Research Article

Selection of bovine oocytes by brilliant cresyl blue staining: effect on meiosis progression, organelle distribution and embryo development

D.S. Silva^a2, P. Rodriguez^a2, A. Galuppo^a2, N.S. Arruda^a2 and J.L. Rodrigues^a1 c1

^a1 Laboratory of Embryology and Biotechnics of Reproduction, Faculty of Veterinary Medicine, UFRGS; Cx. Postal 15004, 91501-970 Porto Alegre, RS, Brasil.

^a2 Laboratory of Embryology and Biotechnics of Reproduction, Faculty of Veterinary Medicine, UFRGS, Brasil.

Summary

The selection of competent oocytes for in vitro maturation is still a major problem during bovine in vitro embryo production. Markers for in vitro cytoplasmic maturation, based on the organization of cortical granule and mitochondria, are lacking. We examined the pre-selection of immature bovine oocytes by brilliant cresyl blue stain (BCB test) based on glucose-6-phosphate dehydrogenase (G6PDH) activity during oocyte development. Oocytes were recovered from ovarian follicles exposed to 26 μM BCB stain and classified according to the aspect of their cytoplasm: BCB⁺ (oocytes with blue cytoplasm) and BCB⁻ (unstained cytoplasm) and then in vitro matured into a conventional in vitro maturation (IVM) medium and standard procedure. In Experiment 1, nuclear maturation was determined by polar body identification, while cytoplasmic maturation was based on cortical granule (CG) migration (peripheral) and mitochondria distribution (central). Evidence of polar body, cortical granule migration and of centrally located mitochondria was significantly (p < 0.05) higher in BCB⁺ oocytes than in BCB⁻ (polar body present: 65% vs 20%; peripheral CG: 72% vs. 14%; and central mitochondria: 85% vs. 19%, respectively). In Experiment 2, the efficiency pre-selection of bovine oocytes by BCB on embryo development in vitro was assessed. Cleavage rates were similar (75%) among control, BCB⁺ and BCB⁻ groups, while blastocyst rates on D7 were (p < 0.05) higher (35%) in BCB⁺ vs BCB⁻ (10%) or control (28%). We showed that the BCB test is efficient to identify competent immature bovine oocytes to undergo synchronous nuclear and cytoplasmic in vitro maturation thus yielding higher in vitro embryo development to blastocyst stage.

(Received December 01 2011)

(Accepted June 06 2011)

Keywords:

• BCB;
• Cattle;
• IVP;
• Oocytes;
• Organelle distribution

Correspondence:

^c1 All correspondence to José Luiz Rodrigues. Laboratory of Embryology and Biotechnics of Reproduction, Faculty of Veterinary Medicine, UFRGS; Cx. Postal 15004, 91501-970 Porto Alegre, RS, Brasil. Tel: +55 5133086126. E-mail: joseluiz.rodrigues@ufrgs.br