Selection of prepubertal goat oocytes using the brilliant cresyl blue test.
Brilliant cresyl blue stain allows us to determine the activity of glucose-6-phosphate dehydrogenase (G6PD), an enzyme synthesized in growing oocytes but with decreased activity in oocytes that have finished their growth phase. The objective of this study was to evaluate the utility of the brilliant cresyl blue (BCB) test as an indirect measure of oocyte growth, in order to select competent prepubertal goat oocytes for in vitro embryo production. Oocytes were exposed to BCB diluted in PBS and were classified according to their cytoplasm coloration: oocytes with a blue cytoplasm or grown oocytes (BCB+) and oocytes without a blue cytoplasm or growing oocytes (BCB-). After exposure to different BCB concentrations, we evaluated in vitro maturation (IVM), in vitro fertilization (IVF) and embryo development parameters. We defined matured oocytes as those oocytes that reached the metaphase II (MII) stage after being cultured for 27 h. Oocytes showing two pronuclei at 20 h post-insemination were classified as normally fertilized oocytes. We assessed embryo development 8 days post-insemination and recorded the percentage of total embryos, morale and blastocysts. The mean percentage of BCB+ oocytes was 29.4%. Mean diameter of BCB+ oocytes (136.6+/-6.3 microm) was higher (P < 0.001) than that of BCB- oocytes (125.5+/-10.2 microm). The percentage of BCB+ oocytes reaching the MII stage (81.4%) was higher (P < 0.05) than that of BCB- (52.5%) and control oocytes (72.4%). Normal fertilization rate of BCB+ oocytes was also higher (23.5%) than that of BCB- (8.2%; P < 0.0001) and control oocytes (11.9%; P < 0.05). The percentages of total embryos undergoing development to >8-cell and the morula plus blastocyst stages were higher (P < 0.05) in the group of BCB+ (41.3 and 12.0%, respectively) than in BCB- oocytes (21.3 and 3.6%, respectively). In conclusion, the BCB test is a useful way to select more competent prepubertal goat oocytes for in vitro embryo production.
Rodríguez-González
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Departament de Ciència Animal i dels Aliments, Facultat de Veterinària, Universitat Autònoma de Barcelona, Spain.
López-Béjar
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Velilla
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Paramio
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MT
D000818:Animals; D001755:Blastocyst / Q000502:physiology; D002478:Cells, Cultured; D004396:Coloring Agents; D046508:Culture Techniques; D003593:Cytoplasm; D005314:Embryonic and Fetal Development; D005260:Female; D005307:Fertilization in Vitro / Q000662:veterinary; D005954:Glucosephosphate Dehydrogenase / Q000032:analysis; D006041:Goats; D009028:Morula / Q000502:physiology; D009865:Oocytes / Q000201:enzymology / Q000502:physiology / Q000648:ultrastructure; D010078:Oxazines; D012741:Sexual Maturation; D020858:Tissue and Organ Harvesting / Q000379:methods / Q000662:veterinary*
D004396:Coloring Agents; D010078:Oxazines; C006797:Brilliant Cresyl Blue; D005954:Glucosephosphate Dehydrogenase