using semen
THERMAL
processed in each of four levels of glycerol and frozen with suboptimal or optimal cooling rates (2). MATERIALS
AND
METHODS
Semen Collection, Evaluation and Processing Seventeen ejaculates from mature Yorkshire boars were collected over a 6-week period by the gloved-hand technique. The sperm-rich fraction of the ejaculates was received in a 400-ml glass beaker covered with a single layer of cheese cloth, held in a thermos bottle containing water at 33°C. The sperm concentration was determined spectrophotometrically. The motility was estimated microscopically using a heated stage as the percentage of motile sperm (O100%) and the kinetic rating which is a measure of the quality of movement exhibited by motile sperm, on a scale of 0 (nonmotile) to 5 (vigorous progressive motility). After being held in the thermos bottle for 2 h, the ejaculates were distributed into centrifuge tubes in portions of 7 x lo9 sperm per tube and centrifuged; the supernatants were discarded and BF5 diluent was added to produce a final volume of 5 ml/tube, as described by Purse1 and Johnson (14). After cooling to 5°C over a period of 2 h, each semen sample was further diluted 1: 1 with one of the BF5 diluents containing 0, 4, 8, or 12% glycerol (v/v) producing final glycerol concentrations of 0, 2, 4, or 6% (v/v). The semen samples were then transferred into 0.5 ml plastic straws color coded according to glycerol concentration, cooled to -60°C at a rate of either l”C/min (suboptimal) or 30Wmin (optimal, (2)) using a programmable freezer (CRYO-MED, 900-4 controller, 972-3 freezing chamber), and plunged into liquid nitrogen for storage. Straws were thawed by immersion for 8 s in water at 60°C (3), then emptied individually into test tubes containing 1 ml of Beltsville Thawing Solution (BTS, (14)) at 33°C. After equilibration for 5 min, the post-thaw motility percentage (FMP) and
STRESS
455
TEST
kinetic rating (FMR, scale of &5) of the thawed sperm were assessed microscopically using a heated microscope stage. Samples were removed and fixed in 1% glutaraldehyde for evaluation of acrosomal integrity (15). Samples from each combination of glycerol concentration and cooling rate were then incubated in a water bath for 3 h at 37”C, or at 42.5”C for 45 min. The percentage of motile sperm following the thermal stress test (TMP) and its postthermal stress kinetic rating (TMR) were then determined and acrosomal integrity was reevaluated. The percentage of boar sperm with NAR was determined using phase contrast microscopy and the scoring system of Purse1 et al. (16). Acrosomal damage was classified, using at least 100 sperm/sample. Statistical
Analysis
Least squares procedures (7) were used in a split-plot analysis of variance of sperm motility and acrosomal integrity before and after the two thermal stress tests, using a linear model which included the effects of boar, ejaculate within boar, glycerol concentration, and interaction of boar with glycerol. Because there was known to be significant interaction between glycerol concentration and freezing velocity (2), the data pertaining to each cooling velocity were analyzed separately. Duncan’s multiple range test was used to evaluate differences in survival according to glycerol level at a given thermal stress test temperature. RESULTS
There were no differences (P > 0.05) between the two thermal stress tests in their capacity to discriminate among glycerol concentrations with respect to the influence on sperm motility after freezing and thawing. In both tests, the percentage of motile sperm increased with increasing glycerol concentration. Also, the superiority of a cooling velocity of 3OWmin versus l”C/min was shown equally well by the two tests
456
FISER ET AL.
(Fig. 1). After either test, the percentage of motile sperm, expressed as a proportion of those surviving after freezing and thawing, was substantially higher in semen frozen by cooling at 30Wmin than in sperm frozen by cooling at l”C/min. The capacity of sperm classified as motile after thawing to survive either thermal stress test was influenced greatly by the original cooling rate (Fig. 1: compare upper and lower panels). Fewer sperm survived freezing after cooling at l”C/min than at 3OWmin and of those that survived, fewer survived the thermal stress test.
50
I
Cooling
velocity
l%/mi]
40 30
nl
n
20 10 0
0
2
4 37’
50
0
6 GLYCEROL
b
2
Cooling
4
6
42.5’
% velocity
30°C/min nl
r-l 40 30 20 10 0
0
2
4 37”
6 GLYCEROL
OFMP
0 %
2
4
6
Both tests showed a lower kinetic rating of motile sperm only in semen cooled at a suboptimal rate in the absence of glycerol. Otherwise, the tests did not discriminate among glycerol concentrations with respect to kinetic rating. The thermal stress tests differed significantly (P < O.Ol), however, in their capacity to reveal differences in the proportions of sperm retaining a normal apical ridge after being frozen and thawed in diluents with various glycerol concentrations. The 425°C test discriminated more readily among various glycerol concentrations with respect to the percentage of sperm with NAR in semen frozen by cooling at either l”C/min or at 30Wmin. This is illustrated in Fig. 3 with the preincubation acrosomal integrity of frozen-thawed sperm shown in Fig. 2. Incubation at 37°C for 3 h revealed no differences between 0,2, or 4% glycerol with respect to the effects on membrane integrity of sperm cooled at either rate (P > 0.05), but showed only a difference between these concentrations and 6%. In comparison, incubation for 45 min at 42.5”C, distinguished between glycerol concentrations of 0 or 2%, 4%, and 6%: the lower the concentration the greater the percentage of sperm with NAR. Sperm always showed a higher percentage NAR after incubation at 42.5”C than at 37°C. There were significant differences among boars with respect to all the semen quality variables assessed. However, such biological differences, although always present (10, 19), did not confound the ability to discriminate among treatment effects using the thermal stress test.
42.5’
RRWTMP
FIG. 1. The effect of two thermal stress tests (37”CI 180 min vs 42SW45 min) on the percentage of motile sperm in boar semen protected by O-6% glycerol frozen at 1 or 30Wmin. FMP = Percentage of sperm motile immediately after freezing and thawing (before incubation); TMP = percentage of frozen-thawed sperm motile after the thermal stress test.
DISCUSSION
The quality as well as the viability of spermatozoa deteriorates as a consequence of freezing and thawing. The degree of deterioration depends on several factors, e.g., freezing and thawing rates (2, 3), the nature and concentration of the cryoprotectant and of other components of the diluent used
THERMAL
%
Cooling
velocity
GLYCEROL
%
Cooling
STRESS
457
TEST
%
l’C/min
%
velocity
37”
3O”Wmin
velocity 1”Clmin
Cooling
%
GLYCEROL % Cooling
42.5’
velocity
3O”Clmin
100
0
0
2
4
GLYCEROL
%
6
0 37”
I
FIG. 2. Preincubation acrosomal integrity of boar sperm frozen in O-6% glycerol at I or 3WUmin. NAR = Normal apical ridge; DAR = damaged apical ridge; MAR = missing apical ridge; LAC = loose acrosomal cap.
for freezing (22), the species, and individual donor (19), to name just a few. In order to evaluate fresh or stored ejaculates or sperm which have undergone freezing and thawing, samples are often subjected to an additional test. This usually involves incubation for several hours at body temperature. The new thermal stress test, described here, entails incubation of diluted boar semen at a higher temperature (42.5”C-the upper extreme of the physiological range) for a considerably shorter time than is required by “standard” incubation tests, which may require incubation for up to 5 h at 37°C (1,8, 11, 21). Comparison with such a test has shown that the new thermal stress test provides the same degree of discrimi-
GLYCEROL
%
2
4
6
42.5’
I
FIG. 3. The effect of two thermal stress tests (37W 180 min vs 42S”C/45 min) on acrosomal integrity of boar sperm frozen in O-6% glycerolated diluents at 1 or 30Wmin. NAR = Normal apical ridge; DAR = damaged apical ridge; MAR = missing apical ridge; LAC = loose acrosomal cap.
nation in assessing the effects of cooling rate and glycerol concentration on maintenance of the motility of boar sperm after freezing and thawing, but requires only a third of the usual time (10, 12, 18). A greater loss of motility during incubation of frozen-thawed sperm has been reported to be associated with the use of increased glycerol levels for freezing (1). This effect was also apparent in the present study, particularly in semen frozen by cooling at the suboptimal rate. The loss of motility during the thermal stress test has been attributed to a toxic effect of dead sperm, associated with the liberation of amino oxidase activity (20).
458
FISER ET AL.
However, it is more likely that latent damage to the sperm at the time of freezing under suboptimal conditions is responsible for the death of sperm during post-thaw incubation, i.e., some sperm which survived suboptimal freezing conditions had sustained membrane damage that was revealed only during subsequent incubation. The prolonged incubation of sperm at 37°C causes deterioration and changes in the acrosomes (17), but may also allow a proportion of the sperm to undergo the normal acrosome reaction (IS). This effect would not be distinguishable from an abnormal loss of the apical ridge. These changes contribute to the generally lower proportions of sperm with NAR following 3-h incubation at 37”C, compared with that after incubation at 42.5”, regardless of the original glycerol concentration, and make the 37” test less discriminating. The duration of motility after thawing is believed to be an indication of the usability of bovine semen (9, 17). It can be assumed that in porcine semen, a thermal stress test result showing more lasting motility in sperm which survived optimal freezing indicates a greater likelihood of such sperm surviving in the female reproductive tract to undergo capacitation and fertilize ova. The new test reveals such endurance more rapidly. We believe that incorporating the new test as a part of semen evaluation in vitro will improve the accuracy of predicting the fertilizing ability of semen. However, we emphasize that in such evaluation, one must not rely on the results of a single type of test. ACKNOWLEDGMENT
2.
3.
4.
5.
6.
7. 8.
9. 10.
11. 12.
13.
This study was supported in part by a grant from the Ontario Ministry of Agriculture and Food under the Ontario Pork Industry Improvement Program (OPIIP). REFERENCES
1. Almlid, T., and Johnson, A. L. Effects of glycerol concentration, equilibration time and temperature of glycerol addition on post-thaw viability
of boar spermatozoa frozen in straws. J. Anim. (1988). Fiser, P. S., and Fairfull, R. W. Combined effect of glycerol concentration and cooling velocity on motility and acrosomal integrity of boar spermatozoa frozen in 0.5 ml straws. Mol. Reprod. Dev. 25, 123-129 (1990). Fiser, P. S., Fairfull, R. W., and Marcus, G. J. The effect of thawing velocity on survival and acrosomal integrity of ram spermatozoa frozen at optimal and suboptimal rates in straws. Cryobiology 23, 141-149 (1986). Foley, C. W., Heidenreich, C. J., Harrington, R. B., Jones H. W., and Erb, R. E. Changes in fructose, lactic acid, pH, and motility of boar semen during incubation at 37°C. J. Anim. Sci. 23, 558-561 (1964). Hamano, S., Naito, K., Fukuda, Y., and Toyoda, Y. In vitro capacitation of boar ejaculated spermatozoa: Effect of conditioned media prepared from preincubated sperm suspension. Gamete Res. 24, 483489 (1989). Hammitt, D. G., and Martin, P. A. Correlations among assays of porcine semen quality following cryopreservation. Theriogenology 32, 369384 (1989). Harvey, W. R. “Least Squares Analysis of Data with Unequal Subclass Numbers.” U.S. Dept. of Agriculture ARS-20-8, 1960. King, G. J., and Macpherson, J. W. Boar semen studies. II. Laboratory and fertility results of a method for deep freezing. Can. J. Comp. Med. Vet. Sci. 31, 46-47 (1967). Kozumplik, J. Vliv modilikatoru povrchoveho napeti na morfologii a motilitu spermii kance po rozmrazeni. Vet. Med. 28, 273-277 (1983). Larsson, K., and Ersmar, M. Laboratory studies on frozen-thawed boar semen in relation to contemporary fertility with liquid semen of AI boars. Zuchthygiene 15, 11l-l 17 (1980). Park, C. S., and Purse], V. G. Effect of freezing rate on boar sperm frozen in maxi-straws. J. Anim. Sci. 61(Suppl. 1), 411 (1985). [Abstract] Paquignon, M., Dacheux, J. L., and Courot, M. Effet de differentes solutions de congelation sur le pourvoir fecondant des spermatozoides de verrat. / Rech. Porcine France 15-18 (1977). Pursel, V. G., and Park, C. S. Duration of thawing on post-thaw acrosome morphology and motility of boar spermatozoa frozen in 5-ml maxi-straws. Theriogenology 28, 683-690 (1987). Purse], V. G., and Johnson, L. A. Freezing of boar spermatozoa: Fertilizing capacity with concentrated semen and a new thawing procedure. J. Anim. Sci. 40, 99-102 (1975). Pursel, V. G., Johnson, L. A., and Schulman, Sci. 66, 2899-2905
14.
15.
THERMAL
L. L. Acrosome morphology of boar spermatozoa during in vitro aging. J. Anim. Sci. 38, 113116 (1974). 16. Pursel, V. G., Johnson, L. A., and Rampacek, G. E. Acrosome morphology of boar spermatozoa incubated before cold shock. J. Anim. Sci. 34, 278-283 (1972). 17. Saacke, R. G., and White, J. M. Semen quality tests and their relationship to fertility. Proc. 4th Tech. Conf. A. I. Reprod. NAAB. Chicago. pp. 22-27, 1972. 18. Salamon, S. Deep freezing of boar semen. III. Effects of centrifugation, diluent and dilution rate, pellet volume, and method of thawing on survival of spermatozoa. Amt. J. Biol. Sci. 26, 23!%247 (1973).
STRESS
TEST
459
19. Schuler, D., Kuepfer, U., and Zimmermann, F. Tiefgefrieren von Ebersperma: Ein vergleich zweier methoden. Schweiz. Arch. Tierheilk. 121, 173-178 (1979). 20. Shannon, P., and Curson, B. Toxic effect and action of dead sperm on diluted bovine semen. J. Dairy Sci. 55, 614-620 (1972). 21. Visser, D., and Salamon, S. Effect of composition of Tris-based diluent on survival of boar spermatozoa following deep freezing. Amt. J. Biol. Sci. 27, 485497 (1974). 22. Waberski, D., Weitze, K. F., Rath, D., and Gallmann, H. P. Wirkung von bovinem serumalbumin und Zwitterionen-puffer auf fluessigkonservierten Ebersamen. Zuchthygiene 24, 128-133 (1989).
28, 454-459 (1991)
New Thermal
Stress Test to Assess the Viability Boar Sperm’
P. S. FISER, Animal
C. HANSEN, Research
Centre,
L. UNDERHILL, Agriculture
Canada,
of Cryopreserved
AND G. J. MARCUS Ottawa,
Ontario
KIA
005
A new, rapid, thermal stress test for assessing the viability of boar semen, requiring only 45 min of incubation at 42.X, was developed and compared with a widely used stress test of 180 min incubation at 37°C. The shorter procedure was found to have the same discriminatory ability as the standard test in assessing the effects of freezing conditions on the percentage of spermatozoa remaining motile. Neither test was able to show differences in the kinetic rating of motile sperm after freezing in relation to the glycerol concentration present during freezing. However, the new test had a greater ability to distinguish the effects of different concentrations of glycerol, over the range of 0 to 6%, and to reveal different degrees of acrosomal damage sustained during freezing. The longer procedure was unable to distinguish among glycerol concentrations from 0 to 4% with respect to acrosomal damage and produced an overall lower proportion of sperm having a normal apical ridge. The new thermal stress test thus has the advantages of greater sensitivity and more rapid execution over the test hitherto in widespread use.
Prediction of the fertilizing potential of fresh or frozen mammalian semen is usually based on in vitro assessment. However, there is no single in vitro test applicable to fresh and frozen mammalian semen in general and porcine semen in particular whose results correlate well with fertility. A fair assessment of semen requires evaluation by two or more different tests in vitro. Two tests commonly used with boar semen evaluate sperm motility and membrane integrity, usually expressed in terms of the proportions of spermatozoa with progressive forward motion and a normal apical acrosoma1 ridge. After processing and initial microscopic evaluation, semen is often subjected to a second test, termed a “thermal stress test,” involving in vitro incubation for several hours followed by reexamination, as an estimate of the capacity of sperm to survive in the female reproductive tract and retain their fertilizing capability (8, 9). Exposure of frozen-thawed sperm to the “thermal stress test” may reveal latent
Received July 30, 1990; accepted January 28, 1991. ’ ARC Contribution No. 16%. 454 001 l-2240/91 $3.00
damage, that is, damage which is not apparent immediately after ejaculation and processing. The test is thus an additional aid in assessing different processing procedures and their effect on sperm survival. Most thermal stress tests entail incubation at 3738°C (1, 4, 6, 10-12) for various times, usually 2 to 5 h. It has been reported (10) that the semen of boars of high fertility shows good motility after a 3-h stress test at 37°C. However, the acrosomal integrity of frozen-thawed sperm, evaluated as a percentage of sperm with a normal apical ridge (NAR), deteriorates substantially after incubation for 3 to 5 h, making discrimination among various treatments more difficult. Some investigators, therefore, have used an incubation of 2 h, which reveals fewer differences in motility, but distinguishes differences in the proportions of sperm with NAR more readily (1, 13). To shorten the test while preserving its discriminatory ability, we increased the incubation temperature to 42.5”C and reduced the incubation period to 45 min. We describe a comparison of the new test with a widely used, standard thermal stress test (incubation for 3 h at 37”C), using semen
THERMAL
processed in each of four levels of glycerol and frozen with suboptimal or optimal cooling rates (2). MATERIALS
AND
METHODS
Semen Collection, Evaluation and Processing Seventeen ejaculates from mature Yorkshire boars were collected over a 6-week period by the gloved-hand technique. The sperm-rich fraction of the ejaculates was received in a 400-ml glass beaker covered with a single layer of cheese cloth, held in a thermos bottle containing water at 33°C. The sperm concentration was determined spectrophotometrically. The motility was estimated microscopically using a heated stage as the percentage of motile sperm (O100%) and the kinetic rating which is a measure of the quality of movement exhibited by motile sperm, on a scale of 0 (nonmotile) to 5 (vigorous progressive motility). After being held in the thermos bottle for 2 h, the ejaculates were distributed into centrifuge tubes in portions of 7 x lo9 sperm per tube and centrifuged; the supernatants were discarded and BF5 diluent was added to produce a final volume of 5 ml/tube, as described by Purse1 and Johnson (14). After cooling to 5°C over a period of 2 h, each semen sample was further diluted 1: 1 with one of the BF5 diluents containing 0, 4, 8, or 12% glycerol (v/v) producing final glycerol concentrations of 0, 2, 4, or 6% (v/v). The semen samples were then transferred into 0.5 ml plastic straws color coded according to glycerol concentration, cooled to -60°C at a rate of either l”C/min (suboptimal) or 30Wmin (optimal, (2)) using a programmable freezer (CRYO-MED, 900-4 controller, 972-3 freezing chamber), and plunged into liquid nitrogen for storage. Straws were thawed by immersion for 8 s in water at 60°C (3), then emptied individually into test tubes containing 1 ml of Beltsville Thawing Solution (BTS, (14)) at 33°C. After equilibration for 5 min, the post-thaw motility percentage (FMP) and
STRESS
455
TEST
kinetic rating (FMR, scale of &5) of the thawed sperm were assessed microscopically using a heated microscope stage. Samples were removed and fixed in 1% glutaraldehyde for evaluation of acrosomal integrity (15). Samples from each combination of glycerol concentration and cooling rate were then incubated in a water bath for 3 h at 37”C, or at 42.5”C for 45 min. The percentage of motile sperm following the thermal stress test (TMP) and its postthermal stress kinetic rating (TMR) were then determined and acrosomal integrity was reevaluated. The percentage of boar sperm with NAR was determined using phase contrast microscopy and the scoring system of Purse1 et al. (16). Acrosomal damage was classified, using at least 100 sperm/sample. Statistical
Analysis
Least squares procedures (7) were used in a split-plot analysis of variance of sperm motility and acrosomal integrity before and after the two thermal stress tests, using a linear model which included the effects of boar, ejaculate within boar, glycerol concentration, and interaction of boar with glycerol. Because there was known to be significant interaction between glycerol concentration and freezing velocity (2), the data pertaining to each cooling velocity were analyzed separately. Duncan’s multiple range test was used to evaluate differences in survival according to glycerol level at a given thermal stress test temperature. RESULTS
There were no differences (P > 0.05) between the two thermal stress tests in their capacity to discriminate among glycerol concentrations with respect to the influence on sperm motility after freezing and thawing. In both tests, the percentage of motile sperm increased with increasing glycerol concentration. Also, the superiority of a cooling velocity of 3OWmin versus l”C/min was shown equally well by the two tests
456
FISER ET AL.
(Fig. 1). After either test, the percentage of motile sperm, expressed as a proportion of those surviving after freezing and thawing, was substantially higher in semen frozen by cooling at 30Wmin than in sperm frozen by cooling at l”C/min. The capacity of sperm classified as motile after thawing to survive either thermal stress test was influenced greatly by the original cooling rate (Fig. 1: compare upper and lower panels). Fewer sperm survived freezing after cooling at l”C/min than at 3OWmin and of those that survived, fewer survived the thermal stress test.
50
I
Cooling
velocity
l%/mi]
40 30
nl
n
20 10 0
0
2
4 37’
50
0
6 GLYCEROL
b
2
Cooling
4
6
42.5’
% velocity
30°C/min nl
r-l 40 30 20 10 0
0
2
4 37”
6 GLYCEROL
OFMP
0 %
2
4
6
Both tests showed a lower kinetic rating of motile sperm only in semen cooled at a suboptimal rate in the absence of glycerol. Otherwise, the tests did not discriminate among glycerol concentrations with respect to kinetic rating. The thermal stress tests differed significantly (P < O.Ol), however, in their capacity to reveal differences in the proportions of sperm retaining a normal apical ridge after being frozen and thawed in diluents with various glycerol concentrations. The 425°C test discriminated more readily among various glycerol concentrations with respect to the percentage of sperm with NAR in semen frozen by cooling at either l”C/min or at 30Wmin. This is illustrated in Fig. 3 with the preincubation acrosomal integrity of frozen-thawed sperm shown in Fig. 2. Incubation at 37°C for 3 h revealed no differences between 0,2, or 4% glycerol with respect to the effects on membrane integrity of sperm cooled at either rate (P > 0.05), but showed only a difference between these concentrations and 6%. In comparison, incubation for 45 min at 42.5”C, distinguished between glycerol concentrations of 0 or 2%, 4%, and 6%: the lower the concentration the greater the percentage of sperm with NAR. Sperm always showed a higher percentage NAR after incubation at 42.5”C than at 37°C. There were significant differences among boars with respect to all the semen quality variables assessed. However, such biological differences, although always present (10, 19), did not confound the ability to discriminate among treatment effects using the thermal stress test.
42.5’
RRWTMP
FIG. 1. The effect of two thermal stress tests (37”CI 180 min vs 42SW45 min) on the percentage of motile sperm in boar semen protected by O-6% glycerol frozen at 1 or 30Wmin. FMP = Percentage of sperm motile immediately after freezing and thawing (before incubation); TMP = percentage of frozen-thawed sperm motile after the thermal stress test.
DISCUSSION
The quality as well as the viability of spermatozoa deteriorates as a consequence of freezing and thawing. The degree of deterioration depends on several factors, e.g., freezing and thawing rates (2, 3), the nature and concentration of the cryoprotectant and of other components of the diluent used
THERMAL
%
Cooling
velocity
GLYCEROL
%
Cooling
STRESS
457
TEST
%
l’C/min
%
velocity
37”
3O”Wmin
velocity 1”Clmin
Cooling
%
GLYCEROL % Cooling
42.5’
velocity
3O”Clmin
100
0
0
2
4
GLYCEROL
%
6
0 37”
I
FIG. 2. Preincubation acrosomal integrity of boar sperm frozen in O-6% glycerol at I or 3WUmin. NAR = Normal apical ridge; DAR = damaged apical ridge; MAR = missing apical ridge; LAC = loose acrosomal cap.
for freezing (22), the species, and individual donor (19), to name just a few. In order to evaluate fresh or stored ejaculates or sperm which have undergone freezing and thawing, samples are often subjected to an additional test. This usually involves incubation for several hours at body temperature. The new thermal stress test, described here, entails incubation of diluted boar semen at a higher temperature (42.5”C-the upper extreme of the physiological range) for a considerably shorter time than is required by “standard” incubation tests, which may require incubation for up to 5 h at 37°C (1,8, 11, 21). Comparison with such a test has shown that the new thermal stress test provides the same degree of discrimi-
GLYCEROL
%
2
4
6
42.5’
I
FIG. 3. The effect of two thermal stress tests (37W 180 min vs 42S”C/45 min) on acrosomal integrity of boar sperm frozen in O-6% glycerolated diluents at 1 or 30Wmin. NAR = Normal apical ridge; DAR = damaged apical ridge; MAR = missing apical ridge; LAC = loose acrosomal cap.
nation in assessing the effects of cooling rate and glycerol concentration on maintenance of the motility of boar sperm after freezing and thawing, but requires only a third of the usual time (10, 12, 18). A greater loss of motility during incubation of frozen-thawed sperm has been reported to be associated with the use of increased glycerol levels for freezing (1). This effect was also apparent in the present study, particularly in semen frozen by cooling at the suboptimal rate. The loss of motility during the thermal stress test has been attributed to a toxic effect of dead sperm, associated with the liberation of amino oxidase activity (20).
458
FISER ET AL.
However, it is more likely that latent damage to the sperm at the time of freezing under suboptimal conditions is responsible for the death of sperm during post-thaw incubation, i.e., some sperm which survived suboptimal freezing conditions had sustained membrane damage that was revealed only during subsequent incubation. The prolonged incubation of sperm at 37°C causes deterioration and changes in the acrosomes (17), but may also allow a proportion of the sperm to undergo the normal acrosome reaction (IS). This effect would not be distinguishable from an abnormal loss of the apical ridge. These changes contribute to the generally lower proportions of sperm with NAR following 3-h incubation at 37”C, compared with that after incubation at 42.5”, regardless of the original glycerol concentration, and make the 37” test less discriminating. The duration of motility after thawing is believed to be an indication of the usability of bovine semen (9, 17). It can be assumed that in porcine semen, a thermal stress test result showing more lasting motility in sperm which survived optimal freezing indicates a greater likelihood of such sperm surviving in the female reproductive tract to undergo capacitation and fertilize ova. The new test reveals such endurance more rapidly. We believe that incorporating the new test as a part of semen evaluation in vitro will improve the accuracy of predicting the fertilizing ability of semen. However, we emphasize that in such evaluation, one must not rely on the results of a single type of test. ACKNOWLEDGMENT
2.
3.
4.
5.
6.
7. 8.
9. 10.
11. 12.
13.
This study was supported in part by a grant from the Ontario Ministry of Agriculture and Food under the Ontario Pork Industry Improvement Program (OPIIP). REFERENCES
1. Almlid, T., and Johnson, A. L. Effects of glycerol concentration, equilibration time and temperature of glycerol addition on post-thaw viability
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