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

Zygote ◽  
2016 ◽  
Vol 25 (1) ◽  
pp. 49-55 ◽  
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.

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.

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.

293 EFFECT OF DIFFERENT CONCENTRATIONS OF LH, FSH, AND E2 ON THE MATURATIONAL RATE OF INDIGENOUS SOUTH AFRICAN CATTLE OOCYTES SELECTED BY BRILLIANT CRESYL BLUE STAINING

2015 ◽  
Vol 27 (1) ◽  
pp. 235
Author(s):  
K. P. M. Lekola ◽  
J. W. Ng'ambi ◽  
N. Nkadimeng ◽  
M. L. Mphaphathi ◽  
T. L. Nedambale
Keyword(s):  
South African ◽  
In Vitro Maturation ◽  
Polar Body ◽  
Blue Staining ◽  
Brilliant Cresyl Blue ◽  
Cresyl Blue ◽  
African Cattle ◽  
Maturation Medium ◽  
Maturation Rate

In vitro maturation of indigenous African cattle oocytes is a major challenge even though different maturation protocols work successfully in other breeds. The objective of this study was to determine the maturation rate of indigenous South African cattle oocytes following in vitro maturation in media supplemented with different concentrations of hormones and selected using brilliant cresyl blue (BCB) staining. Indigenous cattle ovaries were collected from the slaughterhouse and then oocytes were retrieved by aspiration method. A total of 966 oocytes were exposed to 26 µM BCB stain and 700 oocytes were not exposed to the BCB stain. Thereafter, oocytes exposed to the BCB stain were grouped according to the colour of their cytoplasm BCB+ (oocytes with blue cytoplasm, low G6PDH) and BCB– (unstained oocytes, increased G6PDH). The BCB exposed (BCB+ and BCB–) and the oocytes not exposed to BCB were then randomly allocated into tissue culture medium (TCM199) + 10% (vol/vol) fetal bovine serum (FBS) supplemented with 3 different concentrations of hormones as treatments (T). The T1 group was matured in the presence of 0.5 µg mL–1 of FSH, 5 mg mL–1 of LH, and 2 µg mL–1 of E2; the T2 group was matured in the presence of 1 µg mL–1 of FSH, 6 mg mL–1 of LH, and 2.5 µg mL–1 of E2; and the T3 group was matured in the presence of 1.5 µg mL–1 of FSH, 7 mg mL–1 of LH, and 4.5 µg mL–1 of E2. For IVM, 20 to 25 COC were placed in 50-µL droplets of IVM medium containing the 3 different levels of hormones. Maturation rate of oocytes was determined by the extrusion of the first polar body after 24 h of incubation in maturation medium. Data was analysed by ANOVA using SAS with 4 replicates per treatment. Treatment 2 yielded higher maturation rate for both BCB+ (65.6%) and not exposed to BCB (60.3%) oocytes compared to T1 (22, 3.03, and 16% for BCB+, BCB–, and not exposed to BCB, respectively) and T3 (48, 2.2, and 48% for BCB+, BCB–, and not exposed to BCB respectively). However, BCB– oocytes had lower polar body extrusion for T1, T2, and T3 (3.03, 8.1, and 2.2%, respectively) compared to BCB+ oocytes (22, 65.6, and 48% for T1, T2, and T3, respectively). In conclusion, immature oocytes that were cultured into TCM199 supplemented with 10% FBS, 1 µg mL–1 of FSH, 6 mg mL–1 of LH, and 2.5 µg mL–1 of E2 showed maturation rate for BCB+ oocytes and those not exposed to BCB. Oocytes selection using BCB staining was a useful test to classify good quality cattle oocytes. Therefore, it is suggested that treatment 2 is a suitable in vitro-maturation medium to mature indigenous South African cattle oocytes.

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.

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

2007 ◽  
Vol 19 (1) ◽  
pp. 273 ◽  
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, &lt;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 &lt; 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 &lt; 0.01). There were also significant differences (P &lt; 0.01) in the blastocyst development between the BCB+ and BCB− embryos and between the BCB− and the control embryos (P &lt; 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

scholarly journals Resveratrol supplementation during in vitro maturation improves embryo development of prepubertal goat oocytes selected by brilliant cresyl blue staining

2019 ◽  
Vol 65 (2) ◽  
pp. 113-120 ◽  
Author(s):  
Anna-Rita PIRAS ◽  
Irene MENÉNDEZ-BLANCO ◽  
Sandra SOTO-HERAS ◽  
Maria-Gracia CATALÁ ◽  
Dolors IZQUIERDO ◽  
...  
Keyword(s):  
Embryo Development ◽  
Blue Staining ◽  
Brilliant Cresyl Blue ◽  
Cresyl Blue

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

Cell Research ◽  
2007 ◽  
Vol 17 (8) ◽  
pp. 722-731 ◽  
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

scholarly journals 237 INCREASE OF BLASTOCYST DEVELOPMENTAL RATE IN VITRO BY SELECTION OF DEVELOPMENTALLY COMPETENT COW OOCYTES BEFORE IVM USING A STAINING TEST

2005 ◽  
Vol 17 (2) ◽  
pp. 269
Author(s):  
H. Alm ◽  
H. Torner ◽  
B. Loehrke ◽  
T. Viergutz ◽  
I. Ghoneim ◽  
...  
Keyword(s):  
Developmental Rate ◽  
Cell Number ◽  
Developmental Competence ◽  
Control Group ◽  
G6pdh Activity ◽  
Blastocyst Development ◽  
Brilliant Cresyl Blue ◽  
Immature Oocytes ◽  
Cresyl Blue

A large proportion of bovine oocytes fail to develop to blastocyst stage following maturation, fertilization, and culture in vitro. While suboptimal culture conditions undoubtedly contribute to this poor development, it is recognized that immature oocytes, especially from cows with reduced reproductive performance or which are slaughtered on the end of their use, are heterogeneous in quality and developmental competence (Gordon 2003). The aim of the present study was to increase the efficiency of blastocyst production from cows after IVM/IVF by oocyte selection before maturation. Immature oocytes are known to synthesize a variety of proteins (Wassarman PM 1988, Annu. Rev. Biochem. 57, 415–442), among them, glucose-6-phosphate dehydrogenase (G6PDH). This enzyme is active in the growing oocyte, but has decreased activity in oocytes that have finished their growth phase. Brilliant cresyl blue (BCB) has been used to measure G6PDH activity. The BCB test is based on the capability of the G6PDH to convert the BCB stain from blue to colorless (Erisson et al. 1993 Theriogenology 39, 214). The ovaries were obtained from a slaughterhouse and transported to the laboratory; cumulus-oocyte complexes (COCs) were recovered by slicing the surface of the ovary. Only oocytes with a compact cumulus investment were used. Oocytes were placed into three groups: (1) control – placed immediately into culture; (2) holding control – COCs kept in PBS containing 0.4% BSA for 90 min at 38.5°C before placement into culture; and (3) treatment – incubation with brilliant cresyl blue for 90 min at 38.5°C before culture. Treated oocytes were then divided into BCB− (colorless cytoplasm, increased G6PDH) and BCB+ (colored cytoplasm, low G6PDH) on their ability to metabolize the stain. Activity of G6PDH was determined via measurement of NADP reduction in control, BCB−, and BCB+ groups; activity was significantly increased in BCB− COCs in comparison to the control and BCB+ COCs. After IVM, oocytes were fertilized in vitro. Embryos were cultured to Day 8. The rate of maturation to metaphase II was significantly higher for control and BCB+ oocytes (77.1 and 72.5%, respectively) than for BCB− oocytes (58.1%). The BCB+ oocytes yielded a significantly higher proportion of blastocysts (34.1%) than either control group (18.3 and 19.2%); and both controls and BCB+ oocytes had significantly higher blastocyst development than did BCB− oocytes (3.9%). The number of nuclei in the blastocysts was comparable in BCB+ and both control groups (105.5 ± 5.8 and 117.5 ± 8.5, 101.8 ± 6.2, respectively). Blastocysts in the BCB− group had a significantly lower cell number (61.0 ± 2.6) than did controls. The results show that the staining of COCs from cows before IVM may be useful in increasing the efficiency of blastocyst production during standard IVF procedures. In addition, classification of G6PDH activity on the basis of BCB staining may be used to effectively select cow oocytes with further developmental competence. To our knowledge, this is the first study to evaluate the association between G6PDH activity in oocytes and further blastocyst development in cows.

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

2007 ◽  
Vol 19 (1) ◽  
pp. 273 ◽  
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&apos;s t-test. The mean (&plusmn; SEM) percentage of oocytes classified as BCB&plus; in calves was significantly lower than that in cows (34.4 &plusmn; 2.9&percnt; and 69.2 &plusmn; 2.1&percnt;, respectively; P &lt; 0.01). In cows, BCB&plus; oocytes showed significantly higher cleavage and blastocyst formation percentages (72.5&percnt; and 42.4&percnt;, respectively) than those of BCB&minus; 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&plus; oocytes (56.9&percnt; and 25.3&percnt;, respectively) and BCB&minus; oocytes (65.4&percnt; and 22.4&percnt;, respectively). The mean (&plusmn; SEM) numbers of usable oocytes and blastocysts obtained per calf (19.0 &plusmn; 1.5 and 4.5 &plusmn; 0.6, respectively) were similar to those obtained per cow (16.4 &plusmn; 1.1 and 5.2 &plusmn; 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.

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.

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