The highly dynamic plasma membrane exquisitely regulates sperm function. While this membrane serves as the barrier between the sperm and its environment, it also must undergo remarkable post-testicular modifications thought to be required for fertilisation. Indeed, plasma membrane modifications during epididymal transit and capacitation have been well described. In particular, the plasma membrane is enriched in cholesterol in the epididymis, whereas cholesterol efflux has been demonstrated to initiate the intracellular signalling cascades that lead to capacitation, thereby priming the sperm for acrosomal exocytosis.

In animal production, artificial insemination (AI) using semen from genetically-superior males is a well-established strategy for herd improvement that has greatly benefited the agricultural industry worldwide. Similarly, reproductive strategies for conservation of endangered species also rely heavily on AI. The advantages of AI are considerably enhanced by the use of cryopreserved semen. Cryopreservation, however, decreases the viability and motility of sperm from many species, while inducing spontaneous capacitation (“cryocapacitation”) and acrosome reactions (AR). Sperm from species with high membrane cholesterol:phospholipid ratios, however, can resist cooling. Based on the hypothesis that enhancing the cholesterol content of sperm would improve the resistance of mammalian sperm to cryopreservation and using the goat as a model, our team has assessed sperm function after treating with cholesterol-loaded methyl-beta-cyclodextrin (CLC).
Indeed, CLC treatment enriches sperm membrane cholesterol, which increases sperm tolerance to cold shock and osmotic stress. These results suggest that that enriching sperm cholesterol levels using CLC would improve sperm resistance to cryopreservation. Work on other species, however, demonstrates only modest increases in sperm quality and no improvement in fertility following CLC treatment in traditional egg yolk-based extenders. We hypothesised, therefore, that a skim milk-based extender would more effectively permit CLC to exchange with and protect sperm. Without CLC, there was no difference in sperm quality between extenders after thawing. However, the percentages of motile, progressively motile, or uncapacitated sperm increased with CLC in skim milk extender even during incubation after thawing for 3 h at 38.5°C. In contrast, CLC treatment did not alter the post-thaw quality of sperm in egg yolk extender. A pilot AI trial revealed that enriching sperm with cholesterol using CLC in the skim milk extender markedly increases in vivo fertility.
We conclude, therefore, that cyclodextrin-mediated delivery of exogenous cholesterol improves sperm tolerance to cold temperatures and osmotic stress, and that this transfer is biologically more effective in skim milk-based relative to egg yolk-based extenders. Additional research is required to perfect the protocol and to test this strategy on other species whose reproduction would benefit from semen cryopreservation.