Effects of rumen-protected niacin on inflammatory response to repeated intramammary lipopolysaccharide challenges.

Nutritional strategies that improve an animal's resilience to various challenges may improve animal health and welfare. One such nutrient is niacin, which has reduced inflammation in mice, humans, and swine; however, niacin's anti-inflammatory effects have not been investigated in cattle. Our objective was to determine whether rumen-protected niacin (RPN) alters lactating dairy cows' inflammatory response to intramammary LPS challenges, whether RPN resulted in any carryover effects, and whether repeated LPS challenges result in signs of immune tolerance or innate immune training. Twenty healthy, late-lactation Holstein cows (232 ± 65 DIM; 39 ± 5.8 kg/d of milk) were enrolled in a randomized complete block experiment that lasted 70 d. Cows received 26 g/d of RPN or no top-dress (CON) for the first 42 d of the experiment. During the final milking of d 27 and 55, cows were challenged in their rear right (RR) mammary gland with 100 µg of LPS suspended in 5 mL of PBS. Milk yield, milk conductivity, and feed intake were measured daily. Milk composition was measured on d 14, 23, 24, 30, 37, 45, and 52. Blood samples were collected at 0, 8, 12, 24, 48, 72, 96, and 120 h after each LPS challenge, whereas RR quarter milk samples were collected at 0, 8, 16, 24, 48, 72, 96, 120, 144, and 168 h after each LPS challenge. Body temperature was measured continuously during each challenge with an intravaginal thermometer. Linear mixed models with repeated measures were used to analyze the results. Before LPS challenge, RPN did not affect feed intake or milk production, but it reduced SCS (1.24 ± 0.41 [SE] vs. 0.05 ± 0.45). After challenge, RPN did not affect feed intake, milk production, milk composition, SCS, body temperature, plasma glucose, or plasma insulin concentrations. Our results suggest RPN reduced peak plasma haptoglobin and lipopolysaccharide binding protein during the first LPS challenge. Plasma haptoglobin tended to be less after the second challenge for cows previously supplemented RPN, and lipopolysaccharide binding protein was similar for each treatment group after the second challenge. The second LPS challenge resulted in decreased plasma haptoglobin compared with the first LPS challenge, suggestive of tolerance, but it also induced a greater peak SCS than the first LPS challenge. Our results suggest that repeated LPS challenges promote a systemic tolerance but heightened local response to LPS-induced mastitis. Feeding RPN reduced SCS before challenge and reduced plasma acute phase proteins after challenge, suggesting that RPN may reduce systemic inflammation without altering the local inflammatory responses.

Effects of dietary rumen-protected choline supplementation to periparturient dairy cattle on inflammation and metabolism in mammary and liver tissue during an intramammary lipopolysaccharide challenge.

The objective of this experiment was to examine the effects of supplementation and dose of rumen-protected choline (RPC) on markers of inflammation and metabolism in liver and mammary tissue during an intramammary lipopolysaccharide (LPS) challenge. Parous Holstein cows were blocked by calving month and randomly assigned within block to receive 45 g/d of RPC (20.4 g/d of choline ions; CHOL45), 30 g/d of RPC (13.6 g/d of choline ions; CHOL30), or no RPC (CON) as a top-dress starting 24 d before expected calving until 21 d postpartum. Cows were alternately assigned within treatment group to either receive an intramammary LPS challenge (200 µg in each rear quarter; Escherichia coli O111:B4) or not at 17 DIM (CHOL45, n = 9; CHOL45-LPS, n = 9; CHOL30, n = 11; CHOL30-LPS, n = 10; CON, n = 10; CON-LPS, n = 9). Hepatic and mammary tissues were collected from all cows on d 17 postpartum. Hepatic and mammary tissues were collected at ∼7.5 and 8 h, respectively, after the LPS challenge. An additional mammary biopsy was conducted on LPS-challenged cows (CHOL45-LPS, CHOL30-LPS, and CON-LPS) at 48 h postchallenge. Hepatic and mammary RNA copy numbers were quantified for genes involved in apoptosis, methylation, inflammation, oxidative stress, and mitochondrial function using NanoString technology. Targeted metabolomics was conducted only on mammary tissue samples (both 8 and 48 h biopsies) to quantify 143 metabolites including choline metabolites, amino acids, biogenic amines and derivatives, organic acids, carnitines, and glucose. Hepatic IFNG was greater in CHOL45 as compared with CON in unchallenged cows, suggesting an improvement in type 1 immune responses. Hepatic CASP3 was greater in CHOL45-LPS as compared with CON-LPS, suggesting greater apoptosis. Mammary IL6 was reduced in CHOL30-LPS cows as compared with CHOL45-LPS and CON-LPS (8 and 48 h). Mammary GPX4 and COX5A were reduced in CHOL30-LPS as compared with CON-LPS (8 h), and SDHA was reduced in CHOL30-LPS as compared with CON-LPS (8 and 48 h). Both CHOL30-LPS and CHOL45-LPS cows had lesser mammary ATP5J than CON-LPS, suggesting that dietary RPC supplementation altered mitochondrial function following LPS challenge. Treatment did not affect mammary concentrations of any metabolite in unchallenged cows, and only 4 metabolites were affected by dietary RPC supplementation in LPS-challenged cows. Mammary concentrations of isobutyric acid and 2 acyl-carnitines (C4:1 and C10:2) were reduced in CHOL45-LPS as compared with CHOL30-LPS and CON-LPS. Taken together, reductions in medium- and short-chain carnitines along with an increase in long-chain carnitines in mammary tissue from CHOL45-LPS cows suggests less fatty acid entry into the ß oxidation pathway. Although the intramammary LPS challenge profoundly affected markers for inflammation and metabolism in liver and mammary tissue, dietary RPC supplementation had minimal effects on inflammatory markers and the mammary metabolome.

Effects of dietary rumen-protected choline supplementation to periparturient dairy cattle on inflammation, metabolism, and performance during an intramammary lipopolysaccharide challenge.

Recent studies have suggested that dietary rumen-protected choline (RPC) supplementation can modulate immune function, attenuate inflammation, and improve performance in periparturient dairy cattle; however, this has yet to be evaluated during a mastitis challenge. Therefore, the objective of this study was to examine the effects of supplementation and dose of RPC on metabolism, inflammation, and performance during an intramammary lipopolysaccharide (LPS) challenge. Parous Holstein cows (parity, mean ± SD, 1.9 ± 1.1 at enrollment) were blocked by calving month and randomly assigned within block to receive either 45 g/d of RPC (20.4 g/d of choline ions; CHOL45, n = 18), 30 g/d of RPC (13.6 g/d of choline ions; CHOL30, n = 21), or no RPC (CON, n = 19) as a top-dress starting 24 d before expected calving until 21 d postpartum. Cows were alternately assigned within treatment group to either receive an intramammary LPS challenge (200 µg in each rear quarter; Escherichia coli O111:B4) or not at 17 DIM. Before the challenge, CHOL45 and CHOL30 cows produced 3.4 and 3.8 (±1.2 SED) kg/d more milk than CON, respectively. Dietary RPC supplementation did not mitigate the milk loss associated with the intramammary LPS challenge; however, CHOL45 and CHOL30 cows produced 3.1 and 3.5 (±1.4 SED) kg/d more milk than CON, respectively in the carryover period (22 to 84 DIM). Dietary RPC supplementation enhanced plasma ß-hydroxybutyrate (BHB) concentrations before the LPS challenge, and increased plasma nonesterified fatty acids (NEFA) and acetylcarnitine concentrations during the LPS challenge, potentially reflecting greater adipose tissue mobilization, fatty acid transport and oxidation. Aside from trimethylamine N-oxide and sarcosine, which were increased in CHOL45-LPS as compared with CON-LPS, most other choline metabolite concentrations in plasma were unaffected by treatment, likely because more choline was being secreted in milk. Plasma lactic acid concentrations were decreased in CHOL45-LPS and CHOL30-LPS as compared with CON-LPS, suggesting a reduction in glycolysis or an enhancement in the flux through the lactic acid cycle to support gluconeogenesis. Plasma concentrations of fumaric acid, a byproduct of AA catabolism and the urea cycle, were increased in both choline groups as compared with CON-LPS during the LPS challenge. Cows in the CHOL45 group had greater plasma antioxidant potential before the LPS challenge and reduced plasma methionine sulfoxide concentrations during the LPS challenge compared with CON-LPS, suggesting an improvement in oxidant status. Nevertheless, concentrations of inflammatory markers such as haptoglobin and tumor necrosis factor α (TNFα) were not affected by treatment. Taken together, our data suggest that the effects of dietary RPC supplementation on milk yield could be mediated through metabolic pathways and are unlikely to be related to the resolution of inflammation in periparturient dairy cattle. Lastly, dose responses to dietary RPC supplementation were not found for various economically important outcomes including milk yield, limiting the justification for feeding a greater dietary RPC dose in industry.

Effects of dietary rumen-protected choline supplementation on colostrum yields, quality, and choline metabolites from dairy cattle.

Colostrum is a critical nutrient source that provides passive immunity to dairy calves. Choline is a trimethylated molecule that is frequently supplemented in the diet to periparturient dairy cows to support postpartum health and performance. Whereas choline and its metabolites have been characterized in milk, the effects of dietary rumen-protected choline (RPC) supplementation on choline metabolites in colostrum from dairy cattle have yet to be explored. Therefore, the objective of the present study was to assess the effects of dietary supplementation and dose of RPC on colostrum yields, quality, and choline metabolites. Parous Holstein cows were blocked by calving month and randomly assigned within block to receive 45 g/d (20.4 g/d of choline ions) of RPC (CHOL45, n = 22), 30 g/d (13.6 g/d of choline ions) of RPC (CHOL30, n = 20), or no RPC (control, n = 19) starting 24 d before expected calving. The effects of dietary supplementation and dose of RPC were assessed on colostrum yields, component yields, somatic cell score (SCS), quality (as assessed by Brix), and choline metabolites. Data were analyzed using a linear mixed model with the fixed effects of treatment, parity, and the 2-way interaction and the random effect of block. Regardless of dose, dietary RPC supplementation increased colostrum yields and protein yields. No effects of dietary RPC supplementation were found on colostrum component percentages, SCS, or colostrum quality. For choline metabolites, treatment interacted with parity for phosphocholine where colostrum from second-parity CHOL45 and CHOL30 cows had greater concentrations of phosphocholine than colostrum from second-parity control cows, but no treatment effect was seen in the colostrum from 3+ parity cows. Dietary choline supplementation, regardless of dose, increased trimethylamine N-oxide concentrations. Dietary choline supplementation did not affect the concentrations of choline, betaine, glycerophosphocholine, sphingomyelin, phosphatidylcholine, or total choline in colostrum. In conclusion, dietary choline supplementation increased phosphocholine concentrations in colostrum from second-parity cows, enhanced trimethylamine N-oxide concentrations, and increased colostrum yields without affecting colostrum quality.

Effects of prenatal dietary rumen-protected choline supplementation during late gestation on calf growth, metabolism, and vaccine response.

The objective of this study was to examine the effects of prenatal supplementation and dose of rumen-protected choline (RPC) on neonatal calf growth, metabolism, and vaccine response. Parous Holstein cows were blocked by calving month and randomly assigned within block to receive 45 g/d of RPC [20.4 g/d of choline ions (CHOL45), n = 19], 30 g/d of RPC [13.6 g/d of choline ions (CHOL30), n = 22], or no RPC (CON, n = 19) as a top-dress, starting 24 d before expected calving. Calf body weights were recorded for the first 3 wk of life. All calves were fed colostrum replacer (300 g of IgG) at birth, and apparent efficiency of IgG absorption was calculated. On d 1, 7, 14, and 21, blood samples were taken to quantify plasma reactive oxygen and nitrogen species, antioxidant potential, haptoglobin, nonesterified fatty acids (NEFA), ß-hydroxybutyrate, and glucose. Calves received an intranasal vaccine at birth, and nasal secretions were collected on d 0, 7, 10, 14, and 21 to quantify bovine respiratory syncytial virus-specific IgA. Data were analyzed using linear mixed models including the fixed effects of treatment, time (when applicable), calf sex, and prepartum dam data (-24 d) along with interactions. Treatment did not affect calf body weight, ß-hydroxybutyrate, or glucose concentrations. For apparent efficiency of IgG absorption, treatment interacted with the dam's prepartum body condition score. Where the dam's body condition score was ≤3.25, IgG absorption was reduced in calves born from CHOL45 dams as compared with calves from either CHOL30 or CON dams. Calves from CHOL30 dams had a lesser oxidative stress index (OSi; reactive oxygen and nitrogen species/antioxidant potential) than calves from CON dams. Haptoglobin concentrations were less in heifer calves from CHOL45 dams as compared with heifers from CON dams. The dam's prepartum NEFA concentration interacted with treatment. When dam NEFA was minimal, calves from CHOL45 and CHOL30 dams had greater or tended to have greater NEFA, respectively. Conversely, when dam NEFA was greater, calves from CHOL30 and CHOL45 dams had lesser or tended to have lesser NEFA than calves from CON dams, respectively. For vaccine response, treatment interacted with the dam's prepartum OSi. Among calves born from dams with a greater OSi, calves from CHOL45 and CHOL30 dams had lesser bovine respiratory syncytial virus-specific IgA concentrations in nasal secretions as compared with CON. Prenatal RPC supplementation during late gestation affected IgG absorption, neonatal calf metabolism, and vaccine response with some effects dependent on the dam's prepartum parameters.

Effect of Saccharomyces cerevisiae fermentation product on oxidative status, inflammation, and immune response in transition dairy cattle.

Dairy cattle are subjected to oxidative stress, inflammation, and altered immune function during the transition to lactation. The objective of this study was to evaluate the effects of a dietary Saccharomyces cerevisiae fermentation product (SCFP; NutriTek, Diamond V) on oxidative status, inflammation, and innate and adaptive immune responses during the transition period. Holstein cows were blocked by parity, expected calving date, and previous milk yield and then randomly assigned to treatment within block. Treatment was a control total mixed ration (n = 30) or SCFP total mixed ration (n = 34) fed from -29 ± 5 to 42 d relative to calving (RTC). Blood was sampled during wk -4, -2, 1, 2, and 5 and liver tissue at wk -3 and 2 RTC. Oxidative status was evaluated in plasma by retinol, α-tocopherol, and malondialdehyde concentrations, glutathione peroxidase activity, and Trolox equivalent antioxidant capacity, and in liver by mRNA abundance of nuclear factor E2-related factor 2 (NFE2L2), metallothionein 1E (MT1E), and glutathione peroxidase 3 (GPX3). Inflammation was evaluated in plasma by haptoglobin (HP) and serum amyloid A (SAA) concentrations and in liver by mRNA abundance of HP, serum amyloid A3 (SAA3), and nuclear factor kappa-light-chain-enhancer of activated B cells (NFKB1). Innate immune response was measured by stimulated oxidative burst of polymorphonuclear cells (neutrophils) isolated from blood. Ovalbumin (OVA) was administered with adjuvant on d 7 and 21 RTC, and adaptive immune response was evaluated by serum anti-OVA IgG content on d 28 and 35. Mixed models were used to assess effects of treatment, time, parity, and all interactions. We previously reported that SCFP had limited effects on productivity in this cohort, although milk fat yield was transiently increased and subclinical ketosis incidence was increased. Supplementation with SCFP did not affect overall oxidative, inflammatory, or immune parameters. The only treatment × week interaction detected was for plasma α-tocopherol concentration, which tended to be greater in control cows during wk 2 RTC. A tendency for a treatment × parity interaction was detected for serum anti-OVA IgG titer, which tended to be greater for SCFP than for controls among primiparous cows. Plasma inflammatory biomarkers were not affected by SCFP but, unexpectedly, plasma HP was elevated at both prepartum time points and plasma SAA was elevated during wk -2 RTC compared with the expected increases in both biomarkers postpartum. In this cohort of transition cows with low disease incidence, SCFP generally did not affect oxidative, inflammatory, or immune parameters.

Effects of sodium salicylate and time postpartum on mammary tissue proliferation, gene transcript profile, and DNA methylation.

Previous studies have demonstrated nonsteroidal antiinflammatory drug treatment in early lactation had a positive impact on whole-lactation milk production in older cows. The objective of this study was to evaluate proliferative, transcriptional, and epigenetic changes in the mammary gland that could explain increased production responses due to nonsteroidal antiinflammatory drug treatment. Sodium salicylate (SAL; 125 g/d) or water (CON) were administered via oral drench to multiparous Holstein cows (n = 8/treatment) once daily for 3 d beginning approximately 24 h after parturition, and mammary tissue was collected on d 1, 4, and 45 postpartum. Day 1 tissue was collected immediately preceding the initial drench, and d 4 tissue was collected 24 h following the final drench. Blood was collected twice weekly and analyzed for plasma glucose, insulin, ß-hydroxybutyrate, free fatty acids, and prolactin. Cows were milked twice daily until d 7 of lactation, and thrice daily for the remainder of the study. Total RNA extracted from tissue was deep-sequenced and analyzed for differential gene expression using DESeq2. We detected no treatment effect on milk yield or plasma metabolites through 45 d of lactation; additionally, no change in mammary epithelial cell proliferation was detected when assessed by Ki67 labeling. Comparison of SAL versus CON revealed that only 16 of 18,286 genes were differentially expressed (false discovery rate <0.1) in mammary tissue collected on d 45, whereas no differentially expressed genes due to treatment were detected on d 1 or 4. Analysis of transcriptional differences over time showed downregulation of pathways related to immune cell recruitment and differentiation, and extensive overlap with pathways related to cholesterol synthesis and liver X receptor signaling. Global DNA methylation of mammary tissue was decreased for CON compared with SAL. Transcriptome analysis emphasized extensive involvement of immune-related signaling pathways in the switch from lactogenesis to galactopoiesis, and changes in methylation with SAL treatment merit future investigation into epigenetic effects on milk production.

Sodium salicylate reduced mRNA abundance of hypoxia-associated genes in MAC-T cells.

Hypoxia is an oxygen deficiency commonly found in growing tissues and is speculated to occur in the rapidly developing mammary gland in peripartum dairy cattle. Low oxygen concentrations can activate hypoxia-inducible factor-1 (HIF-1), which increases transcription of genes involved in angiogenesis (VEGFA) and glucose transport (GLUT1), among other processes. The mRNA stability of these genes is positively regulated by heterogeneous nuclear ribonucleoprotein D (HNRNPD; also known as AUF1). In our previous research, postpartum administration of sodium salicylate (SS) increased whole-lactation milk yield in multiparous cows but tended to reduce milk yield in primiparous cows. Because rapid mammary tissue development likely occurs in cows approaching first lactation, we hypothesized that SS inhibited the activation of HIF-1α and decreased transcription of downstream targets. MAC-T cells were treated with SS (100 µM) or control medium before incubation under either hypoxic (1% O2) or normoxic conditions for 12 h. Additionally, cells were transfected with either HIF1A small interfering RNA (siRNA) or a scrambled siRNA negative control 48 h before hypoxia treatments. HIF1A, GLUT1, VEGFA, and HNRNPD were quantified relative to the internal control gene NENF. Transcript abundance was assessed using a linear mixed model with the fixed effects of SS, hypoxia, siRNA, and all 2- and 3-way interaction terms and the random effect of plate nested within hypoxia. Treatment with SS interacted with hypoxia for GLUT1, as SS reduced GLUT1 when MAC-T cells were cultured in normoxic conditions; however, no effect of SS was found in hypoxia-treated cells. Regardless of oxygen status, SS reduced HNRNPD and tended to decrease VEGFA mRNA relative to untreated cells. Hypoxia increased GLUT1, yet no effect was observed on VEGFA or HNRNPD. Small interfering RNA knocked down HIF1A, but no effect was found on GLUT1, VEGFA, or HNRNPD. In conclusion, SS reduced transcript abundance of genes involved with mammary gland development but generally did not interact with oxygen status.

Diverging in vitro inflammatory responses toward Streptococcus uberis in mouse macrophages either preconditioned or continuously treated with ß-hydroxybutyrate.

Hyperketonemia is a common condition in early-lactation dairy cows that has been associated with an increase in the risk of infectious disease. Recent mouse studies have elucidated an anti-inflammatory effect of the ketone body ß-hydroxybutyrate (BHB). Therefore, the objective of this study was to determine whether BHB altered inflammatory responses in macrophages challenged with the common mastitis pathogen Streptococcus uberis. A secondary objective was to determine whether the inflammatory response to the S. uberis challenge was dependent on whether BHB was present in the medium during the challenge (i.e., preconditioned vs. continuous treatment). Two cell culture experiments were conducted. In the first experiment, mouse macrophages (RAW 264.7 line) were preconditioned with BHB (0, 0.6, 1.2, and 1.8 mM) for 24 h; the medium was then replaced with a standard cell culture medium, and the cells were challenged or not with S. uberis for an additional 6 h. In the second experiment, a similar protocol was used; however, cells were preconditioned with BHB (0, 0.6, 1.2, and 1.8 mM) for 24 h, the medium was replaced with fresh medium containing the same concentration of BHB, and cells were either challenged or not with S. uberis for 6 h. In both experiments, relative transcript abundance of cell membrane receptors (Tlr2 and Gpr109a), cytokines (Il1b, Il10, Tnf, and Tgfb1), and chemokines (Cxcl2 and Ccl5) were determined using quantitative real-time PCR and normalized against the geometric mean of Hprt and B2m. Data were analyzed using a linear mixed model, and orthogonal contrasts were conducted to examine the effect of S. uberis challenge and BHB treatment. Streptococcus uberis activated the macrophages, noted by greater transcript abundance of analyzed genes. Intriguingly, in both experiments, the S. uberis challenge increased expression of Gpr109a, which encodes a receptor that is ligated by BHB. Paradoxically, preconditioning macrophages with BHB increased transcript abundance of the immunosuppressive cytokine Tgfb1 and increased that of the neutrophil chemoattractant Cxcl2. Preconditioning decreased Tlr2 and tended to decrease Il10 transcript abundance. In opposition to the preconditioning experiment, continuous treatment of BHB during the S. uberis challenge linearly increased abundance of Tlr2 and Il10 transcripts. Continuous BHB treatment also increased expression of Il1b. In conclusion, BHB treatment altered macrophage inflammatory responses during an S. uberis challenge; however, the direction of this response was dependent on whether BHB was added to the medium during the S. uberis challenge. Future studies should be conducted using bovine macrophages and in vivo approaches to examine BHB effects during an S. uberis challenge.

Acute-phase protein α-1-acid glycoprotein is negatively associated with feed intake in postpartum dairy cows.

α-1-Acid glycoprotein (AGP) is an acute-phase protein that may suppress dry matter intake (DMI), potentially by acting on the leptin receptor in the hypothalamus. Our objectives were to characterize plasma AGP concentration and associations with DMI during the transition period, and to determine the utility of AGP to identify or predict cows with low DMI. Plasma samples (n = 2,086) from 434 Holstein cows in 6 studies were analyzed on d -21, -13 ± 2, -3, 1, 3, 7 ± 1, 14 ± 1, and 21 ± 1 relative to parturition. A commercially available ELISA kit specific for bovine AGP was validated, and 2 internal controls were analyzed on each plate with interplate variation of 15.0 and 17.3%, respectively. Bivariate analysis was used to assess the relationship between AGP and DMI. For significant associations, treatment(study) was added to the model, and quadratic associations were included in the model if significant. Plasma AGP concentration (±SEM) increased from 213 ± 37.3 µg/mL on d -3 to 445 ± 60.0 µg/mL on d 14. On d 3, AGP was associated negatively with DMI in a quadratic manner for wk 1 and wk 2 and linearly for wk 3. Day 7 AGP was associated negatively with DMI in a quadratic manner for wk 2 and linearly for wk 3. Similarly, d 14 AGP was negatively associated with DMI for wk 3 and wk 4. As d 3 AGP concentration increased over the interquartile range, a calculated 1.4 (8.5%), 0.5 (2.7%), and 0.4 (1.9%) kg/d reduction in predicted DMI was detected during wk 1, 2, and 3, respectively. Using bivariate analysis, d 3 AGP explained 10% of the variation in DMI during wk 1. We explored the clinical utility of d 3 AGP to diagnose low DMI, defined as wk 1 DMI >1 standard deviation below the mean. Receiver operating characteristic analysis identified a threshold of 480.9 µg/mL, providing 76% specificity and 48% sensitivity (area under the curve = 0.60). Limited associations occurred between AGP and blood biomarkers; however, AGP was associated with plasma haptoglobin concentration postpartum and incidence of displaced abomasum, retained placenta, and metritis. These results demonstrate a negative association between plasma AGP concentration and DMI in early-postpartum dairy cows, although its diagnostic performance was marginal. Further investigation into whether AGP directly suppresses DMI in dairy cattle is warranted.

Development of an in vitro macrophage screening system on the immunomodulating effects of feed components.

BACKGROUND: While feed components capable of modulating the immune system are highly sought after and marketed, often little evidence is available to support functional immune response claims. Thus, a high-throughput in vitro cell screening system was developed to test these compounds for innate immune signaling effects, using Saccharomyces cerevisiae and its cell wall components in addition to lauric acid and its esters as models in two separate experiments. This screening system utilized RAW 264.7 murine macrophages to assess live S. cerevisiae cells and S. cerevisiae-derived cell wall components ß-glucan, mannan, and zymosan (a crude cell wall preparation containing both ß-glucan and mannan). D-mannose was also evaluated as the monomer of mannan. We also examined the effect of a saturated fatty acid (C12:0, lauric acid) and its esters (methyl laurate and glycerol monolaurate) on innate immune cell activation and cellular metabolism. RAW cells were transfected with a vector that drives expression of alkaline phosphatase upon promoter activation of nuclear factor κ-light-chain-enhancer of activated B cells (NFκB), a major inflammatory/immune transcription factor. RAW cells were incubated with 0.01, 0.1 or 1 mg/mL of yeast compounds alone or RAW cells were challenged with LPS and then incubated with yeast compounds. In a separate experiment, RAW cells were incubated with 0, 0.5, 2.5, 12.5, 62.5, and 312.5 µmol/L of lauric acid, methyl laurate, or glycerol monolaurate alone, or RAW cells were challenged with LPS and then incubated with fatty acid treatments. RESULTS: Treatment with zymosan or ß-glucan alone induced NFκB activation in a dose-dependent manner, whereas treatment with D-mannose, mannan, or live S. cerevisiae cells did not. Post-treatment with mannan after an LPS challenge decreased NFκB activation, suggesting that this treatment may ameliorate LPS-induced inflammation. Slight increases in NFκB activation were found when fatty acid treatments were applied in the absence of LPS, yet substantial reductions in NFκB activation were seen when treatments were applied following an LPS challenge. CONCLUSIONS: Overall, this cell screening system using RAW macrophages was effective, high-throughput, and sensitive to feed components combined with LPS challenges, indicating modulation of innate immune signaling in vitro.

Effect of prepartum energy intake and supplementation with ruminally protected choline on innate and adaptive immunity of multiparous Holstein cows.

Objectives were to evaluate the effect of prepartum energy intake and peripartal supplementation of ruminally protected choline (RPC) on select indicators of immune status in blood plasma and on lipopolysaccharide-stimulated blood cells ex vivo. At 47 ± 6 d before the expected calving date, 93 multiparous Holstein cows were assigned randomly to 1 of 4 dietary treatments in a 2 × 2 factorial arrangement. Cows were fed energy to excess [EXE; 1.63 Mcal of net energy for lactation (NEL)/kg of dietary dry matter (DM)] or to maintenance (MNE; 1.40 Mcal of NEL/kg of dietary DM) ad libitum throughout the nonlactating period. The RPC was fed at 0 or 60 g/d to supply 0 or 12.9 g/d of choline ions top-dressed for 17 ± 4.6 d prepartum through 21 d postpartum. After calving, cows were fed the same methionine-supplemented diet, apart from RPC supplementation. During the last 2 wk before calving and during the first 5 wk postpartum, blood was sampled repeatedly and analyzed for cell types, acute-phase proteins, tumor necrosis factor-α (TNFα), and neutrophil function. Samples of whole blood were collected at 3 and 14 DIM and stimulated with 1 µg/mL lipopolysaccharide (LPS) in vitro for 6 and 24 h. After 6 h of LPS exposure, peripheral blood leucocytes (PBL) were harvested, and relative transcript abundance for select cytokines were measured. Supernatant was analyzed for TNFα after 24 h of LPS exposure. The PBL from cows fed EXE diets during the whole dry period had increased transcripts for the proinflammatory cytokines CXCL8 and TNF, although the plasma concentrations of the acute-phase proteins haptoglobin and fibrinogen, and the killing activity of the blood neutrophils in the postpartum period, were not affected by feeding different energy levels prepartum. Feeding RPC to cows overfed energy prepartum modulated their inflammatory state, as evidenced by decreased IL6 in PBL and reduced mean fluorescence intensity of CD14 during the postpartum period, compared with cows not fed RPC. Feeding RPC also decreased TNFα protein production, abundances of IL1B, CXCL8, and TNF transcripts, and mean fluorescence intensity of CD80 of PBL stimulated by LPS, regardless of prepartum energy intake. In contrast, proportions of blood neutrophils undergoing phagocytosis and oxidative burst were increased at 17 d postpartum in cows supplemented with RPC. Collectively, these data indicate that transition cows supplemented with RPC experienced less inflammation, which may partially explain increased milk production in cows supplemented with RPC.

Diet starch concentration and starch fermentability affect markers of inflammatory response and oxidant status in dairy cows during the early postpartum period.

Our objective was to evaluate the effects of diet starch concentration and starch fermentability on inflammatory response markers and oxidant status during the early postpartum (PP) period and its carryover effects. Fifty-two multiparous Holstein cows were used in a completely randomized block design experiment with a 2 × 2 factorial arrangement of treatments. Treatments were starch concentration and starch fermentability of diets; diets were formulated to 22% (low starch, LS) or 28% (high starch, HS) starch with dry-ground corn (DGC) or high-moisture corn (HMC) as the primary starch source. Treatments were fed from 1 to 23 d PP and then switched to a common diet until 72 d PP to measure carryover (CO) effects. Treatment period (TP) diets were formulated to 22% forage neutral detergent fiber and 17% crude protein. The diet for the CO period was formulated to 20% forage neutral detergent fiber, 17% crude protein, and 29% starch. Coccygeal blood was collected once a week during the TP and every second week during the CO period. Liver and adipose tissue biopsies were performed within 2 d PP and at 20 ± 3 d PP. Blood plasma was analyzed for concentrations of albumin, haptoglobin, reactive oxygen and nitrogen species (RONS), and antioxidant potential (AOP), with lipopolysaccharide-binding protein (LBP) and TNFα evaluated during the TP only. Oxidative stress index (OSi) was calculated as RONS/AOP. Abundance of mRNA from genes involved in inflammation and glucose metabolism in liver and genes involved in lipogenesis in adipose tissue were determined. Data were analyzed separately for the TP and CO periods. During the TP, treatments interacted to affect concentrations of TNFα, haptoglobin, and LBP, with HMC increasing their concentrations for HS (9.38 vs. 7.45 pg/mL, 0.45 vs. 0.37 mg/mL, and 5.94 vs. 4.48 µg/mL, respectively) and decreasing their concentrations for LS (4.76 vs. 12.9 pg/mL, 0.27 vs. 0.41 mg/mL, and 4.30 vs. 5.87 µg/mL, respectively) compared with DGC. Effects of treatments diminished over time for LBP and haptoglobin with no differences by the end of the TP and no main CO effects of treatment for haptoglobin. The opposite treatment interaction was observed for albumin, with HMC tending to decrease its concentration for HS (3.24 vs. 3.34 g/dL) and increase its concentration for LS (3.35 vs. 3.29 g/dL) compared with DGC, with no carryover effect. Feeding DGC increased the OSi during the first week of the TP compared with HMC, with this effect diminishing over time; during the CO period HMC increased OSi for HS and decreased it for LS compared with DGC, with this effect diminishing toward the end of CO. Feeding HMC increased the abundance of genes associated with inflammation and gluconeogenesis in liver for HS and decreased it for LS compared with DGC. Feeding HS increased the mRNA abundance of genes associated with adipose tissue lipogenesis compared with LS. Results during the TP suggest that feeding LS-DGC and HS-HMC elicited a more pronounced inflammatory response and induced an upregulation of genes associated with inflammation and gluconeogenesis in liver, without effects on OSi, but effects on plasma markers of inflammation diminished during the CO period.

Associations between body condition score at parturition and microRNA profile in colostrum of dairy cows as evaluated by paired mapping programs.

MicroRNA (miRNA) are abundant in milk, and likely have regulatory activity involving lactation and immunity. The objective of this study was to determine the miRNA profile in colostrum of overconditioned cows compared with cows of more moderate body condition score (BCS) at calving. Multiparous cows with either high (≥4.0 on a scale of 1 to 5; n = 7) or moderate BCS (2.75 to 3.50; n = 9) in the week before parturition were selected from a commercial dairy herd. Blood and colostrum were sampled within 24 h after calving. Blood serum was analyzed for free fatty acid (FFA) concentration. MicroRNA was isolated from colostrum samples after removing milk fat and cells. MicroRNA were sequenced, and reads were mapped to the bovine genome and to the existing database of miRNA at miRBase.org. Two programs, Oasis 2.0 and miRDeep2, were employed in parallel for read alignment, and analysis of miRNA count data was performed using DESeq2. Identification of differentially expressed miRNA from DESeq2 was not affected by the differences in miRNA detected by the 2 mapping programs. Most abundant miRNA included miR-30a, miR-148a, miR-181a, let-7f, miR-26a, miR-21, miR-22, and miR-92a. Large-scale shifts in miRNA profile were not observed; however, colostrum of cows with high BCS contained less miR-486, which has been linked with altered glucose metabolism. Colostrum from cows with elevated serum FFA contained less miR-885, which may be connected to hepatic function during the transition period. Potential functions of abundant miRNA suggest involvement in development and maintenance of cellular function in the mammary gland, with the additional possibility of influencing neonatal tissue and immune system development.

Feed restriction to induce and meloxicam to mitigate potential systemic inflammation in dairy cows before calving.

Most dairy cows experience a transient decrease in feed intake in the 1 to 2 wk before calving, which has been associated with systemic inflammation (SI), indicated by increased blood haptoglobin (Hp) concentration. We aimed to characterize the association between prepartum decrease in feed intake and the onset of SI and, if present, the ability of meloxicam (MEL), a non-steroidal anti-inflammatory drug, to mitigate SI. Holstein cows (n = 45) were assigned to control (n = 13), feed restriction (FR) untreated (FR-U; n = 15), and FR treated with MEL (FR-T; n = 17) groups. Daily feed intake was measured from -22 d from expected parturition until 35 d postpartum. Control cows were fed ad libitum, whereas FR-U and FR-T cows were reduced to 60% of their average intake for 4 consecutive days (-15 to -12 d from expected calving). The FR-T cows received MEL (0.5 mg/kg of body weight) once daily for 4 consecutive days (-13 to -10 d from expected calving). Blood samples were collected -22, -15, -14, -13, -12, -10, -7, -5, -3, 0, 1, 3, 5, 7, 15, 22, and 35 d relative to calving to measure serum concentrations of total calcium, total protein, albumin, globulin, cholesterol, urea, glucose, gamma-glutamyl transferase, aspartate aminotransferase, glutamate dehydrogenase, ß-hydroxybutyrate, nonesterified fatty acids, Hp, and insulin-like growth factor-1. Serum concentrations of lipopolysaccharide-binding protein were measured -22, -15, -14, -13, -12, and -10 d from expected calving. Simplified glucose tolerance tests were performed on -15, -12, -5, 1, and 5 d relative to calving. Mixed linear regression models were used to assess the effects of FR and MEL on each metabolite. The interaction between treatment group and blood sampling day was forced into each model. All models accounted for body condition score, parity, and the cow as a random effect. Nonesterified fatty acids concentrations in both the FR-U and FR-T groups significantly increased from the second until the last day of FR. Feed restriction increased urea concentrations compared with the control group on -14 d but decreased urea concentrations on -10 d from expected calving. Control cows had greater ß-hydroxybutyrate concentrations compared with FR cows on 15, 21, and 35 d postpartum. For all other metabolites, no differences were found. This model of FR produced substantial fat mobilization but based on serum Hp and lipopolysaccharide-binding protein concentrations did not generate measurable SI; therefore, we were unable to evaluate the ability of MEL to mitigate SI.

Effect of Saccharomyces cerevisiae fermentation product on feed intake parameters, lactation performance, and metabolism of transition dairy cattle.

The transition period in dairy cattle is characterized by many stressors, including an abrupt diet change, but yeast product supplementation can alter the rumen environment to increase dairy cattle productivity. Saccharomyces cerevisiae fermentation product (SCFP) was fed from -29 ± 5 to 42 d relative to calving (RTC) to evaluate the effects on feed intake, milk production, and metabolism. Treatments were control (n = 30) or SCFP (n = 34) incorporated into a total mixed ration. Cows were individually fed 3×/d prepartum and 2×/d postpartum. Blood samples were collected once during each of the following time points RTC: d -28 to -24 (wk -4), d -14 to -10 (wk -2), d 3 to 7 (wk 1), d 12 to 16 (wk 2), and d 31 to 35 (wk 5). Liver biopsies were taken once between d -19 and d -12 (wk -3) and at 14 d in milk. Cows were milked 2×/d, and samples were taken 2 d/wk for composition analysis. Dry matter intake did not differ by treatment, but SCFP increased meals per day and decreased time between meals. Body weight (measured at enrollment, d 0, and d 42 RTC) and body condition score (scored weekly) were not affected by treatment. Milk, energy-corrected milk, and fat-corrected milk yields did not differ by treatment. Milk fat concentration was greater for SCFP, with significant differences in wk 4 and 5. Milk lactose concentration tended to be greater for the control and milk urea nitrogen tended to be lesser for the control, but there were no treatment effects on milk protein concentration or somatic cell count. Assuming equal digestibility, energy balance deficit was greater for SCFP than for the control (-6.15 vs. -4.34 ± 0.74 Mcal/d), with significant differences in wk 4 and 5. Plasma concentrations of free fatty acids, ß-hydroxybutyrate, glucose, and insulin did not differ with treatment, but cholesterol was greater for SCFP. Liver triglyceride increased and liver cholesterol decreased with time. Liver triglyceride did not differ by treatment, but liver cholesterol tended to be lesser in SCFP. Relative mRNA abundance of cholesterol-related genes (SREBF2, HMGCS1, HMGCR, MTTP, SPOB100, APOA1), FGF21, and CPT1A did not differ by treatment, but PCK1 tended to be greater for SCFP. The ketogenic transcript HMGCS2 was greater for SCFP, which aligns with SCFP increasing incidence of subclinical ketosis; however, BDH did not differ between treatments. In conclusion, SCFP supplementation increased meals per day with less time between meals, increased milk fat concentration, altered cholesterol metabolism, and increased incidence of subclinical ketosis, but early-lactation milk yield and metabolism were generally unaffected.

Proteomic analysis reveals greater abundance of complement and inflammatory proteins in subcutaneous adipose tissue from postpartum cows treated with sodium salicylate.

This study aimed to investigate sodium salicylate (SS) treatment effects on the proteome of adipose tissue (AT) in postpartum cows. Twenty Holstein cows were assigned to control (CON, n = 10) or SS (n = 10) provided via drinking water (2.3 g/L) during the first 7 d of lactation. Subcutaneous AT was collected on d 7 of treatment and label-free quantitative shotgun proteomics and immunoblotting were analyzed in a subset of 5 AT per group. Eighty out of 1422 proteins (5.6%) were differentially abundant between CON and SS [fold change ±1.5, P < 0.05]. Top canonical pathways differing between CON and SS (Ingenuity) were complement system, interleukin-10 signaling, and acute phase response signaling. The abundances of complement C1r, C1qC, C1qB and C6 were greater in SS than CON. Regarding IL-10 signaling, the abundances of BLVRB, STAT3, and lipopolysaccharide binding protein (LBP) were greater in SS AT compared to CON. Immunoblots revealed increased abundance of paraoxanase-1 and tumor necrosis factor-alpha, as well as a tendency for greater abundance of cluster differentiation 172a in SS AT, which may indicate of increased macrophage infiltration. SS treatment postpartum likely promotes inflammatory signaling in AT of dairy cows, perhaps due to immune cell recruitment. SIGNIFICANCE: This work demonstrates that treating early lactating cows with sodium salicylate, an anti-inflammatory agent that has been shown to have metabolic effects and increase milk production in dairy cows, affects the proteome of subcutaneous adipose tissue in early lactating dairy cows. Unexpectedly, sodium salicylate treatment enriched inflammatory pathways of the complement system, cytokine signaling, and acute phase response, as revealed by proteomic analysis of subcutaneous adipose tissues from cows at 7 d postpartum. These findings imply that SS treatment during the first 7 d of lactation likely promotes inflammatory signaling in AT of the dairy cow, perhaps due to immune cell recruitment. Tissue-specific impacts of systemic sodium salicylate requires further scrutiny.

Dietary supplementation of Scutellaria baicalensis extract during early lactation decreases milk somatic cells and increases whole lactation milk yield in dairy cattle.

Systemic inflammation is common in early lactation dairy cows and is associated with decreased milk production. The Scutellaria baicalensis plant contains flavonoids with anti-inflammatory and anti-oxidative properties, which may counteract the inflammatory state in early lactation dairy cows. The objective of this experiment was to determine whether Scutellaria baicalensis extract (SBE), a source of bioactive flavonoids, would alter the adaptation to lactation. Multiparous Holstein cows (n = 122) were used in a randomized block design to determine the effect of short-term and long-term postpartum administration of SBE on 305-d milk yield, 120-d milk component yield, and early lactation milk markers of inflammation and metabolic function. Treatments were 1) control, 2) short term (5-d) administration of the SBE (SBE5), and 3) long term (60-d) administration of the SBE (SBE60). Treatments were included in a treatment pellet that was identical to a control pellet in ingredient source and composition except for the extract (10 g/d SBE providing 3.3 g/d of the flavonoid baicalin), both provided via an automated milking system beginning on d 1 of lactation. Milk samples were collected on d 1, 3, and once during d 5-12 of lactation, followed by weekly sampling until 120 days in milk (DIM). Milk samples collected in the first 2 wk were used for biomarker analysis (haptoglobin, ß-hydroxybutyrate [BHB], and glucose-6-phosphate [G6P]), and all samples were used for composition analysis. Cows were body condition scored every 2 wk prepartum and postpartum. Milk production, programmed pellet allocation, and actual provision of both pelleted feeds were recorded daily. Treatment effects were evaluated by contrasts between control and SBE5 and control and SBE60 for both the treatment (TP; wk 1-9) and carryover periods (CP; wk 10-37). Total pellet offered was greater for SBE60 in both the TP (P < 0.01) and CP (P = 0.02) but was not different for SBE5 during either period (P ≥ 0.13). No treatment effects were observed for body condition score (BCS), milk haptoglobin, BHB, or G6P. SBE5 did not alter milk yield or milk components. SBE60 increased whole-lactation milk yield by 1,419 kg (13%; P = 0.03). SBE60 increased milk lactose and fat yields (P ≤ 0.04) and tended to increase milk protein yield (P = 0.09) during TP, and each increased during CP (P ≤ 0.04). Somatic cell count decreased by 10% in SBE60 during TP (P = 0.02) but not CP (P = 0.13). Mastitis incidence tended to differ by treatment, being lesser for both SBE5 and SBE60 vs. control (14 and 15% vs. 33%). SBE supplementation did not impact time to pregnancy or hazard of leaving the herd. In conclusion, despite no detected treatment effects on BCS or milk biomarkers of inflammation and metabolic status, supplementation of postpartum dairy cows with Scutellaria baicalensis extract for 60 d was effective at increasing whole lactation milk yield.

Effects of sodium salicylate on glucose kinetics and insulin signaling in postpartum dairy cows.

Low-grade inflammation has been implicated as a contributor to metabolic disease during the transition to lactation. In previous work, administration of sodium salicylate (SS) for 7 d led to hypoglycemia in mature dairy cows in early lactation. The purpose of this study was to identify the mode of action underlying this response to SS. Twenty mature (parity 3) cows were assigned alternately at time of calving to either control or SS treatments; the control received a molasses placebo in drinking water, whereas SS received 2.3 g/L of SS with the molasses carrier in drinking water for 7 d after parturition. Blood samples were collected daily. A glucose turnover assay was performed on d 7, followed by liver, muscle, and adipose tissue biopsies. There were no treatment effects on intake of dry matter or water. Tumor necrosis factor α mRNA abundance tended to be decreased by SS in adipose tissue but not in muscle or liver, and plasma haptoglobin and adiponectin concentrations were not altered by treatment. Treatment did not significantly alter plasma glucose or insulin concentrations, but plasma glucagon concentration tended to be increased by SS and the insulin:glucagon molar ratio was significantly decreased. Cows on SS had a tendency for a 25% decrease in glucose turnover rate compared with control cows. However, there were no differences in transcript abundance of pyruvate carboxylase (PC) or glucose-6-phosphatase (G6PC) in liver or of glucose transporter 4 (GLUT4) in any of the tissues. Finally, SS did not alter insulin receptor substrate-1 phosphorylation in muscle or adipose, but tended to increase phosphorylation of AMP-activated protein kinase and decrease protein kinase B phosphorylation in adipose tissue. These findings may be explained by enhanced hepatic insulin sensitivity leading to posttranscriptional suppression of gluconeogenesis and adaptive responses to decreased glucose supply in the pancreas and adipose tissue.

Exposure to an unpredictable and competitive social environment affects behavior and health of transition dairy cows.

Social factors are important determinants of disease in humans and and laboratory animals, but less research has been done using farm animals. The objective of this study was to determine if an unpredictable and competitive social environment affects behavior and health during the transition period when dairy cows are at high risk of disease. Five weeks before calving, 64 cows were assigned to a predictable and noncompetitive social environment (predictable) or an unpredictable and competitive social environment (unpredictable) using 8 groups of 4 animals per treatment. Each group consisted of 3 multiparous and 1 primiparous cow. At first enrollment (baseline; 5 wk before calving), all groups had access to 4 electronic feed bins. At 4 wk before calving, cows in the predictable groups were given access to 6 feed bins, and cows in the unpredictable groups were moved into a new pen with 4 resident cows each trained to consume feed from one bin. Each cow in the unpredictable group was then provided access to only 1 of the 4 feed bins which they shared with 1 resident cow (resulting in 2 cows/bin), creating a competitive feeding environment. To create an unpredictable environment, access to morning feed was delayed 0, 1, 2, or 3 h every other day. On alternate days, the cows in unpredictable groups were assigned to feed from a new feed bin (and thus had to compete with a new resident partner). Feeding and social behavior were collected electronically from the feed bins. Blood was sampled at baseline (wk -5), wk -2, wk -1, and wk +1 relative to calving to measure inflammatory (haptoglobin and tumor necrosis factor-α) and metabolic (nonesterified fatty acids, ß-hydroxybutyrate, calcium, and glucose) biomarkers. Uterine cytology was performed 3 to 5 wk after calving to diagnose cytological endometritis. Data were analyzed using mixed models including baseline data as a covariate, week as a repeated measure, treatment as a main effect, and a treatment by week interaction. The probability of cytological endometritis at the group level was analyzed using Mann-Whitney U tests. Parity was included in separate models to determine any parity × treatment interactions. Cows from both treatments consumed the same amount of feed, but cows in the unpredictable group spent less time feeding and had a higher rate of feed intake. Cows in the unpredictable groups also visited the feed bins less often, consumed more feed during each visit, and were involved in more social replacements at the feed bin compared with predictable groups. Cows in the unpredictable groups had higher serum concentrations of nonesterified fatty acids and tumor necrosis factor-α, but lower ß-hydroxybutyrate compared with predictable groups. Multiparous cows in unpredictable groups were more likely to be diagnosed with cytological endometritis after calving compared with cows in the predictable groups, but primiparous cows in unpredictable groups showed a tendency for the opposite response. These results suggest that an unpredictable and competitive social environment before calving causes changes in feeding and social behavior, some physiological indicators of metabolism and inflammation, and increases the risk of uterine disease in multiparous cows after calving.