In this study boar sperm mitochondrial activity was studied and deepened in order to delineate the main metabolic strategies used by boar sperm to obtain energy and to link them to sperm function. Boar spermatozoa were collected, diluted at 30 × 106 spz/mL and incubated for 1 h with: Rotenone (ROT), complex I inhibitor, Dimethyl-malonate (DMM), complex II inhibitor, antimycin A (ANTI), complex III inhibitor, oligomycin (OLIGO), ATP synthase inhibitor, Carbonyl cyanide m-chlorophenyl hydrazone (CCCP), uncoupling agent, 2-deoxy-glucose (2DG), glucose agonist, and Dimethyl sulphoxide (DMSO) as control vehicle. Viability and mitochondrial membrane potential (Sybr14/PI/JC1 staining) and sperm motility (using CASA system) were assayed after incubation. ROT, ANTI, OLIGO and CCCP significantly reduced total and progressive motility as well as cell velocities; ANTI and CCCP depressed mitochondrial membrane potential but did not affect cell viability. Cluster analysis of kinematic parameters showed some interesting features of sperm subpopulations: ANTI and CCCP caused a shift in sperm subpopulation towards "slow non progressive" cells, OLIGO and ROT caused a shift towards "average" and "slow non progressive" cells, while DMM and 2DG increased the "fast progressive" cells subpopulation. Sperm mitochondrial respiration and substrate oxidation, assayed polographically and spectrofluorimetrically, respectively pointed out a high ATP turnover and a low spare respiratory capacity, mainly linked to the NADH-O2 oxidase activity. Therefore, boar spermatozoa heavily rely on mitochondrial oxidative phosphorylation, and especially on Complex I activity, to produce ATP and fuel motility.
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