Manipulation of bicarbonate concentration in sperm capacitation media improves in vitro fertilisation output in porcine species

摘要

The in vivo concentration of bicarbonate (HCO3?), one of the essential sperm capacitating effectors, varies greatly in the different environments sperm go through from cauda epididymis to the fertilisation site. On the contrary, porcine in vitro sperm capacitation and fertilisation media usually contains a standard concentration of 25?mmol/L, and one of the main problems presented is the unacceptable high incidence of polyspermy. This work hypothesised that by modifying the HCO3? concentration of the medium, the output of in vitro sperm capacitation and fertilisation could be increased. Once exposed to the capacitation medium, the intracellular pH (pHi) of spermatozoa increased immediately even at low concentrations of HCO3?, but only extracellular concentrations of and above 15?mmol/L increased the substrates protein kinase A phosphorylation (pPKAs). Although with a significant delay, 15?mmol/L of HCO3? stimulated sperm linear motility and increased other late events in capacitation such as tyrosine phosphorylation (Tyr-P) to levels similar to those obtained with 25?mmol/L. This information allowed the establishment of a new in vitro fertilisation (IVF) system based on the optimization of HCO3? concentration to 15?mmol/L, which led to a 25.3% increment of the viable zygotes (8.6% in the standard system vs. 33.9%). Optimising HCO3? concentrations allows for establishing an IVF method that significantly reduced porcine polyspermy and increased the production of viable zygotes. A concentration of 15?mmol/L of HCO3? in the medium is sufficient to trigger the in vitro sperm capacitation and increase the fertilisation efficiency in porcine.