The relationship of excessive energy deficit with milk somatic cell score and clinical mastitis.

Periparturient cows go through a period of immune suppression often marked by immune cell dysfunction. Further exacerbation of this dysfunction through early-lactation excessive energy deficit (EED) has been associated with increased susceptibility to infectious conditions such as mastitis. Our objective was to explore the association of milk somatic cell score (SCS) and clinical mastitis (CM) diagnosis in cows identified with EED, diagnosed using each of the following: blood and milk ß-hydroxybutyrate (BHB), milk predicted blood nonesterified fatty acid (mpbNEFA) concentrations, or milk de novo fatty acid (FA) relative percentages (rel %). We analyzed data collected from 396 multiparous Holstein cows from 2 New York farms in a prospective cohort study. Coccygeal vessel blood samples and composite milk samples were collected twice weekly from 3 to 18 days in milk (DIM) for a total of 4 time points per cow (T1, T2, T3, T4). Blood was analyzed using a hand-held meter, and milk was analyzed using Fourier-transform mid-infrared spectrometry for milk BHB and mpbNEFA concentrations, milk de novo FA rel %, and somatic cell count. Excessive energy deficit was diagnosed as blood BHB ≥ 1.2 mmol/L, milk BHB ≥ 0.14 mmol/L, mpbNEFA ≥ 0.55 mmol/L, or de novo FA ≤ 22.7 rel %, depending on the model. Clinical mastitis cultures were collected from 4 to 60 DIM by on-farm personnel. Incidence of hyperketonemia as determined by blood BHB was 13.4%, and incidence of CM was 23.9%. Separate repeated-measures ANOVA models were developed for each EED diagnostic analyte for parity groups 2, 3, and ≥4 to assess differences in SCS; t-test analyses were similarly used to assess the association of each diagnostic analyte with CM at each time point. For all diagnostic analytes, apart from milk BHB, cows diagnosed with EED tended to have lower SCS than their non-EED counterparts. This was especially apparent at T1 for all parity groups, and at T2, T3, and T4 for blood BHB and mpbNEFA. For EED diagnosis via mpbNEFA, mean SCS were lower in parity ≥4, with a difference in mean SCS between EED and non-EED animals of 0.7 SCS units, equating to a somatic cell count in EED animals approaching half that of non-EED (EED = 67,000 cells/mL, non-EED = 107,000 cell/mL). No important relationships were observed between CM diagnosis and blood BHB, milk BHB, or mpbNEFA. For de novo FA rel %, reductions in this analyte were noted before CM diagnosis at all time points. Although the relationship between EED and CM is still unclear, our findings suggest that cows in EED, diagnosed using blood BHB or mpbNEFA during the first 18 DIM, have a tendency toward lower SCS compared with their non-EED counterparts.

A randomized controlled trial to evaluate propylene glycol alone or in combination with dextrose as a treatment for hyperketonemia in dairy cows.

Our objective was to investigate the effect of i.v. dextrose as an adjunct therapy to oral propylene glycol on the resolution of hyperketonemia (HYK; blood ß-hydroxybutyrate ≥1.2 mmol/L), disease incidence, and early lactation milk yield. Cows (n = 1,249) between 3 and 16 d in milk (DIM) from 4 New York dairy farms were screened once weekly for HYK for 2 wk. Those with HYK and no previous history of retained placenta, metritis, or HYK were randomly assigned to 1 of 3 treatment groups: 300 mL of oral 100% propylene glycol for 3 d (PG3); 300 mL of oral 100% propylene glycol for 3 d plus 500 mL i.v. 50% dextrose on d 1 (PG3D1); or 300 mL of oral 100% propylene glycol for 3 d plus 500 mL i.v. 50% dextrose on all 3 d (PG3D3). Cows with a blood ß-hydroxybutyrate <1.2 mmol/L at initial screening were re-screened the following week and randomly assigned to the above treatment groups if blood ß-hydroxybutyrate was ≥1.2 mmol/L. Cows were assessed for post-treatment HYK resolution 1 and 2 wk after initial HYK diagnosis. We collected farm-diagnosed occurrence of adverse events (sold, died, metritis, displaced abomasum, or ketosis) during the first 60 DIM and milk yield data from the first 10 wk of lactation from herd management software. We used mixed-effects multivariable Poisson regression models to assess the risk of post-treatment HYK resolution at 1 and 2 wk following initial HYK diagnosis and adverse event occurrence among treated cows. We used repeated-measures ANOVA to assess differences in average daily milk yield between treatments. The overall HYK incidence was 30.1% (n = 373). Sixty-four percent of cows (n = 237) were assigned to a treatment group in the first week (3 to 9 DIM), and 36% (n = 136) assigned the second week (10 to 16 DIM). The incidence of 1 or more adverse events during the first 60 DIM was 9.4% (n = 35). We found no effect of treatment on risk of post-treatment HYK resolution at wk 1 (PG3 56.9%, PG3D1 45.0%, PG3D3 50.0%) or wk 2 (PG3 60.0%, PG3D1 52.1%, PG3D3 59.5%) following initial diagnosis, or for risk of adverse event occurrence (PG3 7.4%, PG3D1 8.0%, PG3D3 12.6%). Average daily milk yield (mean ± SE) was similar between treatment groups (PG3: 42.7 ± 0.6 kg/d, PG3D1: 42.4 ± 0.6 kg/d, PG3D3: 42.6 ± 0.6 kg/d). The addition of dextrose for 1 or 3 d provided no improvement in resolution of ketosis assessed once weekly, reduction in adverse events during the first 60 d of lactation, or a difference in average daily milk yield during the first 10 wk of lactation.

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

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

Technical note: Effect of storage time and temperature on total calcium concentrations in bovine blood.

Our objectives were to examine the stability of (1) serum and plasma total calcium (tCa) after delayed separation of bovine whole blood stored at 4°C, and (2) frozen serum and plasma tCa stored at -80°C. Whole blood was collected from 19 cows into vacutainer tubes (Becton Dickinson, Franklin Lakes, NJ) containing no additives (serum) or lithium heparin (plasma). Baseline tCa concentrations (0-h tCa) were determined on serum and plasma harvested within 2 h of blood collection. To evaluate the effect of prolonged whole blood storage at 4°C, serum and plasma were harvested from samples after 6, 24, 48, 72 h, and 7 and 14 d of storage. For evaluation of the stability of frozen serum and plasma stored at -80°C, samples were stored for 7 d and 1, 3, 6, and 12 mo. The median 0-h tCa concentration for serum and plasma was 2.25 mmol/L (range: 1.58 to 2.60 mmol/L) and 2.28 mmol/L (range: 1.60 to 2.65 mmol/L), respectively. The average difference in tCa from 0-h samples ranged from -0.02 to 0.03 mmol/L for all samples stored at either 4 or -80°C. The median within-cow variability for whole blood samples stored at 4°C was 1.1 and 1.0% for serum and plasma, respectively, and for serum and plasma stored at -80°C was 1.1 and 1.3%, respectively. When whole blood was stored at 4°C, no differences in serum or plasma tCa concentrations were observed across the evaluated time points as compared with 0-h concentrations. Similarly, frozen serum and plasma stored at -80°C remained stable up to 12 mo. Therefore, our findings show that whole blood samples may be stored for at least 14 d at 4°C in vacutainer tubes containing no additives or lithium heparin without biologically important changes in tCa concentrations beyond expected analytical variation. Additionally, serum or plasma may be stored at -80°C for up to 12 mo with no effect on tCa concentrations.

Association of mid-infrared-predicted milk and blood constituents with early-lactation disease, removal, and production outcomes in Holstein cows.

Partial least squares regression estimates of milk and blood constituents using Fourier-transform mid-infrared (FTIR) analysis have shown promise as a tool for monitoring early-lactation excessive energy deficit in dairy herds. Our objective was to analyze milk via FTIR to determine the association of early-lactation predicted milk ß-hydroxybutyrate (BHB) concentrations, predicted blood nonesterified fatty acid (NEFA) concentrations, and predicted milk de novo fatty acid (FA) percentages relative to total FA concentrations, with the risk of disease or removal in early lactation (hyperketonemia, displaced abomasum, metritis, culling, or death) and average daily milk yield during the first 15 wk of lactation. We enrolled 517 multiparous Holstein cows from 2 dairy farms in New York. Composite milk samples were collected twice weekly from 3 to 18 DIM for a total of 4 timepoints (T1, T2, T3, T4) and analyzed using FTIR spectrometry for milk BHB and FA composition and predicted blood NEFA. Blood samples were collected for hyperketonemia determination (BHB ≥ 1.2 mmol/L) using a handheld meter, and farm-diagnosed occurrence of disease or removal during the first 30 DIM and average daily milk yield during the first 15 wk of lactation were collected from herd management software. The incidence of disease or removal between 3 and 18 DIM was 20.2%. Explanatory models for disease or removal were developed for each predicted constituent of interest at each timepoint using fixed-effect multivariable Poisson regression. Repeated measures ANOVA models were developed for each predicted constituent to assess differences in average daily milk yield. For all timepoints, increased risk of disease or removal was associated with higher predicted milk BHB [relative risk (RR)T1 = 2.0; RRT2 = 3.4; RRT3 = 5.2; RRT4 = 9.1], higher predicted blood NEFA (RRT1 = 2.7; RRT2 = 2.5; RRT3 = 3.8; RRT4 = 10.0), and lower predicted milk de novo FA relative percentages (RRT1 = 2.9; RRT2 = 3.3; RRT3 = 5.8; RRT4 = 7.2). Average daily milk yield was increased for cows above the cut point for predicted milk BHB (2.1 kg/d) and predicted blood NEFA (3.5 kg/d) and below the cut point for de novo FA relative percentages (2.3 kg/d). Our results suggest that FTIR-predicted milk BHB, blood NEFA, and milk de novo FA relative percentages are promising indicators of subsequent disease or removal in early lactation; their positive relationship with milk yield warrants further exploration.

Epidemiology of subclinical hypocalcemia in early-lactation Holstein dairy cows: The temporal associations of plasma calcium concentration in the first 4 days in milk with disease and milk production.

The objectives of this study were to characterize the epidemiology of subclinical hypocalcemia (SCH) in Holstein dairy cows by assessing the temporal associations of plasma Ca concentrations in the first 4 d in milk (DIM) with the risk of cows being diagnosed with metritis or displaced abomasum (or both), and milk production across the first 15 wk of lactation. A prospective cohort study was conducted in 2 dairy herds in New York State, in which cows had a blood sample collected daily for the first 4 DIM. A total of 396 Holstein cows (137 primiparous and 259 multiparous) were enrolled. Multivariable Poisson regression models were built to evaluate the associations of plasma Ca concentration at each of the 4 d following parturition with the risk of primiparous cows being diagnosed with metritis and multiparous cows being diagnosed with metritis, displaced abomasum, or both. Similarly, generalized linear mixed models were built to evaluate the associations of plasma Ca concentration with milk production across the first 15 wk of lactation. Plasma Ca concentration was assessed on a continuous scale in all models; dichotomization and SCH classification only occurred in the final models if the Ca concentration variable was meaningful by creating an optimized threshold based on receiver operating characteristic curve analysis. Plasma Ca concentration assessed at 1 DIM was not associated with the risk of metritis in primiparous cows, but an association was observed at 2, 3, and 4 DIM (critical thresholds were plasma Ca concentration ≤2.15, 2.10, and 2.15 mmol/L, respectively). Plasma Ca concentration was associated with the risk of metritis or displaced abomasum diagnosis (or both) for 2nd parity animals at 2 DIM (threshold ≤1.97 mmol/L), and at 4 DIM for 3rd and greater lactations (threshold ≤2.20 mmol/L). Reduced plasma Ca concentration was associated with higher milk production when assessed at 1 DIM in primiparous and multiparous cows, and lower milk production when assessed at 4 DIM in multiparous cows only. For primiparous cows, plasma Ca concentration was not associated with lower milk production at any of the DIM assessed. In conclusion, assessments of SCH at the individual cow level must take into account the DIM of Ca concentration measurement and parity of the cow, as the epidemiology of the disorder was demonstrated to be highly dependent on these variables. This study advances the knowledge of the epidemiology of SCH and better establishes thresholds for optimizing SCH diagnosis.

Method comparison and validation of a prototype device for measurement of ionized calcium concentrations cow-side against a point-of-care instrument and a benchtop blood-gas analyzer reference method.

The objective of this study was to assess an optimized ion-selective electrode Ca-module prototype as a potential cow-side device for ionized Ca (iCa) measurements in bovine blood. A linearity experiment showed no deviation from linearity over a range of iCa concentrations compared with a commercial point-of-care (POC) device commonly used in the field (POCVS; VetScan i-STAT, Abaxis North America, Union City, CA) and a laboratory gold standard benchtop blood-gas analyzer [reference analyzer (RA); ABL-800 FLEX, Radiometer Medical, Copenhagen, Denmark]. Coefficient of variation on 3 samples with high, within-range, and low iCa concentrations ranged from 1.0 to 3.9% for the prototype. A follow-up validation experiment was performed, in which our objectives were to (1) assess the performance of the prototype cow-side against the POCVS (farm gold-standard) using fresh non-anticoagulated whole-blood samples; (2) assess the performance of the prototype and the POCVS against the RA in a diagnostic laboratory using blood collected in a heparin-balanced syringe; and (3) assess the agreement of the prototype and POCVS on-farm (fresh non-anticoagulated whole blood) against the RA on heparin-balanced blood. Finally, sensitivity and specificity of the results obtained by the prototype and the POCVS cow-side compared with the results obtained by the laboratory RA using 3 different iCa cut points for classification of subclinical hypocalcemia were calculated. A total of 101 periparturient Holstein cows from 3 dairy farms in New York State were used for the second experiment. Ionized Ca results from the prototype cow-side were, on average, 0.06 mmol/L higher than the POCVS. With heparin-balanced samples under laboratory conditions, the prototype and POCVS measured an average 0.04 mmol/L higher and lower, respectively, compared with the RA. Results from the prototype and POCVS cow-side were 0.01 mmol/L higher and 0.05 mmol/L lower, respectively, compared with results from the laboratory RA on heparinized blood. Sensitivity and specificity for the prototype and the POCVS under farm conditions at 3 potential subclinical hypocalcemia cut points were 100 and ≥93.5%, respectively. This novel ion-selective electrode Ca-module could become a rapid low-cost tool for assessing iCa cow-side, while qualitatively allowing classification of subclinical hypocalcemia on-farm.

Technical note: Comparison of 4 electronic handheld meters for diagnosing hyperketonemia in dairy cows.

The objective of this study was to evaluate 4 handheld ketone meters for use in on-farm ß-hydroxybutyrate (BHB) monitoring of hyperketonemia in transition dairy cows. Blood samples taken from 250 Holstein cows between 262d pregnant and 15d in milk were evaluated on 4 different handheld ketone meters: Precision Xtra (Abbott Laboratories, Abbott Park, IL), TaiDoc (Pharmadoc, Lüdersdorf, Germany), Nova Max (Nova Biomedical, Billerica, MA), and Nova Vet (Nova Biomedical). Samples were screened using the Precision Xtra and tested on the remaining 3 m if the sample BHB concentration fell into predetermined ranges. A total of 89 samples were used for analysis. Performance of each meter was compared with the average of 2 plasma BHB concentrations both determined by a gold standard spectrophotometric Randox assay performed at 2 independent laboratories. Agreement between the 2 laboratories was very strong (Pearson correlation=0.998). All meters had Pearson correlation coefficients greater than 0.95. The Precision Xtra and TaiDoc meters were 100.0% sensitive and 73.5% specific at a BHB concentration cut point of 1.2mmol/L. The Nova Vet and Nova Max meters had sensitivities of 94.9 and 74.4% and specificities of 91.8 and 100.0%, respectively, at the same cut point. Agreement between the gold standard and the handheld meter was the best for the Nova Vet meter when evaluated using a Bland Altman graph with a mean BHB difference of 0.08mmol/L. Trends in bias were noted with the Precision Xtra and Nova Max meters resulting in increasing average discrepancy between the gold standard and the meter for both at higher plasma BHB concentrations and mean BHB differences of -0.34 and 0.26mmol/L, respectively. The coefficient of variation was <10% for the Precision Xtra, TaiDoc, and Nova Vet meters, and <15% for the Nova Max meter. We conclude that the TaiDoc and Nova Vet meters, similar to the already validated Precision Xtra meter, are acceptable for use in on-farm testing for monitoring and treatment of hyperketonemia.

Erratum to "Blood calcium as a prognostic indicator of success after surgical correction of left displaced abomasum" (JDS Commun. 2:207-211).

[This corrects the article DOI: 10.3168/jdsc.2021-0079.].

Diurnal variation of milk fatty acids in early-lactation Holstein cows with and without hyperketonemia.

Estimates of milk constituents by Fourier-transform mid-infrared (FTIR) analysis have been shown to be a useful tool in monitoring energy deficit in early-lactation dairy cows. Our objectives were to describe the diurnal variation in milk fatty acids (FAs) and estimate the association of hyperketonemia with concentrations and diurnal patterns of FTIR estimates of milk FA. Blood samples were collected via jugular catheters bihourly for 5 d from multiparous Holstein cows (n = 28) enrolled between 3 and 9 days in milk. Milk samples were collected thrice daily at 0600, 1400, and 2200 h for d 2, 3, and 4 of the study period. Cows were retrospectively classified as hyperketonemic (HYK; n = 13) or non-HYK (n = 15) based on blood beta-hydroxybutyrate (bBHB) concentrations analyzed during the study period. Cows were classified as HYK if bBHB was ≥ 1.2 mmol/l for ≥ 50% (22/44) of bihourly timepoints; cows were classified as non-HYK if bBHB was ≥ 1.2 mmol/l for < 50% of bihourly timepoints. The HYK cows had bBHB ≥ 1.2 mmol/l for 31.4 ± 6.8 timepoints while the non-HYK cows had bBHB ≥ 1.2 mmol/l for 8.0 ± 3.9 timepoints. We used generalized linear mixed models to analyze concentrations of milk FA over time and differences between HYK groups. The relative percentage of de novo, mixed, and preformed FAs all followed diurnal patterns, however only the yield of preformed FA diurnally cycled, reaching a nadir at 0600 h and peaking at 1400 h. The yield per milking of preformed FA was also greater in the HYK cows than in the non-HYK cows. Oleic acid in milk followed a similar diurnal pattern to the yield of preformed FA, likely driving the cyclical nature of preformed FA. Finally, stearic acid was greater in HYK cows. Our results suggest that FTIR estimates of milk FA offer the potential to provide insight on the energy status of early-lactation cows, and when interested in understanding the absolute concentrations and yields of milk FA, diurnal variation should be considered.

Blood calcium as a prognostic indicator of success after surgical correction of left displaced abomasum.

Subclinical hypocalcemia, a reduction in blood calcium concentrations without apparent clinical signs of milk fever, occurs in 25 to 50% of early-postpartum dairy cows. Research has shown that these cows are at an increased risk of additional early-lactation diseases, including left displaced abomasum (LDA), compared with their normocalcemic counterparts. However, no work has assessed the association of total calcium (tCa) concentration at the time of LDA correction with subsequent milk yield and survival within the herd. Pending future development of an economical and accurate on-farm test for hypocalcemia, the ability to assess LDA prognosis after surgical correction based on precorrection blood tCa concentration is of interest to practitioners. Therefore, our objective was to determine the association of blood tCa concentration at the time of LDA surgical correction with milk yield and herd removal in the 60 d after correction. Nine bovine practitioners, located in New York State, were involved in sample collection from 17 herds for this prospective cohort study. Immediately following LDA diagnosis and before surgical correction, blood samples were collected from 152 dairy cows within the first 30 d in milk for serum tCa determination. The association of tCa with milk yield (n = 110) and herd removal (n = 127) in the first 60 d after LDA surgical correction was analyzed using repeated-measures ANOVA and a generalized linear mixed model, respectively, controlling for parity and days in milk at LDA surgical correction and the random effect of herd or practitioner. We found no evidence to support an association of tCa at time of LDA diagnosis with subsequent milk yield, and when cows were grouped by median tCa into low and high groups (tCa ≤2.1 and >2.1 mmol/L, respectively), subsequent milk production was similar (39.4 ± 1.6 and 40.1 ± 1.4 kg/d for low and high, respectively). Furthermore, we found no evidence to support an association of tCa at time of LDA diagnosis with the odds of herd removal (odds ratio = 0.6; 95% confidence interval = 0.4-1.7). Therefore, our study provided no clear evidence to support the association of tCa at time of LDA diagnosis and correction with either milk yield or herd removal during the 8 wk after correction. However, it did demonstrate low to extremely low tCa concentration in cows with LDA. Whether calcium supplementation would improve health and production outcomes in these cows is unclear and worth investigating further.

Case study: Evaluating quarter and composite milk sampling for detection of subclinical intramammary infections in dairy cattle.

Our objective was to evaluate a 200,000 cells/mL somatic cell count (SCC) cut-point on both the quarter and composite level to determine its effectiveness at identifying subclinical mastitis infections in one commercial dairy herd in Central New York. Milk samples from 107 Holstein cows were used for analysis. All cows were eligible for enrollment provided they had 4 working udder quarters, were >14 and <365 d in milk, and had no clinical mastitis event or treatment with intramammary antibiotics ≤14 d prior to d of sampling. A total of 428 quarter and 107 composite samples from 34 primiparous and 73 multiparous animals were analyzed for total SCC and aerobic culture. Performance of SCC for identification of subclinically infected animals was evaluated against the gold standard aerobic culture. Sensitivities for a 200,000 cells/mL cut-point on both the quarter and composite basis were 28.6%, and specificities were 91.5% and 87.3% on the quarter and composite basis, respectively. Receiver operating characteristic (ROC) curves determined on a quarter basis found the cut-point that optimized the sensitivity and specificity of a positive culture was 32,000 cells/mL, with a sensitivity of 76.2%, a specificity of 62.4%, and an area under the curve (AUC) of 0.73. The ROC curve cut-point that optimized the sensitivity and specificity for the composite samples was 75,000 cells/mL, with a sensitivity of 57.1%, a specificity of 78.9%, and an AUC of 0.67. A large proportion of culture positive primiparous cows (38%) had low SCC (median of 101,000 cells/mL on the quarter and 80,000 cells/mL on the composite level), and therefore, when multiparous cows were examined separately, the cut-point that optimized sensitivity and specificity on the quarter basis increased to 645,000 cells/mL with a corresponding sensitivity of 34.8%, specificity of 97.5%, and AUC of 0.65. On the composite basis, the cut-point based on multiparous cows only was 152,000 cells/mL, with a corresponding sensitivity of 60.0%, and specificity of 82.0%, and an AUC of 0.65. Our data indicate that the 200,000 cells/mL cut-point was inefficient in identifying subclinically infected animals, regardless of whether quarter or composite sampling was used. The low prevalence of subclinical infections as well as the large proportion of minor pathogens, especially in primiparous animals, contributed to this inefficiency. This case study provides evidence that, with continued improvement upon mastitis control and reduction in major mastitis pathogens, blanket cut-points may no longer provide the same diagnostic usefulness as they once did.