Cryopreservation of ram semen for artificial insemination
A theoretical study showed that Al can greatly affect the efficiency of sheep breeding schemes provided fertility is maintained at the highest levels. Factors that affect the survival of ram spermatozoa during preservation were studied. A pH range between 6 and 7 was well tolerated. The addition of 4% (v/v) glycerol to the diluted ram semen in Tris buffer lowered the motility and survival of spermatozoa during 5 hours of storage at 30'C. Following insemination of chilled ram semen, with and without glycerol in the diluent, lambing percentages of 59% and 73% respectively were obtained. Ram semen was frozen in 0.25 ml straws using various cooling combinations. The optimal procedure was found to be to cool rapidly from 5'C to -120'C at -20'C/min. When semen so treated was compared in a fertility trial with semen frozen by the pellet method of Evans and Maxwell (1987), lambing percentages of 14% and 18% respectively were obtained. Attempts were made to formulate a vitrifying diluent for ram semen. A method was developed for the assessment of semen in highly concentrated cryoprotective solutions. Semen tolerated 10% concentrations of each of glycerol, acetamide and propylene glycol applied together, but when concentrations were raised above this level sperm mortality was very high. A simple spectrophotometric procedure for the objective assessment of vigour of ram semen was developed and tested. Raffinose 66 mM in the freezing diluent improved the post-thawing revival rate of spermatozoa from 46% to 71%, and increased the post-thawing recovery of the swimmingup vigour (P< 0.01). Raffinose treatment reduced the ATP content of semen but did not reduce the rate of glucose oxidation by diluted spermatozoa at either the pre-freezing or post-thawing stages. Frozen storage of ram spermatozoa as pellets was best achieved using two volumes of Tris buffer diluent containing 18% (v/v) egg-yolk, 6% (v/v) glycerol and 66 mM raffinose to one volume of semen. The diluted semen was chilled to 5'C and frozen as 0.10 ml pellets on dry ice. For frozen storage of ram semen in 0.25 ml straws, best results were obtained when the Tris buffer diluent contained 18% (v/v) egg-yolk, 9% (v/v) glycerol and 66 mM raffmose, and cooling was at a rate of -30'C/min from 5'C to -120'C. Non-return rates were 21%, 20% and 31% for ewes inseminated with semen samples frozen as standard 0.20 ml pellets, as raffinose containing 0.10 ml pellets, and as raffinose containing 0.25 ml straws respectively. Of the in vitro tests, only the swim-up test was correlated with non-return rates (r=0.904, P< 0.1). Post-thawing survival of the spermatozoa was improved by the addition of raffinose which had no deleterious effect on fertility.