The aim of the present study was to investigate the potential benefit of vitamin E and cholesterol when preloaded in cyclodextrins, alone or in association to protect ram epididymal sperm during the freezing-thawing process. Epididymal sperm were collected from Twenty four testes; sperm from the two testicles of each ram was pooled and divided in 7 aliquots. The control aliquot was diluted with Fraction A (Tris-citric acid-fructose) without further supplementation. The Six (6) other aliquots were diluted with fractions A containing cyclodextrins (CD), cholesterol (CHL), vitamin E (Vit E), cholesterol-loaded cyclodextrins (CD-CHL), vitamin E-loaded cyclodextrins (CD-Vit E) and CD-CHL and CD-Vit E (CD-CHL-Vit E), respectively. After incubation at 22 °C for 15 min and addition of Fraction B (Fraction A-egg yolk-glycerol), all aliquots were equilibrated at 4 °C for 2 h and then frozen in liquid nitrogen. A Computer Aided Semen Analysis (CASA) was used to investigate the impact on different motility parameters and the hypo-osmotic swelling test (HOST) to quantify membrane functionality. The Oxidative stress impact on sperm membrane was investigated through lipid peroxidation (LPO) measurement. After thawing, CD-Vit E and CD-CHL treatments improved significantly (P < 0.05) the total motility, VAP and linearity (LIN), compared to the control, Vit E and CHL samples. However, the association of CD-CHL and CD-Vit E (CD-CHL-Vit E) exhibited a significant effect on total motility, progressive motility, membrane functionality, sperm velocities (VCL, VSL and VAP) and LIN (P < 0.05). Membrane lipid peroxidation was significantly lower in semen pretreated with CD-Vit E than in control and Vit E alone. Among all treatments, the association of CD-CHL and CD-Vit E (CD-CHL-Vit E) showed the highest protection against LPO (P < 0.05). The present results revealed that the significant impact was observed when vitamin E, cholesterol and cyclodextrins are all used in the same treatment, thus demonstrating the complementary effect of solubilized vitamin E and cholesterol in protecting concomitantly spermatozoa against cold shock and oxidative stress.

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