Selected Metabolites Found in Equine Oviductal Fluid do not Modify the Parameters Associated to Capacitation of the Frozen-thawed Equine Spermatozoa In Vitro
2022
Fernández-Hernández, Pablo | García-Marín, Luis Jesús | Bragado, María Julia | Domingo, Andrés | González-Fernández, Lauro | Macías-García, Beatriz
In the horse, a repeatable protocol for in vitro fertilization has not been developed, possibly due to incomplete sperm capacitation. We have previously identified the metabolites present in equine oviductal fluid (OF). We aimed to test the effects of different metabolites found in equine oviductal fluid on quality parameters of frozen-thawed spermatozoa. Different concentrations of myoinositol (5–25 mM), lactate (6–60 mM), glycine (0.1–5 mM), β-alanine (1–6 mM), and histamine (0.05–0.4 mM) were added independently to modified Whitten's medium (pH = 7.25). Thawed equine spermatozoa (three stallions, one ejaculate per stallion, n = 3) were incubated for 2 hours at 37˚C in presence of the selected metabolites. After sperm incubation, total motility (TM), and progressive motility (PM) were evaluated by computer-assisted sperm analysis. Viability (SYBR-14⁺/PI⁻), mitochondrial membrane potential (ΔΨm) (JC-1), acrosome reaction (PNA⁺/PI⁻) and reactive oxygen species (ROS) production (CellRox⁺/PI⁻), were evaluated by flow cytometry. Protein tyrosine phosphorylation (PY) was evaluated by indirect immunofluorescence. Our results show that the addition of the metabolites at the dosages tested does not exert any effect on the sperm parameters analyzed. More research is needed to ascertain if metabolite addition at the dosages found in the equine OF exerts any remarkable effect on in vitro equine sperm capacitation.
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