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Fertility of Bull Spermatozoa in Yolk-Citrate Partially Replaced by Glucose
F E R T I L I T Y OF B U L L S P E R M A T O Z O A I N Y O L K - C I T R A T E P A R T I A L L Y R E P L A C E D BY GLUCOSE J . I . O H M S ~ A.-~]) E . L . W I L L E T T
1
American Foundation for Biological Research, Madison, Wisconsin SUMMARY
Four livability trials and three breeding trials were conducted to compare two diluters: (a) YGC--one part egg yolk, four parts of a 5% solution of glucose, and one part of a 2.9% solution of sodium citrate dihydrate, and (b) YC--one part egg yolk and one part of a 2.9% solution of sodium citrate dihydrate. The means of the progressive motilities for 50 ejaculates stored in these diluters for seven days at 5 ° C. were not significantly different (P > 0.05). After one day of storage, the difference between the average nonreturn rates for the two diluters was not significant (P > 0.05). There was a total of 6,405 first services. After two days of storage, with a total of 1,914 services, the 60- to 90-day nonreturn rate with YGC was 61.7, and with YC, 55.7. The difference is highly significant (P < 0.01). These data support published livability studies which have indicated that a high concentration of electrolytes is detrimental to spermatozoa. Kampsehmidt et al. (1, 2) demonstrated that high concentrations of electrolytes were detrimental to spermatozoa during storage and that the replacement of most of the electrolytes by glucose, a sugar metabolizable by spermatozoa, was beneficial. Livability studies in the present a u t h o r s ' laboratory indicated that replacement of two-thirds of yolk-citrate with glucose might be beneficial. Breeding trials were, therefore, conducted to compare yolk-citrate with yolk-citrateglucose. This paper reports the results. EXPERIMENTAL PROCEDURE I n four separate ]ivability trials, the two diluters to be compared were as follows: (a) One part egg yolk, four parts of a 5% solution of glucose, and one part of a 2.9% solution of sodium citrate dihydrate. This diluter was one studied by Kampschmidt et al. (2). (b) One part egg yolk ancl one part of a 2.9% solution of sodium citrate dihydrate. Dihydrostreptomycin sulfate was added to both diluters at the rate of 500 7 per milliliter. Ill each, semen was diluted to' contain 15 × 106 spermatozoa per milliliter. After storage at 5 ° C. for seven days, motility estimations were made on each sample independently by two workers and the two values were averaged. A comparison of the two diluters was made in three separate breeding trials. The yolk-citrate diluent was routinely employed in the bull stud in which these tests were conducted. I n the first trial, 500 y of dihydrostreptomycin sulfate were added per milliliter of diluent. I n the second and third trials, dihydrostreptomycin sulfate at the rate 500 7 per milliliter and sulfanilamide at the rate of 0.3% were added. Dilution rates varied from 1:76 to 1 : 338, and the number of Received for publication June 17, 1958. 1Present address: Dairy Department, Michigan State University, East Lansing. 1800
1801
F E R T I L I T Y OF S P E R M A T O Z O A IN Y O L K - C I T R A T E AND GLUCOSE
spermatozoa in each milliliter of diluted semen varied from 9 to 34 × 106. Due to the limited number of services, the collections were not split (4). In the first trial, there were two collections from each of eight bulls; in the second, two collections from each o'f 19 bulls, and in the third, six or eight collections from each of eight bulls, with a total of 44 collections. In every instance, the treatments within each bull were alternated in the successive weekly collections. In each week of each experiment, the semen from one-half of the bulls received one treatment and the semen from the o~her half of the bulls received the other treatment, to minimize time effects. Breeding efficiency was measured by nonreturn rates determined from 60 to 90 days after first services. RESULTS AND DISCUSSION The livability data are sunmlarized (Table 1). The difference between the means is not significant (P > 0.05), as established by analysis of variance. These results in conjunction with the p H determinations indicate that the reduced buffer concentration in the yolk-glucose-citrate was still adequate to maintai,1 optimum p H with only 15 × 106 spermatozoa per milliliter. Kampschmidt et al. (1, 2) reported difficulty in maintaining optimum p H in the yolk-glucose-citrate diluter when the semen was diluted 1:10. Also, with the low concentration of sperm employed in the present experiments, and in light of the study of glycolysis by spermatozo'a in yolk-citrate and yolk-glucose-citrate by Salisbury and VanDemark (3), it is doubtful if metabolizable sugar could have been a limiting factor in the yolk-citrate in the present study, although those workers did observe some stimulation of spermatozoa with addition o'f glucose. The number of services and the n o n r e t u r n rates for tile three breeding trials and for all trials conlbined are shown (Table 2). When the data from all three trials are combined, the difference between the means is not significant ( P > 0.05), as established by analysis of variance, for services performed the first day after collection. F o r semen stored two days, the over-all average n o n r e t u r n rate of spermatozoa in yolk-glucose-citrate is highly significantly greater (P < 0.01) TABLE
1
Progressive motility of spermatozoa and p H of semen after storage in yolk-citrate and yolk-ghwose-eitrate for seven days at 5 ° C. Yolk-citrate
Yolk-glucose-citrate
pH Trial
Ejaculates
Motility
(No.)
(Xo.)
(%)
17 18 20 23 Totals and averages
10 13 12 15
31.5 23.8 43.5 34.5
50
33.3
Initial
ptI After storage
Motility
Initial
After storage
(%) 6.84 6.81 ............ ............
6.74 6.63
29.3 33.2 45.0 31.5 34.7
6.94 6.96 ........... ...........
6.78 6.69
J. I.
1802
OHiMS AND E. L. WILLETT
TABLE 2 Sixty- to ninety-day nonreturn rates with spermatozoa stored at ,~° C. for one and ~wo days in yo~k-citrate a~td in yolk-citrate-ghwose Yolk-citrate Services Nonreturns (2¢o.) One-day storage Experiment 1 Experiment 2 Experiment 3 Totals and averages Two-day storage Experiment 1 Experiment 2 Experiment 3 Totals and averages
(%)
Yolk-citrate-glucose Services Nonreturns (No.)
(%)
365 1,678 1,196 3,239
76.7 67.5 6q).5 66.0
405 1,631 1,130 3,166
71.6 72.5 59.8 67.8
318 344 297 959
65.7 53.5 47.5 55.7
330 344 281 955
64.5 68.6 49.8 61.7
t h a n t h a t ill y o l k - c i t r a t e . T h e s e d a t a w e r e a n a l y z e d b y C h i - s q u a r e , b e c a u s e of tlle s m a l l n m n b e r of s e r v i c e s i n m o s t o f t h e c o l l e c t i o n s . T h e r e s u l t s i n d i c a t e t h a t y o l k c i t r a t e as a d i l u t e r f o r b u l l s p e r m a t o z o a is i m p r o v e d b y r e p l a c e m e n t o f t w o - t h i r d s o~ i t b y g l u c o s e . T h e s e b r e e d i n g r e s u l t s s u p p o r t t h e o b s e r v a t i o n s m a d e b y K a m p s c h m i d t et al. (1, 2 ) , t h a t a h i g h c o n c e n t r a t i o n o f e l e c t r o l y t e s is d e t r i m e n t a l t o sp ermato'zoa. ACKNOWLEDGMENT The authors wish to acknowledge the invaluable cooperation of Lynford Tremaine, manager of the American Breeders Service stud at Carmel, Indiana, at the time these breeding trials were conducted. REFERENCES (1) KA~PSCHMIDT, ]~. F., MAYER, D. T., AND HER~[AN, K. A. Viability of Bull Spermatozoa as Influenced by Various Sugars and Electrolytes in the Storage Medium. Missouri Agr. Expt. Sta., Research B~dl. 519. lq53. (2) I~AI~IPSCHI~IIDT,R. F., MAYER, D. T., HERS!AN, I~. A., AXD DICKERSON, G. E. Viability of Bull Spermatozoa as Influence,] by Electrolyte Concentration, Buffer Efficiency, and Added Glucose in Storage Media. J. Dairy Sci., 34: 45. 1951. (3) SALISBUR% @. W., A~]) VANDSMAR~:, N. L. Stimulation of Livability and Glyeolysis by Additions of Glucose to the Egg Yolk-Citrate Diluent for Ejaculated Bovine Semen. A ~ . J. Physiol., 143: 692. 1945. (4) WILLETT, E. L., Ott3,IS, J. I., AND TORRIE, J. H. Factors Influencing Experimental Error in Field Trials in Artificial Insemination. J. Dairy Sci., 38: 1375. 1955.
1
American Foundation for Biological Research, Madison, Wisconsin SUMMARY
Four livability trials and three breeding trials were conducted to compare two diluters: (a) YGC--one part egg yolk, four parts of a 5% solution of glucose, and one part of a 2.9% solution of sodium citrate dihydrate, and (b) YC--one part egg yolk and one part of a 2.9% solution of sodium citrate dihydrate. The means of the progressive motilities for 50 ejaculates stored in these diluters for seven days at 5 ° C. were not significantly different (P > 0.05). After one day of storage, the difference between the average nonreturn rates for the two diluters was not significant (P > 0.05). There was a total of 6,405 first services. After two days of storage, with a total of 1,914 services, the 60- to 90-day nonreturn rate with YGC was 61.7, and with YC, 55.7. The difference is highly significant (P < 0.01). These data support published livability studies which have indicated that a high concentration of electrolytes is detrimental to spermatozoa. Kampsehmidt et al. (1, 2) demonstrated that high concentrations of electrolytes were detrimental to spermatozoa during storage and that the replacement of most of the electrolytes by glucose, a sugar metabolizable by spermatozoa, was beneficial. Livability studies in the present a u t h o r s ' laboratory indicated that replacement of two-thirds of yolk-citrate with glucose might be beneficial. Breeding trials were, therefore, conducted to compare yolk-citrate with yolk-citrateglucose. This paper reports the results. EXPERIMENTAL PROCEDURE I n four separate ]ivability trials, the two diluters to be compared were as follows: (a) One part egg yolk, four parts of a 5% solution of glucose, and one part of a 2.9% solution of sodium citrate dihydrate. This diluter was one studied by Kampschmidt et al. (2). (b) One part egg yolk ancl one part of a 2.9% solution of sodium citrate dihydrate. Dihydrostreptomycin sulfate was added to both diluters at the rate of 500 7 per milliliter. Ill each, semen was diluted to' contain 15 × 106 spermatozoa per milliliter. After storage at 5 ° C. for seven days, motility estimations were made on each sample independently by two workers and the two values were averaged. A comparison of the two diluters was made in three separate breeding trials. The yolk-citrate diluent was routinely employed in the bull stud in which these tests were conducted. I n the first trial, 500 y of dihydrostreptomycin sulfate were added per milliliter of diluent. I n the second and third trials, dihydrostreptomycin sulfate at the rate 500 7 per milliliter and sulfanilamide at the rate of 0.3% were added. Dilution rates varied from 1:76 to 1 : 338, and the number of Received for publication June 17, 1958. 1Present address: Dairy Department, Michigan State University, East Lansing. 1800
1801
F E R T I L I T Y OF S P E R M A T O Z O A IN Y O L K - C I T R A T E AND GLUCOSE
spermatozoa in each milliliter of diluted semen varied from 9 to 34 × 106. Due to the limited number of services, the collections were not split (4). In the first trial, there were two collections from each of eight bulls; in the second, two collections from each o'f 19 bulls, and in the third, six or eight collections from each of eight bulls, with a total of 44 collections. In every instance, the treatments within each bull were alternated in the successive weekly collections. In each week of each experiment, the semen from one-half of the bulls received one treatment and the semen from the o~her half of the bulls received the other treatment, to minimize time effects. Breeding efficiency was measured by nonreturn rates determined from 60 to 90 days after first services. RESULTS AND DISCUSSION The livability data are sunmlarized (Table 1). The difference between the means is not significant (P > 0.05), as established by analysis of variance. These results in conjunction with the p H determinations indicate that the reduced buffer concentration in the yolk-glucose-citrate was still adequate to maintai,1 optimum p H with only 15 × 106 spermatozoa per milliliter. Kampschmidt et al. (1, 2) reported difficulty in maintaining optimum p H in the yolk-glucose-citrate diluter when the semen was diluted 1:10. Also, with the low concentration of sperm employed in the present experiments, and in light of the study of glycolysis by spermatozo'a in yolk-citrate and yolk-glucose-citrate by Salisbury and VanDemark (3), it is doubtful if metabolizable sugar could have been a limiting factor in the yolk-citrate in the present study, although those workers did observe some stimulation of spermatozoa with addition o'f glucose. The number of services and the n o n r e t u r n rates for tile three breeding trials and for all trials conlbined are shown (Table 2). When the data from all three trials are combined, the difference between the means is not significant ( P > 0.05), as established by analysis of variance, for services performed the first day after collection. F o r semen stored two days, the over-all average n o n r e t u r n rate of spermatozoa in yolk-glucose-citrate is highly significantly greater (P < 0.01) TABLE
1
Progressive motility of spermatozoa and p H of semen after storage in yolk-citrate and yolk-ghwose-eitrate for seven days at 5 ° C. Yolk-citrate
Yolk-glucose-citrate
pH Trial
Ejaculates
Motility
(No.)
(Xo.)
(%)
17 18 20 23 Totals and averages
10 13 12 15
31.5 23.8 43.5 34.5
50
33.3
Initial
ptI After storage
Motility
Initial
After storage
(%) 6.84 6.81 ............ ............
6.74 6.63
29.3 33.2 45.0 31.5 34.7
6.94 6.96 ........... ...........
6.78 6.69
J. I.
1802
OHiMS AND E. L. WILLETT
TABLE 2 Sixty- to ninety-day nonreturn rates with spermatozoa stored at ,~° C. for one and ~wo days in yo~k-citrate a~td in yolk-citrate-ghwose Yolk-citrate Services Nonreturns (2¢o.) One-day storage Experiment 1 Experiment 2 Experiment 3 Totals and averages Two-day storage Experiment 1 Experiment 2 Experiment 3 Totals and averages
(%)
Yolk-citrate-glucose Services Nonreturns (No.)
(%)
365 1,678 1,196 3,239
76.7 67.5 6q).5 66.0
405 1,631 1,130 3,166
71.6 72.5 59.8 67.8
318 344 297 959
65.7 53.5 47.5 55.7
330 344 281 955
64.5 68.6 49.8 61.7
t h a n t h a t ill y o l k - c i t r a t e . T h e s e d a t a w e r e a n a l y z e d b y C h i - s q u a r e , b e c a u s e of tlle s m a l l n m n b e r of s e r v i c e s i n m o s t o f t h e c o l l e c t i o n s . T h e r e s u l t s i n d i c a t e t h a t y o l k c i t r a t e as a d i l u t e r f o r b u l l s p e r m a t o z o a is i m p r o v e d b y r e p l a c e m e n t o f t w o - t h i r d s o~ i t b y g l u c o s e . T h e s e b r e e d i n g r e s u l t s s u p p o r t t h e o b s e r v a t i o n s m a d e b y K a m p s c h m i d t et al. (1, 2 ) , t h a t a h i g h c o n c e n t r a t i o n o f e l e c t r o l y t e s is d e t r i m e n t a l t o sp ermato'zoa. ACKNOWLEDGMENT The authors wish to acknowledge the invaluable cooperation of Lynford Tremaine, manager of the American Breeders Service stud at Carmel, Indiana, at the time these breeding trials were conducted. REFERENCES (1) KA~PSCHMIDT, ]~. F., MAYER, D. T., AND HER~[AN, K. A. Viability of Bull Spermatozoa as Influenced by Various Sugars and Electrolytes in the Storage Medium. Missouri Agr. Expt. Sta., Research B~dl. 519. lq53. (2) I~AI~IPSCHI~IIDT,R. F., MAYER, D. T., HERS!AN, I~. A., AXD DICKERSON, G. E. Viability of Bull Spermatozoa as Influence,] by Electrolyte Concentration, Buffer Efficiency, and Added Glucose in Storage Media. J. Dairy Sci., 34: 45. 1951. (3) SALISBUR% @. W., A~]) VANDSMAR~:, N. L. Stimulation of Livability and Glyeolysis by Additions of Glucose to the Egg Yolk-Citrate Diluent for Ejaculated Bovine Semen. A ~ . J. Physiol., 143: 692. 1945. (4) WILLETT, E. L., Ott3,IS, J. I., AND TORRIE, J. H. Factors Influencing Experimental Error in Field Trials in Artificial Insemination. J. Dairy Sci., 38: 1375. 1955.