Developmental competence of bovine oocytes: effects of follicle size and the phase of follicular wave on in vitro embryo production

Developmental competence of bovine oocytes collected from follicles of different size categories (in either the growth or the dominant phase of the first follicular wave) was studied, with the aim of improving in vitro embryo production. Estrus and ovulation of 39 cyclic Holstein dairy cows were synchronized by two prostaglandin F2alpha treatments at 11-day intervals and one hCG treatment on the day of onset of estrus (Day 0). Cows with follicles in either the growth (Day 3, n=25) or the dominant phase (Day 7, n=14) were slaughtered, and follicles >5 mm were counted. Three oocyte populations were recovered separately from large (11-15 mm), medium (6-10 mm) and small (2-5 mm) follicles in both follicular phases. All collected cumulus-oocyte complexes (COC), except for markedly atretic oocytes without cumulus cells, were used in experiments. Oocytes were matured, fertilized and cultured by standard methods. There were no significant differences between the growth and the dominant phases for mean numbers of large follicles, usable oocytes and embryos per donor. Generally, those numbers were low, but the development rates of oocytes into blastocysts were high, particularly in the growth phase (60.0%). Mean (+/- S.E.M.) numbers of medium follicles, oocytes and embryos per donor were higher in the growth as compared with the dominant phase; in the usable oocytes and embryos, this difference was significant (9.6 +/- 1.4 and 3.5 +/- 0.6 versus 3.9 +/- 0.6 and 1.1 +/- 0.3; P<0.01). The development rates of oocytes into blastocysts, however, did not differ significantly between the growth and the dominant phases (36.7% versus 27.8%). Mean numbers of usable oocytes and embryos per donor recovered from small follicles in both follicular wave phases were similar. The development rate of oocytes into blastocysts was generally low, but higher (P<0.01) in the growth than in the dominant phase (24.5% versus 11.7%). Comparison between the two phases showed that mean number of all counted follicles and all usable oocytes collected per donor were similar, but the mean number of embryos per donor and the development rate of oocytes into blastocysts were higher in the growth phase than in the dominant phase (8.0 +/- 1.2 versus 3.8 +/- 2.4; P=0.012 and 30.3% versus 14.9%; P<0.01). The interaction between follicle size and the phase of follicular wave affected the efficiency of embryo production. The yield of embryos was primarily influenced by the number of oocytes collected from medium follicles and the developmental competence of oocytes from small follicles. The growth phase was more effective for oocyte collection; the number of oocytes from medium follicles and the developmental competence of oocytes from small follicles decreased in the dominant phase.     

Superovulation can reduce the developmental competence of bovine embryos

This study was done to determine if different superovulatory regimens could have an effect on the percentage of embryos produced using IVM/IVF/IVC. Cyclic heifers (n = 22) were superovulated between Days 8 and 12 of the estrous cycle with 4, 6 or 8 constant doses of FSH-P (4 mg each, twice daily) +/- the addition of 1 mg prostaglandin 24 h before slaughter. Ovaries from these superovulated cows and from untreated cows were collected and the follicles dissected. Oocytes were classified according to the appearance of their cumulus and cytoplasm. Individual culture as well as group culture were performed but an individual culture reduced the percentage of oocytes developing into embryos for both untreated and superovulated animals. The results indicated that despite the superovulation regimen the developmental competence of the oocytes collected was lower (0 to 15% embryos) than that of oocytes from untreated animals (20 to 34% embryos). Small follicles ( < or = 2.7 mm) yielded mostly oocytes with an incomplete or partially expanded cumulus investment that never developed into an embryo. Differences in the morphology of the oocytes from medium (2.7 to 8 mm) and large ( > or = 8 mm) follicles were apparent, but equal developmental rates were obtained between all classes of oocytes (12 and 8% embryos, respectively). Follicular atresia was reduced significantly after superovulation (81% nonatretic follicles in treated vs 42% nonatretic follicles in untreated animals); however oocytes from atretic and slightly atretic follicles developed similarly to those from nonatretic follicles. These results suggest that although superovulation increases follicular size and decreases atresia, these conditions are not sufficient to confer developmental competence on the oocytes.     

Biochemical and developmental evidence that ooplasmic maturation of prepubertal bovine oocytes is compromised

Our previous studies have shown that oocytes collected from prepubertal calves lack developmental competence. The overall objective of this study was to assess causes by comparing biochemical and physiologic changes during in vitro maturation of oocytes collected from ovaries of adult cattle at slaughter and from superstimulated calves (<6 mo old) by either laporotomy or ultrasound-guided follicular aspiration. Activity and/or concentrations of maturation-promoting factor (MPF), mitogen-activated protein kinase (MAPK), and inositol 1,4,5-trisphosphate receptor (IP(3)R) were determined by measuring phosphorylation of histone H-1 kinase, phosphorylation of myelin basic protein, or Western blotting, respectively, and were compared between oocytes collected from calves and for those collected from cows. The activities of MPF and MAPK and the relative amount of IP(3)R were significantly lower in calf oocytes. The physiologic significance of these observations was determined by assessing the developmental potential of embryos derived by reciprocal transfer of metaphase II (M-II) chromosomes between cow and calf ooplasts and transfer of adult cumulus cells (G0/G1) into cow and calf ooplasts. Procedural controls consisted of transfer of M-II between adult oocytes and parthenogenic activation of adult and calf oocytes. Adult parthenogenically activated oocytes cleaved and developed to blastocysts at a higher rate than did similarly activated calf oocytes (42.1% vs. 3.4%, P < 0.05). Cleavage was also higher in reciprocal M-II transfer embryos containing adult ooplasm (46.2% vs. 12.0%, P < 0.05). Cleavage (66.7% vs. 21.9%, P < 0.05) and development to blastocyst (20.1% vs. 4.8%, P < 0.05) of nuclear transfer embryos reconstructed from adult cumulus cells was higher after transfer to adult ooplasts. Collectively, these results support the hypothesis that lack of developmental competence of calf oocytes is due to their failure or inability to complete ooplasmic maturation.     

Effect of follicular size on meiotic and developmental competence of porcine oocytes

In several species, the developmental competence of the oocyte is acquired progressively during late follicular growth, after the acquisition of the competence to resume and complete meiosis. In the pig, full meiotic competence of the oocyte is reached in ovarian follicles with a diameter of 3 mm or more. However, there is no information about developmental competence acquisition. We analyzed the ability of oocytes from three foll icular size classes to resume and complete meiosis, to be fertilized, and to develop in vitro to the blastocyst stage. A total of 941 follicles were dissected from slaughterhouse gilt ovaries and classified as small (<3 mm, n = 330), medium (3-5 mm, n = 373), or large (>5 mm, n = 238). The cumulus-oocyte complexes recovered from these follicles were submitted to in vitro maturation for 44 h in TCM199 supplemented with 10 ng/ml EGF, 400 ng/ml pFSH and 570 microM cysteamine; in vitro fertilized for 18 h in mTBM with 10(5) frozen-thawed percoll-selected sperms/ml; and developed for 7 days in mSOF. Samples of oocytes or presumptive zygotes were fixed and stained at the end of maturation and fertilization. Groups of oocytes were cultured for 3 h in the presence of 35S-methionine before or after maturation for SDS-PAGE analysis of protein neosynthesis. More oocytes originating from medium and large follicles were competent for maturation than oocytes from small follicles (77 and 86% of metaphase II, respectively, versus 44%, P < 0.05). More oocytes from medium and large follicles werepenetratedby spermatozoa during in vitro fertilization, resulting in significantly more oocytes presenting two or more pronuclei at the end of fertilization (73 and 77% for medium and large follicles, respectively, versus 53% for small follicles, P < 0.05). More oocytes from medium and large follicles developed to the blastocyst stage (14 and 23%, respectively) than those from small follicles (3%, P < 0.05), even if the development rates were corrected by the maturation or fertilization rates. It is concluded that a high proportion of oocytes harvested from follicles of less than 3 mm in the pig are not fully competent for meiosis and are cytoplasmically deficient for development.     

Effect of follicular size on in vitro developmental competence of oocytes and viability of embryos after transfer in the dromedary (Camelus dromedarius)

The aim of this work was to determine the effect of follicle size on camel oocyte quality as measured by developmental competence in vitro and in vivo. Ovaries from a local slaughterhouse were dissected to obtain two classes of follicle size: small (3-6 mm) and large (>6 mm) follicles. Quality of the oocytes was assessed after in vitro maturation (IVM), in vitro fertilization (IVF) and in vitro culture (IVC) of cumulus oocyte complexes (COCs). All cultures were done in four replicates at 38.5 degrees C, under 5% CO(2) and high humidity (>95%). Only COCs with cumulus and homogenous (dark) cytoplasm were used. The COCs were matured for 28 h in TCM-199 medium supplemented with 10% heat-treated fetal calf serum (FCS), 10 ng/mL EGF, and 250 microM cysteamine. Nuclear maturation rate for each class of follicle size was determined by contrast phase microscopy in a sample of COCs (n=30) denuded, fixed and stained with aceto-orcein. In vitro fertilization was performed using fresh semen (0.5 x 10(6)spermatozoa/mL in modified TALP-solution). Fertilized oocytes were cultured in mKSOMaa, under 5% O(2) and 90% N(2). The percentage of COCs reaching metaphase II (MII) after 28 h of maturation was 87% (26/30) and 73% (22/30) for oocytes originating from large and small follicles, respectively (P>0.1). The rate of total cleavage (two cells to blastocyst stage) was greater (P<0.05) for oocytes originating from large follicles (72%; 116/162) than for those derived from small follicles (59%; 140/237). The percentage of fertilized oocytes reaching the blastocyst stage was 35% (57/162) and 20% (48/237) for oocytes collected from large and small follicles, respectively (P<0.05). The viability of in vitro-produced hatched blastocyst from the two groups (15 from 3 to 6mm follicle size and 22 from follicles >6 mm) was assessed by transfer to synchronized recipients. None of the hatched blastocysts from small follicles resulted in a pregnancy whereas 68% (15/22) of the transferred hatched embryos from large follicles developed into a 25-day pregnancy. Of the resulting 15 pregnancies, 53% (n=8) aborted (five between 2 and 4 months and three between 5 and 7 months of pregnancy). The remaining seven pregnant females gave birth to normal healthy offsprings (four females and three males). The present study shows that dromedary oocytes developmental competence is acquired late during the final phase of follicular development and this developmental ability translates into greater pregnancy rates after transfer of in vitro produced hatched blastocysts.     

Bovine oocyte diameter in relation to developmental competence

This study was conducted to determine the diameter of bovine oocytes that were able to attain their full developmental competence to blastocysts. Oocytes were recovered by aspiration of surface-visible follicles (1 to 7 mm in diameter) from slaughterhouse ovaries. Only healthy-looking cumulus-oocyte complexes were used for in vitro maturation, and they were divided into six groups based on diameter: < 110 microm, 110 to < 115 microm, 115 to < 120 microm, 120 to < 125 microm, 125 to < 130 microm and >/= 130 microm. Oocytes were processed through standard procedures for in vitro maturation, fertilization and culture. Following in vitro maturation or fertilization, some oocytes were stained to assess nuclear maturation and penetration rates. The numbers of embryos that cleaved at 42 h post insemination and developed to blastocysts and hatched blastocysts after 8 days of culture were recorded. The mean oocyte diameters were 114.0 +/- 4.8 microm. The oocytes displayed size-related ability to undergo meiotic maturation. The rates of nuclear maturation of oocytes in the greater than 115-microm size range were significantly higher than those of oocytes with diameters < 115 microm. In the < 120 microm diameter groups, the polyspermic fertilization rates of oocytes < 115 microm were significantly higher than those of oocytes 115 to < 120 microm in diameter. The rates of cleavage and development to blastocysts and hatched blastocysts rose as oocyte diameter increased. Among oocytes with a diameter >110 microm, oocytes < 120 microm were found to have significantly lower developmental competence than oocytes 120 to < 130 microm in diameter. These results suggest that bovine oocytes have acquired full meiotic competence at a diameter of 115 microm but not yet attained full developmental competence to blastocysts, and that oocytes have acquired full developmental competence at a diameter of 120 microm.     

In vitro production of bovine embryos: developmental competence is acquired before maturation

Few studies have examined the importance of the time during which oocytes are left in the ovaries following animal slaughter. The objective of this study was to determine the optimal time for retrieving oocytes after slaughter and to ascertain if superovulating cows in association with this optimal time could increase the developmental competence of bovine oocytes. In Experiment 1, oocytes were left in the postmortem ovaries for 2,3,4,5,6 or 7 h and were then transported to the laboratory at approximately 30 degrees C. Recovered oocytes were processed in vitro using standard techniques. In Experiment 2, cyclic heifers (n = 18) were superovulated between Days 8 and 12 of the estrous cycle with 8 constant doses (4 mg each, twice daily) or 8 decreasing doses (2 injections of 4,3,2 and 1 mg every 12 h) of FSH-P +/- 1 mg prostaglandin 24 or 48 h before slaughter. Oocytes were left in the ovaries for 4 h and were classified according to the state of their cumulus and cytoplasm. The results indicated that oocytes aspirated from ovaries collected 4 h after slaughter produced significantly more > or =64-cell embryos after 7 d of in vitro development than those collected 2, 6 or 7 h postslaughter. Oocytes (87%) from superovulated animals had numerous layers of cumulus cells and originated from medium (2.7 to 8 mm) and large (> or =8 mm) follicles. Significantly more oocytes developed from large follicles than from medium follicles. Although individual culture of the oocytes negatively affected the percentage of embryos produced, group culture of oocytes from animals that were superovulated and left in the postmortem ovaries for 4 h resulted in exceptionally high rates of embryos after 5 d of IVD. On average, 60 to 80% of 16-cell embryos were produced, indicating that under the proper conditions, developmental competence is acquired before in vitro maturation.     

Origin of bovine follicular fluid and its effect during in vitro maturation on the developmental competence of bovine oocytes

Protein supplementation during in vitro maturation can profoundly affect both the rate and overall efficiency of the maturation procedure. The present study was conducted to assess the ability of different concentrations (1, 5, and 10%) of bovine follicular fluid (bFF) to support in vitro maturation of oocytes and subsequent developmental capacity. The bFF was derived either from competent follicles ( > 8 mm) obtained by transvaginal recovery following superovulation or from a pool of small follicles (2-5 mm) from abbatoir-derived ovaries. Bovine oocytes were cultured for 24 h in synthetic oviduct fluid medium (m-SOF) supplemented with polyvinylpyrrolidone. Following fertilization and embryo culture, more oocytes (P < 0.05) reached the blastocyst stage when oocytes were cultured with 5% bFF from competent follicles (41 +/- 3.7%) compared with bFF derived from small follicles (16 +/- 2.9%). Estradiol and recombinant human follicle stimulating hormone added to the competent bFF during maturation acted in synergy to increase blastocyst production rate (P < 0.05); this blastocyst production rate (57 +/- 1.2%) was higher than those obtained with the addition of these two hormones to bFF derived from small follicles (26 +/- 2.9%). The quality of blastocysts obtained was reflected by inner cell mass (51.30 +/- 3.5 and 25.50 +/- 3.7) and trophectoderm cell numbers (99.72 +/- 2.5 and 94.80 +/- 4.7) for bFF from competent and small follicles, respectively. In conclusion, follicular fluid originating from competent follicles increased the developmental competence of abbatoir-derived oocytes.     

Comparison of preimplantation developmental competence after mouse oocyte growth and development in vitro and in vivo

Culture systems for oocytes are essential for the experimental analysis of the basic mechanisms of oocyte development and, moreover, they will eventually find wide application in agriculture, the clinic, and wildlife preservation. Here, progress in mouse oocyte growth and development in vitro using oocyte-granulosa cell complexes from preantral follicles is reviewed. Oocyte-granulosa cell complexes were isolated from preantral (secondary) follicles of 12 day old mice, grown in vitro for 10 days, then matured and fertilized in vitro. The developmental competence of these oocytes was compared with oocytes grown in vivo and isolated from 22 day old mice, then matured and fertilized in vitro. In vitro-grown oocytes did not achieve the same size as their in vivo-grown counterparts. However, when oocytes were grown in medium containing fetal bovine serum, their preimplantation developmental competence was equivalent to that of in vivo-grown oocytes. Surprisingly, more blastocysts per animal were produced when oocytes were grown in vitro than in vivo. There was no correlation between oocyte size and either preimplantation developmental competence or number of cells per blastocyst. Oocytes grown in serum-free medium did not achieve the same developmental competence as oocytes grown in medium supplemented with serum. Lastly, the health status as an adult of the only animal born after complete oocyte development in vitro is described and discussed.     

Effects of beta-mercaptoethanol on formation of pronuclei and developmental competence of swamp buffalo oocytes

This study was conducted to determine the effect of supplementing maturation medium with beta-mercaptoethanol (betaME) on pronuclei formation and developmental competence of swamp buffalo oocytes. Buffalo oocytes were matured in TCM199 medium either with 10mM betaME or without betaME supplementation for 24h. In Experiment 1, oocytes were fixed and stained for cytological evaluation after in vitro fertilization (IVF). In Experiment 2, presumptive zygotes were cultured and their developmental competency was assessed. It was found that betaME significantly improved the proportion of oocytes that exhibited synchronous pronuclei formation (31.8+/-5.1% versus 17.9+/-3.3%, P<0.05). There were no significant differences between oocytes matured with or without betaME in their capability of developing into blastocyst-stage embryos (3.0+/-1.3% versus 1.8+/-0.9%). However, blastocysts produced from oocytes matured in the presence of betaME appeared to develop faster than those from oocytes matured in the absence of betaME (P<0.05). Cavitation of embryos from oocytes matured in the presence of betaME occurred at 156 hpi, whereas those matured in the absence of betaME occurred at 180 hpi. Although in vitro production of blastocysts did not increase by addition of betaME to maturation medium, quality of blastocysts produced from oocytes matured in the presence of betaME was improved. This study provides information for further investigations on optimizing a system for in vitro production of swamp buffalo embryos.     

Offspring from one-month-old lambs: studies on the developmental capability of prepubertal oocytes

A wave of follicular growth in lamb ovaries occurs at about 4 weeks of age, generating a life-time peak in follicle numbers. In order to take advantage of the large number of oocytes available, and to substantially decrease the generation interval, embryos were derived from oocytes collected from 1-mo-old lambs. Animals were subjected to one of 3 regimes of hormonal stimulation: groups 1 and 2 were treated to obtain germinal vesicle-stage oocytes, and group 3 to produce mature metaphase II oocytes. Adult sheep stimulated by an appropriate dose of FSH served as control. The developmental ability of collected oocytes was evaluated by either in vivo or in vitro culture to the blastocyst stage after in vitro maturation and/or fertilization. Blastocysts were transferred immediately or after cryopreservation to suitable recipient sheep. In order to investigate the full developmental potential of these embryos, pregnancies were allowed to go to term. The results show significant differences (P < 0.001) between all experimental groups in blastocyst numbers produced. Embryos derived from group 1 animals produced the greatest number of blastocysts, under both in vivo (36. 7%), and in vitro (22.9%) culture systems. Group 2 gave lowest blastocyst production (5.0%), while group 3 yielded 13.2% blastocysts. The number of pregnant recipients carrying to term lamb-derived embryos was severely reduced for both in vivo- (2 of 9; 22.2%) and in vitro-cultured, fresh (3 of 10; 30.0%) and cryopreserved (1 of 6; 16.7%) lamb embryos. This study is the first report of the birth of live lambs derived from oocytes obtained from donors as young as 4 wk. Defects in the competence of lamb-derived embryos may account for the increased fetal loss during pregnancy and the occurrence of mummified fetuses delivered alongside normal healthy lambs.     

Connexin 43 coupling in bovine cumulus cells,during the follicular growth phase,and its relationship to in vitro embryo outcomes

Gap junctional coupling between cumulus cells is required for oocytes to reach developmental competence. Multiple connexins, which form these gap junctions, have been found within the ovarian follicles of several species including bovine. The aim of this study was to determine the role of connexin 43 (CX43) and its relationship to embryo development, after in vitro fertilization (IVF). Cumulus?oocyte complexes (COCs) were obtained from abattoir sourced, mixed breed, bovine ovaries. COCs were isolated from follicles ranging from 2 to 5 mm in size, representing the preselected follicle pool. Immediately after isolation, two cumulus cell biopsies were collected and stored for analysis pending determination of developmental outcomes. Using in vitro procedures, COCs were individually matured, fertilized, and cultured to the blastocyst stage. Biopsies were grouped as originating from COCs that arrested at the two‐cell stage (low developmental competence [LDC]) or having developed to the late morula/blastocyst stage (high developmental competence [HDC]), after IVF and embryo culture. The expression level of CX43 was found to be significantly higher in cumulus cells from COCs that had an HDC when compared with those that had an LDC. Moreover, the gap junctional intercellular coupling rate was significantly higher in cumulus from COCs deemed to have an HDC. Significantly higher expression of the cumulus health markers luteinizing hormone receptor and cytochrome p450 19A1 was found in the cumulus originating from oocytes with HDC, suggesting that this system may provide a mechanism for noninvasively testing for oocyte health in preselected bovine follicles.     

The effects of time of first cleavage, developmental stage, and delipidation of nuclear-transferred porcine blastocysts on survival following vitrification

The effect of removing cytoplasmic lipid droplets (delipidation) at the 2-cell and developmental stages on the survival of porcine somatic cell nuclear-transferred blastocysts developed from the enucleated oocytes receiving somatic cells from kidney of an adult female after cryopreservation was examined. Vitrification was performed using the Cryoloop method with a small volume of medium (0.5 μl). To select 2-cell embryos with a high potential to develop into blastocysts, the relationship between the timing of the first cleavage and the developmental potential was examined. The potential of nuclear-transferred oocytes to develop into blastocysts in the intermediate-cleavage group (20–24 h after activation, 25%) was slightly or significantly (P < 0.05) higher than that in fast-cleavage (<20 h after activation, 13%) and slow-cleavage groups (>24 h after activation, 5%). Most non-delipidated blastocysts did not survive after thawing (0% for early-stage and 9% for advanced-stage blastocysts), but the survival rate of delipidated blastocysts 48 h after culture (54% and 72%, respectively) was not significantly different from that of non-vitrified blastocysts (80% and 92%, respectively). The survival rate of advanced-stage blastocysts after vitrification was slightly higher than that of early-stage blastocysts. The present study demonstrates that somatic cell nuclear-transferred porcine blastocysts developed from embryos selected at the 2-cell stage can be preserved by vitrification with a small volume of medium if the lipid droplets of the embryos are first removed.     

Modification of ovary stock solution with magnesium and raffinose improves the developmental competence of oocytes after long preservation

During ovary storage oocytes lose some of their developmental competence. In the present study, we maintained storage solutions of phosphate-buffered saline (PBS) at various temperatures (20 or 35 degrees C) or supplemented them with magnesium (Mg), raffinose and sucrose. Subsequently, we examined the kinetics of electrolytes in the follicular fluid (FF) during the ovary storage period (9 h), the survival rate of granulosa cells in the follicles, and the developmental competence of oocytes after the storage. Lowering the temperature from 35 to 20 degrees C increased the total cell number of blastocysts that developed at 7 days after in vitro maturation and in vitro fertilization of oocytes. In stock solution with supplements of 15 mM Mg or a combination of 5 mM Mg and 10 mM raffinose or sucrose, a significantly higher number of oocytes developed into blastocysts with a large number of cells in each blastocyst, and a significantly higher number of living granulosa cells were obtained as compared with stock solutions without any supplements. During ovary storage, the concentrations of potassium and chloride in the FF were increased, and the addition of Mg to the stock solution increased the concentration of Mg in the FF. Germinal vesicle breakdown in oocytes that were collected from ovaries stored in the solution supplemented with 15 mM Mg or a combination of 5 mM Mg and 10 mM of raffinose occurred at a slower rate than that in oocytes collected from ovaries stored in PBS alone. On the other hand, the oocytes collected from ovaries stored in the solution supplemented with 15 mM Mg or a combination of 5 mM Mg and 10 mM raffinose reached the metaphase II (MII) stage more rapidly than the oocytes collected from ovaries stored in the PBS alone. In conclusion, the modification of stock solution by the addition of Mg and raffinose improved the developmental competence of oocytes obtained from ovaries preserved for a long period.     

The effects of follicular fluid on in vitro maturation, oocyte fertilization and the development of bovine embryos

We determined the effects of follicular fluid in the maturation medium on bovine oocyte maturation, fertilization and subsequent development, as well as on the number of cells in blastocysts following culture. Fluid and oocytes from bovine follicles less than 5 mm in diameter were collected from the ovaries of slaughtered cows. For the maturation medium, follicular fluid at concentrations of 10, 30 or 60% (v/v) was added to Medium 199 with Earle's salts supplemented with 0.1 microg/ml estradiol-17 beta (E(2), Experiment 1) or 0.1 microg/ml E2 and 100 IU/ml hCG (Experiment 2). The control medium contained polyvinylpyrrolidone (PVP; 3 mg/ml) instead of follicular fluid. After maturation for 24 h, oocytes were fertilized in vitro with bull frozen-thawed spermatozoa and cultured on a monolayer of granulosa cells for 9 d. There were no differences in maturation or fertilization rates of oocytes. In Experiment 1, maturation medium containing 10% follicular fluid did not affect the developmental rate of the oocytes to > 2-cell, 8 to 16-cell, blastocyst and hatched blastocyst stage embryos, respectively; whereas 60% decreased embryonic development (P < 0.05) compared with the control. Blastocysts and hatched blastocysts developed from fertilized oocytes which had been matured in medium containing 10 and 30% follicular fluid/E(2) had more cells than the controls (P < 0.01). In Experiment 2, maturation medium containing 10 or 30% follicular fluid did not affect the development fertilized oocytes to the blastocyst stage compared with the control, but decreased at 60% (P < 0.01). There were no differences in the number of cells from Day 9 blastocysts and hatched blastocysts from fertilized oocytes matured in maturation medium containing follicular fluid and E(2) + hCG. The results of these experiments suggest that the addition of bovine follicular fluid to the maturation medium enhances the cell numbers in blastocysts from bovine follicular oocytes matured in vitro.