Index IntroductionMaterials and methodsPreparation of bitter leaf extractExperimental design and treatmentSample collectionBiochemical analysisHistological examinationStatistical analysisResultDiscussionConclusionIntroductionThe environment we live in has been increasingly flooded from different types of environmental factors toxic substances that have the tendency to cause injury and metabolic stress to plants, animals and humans. Several industrial pollutants, including unrefined petroleum and its related products, such as kerosene, gas flaring, premium motor alcohol, diesel, 3,1-dinitrobenzene or nonylphenol, methanoxyethanol, glycol ether, and brake oils, are known to exert stress oxidative metabolism and testicular atrophy. A significant danger for those exposed to environmental toxicants is the increased risk of infertility, defined as the inability of a sexually active, non-contraceptive person or couple to achieve a spontaneous pregnancy within one year (WHO , 2010; Zegers et al., 2009). Ample evidence from studies reveals that most male infertility problems are the result of testicular oxidative stress which has been reported to affect seminal plasma antioxidants, increased lipid peroxidation (altered sperm morphology, impaired motility of spermatozoa). Say no to plagiarism. Get a tailor-made essay on 'Why violent video games should not be banned'?Download the original essayThe mechanistic defense against oxidative stress depends on the ability of the body and cells to enhance the buffering capacities of antioxidants that help eliminate the generated oxidative radicals by various metabolic processes especially when it comes to the elimination of toxic substances. Today, Vernonia amygdalina, a well-known vegetable common to many tribes of Nigeria, has been elucidated for its antioxidant buffering abilities is well known for its use as an alternative regimen for malaria. It has been used separately as a protective and ameliorative agent for the deleterious effects of many toxic substances such as cyanide, carbon tetrachloride, unrefined petroleum, and cycasin. From the above, there is no doubt that there is ample evidence for the ability of unrefined oil to induce various forms of metabolic oxidative stress. There is little evidence for the possible role of unrefined oil intoxication in inducing testicular damage caused by adulterated foods. with unrefined oil, as well as on the ability of Vernonia amygdalina to induce the possible restoration or control of activated metabolic stress parameters. This study was therefore conducted to clearly show research evidence to cover these existing gaps. Materials and methods Mature bitter leaf (Vernoniaamygdalina Del) was collected from a farm in Abraka, Nigeria and preliminary identification was carried out at the Department of Botany, Delta State University, Abraka, Nigeria by Dr Erhenhi AH The leaf is was subsequently authenticated at the Nigeria Forestry Research Institute, Jericho Hill, Ibadan, Nigeria, where a specimen with the voucher number, F101963 was deposited in the herbarium. Male albino rats (Rattusnorvegicus), thirty-six, an average weight of 150 g-182 g were purchased from the animal shelter at Delta State University, Abraka Nigeria. The rats were placed in a wooden cage and allowed to acclimatise for one week on the grower's feed (a product of Rainbow Feed Limited). The feed composition declared by the manufacturer was previously published by Achuba (2018). Everyonethe other reagents used for biochemical analysis were of analytical grade. Preparation of Bitter Leaf Extract Bitter leaf was washed, chopped and air dried at room temperature for one week in an open space within the laboratory of the Department of Biochemistry, Delta State University, Abraka. After drying, the leaf was bittercut and macerated using a Warren blender until a smooth, dry powder was obtained. Bitter leaf extract was prepared using methanol as described by Yin et al. (2013). One hundred (100 g) powdered leaves were dissolved in 400 ml methanol by sonication for 10 minutes, then filtered with Whatman No.1 using a vacuum pump. The obtained extract is concentrated via rotary evaporator at 40-50℃ under reduced pressure to obtain bitter leaf methanol extract (BLME). The extract was stored at −8℃ until needed. Experimental design and treatment The distribution of six rats per group was carried out according to the following description: Group A = Feed Group B = Feed +100 mgkg-1 body weight of BLME Group C = Feed + 200 mgkg -1 body weight BLMEGroup D = Feed (100g Feed+4ml Unrefined Oil)Group E =Feed (100g Feed+4ml Unrefined Oil) +100 mgkg-1BLMEGroup F= Feed (100g Feed+4ml Unrefined Oil) +200 mgkg -1BLMEL's body weight' Bitter leaf extract used was prepared fresh at the time of administration. To obtain 200 mgml-1, 20 grams of the extract were dissolved in 100 ml of distilled water from which aliquots of the freshly dissolved extract were administered by gavage based on the body weight of the rats once a day. Rats in groups A and D were not administered the extracts while all rats were allowed free access to water. All treatments lasted for a period of 30 days. Sample collection After a 30-day exposure period, rats were sacrificed by cervical decapitation on the 31st day after an overnight fast. Testes were collected in pre-chilled labeled containers. The wet testicular tissue (0.5 g) was homogenized with 9.0 ml normal saline using pre-cooled mortar and pestle, and the obtained supernatant was stored at -8°C in the cold room and used for biochemical analysis within 48 hours. Biochemical analysis Standard methods for the measurement of lipid peroxidation (MDA) level (Gutteridge and Wilkins, 1982) and enzymatic oxidative stress markers were employed as follows; aldehyde oxidase (AO) (Omarov et al. 1998), sulfite oxidase (Macleod et al. 1961); monoamine oxidase (MO) and xanthine oxidase (XO) (McEwen, 1971). The assay of the nonenzymatic antioxidant profile in the testes was determined using the methods of Ellman (1959) for the assay of reduced glutathione, while the assay of vitamin C used the methods reported by Achuba (2008). Tests for specific activities of enzymatic antioxidants were conducted using the methods of Misra and Fredorich (1972) for superoxide dismutase (SOD), Cohen et al., (1972) for Catalase, Habig et al. (1974) for glutathione-s-transferase (GST) and Khan et al. (2009) for glutathione peroxidase (GPx). Histological examination A known portion of the testes of each rat was collected and fixed in 10% saline form for 48 hours and processed for paraffin embedding with an automatic tissue processor by dehydration at 70%, 90%, 95% and two changes of absolute ethanol for 90 minutes each. Clarity was achieved by two changes of xylene for 2 hours each; and infiltrating with two changes of paraffin for 2 hours. Sections were cut at 5 μm with a rotary microtome. The sections were stained withhematoxylin and eosin (H and E) using the method of Odoula et al. (2009), examined and photographed under an optical microscope. Statistical analysis Data analysis was carried out using single-factor analysis of variance (ANOVA) with the help of the Statistical Package for the Social Sciences version 17 (SPSS 17). Post hoc analysis (between-group comparisons) was performed using Bonferroni at a significance level of P < 0.05. Result The results presented revealed a significant increase in lipid peroxidation (MDA) levels in rats administered both doses of BLME without contaminated diets (B and C) compared to positive control (A) fed a normal diet. This did not differ significantly between rats fed contaminated diets without treatment (groups D) and rats fed contaminated diets and given both doses of BLME (groups E and F). Feeding rats diets contaminated with unrefined oil . Furthermore, an increase in AO, SO, MO and significant compared to rats fed unrefined petroleum contaminated feed (group D). Administration of both doses of BLME to rats fed unrefined oil-contaminated feed (groups E and F) significantly increased oxidase activities (AO, SO, MO and XO) compared to rats fed untreated feed (group A) and rats fed unrefined feed contaminated with oil (group D). However, there were no significant differences in groups E and F when relative. As shown, CuZnSOD activities did not significantly increase in rats administered 100 mgKg-1 body weight of BLME (group B) compared to control group A fed normal diets only. However, it increased significantly in rats administered 200 mgKg-1 body weight compared to the control. Furthermore, CuZnSOD activities in rats given both doses were significantly higher than rats fed oil-contaminated diets without treatment and compared to rats fed contaminated diets and treated with both doses of BLME. . It was observed that rats fed only contaminated diets had reduced CuZnSOD activities compared to normal control, but significantly increased compared to those fed contaminated diets and treated with 200 mgKg-1 body weight. MnSOD activities did not change in rats from groups A and B but increased significantly when rats from group C and group A are relative. MnSOD activities were significantly reduced in rats fed contaminated diets in group D compared to normal control and in rats fed normal diets and treated with both doses of BLME in groups B and C. Treatment of rats fed contaminated diets with 100 mgKg-1 and 200 mgKg-1 of BLME showed no significant differences compared to untreated rats in group D. Total SOD activities showed no significant differences between AE groups but significantly reduced in group F which was exposed to contaminated diets and treated with 200 mgKg-1 of BLME compared to AD groups. The results presented reveal that there were no significant changes in vitamin C levels in all groups. It was observed that GSH levels did not present significant changes in rats administered 100 mgKg-1 and 200 mgKg-1 body weight of BLME (B and C) compared to the normal control group A, but increased significantly compared to rats fed dietspolluted by oil. Administration of 100 MgKg-1 body weight of BLME significantly increased GSH levels compared to those fed only contaminated diets, but decreased compared to the normal control (group A). Those fed polluted diets and treated with 200 mgKg-1 body weight of BLME (group E) remained unchanged compared to group D but reduced compared to all other groups. The activity of the antioxidant enzyme catalase was significantly elevated in rats treated with 100 mgKg-1 of body weight but not with 200 mgKg-1 compared to control group A. This was however significantly increased for both doses compared to rats in group D fed contaminated diets without treatment. . treatment with both doses led to a further reduction in catalase activities compared to groups A and D. It was observed that GPX and GST activities had no significant change for rats treated with 100 mgKg-1 wt. body without contamination compared to the control (group A) and significantly reduced for GPX while increasing for GST compared to rats fed only with polluted diets (group D). Treatment with both doses of BLME significantly reduced GSTS activities compared to control group A, while GPx activities were significantly reduced only for the 200 mgKg-1 body weight dose. Compared to group D, however, it was observed that the activity of GSTs remained significantly unchanged at both doses (100 mgKg-1 and 200 mgKg-1) of body weight. GPx, on the other hand, was significantly reduced for both doses. Discussion Unrefined petroleum contamination has remained a significant contributor to several endocrine-induced effects, stress, and metabolic dysfunction. On the other hand, testicular oxidative stress is said to be responsible for most of the numerous cases of infertility across the world. The result presented in this study revealed an increase in MDA levels and oxidative enzyme activities (AO, SO, MO and XO) in the testes of rats fed contaminated diets compared to normal control. Increased MDA levels have been increasingly reported as a powerful indicator of the adverse effects of consuming unrefined petroleum diets and other exposures to unrefined petroleum derivatives. The petroleum-induced increase in tissue peroxidation is said to go hand in hand with the eventual increase in oxidative enzymes needed to initiate the eventual elimination of peroxides and superoxides generated by petroleum contamination. It is important to state that based on the physiological location and nature of the testicles, it is said to be highly vulnerable to toxins, hence there is a built-in enhanced antioxidant buffer due to the presence of non-enzymatic antioxidants (vitamins such as Vitamin C, Vitamin E and GSH ) and enzymatic antioxidants (SOD, GST, GPX and catalase). Therefore, for oxidative damage to occur, there must be overwhelming scientific evidence of the antioxidant defense capabilities of the tissues and biological membranes involved. The observed induction of lipid peroxidation and oxidative enzymes in the testes of rats fed oil-polluted diets without BLME treatment are in agreement with the observed reduced levels of the antioxidant defensive markers SOD and catalase, GPx, GST, GSH, and vitamin C. These observations are similar to previous observations made by Achuba et al., (2016); Achuba (2018a) and Ita et al. (2018). The administration of BLME to these rats was not able to restore the levels of these non-enzymatic and enzymatic antioxidants to a comparable state compared to the control that had not been fed the diets.