Melatonin affects red deer spermatozoa motility and physiology in capacitating and non‐capacitating conditions

Abstract Melatonin affects sperm physiology, possibly through membrane receptors. Effects were tested at low concentrations (1 pM, 100 pM, 10 nM and 1 µM) in red deer epididymal spermatozoa as a model for high‐seasonality species. Samples were incubated with melatonin as uncapacitated or capacitating conditions (heparin) and evaluated for motility and physiology (flow cytometry). Most effects occurred at low concentrations (nM‐pM), mainly protecting from apoptosis and maintaining acrosomal integrity, suggesting a role for membrane receptors rather than a direct antioxidant effect. Intracellular calcium was not affected, differing from other studies and perhaps because of the epididymal origin. This study supports the relevance of melatonin on sperm physiology and could contribute to the application of reproductive technologies in wild ruminants.


| INTRODUC TI ON
Melatonin is a widespread and pleiotropic molecule (Hardeland et al., 2011). Widely studied as a hormone regulating the circadian cycle and seasonality, evidence has mounted on its paracrine activity in reproductive organs (Gonzalez-Arto et al., 2016;Martínez-Marcos et al., 2019) and its membrane receptors MT 1 and MT 2 are present in spermatozoa , 2017. Melatonin affects sperm survival, capacitation and chemotaxis (Casao et al., 2012;Cebrián-Pérez et al., 2014;Espino et al., 2011). This topic has been studied in some domestic ruminants (Fernández-Alegre et al., 2020;, but it could be different for wild ruminants, subjected to pronounced seasonal reproductive rhythms (Martinez-Pastor et al., 2005). Our objective was to test its role on red deer (Cervus elaphus) spermatozoa under non-capacitating or capacitating conditions, with the hypothesis that it could affect them at concentrations compatible with receptor activation, as reported for bull (Fernández-Alegre et al., 2020).

| MATERIAL S AND ME THODS
The experimental design and methods followed our study on bull (Fernández-Alegre et al., 2020) and are expanded in the supple-   17.5 ± 1.9* Note: Results are shown as mean ± SEM. Significant effects on the control in each group are indicated by *p <.05, **p <.01, ***p <.001. Parameters are described in detail in the main text and the supplementary material. 50 × 10 6 /ml. Tubes were split, one receiving 2 U/ml heparin (capacitated) and split again, adding melatonin at 1 µM, 10 nM, 100 pM, 1 pM and control (vehicle, 0.2% DMSO).
The samples were incubated for 4 h (38°C, 5% CO 2 ) and analysed The statistical analysis was performed with R using linear mixedeffects models (treatment and incubation as fixed effects; male as random effect). Results are shown as mean ± SEM.

| DISCUSS ION
Melatonin affected red deer epididymal spermatozoa at the low range of concentrations, consistent with our previous study on bull semen (Fernández-Alegre et al., 2020) and exerting a role on sperm physiology through its membrane receptors. In these conditions, a direct antioxidant effect seems unlikely. Our results support previous evidence on a functional role for MT 1 and MT 2 in spermatozoa (Casao et al., 2012;Fujinoki, 2008;, consistently showing their implication in sperm capacitation modulation keeping motility and preserving viability and oxidation state. This first study in red deer spermatozoa hints at a protective effect, possibly by inhibiting apoptosis-related pathways (Casao et al., 2010;Espino et al., 2011). However, we could not detect intracellular calcium changes, contrarily to studies in ejaculated spermatozoa (Fernández-Alegre et al., 2020;Gimeno-Martos et al., 2019). Epididymal spermatozoa are quiescent, not having contacted seminal plasma (Martínez et al., 2008), and signalling pathways modifying calcium patterns could be inhibited at this stage (Ernesto et al., 2015;Gibbs et al., 2011).
This study suggests differences between species and possibly epididymal and ejaculated spermatozoa and could be helpful for the conservation and application of wild ruminant post-mortem samples.

ACK N OWLED G EM ENTS
The authors thank Dr. Joaquín Vicente and SaBio (IREC, CSIC-UCL-JCMM, Spain) and hunting wardens at Riaño Reserve for providing the samples, and Indira Álvarez-Fernández and Cristina Arija.