ABSTRACT
In adult teleost fi shes, hematopoiesis mainly takes place in the cephalic kidney where stem cells proliferate to give rise to the various blood cell lineages. In addition, the spleen, the major lymphoid organ, also retains the capability to originate lymphocytes from lymphoid cell precursors. Since cephalic kidneys and the spleen regularly produce dividing cells, their tissues are commonly used for preparing fi sh chromosomes through direct in vivo methods (see Chapter 3, 4, 6). However fi sh species adapted to Antarctic waters have a low metabolism and can show complete aplasia, especially after the reproductive season, so the usual in vivo chromosome preparation techniques often do not produce good results. After some unsuccessful attempts to develop an effective
method based on lymphocyte culture (as described in Chapter 9), in these species, we developed a protocol for preparing chromosomes by cell culture from the cephalic kidney and spleen. This protocol has been performed in a fi eld laboratory in Terre Adelie, and has also been tested on board a research vessel in the same sector of the Southern Ocean. The method always produced a higher mitotic index than the direct in vivo methods in the polar species tested. A suspension of cephalic kidney and/or spleen cells is prepared and cultured in L-15 Leibovitz’s culture medium, without bicarbonate, supplemented with L-glutamine, fetal calf serum, lectins and antibiotics, for a period of up to one week at 0° to +2°C. Colchicine is added 6 hrs prior to harvesting cells. This is followed by a hypotonic treatment (1 hr at +2°C) and conventional steps of fi xation. The most effi cient lectins for stimulating cell division in these fi shes are concanavalin A and pokeweed m itogen, used separately or combined together. Phyto-hemaglutinin (PHA) and lipopolysaccharide of Escherichia coli (LPS) did not provide satisfying results.
