Liquid or frozen storage of poultry semen negatively affects the sperm motility and causes nonreversible changes in sperm morphology (1). Phospholipids in chicken sperm membrane are composed of high proportions of polyunsaturated fatty acids which are susceptible to lipid peroxidation (2). Antioxidant supplementation is shown to be effective in decreasing lipid peroxidation during in vitro storage (3). This research was conducted in three experiments to understand the effect of melatonin as an antioxidant, on semen quality and fertility of Fars indigenous roosters of Iran during in vitro semen storage.

Seventy-four 9-month-old native roosters were randomly divided into three equal groups. One group was exposed to 14 hours light and 10 hours darkness as control group (Group C). Another group was exposed to the same lighting schedule as the first group and supplemented with 3 mg/kg body weight edible melatonin daily (Group M). The third group was exposed to 24 hours constant light (Group L). Semen was collected four times at 20 day intervals by using the abdominal massage technique.

In the first experiment semen samples of every 8 roosters were pooled and stored for 6 and 12 hours at 5^ºC. Then, semen quality, malondialdehyde (MDA) concentration, fertility and hatchability were evaluated before and after storage (2,4). In control birds, percentage of viable and motile sperm decreased significantly (P<0.05) after 6 hours storage but in other groups these parameters decreased after 12 hours. Concentration of MDA decreased in Group M compared with Group C. Fertility and hatchability did not changed significantly (P>0.05) in M and L groups compared with C group.

In the second experiment, semen samples of every 8 roosters in each group were pooled. The pooled semen samples of the first group were supplemented with 4 or 8 µg/ml vitamin E or 3 and 6 mM melatonin, respectively. The pooled semen samples were frozen, and then thawed after 2 weeks. Semen quality and MDA concentration were evaluated before freezing and after thawing (2,3). The interaction between treatment and freezing process was significant (P<0.05) only for MDA concentration and sperm viability. Supplementation of semen with melatonin significantly decreased the sperm viability compared with the control sample (P<0.05). Vitamin E at the concentration of 4 µg/ml increased sperm viability after thawing compared with the control sample (P<0.05). The maximum and minimum concentrations of MDA were observed at 6 mM melatonin and 4 µg /ml vitamin E supplementation, respectively.

In the third experiment, 15 roosters were randomly selected from each group. Then, semen quality, serum and seminal plasma concentrations of melatonin, testosterone, and estradiol were measured four times at 20 day intervals for every rooster (5,6). Semen volume, sperm motility and total number of live and normal sperm significantly (P<0.05) increased in L group compared with C group. No significant difference was recovered in semen volume and live sperm between M and C group. Serum concentrations of melatonin, testosterone and estradiol increased (P<0.05) in M group compared with C group. Serum concentration of testosterone decreased (P<0.05) in L group compared with C group. No significant (P>0.05) difference was observed in seminal plasma concentration of melatonin between experimental groups.

In conclusion, edible melatonin administration reduced polyunsaturated fatty acids of sperm membrane lipids and reduced lipid peroxidation during liquid storage of semen but it did not affect fertility. Supplementation of semen with melatonin, reduced lipid peroxidation of sperm during frozen storage but edible melatonin supplementation did not affect sperm lipid peroxidation during frozen storage.