Heat stress (HS) causes reductions in milk production, but it is unclear whether this effect is due to reduced number or functional capacity (or both) of mammary cells. Methionine supplementation improves milk protein, whereas Arg is taken up in excess by mammary cells to produce energy and nonessential AA that can be incorporated into milk protein. To evaluate molecular mechanisms by which mammary functional capacity is affected by HS and Met or Arg, mammary alveolar (MAC-T) cells were incubated at thermal-neutral (37°C) or HS (42°C) temperatures. Treatments were optimal AA profiles (control; Lys:Met = 2.9:1.0; Lys:Arg = 2.1:1.0), control plus Met (Lys:Met = 2.5:1.0), or control plus Arg (Lys:Arg = 1.0:1.0). After incubation for 6 h, cells were harvested and RNA and protein were extracted for quantitative real-time PCR and Western blotting. Protein abundance of mechanistic target of rapamycin (MTOR), eukaryotic initiation factor 2a, serine-threonine protein kinase (AKT), 4E binding protein 1 (EIF4EBP1), and phosphorylated EIF4EBP1 was lower during HS. The lower phosphorylated EIF4EBP1 with HS would diminish translation initiation and reduce protein synthesis. Both Met and Arg had no effect on MTOR proteins, but the phosphorylated EIF4EBP1 decreased by AA, especially Arg. Additionally, Met but not Arg decreased the abundance of phosphorylated eukaryotic elongation factor 2, which could be positive for protein synthesis. Although HS upregulated the heat shock protein HSPA1A, the apoptotic gene BAX, and the translation inhibitor EIF4EBP1, the mRNA abundance of PPARG, FASN, ACACA (lipogenesis), and BCL2L1 (antiapoptotic) decreased. Greater supply of Met or Arg reversed most of the effects of HS occurring at the mRNA level and upregulated the abundance of HSPA1A. In addition, compared with the control, supply of Met or Arg upregulated genes related to transcription and translation (MAPK1, MTOR, SREBF1, RPS6KB1, JAK2), insulin signaling (AKT2, IRS1), AA transport (SLC1A5, SLC7A1), and cell proliferation (MKI67). Upregulation of microRNA related to cell growth arrest and apoptosis (miR-34a, miR-92a, miR-99, and miR-184) and oxidative stress (miR-141 and miR-200a) coupled with downregulation of fat synthesis-related microRNA (miR-27ab and miR-221) were detected with HS. Results suggest that HS has a direct negative effect on synthesis of protein and fat, mediated in part by coordinated changes in mRNA, microRNA, and protein abundance of key networks. The positive responses with Met and Arg raise the possibility that supplementation with these AA during HS might have a positive effect on mammary metabolism.

Systems biology is a computational field that has been used for several years across different scientific areas of biological research to uncover the complex interactions occurring in living organisms. Applications of systems concepts at the mammalian genome level are quite challenging, and new complimentary computational/experimental techniques are being introduced. Most recent work applying modern systems biology techniques has been conducted on bacteria, yeast, mouse, and human genomes. However, these concepts and tools are equally applicable to other species including ruminants (e.g., livestock). In systems biology, both bottom-up and top-down approaches are central to assemble information from all levels of biological pathways that must coordinate physiological processes. A bottom-up approach encompasses draft reconstruction, manual curation, network reconstruction through mathematical methods, and validation of these models through literature analysis (i.e., bibliomics). Whereas top-down approach encompasses metabolic network reconstructions using ‘omics’ data (e.g., transcriptomics, proteomics) generated through DNA microarrays, RNA-Seq or other modern high-throughput genomic techniques using appropriate statistical and bioinformatics methodologies. In this review we focus on top-down approach as a means to improve our knowledge of underlying metabolic processes in ruminants in the context of nutrition. We also explore the usefulness of tissue specific reconstructions (e.g., liver and adipose tissue) in cattle as a means to enhance productive efficiency.

The liver is the main metabolic organ coordinating the adaptations that take place during the peripartal period of dairy cows. A successful transition into lactation, rather than management practices alone, depends on environmental factors such as temperature, season of parturition, and photoperiod. Therefore, we analyzed the effect of calving season on the hepatic transcriptome of dairy cows during the transition period. A total of 12 Holstein dairy cows were assigned into 2 groups based on calving season (6 cows March-April, spring; 6 cows June-July, summer, SU). The RNA was extracted from liver samples obtained at -30, 3, and 35 DIM via percutaneous biopsy and hybridized to the Agilent 44K Bovine (V2) Gene Expression Microarray (Agilent Technologies Inc., Santa Clara, CA). A quantitative PCR on 22 target genes was performed to verify and expand the analyses. A total of 4,307 differentially expressed genes were detected (false discovery rate ≤0.05) in SU compared with spring. Furthermore, 73 unique differentially expressed genes were detected in SU compared with spring cows after applying a fold-change threshold ≥3 and ≤-3. For Kyoto Encyclopedia of Genes and Genomes pathways analysis of differentially expressed genes, we used the dynamic impact approach. Ingenuity Pathway Analysis software was used to analyze upstream transcription regulators and perform gene network analysis. Among metabolic pathways, energy metabolism from lipids, carbohydrates, and amino acids was strongly affected by calving in SU, with a reduced level of fatty acid synthesis, oxidation, re-esterification, and synthesis of lipoproteins, leading to hepatic lipidosis. Glycan-synthesis was downregulated in SU cows probably as a mechanism to counteract the progression of this lipidosis. In contrast, calving in the SU resulted in upregulation of gluconeogenesis but also greater use of glucose as an energy source. Among nonmetabolic pathways, the heat-shock response was obviously activated in SU cows but was also associated with inflammatory and intracellular stress response. Furthermore, data support a recent finding that cows experience endoplasmic reticulum stress around parturition. Transcription regulator analysis revealed how metabolic changes are related to important regulatory mechanisms, including epigenetic modification. The holistic analyses of the liver transcriptome response to calving in the summer at high environmental temperatures underscore how transition cows should be carefully managed during this period, as they experience alterations in liver energy metabolism and inflammatory state increasing susceptibility to health disorders in early postpartum.

The combination of domains in multidomain proteins enhances their function and structure but lengthens the molecules and increases their cost at cellular level. The dependence of domain length on the number of domains a protein holds was surveyed for a set of 60 proteomes representing free-living organisms from all kingdoms of life. Distributions were fitted using non-linear functions and fitted parameters interpreted with a formulation of decreasing returns. We find that domain length decreases with increasing number of domains in proteins, following the Menzerath-Altmann (MA) law of language. Highly significant negative correlations exist for the set of proteomes examined. Mathematically, the MA law expresses as a power law relationship that unfolds when molecular persistence P is a function of domain accretion. P holds two terms, one reflecting the matter-energy cost of adding domains and extending their length, the other reflecting how domain length and number impinges on information and biophysics. The pattern of diminishing returns can therefore be explained as a frustrated interplay between the strategies of economy, flexibility and robustness, matching previously observed trade-offs in the domain makeup of proteomes. Proteomes of Archaea, Fungi and to a lesser degree Plants show the largest push towards molecular economy, each at their own economic stratum. Fungi increase domain size in single domain proteins while reinforcing the pattern of diminishing returns. In contrast, Metazoa, and to lesser degrees Protista and Bacteria, relax economy. Metazoa achieves maximum flexibility and robustness by harboring compact molecules and complex domain organization, offering a new functional vocabulary for molecular biology. The tendency of parts to decrease their size when systems enlarge is universal for language and music, and now for parts of macromolecules, extending the MA law to natural systems.

Using published dairy cattle liver transcriptomics dataset along with novel blood biomarkers of liver function, metabolism, and inflammation we have attempted an integrative systems biology approach applying the classical functional enrichment analysis using DAVID, a newly-developed Dynamic Impact Approach (DIA), and an upstream gene network analysis using Ingenuity Pathway Analysis (IPA). Transcriptome data was generated from experiments evaluating the impact of prepartal plane of energy intake [overfed (OF) or restricted (RE)] on liver of dairy cows during the peripartal period. Blood biomarkers uncovered that RE vs. OF led to greater prepartal liver distress accompanied by a low-grade inflammation and larger proteolysis (i.e., higher haptoglobin, bilirubin, and creatinine). Post-partum the greater bilirubinaemia and lipid accumulation in OF vs. RE indicated a large degree of liver distress. The re-analysis of microarray data revealed that expression of >4,000 genes was affected by diet × time. The bioinformatics analysis indicated that RE vs. OF cows had a liver with a greater lipid and amino acid catabolic capacity both pre- and post-partum while OF vs. RE cows had a greater activation of pathways/functions related to triglyceride synthesis. Furthermore, RE vs. OF cows had a larger (or higher capacity to cope with) ER stress likely associated with greater protein synthesis/processing, and a higher activation of inflammatory-related functions. Liver in OF vs. RE cows had a larger cell proliferation and cell-to-cell communication likely as a response to the greater lipid accumulation. Analysis of upstream regulators indicated a pivotal role of several lipid-related transcription factors (e.g., PPARs, SREBPs, and NFE2L2) in priming the liver of RE cows to better face the early postpartal metabolic and inflammatory challenges. An all-encompassing dynamic model was proposed based on the findings.

Colostrum and milk are essential sources of antibodies and nutrients for the neonate, playing a key role in their survival and growth. Slight abnormalities in the timing of colostrogenesis/lactogenesis potentially threaten piglet survival. To further delineate the genes and transcription regulators implicated in the control of the transition from colostrogenesis to lactogenesis, we applied RNA-seq analysis of swine mammary gland tissue from late-gestation to farrowing. Three 2nd parity sows were used for mammary tissue biopsies on days 14, 10, 6 and 2 before (−) parturition and on day 1 after (+) parturition. A total of 15 mRNA libraries were sequenced on a HiSeq2500 (Illumina Inc.). The Dynamic Impact Approach and the Ingenuity Pathway Analysis were used for pathway analysis and gene network analysis, respectively. A large number of differentially expressed genes were detected very close to parturition (−2d) and at farrowing (+ 1d). The results reflect the extraordinary metabolic changes in the swine mammary gland once it enters into the crucial phases of lactogenesis and underscore a strong transcriptional component in the control of colostrogenesis. There was marked upregulation of genes involved in synthesis of colostrum and main milk components (i.e. proteins, fat, lactose and antimicrobial factors) with a pivotal role of CSN1S2, LALBA, WAP, SAA2, and BTN1A1. The sustained activation of transcription regulators such as SREBP1 and XBP1 suggested they help coordinate these adaptations. Overall, the precise timing for the transition from colostrogenesis to lactogenesis in swine mammary gland remains uncharacterized. However, our transcriptomic data support the hypothesis that the transition occurs before parturition. This is likely attributable to upregulation of a wide array of genes including those involved in ‘Protein and Carbohydrate Metabolism’, ‘Immune System’, ‘Lipid Metabolism’, ‘PPAR signaling pathway’ and ‘Prolactin signaling pathway’ along with the activation of transcription regulators controlling lipid synthesis and endoplasmic reticulum biogenesis and stress response.