DAO 63:89-94 (2005)  -  doi:10.3354/dao063089

Bacterial clearance rate and a new differential hemocyte staining method to assessimmunostimulant activity in shrimp

Kallaya Sritunyalucksana1, Warachin Gangnonngiw1, Somwit Archakunakorn2,Daniel Fegan2, Timothy W. Flegel1,*

1National Center for Genetic Engineering and Biotechnology (BIOTEC) and Centex Shrimp, and
2Shrimp Biotechnology Business Unit (SBBU), Faculty of Science, Mahidol University, Rama VI Road,Bangkok 10400, Thailand
*Corresponding author. Email:

ABSTRACT: New methods were developed to assess immunostimulant efficacy in the black tiger shrimp Penaeus monodon. Test shrimp were fed with 2 or 4% yeast extract (YE)-coated feed while controls were fed non-coated feed. After 4 wk of feeding, individual shrimp were assessed for total hemocyte counts (THC), the number of granular hemocytes (GH) and rate of bacterial clearance. For hemocyte counts, formalin-fixed hemolymph was stained with 1.2% Rose Bengal in 50% ethanol for 20 min at room temperature. Some of this mixture was used for THC with a hemocytometer while some was smeared on a microscope slide and left to dry before counterstaining with hematoxylin for GH counts. By this technique, high quality smears were obtained for accurate differential counts. Bacterial clearance assays were used to assess the sum effect of humoral and cellular defense mechanisms. Vibrio harveyi was injected intramuscularly at 1 × 108 cells per shrimp and hemolymph was collected in anticoagulant at 0, 15, 30 and 60 min post-injection for quadruplicate drop counts (20 µl) on TCBS agar. Total hemocyte counts for shrimp fed with 4% YE were significantly higher (p < 0.05) than those for shrimp fed with non-coated feed. The percentage of granular cells and the rates of bacterial clearance for the YE-fed shrimp were higher than those for shrimp fed the control diet. These 2 methods provide a simple and rapid comparison of shrimp groups for differences in anti-bacterial defense capacity.


KEY WORDS: Black tiger shrimp · Penaeus monodon · Hemocytes · Bacterial clearance · Immunostimulant


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