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Maternal treatment with dexamethasone induces oxidative stress in the reproductive structures of offspring. Consumption of Allium cepa Linn improves antioxidant status. This study was designed to evaluate the protective role of Allium cepa Linn juice on maternal dexamethasone induced alterations in reproductive functions of the female offspring of Wistar rats. Twenty lactating dams (180–200 g) were randomly assigned into four groups (n = 5) on the day of parturition and treated as follows during lactation for 21 days: Control (5 ml/kg BW distilled water); Dexamethasone (60 μg/kg BW); Allium cepa (5 ml/kg BW); Dexamethasone + Allium cepa (60 μg/kg BW + 5 ml/kg BW). The female offspring were separated at birth. Days of vaginal opening and first oestrus cycle, length and frequency of estrous cycle as well as serum hormonal profiles were assessed as measure of reproductive functions. Ovarian superoxide dismutase (SOD) activity, catalase activity and malondialdehyde (MDA) level were measured as indices of oxidative stress. Oestrous cycle length, frequencies of diestrus as well as the Ovarian MDA were significantly increased (p < 0.05) in dexamethasone (DEX) group relative to control group. Serum 17β-oestradiol and corticosterone level in addition to SOD and catalase activities were significantly reduced (p < 0.05) in DEX group relative to control. Co-administration of Dex with Allium cepa Linn juice reduced the oestrous length, frequency of diestrous as well as ovarian MDA. There was also a significant increase in serum 17β-oestradiol, ovarian SOD and catalase activity. The results suggest that Allium cepa could protect against alterations in reproductive functions of offspring induced by maternal treatment with dexamethasone during lactation in Wistar rats. The flavonoid constituent of onion may also help in reducing oxidative stress in the offspring.
Adult male and nulliparous female Wistar rats weighing 200–230 g and 180–200 g respectively were used for the study. The rats were housed in well aerated plastic cages and allowed access to rodent's pelletized feed (Ladokun Feed Mill, Ibadan. Feed Composition: 26.5% protein, 40% carbohydrates, 29% Fat and 4.5% crude fibre) and water ad libitum. All animals were acclimatized to the environmental conditions of the laboratory for two weeks before the commencement of the study. 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. Dexamethasone-BP (DEX) tablets (Xasthen®, Jiangsu Penyao Pharmaceuticals Ltd, China) was suspended in distilled water and administered at an oral dose of 60 μg/kg body weight daily. Allium cepa bulb juice was prepared according to the method described by Ola-Mudathir et al. (2008). The Kano Red Creole variety of fresh Allium cepa bulbs was purchased from Bodija market in Ibadan. It was identified at the herbarium of Botany Department, University of Ibadan and National Institute of Horticultural Studies (NIHORT), Ibadan. The Kano Red Creole species was selected as studies have reported its high antioxidant potency (Denton and Ojeifo, 1990). The fresh Allium cepa bulbs were washed, cut into small pieces and homogenized in a blender. The resultant slurry was squeezed and filtered through a fine cloth 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 in the morning. The determination of oestrous cycle was done using the Marcondes' technique (Marcondes et al., 2002) Oestrous pattern was studied by determining the oestrous phase of each animal every morning between 7:00 am and 8:00 am throughout the study. Vaginal content was collected with a Pasteur pipette containing about 0.1 mL of normal saline (0.9% NaCl) by gently inserting the tip of the pipette into the rat's vagina. The pipette was pressed to release the fluid content 2 or 3 times in order to make a vaginal lavage which contained some of the vaginal cells of the rat. The fluid and its content were thereafter siphoned with pipette and the content was placed on a glass slide. A new and clean glass slide was used for each animal. The slide was viewed using the ×10 and ×40 magnification objective lens of the microscope (Olympus, Japan). The cell types and the proportion among them were used to determine the oestrous cycle phase of the rat. Female rats were paired with male rats at ratio 2:1 (female: male) during the proestrus phase of the female rats' cycle. On the morning after pairing, vaginal lavage was collected with Pasteur pipette filled with about 0.1 mL of normal saline (0.9% NaCl) by gently inserting the tip of the pipette into the rat's vagina. The withdrawn vaginal content was dropped on a glass slide and the smear was spread out evenly. The glass slide was examined using the ×40 objective lens of the light microscope (Olympus, Japan) to determine the presence of spermatozoa. Mating was confirmed by the presence of spermatozoa in the vaginal smear. Confirmed pregnant dams were transferred into nursing cages. Pregnant dams were allowed to litter naturally, and the day of parturition was designated as Post-Natal Day (PND) 1. The litter size was standardized to six pups per litter. Birth weight was measured within 24 h of postnatal life. They were weighed on a sensitive electronic scale (Lisay, China). Twenty (20) lactating dams were divided into four groups (I-IV) of five rats each on PND1 and treated as follows: Group I was administered 5 ml/kg body weight distilled water orally. Group II received 5 ml/kg body weight Allium cepa bulb juice orally (Lee et al., 2013). Group III received 60 μg/kg body weight dexamethasone orally (Amar et al., 2013). Group IV received 60 μg/kg body weight dexamethasone and 5 ml/kg body weight Allium cepa bulb juice orally. Administration was done in each group in the morning within 8–10am daily, and pups were fed by dams ad libitum. Administration was done using an orogastric gavage method with an oral cannula. Administration lasted for 21 days (lactation period) after which the female pups were weaned and transferred into separate cages where they were allowed to mature. The female offspring were weaned on PND 28 (Post-Natal Week [PNW] 4 and pooled into groups depending on maternal lactational treatment. The weaned offspring were fed standard rat chow (Ladokun Feed Limited, Ibadan) and allowed access to water ad libitum. No administration was done on the randomly selected offspring. Body weight was recorded on post-natal weeks 4, 8, and 12. Sexual maturity of the female rodents was determined by vaginal opening. This heralds the attainment of puberty and sexual maturity. The first oestrus is an indication of the ability of the animal to conceive. Beginning from PND 40, the female rats were checked for vaginal opening and commencement of oestrous cycle was monitored. Date of vaginal opening (VOD), first oestrus, oestrous pattern (frequency, length), were monitored for 21 days. On the 14th week of postnatal life during proestrus, the rats were euthanized under sodium thiopental anaesthesia (50 mg/kg, i.p.). They were surgically opened at the linea alba of the anterior abdominal wall to the thoracic cavity to expose the heart and other organs. Using a 5 mL syringe and needle, blood was collected into plain serum bottles through cardiac puncture. The blood was allowed to clot for at least 60 min after which it was centrifuged at 3000 rpm for 10 min. The serum portion which is the supernatant was decanted from the centrifuged blood and stored at −20 °C for hormonal assay. Serum levels of Follicle Stimulating Hormones (FSH), Luteinizing Hormones (LH), oestradiol, and corticosterone were assayed in the female offspring. The ovaries and uteri were harvested and freed of adherent tissues. All harvested organs were fixed in 10% formalin for histological examination. The serum corticosterone, FSH, LH and estradiol levels were determined per animal using the ELISA kit ((Fortress Diagnostics, UK) according to the protocol in respective manufacturer's manual. One of the harvested ovaries was homogenized in phosphate buffer (pH 7.4) and the homogenate was centrifuged at 10,000 g × 15 min at 4 °C. The supernatant was collected for the assay of total protein, malondialdehyde (for lipid peroxidation), superoxide dismutase, catalase, and glutathione peroxidase concentration in the ovaries. All biochemical assays were done within 48 h of sample collection. Ovarian lipid peroxidation was determined by measuring the Thiobarbituric Acid Reactive Substances (TBARS) produced during lipid peroxidation (Ohkawa et al., 1979). This method is based on the reaction between thiobarbituric acid (TBA) and malondialdehyde (MDA), an end product of lipid peroxide during peroxidation. The level of SOD activity was determined by the method of Misra and Fridovich (1972). The ability of SOD to inhibit the autoxidation of epinephrine at pH 10.2 makes this reaction a basis for a simple assay for this dismutase. Superoxide radical generated by the xanthine oxidase reaction caused the oxidation of epinephrine to adrenochrome and the yield of adrenochrome produced per superoxide introduced increased with increasing pH and also increased with increasing concentration of epinephrine. Catalase activity was determined according to the method of Sinha (1972). This method is based on the fact that dichromate in acetic acid is reduced to chromic acetate when heated in the presence of H2O2, with the formation of perchromic acid as an unstable intermediate. The chromic acetate then produced is measured colorimetrically at 570–610 nm. Since dichromate has no absorbency in this region, the presence of the compound in the assay mixture does not interfere at all with the colorimetric determination of chromic acetate. The catalase preparation is allowed to split H2O2 for different periods of time. The reaction is stopped at a particular time by the addition of dichromate/acetic acid mixture and the remaining H2O2 is determined by measuring chromic acetate colorimetrically after heating the reaction mixture. Glutathione peroxidase (GPx) catalyzes the redox reaction between reduced glutathione (GSH) and hydrogen peroxide (H2O2). The amount of GSH utilized is estimated by measuring it in the assay mixture before and after the enzyme activity (Rotruck et al., 1973). GSH reacts with Ellman's reagent (5, 5′-dithiobis-2-nitrobenzoic acid or DTNB) to give a yellow colour which was then measured at 412 nm. Histological assessments were conducted following heamatoxylin and eosin staining techniques. Data are expressed as mean ± Standard Error of Mean (SEM). Differences between mean values were evaluated by analysis of variance (ANOVA), followed by Tukey's post-hoc test for pairwise comparisons. Values of P < 0.05 were considered statistically significant. GraphPad Prism 7.0 software (GraphPad Inc, USA) was used for the statistical analysis.
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