Post-thaw survival and longevity of bull spermatozoa frozen with an egg yolk-based or two egg yolk-free extenders after an equilibration period of 18 h

The aim of the present study was to determine the suitability of using two egg yolk-free commercial extenders, Andromed (R) and Biociphos Plus (R), as compared with the Tris-egg yolk based diluent Biladyl (R), for the cryopreservation of bull spermatozoa when the freezing protocol involved holding the extended semen at 4 degrees C for 18 h before the freezing. Six ejaculates from each of 10 Holstein bulls were collected by using artificial vagina. The ejaculates were evaluated for volume, sperm concentration and motility, divided in to three equal volumes, and diluted, respectively, with the three extenders as specified above. Extended semen was equilibrated for 18 h at 4 degrees C and frozen in 0.25-ml straws. After thawing, 100-mu l aliquots of semen were labelled with SYBR-14, PI and PE-PNA (Phycoerythrin-conjugated Peanut agglutinin) and analysed by flow cytometry at 0, 3, 6 and 9 h after incubation at 37 degrees C. A General Lineal Model procedure for repeated measures was used to determine the effects of extender, bull, replicate and the interaction between them, on sperm viability and acrosomal integrity. Semen samples frozen with Biladyl (R) showed higher (p < 0.001) sperm survival after 0 h (47.9%) and 9 h (30.3%) of incubation than those frozen with Andromed (R) (38.5% and 17.3%, after 0 and 9 h respectively) or Biociphos Plus (R) (34.9% and 21.6%, after 0 and 9 h respectively). The bull and replicate had significant effects (p < 0.001) on both sperm viability and acrosomal integrity, but the interactions between bull and extender and between replicate and extender were not significant. It was concluded that, when holding the semen overnight before freezing, the use of Biladyl (R) results in higher sperm survival and longevity than the use of Andromed (R) or Biociphos Plus (R).