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Hyperketonemia is a common condition in early lactating dairy cows. Usually induced by a maladaptation to the negative energy balance affecting most dairy cows during early lactation, it increases the risk for the development of subsequent diseases. Furthermore, a negative impact on reproduction parameters is proven. As most cows suffering from hyperketonemia do not show clinical signs of ketosis, the diagnosis depends on the measurement of ketone bodies in blood, milk or urine. Laboratory-based measurement of increased blood concentrations of ß-hydroxybutyrate (BHBA) is the gold standard for the diagnosis of hyperketonemia, using cut-points of 1.2 mmol/L or 1.4 mmol/L. In addition, a range of semiquantitative or quantitative cow-side hyperketonemia tests is available. Currently, electronic handheld blood BHBA meters are frequently used in the diagnostic of hyperketonemia.
Strategic hyperketonemia screening protocols have been recommended, addressing 2 main objectives: first to detect and subsequently treat affected cows and second to monitor and evaluate the success of early lactation feeding and management procedures. The overall objective of this thesis was to evaluate factors potentially influencing the diagnostic of hyperketonemia in dairy cows.
The aim of the first study was to investigate the effect of sampling time in the course of a day on blood BHBA concentrations in continuously fed dairy cows. Furthermore, test characteristics of single and repeated BHBA measurements during the day to diagnose subclinical ketosis were determined for 2 frequently used BHBA cut-points (1.2 mmol/L and 1.4 mmol/L). A total of 128 cows (8 to 28 days in milk (DIM) fed with a total mixed ration (TMR) 10 times daily were preselected by blood BHBA concentration, measured with an electronic handheld meter. Cows with BHBA concentrations ≥ 0.8 mmol/L at the initial measurement were enrolled in the trial (n = 92). Subsequent measurements were repeated in intervals of 3 h for a total of 8 measurements during 24 h.
The analysis conducted indicated that sampling time did not affect BHBA concentration (P = 0.23). To evaluate test characteristics of single and repeated BHBA measurements, the average daily BHBA concentration, averaged from the 8 measurements, was used as gold standard. A single BHBA measurement at a random time of the day had a sensitivity of 0.90 and a specificity of 0.88, using a BHBA concentration of 1.2 mmol/L as cut-point. Using 1.4 mmol/L as cut-point, sensitivity and specificity were 0.89 and 0.90, respectively. Repeating measurements after different time intervals in cases of marginal BHBA concentrations (1.0 mmol/L to 1.6 mmol/L) and using the averaged BHBA concentration from both measurements to diagnose hyperketonemia improved test characteristics only slightly. In conclusion, blood samples for measurement of BHBA in continuously TMR fed dairy cows to can be drawn at any time of the day. A single measurement provides very good test characteristics, which can be enhanced slightly by repeating measurements in cows with marginal BHBA concentrations.
The second study was conducted to compare BHBA concentrations in blood samples drawn from 3 in daily practice frequently used sampling locations (A./V. coccygealis, V. jugularis, V. epigastrica cranialis superficialis). Blood samples of 116 early lactating dairy cows were drawn from the 3 sampling locations and tested for BHBA using an electronic handheld meter. Concentrations of BHBA differed between the sampling locations (P = 0.03). Mean BHBA concentration in samples drawn from the V. epigastrica cranialis superficialis was 0.3 mmol/L lower compared with samples drawn from the V. jugularis and 0.4 mmol/L lower compared with samples drawn from the A./V. coccygealis. Mean BHBA concentration did not differ significantly between samples drawn from the V. jugularis and samples drawn from the A./V. coccygealis (P = 0.82). In conclusion, blood samples for measurement of BHBA in lactating dairy cows should be drawn from the V. jugularis or A./V. coccygealis, whereas samples drawn from the V. epigastrica cranialis superficialis.contain significantly lower concentrations of BHBA.
The objective of the third study was to describe factors concerning the timing and setting of hyperketonemia screening protocols during early lactation. A total of 252 cows from 3 dairy farms were tested twice weekly during the first 42 DIM using an electronic handheld BHBA meter. The resulting 12 test results per cow therefore corresponded to lactation weeks 0.5 to 6.0. The time of onset of hyperketonemia, the number of positive hyperketonemia test results, and the duration of the longest hyperketonemic period during the first 42 DIM were investigated. Furthermore, test characteristics of single and repeated BHBA measurements during this period to diagnose hyperketonemia were evaluated to simulate different hyperketonemia screening protocols. In detail, test characteristics of 4 different testing scenarios (testing all cows 1, 2, 3, or 6 times during the first 42 DIM) were calculated for 2 different gold standard definitions (BHBA ≥ 1.2 mmol/L at least once or BHBA ≥ 1.2 mmol/L at least twice during the first 42 DIM).
Mean prevalence of hyperketonemia was 11.8% and ranged between 9.6% in lactation weeks 0.5 and 2.0 and 14.6% in lactation week 5.5. A total of 134 cows (53.2%) had at least 1 positive hyperketonemia test result during the observation period. Of these cows, 47% had the first positive hyperketonemia test later than 14 DIM. The median first positive hyperketonemia test result occurred in lactation week 2.0 (interquartile range (IQR) 1.0 to 3.5). Of all cows with hyperketonemia, 46.3% had only 1 positive test result. Median frequency of positive BHBA test results in cows affected by hyperketonemia was 2 (IQR 1 to 3). The longest hyperketonemic period in affected cows lasted in median 1 examination interval (3-4 days; IQR 1-2 examination intervals).
Test characteristics of the 4 testing scenarios varied depending on the test frequency and the gold standard considered. A single BHBA measurement during the whole 42 day period achieved a sensitivity of 21% and a specificity of 100% considering a minimum of 1 positive hyperketonemia test result during the 42 day period as gold standard. A testing scenario including weekly measurements of BHBA had a sensitivity of 72% and a specificity of 100% considering the same gold standard. Increasing the gold standard to a minimum of 2 positive hyperketonemia test results, i.e. in cases of sustained or repeated hyperketonemia, sensitivity and specificity were 33% and 97% for a single BHBA measurement during the first 42 DIM and 91% and 83% for a testing scenario including weekly measurements, respectively.
These data, in conclusion, show that hyperketonemia affects cows at least over the first 42 DIM. Screening protocols therefore should consider a longer period than the frequently mentioned first 2 weeks of lactation. A moderately laborious screening protocol including a biweekly testing scheme during the first 42 DIM achieves a sensitivity of 69% and a specificity of 91% considering cases of sustained or repeated hyperketonemia. Further research is needed to investigate the effects of an isolated positive BHBA tests during the whole observation period on disease risk, milk yield and reproductive performance.
The objective of the fourth study was to evaluate test characteristics of a new commercially available electronic handheld BHBA meter to diagnose hyperketonemia in dairy cows. Blood BHBA in samples from 155 lactating dairy cows from 3 farms was measured using the handheld device (NovaVet, Nova Biomedical, Waltham, MA, USA). Subsequently, blood was centrifuged and serum analyzed by a commercial laboratory. Concentrations determined by the device and the laboratory were compared. The methods were highly correlated (rs = 0.87; P < 0.05). A difference (median 0.0 mmol/L; IQR -0.1 to 0.2 mmol/L; P < 0.05) was observed between BHBA concentrations measured by the handheld device (median 1.0 mmol/L; IQR 0.7 to 1.3 mmol/L) and the laboratory (median 0.9 mmol/L; IQR 0.7 to 1.1 mmol/L). Sensitivity and specificity of the handheld device were 97% and 82%, respectively, using a cut-point of 1.2 mmol/L to diagnose cows as hyperketonemic. Using a gold standard cut-point of 1.4 mmol/L, the receiver operating characteristic analysis determined an adjusted cut-point of 1.3 mmol/L for the handheld device. Sensitivity and specificity were 96% and 85%, respectively. In conclusion, the handheld device can be recommended for the diagnostic of hyperketonemia in dairy cows. However, due to the specificities of 82% and 85%, respectively, a certain number of false positive results must be considered.