The cytogenetic identification is important for the
characterization of an organism. In amphibians, the direct method is a
technique of routine for chromosomal characterization, but is necessary for the sacrifice of the copies. The aim of this work is
to develop the technique of lymphocyte cultures for the
species Rhinellaarenarum without sacrifice of specimen. Materials and Methods:
Male specimens’ Rhinellaarenarum were collected at San Luis; the blood sample was obtained
by cardiac puncture. The mediums tested were: the culture media: RPMI 1690 with
HEPES and Glutamine, MEM and F10; and different volumes of: phytohemagglutinin,
penicillin-streptomycin, fetal bovine serum and colchicine. Results: For the
standardization of the protocol we rely on cell culture techniques of fish and
human. The following parameters were standardized: volume blood: volume of 100
μl; culture medium was chosen: the best results were observed with RPMI 1640;
fetal bovine serum: he worked with a volume of 500 μl in the case of RPMI 1640
and F10, for MEM 1000 μl was used; phytohemagglutinin: it was observed that
large volumes of this reagent agglutinated cells, and hence the greater number of metaphases was obtained
with 10 μl; colchicine: best chromosome size was observed with 100 μl and an incubation time of 12 h. Conclusion: The standardized protocol is a simple
and inexpensive technique that does not require equipment or facilities of high
complexity and specimen are kept alive.
Cite this paper
Agüero, R. , Marsa, S. M. , Moreno, L. E. and Gomez, M. E. V. (2017). Peripheral Blood Lymphocyte Culture of Rhinella arenarum. Open Access Library Journal, 4, e4212. doi: http://dx.doi.org/10.4236/oalib.1104212.
Hillis, D.M. (1991) The Phylogeny of Amphibians: Current Knowledge and the Role of Cytogenetics. In: Green, D.M. and Sessions, S.K., Eds., Amphibian Cytogenetics and Evo-lution, Academic Press, San Diego, 7-31. https://doi.org/10.1016/B978-0-12-297880-7.50006-8
Aguiar Jr., O., Carvalho, K.A., Giaretta, A.A. and Recco-Pimentel, S.M. (2004) Cytogenetics of Hylodes and Crossodactylus Species (Anura, Leptodactylidae) with Comments on Hylodinae/Dendrobatidae Relationships. Genetica, 121, 43-53. https://doi.org/10.1023/B:GENE.0000019926.50310.26
Morescalchi, A. (1990) Cytogenetics and the Problem of Lissamphibian Relationships. In: Olmo, E., Ed., Cytoge-netics of Amphibians and Reptiles, Birkhauser Verlag, Berlin, 1-19.
Rodriguez Piazze, M.E. (1995) Cariotipo y patrones de bandas C en Bufo spinulosus arequipensis (Amphibia, Anura). Revista de Ecología Latino Americana, 2, 5-11.
King, M. and Rofe, R. (1976) Karyotypic Variation in the Australian Gecko Phyllodactylus marmoratus (Gray) (Gekkonidae: Reptilia). Chromosoma, 54, 75-87. https://doi.org/10.1007/BF00331835
Schmid, M. (1978) Chromosome Banding in Amphibia. I. Constitutive Heterochromatin and Nucleolus Organizer Regions in Bufo and Hyla. Chromosoma, 66, 361-368. https://doi.org/10.1007/BF00328536
Schmid, M. (1978) Chromosome Banding in Amphibia. II. Constitutive Heterochromatin and Nucleolus Organizar Regions in Ramdae, Microhylidae and Rhacophoridae. Chromosoma, 68, 131-148. https://doi.org/10.1007/BF00287145
Schmid, M., Olert, J. and Klett, C. (1979) Chromosome Banding in Amphibia. III. Sex Chromosome in Triturus. Chromosoma, 71, 29-55. https://doi.org/10.1007/BF00426365
Moorhead, P., Nowell, P., Mellman, W., Battips, D. and Hungerford, D. (1960) Chromosome Preparations of Leukocytes Cultures from Human Peripheral Blood. Experimental Cell Research, 20, 613-616. https://doi.org/10.1016/0014-4827(60)90138-5
Garnero, A.V., Boccelli, M., Oliveira, J.C.P., Ledesma, M.A., Montalti, D., Coria, N. and Gunski, R.J. (2013) Chromosomal Characterization of Four Antarctic Procellariiformes. Marine Ornithology, 41, 63-68.
Wójcik, E. and Smalec, E. (2007) Description of the Mallard Duck (Anasplatyrhynchos) Karyotype. Folia Biologica (Kraków), 55, 3-4. https://doi.org/10.3409/173491607781492588
Ramos, M.G., Descailleaux, J.L., Velásquez, M.R., Huanca, W.L., Iannuzz, L. and Perucatti, A. (2014) Adaptation of the Alpacas and Flame Lymphocyte Culture Technique for the Exchange Analysis of Sister Chromatids. Revista de Investigaciones Veterinarias del Perú, 25, 461-467.
Camacho-Garzon, J. and Burbano, C. (1999) Technique for the In Vitro Culture of Fish Lymphocytes. Dahlia-Revista de la asociacion colombiana de ictiólogos, 3, 69-79.
Vaira, M., Akmentins, M., Attademo, M., Baldo, D., Barrasso, D., Barrionuevo, S., Basso, N., Blotto, B., Cairo, S., Cajade, R., Céspedez, J., Corbalán, V., Chilote, P., Duré, P., Falcione, C., Ferraro, D., Gutierrez, R., Ingaramo, M.R., Junges, C., Lajmanovich, R., Lescano, J.N., Marangoni, F., Martinazzo, L., Marti, R., Moreno, L., Natale, G.S., Pérez Iglesias, J.M., Peltzer, P., Quiroga, L., Rosset, S., Sanabria, E., Sanchez, L., Schaefer, E., úbeda, C. and Zaracho, V. (2012) Categorization of the State of Conservation of the Amphibians of the Argentine Republic. Cuadernos de Herpetología, 26, 131-159.
Heyer, W.R., Donnelly, M.A., McDiarmid, R.W., Hayek, L.C. and Foster, M.S. (1994) Measuring and Monitoring Biological Diversity: Standard Methods for Amphibians. Editorial Peter Strupp/Princeton Smithsonian Institution, Washington DC.
Baraquet, M., Valetti, J.A., Salas, N.E. and Martino, A.L. (2011) Redescription of the Karyotype of Five Species of the Family Bufonidae (Amphibia: Anura) from Central Area of Argentina. Biologia, 66, 543-547. https://doi.org/10.2478/s11756-011-0048-8