Anwendung hämatologischer Untersuchungsmethoden für Fischblut und Beeinflussung des Blutbildes von Bachforellen (Salmo Trutta F. Fario) durch Haltungs- und Umwelteinflüsse sowie endogene Faktoren

Abstract

In the present thesis, three different blood withdrawal techniques and several hematological methods were evaluated regarding their applicability to brown trouts (Salmo tutta f. fario) and their blood, respectively. After standardization and optimization of methods it was investigated whether the water quality due to keeping fish in recirculating or flow-through systems had any influence on trout blood parameters. In a further study the variations of the cellular blood composition were evaluated when the fish were subjected to harmfull acute stressors. Resulting longtime shifts in blood composition as well as possible seasonal variations or dependency on fish body weight were monitored over 5 month.

The results are as follows:

In contrast to the blood sampling from the caudal vein or the cardiac puncture the withdrawal from the Ductus Cuvieri provide the most convenient procedure. It was safe for the fish and easy to handle. A adequate volume of blood could be obtained in a short time, no injuries in contrast to the other methods could be detected. A heparin concentration of at least 0,24 mg/ml blood (41 USP-units/ml), solved in phosphate buffered saline (pH 7,05) was necessary to prevent clotting completely. Best results for counting erythrocytes, thrombocytes and leukocytes in the hemocytometer were obtained by blood diluting fluids according to Dacie´s and Natt-Herrick. Thereby it was possible to clearly identify the cell types because of their different staining properties and preserved morphology. For the differential leukocytes counts three different staining methods were evaluated. The modified Pappenheim staining technique gives adequate results concerning the differentiation between mature and immature granulocytes, monocytes, lymphocytes and thrombocytes. The different cell types could be distinguished by their colorability and preserved morphology. The resulting color intensity was strongly influenced by the pH value, incubation time and the concentrations of the staining solutions. Incubation of blood smears for 1,5 min in a concentrated May-Grünwald and a Giemsa solution diluted 1:50 in phosphate buffered A. bidest. (pH 6,5-6,8) each yielded a distinguishing coloration of cells. The investigation of the erythrocyte fragilities revealed similiar values in comparison with erythrocytes of birds, probably because of similiar membrane properties due to their nucleated cells. Hematocrit values became reproducible by centrifugation of the blood at 12500 rpm for 8 to 10 minutes in standard microhematocrit tubes. The hematocrit and pH values, hemoglobin concentration and the osmolality of the blood, all of them important diagnostic parameters, were influenced by the choice of the anticoagulant, its concentration and the sampled blood volume. This could be attributed to a diluting effect of heparin, the concentration of EDTA and to the changing blood osmolality. Unlike EDTA, heparin with its isoosmolality to fish blood caused only minor changes of blood parameters investigated. Given that the use of an anticoagulant is indispensible, the smallest possible amount of heparin solution should be applied. The use of EDTA should be avoided because of its strong influence on the above cited blood parameters. Hemoglobin concentrations were measured by the cyanmethemoglobin method. The calculation of the hemoglobin values from the standard curve gave significantly higher concentrations in comparison with the determination with 36.8, a factor which is used for calculating the hemoglobin content of human blood.

In comparison with fish which were reared in flow-trough systems, the blood picture of fish kept in recirculating systems revealed changes of several blood parameters. This could be attributed to the poor water quality in the recirculation unit. By the nitrification process, ammonia, nitrite, and nitrate as well as phosphate, sulphate, chloride, and calcium ions accumulated, thus lowering the carbonate hardness. Most striking was the elevated nitrate concentration, 25 to 44 times higher than in the flow-through system. Other ions in the recirculating water were raised 1-2 fold (sulphate), 4-8 fold (phosphate), and 2 fold (chloride). The toxicologically relevant ammonia and nitrite concentrations were here 5 to 7 times higher but, like nitrate values, did not reach toxic limits published for trouts. The stress-associated response consisted of a neutrophilia, lymphopenia and an increase of the blastogenic stages of the granulocytes. Furthermore, a hemoconcentration could be observed. On the other hand, no mortalities could be observed during the whole examination period. Therefore, the changed blood parameters indicated only mild stress for the fish without reaching exhaustion level as a part of the General Adaptation Syndrome. Acute stressors like transport and confinement of the fish, resulted in long lasting blood changes. 5 to 10 days after stress the absolute numbers of erythrocytes and leukocytes (lympho- and monocytes) increased 1,5 and 2 times, respectively, and declined again until day 40 without reaching the prestressed level again. Thrombocyte and granulocyte numbers followed a reversed course, thrombocyte counts were reduced by half, granulocytes to 1/6 after 5 days. Thereafter, all cell numbers kept nearly constant.

With maturation of the gonadal products in august/september leukocytes and thrombocytes decline, whereas erythrocyte and monocyte counts changed only marginally. Increase of sexual hormones in this time of the year was the most probable reason for the alterations observed. Any influence of fish body weight or variable environmental conditions was excluded.