Effect of supplementing live Saccharomyces cerevisiae yeast on performance, rumen function, and metabolism during the transition period in Holstein dairy cows.

Dairy cows have to face several nutritional challenges during the transition period, and live yeast supplementation appears to be beneficial in modulating rumen activity. In this study, we evaluated the effects of live yeast supplementation on rumen function, milk production, and metabolic and inflammatory conditions. Ten Holstein multiparous cows received either live Saccharomyces cerevisiae (strain Sc47; SCY) supplementation from -21 to 21 d from calving (DFC) or a control diet without yeast supplementation. Feed intake, milk yield, and rumination time were monitored until 35 DFC, and rumen fluid, feces, milk, and blood samples were collected at different time points. Compared with the control diet, SCY had increased dry matter intake (16.7 vs. 19.1 ± 0.8 kg/d in wk 2 and 3) and rumination time postpartum (449 vs. 504 ± 19.9 min/d in wk 5). Milk yield tended to be greater in SCY (40.1 vs. 45.2 ± 1.7 kg/d in wk 5), protein content tended to be higher, and somatic cell count was lower. In rumen fluid, acetate molar proportion was higher and that of propionate lower at 21 DFC, resulting in increased acetate:propionate and (acetate + butyrate):propionate ratios. Cows in the SCY group had lower fecal dry matter but higher acetate and lower propionate proportions on total volatile fatty acids at 3 DFC. Plasma analysis revealed a lower degree of inflammation after calving in SCY (i.e., lower haptoglobin concentration at 1 and 3 DFC) and a likely better liver function, as suggested by the lower γ-glutamyl transferase, even though paraoxonase was lower at 28 DFC. Plasma IL-1ß concentration tended to be higher in SCY, as well as Mg and P. Overall, SCY supplementation improved rumen and hindgut fermentation profiles, also resulting in higher dry matter intake and rumination time postpartum. Moreover, the postcalving inflammatory response was milder and liver function appeared to be better. Altogether, these effects also led to greater milk yield and reduced the risk of metabolic diseases.

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 ß-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.

Methyl donor supply to heat stress-challenged polymorphonuclear leukocytes from lactating Holstein cows enhances 1-carbon metabolism, immune response, and cytoprotective gene network abundance.

Mechanisms controlling immune function of dairy cows are dysregulated during heat stress (HS). Methyl donor supply-methionine (Met) and choline (Chol)-positively modulates innate immune function, particularly antioxidant systems of polymorphonuclear leukocytes (PMN). The objective of this study was to investigate the effect of Met and Chol supply in vitro on mRNA abundance of genes related to 1-carbon metabolism, inflammation, and immune function in short-term cultures of PMN isolated from mid-lactating Holstein cows in response to heat challenge. Blood PMN were isolated from 5 Holstein cows (153 ± 5 d postpartum, 34.63 ± 2.73 kg/d of milk production; mean ± SD). The PMN were incubated for 2 h at thermal-neutral (37°C; TN) or heat stress (42°C; HS) temperatures with 3 levels of Chol (0, 400, or 800 µg/mL) or 3 ratios of Lys:Met (Met; 3.6:1, 2.9:1, or 2.4:1). Supernatant concentrations of IL-1ß, IL-6, and tumor necrosis factor-α were measured via bovine-specific ELISA. Fold-changes in mRNA abundance were calculated separately for Chol and Met treatments to obtain the fold-change response at 42°C (HS) relative to 37°C (TN). Data were subjected to ANOVA using PROC MIXED in SAS (SAS Institute Inc., Cary, NC). Orthogonal contrasts were used to determine the linear or quadratic effect of Met and Chol for mRNA fold-change and supernatant cytokine concentrations. Compared with PMN receiving 0 µg of Chol/mL, heat-stressed PMN supplemented with Chol at 400 or 800 µg/mL had greater fold-change in abundance of CBS, CSAD, GSS, GSR, and GPX1. Among genes associated with inflammation and immune function, fold-change in abundance of TLR2, TLR4, IRAK1, IL1B, and IL10 increased with 400 and 800 µg of Chol/mL compared with PMN receiving 0 µg of Chol/mL. Fold-change in abundance of SAHH decreased linearly at increasing levels of Met supply. A linear effect was detected for MPO, NFKB1, and SOD1 due to greater fold-change in abundance when Met was increased to reach Lys:Met ratios of 2.9:1 and 2.4:1. Although increasing Chol supply upregulated BAX, BCL2, and HSP70, increased Met supply only upregulated BAX. Under HS conditions, enhancing PMN supply of Chol to 400 µg/mL effectively increased fold-change in abundance of genes involved in antioxidant production (conferring cellular processes protection from free radicals and reactive oxygen species), inflammatory signaling, and innate immunity. Although similar outcomes were obtained with Met supply at Lys:Met ratios of 2.9:1 and 2.4:1, the response was less pronounced. Both Chol and Met supply enhanced the cytoprotective characteristics of PMN through upregulation of heat shock proteins. Overall, the modulatory effects detected in the present experiment highlight an opportunity to use Met and particularly Chol supplementation during thermal stress.

Feeding synthetic zeolite to transition dairy cows alters neutrophil gene expression.

Synthetic zeolites are used to control the availability of dietary minerals (e.g., Ca, Mg, and P) in dairy cows. Due to calcium demand increasing with lactation onset, most cows become hypocalcemic immediately postpartum, which likely contributes to poorer immune function because calcium is important for immune cell signaling. To overcome postpartum hypocalcemia, we fed transition cows synthetic zeolite A (sodium aluminosilicate) precalving and hypothesized that it would alter calcium and thus neutrophil function during the transition period. Multiparous Holstein-Friesian cows in late gestation were randomly allocated to an untreated control group (n = 10) or a treatment group in which each cow received 500 g of zeolite A daily (n = 10) for 14 d prior to the expected calving date (actual duration = 17 ± 3 d prepartum). The cows grazed pasture, and each was supplemented with 2 kg/d of maize silage (dry matter basis), with or without zeolite, until calving. Blood samples for neutrophil isolation and analysis of plasma indicators of mineral status, energy status, liver function, and inflammation were collected pretreatment (covariate; d -19); on d -14 and -7 precalving; on the day of calving (d 0); and on d 1, 4, 7, and 28 postcalving. Neutrophils were isolated and gene expression was analyzed using microfluidic gene expression arrays. Neutrophil respiratory burst was assessed using stimulation with phorbol 12-myristate 13-acetate and flow cytometry. Plasma calcium and phosphorus revealed a treatment by time interaction; cows offered zeolite had greater plasma calcium concentrations at d 0, 1, and 4 postcalving and plasma phosphorus concentrations were lower in zeolite-treated cows during the precalving period until d 1 postcalving compared with control animals. Zeolite treatment downregulated neutrophil gene expression of CXCR4 and S100A8 and tended to lower gene expression for other immune mediators (CXCR1, IFNG, S100A12, and S100A9) compared with the control. Zeolite treatment did not affect neutrophil respiratory burst or expression of the other genes investigated. Plasma concentrations of cytokine IL-6 were reduced with zeolite treatment, which was most evident immediately postcalving (d 0, 1, and 7). Overall, feeding zeolite precalving had few effects on neutrophil gene expression and function; however, the lower gene expression of neutrophil inflammatory mediators may be due to altered availability of dietary minerals prepartum and indicates that zeolite A may control inflammation during the transition period.

Inflammation and oxidative stress transcription profiles due to in vitro supply of methionine with or without choline in unstimulated blood polymorphonuclear leukocytes from lactating Holstein cows.

Neutrophils are the most important polymorphonuclear leukocytes (PMNL), representing the front-line defense involved in pathogen clearance upon invasion. As such, they play a pivotal role in immune and inflammatory responses. Isolated PMNL from 5 mid-lactating Holstein dairy cows were used to evaluate the in vitro effect of methionine (Met) and choline (Chol) supplementation on mRNA expression of genes related to the Met cycle and innate immunity. The target genes are associated with the Met cycle, cell signaling, inflammation, antimicrobial and killing mechanisms, and pathogen recognition. Treatments were allocated in a 3 × 3 factorial arrangement, including 3 Lys-to-Met ratios (L:M, 3.6:1, 2.9:1, or 2.4:1) and 3 levels of supplemental Chol (0, 400, or 800 µg/mL). Three replicates per treatment group were incubated for 2 h at 37°C and 5% atmospheric CO2. Both betaine-homocysteine S-methyltransferase and choline dehydrogenase were undetectable, indicating that PMNL (at least in vitro) cannot generate Met from Chol through the betaine pathway. The PMNL incubated without Chol experienced a specific state of inflammatory mediation [greater interleukin-1ß (IL1B), myeloperoxidase (MPO), IL10, and IL6] and oxidative stress [greater cysteine sulfinic acid decarboxylase (CSAD), cystathionine gamma-lyase (CTH), glutathione reductase (GSR), and glutathione synthase (GSS)]. However, data from the interaction L:M × Chol indicated that this negative state could be overcome by supplementing additional Met. This was reflected in the upregulation of methionine synthase (MTR) and toll-like receptor 2 (TLR2); that is, pathogen detection ability. At the lowest level of supplemental Chol, Met downregulated GSS, GSR, IL1B, and IL6, suggesting it could reduce cellular inflammation and enhance antioxidant status. At 400 µg/mL Chol, supplemental Met upregulated PMNL recognition capacity [higher TLR4 and L-selectin (SELL)]. Overall, enhancing the supply of methyl donors to isolated unstimulated PMNL from mid-lactating dairy cows leads to a low level of PMNL activation and upregulates a cytoprotective mechanism against oxidative stress. Enhancing the supply of Met coupled with adequate Chol levels enhances the gene expression of PMNL pathogen-recognition mechanism. These data suggest that Chol supply to PMNL exposed to low levels of Met effectively downregulated the entire repertoire of innate inflammatory-responsive genes. Thus, Met availability in PMNL during an inflammatory challenge may be sufficient for mounting an appropriate biologic response.

Short communication: Supply of methionine during late pregnancy enhances whole-blood innate immune response of Holstein calves partly through changes in mRNA abundance in polymorphonuclear leukocytes.

The supply of methionine (Met) in late pregnancy can alter mRNA abundance of genes associated with metabolism and immune response in liver and polymorphonuclear leukocytes (PMN) of the neonatal calf. Whether prenatal supply of Met elicits postnatal effects on systemic inflammation and innate immune response of the calf is not well known. We investigated whether enhancing the maternal supply of Met via feeding ethyl-cellulose rumen-protected Met (RPM) was associated with differences in calf innate immune response mRNA abundance in PMN and systemic indicators of inflammation during the first 50 d of life. Calves (n = 14 per maternal diet) born to cows fed RPM at 0.09% of diet dry matter per day (MET) for the last 28 ± 2 d before calving or fed a control diet with no added Met (CON) were used. Blood for biomarker analysis and isolation of PMN for innate immune function assays and mRNA abundance was harvested at birth (before colostrum feeding) and at 7, 21 and 50 d of age. Whole blood was challenged with enteropathogenic bacteria (Escherichia coli 0118:H8) and phagocytosis and oxidative burst of neutrophils and monocytes were quantified via flow cytometry. Although concentration of haptoglobin and activity of myeloperoxidase among calves from both maternal groups increased markedly between 0 and 7 d of age followed by a decrease to baseline at d 21 the responses were lower in MET compared with CON calves. Nitric oxide concentration decreased markedly between 0 and 7 d regardless of maternal group but MET calves tended to have lower overall concentrations during the study. In vitro phagocytosis in stimulated neutrophils increased markedly over time in both CON and MET calves but responses were overall greater in MET calves. Oxidative burst in both neutrophils and monocytes increased over time regardless of maternal treatment. The mRNA abundance of lactate dehydrogenase (LDHA) signal transducer and activator of transcription 3 (STAT3) and S100 calcium binding protein A8 (S100A8) in PMN was overall greater in MET calves. Overall data suggest that increasing the maternal supply of Met during late pregnancy could affect the neonatal calf inflammatory status and innate immune response. Although changes in mRNA abundance could play a role in coordinating the immune response the exact mechanisms merit further study.

Evaluation of the capillary electrophoresis method for measurement of immunoglobulin concentration in ewe colostrum.

Capillary electrophoresis (CE) is a technique routinely used in clinical laboratories that allows the separation and quantification of blood serum proteins in a rapid, precise, accurate, and inexpensive manner. Recently, CE has been proposed to separate and measure colostral proteins, but an evaluation of the agreement between CE and radial immunodiffusion (RID) method, currently used to quantify IgG in colostrum, is still lacking. The purpose of this study was to test the ability of a CE instrument, normally used in blood serum protein analysis, to realize the correct quantification of total Ig concentration in ewe colostrum, using RID assay as reference. Colostrum samples (n = 68) were collected from 35 multiparous Sarda ewes at first milking (n = 33) and at 24 h postpartum (n = 35). The mean ± standard deviation of IgG concentration measured by RID and whey colostrum total Ig concentration measured by CE were 54.76 ± 41.82 g/L and 54.70 ± 41.43 g/L, respectively. Lin's concordance correlation coefficient (r = 0.993; 95% confidence interval = 0.989 to 0.996) and linear regression analysis results (RID = 1.0022CE - 0.063; R2 = 0.986) showed an excellent agreement between these 2 methods. Bland-Altman analysis confirmed that CE method can be a suitable alternative to RID: the mean of the differences between CE and RID was -0.055 ± 4.95 g/L (95% confidence interval = -1.25 to 1.14 g/L) and the agreement limits were -9.75 to 9.60 g/L (low limit 95% confidence interval = -11.82 to -7.68 g/L; high limit 95% confidence interval = 7.57 to 11.72 g/L). In conclusion, the current study indicates that CE method may be a reliable tool for the quantification of the total Ig concentration in ewe colostrum.