- Email: [email protected]
Effect of in vitro selenium supplementation on bovine sperm motility
THERIOGENOLOGY
EFFECT OF -IN VITRO SELENIUM SUPPLEMENTATION ON BOVINE SPEPtiMOTILITYasb Rebecca B. Siegel', Finnie A. Murray, W. E. Juliend, A. L. Moxon and H. R. Conrad Department of Animal Science and Department of Dairy Science Ohio Agricultural Research and Development Center Wooster, Ohio 44691 Received for publication:
2/28/80
Abstract The effects of selenium (Se) on motility of bovine spermatozoa were evaluated in two experiments. In Experiment 1, increasing doses of Se alone or with vitamin E were found to increase motility in Se concentrations up to 1 pig/ml. However, Se alone had a greater effect than did Se with vitamin E. The sperm motility response to Se concentrations from background to 1 ng/ml on motility best fit a quadratic equation with a maximum at about .65 pg Se/ml. In Experiment 2, the effect of Se addition before and after freezing was evaluated. This experiment demonstrated that treatment of semen with Se before freezing significantly increased the post-thaw motility as compared with portions of the same ejaculate which were not treated. Treatment after thawing produced motilities which were intermediate and not significantly different from either pre-freezing supplementation or no supplementation at all. These data indicate that some semen exhibits a greater percentage of motile sperm when Se is added prior to freezing, however, no information is yet available on the fertility of Se-treated semen.
a Approved for publication as Journal Article No. 152-78 of the Ohio Agricultural Research and Development Center, Wooster, Ohio 44691. b Supported by a gift from Select Sires, Inc., Plain City, Ohio 43064, and a gift of semen from NOBA Animal Industry Services, Tiffin, Ohio 44883. We appreciate help and cooperation from Clifford Marshall, Max Drake and B. W. Kagy. ' Present address: Department of Genetics and Development, University of Illinois, Urbana, Illinois 61801. d Present address: U.S. Feed Grains Council, Gatterburgasse 18/2/3A, A-1190 Vienna, Austria.
MAY 1980 VOL. 13 NO. 5
357
THERIOGENOLOGY
Introduction Feedstuffs produced in many parts of the world are deficient in Se, an element necessary in trace amounts for optimal livestock performance. A number of Se deficiency syndromes in cattle, poultry, horses, sheep and swine are known (1). Recently several reproductive problems in cattle have been shown to be responsive to Se treatment. These include decreased incidence of retained placenta in dairy cattle (2,3) and improved fertility of ova in beef cattle (4). Furthermore, McCoy and Weswig (5) found that male offspring of Se deficient, vitamin E adequate female rats produced non-motile sperm. Wu -et al. (6) found that Se deficient rats produced sperm with poor motility and with a high proportion of broken tails. These effects could not be overcome with vitamin E. Recently Smith et al. (7,8) found that after a single i.v. injection of 75Se into bulls, -nSe was incorporated into insoluble structural components of spermatozoa. Furthermore an association of Se with a small structural protein in rat (9,lO) and bull (11) sperm mitochondria has been reported. In view of these results we thought it important to determine the effects of Se on bull sperm motility and viability. The objective of this study was to determine whether Se added to diluted bull semen affects motility and viability of fresh or frozen semen. Materials and Methods Semen was obtained from dairy bulls held at commercial semen collection and storage laboratories. In all cases the semen was diluted with the standard yolk-citrate diluent used by one of these commercial laboratories. Additional quantities of this diluent were made available for Se analyses and further semen dilution. The composition of the diluent is provided in table I. Experiment 1. Fresh Semen. Three ejaculates from a Jersey bull and four ejaculates from a Holstein bull, both housed at NOBA Animal Industry Services, Inc., Tiffin, Ohio, were used for this experiment. The semen was diluted for commercial use and an aliquot containing approximately 20x106 motile sperm/ml was transported under refrigeration to this laboratory. All handling of sperm in the laboratory was performed under a laminar flow biological safety cabinet to minimize microbial contamination. The semen was further diluted (1:20) with the yolk-citrate diluent containing various concentrations of Se as NaZSe03 or in the product Muse (Burns Biotec Lab., Oakland, California) which contains 13.6 I.U. vitamin E/mg Se. Sperm in an aliquot of each ejaculate were counted at the time Se was added to determine initial motility. Motility counts were performed with a hemacytometer at 22'C, and sperm showing any type of active motion were classed as motile. The sperm preparations were incubated in duplicate in sterile Falcon Micro-Test II plates and maintained at 22OC for 24 hours (h), at which time at least 100 sperm were counted by the observer for each well to determine motility. The mean number of motile sperm divided by the mean
358
MAY
1980 VOL. 13 NO. 5
THERIOGENOLOGY
number of total sperm multiplied by 100 yields the percent motile sperm. For uncertain reasons the percent motile sperm in the control cultures varied greatly within and between bulls and among ejaculates. To reduce this heterogeneity in the experiment, all data were expressed as percents of the appropriate control. This reduced the heterogeneity and made statistical analysis possible. Within each experiment a dose response curve of increasing levels of Se as Na2SeO3 or Muse was performed for each bull. Multiple linear regression analysis was performed on data from each bull as well as the pooled data (12). Se analysis was by the method of Olson (13). Experiment 2. Frozen Semen. Semen from six dairy bulls (four Holstein and two Brown Swiss) housed at Select Sires, Inc., Plain City, Ohio, was used for this experiment. The bulls were selected by Select Sires, Inc. to give 2 bulls each which were good, medium and poor with respect to semen quality. Two ejaculates collected at a 7 day interval were obtained from each bull. Each ejaculate was split and one portion of semen was diluted with a diluent which contained Na2SeO3 so that the final Se concentration was 1 ug/ml above the endogenous concentration, while the second portion of semen was diluted with diluent containing only endogenous Se. The semen was added to .5 ml straws and sealed automatically. The straws were frozen in liquid nitrogen vapor and stored in liquid nitrogen by standard methods in use at Select Sires, Inc. The semen was maintained in liquid N2 until motility and live-dead analyses were performed in this laboratory. Upon thawing the semen was further diluted with the yolk-citrate buffer 1:lO. Semen frozen in diluent containing 1 ug Se/ml was further diluted with diluent containing 1 ug Se/ml. Semen containing no additional Se was split after thawing, and one portion was diluted with diluent containing supplemental Se to give a final supplemental Se content of 1 ug/ml. Thus, three conditions with regard to Se supplementation were achieved: 1 pg Se/ml prior to freezing, 1 pg Se/ml upon thawing and no Se. All treatments were incubatad in quadruplicate prior to motility and viability determinations. Tw;,incubation conditions were employed: 4 h at 37'C in 95 percent air, 5 percent CO2 and 6 h at 20°C in air. Incubation was carried out in Falcon Micro Test Plates II, and semen was handled prior to counting in a laminar flow biological safety cabinet to prevent microbial contamination. Motility counts were performed as described for Experiment 1, and the 37'C cultures were allowed to rest at 20°C for 5 minutes or more before counting. Viability determinations were made using eosin bluish-fast green stain as described by Sorenson (14). Data in Experiment 2 were subjected to least squares analysis of variance (15) and, where appropriate, to linear regression. Results Experiment 1.
MAY
The addition of increasing concentrations of Se
1980 VOL. 13 NO. 5
359
THERIOGENOLOGY in diluted bull semen increased sperm motility at 24 h as compared with controls receiving no added Se. However, at Se doses greater than 1 ng/ml a reduction in motility appeared such that at 5 ug/ml the motility values were significantly lower than controls (65.6 C 32.4 vs 100.0 + 0.0, lOdf, Pc.01). The responses to both Se treatments(Se alone and Muse) fit quadratic equations with maxima at approximately 2 pg Se/ml. Since the experimental design used Se concentrations at l/2 log intervals, no data points were available between 1 ng Se/ml and 5 ng Se/ml except for one ejaculate. Therefore, data points for Se concentrations greater than 1 ng/ml were omitted and the curve best fitting the data was computed. Quadratic equations provided the highest coefficient of determination (R2 = -532 for Se alone, R2 = ,330 for Muse), and revealed maxima at approximately 0.65 ng Se/ml for both Se and Muse (figure 1). However, the maximum for the Se equation (Y = 92.87 + 188.52X - 143.18X2) was higher (Pc.02) than that for the Muse equation (Y = 101.97 + 131.50X - 103.86X2). Thus the presence of vitamin E in the Muse preparation was not beneficial to sperm motility as compared with Se alone, yet both treatments produced significant increases in the motility of bull spermatozoa -in vitro. Experiment 2. When bull sperm were treated with 1 ng Se/ml before freezing, upon thawing the sperm exhibited greater (Pc.05) motility than untreated semen (table II) but no differences in sperm viability were evident. Addition of Se after thawing did not significantly affect motility. Interactions among the following sources of variation were found to be statistically non-significant: Se treatment X ejaculate, Se treatment X bull, Se treatment X incubation condition, ejaculate X incubation condition and bull X ejaculate. There were differences among bulls with regard to motility after culture (table III) and these differences correspond generally to the semen quality of the bulls, i.e., bulls 5 and 6 were of high quality, while bulls 3 and 4 were of poor quality. Although the least square analysis of variance revealed no Se treatment X bull interaction, linear regression of the difference between the mean percent motility of semen treated with Se before freezing and the mean percent motility of control semen for each bull on the number of motile sperm per culture well demonstrated that the response to Se treatment was greatest in semen of bulls producing the greatest number of motile sperm (r = .87, Pc.05). This relationship is depicted in figure 2. This suggests that sperm from certain ,bulls were more competent to respond to Se supplementation than are sperm from other bulls. There was no relationship between the blood Se levels and response of sperm from the various bulls to supplemental Se in the diluent. However, the serum Se values were similar among the six bulls used in this study (.068 + .008 ng Se/ml, mean + S.E.M.), and it is possible that a relationship might be observed if bulls differing to a greater extent in Se status were used. Discussion
360
MAY
1980 VOL. 13 NO. 5
THERIOCENOLOGY
The data presented above indicate that Na2Se08 significantly stimulates motility of both freshly ejaculated, as well as frozen bull sperm, in concentrations up to about 1 vg Se/ml without affecting viability. Thus, Se promotes motility in sperm which would otherwise be non-motile, but still remain alive. Least squares analysis of variance revealed no bull X Se treatment interaction, while a positive correlation was observed between the response of sperm from the various bulls to Se and semen quality of the bulls (as measured by number of motile sperm per unit volume). This discrepancy appears to be related to the variation among ejaculates, since the interaction became evident only when the mean difference between treated and control sperm is used in the regression. Thus, sperm produced by certain bulls does not exhibit increased motility in the presence of Se, suggesting that such semen contains a smaller proportion of "marginal" sperm, which remain alive, but are non-motile. Another possibility is that the metabolic activity of sperm from some bulls is not responsive to Se. The data obtained in Experiment 1 might suggest an interference of vitamin E with the motility response to Se, since Se produced significantly greater stimulation than Muse. However, other possible explanations for this difference are: 1) Muse contains an emulsifying agent (polysorbate 80) and an antibiotic, and 2) it is likely to differ from the diluent in ionic strength, in viscosity, and other physical properties. Thus the smaller effect of Muse may be the result of factors other than vitamin E. A number of studies provide evidence tha;SSe is associated with spermatozoa. Brown and Burk,$16) found that Se0jm2 - treated r;;Tth Se was localized at the midpiece. produced sperm in which the et al. (7,8) found intravenously injected 75Se to be associated with -bovine seminal plasma and spermatozoa and the evidence indicated the possibility that Se may have a role in the spermatozoa as a structural component. Furthermore, Calvin (9) found 75Se incorporation into a polypeptide localized in a keratinous fraction of a sperm tail after intratesticular administration of 75Se0Sm2. Autoradiographic studies with spermatozoa from 75Se03-2 - treated rats (16), indicate an association of Se with the midpiece, and Calvin and Cooper (10) recently reported the association of Se with the keratinous outer membrane of sperm mitochondria. These observations are in accordance with the possibility that Se is involved in metabolic functions, a concept derived from the work of Whanger -et al. (17,18) in which a Se-binding protein from lamb muscle was observed to have properties similar to cytochromes c and bg. Although the data reported herein do not provide evidence for or against incorporation of Se into proteins of spermatozoa, they do support the concept that Se may be involved in metabolic activity of spermatozoa, since motility is increased. Recent additional data from this laboratory (19) provides direct evidence for such an involvement, since bull spermatozoa oxygen consump-
MAY
1980 VOL. 13 NO. 5
361
THERIOGENOLOGY tion was increased by addition of Se to the diluent. The authors are cognizant of the fact that motility evaluations of sperm do not provide the highest correlations with fertility. Saacke and White (20) demonstrated that percent intact acrosomes provides a higher correlation with fertility in bull sperm. Such determinations are beyond the expertise and facilities of the present investigators and were, therefore, not attempted. It is hoped that investigators having expertise, facilities and interest to perform more sophisticated evaluations of spermatozoa than those reported herein will further investigate the direct effects of Se on spermatozoa.
362
MAY 1980VOL. 13 NO. 5
THERIOGENOLOGY
Figure 1.
Figure 2.
MAY
Regression of sperm motility (expressed as a percent of the control) on Se content of the diluent.
Regression of mean increase in sperm (mean of Se-treated semen minus mean each bull) on number of motile sperm II well. Each well contained 200 ~1 represents a 20-fold dilution of the frozen.
1980VOL.13NO.5
motility due to Se of control semen for per Micro Test plate total volume; this semen as it had been
363
THERIOGENOLOGY Table I.
Stock Solution
Composition of Semen Diluent*
212.12 .g 68.00 g
Citric acid
50.00 g
Fructose
3642 ml
Extender
Tris-(hydroxymethyl)-aminomethane
Distilled, deionized Hz0
394 ml
Glycerine is then added to the above
200 ml
Egg yolk
800 ml
Stock solution
1.5x106 units
Penicillin G
3 ml
Linco-Spectin
1S
Streptomycin
5X1.05units
Polymyxin B
*Prepared by NOBA, Inc., Tiffin, Ohio.
364
MAY
1980VOL.13NO.5
THERIOGENOLOGY Table II.
Effect of Supplemental Selenium (1 ug/ml) on Post-Thaw Sperm Motility -in vitro
Treatment
N
Percent Motile (LSM+SEM)
Se before freezing
108
21.4 rt .0808a
Se after thawing
108
19.2 _c .08C18~'~
No supplemental Se
108
18.6 i .0808b
aab Treatments with different superscripts are significantly different from each other (P
Table III.
Differences in Sperm Motility Due to Bulls
Bull
N
Percent Motile Sperm (LSM f SEM)
1
54
19.8 f .15b'C'Y
2
54
20.1 f .18byc,y
3
54
16.5 f .18a,b,x
4
54
15.1 !z .lgasx
5
54
23.0 i .16c'y'x
6
54
24.0 f .18cJz
Means having different superscripts are significantly different from each other (abc, WO.01); (xyz, P
MAY
1980 VOL. 13 NO. 5
365
THERIOGENOLOGY
References
1. Hartley, W. J. and Grant, A. B. A review of selenium responsive diseases of New Zealand livestock. Fed. Proc. 20:679-688 (1961) 2. Julien, W. E., Conrad, H. R., Jones, J. .E., and Moxon, A. L. Selenium and vitamin E and incidence of retained placenta in parturient dairy cows. J. Dairy Sci. 59:1954-1959 (1976) 3. Julien, W. E., Conrad, H. R., and Moxon, A. L. Selenium and vitamin E and incidence of retained placenta in parturient dairy cows. II. Prevention in commercial herds with prepartum treatment. J. Dairy Sci. 59:1960-1962 (1976) 4. Segerson, E. C., Murray, F. A., Moxon, A. L., Redman, D. R., and Conrad, H. R. Selenium/Vitamin E: Role in fertilization of bovine ova. J. Dairy Sci. 60(6):1001-1005 (1977) 5. McCoy, K. E. M. and Weswig, P. H. Some selenium responses in the rat not related to vitamin E. J. Nutr. 98:383-389 (1969) 6. Wu, S. H., Oldfield, J. E., Whanger, P. D., and Weswig, P. H. Effect of selenium, vitamin E and anti-oxidants on testicular function in rats. Biol. Reprod. 8:625-629 (1973) 7. Smith, D. G., Senger, P. L., McCutchan, J. F., and Landa, C. F. Selenium and glutathione peroxidase distribution in bovine semen. Amer. Sot. Animal Sci. Annual Meeting. East Lansing, Mich. July 9-13. Abstract no. 435 (1978) 8. Smith, D. G. and Senger, P. L. Selenium retention by the tissues of the reproductive tract in the bull. Amer. Sot. Animal Sci. Annual Meeting. East Lansing, Michigan. July 9-13. Abstract no. 436 (1978) 9. Calvin, H. I. Selective incorporation of Selenium-75 into a polypeptide of the rat sperm tail. J. Exp. 2001. 204:445-452 (1978) 10. Calvin, H. I., and Cooper, G. W. A specific selenopolypeptide associated with the outer membrane of rat sperm mitochondria. In: The Spermatozoon (Fawcett, D. W. and Bedford, J. M., eds.). Urban and Schwarzenberg, Inc. Baltimore-Munich. pp 135-140 (1979) 11. Pallini, V. and Bacci, E. Bull sperm selenium is bound to a structural protein of mitochondria. J. Submicrosc. Cytol. 11:165-170 (1979) 12. Steele, G. D. and Torrie, J. H. Principles and Procedures of Statistics. McGraw-Hill Book Co., Inc. N.Y. (1960)
366
MAY
1980 VOL. 13 NO. 5
THERIOGENOLOGY
13.
Olson, 0. E., Palmer, I. S., and Cary, E. E. Modification of the official fluorometric method for selenium in plants. J. A.O.A.C. 58:117-121 (1975)
14. Sorenson, A. M. Reproduction Laboratory 2nd ed. Animal Science Department, Texas A and M University. Kendall/Hunt Publishing Co. Iowa (1973) 15. Harvey, W. K. Least squares analysis of data with unequal subclass numbers. USDA, ARS 20-8 (1960) 16. Brown, D. G. and Burk, R. F. Selenium retention in tissues and sperm of rats fed a torula yeast diet. J. Nutrition. 103:102-108 (1973) 17. Whanger, P. D., Pedersen, N. D., and Weswig, P. H. Characteristics of a selenium binding protein from lamb muscle. Fed. Proc. 31:691 (1972) 18. Whanger, P. D., Pedersen, N. D., and Weswig, P. H. Selenium proteins in ovine tissues II. Spectral properties of a 10,000 molecular weight selenium protein. Bioch. and Biophys. Res. Comm. 53(3):1031-1035 (1973) 19.
Pratt, .W. D., Murray, F. A., Moxon, A. L., Conrad, H. R., and Kinder, J. E. Effects of selenium supplementations on bull sperm metabolism -in vitro. Theriogenology (submitted 1980)
20. Saacke, R. G. and White, 3. M. Semen quality tests and their relationship to fertility. Proc. 4th Tech. Conf. Artif. Insem. and Reprod. Feb. 17-19. National Assoc. Animal Breeders, Inc. Columbia, Missouri. pp 22-27 (1972)
MAY
1980 VOL. 13 NO. 5
367
EFFECT OF -IN VITRO SELENIUM SUPPLEMENTATION ON BOVINE SPEPtiMOTILITYasb Rebecca B. Siegel', Finnie A. Murray, W. E. Juliend, A. L. Moxon and H. R. Conrad Department of Animal Science and Department of Dairy Science Ohio Agricultural Research and Development Center Wooster, Ohio 44691 Received for publication:
2/28/80
Abstract The effects of selenium (Se) on motility of bovine spermatozoa were evaluated in two experiments. In Experiment 1, increasing doses of Se alone or with vitamin E were found to increase motility in Se concentrations up to 1 pig/ml. However, Se alone had a greater effect than did Se with vitamin E. The sperm motility response to Se concentrations from background to 1 ng/ml on motility best fit a quadratic equation with a maximum at about .65 pg Se/ml. In Experiment 2, the effect of Se addition before and after freezing was evaluated. This experiment demonstrated that treatment of semen with Se before freezing significantly increased the post-thaw motility as compared with portions of the same ejaculate which were not treated. Treatment after thawing produced motilities which were intermediate and not significantly different from either pre-freezing supplementation or no supplementation at all. These data indicate that some semen exhibits a greater percentage of motile sperm when Se is added prior to freezing, however, no information is yet available on the fertility of Se-treated semen.
a Approved for publication as Journal Article No. 152-78 of the Ohio Agricultural Research and Development Center, Wooster, Ohio 44691. b Supported by a gift from Select Sires, Inc., Plain City, Ohio 43064, and a gift of semen from NOBA Animal Industry Services, Tiffin, Ohio 44883. We appreciate help and cooperation from Clifford Marshall, Max Drake and B. W. Kagy. ' Present address: Department of Genetics and Development, University of Illinois, Urbana, Illinois 61801. d Present address: U.S. Feed Grains Council, Gatterburgasse 18/2/3A, A-1190 Vienna, Austria.
MAY 1980 VOL. 13 NO. 5
357
THERIOGENOLOGY
Introduction Feedstuffs produced in many parts of the world are deficient in Se, an element necessary in trace amounts for optimal livestock performance. A number of Se deficiency syndromes in cattle, poultry, horses, sheep and swine are known (1). Recently several reproductive problems in cattle have been shown to be responsive to Se treatment. These include decreased incidence of retained placenta in dairy cattle (2,3) and improved fertility of ova in beef cattle (4). Furthermore, McCoy and Weswig (5) found that male offspring of Se deficient, vitamin E adequate female rats produced non-motile sperm. Wu -et al. (6) found that Se deficient rats produced sperm with poor motility and with a high proportion of broken tails. These effects could not be overcome with vitamin E. Recently Smith et al. (7,8) found that after a single i.v. injection of 75Se into bulls, -nSe was incorporated into insoluble structural components of spermatozoa. Furthermore an association of Se with a small structural protein in rat (9,lO) and bull (11) sperm mitochondria has been reported. In view of these results we thought it important to determine the effects of Se on bull sperm motility and viability. The objective of this study was to determine whether Se added to diluted bull semen affects motility and viability of fresh or frozen semen. Materials and Methods Semen was obtained from dairy bulls held at commercial semen collection and storage laboratories. In all cases the semen was diluted with the standard yolk-citrate diluent used by one of these commercial laboratories. Additional quantities of this diluent were made available for Se analyses and further semen dilution. The composition of the diluent is provided in table I. Experiment 1. Fresh Semen. Three ejaculates from a Jersey bull and four ejaculates from a Holstein bull, both housed at NOBA Animal Industry Services, Inc., Tiffin, Ohio, were used for this experiment. The semen was diluted for commercial use and an aliquot containing approximately 20x106 motile sperm/ml was transported under refrigeration to this laboratory. All handling of sperm in the laboratory was performed under a laminar flow biological safety cabinet to minimize microbial contamination. The semen was further diluted (1:20) with the yolk-citrate diluent containing various concentrations of Se as NaZSe03 or in the product Muse (Burns Biotec Lab., Oakland, California) which contains 13.6 I.U. vitamin E/mg Se. Sperm in an aliquot of each ejaculate were counted at the time Se was added to determine initial motility. Motility counts were performed with a hemacytometer at 22'C, and sperm showing any type of active motion were classed as motile. The sperm preparations were incubated in duplicate in sterile Falcon Micro-Test II plates and maintained at 22OC for 24 hours (h), at which time at least 100 sperm were counted by the observer for each well to determine motility. The mean number of motile sperm divided by the mean
358
MAY
1980 VOL. 13 NO. 5
THERIOGENOLOGY
number of total sperm multiplied by 100 yields the percent motile sperm. For uncertain reasons the percent motile sperm in the control cultures varied greatly within and between bulls and among ejaculates. To reduce this heterogeneity in the experiment, all data were expressed as percents of the appropriate control. This reduced the heterogeneity and made statistical analysis possible. Within each experiment a dose response curve of increasing levels of Se as Na2SeO3 or Muse was performed for each bull. Multiple linear regression analysis was performed on data from each bull as well as the pooled data (12). Se analysis was by the method of Olson (13). Experiment 2. Frozen Semen. Semen from six dairy bulls (four Holstein and two Brown Swiss) housed at Select Sires, Inc., Plain City, Ohio, was used for this experiment. The bulls were selected by Select Sires, Inc. to give 2 bulls each which were good, medium and poor with respect to semen quality. Two ejaculates collected at a 7 day interval were obtained from each bull. Each ejaculate was split and one portion of semen was diluted with a diluent which contained Na2SeO3 so that the final Se concentration was 1 ug/ml above the endogenous concentration, while the second portion of semen was diluted with diluent containing only endogenous Se. The semen was added to .5 ml straws and sealed automatically. The straws were frozen in liquid nitrogen vapor and stored in liquid nitrogen by standard methods in use at Select Sires, Inc. The semen was maintained in liquid N2 until motility and live-dead analyses were performed in this laboratory. Upon thawing the semen was further diluted with the yolk-citrate buffer 1:lO. Semen frozen in diluent containing 1 ug Se/ml was further diluted with diluent containing 1 ug Se/ml. Semen containing no additional Se was split after thawing, and one portion was diluted with diluent containing supplemental Se to give a final supplemental Se content of 1 ug/ml. Thus, three conditions with regard to Se supplementation were achieved: 1 pg Se/ml prior to freezing, 1 pg Se/ml upon thawing and no Se. All treatments were incubatad in quadruplicate prior to motility and viability determinations. Tw;,incubation conditions were employed: 4 h at 37'C in 95 percent air, 5 percent CO2 and 6 h at 20°C in air. Incubation was carried out in Falcon Micro Test Plates II, and semen was handled prior to counting in a laminar flow biological safety cabinet to prevent microbial contamination. Motility counts were performed as described for Experiment 1, and the 37'C cultures were allowed to rest at 20°C for 5 minutes or more before counting. Viability determinations were made using eosin bluish-fast green stain as described by Sorenson (14). Data in Experiment 2 were subjected to least squares analysis of variance (15) and, where appropriate, to linear regression. Results Experiment 1.
MAY
The addition of increasing concentrations of Se
1980 VOL. 13 NO. 5
359
THERIOGENOLOGY in diluted bull semen increased sperm motility at 24 h as compared with controls receiving no added Se. However, at Se doses greater than 1 ng/ml a reduction in motility appeared such that at 5 ug/ml the motility values were significantly lower than controls (65.6 C 32.4 vs 100.0 + 0.0, lOdf, Pc.01). The responses to both Se treatments(Se alone and Muse) fit quadratic equations with maxima at approximately 2 pg Se/ml. Since the experimental design used Se concentrations at l/2 log intervals, no data points were available between 1 ng Se/ml and 5 ng Se/ml except for one ejaculate. Therefore, data points for Se concentrations greater than 1 ng/ml were omitted and the curve best fitting the data was computed. Quadratic equations provided the highest coefficient of determination (R2 = -532 for Se alone, R2 = ,330 for Muse), and revealed maxima at approximately 0.65 ng Se/ml for both Se and Muse (figure 1). However, the maximum for the Se equation (Y = 92.87 + 188.52X - 143.18X2) was higher (Pc.02) than that for the Muse equation (Y = 101.97 + 131.50X - 103.86X2). Thus the presence of vitamin E in the Muse preparation was not beneficial to sperm motility as compared with Se alone, yet both treatments produced significant increases in the motility of bull spermatozoa -in vitro. Experiment 2. When bull sperm were treated with 1 ng Se/ml before freezing, upon thawing the sperm exhibited greater (Pc.05) motility than untreated semen (table II) but no differences in sperm viability were evident. Addition of Se after thawing did not significantly affect motility. Interactions among the following sources of variation were found to be statistically non-significant: Se treatment X ejaculate, Se treatment X bull, Se treatment X incubation condition, ejaculate X incubation condition and bull X ejaculate. There were differences among bulls with regard to motility after culture (table III) and these differences correspond generally to the semen quality of the bulls, i.e., bulls 5 and 6 were of high quality, while bulls 3 and 4 were of poor quality. Although the least square analysis of variance revealed no Se treatment X bull interaction, linear regression of the difference between the mean percent motility of semen treated with Se before freezing and the mean percent motility of control semen for each bull on the number of motile sperm per culture well demonstrated that the response to Se treatment was greatest in semen of bulls producing the greatest number of motile sperm (r = .87, Pc.05). This relationship is depicted in figure 2. This suggests that sperm from certain ,bulls were more competent to respond to Se supplementation than are sperm from other bulls. There was no relationship between the blood Se levels and response of sperm from the various bulls to supplemental Se in the diluent. However, the serum Se values were similar among the six bulls used in this study (.068 + .008 ng Se/ml, mean + S.E.M.), and it is possible that a relationship might be observed if bulls differing to a greater extent in Se status were used. Discussion
360
MAY
1980 VOL. 13 NO. 5
THERIOCENOLOGY
The data presented above indicate that Na2Se08 significantly stimulates motility of both freshly ejaculated, as well as frozen bull sperm, in concentrations up to about 1 vg Se/ml without affecting viability. Thus, Se promotes motility in sperm which would otherwise be non-motile, but still remain alive. Least squares analysis of variance revealed no bull X Se treatment interaction, while a positive correlation was observed between the response of sperm from the various bulls to Se and semen quality of the bulls (as measured by number of motile sperm per unit volume). This discrepancy appears to be related to the variation among ejaculates, since the interaction became evident only when the mean difference between treated and control sperm is used in the regression. Thus, sperm produced by certain bulls does not exhibit increased motility in the presence of Se, suggesting that such semen contains a smaller proportion of "marginal" sperm, which remain alive, but are non-motile. Another possibility is that the metabolic activity of sperm from some bulls is not responsive to Se. The data obtained in Experiment 1 might suggest an interference of vitamin E with the motility response to Se, since Se produced significantly greater stimulation than Muse. However, other possible explanations for this difference are: 1) Muse contains an emulsifying agent (polysorbate 80) and an antibiotic, and 2) it is likely to differ from the diluent in ionic strength, in viscosity, and other physical properties. Thus the smaller effect of Muse may be the result of factors other than vitamin E. A number of studies provide evidence tha;SSe is associated with spermatozoa. Brown and Burk,$16) found that Se0jm2 - treated r;;Tth Se was localized at the midpiece. produced sperm in which the et al. (7,8) found intravenously injected 75Se to be associated with -bovine seminal plasma and spermatozoa and the evidence indicated the possibility that Se may have a role in the spermatozoa as a structural component. Furthermore, Calvin (9) found 75Se incorporation into a polypeptide localized in a keratinous fraction of a sperm tail after intratesticular administration of 75Se0Sm2. Autoradiographic studies with spermatozoa from 75Se03-2 - treated rats (16), indicate an association of Se with the midpiece, and Calvin and Cooper (10) recently reported the association of Se with the keratinous outer membrane of sperm mitochondria. These observations are in accordance with the possibility that Se is involved in metabolic functions, a concept derived from the work of Whanger -et al. (17,18) in which a Se-binding protein from lamb muscle was observed to have properties similar to cytochromes c and bg. Although the data reported herein do not provide evidence for or against incorporation of Se into proteins of spermatozoa, they do support the concept that Se may be involved in metabolic activity of spermatozoa, since motility is increased. Recent additional data from this laboratory (19) provides direct evidence for such an involvement, since bull spermatozoa oxygen consump-
MAY
1980 VOL. 13 NO. 5
361
THERIOGENOLOGY tion was increased by addition of Se to the diluent. The authors are cognizant of the fact that motility evaluations of sperm do not provide the highest correlations with fertility. Saacke and White (20) demonstrated that percent intact acrosomes provides a higher correlation with fertility in bull sperm. Such determinations are beyond the expertise and facilities of the present investigators and were, therefore, not attempted. It is hoped that investigators having expertise, facilities and interest to perform more sophisticated evaluations of spermatozoa than those reported herein will further investigate the direct effects of Se on spermatozoa.
362
MAY 1980VOL. 13 NO. 5
THERIOGENOLOGY
Figure 1.
Figure 2.
MAY
Regression of sperm motility (expressed as a percent of the control) on Se content of the diluent.
Regression of mean increase in sperm (mean of Se-treated semen minus mean each bull) on number of motile sperm II well. Each well contained 200 ~1 represents a 20-fold dilution of the frozen.
1980VOL.13NO.5
motility due to Se of control semen for per Micro Test plate total volume; this semen as it had been
363
THERIOGENOLOGY Table I.
Stock Solution
Composition of Semen Diluent*
212.12 .g 68.00 g
Citric acid
50.00 g
Fructose
3642 ml
Extender
Tris-(hydroxymethyl)-aminomethane
Distilled, deionized Hz0
394 ml
Glycerine is then added to the above
200 ml
Egg yolk
800 ml
Stock solution
1.5x106 units
Penicillin G
3 ml
Linco-Spectin
1S
Streptomycin
5X1.05units
Polymyxin B
*Prepared by NOBA, Inc., Tiffin, Ohio.
364
MAY
1980VOL.13NO.5
THERIOGENOLOGY Table II.
Effect of Supplemental Selenium (1 ug/ml) on Post-Thaw Sperm Motility -in vitro
Treatment
N
Percent Motile (LSM+SEM)
Se before freezing
108
21.4 rt .0808a
Se after thawing
108
19.2 _c .08C18~'~
No supplemental Se
108
18.6 i .0808b
aab Treatments with different superscripts are significantly different from each other (P
Table III.
Differences in Sperm Motility Due to Bulls
Bull
N
Percent Motile Sperm (LSM f SEM)
1
54
19.8 f .15b'C'Y
2
54
20.1 f .18byc,y
3
54
16.5 f .18a,b,x
4
54
15.1 !z .lgasx
5
54
23.0 i .16c'y'x
6
54
24.0 f .18cJz
Means having different superscripts are significantly different from each other (abc, WO.01); (xyz, P
MAY
1980 VOL. 13 NO. 5
365
THERIOGENOLOGY
References
1. Hartley, W. J. and Grant, A. B. A review of selenium responsive diseases of New Zealand livestock. Fed. Proc. 20:679-688 (1961) 2. Julien, W. E., Conrad, H. R., Jones, J. .E., and Moxon, A. L. Selenium and vitamin E and incidence of retained placenta in parturient dairy cows. J. Dairy Sci. 59:1954-1959 (1976) 3. Julien, W. E., Conrad, H. R., and Moxon, A. L. Selenium and vitamin E and incidence of retained placenta in parturient dairy cows. II. Prevention in commercial herds with prepartum treatment. J. Dairy Sci. 59:1960-1962 (1976) 4. Segerson, E. C., Murray, F. A., Moxon, A. L., Redman, D. R., and Conrad, H. R. Selenium/Vitamin E: Role in fertilization of bovine ova. J. Dairy Sci. 60(6):1001-1005 (1977) 5. McCoy, K. E. M. and Weswig, P. H. Some selenium responses in the rat not related to vitamin E. J. Nutr. 98:383-389 (1969) 6. Wu, S. H., Oldfield, J. E., Whanger, P. D., and Weswig, P. H. Effect of selenium, vitamin E and anti-oxidants on testicular function in rats. Biol. Reprod. 8:625-629 (1973) 7. Smith, D. G., Senger, P. L., McCutchan, J. F., and Landa, C. F. Selenium and glutathione peroxidase distribution in bovine semen. Amer. Sot. Animal Sci. Annual Meeting. East Lansing, Mich. July 9-13. Abstract no. 435 (1978) 8. Smith, D. G. and Senger, P. L. Selenium retention by the tissues of the reproductive tract in the bull. Amer. Sot. Animal Sci. Annual Meeting. East Lansing, Michigan. July 9-13. Abstract no. 436 (1978) 9. Calvin, H. I. Selective incorporation of Selenium-75 into a polypeptide of the rat sperm tail. J. Exp. 2001. 204:445-452 (1978) 10. Calvin, H. I., and Cooper, G. W. A specific selenopolypeptide associated with the outer membrane of rat sperm mitochondria. In: The Spermatozoon (Fawcett, D. W. and Bedford, J. M., eds.). Urban and Schwarzenberg, Inc. Baltimore-Munich. pp 135-140 (1979) 11. Pallini, V. and Bacci, E. Bull sperm selenium is bound to a structural protein of mitochondria. J. Submicrosc. Cytol. 11:165-170 (1979) 12. Steele, G. D. and Torrie, J. H. Principles and Procedures of Statistics. McGraw-Hill Book Co., Inc. N.Y. (1960)
366
MAY
1980 VOL. 13 NO. 5
THERIOGENOLOGY
13.
Olson, 0. E., Palmer, I. S., and Cary, E. E. Modification of the official fluorometric method for selenium in plants. J. A.O.A.C. 58:117-121 (1975)
14. Sorenson, A. M. Reproduction Laboratory 2nd ed. Animal Science Department, Texas A and M University. Kendall/Hunt Publishing Co. Iowa (1973) 15. Harvey, W. K. Least squares analysis of data with unequal subclass numbers. USDA, ARS 20-8 (1960) 16. Brown, D. G. and Burk, R. F. Selenium retention in tissues and sperm of rats fed a torula yeast diet. J. Nutrition. 103:102-108 (1973) 17. Whanger, P. D., Pedersen, N. D., and Weswig, P. H. Characteristics of a selenium binding protein from lamb muscle. Fed. Proc. 31:691 (1972) 18. Whanger, P. D., Pedersen, N. D., and Weswig, P. H. Selenium proteins in ovine tissues II. Spectral properties of a 10,000 molecular weight selenium protein. Bioch. and Biophys. Res. Comm. 53(3):1031-1035 (1973) 19.
Pratt, .W. D., Murray, F. A., Moxon, A. L., Conrad, H. R., and Kinder, J. E. Effects of selenium supplementations on bull sperm metabolism -in vitro. Theriogenology (submitted 1980)
20. Saacke, R. G. and White, 3. M. Semen quality tests and their relationship to fertility. Proc. 4th Tech. Conf. Artif. Insem. and Reprod. Feb. 17-19. National Assoc. Animal Breeders, Inc. Columbia, Missouri. pp 22-27 (1972)
MAY
1980 VOL. 13 NO. 5
367