Background: 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.Materials, Methods & Results: 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 1.55 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 (5.1-8.3) 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 analytesdecreasesduring 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).Discussion: 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.
Abstract:The aim of this study is to determine correlation between change in milk production and metabolic adaptation of cows during heat-stress. Experiment included 30 Holstein-Frisian cows. Cows exposed to heat-stress, which is expressed in high THI index, have lower milk production, higher concentration of insulin and lover concentrations of glucose and NEFA. Indexes of insulin resistance-RQUICKI, insulin: glucose and insulin: NEFA relations are higher in cows under heat stress. Grouping cows in accordance to reduced milk yield have showed that trend of reduction of glucose and NEFA levels, followed by increased insulin level, higher RQUICKI, greater insulin: glucose and insulin: NEFA ratios, started and increased during the reduction in milk production. Among this, significant correlation was founded between these parameters and milk yield. Significant correlation was also noted between those parameters. Change in metabolic value is of great importance for prediction of cows that would have decreased milk yield (above 18%). With almost 90% certainty cows with great reduction in milk yield can be detected. These cows have had following relative changes in values of metabolic parameters during heat-stress compared to thermo-neutral period: insulin increased for ≥12.5%, reduction in NEFA values for ≤14.1%, reduction in glucose for 21.5%, RQUICKI index increased for ≥9.6%, insulin: NEFA ratio increased for ≥20.1% and insulin: glucose for ≥20.3%. Dynamical changes in metabolites and insulin resistance Received 12 October 2017 Accepted 19 October 2017Acta Agriculturae Serbica, Vol. XXII, 44(2017); 123-131 124 values have great influence on milk yield in cows under heat stress. Decreasing in glucoses followed by increased insulin level and increased insulin sensitivity indicate that glucose is transferred from udder to other tissues which can cause decreased milk production.
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.
Peripartal metabolic stress is characterized by increased lipid mobilization, when non-esterified fatty acids (NEFA) are increased, as well as by increased ketogenesis, when the concentration of beta-hydroxybutyrate (BHB) is increased. NEFA are metabolized in all tissues but the main organ is the liver. Possible processes are: a) complete oxidation of NEFA, b) partial oxidation and synthesis of ketone bodies (BHB), c) development of triglycerides from NEFA that can be transported or stay in the liver when fatty liver is apparent. Moreover, increased lipolysis and ketogenesis can cause oxidative stress because concentrations of MDA and/or TBARS are positively correlated with NEFA and BHB concentrations. The increase in NEFA during the peripartal period affects the cellular immunologic response by changing intracellular signals, gene expressing control, activation of transcriptional factors, apoptosis induction and by modifying mediators of lipid production. Increased proportion of cows with high NEFA and BHB concentrations in the herd can cause reduced milk yield at the end of a standard 305-day lactation. NEFA concentrations can be related to postpartal ovarian activity, especially given that blood NEFA concentrations represent NEFA concentrations in the ovarian follicular fluid. Cows on farms with lower scores of animal welfare and nutrition have higher concentrations of cortisol, NEFA, BHB, bilirubin, glucose and urea. NEFA and BHB concentrations in early lactation can be used for estimating metabolic adaptation in the first 8 weeks after calving. For the estimation of metabolic adaptation, increased lipolysis has a greater significance than decreased anabolic parameters.
The aim of this study is to determine value of temperature-humidity index (THI) in period of 2005-2016. in Vojvodina and their correlations with milk production and temperature of body surface of animals. THI index is calculated according to the following formula THI=(1,8×Temperature)-(1-Relative Humidity) × (Temperature-14,3)+32. Temperature and humidity data were collected directly from Hydrometeorological Institute of Serbia. Average THI values were calculated for every month for every year from 2005 to 2016. Measuring spots were cities Vršac, Zrenjanin, Kikinda, Palić, Sombor, Rimski Šančevi and Sremska Mitrovica. Temperature and humidity are noted at 14 h , so they represented maximal average values of THI index. Trend of THI values was represented for each day in every month in years from 2005. to 2016. Average production of drinking milk for year of 2016 was calculated. This year was chosen because is the last year of research and represents cumulative effect of heat stress in cows. Data were collected from Statistic Institute of R. Serbia. Thermo-vision camera was used for determining skin temperature of udder and skin in eye region. Results were showed that temperature ranged from 40.3 in January to 77.5 in July. Average maximal THI value in period of 2005-2016 showed that heat stress was presented in May, June, July and August. Drinking milk production was the greatest in January. After that it started dropping. The lowest production of drinking milk was noted in August with an increase afterwards till December. Body surface temperature was ranged from 34.1 to 38.5ºC. The highest body temperature was measured at August and the lowest in winter time. Significant negative correlation between ten-year average THI value and milk production was noted. Positive correlation was noted between THI and body temperature. From 2005 to 2016 positive linear trend of THI values that were measured in hottest period of day was noted. In every month statistically significant linear trend of rising THI value was founded, except in January, October and November. Extremely hot summers in Serbia appear in every five years when cows are affected the most. Showed data indicated global warming and climate changes which affect biological adaptation of cows.
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