Epsilon Open Archive

Abstract

There is considerable variability in the ability of spermatozoa from different stallions to withstand the freezing process. As a result, stallions are usually categorized as good or bad freezers. In the equine industry, the demand for frozen semen for artificial insemination is increasing, resulting in a need for optimising cryopreservation techniques. Previously the removal of seminal plasma (SP) prior to cryopreservation was shown to improve post-thaw sperm quality, whereas the effect of adding SP on spermatozoa varied among studies. Single Layer Centrifugation (SLC) is a technique that has been used to select the most robust spermatozoa from the ejaculate and prolong their viability, as well as separating them from SP. The aims of this thesis were: 1) to study the effect of combining SLC with addition of SP from stallions of known freezability on sperm cryosurvival, including their ability to bind to heterologous oocytes; 2) To explore the differences in the major protein groups in SP between the breeding and non-breeding seasons in relation to sperm quality. Selecting robust spermatozoa using SLC prior to freezing improved post-thaw sperm quality, whereas addition of pooled SP from good or bad freezers to the selected sperm samples did not have an additional beneficial effect. Sperm quality was adversely impaired after restoring pooled SP to SLC-selected spermatozoa after thawing, independently of whether the SP came from a good or a bad freezer stallion, suggesting an inability of SP to repair stallion sperm cryoinjuries in the presence of an egg yolk-containing extender. Sperm binding affinity to the zona pellucida (ZP) of bovine oocytes was reduced after adding SP from good freezer stallions prior to cryopreservation. However, when SP was added after thawing, the mean number of bound spermatozoa was higher for the group treated with SP from good freezers than for the group treated with SP from bad freezers. Minor differences in protein composition were observed between seasons as well as between good and bad freezers. In the nonbreeding season there was a higher content of heparin-binding proteins and also of a subset of non-heparin binding proteins compared to the breeding season; these proteins may be linked to fertility. Sperm quality did not decline during the non-breeding season and SP-testosterone levels did not vary between seasons. Customization of the cryopreservation protocol for individual stallions, including studying the effect of adding SP from individual stallions, would be required to optimize the effect on sperm cryosurvival. Global proteome investigation of SP proteins could help to identify cryosurvival biomarkers for stallion spermatozoa.