DNA extraction methods of canine sperm cells to sexing by quantitative PCR





Chromosome; Dog; Phenol-chloroform; Semen; Sexing sperm.


Despite similarity to other cell types, the sperm has a compacted chromatin which makes DNA extraction more difficult. Based on this, this study compared different sperm concentrations and three DNA extraction techniques to use in qPCR. Four protocols were tested: a non-commercial using phenol:chloroform (M1) alone or associated with a preparatory kit (Differex-System-Kit-M2), and two commercial with mini-columns extraction: M3 (Illustra-Blood-Genomic-Prep-Mini-Spin) and M4 (Wizard®-Genomic-DNA-Purification). Four sperm concentrations were used (1, 10, 30 and 50x106) from ejaculates of 3 dogs. After extraction, the DNA was used for qPCR with primers directed against X and Y chromosomes. M1 and M2 protocols recovered high DNA concentration independently of initial sperm concentration, with a satisfactory ratio of 260/280 absorbances (mean 1.80 and 2.10, M1 and M2 respectively). In contrast, other methods recovered low DNA concentration and with poor quality in M3 (mean 1.60 in M3 and 1.85 in M4). The DNA extraction of canine sperm cells using protocols with phenol:chloroform (M1 and M2) was efficient. The M1 protocol notably allowed the quantification of X and Y chromosomes in qPCR using low number of sperm cells (1x106). This study provides the first comparison of DNA extraction techniques of canine sperm and it is concluded that independent of sperm concentration used, including use of only 1x106 sperm, DNA extraction from these cells using phenol:chloroform has satisfactory results regarding the quantity and quality of the extracted sample, and enabled quantitation of target gene sequences in qPCR, allowing subsequent use in sperm sexing and other biotechnologies.


Abu-Amero, K. K., Jaeger, M., Plantinga, T., Netea, M. G., & Hassan, H. Y. (2013). Genetic variation of TLR2 and TRL4 among the Saudi Arabian population: insight into the evolutionary dynamics of the Arabian Peninsula. Genet. Test. Mol. Bioma, 17(2), 166-169.

Almeida, D. B., Santos, A. G., Costa, M. A. P., Oliveira, P. A., Bassini, L. N., Moreira, C. G. A., Carla, G. A., Tavares, R. A., & Moreira, H. L. M. (2009). Utilização de um novo protocolo de extração de DNA em tilápias (Oreochromisniloticus). In: XI Encontro de Pós-Graduação. Evoluir sem Extinguir: por uma ciência do devir. Pelotas: Editora e Gráfica Universitária.

Alvarez, M. J., Vila-Ortiz, G. J., Salibe, M. C., Podhajcer, O. L., Pitossi, F. J. (2007). Model based analysis of real-time PCR data from DNA binding dye protocols. BMC Bioinformatics, 8(85), 1-10.

Bueno, V. (2004). DNA e aperfeiçoamento das técnicas de extração. Rev. Bras. Hematol. Hemoter, 26(4), 233-234.

Coelho, E. G. A., Oliveira, D. A. A., Teixeira, C. S., Sampaio, I. B. M., Rodrigues, S. G., & Alves, C. (2004). Comparação entre métodos de estocagem de DNA extraído de amostras de sangue, sêmen e pelos e entre técnicas de extração. Arq. Bras. Med. Vet. Zootec, 56(1), 111-115.

Crowe, J. S., Cooper, H. J., Smith, M. A., Sims, M. J., Parker, D., & Gewert, D. (1991). Improved cloning efficiency of polymerase chain reaction (PCR) product safter proteinase K digestion. Nucleic Acids Res, 19(1), 184-186.

Eddy, E. M. (2006). The spermatozoon. In EIL, J.D. Ed(s) Knobil and Neill’ Phisiology of Reproduction. United States of America: Elsevier, 3-54.

Edwards, K. T., Goddard, J., Jones, T. L., Paddock, C. D., & Varela-Stokes, A. S. (2011). Cattle and the natural history of rickettsia parkeri in Mississipi. Vec. Bor. Zoo. Dis, 11(5), 485-491.

Fernandes, J. V., Meissner, R. V., Fernandes, T. A. A. M., Rocha, L. R. M., Cabral, M. C., & Villa, L. L. (2004). Comparação de três protocolos de extração de DNA a partir de tecido fixado em formol e incluído em parafina. J. Bras. Patol. Med. Lab, 40(3), 141-146.

GE Manual. (2007). Illustra blood genomic Prep Mini Spin Kit. Available: https://au.vwr.com/assetsvc/asset/en_AU/id/17887080/contents

Hanson, E. K., & Ballantyne, J. (2004). A highly discriminating 21 locus Y-STR “megaplex” system designed to augment the minimal haplotype loci for forensic casework. J. Forensic Science, 49, 40-51.

Kamani, J., Baneth, G., Mumcuoglu, K. Y., Waziri, N. E., Eyal, O., Guthmann, Y., & Harrus, S. (2013). Molecular detection and characterization of tick-borne pathogens in dogs and ticks from Nigeria. PLOS Negl. Trop. Dis., 7(3), 1-7.

Kuretake, S., Kimura, Y., Hoshi, K., & Yanagimachi, R. (1996). Fertilization and development of mouse oocytes injected with isolated sperm heads. Biol Reprod, 55, 789-95.

Lalam, N. (2006). Estimation of the reaction efficiency in polymerase chain reaction. J. Theor. Biol, 242(4), 947–953.

Lee, K., Haugen, H. S., Clegg, C. H., & Braun, R. E. (1995). Premature translation of protamine 1mRNA causes precocious nuclear condensation and arrests spermatid differentiation in mice. Proc. Natl. Acad. Sci. USA, 92, 12451-55.

Lee, Y. K., Kim, H. W., Liu, C. L., Lee, H. K. A. (2003). A simple method for DNA extraction from marine bacterium that produce extracellular materials. J. Micro. Methods, 52, 245-250.

Liss, B. (2002). Improved quantitative real-time RT-PCR for expression profiling of individual cells. Nucleic Acids Res, 30(17), 1-9.

Marushige, Y., & Marushige, K. (1975). Transformation of sperm histone during formation and maturation of rat spermatozoa. J. Biol. Chem, 250, 39-45.

Mothé, G. B., Scott, C., & Souza, F. F. (2018a). Centrifugação em gradiente de densidade: alternativa para sexagem espermática em cães? Vet. e Zootec, 25, 9-19.

Mothé, G. B., Scott, C., Sicherle, C., Guaitolini, C. R. F., Freitas Dell’Aqua, C. P., Malossi, C. D., Araújo-Junior, J. P., & Souza, F. F. (2018b). Sperm sexing with density gradient centrifugation in dogs. Anim. Reprod. Sci., 199, 84-92.

Namba, H., Nakashima, M., Hayashi, T., Hayashida, N., Maeda, S., Rogounovitch, T. I., Ohtsuru, A., Saenko, V. A., Kanematsu, T., & Yamashita, S. (2003). Clinical implication of hot spot BRAF mutation, V599E, in papillary thyroid cancers. J. Clin. Endocrinol. Metab., 88, 4393–4397.

Oliveira, M. C. S., Regitano, L. C. A., Roese, A. D., Anthonisen, D. G., Patrocínio, E., Parma, M. M., Scagliusi, S. M. M., Timóteo, W. H. B., & Jardim, S. N. (2007). Fundamentos teórico-práticos e protocolos de extração e de amplificação de DNA por meio de reação em cadeia de polimerase. Embrapa Pecuária Sudeste. 12-17.

Pereira, A. S., Shitsuka, D. M., Parreira, F. J., & Shitsuka, R. (2018). Metodologia da pesquisa científica. UFSM.

Pógany, G. C., Corzett, M., Weston, S., Balhorn, R. (1981). DNA and protein content of mouse sperm. Implications regarding sperm chromatin structure. Exp. Cell Res, 136(1), 127-136.

Scott, C., Lourenço, T. T., Moira, D. S., Moscardini, M. M., Honsho, C. S., Souza, F. F. (2016). Viabilidade de espermatozoides criopreservados de touros colhidos do epidídimo após 12 horas a 4ºC. Rev. Bras. Ci. Vet., 23, 191-195.

Scott, C., Souza, F. F, Mothé, G. B., Aristizabal, V. H. V., & Dell’Aqua Junior, J. A. (2018). Estudo sobre as diferentes técnicas de sexagem de espermatozoides. Vet. e Zootec, 25, 9-17.

Seager, S. W. J., & Platz, C. C. (1977). Artificial insemination and frozen semen in the dog. Vet. Clin. North Am, 7, 757-64.

Silva, E. C. B., Pelinca, M. A., Acosta, A. C., Silva, D. M. F., Gomes Filho, M. A., & Guerra, M. M. P. (2014). Comparative study of DNA extraction methodologies from goat sperm and its effects on polymerase chain reaction analysis. Genet. Mol. Res, 13(3), 6070-78.

Svec, D., Tichopad, A., Novosadova, V., Pfaffl, M. W., & Kubista, M. (2015). How good is a PCR efficiency estimate: Recommendations for precise and robust qPCR efficiency assessments? Biomol. Detect. Quantif, 3, 9-16.

Taylor, T. M. (2005). Comparing calf sex ratio and semen sex ratio determined by conventional PCR. Dissertation of Master, Louisiana State University and Agricultural and Mechanical College, Program in Animal and Dairy Sciences, Southern Arkansas University, 10-11.

Tsukada, K., Asamura, H., Ota, M., Kobayashi, K., & Fukushima, H. (2006). Sperm DNA extraction from mixed stains using Differex System. Elsevier, International Congress Series, 1288, 700-703.

Wittwer, C. T., Herrmann, M. G., Moss, A. A., & Rasmussen, R. P. (1997). Continuous fluorescence monitoring of rapid cycle DNA amplification. Biotechniques, 22(1), 130-138.




How to Cite

MOTHÉ, G. B. .; SCOTT, C.; MALOSSI, C. D. .; ARAÚJO-JUNIOR, J. P. .; SOUZA, F. F. de . DNA extraction methods of canine sperm cells to sexing by quantitative PCR. Research, Society and Development, [S. l.], v. 12, n. 4, p. e3612440769, 2023. DOI: 10.33448/rsd-v12i4.40769. Disponível em: https://rsdjournal.org/index.php/rsd/article/view/40769. Acesso em: 20 may. 2024.



Agrarian and Biological Sciences