| Dairy cows ketosis is a metabolic diseases that carbohydrates and fat metabolic disorder caused by a systemic dysfunction, It is one of major diseases which are harmful to dairy industry. The disease usually take place in high-yielding cow which day milk production are more than 30 Kg, the main pathology is characterized by low blood sugar, high blood ketone. Because of the introduction of high-yielding varieties, cultivation, high-energy feed, the development, use, large-scale breeding and improvement in the level and many other reasons, making the incidence of the disease has long been high. Ketosis in dairy cattle in our country the incidence of lactational cow account to15%-30%, 5.0% in American, 15.4% in Iran,14.69%in India, while has up to 43.1% in Japan. The disease cause huge economic losses to the dairy industry, is an important factor that constrain the development of dairy industry. It is reported that a clinical treatment of ketosis in dairy cattle only drugs and the decline in milk production losses, which amounts to 145 American dollars. Ketosis in dairy cattle caused not only the economic loss but also displaced abomasums, retained placenta, decreasing of reproductive performance, perinatal paralysis et al. Therefore, ketosis in dairy cattle have been classified as the most important dairy cattle diseases to study in many countries.Ketosis in dairy cattle according to their availability of obvious clinical symptoms can be classified into clinical and subclinical ketosis. Clinical ketosis in accordance with its obvious clinical symptoms (such as loss of appetite, constipation, fecal mucus overlying have mental gloomy or mental excitement, milk yield decreased, exhaled gas has ketone flavor) is not difficult to make a diagnosis, and is relative rare in clinical practice. Subclinical ketosis without obvious clinical symptoms, it is reported that each of the dairy cow with subclinical ketosis caused economic losses of about 80 American dollars. Ketones are the material produced by partial oxidation of liver nonesterified fatty acids (NEFA).Animals includes three ketones in vivo, acetone, acetoacetate andβ-hydroxybutyric acid (BHBA). Detection of ketones are diagnostic indicators of subclinical ketosis in cows. BHBA relatively stable, it is often used as a diagnostic indicator of ketosis, and acetoacetate can decompose into acetone, and acetone with volatility. Ketone detection including blood ketone detection, urine ketone testing and milk ketone body detection. Milk ketone body detection is easy to implement, cost far less than the blood ketone body detection; It is reported that the milk ketone body and blood ketone body content has strong correlation, milk ketone tests can be used to diagnose cows subclinical ketosis.At present, blood BHBA>1200μmmol/L is used as the diagnostic criteria for the dairy cows subclinical disease in the international arena. Reported diagnosis methods for subclinical ketosis include ketone powder, urine analysis strip, milk BHBA test strip and ketone tablet et al.As the milk samples are easier collected than urine and blood collection, it is a very convenient way for routine monitoring subclinical ketosis or diagnosis of subclinical ketosis in dairy cows. Study showed that detection sensitivity and specificity of the milk BHBA test strip were the best. Foreign milk BHBA test strip includes Ketolac strip, it is expensive, Ketolac strip can not be bought in domestic market so far also and is still at the blank stage. In this study, we first developed a milk BHBA detect strip strip for the diagnosis of subclinical ketosis in dairy cattle at the domestic, and carried out a preliminary clinical application research on the strip.Firstly, we set up the reaction principle of strip for detection of milk BHBA, the main reagent composition is dihydrouracil dehydrogenase (NAD), nitroblue tetrazolium (NBT), BHBA dehydr- odehydrogenase and diaphorase. Through comparison test between three commonly used buffer, sodium-hydrochloric acid buffer, Tris-hydrochloric acid buffer and borax-hydrochloric acid buffer, which the range of pH was 7.0-9.0, and determined to use the Tris-hydrochloride buffer as buffer of the test strip. The results of different concentration and pH experiments showed that optimum concentration and pH of Tris-hydrochloric acid buffer solution for preparation strip reaction solution were 0.01 mol/L and 8.5 respectively. Determining the sensitizer was Tween-20 based on different sensitizer test, and its optimum concentration was 0.1%. The study of effect of several metal ions on the reaction results showed that K+, Na+, NH4+, Mn2+, Zn2+, Ca2+, Ba2+ and Mg2+ inhibited differently degrees the reaction, the inhibition order was K+1200μmol/L as diagnostic criteria for subclinical ketosis, and analyzing the sensitivity and specificity of detection of self-test strip, congeneric foreign strip, ketones powder and urine analysis strip. The results showed that the sensitivity and specificity of milk test strip were higher than ketone powder and urine analysis strip; the sensitivity and specificity of self-made strip test reached to the level of foreign congeneric strip. The correlation of BHBA milk with blood BHBA was 63%, which maybe the main influencing factor for the sensitivity and specificity of self-made milk BHBA test strip and Ketolac strip, belonging to the limitations of detection methods.Self-made milk BHBA test strip is easy-to-use and inexpensive, detection performance of it reaches to the same level of foreign congeneric strip, can be applied to diagnose subclinical ketosis of dairy cows.
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