Zbi. Vet. Med. A, 23, 556-561
(1976) @ 1976 Verlag Paul Parey, Berlin und H a m b u r g
ISSN 0300-871 l/ASTM-Coden: Z V R A A X
Cattedra di Scienza eTecnica della Fecondazione Artificiale Istituto di Ostetricia e Ginecologia Veterinaria, Universitd degli Studi di Sassari Direclor: P r o f . U.Sbernardori
Effect of Different Semen Diluents on Survival of Ram Spermatozoa at 5 “C BY V. PETRUZZI, S. TARANTINI and P. N. ROYCHOUDHURY:~ W i t h one table (Received for publication August 29, 1975)
Introduction Difficulties in the preservation and deep-freezing of diluted ram semen show that ram spermatozoa are susceptible to the effects of different diluents and are very sensitive to the freezing techniques. The main aim of dilution is to reduce the sperm concentration so that an ejaculate can be more easily divided into a large number of doses. Dilution of ram semen is reported to be beneficial for storage in some cases but it may also result in lower conception rates. ANToNjAN and KAMALJAN (1970) reported that the conception rates of ewes inseminated with freshly diluted semen was 74 O / n to 40 O / o when the diluted semen had been stored a t O OC for 24-28 hours. (1939) has The egg yolk buffer mixture developed by LARDYand PHILLIPS been a satisfactory diluent for ram semen. Various improvements and changes have been made by many workers (AHMED,1955; ROY et al., 1956; PRASAD and NORMAN, 1969; TAMHAN and SINGH,1972; BRUCKNER and BAUER,1973). Milk diluents appear to be useful as a ram semen extender and have been widely used in different parts of the world (ISTVAN, 1956; FILIMON et al., 1956; HILLet al., 1958; BLACKSHAW, 1960; EMMENS and ROBINSON,1962; JONESand MARTIN,1965; SAXENAand SINGH, 1967; JONES,1968, 1969; TEWARI et al., 1968). More recent semen extenders like TRIS - and TEST buffers which are satisfactory for diluting and deep-freezing bull semen are still in an experimental stage for ram semen dilution (LAPWOOD and MARTIN, 1972; GRAHAM et al., 1973; SCHLEICHER and BRUCKNER, 1973). ‘) Istituto Sperimentale Iraliano “L. Spallanzani” per la Fecondazioe Artificiale Milano (Director: Prof. T. BONAUONNA). Present address: Senior Project Executive (A. H.), National Dairy Development Board, Anand, Gujarat, India.
Effect of Different Semen Diluents on Survival of Ram Spermatozoa a t 5 OC
557
Contributions on the dilution and storage of ram semen in various diluters at different temperatures (0 to 20 "C) and also the difficulties in deep(1974) freezing ram spermatozoa have been recently discussed by INSKEEP and BONADONNA (1974). Considering the short-term storage of ram semen and the increased susceptibility of spermatozoa to different semen diluents, the present work was undertaken to study the survival of ram spermatozoa at 5 O C in six different semen extenders.
Material and Methods Semen samples were collected by electrical stimulation from 6 adult rams of Sardinian breed. The semen diluents used were milk powder (PM), skim cow milk (SM), milk powder with egg-yolk( EYPM), skim cow milk with egg-yolk (EYSM), egg-yolk citrate (EYC) and Tris-citric acid-fructose (EYT) extenders. The milk powder diluter was a 10 O / o solution of the milk powder containing approximately 0.5-1.0 O / o fat. The skim milk used was homogenised and pasteurized cow milk containing approximately 1.8 O/o fat. The milk powder and the skim milk diluters were also used with 20 O/u egg yolk. The yolk-citrate extender was prepared according to SALISBURY et al. (194 1). The Tris (hydroxymethyl) aminomethane-yolk diluter was (1967). prepared according to STEINBACH and FOOTE After examining the initial sperm motility, the neat semen samples were diluted in two steps. The first dilution was made at 37 O C and then the Samples were brought to 20 OC a t the rate of 1 OC/min. The second dilution was completed at 20 O C . The dilution rate with each semen extender was 1 : 10. The diluted samples were preserved at 5 O C and the aliquots were examined at 4, 24, 48, 72, 96 and 120 hours of storage. Percent motile spermatozoa of each sample was examined microscopically by two workers and the mean value was noted. The data were subjected to statistical analysis according to SNEDECOR (1956). For the analysis of variance, the observations of the percent motile spermatozoa were converted to corresponding values by angular transformation.
Results and Discussion The mean percent motile spermatozoa with standard deviations are shown in Table I , which shows that the mean percent motile spermatozoa is higher in E Y T diluter than in the other five diluters. Not much difference in sperm motility was found between EYC diluter and the two milk diluters containing 20 O / o egg yolk. The mean percent motile spermatozoa in milk powder and skim milk diluters was slightly lower than the mean percent motile spermatozoa in EYPM and EYSM diluents. It was possible to preserve ram spermatozoa for 120 hours with 45 O / o and 26 O / o motility in EYT and EYC extenders respectively. In milk diluents containing egg yolk, the mean percent motile spermatozoa was almost equal (23 O / o ) , whereas no sperm motility was found after 120 hours in milk powder and skim milk extenders. The data were also analysed to find the differences in the mean percent motile spermatozoa between the different diluters a t the same period of storage. The results are shown in Table 1 with superscript letters. No significant difference was found in the mean percent motile spermatozoa between the six semen diluters after 4 hours of storage at 5 O C . The differences in the mean percent motile spermatozoa between EYT diluter and other semen diluters
558
PETRUZZI,
TARANTINI and KOYCHOUDHURY
after 48 hours of conservation were highly significant (P < 0.01). Whereas the differences in the mean percent motile spermatozoa between the milk powder or skim milk extenders each containing 20 O/o egg yolk and the EYC diluter were highly significant (P < 0.01) only at some periods of storage. There was not much difference in percent motile spermatozoa between milk powder and skim milk extenders except at 96 hours of storage (P < 0.01). SAXENA and SINGH(1967) used skim milk-yolk, egg yolk-glucose-bicarbonate, egg yolk-citrate and Cornell University Extender, each containing 'I different percentage of egg yolk for the dilution and preservation of ram spermatozoa. Differences due to diluent were significant at 72, 120 and 168 hours for motility and percentage live spermatzoa. Motility and percentage live spermatozoa were highest in skim milk-yolk followed by eggyolk-glucoseet al. (1968) reported ram sperm bicarbonate up to 168 hours storage. TEWAKI motility of 27.65 '. 2.0, 52.43 k 2.3, 64.22 f 2.0 and 72.34 i 2.2 at 6 hr storage (5-7 "C) in the four diluent rates (1 : 1, 1 : 3 , 1 : 5 and 1 : 10) respectively and 6.25 t- 1.2, 32.34 t- 2.5, 45.93 i. 2.8 and 54.53 i 2.7 at 30 hr storage in cow milk diluent. The differences between dilution rates as well as between storage intervals were highly significant. In the present study the mean percent motile spermatozoa in the skim cow milk diluent with and without egg yolk was found to be 55.22 f 12.70 and 45.74 t- 15.36 respectively at 48 hr storage in the dilution rate of 1 : 10. JONES et al. (1969) reported only 8.8 O / o fertility with ram spermatozoa diluted (1 : 10) with skim milk and stored at 5 "C for approximately 36 hours. The ability of four extenders (egg yolk-citrate, egg yolk-Tris, Cornell University Extender (CU-16) and Coconut Milk Extender) to maintain ram spermatozoa at variable temperatures (5 OC, 24 "C-34 "C) was rated as follows: CME > CU-16 > EYT > EYC (PRASAD and NORMAN,1969). SAHNIand ROY (1969) observed the highest sperm motility when diluted ram spermatozoa was preserved at 5 O C in Cornell University Extender (CUE) and cow milk, followed by milk and milk-containing diluents which were significantly superior to all egg yolk-containing diluents. The results of this experiment did not agree with the findings of SAHNIand ROY(loc. cit); in this study addition of egg yolk in the milk diluents improved the mean percent motile spermatozoa (Table). Table I Hours of conservation at 5OC
4 Hours
1 Percent
I
motile spermatozoa in different semen diluters (Dilution rate 1 : 10)
Powder milk
(PM)
1
Skim milk (SM)
I
Powder milk + ZpETPY ;
I
Skim milk
Y$s;+2;
Na Citrate ldilu;gY,,
I
Tris Diluter
(EYT)
66.85r 7.35N.S.64.64?: 10.77N.S,66.66r 8.52N.5. 66.00t 8.36N.5.69.11 f 7.9gN.'. 70.442 7.21N.'. (27) (27) (45) 145) (45) 145)
I
1
Effect of Different Semen Diluents on Survival of Ram Spermatozoa at 5 'C
559
DESSOUKY et al. (1970) found that egg-yolk-citrate gave the best results in terms of motility, followed by egg-yolk-citrate-glycerol and egg-yolkand SINGH(1972) reported no significant differences in skim milk. TAMHAN sperm motility and percentage of live spermatozoa in ejaculates diluted with yolk-citrate, yolk-citrate-glucose-glycine, skim milk-yolk-glucose and yolktomato juice extenders and stored at 5 O C , 10 O C , 15 OC or 25 OC for 0, 24, 48, 72, 96, 120 or 144 hrs. A storage temperature of 25 O C proved unsatisfactory for all diluents. In the German Democratic Republic A.I. of ewes is practised using glucose-egg yolk-citrate extender and storing the diluted semen up to 24 hrs a t 3-4 "C (BRUCKNER and BAUER,1973). The results of the present experiment thus agree with those findings of some workers in maintaining sperm motility for different periods at 5 O C in milk diluents, milkyolk diluents and yolk-citrate diluent. Contributions on the use of Tris-buffer as ram semen diluent are limited. SCHLEICHER and BRUCKNER (1973) preserved ram semen at 2-4 OC for up to 216 hrs in egg yolk-citrate, egg yolk-phosphate or Tris-buffer solutions. Under the electron microscope fewer structurally altered spermatozoa were reported after 72 hrs in Tris diluent than after 24 hrs in either of the other two diluents. Recently KOUTSOURIS and VAUPEL(1973) used a Tris diluent and a modified raffinose-yolk-glycerol diluent for deep-freezing ram spermatozoa and reported that the type of diluent had no significant effect on forward motility after thawing. As is evident from our experiment (Table l), higher mean percent motile spermatzoa were found in Tris extender when ram semen was diluted (1 : 10) and stored at 5 "C. Percent motile spermatozoa in EYT, EYC, EYSM and EYPM diluents were 45.00, 26.00, 23.33 and 23.33 O/o, respectively after 120 hours of preservation at 5 O C . It was not possible to preserve ram spermatozoa for 120 hrs in skim milk and powder milk diluents without egg yolk. Based on the results of mean percent motile spermatozoa, the ability of the six semen extenders to preserve ram spermatozoa at 5 O C was thus rated as follows: EYT > EYC > EYSM > EYPM > SM > PM. Summary Semen was collected by electrical stimulation from 6 adult Sardinian rams. Semen samples were diluted with 10 O/o milk powder solution (PM), pasteurized skim cow milk (SM), powder milk with 20 O / o egg yolk (EYPM), skim milk with 20 O/o egg yolk (EYSM), egg yolk-citrate (EYC) and egg yolkTris-citric acid-fructose (EYT) extenders. The semen diluents were added in two steps and the diluted samples were preserved at 5 OC. The dilution rate was 1 : 10 with each diluter. The mean percent motile spermatozoa was higher in EYT during different periods of preservation at 5 OC. The E Y C and milk diluents contaning egg yolk had more or less similar effects on the percent motile spermatozoa. The milk powder and skim milk diluters gave poor results in comparison to others. Based on mean readings and statistical analysis of percent motile spermatozoa, the six semen extenders are rated as follows: EYT > E Y C > EYSM > EYPM > SM > PM, considering both storage period and sperm motility. Zusammenfassung
EinfluS verschiedener Verdiinner auf die Uberlebenszeit von Schaf'spermienbei 5 OC Von 6 sardinischen Schafen wurde mittels Elektroejakulation Sperma gewonnen. Die Verdunner wiesen folgende Zusammensetzung auf: 10 O/o
560
PETRUZZJ, TARANTINI and ROYCHOUDHURY
Milchpulverlosung (PM), pasteurisierte Kuhmagermilch (SM); Milchpulver und 20 O / o Eigelb (EYPM); Magermilch mit 20 O/o Eigelb (EYSM), EigelbCitrat-Verdunner (EYC), Eigelb-Tris-Citrat-Fructose-Verdiinner(EYT). Die Verdunner wurden dem Sperma in zwei Schritten zugefugt. Der Verdunnungsgrad betrug in allen Fallen 1 : 10. Die verdunnten Proben wurden bei 5 OC aufbewahrt. Der Prozentsatz der beweglichen Spermien war im EYT Verdunner bei den verschiedenen Konservierungszeiten am hochsten. Die EY C und Milchverdiinner mit Eigelb hatten etwa den gleichen Einflufi auf den Prozentsatz beweglicher Spermien. Die Milchpulver- und Magermilchverdiinnerer gaben schlechtere Ergebnisse. Die Qualitat der Verdunner nahm in nachstehender Reihenfolge ab: EYT > EYC > EYSM EYPM > SM > PM.
>
Resume Influence de differents diluants sur le temps de survie des spermatozoi‘des de mouton A 5 OC Du sperme fut obtenu par klectrokjaculation chez 6 moutons sardes. Les diluants prksentkrent les formules suivantes: solution de poudre de lait (PM) ; petit lait bovin pasteurisk (SM); poudre de lait et 20 O / o de jaune d’oeuf (EYPM; petit lait avec 20 O / o de jaune d’oeuf (EYSM); jaune d’oeuf - citrate (EYC); jaune d’oeuf - tris - citrate - fructose (EYT). Les diluants ont ktk ajoutks au sperme en deux temps. Le degrk de dilution fut toujours de 1 : 10. Les kchantillons diluks furent conservks B 5 O C . Le pourcentage de mobilitk fut le plus klevk dans les diffCrents temps de conservation avec le diluant EYT. EYC et le diluant lactk avec jaune d’oeuf ont eu B peu prks la meme influence sur la mobilitk. La poudre de lait et le petit lait ont donnk de mauvais rksultats. La qualitk des diluants fut par ordre dkcroissant la suivante: EYT > EYC > EYSM > EYPM > SM > PM. Resumen Influjo de varios diluyentes sobre el tiempo de supervivencia de 10s espermatozoides de morueco a 5 OC Se obtuvo esperma mediante electroeyaculaci6n de 6 moruecos sardos. Las muestras de semen se diluian con soluci6n a1 10 O / o de leche en polvo (PM), leche descremada pasteurizada de vaca (SM); leche en polvo y 20 O / o de yema de huevo (EYPM), leche descremada con 2 O o / o de yema (EYSM), diluyente yema-citrato (EYC), diluyente yema-tris-citrato-fructosa (EYT). Se afiadieron 10s diluyentes a1 esperma en dos fases. El grado de diluci6n era siempre def orden de 1 : 10. Las muestras diluidas se conservaban a 5 O C . El porcentaje de espermatozoides m6viles era m4ximo en el diluyente EYT en tiempos diferentes de conservaci6n. El EYC y el diluyente a base de leche con yema ejercian casi el mismo influjo sobre el porcentaje de espermatozoos m6viles. Los diluyentes de leche en polvo y leche desnatada ofrecian resultados inferiores. La calidad de 10s diluyentes disminuia en el orden decreciente siguiente: EYT > EYC > EYSM > EYPM > SM > PM, considerhndose tan0 el period0 de almacenamiento como la motilidad del esperma. References 1. AHMED, S. J., 1955: Effect of glycinc on storage of ram semen. 1. Agri. Sci. 46, 164. 2. ANTONJAN, A. S., and V. S. KAMALJAN, 1970: Effect of sperm numbers and quality on conception rate in the dam and on some characters in the progeny. Biol. Zh. Armenii. 23, 104 (Anim. Breed. Abstr. Annotated Bibliography No. 55 B on Dilution and storage of ram scmen). 3. BLACKSHAW, A. W., 1960: The effects of milk diluents on the viability of ram spermatozoa and their revival after freezing. Aust. Vet. J. 36, 432.
Effect of Different Semen Diluents on Survival of Ram Spermatozoa at 5 "C
561
4 BONADONNA, T., 1974: La fecondazione strumentale negli ovini e nei caprini. In Riproduzione Animale e Fecondazione Artificiale. Edited by UTET, Torino, Italy. p. 342. 5. BRUCKNER,G., and H. J. BAUER,1973: Aspects of the application of scientific and technical advances in the artificial insemination of sheep. Archiv. fur Tierzucht. 16, 251. 6 DESSOUKY, F., M. K. AL-HAKIN,K. H. JUMA and S. M. A. FARHAM,1970: A comparative study on livability of Awassi sperms in different extenders. J. Egypt. Vet. med. Ass., 30, 43. 7. EMMENS, C. W., and T. J. ROBINSON, 1962: Artificial insernination in the sheep. I n Semen of Animals and Artificial Insemination. Edited by J. P. Maule. Tech. Commun. no. 15 of the Commonwealth Bureau of Animal Breeding and Genetics, Edinburgh. .205. 8. FILIMON,S., N. LUNCA,I. BRATESCUand V. OTEL,1956: The dilution a n l s t o r a g e of ram and bull semen. Anim. Breed. Abstr. 25, 401. 9. GRAHAM, E. F., B. G. CRABOand M. K. L. SCHMEHL, 1973: Utilization of enzyme assay in developing techniques for freezing semen. Proc. VIII Inter. Zootechny Symposium, Milano. p. 95. 10. HILL,J. R., V. HURST and W. C. GODLEY,1958: A comparison of reconstituted skim milk and egg yolk sodium citrate as extenders for ram semen. Am. J. Vet. Res. 19, 132. 11. INSkEEP, E. K., 1974: Artifical Insemination and Preservation of ram semen. Bulletin 629, West Virginia University, Agricultural Experiment Station. p. 5. S.. 1957: The effectiveness of usinr milk as a diluent for ram semen. Anim. 12. ISTVAN. Breed. Abstr. 25, 231. 13. TONES, R. C.. and I. C. A. MARTIN.1965: Deeu-freezing ram suermatozoa: the effects of " milk yolk-citrate and synthetic diluents contahing sugar. J. Riprod. Fert. 10, 413. 14. TONES, R. C.. 1968: Survival of bull and ram spermatozoa in preuarations from skim " milk. ,J. Dairy Sci. 51, 1288. 15. TONES. R. C.. 1969: Studies of the suitability of meparations of ewe and cow milk for " storing ram'spermatozoa a t 37,5 and - 79 'C. AGst: J. Biol. Sci. 22, 983. 16. JONES,R. C., I. C. A. MARTINand K. R. LAPWOOD,1969: Studies of the artifical insemination of sheep: the effects on fertility of diluting ram semen, stage of Oestrus of the ewe at insemination and injection of synthetic oxytocin. Aust. J. Agric. Res. 20, 141. 17. KOUTSOURIS, C. D., and H. VAUPEL,1973: Deep-freezing of ram semen in pellets with different pre-freezing cooling procedures. Zuchthygiene. 8 , 163. 18. LARDY,H. A., and R. H. PHILLirs, 1939: Preservation of spermatozoa. Proc. Am. SOC. Anim. Prod. 32nd. Ann. Meet. p. 219. 19. LAPWOOD, K. R., and I. C. A. MARTIN,1972: Effects of some buffers and inorganic and organic sodium salts in synthetic diluents for the storage of ram spermatozoa at 37 OC or 5 OC. Aust. 1. Biol. Sci. 25, 367. 20. PRASAD, M. M., and C. NORMAN, 1969: Conservation of ram semen at room temperatures following storage at 5 O C . Proc. 2nd. Wld. Conf. Anim. Prod. p. 431. 21, ROY. A.. H . C. GUPTA.R. K. SRIVASTAVA and M. D. PANDEY. 1956: Preservation in glycine-egg yolk medium. Indian Vet. J. 33, 18. 22. SAXENA,V. B., and G. SINGH, 1967: Preservation of ram spermatozoa. I. Effects on motility, livability and morphology of ram spermatozoa in four semen diluents. J. Anim. Morph. Physiol. 14, 114. 23. SALISBURY, G. W., H. K. FULLERand E. WILLETT,1941: Preservation of bovine spermatozoa in yolk-citrate diluent and field results from its use. J. Dairy Sci. 24, 905. 24. SAHNI,K. L., and A. ROY, 1969: Influence of season on semen quality of rams and effects of dilutors and dilutions on in vitro preservation. Indian J. Anim. Sci. 39, 1. 25. SCHLEICHER, J., and G. BRUCKNER, 1973 : Ultrastructural findings on ram spermatozoa stored in liquid form in different buffers. Monatshefte fur Veterinarmedizin. 28, 391. 26. SNEDECOR, G. W., 1956: Statistical Methods. 5th Ed., Iowa State College Press, Ames, Iowa. 27. STEINBACH, J., and R. H. FOOTE,1967: Osmotic pressure and pH effects of survival of frozen bovine spermatozoa. J. Dairy Sci., 50, 205. 28. TAMHAN, S. S., and G. SINGH, 1972: Semen extenders and preservation temperature for ram spermatozoa. Indian J. Anim. Prod. 3, 51. 29. TEWARI, S. B., R. P. SHARMA and A. ROY,1968: Effects of dilution on the preservation of ram and goat spermatozoa1 viability. Indian J. Vet. Sci. 38, 567.
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A
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Author's address: Dr. V. PETRUZZI, Istituto di Patologia Speciale e Clinica Chirurgica Veterinaria, Universiti degli Studi di Sassari, Piazza Conte di Moriana, 3 . 07100 Sassari.
Zbl. Vrr. Mcd., Rrihe A , Bd. 23, H c f r 7
38
(1976) @ 1976 Verlag Paul Parey, Berlin und H a m b u r g
ISSN 0300-871 l/ASTM-Coden: Z V R A A X
Cattedra di Scienza eTecnica della Fecondazione Artificiale Istituto di Ostetricia e Ginecologia Veterinaria, Universitd degli Studi di Sassari Direclor: P r o f . U.Sbernardori
Effect of Different Semen Diluents on Survival of Ram Spermatozoa at 5 “C BY V. PETRUZZI, S. TARANTINI and P. N. ROYCHOUDHURY:~ W i t h one table (Received for publication August 29, 1975)
Introduction Difficulties in the preservation and deep-freezing of diluted ram semen show that ram spermatozoa are susceptible to the effects of different diluents and are very sensitive to the freezing techniques. The main aim of dilution is to reduce the sperm concentration so that an ejaculate can be more easily divided into a large number of doses. Dilution of ram semen is reported to be beneficial for storage in some cases but it may also result in lower conception rates. ANToNjAN and KAMALJAN (1970) reported that the conception rates of ewes inseminated with freshly diluted semen was 74 O / n to 40 O / o when the diluted semen had been stored a t O OC for 24-28 hours. (1939) has The egg yolk buffer mixture developed by LARDYand PHILLIPS been a satisfactory diluent for ram semen. Various improvements and changes have been made by many workers (AHMED,1955; ROY et al., 1956; PRASAD and NORMAN, 1969; TAMHAN and SINGH,1972; BRUCKNER and BAUER,1973). Milk diluents appear to be useful as a ram semen extender and have been widely used in different parts of the world (ISTVAN, 1956; FILIMON et al., 1956; HILLet al., 1958; BLACKSHAW, 1960; EMMENS and ROBINSON,1962; JONESand MARTIN,1965; SAXENAand SINGH, 1967; JONES,1968, 1969; TEWARI et al., 1968). More recent semen extenders like TRIS - and TEST buffers which are satisfactory for diluting and deep-freezing bull semen are still in an experimental stage for ram semen dilution (LAPWOOD and MARTIN, 1972; GRAHAM et al., 1973; SCHLEICHER and BRUCKNER, 1973). ‘) Istituto Sperimentale Iraliano “L. Spallanzani” per la Fecondazioe Artificiale Milano (Director: Prof. T. BONAUONNA). Present address: Senior Project Executive (A. H.), National Dairy Development Board, Anand, Gujarat, India.
Effect of Different Semen Diluents on Survival of Ram Spermatozoa a t 5 OC
557
Contributions on the dilution and storage of ram semen in various diluters at different temperatures (0 to 20 "C) and also the difficulties in deep(1974) freezing ram spermatozoa have been recently discussed by INSKEEP and BONADONNA (1974). Considering the short-term storage of ram semen and the increased susceptibility of spermatozoa to different semen diluents, the present work was undertaken to study the survival of ram spermatozoa at 5 O C in six different semen extenders.
Material and Methods Semen samples were collected by electrical stimulation from 6 adult rams of Sardinian breed. The semen diluents used were milk powder (PM), skim cow milk (SM), milk powder with egg-yolk( EYPM), skim cow milk with egg-yolk (EYSM), egg-yolk citrate (EYC) and Tris-citric acid-fructose (EYT) extenders. The milk powder diluter was a 10 O / o solution of the milk powder containing approximately 0.5-1.0 O / o fat. The skim milk used was homogenised and pasteurized cow milk containing approximately 1.8 O/o fat. The milk powder and the skim milk diluters were also used with 20 O/u egg yolk. The yolk-citrate extender was prepared according to SALISBURY et al. (194 1). The Tris (hydroxymethyl) aminomethane-yolk diluter was (1967). prepared according to STEINBACH and FOOTE After examining the initial sperm motility, the neat semen samples were diluted in two steps. The first dilution was made at 37 O C and then the Samples were brought to 20 OC a t the rate of 1 OC/min. The second dilution was completed at 20 O C . The dilution rate with each semen extender was 1 : 10. The diluted samples were preserved at 5 O C and the aliquots were examined at 4, 24, 48, 72, 96 and 120 hours of storage. Percent motile spermatozoa of each sample was examined microscopically by two workers and the mean value was noted. The data were subjected to statistical analysis according to SNEDECOR (1956). For the analysis of variance, the observations of the percent motile spermatozoa were converted to corresponding values by angular transformation.
Results and Discussion The mean percent motile spermatozoa with standard deviations are shown in Table I , which shows that the mean percent motile spermatozoa is higher in E Y T diluter than in the other five diluters. Not much difference in sperm motility was found between EYC diluter and the two milk diluters containing 20 O / o egg yolk. The mean percent motile spermatozoa in milk powder and skim milk diluters was slightly lower than the mean percent motile spermatozoa in EYPM and EYSM diluents. It was possible to preserve ram spermatozoa for 120 hours with 45 O / o and 26 O / o motility in EYT and EYC extenders respectively. In milk diluents containing egg yolk, the mean percent motile spermatozoa was almost equal (23 O / o ) , whereas no sperm motility was found after 120 hours in milk powder and skim milk extenders. The data were also analysed to find the differences in the mean percent motile spermatozoa between the different diluters a t the same period of storage. The results are shown in Table 1 with superscript letters. No significant difference was found in the mean percent motile spermatozoa between the six semen diluters after 4 hours of storage at 5 O C . The differences in the mean percent motile spermatozoa between EYT diluter and other semen diluters
558
PETRUZZI,
TARANTINI and KOYCHOUDHURY
after 48 hours of conservation were highly significant (P < 0.01). Whereas the differences in the mean percent motile spermatozoa between the milk powder or skim milk extenders each containing 20 O/o egg yolk and the EYC diluter were highly significant (P < 0.01) only at some periods of storage. There was not much difference in percent motile spermatozoa between milk powder and skim milk extenders except at 96 hours of storage (P < 0.01). SAXENA and SINGH(1967) used skim milk-yolk, egg yolk-glucose-bicarbonate, egg yolk-citrate and Cornell University Extender, each containing 'I different percentage of egg yolk for the dilution and preservation of ram spermatozoa. Differences due to diluent were significant at 72, 120 and 168 hours for motility and percentage live spermatzoa. Motility and percentage live spermatozoa were highest in skim milk-yolk followed by eggyolk-glucoseet al. (1968) reported ram sperm bicarbonate up to 168 hours storage. TEWAKI motility of 27.65 '. 2.0, 52.43 k 2.3, 64.22 f 2.0 and 72.34 i 2.2 at 6 hr storage (5-7 "C) in the four diluent rates (1 : 1, 1 : 3 , 1 : 5 and 1 : 10) respectively and 6.25 t- 1.2, 32.34 t- 2.5, 45.93 i. 2.8 and 54.53 i 2.7 at 30 hr storage in cow milk diluent. The differences between dilution rates as well as between storage intervals were highly significant. In the present study the mean percent motile spermatozoa in the skim cow milk diluent with and without egg yolk was found to be 55.22 f 12.70 and 45.74 t- 15.36 respectively at 48 hr storage in the dilution rate of 1 : 10. JONES et al. (1969) reported only 8.8 O / o fertility with ram spermatozoa diluted (1 : 10) with skim milk and stored at 5 "C for approximately 36 hours. The ability of four extenders (egg yolk-citrate, egg yolk-Tris, Cornell University Extender (CU-16) and Coconut Milk Extender) to maintain ram spermatozoa at variable temperatures (5 OC, 24 "C-34 "C) was rated as follows: CME > CU-16 > EYT > EYC (PRASAD and NORMAN,1969). SAHNIand ROY (1969) observed the highest sperm motility when diluted ram spermatozoa was preserved at 5 O C in Cornell University Extender (CUE) and cow milk, followed by milk and milk-containing diluents which were significantly superior to all egg yolk-containing diluents. The results of this experiment did not agree with the findings of SAHNIand ROY(loc. cit); in this study addition of egg yolk in the milk diluents improved the mean percent motile spermatozoa (Table). Table I Hours of conservation at 5OC
4 Hours
1 Percent
I
motile spermatozoa in different semen diluters (Dilution rate 1 : 10)
Powder milk
(PM)
1
Skim milk (SM)
I
Powder milk + ZpETPY ;
I
Skim milk
Y$s;+2;
Na Citrate ldilu;gY,,
I
Tris Diluter
(EYT)
66.85r 7.35N.S.64.64?: 10.77N.S,66.66r 8.52N.5. 66.00t 8.36N.5.69.11 f 7.9gN.'. 70.442 7.21N.'. (27) (27) (45) 145) (45) 145)
I
1
Effect of Different Semen Diluents on Survival of Ram Spermatozoa at 5 'C
559
DESSOUKY et al. (1970) found that egg-yolk-citrate gave the best results in terms of motility, followed by egg-yolk-citrate-glycerol and egg-yolkand SINGH(1972) reported no significant differences in skim milk. TAMHAN sperm motility and percentage of live spermatozoa in ejaculates diluted with yolk-citrate, yolk-citrate-glucose-glycine, skim milk-yolk-glucose and yolktomato juice extenders and stored at 5 O C , 10 O C , 15 OC or 25 OC for 0, 24, 48, 72, 96, 120 or 144 hrs. A storage temperature of 25 O C proved unsatisfactory for all diluents. In the German Democratic Republic A.I. of ewes is practised using glucose-egg yolk-citrate extender and storing the diluted semen up to 24 hrs a t 3-4 "C (BRUCKNER and BAUER,1973). The results of the present experiment thus agree with those findings of some workers in maintaining sperm motility for different periods at 5 O C in milk diluents, milkyolk diluents and yolk-citrate diluent. Contributions on the use of Tris-buffer as ram semen diluent are limited. SCHLEICHER and BRUCKNER (1973) preserved ram semen at 2-4 OC for up to 216 hrs in egg yolk-citrate, egg yolk-phosphate or Tris-buffer solutions. Under the electron microscope fewer structurally altered spermatozoa were reported after 72 hrs in Tris diluent than after 24 hrs in either of the other two diluents. Recently KOUTSOURIS and VAUPEL(1973) used a Tris diluent and a modified raffinose-yolk-glycerol diluent for deep-freezing ram spermatozoa and reported that the type of diluent had no significant effect on forward motility after thawing. As is evident from our experiment (Table l), higher mean percent motile spermatzoa were found in Tris extender when ram semen was diluted (1 : 10) and stored at 5 "C. Percent motile spermatozoa in EYT, EYC, EYSM and EYPM diluents were 45.00, 26.00, 23.33 and 23.33 O/o, respectively after 120 hours of preservation at 5 O C . It was not possible to preserve ram spermatozoa for 120 hrs in skim milk and powder milk diluents without egg yolk. Based on the results of mean percent motile spermatozoa, the ability of the six semen extenders to preserve ram spermatozoa at 5 O C was thus rated as follows: EYT > EYC > EYSM > EYPM > SM > PM. Summary Semen was collected by electrical stimulation from 6 adult Sardinian rams. Semen samples were diluted with 10 O/o milk powder solution (PM), pasteurized skim cow milk (SM), powder milk with 20 O / o egg yolk (EYPM), skim milk with 20 O/o egg yolk (EYSM), egg yolk-citrate (EYC) and egg yolkTris-citric acid-fructose (EYT) extenders. The semen diluents were added in two steps and the diluted samples were preserved at 5 OC. The dilution rate was 1 : 10 with each diluter. The mean percent motile spermatozoa was higher in EYT during different periods of preservation at 5 OC. The E Y C and milk diluents contaning egg yolk had more or less similar effects on the percent motile spermatozoa. The milk powder and skim milk diluters gave poor results in comparison to others. Based on mean readings and statistical analysis of percent motile spermatozoa, the six semen extenders are rated as follows: EYT > E Y C > EYSM > EYPM > SM > PM, considering both storage period and sperm motility. Zusammenfassung
EinfluS verschiedener Verdiinner auf die Uberlebenszeit von Schaf'spermienbei 5 OC Von 6 sardinischen Schafen wurde mittels Elektroejakulation Sperma gewonnen. Die Verdunner wiesen folgende Zusammensetzung auf: 10 O/o
560
PETRUZZJ, TARANTINI and ROYCHOUDHURY
Milchpulverlosung (PM), pasteurisierte Kuhmagermilch (SM); Milchpulver und 20 O / o Eigelb (EYPM); Magermilch mit 20 O/o Eigelb (EYSM), EigelbCitrat-Verdunner (EYC), Eigelb-Tris-Citrat-Fructose-Verdiinner(EYT). Die Verdunner wurden dem Sperma in zwei Schritten zugefugt. Der Verdunnungsgrad betrug in allen Fallen 1 : 10. Die verdunnten Proben wurden bei 5 OC aufbewahrt. Der Prozentsatz der beweglichen Spermien war im EYT Verdunner bei den verschiedenen Konservierungszeiten am hochsten. Die EY C und Milchverdiinner mit Eigelb hatten etwa den gleichen Einflufi auf den Prozentsatz beweglicher Spermien. Die Milchpulver- und Magermilchverdiinnerer gaben schlechtere Ergebnisse. Die Qualitat der Verdunner nahm in nachstehender Reihenfolge ab: EYT > EYC > EYSM EYPM > SM > PM.
>
Resume Influence de differents diluants sur le temps de survie des spermatozoi‘des de mouton A 5 OC Du sperme fut obtenu par klectrokjaculation chez 6 moutons sardes. Les diluants prksentkrent les formules suivantes: solution de poudre de lait (PM) ; petit lait bovin pasteurisk (SM); poudre de lait et 20 O / o de jaune d’oeuf (EYPM; petit lait avec 20 O / o de jaune d’oeuf (EYSM); jaune d’oeuf - citrate (EYC); jaune d’oeuf - tris - citrate - fructose (EYT). Les diluants ont ktk ajoutks au sperme en deux temps. Le degrk de dilution fut toujours de 1 : 10. Les kchantillons diluks furent conservks B 5 O C . Le pourcentage de mobilitk fut le plus klevk dans les diffCrents temps de conservation avec le diluant EYT. EYC et le diluant lactk avec jaune d’oeuf ont eu B peu prks la meme influence sur la mobilitk. La poudre de lait et le petit lait ont donnk de mauvais rksultats. La qualitk des diluants fut par ordre dkcroissant la suivante: EYT > EYC > EYSM > EYPM > SM > PM. Resumen Influjo de varios diluyentes sobre el tiempo de supervivencia de 10s espermatozoides de morueco a 5 OC Se obtuvo esperma mediante electroeyaculaci6n de 6 moruecos sardos. Las muestras de semen se diluian con soluci6n a1 10 O / o de leche en polvo (PM), leche descremada pasteurizada de vaca (SM); leche en polvo y 20 O / o de yema de huevo (EYPM), leche descremada con 2 O o / o de yema (EYSM), diluyente yema-citrato (EYC), diluyente yema-tris-citrato-fructosa (EYT). Se afiadieron 10s diluyentes a1 esperma en dos fases. El grado de diluci6n era siempre def orden de 1 : 10. Las muestras diluidas se conservaban a 5 O C . El porcentaje de espermatozoides m6viles era m4ximo en el diluyente EYT en tiempos diferentes de conservaci6n. El EYC y el diluyente a base de leche con yema ejercian casi el mismo influjo sobre el porcentaje de espermatozoos m6viles. Los diluyentes de leche en polvo y leche desnatada ofrecian resultados inferiores. La calidad de 10s diluyentes disminuia en el orden decreciente siguiente: EYT > EYC > EYSM > EYPM > SM > PM, considerhndose tan0 el period0 de almacenamiento como la motilidad del esperma. References 1. AHMED, S. J., 1955: Effect of glycinc on storage of ram semen. 1. Agri. Sci. 46, 164. 2. ANTONJAN, A. S., and V. S. KAMALJAN, 1970: Effect of sperm numbers and quality on conception rate in the dam and on some characters in the progeny. Biol. Zh. Armenii. 23, 104 (Anim. Breed. Abstr. Annotated Bibliography No. 55 B on Dilution and storage of ram scmen). 3. BLACKSHAW, A. W., 1960: The effects of milk diluents on the viability of ram spermatozoa and their revival after freezing. Aust. Vet. J. 36, 432.
Effect of Different Semen Diluents on Survival of Ram Spermatozoa at 5 "C
561
4 BONADONNA, T., 1974: La fecondazione strumentale negli ovini e nei caprini. In Riproduzione Animale e Fecondazione Artificiale. Edited by UTET, Torino, Italy. p. 342. 5. BRUCKNER,G., and H. J. BAUER,1973: Aspects of the application of scientific and technical advances in the artificial insemination of sheep. Archiv. fur Tierzucht. 16, 251. 6 DESSOUKY, F., M. K. AL-HAKIN,K. H. JUMA and S. M. A. FARHAM,1970: A comparative study on livability of Awassi sperms in different extenders. J. Egypt. Vet. med. Ass., 30, 43. 7. EMMENS, C. W., and T. J. ROBINSON, 1962: Artificial insernination in the sheep. I n Semen of Animals and Artificial Insemination. Edited by J. P. Maule. Tech. Commun. no. 15 of the Commonwealth Bureau of Animal Breeding and Genetics, Edinburgh. .205. 8. FILIMON,S., N. LUNCA,I. BRATESCUand V. OTEL,1956: The dilution a n l s t o r a g e of ram and bull semen. Anim. Breed. Abstr. 25, 401. 9. GRAHAM, E. F., B. G. CRABOand M. K. L. SCHMEHL, 1973: Utilization of enzyme assay in developing techniques for freezing semen. Proc. VIII Inter. Zootechny Symposium, Milano. p. 95. 10. HILL,J. R., V. HURST and W. C. GODLEY,1958: A comparison of reconstituted skim milk and egg yolk sodium citrate as extenders for ram semen. Am. J. Vet. Res. 19, 132. 11. INSkEEP, E. K., 1974: Artifical Insemination and Preservation of ram semen. Bulletin 629, West Virginia University, Agricultural Experiment Station. p. 5. S.. 1957: The effectiveness of usinr milk as a diluent for ram semen. Anim. 12. ISTVAN. Breed. Abstr. 25, 231. 13. TONES, R. C.. and I. C. A. MARTIN.1965: Deeu-freezing ram suermatozoa: the effects of " milk yolk-citrate and synthetic diluents contahing sugar. J. Riprod. Fert. 10, 413. 14. TONES, R. C.. 1968: Survival of bull and ram spermatozoa in preuarations from skim " milk. ,J. Dairy Sci. 51, 1288. 15. TONES. R. C.. 1969: Studies of the suitability of meparations of ewe and cow milk for " storing ram'spermatozoa a t 37,5 and - 79 'C. AGst: J. Biol. Sci. 22, 983. 16. JONES,R. C., I. C. A. MARTINand K. R. LAPWOOD,1969: Studies of the artifical insemination of sheep: the effects on fertility of diluting ram semen, stage of Oestrus of the ewe at insemination and injection of synthetic oxytocin. Aust. J. Agric. Res. 20, 141. 17. KOUTSOURIS, C. D., and H. VAUPEL,1973: Deep-freezing of ram semen in pellets with different pre-freezing cooling procedures. Zuchthygiene. 8 , 163. 18. LARDY,H. A., and R. H. PHILLirs, 1939: Preservation of spermatozoa. Proc. Am. SOC. Anim. Prod. 32nd. Ann. Meet. p. 219. 19. LAPWOOD, K. R., and I. C. A. MARTIN,1972: Effects of some buffers and inorganic and organic sodium salts in synthetic diluents for the storage of ram spermatozoa at 37 OC or 5 OC. Aust. 1. Biol. Sci. 25, 367. 20. PRASAD, M. M., and C. NORMAN, 1969: Conservation of ram semen at room temperatures following storage at 5 O C . Proc. 2nd. Wld. Conf. Anim. Prod. p. 431. 21, ROY. A.. H . C. GUPTA.R. K. SRIVASTAVA and M. D. PANDEY. 1956: Preservation in glycine-egg yolk medium. Indian Vet. J. 33, 18. 22. SAXENA,V. B., and G. SINGH, 1967: Preservation of ram spermatozoa. I. Effects on motility, livability and morphology of ram spermatozoa in four semen diluents. J. Anim. Morph. Physiol. 14, 114. 23. SALISBURY, G. W., H. K. FULLERand E. WILLETT,1941: Preservation of bovine spermatozoa in yolk-citrate diluent and field results from its use. J. Dairy Sci. 24, 905. 24. SAHNI,K. L., and A. ROY, 1969: Influence of season on semen quality of rams and effects of dilutors and dilutions on in vitro preservation. Indian J. Anim. Sci. 39, 1. 25. SCHLEICHER, J., and G. BRUCKNER, 1973 : Ultrastructural findings on ram spermatozoa stored in liquid form in different buffers. Monatshefte fur Veterinarmedizin. 28, 391. 26. SNEDECOR, G. W., 1956: Statistical Methods. 5th Ed., Iowa State College Press, Ames, Iowa. 27. STEINBACH, J., and R. H. FOOTE,1967: Osmotic pressure and pH effects of survival of frozen bovine spermatozoa. J. Dairy Sci., 50, 205. 28. TAMHAN, S. S., and G. SINGH, 1972: Semen extenders and preservation temperature for ram spermatozoa. Indian J. Anim. Prod. 3, 51. 29. TEWARI, S. B., R. P. SHARMA and A. ROY,1968: Effects of dilution on the preservation of ram and goat spermatozoa1 viability. Indian J. Vet. Sci. 38, 567.
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A
.
Author's address: Dr. V. PETRUZZI, Istituto di Patologia Speciale e Clinica Chirurgica Veterinaria, Universiti degli Studi di Sassari, Piazza Conte di Moriana, 3 . 07100 Sassari.
Zbl. Vrr. Mcd., Rrihe A , Bd. 23, H c f r 7
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