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The effect of ions and caffeine on the maintenance of motility of bovine spermatozoa diluted in egg yolk buffers
Animal Reproduction Science, 15 (1987) 161-168
161
Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands
The Effect of Ions and C a f f e i n e on the M a i n t e n a n c e of Motility of B o v i n e S p e r m a t o z o a D i l u t e d in E g g Yolk B u f f e r s P. SHANNON and B. CURSON
New Zealand Dairy Board, Private Bag, Hamilton (New Zealand) (Accepted 28 August 1987)
ABSTRACT Shannon, P. and Curson, B., 1987. The effect of ions and caffeine on the maintenance of motility of bovine spermatozoa diluted in egg yolk buffers. Anim. Reprod. Sci., 15: 161-168. The effect on the duration of bovine sperm motility resulting from the inclusion of Ca, Mg, and/or K ions, and caffeine in either whole or dialysed egg yolk diluents was tested in four trials. The effect of these ions and caffeine on the intensity of motility was determined in two trials. The duration of motility of sperm incubated at 37 ° C in a 5% dialysed egg yolk buffer with Na as the only metal ion (19.5 h), was significantly less ( P < 0.0001 ) than when sperm was diluted in 5% whole egg yolk buffer (48 h) or a 5% dialysed egg yolk buffer with Ca, Mg and K ions (49.7 h). When Ca, Mg and K ions were added singly to a 5% dialysed egg yolk buffer only K ions significantly increased survival at 37 °C (increase in survival 30.5 h, P < 0.001 ). The K ion concentration in 5% whole egg yolk buffer appeared to be close to optimum as addition of K ions to this diluent had no significant effect on duration of motility. There was no significant difference in motility scores between sperm suspended in whole egg yolk buffers or dialysed egg yolk buffers plus K +. Caffeine increased the motility score of sperm diluted in dialysed egg yolk buffers at 6 h of incubation and of sperm diluted in whole egg yolk or dialysed egg yolk with K ion buffers at 24 h of incubation (P < 0.001 ). It increased the duration of motility of sperm diluted in a dialysed egg yolk buffer by 16 h ( P < 0.001), but depressed the duration of motility when sperm were diluted in whole egg yolk or dialysed egg yolk buffers with K ions by 10 h (P < 0.001 ).
INTRODUCTION
Semen diluents for A.I. depend, for their success, on the inclusion of organic substances, such as egg yolk and milk, the properties of which are imperfectly understood. Egg yolk has been a common constituent of commercial semen diluents since the discovery of its protective action against cold shock ( Philips, 1939; Mann, 1964 ). Subsequently other beneficial properties of egg yolk have been reported, 0378-4320/87/$03.50
© 1987 Elsevier Science Publishers B.V.
162
namely its protective action against the toxic effects of bovine seminal plasma ( S h a n n o n and Curson, 1972 ) and its ability to sustain motility ( S h a n n o n et al., 1983). This latter property is more fully explored in this paper. S h a n n o n et al. (1983) observed that bovine spermatozoa, incubated at 37 ° C, in bovine serum albumin plus buffer containing Na as its only metal ion, became immotile after 1-4 h; at this point motility could be reinitiated by the addition of the diffusate of dialysed egg yolk. B a a s e t al. (1983) have reported a similar reactivation following the addition of the phosphodiesterase inhibitor, theophylline. The sustaining compound ( s ) in egg yolk is of low molecular weight. Three possible reasons for its action are: (i) the presence of an activator such as the sperm motility factor (Bavister, 1975; Bavister and Yanagimachi, 1977) or caffeine ( Garbers and Kopf, 1980 ) ; (ii) the provision of ions, other than those in the basic buffer, essential for motility ( M a n n and Lutwak Mann, 1981 ) ; or (iii) a combination of (i) and ( ii ). The principal objective of the trials reported herein was to determine which, if any, of these factors account for the motility-sustaining properties of egg yolk. Because egg yolk is a common constituent of commercial semen diluents, egg yolk, either whole or dialysed to remove all motility-sustaining compound ( s ), was included in all buffers. These were supplemented with Mg, Ca, and/or K ions, either singly or in combination, and caffeine. Duration of motility was measured at 3'7 ° C. As duration of motility depends not only on the presence of motility-sustaining compounds but also on viability of the sperm, the experiments indirectly measured whether other low molecular weight compounds that affect viability of the sperm are lost on dialysis. MATERIALS AND METHODS
Sperm. Semen was collected from bulls using an artificial vagina and ejaculates were evaluated for percentage motile sperm and concentration. Ejaculates containing less t h a n 60% live sperm were rejected. Dialysed egg yolk. The control diluent was 5% v/v egg yolk in 14GC 1 (Shannon, 1965). One hundred ml of the control diluent was ptaeed in a dialysis sac and dialysed for 48 h, at 5 °C, against thi"ee changes of 500 ml 14GC; the interval between changes was 12 h. In trial 3, semen from two bulls was diluted in dialysed egg yolk which had been dialysed for 12 h only without a change of dialysis fluid.
Buffers. The basic buffer was 14GC (Shannon, 1965) which contained Na + as its only metal ion. The additional ions and their molar concentration in the diluent were: Mg 2+ 2.2 X 10 -3 M as Mg SO4; Ca 2+ at 2.5 × ] 0-3 M as CaC12; ~Formula: 2% tri-sodium citrate (7 H20), 1% glycine, 1% glycerol, 0.025% caproic acid plus 1000 units penicillin and ] 00O milts streptomycin per ml.
i63 and K + at 3.5 × 10 .3 M as KeHP04. In one trial KeHP04 was compared with KC1 and Na2HP04, all three at 3.5× 10 -3 M. Trials. Four trials were conducted. These were designed to test: (i) the modifying effect of Mg, Ca, and K ions on the duration of motility of sperm suspended in a dialysed egg yolk diluent; (ii) which of the ions significantly affected the duration of motility; (iii) the effect of caffeine and ions on the duration of motility; and (iv) the effect of the substitution of KCI or Na2HPO4 for KzHPO4 on the duration of motility. Caffeine (0.1%) was included in all diluents in trials 1 and 2. In every trial each ejaculate was diluted in each of the experimental diluents, at 20 ° C, to a concentration of 10 × 106 total sperm/ml. Immediately after dilution 5 ml aliquots were placed in 10 ml test tubes, corked, and then transferred to an incubator at 37°C. Duration of motility was estimated by the method of Shannon (1965). Motility scores (i.e. velocity), were estimed microscopically at 37 ~C. Scoring was in the range of 0 to 9, at unit intervals, 0 representing no forward motility, I very slow progressive motility, and 9 very rapid forward motility. These estimates were made only in trials 3 and 4. Statistical methods. Data were analysed using analysis of variance techniques outlined by Snedecor and Cochran (1972).
RESULTS Trial 1
The average duration of motility in the three experimental diluents (n = 8) was: (i) whole egg yolk and caffeine, 48 h; (ii) dialysed egg yolk and caffeine 19.5 h; and (iii) diluent (ii) plus Mg, Ca, and K ions, 49.7 h. Duration of motility for sperm diluted in dialysed egg yolk was significantly less than for sperm suspended in either whole egg yolk or dialysed egg yolk with additional ions ( P < 0.0001). Duration of motility of sperm in these latter two diluents did not differ ( P > 0.25). Triai 2
Results from trial 2 (see Table 1) demonstrate the considerable effect of K ions on sustaining motility in semen suspended in dialysed egg yolk diluents. Duration of motility in the dialysed egg yolk diluent was 18.5 h and this was increased by between 30.5 and 34.5 h by K ions, independently of other ions ( P < 0.001 ). W h e n dialysed egg yolk included K ions it did not differ from the whole egg yolk diluent. The effect of Mg and Ca ions was not significant.
164 TABLE 1 T h e effect of various ions on the duration of motility ( h ) for sperm diluted in dialysed egg yolk plus caffeine ( n = 4) Added ions 0 Mg ~+ Ca 2+
Added ions 0
Mg 2+
Ca 2+
K+
Mg 2+ Ca 2+ K ÷
18.3 b
20.3 b
27.05 27.55
48.8 a 51.0 a 52.5 a
53.0 ~
Diagonals are c o m b i n a t i o n s of ions. abMeans with different superscripts are different ( P < 0.001 ). M e a n survival for sperm diluted in whole egg buffer with caffeine was 49.5 h.
Trial 3
Duration of motility results are shown in Table 2. Significant features of Table 2 are that while all main effects were significant, i.e. whole egg yolk was superior to dialysed egg yolk, the added cations increased and caffeine decreased duration of motility (P < 0.0001 ), these were modified by a number of interactions. Cations significantly increased the duration of motility only in dialysed egg yolk diluents (egg yolk type (i.e. whole or dialysed) × cations, P < 0.001 ). Duration of motility did not differ between sperm diluted in either whole or dialysed egg yolk buffers when the latter contained Mg, Ca, and K ions. Caffeine depressed duration of motility for all but the dialysed egg yolk treatment (egg yolk type × cations × caffeine, P < 0.025) in which treatment it increased the duration of motility. TABLE2 T h e effect of cations a n d caffeine on duration of motility ( h ) of sperm diluted in egg yolk diluents (n.=8) Caffeine (5)
Whole egg yolk
Dialysed egg yolk Ions added
0 0 0.1 Av. Av.
Mg 2+ Ca 2+ K +
73.8 52.5 63.2
73.8 53.4 63.6 63.4
0
M g 2+ Ca 2+ K +
26.4 28.4 27.4
76.3 51.5 63.9 45.7
Significant effects: (i) t r e a t m e n t s , egg yolk type (i.e. whole or dialysed), caffeine, cations ( P < 0.0001 ); (ii) interactions, egg yolk type × cations ( P < 0.0001 ) egg yolk type × caffeine X cations ( P < 0 . 0 2 5 ) .
165 TABLE3 Motility scores at 24 h of semen in diluents with a n d w i t h o u t caffeine ( n = 8 ) Whole egg yolk Caffeine ( % )
0 0.1 Av.
Dialysed egg yolk Cations added
0
Mg 2+ Ca z+ K +
M g 2+ Ca 2+ K +
Av.
5.3 6.9 6.1
5.5 7.3 6.4
4.8 6.5 5.6
5.2 6.9 6.0
Caffeine P < 0.001.
The 2-h advantage for caffeine in the latter treatment is probably an underestimate. Semen from two bulls was diluted in egg yolk dialysed for only 12 h. Duration of motility for sperm of these bulls was 56 h with no caffeine compared to 47 h with caffeine; for a further two bulls for first examination for motility was at 24 h and estimates of duration of motility for this treatment were subject to error. When these bulls were excluded the duration of motility was 14 h without caffeine and 24 h with caffeine. Motility score estimates presented some difficulties. Motility had ceased at 24 h in many of the nil cation dialysed egg yolk samples; likewise at 48 h motility had ceased in a number of caffeine whole egg yolk and caffeine dialysed egg yolk plus cation buffers. Comparative motility scores were therefore restricted to 6 h for the dialysed egg yolk nil cation samples and 24 h for all others. The motility score at 6 h for sperm in the nil electrolyte dialysed egg yolk buffers was ( n = 4 , 4 bulls omitted, see above), no caffeine 1.3, plus caffeine 6.5 ( P < 0.001 ). Motility scores for sperm in other buffers are shown in Table 3. There were no significant differences between motility scores for sperm suspended in different egg yolk buffers ( P = 0.12). However, caffeine did stimulate motility ( P < 0.001 ). Mean motility score for sperm suspended in diluents containing caffeine was 6.9 compared to 5.2 for those suspended in diluents without caffeine.
Trial 4 Initial analysis showed that phosphate ions had no effect on the duration of motility ( P = 0.95 ). Phosphate results were therefore absorbed into the appropriate metal ion treatment, being treated as replicates within treatment. Results of the trial are shown in Table 4. They are similar to those obtained in Trial 3; namely, caffeine depressed the duration of motility, by approximately 10 h, in all diluents except dialysed egg yolk, in which diluent it increased the duration of motility by 16 h (egg yolk t y p e × c a f f e i n e × K ions,
166 TABLE 4 The effec~ of K ~ cn duration of sperm motility (h) in egg yolk diluents (n = 8) Whole egg yolk Caffeine (%)
0 0.1 Av. Av.
Dialysed egg yolk Cations added
0
K+
0
K+
Av.
60 49.8 54.9
62.0 51.0 56.5
2.5 18.3 10.4
59.8 49.5 54.6
44.0 42.1 43,1
55.7
32.5
Significant effects: (i) treatments, egg yolk type, K +, caffeine (P < 0.0001 ) ; (ii) interactions, egg yolk type X K + ( P < 0.0001 ), e gg yolk type X caffeine X K + ( P < 0.01 ). TABLE5 Motility scores at 24 h of sperm in diluents with and without caffeine Whole egg yolk
Dialysed egg yolk Cations added
Caffeine ( % )
0 0.1 Av.
0
K+
K+
Av.
5.4 6.9 6.1
5.5 7.1 6.3
5.5 6.9 6.2
5.5 7.0 6.3
Significant: caffeine (P < 0.001 ).
P < 0.01); and K ions enhanced duration of motility only in the dialysed egg yolk buffer, in which diluent it increased the duration by 44 h (egg yolk type × K ions, P < 0.0001 ). The duration of motility of sperm suspended in dialysed egg yolk plus K ions did not differ significantly from that of sperm suspended in whole egg yolk buffers. Motility score at 6 h for semen diluted in dialysed egg yolk without caffeine was 0 compared to 6.3 when caffeine was present. Motility at 24 h for the other diluents is shown in Table 5. Caffeine significantly increased motility (P<0.001). No other differences were significant. DISCUSSION
The inclusion of K, but not Mg or Ca ions, in dialysed egg yolk buffers significantly extended the duration of sperm motility. Additional K ions had no significant effect in whole egg yolk buffers. Duration of motility, and motility score, of sperm diluted in dialysed egg yolk buffers, or whole egg yolk with or
167
without K ions, did not differ. These results show that the motility-sustaining property of egg yolk ( Shannon et al., 1983) is due to K ions. They negate the possibility that egg yolk contains low molecular weight compounds that increase viability or stimulate motility, such as sperm motility factors (Bavister, 1975), when freshly collected sperm are stored at 37°C. Further, the lack of difference between whole egg yolk and whole egg yolk plus K ions indicates that K ions are close to or exceed optimum threshold concentration in 5% whole egg yolk buffers. The importance of ions in the enzymatic processes that provide energy for motility is well documented ( Mann and Lutwak Mann, 1981 ). The necessity for such ions in the extracellular media will depend on exchange of these ions between the sperm cell and the media; and whether, as a result of this exchange, intracellular concentrations of the ions fall below critical levels for enzyme activity. Ca, Na and K ions are readily exchanged between the cell and the media: Ca ion uptake is controlled by a mitochondrial pump, and effiux by a plasma membrane pump (Bradley et al., 1979; Bradley and Forrester, 1980); Na and K ion translocation is governed by the Na- and K-activated Mg-dependent ATP-ase (Quinn and White, 1968; Petersen and Freund, 1975). Over the time-span of this experiment it is evident that any efflux of Mg or Ca ions was insufficient to reduce intracellular concentrations to critical levels. Reduction of intracellular concentrations of K ions to below critical levels was comparatively rapid. The loss of motility was probably due to reduced activity of the Na +/K + Mg 2+-dependent ATP-ase, inhibition of which has been shown to impair motility (Petersen and Freund, 1975 ). Caffeine initially stimulated motility of diluted sperm, which is in agreement with the results of Garbers and Kopf (1980). It increased the duration of motility of sperm diluted in dialysed egg yolk buffer, but depressed duration of motility in all other buffers. The improvement occasioned by caffeine in the dialysed egg yolk buffer is possibly due to its phosphodiesterase activity (Mann and Lutwak-Mann, 1981 ) allowing the accumulation of sufficient cyclic AMP to support motility at low ATP-ase activity. However, even with caffeine present, the duration of motility in the dialysed egg yolk buffer was considerably less than sperm diluted in buffers with K ions. In these buffers the reduction in duration of motility due to caffeine may be due to ultrastructural damage (Traub et al., 1982). We conclude that extracellular K ions are an essential diluent component when sperm are stored at 37 ° C. Whether K or other ions are essential, and at what concentration, at other storage temperatures remains to be determined. In this respect it is interesting to note that washed sperm stored for 8 days at ambient temperatures (16-20°C) became totally immotile but were reactivated by incubation at 37°C for several hours in an egg yolk buffer ( Shannon et al., 1983).
168 REFERENCES Baas, J.W., Molan, P.C. and Shannon, P., 1983. Factors in seminal plasma of bulls that affect the viability and motility of spermatozoa. J. Reprod. Fertil., 68: 275-280. Bavister, B.D., 1975. Properties of the sperm motility-stimulating component derived from human serum. J. Reprod. Fertil., 43: 363-366. Bavister, B.D. and Yanagimachi, R., 1977. The effects of sperm extracts and energy sources on the motility and acrosome reaction of hamster spermatozoa in vitro. Biol. Reprod., 16: 228-237. Bradley, M.P. and Forester, I.T., 1980. A [Ca 2+ + M g 2+ ] -ATP-ase and active Ca 2+ transport in the plasma membranes isolated from ram sperm flagella. Cell Calcium, 1: 381-390. Bradley, M.P., Van Eerton, M.T.W. and Forrester, I.T., 1979. The energy dependent uptake of Ca 2+ by mammalian spermatozoa. Proc. Univ. Otago Med. Sch., 57: 5-7. Garbers, D.L. and Kopf, G.S., 1980. The regulation of spermatozoa by calcium and cyclic nucleotides. Adv. Cyclic Nucleotide Res., 13: 261-306. Mann, T., 1964. The Biochemistry of Semen and of the Male Reproductive Tract. Methuen, London, 493 pp. Mann, T. and Lutwak Mann, C., 1981. Male Reproductive Function and Semen. Springer-Verlag, Berlin, Heidelberg and New York, 495 pp. Petersen, R.N. and Freund, M., 1975. Metabolism of human spermatozoa. In: E.S.E. Hafez (Editor), Human Sperm and Fertility Regulation in Men. Mosby, St. Louis, MO, pp. 176-186. Philips, P.H., 1939. The preservation of bull semen. J. Biol. Chem., 130: 415. Quinn, P.J. and White, I.G., 1968. Distribution of adenosinetriphosphatase in ram and bull spermatozoa. J. Reprod. Fertil., 15: 449-452. Shannon, P., 1965. Contribution of seminal plasma, sperm numbers, and gas phase to dilution effects of bovine spermatozoa. J. Dairy Sci., 48: 1357-1361. Shannon, P. and Curson, B., 1972. The effect of egg yolk levels on bovine seminal plasma toxins. Proc. 7th Int. Cong. Anim. Reprod. A.I., Munich, Vol. 4, pp. 1360-1362. Shannon, P., Curson, B., Pitt, C.J. and Lai, K.C., 1983. Effects of various fractions of egg yolk, BSA, and ovalbumin on sperm motility, and effect of storage on restoration of motility. N.Z.J. Agric. Res., 26: 297-302. Snedecor, G.W. and Cochran, W.G., 1972. Statistical Methods. Iowa State College Press, Ames, IA, 593 pp. Traub, A.I., Earnshaw, J.C., Brannigan, P.D. and Thompson, W., 1982. A critical assessment of the response to caffeine of human sperm motility. Fertil. Steril., 37: 436-437.
161
Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands
The Effect of Ions and C a f f e i n e on the M a i n t e n a n c e of Motility of B o v i n e S p e r m a t o z o a D i l u t e d in E g g Yolk B u f f e r s P. SHANNON and B. CURSON
New Zealand Dairy Board, Private Bag, Hamilton (New Zealand) (Accepted 28 August 1987)
ABSTRACT Shannon, P. and Curson, B., 1987. The effect of ions and caffeine on the maintenance of motility of bovine spermatozoa diluted in egg yolk buffers. Anim. Reprod. Sci., 15: 161-168. The effect on the duration of bovine sperm motility resulting from the inclusion of Ca, Mg, and/or K ions, and caffeine in either whole or dialysed egg yolk diluents was tested in four trials. The effect of these ions and caffeine on the intensity of motility was determined in two trials. The duration of motility of sperm incubated at 37 ° C in a 5% dialysed egg yolk buffer with Na as the only metal ion (19.5 h), was significantly less ( P < 0.0001 ) than when sperm was diluted in 5% whole egg yolk buffer (48 h) or a 5% dialysed egg yolk buffer with Ca, Mg and K ions (49.7 h). When Ca, Mg and K ions were added singly to a 5% dialysed egg yolk buffer only K ions significantly increased survival at 37 °C (increase in survival 30.5 h, P < 0.001 ). The K ion concentration in 5% whole egg yolk buffer appeared to be close to optimum as addition of K ions to this diluent had no significant effect on duration of motility. There was no significant difference in motility scores between sperm suspended in whole egg yolk buffers or dialysed egg yolk buffers plus K +. Caffeine increased the motility score of sperm diluted in dialysed egg yolk buffers at 6 h of incubation and of sperm diluted in whole egg yolk or dialysed egg yolk with K ion buffers at 24 h of incubation (P < 0.001 ). It increased the duration of motility of sperm diluted in a dialysed egg yolk buffer by 16 h ( P < 0.001), but depressed the duration of motility when sperm were diluted in whole egg yolk or dialysed egg yolk buffers with K ions by 10 h (P < 0.001 ).
INTRODUCTION
Semen diluents for A.I. depend, for their success, on the inclusion of organic substances, such as egg yolk and milk, the properties of which are imperfectly understood. Egg yolk has been a common constituent of commercial semen diluents since the discovery of its protective action against cold shock ( Philips, 1939; Mann, 1964 ). Subsequently other beneficial properties of egg yolk have been reported, 0378-4320/87/$03.50
© 1987 Elsevier Science Publishers B.V.
162
namely its protective action against the toxic effects of bovine seminal plasma ( S h a n n o n and Curson, 1972 ) and its ability to sustain motility ( S h a n n o n et al., 1983). This latter property is more fully explored in this paper. S h a n n o n et al. (1983) observed that bovine spermatozoa, incubated at 37 ° C, in bovine serum albumin plus buffer containing Na as its only metal ion, became immotile after 1-4 h; at this point motility could be reinitiated by the addition of the diffusate of dialysed egg yolk. B a a s e t al. (1983) have reported a similar reactivation following the addition of the phosphodiesterase inhibitor, theophylline. The sustaining compound ( s ) in egg yolk is of low molecular weight. Three possible reasons for its action are: (i) the presence of an activator such as the sperm motility factor (Bavister, 1975; Bavister and Yanagimachi, 1977) or caffeine ( Garbers and Kopf, 1980 ) ; (ii) the provision of ions, other than those in the basic buffer, essential for motility ( M a n n and Lutwak Mann, 1981 ) ; or (iii) a combination of (i) and ( ii ). The principal objective of the trials reported herein was to determine which, if any, of these factors account for the motility-sustaining properties of egg yolk. Because egg yolk is a common constituent of commercial semen diluents, egg yolk, either whole or dialysed to remove all motility-sustaining compound ( s ), was included in all buffers. These were supplemented with Mg, Ca, and/or K ions, either singly or in combination, and caffeine. Duration of motility was measured at 3'7 ° C. As duration of motility depends not only on the presence of motility-sustaining compounds but also on viability of the sperm, the experiments indirectly measured whether other low molecular weight compounds that affect viability of the sperm are lost on dialysis. MATERIALS AND METHODS
Sperm. Semen was collected from bulls using an artificial vagina and ejaculates were evaluated for percentage motile sperm and concentration. Ejaculates containing less t h a n 60% live sperm were rejected. Dialysed egg yolk. The control diluent was 5% v/v egg yolk in 14GC 1 (Shannon, 1965). One hundred ml of the control diluent was ptaeed in a dialysis sac and dialysed for 48 h, at 5 °C, against thi"ee changes of 500 ml 14GC; the interval between changes was 12 h. In trial 3, semen from two bulls was diluted in dialysed egg yolk which had been dialysed for 12 h only without a change of dialysis fluid.
Buffers. The basic buffer was 14GC (Shannon, 1965) which contained Na + as its only metal ion. The additional ions and their molar concentration in the diluent were: Mg 2+ 2.2 X 10 -3 M as Mg SO4; Ca 2+ at 2.5 × ] 0-3 M as CaC12; ~Formula: 2% tri-sodium citrate (7 H20), 1% glycine, 1% glycerol, 0.025% caproic acid plus 1000 units penicillin and ] 00O milts streptomycin per ml.
i63 and K + at 3.5 × 10 .3 M as KeHP04. In one trial KeHP04 was compared with KC1 and Na2HP04, all three at 3.5× 10 -3 M. Trials. Four trials were conducted. These were designed to test: (i) the modifying effect of Mg, Ca, and K ions on the duration of motility of sperm suspended in a dialysed egg yolk diluent; (ii) which of the ions significantly affected the duration of motility; (iii) the effect of caffeine and ions on the duration of motility; and (iv) the effect of the substitution of KCI or Na2HPO4 for KzHPO4 on the duration of motility. Caffeine (0.1%) was included in all diluents in trials 1 and 2. In every trial each ejaculate was diluted in each of the experimental diluents, at 20 ° C, to a concentration of 10 × 106 total sperm/ml. Immediately after dilution 5 ml aliquots were placed in 10 ml test tubes, corked, and then transferred to an incubator at 37°C. Duration of motility was estimated by the method of Shannon (1965). Motility scores (i.e. velocity), were estimed microscopically at 37 ~C. Scoring was in the range of 0 to 9, at unit intervals, 0 representing no forward motility, I very slow progressive motility, and 9 very rapid forward motility. These estimates were made only in trials 3 and 4. Statistical methods. Data were analysed using analysis of variance techniques outlined by Snedecor and Cochran (1972).
RESULTS Trial 1
The average duration of motility in the three experimental diluents (n = 8) was: (i) whole egg yolk and caffeine, 48 h; (ii) dialysed egg yolk and caffeine 19.5 h; and (iii) diluent (ii) plus Mg, Ca, and K ions, 49.7 h. Duration of motility for sperm diluted in dialysed egg yolk was significantly less than for sperm suspended in either whole egg yolk or dialysed egg yolk with additional ions ( P < 0.0001). Duration of motility of sperm in these latter two diluents did not differ ( P > 0.25). Triai 2
Results from trial 2 (see Table 1) demonstrate the considerable effect of K ions on sustaining motility in semen suspended in dialysed egg yolk diluents. Duration of motility in the dialysed egg yolk diluent was 18.5 h and this was increased by between 30.5 and 34.5 h by K ions, independently of other ions ( P < 0.001 ). W h e n dialysed egg yolk included K ions it did not differ from the whole egg yolk diluent. The effect of Mg and Ca ions was not significant.
164 TABLE 1 T h e effect of various ions on the duration of motility ( h ) for sperm diluted in dialysed egg yolk plus caffeine ( n = 4) Added ions 0 Mg ~+ Ca 2+
Added ions 0
Mg 2+
Ca 2+
K+
Mg 2+ Ca 2+ K ÷
18.3 b
20.3 b
27.05 27.55
48.8 a 51.0 a 52.5 a
53.0 ~
Diagonals are c o m b i n a t i o n s of ions. abMeans with different superscripts are different ( P < 0.001 ). M e a n survival for sperm diluted in whole egg buffer with caffeine was 49.5 h.
Trial 3
Duration of motility results are shown in Table 2. Significant features of Table 2 are that while all main effects were significant, i.e. whole egg yolk was superior to dialysed egg yolk, the added cations increased and caffeine decreased duration of motility (P < 0.0001 ), these were modified by a number of interactions. Cations significantly increased the duration of motility only in dialysed egg yolk diluents (egg yolk type (i.e. whole or dialysed) × cations, P < 0.001 ). Duration of motility did not differ between sperm diluted in either whole or dialysed egg yolk buffers when the latter contained Mg, Ca, and K ions. Caffeine depressed duration of motility for all but the dialysed egg yolk treatment (egg yolk type × cations × caffeine, P < 0.025) in which treatment it increased the duration of motility. TABLE2 T h e effect of cations a n d caffeine on duration of motility ( h ) of sperm diluted in egg yolk diluents (n.=8) Caffeine (5)
Whole egg yolk
Dialysed egg yolk Ions added
0 0 0.1 Av. Av.
Mg 2+ Ca 2+ K +
73.8 52.5 63.2
73.8 53.4 63.6 63.4
0
M g 2+ Ca 2+ K +
26.4 28.4 27.4
76.3 51.5 63.9 45.7
Significant effects: (i) t r e a t m e n t s , egg yolk type (i.e. whole or dialysed), caffeine, cations ( P < 0.0001 ); (ii) interactions, egg yolk type × cations ( P < 0.0001 ) egg yolk type × caffeine X cations ( P < 0 . 0 2 5 ) .
165 TABLE3 Motility scores at 24 h of semen in diluents with a n d w i t h o u t caffeine ( n = 8 ) Whole egg yolk Caffeine ( % )
0 0.1 Av.
Dialysed egg yolk Cations added
0
Mg 2+ Ca z+ K +
M g 2+ Ca 2+ K +
Av.
5.3 6.9 6.1
5.5 7.3 6.4
4.8 6.5 5.6
5.2 6.9 6.0
Caffeine P < 0.001.
The 2-h advantage for caffeine in the latter treatment is probably an underestimate. Semen from two bulls was diluted in egg yolk dialysed for only 12 h. Duration of motility for sperm of these bulls was 56 h with no caffeine compared to 47 h with caffeine; for a further two bulls for first examination for motility was at 24 h and estimates of duration of motility for this treatment were subject to error. When these bulls were excluded the duration of motility was 14 h without caffeine and 24 h with caffeine. Motility score estimates presented some difficulties. Motility had ceased at 24 h in many of the nil cation dialysed egg yolk samples; likewise at 48 h motility had ceased in a number of caffeine whole egg yolk and caffeine dialysed egg yolk plus cation buffers. Comparative motility scores were therefore restricted to 6 h for the dialysed egg yolk nil cation samples and 24 h for all others. The motility score at 6 h for sperm in the nil electrolyte dialysed egg yolk buffers was ( n = 4 , 4 bulls omitted, see above), no caffeine 1.3, plus caffeine 6.5 ( P < 0.001 ). Motility scores for sperm in other buffers are shown in Table 3. There were no significant differences between motility scores for sperm suspended in different egg yolk buffers ( P = 0.12). However, caffeine did stimulate motility ( P < 0.001 ). Mean motility score for sperm suspended in diluents containing caffeine was 6.9 compared to 5.2 for those suspended in diluents without caffeine.
Trial 4 Initial analysis showed that phosphate ions had no effect on the duration of motility ( P = 0.95 ). Phosphate results were therefore absorbed into the appropriate metal ion treatment, being treated as replicates within treatment. Results of the trial are shown in Table 4. They are similar to those obtained in Trial 3; namely, caffeine depressed the duration of motility, by approximately 10 h, in all diluents except dialysed egg yolk, in which diluent it increased the duration of motility by 16 h (egg yolk t y p e × c a f f e i n e × K ions,
166 TABLE 4 The effec~ of K ~ cn duration of sperm motility (h) in egg yolk diluents (n = 8) Whole egg yolk Caffeine (%)
0 0.1 Av. Av.
Dialysed egg yolk Cations added
0
K+
0
K+
Av.
60 49.8 54.9
62.0 51.0 56.5
2.5 18.3 10.4
59.8 49.5 54.6
44.0 42.1 43,1
55.7
32.5
Significant effects: (i) treatments, egg yolk type, K +, caffeine (P < 0.0001 ) ; (ii) interactions, egg yolk type X K + ( P < 0.0001 ), e gg yolk type X caffeine X K + ( P < 0.01 ). TABLE5 Motility scores at 24 h of sperm in diluents with and without caffeine Whole egg yolk
Dialysed egg yolk Cations added
Caffeine ( % )
0 0.1 Av.
0
K+
K+
Av.
5.4 6.9 6.1
5.5 7.1 6.3
5.5 6.9 6.2
5.5 7.0 6.3
Significant: caffeine (P < 0.001 ).
P < 0.01); and K ions enhanced duration of motility only in the dialysed egg yolk buffer, in which diluent it increased the duration by 44 h (egg yolk type × K ions, P < 0.0001 ). The duration of motility of sperm suspended in dialysed egg yolk plus K ions did not differ significantly from that of sperm suspended in whole egg yolk buffers. Motility score at 6 h for semen diluted in dialysed egg yolk without caffeine was 0 compared to 6.3 when caffeine was present. Motility at 24 h for the other diluents is shown in Table 5. Caffeine significantly increased motility (P<0.001). No other differences were significant. DISCUSSION
The inclusion of K, but not Mg or Ca ions, in dialysed egg yolk buffers significantly extended the duration of sperm motility. Additional K ions had no significant effect in whole egg yolk buffers. Duration of motility, and motility score, of sperm diluted in dialysed egg yolk buffers, or whole egg yolk with or
167
without K ions, did not differ. These results show that the motility-sustaining property of egg yolk ( Shannon et al., 1983) is due to K ions. They negate the possibility that egg yolk contains low molecular weight compounds that increase viability or stimulate motility, such as sperm motility factors (Bavister, 1975), when freshly collected sperm are stored at 37°C. Further, the lack of difference between whole egg yolk and whole egg yolk plus K ions indicates that K ions are close to or exceed optimum threshold concentration in 5% whole egg yolk buffers. The importance of ions in the enzymatic processes that provide energy for motility is well documented ( Mann and Lutwak Mann, 1981 ). The necessity for such ions in the extracellular media will depend on exchange of these ions between the sperm cell and the media; and whether, as a result of this exchange, intracellular concentrations of the ions fall below critical levels for enzyme activity. Ca, Na and K ions are readily exchanged between the cell and the media: Ca ion uptake is controlled by a mitochondrial pump, and effiux by a plasma membrane pump (Bradley et al., 1979; Bradley and Forrester, 1980); Na and K ion translocation is governed by the Na- and K-activated Mg-dependent ATP-ase (Quinn and White, 1968; Petersen and Freund, 1975). Over the time-span of this experiment it is evident that any efflux of Mg or Ca ions was insufficient to reduce intracellular concentrations to critical levels. Reduction of intracellular concentrations of K ions to below critical levels was comparatively rapid. The loss of motility was probably due to reduced activity of the Na +/K + Mg 2+-dependent ATP-ase, inhibition of which has been shown to impair motility (Petersen and Freund, 1975 ). Caffeine initially stimulated motility of diluted sperm, which is in agreement with the results of Garbers and Kopf (1980). It increased the duration of motility of sperm diluted in dialysed egg yolk buffer, but depressed duration of motility in all other buffers. The improvement occasioned by caffeine in the dialysed egg yolk buffer is possibly due to its phosphodiesterase activity (Mann and Lutwak-Mann, 1981 ) allowing the accumulation of sufficient cyclic AMP to support motility at low ATP-ase activity. However, even with caffeine present, the duration of motility in the dialysed egg yolk buffer was considerably less than sperm diluted in buffers with K ions. In these buffers the reduction in duration of motility due to caffeine may be due to ultrastructural damage (Traub et al., 1982). We conclude that extracellular K ions are an essential diluent component when sperm are stored at 37 ° C. Whether K or other ions are essential, and at what concentration, at other storage temperatures remains to be determined. In this respect it is interesting to note that washed sperm stored for 8 days at ambient temperatures (16-20°C) became totally immotile but were reactivated by incubation at 37°C for several hours in an egg yolk buffer ( Shannon et al., 1983).
168 REFERENCES Baas, J.W., Molan, P.C. and Shannon, P., 1983. Factors in seminal plasma of bulls that affect the viability and motility of spermatozoa. J. Reprod. Fertil., 68: 275-280. Bavister, B.D., 1975. Properties of the sperm motility-stimulating component derived from human serum. J. Reprod. Fertil., 43: 363-366. Bavister, B.D. and Yanagimachi, R., 1977. The effects of sperm extracts and energy sources on the motility and acrosome reaction of hamster spermatozoa in vitro. Biol. Reprod., 16: 228-237. Bradley, M.P. and Forester, I.T., 1980. A [Ca 2+ + M g 2+ ] -ATP-ase and active Ca 2+ transport in the plasma membranes isolated from ram sperm flagella. Cell Calcium, 1: 381-390. Bradley, M.P., Van Eerton, M.T.W. and Forrester, I.T., 1979. The energy dependent uptake of Ca 2+ by mammalian spermatozoa. Proc. Univ. Otago Med. Sch., 57: 5-7. Garbers, D.L. and Kopf, G.S., 1980. The regulation of spermatozoa by calcium and cyclic nucleotides. Adv. Cyclic Nucleotide Res., 13: 261-306. Mann, T., 1964. The Biochemistry of Semen and of the Male Reproductive Tract. Methuen, London, 493 pp. Mann, T. and Lutwak Mann, C., 1981. Male Reproductive Function and Semen. Springer-Verlag, Berlin, Heidelberg and New York, 495 pp. Petersen, R.N. and Freund, M., 1975. Metabolism of human spermatozoa. In: E.S.E. Hafez (Editor), Human Sperm and Fertility Regulation in Men. Mosby, St. Louis, MO, pp. 176-186. Philips, P.H., 1939. The preservation of bull semen. J. Biol. Chem., 130: 415. Quinn, P.J. and White, I.G., 1968. Distribution of adenosinetriphosphatase in ram and bull spermatozoa. J. Reprod. Fertil., 15: 449-452. Shannon, P., 1965. Contribution of seminal plasma, sperm numbers, and gas phase to dilution effects of bovine spermatozoa. J. Dairy Sci., 48: 1357-1361. Shannon, P. and Curson, B., 1972. The effect of egg yolk levels on bovine seminal plasma toxins. Proc. 7th Int. Cong. Anim. Reprod. A.I., Munich, Vol. 4, pp. 1360-1362. Shannon, P., Curson, B., Pitt, C.J. and Lai, K.C., 1983. Effects of various fractions of egg yolk, BSA, and ovalbumin on sperm motility, and effect of storage on restoration of motility. N.Z.J. Agric. Res., 26: 297-302. Snedecor, G.W. and Cochran, W.G., 1972. Statistical Methods. Iowa State College Press, Ames, IA, 593 pp. Traub, A.I., Earnshaw, J.C., Brannigan, P.D. and Thompson, W., 1982. A critical assessment of the response to caffeine of human sperm motility. Fertil. Steril., 37: 436-437.