The effect of regular resistance exercise, vitamin D, and calcium supplements on the gastrocnemius muscle in rats in the post-menopausal period: An experimental study

Abstract Background Menopause is the natural termination of menstruation which affects the quality and important aspects of women's life. Objective To evaluate the effect of regular resistance training (Ex) with vitamin D (Vit. D) and calcium (Ca) supplements in the postmenopausal period on muscle tissue in rats. Materials and Methods In this experimental study, 72 female Wistar rats (8-10-wk old) were randomly divided into control, placebo, Vit. D, Ca, Ex, Ca + Vit. D, Ex + Ca, Ex + Vit. D, and Ex + Ca + Vit. D groups. Control and placebo groups were fed with a standard diet and sesame oil, respectively. Two month after the ovariectomy, Ex, Ca (35 mg/kg), and Vit. D (10000 IU) were administred in all groups except the control. The number of muscle and inflammatory cells, fiber diameter, endomysium thickness, and degenerative collagen fiber area were assessed through hematoxylin-eosin staining. Results Muscle cell number was increased in the Ex + Vit. D + Ca, Vit. D + Ex, and Vit. D groups compared to the control group; also, inflammatory cell number showed significant increase in the Ex + Vit. D + Ca (12 ± 5.46), Vit. D + Ex (14 ± 3.25), Ex (13 ± 4.08), Vit. D (11 ± 3.26), Ca + Vit. D (10 ± 1.01), and Ca + Ex (9 ± 2.87) groups. Muscle fiber diameter in the Ex + Vit. D + Ca and Vit. D + Ex groups was higher than the other groups. Endomysium thickness was significantly decreased in the Ex + Vit. D + Ca and Vit. D + Ex groups compared to the control and placebo groups (p < 0.001). Degenerative collagen fiber area showed a significant increase in the Ex + Vit. D + Ca and Vit. D + Ex groups (p ≤ 0.001) comparison with the control group. Conclusion Regular resistance exercise, Vit. D, and Ca supplements can improve muscle morphological features in the postmenopausal period.


Introduction
Women naturally spend a third of their lives in a stage called menopause (1). The average age of menopause in developed countries is 51 years, and approximately 4% of women experience menopause before the age of 40 (early menopause) (2). Physiologically, menopause is associated with a decrease in estrogen hormone secretion due to lack of follicular function and is characterized by symptoms such as exhaustion, depression, and muscle pain that have negative effects on quality of life (3).
Aging is associated with loss of muscle mass and muscle strength and function of the musculoskeletal system that is clinically known as sarcopenia (4). Although sarcopenia is characterized by loss and atrophy of muscle fibers, there is no identified scientific evidence for the cause of sarcopenia (5). Experiments have shown that postmenopausal women lose their muscle tissue rapidly within 10 years (6,7). Studies have also shown that exercise (Ex) in old age can improve physiological conditions, increase protein biosynthesis, increase muscle strength and function, glycolytic fiber hypertrophy, and reduce or prevent the prevalence of sarcopenia (8,9). Although physical activity has many benefits for postmenopausal women, studies available in this field are limited and have reported conflicting results. Aubertin-Leheudre and others, and Liberman and coworkers in two separate studies evaluated the effect of Ex on muscle size in the elderly (10). Although they did not show the effect of Ex on the muscle size in the postmenopausal period, they confirmed that there were evidences that Ex could improve muscle function.
Vitamin D (Vit. D), calcium (Ca), and parathormone metabolism are altered during menopause (11). Menopause is associated with changes in Ca metabolism. In recent years, bone mineral density test, has been able to show the sufficiency of Ca and serum Vit. D levels in human body (12). Gimigliano and colleagues found a positive relationship between Vit. D levels and muscle mass in the elderly. Also, they concluded that people with low Vit. D levels are at a higher risk of musculoskeletal disorders (6). Changes in skeletal muscles mass have significant negative effects on the quality of life.
In addition, the burden of diseases increases with age, especially musculoskeletal disorders, so it is necessary to find a suitable method to preserve the strength of muscles in old age.
Because women are more susceptible than men to age-related musculoskeletal disorders, it is important to investigate the factors of age-related musculoskeletal disorders in women. Since ex and good nutrition are two identified factors for the health of the musculoskeletal system, reports show that 81.3% of Iran's urban population has Vit. D deficiency and that this is higher in women than men (13,14). Also, the prevalence of inactivity in women is higher in developing countries and this is associated with a high prevalence of musculoskeletal disorders (15). Therefore, the present study aimed to investigate the effect of resistance Ex with Vit. D and Ca supplementations on the gastrocnemius muscle in rats in post-menopausal period.

Animal housing and treatment
In this experimental study, 72 female Wistar rats (8-10-wk old, weighing 250 ± 15 gr) were randomly divided into control, placebo, Vit. D, Ca, International Journal of Reproductive BioMedicine Post-menopausal exercise, vitamin D, and calcium intake's effect on muscle in rats Ca + Vit. D, Ex, Ex + Ca, Ex + Vit. D, and Ex + Ca + Vit. D groups. Animal house temperature (21 ± 2°C), relative humidity (30-40%), and 12hr light-dark cycle were controlled and the rats were allowed free access to food and drinking water.
The different treatments were initiated 2 wk after the adaptation of animals. In all groups except the control and placebo groups, rats were ovariectomized and fed with a standard diet for two months. In order to make the ovariectomy, after the suture of the vascular plexus with fine linen thread, both ovaries were removed (16).

Gastrocnemius muscle assessment
Fresh gastrocnemius muscle was rinsed with PBS buffer, and then tissues fixed in a 10% formalin

Statistical analysis
The results of the experiments were expressed as mean and standard deviations of the different variables. The statistical analysis was done initially by SPSS, version 20 and one-way test. P < 0.05 and p < 0.001 were accepted as denoting a significant difference. Table I shows

Discussion
Menopause is associated with a decrease in the

Conclusion
Our findings demonstrated that resistance Ex in