DOUBLE CONGELATION DE LA SEMENCE EQUINE (Effet de la concentration en glycérol et de la dilution finale sur la qualité spermatique)

Abstract

The objective of this study is to evaluate the effect of glycerol concentration, final sperm concentration, and treatment with cholesterol and α-tocopherol loaded cyclo-dextrins on equine sperm quality across two cryopreservation cycles. Two experiments were carried out. In experiment1, eighteen ejaculates harvested from six stallions were used. Each ejaculate was evaluated initially (CASA & CMF), then frozen in 1 commercial diluent (INRA FREEZE®, 2.5% glycerol) and four experimental diluents composed from (INRA96®) supplemented with 1%, 2%, 3% and 4% glycerol respectively thawed and evaluated again. A second freeze-thaw cycle was performed. The glycerol concentration giving the best result will be retained for the experiment2. In experiment 2, straws previously frozen in INRA-Freeze® were thawed and refrozen in three dilutions (1: 10, 1: 20, 1: 100) treated or not with cholesterol and α-tocopherol loaded cyclo-dextrins. An analysis (CASA and CMF) was performed after thawing. The concentration of 2.5% glycerol (INRA-Freeze ®) was optimal for a refreezing of equine spermatozoa. Centrifugation of the equine seed before refreezing (600xg, 10mn on a "Maxifreeze®" cushion) reduced the rate of cell loss to 12% associated with good acrosome viability. Treatment of the cholesterol and α-tocopherol loaded cyclo-dextrins(CLC + TLC) significantly improved the quality of equine spermatozoa (mobility, membrane and acrosomal integrity, and oxidative status) refreezed at low concentration of 1 million spermatozoa per milliliter, allowing production of 100 to 300 ICSI flakes from a conventional flake of AI.