| During transition period,large amount of calcium need to be transported into milk from blood in dairy goat.Calcium in the blood was mainly derived from osteolysis.However,there is a hysteresis in this process,calcium in the blood can't be replenished in time.Dairy goats are prone to clinical or subclinical hypocalcemia during this period.This disease can easily lead to mastitis,ketosis,metabolic alkalosis and many other diseases,also can lead to lower milk yield,lower milk quality,blocked lamb growth and death of ewe.Hypocalcemia causes huge economic losses in production and has serious harm to the development of dairy goat industry.5-HT,also known as serotonin,is synthetisedfrom L-tryptophan andtryptophan hydroxylase 1 is the rate-limiting enzyme in this process.5-HT is a kind of monoamine neurotransmitter,which is considered as a regulator of bone turnover in lactation,playing important roles in inducing bone regulating factor,maintaining bone density and inducing bone remodeling.It is speculated thatinjection of 5-HTP or pre-partum milking may help maintain the calcium metabolism balance of dairygoats during transition period and reduce the incidence of hypocalcemia.However,the related studies have not yet been reported.The purpose of this study was to investigate the effect of 5-HT on calcium circulation and its molecular mechanism in dairy goats during transition period by in vivo and in vitro,and to elucidate the regulatory mechanism of 5-HT on the concentration of Ca in blood of dairy goats during perinatal period,also to find a new effective method to prevent perinatal hypocalcemia in dairy goats.Firstly,the dairy goats were injected with 5-HTP or pre-partum milking one week before birth.The concentrations of 5-HT,Ca,PTHrP in serum and milk of dairy goats were detected and compared,and the correlations among them were analyzed.Then the goat mammary epithelial cell line TPH1 gene knocked out was constructed by using CRISPR/CAS technique.Wild typegoat mammary epithelial cells and goat mammary epithelial cell lines with TPH1 gene knocked out were used as experimental materials.RT-PCR was used to detect the expression abundance of key genes related to calcium transport in goat mammary epithelial cells at different 5-HT levels.The content of Ca in the cells was detected by confocal laser imaging.The main results obtained are as follows:1.There were significant correlations between concentrations of 5-HT,PTHrP and Ca in serum and milk of dairy goatsduring transitionperiod.After injection of 5-HTP,the concentrations of 5-HT and PTHrP in the blood wasere higher than those of the control group.Also the concentrations of PTHrP and Ca in milk werehigher than those of the control group.After pre-partum milking,the concentrations of PTHrP in the serum was higher than that of the control group.2.Goat mammary epithelial cell line with TPH1 gene knocked out was obtained by using CRISPR/CAS technique.The exon 1 of goat TPH1 gene was successfully targeted.3.In vitro experiments showed that the addition of 5-HTP to wild typegoat mammary epithelial cells could promote the m RNA expression of SPCA2 and decrease the mRNA expression PMCA1,while the expression of PTHrP?SPCA1 and PMCA1 mRNA expression decreased inTPH1 gene knockout cells.The expression of PMCA1 decreased whenthe cells THP1 gene knocked outwwere treated with 5-HTP,the mRNA expression of SPCA1 was rescued.The results of laser confocal imaging showed that 5-HT could promote calcium excretion in goat mammary epithelial cells.In conclusion,5-HT plays important roles in the regulation of calcium circulatory metabolismof dairy goats during transition period.Injection of 5-htp or pre-partum milking can increase the concentration of PTHrP in serum.they may be potential methods to prevent hypocalcemia in dairy goats.5-HT may promote intracellular Ca efflux by regulating the expression of PTHrP,PMCA1,SPCA1 and SPCA2,which are calcium transport key genes in mammary epithelial cells. |
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