Ketosis in dairy cows during early lactation - detection in pooled blood serum samples

Background: Ketosis is the most important metabolic disease with prevalence from 15 to 45%. Ketosis is diagnosed using a metabolic profile. Due to the high prevalence, it is necessary to determine a large number of metabolic profiles within farm, which represents an additional cost, so the implementation of pooled serum in assessing the metabolic status of cows was examined. The aim of this study was to validate and evaluate the influence of the relative position (Z-score) of the value of pooled sample metabolic parameters within the known reference value of healthy cows in the detection of ketosis in herd during early lactation. Materials, Methods & Results: The experiment has been carried out using blood samples collected by puncture of coccygeal vein from 50 ketotic and 50 healthy cows. Laboratory analysis includes determination of beta-hydroxybutyrate-BHB, non-esterified fatty acids-NEFA, cholesterol-CHOL, triglycerides-TGC, glucose-GLU, albumin-ALB, total protein-TPROT, UREA, Ca, P, total bilirubin-TBIL and aspartat aminotransferase-AST. The pooled serum was made from 10 individual samples originating from 10 different cows. A serum aliquot of 0.1 mL was taken from each sample, and a 1 mL volume of pooled serum was finally formed. Three types of serum pools were made: 1) 30 pooled sample were from ketosis; 2) 30 pooled sample were from healthy cows and 3) 60 pooled samples containing mixed sera of healthy cows and cows with ketosis were made as follows: 10 pools contain 10% to 60% of ketotic cows (1/10 to 6/10 samples). Statistical analysis includes: a) difference in metabolite concentration and Z-score in pooled sample and arithmetic mean individual sample in healthy and ketotic cow, b) correlation between Z-score of pooled sample and arithmetic mean of individual sample, c) ability of Z-score of metabolite to divide ketotic from healthy cow, d) correlation between Z-score and % of ketotic cow in pooled sample; and e) calculation of 95%CI of pooled sample Z-scores for each % of ketotic cow in pools. Z-score and all analysis were calculated for each metabolic parameter. The results of the study show that the mean values and Zscores of the pool and the calculated average value of the individual samples participating in that pool differ significantly in healthy cows and cows in ketosis, except for TPROT and Ca. A higher value and a higher Z-score were found for BHB, NEFA, UREA, TBIL and AST, and a lower value and a lower Z-score for TGC, CHOL, GLU, ALB and P in ketotic cows compared to healthy cows. The value of the Z-score of the pooled sample and the calculated mean values of individual samples participating in the pool are highly correlated with each other (coefficient of determination over 99%). Z-score of metabolites in the pooled sample can be used to distinguish healthy from ketotic cows (ROC AUC= 0.711 to 0.989), except for TPROT and Ca. The Z-score value of the pooled sample shows a linear correlation with the percentage of ketotic cows in the pool and the reference ranges of Z-scores change significantly as a function of the percentage of ketosis cows. Discussion: Modern research on the metabolic profile in cows requires obtaining a large amount of information from as few samples as possible. The advantages of using the Z-score are reflected in the following: this score does not depend on the absolute value of the metabolite, but on the position within the known population reference value, Z-score of sample and the arithmetic mean of individual samples included in the same pool are almost identical, the Z-score of these 2 groups of results is ideally correlated, the Z-score significantly correlates with the % of ketosis samples in the pooled sample. The use of pooled sample Z-score can be a useful in a herd level assessment of metabolic status and detection of ketosis as most important metabolic disease in dairy cows.

Genotype profiles of Actinobacillus pleuropneumoniae isolated from pigs in Vojvodina, Serbia

Background: Actinobacillus pleuropneumoniae (A. pleuropneumoniae) is one of the most important bacterial respiratory pathogens. It is the only etiological agent of porcine pleuropneumonia (PPP) or it appears as a secondary bacterial infection in the swine respiratory disease complex (PRDC). In Serbia, apart from the identification of serotype 2, no tests have been performed to establish the presence of other A. pleuropneumoniae serotypes in the pig population. The aim of this study was to perform genotyping of A. pleuropneumoniae isolates originating from pig farms in Serbia by apx genes and using multiplex polymerase chain reaction (mPCR). Materials, Methods & Results: Isolates of A. pleuropneumoniae examined in this study were obtained from lungs with macroscopically visible alterations characteristic of a A. pleuropneumoniae. A total of 46 isolates were examined. They were extracted from the lung tissue samples of pig carcasses from 9 farms across different parts of Serbia. Genotyping of isolates was performed in the previously described manner. Briefly, 5 pairs of oligonucleotide primers were used for amplification of 4 different apx genes which encode synthesis of exotoxins (ApxI , ApxII , ApxIII i ApxIV) characteristic for all A. pleuropneumoniae serotypes and biovars. Amplification of appropriate genome parts was performed with a reaction chain polymerase (PCR) in multiplex (m) format using appropriate diagnostic kits to extract DNA from bacteria and perform mPCR reaction. The results of genotyping of 46 isolates of A. pleuropneumoniae indicate the existence of a large number of different serotypes of A. pleuropneumoniae on the studied farms or that different serotypes of this microorganism circulate in the pig population in Serbia. In addition to the detection of dominant serotype 2, which was established on 7 farms, of which in 4 farms it was the only detected serotype, in the examined pig population the presence of serotypes 3, 5, 6, 7 and 9 was also found. Furthermore, the presence of 2 different serotypes of A. Pleuropneumoniae was also detected on 3 farms; on the first farm serotypes 2 and 3, on the second farm serotypes 2 and 6, and on the third farm serotypes 2 and 7. Discussion: Although the research was done with a relatively small number of isolates of A. pleuropneumoniae, comparing the obtained results with the results on the presence and prevalence of appropriate serotypes from other countries, we concluded that there is significant diversity of this pathogen in the pig population in farms of Serbia. Detection of different serotypes of A. pleuropneumoniae in the pig population and the presence of several different serotypes on 1 farm was established for the very first time in Serbia. All isolates from our study can be characterized as highly virulent, considering that the clinical symptoms, pathological findings and the results of bacteriological examination indicated A. pleuropneumoniae to be the cause of animal death. Like in the neighbouring countries, the strongly pathogenic serotype 9 and the less pathogenic serotype 2 are the most frequently identified causative agents of porcine pleuropneumonia in the Autonomous Province of Vojvodina, Republic of Serbia. The necessity to establish the presence of all A. pleuropneumoniae serotypes in the pig population, and in particular to determine the presence of different serotypes on individual farms, is crucial for several reasons: making a definitive e diagnosis; development of prophylactic strategies for medicines; implementation of immunoprophylactic vaccination.

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)

Effects of ketoprofen administration on relation between acute phase proteins and metabolic parameters in cows during early lactation

Background: Acute phase proteins (APPs) are clinically useful parameters for measuring the occurrence and severity ofinflammatory responses in cattle. As large group of proteins, APPs are mainly secreted by hepatocytes whose concentrationincrease or decrease as response to tissue injury, inflammation or infection. The transition from late pregnancy to earlylactation is associated with a compromised immune status coupled with increased acute phase response. Non-steroidalanti-inflammatory drugs (NSAIDs) are used in treatment of inflammatory conditions. The main objective of the presentstudy was to analyze the effects of a preventive administration of the NSAID (ketoprofen) in post-calving dairy cows onconcentration of APPs (haptoglobin and fibrinogen) and their relationship with indicators of liver function.Materials, Methods & Results: The treatment group (n = 15) was given an intramuscular injection of ketoprofen in theconcentration of 3 mg/kg during three consecutive days after calving. The control group (n = 15) was not treated withketoprofen. Blood samples were taken from coccygeal vein, on the first day of treatment and in the first and second weekpostpartum and they were analyzed for metabolic parameters (albumin and aspartat aminotransferaze) and APPs such ashaptoglobin and fibrinogen. Compared with control, ketoprofen administration decrease the levels of haptoglobin and fibrinogen and AST activity. Increase in albumin concentration was recorded in experimental group of cows compared withcontrol. Negative correlations (P < 0.01) were found between the haptoglobin concentration and albumin concentrationand aspartate aminotransferaze activity. Discussion: As inflammatory indicator in dairy cows, it is recorded that serum concentrations of haptoglobin is increasedduring mastitis. Beside haptoglobin, fibrinogen represents one of the APPs whose serum concentration increases during response in the acute phase. During the first and second week...

Estimation of metabolic status in high yielding dairy cows during transition period and full lactation

Background: Major changes in the metabolic functions in high-yielding dairy cows occur during the transitional periodand during lactation. Parturition and lactogenesis are accompanied by many physiological changes that facilitate themaintenance of homeostasis Consequently, physiological situations leading to a negative energy balance are coupled to anincreased uncontrolled rate of body fat mobilisation and the increased fatty acids accumulation in hepatocytes and bloodketone bodies, resulting in disturbances of the morphological and physiological liver integrity. The objective of the presentstudy was to estimate metabolic status in late pregnant, early lactation and full lactation Holstein dairy cows on the basischanges of blood concentrations of selected biochemical markers.Materials, Methods & Results: The experiment included 36 Holstein cows. Three groups of clinically healthy cows werechosen from the herd. Group 1 consisted of late pregnant cows (n = 12) from 30 to 1 day (20 ± 15) to partus; Group 2comprised early lactation cows (n = 12) in the first month of lactation (15 ± 12 days), and Group 3 included full lactationcows (n = 12) between 60 to 90 days of lactation (81 ± 30 days). Blood samples were collected from all cows, by punctionof the jugular vein. Biochemical testing for markers in the blood serum showed significantly lower values (P < 0.05) ofglucose, TG, Tchol. and urea in early cows than in full lactation and late-pregnant cows. The blood concentration of NEFAand BHB was significantly increased (P < 0.05) in the group of cows in early lactation compared to the other groups ofcows. The mean tBIL. concentration and the serum AST, GGT and ALT activities were markedly increased (P < 0.01)in the lactation cows compared to the late pregnant cows. Furthermore, the intensity of lipomobilisation (NEFA or BHBconcentrations) correlated positively (P < 0.05) with the markers of cell damage or liver function impairment (tBIL., serumAST...

Estimation of metabolic status in high yielding dairy cows during transition period and full lactation

Background: Major changes in the metabolic functions in high-yielding dairy cows occur during the transitional periodand during lactation. Parturition and lactogenesis are accompanied by many physiological changes that facilitate themaintenance of homeostasis Consequently, physiological situations leading to a negative energy balance are coupled to anincreased uncontrolled rate of body fat mobilisation and the increased fatty acids accumulation in hepatocytes and bloodketone bodies, resulting in disturbances of the morphological and physiological liver integrity. The objective of the presentstudy was to estimate metabolic status in late pregnant, early lactation and full lactation Holstein dairy cows on the basischanges of blood concentrations of selected biochemical markers.Materials, Methods & Results: The experiment included 36 Holstein cows. Three groups of clinically healthy cows werechosen from the herd. Group 1 consisted of late pregnant cows (n = 12) from 30 to 1 day (20 ± 15) to partus; Group 2comprised early lactation cows (n = 12) in the first month of lactation (15 ± 12 days), and Group 3 included full lactationcows (n = 12) between 60 to 90 days of lactation (81 ± 30 days). Blood samples were collected from all cows, by punctionof the jugular vein. Biochemical testing for markers in the blood serum showed significantly lower values (P < 0.05) ofglucose, TG, Tchol. and urea in early cows than in full lactation and late-pregnant cows. The blood concentration of NEFAand BHB was significantly increased (P < 0.05) in the group of cows in early lactation compared to the other groups ofcows. The mean tBIL. concentration and the serum AST, GGT and ALT activities were markedly increased (P < 0.01)in the lactation cows compared to the late pregnant cows. Furthermore, the intensity of lipomobilisation (NEFA or BHBconcentrations) correlated positively (P < 0.05) with the markers of cell damage or liver function impairment (tBIL., serumAST...(AU)

Effects of ketoprofen administration on relation between acute phase proteins and metabolic parameters in cows during early lactation

Background: Acute phase proteins (APPs) are clinically useful parameters for measuring the occurrence and severity ofinflammatory responses in cattle. As large group of proteins, APPs are mainly secreted by hepatocytes whose concentrationincrease or decrease as response to tissue injury, inflammation or infection. The transition from late pregnancy to earlylactation is associated with a compromised immune status coupled with increased acute phase response. Non-steroidalanti-inflammatory drugs (NSAIDs) are used in treatment of inflammatory conditions. The main objective of the presentstudy was to analyze the effects of a preventive administration of the NSAID (ketoprofen) in post-calving dairy cows onconcentration of APPs (haptoglobin and fibrinogen) and their relationship with indicators of liver function.Materials, Methods & Results: The treatment group (n = 15) was given an intramuscular injection of ketoprofen in theconcentration of 3 mg/kg during three consecutive days after calving. The control group (n = 15) was not treated withketoprofen. Blood samples were taken from coccygeal vein, on the first day of treatment and in the first and second weekpostpartum and they were analyzed for metabolic parameters (albumin and aspartat aminotransferaze) and APPs such ashaptoglobin and fibrinogen. Compared with control, ketoprofen administration decrease the levels of haptoglobin and fibrinogen and AST activity. Increase in albumin concentration was recorded in experimental group of cows compared withcontrol. Negative correlations (P < 0.01) were found between the haptoglobin concentration and albumin concentrationand aspartate aminotransferaze activity. Discussion: As inflammatory indicator in dairy cows, it is recorded that serum concentrations of haptoglobin is increasedduring mastitis. Beside haptoglobin, fibrinogen represents one of the APPs whose serum concentration increases during response in the acute phase. During the first and second week...(AU)

Hematological parameters in cows in early lactation treated with ketoprofen and their relationship with lipid mobilization and ketogenesis

Background: Dairy cows are exposed to numerous hematological and biochemical changes, what is bringing cows into the state of increased metabolic activity and physiological adaptations. These adaptive processes have resulted in increased lipid mobilization and ketogenesis with increased concentration of non-esterified fatty acids (NEFA) and beta-hydroxybutyrate (BHB). As a non steroidal anti-inflammatory drug, ketoprofen produces anti-inflammatory effects. The main objective of the present study was to found relationship between hematological parameters and ketoprofen administration and high lipid mobilization and ketogenesis in cows treated with ketoprofen immediately after calving. Materials, Methods & Results: Ketoprofen was used (3 mg/kg body weight) intramuscularly for three consecutive days post-partum on 15 cows of Holstein-Friesian breed. Cows of the control group (n = 15) were not treated with ketoprofen. Blood samples were collected at the day of calving, in the first and in the second week after parturition from the coccygeal vein of the both groups. Hematological parameters (erythrocytes, hemoglobin, neutrophils, lymphocytes and mean platelet volume) were determined on the automatic hematological counter. Metabolic parameters (NEFA, BHB) were determined by standard colorimetric kits using a semi-automatic biochemistry analyzer. Students t-test was used to [...](AU)

Effects of iron administration method on anemia prevention and production performance of piglets

Background: Anemia caused by iron deficiency can lead to increased susceptibility to infection, lower production performance and higher mortality rates. Modern piglets grow rapidly, as within a few days of birth they double their body weight, thereby increasing blood volume. This increasing amount of blood naturally increases the requirement for hemoglobin (Hb) production, each molecule of which contains iron. This study examined the impact of different an iron application method on hematological values of iron (Fe), hemoglobin (Hb), hematocrit (Hct) and production performance of piglets in the suckling and nurturing stages. Materials, Methods & Results: The study was performed on two farrow-to-finish farms (I and II) of capacities of 2500 and 900 sows, respectively. Piglets were divided into four experimental groups: the IM group was given iron intramuscularly on the third day of life; the SC group was given iron subcutaneously on the third day of life; the PO group was given iron perorally on the third day of life; and the control group C was not given any iron. On the third day of life and at weaning (day 28) piglets body weights were measured and their blood was taken for analysis, while at the end of the nurturing stage (day 70), only body weights were measured. The influence of the farm was tested, as well as the impact of the day of blood sample collection and the [...](AU)

Effects of iron administration method on anemia prevention and production performance of piglets

Background: Anemia caused by iron deficiency can lead to increased susceptibility to infection, lower production performance and higher mortality rates. Modern piglets grow rapidly, as within a few days of birth they double their body weight, thereby increasing blood volume. This increasing amount of blood naturally increases the requirement for hemoglobin (Hb) production, each molecule of which contains iron. This study examined the impact of different an iron application method on hematological values of iron (Fe), hemoglobin (Hb), hematocrit (Hct) and production performance of piglets in the suckling and nurturing stages. Materials, Methods & Results: The study was performed on two farrow-to-finish farms (I and II) of capacities of 2500 and 900 sows, respectively. Piglets were divided into four experimental groups: the IM group was given iron intramuscularly on the third day of life; the SC group was given iron subcutaneously on the third day of life; the PO group was given iron perorally on the third day of life; and the control group C was not given any iron. On the third day of life and at weaning (day 28) piglets body weights were measured and their blood was taken for analysis, while at the end of the nurturing stage (day 70), only body weights were measured. The influence of the farm was tested, as well as the impact of the day of blood sample collection and the [...]

Hematological parameters in cows in early lactation treated with ketoprofen and their relationship with lipid mobilization and ketogenesis

Background: Dairy cows are exposed to numerous hematological and biochemical changes, what is bringing cows into the state of increased metabolic activity and physiological adaptations. These adaptive processes have resulted in increased lipid mobilization and ketogenesis with increased concentration of non-esterified fatty acids (NEFA) and beta-hydroxybutyrate (BHB). As a non steroidal anti-inflammatory drug, ketoprofen produces anti-inflammatory effects. The main objective of the present study was to found relationship between hematological parameters and ketoprofen administration and high lipid mobilization and ketogenesis in cows treated with ketoprofen immediately after calving. Materials, Methods & Results: Ketoprofen was used (3 mg/kg body weight) intramuscularly for three consecutive days post-partum on 15 cows of Holstein-Friesian breed. Cows of the control group (n = 15) were not treated with ketoprofen. Blood samples were collected at the day of calving, in the first and in the second week after parturition from the coccygeal vein of the both groups. Hematological parameters (erythrocytes, hemoglobin, neutrophils, lymphocytes and mean platelet volume) were determined on the automatic hematological counter. Metabolic parameters (NEFA, BHB) were determined by standard colorimetric kits using a semi-automatic biochemistry analyzer. Students t-test was used to [...]