Varying dietary protein and fat elicits differential transcriptomic expression within stress response pathways in preweaned Holstein heifers

Abstract

Increases in milk replacer dietary energy subsequently increase growth and weight in preweaned dairy heifers. However, the underlying effects of dietary component increases on key functional pathways have yet to be fully investigated. Elucidating these relationships may provide insights into the mechanisms through which protein and fat are partitioned for tissue growth and metabolism. We hypothesized that genes within key growth and metabolic pathways would be differentially expressed between calves fed a proteinand fat-restricted diet and calves fed a protein-and fat-enhanced diet. The objectives of this study were to (1) identify genes differentially expressed between dietary restricted calves and enhanced calves and (2) determine the key regulatory pathways influenced by these genes. Preweaned Holstein heifers (n = 12; 6 ± 0.02 d of age) were randomly assigned to 1 of 2 milk replacer diets: enhanced (28.9% crude protein, 26.2% fat; n = 6) or restricted (20.9% crude protein, 19.8% fat; n = 6). Growth measures included average daily gain and gain-to-feed ratio. After 56 d, calves were killed for tissue collection. Samples from longissimus dorsi, adipose, and liver tissues were collected and RNA was isolated for RNA sequencing analysis. The MIXED procedure of SAS (SAS Institute Inc., Cary, NC) was used to evaluate relationships of growth with dietary energy. Fixed effects included date of collection and time (day). Random effects included sire and birth weight. The RNA sequencing analysis was performed using CLC Genomics Workbench (Qiagen, Germantown, MD), and the Robinson and Smith exact test was used to identify differentially expressed genes between diets. The Protein Analysis Through Evolutionary Relationships (PANTHER) database was then used to identify functional categories of differentially expressed genes. Enhanced calves had increased growth rates and feed efficiency compared with restricted calves (average daily gain = 0.76 and 0.22, respectively; gain-to-feed ratio = 0.10 and 0.06, respectively). There were 238 differentially expressed genes in adipose, 227 in longissimus dorsi, and 40 in liver. We identified 10 genes concordant among tissues. As expected, functional analyses suggested that the majority of genes were associated with metabolic or cellular processes, predominantly cell communication and cell cycle. Overall, it appears that varying levels of dietary protein and fat influence calf growth and development through metabolic processes, including oxidative phosphorylation and glyceroneogenesis. However, protein-and fat-restricted calves appeared to experience metabolic stress at a cellular level, as evidenced by an upregulation in stress response pathways, including genes in the p53 pathway. Calves could be fed at a higher level of protein and fat to decrease the prevalence of metabolic stress at the cellular level, but evidence indicating the presence of inflammatory stress and adipose fibrosis in enhanced calves prompts further investigation of the effects of milk replacer component levels.