Introduction
Melatonin is widely present in nature and is commonly known for the regulation of circadian rhythms
(Falcón et al., 2010)
. For many years, it was thought to be produced only by the pineal organ, but both autocrine and paracrine mechanisms of action are already described in different organs
. Its important role in fish gonad maturation have been studied in fish, providing evidences of antioxidant capacity
. Therefore, our hypothesis is that melatonin antioxidant properties may provide additional protection to sperm during cryopreservation, reducing oxidative stress. Cryopreservation has been used as a tool to help in artificial reproduction in many aquaculture species. Since these protocols for gilthead seabream ( Sparus aurata) are well stablished
, we aim to understand the effect of endogenously produced melatonin by night at the spermatozoa quality level and its effect as a supplemented antioxidant on a cryopreservation medium.
Methodology
For this experiment, gilthead seabream broodstock was acquired from a semi-in tensive aquaculture, Aqualvor (Portugal) and maintained at the Ramalhete station at the University of Algarve. Fish had a mean body weight of 520 ± 14g and were kept under a controlled short photoperiod of 8h light: 16h darkness, in order to simulate the normal environmental conditions of the reproductive season
. Sperm samples were collected by abdominal massage at two different points, mid light (ML) and mid dark (MD) (6 males at each point), avoiding any contamination, and kept at 4ºC until analysis. Afterwards, a cryopreservation protocol developed by our group was used (Cabrita et al., 2005), supplemented with melatonin (MEL) to evaluate its protection role. According to the available literature, three different MEL concentrations were chosen: 0.001 mM, 0.01 mM and 0.1mM melatonin , together with a control group without MEL. Sperm motility and concentration were assessed using a Computer Assisted Sperm Analysis software (CASA) to check potential differences in fresh and cryopreserved sperm quality between day and night . On a second experiment, cell viability and DNA fragmentation, by Comet assay, were tested (N=8) as described by
for gilthead seabream sperm. For cell viability, samples were diluted 1:100 in 1% NaCl and a mix of Propidium Iodide (PI) and SYBR-green dyes were used to stain non-viable and viable cells, respectively. Three photos of different fields were taken per sample, and at least 100 cells per field were counted under a fluorescent microscope (Nikon E200, Tokyo, Japan) using the “cell counter” feature from ImageJ (Java) software. Results were expressed as percentage of viable cells. Statistical analysis was performed using SPSS software (IBM). D ata that assumed normality and homogeneity of variance was analyzed with a T-test or One-way ANOVA , followed by a Student–Newman–Keuls (SNK) post-hoc test to identify statistical differences between groups. Mann-Whytney or Kruskal-Wallis nonparametric tests were respectively applied to data that did not assume the above mentioned principles.
Results
Results from day and night cryopreservation experiment revealed that cell concentration at MD was higher than ML, and all motility parameters analyzed, total motility (TM), progressive motility (PM), curvilinear velocity (VCL), straight line velocity (VSL) and linearity (LIN) , revealed to be influenced by melatonin . Although TM in fresh samples from ML and MD points did not differ, all cryopreserved treatments, including the control only with DMSO, had higher TM and VCL values at nighttime. Regarding DNA fragmentation, no differences were observed between treatments in Tail DNA (%), but Olive Tail Moment (OTM) was consistently higher at ML.
Discussion
The present results can be explained by the potential antioxidant activity of melatonin
when supplemented in the cryoprotectant medium , as described by
for paddlefish (Polyodon spathula ) sperm. However, the fact that control group also revealed an increase in TM and VCL at MD may suggest that the higher levels of melatonin endogenous production at night
can have an important role in spermatozoa protection , especially during cryopreservation (Len et al., 2019 ). Further research is needed in order to understand this melatonin mechanism of action
(Zhao et al., 2019)
and either if it is produced locally in the testis or absorbed by the blood stream
(Acuna-Castroviejo et al., 2007; Falcón et al., 2010)
.
Acknowledgments
This work is part of a PhD programme funded by Portuguese national funds from FCT through the grant SFRH/BD/148280/2019 to F.F and contract DL 57/2016/CP1361/CT0007 to C.C.V.O. The work was funded through projects UIDB/04326/2020 and PTDC/CVT-CVT/4109/2020 ( FCT, SpermAntiOx) , ASSEMBLE+ JRA2-H2020-INFRAIA-2016-2017 (No 730984), EBB-EAPA_501/2016 (Interreg Atlantic Area) and CCMAR/Multi/04326/2021 (FCT).
References