Effects of Three Different Anticoagulants on Blood Cell Morphology, Anticoagulation, and Hematological Parameters in Turbot (Scophthalmus maximus)

Abstract

Fish blood tends to coagulate due to its specialized characteristics. In general, the detection of blood physiological indices using anticoagulants reflects normal physiological status. However, the selection and application of anticoagulants have no uniform standards because of the specificity and diversity of fish species. In the present study, the effects of three different anticoagulants (sodium heparin, K2EDTA, and sodium citrate) were investigated on coagulation, blood cell type, hematological parameters (white blood cells, red blood cells, hematocrit, and hemoglobin), and plasma cortisol and glucose contents in turbot during acute hypoxic stress. The choice of anticoagulants may result in significant differences in turbot blood physiology and chemistry. To supply technological support in turbot hematological research and best-practice aquaculture, this study compared the effects of three common anticoagulants between normal dissolved oxygen and acute hypoxic states. The recirculating aquaculture system is the main culture model for turbot; water temperature and dissolved oxygen levels are important environmental factors, especially in high-density industrial systems. Sufficient oxygen is key to maintaining normal metabolism in turbot. Experimental procedures were designed for control and treatment groups, each group had three parallels, and 54 turbots were studied in all. In the control group, 200 mg/L MS-222 was used to anesthetize a specimen before collecting 5 mL of blood from the caudal vein; the blood was transferred through a needle and vacuum tubes containing three different anticoagulants, then the vacuum tubes were stored for evaluation after 6 h and 12 h. In the treatment group, nitrogen gas was used to rapidly reduce the concentration of dissolved oxygen in the container to (1.2±0.3) mg/L (measured with a dissolved oxygen meter), after which the same procedure of anesthesia and blood collection was conducted. For blood cell type and morphology studies, the Giemsa staining method was used to make blood smears. The original solution of Giemsa was diluted before use. The slide was sterilized before a drop of blood was placed on one side of the slide; one edge of a cover glass was placed in contact with the drop of blood and pushed to the opposite side of the slide at a constant velocity; the slide was then fixed with methanol for 10~15 min and stained for 15~30 min. The smears were washed and dried after staining. Finally, the stained smears were observed under a Leica microscope to determine blood cell morphology, and images were taken. Blood samples were divided into two parts for detecting physiological and biochemical indices: one was centrifuged at 3500 r/min for 10 min, and the supernatant was stored at –80℃ for the detection of plasma glucose and cortisol content using commercial kits. The residual blood samples were stored in a refrigerator at 4℃ for the detection of physiological indices using an automatic blood cell analyzer. The anticoagulant effect of K2EDTA was most effective under a normal dissolved oxygen state, and the anticoagulant effect of K2EDTA and heparin sodium were most effective after 6 h and 12 h of exposure to acute hypoxia stress, respectively. In addition to coagulation, blood cell morphology showed that binucleate cells occurred through sodium heparin, eosinophils, and basophils through sodium citrate and basophils through K2EDTA in the control. Three nucleus abnormality types were identified in the smears: micronucleus cells, binucleate cells, and erythroplastids. Binucleate cells were found using sodium heparin in the control group. In addition, binucleate cells were observed under three different anticoagulants in the treatment group, and micronucleus cells and erythroplastids were observed in the treatment group. The hematological index showed that the number of white blood cells was significantly increased by treatment with three anticoagulants during acute hypoxic stress (P < 0.05), and the number of red blood cells and hemoglobin content was significantly increased by treatment with K2EDTA and sodium heparin (P < 0.05). However, they were significantly reduced after treatment with sodium citrate. In addition, the plasma glucose and cortisol content were significantly increased when subjected to acute hypoxic stress, but the content of sodium heparin was significantly lower than that of both sodium citrate and K2EDTA. In summary, K2EDTA showed less blood coagulation than other anticoagulants, sodium heparin caused binucleate cells and decreased plasma glucose and cortisol, and sodium citrate affected the number of red blood cells and hemoglobin content. K2EDTA is the more promising anticoagulant compared to sodium heparin and sodium citrate for blood analysis of turbot and promotes the precision of turbot hematological studies under acute hypoxic conditions.

Keywords

• scophthalmus maximus
• anticoagulants
• anticoagulant effect
• blood cell morphology
• blood physiology and biochemistry