Danielle N. Coleman

Heat stress (HS) during the transition period disrupts metabolic adaptation and mammary gland (MG) remodeling. Methionine, a rate-limiting amino acid, supports cellular growth and stress responses through one-carbon metabolism pathways. We evaluated whether rumen-protected methionine (RPM) supplementation during the transition period preserves MG structure and function in heat-stressed multiparous Holstein cows. We hypothesized that RPM would mitigate the negative effects of HS on MG microstructure, gene expression, and cellular turnover. A total of 53 multiparous Holstein cows were enrolled at dry-off and assigned to diets with or without RPM (MET, CON). Four weeks prepartum, all MET and half of CON cows were fitted with electric heat blankets to induce HS (METHS, CONHS), while remaining CON cows were thermoneutral (CONTN). MG biopsies (8 and 28 days in milk; DIM) and milk samples were collected to assess microstructure, cellular turnover, gene expression, and mammary epithelial integrity. HS appeared to impair MG remodeling, with fewer but larger alveoli and a thicker epithelial layer, with a slight increase in epithelial cell proliferation, increased occludin fluorescence intensity, and increased milk concentration of K⁺. Meanwhile, RPM under HS increased stromal proliferation, lower occludin fluorescence intensity, and a reduced Na⁺:K⁺ ratio, indicating overall improved mammary epithelial integrity. Additionally, RPM partially promoted greater alveolar organization early postpartum and reduced lumen expansion, with coordinated changes in one-carbon metabolism, amino acid transport, and mTOR-related pathways. Overall, HS disrupts MG cellular dynamics, whereas close-up methionine supplementation partially improves mammary adaptability under chronic transition period HS.

We investigated effects of rumen-protected Met (RPM) during a heat stress (HS) challenge on (1) hepatic abundance of mTOR, insulin, and antioxidant signaling proteins, (2) enzymes in 1-carbon metabolism, and (3) innate immunity. Holstein cows (n = 32; mean ± standard deviation, 184 ± 59 d in milk) were randomly assigned to 1 of 2 environmental groups, and 1 of 2 diets [total mixed ration (TMR) with RPM (Smartamine M; 0.105% dry matter as top-dress) or TMR without (CON); n = 16/diet] in a split-plot crossover design. There were 2 periods with 2 phases. During phase 1 (9 d), all cows were in thermoneutral conditions (TN; temperature-humidity index = 60 ± 3) and fed ad libitum. During phase 2 (9 d), half the cows (n = 8/diet) were exposed to HS using electric heat blankets. The other half (n = 8/diet) remained in TN, but was pair-fed to HS counterparts. After a 14-d washout and 7-d adaptation period, the study was repeated (period 2) and environmental treatments were inverted relative to phase 2, but dietary treatments were the same. Blood was collected on d 6 of each phase 2 to measure immune function and isolate whole-blood RNA. Liver biopsies were performed at the end of each period for cystathione β-synthase (CBS) and methionine adenosyltransferase activity, glutathione concentration, and protein abundance. Data were analyzed using PROC MIXED in SAS. Abundance of CUL3, inhibitor of antioxidant responses, tended to be downregulated by HS suggesting increased oxidative stress. Heat-shock protein 70 abundance was upregulated by HS. Phosphorylated mTOR abundance was greater overall with RPM, suggesting an increase in pathway activity. An environment × diet (E × D) effect was observed for protein kinase B (AKT), whereas there was a tendency for an interaction for phosphorylated AKT. Abundance of AKT was upregulated in CON cows during HS versus TN, this was not observed in RPM cows. For phosphorylated AKT, tissue from HS cows fed CON had greater abundance compared with all other treatments. The same effect was observed for EIF2A (translation initiation) and SLC2A4 (insulin-induced glucose uptake). An E × D effect was observed for INSR due to upregulation in CON cows during HS versus TN cows fed CON or RPM. There was an E × D effect for CBS, with lower activity in RPM versus CON cows during HS. The CON cows tended to have greater CBS during HS versus TN. An E × D effect was observed for methionine adenosyltransferase, with lower activity in RPM versus CON during HS. Although activity increased in CON during HS versus TN, RPM cows tended to have greater activity during TN. Neutrophil and monocyte oxidative burst and monocyte phagocytosis decreased with HS. An (E × D) effect was observed for whole-blood mRNA abundance of CBS, SOD1 and CSAD; RPM led to upregulation during TN versus HS. Regardless of diet, CDO1, CTH, and SOD1 decreased with HS. Although HS increased hepatic HSP70 and seemed to alter antioxidant signaling, feeding RPM may help cows maintain homeostasis in mTOR, insulin signaling, and 1-carbon metabolism. Feeding RPM also may help maintain whole-blood antioxidant response during HS, which is an important aspect of innate immune function.

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.

The objective was to perform a proof-of-principle study to evaluate the effects of methionine (Met) and arginine (Arg) supply on protein abundance of amino acid, insulin signaling, and glutathione metabolism-related proteins in subcutaneous adipose tissue (SAT) explants under ceramide (Ce) challenge. SAT from four lactating Holstein cows was incubated with one of the following media: ideal profile of amino acid as the control (IPAA; Lys:Met 2.9:1, Lys:Arg 2:1), increased Met (incMet; Lys:Met 2.5:1), increased Arg (incArg; Lys:Arg 1:1), or incMet plus incArg (Lys:Met 2.5:1 Lys:Arg 1:1) with or without 100 μM exogenous cell-permeable Ce (N-Acetyl-d-sphingosine). Ceramide stimulation downregulated the overall abundance of phosphorylated (p) protein kinase B (AKT), p-mechanistic target of rapamycin (mTOR), and p-eukaryotic elongation factor 2 (eEF2). Without Ce stimulation, increased Met, Arg, or Met + Arg resulted in lower p-mTOR. Compared with control SAT stimulated with Ce, increased Met, Arg, or Met + Arg resulted in greater activation of mTOR (p-mTOR/total mTOR) and AKT (p-AKT/total AKT), with a more pronounced response due to Arg. The greatest protein abundance of glutathione S-transferase Mu 1 (GSTM1) was detected in response to increased Met supply during Ce stimulation. Ceramide stimulation decreased the overall protein abundance of the Na-coupled neutral amino acid transporter SLC38A1 and branched-chain alpha-ketoacid dehydrogenase kinase (BCKDK). However, compared with controls, increased Met or Arg supply attenuated the downregulation of BCKDK induced by Ce. Circulating ceramides might affect amino acid, insulin signaling, and glutathione metabolism in dairy cow adipose tissue. Further in vivo studies are needed to confirm the role of rumen-protected amino acids in regulating bovine adipose function.

Managing body condition in dairy cows during the close-up period could alter the availability of nutrients to the fetus during the final growth stages in utero. We investigated how maternal body condition score (BCS) in late pregnancy affected calf whole-blood mRNA abundance and IL-1β concentrations after ex vivo lipopolysaccharide (LPS) challenge. Thirty-eight multiparous Holstein cows and their calves from a larger cohort were retrospectively grouped by prepartal BCS as normal BCS (≤3.25; n = 22; NormBCS) and high BCS (≥3.75; n = 16; HighBCS). Calf blood samples collected at birth (before receiving colostrum, d 0) and at ages 21 and 42 d (at weaning) were used for ex vivo whole-blood challenge with 3 µg/mL of LPS before mRNA isolation. Target genes evaluated by real-time quantitative PCR were associated with immune response, antioxidant function, and 1-carbon metabolism. Plasma IL-1β concentrations were also measured. Responses in plasma IL-1β and mRNA abundance were compared between LPS-challenged and nonchallenged samples. Statistical analyses were performed at all time points using a MIXED model in SAS 9.4. Neither birth body weight (NormBCS = 43.8 ± 1.01 kg; HighBCS = 43.9 ± 1.2 kg) nor colostrum IgG concentration (NormBCS = 70 ± 5.4 mg/mL; HighBCS = 62 ± 6.5 mg/mL) differed between groups. At birth, whole blood from calves born to HighBCS cows had greater mRNA abundance of IL1B, NFKB1, and GSR and lower GPX1 and CBS abundance after LPS challenge. The longitudinal analysis of d 0, 21, and 42 data revealed a BCS × age effect for SOD2 and NOS2 due to lower mRNA abundance at 42 d in the HighBCS calves. Regardless of maternal BCS, mRNA abundance decreased over time for genes encoding cytokines (IL1B, IL6, IL10, TNF), cytokine receptors (IRAK1, CXCR1), toll-like receptor pathway (TLR4, NFKB1), adhesion and migration (CADM1, ICAM1, ITGAM), and antimicrobial function (MPO). Concentration of IL-1β after LPS challenge was also markedly lower at 21 d regardless of maternal BCS. Overall, results suggested that maternal BCS in late prepartum influences the calf immune system response to an inflammation challenge after birth. Although few genes among those studied were altered due to maternal BCS, the fact that genes related to oxidative stress and 1-carbon metabolism responded to LPS challenge in HighBCS calves underscores the potential role of methyl donors (e.g., methionine, choline, and folic acid) in the early-life innate immune response.

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.

Mastitis, inflammation of the udder, is one of the most common diseases hampering milk yield of dairy cows. Methionine (Met) and arginine (Arg) are key nutrients with potential to regulate inflammation and oxidative stress. The aim of this study was to evaluate the effect of increased supply of Met and Arg on mRNA and protein abundance associated with innate immune response and redox balance during lipopolysaccharide (LPS) stimulation in primary bovine mammary epithelial cells (BMEC). Primary BMEC (n = 4 replicates per treatment) were pre-incubated for 12 h in media with the following amino acid combinations: ideal profile of amino acids (control; Con), increased Met supply (incMet), increased Arg supply (incArg), and increased supply of Met and Arg (incMetArg). Subsequently, cells were challenged with or without LPS (1 µg/mL) and incubated for 6 h. Data were analyzed as a 2 × 2 × 2 factorial using the MIXED procedure of SAS 9.4 (SAS Institute Inc., Cary, NC). The downregulation of SLC36A1 and SLC7A1 mRNA abundance induced by LPS was attenuated in the incArg cultures. Although challenge with LPS led to lower abundance of proteins related to the antioxidant response (NFE2L2, NQO1, GPX1), lower levels of ATG7, and lower mRNA abundance of GPX3, we found little effect in cultures with incMet or incArg. Cultures with incMet, incArg, or incMetArg led to attenuation of the upregulation of SOD2 and NOS2 induced by LPS. Abundance of phosphorylated p65 (RELA) was greater after LPS stimulation, but the response was attenuated in cultures with incMet. The greater ratio of pRELA to total RELA in responses to LPS was also attenuated in cultures with incMetArg. The greater mRNA abundance of the proinflammatory cytokine IL1B induced by LPS was attenuated in cultures with incMet, and the same trend induced by LPS on CXCL2 was also alleviated in cultures with incArg. Overall, the data suggest that greater supply of Met and Arg alleviated the proinflammatory responses triggered by LPS through controlling the abundance of proinflammatory cytokines and chemokines and activity of NF-κB. Little benefit on oxidative stress induced by LPS challenge in BMEC was detected with greater supply of Met and Arg.