Analytical and Clinical Interference of Sample Hemolysis in Evaluating Blood Biochemical and Endocrine Parameters in Cows.

Hemolysis is a common cause of errors in laboratory tests as it affects blood parameters and leads to a positive or negative bias. This study aims to examine the relationship between the level of hemolysis (expressed as cell-free hemoglobin concentration, g/L) and the variability of metabolic and endocrine parameters and to determine the threshold level of hemolysis that causes an analytically and clinically significant bias for the twenty most frequently examined blood parameters in cows. Paired blood samples of 10 mL each were obtained from 30 cows. One was subjected to mechanical trauma and plasma was extracted directly from the other. Hemolyzed and non-hemolyzed samples from the same animal were mixed to obtain final samples with cell-free hemoglobin concentrations of 0, 1, 2, 4, 6, 8, and 10 g/L. Metabolic and endocrine parameters were measured in the samples and their deviation and the linear equation between the level of hemolysis and the deviation were determined. The following threshold values of hemolysis were determined, which correspond to the acceptable analytical (lower value) and clinical (upper value) levels of parameter variability: BHB 0.96 and 4.81; NEFA 0.39 and 3.31; GLU 0.38 and 3.90; ALB 1.12 and 6.11; TPROT 1.40 and 6.80; UREA 6.62 and 20.1; TBIL 0.75 and 5.65; AST 0.11 and 2.18; GGT 1.71 and 8.90, LDH 0.01 and 0.11, ALP 0.97 and 2.95; TGC 1.56 and 15.5; CHOL 1.29 and 8.56; Ca 5.68 and 25.7; P 0.57 and 8.43; Mg 1.10 and 8.47; INS 1.15 and 3.89; T3 8.19 and 15.6; T4 8.97 and 18.5; and CORT 2.78 and 11.22 g/L cell-free hemoglobin. Three decision levels are available for each metabolic and endocrine parameter: if hemolysis is below the lower (analytical) threshold value, results can be reported without restriction; if hemolysis is between the lower and upper thresholds, the results can be issued with guidance in the form of corrective linear equations; and if hemolysis is above the upper (clinical) threshold, the results and sample must be discarded. This method contributes to an optimal approach to hemolysis interference with metabolic profile parameters in blood samples from cows.

Influence of Heat Stress on Body Temperatures Measured by Infrared Thermography, Blood Metabolic Parameters and Its Correlation in Sheep.

The aim of this research is to examine the influence of heat stress (HS) on body temperature (BT) measured rectally (RT) or by infrared thermography (IRT) of the nose (NT), eye (ET), leg (LT) and abdominal (AT) regions in intensively and extensively breed sheep and to detect a correlation between body temperature and metabolic response in sheep. A total of 33 Wurttemberg × Sjenica Pramenka sheep breeds were examined, 17 ewes were from outdoors and 16 were from indoor housing systems during three experimental periods (thermoneutral period, severe HS and moderate HS). Sheep under HS have a higher BT, and the magnitude of BT measured by infrared thermography (IRT) was higher than RT. LT and AT showed positive linear correlations with the temperature-humidity index (THI), while other ways of measuring BT did not give statistically significant correlations. Sheep under HS showed higher cortisol, insulin, total protein, albumin, urea, creatinine, bilirubin, aspartate aminotransferase, alanine aminotransferase, gamma-glutamyl transferase, alkaline phosphatase, lactate dehydrogenase, creatine kinase and index of insulin resistance, with lower values of triiodothyronine (T3), thyroxine (T4), non-esterified fatty acids, beta-hydroxybutyrate (BHB), glucose, calcium, inorganic phosphates, magnesium and cholesterol. BT and metabolic response were different in the function of the housing method of sheep. LT and AT showed a significant correlation with almost all blood parameters, and the strongest connections were made with T3, T4, BHB and the revised quantitative insulin sensitivity check index of insulin resistance. The abdomen and legs are good thermal windows because LT and AT are good summative responses to external ambient THI and internal metabolic changes in sheep under heat stress.

The Influence of Energy Balance, Lipolysis and Ketogenesis on Metabolic Adaptation in Cows Milked Twice and Three Times Daily.

Increasing milk production requires increasing milking frequency (MF) from two times daily (2X) to three (3X) or more. High milk production leads to negative energy balance (NEB) and homeorhesis, characterized by lipolysis, ketogenesis, and endocrinological changes. The relationship among energy balance (EB), lipolysis, and ketogenesis with endocrine and metabolic parameters in blood of cows milked 2X and 3X daily was studied. Holstein Friesian cows milked 2X (n = 45) and 3X (n = 45) were analyzed, with approximately 50% of cows in each group in positive EB (PEB) and 50% in NEB. After determining EB, blood samples were collected from all cows and blood serum was analyzed for non-esterified fatty acids (NEFA), beta-hydroxybutyrate (BHB), glucose (GLU), cholesterol (CHOL), triglycerides (TGC), total bilirubin (TBIL), aspartate aminotransferase (AST), gamma-glutamyl transferase (GGT), calcium (Ca), inorganic phosphate (P), total protein (TPROT), albumin (ALB), urea, insulin (INS), T3, T4, and cortisol (CORT), and the RQUICKIBHB index of insulin resistance was calculated. Cows milked 3X in NEB represent a special cluster that partially overlaps with cows milked 2X in NEB and has no contact points with cows in PEB. Cows milked 3X had higher levels of NEFA, BHB, AST, GGT, TBIL, and CORT and lower levels of GLU, Ca, INS, and T4. Cows milked 3X in NEB had higher levels of NEFA, BHB, AST, GGT, TBIL, and CORT and lower levels of GLU, CHOL, TGC, TPROT, P, INS, RQUICKIBHB, and T3 compared with cows milked 2X in NEB and cows in PEB. In cows milked 3X, lipolysis and ketogenesis were much more prominent, and EB levels were lower, implying a pronounced shift in homeorhesis. Metabolic and endocrinology parameters were determined mainly by the values of EB, NEFA, and BHB in cows milked 3X in NEB compared with other categories of cows. The results confirm the peculiarity of metabolic adaptation in cows with increased MF, characterized not only by differences in the concentration of metabolites but also in their interactions.

Relationships between Milk and Blood Biochemical Parameters and Metabolic Status in Dairy Cows during Lactation.

This study aimed to determine blood and milk metabolic parameters and their correlations for the purpose of evaluating metabolic status in dairy cows. Blood and milk samples were collected from 100 Holstein dairy cows during morning milking. The cows were allocated to four groups according to the production period, including cows in early (n = 18), full (n = 26), mid (n = 25) and late (n = 31) lactation. The value of non-esterified fatty acids (NEFA), ß-hydroxybutyrate (BHB), glucose, triglycerides (TG), total cholesterol (TChol), total protein (TP), albumin, globulin, urea, total bilirubin (TBil), aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), gamma-glutamyl transferase (GGT), and lactate dexydrogenase (LDH) in the blood were determined. The following milk parameters were measured: fat, protein, lactose, urea, AST, ALT, ALP, GGT, LDH and BHB. Blood serum NEFA, BHB, TBil, AST, ALT, ALP and LDH were higher in early lactation cows, whereas glucose, TP, globulin and urea levels were significantly lower in early lactation cows. Milk fat and lactose levels were lower in early lactation cows, whereas milk protein and the activities of AST, ALT, ALP and LDH in milk were highly greater in early lactation cows. Milk fat was positively correlated with glucose, TP and TG, and negatively correlated with BHB, NEFA, TBil, ALT, LDH and ALP levels in the blood. Enzyme activities in milk were positively correlated with those in blood and with blood NEFA, BHB and TBil levels, and negatively correlated with blood glucose, TChol and TG. A significant positive correlation existed between blood and milk BHB values. Many correlations showed the same slope during all lactation periods. In conclusion, similar changes in blood and milk metabolite concentration during lactation and milk to blood correlations confirm that milk has great potential in predicting of blood metabolites and metabolic status of cows.

Niacin Status Indicators and Their Relationship with Metabolic Parameters in Dairy Cows during Early Lactation.

Previous experimental models on cows have examined the difference in the metabolic adaptation in cows after niacin administration, without identifying the most important mediators between niacin administration and its biological effects, namely active forms of niacin. All tissues in the body convert absorbed niacin into its main metabolically active form, the coenzyme nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP). The aim of this study was to determine the influence of niacin administration in periparturient period on NAD, NADP and the NAD:NADP ratio and to determine relationship between these indicators of an active form of niacin with metabolic parameters in cow blood. The study included 90 healthy cows: 45 cows receiving niacin and 45 cows were negative control. The niacin group was treated with nicotinic acid for two weeks before, as well as two weeks after parturition. Nicotinic acid was applied per os with feed. In cows receiving niacin, there was a significantly higher concentration of NAD and NADP, but the NAD:NADP ratio did not differ compared with control. All three indicators were able to separate cows who received and who did not receive additional niacin. NAD and NADP are good indicators of the availability of niacin from additional sources. The NAD:NADP ratio is a good indicator of the biological effect of applied niacin on metabolites in cows due to its correlation with a number of metabolites: positive correlation with glucose, insulin, glucose to insulin ratio and the revised quantitative insulin sensitivity check index (RQUICKIBHB) of insulin resistance, triglycerides and cholesterol, and a negative correlation with nonesterified fatty acid (NEFA), beta hydroxybutyrate (BHB), gamma-glutamyltranspherase (GGT) and urea in cows receiving niacin. The same amount of added niacin in feed can produce different concentrations of NAD, NADP and NAD:NADP in the blood, and this was not related to their concentration before the addition of niacin. The change in the concentration of the active form of niacin (NAD, NADP and NAD:NADP) further correlates with the concentration of metabolic parameters, which indicates that the intensity of the biological effect of additional niacin can be accurately determined only if we know the concentrations of its active forms in blood. Under basal conditions (without additional niacin), active forms of niacin that already exist in the blood do not show significant correlations with metabolic parameters.

The Correlation between Extracellular Heat Shock Protein 70 and Lipid Metabolism in a Ruminant Model.

Metabolic stress in early lactation cows is characterized by lipolysis, ketogenesis, insulin resistance and inflammation because of negative energy balance and increased use of lipids for energy needs. In this study the relationship between lipid metabolite, lipid-based insulin resistance, and hepatocyte functionality indexes and tumor necrosis factor alpha (TNF-α) with extracellular heat shock protein 70 (eHsp70) was investigated. The experiment included 50 cows and all parameters were measured in blood serum. In cows with a more pronounced negative energy balance, the following was determined: a higher concentration of eHsp70, TNF-α, non-esterified fatty acid (NEFA), beta-hydroxybutyrate (BHB), NEFA to insulin and NEFA to cholesterol ratio and lower concentration of cholesterol, very low-density lipoproteins (VLDL), low density lipoproteins (LDL) and liver functionality index (LFI). The eHsp70 correlated negatively with the values of cholesterol, VLDL, LDL, and triglycerides, while correlated positively with the level of NEFA and BHB. A higher concentration of eHsp70 suggests the development of fatty liver (due to a higher NEFA to cholesterol ratio and lower LFI) and insulin resistance (due to a lower revised quantitative insulin sensitivity check index RQUICKI-BHB and higher NEFA to insulin ratio). The eHsp70 correlated positively with TNF-α. Both TNF-α and eHsp70 correlated similarly to lipid metabolites. In cows with high eHsp70 and TNF-α values we found higher concentrations of NEFA, BHB, NEFA to insulin and NEFA to cholesterol ratio and a lower concentration of triglycerides and VLDL cholesterol compared to cows that had only high TNF-α values. Based on the positive correlation between eHsp70 and TNF-α, their similar relations, and the additional effect of eHsp70 (high TNF-α + eHsp70 values) on lipid metabolites we conclude that eHsp70 has pro-inflammatory effects implicating lipolysis, fatty liver, and fat tissue insulin resistance.