Effect of ooplast activation on the development of oocytes following nucleus transfer in cattle

We assessed the effect of ooplast (enucleated oocytes) activation prior to receiving a donor nucleus on the development of nucleus transferred oocytes in cattle. The ooplasts were activated by electric stimulus at 30, 33, 36 and 39 h after being placed in culture medium for meiotic maturation. The activated ooplasts were further cultured in vitro, for a total 42 h from the beginning of maturation, 16- to 32-cell stage embryos produced by in vitro fertilization were used as donor embryos. The nucleus transferred oocytes were co-cultured with bovine oviductal epithelial cells in vitro. The fusion rate was not different between the activated (90%) and aged (94%) ooplasts 42 h after culture. Activated ooplasts receiving a donor nucleus showed a higher developmental rate than the aged ooplasts. Maximal development of the oocytes was obtained if the ooplast was activated at 9 h prior to receiving a donor nucleus. Thirty-nine percent developed to morulae and 24% to blastocysts. This compares (P<0.01) with 13% of the aged ooplasts developing to morulae and 8% to blastocysts. Of the activated ooplasts at 3, 6 and 12 h prior to fusion with a donor blastomere, 12, 16 and 13% developed to blastocysts, respectively. Of the 17 recipient cows receiving nucleus transferred embryos, 9 (53%) were diagnosed pregnant by palpation per rectum examination, and 3 normal offspring were obtained.     

Pregnancies after co-culture of cumulus cells with bovine embryos derived from in-vitro fertilization of in-vitro matured follicular oocytes

Bovine follicular oocytes surrounded by cumulus cells for more than one-third of their surface were matured, fertilized and developed in vitro utilizing a co-culture system with bovine cumulus cells. Embryos developed into blastocysts were non-surgically transferred to the uteri of cows at Day 6, 7 or 8 (Day 0 = oestrus). Out of 6 recipient cows (19 blastocysts transferred), 3 became pregnant. One of the 3 pregnant cows carried twins. The results of this study demonstrated the viability of embryos obtained from in-vitro maturation of bovine oocytes followed by in-vitro fertilization and culture to the blastocyst stage in vitro.     

Developmental potential of selectively enucleated immature mouse oocytes upon nuclear transfer

Selective enucleation (SE) was applied to germinal vesicle (GV) oocytes by removing the chromatin attached to nuclear envelope, and leaving the liquid contents of GV in the cytoplast. However, after reconstruction with 1/8 blastomeres or fetal fibroblasts (FFs) neither the maturation efficiency nor the frequency of normal (asymmetric) division was improved as compared with completely enucleated (CE) oocytes. Chromosomal aberrations introduced with somatic nuclei were not rescued in SE oocytes either. On the other hand, timing of maturation division in SE GV oocytes, but not in CE GV oocytes, reconstructed with GV-karyoplasts was like in the control. After maturation and fertilization in vitro, SE oocytes reconstructed with 1/8 blastomeres developed nucleolated donor pronuclei, contrary to CE oocytes. The latter could be rescued with nucleoli-containing nucleus, but not anucleolate nucleus, from a 1/2 blastomere. SE oocytes reconstructed with FFs contained nucleolated pronuclei upon activation, unlike CE GV oocytes. These experiments show that the ooplast nucleolar material and/or embryonic nucleolus are indispensable for pronuclei formation. SE oocytes reconstructed with 1/8 blastomeres or FFs failed to cleave after activation or in vitro fertilization. Control GV oocytes enucleolated before fertilization seized cleavage at the 6-cell stage, as oppose to intact GV oocytes, which in 50.9% yielded morulae/blastocysts. These results suggest that ooplast nucleolar material is essential for the cleavage divisions. Activation of cumulus-enclosed SE GV oocytes matured in hormone-supplemented medium and fused to 1/2 blastomere-karyoplasts, yielded morulae, and blastocysts in 45.5% and 23.4% of reconstructed oocytes, respectively.     

In vitro fertilization and development of frozen-thawed bovine oocytes.

Bovine oocytes were vitrified (V-oocytes) or frozen slowly (S-oocytes) at the germinal vesicle (GV) stage or after maturation in vitro (IVM) and their survival assessed morphologically and also by in vitro fertilization (IVF) and culture. The morphological survival of S-oocytes was 30.7% after freezing at the GV stage and 53.3% after IVM. The corresponding survival rates of V-oocytes were significantly lower, viz. 14.6 and 14.0%, respectively. The fertilization rate of S-oocytes frozen after IVM (51.0%) was lower than that of unfrozen controls (75.8%), but higher than after other treatments. Development continued in 16.0% of the fertilized S-oocytes, compared to 39.4% of control IVF zygotes and 1.6% developed into morulae or blastocysts (4.5% in controls). Only 0.8% of frozen-thawed GV stage oocytes and 4.6% of post-IVM V-oocytes cleaved after IVF and none formed morulae or blastocysts. Transfer of four embryos (two morulae and two blastocysts) derived from post-IVM S-oocytes into a recipient heifer resulted in pregnancy and the birth of twin calves.     

Effect of telophase enucleation on bovine somatic nuclear transfer

Telophase enucleation has been proven to be an efficient method for preparing recipient cytoplasts in bovine embryonic nuclear transfer (2, 11). This research was designed to study in vitro development of bovine oocytes containing transferred somatic cell nuclei, reconstructed by using enucleated in vitro-matured oocytes 32 h of age at telophase II stage as recipient cytoplasts, compared with those 24 h of age at metaphase II stage. Two protocols for donor cell injection were adopted, i.e., subzonal injection (SUZI) and intracytoplasmic injection (ICI). Bovine oviduct epithelial cells (BOECs) and bovine cumulus cells (BCCs) from an adult cow were used as nuclear donors for these experiments. In SUZI groups, the fusion rate of donor cells, both BOECs and BCCs, with MII enucleated oocytes were higher than those with TII enucleated oocytes (54% vs. 41% and 53% vs. 39%, respectively; P<0.05), but the development rates to morula plus blastocyst stage in MII groups were lower than those in TII groups (22% vs. 39% and 21% vs. 41%, respectively; P<0.05). In ICI groups, about 26% of enucleated MII oocytes injected with BOECs or BCCs cleaved and only small parts of them developed to blastocyst stage (4% and 3%, respectively; P>0.05). When BOECs or BCCs were intracytoplasmically injected into oocytes enucleated at TII stage, no blastocyst was formed in either donor cell group and no cleavage occurred in BOEC group. Our data demonstrated that telophase enucleation is beneficial to early embryo development when bovine somatic nuclei are transferred by subzonal injection. However, it is harmful when donor cells are directly injected into the cytoplast of the enucleated oocytes.     

卵母细胞的生发泡移植

生发泡(germinal vesicle,GV)移植到去核的GV期卵母细胞后,获得重构卵,重构卵在体外能成熟,受精和进行胚胎发育。GV移植到去核的第二次减数分裂中期(metaphase Ⅱ,MII)卵母细胞后,重构卵能发生GV破裂,但难以排出第一极体。GV移植后,通过连续核移植,重构合子具有发育到终期的能力。GV移植为研究卵母细胞的发育提供了一种重要工具。     

Developmental capacity of bovine oocytes cryopreserved after maturation in vitro and of frozen-thawed bovine embryos derived from frozen mature oocytes

The present study was conducted 1) to investigate the post-thaw developmental capacity of in vitro mature bovine oocytes (Metaphase II) frozen by 1.6 M of 1,2-propanediol and 2) to confirm the viability of frozen bovine embryos derived from frozen mature oocytes. The cleavage and developmental rates to the blastocyst stage of frozen-thawed mature oocytes were significantly lower (P<0.01) than that of nonfrozen oocytes. When mature oocytes were treated with hyaluronidase, trypsin, or base solution (solution control) before processing to remove the cumulus cells, the developmental rates to the blastocyst stage of frozen-thawed oocytes were 2.8% (5 180 ), 3.1% (9 295 ) and 1.1% (1 89 ), respectively. The viability and developmental capacity of frozen-thawed bovine embryos derived from frozen mature oocytes were not different from those of frozen-thawed bovine embryos derived from nonfrozen mature oocytes (control). Furthermore, nonfrozen and frozen-thawed embryos derived from frozen-thawed mature oocytes were nonsurgically transferred to recipient cows. One of the four and one of the two recipient cows became pregnant, respectively. The results of this study demonstrated the viability of embryos obtained from frozen-thawed bovine oocytes at Metaphase II followed by in vitro fertilization and culture to the blastocyst stage in vitro.     

Heterogeneous nuclear transfer embryos reconstructed by bovine oocytes and camel (Camelus bactrianus) skin fibroblasts and their subsequent development

Summary This study reconstructed heterogeneous embryos using camel skin fibroblast cells as donor karyoplasts and the bovine oocytes as recipient cytoplasts to investigate the reprogramming of camel somatic cell nuclei in bovine oocyte cytoplasm and the developmental potential of the reconstructed embryos. Serum-starved skin fibroblast cells, obtained from adult camel, were electrically fused into enucleated bovine metaphase II (MII) oocytes that were matured in vitro. The fused eggs were activated by Inomycin with 2 mM/ml 6-dimethylaminopurine. The activated reconstructed embryos were cocultured with bovine cumulus cells in synthetic oviduct fluid supplemented with amino acid (SOFaa) and 10% fetal calf serum for 168 h. Results showed that 53% of the injected oocytes were successfully fused, 34% of the fused eggs underwent the first egg cleavage, and 100% of them developed to four- or 16-cell embryo stages. The first completed cleavage of xenonuclear transfer camel embryos occurred between 22 and 48 h following activation. This study demonstrated that the reconstructed embryos underwent the first embryonic division and that the reprogramming of camel fibroblast nuclei can be initiated in enucleated bovine MII oocytes.     

Pregnancies and live birth from in vitro fertilization of calf oocytes collected by laparoscopic follicular aspiration

Oocytes were recovered by laparoscopic aspiration from 3- to 8-week-old calves treated with follicle-stimulating hormone (FSH) followed by human chorionic gonadotropin (hCG) to induce follicular growth and oocyte maturation in vivo. Most of the recovered oocytes either had resumed meiotic maturation at the time of aspiration or were competent to undergo maturation during subsequent culture in vitro. Oocytes matured in vivo following FSH and hCG treatment underwent in vitro fertilization (70%) at rates not significantly different from those of control oocytes recovered from adult cow ovaries at abattoirs and matured in vitro (75%). Calf oocytes that were immature at aspiration exhibited lower fertilization rates after in vitro maturation (36%) but their rate of development to morulae and blastocysts did not differ from that of mature oocytes at aspiration. A total of 91% of the zygotes produced from calf oocytes developed to morula and 27% to blastocyst stages during 6 days of culture. The proportion developing to morulae was significantly higher (P<0.05) than that observed for zygotes resulting from in vitro maturation and fertilization of oocytes recovered from cow ovaries obtained at an abattoir and processed concomitantly (59% to morulae and 18% to blastocysts). Morulae or blastocysts developed from oocytes from 5 to 6-week-old calves, when transferred to synchronized recipient heifers, resulted in 2 confirmed pregnancies, one of which produced a single full-term live calf. The ability to produce embryos from oocytes recovered from newborn or prepubertal calves offers the potential for markedly reducing the generation interval in cattle, thereby substantially accelerating the rate of genetic gain that can be achieved through embryo transfer.     

Viable embryos and normal calves after nuclear transfer into Hoechst stained enucleated demi-oocytes of cows.

Bovine oocytes were bisected, stained with Hoechst 33342 and observed under a fluorescent microscope to identify nucleated and enucleated demi-oocytes. Other oocytes were bisected but not stained, or bisected and only half of each oocyte stained, and viewed under a fluorescent microscope. The oocytes were then used for nuclear transfer by fusing them with embryonic blastomeres from a 5-6 day bovine embryo. The fusion rate and proportion developing into compact morulae or blastocysts was compared among different types of demi-oocytes. Expt 1 examined the effect of staining and indicated no effect on either fusion rate or embryonic development whether or not the oocytes were stained. In Expt 2, stained and unstained nucleated and enucleated oocytes were compared. As in the first experiment, there were no differences between stained and unstained demi-oocytes. There was no difference between fusion rates of nucleated and enucleated oocytes. However, there was a significant difference in embryonic development between nucleated (10.4%) and enucleated (22.6%) demi-oocytes (P less than 0.05). In a final experiment, stained and unstained enucleated oocytes were used for nuclear transfer and the resulting embryos transferred into recipient cows. There was no difference in pregnancy rates or in the number of normal calves born whether stained or unstained recipient oocytes were used. Results from these experiments indicate that Hoechst staining and fluorescent microscopy can be used to identify enucleated demi-oocytes, and that these can be used for nuclear transfer, and result in viable embryos and normal calves.(ABSTRACT TRUNCATED AT 250 WORDS)     

Blastocyst formation by pig embryos resulting from in-vitro fertilization of oocytes matured in vitro

Follicular oocytes collected from prepubertal gilts at a local slaughter house were matured (36 h), fertilized and developed in vitro. Of 785 embryos, 190 (24%) embryos cleaved to the 2-4 cell stages with blastomeres of regular size by 33 h after insemination. These cleaved embryos were surgically transferred into the oviducts of 4 synchronized recipient gilts and recovered from the uterine horns 4 or 7 days later: 13 morulae, 2 blastocysts and 1 expanded blastocyst were recovered after 4 days and 3 hatched blastocysts were recovered 7 days after transfer. Re-culture in vitro sustained further development of morulae recovered 4 days after transfer: 11 of 13 morulae had developed to the blastocyst/hatched blastocyst stages. Overall, 17 of 190 (9%) embryos developed to the blastocyst stage. The results indicate that pig oocytes can be matured and fertilized in vitro, and subsequently develop to the blastocyst stage.     

Porcine nuclei in early growing stage do not possess meiotic competence in matured oocytes

To determine whether the nuclei of early growing stage porcine oocytes can mature to the MII stage, we examined meiotic competence of nuclei that had been fused with enucleated GV oocytes using the nuclear transfer method. In vitro matured oocytes were enucleated and then fused with early growing oocytes (30-40 μm in diameter) from 5 to 7-wk-old piglets using the hemagglutinating virus of Japan (HVJ). Reconstructed oocytes were cultured for 24 h to the MII stage. Although these oocytes extruded the first polar body, they did not contain normal haploid chromosomes, and the spindles were misaligned or absent at the metaphase II (MII) stage. Furthermore, maturation promoting factor (MPF) activity levels were low in oocytes reconstructed with early growing oocytes at metaphase I (MI) and MII. In contrast, mitogen-activated protein kinase (MAPK) activity was detected between the MI and MII stages, although at slightly lower levels. In conclusion, the nuclei of early growing oocytes did not accomplish normal meiotic division in matured oocytes due to misaligned or absent spindle formation.     

Decreased embryonic survival of in-vitro fertilized oocytes in rats is due to retardation of preimplantation development

Immature female rats (60-65 g) were injected with 4 i.u. PMSG on Day -2 and allocated to 3 groups. On the evening of Day 0, rats in Groups I and II were allowed to mate. Embryos were collected on Day 4 (Group I, control morulae) or Day 5 (Group II, control blastocysts) and were transferred into the oviduct or uterine horn of Day-4 pregnant recipient rats. On the transfer side of the recipients, the bursa had been peeled from around the ovary to prevent endogenous oocytes from entering the oviduct. For Group III, unmated donors were killed 65-67 h after PMSG injection. Ovulated oocytes recovered from the oviducts were fertilized in vitro and transferred 16-18 h later. Embryos developing from in-vitro fertilized (IVF) oocytes were recovered on Day 5, separated into morulae (Group IIIm) and blastocysts (Group IIIb) and transferred into Day-4 pregnant recipients similar to control embryos. Some embryos from each group were used to determine the mean number of cells/embryo. Embryo recipients were killed on Day 20. After transfer, the development of IVF oocytes was retarded compared to control embryos. IVF morulae contained significantly fewer cells/embryo than did control morulae but were able to implant and grow to fetuses, in proportions similar to controls, if transferred into the oviduct of the recipients. These results suggest that the developmental potential of rat oocytes fertilized in vitro is limited due to asynchrony between the embryo and the uterine environment at the time of implantation, rather than possible defects incurred by the oocyte during the fertilization procedure.     

Cloning of bovine embryos by multiple nuclear transfer

The in vitro development of multiple generation bovine nuclear transferred embryos to blastocysts and their survival ability after freezing and thawing were examined. Parent donor embryos which had 20 to 50 cells were recovered from superovulated cows. Follicular oocytes matured in vitro were used as recipient oocytes. The recipient oocytes enucleated at 22 to 24 h after the onset of maturation were preactivated at 33 h. Enucleated oocytes with a donor blastomere were fused 9 h after activation by an electric stimulus and the fused oocytes were cultured in vitro (first generation). Reconstituted oocytes that had developed to the 8- to 16-cell stage 3 to 4 d after fusion were used as donor embryos for the next generation. Recloning procedures were performed twice (second and third generations). The proportion of recipient oocytes successfully fused with a blastomere increased with the cycle of nuclear transfer. Eighty to 86% of fused oocytes developed to the 2-cell stage and there was no significant difference with the generation. The proportion of reconstituted embryos receiving blastomeres derived from first generation embryos had higher developmental ability in vitro, than those derived from other generations (43 vs 31% for 8 to 16-cell stage, 37 vs 20 and 21% for blastocyst stage). The number of cloned blastocysts increased with repeated nuclear transfer (once: 6.2 +/- 4.3, twice: 19.8 +/- 9.2 and three times: 30.0 +/- 14.7) but varied greatly with each parent donor embryo. The in vitro viability of cloned blastocysts after freezing and thawing (59%) was low but not significantly different from that obtained for in vitro fertilized blastocysts (72%). After transfer of either fresh or frozen-thawed cloned blastocysts to 21 recipients, 10 of them were pregnant on Day 60. Four and 3 offspring were produced from 20 fresh and 14 frozen-thawed blastocysts,respectively.     

Development of bovine oocytes reconstructed with different donor somatic cells with or without serum starvation

We conducted this study to examine whether serum starvation in culture contributes to better development of bovine reconstructed oocytes and to evaluate which serum-starved somatic cell is the most effective for cloned calf production. In Experiment 1, donor cells of four different types (cumulus cells, ear fibroblasts, oviduct cells and uterine cells) were either serum-starved or not before fusion with enucleated oocytes, and reconstructed oocytes were further cultured for 168 h. Regardless of serum starvation, cumulus cells or ear fibroblasts yielded higher (P < 0.05) rates of fusion than other cells (62.6-69.3 versus 33.3-38.7%). In the serum-starved group, the first cleavage after reconstruction was significantly increased in cumulus cells and ear fibroblasts, compared with oviduct cells (93.4-94.3 versus 78.8-86.0%), and oocytes reconstructed with either of these yielded more blastocysts than oocytes reconstructed with oviduct or uterine cells (40.6-43.8 versus 20.3-19.0%). We observed a similar pattern in the non-starved group, but we found a significant increase in blastocyst formation was found only in cumulus cells compared with other donor cells (42.6 versus 15.4-27.7%). Overall comparison showed that serum starvation increased the rates of cleavage and development to the blastocyst stage, but we found a statistical significance only in the cleavage rate (80.0 versus 89.5%). In Experiment 2, we transferred randomly selected 59 blastocysts that were developed from oocytes reconstructed with serum-starved cells to 44 synchronised recipients. Of those recipients, 23 became pregnant on Day 60 after transfer (52.3%) and 12 (27.3%) delivered cloned calves. The mean gestation length and birth weight was 275 +/- 8 days and 39.6 +/- 15.6 kg, respectively. Although there was no significant difference among donor cells, blastocysts that were derived from oocytes reconstructed with ear fibroblasts yielded the highest rates of pregnancy (50.0%) and delivery (27.3%). In conclusion, serum starvation is effective for improving preimplantation development of oocytes reconstructed with cumulus or ear fibroblast cells and it may positively influence on obtaining better pregnancy outcome.     

Hypertonic medium treatment for localization of nuclear material in bovine metaphase II oocytes

Oocytes enucleated at the second metaphase stage (MII) are often used as recipient cytoplasts for nuclear transfer. The oocyte's nuclear material has been traditionally removed blindly by aspirating the first polar body (Pb1) along with a portion of the cytoplasm. However, the Pb1-guided enucleation method is unreliable because the position of the Pb1 is variable. A previous study showed that pretreatment of mouse oocytes with 3% (0.09 M) sucrose allowed visualization of the metaphase spindle and chromosomes under standard light microscopy and led to a 100% enucleation rate. The same sucrose treatment, however, did not produce the same effect in bovine oocytes. In this study, we increased the concentration of sucrose to 0.3-0.9 M in PBS containing 20% fetal bovine serum (SPF) and found that the majority of the treated bovine oocytes (75%-86%) formed a small transparent bud into the perivitelline space, as compared with the 0.1 M sucrose (6%) or the no sucrose (0%) control groups. Staining of DNA with Hoechst 33342 revealed that these projections coincided with the position of the metaphase chromosomes in 100% of sucrose-treated oocytes, whereas only 31% of oocytes showed alignment of the position of Pb1 with their nuclear materials. Furthermore, 95% of oocytes treated in 0.3 M SPF were successfully enucleated by removing a small amount of cytoplasm adjacent to the projection. This is a significantly higher enucleation rate than that obtained by conventional Pb1-guided enucleation, even when a larger amount of cytoplasm was removed. For nuclear transfer, the enucleated oocytes treated with sucrose did not differ from the control oocytes in rates of fusion, cleavage, or development to blastocysts, or in the average cell numbers in blastocysts. This study demonstrated that 0.3 M sucrose treatment of bovine oocytes facilitates the localization of metaphase chromosomes under normal light microscopy and hence increases enucleation efficiency without compromising the in vitro development potential of cloned embryos by nuclear transfer.     

Development of rabbit parthenogenetic oocytes and nuclear-transferred oocytes receiving cultured cumulus cells

The present study determined a suitable parthenogenetic activation procedure for rabbit oocytes and examined the developmental potential of enucleated oocytes receiving cultured cumulus cells. Unfertilized oocytes recovered from superovulated rabbits were activated with one or two sets of electrical pulses, with or without subsequent administration of 6-dimethylaminopurine (6-DMAP). The proportion of oocytes treated with one or two sets of electrical pulses and 6-DMAP that cleaved (87% and 98%, respectively) and developed into blastocysts (77% and 85%, respectively) was significantly higher (P < 0.05) than those activated with electrical pulses alone (30% and 42% for cleavage, 7% and 17% for blastocysts). Cumulus cells separated from ovulated oocytes obtained from mature rabbits were cultured for three to five passages and then induced to quiescence by serum starvation before nuclear transfer. The enucleated oocytes receiving cumulus cells were activated with electrical pulses followed by the addition of 6-DMAP, and cultured in vitro for 5 to 6 d or transferred to pseudopregnant recipient females 1 d after activation. Of 186 nuclear-transferred oocytes, 123 (66%) cleaved and 42 (23%) developed into blastocysts. After transfer of 174 nuclear-transferred oocytes to 8 recipient females, a total of 3 implantation sites were observed in 3 recipient females but no fetuses were obtained.     

In vivo aging of oocytes influences the behavior of nuclei transferred to enucleated rabbit oocytes

The present study examined nuclear remodeling in rabbit nuclear transfer (NT) embryos formed from metaphase II (MII) oocytes aged in vivo until 19 hr postcoitum (hpc), enucleated, and fused at 22–26 hpc with 32-cell morula blastomeres by means of electric fields, which also induced recipient oocyte activation. Post-activation events observed during the first hour following the fusion/activation pulse were studied in terms of chromatin, lamins, and micro-tubules, and revealed that transferred nuclei underwent premature chromosomes condensation (PCC) in only one-third of NT embryos and remained in interphase in others. Recipient oocytes were mostly not activated by manipulations performed before the fusion/activation pulse. The persistance of transferred nuclei in interphase resulted from the rapid progression of recipient oocytes to interphase after activation, suggesting that the cytoplasmic state of MII oocytes aged in vivo was poised for the approach to interphase. Studying micro-tubular organization in MII oocytes before nuclear transfer manipulations, we found that 19 hpc MII oocytes aged in vivo differed from 14 hpc MII oocytes (freshly ovulated) and from 19-hpc MII oocytes aged in vitro (collected at 14 hpc and cultured for 5 hr), notably by the presence of microtubule asters and tubulin foci or only tubulin foci dispersed throughout the cytoplasm. When PCC was avoided, remodeling of the transferred nucleus was well advanced 1 hr after nuclear transfer, and NT embryos developed better to the blastocyst stage. Mol. Reprod. Dev. 46:325–336, 1997. © 1997 Wiley-Liss, Inc.     

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.