EDTA-Na<sub>2</sub> - anticoagulant for hemolymph of australian red claw crayfish (cherax quadricarinatus)
Authors: Skafar D. , Shumeyko D. 
Published: August 2022

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.