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Treatment with dams with dexamethasone during lactation has been reported to induce oxidative stress in the testis of the offspring. Allium cepa L (Red Onion) is known to be a potent free radical scavenger. The protective role of Allium cepa against oxidative stress induced in testis following treatment with dexamehasone during lactation in Wistar rats was assessed. Twenty female rats were assigned into four groups (n = 5) during lactation and they were treated as follows: Group 1 serve as Control (distilled water), Group 2, 3, and four were admistered dexamethasone (60 μg/kg), Allium cepa (5 ml/kg) and dexamethasone + Allium cepa respectively. Testicular descent, pubertal age, sperm quality indices, and serum hormonal profile were assessed as indices of reproductive function. Testicular malondialdehyde (MDA) reduced glutathione (GSH) as well as superoxide dismutase (SOD) and catalase activities were assessed as measures of oxidative stress. Results obtained showed that dexamethasone caused significant (P < 0.05) reduction in testes weights, indices of sperm quality, serum testosterone, FSH, LH levels and testicular antioxidant enzyme activities. There was significant delay (P < 0.05) in days of testes descent, preputial separation and increase in testicular MDA. However, maternal treatment with Allium cepa Linn juice significantly (P < 0.05) improved both indices of reproductive function and testicular antioxidant enzymes. These findings suggest that Allium cepa Linn has a protective effect against testicular oxidative stress and reproductive dysfunction following treatment of dams with dexamethasone during lactation.
Dexamethasone-BP (DEX) tablets (Xasthen®, Jiangsu Penyao Pharmaceuticals Ltd, China) was suspended in distilled water in a dose of 60 μg/kg. Allium cepa Linn. juice was prepared according to the method of Ola-Mudathir et al. (2008). The Kano Red Creole variety of fresh Allium cepa bulbs was obtained. It was identified at the herbarium of Botany Department, University of Ibadan and National Institute of Horticultural Studies (NIHORT), Ibadan. The fresh (100 g) Allium cepa bulbs were washed, cut into small pieces and homogenized in a blender. The resultant slurry was filtered and the filtrate was used. The Allium cepa bulb extract was administered to the rats with the aid of a flexible oral gavage tube throughout the course of the study. Fresh juice was prepared daily every morning. Twenty virgin female (180–200 g) and ten male Wistar rats (200–250 g) (12 weeks of age) were obtained from the Central Animal House, College of medicine, University of Ibadan. The animals were housed in well ventilated cages with access to adequate rodent's feed and water ad libitum. Acclimatization took place for two weeks. Female rats were mated overnight during their proestrous phase with proven male breeders in ratio 2:1 (female to male). After mating had been established, female rats were randomly divided into four groups of five animals each and were treated during lactation (Table 1). Parturition was allowed to occur naturally and maternal dexamethasone administration commenced on Postnatal Day (PND) 1. Litter size was standardized to six pups per litter. All animal experiments were conducted in accordance with the International Ethical Norms on Animal Care and Use as contained in NIH publication/85-23, revised in 1985. The study was approved by Ethical committee on the use of laboratory animals, Department of Physiology, Cross River University of Technology, Calabar, Nigeria. Experimental groups. n=5. Treatment lasted for 21 days PND 1–21. The male pups were weaned on PND 28. Testis descent and preputial separation were monitored starting from PND 21 and PND 42 respectively. Body weight was measured at PND 56 (8 weeks) and PND 84 (12 weeks) to examine the progression in growth rate. The animals were sacrificed under Sodium thiopentone anesthesia (50 mg/kg, i. p.) at the end of PND 96 (14 weeks) (Pereda et al., 2006). Blood samples were collected via cardiac puncture and serum was obtained for hormonal analysis. The testis was harvested, weighed and placed in phosphate buffer solution for oxidative status analysis. Blood was obtained via cardiac puncture (PND 96) into polythene tubes and allowed to clot for 1 h. The blood samples were centrifuged at 3000 rpm for 10 min. Serum was aspirated and stored at 4 °C. Serum testosterone, FSH, LH and corticosterone were assayed using enzyme-linked immunosorbent assay (ELISA) kits (Fortress Diagnostics, UK) according to the protocol in respective manufacturer's manual. The testis was harvested, weighed and homogenized in phosphate buffer solution for oxidative status analysis. The homogenate was centrifuge at 10,000 rpm in a cold centrifuge. The supernatant was aspirated into a plane tube, stored at 4 °C until use. The assays were done within 48 h of collecting the sample. Level of Lipid peroxidation was evaluated by method of Buege and Aust (1978). Catalase activity was evaluated by method of Sinha (1972). The SOD activity was evaluated by method of Misra and Fridovich (1972). The GST activity was evaluated by the method of Habig et al. (1974). Protein estimation was done by method of Lowrey et al. (1951). GSH content was evaluated by the method of Beutler et al. (1963). Sperm analysis was done by microscopy as previously described (Raji and Bolarinwa, 1997; Raji et al., 2003). Epididymal spermatozoa were obtained by mincing the epididymis with anatomical scissors in 5ml of pre-warmed physiological saline and incubated for 2 min. An aliquot of this solution was placed in improved Neubauer counting hemocytometer and motile sperm were counted by using microscope at 400× magnification. Non-motile sperm numbers were first determined, followed by counting of total sperm. Sperm motility was expressed as a percentage of motile sperm of the total sperm counted. Percentage of morphologically abnormal spermatozoa was determined by preparing two slides with Hemaoxylin and Eosin stains for morphological examination of live–dead ratio. A total of 400 sperm cells were counted on each slide under light microscope at 400× magnifications. Sperms with abnormal head and/or tail were considered abnormal. Sperm motility, viability and count were done immediately and quickly. A sperm viability test was done using eosin/negrosin stain (containing 1 g of Eosin and 4 g of Negrosin in 100 ml phosphate buffer). A drop of the epididymal fluid was placed on the slide and two drops of the stain was added. A thick smear was made from this and dried. After this, the slide was studied under light microscope using 40x objective lens. The unstained spermatic cells were considered as live sperms while the stained ones was considered as dead sperm. A minimum of 100 spermatic cells (both stained and unstained) was counted and an average was taken for the percentage live sperm. Data were presented as mean ± SEM and Anova was used for comparison of results, followed by Tukey's post-hoc test to compare the differences in means of the different treatment groups. P < 0.05 was considered significant. The data analysis was done with the use of Graphpad Prism Version 7.0 for Windows.
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