The present study found that training regimen of high intensity jumping exercise containing 80 jumps per day combined with 1 g per kg body weight of daily honey supplementation elicited more discernable beneficial effects on tibial and femoral mass compared to low intensity jumping exercise containing 20 jumps per day combined with honey supplementation, jumping exercise alone and honey supplementation alone in young female rats. We observed that bone wet and fat free dry weights as indicators of bone mass with both organic and mineral phases in high intensity jumping combined with honey supplementation group was significantly greater than all the other experimental groups. Meanwhile honey supplementation alone, and both low and high intensity jumping exercises with or without honey supplementation elicited beneficial effects on enhancing bone mass compared to sedentary without honey supplementation control rats. The present study also showed that high intensity jumping exercise combined with honey may elicit more beneficial effects on tibial mass compared to low intensity jumping exercise combined with honey supplementation.
The osteogenic effects of mechanical loading have been manifested by many studies. Loading is known as an essential factor in the bone formation . Forwood  has suggested that mechanical loads greater than common force which met by skeleton can increase bone mass as result of increasing bone formation and reduction of bone turnover. It has been proven by animal studies that jump exercise as a high impact loading exercise model gives large ground reaction and muscular contraction forces to lower limb bones during landing [3, 30]. Similarly, the present study finding was consistent with previous jumping exercise studies which reported positive effects of this type of exercise on bone mass in rats [3, 5]. It is postulated that the rhythmic nature of dynamic loading in this type of high impact loading exercise may increase delivered blood volume to working muscle compared to low impact loading exercise , therefore beneficial effects on bone mass was observed in the present study. It was reported in a previous study that bone mass increased significantly with only 5 or 10 jumps per day, and high amount of loadings per day was not necessarily for increasing bone health . Two other human studies also showed the efficiency of high-impact and low-repetition exercises on bone mineral density [32, 33]. Our results are in agreement with these aforementioned previous studies with the evidences that jumping exercise as low as 20 jumps per day at 5 days per week for 8 weeks may have potential to increase bone mass in young rats.
Regarding the present positive finding of honey supplementation on bone mass, it was reported that ingestion of carbohydrates is effective in increasing calcium absorption and suppressing bone resorption in human  and animal , implying that carbohydrate ingestion may enhance bone mineralization and organic matrix reflected by increased bone mass, as observed in the present study among honey fed rats. In a previous study, Ariefdjohan and colleagues  have shown that acute feeding of honey could increase calcium absorption in rats. Therefore the finding of large increased in bone mass as shown by the great increased bone wet weight in rats with combination of honey supplementation and high intensity of 80 jumps per day in this study might be due to increased calcium absorption with honey supplementation. The present finding may reflect that high intensity jumping exercise with 80 jumps per day combined with honey supplementation could cause enhancement of blood flow to working muscle for supplying nutrients contained in honey such as calcium, phosphorus, magnesium and vitamins to the bone during exercise.
Regarding bone metabolism markers, it was found that serum total calcium was significantly higher in high intensity jumping combined with honey supplementation than honey alone group. This observation implies that 80 jumps per day combined with honey feeding were most effective in enhancing serum total calcium level in the rats. It is speculated that honey feeding and its carbohydrate constituents such as glucose, fructose, and raffinose enhanced calcium absorption from intestinal tract  through the large volume of blood delivered to the muscle and subsequently to the bone by performing jumping exercise .
Serum alkaline phosphatase, a bone formation marker, was significantly greater in high exercise intensity combined with honey group and honey supplementation alone group compared to sedentary without honey supplementation controls. Additionally, serum alkaline phosphatase was significantly higher in low and high intensity jumping exercise combined with honey supplementation group and honey supplementation group compared to low intensity jumping exercise alone group. It seems that jumping exercise in both high and low levels of intensity combined with honey supplementation may elicit beneficial effects on bone by increasing level of bone formation marker, nevertheless, more discernable effect was observed in combined 80 jumps per day with honey group. The positive finding in combined 80 jumps per day with honey group in serum formation marker was consistent with serum total calcium mentioned earlier. In a recent human study carried out by the present researcher, it was also observed that aerobic dance exercise combined with honey supplementation could increase level of bone formation marker in young woman . Interestingly, the present study also found that honey supplementation alone was effective in increasing serum alkaline phosphatase, this observation may reflect that the vital components contained in honey such as sugar, i.e. fructose, glucose, maltose and sucrose; enzymes, flavonoids, minerals, oxidants, organic acids, proteins, phenolic acids and vitamins  may have played their roles in enhancing bone formation.
In bone resorption marker, it was evidenced in the present study that serum 1CTP levels were significantly lower in honey alone group, low and high intensity jumping exercise alone groups and combined 20 and 80 jumps per day with honey group when compared to sedentary without honey supplementation control group. Nevertheless, combined 80 jumps per day with honey group elicited more discernable effect on reducing the level of serum bone resorption marker than 20 jumps per day combined with honey supplementation.
The beneficial effects of honey alone on bone resorption revealed that the vital components contained in honey such as carbohydrates, minerals, vitamins and flavonoids  may have potential in reducing bone resorption in the present study. Similar findings of reduction of bone resorption marker as results of exercise as observed in the present study has been reported by Kishimoto et al. which found that 10 jumps per day for 5 days per week decreased level of serum 1CTP after 2 weeks of training in female college-aged non-athletes. We also found that combined 80 jumps per day with honey elicited discernable effect on reducing the level of serum 1CTP. Nevertheless, a recent human study done by Ooi et al. found that aerobic dance exercise consisted of 1 hour per day, 3 times per week combined with 20 g of Gelam honey for 7 days per week for a duration of 6 weeks did not show any significant effect on serum C-terminal telopeptide of type 1 collagen level in young female. The discrepancy between the present study and the aforementioned previous study results maybe due to variation in type and duration of the physical trainings and kind of recruited subjects in both studies. The reduction in bone resorption marker in combined exercise with honey group of the present study may be due to the effect of jumping exercise on enhancement of blood volume and osteoblastic activity, and there was suppression of parathyroid hormone-vitamin D mechanism subsequent of increased calcium absorption after honey feeding . Additionally, it was reported by previous studies that kaempferol, a flavonoid, has osteogenic and anti-osteoclastogenic effects on rats [40, 41]. Flavonoid is effective in bone loss reduction by increase the numbers of osteoprogenitor cells and inhibit osteoclastic activity . Therefore, the significant reduction in bone resorption marker in combined honey with jumping exercise group may be due to increase in supply of flavonoids component of Tualang honey, i.e. kaempferol  through the increased of blood flow to the working muscle induced by jumping exercise.
In the present study, levels of FSH were lower in low and high intensity jumping exercise alone groups and honey supplementation alone group compared to sedentary without honey supplementation control group. However, there were no significant differences in both combined low and high intensity jumping with honey groups compared to sedentary without honey supplementation control group. These findings imply that honey combined with low and high intensity jumping exercise can maintain FSH level as sedentary without honey supplementation group. In a human study done by Valentino et al. , it was reported that intense ballet training was accompanied with lower level of reproductive hormone, i.e. FSH in young women. Meanwhile, it was reported by Williams and colleagues  that low energy availability and negative energy balance, i.e. dietary energy intake minus exercise energy expenditure following vigorous and regular exercise regimen reduces gonadotrophins secretion from anterior pituitary gland via hypothalamic-pituitary pathway. They have shown that calories supplementation which contained one quarter piece of fresh fruit, i.e. 25 kcal that was equal to 138–181% of calorie intake, during amenorrhea induced by strenuous exercise, caused increases in reproductive hormone levels, i.e. FSH and LH, and reestablished ovulatory cycles in female cynomolgus monkeys. Their finding confirmed that exercise-induced suppression of reproductive function is caused by the energy expenditure associated with vigorous regular exercise.
Our data revealed that honey supplementation alone was accompanied with low level of FSH. Similarly, Zaid et al. reported that administration of low dose of 0.2 g per kg, medium dose of 1.0 g per kg and high dose of 2.0 g per kg Tualang honey were accompanied with nonsignificant lower level of serum FSH in honey treatment groups compared to ovariectomised control rats. The present study found that both low and high intensities of jumping exercise may have elicited negative effects on FSH level and the combination of honey supplementation and exercise in both levels of intensities could improve the adverse effects induced by jumping exercise on FSH level.
In the present study, LH level was significantly lower in all the experimental groups compared to sedentary without supplementation control group, with exception of higher intensity jumping exercise combined with honey and lower intensity jumping exercise. Moreover, level of LH were significantly higher in both higher and lower intensities jumping combined with honey and lower intensity jumping compared to high intensity jumping without honey supplementation. As we explained before, exercise especially high intense exercise can affect release of the amount of hypothalamic hormone and reduction in the level of gonadotrophins, i.e. LH [44, 45]. It was also reported that restricted energy avaibility due to intense exercise via disruption in gonadotropin releasing hormone can cause reduction in LH concentration in female [43, 46]. In the present study, lower level of serum LH in the rats of honey supplementation group compared to sedentary without supplementation group may be implies that rats in the honey group were under stress due to force feeding and honey alone did not have protective effects on luteinizing hormone. Nevertheless, the precise mechanism of lower level of LH in HJ20 group compared to sedentary group is unknown and further investigation is needed. The present finding of lower level of serum LH in the J80 compared to J20, HJ20 and HJ80 implied that high intensity jumping exercise without honey supplementation may have elicited negative effects on serum LH. This result implies that honey, a high concentrated source of energy with 313 calories per 100 g and antioxidants such as flavonoids and phenolic acid  may have potential to elicit beneficial effects on reduction of adverse effect induced by high intensity jumping exercise.
Body composition such as fat or muscle mass are potent stimulators of bone mass. One of the limitations of the present study was that fat mass was not measured, and the lower limb muscle of the rats were not collected and weighed for adjusting the bone mass value of the rats. The present finding may reflect the potential use of honey for maintaining bone health and reproductive functions. However, it is suggested to monitor insulin sensitivity for detecting the possible metabolic side effects due to an overload of sugar amount for those who are supplemented with honey in their daily life. Nevertheless, further studies are needed to elucidate the exact mechanism of honey in improving bone metabolism and reproductive hormone levels.