Because hydrostatic pressure treatments seldom result in 100 percent triploid grass carp, each individual must be verified triploid before they can be stocked in the waters of many states.
Triploidy in grass carp is commonly verified by taking a blood sample and analyzing the volume of red blood cells, using an electronic particle counter. Blood samples can be taken from grass carp greater than 50 millimeters (2 inches) long.
Fish maybe anesthetized if necessary. The head of the fish is tilted back and the branchial artery on the isthmus is punctured with a blood lancet rinsed in EDTA between samples.
Approximately 1 microliter of blood is withdrawn using either a positive displacement micropipette with disposable polypropylene tips (Figure 2) or disposable hematocrit capillary tubes.
The blood sample is immediately placed in a vial containing an electrolyte solution (e.g., Hematall Azide-Free Isotonic Diluent, Fisher Scientific.The cell membrane of the red blood cells are dissolved (lysed) with a lysing agent (e.g., Hematall LA-Hgb Reagent, Fisher Scientific),leaving the nuclei.
The lysed blood cells are immediately scanned for ploidy determination with an electronic particle counter, calibrated to read both diploid and triploid red blood cell nuclei volumes. Diploid grass carp have a red blood cell nuclei volume of 10.06 cubic micrometers (µm3), while the mean volume of triploid red blood cell nuclei is 14.82 µm3.
Conclusions:
Sexually sterile fish are advantageous for controlling reproduction of exotic species, preventing the potential backcross of hybrids with either parent species resulting in intermingling of genetic material, and improving growth of aquiculture species.
Interest in sexually sterile fish has prompted research into production of triploid fish. Triploids have been produced by preventing the second meiotic division after the sperm enters the egg, thereby resulting in two sets of chromosomes from the female and one set from the male.
Triploid induction technology is used for wide-spread commercial production of grass carp and rainbow trout. Hydrostatic pressure is presently the most consistent method for the commercial production of triploid grass carp. Because these treatments seldom result in 100 percent triploid fish, each individual must be verified triploid before they can be stocked.
Authors:
R.W. Rottmann, J.V. Shireman, and F.A. Chapman
