Research On The Regulation Mechanism Of Lipid Formation In Pigeon Milk

In the current study,we used the American king pigeon as the experimental modle.Here,we first investigated the dynamic changes in histomorphology of pigeon crop and the nutrient metabolism in parental pigeons during the breeding cycle.Futhermore,we analyzed the profiles of hormones,growth factors and mRNA expression of the related receptors,the expression of fatty acid transporters and fatty acid synthesis-related genes and expression patterns of Akt/mTOR signaling pathway-related proteins in crops tissues,aiming to determine the molecular mechanism of lipid formantion in crop milk;Using the established organ-cultured pigeon crops,the effects of prolactin(PRL)on lipid accumulation of pigeon crop,the expression of fatty acid transporters and fatty acid synthesis-related genes and expression patterns of Akt/mTOR signaling pathway-related proteins in pigeon crops were investigated.After treating with the inhibitors of Akt/mTOR pathway,PRL-induced activity of Akt/mTOR signaling and the lipid accumulaition in the organ-cultured pigeon crops were measured.Our results are expected to provide important data for clarifying the regulation mechanism of lipid formation in crop milk,aiming to provide theoretical basis for crop milk lipid formation and the nutrition regulation in the squabs.The results were shown as below:1 Research on the changes in histomorphology of crop tissues in parental pigeons during the breeding cycleIn this study,we investigated the histomorphological changes of pigeon crop epithelium and lipid dropletsin crop tissues in male and famle pigeons during the incubation(Ⅰ)and chick-rearing(R)period,as well as the ultrastructure of crop tissues.The results showed the many spikes called rete pegs existed at the surface of pigeon crop at the day 10 of incubation(110),the crop epithelial thickness increased dramatically at day 1 of chick-rearing(R1)(P<0.05),and the space of adjacentrete pegs of crop inner wall decreased significantly in both male and female pigeons(P<0.05).At R1,the adjacent hyperplasia rete pegs combine together,desquamation formed.The rete pegs in females and males began to degenerate at R7 and R10,respectively.At R1,eosinophilic cells displayed with a ground-glass appearance,and the shape was bigger than that at 12,this was with no sexual effects of parental pigeons.In both sexes,pigeon crops demonstrate the appearance of sparse lipid drops at the initial phase of incubation,and then the density and sizes of lipid droplets increased dramatically at 117 and Rl.Density of lipid droplets decreased at R7 in both sexes.There existed difference in lipid droplets distribution between males and females at R1 and R7.Less lipid droplets and more desmosome were found in the cytoplasm of pigeon crop at R25 as compared to that at R1.2 Research on the metabolism of parental pigeons during the breeding cycleThe body weight,relative organ weigh,serum biochemistry and glucose and lipid metabolism-related parameters in liver were studied at 14,110,117,R1,R7,R15,R25 in both male and female pigeons.The results showed that body weight of female pigeons and male pigeons was the greatest at 117(P<0.05).The relative weight of breast and abdominal fat in both sexes and thighs in the male pigeons got the lowest level at R7(P<0.05).At 117,serum total protein,albumin,and globulin concentrations were the highest in both sexes(P<0.05);In female pigeons,total cholesterol,triglyceride,and low-density lipoprotein were maximized at R25,while they were maximized at 117 in male pigeons(P<0.05).At the teriminal phase of incubation and the initial phase of chick-rearing,fat content,glycogen content,gene expression of fatty acid synthase(FAS)and acetyl-CoA carboxylase(ACC)and hexokinase activity in livers were increased dramatically in both sexes(P<0.05);The expressions of fructose-1,6-bisphosphatase and glucose-6-phosphatase genes were maximized at R15(P<0.05);The activity of 6-phosphofructokinase and expressions of pyruvate kinase and carnitine palmitoyltransferase 1 genes were the highest at R7(P<0.05);In females,hepatic expressions of glucokinase and phosphoenolpyruvate carboxykinase cytosolic genes were the greatest at R7 and 117,respectively;however,in males,both of them were maximized at R15(P<0.05).3 Reseach on profiles of hormones,growth factors and mRNA expression of therelated receptors in parental pigeons during the breeding cycle In this study,the concentrations of serum prolactin(PRL),estradiol(E2)andprogesterone(P),as well as epidermal growth factor(EGF)and insulin-like growth factor-1(IGF-1)concentrations in the crop tissue were studied at 12,14,16,110,114,117,R1,R4,R7,R10,R15,R20,R25.Furthermore,the gene expression of the PRL receptor(PRLR),E2 receptor(E2R),P receptor(PR),GH receptor(GHR),insulin receptor(INR),IGF-1 receptor(IFG-1)and EGF receptor(EGFR)were analyzed.The results showed that PRL and IGF-1 concentrations were the highest at R1 in both sexes(P<0.05),and E2 concentration was the highest at R15(P<0.05).EGF concentration remained at a low level at 114,and then it increased by 71%at R1 in males and by 68.3%at 117 in females,respectively(P<0.05).Gene expression of PRLR in male and female pigeons reached peak values at 117 and 114,respectively(P<0.05).Both of E2R and PR gene expressions were significantly repressed at R1 in both male and female pigeons(P<0.05).GHR mRNA reached the lowest level at R4 in both sexes(P<0.05).During the chick-rearing period,INR gene expression at R4 was significantly higher than other time points in female pigeons(P<0.05),and no changes were found in male pigeons(P>0.05).Gene expression of EGFR reached its highest value at R1 in female pigeons and at 117 in male pigeons(P<0.05);IGF-1 receptor gene expression was the highest at 117 in female pigeons,while it increased significantly at R7 in males(P<0.05).4 Research on expression of fatty acid transporters and fatty acid synthesis-related genes in crop tissues of parental pigeons during the breeding cycleThe results showed that lipid content of crop milk decreased with progression of chick-rearing period(P<0.05);At R4,Lipid content of crop milk from male parental pigeons was significantly higher than that from female parental pigeons(P<0.05).At 117,the gene expression of fatty acid translocase(FAT/CD36),fatty acid binding protein 5(EFABP)and acyl-CoA binding protein(ACBP)in the crop tissue of male pigeons got the highest level(P<0.05);In female pigeons,the gene expression of FAT/CD36 got the peak value at 114,while both EFABP and ACBP gene expression were significantly up-regulated at R7(P<0.05).For the gene expression of ACC and FAS,both of them were notably enhanced at R1 in crop tissues of male pigeons(P<0.05),whereas they got the peak level at 114 and 117 in female crop tissues(P<0.05),respectively.No regular changing pattern was found in PPARa gene expression in crop tissues of both sexes.For PPARy expression,it was the highest at 117 in the males and at 114 in the females(P<0.05).5 Research on the mechanism of Akt/mTOR signal pathway on the regulation of lipid biosynthesis in pigeon crops during the breeding cycle(1)Expression patterns of Akt/mTOR signaling pathway-related proteins were studied in crop tissues of male and female pigeons at 14,I10,117,R1,R7,R15,R25 by Western blot.The results showed that the expressions of Akt,phosphorylated Akt(P-Akt(T308)),phosphorylated Akt(P-Akt(S473)),mTOR and phosphorylated mTOR(P-mTOR)protein were dramatically up-regulated at R1 in both sexes(P<0.05);P-Akt(T308)in both sexes and P-Akt(S473)in females decreased remarkably at R7(P<0.05);P-Akt(S473)in males maintained at a high level during the chick-rearing period;Expressions of mTOR and P-mTOR protein at 117 were significantly higher than those at other time points in males,while both of them were maximized at R1 in females(P<0.05).(2)The present study evaluated the histological structure,enzymes acti’vities and genes expressions of organ-cultued pigeon crops over a 7-day period in vitro to determine the change in viability of crops and the optimal culture period.Adult pigeons at 110 and R8 were the source for this experiment.The results showed that the viability of crops from parental pigeons at I10 and R8 show a similar trend under the in vitro contition.Structure of crops were integrated from 0 to 3 days after culture in vitro;After culturing for 1 day in vitro,activities of enzymes and genes expression in pigeon crops varied significantly,and then these parameters remained stable during day 2-3 culture in vitro;After culturing for 2-3 days in vitro,Caspase-3 activity and Bakl gene expression remained at a lower level,while activities of succinate dehydrogenase,Na+-K+-ATPase,Ca2+-Mg2+-ATPase,Total ATPase and gene expressions of Bcl-2 and CK-19 stayed at a higher level.(3)Using the established in vitro organ-cultured pigeon crop as model,the morphology of lipid droplets,lipid content,the fatty acid transporters and fatty acid synthesis-related genes expressions and expression patterns of Akt/mTOR signaling pathway-related proteins were analyzed after treating with 25,50 and 100 ng/mL PRL.The results showed that the crops with PRL supplements showed higher amounts of lipid droplets in the basal nutritive layer of epitheliums compared to the controls across the experiments;After incubating for 24 h,the lipid content in 100 ng/mL PRL groups was higher than that in other groups(P<0.05);After incubating for 36 h,groups with 50 ng/mLPRL displayed the highest lipid content in this study(P<0.05).Compared with the controls,25,50 ng/mL PRL dramatically increased the expression of ACC,FAS,FAT/CD36,ACBP,EFABP and PPARγ genes(P<0.05),and the relative protein levels for Akt,P-Akt(S473),P-Akt(T308),mTOR and P-mTOR were significantly up-regulated by 50 ng/mL PRL(P<0.05).The stimulatory effect of PRL on Akt/mTOR signaling pathway-related proteins and lipid accumulation was significantly decreased by treatment with the PI3K hibitor(LY294002)or Akt hibitor(MK-2206)(P<0.05).In conclusion,the changes in histomorphology of parental pigeons crops is the physiological basis for crop milk production during the breeding cycle.The regulation of metabolism and nutrition provided the nutritional and energetic support for the crop milk formation.We suggested that the hormones,growth factors and their receptors potentially underlay pigeon crop tissue development and crop milk formation.The lipid content in crop milk decreased as the progression of chick-rearing,and the expressions of fatty acid transporters and fatty acid synthesis-related genes were repressed significantly,suggesting that fatty acids used in lipid biosynthesis of crop milk originated from both exogenous supply and de novo synthesis.The Akt/mTOR signal pathway in pigeon crops was activiated at the terminal phase of incubation and at the initial phase of chick-rearing period,as well as in the model of PRL-induced lipid accumulation.Furthermore,the inhibition of Akt/mTOR signal pathway significantly decreased the lipid accumulation induced by PRL.Our results indicated that Akt/mTOR signal pathway plays an important role in lipid formation in crop milk.Different physiological conditions of male and female pigeons during the breeding cycle may affect the process of crop milk formation.