Cytogenetics of small mammals from bone marrow extraction: a more detailed description of the in vivo technique with adjustments

Abstract

Cytogenetics is an effective tool for identifying species and solving taxonomic problems in small mammals. In vivo protocols are useful due to their low cost, but they present methodological divergence and need to be frequently adjusted. In this study, we present a standardized method for the cytogenetic analysis of small mammals from femoral bone marrow extraction, and demonstrate the importance of this standardization in contrast to other approaches.  We applied the technique of Ford and Hamerton (1956)  with the following updates: 1) adjustment of the quantities and concentrations of solutions used (colchicine and hypotonic solution); 2) detailed description of the steps; 3) use of centrifugation as a separation method; 4) addition of a slide mounting procedure, which improves visualization of metaphases. Six cytogenetic protocols using bone marrow were compared in important stages of the sample preparation phase.  There were differences in the concentration of colchicine solutions between all the methods. The action time of the solution also varied (from 15 minutes to 6 hours). The time and concentration of the colchicine solutions are crucial stages for chromosome condensation and the generation of good metaphases. In this way, we highlight that the methodology presented in our study contributes to the standardization of an in vivo cytogenetic protocol suitable for small mammals, which makes it possible to use the material and reduce the number of animals sacrificed for methodology adjustments, resulting in viable samples from the very first attempts.