Caulerpa lentillifera Polysaccharides-Rich Extract Reduces Oxidative Stress and Proinflammatory Cytokines Levels Associated with Male Reproductive Functions in Diabetic Mice

Diabetes mellitus is a chronic metabolic disease that is positively correlated with reproductive dysfunction. Caulerpa lentillifera is an edible green alga with antioxidant and anti-diabetic properties. This study aims to evaluate the ameliorative effects of a polysaccharides-rich extract from C. lentillifera on the reproductive dysfunctions of diabetic male BALB/c mice induced by a high-fat diet (HFD) supplemented with intraperitoneal injections of streptozotocin (STZ). C. lentillifera was obtained from hot water and converted into a powder form (C. lentillifera extract (CLE)) by freeze drying. Mice were fed an HFD for 4 weeks before supplementing with STZ (30 mg/kg). The diabetic mice were divided into five groups, including a control group, a diabetic (DM) group, a DM with administration of a low-dose CLE treatment (DM+CLE1, 600 mg/kg), a DM with administration of a high-dose of CLE (DM+CLE2, 1000 mg/kg) and a DM with metformin treatment as a positive control (DM+Met, 200 mg/kg) for 6 weeks. The results showed that the CLE administration improved hyperglycemia and insulin resistance. Proinflammatory cytokines such as interleukin-1β and tumor necrosis factor-α were found to decrease in the CLE-treated groups. Additionally, CLE was shown to improve sperm motility and testis morphology. Based on the results, it was confirmed that the polysaccharides-rich extract from C. lentillifera extract was able to prevent diabetes-induced male reproductive dysfunction.


Introduction
Diabetes mellitus is a type of metabolic disorder that is considered a serious health problem worldwide. Diabetes is caused either as a result of insufficient insulin secretion (type-1 diabetes) or failure of insulin action (type-2 diabetes). It is characterized by a high glucose level in the blood (hyperglycemia) [1], and proinflammatory cytokines such as interleukin (IL)-1β, IL-6, and IL-2 are increased in the hyperglycemia condition [2]. Dysregulation of tumor necrosis factor-α production is also associated with diabetes [3]. Additionally, hyperglycemia causes an increase in oxidative stress and has a positive association with cell dysfunction in diabetic patients [4,5]. This disease also causes an adverse effect on some organs such as the testis, kidney, liver, and pancreas [6,7]. Reduced levels of some essential hormones, such as luteinizing hormone, follicle-stimulating hormone, and testosterone, as well as low sperm count and motility, have also been seen in diabetic patients [8,9]. supernatant. Then, the supernatant was filtered using a paper filter and dried by using a freeze dryer to obtain the C. lentillifera extract (CLE) powder.

Characterization of Caulerpa Lentillifera Extract
A proximate analysis was performed to determine the content of the CLE. Moisture content was determined in triplicate by drying at 105 • C in an oven to obtain a constant dry weight. The protein content was determined by the Kjeldahl method (Association of Official Analytical Chemists, AOAC method 981.10), and crude fat content was determined by Soxhlet extraction with ethyl ether (AOAC method 991.36). The ash content was determined by complete incineration of the sample in a muffle furnace at 550 • C (AOAC method 930.05). Total sugar was determined by a phenol-sulfuric acid assay according to a previous method [26]. Briefly, the glucose standard was prepared into serial concentrations. The glucose standard and sample extract solutions were added as 1 mL of 80% phenol and 500 µL of 95% sulfuric acid (H 2 SO 4 ), mixed well and incubated at room temperature for 10 min. After cooling, 200 µL of the solution was pipetted into a 96-well microplate and absorbance was immediately measured at 490 nm using a spectrophotometer.

Animal Experiment
The animal experiment was conducted according to a previous method with some modifications [27]. Six-week-old male BALB/c mice (total of mice, N = 25) were used for the diabetes model. The animals had maintained living conditions (room temperature of 25 • C, 45-50% relative humidity and 12/12-h dark/light cycle) in the Animal Research Center, College of Life Science, National Taiwan Ocean University. All procedures followed the standard of the Institutional Animal Care and Use Committee (IACUC Approval No. 1088011, Date: 23 May 2019) of the National Taiwan Ocean University, Taiwan. Briefly, after a week's acclimatization, the mice were divided into two main groups: a control group fed a standard commercial diet (five mice) and a diabetes group fed a high-fat diet (HFD, 20 mice) for 4 weeks, as shown in Figure 1. Mice in the diabetes group were intraperitoneally injected with streptozotocin (STZ, 30 mg/kg in citrate buffer, pH 4.5) and divided into four subgroups: a diabetes group (DM, daily oral gavage with water), a DM with a low dose of CLE treatment (CLE1, 600 mg/kg of body weight), a DM with a high dose of CLE (CLE2, 1000 mg/kg), and a DM with metformin as a positive control (metformin, 200 mg/kg) [28]. The mice were treated for 6 weeks before sacrifice, during which time they were placed in an empty chamber and euthanized via CO 2 exposure. After sacrifice, whole blood and organs (liver, testis, and hypothalamus) were collected for future analysis.
Appl. Sci. 2020, 10, x FOR PEER REVIEW  3 of 14 the supernatant. Then, the supernatant was filtered using a paper filter and dried by using a freeze dryer to obtain the C. lentillifera extract (CLE) powder.

Characterization of Caulerpa Lentillifera Extract
A proximate analysis was performed to determine the content of the CLE. Moisture content was determined in triplicate by drying at 105 °C in an oven to obtain a constant dry weight. The protein content was determined by the Kjeldahl method (Association of Official Analytical Chemists, AOAC method 981.10), and crude fat content was determined by Soxhlet extraction with ethyl ether (AOAC method 991.36). The ash content was determined by complete incineration of the sample in a muffle furnace at 550 °C (AOAC method 930.05). Total sugar was determined by a phenol-sulfuric acid assay according to a previous method [26]. Briefly, the glucose standard was prepared into serial concentrations. The glucose standard and sample extract solutions were added as 1 mL of 80% phenol and 500 µL of 95% sulfuric acid (H2SO4), mixed well and incubated at room temperature for 10 min. After cooling, 200 µL of the solution was pipetted into a 96-well microplate and absorbance was immediately measured at 490 nm using a spectrophotometer.

Animal Experiment
The animal experiment was conducted according to a previous method with some modifications [27]. Six-week-old male BALB/c mice (total of mice, N = 25) were used for the diabetes model. The animals had maintained living conditions (room temperature of 25 °C, 45-50% relative humidity and 12/12-h dark/light cycle) in the Animal Research Center, College of Life Science, National Taiwan Ocean University. All procedures followed the standard of the Institutional Animal Care and Use Committee (IACUC Approval No. 1088011, Date: 23 May 2019) of the National Taiwan Ocean University, Taiwan. Briefly, after a week's acclimatization, the mice were divided into two main groups: a control group fed a standard commercial diet (five mice) and a diabetes group fed a highfat diet (HFD, 20 mice) for 4 weeks, as shown in Figure 1. Mice in the diabetes group were intraperitoneally injected with streptozotocin (STZ, 30 mg/kg in citrate buffer, pH 4.5) and divided into four subgroups: a diabetes group (DM, daily oral gavage with water), a DM with a low dose of CLE treatment (CLE1, 600 mg/kg of body weight), a DM with a high dose of CLE (CLE2, 1000 mg/kg), and a DM with metformin as a positive control (metformin, 200 mg/kg) [28]. The mice were treated for 6 weeks before sacrifice, during which time they were placed in an empty chamber and euthanized via CO2 exposure. After sacrifice, whole blood and organs (liver, testis, and hypothalamus) were collected for future analysis.

Plasma Collection and Homogenized Testis Tissue Preparation
Whole blood was collected on the day of sacrifice using a heparinized syringe to collect it into tubes. The whole blood was then centrifuged at 1000× g for 15 min at 4 • C to separate the plasma. The supernatant (plasma) was pipetted into new collection tubes (1.5 mL conical microtube) and retained for future analysis [29].
The homogenized testis tissue was prepared using a previously published freeze-thaw method [30]. Briefly, the testis tissue (100 mg) was suspended in cold phosphate-buffered saline (PBS, 900 µL). The suspension was homogenized using a micro-tube homogenizer and stored at −20 • C. After 24 h, the freeze-homogenized tissue was thawed at room temperature before use and centrifuged at 5000 rpm for 25 min. The supernatant of the homogenized tissue was collected for future analysis.

Glucose and Plasma Biochemical Analysis
The plasma glucose concentration was measured by commercial glucose enzymatic kits. Plasma insulin concentration was tested by an insulin ELISA kit. Homeostasis Model Assessment-Insulin Resistance (HOMA-IR) was determined by multiplying the fasting plasma glucose (mmol/L) by the fasting plasma insulin (mU/L), then dividing by 22.5 [31]. The proinflammatory cytokines (i.e., TNF-α and IL-1, kisspeptin (kiss-1)) and the reproductive hormones (i.e., luteinizing hormone, follicle-stimulating hormone, and testosterone) were analyzed by ELISA kits according to their manufacturers' protocols.

Enzymatic Antioxidants and Oxidative Stress Assay
The activity of enzymatic antioxidants, such as superoxide dismutase (SOD), catalase (Cat), and glutathione peroxidase (GPx), were measured using commercial kits. The malondialdehyde (MDA) level of the homogenized testis tissue supernatant was used to evaluate lipid peroxidation and was determined according to a previously reported method using MDA reagent (15% (w/v) trichloroacetic acid in 0.25 N HCl and 0.375% (w/v) thiobarbituric acid in 0.25 185 N HCl) [32]. A nitro blue tetrazolium (NBT) test was used to determine the superoxide anion (O 2− ) level in the sperm [33]. Briefly, the sperm cells were added to 0.2 mL of NBT solution (0.1 mg/mL NBT, 5% Fetal bovine serum, 3% Dimethyl sulfoxide) in the tubes. The mixture was incubated in 10 mL RPMI for 60 min at 37 • C then centrifuged at 500× g for 10 min. The supernatant was removed from the tubes, then the tubes were washed with PBS. Next, 200 µL of DMSO was added to dissolve the purple crystals in the cells, which were placed in an ultrasonic oscillator for 15 min, and 200 µL of the purple solution was pipetted to a 96-well microplate to measure the absorbance at 570 nm using a spectrophotometry. The nitric oxide (NO) assay was conducted based on a previously reported protocol [34]. Briefly, the sperm cells or supernatant of homogenized testis were added to Griess reagent (sulfanilamide dissolved in 5% H 3 PO 4 and N-(1-naphthyl)ethylenediamine dissolved in H 2 O) at a 1:1 volume ratio. The solution was allowed to react for 15 min at room temperature, then the absorbance was measured at 540 nm using a spectrophotometer.

Sperm Analysis
The swim-up method was used to collect the sperm from the epididymis, as described previously [35]. Briefly, the epididymis was put into RPMI medium, then the cauda epididymis was cut and shaken at 120 rpm for 10 min. It was centrifuged at 200× g for 5 min, then incubated at 37 • C for 30 min. The liquid in the upper layer was taken and transferred into a new collection tube for future analysis. The sperm count, motility, and sperm abnormality were observed using a hemocytometer under a light microscope according to previous methods [36,37]. Sperm count is the total number of sperm, and sperm motility is the total sperm with the ability to move divided by total sperm count then multiplied by 100. Sperm abnormality is the total sperm with an abnormal form (such as a double head or double tail) divided by the total sperm then multiplied by 100.

Histopathology Analysis
After sacrifice, one testis was fixed with 4% formaldehyde solution for 2 days. After decalcification, the sections were cut (paraffin-embedded, 5 µm) and stained with hematoxylin and eosin (H&E). The staining process was performed by Li Pie Co. Ltd. (Taichung, Taiwan). The stained section was observed under a light microscope.

Statistical Analysis
All values were given as mean ± standard deviation (SD). All statistical calculations were done by the SPSS statistics v22.0 (SPSS for Windows Inc., version 22; Chicago, IL, USA) system. One-way ANOVA was used to examine the overall differences between groups, and a Duncan's multiple range test was used to identify significant differences (p < 0.05) between the groups. All figures produced were drawn using GraphPad Prism 5 (GraphPad Software Inc., San Diego, CA, USA).

Caulerpa Lentillifera Extract Chemical Compositions
The C. lentillifera extract was composed of carbohydrate (69.75 ± 0.86%), protein (10.5 ± 0.77%), lipid (0.83 ± 0.53%), ash (12.5 ± 0.76%), and moisture (6.42 ± 0.53%). The total sugar content of the extract was about 12.49 ± 0.77 µg/mL. Table 1 shows that there was no significant difference in the initial body weight of the mice between all groups, and that C. lentillifera extract (CLE) significantly protected the body weight loss after 6 weeks' treatment. CLE also significantly reduced kidney and abdominal fat weights in diabetic mice, whereas there were no significant effects on spleen and liver weights between the control, DM, and treated groups. Data are shown as mean ± SD (n = 5). The values with different letters (a-c) in same row represent significant difference (p < 0.05) as analyzed by a Duncan's multiple range test. BW, body weight; DM, diabetes; DM+CLE1, diabetes and 600 mg/kg of C. lentillifera; DM+CLE2, diabetes and 1000 mg/kg of C. lentillifera; DM+Met, diabetes and 200 mg/kg of metformin.

Caulerpa Lentillifera Extract Improves Plasma Glucose, Insulin, and HOMA-IR Levels in Diabetic Mice
High levels of glucose, insulin, and homeostasis model assessment-insulin resistance (HOMA-IR) were observed in the diabetes (DM) group, as shown in Figure 2. Hypoglycemic effects were observed after treatment with CLE (especially at high doses), which was determined from the decreased level of abovementioned factors. As a positive control, metformin also showed a lowering effect on glucose level. However, there was no significant reduction in glucose level when compared to the DM group. On the other hand, insulin and HOMA-IR levels decreased significantly after metformin treatment. Appl. Sci. 2020, 10, x FOR PEER REVIEW 6 of 14

Caulerpa Lentillifera Extract Ameliorates Oxidative Stress in Diabetic Male Mice
The effect of C. lentillifera extract (CLE) on the activities of plasma enzymatic antioxidants such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) are showed in Figure 3. The diabetes (DM) group was characterized by low levels of enzymatic antioxidants, CLE administration significantly enhanced SOD activity, especially in high-dose CLE, and there were no effects of CLE administration on CAT activity. On the other hand, GPx activity increased after CLE treatments. Ultimately, there was no significant difference between CLE-treated and DM groups.
Lipid peroxidation (MDA) in the testis was determined after 6 weeks treatment with C. lentillifera and metformin, as shown in Figure 4. The high value of MDA was shown in the diabetes (DM) group. The CLE treatment group significantly reduced MDA levels compared to the DM group, whereas metformin showed no significant difference from the DM group. The level of nitro blue tetrazolium (NBT) reduction was a quantitative assay for superoxide anion (O2ˉ) production, and nitric oxide (NO) increased in the DM group ( Figure 4). The level of NO in the testis and sperm was significantly reduced after 6 weeks treatment with CLE.

Caulerpa Lentillifera Extract Ameliorates Oxidative Stress in Diabetic Male Mice
The effect of C. lentillifera extract (CLE) on the activities of plasma enzymatic antioxidants such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) are showed in Figure 3. The diabetes (DM) group was characterized by low levels of enzymatic antioxidants, CLE administration significantly enhanced SOD activity, especially in high-dose CLE, and there were no effects of CLE administration on CAT activity. On the other hand, GPx activity increased after CLE treatments. Ultimately, there was no significant difference between CLE-treated and DM groups.

Caulerpa Lentillifera Extract Ameliorates Oxidative Stress in Diabetic Male Mice
The effect of C. lentillifera extract (CLE) on the activities of plasma enzymatic antioxidants such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) are showed in Figure 3. The diabetes (DM) group was characterized by low levels of enzymatic antioxidants, CLE administration significantly enhanced SOD activity, especially in high-dose CLE, and there were no effects of CLE administration on CAT activity. On the other hand, GPx activity increased after CLE treatments. Ultimately, there was no significant difference between CLE-treated and DM groups.
Lipid peroxidation (MDA) in the testis was determined after 6 weeks treatment with C. lentillifera and metformin, as shown in Figure 4. The high value of MDA was shown in the diabetes (DM) group. The CLE treatment group significantly reduced MDA levels compared to the DM group, whereas metformin showed no significant difference from the DM group. The level of nitro blue tetrazolium (NBT) reduction was a quantitative assay for superoxide anion (O2ˉ) production, and nitric oxide (NO) increased in the DM group (Figure 4). The level of NO in the testis and sperm was significantly reduced after 6 weeks treatment with CLE.  Lipid peroxidation (MDA) in the testis was determined after 6 weeks treatment with C. lentillifera and metformin, as shown in Figure 4. The high value of MDA was shown in the diabetes (DM) group. The CLE treatment group significantly reduced MDA levels compared to the DM group, whereas metformin showed no significant difference from the DM group. The level of nitro blue tetrazolium (NBT) reduction was a quantitative assay for superoxide anion (O 2− ) production, and nitric oxide (NO) increased in the DM group (Figure 4). The level of NO in the testis and sperm was significantly reduced after 6 weeks treatment with CLE. Appl. Sci. 2020, 10, x FOR PEER REVIEW 7 of 14

Caulerpa Lentillifera Extract Reduces Interleukin-1 Beta and Tumor Necrosis Factor-Alpha
The interleukin (IL)-1β and tumor necrosis factor (TNF)-α levels in plasma were increased in the diabetes (DM) group when compared to the control group ( Figure 5). The expression of IL-1β in highdose CLE and metformin treatments were reduced when compared to the DM group. However, there were no significant effects. The TNF-α expression was also significantly reduced in the high-dose CLE group compared to the DM group.

Caulerpa Lentillifera Extract Reduces Interleukin-1 Beta and Tumor Necrosis Factor-Alpha
The interleukin (IL)-1β and tumor necrosis factor (TNF)-α levels in plasma were increased in the diabetes (DM) group when compared to the control group ( Figure 5). The expression of IL-1β in high-dose CLE and metformin treatments were reduced when compared to the DM group. However, there were no significant effects. The TNF-α expression was also significantly reduced in the high-dose CLE group compared to the DM group.

Caulerpa Lentillifera Extract Reduces Interleukin-1 Beta and Tumor Necrosis Factor-Alpha
The interleukin (IL)-1β and tumor necrosis factor (TNF)-α levels in plasma were increased in the diabetes (DM) group when compared to the control group ( Figure 5). The expression of IL-1β in highdose CLE and metformin treatments were reduced when compared to the DM group. However, there were no significant effects. The TNF-α expression was also significantly reduced in the high-dose CLE group compared to the DM group.

Caulerpa Lentillifera Extract Administration Improves Levels of Kisspeptin-1 and Reproductive Hormones
The level of kisspeptin-1 was significantly lower in the diabetes (DM) group than the control group. However, this level significantly increased after treatment for 6 weeks with CLE ( Figure 6). The levels of follicle-stimulating hormone (FSH) and testosterone in the DM group were also significantly lower than the control group. Oral supplementation with CLE for 6 weeks successfully restored the levels of FSH and testosterone, especially in high doses of CLE. The CLE treatments showed no effects on LH levels when compared to the DM group.

Caulerpa Lentillifera Extract Administration Improves Levels of Kisspeptin-1 and Reproductive Hormones
The level of kisspeptin-1 was significantly lower in the diabetes (DM) group than the control group. However, this level significantly increased after treatment for 6 weeks with CLE ( Figure 6). The levels of follicle-stimulating hormone (FSH) and testosterone in the DM group were also significantly lower than the control group. Oral supplementation with CLE for 6 weeks successfully restored the levels of FSH and testosterone, especially in high doses of CLE. The CLE treatments showed no effects on LH levels when compared to the DM group.

Effects of Caulerpa Lentillifera on the Sperm and Seminiferous Tubules Morphology
The total sperm and sperm motility of the diabetes (DM) group was significantly lower than the control group, whereas sperm abnormality was significantly higher in the DM group compared to the control and treated groups (Figure 7). Treatment with CLE for 6 weeks not only significantly increased sperm motility, but also significantly reduced sperm abnormality. Hematoxylin and eosin (H&E) staining was used to evaluate seminiferous tubules morphology. The seminiferous tubule structure in the DM group appeared separated when compared to the other groups ( Figure 8). Whereas, the high-dose CLE and metformin treatments successfully ameliorated the seminiferous tubule morphology.

Effects of Caulerpa Lentillifera on the Sperm and Seminiferous Tubules Morphology
The total sperm and sperm motility of the diabetes (DM) group was significantly lower than the control group, whereas sperm abnormality was significantly higher in the DM group compared to the control and treated groups (Figure 7). Treatment with CLE for 6 weeks not only significantly increased sperm motility, but also significantly reduced sperm abnormality. Hematoxylin and eosin (H&E) staining was used to evaluate seminiferous tubules morphology. The seminiferous tubule structure in the DM group appeared separated when compared to the other groups ( Figure 8). Whereas, the high-dose CLE and metformin treatments successfully ameliorated the seminiferous tubule morphology.

Discussion
In this study, a diabetic male mouse model induced via a high-fat diet and intraperitoneal injections with streptozotocin was administered C. lentillifera extract for 6 weeks, which successfully ameliorated oxidative stress and some cytokine-related reproductive dysfunction. The C. lentillifera extract contained a high level of carbohydrates. Polysaccharides (a long chain of carbohydrates) are in the major constituent of seaweed, and polysaccharides from marine organisms have been widely used for pharmaceutical, nutraceutical, and cosmetic applications [38]. Seaweed polysaccharides have also been used for the potential treatment of inflammatory bowel disease (IBD), osteoarthritis, and diabetes [12,30,39].
The abdominal fat weight in the diabetes (DM) group was significantly increased compared to the control group. The high fat weight observed was the result of rats fed with a high-fat diet (HFD). However, the CLE treatment significantly reduced abdominal fat weight to a level that was similar to the control group. Previous studies reported increases in the total fat, subcutaneous fat, and

Discussion
In this study, a diabetic male mouse model induced via a high-fat diet and intraperitoneal injections with streptozotocin was administered C. lentillifera extract for 6 weeks, which successfully ameliorated oxidative stress and some cytokine-related reproductive dysfunction. The C. lentillifera extract contained a high level of carbohydrates. Polysaccharides (a long chain of carbohydrates) are in the major constituent of seaweed, and polysaccharides from marine organisms have been widely used for pharmaceutical, nutraceutical, and cosmetic applications [38]. Seaweed polysaccharides have also been used for the potential treatment of inflammatory bowel disease (IBD), osteoarthritis, and diabetes [12,30,39].
The abdominal fat weight in the diabetes (DM) group was significantly increased compared to the control group. The high fat weight observed was the result of rats fed with a high-fat diet (HFD). However, the CLE treatment significantly reduced abdominal fat weight to a level that was similar to the control group. Previous studies reported increases in the total fat, subcutaneous fat, and

Discussion
In this study, a diabetic male mouse model induced via a high-fat diet and intraperitoneal injections with streptozotocin was administered C. lentillifera extract for 6 weeks, which successfully ameliorated oxidative stress and some cytokine-related reproductive dysfunction. The C. lentillifera extract contained a high level of carbohydrates. Polysaccharides (a long chain of carbohydrates) are in the major constituent of seaweed, and polysaccharides from marine organisms have been widely used for pharmaceutical, nutraceutical, and cosmetic applications [38]. Seaweed polysaccharides have also been used for the potential treatment of inflammatory bowel disease (IBD), osteoarthritis, and diabetes [12,30,39].
The abdominal fat weight in the diabetes (DM) group was significantly increased compared to the control group. The high fat weight observed was the result of rats fed with a high-fat diet (HFD). However, the CLE treatment significantly reduced abdominal fat weight to a level that was similar to the control group. Previous studies reported increases in the total fat, subcutaneous fat, and visceral fat of mice after being fed an HFD [40,41]. In contrast, total body weight was significantly lowered in the DM group compared to the control and treatment groups. A previous study reported that BALB/c mice do not store excess lipids such as diacylglycerols and triacylglycerols in the liver. This condition is due to differences in lipid oxidation or lipogenesis, which are higher than fatty acid uptake and result in higher body weight [42]. Additionally, early literature also reported that diabetic BALB/c mice showed weight loss 1 week after streptozotocin injection [43].
The present study showed significantly increased levels of fasting plasma glucose and homeostasis measurements of insulin resistance (HOMA-IR) in the diabetes (DM) group compared to the control and treated groups. Type-2 diabetes is characterized by high levels of glucose and insulin resistance, as reported in previous studies [43,44]. Rising insulin concentrations are recognized as insulin resistance (IR) in type-2 diabetes. HOMA-IR has been used previously for IR estimation [45,46]. In this study, the levels of glucose and HOMA-IR were reduced after 6 weeks of oral administration of CLE. According to these data, CLE treatments showed a hypoglycemic effect. Previous studies also showed that seaweed extracts possess hypoglycemic effects, such as the extracts of S. ringgoldianum, S. polycystum, and I. okamurae [19][20][21].
Low levels of enzymatic antioxidants such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) were observed in the DM group compared to the control and treated groups. The DM group also showed high levels of lipid peroxidation products and superoxide anions, as analyzed by malondialdehyde (MDA) and nitro-tetrazolium (NBT) assays, respectively. These conditions proved that diabetes is characterized by increasing oxidative stress. A review study reported that oxidative stress can be induced by high glucose levels (hyperglycemia) in diabetic complications [4]. The level of nitric oxide (NO) also increased in the DM group compared to the control and treated-groups. Nitric oxide has been reported as an oxidative stress indicator [47]. The oral administration of CLE for 6 weeks successfully enhanced enzymatic antioxidant levels, especially for SOD and GPx, while reducing lipid peroxidation products and nitric oxide levels.
Increases in pro-inflammatory cytokines such as interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α have been evaluated in diabetes conditions [48,49]. The present study also recorded increases in pro-inflammatory cytokines such as IL-1β and TNF-α in the DM group. A previous study reported that the levels of these cytokines significantly are increased in a hyperglycemic condition compared to a healthy condition [48], whereas TNF-α is also recognized as an inflammatory mediator to promote IR [50].
Kisspeptin (kiss-1 protein) plays important roles in reproductive function. It is also recognized as an upstream regulator of gonadotropin-releasing hormone (GnRH) in the hypothalamic-pituitary-gonadal (HPG) axis [51]. In the present study, the low levels of kisspeptin-1 and reproductive hormones such as luteinizing hormone (LH), follicle-stimulating hormone (FSH), and testosterone were observed in the DM group. A previous study reported that a low level of the kiss-1 protein was evaluated in a diabetes model. Additionally, levels of LH and FSH were also reduced after STZ injections in the diabetic animal model [52]. The disruption of kiss-1 expression was associated with a low level of gonadotrophin (hypogonadotropic hypogonadism) [53]. The oral administration of CLE for 6 weeks successfully increased kiss-1 protein, FSH, and testosterone levels.
The present study showed low total sperm and sperm motility as well as high sperm abnormality in the DM group compared to the control group. This result proves that disruption of the upstream regulator such as kiss-1 protein and certain male reproductive hormones results in low sperm quality. In men, LH and FSH play an important role in spermatogenesis and maintaining their level of testosterone [54]. A previous study reported that hyperglycemia impairs male reproductive function [55]. In the present study, men in a diabetic condition showed a higher level of sperm DNA fragmentation compared to healthy men, due to the oxidative process, whereas damage to sperm DNA was associated with a reduction in the quality of the sperm [56]. The present study also showed a decrease in seminiferous tubule diameter in the DM group. Previous studies have reported that diabetes can result in a decreased diameter of the seminiferous tubule [57]. In diabetes, testicular dysfunction is caused by seminiferous tubule atrophy, decreased diameter of the tubule, spermatogenesis cell reduction, and cell apoptosis in germ cells [58]. The oral administration of CLE for 6 weeks successfully ameliorated testicular properties, such as increases in sperm motility and reduced sperm abnormality, while protecting seminiferous tubule morphology.
Overall, this study shows that CLE not only possesses an antidiabetic effect, but also improves some proinflammatory cytokines related to male reproductive function. Previous studies have reported that seaweed extracts showed ameliorative effects on reproductive function not only in diabetic models, including dietary supplementation of U. lactuca, fucoxanthin-rich extract from L. japonica, and S. angustifolium [22][23][24]59].

Conclusions
C. lentillifera extract (CLE) is rich in polysaccharide. CLE administration was able to reduce pro-inflammatory cytokines, lipid peroxidation, oxidative stress, and insulin resistance. Additionally, CLE showed improved sperm motility and seminiferous tubule morphology. According to these results, C. lentillifera extract successfully prevented diabetes-induced male reproductive dysfunction in mice models. This extract can also be used as a dietary supplement and an alternative treatment for diabetes management.