Pentoxifylline and EDTA can modulate the capacitation of frozen/thawed donkey sperm

Capacitation is a process in which sperm undergo a series of modifications that allow it to fertilize the oocyte. Capacitation inducers are agents or substances that promote these changes essential for in vitro fertilization.

To evaluate the use of capacitation inducers in frozen/thawed donkey semen.

Fifteen semen samples from five Creole donkeys were frozen. Post-thawing, semen samples were supplemented with pentoxifylline (PTX) at 2.5, 5.0, and 7.5 mM; ethylenediaminetetraacetic acid (EDTA) at 0.5, 1.0, and 1.5 mM; heparin (HEP) at 1.1 mM (positive control). A non-supplemented sample (negative control) was included. Sperm motility and kinematics, sperm viability (SV), and acrosome integrity (AI) were evaluated using an IVOS sperm analyzer, and fluorescence microscopy with the SYBR14/PI and FITC-PSA probes, respectively. Capacitation, mitochondrial membrane potential (ΔΨM), and intracellular calcium (Ca²⁺) concentration were assessed using flow cytometry.

The use of 2.5 mM PTX showed an increase in total motility (TM), while the addition of 5.0 mM PTX improved the SV of the thawed semen. EDTA was found to reduce motility and some kinematic parameters of post-thawed sperm at concentrations starting at 0.5 mM. The addition of 1.0 and 1.5 mM EDTA, and 5.0 and 7.5 mM PTX increased capacitation (membrane destabilization). Furthermore, EDTA at 0.5 mM, and PTX at 2.5, 5.0, and 7.5 mM reduced intracellular Ca²⁺ concentrations.

Motility, kinematics, and viability of frozen/thawed donkey sperm, as well as other events associated with their capacitation such as membrane stability and Ca²⁺ influx, can be modulated by supplementation with PTX and EDTA.