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The Effect of Glycerol Equilibration Time on the Freezing of Bovine Spermatozoa in Egg Yolk–Sodium Citrate and Skimmilk Semen Extenders1,2
THE
EFFECT
OF GLYCEROL E Q U I L I B R A T I O N TIME
ON T H E
F R E E Z I N G OF BOVINE SPERMATOZOA IN EGG YOLK-SODIUM C I T R A T E A N D S K I M M I L K S E M E N E X T E N D E R S 1'2 GLEN D. O'DELL ANDVICTOR HURST Dairy Department, South Carolina Agricult~lral Experiment Station, Clemson
There is some difference of opinion among experimenters as to the significance of the length of time that sperm cells remain in a glycerolated extender prior to freezing. The basis of this study was a comparison of results of using 0.5-hour and 18-hour equilibration times on bovine semen in egg yolk-sodium citrate and skimmilk semen extenders. The results were measured in terms of the percentage o f motile sperm after thawing as compared w i t h the percentage observed prior to freezing. The study made possible a determination of the differences between individual bulls in relation to recovery percentage and the significance of the length of storage period in evaluating semen samples. Editor.
The time of the glycerol equilibration period is a matter of controversy among workers in the field of freezing bovine semen. The equilibration period may be defined as the time that sperm cells remain in a glycerolated extender prior to freezing. Polge and Rowson (4) recommended an equilibration time r a n g i n g between 15 and 20 hours when using an egg yolk extender. Miller and V a n D e m a r k (2), comparing 2-, 6-, and 18-hour equilibration times when using an egg yolk extender, f o u n d the 6-hour period to be superior in terms of percentage of motile sperm cells surviving after freezing. Cragle et al. (1), also using an egg yolk extender, found 14.9 hours to be the most favorable equilibration time between 4 and 28 hours. Saroff and Mixner (5) showed a progressive increase in sperm cell survival in an egg yolk extender as the equilibration time was increased from 2 to 18 hours. O'Dell and Almquist (3), using a heated skimmilk extender, did not observe a n y significant difference ill sperm cell survival after freezing when using 0.5-, 4-, and 18-hour equilibration times. The studies reported here compare the effects of 0.5-hour and 18-hour equilibration times on the freezing of bovine spermatozoa in egg yolk-sodium citrate and skimmilk semen extenders. Received for publication December 9, 1955. 1Technical Contribution No. 246, South Carolina Agricultural Experiment Station. -"Data in this publication are taken from a thesis submitted by the senior author to the Graduate School of the Clemson Agricultural College, Clemson, S. C. in partial fulfillment of ~the requirements for the M.S. degree. 1156
GLYCEROL EQUILIBRATION
EXPERIMENTAL
T I M E AND S P E R M S U R V I V A L
1157
PROCEDURE AND RESULTS
The two extenders used in this work were egg yolk-citrate and skimmilk. The composition of the egg yolk-citrate extender was 20% egg yolk and 80% of a 3% aqueous solution of sodium citrate dihydrate. The skimmilk was separated not later than one day prior to its use, and a Babcock b u t t e r f a t test was conducted to insure that no skimmilk would be used testing higher than 0.04% butterfat. The skimmilk was heated to 92-95 ° C. for 3 minutes and then cooled to 5 ° C. Streptomycin sulfate and potassium penicillin G were added to both extenders at the rate of 500 units each per milliliter. Split samples of the same ejaculate were extended at a rate of 1 to 25. Glycerol was added to separate containers of extender at double the desired percentage level and then an equal volume of this was added to the extended semen to give a final dilution of I to 50. A t the same time the glycerol level was reduced to the desired percentage. This glycerolated extender was added to the extended semen in five steps, each at 6-minute intervals. A f t e r giving the samples the desired equilibration period, freezing was conducted at a rate of 0.8 ° C. per minute in a Dewar flask with ethanol and d r y ice from 5 ° to -15 ° C. and then at approximately 3.5 ° C. per minute down to -65 ° C. F r o m this point the reduction in temperature was carried out as r a p i d l y as possible to -75 ° C. The samples were then placed in a storage cabinet for the final reduction to -79 ° C. I n thawing, the samples were removed from the storage cabinet and immediately submerged in containers of ice water, where they remained until completely thawed. The basis of evaluation of this work was the percentage of motile sperm observed after thawing as compared to the percentage observed prior to freezing. This was expressed as per cent recovery and was obtained by use of the following formula : Per cent of motile sperm after thawing × 100 ----per cent recovery P e r cent of motile sperm prior to freezing This work was divided into two phases, which will be discussed separately. EXPERIMENT
]
Preliminary trials involving a study of the effect of different glycerol levels on the effectiveness of skimmilk as a frozen semen extender. Single ejaculations from 18 bulls were split into each of three skimmilk extenders to contain a final volume of 8, 10, and 12% glycerol. These samples were then frozen as outlined above. Samples of each group were stored for periods of 24 to 48 hours and then thawed. The results of this work are shown in Table ]. A n analysis of variance (6) of the combined recovery percentages for both 24 and 48 hours after freezing showed that the differences in recovery percentages were highly significant. F r o m these data it was concluded that an 8% level of glycerol is the superior level insofar as these studies were concerned.
1158
GLEN D. O'DELL AND VICTOR I-IURST
TABLE 1 R e c o v e r y o f s p e r m cells dil~ted w i t h three levels of glycerol in s k i m m i l k and stored at - 7 9 ° C. Glycerol level
Recovery a f t e r being frozen for 24 hours
Recovery a f t e r being frozen for 48 hours
(%)
(%)
(%)
8 10 12
62.7 46.8 50.5
55.8 45.1 45.0
EXPERIMENT 2
Trials involving 0.5-hour and 18-hour equilibration times with egg yolk and skimmilk extenders. Twelve bulls were selected from Experiment 1 for these studies. They were selected on the basis of past histories of high initial motility of their semen and on the ability of their semen to freeze successfully. One ejaculation was collected from each bull weekly for a period of 4 weeks. On this basis, all initial motilities ranged between 50 and 70% except for one bull that had three initial ejaculation motilities of 40%. The level of glycerol used in the second experiment was 8% by volume for both the skimmilk and egg yolk-citrate. The citrate level was reduced to 2.76% by the addition of the glycerol. Each of the 48 ejaculations was split in the following eight ways: egg yolkcitrate (EYC), 0.5-hour equilibration with 48 hours storage; EYC, 0.5-hour equilibration with 10 days storage; EYC, 18 hours equilibration with 48 hours storage; EYC, 18-hour equilibration with 10 days storage; skimmilk (SM), 0.5-hour equilibration with 48 hours storage; SM, 0.5-hour equilibration with 10 days storage; SM, 18 hours equilibration with 48 hours storage, and SM, 18 hours equilibration with 10 days storage. Results are shown in Table 2. An analysis of variance test was conducted on the recovery percentage figures. Statistically significant differences in per cent recovery of semen were found at the 5% level among bulls and between the equilibration times. Highly statistical significance at the 1% level was found between differences in storage periods. Highly significant interactions were found between bulls × ejaculations, bulls × extenders, and bulls × equilibration times. The high significance of these interactions confirms the observations of the raw data in which the freezing of a given bull's semen is affected by the type of extender used and the length of TABLE 2 ~ e e o v e r y o f s p e r m cells o f 48 ejaculations f r o m 12 bulls at the end o f 48 hours a n d 10 d a y s s t o r a g e at - 7 9 ° C. E g g yolk-citrate extender Time frozen
48 hours 10 days
Skimmilk extender
Recovery a f t e r 0.5-hour equilibration
Recovery a f t e r 18 hours equilibration
l~ecovery a f t e r O.5-hour equilibration
Recovery after 18 hours equilibration
(%J
(%)
(%)
(%)
78.5 74.4
76.7 72.2
78.8 73.9
75.6 68.6
1159
GLYCEROL EQUILIBRATION TIME AND SPERm SURVIVAL
the equilibration time. The interactions ejaculations × equilibration times and ejaculations × storage were significant at the 5% level. Retention of motility of samples thawed 48 hours after freezing ill Experiment 2 was followed until death of all cells occurred. The average daily readings on all samples are shown in Table 3. It is obvious from the inspection of these data that none of the thawed semen maintained good motility in storage. TABLE 3 t~eeovery retained by sperm ce~Is a f t e r thawing f r o m -79 ° C. to 5 ° C. and holding at this temperature f o r 5 days Egg yolk-citrate extender Day
1 2 3 4 5
Skimmilk extender
0.5-hour equilibration
18 h o u r s equilibration
0.5-hour equilibration
18 h o u r s equilibration
(%)
(%)
(%)
(%)
78.5 59.6 37.0 23.9 13.8
76.7 54.8 37.2 23.8 12.6
78.8 61.1 42.3 27.9 16.5
75.6 57.1 40.2 24.7 15.7
DISCUSSION
In freezing bovine semen the emphasis should be placed on each individual bull. A determilmtion of the optimal conditions for a p a r t i c u l a r bull must be made if satisfactory results are to be obtained. In this work semen from 18% of 28 bulls initially studied did not freeze successfully in E x p e r i m e n t 1. F r o m these studies it appears that all work reported in this field should acknowledge whether bulls producing poor freezing semen had to be eliminated before final experimental data are reported. There was a drop in recovery percentage of frozen semen samples a f t e r 10 days storage as compared to samples stored for 48 hours. Since these differences in recovery percentages were highly significant, it is recommended that frozen semen samples be evaluated after a 10-day storage period following freezing. This should give a more accurate appraisal of the stored samples while still providing a test within practical time limits a f t e r freezing. The data clearly show that frozen semen, once thawed, should not be used for artificial insemination beyond the day of thawing. Loss of motility on samples stored after thawing is far more rapid than would be anticipated with samples of unfrozen semen. SUMMARY
A study was made of the effect of glycerol equilibration time on the freezing of bovine spermatozoa in egg yolk-citrate and skimmilk extenders.
1160
GLEN
D. O'DELL
AND ~rlCTOR HURST
A n a n a l y s i s of v a r i a n c e i n d i c a t e d t h a t t h e d i f f e r e n c e s i n s u r v i v a l m o t i l i t y b e t w e e n 0.5-hour a n d 18-hour e q u i l i b r a t i o n t i m e s w e r e s i g n i f i c a n t a t t h e 5 % level a n d w e r e i n f a v o r o f t h e 0.5-hour p e r i o d . T h i s s h o u l d n o t be c o n s t r u e d to m e a n t h a t a 0.5-hour e q u i l i b r a t i o n t i m e is o p t i m a l f o r all semen, since i t was f o u n d t h a t s e m e n f r o m s e v e r a l b u l ls r e s p o n d e d b e t t e r to a n 18-hour p e r i o d . I t is i n d i c a t e d t h a t t h e r e is c o n s i d e r a b l e v a r i a t i o n a m o n g b u l l s i n t h e a b i l i t y of t h e i r s e m e n to f r e e z e s u c c e s s f u l l y . ACKNOWLEDGMENT The authors acknowledge the assistance of E. It. Stewart, Agronomy Department, South Carolina Agricultural Experiment Station, in making the statistical analysis. REFERENCES (1) CRAGLE~R. G., MEYF_~S,R. M., WAUGH~R. K., HUNTER, J. S., AND ANDEaSON, R. L. The Effects of Various Levels of Sodium Citrate, Glycerol, and Equilibration Time on Survival of Bovine Spermatozoa After Storage at -79 ° C. J. Dairy Sci., 38: 508. 1955. (2) MILLER,W. J., AND VANDEMARK,N. L. The Influence of Glycerol Level, Various Temperature Aspects and Certain Other Factors on the Survival of Bull Spermatozoa at SubZero Temperatures. J. Dairy Sci., 37: 45. 1954. (3) O'Dv.LL, W. T., AND ALMQUIST, J. O. Techniques for Freezing Bull Spermatozoa in Heated Milk and Preliminary Breeding Results. J. Dairy Sci., 37: 652. 1954. (4) POLG•, C., AND ROWSON, L. E. A. Fertility Capacity of Bull Spermatozoa After Freezing at -79 ° C. Nature, 159: 626. 1952. (5) SAROFF, J., AND MIXNE•, J. P. The Relationship of Diluter Composition and Glycerol Equilibration Time to Survival of Bull Spermatozoa After Freezing to -79 ° C. J. Dairy Sci., 37: 651. 1954. (6) SN~.D~COR~G. W. Statistical Methods. 4th ed. The Iowa State College Press, Ames. 1946.
EFFECT
OF GLYCEROL E Q U I L I B R A T I O N TIME
ON T H E
F R E E Z I N G OF BOVINE SPERMATOZOA IN EGG YOLK-SODIUM C I T R A T E A N D S K I M M I L K S E M E N E X T E N D E R S 1'2 GLEN D. O'DELL ANDVICTOR HURST Dairy Department, South Carolina Agricult~lral Experiment Station, Clemson
There is some difference of opinion among experimenters as to the significance of the length of time that sperm cells remain in a glycerolated extender prior to freezing. The basis of this study was a comparison of results of using 0.5-hour and 18-hour equilibration times on bovine semen in egg yolk-sodium citrate and skimmilk semen extenders. The results were measured in terms of the percentage o f motile sperm after thawing as compared w i t h the percentage observed prior to freezing. The study made possible a determination of the differences between individual bulls in relation to recovery percentage and the significance of the length of storage period in evaluating semen samples. Editor.
The time of the glycerol equilibration period is a matter of controversy among workers in the field of freezing bovine semen. The equilibration period may be defined as the time that sperm cells remain in a glycerolated extender prior to freezing. Polge and Rowson (4) recommended an equilibration time r a n g i n g between 15 and 20 hours when using an egg yolk extender. Miller and V a n D e m a r k (2), comparing 2-, 6-, and 18-hour equilibration times when using an egg yolk extender, f o u n d the 6-hour period to be superior in terms of percentage of motile sperm cells surviving after freezing. Cragle et al. (1), also using an egg yolk extender, found 14.9 hours to be the most favorable equilibration time between 4 and 28 hours. Saroff and Mixner (5) showed a progressive increase in sperm cell survival in an egg yolk extender as the equilibration time was increased from 2 to 18 hours. O'Dell and Almquist (3), using a heated skimmilk extender, did not observe a n y significant difference ill sperm cell survival after freezing when using 0.5-, 4-, and 18-hour equilibration times. The studies reported here compare the effects of 0.5-hour and 18-hour equilibration times on the freezing of bovine spermatozoa in egg yolk-sodium citrate and skimmilk semen extenders. Received for publication December 9, 1955. 1Technical Contribution No. 246, South Carolina Agricultural Experiment Station. -"Data in this publication are taken from a thesis submitted by the senior author to the Graduate School of the Clemson Agricultural College, Clemson, S. C. in partial fulfillment of ~the requirements for the M.S. degree. 1156
GLYCEROL EQUILIBRATION
EXPERIMENTAL
T I M E AND S P E R M S U R V I V A L
1157
PROCEDURE AND RESULTS
The two extenders used in this work were egg yolk-citrate and skimmilk. The composition of the egg yolk-citrate extender was 20% egg yolk and 80% of a 3% aqueous solution of sodium citrate dihydrate. The skimmilk was separated not later than one day prior to its use, and a Babcock b u t t e r f a t test was conducted to insure that no skimmilk would be used testing higher than 0.04% butterfat. The skimmilk was heated to 92-95 ° C. for 3 minutes and then cooled to 5 ° C. Streptomycin sulfate and potassium penicillin G were added to both extenders at the rate of 500 units each per milliliter. Split samples of the same ejaculate were extended at a rate of 1 to 25. Glycerol was added to separate containers of extender at double the desired percentage level and then an equal volume of this was added to the extended semen to give a final dilution of I to 50. A t the same time the glycerol level was reduced to the desired percentage. This glycerolated extender was added to the extended semen in five steps, each at 6-minute intervals. A f t e r giving the samples the desired equilibration period, freezing was conducted at a rate of 0.8 ° C. per minute in a Dewar flask with ethanol and d r y ice from 5 ° to -15 ° C. and then at approximately 3.5 ° C. per minute down to -65 ° C. F r o m this point the reduction in temperature was carried out as r a p i d l y as possible to -75 ° C. The samples were then placed in a storage cabinet for the final reduction to -79 ° C. I n thawing, the samples were removed from the storage cabinet and immediately submerged in containers of ice water, where they remained until completely thawed. The basis of evaluation of this work was the percentage of motile sperm observed after thawing as compared to the percentage observed prior to freezing. This was expressed as per cent recovery and was obtained by use of the following formula : Per cent of motile sperm after thawing × 100 ----per cent recovery P e r cent of motile sperm prior to freezing This work was divided into two phases, which will be discussed separately. EXPERIMENT
]
Preliminary trials involving a study of the effect of different glycerol levels on the effectiveness of skimmilk as a frozen semen extender. Single ejaculations from 18 bulls were split into each of three skimmilk extenders to contain a final volume of 8, 10, and 12% glycerol. These samples were then frozen as outlined above. Samples of each group were stored for periods of 24 to 48 hours and then thawed. The results of this work are shown in Table ]. A n analysis of variance (6) of the combined recovery percentages for both 24 and 48 hours after freezing showed that the differences in recovery percentages were highly significant. F r o m these data it was concluded that an 8% level of glycerol is the superior level insofar as these studies were concerned.
1158
GLEN D. O'DELL AND VICTOR I-IURST
TABLE 1 R e c o v e r y o f s p e r m cells dil~ted w i t h three levels of glycerol in s k i m m i l k and stored at - 7 9 ° C. Glycerol level
Recovery a f t e r being frozen for 24 hours
Recovery a f t e r being frozen for 48 hours
(%)
(%)
(%)
8 10 12
62.7 46.8 50.5
55.8 45.1 45.0
EXPERIMENT 2
Trials involving 0.5-hour and 18-hour equilibration times with egg yolk and skimmilk extenders. Twelve bulls were selected from Experiment 1 for these studies. They were selected on the basis of past histories of high initial motility of their semen and on the ability of their semen to freeze successfully. One ejaculation was collected from each bull weekly for a period of 4 weeks. On this basis, all initial motilities ranged between 50 and 70% except for one bull that had three initial ejaculation motilities of 40%. The level of glycerol used in the second experiment was 8% by volume for both the skimmilk and egg yolk-citrate. The citrate level was reduced to 2.76% by the addition of the glycerol. Each of the 48 ejaculations was split in the following eight ways: egg yolkcitrate (EYC), 0.5-hour equilibration with 48 hours storage; EYC, 0.5-hour equilibration with 10 days storage; EYC, 18 hours equilibration with 48 hours storage; EYC, 18-hour equilibration with 10 days storage; skimmilk (SM), 0.5-hour equilibration with 48 hours storage; SM, 0.5-hour equilibration with 10 days storage; SM, 18 hours equilibration with 48 hours storage, and SM, 18 hours equilibration with 10 days storage. Results are shown in Table 2. An analysis of variance test was conducted on the recovery percentage figures. Statistically significant differences in per cent recovery of semen were found at the 5% level among bulls and between the equilibration times. Highly statistical significance at the 1% level was found between differences in storage periods. Highly significant interactions were found between bulls × ejaculations, bulls × extenders, and bulls × equilibration times. The high significance of these interactions confirms the observations of the raw data in which the freezing of a given bull's semen is affected by the type of extender used and the length of TABLE 2 ~ e e o v e r y o f s p e r m cells o f 48 ejaculations f r o m 12 bulls at the end o f 48 hours a n d 10 d a y s s t o r a g e at - 7 9 ° C. E g g yolk-citrate extender Time frozen
48 hours 10 days
Skimmilk extender
Recovery a f t e r 0.5-hour equilibration
Recovery a f t e r 18 hours equilibration
l~ecovery a f t e r O.5-hour equilibration
Recovery after 18 hours equilibration
(%J
(%)
(%)
(%)
78.5 74.4
76.7 72.2
78.8 73.9
75.6 68.6
1159
GLYCEROL EQUILIBRATION TIME AND SPERm SURVIVAL
the equilibration time. The interactions ejaculations × equilibration times and ejaculations × storage were significant at the 5% level. Retention of motility of samples thawed 48 hours after freezing ill Experiment 2 was followed until death of all cells occurred. The average daily readings on all samples are shown in Table 3. It is obvious from the inspection of these data that none of the thawed semen maintained good motility in storage. TABLE 3 t~eeovery retained by sperm ce~Is a f t e r thawing f r o m -79 ° C. to 5 ° C. and holding at this temperature f o r 5 days Egg yolk-citrate extender Day
1 2 3 4 5
Skimmilk extender
0.5-hour equilibration
18 h o u r s equilibration
0.5-hour equilibration
18 h o u r s equilibration
(%)
(%)
(%)
(%)
78.5 59.6 37.0 23.9 13.8
76.7 54.8 37.2 23.8 12.6
78.8 61.1 42.3 27.9 16.5
75.6 57.1 40.2 24.7 15.7
DISCUSSION
In freezing bovine semen the emphasis should be placed on each individual bull. A determilmtion of the optimal conditions for a p a r t i c u l a r bull must be made if satisfactory results are to be obtained. In this work semen from 18% of 28 bulls initially studied did not freeze successfully in E x p e r i m e n t 1. F r o m these studies it appears that all work reported in this field should acknowledge whether bulls producing poor freezing semen had to be eliminated before final experimental data are reported. There was a drop in recovery percentage of frozen semen samples a f t e r 10 days storage as compared to samples stored for 48 hours. Since these differences in recovery percentages were highly significant, it is recommended that frozen semen samples be evaluated after a 10-day storage period following freezing. This should give a more accurate appraisal of the stored samples while still providing a test within practical time limits a f t e r freezing. The data clearly show that frozen semen, once thawed, should not be used for artificial insemination beyond the day of thawing. Loss of motility on samples stored after thawing is far more rapid than would be anticipated with samples of unfrozen semen. SUMMARY
A study was made of the effect of glycerol equilibration time on the freezing of bovine spermatozoa in egg yolk-citrate and skimmilk extenders.
1160
GLEN
D. O'DELL
AND ~rlCTOR HURST
A n a n a l y s i s of v a r i a n c e i n d i c a t e d t h a t t h e d i f f e r e n c e s i n s u r v i v a l m o t i l i t y b e t w e e n 0.5-hour a n d 18-hour e q u i l i b r a t i o n t i m e s w e r e s i g n i f i c a n t a t t h e 5 % level a n d w e r e i n f a v o r o f t h e 0.5-hour p e r i o d . T h i s s h o u l d n o t be c o n s t r u e d to m e a n t h a t a 0.5-hour e q u i l i b r a t i o n t i m e is o p t i m a l f o r all semen, since i t was f o u n d t h a t s e m e n f r o m s e v e r a l b u l ls r e s p o n d e d b e t t e r to a n 18-hour p e r i o d . I t is i n d i c a t e d t h a t t h e r e is c o n s i d e r a b l e v a r i a t i o n a m o n g b u l l s i n t h e a b i l i t y of t h e i r s e m e n to f r e e z e s u c c e s s f u l l y . ACKNOWLEDGMENT The authors acknowledge the assistance of E. It. Stewart, Agronomy Department, South Carolina Agricultural Experiment Station, in making the statistical analysis. REFERENCES (1) CRAGLE~R. G., MEYF_~S,R. M., WAUGH~R. K., HUNTER, J. S., AND ANDEaSON, R. L. The Effects of Various Levels of Sodium Citrate, Glycerol, and Equilibration Time on Survival of Bovine Spermatozoa After Storage at -79 ° C. J. Dairy Sci., 38: 508. 1955. (2) MILLER,W. J., AND VANDEMARK,N. L. The Influence of Glycerol Level, Various Temperature Aspects and Certain Other Factors on the Survival of Bull Spermatozoa at SubZero Temperatures. J. Dairy Sci., 37: 45. 1954. (3) O'Dv.LL, W. T., AND ALMQUIST, J. O. Techniques for Freezing Bull Spermatozoa in Heated Milk and Preliminary Breeding Results. J. Dairy Sci., 37: 652. 1954. (4) POLG•, C., AND ROWSON, L. E. A. Fertility Capacity of Bull Spermatozoa After Freezing at -79 ° C. Nature, 159: 626. 1952. (5) SAROFF, J., AND MIXNE•, J. P. The Relationship of Diluter Composition and Glycerol Equilibration Time to Survival of Bull Spermatozoa After Freezing to -79 ° C. J. Dairy Sci., 37: 651. 1954. (6) SN~.D~COR~G. W. Statistical Methods. 4th ed. The Iowa State College Press, Ames. 1946.