Subclinical ketosis (SCK) is defined as an abnormal concentration of blood circulating ketone bodies without clinical signs of ketosis [1]. It is a common and important metabolic disease in high-lactating dairy cows which typically occurs in the first 60 d post-partum [2]. In the same period, the overall prevalence of subclinical ketosis ranges from 6.9 to 34% [3-7] with a peak observed within 14 d [8]. Sub clinical ketosis causes economic losses in the dairy industry as a result of impaired milk production and longer calving intervals [3,6,9-11]. Besides, cows with subclinical ketosis are at increased risk of developing cystic ovaries, clinical ketosis, and displaced abomasum [3,6,9-11]. In the light of these shortfalls, the cost of SCK per cow is estimated to be 78 dollars, whereas one case of clinical ketosis can induce economic losses of up to 145 [12,13]. It has been postulated that early diagnosis of individual cases of subclinical ketosis would permit for early treatment and may help mitigate further losses [14,12].

Elevated concentrations of ketone bodies [acetone (Ac), acetoacetate (AcAc), and BHBA] in blood, urine, milk, and other body fluids without any clinical signs are the main characteristic of SCK, and various thresholds have been used to define SCK [2,6,15,16]. The gold standard diagnostic test for SCK is measuring BHBA concentration in serum or plasma because it is stable [13,17,18]. Threshold values of either 1.2 mmol/L [19,20] or 1.4 mmol/L [6,12,16,17] of BHBA in serum or plasma have been recommended to distinguish between normal and cows suffering from SCK. Measuring serum concentration of BHBA is useful for examining individual cows, evaluating herd health and monitoring feeding management [21]. However, the quantitative determination of BHBA depends on special laboratory equipment and requires blood sampling, centrifugation, freezing of plasma or serum samples, and shipping of frozen samples to the laboratory [21]. These methods are thus neither convenient nor costeffective for routine cowside diagnostic test in early detection of SCK [2]. Several rapid cowside tests have been developed to try and abate these inconveniences, reduce laboratory costs, and provide results immediately after sampling. However, certain limitations in their use exist, such as inadequate sensitivities for milk tests, suboptimal specificities for urine tests and the urine sampling requirements [22,23]. Oetzel [20] reported that cowside tests in blood BHBA had better sensitivity and specificity compared to urine and milk. Thus, blood ketone test methods that quantify BHBA have analytical, clinical and technical advantages over the cowside milk and urine tests in diagnosis or monitoring of SCK [2].

Hand-held meter tests for BHBA have been useful in diagnosis and monitoring of diabetic ketosis in humans because of its high sensitivity and specificity [24,25]. Few studies have shown that hand-held meters are useful in detecting subclinical ketosis of dairy cows [21,2]. Therefore, the objective of this study was to evaluate the performance of a new hand-held meter (TNN, Yicheng, Beijing,China) which is used to diagnose SCK in dairy cows by comparing its accuracy with laboratory methods for determining BHBA concentration in blood, milk and urine samples.

The experiment was conducted in accordance with the established standards outlined in the Guide for the Care and Use of Agriculture Animals in Agriculture Research and Teaching [9]. A total of 298 Holstein cows from three commercial farms (2 in Beijing and 1 in AnHui province, China) were included in this study. Based on clinical examinations, all cows were healthy on the sampling days (d 1 to 58 post-partum). Sampling was carried out from September to October 2012.

The blood samples were collected from the coccygeal vessels using K3-EDTA-coated tubes before morning feeding (6.30 am to 7.30 am). Urine and milk were sampled on the same day. Blood, urine and milk were tested by cowside test immediately after sampling. Milk was tested with BHBA testing strips (Shanghai Bright, China). Urine was tested with URS (Urine Reagent Strip) strips (Nantong Diagnos Biotechnology Co., Ltd. Jiangsu, China). Blood was tested with TNN hand-held meter (Cheng, Beijing, China). The TNN device measures BHBA in whole blood by an enzyme-based electrochemical technique. When the blood sample is applied on the β-ketone test strip, the BHBA in the blood reacts with the chemical in the test strip producing a small electrical current. The current is measured by a sensor and the results are digitally displayed. The amount of current correlates with the amount of BHBA. According to the manufacturer, the results are shown within 30s and amount of BHBA that can be detected is given in the range of 0-6.0 mmol/L. After the BHBA test, the rest of the blood samples were centrifuged at 3000 g for 10 min and plasma was separated, stored in centrifugal tubes and stored at -20 pending further analysis. The plasma concentration of BHBA was measured using a D-3-hydroxybutyrate dehydrogenase enzymatic method (Hitachi 7020 auto-biochemistry, Randox Laboratories Ltd., Co. Antrim, UK). The enzymatic analysis was used as the laboratory reference method for validation purposes.

Data were analyzed using SPSS for Windows (Version 20.0). The significant differences between the means of BHBA in blood measured by the two methods was evaluated by Student’s t-test for paired samples. The correlation coefficients (Pearson) were calculated between BHBA concentrations in plasma measured by the laboratory method and the values obtained by TNN device for the whole blood. Agreement between the two blood testing methods was verified using the Bland-Altman difference plot and Passing-Bablok regression using the laboratory methods as the comparison method. Significance was declared at P ≤ 0.05.

Using plasma concentrations of BHBA as the standard for determination of SCK, the three cowside tests were evaluated by comparing their sensitivities, specificities, positive predictive values and negative predictive values.

In this study, SCK was defined as plasma BHBA concentrations either ≥ 1.2 or ≥ 1.4 mmol/L. Of the 298 cows studied, 13.1 (39/298) and 11.4% (34/298) had SCK of ≥ 1.2 and ≥ 1.4 mmol BHBA/L, respectively in the whole blood measured using the hand-held device. On the other hand, laboratory method reported 11.7 and 10.4% for ≥ 1.2 and ≥ 1.4 mmol BHBA/L, respectively. Majority of SCK occurred within the first 3 wk of lactation, with few cases thereafter.

The means for BHBA concentrations measured using two different methods are shown in Table 1. Plasma concentrations of BHBA ranged from 0.1 to 5.8 mmol/L with the hand-held device and from 0.2 to 5.9 mmol/L using the laboratory method. There were no significant differences between the two methods (P=0.11). Comparison of BHBA data from the hand-held meter and the laboratory method revealed a high correlation between the two methods (r=0.98).

Table 1: Mean ± SD of BHBA concentrations measured using two different methods.

There was a linear relationship between the two methods in determining plasma concentration of BHBA (Figure 1). Examination of the difference plot showed a small positive bias of -0.02 mmol/L and the central 0.95 interval indicating a fair agreement between the applied methods for BHBA measurement (Figure 2). Thirty outliers (4.4%) were detected.

Figure 1: Passing-Bablok’s regression between the laboratory method and the hand-held meter.

Figure 2: Bland-Altman’s different plot of the laboratory method and the hand-held meter. Note: Hand-Lab means the difference between the concentration of BHBA measured by the laboratory method and the concentration of BHBA measured by the hand-held meter.

The test performance of the three cowside tests relative to plasma BHBA ≥ 1.2 mmol/L and ≥ 1.4 mmol/L for detection of SCK is shown in Table 2. Compared to urine and milk tests, using blood sample test had a better accuracy. The sensitivities and specificities of urine and milk tests in measuring BHBA concentration were lower than those obtained by the hand-held meter. The sensitivity and specificity for hand-held meter at the threshold of 1.2 mmol/L BHBA were 91.4 and 97.3%, respectively. The sensitivity of the hand-held meter was higher in blood than both urine (71.4%) and milk (57.1%) tests (Table 2). Similarly, the specificity of hand-held meter was also higher. When the threshold of detection of SCK increased to 1.4 mmol/L BHBA, the sensitivity and specificity of the hand-held meter increased to 96.8 and 98.5%, respectively (Table 2).

Table 2: Performance of 3 cowside tests for detection subclinical ketosis.

The threshold values for BHBA in serum or plasma of cows suffering from SCK has been defined either as 1.2 mmol/L or 1.4 mmol by different researchers [6,14,16,17,19]. The proportion of cows diagnosed with SCK was lower in this study compared to others [2,16,21,23] which showed that SCK was not a serious problem in the farms studied.

The BHBA concentrations were similar between the methods for the blood samples. The results were not consistent with the findings of Voyvoda and Erdogan [2] who reported that hand-held meter gave significantly higher values for plasma BHBA compared to laboratory methods. The correlation coefficient between the BHBA values obtained from the laboratory method compared to those obtained from the hand-held meter was 0.98 indicating that the hand-held method of testing SCK was as effective and accurate as the laboratory method. The results were in agreement with the findings of other researchers who reported that the coefficient of correlation between the laboratory methods and a hand-held meter in measuring plasma concentration for BHBA varied from 0.92 to 0.97 [2,21,26]. The agreement between the two methods was good since the bias was -0.02 which was lower than 0.036 reported by Voyvoda and Erdogan [2]. In the previous reports the percentages of outliers were between 1.3% [2] and 3.0% [2]. The similarity in outliers with those reported by lwersen [2] might be attributed to the number of animals sampled (298 vs. 196 in the current and Iwersen’s studies, respectively). The lower percentage of Voyvoda and Erdogan [2] study could be due to fewer number of animals (78 cows) used. The high correlation and agreement between the two methods supports our hypothesis that the hand-held meter can be used as an alternative to the laboratory method in measuring plasma concentration of BHBA.

The sensitivities and specificities of urine and milk tests in measuring BHBA concentration were lower than those obtained by the hand-held meter using blood samples. This was in agreement with the results of Oetzel [22] who reported that the hand-held meter can be used as a cow-side blood test for detecting SCK because of its higher sensitivity and specificity compared to the available urine and milk cowside tests. The sensitivity and specificity at the theshold of 1.2 mmol/L of plasma BHBA for hand-held meter in the current study were higher than those reported by Voyvoda and Erdogan [2]. At a threshold of 1.4 mmol/L BHBA for SCK, the sensitivity and specificity were in agreement with previous studies in which hand-held meter results ranged from 85 to 100%, and from 97 to 100%, respectively [2,21,27].

The results of this study showed that TNN hand-held meter can be used as a rapid and useful tool for detecting SCK because of its high accuracy in detecting blood BHBA concentration. Furthermore, blood samples were better than milk and urine samples in diagnosis of SCK.

Funding for this experiment was provided by National Nature Science Foundation of China (31372334).