Effects of a wide range of dietary forage-to-concentrate ratios on nutrient utilization and hepatic transcriptional profiles in limit-fed Holstein heifers

Abstract

Background: Improving the efficiency of animal production is a relentless pursuit of ruminant producers. Energy utilization and partition can be affected by dietary composition and nutrient availability. Furthermore, the liver is the central metabolic intersection in cattle. However, the specific metabolic changes in the liver under conditions of limit-feeding remain unclear and require further study. The present study aimed to elucidate the effects of a wide range of dietary forage:concentrate ratios (F:C) on energy utilization, and identify potential changes in molecular metabolism by analyzing hepatic transcriptional profiles. Twenty-four half-sib Holstein heifers were fed four F:C diets (20:80, 40:60, 60:40, and 80:20 on a dry matter basis), with similar intake levels of metabolizable energy (ME) and crude protein. Liver biopsy samples were obtained and RNA sequencing was conducted to identify the hepatic transcriptomic changes. Moreover, the ruminal fermentation profiles, growth characteristics, and levels of metabolites in the liver and plasma of the heifers were monitored.

Results: The proportion of acetate showed a linear increase (P < 0.01) with increasing dietary forage levels, whereas the proportion of propionate showed a linear decline (P ≤ 0.01). Lower levels of average daily gain and feed efficiency (P < 0.01) were observed in heifers fed high levels of forage, with a significant linear response. Using the Short Time-series Expression Miner software package, the expression trends of significant differentially expressed genes (DEGs) were generally divided into 20 clusters, according to their dynamic expression patterns. Functional classification analysis showed that lipid metabolism (particularly cholesterol and steroid metabolism which were in line with the cholesterol content in the liver and plasma) was significantly increased with increasing dietary forage levels and slightly reduced by the 80% forage diet. Nine DEGs were enriched in the related pathways, namely HMGCS1, HMGCR, MSMO1, MVK, MVD, IDI1, FDPS, LSS, and DHCR7.

Conclusions: The ruminal fermentation and feed efficiency results suggest that different mechanisms of energy utilization might occur in heifers fed different F:C diets with similar levels of ME intake. Increased cholesterol synthesis from acetate might be responsible for the reduced efficiency of energy utilization in heifers fed high-forage diets.

Keywords: Energy utilization; Forage level; Heifer; Lipid metabolism; Liver; RNA-Seq.

Fig. 1

Comparison of qRT-PCR and RNA-Seq expression ratios for selected genes. Red curves represent results of qRT-PCR; blue curves represent results from RNA sequence. FPKM, fragments per kilobase of exon per million fragments mapped

Fig. 2

Heatmap showing hierarchical clustering of 532 differentially expressed genes among treatments. The log2 ratio values of DEG abundance were used for hierarchical cluster analysis with the R pheatmap package. Red and blue indicate relative over- or under-expression of genes, respectively. Dietary treatments were corn silage-based diets that consisted of 20%, 40%, 60%, and 80% corn silage (on a DM basis, namely the S20, S40, S60, and S80 groups)

Fig. 3

KEGG pathway enrichment analysis of differentially expressed genes (top 14 pathways listed according to P-value)

Fig. 4

Dynamic expression pattern profiles of differentially expressed genes among treatments. Short Time-series Expression Miner (STEM) clustering analysis was performed to identify clusters; each cluster contained various numbers of DEGs with similar expression patterns. The top left-hand corner indicates the cluster ID. The lower left-hand corner contains the P-value of the number of assigned genes compared with the expected value. The black lines show model expression profiles. The red lines represent all individual gene expression profiles. The x-axis represents the dietary corn silage inclusion levels. The time series was log-normalized to start at 0. The y-axes of all genes in a cluster box are at the same scale

Fig. 5

GO analysis of differentially expressed genes (P < 0.05) in profiles 17, 19, and 4. Red bars represent molecular function (MF) terms; blue bars represent biological process (BP) terms; green bars represent cellular component (CC) terms. Asterisks represent significantly enriched terms (FDR < 0.05). GO, Gene Ontology

Fig. 6

Regulation of hepatic cholesterol biosynthesis in different forage-to-concentrate ratios fed to heifers. Major metabolic intermediates are shown in red font and genes are shown in black font. Line charts represent the significant differentially expressed genes associated with cholesterol biosynthesis in this study, and mean values of FPKM are displayed. S20, 20% forage in diets; S40, 40% forage in diets; S60, 60% forage in diets; S80, 80% forage in diets. HMGCS1, 3-hydroxy-3-methylglutaryl-CoA; HMGCR, 3-hydroxy-3-methylglutaryl-CoA reductase; MSMO1, methylsterol monooxygenase 1; MVK, mevalonate kinase; MVD, mevalonate diphosphate decarboxylase; IDI1, isopentenyl-diphosphate delta isomerase 1; FDPS, farnesyl diphosphate synthase; LSS, lanosterol synthase; DHCR7, 7-dehydrocholesterol reductase; FPKM, fragments per kilobase of exon per million fragments mapped