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Comparison of CellRox green fluorescence upon thawing on in vitro Bos taurus and Bos indicus embryos cryopreserved by slow freezing or vitrification

Published online by Cambridge University Press:  18 September 2024

David Osornio ,
David Alejandro Contreras ,
Edgar Jimenez-Diaz ,
Tatiana Fiordelisio Open the ORCID record for Tatiana Fiordelisio [Opens in a new window] ,
Patricia López-Damian ,
José Francisco Martínez  and
Carlos Salvador Galina
David Osornio
Affiliation:
Departamento de Medicina y Zootecnia de Rumiantes, Facultad de Medicina Veterinaria y Zootecnia
David Alejandro Contreras
Affiliation:
Departamento de Medicina y Zootecnia de Rumiantes, Facultad de Medicina Veterinaria y Zootecnia
Edgar Jimenez-Diaz
Affiliation:
Unidad de Imagenologia Cuantitativa, Laboratorio Nacional de Soluciones Biomiméticas para Diagnóstico y Terapia LaNSBioDyT
Tatiana Fiordelisio*
Affiliation:
Unidad de Imagenologia Cuantitativa, Laboratorio Nacional de Soluciones Biomiméticas para Diagnóstico y Terapia LaNSBioDyT Laboratorio de Neuroendocrinología Comparada, Departamento de Ecología y Recursos Naturales, Biología, Facultad de Ciencias
Patricia López-Damian
Affiliation:
Laboratorio de Neuroendocrinología Comparada, Departamento de Ecología y Recursos Naturales, Biología, Facultad de Ciencias
José Francisco Martínez
Affiliation:
Departamento de Reproducción, Facultad de Medicina Veterinaria y Zootecnia, Universidad Nacional Autónoma de México, Mexico City, Mexico
Carlos Salvador Galina
Affiliation:
Departamento de Reproducción, Facultad de Medicina Veterinaria y Zootecnia, Universidad Nacional Autónoma de México, Mexico City, Mexico
*
Corresponding author: Tatiana Fiordelisio; Email: [email protected]
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Summary

The aim of this study was to compare the levels of reactive oxygen species (ROS) in Bos taurus and Bos indicus in vitro embryos cryopreserved using either slow freezing or vitrification. Embryos were divided into four groups based on subspecies and freezing method: Bos indicus slow freezing (BiSF; n = 8), Bos indicus vitrification (BiVT; n = 10), Bos taurus slow freezing (BtSF; n = 9), and Bos taurus vitrification (BtVT; n = 6). After thawing, the embryos were incubated with CellRox Green and images were obtained using a confocal microscope. The fluorescence intensity of each cell was measured and expressed as arbitrary units of fluorescence (auf) and compared using a multiple regression and unpaired t-test with α = 0.05. Results showed that subspecies and the freezing method significantly affected auf (P < 0.001; R2 = 0.1213). Bos indicus embryos had higher auf than Bos taurus embryos, whether frozen by slow freezing (67.05 ± 23.18 vs 51.30 ± 16.84, P < 0.001) or vitrification (64.44 ± 23.32 vs 47.86 ± 17.53, P < 0.001). Slow freezing induced higher auf than vitrification in both Bos taurus (51.30 ± 16.84 vs 47.86 ± 17.53, P < 0.001) and Bos indicus (67.05 ± 23.18 vs 64.44 ± 23.32, P < 0.014). In conclusion, Bos taurus embryos had lower ROS levels when frozen using vitrification, while Bos indicus embryos had consistent ROS patterns regardless of the freezing method. However, Bos indicus embryos frozen by slow freezing tended to have a higher number of cells with elevated ROS levels.

Keywords

BovineEmbryosROSSlow freezingVitrification
Type
Research Article
Information
Zygote , Volume 32 , Issue 3 , June 2024 , pp. 243 - 249
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Copyright
© The Author(s), 2024. Published by Cambridge University Press

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References

Agarwal, A., Said, T. M., Bedaiwy, M. A., Banerjee, J. and Alvarez, J. G. (2006). Oxidative stress in an assisted reproductive techniques setting. Fertility and Sterility, 86(3), 503–512. doi: 10.1016/j.fertnstert.2006.02.088 CrossRefGoogle Scholar
Aguilar, M. M., Galina, C. S., Merchant, H., Montiel, F., Canseco, R. and Márquez, Y. C. (2002). Comparison of stereoscopy, light microscopy and ultrastructural methods for evaluation of bovine embryos. Reproduction in Domestic Animals, 37(6), 341–346. doi: 10.1046/j.1439-0531.2002.t01-1-00379.x CrossRefGoogle Scholar
Arshad, U., Sagheer, M., González-Silvestry, F. B., Hassan, M. and Sosa, F. (2021). Vitrification improves in-vitro embryonic survival in Bos taurus embryos without increasing pregnancy rate post embryo transfer when compared with slow-freezing: A systematic meta-analysis. Cryobiology, 101, 1–11. doi: 10.1016/j.cryobiol.2021.06.007 CrossRefGoogle Scholar
Bó, G. A. and Mapletoft, R. J. (2018). Evaluation and classification of bovine embryos. Animal Reproduction, 10, 344–348. https://www.animal-reproduction.org/article/5b5a604cf7783717068b46a2 Google Scholar
Chatterjee, S. and Gagnon, C. (2001). Production of reactive oxygen species by spermatozoa undergoing cooling, freezing and thawing. Molecular Reproduction and Development, 59(4), 451–458. doi: 10.1002/mrd.1052 CrossRefGoogle Scholar
Contreras, D. A., Galina, C. S. and Chenoweth, P. (2021). Prospects for increasing the utilization of cattle embryo transfer by small-scale milk and meat producers in tropical regions. Reproduction in Domestic Animals, 56(12), 1479–1485. doi: 10.1111/rda.14015 CrossRefGoogle Scholar
Do, V. H., Catt, S., Kinder, J. E., Walton, S. and Taylor-Robinson, A. W. (2019). Vitrification of in vitro-derived bovine embryos: Targeting enhancement of quality by refining technology and standardising procedures. Reproduction, Fertility and Development, 31(5), 837–846. doi: 10.1071/RD18352 CrossRefGoogle Scholar
Dorland, M. (1994). Serum in synthetic oviduct fluid causes mitochondrial degeneration in ovine embryos. Journal of Reproduction and Fertility. Abstract Series, 13, 25.Google Scholar
Gardner, D. K. Lane, M. and Watson, A. J. (Eds.). (2004). A Laboratory Guide to Mammalian Embryo. Oxford University Press.CrossRefGoogle Scholar
Godinez, B., Galina, C. S., Leon, H., Gutierrez, M. and Moreno-Mendoza, N. (2013). Assessment of the viability of embryos stored in liquid nitrogen produced commercially using culture medium as a complementary test for stereoscopic microscopy. Zygote, 21(2), 110–114. doi: 10.1017/S0967199411000669 CrossRefGoogle Scholar
Gupta, K., Donlan, J. A. C., Hopper, J. T. S., Uzdavinys, P., Landreh, M., Struwe, W. B., Drew, D., Baldwin, A. J., Stansfeld, P. J. and Robinson, C. V. (2017). The role of interfacial lipids in stabilizing membrane protein oligomers. Nature, 541(7637), 421–424. doi: 10.1038/nature20820 CrossRefGoogle ScholarPubMed
Hochachka, P. W. (1986). Defense strategies against hypoxia and hypothermia. Science, 231(4735), 234–241. doi: 10.1126/science.2417316 CrossRefGoogle Scholar
Holm, P., Booth, P. J., Schmidt, M. H., Greve, T. and Callesen, H. (1999). High bovine blastocyst development in a static in vitro production system using SOFaa medium supplemented with sodium citrate and myo-inositol with or without serum-proteins. Theriogenology, 52(4), 683–700. doi: 10.1016/S0093-691X(99)00162-4 CrossRefGoogle Scholar
Kobayashi, K., Yamashita, S. and Hoshi, H. (1994). Influence of epidermal growth factor and transforming growth factor-α on in vitro maturation of cumulus cell-enclosed bovine oocytes in a defined medium. Journal of Reproduction and Fertility, 100(2), 439–446. doi: 10.1530/jrf.0.1000439 CrossRefGoogle Scholar
Lamb, G. C., Mercadante, V. R. G., Henry, D. D., Fontes, P. L. P., Dahlen, C. R., Larson, J. E. and DiLorenzo, N. (2016). Invited review: Advantages of current and future reproductive technologies for beef cattle production. The Professional Animal Scientist, 32(2), 162–171. doi: 10.15232/pas.2015-01455 CrossRefGoogle Scholar
López-Damián, E. P., Jiménez-Medina, J. A., Alarcón, M. A., Lammoglia, M. A., Hernández, A., Galina, C. S. and Fiordelisio, T. (2020). Cryopreservation induces higher oxidative stress levels in Bos indicus embryos compared with Bos taurus. Theriogenology, 143, 74–81. https://doi.org/10.1016/j.theriogenology.2019.12.001 CrossRefGoogle Scholar
López-Damián, E. P., Jiménez-Medina, J. A., Lammoglia, M. A., Pimentel, J. A., Agredano-Moreno, L. T., Wood, C., Galina, C. S. and Fiordelisio, T. (2018). Lipid droplets in clusters negatively affect Bos indicus embryos during cryopreservation. Anatomia, Histologia, Embryologia, 47(5), 435–443. doi: 10.1111/ahe.12382 CrossRefGoogle Scholar
Marquez, Y. C., Galina, C. S., Moreno, N., Ruiz, H., Ruiz, A. and Merchant, H. (2005). Seasonal effect on zebu embryo quality as determined by their degree of apoptosis and resistance to cryopreservation. Reproduction in Domestic Animals, 40(6), 553–558. doi: 10.1111/j.1439-0531.2005.00632.x CrossRefGoogle ScholarPubMed
McCord, J. M. (2000). The evolution of free radicals and oxidative stress. The American Journal of Medicine, 108(8), 652–659. doi: 10.1016/s0002-9343(00)00412-5 CrossRefGoogle ScholarPubMed
McEvoy, T. G., Robinson, J. J. and Sinclair, K. D. (2001). Developmental consequences of embryo and cell manipulation in mice and farm animals. Reproduction, 122(4), 507–518. doi: 10.1530/rep.0.1220507 CrossRefGoogle ScholarPubMed
Mucci, N., Aller, J., Kaiser, G. G., Hozbor, F., Cabodevila, J. and Alberio, R. H. (2006). Effect of estrous cow serum during bovine embryo culture on blastocyst development and cryotolerance after slow freezing or vitrification. Theriogenology, 65(8), 1551–1562. doi: 10.1016/j.theriogenology.2005.08.020 CrossRefGoogle Scholar
Stinshoff, H., Wilkening, S., Hanstedt, A., Brüning, K. and Wrenzycki, C. (2011). Cryopreservation affects the quality of in vitro produced bovine embryos at the molecular level. Theriogenology, 76(8), 1433–1441. doi: 10.1016/j.theriogenology.2011.06.013 CrossRefGoogle Scholar
Sudano, M. J., Caixeta, E. S., Paschoal, D. M., Martins, A., Machado, R., Buratini, J. and Landim-Alvarenga, F. D. C. (2014). Cryotolerance and global gene-expression patterns of Bos taurus indicus and Bos taurus taurus in vitro- and in vivo-produced blastocysts. Reproduction, Fertility, and Development, 26(8), 1129–1141. doi: 10.1071/RD13099 CrossRefGoogle Scholar
Tatone, C., Di Emidio, G., Vento, M., Ciriminna, R. and Artini, P. G. (2010). Cryopreservation and oxidative stress in reproductive cells. Gynecological Endocrinology, 26(8), 563–567. doi: 10.3109/09513591003686395 CrossRefGoogle Scholar
Viana, J. H. M., Siqueira, L. G. B., Palhao, M. P. and Camargo, L. S. A. (2018). Features and perspectives of the Brazilian in vitro embryo industry. Animal Reproduction (AR), 9, 12–18.Google Scholar
Visintin, J. A., Martins, J. F. P., Bevilacqua, E. M., Mello, M. R. B., Nicácio, A. C. and Assumpção, M. E. O. A. (2002). Cryopreservation of Bos taurus vs Bos indicus embryos: Are they really different? Theriogenology, 57(1), 345–359. doi: 10.1016/s0093-691x(01)00675-6 CrossRefGoogle Scholar
Walton, S., Catt, S. and Taylor-Robinson, A. W. (2017). A comparative analysis of the efficacy of three cryopreservation protocols on the survival of in vitro-derived cattle embryos at pronuclear and blastocyst stages. Cryobiology, 77, 58–63. doi: 10.1016/j.cryobiol.2017.05.007 Google Scholar
Zárate-Guevara, O. E., Prado, J. L. C., Canseco-Sedano, R., Palacios, F. M. and García, A. A. C. (2018). Transferencia de embriones bovinos criopreservados: Efecto de la blastocentesis. Agrociencia, 52, 21–32. https://agrociencia-colpos.org/index.php/agrociencia/article/view/1734 Google Scholar