Study of Antioxidant Properties of Silymarin in Dealing with the Toxicity of Lithium Chloride in Ram Sperm

Introduction Lithium as a toxic and heavy metal and environmental contaminant could be a risk factor for male fertility. Lithium can induce male reproductive toxicity through damage in testes structure, sex hormones imbalance and decrease in testes and accessory sex organ weights as well as reduction in epididymal sperm count, normal morphology, viability and motility. Lithium is proposed to exert its cytotoxicity by free radicals generation and the activation of oxidative sensitive signaling pathways. Oxidative stress is metabolic and physiologic status caused by imbalance between free radical production and antioxidant defense of body. Therefore, the use of natural antioxidants could be a possible strategy for reducing oxidative stress in body. Silymarin is extracted from milk thistle (Silybum marianum) seeds. This compound is a polyphenolic flavonoid with a potent antioxidant property which not only acts as free radical scavenger but also increases the capacity of cell antioxidant enzymes. This study aimed to evaluate the harmful effects of lithium on sperm of Farahani’s ram and to know the protective effect of silymarin on protection against to toxic effects of lithium.
Materials and Methods In this experimental study, Farahani's ram testes were received from Arak slaughterhouse and transferred to the research laboratory under standard conditions. The experiment funding was approved by the ethical committee at Arak University. A few incisions were made in the caudal epididymis and spermatozoa were then washed into a sterile Falcon tube by Ham's F10 medium (Sigma, USA). Firstly sperm number and sperm motility were determined, according to World Health Organization protocol (WHO), to estimate sperm quality. High quality sperm samples were then used for experiments. The sperm samples were separated in eppendorf tubes as each tube contained 5×106 spermatozoa and divided into four groups to assess sperm viability. The MTT, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide assay was used to assess viability. In brief, 10 μl of MTT (Sigma, USA) stock solution (5 mg/ml ham's F10) was added to each tube containing sperm suspension and incubated at 37PoPC in COR2R incubator for 1 hr. The tubes centrifuged at 6000 rpm for 6 min and the precipitate was dissolved in 200 μl dimethyl sulfoxide (DMSO). The solution was then centrifuged at 4000 rpm for 4 min. 100 μl of the purple solution were transferred into a 96-well plate and absorbance was measured using ELISA reader (SCO diagnostic, Germany) at 505 nm. Optical density of sample was then used for calculating sperm viability percentage Evaluation of sperm motility was done according to WHO guidelines. Minimum of five microscopic fields was evaluated to estimate sperm motility on at least 200 spermatozoa for each sample. The percentage of sperm motility was evaluated for following motion patterns: progressive motile sperm (PMS), non-progressive motile sperm (NPMS) and non-motile sperm (NMS). Evaluation of sperm motility was done according to WHO guidelines. In brief, 10 μl of sperm suspension was placed on semen analysis chamber. Minimum of five microscopic fields was evaluated to estimate sperm motility on at least 200 spermatozoa for each sample. The percentage of sperm motility was evaluated for following motion patterns: progressive motile sperm (PMS), non-progressive motile sperm (NPMS) and non-motile sperm (NMS). One-Way analysis of variance (ANOVA) followed by Tukey's test was used to assess data statistical significance. p