Beta-hydroxybutyrate in milk as screening test for subclinical ketosis in dairy cows

Polish Journal of Veterinary Sciences Vol. 20, No. 3 (2017), 507–512 DOI 10.1515/pjvs-2017-0061 Original article Beta-hydroxybutyrate in milk as screening test for subclinical ketosis in dairy cows J. Ježek1, M.R. Cincović2, M. Nemec1, B. Belić2, R. Djoković3, M. Klinkon1, J. Starič1 1 University of Ljubljana, Veterinary Faculty, Gerbičeva 60, Ljubljana, Slovenia University of Novi Sad, Faculty of Agriculture, Department of Veterinary Medicine, Trg D. Obradovića 8, Novi Sad, Serbia 3 University of Kragujevac, Faculty of Agronomy, Department of Animal Science, Cara Dušana 34, Čačak, Serbia 2 Abstract Ketosis is a very frequent metabolic disease in dairy cows, resulting in lower milk production, impaired fertility and increased frequency of other diseases. The course of the disease is often subclinical, so early detection is very important. The aim of the study was to investigate the relation between the concentration of beta-hydroxybutyrate in blood and milk and to determine the cut-off value in milk for detection of subclinical ketosis. The study included 94 cows, which were in the first third of lactation. Beta-hydroxybutyrate (BHB) concentrations were measured in blood and milk serum using a biochemical analyser. The average concentration of BHB in the blood serum samples was 1.14 mmol/L while in the milk it was about ten times lower at 0.117 mmol/L. A statistically significant positive correlation between the concentration of BHB in blood and milk (r=0.705, p<0.001) was found. In cows with BHB in blood below 2.0 mmol/L a stronger correlation between blood and milk BHB was established (r=0.658, p<0.001) than in cows with blood BHB above 2.0 mmol/L (r=-0.292, p=0.206). Therefore, BHB in milk is a very suitable indicator in the diagnosis of subclinical ketosis as there is a good correlation between BHB in the blood and milk of cows with subclinical ketosis. The cut-off concentration of BHB in milk set at ≥0.080 mmol/L (AUC=0.91±0.03; p<0.001) is a significant indicator for subclinical ketosis in dairy cows. The sensitivity of the test was 94% and specificity 74%. Beta-hydroxybutyrate in milk is a good indicator of subclinical ketosis in dairy cows and can be measured accurately with a biochemical analyser. Key words: subclinical ketosis, biochemical analyser, BHB, milk, cows Introduction Ketosis is a common disease in dairy herds, associated with high milk production and a negative energy balance. Most cases occur in the first 6 weeks to Correspondence to: J. Ježek, e-mail: 2 months after calving (Herdt 2000, Enjalbert et al. 2001, Fleming 2002). Ketosis results in lower milk yield, lower fertility and increased frequency of other diseases (abomasal displacement) (McArt et al. 2012, Suthar et al. 2013). Subclinical ketosis causes greater 508 losses than clinical ketosis because it occurs more frequently (Geishauser et al. 2000) and often cannot be detected by the farmers. On average 40% of the cows have subclinical ketosis at least once during lactation (Dirksen et al. 1997, Berg and Vert 2014), while clinical ketosis affects on average 5% of cows (Kelton and et al. 1998, Oetzel 2004, Koeck et al. 2013). The prevalence of subclinical ketosis in ten European countries was on average 21.8% (from 11.2 to 36.6%), and clinical ketosis was 3.7% (0.4 to 11.1%). The average prevalence of subclinical ketosis in 24 herds in Slovenia was 24%, while in Serbia it was up to 19.5% in 42 herds (Suthar et al. 2013). In view of the above mentioned facts, it is crucial to detect the disease as soon as possible, begin to treat it and to introduce preventive measures. The most reliable way to establish subclinical ketosis is measuring the concentration of beta-hydroxybutyrate (BHB) in the blood serum (Duffield 2000) or in the whole blood. The most accurate method is measuring BHB concentration in the laboratory with a biochemical analyser. In the literature different thresholds for the determination of subclinical ketosis are indicated: 1.40 mmol/L (Oetzel 2004), 1.20 mmol/L (Asl et al. 2011) and 1.00 mmol/L (Ospina et al. 2010, Whitaker 1997). Values of BHB above 2.99 mmol/L are most often associated with the clinical form of ketosis (Oetzel 2004). Milk is a very convenient sample for the determination of ketosis because it is easily accessible also by farm personnel. In the case of sub-clinical ketosis the content of BHB in the milk is elevated but the concentrations are lower than in the blood. BHB concentration in milk can be measured in the field by using a semi quantitative colorimetric dipstick test. The cut-off value is 100 to 200 μmol/L, higher values indicate ketosis. Carrier (2004) found in his study that the test (KetoTest strip) detected 73% of the actual positive cows and 96% of the actually negative. When the BHB level in milk exceeds 100 μmol/L the risk of clinical ketosis increases (Francos et al. 1997). The Fossomatic milk analyser is widely used for the testing of milk samples and has the possibility to test for BHB with Fourier transform infrared spectrometry (FTIR) as well. The results of Wilson and Goodel (2013) show that the BHB test methods agreed well for most non-ketotic cows, but the test did not agree well on classification of ketotic cows. They concluded that calibration improvements are necessary for improved testing of BHB in milk. Biochemical analysers are routinely used and they are very accurate for measuring of BHB in blood serum. Regarding the producer instructions the RX Daytona biochemical analyser can be used also for biochemical analysis of milk serum. For this reason J. Ježek et al. BHB was measured in blood and milk serum with the same analyser in this study. The authors were not able to find any data in the literature concerning the measurement of BHB in milk with a biochemical analyser. The aim of this study was to assess the agreement between the concentration of BHB in blood and milk of cows measured by biochemical analyser. Additionally, the cut-off value of BHB in milk serum for identifying subclinical ketosis was calculated. Materials and Methods Ninety-four samples of blood and milk of cows were analysed. The cows were Holstein Friesian breed and in the first third of lactation. Average production of milk was 30.3±8.7 kg/day. Cows were milked twice a day and fed according to requirements. Blood was taken approximately four hours after the morning feed from the tail vein (vena caudalis mediana), and milk samples were collected at the same time. To obtain milk serum for biochemical analysis the milk samples were centrifuged at 4500 rotations for 15 minutes to obtain skimmed milk. Skimmed milk samples were centrifuged in Eppendorf tubes for 30 minutes at 13000 rotations to obtain a milk serum. BHB concentrations were measured in blood serum and milk serum using a RX Daytona biochemical analyser (RANDOX Laboratories Ltd., UK) and Rayto 1904cv (Rayto Electronics Inc. Shenzhen, China). A kinetic enzyme test with BHB dehydrog (...truncated)