Glycerol addition and conservation of fresh and cryopreserved ram spermatozoa

Fresh extended ram semen has a short fertile lifespan whereas acceptable fertility with cryopreserved semen is achieved only by laparoscopy, which limits widespread artificial insemination in sheep. Although glycerol is considered essential for freezing spermatozoa, it is often included in extenders for short-term storage at above-freezing temperatures. To test the hypothesis that glycerol reduces the function of fresh sperm, ram semen was divided into two aliquots and diluted with commercial extenders that were identical, except that one contained 7% glycerol (n = 6). In a second experiment, ram semen was prepared for cryopreservation by a one-step dilution with a 7% glycerol extender or gradually, with a two-step protocol, to test the hypothesis that the method and time of glycerol addition affects sperm quality after freezing and thawing (n = 7). For both experiments, semen was diluted in a synthetic oviductal fluid (SOF-m) and sperm quality was assessed by computer-assisted motility, viability and chlortetracycline fluorescence (CTC) patterns (an indicator of capacitation status). The presence of glycerol did not affect the quality of fresh sperm (P > 0.27). For cryopreserved sperm, the method of glycerol addition also did not affect thawed sperm. However, a decrease in sperm motility and viability, and different distribution of CTC patterns occurred due to the duration of time in extender and in SOF-m (P ≤ 0.0002). Cryo-capacitation was also observed. In conclusion, the presence of glycerol in the extender did not reduce ram sperm quality during conservation of the semen at 5°C or when it was used to completely and rapidly dilute the semen before cooling for cryopreservation. Key words: Sheep, Triladyl, Biladyl, chlortetracycline, artificial insemination, spermatozoa.