Ndled in accordance with the ethical suggestions approved by the Animal Ethics Committee of Universiti Sains Malaysia with reference no. USM/IACUC/2018/(112)(921) and also 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) had been further 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 have been administered by means of oral forced feeding making use of needle gavage for 28 consecutive days among 09:00 a.m. and 10:00 a.m. [12]. Soon after four weeks of remedy, two verified fertile female rats in the oestrous phase had been paired with every male rat. Nevertheless, prior to pairing together with the male rats, the female rats were first MMP-14 MedChemExpress screened for two consecutively common oestrous cycles. They have been paired through the dark phase from the reversed light/dark cycle in between 9:002:00 h for 3 h per day [36]. Immediately after the mating period, by using a light microscope (Olympus BX41, Olympus Corporation, Tokyo, Japan), vaginal smears have been 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, 9,4 ofvaginal smear [37]. Female rats were left till giving birth to progeny, starting from day 21. Soon after mating, the male rats had 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 had been 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 till postnatal day 70 for evaluation of developmental landmarks. At the BRD9 Storage & Stability finish of the study, the chosen organs from both sexes of F1 rats had been utilized for histomorphometric evaluation. 2.four. Sperm Traits Evaluation Immediately after dissection, the sperm was collected quickly and was suspended in Hank’s balanced salt option (HBSS) with 298 mOsmol/kg, pH 7.4. For the epididymal sperm count and motility evaluation, a total of ten 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 when sperm count was expressed as million sperm cells per ml of suspension. Meanwhile, for sperm viability assessment, a thick smear was performed utilizing 10 of sperm suspension and adding ten of eosin-nigrosin stain around the slides. The dead sperm will take up the eosin stain and appear pinkish even though standard live sperm is not going to take up the eosin stain and appear white in color. As a way to assist the observation, a thin smear of sperm suspension making use of a Diff-Quik staining kit was done and also the percentage of abnormal sperm morphology was calculated. The morphological abnormalities of 200 sperms had been examined per slide beneath oil immersion. The data are obtainable as a percentage of abnormal sperm morphology. The sperm characteristics evaluation was performed in triplicate per rat in accordance using the recommendations by [38] whilst suggestions by [39] were made use of to analyze the rat sperm abnormal morphology. two.five. Sperm DNA Fragmentation Analysis Sperm smears have been air-dried at space temperature for 1 h on glass slides and fixed in Carnoy’s solu.