Comparison of chromosome preparation methods for the different developmental stages of the half-smooth tongue sole, Cynoglossus semilaevis

Chromosome preparation in fish: effects of fish species and larval age

“…This study showed that using 11 % of Giemsa solution for 45 min could lead to better quality of stained chromosomes in both fish species and in the polyploid fish. Most of the previous methods employed concentrations similar to this study but with a lower incubation period (e.g., Hussain and McAndrew 1994;Pradeep et al 2011;Shao et al 2010). Some other studies used lower concentrations such as 4-5 % (FoppBayat and Woznicki 2006;Inokuchi et al 1994;Kligerman and Bloom 1977) or higher concentrations like 14 % (Karami et al 2010).…”

“…In turn, mitotic rate is a factor influenced by species and environmental parameters (Shelton et al 1997). By comparing metaphase chromosome numbers and mitotic rates in different life stages of Cynoglossus semilaevis, Shao et al (2010) found a higher mitotic rate in the larvae compared to the juveniles and adults. In a similar way, differences in mitotic division rates may explain why in this study different aged C. gariepinus and D. rerio larvae responded differently to the colchicine concentration and duration, and to the hypotonic solutions.…”

“…Larvae were incubated in 0.01, 0.025, or 0.05 % colchicine for 3 or 5 h. The procedure was followed as described by Shao et al 2010; Table 1). Finally, on each slide, clear metaphase chromosome spreads were counted.…”

“…Nevertheless, methods targeting embryonic and larval stages of fish have faced difficulties in achieving a reliable number of identifiable and wide-spread metaphase chromosomes probably due to the variations in mitotic cell division rates among different fish species (Shao et al 2010). In conventional chromosome preparation protocols, a spindle poison (e.g., colchicine) is employed to arrest the cells at their metaphase stage (Kligerman and Bloom 1977).…”

“…or 0.075 M KCl solution(Pradeep et al 2011) for 30 min, followed by the procedure ofShao et al (2010; Table 1). Finally, clear chromosome spreads were counted on each slide.FixationFollowing the optimization of colchicine concentration and incubation period, and hypotonic solution for the different larval age in each species, larvae were fixed in chilled Carnoy's fixative solution (ethanol:glacial acetic acid 3:1 vol/vol) for a total of 40 min.…”