Matko Kardum

Supplementation with imuno-2865® in gilthead sea bream (Sparus aurata Linnaeus, 1758): Effects on hematological and antioxidant parameters

The aim of this study was to evaluate the effects of IMUNO-2865(®) on hematological and antioxidative parameters in sea bream. Total of 640 sea bream were fed with diets containing 0 (Group 1), 1 (Group 2), 10 (Group 3) and 25 (Group 4) g of IMUNO-2865(®) kg(-1) feed during 90 days. Samples were taken each month and three months after the supplementation. A significant heterophils increase was observed in group 4 compared to group 1 after two months, and an increase in monocytes number was observed in group 4 compared to the other groups after one month. Glutathione peroxidase (GSH-Px) and paraoxonase-1 (PON1) were significantly increased in groups 3 and 4 compared to the control group three months into the experiment. Superoxide dismutase (SOD) was increased in group 4 compared to the control group from day 60 until the end of the experiment, and in groups 2 and 3 compared to the control after three months. Based on the differences in the cellular immunity and oxidative stress parameters, with an overall absence of mortality, the results of this study suggest that the use of IMUNO-2865(®) in aquaculture is safe and possess a cumulative immunostimulatory effect on sea bream.

The Effects of Splenectomy and Autologous Spleen Transplantation on Complete Blood Count and Cell Morphology in a Porcine Model

Spleen, as a part of hematopoietic and immune system, plays an important role in the life cycle of blood cells. There are three major functions of the spleen and these are handled by three different tissues within the spleen. Reticuloendothelial tissue is responsible for removing old or damaged erythrocytes and cell debris from the blood stream. This same tissue may participate in haemopoiesis when there is an increased need for red blood cells and is a place where young erythrocytes produced in bone marrow undergo the process of maturation before they are released into the blood stream. Venous sinusoids along with the ability of the spleen to contract, provides a means for expelling the contained blood to meet increased circulatory demands in certain animals. White pulp provides lymphocytes and a source of plasma cells and hence antibodies for the cellular and humoral specific immune defenses. Splenectomy is surgical removal of the spleen, it may be carried out in patients whose spleen has been ruptured by trauma or damaged by other pathological processes such as cancer, infections or some autoimmune diseases. However, total removal of the spleen may lead to side effects such as postsplenectomy infections and sepsis, due to the decreased production of antibodies and phagocytes, or thrombosis, due to elevated platelet count in blood. Also, many studies report increased count of morphologically abnormal erythrocytes, immature red blood cells and pathologic erythrocyte inclusions in the peripheral blood of various species following splenectomy as a result of the loss of splenic filtrating function. In addition, it has been reported that removal of the spleen causes significantly higher increase of retikulocyte count than other surgeries. This suggests that spleen may somehow hormonally regulate release of red blood cells into circulation, thus after removal of spleen bone marrow releases more red blood cells as well as more immature erythrocytes into the blood stream. Severe postoperative infections after removal of the spleen prompted a development of alternative methods to conserve functions of the spleen. Autlogous spleen transplantation is a method of choice after total splenectomy in order to preserve splenic immune and hematopoietic functions. The effectiveness of splenic autotransplant depends on many factors and is still controversial. Studies done on rats, mice, rabbits and men report that autotransplantats capability of recovering its primary function highly depends on the volume of transplanted spleen tissue. The aim of this study was to evaluate the effects of total splenectomy and autologous spleen transplantation in porcine model on complete blood count and cell morphology. Also, we aimed to determine the functional effectiveness of autotransplanted splenic tissue by its capacity to remove erythrocyte hawing Howell-Yolly bodies. Twenty Landras pigs aged three months observed in the research were randomly divided into three groups. The first (the control) group underwent sham surgery, second group was splenectomized and the third group, after being splenectomized, had 20% of autologous spleen tissue transplanted into the greater omentum. Blood was sampled prior to surgery, and then again on the 1st, 5th, 12th and 26th day after surgery. Blood samples were taken from each animal from (v. auricularis lateralis). Three milliter of blood was taken from each piglet using sterile tubes containing 7.5% K3E anticoagulant. Total leukocyte count, erythrocyte and platelet count, as well as hemoglobin level and hematocrit were determined using automated cell counter (SERONO – 9120 Baker System). A drop of anticoagulant free blood was taken from each piglet and used to make blood smears which were stained with May Grunwald – Giemsa method for differential leukocyte count and new methylene blue for reticulocyte count using immersion objective with 15000× enlargement of the light microscope (Olympus BX 41). Also, changes in cell morphology and the presence of immature cells were observed and documented. Following the operation, all groups showed leukocytosis with an increase of the absolute neutrophil number and initial decrease of the lymphocyte number which was followed by the later recovery. Noted leukocytosis was accompanied with increased band neutrophil number up to 12th postoperative day. At the 12th postoperative day reactive reactive lymphocytes were frequently found in the autotransplantated group. Erythrocyte count was significantly decreased postoperatively in sham-operated pigs (on the 5th day) and in autotransplanted pigs (on days 5 and 12) while in splenectomized pigs significant decrease of erythrocyte count occurred on day 1 after the surgery and maintained on the significantly low values through all days of the experiment. Hgb values and Htc were changing accordingly with changes of red blood cell count during whole experiment. In splenectomized pigs values of all erythrocyte indices were significantly increased postoperatively (MCV on the 12th and 26th day, MCH and MCHC on day 5). In contrast, autotransplanted pigs had significantly lower values of MCV (on days 1, 5 and 12) and MCH (on days 1 and 12), but significantly higher value of MCHC (on the 5th day). Platelet count was significantly increased on the 12th day in splenectomized group of pigs while there were no changes of platelet count in other two groups of pigs. Percentage of reticulocytes in blood including polychromasia was significantly increased in all three groups of pigs during all postoperative days of experiment. In conclusion, anemia and retikulocytosis found in blood samples of all three groups of pigs may have been physiological results showing the classical postoperative organism reaction to blood loss and surgical trauma. On the other side, frequenter appearance of variations in red blood cell morphology such as appearance of leptocytes, codocytes and Howell-Jolly bodies on the blood smears of splenectomized pigs when compared with other two experimental groups suggests that this was a change specific for splenectomy. More frequent occurance of morphologicaly abnormal red blood cells on the blood films of autotransplanted pigs compared with sham-operated pigs suggests that the autologous spleen tissue wasn’t able to effectively filtrate the blood.