Substitution of egg yolk by a cyclodextrin-cholesterol complex allows a reduction of the glycerol concentration into the freezing medium of equine sperm

The aim of this work was to completely replace the egg yolk a classical diluent for freezing equine semen by a cyclodextrin-cholesterol complex. At the same time, the reduction in the glycerol content used for cryopreservation and the incubation time between sperm and the freezing media were evaluated. Horse ejaculates were frozen with four different freezing extenders: a frozen reference medium (IF) containing egg yolk and 2.5% glycerol and media without egg yolk but supplemented with 1.5 mg 2-hydroxypropyl-beta-cyclodextrin cholesterol (HP beta CD-C) complex and containing either 1% (G1), 2% (G2) or 3% glycerol (G3). Three incubation times (90, 120 and 180 min) at 4 C between the fresh semen and the different media were tested before freezing. Viability and motility analyses were performed with computer assisted semen analysis (CASA). Results showed that the freezing media containing the HP beta CD-C complex with 1%, 2% and 3% glycerol significantly improve the 3 in vitro parameters of post thawing semen quality (viability, progressive and total mobilities) compared to IF. The best improvement of the parameters was obtained with Cl medium and the longest contact time. The substitution of egg yolk by HP beta CD-C complex allows the decrease of protein charge of the medium while favouring the cholesterol supply to membrane spermatozoa offering it a better resistance to osmotic imbalance and a better tolerance to the glycerol toxicity. Our results highlight that the egg yolk of an extender for the freezing of horse semen can be completely substituted by HP beta CD-C complex. (C) 2015 Published by Elsevier Inc.