Effect of incubation of thawed equine spermatozoa under different capacitating conditions and binding to the Zona Pellucida
of mare oocytes.

Introduction: Eyaculated mammalian sperm accuire their ability to fertilize an oocyte during transit through the female reproductive tract, a process known as sperm capacitation. Induction of capacitation in vitro can be achieved by incubating spermatozoa with bicarbonate, calcium and albumin. However, in the equine species, in vitro sperm capacitation is not efficient. The aim of this study was to evaluate the effect of incubating thawed equine spermatozoa in SP-TALP medium with different capacitation inducers and assess the binding to the zona pellucida of these spermatozoa in mares oocytes.

MATERIALS AND METHODS:Spermatozoa were incuvated for 120 minutes at 38,50 C in SP-TALP base medium supplemented with MßCD, IBMX, dbAMPc. SP-TALP without bicarbonate and albumin was used as non-capacitating control. Acrosome reaction (PNA FITC), membrane fluidity (MC540) and intracellular Ca+2 ( Fluo-3) were analyzed by flow cytrometry. Capacitated sperm (1x 10 6 per ml) were taken and deposited with the oocytes for one hour, and then fixed stained to count the numbre bound sperm.

Results: Incubation of spermatozoa in capacitating conditions showed no differences in the acrosome reaction, howevwe, greater membrane fluidity and intracellular Ca+2 levels was observed in the treatments with capacitation inducers compared to the control. Additionally, a higher binding of spermatozoa to the zona pellucida was observed whith MßCD treatment.

Conclusions: Addition of capacitation inducers to the SP-TALP medium imporved the membrane fluidity and intracellular Ca+2 levels, without compromising the integrity of the plasma membrane, wich is a consistent with an increase in the capacitation of equine spermatozoa under these conditions. Additionally, binding to the zon pellucida of capacitated spermatozoa is higher in these capacitating conditions. Future research wil focus on analyzing the efect of these treatments on ICSI or IVF.

References

Maintan et al. Henning H. Bicarbonate-Stumulated Membrane Reorganization in Stallion Spermatozoa. Front Cell Boil Vol 9 772254. 2021.