Five factorial experiments were conducted to examine the effects of different freezing methods on the survival of ram spermatozoa following the freeze-thawing procedures Semen was frozen in pellet form and in straws on either dry ice or in liquid nitrogen. Freezing of semen
in pellet form on dry ice gave better results than pelleting in liquid nitrogen or freezing in straws. Recovery of spermatozoa was poor when the diluted, cooled semen was dropped directly into liquid nitrogen and the pellets allowed to sink in the freezing fluid. Cell survival was substantially improved by allowing the semen droplets to float for only five seconds in the nitrogen before transferring into dry tubes held in liquid nitrogen vapour. Best results with this method were obtained when the diluted semen contained only 2% glycerol Increasing the glycerol concentration to 8% had a deleterious effect on cell survival, particularly when freezing in liquid nitrogen with complete sinking of the pellets. Freezing ram semen in straws on dry ice gave poor results, but recovery and survival of spermatozoa was improved by freezing the straws in liquid nitrogen vapour at -80 or -160°C Tris-glucose was a more suitable freezing medium than raffinose-citrate and results were particularly low when the semen frozen in the latter diluent was thawed in dry tubes and a solution was not added prior to incubation. The best alternative procedure to pellet·freezing on dry ice was to freeze the semen in straws in liquid nitrogen vapour. When applying this method recovery and survival of cells was satisfactory following dilution (1:4) and freezing in Tris (300 mM)-citric acid (94,7 mM)-glucose (27,75 mM)-egg yolk (15 %. v/v)-glycerol (5 %, v/v) diluent, with or without addition of Tris-fructose solution after thawing.