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Effect of thaw rates on motility, survival and acrosomal integrity of buffalo spermatozoa frozen in medium French straws
Animal Reproduction Science, 12 (1986) 123--129 Elsevier Science Publishers B.V., Amsterdam -- Printed in The Netherlands
123
EFFECT OF THAW RATES ON MOTILITY, SURVIVAL AND ACROSOMAL INTEGRITY OF BUFFALO SPERMATOZOA FROZEN IN MEDIUM FRENCH STRAWS
A.V. NARASIMHA RAO, G.B. HARANATH, G. SOMA SEKHARAM ~ and J. RAMAMOHANA RAO
Indo-Swiss Project, Visakhapatnam, Andhra Pradesh (India) College of Veterinary Science, Tirupathi, Andhra Pradesh (India) (Accepted 21 January 1986)
ABSTRACT Rao, A.V.N., Haranath, G.B., Soma Sekharam, G. and Rao, J.R., 1986. Effect of thaw •rates on motility, survival and acrosomal integrity of buffalo spermatozoa frozen in medium French straws. Anita. Reprod. Sei~, 12: 123--129. The objective was to determine the effect of different thaw rates on motility, survival and acrosomal integrity of buffalo spermatozoa frozen in medium French straws. Sixteen ejaculates from four mature buffalo bulls of Murrah breed were tested in a 4 X 4 X 4 factorial combination. Semen was extended in Tris--egg yolk--glycerol extender, frozen in 0.5 ml polyvinyl chloride straws in liquid nitrogen vapour and stored in liquid nitrogen for 24 h. Straws were thawed at water bath temperatures of 30 °, 37 ° or 75°C for 30 s, 15 or 30 s, and 9 s respectively, Semen was incubated at 37°C for 6 h and evaluated at hourly intervals for percentage of motile spermatozoa (% MOT), percentage of total spermatozoa with intact acrosomes (PIA) and percentage of spermatozoa with intact, healthy acrosomes (PIHA) after 0 and 3 h of incubation. The initial post-thaw motility (0 h) averaged 66.9, 66.6, 72.1 and 64.6% for the four thaw rates respectively. Differences were significant between thaw rates for % MOT at 0 h (P ~ 0.05) and 1 h (P ~ 0.01) evaluation, post-thaw sperm survival at 37 ° C and absolute index of sperm survival. Bull~ also differed (P ~ 0.01) for % MOT at 1, 2, 3 and 4 h evaluation, post-thaw sperm survival at 37°C and absolute index of sperm survival. Significant (P ~ 0.01)interaction of thaw rate x bull for % MOT at 1 h evaluation was observed. Neither treatments nor bulls had any significant effect on PIA and PIHA after 0 and 3 h incubation. Thaw rate of 37 ° C for 30 s was comparatively superior to other rates studied.
INTRODUCTION T h e successful freezing of bull s e m e n b y Polge a n d R o w s o n ( 1 9 5 2 ) has given a new dimension to artificial insemination of cattle culminating in w i d e s p r e a d use o f f r o z e n s e m e n in t h e A.I. i n d u s t r y for rapid genetic improvement. This revolutionary biotechnique has since been extended to o t h e r d o m e s t i c s p e c i e s i n c l u d i n g t h e r i v e r i n e b u f f a l o (Bubalus bubalis), a predominant dairy animal in India.
0378-4320/86/$03.50
© 1986 Elsevier Science Publishers B.V.
124 Although the use o f frozen semen in A.I. programmes for buffaloes in the c o u n t r y is extensive, apparently it has n o t met with the same success as in cattle. The buffalo spermatozoa have certain distinctive features and among other things, the suitability o f the handling m e t h o d s devised for cattle semen has to be assessed. Thus there is a highly imperative need to evolve suitable semen freezing and handling techniques consistent with attainment of an acceptable level of fertility through A.I. in this species. Reports on deep freezing of buffalo semen using different extenders, glycerol levels, equilibration periods and freezing times are available (Rathore, 1965; R o y and Ansari, 1973; Roy, 1974; Fluekiger et al., 1976; Matharoo and Singh, 1980). A h m e d (1984) found that thaw rate of 75°C for 9 s was superior to 37°C for 15 s or 0°C for 2 min for spermatozoa of Nili-Ravi buffalo bulls extended in lactose--fructose--egg y o l k - g l y c e r o l extender. However, no information is available on the thaw rate of 37°C for 30 s which was reported to result in better conception rates in cattle and on the effect of various thawing procedures on acrosomal integrity of spermat o z o a of Murrah buffalo bulls extended in Tris--egg yolk--glycerol extender. The objective of the present work, therefore, was to study the comparative efficiency of four thawing procedures on post-thaw motility, survival and acrosomal integrity of spermatozoa of Murrah bulls extended in Tris-egg yolk--glycerol extender and deep frozen in m e d i u m French straws. MATERIALS AND METHODS F o u r healthy Murrah bulls of 6 to 9 years of age maintained as regular A.I. sires at the bull station of the Frozen Semen Bank, Indo-Swiss Project, Visakhapatnam (18°N, 83°E), India were used in the study. Semen was collected twice a w e e k using an artificial vagina (42--45°C) and each collection was preceded b y teasing in the form of two false mounts. After recording the physical characteristics (volume, colour, consistency and pH) the semen was evaluated for mass activity, percent motile spermatozoa and sperm concentration. F o u r ejaculates from each bull with at least 70% progressively motile spermatozoa and a minimum concentration of 1.0 × 109/mt were extended to contain 60 × 1 0 6 spermatozoa/ml in Tris--egg yolk--glycerol extender (Tris 2.42%, citric acid 1.36%, fructose 1%, glycerol 6.4%, egg yolk 20%, penicillin 500 i.u. and streptomycin 500 mg per ml of extender). This extender was earlier found to be superior to other extenders for preservation o f buffalo spermatozoa. Semen samples were extended in a water bath at 37°C, packaged in 0.5 ml French straws, sealed with polyvinyl chloride powder and immersed in water at 37°C. The straws were then transferred into a cooling cabinet (4°C) and equilibrated for 6 h. Freezing was done b y arranging t h e dried straws horizontally on a steel rack which was held in static liquid nitrogen vapour 3 cm above the level of liquid nitrogen for 8 min and then stored in liquid nitrogen for 24 h.
125 Straws were thawed by plunging at water bath temperatures o f 30, 37 or 75°C for 30 s, 15 s and 30 s, and 9 s, respectively. F o u r straws per treatment within each ejaculate were thawed and pooled in 12 X 75 m m test tubes and incubated in a water bath at 37°C. Each sample was evaluated for percentage of progressively motile spermatozoa (% MOT) immediately after thawing and at hourly intervals of incubation at 37°C until no sperm remained motile (post-thaw sperm survival at 37°C) in a phase contrast microscope fitted with a stage w a r m e r at 37°C. The percentage o f spermatozoa with intact acrosomes (PIA) and the percentage o f intact h e a l t h y acrosomes (PIHA) were estimated after 0 and 3 h of incubation from fixed smears stained with Giemsa stain. The PIA were the total sperm with intact apical ridges, while PIHA were the PIA w i t h o u t showing signs of acrosomal damage such as swollen and ruffled acrosomes, presence o f equatorial segment and loss of stainable material. Samples were evaluated by a single observer and were coded to minimise observer bias. Absolute index o f post-thaw sperm survival at 37°C was constructed using the formula o f Milovanof (1962) Ia = ~ ( T X R) Where:
Ia = absolute index o f survival T = time interval between motility evaluation and R = average o f every two consecutive motility evaluations. A randomized block design was used and the overall evaluation means of each variable were analysed after arc sine transformation by analysis of variance (Snedecor and Cochran, 1980) with thaw rates and bulls as fixed variables and bull replicates as random variables in 4 × 4 X 4 factorial arrangement, The thaw rate and bull means were compared by Duncan's multiple range test (1955). RESULTS The average seminal volume for 16 ejaculates from 4 bulls was 2.78 + 0.32 ml with 78.1 -+ 2 . 1 % motility, and 1.31 + 0.42 X 109 sperm/ml. The percentage of intact acrosomes and percentage of intact healthy acrosomes were 91.2 -+ 4.8 and 87.2 + 4.5 respectively. The prefreezing percentage of motility averaged 72.4 -+ 1.8, while the PIA and PIHA were 88.3 + 3.9 and 72.1 + 3.6 respectively. A s u m m a r y o f effects o f thaw rates on % MOT after initial thawing and at hourly intervals for 6 h o f incubation at 37°C, post-thaw sperm survival at 37°C, and absolute index of sperm survival is presented in Table 1 and on PIA and PIHA in Table 2. The highest rating o f each o f these measures was obtained by thawing straws at 37°C for 30 s. Thaw rates differed for % MOT at 0 h (P < 0.05), at 1 h evaluation (P < 0.01) and the overall mean (P < 0.05), post thaw sperm survival at 37°C (P < 0.01) and absolute index of sperm survival (P < 0.05). Differ-
126 TABLE 1 E f f e c t o f t h a w rates o n p e r c e n t a g e o f m o t i l e s p e r m a t o z o a , s p e r m survival at 37°C and a b s o l u t e i n d e x o f s p e r m survival o f b u f f a l o s p e r m a t o z o a f r o z e n in straws (16 ejaculates) Criteria
T h a w rates 30°C f o r 30 s
37°C f o r 15 s
% MOT after post-thaw incubation at 37°C for Oh lh 2h 3h 4h 5h 6h
66.9 a +- 1.5 55.6 ac 36.6 13.8 7.9 3.4 1.2
66.6 a 54,6 a 33.9 13.4 4.7 0.7 0.4
Overall m e a n
26.6 ab
24.9 a
Post-thaw sperm survival at 37°C (h) Absolute index of s p e r m survival
4.69 ab +- 0.62 152.0 ab
+
1.28
+
1.16
75°C f o r 9 s
72.1 b -+ 0.56 59.4 c 39.6 25.5 12.7 4.1 1.6
64.6 a -+ 1.12 49.1 a 35.9 19.1 7.6 2.0 1.4
30.7 b
4.06 a + 0.32 139.6 a
37°C for 30 s
+
0.82
5.38 b
25.7 a +
0.39
180.7 b + 1.11
4.06 a + 0.35 144.6 a + 2.28
a ' b M e a n s in t h e s a m e r o w w i t h d i f f e r e n t s u p e r s c r i p t s are d i f f e r e n t (P ~ 0.05). a'CMeans in t h e s a m e r o w w i t h d i f f e r e n t s u p e r s c r i p t s are d i f f e r e n t (P
Thaw rates 30°C f o r 30 s
37°C f o r 15 s
37°C for 30 s
75°C f o r 9 s
Percentage of intact acrosomes after postt h a w i n c u b a t i o n at 37°C f o r 0 h 3 h
78.0 a 68.8
79.0 73.6
82.8 77.1
77.7 74.6
Overall m e a n
73.4
76.3
79.9
76.2
Percentage of intact healthy acrosomes after post-thaw i n c u b a t i o n at 37 ° C f o r 0 h 3 h
58.6 55.7
61.3 55.4
59.1 57.5
58.1 54.0
Overall m e a n
57.1
58.4
58.3
56.1
aMeans in t h e s a m e r o w are n o n - s i g n i f i c a n t .
127
ences among bulls were highly significant (P < 0.01) for % MOT at 1, 2, 3, and 4th evaluation, post-thaw sperm survival and absolute index of sperm survival. Ejaculate differences were significant for % MOT at 0 h (P < 0.01) and at t h e 4th evaluation (P < 0.05), while significant ( P < 0 . 0 1 ) t h a w rate × bull interaction existed for % MOT at 1 h evaluation. The thawing o f spermatozoa at 37°C for 30 s resulted in higher % MOT at all evaluations, better post-thaw sperm survival at 37°C and absolute index of sperm survival, while 30°C for 30 s was found to be the next in order of merit. R e d u c t i o n o f motility immediately after thawing and after 1 h incubation was most evident when spermatozoa were t h a w e d at 75°C for 9 s. In contrast, effect of thaw rate o f 37°C for 15 s was latent in that the % MOT was lower at 4, 5 and 6 h evaluation. However the effect of thaw rates o f 30°C for 30 s, 37°C for 15 s and 75°C for 9 s on motility and survival o f spermatozoa was non-significant. The acrosomal integrity (percentage of spermatozoa with intact acrosomes after 0 and 3 h o f incubation at 37°C) was also higher for the thaw rate of 37°C for 30 s. The incidence o f acrosomal injuries following thawing varied from 16.3 to 21.6% for t h e different thaw rates. However, the effect of four thaw rates tested on either PIA or PIHA was non-significant. DISCUSSION
The motility scores obtained in this study at different post-thaw time evaluations were higher than those reported earlier for buffalo spermatozoa e x t e n d e d in Tris--egg yolk--glycerol extender (Matharoo and Singh, 1980; Tuli et al., 1981), probably due to higher average initial motility (78.1%) of the ejaculates selected for the study. O f the f o u r thawing procedures' tested, thaw rate o f 37°C for 30 s was f o u n d to be the best b o t h in respect of improved motility and survival, and acrosomal integrity as compared to 30°C for 30 s, or 37°C for 15 s or 75°C for 9 s, while the latter did n o t differ in any one o f these criteria. F o r cattle semen, fast thawing b y immersing straws in warm water (35--40°C) has been shown to be consistently ideal under field conditions. Almquist and Wiggin (1973) and Berndtson and Pickett (1981) have reported improved post-thaw motility, acrosomal integrity and fertility following fast thawing o f bovine spermatozoa packaged in plastic straws. In contrast, acrosomal integrity was lower for semen thawed at 5°C for 3 min and spermatozoal damage occurred on exposure to warm post-thaw temperatures (Senger et al., 1976). Sperm motility was improved b y increasing the thawing bath temperature from 5 to 55°C for bovine semen frozen in ampoules (Rodriguez et al., 1975) b u t no further increase of sperm motility resulted b y increasing thawing bath temperature above 55°C. Similar observations were made b y Jainudeen and Dass (1982) in swamp buffaloes with improved post-thaw motility and acrosomal retention b y increasing the thawing temperature from 5 to 50°C. Ahmed (1984) observed significantly higher motil-
128
ity and sperm survival for the buffalo semen thawed at 75°C for 9 s as compared to thaw rates o f either 30°C for 30 s or 0°C for 2 min. The results showing significantly improved motility and survival by increasing the thawing time from 15 s to 30 s at 37°C are consistent with those of Almquist et al. (1982) who f o u n d t h a t even durations of exposure o f spermatozoa at the same thawing temperature affected fertility rates of cattle. According to Brown et al. (1982) semen straws removed from the 35°C water bath after 12 s had an internal temperature of approximately --4°C, while straws removed after 1 min had reached 35°C. It is suggested t h a t the observed spermatozoal damage with shorter thawing time at the same temperature might be due to cold shock resulting from delay in complete thawing o f semen. De Abreu et al. (1979) reported t h a t post-thaw warming damage resulted when the temperature of semen was increased from 5 to 35°C following a rapid thaw. When semen is thawed very fast at 75°C for 9 s, post-thaw warming damage may occur. This is in contrast with the observation of A h m e d (1984) who reported best results by thawing buffalo spermatozoa at 75°C for 9 s. However, it is difficult to compare our results since the breed, extender and thawing procedures tested were different and the thaw rate o f 37°C for 30 s was n o t included in t h a t study. Therefore, it appears beneficial to buffalo spermatozoal viability to use longer thawing time i.e. higher temperature of semen (37°C for 30 s). Furthermore, FCrde and Gravir (1973), Almquist et al. (1979), Berndtson and Pickett (1981) and Gaillard and Kupferschmied (1982) f o u n d that conception rate was significantly higher for semen thawed at 35°C for 30 s than for semen thawed at the same temperature for 12 s. Gaillard and Kupferschmied (1982) and Almquist et al. (1982) have also shown t h a t semen warmed up to b o d y temperature was more resistant to different environments t h a t semen warmed up to 5°C by l i m i t a t i o n of the thawing time. Since our observations agree w i t h previous reports, it seems t h a t the behaviour patterns of buffalo and cattle spermatozoa are analogous during thawing of the deep frozen semen packaged in plastic straws. However, low fertility rates with frozen semen in water buffaloes m a y be due to factors other than fertilising ability of semen. The improved motility and survival of spermatozoa at thaw rate of 37°C for 30 s m a y result from minimal sperm losses either from cold shock or warm shock. REFERENCES Ahmed, K., 1984. Effect of t h a w rates on survival of buffalo spermatozoa frozen in straws. J. Dairy Sci., 67: 1535--1538. Almquist, J.O., Gruba, K.E. and Rosenberger, J.L., 1982. Effect of thawing time on fertility of bovine spermatozoa in French straws. J. Dairy Sci., 65 : 824--827. Almquist, J.O., Rosenberger, J.L. and Branas, R,J., 1979. Effect of thawing time in
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warm water on fertility of bovine spermatozoa in plastic straws. J. Dairy Sci., 62: 722--728. Almquist, J.O. and Wiggin, H.B., 1973. Survival of bull spermatozoa frozen and thawed by different methods in plastic straws. A.I. Digest, 21 : 12. Berndtson, W.E. and Pickett, B.V., 1981. Factors affecting fertility in an artificial insemination program for beef cattle. Colorado State Univ. Exp. Sta., Anita. Reprod. Lab., General Series, 999: 13. Brown, J.L., Senger, P.L. and Hillers, J.K., 1982. Influence of thawing time and postthaw temperature on acrosomal maintenance and motility of bovine spermatozoa frozen in 0.5 ml French straws. J. Anita. Sci., 54: 938--944. De Abreu, R.M., Berndtson, W.E., Smith, R.L. and Pickett, B.W., 1979. Effect of postthaw warming on viability of bovine spermatozoa thawed at different rates in French straws. J. Dairy Sci., 62: 1449--1454. Duncan, D.B., 1955. Multiple range and multiple F tests. Biometrics, 11 : 1. Fluekiger, A., Singh Sal, H. and Wieser, M.F., 1976. Deep freezing of buffalo bulls semen using four different diluents. VIII Int. Congr. Anita. Reprod. A.I., Kracow, IV: 798-801. F~rde, D. and Gravir, K., 1973. En ensartet metode for opptining av dypfrossen oksessed. Nor. Vet. Tidsskr., 85: 146--148. Gaillard, C. and Kupferschmied, H., 1982. Thawing time and non-return rate of bovine semen frozen in fine French straws. Theriogenology, 18: 487--495. Jainudeen, M.R. and Dass, S., 1982. Effect of level of glycerol, rate of freezing and thawing on survival of buffalo spermatozoa in.straws. In: Anita. Prod. and Health in the Tropics, pp. 409--411. Matharoo, J.S. and Singh, M., 1980. Revivability of buffalo spermatozoa after deep freezing the semen using various extenders. Zentralbl. Veterin~irmed. A., 27 : 385--391. Milovanof, V.K., 1962. In: Biology of Reproduction. 1st Edn. Publ. for Agric. Lit. J. Pamph. Moscow, 451 pp. Polge, C. and Rowson, L.E.A., 1952. Fertilizing capacity of bull spermatozoa after freezing at--79°C. Nature, 169: 626--627. Rathore, A.K., 1965. Effect of instantaneous deep freezing (--196°C) on survival of buffalo bull spermatozoa with two levels of glycerol. Indian J. Vet. Sci., 42: 680-685. Rodriguez, O.L., Berndtson, W.E., Ennen, D.B. and Pickett, B.W., 1975. Effect of rate of freezing, thawing and level of glycerol on the survival of bovine spermatozoa in straws. J. Anita. Sci., 41: 129--135. Roy, D.J., 1974. A new field technique for deep freezing of buffalo semen in Tupol. Indian Vet. J., 51 : 249--256. Roy, D.J. and Ansari, M.R., 1973. Studies on freezability of buffalo spermatozoa using two techniques. Indian J. Anita. Sci., 43: 1031--1033. Senger, P.L., Becker, W.C. and Hillers, J.K., 1976. Effect of thawing rate and post-thaw temperature on motility and acrosomal maintenance in bovine semen frozen in plastic straws. J. Anita. Sci., 42: 932--937. Snedecor, G.W. and Cochran, W.G., 1980. Statistical Methods. 7th Edn. Iowa State Univ. Press, Ames, IA, pp. 359--364. Tuli, R.K., Singh, M. and Matharoo, J.S., 1981. Effect of different equilibration times and extenders on deep freezing of buffalo semen. Theriogenology, 16: 99--104.
123
EFFECT OF THAW RATES ON MOTILITY, SURVIVAL AND ACROSOMAL INTEGRITY OF BUFFALO SPERMATOZOA FROZEN IN MEDIUM FRENCH STRAWS
A.V. NARASIMHA RAO, G.B. HARANATH, G. SOMA SEKHARAM ~ and J. RAMAMOHANA RAO
Indo-Swiss Project, Visakhapatnam, Andhra Pradesh (India) College of Veterinary Science, Tirupathi, Andhra Pradesh (India) (Accepted 21 January 1986)
ABSTRACT Rao, A.V.N., Haranath, G.B., Soma Sekharam, G. and Rao, J.R., 1986. Effect of thaw •rates on motility, survival and acrosomal integrity of buffalo spermatozoa frozen in medium French straws. Anita. Reprod. Sei~, 12: 123--129. The objective was to determine the effect of different thaw rates on motility, survival and acrosomal integrity of buffalo spermatozoa frozen in medium French straws. Sixteen ejaculates from four mature buffalo bulls of Murrah breed were tested in a 4 X 4 X 4 factorial combination. Semen was extended in Tris--egg yolk--glycerol extender, frozen in 0.5 ml polyvinyl chloride straws in liquid nitrogen vapour and stored in liquid nitrogen for 24 h. Straws were thawed at water bath temperatures of 30 °, 37 ° or 75°C for 30 s, 15 or 30 s, and 9 s respectively, Semen was incubated at 37°C for 6 h and evaluated at hourly intervals for percentage of motile spermatozoa (% MOT), percentage of total spermatozoa with intact acrosomes (PIA) and percentage of spermatozoa with intact, healthy acrosomes (PIHA) after 0 and 3 h of incubation. The initial post-thaw motility (0 h) averaged 66.9, 66.6, 72.1 and 64.6% for the four thaw rates respectively. Differences were significant between thaw rates for % MOT at 0 h (P ~ 0.05) and 1 h (P ~ 0.01) evaluation, post-thaw sperm survival at 37 ° C and absolute index of sperm survival. Bull~ also differed (P ~ 0.01) for % MOT at 1, 2, 3 and 4 h evaluation, post-thaw sperm survival at 37°C and absolute index of sperm survival. Significant (P ~ 0.01)interaction of thaw rate x bull for % MOT at 1 h evaluation was observed. Neither treatments nor bulls had any significant effect on PIA and PIHA after 0 and 3 h incubation. Thaw rate of 37 ° C for 30 s was comparatively superior to other rates studied.
INTRODUCTION T h e successful freezing of bull s e m e n b y Polge a n d R o w s o n ( 1 9 5 2 ) has given a new dimension to artificial insemination of cattle culminating in w i d e s p r e a d use o f f r o z e n s e m e n in t h e A.I. i n d u s t r y for rapid genetic improvement. This revolutionary biotechnique has since been extended to o t h e r d o m e s t i c s p e c i e s i n c l u d i n g t h e r i v e r i n e b u f f a l o (Bubalus bubalis), a predominant dairy animal in India.
0378-4320/86/$03.50
© 1986 Elsevier Science Publishers B.V.
124 Although the use o f frozen semen in A.I. programmes for buffaloes in the c o u n t r y is extensive, apparently it has n o t met with the same success as in cattle. The buffalo spermatozoa have certain distinctive features and among other things, the suitability o f the handling m e t h o d s devised for cattle semen has to be assessed. Thus there is a highly imperative need to evolve suitable semen freezing and handling techniques consistent with attainment of an acceptable level of fertility through A.I. in this species. Reports on deep freezing of buffalo semen using different extenders, glycerol levels, equilibration periods and freezing times are available (Rathore, 1965; R o y and Ansari, 1973; Roy, 1974; Fluekiger et al., 1976; Matharoo and Singh, 1980). A h m e d (1984) found that thaw rate of 75°C for 9 s was superior to 37°C for 15 s or 0°C for 2 min for spermatozoa of Nili-Ravi buffalo bulls extended in lactose--fructose--egg y o l k - g l y c e r o l extender. However, no information is available on the thaw rate of 37°C for 30 s which was reported to result in better conception rates in cattle and on the effect of various thawing procedures on acrosomal integrity of spermat o z o a of Murrah buffalo bulls extended in Tris--egg yolk--glycerol extender. The objective of the present work, therefore, was to study the comparative efficiency of four thawing procedures on post-thaw motility, survival and acrosomal integrity of spermatozoa of Murrah bulls extended in Tris-egg yolk--glycerol extender and deep frozen in m e d i u m French straws. MATERIALS AND METHODS F o u r healthy Murrah bulls of 6 to 9 years of age maintained as regular A.I. sires at the bull station of the Frozen Semen Bank, Indo-Swiss Project, Visakhapatnam (18°N, 83°E), India were used in the study. Semen was collected twice a w e e k using an artificial vagina (42--45°C) and each collection was preceded b y teasing in the form of two false mounts. After recording the physical characteristics (volume, colour, consistency and pH) the semen was evaluated for mass activity, percent motile spermatozoa and sperm concentration. F o u r ejaculates from each bull with at least 70% progressively motile spermatozoa and a minimum concentration of 1.0 × 109/mt were extended to contain 60 × 1 0 6 spermatozoa/ml in Tris--egg yolk--glycerol extender (Tris 2.42%, citric acid 1.36%, fructose 1%, glycerol 6.4%, egg yolk 20%, penicillin 500 i.u. and streptomycin 500 mg per ml of extender). This extender was earlier found to be superior to other extenders for preservation o f buffalo spermatozoa. Semen samples were extended in a water bath at 37°C, packaged in 0.5 ml French straws, sealed with polyvinyl chloride powder and immersed in water at 37°C. The straws were then transferred into a cooling cabinet (4°C) and equilibrated for 6 h. Freezing was done b y arranging t h e dried straws horizontally on a steel rack which was held in static liquid nitrogen vapour 3 cm above the level of liquid nitrogen for 8 min and then stored in liquid nitrogen for 24 h.
125 Straws were thawed by plunging at water bath temperatures o f 30, 37 or 75°C for 30 s, 15 s and 30 s, and 9 s, respectively. F o u r straws per treatment within each ejaculate were thawed and pooled in 12 X 75 m m test tubes and incubated in a water bath at 37°C. Each sample was evaluated for percentage of progressively motile spermatozoa (% MOT) immediately after thawing and at hourly intervals of incubation at 37°C until no sperm remained motile (post-thaw sperm survival at 37°C) in a phase contrast microscope fitted with a stage w a r m e r at 37°C. The percentage o f spermatozoa with intact acrosomes (PIA) and the percentage o f intact h e a l t h y acrosomes (PIHA) were estimated after 0 and 3 h of incubation from fixed smears stained with Giemsa stain. The PIA were the total sperm with intact apical ridges, while PIHA were the PIA w i t h o u t showing signs of acrosomal damage such as swollen and ruffled acrosomes, presence o f equatorial segment and loss of stainable material. Samples were evaluated by a single observer and were coded to minimise observer bias. Absolute index o f post-thaw sperm survival at 37°C was constructed using the formula o f Milovanof (1962) Ia = ~ ( T X R) Where:
Ia = absolute index o f survival T = time interval between motility evaluation and R = average o f every two consecutive motility evaluations. A randomized block design was used and the overall evaluation means of each variable were analysed after arc sine transformation by analysis of variance (Snedecor and Cochran, 1980) with thaw rates and bulls as fixed variables and bull replicates as random variables in 4 × 4 X 4 factorial arrangement, The thaw rate and bull means were compared by Duncan's multiple range test (1955). RESULTS The average seminal volume for 16 ejaculates from 4 bulls was 2.78 + 0.32 ml with 78.1 -+ 2 . 1 % motility, and 1.31 + 0.42 X 109 sperm/ml. The percentage of intact acrosomes and percentage of intact healthy acrosomes were 91.2 -+ 4.8 and 87.2 + 4.5 respectively. The prefreezing percentage of motility averaged 72.4 -+ 1.8, while the PIA and PIHA were 88.3 + 3.9 and 72.1 + 3.6 respectively. A s u m m a r y o f effects o f thaw rates on % MOT after initial thawing and at hourly intervals for 6 h o f incubation at 37°C, post-thaw sperm survival at 37°C, and absolute index of sperm survival is presented in Table 1 and on PIA and PIHA in Table 2. The highest rating o f each o f these measures was obtained by thawing straws at 37°C for 30 s. Thaw rates differed for % MOT at 0 h (P < 0.05), at 1 h evaluation (P < 0.01) and the overall mean (P < 0.05), post thaw sperm survival at 37°C (P < 0.01) and absolute index of sperm survival (P < 0.05). Differ-
126 TABLE 1 E f f e c t o f t h a w rates o n p e r c e n t a g e o f m o t i l e s p e r m a t o z o a , s p e r m survival at 37°C and a b s o l u t e i n d e x o f s p e r m survival o f b u f f a l o s p e r m a t o z o a f r o z e n in straws (16 ejaculates) Criteria
T h a w rates 30°C f o r 30 s
37°C f o r 15 s
% MOT after post-thaw incubation at 37°C for Oh lh 2h 3h 4h 5h 6h
66.9 a +- 1.5 55.6 ac 36.6 13.8 7.9 3.4 1.2
66.6 a 54,6 a 33.9 13.4 4.7 0.7 0.4
Overall m e a n
26.6 ab
24.9 a
Post-thaw sperm survival at 37°C (h) Absolute index of s p e r m survival
4.69 ab +- 0.62 152.0 ab
+
1.28
+
1.16
75°C f o r 9 s
72.1 b -+ 0.56 59.4 c 39.6 25.5 12.7 4.1 1.6
64.6 a -+ 1.12 49.1 a 35.9 19.1 7.6 2.0 1.4
30.7 b
4.06 a + 0.32 139.6 a
37°C for 30 s
+
0.82
5.38 b
25.7 a +
0.39
180.7 b + 1.11
4.06 a + 0.35 144.6 a + 2.28
a ' b M e a n s in t h e s a m e r o w w i t h d i f f e r e n t s u p e r s c r i p t s are d i f f e r e n t (P ~ 0.05). a'CMeans in t h e s a m e r o w w i t h d i f f e r e n t s u p e r s c r i p t s are d i f f e r e n t (P
Thaw rates 30°C f o r 30 s
37°C f o r 15 s
37°C for 30 s
75°C f o r 9 s
Percentage of intact acrosomes after postt h a w i n c u b a t i o n at 37°C f o r 0 h 3 h
78.0 a 68.8
79.0 73.6
82.8 77.1
77.7 74.6
Overall m e a n
73.4
76.3
79.9
76.2
Percentage of intact healthy acrosomes after post-thaw i n c u b a t i o n at 37 ° C f o r 0 h 3 h
58.6 55.7
61.3 55.4
59.1 57.5
58.1 54.0
Overall m e a n
57.1
58.4
58.3
56.1
aMeans in t h e s a m e r o w are n o n - s i g n i f i c a n t .
127
ences among bulls were highly significant (P < 0.01) for % MOT at 1, 2, 3, and 4th evaluation, post-thaw sperm survival and absolute index of sperm survival. Ejaculate differences were significant for % MOT at 0 h (P < 0.01) and at t h e 4th evaluation (P < 0.05), while significant ( P < 0 . 0 1 ) t h a w rate × bull interaction existed for % MOT at 1 h evaluation. The thawing o f spermatozoa at 37°C for 30 s resulted in higher % MOT at all evaluations, better post-thaw sperm survival at 37°C and absolute index of sperm survival, while 30°C for 30 s was found to be the next in order of merit. R e d u c t i o n o f motility immediately after thawing and after 1 h incubation was most evident when spermatozoa were t h a w e d at 75°C for 9 s. In contrast, effect of thaw rate o f 37°C for 15 s was latent in that the % MOT was lower at 4, 5 and 6 h evaluation. However the effect of thaw rates o f 30°C for 30 s, 37°C for 15 s and 75°C for 9 s on motility and survival o f spermatozoa was non-significant. The acrosomal integrity (percentage of spermatozoa with intact acrosomes after 0 and 3 h o f incubation at 37°C) was also higher for the thaw rate of 37°C for 30 s. The incidence o f acrosomal injuries following thawing varied from 16.3 to 21.6% for t h e different thaw rates. However, the effect of four thaw rates tested on either PIA or PIHA was non-significant. DISCUSSION
The motility scores obtained in this study at different post-thaw time evaluations were higher than those reported earlier for buffalo spermatozoa e x t e n d e d in Tris--egg yolk--glycerol extender (Matharoo and Singh, 1980; Tuli et al., 1981), probably due to higher average initial motility (78.1%) of the ejaculates selected for the study. O f the f o u r thawing procedures' tested, thaw rate o f 37°C for 30 s was f o u n d to be the best b o t h in respect of improved motility and survival, and acrosomal integrity as compared to 30°C for 30 s, or 37°C for 15 s or 75°C for 9 s, while the latter did n o t differ in any one o f these criteria. F o r cattle semen, fast thawing b y immersing straws in warm water (35--40°C) has been shown to be consistently ideal under field conditions. Almquist and Wiggin (1973) and Berndtson and Pickett (1981) have reported improved post-thaw motility, acrosomal integrity and fertility following fast thawing o f bovine spermatozoa packaged in plastic straws. In contrast, acrosomal integrity was lower for semen thawed at 5°C for 3 min and spermatozoal damage occurred on exposure to warm post-thaw temperatures (Senger et al., 1976). Sperm motility was improved b y increasing the thawing bath temperature from 5 to 55°C for bovine semen frozen in ampoules (Rodriguez et al., 1975) b u t no further increase of sperm motility resulted b y increasing thawing bath temperature above 55°C. Similar observations were made b y Jainudeen and Dass (1982) in swamp buffaloes with improved post-thaw motility and acrosomal retention b y increasing the thawing temperature from 5 to 50°C. Ahmed (1984) observed significantly higher motil-
128
ity and sperm survival for the buffalo semen thawed at 75°C for 9 s as compared to thaw rates o f either 30°C for 30 s or 0°C for 2 min. The results showing significantly improved motility and survival by increasing the thawing time from 15 s to 30 s at 37°C are consistent with those of Almquist et al. (1982) who f o u n d t h a t even durations of exposure o f spermatozoa at the same thawing temperature affected fertility rates of cattle. According to Brown et al. (1982) semen straws removed from the 35°C water bath after 12 s had an internal temperature of approximately --4°C, while straws removed after 1 min had reached 35°C. It is suggested t h a t the observed spermatozoal damage with shorter thawing time at the same temperature might be due to cold shock resulting from delay in complete thawing o f semen. De Abreu et al. (1979) reported t h a t post-thaw warming damage resulted when the temperature of semen was increased from 5 to 35°C following a rapid thaw. When semen is thawed very fast at 75°C for 9 s, post-thaw warming damage may occur. This is in contrast with the observation of A h m e d (1984) who reported best results by thawing buffalo spermatozoa at 75°C for 9 s. However, it is difficult to compare our results since the breed, extender and thawing procedures tested were different and the thaw rate o f 37°C for 30 s was n o t included in t h a t study. Therefore, it appears beneficial to buffalo spermatozoal viability to use longer thawing time i.e. higher temperature of semen (37°C for 30 s). Furthermore, FCrde and Gravir (1973), Almquist et al. (1979), Berndtson and Pickett (1981) and Gaillard and Kupferschmied (1982) f o u n d that conception rate was significantly higher for semen thawed at 35°C for 30 s than for semen thawed at the same temperature for 12 s. Gaillard and Kupferschmied (1982) and Almquist et al. (1982) have also shown t h a t semen warmed up to b o d y temperature was more resistant to different environments t h a t semen warmed up to 5°C by l i m i t a t i o n of the thawing time. Since our observations agree w i t h previous reports, it seems t h a t the behaviour patterns of buffalo and cattle spermatozoa are analogous during thawing of the deep frozen semen packaged in plastic straws. However, low fertility rates with frozen semen in water buffaloes m a y be due to factors other than fertilising ability of semen. The improved motility and survival of spermatozoa at thaw rate of 37°C for 30 s m a y result from minimal sperm losses either from cold shock or warm shock. REFERENCES Ahmed, K., 1984. Effect of t h a w rates on survival of buffalo spermatozoa frozen in straws. J. Dairy Sci., 67: 1535--1538. Almquist, J.O., Gruba, K.E. and Rosenberger, J.L., 1982. Effect of thawing time on fertility of bovine spermatozoa in French straws. J. Dairy Sci., 65 : 824--827. Almquist, J.O., Rosenberger, J.L. and Branas, R,J., 1979. Effect of thawing time in
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warm water on fertility of bovine spermatozoa in plastic straws. J. Dairy Sci., 62: 722--728. Almquist, J.O. and Wiggin, H.B., 1973. Survival of bull spermatozoa frozen and thawed by different methods in plastic straws. A.I. Digest, 21 : 12. Berndtson, W.E. and Pickett, B.V., 1981. Factors affecting fertility in an artificial insemination program for beef cattle. Colorado State Univ. Exp. Sta., Anita. Reprod. Lab., General Series, 999: 13. Brown, J.L., Senger, P.L. and Hillers, J.K., 1982. Influence of thawing time and postthaw temperature on acrosomal maintenance and motility of bovine spermatozoa frozen in 0.5 ml French straws. J. Anita. Sci., 54: 938--944. De Abreu, R.M., Berndtson, W.E., Smith, R.L. and Pickett, B.W., 1979. Effect of postthaw warming on viability of bovine spermatozoa thawed at different rates in French straws. J. Dairy Sci., 62: 1449--1454. Duncan, D.B., 1955. Multiple range and multiple F tests. Biometrics, 11 : 1. Fluekiger, A., Singh Sal, H. and Wieser, M.F., 1976. Deep freezing of buffalo bulls semen using four different diluents. VIII Int. Congr. Anita. Reprod. A.I., Kracow, IV: 798-801. F~rde, D. and Gravir, K., 1973. En ensartet metode for opptining av dypfrossen oksessed. Nor. Vet. Tidsskr., 85: 146--148. Gaillard, C. and Kupferschmied, H., 1982. Thawing time and non-return rate of bovine semen frozen in fine French straws. Theriogenology, 18: 487--495. Jainudeen, M.R. and Dass, S., 1982. Effect of level of glycerol, rate of freezing and thawing on survival of buffalo spermatozoa in.straws. In: Anita. Prod. and Health in the Tropics, pp. 409--411. Matharoo, J.S. and Singh, M., 1980. Revivability of buffalo spermatozoa after deep freezing the semen using various extenders. Zentralbl. Veterin~irmed. A., 27 : 385--391. Milovanof, V.K., 1962. In: Biology of Reproduction. 1st Edn. Publ. for Agric. Lit. J. Pamph. Moscow, 451 pp. Polge, C. and Rowson, L.E.A., 1952. Fertilizing capacity of bull spermatozoa after freezing at--79°C. Nature, 169: 626--627. Rathore, A.K., 1965. Effect of instantaneous deep freezing (--196°C) on survival of buffalo bull spermatozoa with two levels of glycerol. Indian J. Vet. Sci., 42: 680-685. Rodriguez, O.L., Berndtson, W.E., Ennen, D.B. and Pickett, B.W., 1975. Effect of rate of freezing, thawing and level of glycerol on the survival of bovine spermatozoa in straws. J. Anita. Sci., 41: 129--135. Roy, D.J., 1974. A new field technique for deep freezing of buffalo semen in Tupol. Indian Vet. J., 51 : 249--256. Roy, D.J. and Ansari, M.R., 1973. Studies on freezability of buffalo spermatozoa using two techniques. Indian J. Anita. Sci., 43: 1031--1033. Senger, P.L., Becker, W.C. and Hillers, J.K., 1976. Effect of thawing rate and post-thaw temperature on motility and acrosomal maintenance in bovine semen frozen in plastic straws. J. Anita. Sci., 42: 932--937. Snedecor, G.W. and Cochran, W.G., 1980. Statistical Methods. 7th Edn. Iowa State Univ. Press, Ames, IA, pp. 359--364. Tuli, R.K., Singh, M. and Matharoo, J.S., 1981. Effect of different equilibration times and extenders on deep freezing of buffalo semen. Theriogenology, 16: 99--104.