Abstract
BackgroundThe liver is central to most economically important metabolic processes in cattle. However, the changes in expression of genes that drive these processes remain incompletely characterised. RNA-seq is the new gold standard for whole transcriptome analysis but so far there are no reports of its application to analysis of differential gene expression in cattle liver. We used RNA-seq to study differences in expression profiles of hepatic genes and their associated pathways in individual cattle in either mild negative energy balance (MNEB) or severe negative energy balance (SNEB). NEB is an imbalance between energy intake and energy requirements for lactation and body maintenance. This aberrant metabolic state affects high-yielding dairy cows after calving and is of considerable economic importance because of its negative impact on fertility and health in dairy herds. Analysis of changes in hepatic gene expression in SNEB animals will increase our understanding of NEB and contribute to the development of strategies to circumvent it.ResultsRNA-seq analysis was carried out on total RNA from liver from early post partum Holstein Friesian cows in MNEB (n = 5) and SNEB (n = 6). 12,833 genes were deemed to be expressed (>4 reads per gene per animal), 413 of which were shown to be statistically significantly differentially expressed (SDE) at a false discovery rate (FDR) of 0.1% and 200 of which were SDE (FDR of 0.1%) with a ≥2-fold change between MNEB and SNEB animals. GOseq/KEGG pathway analysis showed that SDE genes with ≥2- fold change were associated (P <0.05) with 9 KEGG pathways. Seven of these pathways were related to fatty acid metabolism and unexpectedly included ‘Steroid hormone biosynthesis’, a process which mainly occurs in the reproductive organs rather than the liver.ConclusionsRNA-seq analysis showed that the major changes at the level of transcription in the liver of SNEB cows were related to fat metabolism. 'Steroid hormone biosynthesis', a process that normally occurs in reproductive tissue, was significantly associated with changes in gene expression in the liver of SNEB cows. Changes in gene expression were found in this pathway that have not been previously been identified in SNEB cows.
Highlights
The liver is central to most economically important metabolic processes in cattle
Library preparation and sequencing of polyA mRNA-seq libraries Twelve polyA RNA-seq single read libraries that had been prepared from total RNA extracted from liver from 6 mild negative energy balance (MNEB) animals and 6 severe negative energy balance (SNEB) animals were run as 36 bp single reads on 21 lanes randomly distributed across 3 flowcells (Table 1)
We found that in the liver of cows in negative energy balance (NEB), on average 16% of the total RNA-seq reads aligned to only 10 genes and 30% of the RNA-seq reads mapped to just 619 genes (Figure 1A) per lane
Summary
The liver is central to most economically important metabolic processes in cattle. the changes in expression of genes that drive these processes remain incompletely characterised. Selective breeding for high milk yield in dairy cows has led to breeds in which the nutritional demands of the very high lactation rates following calving are in excess of that which the animal can metabolise from ingested feed [1] This aberrant physiological state is known as negative energy balance (NEB) and is of particular. There are strategies to prevent and treat NEB, such as management of diet and inclusion of dietary supplements [6], [7] NEB and ketosis continue to have a significant negative economic impact in dairy cattle This has led to an effort to understand NEB at the level of genes and their expression [8]
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