Field and in vitro assay of three methods for freezing ram semen

Abstract 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 0.25 ml straws (final cellular concentration: 100×10 6  spz/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 2 h. 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 2 h. After that, it was frozen in nitrogen vapours. Method M3 : Semen was diluted 1:1 in a specific solution (concentration of glycerol 2%) and cooled to 5 °C (−0.25 °C/min). The sample was then diluted again in the same solution to the final cellular concentration (final concentration of glycerol 4%). It was left for 1 h at 5 °C and then frozen in a programmable biofreezer (−20 °C/min down to −100 °C). Best total motility (TM) and progressive motility (PM) (75.8 and 55.18%) were obtained using Method M3. Methods M1 and M3 gave significantly higher values ( P P