Aquaculture America 2024

February 18 - 21, 2024

San Antonio, Texas

COMPARISON OF HEMOLYMPH COLLECTED WITH COMMERCIAL AND LAB-MADE ANTICOAGULANT

Susan Laramore* ,  Lindee Mason and Ahmed Mustafa

 

 Aquaculture and Stock Enhancement Program

 5600 US 1 North

 Fort Pierce, FL 34946 USA

 slaramo1@fau.edu

 



 Shrimp, as well as other invertebrate species, have extremely quick hemolymph clotting times.  A non-clotted sample is optimal to effectively measure most hematological parameters.  Syringes are  typically  coated with some form of anticoagulant  prior to hemolymph collection  to delay  or prevent  clotting  long enough to allow time for such analysis to be conducted.  During a recent experiment we used  a commercial ly available anticoagulant, containing dipotassium ethylenediamine tetraacetate,  that had been successfully  used  to collect  hemolymph  from other invertebrates  for Litopenaeus vannamei; however, the hemolymph clotted  too fast to be used for the intended analysis . To preserve the remaining samples, a solution of lab-made anticoagulant , containing sodium citrate,  was used in place of  the commercial anticoagulant . There were observable differences between the hemolymph collected with the two anticoagulants, including s ample  clotting time and sample hue. The hemolymph collected with the commercial anticoagulant clotted within seconds and had a cloudy, slightly purple color, while the lab-made anticoagulant did not clot and was clear in appearance. Due to the increased clotting and the cloudy appearance of the hemolymph when using  the commercial anticoagulant, it was difficult  to obtain differential hemocyte counts using a hemocytometer . When using the hemolymph collected with  the lab-made anticoagulant the samples were clear  and the different cell types were easily distinguished  under the microscope. Measuring phagocytic capacity of hemolymph collected with the commercial anticoagulant was also challenging. The sample was too thick and the unadhered hemocytes did not wash off the slides, creating a dense mound that became saturated with dye when staining , making it difficult to visualize the  phagocytizing hemocytes. When using the lab-made anticoagulant , the unadhered hemocytes were readily washed away, and staining was even . This allowed for  easier visualization of phagocytic activity.  Due to these observable differences, the use of the commercially available anticoagulant  initially used is not advocated for the  collection of hemolymph from L. vannamei .  Although  this product  may work well as an anticoagulant for some invertebrate species we recommend the use of  sodium citrate for L. vannamei