, Ltd., Shanghai, China), special formula with oligosaccharides and glucose10 and 11 (QS130123010057; Hebei Hua Chen Starch Sugar Co., Ltd, Gaoyi, China) were used as additional energy supplements. This study has been approved

by Shanghai Institute of Nutrition (Ethical selleck products Certification 2013-002). Prior to the experiment, all rats were examined for vaginal cytology twice per day for two continuous estrous cycles to monitor menstrual cycles in all experimental rats.12 At the end of experiments, all rats were anesthetized and the ovaries were removed for electron microscopy study. Blood was collected from inferior vena cava for detecting serum levels of 17β-estradiol and progesterone, respectively. In order to compare the levels of ovarian hormone and microstructure of ovary at same stage of menstrual cycle, all rats were sacrificed during anestrous phase determined by vaginal smears. As shown in Fig. 1, rats Icotinib molecular weight were randomly divided into five groups, namely, normal controls (C) with no excise, EAMD model (E) having 6 weeks treadmill running with increasing work load followed by 3 weeks running with constant speed (Table 1), post-EAMD recovery model (R) having 3 weeks without

exercise after 6 weeks running, and 3 weeks carbohydrate supplements, oligosaccharide (O) or glucose (G), after 6 weeks running. Vaginal cytology examination was performed daily during the 9-week study to monitor the estrous cycle of each rat. All rats were fed with regular food chew throughout the study, except rats in groups O and G who received oligosaccharide and glucose for 3 weeks prior CYTH4 the end of the 9th week, respectively. We have previously identified that adult female rats developed EAMD after 6 weeks of exercise with incremental load with delayed the onset of estrus phase, much longer anestrous time, and irregular menstrual cycling (data not published). All rats were sacrificed at the end of the 9 weeks of exercise with 12 h fast. A Duanshi PT98-type treadmill (Xin Ruan Information

Technology Ltd., Shanghai, China) was used in this experiment. Animals were trained according to the Bedford animal training load standard13 described in Table 1 and Fig. 1. After 1 week of adaptation training, rats were exercising twice a day, one time in the morning and one time in the afternoon with minimum 5 h rest between the two exercise segments. Total of 9 weeks exercise with each training cycle lasted for 1 week (6 days exercise, 1 day rest). Vaginal smear was performed daily to monitor menstrual cycle for each rat throughout the 9-week study.12 Menstrual cycle dysfunction was diagnosed by the onset of estrus phase or length change of anestrus phase in menstrual cycle as described previously.14 and 15 The serum levels of 17β-estradiol were detected by immunochemistry luminescence kit following the instruction provided by manufactory (Beckman Coulter Inc.