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.

Subcutaneous lysophosphatidylcholine administration promotes a febrile and immune response in Holstein heifer calves.

Lysophosphatidylcholine (LPC) is immunomodulatory in nonruminants; however, the actions of LPC on immunity in cattle are undefined. Our objective was to study the effects of LPC administration on measures of immunity, liver health, and growth in calves. Healthy Holstein heifer calves (n = 46; age 7 ± 3 d) were randomly assigned to 1 of 4 treatments (n = 10 to 11 calves/treatment): a milk replacer diet unsupplemented with lecithin in the absence (CON) or presence of subcutaneously (s.c.) administered mixed (mLPC; 69% LPC-16:0, 25% LPC-18:0, 6% other) or pure LPC (pLPC; 99% LPC-18:0), or a milk replacer diet supplemented with 3% lecithin enriched in lysophospholipids containing LPC in the absence of s.c.-administered LPC (LYSO) for 5 wk. Calves received 5 s.c. injections of vehicle (10 mL of phosphate-buffered saline containing 20 mg of bovine serum albumin/mL; CON and LYSO) or vehicle containing mLPC or pLPC to provide 10 mg of total LPC per kilogram of BW per injection every 12 h during wk 2 of life. Calves were fed a milk replacer containing 27% crude protein and 24% fat at 1.75% of BW per day (dry matter basis) until wk 6 of life (start of weaning). Starter grain and water were provided ad libitum. Body measurements were recorded weekly, and clinical observations were recorded daily. Blood samples were collected weekly before morning feeding and at 0, 5, and 10 h, relative to the final s.c. injection of vehicle or LPC. Data were analyzed using a mixed model, with repeated measures including fixed effects of treatment, time, and their interaction. Dunnett's test was used to compare treatments to CON. Peak rectal temperatures were higher in mLPC or pLPC, relative to CON. Plasma LPC concentrations were greater in mLPC and LYSO calves 5 h and 10 h after the final injection, relative to CON. Calves receiving mLPC and pLPC also had higher circulating serum amyloid A concentrations, relative to CON. Calves receiving mLPC had greater serum aspartate aminotransferase, γ-glutamyltransferase, and glutamate dehydrogenase concentrations, relative to CON. Calves provided mLPC experienced lower average daily gain (ADG) after weaning, relative to CON. The LYSO treatment did not modify rectal temperatures, ADG, or measures of liver health, relative to CON. We conclude that LPC administered as s.c. injections induced an acute febrile response, modified measures of liver and immune function, and impaired growth in calves.

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.

The effects of feeding α-amylase-enhanced corn silage with different dietary starch concentrations to lactating dairy cows on milk production, nutrient digestibility, and blood metabolites.

Corn silage is one of the most common ingredients fed to dairy cattle. Advancement of corn silage genetics has improved nutrient digestibility and dairy cow lactation performance in the past. A corn silage hybrid with enhanced endogenous α-amylase activity (Enogen, Syngenta Seeds LLC) may improve milk production efficiency and nutrient digestibility when fed to lactating dairy cows. Furthermore, evaluating how Enogen silage interacts with different dietary starch content is important because the ruminal environment is influenced by the amount of rumen fermentable organic matter consumed. To evaluate the effects of Enogen corn silage and dietary starch content, we conducted an 8-wk randomized complete block experiment (2-wk covariate period, 6-wk experimental period) with a 2 × 2 factorial treatment arrangement using 44 cows (n = 11/treatment; 28 multiparous, 16 primiparous; 151 ± 42 d in milk; 668 ± 63.6 kg of body weight). Treatment factors were Enogen corn silage (ENO) or control (CON) corn silage included at 40% of diet dry matter and 25% (LO) or 30% (HI) dietary starch. Corn silage used in CON treatment was a similar hybrid as in ENO but without enhanced α-amylase activity. The experimental period began 41 d after silage harvest. Feed intake and milk yield data were collected daily, plasma metabolites and fecal pH were measured weekly, and digestibility was measured during the first and final weeks of the experimental period. Data were analyzed using a linear mixed model approach with repeated measures for all variables except for body condition score change and body weight change. Corn silage, starch, week, and their interactions were included as fixed effects; baseline covariates and their interactions with corn silage and starch were also tested. Block and cow served as the random effects. Plasma glucose, insulin, haptoglobin, and serum amyloid A concentrations were unaffected by treatment. Fecal pH was greater for cows fed ENO versus CON. Dry matter, crude protein, neutral detergent fiber, and starch digestibility were all greater for ENO than CON during wk 1, but differences were less by wk 6. The HI treatments depressed neutral detergent fiber digestibility compared with LO. Dry matter intake (DMI) was not affected by corn silage but was affected by the interaction of starch and week; in wk 1, DMI was similar but by wk 6, cows fed HI had 1.8 ± 0.93 kg/d less DMI than LO cows. Milk, energy-corrected milk, and milk protein yields were 1.7 ± 0.94 kg/d, 1.3 ± 0.70 kg/d, and 65 ± 27 g/d greater for HI than LO, respectively. In conclusion, ENO increased digestibility but it did not affect milk yield, component yields, or DMI. Increasing dietary starch content improved milk production and feed efficiency without affecting markers of inflammation or metabolism.

Meta-analysis of the effects of sulfate versus hydroxy trace mineral source on nutrient digestibility in dairy and beef cattle.

Trace mineral (TM) source can potentially alter nutrient digestibility through effects on microbial populations. A meta-analysis was conducted to determine whether sulfate versus hydroxy (IntelliBond) sources of supplemental Cu, Zn, and Mn had any effect on dry matter intake (DMI), dry matter digestibility, and neutral detergent fiber (NDF) digestibility. All available cattle studies (8 studies, 12 comparisons) were used to estimate the effect size (hydroxy mean - sulfate mean). Factors included in the analysis were method of digestibility analysis (total collection, marker-based, or 24 h in situ), study design (randomized design or Latin square), beef (n = 5) versus dairy (n = 7) cattle, and days on treatment; these factors were retained when P < 0.05. Dry matter digestibility was increased by hydroxy TM in beef (1.64 ± 0.35 units) but not in dairy models (0.16 ± 0.13 units) relative to sulfate TM. The NDF digestibility increased significantly with hydroxy versus sulfate TM, but digestibility assessment method influenced this response. Studies using total collection or undigested NDF as a flow marker showed a significant increase (2.68 ± 0.40 units and 1.08 ± 0.31 units, respectively) in NDF digestibility for hydroxy versus sulfate TM; but studies utilizing 24-h in situ incubation did not detect any change (-0.03 ± 0.23 units). These observations may reveal differences in precision of measurement or may indicate mineral effects beyond the rumen; total collection is considered the gold standard method. Hydroxy TM did not affect DMI per animal or per unit of body weight relative to sulfate TM. In conclusion, feeding hydroxy versus sulfate TM does not appear to affect DMI but, depending on type of cattle and method of measurement, can increase dry matter digestibility and NDF digestibility, which may be explained by differences in solubility of the TM sources in rumen, differentially affecting fermentation.

Does feeding starch contribute to the risk of systemic inflammation in dairy cattle?

In the high-producing dairy cow, providing an adequate supply of digestible energy is essential. One strategy to meet this need is to provide fermentable starch from cereal grains or silages like corn, barley, or wheat. Unfortunately, excess dietary starch increases the risk of rumen acidosis. Rumen acidosis challenge models using high-grain diets, particularly with wheat and barley, have demonstrated that a sudden change in starch concentration or digestibility leads to the breakdown of the rumen epithelial barrier. As a result, increases in circulating lipopolysaccharide (a marker of bacterial translocation) and acute phase proteins (APP) have been observed. Feeding increasing amounts of starch in chronic feeding studies does not appear to consistently modulate inflammation in early-lactation cows that already experience inflammation. In mid- and late-lactation cows, increasing starch above 30% may increase APP, but the response is inconsistent and has not been investigated using different grains or differently processed starch sources. Abomasal starch infusion experiments indicate that increasing the intestinal starch supply consistently reduces fecal pH but does not lead to an APP response or changes in gut integrity. Increasing intestinal starch supply increases fecal butyrate concentrations, and butyrate has had positive effects on gut health and integrity in other species and experimental models. More chronic feeding experiments are needed to investigate how starch concentrations, sources, processing methods, and interactions affect inflammation and gut integrity. There is a paucity of data investigating the role that carbohydrate concentrations and sources play on ruminant hindgut health, integrity, function, structure, or microbiome. Currently, data indicate that feeding diets with less than 30% starch to lactating dairy cows does not contribute to systemic inflammation.

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.

Invited review: Mechanisms of hypophagia during disease.

Suppression of appetite, or hypophagia, is among the most recognizable effects of disease in livestock, with the potential to impair growth, reproduction, and lactation. The continued evolution of the field of immunology has led to a greater understanding of the immune and endocrine signaling networks underlying this conserved response to disease. Inflammatory mediators, especially including the cytokines tumor necrosis factor-α and interleukin-1ß, are likely pivotal to disease-induced hypophagia, based on findings in both rodents and cattle. However, the specific mechanisms linking a cytokine surge to decreased feeding behavior are more difficult to pin down and likely include direct effects on appetite centers in the brain, alteration of gastric motility, and modulation of other endocrine factors that influence appetite and satiety. These insights into the mechanisms for disease-induced hypophagia have great relevance for management of neonatal calves, mature cows transitioning to lactation, and cows experiencing mastitis; however, it is not necessarily the case that increasing feed intake by any means possible will improve health outcomes for diseased cattle. We explore conflicting effects of hypophagia on immune responses, which may be impaired by the lack of specific substrates, versus apparent benefits for controlling the growth of some pathogens. Anti-inflammatory strategies have shown promise for promoting recovery of feed intake following some conditions but not others. Finally, we explore the potential for early disease detection through automated monitoring of feeding behavior and consider which strategies may be implemented to respond to early hypophagia.

First postpartum ovulation, metabolites and hormones in follicular fluid and blood in transition dairy cows supplemented with a Saccharomyces cerevisiae fermentation product.

We hypothesized that feeding a Saccharomyces cerevisiae fermentation product (SCFP) from -4 through +7 wk (calving = Day 0) facilitates early first postpartum ovulation and alters blood and follicular fluid concentrations of glucose, beta-hydroxybutyrate (BHB), free fatty acids (FFA), and steroid hormones favorable to subsequent fertility. Holstein cows were fed individually a SCFP product (n = 24) or served as controls (n = 23). Blood samples were collected at wk -4 and -2 from expected calving and at 1, 2, 5, and 7 wk postpartum to determine plasma concentrations of FFA and BHB. Early spontaneous ovulation (progesterone > 1 ng/mL or corpus luteum presence by postpartum median Day 33) or late ovulation was determined. Plasma FFA in weekly samples was not affected by SCFP supplementation, but FFA was greater (P < 0.01; week by ovulation status) in late compared with early ovulating cows during and after postpartum wk 2. Plasma BHB in weekly samples was greater (P = 0.03) in SCFP than control cows and tended (P = 0.06) to be greater in late than early ovulating cows. Cows were exposed to ovulation synchronization (GnRH, PGF2α, and GnRH on Days 33, 40, and 43 ± 3, respectively). Transvaginal dominant follicle aspiration was conducted at Day 50, 7 d after GnRH on Day 43. Metabolites (FFA, BHB, and glucose) and steroid hormones (progesterone, androstenedione, and estradiol) measured in follicular fluid and blood samples collected at aspiration revealed that androstenedione in serum was numerically less (P = 0.11) in SCFP-treated compared with control cows, whereas androstenedione in serum was less (P < 0.05) in late than early ovulating cows. Concentrations of BHB (r = 0.75) and glucose (r = 0.52) in follicular fluid were positively correlated (P < 0.01) with those in blood. Body weight at calving and Day 42 was less (P ≤ 0.05), and energy balance through Days 28 and 42 was more positive (P < 0.05) in early than late ovulating cows and in SCFP-supplemented compared with control cows (P < 0.05). Dry matter intake, daily milk yield, and yields of fat, protein, lactose, and total solids were less (P < 0.01) in early compared with late ovulating cows, whereas milk fat percentage was increased (P < 0.01) by SCFP supplementation. We conclude that elevated postpartum BHB and FFA in plasma, greater negative energy balance, and greater milk yield and components were associated with later postpartum ovulation, but metabolites and steroid hormones in blood and follicular fluid were unaffected by SCFP treatment or ovulation status except for androstenedione.

Intergenerational cycle of disease: Maternal mastitis is associated with poorer daughter performance in dairy cattle.

Adverse prenatal environments, such as maternal stress and infections, can influence the health and performance of offspring. Mastitis is the most common disease in dairy cattle, yet the intergenerational effects have not been specifically investigated. Therefore, we examined the associations between the dam's mammary gland health and daughter performance using somatic cell score (SCS) as a proxy for mammary health. Using data obtained from Dairy Records Management Systems (Raleigh, NC), we linked daughter records with their dam's records for the lactation in which the daughter was conceived. Linear and quadratic relationships of dam mean SCS with the daughter's age at first calving (AFC; n = 15,992 daughters, 4,366 herds), first- (n = 15,119 daughters, 4,213 herds) and second-lactation SCS (n = 3,570 daughters, 1,554 herds), first- and second-lactation mature-equivalent 305-d milk yield, and milk component yields were assessed using mixed linear regression models. We uncovered a phenomenon similar to those found in human and mouse models examining prenatal inflammation effects, whereby daughters born from dams with elevated SCS had poorer performance. Dam mean SCS was positively associated with daughter's AFC and first- and second-lactation mean SCS. Furthermore, for every 1-unit increase in dam mean SCS, daughter's first- and second-lactation mature-equivalent fat yield declined by 0.34% and 0.91% (-1.6 ± 0.49 kg, -4.0 ± 1.0 kg, respectively), although no effect was found on first- or second-lactation milk or milk protein yield. When accounting for genetics, daughter SCS, and AFC (first lactation only), dam mean SCS was associated with reduced second-lactation milk fat yield (-3.5 ± 1.8 kg/unit SCS), and a tendency was found for first-lactation milk fat yield (-1.9 ± 1.0 kg/unit SCS). Taken together, the association of greater dam mean SCS with lesser daughter milk fat yield is likely due to a few underlying mechanisms, in particular, a predisposition for mastitis and alterations in the epigenome controlling milk fat synthesis. As such, future studies should examine epigenetic mechanisms as a potential underpinning of this phenomenon.

Effects of milk feeding strategies on short- and long-term productivity of Holstein heifers.

Research investigating the effects of feeding raw or pasteurized nonsaleable milk (NSM) on heifers' performance beyond the period of supplementation is limited. This study aimed to examine the effects of type of milk [NSM or milk replacer (MR)] and pasteurization of NSM on preweaning and first-lactation performance of heifers born with low (<36.3 kg) or normal birth weight (≥36.3 kg). Holstein heifers (n = 154) were sequentially assigned to 1 of 3 treatments: MR, pasteurized NSM, or raw NSM. Heifers assigned to raw NSM were fed raw colostrum, whereas heifers assigned to MR and pasteurized NSM were fed pasteurized colostrum. The low birth weight heifers were fed 1.4 L at each feeding until they reached 36.3 kg body weight, whereas normal birth weight heifers were fed 1.9 L at each feeding. A grain mix starter was offered throughout the study. Heifers were weaned ≥42 d old if consuming at least 0.9 kg/d of starter for 3 consecutive days. Data were analyzed with the MIXED procedure of SAS (SAS Institute Inc., Cary, NC), and the basic model included milk treatments, birth weight group, and treatment × birth weight group. The low birth weight heifers fed raw colostrum and NSM versus pasteurized colostrum and NSM had lower serum protein concentrations. Heifers fed MR versus NSM had or tended to have greater concentrations of hematocrit, red blood cells, and eosinophils but lesser concentrations of platelets, although some of those responses were temporary. Pasteurization tended to increase blood lymphocyte concentrations. Heifers with normal birth weight had greater concentrations of blood neutrophils, lymphocytes, and monocytes, compared with low birth weight heifers. For the first 42 d of life, low birth weight heifers fed pasteurized versus raw NSM had greater weight gain, grain intake, and feed efficiency and were weaned earlier (hazard ratio for weaning by 56 d: 2.90). These pasteurization effects for low birth weight heifers tended to be sustained through 24 wk of age, indicated by greater weight gain and hip height growth. In their first lactation, low birth weight heifers produced less mature-equivalent (MEq) protein and tended to produce less MEq milk and fat than normal birth weight heifers. However, the negative effects of low birth weight on MEq milk and fat yield was only evident in heifers fed raw NSM, whereas the performance of low birth weight heifers was similar to that of normal birth weight when fed MR or pasteurized NSM. These findings confirm that calf management practices influence future performance; in this case, failing to pasteurize milk and colostrum for low birth weight heifers had effects that remained apparent for more than 2 years.

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.

Relative availability of metabolizable methionine from 2 ruminally protected sources of methionine fed to lactating dairy cattle.

Our objective was to evaluate the lactational responses of dairy cows to methionine provided from 2 ruminally protected sources of methionine activity. Twenty-one Holstein dairy cows [11 primiparous (634 kg of body weight, 140 d in milk) and 10 second-parity (670 kg of body weight, 142 d in milk)] were assigned to a treatment sequence in 4 replicated 5 × 5 Latin squares plus 1 cow, with 14-d periods. Treatments were as follows: control; 7.5 or 15 g/d of a ruminally protected product of 2-hydoxy-4-methylthio-butyric acid (NTP-1401; Novus International Inc., St. Charles, MO); or 7.5 or 15 g/d of a ruminally protected dl-methionine product (Smartamine M; Adisseo, Alpharetta, GA). The diet was predicted to meet metabolizable protein and energy requirements. Diets contained 16.1% crude protein, and the control diet was predicted to be deficient in metabolizable methionine (1.85% of metabolizable protein) but sufficient in lysine (6.8% of metabolizable protein). Feed intake and milk yield were measured on d 11 to 14. Blood was collected on d 14. Dry matter intake, milk yield, energy-corrected milk, milk fat yield and percentage, and efficiencies of milk and energy-corrected milk yield were not affected by treatment. Milk protein percentage and milk protein yield increased linearly with supplementation, without differences between methionine sources or interactions between source and level. Linear regressions of milk protein percentage and milk protein yield against supplement amount within source led to slope ratios (NTP-1401:Smartamine M) of 95% for protein percentage and 84% for protein yield, with no differences between sources for increasing milk protein. Plasma methionine concentrations were increased linearly by methionine supplementation; the increase was greater for Smartamine M than for NTP-1401. Plasma d-methionine was increased only by Smartamine M. Plasma 2-hydoxy-4-methylthio-butyric acid was increased only by NTP-1401. Our data demonstrated that supplementation with these methionine sources can improve milk protein percentage and yield, and the 2 methionine sources did not differ in their effect on lactation performance or milk composition.

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.

Do biological and management reasons for a short or long dry period induce the same effects on dairy cattle productivity?

The dry period is a well-established factor that determines lactation success. A retrospective observational study used 32,182 lactations from 16 farms to determine whether management versus biological reasons for deviations from the targeted 60-d dry period have the same associations with subsequent lactation performance. Herd inclusion criteria were Holstein cows, herd size ≥900 cows, breeding by artificial insemination, and (minimally) bimonthly milk testing. Dry period length (DPL) and gestation length (GL) were each categorized as short [>1 standard deviation (SD) below mean within herd; means 45 d DPL, 269 d GL] or long (>1 SD above mean within herd; means 73 d DPL, 284 d GL) and combined to generate the following 7 study groups: short DPL, short GL (SDSG, n = 2,123); short DPL, average GL (SDAG, n = 1,418); average DPL, short GL (ADSG, n = 1,759); average DPL, average GL (ADAG, n = 19,265); average DPL, long GL (ADLG, n = 3,325); long DPL, average GL (LDAG, n = 2,573); and long DPL, long GL (LDLG, n = 1,719). Responses evaluated included milk and component yields at first test and over the whole lactation, days to first service, first service conception risk, days open, and herd retention through 60 and 365 d postpartum. Continuous data were analyzed by mixed models and time to event data by Cox proportional hazard models, both accounting for clustering at the herd level. First test and whole-lactation milk and component yields were lowest for SDSG. Within cows that experienced calving difficulty, rates of receiving first service were 13 and 20% less for SDSG and ADSG compared with ADAG. Hazard of leaving the herd by 60 d in milk (DIM) was 34% greater for ADSG than ADAG. Similar effects between SDSG and ADSG but not SDAG indicated that short GL was a greater contributor to poor performance than DPL itself. Overall production was similar between ADAG and SDAG; however, somatic cell linear score at first test was greater for SDAG, and milk yield at first test was lesser for SDAG cows with greater milk at last test before dry-off. Although short DPL might be a successful strategy for some herds or cows, cows with high milk yield at dry-off should not be subjected to a short dry period. Long DPL or GL did not influence early-lactation or whole-lactation milk yield. Cows with a long DPL due to early dry-off (LDAG) likely experienced issues related to excessive lipid mobilization, as milk fat concentration and fat:protein ratio at first test were greater and hazard of leaving the herd was 30 and 24% greater compared with ADAG by 60 and 365 DIM, respectively. We conclude that deviations in DPL length caused by biology (short GL) were associated with greater effects than management causes of short DPL, whereas management reasons for long DPL were associated with more negative outcomes than long GL.

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.