Kinetic activity, membrane mitochondrial potential, lipid peroxidation, intracellular pH and calcium of frozen/thawed bovine spermatozoa treated with metabolic enhancers

Owing to the progressive decline of sperm motility during storage there is a need to find substances capable of enhancing sperm energy metabolism and motility and/or preserving it from oxidative damage. The aim of this study was to evaluate in frozen/thawed bovine spermatozoa the effect of several compounds, such as myo-inositol, pentoxifylline, penicillamine + hypotaurine + epinephrine mixture (PHE), caffeine and coenzyme Q10+ zinc + d-aspartate mixture (CZA), on either kinetic or metabolic parameters. Sperm kinetics was evaluated by Sperm Class Analyser whereas specific fluorochromes were used to evaluated mitochondrial membrane potential (MMP), intracellular pH, intracellular calcium concentration and lipid peroxidation. Lipid peroxidation was also evaluated by TBARS analysis. Treatments significantly affected total and progressive motility with different dynamics in relation to the incubation time. After the first hour of incubation, CZA treatment produced the best performance in total and progressive sperm motility as well as in curvilinear velocity, average path velocity and amplitude of head displacement, whereas pentoxifylline stimulated the highest straight-line velocity. MMP showed higher values (p<0.01) after treatment with pentoxifylline and PHE. Intracytoplasmic calcium concentration and lipid peroxidation were significantly (p<0.05) affected by the incubation time rather than the treatments. Intracellular pH varied significantly (p<0.01) in relation to either the incubation time or treatments. In particular, it showed a progressive increase throughout incubation with values in control group significantly higher than in myo-inositol, PHE, caffeine, pentoxifylline and CZA groups (7.37 +/- 0.03 vs. 7.29 +/- 0.03, 7.28 +/- 0.03, 7.26 +/- 0.03, 7.22 +/- 0.03 and 7.00 +/- 0.03, respectively; p<0.01).; however, among treatments, CZA displayed the lowest values. Significant correlations were found between sperm kinetic and metabolic parameters. These findings provide new comparative information on the effects of putative metabolic enhancers on kinetics and metabolic activities of bovine spermatozoa. In this study, a rapid methodological approach for evaluating sperm quality is proposed.