The Complementary Effect of Cholesterol and Vitamin E Preloaded in Cyclodextrins on Frozen Bovine Semen: Motility Parameters, Membrane Integrity and Lipid Peroxidation

During cryopreservation sperm cells suffer from two major deleterious impacts: oxidative stress and cold shock.

To investigate in bovine species the benefit of cholesterol and vitamin E, both loaded in cyclodextrins, as a double protection against oxidative stress and cold shock.

Semen was collected from nine mature bulls using an artificial vagina and each ejaculate was split into four equal aliquots. The control aliquot was diluted with Fraction A (Tris+citric acid+fructose+penicillin) without further supplementation; the treated samples were diluted in Fraction A supplemented with cholesterol-loaded cyclodextrins (CD-CHL), vitamin E-loaded cyclodextrins (CD-Vit E) or CD-CHL in association with CD-Vit E (CD-CHL-VitE). After incubation at 22°C for 15 min and addition of Fraction B (Fraction A+egg yolk+glycerol), all aliquots were frozen in 0.25 ml straws. Straws were then thawed individually at 37C for 30 seconds in a water bath and immediately analyzed for motility, using Computer Aided Semen Analysis (CASA), membrane integrity and oxidative stress status.

The results showed that samples treated with CD-CHL and CD-Vit E were protected against the deleterious impact of freezing thawing process. However, the optimal protection was observed when the two complexes CD-CHL and CD-Vit E were simultaneously used. All analysed semen parameters including motility, membrane integrity and oxidative stress status were significantly improved in CD-CHL-Vit E compared to all other treatments.

Cholesterol and vitamin E, both preloaded in cyclodextrins to increase their solubility, appeared as a powerful protection in cryopreserved bovine semen to fight simultaneously cold shock and oxidative stress.