Ndled in accordance with the ethical Casein Kinase site guidelines authorized by the Animal Ethics Committee of Universiti Sains Malaysia with reference no. USM/IACUC/2018/(112)(921) and the Universiti Kebangsaan Malaysia Animal Ethics Committee (UKMAEC) with reference no. FSK/2016/IZATUS/23-NOV./807-NOV.-2016-FEB.-2019. two.3. Experimental Style Randomly, all male rats (F0) were additional divided into 3 groups with eight rats per group. The control group (C) received corn oil in the dose of 1 mL/kg; whereas FNT-10 received 10 mg/kg/day FNT (1/60 LD50) [35] and FNT-20 received 20 mg/kg/day FNT (1/30 LD50) [12]. All the substances were administered via oral forced feeding using needle gavage for 28 consecutive days in between 09:00 a.m. and ten:00 a.m. [12]. Just after 4 weeks of Ras Inhibitor Accession treatment, two confirmed fertile female rats inside the oestrous phase had been paired with each male rat. Nevertheless, ahead of pairing together with the male rats, the female rats had been very first screened for two consecutively normal oestrous cycles. They have been paired through the dark phase with the reversed light/dark cycle amongst 9:002:00 h for three h each day [36]. Immediately after the mating period, by utilizing a light microscope (Olympus BX41, Olympus Corporation, Tokyo, Japan), vaginal smears were observed for the presence of spermatozoa. The day was recorded as day 0 of pregnancy (confirmed mating) when there was spermatozoon optimistic in theToxics 2021, 9,four ofvaginal smear [37]. Female rats were left till providing birth to progeny, beginning from day 21. Following mating, the male rats have been anaesthetized with a single intraperitoneal injection of ketamine and xylazine cocktail (KTX) prior to getting sacrificed [14]. For evaluating the sperm qualities and DNA fragmentation, the sperm were collected in the cauda epididymis of F0 male rats. Meanwhile, the rat’s progeny (F1), namely pControl, pFNT-10, and pFNT-20, have been left to grow until postnatal day 70 for evaluation of developmental landmarks. In the finish on the study, the chosen organs from both sexes of F1 rats were utilised for histomorphometric analysis. 2.4. Sperm Traits Evaluation After dissection, the sperm was collected straight away and was suspended in Hank’s balanced salt option (HBSS) with 298 mOsmol/kg, pH 7.four. For the epididymal sperm count and motility analysis, a total of 10 of sperm suspension was placed on a Makler counting chamber (Sefi-Medical Instruments, New York, NY, USA). The sperm motility was expressed in percentage of motile sperm although sperm count was expressed as million sperm cells per ml of suspension. Meanwhile, for sperm viability assessment, a thick smear was done making use of 10 of sperm suspension and adding 10 of eosin-nigrosin stain around the slides. The dead sperm will take up the eosin stain and seem pinkish though regular live sperm won’t take up the eosin stain and appear white in colour. To be able to help the observation, a thin smear of sperm suspension utilizing a Diff-Quik staining kit was carried out along with the percentage of abnormal sperm morphology was calculated. The morphological abnormalities of 200 sperms have been examined per slide below oil immersion. The data are obtainable as a percentage of abnormal sperm morphology. The sperm characteristics analysis was performed in triplicate per rat in accordance with the guidelines by [38] though suggestions by [39] have been utilised to analyze the rat sperm abnormal morphology. 2.five. Sperm DNA Fragmentation Analysis Sperm smears were air-dried at room temperature for 1 h on glass slides and fixed in Carnoy’s solu.
