Sperm fertility decreases significantly after freezing. Providing a suitable and useful diluent compound for freezing ram sperm can increase the efficiency of artificial insemination and consequently, the reproductive performance of sheep. Various biological properties such as antibacterial, anti-cancer, immunosuppressive, antioxidant and reproductive properties of royal jelly (RJ) are well known. The aim of this study was to investigate the possible synergistic effect of royal jelly in combination with glycerol and dimethyl sulfoxide (DMSO) in sperm cryopreservation extender of Romanov ram. The pooled semen samples from 5 Romanov rams were allocated into 3 experiments. The effect of 6% DMSO, 6% glycerol and a combination of 3% DMSO +3% glycerol co-supplemented with 1, 2 and 3% RJ was evaluated in 3 experiments. Samples were frozen by conventional slow freezing method and post-thaw parameters of total motility, progressive motility, plasma membrane integrity, DNA damage, apoptosis, enzyme activity of superoxide dismutase (SOD) and glutathione peroxidase (GPx), total antioxidant capacity (TAC) and malondialdehyde (MDA) were evaluated. The results showed that the percentage of motility, progressive motility, TAC, GPx, SOD and all sperm kinematic parameters except LIN in the group containing 2% RJ + 6% DMSO was higher than the control group (p < 0.05). Some parameters such as progressive motility, sperm membrane integrity, TAC, GPx, VAP, VCL, STR and SRT in the group containing 2% RJ + 6% DMSO were more than (p < 0.05) in the sperm group containing 1% RJ + 6% DMSO. MDA values in sperm groups containing 2% RJ + 6% DMSO were significantly (p < 0.05) lower than the sperm containing 1% royal jelly and the control group. In the sperm group containing 2% RJ + 6% glycerol, sperm membrane integrity, TAC, GPx, SOD, progressive motility and all sperm kinematic parameters except VAP were higher and MDA values and sperm abnormalities were lower than the control group (p < 0.05). The sperm group containing 1% RJ and 3% DMSO +3% glycerol had higher motility, progressive motility, membrane integrity, and all sperm kinematic parameters except VSL; and lower sperm abnormalities, DNA damage, apoptosis and MDA than the control group (p < 0.05). As a general conclusion of this study, the addition of 2% RJ + 3% DMSO and 3% glycerol to the freezing extender improved microscopic and biochemical ram sperm parameters after the freeze-thaw process. Hence, moderate concentrations of royal jelly (2%) are sufficient to protect sperm from freezing damage, and high (3%) and low (1%) concentrations do not have a good cryoprotective effect.
Keywords: DMSO; Glycerol; Romanov; Royal jelly; Sperm extender; Synergistic.
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