The onset of lactation in dairy cows is characterized by severe negative energy and protein balance. Methionine availability during this time for milk production, hepatic lipid metabolism, and immune function may be limiting. Supplementing Met to peripartal diets with adequate Lys in metabolizable protein (MP) to fine-tune the Lys:Met ratio may be beneficial. Fifty-six multiparous Holstein cows were fed the same basal diet from 50 d before expected calving to 30 d in milk. From -50 to -21 d before expected calving, all cows received the same diet [1.24 Mcal/kg of dry matter (DM), 10.3% rumen-degradable protein, and 4% rumen-undegradable protein] with no Met supplementation. From -21 d to expected calving, the cows received diets (1.54 Mcal/kg of DM, 10% rumen-degradable protein, and 5.1% rumen-undegradable protein) with no added Met (control, CON; n=14), CON plus MetaSmart (MS; Adisseo Inc., Antony, France; n=12), or CON plus Smartamine M (SM; Adisseo Inc.; n=12). From calving through 30 d in milk, the cows received the same postpartum diet (1.75 Mcal/kg of DM and 17.5% CP; CON), or the CON plus MS or CON plus SM. The Met supplements were adjusted daily and top-dressed over the total mixed ration at a rate of 0.19 or 0.07% (DM) of feed for MS or SM. Liver tissue was collected on -10, 7, and 21 d, and blood samples more frequently, from -21 through 21 d. Data were analyzed using the MIXED procedure of SAS (SAS Institute Inc., Cary, NC) with the preplanned contrasts CON versus SM + MS and SM versus MS. No differences in prepartal DM intake (DMI) or body condition score were observed. After calving, body condition score was lower (2.6 vs. 2.8), whereas DMI was greater (15.4 vs. 13.3 kg/d) for Met-supplemented cows. Postpartal diet × time interactions were observed for milk fat percentage, milk fat yield, energy-corrected milk:DMI ratio, and energy balance. These were mainly due to changes among time points across all treatments. Cows supplemented with either Met source increased milk yield, milk protein percentage, energy-corrected milk, and milk fat yield by 3.4 kg/d, 0.18% units, 3.9 kg/d, and 0.18 kg/d, respectively. Those responses were associated with greater postpartum concentration of growth hormone but not insulin-like growth factor 1. There was a diet × time effect for nonesterified fatty acid concentration due to greater values on d 7 for MS; however, liver concentration of triacylglycerol was not affected by diet or diet × time but increased postpartum. Blood neutrophil phagocytosis at 21 d was greater with Met supplementation, suggesting better immune function. Supplemental MS or SM resulted in a tendency for lower incidence of ketosis postpartum. Although supplemental MS or SM did not decrease liver triacylglycerol, it improved milk production-related traits by enhancing voluntary DMI.

Osorio JS, Lohakare J, Bionaz M. Biosynthesis of milk fat, protein, and lactose: roles of transcriptional and posttranscriptional regulation.

Mechanisms regulating subcutaneous adipose tissue (SAT) insulin sensitivity and gene network expression during the peripartal period were evaluated in cows fed to meet or exceed prepartal energy requirements. Holstein cows were dried off at -50 d relative to expected parturition and fed a controlled-energy diet [CON; net energy for lactation=1.24 Mcal/kg of dry matter (DM); 36% of DM as wheat straw] until -21 d. Cows were then randomly assigned (n=7/diet) to either the same CON diet or a moderate-energy close-up diet (OVE; net energy for lactation=1.47 Mcal/kg of DM) until parturition. Biopsies of SAT were harvested at -10, 7, and 21 d for mRNA expression of 48 genes associated with insulin signaling, adipogenesis, and lipolysis. In vitro basal and insulin-stimulated insulin receptor substrate 1 tyrosine phosphorylation (IRS1-PY) was assessed at -10 and 7 d. The OVE led to more positive energy balance and greater serum insulin concentration prepartum. Compared with CON, OVE led to a more drastic increase in serum NEFA and also greater overall serum BHBA postcalving, both of which were associated with greater hepatic total lipid and triacylglycerol concentration. Close-up OVE did not improve any aspect of performance. In prepartal SAT, insulin-stimulated IRS1-PY was greater in OVE than in CON. However, IRS1-PY, serum insulin, and GLUT4 expression decreased postpartum regardless of prepartal treatment, suggesting a more severe state of insulin resistance. The expression of all genes encoding adipogenic regulators (PPARG and ZFP423), most lipogenic enzymes/inducers (FASN, SCD, DGAT2, and INSIG1), and basal-lipolysis regulators (ATGL and ABDH5) was greater at -10 d in OVE than in CON. Whereas adipogenic and basal lipolysis regulator expression remained greater in cows fed OVE by 7 d postpartum, expression of all lipogenic enzymes decreased regardless of diet. Despite those responses, the approximately 3-fold increase in expression of IRS1 and ZFP423 between 7 and 21 d suggested that insulin responsiveness and adipogenic capacity of SAT were partially restored. Expression of the preadipocyte marker DLK1, adiponutrin (PNPLA3), and fibroblast growth factor 21 (FGF21) was undetectable. Results suggested that close-up energy overfeeding did not exacerbate insulin resistance in SAT. Signs of restored insulin responsiveness (upregulation of IRS1, INSIG2, SREBF1, and ZFP423) were apparent as early as 3 wk postpartum. Thus, identifying specific nutrients capable of activating PPARγ after calving in AT might help accelerate its replenishment. A regulatory network encompassing the genes and physiological measurements obtained is proposed.

The peripartal dairy cow experiences a state of reduced liver function coupled with increased inflammation and oxidative stress. This study evaluated the effect of supplementing basal diets with rumen-protected Met in the form of MetaSmart (MS) or Smartamine M (SM) (both from Adisseo Inc., Antony, France) during the peripartal period on blood and hepatic biomarkers of liver function, inflammation, and oxidative stress. Thirty-seven multiparous Holstein cows were fed the same basal diet from -50 to -21 d relative to expected calving [1.24 Mcal/kg of dry matter (DM); no Met supplementation]. From -21 d to calving, the cows received diets (1.54 Mcal/kg of DM) with no added Met (control, CON; n=13), CON plus MS (n=11), or CON plus SM (n=13). From calving through 30 d in milk (DIM), the cows received the same postpartal diet (1.75 Mcal/kg of DM; CON), or CON plus MS or CON plus SM. Liver and blood samples were harvested at various time points from -21 to 21 d relative to calving. Preplanned contrasts of CON versus SM + MS during prepartum (-21 and -10 d before calving) and postpartum (7, 14, and 21 d after calving) responses were evaluated. Cows fed MS or SM compared with CON had lower overall concentrations of plasma ceruloplasmin and serum amyloid A (SAA). Compared with CON, Met-supplemented cows had greater overall plasma oxygen radical absorbance capacity. Liver concentrations of glutathione and carnitine also were greater overall with Met supplementation. Milk choline and liver phosphatidylcholine were lower overall in cows fed Met compared with controls. Liver tissue choline concentrations did not differ. Data indicate that supplemental Met enhanced de novo glutathione and carnitine synthesis in liver and, thus, increased antioxidant and β-oxidation capacity. The greater decrease of IL-6 after calving coupled with lower ceruloplasmin and SAA in Met-supplemented cows indicated a reduction in proinflammatory signaling within liver. The lower hepatic phosphatidylcholine in Met-supplemented cows might have been associated with greater assembly or export of very low density lipoproteins. Overall, biomarker analyses in blood and tissue indicate that the beneficial effect of feeding SM and MS on postpartal cow performance is due in part to a better immunometabolic status.

Nutrigenomics in dairy cows is a relatively new area of research. It is defined as the study of the genomewide influences of nutrition altering the expression of genes. Dietary compounds affect gene expression directly or indirectly via interactions with transcription factors. Among those, the most relevant for nutrigenomics are ligand-dependent nuclear receptors, especially peroxisome proliferator-activated receptors (PPAR) and liver X receptor. Among other transcription factors, a prominent nutrigenomic role is played by the sterol regulatory element-binding protein 1 (SREBP1). Data from studies on dairy cows using gene expression and gene reporters among the main molecular methods used to study nutrigenomics in dairy cows are indicative of a network of multiple transcription factors at play in controlling the nutrigenomic responses. Fatty acids, AA, and level of feed and energy intake have the strongest nutrigenomic potential. The effect of 10,12 CLA on depressing milk fat synthesis via inhibition of SREBP1 was among the first and likely the best-known nutrigenomic example in dairy cows. Although long-chain fatty acids (LCFA) are clearly the most potent, a nutrigenomic role for short-chain fatty acids is emerging. Available data indicate that saturated compared with unsaturated LCFA have a more potent nutrigenomic effect in vitro, likely through PPAR. In vivo, the effect of saturated LCFA is more modest, with contrasting effects among tissues. Nutrigenomic effects of AA are emerging, particularly for the regulation of milk protein synthesis-associated genes. The level of energy in the diet has a strong and broad nutrigenomic effect and appears to "prime" tissue metabolism, particularly liver. We are at the frontier of the nutrigenomics era in ruminants and initial data strongly indicate that this scientific branch (and spinoffs such as nutriepigenomics) can play a critical role in future strategies to better feed dairy cattle.

Peripartal cows likely require greater amounts of Met not only at the tissue and cell level for methylation reactions but also for milk protein synthesis after calving. Thirty-nine Holstein cows were fed throughout the peripartal period (-21 d to 30 d in milk) a basal control (CON) diet (n=14) with no Met supplementation, CON plus MetaSmart (MS; Adisseo Inc., Antony, France; n=12), or CON plus Smartamine M (SM; Adisseo Inc.; n=13). The Met supplements were adjusted daily and top-dressed over the total mixed ration at a rate of 0.19 or 0.07% (dry matter) of feed for MS or SM. Liver tissue was collected on -10, 7, and 21 d for transcriptome profiling of genes associated with Met and glutathione metabolism as well as components of the inflammation, oxidative stress, growth hormone/insulin-like growth factor-1 axis, and DNA methylation pathways. Data were analyzed using PROC MIXED of SAS (SAS Institute Inc., Cary, NC) with the preplanned contrasts CON versus SM + MS and SM versus MS. The S-adenosylhomocysteine hydrolase (SAHH) gene was the most abundant among all genes evaluated, with overall greater expression in Met-supplemented cows than CON, and in SM than MS. Expression of Met adenosyltransferase 1A (MAT1A) was greater in Met-supplemented cows than CON by 21 d postpartum. A greater overall expression of 5-methyltetrahydrofolate-homocysteine methyltransferase (MTR) occurred in Met-supplemented cows than CON. In contrast, the expression of glutathione synthase (GSS); glutamate-cysteine ligase, catalytic subunit (GCLC); and superoxide dismutase 1, cytosolic (SOD1) was lower in Met-supplemented cows than CON. A greater overall expression of nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 (NFKB1) and greater upregulation of haptoglobin (HP) on d 7 occurred in Met-supplemented cows than CON. Expression of DNA cytosine-5-methyltransferase 3 alpha (DNMT3A) was greater but expression of DNMT1 was lower in Met-supplemented cows than CON. The response observed in SAHH reflects its importance to Met supplementation during the peripartum period. Despite greater HP expression after calving, the lower expression of glutathione (GSS and GCLC) metabolism genes and SOD1 due to Met reflect a lower oxidative stress and mild inflammatory status. The extent to which changes in expression of DNMT3A and DNMT1 result in epigenetic effects partly responsible for the previously observed enhanced performance in Met-supplemented cows remains to be examined. Increasing the supply of Met as SM or MS can affect expression of genes in the Met cycle to various extents and, hence, the supply of methyl donors such as S-adenosylmethionine and antioxidants such as glutathione. These compounds likely are in high demand during the peripartum period.

Organic trace mineral (ORG) supplementation to dairy cows in substitution of sulfate (INO) sources has been associated with improvement in immune function during stressful states such as the peripartal period. However, the effect of supplemental ORG during pregnancy on the neonatal calf is unknown. Therefore, our aim was to investigate the effects of ORG supplementation during late pregnancy on the immune system and growth of the neonatal calf. Of specific interest was the evaluation of inflammation-related microRNA (miRNA) and target gene expression in blood neutrophils as indicators of possible nutritional programming. Forty multiparous cows were supplemented for 30d prepartum with 40 mg/kg of Zn, 20 mg/kg of Mn, 5 mg/kg of Cu, and 1mg/kg of Co from either organic (ORG) or sulfate (INO) sources (total diet contained supplemental 75 mg/kg of Zn, 65 mg/kg of Mn, 11 mg/kg of Cu, and 1 mg/kg of Co, and additional Zn, Mn, and Co provided by sulfates), and a subset of calves (n=8/treatment) was used for blood immunometabolic marker and polymorphonuclear leukocyte (PMNL) gene and miRNA expression analyses. Samples were collected at birth (before colostrum feeding), 1d (24 h after colostrum intake), and 7 and 21d of age. Data were analyzed as a factorial design with the PROC MIXED procedure of SAS. No differences were detected in BW, but maternal ORG tended to increase calf withers height. Calves from INO-fed cows had greater concentrations of blood glucose, GOT, paraoxonase, myeloperoxidase, and reactive oxygen metabolites. Antioxidant capacity also was greater in INO calves. The PMNL expression of toll-like receptor pathway genes indicated a pro-inflammatory state in INO calves, with greater expression of the inflammatory mediators MYD88, IRAK1, TRAF6, NFKB, and NFKBIA. The lower expression of miR-155 and miR-125b in ORG calves indicated the potential for maternal organic trace minerals in regulating the PMNL inflammatory response at least via alterations in mRNA and miRNA expression. Overall, these results indicate that maternal nutrition with organic trace minerals could alter the neonatal innate immune response at least in part via changes in gene and miRNA expression. Further studies involving inflammatory challenges during the neonatal period should be performed to determine the functional benefit of maternal organic trace minerals on the neonatal immune response.

The physiologic and metabolic stresses that dairy cows experience during the transition into early lactation can promote oxidative stress, inflammation, and immune dysfunction. Optimal supply of micronutrients such as trace minerals (e.g., Zn, Mn, Cu, and Co) via more bioavailable forms (e.g., AA complexes) might minimize these negative effects. Multiparous Holstein cows were enrolled at 60 d before dry-off (~110 d before calving) and remained on experiment until 30 d in milk (DIM). Cows were offered a common diet supplemented entirely with inorganic trace minerals (INO) from -110 to -30 d before calving. From -30 to calving cows received a common prepartal [1.5 Mcal/kg of dry matter (DM), 15% crude protein] diet, and from calving to 30 DIM a common postpartal (1.76 Mcal/kg of DM, 18% crude protein) diet. Both diets were partially supplemented with an INO mix of Zn, Mn, and Cu to supply 35, 45, and 6 mg/kg, respectively, of the total diet DM. Cows were assigned to treatments in a randomized complete block design to receive an oral bolus with a mix of INO (n=21) or organic AA complexes (AAC; n=16) of Zn, Mn, Cu, and Co to achieve supplemental levels of 75, 65, 11, and 1mg/kg, respectively, in the total diet DM. Inorganic trace minerals were provided in sulfate form and AAC were supplied via Availa Zn, Availa Mn, Availa Cu, and COPRO (Zinpro Corp., Eden Prairie, MN). Liver tissue was harvested on -30, -15, 10, and 30 d, and blood samples for biomarker analyses were obtained more frequently from -30 to 30 DIM. Short-term changes in blood ketones were measured via Precision Xtra (Abbott Diabetes Care, Alameda, CA) every other day from 1 to 15 d postpartum. Prepartal DM intake was lower in AAC cows. In contrast, a tendency for a diet by time (D × T) interaction resulted in greater postpartal DM intake of approximately 2 kg/d in cows fed AAC. Milk and milk protein yield had a D × T interaction because AAC cows produced approximately 3.3 kg/d more milk and 0.14 kg/d more protein during the first 30 DIM. Although blood glucose, fatty acids, and liver triacylglycerol were not affected by diet, the Precision Xtra ketones (1.44 vs. 2.18 mmol/L) and γ-glutamyltransferase (liver function biomarker) were lower in AAC than INO. Furthermore, feeding AAC increased (D × T) polymorphonuclear neutrophilic lymphocyte phagocytosis, antioxidant capacity postpartum, and overall concentration of liver tissue Co and Cu. Overall, the positive response in milk yield and milk protein in AAC cows might be partly explained by the beneficial effects of AAC on postpartal DM intake driven at least in part by better liver and immune function as a result of improved antioxidant status.