| The cyclical changes in animal reproduction begin with oestrus,the initiation mechanism of which is closely linked to the nutrient base.Copper,an essential trace element for animals,acts as a cofactor for many enzymes,promotes growth and participates in the metabolism and immune regulation of the body.Recent studies have shown that copper plays an important role in the regulation of reproduction in female mammals by regulating the synthesis and secretion of oestrus hormones and related peptides on the HPOA axis,which in turn affects a series of physiological processes such as oestrus,ovarian ovulation and the secretion of sex hormones in female animals.The initiation of oestrus is an important marker of the onset of reproduction in animals,and its initiation mechanism is complex.There are no studies on the effect of copper on this particular phase of oestrus initiation in animals.Therefore,the aim of this experiment was to investigate the effect of dietary copper on the initiation of oestrus in female SD rats and its possible pathways of action,in order to provide a basis for the rational use of copper in breeding animals.In this experiment,50 weaned female SD rats of 21 days old from the same batch,healthy and of similar body weight（44.24±2.95g）were selected in a completely randomized design and randomly divided into 5 treatment groups A,B,C,D and E.Five replicates of each treatment,2 rats per replicate,were fed and watered ad libitum for 6 d during the pre-feeding period.Groups A,C,D and E were fed with CuSO4·5H2O containing 15,30,45 and 60 mg/kg of copper ions on top of the basal diet,and the oestrus cycle was measured and recorded by vaginal smear.The first oestrus was initiated by vaginal smear and the time of oestrus initiation was recorded.The animals were fasted for 8 h before slaughter,anaesthetized and blood was collected from the heart,and the hypothalamus,pituitary and ovary tissues were removed and frozen at-80℃ for examination.In order to determine the effect of dietary copper on estrus-related hormones in animals at the beginning of estrus,this experiment was carried out by ELISA to detect the levels of GnRH,FSH,LH,E2 and P4 in the serum of SD rats in each treatment group;fluorescence quantitative PCR and Western blot were used to detect the relative mRNA expression of estrus-related hormones and their receptors in the HPOA axis such as GnRH,LH and FSH.The relative mRNA expression levels and protein expression of estrus-related hormone receptors in the HPOA axis,including GnRH,LH and FSH,were measured by fluorescence PCR and Western blot.The relative mRNA and protein expression of the KISS-1/GPR54 system and PAM in the hypothalamus of rats were also examined to further investigate the possible mechanisms of copper regulation of GnRH secretion.The results showed that:（1）Compared with the control group,with the increase of copper addition,the rats showed an overall trend of earlier age of first estrus,among which the rats in test group D showed the earliest age of first estrus,which was not significantly different from group C（P≥0.05）and significantly earlier than the control group（P<0.05）.（2）Compared with the control group,the serum levels of GnRH,FSH,LH,E2 and P4 in all groups showed a trend of increasing and then decreasing with the increase of copper addition,and all of them were significantly higher than the control group.The hormone levels in group D were the highest,significantly higher than those in groups B and E（P < 0.05）.（3）Fluorescence quantification results showed that the relative expression of GnRH,FSH,LH and E2 in the HPOA axis tended to increase and then decrease with the increase of copper addition level.The relative mRNA expressions of KISS-1,GPR54 and PAM in the hypothalamus of rats all showed a trend of increasing and then decreasing.Compared with the control group,the relative mRNA expressions of Group C and Group D were significantly increased（P<0.05）,and the difference between Group C and Group D was not significant（P≥0.05）.（4）The Western blot results showed that the relative protein expression of GnRHR,LHR,FSHR,E2 R and GPR54 in the HPOA axis and the protein concentrations of Kisspeptin and PAM in the hypothalamus showed a trend of first increasing and then decreasing with the increase of copper addition level,compared with the control group,the relative protein expression was significantly increased in groups C and D（P < 0.05）,and the difference between groups C and D was not significant（P > 0.05）.In conclusion,compared with the control group,CuSO4·5H2O added to the basal diet at a concentration of 3045 mg/kg of copper was the most effective in promoting the initiation of estrus in female SD rats at an earlier day of estrus,presumably because copper affects estrus in animals by promoting the KISS-1/GPR54-GnRH pathway and thus the secretion of hormones related to estrus on the HPOA axis. |
PDF Full Text Request