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.

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.

Blood serum stability limit and maximum storage time of bovine samples

Measuring metabolic parameters in the blood has been an indispensable tool for assessing the productive and health status of dairy cows for more than 100 years. The values of laboratory parameters depend on various preanalytical, analytical and postanalytical factors. The most important preanalytical factors are sample transport time and temperature, hemolysis, anticoagulant type, and sample volume. Preanalytical factors can lead to reduced stability of the analyte in the sample, which changes their concentration. Loss of stability changes the time of storage and manipulation of the sample, which determines the criteria for its acceptance or rejection. The two stability indicators are stability limit and maximum permissible instability. A stability limit (SL) is defined as the period of time in which a property variation does not exceed a maximum permissible instability (MPI). The aim of this study was to determine the SL and MPI for each analyte in the blood serum of cows and to determine whether SL differs in the function of the presence of preanalytical errors in the blood sample. Three hundred samples of dairy cow origin in different periods of lactation participated in this research. They were classified into 6 groups of 50 samples: according to the time from sampling to processing in the laboratory (0-4 h, 4-8 h and over 8 h; all transported on dry ice, protected from environmental factors, without preanalytical errors) and according to the presence of preanalytical errors (group with hemolysis, a group transported at ambient temperature and a group with a small sample volume). Each sample was aliquoted in two portions. One portion was left at +4°C and tested once a day for 6 days of sample storage, and the second portion, placed at -20°C, was tested once a month for 6 months. The MPI had a value ranging from 1.51 to 8.4. Metabolic profile analytes with lower MPI values (1.51-3.22) were albumin (ALB), total protein (TPROT), UREA, glucose (GLU), calcium (Ca), and phosphorus (P). Higher MPI values (5.1-8.4) were found for nonesterified fatty acids (NEFA), beta-hydroxybutirate (BHB), cholesterol (CHOL), triglycerides (TGC), total bilirubin (TBIL) and aspartat aminotransferase (AST). For most parameters, we can conclude that their PD% changed faster in storage conditions at +4°C compared to the regime of -20°C. The largest number of biochemical analytes in bovine blood serum shows preserved stability in the first 6 days at +4°C or 6 months at -20°C if transported to the laboratory within 8 h after sampling in ideal conditions and without the action of preanalytical errors. Prolonged transport under ideal conditions or the existence of preanalytical errors such as transport at room temperature, hemolysis or small sample volume shorten the stability of the ALB, NEFA, GLU, UREA and P. Concentration of all analytes decreases during the stability test except for UREA, NEFA, BHB and for CHOL and TGC in some groups. Variations in parameters such as BHB, NEFA, TBIL, AST, and Ca have shown potential clinical significance. At storage conditions at +4°C, clinically significant variations at at least one measurement point were found for AST (7.5% of samples), BHB (6.1% of samples), NEFA (9.9% of samples) and for TBIL (in 7% of samples). This study can help define acceptable delay times and storage conditions for bovine blood samples, which is of great importance because in working with farm animals it is often not possible to take samples in a short time and deliver them to the laboratory, and samples are often burdened with certain preanalytical errors with limited possibilities of re-sampling.(AU)

[Impact of oxygen toxic action on the erythrocyte membrane and possibility of estimating central nervous system function disturbances].

UNLABELLED: BACKGROUND/AIM; Prolonged exposure to hyperbaric oxygen leads to changes of erythrocytes shape as a consequence of toxic effects of oxygen on the erythrocyte membrane. The aim of this study was to examine the association between occurance of pathological forms of erythrocytes at different time from the start of hyperbaric oxygenation and the moment of convulsions occurrence, an interrelationship of different pathological forms of erythrocytes during exposure to hyperbaric oxygenation, as well as the correlation between the presence of ruptured erythrocytes and function of central nervous system (CNS) after completion of hyperbaric treatment. METHODS: Sixty laboratory mice, Mus musculus, were exposed to the wholly-oxygen pressure of 3.5 absolute atmospheres (ATA). Blood was collected at the 32nd, 34th, 36th, 38th and 40th minutes after the exposure to oxygen. Pathological forms of erythrocytes were examined by electron microscopy. A moment of convulsions occurrence was registered in all animals. After decompression neurological examinations of experimental animals were perfomed. The Pearson's coefficient of correlation, and linear regression equations for the parameters outlined in the aim of the study were calculated. RESULTS: Hyperbaric oxygen caused damages of erythrocytes at the 34th minute after beginning of the treatment. Various forms of abnormal red blood cells occured, and immediately before the occurrence of irreversible changes (erythrocyte membrane rupture) echinocyte shape was dominated. A significant correlation between the number of damaged red blood cells at 34th minute and their number at the 36th, 38th and 40th minute was found. Convulsions were diagnosed significantly earlier in mice with a greater number of damaged red blood cells (p < 0.01). There was a negative correlation between the number of irreversiblly damaged red blood cells (ruptured) at the 40th minute and neurological score in the studied animals (p < 0.05). CONCLUSION: The analysis of altered erythrocytes during hyperbaric oxygenation could predict a moment of seizures occurrence, and therefore the duration of the therapy with hyperbaric oxygen. Ehinocytes indicate impending rupture of red blood cells and a possible occurrence of seizures. An increased number of ruptured red blood cells may also even indicate the potential burden of CNS after cessation of hyperbaric oxygenation.