Ameliorative effects of Allium cepa Linn. scaly leaves extract on reproductive dysfunctions in streptozotocin-induced diabetic Wistar rats

Diabetes mellitus, an endocrine and metabolic disorder characterized by hyperglycemia and low blood insulin or target organs insensitivity to insulin affects life quality due to its complications. Infertility is a complication in diabetes. Various agents have been used for research on diabetes-induced infertility globally, but there are little documented treatments for diabetes associated infertility. Allium cepa scaly leaves extract (ACSLE) possess anti-oxidant and anti-diabetic activities. This study investigated the effects of ACSLE on reproductive dysfunctions in male diabetic rats. Twenty eight male rats were assigned to 4 groups (n=7): CT (control); DNT (streptozotocin, 60 mg/kg once, intraperitoneal), DT1 and DT2 (streptozotocin, 60 mg/kg once, intraperitoneal, ACSLE 125 and 250 mg/kg rat/day respectively). Organ samples were obtained after 2 weeks and testicular weights recorded. Fasting blood glucose was determined using a digital glucometer. Sperm count, motility, viability and morphology were assessed microscopically. Testes were histologically evaluated. Glucose levels were reduced in DT1 and DT2 compared with DNT. Testes weights increased in DT1and DT2 compared with DNT. Sperm concentration increased in DT1 and DT2 compared with DNT motility increased in DT1 and DT2 compared with DNT; viability increased significantly in DT1 and DT2 compared with DNT. Abnormal sperm morphology decreased in DT1 and DT2 compared with DNT. Testes showed degenerated cells in DNT and no lesions in DT1 and DT2. Reduced blood glucose, improved testicular functions and morphology showed that ACSLE ameliorated reproductive dysfunctions associated with streptozotocin-induced diabetes in male Wistar rats.

Diabetes mellitus (DM), a disorder in which the body is unable to properly metabolize carbohydrates is increasing globally and now affects 9.3 % of the world's adult population (Saeedi et al., 2019). It is estimated that 463 million people had diabetes in 2019 (Saeedi et al., 2019) and without urgent and sufficient actions, it is predicted that 578 million people will have diabetes in 2030 and the number will increase by 51% (700 million) in 2045 (Saeedi et al., 2019).
The disease is characterized by excessive amounts of sugar in the blood and urine; inadequate production and / or utilization of insulin; thirst, hunger and loss of weight (Maiti et al., 2004). DM is a complex metabolic Introduction disorder that results from defects in insulin secretion, action, or a combination of both (Akkati et al., 2011). DM is grouped into two main types, type I diabetes mellitus (T1DM) and type II diabetes mellitus (T2DM) (American Diabetes Association, 2019).
T2DM is characterized by persistently high blood glucose caused by insulin resistance and usually with relative insulin deficiency (Butler I., 2003;Akkati I., 2011). Risk factors for T2DM are genetic predisposition, hyperlipidemia, lifestyle factors (including obesity, lack of physical activity, poor diet, stress, urbanization) and history of gestational diabetes (Kahn and Hull, 2006;Riserus et al., 2009;Akkati et al., 2011).
Diabetes is linked with increased oxidative stress (Freitas et al., 1997;Abou-Seif and Youssef, 2004) which may play an important role in the cause of diabetic complications.
Physiologically, the balance between reactive oxygen species (ROS) production and antioxidant activity is maintained in the body, but when ROS accumulates in excessive amounts either due to increased generation or impaired clearance, they can cause deleterious effects on sperm cells which can lead to sperm dysfunction (Marti et al., 2007;Agarwal et al., 2008;Tremellen, 2008).
The management of DM without negative effects is still challenging to the medical system, which has increased demands for natural and other products with anti-diabetic activity and little or no side effects (Kameswara et al., 1999;Philippe and Raccah, 2009;Akkati et al., 2011). Plants, plants extract, and the active compound from plants usage to manage diseases are crucial in new drugs discovery (Rupeshkumar et al., 2014).
Flavonoids are the major phenolics in onions, which can be classified to different subclasses (flavones, flavanones, flavonols, isoflavones, flavanonols, flavanols, chalcones, and anthocyanins) on the basis of the degree of unsaturation and the degree of oxidation of the central ring (P´erez-Gregorio et al., 2010). Flavonoids subclasses can be further differentiated on the basis of the number and nature of substituent groups attached to the rings (P´erez-Gregorio et al., 2010). Flavonols are the most abundant in onions, present as their glycosides, that is, quercetin and kaempferol (Prakash et al., 2007;Santas et al., 2010;Nile et al., 2020) in higher concentration (280-400 mg/kg) than other vegetables (i.e., 100 mg/kg in broccoli, 50 mg/kg in apple) (de Ancos et al., 2015). Anthocyanins, belonging to anthocyanidins, are mainly present in red onions (250 mg/kg), besides having a composition rich in flavonols as yellow onions (de Ancos et al., 2015).
Quercetin which is one of the most common flavonoids in A. cepa has been reported to improve diabetic status by decreasing oxidative stress (Mahesh and Menon, 2004;Coskun et al., 2005;Dias et al., 2005) and by reducing the disturbance of hepatic gene expressions (Kobori et al., 2009;Jiet al., 2011).
The objectives of this study were to study the anti -diabetic, antioxidant activity and free radical scavenging effects of Allium cepa scaly leaf extract on high blood glucose level and impaired testicular functions and morphology associated with streptozotocin-induced diabetic male Wistar rats.

Materials and methods
Ethical statement: This experiment was conducted in the experimental animal house at the College of Veterinary Medicine, Federal University of Agriculture, Abeokuta, Nigeria according to the guidance and approval of the committee on animal care ethics and use, Federal University of Agriculture, Abeokuta (CACEU/2019/3224). Animals: Twenty eight male Wistar rats (150-200g) were used for this study. The rats were housed in the experimental animal house of the college. They were kept in well ventilated standard rat cages at ambient temperature and 12 hour light/darkness period was maintained. The rats were fed standard pelleted rat chow and clean water was given ad libitum. They were kept for 10 days for adaptation prior to start of the experiment. Plant material: Allium cepa L. (onion) were obtained from a market in Abeokuta, Ogun State. Identification and authentication was done at Department of Pure and Applied Botany, College of Biosciences, Federal University of Agriculture, Abeokuta (FUNAABH0029). Plant extraction: Allium cepa L. scaly leaves were removed from the onion and reduced into coarse particles. The coarse particles were soaked in 97% ethanol for 72 hours at room temperature. The mixture was filtered using Whatman's filter paper and the filtrate was evaporated at 600C using a vacuum rotary evaporator. The wet brown residue i.eAllium cepa scaly leaves extract (ACSLE) was allowed to evaporate in vacuo and stored in the refrigerator (40C) until ready to use. Thereafter, 1 g of the residue was dissolved in 20 mL of distilled water to give a concentration of 50 mg/mL. Plant phytochemical analysis: Standard screening tests for the extract was performed for various plant constituents. Presence or absence of secondary metabolites such as alkaloids, steroidal compounds, phenolic compounds, flavonoids, saponins, tannins, and anthraquinones were screened for using standard procedures (Edeogaet al., 2005). Diabetes induction: Streptozotocin (STZ) (Sigma Chemical Co., St. Louis, MO, USA) was used to induce diabetes in rats. It was injected intra-peritoneally at a single dose of 60 mg/kg of body weight after being freshly prepared in ice-cold citrate buffer of pH 4.5 (Akbarzadeh et. al., 2007). Animals with glucose levels above 300 mg/dL were considered diabetic and were included in the study. Experimental Procedure: The rats were randomly distributed into 4 groups (n=7) after diabetes induction and treated as listed in Table 1. Blood glucose measurement Fasting blood glucose levels were estimated 3 times within a week during the treatment period of two weeks using blood sample obtained from the tail vein of the rats and determined in mg/dL by a digital glucometer (Accu-chek Advantage, Roche Diagnotics, Germany). The animals were fasted for a period of 16 hours before their blood glucose levels were measured.
At the end of the 14 days treatment, the rats were euthanized with thiopental injection (50 mg/kg) and a ventral midline abdominal incision was made to expose the reproductive organs. The testes were identified, carefully removed and processed further for sperm evaluation; sperm motility, concentration, viability and morphology as described by Jequier, (2010) and testicular histology. Statistical Analysis: The differences between the means were analyzed statistically with one-way analysis of variance, followed by Tukey's comparison test (ANOVA; 95% confidence interval). Values of P<0.05 were taken to imply statistical significance. Results were expressed as mean and standard error of mean and were presented in bar charts. Statistical Package for the Social Science (SPSS) software (version 16.0; SPSS Inc., USA) was used in all data analysis.

Discussion
Diabetes mellitus is a global health problem due to its serious health complications which includes hyperglycaemia and male infertility (Melendez-Ramirez et al., 2010). Streptozotocin-induced diabetes in laboratory animals has been widely used for research on diabetes mellitus (Tamtaji et al., 2017), which was employed in this present study and results from this study aligned with this documented finding. The blood glucose level of the diabetic non treated group was significantly increased compared to the control group (Figure 1).
Administration of Streptozotocin within one week to ten days induces stable diabetes which indicates irreversible destruction of Langerhans islets cells (Akbarzadeh et al., 2007) and diabetic patients have been documented to have increased reactive oxygen species in circulation which ultimately makes them suffer from an increased risk of free radical-mediated damage (Maxwell et al., 1997). The levels of plasma lipid peroxide products, including malondialdehyde in diabetic patients, increased compared to control subjects as documented by Freitas et al. (1997) and this may be one of the reasons for increased glucose concentrations in DNT.
Previous studies have suggested that quercetin derivatives in Allium cepa possess a strong antioxidant activity (Dias et al., 2005;Machavarapu et al., 2013), which is responsible for its beneficial effect on hyperglycemia and defects in male fertility caused by diabetes mellitus (Smith et al., 2003;Khaki et al., 2010;Chae et al., 2017). Daily oral administration of 125 mg/kg and 250 mg/kg of ACSLE to diabetic rats for 14 consecutive days caused a statistically significant decrease in the blood glucose level of diabetic animals ( Figure 1). This decrease in glucose level may be due to the potent antioxidant actions of ACSLE.
High glucose level impairs male fertility, affecting quality of sperm (Scarano et al. 2006;Kim and Moley 2008;Navarro-Casado et al. 2010;Amaral et al., 2014), multiplication rate or inhibits Sertoli cells maturation, and eventually the functionality having repercussions on spermatogenesis and spermatozoa (Tavareset al, 2017). Sertoli cells contain insulin receptors (Oonk and Grootegoed, 1987) which have effects on their metabolic functions (Oonk et al. 1985;Oliveira et al. 2012). Therefore, distortions in concentrations of insulin because of high blood glucose affect functions of Sertoli cell and spermatogenesis negatively (Schoeller et al. 2012).
The results of this present study indicated that there was a significant improvement in the sperm parameters (motility, viability, morphology and sperm concentration) of streptozotocin-induced diabetic rats by ACSLE administration (Table 3), this is in accordance with Khaki et al. (2010) who documented that Allium cepa increases the epididymal sperm number and the percentages of motile and viable spermatozoa in streptozotocin-induced diabetic rats. Also, Chae et al., (2017) documented that onion peel extract possesses beneficial properties on sperm motility that could be used in development of drugs to  (Table 3). Therefore, ACSLE administration was observed to ameliorate the deleterious effects of diabetes mellitus on sperm variables. These deleterious effects could be due to direct actions of high glucose concentration in circulation because abnormal glucose homeostasis has adverse outcomes for the reproductive function in the male gametes (Agbaje et al., 2007) or effect of reactive oxygen species (ROS) generation on sperm cells because there are documentations showing  (Freitas et al., 1997;Abou-Seif and Youssef, 2004;Palsamy and Subramanian, 2010;Folli et al., 2011;Muthukumaran et al., 2018). Energy in sperm cells is mainly used to maintain the motility to complete capacitation and subsequent acrosome reaction (Yanagimachi, 1994;Flesch and Gadella, 2000;Harrison and Gadella, 2005). Quercetin, the major derivative in Allium cepa is a strong antioxidant (Dias et al., 2005;Machavarapu et al., 2013;Nile et al., 2020), which help to ameliorate defects in male fertility arising from diabetes mellitus (Smith et al., 2003;Khaki et al., 2010;Jiet al., 2011). This potent antioxidant action of ACSLE may be the mechanism of ameliorating diabetic complications seen in the reproductive system. There was a significant increase in the testicular weight of streptozotocin-induced diabetic rats administered with ACSLE in this study. Daily oral administration of 125 mg/kg and 250 mg/kg of ACSLE to diabetic rats for 14 consecutive days caused a statistically significant increase in testicular weight when compared to that of the diabetic non-treated group. The reduction in weight of testes is often attributed to decreased population of germ cells including spermatogonia, spermatids and spermatocytes at various stages (Kanter et al., 2013). Studies have shown that Allium cepa improves testicular weight by increasing the number of germ cells through reduction of oxidative stress caused by diabetes mellitus (Mahesh and Menon, 2004) which suggested why there was a significant increase in testicular weight of diabetic rats treated by ACSLE.
Finally this present study revealed normal histological structure of most of the seminiferous tubules with normal spermatogenic series of the testes under microscopic examination after 14 days of treatment with ACSLE at 125 mg/kg and 250 mg/kg to streptozotocin-induced diabetic rats (Figure 2). The diabetic non treated group revealed severe degeneration and necrosis of germinal epithelial cells of the seminiferous tubules. This variation shows that ACSLE significantly improved histological testicular degeneration observed in streptozotocin-induced diabetic rats and this complies with previous reports by Park et al. (2007) and Khaki et al. (2010).
The ameliorative effect of ACSLE may be due to its high concentration of quercetin derivatives especially in the outer layers of Allium cepa (Park et al., 2007;Melendez-Ramirez et al., 2010), which was why ACSLE was used in this present study. Allium cepa improves diabetic status of animals by decreasing oxidative stress (Coskun et al., 2005) and by also reducing the disturbance of hepatic gene expressions (Kobori et al., 2009).
In conclusion, the results of the present study showed that ACSLE possesses ameliorative effects on high blood glucose level and impaired testicular functions and morphology associated with diabetes mellitus.

Recommendations
The whole data made available in this work supports the use of ACSLE has an antidiabetic agent with low reproduction complications. However, further investigations are required to study the exact mechanism of the ameliorative actions of ACSLE on reproductive dysfunctions in streptozotocin-induced diabetic Wistar rats with great emphasis on quercetin.