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Sperm sorting by flow cytometry is a useful technology in the bovine industry, but the conception rates after artificial insemination using sex-sorted sperm are lower than when using the un-sorted sperm. In this study, we have investigated the causes for these low conception rates. We have focused on changes caused by flow cytometry to the glycocalyx, which forms the outermost surface of the sperm membrane. We have also evaluated the effects of capacitation on the glycocalyx since capacitation involves a redistribution of the sperm membrane that is vital for successful fertilization and conception. Lectin histochemistry was used to visualize the structure of the sperm glycocalyx. Lectin-staining sites were examined in non-treated sperm, sex-sorted sperm, and capacitated sperm. We have detected six different staining patterns related to different labeling regions of the sperm. Phaseolus vulgaris-erythroagglutinin (PHA-E) lectin-staining patterns of non-treated sperm were very different from those observed for sex-sorted sperm or capacitated sperm, suggesting that both, sex sorting by flow cytometry and the capacitation process affected the glycocalyx structures in the sperm. In addition, the total tyrosine-phosphorylation level in sex-sorted sperm was significantly higher than that in the non-treated sperm. Therefore, we concluded that the unexpected capacitation of bovine sperm during flow cytometry is associated with changes in the glycocalyx. Since premature capacitation leads to low conception rates, this unexpected capacitation could be a cause of low conception rates after artificial insemination using sex-sorted sperm.

Effects on glycocalyx structures of frozen-thawed bovine sperm induced by flow cytometry and artificial capacitation