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

Sheep oocytes derived from the ovaries collected from the slaughterhouse are often used for research onin vitroembryo production, animal cloning, transgenesis, embryonic stem cells, and other embryo biotechnology aspects. Improving thein vitroculture efficiency of oocytes can provide more materials for similar studies. Generally, determination of oocyte quality is mostly based on the layers of cumulus cells and cytoplasm or cytoplasm uniformity and colors. This requires considerable experience to better identify oocyte quality because of the intense subjectivity involved (Gordon (2003), Madison et al. (1992) and De Loos et al. (1992)). BCB staining is a function of glucose-6-phosphate dehydrogenase (G6PD) activity, an enzyme synthesized in developing oocytes, which decreases in activity with maturation. Therefore, unstained oocytes (BCB−) are high in G6PD activity, while the less mature oocytes stains are deep blue (BCB+) due to insuffcient G6PD activity to decolorize the BCB dye.

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315 SELECTION OF BOVINE OOCYTES BY BRILLIANT CRESYL BLUE BEFORE IN VITRO MATURATION IMPROVES BLASTOCYST DEVELOPMENT

Reproduction Fertility and Development ◽

10.1071/rdv19n1ab315 ◽

2007 ◽

Vol 19 (1) ◽

pp. 273 ◽

Cited By ~ 2

Author(s):

A. Sugulle ◽

S. Katakawa ◽

S. Yamamoto ◽

S. Oomori ◽

I. Itou ◽

...

Keyword(s):

Embryo Development ◽

In Vitro Maturation ◽

Control Group ◽

Blastocyst Development ◽

Cell Stage ◽

Brilliant Cresyl Blue ◽

Cresyl Blue ◽

Bovine Oocytes ◽

Selection Of

The morphological identification of immature oocytes has commonly been used to select the bovine oocytes for IVF. However, <30% of the recovered oocytes reach the blastocyst stage after fertilization, and this is probably due to the quality of the oocytes at the beginning of maturation. The brilliant cresyl blue (BCB) stain determines the activity of glucose-6-phosphate dehydrogenase, an enzyme synthesized in growing oocytes. The aim of this study was to evaluate the effect of the BCB stain on the selection of bovine oocytes and on the subsequent embryo development for in vitro production (IVP). Cumulus–oocyte complexes (COCs) were collected by the aspiration of 2- to 6-mm follicles. A total of 559 oocytes were divided into 2 groups: (1) a control group, immediately cultured, and (2) a BCB-incubated group. After 90 min of BCB staining (Pujol et al. 2004 Theriogenology 61, 735–744), the oocytes were divided into oocytes with blue cytoplasm (BCB+) and oocytes without blue cytoplasm (BCB−). The COCs were matured for 20 h in TCM-199 supplemented with 5% calf serum (CS) and 0.02 mg mL−1 FSH at 38.5°C under an atmosphere of 5% CO2 in air. The matured COCs were inseminated with 5 × 106 sperm mL−1. After 18 h of gamete co-culture, the presumed zygotes were cultured in CR1aa supplemented with 5% CS for 9 days at 38.5°C under an atmosphere of 5% CO2, 5% O2, and 90% N2. Embryonic development was evaluated at 48 h after IVF (proportion of ≥5-cell stage, the total cleavage rates) and on Days 7 to 9 (blastocyst rate). The experiment was replicated 5 times, and the data were analyzed by a chi-square test and ANOVA. The results are presented in Table 1. The proportion of embryos with ≥5-cell stage was significantly higher (P < 0.01) in the BCB+ group than in the BCB− group, but not in the control group. The total cleavage rate for the BCB+ embryos was significantly higher than that of either the BCB− or the control group (P < 0.01). There were also significant differences (P < 0.01) in the blastocyst development between the BCB+ and BCB− embryos and between the BCB− and the control embryos (P < 0.05). This result showed that the selection of bovine oocytes by BCB staining before in vitro maturation may be useful for selecting oocytes that are developmentally competent up to Day 9 for IVP. Table 1.Effect of selection of oocytes by brilliant cresyl blue (BCB) staining on the subsequent embryo development of in vitro-matured/in vitro-fertilized bovine embryos

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Selection of Rattus norvegicus oocytes for in vitro maturation by brilliant cresyl blue staining

Zygote ◽

10.1017/s0967199411000463 ◽

2011 ◽

Vol 21 (3) ◽

pp. 238-245 ◽

Cited By ~ 8

Author(s):

Diego Duarte Alcoba ◽

Bianca Letícia da Rosa Braga ◽

Nathallie Louise Sandi-Monroy ◽

Letícia Auler Proença ◽

Rui Fernando Felix Lopes ◽

...

Keyword(s):

Rattus Norvegicus ◽

Blue Staining ◽

Control Group ◽

Brilliant Cresyl Blue ◽

Cresyl Blue ◽

Nuclear Maturation ◽

Effective Evaluation ◽

Maturation Rate ◽

Selection Of

SummaryThe objective of this work was to evaluate the rate of meiosis resumption and nuclear maturation of rat (Rattus norvegicus) oocytes selected for in vitro maturation (IVM) after staining of cumulus–oocyte complexes (COCs) with blue cresyl brilliant (BCB) using different protocols: exposure for 30, 60 or 90 min at 26 μM BCB (Experiment 1), and exposure for 60 min at 13, 20 or 26 μM BCB (Experiment 2). In Experiment 1, the selection of oocytes exposed to BCB for 60 min was found to be the most suitable, as meiosis resumption rates in the BCB+ group (n = 35/61; 57.37%) were the closest to the observed in the control (not exposed) group (n = 70/90; 77.77%) and statistically higher than the values observed for the BCB− group (n = 3/41; 7.32%). Additionally, the more effective evaluation of diagnostic tests (sensitivity and negative predictive value 100%) was observed in COCs exposed for 60 min. In Experiment 2, the 13 μM BCB+ group presented rates of meiosis resumption (n = 57/72; 72.22%) similar to the control group (n = 87/105; 82.86%) and higher than other concentration groups. However, this results of the analysis between BCB− oocytes was also higher in the 13 μM BCB group (n = 28/91; 30.78%) when compared with BCB− COCs exposed to 20 μM (n = 3/62; 4.84%) or 26 μM (n = 3/61; 4.92%) BCB. The nuclear maturation rate in the 13 μM BCB group was similar between BCB+ or BCB− oocytes. The 20 μM BCB group had a lower rate of nuclear maturation of BCB− oocytes than other groups. Thus, our best results in the selection of Rattus norvegicus oocytes by staining with BCB were obtained using the concentration of 13 μM and 20 μM, and an incubation period of 60 min.

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Selection of porcine oocytesin vitrothrough brilliant cresyl blue staining in distinct incubation media

Zygote ◽

10.1017/s0967199416000319 ◽

2016 ◽

Vol 25 (1) ◽

pp. 49-55 ◽

Cited By ~ 3

Author(s):

Elisa C.S Santos ◽

Jorgea Pradieé ◽

Elisângela M. Madeira ◽

Miriane M. Pereira ◽

Bruna Mion ◽

...

Keyword(s):

Dna Fragmentation ◽

Blue Staining ◽

Dna Integrity ◽

Cortical Granules ◽

Blastocyst Development ◽

Brilliant Cresyl Blue ◽

Cresyl Blue ◽

Nuclear Maturation ◽

Selection Medium

SummaryStaining with brilliant cresyl blue (BCB) may be used for oocyte selection, but BCB staining itself and the most commonly used selection medium (DMPBS) may compromise the development of porcine oocytesin vitro.This study evaluated DNA fragmentation, nuclear maturation, the area of migration of cortical granules (CG) and embryo development for stained (BCB+) and unstained (BCB−) oocytes incubated in DMPBS and in a modified medium (ReproPel) tested for the first time. Unexposed (UN), BCB+ and BCB− oocytes were incubated composing six groups: DMPBS/UN; DMPBS/BCB+; DMPBS/BCB−; ReproPel/UN; ReproPel/BCB+; and ReproPel/BCB−. There were more BCB+ oocytes in ReproPel than in DMPBS (P< 0.05). The DNA fragmentation was evaluated for oocytes in DMPBS/BCB+, DMPBS/BCB−, ReproPel/BCB+, ReproPel/BCB− and in porcine follicular fluid (control). The frequency of oocytes with no DNA fragmentation was greatest (64.6%) in DMPBS/BCB+ and lowest in ReproPel/BCB+ and ReproPel/BCB− (26.8 and 34.1%, respectively) (P< 0.05). Nuclear maturation rates were greater (P< 0.05) for DMPBS/BCB+ (63.1%), ReproPel/UN (55.1%) and ReproPel/BCB+ (50.2%) than for DMPBS/UN (40.8%) and ReproPel/BCB− (35.5%). The area of CG was greater (P< 0.05) for ReproPel/BCB− (80.7%) and DMPBS/UN (77.6%) than for ReproPel/UN (34.7%). Cleavage rates for DMPBS/BCB+ and ReproPel/BCB+ were greater than for DMPBS/UN (P< 0.05). Blastocyst development rates were greatest (P< 0.05) for ReproPel/UN and ReproPel/BCB+. In both media, BCB staining was apparently unable to select competent oocytes, which likely occurred due to toxicity. Despite the similar nuclear maturation and area of CG compared with DMPBS, oocytes selected in ReproPel presented impaired DNA integrity.

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Selection of oocytes for in vitro maturation by brilliant cresyl blue staining: a study using the mouse model

Cell Research ◽

10.1038/cr.2007.66 ◽

2007 ◽

Vol 17 (8) ◽

pp. 722-731 ◽

Cited By ~ 51

Author(s):

Yan-Guang Wu ◽

Yong Liu ◽

Ping Zhou ◽

Guo-Cheng Lan ◽

Dong Han ◽

...

Keyword(s):

Mouse Model ◽

In Vitro Maturation ◽

Blue Staining ◽

Brilliant Cresyl Blue ◽

Cresyl Blue ◽

Selection Of

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316 DEVELOPMENTAL COMPETENCE OF OOCYTES SELECTED BY THE BRILLIANT CRESYL BLUE STAINING IN PREPUBERTAL AND ADULT CATTLE

Reproduction Fertility and Development ◽

10.1071/rdv19n1ab316 ◽

2007 ◽

Vol 19 (1) ◽

pp. 273 ◽

Cited By ~ 2

Author(s):

M. Tagawa ◽

S. Matoba ◽

M. Okada ◽

K. Metoki ◽

K. Imai

Keyword(s):

Farm Animals ◽

Blue Staining ◽

Blastocyst Formation ◽

Adult Cattle ◽

Brilliant Cresyl Blue ◽

Cresyl Blue ◽

Significant Difference ◽

The Mean ◽

Selection Of

Transvaginal ultrasound guided ovum pickup (OPU) can be carried out repeatedly not only in adult cows but also in prepubertal calves. Moreover, recently the brilliant cresyl blue (BCB) staining has been used successfully to select oocytes homologous stained with BCB as an indirect measure of oocyte growth for in vitro fertilization (IVF) in several farm animals. The aim of this study was to evaluate the effectiveness of BCB staining on the selection of developmentally competent oocytes, collected from cows and calves by OPU, before in vitro maturation by assessing the embryonic development to the blastocyst stage after IVM/IVF. OPU was performed once weekly for a period of 7 weeks on Japanese Black prepubertal calves (9 months old, n = 4) and adult cows (n = 4) without gonadotropin stimulation. Calf and cow oocytes with homogeneous cytoplasm were exposed to 26 �M BCB for 90 min and classified according to their cytoplasmic coloration: blue coloration (BCB+) or colorless (BCB-). Classified oocytes were then matured for 20 h in TCM-199 supplemented with 5% calf serum (CS). Matured oocytes were inseminated with Percoll-separated spermatozoa (3 � 106 mL) for 6 h in BO solution supplemented with 5 mM hypotaurine and 2 U mL-1 heparin. Presumptive zygotes were cultured in CR1aa supplemented with 5% CS for 8 days. Data were analyzed by Student's t-test. The mean (± SEM) percentage of oocytes classified as BCB+ in calves was significantly lower than that in cows (34.4 ± 2.9&percnt; and 69.2 ± 2.1&percnt;, respectively; P < 0.01). In cows, BCB+ oocytes showed significantly higher cleavage and blastocyst formation percentages (72.5&percnt; and 42.4&percnt;, respectively) than those of BCB− oocytes (47.0&percnt; and 13.0&percnt;, respectively). In contrast, in calves there were no significant differences in cleavage and blastocyst formation percentages between BCB+ oocytes (56.9&percnt; and 25.3&percnt;, respectively) and BCB− oocytes (65.4&percnt; and 22.4&percnt;, respectively). The mean (± SEM) numbers of usable oocytes and blastocysts obtained per calf (19.0 ± 1.5 and 4.5 ± 0.6, respectively) were similar to those obtained per cow (16.4 ± 1.1 and 5.2 ± 0.6, respectively). No significant difference was observed in the numbers between calves and cows. These results indicate that the selection of developmentally competent oocytes before IVM/IVF, using the BCB staining, was effective for cow oocytes but not for calf oocytes, and that blastocysts could be produced by OPU-IVF of oocytes from 9-month-old prepubertal calves at an efficiency equivalent to that achieved from adult cows.

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Selection of developmentally competent oocytes through brilliant cresyl blue stain enhances blastocyst development rate after bovine nuclear transfer

Theriogenology ◽

10.1016/j.theriogenology.2006.08.006 ◽

2007 ◽

Vol 67 (2) ◽

pp. 341-345 ◽

Cited By ~ 59

Author(s):

S. Bhojwani ◽

H. Alm ◽

H. Torner ◽

W. Kanitz ◽

R. Poehland

Keyword(s):

Nuclear Transfer ◽

Development Rate ◽

Blastocyst Development ◽

Brilliant Cresyl Blue ◽

Cresyl Blue ◽

Blue Stain ◽

Selection Of

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Selection of developmentally competent immature equine oocytes by brilliant cresyl blue staining prior to maturation with equine growth hormone in vitro.

Animal Reproduction Science ◽

10.1016/j.anireprosci.2010.04.149 ◽

2010 ◽

Vol 121 (1-2) ◽

pp. 248-249

Keyword(s):

Growth Hormone ◽

Blue Staining ◽

Brilliant Cresyl Blue ◽

Cresyl Blue ◽

Selection Of

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

Zygote ◽

10.1017/s0967199411000487 ◽

2011 ◽

Vol 21 (3) ◽

pp. 250-255 ◽

Cited By ~ 24

Author(s):

D.S. Silva ◽

P. Rodriguez ◽

A. Galuppo ◽

N.S. Arruda ◽

J.L. Rodrigues

Keyword(s):

Embryo Development ◽

Polar Body ◽

Blue Staining ◽

Cortical Granule ◽

Brilliant Cresyl Blue ◽

Cresyl Blue ◽

Cytoplasmic Maturation ◽

Bovine Oocytes ◽

Selection Of

SummaryThe 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.

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