Vol. 26, No. 2, February 1975 Printed in U.S.A.
FERTILITY AND STERILITY Copyright c 1975 The American Fertility Society
EFFECT OF CENTRIFUGATION AND SEMINAL PLASMA ON MOTILITY AND FERTILITY OF STALLION AND BULL SPERMATOZOA B. W. PICKETT, PH.D., J. J. SULLNAN, PH.D.,* W. W. BYERS, M.S.,t M. M. PACE, PH.D.,* AND E. E. REMMENGA, PH.DJ Animal Reproduction Laboratory, Department of Physiology and Biophysics, Colorado State University, Fort Collins 80523, and American Breeders Service, DeForest, Wisconsin 53532
Low concentrations of spennatozoa in some stallion ejaculates necessitate removal of seminal plasma by centrifugation to maintain desired insemination volume. 1 • 8 Low centrifugal forces did not adversely affect stallion spermatozoa. 9 Dilution of semen or removal of seminal plasma is believed to be necessary for prolonged storage, 10' 14 but motility is depressed by dilution above or below 1:4.15-18 The objectives of this research were to determine the effect of centrifuging diluted (1:2) and undiluted semen on bovine and equine spennatozoan motility and fertility, and the effect of seminal plasma and dilution on equine spennatozoan survival during incubation before and after freezing. MATERIALS AND METHODS
I. Influence of centrifugal force, washing, and seminal plasma on prefreeze and postfreeze motility of stallion spermatozoa. The stallions (Quarter Horses or thoroughbreds) were stabled at Colorado State University and ranged in age from five to 12 years. Semen was collected with an artificial vagina. 19 Sudden temperature changes were avoided. After final dilution, all samples were Received April 1, 1974. *American Breeders Service. tPresent address: Sandarac Arabian Horse Farm, R.R. 2, Wolf Road, Geneseo, Illinois 61254. *Department of Statistics, Colorado State University.
cooled to 5 C in approximately one hour and placed in 1.0 ml ampules. Two drops of extended semen were placed on opposite ends of a glass slide at '38 C and covered; motility was estimated under phase-contrast microscopy (x 200). Semen was frozen in liquid-nitrogen vapor. 20 After freezing, the ampules were thawed in a 38 C water bath for 1.5 minutes and then transferred to a 1 C water bath for the experiment. A randomized block design was used and the data were analyzed by analysis ofvariance. 21 The percent motility values were transformed to angles (arcsin V"%) prior to analysis. Transformed means were tested by the Honest significant difference test which is based on Student's range. However, nontransformed meaits are presented here. Experiment 1. Diluted samples.-Three ejaculates were collected from each of four stallions; 4 ml of semen from each ejaculate was placed in each of five 17-ml centrifuge tubes. Eight milliliters of an EYT extender (Tris [hydroxymethyl] aminomethane [W/V = 0.24% ], glucose [W/ V=4.08%], glycerol [VN=4.70%], citric acid [W/V=0.1%], egg yolk [V/V=20%] in deionized, autoclaved water) was added. One of the tubes (control) was not centrifuged, but was incubated at 38 C while the others were processed. Two tubes each were centrifuged at 370 g and 829 g for five minutes. Following centrifugation, 10 ml of supernatant was removed from one tube
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centrifuged at each of the centrifugal forces and the spermatozoa resuspended in fresh extender to a volume of 40 ml. The final dilution was 1:9. The spermatozoa in each of the remaining tubes were resuspended in their supernatant and diluted 1:9. Thus, one sample processed at each g force contained 2% seminal plasma, while the others contained 10%. The change in osmotic pressure was minimal, since the extender osmotic pressure was very close to that of seminal plasma. Undiluted samples.-Five additional tubes containing 4 ml of raw semen from each ejaculate were processed in a similar manner. Prefreeze motility was estimated from two randomly selected ampules 90 minutes after dilution. Postfreeze motility was estimated immediately after thawing and at two-hour intervals for six hours. Experiment 2. The effects of 0, 2%, and 10% seminal plasma on prefreeze and postfreeze spermatozoan motility after centrifugation at 956 g were examined. In experiment 1, the .final concentration of extender ingredients in the diluted semen changed when different amounts of seminal plasma were removed. Thus, in this experiment the EYT extenders were adjusted to eliminate this variable. Samples of three ejaculates from each of four stallions were treated as follows: (1) diluted 1:9 and cooled to 5 C; (2) incubated at 38 C, then diluted 1:9; (3) centrifuged at 956 g for three minutes followed by removal of seminal plasma, resuspension of spermatozoa, and 1:9 dilution; (4) centrifuged, followed by resuspension of spermatozoa in 2% seminal plasma when diluted 1:9; (5) centrifuged, followed by resuspension of spermatozoa in 10% seminal plasma when diluted 1:9. Prefreeze motility was estimated 7 5 minutes after dilution, while postfreeze
February 1975
motility was estimated immediately and two hours after thawing. Experiment 3. The effect of dilution ratios of1:1, 1:2, 1:4, 1:8, 1:16, and 1:32 on prefreeze and postfreeze spermatozoan motility was examined. An extender composed of dried skim milk (W/V=5.02%), glucose (W/V=5.02%), glycerol (V/V= 5.02%) in deionized, autoclaved water was used. Since the dilution ratio varied from 1:1 to 1:32, the seminal plasma concentration varied from 50% to 3%. Two ejaculates from each of five stallions were used. Prefreeze motility was estimated immediately, and at eight, 16, and 24 hours after dilution, while postfreeze motility was estimated immediately and at two hours after thawing. Experiment 4. To establish the concentration of seminal plasma required for maximum motility, an effort was made to eliminate the ingredient concentration variable in experiment 3. The concentrations of skim milk and glycerol were maintained constant at 5% while glucose was varied from 2.52% to 4.95% to compensate for the osmotic pressure when 0, 5%, 10%, 20%, 30%, or 50% seminal plasma replaced water in the extender. Seminal plasma was obtained from first and second ejaculates collected on the same day and the semen was centrifuged at 26,650 g for ten minutes. The supernatants were pooled and maintained at -20 c. Two ejaculates from each of four stallions were used. Samples (4 ml) from individual ejaculates were centrifuged for three minutes at 956 g. The seminal plasma was decanted and the spermatozoa resuspended in the appropriate diluent. Prefreeze and postfreeze motility was estimated as in experiment 3. II. Influence of centrifugation on fertility of stallion and bull spermatozoa. Stallions were of the Arabian, Connemara, Paint, Quarter Horse, and Thoroughbred breeds. Holstein bulls were used. Stal-
Vol. 26, No.2
MOTILITY AND FERTILITY OF SPERMATOZOA
lions, bulls, and mares were stabled at American Breeders Service. Semen was collected as in part I. Motilities were estimated under phase-contrast microscopy (x 200) immediately after collection. Postfreeze motilities were evaluated photometrically (x 180).22 The mares were inseminated daily during estrus beginning on day 2. The fertility data were analyzed by analysis of variance. 21 Experiment 1. Unfrozen stallion semen.-Undiluted semen (5 to 15 ml) were centrifuged at 310 g for 3.5 minutes; the spermatozoa were then resuspended. Aliquots containing 500 x 106 motile spermatozoa were used for each insemination. The volume inseminated ranged from 1.7 to 33.2 ml. Time from collection to insemination ranged from 20 to 40 minutes. Experiment 2. Frozen stallion semen.Centrifugation was performed as in part II, experiment 1, except that up to 80% of the seminal plasma was removed. Spermatozoa were diluted no less than 1:9 and concentrations were adjusted prior to freezing to provide 80 to 150 x 106 motile spermatozoa/10 ml insemination unit after freezing and thawing. After collection and dilution, the semen was cooled to 5 C in one hour, frozen one hour later, and stored at -196 C from one to five months. It was thawed in 32 C to 35· C water just before insemination. Experiment 3. Frozen bull semen.Semen was collected from five mature Holstein bulls twice a week for 15 collections. Ejaculates were randomly treated as follows: (1) semen not centrifuged, held three to four minutes at room temperature, diluted 1:2, and cooled to 5 C; (2) semen centrifuged at 270 g for three minutes, spermatozoa resuspended, diluted 1:2, and cooled to 5 C; (3) semen diluted 1:2, centrifuged at 270 g for three minutes, spematozoa resuspended, and cooled to 5 C.
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After cooling about two hours, the semen was further extended to provide between 5 x 10 6 and 14 x 10 6 motile spermatozoa/0.5 ml insemination unit after freezing and thawing. The extender was composed of sodium citrate (WN= 2.7%), egg yolk (V/V=20%), glycerol (VN = 7% ), antibiotics, and deionized, autoclaved water. The semen was frozen four to eight hours after collection and stored at -196 C. Fertility was evaluated by 60- to 90-day nonreturns to estrus. RESULTS
I. Experiment 1. The mean prefreeze and postfreeze motility of stallion spermatozoa subjected to dilution, combinations of centrifugal force, and seminal plasma concentration are presented in Table 1. Diluted samples.-Centrifugation at 370 g or 829 g was not detrimental {P>0.05) to prefreeze or postfreeze motilTABLE 1.-Mean Prefreeze and Postfreeze Motility of Stallion Spermatozoa Subjected to Combinations of Centrifugal Force, Dilution, and Seminal Plasma Concentration for Part I, Experiment 1 Treatment Semen
Diluteda Diluted Diluted Diluted Diluted Undiluted Undiluted Undiluted Undiluted Undiluted
Centrifugation
Not centrifuged 370 g 370 g 829 g 829 g Not centrifuged 428 g 428 g 956 g 956 g
Plasmab
Motility(%) PrePostfreezec freezed
69"
19•
Removed Retained Removed Retained
481 69• 44f 68• 73•
14f 20" 151 21• 221
Removed Retained Removed Retained
70"·f 73• 661 6gf·•
29" 28• 27• 26•
•Diluted= 1:2. bPlasma removed=2% seminal plasma sample; plasma retained= 10% seminal plasma sample. cMean of two samples estimated 90 min after initial dilution. dMean of estimates made at 0, 2, 4, and 6 hr after thawing. •· 1·•Means for prefreeze or postfreeze motility bearing different symbols differ significantly (P
FERTILITY AND STERILITY Copyright c 1975 The American Fertility Society
EFFECT OF CENTRIFUGATION AND SEMINAL PLASMA ON MOTILITY AND FERTILITY OF STALLION AND BULL SPERMATOZOA B. W. PICKETT, PH.D., J. J. SULLNAN, PH.D.,* W. W. BYERS, M.S.,t M. M. PACE, PH.D.,* AND E. E. REMMENGA, PH.DJ Animal Reproduction Laboratory, Department of Physiology and Biophysics, Colorado State University, Fort Collins 80523, and American Breeders Service, DeForest, Wisconsin 53532
Low concentrations of spennatozoa in some stallion ejaculates necessitate removal of seminal plasma by centrifugation to maintain desired insemination volume. 1 • 8 Low centrifugal forces did not adversely affect stallion spermatozoa. 9 Dilution of semen or removal of seminal plasma is believed to be necessary for prolonged storage, 10' 14 but motility is depressed by dilution above or below 1:4.15-18 The objectives of this research were to determine the effect of centrifuging diluted (1:2) and undiluted semen on bovine and equine spennatozoan motility and fertility, and the effect of seminal plasma and dilution on equine spennatozoan survival during incubation before and after freezing. MATERIALS AND METHODS
I. Influence of centrifugal force, washing, and seminal plasma on prefreeze and postfreeze motility of stallion spermatozoa. The stallions (Quarter Horses or thoroughbreds) were stabled at Colorado State University and ranged in age from five to 12 years. Semen was collected with an artificial vagina. 19 Sudden temperature changes were avoided. After final dilution, all samples were Received April 1, 1974. *American Breeders Service. tPresent address: Sandarac Arabian Horse Farm, R.R. 2, Wolf Road, Geneseo, Illinois 61254. *Department of Statistics, Colorado State University.
cooled to 5 C in approximately one hour and placed in 1.0 ml ampules. Two drops of extended semen were placed on opposite ends of a glass slide at '38 C and covered; motility was estimated under phase-contrast microscopy (x 200). Semen was frozen in liquid-nitrogen vapor. 20 After freezing, the ampules were thawed in a 38 C water bath for 1.5 minutes and then transferred to a 1 C water bath for the experiment. A randomized block design was used and the data were analyzed by analysis ofvariance. 21 The percent motility values were transformed to angles (arcsin V"%) prior to analysis. Transformed means were tested by the Honest significant difference test which is based on Student's range. However, nontransformed meaits are presented here. Experiment 1. Diluted samples.-Three ejaculates were collected from each of four stallions; 4 ml of semen from each ejaculate was placed in each of five 17-ml centrifuge tubes. Eight milliliters of an EYT extender (Tris [hydroxymethyl] aminomethane [W/V = 0.24% ], glucose [W/ V=4.08%], glycerol [VN=4.70%], citric acid [W/V=0.1%], egg yolk [V/V=20%] in deionized, autoclaved water) was added. One of the tubes (control) was not centrifuged, but was incubated at 38 C while the others were processed. Two tubes each were centrifuged at 370 g and 829 g for five minutes. Following centrifugation, 10 ml of supernatant was removed from one tube
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centrifuged at each of the centrifugal forces and the spermatozoa resuspended in fresh extender to a volume of 40 ml. The final dilution was 1:9. The spermatozoa in each of the remaining tubes were resuspended in their supernatant and diluted 1:9. Thus, one sample processed at each g force contained 2% seminal plasma, while the others contained 10%. The change in osmotic pressure was minimal, since the extender osmotic pressure was very close to that of seminal plasma. Undiluted samples.-Five additional tubes containing 4 ml of raw semen from each ejaculate were processed in a similar manner. Prefreeze motility was estimated from two randomly selected ampules 90 minutes after dilution. Postfreeze motility was estimated immediately after thawing and at two-hour intervals for six hours. Experiment 2. The effects of 0, 2%, and 10% seminal plasma on prefreeze and postfreeze spermatozoan motility after centrifugation at 956 g were examined. In experiment 1, the .final concentration of extender ingredients in the diluted semen changed when different amounts of seminal plasma were removed. Thus, in this experiment the EYT extenders were adjusted to eliminate this variable. Samples of three ejaculates from each of four stallions were treated as follows: (1) diluted 1:9 and cooled to 5 C; (2) incubated at 38 C, then diluted 1:9; (3) centrifuged at 956 g for three minutes followed by removal of seminal plasma, resuspension of spermatozoa, and 1:9 dilution; (4) centrifuged, followed by resuspension of spermatozoa in 2% seminal plasma when diluted 1:9; (5) centrifuged, followed by resuspension of spermatozoa in 10% seminal plasma when diluted 1:9. Prefreeze motility was estimated 7 5 minutes after dilution, while postfreeze
February 1975
motility was estimated immediately and two hours after thawing. Experiment 3. The effect of dilution ratios of1:1, 1:2, 1:4, 1:8, 1:16, and 1:32 on prefreeze and postfreeze spermatozoan motility was examined. An extender composed of dried skim milk (W/V=5.02%), glucose (W/V=5.02%), glycerol (V/V= 5.02%) in deionized, autoclaved water was used. Since the dilution ratio varied from 1:1 to 1:32, the seminal plasma concentration varied from 50% to 3%. Two ejaculates from each of five stallions were used. Prefreeze motility was estimated immediately, and at eight, 16, and 24 hours after dilution, while postfreeze motility was estimated immediately and at two hours after thawing. Experiment 4. To establish the concentration of seminal plasma required for maximum motility, an effort was made to eliminate the ingredient concentration variable in experiment 3. The concentrations of skim milk and glycerol were maintained constant at 5% while glucose was varied from 2.52% to 4.95% to compensate for the osmotic pressure when 0, 5%, 10%, 20%, 30%, or 50% seminal plasma replaced water in the extender. Seminal plasma was obtained from first and second ejaculates collected on the same day and the semen was centrifuged at 26,650 g for ten minutes. The supernatants were pooled and maintained at -20 c. Two ejaculates from each of four stallions were used. Samples (4 ml) from individual ejaculates were centrifuged for three minutes at 956 g. The seminal plasma was decanted and the spermatozoa resuspended in the appropriate diluent. Prefreeze and postfreeze motility was estimated as in experiment 3. II. Influence of centrifugation on fertility of stallion and bull spermatozoa. Stallions were of the Arabian, Connemara, Paint, Quarter Horse, and Thoroughbred breeds. Holstein bulls were used. Stal-
Vol. 26, No.2
MOTILITY AND FERTILITY OF SPERMATOZOA
lions, bulls, and mares were stabled at American Breeders Service. Semen was collected as in part I. Motilities were estimated under phase-contrast microscopy (x 200) immediately after collection. Postfreeze motilities were evaluated photometrically (x 180).22 The mares were inseminated daily during estrus beginning on day 2. The fertility data were analyzed by analysis of variance. 21 Experiment 1. Unfrozen stallion semen.-Undiluted semen (5 to 15 ml) were centrifuged at 310 g for 3.5 minutes; the spermatozoa were then resuspended. Aliquots containing 500 x 106 motile spermatozoa were used for each insemination. The volume inseminated ranged from 1.7 to 33.2 ml. Time from collection to insemination ranged from 20 to 40 minutes. Experiment 2. Frozen stallion semen.Centrifugation was performed as in part II, experiment 1, except that up to 80% of the seminal plasma was removed. Spermatozoa were diluted no less than 1:9 and concentrations were adjusted prior to freezing to provide 80 to 150 x 106 motile spermatozoa/10 ml insemination unit after freezing and thawing. After collection and dilution, the semen was cooled to 5 C in one hour, frozen one hour later, and stored at -196 C from one to five months. It was thawed in 32 C to 35· C water just before insemination. Experiment 3. Frozen bull semen.Semen was collected from five mature Holstein bulls twice a week for 15 collections. Ejaculates were randomly treated as follows: (1) semen not centrifuged, held three to four minutes at room temperature, diluted 1:2, and cooled to 5 C; (2) semen centrifuged at 270 g for three minutes, spermatozoa resuspended, diluted 1:2, and cooled to 5 C; (3) semen diluted 1:2, centrifuged at 270 g for three minutes, spematozoa resuspended, and cooled to 5 C.
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After cooling about two hours, the semen was further extended to provide between 5 x 10 6 and 14 x 10 6 motile spermatozoa/0.5 ml insemination unit after freezing and thawing. The extender was composed of sodium citrate (WN= 2.7%), egg yolk (V/V=20%), glycerol (VN = 7% ), antibiotics, and deionized, autoclaved water. The semen was frozen four to eight hours after collection and stored at -196 C. Fertility was evaluated by 60- to 90-day nonreturns to estrus. RESULTS
I. Experiment 1. The mean prefreeze and postfreeze motility of stallion spermatozoa subjected to dilution, combinations of centrifugal force, and seminal plasma concentration are presented in Table 1. Diluted samples.-Centrifugation at 370 g or 829 g was not detrimental {P>0.05) to prefreeze or postfreeze motilTABLE 1.-Mean Prefreeze and Postfreeze Motility of Stallion Spermatozoa Subjected to Combinations of Centrifugal Force, Dilution, and Seminal Plasma Concentration for Part I, Experiment 1 Treatment Semen
Diluteda Diluted Diluted Diluted Diluted Undiluted Undiluted Undiluted Undiluted Undiluted
Centrifugation
Not centrifuged 370 g 370 g 829 g 829 g Not centrifuged 428 g 428 g 956 g 956 g
Plasmab
Motility(%) PrePostfreezec freezed
69"
19•
Removed Retained Removed Retained
481 69• 44f 68• 73•
14f 20" 151 21• 221
Removed Retained Removed Retained
70"·f 73• 661 6gf·•
29" 28• 27• 26•
•Diluted= 1:2. bPlasma removed=2% seminal plasma sample; plasma retained= 10% seminal plasma sample. cMean of two samples estimated 90 min after initial dilution. dMean of estimates made at 0, 2, 4, and 6 hr after thawing. •· 1·•Means for prefreeze or postfreeze motility bearing different symbols differ significantly (P
