Transcriptomic and metagenomic impacts of dietary energy of milk replacer in pre-weaned Holstein heifers

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Virginia Tech


The variability in calf management can change the physiological state of the calf as they are weaned or attain puberty. It is up to the producer to ensure that the calves develop properly to meet their expected needs on the farm. While there are guidelines from the NRC in place, there is a substantial range in the amount of protein and fat that a calf can be fed. This physiological state can be reflected in the proteins produced in tissues, the expression of gene regulatory pathways, or even the microbes present in the gut. The purpose of this study was to examine how an increase in dietary energy in milk replacer of pre-weaned Holstein heifers impacts the microbial profile of the rumen as well as the transcriptome in tissues related to growth and metabolism. Our hypothesis was that pre-weaned Holstein heifers on milk replacer diets with lower dietary energy will have a different rumen microbiome composition and a different transcriptome in growth related tissues.

Holstein heifer calves (n = 36) were assigned randomly to 1 of 2 milk replacer diets: restricted (R; 20.9% CP, 19.8% Fat; n = 18) or enhanced (E; 28.9% CP, 26.2% Fat; n = 18). Calves were euthanized and rumen fluid was collected at pre-weaning (8 wks; n = 6) or post-weaning (10 wks; n = 6). Liver (L), adipose (A), and longissimus dorsi (LD) tissues were collected at pre-weaning (8 wks; n = 12). Average daily gain (ADG) and gain-to-feed ratio (G:F) were calculated for each calf. Analysis of ADG and G:F was performed using a PROC GLM in SAS with diet as the main effect; E calves had increased ADG and G:F compared to R calves.

For rumen samples, libraries were constructed from extracted DNA and DNASeq was conducted using a paired-end analysis at 100 bp using Illumina HiSeq 2500. Operational taxonomic unit (OTU) clustering analysis was conducted using the 16s rRNA Greengenes reference. A PERMANOVA analysis was conducted in R to determine OTU populations for age and treatment. There was no difference in microbiome composition between pre-weaning and post-weaning calves (P = 0.761). Microbiome composition differed between E and R calves (P < 0.001). Bacteroidetes and Firmicutes represented the most abundant phyla for both E and R calves. Enhanced calves had 49.4% (5141 reads) Bacteriodetes and 36.4% (3789 reads) Firmicutes; whereas, R calves had 31.6% (2491 reads) Bacteriodetes and 41.1% (3236 reads) Firmicutes.

For L, A, and LD samples, libraries were constructed from extracted RNA for RNA-Seq analyses. RNA-Seq analysis was performed using CLC Genomics Workbench and the Robinson and Smith Exact Test was used to identify differentially expressed genes between diets. There were 238 differentially expressed genes in A, 227 in LD, and 40 in L. Of the differentially expressed genes, 10 appeared in at least 2 tissues. PANTHER was used to identify functional categories of differentially expressed genes. The majority of genes were associated with metabolic processes (A = 112, 26.7%; L = 16, 32.0%; LD = 81, 34.0%) or cellular processes (A = 93, 22.1%; L = 13, 26.0%; LD = 73, 30.7%). In E calves, upregulated genes included those regulating NADH dehydrogenation (LD = 17, A = 5; i.e. ND1, ND4), gluconeogenesis (LD = 2, A = 6; i.e. ALDOB, PCK2), and cell proliferation (LD = 2, A = 3; i.e. GADD45A, CDKN1A).

There was a difference in both the transcriptome and rumen microbiome of calves fed differing levels of dietary energy. The calves on the R diet had a rumen microbial composition more similar to a younger calf, while the composition of E calves was more similar to a mature calf. The change in regulation of genes involved in the cell cycle and ATP synthesis in response to dietary energy could explain the change in ADG between diets. Because the R calves appeared to have stunted development of their microbiomes and an expression profile similar to oxidative stress, it is possible that the R diet did not meet the nutritional requirements of that calves.



Dairy, Calf management, Rumen microbiome, Transcriptome, Growth and development