antioxidants Article Sperm Motility, Oxidative Status, and Mitochondrial Activity: Exploring Correlation in Different Species Alessandra Gallo 1, * , Maria Consiglia Esposito 1 , Elisabetta Tosti 1 and Raffaele Boni 1,2, *   Citation: Gallo, A.; Esposito, M.C.; Tosti, E.; Boni, R. Sperm Motility, Oxidative Status, and Mitochondrial Activity: Exploring Correlation in Different Species. Antioxidants 2021, 10, 1131. https://doi.org/10.3390/ antiox10071131 Academic Editor: Marco G. Alves Received: 22 June 2021 Accepted: 13 July 2021 Published: 16 July 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). 1 Department of Biology and Evolution of Marine Organisms, Stazione Zoologica Anton Dohrn, Villa Comunale, 80121 Naples, Italy; mariaconsiglia.esposito@szn.it (M.C.E.); tosti@szn.it (E.T.) 2 Department of Sciences, University of Basilicata, 85100 Potenza, Italy * Correspondence: alessandra.gallo@szn.it (A.G.); raffaele.boni@unibas.it (R.B.); Tel.: +39-081-5833233 (A.G.); +39-0971-205017 (R.B.) Abstract: Sperm quality assessment is the first step for evaluating male fertility and includes the estimation of sperm concentration, motility, and morphology. Nevertheless, other parameters can be assessed providing additional information on the male reproductive potential. This study aimed to evaluate and correlate the oxidative status, mitochondrial functionality, and motility in sperma- tozoa of two marine invertebrate (Ciona robusta and Mytilus galloprovincialis) and one mammalian (Bos taurus) species. By combining fluorescent staining and spectrofluorometer, sperm oxidative status was evaluated through intracellular reactive oxygen species (ROS) and plasma membrane lipid peroxidation (LPO) analysis. Mitochondrial functionality was assessed through the mitochondrial membrane potential (MMP). In the three examined species, a negative correlation emerged between sperm motility vs ROS levels and LPO. Sperm motility positively correlated with MMP in bovine, whereas these parameters were not related in ascidian or even negatively related in mussel sper- matozoa. MMP was negatively related to ROS and LPO levels in ascidians, only to LPO in bovine, and positively related in mussel spermatozoa. These results suggest that energy sources for sperm motility vary between species and that ROS causes a decline in sperm motility via oxidative damage of membrane lipids. Overall, this study validates the use of fluorescent probes in combination with spectrofluorometer as a simple and powerful methodology for supplementary evaluation of sperm quality shedding light on new potential quality markers and provided relevant information on sperm energetic metabolism. Keywords: ascidian; bovine; fluorescent probe; intracellular reactive oxygen species; mitochondrial activity; mussel; oxidative status; plasma membrane lipid peroxidation; sperm motility; spermatozoon 1. Introduction The spermatozoon is a highly specialized haploid cell generated in the male gonad through a differentiation process called spermatogenesis. The function of spermatozoon is to deliver the paternal genome into the female gamete during fertilization, the very special event in which the two gametes fuse their genomes originating a zygote, the first cell of a new diploid individual. To accomplish this task, spermatozoa are equipped with specific structures: a flagellum, which generates the movement, and a very compact nucleus that ensures protection of the paternal genome. Sperm motility is an essential requirement to ensure the fertilization process and its activation is induced by ionic changes or compounds released from the oocyte or female reproductive tract and implicates the activation of both the motility apparatus and energy metabolism. The motility apparatus is localized into the flagellum and consists of a highly organized microtubule-based structure called axoneme; this organization is well conserved through evolution [1]. Flagellar movement requires an adequate supply of energy in the form of ATP, which is used by the flagellar dynein-ATPase localized into the axoneme along the entire length of the flagellum [2]. A relevant source of ATP Antioxidants 2021, 10, 1131. https://doi.org/10.3390/antiox10071131 https://www.mdpi.com/journal/antioxidants