Transition Cow Nutrition and Management Strategies of Dairy Herds in the Northeastern United States: Associations of Nutritional Strategies with Analytes, Health, Milk Yield, and Reproduction.

The objective was to identify relationships between transition cow nutritional strategies and the prevalence of elevated analytes (nonesterified fatty acids (NEFA), ß-hydroxybutyrate (BHB), and haptoglobin (Hp)), disorder incidence (DI), milk yield, and reproductive performance. Multiparous and primiparous cows from 72 farms in the northeastern US were enrolled in a herd-level cohort study. Farms were dichotomized within parity into a nutritional strategy within each period; far-off: controlled energy (CE; <16.5% starch and ≥40% forage neutral detergent fiber (FNDF)) or not CE (NCE; ≥16.5% starch or <40% FNDF or both), close-up: high FNDF (HF; ≥40% FNDF) or low FNDF (LF; <40% FNDF), and fresh: low starch (LS; <25.5% starch) or high starch (HS; ≥25.5% starch). No evidence existed that transition cow nutritional strategies were associated with milk yield outcomes (p ≥ 0.20). In general, our results support feeding multiparous cows HF close-up and HS fresh to minimize excessive BHB and DI; however, multiparous cows fed LF close-up had a higher pregnancy rate, and lower prepartum NEFA and Hp. Similarly, our results support feeding primiparous cows CE far-off, HF close-up, and HS fresh to maximize reproductive performance, and minimize BHB and DI; however, herds fed HF close-up or HS fresh had higher Hp.

Effects of dietary branched-chain amino acid supplementation on serum and milk metabolome profiles in dairy cows during early lactation.

The 3 branched-chain AA (BCAA), Val, Leu, and Ile, are essential AA used by tissues as substrates for protein synthesis and energy generation. In addition, BCAA are also involved in modulating cell signaling pathways, such as nutrient sensing and insulin signaling. In our previous study, dietary BCAA supplementation was shown to improve protein synthesis and glucose homeostasis in transition cows. However, a more detailed understanding of the changes in metabolic pathways associated with an increased BCAA availability is desired to fine-tune nutritional supplementation strategies. Multiparous Holstein cows (n = 20) were enrolled 28 d before expected calving and assigned to either the BCAA treatment (n = 10) or the control group (n = 10). Cows assigned to BCAA were fed 550 g/d of rumen-protected BCAA mixed with 200 g/d of dry molasses from calving until 35 DIM, whereas the cows assigned to the control were fed only 200 g/d of dry molasses. Serum samples were collected on d 10 before expected calving, as well as on d 4 and d 21 postpartum. Milk samples were collected on d 14 postpartum. From a larger cohort, we selected 20 BCAA-supplemented cows with the greatest plasma urea nitrogen concentration, as an indicator for greater BCAA availability, for the metabolomics analysis herein. Serum and milk samples were subjected to a liquid chromatography-mass spectrometry-based assay, detecting and measuring the abundance of 241 serum and 211 milk metabolic features, respectively. Multivariable statistical analyses revealed that BCAA supplementation altered the metabolome profiles of both serum and milk samples. Increased abundance of serum phosphocholine and glutathione and of milk Val, Ile, and Leu, and decreased abundance of milk acyl-carnitines were associated with BCAA supplementation. Altered phosphocholine and glutathione abundances point to altered hepatic choline metabolism and antioxidant balance, respectively. Altered milk acyl-carnitine abundances suggest changes in mammary fatty acid metabolism. Dietary BCAA supplementation was associated with a range of alterations in serum and milk metabolome profiles, adding to our understanding of the role of BCAA availability in modulating dairy cow protein, lipid, and energy metabolism on a whole-body level and how it affects milk composition.

The effect of ex vivo lipopolysaccharide stimulation and nutrient availability on transition cow innate immune cell AKT/mTOR pathway responsiveness.

Postpartum dairy cows experience a heightened inflammatory state coinciding with the time of greatest nutrient deficit. Nutrient availability is sensed on the cellular level by nutrient sensing kinases, such as the PI3K/AKT/mTOR (mTOR) pathway, a key orchestrator of immune cell activation and inflammatory balance. Our objective was to determine the responsiveness of this pathway to inflammatory stimulation with and without nutrient supplementation ex vivo. Blood samples were collected from Holstein cows (n = 14) at -42, -14, 7, 21, and 42 d relative to calving. Control samples and samples pretreated with a mixture of amino acids, glucose, and insulin (AAM) were stimulated with 100 ng/mL E. coli lipopolysaccharide (LPS; LPS, AAMLPS) or left unstimulated (control, AAM). After 1 h, ratios of mean fluorescence intensity for phosphorylated to total protein of AKT and mTORC1 substrates S6RP and 4EBP1 were analyzed in polymorphonuclear cells (PMN), and monocytes by flow cytometry. A separate aliquot was stimulated with LPS for 2 h and relative mRNA abundance of IL10, IL12A, IL12B, and TNFA in whole blood leukocytes from 10 cows was measured by reverse-transcription quantitative PCR. Repeated measures ANOVA was performed with fixed effects of time, treatment, and their interaction. Cells had different ratios of pathway proteins with PMN having the highest phosphorylation of AKT, S6RP, and 4EBP1. Stimulation with LPS consistently activated mTOR signaling in PMN regardless of nutrient supplementation except for postpartum 4EBP1, which increased in response to nutrients alone. In monocytes, AKT baseline phosphorylation was lower and activation could not be induced by either treatment, whereas activation of 4EBP1 responded to nutrient supplementation. Treatment with LPS increased phosphorylation of S6RP in both innate immune cell types. Nutrient supplementation increased baseline IL10 expression and decreased baseline as well as LPS-induced IL12B and TNFA expression. We conclude that the mTOR pathway in bovine innate immune cells can be differentially activated in response to inflammatory stimulation and nutrient supplementation in monocytes versus PMN. Effects of nutrient supplementation on cytokine mRNA abundance are likely specific to immune cell type.

Longitudinal Phenotypes Improve Genotype Association for Hyperketonemia in Dairy Cattle.

The objective of our study was to identify genomic regions associated with varying concentrations of non-esterified fatty acid (NEFA), ß-hydroxybutyrate (BHB), and the development of hyperketonemia (HYK) in longitudinally sampled Holstein dairy cows. Our study population consisted of 147 multiparous cows intensively characterized by serial NEFA and BHB concentrations. To identify individuals with contrasting combinations in longitudinal BHB and NEFA concentrations, phenotypes were established using incremental area under the curve (AUC) and categorized as follows: Group (1) high NEFA and high BHB, group (2) low NEFA and high BHB), group (3) low NEFA and low BHB, and group (4) high NEFA and low BHB. Cows were genotyped on the Illumina Bovine High-density (777K) beadchip. Genome-wide association studies using mixed linear models with the least-related animals were performed to establish a genetic association with HYK, BHB-AUC, NEFA-AUC, and the comparisons of the 4 AUC phenotypic groups using Golden Helix software. Nine single-nucleotide polymorphisms were associated with high longitudinal concentrations of BHB and further investigated. Five candidate genes related to energy metabolism and homeostasis were identified. These results provide biological insight and help identify susceptible animals thus improving genetic selection criteria thereby decreasing the incidence of HYK.

Associations of peripartum markers of stress and inflammation with milk yield and reproductive performance in Holstein dairy cows.

The objective was to evaluate the association of peripartum concentrations of fecal cortisol metabolites (11,17-dioxoandrostane; 11,17-DOA), plasma cortisol and haptoglobin (Hp), as well as two markers of negative energy balance, non-esterified fatty acid (NEFA) and postpartum ß-hydroxybutyrate (BHBA), with milk yield and reproductive performance. Blood and fecal samples were collected weekly from 412 Holstein dairy cows from wk -3 through wk +1 relative to calving. Pregnancies by 150 days in milk (DIM) and projected 305-d mature equivalent (305ME) milk yield based on the 3rd Dairy Herd Improvement (DHI) test day (mean±SD; 102±17 DIM) were measured. Multivariable linear regression models were used to describe the associations of metabolites with 305ME milk yield. Semiparametric proportional hazards models were used to describe associations of the same metabolites with risk of conception by 150 DIM. Negative associations with milk yield were found for prepartum Hp in wk -2, -1, and +1 relative to calving (estimate±SE: 490±251, 564±259, and 464±136kg lower yield for every increase in Hp concentration by 1g/L, respectively) as well as with NEFA concentration in wk -2 (estimate±SE: 1465±541kg lower milk yield for an increase in NEFA concentration by 1mEq/L). Postpartum associations of NEFA with milk yield depended on parity; NEFA was associated with an increase in milk yield in primiparous animals only (estimate±SE: 1548±510kg increase for an increase in NEFA concentration by 1mEq/L). An increase in plasma cortisol concentration by 1µg/dL in wk +1 relative to calving was associated with an increase in milk yield (estimate±SE: 580±176kg). Prepartum 11,17-DOA was associated in all three prepartum sampling weeks with a reduced hazard ratio (HR) of conception (HR [95% CI]: 0.81 [0.67-0.97], 0.85 [0.72-0.99], and 0.85 [0.75-0.97] for every increase in concentration by 1mg/g fecal dry matter (DM) in wk -3, -2, and -1 relative to calving, respectively). Increased cortisol concentrations in wk -3 and -1 relative to calving were associated with decreased hazard of conception in primiparous animals only (HR [95% CI]: 0.54 [0.32-0.92] and 0.59 [0.35-0.99], respectively. Increases in postpartum metabolites Hp and BHBA had a negative association with hazard of conception (HR [95% CI]: 0.81 [0.70-0.97], 0.74 [0.56-0.98], respectively). Biomarkers of inflammation and stress around calving may be useful to assess opportunities for improved milk yield and reproduction.

Elevated non-esterified fatty acids and ß-hydroxybutyrate and their association with transition dairy cow performance.

Dairy cows pass through a period of negative energy balance as they transition from late gestation to early lactation. Poor adaptation through this period, expressed as excessively elevated concentrations of non-esterified fatty acids (NEFAs) pre- or post-partum and elevated concentrations of ß-hydroxybutyrate post-partum, increases an individual animal's risk of post-partum disease, removal from the herd, reproductive difficulty, and reduced milk production. Field studies have shown that subclinical ketosis often affects 40% of cows in a herd although the incidence can be as high as 80%. Peak incidence occurs at 5 days in milk, and cows that develop subclinical ketosis in the first week of lactation have a higher risk of negative effects and reduced milk production than cows that develop subclinical ketosis in the second week of lactation. Herds with more than a 15-20% prevalence of excessively elevated concentrations of NEFAs and ß-hydroxybutyrate in early lactation have higher rates of negative subsequent events, poorer reproduction, and lower milk yield than herds with a lower prevalence of negative energy balance. This paper reviews (1) strategies for testing of energy-related metabolites, (2) consequences of poor adaptation to negative energy balance (for individual animals and for herds), (3) treatment approaches for affected cows, and (4) economic considerations for testing and treating cows with poor adaptation to negative energy balance.

Using nonesterified fatty acids and ß-hydroxybutyrate concentrations during the transition period for herd-level monitoring of increased risk of disease and decreased reproductive and milking performance.

Dairy cows visit a state of negative energy balance (NEB) as they transition from late gestation to early lactation. At the individual level, there are several metabolic adaptations to manage NEB, including mobilization of nonesterified fatty acids (NEFA) from body fat reserves and glucose sparing for lactogenesis. Based on current pen-level feeding and management practices, strategies to minimize excessive NEB in both the individual and herd should focus on herd-level testing and management. This article reviews strategies for testing and monitoring of excessive NEB at the herd level through individual testing of 2 energy markers: NEFA and ß-hydroxybutyrate.

Dairy cows experience selective reduction of the hepatic growth hormone receptor during the periparturient period.

At parturition, dairy cows experience a 70% reduction in plasma IGF-I. This reduction coincides with decreased abundance of GHR1A, the liver-specific transcript of the growth hormone receptor (GHR) gene, suggesting impaired growth hormone-dependent synthesis of IGF-I. It is not immediately obvious that the periparturient reduction in GHR1A is sufficient to reduce hepatic GHR abundance. This is because approximately 50% of total GHR mRNA abundance in prepartum liver is accounted for by ubiquitously expressed transcripts which remain collectively unchanged at parturition. In addition, the possibility that parturition alters GHR expression in other growth hormone target tissue has not been examined. To address these questions, we measured GHR gene expression and GHR protein in liver and skeletal muscle of four dairy cows on days -35,+3 and+56 (relative to parturition on day 0). Hepatic GHR abundance and GHR1A transcripts were lower on day+3 than on day -35 and returned to late pregnancy value by day+56. Additional studies in two other groups of cows indicated that the hepatic levels of the GHR protein recovered substantially within 10 days after parturition. These changes occurred without variation in the abundance of HNF4, a liver-enriched transcription factor activating the promoter responsible for GHR1A synthesis. In contrast to liver, levels of GHR gene expression and GHR protein were identical on days -35,+3 and+56 in skeletal muscle. These data suggest a role for the GHR in regulating tissue-specific changes in growth hormone responsiveness in periparturient dairy cows.

Development and testing of a high-density cDNA microarray resource for cattle.

A cDNA microarray resource has been developed with the goal of providing integrated functional genomics resources for cattle. The National Bovine Functional Genomics Consortium's (NBFGC) expressed sequence tag (EST) collection was established in 2001 to develop resources for functional genomics research. The NBFGC EST collection and microarray contains 18,263 unique transcripts, derived from many different tissue types and various physiologically important states within these tissues. The NBFGC microarray has been tested for false-positive rates using self-self hybridizations and was shown to yield robust results in test microarray experiments. A web-accessible database has been established to provide pertinent data related to NBFGC clones, including sequence data, BLAST results, and ontology information. The NBFGC microarray represents the largest cDNA microarray for a livestock species prepared to date and should prove to be a valuable tool in studying genome-wide gene expression in cattle.