Effect of bicarbonate and polyvinyl alcohol on in vitro capacitation and fertilization ability of cryopreserved equine spermatozoa

Abstract

Background: Factors contributing to the limited success of in vitro fertilization inhorses remain to be studied. In this work, we elucidated the effect of different essentialcapacitation media components, bicarbonate, and bovine serum albumin or polyvinylalcohol, and the incubation microenvironment on sperm parameters associated withcapacitation, acrosome reaction, and their ability to activate oocytes via heterologousintracytoplasmic spermatozoa injection in equine cryopreserved spermatozoa.Methods: Frozen–thawed spermatozoa underwent incubation at different time intervals in either Tyrode’s albumin lactate pyruvate medium (non-capacitating; NC) orTyrode’s albumin lactate pyruvate supplemented with bicarbonate, bicarbonate andpolyvinyl-alcohol, bicarbonate and bovine serum albumin, polyvinyl-alcohol and bovineserum albumin alone. Protein kinase A-phosphorylated substrates and tyrosine phosphorylation levels, sperm motility, and acrosome reaction percentages were evaluated.After determining the best condition media (capacitating; CAP), heterologous intracytoplasmic spermatozoa injection on pig oocytes was performed and the phospholipaseC zeta sperm localization pattern was evaluated.Results: Incubation of frozen–thawed equine spermatozoa with bicarbonate andpolyvinyl-alcohol in atmospheric air for 45 min induced an increase in protein kinaseA-phosphorylated substrates and tyrosine phosphorylation levels compared to NCcondition. Sperm incubation in bicarbonate and polyvinyl-alcohol medium showedan increase in total motility and progressive motility with respect to NC (p ≤0.05). Interestingly, three parameters associated with sperm hyperactivation weremodulated under bicarbonate and polyvinyl-alcohol conditions. The kinematic parameters curvilinear velocity and amplitude of lateral head displacement significantlyincreased, while straightness significantly diminished (curvilinear velocity: bicarbonate and polyvinyl-alcohol = 120.9 ± 2.9 vs. NC = 76.91 ± 6.9 µm/s) (amplitudeof lateral head displacement: bicarbonate and polyvinyl-alcohol = 1.15 ± 0.02 vs NC = 0.77 ± 0.03 µm) (straightness: bicarbonate and polyvinyl-alcohol = 0.76 ± 0.01vs. NC = 0.87 ± 0.02) (p ≤ 0.05). Moreover, the spontaneous acrosome reaction significantly increased in spermatozoa incubated in this condition. Finally, bicarbonate andpolyvinyl-alcohol medium was established as CAP medium. Although no differenceswere found in phospholipase C zeta localization pattern in spermatozoa incubatedunder CAP, equine spermatozoa pre-incubated in CAP condition for 45 min showedhigher fertilization rates when injected into matured pig oocytes (NC: 47.6% vs. CAP76.5%; p ≤ 0.05).Conclusion: These findings underscore the importance of bicarbonate and polyvinylalcohol in supporting critical events associated with in vitro sperm capacitationin the horse, resulting in higher oocyte activation percentages following heterologous intracytoplasmic spermatozoa injection. This protocol could have an impact onreproductive efficiency in the equine breeding industry.