Effects Of β-hydroxybutyric Acid On Gene Expression And Metabolism Of Rumen Epithelium In Goat Kids

Rumen is the key digestive organ for ruminants to convert roughage into high-quality animal products.At present,there is still insufficient understanding of the timing characteristics and regulation mechanism of rumen development in young ruminants.First,this paper studied the changes of rumen fermentation,tissue morphology and related gene expression of goat kids from birth to weaning,and identified the key nodes of rumen development of goats.Then transcriptome and metabolomics techniques were used to reveal the mechanism of β-hydroxybutyric acid(BHBA)on gene expression and metabolic pattern in rumen epithelium of goat kids,to provide data support for enriching rumen development regulation theory and developing new feed additives in goats.This study is divided into three parts:Experiment 1: study on the law of rumen development and metabolism of goat kids at different ages.A total of 48 goat kids of 1,7,14,28,42,56,70 and 84 days old were selected.The kids from birth to 30 days old were breast-fed with their dams,and the kids were fed with pellet feed at 30 days old.The kids were weaned at 60 days old and only fed with pellets at the age of 60-84 days.The kids were slaughtered at the above eight days old,and the indexes of rumen fermentation,tissue morphology and related gene expression were determined.The results showed that the contents of acetic acid,butyric acid and ammonia nitrogen in rumen of 56-,70-and 84-day-old goats were significantly higher than those of28-and 42-day-old kids(P < 0.05).The length,width,and stratum corneum thickness of rumen papilla in70-and 84-day-old groups were significantly higher than those in 7-,14-and 28-day-old groups(P < 0.05).The expression levels of HMGCS2,HMGCL,BDH1,PPARA,PPARG,SLC16A1 and SLC14A1 in the rumen epithelium of goat kids increased significantly after 28 days of age,and reached the peak at 56 days of age.Experiment 2: effect of β-hydroxybutyric acid on gene expression in rumen epithelium of goat kids Sixty-four 30-day-old goat kids were selected and divided into four groups and fed with 0g,3G,6g and9g/ BHBA additive per day respectively.At the age of 60 days and 90 days,6 kids in each group were slaughtered,and rumen epithelial samples were collected for transcriptional sequencing and RT-PCR detection.The results showed that there were 58,44 and 250 differential genes between group L,M,H and group C at 60 and 90 days old,respectively.The results of GO enrichment of differential genes in 60-day-old kids showed that the differential genes in group L and C were enriched in secretory signal,the regulation of peptidase activity,the positive regulation of cell proliferation and migration,the differential genes in group M and C were enriched in the process of tissue regeneration and intracellular amino acid metabolism,and the differential genes in group H and C were enriched in the process of lactic acid decomposition and purinergic nucleotide receptor signal pathway.The differential genes of group L and C of 90-day-old kids were enriched in half-desmosome assembly and RNA polymerase regulation,while those of group M and C and H and C were enriched in respiratory chain complex I assembly and redox process.The KEGG results showed that the differential genes in 60-day-old kids were enriched in propionic acid metabolism and pyruvate metabolism,while those in 90-day-old kids L and C90 were enriched in linoleic acid metabolism and PPAR signal regulation pathways.The differential genes in M and C groups were enriched in oxidative phosphorylation and cholesterol metabolism,and the differential genomes in H and C groups were enriched in Th17 cell proliferation,differentiation,and purine metabolism.The results of RT-PCR showed that at 60 days of age,the relative expression of SLC16A1 in M group was significantly higher than that in C group,the relative expression of BDH1,HMGCS2 and SLC16A3 in M and H groups was significantly higher than that in C group,while at 90 days old,the relative expression of SLC16A3,PPARA and BDH1 in three groups was significantly higher than that in C group(P < 0.05).The relative expression of SLC16A1 and HMGCS2 in H group was also significantly higher than that in C group.Experiment 3: effect of β-hydroxybutyric acid on rumen epithelial metabolism of goat kidsMetabolomics technique was used to quantitatively detect the metabolites of rumen epithelium.The results of PCA analysis showed that at 60 and 90 days of age,the metabolites of L,M and H groups were significantly different from those of C groups,the differential metabolites of L and C groups mainly included succinic acid,pyruvate and ADP,M and C groups mainly included L-malic acid and decanoic acid,H and C groups included decanoic acid and L-glutamic acid.The differential metabolites of 90-dayold kids in group L and C mainly included palmitic acid and D-glucose,while those in groups M and C,H and C also included butyric acid and aspartyl.The functional enrichment analysis of differential metabolites in 60-day-old kids showed that the differential metabolites in group L and C were mainly enriched in fatty acid oxidation and glycolysis,while those in group M and C were enriched in functional pathways such as glycerol ester metabolism and tricarboxylic acid cycle.The differential metabolites in H and C60 groups were enriched in functional pathways such as electron transport chain and tricarboxylic acid cycle.The differential metabolites in group L and C of 90-day-old kids were mainly enriched in functional pathways such as fatty acid biosynthesis and glycolysis,while those in group M and C were enriched in functional pathways such as fatty acid oxidation and gluconeogenesis.The differential metabolites in group H and C were mainly enriched in fatty acid oxidation,glycolysis,and other biological pathways.To sum up,the rumen development of goat kids matured gradually with the increase of age,and the rumen fermentation ability,tissue morphology and the expression of related lipid metabolism genes increased significantly after 30 days of age.β-hydroxybutyric acid increased the energy production of mitochondrial oxidative phosphorylation by promoting the gene expression of lipid metabolism pathway in rumen epithelium(HMGCS2,BDH1,HMGCL,PPARA,etc.),and stimulated rumen development.The addition of BHBA significantly increased the abundance of sugars,lipid metabolites,amino acids,and nucleotides in rumen epithelial cells.This study will further explore the genes related to the biosynthesis of secondary metabolites during rumen development in kids,and provide candidate genes and corresponding theoretical basis for the regulation of rumen metabolites.