Numerous factors (e.g., stress, vitamin K concentration, liver damage) can influence hemostasis in fish . Despite being relatively understudied, the hemostasis of bony fish shows striking parallels to that of other vertebrates, such as mammals, encompassing both intrinsic and extrinsic pathways. This study aimed to measure the prothrombin time (PT), activated partial thromboplastin time (aPTT ), fibrinogen concentration (Fib), and antithrombin activity (AT), in plasma samples collected from bony fishes using an automated coagulation analyzer.
We examined K3 EDTA plasma samples obtained from 90 European seabass (weight 154.9 ± 34.1 gr ) reared in a recirculation aquaculture system (temperature 23.0 ± 2.5 °C) at the Laboratory of Aquaculture of the University of Bologna, Cesenatico, Italy. Blood s amples were collected by venipuncture from the caudal vein, centrifuged at 3,000x g within 2 hours and analyzed within 12 hours from collection using an automated analyzer (Siemens BCS XP + manufacturer’s reagents , Siemens Healthineers , Marburg, Germany). Plasma samples were stored at 0 - + 4°C until analysis. PT was measured using 50 μl of plasma incubated with Thromborel S (Human thromboplastin containing Calcium). The aPTT was measured by incubating plasma sample ( 50 μl ) with Dade Actin Activated Cephaloplastins r eagent to allow the phospholipid and surface activator interaction, followed by the addition of calcium chloride solution as a trigger for the coagulation. For AT analysis, the Innovance Antithrombin kit was used. It consists of adding an excess amount of factor Xa (FXa ) to 10 μl of a 1:4 prediluted plasma with a buffer. Subsequently, the factor Xa is complexed and inactivated by the AT present in the sample The remaining, unbound FXa was cleaved into a specific chromogenic substrate and then releasing a dye . Fib was measured following the Clauss method, by adding the Dade Thrombin reagent to the plasma sample ( 50 μl) , prediluted 1:10 with Dade Owren’s Veronal buffer . All analyses were conducted at a temperature of +37 °C (default setting) and coagulometric and colorimetric readings were performed at a wavelength of 405 nm . A total of 160 μl of the sample was required for the 4 analyses. All methods used were fully automated and results were available in 9 min.
The analyzer successfully measured aPTT , Fib and AT which results were similar to those found in mammal species (Table 1). In this study, we demonstrated that the intrinsic coagulation pathway, AT, and Fib can be evaluated in the plasma of European Seabass with a commercially available automated coagulation analyzer . PT analysis was unsuccessful probably because this variable is affected by changes in temperature, likely due to the presence of heat-sensitive clotting factors in the extrinsic coagulation pathway as previously reported . The analytical methods used in this study seem promising for prospective research studies concerning hemostasis in bony fish by rapidly processing a high number of samples. An extensive validation of this method is necessary for European seabass, evaluating the repeatability of the results , stability of the samples for analysis and species-specific variations.