Modifications of bull spermatozoa induced by three extenders: Biociphos, low density lipoprotein and Triladyl, before, during and after freezing and thawing

The success of artificial insemination with frozen semen implies the reduction of the deleterious effects on the cells induced by this technique. These effects can occur as early as during the first dilution in an extender, as well as at any step, during or after the freezing process. In this work, we have compared the modifications induced by Triladyl, low density lipoproteins (LDL) and Biociphos extenders, after dilution and cooling to 4 °C for 1, 4 and 24 h. Alterations in the cell structures were visualized by electron microscopy (EM). More than 80% of spermatozoa were injured after incubation for 4 h in Triladyl, while 3% and 47% were counted in LDL and Biociphos respectively. This latter extender was deleterious to cell membrane integrity after incubation for 4 h or longer. The ultrastructure of frozen spermatozoa was studied by EM of cryofixed-cryosubstituted samples obtained from regular 0.5 ml French straws frozen using our usual protocol. The main differences between samples concerned the size and appearance of the frozen extender veins, while very few cell defects were found to be added by the freezing process at any depth in the straws. After thawing, semen motility was twofold higher (P < 0.05) in Biociphos (64%) and LDL (61%) than in Triladyl (32%) and the cells were less altered in LDL. We concluded that the LDL extender offers a better protection for storage of frozen spermatozoa, and can probably also be used for the preservation of fresh semen for short periods.