Nitric oxide in frozen-thawed equine sperm: Effects on motility, membrane integrity and sperm capacitation

• Published in: Animal reproduction science 2018-08, Vol.195, p.176-184
• Main Authors: de Andrade, André F.C., Arruda, Rubens P., Torres, Mariana A., Pieri, Naira C.G., Leite, Ticiano G., Celeghini, Eneiva Carla C., Oliveira, Leticia Z., Gardés, Thayna P., Bussiere, Maria Clara C., Silva, Daniela F.
• Format: Article
• Language: English
• Subjects: Capacitation; L-arginine; L-NAME; Nitric oxide; Spermatozoa
• ISSN: 0378-4320; 1873-2232
• DOI: 10.1016/j.anireprosci.2018.05.022

•Nitric oxide (NO) regulate of motility, capacitation and acrosome reaction in post-thawed equine sperm.•The effects of L-arginine and L-NAME of NO synthesis and scavenge of free radical on equine sperm during in vitro capacitation.•NO is involved in regulation of several variables (MOT, PROG, VCL, BCF, Hyper and ARIM) in the process of equine spermatozoa. Nitric oxide (NO) is a reactive nitrogen species (RSN) that, over the years, has been shown to be integrated with biological and physiological events, including reproductive processes. NO can affect the functionality of spermatozoa through free radical scavenging, deactivating and inhibiting the production of superoxide anions (O2.−). However, the role of NO in mammalian spermatozoa physiology seems paradoxical. The aim of this study was to investigate the effects of NO on motility, hyperactivation, membrane integrity, peroxidation, and capacitation in cryopreserved equine sperm. Ejaculates were collected and cryopreserved. After thawing, samples were centrifuged, suspended in an in vitro fertilization (IVF) medium and incubated with the following treatments: 1) C = control (IVF); 2) A = l-arginine (10 mM – In); 3) L = L-NAME (1 mM – Ih); 4) M = methylene blue (100 mM – Re); 5) AL = L-arginine + L-NAME (In + Ih); 6) AM = L-arginine + methylene blue (In + Re). The samples were evaluated for spermatic kinetics by CASA and other analyses [plasma and acrosomal membranes used the propidium iodide (PI) and Pisum sativum agglutinin (PSA), detection of tyrosine residues phosphorylation in the membrane (F0426), nitric oxide (DAF-2/DA), lipid peroxidation (C11-BODIPY581/591)] by flow cytometry. The l-arginine treatments reduced MOT, PROG, RAP and LIN only at time 0 min compared to the control and L-NAME. These treatments (MT and MP, VAP, VSL, LIN, RAP) also reduced the sperm movement characteristics but only at the beginning of the incubation period. After this period of incubation, motility recovered. NO removal by methylene blue almost completely inhibited sperm motility, but these treatments had the highest percentages of intact membranes. l-arginine treatments improved acrosome reactions and differed from M and AM. NO production, tyrosine phosphorylation and lipid peroxidation did not differ among treatments, except for M and AM, where a reduction in these variables was detected. Therefore, equine sperm capacitation and the acrosome reaction are part of an oxidative process that involves the participation of R