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Transition cow nutrition and management strategies of dairy herds in the northeastern United States: Part II—Associations of metabolic- and inflammation-related analytes with health, milk yield, and reproduction

Open AccessPublished:April 22, 2022DOI:https://doi.org/10.3168/jds.2021-20863

      ABSTRACT

      The objectives were as follows: (1) establish cow-level thresholds for prepartum nonesterified fatty acids (NEFA) and postpartum NEFA, β-hydroxybutyrate (BHB), and haptoglobin (Hp) concentrations associated with negative health events; (2) evaluate cow-level associations between biomarkers and 305-d mature equivalent milk at the fourth test day (ME305) and reproductive performance; and (3) identify herd-alarm levels (proportion of cows sampled above the critical threshold) for biomarkers that are associated with herd-level changes in disorder incidence (displaced abomasum and clinical ketosis), reproductive performance, and ME305. In a prospective cohort study, 1,473 cows from 72 farms were enrolled from the northeastern United States. Blood samples were collected from the same 11 to 24 cows per herd during the late-prepartum and early-postpartum periods. Whole blood was analyzed for postpartum BHB concentrations; plasma was analyzed for prepartum and postpartum NEFA and postpartum Hp concentrations. Critical thresholds for the biomarkers associated with health events for all cows were established using a receiver operating characteristic curve analysis. Poisson, linear mixed effects, and Cox proportional hazards models investigated the association of the biomarkers with health and performance. The prepartum NEFA and Hp threshold associated with culling was ≥0.17 mmol/L and 0.45 g/L, respectively. The postpartum NEFA and BHB thresholds associated with diagnosis of metritis, displaced abomasum, or clinical ketosis were ≥0.46 mmol/L and ≥0.9 mmol/L, respectively. Multiparous cows with prepartum NEFA concentration ≥0.17 mmol/L produced 479 kg less ME305. Multiparous and primiparous cows with postpartum NEFA concentration ≥0.46 mmol/L produced 280 kg less and 446 kg more ME305, respectively. Cows with BHB concentration ≥0.9 and ≥1.1 mmol/L produced 552 kg more ME305 and had a 20% decreased risk of pregnancy within 150 d in milk, respectively; however, multiparous cows with BHB concentration ≥1.5 mmol/L produced 376 kg less ME305. Cows with Hp concentration ≥0.45 g/L produced 492 kg less ME305 and had 28% decreased risk of pregnancy within 150 DIM. Cows with Hp concentration ≥0.45 g/L had 19% decreased pregnancy risk to first service (PRFS). Herds above the herd-alarm levels for prepartum NEFA had a 6.0-percentage unit increase in disorder incidence and a 6.0-percentage unit decrease in 21-d pregnancy rate (PR) for multiparous cows, a 3.9-percentage unit increase in PR and a 5.8-percentage unit increase in the probability of pregnancy for primiparous cows. Herds above the herd-alarm levels for postpartum NEFA had a 5.8- and 4.2-percentage unit increase in disorder incidence for multiparous and primiparous cows, respectively, a 789 kg decrease in ME305 for multiparous cows, and a 6.8- and 6.3-percentage unit decrease and increase in PR and PRFS for multiparous cows, respectively. Herds above the herd-alarm levels for BHB had an 8.5-percentage unit increase in disorder incidence, a 332 and 229 kg increase in ME305 for primiparous and multiparous cows, respectively, and a 3.2-, 5.2-, and 7.0-percentage unit decrease in PR, probability of pregnancy, and PRFS, respectively. Herds above the herd-alarm levels for postpartum Hp had a 5.3-percentage unit increase in disorder incidence. At the cow level and herd level, elevated biomarker concentrations were associated with an increased disorder risk and varied performance responses.

      Key words

      INTRODUCTION

      Given an increase in nutrient requirements for milk production, decrease in DMI, parturition, and management factors such as frequent pen moves, the transition period is arguably the most demanding time in a dairy cow's life. Increased blood concentrations of nonesterified fatty acids (NEFA) and BHB are a result of adipose tissue being mobilized in response to meeting the energy demands of the conceptus or calf, via milk production, as well as partial oxidation of NEFA by the liver (
      • Bell A.W.
      Regulation of organic nutrient metabolism during transition from late pregnancy to early lactation.
      ). In addition, haptoglobin (Hp), a positive acute phase protein produced in the liver, may be elevated in the postpartum cow due to inflammation (
      • Uchida E.
      • Katoh N.
      • Takahashi K.
      Appearance of haptoglobin in serum from cows at parturition.
      ;
      • Bradford B.J.
      • Yuan K.
      • Farney J.K.
      • Mamedova L.K.
      • Carpenter A.J.
      Invited review: Inflammation during the transition to lactation: New adventures with an old flame.
      ). Inflammation may occur in the transition cow due to tissue damage associated with parturition (
      • Medzhitov R.
      Origin and physiological roles of inflammation.
      ), uterine infection (
      • Skinner J.G.
      • Brown R.A.
      • Roberts L.
      Bovine haptoglobin response in clinically defined field conditions.
      ;
      • Huzzey J.M.
      • Nydam D.V.
      • Grant R.J.
      • Overton T.R.
      Associations of prepartum plasma cortisol, haptoglobin, fecal cortisol metabolites, and nonesterified fatty acids with postpartum health status in Holstein dairy cows.
      ), insults to the gastrointestinal tract causing gastrointestinal barrier dysfunction (
      • Horst E.A.
      • Kvidera S.K.
      • Baumgard L.H.
      Invited review: The influence of immune activation on transition cow health and performance – A critical evaluation of traditional dogmas.
      ), or potentially through infiltration of excessive NEFA in the liver (
      • Katoh N.
      Relevance of apolipoproteins in the development of fatty liver and fatty liver-related peripartum diseases in dairy cows.
      ). Environmental stressors, such as transportation, may also trigger an acute phase response (
      • Lomborg S.R.
      • Nielsen L.R.
      • Heegaard P.M.
      • Jacobsen S.
      Acute phase proteins in cattle after exposure to complex stress.
      ;
      • Cooke R.F.
      • Bohnert D.W.
      • Moriel P.
      • Hess B.W.
      • Mills R.R.
      Effects of polyunsaturated fatty acid supplementation on ruminal in situ forage degradability, performance, and physiological responses of feeder cattle.
      ), though this has not been investigated in transition cows. It is of interest to investigate Hp because it is observed at negligible blood concentrations in a healthy dairy cow but can increase over 100-fold at the onset of an inflammation event (
      • Eckersall P.D.
      Recent advances and future prospects for the use of acute phase proteins as markers of disease in animals.
      ).
      Cows with elevated circulating NEFA, BHB, and Hp during the first couple weeks of lactation have an increased risk of developing disorders (
      • Huzzey J.M.
      • Duffield T.F.
      • LeBlanc S.J.
      • Veira D.M.
      • Weary D.M.
      • von Keyserlingk M.A.G.
      Short communication: Haptoglobin as an early indicator of metritis.
      ;
      • Ospina P.A.
      • Nydam D.V.
      • Stokol T.
      • Overton T.R.
      Evaluation of nonesterified fatty acids and β-hydroxybutyrate in transition dairy cattle in the northeastern United States: Critical thresholds for prediction of clinical diseases.
      ;
      • Chapinal N.
      • Carson M.
      • Duffield T.F.
      • Capel M.
      • Godden S.
      • Overton M.
      • Santos J.E.P.
      • LeBlanc S.J.
      The association of serum metabolites with clinical disease during the transition period.
      ), decreased milk production (
      • Ospina P.A.
      • Nydam D.V.
      • Stokol T.
      • Overton T.R.
      Associations of elevated nonesterified fatty acids and β-hydroxybutyrate concentrations with early lactation reproductive performance and milk production in transition dairy cattle in the northeastern United States.
      ;
      • Chapinal N.
      • Carson M.E.
      • LeBlanc S.J.
      • Leslie K.E.
      • Godden S.
      • Capel M.
      • Santos J.E.P.
      • Overton M.W.
      • Duffield T.F.
      The association of serum metabolites in the transition period with milk production and early-lactation reproductive performance.
      ;
      • Huzzey J.M.
      • Mann S.
      • Nydam D.V.
      • Grant R.J.
      • Overton T.R.
      Associations of peripartum markers of stress and inflammation with milk yield and reproductive performance in Holstein dairy cows.
      ), and reduced reproductive performance (
      • Ospina P.A.
      • Nydam D.V.
      • Stokol T.
      • Overton T.R.
      Associations of elevated nonesterified fatty acids and β-hydroxybutyrate concentrations with early lactation reproductive performance and milk production in transition dairy cattle in the northeastern United States.
      ;
      • Garverick H.A.
      • Harris M.N.
      • Vogel-Bluel R.
      • Sampson J.D.
      • Bader J.
      • Lamberson W.R.
      • Spain J.N.
      • Lucy M.C.
      • Youngquist R.S.
      Concentrations of nonesterified fatty acids and glucose in blood of periparturient dairy cows are indicative of pregnancy success at first insemination.
      ;
      • Huzzey J.M.
      • Mann S.
      • Nydam D.V.
      • Grant R.J.
      • Overton T.R.
      Associations of peripartum markers of stress and inflammation with milk yield and reproductive performance in Holstein dairy cows.
      ).
      Thresholds have been established for prepartum NEFA and postpartum NEFA and BHB concentrations (
      • Ospina P.A.
      • Nydam D.V.
      • Stokol T.
      • Overton T.R.
      Evaluation of nonesterified fatty acids and β-hydroxybutyrate in transition dairy cattle in the northeastern United States: Critical thresholds for prediction of clinical diseases.
      ;
      • Chapinal N.
      • Carson M.
      • Duffield T.F.
      • Capel M.
      • Godden S.
      • Overton M.
      • Santos J.E.P.
      • LeBlanc S.J.
      The association of serum metabolites with clinical disease during the transition period.
      ). Measuring these biomarkers in fresh cows can help identify individual cows that are at a greater risk of adapting poorly to lactation. In addition, these biomarkers can be used as a herd-level monitoring tool for improving a farm's health-management program. We wanted to reevaluate these metabolites using pragmatic models to further investigate their association with health and performance in an independent population and identify associations between the prevalence of elevated biomarkers and nutritional and management strategies. Although there have been large, epidemiological studies evaluating metabolic-related analytes (
      • Ospina P.A.
      • Nydam D.V.
      • Stokol T.
      • Overton T.R.
      Association between the proportion of sampled transition cows with increased nonesterified fatty acids and β-hydroxybutyrate and disease incidence, pregnancy rate, and milk production at the herd level.
      ;
      • Chapinal N.
      • LeBlanc S.J.
      • Carson M.E.
      • Leslie K.E.
      • Godden S.
      • Capel M.
      • Santos J.E.P.
      • Overton M.W.
      • Duffield T.F.
      Herd-level association of serum metabolites in the transition period with disease, milk production, and early lactation reproductive performance.
      ), Hp thresholds have previously been identified in controlled research trials rather than cohort studies of large populations. It is of interest to evaluate Hp as it may provide a unique opportunity to address nutritional and nonnutritional challenges, such as management and environmental aspects that may be related to inflammation (
      • Huzzey J.M.
      • Mann S.
      • Nydam D.V.
      • Grant R.J.
      • Overton T.R.
      Associations of peripartum markers of stress and inflammation with milk yield and reproductive performance in Holstein dairy cows.
      ).
      We hypothesized that (1) elevated prepartum NEFA and postpartum NEFA, BHB, and Hp concentrations are associated with negative health events, (2) elevated prepartum NEFA and postpartum NEFA, BHB, and Hp concentrations are associated with decreased milk production and reproductive performance, and (3) herds above the herd-alarm level identified for prepartum NEFA and postpartum NEFA, BHB, and Hp concentrations will have increased disorder incidence, decreased milk production, and decreased reproductive performance. Therefore, the objectives were as follows: (1) establish cow-level thresholds for prepartum NEFA and postpartum NEFA, BHB, and Hp concentrations that are associated with negative health events in the first 30 DIM, (2) evaluate the association between the identified prepartum NEFA and postpartum NEFA, BHB, and Hp concentration thresholds with milk production and reproductive performance at the cow level, and (3) identify the herd-alarm levels for prepartum NEFA and postpartum NEFA, BHB, and Hp concentrations that are associated with herd-level changes in disorder incidence, reproductive performance, and milk production.

      MATERIALS AND METHODS

      Study Population and Study Design

      A more complete description of the study design and study population can be found in
      • Kerwin A.L.
      • Burhans W.S.
      • Mann S.
      • Tetreault M.
      • Nydam D.V.
      • Overton T.R.
      Transition cow nutrition and management strategies of dairy herds in the Northeastern United States: Part I—Herd description and performance characteristics.
      . Briefly, a prospective cohort study was conducted from a convenience sample of 72 farms located in New York and Vermont between November 2012 and August 2015. All procedures involving animals in this study were approved by the Cornell University Institutional Animal Care and Use Committee, protocol # 2012–0124. Inclusion criteria for farms were as follows: (1) Holstein herds, (2) ≥400 milking cows, (3) freestall housing, (4) TMR-fed herds, and (5) enrolled in monthly DHI testing or have on-farm milk recording with record management by Dairy Comp 305 (DairyComp 305, Valley Ag Software) or PCDART (PCDART, Dairy Records Management System). The inclusion criteria represented the majority of cows within New York and provided us with an adequate sample size for blood sample collection.
      Detail on blood sampling and analytical methods can be found in
      • Kerwin A.L.
      • Burhans W.S.
      • Mann S.
      • Tetreault M.
      • Nydam D.V.
      • Overton T.R.
      Transition cow nutrition and management strategies of dairy herds in the Northeastern United States: Part I—Herd description and performance characteristics.
      . Briefly, blood from a convenience sample of 11 to 24 cows was collected twice, once during the close-up dry period when they were approximately 0 to 17 d (0–21 d for the first 10 herds enrolled) from expected calving and once during the fresh period when they were 0 to 17 DIM (0–21 DIM for the first 10 herds enrolled). Approximately one-third of the cows sampled were primiparous cows to reflect typical dairy herd demographics. Primiparous cows were defined as cows entering their first lactation, and multiparous cows were defined as cows entering their second or greater lactation. Whole blood from the postpartum period was analyzed for BHB concentrations. Plasma from the prepartum and postpartum periods were analyzed for NEFA concentrations, and plasma from the postpartum period was analyzed for Hp concentrations for cows 0 to 12 DIM. Cows were observed for disorders of interest by the farm personnel, and these were described previously (
      • Kerwin A.L.
      • Burhans W.S.
      • Mann S.
      • Tetreault M.
      • Nydam D.V.
      • Overton T.R.
      Transition cow nutrition and management strategies of dairy herds in the Northeastern United States: Part I—Herd description and performance characteristics.
      ). Locomotion scores (LS) and BCS were assigned to each cow at each visit by a single investigator and described previously (
      • Kerwin A.L.
      • Burhans W.S.
      • Mann S.
      • Tetreault M.
      • Nydam D.V.
      • Overton T.R.
      Transition cow nutrition and management strategies of dairy herds in the Northeastern United States: Part I—Herd description and performance characteristics.
      ). Health event records, reproductive records, and milk records were acquired through the farm's record management software program when all cows observed were at least 150 DIM.

      Statistical Analysis

      A sample size calculation was conducted to estimate the prevalence of cows with hyperketonemia within a herd (≥1.2 mmol/L;
      • McArt J.A.A.
      • Nydam D.V.
      • Oetzel G.R.
      Epidemiology of subclinical ketosis in early lactation dairy cattle.
      ) using a 2-stage cluster sampling (
      • Thrusfield M.V.
      Veterinary Epidemiology.
      ). With a 95% confidence interval, precision of ±3.5%, expected prevalence of 17.5%, a calculated between-cluster variance of 0.01 (
      • Ospina P.A.
      • Nydam D.V.
      • Stokol T.
      • Overton T.R.
      Evaluation of nonesterified fatty acids and β-hydroxybutyrate in transition dairy cattle in the northeastern United States: Critical thresholds for prediction of clinical diseases.
      ), and a fixed cluster size of 72 farms, we calculated that we needed 12 cows per herd. A precision of 3.5% was chosen as it is one-fifth of the estimated prevalence (
      • Pourhoseingholi M.A.
      • Vahedi M.
      • Rahimzadeh M.
      Sample size calculation in medical studies.
      ). With a precision of ±3.0%, we needed 23 cows per herd. We attempted to sample 24 cows per herd during the close-up visit to account for attrition rate of cows that were excluded from further sampling if they had not calved by the fresh visit.
      Raw data were entered into Microsoft Excel (Microsoft Corp.). All statistical analyses were performed using SAS software (SAS 9.4, SAS Institute Inc.). Before statistical analysis, data cleaning was conducted to correct human data recording errors.
      To determine whether quadratic relationships existed between the biomarkers and our cow-level outcomes, we used a logistic regression using PROC GENMOD with a logit-link function, p-scale option for overdispersion correction, an exchangeable correlation matrix, and farm as a random effect for binary outcomes and a mixed linear regression model using PROC MIXED with herd as a random effect for continuous outcomes. We did observe a quadratic effect (P < 0.05) for some of the negative health event models [BHB: clinical ketosis (CK); prepartum NEFA: CK; postpartum NEFA: metritis (MET), CK, and 1 or more of MET, displaced abomasum (DA), and CK; and Hp: culling within 30 DIM (CULL30)] and 305-d mature equivalent milk yield at the fourth test day (ME305; BHB); however, we identified very high inflection points with <10% of observations above the inflection point. It is difficult to make any inferences regarding the quadratic effect due to few observations above the inflection point; therefore, a linear relationship was assumed between the biomarkers and the outcomes of interest, and pragmatic models were used in the statistical analysis described in the following paragraphs for the different outcomes.

      Biomarker Threshold Analysis

      Poisson regression models using PROC GENMOD with a log-link function, p-scale option for overdispersion correction, an exchangeable correlation matrix, and farm as a random effect were developed to determine if postpartum (1) NEFA, (2) BHB, and (3) Hp as independent variables were associated with the following dependent binary variables: (1) MET, (2) DA, (3) CK, (4) 1 or more of the disorders (MET, DA, CK), or (5) CULL30. Additional models were developed to determine if prepartum NEFA as an independent variable was associated with the following dependent binary variables: (1) MET or retained placenta (RP), (2) DA, (3) CK, (4) 1 or more of the disorders (MET, RP, DA, CK), or (5) CULL30. The unit of observation was the cow. A farm was removed from the analysis for the specific dependent variable if the disorder was not recorded, but was retained for the other analyses. A cow was removed from the analysis for the specific dependent variable if the outcome of interest occurred before the blood sample was acquired. Cows with missing covariate information were not included in the analysis unless the covariate was not included in the final model. Categorical covariates were screened and included in the full model if a simple logistic regression model (PROC LOGISTIC) yielded P < 0.20. Covariates considered in the full model included the following binary variables: parity group (primiparous vs. multiparous), calving difficulty (calving score <3 vs. calving score ≥3 or twins; calving score described previously in
      • Kerwin A.L.
      • Burhans W.S.
      • Mann S.
      • Tetreault M.
      • Nydam D.V.
      • Overton T.R.
      Transition cow nutrition and management strategies of dairy herds in the Northeastern United States: Part I—Herd description and performance characteristics.
      ), LS at time of blood sample collection (<3 vs. ≥ 3), BCS at time of blood sample collection (close-up visit: ≤3.25 vs. >3.25 or fresh visit: ≤3.0 vs. >3.0), change in BCS from previous visit to blood sample collection visit (far-off to close-up visit: ≥0 vs. <0, or close-up to fresh visit:≥−0.25 vs. <−0.25), and calving season [cool (October through April) vs. warm (May through September)]. Multicollinearity was assessed and determined to be present if a covariate that was included in the full model had a tolerance <0.40 (
      • Allison P.D.
      Logistic Regression using SAS: Theory and Application.
      ). Days relative to parturition was forced into all models as a continuous covariate to control for variability within the sampled cohort. The main effect of biomarker was initially forced into the model as a continuous variable, and manual backward stepwise elimination was used to remove variables with P ≥ 0.10. Biologically plausible interactions between the main effect and covariates were then assessed, and manual backward stepwise elimination was used to remove any variable with P ≥ 0.05. If the biomarker remained in the model, a generalized linear regression using the GLIMMIX procedure with farm as a random effect was used to obtain predicted probabilities. A simple linear regression using the LOGISTIC procedure was then used to perform a receiver operating characteristic (ROC) curve analysis using the predicted probabilities from the generalized linear regression for determining the critical threshold associated with the health event of interest. If the interaction between the biomarker and parity was assessed and had a P < 0.05, the ROC analysis was conducted separately for each parity group (postpartum NEFA ketosis model). The point on the curve with the maximum Youden's index was considered the critical threshold. Positive likelihood ratios were evaluated using the sensitivity (Se) and specificity (Sp) values obtained from the ROC analysis. The continuous variables were then categorized based on the critical threshold from the ROC analysis, and the reduced Poisson regression model was reassessed to obtain risk ratios (RR;
      • Ospina P.A.
      • Nydam D.V.
      • DiCiccio T.J.
      Technical note: The risk ratio, an alternative to the odds ratio for estimating the association between multiple risk factors and a dichotomous outcome.
      ). Type III P-values are reported, and the standard error and RR confidence intervals were rescaled to reflect the type III score statistic (
      • Stokes M.E.
      • Davis C.S.
      • Koch G.G.
      Categorical Data Analysis using SAS.
      ).

      Milk and Reproduction Analysis

      The outcomes of interest in this next step were as follows: (1) ME305 (mean ± SD: 114 ± 13 DIM), (2) time to pregnancy with censoring at 150 DIM, and (3) pregnancy risk at first service (PRFS). Models were developed for each outcome with each of the following main independent effects: (1) prepartum NEFA, (2) postpartum NEFA, (3) postpartum BHB, and (4) postpartum Hp in the binary categorization derived from thresholds determined in the biomarker analysis described above. The unit of observation was the cow. Cows with missing covariate information were not included in the analysis unless the covariate was not included in the final model. For the reproductive performance outcomes, farms were removed from the analysis due to using natural service or because the farm's reproductive management was altered during the period of data collection. For the NEFA and BHB models, primiparous and multiparous cows were initially analyzed separately; however, if the magnitude of the model P-values for associations between the main effect and outcome were similar, primiparous and multiparous cows were combined for the final analysis and parity group, and the interaction between parity group and the biomarker were included as covariates but removed if P ≥ 0.05. Due to a limited sample size for primiparous cows and to decrease risk for type II error, primiparous and multiparous cows were grouped in the Hp analysis a priori with parity included as a covariate. The final model included the smallest biomarker threshold that resulted in the largest estimate and smallest chance of committing a type I error.
      Herds and cows with missing ME305 milk were removed from the analysis. A linear mixed effects model using the MIXED procedure with farm included as a random effect was used to analyze ME305. Binary covariates included in the milk production models were LS at time of blood sample collection, BCS at time of blood sample collection, change in BCS from previous visit to blood sample collection visit, and season. The main effect was initially forced into the model, and manual backward stepwise elimination was used to remove variables with P ≥ 0.10. If the parity interaction had a P < 0.05, an F-test using the SLICE option was used in the LSMEANS statement to determine at which parity level the biomarker groups differed. Type III P-values are reported for the ME305 milk analysis.
      Pregnancy within 150 DIM was analyzed by a Cox proportional hazards model using the PHREG procedure. Cows that died or were sold before the end of the farm's voluntary waiting period (VWP) were not included in the analysis as they were not eligible to be bred. Cows that died or were sold after the VWP or not pregnant by 150 DIM were right censored. All potential covariates were first assessed using a bivariate analysis, and if the association between the covariate and the outcome was P < 0.2, then they were offered to the model and manual backward stepwise elimination was used to remove any variable with P ≥ 0.05. Binary covariates included in the models were ME305 (above vs. below median production of the sampled cohort within each herd) and BCS at time of blood sample collection. Farm was included as a random effect except for the primiparous cow models, as farm did not contribute to the variation observed and was thus removed. The proportional hazards assumption was checked statistically for the time-dependent covariates (
      • Allison P.D.
      Survival Analysis using SAS: A Practical Guide.
      ), and a sensitivity analysis was performed to evaluate noninformative censoring. The final model included the biomarker threshold that resulted in the largest estimate and smallest chance of committing a type I error. Kaplan-Meier estimator graphs of time to pregnancy within 150 DIM were created for cows above and below the chosen threshold for the biomarker of interest using the LIFETEST procedure, and the survival curves were compared using the log-rank test.
      Pregnancy risk to first service was analyzed with a Poisson regression model using the GENMOD procedure with a log-link function, p-scale option for correcting for overdispersion, an exchangeable correlation matrix, and farm as a random effect. Cows that were never bred were not included in the analysis. Binary covariates included ME305, LS at time of blood sample collection, BCS at time of blood sample collection, change in BCS from previous visit to blood sample collection visit, and season. Covariates were removed if P ≥ 0.10 using a manual backward stepwise elimination process. Type III P-values are reported for the PRFS analysis.

      Herd-Alarm Levels

      The outcomes of interest in this next step were as follows: (1) incidence of DA, CK, or both, (2) ME305 milk yield, (3) 21-d herd pregnancy rate (PR), (4) probability of pregnancy (PP), and (5) PRFS. Models were developed for each outcome with each of the following main independent effects: (1) prepartum NEFA, (2) postpartum NEFA, (3) postpartum BHB, and (4) postpartum Hp. The unit of observation was the herd. For the NEFA and BHB models, primiparous and multiparous cows were initially analyzed separately; however, if the associations between the main effect and outcome were similar, the primiparous and multiparous cows were combined in the final analysis. Due to a limited sample size for primiparous cows, primiparous and multiparous cows were grouped a priori in the Hp analysis. The herd-alarm level for all outcomes was evaluated with mixed effects models using PROC MIXED. Milking herd size was included as a covariate. Milking herd size was divided by 100, reflecting a herd size change of 100 cows for every 1-unit change in the model. Farms were removed from the disorder incidence analysis if at least one of the disorders was not recorded on the farm. Farms with missing ME305 milk were removed from the ME305 analysis. Farms were removed from the 21-d PR, PP, and PRFS analyses due to using natural service or because the farm's reproductive management was altered during the period of data collection.
      The herd PP was determined by averaging the PP for the first 2 estrus cycles after the herd VWP for the group of cows that calved within the same calving date range as the cows observed using Dairy Comp software calculations (the percent of services with confirmed pregnancy;
      • Eicker S.
      • Stewart S.
      • Rapnicki P.
      Dairy herd reproductive records.
      ). The 21-d herd PR was determined by averaging the two 21-d periods after the farm VWP for the group of cows that calved within the same calving date range as the cows observed using Dairy Comp software calculations (calculated as the heat detection rate × PP;
      • Eicker S.
      • Stewart S.
      • Rapnicki P.
      Dairy herd reproductive records.
      ). The PRFS was calculated for each herd from the cohort of cows that were blood-sampled, and cows never bred were not included in the analysis.
      Similar to
      • Ospina P.A.
      • Nydam D.V.
      • Stokol T.
      • Overton T.R.
      Association between the proportion of sampled transition cows with increased nonesterified fatty acids and β-hydroxybutyrate and disease incidence, pregnancy rate, and milk production at the herd level.
      , the herd-alarm level was based on the following: (1) the biomarker threshold and (2) the proportion of cows within a herd with elevated biomarker concentrations that is associated with the outcome of interest. The biomarker thresholds tested were based on the thresholds established in the first part of this study as follows: prepartum NEFA, 0.17 mmol/L; postpartum NEFA, 0.46 to 0.59 mmol/L; postpartum BHB, 0.9 to 1.2 mmol/L; and postpartum Hp, 0.45 to 0.96 g/L. The proportion of cows above the thresholds were determined, and 30% was the initial proportion evaluated. To increase the sensitivity, the proportion of cows evaluated was decreased in 5% increments until the highest estimate and lowest P-value was attained. The lowest biomarker threshold that resulted in the greatest change in the outcome of interest with the lowest P-value was kept in the final model. A herd-alarm level was determined if P ≤ 0.10.

      Prevalence

      Herds were categorized into risk groups based on the proportion of cows above the herd-alarm level associated with negative outcomes for the biomarkers (prepartum NEFA, postpartum NEFA, BHB and Hp: ≥30% of multiparous cows sampled with prepartum NEFA concentrations ≥0.17 mmol/L, ≥15% of cows sampled with postpartum NEFA concentrations ≥0.59 mmol/L, ≥15% of cows sampled with postpartum BHB ≥1.2 mmol/L, and ≥ 20% of cows sampled with postpartum Hp ≥ 0.45 g/L). Bar graphs were created for the proportion of cows above the biomarker threshold.

      RESULTS

      Descriptive Results and Study Population

      Negative health event incidence and the number of cows included in each model are reported in Supplemental Table S1 (https://doi.org/10.17632/n9r22yzj5d.2). A more complete description of the study population has been reported previously (
      • Kerwin A.L.
      • Burhans W.S.
      • Mann S.
      • Tetreault M.
      • Nydam D.V.
      • Overton T.R.
      Transition cow nutrition and management strategies of dairy herds in the Northeastern United States: Part I—Herd description and performance characteristics.
      ). The (mean ± SD) ME305 for primiparous and multiparous cows was 12,711 ± 2,224 kg (n = 435) and 12,614 ± 2,030 kg (n = 882), respectively. The herd 21-d PR for primiparous and multiparous cows was 30.1 ± 8.0% (n = 66) and 23.7 ± 6.9% (n = 68), respectively. The herd PP for primiparous and multiparous cows was 30.0 ± 9.9% (n = 66) and 36.3 ± 9.2% (n = 68), respectively. The herd PRFS for primiparous and multiparous cows was 39.4 ± 20.1% (n = 66) and 32.1 ± 15.5% (n = 68), respectively.
      Samples showing moderate or severe hemolysis, as defined by the laboratory where analysis was performed, were removed from the Hp analysis (n = 1). Samples with severe hemolysis were removed from the prepartum NEFA analysis (n = 3). For the biomarker threshold analysis, there were 72 farms included in the CULL30 and DA models, 71 farms in the CK models, 67 farms in the MET models, and 66 farms in the 1 or more of the disorders models. Cows were removed from the DA, CK, MET, and 1 or more of the disorders models if the cow was diagnosed with the disorder before blood sample collection (postpartum NEFA and BHB: n = 12, 51, 84, and 131 cows respectively; postpartum Hp: n = 7, 35, 55, and 87 cows respectively). For the cow-level ME305 analysis, 2 farms, representing 34 cows, and individual cows from remaining farms (n = 131; n = 101 for Hp) were removed due to missing records. For the cow-level pregnancy within 150 DIM analysis, farms were removed due to using natural service or because the farm's reproductive management was altered during the period of data collection (n = 3 farms representing 45 cows), and individual cows from remaining farms were removed if they were culled before the end of the farm's VWP (n = 64; n = 51 for Hp) and due to missing ME305 at the fourth test day (n = 84; n = 62 for Hp). For the cow-level PRFS analysis, farms were removed due to using natural service or because the farm's reproductive management was altered (n = 3 farms representing 45 cows) or if ME305 at the fourth test day (∼120 DIM) was missing (n = 2 farms representing 25 cows). Cows were removed if they died or left the farm before the end of the farm's VWP (n = 64; n = 51 for Hp) or if they were never bred (n = 51; n = 30 for Hp). Cows were right censored if they left the herd after the end of the farm's VWP or if they were not pregnant by 150 DIM (n = 432 or 33.8% for prepartum NEFA; n = 433 or 33.8% for postpartum NEFA and BHB analysis; n = 275 or 32.6% for Hp analysis). For the herd-level analysis, 1 farm was removed from the disorder analysis due to incomplete disorder records, 2 farms were removed from the ME305 analysis due to missing records, and 4 farms were removed from the 21-d PR, PP, and PRFS analyses due to missing records, use of natural service, or altered reproductive management.

      Biomarker Thresholds and Negative Health Event Association

      Results for biomarkers that remained in the multivariable models and that were subjected to ROC analysis to establish critical thresholds are reported in Table 1. The generalized linear models were reassessed with the dichotomized variables, and the results from the final models are reported in Table 2, along with measures of association (RR) between the variables and health event outcomes.
      Table 1Receiver operator characteristic (ROC) curve determination of critical thresholds for biomarkers associated with transition cow negative health events for a prospective cohort study involving 72 farms across the northeastern United States
      The ROC analysis was performed if the biomarker (P < 0.05) remained in the logistic regression model with the negative health event as the outcome after manual backward stepwise elimination. To account for herd as a random effect in the ROC analysis, a generalized linear regression model was used to obtain predicted probabilities, which were used for the ROC analysis. The population incidence reported are the raw values.
      Negative health event
      NEFA = nonesterified fatty acids; CULL30 = culling within 30 DIM; MET = metritis; DA = displaced abomasum; CK = clinical ketosis; Any 3 = 1 or more of the disorders (DA, CK, MET).
      Critical threshold
      Critical threshold is the point on the ROC curve with the highest combined sensitivity and specificity (i.e., Youden's index). Units of measure for NEFA and BHB are mmol/L and for Hp is g/L.
      Population incidence, n (%)Se
      Se = sensitivity.
      Sp
      Sp = specificity.
      PPV
      PPV = positive predictive value.
      NPV
      NPV = negative predictive value.
      AUC
      AUC = area under the curve.
      AUC CI
      AUC CI = area under the curve 95% confidence interval.
      LR+
      LR+ = likelihood ratio positive. Calculated by Se/(100 − Sp).
      P-value
      Below thresholdAt or above threshold
      Prepartum NEFA model
       CULL300.1714/739 (1.9%)23/729 (3.2%)92720.081.000.860.83–0.903.3<0.001
      Postpartum NEFA models
       MET0.487/787 (0.9%)17/503 (3.4%)96800.091.000.920.89–0.954.90.006
       DA0.594/1,101 (0.4%)17/360 (4.7%)95850.091.000.930.90–0.976.4<0.001
       CK, multiparous0.4811/531 (2.1%)33/408 (8.1%)93750.151.000.900.87–0.943.7<0.001
       CK, primiparous0.461/314 (0.3%)7/145 (4.8%)100920.181.000.980.96–1.012<0.001
       Any 30.4619/722 (2.6%)50/497 (10.1%)84740.160.990.870.83–0.903.3<0.001
      Postpartum BHB models
       DA1.27/1,226 (0.6%)14/235 (6.0%)90820.071.000.920.88–0.965.1<0.001
       CK1.113/1,137 (1.1%)39/261 (14.9%)87860.190.990.930.91–0.966.0<0.001
       Any 30.925/869 (2.9%)44/350 (12.6%)91700.150.990.880.85–0.923.1<0.001
      Postpartum Hp
      Hp = haptoglobin.
      models
       MET0.967/647 (1.1%)14/218 (6.4%)90880.161.000.950.92–0.987.7<0.001
       CULL300.457/550 (1.3%)25/438 (5.7%)97580.071.000.830.78–0.882.3<0.001
      1 The ROC analysis was performed if the biomarker (P < 0.05) remained in the logistic regression model with the negative health event as the outcome after manual backward stepwise elimination. To account for herd as a random effect in the ROC analysis, a generalized linear regression model was used to obtain predicted probabilities, which were used for the ROC analysis. The population incidence reported are the raw values.
      2 NEFA = nonesterified fatty acids; CULL30 = culling within 30 DIM; MET = metritis; DA = displaced abomasum; CK = clinical ketosis; Any 3 = 1 or more of the disorders (DA, CK, MET).
      3 Critical threshold is the point on the ROC curve with the highest combined sensitivity and specificity (i.e., Youden's index). Units of measure for NEFA and BHB are mmol/L and for Hp is g/L.
      4 Se = sensitivity.
      5 Sp = specificity.
      6 PPV = positive predictive value.
      7 NPV = negative predictive value.
      8 AUC = area under the curve.
      9 AUC CI = area under the curve 95% confidence interval.
      10 LR+ = likelihood ratio positive. Calculated by Se/(100 − Sp).
      11 Hp = haptoglobin.
      Table 2Risk ratios (RR) of negative health events for biomarkers categorized based on the critical thresholds established by the receiver operator characteristic analysis and other covariates for a prospective cohort study involving 72 farms across the northeastern United States
      Negative health event and variable
      MET = metritis; DA = displaced abomasum; CK = clinical ketosis; CULL30 = culled within 30 DIM; Any 3 = 1 or more of the disorders (MET, DA, CK).
      ComparisonEstimateSEP-valueRR95% CI
      Confidence interval for the risk ratio.
      Prepartum NEFA model
      NEFA = nonesterified fatty acids.
       CULL30
      NEFA≥0.17 vs. <0.17 mmol/L0.830.360.022.31.1–4.6
      LS
      LS = locomotion score.
      ≥3 vs. <30.710.340.042.01.0–4.0
      BCS change
      BCS change = change in BCS from the far-off to close-up period visits.
      <0 vs. ≥00.920.400.022.51.1–5.5
      Postpartum NEFA models
       MET
      NEFA≥0.48 vs. <0.48 mmol/L1.070.440.022.91.2–7
       DA
      NEFA≥0.59 vs. <0.59 mmol/L2.350.760.002112.4–47
      ParityMultiparous vs. primiparous1.160.570.043.21.0–10
       CK
      NEFAElevated vs. not elevated
      Threshold used for primiparous cows was 0.46 mmol/L and 0.48 mmol/L for multiparous cows.
      1.500.42<0.0014.52.0–10
      ParityMultiparous vs. primiparous0.820.330.012.31.2–4.3
      SeasonWarm vs. cool1.140.440.0093.11.3–7.3
       Any 3
      NEFA≥0.46 vs. <0.46 mmol/L1.160.34<0.0013.21.6–6.2
      BCS change
      BCS change = change in BCS from the close-up to fresh period visits.
      <-0.25 vs. ≥−0.250.430.210.051.51.0–2.3
      SeasonWarm vs. cool0.890.330.0082.41.3–4.7
      Postpartum BHB models
       DA
      BHB≥1.2 vs. <1.2 mmol/L2.380.820.004112.2–54
       CK
      BHB≥1.1 vs. <1.1 mmol/L2.310.62<0.001103.0–34
      ParityMultiparous vs. primiparous0.650.320.041.91.0–3.6
       Any 3
      BHB≥0.9 vs. <0.9 mmol/L1.330.35<0.0013.81.9–8
      Postpartum Hp models
      Hp = haptoglobin.
       MET
      Hp≥0.96 vs. <0.96 g/L1.550.520.0034.71.7–13
       CULL30
      Hp≥0.45 vs. <0.45 g/L1.430.440.0014.21.8–10
      LS≥3 vs. <31.260.36<0.0013.51.7–7.1
      1 MET = metritis; DA = displaced abomasum; CK = clinical ketosis; CULL30 = culled within 30 DIM; Any 3 = 1 or more of the disorders (MET, DA, CK).
      2 Confidence interval for the risk ratio.
      3 NEFA = nonesterified fatty acids.
      4 LS = locomotion score.
      5 BCS change = change in BCS from the far-off to close-up period visits.
      6 Threshold used for primiparous cows was 0.46 mmol/L and 0.48 mmol/L for multiparous cows.
      7 BCS change = change in BCS from the close-up to fresh period visits.
      8 Hp = haptoglobin.

      Prepartum NEFA

      Prepartum NEFA only remained in the CULL30 model. Prepartum samples with a NEFA concentration ≥0.17 mmol/L were 3.3 times as likely to come from a cow that was culled within 30 DIM than from a cow not culled within 30 DIM (positive likelihood ratio = 3.3; P < 0.001; Table 1). The multivariable analysis was reevaluated with the dichotomized biomarker based on the established critical threshold, and RR were calculated and are reported in Table 2. When controlling for LS and BCS change from the far-off to the close-up period visit and days relative to parturition, cows with prepartum NEFA concentrations ≥0.17 mmol/L were 2.3 times more likely to be culled within 30 DIM than cows with a NEFA concentrations <0.17 mmol/L (P = 0.02).

      Postpartum NEFA

      Postpartum NEFA remained in the MET, DA, CK, and the 1 or more of the disorders (MET, DA, CK) models, and the critical thresholds for NEFA ranged from 0.46 to 0.59 mmol/L (Table 1). When controlling for calving season, change in BCS from the close-up to the fresh period visit, and DIM, cows with postpartum NEFA concentrations ≥0.46 mmol/L were 3.2 times more likely to be later diagnosed with any of the disorders (MET, DA, CK) than cows with NEFA concentrations <0.46 mmol/L (P < 0.001; Table 2). For the CK model, we identified a threshold of 0.48 and 0.46 mmol/L for multiparous and primiparous cows, respectively (Table 1); however, the interaction between parity and NEFA did not remain in the final model (Table 2). Cows with elevated NEFA concentrations were 4.5 times more likely to be later diagnosed with CK than cows with NEFA concentrations below the threshold identified (Table 2). Critical thresholds and RR for the MET and DA outcomes are reported in Table 2.

      Postpartum BHB

      β-Hydroxybutyrate remained in the DA, CK, and the 1 or more of the disorders (MET, DA, CK) models, and the critical thresholds for BHB ranged from 0.9 to 1.2 mmol/L (Table 1). For the association with 1 or more of the disorders, the ROC analysis yielded a BHB critical threshold of 0.9 mmol/L (Table 1). Cows with BHB concentrations ≥0.9 mmol/L were 3.8 times more likely to be subsequently diagnosed with any of the disorders (MET, DA, CK) compared with cows with BHB concentrations <0.9 mmol/L (P < 0.001; Table 2). Critical thresholds and RR for the DA and CK outcomes are reported in Table 2.

      Postpartum Haptoglobin

      Haptoglobin remained in the MET and CULL30 models (Table 2). For the association with MET and CULL30, the ROC analysis yielded a Hp critical threshold of 0.96 and 0.45 g/L, respectively (Table 1). Cows with Hp concentrations ≥0.96 g/L were 4.7 times more likely to be diagnosed with MET compared with cows with Hp concentrations <0.96 g/L (P = 0.003; Table 2). When controlling for LS, cows with Hp concentrations ≥0.45 g/L were 4.2 times more likely to be culled within 30 DIM compared with cows with Hp concentrations <0.45 g/L (P = 0.001; Table 2).

      Milk and Reproduction

      Milk Production

      Results from the milk production models are reported in Table 3. When controlling for LS and BCS at the fresh period visit, multiparous cows with prepartum NEFA concentrations ≥0.17 mmol/L produced 479 kg less ME305 milk than cows with prepartum NEFA concentrations <0.17 mmol/L (P < 0.001). We did not observe an association between prepartum NEFA and ME305 for primiparous cows. Data from primiparous and multiparous cows were combined in the postpartum NEFA model, as the association between NEFA and milk production were similar for both parity groups. There was an interaction between NEFA and parity (P = 0.002) such that primiparous and multiparous cows with NEFA concentrations ≥0.46 mmol/L produced 446 kg more (P = 0.02) and 280 kg less (P = 0.03) ME305 milk compared with primiparous and multiparous cows with NEFA concentrations <0.46 mmol/L, respectively. Primiparous cows with BHB concentrations ≥0.9 mmol/L produced 552 kg more ME305 milk than primiparous cows with BHB concentrations <0.9 mmol/L (P = 0.04). We did not observe an association between BHB concentrations and ME305 for multiparous cows. Cows with Hp concentrations ≥0.45 g/L produced 492 kg less ME305 milk than cows with Hp concentrations <0.45 g/L (P < 0.001).
      Table 3Cow-level differences in LSM of 305-d mature equivalent milk production at approximately 120 DIM (ME305) for the main effect of prepartum nonesterified fatty acids (NEFA), postpartum NEFA, BHB, or haptoglobin (Hp) and associated covariates for a prospective cohort study involving 72 farms across the northeastern United States
      VariableDifference in ME305, kgSEP-value
      Prepartum NEFA model for multiparous cows
      Prepartum NEFA dichotomized into ≥0.17 vs. <0.17 mmol/L (n = 878).
       NEFA−479162<0.001
       LS
      Locomotion score (LS) was dichotomized into lame (LS ≥3) vs. not lame (LS <3).
      −474161<0.001
       BCS
      Body condition score at corresponding blood sampling visit dichotomized as ≥3.5 vs. <3.5.
      −2681690.05
      Postpartum NEFA model
      Postpartum NEFA dichotomized into ≥0.46 vs. <0.46 mmol/L (n = 1,305).
       NEFA831610.49
       LS−3031550.006
       Parity
      Parity was dichotomized into multiparous vs. primiparous cows.
      −711630.54
      NEFA × parity0.002
       Primiparous
      F-test P-value reported.
      4462080.02
       Multiparous
      F-test P-value reported.
      −2801610.03
      BHB model for primiparous cows
      BHB dichotomized into ≥0.9 vs. <0.9 mmol/L (n = 435).
       BHB5522750.04
      Hp model
      Hp dichotomized into ≥0.45 vs. <0.45 g/L (n = 868).
       Hp−492165<0.001
      1 Prepartum NEFA dichotomized into ≥0.17 vs. <0.17 mmol/L (n = 878).
      2 Locomotion score (LS) was dichotomized into lame (LS ≥3) vs. not lame (LS <3).
      3 Body condition score at corresponding blood sampling visit dichotomized as ≥3.5 vs. <3.5.
      4 Postpartum NEFA dichotomized into ≥0.46 vs. <0.46 mmol/L (n = 1,305).
      5 Parity was dichotomized into multiparous vs. primiparous cows.
      6 F-test P-value reported.
      7 BHB dichotomized into ≥0.9 vs. <0.9 mmol/L (n = 435).
      8 Hp dichotomized into ≥0.45 vs. <0.45 g/L (n = 868).
      Because we unexpectedly did not observe a milk response for multiparous cows with elevated BHB concentrations using a threshold range of 0.9 to 1.2 mmol/L and we observed a positive milk response for primiparous cows, we repeated the analysis for a BHB range of 0.7 to 1.6 mmol/L at 0.1 mmol/L increments for primiparous and multiparous cows. We observed a positive ME305 response for all BHB thresholds for primiparous cows (ME305 range: 551–1,164 kg; P < 0.10); however, for multiparous cows, we observed a positive ME305 response using a threshold of ≥0.7 mmol/L (+363 kg) and a negative ME305 response using a BHB threshold of ≥1.5 mmol/L (−376 kg; Table 4). Although differences in ME305 milk evaluated using BHB concentration thresholds 0.9 to 1.4 mmol/L were not different from 0, we observed an apparent inflection point using a threshold of 1.2 mmol/L.
      Table 4Cow-level differences in LSM and SE of 305-d mature equivalent milk production at approximately 120 DIM (ME305) for multiparous cows, 0 to 21 DIM, at or above the threshold versus below the threshold for a range of postpartum BHB concentrations (n = 870) for cows enrolled in a prospective cohort study involving 72 farms across the northeastern United States
      BHB threshold, mmol/LDifference in ME305, kgSEP-value
      0.73631320.006
      0.82801310.03
      0.91641380.24
      1.01061490.48
      1.11291570.41
      1.2−21720.99
      1.3−2741840.14
      1.4−3081920.11
      1.5−3761960.06

      Reproduction

      Results from the pregnancy within 150 DIM analysis for postpartum biomarkers are reported in Table 5. We did not observe an association between prepartum NEFA concentrations and pregnancy by 150 DIM for multiparous or primiparous cows nor between postpartum NEFA concentrations and pregnancy by 150 DIM for primiparous cows. Multiparous cows with postpartum NEFA concentrations ≥0.46 mmol/L had a 20% decreased hazard of pregnancy by 150 DIM compared with multiparous cows with NEFA concentrations <0.46 mmol/L [hazard ratio (HR) = 0.80; P = 0.02]. The Kaplan-Meier analysis yielded a median time to pregnancy of 117 d for multiparous cows with elevated postpartum NEFA versus 114 d for multiparous cows with a NEFA concentration <0.46 mmol/L, and there was no evidence that the survival curves were different (P = 0.15; Figure 1). Primiparous and multiparous cows were combined in the BHB model because the association between BHB and pregnancy within 150 DIM were similar between parities. Cows sampled with a BHB concentration ≥1.1 mmol/L had a 20% decreased hazard of pregnancy by 150 DIM compared with cows with BHB concentration <1.1 mmol/L (HR = 0.80; P = 0.02). The Kaplan-Meier analysis yielded a median time to pregnancy of 115 d for cows with elevated BHB concentrations versus 107 d for multiparous cows with BHB concentrations <1.1 mmol/L, and the survival curves were different (P = 0.02; Figure 1). Cows sampled with a Hp concentration ≥0.45 g/L had a 28% decreased hazard of pregnancy within 150 DIM compared with cows with Hp concentration <0.45 g/L (HR = 0.72; P < 0.001). The Kaplan-Meier analysis yielded a median time to pregnancy of 115 d for cows with elevated Hp concentrations versus 100 d for cows with a Hp concentration <0.45 g/L, and there was evidence that the survival curves were different (P < 0.001; Figure 1).
      Table 5Hazard ratios (HR) from the Cox proportional hazards model for the main effect of postpartum nonesterified fatty acids (NEFA), BHB, or haptoglobin (Hp) and covariates on days to pregnancy within 150 DIM for a prospective cohort study involving 72 farms across the northeastern United States
      VariableEstimateSEP-valueHR95% CI
      Confidence interval for the hazard ratio.
      NEFA model for multiparous cows
      NEFA dichotomized into ≥0.46 vs. <0.46 mmol/L (n = 849).
       NEFA−0.220.090.020.800.67–0.97
       ME305
      Mature equivalent 305-d milk yield at 120 DIM (ME305) was dichotomized into greater than or equal to vs. less than the median ME305 of the sampled animals within herd.
      0.120.090.161.130.95–1.34
       BCS
      BCS was dichotomized into >3.0 vs. ≤3.0.
      0.160.100.091.170.97–1.42
      BHB model
      BHB dichotomized into ≥1.1 vs. <1.1 mmol/L (n = 1,271).
       BHB−0.220.100.020.800.66–0.97
       ME3050.200.070.0041.221.07–1.40
       BCS0.140.070.061.150.99–1.33
       Parity
      Parity was dichotomized into multiparous vs. primiparous cows.
      −0.220.070.0020.800.69–0.92
      Hp model
      Hp dichotomized into ≥0.45 vs. <0.45 g/L (n = 840).
       Hp−0.330.09<0.0010.720.61–0.86
       ME3050.230.090.0071.261.06–1.49
       BCS0.190.090.031.211.02–1.44
       Parity−0.240.090.0080.790.66–0.94
      1 Confidence interval for the hazard ratio.
      2 NEFA dichotomized into ≥0.46 vs. <0.46 mmol/L (n = 849).
      3 Mature equivalent 305-d milk yield at 120 DIM (ME305) was dichotomized into greater than or equal to vs. less than the median ME305 of the sampled animals within herd.
      4 BCS was dichotomized into >3.0 vs. ≤3.0.
      5 BHB dichotomized into ≥1.1 vs. <1.1 mmol/L (n = 1,271).
      6 Parity was dichotomized into multiparous vs. primiparous cows.
      7 Hp dichotomized into ≥0.45 vs. <0.45 g/L (n = 840).
      Figure thumbnail gr1
      Figure 1Kaplan-Meier estimator graphs for time to pregnancy for cows with (A) nonesterified fatty acid (NEFA) concentrations ≥0.46 mmol/L or <0.46 mmol/L for multiparous cows (log-rank P = 0.15), (B) BHB concentrations ≥1.1 mmol/L or <1.1 mmol/L (log-rank P = 0.02), and (C) haptoglobin (Hp) concentrations ≥0.45 g/L or <0.45 g/L (log-rank P < 0.001), for a prospective cohort study involving 72 farms across the northeastern United States.
      Results from the PRFS analysis are reported in Table 6. Haptoglobin was the only biomarker associated with PRFS. Cows with a Hp concentration ≥0.45 g/L were 0.81 times as likely to become pregnant to first service compared with cows with Hp concentrations <0.45 g/L (P = 0.03).
      Table 6Risk ratios (RR) of pregnancy to first service for the main effect of haptoglobin (Hp) and covariates for a prospective cohort study involving 72 farms across the northeastern United States
      VariableEstimateSEP-valueRR95% CI
      Confidence interval for the risk ratio.
      Hp
      Hp dichotomized into ≥0.45 vs. <0.45 g/L (n = 833).
      −0.210.090.030.810.68–0.98
      ME305
      Mature equivalent 305-d milk yield at 120 DIM (ME305) was dichotomized into greater than or equal to vs. less than the median ME305 of the sampled animals within herd.
      0.170.090.071.190.99–1.42
      Season
      Calving season was dichotomized into warm (May–September) vs. cool (October–April).
      −0.250.100.020.780.64–0.95
      Parity
      Parity was dichotomized into multiparous vs. primiparous cows.
      −0.160.080.070.850.72–1.00
      1 Confidence interval for the risk ratio.
      2 Hp dichotomized into ≥0.45 vs. <0.45 g/L (n = 833).
      3 Mature equivalent 305-d milk yield at 120 DIM (ME305) was dichotomized into greater than or equal to vs. less than the median ME305 of the sampled animals within herd.
      4 Calving season was dichotomized into warm (May–September) vs. cool (October–April).
      5 Parity was dichotomized into multiparous vs. primiparous cows.

      Herd-Alarm Levels

      Prepartum NEFA

      The herd-level associations of increased prepartum NEFA concentrations on disorder incidence, 21-d PR, and PP are reported in Table 7. We did not identify a herd-alarm level for ME305 or PRFS. We only identified a herd-alarm level for disorder incidence for multiparous cows and was defined as ≥30% of sampled multiparous cows with prepartum NEFA concentrations of ≥0.17 mmol/L. Herds above this herd-alarm level had a 6.0-percentage unit increase in disorder incidence for multiparous cows (P = 0.05). The 21-d PR herd-alarm level for multiparous and primiparous cows was defined as ≥15% of sampled multiparous cows (P = 0.009) and ≥40% of sampled primiparous cows (P = 0.09) with prepartum NEFA concentrations ≥0.17 mmol/L. Herds above these herd-alarm levels had a 6.0-percentage unit decrease and 3.9-percentage unit increase in 21-d PR for multiparous and primiparous cows, respectively. We only identified a herd-alarm level for PP for primiparous cows and was defined as ≥40% of sampled primiparous cows with prepartum NEFA concentrations ≥0.17 mmol/L (P = 0.04). Herds above this herd-alarm level had a 5.8-percentage unit increase in PP for primiparous cows.
      Table 7Herd-level associations of increased prepartum nonesterified fatty acids (NEFA) concentrations on disorder incidence (DI; displaced abomasum, clinical ketosis, or both), 21-d pregnancy rate (PR), and probability of pregnancy (PP) for a prospective cohort study involving 72 farms across the northeastern United States
      Association and variableChangeSEP-value
      Association with DI in multiparous cows,
      The association with disorder incidence if ≥30% of multiparous cows sampled had a prepartum NEFA concentration ≥0.17 mmol/L (n = 71).
      %
       Herd-alarm level6.03.00.05
       Herd size
      A 1-unit change in herd size is equivalent to 100 cows.
      0.20.30.44
      Association with herd PR in multiparous cows,
      The association with herd PR for multiparous cows if ≥30% of multiparous cows sampled had a prepartum NEFA concentration ≥0.17 mmol/L (n = 66). Herd PR was measured as the average of the two 21-d periods after the farm voluntary waiting period for the multiparous cows that calved during the same time frame as the multiparous cows observed using Dairy Comp (DairyComp 305, Valley Ag Software) software calculations (calculated as the heat detection rate × PP; Eicker et al., 2000).
      %
       Herd-alarm level−6.02.20.009
       Herd size0.20.10.21
      Association with herd PR in primiparous cows,
      The association with herd PR for primiparous cows if ≥40% of primiparous cows sampled had a prepartum NEFA concentration ≥0.17 mmol/L (n = 66). Herd PR was measured as the average of the two 21-d periods after the farm voluntary waiting period for the primiparous cows that calved during the same time frame as the primiparous cows observed using Dairy Comp software calculations (calculated as the heat detection rate × PP; Eicker et al., 2000).
      %
       Herd-alarm level3.92.30.09
       Herd size0.10.20.42
      Association with herd PP in primiparous cows,
      The association with herd PP for primiparous cows if ≥40% of primiparous cows sampled had a prepartum NEFA concentration ≥0.17 mmol/L (n = 66). The herd PP was determined by averaging the PP for the first 2 estrus cycles after the farm voluntary waiting period for the group of primiparous cows that calved within the same calving date range as the primiparous cows observed using Dairy Comp software calculations (the percent of services with confirmed pregnancy; Eicker et al., 2000).
      %
       Herd-alarm level5.82.80.04
       Herd size0.00.20.83
      1 The association with disorder incidence if ≥30% of multiparous cows sampled had a prepartum NEFA concentration ≥0.17 mmol/L (n = 71).
      2 A 1-unit change in herd size is equivalent to 100 cows.
      3 The association with herd PR for multiparous cows if ≥30% of multiparous cows sampled had a prepartum NEFA concentration ≥0.17 mmol/L (n = 66). Herd PR was measured as the average of the two 21-d periods after the farm voluntary waiting period for the multiparous cows that calved during the same time frame as the multiparous cows observed using Dairy Comp (DairyComp 305, Valley Ag Software) software calculations (calculated as the heat detection rate × PP;
      • Eicker S.
      • Stewart S.
      • Rapnicki P.
      Dairy herd reproductive records.
      ).
      4 The association with herd PR for primiparous cows if ≥40% of primiparous cows sampled had a prepartum NEFA concentration ≥0.17 mmol/L (n = 66). Herd PR was measured as the average of the two 21-d periods after the farm voluntary waiting period for the primiparous cows that calved during the same time frame as the primiparous cows observed using Dairy Comp software calculations (calculated as the heat detection rate × PP;
      • Eicker S.
      • Stewart S.
      • Rapnicki P.
      Dairy herd reproductive records.
      ).
      5 The association with herd PP for primiparous cows if ≥40% of primiparous cows sampled had a prepartum NEFA concentration ≥0.17 mmol/L (n = 66). The herd PP was determined by averaging the PP for the first 2 estrus cycles after the farm voluntary waiting period for the group of primiparous cows that calved within the same calving date range as the primiparous cows observed using Dairy Comp software calculations (the percent of services with confirmed pregnancy;
      • Eicker S.
      • Stewart S.
      • Rapnicki P.
      Dairy herd reproductive records.
      ).

      Postpartum NEFA

      The herd-level associations of increased postpartum NEFA concentrations on disorder incidence, ME305 milk, 21-d PR, and PRFS are reported in Table 8. Primiparous and multiparous cows were separated for the ME305 model due to dissimilar results. The herd-alarm level for disorder incidence was defined as ≥15% of sampled multiparous or primiparous cows with postpartum NEFA concentrations of ≥0.59 mmol/L. Herds above this herd-alarm level had a 5.8- and 4.2-percentage unit increase in disorder incidence for multiparous (P = 0.04) and primiparous cows (P = 0.02), respectively. We did not identify a herd-alarm level associated with ME305 milk for primiparous cows; however, the ME305 milk herd-alarm level for multiparous cows was defined as ≥30% of sampled multiparous cows with postpartum NEFA concentrations ≥0.48 mmol/L. Multiparous cows in herds above this herd-alarm level had, on average, a 789-kg decrease in ME305 milk (P = 0.01). We did not identify a herd-alarm level associated with PR or PRFS for primiparous cows; however, the herd-alarm level for 21-d PR and PRFS was defined as ≥10% and ≥20% of sampled multiparous cows with postpartum NEFA concentrations ≥0.48 mmol/L, respectively. Herds above these herd-alarm levels had a 6.8-percentage unit decrease in 21-d PR (P = 0.05) and a 6.3-percentage unit increase in PRFS (P = 0.07) for multiparous cows. The disorder incidence and PRFS models had herd size with P ≤ 0.10 such that an additional 100 milking cows resulted in a 0.3- and 0.7-percentage unit increase in disorder incidence and PRFS, respectively.
      Table 8Herd-level associations of increased postpartum nonesterified fatty acids (NEFA) concentrations on disorder incidence (DI; displaced abomasum, clinical ketosis, or both), mature equivalent 305-d milk production at 120 DIM (ME305), 21-d pregnancy rate (PR), and pregnancy risk at first service (PRFS) for a prospective cohort study involving 72 farms across the northeastern United States
      Association and variableChangeSEP-value
      Association with DI in multiparous cows,
      The association with disorder incidence if ≥15% of multiparous cows sampled had a postpartum NEFA concentration ≥0.59 mmol/L (n = 71).
      %
       Herd-alarm level5.82.80.04
       Herd size
      A 1-unit change in herd size is equivalent to 100 cows.
      0.30.30.30
      Association with DI in primiparous cows
      The association with disorder incidence if ≥15% of primiparous cows sampled had a postpartum NEFA concentration ≥0.59 mmol/L (n = 69).
       Herd-alarm level4.21.80.02
       Herd size0.30.20.08
      Association with average ME305 milk in multiparous cows,
      The association with average ME305 milk in multiparous cows if ≥30% of multiparous cows sampled had a postpartum NEFA concentration ≥0.48 mmol/L (n = 70).
      kg
       Herd-alarm level−7893110.01
       Herd size22260.39
      Association with herd PR for multiparous cows,
      The association with herd PR if ≥10% of multiparous cows sampled had a postpartum NEFA concentration ≥0.48 mmol/L (n = 68). Herd PR was measured as the average of the two 21-d periods after the farm voluntary waiting period for the multiparous cows that calved during the same time frame as the cows observed using Dairy Comp software calculations (calculated as the heat detection rate × PP; Eicker et al., 2000; DairyComp 305, Valley Ag Software).
      %
       Herd-alarm level−6.83.50.05
       Herd size0.20.10.25
      Association with PRFS for multiparous cows,
      The association with PRFS if ≥25% of multiparous cows sampled had a postpartum NEFA concentration ≥0.48 mmol/L (n = 68).
      %
       Herd-alarm level6.33.40.07
       Herd size0.70.30.02
      1 The association with disorder incidence if ≥15% of multiparous cows sampled had a postpartum NEFA concentration ≥0.59 mmol/L (n = 71).
      2 A 1-unit change in herd size is equivalent to 100 cows.
      3 The association with disorder incidence if ≥15% of primiparous cows sampled had a postpartum NEFA concentration ≥0.59 mmol/L (n = 69).
      4 The association with average ME305 milk in multiparous cows if ≥30% of multiparous cows sampled had a postpartum NEFA concentration ≥0.48 mmol/L (n = 70).
      5 The association with herd PR if ≥10% of multiparous cows sampled had a postpartum NEFA concentration ≥0.48 mmol/L (n = 68). Herd PR was measured as the average of the two 21-d periods after the farm voluntary waiting period for the multiparous cows that calved during the same time frame as the cows observed using Dairy Comp software calculations (calculated as the heat detection rate × PP;
      • Eicker S.
      • Stewart S.
      • Rapnicki P.
      Dairy herd reproductive records.
      ; DairyComp 305, Valley Ag Software).
      6 The association with PRFS if ≥25% of multiparous cows sampled had a postpartum NEFA concentration ≥0.48 mmol/L (n = 68).

      Postpartum BHB

      The herd-level associations of increased BHB concentrations on disorder incidence, ME305 milk, 21-d PR, PP, and PRFS are reported in Table 9. Primiparous and multiparous cows were combined in the disorder incidence, 21-d PR, PP, and PRFS but were separated for the ME305 models due to dissimilar results. The herd-alarm level for disorder incidence was defined as ≥15% of sampled cows with BHB concentrations ≥1.2 mmol/L. Herds above this herd-alarm level had an 8.5-percentage unit increase in disorder incidence (P < 0.001). The herd-alarm level associated with ME305 milk was separated by parity and defined as ≥20% of sampled primiparous cows with BHB concentrations ≥0.9 mmol/L and ≥10% of sampled multiparous cows with BHB concentrations ≥1.2 mmol/L. Primiparous and multiparous cows in herds above these herd-alarm levels had a 332-kg (P = 0.04) and 229-kg (P = 0.09) increase in ME305 milk, respectively. The herd-alarm level for 21-d PR was defined as ≥15% of sampled cows with BHB concentrations ≥0.9 mmol/L, and the herd-alarm level for PP and PRFS was defined as ≥10% of sampled cows with BHB concentrations ≥1.1 mmol/L. Herds above these herd-alarm levels had a 3.2-percentage unit decrease in 21-d PR (P = 0.05), a 5.2-percentage unit decrease in PP (P = 0.009), and a 7.0-percentage unit decrease in PRFS (P = 0.03).
      Table 9Herd-level associations of increased BHB concentrations on disorder incidence (DI; displaced abomasum, clinical ketosis, or both), mature equivalent 305-d milk production at 120 DIM (ME305), 21-d pregnancy rate (PR), probability of pregnancy (PP), and pregnancy risk at first service (PRFS) for a prospective cohort study involving 72 farms across the northeastern United States
      Association and variableChangeSEP-value
      Association with DI,
      The association with disorder incidence if ≥15% of cows sampled had a BHB concentration ≥1.2 mmol/L (n = 71).
      %
       Herd-alarm level8.52.0<0.001
       Herd size
      A 1-unit change in herd size is equivalent to 100 cows.
      0.30.20.11
      Association with average ME305 milk in primiparous cows,
      The association with average ME305 milk in primiparous cows if ≥20% of primiparous cows sampled had a BHB concentration ≥0.9 mmol/L (n = 69).
      kg
       Herd-alarm level3321580.04
       Herd size−1140.93
      Association with average ME305 milk in multiparous cows,
      The association with average ME305 milk in multiparous cows if ≥10% of primiparous cows sampled had a BHB concentration ≥1.2 mmol/L (n = 70).
      kg
       Herd-alarm level2291330.09
       Herd size7120.57
      Association with herd PR,
      The association with herd PR if ≥15% of cows sampled had a BHB concentration ≥0.9 mmol/L (n = 68). Herd PR was measured as the average of the two 21-d periods after the farm voluntary waiting period for the cows that calved during the same time frame as the cows observed using Dairy Comp software calculations (calculated as the heat detection rate × PP; Eicker et al., 2000; DairyComp 305, Valley Ag Software).
      %
       Herd-alarm level−3.21.60.05
       Herd size0.20.10.18
      Association with herd PP,
      The association with herd PP if ≥10% of cows sampled had BHB concentration ≥1.1 mmol/L (n = 68). The herd PP was determined by averaging the PP for the first 2 estrus cycles after the farm voluntary waiting period for the group of cows that calved within the same calving date range as the cows observed using Dairy Comp software calculations (the percent of services with confirmed pregnancy; Eicker et al., 2000).
      %
       Herd-alarm level−5.21.90.009
       Herd size0.00.20.97
      Association with PRFS,
      The association with PRFS if ≥10% of cows sampled had a BHB concentration ≥1.1 mmol/L (n = 66).
      %
       Herd-alarm level−7.03.20.03
       Herd size0.50.30.08
      1 The association with disorder incidence if ≥15% of cows sampled had a BHB concentration ≥1.2 mmol/L (n = 71).
      2 A 1-unit change in herd size is equivalent to 100 cows.
      3 The association with average ME305 milk in primiparous cows if ≥20% of primiparous cows sampled had a BHB concentration ≥0.9 mmol/L (n = 69).
      4 The association with average ME305 milk in multiparous cows if ≥10% of primiparous cows sampled had a BHB concentration ≥1.2 mmol/L (n = 70).
      5 The association with herd PR if ≥15% of cows sampled had a BHB concentration ≥0.9 mmol/L (n = 68). Herd PR was measured as the average of the two 21-d periods after the farm voluntary waiting period for the cows that calved during the same time frame as the cows observed using Dairy Comp software calculations (calculated as the heat detection rate × PP;
      • Eicker S.
      • Stewart S.
      • Rapnicki P.
      Dairy herd reproductive records.
      ; DairyComp 305, Valley Ag Software).
      6 The association with herd PP if ≥10% of cows sampled had BHB concentration ≥1.1 mmol/L (n = 68). The herd PP was determined by averaging the PP for the first 2 estrus cycles after the farm voluntary waiting period for the group of cows that calved within the same calving date range as the cows observed using Dairy Comp software calculations (the percent of services with confirmed pregnancy;
      • Eicker S.
      • Stewart S.
      • Rapnicki P.
      Dairy herd reproductive records.
      ).
      7 The association with PRFS if ≥10% of cows sampled had a BHB concentration ≥1.1 mmol/L (n = 66).

      Postpartum Haptoglobin

      The herd-level associations of increased Hp concentrations on disorder incidence are reported in Table 10. There was not a herd-alarm level associated with ME305 milk, 21-d PR, PP, or PRFS. The herd-alarm level for disorder incidence was defined as ≥20% of sampled cows with Hp concentrations ≥0.45 g/L. Herds above this herd-alarm level had a 5.3-percentage unit increase in disorder incidence (P = 0.05).
      Table 10Herd-level associations of increased haptoglobin (Hp) concentrations on disorder incidence (DI; displaced abomasum, clinical ketosis, or both) for a prospective cohort study involving 72 farms across the northeastern United States
      Association and variableChange in DI, %SEP-value
      Association with DI
      The association with disorder incidence if ≥20% of cows sampled had a Hp concentration ≥0.45 g/L (n = 71).
       Herd-alarm level5.32.60.05
       Herd size
      A 1-unit change in herd size is equivalent to 100 cows.
      0.20.20.44
      1 The association with disorder incidence if ≥20% of cows sampled had a Hp concentration ≥0.45 g/L (n = 71).
      2 A 1-unit change in herd size is equivalent to 100 cows.

      Prevalence

      We determined the prevalence of herds above the biomarker herd-alarm levels for the 72 farms included in this study as follows: 71% of herds had ≥30% of sampled multiparous cows with prepartum NEFA concentrations ≥0.17 mmol/L; 61% of herds had ≥15% of sampled cows with postpartum NEFA concentrations ≥0.59 mmol/L; 46% of herds had ≥15% of sampled cows with BHB concentrations ≥1.2 mmol/L; and 90% of herds had ≥20% of sampled cows with Hp concentrations ≥0.45 g/L (Figure 2).
      Figure thumbnail gr2
      Figure 2The prevalence of herds in which at least (A) 30% of sampled multiparous cows had prepartum nonesterified fatty acid (NEFA) concentration ≥0.17 mmol/L, (B) 15% of sampled cows had postpartum NEFA concentration ≥0.59 mmol/L, (C) 15% of sampled cows had postpartum BHB concentration ≥1.2 mmol/L, and (D) at least 20% of sampled cows had postpartum haptoglobin concentration ≥0.45 g/L, for a prospective cohort study involving 72 farms across the northeastern United States.

      DISCUSSION

      This study investigated critical thresholds for prepartum NEFA and postpartum NEFA, BHB, and Hp and their association with negative health events, milk production, and reproductive performance at the cow and herd levels for farms in the northeastern United States. Although previous studies have identified thresholds for prepartum NEFA and postpartum NEFA and BHB, this study added to the body of literature, particularly for the northeastern United States. This is the first large, epidemiological study to identify Hp thresholds.

      Cow-Level Biomarker Thresholds and Disorder Association

      We determined that there was an association between elevated prepartum and postpartum NEFA and postpartum BHB with negative health events at the cow level. We found that elevated prepartum NEFA was only associated with a higher risk of being culled within 30 DIM. This is in agreement with
      • Roberts T.
      • Chapinal N.
      • LeBlanc S.J.
      • Kelton D.F.
      • Dubuc J.
      • Duffield T.F.
      Metabolic parameters in transition cows as indicators for early-lactation culling risk.
      , who reported that cows in wk −1 relative to parturition with a prepartum NEFA concentration ≥0.4 mmol/L were at greater odds of being culled within 60 DIM. Our study had a higher Se and Sp and lower threshold identified compared with
      • Roberts T.
      • Chapinal N.
      • LeBlanc S.J.
      • Kelton D.F.
      • Dubuc J.
      • Duffield T.F.
      Metabolic parameters in transition cows as indicators for early-lactation culling risk.
      . This difference may be explained by the use of different analytical assays, sample population, or difference in statistical analysis. To our knowledge, this is the first study to account for farm as a random effect when identifying thresholds in the ROC analysis. Despite not finding evidence of an association between prepartum NEFA and other disorders, other studies have reported an association between elevated prepartum NEFA (range ≥0.27 to ≥0.6 mmol/L) and negative health events such as RP, MET, DA, and CK (
      • Dubuc J.
      • Duffield T.F.
      • Leslie K.E.
      • Walton J.S.
      • LeBlanc S.J.
      Risk factors for postpartum uterine diseases in dairy cows.
      ;
      • Ospina P.A.
      • Nydam D.V.
      • Stokol T.
      • Overton T.R.
      Evaluation of nonesterified fatty acids and β-hydroxybutyrate in transition dairy cattle in the northeastern United States: Critical thresholds for prediction of clinical diseases.
      ;
      • Chapinal N.
      • Carson M.
      • Duffield T.F.
      • Capel M.
      • Godden S.
      • Overton M.
      • Santos J.E.P.
      • LeBlanc S.J.
      The association of serum metabolites with clinical disease during the transition period.
      ). It is important to note that transition cow disorders are often interrelated;
      • Curtis C.R.
      • Erb H.N.
      • Sniffen C.J.
      • Smith R.D.
      • Kronfeld D.S.
      Path analysis of dry period nutrition, postpartum metabolic and reproductive disorders, and mastitis in Holstein cows.
      observed an increased risk of ketosis when the cow was previously diagnosed with RP, left DA, and parturient paresis.
      We identified a very narrow range of postpartum NEFA concentration thresholds associated with MET, DA, CK, and 1 or more of the disorders (0.46 to 0.59 mmol/L) compared with
      • Ospina P.A.
      • Nydam D.V.
      • Stokol T.
      • Overton T.R.
      Evaluation of nonesterified fatty acids and β-hydroxybutyrate in transition dairy cattle in the northeastern United States: Critical thresholds for prediction of clinical diseases.
      ; 0.36–0.72 mmol/L). We identified lower thresholds for postpartum NEFA concentrations associated with DA, CK, and 1 or more of the disorders, and reported a higher Se, Sp, and area under the curve (AUC) compared with
      • Ospina P.A.
      • Nydam D.V.
      • Stokol T.
      • Overton T.R.
      Evaluation of nonesterified fatty acids and β-hydroxybutyrate in transition dairy cattle in the northeastern United States: Critical thresholds for prediction of clinical diseases.
      ; however,
      • Ospina P.A.
      • Nydam D.V.
      • Stokol T.
      • Overton T.R.
      Evaluation of nonesterified fatty acids and β-hydroxybutyrate in transition dairy cattle in the northeastern United States: Critical thresholds for prediction of clinical diseases.
      reported a low threshold for MET with a similar Se and much lower Sp than our study.
      • Chapinal N.
      • Carson M.
      • Duffield T.F.
      • Capel M.
      • Godden S.
      • Overton M.
      • Santos J.E.P.
      • LeBlanc S.J.
      The association of serum metabolites with clinical disease during the transition period.
      identified a higher threshold and lower Se and Sp for postpartum NEFA concentrations associated with DA compared with our study; however, the authors did not identify an association between postpartum NEFA and MET.
      • Dubuc J.
      • Duffield T.F.
      • Leslie K.E.
      • Walton J.S.
      • LeBlanc S.J.
      Risk factors for postpartum uterine diseases in dairy cows.
      identified a higher threshold and lower Se and Sp for postpartum NEFA concentrations during the first and second week of lactation that was associated with MET compared with our study. Although we did not identify a postpartum NEFA concentration threshold associated with CULL30,
      • Roberts T.
      • Chapinal N.
      • LeBlanc S.J.
      • Kelton D.F.
      • Dubuc J.
      • Duffield T.F.
      Metabolic parameters in transition cows as indicators for early-lactation culling risk.
      reported that animals in the first 2 wk of lactation with postpartum NEFA concentrations ≥0.8 mmol/L were at greater odds of being culled within 60 DIM.
      • Ospina P.A.
      • Nydam D.V.
      • Stokol T.
      • Overton T.R.
      Evaluation of nonesterified fatty acids and β-hydroxybutyrate in transition dairy cattle in the northeastern United States: Critical thresholds for prediction of clinical diseases.
      reported a similar range of thresholds (7–10 mg/dL) for BHB compared with our study. Unlike
      • Ospina P.A.
      • Nydam D.V.
      • Stokol T.
      • Overton T.R.
      Evaluation of nonesterified fatty acids and β-hydroxybutyrate in transition dairy cattle in the northeastern United States: Critical thresholds for prediction of clinical diseases.
      and
      • Dubuc J.
      • Duffield T.F.
      • Leslie K.E.
      • Walton J.S.
      • LeBlanc S.J.
      Risk factors for postpartum uterine diseases in dairy cows.
      , we did not identify an association between BHB concentrations and MET; however, we reported slightly higher thresholds, Se, Sp, and AUC for the association of BHB with DA and CK than
      • Ospina P.A.
      • Nydam D.V.
      • Stokol T.
      • Overton T.R.
      Evaluation of nonesterified fatty acids and β-hydroxybutyrate in transition dairy cattle in the northeastern United States: Critical thresholds for prediction of clinical diseases.
      . We also reported a slightly lower threshold, higher Se, lower Sp, and higher AUC for the association of BHB concentrations with 1 or more of the disorders. Similar to
      • Chapinal N.
      • Carson M.
      • Duffield T.F.
      • Capel M.
      • Godden S.
      • Overton M.
      • Santos J.E.P.
      • LeBlanc S.J.
      The association of serum metabolites with clinical disease during the transition period.
      , we did not identify an association between BHB and MET; however, we did identify a higher threshold, Se, and Sp for the association between BHB and DA.
      Our study differs from
      • Ospina P.A.
      • Nydam D.V.
      • Stokol T.
      • Overton T.R.
      Evaluation of nonesterified fatty acids and β-hydroxybutyrate in transition dairy cattle in the northeastern United States: Critical thresholds for prediction of clinical diseases.
      as we did not sample only apparently healthy cows but followed a cohort from the prepartum to the postpartum periods; therefore, cows were included in the study regardless of their health status at the time of sampling. Consequently, our results likely better reflect the true incidence of disorders in the sampled population and prevalence of elevated biomarkers. However, the disorder incidence we reported for the prepartum NEFA model was very similar to
      • Ospina P.A.
      • Nydam D.V.
      • Stokol T.
      • Overton T.R.
      Evaluation of nonesterified fatty acids and β-hydroxybutyrate in transition dairy cattle in the northeastern United States: Critical thresholds for prediction of clinical diseases.
      , and we had lower disorder incidence for our postpartum biomarker models. This is due to the sampling scheme of this study, which resulted in sampling cows further away from parturition (median DIM = 10) and removing cows already diagnosed with the negative health event from the analysis.
      We found there was an association between elevated Hp concentrations with negative health events at the cow level. We observed cows with elevated Hp were more likely to be culled within 30 DIM and diagnosed with MET. Our study was unique in that it was the first study to our knowledge to evaluate the association between culling and Hp. Previous research has observed an association between Hp and MET (
      • Huzzey J.M.
      • Duffield T.F.
      • LeBlanc S.J.
      • Veira D.M.
      • Weary D.M.
      • von Keyserlingk M.A.G.
      Short communication: Haptoglobin as an early indicator of metritis.
      ;
      • Dubuc J.
      • Duffield T.F.
      • Leslie K.E.
      • Walton J.S.
      • LeBlanc S.J.
      Risk factors for postpartum uterine diseases in dairy cows.
      ;
      • Pohl A.
      • Burfeind O.
      • Heuwieser W.
      The associations between postpartum serum haptoglobin concentration and metabolic status, calving difficulties, retained fetal membranes, and metritis.
      ). For the association between Hp and MET, we observed a very similar threshold and Sp, with a higher Se compared with
      • Huzzey J.M.
      • Duffield T.F.
      • LeBlanc S.J.
      • Veira D.M.
      • Weary D.M.
      • von Keyserlingk M.A.G.
      Short communication: Haptoglobin as an early indicator of metritis.
      ;
      • Huzzey J.M.
      • Duffield T.F.
      • LeBlanc S.J.
      • Veira D.M.
      • Weary D.M.
      • von Keyserlingk M.A.G.
      Short communication: Haptoglobin as an early indicator of metritis.
      reported a threshold of 1.0 g/L. Compared with other studies (
      • Humblet M.-F.
      • Guyot H.
      • Boudry B.
      • Mbayahi F.
      • Hanzen C.
      • Rollin F.
      • Godeau J.-M.
      Relationship between haptoglobin, serum amyloid A, and clinical status in a survey of dairy herds during a 6-month period.
      ;
      • Burfeind O.
      • Sannmann I.
      • Voigtsberger R.
      • Heuwieser W.
      Receiver operating characteristic curve analysis to determine the diagnostic performance of serum haptoglobin concentration for the diagnosis of acute puerperal metritis in dairy cows.
      ), we observed greater combined Se and Sp using a Hp threshold of ≥0.96 g/L associated with MET. This may be due to the higher threshold identified compared with previous studies reporting Hp thresholds (
      • Humblet M.-F.
      • Guyot H.
      • Boudry B.
      • Mbayahi F.
      • Hanzen C.
      • Rollin F.
      • Godeau J.-M.
      Relationship between haptoglobin, serum amyloid A, and clinical status in a survey of dairy herds during a 6-month period.
      ), our sample being more representative of the population, or farms diagnosing clinical metritis versus acute puerperal metritis diagnosed by vaginal palpation. It is important to note that
      • Humblet M.-F.
      • Guyot H.
      • Boudry B.
      • Mbayahi F.
      • Hanzen C.
      • Rollin F.
      • Godeau J.-M.
      Relationship between haptoglobin, serum amyloid A, and clinical status in a survey of dairy herds during a 6-month period.
      used a predetermined Hp threshold based on previous studies (0.15 g/L).
      • Burfeind O.
      • Sannmann I.
      • Voigtsberger R.
      • Heuwieser W.
      Receiver operating characteristic curve analysis to determine the diagnostic performance of serum haptoglobin concentration for the diagnosis of acute puerperal metritis in dairy cows.
      identified 3 thresholds, 0.93, 1.39, and 0.77 g/L, at 2, 5, and 10 DIM, respectively.
      • Skinner J.G.
      • Brown R.A.
      • Roberts L.
      Bovine haptoglobin response in clinically defined field conditions.
      observed higher mean Hp concentrations in cows diagnosed with acute severe MET and RP, but did not observe higher mean Hp concentrations in cows diagnosed with CK compared with healthy cows. We also did not observe a direct association between Hp and CK; however, we cannot disregard the possible association between Hp and excessive fatty acid mobilization. It has been proposed that cows with fatty liver would have elevated Hp due to stimulated liver parenchymal cells from excessive lipid accumulation (
      • Katoh N.
      Relevance of apolipoproteins in the development of fatty liver and fatty liver-related peripartum diseases in dairy cows.
      ). In addition, an increase in NEFA can cause stress on the endoplasmic reticulum in the hepatocytes, stimulating an inflammatory response as a result of increased reactive oxygen species; however, an inflammatory response depends on the antioxidant status of the cow (
      • Sordillo L.M.
      • Contreras G.A.
      • Aitken S.L.
      Metabolic factors affecting the inflammatory response of periparturient dairy cows.
      ;
      • Bradford B.J.
      • Yuan K.
      • Farney J.K.
      • Mamedova L.K.
      • Carpenter A.J.
      Invited review: Inflammation during the transition to lactation: New adventures with an old flame.
      ;
      • Ringseis R.
      • Gessner D.K.
      • Eder K.
      Molecular insights into the mechanisms of liver-associated diseases in early-lactating dairy cows: hypothetical role of endoplasmic reticulum stress.
      ). An inflammatory response has been associated with anorexic effects (
      • Kushibiki S.
      • Hodate K.
      • Shingu H.
      • Obara Y.
      • Touno E.
      • Shinoda M.
      • Yokomizo Y.
      Metabolic and lactational responses during recombinant bovine tumor necrosis factor-alpha treatment in lactating cows.
      ), and depressed intakes around parturition have been observed in cows with increased Hp concentrations (
      • Trevisi E.
      • Han X.-T.
      • Piccioli-Cappelli F.
      • Bertoni G.
      Dry matter intake reduction before calving in dairy cows: Relationship with immune system and metabolism conditions.
      ). It has been proposed by
      • Horst E.A.
      • Kvidera S.K.
      • Baumgard L.H.
      Invited review: The influence of immune activation on transition cow health and performance – A critical evaluation of traditional dogmas.
      that decreased DMI, increased NEFA concentrations, and hyperketonemia may be a result of immune activation. Observing the association between elevated Hp with elevated NEFA or BHB was not one of the objectives of this study but warrants further investigation.
      Haptoglobin is part of the acute phase response, which may be activated from the act of parturition (
      • Uchida E.
      • Katoh N.
      • Takahashi K.
      Appearance of haptoglobin in serum from cows at parturition.
      ). Therefore, it is difficult to distinguish whether elevated Hp in the immediate postpartum period is a result of the acute phase response due to parturition or if it is a result of inflammation from an infection or disease.
      • Humblet M.-F.
      • Guyot H.
      • Boudry B.
      • Mbayahi F.
      • Hanzen C.
      • Rollin F.
      • Godeau J.-M.
      Relationship between haptoglobin, serum amyloid A, and clinical status in a survey of dairy herds during a 6-month period.
      proposed that it may be difficult to distinguish between parturition and disease as the root cause of elevated Hp due to interindividual variability in the acute phase response at parturition.
      • Humblet M.-F.
      • Guyot H.
      • Boudry B.
      • Mbayahi F.
      • Hanzen C.
      • Rollin F.
      • Godeau J.-M.
      Relationship between haptoglobin, serum amyloid A, and clinical status in a survey of dairy herds during a 6-month period.
      observed elevated Hp concentrations in the first week postpartum in clinically healthy and diseased cows but reported that Hp concentrations in the first week after parturition were higher in diseased cows than healthy cows.
      • Skinner J.G.
      • Brown R.A.
      • Roberts L.
      Bovine haptoglobin response in clinically defined field conditions.
      did not observe an increase in Hp in cows with normal calving, indicating that an increase in Hp was a result of infection and not from calving trauma. Our study only evaluated the association between negative health events of interest with Hp; therefore, we did not evaluate cow-level factors that may be associated with elevated Hp; however, our models initially included CD as a covariate, but the variable was removed because P > 0.05. The association between cow-level factors and elevated Hp should be investigated in the future.
      To our knowledge, this is the first study to have reported positive and negative predicted values for thresholds identified. It is of note that the positive predicted values were very low for all the thresholds as a result of the low disorder incidence, indicating that many individual cows that had a biomarker concentration above the threshold were not diagnosed with the negative health event of interest. Biomarker concentrations should not be used exclusively for diagnosing cows with negative health events but can be used to aid in the management decisions.
      A lot of variation exists when reviewing thresholds identified in this study and the others studies discussed. This may be due to differences in study population, sample size, variation in the identification of disorders, assay variation, or the statistical analysis approach.

      Cow-Level Associations with Milk Production and Reproduction

      We found an association between multiparous cows with elevated prepartum and postpartum NEFA concentrations and cows with elevated postpartum Hp concentrations with decreased subsequent milk yield; however, we observed an association between primiparous cows with elevated postpartum NEFA and BHB concentrations with increased subsequent milk yield.
      • Ospina P.A.
      • Nydam D.V.
      • Stokol T.
      • Overton T.R.
      Associations of elevated nonesterified fatty acids and β-hydroxybutyrate concentrations with early lactation reproductive performance and milk production in transition dairy cattle in the northeastern United States.
      and
      • Huzzey J.M.
      • Mann S.
      • Nydam D.V.
      • Grant R.J.
      • Overton T.R.
      Associations of peripartum markers of stress and inflammation with milk yield and reproductive performance in Holstein dairy cows.
      did not observe a parity effect on milk production for prepartum NEFA concentrations.
      • Ospina P.A.
      • Nydam D.V.
      • Stokol T.
      • Overton T.R.
      Associations of elevated nonesterified fatty acids and β-hydroxybutyrate concentrations with early lactation reproductive performance and milk production in transition dairy cattle in the northeastern United States.
      reported a 683-kg decrease in ME305 milk for cows with a prepartum NEFA concentration ≥0.33 mmol/L.
      • Huzzey J.M.
      • Mann S.
      • Nydam D.V.
      • Grant R.J.
      • Overton T.R.
      Associations of peripartum markers of stress and inflammation with milk yield and reproductive performance in Holstein dairy cows.
      reported a 1,465-kg decrease in ME305 at the third DHI test day, which occurred at 102 ± 17 DIM, for every 1 mmol/L increase in prepartum NEFA concentration during wk −2 relative to parturition.
      • Huzzey J.M.
      • Mann S.
      • Nydam D.V.
      • Grant R.J.
      • Overton T.R.
      Associations of peripartum markers of stress and inflammation with milk yield and reproductive performance in Holstein dairy cows.
      did not observe an association between prepartum NEFA concentrations and ME305 milk during wk −1 relative to parturition. Similar to our results,
      • Chapinal N.
      • Carson M.E.
      • LeBlanc S.J.
      • Leslie K.E.
      • Godden S.
      • Capel M.
      • Santos J.E.P.
      • Overton M.W.
      • Duffield T.F.
      The association of serum metabolites in the transition period with milk production and early-lactation reproductive performance.
      observed a parity effect for prepartum NEFA concentrations such that only multiparous cows had a decrease in milk yield (−1.6 kg of milk/d across the first 4 DHIA test days) if prepartum NEFA (−1 wk relative to parturition) concentrations were ≥0.5 mmol/L.
      • Ospina P.A.
      • Nydam D.V.
      • Stokol T.
      • Overton T.R.
      Associations of elevated nonesterified fatty acids and β-hydroxybutyrate concentrations with early lactation reproductive performance and milk production in transition dairy cattle in the northeastern United States.
      and
      • Huzzey J.M.
      • Mann S.
      • Nydam D.V.
      • Grant R.J.
      • Overton T.R.
      Associations of peripartum markers of stress and inflammation with milk yield and reproductive performance in Holstein dairy cows.
      reported a 647-kg and 529-kg decrease in milk yield for multiparous cows with elevated postpartum NEFA concentrations, respectively.
      • Chapinal N.
      • Carson M.E.
      • LeBlanc S.J.
      • Leslie K.E.
      • Godden S.
      • Capel M.
      • Santos J.E.P.
      • Overton M.W.
      • Duffield T.F.
      The association of serum metabolites in the transition period with milk production and early-lactation reproductive performance.
      reported a decrease of 1.8 kg/d of milk at the first DHIA test for multiparous cows with elevated postpartum NEFA concentrations at 1 wk postpartum (≥0.7 mmol/L).
      • Chapinal N.
      • Carson M.E.
      • LeBlanc S.J.
      • Leslie K.E.
      • Godden S.
      • Capel M.
      • Santos J.E.P.
      • Overton M.W.
      • Duffield T.F.
      The association of serum metabolites in the transition period with milk production and early-lactation reproductive performance.
      did not observe an association between postpartum NEFA concentrations and milk yield for primiparous cows. Two studies have also observed increased milk yield for primiparous cows with elevated postpartum NEFA and BHB concentrations (
      • Ospina P.A.
      • Nydam D.V.
      • Stokol T.
      • Overton T.R.
      Associations of elevated nonesterified fatty acids and β-hydroxybutyrate concentrations with early lactation reproductive performance and milk production in transition dairy cattle in the northeastern United States.
      ;
      • Huzzey J.M.
      • Mann S.
      • Nydam D.V.
      • Grant R.J.
      • Overton T.R.
      Associations of peripartum markers of stress and inflammation with milk yield and reproductive performance in Holstein dairy cows.
      ). This further suggested that primiparous cows may use homeorhetic mechanisms more readily than cows for mobilizing nutrient resources to meet milk production demands while balancing for maintenance and growth (
      • Bauman D.E.
      • Currie B.W.
      Partitioning of nutrients during pregnancy and lactation: A review of mechanisms involving homeostasis and homeorhesis.
      ). There is a clear linear relationship between ME305 and BHB concentrations for multiparous cows (x-intercept would be approximately 1.2 mmol/L), as demonstrated in Table 4. By dichotomizing BHB concentrations, we were able to observe a positive effect on milk production with lower BHB concentrations and detrimental effects on milk production with higher BHB concentrations. This observed relationship supports the hypothesis that moderate increases in BHB may be beneficial and a necessary physiological response for supporting the energy demands of milk production but may be detrimental when in excess. For this reason, the statistical approach of dichotomizing BHB concentrations is more applicable at the farm level. Analyzing BHB as a continuous variable would result in a negative relationship between BHB concentrations and ME305 (as demonstrated in Table 4), resulting in the conclusion that any increase in BHB concentration would be detrimental to ME305.
      • Duffield T.F.
      • Lissemore K.D.
      • McBride B.W.
      • Leslie K.E.
      Impact of hyperketonemia in early lactation dairy cows on health and production.
      and
      • Chapinal N.
      • Carson M.E.
      • LeBlanc S.J.
      • Leslie K.E.
      • Godden S.
      • Capel M.
      • Santos J.E.P.
      • Overton M.W.
      • Duffield T.F.
      The association of serum metabolites in the transition period with milk production and early-lactation reproductive performance.
      evaluated a series of postpartum BHB concentration thresholds and observed a positive association with DHI milk yield at the first test day for cows with BHB concentrations ≥0.6 mmol/L during wk 1 and 2 of lactation and a positive association on DHIA milk yield over the first 4 DHIA tests for cows with BHB concentrations ≥0.8 mmol/L during wk 1 and 2 of lactation, respectively. β-Hydroxybutyrate can serve as an energy substrate and as a substrate for de novo fatty acid synthesis by the mammary gland (
      • White H.M.
      The role of TCA cycle anaplerosis in ketosis and fatty liver in periparturient dairy cows.
      ), but can also be used as an energy source by other tissues, thus sparing glucose for lactose production (
      • Herdt T.H.
      Ruminant adaptation to negative energy balance.
      ). This sparing effect may explain the increase in milk yield when moderate increases in BHB concentration were observed in this study and studies by
      • Duffield T.F.
      • Lissemore K.D.
      • McBride B.W.
      • Leslie K.E.
      Impact of hyperketonemia in early lactation dairy cows on health and production.
      and
      • Chapinal N.
      • Carson M.E.
      • LeBlanc S.J.
      • Leslie K.E.
      • Godden S.
      • Capel M.
      • Santos J.E.P.
      • Overton M.W.
      • Duffield T.F.
      The association of serum metabolites in the transition period with milk production and early-lactation reproductive performance.
      ; however, when in excess, BHB can have deleterious effects on the health and productivity of the cow (
      • Chapinal N.
      • Carson M.E.
      • LeBlanc S.J.
      • Leslie K.E.
      • Godden S.
      • Capel M.
      • Santos J.E.P.
      • Overton M.W.
      • Duffield T.F.
      The association of serum metabolites in the transition period with milk production and early-lactation reproductive performance.
      ;
      • White H.M.
      The role of TCA cycle anaplerosis in ketosis and fatty liver in periparturient dairy cows.
      ). Our results for the Hp concentration analysis are in agreement with 2 other studies (
      • Huzzey J.M.
      • Mann S.
      • Nydam D.V.
      • Grant R.J.
      • Overton T.R.
      Associations of peripartum markers of stress and inflammation with milk yield and reproductive performance in Holstein dairy cows.
      ;
      • Shin D.H.
      • Jeong J.K.
      • Choi I.S.
      • Moon S.H.
      • Lee S.C.
      • Kang H.G.
      • Park S.B.
      • Kim I.H.
      Associations between serum haptoglobin concentration and peri- and postpartum disorders, milk yield, and reproductive performance in dairy cows.
      ).
      • Huzzey J.M.
      • Mann S.
      • Nydam D.V.
      • Grant R.J.
      • Overton T.R.
      Associations of peripartum markers of stress and inflammation with milk yield and reproductive performance in Holstein dairy cows.
      observed a 464-kg decrease in 305-d mature equivalent milk at the third test day (∼90 DIM) for every 1 g/L increase in Hp during the first week of lactation.
      • Shin D.H.
      • Jeong J.K.
      • Choi I.S.
      • Moon S.H.
      • Lee S.C.
      • Kang H.G.
      • Park S.B.
      • Kim I.H.
      Associations between serum haptoglobin concentration and peri- and postpartum disorders, milk yield, and reproductive performance in dairy cows.
      also reported a decrease in milk yield at 1 and 2 mo postpartum for cows with elevated Hp concentrations (>0.1 g/L).
      Multiparous cows with elevated postpartum NEFA concentrations and cows with elevated postpartum BHB and Hp concentrations had a lower hazard of pregnancy within 150 DIM, and cows with elevated Hp concentrations were also less likely to become pregnant to first service. We did not observe an association between prepartum NEFA concentrations and pregnancy within 150 DIM, nor did we observe an association between prepartum NEFA and postpartum NEFA or BHB concentrations on PRFS. Similar to our results,
      • Huzzey J.M.
      • Mann S.
      • Nydam D.V.
      • Grant R.J.
      • Overton T.R.
      Associations of peripartum markers of stress and inflammation with milk yield and reproductive performance in Holstein dairy cows.
      did not observe an association between prepartum NEFA concentrations and risk of pregnancy by 150 DIM, and
      • Chapinal N.
      • Carson M.E.
      • LeBlanc S.J.
      • Leslie K.E.
      • Godden S.
      • Capel M.
      • Santos J.E.P.
      • Overton M.W.
      • Duffield T.F.
      The association of serum metabolites in the transition period with milk production and early-lactation reproductive performance.
      reported no evidence of prepartum NEFA concentrations being associated with the odds of pregnancy to first service.
      • Ospina P.A.
      • Nydam D.V.
      • Stokol T.
      • Overton T.R.
      Associations of elevated nonesterified fatty acids and β-hydroxybutyrate concentrations with early lactation reproductive performance and milk production in transition dairy cattle in the northeastern United States.
      also evaluated the association of elevated postpartum (3–14 DIM) NEFA and BHB concentrations on pregnancy within 70 d post VWP. The authors reported cows with postpartum NEFA concentrations ≥0.72 mmol/L had a 16% decreased risk of pregnancy (HR = 0.84; P = 0.05), and cows with postpartum BHB concentrations ≥1.0 mmol/L had a 13% decreased risk of pregnancy (HR = 0.87; P = 0.1).
      • Walsh R.B.
      • Walton J.S.
      • Kelton D.F.
      • LeBlanc S.J.
      • Leslie K.E.
      • Duffield T.F.
      The effect of subclinical ketosis in early lactation on reproductive performance of postpartum dairy cows.
      reported a lower odds of pregnancy to first service for cows with BHB concentrations ≥1.0 mmol/L during the first week of lactation (odds ratio = 0.7; P = 0.04).
      • Huzzey J.M.
      • Mann S.
      • Nydam D.V.
      • Grant R.J.
      • Overton T.R.
      Associations of peripartum markers of stress and inflammation with milk yield and reproductive performance in Holstein dairy cows.
      reported a 26% decreased risk of pregnancy within 150 DIM for every 1 mmol/L unit increase in BHB concentration [HR (95% CI) = 0.74 (0.56–0.98); P = 0.04] for cows 3 to 10 DIM. The association between prepartum NEFA, postpartum NEFA, and postpartum BHB concentrations with reproductive outcomes is not consistent throughout the literature.
      • Ospina P.A.
      • Nydam D.V.
      • Stokol T.
      • Overton T.R.
      Associations of elevated nonesterified fatty acids and β-hydroxybutyrate concentrations with early lactation reproductive performance and milk production in transition dairy cattle in the northeastern United States.
      reported a 19% decrease risk of pregnancy within 70 d after the VWP for cows with prepartum NEFA concentrations ≥0.27 mmol/L (HR = 0.81; P = 0.01).
      • Huzzey J.M.
      • Mann S.
      • Nydam D.V.
      • Grant R.J.
      • Overton T.R.
      Associations of peripartum markers of stress and inflammation with milk yield and reproductive performance in Holstein dairy cows.
      did not observe an association between postpartum NEFA concentrations and pregnancy within 150 DIM. Likewise,
      • Chapinal N.
      • Carson M.E.
      • LeBlanc S.J.
      • Leslie K.E.
      • Godden S.
      • Capel M.
      • Santos J.E.P.
      • Overton M.W.
      • Duffield T.F.
      The association of serum metabolites in the transition period with milk production and early-lactation reproductive performance.
      did not observe an association between postpartum NEFA or BHB concentrations on pregnancy to first service.
      • McArt J.A.A.
      • Nydam D.V.
      • Oetzel G.R.
      Epidemiology of subclinical ketosis in early lactation dairy cattle.
      sampled 751 cows across 3 farms and did not observe an association between elevated BHB concentrations (≥1.2 mmol/L; 3–16 DIM) and pregnancy to first service (RR = 0.9; P = 0.55) or pregnancy within 150 DIM (HR = 0.9; P = 0.40). Our results for the Hp analysis are in agreement with previous studies.
      • Nightingale C.R.
      • Sellers M.D.
      • Ballou M.A.
      Elevated plasma haptoglobin concentrations following parturition are associated with elevated leukocyte responses and decreased subsequent reproductive efficiency in multiparous Holstein dairy cows.
      reported multiparous Holstein cows 2 to 8 DIM with high Hp concentrations (≥459 μg/L) had a different survival curve for days to pregnancy than cows with moderate (8.4 μg/L ≤ Hp ≤458 μg/L; P = 0.044) or low Hp concentrations (Hp <8.4 μg/L; P = 0.09). The mean days for 75% of cows to become pregnant was 183 d for high-Hp cows compared with 139 d and 123 d for moderate- and low-Hp cows, respectively.
      • Huzzey J.M.
      • Mann S.
      • Nydam D.V.
      • Grant R.J.
      • Overton T.R.
      Associations of peripartum markers of stress and inflammation with milk yield and reproductive performance in Holstein dairy cows.
      reported a 19% decreased risk of pregnancy by 150 DIM for every 1 g/L unit increase in Hp concentration [HR (95% CI) = 0.81 (0.70–0.97); P = 0.02] for cows 3 to 10 DIM.

      Herd-Alarm Levels

      We identified herd-alarm levels for prepartum NEFA concentrations and postpartum NEFA, BHB, and Hp concentrations associated with changes in disorder incidence, ME305, and reproductive performance. Herds above the identified herd-alarm level for all biomarkers had an increase in disorder incidence. We also determined that herds above the identified herd-alarm level for postpartum NEFA produced less ME305 for multiparous cows, whereas herds above the herd-alarm level for BHB produced more ME305 for primiparous and multiparous cows. Herds above the herd-alarm level for prepartum NEFA had lower PR for multiparous cows, and higher PR and PP for primiparous cows. Herds above the herd-alarm level for postpartum NEFA had lower PR but higher PRFS for multiparous cows. Herds above the herd-alarm level for BHB had a lower PR, PP, and PRFS compared with herds below the identified herd-alarm level.
      In general, the cow- and herd-level analyses were in agreement with each other except for some outcomes of interest. Although we observed prepartum NEFA concentrations for multiparous cows, postpartum NEFA concentrations for primiparous cows, and Hp concentrations were associated with ME305 at the cow level, we did not identify a herd-alarm level for ME305. Conversely, there was no evidence that prepartum NEFA concentration was associated with reproductive outcomes at the cow level; however, we did identify prepartum NEFA herd-alarm levels for reproductive outcomes at the herd level. In addition, the cow- and herd-level analysis for postpartum NEFA had slightly differing results for reproductive performance outcomes. At the cow level, we did not observe an association between postpartum NEFA concentrations and PRFS, but multiparous cows with elevated postpartum NEFA (≥0.46 mmol/L) had a decreased risk of pregnancy within 150 DIM. Conversely, at the herd level, herds that had at least 10% and 25% of their multiparous cows with NEFA concentrations ≥0.48 mmol/L had a 6.8-percentage unit decrease in PR and a 6.3-percentage unit increase in PRFS, respectively, compared with herds with less than 10% and 25% of their multiparous cows with NEFA concentrations ≥0.48 mmol/L, respectively. This difference may reflect the short and long-term reproductive outcomes. The 21-d PR and PP outcomes assess reproductive success immediately after the VWP, whereas risk of pregnancy within 150 DIM can be used to assess long-term reproductive success. However, the only outcome evaluating only the cows sampled were the PRFS and risk of pregnancy within 150 DIM outcomes, which may also explain the discrepancy between the herd-alarm level differences for postpartum NEFA. In addition, there was a wide range in the DIM for the first breeding (range: 35–150 DIM, mean ± SD: 70 ± 12 DIM;
      • Kerwin A.L.
      • Burhans W.S.
      • Mann S.
      • Tetreault M.
      • Nydam D.V.
      • Overton T.R.
      Transition cow nutrition and management strategies of dairy herds in the Northeastern United States: Part I—Herd description and performance characteristics.
      ); therefore, it is plausible we may have observed a negative association with PRFS for the prevalence of elevated postpartum NEFA concentrations if we eliminated cows that weren't bred for the first time until later in lactation. Pre- and postovulatory failure has been associated with the severity of negative energy balance during the early-lactation period because it coincides with follicular development (
      • Roche J.R.
      • Burke C.R.
      • Crookenden M.A.
      • Heiser A.
      • Loor J.L.
      • Meier S.
      • Mitchell M.D.
      • Phyn C.V.C.
      • Turner S.A.
      Fertility and the transition dairy cow.
      ). Negative energy balance during the early-lactation period can have a negative effect on reproductive success after the VWP as follicles and oocytes will ovulate 50 to 60 d after development (
      • Santos J.E.P.
      • Staples C.R.
      Feeding the herd for maximum fertility.
      ). When observing the Kaplan-Meier graph for postpartum NEFA concentrations (Figure 1), the lines do not start to diverge until ∼120 DIM, indicating that there is little difference in the proportion of cows becoming pregnant before 120 DIM. The differences in the cow and herd-level analysis for the biomarkers discussed may be due to other herd-level factors, such as nutrition or management, which may have a greater effect on milk production and reproductive success.
      There have only been 2 studies that have identified herd-alarm levels for metabolic-related analytes (
      • Ospina P.A.
      • Nydam D.V.
      • Stokol T.
      • Overton T.R.
      Association between the proportion of sampled transition cows with increased nonesterified fatty acids and β-hydroxybutyrate and disease incidence, pregnancy rate, and milk production at the herd level.
      ;
      • Chapinal N.
      • LeBlanc S.J.
      • Carson M.E.
      • Leslie K.E.
      • Godden S.
      • Capel M.
      • Santos J.E.P.
      • Overton M.W.
      • Duffield T.F.
      Herd-level association of serum metabolites in the transition period with disease, milk production, and early lactation reproductive performance.
      ); however, this is the first study to identify herd-alarm levels for a biomarker related to inflammation.
      • Ospina P.A.
      • Nydam D.V.
      • Stokol T.
      • Overton T.R.
      Association between the proportion of sampled transition cows with increased nonesterified fatty acids and β-hydroxybutyrate and disease incidence, pregnancy rate, and milk production at the herd level.
      observed 60 farms for the herd-level analysis, and
      • Chapinal N.
      • LeBlanc S.J.
      • Carson M.E.
      • Leslie K.E.
      • Godden S.
      • Capel M.
      • Santos J.E.P.
      • Overton M.W.
      • Duffield T.F.
      Herd-level association of serum metabolites in the transition period with disease, milk production, and early lactation reproductive performance.
      observed 55 farms. We identified a lower threshold but higher proportion for the prepartum NEFA herd-alarm level that was associated with disorder incidence compared with
      • Ospina P.A.
      • Nydam D.V.
      • Stokol T.
      • Overton T.R.
      Association between the proportion of sampled transition cows with increased nonesterified fatty acids and β-hydroxybutyrate and disease incidence, pregnancy rate, and milk production at the herd level.
      ; ≥15% of animals with a prepartum NEFA concentration ≥0.27 mmol/L).
      • Ospina P.A.
      • Nydam D.V.
      • Stokol T.
      • Overton T.R.
      Association between the proportion of sampled transition cows with increased nonesterified fatty acids and β-hydroxybutyrate and disease incidence, pregnancy rate, and milk production at the herd level.
      reported that herds above the herd-alarm level had a 3.6% increase in disorder incidence (DA, CK, or both), 1.2% decrease in 21-d PR, and 282-kg decrease in ME305 milk. The identified prepartum NEFA herd-alarm levels by
      • Chapinal N.
      • LeBlanc S.J.
      • Carson M.E.
      • Leslie K.E.
      • Godden S.
      • Capel M.
      • Santos J.E.P.
      • Overton M.W.
      • Duffield T.F.
      Herd-level association of serum metabolites in the transition period with disease, milk production, and early lactation reproductive performance.
      had a higher prepartum NEFA concentration threshold (≥0.5 mmol/L) and proportion (≥30% and 50%) compared with our study or
      • Ospina P.A.
      • Nydam D.V.
      • Stokol T.
      • Overton T.R.
      Association between the proportion of sampled transition cows with increased nonesterified fatty acids and β-hydroxybutyrate and disease incidence, pregnancy rate, and milk production at the herd level.
      ; however, the reported decrease in milk yield and odds of pregnancy to first service was only observed for multiparous cows and not primiparous cows. In our study, we only observed an association between the prepartum NEFA herd-alarm level with disorder incidence, 21-d PR for primiparous and multiparous cows, and PP for primiparous cows; however, we observed the opposite association on 21-d PR for primiparous cows compared with
      • Ospina P.A.
      • Nydam D.V.
      • Stokol T.
      • Overton T.R.
      Association between the proportion of sampled transition cows with increased nonesterified fatty acids and β-hydroxybutyrate and disease incidence, pregnancy rate, and milk production at the herd level.
      . The differences observed in our study compared with
      • Ospina P.A.
      • Nydam D.V.
      • Stokol T.
      • Overton T.R.
      Association between the proportion of sampled transition cows with increased nonesterified fatty acids and β-hydroxybutyrate and disease incidence, pregnancy rate, and milk production at the herd level.
      may be due to our study having a lower prevalence of elevated NEFA (≥0.27 mmol/L;
      • Kerwin A.L.
      • Burhans W.S.
      • Mann S.
      • Tetreault M.
      • Nydam D.V.
      • Overton T.R.
      Transition cow nutrition and management strategies of dairy herds in the Northeastern United States: Part I—Herd description and performance characteristics.
      ). Similar to
      • Ospina P.A.
      • Nydam D.V.
      • Stokol T.
      • Overton T.R.
      Association between the proportion of sampled transition cows with increased nonesterified fatty acids and β-hydroxybutyrate and disease incidence, pregnancy rate, and milk production at the herd level.
      , we identified a lower postpartum NEFA threshold (0.48 mmol/L) that was associated with reproductive performance compared with the threshold identified for disorder incidence (0.59 mmol/L). As hypothesized by
      • Ospina P.A.
      • Nydam D.V.
      • Stokol T.
      • Overton T.R.
      Association between the proportion of sampled transition cows with increased nonesterified fatty acids and β-hydroxybutyrate and disease incidence, pregnancy rate, and milk production at the herd level.
      , the mechanism associated with DA and CK may not be as sensitive to elevated postpartum NEFA concentrations as the reproductive system.
      There were other limitations to this study. This study aimed to take one-third of blood samples from primiparous cows and two-third of blood samples from multiparous cows to reflect typical herd demographics in the northeastern United States. This study design allowed the results to be interpretable at the herd level for monitoring purposes; however, the herd-alarm level analysis was stratified by parity for certain outcomes. This reduced our sample size, particularly for the primiparous cow analysis, and thus decreased our confidence that our sample reflected the true herd incidence adequately. Therefore, future studies should aim to investigate these metabolites with outcomes of interest with an adequate sample size for primiparous cows. It is difficult to develop a test at the cow level that is applicable at the herd level due to normal biological variation and sample size constraints (
      • Oetzel G.R.
      Monitoring and testing dairy herds for metabolic disease.
      ). In addition, the accuracy of the herd-level test [i.e., whether the herd is truly above the herd-alarm level (herd-level Se), or truly not (herd-level Sp)] is influenced by the Se and Sp of the cow-level test (
      • Ospina P.A.
      • McArt J.A.
      • Overton T.R.
      • Stokol T.
      • Nydam D.V.
      Using nonesterified fatty acids and β-hydroxybutyrate concentrations during the transition period for herd-level monitoring of increased risk of disease and decreased reproductive and milking performance.
      ). We also followed the same cohort of cows throughout this study, which will allow us to investigate associations with pen- and herd-level factors in other studies; however, for this part of the study, we observed a slight skewness in the days relative to parturition relative to the prepartum and postpartum sampling visits. The cows observed were closer to parturition during the prepartum visit (median = −7 d) and further away from parturition at the postpartum visit (median = +10 d). For the disorder analysis at the cow and herd levels, this potentially led us to removing more cows in the postpartum analysis due to the cows being diagnosed with the disorder of interest before we took the blood sample. Although our sampling scheme estimated herd-level prevalence of elevated biomarkers, it did not allow us to identify when the cow first had the elevated biomarker of interest relative to parturition; therefore, misclassification may have occurred. Our analysis removed cows that were diagnosed with the disorder of interest before blood sample collection, leading to potential selection bias; however, this approach resulted in more conservative values, the prevention of potential confounders (i.e., the influence of treatment for the disorder), and maintenance of the temporal association and true sequence of events (
      • Hill A.B.
      The environment and disease: Association or causation?.
      ). By removing cows that were previously diagnosed with the disorder of interest, we may not have observed an association between the biomarker and the disorder, which may explain the variation observed across the literature (i.e., why some studies reported an association and others did not). We initially identified quadratic relationships between the biomarkers and some of our cow-level outcomes; however, due to few observations above the inflection point, we were unable to make strong conclusions regarding the meaning of this relationship, but this relationship should be considered in the future. In addition, because this was an observational study, there were many sources of variation that were not accounted for in the models, such as differences in nutrition or management. We also relied on the farm staff to record our outcomes of interest. Although we tried to standardize the disorder definitions between farms, farms have different protocols for identifying sick cows and for fresh cow health checks (
      • Kerwin A.L.
      • Burhans W.S.
      • Mann S.
      • Tetreault M.
      • Nydam D.V.
      • Overton T.R.
      Transition cow nutrition and management strategies of dairy herds in the Northeastern United States: Part I—Herd description and performance characteristics.
      ). Farm staff may interpret the definitions differently, which likely adds to variation observed.

      ACKNOWLEDGMENTS

      The authors acknowledge and thank the farms, nutritionists, and veterinarians who participated and the numerous people who assisted with data collection. This project was partially funded by the New York Farm Viability Institute (Syracuse, NY), Poulin Grain (Newport, VT), Elanco US (Greenfield, IN), Phibro Animal Health (Teaneck, NJ), and a USDA-NIFA (Washington, DC) Multi-State Hatch Project. The authors have not stated any conflicts of interest.

      REFERENCES

        • Allison P.D.
        Survival Analysis using SAS: A Practical Guide.
        2nd ed. SAS Institute Inc, 2010
        • Allison P.D.
        Logistic Regression using SAS: Theory and Application.
        2nd ed. SAS Institute Inc, 2012
        • Bauman D.E.
        • Currie B.W.
        Partitioning of nutrients during pregnancy and lactation: A review of mechanisms involving homeostasis and homeorhesis.
        J. Dairy Sci. 1980; 63 (7000867): 1514-1529
        • Bell A.W.
        Regulation of organic nutrient metabolism during transition from late pregnancy to early lactation.
        J. Anim. Sci. 1995; 73 (8582872): 2804-2819
        • Bradford B.J.
        • Yuan K.
        • Farney J.K.
        • Mamedova L.K.
        • Carpenter A.J.
        Invited review: Inflammation during the transition to lactation: New adventures with an old flame.
        J. Dairy Sci. 2015; 98 (26210279): 6631-6650
        • Burfeind O.
        • Sannmann I.
        • Voigtsberger R.
        • Heuwieser W.
        Receiver operating characteristic curve analysis to determine the diagnostic performance of serum haptoglobin concentration for the diagnosis of acute puerperal metritis in dairy cows.
        Anim. Reprod. Sci. 2014; 149 (25128191): 145-151
        • Chapinal N.
        • Carson M.
        • Duffield T.F.
        • Capel M.
        • Godden S.
        • Overton M.
        • Santos J.E.P.
        • LeBlanc S.J.
        The association of serum metabolites with clinical disease during the transition period.
        J. Dairy Sci. 2011; 94 (21943741): 4897-4903
        • Chapinal N.
        • Carson M.E.
        • LeBlanc S.J.
        • Leslie K.E.
        • Godden S.
        • Capel M.
        • Santos J.E.P.
        • Overton M.W.
        • Duffield T.F.
        The association of serum metabolites in the transition period with milk production and early-lactation reproductive performance.
        J. Dairy Sci. 2012; 95 (22365212): 1301-1309
        • Chapinal N.
        • LeBlanc S.J.
        • Carson M.E.
        • Leslie K.E.
        • Godden S.
        • Capel M.
        • Santos J.E.P.
        • Overton M.W.
        • Duffield T.F.
        Herd-level association of serum metabolites in the transition period with disease, milk production, and early lactation reproductive performance.
        J. Dairy Sci. 2012; 95 (22863094): 5676-5682
        • Cooke R.F.
        • Bohnert D.W.
        • Moriel P.
        • Hess B.W.
        • Mills R.R.
        Effects of polyunsaturated fatty acid supplementation on ruminal in situ forage degradability, performance, and physiological responses of feeder cattle.
        J. Anim. Sci. 2011; 89 (21680784): 3677-3689
        • Curtis C.R.
        • Erb H.N.
        • Sniffen C.J.
        • Smith R.D.
        • Kronfeld D.S.
        Path analysis of dry period nutrition, postpartum metabolic and reproductive disorders, and mastitis in Holstein cows.
        J. Dairy Sci. 1985; 68 (4067048): 2347-2360
        • Dubuc J.
        • Duffield T.F.
        • Leslie K.E.
        • Walton J.S.
        • LeBlanc S.J.
        Risk factors for postpartum uterine diseases in dairy cows.
        J. Dairy Sci. 2010; 93 (21094748): 5764-5771
        • Duffield T.F.
        • Lissemore K.D.
        • McBride B.W.
        • Leslie K.E.
        Impact of hyperketonemia in early lactation dairy cows on health and production.
        J. Dairy Sci. 2009; 92 (19164667): 571-580
        • Eckersall P.D.
        Recent advances and future prospects for the use of acute phase proteins as markers of disease in animals.
        Rev. Med. Vet. 2000; 151: 577-584
        • Eicker S.
        • Stewart S.
        • Rapnicki P.
        Dairy herd reproductive records.
        in: Minnesota Dairy Health Conf. University of Minnesota, 2000: 183-194
        • Garverick H.A.
        • Harris M.N.
        • Vogel-Bluel R.
        • Sampson J.D.
        • Bader J.
        • Lamberson W.R.
        • Spain J.N.
        • Lucy M.C.
        • Youngquist R.S.
        Concentrations of nonesterified fatty acids and glucose in blood of periparturient dairy cows are indicative of pregnancy success at first insemination.
        J. Dairy Sci. 2013; 96 (23141836): 181-188
        • Herdt T.H.
        Ruminant adaptation to negative energy balance.
        Vet. Clin. North Am. Food Anim. Pract. 2000; 16 (11022337): 215-230
        • Hill A.B.
        The environment and disease: Association or causation?.
        Proc. R. Soc. Med. 1965; 58 (14283879): 295-300
        • Horst E.A.
        • Kvidera S.K.
        • Baumgard L.H.
        Invited review: The influence of immune activation on transition cow health and performance – A critical evaluation of traditional dogmas.
        J. Dairy Sci. 2021; 104 (34053763): 8380-8410
        • Humblet M.-F.
        • Guyot H.
        • Boudry B.
        • Mbayahi F.
        • Hanzen C.
        • Rollin F.
        • Godeau J.-M.
        Relationship between haptoglobin, serum amyloid A, and clinical status in a survey of dairy herds during a 6-month period.
        Vet. Clin. Pathol. 2006; 35 (16783711): 188-193
        • Huzzey J.M.
        • Duffield T.F.
        • LeBlanc S.J.
        • Veira D.M.
        • Weary D.M.
        • von Keyserlingk M.A.G.
        Short communication: Haptoglobin as an early indicator of metritis.
        J. Dairy Sci. 2009; 92 (19164673): 621-625
        • Huzzey J.M.
        • Mann S.
        • Nydam D.V.
        • Grant R.J.
        • Overton T.R.
        Associations of peripartum markers of stress and inflammation with milk yield and reproductive performance in Holstein dairy cows.
        Prev. Vet. Med. 2015; 120 (25957974): 291-297
        • Huzzey J.M.
        • Nydam D.V.
        • Grant R.J.
        • Overton T.R.
        Associations of prepartum plasma cortisol, haptoglobin, fecal cortisol metabolites, and nonesterified fatty acids with postpartum health status in Holstein dairy cows.
        J. Dairy Sci. 2011; 94: 5878-5889
        • Katoh N.
        Relevance of apolipoproteins in the development of fatty liver and fatty liver-related peripartum diseases in dairy cows.
        J. Vet. Med. Sci. 2002; 64 (12014573): 293-307
        • Kerwin A.L.
        • Burhans W.S.
        • Mann S.
        • Tetreault M.
        • Nydam D.V.
        • Overton T.R.
        Transition cow nutrition and management strategies of dairy herds in the Northeastern United States: Part I—Herd description and performance characteristics.
        J. Dairy Sci. 2021; 105: 5327-5348
        • Kushibiki S.
        • Hodate K.
        • Shingu H.
        • Obara Y.
        • Touno E.
        • Shinoda M.
        • Yokomizo Y.
        Metabolic and lactational responses during recombinant bovine tumor necrosis factor-alpha treatment in lactating cows.
        J. Dairy Sci. 2003; 86 (12703618): 819-827
        • Lomborg S.R.
        • Nielsen L.R.
        • Heegaard P.M.
        • Jacobsen S.
        Acute phase proteins in cattle after exposure to complex stress.
        Vet. Res. Commun. 2008; 32 (18461465): 575-582
        • McArt J.A.A.
        • Nydam D.V.
        • Oetzel G.R.
        Epidemiology of subclinical ketosis in early lactation dairy cattle.
        J. Dairy Sci. 2012; 95 (22916909): 5056-5066
        • Medzhitov R.
        Origin and physiological roles of inflammation.
        Nature. 2008; 454 (18650913): 428-435
        • Nightingale C.R.
        • Sellers M.D.
        • Ballou M.A.
        Elevated plasma haptoglobin concentrations following parturition are associated with elevated leukocyte responses and decreased subsequent reproductive efficiency in multiparous Holstein dairy cows.
        Vet. Immunol. Immunopathol. 2015; 164 (25613776): 16-23
        • Oetzel G.R.
        Monitoring and testing dairy herds for metabolic disease.
        Vet. Clin. North Am. Food Anim. Pract. 2004; 20 (15471629): 651-674
        • Ospina P.A.
        • McArt J.A.
        • Overton T.R.
        • Stokol T.
        • Nydam D.V.
        Using nonesterified fatty acids and β-hydroxybutyrate concentrations during the transition period for herd-level monitoring of increased risk of disease and decreased reproductive and milking performance.
        Vet. Clin. North Am. Food Anim. Pract. 2013; 29 (23809897): 387-412
        • Ospina P.A.
        • Nydam D.V.
        • DiCiccio T.J.
        Technical note: The risk ratio, an alternative to the odds ratio for estimating the association between multiple risk factors and a dichotomous outcome.
        J. Dairy Sci. 2012; 95 (22541486): 2576-2584
        • Ospina P.A.
        • Nydam D.V.
        • Stokol T.
        • Overton T.R.
        Association between the proportion of sampled transition cows with increased nonesterified fatty acids and β-hydroxybutyrate and disease incidence, pregnancy rate, and milk production at the herd level.
        J. Dairy Sci. 2010; 93 (20655428): 3595-3601
        • Ospina P.A.
        • Nydam D.V.
        • Stokol T.
        • Overton T.R.
        Associations of elevated nonesterified fatty acids and β-hydroxybutyrate concentrations with early lactation reproductive performance and milk production in transition dairy cattle in the northeastern United States.
        J. Dairy Sci. 2010; 93 (20338437): 1596-1603
        • Ospina P.A.
        • Nydam D.V.
        • Stokol T.
        • Overton T.R.
        Evaluation of nonesterified fatty acids and β-hydroxybutyrate in transition dairy cattle in the northeastern United States: Critical thresholds for prediction of clinical diseases.
        J. Dairy Sci. 2010; 93 (20105526): 546-554
        • Pohl A.
        • Burfeind O.
        • Heuwieser W.
        The associations between postpartum serum haptoglobin concentration and metabolic status, calving difficulties, retained fetal membranes, and metritis.
        J. Dairy Sci. 2015; 98 (25912860): 4544-4551
        • Pourhoseingholi M.A.
        • Vahedi M.
        • Rahimzadeh M.
        Sample size calculation in medical studies.
        Gastroenterol. Hepatol. Bed Bench. 2013; 6 (24834239): 14-17
        • Ringseis R.
        • Gessner D.K.
        • Eder K.
        Molecular insights into the mechanisms of liver-associated diseases in early-lactating dairy cows: hypothetical role of endoplasmic reticulum stress.
        J. Anim. Physiol. Anim. Nutr. (Berl.). 2015; 99 (25319457): 626-645
        • Roberts T.
        • Chapinal N.
        • LeBlanc S.J.
        • Kelton D.F.
        • Dubuc J.
        • Duffield T.F.
        Metabolic parameters in transition cows as indicators for early-lactation culling risk.
        J. Dairy Sci. 2012; 95 (22612941): 3057-3063
        • Roche J.R.
        • Burke C.R.
        • Crookenden M.A.
        • Heiser A.
        • Loor J.L.
        • Meier S.
        • Mitchell M.D.
        • Phyn C.V.C.
        • Turner S.A.
        Fertility and the transition dairy cow.
        Reprod. Fertil. Dev. 2017; 30 (29539306): 85-100
        • Santos J.E.P.
        • Staples C.R.
        Feeding the herd for maximum fertility.
        in: Beede D.K. Large Dairy Herd Management. 3rd ed. American Dairy Science Association, 2017: 799-811
        • Shin D.H.
        • Jeong J.K.
        • Choi I.S.
        • Moon S.H.
        • Lee S.C.
        • Kang H.G.
        • Park S.B.
        • Kim I.H.
        Associations between serum haptoglobin concentration and peri- and postpartum disorders, milk yield, and reproductive performance in dairy cows.
        Livest. Sci. 2018; 213: 14-18
        • Skinner J.G.
        • Brown R.A.
        • Roberts L.
        Bovine haptoglobin response in clinically defined field conditions.
        Vet. Rec. 1991; 128 (1903006): 147-149
        • Sordillo L.M.
        • Contreras G.A.
        • Aitken S.L.
        Metabolic factors affecting the inflammatory response of periparturient dairy cows.
        Anim. Health Res. Rev. 2009; 10 (19558749): 53-63
        • Stokes M.E.
        • Davis C.S.
        • Koch G.G.
        Categorical Data Analysis using SAS.
        3rd ed. SAS Institute Inc, 2012
        • Thrusfield M.V.
        Veterinary Epidemiology.
        3rd ed. Blackwell Science, 2005
        • Trevisi E.
        • Han X.-T.
        • Piccioli-Cappelli F.
        • Bertoni G.
        Dry matter intake reduction before calving in dairy cows: Relationship with immune system and metabolism conditions.
        in: Proc. 53rd Annual Meet. EAAP. European Federation of Animal Science, 2002: 54
        • Uchida E.
        • Katoh N.
        • Takahashi K.
        Appearance of haptoglobin in serum from cows at parturition.
        J. Vet. Med. Sci. 1993; 55 (8286556): 893-894
        • Walsh R.B.
        • Walton J.S.
        • Kelton D.F.
        • LeBlanc S.J.
        • Leslie K.E.
        • Duffield T.F.
        The effect of subclinical ketosis in early lactation on reproductive performance of postpartum dairy cows.
        J. Dairy Sci. 2007; 90 (17517719): 2788-2796
        • White H.M.
        The role of TCA cycle anaplerosis in ketosis and fatty liver in periparturient dairy cows.
        Animals (Basel). 2015; 5 (26479386): 793-802

      Linked Article

      • Transition cow nutrition and management strategies of dairy herds in the northeastern United States: Part I—Herd description and performance characteristics
        Journal of Dairy ScienceVol. 105Issue 6
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          Our objective was to describe management and herd characteristics of the transition period on freestall dairy herds in the northeastern United States using an on-farm survey and prospective cohort design. Enrolled herds (n = 72) had a median of 900 milking cows (range: 345–2,900) and a rolling herd average of 12,674 kg (standard deviation ± 1,220 kg), and 87.2% (n = 82/94) of fresh pens were milked at least 3×/d. The prevalence of herds with ≥15% of sampled cows with elevated concentrations of nonesterified fatty acids prepartum (≥0.27 mmol/L, 2–14 d before parturition) and postpartum [primiparous: ≥0.60 mmol/L, multiparous: ≥0.70 mmol/L, 3–14 d in milk (DIM)], β-hydroxybutyrate postpartum (≥1.2 mmol/L, 3–14 DIM), and haptoglobin postpartum (≥1 g/L, 0–12 DIM) was 51%, 51%, 51%, and 57%, respectively.
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