Function of ram spermatozoa frozen in diluents supplemented with casein and vegetable oils

The aim of this study was to assess biologically safer components as alternatives to egg yolk for the frozen storage of ram semen using casein, coconut or palm oil in either Salamon's diluent (S) or a swim-up medium (SU). Ejaculates were frozen as pellets and sperm motility (subjectively) and acrosome integrity (FITC-PNA/PI) by flow cytometry were assessed at 0, 3 and 6h after thawing and incubation at 37°C. Three experiments were done: different concentrations of palm oil (5%, 10% and 20%); casein added as emulsifier and protective agent; and differences between egg yolk, coconut and palm oil in S and SU. 20% of oil added to SU accounted for a lesser percentage (P<0.05) of motile cells compared to rest while no differences were found between different oil levels on viable cells. When casein was added to diluents containing 5% of palm oil, no differences were found between palm or casein (P>0.05). No differences were found when S and SU were compared neither as groups nor between S alone and containing coconut or palm oil; however, SU alone yielded less motility than SU 5% coconut. However, in both groups, S and SU, egg yolk accounted for the greatest values in both bases. These results indicate that none of biologically safer media components (casein, palm or coconut oil) used in this study maintained the function of ram spermatozoa after freeze-thawing better than S-containing egg yolk. The application of vegetable oils as substitutes for egg yolk in diluents for the cryopreservation of ram spermatozoa requires further research.