Effect of rosemary extract on DNA damage and some parameters of rooster semen in liquid storage condition

Introduction: The increasing use of artificial insemination (AI) in the poultry industry emphasizes the need for the sharing of good quality sperm. The evaluation of semen quality characteristics of poultry gives an excellent sign of their reproductive potential and has been reported to be a major determinant of fertility and subsequent hatchability of eggs. In poultry industry in order to take advantage of new AI techniques, proper storage of poultry semen is needed. As poultry semen is viscous, highly concentrated, with low volume, containing 6 (chicken) to 12 (turkey) billion spermatozoa/ mL, the extension of good semen with a proper diluent is required prior to AI and storage. Reproduction performance is affected some factors include; nutrition, genetic, management, and environment. One of the fundamental aim in poultry industry is production of fertile eggs with high hatchability. In reproduction physiology, male and female are important, but sire effect is more than dam because of breeding program, semen samples could be applied for females. In layer industry, number of females are more than males; so, artificial insemination is an important tool for genetic diversity and high reproductive performance. One of the major problems in artificial insemination is low quality of poultry semen. Freezing and thawing have adverse effects on sperm viability. Since lipids (poly unsaturated fatty acids) are important part of plasma membrane of sperm cells, it could be destroyed in short times of storage in a liquid condition. It has been reported that antioxidants can be employed to prevent sperm damage during chilling storage condition (Malo et al. 2011). It is well known that sperm of farm animals is highly sensitive to oxidative stress (Surai et al. 1998). The high concentration of polyunsaturated fatty acids in the cell membrane of the sperm is also considered as another key factor for susceptibility to the oxidative stress. The process of semen production of reactive oxygen species (ROS) may lead to impairment of sperm morphology, motility, and finally viability. Pharmacological plants have some antioxidant components, which decrease free radical levels in organism. One of the major problems in sperm viability is sperm fragmentation. In this case, nucleus chromatin of sperm damages or splits. In some cases, the motility and normality of the semen analysis is acceptable, but the sperm nucleus of sample is damaged.  Different tests are available for detection of sperm DNA damages like COMET assay, TUNEL assay, SCSA, and Sperm Chromatin Dispersion (SCD) test. Among these assays, the SCD test is a simple and low-cost method for the analysis of sperm DNA fragmentation (Shanmugam et al., 2014). This method is based on the principle that sperm with DNA fragmentation fails to produce halo of dispersed DNA loops when mixed with agarose followed by acid denaturation and nuclear protein removal. The halos can be visualized using bright-field microscopy after staining with Wright’s stain. This study was designed to evaluate the effects of Rosemary extract on rooster semen parameters during chilling storage condition at 4ºC.
Material and methods: Dimension of individual cages were 85×70×70cm. Photoperiod program was applied based on 10 h darkness and 14h light. Water and feed were ad libitum. Rooster were fed corn and soybean meals based on 2107 kcal and 12% protein per Kg of dry matter. The semen was collected twice a week for six times from 10 mature roosters using abdominal rubbing method. As the rooster responded to abdominal rubbing by everting its cloaca, the ejaculate was collected using a graduated pipette; then, total volume was recorded. Urine or fecal contamination was discarded. Fresh semen was evaluated for sperm concentration using a haemocytometer. The semen samples were diluted by Sexton extender and evaluated at 25-27 °C. Then, the samples were divided into six equal parts. Different treatments were including: 0(control), 15, 25, 35, 45 and 60 µg/ml of Rosemary extract. Rosemary extract were added to each part and kept at 4^oC for 72h. The parameters included motility, viability and plasma membrane integrity were evaluated at 0, 24, 48 and 72 h after storage. Also, DNA fragmentation was evaluated 48h after storage at 4 ^oC. In this method, sample is affected by acid treatment and denatures sperm chromatin. For elimination of proteins from chromatin, DNA strands are scattered around the sperm head and then, a halo forms around the sperm head.  With staining, halo is visible under the microscope. Halo in fragmented sperm is very small.   Data of the experiment were analyzed by Proc ANOVA SAS software (repeated measurement) and means were compared by Duncan's multiple range test.
Results and discussion: Sperm during time of storage undergoes different changes and potentially undergoes mechanical and oxidative damages. At the time of storage in liquid condition, free radicals are released and sperm performance decreases significantly. The results of this experiment revealed that applying 35 µg/ml Rosemary extract could decrease DNA fragmentation (p < 0.05). It seems that this concentration of Rosemary extract has antioxidant properties in rooster semen. Avian sperm have high concentration of long chain polyunsaturated fatty acids within the phospholipids and therefore, are prone to peroxidation damage (Blesbois et al. 1997). Also, after 48h of storage, motility, viability and sperm plasma membrane integrity were lower in treatment 60µg/ml Rosemary extract than control group (p < 0.05). Finally, it was demonstrated that 48h after storage, motility, viability and sperm plasma membrane integrity in samples treated by 35µg/ml Rosemary extract were higher than control group (p < 0.05). High level of reactive oxygen species (ROS) in oxidative stress is related to DNA fragmentation. Free radicals oxidize purine and pyrimidine bases of DNA structure of sperm and it causes damage DNA structure. The prevention of free radicals is the first defense line of antioxidants against oxidative damage. Rosemarry extract improved rooster sperm quality after thawing (Shafigh et al. 2016).  This is the first study to report the use of DNA fragmentation test in rooster sperm in Iran. This is a simple technique requiring no sophisticated instruments when Wright stain is used for visualization of the halos. According to the results of this study, it seems that applying 35µg/ml Rosemary extract can be useful in rooster semen at 4 ^oC. Rosemary extract effectively reduced DNA fragmentation. This effect seems to be associated with the high motility of treatment (35µg/ml Rosemary extract) compare with other treatments.