Investigation of HCAR2 antagonists as a potential strategy to modulate bovine leukocytes.

BACKGROUND: Dairy cows experiencing ketosis after calving suffer greater disease incidence and are at greater risk of leaving the herd. In vitro administration of beta-hydroxybutyric acid (BHBA; the primary blood ketone) has inhibitory effects on the function of bovine leukocytes. BHBA is a ligand of HCAR2 and the activation of these receptors promotes an anti-inflammatory response which may be related with immunosuppression observed in transition dairy cattle. The objective of this study was to identify and test antagonists for HCAR2 in bovine immune cells cultured with BHBA. RESULTS: We observed expression of HCAR2 at the protein level within lymphocytes, monocytes, and granulocytes. The proportion of cells expressing HCAR2 tended to be greater in mid-lactation compared to early lactation cows; the increase was a result of increased proportion of T and B cells expressing HCAR2. Stimulation of HCAR2 with niacin or BHBA promoted Ca2+ mobilization in neutrophils and mononuclear cells. Mononuclear cells treated with BHBA had diminished intracellular Ca2+ responses when HCAR2 was knocked down by siRNA silencing, indicating Ca2+ mobilization was mediated by HCAR2 signaling. Two candidate antagonists for HCAR2, synthesized from niacin (NA-1 and NA-5), were tested; monocytes and neutrophils pre-treated with NA-1 and NA-5 had reduced Ca2+ mobilization after incubation with BHBA. Furthermore, NA-5 but not NA-1 prevented BHBA-associated reductions in cyclic AMP. CONCLUSIONS: We demonstrated that HCAR2 is present on bovine leukocytes and has greater expression later in lactation. We confirmed that BHBA and niacin derived HCAR2 antagonists alter bovine leukocyte activity. Our results demonstrate that both BHBA and niacin affect bovine leukocyte Ca2+ mobilization in a HCAR2-dependent manner.

Adipose Tissue Inflammation: Linking Physiological Stressors to Disease Susceptibility.

The study of adipose tissue (AT) is enjoying a renaissance. White, brown, and beige adipocytes are being investigated in adult animals, and the critical roles of small depots like perivascular AT are becoming clear. But the most profound revision of the AT dogma has been its cellular composition and regulation. Single-cell transcriptomic studies revealed that adipocytes comprise well under 50% of the cells in white AT, and a substantial portion of the rest are immune cells. Altering the function of AT resident leukocytes can induce or correct metabolic syndrome and, more surprisingly, alter adaptive immune responses to infection. Although the field is dominated by obesity research, conditions such as rapid lipolysis, infection, and heat stress impact AT immune dynamics as well. Recent findings in rodents lead to critical questions that should be explored in domestic livestock as potential avenues for improved animal resilience to stressors, particularly as animals age.

Analysis of Jersey versus Holstein breed profitability on north central US dairies.

With over 9 million cows in the United States, Holstein is the dominant breed in the US dairy population; however, the US Jersey population is growing. The objective of this study was to determine the profitability of Holstein and Jersey cows managed similarly on the same farms. Holstein and Jersey economic performance was compared within 3 north central US dairies, each milking more than 500 cows. The herds' average distribution was 21% Jersey (27 ± 0.67 kg/d milk, 4.92% ± 0.24 fat, 3.72% ± 0.03 protein) and 79% Holstein (37 ± 1.98 kg/d milk, 3.85% ± 0.21 fat, 3.17% ± 0.17 protein). A comparative budget approach was used to assess economic factors that differed between the breeds on a per cow annual basis, based on the assumption that an existing farm would be constrained by stalls and parlor to an equal number of Jersey and Holstein cows. Data from 2020 were gathered from farm management software, on-farm evaluations, and producer interviews. Sensitivity analysis was performed to determine which conditions would lead to different conclusions. Factors considered in the analysis included milk and component production, milk bonuses, ration prices, and dry matter (DM) intake. In a 2021 price scenario, Holstein cows ranged from $345 to $601 more profitable than Jersey cows on a per cow annual basis. Although Jersey cows had an advantage in component concentration, Holstein cows produced 13 ± 4.7% more fat and 22 ± 6.6% more protein annually due to greater milk yield. This accounted for most of the profitability advantage for Holsteins; 78% of the revenue advantage for Holstein cows came from increased component production. Few health and reproductive differences were found. The sensitivity analysis revealed, if all other factors remained the same, Jersey profitability would equal that of Holstein if any of the following changes occurred (assuming no change in Holstein metrics): mean Jersey milk production increased to 31 kg/d; milk price adjustments decreased from -$0.008 to -$0.11 per kg fluid milk; lactating cow ration price increased from $0.27 per kg DM to $0.53 per kg DM; or Jersey DM intake decreased from 20 to 15 kg/d. The study did not consider crossbred profitability or new infrastructure investments. In conclusion, Holstein cows were more profitable than Jersey cows on these 3 north central US dairies.

Effects of substituting noug seed cake with pigeon pea leaves or desmodium hay on performance of male dairy calves.

This experiment was conducted to evaluate the effects of substituting 50% of noug seed cake (NSC) in a concentrate mixture with pigeon pea leaves (PPL) or desmodium hay (DH) on feed intake, digestibility, body weight gain, carcass composition, and meat quality of crossbred male dairy calves. Twenty-seven male dairy calves at 7-8 months of age with an average initial body weight of 150 ± 31 kg (mean ± SD) were assigned to 3 treatments in a randomized complete block design with 9 replications. Calves were blocked based on their initial body weight and assigned to the 3 treatments. All calves were fed native pasture hay ad libitum (at ⁓10% refusal) supplemented with a concentrate containing 24% NSC (treatment 1) or supplemented with a concentrate where 50% of NSC was replaced with PPL (treatment 2) or a concentrate where 50% of NSC was replaced with DH (treatment 3). Feed and nutrient intake, apparent nutrient digestibility, body weight gain, feed conversion ratio, carcass composition, and meat quality (except texture) were similar (P > 0.05) among treatments. Treatments 2 and 3 had more (P < 0.05) tender loin and rib meat than treatment 1. It can be concluded that 50% of NSC in the concentrate mixture can be replaced with either PPL or DH in growing male crossbred dairy calves to achieve similar growth performance and carcass characteristics. Since the substitution of 50% NSC either with PPL or DH resulted in similar outcomes in almost all responses measured, it is recommended to evaluate the complete substitution of NSC either with PPL or DH on the performance of calves.

Symposium review: Fueling appetite: Nutrient metabolism and the control of feed intake.

Conceptual models developed over the past century describe 2 key constraints to feed intake (FI) of healthy animals: gut capacity and metabolic demand. Evidence that greater energy demands (e.g., greater milk production) drive a corresponding increase in caloric intake led to the dominant concept that animals "eat to energy requirements." Although this model provides reasonable initial estimates of FI, it lacks a proposed physiological basis for the control system, does not consider nutrient constraints beyond energy, and fails to explain differential energy intake responses to different fuels. To address these gaps, research has focused on mechanisms for sensing nutrient availability and providing feedback to hypothalamic centers that integrate signals to control feeding behavior. The elimination of FI response to certain nutrients by vagotomy suggests that peripheral tissues play a role in nutrient sensing. These findings and the central role of the liver in metabolic flux led to the development of the hepatic oxidation theory (HOT). According to the HOT, liver energy charge is the regulated variable that induces dietary intake changes and consequently affects whole-body energy balance. Evidence in support of HOT includes associations between hepatic energy charge and meal patterns, increased FI in response to phosphate trapping, and reduced FI in response to phosphate loading. In accordance with the HOT, infusion studies in dairy cattle have consistently demonstrated that providing fuels that either oxidize or stimulate oxidation in the liver decreases FI and energy intake to a greater extent than fuels that bypass the liver. Importantly, this holds true for glucose, which is readily oxidized by nerve cells, but is rarely taken up by the bovine liver. Although the brain integrates multiple signals including those related to gastric distention and illness, the HOT provides a physiological framework for understanding the dominant role the liver likely plays in sensing short-term energy status. Understanding this model provides insights into how to use or bypass the regulatory system to manage FI of animals.

Evaluation of reticuloruminal temperature for the prediction of clinical mastitis in dairy cows challenged with Streptococcus uberis.

Automated monitoring devices have become increasingly utilized in the dairy industry, especially for monitoring or predicting disease status. While multiple automated monitoring devices have been developed for the prediction of clinical mastitis (CM), limitations in performance or applicability remain. The aims of this study were to (1) detect variations in reticuloruminal temperature (RRT) relative to an experimental intramammary challenge with Streptococcus uberis and (2) evaluate alerts generated automatically based on variation in RRT to predict initial signs of CM in the challenged cows based on severity of clinical signs and the concentration of bacteria (cfu/mL) in the infected quarter separately. Clinically healthy Holstein cows without a history of CM in the 60 d before the experiment (n = 37, parity 1 to 5, ≥120 d in milk) were included if they were microbiologically negative and had a somatic cell count under 200,000 cells/mL based on screening of quarter milk samples 1 wk before challenge. Each cow received an intra-reticuloruminal automated monitoring device before the trial and was challenged with 2,000 cfu of Strep. uberis 0140J in 1 rear quarter. Based on interrupted time series analysis, intramammary challenge with Strep. uberis increased RRT by 0.54°C [95% confidence interval (CI): 0.41, 0.66] at 24 h after the challenge, which remained elevated until the end of the study. Alerts based on RRT correctly classified 78.3% (95% CI: 65.8, 87.9) of first occurrences of CM at least 24 h in advance, with a sensitivity of 70.0% (95% CI: 50.6, 85.3) and a specificity of 86.7% (95% CI: 69.3, 96.2). The accuracy of CM for a given severity score was 90.9% (95% CI: 70.8, 98.9) for mild cases, 85.2% (95% CI: 72.9, 93.4) for moderate cases, and 92.9% (95% CI: 66.1, 99.8) for severe cases. Test characteristics of the RRT alerts to predict initial signs of CM improved substantially after bacterial count in the challenged quarter reached 5.0 log10 cfu/mL, reaching a sensitivity of 73.5% (95% CI: 55.6, 87.1) and a specificity of 87.5% (95% CI: 71.0, 96.5). Overall, the results of this study indicated that RRT was affected by the intramammary challenge with Strep. uberis and the RRT-generated alerts had similar accuracy as reported for other sensors and algorithms. Further research that includes natural infections with other pathogens as well as different variations in RRT to determine CM status is warranted.

The application, value, and impact of outcomes research in animal health and veterinary medicine.

Outcomes research is a relatively recent field of study in animal health and veterinary medicine despite being well-established in human medicine. As the field of animal health is broad-ranging in terms of animal species, objectives, research methodologies, design, analysis, values, and outcomes, there is inherent versatility in the application and impact of the discipline of outcomes research to a variety of stakeholders. The major themes of outcomes relevant to the animal health industry have been distilled down to include, but are not limited to, health, production, economics, and marketing. An outcomes research approach considers an element of value along with an outcome of interest, setting it apart from traditional research approaches. Elements of value are determined by the stakeholders' use of products and/or services that meet or exceed functional, emotional, life-changing, and/or societal needs. Stakeholder perception of value depends on many factors such as the purpose of the animal (e.g., companion vs. food production) and the stakeholder's role (e.g., veterinarian, client, pet-owner, producer, consumer, government official, industry representative, policy holder). Key areas of application of outcomes research principles include comparative medicine, veterinary product development, and post-licensure evaluation of veterinary pharmaceuticals and/or biologics. Topics currently trending in human healthcare outcomes research, such as drug pricing, precision medicine, or the use of real-world evidence, offer novel and interesting perspectives for addressing themes common to the animal health sector. An approach that evaluates the benefits of practices and interventions to veterinary patients and society while maximizing outcomes is paramount to combating many current and future scientific challenges where feeding the world, caring for our aging companion animals, and implementing novel technologies in companion animal medicine and in production animal agriculture are at the forefront of our industry goals.

Enhancing untargeted metabolomics using metadata-based source annotation.

Human untargeted metabolomics studies annotate only ~10% of molecular features. We introduce reference-data-driven analysis to match metabolomics tandem mass spectrometry (MS/MS) data against metadata-annotated source data as a pseudo-MS/MS reference library. Applying this approach to food source data, we show that it increases MS/MS spectral usage 5.1-fold over conventional structural MS/MS library matches and allows empirical assessment of dietary patterns from untargeted data.

Proteome dataset of liver from dairy cows experiencing negative or positive energy balance at early lactation.

This article contains raw and processed data related to research published by Swartz et al. [1]. We present proteomics data from liver of postpartum dairy cows that were obtained by liquid chromatography-mass spectrometry following protein extraction. Differential abundance between liver of cows experiencing either negative energy balance (NEB, n = 6) or positive energy balance (PEB, n = 4) at 17 ± 3 days in lactation was quantified using MS1 intensity based label-free. There is a paucity of studies examining the associations of NEB with the liver proteome in early lactation dairy cows. Therefore, our objective was to examine the differences in the liver proteome in periparturient dairy cows experiencing naturally occurring NEB compared to cows in PEB. In this study, multiparous Holstein dairy cows were milked either 2 or 3 times daily for the first 30 days in milk (DIM) to alter energy balance, and were classified retrospectively as NEB (n = 18) or PEB (n = 22). We collected liver biopsies from 10 cows (n = 5 from each milking frequency), that were retrospectively classified according to their energy balance (NEB, n = 6; PEB, n = 4). The liver proteome was characterized using label-free quantitative shotgun proteomics. This novel dataset contains 2,741 proteins were identified, and 68 of those were differentially abundant between NEB and PEB (P ≤ 0.05 and FC± 1.5); these findings are discussed in our recent research article [1]. The present dataset of liver proteome can be used as either biological markers for disease or therapeutic targets to improve metabolic adaptations to lactation in postpartum dairy cattle. Data are available via ProteomeXchange with identifier PXD028124.

Connecting Metabolism to Mastitis: Hyperketonemia Impaired Mammary Gland Defenses During a Streptococcus uberis Challenge in Dairy Cattle.

ß-hydroxybutyrate (BHB) has been associated with disease incidence in early lactation dairy cattle, but such associations do not demonstrate causation. Therefore, the objective of this study was to examine the effects of BHB during an intramammary Streptococcus uberis challenge. A secondary objective was to elucidate the mechanisms behind BHB effects on cytokine transcript abundance using the RAW 264.7 cell line. Late lactation multiparous dairy cows (n = 12) were continuously infused intravenously with either BHB to induce hyperketonemia (target concentration: 1.8 mM) or with saline (CON) for 72 h during a S. uberis intramammary challenge. Body temperature, dry matter intake (DMI), milk production, and milk S. uberis cfu were measured daily until one week post-challenge. Blood samples were collected during infusion to assess changes in metabolism (glucose, insulin, glucagon, NEFA, and cortisol) and systemic inflammation (IL-1ß and SAA). Mammary biopsies were conducted at 72 h post-challenge to assess transcript abundance of inflammation-associated genes. BHB-infused cows exhibited a delayed febrile response, noted by a lesser vaginal temperature during the final day of infusion, followed by a greater vaginal temperature 6 d post-challenge. Consequently, BHB-infused cows had greater S. uberis cfu on d 4, 6, and 7 as compared to CON. Accordingly, BHB-infused cows consumed less DM, produced less milk, had reduced blood glucose, and had increased cortisol concentrations, however, no effects were seen on other systemic parameters or transcript abundance of inflammation-related genes in mammary tissue. To elucidate mechanisms behind the impaired immune defenses, RAW 264.7 cells were transfected with a GPR109A siRNA for 24 h and then treated with or without 1.8 mM BHB and challenged or left unchallenged with S. uberis for an additional 3 h. Transfection with siRNA reduced Gpr109a by 75%. Although BHB treatment did not significantly increase Il10, GPR109A knockdown as compared to the scrambled control reduced Il10 by 90% in S. uberis challenged macrophages treated with BHB, suggesting that macrophage immune responses to S. uberis can be altered via a GPR109A-dependent mechanism. Taken together, these data suggest that BHB altered the immune response promoting tolerance toward S. uberis rather than resistance.

Effects of cultivar and harvest days after planting on dry matter yield and nutritive value of teff.

One of the most pressing issues facing the dairy industry is drought. In areas where annual precipitation is low, irrigation for growing feed presents the greatest water-utilization challenge for dairy producers. Here, we investigated the effects of cultivar and harvest days after planting (DAP) on dry matter (DM) yield and nutritive value of teff (Eragrostis tef), a warm-season annual grass native to Ethiopia that is well adapted to drought conditions. Eighty pots were blocked by location in a greenhouse and randomly assigned to four teff cultivars (Tiffany, Moxie, Corvallis, and Dessie) and to five harvest times (40, 45, 50, 55, or 60 DAP). Cultivars had no effect on DM yield and nutritive value. As harvest time increased from 40 to 60 DAP, DM yield and ash-free neutral detergent fiber (aNDFom) concentrations increased, while crude protein (CP) concentrations and in vitro NDF digestibility decreased. To assess carryover effects of time of harvest on yield and nutritive value, two additional cuttings were taken from each pot. Increasing first-cutting harvest time decreased CP concentrations in the second cutting and reduced DM yields in the second and third cutting. Harvesting teff between 45 and 50 DAP best optimized forage yield and nutritive value in the first and subsequent cuttings.

Characterization of the liver proteome in dairy cows experiencing negative energy balance at early lactation.

Negative energy balance (NEB) is associated with metabolic disorders in early lactation dairy cows. Therefore, our objective was to characterize the liver proteome in cows experiencing either NEB or positive energy balance (PEB). Forty-two multiparous Holstein dairy cows were milked either 2 or 3 times daily for the first 30 days in milk (DIM) to alter EB, and were classified retrospectively as NEB (n = 18) or PEB (n = 22). Liver biopsies were collected from 10 cows (n = 5 from each milking frequency) at 17 ± 3 DIM (NEB, n = 6; PEB, n = 4). The liver proteome was characterized using label-free quantitative shotgun proteomics and Ingenuity Pathway Analysis used to identify key affected canonical pathways. Overall, 2741 proteins were identified, and 68 of those were differentially abundant (P ≤ 0.05 and FC ± 1.5). ENO3 (FC = 10.3, P < 0.01) and FABP5 (FC = -12.5, P = 0.045) were the most dramatically upregulated and downregulated proteins, respectively, in NEB cows. Numerous mitochondrial proteins (NDUFA5, NDUFS3, NDUFA6, COX7A2L, COX6C, and COA5) were differentially abundant. Canonical pathways associated with NEB were LPS/IL-1 mediated inhibition of RXR function, oxidative phosphorylation, and mitochondrial dysfunction. Additionally, cows experiencing NEB had less hepatic IL10 transcript abundance than PEB. Together, NEB was associated with altered hepatic inflammatory status, likely due to oxidative stress from mitochondrial dysfunction. SIGNIFICANCE: Our manuscript describes the associations of negative energy balance with the liver proteome in early lactation dairy cows, when metabolic stress and the incidence of diseases is increased. Specifically, we found associations of negative energy balance with shifts in hepatic protein abundance involved in fatty acid uptake, impaired anti-inflammatory responses, and mitochondrial dysfunction. Moving forward, differentially abundant proteins found in this study may be useful as either biological markers for disease or therapeutic targets to improve metabolic adaptations to lactation in postpartum dairy cattle.

Postpartum meloxicam administration alters plasma haptoglobin, polyunsaturated fatty acid, and oxylipid concentrations in postpartum ewes.

BACKGROUND: Postpartum inflammation is a natural and necessary response; however, a dysfunctional inflammatory response can be detrimental to animal productivity. The objective of this study was to determine the effects of a non-steroidal anti-inflammatory drug (meloxicam) on ewe postpartum inflammatory response, ewe plasma polyunsaturated fatty acid and oxylipid concentrations, and lamb growth. RESULTS: After lambing, 36 Hampshire and Hampshire × Suffolk ewes were sequentially assigned within type of birth to control (n = 17) or meloxicam orally administered on d 1 and 4 of lactation (MEL; 90 mg, n = 19). Milk and blood samples were collected on d 1 (prior to treatment) and d 4. Milk glucose-6-phosphate was not affected by MEL. Plasma haptoglobin (Hp) concentrations were less for MEL ewes; control ewes with greater d 1 Hp concentrations had elevated Hp on d 4, but this was not the case for MEL-treated ewes. Treatment with MEL increased plasma arachidonic acid concentration by more than 4-fold in ewes rearing singles but decreased concentrations of 9,10-dihydroxyoctadecenoic acid, prostaglandin F2α, 8-iso-prostaglandin E2, and 8,9-dihydroxyeicosatetraenoic acid. Nine oxylipids in plasma had interactions of treatment with d 1 Hp concentration, all of which revealed positive associations between d 1 Hp and d 4 oxylipid concentrations for CON, but neutral or negative relationships for MEL. MEL decreased 13-hydroxyoctadecadienoic acid:13-oxooctadecadienoic acid ratio and tended to increase 9-hydroxyoctadecadienoic acid:9-oxooctadecadienoic acid ratio (both dependent on d 1 values), indicating progressive metabolism of linoleic acid-derived oxylipids occurred by enzymatic oxidation after MEL treatment. Meloxicam reduced oxylipids generated across oxygenation pathways, potentially due to an improved redox state. CONCLUSIONS: Postpartum MEL treatment of ewes decreased plasma concentrations of Hp and several oxylipids, with the greatest impact in ewes with biomarkers reflecting a greater inflammatory state before treatment. Anti-inflammatory strategies may help resolve excessive postpartum inflammation in some dams.

Comparison of ruminal digestibility of Origanum onites L. leaves in dairy buffalo and cows.

This experiment evaluated the ruminal digestibility of Turkish oregano leaves in dairy buffalo and cows. Ruminally cannulated, multiparous Brown Swiss cows (n = 3) and water buffalo (Bubalus bubalis; n = 3) were used in the experiment. The ad libitum basal diet was balanced to NRC requirements for a dry, nonpregnant multiparous dairy cow (680 kg) and consuming 12.8 kg of DM/day. Air-dried, ground, weighed oregano leaves were inserted in the rumen of all animals before the morning feeding within heat-sealed nylon bags (4 replicates per treatment and time point). After incubation (4, 8, 24, 48, and 72 h), dry matter (DM), crude protein (CP), aNDFom, and ADFom concentrations were determined and compared to initial leaf chemical composition. The means for in situ disappearance (ISD, %) of DM, aNDFom, and ADFom did not differ between the species, but ISD of CP was greater in buffalo than the cows (P = 0.05) after 72 h incubation. The lag time of kinetic degradation curves, the potentially degradable fraction (b), hourly degradation rate (c), and undegradable fraction were similar between species. The immediately degraded fraction (a) and effective digestibility (ED) of CP were greater in buffalo than the cows (P = 0.04), but the a fraction and ED of other nutrients showed no differences between the species. In conclusion, CP of oregano leaves are degraded more rapidly in the rumen of dairy buffalo than cows, primarily due to a large difference in the rapidly soluble CP fraction in the buffalo.

Dietary Zinc-Amino Acid Complex Does Not Affect Markers of Mammary Epithelial Integrity or Heat Stability of Milk in Mid-Lactating Cows.

Supplying dietary zinc in excess of traditional requirements has clear impacts on the gut epithelium, but little research has explored whether similar impacts on the mammary epithelium may occur. Our objective was to determine the effects of supplemental Zn sources, in excess of minimal requirements, on markers of mammary epithelial integrity in blood and in milk as well as the heat stability of milk in mid-lactation cows. Twelve multiparous Holstein cows (132 ± 21 days in milk and 51 ± 3 kg/day milk) were blocked according to milk yield and enrolled in a replicated 3 × 3 Latin square experiment. Experimental periods were 21 days, with 17 days allowed for diet adaptation and 4 days for sampling. Treatment sequences were randomly assigned to animals and treatments were as follows: (1) 0.97 g Zn/day provided as ZnSO4 (34.5 mg supplemental Zn/kg diet DM; 30-ZS), (2) 1.64 g Zn/day provided as ZnSO4 (56.5 mg supplemental Zn/kg diet DM; 60-ZS), and (3) 0.55 g Zn/day provided as ZnSO4 plus 1.13 g Zn/day provided as a zinc-methionine complex (58.2 mg supplemental Zn/kg diet DM; 60-ZM). Treatments were administered once daily as an oral bolus containing all supplemental trace minerals. Rumen-bypass methionine was also included in the 30-ZS and 60-ZS boluses to provide metabolizable methionine equivalent to that provided in 60-ZM rations. Milk samples were assessed for electrolytes, somatic cell transcript abundance of genes related to zinc metabolism, and heat coagulation time. Whole blood samples were analyzed for Na and K concentrations, and plasma samples were analyzed for lactose concentration. Cows fed 60-ZS or 60-ZM had greater zinc intake compared to 30-ZS. Dry matter intake and milk fat content tended to be greater in 60-ZS and 60-ZM cows compared to 30-ZS. Somatic cell linear score was similar among treatments. Treatments neither affected markers of mammary epithelial integrity in blood nor in milk of cows, including plasma concentration of lactose, milk concentrations of Na+ and K+, and SLC30A2 and CLU transcript abundance. Treatments had no effect on milk N fractions or heat coagulation time. This study provided no evidence that supplemental Zn above the established requirements can improve blood-milk epithelial barrier or heat stability of milk in healthy mid-lactation dairy cows.

Choline Regulates the Function of Bovine Immune Cells and Alters the mRNA Abundance of Enzymes and Receptors Involved in Its Metabolism in vitro.

Dietary choline can impact systemic immunity, but it remains unclear whether this is primarily via direct impacts on immune cells or secondary effects of altered metabolic function. To determine whether increased choline concentrations (3.2, 8.2, 13.2 µM) in cell culture alter the function of bovine innate and adaptive immune cells, we isolated cells from dairy cows in early and mid-lactation as models of immuno-compromised and competent cells, respectively. Phagocytic and killing capacity of isolated neutrophils were linearly diminished with increasing doses of choline. In contrast, lymphocyte proliferation was linearly enhanced with increasing doses of choline. Furthermore, increasing doses of choline increased the mRNA abundance of genes involved in the synthesis of choline products (betaine, phosphatidylcholine, and acetylcholine) as well as muscarinic and nicotinic acetylcholine receptors in a quadratic and linear fashion for neutrophils and monocytes, respectively. Phagocytic and killing capacity of neutrophils and proliferation of lymphocytes were not affected by stage of lactation or its interaction with choline or LPS. In neutrophils from early lactation cows, choline linearly increased the mRNA abundance of muscarinic and nicotinic cholinergic receptors, whereas choline-supplemented monocytes from mid-lactation cows linearly increased the mRNA abundance of several genes coding for choline metabolism enzymes. These data demonstrate that choline regulates the inflammatory response of immune cells and suggest that the mechanism may involve one or more of its metabolic products.

High-Throughput Production of Chromium(III) Complexes for Antibody Immobilization.

A robot-assisted high-throughput methodology was employed to produce chromium(III) complexes suitable for the surface modification of the commercially available PerkinElmer Optiplate96 well plate for use in enzyme-linked immunosorbent assays (ELISAs). The complexes were immobilized to the native functionality of the well plate and first screened using a horseradish-peroxidase-tagged (HRP) mouse antibody to quantify binding. The top "hits" were further assessed for their ability to present the antibody in a functional state using an ELISA. "Hits" from the second screen yielded four complexes capable of improving the signal intensity of the ELISA by greater than 500%. The metal/base ratio of these complexes was also investigated, and we isolated the most stable and reproducible candidate, [Cr(OH)6]3-, which was formed from chromium(III) perchlorate and pH adjusted with ethylenediamine. This chromium solution was employed in a clinically relevant setting for the detection of bovine TNFα producing up to a 200% increase in signal intensity.

Invited review: Recommendations for reporting intervention studies on reproductive performance in dairy cattle: Improving design, analysis, and interpretation of research on reproduction.

Abundant evidence from the medical, veterinary, and animal science literature demonstrates that there is substantial room for improvement of the clarity, completeness, and accuracy of reporting of intervention studies. More rigorous reporting guidelines are needed to improve the quality of data available for use in comparisons of outcomes (or meta-analyses) of multiple studies. Because of the diversity of factors that affect reproduction and the complexity of interactions between these, a systematic approach is required to design, conduct, and analyze basic and applied studies of dairy cattle reproduction. Greater consistency, clarity, completeness, and correctness of design and reporting will improve the value of each report and allow for greater depth of evaluation in meta-analyses. Each of these benefits will improve understanding and application of current knowledge and better identify questions that require additional modeling or primary research. The proposed guidelines and checklist will aid in the design, conduct, analysis, and reporting of intervention studies. We propose an adaptation of the REFLECT (Reporting Guidelines for Randomized Controlled Trials for Livestock and Food Safety) statement to provide guidelines and a checklist specific to reporting intervention studies in dairy cattle reproduction. Furthermore, we provide recommendations that will assist investigators to produce studies with greater internal and external validity that can more often be included in systematic reviews and global meta-analyses. Such studies will also assist the development of models to describe the physiology of reproduction.