Effects Of Dietary Valine-to-lysine Ratio To Gilts During Late Gestation On The Development Of Mammary Gland And Lactation And The Underlying Mechanism

The crucial factors determining the synthesis of colostrum depend largely on the development of mammary gland during late pregnancy.The composition and yield of gilts'colostrum are major limiting factors for maximal pre-weaning growth and survival of offspring.Among the three BCAA,valine is transported with highest efficiency and is extensively consumed by the porcine mammary gland.Valine is not just a substrate for protein synthesis,evidence in rats and human have identified a strong relationship between valine metabolism and fat metabolism,cell proliferation and signaling pathway activation.However,the role of valine in regulating mammary development and secretion in porcine mammary glands during late gestation remains to be determined.Therefore,the objectives of this study were to investigate the roles and potential mechanism of valine on milk protein,milk fat and mammary development in gilts.The main contents and results are presented as follows.Experiment 1 Effect of dietary valine-to-lysine ratio during late gestation on colostrum composition of primiparous giltsThe colostrum composition is mainly affected by nutritional regulation and development of mammary gland during late gestation.Previous studies of our group showed that dietary valine-to-lysine ratio during late gestation of gilts significantly increased the weaning weight of piglets.So,this study focused on the effect of valine on colostrum quality in sows during late gestation.According to valine-to-lysine ratio,sixty pregnant gilts were randomly allocated to 0.57 group and 0.87 group,each group contained 30 gilts.During lactation,all gilts were fed the same commercial gestation diet.This experiment lasted from day 85 of gestation to day 21 of lactation.The results showed that:(1)Compared to 0.57 group,those offered the 0.87 group diet during late gestation had an increased yield of milk and average daily gain of piglets(P<0.05),as well as feed intake of gilts in the first two weeks of lactation(P<0.05).In the whole experiment period,treatment had no effect on gilts backfat thickness(P>0.05).(2)Compared to 0.57 group,the contents of leucine,isoleucine,threonine and serine were increased significantly(P<0.05)in 0.87 group.Meanwhile,the contents of glutamate and arginine,alanine,glycine and histidine were increased in 0.87 group,but that were not significant(0.05<P<0.1).(3)Targeted metabolomics assays showed that 40 molecules differed related to amino acids metabolism in colostral samples between gilts fed the 2 diets;37 had higher contents and 3 had lower contents in 0.87 group,compared with the 0.57 group.These upregulated molecules included essential amino acids(lysine,valine and leucine)and nonessential amino acids(glutamine,glutamate and aspartate);these downregulated molecules related to nucleotide metabolism.(4)Higher proportions of C18:2(n-6),C18:3(n-3),C20:4(n-6)and total polyunsaturated fatty acids were observed in the colostrum of the 0.87 group compared with the 0.57 group(P<0.05).(5)Targeted metabolomics assays showed that 9 molecules differed related to fatty acids metabolism in colostral samples between gilts fed the 2 diets.All differential metabolites included sphingomyelin and triglycerides had higher contents in 0.87 group,compared with the 0.57 group.Among these differential metabolites,the change multiple of TG(16:0/18:2/18:3)+NH4 reached 4.69 times.In summary,this study indicated that supplementing gilts diets 0.87 of valine-to-lysine ratio during late gestation significantly increased growth performance of piglets and improved the contents of free amino acids and polyunsaturated fatty acids in colostrum.Experiment 2 Effect of dietary valine-to-lysine ratio during late gestation on mammary gland development and primiparous gilts performanceExperiment 1 confirmed that increasing dietary valine-to-lysine ratio in gilts during late gestation significantly improved the quality of colostrum.However,the role of valine in regulating development and secretion in porcine mammary glands remains to be determined.According to valine-to-lysine ratio,30 gilts(Yorkshire × Landrace)pregnant gilts were randomly allocated to one of three valine nutritional groups with 10 repeats for each group:0.63 group,0.73 group and 0.93 group at day 75 of gestation.The 0.63 group diet contained 0.54%valine by analysis and total valine:lysine=0.63:1;The 0.73 group diet contained 0.62%valine and total valine:lysine=0.73:1;The 0.93 group diet contained 0.79%valine and total valine:lysine=0.93:1.During lactation,all gilts were fed the same commercial lactation diet.This experiment lasted from day 75 of gestation to day 21 of lactation.The results showed that:(1)No significant effect was observed for the number of piglets born alive,average individual weight and litter weight at birth among the three treatment groups(P>0.05),however,the piglet weight at weaning,litter weight and average daily gain at day 21 increased linearly(Linear,P<0.05)with the increase in valine-to-lysine ratio of diet during late gestation.Compared to 0.73 group,the gilts in 0.93 treatment group had an increased feed intake during first week of lactation(P<0.05).In the whole experiment period,treatment had no effect on gilts backfat thickness(P>0.05).(2)At day 1 and 21 of lactation,the area of lumen of alveolus of mammary tissue was larger in 0.93 group than 0.73 and 0.63 group.At day 1 of lactation,the content of DNA,RNA and protein expression of Cyclin D1 and Stat 5 of mammary tissue increased linearly(Linear,P<0.05)with the increase in valine-to-lysine ratio of diet during late gestation.However,no significant effected on the expression of protein related to cell apoptosis,as well as day 21 of lactation.(3)The milk yield of gilts increased linearly(Linear,P<0.05)with the increase in valineto-lysine ratio of diet.However,by 5,7,10,14,17,and 21 day,treatment groups had no significantly effected on milk yield(P>0.05),although all values were the highest in 0.93 group.In addition,with the increase in valine-to-lysine ratio of diet,the content of milk protein,milk fat and solids-not-fat in colostrum were increased linearly(Linear,P<0.05).However,no significant effected on the composition of milk.(4)At day 1 of lactation,the protein expression levels of ?-casein,related to mTOR signaling pathway(p-mTOR,p-S6),related to fatty acids synthesis(N-SREBP1,ACC,FASN,FABP3)increased linearly(Linear,P<0.05)with the increase in valine-to-lysine ratio of diet during late gestation.However,the protein expression related to synthesis of milk protein and fat were no significant difference among the three groups at day 21 of lactation(P>0.05).In summary,dietary valine supplementation in gilts during late gestation to valine:lysine to 0.93:1 improved the growth performance of piglets by increased the development of mammary gland,content of protein and fat in colostrum.Experiment 3 Effect of valine on cell proliferation and milk protein synthesis and the underlying mechanism in porcine mammary gland epithelial cells.Experiment 1 and 2 confirmed that increasing dietary valine-to-lysine ratio in gilts during late gestation significantly improved the mammary gland development and colostral protein synthesis.So,this experiment was to investigate the appropriate concentration of valine and its molecular mechanism in regulating cell proliferation and milk protein synthesis in porcine mammary gland epithelial cells(PMECs).PMECs were isolated by tissue block enzymolytic method.PMECs were cultured in valine-free dulbecco's modified eagle medium containing different concentrations of valine(0.1-7.2 mM)or rapamycin(40 nM).PMECs were collected for determining cell proliferation,cell cycle and related protein expression levels.The results showed that:(1)We established a high purity and proliferation rate PMECs line by tissue block enzymolytic method,these cells present the typical morphological characters of epithelial cells.The PMECs maintained their cell morphology and the potential for differentiation after at least 10 passages in culture.(2)Exposure of PMECs to 0.1 mM to 0.9 mM valine dose-dependently increased live cell numbers,as well as percentage of cells in the G2 and S phases.The protein expression of Cyclin D1 and Stat 5 were significantly increased in PMECs treated with 0.9 mM valine than exposure to low concentrations of valine(P<0.05).However,the abundance of Active caspase-3 in PMECs exposed to 3.6 and 7.2 mM valine was increased.(3)Exposure of PMECs to 0.1 mM to 1.8 mM valine dose-dependently increased ?-casein while higher concentrations(3.6 and 7.2 mM)decreased ?-casein from its maximum,obtained with 0.9 mM valine.The abundance of ?-casein in PMECs exposed to 0.1,0.9 and 7.2 mM valine was evaluated by immunofluorescence microscopy to confirm the Western blot data and,again,treatment with 0.9 mM showed the strongest fluorescence(P<0.05).(4)The phosphorylation of mTOR increased in a log-dose dependent manner during exposure of PMECs to 0.1 mM to 1.8 mM valine,but sharply decreased with higher concentrations.The phosphorylation of S6 changed with increased concentrations of valine and 4EBP1 was different with lower concentrations of valine(0.1 to 0.45 mM)being similar and with greater phosphorylation for all higher levels of valine.However,the phosphorylation of mTOR,S6 and 4EBP1 all decreased by co-treatment with 40 nM rapamycin(P<0.05).(5)Compared PMECs exposure to 0.1 and 7.2 mM valine,the utilization of leucine,isoleucine,lysine and methionine were increased in PMECs exposed to 0.9 mM valine,as well as the mRNA expression of SLC7A7 and SLC7A1(P<0.05).In summary,this study demonstrated that optimal exposure to valine improved PMECs viability and proliferation,as well as the production of a milk-specific protein via activation of the mTOR pathway and its downstream signaling proteins.Experiment 4 Effect of valine on fatty acids synthesis and the underlying mechanism in porcine mammary gland epithelial cells.Experiment 1 and 2 confirmed that increasing dietary valine-to-lysine ratio in gilts during late gestation significantly improved the colostrum fat synthesis.So,the objectives of this study were to investigate the roles and potential mechanism of valine on fatty acid synthesis in porcine mammary gland epithelial cells(PMECs).PMECs were cultured in valine-free dulbecco's modified eagle medium containing different doses of valine(0.1-1.8 mM)and using rapamycin to inhibit mTOR or LY294002 to inhibit PI3K.In a separate experiment,PMECs were transfected SREBP1 siRNA for 48 h.PMECs were collected for determining concentration of triglycerides(TGs)and related protein expression levels.The results showed that:(1)Compared to PMECs exposure to 0.1 mM valine,our results showed that 0.9 mM of valine significantly increased the levels of TGs in the medium(P<0.05);These results were further supported by cellular staining with the lipophilic dye BODIPY 493/503;The protein expression levels of FAs synthesis enzymes,including ACC,FASN,DGAT,and FABP3,were significantly up-regulated(P<0.05).However,the protein expression of enzymes involved in FAs ?-oxidation,including CPT1-B,ACSL and HADH did not differ among groups.In addition,phosphorylation of Akt and mTOR were significantly increased in PMECs treated with 0.9 mM valine compared with 0.1 mM valine(P<0.05).Although the total and cytoplasmic abundance of SREBP1 in PMECs exposed to 0.9 mM valine were not increased,the SREBP1 levels in cell nuclei were significantly(P<0.01)higher in cells exposed to 0.9 mM valine compare with 0.1 mM valine.(2)When SREBP1 was knocked down by siRNA,there was a significant decrease in the TGs concentration of the medium(P<0.05)as well as the abundance of lipids in PMECs as demonstrated by the lipophilic dye BODIPY 493/503,when compared with related controls.As expected,silencing of SREBP1c by siRNA resulted in a marked reduction in the expressions of ACC,FASN,DGAT,and FABP3 in PMECs(P<0.05).(3)After incubating PMECs with a specific inhibitor of mTOR rapamycin,the effects of valine on cellular TGs content were significantly abolished,accompanied by the reduced intensity of BODIPY staining,and the decreased the phosphorylation of mTOR.Moreover,the protein expression of SREBP1 in cell nuclei were significantly(P<0.01)lower in PMECs exposed to rapamycin relative to those in the control.Furthermore,the protein expression of ACC,FASN,and FABP3 was markedly decreased(P<0.05).(4)On treatment with LY294002,we observed a decrease in the contents of intracellular TGs and BODIPY staining of neutral lipids.Furthermore,the levels of p-Akt and p-mTOR were markedly decreased in PMECs treated with the PI3K inhibitor,LY294002(P<0.05).Moreover,the inhibition of Akt by LY294002 decreased the expression of SREBP1(in the nucleus and cytoplasm),ACC,and FASN(P<0.05).In summary,we demonstrated that valine plays an important role in regulating milk fat synthesis via activation of the Akt/mTOR/SREBP1 signaling pathway in PMECs.In conclusion,our research demonstrated that appropriate improved dietary valine-tolysine ratio to gilts during late gestation significantly increased lactation performance by improved development of mammary gland;Dietary valine enhanced milk protein and fat synthesis of colostrum in porcine mammary glands via activation of the mTOR and Akt/mTOR/SREBP1 signaling pathway,respectively.