Melatonin regulates male reproductive function in seasonal and non-seasonal breeder mammals. The presence of melatonin membrane receptors (MT1 and MT2) in the testis and epididymis has been demonstrated in several species. Wild roe deer are a short-day breeding species characterised by a short rutting season lasting from mid-July to mid-August. The aim of this study was to determine the concentration of melatonin in the peripheral blood and the presence of MT1 and MT2 receptors in the testis and epididymis in male roe deer during the pre-rut (May), rut (July/August) and post-rut (September) periods. The melatonin concentration was higher in May (522.50 ± 54.20 pg/mL) compared to July/August (258.50 ± 36.82 pg/mL; P < 0.05). During September, the melatonin concentration was higher (393.50 ± 36.77 pg/mL) than in July/August (P < 0.05) but lower than in May (P < 0.05). Immunohistochemical analysis showed the presence of MT1 and MT2 receptors in Leydig cells, Sertoli cells and germ cells in the testis, in addition to the epithelial cells of the epididymis caput, corpus and cauda. MT1 and MT2 receptor expression in the testis and epididymis, assessed by Western blot, was higher in May and July/August (when spermatogenic and steroidogenic activity restarts and reaches its peak, respectively) compared to September (when spermatogenic and steroidogenic activity decreases). This could indicate a stimulatory effect of melatonin on testicular (i.e., steroidogenesis and spermatogenesis) and epididymal (i.e., spermatozoa maturation) function in male roe deer through the MT1 and MT2 receptors. Our results form the basis for further studies into the detailed mechanism of action of melatonin through MT1 and MT2 receptors for optimal reproductive activity in male roe deer and other mammals.

The aim of this study was to analyse the morphology of the epididymal spermatozoa of male roe deer obtained postmortem at the beginning (May), peak (July/August) and the end (September) of the reproductive season. Spermatozoal abnormalities were divided into major (associated with impaired fertility) and minor (not associated with impaired fertility) defects. The highest percentage of abnormal spermatozoa was observed in May (17.78 ± 1.88%), with a much higher proportion of major (12.35 ± 1.11%) than minor defects (5.43 ± 1.59%) being observed. The percentage of abnormal spermatozoa was lowest during the peak of the reproductive season (4.97 ± 1.13%), with the proportion of major (2.68 ± 0.78%) and minor defects (2.28 ± 0.45%) being comparable during this period. The percentage of abnormal spermatozoa increased again in September (11.05 ± 1.60%), with the major defects (6.15 ± 1.04%) slightly surpassing the minor defects (4.90 ± 0.77%); however, total abnormalities still remained lower than those found in May. These differences were statistically significant, with the exception of the difference in minor defects between the pre-rut and post-rut periods. These results indicate that the best period to collect epididymal spermatozoa from roe deer postmortem is the peak of the reproductive season (July/August); however, they can also be recovered at the end of the reproductive season (September), as the percentage of major defects is relatively low at this time. This study provides the basis for further research to determine optimal methods for the storage and cryopreservation of epididymal spermatozoa in this species.