scholarly journals Three-step in vitro maturation culture of bovine oocytes imitating temporal changes of estradiol-17<i>β</i> and progesterone concentrations in preovulatory follicular fluid

2017 ◽  
Vol 60 (4) ◽  
pp. 385-390 ◽  
Author(s):  
Minami Matsuo ◽  
Kazuma Sumitomo ◽  
Chihiro Ogino ◽  
Yosuke Gunji ◽  
Ryo Nishimura ◽  
...  
Keyword(s):  
Follicular Fluid ◽  
Culture System ◽  
In Vitro Maturation ◽  
Formation Rate ◽  
Temporal Changes ◽  
Control Group ◽  
Bovine Embryo ◽  
Developmental Potential ◽  
Bovine Oocytes

Abstract. The objective of the article is to evaluate the effect of three-step in vitro maturation (IVM) culture system imitating estradiol-17β (E2) and progesterone (P4) concentrations in preovulatory follicles on in vitro bovine embryo production. The cumulus–oocyte complexes (COCs) were collected from follicles (2 to 8 mm in diameter) of bovine ovaries obtained from a local slaughterhouse. For IVM, the COCs were cultured for 22 h in a three-step system: (1) culture in medium 199, containing 700 ng mL−1 E2 and 50 ng mL−1 P4, for 5 h, followed by the medium containing 150 ng mL−1 E2 and 150 ng mL−1 P4 for 11 h, and then the medium containing 20 ng mL−1 E2 and 300 ng mL−1 P4 for 6 h (EP group); (2) culture in the medium containing 700 ng mL−1 E2 for 5 h, followed by the medium containing 150 ng mL−1 E2 for 11 h, and then the medium containing 20 ng mL−1 E2 for 6 h (E group); or (3) culture in the medium containing 50 ng mL−1 P4 for 5 h, followed by the medium containing 150 ng mL−1 P4 for 11 h, and then the medium containing 300 ng mL−1 P4 for 6 h (P group). The COCs were cultured in the medium containing 1000 ng mL−1 E2 for 22 h (control group). After IVM, the COCs were co-incubated with sperm and further cultured. At 48 h after insemination, the cleavage rate of embryos was not different among the groups. At 192 h after insemination, the blastocyst formation rate of EP group was significantly higher than that of the other groups. The total cell number of blastocysts did not differ among the groups. In conclusion, these results demonstrate that the three-step IVM culture system of bovine oocytes imitating temporal changes of E2 and P4 concentrations in preovulatory follicular fluid improves the developmental potential of embryos in vitro.

scholarly journals LÍQUIDO FOLICULAR EQÜINO NA MATURAÇÃO IN VITRO DE OÓCITOS BOVINOS

2004 ◽  
Vol 9 (1) ◽  
Author(s):  
M.G.L. PINTO ◽  
M.I.B. RUBIN ◽  
C.A.M. SILVA ◽  
T.F. HILGERT ◽  
M.F. SÁ FILHO ◽  
...  
Keyword(s):  
Follicular Fluid ◽  
In Vitro Maturation ◽  
Mineral Oil ◽  
Control Group ◽  
Sodium Pyruvate ◽  
Vitro Fertilization ◽  
Treatment Groups ◽  
Maturation Medium ◽  
Bovine Oocytes

O desenvolvimento embrionário de oócitos bovinos maturados in vitro (MIV) foi avaliado em meio suplementado com líquido folicular eqüino (Lfe). Foram distribuídos 1045 oócitos em 11 repetições formando três grupos tratamentos (T1, T2, T3) e um controle (C). O meio de maturação utilizado foi o TCM-199 acrescido de piruvato de sódio, hormônio folículo estimulante recombinante (rFSHh) e hormônio luteinizante equino (LHe). Suplementou-se esse meio com 10% de soro de égua em estro para o grupo controle e para T1, T2 e T3, o meio foi suplementado com 5, 10, e 20% de LFe, respectivamente. Os oócitos foram maturados in vitro (MIV) por 24h. A fecundação in vitro (FIV) foi realizada em meio Talp-Fert. A MIV e a FIV foram realizadas em estufa a 39ºC com 5% de CO2 em ar e umidade saturada. Os zigotos foram cultivados em meio SOFaaci, sob óleo mineral no interior de bolsas plásticas gaseificadas. As taxas de clivagem e de blastocistos foram observadas diariamente (D), e em D7, foram superiores (P0,05) às do grupo controle. Em D9, a taxa de blastocistos do T2 foi superior (P0,05). O LFe, na concentração de 10% pode ser utilizado, em substituição ao soro de égua em estro para suplementar o meio de MIV de oócitos bovinos. Equine follicular fluid on in vitro maturation of bovine oocytes Abstract Embryo development of bovine oocytes was evaluated using maturation medium supplemented with equine follicular fluid (eFF). One thousand and forty five (1045) oocytes were distributed in 11 replications forming three treatment groups (T1, T2 e T3) and one Control (C). TCM-199 added with sodium pyruvate, rFSHh and LHe was used as maturation medium. This medium was supplemented with 10% estrous mare serum for Control group, and 5, 10, and 20% eFF, respectively, for T1, T2 e T3 groups. In vitro maturation (IVM) of all groups was performed during 24h. In vitro fertilization (IVF) was performed in TALP-FERT medium. IVM and IVF were carried out in an incubator at 39ºC with 5% CO2 in air and saturated humidity. Zygotes were cultured in SOFaaci medium, under mineral oil in gasified bags. Cleavage and blastocyst rates were daily observed (D), and at D7, were higher (P0.05) for those from control group. At D9, blastocyst rate of T2 was higher (P0.05). The eFF, at a 10% concentration, can replace the use of estrous mare serum to supplement the IVM medium of bovine oocytes.

286 EFFECTS OF PORCINE FOLLICULAR FLUID DURING IN VITRO MATURATION OF OOCYTES ON IN VITRO FERTILIZATION AND EMBRYONIC DEVELOPMENT

2007 ◽  
Vol 19 (1) ◽  
pp. 258
Author(s):  
B. Agung ◽  
T. Otoi ◽  
D. Fuchimoto ◽  
S. Senbon ◽  
A. Onishi ◽  
...  
Keyword(s):  
In Vitro Fertilization ◽  
Embryonic Development ◽  
Follicular Fluid ◽  
Culture System ◽  
In Vitro Maturation ◽  
Control Group ◽  
Rotating System ◽  
Vitro Fertilization ◽  
Maturation Medium

When used as a solo maturation medium for oocytes, porcine follicular fluid (pFF) promoted male pronucleus formation (MPF) of oocytes after in vitro maturation (IVM), using a static system, and in vitro fertilization (IVF) in pigs (Naito et al. 1988 Gamete Res. 21, 289–295). However, the developmental competence of oocytes matured in pFF after IVM/IVF has not been reported. This study was conducted to assess the ability of pFF as a maturation medium to support IVM/IVF of porcine oocytes and their subsequent in vitro development. pFF, including cumulus–oocyte complexes (COCs), was aspirated from follicles (2–5 mm in diameter) of prepubertal crossbred gilt ovaries, and large clusters of follicular cells (FC) were removed from pFF by filtration through 212 �m of mesh. All of the COCs in filtered pFF were collected, and COCs with compact cumulus cells were selected for IVM. Also, small clusters of FC were collected by centrifugation of the filtered pFF, and pFF without any cells was prepared by centrifugation and used as a maturation medium (MpFF) after supplementation with FSH and antibiotics. COCs were transferred to 3.5 mL (in a 15-mL test tube) of MpFF with FC (5.2 � 106 cells mL-1) and cultured for 44–48 h at 38.5�C in 5% O2 and 5% CO2 using the rotating culture system. As a control group, COCs were cultured in 2 mL of MpFF without FC in a 35-mm Petri dish by the standard static culture system. After maturation, culture oocytes were co-incubated (IVF) for 5 h with frozen–thawed sperm in vitro, as reported previously (Kikuchi et al. 2002 Biol. Reprod. 66, 1033–1041), and then some of them were fixed 10 h after IVF to assess the fertilization status; the rest of them were cultured in PZM (Yoshioka et al. 2002 Biol. Reprod. 60, 112–119) for 7 days to assess their early embryonic development. All of the data were analyzed by ANOVA. Oocytes cultured with FC in the rotating system (R group) showed significantly higher sperm penetration (71.0%), MPF formation (70.5%), and normal fertilization (monospermic fertilization with female and male pronuclei; 31.5%) rates than those in the control group (56.0%, 56.9%, and 17.6%, respectively; P &lt; 0.05). Also, the R group showed significantly higher rates of 8-cell embryos at 2 days after IVF and blastocyst formation at 7 days after IVF than those of the control group (17.2% vs. 8.3% and 10.9% vs. 4.5%, respectively; P &lt; 0.05). These results indicate that porcine oocytes matured in pFF supplemented with FC using the rotating system have the ability to be penetrated by sperm and form MPF, and to develop to the blastocyst stage at higher rates, than oocytes cultured in the standard static maturation culture system. In conclusion, the pFF can be a sole and simple maturation culture medium useful for the in vitro production of blastocysts in pigs.

scholarly journals LÍQUIDO FOLICULAR FRESCO OU CONGELADO NA PRODUÇÃO IN VITRO DE EMBRIÕES BOVINOS

2003 ◽  
Vol 8 (1) ◽  
Author(s):  
L.P. RAUBER ◽  
D.F. ALVES ◽  
F.D. MOZZAQUATRO ◽  
J.V. TESSMANN ◽  
M.L. BERNARDI ◽  
...  
Keyword(s):  
Follicular Fluid ◽  
Freezing Process ◽  
Control Group ◽  
Hatching Rate ◽  
Bovine Embryo ◽  
Cumulus Oocyte Complex ◽  
Follicle Size ◽  
Blastocyst Rate ◽  
Bovine Oocytes

A manutenção dos complexos cumulus-oócitos (CCO) em líquido folicular (LF) antes da sua maturação, além de visar a capacitação, viabiliza o transporte até o laboratório por ser de baixo custo, de fácil aquisição e o congelamento do LF permite seu armazenamento para futura utilização. Neste experimento avaliou-se o efeito do congelamento do LF obtido de folículos de 2-8mm e de folículos >8mm, sobre a taxa de produção embrionária. Oócitos foram aspirados de folículos de 2 a 8mm de ovários provenientes de abatedouro. No grupo controle (n=295) os CCO foram maturados por 24h. Nos tratamentos GF (n=297) e GC (n=282), os CCO foram mantidos por 6h a 30ºC em LF fresco ou congelado, respectivamente, de folículos >8mm. Já no tratamento PF(n=278) e PC (n=281), os CCO foram mantidos em LF fresco ou congelado, respectivamente, de folículos de 2-8mm. Posteriormente, os CCO dos tratamentos GF, GC, PF e PC foram maturados por 18h. Não houve efeito negativo do congelamento do líquido folicular e nem do tamanho dos folículos sobre as taxas de clivagem e produção embrionária em D7 e D9 (P>0,05). No entanto, o congelamento do LF de folículos de 2 a 8mm resultou em redução da taxa de eclosão e do número de células dos blastocistos. A manutenção de oócitos bovinos por 6h a 30ºC, antes da maturação, pode ser efetuada em líquido folicular de folículos >8mm, fresco ou congelado. Fresh or frozen follicular fluid in vitro bovine embryo production Abstract In addition to the capacitation, the maintenance of cumulus-oocyte complex (COC) in follicular fluid (FF) before maturation, allows the transport to the laboratory, being a practical and less expensive media. The FF can be stored after freezing to future use. Oocytes aspirated from bovine slaughterhouse ovaries, were used to evaluate the effect of maintaining the oocytes in fresh or frozen bovine FF (from 2-8mm and >8mm follicles) on the blastocyst rate. In the control group (n=259) the COC were matured for 24h. On treatments GF (n=297) and GC (n=282) the COC were held for 6h at 30°C in fresh or frozen FF from >8mm follicles, respectively. In treatments PF (n=278) and PC (n=281) the COC were held in fresh or frozen FF from 2-8mm follicles, respectively. Later, the COC from GF, GC, PF and PC were matured for 18h. The freezing process as well as the follicle size had no effect on the cleavage, D7 or D9 blastocyst rates (P>0,05). Nevertheless, the frozen FF from 2-8mm follicles resulted in a reduced hatching rate and lower ICM cells. Fresh or frozen follicular fluid of >8mm follicles could be used for a 6h transport of bovine oocytes before maturation for 18h.

scholarly journals Follicular Fluid from Infertile Women with Mild Endometriosis Impairs In Vitro Bovine Embryo Development: Potential Role of Oxidative Stress

2021 ◽  
Author(s):  
Vanessa Silvestre Innocenti Giorgi ◽  
Rui Alberto Ferriani ◽  
Paula Andrea Navarro
Keyword(s):  
Oxidative Stress ◽  
Embryo Development ◽  
Follicular Fluid ◽  
Formation Rate ◽  
Fertilization In Vitro ◽  
Hatching Rate ◽  
Bovine Embryo ◽  
Infertile Women ◽  
Bovine Oocytes

Abstract Objective To investigate whether follicular fluid (FF) from infertile women with mild endometriosis (ME) alters in vitro bovine embryo development, and whether the antioxidants N-acetyl-cysteine (NAC) and/or L-carnitine (LC) could prevent such damages. Methods Follicular fluid was obtained from infertile women (11 with ME and 11 control). Bovine oocytes were matured in vitro divided in: No-FF, with 1% of FF from control women (CFF) or ME women (MEFF); with 1.5 mM NAC (CFF + NAC, MEFF + NAC), with 0.6 mg/mL LC (CFF + LC, MEFF + LC), or both antioxidants (CFF + NAC + LC, MEFF + NAC + LC). After in vitro fertilization, in vitro embryo culture was performed for 9 days. Results A total of 883 presumptive zygotes were cultured in vitro. No differences were observed in cleavage rate (p = 0.5376) and blastocyst formation rate (p = 0.4249). However, the MEFF group (12.5%) had lower hatching rate than the No-FF (42.1%, p = 0.029) and CFF (42.9%, p = 0.036) groups. Addition of antioxidants in the group with CFF did not alter hatching rate (p ≥ 0.56), and in groups with MEFF, just NAC increased the hatching rate [(MEFF: 12.5% versus MEFF + NAC: 44.4% (p = 0.02); vs MEFF + LC: 18.8% (p = 0.79); versus MEFF + NAC + LC: 30.8% (p = 0.22)]. Conclusion Therefore, FF from infertile women with ME added to medium of in vitro maturation of bovine oocytes impairs hatching rate, and NAC prevented these damages, suggesting involvement of oxidative stress in worst of oocyte and embryo quality of women with ME.

189 THE EFFECTS OF RESVERATROL ON PORCINE OOCYTES IN VITRO MATURATION AND SUBSEQUENT DEVELOPMENT AFTER PARTHENOGENETIC ACTIVATION AND IN VITRO FERTILIZATION

2012 ◽  
Vol 24 (1) ◽  
pp. 207 ◽  
Author(s):  
S. S. Kwak ◽  
S. A. Jeong ◽  
Y. B. Jeon ◽  
S. H. Hyun
Keyword(s):  
Gene Expression ◽  
Embryonic Development ◽  
In Vitro Maturation ◽  
Formation Rate ◽  
Control Group ◽  
Developmental Potential ◽  
Parthenogenetic Activation ◽  
Nuclear Maturation ◽  
Significant Difference

The present study investigated the effects of resveratrol (a phytoalexin with various pharmacological activities) during in vitro maturation (IVM) of porcine oocytes on nuclear maturation, intracellular glutathione (GSH) and reactive oxygen species (ROS) levels, gene expression in matured oocytes and subsequent embryonic development after parthenogenetic activation (PA) and IVF. Data were analyzed with SPSS 17.0 using Duncan's multiple range test. In experiment 1, a total of 1146 cumulus–oocyte complexes (COC) were divided into 5 groups (0, 0.1, 0.5, 2.0 and 10.0 μM resveratrol). In the nuclear maturation after 44-h IVM, the groups of 0.1, 0.5 and 2.0 μM (83.0, 84.1 and 88.3%, respectively) had no significant difference compared to the control group (84.1%). The group of 10.0 μM decreased the nuclear maturation (75.0%) significantly (P < 0.05). In experiment 2, a total of 300 matured oocytes were examined for the effects of different resveratrol concentrations (0, 0.5, 2.0 and 10.0 μM) on porcine oocyte intracellular GSH and ROS levels. The groups of 0.5 and 2.0 μM showed a significant (P < 0.05) increase in intracellular GSH levels (1.3 and 1.3, respectively) compared with the control and 10.0 μM groups (1.0 and 1.0, respectively). The intracellular ROS level of oocytes matured with 2.0 μM resveratrol (0.4) was significantly (P < 0.05) decreased compared to other groups (control: 1.0; 0.5 μM: 0.6; and 10.0 μM: 0.7). In experiment 3, lower expression of apoptosis-related genes (Bax, Caspase-3 and Bak) was observed in matured oocytes treated with 2.0 μM resveratrol when compared with that of the control (P < 0.05). In experiment 4, a total of 728 oocytes were divided into 4 groups (control, 0.5, 2.0 and 10.0 μM) and examined subsequent to embryonic development after PA. Oocytes treated with 2.0 μM resveratrol during IVM had a significantly higher cleavage (CL) rate, blastocyst (BL) formation rate and total cell numbers (TCN) after PA compared with those of the control (2.0 μM: 96.6%, 62.1% and 49.1 vs control: 88.3%, 48.8% and 41.4, respectively) and the 10.0 μM groups (87.3%, 41.4% and 40.9, respectively). Oocytes treated with 0.5 μM resveratrol (87.2%, 50.5% and 48.6, respectively) during IVM had significantly higher TCN, but there were no differences in CL and BL formation rates. In experiment 5, a total of 935 oocytes in 3 groups (control, 2.0 and 10.0 μM resveratrol) were conducted in IVF. The BL formation rate and TCN were significantly higher in the group of 2.0 μM resveratrol (20.5% and 54.0, respectively) than the control (11.0% and 43.4, respectively) and 10.0 μM group (11.7% and 45.0, respectively), but there was no significant difference in CL rate. In conclusion, 2.0 μM resveratrol supplementation during IVM improved the developmental potential of PA and IVF in porcine embryos by increasing the intracellular GSH concentration, decreasing the ROS level and decreasing apoptosis-related gene expression during oocyte maturation. This work was supported by a grant from the Next-Generation BioGreen 21 Program (No. PJ008121), Rural Development Administration, Republic of Korea.

PSX-B-11 Comparing effects of FGF2 and IGF1 on the development competence of parthenogenetic activated bovine oocytes maturing in vitro

2021 ◽  
Vol 99 (Supplement_3) ◽  
pp. 327-328
Author(s):  
Galina Singina
Keyword(s):  
Growth Factor ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Total Cell ◽  
Control Group ◽  
Total Cell Number ◽  
Developmental Potential ◽  
Maturation Medium ◽  
Bovine Oocytes

Abstract The oocyte quality acquired during in vitro maturation (IVM) are the main limitative factors affecting the embryo production. The aim of the present research was to study effects of fibroblast growth factor 2 (FGF2) and insulin-like growth factor 1 (IGF1) during IVM of bovine oocytes on their developmental potential after parthenogenetic activation. Bovine cumulus-oocyte complexes (COC; n = 1176) were cultured for 22h in either standard maturation medium (TCM-199 supplemented with 10% fetal calf serum (FCS), 0.2 mM sodium pyruvate, 10 μg/ml FSH and 10 μg/ml LH; Control) or maturation medium supplemented with different concentrations (5–160 ng/ml) of FGF2 and IGF1. After IVM, matured oocytes activated by sequential treatment with ionomycin followed by DMAP and cyclohexamide and then cultured up to the blastocyst stage. The obtained blastocysts were fixed, and the total cell number and the level of apoptosis were determined using DAPI and TUNEL staining. The data from 4 replicates (77–91 oocytes per treatment) were analyzed by ANOVA. Cleavage rates of activated oocytes did not differ between groups and ranged from 63.7 to 68.1%. The addition of 10, 20 and 40 ng/ml of FGF2 to the IVM medium led to an increase in the yield of blastocysts [from 19.6±1.8% (Control) to 35.2±3.4, 29.8±1.9 and 31.1±2.1%, respectively (P&lt;0.05)] and in the total cell number in embryos that developed to the blastocyst stage (P&lt;0.05). Meanwhile, the blastocyst yield and the total cell number in blastocysts in the IGF1-treated groups were similar to that in the control group. No effects of both growth factors on the proportion of apoptotic nuclei in blastocysts (5.3–7.1%) were observed. Thus, FGF2 (but not IGF1) are able to maintain competence for parthenogenetic development of bovine COC during their maturation invitro. Supported by RFBR (18-29-07089) and the Ministry of Science and Higher Education of Russia.

Treatment with roscovitine and butyrolactone I prior to in vitro maturation alters blastocyst production

Zygote ◽  
2019 ◽  
Vol 28 (1) ◽  
pp. 24-31 ◽  
Author(s):  
Rosiara Rosária Dias Maziero ◽  
Carlos Renato de Freitas Guaitolini ◽  
Daniela Martins Paschoal ◽  
André Maciel Crespilho ◽  
Bianca Andriolo Monteiro ◽  
...  
Keyword(s):  
In Vitro Maturation ◽  
Study Groups ◽  
Embryo Cryopreservation ◽  
Control Group ◽  
Bovine Embryos ◽  
Nuclear Maturation ◽  
Oocyte Meiosis ◽  
Maturation Index ◽  
Bovine Oocytes

SummaryThis study evaluated the effects of oocyte meiosis inhibitors roscovitine (ROS) and butyrolactone I (BL-I) on in vitro production of bovine embryos. Bovine oocytes were maintained in pre in vitro maturation (pre-IVM) with 25 µM ROS or 100 µM BL-I for 24 h to delay meiosis and for 24 h in in vitro maturation (IVM). Following this treatment, the nuclear maturation index was evaluated. All embryos degenerated following this procedure. In the second set of experiments, oocytes were maintained for 6 or 12 h in pre-IVM with the following three treatments: ROS (25 µM or 12.5 µM), BL-I (100 µM or 50 µM) or a combination of both drugs (6.25 µM ROS and 12.5 µM BL-I). Oocytes were cultivated for 18 or 12 h in IVM. When a meiosis-inducing agent was used during pre-IVM for 24 h, more degenerated oocytes were observed at the end of the IVM period. This effect decreased when the meiotic blocking period was reduced to 6 or 12 h. No significant differences were observed in the blastocyst production rate of oocytes in pre-IVM for 6 h with ROS, BL-I, or ROS + BL-I compared with that of the control group (P > 0.05). However, inhibition of oocytes for 12 h resulted in decreased embryo production compared with that in the controls (P < 0.05). There was no difference in the post-vitrification embryo re-expansion rate between the study groups, showing that the meiotic inhibition for 6 or 12 h did not alter the embryo cryopreservation process.

346 BOVINE EMBRYO DEVELOPMENT AFTER IVM/IVF/IVC OF OOCYTES STORED FOR 22 H IN VARIOUS MEDIA

2007 ◽  
Vol 19 (1) ◽  
pp. 288
Author(s):  
C. Kubota ◽  
T. Kojima ◽  
T. Nagai ◽  
X. Tian ◽  
X. Yang
Keyword(s):  
Subsequent Development ◽  
Industrial Applications ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Control Group ◽  
Bovine Embryo ◽  
Becton Dickinson ◽  
In Vitro Storage ◽  
Bovine Oocytes

The timing of IVM–IVF–IVC is restricted by the onset of oocyte maturation, and sometimes oocytes must be treated at midnight. If we could regulate the timing of IVM of oocytes without decreasing their developmental competence, the IVM–IVF–IVC system could be a more applied technology. The present study was performed to examine the effects of in vitro storage of bovine oocytes in simple media prior to maturation culture to manipulate the start of IVM. Bovine follicular fluid (bFF), Dulbecco&apos;s PBS (PBS), M199 Earle salts (M199), and Earle salts supplemented with 5 mM NaHCO3 (M199A) were used as the fundamental media, after an addition of antibiotics, for in vitro storage of bovine cumulus&ndash;oocyte complexes (COCs) collected from ovaries obtained at the slaughterhouse. The fundamental media except for bFF were supplemented with 10&percnt; fetal bovine serum (FBS) or 1 mg mL&minus;1 polyvinyl alcohol (PVA). COCs were collected from follicles (3&ndash;8 mm in diameter) and washed twice in each medium; then approximately 50 COCs were submerged in 1 mL of each medium in cryotubes (Falcon #2812, 2.5 mL; Becton Dickinson Labware, Lincoln, NJ, USA), which were stored in a container kept at 38.5&deg;C for 22 h under air-closed condition (in vitro storage: IVS). Subsequently, the stored COCs were in vitro-matured (IVM) for 22 h in M199 with 10&percnt; FBS and 20 &micro;g mL&minus;1 estradiol, fertilized (IVF), and cultured in CR1aa (IVC) for examination of their development to the blastocyst stage (Kubota et al. 1998 Mol. Reprod. Dev. 51, 281&ndash;286). Fresh oocytes without IVS were used as controls. The nuclear status of oocytes after IVS&ndash;IVM was compared to that of control oocytes by aceto-orcein stain. Their developmental rates to the blastocyst stage after IVM&ndash;IVF&ndash;IVC were compared between experimental and control groups. The experiment was repeated more than 3 times, and results were statistically analyzed using Student&apos;s t-test. When bFF and PBS supplemented with FBS or PVA were used for IVS, the rates of survived COCs after IVS and the development to the blastocyst stage after IVM&ndash;IVF&ndash;IVC (bFF (n &equals; 87): 0&percnt;, 0&percnt;; PBS/FBS (n &equals; 72): 84&percnt;, 1&percnt;; and PBS/PVA (n &equals; 81): 89&percnt;, 6&percnt;, respectively) were significantly lower than those of the control group (n &equals; 406; 97&percnt; and 29&percnt;, respectively). On the other hand, when M199A supplemented with FBS or PVA was used for IVS, the survival rate after IVS and the developmental rate to the blastocyst stage after IVS&ndash;IVM&ndash;IVF (M199A/FBS (n &equals; 97): 82&percnt;, 28&percnt;; and M199A/PVA (n &equals; 111): 98&percnt;, 31&percnt;, respectively) did not differ from those of the control group. After IVS, cumulus expansion was not seen and most of the oocyte nuclei reached the GVBD stage. These results suggest that the nuclear maturation progress of bovine oocytes can be regulated for at least 22 h in M199A without any deleterious influence on the number of oocytes surviving at an immature state after the storage and their subsequent development to the blastocyst stage after IVM&ndash;IVF&ndash;IVC. The delayed maturation allows a flexible fertilization schedule which is advantageous in research and industrial applications.

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