Mammalian target of rapamycin signaling and ubiquitin proteasome–related gene expression in 3 different skeletal muscles of colostrum- versus formula-fed calves

The rates of protein turnover are higher during the neonatal period than at any other time in postnatal life. The mammalian target of rapamycin (mTOR) and the ubiquitin-proteasome system are key pathways regulating cellular protein turnover. The objectives of this study were (1) to elucidate the effect of feeding colostrum versus milk-based formula on the mRNA abundance of key components of the mTOR pathway and of the ubiquitin-proteasome system in skeletal muscle of neonatal calves and (2) to compare different muscles. German Holstein calves were fed either colostrum (COL; n = 7) or milk-based formula (FOR; n = 7) up to 4 d of life. The nutrient content in formula and colostrum was similar, but formula had lower concentrations of free branched-chain AA (BCAA) and free total AA, insulin, and insulin-like growth factor (IGF)-I than colostrum. Blood samples were taken from d 1 to 4 before morning feeding and before and 2 h after the last feeding on d 4. Muscle samples from M. longissimus dorsi (MLD), M. semitendinosus (MST), and M. masseter (MM) were collected after slaughter on d 4 at 2 h after feeding. The preprandial concentrations of free total AA and BCAA, insulin, and IGF-I in plasma changed over time but did not differ between groups. Plasma free total AA and BCAA concentrations decreased in COL, whereas they increased in FOR after feeding, resulting in higher postprandial plasma total AA and BCAA concentrations in FOR than in COL. Plasma insulin concentrations increased after feeding in both groups but were higher in COL than in FOR. Plasma IGF-I concentrations decreased in COL, whereas they remained unchanged in FOR after feeding. The mRNA abundance of mTOR and ribosomal protein S6 kinase 1 (S6K1) in 3 different skeletal muscles was greater in COL than in FOR, whereas that of eukaryotic translation initiation factor 4E binding protein 1 (4EBP1) was unaffected by diet. The mRNA abundance of ubiquitin activating enzyme (UBA1) and ubiquitin conjugating enzyme 1 (UBE2G1) enzymes was not affected by diet, whereas that of ubiquitin conjugating enzyme 2 (UBE2G2) was greater (MLD) or tended to be greater (MM) in COL than in FOR. The mRNA abundance of atrogin-1 in MLD and MST was lower in COL than in FOR, whereas that of muscle ring finger protein-1 (MuRF1) was greater (MST) or tended to be greater (MLD). The abundance of MuRF1 mRNA was highest in MST, followed by MLD, and was lowest in MM. The results indicate that colostrum feeding may stimulate protein turnover that may result in a high rate of protein deposition in a muscle type–specific manner. Such effects seem to be mediated by the postprandial increase in plasma insulin.     

Maturation of endogenous glucose production in preterm and term calves

Glucose disposability is often impaired in neonatal calves and even more in preterm calves. The objective of this study was to investigate ontogenic maturation of endogenous glucose production (eGP) in calves and its effects on postnatal glucose homeostasis. Calves (n = 7 per group) were born preterm (PT; delivered by section 9 d before term) or at term (T; spontaneous vaginal delivery), or spontaneously born and fed colostrum for 4 d (TC). Blood samples were taken immediately after birth and before and 2 h after feeding at 24 h after birth (PT; T) or on d 4 of life (TC) to determine metabolic and endocrine changes. After birth (PT and T) or on d 3 of life (TC), fasted calves were gavaged with deuterium-labeled water to determine gluconeogenesis (GNG) and intravenously infused with [U¹³C]-glucose to measure eGP and glucose oxidation (GOx) in blood plasma. After slaughter at 26 h after birth (PT, T) or on d 4 of life (TC), glycogen concentrations in liver and hepatic mRNA concentrations and enzyme activities of pyruvate carboxylase, phosphoenolpyruvate carboxykinase (PEPCK), and glucose-6-phosphatase were measured. Preterm calves had the lowest plasma concentrations of cortisol and 3,5,3′-triiodothyronine at birth. Plasma glucose concentrations from d 1 to 2 decreased more, but plasma concentrations of lactate and urea and glucagon:insulin ratio were higher in PT than in T and TC calves. The eGP, GNG, GOx, as well as hepatic glycogen concentrations and PEPCK activities, were lowest in PT calves. Results indicate impaired glucose homeostasis due to decreased eGP in PT calves and maturation of eGP with ontogenic development.     

Changes in tissue abundance and activity of enzymes related to branched-chain amino acid catabolism in dairy cows during early lactation

Branched-chain α-keto acid dehydrogenase (BCKDH) complex catalyzes the irreversible oxidative decarboxylation of branched-chain α-keto acids. This reaction is considered as the rate-limiting step in the overall branched-chain amino acid (BCAA) catabolic pathway in mammals. For characterizing the potential enzymatic involvement of liver, skeletal muscle, adipose tissue (AT), and mammary gland (MG) in BCAA metabolism during early lactation, tissue and blood samples were examined on d 1, 42, and 105 after parturition from 25 primiparous Holstein cows. Serum BCAA profiles were analyzed and the mRNA and protein abundance as well as the activity in the different tissues were assessed for the BCAA catabolic enzymes, partly for the branched-chain aminotransferase and completely for BCKDH. Total BCAA concentration in serum was lowest on d 1 after parturition and increased thereafter to a steady level for the duration of the experiment. Pronounced differences between the tissues were observed at all molecular levels. The mRNA abundance of the mitochondrial isoform of branched-chain aminotransferase (BCATm) was greatest in AT as compared with the other tissues studied, indicating that AT might be an important contributor in the initiation of BCAA catabolism in dairy cows. From the different subunits of the BCKDH E1 component, only the mRNA for the β polypeptide (BCKDHB), not for the α polypeptide (BCKDHA), was elevated in liver. The BCKDHA mRNA abundance was similar across all tissues except muscle, which tended to lower values. Highest BCKDHA protein abundance was observed in both liver and MG, whereas BCKDHB protein was detectable in these tissues but could not be quantified. Adipose tissue and muscle only displayed abundance of the α subunit, with muscle having the lowest BCKDHA protein of all tissues. We found similarities in protein abundance for both BCKDH E1 subunits in liver and MG; however, the corresponding overall BCKDH enzyme activity was 7-fold greater in liver compared with MG, allowing for hepatic oxidation of BCAA transamination products. Reduced BCKDH activity in MG associated with no measurable activity in AT and muscle may favor sparing of BCAA for the synthesis of the different milk components, including nonessential AA. Deviating from previously published data on BCAA net fluxes and isotopic tracer studies in ruminants, our observed results might in part be due to complex counter-regulatory mechanisms during early lactation.     

Effects of colostrum feeding on the mRNA abundance of genes related to toll-like receptors,key antimicrobial defense molecules,and tight junctions in the small intestine of neonatal dairy calves

The objective of the present study was to elucidate the effect of feeding either colostrum or milk-based formula on the mRNA abundance of genes related to pathogen recognition [toll-like receptors (TLR1–10)], antimicrobial defense [β-defensin 1 (DEFB1) and peptidoglycan recognition protein 1 (PGLYRP1)], and tight junctions (claudin 1 = CLDN1, claudin 4 = CLDN4, and occludin = OCLN) in different sections of the small intestine of neonatal calves at d 4 of life. Holstein dairy calves were fed either colostrum (COL; n = 7) or milk-based formula (FOR; n = 7) with comparable nutrient composition but lower contents of several bioactives in the formula than in the respective colostrum group until d 4 of life. Following euthanasia on d 4 (2 h after feeding), tissue samples from the duodenum, jejunum (proximal, middle, and distal), and ileum were collected. The mRNA abundance of the target genes was quantified by quantitative PCR. The mRNA abundance of TLR1, TLR6, TLR9, and TLR10 were greater in COL than in FOR calves. However, the mRNA abundance of TLR2, TLR3, TLR4, TLR5, and TLR7 did not differ between groups. A group × gut region interaction was observed for the mRNA abundance of TLR8 with greater values in duodenum and proximal jejunum of COL than in FOR calves but in the more distal regions, in mid and distal jejunum, and ileum, this diet effect disappeared or was reversed. We observed greater mRNA abundance of TLR1 in the jejunum (middle and distal) and ileum, TLR2, TLR4, TLR6, and TLR9–10 in the distal jejunum and ileum, and of TLR3 in the distal jejunum, and TLR5, TLR7, and TLR8 in the ileum compared with the other gut regions. The mRNA abundance of PGLYRP1, DEFB1, and OCLN did not differ between groups. The mRNA abundance of CLDN1 was greater, but the CLDN4 mRNA tended to be lower in COL than in FOR calves. The mRNA abundance of PGLYRP1 was lower in the distal jejunum and DEFB1 mRNA in the middle jejunum compared with the other gut regions. The mRNA abundances of OCLN and CLDN4 were greater in the duodenum, and of CLDN1 in the middle and proximal jejunum compared with the other gut regions. Overall, the greater mRNA abundance of 5 different TLR, and CLDN1 in most intestinal sections of the COL calves may suggest that feeding colostrum improves immune responsiveness and epithelial barrier function in neonatal calves.     

Effects of branched-chain amino acids on glucose uptake and lactose synthesis rates in bovine mammary epithelial cells and lactating mammary tissue slices

Even though supplementations of essential AA (EAA) are often related to increased lactose yields in dairy cows, underlying mechanisms connecting EAA availability to the mammary glands and lactose synthesis are poorly understood. The objective of this study was to examine the effects of branched-chain AA (BCAA) including Leu, Ile, and Val on (1) glucose transporter (GLUT1) abundance and glucose uptake, (2) the abundance of proteins regulating lactose synthesis pathway, and (3) fractional synthesis rates of lactose (FSR) using bovine mammary epithelial cells (BMEC) and mammary tissues slices (MTS). The BMEC (n = 4) were allocated randomly to regular Dulbecco's Modified Eagle Medium with Ham's F12 (DMEM/F12) medium (+EAA) or +EAA deficient (by 90%) in all EAA (?EAA), all BCAA (?BCAA), only Leu (?Leu), only Ile (?Ile) or only Val (?Val). Western immunoblotting analyses, depletion of glucose in media, and a proteomic analysis were performed to determine the abundance of GLUT1 in the cell membrane, net glucose uptake, and the abundance of enzymes involved in lactose synthesis pathway in BMEC, respectively. The MTS (n = 6) were allocated randomly to DMEM/F12 medium having all EAA and ¹³C-glucose at concentrations similar to plasma concentrations of cows (+EAA_p), and +EAA_p deprived of all BCAA (?BCAA_p) or only Leu (?Leu_p) for 3 h. The ¹³C enrichments of free glucose pool in MTS (E_Glu-free) and the enrichments of glucose incorporated into lactose in MTS and media [E_{Lactose-bound (T&M)}] were determined and used in calculating FSR. In BMEC, ?BCAA increased the fraction of total GLUT1 translocated to the cell membrane and the fraction that was potentially glycosylated compared with +EAA. Among individual BCAA, only ?Leu was associated with a 63% increase in GLUT1 translocated to the cell membrane and a 40% increase in glucose uptake of BMEC. The ?BCAA tended to be related to a 75% increase in the abundance of hexokinase in BMEC. Deprivation of Leu tended to increase glucose uptake of MTS but did not affect E_Glu-free, E_{Lactose-bound (T&M)}, or FSR relative to +EAA_p. On the other hand, ?BCAAp did not affect glucose uptake of MTS but was related to lower E_{Lactose-bound (T&M)}, or FSR relative to +EAA_p. Considering together, decreasing Leu supply to mammary tissues enhances GLUT1 and thus glucose uptake, which, however, does not affect lactose synthesis rates. Moreover, the deficiency of other BCAA, Ile, and Val alone or together with the deficiency of Leu seemed to decrease lactose synthesis rates without affecting glucose uptake. The data also emphasize the importance of addressing the effect of the supply of other nutrients to the mammary glands than the precursor supply in describing the synthesis of a milk component.     

Alterations in immune and antioxidant gene networks by gamma-d-glutamyl-meso-diaminopimelic acid in bovine mammary epithelial cells are attenuated by in vitro supply of methionine and arginine

Nucleotide-binding oligomerization domain (NOD)-like receptor 1 (NOD1) is a cytosolic pattern recognition receptor with a crucial role in the innate immune response of cells triggered by the presence of compounds such as gamma-d-glutamyl-meso-diaminopimelic acid (iE-DAP) present in the peptidoglycan of all gram-negative and certain gram-positive bacteria. Methionine (Met) and arginine (Arg) are functional AA with immunomodulatory properties. In the present study, we aimed to assess the effect of increased Met and Arg supply on mRNA abundance of genes associated with innate immune response, antioxidant function, and AA metabolism during iE-DAP challenge in bovine mammary epithelial cells (BMEC). Primary BMEC (n = 4 per treatment) were precultured in modified medium for 12 h with the following AA formulations: ideal profile of AA (control), increased Met supply (incMet), increased Arg supply (incArg), or increased supply of Met plus Arg (incMetArg). Subsequently, cells were challenged with or without iE-DAP (10 μg/mL) for 6 h. Data were analyzed as a 2 × 2 × 2 factorial using the MIXED procedure of SAS 9.4. Greater mRNA abundance of NOD1, the antioxidant enzyme SOD1, and AA transporters (SLC7A1 and SLC3A2) was observed in the incMet cells after iE-DAP stimulation. Although increased Met alone had no effect, incMetArg led to greater abundance of the inflammatory cytokine IL-6, and the antioxidant enzyme GPX1 after iE-DAP stimulation. The increased Arg alone downregulated NOD1 after iE-DAP stimulation, coupled with a downregulation in the AA transporters mRNA abundance (SLC7A1, SLC7A5, SLC3A2, and SLC38A9), and upregulation in GSS and KEAP1 mRNA abundance. Overall, the data indicated that increased supply of both Met and Arg in the culture medium were more effective in modulating the innate immune response and antioxidant capacity of BMEC during in vitro iE-DAP stimulation.     

Role of sortilin 1 (SORT1) on lipid metabolism in bovine liver

High circulating concentrations of fatty acids cause triacylglycerol (TAG) accumulation in hepatocytes of dairy cows, a common metabolic disorder after calving. Low secretion of apolipoprotein B (APOB) and very low density lipoprotein (VLDL) are thought to be the major factors for TAG accumulation in hepatocytes. Recent data in nonruminant models revealed that sortilin 1 (SORT1) is a key regulator of VLDL secretion in part due to its ability to bind APOB. Thus, SORT1 could play a role in the susceptibility of dairy cows to develop fatty liver. To gain mechanistic insights in vivo and in vitro, we performed experiments using liver biopsies or isolated primary hepatocytes. For the in vivo study, blood and liver samples were collected from healthy multiparous dairy cows (n = 6; 9.0 ± 2.1 d in milk) and cows with fatty liver (n = 6; 9.7 ± 2.2 d in milk). In vitro, hepatocytes isolated from 4 healthy female calves (1 d old, 42–51 kg) were challenged with (fatty acids) or without (control) a 1.2 mM mixture of fatty acids in an attempt to induce metabolic stress. Furthermore, hepatocytes were treated with empty adenovirus vectors (Ad-GFP) or SORT1 overexpressing adenovirus (Ad-SORT1) for 6 h, or SORT1 inhibitor for 2 h followed by a challenge with (Ad-GFP + fatty acids, Ad-SORT1 + fatty acids, or SORT1 inhibitor + fatty acids) or without (Ad-GFP, Ad-SORT1, or SORT1 inhibitor) the 1.2 mM mixture of fatty acids for 12 h. Data from liver biopsies were compared using a 2-tailed unpaired Student's t-test. Data from calf hepatocytes were analyzed by one-way ANOVA. Data revealed that both fatty liver and in vitro challenge with fatty acids were associated with greater concentrations of TAG and mRNA and protein abundance of SORT1, SREBF1, FASN, and ACACA. In contrast, mRNA and protein abundance of CPT1A and APOB, and mRNA abundance of MTTP were markedly lower. Compared with fatty acid challenge alone, SORT1 overexpression led to greater concentration of TAG and mRNA abundance of SREBF1, FASN, ACACA, DGAT1, and DGAT2, and protein abundance of SREBF1, FASN, and ACACA. In contrast, concentration of secreted VLDL-APOB and mRNA abundance of APOB and MTTP, and protein abundance of CPT1A, APOB, and MTTP were lower. Compared with fatty acid challenge alone, SORT1 inhibitor + fatty acids led to lower concentrations of TAG and mRNA abundance of SREBF1, FASN, and DGAT2, and protein abundance of FASN, ACACA, and DGAT1. Concentrations of secreted VLDL-APOB and mRNA abundance of CPT1A and protein abundance of CPT1A and APOB were greater. Overall, in vitro data suggested that greater SORT1 abundance induced by exogenous fatty acids caused a reduction in VLDL-APOB secretion and increased hepatocyte TAG synthesis. Such mechanism was also apparent in tissue from cows with fatty liver. Thus, targeted downregulation of hepatic SORT1 could represent a viable mechanism to unload lipid during conditions where the influx of fatty acids increases markedly.     

Effect of postruminal glutamine supplementation on immune response and milk production in dairy cows

Seventeen multiparous Holstein cows were used to examine the effect of an increased duodenal supply of Gln on immune function and production. Cows received continuous abomasal infusions of water (control: n = 8) or 300 g/d of Gln (n = 9) for 21 d starting within 48 h of calving. There were nonsignificant increases in milk and milk protein yields in response to Gln supplementation. Glutamine treatment had no effect on plasma glucose, nonesterified fatty acids (NEFA), or β-hydroxybutyrate (BHBA) concentrations but did tend to increase plasma urea N concentration. The Gln treatment resulted in an increase of 108 μM in the plasma Gln concentration. Total essential AA concentrations decreased with the Gln treatment, whereas total nonessential AA concentrations were unaffected. T Lymphocyte proliferation did not differ between the control and Gln-treated cows. Treatment had no effect on the relative abundance of CD8 T cells but did increase the abundance of CD4 T cells. Cytokine production, as measured by IFN-γ concentration determined in vitro in concanavalin-A-stimulated peripheral blood mononuclear cells, was similar between the treatments. Over the first 3 wk following calving, Gln supplementation had limited effects on milk production, metabolic parameters, and immune function.     

Role of diacylglycerol O-acyltransferase (DGAT) isoforms in bovine hepatic fatty acid metabolism

Fatty acid accumulation in hepatocytes induced by high concentrations of fatty acids due to lipolysis and the associated oxidative damage they cause occur most frequently after calving. Because of their role in esterification of fatty acids, diacylglycerol acyltransferase isoforms (DGAT1 and DGAT2) could play a role in the susceptibility of dairy cows to develop fatty liver. To gain mechanistic insights, we performed in vivo and in vitro analyses using liver biopsies or isolated primary hepatocytes. The in vivo study (n = 5 cows/group) involved healthy cows [average liver triacylglycerol (TAG) = 0.78%; 0.58 to 0.93%, ratio of triglyceride weight to wet liver weight] or cows diagnosed with fatty liver (average TAG = 7.60%; 5.31 to 10.54%). In vitro, hepatocytes isolated from 3 healthy female calves (1 d old, 44 to 53 kg) were challenged with (fatty acids) or without (control) a 1.2 mM mixture of fatty acids in an attempt to induce metabolic stress. Furthermore, hepatocytes were treated with DGAT1 inhibitor or DGAT2 inhibitor for 2 h followed by a challenge with (DGAT1 inhibitor + fatty acids or DGAT2 inhibitor + fatty acids) or without (DGAT1 inhibitor or DGAT2 inhibitor) the 1.2 mM mixture of fatty acids for 12 h. Data analysis of liver biopsies was compared using a 2-tailed unpaired Student's t-test. Data from calf hepatocyte treatment comparisons were assessed by one-way ANOVA, and multiplicity for each experiment was adjusted by the Holm's procedure. Data indicated that both fatty liver and in vitro challenge with fatty acids were associated with greater mRNA and protein abundance of SREBF1, FASN, DGAT1, and DGAT2. In contrast, mRNA and protein abundance of CPT1A and very low-density lipoprotein synthesis-related proteins MTTP and APOB were markedly lower. However, compared with fatty acid challenge alone, DGAT1 inhibitor + fatty acids led to greater mRNA and protein abundance of CPT1A and APOB, and greater mRNA abundance of SREBF1 and MTTP. Furthermore, this treatment led to lower mRNA abundance of FASN and DGAT2 and TAG concentrations. Compared with fatty acid challenge alone, DGAT2 inhibitor + fatty acids led to greater mRNA and protein abundance of CPT1A, MTTP, and APOB, and lower mRNA and protein abundance of SREBF1 and FASN. In addition, compared with control and fatty acids, there was greater protein abundance of GRP78 and PERK in both DGAT1 and DGAT2 inhibitor with or without fatty acids. Furthermore, compared with control and fatty acids, reactive oxygen species concentrations in the DGAT1 inhibitor with or without fatty acid group was greater. Overall, data suggested that DGAT1 is particularly relevant in the context of hepatocyte TAG synthesis from exogenous fatty acids. Disruption of both DGAT1 and DGAT2 altered lipid homeostasis, channeling fatty acids toward oxidation and generation of reactive oxygen species. Both DGAT isoforms play a role in promoting fatty acid storage into TAG and lipid droplets to protect hepatocytes from oxidative damage.     

Influence of ad libitum milk replacer feeding and butyrate supplementation on the systemic and hepatic insulin-like growth factor I and its binding proteins in Holstein calves

Ad libitum milk feeding and butyrate (B) supplementation have the potential to stimulate postnatal growth and development in calves. The somatotropic axis is the main endocrine regulator of postnatal growth and may be affected by both ad libitum milk replacer (MR) feeding and B supplementation in calves. We hypothesized that ad libitum MR feeding and B supplementation stimulate systemic and hepatic insulin-like growth factor (IGF)-I and IGF binding proteins (IGFBP) in preweaning calves. Sixty-four (32 male, 32 female) Holstein calves were examined from birth until wk 11 of life. Calves received MR either ad libitum (Adl) or restrictively (6 L/d; Res). In each feeding group half of the calves received a MR with 0.24% butyrate and the other half received same MR without butyrate. Ad libitum MR feeding was performed from d 4 until wk 8 of age. From wk 9 to 10, Adl and Res calves were gradually weaned and were fed 2 L/d until the end of the trial. Concentrate, hay, and water were freely available. Feed intake was measured daily and body weight weekly. Blood samples for analyzing plasma concentrations of glucose, insulin, IGF-I, and IGFBP-2, -3, and -4 were taken on d 1, 2, 4, and 7, then weekly or every other week (IGFBP) until wk 11 of life. Liver samples were taken on d 50 and at the end of the study (d 80) to measure gene expression of the growth hormone receptor 1A (GHR1A), IGF1, IGFBP1 to 4, and of the IGF Type 1 and insulin receptor in the liver. Intake of MR and body weight were greater, but concentrate intake was lower in Adl than in Res. Plasma concentrations of IGF-I and IGFBP-3 were greater and plasma concentration of IGFBP-2 was lower in Adl than in Res during the ad libitum milk feeding period. After reduction of MR in both groups to 2 L/d plasma concentrations of IGF-I and IGFBP-4 were lower and plasma concentration of IGFBP-2 was higher in Adl than in Res. Supplementation of B depressed plasma IGF-I from wk 1 to 4 and in wk 9. On d 50, mRNA abundance of the GHR1A and IGF1 was greater and of IGFBP2 mRNA was lower in Adl than in Res. At d 80, IGFBP2 mRNA was greater in Adl than in Res, and IGFBP2 mRNA increased with B supplementation. Ad libitum MR feeding stimulated the systemic and hepatic IGF system and mirrored the greater growth rate during the ad libitum MR feeding, whereas butyrate supplementation partly reduced the systemic and hepatic IGF system.     

Maternal supplementation with cobalt sources,folic acid,and rumen-protected methionine and its effects on molecular and functional correlates of the immune system in neonatal Holstein calves

Calves born to multiparous Holstein cows fed during the last 30 d of pregnancy 2 different cobalt sources [cobalt glucoheptonate (CoPro) or cobalt pectin (CoPectin)], folic acid (FOA), and rumen-protected methionine (RPM) were used to study neonatal immune responses after ex vivo lipopolysaccharide (LPS) challenge. Groups were (n = 12 calves/group) CoPro, FOA+CoPro, FOA+CoPectin, and FOA+CoPectin+RPM. Calves were weighed at birth and blood collected at birth (before colostrum), 21 d of age, and 42 d of age (at weaning). Growth performance was recorded once a week during the first 6 wk of age. Energy metabolism, inflammation, and antioxidant status were assessed at birth through various plasma biomarkers. Whole blood was challenged with 3 µg/mL of LPS or used for phagocytosis and oxidative burst assays. Target genes evaluated by real-time quantitative PCR in whole blood samples were associated with immune response, antioxidant function, and 1-carbon metabolism. The response in mRNA abundance in LPS challenged versus nonchallenged samples was assessed via Δ = LPS challenged ? LPS nonchallenged samples. Phagocytosis capacity and oxidative burst activity were measured in neutrophils and monocytes, with data reported as ratio (percentage) of CD14 to CH138A-positive cells. Data including all time points were subjected to ANOVA using PROC MIXED in SAS 9.4 (SAS Institute Inc.), with Treatment, Sex, Age, and Treatment × Age as fixed effects. A 1-way ANOVA was used to determine differences at birth, with Treatment and Sex as fixed effects. Calf birth body weight and other growth parameters did not differ between groups. At birth, plasma haptoglobin concentration was lower in FOA+CoPro compared with CoPro calves. We detected no effect for other plasma biomarkers or immune function due to maternal treatments at birth. Compared with CoPro, in response to LPS challenge, whole blood from FOA+CoPectin and FOA+CoPectin+RPM calves had greater mRNA abundance of intercellular adhesion molecule 1 (ICAM1). No effect for other genes was detectable. Regardless of maternal treatments, sex-specific responses were observed due to greater plasma concentrations of haptoglobin, paraoxonase, total reactive oxygen metabolites, nitrite, and β-carotene in female versus male calves at birth. In contrast, whole blood from male calves had greater mRNA abundance of IRAK1, CADM1, and ITGAM in response to LPS challenge at birth. The longitudinal analysis of d 0, 21, and 42 data revealed greater bactericidal permeability-increasing protein (BPI) mRNA abundance in whole blood from FOA+CoPectin versus FOA+CoPro calves, coupled with greater abundance in FOA+CoPro compared with CoPro calves. Regardless of maternal treatments, most genes related to cytokines and cytokine receptors (IL1B, IL10, TNF, IRAK1, CXCR1), toll-like receptor pathway (TLR4, NFKB1), adhesion and migration (ICAM1, ITGAM), antimicrobial function (MPO), and antioxidant function (GPX1) were downregulated over time. Phagocytosis capacity and oxidative burst activity in both neutrophils and monocytes did not differ due to maternal treatment. Regardless of maternal treatments, we observed an increase in the percentage of neutrophils capable of phagocytosis and oxidative burst activity over time. Overall, these preliminary assessments suggested that maternal supplementation with FOA and Co combined with RPM had effects on a few plasma biomarkers of inflammation at birth and molecular responses associated with inflammatory mechanisms during the neonatal period.     

Effects of polymerization of casein and sources of lysine on amino acid bioavailability among calves fed liquid-based diets

Two experiments were conducted to evaluate the bioavailability of AA between polymerized and less polymerized or unpolymerized sources of AA. In the first experiment, 6 bull calves (53.8 ± 0.6 kg of body weight) were bottle-fed milk replacer that contained 0, 60, or 120 additional grams of AA from casein or acid hydrolyzed casein every 12 h. Plasma essential AA increased linearly with increasing intake of casein from either source. Branched-chain amino acids accounted for 74% of increases in essential AA, regardless of source of AA. Concentrations of nonessential AA increased linearly with increased intake of AA from acid hydrolyzed casein but only tended to increase in response to casein. Also, the rate of increase in total plasma AA concentration in response to acid hydrolyzed casein (4.3 µM increase per g of supplemental AA) tended to be 145% greater than casein (3.0 µM per g of supplemental AA). In a separate experiment, 6 additional bull calves (52.1 ± 0.9 kg of body weight) were bottle-fed milk replacer that contained 0, 4.8, or 9.6 additional grams of Lys from ε-polylysine or Lys-HCl each 12 h to measure Lys bioavailability between a polymerized and unpolymerized source of Lys. Plasma Lys concentrations increased linearly in response to greater Lys intake from Lys-HCl (slope = 13.51 µM/g Lys,), but plasma Lys concentrations did not change in response to increased intake of Lys from ε-polylysine. Plasma concentrations of Thr, Met, Glu, and Gln decreased linearly with increasing ε-polylysine intake, whereas concentrations of His, Val, Leu, and Ile increased linearly with increasing ε-polylysine intake. Data from these experiments suggest that the form of AA provided to calves should be considered when formulating diets to meet AA requirements.     

Maternal supplementation with rumen-protected methionine increases prepartal plasma methionine concentration and alters hepatic mRNA abundance of 1-carbon,methionine, and transsulfuration pathways in neonatal Holstein calves

An important mechanism of nutritional “programming” induced by supplementation with methyl donors during pregnancy is the alteration of mRNA abundance in the offspring. We investigated the effects of rumen-protected Met (RPM) on abundance of 17 genes in the 1-carbon, Met, and transsulfuration pathways in calf liver from cows fed the same basal diet without (control, CON) or with RPM at 0.08% of diet dry matter/d (MET) from ?21 through +30 d around calving. Biopsies (n = 8 calves per diet) were harvested on d 4, 14, 28, and 50 of age. Cows fed RPM had greater plasma concentration of Met (17.8 vs. 28.2 μM) at ?10 d from calving. However, no difference was present in colostrum yield and free AA concentrations. Greater abundance on d 4 and 14 of betaine-homocysteine S-methyltransferase 2 (BHMT2), adenosylhomocysteinase (AHCY; also known as SAHH), and cystathionine-β-synthase (CBS) in MET calves indicated alterations in Met, choline, and homocysteine metabolism. Those data agree with the greater abundance of methionine adenosyltransferase 1A (MAT1A) in MET calves. Along with CBS, the greater abundance of glutamate-cysteine ligase (GCLC) and glutathione reductase (GSR) on d 4 in MET calves indicated a short-term postnatal alteration in the use of homocysteine for taurine and glutathione synthesis (both are potent intracellular antioxidants). The striking 7-fold upregulation at d 50 versus 4 of cysteine sulfinic acid decarboxylase (CSAD), catalyzing the last step of taurine synthesis, in MET and CON calves underscores an important role of taurine during postnatal calf growth. The unique role of taurine in the young calf is further supported by the upregulation of CBS, GCLC, and GSR at d 50 versus 14 and 28 in MET and CON. Although betaine-homocysteine S-methyltransferase (BHMT) activity did not differ in MET and CON, it increased ～50% at d 14 and 28 versus 4. A significant positive correlation (r = 0.79) was present between BHMT abundance and BHMT activity regardless of treatment. The gradual upregulation over time of BHMT2 and SAHH coupled with the gradual upregulation of MAT1A and the DNA (cytosine-5-)-methyltransferases (DNMT1, DNMT3A, DNMT3B) in MET and CON calves was indicative of adaptations potentially driven by differences in intake of milk replacer and starter feed as calves grew. In that context, the ～2.5-fold increase in abundance of DNMT3B at d 50 versus 4 in MET and CON indicate that DNA methylation might be an important component of the physiologic adaptations of calf liver. The data indicate that calves from MET-supplemented cows underwent alterations in Met, choline, and homocysteine metabolism partly to synthesize taurine and glutathione, which would be advantageous for controlling metabolic-related stress. Whether the effects in MET calves were directly related to increased Met supply in utero remains to be determined.     

Expression of genes related to energy metabolism and the unfolded protein response in dairy cow mammary cells is affected differently during dietary supplementation with energy from protein and fat

Secretory capacity of bovine mammary glands is enabled by a high number of secretory cells and their ability to use a range of metabolites to produce milk components. We isolated RNA from milk fat to measure expression of genes involved in energy-yielding pathways and the unfolded protein response in mammary glands of lactating cows given supplemental energy from protein (PT) and fat (FT) tested in a 2 × 2 factorial arrangement. We hypothesized that PT and FT would affect expression of genes in the branched-chain AA catabolic pathway and tricarboxylic acid (TCA) cycle based on the different energy types (aminogenic versus lipogenic) used to synthesize milk components. We also hypothesized that the response of genes related to endoplasmic reticulum (ER) homeostasis via the unfolded protein response would reflect the increase in milk production stimulated by PT and FT. Fifty-six multiparous Holstein-Friesian dairy cows were fed a basal total mixed ration (34% grass silage, 33% corn silage, 5% grass hay, and 28% concentrate on a dry matter basis) for a 28-d control period. Experimental rations were then fed for 28 d, consisting of (1) low protein, low fat (LP/LF); (2) high protein, low fat (HP/LF); (3) low protein, high fat (LP/HF); or (4) high protein and high fat (HP/HF). To obtain the high-protein (HP) and high-fat (HF) diets, intake of the basal ration was restricted and supplemented isoenergetically (net energy basis) with 2.0 kg/d rumen-protected protein (soybean + rapeseed, 50:50 mixture on dry matter basis) and 0.68 kg/d hydrogenated palm fatty acids on a dry matter basis. RNA from milk fat samples collected on d 27 of each period underwent real-time quantitative PCR. Energy from protein increased expression of BCAT1 (branched-chain amino acid transferase 1) mRNA, but only at the LF level, and tended to decrease expression of mRNA encoding the main subunit of the branched-chain keto-acid dehydrogenase complex. mRNA expression of malic enzyme, a proposed channeling route for AA though the TCA cycle, was decreased by PT, but only at the LF level. Expression of genes associated with de novo fatty acid synthesis was not affected by PT or FT. Energy from fat had no independent effect on genes related to ER homeostasis. At the LF level, PT activated XBP1 (X-box binding protein 1) mRNA. At the HF level, PT increased mRNA expression of the gene encoding GADD34 (growth arrest and DNA damage-inducible 34). These findings support our hypothesis that mammary cells use aminogenic and lipogenic precursors differently for milk component production when dietary intervention alters AA and fatty acid supply. They also suggest that mammary cells respond to increased AA supply through mechanisms of ER homeostasis, dependent on the presence of FT.     

Short communication: Relationship between lysine/methionine ratios and glucose levels and their effects on casein synthesis via activation of the mechanistic target of rapamycin signaling pathway in bovine mammary epithelial cells

The synthesis of protein requires the availability of specific AA and a large supply of energy in bovine mammary epithelial cells (BMEC). Whether an interaction exists between Lys/Met ratio and glucose level on milk protein synthesis and its potential regulatory mechanism is unclear. We investigated the effects of different Lys/Met ratios and glucose levels on casein synthesis-related gene expression in BMEC to elucidate the underlying molecular mechanisms. Primary BMEC were subjected to 4 treatments for 36 h, arranged in a 2 × 2 factorial design with Lys/Met ratios of 3:1 (1.2:0.4 mM, LM3.0; total AA = 8.24 mM) and 2.3:1 (1.4:0.6 mM, LM2.3; total AA = 8.64 mM) and glucose levels of 17.5 mM (high glucose level) and 2.5 mM (low glucose level). No interactions between Lys/Met ratio and glucose level on cell viability, cell cycle progression, mRNA, or protein expression levels were found. High glucose level increased cell proliferation and promoted cell cycle transition from intermediate phase (G1 phase) to synthesis (S phase) by approximately 50%, whereas Lys/Met ratio had no effect. Both mRNA and protein abundance of α_S1-casein and β-casein were positively affected by LM3.0, whereas a high glucose level increased protein abundance of α_S1-casein and β-casein and increased gene expression of CSN1S1 but not of CSN2. Furthermore, high glucose increased the mRNA abundance of ELF5 and decreased that of GLUT8, enhanced protein expression of total and phosphorylated mechanistic target of rapamycin (mTOR), and decreased phosphorylated AMP-activated protein kinase (AMPK) levels. Treatment LM3.0 had a stimulatory effect on total and phosphorylated mTOR but did not affect AMPK phosphorylation. The mRNA levels of JAK2, ELF5, and RPS6KB1 were upregulated and mRNA levels of EIF4EBP1 were downregulated with LM3.0 compared with LM2.3. Our results indicate that casein synthesis was regulated by Lys/Met ratio via JAK2/ELF5, mTOR, and its downstream RPS6KB1 and EIF4EBP1 signaling. In contrast, glucose regulated casein synthesis through promoting cell proliferation, accelerating cell cycle progression, and activating the ELF5 and AMPK/mTOR signaling pathways. Within the range of substrate levels in the present study, a change in Lys/Met ratio had a stronger effect on abundance of α_S1-casein and β-casein than a change in glucose level.     

Feeding rumen-protected lysine to dairy cows prepartum improves performance and health of their calves

Providing adequate concentrations of AA in the prepartum diet is pivotal for the cow's health and performance. However, less is known about the potential in utero effects of particular AA on early-life performance of calves. This experiment was conducted to determine the effects on dairy calves when their dams were fed rumen-protected lysine (RPL; AjiPro-L Generation 3, Ajinomoto Heartland Inc.; 0.54% dry matter of total mixed ration as top dress) from 26 ± 4.6 d (mean ± standard deviation) before calving until calving. Seventy-eight male (M) and female (F) Holstein calves were assigned to 2 treatments based on their dams' prepartum treatment, RPL supplementation (PRE-L) or without RPL (CON). At the time of birth (0.5–2 h after calving), before colostrum was fed, blood samples were collected. An initial body weight was obtained at 1 to 3 h after birth. Calves were fed 470 g of colostrum replacer (Land O'Lakes Bovine IgG Colostrum Replacer, Land O'Lakes, Inc.) diluted in 3.8 L of water. Calves were provided water ad libitum and fed milk replacer (Advance Excelerate, Milk Specialties Global Animal Nutrition; 28.5% crude protein, 15% fat) at 0600 h and 1700 h until 42 d of age. Calves were measured weekly, at weaning (d 42), and at the end of the experimental period (d 56). Plasma concentrations of AA were measured on d 0, 7, and 14 d using ultra-performance liquid chromatography–mass spectrometry (Waters) with a derivatization method (AccQ-Tag Derivatization). Final body weight was greater for M (87 ± 11 kg) than F (79 ± 7 kg). Calves in PRE-L tended to have greater dry matter (814 ± 3 g/d) and crude protein (234 ± 6 g/d) intakes than those in CON (793 ± 9 g/d and 228 ± 11 g/d, respectively). Calves in PRE-L had greater average daily gain (0.96 ± 0.04 kg/d) than calves in CON (0.85 ± 0.03 kg/d) during wk 6 to 8. Calves in PRE-L tended to be medicated fewer days than CON (4.7 ± 1.2 d vs. 6.2 ± 3.4 d, respectively). Calves in PRE-L-M and CON-F (2,916 ± 112 µM and 2,848 ± 112 µM, respectively) had greater total AA concentration in plasma than calves in PRE-L-F and CON-M (2,684 ± 112 µM and 2,582 ± 112 µM, respectively). Calves in PRE-L-F and CON-M (4.09 ± 0.11% and 4.16 ± 0.11%, respectively) had greater concentration of Lys as a percentage of total AA compared with calves in CON-F and PRE-L-M (3.91 ± 0.11% and 3.90 ± 0.11%, respectively). Calves in PRE-L tended to have greater percentage of phagocytic neutrophils (39.6 ± 1.59%) than calves in CON (35.9 ± 1.59%). In conclusion, increasing the metabolizable lysine provided to prepartum dairy cows had modest effect over offspring performance, with the major result being a greater average daily gain for calves in PRE-L during the preweaning phase (wk 6–8).