The objective of this study was to characterize the mRNA expression of metabolic and proliferative genes in the rumen epithelium during ruminal acidosis. To meet our objectives, 16 rumen-fistulated, lactating Holstein dairy cattle (618±35 kg of body weight, 221±32 d in milk) were used in a randomized complete block design. All cattle were fed a high-forage diet (HF; 88.9% of dry matter) for 5 wk before the experiment. After the baseline week (wk 0), half of the cattle were randomly assigned and transitioned to a high-concentrate diet (HC; 62.2% of dry matter) which was fed for 3 wk (wk 1, 2, and 3). For the last 48 h of each week, continuous ruminal pH, short-chain fatty acids, and plasma β-hydroxybutyrate were assessed, followed by a rumen papillae biopsy. Milk production was higher in HC cattle compared with HF during wk 1, 2, and 3 (17.4±0.5 vs. 23.4±0.9 kg/d, respectively); however, the mean ruminal pH was decreased (5.75±0.03 vs. 6.30±0.02). The HC cattle spent more time below pH 5.6 (594±54 vs. 3±3 min/d) and displayed greater concentrations of ruminal butyrate (15.8±0.9 vs. 10.2±0.4 mmol) and plasma β-hydroxybutyrate (1,036±63 vs. 778±20 μM) compared with the HF cattle. The mRNA expression of genes involved in ketogenesis (HMGCS2 and PPARA) and short-chain fatty acid transport (MCT1) was unchanged by treatment. However, a downregulation in HMGCS1 (0.72±0.09), one of the cholesterol biosynthesis genes, was observed in HC cattle during wk 1 of the grain challenge. In addition, the relative mRNA expression value of insulin-like growth factor-binding protein 3 was lower (0.78±0.06), whereas insulin-like growth factor-binding protein 5 was higher (1.79±0.15) in HC compared with HF cattle. These results suggest that grain-induced ruminal acidosis alters the mRNA expression of IGF-binding proteins and a cholesterolgenic enzyme in the rumen epithelium of lactating dairy cattle.

To determine how glucose modulates protein synthesis when essential AA are in abundant supply, 5 early-lactation, rumen-fistulated Holstein dairy cows were fed a diet containing 6.95 MJ/kg of net energy for lactation and 12.4% crude protein and abomasally infused for 5 d with saline, 844 or 1,126 g/d of a complete essential AA mix, with and without the inclusion of 1,000 g/d of glucose, in a 5×5 Latin square design. Infusion of essential AA increased milk yield by 4.1 kg/d, milk protein by 256 g/d, milk fat by 95 g/d, and milk urea nitrogen by 70% compared with saline, with no differences between the level of essential AA infusion. The addition of glucose to essential AA infusate did not stimulate milk protein yield or concentration, but reduced milk urea nitrogen by 17% and decreased milk fat yield. Arterial concentrations of total essential AA increased 3- to 4-fold, mammary clearance decreased 61%, and mammary uptake of essential AA increased 65% in response to essential AA infusion. Arterial branched-chain AA concentrations declined 29% in response to glucose and mammary clearance increased 48%, but mammary AA uptake was unchanged. Essential AA infusion increased plasma 3-methylhistidine by 50% and reduced muscle branched-chain α-keto acid dehydrogenase kinase abundance by 14%, indicating stimulation of muscle protein turnover and branched-chain AA catabolism, respectively. Glucose had no further effect on muscle branched-chain α-keto acid dehydrogenase kinase abundance but decreased mRNA expression of branched chain aminotransferase 1. Lack of further increases in plasma 3-methylhistidine or greater stimulation of muscle branched-chain AA catabolism indicates that muscle protein degradation was unchanged with glucose but that accretion may have been stimulated. The decrease in circulating branched-chain AA concentrations and nitrogen excretion in response to glucose suggests that surplus essential AA were redirected to peripheral, extra-mammary tissues.

The phosphorylation of mammalian target of rapamycin complex 1 (mTORC1) components and integrated stress response networks in the mammary glands of lactating cows have not accounted for the stimulation of milk protein yield by chronic supplementation with AA or glucose. Faster milk protein synthesis could be a consequence of increased milk protein mRNA per cell, the number of ribosomes per cell, the secretory capacity of cells, or the mammary cell number. To investigate these 4 possibilities using a translational and transcriptional approach, we performed protein and gene expression analyses of mammary and longissimus dorsi tissue collected from lactating dairy cows after 5 d of abomasal infusion with saline or 844 or 1,126 g/d of an essential AA (EAA) mixture, with and without 1,000 g/d glucose. Infusion with EAA increased milk protein yield but did not affect the phosphorylation of mTORC1-related proteins in the mammary gland. In skeletal muscle, phosphorylation of 4EBP1 (eIF4E-binding protein 1) increased in response to both EAA and glucose, and phosphorylated S6K1 (70-kDa ribosomal protein S6 kinase) increased with glucose. In response to EAA, mammary mRNA expression of the marker genes for milk proteins, ribosome biogenesis, and cell proliferation were not upregulated. Instead, reciprocal regulation of 2 arms of the unfolded protein response occurred. Infusion of EAA for 5 d activated XBP1 (X-box binding protein 1) mRNA, encoding a transcription factor for endoplasmic reticulum biogenesis, and it decreased the mRNA expression of genes encoding pro-apoptotic protein CHOP (C/EBP homologous protein) and downstream GADD34 (growth arrest and DNA damage-inducible 34). These findings implicate non-stress-related, adaptive capabilities of the unfolded protein response in the long-term nutritional regulation of milk protein yield in lactating dairy cows.

Essential amino acid (EAA) deficiencies and imbalances were created in lactating cows by using an infusion subtraction protocol to explore effects on milk protein yield and activity state of regulators of mRNA translation in the mammary glands. Six lactating cows on a diet of 11.2% protein were infused abomasally for 5d with saline, 563g/d of a complete EAA mix, or EAA without His, Met, Phe, or Trp in a 6×6 Latin square design. Infusion of complete and imbalanced EAA solutions increased mammalian target of rapamycin (mTOR) signaling in the mammary glands, as evidenced by higher ribosomal S6 kinase 1 (S6K1) phosphorylation compared with saline infusion. Total S6K1 abundance was decreased by imbalanced AA infusions. Except for the mixture lacking Phe, infusion of EAA, whether imbalanced or not, increased abundance of total eukaryotic initiation factor 2Bε (eIF2Bε). A correlation of 0.33 between phosphorylation state of S6K1 and total eIF2Bε abundance suggests that an mTOR-mediated upregulation of eIF2Bε translation occurred. Despite increased mTOR/eIF2Bε signaling, milk protein yields increased only with the complete EAA mixture compared with saline. Low plasma concentrations of His, Met, and Phe during their respective imbalances likely interfered with protein synthesis. Total abundance and phosphorylation state of eukaryotic initiation factor 2α were not responsible for the interference. Further study of eIF2Bε as a regulator of milk protein yield is warranted.