Gum Arabic Improves Reproductive Function Associated with Alteration of Testicular Visfatin mRNA Expression in Alloxan Induced Diabetic Rats

Objectives: Diabetes mellitus (DM), an endocrine disorder, epidemic all over the world which causes dysfunction of reproductive capacity. Gum Arabic (GA), a dietary fibre has potential health effects on reproductive system. Visfatin is one of the adipokines which has a crucial role in reproductive function. Aims: We hypothesized that the testicular Visfatin may be affected by administration of GA in diabetic rat. Material and Methods: Sixty male Sprague-Dawley rats were divided into 3 groups (n=20 of each): control group, diabetic group which were injected with Alloxan, and diabetic group given 10% GA in the form of drinking water for 8 weeks. The sperm quality, blood glucose, serum testosterone were measured. In addition, both testicular mRNA and protein expression of Visfatin were measured. Moreover, testicular histopathological features were examined. Results: The treatment of GA significantly (P<0.05) improved semen quality compared the diabetic and control groups. The treatment of GA significantly (P<0.05) decreased blood glucose concentrations whereas, increased serum testosterone concentrations when compared to diabetic and control groups. In addition, treatment of GA significantly (P<0.05) increased testicular Visfatin mRNA expression associated with upregulation of Visfatin protein expression compared to the diabetic group. Testis of diabetic rats showed obvious degeneration whereas; slight degeneration was seen in GA treated rats compared to control. Visfatin mRNA expression revealed a positive correlation with body, testis weight and serum testosterone concentrations, while a negative correlation was observed with blood glucose concentrations. Conclusion: Our findings conclude that GA may improve reproductive performance and it may be useful to meliorate the diabetic fertility complications in male.


Introduction
Bioactive natural principles are reported used as a source of medicinal agents for several decades [1,2]. Herbal-based principles play vital roles in the treatment the prevention of diabetes mellitus (DM) [3,4]. In developing countries, herbal products considered as a traditional medicine for meliorating diabetes complications [5,6]. Approximately, fifty millions of couples worldwide are experience infertility problems [7,8], mainly through comparative contributions in change of lifestyle [9] and metabolic disorder factors [10]. Herbal bioactive agents are principally used worldwide to improve reproductive capacity [11]. Various types of medicinal plants are reported to be used in mixture with assisted reproductive technologies [11,12] to decrease the cost [13,14] and boost infertility success rate [15]. DM is a group of metabolic disorders that characterized by hyperglycemia [16] [59], improves antioxidant capacity [60] decreases blood glucose [61], decreases blood pressure in patients with type 2 DM patients [62]. However, the effects of GA on Visfatin in testis of type I diabetic rats have not been reported. In addition, it is less clear whether GA may modify testosterone levels in type I diabetic rats. Therefore, in the present study, we induced type I diabetic in rat to elucidate our hypothesis that the treatment of GA to type I diabetic rat may change blood Visfatin and its mRNA and protein in the testis of diabetic rat and the changes in Visfatin may correlate with blood glucose, testosterone concentrations, testis and body weight in diabetic rat.

Animals and Experimental Design
Male Sprague-Dawley (SD) rats weighing 200±10g were purchased Sudanese National Center for Research and housed in a controlled environment with a 12h light-dark cycle. Animals were acclimatized for one week before the study and had free access to water and standard rat chow throughout the experimental period. The rats were divided into 3 groups: control group (n=20) given standard animal pellet and water ad libitum; diabetic group (n=20); and diabetic group (n=20) given 10% Gum Arabic in drinking water for 8 weeks. Type I diabetes mellitus was induced as described by Adeyi, et al. [63]. Briefly, Alloxan monohydrate was purchased from Sigma-Aldrich China (Shanghai, China), and type I diabetes mellitus was induced by single intraperitoneal injection of 150mg/kg of Alloxan monohydrate dissolved in normal saline after an overnight fast. Surviving rats after 3 days with blood glucose concentration more than 200mg/dl of blood were considered as Alloxan-induced diabetic rats and used as type I diabetic models rat for further study. All rats were euthanized after 8 weeks of treatment. On day 70, the rats were fasted overnight, urine and blood samples were collected prior to euthanasia. Body weights and organ weights were recorded; blood and tissue samples were collected and stored at -80 °C until analyzed. The experiment procedures were approved by the Animal Ethics Committee of University of Nyala.

Sperm Analysis
Testes with epididymis were removed, and the caudal epididymidis were separated from the testis and the semen was collected. Squeezed semen was incubated in buffer containing BSA at 37 °C for 30 minutes. The normal morphology of sperm, motility, sperm count and its viability were measured in groups of experimental rats. We used Makler Chamber and light microscopy (Olympus Co., Tokyo, Japan) for sperm movement analysis. The The data were presented as percentage of morphological normal sperm.

Blood Glucose
Serum was obtained from blood by centrifugation (at 3000rpm for 15min) and stored at -20 ºC until analyzed. Blood glucose was measured using assay kits (Nanjing Jiancheng Bioengineering Company, Nanjing, China), according to the manufacturers' instructions.

Plasma Testosterone
After decapitation of rats, blood was collected from the ruptured cervical vessels in new heparinized tubes for plasma testosterone measurement. The plasma was obtained after centrifugation (2,400rpm, 20min, 3.5 °C) in a refrigerated device and frozen at -20 °C until measurement of hormone levels. Plasma testosterone concentrations were measured by radioimmunoassay method, using the Testosterone Maia® kit (Biochem Immuno System) according to the manufacturers' instructions. All plasma samples were dosed in the same assay, to avoid inter-assay errors. The lower detection limit for testosterone was 0.064ng/mL, with a 4% intraassay error.

Real-Time PCR and Gene Expression
About 100mg of liver was ground in liquid N 2 , and a portion of about 50mg was used for RNA extraction using TRIzol total RNA kit (Invitrogen, Biotechnology Co, Ltd, Carlsbad, CA, USA) according to the manufacturer's instruction. Two approaches were taken to ensure that all the total RNA preparations are free of genomic DNA contamination. Firstly, total RNAs were treated with 10 U DNase I The melting curves were performed to insure a single specific PCR product for each gene. The PCR products were sequenced to validate the identity of the amplicons. Primers specific for Visfatin (Table 2) was synthesized by Geneary (Shanghai, China). Rat GAPDH was used as a reference gene for normalization purpose.
The method of 2 -ΔΔCt was used to analyze the real-time PCR data [65]. The mRNA abundances were presented as the fold change relative to the average level of the control group.

Protein Extraction and Western Blot Analysis
Protein extracts from 50mg of frozen liver tissue were prepared as described in our previous publication [66]. The

Statistical Analysis
Descriptive statistics analysis was made to check the normality and homogeneity of variances before parametric analyses. Body weight, organs weight, semen parameters, blood glucose, plasma testosterone, relative quantitative data of gene expression, in addition to western blot analysis data were statistically analyzed by one-way ANOVA using IBM SPSS statistics 21.0 for Windows, followed by a least-significant difference (LSD) test for individual comparisons. A P-value ≤0.05 was considered significant.

Effect of GA Treatment on Food Intake, Body Weight and Organs Weight
In the present study, Alloxan induced diabetic rat groups showed significantly increases in food intake compared to the control group. The treatment of GA significantly (P< 0.05) decreased food intake ( Figure 1A). No significant differences were observed final body weight ( Figure 1B), testis weight ( Figure 1C) and epididymis weight ( Figure 1D) regarding the treatment of GA.  (Figure 2A). In contrast, the treatment of GA significantly (P<0.05) increased serum testosterone concentration compared to the diabetic rat group ( Figure 2B). Likewise, the treatment of GA significantly increased serum Visfatin concentration compared to the diabetic rat group ( Figure 2C).

Parameters Correlations
The present study, expression of Visfatin mRNA revealed a positive correlation with body weight (Figure 3A), testis weight ( Figure 3B), and serum testosterone concentration ( Figure 3D).
However, the expression of Visfatin showed negative correlation with blood glucose concentrations ( Figure 3C).

Effect of GA Treatment on Semen Quality
Alloxan induced diabetic rat group showed significant decreases in sperm count, sperm rapid mobility, slow motility, non-progress mobility, total motility and sperm vitality compared to the control.
However, the treatment of GA significantly (P < 0.05) improved the above mentioned sperm quality parameters. On the other hand, diabetic rat group showed significant increases in immotile sperm compared to the control group. But the treatment of GA significantly (P < 0.05) reduced immotile sperm compared to that in diabetic rat (Table 1).

Testicular Visfatin mRNA Expression
In the current study, the treatment of Alloxan significantly reduced testicular mRNA expression of Visfatin compared to the control group. But the treatment of GA significantly (P<0.05) upregulated testicular Visfatin mRNA expression compared to the diabetic rat group ( Figure 4A).

Testicular Visfatin Protein Expression
In the present study, Alloxan treated group rats significantly

Histopathological Features
Histological features in testis of the control group revealed normal cytology of the testis with no visible degenerative changes ( Figure 6A). The testis of diabetic rats showed obvious degenerative changes with vacoulations ( Figure 6B). However, the treatment of GA slightly protected the testis of diabetic rats from degenerative damages compared to control and diabetic rats ( Figure 6C).

Discussion
Diabetes mellitus (DM) is a potential pandemic metabolic disorder causes dysfunction of reproductive performance [67]. In the present study, the treatment of Gum Arabic (GA) decreased food intake associated with decreases in body weight and testis weight. These findings are in line with previous studies that the treatment of dietary fibre decreased food intake and decreased body weight in mice [67][68][69]. The decreases in food intake or body weight by GA could be due to the fact that a number of studies revealed that the dietary fiber enhanced satiation and satiety [70], changed the glycaemic index [71], influenced the gastric emptying, and secretion of gastric hormone [72] thus, it reduces body weight [67]. In addition, the supplementation of GA in the form of drinking water decreased blood glucose. These findings are consistent with our earlier studies that GA decreased blood glucose both in normal and diabetic rat [73,74] or normal mice [61]. It is well documented that the consumption of GA inhibits absorption of glucose in the intestine through interaction of membrane abundance in sodium-glucose transporter 1(SGLT1) in mice [75]. However, the mechanism of underlying the reduction of blood glucose by GA is not yet fully elucidated, due to the lack of researches in this field.
Several experimental studies documented that the induction of diabetes in animal models has impaired reproductive performance via decreasing testicular function thus caused male fertility [76].
In the present study, the treatment of GA increased sperm quality parameters in diabetic rat. These results confirm our previous studies which showed the improvement of semen quality by GA in diabetic rat model [77]. In addition, it was report that ginger (Zingiber officinale) a dietary fibre enhanced reproductive capacity via increasing semen quantity in diabetic rat [78]. Moreover, the histological changes of the testis showed marked degeneration in the testis of Alloxan-induced diabetic rats. However, the treatment of GA significantly protected the testis of diabetic rats from  [79,80], hepatotoxicity [81] and testis [77] in rat.
Visfatin, one of the adipokines [82], is present in a variety of tissues including the testis [83]. In the present study, the treatment of GA decreased blood glucose concentrations whereas, increased serum testosterone concentrations. Our findings are in line with previous studies that GA treatment decreased glucose concentration and production in rats [60,61]. In addition, lowfat intervention together with high-fiber increased testosterone levels after in human [84]. Conversely, intervention of low fat diet simultaneously with high fiber diet decreased serum and urine androgens in men [85]. In addition, treatment of GA significantly (P<0.05) increased testicular Visfatin mRNA expression associated with upregulation of Visfatin protein expression compared to the diabetic group. Testis of diabetic rats showed obvious degeneration whereas; slight degeneration was seen in GA treated rats compared to control. Visfatin mRNA expression revealed a positive correlation with body, testis weight and serum testosterone concentrations, while a negative correlation was observed with blood glucose concentrations.

Conclusion
Our findings conclude that GA may improve reproductive performance and it may be useful to meliorate the diabetic infertility complications in male.

Conflict of Interest Statement
All authors declare that they do not have any conflict of interest