Displaced abomasum is more frequent in high producing cows with more than 80% of cases occuring within 3–4 weeks postpartum  a period that coincides with negative energy balance (NEB) resulting from increased energy needs and reduced dry matter intake. These have been suggested to be a risk factor for LDA . The consensus of opinion has it that increased serum NEFA and BHBA levels, as blood characteristics of NEB cows, are associated with an increased risk of LDA [12–14]. Consistent with the previous studies , we confirmed the strong increase in serum NEFA and BHBA levels in LDA cows. Findings regarding GLU and INS levels in the published literature have been inconsistent in LDA cows. For instance, it has been reported that cows with displaced abomasum had elevated serum GLU and INS levels [5, 14]. The reason why abomasal displacement in these studies led to hyperglycemia is still unknown. Van Winden et al.  found decreased serum INS and GLU levels in cows developing abomasum displacement after 10 days from calving. The state of NEB is characterized by low serum GLU and INS levels along with elevated serum NEFA and BHBA levels . Possible explanations for these opposite results include the uncertain duration of LDA onset at the detection and the additional environmental stress during transportation .
In the present study, the environmental interference was reduced, and serum GLU level in healthy and LDA cows agree with . The lower RQUICKI in LDA cows in our study suggested they experienced a more pronounced degree of insulin resistance. The RQUICKI, an index based on serum NEFA, GLU, and INS, was used to determine insulin sensitivity in both humans [17, 18] and cows [16, 19, 20]. Thus, the RQUICKI can be a suitable candidate for LDA identification.
Metabolism of circulating NEFA occurs mainly in the liver, where they can be completely oxidized for energy, exported as lipoproteins, or partially oxidized into ketone bodies such as BHBA [21, 22]. Enhanced lipid mobilization will result in an overload of NEFA in the liver and further accumulation of ketone bodies, eventually leading to subclinical or clinical ketosis . High serum NEFA or ketone bodies induce hepatocyte damage [24–26]. The activities of ALT, AST, GGT and LDH reflect the integrity of hepatocytes and are often regarded as sensitive indicators of liver injury [27, 28]. Thus, the greater activities of all these enzymes in LDA cows suggested the existence of hepatocytes damage, which is consistent with previous studies [4, 8, 16].
Bilirubin is another sensitive parameter for evaluating functional capacity of the liver. The concentration of serum bilirubin is closely related to liver injury or bile duct abnormality. For example, dilation of the gallbladder duct is often detected in cows with LDA [14, 29]. Thus, the greater serum TBIL, DBIL and IBIL in LDA cows was consistent with previous studies, underscoring the existence of liver damage or bile duct obstruction in cows with LDA . One explanation is that hyperbilirubinemia in LDA cows may be caused by biliary traction due to the change of duodenal position . The level of serum TP can reflect the state of water/salt metabolism and the reserve capacity of the liver. Decreased reserve capacity of the liver often follows liver damage, which can be manifested by a decrease of serum TP and ALB levels. Thus, the lower TP and ALB levels in cows with LDA in the present study were consistent with the literature .
BUN and CREA are the final product of protein metabolism and they are often regarded as indicators of renal function. The greater BUN and CREA in LDA cows was consistent with the findings of de Cardoso et al. . The nephrogenic increase of both happens only when the glomerular filtration rate falls below 50%. We are unaware of previous studies reporting impaired renal function in cows with LDA. Alternatively, the increase of BUN and CREA can also result from increased protein decomposition or dehydration, which is more likely in the present study as judged by haemoconcentration and dehydration in cows with LDA . Overall the present and previous data indicate that liver- or renal-related biomarkers are tightly associated with LDA in dairy cows. Thus, they should be under consideration as serum indicators for early warning.
High amounts of K are lost through milk production (1.4 g K per liter of milk), which is a reason why high-yielding dairy cows usually have low serum K concentrations [31, 32]. Alternatively, K also participates in the metabolism and synthesis of glycogen and protein , and these processes are enhanced in LDA dairy cows undergoing NEB . It has been shown that K and Cl are not transported from the abomasum into the duodenum, but flow back into the forestomach in cows with LDA . In addition to the above factors, reduced food intake in LDA cows, resulting in decreased K intake, should also be taken into consideration.
A strong positive correlation between serum Cl level and K levels in dairy cows with LDA has been reported . Blockage of abomasal emptying in LDA cows will result in accumulation of Cl in the rumen, which impairs the absorption of Cl and leads to alkalemia [35, 36]. The reduction of serum Cl level in LDA cows is also associated with decreased food intake. A lower serum Ca concentration in LDA cows was reported in a previous study . In addition, serum Ca concentration was positively correlated with serum K concentration, both of which were negatively correlated with serum bilirubin . Disequilibrium of milk-production-associated Ca consumption, dietary intake and bone Ca mobilization likely are the main causes of reductions in circulating Ca concentrations in cows with LDA. The lower serum Ca levels in cows with LDA is consistent with the study of Mokhber Dezfouli et al. , supporting the theory of disturbed Ca homeostasis. It is generally accepted that low serum Ca inhibits abomasal motility and promotes the occurrence of LDA in dairy cows . Thus, it is meaningful to verify whether these sensitive ions can be used as indicators for LDA identification and early warning.
The most common diagnostic physical finding of LDA in cows is a pinging sound through simultaneous auscultation and percussion of the abdomen area marked by a line from the tuber coxae to the point of the elbow [5, 30, 39]. The analysis of hematological biomarkers is also regarded as a useful diagnostic method in abdominal disorders of dairy cows because these biomarkers could reflect the conditions of metabolism, stress, injury, and inflammation. These are of great significance in the rapid early risk warning and diagnosis of LDA, especially in large-scale dairy farms. In present study, the ROC method indicated that energy metabolism-related biomarkers including NEFA, INS, and RQUICKI; liver/renal function-related parameters including ALT, AST, GGT, ALB, and CREA; as well as minerals including Ca and Cl are potentially effective for LDA identification. Cut-off points also were identified. For example, serum Ca had the highest AUC (0.94) and together with RQUICKI (0.89), ALT (0.89), INS (0.89), GGT (0.88) and CREA (0.86) can be used as preferred indicators for LDA identification.
Several electronic hand-held devices have become available for easy detection of hematological biomarkers, hence, they represent excellent tools for comparing the correlation between laboratory detected data and hand-held meter measured data [40–42]. In the future, it is likely that more precise hand-held devices will be developed for specific applicability to a variety of hematological biomarkers. Our findings also provide data support for the development of these devices for LDA diagnosis and early warning.