Assessment of boar sperm intracellular Ca²⁺ level and motility characters by flow cytometry and CASA. Optimizing the protocol for Fluo-4 assessment of sperm intracellular Ca²⁺level by flow cytometry and investigation of relationships between storage time, intracellular Ca²⁺ and sperm motility characters

Abstract

Porcine production industry demand effective artificial insemination for future challenges. Variation in field fertility and litter size not only caused by sows or farm management but also affected by semen and boar related parameters. Assessment of semen using objective methods provides an effective tool for better and precise assessment of sperm quality during the collection until consumption and leads to prediction of field fertility and genetic selection. In order to evaluate sperm intracellular Ca²⁺ level by flow cytometry, a Fluo-4 assay was successfully adapted for boar spermatozoa by minimizing the handling effect and optimizing the Fluo-4 incubation and concentration. Clear different Fluo-4 patterns were observed for spermatozoa with low and high intracellular Ca²⁺ by fluorescence microscope and flow cytometry. In the present study, semen samples from Norwegian Landrace and Duroc were analyzed by flow cytometry for assessment of sperm intracellular Ca²⁺ level and CASA for sperm motility parameters on the day of collection and after 4 days liquid preservation at 18 °C. Results showed that in both Duroc and Landrace, proportion of sperm cells with high intracellular Ca²⁺ were increased during the storage time. The amount of motile sperm cells was remained constant during the preservation in Landrace and decreased in Duroc. Furthermore, high degree of hyperactivated sperm cells were detected in Duroc on the day of collection and hyperactivited sperm population decreased after 4 days preservation while, Landrace sperm motility pattern developed toward hyperactivation. In addition, for Duroc semen, hyperactivation significantly affected positively by motility and negatively by intracellular Ca²⁺ . While, Landrace semen hyperactivity level was affected just by intracellular Ca²⁺ level. The present finding provides further evidence supporting the effect of breed on sperm physiology and behavior. Moreover, this study has shown that objective methods for assessment of semen quality could count as valid, rapid and precise methods in sperm evaluation.