205 GLUCOSE-6-PHOSPHATE DEHYDROGENAZE ACTIVITY IN OVINE OOCYTES IS ASSOCIATED WITH PRE-IMPLANTATION EMBRYONIC DEVELOPMENT IN VITRO

2011 ◽  
Vol 23 (1) ◽  
pp. 201 ◽  
Author(s):  
F. Asghari ◽  
M. Shahidi ◽  
Y. Chashnidel ◽  
H. Deldar ◽  
Z. Ansari-Pirsaraei ◽  
...  
Keyword(s):  
Oocyte Quality ◽  
G6pd Activity ◽  
Blue Staining ◽  
Control Group ◽  
Embryo Production ◽  
Brilliant Cresyl Blue ◽  
Cresyl Blue ◽  
In Vitro Embryo Production ◽  
Humidified Air

A large proportion of ovine oocytes fail to develop into viable embryos following maturation, fertilization, and culture in vitro. Accurate, fast, and noninvasive predictors of ovine oocyte quality are therefore in urgent need for oocyte selection before in vitro maturation (IVM). Recent studies have shown that oocyte competence can be predicted through the presence of the glucose-6-phosphate dehydrogenase (G6PD) enzyme, as indicated by brilliant cresyl blue (BCB), a dye that can be degraded by G6PD. Thus, oocytes that have completed their growth phase show decreased G6PD activity and exhibit cytoplasm with a blue colouration (BCB+), whereas growing oocytes are expected to have a high level of G6PD, which results in colourless cytoplasm (BCB–). The brilliant cresyl blue staining test, as a noninvasive intrinsic criterion, has been successfully used to identify the more competent oocytes in various species. Therefore, this study aimed to investigate whether BCB staining, as an indicator of G6PD activity, can be used to select developmentally competent ovine oocytes before IVM and thereby increase the efficiency of in vitro embryo production. Ovine ovaries were obtained from a local slaughterhouse and transported to the laboratory, where cumulus–oocyte complexes (COC) were recovered by slicing the ovaries. Only oocytes with one or more complete layers of unexpanded cumulus cells and a homogeneous cytoplasm were used. The COC were exposed to 26 mM BCB diluted in modified Dulbecco’s PBS for 90 min at 39°C in humidified air. After BCB exposure, the COC were examined under a stereomicroscope and divided into 2 groups: BCB+ (blue cytoplasm, low G6PD activity) and BCB– (colourless cytoplasm, high G6PD activity). Cumulus–oocyte complexes in the control group were incubated for IVM directly after selection, without exposure to BCB dye. After IVM, oocytes were subjected to IVF followed by embryo culture for 7 days (5% CO2, 39°C, humidified air). Results were analysed by a chi-square test, and P < 0.05 was considered statistically significant. The proportion of oocytes that cleaved by Day 2 after insemination was significantly (P < 0.05) higher for the control and BCB+ groups [67.3% (68/101) and 71.7% (81/113), respectively] than for the BCB– group [50.5% (46/91)]. Significant differences among groups were also observed on Day 7 after fertilization, when the embryos reached the blastocyst stage of development. The BCB+ group yielded a significantly (P < 0.05) higher proportion of blastocysts [34.5% (39/113)] than both the control [20.8% (21/101)] and BCB– [4.3% (4/93)] groups. In addition, the blastocyst rate of development in the control group was significantly (P < 0.05) higher than that for the BCB– group. In conclusion, results of this study show that selection of ovine oocytes based on G6PD activity through the BCB test can be used as an efficient predictor of in vitro embryonic developmental competence. This positive predictive parameter of oocyte quality may also be useful in increasing the efficiency of blastocyst production during in vitro embryo production procedures in the ovine.

scholarly journals Selection of Ovine Oocytes by Brilliant Cresyl Blue Staining

2012 ◽  
Vol 2012 ◽  
pp. 1-7 ◽  
Author(s):  
Liqin Wang ◽  
Jiapeng Lin ◽  
Juncheng Huang ◽  
Jing Wang ◽  
Yuncheng Zhao ◽  
...  
Keyword(s):  
Embryonic Stem ◽  
Cumulus Cells ◽  
Oocyte Quality ◽  
G6pd Activity ◽  
Blue Staining ◽  
Animal Cloning ◽  
Cresyl Blue ◽  
Deep Blue ◽  
Glucose 6 Phosphate Dehydrogenase

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.

scholarly journals Use of oocytes selected by brilliant cresyl blue staining enhances rabbit cloned embryo development in vitro

Zygote ◽  
2019 ◽  
Vol 27 (3) ◽  
pp. 166-172 ◽  
Author(s):  
Linying Jia ◽  
Bo Ding ◽  
Chong Shen ◽  
Shiwei Luo ◽  
Yanru Zhang ◽  
...  
Keyword(s):  
Cell Mass ◽  
Developmental Competence ◽  
Blue Staining ◽  
Control Group ◽  
Control Groups ◽  
Brilliant Cresyl Blue ◽  
Positive Group ◽  
Cresyl Blue ◽  
And Control

SummaryRabbits play an important role in people’s lives due to their high nutritional value and high-quality hair that can be used as raw material for textiles. Furthermore, rabbits are an important animal model for human disease, as genome-edited animals are particularly valuable for studying gene functions and pathogenesis. Somatic cell nuclear transfer (SCNT) is an important technique for producing genome-edited animals and it has great value in saving endangered species and in clone stem cell therapy. However, the low efficiency of SCNT limits its application, with the selection of suitable rabbit oocytes being crucial to its success. In the present study, we collected oocytes from ovarian follicles and stained them with 26 μM brilliant cresyl blue (BCB). We then matured the oocytes in vitro and used them for SCNT. Comparison of the BCB-positive oocytes with BCB-negative oocytes and the control group showed that the BCB-positive group had a significantly higher maturation rate (81.4% vs. 48.9% and 65.3% for the negative and control groups, respectively), cleavage rate (86.6% vs. 67.9% and 77.9%), blastocyst rate (30.5% vs. 12.8% and 19.6%), total number of blastocysts (90±7.5 vs. 65.3±6.3 and 67.5±5.7), and inner cell mass (ICM)/ trophectoderm (TE) index (42.3±4.2 vs. 30.2±2.1 and 33.9±5.1) (P<0.05). The BCB-positive group had a significantly lower apoptosis index (2.1±0.6 vs. 8.2±0.9 and 6.7±1.1 for the negative and control groups, respectively) (P<0.05). These findings demonstrate that BCB-positive oocytes have a higher maturation ability and developmental competence in vitro, indicating that BCB staining is a reliable method for selecting oocytes to enhance the efficiency of SCNT.

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

Zygote ◽  
2011 ◽  
Vol 21 (3) ◽  
pp. 238-245 ◽  
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.

scholarly journals Selection of Immature Cat Oocytes with Brilliant Cresyl Blue Stain Improves In Vitro Embryo Production during Non-Breeding Season

Animals ◽  
2020 ◽  
Vol 10 (9) ◽  
pp. 1496
Author(s):  
Anna Rita Piras ◽  
Federica Ariu ◽  
Maria-Teresa Zedda ◽  
Maria-Teresa Paramio ◽  
Luisa Bogliolo
Keyword(s):  
Breeding Season ◽  
Domestic Cat ◽  
Hatching Rate ◽  
Domestic Cats ◽  
Embryo Production ◽  
Brilliant Cresyl Blue ◽  
Cresyl Blue ◽  
In Vitro Embryo Production ◽  
Blastocyst Rate

In domestic cats, the maturation, fertilization, and development potential in vitro decreases during the non-breeding season. This study aims at evaluating the efficacy of Brilliant Cresyl Blue (BCB) staining in selecting developmentally competent oocytes to be used in in vitro embryo production (IVEP) programs in order to overcome the season variability in blastocyst yield. Cumulus-oocytes complexes (COCs) collected from antral follicles of domestic cat ovaries during the anestrus phase (July to November) were selected by BCB staining and classified as BCB+ (colored cytoplasm) and BCB− (colorless cytoplasm). COCs not exposed to BCB staining were used as control. Before and after in vitro maturation mitochondrial activity and reactive oxygen species (ROS) were measured. Following in vitro fertilization, blastocyst rate, hatching rate, and blastocyst cell numbers were recorded. The results show that BCB staining did not alter the mitochondrial function and ROS production in cat oocytes. BCB+ oocytes presented a higher (p < 0.05) blastocyst rate, hatching rate, and blastocyst cell number than BCB− and control oocytes. In conclusion, BCB staining does not affect the bioenergetic/oxidative status of the oocyte while being a useful tool for selecting good quality oocytes to increase IVEP in domestic cats during non-breeding season.

Brilliant cresyl blue staining negatively affects mitochondrial functions in porcine oocytes

Zygote ◽  
2013 ◽  
Vol 23 (3) ◽  
pp. 352-359 ◽  
Author(s):  
E.C.S. Santos ◽  
D. Sato ◽  
T. Lucia ◽  
H. Iwata
Keyword(s):  
Early Embryo ◽  
Blue Staining ◽  
Control Group ◽  
Early Developmental Stage ◽  
Atp Content ◽  
Brilliant Cresyl Blue ◽  
Cresyl Blue ◽  
Mitochondrial Dna Copy Number ◽  
Mitochondrial Functions

SummaryThe aim of the present study was to examine the effects of brilliant cresyl blue (BCB) staining on mitochondrial functions in porcine oocytes. Cumulus–oocyte complexes (COCs) collected from slaughterhouse-derived porcine ovaries were cultured with (13 μM) or without (0 μM, control) BCB for 60 min. Mitochondrial functions in oocytes were examined immediately after staining or after in vitro maturation. The BCB-stained oocytes produced reactive oxygen species (ROS) at higher levels than control oocytes immediately after staining (2.2-fold, P < 0.001) and after maturation (1.7-fold, P < 0.001). The adenosine triphosphate (ATP) content and mitochondrial membrane potential (MMP) in oocytes were similar for the two groups immediately after staining. However, ATP and relative MMP levels were significantly (P < 0.05) lower in BCB-treated oocytes than in the control (2.18 versus 2.83 pM and 0.82 versus 1.0, respectively). There was no difference in mitochondrial DNA copy number between the two groups after maturation. The ATP content in early developmental stage embryos (3 days after parthenogenetic activation) was lower in the BCB-stained group than that in the control group but the difference was not significant. In conclusion, BCB staining of oocytes at the immature stage compromises mitochondrial functions throughout oocyte maturation, but function is restored during early embryo development.

Effects of Myo-inositol on Physiological and Reproductive Traits through Blood Parameters, Oocyte Quality and Embryo Transfer in Mice

2020 ◽  
Author(s):  
Abd El-Nasser Ahmed Mohammed ◽  
Tarek Alshaheen ◽  
Shaker Al-Suwaiegh
Keyword(s):  
Embryo Transfer ◽  
Total Protein ◽  
Virgin Female ◽  
Reproductive Traits ◽  
Oocyte Quality ◽  
Blood Parameters ◽  
Blue Staining ◽  
Control Group ◽  
Brilliant Cresyl Blue ◽  
Cresyl Blue

Background: The beneficial effects of myo-inositol to mammals on physiological and reproductive functions and treatments of malfunctions have been reported. The aims of present study were to assess the efficacy of myo-inositol supplementation on physiological and reproductive performances through blood parameters, oocyte quality and embryo transfer in mice. Methods: Seventy-five virgin female mice were classified into three equal groups; control group (T1) versus two myo-inositol groups (T2 receive 30 mmol/l and T3 receive 60 mmol/l). Changes of body temperature, blood oxygen and glucose, heart rate, blood parameters (RBCs, WBCs, hematocrit, glucose and total protein), oocyte quality (cumulus enclosed, diameter and brilliant cresyl blue stain) and reproductive performances (litter weight and size) were determined. Result: The results indicated that myo-inositol supplementation resulted in significant increase in values of RBCs, WBCs, hematocrit and total protein in addition to significant hypoglycemia. The quality of oocytes in case of cumulus enclosed, diameter and brilliant cresyl blue staining and reproductive performances were improved due to myo-inositol supplementation. Furthermore, myo-inositol supplementation effectively alleviated hypothermia and hyperglycemia following general anaesthesia.

Safety of brilliant cresyl blue staining protocols on human granulosa and cumulus cells

Zygote ◽  
2015 ◽  
Vol 24 (1) ◽  
pp. 83-88 ◽  
Author(s):  
Diego Duarte Alcoba ◽  
Maiara Conzatti ◽  
Gustavo Dias Ferreira ◽  
Anita Mylius Pimentel ◽  
Ana Paula Kussler ◽  
...  
Keyword(s):  
Cumulus Cells ◽  
Blue Staining ◽  
Control Group ◽  
Phenol Red ◽  
Brilliant Cresyl Blue ◽  
Human Oocytes ◽  
Cresyl Blue ◽  
Cumulus Oocyte Complex ◽  
Close Relationship

SummaryThe selection of human immature oocytes destined for in vitro maturation (IVM) is performed according to their cumulus–oocyte complex (COC) morphology. In animal models, oocyte pre-selection with brilliant cresyl blue (BCB) staining improves fertilization and blastocyst rates and even increases the number of calves born. As the granulosa cells and cumulus cells (GCs and CCs) have a close relationship with the oocyte and are available in in vitro fertilization (IVF) programs, applying BCB staining to these cells may help to elucidate whether BCB shows toxicity to human oocytes and to determine the safest protocol for this dye. GCs and CCs were isolated from 24 patients who underwent controlled ovarian stimulation. After 48 h, cells were exposed to: Dulbecco's Modified Eagle Medium (DMEM) with or without phenol red, DPBS and mDPBS for 60 min; 13, 20 and 26 μM BCB for 60 min; and 60, 90 or 120 min to 13 μM BCB. Cellular viability was tested using 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) and trypan blue assays. The 20 and 26 μM BCB exposures resulted in lower cell viability, similar to when cells were exposed to BCB for 90 or 120 min. GCs and CCs viabilities were equal among control group and 13 μM BCB group after 60 min. BCB staining was not toxic to GCs and CCs when the regime of 13 μM BCB for 60 min was used. Due to the close molecular/biochemical relationship between these cells and the gamete, we propose that it is unlikely that the use of BCB could interfere with the viability/health of human oocytes.

scholarly journals Effect of adding growth factors during in vitro maturation on the developmental potentials of ewe oocytes selected by brilliant cresyl blue staining

2021 ◽  
Vol 14 (2) ◽  
pp. 452-456
Author(s):  
Mohamed Fathi ◽  
Amr F. Elkarmoty
Keyword(s):  
Growth Factors ◽  
Growth Factor ◽  
Embryo Development ◽  
In Vitro Maturation ◽  
Blue Staining ◽  
Brilliant Cresyl Blue ◽  
Cresyl Blue ◽  
Nuclear Maturation ◽  
Maturation Medium

Aim: Several factors had been concerned with the developmental competence of the sheep oocyte. This study aims to investigate the effect of adding growth factors (insulin-like growth factor 1 [IGF-1] and epidermal growth factor [EGF]) in the maturation medium of ewe oocytes selected based on brilliant cresyl blue (BCB) screening on in vitro maturation (IVM), fertilization, and pre-implantation embryo development. Materials and Methods: Cumulus-oocyte complexes (COCs) were obtained from the ovaries of slaughtered ewes by either aspiration or slicing techniques. COCs were in vitro matured in a medium containing IGF-1 and EGF (control group). For BCB screening, oocytes were stained and divided into BCB+ oocytes that matured in the same maturation conditions without adding growth factors (Group 2) or in the presence of growth factors (Group 3), and BCB– oocytes that matured in medium without growth factors (Group 4) or with growth factors (Group 5). Results: The supplementation of the maturation medium with growth factors during IVM of (BCB+) oocytes resulted in a significant increase in nuclear maturation rate (90.9%), fertilization rate (75.6%), and embryo developmental rates (60.0%, 46.7%, and 33.3% for cleavage, morula, and blastocyst, respectively). Conclusion: Culturing BCB+ oocytes in a maturation medium containing both EGF and IGF-1 showed a significant improvement in nuclear maturation, fertilization, and pre-implantation embryo development in vitro.

195 RELATIVE mRNA EXPRESSION OF 4 CANDIDATES IN LAMB OOCYTES SELECTED BY BRILLIANT CRESYL BLUE STAINING

2013 ◽  
Vol 25 (1) ◽  
pp. 246
Author(s):  
M. G. Catalá ◽  
M. Roura ◽  
D. Izquierdo ◽  
S. Hammammi ◽  
S. Uzbekova ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Relative Concentration ◽  
Rna Extraction ◽  
Melting Curve ◽  
Elongation Factor ◽  
Oocyte Quality ◽  
Blue Staining ◽  
Melting Curve Analysis ◽  
Brilliant Cresyl Blue ◽  
Cresyl Blue

Brilliant cresyl blue (BCB) staining determines the activity of glucose-6-phosphate dehydrogenase (G6PDH), an enzyme in which activity decreases as the oocytes reach their growth phase. We have previously shown in lamb the effectiveness of this stain in selecting the largest and most competent oocytes to develop up to the blastocyst stage (Catala et al. 2011 Reproduction 142, 517–527). The aim of this study was to analyse the expression of 4 genes related to oocyte quality in BCB-selected oocytes. The 4 genes analyzed in this study were selected after a literature review in which they were associated with a better oocyte quality or embryo development; 2 of these genes were related to cell cycle: nuclear auto-antigenic sperm protein (NASP) and histone H2A (H2A.Z), and 2 genes with enzymatic functions: peroxiredoxin (PRDX1) and elongation factor-A1 (EEF1A1). Oocytes were recovered after slicing the surface of lamb ovaries (3 to 5 months old) obtained from a local slaughterhouse. Oocytes with more than 3 compact cumulus layers and homogenic cytoplasm were selected and exposed to 13 µM BCB during 1 h before IVM and classified according to oocyte coloration: oocytes with blue cytoplasm or grown oocytes (BCB+) and oocytes not coloured or growing oocytes (BCB–). Cumulus–oocyte complexes were then matured in conventional TCM199 medium supplemented with 10% fetal bovine serum and hormones during 24 h in a controlled atmosphere. Groups of 15 denuded oocytes (3 replicates) were taken before (0 h) and after maturation (24 h) and stored at –80°C in 100 mL of Trizol until use. After RNA extraction following manufacturer’s protocol (Promega, Madison, WI, USA), reverse transcription was performed by extended cDNA using Oligo (dT) primers during 5 min at 70°C and 1 h at 65°C using superscript III (200 U mM–1; Invitrogen). Relative qualitative PCR analyses were performed in duplicate using SYBR Green Fluorophore kit (Bio-Rad, Hercules, CA, USA). The specificity of each PCR product was determined by a melting curve analysis and the amplicon size determination by an agarose gel. A standard curve was also included, consisting of corresponding plasmid DNA fragments from 1 pg to 0.1 fg, purified with QIAquick PCR Purification Kit (Qiagen, Valencia, CA, USA). Correlation coefficients and PCR efficiencies were considered between 85 and 100%. Finally, the results for mRNA were normalized according to the relative concentration of the internal and external gene (luciferase and 18S, respectively). The statistical analysis was performed by One-way ANOVA in GraphPad Prism v 3 (GraphPad Software, San Diego, CA, USA). The analysis of gene expression showed no differences in relation to BCB classification. The only difference we found is a significant decrease (P ≤ 0.05) in the RE of PRDX observed in matured oocytes compared with immature ones. In conclusion, the expression of the mRNA expression of NASP, H2A.Z, PRDX1, and EEF1A1 were not affected by oocyte quality.

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