The optimal formula for calculating the total number of hemocytes (HTN) of the Australian red-claw crayfish (Cherax quadricarinatus) has been established, which allows optimizing the time of counting HTN without re-ducing accuracy. Methodical recommendations on preparing a hemolymph sample, HTN calculation and differentiated calculation of hemocytes are given. It was found out that the differences in HTN are minimal when using different formulas, which is con-firmed by the analysis of variance F = 0.011, p = 0.998, the average values of HTN range from 2 384 to 2 427 cell/µl. Conducting multiple comparisons also demonstrated that the differences between the formulas are minimal: the significance levels between the formulas were p = 0.99. The optimal formula for the Australian red-claw crayfish is: HTN in 1 µl = N • 10, where N is the total number of hemocytes in 25 large squares of Gorjaev’s chamber. The counting chamber should be clean, since when even small dust particles are found on the grid, hemocyte clusters can form and impede counting, or the result of the calculation will be incorrect, because the distribution of hemocytes on the grid will not be uniform. After filling the chamber with hemolymph, it is recommended to wait 1-1.5 minutes so that the movement of the hemolymph stops and the hemocytes stop moving. It is recommended to use an anticoagulant, which prevents the rapid decay of cells and promotes their uniform distribution on the grid of the chamber without developing clots (clusters) that affect the accuracy of counting. Working with the whole hemolymph implies the speed and accuracy of the researcher's actions, since it quickly coagulates and forms clusters of hemocytes. In order to avoid confusion when counting cells located on the border between squares, Egorov’s rule is applied. The optimal increase for counting is a 400-fold increase.
Purpose: selection of anticoagulants based on EDTA-Na2 and methods of their use for working with the hemolymph of the Australian red claw crayfish (C. quadricarinatus)Materials and methods. For the study, different-sized males and females of the australian red-clawed crayfish (Cherax quadricarinatus (Von Martens, 1868)) were used. EDTA-Na2 was used as an anticoagulant, the concentrationof which was 4%. A 2 ml syringe with a 23G needle for hemolymph removal was pre-washed with anticoagulant remaining in the needle and the needle cone (about 1/3 of the volume of the needle cone was filled with a solution). The experiment is presented in the following series: 1. differences between the total hemocytes number (THC) and the proportion of granulocytes in native hemolymph and treated with a small amount of EDTA-Na2; 2. differences in the content of total hemolymph protein (THP) in native and treated with a small amount of EDTA-Na2 hemolymph; 3. changes in THC and the proportion of granulocytes in hemolymph treated with a small amount the amount of EDTA-Na2 immediately after sampling and a day later. To carry out the work, samples of 20 individuals were used, while for the first two series, two samples of hemolymph were taken from each, and one from the third. Syringes with hemolymph treated with anticoagulant were stored in the refrigerator at a temperature of 8.5 °C. Additionally, similar studies were carried out on differences in the THC and proportion of granulocytes in the hemolymph immediately after sampling with treatment with a multicomponent anticoagulant consisting of 4 g of EDTA-Na2, 3 g of sodium citrate, 2 g of glucose and 1 g of NaCl per 100 ml of distilled water. Hemolymph was taken with a syringe from the ventral sinus. Three indicators were determined: the total number of hemocytes (THC), the proportion of granulocytes and the total protein content (THC). THC and proportion of granulocytes was determined in the Goryaev chamber under a light microscope. THP was determined by the refractometric method.Results. The study revealed significant differences in the proportion of granulocytes (p<0.05), which are 32% more in the anticoagulant treated than in the native hemolymph, which can be explained by the uniform distribution of all types of hemocytes in the sample. There were no significant differences in the remaining indicators of all series. Studies of hemolymph treated with a multicomponent anticoagulant showed a low level of hemocyte preservation (p<0.05) and the proportion of granulocytes compared to the indicators immediately after selection, therefore, they are not reflected in the work. Conclusion. The data obtained indicate that there is a possibility of using four percent EDTA-Na2 when working with hemolymph. The anticoagulant prevents the formation of gel and the rapid destruction of hemocytes, promotes the uniform distribution of cells in the Goryaev chamber and allows the use of a refractometer in determining the total blood protein. The use of an anticoagulant contributes to the preservation of hemocytes during the day, at a level that allows the use of EDTA-Na2 in practice. At the same time, work should continue on the development of methods for storing and transporting blood treated with an anticoagulant.
Purpose: to study the effect of ethanol on the parameters of THC, the percentage of granulocytes and total protein in the hemolymph of the Red claw crayfish (Cherax quadricarinatus).Materials and methods. The object of this experiment was 26 males of the Australian red-clawed crayfish (Cherax quadricarinatus) weighing from 23 to 83 g. The individuals were evenly divided into two experimental groups - with an injection of ethanol and a control group without an injection of 13 crayfish for each group. The injection dose was 2515 mg per 100 g of body weight. A day after the introduction of ethanol, hemolymph was taken with a syringe from the ventral sinus, the syringe was pre-washed with a 4% EDTA-Na2 solution. Three parameters were determined: the total hemocyte count (THC), percent granulocytes and percent total protein content. Counting of hemocytes and determination of granulocytes were performed in a Goryaev chamber under a light microscope. The total protein was determined by the refractometric method.Results. Differences in THC and total protein between the groups were statistically unreliable (p>0,05). THC in the experimental group is 36% more than in the control group. The total protein after the introduction of ethanol actually increased by 0,7%, and relatively by 14%. There were statistically different indicators of the proportion of granulocytes (p<0,05) - the average value of 33,1% in the experimental group versus 24,5% in the control group. A reliable (p<0,05) strong feedback was revealed between the total protein and the mass of individuals in both experimental groups, while in the experimental group there is a visible shift in the values of dependent hemolymph indicators towards an increase in smaller individuals.Conclusion. A single injection of ethyl alcohol with a dosage of 2515 mg per 100 g of body weight into the hemolymph of C. quadricarinatus does not cause significant changes in the THC and total protein after 24 hours. At the same time, the proportion of granulocytes actually increases by 9%, relative to 37%. This may indicate that granulocytes are involved in the formation of cancer defense mechanisms when exposed to toxic substances. The effect of different dosages of ethanol injections and the duration of its effect on hematological parameters requires additional consideration. It is necessary to investigate its effect on other indicators, such as the pH and buffer capacity of the hemolymph, the concentration of hemocyanin, glucose, lactates and calcium.
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