Sperm quality changes during equilibration time at 4 °C before cryopreservation of chicken semen

Cryopreservation procedure includes many steps and each one may affect sperm quality and then sperm viability after thawing. The aim of our experiment was to study if sperm quality changes occur during the initial steps of the cryopreservation pellet procedure in chicken semen. The initial steps, dilution rate and equilibration time at 4 °C, were studied. Ejaculates were collected from Mericanel della Brianza male breeders (n. 18), pooled and splitted into two aliquots, one diluted 1:2 and the other one 1:3 in prefreezing Lake’s diluent. Diluted aliquots were equilibrated at 4 °C for 40 minutes. The following semen parameters were measured on fresh semen (T0), after 20 (T20) and 40 (T40) minutes: motility (%), VCL (µm/sec), VSL (µm/sec), LIN (%), ALH (µm), normal and abnormal viable sperm, dead sperm and damaged sperm (%). The main effects, dilution rate and equilibration time, and the relative interaction were studied by repeated measure analysis of variance. Sperm quality was significantly affected by the equilibration time, whereas the dilution rate and the interaction were not significant. The proportion of abnormal viable sperm significantly increased from T0 to T20 (15 to 21%). Other changes occurred from T20 to T40: the proportion of viable normal sperm significantly decreased compared to T0 (69 to 62%), and the VCL (131 to 119 µm/sec) and ALH (11 to 9 µm) values also significantly decreased. No further decrease in the proportion of abnormal viable sperm from T20 to T40 was recorded. Very similar trend in sperm quality was found with both dilutions during equilibration time at 4 °C. The variability found in semen concentration among ejaculates suggests the need to improve the criteria of dilution to standardize semen processing. In conclusion, short equilibration time, such as 20 minutes, in prefreezing diluent at 4 °C is recommended to prevent a decrease in sperm quality during chicken semen processing for cryopreservation.