Comparative proteomic analysis of the effects of high-concentrate diet on the hepatic metabolism and inflammatory response in lactating dairy goats

Background： To understand the impact of feeding a high-concentrate diet to mid-lactating goats for a long time on liver metabolism and inflammatory response, two dimensional polyacrylamide gel electrophoresis（2-DE） and real-time PCR method were employed to detect proteins differentially expressed in liver and their m RNAs expression in goats fed high concentrate diet（HC） or low concentrate diet（LC）. Twelve lactating dairy goats were randomly assigned to either a HC diet group（65 % concentrate of dry matter; n = 6） or a LC diet group（35 % concentrate of dry matter; n = 6） for 10 wk.Results： Twenty differentially expressed spots（≥2.0-fold changes） in the hepatic tissues were excised and successfully identified using MALDI TOF/TOF. Of these, 8 proteins were up-regulated, while the rest 12 proteins were down-regulated in HC goats compared to LC. Differential expressed proteins including alpha enolase 1（ENO1）, glutamate dehydrogenase 1（GLUD1）, glutathione S-transferase A1（GSTA1）, ATP synthase subunit 5β（ATP5β）, superoxide dismutase [Cu-Zn]（SOD1）, cytochrom c oxidase subunit Via（COX6A1） and heat shock protein 60（HSP60） were further verified by real-time PCR and/or western blot at m RNA or protein expression level. Consistent with the 2-DE results, a significant decrease of β-actin protein expression and SOD enzyme activity was observed in liver of HC goats（P 〈 0.05）, while ENO1 protein expression was significantly up-regulated in HC compared to LC goats（P 〈 0.05）. However, western blot analysis did not show a significant difference of hepatic HSP60 protein between HC and LC group, which did not match the decrease of HSP60 content detected by 2-DE analysis. Real-time PCR showed that glutathione S-transferase P1（GSTP1） and SOD1 m RNA expression was significantly decreased in liver of HC goats, while cytochrom c oxidase（COX3） and ATPase 8（ATP8） m RNAs expression were markedly increased compared to LC（P 〈 0.05）. Gene Ontology（GO） analysis revealed that HC diet resulted in altered expression of proteins related to catalytic and mitochondrial metabolism in the liver, and may increase the stress response with up-regulating the expression of differentiation 14（CD14） cluster and serum amyloid A（SAA） as well as C-reactive protein（CRP） in the liver.Conclusions： These results suggest that feeding high concentrate diet to lactating goats for 10 wk leads to the activation of the inflammatory response, and decreases the anti-oxidant capacity, and subsequently impairs the mitochondrial function in the liver.

Negative effects of long-term feeding of high-grain diets to lactating goats on milk fat production and composition by regulating gene expression and DNA methylation in the mammary gland

Background： It is well known that feeding a high concentrate（HC） diet to lactating ruminants likely induces subacute ruminal acidosis（SARA） and leads to a decrease in milk fat production. However, the effects of feeding a HC diet for long periods on milk fatty acids composition and the mechanism behind the decline of milk fat still remains poorly understood. The aim of this study was to investigate the impact of feeding a HC diet to lactating dairy goats on milk fat yield and fatty acids composition with an emphasis on the mechanisms underlying the milk fat depression. Seventeen mid-lactating dairy goats were randomly allocated to three groups. The control treatment was fed a low-concentrate diet（35% concentrate, n = 5, LC） and there were two high-concentrate treatments（65% concentrate, HC）, one fed a high concentrate diet for a long period（19 wks, n = 7, HL）; one fed a high concentrate diet for a short period of time（4 wk, n = 5, HS）. Milk fat production and fatty acids profiles were measured. In order to investigate the mechanisms underlying the changes in milk fat production and composition,the gene expression involved in lipid metabolism and DNA methylation in the mammary gland were also analyzed.Results： Milk production was increased by feeding the HC diet in the HS and HL groups compared with the LC diet（P 〈 0.01）, while the percentage of milk fat was lower in the HL（P 〈 0.05） but not in the HS group. The total amount of saturated fatty acids（SFA） in the milk was not changed by feeding the HC diet, whereas the levels of unsaturated fatty acids（UFA） and monounsaturated fatty acids（MUFA） were markedly decreased in the HL group compared with the LC group（P 〈 0.05）. Among these fatty acids, the concentrations of C15：0（P 〈 0.01）, C17：0（P 〈 0.01）, C17：1（P 〈 0.01）, C18：1 n-9 c（P 〈 0.05）, C18：3 n-3 r（P 〈 0.01） and C20：0（P 〈 0.01） were markedly lower in the HL group, and the concentrations of C20：0（P 〈 0.05） and C18：3 n-3 r（P 〈 0.01） were lower in the HS group compared with the LC group. However, the concentrations of C18：2 n-6 c（P 〈 0.05） and C20：4 n-6（P 〈 0.05） in the milk fat were higher in the HS group. Real-time PCR results showed that the m RNA expression of the genes involved in milk fat production in the mammary gland was generally decreased in the HL and HS groups compared with the LC group. Among these genes, ACSL1, ACSS1 ＆ 2, ACACA, FAS, SCD, FADS2, and SREBP1 were downregulated in the mammary gland of the HL group（P 〈 0.05）, and the expressions of ACSS2, ACACA, and FADS2 m RNA were markedly decreased in the HS goats compared with the LC group（P 〈 0.05）. In contrast to the gene expression, the level of DNA methylation in the promoter regions of the ACACA and SCD genes was increased in the HL group compared with the LC group（P 〈 0.05）. The levels of ACSL1 protein expression and FAS enzyme activity were also decreased in the mammary gland of the HL compared with the LC group（P 〈 0.05）.Conclusions： Long-term feeding of a HC diet to lactating goats induced milk fat depression and FAs profile shift with lower MUFAs but higher SFAs. A general down-regulation of the gene expression involved in the milk fat production and a higher DNA methylation in the mammary gland may contribute to the decrease in milk fat production in goats fed a HC diet for long time periods.

Whole transcriptome analysis of RNA expression profiles reveals the potential regulating action of long noncoding RNA in lactating cows fed a high concentrate diet

Subacute ruminal acidosis(SARA)is a common metabolic disease in the dairy farming industry which is usually caused by an excessive amount of high concentrate diet.SARA not only threatens animal welfare but also leads to economic losses in the farming industry.The liver plays an important role in the distribution of nutritional substances and metabolism;however,a high concentrate diet can cause hepatic metabolic disorders and liver injury.Recently,noncoding RNA has been considered as a critical regulator of hepatic disease,however,its role in the bovine liver is limited.In this study,12 mid-lactating dairy cows were randomly assigned to a control(CON)group(40% concentrate of dry matter,n=6)and a SARA group(60% concentrate of dry matter,n=6).After 21 d of treatment,all cows were sacrificed,and liver tissue samples were collected.Three dairy cows were randomly selected from the CON and SARA groups respectively to perform whole transcriptome analysis.More than 20,000 messenger RNA(mRNA),10,000 long noncoding RNA(lncRNA),3,500 circular RNA(circRNA)and 1,000 micro RNA(miRNA)were identified.Furthermore,43 mRNA,121 lncRNA and 3 miRNA were differentially expressed,whereas no obvious differentially expressed circRNA were detected between the 2 groups.Gene Ontology(GO)annotation revealed that the differentially expressed genes were mainly enriched in oxidoreductase activity,stress,metabolism,the immune response,cell apoptosis,and cell proliferation.Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis showed that the deferentially expressed genes were highly enriched in the phosphatidylinositol 3 kinase(PI3) K-serine/threonine kinase(AKT)signaling pathway(P<0.05).According to KEGG pathway analysis,the differentially expressed lncRNA(DElncRNA)target genes were mainly related to proteasomes,peroxisomes,and the hypoxia-inducible factor-1 signaling pathway(P<0.005).Further bioinformatics and integrative analyses revealed that the lncRNA were strongly correlated with mRNA;therefore,it is reasonable to speculate that lncRNA potentially play important roles in the liver dysfunction induced by SARA.Our study provides a valuable resource for future investigations on the mechanisms of SARA to facilitate an understanding of the importance of lncRNA,and offer functional RNA information.