Graduate Thesis Or Dissertation
 

Cryogenic preservation of ram spermatozoa

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  • Investigations with ram semen were conducted to develop procedures of cyrogenic preservation that would provide long-term retention of fertilizing capacity in ram spermatozoa. An isotonic extender consisting of egg yolk, sodium citrate, lactose, glycerol, and a broad spectrum antibiotic was selected from among several extenders tested in pilot studies. Using this extender, ram semen collected by artificial vagina was frozen in glass ampules at controlled rates in a Biological Freezing unit, in glass capillary tubes on dry ice, in plastic straws on dry ice and in pellet form on dry ice. Factorial experiments were conducted to compare: (1) freezing containers, (2) freezing in ampules in a biological freezing unit at controlled rates with the freezing of pellets on dry ice, (3) the effect of enzymes added to the extender, (4) the effect of holding sperm in seminal plasma prior to extension, (5) the influence of glycerol on acrosomal mophology of frozen semen, and (6) ram effects and interactions. The viability of spermatozoa held in liquid nitrogen storage 18 to 21.8 months was examined. Fertilizing capacity of frozen semen was tested with ewes in natural estrus and with ewes in estrus induced by hormonal treatment. Freezing in glass ampules at an optimum controlled rate and freezing in plastic straws on dry ice gave significantly higher postfreeze motility and percent survival than other methods used. Semen frozen in ampules at the controlled rate and thawed in ice water showed no apparent loss of viability compared to that thawed at 40°C. Semen frozen in plastic straws on dry ice and thawed at 75°C for 12 seconds gave viability equal to that frozen in glass ampules at the optimum controlled rate of freezing. Alpha amylase, beta amylase, and beta glucuronidase had no significant effect upon postfreeze viability when added to the extender at the rate of 10 microgram per ml. Holding ram spermatozoa in seminal plasma for four hours prior to extension caused no significant change in postfreeze motility or percent survival, The addition of glycerol to the freezing medium caused a significant increase in acrosomal damage. The addition of glycerol was also found necessary to obtain postfreeze motility. The insemination of a limited number of ewes with semen frozen and stored at -196°C for 30 days to 120 days failed to produce observed pregnancies. The probable causes of this are discussed. The techniques developed in this study allowed ram spermatozoa to be frozen and to retain motility and percent survival in storage at -196°C for periods up to 21.8 months equal to that observed at one week of frozen storage.
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