Invited review: Ketone biology-The shifting paradigm of ketones and ketosis in the dairy cow.

Ketosis is currently regarded as a major metabolic disorder of dairy cows, reflective of the animal's efforts to adapt to energy deficit while transitioning into lactation. Currently viewed as a pathology by some, ketosis is associatively implicated in milk production losses and peripartal health complications that increase the risk of early removal of cows from the herd, thus carrying economic losses for dairy farmers and jeopardizing the sustainability of the dairy industry. Despite decades of intense research in the mitigation of ketosis and its sequelae, our ability to lessen its purported effects remains limited. Moreover, the association of ketosis to reduced milk production and peripartal disease is often erratic and likely mired by concurrent potential confounders. In this review, we discuss the potential reasons for these apparent paradoxes in the light of currently available evidence, with a focus on the limitations of observational research and the necessary steps to unambiguously identify the effects of ketosis on cow health and performance via controlled randomized experimentation. A nuanced perspective is proposed that considers the dissociation of ketosis-as a disease-from healthy hyperketonemia. Furthermore, in consideration of a growing body of evidence that highlights positive roles of ketones in the mitigation of metabolic dysfunction and chronic diseases, we consider the hypothetical functions of ketones as health-promoting metabolites and ponder on their potential usefulness to enhance dairy cow health and productivity.

Milk component ratios and their associations with energy balance indicators and serum calcium concentration in early-lactation spring-calving pasture-based dairy cows.

Indirect assessment of metabolic status using milk samples provides a non-invasive and objective tool for cow-level health monitoring. Milk fat-to-protein ratio (FPR) has been commonly evaluated as an indirect measure for negative energy balance (EB) in confined dairy cows. However, milk component ratios have not been explored for their association with pasture-based cows' metabolic status. The objectives of this observational study were to 1) describe milk component ratios from 0 to 45 d postpartum, 2) evaluate the associations between milk component ratios [FPR, fat-to-lactose (FLR), protein-to-lactose (PLR)] and indicators of EB (serum ß-hydroxybutyrate (BHB) concentration at 5-45 d postpartum and body condition score (BCS) change during the transition period), and 3) evaluate the associations between milk component ratios and serum Ca concentration 0-4 d postpartum in spring-calving dairy cows from pasture-based commercial farms. Milk component ratios were determined on samples collected before AM or PM milkings from 548 cows at 0-45 d postpartum (n = 970). Serum BHB and Ca determinations were performed in blood samples collected at the time of milk sample collection at 5-45 d postpartum (n = 918) and 0-4 d postpartum (n = 50), respectively; and BCS change was calculated using BCS assigned between 29 d prepartum and 45 d postpartum (n = 851). Cows' calving date, parity (1st, 2nd-3rd or ≥ 4th) and breed (Holstein-Friesian or dairy crossbred) information was obtained from the farm records. Data was analyzed by multiple linear regression. Average milk FPR, FLR and PLR were 0.70, 0.53 and 0.72, respectively. Milk FPR linearly increased while milk FLR linearly decreased postpartum both at a rate of 0.004 units per day; milk PLR decreased 0.05 units per day for the first 30 d postpartum and moderately increased afterward. Milk FPR and FLR were 0.71 and 0.52 units lower before AM than PM milking, respectively; while milk PLR was similar before AM and PM milking. Milk FPR and FLR were 0.07 to 0.10 units higher for 2nd-3rd compared with 1st and ≥ 4th parity cows. Milk PLR was 0.03 units greater for ≥ 4th compared with 2nd-3rd and 1st parity cows. Further, crossbred cows had 0.07, 0.08 and 0.03 higher milk FPR, FLR and PLR than Holstein-Friesian cows, respectively. Moderate to high P-values along with moderate to small estimated slopes and wide 95% confidence intervals were observed for the associations between milk component ratios and indicators of EB. A positive linear association was observed between milk FPR and serum Ca concentration within 4 d postpartum; milk FPR increased 0.31 units per each mmol/L increase in serum Ca concentration. Cows with low serum Ca concentration within 4 d postpartum had 0.27 units lower milk FPR compared with cows at or above the threshold (2.12 mmol/L), and tended to have 0.15 units lower milk FPR compared with cows at or above the threshold (2.00 mmol/L). In conclusion, further research is needed to reach conclusions on the association between milk component ratios determined before milking and EB indicators. The potential of milk FPR for monitoring blood Ca status warrants further investigation in early-lactation pasture-based dairy cows.

Untargeted metabolomics and lipidomics to assess plasma metabolite changes in dairy goats with subclinical hyperketonemia.

Subclinical hyperketonemia (SCHK) is the major metabolic disease observed during the transition period in dairy goats, and is characterized by high plasma levels of nonesterified fatty acids (NEFA) and ß-hydroxybutyrate (BHB). However, no prior study has comprehensively assessed metabolomic profiles of dairy goats with SCHK. Plasma samples were collected within 1 h after kidding from SCHK goats (BHB concentration >0.8 mM, n = 7) and clinically healthy goats (BHB concentration <0.8 mM, n = 7) with similar body condition score (2.75 ± 0.15, mean ± standard error of the mean) and parity (primiparous). A combination of targeted and untargeted mass spectrometric approaches was employed for analyzing the various changes in the plasma lipidome and metabolome. Statistical analyses were performed using the GraphPad Prism 8.0, SIMCA-P software (version 14.1), and R packages (version 4.1.3). Plasma aminotransferase, nonesterified fatty acids, and BHB concentrations were greater in the SCHK group, but plasma glucose concentrations were lower. A total of 156 metabolites and 466 lipids were identified. The analysis of untargeted metabolomics data by principal component analysis and orthogonal partial least squares discriminant analysis revealed a separation between SCHK and clinically healthy goats. According to the screening criteria (unpaired t-test, P < 0.05), 30 differentially altered metabolites and 115 differentially altered lipids were detected. Pathway enrichment analysis identified citrate cycle, alanine, aspartate and glutamate metabolism, glyoxylate and dicarboxylate metabolism, and phenylalanine metabolism as significantly altered pathways. A greater concentration of plasma isocitric acid and cis-aconitic acid levels was observed in SCHK goats. In addition, AA such as lysine and isoleucine were greater, whereas alanine and phenylacetylglycine were lower in SCHK dairy goats. Dairy goats with SCHK also exhibited greater oleic acid, acylcarnitine, and phosphatidylcholine and lower choline and sphingomyelins. Acylcarnitines, oleic acid, and tridecanoic acid displayed positive correlations with several lipid species. Alanine, hippuric acid, and histidinyl-phenylalanine were negatively correlated with several lipids. Overall, altered metabolites in SCHK dairy goats indicated a more severe degree of negative energy balance. Data also indicated an imbalance in the tricarboxylic acid (TCA) cycle, lipid metabolism, and AA metabolism. The findings provide a more comprehensive understanding of the pathogenesis of SCHK in dairy goats.

The association of hyperketonemia with fecal and rumen microbiota at time of diagnosis in a case-control cohort of early lactation cows.

BACKGROUND: Many dairy cows experience a state of energy deficit as they transition from late gestation to early lactation. The aims of this study were to 1) determine if the development of hyperketonemia in early lactation dairy cows is indicated by their gut microbiome, and 2) to identify microbial features which may inform health status. We conducted a prospective nested case-control study in which cows were enrolled 14 to 7 days before calving and followed through their first 14 days in milk (DIM). Hyperketonemic cows (HYK, n = 10) were classified based on a blood ß-hydroxybutyrate (BHB) concentration 1.2 mmol/L within their first 14 DIM. For each HYK cow, two non-HYK (CON, n = 20) cows were matched by parity and 3 DIM, with BHB < 1.2 mmol/L. Daily blood BHB measures were used to confirm CON cows maintained their healthy status; some CON cows displayed BHB 1.2 mmol/L after matching and these cows were reclassified as control-HYK (C-HYK, n = 9). Rumen and fecal samples were collected on the day of diagnosis or matching and subjected to 16S rRNA profiling. RESULTS: No differences in taxa abundance, or alpha and beta diversity, were observed among CON, C-HYK, and HYK health groups for fecal microbiomes. Similar microbiome composition based on beta diversity analysis was detected for all health statuses, however the rumen microbiome of CON and HYK cows were found to be significantly different. Interestingly, highly similar microbiome composition was observed among C-HYK cow rumen and fecal microbiomes, suggesting that these individual animals which initially appear healthy with late onset of hyperketonemia were highly similar to each other. These C-HYK cows had significantly lower abundance of Ruminococcus 2 in their rumen microbiome compared to CON and HYK groups. Multinomial regressions used to compute log-fold changes in microbial abundance relative to health status were not found to have predictive value, therefore were not useful to identify the role of certain microbial features in predicting health status. CONCLUSIONS: Lower relative abundance of Ruminococcus 2 in C-HYK cow rumens was observed, suggesting these cows may be less efficient at degrading cellulose although the mechanistic role of Ruminococcus spp. in rumen metabolism is not completely understood. Substantial differences in fecal or rumen microbiomes among cows experiencing different levels of energy deficit were not observed, suggesting that hyperketonemia may not be greatly influenced by gut microbial composition, and vice versa. Further studies using higher resolution -omics approaches like meta-transcriptomics or meta-proteomics are needed to decipher the exact mechanisms at play.

An investigation of blood, milk, and urine test patterns for the diagnosis of ketosis in dairy cows in early lactation.

Ketosis in dairy cattle is primarily diagnosed based on the concentrations of ketone bodies in the blood, milk, or urine. Cow-side tests using these fluids are available for rapid detection of elevated concentrations of ketone bodies. Although these tests have been extensively validated, the performance of different tests has not been compared over time. Our objectives were to investigate the relationship between point-of-care diagnostic tests measuring the concentrations of ß-hydroxybutyrate (BHB) in blood (BT; Precision Xtra, Abbott Laboratories), BHB in milk (MT; Keto-Test, Elanco), and acetoacetate (AcAc) in urine (UT; Ketostix, Bayer Corporation) through cases of ketosis. Holstein cows (n = 148) were screened daily for hyperketonemia (HYK; blood BHB ≥1.2 mmol/L) from 3 to 16 d in milk (DIM); moreover, milk and urine samples were collected concomitantly and tested for ketones (ketosis thresholds: 100 µmol/L milk BHB and 5 mg/dL urine AcAc). Of the animals screened (n = 148), 74% were diagnosed with HYK. When diagnosed with HYK, cows were treated with propylene glycol orally once daily for 5 d. After the day of diagnosis (d 0), hyperketonemic cows were retested with BT, MT, and UT for 3 d (d 1, 2, and 3). We assessed the diagnostic test performance and time to ketosis (survival analyses and Cox proportional hazards models) of MT and UT compared with BT. Considering all paired samples (before and after diagnosis of HYK), MT had 61% sensitivity and 91% specificity, whereas the UT had 77% sensitivity and 94% specificity compared with BT. The specificity of MT and UT increased from d 0 to d 1, decreased on d 2, and increased on d 3. The median time to diagnosis of ketosis in blood was 5 DIM (95% CI 5 to 7 DIM); moreover, MT and UT had 2 d greater median time to diagnosis of ketosis compared with the BT [7 DIM (6 to 11 d); and 7 DIM (6 to 13 d), respectively]. We concluded that the UT is a more sensitive predictor of blood BHB concentration than the MT. The UT and MT tests diagnosed ketotic cows approximately 2 d later than the BT. The possible consequences of delay in detection of ketosis in milk and urine should be investigated.

Assessment of milk yield and composition, early reproductive performance, and herd removal in multiparous dairy cattle based on the week of diagnosis of hyperketonemia in early lactation.

The purpose of this retrospective cohort study was to evaluate the effects of the timing of hyperketonemia (HYK) diagnosis during early lactation on milk yield and composition, reproductive performance, and herd removal. Plasma ß-hydroxybutyrate (BHB) was measured twice a week during the first 2 wk of lactation in 362 multiparous Holstein cows for the diagnosis of HYK. In each week, cows were diagnosed as HYK positive (HYK+) if the plasma BHB concentrations were ≥1.2 mmol/L in at least one of the tests for the week evaluated. Milk-related outcomes (first 10 monthly milk tests) included milk yield, milk fat and protein content, milk urea nitrogen (MUN), and linear score of somatic cell count. Other performance outcomes of interest included risk of pregnancy within 150 d in milk (DIM) and herd removal (i.e., culling or death) within 300 DIM. Statistical models were built separately for cows diagnosed with HYK during the first week of lactation (wk1) and for cows diagnosed during the second week of lactation (wk2). All models for wk2 were adjusted by HYK diagnosed in wk1, along with other potential confounder variables. The association between HYK in each week and milk-related outcomes was assessed using generalized estimated equation models that accounted for repeated measures. Time to pregnancy and time to herd removal were analyzed using Cox's proportional hazard regression models. Seventy-eight cows (21.5%) tested positive for HYK during wk1, 60 cows (16.6%) in wk2, and 29 cows (8.0%) in both weeks. Hyperketonemia during wk1 was associated with a milk yield reduction of 3.7 kg [95% confidence interval (CI): -6.67 to -0.76] per cow per day throughout the lactation. Meanwhile, we did not observe evidence of an association between HYK diagnosed during wk2 and milk yield. During the first 2 monthly milk tests, cows diagnosed as HYK+ in wk1 had greater fat (0.42%; 95% CI: 0.16 to 0.67) and MUN (0.75 mg/dL; 95% CI: 0.26 to 1.24) content in milk than HYK-negative (HYK-) cows. We did not detect any evidence of an association between HYK diagnosed in wk2 and these outcomes. The HYK+ cows in wk1 had a 30% [hazard ratio (HR) = 0.70; 95% CI: 0.48 to 1.01] lower risk of pregnancy within 150 DIM and 2.48 times (95% CI: 1.63 to 2.89) higher risk of herd removal within 300 DIM than HYK- cows. Conversely, no evidence of association was observed between HYK+ cows in wk2 and risk of pregnancy by 150 DIM (HR = 0.98; 95% CI: 0.64 to 1.51) or removal from the herd within 300 DIM (HR = 0.91; 95% CI: 0.52 to 1.60). Our findings indicate that HYK diagnosed during wk1 of lactation is associated with negative performance in terms of milk yield, reproduction, and herd removal. No evidence of association was found for the same outcomes when HYK was diagnosed in wk2. Our results suggest the need to consider the timing when HYK is diagnosed when investigating its association with performance outcomes.

A systematic review of the cost of ketosis in dairy cattle.

A systematic review was conducted to assess the cost of ketosis in dairy cattle, and to elucidate how ketosis cost is estimated in each of the studies. Scientific papers addressing the economic impact of ketosis in dairy cows were identified through a search in 4 databases (Medline, ISI Web of Science, CAB Abstracts, and Agricola). The literature search was conducted with no restrictions on the date of study publication, publication type, or language. The methodological quality of the studies was assessed regarding study design, data collection, and analysis and interpretation of the study results. Of 531 identified records, 10 were selected, of which 9 were published from 2015 onward. Of the 10 studies reviewed, 9 report cost of a case of ketosis, and the estimates vary widely, with values ranging from €19 to €812. Two studies report ketosis cost at a farm level (€3.6-€29/cow per year). Among the studies, we observed great variation not only in the estimation models and inputs used (costs and losses associated with the disease) but also in the definition of ketosis and its prevalence or incidence figures. Moreover, the cost of ketosis was estimated for dairy farms in the United States, Canada, the Netherlands, Denmark, France, Germany, Spain, Sweden, Norway, and India. Consequently, there was great heterogeneity regarding herd characteristics, milk production, milk prices, culled cows' value, feed prices, and costs of veterinary services. Ketosis cost estimates vary as a consequence of all these aspects. Therefore, although most of the studies were well-designed and used high-quality data, the systematic approach review does not allow combination of the cost estimates of into a single figure. In conclusion, our review highlights an overall considerable economic impact of ketosis in dairy cattle. Economic prevention and mitigation strategies should be taken according to herd- and country-specific conditions. Ketosis cost figures reported in economic studies should always be considered carefully and interpreted with appropriate consideration of the inputs of the estimation, country context, and herd parameters.

Reducing milking frequency from twice to once daily as an adjunct treatment for ketosis in lactating dairy cows-A randomized controlled trial.

This randomized controlled trial investigated the effects of temporarily reducing milking frequency (MF) on the resolution of ketosis and milk production in dairy cows in early lactation. To detect ketosis [blood ß-hydroxybutyrate (BHB) ≥1.2 mmol/L], Holstein cows were screened daily from 3 to 16 d in milk using a cow-side meter. Cows diagnosed with ketosis (n = 104) were randomly assigned to twice-daily milking (TDM) or reduced to once-daily milking (ODM) for 2 wk, then returned to twice-daily milking. Both treatment groups received a 5-d treatment of an oral propylene glycol drench (PG; 300 g) beginning on the afternoon of the diagnosis; cows received additional 5-d PG treatments if they had a ketotic test result (blood BHB ≥1.2 mmol/L) at least 4 d after finishing the first PG treatment. Blood BHB tests were conducted for the first 3 d after ketosis diagnosis, and then once every 3 d for 21 d of trial (DOT). Milk and milk component data were collected weekly for 15 wk following trial enrollment. The ODM group showed rapidly and markedly decreased blood BHB concentrations (primiparous cows: 1 DOT, 0.92 ODM vs. 1.22 TDM, 15 DOT, 0.55 vs. 0.81 mmol/L; multiparous cows: 1 DOT, 1.01 vs. 1.40, 15 DOT, 0.78 vs. 1.65 mmol/L). In addition, a logistic regression model indicated that ODM cows were less likely to have blood BHB concentrations ≥1.2 mmol/L [primiparous cows: 3 DOT: ODM 1% (95% confidence interval: 0-10%) vs. TDM 43% (30-58%), 15 DOT ODM 0% (0-0.2%) vs. TDM 22% (13-36%); multiparous cows: 3 DOT: ODM 33% (24-44%) vs. TDM 59% (48-69%), 15 DOT ODM 20.9% (13-31%) vs. TDM 64% (53-74%)]. The proportion of ODM cows that required additional treatments of PG were substantially lower than the TDM group (ODM: 39%; TDM: 64%) than the TDM cows during the initial 21-d period. However, during the 2-wk treatment period, cows in the ODM group produced 26% less milk and 25% less energy-corrected milk than the TDM cows. During wk 3 to 15, when all cows were milked twice daily, ODM cows produced less milk (-14%) and energy-corrected milk (-12%) compared with the TDM group. Milk protein percentage was greater, and milk fat percentage and linear score tended to be greater in the ODM group over 15 wk. In conclusion, a 2-wk reduction of MF in ketotic cows from twice to once daily with treatment with PG resolved ketosis and decreased blood BHB concentrations more effectively than treating TDM cows with PG alone. However, the 2-wk MF reduction had immediate and long-term (up to 13 wk after cessation of MF reduction) negative effects on milk production.

Effect of hyperketonemia on the diurnal patterns of energy-related blood metabolites in early-lactation dairy cows.

Most dairy cows experience a period of energy deficit in early lactation, resulting in increased plasma concentrations of nonesterified fatty acids (NEFA) and ß-hydroxybutyrate (BHB). Our objectives were to determine (1) the diurnal variation in plasma BHB and NEFA, (2) the correlation between plasma NEFA and BHB when accounting for diurnal changes, and (3) the effect of hyperketonemia (HYK) on the diurnal pattern of blood metabolites. Jugular catheters were placed in 28 multiparous Holstein cows between 3 and 9 days in milk, and blood samples were collected every 2 h for 96 h. Cows were retrospectively classified as HYK positive (HYK; n = 13) if they had plasma BHB concentrations ≥1.2 mmol/L for ≥3 study days, or HYK negative (non-HYK; n = 15) if they had plasma BHB concentrations ≥1.2 mmol/L for ≤2 study days. Generalized linear mixed models were used to analyze concentrations of analytes over time and differences in metabolites between HYK groups. The correlation between total plasma NEFA and BHB was analyzed by calculating the area under the curve for plasma NEFA and BHB for all cows. Plasma NEFA reached a peak approximately 2 h before morning feed delivery, falling to a nadir in the late evening. Plasma BHB was at a nadir at the time of morning feed delivery, peaking 4 h later. We observed a strong positive correlation between daily plasma NEFA and BHB. Additionally, HYK cows had greater concentrations of plasma NEFA and BHB than non-HYK cows. The HYK cows also experienced a greater magnitude of change in BHB throughout the day than the non-HYK cows. Our results suggest that the time relative to feeding should be considered when analyzing plasma metabolites, as classification of energy status may change throughout a day.

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.

Increased autophagy mediates the adaptive mechanism of the mammary gland in dairy cows with hyperketonemia.

Hyperketonemia is a metabolic disease in dairy cows, associated with negative nutrition balance (NNB) induced by low dry matter intake (DMI) and increased nutrient requirements. Hyperketonemia could induce metabolic stress, which might indirectly affect mammary tissue. Autophagy is a highly conserved physiological process that results in the turnover of intracellular material, and is involved in maintaining cellular homeostasis under the challenge of metabolic stress induced by NNB. The aim of this study was to investigate the autophagy status and autophagy-related pathways AMP-activated kinase α (AMPKα) and mechanistic target of rapamycin (mTOR) in the mammary glands of dairy cows with hyperketonemia. Cows with hyperketonemia [CWH, n = 10, blood ß-hydroxybutyrate (BHB) concentration 1.2 to 3.0 mmol/L] and cows without hyperketonemia (CWOH, n = 10, BHB < 1.2 mmol/L) from 3 to 12 DIM were randomly selected from the herd. The mammary tissue and blood samples were collected from these cows between 0630 and 0800 h, before feeding, at 3 to 12 d in milk. Serum concentrations of glucose, BHB, and fatty acids were determined using an autoanalyzer with commercial kits between 0630 and 0800 h, before feeding. Concentrations of fatty acids, BHB (median and interquartile range: CWH, 2.44 and 1.3, 2.82 mM; CWOH, 0.49 and 0.41, 0.57 mM), and milk fat were greater in CWH. The DMI, glucose concentration, milk production, and milk protein levels were lower in CWH. The mRNA abundance of autophagosome formation-related gene, beclin 1 (BECN1), phosphatidylinositol 3-kinase catalytic subunit type 3 (PIK3C3), autophagy-related gene (ATG) 5, ATG7, ATG12, microtubule-associated protein 1 light chain 3 (MAP1LC3, also called LC3) and sequestosome-1 (SQSTM1, also called p62) were greater in the mammary glands of CWH. The protein abundance of LC3-II and phosphorylation level of Unc-51-like kinase 1 (ULK1) were greater in CWH, but the total ubiquitinated proteins and protein abundance of p62 were lower. Transmission electron microscopy showed an increased number of autophagosomes in the mammary glands of CWH. Furthermore, the phosphorylation of AMPKα was greater, but the phosphorylation of mTOR was lower in the mammary glands of CWH. These results indicate that activity of mTOR pathways and autophagy activity, and upregulation of AMPKα, may be response mechanisms to mitigate metabolic stress induced by hyperketonemia in the mammary glands of dairy cows.

Short communication: Assessing the accuracy of inline milk fat-to-protein ratio data as an indicator of hyperketonemia in dairy cows in herds with automated milking systems.

The objective of this study was to evaluate the accuracy of inline milk fat-to-protein (F:P) data to detect hyperketonemia (HYK) in herds with automated milking systems (AMS). The F:P ratio has been investigated as a tool for detecting HYK with moderate accuracy in past studies, but inline F:P data in AMS may also be useful for HYK screening. To assess the accuracy of these data in commercial settings, we monitored 484 cows from 9 AMS herds for their first 3 wk of lactation, taking blood samples once per week (n = 1,427). Positive cases of HYK were defined by whole-blood ß-hydroxybutyrate (BHB) concentrations ≥1.2 or ≥1.4 mmol/L. Milk data were collected from the AMS software on each farm for each cow and converted into 4 different F:P values: (1) value from the same day as the BHB test; (2) 5-d centered-moving average (CMA); (3) 5-d backward-moving average (BMA); (4) 5-d forward-moving average (FMA). In linear regression models, all 4 values were associated with BHB, but slope estimates varied and R2 were low: same day (slope = 0.95, R2 = 0.07), CMA (slope = 1.05, R2 = 0.07), BMA (slope = 0.65, R2 = 0.04), and FMA (slope = 1.23, R2 = 0.09). In logistic regression models, the odds of having HYK (BHB ≥1.2 mmol/L) increased with every 0.1-unit increase from the mean F:P ratio (1.16) using same-day values (odds ratio = 1.35, 95% confidence interval = 1.25-1.47) and CMA (odds ratio = 1.39, 95% confidence interval = 1.27-1.51). The same increase in F:P from mean BMA (1.14) and FMA (1.17) was associated with 1.22 and 1.49 times the odds of HYK, respectively. For all 4 F:P variations, we evaluated the sensitivity, specificity, positive predictive value, and negative predictive value of different F:P thresholds with HYK status. As the F:P threshold increased from 1.17 to 1.50, sensitivity decreased (range: 77 to 9%) but specificity increased (range: 58 to 96%). Same-day and CMA F:P cutoffs at which a balance was reached between sensitivity and specificity ranged from 1.18 to 1.22; however, even at these values we found high rates of false positives and negatives (range: 31-39%). These results suggest that inline milk F:P data from inconsistently calibrated sensors should not be used alone to detect HYK in AMS herds.

Postpartum supplementation of fermented ammoniated condensed whey improved feed efficiency and plasma metabolite profile.

Postpartum dietary supplementation of gluconeogenic precursors may improve the plasma metabolite profile of dairy cows, reducing metabolic disorders and improving lactation performance. The objective of this trial was to examine the effects of supplementation with fermented ammoniated condensed whey (FACW) postpartum on lactation performance and on profile of plasma metabolites and hormones in transition dairy cows. Individually fed multiparous Holstein cows were blocked by calving date and randomly assigned to control (2.9% dry matter of diet as soybean meal; n = 20) or FACW (2.9% dry matter of diet as liquid GlucoBoost, Fermented Nutrition, Luxemburg, WI; n = 19) dietary treatments. Treatments were offered from 1 to 45 d in milk (DIM). Cows were milked twice a day. Dry matter intake and milk yield were recorded daily and averaged weekly. Individual milk samples from 2 consecutive milkings were obtained once a week for component analysis. Rumen fluid was collected (n = 3 cows/treatment) at 4 time points per day at 7 and 21 DIM. Blood samples were collected within 1 h before feeding time for metabolite analysis and hyperketonemia diagnosis. Supplementation of FACW improved feed efficiency relative to control; this effect may be partially explained by a marginally significant reduction in dry matter intake from wk 3 to 7 for FACW-supplemented cows with no detected FACW-driven changes in milk yield, milk protein yield, and milk energy output compared with control. Also, there was no evidence for differences in intake of net energy for lactation, efficiency of energy use, energy balance, or body weight or body condition score change from calving to 45 DIM between treatments. Supplementation of FACW shifted rumen measures toward greater molar proportions of propionate and butyrate, and lesser molar proportions of acetate and valerate. Cows supplemented with FACW had greater plasma glucose concentrations in the period from 3 to 7 DIM and greater plasma insulin concentrations compared with control. Plasma nonesterified fatty acid and ß-hydroxybutyrate concentrations were decreased in cows supplemented with FACW compared with control cows in the period from 3 to 7 DIM. These findings indicate that FACW may have improved the plasma metabolite profile immediately postpartum in dairy cows. Additionally, supplementation of FACW resulted in improved feed efficiency as accessed by measures of milk output relative to feed intake.

Short communication: Cow- and herd-level prevalence of hypoglycemia in hyperketonemic postpartum dairy cows.

The objective of this study was to quantify the prevalence of hypoglycemia in hyperketonemic dairy cows during the early postpartum period. A prospective observational study was conducted in 100 dairy herds selected by convenience. Within all participating herds, 40 cows (or the entire herd if smaller than 40 cows) were enrolled in the study (total of 3,776 enrolled cows). Herds were visited every 2 wk by an animal health technician. Cows were bled from their coccygeal vessels once between 1 and 14 d in milk, and cow-side testing was performed for ketonemia and glycemia using a device validated in cattle (Precision Xtra, Abbott, Mississauga, ON, Canada). Hyperketonemia was defined as ß-hydroxybutyrate ≥1.4 mmol/L, and hypoglycemia was defined as glucose ≤2.2 mmol/L. Descriptive statistics were computed at the cow and herd levels. The cow-level prevalence of hyperketonemia, hypoglycemia, and simultaneous hypoglycemia and hyperketonemia was 20.0% (757/3,776), 13.8% (642/3,776), and 6.2% (235/3,776), respectively. Within the subset of hyperketonemic cows only, the prevalence of hypoglycemia was 31.0% (235/757). At the herd level, the median prevalence was 17.5% (minimum: 5.0%, first quartile: 10.0%, third quartile: 22.5%, maximum: 77.5%) for hyperketonemia, 15.0% (minimum: 5.0%, first quartile: 12.5%, third quartile: 20.0%, maximum: 47.5%) for hypoglycemia, and 7.5% (minimum: 2.5%, first quartile: 5.0%, third quartile: 12.5%, maximum: 17.5%) for simultaneous hypoglycemia and hyperketonemia. The herd-level median prevalence of hypoglycemia within the subset of hyperketonemic cows only was 30.6% (minimum: 2.5%, first quartile: 20.0%, third quartile: 39.1%, maximum: 63.0%). The results from this study show that the prevalence of simultaneous hyperketonemia and hypoglycemia is relatively low in the overall early postpartum cow population but also that approximately one-third of hyperketonemic cows are hypoglycemic, which might represent an opportunity to improve their management on the farm.

Short communication: The association of adiponectin and leptin concentrations with prepartum dietary energy supply, parity, body condition, and postpartum hyperketonemia in transition dairy cows.

Adipokines-hormones produced by adipose tissue-have important regulatory functions, and their concentrations can change around the time of calving when energy balance rapidly decreases. Hence, energy balance may be an important factor in determining the circulating concentrations of adipokines, particularly adiponectin and leptin. The objective of our study was to investigate the association between the level of energy fed to prepartum Holstein cows and circulating concentrations of adiponectin and leptin before and after calving. Holstein dairy cows entering second or greater lactation were fed either a controlled-energy diet formulated to supply approximately 100% of energy requirements (n = 28) or a high-energy diet formulated to supply approximately 150% of energy requirements throughout the entire dry period (n = 28). Serum samples were analyzed for adiponectin and leptin concentrations at 56, 28, 10, and 1 d prepartum as well as on d 1, 10, 21, and 42 postpartum using ELISA. Parity was dichotomized into cows entering second versus higher parity. Average peripartum body condition score (BCS) was computed from weekly measurements and dichotomized into animals with an average BCS of ≤3.25 and >3.25. In addition, cows were classified according to the occurrence of hyperketonemia (ß-hydroxybutyrate concentrations ≥1.2 mmol/L at any time between 3 and 21 d in milk). Data were analyzed using repeated-measures ANOVA. Serum leptin but not adiponectin concentrations were associated with prepartum feeding level such that leptin concentrations increased transiently during the dry period in cows overfed energy, but concentrations were not different postpartum. Cows entering second parity had higher adiponectin and lower leptin concentrations compared with cows in higher parities. Cows that developed hyperketonemia postpartum had consistently lower adiponectin concentrations during the study period. Cows with average BCS >3.25 had higher leptin concentrations during the dry period only, but adiponectin concentrations were not associated with BCS. In conclusion, prepartum energy level had only transient effects on leptin concentrations and did not lead to changes in adiponectin concentrations.

Technical note: Evaluation of the diagnostic accuracy of 2 point-of-care ß-hydroxybutyrate devices in stored bovine plasma at room temperature and at 37°C.

The use of point-of-care (POC) devices to measure blood metabolites, such as ß-hydroxybutyrate (BHB), on farm have become an important diagnostic and screening tool in the modern dairy industry. The POC devices allow for immediate decision making and are often more economical than the use of laboratory-based methods; however, precision and accuracy may be lower when measurements are performed in an uncontrolled environment. Ideally, the advantages of the POC devices and the standardized laboratory environment could be combined when measuring samples that do not require an immediate result-for example, in research applications or when immediate intervention is not the goal. The objective of this study was to compare the capability of 2 POC devices (TaiDoc, Pharmadoc, Lübeck, Germany; Precision Xtra, Abbott Diabetes Care, Abingdon, UK) to measure BHB concentrations either at room temperature (RT; 20-22°C) or at 37°C compared with the gold standard test in stored plasma samples. Whole blood from multiparous Holstein dairy cows (n = 113) was sampled from the coccygeal vessels between 28 d before expected calving and 42 DIM. Whole-blood BHB concentrations were determined cow-side using the TaiDoc POC device. Plasma was separated within 1 h of collection and stored until analysis. A subset of stored plasma samples (n = 100) consisting of 1 sample per animal was chosen retrospectively based on the BHB concentrations in whole blood within the range of 0.2 to 4.0 mmol/L. The samples were analyzed for BHB plasma concentration using an automated chemistry analyzer (Hitachi 917, Hitachi, Tokyo, Japan), which was considered the gold standard. On the same day, the samples were also measured with the 2 POC devices, with samples either at RT or heated up to 37°C. Our study showed high Spearman correlation coefficients (>0.99) using either device and with samples at both temperatures compared with the gold standard. Passing-Bablok regression revealed a very strong correlation (>0.99), indicating good agreement between both POC devices and the gold standard method. For hyperketonemia detection, defined as BHB concentration ≥1.2 mmol/L, the sensitivity for both POC devices at RT and 37°C was equally high at 100%. Specificity was lowest (67.4%) for the TaiDoc used with plasma at RT and was highest (86.5%) when plasma was measured at 37°C with the Precision Xtra meter. Bland-Altman plots revealed a mean bias of 0.25 and 0.4 mmol/L for the Precision Xtra meter and TaiDoc, respectively, when tested on plasma at 37°C. Our data showed that both POC devices are suitable for measuring BHB concentration in stored bovine plasma, and accuracy was highest when samples were heated to 37°C compared with RT.

Effect of rumen-protected B vitamins and choline supplementation on health, production, and reproduction in transition dairy cows.

The objectives were to determine the effects of a rumen-protected blend of B vitamins and choline (RPBC) on the incidence of health disorders, milk yield, and reproduction in early lactation and the effects on gene expression and liver fat infiltration. A randomized controlled trial in 3 commercial dairy herds (n = 1,346 cows with group as the experimental unit; experiment 1) and a university research herd (n = 50 cows with cow as the experimental unit; experiment 2) evaluated the use of 100 g/cow per d of commercially available proprietary RPBC supplement (Transition VB, Jefo, St. Hyacinthe, Quebec, Canada), or a placebo, fed 3 wk before to 3 wk after calving. In experiment 2 liver biopsies were taken at 4 and 14 ± 1 d in milk to measure triacylglycerol concentrations and expression of 28 genes selected to represent relevant aspects of liver metabolism. Treatment effects were assessed using multivariable mixed logistic regression models for binary health and reproductive outcomes; linear regression models for milk yield, dry matter intake, and liver outcomes; and survival analysis for time insemination and pregnancy. In experiment 1, treatment did not have an effect on the incidence of hyperketonemia (blood ß-hydroxybutyrate ≥ 1.2 mmol/L; cumulative incidence to 3 wk postpartum of 28 to 30%), clinical health disorders, or udder edema. The prevalence of anovulation at 8 wk postpartum was 11% in the treatment group and 23% in the control but did not differ statistically given group-level randomization. Pregnancy at first insemination (33 and 35%) and median time to pregnancy to 200 d in milk (96 and 97 d) were not different between treatment and control, respectively. No difference was observed between treatment groups in milk yield or components through the first 3 Dairy Herd Improvement Association test days (44 kg/d in both groups, accounting for parity and components). In experiment 2, there were no differences between treatment groups in feed intake. Mean blood ß-hydroxybutyrate was lower at wk 3 in RPBC (0.6 vs. 0.9 ± 0.12 mmol/L) with no difference between treatments for mean blood concentrations of fatty acids (wk -1 or 1) and ß-hydroxybutyrate at wk 1 or 2. The gene for acyl-CoA oxidase 1 (ACOX1) had lower mRNA abundance in RPBC with no difference between treatments for the other genes, but the expression of half of the genes assessed differed with days in milk. Liver triacylglycerol was lower in primiparous cows at 4 d in milk in RPBC (2.0 vs. 4.4 ± 1.2%) but not at 14 d in milk (2.2 vs. 3.2 ± 0.97%) with no treatment effect in multiparous cows (4.6 ± 0.8%). Accounting for parity, days in milk, fat and protein percentages, repeated test days, and a random effect of cow, no significant difference was observed between treatments in milk yield across the first 3 Dairy Herd Improvement Association tests (41.2 ± 1.3 in RPBC vs. 38.0 ± 1.4 kg/d in control). Under the diet and management conditions of the field study including low prevalence of clinical health disorders, in experiment 1 we did not detect a benefit of RPBC, but in experiment 2 liver fat content decreased in primiparous cows.

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.

The hypophagic response to heat stress is not mediated by GPR109A or peripheral ß-OH butyrate.

Rising temperatures resulting from climate change will increase the incidence of heat stress, negatively impacting the labor force and food animal production. Heat stress elevates circulating ß-OH butyrate, which induces vasodilation through GPR109a. Interestingly, both heat stress and intraperitoneal ß-OH butyrate administration induce hypophagia. Thus, we aimed to investigate the role of ß-OH butyrate in heat stress hypophagia in mice. We found that niacin, a ß-OH butyrate mimetic that cannot be oxidized to generate ATP, also reduces food intake. Interestingly, the depression in food intake as a result of 8-h intraperitoneal niacin or 48-h heat exposure did not result from changes in hypothalamic expression of orexigenic or anorexigenic signals (AgRP, NPY, or POMC). Genetically eliminating GPR109a expression did not prevent the hypophagic response to heat exposure, intraperitoneal ß-OH butyrate (5.7 mmol/kg), or niacin (0.8 mmol/kg). Hepatic vagotomy eliminated the hypophagic response to ß-OH butyrate and niacin but did not affect the hypophagic response to heat exposure. We subsequently hypothesized that the hypophagic response to heat stress may depend on direct effects of ß-OH butyrate at the central nervous system: ß-OH butyrate induced hormonal changes (hyperinsulinemia, hypercorticosteronemia, and hyperleptinemia), or gene expression changes. To test these possibilities, we blocked expression of hepatic hydroxyl methyl glutaryl CoA synthase II (HMGCS2) to prevent hepatic ß-OH butyrate synthesis. Mice that lack HMGCS2 maintain a hypophagic response to heat stress. Herein, we establish that the hypophagia of heat stress is independent of GPR109a, the hepatic vagus afferent nerve, and hepatic ketone body synthesis.

A randomized controlled trial of dexamethasone as an adjunctive therapy to propylene glycol for treatment of hyperketonemia in postpartum dairy cattle.

Treatment of hyperketonemia with oral propylene glycol has proven efficacy but the cure rate remains moderate. Dexamethasone has long been suggested as a treatment for hyperketonemia, even though evidence of its efficacy is contradictory. The objective of this randomized controlled trial was to evaluate the effect of adding a single intramuscular injection of 20mg of dexamethasone to oral propylene glycol therapy for hyperketonemia [blood ß-hydroxybutyrate (BHB) ≥1.2mmol/L]. All cows between 3 and 16d in milk on 4 dairy farms in New York State were tested once weekly for hyperketonemia using a handheld ketone meter. All enrolled animals received 312g (300mL) of propylene glycol orally once daily for 4d and either a single injection of dexamethasone or an equivalent volume of sterile saline. A total of 509 animals were enrolled, with 254 and 255 in the placebo and dexamethasone groups, respectively. Treatment with dexamethasone decreased the odds of being hyperketonemic in the second week posttreatment; however, the odds of hyperketonemia in the first week posttreatment only decreased in those animals that were treated at a BHB blood concentration between 1.2 and 1.5mmol/L. For the 8% of cows with blood BHB >3.2mmol/L at enrollment, receiving dexamethasone increased the odds of being hyperketonemic the following week. We detected no difference between treatment groups in the odds of postpartum disease or in milk production. For cows with initial BHB of 1.2 to 1.5mmol/L, treatment with dexamethasone tended to reduce the odds of pregnancy at first insemination. Based on the small and conditional benefits of dexamethasone and a lack of difference in milk yield or disease incidence, we do not recommend the use of dexamethasone to treat hyperketonemia.