3种抗凝剂对大菱鲆血液抗凝效果、血细胞形态和血液生理生化指标的影响

Effects of three different anticoagulants on blood cell morphology, anticoagulation, and hematological parameters in turbot (Scophthalmus maximus)

中文关键词: 大菱鲆  抗凝剂  抗凝效果  血细胞形态  血液生理生化

英文关键词: Scophthalmus maximus  Anticoagulants  Anticoagulant effect  Blood cell morphology  Blood physiology and biochemistry

中文摘要:

      鱼类血液极易凝结，经抗凝剂处理后的血液可充分满足各项生理指标检测，为衡量鱼体各项生理机能提供重要参考，同时，鱼类种属多样，在抗凝剂的选择和使用上存在一定差异。基于此，本研究以欧亚养殖良种大菱鲆(Scophthalmus maximus)为研究对象，选用乙二胺四乙酸二钾、肝素钠和柠檬酸钠3种抗凝剂，通过对大菱鲆正常状态和急性低氧胁迫条件下[溶解氧浓度为(1.2±0.3) mg/L]抗凝效果评价、血细胞形态观察和血液生理生化指标分析，筛选急性低氧胁迫条件下最佳血液抗凝剂。结果显示，正常溶解氧状态下，乙二胺四乙酸二钾抗凝效果显著，急性低氧胁迫下静置6 h和12 h后，抗凝效果显著抗凝剂分别为乙二胺四乙酸二钾和肝素钠。正常溶解氧状态肝素钠抗凝剂下血细胞出现双核现象，柠檬酸钠和乙二胺四乙酸二钾抗凝剂下分别出现嗜酸性、嗜碱性粒细胞和嗜碱性粒细胞，急性低氧胁迫导致乙二胺四乙酸二钾、肝素钠、柠檬酸钠抗凝剂均观察到双核血细胞，其中，肝素钠和柠檬酸钠抗凝剂还分别出现微核和无核血细胞。急性低氧胁迫处理后，3种抗凝剂作用下血液白细胞数目均显著增高(P<0.05)，乙二胺四乙酸二钾和肝素钠抗凝作用下，红细胞数目和血红蛋白含量显著升高(P<0.05)，而柠檬酸钠抗凝剂作用下，红细胞数目和血红蛋白含量显著降低(P<0.05)。3种抗凝剂作用下血浆葡萄糖和皮质醇浓度显著增高(P<0.05)，但肝素钠抗凝剂作用下，葡萄糖和皮质醇浓度显著低于柠檬酸钠和乙二胺四乙酸二钾(P<0.05)。综上所述，乙二胺四乙酸二钾可作为急性低氧胁迫条件下大菱鲆血液学分析的首选抗凝剂。

英文摘要:

      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.