Overheating is detrimental to meiotic spindles within in vitro matured human oocytes

The present study was designed to examine the effects of overheating on meiotic spindle morphology within in vitro matured human oocytes using a polarized light microscope (Polscope). Immature human oocytes at either germinal vesicle or metaphase I stage were cultured in vitro for 24-36 h until they reached metaphase II (M-II) stage. After maturation, oocytes at M-II stage were imaged in the living state with the Polscope at 37, 38, 39 and 40 degrees C for up to 20 min. After heating, oocytes were returned to 37 degrees C and then imaged for another 20 min at 37 degrees C. The microtubules in the spindles were quantified by their maximum retardance, which represents the amount of microtubules. Spindles were intact at 37 degrees C during 40 min of examination and their maximum retardance (1.72-1.79) did not change significantly during imaging. More microtubules were formed in the spindles heated to 38 degrees C and the maximum retardance was increased from 1.77 before heating to 1.95 at 20 min after heating. By contrast, spindles started to disassemble when the temperature was increased to 39 degrees C for 10 min (maximum retardance was reduced from 1.76 to 1.65) or 40 degrees C for 1 min (maximum retardance was reduced from 1.75 to 1.5). At the end of heating (20 min), fewer microtubules were present in the spindles and the maximum retardance was reduced to 0.8 and 0.78 in the oocytes heated to 39 degrees C and 40 degrees C, respectively. Heating to 40 degrees C also induced spindles to relocate in the cytoplasm in some oocytes. After the temperature was returned to 37 degrees C, microtubules were repolymerized to form spindles, but the spindles were not reconstituted completely compared with the spindles imaged before heating. These results indicate that spindles in human eggs are sensitive to high temperature. Moreover, maintenance of an in vitro manipulation temperature of 37 degrees C is crucial for normal spindle morphology.     

Effect of the cumulus on in vitro fertilization of bovine matured oocytes

The effect of the cumulus on in vitro fertilization in bovines was examined. Follicular oocytes were cultured in medium 199 plus OCS and extra granulosa cells. Frozen-thawed bovine spermatozoa was separated by the swim-up technique, suspended in Talp medium and capacitated with heparin. Fresh sheep and goat semen was incubated for 4 h at room temperature, washed and spermatozoa were then suspended in Talp medium and capacitated by incubation at 38.5 °C and 5% CO₂ in air and heparin.

In experiment 1, cumulus-enclosed oocytes, denuded oocytes and denuded oocytes plus additional cumulus cells were incubated with a reduced concentration of bovine spermatozoa for 8 or 18 h. In Experiment 2, cumulus enclosed and denuded oocytes were incubated with bovine spermatozoa for 4, 6, 8 and 18 h using a sperm concentration adjusted to secure high fertilization rates. In Experiment 3, cumulus-enclosed and denuded bovine oocytes were incubated with either sheep or goat spermatozoa for 18 h. Fertilization rates were then calculated and compared statistically. The results showed that 1) the cumulus improved the fertilization rate only when cumulus cells were associated with the oocytes 2) the timing of sperm penetration was not modified by the cumulus and started at 4 h after sperm incubation and 3) the presence of the cumulus improved the heterologous fertilization rate only when sheep spermatozoa were used. The results suggest that the cumulus improves fertilization rate by providing a capacitation-inducing mechanism and by facilitating the interaction between capacitated spermatozoa and the zona pellucida surface.     

In vitro fertilization and cleavage capability of bovine follicular oocytes classified by cumulus cells and matured in vitro

Bovine oocytes were collected from ovaries obtained from an abattoir. They were classified according to the character of the cumulus cells using a stereomicroscope, and cultured in 25 mM Hepes buffered Tissue Culture Medium 199 supplemented with 10% fetal calf serum at 39 degrees C and inseminated by capacitated sperm. Maturation rates of Class A oocytes, with compact, dense cumulus cells; Class B, partially naked oocytes with thin cumulus layers or small remnants of cumulus cells and Class C, naked oocytes were 97.4% (38/39), 89.8% (106/118) and 52.9% (45/85), respectively. The fertilization rates for the three classes were 86.8%, 85.8% and 53.3%, respectively. The naked oocytes had a significantly lower fertilization rate than oocytes of the other two classes. Significantly more Class A oocytes cleaved (63.7%, 232/364) than those of Class B (29.5%, 36/122) and Class C (17.7%, 28/158).     

Developmental competence of bovine oocytes is not related to apoptosis incidence in oocytes, cumulus cells and blastocysts

The number of follicles undergoing atresia in an ovary is very high, and isolation of cumulus-oocyte complexes (COCs) from such atretic follicles may impair subsequent embryo development in vitro. Our aim was to study if stringent selection by morphological assessment of COCs can improve embryo development, and to evaluate whether oocyte diameter is related with apoptotic ratio in oocytes and blastocysts. COCs from slaughtered cattle were recovered by follicle aspiration and classified depending on oocyte diameter: (A) <110 microm; (B) 110-120 microm; (C) >120 microm. COCs were matured, fertilized and cultured in vitro. Early and late stages of apoptosis were detected by Annexin-V and TUNEL staining, respectively, in denuded oocytes, COCs and blastocysts. Immature oocytes from Group A showed higher apoptotic ratio assessed by TUNEL assay, and the COCs corresponding to this group also showed a higher proportion of apoptotic cumulus cells. After maturation, no differences were present in the incidence of apoptosis among oocytes from different groups, but COCs corresponding to the largest diameter showed less apoptotic cumulus cells. In addition, the percentage of apoptotic oocytes decreased during in vitro maturation in all groups. Apoptotic cell ratio (ACR) in blastocysts was not related to oocyte diameter. In conclusion, oocyte selection and oocyte morphological evaluation prior to maturation was not sufficient to select non-atretic oocytes. When oocyte diameter was used as an additional selection the embryonic developmental potential increased together with oocyte diameter, but this improvement was not related to a lower incidence of apoptosis in the largest oocytes.     

Cytoplasmic changes and developmental competence of bovine oocytes cryopreserved without cumulus cells

The cryopreservation of female gametes is still an open problem because of their structural sensitivity to the cooling-and-freezing process and to the exposure to cryoprotectants. The present work was aimed to study the effect of vitrification on immature bovine oocytes freed of cumulus cell investment before freezing. To verify the feasibility and efficiency of denuded oocyte (DO) cryopreservation, the cytoplasmic alterations eventually induced either by cell removal or by the vitrification process were analyzed. In particular, the migration of cortical granules and Ca++ localization were studied. In addition, the localization and distribution of microtubules and microfilaments in immature fresh and vitrified DOs were evaluated. Finally, to establish whether the removal of cumulus cells influenced developmental competence, DOs were thawed after vitrification, matured in vitro and fertilized; then presumptive zygotes were cultured to reach the blastocyst stage. The results indicate that mechanical removal of cumulus cells from immature bovine oocytes does not affect their maturation competence but reduces the blastocyst rate when compared with intact cumulus oocyte complexes (COCs). The findings indicate further that the vitrification process induces changes of cytoplasmic components. However, the composition of the manipulation medium used to remove cumulus cells plays a crucial role in reducing the injuries caused by cryopreservation in both cytoplasmic and nuclear compartments. In fact, the presence of serum exerts a sort of protection, significantly improving both oocyte maturation and blastocyst rates. In conclusion, we demonstrate that denuded immature oocytes can be vitrified after cumulus cells removal and successfully develop up, after thawing, to the blastocyst stage, following in vitro maturation and fertilization.     

Follicular size affects the meiotic competence of in vitro matured prepubertal and adult oocytes in sheep.

Non-atretic follicles dissected from prepubertal and adult ovaries were allocated in three groups: a) < 1 mm; b) 1-2 mm; c) > 2 mm. After 24 h of maturation, a lower percentage of adult oocytes from group a (P < 0.01) reached metaphase II than those from groups b and c (70.4 versus 89.5 and 95.5). Prepubertal oocytes showed similar results (P < 0.01; 27.2 versus 79.8 and 81.8). There was a significant difference (P < 0.01) in meiotic progression between prepubertal and adult oocytes of < 1 mm follicles. The diameter of prepubertal oocytes derived from group a was significantly lower (P < 0.01) compared to groups b and c (138.1 versus 142.1 and 145.6); adult oocytes showed similar results (P < 0.01; 142.2 versus 157.2 and 158.1). Oocytes with the same diameter derived from different follicles of prepubertal and adult ovaries showed similar meiotic progression rates.     

Cytogenetic study of bovine oocytes matured in vitro

Described in the present paper is a cytogenetic study of bovine oocytes matured in vitro. The cumulus-oocyte complexes (COC), punctured from ovaries recovered in a local slaughterhouse, were classified into 3 groups according to follicular diameter 1 to 4mm, 5 to 8mm and 9 to 13 mm. Metaphases available for observation were classified as metaphase I, haploid and diploid metaphase II. High levels of haploid metaphases II (90.6, 86.9 and 94.4 %) among the 3 groups of follicular sizes indicated successful meiotic resumption during in vitro maturation and suggested that cytoplasmic maturation may be responsible for low developmental rate after IVM, IVF and in vitro development (IVD).     

Chromosome configurations and time sequence of the first meiotic division in bovine oocytes matured in vitro

Bovine oocytes aspirated from small follicles were cultured for various time periods. Subsequently, the oocytes were fixed and stained with Giemsa and analyzed for their chromosome configuration. The appearance of the various chromosome configurations and their time sequence were documented. The influence of the presence or absence of follicle-stimulating hormone (FSH) during culture was studied. No difference was found in the proportion of oocytes completing meiotic maturation in the presence or absence of FSH. On average, 75% of the oocytes reached metaphase II after 20 h. FSH showed two main effects: 1) all the cumulus oocyte complexes incubated longer than 10 h in the presence of FSH showed cumulus expansion, and 2) the time period required for chromosome condensation was prolonged for 3 h in the presence of FSH. However, the time sequence in vitro in the presence as well as in the absence of FSH paralleled the time sequence found in vivo, where variations of several hours have been reported. The delay in chromosome condensation in the presence of FSH was assumed to be due to a transient increase in cyclic adenosine 3',5'-monophosphate in the cumulus oocyte complexes. As demonstrated for FSH, the described culture system allowed the study of individual factors for their influence on meiotic maturation of bovine oocytes.     

Effect of cumulus cell removal of in vitro matured bovine oocytes prior to in vitro fertilization on subsequent cleavage rate

The aim of this study is to identify the effect of cumulus cells removal prior to the in vitro fertilization of matured bovine oocytes on cleavage rate. Denuded, matured oocytes were fertilized in presence or absence of loose cumulus cells, cumulus cell conditioned IVF medium (CCCM), charcoal-treated CCCM and charcoal-treated CCCM supplemented with progesterone at a final concentration of 150 ng/ml. After 18 h of incubation with sperm, the presumptive embryos were cultured on a BRL monolayer and the percentage of cleaved embryos was evaluated on Day 4. Removal of cumulus cells prior to IVF significantly reduced the cleavage rate (25% for denuded oocytes versus 56% for cumulus-oocyte complexes (COCs)). The addition of loose cumulus cells partially restored the effect of denudation (cleavage rate: 37% for denuded oocytes supplemented with loose cumulus cells versus 27% for denuded oocytes and 58% for COCs). CCCM also had a positive effect on the cleavage rate of oocytes denuded prior to IVF (36% for denuded oocytes fertilized in CCCM versus 14% for denuded oocytes). Treating the CCCM with charcoal resulted in complete loss of its effect on cleavage rate (18% for denuded oocytes fertilized in charcoal-treated CCCM versus 34% for denuded oocytes fertilized in CCCM). The addition of progesterone to charcoal-treated CCCM partially restored the reduction of the cleavage rate caused by charcoal treatment (27% for denuded oocytes fertilized in charcoal-treated CCCM supplemented with progesterone versus 14% for denuded oocytes fertilized in charcoal-treated CCCM and 36% for denuded oocytes fertilized in CCCM). In conclusion, removal of cumulus cells prior to IVF adversely affects the cleavage rate through loss of a factor secreted by these cells. This factor probably is progesterone.     

Developmental competence of pig oocytes matured and fertilized in vitro

Pig follicles 3 to 6 mm in diameter were everted and matured for 44 h. The oocytes were then collected and exposed to capacitated boar sperm purified by centrifugation in a two step (65 and 70%) Percoll gradient. Of 110 ova fixed 14 h after in vitro fertilization, 78% were penetrated and 47% were monospermic. Next, 681 oocytes were cultured in vitro for 44 h after in vitro fertilization and the 266 embryos which had reached the two- to four-cell stage were transferred into the oviducts of 12 synchronized recipient gilts. Four days later, 211 embryos (79%) were recovered by uterine flushing. 40.7% of these were at the blastocyst stage, and 20% were at the morula stage. In a final experiment, four out of eight gilts which had received 40 to 50 two- to four-cell embryos, were diagnosed pregnant 30 and 37 d after in vitro fertilization. One sow farrowed nine live piglets and one stillborn, two pregnancies were in progress, while one sow returned to estrus 47 d after in vitro fertilization. These results demonstrate that pig oocytes matured and fertilized in vitro can develop to the blastocyst stage and establish a normal pregnancy resulting in the birth of live piglets.     

The ploidy of in vitro matured bovine oocytes is related to the diameter

The aim of the present study was to investigate a possible relationship between bovine oocyte diameter and the ploidy after maturation in vitro. The cumulus-oocyte-complexes (COCs) were collected by slicing slaughterhouse ovaries and were matured in vitro in standard conditions. Oocytes were collected separately from each ovary and then processed in groups according to their origin. After maturation, the inside zona pellucida diameter of each cell was measured and cytogenetic slides were made. Four size categories were distinguished: <110, 110-115, 115-120 and >120 microm. Altogether, 600 oocytes derived from single ovaries of 50 donors were measured and cytogenetically analyzed. The diploid chromosome number (2MII) was found for 8.4% of oocytes (36/427) and for 44% of donors (22/50). The observed number of 2MII cells varied between 1 and 6 per donor. The size of secondary oocytes with unreduced chromosome numbers was significantly smaller (P < 0.01) than the haploid ones. We conclude that bigger oocytes underwent normal meiotic division, whereas their smaller counterparts tended to follow an abnormal path of maturation.     

Effects of cumulus cell density during in vitro maturation of the developmental competence of bovine oocytes

To determine the role of cumulus cells in oocyte maturation, we carried out an investigation on the effects of addition of cumulus cells to the maturation medium on the developmental competence of corona-enclosed oocytes and oocytes denuded from their somatic cells. The addition of cumulus cell (1.6 x 10(6) cells/mL) improved the development of bovine corona-enclosed oocytes, however, addition of a similar number of cumulus cells as cumulus-oocyte-complexes (COCs, cumulus cell density: 4.2 x 10(6) cells/mL) had no effect on the development of oocytes denuded from their somatic cells. To determine if corona-enclosed oocytes can obtain developmental competence without the addition of extra cumulus cells, the effects of cell density during in vitro maturation on the developmental competence were studied. A density of 1.6 to 3.2 x 10(6) cumulus cells/mL was the most effective for in vitro maturation of oocytes with intact gap junctions. The effects of the medium conditioned by COCs on the developmental competence of oocytes was also examined. It was demonstrated that COC-conditioned medium improved the development of bovine oocytes to the blastocyst stage. These data suggest that the developmental competence of bovine oocytes surrounded with corona cells is supported in a cell density-dependent manner in the maturation medium. In addition, the data indicate that cumulus cells benefit bovine oocyte development either by secreting soluble factors which induce developmental competence or by removing an embryo development-suppressive component from the medium.     

Parthenogenetic development of activated in vitro matured bovine oocytes

Bovine oocytes matured in vitro for 26 hours were electrically stimulated 1) by a single pulse (Treatment A); 2) by 3 pulses 30 minutes apart (Treatment B); 3) by a single pulse followed by 5 minutes of incubation in the stimulation medium (Treatment C); or 4) by a single pulse at 27 hours of maturation (Treatment D). The oocytes were then cultured for up to 8 days to assess parthenogenetic activation and development. Each electrical stimulation consisted of a 60-mus square wave pulse of 2.5 or 3.6 kV/cm. Treatment A was less effective than the other treatments (P<0.05), activating 47 or 59% of oocytes at 2.5 or 3.6 kV/cm, respectively. However, there were no differences due to voltage nor among the other treatments, which activated 64 to 78% of the oocytes. The cleavage rate, 28 to 38%, was not affected by the activation treatment, but development to the 8-cell stage or beyond was greater after activation with the higher voltage. While the numbers of morulae or blastocysts resulting from any given treatment were too small to support meaningful statistical comparison, the results indicate that bovine parthenogenotes produced in vitro are capable of development to the blastocyst stage.     

Cumulus cell layers as a critical factor in meiotic competence and cumulus expansion of ovine oocytes

A critical stage in the optimization of in vitro maturation (IVM) is the selection of good quality oocytes. There exists a relationship between the size of the cumulus investment and the in vitro developmental ability of the cumulus–oocyte complex (COC), which provides a basis for the selection of the COCs. This study was designed to evaluate the effect of the number of cumulus cell layers which enclose the oocytes, on the in vitro maturation, cytoplasm quality and cumulus expansion of the ovine oocytes. Ovaries were obtained from an abattoir and transported to the laboratory within 1–2 h, at 37 °C. Oocytes (n = 535) were recovered by means of an aspiration pump (set at a flow rate of 10 mL H₂O/min), with a disposable 20 G needle attached. Oocytes were divided into four classes (classes I to IV – with more than 5, 3–4, 1–2 and no cumulus cell layers, respectively) and separately cultured in a TCM199 medium for 24 h. The morphology of oocytes was evaluated following in vitro culture (IVC) to assess cumulus expansion, cytoplasm quality (score I with a homogenous cytoplasm and II with granulated cytoplasm) and nuclear maturation stage. The percentage of maximum cumulus expansion for classes I to III oocytes were 53.0 ± 1.0, 36.3 ± 2.2 and 16.3 ± 1.8% respectively. The rate of meiotic resumption of oocytes in classes I to IV were 77.0 ± 2.7, 77.2 ± 1.9, 53.0 ± 2.1 and 2.7 ± 1.1% respectively. The proportion of oocytes with a cytoplasm quality I in oocyte classes I to IV were 62.8 ± 1.5, 59.4 ± 1.2, 36.4 ± 2.1 and 0.5 ± 1.1%, respectively. Results showed that the presence of ≥3 cumulus cell layers in the COC prior to IVM led to a better (p < 0.05) cumulus expansion, meiotic resumption and cytoplasmic maturation rate. Thus the morphological grading of immature ovine oocytes may be an appropriate selection criterion regarding their developmental ability.     

Effect of follicle cells on the acrosome reaction, fertilization, and developmental competence of bovine oocytes matured in vitro

The role of follicle cells in the acrosome reaction of frozen-thawed bovine spermatozoa, in vitro fertilization, cleavage, and development in vitro was investigated. Cumulus-oocyte complexes were cocultured and matured in vitro with additional granulosa cells for 24 hr. Immediately before in vitro insemination, the oocytes were divided into three types with different follicle cells: denuded and corona- and cumulus-enclosed oocytes. The proportion of live, acrosome-reacted spermatozoa significantly increased at 3 and 6 hr after insemination in all types of oocytes. However, the mean proportion of live, acrosome-reacted spermatozoa that inseminated cumulus-enclosed oocytes at 6 hr after insemination was significantly higher than that of spermatozoa inseminating denuded oocytes (18.3% and 13.3%, respectively). The frequency of in vitro fertilization was significantly higher for cumulus-enclosed oocytes (65.4%) than for denuded and corona-enclosed oocytes (30.8% and 39.4%, respectively). Cumulus-enclosed oocytes when cocultured with oviduct epithelial cells also had significantly higher rates of cleavage (two- to eight-cell, 59.8%; eight-cell, 22.4%) and blastocyst formation (7.7%) than denuded and corona-enclosed oocytes. No eight-cell embryos or more advanced stages of embryonic development were observed in either denuded or corona-enclosed oocytes without the coculture. The present results indicate that cumulus cells at fertilization play an important role in inducing the acrosome reaction and promoting a high fertilization rate, cleavage, and development into blastocysts in vitro.     

Demonstration of a suppressive effect of inhibin alpha-subunit on the developmental competence of in vitro matured bovine oocytes.

Changes in intrafollicular concentrations of different forms of inhibin (free alpha-subunits and alpha beta dimers) occur during follicle development and may influence the oocyte maturation process. The aim of this study was to investigate the effects of inhibin A and free alpha-subunit (pro-alpha C) isolated from bovine follicular fluid on maturation of bovine cumulus-oocyte complexes, as reflected by their competence for embryo development after in vitro fertilization. Bovine cumulus-oocyte complexes were isolated from ovaries obtained from an abattoir and were cultured for 22-24 h at 38.5 degrees C in TCM-199 medium supplemented with 10% oestrous cow serum, pregnant mares' serum gonadotrophin (2.5 iu ml-1) and either inhibin A (0, 0.2 and 1.0 microgram ml-1) or pro-alpha C (0, 2 and 10 micrograms ml-1). Neither inhibin A nor free alpha-subunit affected the cleavage rate of cumulus-oocyte complexes after fertilization (approximately 60%). Inhibin A reduced the proportion of cleaved oocytes reaching the eight-cell stage by 19% (P < 0.05), but did not affect the yield of blastocysts. However, pro-alpha C decreased the proportion of cleaved oocytes that reached the eight-cell (25%; P < 0.05) and blastocyst (28%; P < 0.05) stages. In addition, a negative correlation (r = -0.55, P < 0.001) was found between concentrations of total immunoreactive (ir) alpha-inhibin (measured by radioimmunoassay) produced by untreated control cumulus-oocyte complexes and their post-cleavage development to the blastocyst stage. In a second experiment, mouse monoclonal antibodies (20 micrograms ml-1) against two different regions of the inhibin alpha-subunit precursor (pro-region and alpha C fragment) were tested for their ability to neutralize endogenous inhibin alpha-subunit-related molecules produced by cumulus cells; control cumulus-oocyte complexes were treated with normal mouse IgG (20 micrograms ml-1). Although the cleavage rate was not affected, the yield of blastocysts was significantly higher in the presence of mouse monoclonal antibodies to both pro-alpha (77% increase; P < 0.05) and alpha C (48% increase; P < 0.05). None of the treatments tested affected endogenous production of activin-A or follistatin by cumulus-oocyte complexes. Overall, these results indicate that the inhibin alpha-subunit (pro-alpha C) has an inhibitory role in oocyte maturation which is independent of the modulatory effects of activin and follistatin.     

Apoptosis in cumulus cells, but not in oocytes, may influence bovine embryonic developmental competence

Aim of our study was to clarify if the occurrence of apoptosis in oocytes and cumulus cells is correlated to bovine oocyte developmental competence. The cumulus-oocyte complexes (COCs) were selected according to cumulus status: G1 with more than five layers of compact cumulus cells, G2 with one to five layers of compact cumulus cells and G3 with expanded cumulus cells. The degree of apoptosis in cumulus cells and oocytes measured by caspase staining and TUNEL assay before and after maturation, and 24 h post-insemination was compared to the cleavage, blastocyst formation and hatching rates of each group. Highest cleavage, blastocyst and hatching rates were found in cumulus-oocyte complexes with more than five layers of compact cumulus cells, but no apoptosis was detected in immature or in vitro matured oocytes, regardless of the cumulus status. Many cumulus cells contained active caspases before maturation, but caspase activity declined dramatically after maturation. TUNEL positive cells were rarely observed in each cumulus-oocyte complex upon oocyte recovery, but a huge increase of them was seen after in vitro maturation. Significantly more TUNEL and caspase positive cells were found in G2 cumulus-oocyte complexes. Our results suggest that: (i) oocyte apoptosis does not account for the inferior oocyte quality of G2 and G3; (ii) apoptosis occurs in cumulus cells regardless of the number and compactness of cumulus cells; and (iii) the degree of apoptosis in the compact cumulus-oocyte complexes (G1 and G2) is negatively correlated to the developmental competence of oocyte.     

Efficiency and kinetics of the in vitro fertilization process in bovine oocytes with different meiotic competence

The aim of the study was to investigate the efficiency and kinetics of fertilization in oocytes with different meiotic competence, as defined by the phase of the follicular wave and follicle size. Oocytes were recovered from cows with synchronized estrus cycles, slaughtered in either the growth (day 3) or the dominant (day 7) phase, separately from large, medium and small follicles. The oocytes were matured and fertilized by a standard protocol. Twenty-four hours after fertilization, the oocytes were denuded from cumulus cells, fixed and stained with bisbensimid Hoechst-PBS. Fertilization was more efficient and the first cleavage was accelerated in growth phase-derived oocytes, as shown by significantly higher (p < or = 0.01) proportions of both normally fertilized and cleaved oocytes (68.8 and 25.1%), in comparison with dominant phase-derived oocytes (44.2 and 10.3%). In the growth-phase derived oocytes, proportions of normally fertilized and cleaved oocytes were significantly higher (p < or = 0.01) in oocytes from large (100.0 and 36.4%) and medium (83.3 and 36.5%) follicles than in those from small (54.8 and 14.6%) follicles. The dominant phase-derived oocytes showed higher proportions of normally fertilized and cleaved oocytes in the populations recovered from small (51.5 and 10.0%) and medium (43.1 and 12.0%) follicles than in those from large (25.0 and 0%) follicles; however, the differences were not significant. It can be concluded that: (i) efficiency and kinetics of fertilization differ in relation to oocyte's meiotic competence; (ii) improved development of embryos from oocytes with greater meiotic competence is associated with a more effective fertilization process.