¹Animal Biotechnologies Laboratory (LBA), Institute of Veterinary Sciences, Saad Dahleb University, BP270, Soumaa, 09000, Blida, ALGERIA
²Biotechnologies Platform for Animal Medicine & Reproduction (BIOMERA), Saad Dahleb Blida University 1, ALGERIA
³Iowa State University, Department of Animal Science, Ames, Iowa, USA
⁴Higher National Veterinary School (ENSV), Issad Abbes, Algiers, ALGERIA DOI : 10.9775/kvfd.2026.36549 The present study evaluated the effect of glycerol concentration, treatment with cholesterol andα-tocopherol loaded into methyl-β-cyclodextrin, and sperm cell concentration on the quality of equine spermatozoa after two cycles of cryopreservation. Two experiments were conducted. The first trial utilized 18 ejaculates collected from six stallions. Each ejaculate was evaluated initially using computer assisted sperm analysis (CASA) and flow cytometry (FCM), and spermatozoa were subsequently frozen in a commercial diluent (containing 2.5% glycerol: control) or in one of four experimental diluents (each supplemented with 1%, 2%, 3% or 4% glycerol). Frozen samples were thawed and evaluated again prior to a second freeze-thaw cycle. The glycerol concentration that produced the best result was further investigated in experiment 2 where spermatozoa previously frozen in 2.5% glycerol were thawed and refrozen in one of three sperm cell concentrations (1, 5, 05 10 x106 spermatozoa/mL) treated or not treated with cholesterol and α-tocopherol incorporated into methyl-β-cyclodextrin. Semen analysis (CASA and FCM) was repeated after the second freezing and thawing. Results revealed that the 2.5% concentration of glycerol was optimal for refreezing equine spermatozoa. Centrifugation (900xg for 10 minutes) of the equine semen before refreezing resulted in a sperm loss rate of 12% but was associated with good viability and acrosomal integrity. Treatment of semen with cholesterol and α-tocopherol loaded into methyl-β-cyclodextrin before refreezing improved (P<0.05) the post-thaw quality of spermatozoa (motility, membrane and acrosome integrity, and oxidative status) refrozen at 1×10⁶ spermatozoa/mL, thus allowing production of 100 to 300 ICSI straws from a standard 0.5 mL equine artificial insemination straw. Keywords : Cryopreservation, Sperm, Glycerol, Methyl-Β-cyclodextrin, Stallion, Vitamin E