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The effects of egg yolk, the low density lipoprotein fraction of egg yolk, and three monosaccharides on the survival of cat (Felis catus) spermatozoa stored at 5°C
Animal Reproduction Science, 13 (1987) 229-237
229
Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands
The Effects of E g g Yolk, the L o w D e n s i t y L i p o p r o t e i n Fraction of E g g Yolk, and T h r e e M o n o s a c c h a r i d e s on the S u r v i v a l of Cat (Fells catus) S p e r m a t o z o a Stored at 5°C T.E. GLOVER and P.F. WATSON
Department of Physiology, Royal Veterinary College, London NW10TU (Great Britain) (Accepted 10 December 1986)
ABSTRACT Glover, T.E. and Watson, P.F., 1987. The effects of egg yolk, the low density lipoprotein fraction of egg yolk, and three monosaccharides on the survival of cat (Felis catus) spermatozoa stored at 5°C. Anim. Reprod. Sci., 13: 229-237. The survival of cat spermatozoa at 5 ° C was studied in the presence of egg yolk, the low density fraction (LDF) of egg yolk, glucose, fructose and galactose each made up to the desired concentration in 325 mOsm Tes-Tris buffer at pH 7.5. Increasing the egg yolk concentration (v/v) from 2 to 20% significantly decreased both the percentage of motile cells and the quality of motility score, but did not significantly increase the number of cells staining with a supravital stain. In contrast, increasing concentrations of LDF neither significantly decreased the percentage of motile cells nor increased the number of cells staining with a supravital stain, and significantly increased the motility score. However, the protection provided by the higher concentration of LDF (20%) was no more effective than that provided by the lower concentration of egg yolk (2%). It is contended that the currently high levels of egg yolk (20%) used in the preservation of cat spermatozoa may have contributed to the very poor fertility reported for preserved semen. The effect of glucose, fructose and galactose was examined at concentrations of 0.5 and 1.5 mg/ml. All three sugars significantly decreased the percentage of motile spermatozoa when used at the higher concentration, although for glucose alone the quality of motility was not significantly poorer than that in either the lower concentration or the control. In all experiments, motility declined significantly, and, where it was studied, the number of cells staining with a vital stain increased significantly, as a result of cooling and storage for up to 1 week. There were also significant differences between ejaculates in all experiments.
INTRODUCTION Egg yolk has been a common constituent of diluents used in the preservation of mammalian spermatozoa at low temperature since it was first shown to exert a protective effect on the motility and fertility of bull spermatozoa cooled to
0378-4320/87/$03.50
© 1987 Elsevier Science Publishers B.V.
230 10 °C (Phillips and Lardy, 1940). However, the effects of egg yolk are not all beneficial, either the survival and/or fertility of the spermatozoa of several species being reducedby increasing concentration ( b u l l - - S h a n n o n , 1972; ram Watson and Martin, 1976; brindled gnu, yak, American bison - - Watson, 1979; chicken - - Fewlass et al., 1975). Diluents used in the preservation of cat spermatozoa have been derived from those used for bull semen and include egg yolk as a substantial component (Sojka and Jennings, 1973; Platz et al., 1978). There have been no reports on the effect of egg yolk concentration on the survival of cat spermatozoa, nor on the effect of the isolated low density lipoprotein fraction (LDF) of egg yolk which has been shown in other species to possess the cryoprotective properties of whole egg yolk (Watson, 1976; Foulkes, 1977; Foulkes and Stewart, 1977). Sugars have been included in semen diluents as exogenous energy substrates, as osmotic components and as cryoprotective agents (Watson, 1979). Spermatozoa are capable of metabolizing glucose and fructose (Mann, 1964) which are thus potential energy sources. Galactose, on the other hand, although it has been shown to enter bull spermatozoa readily, is only slowly metabolized (Flipse, 1962 ), and might therefore be expected to provide little benefit. Lapwood and Martin (1966), however, studying the effects of sugars on ram spermatozoa, reported that galactose was beneficial especially during storage at 5 ° C, albeit at a relatively high concentration. This report concerns the effect of the concentration of egg yolk or LDF on the survival of cat spermatozoa stored at 5 ° C. Additionally, initial observations on the effect of low concentrations of the monosaccharides glucose, fructose and galactose are presented. MATERIALSAND METHODS The males were housed individually in a naturally lighted room and fed a commercial canned cat food with free access to water and litter as described previously (Glover et al., 1985). Semen was collected in an artificial vagina after the method of Sojka et al. (1970). Upon collection the sample was maintained at 37 °C and examined within 2 min for volume, colour and motility; only samples of volume > 25 #l, good colour and a progressive motility of 70% or greater were used for experiments. Samples were extended 1:3 (semen:diluent), cooled over 40 min to 5 ° C and stored for up to 1 week (Glover and Watson, 1985 ). All samples were assessed subjectively in random order on a warm stage at 37 ° C for the percentage of motile spermatozoa and a motility score (0-5 scale); supravital staining was performed with eosin/nigrosin (Hancock, 1957 ) and the percentage of stained cells counted. The low density lipoprotein fraction of egg yolk (LDF) was prepared as previously described (Watson, 1976) and was dispersed in buffer equivalent to its original concentration in egg yolk. The buffer was made by titrating 325
231 m M N-tris (hydroxymethyl) -methyl-2-aminomethane-sulphonic acid (Tes) against 325 m M tris (hydroxymethyl) aminomethane (Tris) to pH 7.4. Antibiotics, penicillin B (1000 i.u./ml) and streptomycin (1 m g / m l ) , were added to all diluents, and egg yolk and LDF were added at the stated concentrations (v/v). Egg yolk diluents were then centrifuged for 20 rain at 1500g and the supernatant was used for experiments. Sugars (fructose, glucose or galactose) were added during buffer preparation at concentrations of 0.5 or 1.5 mg/ml (2.7 or 8.3 mM, respectively). The experimental design involved a split ejaculate principle, all treatments being represented within a single ejaculate. A m i n i m u m of four males, selected randomly, was used for each experiment. Data were subjected to computerised analysis of variance after transformation of percentage data to angles; single degree of freedom contrasts were achieved by the use of orthogonal polynomial coefficients. RESULTS In the first experiment (Experiment 1A) the effect of egg yolk concentrations of 5, 10 and 20% (v/v) was examined on the survival of spermatozoa at 5 °C up to 1 week. The experiment was repeated (Experiment 1B) with egg yolk concentrations of 2, 5 and 8% (v/v). The results (Table 1 ) showed significant decreases in the percentage of motile spermatozoa and the motility score with increasing concentrations of egg yolk. Both cooling and storage caused a significant decline in both the percentage of motile spematozoa and the motility score, but only in Experiment 1B was the storage effect significant during the first 72 h of storage. By i week, however, both experiments revealed a significant loss of sperm motility. With the exception of the motility score in Experiment 1A, significant variability between ejaculates was observed in both experiments. The only significant interactions were between ejaculates and other factors in Experiment lB. In the second experiment, LDF concentrations of 5, 10 and 20% were considered. Increasing LDF concentrations (Table 2) caused no decline in the percentage of motile cells, and significantly improved the motility. There was a progressive loss of motility as a result of cooling and at all stages of storage. A significant interaction between LDF concentration and storage time was observed; there was a markedly lower percentage of motile spermatozoa in the 5 % LDF after storage for 1 week compared with that in the diluents containing 10 or 20% L D F . Significant variability between ejaculates was also observed. In Experiment 3 (Table 3 ) the effects were compared of two concentrations of egg yolk and LDF ( 2 and 20 % ) on motility ( Experiment 3A) and supravital staining (Experiment 3B). Egg yolk was significantly superior to LDF as judged by motility score; however, there was no significant difference between egg yolk and LDF for either the percentage of motile spermatozoa or number of cells
232 TABLE
1
The effect of egg yolk concentration on the survival of cat spermatozoa at 5 ° C ( m e a n values for five animals, one ejaculate per animal) Experiment 1A Motility
% Motile
Motility
-45.2 -39.2 37.8
-2.78 -2.92 3.10
46.1 41.8 39.9 ---
3.10 2.85 2.85 ---
54.7 40.8 41.5 39.7 26.9
3.60 2.97 2.93 2.83 2.33
60.3 49.0 46.2 35.9 21.6
3.96 3.38 3.13 2.71 1.50
31.9-48.2
2.69-3.06
39.8-47.3
2.67-3.17
% A.
Motile
Experiment 1B
Egg yolk concentration ( % ) 2 5 8 10 20
Cooling and storage at 5 ° C Before cooling After 24 h s t o r a g e After 4 8 h s t o r a g e After 72 h s t o r a g e After 168 I1 s t o r a g e C. Ejaculates Range of means B.
Summary of significant contrasts from the analyses of variance A. Egg yolk concentration 2 vs. 5 a n d 8 % 5 v s . 10 a n d 2 0 %
-P<0.0Ol
---
P<0.01 --
P<0.05 --
24 v s . 72 h 2 4 - 7 2 v s . 168 h C. E j a c u l a t e s
P < 0.001 -P<0.001 P < 0.01
P < 0.001 -P < 0.01 --
P < 0.001 P<0.001 P<0.001 P < 0.01
P < 0.001 P<0.01 P < 0.001 P < 0.05
AxC B×C
---
---
P<0.05 P<0.01
-P<0.05
B.
Storage time Control vs. cooled and stored
staining with a supravital stain. Both cooling and storage for 96 h significantly reduced the percentage of motile spermatozoa and the motility score, and significantly increased the number of cells staining with a supravital stain. Significant interactions were observed between additive and concentration and between concentration and storage time, reflecting the fact that the higher level of LDF (20%) provided greater support for spermatozoa during storage than the other treatments. Again, there were significant variations between ejaculates. The effects of the addition of any of three monosaccharides are shown in Table 4. With the exception of the motility score in the presence of glucose, both motility and the percentage of motile cells were significantly reduced by the addition to the diluent of 1.5 mg sugar/ml, the effect being more pronounced for fructose and galactose. A significant interaction between glucose concentration and storage time was caused by a decline in motility during storage only at the higher concentration of 1.5 mg/ml.
233 TABLE 2
The effect of the low density lipoprotein fraction of egg yolk (LDF) on the survival of cat spermatozoa at 5 °C (mean value for five animals, one ejaculate per animal)
A. L D F concentration (%) 5 10 20 B. Cooling and storage at 5 ° C
Before cooling After 24 h storage After 48 h storage After 72 h storage After 168 h storage C. Ejaculates Range of means
% Motile
Motility score
43.5 45.5 44.2
2.67 3.12 2.97
58.9 50.1 44.6 41.7 26.7
3.97 3.50 2.90 2.53 1.70
41.9-49.0
2.43-3.40
Summary of significant contrasts from the analyses of variance A. L D F concentation 5 vs. 10 and 20% " B. Storage time at 5 ° C
--
P<0.05
24 vs. 72 h 24-72 vs. 168 h C. Ejaculates
P < 0.001 P < 0.01 P<0.01 --
P < 0.001 P < 0.001 P<0.001 P < 0.01
AXB
P<0.05
--
Control vs. cooled and stored
DISCUSSION
Our primary interest lies in developing diluents appropriate to the shortterm storage of cat semen; the purpos e of this study was to examine the benefits to cat spermatozoa of several diluent components used in the preservation of semen of other species. Egg yolk did not increase the survival of cat spermatozoa at 5°C, unlike its effect on ram and bull spermatozoa (Watson, 1976). Indeed, it had an opposite effect, with a reduced motility resulting from an increased concentration of egg yolk, a phenomenon previously observed in several other species (Watson, 1979). The only beneficial effect of egg yolk was seen during storage in the presence of 20% egg yolk or LDF. The low density fraction includes the protective lipoproteins of egg yolk (Watson, 1976). It appeared to be better tolerated by cat spermatozoa, resulting in no loss of motility as the concentration in the diluent was increased to 20%. However, in a direct comparison with egg yolk, it yielded a survival no greater than that achieved with only 2% egg yolk. The toxicity of egg yolk has been attributed to the accumulation of hydrogen
234 TABL E 3 Effect of two concentrations of egg yolk and low density lipoprotein fraction on the survival (Expt. 3A) and supravital staining (Expt. 3B ) of cat spermatozoa stored at 5 °C (mean values for five animals, one ejaculate
per animal) Egg yolk % Motile
LDF Motility
%
% Motile
Motility
% Stained cells
Stained cells Additive
Cooling/storage
conc.
(%) 2
Before cooling 24 h storage 96 h storage
70.0 57.4 33.3 52.6
4.0 3.5 2.2 3.2
16.6 25.0 33.4 28.5
63.6 46.9 36.2 48.2
3.9 2.8 2.1 2.9
18.4 27.8 49.2 35.2
Before cooling 24 h storage 96 h storage
Mean
60.2 37.1 36.0 44.4
4.2 3.6 2.9 3.6
16.2 25.4 41.6 31.6
57.6 48.5 45.6 50.5
3.8 2.8 2.9 3.2
12.2 24.4 46.2 32.4
Overall mean
48.5
3.4
30.1
49.7
3.1
33.8
% Motile
Motility
Mean 20
%
Stained cells Ejaculates
Range of means
43;9-55.7
2.8-3.9
23.2-37.7
Summary of significant contrasts from analyses of variance A. Additive B. Concentration C. Storage time at 5 ° C Control vs. cooled and stored 24 vs. 96 h storage D. Ejaculates
---
P < 0.05 --
P < 0.001 P < 0.01 P<0.05
P < 0.001 P < 0.01 P<0.01
AXB
P<0.05
--
BXC
P<0.05
--
m
P < 0.001 P < 0.001 P<0.01
peroxide, a spermicidal product of metabolism of certain amino acids (Mann, 1964) or to itsenhancing the toxicity of dead spermatozoa (Shannon, 1972). In both instances, catalase, which destroys hydrogen peroxide, has been claimed to be of benefit (VanDemark et al., 1949; Shannon and Curson, 1972). VanDemark et al. (1949), however, found no significant benefit from the presence of catalase in diluents for semen at low dilution cooled to 5 ° C until after 4 days of storage. Preliminary trials using catalase under similar conditions failed to demonstrate any advantage for c a t spermatozoa stored over 3 days at 5°C.
235 TABLE 4
The effect o f the concentration of glucose, fructose and galactose on the motility of cat spermatozoa stored at 5 ° C (mean values, four (fructose and galactose) or six (glucose) animals, one ejaculate per animal) Glucose % Motile
A. Sugar concentration ( m g / m l ) 0 46.5 0.5 46.4 1.5 40.1 B. Cooling and storage at 5 ° C Before cooling 55.3 After 2 4 h s t o r a g e 47.2 After 48 h s t o r a g e 45.8 After 72 h s t o r a g e 41.3 After 168 h s t o r a g e 32.2 C. Ejaculates Range of means 37.7-51.0
Fructose
Galactose
Motility score
% Motile
Motility score
% Motile
Motility score
3,17 3.17 3,02
43.3 41.4 34.1
3.33 3.18 2.85
43.3 42.2 30.8
3.10 3.35 2.48
3.50 3.33 3.36 2.97 2.42
54.7 37.4 37.8 36.4 31.8
3.63 3.13 3,13 3,08 2,63
50.4 39.1 39.5 39.6 25.2
3.83 3.33 2.79 2.88 2.04
2.72-3.52
34.2-47.4
2,90-3.40
34.9-42.4
2.70-3.30
S u m m a r y o f significant contrasts from the analyses of variance A. Sugar concentration +vs.0.5 vs. 1.5 m g / m l
-P<0.05
---
P<0,01 P<0.01
P<0.01 P<0.01
P<0.05 P<0.01
-P<0.001
P < 0.001 P<0.001
P < 0.05 P<0.01
P<0.001 P<0.05
P < 0.001 P < 0.001
P < 0.001 P<0.001
P < 0.001 P<0.001
--
--
P<0.01
P<0.05
--
--
AXB
P<0.05
--
AxC
--
--
C. Storage time Fresh vs. stored 24, 48 a n d 72 h vs. 168 h C. E j a c u l a t e s
P<0.05
Egg yolk or LDF is usually included in semen diluents primarily to protect spermatozoa against cooling damage, being particularly valuable in those species which are highly susceptible to cold shock. Cat spermatozoa are less susceptible to this stress (Glover and Watson, 1985) and have, therefore, less need of egg yolk lipoproteins. In view of the observation that egg yolk adversely affects fertility (Shannon, 1972; Watson and Martin, 1976) it is possible that the poor fertility obtained previously with preserved cat spermatozoa (Sojka and Jennings, 1973; Platz et al., 1978) is partly due to the presence of high levels of egg yolk. The results with the monosaccharide sugars were surprising. The inclusion of glucose or fructose would have provided an exogenous energy substrate, but no advantage was evident over galactose, a non-metabolisable sugar. Cooled cat spermatozoa either have a limited metabolic activity and/or have sufficient endogenous energy substrates. The distinct reduction in motility with the higher
236 c o n c e n t r a t i o n o f all t h r e e s u g a r s s u g g e s t s a n o n - m e t a b o l i c effect. T h e c o n c e n t r a t i o n of m o n o s a c c h a r i d e u s e d (1.5 m g / m l ) w o u l d h a v e c o n t r i b u t e d less t h a n 10 m O s m / k g to t h e o s m o l a r i t y o f t h e d i l u e n t w h i c h is u n l i k e l y to h a v e c a u s e d t h e o b s e r v e d effects. A l t e r n a t i v e l y , t o x i n s r e s u l t i n g f r o m t h e p r o l i f e r a t i o n of a bacterial organism resistant to penicillin and streptomyciri might have been r e s p o n s i b l e , b u t it is u n l i k e l y t h a t s u c h a n o r g a n i s m w o u l d h a v e p r o l i f e r a t e d a t t h e s a m e r a t e in all t h r e e s u g a r s o l u t i o n s . M o r e o v e r , L a p w o o d a n d M a r t i n (1966) f o u n d in t h e i r e x a m i n a t i o n of a wide r a n g e o f s u g a r s for t h e p r e s e r v a t i o n o f r a m s p e r m a t o z o a t h a t t h e e f f e c t of a s u g a r w a s i n f l u e n c e d b y t h e t e m p e r a t u r e , b u t c o u l d offer n o s i m p l e e x p l a n a t i o n . P e r h a p s a s i m i l a r s i t u a t i o n exists w i t h c a t s p e r m a t o z o a ; w h e r e a s glucose a n d f r u c t o s e m a y be b e n e f i c i a l a t b o d y t e m p e r a t u r e , n o n e o f t h e t h r e e m o n o s a c c h a r i d e s offers a n y a d v a n t a g e d u r i n g s t o r a g e a t 5 ° C. I n t h e light o f t h e s e o b s e r v a t i o n s a s i m p l e T e s - T r i s b u f f e r w i t h o u t egg y o l k or s u g a r a p p e a r s to be a n a d e q u a t e e x t e n d e r for c a t s e m e n cooled to 5 ° C for s h o r t - t e r m storage. ACKNOWLEDGEMENTS W e w i s h to t h a n k M r s . A. A n d e r s o n a n d M r . I.T. B a i l e y for t h e i r e x p e r t a n i m a l care, a n d Mr. W . J . A n d e r s o n for his skilful l a b o r a t o r y a s s i s t a n c e .
REFERENCES Fewlass, T.A., Sexton, T.J. and Shaffner, C.S., 1975. Effect of various levels of egg yolk, milk, seminal plasma or blood serum on the respiration and reproductive efficiency of chicken spermatozoa. Poultry Sci., 54: 346-349. Flipse, R.J., 1962. Metabolism of bovine sperm: galactose as an indicator of hexose transport by bovine spermatozoa. J. Dairy Sci., 45: 1083-1086. Foulkes, J.A., 1977. Separation of lipoproteins from egg yolk and their effect on the motility and integrity of bovine spermatozoa. J. Reprod. Fertil., 49: 277-284. Foulkes, J.A. and Stewart, D.L., 1977. Fertility of dairy cattle after artificial insemination with semen frozen in a lipoprotein diluent. J. Reprod. Fertil., 51: 175-177. Glover, T.E. and Watson, P.F., 1985. Cold shock and its prevention by egg yolk in spermatozoa of the cat (Fells catus). Cryo-Letters, 6: 239-244. Glover, T.E., Watson, P.F. and Bonney, R.C., 1985. Observations on variability in LH release and fertility during oestrus in the domestic cat (Fells catus). J. Reprod. Fertil., 75: 145-152. Hancock, J.L., 1957. The morphology of boar spermatozoa. J. R. Microsc. Soc., 76: 84-97. Lapwood, K.R. and Martin, I.C.A., 1966. The use of monosaccharides, disaccharides and trisaccharides in synthetic diluents for the storage of ram spermatozoa at 37°C and 5°C. Aust. J. Biol. Sci., 19: 655-671. Mann, T., 1964. The Biochemistry of Semen and of the Male Reproductive Tract. 2nd Edn. Methuen, London, 493 pp. Phillips, P.H. and Lardy, H.A., 1940. A yolk-buffer pabulum for the preservation of bull sperm. J. Dairy Sci., 23: 399-404. Platz, C.C., Wildt, D.E. and Seager, S.W.J., 1978. Pregnancy in the domestic cat after artificial insemination with previously frozen spermatozoa. J. Reprod. Fertil., 52: 279-282.
237 Shannon, P., 1972. The effect of egg yolk level and dose rate on conception rate of semen diluted in Caprogen. In: Proceedings, VIIth International Congress on Animal Reproduction and Artificial Insemination (Munich), Vol. 2: 1440-1442. Shannon, P. and Curson, B., 1972. Toxic effect and action of dead sperm on diluted bovine semen. J. Dairy Sci., 55: 614-620. Sojka, N.J. and Jennings, L.L., 1973. Collection and storage of cat semen for artificial insemination. Va. J. Sci., 24: 166. Sojka, N.J., Jennings, L.L. and Hamner, C.E., 1970. Artificial insemination in the cat (Felis catus). Lab. Anim. Care, 20: 198-204. VanDemark, N.L., Salisbury, G.W. and Bratton, R,W., 1949. Oxygen damage to spermatozoa and its prevention by catalase. J. Dairy Sci., 32: 353-360. Watson, P.F., 1976. The protection of ram and bull spermatozoa by the low density lipoprotein fraction of egg yolk during storage at 5 °C and deep freezing. J. Thermal Biol., 1: 137-141. Watson, P.F., 1979. The preservation of semen in mammals. In: C.A. Finn (Editor), Oxford Reviews of Reproductive Biology, Vol. 1. Oxford University Press, Oxford, pp. 283-351. Watson, P.F. and Martin, I.C.A., 1976. Artificial inseminationof sheep: the effect of semen diluents containing egg yolk on the fertility of ram semen. Theriogenology, 6: 559-564.
229
Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands
The Effects of E g g Yolk, the L o w D e n s i t y L i p o p r o t e i n Fraction of E g g Yolk, and T h r e e M o n o s a c c h a r i d e s on the S u r v i v a l of Cat (Fells catus) S p e r m a t o z o a Stored at 5°C T.E. GLOVER and P.F. WATSON
Department of Physiology, Royal Veterinary College, London NW10TU (Great Britain) (Accepted 10 December 1986)
ABSTRACT Glover, T.E. and Watson, P.F., 1987. The effects of egg yolk, the low density lipoprotein fraction of egg yolk, and three monosaccharides on the survival of cat (Felis catus) spermatozoa stored at 5°C. Anim. Reprod. Sci., 13: 229-237. The survival of cat spermatozoa at 5 ° C was studied in the presence of egg yolk, the low density fraction (LDF) of egg yolk, glucose, fructose and galactose each made up to the desired concentration in 325 mOsm Tes-Tris buffer at pH 7.5. Increasing the egg yolk concentration (v/v) from 2 to 20% significantly decreased both the percentage of motile cells and the quality of motility score, but did not significantly increase the number of cells staining with a supravital stain. In contrast, increasing concentrations of LDF neither significantly decreased the percentage of motile cells nor increased the number of cells staining with a supravital stain, and significantly increased the motility score. However, the protection provided by the higher concentration of LDF (20%) was no more effective than that provided by the lower concentration of egg yolk (2%). It is contended that the currently high levels of egg yolk (20%) used in the preservation of cat spermatozoa may have contributed to the very poor fertility reported for preserved semen. The effect of glucose, fructose and galactose was examined at concentrations of 0.5 and 1.5 mg/ml. All three sugars significantly decreased the percentage of motile spermatozoa when used at the higher concentration, although for glucose alone the quality of motility was not significantly poorer than that in either the lower concentration or the control. In all experiments, motility declined significantly, and, where it was studied, the number of cells staining with a vital stain increased significantly, as a result of cooling and storage for up to 1 week. There were also significant differences between ejaculates in all experiments.
INTRODUCTION Egg yolk has been a common constituent of diluents used in the preservation of mammalian spermatozoa at low temperature since it was first shown to exert a protective effect on the motility and fertility of bull spermatozoa cooled to
0378-4320/87/$03.50
© 1987 Elsevier Science Publishers B.V.
230 10 °C (Phillips and Lardy, 1940). However, the effects of egg yolk are not all beneficial, either the survival and/or fertility of the spermatozoa of several species being reducedby increasing concentration ( b u l l - - S h a n n o n , 1972; ram Watson and Martin, 1976; brindled gnu, yak, American bison - - Watson, 1979; chicken - - Fewlass et al., 1975). Diluents used in the preservation of cat spermatozoa have been derived from those used for bull semen and include egg yolk as a substantial component (Sojka and Jennings, 1973; Platz et al., 1978). There have been no reports on the effect of egg yolk concentration on the survival of cat spermatozoa, nor on the effect of the isolated low density lipoprotein fraction (LDF) of egg yolk which has been shown in other species to possess the cryoprotective properties of whole egg yolk (Watson, 1976; Foulkes, 1977; Foulkes and Stewart, 1977). Sugars have been included in semen diluents as exogenous energy substrates, as osmotic components and as cryoprotective agents (Watson, 1979). Spermatozoa are capable of metabolizing glucose and fructose (Mann, 1964) which are thus potential energy sources. Galactose, on the other hand, although it has been shown to enter bull spermatozoa readily, is only slowly metabolized (Flipse, 1962 ), and might therefore be expected to provide little benefit. Lapwood and Martin (1966), however, studying the effects of sugars on ram spermatozoa, reported that galactose was beneficial especially during storage at 5 ° C, albeit at a relatively high concentration. This report concerns the effect of the concentration of egg yolk or LDF on the survival of cat spermatozoa stored at 5 ° C. Additionally, initial observations on the effect of low concentrations of the monosaccharides glucose, fructose and galactose are presented. MATERIALSAND METHODS The males were housed individually in a naturally lighted room and fed a commercial canned cat food with free access to water and litter as described previously (Glover et al., 1985). Semen was collected in an artificial vagina after the method of Sojka et al. (1970). Upon collection the sample was maintained at 37 °C and examined within 2 min for volume, colour and motility; only samples of volume > 25 #l, good colour and a progressive motility of 70% or greater were used for experiments. Samples were extended 1:3 (semen:diluent), cooled over 40 min to 5 ° C and stored for up to 1 week (Glover and Watson, 1985 ). All samples were assessed subjectively in random order on a warm stage at 37 ° C for the percentage of motile spermatozoa and a motility score (0-5 scale); supravital staining was performed with eosin/nigrosin (Hancock, 1957 ) and the percentage of stained cells counted. The low density lipoprotein fraction of egg yolk (LDF) was prepared as previously described (Watson, 1976) and was dispersed in buffer equivalent to its original concentration in egg yolk. The buffer was made by titrating 325
231 m M N-tris (hydroxymethyl) -methyl-2-aminomethane-sulphonic acid (Tes) against 325 m M tris (hydroxymethyl) aminomethane (Tris) to pH 7.4. Antibiotics, penicillin B (1000 i.u./ml) and streptomycin (1 m g / m l ) , were added to all diluents, and egg yolk and LDF were added at the stated concentrations (v/v). Egg yolk diluents were then centrifuged for 20 rain at 1500g and the supernatant was used for experiments. Sugars (fructose, glucose or galactose) were added during buffer preparation at concentrations of 0.5 or 1.5 mg/ml (2.7 or 8.3 mM, respectively). The experimental design involved a split ejaculate principle, all treatments being represented within a single ejaculate. A m i n i m u m of four males, selected randomly, was used for each experiment. Data were subjected to computerised analysis of variance after transformation of percentage data to angles; single degree of freedom contrasts were achieved by the use of orthogonal polynomial coefficients. RESULTS In the first experiment (Experiment 1A) the effect of egg yolk concentrations of 5, 10 and 20% (v/v) was examined on the survival of spermatozoa at 5 °C up to 1 week. The experiment was repeated (Experiment 1B) with egg yolk concentrations of 2, 5 and 8% (v/v). The results (Table 1 ) showed significant decreases in the percentage of motile spermatozoa and the motility score with increasing concentrations of egg yolk. Both cooling and storage caused a significant decline in both the percentage of motile spematozoa and the motility score, but only in Experiment 1B was the storage effect significant during the first 72 h of storage. By i week, however, both experiments revealed a significant loss of sperm motility. With the exception of the motility score in Experiment 1A, significant variability between ejaculates was observed in both experiments. The only significant interactions were between ejaculates and other factors in Experiment lB. In the second experiment, LDF concentrations of 5, 10 and 20% were considered. Increasing LDF concentrations (Table 2) caused no decline in the percentage of motile cells, and significantly improved the motility. There was a progressive loss of motility as a result of cooling and at all stages of storage. A significant interaction between LDF concentration and storage time was observed; there was a markedly lower percentage of motile spermatozoa in the 5 % LDF after storage for 1 week compared with that in the diluents containing 10 or 20% L D F . Significant variability between ejaculates was also observed. In Experiment 3 (Table 3 ) the effects were compared of two concentrations of egg yolk and LDF ( 2 and 20 % ) on motility ( Experiment 3A) and supravital staining (Experiment 3B). Egg yolk was significantly superior to LDF as judged by motility score; however, there was no significant difference between egg yolk and LDF for either the percentage of motile spermatozoa or number of cells
232 TABLE
1
The effect of egg yolk concentration on the survival of cat spermatozoa at 5 ° C ( m e a n values for five animals, one ejaculate per animal) Experiment 1A Motility
% Motile
Motility
-45.2 -39.2 37.8
-2.78 -2.92 3.10
46.1 41.8 39.9 ---
3.10 2.85 2.85 ---
54.7 40.8 41.5 39.7 26.9
3.60 2.97 2.93 2.83 2.33
60.3 49.0 46.2 35.9 21.6
3.96 3.38 3.13 2.71 1.50
31.9-48.2
2.69-3.06
39.8-47.3
2.67-3.17
% A.
Motile
Experiment 1B
Egg yolk concentration ( % ) 2 5 8 10 20
Cooling and storage at 5 ° C Before cooling After 24 h s t o r a g e After 4 8 h s t o r a g e After 72 h s t o r a g e After 168 I1 s t o r a g e C. Ejaculates Range of means B.
Summary of significant contrasts from the analyses of variance A. Egg yolk concentration 2 vs. 5 a n d 8 % 5 v s . 10 a n d 2 0 %
-P<0.0Ol
---
P<0.01 --
P<0.05 --
24 v s . 72 h 2 4 - 7 2 v s . 168 h C. E j a c u l a t e s
P < 0.001 -P<0.001 P < 0.01
P < 0.001 -P < 0.01 --
P < 0.001 P<0.001 P<0.001 P < 0.01
P < 0.001 P<0.01 P < 0.001 P < 0.05
AxC B×C
---
---
P<0.05 P<0.01
-P<0.05
B.
Storage time Control vs. cooled and stored
staining with a supravital stain. Both cooling and storage for 96 h significantly reduced the percentage of motile spermatozoa and the motility score, and significantly increased the number of cells staining with a supravital stain. Significant interactions were observed between additive and concentration and between concentration and storage time, reflecting the fact that the higher level of LDF (20%) provided greater support for spermatozoa during storage than the other treatments. Again, there were significant variations between ejaculates. The effects of the addition of any of three monosaccharides are shown in Table 4. With the exception of the motility score in the presence of glucose, both motility and the percentage of motile cells were significantly reduced by the addition to the diluent of 1.5 mg sugar/ml, the effect being more pronounced for fructose and galactose. A significant interaction between glucose concentration and storage time was caused by a decline in motility during storage only at the higher concentration of 1.5 mg/ml.
233 TABLE 2
The effect of the low density lipoprotein fraction of egg yolk (LDF) on the survival of cat spermatozoa at 5 °C (mean value for five animals, one ejaculate per animal)
A. L D F concentration (%) 5 10 20 B. Cooling and storage at 5 ° C
Before cooling After 24 h storage After 48 h storage After 72 h storage After 168 h storage C. Ejaculates Range of means
% Motile
Motility score
43.5 45.5 44.2
2.67 3.12 2.97
58.9 50.1 44.6 41.7 26.7
3.97 3.50 2.90 2.53 1.70
41.9-49.0
2.43-3.40
Summary of significant contrasts from the analyses of variance A. L D F concentation 5 vs. 10 and 20% " B. Storage time at 5 ° C
--
P<0.05
24 vs. 72 h 24-72 vs. 168 h C. Ejaculates
P < 0.001 P < 0.01 P<0.01 --
P < 0.001 P < 0.001 P<0.001 P < 0.01
AXB
P<0.05
--
Control vs. cooled and stored
DISCUSSION
Our primary interest lies in developing diluents appropriate to the shortterm storage of cat semen; the purpos e of this study was to examine the benefits to cat spermatozoa of several diluent components used in the preservation of semen of other species. Egg yolk did not increase the survival of cat spermatozoa at 5°C, unlike its effect on ram and bull spermatozoa (Watson, 1976). Indeed, it had an opposite effect, with a reduced motility resulting from an increased concentration of egg yolk, a phenomenon previously observed in several other species (Watson, 1979). The only beneficial effect of egg yolk was seen during storage in the presence of 20% egg yolk or LDF. The low density fraction includes the protective lipoproteins of egg yolk (Watson, 1976). It appeared to be better tolerated by cat spermatozoa, resulting in no loss of motility as the concentration in the diluent was increased to 20%. However, in a direct comparison with egg yolk, it yielded a survival no greater than that achieved with only 2% egg yolk. The toxicity of egg yolk has been attributed to the accumulation of hydrogen
234 TABL E 3 Effect of two concentrations of egg yolk and low density lipoprotein fraction on the survival (Expt. 3A) and supravital staining (Expt. 3B ) of cat spermatozoa stored at 5 °C (mean values for five animals, one ejaculate
per animal) Egg yolk % Motile
LDF Motility
%
% Motile
Motility
% Stained cells
Stained cells Additive
Cooling/storage
conc.
(%) 2
Before cooling 24 h storage 96 h storage
70.0 57.4 33.3 52.6
4.0 3.5 2.2 3.2
16.6 25.0 33.4 28.5
63.6 46.9 36.2 48.2
3.9 2.8 2.1 2.9
18.4 27.8 49.2 35.2
Before cooling 24 h storage 96 h storage
Mean
60.2 37.1 36.0 44.4
4.2 3.6 2.9 3.6
16.2 25.4 41.6 31.6
57.6 48.5 45.6 50.5
3.8 2.8 2.9 3.2
12.2 24.4 46.2 32.4
Overall mean
48.5
3.4
30.1
49.7
3.1
33.8
% Motile
Motility
Mean 20
%
Stained cells Ejaculates
Range of means
43;9-55.7
2.8-3.9
23.2-37.7
Summary of significant contrasts from analyses of variance A. Additive B. Concentration C. Storage time at 5 ° C Control vs. cooled and stored 24 vs. 96 h storage D. Ejaculates
---
P < 0.05 --
P < 0.001 P < 0.01 P<0.05
P < 0.001 P < 0.01 P<0.01
AXB
P<0.05
--
BXC
P<0.05
--
m
P < 0.001 P < 0.001 P<0.01
peroxide, a spermicidal product of metabolism of certain amino acids (Mann, 1964) or to itsenhancing the toxicity of dead spermatozoa (Shannon, 1972). In both instances, catalase, which destroys hydrogen peroxide, has been claimed to be of benefit (VanDemark et al., 1949; Shannon and Curson, 1972). VanDemark et al. (1949), however, found no significant benefit from the presence of catalase in diluents for semen at low dilution cooled to 5 ° C until after 4 days of storage. Preliminary trials using catalase under similar conditions failed to demonstrate any advantage for c a t spermatozoa stored over 3 days at 5°C.
235 TABLE 4
The effect o f the concentration of glucose, fructose and galactose on the motility of cat spermatozoa stored at 5 ° C (mean values, four (fructose and galactose) or six (glucose) animals, one ejaculate per animal) Glucose % Motile
A. Sugar concentration ( m g / m l ) 0 46.5 0.5 46.4 1.5 40.1 B. Cooling and storage at 5 ° C Before cooling 55.3 After 2 4 h s t o r a g e 47.2 After 48 h s t o r a g e 45.8 After 72 h s t o r a g e 41.3 After 168 h s t o r a g e 32.2 C. Ejaculates Range of means 37.7-51.0
Fructose
Galactose
Motility score
% Motile
Motility score
% Motile
Motility score
3,17 3.17 3,02
43.3 41.4 34.1
3.33 3.18 2.85
43.3 42.2 30.8
3.10 3.35 2.48
3.50 3.33 3.36 2.97 2.42
54.7 37.4 37.8 36.4 31.8
3.63 3.13 3,13 3,08 2,63
50.4 39.1 39.5 39.6 25.2
3.83 3.33 2.79 2.88 2.04
2.72-3.52
34.2-47.4
2,90-3.40
34.9-42.4
2.70-3.30
S u m m a r y o f significant contrasts from the analyses of variance A. Sugar concentration +vs.0.5 vs. 1.5 m g / m l
-P<0.05
---
P<0,01 P<0.01
P<0.01 P<0.01
P<0.05 P<0.01
-P<0.001
P < 0.001 P<0.001
P < 0.05 P<0.01
P<0.001 P<0.05
P < 0.001 P < 0.001
P < 0.001 P<0.001
P < 0.001 P<0.001
--
--
P<0.01
P<0.05
--
--
AXB
P<0.05
--
AxC
--
--
C. Storage time Fresh vs. stored 24, 48 a n d 72 h vs. 168 h C. E j a c u l a t e s
P<0.05
Egg yolk or LDF is usually included in semen diluents primarily to protect spermatozoa against cooling damage, being particularly valuable in those species which are highly susceptible to cold shock. Cat spermatozoa are less susceptible to this stress (Glover and Watson, 1985) and have, therefore, less need of egg yolk lipoproteins. In view of the observation that egg yolk adversely affects fertility (Shannon, 1972; Watson and Martin, 1976) it is possible that the poor fertility obtained previously with preserved cat spermatozoa (Sojka and Jennings, 1973; Platz et al., 1978) is partly due to the presence of high levels of egg yolk. The results with the monosaccharide sugars were surprising. The inclusion of glucose or fructose would have provided an exogenous energy substrate, but no advantage was evident over galactose, a non-metabolisable sugar. Cooled cat spermatozoa either have a limited metabolic activity and/or have sufficient endogenous energy substrates. The distinct reduction in motility with the higher
236 c o n c e n t r a t i o n o f all t h r e e s u g a r s s u g g e s t s a n o n - m e t a b o l i c effect. T h e c o n c e n t r a t i o n of m o n o s a c c h a r i d e u s e d (1.5 m g / m l ) w o u l d h a v e c o n t r i b u t e d less t h a n 10 m O s m / k g to t h e o s m o l a r i t y o f t h e d i l u e n t w h i c h is u n l i k e l y to h a v e c a u s e d t h e o b s e r v e d effects. A l t e r n a t i v e l y , t o x i n s r e s u l t i n g f r o m t h e p r o l i f e r a t i o n of a bacterial organism resistant to penicillin and streptomyciri might have been r e s p o n s i b l e , b u t it is u n l i k e l y t h a t s u c h a n o r g a n i s m w o u l d h a v e p r o l i f e r a t e d a t t h e s a m e r a t e in all t h r e e s u g a r s o l u t i o n s . M o r e o v e r , L a p w o o d a n d M a r t i n (1966) f o u n d in t h e i r e x a m i n a t i o n of a wide r a n g e o f s u g a r s for t h e p r e s e r v a t i o n o f r a m s p e r m a t o z o a t h a t t h e e f f e c t of a s u g a r w a s i n f l u e n c e d b y t h e t e m p e r a t u r e , b u t c o u l d offer n o s i m p l e e x p l a n a t i o n . P e r h a p s a s i m i l a r s i t u a t i o n exists w i t h c a t s p e r m a t o z o a ; w h e r e a s glucose a n d f r u c t o s e m a y be b e n e f i c i a l a t b o d y t e m p e r a t u r e , n o n e o f t h e t h r e e m o n o s a c c h a r i d e s offers a n y a d v a n t a g e d u r i n g s t o r a g e a t 5 ° C. I n t h e light o f t h e s e o b s e r v a t i o n s a s i m p l e T e s - T r i s b u f f e r w i t h o u t egg y o l k or s u g a r a p p e a r s to be a n a d e q u a t e e x t e n d e r for c a t s e m e n cooled to 5 ° C for s h o r t - t e r m storage. ACKNOWLEDGEMENTS W e w i s h to t h a n k M r s . A. A n d e r s o n a n d M r . I.T. B a i l e y for t h e i r e x p e r t a n i m a l care, a n d Mr. W . J . A n d e r s o n for his skilful l a b o r a t o r y a s s i s t a n c e .
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237 Shannon, P., 1972. The effect of egg yolk level and dose rate on conception rate of semen diluted in Caprogen. In: Proceedings, VIIth International Congress on Animal Reproduction and Artificial Insemination (Munich), Vol. 2: 1440-1442. Shannon, P. and Curson, B., 1972. Toxic effect and action of dead sperm on diluted bovine semen. J. Dairy Sci., 55: 614-620. Sojka, N.J. and Jennings, L.L., 1973. Collection and storage of cat semen for artificial insemination. Va. J. Sci., 24: 166. Sojka, N.J., Jennings, L.L. and Hamner, C.E., 1970. Artificial insemination in the cat (Felis catus). Lab. Anim. Care, 20: 198-204. VanDemark, N.L., Salisbury, G.W. and Bratton, R,W., 1949. Oxygen damage to spermatozoa and its prevention by catalase. J. Dairy Sci., 32: 353-360. Watson, P.F., 1976. The protection of ram and bull spermatozoa by the low density lipoprotein fraction of egg yolk during storage at 5 °C and deep freezing. J. Thermal Biol., 1: 137-141. Watson, P.F., 1979. The preservation of semen in mammals. In: C.A. Finn (Editor), Oxford Reviews of Reproductive Biology, Vol. 1. Oxford University Press, Oxford, pp. 283-351. Watson, P.F. and Martin, I.C.A., 1976. Artificial inseminationof sheep: the effect of semen diluents containing egg yolk on the fertility of ram semen. Theriogenology, 6: 559-564.