Combination of oocyte and zygote selection by brilliant cresyl blue (BCB) test enhanced prediction of developmental potential to the blastocyst in cattle

The cumulus oocyte complexes (COCs) were obtained from local abattoir. After aspiration, the COCs were allotted into four treatments to evaluation of brilliant cresyl blue (BCB) test. Control treatment (C): oocytes were cultured directly (without exposure to BCB) after recovery in in vitro production (IVP) process. Oocyte treatment (OBCB): immediately after aspiration, COCs were incubated in modified Dulbecco's phosphate-buffered saline (mDPBS) supplemented with 26 μM of BCB for 90 min and classified into two classes: oocytes with blue cytoplasm coloration (OBCB+: more competent oocytes) and oocytes without blue cytoplasm coloration (OBCB−: low competent oocytes). Directly after classification, the oocytes were maintained undisrupted in the IVP process. Zygote treatment (ZBCB): After oocyte collection, maturation and fertilization, zygotes were stained with BCB for 10 min and categorized into three ways, according to whether they were highly stained (ZBCB++: low competent zygotes), moderately stained (ZBCB+: moderate competent zygotes) and unstained (ZBCB−: more competent zygotes). Directly after classification, the zygotes were maintained undisrupted in the culture process. Oocyte and zygote treatments (OBCB/ZBCB): COCs were stained with BCB after recovery and classified into two classes (OBCB+ and OBCB−). After fertilization, the zygotes produced from OBCB+ and OBCB− oocytes were further stained with BCB for 10 min and categorized six ways (OBCB+/ZBCB++, OBCB+/ZBCB+, OBCB+/ZBCB−, OBCB−/ZBCB++, OBCB−/ZBCB+ and OBCB−/ZBCB−). Directly after classification, the zygotes were maintained undisrupted in the culture process. The selection rate produced from OBCB treatment (OBCB+; 54.3%) was greater (P < 0.05) than ZBCB treatment (ZBCB−; 44.3%). In addition, the selection rate produced from double application (combination of oocyte and zygote selection) of BCB test (OBCB+/ZBCB−: 28.8%) was less (P < 0.01) than single application of BCB test (ZBCB−: 44.3%or OBCB+: 54.3%). The percentage of blastocyst production from OBCB+ oocytes (35.7%) and ZBCB− zygotes (36.6%) were greater (P < 0.05) than that from C oocytes (25.7%), OBCB− oocytes (16.5%), ZBCB++ (13.5%) and ZBCB+ zygotes (21.3%). However, there were no significant differences (P > 0.05) in the percentages of blastocyst production between OBCB+ oocytes (35.7%) and ZBCB− zygotes (36.6%). The proportion of blastocyst production from double application of BCB test (OBCB+/ZBCB−: 48.0%) was greater (P < 0.05) than that from single application of BCB test (OBCB+: 35.7% or ZBCB−: 36.6%). In conclusion, current results confirmed that combination of oocyte and zygote selection by BCB test enhanced the efficiency of selecting for high quality embryos, compared to the single BCB test.     

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

Selecting oocytes that are most likely to develop is crucial for in vitro fertilization and animal cloning. Brilliant cresyl blue （BCB） staining has been used for oocyte selection in large animals, but its wider utility needs further evaluation. Mouse oocytes were divided into those stained （BCB＋） and those unstained （BCB-） according to their ooplasm BCB coloration. Chromatin configurations, cumulus cell apoptosis, cytoplasmic maturity and developmental competence were compared between the BCB＋ and BCB- oocytes. The effects of oocyte diameter, sexual maturity and gonadotropin stimulation on the competence of BCB＋ oocytes were also analyzed. In the large- and medium-size groups, BCB＋ oocytes were larger and showed more surrounded nucleoli （SN） chromatin configurations and higher frequencies of early atresia, and they also gained better cytoplasmic maturity （determined as the intracellular GSH level and pattern of mitochondrial distribution） and higher developmental potential after in vitro maturation （IVM） than the BCB-oocytes. Adult mice produced more BCB＋ oocytes with higher competence than the prepubertal mice when not primed with PMSG. PMSG priming increased both proportion and developmental potency of BCB＋ oocytes. The BCB＋ oocytes in the large-size group showed more SN chromatin configurations, better cytoplasmic maturity and higher developmental potential than their counterparts in the medium-size group. It is concluded that BCB staining can be used as an efficient method for oocyte selection, but that the competence of the BCB＋ oocytes may vary with oocyte diameter, animal sexual maturity and gonadotropin stimulation. Taken together, the series of criteria described here would allow for better choices in selecting oocytes for better development.     

Developmental competence of heifer oocytes selected using the brilliant cresyl blue (BCB) test

The aim of this study was to evaluate the usefulness of the brilliant cresyl blue (BCB) test in the selection of more competent heifer oocytes for in vitro embryo production (IVEP). IVEP from selected BCB heifer oocytes was compared to IVEP from morphologically selected heifer (control group) and cow oocytes. BCB staining determines the activity of glucose-6-phosphate dehydrogenase (G6PD), an enzyme synthesized in growing oocytes but with less activity in grown oocytes. Six hundred and fifty seven heifer cumulus-oocyte complexes (COC) were classified morphologically as Grade 1-3 and exposed to 26 microM of BCB and classified as: blue (or grown) oocytes (BCB+) or unstained oocytes or growing oocytes (BCB-). Grade 1-3 heifer oocytes showed significantly different percentages of BCB+ oocytes (78.6, 66.2, and 51.1%, respectively; P<0.05). The diameter of BCB+ oocytes was significantly higher than BCB- oocytes (152.6+/-5.8 microm and 147+/-5.9 microm, respectively; P<0.001). The percentage of BCB+ oocytes reaching the blastocyst stage was significantly higher than those of BCB- and control heifer oocytes (12.3, 1.6, and 5.2%, respectively; P<0.05), but lower than those of cow oocytes (30.0%; P<0.05). In conclusion, heifer oocytes selected by the BCB test (BCB+) are larger and more competent for IVEP than control heifer oocytes. However, fewer heifer oocytes selected using the BCB test develop to blastocyst stage compared to cow oocytes.     

Gene expression pattern of adiponectin and adiponectin receptors in dominant and atretic follicles and oocytes screened based on brilliant cresyl blue staining

Adiponectin and its receptors (AdipoR1 and AdipoR2) are novel endocrine systems that act at various levels to control male and female fertility. The aim of this study was to determine whether adiponectin and its receptors gene expression levels differ between dominant follicle (DF) and atretic follicle (AF) and also between oocytes which were stained positively and negatively with brilliant cresyl blue (BCB(+) and BCB(-)). Based on estradiol/progesterone ratio, follicles from ovaries were classified as AFs and DFs. The stages of estrous cycle (follicular or luteal phases) were defined by macroscopic observation of the ovaries and the uterus. Oocytes were stained with BCB for 90 min. The relative expression of adiponectin, AdipoR1 and AdipoR2 mRNA in theca and cumulus cells and oocytes of different follicles were determined by quantitative real time PCR. Adiponectin and its receptors genes were clearly expressed higher (P<0.05) in theca and cumulus cells and oocytes of DFs than those of AFs during the follicular and luteal phases. BCB(+) oocytes showed a higher (P<0.05) expression of adiponectin and its receptors compared with their BCB(-) counterparts. Positive correlation (r>0.725, P<0.001) was observed between adiponectin mRNA level in ovarian cells of DFs and follicular fluid E2 concentration in follicular phase. Adiponectin mRNA abundance in ovarian cells of AFs showed a significant negative correlation with follicular fluid progesterone concentration in follicular and luteal phases (r<-0.731, P<0.001). This work has revealed the novel association of adiponectin and its receptors genes with follicular dominance and oocyte competence, thereby opening several new avenues of research into the mechanisms of dominance and competence in animal and human.     

Selection of developmentally competent buffalo oocytes by brilliant cresyl blue staining before IVM

The brilliant cresyl blue (BCB) test determines the activity of glucose-6-phosphate dehydrogenase (G6PDH); the activity of this enzyme is greatest in growing oocytes, but it declines as oocytes mature. The objective was to develop and evaluate this test for assessing development of buffalo oocytes (to select developmentally competent oocytes for increased in vitro embryo production). Oocytes were exposed to BCB stain diluted in mDPBS (DPBS with 0.4% BSA) for 90 min at 38.5 °C in a humidified air atmosphere; those with or without blue coloration of the cytoplasm were designated as BCB+ and BCB−, respectively. In Experiment 1, oocytes were exposed to 13, 26, or 39 μM BCB. There were fewer BCB+ oocytes after exposure to 13 μM BCB (10%) than after exposure to 26 or 39 μM BCB (57.2 and 61.8%; P < 0.05), but there was no significant difference among treatments for blastocyst production rate. In Experiment 2, the diameter of BCB+ oocytes (144.4 ± 4.2 μm; mean ± S.E.M.) was higher (P < 0.05) than that of BCB− oocytes (136.8 ± 4.6 μm). In Experiment 3, oocytes were allocated into three groups: control (immediately cultured); holding-control (kept in mDPBS for 90 min before cultured); and treatment-incubation (incubated with 26 μM BCB). After IVM, oocytes were fertilized in vitro and cultured on an oviductal monolayer. The nuclear maturation rate was higher (P < 0.05) in BCB+ (86.2%), control (83.4%) and holding-control (82.6%) oocytes than BCB− (59.2%) oocytes. The BCB+ oocytes yielded more blastocysts than control or holding-control oocytes (33.4, 20.2, and 21.0%, P < 0.05); blastocyst development was lowest in BCB− oocytes (5.2%). In conclusion, staining of buffalo oocytes with BCB before IVM may be used to select developmentally competent oocytes for increased in vitro embryo production.     

Ca2+ release from intracellular stores of pig oocytes during different stages of growth

Ca2+ release from intracellular stores of pig oocytes was investigated using the Ca(2+)-sensitive fluorescent dye chlorotetracycline. Oocytes were divided into growing ones and those that completed their growth using brilliant cresyl clue (BCB) staining. The stained oocytes (BCB "+") were determined as the ones that completed their growth, while the stainless ones (BCB "-") were determined as those in the final stages of growth. In the BCB "+" and BCB "-" oocytes, prolactin, theophylline, GTP, and GDP cause Ca2+ to exit intracellular stores. In the oocytes that completed their growth, joint action of prolactin and GTP activates additional release of Ca2+, in which protein kinase C takes part. In growing oocytes, joint action of prolactin and GTP does not lead to additional release of Ca2+. Joint action of theophylline and GDP in growing oocytes and oocytes that completed the growth stage promotes additional Ca2+ exit from intracellular stores. This exit is regulated by protein kinase A. The obtained data show that there various routes of Ca2+ release from intracellular stores in growing and grown pig oocytes.     

Ca<Superscript>2+</Superscript> release from intracellular stores of pig oocytes at different stages of growth

Ca²⁺ release from intracellular stores of pig oocytes was investigated using the Ca²⁺-sensitive fluorescent dye chlorotetracycline. Oocytes were divided into growing ones and those that completed their growth using brilliant cresyl blue (BCB) staining. The stained oocytes (BCB “+”) were determined as the ones that completed their growth, while the stainless ones (BCB “−”) were determined as those in the final stages of growth. In the BCB “+” and BCB “−” oocytes, prolactin, theophylline, GTP, and GDP cause Ca²⁺ to exit intracellular stores. In the oocytes that completed their growth, joint action of prolactin and GTP activates additional release of Ca²⁺, in which protein kinase C takes part. In growing oocytes, joint action of prolactin and GTP does not lead to additional release of Ca2⁺. Joint action of theophylline and GDP in growing oocytes and oocytes that completed the growth stage promotes additional Ca²⁺ exit from intracellular stores. This exit is regulated by protein kinase A. The obtained data show that there various routes of Ca²⁺ release from intracellular stores in growing and grown pig oocytes.     

Selection of developmentally competent buffalo oocytes by brilliant cresyl blue staining before IVM

The brilliant cresyl blue (BCB) test determines the activity of glucose-6-phosphate dehydrogenase (G6PDH); the activity of this enzyme is greatest in growing oocytes, but it declines as oocytes mature. The objective was to develop and evaluate this test for assessing development of buffalo oocytes (to select developmentally competent oocytes for increased in vitro embryo production). Oocytes were exposed to BCB stain diluted in mDPBS (DPBS with 0.4% BSA) for 90 min at 38.5 degrees C in a humidified air atmosphere; those with or without blue coloration of the cytoplasm were designated as BCB+ and BCB-, respectively. In Experiment 1, oocytes were exposed to 13, 26, or 39 microM BCB. There were fewer BCB+ oocytes after exposure to 13 microM BCB (10%) than after exposure to 26 or 39 microM BCB (57.2 and 61.8%; P<0.05), but there was no significant difference among treatments for blastocyst production rate. In Experiment 2, the diameter of BCB+ oocytes (144.4+/-4.2 microm; mean+/-S.E.M.) was higher (P<0.05) than that of BCB- oocytes (136.8+/-4.6 microm). In Experiment 3, oocytes were allocated into three groups: control (immediately cultured); holding-control (kept in mDPBS for 90 min before cultured); and treatment-incubation (incubated with 26 microM BCB). After IVM, oocytes were fertilized in vitro and cultured on an oviductal monolayer. The nuclear maturation rate was higher (P<0.05) in BCB+ (86.2%), control (83.4%) and holding-control (82.6%) oocytes than BCB- (59.2%) oocytes. The BCB+ oocytes yielded more blastocysts than control or holding-control oocytes (33.4, 20.2, and 21.0%, P<0.05); blastocyst development was lowest in BCB- oocytes (5.2%). In conclusion, staining of buffalo oocytes with BCB before IVM may be used to select developmentally competent oocytes for increased in vitro embryo production.     

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

The aim of the present investigation was to study the effect of oocyte selection on the efficiency of bovine nuclear transfer in terms of increased blastocyst production. For this purpose, prior to in vitro maturation (IVM), oocytes were selected for their developmental competence on the basis of glucose-6-phosphate dehydrogenase (G6PDH) activity indicated by brilliant cresyl blue (BCB) staining. It has been hypothesized that growing oocytes have a higher level of active G6PDH in comparison to the mature oocytes. Compact cumulus oocyte complexes (COCs) were recovered from slaughterhouse-collected bovine ovaries and classified either as control group, which were placed immediately into culture without exposure to BCB stain, or treatment group, which were stained with BCB for 90min before culture. Treated oocytes were then divided into BCB- (colourless cytoplasm, increased G6PDH) and BCB+ (coloured cytoplasm, low G6PDH) based on their ability to metabolize the stain. After IVM, oocytes were subjected to nuclear transfer procedure for the production of cloned embryos which were then cultured for a period of 8 days to determine the blastocyst rate. The BCB+ oocytes yielded a significantly higher blastocyst rate (39%) than the control (21%) or BCB- oocytes (4%). These results show that the staining of bovine cumulus-oocyte complexes with BCB before in vitro maturation could be used to select developmentally competent oocytes for nuclear transfer. In addition, G6PDH activity could prove to be a useful marker for determining the oocyte quality in future.     

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.     

The quality of porcine oocytes is affected by sexual maturity of the donor gilt

Although differences in the quality of oocytes derived from young gilts and adult sows are well documented, evidence concerning gametes of pre-pubertal and cycling gilts is scarce and inconsistent. The aim of this work was to establish whether sexual maturity of gilts affects the quality of their oocytes with the use of the brilliant cresyl blue (BCB) test, oocyte diameter and apoptosis. Ovarian morphology was evaluated, and gonads with corpus luteum or albicans were recognized as originating form cycling gilts (C) and those with follicles as originating form pre-pubertal females (P). Altogether 952 cumulus-oocyte complexes (COCs; group P: 554; group C: 398) were examined, whereas 149 COCs, not subjected to BCB test, served as a control for TUNEL. COCs of proper morphology were evaluated by the BCB test which differentiated two categories of gametes: more competent, BCB+, and less competent BCB- oocytes. The control group comprised oocytes of proper morphology aspirated from ovaries of P and C gilts not subjected to BCB test. Finally five groups of COCs were matured in vitro: 1/P-BCB+, 2/P-BCB-, 3/C-BCB+, 4/ C-BCB- and 5/ control. Significantly more large oocytes (≥ 120 μm), more BCB+ oocytes and more high quality (both BCB+ and ≥ 120 μm) oocytes originated from ovaries of cycling gilts than pre-pubertal gilts (p<0.001). The rate of mature oocytes at the MII stage differed significantly between C-BCB+ (68.5%) and P-BCB+ (32.9%) oocytes. The incidence of apoptosis among BCB-treated oocytes after in vitro maturation was 21.4% and was similar to that observed in control oocytes (17.4%). BCB+ oocytes from cycling gilts showed significantly higher (28.7%) incidence of apoptosis than that of the group P (16.2%). Interestingly, high quality oocytes displayed a similar level of apoptosis regardless of the donor puberty. We demonstrated that C gilts provided more BCB+ oocytes as well as more large oocytes than P gilts, although C-BCB+ oocytes showed higher apoptotic rate. In conclusion, high incidence of apoptosis and a big variation in the diameter of more competent BCB+ oocytes make the BCB test a less effective selection tool than previously reported.     

Mitochondrial aggregation patterns and activity in in vitro cultured bovine oocytes recovered from early antral ovarian follicles

The low developmental competence seen in in vitro cultured oocytes collected from early antral follicles may be related to their mitochondrial status. The aim of this study was to examine the chromatin configuration, pattern of mitochondrial aggregation and mitochondrial activity of non-cultured and in vitro-cultured bovine oocytes originating from early antral ovarian follicles. Cumulus-oocyte complexes with adjacent granulosa cells (COCGs) were recovered from early antral follicles of 0.4 to 0.8 mm diameter. Control (Day 0) oocytes were recovered from freshly collected COCGs and fixed and stained. Selected COCGs were placed in growth culture for 7 days (Day 7) or 14 days (Day 14). Following growth culture, COCs with normal appearance were placed in maturation medium (IVM) for 24 h and then fixed and stained with MitoTracker CMTM Ros Orange and Hoechst 33258. The percentage of oocytes with an immature meiotic configuration after growth culture decreased with the time of growth culture, being 96.7; 72.5 and 35.4% respectively for Day 0, Day 7 and Day 14 of culture; the remaining oocytes were degenerating or resuming meiosis. After subsequent IVM the highest proportion of oocytes in diakinesis or metaphase I was found in the D7+IVM group (59.4%). When growth culture was prolonged to day 14 and IVM, the number of degenerated oocytes increased dramatically after IVM. The mitochondrial distribution in the oocytes changed from homogeneous to heterogeneous as growth culture time increased. The respiratory activity as measured by fluorescence intensity increased over the time of growth culture, and was highest in oocytes that had resumed GVBD. In conclusion, for oocytes in isolated COCGs from early antral follicles, culture conditions longer than 7 days should be more adapted for a slow nuclear maturation accompanied by a decreased energy metabolism to prevent chromatin pycnosis.     

Developmental competence of prepubertal goat oocytes selected with brilliant cresyl blue and matured with cysteamine supplementation

The aim of this study was to assess the effect of oocyte selection using the brilliant cresyl blue (BCB) test plus the addition of cysteamine to the in vitro maturation (IVM) medium to improve the in vitro embryo development of prepubertal goat oocytes. The oocytes were exposed to 26 microM BCB and classified according to their cytoplasm coloration: BCB+ (oocytes with blue cytoplasm) and BCB- (unstained oocytes). The oocytes were matured in a conventional IVM medium supplemented with cysteamine 100 microM. The control group consisted of oocytes not exposed to BCB and matured without cysteamine. The IVM-oocytes were inseminated and cultured in synthetic oviductal fluid (SOF) for 7 days. The normal fertilisation rate (oocytes showing 2 pronuclei and 1 sperm tail) of BCB+ oocytes (40%) was higher than those of BCB- (21%) and control oocytes (22%). The percentage of morulae plus blastocysts was higher (P < 0.05) in the BCB+ group than in the BCB- group (23.8 vs. 5.1%, respectively). In conclusion, the integration of the BCB test and the addition of cysteamine in the protocol of in vitro embryo production from prepubertal goat oocytes has improved the developmental rates of embryo development.     

Effects of single and double exposure to brilliant cresyl blue on the selection of porcine oocytes for in vitro production of embryos

The aim of this study was to evaluate the effectiveness and toxicity of single and double application of the brilliant cresyl blue (BCB) test on the selection of porcine oocytes as an indirect measure of oocyte growth for in vitro fertilization (IVF) and nuclear transfer. In the first experiment, oocytes were exposed to BCB before and after maturation culture and classified according to their cytoplasmic coloration: blue coloration and colorless. The classified oocytes were fertilized with spermatozoa and then cultured for 7 days. The percentages of maturation to metaphase II in blue oocytes at the start of maturation culture were higher than those of colorless oocytes (68.7-70.1% versus 0.8-3.6%, P < 0.05). However, double application of BCB test before and after maturation culture had a toxic effect on fertilization and embryonic development. No significant differences in the blastocyst formation were found between blue oocytes without double application of BCB test and control oocytes without any application of BCB test, whereas the total cell number per blastocyst from the blue oocytes was higher than that from the control oocytes (48.0 versus 34.2, P < 0.05). In the second experiment, oocytes were exposed to the BCB test before or after maturation culture, and then the matured oocytes were activated to evaluate the ability of parthenogenetic development. The proportion of blastocyst formation of blue oocytes classified after maturation culture was lower than that of blue oocytes classified before maturation culture (10.0% versus 27.0%, P < 0.05). Therefore, double application of the BCB test before and after maturation culture impaired fertilization and embryonic development, whereas a single application before maturation culture was efficient to select oocytes for IVF and nuclear transfer.     

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

The aim of this present study was to increase the efficiency of blastocyst production from cows after in vitro maturation/fertilization (IVM/IVF) by oocyte selection before maturation. Oocytes were selected on the basis of brillant cresyl blue (BCB) staining, used to indicate glucose-6-phosphate dehydrogenase (G6PDH) activity. To re-valuate the hypothesis that growing oocytes are expected to have a high level of active G6PDH, while mature oocytes have low G6PDH activity, cumulus oocyte complexes (COCs) were recovered from slaughterhouse ovaries 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 before placement into culture; and (3) treatment--incubation with BCB for 90 min 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 induced by G6P as substrate oxidized by G6PDH in the cytosol of control, BCB- and BCB+ groups; G6PDH activity was significant higher in BCB- COCs than in 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 than for BCB- oocytes. The BCB+ oocytes yielded a significantly higher proportion of blastocysts (34.1%) than did control or holding control oocytes (18.3 and 19.2%); and both controls and BCB+ oocytes had significantly higher blastocyst development than did BCB- oocytes (3.9%). These results show that the staining of bovine cumulus oocyte complexes with BCB before in vitro maturation may be used to select developmentally competent oocytes for IVF. In addition, G6PDH activity may be useful as a marker for oocyte quality in future studies on factors affecting developmental competence.