| Improving the reproductive potential of animals and producing more high-quality meat,eggs,and dairy products to meet the growing material needs of increasing human population is a goal pursued by many researchers in the fields of animal genetics,breeding and reproduction.In particular,improvements in animal breeding and reproduction efficiency can greatly improve the production efficiency of important economical livestock and can potentially solve the rising problems related to food-supply shortage.In intensive livestock production systems,nutrition of farmed animals mainly comes from allocated feed and daily drinking water.Evidence from mainstream literature indicates that supplementation of feed and drinking water with essential ingredients at appropriate levels can help optimize the reproductive efficiency of animals.Melatonin(MT)is mainly secreted by the pineal gland and has implications in regulating the circadian rhythm and reproductive cycle in animals In this study,female ICR mice were used as a model to investigate the effects of oral administration of different concentrations of MT on the female reproductive systemIn this study,six-week-old female ICR mice were divided into four groups and received one of the four MT concentrations(0μg/mL,3 μg/mL,30 μg/mL,and 300 μg/mL)via drinking water for 21 days.At the end of MT supplementation trial(day 21),the mice in all groups were used for evaluating the effect of MT on litter size and litter birth weight following natural mating some of the mice in all groups and part of the mice were sacrificed and ovaries were collected immediately and utilized for further experiments:analysis of ovarian follicle development(H&E staining).On days 7,14,and 21 following MT addition(MT addition for the first time was recorded as the first day),blood samples were collected at eight different time points i.e.,09:00,13:00,17:00,21:00;23:00,01:00,03:00,05:00,and assayed for estimation of serum levels of MT,follicle-stimulating hormone(FSH),and Estradiol(E2).Moreover,analysis of mRNA expression of oocyte maturation-related genes(FSHr,Gdf9,Bmp15,Grim 19 and Nlrp5)in oocytes via RT-qPCR perfomed on day 21The higher litter size was observed in mice treated with 3 μg/mL MT,whereas no significant difference was observed in litter birth weight between the groups(P≥0.05).The histological assessment of H&E stained ovarian sections revealed that the percentage of antral follicles in 3 μg/mL MT-treated group was significantly higher(P<0.05)compared to the control group(43.46±7.58%vs.29.62±4.01%).The percentage of secondary follicles in 3 μg/mL MT-treated group was significantly lower(P<0.05)than that of the control group(29.33±7.84%vs.43.08±2.12%;).The percentage of primary follicle was significantly higher(P<0.05)in 30 μg/mL MT-treated group compared to the control(36.09±1.84%vs.27.30±2.46%).Regardless of MT addition in drinking water,the serum MT levels were low during daytime(210.64-274.89 pg/mL)and high during night(236.96-336.63 pg/mL)When 30 μg/mL MT was added to drinking water,peak MT secretion during night phase(333.45 pg/mL)was observed at 1 am;two hours later than that of other treatment and control groups(control group:336.63 pg/mL;3 μg/mL T treatment group:321.25 pg/mL;300 μg/mL MT treatment group:314.60 μg/mL).The level of FSH in 300 μg/mL MT-treated group fluctuated greatly(95.11-161.89 mIU/mL)and also exhibited a higher level compared to the control group on days 7,14,and 21.Mice in other two MT treatment groups(i.e.,30μg/mL,and 3 μg/mL)also showed a significant increase(P<0.05)in FSH levels on day 21 However,the levels of serum E2 showed an opposite trend compared to the FSH levels.In 300 μg/mL MT-treated group,the levels of serum E2 decreased significantly on days 7 and 14 compared to the control group(p<0.05),and then retuned back to a normal level on day 21.The results of RT-qPCR showed that the expression of Bmp 15 and FSHr in GV oocytes retrieved from mice treated with 3 μg/mL MT was significantly higher than that of the control group(P<0.05),while the expression of Nlrp5 and Grim19 showed no significant differences between the groups(P≥0.05)In summary,the addition of MT in daily drinking water of mice affected the levels of serum FSH and E2,which may have had an impact on the reproductive system in mice.Mice treated with 3 μg/mL MT showed the higher percentage of dominant ovarian follicles as well as the expression of oocyte growth-related genes FSHr and Bmp15.Therefore,in light of above findings,it is reasonable to imply that the improved litter size in mice treated with 3μg/ml MT maybe attributable to the MT-based modulation of above-mentioned biomarkers In addidtion,it should be noted that the high dose of MT(300 μg/ml)did not negatively affect the follicular growth and female reproductive efficiency in mice. |
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