the effect of adding ascorbic acid on sperm quality, lipid peroxidation and antioxidant enzyme activities of ram seminal plasma in outside of breeding season

introduction the cryopreservation of spermatozoa has provided special opportunities for the preservation of genetic resources and improving breed programs by the artificial insemination technique (holt, 1996). sperm cryopreservation stimulates intracellular ice crystals formation, increasing osmotic and chilling injury that causes sperm cell damage (isachenko et al. 2003). freezing and thawing processes impose physical and chemical insults on the sperm membrane that decrease sperm viability and fertilizing ability (alvarez and storey, 1992). both damages are associated with excessive generation of reactive oxygen species (ros) and peroxidation of the phospholipids in the membrane (wang et al. 1997; lasso et al. 1994). high concentrations of ros have a negative effect on sperm quality and increased degradation of dna, lipid peroxidation and oxidative stress which inhibits sperm motility and changes the structure of sperm and finally the reduces the fertility (aitken et al., 1997; agarwal and saleh, 2002; khosrowbeygi and zarghami, 2007; zalata et al., 1995). seminal plasma contains enzymes such as superoxide dismutase (sod), glutathione peroxidase (gpx) and catalase (cat) which have an important role in the inhibition of the deleterious effects of ros. lipid peroxidation may be done due to lack of coordination between sod, gpx, and cat in seminal plasma or deficiency of total antioxidant capacity of the cell (tavilani et al., 2008). ascorbic acid is the main water-soluble antioxidant which acts as a scavenger for a whole range of reactive oxygen species. ascorbic acid acts as a strong electron donor reacts with superoxide, peroxide and hydroxyl to form dehydroascorbic acid. ascorbic acid protects sperm dna against damage caused by h2o2 radical and reduces the amount of nitrite. this study was conducted to study the effects of adding ascorbic acid on sperm quality, plasma lipid peroxidation and antioxidant enzyme activities of ram semen after freeze- thawing process. material and methods five sexually mature ghezel rams (3 to 4 years of age) were used. semen samples were collected every three days using an artificial vagina. in order to eliminate the individual differences, ejaculates containing sperm with >80% progressive motility, volume of 0.75 to 2 ml, sperm concentrations greater than 3 × 109 sperm/ml and sperm abnormalities of less than 10%, were pooled. different levels of ascorbic acid (0, 0.5, 1, 1.5 and 2 mg ml-1) were added in tris-egg yolk based diluent. after processing and freezing, the samples were stored in liquid nitrogen until the time of evaluation. after thawing of semen samples, sperm motility characteristics were analyzed using computer-assisted sperm analysis. the sperm viability was determined by means of the nigrosin–eosin staining method. the hypo-osmotic swelling test (hos-test) was used to evaluate functional sperm plasma membrane integrity after freeze-thawing. sperm abnormalities were assessed using hancock solution. lipid peroxidation was measured with determining malondialdehyde and the seminal plasma antioxidant enzymes of glutathione peroxidase, superoxide dismutase and total antioxidant capacity using the randox laboratories kit. statistical analyses were performed using sas software (9.1.3) software using glm procedures. the least square means were calculated to determine the differences between the experimental treatments for the post-thaw evaluation times. results and discussion the results showed that the 1 and 1.5 mg/ml ascorbic acid significantly improved the total motility (tm), progressive motility (pm), curvilinear velocity (vcl) compared to the control group (p< 0.05). average path velocity (vap) and straight line velocity (vsl) in semen samples receiving 1.5 mg ml-1 ascorbic acid was increased compared to control group (p< 0.05). but the linearity percentage (lin) and sperm track straightness (str) was not significant compared to the control group. viability and integrity of the plasma membrane parameters were improved in groups receiving 1 and 1.5 mg/ml ascorbic acid compared to the control group. the percentage of sperm with abnormal morphology in 1.5 mg ml-1 ascorbic acid significantly was decreased than the control group (p<0.05). malondialdehyde production in 1.5 mg ml-1 ascorbic acid treatment tended to have decreased (p<0.05). superoxide dismutase and glutathione peroxidase enzyme activity were significantly increased in treatments with 1 and 1.5 mg/ml ascorbic acid compared to control (p<0.05). total antioxidant capacity in groups receiving 0.5 and 1.5 mg ml-1 ascorbic acid showed a significant improvement compared to the control group (p<0.05). conclusion the findings of this study was showed that the diluent contains 1.5 mg/ml ascorbic acid significantly improved sperm parameters compared to other levels, after freezing-thawing process.