283 EFFECTS OF CUMULUS CELL REMOVAL ON IN VITRO OOCYTE MATURATION, FERTILIZATION, AND EMBRYONIC DEVELOPMENT IN PIGS

2006 ◽  
Vol 18 (2) ◽  
pp. 249 ◽  
Author(s):  
N. Maedomari ◽  
N. Kashiwazaki ◽  
M. Ozawa ◽  
A. Takizawa ◽  
J. Noguchi ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Oocyte Maturation ◽  
Polar Body ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Blastocyst Stage ◽  
Control Group ◽  
Nuclear Maturation ◽  
First Polar Body

It is generally accepted that cumulus cells (CCs) support the nuclear maturation of immature oocytes in mammals. However, the precise mechanism of interaction between cumulus cells and oocytes has not been clarified. Furthermore, the role of cumulus cells in embryonic development has not been reported. In the present study, the effect of denuding cumulus cells from porcine oocytes on oocyte maturation, ertilization, and their subsequent development to the blastocyst stage was examined in vitro. In vitro maturation, fertilization, and culture were carried out as previously reported (Kikuchi et al. 2002 Biol. Reprod. 66, 1033-1041). Porcine cumulus-oocyte complexes (COCs) were collected; some of them were completely denuded of cumulus cells immediately after the collection (DO-0 group). The remaining intact COCs and the DO-0 oocytes were cultured for 24 h in the presence of dbcAMP and hormones. After the initial culture, some of the intact COCs were denuded either completely (DO-24 group) or partially (H-DO-24 group). Additionally, some of DO-24 oocytes were co-cultured with the cumulus cells removed at 0 h and pre-cultured for 24 h (DO-24 + CCs group). The denuded oocytes in each experimental group and intact COCs (control) were further cultured for total 46 h. The remaining oocytes with a first polar body were either examined for the levels of intracellular glutathione (GSH) or fertilized in vitro with frozen-thawed boar spermatozoa. The inseminated oocytes were cultured and examined for their fertilization status after 10 h and for their developmental competence after 6 days. Data were analyzed by ANOVA, followed by the Duncan's multiple range tests. The maturation rates of all denuded groups were significantly lower (P < 0.05; 34.3 to 45.0%) than that of the control group (64.5%). Intracellular GSH concentrations of all denuded groups were also significantly lower (P < 0.05; 4.03 to 7.00 pmol/oocyte) than that of the control group (9.60 pmol/oocyte); however, the GSH level of H-DO-24 oocytes was significantly higher (P < 0.05) than the GSH levels in the other denuded groups. Male pronuclear formation rates of completely denuded oocytes (DO-0, DO-24, and DO-24 + CCs groups) were significantly lower (P < 0.05; 41.4 to 59.3%) than those of the control (89.4%) and the H-DO-24 (80.0%) groups. The blastocyst rate of the control group was significantly higher (P < 0.05; 19.9%) than that of H-DO-24 group (11.6%), and these rates were significantly higher (P < 0.05) than those of the completely denuded groups (3.0 to 4.5%). The results suggest that the presence of cumulus cells during maturation culture improves nuclear maturation of oocytes and plays an important role in embryonic development to the blastocyst stage in vitro.

Co-culture embedded in cumulus clumps promotes maturation of denuded oocytes and reconstructs gap junctions between oocytes and cumulus cells

Zygote ◽  
2012 ◽  
Vol 21 (3) ◽  
pp. 231-237 ◽  
Author(s):  
Guixue Feng ◽  
Deshun Shi ◽  
Shufang Yang ◽  
Xiaoli Wang
Keyword(s):  
Gap Junctions ◽  
Lucifer Yellow ◽  
Ovarian Tissue ◽  
Polar Body ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Control Group ◽  
First Polar Body ◽  
Cytoplasmic Maturation

SummaryThe present study was undertaken to establish an effective method for in vitro maturation (IVM) of denuded oocytes (DOs) by simulating the ovarian three-dimensional status in vivo using buffalo ovarian tissues or cumulus cells, so as to provide a model for investigating the mechanisms of oocyte maturation. Buffalo cumulus–oocyte complexes from ovaries taken at slaughter were denuded by pipetting, and then allocated randomly into four groups for IVM by direct culture in maturation medium (M1, control group), co-culture with a monolayer of cumulus cells (M2), embedded in cumulus cell clumps (M3) and ovarian tissue (M4) for 24 h. The nuclear maturation of DOs was assessed by the extrusion of the first polar body and the cytoplasmic maturation was evaluated by subsequently developmental capacity after parthenogenetic activation. More DOs matured to MII (56.89%) and developed to blastocysts (25.75%) when they were matured in vitro with M3 in comparison with DOs matured in vitro with M1 (45.14 and 15.97%) and M4 (40.48 and 13.49%). Further detection of gap junctions by injecting Lucifer yellow directly into cytoplasm of matured DOs with adherent cumulus cells and scanning with confocal microscope showed that Lucifer yellow were found in nine out of 11 the adherent cumulus cells in M3, indicating that the gap junctions between oocytes and cumulus cells was reconstructed in vitro. These results indicate that co-culture of DOs embedded in cumulus cell clumps can improve their nuclear and cytoplasmic maturation of DOs, possibly through the reconstruction of gap junctions in vitro.

scholarly journals Progesterone influences cytoplasmic maturation in porcine oocytes developingin vitro

PeerJ ◽  
2016 ◽  
Vol 4 ◽  
pp. e2454 ◽  
Author(s):  
Bao Yuan ◽  
Shuang Liang ◽  
Yong-Xun Jin ◽  
Jeong-Woo Kwon ◽  
Jia-Bao Zhang ◽  
...  
Keyword(s):  
Oocyte Maturation ◽  
Polar Body ◽  
Specific Inhibitor ◽  
Blastocyst Stage ◽  
Female Reproduction ◽  
Nuclear Maturation ◽  
First Polar Body ◽  
Porcine Oocyte ◽  
Cytoplasmic Maturation

Progesterone (P4), an ovarian steroid hormone, is an important regulator of female reproduction. In this study, we explored the influence of progesterone on porcine oocyte nuclear maturation and cytoplasmic maturation and developmentin vitro. We found that the presence of P4 during oocyte maturation did not inhibit polar body extrusions but significantly increased glutathione and decreased reactive oxygen species (ROS) levels relative to that in control groups. The incidence of parthenogenetically activated oocytes that could develop to the blastocyst stage was higher (p< 0.05) when oocytes were exposed to P4 as compared to that in the controls. Cell numbers were increased in the P4-treated groups. Further, the P4-specific inhibitor mifepristone (RU486) prevented porcine oocyte maturation, as represented by the reduced incidence (p< 0.05) of oocyte first polar body extrusions. RU486 affected maturation promoting factor (MPF) activity and maternal mRNA polyadenylation status. In general, these data show that P4 influences the cytoplasmic maturation of porcine oocytes, at least partially, by decreasing their polyadenylation, thereby altering maternal gene expression.

H1foo is essential for in vitro meiotic maturation of bovine oocytes

Zygote ◽  
2014 ◽  
Vol 23 (3) ◽  
pp. 416-425 ◽  
Author(s):  
Yan Yun ◽  
Peng An ◽  
Jing Ning ◽  
Gui-Ming Zhao ◽  
Wen-Lin Yang ◽  
...  
Keyword(s):  
Oocyte Maturation ◽  
Polar Body ◽  
Linker Histone ◽  
Meiotic Maturation ◽  
Control Group ◽  
Bovine Oocyte ◽  
First Polar Body ◽  
Effective Sirnas ◽  
Bovine Oocytes

SummaryOocyte-specific linker histone, H1foo, is localized on the oocyte chromosomes during the process of meiotic maturation, and is essential for mouse oocyte maturation. Bovine H1foo has been identified, and its expression profile throughout oocyte maturation and early embryo development has been established. However, it has not been confirmed if H1foo is indispensable during bovine oocyte maturation. Effective siRNAs against H1foo were screened in HeLa cells, and then siRNA was microinjected into bovine oocytes to down-regulate H1foo expression. H1foo overexpression was achieved via mRNA injection. Reverse transcription polymerase chain reaction (RT-PCR) results indicated that H1foo was up-regulated by 200% and down-regulated by 70%. Based on the first polar body extrusion (PB1E) rate, H1foo overexpression apparently promoted meiotic progression. The knockdown of H1foo significantly impaired bovine oocyte maturation compared with H1foo overexpression and control groups (H1foo overexpression = 88.7%, H1foo siRNA = 41.2%, control = 71.2%; P < 0.05). This decrease can be rescued by co-injection of a modified H1foo mRNA that has escaped from the siRNA target. However, the H1e (somatic linker histone) overexpression had no effect on PB1E rate when compared with the control group. Therefore we concluded that H1foo is essential for bovine oocyte maturation and its overexpression stimulates the process.

40 TRICHOSTATIN A TREATMENT EFFECTS ON IN VITRO DEVELOPMENT OF INTERSPECIES NUCLEAR TRANSFER CAT EMBRYOS DEPEND ON RECIPIENT CYTOPLASM SPECIES

2015 ◽  
Vol 27 (1) ◽  
pp. 113
Author(s):  
L. T. K. Do ◽  
Y. Sato ◽  
M. Taniguchi ◽  
T. Otoi
Keyword(s):  
Nuclear Transfer ◽  
Treatment Effects ◽  
Trichostatin A ◽  
Polar Body ◽  
Blastocyst Stage ◽  
Control Group ◽  
Fluid Medium ◽  
First Polar Body ◽  
Bovine Oocytes

The developmental ability of interspecies somatic cell nuclear transfer (iSCNT) embryos decreases as the taxonomic distance between the donor and recipient species increases. Treatment of cat iSCNT embryos using bovine oocytes with 50 nM of trichostatin A (TSA) improves in vitro embryonic development (Wittayarat et al. 2013 Cell. Reprogram. 15, 301–308). This study investigated whether the TSA treatment effects differ between the development of cat iSCNT embryos reconstructed with porcine and bovine oocytes. Porcine and bovine cumulus-oocyte complexes were in vitro matured for 44 h and 24 h, respectively. After cumulus cell removal, enucleation was performed by aspiration of the metaphase II plate and the first polar body using a piezo-driven pipette. A cat fibroblast cell was then injected into cytoplasm of successfully enucleated oocyte. Reconstructed cybrids were electrically activated by a single 1.5 kV cm–1 pulse for 100 µs (pig-cat embryos), or a 2.3 kV cm–1 pulse for 30 µs (cow-cat embryos). Pig-cat and cow-cat embryos were cultured in porcine zygote medium (PZM)-5 and modified synthetic oviducal fluid medium (mSOF), respectively. After electrical activation, pig-cat and cow-cat embryos were cultured in medium supplemented with 5 µg mL–1 cytochalasin B + 50 nM TSA (TSA group) or without TSA (control group), and the cow-cat embryo medium was also supplemented with 10 µg mL–1 cycloheximide. After 2 h, TSA-treated pig-cat and cow-cat embryos were incubated in medium supplemented with TSA for 22 h, followed by 48 h incubation without TSA. Pig-cat and cow-cat control embryos were cultured in medium without TSA for 70 h after activation. Then, all pig-cat and cow-cat embryos were cultured in porcine blastocyst medium (PBM) or mSOF medium supplemented with 5% fetal bovine serum, respectively, for 5 additional days. Four to seven replicates were performed for each experiment. Data were analysed using Student's t-test. For pig-cat embryos, no difference was observed in cleavage rates between both groups, but development to the blastocyst stage was higher in the pig control group (n = 147, 8.0%) than that of pig TSA group (n = 131, 0.7%; P < 0.05). In contrast, development to the blastocyst stage in cow-cat embryos was not observed in the cow control group (n = 125, 0%), but it was observed in cow TSA group (n = 136, 3.7%). These results indicate that TSA treatment effects are species-specific, but those effects remain to be clarified.

60 EFFECTS OF AY9944 A-7 ON MEIOTIC RESUMPTION OF PORCINE OOCYTES AND CUMULUS CELL EXPANSION

2016 ◽  
Vol 28 (2) ◽  
pp. 160
Author(s):  
S. Lee ◽  
C. Khoirinaya ◽  
J.-X. Jin ◽  
G. A. Kim ◽  
B.-C. Lee
Keyword(s):  
Gene Expression ◽  
Oocyte Maturation ◽  
Cell Expansion ◽  
Cholesterol Biosynthesis ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Mitogen Activated Protein Kinase ◽  
Control Group ◽  
Cumulus Expansion

In vitro studies on mammalian oocytes have shown that follicular fluid-meiosis activating sterol (FF-MAS) can overcome the inhibitory effect of hypoxanthine (Hx) on the resumption of meiosis. FF-MAS, an intermediate in the cholesterol biosynthesis pathway, is converted to testis meiosis–activating sterol by a sterol Δ14-reductase. AY9944 A-7, an inhibitor of Δ14-reductase and Δ7-reductase, induces accumulation of FF-MAS by inhibiting its metabolism. The aim of this study was to evaluate the effects of AY9944 A-7 on meiotic resumption of porcine oocytes, cumulus cell expansion, and gene expression related to M-phase-promoting factor (MPF), mitogen-activated protein kinase (MAPK), and oocyte maturation in oocytes and related to cumulus expansion in cumulus cells. In experiment 1, 1136 cumulus-oocyte complexes (COCs) were cultured in IVM media with 4 different concentrations (0, 10, 20, and 40 μM) of AY9944 A-7 in addition to a meiotic inhibitor (Hx, 4 mM) for 44 h. Oocytes treated with 10 and 20 μM AY9944 A-7 in the presence of Hx had significantly higher GVBD and M2 rates than the control group. However, 40 μM AY9944 A-7 significantly decreased GVBD and M2 rates and increased degeneration of oocytes compared with other groups. In experiment 2, 600 COCs were cultured in IVM media with 4 different concentrations (0, 10, 20, and 40 μM) of AY9944 A-7 in the absence of Hx for 44 h. Cumulus expansion of 40 μM AY9944 A-7 treated group was significantly decreased compared with other groups. In experiment 3, we evaluate the effects of AY9944 A-7 on gene expression, and the experiment was replicated four times. Data on gene expression were analysed using Student’s t-test. Oocytes treated with 10 μM AY9944 A-7 increased expression of genes involved in MPF (Cyclin B and Cdc2), MAPK (C-mos), and oocyte maturation (GDF9 and BMP15). Cumulus cells treated with 10 μM AY9944 A-7 decreased cumulus expansion-related genes (Has2, Tnfaip6, Ptgs2, and Ptx-3). In conclusion, our results suggest that although 10 μM AY9944 A-7 decreased cumulus expansion-related genes, there was no difference in cumulus expansion and it induced meiotic resumption of porcine oocytes with increased MPF, MAPK, and oocyte maturation-related genes. Further studies are needed to evaluate the effect of AY9944 A-7 on porcine embryo development. This study was supported by Ministry Of Trade, Industry & Energy (#10048948), Korea IPET (#114059–3), Research Institute for Veterinary Science, TS Corporation, and the BK21 plus program.

scholarly journals Effect of epidermal growth factor-like peptides on pig cumulus cell expansion, oocyte maturation, and acquisition of developmental competence in vitro: comparison with gonadotropins

Reproduction ◽  
2011 ◽  
Vol 141 (4) ◽  
pp. 425-435 ◽  
Author(s):  
Radek Procházka ◽  
Michal Petlach ◽  
Eva Nagyová ◽  
Lucie Němcová
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Oocyte Maturation ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Blastocyst Stage ◽  
Human Chorionic Gonadotrophin ◽  
Developmental Competence ◽  
Epidermal Growth

The aim of this work was to assess the FSH-stimulated expression of epidermal growth factor (EGF)-like peptides in cultured cumulus–oocyte complexes (COCs) and to find out the effect of the peptides on cumulus expansion, oocyte maturation, and acquisition of developmental competencein vitro. FSH promptly stimulated expression of amphiregulin (AREG) and epiregulin (EREG), but not betacellulin (BTC) in the cultured COCs. Expression ofAREGandEREGreached maximum at 2 or 4 h after FSH addition respectively. FSH also significantly stimulated expression of expansion-related genes (PTGS2,TNFAIP6, andHAS2) in the COCs at 4 and 8 h of culture, with a significant decrease at 20 h of culture. Both AREG and EREG also increased expression of the expansion-related genes; however, the relative abundance of mRNA for each gene was much lower than in the FSH-stimulated COCs. In contrast to FSH, AREG and EREG neither stimulated expression ofCYP11A1in the COCs nor an increase in progesterone production by cumulus cells. AREG and EREG stimulated maturation of oocytes and expansion of cumulus cells, although the percentage of oocytes that had reached metaphase II was significantly lower when compared to FSH-induced maturation. Nevertheless, significantly more oocytes stimulated with AREG and/or EREG developed to blastocyst stage after parthenogenetic activation when compared to oocytes stimulated with FSH alone or combinations of FSH/LH or pregnant mares serum gonadotrophin/human chorionic gonadotrophin. We conclude that EGF-like peptides do not mimic all effects of FSH on the cultured COCs; nevertheless, they yield oocytes with superior developmental competence.

scholarly journals Pengaruh urea dalam media maturasi in vitro terhadap tingkat maturasi oosit sapi

2021 ◽  
Vol 10 (2) ◽  
pp. 46
Author(s):  
Sepvian Dewi Kurniawati ◽  
Suryanie Sarudji ◽  
Widjiati Widjiati
Keyword(s):  
Oocyte Maturation ◽  
Polar Body ◽  
Metaphase Plate ◽  
Petri Dish ◽  
Control Group ◽  
Maturation Medium ◽  
First Polar Body ◽  
Maturation Rate ◽  
Follicle Aspiration

This study was aimed to determine the effect of urea in maturation medium on in vitro oocyte maturation rate. The medium used was TCM-199 added with Hepes, NaHCO3, Kanamycin 0.15 IU/mL, PMSG, 0.15 IU/mL hCG, and 10% FBS. Cumulus oocyte complexes (COCs) of cows derived from follicle aspiration were divided into three groups. In control group (P0), the COCs were matured in vitro in a maturation medium without urea addition, meanwhile in the P1 and P2 groups, the medium was added with urea 20 and 40 mg/dL, respectively. Each petri dish contained three drops of maturation medium (300 µl/drops) according to the groups. Microdrops were coated with mineral oil and then incubated in a 5% CO2 incubator, at 39 ˚C with maximum humidity. Aceto-orcein staining was conducted to evaluate the maturation of oocytes based on the achievement of metaphase II phase that is indicated by the presence of metaphase plate and/or first polar body. The result showed that the oocyte maturation rates of P0, P1, and P2 were 51.25, 52.43 (p >0.05), and 46.88 % (p <0.05) respectively. It could be concluded that the presence of urea at 40 mg/dL in maturation medium reduced the percentage of bovine oocyte maturation in vitro.

scholarly journals In vitro maturation of domestic cat oocytes subjected to different incubation times

2021 ◽  
Vol 10 (3) ◽  
pp. e15710313074
Author(s):  
Denilsa Pires Fernandes ◽  
Fernanda Araujo dos Santos ◽  
Luã Barbalho de Macêdo ◽  
Roberta Gonçalves Izzo ◽  
Brenna de Sousa Barbosa ◽  
...  
Keyword(s):  
Incubation Period ◽  
Cell Expansion ◽  
In Vitro Maturation ◽  
Polar Body ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Domestic Cat ◽  
First Polar Body ◽  
The Ideal

The aim of this study was to evaluate the effect of three different incubation times on in vitro maturation of domestic cat oocytes. Thus, ovaries (n = 42) were submitted to slicing procedure and the oocytes recovered were classified; only good quality oocytes (Grade I and II) underwent in vitro maturation for three different periods (24 vs. 30 vs. 36 h) in supplemented TCM-99 medium. After, oocytes were evaluated for cumulus cell expansion and presence of the first polar body. After six replicates (7 ± 1,7 ovaries per replicate), a total of 334 viable oocytes were recovered. Differences (p <0.05) were observed regarding the percentage of oocytes presenting expansion of the cumulus cells, where higher values were observed in the group of oocytes incubated for 36 h (84.3%), when compared to 30 (73.4%) and 24 h (71.0%). Moreover, differences were also observed regarding the presence of the first polar body (24 h: 29.7%; 30 h: 58.2%; 36 h: 69.8%). We conclude that the incubation period influenced the maturation rates, indicating 36 h as the ideal period for the in vitro maturation of domestic cat oocytes in supplemented TCM-199 medium.

scholarly journals The Number of LH Receptor could Predict the Success of Oocyte Maturity in the Process of In Vitro Maturation

2016 ◽  
Author(s):  
Adek Amansyah
Keyword(s):  
In Vitro Maturation ◽  
Clinical Laboratory ◽  
Polar Body ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Oocyte Maturity ◽  
Lh Receptor ◽  
First Polar Body ◽  
Significant Difference

Objective: To evaluate the relationship between the number of LH receptor and the success of oocyte maturity in the process of in vitro maturation (IVM). Method: This experimental study was conducted in the Permata Hati Infertility Clinical Laboratory, Dr. Sardjito General Hospital, Yogyakarta, with the samples of 300 oocytes obtained through collecting immature cow’s oocytes from the abattoir and grouped the oocytes into 3 (three) groups based on the pattern of oocyte cumulus cells on the vesicle germinal stage 2 - 8 mm with three layers of cumulus cell. The sample of the cumulus cells from these three groups were taken and the LH receptor examination was done with immunohistochemistry. After that, the IVM process was performed to the three groups and its development for 24 hours was evaluated. Its maturation quality was evaluated with the emergence of the first polar body (1PB) and compared to the other groups and related to the number of LH receptor in the three groups. Result: The result of this study indicated that the oocyte cumulus cells showed a difference of function during IVM process. The maturity rate in this study showed that the number of LH receptor was related to the morphological pattern of oocyte cumulus cells with oocyte maturity. The maturity of the cumulus cells which 100% covered the oocyte was higher than that of the cumulus cells which > 50% and < 30% covered the oocytes, namely, 74% compared to 60% and 12%. The result of this study also showed that the average number of LH receptors in the three groups (A, B, and C) was 183.4, 78.8, and 24.0 respectively. A significant difference was found in the three groups (p < 0.0001). When related to IVM maturity, this difference showed that the bigger number of oocyte cumulus cells influenced the oocyte maturity. Conclusion: The number of LH receptor can be used as a prediction to determine the success of oocyte maturation in the process of in vitro maturation. [Indones J Obstet Gynecol 2013; 1-4:183-7] Keywords: IVM, LH receptor, oocyte cumulus cell

29 PRODUCTION OF BOVINE CLONED EMBRYOS BY NUCLEAR TRANSFER USING VITRIFIED IMMATURE OOCYTES AS RECIPIENT CYTOPLASTS

2009 ◽  
Vol 21 (1) ◽  
pp. 115
Author(s):  
F. Forell ◽  
C. Feltrin ◽  
L. C. Santos ◽  
A. D. Vieira ◽  
U. M. Costa ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Technological Development ◽  
Polar Body ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Blastocyst Stage ◽  
National Council ◽  
Immature Oocytes ◽  
Oocyte Recovery

The cryopreservation of immature oocytes is a logistic alternative to make cytoplasts available throughout the year for cloning by somatic cell nuclear transfer (SCNT). Oocyte cryopreservation will help to overcome hurdles related to oocyte availability, seasonality, or sanitary constraints. The objective of this experiment was to determine the efficiency of vitrification of bovine immature oocytes for use as cytoplasts to produce clone embryos. Cumulus–oocyte complexes (COCs) obtained from bovine ovaries by slicing from a local abattoir were selected and vitrified prior to maturation. Vitrification and warming solutions and exposure times were as previously described (Vieira AD et al. 2008 Rep. Dom. Anim. 43, 314–318) with minor modifications. Groups of 15 COCs were loaded in a 5-μL vitrification solution microdrop in beveled-cut straws (0.5 mL), which were plunged into N2L. Following warming, vitrified and control (non-vitrified) oocytes were in vitro-matured for 22 h and 17 h, respectively (Oliveira ATD et al. 2005 Theriogenology 64, 1559–1572). After maturation, cumulus cells were removed and oocytes were selected by the presence of a polar body. Embryo reconstruction by SCNT, carried out by standard micromanipulation procedures using fibroblast cells from adult origin, and in vitro culture to the blastocyst stage (Day 7) were based on our established procedures (Forell F et al. 2008 Acta Sci. Vet. 36, 141–148). Data regarding oocyte recovery following cumulus cell removal, oocyte survival after micromanipulation, and maturation, fusion, cleavage (Day 2), and blastocyst (Day 7) rates were analyzed by the chi-square test. Oocyte recovery (73.0%, n = 558/764 v. 91.4%, n = 529/579), maturation (46.8%, n = 261/558 v. 65.8%, n = 348/529) and cleavage (47.2%, n = 60/127 v. 60.2%, n = 77/128) rates were lower in the vitrified than in the non-vitrified group, respectively (P < 0.05). Conversely, oocyte survival after micromanipulation (77.8% and 78.4%) and fusion (82.1% and 82.3%) and blastocyst (16.7%, 10/60 v. 23.4%, n = 18/77) rates were similar between vitrified and non-vitrified groups. However, the overall efficiency (blastocysts produced from selected COCs) was 3.4-fold lower for vitrified oocytes than controls. In conclusion, the vitrification of immature bovine oocytes was proven as a valuable procedure for the production of blastocysts by SCNT, providing that a strict selection is made following warming, being an alternative resource either for the use of large numbers of oocytes obtained from slaughterhouse ovaries or to overcome seasonal variations in oocyte supply for use in animal cloning. This work was supported by the Brazilian National Council for Scientific and Technological Development (CNPq).

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