Prediction of Liver Triglyceride Content in Early Lactation Multiparous Holstein Cows Using Blood Metabolite, Mineral, and Protein Biomarker Concentrations.

Bovine fatty liver syndrome (bFLS) is difficult to diagnose because a liver tissue biopsy is required to assess liver triglyceride (TG) content. We hypothesized that a blood biomarker panel could be a convenient alternative method of liver TG content assessment and bFLS diagnosis. Our objectives were to predict liver TG using blood biomarker concentrations across days in milk (DIM; longitudinal, LT) or at a single timepoint (ST; 3, 7, or 14 DIM), as well as different biomarker combination based on their perceived accessibility. Data from two separate experiments (n = 65 cows) was used for model training and validation. Response variables were based on the maximum liver TG observed in 1 and 14 DIM liver biopsies: Max TG (continuous), Low TG (TG > 13.3% dry matter; DM), Median TG (TG > 17.1% DM), and High TG (TG > 22.0% DM). Model performance varied but High TG was well predicted by sparse partial least squares­discriminate analysis models using LT and ST data, achieving balanced error rates ≤ 15.4% for several model variations during cross-validation. In conclusion, blood biomarker panels using 7 DIM, 14 DIM, or LT data may be a useful diagnostic tool for bFLS in research and field settings.

Circulating Metabolites Indicate Differences in High and Low Residual Feed Intake Holstein Dairy Cows.

Selection for more feed efficient dairy cows is key to improving sustainability and profitability of dairy production; however, underlying mechanisms contributing to individual animal feed efficiency are not fully understood. The objective of this study was to identify circulating metabolites, and pathways associated with those metabolites, that differ between efficient and inefficient Holstein dairy cows using targeted metabolite quantification and untargeted metabolomics. The top and bottom fifteen percent of cows (n = 28/group) with the lowest and highest residual feed intake in mid-lactation feed efficiency trials were grouped retrospectively as high-efficient (HE) and low-efficient (LE). Blood samples were collected for quantification of energy metabolites, markers of hepatic function, and acylcarnitines, in addition to a broader investigation using untargeted metabolomics. Short-chain acylcarnitines, C3-acylcarnitine, and C4-acylcarntine were lower in HE cows (n = 18/group). Untargeted metabolomics and multivariate analysis identified thirty-nine differential metabolites between HE and LE (n = 8/group), of which twenty-five were lower and fourteen were higher in HE. Pathway enrichment analysis indicated differences in tryptophan metabolism. Combined results from targeted metabolite quantification and untargeted metabolomics indicate differences in fatty acid and amino acid metabolism between HE and LE cows. These differences may indicate post-absorptive nutrient use efficiency as a contributor to individual animal variation in feed efficiency.

Novel Facets of the Liver Transcriptome Are Associated with the Susceptibility and Resistance to Lipid-Related Metabolic Disorders in Periparturient Holstein Cows.

Lipid-related metabolic disorders (LRMD) are prevalent in early lactation dairy cows, and have detrimental effects on productivity and health. Our objectives were to identify cows resistant or susceptible to LRMD using a ketosis induction protocol (KIP) to discover differentially expressed liver genes and metabolic pathways associated with disposition. Clustering cows based on postpartum lipid metabolite concentrations within dietary treatments identified cows more or less susceptible (MS vs. LS) to LRMD within the control treatment, and more or less resistant (MR vs. LR) within the KIP treatment. Whole-transcriptome RNA sequencing was performed on liver samples (-28, +1, and +14 days relative to calving) to assess differential gene and pathway expression (LS vs. MS; MR vs. LR; n = 3 cows per cluster). Cows within the MS and LR clusters had evidence of greater blood serum ß-hydroxybutyrate concentration and liver triglyceride content than the LS and MR clusters, respectively. The inferred metabolism of differentially expressed genes suggested a role of immune response (i.e., interferon-inducible proteins and major histocompatibility complex molecules). Additionally, unique roles for glutathione metabolism and eicosanoid metabolism in modulating susceptibility and resistance, respectively, were implicated. Overall, this research provides novel insight into the role of immunometabolism in LRMD pathology, and suggests the potential for unique control points for LRMD progression and severity.

Hyperketonemia Predictions Provide an On-Farm Management Tool with Epidemiological Insights.

Prediction of hyperketonemia (HYK), a postpartum metabolic disorder in dairy cows, through use of cow and milk data has allowed for high-throughput detection and monitoring during monthly milk sampling. The objective of this study was to determine associations between predicted HYK (pHYK) and production parameters in a dataset generated from routine milk analysis samples. Data from 240,714 lactations across 335 farms were analyzed with multiple linear regression models to determine HYK status. Data on HYK or disease treatment was not solicited. Consistent with past research, pHYK cows had greater previous lactation dry period length, somatic cell count, and dystocia. Cows identified as pHYK had lower milk yield and protein percent but greater milk fat, specifically greater mixed and preformed fatty acids (FA), and greater somatic cell count (SCC). Differential somatic cell count was greater in second and fourth parity pHYK cows. Culling (60d), days open, and number of artificial inseminations were greater in pHYK cows. Hyperketonemia prevalence decreased linearly in herds with greater rolling herd average milk yield. This research confirms previously identified risk factors and negative outcomes associated with pHYK and highlights novel associations with differential SCC, mixed FA, and preformed FA across farm sizes and production levels.

Greater liver PNPLA3 protein abundance in vivo and in vitro supports lower triglyceride accumulation in dairy cows.

Fatty liver syndrome is a prevalent metabolic disorder in peripartum dairy cows that unfavorably impacts lactation performance and health. Patatin-like phospholipase domain-containing protein 3 (PNPLA3) is a lipase that plays a central role in human non-alcoholic fatty liver disease etiology but has received limited attention in bovine fatty liver research. Thus, we investigated the relationship between tissue PNPLA3 expression and liver triglyceride accumulation in vivo via a ketosis induction protocol in multiparous dairy cows peripartum, as well as in vitro via small interfering RNA knockdown of PNPLA3 mRNA expression in bovine primary hepatocytes. Results demonstrated a negative association (P = 0.04) between liver PNPLA3 protein abundance and liver triglyceride content in peripartum dairy cows, while adipose PNPLA3 protein abundance was not associated with liver triglyceride content or blood fatty acid concentration. Knockdown of PNPLA3 mRNA resulted in reduced PNPLA3 protein abundance (P < 0.01) and greater liver triglyceride content (P < 0.01). Together, these results suggest greater liver PNPLA3 protein abundance may directly limit liver triglyceride accumulation peripartum, potentially preventing bovine fatty liver or accelerating recovery from fatty liver syndrome.

Hyperketonemia GWAS and parity-dependent SNP associations in Holstein dairy cows intensively sampled for blood ß-hydroxybutyrate concentration.

Hyperketonemia (HYK) is a metabolic disorder that affects early postpartum dairy cows; however, there has been limited success in identifying genomic variants contributing to HYK susceptibility. We conducted a genome-wide association study (GWAS) using HYK phenotypes based on an intensive screening protocol, interrogated genotype interactions with parity group (GWIS), and evaluated the enrichment of annotated metabolic pathways. Holstein cows were enrolled into the experiment after parturition, and blood samples were collected at four timepoints between 5 and 18 days postpartum. Concentration of blood ß-hydroxybutyrate (BHB) was quantified cow-side via a handheld BHB meter. Cows were labeled as a HYK case when at least one blood sample had BHB ≥ 1.2 mmol/L, and all other cows were considered non-HYK controls. After quality control procedures, 1,710 cows and 58,699 genotypes were available for further analysis. The GWAS and GWIS were performed using the forward feature select linear mixed model method. There was evidence for an association between ARS-BFGL-NGS-91238 and HYK susceptibility, as well as parity-dependent associations to HYK for BovineHD0600024247 and BovineHD1400023753. Candidate genes annotated to these single nuclear polymorphism associations have been previously associated with obesity, diabetes, insulin resistance, and fatty liver in humans and rodent models. Enrichment analysis revealed focal adhesion and axon guidance as metabolic pathways contributing to HYK etiology, while genetic variation in pathways related to insulin secretion and sensitivity may affect HYK susceptibility in a parity-dependent matter. In conclusion, the present work proposes several novel marker associations and metabolic pathways contributing to genetic risk for HYK susceptibility.

Prepartum supplementation of conjugated linoleic acids (CLA) increased milk energy output and decreased serum fatty acids and ß-hydroxybutyrate in early lactation dairy cows.

Prepartum supplementation with conjugated linoleic acid (CLA) may influence lipolysis and hyperketonemia in dairy cows. The objective of this study was to examine the effect of prepartum CLA supplementation on lactation performance and serum fatty acids (FA) and ß-hydroxybutyrate (BHB) in early lactation dairy cows, and secondarily on reproductive performance. Multiparous cows were enrolled in the study at 18 days prior to expected calving date, and randomly assigned 100 g/day of Lutrell Pure (BASF, Ludwigshafen, Germany; 75% FA), providing 10 g/day of each CLA isomer (trans-10 cis-12 and cis-9 trans-11 CLA) or equivalent amount of rumen inert fatty acids as control (78 g/day of Energy Booster 100; Milk Specialties Global, Eden Prairie, MN). Treatments were top dressed daily to individual cows from enrollment to calving and all cows were offered the same ration. Blood samples were collected on the first day of supplementation, 10 days prepartum, and 1, 7, 14, and 30 days postpartum. Hyperketonemia was defined as serum BHB ≥ 1.2 mM. Milk yield was recorded daily until 60 days postpartum and averaged weekly. Milk samples were obtained weekly for component analysis. Prepartum CLA supplementation tended to increase serum concentration of cis-9, trans-11 CLA and increased trans-10, cis-12 CLA prepartum. Cows supplemented with CLA had increased milk protein yield and tended to have increased milk fat yield and milk yield, which together resulted in greater energy content of milk. Cows supplemented with CLA had lower serum FA on day 1 and 7 postpartum and overall lower serum BHB postpartum, which resulted in decreased prevalence of hyperketonemia on day 14 postpartum. There were no differences in body condition score change, other health disorders, or reproductive outcomes by treatment. Together, these findings indicate that prepartum CLA supplementation may be a plausible strategy to positively influence postpartum performance.

Relationships between body condition score change, prior mid-lactation phenotypic residual feed intake, and hyperketonemia onset in transition dairy cows.

Extensive efforts have been made to identify more feed-efficient dairy cows, yet it is unclear how selection for feed efficiency will influence metabolic health. The objectives of this research were to determine the relationships between residual feed intake (RFI), a measure of feed efficiency, body condition score (BCS) change, and hyperketonemia (HYK) incidence. Blood and milk samples were collected twice weekly from cows 5 to 18 d postcalving for a total of 4 samples. Hyperketonemia was diagnosed at a blood ß-hydroxybutyrate (BHB) ≥1.2 mmol/L and cows were treated upon diagnosis. Dry period, calving, and final blood sampling BCS was recorded. Prior mid-lactation production, body weight, body weight change, and dry matter intake (DMI) data were used to determine RFI phenotype, calculated as the difference between observed DMI and predicted DMI. The maximum BHB concentration (BHBmax) for each cow was used to group cows into HYK or not hyperketonemic. Lactation number, BCS, and RFI data were analyzed with linear and quadratic orthogonal contrasts. Of the 570 cows sampled, 19.7% were diagnosed with HYK. The first positive HYK test occurred at 9 ± 0.9 d postpartum and the average BHB concentration at the first positive HYK test was 1.53 ± 0.14 mmol/L. In the first 30 d postpartum, HYK-positive cows had increased milk yield and fat concentration, decreased milk protein concentration, and decreased somatic cell count. Cows with a dry BCS ≥4.0, or that lost 1 or more BCS unit across the transition to lactation period, had greater BHBmax than cows with lower BCS. Prior-lactation RFI did not alter BHBmax. Avoiding over conditioning of dry cows and subsequent excessive fat mobilization during the transition period may decrease HYK incidence; however, RFI during a prior lactation does not appear to be associated with HYK onset.