EFFECT OF HESPERETIN TREATMENT ON BLOOD GLUCOSE LEVEL, SPERMATOZOA QUALITY, AND SPERMATOZOA QUANTITY IN ALLOXAN-INDUCED DIABETIC MICE

Male infertility has occurred rapidly in the last few decades, primarily in developing countries. An antioxidant, hesperetin is a flavonoid that is found in abundance in orange peels. The aims of this research were to determine the effect of hesperetin on blood sugar levels, spermatozoa quality, and spermatozoa quantity. The research structure included induction of diabetes mellitus and treatment for 8 weeks, followed by determination of blood sugar levels, spermatozoa quality, and spermatozoa quantity. Hesperetin has the ability to restore blood sugar levels, spermatozoa quantity, seminiferous tubules diameter, and testicular weight, volume, and germinal epithelial layer thickness with significant difference from the normal control group. Hesperetin did not fully restore spermatozoa motility, viability, and morphology with significant difference from the normal control group, nor from the positive control group. However, overall, hesperetin decreased blood glucose levels, increased spermatozoa quantity, and improved the spermatozoa quality in alloxan-induced diabetes mellitus mice. Dose-dependent activity was observed with the optimum dose at 200 mg/kg body weight. ____________________________________________________________________________________________________________________


INTRODUCTION
Diabetes mellitus is a chronic disease and one of the most important public health problems to be resolved globally. Diabetes mellitus has a very high prevalence in Indonesia (Ligita et al., 2019). Diabetes mellitus increases morbidity and mortality along with complications in various organs. Malfunction of male reproductive organs is one of the most widely reported complications in people with diabetes mellitus and can cause infertility in men and women (Deyhoul et al., 2017). Previous research on diabetes mellitus finds that decreases in the hormone testosterone and disruption of spermatogenesis are closely related to uncontrolled increases in blood glucose levels (Salimnejad et al., 2017;Soliman et al., 2019).
Hyperglycemia can also interfere with hypothalamus-pituitary-gonad axis function and cause local damage to the proliferation of Leydig and Sertoli cells. Leydig and Sertoli cells are intratesticular functional cells that have a role in testosterone biosynthesis and spermatogenesis (Petersen et al., 2015). Flavonoid is a compound which is found to reduce blood glucose level by antioxidant activity, recovering spermatozoa abnormalies caused by diabetes mellitus (Sarian et al., 2017;Jalili et al., 2018). Hesperetin is a class of flavonoid that can reduce blood sugar levels and can be extracted abundantly from citrus fruit peels (Meneguzzo et al., 2020). However, the effect of hesperetin on spermatozoa, and spermatozoa disorders caused by alloxan-induced diabetes mellitus in testes, is not yet known. Based on the findings above, the researchers are interested to examine the effect of hesperetin treatment on blood glucose level, spermatozoa quality, and spermatozoa quantity in alloxan-induced diabetes mellitus mice (Mus musculus L.).

MATERIALS AND METHODS
This study used 48 male mice obtained from the Department of Pharmacy at Universitas Sumatera Utara, Medan, Indonesia. Average age and weight for the mice were 60 days ±5 days and 30±5 g, respectively. Mice were placed in a plastic cage covered with husks; husks were changed twice a week. The cage was covered with wire and the mice were given food and drink ad libitum every day in accordance with the Ethical Clearance from the Department of Biology at Universitas Sumatera Utara.
The experiment used a completely randomized design with 8 treatments composed of 3 controls (normal, negative, and positive) and 5 treatments of hesperetin (Sigma Aldrich) in various concentrations. The number of treatments and replications in both tests fulfilled the Federer formula, (t-1) × (n-1) ≥ 15. Each treatment was replicated 6 times. The distribution of each treatment is C(0): normal control (without alloxan (Sigma Aldrich) induction and without treatment); C(-): negative control (with alloxan induction and without treatment); C(+): positive control (with alloxan induction and 150 mg/kg metformin treatment); T(1): treatment one (with alloxan induction and 50 mg/kg hesperetin treatment ; T(2): treatment two (with alloxan induction and 100 mg/kg hesperetin treatment); T(3): treatment three (with alloxan induction and 150 mg/kg hesperetin treatment); T(4): treatment four (with alloxan induction and 200 mg/kg hesperetin treatment); and T(5): treatment five (with alloxan induction and 250 mg/kg hesperetin treatment).
Alloxan solution in water was administered to the animals by intramuscular induction with a dose of 150 mg/kg body weight. Blood glucose level was checked on the 7 th day after induction using a glucometer. The tail of each mouse was cleaned with cotton dipped in warm water. Blood from the tail tip dripped onto a strip test and was read with a glucometer. Mice with blood glucose levels >200 mg/dL were diabetic and ready to be tested. The test-ready mice were given an extract according to the treatment dose. Hesperetin was dissolved with water and given to the mice orally at 0.3 mL for 8 weeks. During 8 weeks trial, the blood glucose level of mice was measured once a week after the mice fasted for 24 hours.

The Examination of Sperm Quality
Mice were killed by anaesthetic with ether and neck dislocation before being operated on to isolate the testes and epididymis. The epididymis was taken from the testes, and spermatozoa samples were taken immediately after operating by slicing and pressing the cauda epididymis slowly. Spermatozoa motility, viability, morphology, and quantity were then observed. Testicular weight and volume, diameter of seminiferous tubules, and thickness of testicular germinal epithelial layer were also determined. These methods were modified from Nak-Ung et al. Observation of spermatozoa motility was performed using a 100 fold magnification microscope. Spermatozoa motility was calculated by the formula (A + B) = (A + B + C + D) × 100. The motility of spermatozoa was grouped into the categories: fast progressive (A), slow progressive (B), non-progressive (C), and immotile spermatozoa (D) as they were counted. Observations of spermatozoa viability was conducted by placing one drop of spermatozoa sample in a petri dish. One drop of 0.9% NaCl solution was added and mixed using a glass rod. The suspension was observed.
Morphology of spermatozoa was examined as follows: spermatozoa suspensions were placed on a glass object, to which 1 drop of 70% methanol was added. This was fixed to dry with Canadian balsam and covered with a glass cover. The number of normal spermatozoa and abnormal spermatozoa was observed under a microscope with a 100-fold magnification microscope. The results obtained were calculated with the formula a= (a + b) × 100. Spermatozoa morphology was grouped into the categories normal morphology (a) and abnormal morphology (b). Normal mouse spermatozoa consist of a head (caput) that forms a hook like head, a short middle section, and a very long cauda.
For measuring the spermatozoa quantity, suspended spermatozoa were homogenized with 0.9% NaCl. Then 0.1 mL of the sample was added to the Neubauer improved hemocytometer and topped with a glass cover. Under a light microscope with a 100-fold magnification, a hemocytometer was placed and the number of spermatozoa was counted in rooms 1, 2, 3, 4, and 5. The spermatozoa quantity was calculated by N = 2 × 10 5 . N is the number of spermatozoa calculated in boxes 1, 2, 3, 4, and 5 on the Neubauer improved hemocytometer.

The Examination of Testicle
The testicular weight was measured by weighing the testes. The testicular volume was calculated by measuring the length and width of the testes, using a calliper and millimetre paper. The testicular volume was calculated according to the formula (π × W2 × L) = 6. The testicular volume in mm 3 was determined with π, testicular width (W), and testicular length (L).
Preparations of testicular histology were carried out using the paraffin method with hematoxylin eosin double stain. The paraffin method required fixation, washing, dehydration, purification, paraffin infiltration, implantation, incision, sticking, deparaffinization, staining, closing, and labelling.
The diameter of the seminiferous tubules and the thickness of the testicular germinal epithelial layer were determined using a 100x microscope and computer program Axiocam 8.0. Measurements were conducted on seminiferous tubules which were round or close to each other. Figure 1 shows the overview of the seminiferous tubules diameter and testicular germinal epithelial layer thickness. D is the diameter of the seminiferous tubules; E is the thickness of the testicular germinal epithelial layer with Hematoxylin Eosin and 100x magnification (Altoe et al., 2014).

Data Analysis
Data were arranged with the software Statistical Product and Service Solutions. The observed data were analyzed statistically using the parametric analysis of variance test. If the results were significant, analysis continued via Duncan test with degree of significance α = 0.05.

RESULTS AND DISCUSSION
The blood glucose level in the negative control group, positive control group, and the treatment group after alloxan induction was greater compared to the normal group without alloxan induction. This might be observed because alloxan compound is toxic and can damage pancreatic ß cells which produce insulin (Lenzen, 2008). Figure 2 shows the blood glucose level with various treatments from the first week to the eighth week.
After alloxan induction, blood glucose levels increased in the negative control group, positive control group, and treatment groups. Hesperetin and metformin treatments in alloxan-induced mice resulted in lower blood glucose levels than in the negative alloxaninduced control group. In the final week, it was found that the hesperetin and metformin treatment groups had significantly reduced blood glucose levels (P<0.05). The antidiabetic effect of hesperetin at 200 mg/kg body weight was not significantly different from the antidiabetic effect of metformin with dose 150 mg/kg body weight. In the eighth week, antidiabetic effects of hesperetin at all doses were not significantly different from the normal control group. Flavonoid acted as an active antidiabetic compound triggered insulin production and reduced blood glucose levels (Al-Ishaq et al., 2019;Caro-Ordieres et al., 2020).
All hesperetin treatment groups and metformin treatment groups declined in blood glucose level from the first week (W1). The greatest decrease in glucose level occurred in the 250 mg/kg hesperetin treatment group. The higher the dose, the greater the antidiabetic effect, indicating that flavonoid antidiabetic activity is dose dependent (Ahmed et al., 2012). As antioxidants, flavonoids play a role in lowering blood glucose levels (Banjarnahor and Artanti, 2014). Flavonoids can also prevent damage to insulin-producing β-cells by suppressing β cell apoptosis without changing the proliferation of pancreatic β cells (Gregory and Kelly, 2011). Oral antidiabetic drugs can trigger diabetic complications, including reproductive system disorders (Maiorino et al., 2014;Bahar et al., 2020). Therefore, antidiabetic drugs also have the potential to treat reproductive system disorders for people with diabetes (Meneses et al., 2015).
The spermatozoa quality and quantity decreased after alloxan induction. High blood glucose level disrupts blood flow due to vessel damage in several parts of the body, affecting spermatogenesis (Mangoli et al., 2013). Table 1 shows spermatozoa motility, viability, normal morphology, and quantity in the eight week, with various treatments. Table 2 shows the testicular weight, volume, and germinal epithelium thickness, along with the diameter of seminiferous tubules in the eight week, with various treatments.
Compared to the negative control group, the hesperetin treatment groups significantly affected the percentage of spermatozoa motility, viability, normal morphology, and quantity, as well as testicular weight, Hesperetin treatment fully recovered the spermatozoa quantity, along with seminiferous tubules diameter and testicular weight, volume, and germinal epithelial layer thickness compared to the normal control. Hesperetin treatment could not fully recover but closely resembled the spermatozoa motility, viability, and normal morphology of the normal control. However, the effect of 200 mg/kg hesperetin treatment was not significantly different from 150 mg/kg metformin treatment. Hesperetin treatment improved spermatozoa quantity and quality compared to the negative control. This corroborates previous studies which reveal that plant extracts with high flavonoid content can significantly improve spermatozoa quality and quantity (Benko et al., 2019).
Flavonoids can affect the process of spermatozoa transport to increase motility (Jofré et al., 2019). Adenosine triphosphatase enzyme maintains internal homeostasis for sodium and potassium ions. Ion concentrations maintain the ability of the membrane to transport dissolved materials across the cell membrane. A disrupted sperm membrane interferes with the transport of spermatozoa essential nutrients (Saez et al., 2011;Delpire and Gagnon, 2018). Alloxan induction causes damage to the cells of the pancreas, interferes with insulin production, causes hyperglycemia, and increases reactive oxygen species. Reactive oxygen species damage the mitochondrial membrane, potentially causing a loss of function which can induce sperm cell apoptosis (Loeken, 2012;Noh et al., 2020).
Flavonoids thought to increase testosterone production play an important role in spermatozoa viability. They maintain the epididymis, which is a temporary storage area for mature spermatozoa, allowing them to survive until they are excreted through the penis (Martin and Touaibia, 2020). Good epididymis function increases the percentage of spermatozoa viability. When dye is applied, viable sperm will appear colourless, however, non-viable sperm will appear red due to the entry of dye across the damaged sperm head membrane (Popal and Nagy, 2013).
Increased spermatozoa abnormalities may also occur due to various disturbances, particularly during spermatogenesis at spermiogenesis (Abdelhamid et al., 2019). The shape of the spermatozoa changes based on decreased testosterone levels, on which the epididymis also depends. Thus decreased testosterone levels cause abnormal spermatozoa morphology (Orieke et al., 2019). Flavonoid can repair spermatozoa morphology by decreasing free radical activity. This increases fertility and decreases the percentage of abnormal spermatozoa. It produces a positive feedback loop that also leads to increased testosterone levels (Olayinka et al., 2019). The normal shape of the spermatozoa will directly affect the spermatozoa motility and integrity (Jakubik-Uljasz et al., 2020). Spermatozoa morphology also plays a role in fertilization, wherein if the abnormal number of spermatozoa is too high, fertility will decrease (Alahmar, 2019).
Flavonoid increases spermatozoa quantity because of hypoglycemic activity; flavonoid reduces the blood glucose level in diabetic mice (Shi et al., 2019). Phytochemicals such as flavonoids increase the number of spermatozoa in mice due to antioxidant activity in which they neutralize free radicals (Biagi et al., 2019). Antioxidants are hydrogen and electron donors for free    (Aryal et al., 2019). Increased testicular weight is probably due to the increased activity of spermatogenic cells that make up the structure and the weight of the testicles. Spermatogenic cells also increase spermatogonia and increase testicular weight (Parhizkar et al., 2014). Flavonoid increases testicular weight by increasing secretion of gonadotropin releasing hormone that can affect the anterior pituitary, which in turn secretes follicle stimulating hormone and luteinizing hormone (Das and Kumar, 2018). Luteinizing hormone maintains the growth of Leydig cells, increases follicle stimulating hormone, maintains spermatogenesis, and increases testicular weight (Ramaswamy and Weinbauer, 2014).
Citrus flavonoid has antioxidant properties that restore testicular volume after exposure to certain substances (Gandhi et al., 2020). Testicular volume is related to semen quality, which is influenced by male sexual libido. The existence of repeated stimulation increases the gonadotropin hormone, which induces the hormone testosterone for optimum spermatogenesis (Dutta et al., 2019;Swee and Quinton, 2019). Semen fluid is secreted from the accessory gland stimulated by testosterone. Semen production and the volume are related to testicular volume (Noda, 2019).
Diameter and thickness of seminiferous tubules are determined by the interaction between follicle stimulating hormone and luteinizing hormone (Oduwole et al., 2018;Kalwar et al., 2019). If follicle stimulating hormone is not produced, luteinizing hormone fails to maintain homeostasis, causing the diameter of the seminiferous tubules to shrink. Normal levels of follicle stimulating hormone maintain the diameter and the thickness of the seminiferous tubules (Gupta and Verma, 2018). A diabetic condition can inhibit supply of nutrients to the testicles and disrupt spermatogenesis (Ding et al., 2015). Flavonoids provide antioxidant effects which restore the diameter and thickness of seminiferous tubules because antioxidant compounds neutralize free radicals (Gayatri et al., 2017).

CONCLUSION
Hesperetin decreases blood glucose levels, increases spermatozoa quantity, and improves spermatozoa quality in alloxan-induced diabetes mellitus mice. Activity was dose-dependent with the optimum dose observed at 200 mg/kg body weight. It is recommended that the next study be conducted with a longer treatment period to see the extent of damage to the reproductive organs due to complications of alloxan-induced diabetes.