The free radicals are atoms or molecules possessing in the outer shell of electronic envelope one or more unpaired electrons which makes them in biological sense extraordinary non stable and reactive. In this regard, it is well known that one of the most important factors which may predispose to their lower motility and fertilization capability of spermatozoa are their oxidative damages which are the consequence of activity of an excessive quantity of free radicals, particularly reactive oxygen species (ROS). Almost all cells of an organism, but primarily reproductive cells are constantly exposed to such excessive quantity of the ROS which are produced during process of manipulation and cryopreservation of spermatozoa and in the case of inflammatory states of genital organs. The counterbalance to such damaging ROS activities in an organism are antioxidant substances present in seminal plasma and spermatozoa. Disproportional production of free radicals and antioxidative protection of an organism is called oxidative stress which may lead to cell damage and consequently to infertility. The melatonin is the molecule with the capability of direct and indirect antioxidative protection. It also prevents lipid peroxidation of cell membranes, apoptosis of spermatozoa, and protects spermatozoa DNA and mitochondria from damage which may happen due to negative effects of the ROS. Also, in sheep and goats as polyestrous seasonal animals, melatonin is participating in the regulation of sexual cycle. The aim of this study was to determine the effect of exogenous melatonin on antioxidative protection of bucks ejaculate in non-breeding season by monitoring of antioxidative enzymatic activity in semen as well as various parameters of ejaculate quality before and after deep freezing, and to evaluate the effectiveness of supplementation of the commercial extender with vitamins E and C in particular doses before deep freezing. In this study 12 bucks of French Alpine breed, aged from 1.5 to 4 years were included into 6 experimental periods (duration of each period was 2 weeks) out of reproductive season. The bucks were randomly assigned into two groups: the control (n = 6) and melatonin treated (n = 6) group. All bucks were in a good reproductive condition, kept and fed in almost identical macroclimatic and microclimatic conditions. At the end of second period the melatonin treated group of bucks received 4 implants of 18 mg of melatonin subcutaneously in the ear basis. The ejaculate was taken from the bucks on a weekly basis by artificial vagina (for 12 weeks in total) during morning hours and their libido was also assessed. For obtaining the ejaculate the synchronization of oestrus was performed in goats and they served as phantoms. In order to obtain the higher volumes of seminal plasma the ejaculate from each buck was taken two times consecutively within the interval not longer than 15 minutes. The ejaculates were collected into sterile graduated glass tubes. After sampling of the ejaculates, their volume and concentration, motility, proportions of vital and abnormal spermatozoa were determined. The concentration of spermatozoa was determined by electronic counter, their motility was assessed natively under binocular microscope with built-in sperm heater plate, proportions of live and dead spermatozoa were assessed based on supravital staining after Bloom, and for determining morphologically normal and pathologic forms of spermatozoa a commercial staining set after Farelly was used. After 10 minutes from taking of the ejaculates both were centrifuged in a shaded incubator (at 21°C) for 5 minutes at 2400 g. The supernatants from both ejaculates were taken by pipette, mixed and again centrifuged for 15 minutes at 2400 g. Following the second centrifugation the supernatant, i.e. seminal plasma was taken by pipette and cooled off to + 4°C, and soon after that to – 80°C in the freezer until analysed. The remaining spermatozoa were washed 3 times with saline and after each washing they were centrifuged for 5 minutes at 500 g. The activities of glutathione-reductase (GR), glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), catalase (CAT) and concentration of malondialdehyde (MDA) were determined. After the first centrifugation the rest of spermatozoa were added commercial synthetic diluent with or without supplementation of vitamins E and C, cooled off at + 4°C, transported to the Centre for artificial insemination where after equilibration for 24 hours the spermatozoa were deep frozen within the apparatus for automated deep freezing. After thawing of frozen spermatozoa their motility and velocity parameters were determined by the computer analyses of spermatozoa. The proportion of the cells with damaged plasma and acrosome membrane, damaged DNA, damages and depolarization of mitochondria were assessed by the flow cytometry. The proportions of live spermatozoa and spermatozoa with different forms of abnormalities were determined by the cytologicalmorphological examination. In the melatonin treated group of bucks non-significant higher level of libido was determined. These bucks had significantly better motility of spermatozoa and a higher proportion of live spermatozoa during the 3rd and 4th period of the experiment. The melatonin treated group of bucks had significantly lower values of GR in the spermatozoa and the seminal plasma during almost all periods of experiment. In addition, significantly lower activity of GSH-Px in the spermatozoa and higher in the seminal plasma in the last period of the experiment as well as significantly lower value of SOD in spermatozoa during the last three periods of the experiment were determined. The group of bucks that received melatonin had significantly higher values of the ratios: CAT/SOD, GSH-Px/SOD in the seminal plasma and spermatozoa during 6th period of the experiment. In addition, same group had significantly lower values of the ratio GR/GSH-Px in the spermatozoa during 6th period and in the seminal plasma during 5th period of the experiment. The melatonin treated group of bucks had better motility, progressive motility, proportions of the live spermatozoa and the live spermatozoa with intact acrosome during 3rd period after thawing of the semen. Furthermore, the melatonin treated group of bucks had a higher percentage of beat cross frequency in the 4th period of the experiment. The thawed ejaculates with extender supplemented with vitamins E and C had a higher proportion of the spermatozoa with damaged plasma membrane as well as a lower total proportion of the live spermatozoa and the live spermatozoa with intact acrosome for the whole experimental period. However, when each of the 6 periods was separately observed there were no differences among ejaculates with or without the supplemented vitamins. According to the obtained results it could be concluded that the exogenous melatonin had a positive effect on motility and proportion of live spermatozoa in fresh and thawed semen immediately after application of melatonin. The exogenous melatonin changed the activities of particular antioxidative enzyme in certain periods of the experiment, especially on GR and GSH-Px in the seminal plasma and spermatozoa and SOD in spermatozoa. Also, the exogenous melatonin had an significant influence on the ratios of antioxidative enzymes in the seminal plasma and the spermatozoa in the same experimental period, and thus, the precise determination of these ratios in the future could be considered as a better indicator of oxidative stress which may provide a better insight into adaptation and antioxidative status in regard to activities of single antioxidative enzymes. In this study the antioxidative status in French Alpine buck spermatozoa was established for the first time and the positive correlation of CAT, GR and SOD activities with DNA damage in the spermatozoa was recorded. Vitamin supplementation of extender before freezing did not improve the quality of ejaculates which could be the consequence of various endogenous and exogenous factors. Accordingly, it could be assumed that in this study the doses of applied vitamins E and C were not properly assessed, and thus the proper calibration of their doses should be emphasized in further research.