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.     

Prepubertal goat oocytes from large follicles result in similar blastocyst production and embryo ploidy than those from adult goats

Developmental competence of oocytes from prepubertal females is lower than those from adult females. Oocyte development competence is positively related to follicular diameter. Most of the follicles of prepubertal goat ovaries are smaller than 3 mm. The aim of this study was to compare oocytes of two follicle sizes (< 3 mm and ≥ 3 mm) from prepubertal goats with oocytes from adult goats in relation to their in vitro production and quality of blastocysts. Oocytes from prepubertal goats were obtained from slaughterhouse ovaries and selected according to the follicle diameter whereas oocytes from adult goats were recovered in vivo by LOPU technique without prior selection of follicle size. COCs were IVM for 27 h, IVF at the conventional conditions with fresh semen and presumptive zygotes were cultured in SOF medium for 8 days. Blastocysts obtained were vitrified and after warming their blastocoele re-expansion and the ploidy by FISH technique were assessed. We found significant differences between blastocysts yield of oocytes recovered from follicles smaller than 3 mm of prepubertal goats compared to those from adult goats (5.45% vs 20. 83%, respectively) however, these differences disappear if oocytes were recovered form large follicles (18.07%). A total of 28 blastocysts were analysed and 96.43% showed mixoploidy. Age did not affect the number of embryos with abnormal ploidy or blastocyst re-expansion after warming. Furthermore, the percentage of diploid blastomeres per embryo was similar in the 3 groups studied, adult, prepubertal from follicles ≥ 3 mm and < 3 mm (68.6%, 80.8% and 73.6%, respectively). In conclusion, IVP of blastocysts coming from follicles larger than 3 mm of goats 45 days old were not different to the blastocysts produced from adult goats, both in terms of quantity and quality.     

Effects of follicular atresia and size on the developmental competence of bovine oocytes: a study using the well-in-drop culture system

The effect of granulosa cell (GC) apoptosis and follicle size on the competence of bovine oocytes were studied using a well-in-drop (WID) oocyte/embryo culture system, which allows identification of follicular origin. Hatching rates of blastocysts did not differ (P>0.05) between oocytes cultured in the WID system (13%) and those cultured in the conventional group system (16%). Hatching rates of blastocysts were higher (P<0.05) in early atretic (17%) than in non-atretic (8%) and late atretic follicles (10%) of the same size (4-8mm), and in 6-8mm (22%) than in 4-5mm follicles (15%) at the early atretic stage. More oocytes (P<0.05) from late atretic (17%) than from non-atreteic (7%) or early atretic follicles (9%) of the same size (4-8mm) were arrested at Grade 1 cumulus expansion (only cells in the peripheral two layers began to expand). Similarly, more oocytes from 2 to 3mm follicles (30%) than from 6 to 8mm follicles (21%) at the same (late) atretic stage had Grade 1 cumulus expansion (P<0.05). Hatching blastocyst percentages of oocytes with Grade 3 (all layers of the cumulus except corona radiate cells expanded) or Grade 4 (full) cumulus expansion were higher in early atretic (20%) than in non-atretic (13%) or late atretic follicles (12%). Hatching blastocyst percentages of oocytes from follicles at the early atretic stage increased as cumulus expanded from Grade 2 (9%) to Grade 4 (27%). Regardless of the degree of follicle atresia, 72-76% of the floating cells in the follicular fluid (FF) were undergoing apoptosis. The floating cell density in FF was highly (r=0.6-0.7) correlated with oocyte developmental potency. In conclusion, the WID culture system was as efficient as group culture and allowed identification of follicular origin. Furthermore, the developmental potential of oocytes was affected by GC apoptosis, follicle size and cumulus expansion, and the floating cell density in FF could be used as a simple and non-invasive marker of oocyte quality.     

Storage of bovine isolated follicles: a new alternative way to improve the recovery rate of viable embryos from ovarian follicles of slaughtered cows

The vitality of bovine oocytes stored in isolated follicles was examined. The aim of this work was to prolong the time of in vitro manipulation of oocytes before their maturation and develop a new alternative of oocyte "capacitation" to improve the quality of in vitro produced embryos. Follicles were dissected from the ovaries of slaughtered cows; subsequently, follicles were divided according to their diameter into three categories (2-3, 3-4 and 4-6 mm), and stored at 17-18 degrees C for 24 or 48 h in a modified tissue culture medium-199 (TCM-199) with reduced pH. After that time, the cumulus-oocyte complexes (COCs) were isolated, matured, fertilized, and embryos cultured in vitro for a total of 9 days. The percentage of total blastocysts, and hatched blastocysts developed from oocytes, initially kept ("capacitated") for 24h at 17-18 degrees C, within follicles of 3-6mm size categories, were significantly higher than that oocytes of the control [of control oocytes] (44.9 and 30.3% versus 36.2 and 20.4%, respectively). The oocytes of follicles stored for 48 h at 17-18 degrees C already had decreased developmental capacity. Interesting data were obtained when COCs of the 3-4 and 4-6 categories were additionally divided into two subgroups according to their presumed developmental history (originating from the supposed growing "fit" in contrast to the supposed regressing "unfit" follicles). The higher improvement in the rate of hatched blastocysts from 24h stored oocytes was observed only in the subgroup originated from "fit" COCs (15.3 versus 25.0%, and 20.0 versus 34.4%, in the 3-4 and 4-6mm categories, respectively). The transfer of 26 blastocysts (developed of follicles kept for 24h at 17-18 degrees C) to 26 recipient heifers resulted in 18 pregnancies. Storage of follicles at 17-18 degrees C in vitro resulted not only in recovery of higher numbers of blastocysts of better quality but also facilitated the safe transport of follicles for a long distance. The extended, time of follicle storage before the proper oocyte maturation allowed also the synchronization of an appropriate number of recipient animals according to the number of isolated follicles.     

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.     

Developmental competence of juvenile calf oocytes in vitro and in vivo: influence of donor animal variation and repeated gonadotropin stimulation

Juvenile calf oocytes represent an untapped source of germ plasm for reproduction. Reports on the developmental competence of calf oocytes have been controversial. In this research, oocytes were recovered after gonadotropin stimulation from Holstein calves (N = 10) at 2-3 mo of age (2-mo cycle) and again at 4-5 mo of age (4-mo cycle). The in vitro developmental competence was measured, and prestimulation follicle numbers (for 2-mo cycle) and poststimulation follicle numbers (both cycles) were obtained. The number of antral follicles doubled after stimulation (23.4 +/- 6.1 vs. 55.1 +/- 16.1) for the 2-mo cycle and for the 4-mo cycle (47.4 +/- 12.4). The number of follicles observed prior to stimulation in the 2-mo cycle was found to be highly correlated with the poststimulation oocyte recovery for both collection cycles (r = 0.95, 2-mo cycle; r = 0.81, 4-mo cycle). The majority (90-96%) of recovered oocytes were found to be usable for in vitro maturation and fertilization; of these, 41-42% cleaved and 10-11% developed to morulae or blastocysts. Eighty-four in vitro-produced embryos were transferred to synchronized recipients and resulted in 11 pregnancies, leading to 7 live (4 males, 3 females) and 2 dead (one male, one female) calves at full term. No significant differences were observed between the 2-mo and 4-mo collection cycles; however, 73% of the total pregnancies resulted from the 2-mo cycle. All pregnancies resulted from embryos of high-responding donors. The high correlation between the number of follicles prior to stimulation and the poststimulation response suggests the possibility of screening calves prior to stimulation for routine embryo production.     

Changes in ovarian, follicular, and oocyte morphology immediately after the onset of puberty are not accompanied by an increase in oocyte developmental competence in the pig

This study was conducted to determine whether ovarian morphology and developmental competence of in vitro-matured (IVM) oocytes is immediately affected by the onset of puberty in the pig. Ovaries of peri-pubertal pigs were sorted into two groups according to the presence or absence of corpora lutea presence (CL and NCL, respectively. Ovary dimensions, follicle diameter and number, and oocyte diameter (with and without zona pellucidae) were determined. The developmental competence of in vitro-matured oocytes from these two groups was evaluated following parthenogenetic activation and culture in vitro. CL ovaries were significantly (P<0.01) larger than NCL ovaries (width: 22.3+/-0.9 mm versus 15.9+/-0.4 mm, length: 33.2+/-1 mm versus 24.1+/-0.4 mm). Although CL ovaries had fewer antral follicles in total compared with NCL ovaries (21.1+/-1.8 mm versus 46.8+/-2.2 mm), they had a similar number of follicles 3-8mm in diameter. The mean diameter of follicles that were aspirated was greater for CL ovaries than for NCL ovaries (4.5+/-0.1 mm versus 3.3+/-0.02 mm). Oocytes from CL ovaries were greater in diameter compared with those from NCL ovaries (zona retained: 159+/-1.3 microm versus 146.1+/-1.5 microm, zona free: 124.7+/-1.8 microm versus 113.1+/-1.6 microm). No differences were found between oocytes from CL and NCL ovaries for rates of meiotic maturation (91.6+/-3.2% versus 92.4+/-3.2%), cleavage (88.4+/-11% versus 90.7+/-2.6%) and blastocyst formation (21.0+/-3.7% versus 23.7+/-5.7%). Therefore, the onset of puberty coincides with immediate changes in ovarian morphology, increased ovary size, follicle and oocyte diameter, but not with improved oocyte developmental competence. This suggests that the higher developmental competence usually observed in adult oocytes is acquired gradually and requires exposure to multiple estrus cycles.     

卵泡内环境对猪卵泡卵体外成熟和发育的影响

研究卵泡内环境对猪卵母细胞体外成熟、受精及受精卵体外发育的影响。主要结果如下：直径≥5mm、4－4.9mm、3－3.9mm和2－2.9mm的卵泡卵母细胞体外成熟率分别为90.5%、89.7%、85.4%和67.4%,体外受精后,卵母细胞的发育能力随卵泡直径的增大而增强,直径≥5mm和4－4.9mm卵泡卵的2－细胞、3－4－细胞发育率显著高于直径2－2.9mm的卵泡卵（P＜0.05或0.01）。体外成熟培养36h、42h和48h,直径2－2.9mm卵泡卵的体外成熟率,体外受精后的卵裂率差异不显著（P＞0.05）。在体外成熟培养液中添加5％或15％的不同直径卵泡的卵泡液,各组间卵母细胞的体外成熟率,受精卵的体外发育率均无显著差异,结果表明：卵泡大小对猪卵母细胞体外成熟、受精及受精卵体外发育有重要影响。     

Influence of the dominant follicle on oocytes from subordinate follicles

As the oocyte grows within the follicle, a number of factors influence its health and developmental competence. These factors include follicle size, day of estrous cycle, level of atresia and influence of other follicles such as the dominant follicle. Follicles were dissected from ovaries of synchronized dairy cows on four days during the estrous cycle, and the oocyte from each follicle collected, matured, fertilized and cultured singly until Day 8. Development to blastocyst was greater in oocytes collected during phases of follicular growth than those collected during phases of follicular dominance (P<0.001) over all follicle size categories. Oocyte competence tended to increase with increasing follicle size (P<0.1). Follicular cells analyzed by flow cytometry showed an increase in proportion of apoptotic cells in subordinate follicles during the dominant phase compared to growth phase (P<0.05). Thus, the dominant follicle on both oocyte competence and levels of atresia. Further studies on the effect of dominance has shown that lactate production in cumulus-oocyte-complexes (COCs) from medium-sized follicles collected during a dominance phase and small follicles collected during a growth phase are no different from other follicles, despite having significantly lower uptake of glucose (P<0.1). Thus, COCs from different follicle subclasses differ in their nutrient requirements, and current IVM technology needs further improvement to better assist those oocytes that are developmentally challenged.     

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.     

Aspects of follicle and oocyte stage that affect in vitro maturation and development of bovine oocytes

Success of in vitro maturation (IVM) and production of bovine embryos as related to aspects of follicle source and oocyte size were evaluated. First, it was determined that bovine oocytes continue growing in all follicular sizes studied, including >1- to 15-mm follicles. Populations of oocytes were collected from surface visible (peripheral) and cortical follicles from the same ovaries. When the number of oocytes from both peripheral and cortical follicles was combined, the yield of oocytes was approximately double that collected from 1 ovarian site alone. Oocytes from cortical follicles were smaller than those from the surface population, and the smaller cortical oocytes had a lower potential for both meiotic maturation and embryo development Only cortical oocytes with the largest diameters underwent IVM and subsequently developed to blastocysts at rates comparable to oocytes from peripheral follicles. As the diameter of the oocytes recovered from peripheral follicles increased, so did their developmental potential. When the stage of the estrous cycle was observed, it was found to have no effect on developmental potential. Finally, oocytes which extruded polar bodies at an earlier time during maturation were, on average, larger than those which extruded polar bodies later. The results serve a practical purpose in assisting selection of oocytes capable of developing into blastocysts and they give useful correlates of oocyte competencies based on knowledge of follicle source and oocyte stage.     

Developmental stage of the oocyte during antral follicle growth and cumulus investment determines in vitro embryo development of sow oocytes

The aim of the study was to determine the contribution of cumulus cells on the developmental competence of porcine oocytes during follicle growth. Oocytes from large (5-8mm) and small (2-3mm) follicles were cultured with or without follicle stimulating hormone (FSH), subsequently examined for nuclear stage and spindle morphology, or fertilized and cultured for embryo development, or analyzed for glutathione content. Additionally, the significance of cumulus investment, corona radiata cells, cumulus cell number and origin of cumulus cells for oocyte maturation were investigated. Small follicle oocytes cultured without FSH exhibited the highest incidence of spindle aberrations. Oocytes cultured without FSH exhibited reduced sperm penetration and blastocyst rates, and a higher proportion monospermic oocytes developed to the blastocyst stage when derived from large follicles. The glutathione content in oocytes increased during follicle growth and oocyte maturation, but no direct correlation between oocyte glutathione content and oocyte developmental capacity was observed. Oocytes with a bigger cumulus investment exhibited better embryo development. Oocytes with a single corona radiata cell layer (CROs) exhibited similar progression through meiosis to oocytes with more cumulus cell layers, but showed reduced embryo development. More blastocysts were observed when CROs were cultured with disconnected cumulus cells during IVM, but no blastocyst increase was observed when CROs were cocultured with a higher number of cumulus cells or with cumulus cells from large follicles. We conclude that increased developmental capacity of oocytes during follicle growth is intrinsic and whether cumulus cells originate from large or small follicles, their contribution to oocyte maturation remains unchanged. Further, cumulus investment can be used as a variable to predict oocyte developmental capacity.     

Effect of follicle diameter on oocyte apoptosis, embryo development and chromosomal ploidy in prepubertal goats

The aim of this study was to assess the following parameters in prepubertal goat oocytes of different follicle diameter (≥3 mm, <3 mm, control): oocyte diameter, early (Annexin-V) and late (TUNEL) apoptosis, embryo development and chromosomal ploidy of these blastocysts using Fluorescence In Situ Hybridization (FISH). Before in vitro maturation, oocytes were measured and stained with Annexin-V or TUNEL. The rest of the oocytes were matured, fertilized, and cultured in vitro for 8 days. Oocytes from follicles of ≥3 mm showed greater mean oocyte diameter (128.27 ± 7.20 μm vs. 125.35 ± 7.59 μm), higher percentages of TUNEL positive (42.86 vs. 24.23%), higher cleavage (47.85 ± 3.98 vs. 23.07 ± 2.44 %) and blastocyst rates (19.77 ± 3.04 vs. 4.11 ± 1.10 %) than oocytes from follicles of <3 mm.. Blastocyst mean cell numbers did not show differences between follicular groups (123.83 ± 49.62 vs. 104.29 ± 36.09 for follicles of ≥3 mm and <3 mm, respectively). A total of 54 blastocysts with 7084 nuclei were hybridized with specific probes to chromosomes X and Y. Ninety-eight percent (98%) of the embryos presented at least one cell carrying an abnormal number of chromosomes, but 78% of them presented less than 25% of chromosomal abnormal cells. No differences in the percentage of blastocysts with abnormal ploidy were found in embryos produced from oocytes of different follicle diameter.     

Effects of follicular size of cytoplast donor on the efficiency of cloning in cattle

In cattle, oocytes obtained from follicles smaller than 3 mm in diameter can undergo maturation in vitro, progressing to MII and undergoing fertilization, but are developmentally incompetent. Cytoplasts were prepared from in vitro matured oocytes aspirated from small (1-3 mm) or large (6-12 mm) follicles and fused to serum starved mural granulosa cells. Following activation, reconstructed embryos were cultured for 7 days and classified G1 to G4, before being processed for nuclei counting or transferred to synchronized recipients. Oocytes from small follicles had lower rates of polar body extrusion (59.6 vs. 69%; 731/1230 vs. 608/857) and fusion (71.4 vs. 78.8%; 360/497 vs. 364/465; P < 0.06). There were no differences in total rate of blastocysts development (60 vs. 59.8%; small vs. large), or any grade classification. A significant interaction was detected between follicle size and embryo grade with G3 embryos from small follicles having a greater cell number. Developmental competence of G1 and G2 embryos did not differ at day 27 (48 vs. 46%; 16/33 vs. 17/37; small vs. large). Although there were no differences in fetal size between the two groups, differences in allantois length (53 vs. 86 mm; small vs. large; P < 0.002) and allantois width (9.5 vs. 13 mm; small vs. large; P < 0.06) were seen. No differences in survival to term (2/13 in each group) were observed. These results indicate that cytoplasts from follicles of 1-3 and 6-12 mm in diameter are equally developmentally competent when used in a nuclear transfer procedure.     

Oocyte secreted factors improve embryo developmental competence of COCs from small follicles in prepubertal goats

Oocytes secrete soluble paracrine factors called Oocyte Secreted Factors (OSFs) which regulate the cumulus cell phenotype. Follicle populations in ovaries from prepubertal females have smaller diameters than their adult counterparts. Oocytes from small follicles are less competent than those from large follicles. The aim of this study was to investigate, in prepubertal goats, the effect of OSFs secreted by denuded oocytes (DOs) from small (<3 mm) or large (≥3 mm) follicles during IVM on embryo development and the blastocyst quality of cumulus-oocyte complexes (COCs) from small follicles and to determine if GDF9 participates in this process. Treatment groups were: (A) COCs non selected by their follicle size (control group); (B) cumulus oocytes complexes from small follicles (SFCOCs), (C) cumulus oocytes complexes from small follicles co-cultured with denuded oocytes from small follicles (SFCOCs + SFDOs), and (D) cumulus oocytes complexes from small follicles co-cultured with denuded oocytes from large follicles (SFCOCs + LFDOs). The effect of the addition of kinase inhibitor SB-431542, which antagonizes GDF9, was tested in A, C, and D treatment groups. Co-cultured SFCOCs with SFDOs or LFDOs significantly augmented the blastocyst rate in comparison to SFCOCs alone (15.77%, 17.39% vs. 10.31%, respectively). Blastocysts from SFCOCs + LFDOs group showed higher rates of tetraploid nuclei than blastocysts from SFCOCs and the control group (14.43% vs. 5.45% and 5.24%, respectively; P < 0.05). However, we did not observe differences in the hatching rate, mean cell number or embryo cryotolerance (P > 0.05) between the four treatment groups. The addition of SB-431542 during IVM did not have any effect on blastocyst rate (P > 0.05). In conclusion, in prepubertal goats, COCs with a low embryo developmental competence as a consequence of follicle size can be improved by coculturing them with denuded oocytes from both small and large follicles. GDF9 does not seem play a role in this improvement.     

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.     

Different intervals of ovum pick-up affect the competence of oocytes to support the preimplantation development of cloned bovine embryos

The objective of this study was to determine the effect of different frequencies of transvaginal ovum pick-up (OPU) on the quantity of recovered cumulus oocyte complexes (COCs) and subsequently the competence of matured oocytes to support the preimplantation development of cloned bovine embryos. The COCs were aspirated from the ovaries of 6 Chinese Holstein cows by transvaginal follicle aspiration twice a week (every 3 or 4 days) (Group I), every 5 days (Group II), once a week (every 7 days) (Group III), every 10 days (Group IV), and once every 2 weeks (every 14 days) (Group V). The developmental stages of the follicles were confirmed by the diameter of the dominant follicle (DF) and harvested COCs, and the dynamics of the follicular wave were clarified. In addition, extrusions of the first polar body (PB I) from the oocytes were observed at different time intervals after the initiation of in vitro maturation (IVM) to identify the appropriate culture time window for somatic cell nuclear transfer. Matured oocytes were used to produce cloned bovine embryos that were subsequently cultured in the goat oviduct. After 7 days, the embryos were flushed out, and the developmental rates of the blastocysts were compared among the five groups. The results showed that the aspirations of all follicles >or=3 mm in diameter (D1) induced and synchronized the dynamics of the follicular wave, and the subordinate follicles became atretic after 4 days (D5). Another follicular wave started between D7 and D10, and atresia in the subordinate follicles in the second follicular wave began on D14. The timing of meiotic progression (from the initiation of IVM to the extrusion of PB I) in the oocytes obtained by OPU was later than that of the oocytes obtained from the abattoir. Between 20 and 24 hr after the initiation of IVM, 20% of the oocytes extruded their PB I. Further, 80% (520/650) of the harvested COCs were arrested at metaphase II (MII) by 22 hr of the initiation of IVM and were used as cytoplast donors. The rates of development of the reconstituted embryos to the blastocyst stage were 23.1% (Group I), 15.0% (Group II), 10.9% (Group III), 4.9% (Group IV), and 29.0% (Group V). The results indicate that the developmental potential of follicles from the same living donors were different when different intervals of OPU were adopted and early atretic follicles from the second follicular wave had higher competence to support the early development of cloned bovine embryos.     

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.     

Relationship between follicle size and ultrasound-guided transvaginal oocyte recovery.

We evaluated the relationship between follicle size and oocyte recovery (OR) using ultrasound-guided follicle aspiration. Thirty Holstein cows were subjected to OR without gonadotrophic therapy. Oocytes were recovered two to four times from each cow in a total of 67 aspiration sessions. Ovarian follicles with diameters < or =4 mm and >4 mm were aspirated in separated groups. Recovered oocytes from each group were kept separate and submitted to in vitro maturation, fertilization, and culture to the blastocyst stage. A total of 430 follicles were aspirated, of which 154 (35.8%) were from follicles >4 mm and 276 (64.2%) were from follicles < or =4 mm. Seventy-seven oocytes (50%) were recovered from follicles >4 mm and 200 (72.2%) were from follicles < or =4 mm. Nineteen blastocysts were obtained from follicles >4 mm, whereas 45 blastocysts were obtained from follicles < or =4 mm. Recovery rate was greater (P<0.01) in follicles < or =4 mm. Oocyte quality, cleavage rate and blastocyst development did not differ between different follicle sizes. Routine aspiration of small follicles (< or =4 mm) could increase the number of oocytes available for in vitro development.