Evaluation of in vitro production rates of bovine embryos using melatonin-supplemented culture medium





Blastocysts; Bovine; In vitro culture; Melatonin; In vitro embryo production.


In this study, we aimed to evaluate the rate of bovine embryo production by using 50 ng/mL melatonin supplementation in in vitro culture medium. For this, oocytes from slaughterhouse ovaries were matured in vitro in TCM-199 medium with Earle’s balanced salt solution + 10% SFB, FSH, and LH in an atmosphere of 5% CO2. Twenty-four hours after IVM, the oocytes underwent in vitro fertilization in human tubal fluid under the same conditions as above, for 18 h. Semen was fractionated by Percoll gradient centrifugation and the concentration of sperm was adjusted to 1 × 106/mL. Probable zygotes were then divided into two groups: the control group grown in drops of 90 μL SOFaa medium + 0.6% BSA + 2.5% SFB, in an atmosphere of 5% CO2, 90% N2, and a melatonin group (Mel), similarly cultured in 90 μL drops of SOFaa medium + 0.6% BSA + 2.5% SFB + 50 ng/mL melatonin. Cleavage rates were assessed on day 3 (D3). On D7, blastocyst formation rates were evaluated. Eight routines were performed (320 oocytes per routine). Data were analyzed with ANOVA, followed by Tukey’s range test using a general linear model. The level of statistical significance was set at 5%. There were no differences in the rates of cleavage or blastocyst formation between the control and melatonin groups (P > 0.05). Thus, under the conditions used in this study, supplementation with melatonin did not yield benefits in increasing the rate of in vitro bovine embryo production.

Author Biographies

Ricardo Magalhães, Paranaense University

Animal Reproduction

Carlos Renato de Freitas Guaitolini, Paranaense University

Animal Reproduction

Marcio Luiz Denck Tramontin, Paranaense University

Animal Reproduction

Danielle Andressa Oliveira Sestari, Paranaense University

Animal Reproduction

Bruno Argenton de Barros, Paranaense University

Animal Reproduction


Adona, P. R.; Pires, P. R. L.; Quetglas, M. D.; Schwarz, K. R. L.; & Leal, C. L. V. (2008). Nuclear maturation kinetics and in vitro embryo development of cattle oocytes prematured with butyrolactone. I. combined or not combined with roscovitine. Animal Reproduction Science 104: 389-397.

Asgari, Z.; Ghasemian, F.; Ramezani, M.; & Bahadori, M. H. (2012). The effect of melatonin on the developmental potential and implantation rate of mouse embryos. Cell Journal 14(3): 203-208.

Assis, P. M. (2014). Melatonina no meio de cultivo in vitro de embriões bovinos: dinâmica e ação antioxidante. Dissertação. Pós-Graduação em Reprodução Animal da Faculdade de Medicina Veterinária e Zootecnia. Universidade de São Paulo. 2014.

Cebrian-Serrano, A.; Salvador, I.; Raga, E.; Dinnyes, A.; & Silvestre, A. (2013). Beneficial Effect of Melatonin on Blastocyst In Vitro Production from Heat-Stressed Bovine Oocytes. Reproduction in Domestic Animals 48:738-746.

Corrêa, G. A.; Rumpf, R.; Mundim, T. C. D.; Franco, M. M.; & Dode, M. A. N. (2008). Oxygen tension during in vitro culture of bovine embryos: effect in production and expression of genes related to oxidative stress. Animal Reproduction Science 104:132–142.

El-Raey, M.; Geshi, M.; Somfai, T.; Kaneda, M.; Hirako, M.; Abdel- Ghaffar, A. E.; Sosa, G. A.; El-Roos, M. E. A. A.; & Nagai, T. (2011). Evidence of melatonin synthesis in the cumulus oocyte complexes and its role in enhancing oocyte maturation in vitro in cattle. Molecular, Reproduction and Development 78(4):250–262.

Gonçalves, R. L. R. & Vianna, J. H. M. (2019). Situação atual da produção de embriões bovinos no Brasil e no mundo. Anais do XXIII Congresso Brasileiro de Reprodução Animal (CBRA-2019), Gramado, RS, 2019.

Guérin, P.; El, Mouatassim. S.; & Ménézo, Y. (2001). Oxidative stress and protection against reactive oxygen species in the preimplantation embryo and its surroundings. Human Reproduction Update 7:175-189.

Holm, P.; Booth, P. J.; Schimid, T. M. T.; Greve, T.; & 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: 683-700.

Iwasaki, S.; Nakazawa, K.; Sakai, J.; Kometani, K.; Iwashita, M.; Yoshimura, Y.; & Maruyama, T. (2005). Melatonin as a local regulator of human placental function. Journal of Pineal Research 39:261–265.

Kang, J. T.; Koo, O. J.; Kwon, H. J.; Park, H. J.; Jang, G.; Kang, S. K.; & Lee, S. C. (2009). Effects of melatonin on in vitro maturation of porcine oocyte and expression of melatonin receptor RNA in cumulus and granulosa cells. Journal of Pineal Research 46: 22-28.

Lira, A. S.; Chaves, R. M.; Moraes Junior, F. J.; Costa Junior, S. H.; Amaral, B. K. L.; & Trovão, H. M. P. (2020). Use of melatonin in the in vitro production of bovine embryos. Revista Brasileira em Saúde e Produção Animal 21(1-9): e210322020.

Lonergan, P.; Fair, T.; Corcoran, D.; & Evans, A. C. O. (2006). Effect of culture environment on gene expression and developmental characteristics in IVF-derived embryos. Theriogenology 65:137-152.

Manjunatha, B. M.; Devaraj, M.; Gupta, P. S. P.; Ravindra, J. P.; & Nandi, S. (2009). Effect of taurine and melatonin in the culture medium on buffalo in vitro embryo development. Reproduction in Domestic Animal 44:12–16.

Mayo, J. C.; Aguado, A.; Cernuda-Cernuda, R.; Artime, A. A.; Cepas, V.; Quiros-González, I.; Hevia, D.; & Sáinz, R. M. (2018). Melatonin Uptake by Cells: An Answer to Its Relationship with Glucose? Molecules 23(8):1999.

Maziero, R. R. D.; Guaitolini, C. R. F.; Paschoal, D. M.; Crespilho, A. M.; Sestari, D. A. O.; Dode, M. A. N.; & Landim-Alvarenga, F. C. (2020). Effects of the addition of oocyte meiosis-inhibiting drugs on the expressionof maturation-promoting factor components and organization ofcytoplasmic organelles. Reproductive Biology 20:48-62.

Maziero, R. R. D.; Guaitolini, C. R. F.; Paschoal, D. M.; Kievitsbosch, T.; Guastali, M. D.; Moraes, C. N.; & Landim-Alvarenga, F. C. (2016). Effect of temporary meiotic attenuation of oocytes with butyrolactone-I and roscovitine in resistance to bovine embryos on vitrification. Reproduction in Domestic Animal 51: 204-2011.

Papis, K.; Poleszczuk, O.; Wenta-Muchalska, E.; & Mondlinski, J. A. (2007). Melatonin effect on bovine embryo development in vitro in relation to oxygen concentration. Journal of Pineal Research 43:321-326.

Parrish, J. J.; Krogenaes, A.; & Susko-Parrish, J. L. (1994). Effect of bovine sperm separation by either swim-up and percoll method on success of in vitro fertilization and early embryonic development. Theriogenology 44: 859-869.

Paula, F. A. P. (2017). Efeito da melatonina na maturação in vitro sobre a expressão gênica e a qualidade de oócitos bovinos sob choque térmico. Dissertação (Mestrado) apresentada à Universidade Federal de Lavras, 98p., 2017.

Pontes, J. H. F.; Nonato-Junior, I.; Sanches, B. V.; Ereno-Junior, J. C.; Uvo, S.; Barreiros, T. R. R.; Oliveira, J. A.; Hasler, J. F.; & Seneda, M. M. (2011). Comparison of embryo yield and pregnancy rate between in vivo and in vitro methods in the same Nelore (Bos indicus) donor cows. Theriogenology, 71(4):690–697.

Rizos, D.; Ward, F.; Duffy, P.; Boland, M. P.; & Lonergan, P. (2002). Consequences of bovine oocyte maturation, fertilization and early embryo development in vitro versus in vivo: implication for blastocyst yield and blastocyst quality. Molecular, Reproduction and Development 61:234-248.

Rocha-Frigoni, N. A. D. S.; Leão, B. C. D. S.; Nogueira, E.; Accorsi, M. F.; & Mingoti, G. Z. (2013). Effects of gaseous atmosphere and antioxidants on the development and cryotolerance of bovine embryos at different periods of in vitro culture. Zygote 41:1–10.

Rodriguez-Osorio, N.; Kim, I. J.; Wang, H.; Kaya, A.; & Memili, E. (2007). Melatonin increases cleavage rate of porcine preimplantation embryos in vitro. Journal of Pineal Research 43(3): 283–288.

Sagirkaya, H.; Misirlioglu, M.; Kaya, A.; First, N. L.; Parrish, J. J.; & Memili, E. (2007). Developmental potential of bovine oocytes cultured in different maturation and culture conditions. Animal Reproduction Science 101:225-240.

Sudano, M. J.; Paschoal, D. M.; Rascado, T. S.; Magalhães, L. C. O.; Crocomo, L. F.; Lima-Neto, J. F.; & Landim-Alvarenga, F. C. (2011). Lipid content and apoptosis of in vitro-produced bovine embryos. Theriogenology 5:1211-1220.

Takada, L.; Junior, A. M.; Mingoti, G. Z.; Balieiro, J. C.; Cipolla-Neto, J.; & Coelho, L. A. (2012). Effect of melatonin on DNA damage of bovine cumulus cells during in vitro maturation (IVM) and on in vitro embryo development. Research and Veterinary Science 92:124–127.

Takada, L.; Martins Junior, A.; Mingoti, G. Z.; Balieiro, J. C. J.; & Coelho, L. A. (2010). Melatonin in maturation media fails to improve oocyte maturation, embryo development rates and DNA damage of bovine embryos. Scientia Agricola 67(1): 393-398.

Tsantarioutou, M. P.; Altanasio, L.; De Rosa, A.; Boccia, L.; Pellerano, G.; & Gasparrini, B. (2007). The effect of melatonin on bovine in vitro embryo development. Italian Journal of Animal Science 6:488-489.

Viana, J. H. M. (2018). Statistics of embryo production and transfer in domestic farm animals: Is it a turning point? In 2017 more in vitro-produced than in vivo-derived embryos were transferred worldwide. Embryo Transfer Newsletters 36(4):8-25.

Zhao, X. M.; Min, J. T.; Du, W. H.; Hao, H. S.; Liu, Y.; Qin, T.; Wang, D.; & Zhu, H. B. (2015). Melatonin enhances the in vitro maturation and developmental potential of bovine oocytes denuded of the cumulus oophorus. Zygote 23(4): 1–12.




How to Cite

MAGALHÃES, R. .; GUAITOLINI, C. R. de F.; TRAMONTIN, M. L. D.; SESTARI, D. A. O.; BARROS, B. A. de; ELIAS, A. S. de L.; ARAUJO, A. L.; GIOSA, D. C.; TRINDADE, A. B.; MAZIERO, R. R. D. . Evaluation of in vitro production rates of bovine embryos using melatonin-supplemented culture medium. Research, Society and Development, [S. l.], v. 10, n. 6, p. e19010615544, 2021. DOI: 10.33448/rsd-v10i6.15544. Disponível em: https://rsdjournal.org/index.php/rsd/article/view/15544. Acesso em: 23 jun. 2021.



Agrarian and Biological Sciences