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Use of Cholesterol in Sperm Cryopreservation: Present Moment and Perspectives to Future E Moce ?

1 , E Blanch1 , C Toma ? s1 and JK Graham2

1 Centro de Tecnolog??a Animal C Instituto Valenciano de Investigaciones Agrarias (CITA-IVIA), Pol??gono '

La Esperanza'

, Segorbe, Castello ?n, Spain;

2 Department of Biomedical Sciences, Colorado State University, Fort Collins, CO, USA Contents Sperm cryosurvival rates are not optimal for most species.

Therefore, new cryopreservation strategies are needed with the objective of increasing the number of surviving sperm and the quality of those sperm after thawing. Cholesterol plays important roles in many sperm functions, including e?ects on membrane properties. One of these e?ects is to stabilize membranes at low temperatures. Thus, species that produce sperm which possess high membrane cholesterol : phospho- lipid ratios are more resistant to cold shock than sperm with low cholesterol : phospholipid ratios. Therefore, increasing the cholesterol content of sperm membranes may be a strategy that can improve sperm quality after freeze-thawing. In this review, information is presented related to using cyclodextrins pre-loaded with cholesterol for cryopreserving sperm from di?erent species. The topics discussed include both in vitro and in vivo assessments of sperm quality after cryopreservation, as well as how increasing sperm cholesterol content a?ects other sperm functions. Introduction Although many years have passed since glycerol was discovered to be an e?ective cryoprotectant for sperm cryopreservation (Polge et al. 1949), protocols for freezing sperm still result in relatively few surviving sperm for many species. Numerous studies, conducted over the years, have had the objective of increasing the quantity and quality of the sperm recovered after cryopreservation and, notwithstanding the advances made, most of the improvements observed in vitro have not resulted in increased fertilizing ability in vivo. Cryopreservation induces damage to all sperm com- partments, which have been reviewed previously (Bailey et al. 2000;

Medeiros et al. 2002;

among others). This damage is responsible for the loss of sperm motility, viability, acrosomal integrity and of course the fertiliz- ing capacity of frozen-thawed sperm (Holt 2000). However, cryopreserved sperm can be e?ectively used for arti?cial insemination (AI) in several species, when the AI techniques are modi?ed. In general, by increasing the number of sperm in the insemination, depositing the inseminate deeper into the female reproductive tract (the uterus or oviduct) and reducing the time interval between insemination and ovulation, pregnancy rates using cryopreserved sperm are improved (Watson 2000). Despite the improved fertility, these strategies require changing in the management schemes used by commer- cial farms, for many species, and these increased management costs combined with lower fertility, inhibit the extensive use of cryopreserved sperm for many farms. The ultimate goal for frozen semen is to obtain similar fertility rates with cryopreserved sperm as with fresh sperm, using low sperm numbers and using traditional AI techniques. Only when this objective is achieved, will cryopreserved sperm be attractive to both farmers and AI centres. Sperm cryopreservation is a multi-factorial problem, where the diluents, the protocols, the species, breed and the individual sires within each breed are merely some of the many parameters that need to be included in the overall equation for success. Each of these parameters includes a variety of factors and each in?uences the other parameters. Obviously, testing all factors at the same time to optimize a freezing protocol is technically impossible;