In vitro embryos production of oocytes selected based on follicular size and by brilliant cresyl blue staining

Fabiana Dakkach de Almeida Barros, Paulo Roberto Adona, Samuel Guemra, Tiago Henrique Camara De Bem, Paulo Sergio Monzani, Claudia Lima Verde Leal


We evaluated the competence of bovine oocytes via morphological selection associated with follicular diameter and by brilliant cresyl blue (BCB) staining, in order to improve oocyte selection and the efficiency of in vitro embryo production. Follicles with small (2 < 4 mm), large (4–8 mm) or mixed (2–8 mm) diameters were aspirated from slaughterhouse ovaries. Next, the morphologically categorized oocytes were stained and evaluated with 26 ?M of BCB (90 min), followed by an assessment of in vitro maturation (MII) and blastocyst production. Oocytes from the large (61.1 ± 3.9%) and mixed (52.9 ± 4.6%) follicular groups showed comparable (p > 0,05) BCB+ rates, but both differed (p < 0.05) as compared to the small-diameter group (41.0 ± 4.5%). Between the BCB+ and BCB – oocytes, there was no difference (p > 0,05) in MII rate in the mixed (respectively 86.7 ± 3.4 and 75.8 ± 2.2), large (respectively 89.4 ± 2.6 and 79.7 ± 2.3) and small (respectively 76.7 ± 2.9 and 67.2 ± 2.5) diameter groups. Comparing BCB (+ and -) oocytes, we observed (p > 0,05) no change in the percentage of embryos in the large group (respectively 67.3 ± 3.3 and 53.5 ± 5.8) and in the small group (respectively 43.8 ± 5.3 and 33.9 ± 2.2), but there was a difference (p < 0.05) in the mixed group (respectively 57.8 ± 3.9 and 39.6 ± 7.5). Furthermore, when we compared between follicular groups, the BCB+/large-diameter group (61.1 ± 3.9%) had a significant higher percentage of embryos (p < 0.05) than the small-diameter BCB+ (43.8 ± 5.3) groups. To conclude, selection procedures that use follicle size and/or BCB staining may be used as an additional tool to improve the in vitro production of embryos.


Blastocysts; Competence; Follicle; Maturation; Morphology.

Full Text:



ADONA, P. R.; LEAL, C. L.; BIASE, F. H.; DE BEM, T. H.; MESQUITA, L. G.; MEIRELLES, F. V.; FERRAZ, A. L.; FURLAN, L. R.; MONZANI, P. S.; GUEMRA, S. In vitro maturation alters gene expression in bovine oocytes. Zygote, Cambridge, v. 24, n. 4, p. 624-633, 2016. DOI: 10.1017/S0967199415000672

ADONA, P. R.; PIRES, P. R.; QUETGLAS, M. D.; SCHWARZ, K. R.; LEAL, C. L. Prematuration of bovine oocytes with butyrolactone I: effects on meiosis progression, cytoskeleton, organelle distribution and embryo development. Animal Reproduction Science, Netherlands, v. 108, n. 1, p. 49-65, 2008. DOI: 10.1016/j.anireprosci.2007.07.002

ALM, H.; TORNER, H.; LOHRKE, B.; VIERGUTZ, T.; GHONEIM, I. M.; KANITZ, W. Bovine blastocyst development rate in vitro is influenced by selection of oocytes by brillant cresyl blue staining before IVM as indicator for glucose-6-phosphate dehydrogenase activity. Theriogenology, New York, v. 63, n. 8, p. 2194-2205, 2005. DOI: 10.1016/j.theriogenology.2004.09.050

BAKRI, N. M.; IBRAHIM, S. F.; OSMAN, N. A.; HASAN, N.; JAFFAR, F. H.; RAHMAN, Z. A.; OSMAN, K. Embryo apoptosis identification: Oocyte grade or cleavage stage? Saudi Journal Biological Sciences, Riyad?, v. 23, n. 1, p. 50-55, 2016. DOI: 10.1016/j.sjbs.2015.10.023

BLONDIN, P.; BOUSQUET, D.; TWAGIRAMUNGU, H.; BARNES, F.; SIRARD, M. A. Manipulation of follicular development to produce developmentally competent bovine oocytes. Biology Reproduction, New York, v. 66, n. 1, p. 38-43, 2002.

BOLS, P.; JORSSEN, E.; GOOVAERTS, I.; LANGBEEN, A.; LEROY, J. High throughput non-invasive oocyte quality assessment: the search continues. Animal. Reproduction, Belo Horizonte, v. 9, n. 3, p. 420-425, 2012.

BRAGANCA, G. M.; BATISTA, R.; SOUZA-FABJAN, J. M. G.; ALFRADIQUE, V. A. P.; ARASHIRO, E. K. N.; COSENTINO, I. O.; PINTO, P. H. N.; CAMARGO, L. S. A.; FONSECA, J. F. da; BRANDAO, F. Z. Dose and administration protocol for FSH used for ovarian stimulation affect gene expression in sheep cumulus-oocyte complexes. Reproduction Fertility and Development, East Melbourne, v. 30, n. 9, p. 1234-1244, 2018. DOI: 10.1071/RD17337

CASTANEDA, C. A.; KAYE, P.; PANTALEON, M.; PHILLIPS, N.; NORMAN, S.; FRY, R.; D'OCCHIO, M. J. Lipid content, active mitochondria and brilliant cresyl blue staining in bovine oocytes. Theriogenology, New York, v. 79, n. 3, p. 417-422, 2013. DOI: 10.1016/j.theriogenology.2012.10.006

CATALA, M. G.; IZQUIERDO, D.; UZBEKOVA, S.; MORATO, R.; ROURA, M.; ROMAGUERA, R.; PAPILLIER, P.; PARAMIO, M. T. Brilliant Cresyl Blue stain selects largest oocytes with highest mitochondrial activity, maturation-promoting factor activity and embryo developmental competence in prepubertal sheep. Reproduction, Bristol, v. 142, n. 4, p. 517-527, 2011. DOI: 10.1530/REP-10-0528

DE BEM, T.; ADONA, P. R.; BRESSAN, F. F.; MESQUITA, L. G.; CHIARATTI, M. R.; MEIRELLES, F. V.; LEAL, C. The influence of morphology, follicle size and Bcl-2 and Bax transcripts on the developmental competence of bovine oocytes. Reproduction in Domestic Animals, Berlin v. 49, n. 4, p. 576-583, 2014. DOI: 10.1111/rda.12325

DE LOOS, F.; VAN MAURIK, P.; VAN BENEDEN, T.; KRUIP, T. A. Structural aspects of bovine oocyte maturation in vitro. Molecular Reproduction and Development, Hoboken, v. 31, n. 3, p. 208-214, 1992. DOI: 10.1002/mrd.1080310308

DUARTE ALCOBA, D.; GONSALES VALERIO, E.; CONZATTI, M.; SCHNEIDER, J.; CAPP, E.; VON EYE CORLETA, H.; BRUM, I. S. Selection of developmentally competent human oocytes aspirated during cesarean section. The Journal of Maternal-Fetal & Neonatal Medicine, London, v. 31, n. 6, p. 735-739, 2018. DOI: 10.1080/14767058.2017.1297405

FANG, X.; XIA, W.; CAO, H.; GUO, Y.; WANG, H.; ZHANG, X.; WAN, P.; LIU, C.; WEI, Q.; SUN, S.; TIAN, S.; LI, J.; WANG, Z. Effect of supplemetation of Zebularine and Scriptaid on efficiency of in vitro developmental competence of ovine somatic cell nuclear transferred embryos. Animal Biotechnology, Abingdon, v. 30, n. 8, p. 1-9, 2019. DOI: 10.1080/10495398.2018.1559846

FATHI, M.; ASHRY, M.; SALAMA, A.; BADR, M. R. Developmental competence of Dromedary camel (Camelus dromedarius) oocytes selected using brilliant cresyl blue staining. Zygote, Cambridge, v. 25, n. 4, p. 529-536, 2017. DOI: 10.1017/S0967199417000387

GANDOLFI, T. A.; GANDOLFI, F. The maternal legacy to the embryo: cytoplasmic components and their effects on early development. Theriogenology, New York, v. 55, n. 6, p. 1255-1276, 2001. DOI: 10.1016/S0093-691X(01)00481-2

HOLM, P.; BOOTH, P. J.; SCHMIDT, M. H.; GREVE, T.; CALLESEN, H. 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, New York, v. 52, n. 4, p. 683-700, 1999. DOI: 10.1016/S0093-691X(99)00162-4

HYTTEL, P.; FAIR, T.; CALLESEN, H.; GREVE, T. Oocyte growth, capacitation and final maturation in cattle. Theriogenology, New York, v. 47, n. 1, p. 23-32, 1997. DOI: 10.1016/S0093-691X(96)00336-6

IWASAKI, W.; YAMANAKA, K.; SUGIYAMA, D.; TESHIMA, Y.; BRIONES-NAGATA, M. P.; MAEKI, M.; YAMASHITA, K.; TAKAHASHI, M.; MIYAZAKI, M. Simple separation of good quality bovine oocytes using a microfluidic device. Scientific Reports, London, v. 8, n. 1, p. 142-173, 2018. DOI: 10.1038/s41598-018-32687-6

KARAMI SHABANKAREH, H.; AZIMI, G.; TORKI, M. Developmental competence of bovine oocytes selected based on follicle size and using the brilliant cresyl blue (BCB) test. Iranian Journal of Reproductive Medicine, Yazd, v. 12, n. 11, p. 771-778, 2014.

KAUFFOLD, J.; AMER, H. A.; BERGFELD, U.; WEBER, W.; SOBIRAJ, A. The in vitro developmental competence of oocytes from juvenile calves is related to follicular diameter. Journal of Reproduction and Development, Tokyo, v. 51, n. 3, p. 325-332, 2005. DOI:10.1262/jrd.17002

KIM, J.; YOU, J.; HYUN, S. H.; LEE, G.; LIM, J.; LEE, E. Developmental competence of morphologically poor oocytes in relation to follicular size and oocyte diameter in the pig. Molecular Reproduction and Development, Hoboken, v. 77, n. 4, p. 330-339, 2010. DOI: 10.1002/mrd.21148

LABRECQUE, R.; FOURNIER, E.; SIRARD, M. A. Transcriptome analysis of bovine oocytes from distinct follicle sizes: Insights from correlation network analysis. Molecular Reproduction and Development, Hoboken, v. 83, n. 6, p. 558-569, 2016. DOI: 10.1002/mrd.22651

LIU, X. M.; WANG, Y. K.; LIU, Y. H.; YU, X. X.; WANG, P. C.; LI, X.; DU, Z. Q.; YANG, C. X. Single-cell transcriptome sequencing reveals that cell division cycle 5-like protein is essential for porcine oocyte maturation. Journal of Biological Chemistry, Baltimore, v. 293, n. 5, p. 1767-1780, 2018. DOI: 10.1074/jbc.M117.809608

MIRSHAMSI, S. M.; KARAMISHABANKAREH, H.; AHMADI-HAMEDANI, M.; SOLTANI, L.; HAJARIAN, H.; ABDOLMOHAMMADI, A. R. Combination of oocyte and zygote selection by brilliant cresyl blue (BCB) test enhanced prediction of developmental potential to the blastocyst in cattle. Animal Reproduction Science, Amsterdam, v. 136, n. 4, p. 245-251, 2013. DOI: 10.1016/j.anireprosci.2012.11.002

MOHAMMADI-SANGCHESHMEH, A.; SOLEIMANI, M.; DELDAR, H.; SALEHI, M.; SOUDI, S.; HASHEMI, S. M.; SCHELLANDER, K.; HOELKER, M. Prediction of oocyte developmental competence in ovine using glucose-6-phosphate dehydrogenase (G6PDH) activity determined at retrieval time. Journal of Assisted Reproduction Genetics, Amsterdam, v. 29, n. 2, p. 153-158, 2012. DOI: 10.1007/s10815-011-9625-6

OPIELA, J.; KATSKA-KSIAZKIEWICZ, L. The utility of Brilliant Cresyl Blue (BCB) staining of mammalian oocytes used for in vitro embryo production (IVP). Reproductive Biology, Olsztyn, v. 13, n. 3, p. 177-183, 2013. DOI: 10.1016/j.repbio.2013.07.004

PARRISH, J. J. Bovine in vitro fertilization: in vitro oocyte maturation and sperm capacitation with heparin. Theriogenology, New York, v. 81, n. 1, p. 67-73, 2014. DOI: 10.1016/j.theriogenology.2013.08.005

PONTES, J. H.; MELO STERZA, F. A.; BASSO, A. C.; FERREIRA, C. R.; SANCHES, B. V.; RUBIN, K. C.; SENEDA, M. M. Ovum pick up, in vitro embryo production, and pregnancy rates from a large-scale commercial program using Nelore cattle (Bos indicus) donors. Theriogenology, New York, v. 75, n. 9, p. 1640-1646, 2011. DOI: 10.1016/j.theriogenology.2010.12.026

PUJOL, M.; LOPEZ-BEJAR, M.; PARAMIO, M. T. Developmental competence of heifer oocytes selected using the brilliant cresyl blue (BCB) test. Theriogenology, New York, v. 6, n. 4, p. 735-744, 2004. DOI: 10.1016/S0093-691X(03)00250-4

RODRIGUEZ-GONZALEZ, E.; LOPEZ-BEJAR, M.; VELILLA, E.; PARAMIO, M. T. Selection of prepubertal goat oocytes using the brilliant cresyl blue test. Theriogenology, New York, v. 57, n. 5, p. 1397-1409, 2002. DOI: 10.1016/S0093-691X(02)00645-3

SOARES, M. M.; CÁSSIA ANTONINO, D. de; OLIVEIRA, M.; MELO JUNIOR, J.; PEIXOTO, L. R.; MAIA, T. S.; ALVES, K. A.; JACOMINI, J. O.; SANTOS, R. M. dos; MACEDO, G. G. The role of Kisspeptin in bovine in vitro embryo production. Semina: Ciências Agrárias, Londrina, v. 39, n. 2, p. 621-630, 2018. DOI: 10.5433/1679-0359.2018v39n2p621

SOVERNIGO, T. C.; ADONA, P. R.; MONZANI, P. S.; GUEMRA, S.; BARROS, F.; LOPES, F. G.; LEAL, C. Effects of supplementation of medium with different antioxidants during in vitro maturation of bovine oocytes on subsequent embryo production. Reproduction Domestic Animals, Berlin, v. 52, n. 4, p. 561-569, 2017. DOI: 10.1111/rda.12946

SU, J.; WANG, Y.; LI, R.; PENG, H.; HUA, S.; LI, Q.; QUAN, Q.; GUO, Z.; ZHANG, Y. Oocytes selected using BCB staining enhance nuclear reprogramming and the in vivo development of SCNT embryos in cattle. PLoS One, San Francisco, v. 7, n. 4, p. 361-381, 2012. DOI: 10.1371/journal.pone.0036181


Semina: Ciênc. Agrár.
Londrina - PR
E-ISSN 1679-0359
DOI: 10.5433/1679-0359
Este obra está licenciado com uma Licença Creative Commons Atribuição-NãoComercial 4.0 Internacional