Effect of combination of MitoQ as a targeted antioxidant and Pentoxifylline as a non-targeted in Lake based extender on functional quality of rooster sperm during chilled storage on 4◦C

Introduction:
Cooling storage is a common method for reduction of sperm metabolism to save sperm quality during in vitro storage. Different studies have attempted to optimize quality of stored rooster semen for periods more than 24 h, but the obtained reproductive efficiency was not satisfactory. Deleterious effects of chilling storage on the rooster sperm may cause a reduction in reproductive performance. It seems that improvement and enrichment of cooling storage methods are required in order to recover the highest quality of sperm before using in assisted reproductive technique (ART). But this process affects sperm function and quality by producing reactive oxygen species and reducing antioxidant activity. The plasma membrane of sperm contains high unsaturated fatty acids that are prone to peroxidative damage and this can reduce membrane integrity, decrease kinetic and sperm fertility for artificial insemination (Ahmadi and Zamiri 2007)..The targeted antioxidant MitoQ exerts its antioxidant defense by increasing ATP production and reducing ROS production. Experiments have clearly shown that MitoQ accumulated in mitochondria can be restored after oxidation by the electron chain and protect the cell against oxidative stress (Skulachev et al 2009). This antioxidant protects mitochondria from oxidative damage by protecting the mitochondrial membrane potential (Murphy and Smith, 2000). Effect of pentoxifylline on sperm function can be summarized in several categories: inhibition of phosphodiesterase activity, increased conversion of ATP to cAMP, effect on intracellular calcium transport and elimination of free radicals, induced by spermatozoa, and decreased oxidative activity (Esteves et al 2007). Purpose: In this study, the effects of combined targeted and non-target antioxidant were used to reduce oxidative stress and improve rooster sperm quality. Material and methods: This study was carried out in the poultry unit of Tabriz University research station. For this purpose, 15 adult Ross breeds aged 30 weeks were used. The roosters were housed in individual cages 70 × 70 × 85 cm under 15 h light and 9 h dark conditions with the same diet of 150 g (50.8% corn, 8.95% soybean, 20% wheat, Wheat bran 14%, dicalcium phosphate 0.74%, limestone 1.8%, salt 0.38%, lysine 0.08%, methionine 0.17% and vitamin and mineral supplements (0.5%) per day for each rooster and they had free access to water. Sperm collection was done by abdominal dorsal abdominal method. Sperm samples were transferred to the laboratory immediately after collection at 37 ° C. To eliminate individual differences and obtain sufficient sperm for analysis, in each replicate, the ejaculates of the 15 roosters were briefly inspected and ejaculates with ≥ 300 × 106 spermatozoa/mL, ≥ 90% normal morphology and ≥ 80% motility were then pooled. In this study, the combined antioxidant effects of targeted antioxidants of mitoQ and non-target pentoxifylline were evaluated in Lake Extender based on lecithin After diluting the samples and adding different levels of both antioxidants and cooling, the samples were stored in the refrigerator at 4 ° C for 48 hours and the samples were evaluated at 1, 24 and 48 hours. Motility, viability, plasma membrane integrity were evaluated at 1, 24 and 48 hours, and lipid peroxidation and percentage of sperms with normal morphology were evaluated at 48 hours. Results and discussion: Treatment 1 (0.1 nm mitoQ+ 0.5 µM Pentoxifylline) increased sperm viability in the control group one hour after cooling (p < 0.05). Treatment 1(0.1 nm mitoQ+ 0.5 µM Pentoxifylline), Treatment 4 (0.2 nm mitoQ+ 0.5 µM Pentoxifylline) and Treatment 5 (0.2 nm mitoQ+ 0.75 µM Pentoxifylline) increased the progressive motility compared to the control group at 24 h after cooling (p < 0.05). Treatments 4(0.2 nm mitoQ+ 0.5 µM Pentoxifylline) and 4 (0.2 nm mitoQ+ 0.5 µM Pentoxifylline) increased this parameter 48 h after cooling (p < 0.05). Treatment 4 (0.2 nm mitoQ+ 0.5 µM Pentoxifylline) and Treatment 5 (0.2 nm mitoQ+ 0.75 µM Pentoxifylline) significantly reduced malondialdehyde concentration at 48 h after cooling. Treatment 5 (0.2 nm mitoQ+ 0.75 µM Pentoxifylline) significantly increased viability and sperm with intact membrane integrity compared to control group (p < 0.05). All treatments except for treatment 2(0.1 nm mitoQ+ 0.75 µM Pentoxifylline) showed less unhealthy sperm compared to the control group (p < 0.05).To improve artificial insemination in the poultry industry, improving short-term sperm storage methods is critical for maintaining fertility for 6 to 24 hours under field conditions (Zhandi and sharafi 2016). One of the reasons for reduced fertility of frozen sperm in addition to cold shock is the occurrence of oxidative stress (Łukaszewicz et al 2008). The plasma membrane of sperm contains high unsaturated fatty acids that are prone to peroxidative damage and this can reduce membrane integrity, decrease kinetic and sperm fertility for artificial insemination (Fang et al. 2014). Despite many advances in the field of sperm cryopreservation, this process has the potential to affect yield and function due to the introduction of mechanical and chemical damages (Wang et al., 1991), production of reactive oxygen species, oxidative stress, and reduced antioxidant activity (Ahmadi and Zamiri 2007). The freezing process causes significant damage to the sperm, causing the release of several compounds, including cAMP, causing the sperm to lose motility and thereby reduce their fertility (Chaudhry et al., 1975). Due to the positive effect of targeted and non-targeted antioxidants on improving sperm motility parameters, this study was the first to use a combination of these antioxidants in freezing semen. Conclusion: The results of this study showed that the addition of treatment 5(0.2 nm mitoQ+ 0.75 µM Pentoxifylline) to the semen sample had the best performance during 48 h of cooling.
Keywords: Mitochondria , Lipid peroxidation, targeted antioxidants, Pentoxyfyline, Rooster sperm