An efficient protocol for chromosome isolation from sterlet (A. ruthenus) embryos and larvae

Abstract

In the present study, the development of an efficient and feasible protocol for chromosome preparation from sterlet (A. ruthenus) embryos and larvae was carried out. In the established protocol, the mean efficiency of chromosome extraction ranged from 70 to 100%. The average number of recorded metaphases per slide was between 9 to 15. In general, the most satisfactory results were obtained for embryos at 6 dpf and larvae at the age of up to 7 dph. In the 24 dpf group, chromosome isolation was possible without immersion in spindle poison, however; in successive developmental stages, the minimal immersion time exceeded 1.5 hours, regardless of chorionation. Immersion for 14 hours did not compromise the efficacy of chromosome isolation. In the current study, successful chromosome isolation was determined mainly by hypotonization conditions. Younger developmental stages generally require the shortest hypotonization times, whereas older larvae require longer hypotonization times. The optimal hypotonization period is 5-15 minutes for embryos at 24 dpf, 40 minutes for embryos at 4dpf, and 50-60 minutes for fish at 6 dpf-7 dph. The only exception was the 24 hpf group where only blastula cells were used. An additional overnight fixation step significantly enhanced chromosome quality and supported chromosome counting especially in the 24 dpf group. The quality and quantity of chromosome slides were also significantly determined by tissue type, and the slides prepared from heads (gill cells) produced the best results.