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Glycerol has been the most widely used cryopreservation agent for spermatozoa and a wide range of factors affect its action on sperm viability and fertilizing capacity. We tested three methods for freezing ram semen packed in 0.25ml straws (final cellular concentration: 100×10⁶spz/ml). Method M1: Two-thirds of the final volume of diluent was added as solution A (without glycerol) to the pure semen at 35°C. The sample was cooled to 5°C (−0.30°C/min), one-third of final diluent volume was added as solution B (final concentration of glycerol 4%) and the sample was maintained at 5°C for 2h. It was then frozen in a programmable biofreezer (−20°C/min down to −100°C). Method M2: The sample was diluted with a specific solution at 35°C (final concentration of glycerol 3%), cooled to 5°C (−0.20°C/min) and left for 2h. After that, it was frozen in nitrogen vapours. Method M3: Semen was diluted 1:1 in a specific solution …