Melatonin protects ram spermatozoa from cryopreservation injuries in a dose-dependent manner Introduction Reproductive activity is governed, in most of the animal species, by seasonal variations in the frequency of concep- tion. Photoperiodism via the circadian melatonin rhythm registers changes in the annual cycle of day-length, and this signal is used to synchronize seasonal reproductive fluctu- ations [1]. Certain breeds of sheep are highly seasonal in terms of reproductive capability, regulated by photoperiod and melatonin secretion [2, 3]. Therefore, seasonal melatonin variation has been thoroughly studied in this specie, and although seasonality is less marked in males than in females, changes in testicular volume, hormonal profiles, sexual behavior, and semen quality that affect the repro- ductive performance of rams have been reported [4–8]. Melatonin administration was reported to improve sperm quality in rams in the nonbreeding season [9–11]. In particular, beneficial effects of both short days or melatonin treatment in the nonbreeding season may be because of the melatonin regulatory effect on the hypothalamus-pituitary- testicular axis [12], modulating GnRH pulsatility[7] and gonadotropin and testosterone production [13]. On the other hand, a direct effect of melatonin on semen quality cannot be ruled out considering the presence of melatonin receptors in epididymal tissue of the rat [14] and human [15] spermatozoa and the presence of melatonin in human [16] and ram [17] seminal plasma. Reports about the beneficial effects of melatonin in protecting spermatozoa from different kind of injuries have emerged [18–22]. Cold shock can injure spermatozoa in different levels structures, such as mitochondria [23] or plasma and acrosome membranes [24], and can alter spermatozoa functional integrity hence reducing its fertil- izing ability [25]. In addition, a significant reduction in the level of spermatozoa antioxidants has been reported as one of the causes of the enhanced susceptibility of these cells to peroxidative injuries after cryopreservation [26]. Thus, this study determined whether melatonin, thanks to the well- known cytoprotective and antioxidant effects of the indol- amine as well as of its metabolites [27, 28], exerts a beneficial role during semen cryopreservation. Recom- mended ram extender was supplemented with different melatonin concentrations, and its effects on spermatozoa Abstract: Cryopreservation harms spermatozoa at different levels and thus impairs their fertilizing ability. The role of melatonin in protecting spermatozoa from different kind injuries has been widely reported. Thus, this study tested whether the addition of melatonin to ram semen freezing extender could exert a protective effect and ameliorate postthawing sperm function. Melatonin was added to recommended ram extender to yield five different final concentrations: 0.001, 0.01, 0.1, 1, and 10 mm. A control group without melatonin supplementation was included. Spermatozoa viability, motility parameters, and intracellular ATP concentrations were evaluated both before and after cryopreservation, while DNA integrity and in vitro fertilizing ability were evaluated only after thawing. Obtained results showed that the concentration of 1 mm melatonin led to higher viability rates, higher percentages of total motile and progressive motile spermatozoa, higher percentages of spermatozoa with average rapid and medium velocity, higher intracellular ATP concentrations, and higher DNA integrity among semen frozen in control and melatonin-supplemented extenders (P < 0.05). In addition, results obtained after the IVF test showed that at 1 mm concentration, melatonin led to a faster first embryonic division and to higher total cleavage rates compared to the other experimental groups (P < 0.05). No difference in embryo output was observed among the six experimental groups. In conclusion, the addition of melatonin to ram semen freezing extender protected spermatozoa during cryopreservation in a dose- dependent manner. These results are likely to be mediated by its well-known antioxidant properties, even if a direct action of the indolamine cannot be ruled out. Sara Succu 1 , Fiammetta Berlinguer 1 , Valeria Pasciu 2 , Valentina Satta 1 , Giovanni G Leoni 3 and Salvatore Naitana 1 1 Department of Animal Biology, University of Sassari; 2 Presidenza, Biblioteca Veterinaria, Faculty of Veterinary Medicine, University of Sassari; 3 Department of Physiological, Biochemical and Cellular Science, University of Sassari, Sassari, Italy Key words: adenosine triphosphate, DNA integrity, melatonin, ram, semen cryopreservation Address reprint requests to Fiammetta Berlin- guer, Department of Animal Biology, University of Sassari, Via Vienna, 2; 07100 Sassari, Italy. E-mail: berling@uniss.it Received September 29, 2010; accepted November 19, 2010. J. Pineal Res. 2011; 50:310–318 Doi:10.1111/j.1600-079X.2010.00843.x Ó 2011 The Authors Journal of Pineal Research Ó 2011 John Wiley & Sons A/S Journal of Pineal Research 310 Molecular, Biological, Physiological and Clinical Aspects of Melatonin