The Effect of Adding b-Cyclodextrin and Cholesterol during Cyropreservation of Stallion Spermatozoa on Post-thaw Motility, Viability, Capacitation, and the Acrosome Reaction

University of Pennsylvania
School of Veterinary Medicine
 

      In the horse, the consistently lower pregnancy rates obtained by insemination of frozen-thawed sperm than are achieved using fresh or cooled semen indicate that current cryopreservation technologies for equine sperm require improvement.  Poor outcomes result largely from failure of equine sperm to survive the cryopreservation process and from reduced longevity of spermatozoa that maintain viability.  Both decreased viability and reduced lifespan due to premature capacitation are consequences of “cold shock”, the disruption of plasma and acrosomal membranes during rapid cooling.  Within the sperm cell plasma membrane, cholesterol molecules impart stability to the lipid bilayer by maintaining a random arrangement of phospholipids and transmembrane proteins.  Removal of cholesterol from the plasma membrane allows for re-organization of phospholipids and transmembrane proteins and contributes to capacitation.  During freezing, cholesterol impedes aggregation of phospholipids into crystalline arrays and, in doing so, ameliorates the effects of “cold shock” by slowing the phase transition of the membrane.  Cryopreservation of bovine and porcine sperm in the presence of cholesterol along with cyclodextrin, a cholesterol acceptor, has resulted in increased post-thaw viability and improved integrity of plasma and acrosomal membranes.  Treatment of equine sperm with cyclodextrin-cholesterol complexes has also resulted in improved post-thaw viability and membrane integrity; however, a tendency towards pathological stabilization of the plasma membrane resulting in impaired capacitation and acrosome reaction has been noted.  In the absence of exogenous cholesterol, cyclodextrin extracts cholesterol from sperm plasma membranes, contributing to capacitation.  Thus, relative concentrations of cyclodextrin and cholesterol must be carefully balanced.  The ultimate goal of this study is to develop a protocol for supplementation of a defined freezing extender using concentrations of cyclodextrin and cholesterol that maximize motility and viability while maintaining the ability of the sperm to capacitate and acrosome react in response to physiologic stimuli.  A preliminary study using multiple ejaculates from 2 stallions will be conducted to determine the range of concentrations of cyclodextrin and cholesterol that are non-spermiotoxic and produce the expected effect upon exposed sperm.  Using 3 ejaculates from 6 stallions, this study will then investigate the effect of various concentrations of cyclodextrin and cholesterol on post-thaw motility, viability, capacitation and acrosome reaction of frozen sperm of stallions of variable but known fertility.  From these data, concentrations of cyclodextrin and cholesterol that optimize post-thaw sperm parameters will be determined.

This project has been funded by a grant from the Raymond Firestone Trust.