Investigation of Risk Factors of Subclinical Ketosis in Dairy Cows in Central Taiwan Using Ketone Body Assay

酮症 (ketosis) 為泌乳牛所好發的代謝疾病之一，是重要的產媷相關代謝異常疾病 (periparturient disease)。就血液生化檢測結果，當乳牛血液中的酮體含量超過1.4 mM時將之判斷為潛伏性酮症，因為超過此標準值以上的牛隻，有較大的風險演變成臨床性酮症。潛伏性酮症會造成泌乳牛乳汁產量下降並繼發其他與產媷相關疾病，如皺胃異位 (displaced abomasum) 與胎衣滯留 (retained placenta) 等代謝疾病，且會造成泌乳牛每天損失1到4公升的乳汁產量，而使酪農戶蒙受極大的經濟損失。酮體檢驗的黃金標準 (golden standard) 為酵素檢測分析法，儘管現今市面上有諸多用以分析體液中酮體含量的酮體檢驗商業套組，但商業套組價格昂貴，且多半經過長時與長程運送，過程中套組內的藥劑反覆冷凍又解凍，容易造成檢驗品質不佳且操作反應不完全，進而影響實驗的準確性。有鑒於此，本試驗自行開發配製酮體檢測套組的方法。此方法藉由BHBA脫氫酶 (β-Hydroxybutyrate dehydrogenase) 將BHBA (β-hydroxybutyrate) 氧化成酮醋酸的過程中，將NAD+轉變為NADH。接著藉由diaphorase將NADH氧化成NAD+的過程中，將WST-1轉變成WST-1 formazan。利用分光光度計於可見光450 nm的波長下可偵測WST-1 formazan的吸光值，利用此吸光值和標準曲線做比對，可以測得樣品中BHBA的含量。本研究所開發之BHBA酵素檢測分析法可得到標準曲線相關係數為0.9993，分析範圍為0.02 ~ 5.0 mmol/L，分析時間僅需30分鐘。於標準品中添加acetoacetate、lactate及dextrose各三個濃度，檢測結果誤差都不超過上下0.05 mmol/L，顯示本分析法有高度的專一性，不易受到干擾物質影響分析套組當中的酵素成分。靈敏度為3 μmol/L，可對於低BHBA含量之血液與體液進行分析。由精密度得知本分析法在分析內與分析間操作下CV都小於10%以下，即為檢測值之間相當接近。準確度結果可以得到平均97.6%的回收率，相當接近於100%。本研究所開發之分析法可以得到相當精準的檢測值，藉由此法可以精準的檢測乳牛血液以及體液當中BHBA的含量。 本研究調查期間為2011年10月到2011年12月，採集台灣中部地區之八個牧場，總共採樣頭數為228頭，總計採集497次測量數據。選用年齡7歲以內、懷孕過1 ~ 6次之荷蘭牛以及娟姍牛，乳汁平均產量為每頭每天21.0 ~ 26.0 公斤。採樣時間點為預產期前1週、分娩後1週到分娩後4週。本試驗中測量數據中的酮體含量範圍為0.31 ~ 3.73 mmol/L。調查發現台灣中部地區發生潛伏性酮症的發生率為23%。調查期間任何時間點有發生過潛伏性酮症的牛隻，其分娩後3 ~ 4週 (1.99 ± 0.81 mmol/L) 會較分娩後1 ~ 2週 (1.66 ± 0.75 mmol/L) 血液中酮體含量平均值來的高。表示有潛伏性酮症發生時，酮體的含量是會有進程的，一旦發生而沒有治療，就會有持續升高的趨勢。計算風險因子的勝算比，發現分娩為潛伏性酮症之風險因子 (OR: 15.282, 95% CI: 4.742 – 49.250)，其他如品種中的娟姍牛相對於荷蘭牛 (OR: 1.213, 95% CI: 0.423 – 3.481)、牧場規模中的大場相對於小場 (OR: 1.151, 95% CI: 0.653 – 2.027) 及飼養密度中的低密度相對於高密度 (OR: 1.046, 95% CI: 0.630 – 1.737)，皆未構成統計上風險因子的成立要件。另外我們比較管理模式不同是否會造成前後兩週血液中酮體含量的差異，將八個調查的場別，區分為「勤於管理」的四個場，其酪農勤於更換並定時補充新鮮的草料。除了原本一天兩次的飼料放置時間之外，管理人員於其間會定時觀察草料的保存狀況與存量。另外四個牧場，僅做定時的飼料放置的「基礎管理」，管理人員鮮少觀察牛隻吃料情形且並沒有將陳舊草料清除與更換。研究結果發現，勤於管理與基礎管理的牧場，其泌乳牛於分娩前1週的平均血液中酮體含量分別是0.78 ± 0.01 mmol/L與0.94 ± 0.25 mmol/L，於t test統計結果沒有顯著差異。而在分娩後1 ~ 2週，我們發現勤於管理的牧場泌乳牛血液中酮體含量的平均值為0.92 ± 0.03 mmol/L，而基礎管理的牧場泌乳牛血液中酮體含量的平均值為1.44 ± 0.29 mmol/L，於t test統計結果有顯著差異 (p < 0.05)。顯示飼養管理亦為台灣現今潛伏性酮症之風險因子之一。 Ketosis is an important metabolic disease in dairy cows during early lactation and is associated with losses in milk production and several other periparturient diseases. Cows are defined subclinical ketosis when their concentration of blood BHBA (β-hydroxybutyrate) was over 1.4 mmol/L. Subclinical and clinical ketosis differ in prevalence, course of development, and clinical findings. Cows were at significantly higher risk of developing clinical ketosis than those cows with their BHBA less than 1.4 mmol/L. Subclinical ketosis in dairy cows can lead to economic losses through decreased milk production, decreased reproductive performance, increased risk of displaced abomasum, and increased risk of clinical ketosis. When subclinical ketosis is diagnosed, the cow can be treated and monitored during the following days. Therefore, determination of BHBA in blood samples is an important tool in the diagnosis of ketosis in dairy cows. We developed an assay system to determine BHBA in our lab. This method is based on the oxidation of BHBA to acetoacetate by the enzyme s-hydroxybutyrate dehydrogenase. During this reaction, an equimolar amount of nicotinamide adenine dinucleotide (NAD+) is reduced to nicotine adenine dinucleotide (NADH). During the oxidation of NADH to NAD+ by diaphorase, WST-1 was reduced to WST-1 formaiton. The WST-1 fromation absorbs light at 450 nm, and the increase in absorbance is directly proportional to the BHBA concentration in the sample. In this study, the BHBA assay described gave a linear standard curve between 0.02~5.0 mmol/L. We evaluated the specificity of our assays with respect to acetoacetone, lactose and glucose. Sensitivity is 3 μmol/L. All specimens were analyzed within the stability periods in within-run and between-run procession. It’s highly precision in our assay. To assess the accuracy of our assay, we added known amounts of BHBA to plasma samples and we got the analytical recovery, 97.6%. A total of 228 Holstein and Jersey cows (under 7 years old) from eight commercial dairy herds in central Taiwan were included in the study. The cows had one to six lactation, with milk yield ranging from 21.0~26.0 kg/day during the study. Sampling was carried out during the winter housing period from October 2011 until December 2011. The samples were taken during 1 week pre parturition and 1~4 weeks post parturition. The analytic measurement (497 measurements) range is given as 0.31~3.73 mmol/L for BHBA. Average incidence rate of subclinical ketosis in dairy herds in central Taiwan is 23%. Ketone body titer in dairy cows, which have occurred subclinical ketosis, of 1~2 weeks and 3~4 weeks post parturition were 1.66 ± 0.75 mmol/L and 1.99 ± 0.81 mmol/L, respectively. It is indicated that during subclinical ketosis occurring in cows, the ketone body titer will gradually increase. If no treatment has been performed, the ketone body titer will go higher. Parturition has been regarded as a risk factor of subclinical ketosis (OR: 15.282, 95% CI: 4.742-49.250). However, breed, farm size and herd density in this study has not been considered as risk factors of subclinical ketosis. In this report, management factors affect disease incidence. Ketone body titer in dairy cows at 1 week prior of parturition in diligent management and idle management farms were 0.78 ± 0.01 and 0.94 ± 0.25 mmol/L, respectively. The titer of 1~2 weeks post parturition increased to 0.92 ± 0.03 and 1.44 ± 0.29 mmol/L in diligent management and idle management farms. A statistically significant difference was found between them (p < 0.05), therefore, type of management factors has been recognized as an important risk factor of subclinical ketosis in Taiwan