Resveratrol-supplemented holding or re-culture media improves viability of fresh or vitrified-warmed in vitro-derived bovine embryos




Antioxidant effect; Embryonic development; Holding; In vitro production; Warming.


The effect of resveratrol supplementation on fresh (E1) or vitrified/warmed (E2) in vitro produced bovine embryos was investigated by evaluating the time-dependent response. After in vitro production, resveratrol (0.5 µM) was added to the incubation media and after two incubation periods with or without resveratrol, blastocysts were re-cultured for 24h. The rates of re-expansion, hatching, total cell number (TCN), apoptotic cells (ACN), reactive oxygen species (ROS) and intracellular glutathione (GSH) content were evaluated. For E1, the re-expansion rate differed at 6 and 10h within and between treatments (P<0.05), as did the re-expansion rate after 24h (P<0.01). The hatching rate increased after 10h with resveratrol (P<0.01) with differences within (P<0.05), but not between treatments after 24h of re-cultivation. At E2, hatching rate differed between treatments at 24h (P<0.01), with higher TCN in resveratrol-treated blastocysts after 10h (P<0.01). Resveratrol supplementation reduced ROS generation in E1 and E2 after 10h of incubation and increased GSH content (P<0.01). These results indicate that supplementation of holding re-cultivation medium with resveratrol for treatment of fresh or vitrified/warmed in vitro produced bovine embryos has a positive and time-dependent effect. The reduction of ROS content, the increase of GSH and the anti-apoptotic ability of resveratrol are responsible for its protective effects, allowing an extension of embryo storage time before transfer to recipients.


Al-Gubory, K. H., Fowler, P. A., & Garrel, C. (2010). The roles of cellular reactive oxygen species, oxidative stress and antioxidants in pregnancy outcomes. International Journal of Biochememistry and Cell Biology, 42, 1634-50.

Block, J., Bonilla, L., & Hansen, P. J. (2010). Efficacy of in vitro embryo transfer in lactating dairy cows using fresh or vitrified embryos produced in a novel embryo culture medium. Journal of Dairy Science, 93, 5234-42.

Block, J., Fischer-Brown, A. E., Rodina, T. M., Ealy, A. D., & Hansen, P. J. (2007). The effect of in vitro treatment of bovine embryos with IGF-1 on subsequent development in utero to day 14 of gestation. Theriogenology, 68, 153-61.

Bó, G., & Mapletoft, R. J. (2013). Evaluation and classification of bovine embryos. Animal Reproduction, 10, 344-8.

Cagnone, G., & Sirard, M. A. (2016). The embryonic stress response to in vitro culture: insight from genomic analysis. Reproduction, 152, 247-61.

Cavalieri, F. L. B., Andreazzi, M. A., Colombo, A. H. B., Emanuelli, I. P., Moreski, D. A. B., & Silva, W. M. (2015). Studies on in vitro bovine embryos during their transport. Ars Veterinaria, 31, 7-11. https://doi: 10.15361/2175-0106.2015v31n1p07-11.

Dalvit, G. C., Cetica, P. D., Pintos, L. N., & Beconi, M. T. (2005). Reactive oxygen species in bovine embryo in vitro production. Biocell , 29, 209-12.

Do, V. H., Catt, S., Amaya, G., Batsiokis, M., Walton, S., & Taylor-Robinson, A. W. (2018). Comparison of pregnancy in cattle when non-vitrified and vitrified in vitro-derived embryos are transferred into recipients. Theriogenology, 15, 105-10.

Ferraz, P. A., Burnley, C., Karanjam, J., Vieira-Neto, A., Santos, J. E., Chebel, R. C., & Galvão, K. N. (2016). Factors affecting the success of a large embryo transfer program in Holstein cattle in a commercial herd in the southeast region of the United States. Theriogenology, 86, 1834-41.

Hansen, P. J., Block, J., Loureiro, B., Bonilla, L., & Hendricks, K. E. M. (2010). Effects of gamete source and culture conditions on the competence of in vitro-produced embryos for post-transfer survival in cattle. Reproduction, Fertility, Development, 22, 59-66.

Hara, T., Kin, A., Aoki, S., Nakamura, S., Shirasuna, K., Kuwayama, T., & Iwata, H. (2018). Resveratrol enhances the clearance of mitochondrial damage by vitrification and improves the development of vitrified-warmed bovine embryos. PloS One, 13(10), e0204571.

Hayashi, T., Kansaku, K., Abe, T., Ueda, S., & Iwata, H. (2019). Effects of Resveratrol treatment on mitochondria and subsequent embryonic development of bovine blastocysts cryopreserved by slow freezing. Animal Science Journal, 90, 849– 856.

Hayashi, T., Ueda, S., Mori, M., Baba, T., Abe, T., & Iwata, H. (2018). Influence of Resveratrol pretreatment on thawed bovine embryo quality and mitochondrial DNA copy number. Theriogenology, 106, 271-78.

Ideta, A., Aoyagi, Y., Tsuchiya, K., Kamijima, T., Nishimiya, Y., & Tsuda, S. (2013). A simple medium enables bovine embryos to be held for seven days at 4°C. Science Reproduction, 3, 1173.

Lee, T. H., Lee, M. S., Liu, C. H., Tsao, H. M., Huang, C. C., & Yang, Y. S. (2012). The association between microenvironmental reactive oxygen species and embryo development in assisted reproduction technology cycles. Reproduction Science, 19, 725–32.

Madrid Gaviria, S., López Herrera, A., Urrego, R., Restrepo Betancur, G., & Echeverri Zuluaga, J. J. (2019). Effect of Resveratrol on vitrified in vitro produced bovine embryos: Recovering the initial quality. Cryobiology, 89, 42–50.

Madrid Gaviria, S., Morado, S. A., López Herrera, A., Restrepo Betancur, G., Urrego, R., Echeverri Zuluaga, J., & Cética, P. D. (2018). Resveratrol supplementation promotes recovery of lower oxidative metabolism after vitrification and warming of in vitro-produced bovine embryos. Reproduction, Fertility, Development, 31, 521-528.

Marinho, L. S. R., Untura, R. M., Morotti, F., Moino, L. L., Rigo, A. G., Sanches, B. V., Pontes, J. H. F., Seneda, M. M. (2012). Large-scale programs for recipients of in vitro-produced embryos. Animal Reproduction, 9, 323-8.

Marques, T. C., Silva, E. C., Diesel, T. O., Leme, L. O., Martins, C. F., Dode, M., Alves, B. G., Costa, F., de Oliveira, E. B., & Gambarini, M. L. (2018). Melatonin reduces apoptotic cells, SOD2 and HSPB1 and improves the in vitro production and quality of bovine blastocysts. Reproduction in Domestic Animals, 53, 226–36.

Marques, T. C., Santos, E., Diesel, T. O., Martins, C. F., Cumpa, H., Leme, L. O., Dode, M., Alves, B. G., Costa, F., Oliveira, E. B., & Gambarini, M. L. (2021). Blastocoel fluid removal and melatonin supplementation in the culture medium improve the viability of vitrified bovine embryos. Theriogenology, 160, 134–141.

Marsico, T. V., Camargo, J., Valente, R. S., & Sudano, M. J. (2019). Embryo competence and cryosurvival: Molecular and cellular features. Animal Reproduction, 16, 423-39.

Morado, S. A., Cetica, P. D., Beconi, M. T., & Dalvit, G. C. (2009). Reactive oxygen species in bovine oocyte maturation in vitro. Reproduction, Fertility, Development, 21, 608–14.

Mori M, Otoi T, & Suzuki T. (2008). Correlation between the Cell Number and Diameter in Bovine Embryos Produced in vitro. Reproduction of Domestic Animals, 37, 181-4.

Nedambale, T. L., Dum, F., Yang, X., & Tian, X. C. (2006). Higher survival rate of vitrified and thawed in vitro produced bovine blastocysts following culture in defined medium supplemented with beta-mercaptoethanol. Animal Reproduction Science, 93, 61–75.

Papaioannou, V. E., & Ebert, K. M. (1988). The preimplantation pig embryo: cell number and allocation to trophectoderm and inner cell mass of the blastocyst in vivo and in vitro. Development, 102, 793-803.

Romek M, Gajda B, Krzysztofowicz E, & Smorag Z. (2010). Changes of lipid composition in non-cultured and cultured porcine embryos. Theriogenology, 74, 265-76.

Salzano A, Albero G, Zullo G, Neglia G, Abdel-Wahab A, Bifulco G, & Zicarelli L, Gasparrini B. (2014). Effect of Resveratrol supplementation during culture on the quality and cryotolerance of bovine in vitro produced embryos. Animal Reproduction Science,151 (3-4), 91-6.

Sanches B. V., Zangirolamo A. F., & Seneda M. M. (2019). Intensive use of IVF by large-scale dairy programs. Animal Reproduction, 16, 394-401.

Silveira, M. M., Marques, T. C., Silva M. A. P., & Leão K. M. (2020). Development stag e at packaging affects viability of in vitro produced bovine embryos. Research, Society and Development, 9: e13496361.

Sovernigo, T C., Adona, P. R, Monzani, P. S., Guemra, S, Barros, F, Lopes, F. G., & Leal, C. (2017). Effects of supplementation of medium with different antioxidants during in vitro maturation of bovine oocytes on subsequent embryo production. Reproduction of Domestic Animals, 52, 561-9.

Sprícigo, J. F., Morató, R, Arcarons, N., Yeste, M., Dode, M. A., López-Bejar, M., & Mogas, T. (2017). Assessment of the effect of adding L-carnitine and/or Resveratrol to maturation medium before vitrification on in vitro-matured calf oocytes. Theriogenology, 89, 47-57.

Sudano, M. J., Caixeta, E. S., Paschoal, D. M., Martins, A., Machado, R., Buratini, J., & Landim-Alvarenga, F. D. C. (2013). Cryotolerance and global gene-expression patterns of Bos taurus indicus and Bos taurus taurus in vitro- and in vivo-produced blastocysts. Reproduction, Fertility, Development, 26, 1129-41. 10.1071/RD13099

Taniyama, A., Watanabe, Y., Nishino, Y., & Inoue, T. (2011). Assisted hatching of poor-quality bovine embryos increases pregnancy. Journal of Reproduction and Development, 57, 543–6.

Ufer, C., Wang, C. C., Borchert, B., Heydeck, D., & Kuhn, H. (2010). Redox control in mammalian embryo development. Antioxidant Redox Sign, 13, 833–75.

Wang, F., Tian, X., Zhang, L., He, C., Ji, P., Li, Y., Tan, D., & Liu, G. (2014). Beneficial effect of Resveratrol on bovine oocyte maturation and subsequent embryonic development after in vitro fertilization. Fertility and Sterility, 101, 577–86 10.1016/j.fertnstert.2013.10.041.

Yu, S., Long, H., Lyu, Q. F., Zhang, Q. H., Yan, Z. G., Liang, H. X., & Qi, C. (2014). Protective effect of quercetin on the development of pre-implantation mouse embryos against hydrogen peroxide- induced oxida-tive injury. PLoS One, 9: e89520.




How to Cite

SILVA, A. R. N. .; MARQUES, T. C. .; SANTOS, E. C. S. .; DIESEL, T. O. .; MACEDO , I. M. .; TEIXEIRA , R. C. .; MARTINS, C. F. .; ALVES , B. G. .; GAMBARINI, M. L. Resveratrol-supplemented holding or re-culture media improves viability of fresh or vitrified-warmed in vitro-derived bovine embryos. Research, Society and Development, [S. l.], v. 10, n. 14, p. e367101422097, 2021. DOI: 10.33448/rsd-v10i14.22097. Disponível em: Acesso em: 24 jun. 2024.



Agrarian and Biological Sciences