Protective Role of Nigerian Honey on Sperm indices and Testis in Sucrose-Fed Rats

Introduction: The available information on the effect of high sugar on male reproductive function is inadequate, though consumption of sweetened foods is not uncommon in many geographical areas of the World . As a reflection of global changes in dietary behavior, the prevalence of unhealthy diets characterized by high intake of food rich in fats and sugar has increased in women and men within the reproductive age range. Indeed, lifestyle factors arising from unhealthy dietary habit exert a profound influence on reproductive function. A number of studies have shown that various physiologic processes which include reproductive function can be affected by dietary manipulation. For instance, a high fat diet has been shown to impact negatively on both male and female reproductive function. Meanwhile, the possibility of a high sugar diet affecting reproductive function has not been verified experimentally to the best of our knowledge although consumption of a high-sugar diet has been associated with the development of obesity, insulin resistance, diabetes, dyslipidemia, fatty liver and high blood pressure. For a long time, honey has been the only available sweetener until its replacement by industrial sugar after 1800. Honey, a natural mixture of fructose, glucose, and more complex sugars 9 is not associated with the same metabolic effect as sucrose, thus its nutritional benefits . The physiological importance of honey includes its antioxidant, anti-inflammatory, antibacterial and antitumor activity among others 13, 14, . As far as reproduction is concerned, honey from different parts of the world has increased

the sperm count in rats and monkeys and increased vaginal wall epithelium and muscle thickness, without showing any effect on circulating gonadotropins or testosterone 16 .Honey has also been reported to enhance spermatogenesis in rats if given at the appropriate dose 17 and to reduce the toxic effects of cigarette smoke on spermatogenesis 18 .In lay press, sucrose has been assumed to have negative implications on the male reproduction.With this background, this study attempts to investigate whether high sucrose intake will impact negatively on the male reproductive function and if Nigerian honey could exert a protective role.

Materials and Methods: Honey Sample
Honey was obtained from a reliable supplier in Oyo State who freshly collected honey from sealed honeycombs, and the honey was certified pure 14 .Each rat on honey received a daily dose of 10ml honey/kg/5ml of distilled water 19 by oral gavage.

Animals
Male Sprague-Dawley rats weighing 120-150g were obtained from the Laboratory Animal facility of the College of Medicine of the University of Lagos.Animals were housed in plastic cages with ad libitum access to commercial rodent pellet diet (Livestock Feed, Lagos, Nigeria).The light cycle was maintained at 12 h of dark, followed by 12 h of light.The room temperature was maintained at 24±2 °C and humidity of 50% -64%.The rats were allowed to acclimatize for a period of two weeks before the commencement of the experiment.All animal handling and experimental protocols adopted in this study complied with the international principles for laboratory animals as obtained in the Helsinki's Declaration.

Study Design
Twenty-four (24) rats were randomly divided into four groups of six animals each.The groups are as follows: Control: was administered water; H-was administered honey once daily by oral gavage and treatment lasted for 4 weeks ; HSS-had free access to high sucrose solution (HSS) (30 % w/v) as drinking water according to the method of 20 with slight modification and treatment lasted for 4 weeks; HSS+H-had free access to high sucrose solution (HSS) (30 % w/v) as drinking water as well as administration of honey (H) once daily by oral gavage and treatment lasted simultaneously for 4 consecutive weeks.

Determination of food intake, body and organ weights
Food intake was measured at 0800hrs daily.Body weight was determined once a week using a digital weighing scale.On the last day of administration, the reproductive organs; testis, caudal epididymis, seminal vesicle and prostate gland were weighed.

Blood glucose measurement
The experimental rats were fasted overnight before blood samples were collected via tail tipping for blood glucose measurement at the 4 th week of treatment.The glucose levels were measured using a blood glucose monitoring system (Accu-Chek Glucometer, Roche, Germany).

Sperm Analysis
The testes from each rat were carefully exposed and one of them was removed together with its epididymis.The epididymis was separated and the epididymis fluid was collected while the progressive sperm motility, sperm count and sperm viability was determined 21 .

Hormonal Assay
Testosterone, Luteinizing hormone (LH) and Follicle-Stimulating Hormone (FSH) in rat serum were measured by enzyme immunoassays using commercially available kits form Endocrine Technologies (USA), Uscnlife (China) and Biocodehycel (Belgium), respectively.Samples were run in the same assay to avoid inter-assay variations.

Lipid peroxidation level
As a marker of lipid peroxidation, the level of malondialdehyde (MDA) in the liver and testes was measured by the method of 22 as thiobarbituric acid reactive substances (TBARS).The development of a pink complex with absorption maximum at 535nm is taken as an index of lipid peroxidation.

Antioxidant activities
Oxidative analyses of the liver and testes were carried out using previously described standard methods.The reduced glutathione (GSH) was determined using the method described by Van Dooran 23 .The GSH determination method is based on the reaction of Ellman's reagent 5,5' dithiobis (2-nitrobenzoic acid) DNTB) with the thiol group of GSH at pH 8.0 to produce 5-thiol-2-nitrobenzoate which is yellow at 412nm.Activity of the superoxide dismutase (SOD) was also determined according to the method described bySun and Zigman 24 .The reaction was carried out in 0.05m sodium carbonate buffer pH 10.3 and was initiated by the addition of epinephrine in 0.005N HCl.Catalase (CAT) activity was determined by measuring the exponential disappearance of H 2 O 2 at 240nm and expressed in units/mg of protein as described byAebi 25 .Absorbance was recorded using Shimadzu recording spectrophotometer (UV 160) in all measurements.

Statistical Analysis
Data are expressed as mean ± standard error of mean (SEM) and analysed using the ANOVA followed by SNK post-hoc test.P < 0.05 was accepted as significant.All the analyses were carried out using the GraphPad Instat Version 3.05 for Window Vista, GraphPad Software, San Diego California, USA.

Results: Food Intake
The food intake of all rats measured and recorded from week 1 to 4 was shown in Figure 1.At the first week of study, HSS fed rats had reduced food intake (P<0.05)compared with control.Meanwhile, the H fed rats had a significant increase in food intake compared with the other experimental groups.The food intake in the HSS fed rats at week 2 was also significantly reduced compared with control, while H fed rats maintained an increase in food intake when compared with HSS and HSS+H fed rats.However, at week 3, both the HSS and the H fed rats had a significant reduction in food intake compared with control.At week 4, the HSS fed rat still had a significant decrease in food intake when compared with control, but this was however significantly lower (P<0.05)than the HSS+H fed rats.The food intake was reduced (P<0.05) in the H fed rats compared with control, nevertheless, the food intake was significantly higher than that of the HSS and HSS+H fed rats.

Body weight
The body weight was measured and recorded weekly as shown in Table I.The H-fed rats had a significant increase (P<0.05) in body weight at week 1, 2 and 4 of treatment when compared with the control and HSS group as indicated.

Fasting blood glucose
Table II shows the fasting blood glucose of the experimental rats taken at the end of the 4-week treatment.Sucrose and honey consumption did not result in any significant change in the blood glucose of the experimental rats for all comparison.

Reproductive Organ weights
Table III shows the weight of the reproductive organs recorded at the end of the experiment.The weight of the testes and seminal vesicle was significantly increased (P<0.05) in HSS+H and H-fed rats compared with HSS fed rats.The weight of the epi-  didymis was significantly increased in the H-fed rats compared with HSS fed rats.

Table III: Reproductive organ weights (g) of rats following administration of treatments for four weeks
Values are Mean±SEM; n=5.# P < 0.05, compared to HSS .HSS-high sucrose solution, HSS+H-high sucrose solution+ honey, H-honey

Sperm indices
Epididymis sperm count and motility were significantly reduced (P<0.05) in the HSS fed rats compared with control, while, the sperm count and motility were significantly increased (P<0.05) in the H fed rats and HSS+H fed compared with the HSS rats (Table IV).The Epididymis volume was not significantly different across treatment groups.The most common abnormalities encountered during the morphological examination of the sperms was the 'curved tail' and 'curved mid-piece' in the rats that received the HSS treatment.The H group however showed fewer occurrences of the morphological aberration as recorded in Table V.

Table IV: Sperm indices of rats following administration of treatments for four weeks
Values are Mean±SEM; n=5.*-compared with control; # -compared with sucrose P < 0.05.HSS-high sucrose solution, HSS+H-high sucrose solution+ honey, H-honey

Hormonal Analysis
The Luteinizing Hormone (LH) level was significantly reduced (P<0.05) in the H fed rats compared with control and HSS fed rats.There was no significant difference in LH level in the HSS rats compared with control.The HSS+H fed rats however, had a significant reduction in LH level compared with the HSS fed rats.The H fed rats had a significantly reduced (P<0.05)Follicle-Stimulating Hormone (FSH) level compared with control.However the testosterone level of the H fed rats was significantly higher than in the control as well as the HSS rats (Table VI).

Table VI: Hormonal levels of rats following administration of treatments for four weeks
Values are Mean±SEM; n=5.*-compared with control; # -compared with sucrose P < 0.05.HSS-high sucrose solution, HSS+H-high sucrose solution+ honey, H-honey

Malonhydialdehyde (MDA) estimation and antioxidant activity in the testis
There was no significant difference observed in MDA level in the testes when compared with control.The Superoxide dismutase (SOD) activity as well as the GSH level was not significantly different in the testes of all the experimental rats.Catalase activity (CAT) was significantly reduced (P<0.05) in the testes of HSS fed rats compared with control while the H fed rats had a significantly increased (P<0.05) level when compared with HSS and HSS+H fed rats (Table VII).In the liver of HSS fed rats, there was a significant increase (P<0.05) in MDA level compared with control.The activities of SOD was significantly higher (P<0.05) in the liver of HSS+H fed rats compared with HSS fed rats.CAT and GSH activities in the liver were not significantly different among the experimental groups (Table VIII).The pattern of food intake was affected by HSS feeding throughout the period of the study.Most of the literatures have shown that adult and children are satiated by sucrose with satiety being prolonged when larger amount of sucrose is consumed 31 .Another study reported that sucrose feeding in rats increased serum leptin concentration 32 , thus the observed reduction in food intake in the sucrose-fed rats may be due to the central effect of leptin initiating satiety.Previous studies have shown that honey supplementation decreases energy/food intake in rats 10 .However, the increase in food intake in the honey fed rats from the 1 st to 2 nd week of treatment followed by reduction in food intake from the 3 rd to 4 th week of treatment, suggests a prolonged initiation of satiety as against that of sucrose which was noticeable from the beginning of the study.

Table VII: Malonhydialdehyde (MDA) estimation and antioxidant activity in the testis following administration of treatments for four weeks
Interestingly, by the 4 th week of treatment the HSS+H fed rats reduced their food intake to a considerable level lower than that of the HSS-fed rats, this could possibly indicate a synergetic action of both honey and sucrose on food intake.Oliogosaccharides such as palatinose (isomaltulose) present in honey have been reported to delay digestion and intestinal absorption of glucose resulting in reduced glycemia 33, 34 .Asides delaying absorption, report proposes that fructose consumption lowers food intake 35 and this reduced food intake is attributed to delay gastric emptying 36 .This might be the reason for the reduced food intake in the H rats compared with the control.
There was a significant increase in the body weights of the animals in the honey (H) group.This is consistent with a study 14 .Some studies have also found that a composition of honey i.e. fructose consumption at high doses is associated with increased weight gain 37, 38 .
Consumption of a high sucrose solution has been implicated in the development of insulin resistance and glucose intolerance in rats 39 .In the present study, there was however no significant difference in fasting blood glucose among the experimental rats.Meanwhile, the duration of treatment may be an important factor to consider since most studies that have reported metabolic disorders due to high sucrose feeding were carried out over a longer duration of eight weeks and over 40 .The short duration of high sucrose feeding (i.e.4weeks) in this study may have hampered the metabolic changes that become pronounced over time.The fasting blood glucose level of the sucrose fed rats may have appeared to be reduced because serum glucose levels is known to be reduced in starved rats and animals were starved overnight 41 .Researchers have The increased lipid peroxidation in the liver and not in the testes of the HSS fed rats makes it difficult to attribute the alteration of these sperm parameters to the oxidative status of the reproductive organ.However, it brings to fore, that metabolic insult was elicited by HSS feeding regardless on the fasting blood glucose; as the sucrose fed rats did not present hyperglycemia.Meanwhile,, the increased CAT activity in the testes of the H fed rats demonstrates the antioxidant potential of honey 54, 55, 56 on reproductive organs.Enzymatic scavengers like SOD, CAT and GSH protect the system from deleterious effects of ROS.The improved antioxidant status of the H fed rats in this study may have impacted on the sperm function according to the antioxidative nature of honey 54, 57,58,59,60 since spermatozoa have little defence against oxidative damage and are highly sensitive to free radicals.Previous reports have shown that honeys with a high content of polyphenolic compounds have the capacity to decrease significantly the concentration of lipid hydro-peroxides produced during the lipid peroxidation process, in a process similar to that of other antioxidants like melatonin and vitamin E. The liver is susceptible to oxidative stress and damage; and the beneficial effect of antioxidants on hepatic oxidative stress has been documented 61, 62 .In the liver of young and middle-aged rats, honey supplementation was reported to restore activities of CAT and GPx 63 , although in this study the activity of SOD was improved in the HSS+H rats compared to the HSS rats.

Conclusion:
Although several contributory factors have been implicated, the possible role of a sugar-driven dietary lifestyle as a causative factor has gained little or no attention.The result of the present study indeed indicates that a high sugar diet could affect sperm function as evidenced by decreased sperm motility and count in the sucrose fed rats.The observations of this study affirm the statement of hypothesis that a high sucrose diet will impact negatively on male reproductive function while honey consumption will confer a protective effect.The reproductive capacity of male rats is known to be higher than that of men 6 ; therefore, such a decrease in sperm quality as observed in the sucrose fed rats may be enough to alter fertility among human males, thus contributing to the alarming incidence of male infertility.Nigerian honey on the other hand appears to be a credible alternative sweetener and exhibits protective function against reproductive dysfunction via high sucrose feeding.

MDA) estimation and antioxidant activity in the liver
Role of Nigerian Honey on Sperm indices and Testis in Sucrose-Fed Rats