HEMATOLOGICAL AND BIOCHEMICAL PROFILE OF RAINBOW-TROUT ONCORHYNCHUS-MYKISS DURING TESTING THE BUTLYHYDROXYTOLUOL (BHT) BASED ANTIOXIDANT NEOX

Physiological safety of the butylhydroxytoluol (BHT) based antioxidant Neox was tested in 1+ rainbow trout Oncorhynchus mykiss at the weight of 207 to 814 g in four pilot trials in flow-through laminated plasti c tanks at temperature ranges of water 7 to 14-degrees-C, oxygen regim e 9.1 to 10.9 mg O2/l and O2 saturation of water 81 to 89.8%. Dry pell eted food with pellets 4 and 5 mm in size and with gradated doses of 1 00, 200 and 300 mg Neox and 100 mg Neox forte per kg was produced in t hree batches (AB, C, D) with fat content of 3.48 to 7.15%, while dry p ellets with 200 mg Kurasan (Ethoxyquin) per kg were used as positive c ontrol. Diagram of trials A, C and D is shown on pages 517 and 518. He matological and biochemical analysis of heparinized peripheral blood a nd plasma samples taken by puncturing the caudal veins was a testing c riterion. These parameters were evaluated: red blood cell count (RBC i n T/l), hematocrit (PCV in l/l), hemoglobin (Hb in g/l), mean corpuscu lar volume (MCV in fl), mean corpuscular hemoglobin (MCH in pg), mean corpuscular hemoglobin concentration (MCHC in l/l), total protein (TP in g/l), glucose (GL in mmol/l), cholesterol (CHOL in mmol/l), urea (B UN in mmol/l), uric acid (UA in mumol/l), creatinine (CREA in mumol/l) , triacylglycerole (TG in mmol/l), total bilirubin (T - BIL in mumol/l ), inorganic phosphate (P in mmol/l), calcium (Ca in mmol/l), activiti es of alanine aminotransferase (ALT in mucat/l), aspartate aminotransf erase (AST in mucat/l), alkaline phosphatase (ALP in mucat/l), lactate dehydrogenase (LDH in mukat/l) and hydroxybutyryl dehydrodenase (HBD in mucat/l). Individually decreased values of RBC from 0.81 to 0.67, t o 56 in Hb and to 0.287 in PCV were determined in trial A (Tab. 1) in the lst and 2nd experimental groups (100 and 200 mg Neox/kg). This tre nd approached critical values in one case of the negative control (wit hout antioxidant) in which the hematological profile (RBC 0.46; PCV 0. 157; Hb 26.1; MCH 57; and MCHC 0.17) and the patho-anatomic finding in liver confirmed severe microcytic hypochromous anemia and lipoid live r degeneration (LLD) as a result of strongly oxidized fat uptake. Thes e lesions were accompanied by the increased activity of ALT and AST in all cases, the values of which were higher in the experimental groups with Neox (ALT 0.67 and AST 53.8) in comparison with the negative con trol (ALT 0.35 and AST 18.02). LDH activity culminated in the fish wit h anemia disposition in which it had the values 37 to 85, but in did n ot increase in the LLD case. The goal of trial B was to assess hematol ogical response depending upon time in the course of 105-day investiga tion split into five time intervals (Tab II). The trial involved an ex perimental group with 200 mg Neox/kg and negative control without anti oxidant. Fig. 1 shows water temperature, dissolved O2 content and perc ent O2 saturation of water at the moment of blood sampling. The data i n tables indicate that as soon as in 7 days after feeding pellets with Neox a highly significant (P < 0.01) increase in MCV (438 vs. 357) an d significant (P < 0.05) increase in MCH (81 vs. 69) occured in compar ison with the control group of fish. The continuation of the trial bro ught about a highly significant (P < 0.01) and significant (P < 0.05) decrease in the values of both indicators depending upon time, which i s evident from graphs of linear regression in Figs. 2 and 3 (r(MCV) = -0.988* vs. r(MCV) = -0.316 and r(MCH) = -0.925* vs. r(MCH) = -0.622) . On day 63, a twofold significant increase (P < 0.05) of AST activity (14.22 vs. 6.41) and a significant (P < 0.05) decrease in P (6.27 vs. 7.62) in the blood plasma were observed. In the last time interval, t here was an obvious significant (P < 0.05) decrease in PCV (0.362 vs. 0.413) and a significant (P < 0.05) decrease in ALP activity (5.36 vs. 8.64). The significant parabolic dependence of AST activity upon time (I(AST) = 0.942 vs. I(AST) = 0.622) in Fig. 4 demonstrates the dynam ics of this aminotransferase in the course of 105 days and proves at t he same time that its increased activity with the peak in the middle o f the period of investigation is not only a reaction to the presence o f oxidized fats as found out in individual cases in trial A. A signifi cant parabolic variation of Ca level (I(Ca) = 0.957 vs. I(Ca) = 0.828 ) as shown in Fig. 5 was recorded within the range of the values 3.0 - 3.5. On day 29, a large elevation of UA concentration occurred in com parison with the negative control, as seen in Fig. 6. This trend was a lso demonstrated in trial C (Tab. III) in the fish which received pell ets stabilized with Neox. UA concentration was higher in all the exper imental Neox groups and in one experimental Kurasan group if compared with the negative control; it was statistically significant (P < 0.05) higher in the 1st experimental group (172.8 vs. 141) and highly signi ficantly (P < 0.01) higher in the 3rd (173.4), 5th (207.1) and 6th (20 5.7) experimental groups. This increase was most marked in the fish on lecithin diet. A significant increase (P < 0.05) in CREA level agains t the negative control was determined in the 5th experimental group (2 8 vs. 24). Analysis of the results obtained in trial D (Tab. IV) showe d a significant (P < 0.05) increase in RBC in comparison with the posi tive control (1.15 vs. 0.95) and a decrease in ALP activity in compari son with the negative control (5.0 vs. 7.06) in the only tested Neox t reatment (300 mg/kg after 91 days of the trial). Evaluation of the red blood count in these trials has shown a remarkable MCV decrease which exceeds not only the lower boundary of the average values recorded du ring BHT testing at a concentration of 0.01 to 0.04% after 60 days of the trial (Rehulka, 1989) but also it differs from the average referen ce values of our laboratory for the same age class of fish under the s ame conditions. Evaluation of the tested preparation must also involve the observed trend of MCH, the values of which are above the average level of this indicator in the experimental group without Neox. Our op inion of the AST activity in relation to Neox toxic action is reserved in this stage of research. It will be necessary to seek for the answe r to this problem in further experimental testing of still higher anti oxidant concentrations. Our experience has confirmed the increased act ivities of AST and ALT at LLD (Rehulka, 1990). The repeatedly determin ed relatively high UA level in the blood plasma of fish on N