Feeding a Saccharomyces cerevisiae fermentation product improves udder health and immune response to a Streptococcus uberis mastitis challenge in mid-lactation dairy cows.

BACKGROUND: We aimed to characterize the protective effects and the molecular mechanisms of action of a Saccharomyces cerevisiae fermentation product (NTK) in response to a mastitis challenge. Eighteen mid-lactation multiparous Holstein cows (n = 9/group) were fed the control diet (CON) or CON supplemented with 19 g/d NTK for 45 d (phase 1, P1) and then infected in the right rear quarter with 2500 CFU of Streptococcus uberis (phase 2, P2). After 36-h, mammary gland and liver biopsies were collected and antibiotic treatment started until the end of P2 (9 d post challenge). Cows were then followed until day 75 (phase 3, P3). Milk yield (MY) and dry matter intake (DMI) were recorded daily. Milk samples for somatic cell score were collected, and rectal and udder temperature, heart and respiration rate were recorded during the challenge period (P2) together with blood samples for metabolite and immune function analyses. Data were analyzed by phase using the PROC MIXED procedure in SAS. Biopsies were used for transcriptomic analysis via RNA-sequencing, followed by pathway analysis. RESULTS: DMI and MY were not affected by diet in P1, but an interaction with time was recorded in P2 indicating a better recovery from the challenge in NTK compared with CON. NTK reduced rectal temperature, somatic cell score, and temperature of the infected quarter during the challenge. Transcriptome data supported these findings, as NTK supplementation upregulated mammary genes related to immune cell antibacterial function (e.g., CATHL4, NOS2), epithelial tissue protection (e.g. IL17C), and anti-inflammatory activity (e.g., ATF3, BAG3, IER3, G-CSF, GRO1, ZFAND2A). Pathway analysis indicated upregulation of tumor necrosis factor α, heat shock protein response, and p21 related pathways in the response to mastitis in NTK cows. Other pathways for detoxification and cytoprotection functions along with the tight junction pathway were also upregulated in NTK-fed cows. CONCLUSIONS: Overall, results highlighted molecular networks involved in the protective effect of NTK prophylactic supplementation on udder health during a subclinical mastitic event.

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.

Molecular networks of insulin signaling and amino acid metabolism in subcutaneous adipose tissue are altered by body condition in periparturient Holstein cows.

Peripartal cows mobilize not only body fat but also body protein to satisfy their energy requirements. The objective of this study was to determine the effect of prepartum BCS on blood biomarkers related to energy and nitrogen metabolism, and mRNA and protein abundance associated with AA metabolism and insulin signaling in subcutaneous adipose tissue (SAT) in peripartal cows. Twenty-two multiparous Holstein cows were retrospectively classified into a high BCS (HBCS; n = 11, BCS ≥ 3.5) or normal BCS (NBCS; n = 11, BCS ≤ 3.17) group at d 28 before expected parturition. Cows were fed the same diet as a total mixed ration before parturition and were fed the same lactation diet postpartum. Blood samples collected at -10, 7, 15, and 30 d relative to parturition were used for analyses of biomarkers associated with energy and nitrogen metabolism. Biopsies of SAT harvested at -15, 7, and 30 d relative to parturition were used for mRNA (real time-PCR) and protein abundance (Western blotting) assays. Data were subjected to ANOVA using the MIXED procedure of SAS (v. 9.4; SAS Institute Inc., Cary, NC), with P ≤ 0.05 being the threshold for significance. Cows in HBCS had greater overall plasma nonesterified fatty acid concentrations, due to marked increases at 7 and 15 d postpartum. This response was similar (BCS × Day effect) to protein abundance of phosphorylated (p) protein kinase B (p-AKT), the insulin-induced glucose transporter (SLC2A4), and the sodium-coupled neutral AA transporter (SLC38A1). Abundance of these proteins was lower at -15 d compared with NBCS cows, and either increased (SLC2A4, SLC38A1) or did not change (p-AKT) at 7 d postpartum in HBCS. Unlike protein abundance, however, overall mRNA abundances of the high-affinity cationic (SLC7A1), proton-coupled (SLC36A1), and sodium-coupled amino acid transporters (SLC38A2) were greater in HBCS than NBCS cows, due to upregulation in the postpartum phase. Those responses were similar to protein abundance of p-mTOR, which increased (BCS × Day effect) at 7 d in HBCS compared with NBCS cows. mRNA abundance of argininosuccinate lyase (ASL) and arginase 1 (ARG1) also was greater overall in HBCS cows. Together, these responses suggested impaired insulin signaling, coupled with greater postpartum AA transport rate and urea cycle activity in SAT of HBCS cows. An in vitro study using adipocyte and macrophage cocultures stimulated with various concentrations of fatty acids could provide some insights into the role of immune cells in modulating adipose tissue immunometabolic status, including insulin resistance and AA metabolism.

Body condition alters glutathione and nuclear factor erythroid 2-like 2 (NFE2L2)-related antioxidant network abundance in subcutaneous adipose tissue of periparturient Holstein cows.

Dairy cows with high body condition score (BCS) in late prepartum are more susceptible to oxidative stress (OS). Nuclear factor erythroid 2-like 2 (NFE2L2) is a major antioxidant transcription factor. We investigated the effect of precalving BCS on blood biomarkers associated with OS, inflammation, and liver function, along with mRNA and protein abundance of targets related to NFE2L2 and glutathione (GSH) metabolism in s.c. adipose tissue (SAT) of periparturient dairy cows. Twenty-two multiparous Holstein cows were retrospectively classified into a high BCS (HBCS; n = 11, BCS ≥3.5) or normal BCS (NBCS; n = 11, BCS ≤3.17) on d 28 before parturition. Cows were fed a corn silage- and wheat straw-based total mixed ration during late prepartum, and a corn silage- and alfalfa hay-based total mixed ration postpartum. Blood samples obtained at -10, 7, 15, and 30 d relative to parturition were used for analyses of biomarkers associated with inflammation, including albumin, ceruloplasmin, haptoglobin, and myeloperoxidase, as well as OS, including ferric reducing ability of plasma (FRAP), reactive oxygen species (ROS), and ß-carotene. Adipose biopsies harvested at -15, 7, and 30 d relative to parturition were analyzed for mRNA (real-time quantitative PCR) and protein abundance (Western blotting) of targets associated with the antioxidant transcription regulator nuclear factor, NFE2L2, and GSH metabolism pathway. In addition, concentrations of GSH, ROS and malondialdehyde were measured. High BCS cows had lower prepartum dry matter intake expressed as a percentage of body weight along with greater BCS loss between -4 and 4 wk relative to parturition. Plasma concentrations of ROS and FRAP increased after parturition regardless of treatment. Compared with NBCS, HBCS cows had greater concentrations of FRAP at d 7 postpartum, which coincided with peak values in those cows. In addition, NBCS cows experienced a marked decrease in plasma ROS after d 7 postpartum, while HBCS cows maintained a constant concentration by d 30 postpartum. Overall, ROS concentrations in SAT were greater in HBCS cows. However, overall mRNA abundance of NFE2L2 was lower and cullin 3 (CUL3), a negative regulator of NFE2L2, was greater in HBCS cows. Although HBCS cows had greater overall total protein abundance of NFE2L2 in SAT, ratio of phosphorylated NFE2L2 to total NFE2L2 was lower, suggesting a decrease in the activity of this antioxidant system. Overall, mRNA abundance of the GSH metabolism-related genes glutathione reductase (GSR), glutathione peroxidase 1 (GPX1), and transaldolase 1 (TALDO1), along with protein abundance of glutathione S-transferase mu 1 (GSTM1), were greater in HBCS cows. Data suggest that HBCS cows might experience greater systemic OS after parturition, while increased abundance of mRNA and protein components of the GSH metabolism pathway in SAT might help alleviate tissue oxidant status. Data underscored the importance of antioxidant mechanisms at the tissue level. Thus, targeting these pathways in SAT during the periparturient period via nutrition might help control tissue remodeling while allowing optimal performance.

Methionine and arginine supplementation alter inflammatory and oxidative stress responses during lipopolysaccharide challenge in bovine mammary epithelial cells in vitro.

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.

Methionine and choline supply alter transmethylation, transsulfuration, and cytidine 5'-diphosphocholine pathways to different extents in isolated primary liver cells from dairy cows.

Insufficient supply of Met and choline (Chol) around parturition could compromise hepatic metabolism and milk protein synthesis in dairy cows. Mechanistic responses associated with supply of Met or Chol in primary liver cells enriched with hepatocytes (PHEP) from cows have not been thoroughly ascertained. Objectives were to isolate and culture PHEP to examine abundance of genes and proteins related to transmethylation, transsulfuration, and cytidine 5'-diphosphocholine (CDP-choline) pathways in response to Met or Chol. The PHEP were isolated from liver biopsies of Holstein cows (160 d in lactation). More than 90% of isolated cells stained positively for the hepatocyte marker cytokeratin 18. Cytochrome P450 (CYP1A1) mRNA abundance was only detectable in the PHEP and liver tissue compared with mammary tissue. Furthermore, in response to exogenous Met (80 µM vs. control) PHEP secreted greater amounts of albumin and urea. Subsequently, PHEP were cultured with Met (40 µM) or Chol (80 mg/dL) for 24 h. Compared with control or Chol, mRNA and protein abundance of methionine adenosyltransferase 1A (MAT1A) and phosphatidylethanolamine methyltransferase (PEMT) were greater in PHEP treated with Met. The mRNA abundance of S-adenosylhomocysteine hydrolase (SAHH), betaine-homocysteine methyltransferase (BHMT), and sarcosine dehydrogenase (SARDH) was greater in Met-treated PHEP compared with control or Chol. Compared with control, greater expression of 5-methyltetrahydrofolate-homocysteine methyltransferase (MTR), betaine aldehyde dehydrogenase (BADH), and choline dehydrogenase (CHDH) was observed in cells supplemented with Met and Chol. However, Chol led to the greatest mRNA abundance of CHDH. Abundance of choline kinase α (CHKA), choline kinase ß (CHKB), phosphate cytidylyltransferase 1 α (PCYT1A), and choline/ethanolamine phosphotransferase 1 (CEPT1) in the CDP-choline pathway was greater in PHEP treated with Chol compared with control or Met. In the transsulfuration pathway, mRNA and protein abundance of cystathionine ß-synthase (CBS) was greater in PHEP treated with Met compared with control or Chol. Similarly, abundance of cysteine sulfinic acid decarboxylase (CSAD), glutamate-cysteine ligase, catalytic subunit (GCLC), and glutathione reductase (GSR) was greater in response to Met compared with control or Chol. Overall, these findings suggest that transmethylation and transsulfuration in dairy cow primary liver cells are more responsive to Met supply, whereas the CDP-choline pathway is more responsive to Chol supply. The relevance of these data in vivo merit further study.