الفهرس 
DEEDDIICCAATTIIOONNOnly 14 pages are availabe for public view
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Abstract
The present study was carried out to elucidate the role of phosphorus, erythrocytic energy and oxidative stress in the integrity of cell membrane of RBCs and development of PPH in dairy cattle and buffaloes through evaluation of important blood biomarkers including blood phosphorus, calcium and parathermone, erythrocytic energy and oxidative stress biomarkers (G6PD, GSHPX, MDA, and NO). The present study also aimed to study the impact of the status of phosphorus, erythrocytic energy and oxidative stress biomarkers on the integrity of cell membrane of RBCs in PPH via studying osmotic fragility index. Correlation study of the investigated parameters was also aimed.
Materials and Methods: To carry out the study, a total number of 58 cow cattle and buffaloes were included and categorized as followings:
• Control healthy animals (n= 10 buffaloes and 10 cows). Animals were proved to be healthy and free from any internal, external parasites and blood parasites by both clinical and laboratory examination.
• Diseased animals (n=38) suffered from post parturient hemoglobinurea and classified according to clinical signs and packed cell volume (above or below 20%) into mild-moderate (PCV >20%) and severe (PCV <20%).
Categorization of the investigated animals and their data about age, parity body weight, localities and time point of sample collection post parturition as well as seasonal occurrence of parturient hemoglobinurea were recorded. All animals in the present study were fed on berseem and cruciferous plants as a main source of feeding and subjected for thorough clinical examination. Blood samples were collected from all animals and prepared as whole blood, hemolysate, serum and plasma samples. Whole blood sample were used for complete blood picture (RBCs, TWBCs, Hb, PCV, MCV, MCH and MCHC), osmotic fragility and G6PDH. Blood film was prepared for DLC and reticulocyte count. Serum samples were used for estimation of bood phosphorus, calcium, parathermone, nitric oxide levels and plasma for determination of Malondialdehyde. Erythrocyte lysates were prepared and used for determination of GSHPx. The obtained data were analyzed statistically using proper tests and software.
Results and discussion: Clinical observations of diseased animals revealed that passage of red to coffee colored urine was the most prominent clinical sign. Anorexia and low milk yield became evident on third or fourth day of illness. There was straining at the time of defecation decreased to depraved appetite, anemia represented in pale mucous membrane then became icteric, increased in respiratory and heart rate with normal to subnormal body temperature.
Screening of hematological data revealed significant reduction (P<0.05) in the RBCs count (106/mm3), hemoglobin concentration (g/dl) and PCV (%) in the diseased animals. This reduction was largely attributed to intravascular hemolysis that led to macrocytic hypochromic anemia (regenerative type). This was evident by significant increase in the MCV (fl) and significant increase in TWBCs with neutrophilia and lymphopenia. Additionally, cytomorphological study of RBCs revealed strong evidences of regenerative type of anemia in PPH. Presence of an increased number of reticulocytes and nucleated RBCs in peripheral blood strongly suggests regenerative anemia in PPH. The current study demonstrated a marked increased osmotic fragility of RBCs in PPH animals compared with healthy ones. The increased fragility of RBCs leads to intravascular hemolysis with subsequent hemoglobinemia, hemoglobinuria, and hemolytic anemia.
Obtained result of blood phosphorus level (mg/dl) showed significantly decrease (P<0.05) in the serum phosphorus level in mild-moderate and severe PPH affected buffaloes (1.85 ± 0.21 and 1.92 ± 0.28, respectively) and cattle (1.14±0.16 and 1.45±0.12, respectively). Correlation study of osmotic fragility of RBCS revealed that there was strong negative correlation between blood phosphorus level and osmotic fragility suggesting the importance of phosphorus in PPH.
Blood calcium level (mg/dl) showed significant decrease (p≤0.05) in mild-moderate and severe PPH buffaloes (9.36±0.36 and 8.57±0.5) compared to control one (10.48 ± 0.71). In diseased cattle, blood calcium level showed significant increase (p≤0.05) in mild-moderate (9.18±0.64) while it showed no significant changes in severe group (8.70±0.47) compared to control one (8.72±0.22).
The present study showed significant decrease (P<0.05) in the serum PTH (pg/ml) in mild-moderate and severe groups of diseased buffaloes (36.36 ± 7.48.26 and 23.53 ± 6.22) and cattle (20.08±6.05 and 37.85±7.32, respectively) compared to control buffaloes and cattle (51.1± 7.41 and 121.9±24.16, respectively).
Obtained result of erythrocytic G6PD (U/g Hb) showed significant decrease (P<0.05) in mild-moderate and severe PPH affected buffaloes (7.07±1.01 and 7.58 ± 1.04, respectively) compared to control one (17.45 ± 1.67) while in diseased cattle, showed that the activity of erythrocytic G6PD in mild-moderate and severe PPH was lower (6.90 ± 1.79 and 6.15 ± 0.83, respectively) compared to control cattle (9.81 ± 1.02). These finding suggested that decreased erythrocytic G6PD activity in PPH animals may be partially responsible for the decrease in reduced glutathione, thereby causing oxidative stress to erythrocytes, which leads to hemolytic syndrome. It worthwhile to mention that there was strong positive correlation between blood phosphorus level and erythrocytic G6PD. Both blood phosphorus level and erythrocytic G6PD had strong negative correlation with osmotic fragility suggested the importance role of G6PD and the level of RBCs energy in maintaining the integrity of RBCs cell membrane. Suboptimal level of erythrocytic G6PD may be partially responsible for a decrease in reduced glutathione, thereby causing oxidative stress to erythrocytes, which results in hemolytic syndrome.
Screening data of GSH-Px level (mU/ml) in the hemolysate in mild-moderate and severe PPH affected buffaloes revealed significantly decreased (P<0.05) (285.50±25.96 and 188.14±28.08, respectively) compared control one (400.22±21.49). In diseased cattle, GSH-Px level showed significant decrease in both mild-moderate and severe groups (241.20±31.55 and 181.45±15.23, respectively) which might be due to the decreased production or increased depletion. There was negative association between GSH-Px and osmotic fragility index. Suboptimal level of GSH-Px causes oxidative stress to erythrocytes, which results in hemolytic syndrome
Obtained data of blood level of MDA (nmol/l) in the present study revealed significant increase (P<0.05) in mild-moderate and severe diseased buffaloes (10.55 ± 1.60 and 13.12±1.72, respectively) compared to healthy ones (7.62 ± 1.35). In PPH- affected cattle, MDA showed also significant increase in mild-moderate and severe groups (10.54±1.95 and 13.46±2.92, respectively) compared to control one (5.99±1.26). These findings indicate that there was enhanced lipid peroxidation in red cell membranes of PPH affected animals.
It important to pinpoint that there was strong positive correlation between blood MDA level and osmotic fragility suggested the importance role of oxidative stress and the integrity of RBCs cell membrane. Increased level of MDA may be partially caused by suboptimal level of reduced glutathione, thereby causing oxidative stress to erythrocytes, which results in hemolytic syndrome. There is a slightly increase in nitric oxide level (µmol/l) in diseased buffaloes which is also biomarker of lipid peroxidation and formation of free radicals which caused oxidative stress.