scholarly journals Usefulness of modified Medium RD as a chemically defined medium for in vitro maturation of bovine oocytes

2020 ◽  
Vol 19 (4) ◽  
pp. 365-371
Author(s):  
Kenji Momozawa
Keyword(s):  
In Vitro Maturation ◽  
Defined Medium ◽  
Chemically Defined Medium ◽  
Bovine Oocytes

Application of Chemically Defined Medium RD for In Vitro Maturation of Bovine Oocytes.

2011 ◽  
Vol 85 (Suppl_1) ◽  
pp. 258-258
Author(s):  
Kenji Momozawa ◽  
Kaori Ooya ◽  
Rie Saito ◽  
Kanako Morohaku ◽  
Hiroshi Sasada
Keyword(s):  
In Vitro Maturation ◽  
Defined Medium ◽  
Chemically Defined Medium ◽  
Bovine Oocytes

6 TIMING OF DEADENYLATION OF GDF-9 AND CYCLIN 3′ UTR CONSTRUCTS IN BOVINE OOCYTES

2007 ◽  
Vol 19 (1) ◽  
pp. 121
Author(s):  
D. J. Walker ◽  
C. J. Wilusz ◽  
G. E. Seidel Jr
Keyword(s):  
In Vitro Maturation ◽  
Control Point ◽  
Tail Length ◽  
Cumulus Cells ◽  
Cyclin B1 ◽  
Defined Medium ◽  
Untranslated Regions ◽  
Labeled Rna ◽  
Bovine Oocytes

The maternal pool of mRNA undergoes major changes during oocyte maturation and early embryonic development. Specific genes are activated or degraded in response to changes in poly-(A) tail length. However, little is known about how the oocyte targets specific transcripts for degradation or translation in a timely manner. The objective of this study was to determine how poly-(A) tail length of different transcripts is affected in bovine oocytes by time of in vitro maturation. Cyclin B1 and GDF-9 32 untranslated regions (UTRs) were cloned into modified p-GEM plasmids containing a poly-(A) tract of 60 or 0 adenosines (A60 or A0, respectively). Each 32 UTR was transcribed in vitro with (A60) or without (A0) a poly-(A) tail to generate UTP32-labeled RNA. Transcriptions producing at least 200 000 counts per min (cpm) per �L were used for subsequent injections into denuded bovine oocytes. Cumulus-oocyte complexes (COCs) recovered from slaughterhouse-derived ovaries (n = 216) were vortexed to remove cumulus cells immediately after aspiration, after 3 h of in vitro maturation, or after 19 h of maturation in a chemically defined medium supplemented with FSH, LH, EGF, and cysteamine. After vortexing, denuded oocytes were injected and snap frozen, or matured in vitro for 1 or 3 h. Eight oocytes were injected with ~0.5 nL (~100 cpm/oocyte) labeled RNA at each time point in 3 replicates. Total RNA was isolated from injected oocyte pools and loaded onto a 5% denaturing acrylamide gel for size separation. Radiolabeled A0 was used as a control point of reference for deadenylation. Gels were dried, and RNA was visualized on a phosphoimager after 24 h exposure to a phosphor screen. Changes in polyadenylation status (transcript size) were evaluated by comparing shifts in bands from gene-specific A60

scholarly journals In vitro production of cattle blastocysts in chemically defined medium with or without insulin supplementation

1996 ◽  
Vol 5 (5) ◽  
pp. 509-514 ◽  
Author(s):  
Kristiina Bredbacka ◽  
Peter Bredbacka
Keyword(s):  
Bovine Serum Albumin ◽  
Culture Medium ◽  
Defined Medium ◽  
Blastocyst Stage ◽  
Chemically Defined Medium ◽  
In Vitro Production ◽  
Cell Numbers ◽  
Vitro Production ◽  
Bovine Oocytes

In this study we evaluated the use of a chemically defined medium in the production of blastocysts from bovine oocytes fertilized in vitro. As culture medium we used CRI-PVP, a modification of CRlaa medium with bovine serum albumin replaced by polyvinylpyrrolidone. After 168 h of culture (192 h after insemination) 8.7%, 10.5 and 12.8% of the cleaved embryos developed to the blastocyst stage in the presence of 0, 2 or 200 nM insulin, respectively. The supplementation of 200 nM insulin tended to increase cell numbers in morulae and blastocysts (P=0.10). It is concluded that CRI-PVP can be used as a chemically defined medium in the production of blastocysts from bovine 1-cell embryos. However, further modifications are needed, and the insulin concentrations used may be below the optimum for blastocyst production.

241 DIBUTYRYL CYCLIC AMP AND EGF-LIKE FACTORS SUPPORT IN VITRO MATURATION OF PORCINE OOCYTES IN A CHEMICALLY DEFINED MEDIUM WITHOUT GONADOTROPINS

2009 ◽  
Vol 21 (1) ◽  
pp. 218
Author(s):  
Y. Akaki ◽  
K. Yoshioka ◽  
H. Funahashi
Keyword(s):  
Cyclic Amp ◽  
In Vitro Maturation ◽  
Defined Medium ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Chemically Defined Medium ◽  
Dibutyryl Cyclic Amp ◽  
Vitro Fertilization ◽  
Porcine Oocyte

Exposure of porcine oocyte–cumulus complexes (OCC) to gonadotropins induces meiotic resumption, but the details of this mechanism are still unknown. The present study was undertaken to examine combinational effects of EGF-like factors and dibutyryl cyclic AMP (dbcAMP) in a chemically defined medium on in vitro maturation (IVM) of porcine oocytes. The OCC were aspirated from 3- to 6-mm-diameter follicles of prepuberal ovaries and used in the current study. The basic culture medium was a chemically defined medium, Porcine Oocyte Medium (POM; Research Institute for the Functional Peptides, Yamagata, Japan). In the first experiment, various concentrations (0, 10, and 1000 ng mL–1) of EGF-like factors (EGF, amphiregulin, and betacellulin) were added to POM during an entire IVM period (44 h). In the second experiment, to determine the additive effect of EGF-like factors, each EGF-like factor with an effective concentration was combined with the others. In the last experiment, to examine the combined effect with dbcAMP, OCC were exposed to EGF (10 ng mL–1), amphiregulin (1000 ng mL–1), and dbcAMP (1 mm) during the first 20 h of IVM and then the culture was continued in the absence of EGF-like factors and dbcAMP. After culture, in all experiments, meiotic resumption and the progress of oocytes were examined after denuding, fixing, and staining. Statistical analyses was performed by ANOVA with a Bonferroni-Dunn post hoc test (significance, P < 0.05). In the first experiment, all treatments without supplementation with 10 ng mL–1 amphiregulin increased the incidence of oocytes maturing to the MII phase, as compared with controls (29.1 to 39.3% v. 11.1%, P < 0.05). In the second experiment, combinations with 2 kinds of EGF-like factor slightly (but not significantly) improved the percentage of oocytes at the MII stage (37.7 to 47.4%). In the last experiment, supplementation with 1 mm dbcAMP during the first 20 h of IVM, regardless of the presence of EGF-like factors, significantly increased the incidence of MII oocytes as compared with controls, whereas the incidence was the highest when 1 mm dbcAMP, 10 ng mL–1 EGF, and 1000 ng mL–1 amphiregulin were supplemented (75.5%). When those oocytes were cultured in a chemically defined medium after in vitro fertilization, the developmental competence of oocytes to the blastocyst stage (25.0%) was not different from oocytes matured in the presence of gonadotropins and dbcAMP during the first 20 h of IVM (17.3%). These observations indicate that supplementation of a chemically defined maturation medium with EGF-like factors and dbcAMP during the first 20 h of IVM can support the meiotic progress and developmental competence of porcine oocytes well. Currently, we are examining the developmental competence of those oocytes after embryo transfer. The results will be presented at the meeting. This study was supported by MAFF AgriBio1605.

Additional effect of epidermal growth factor during in vitro maturation for individual bovine oocytes using a chemically defined medium

Zygote ◽  
2004 ◽  
Vol 12 (2) ◽  
pp. 143-150 ◽  
Author(s):  
Takahiro Oyamada ◽  
Hiroshi Iwayama ◽  
Yutaka Fukui
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Defined Medium ◽  
Control Medium ◽  
Chemically Defined Medium ◽  
Blastocyst Formation ◽  
Epidermal Growth ◽  
The Individual ◽  
Bovine Oocytes

This study was performed to establish an individual bovine oocyte-IVP system using a chemically defined simple medium (mSOFaa containing 1 mg/ml polyvinyl alcohol: PVA) and to investigate the effects of epidermal growth factor (EGF) during oocyte maturation on in vitro maturation, fertilization and embryonic development. Cumulus–oocyte complexes were collected from bovine ovaries and were matured in mSOFaa containing PVA (control medium) supplemented with 0, 1, 10 or 50 ng/ml of EGF. Two further groups (TCM199 and mSOFaa, supplemented with 10% fetal calf serum were also included. In this study, mSOFaa containing PVA were used as a basic medium for fertilization and embryo development in vitro. Experiments were conducted in both group- and individual-IVP systems. In the group-IVP system, the proportion of matured oocytes (MII) in the control medium (62.7%±5.0%) was significantly (p<0.05) lower than in all other treatments, and in the individual-IVP system, the addition of 1 ng/ml EGF significantly (p<0.05) increased the maturation rate (1 ng/ml EGF vs control: 76.2%±5.4% vs 57.1%±14.4%). The addition of EGF did not affect the proportions of penetrated and normally fertilized oocytes in either individual- or group-culture systems. In the group-IVP system, no significant difference among treatments was found in the rate of blastocyst formation, whereas in the individual-IVP system the control medium supplemented with 10 ng/ml EGF resulted in a significantly (p<0.05) higher the rate of blastocyst formation (20.0±5.2%) than that in the control medium (6.2%±3.5%). These results indicate that bovine oocytes can successfully develop to blastocysts in an individual-IVP system using a single chemically defined medium, and that the group-IVP system also resulted in a similar level of blastocyst formation to that in a standard multiple-media system in our laboratory. The effect of EGF during oocyte maturation medium differed depending on whether embryos were cultured individually or in groups.

In vitro maturation and in vitro fertilization of bovine oocytes cultured in a chemically defined, protein-free medium: effects of carbohydrates and amino acids

1999 ◽  
Vol 11 (2) ◽  
pp. 127 ◽  
Author(s):  
J. M. Lim ◽  
B. C. Lee ◽  
E. S. Lee ◽  
H. M. Chung ◽  
J. J. Ko ◽  
...  
Keyword(s):  
Amino Acids ◽  
In Vitro Fertilization ◽  
In Vitro Maturation ◽  
Essential Amino Acids ◽  
Defined Medium ◽  
Blastocyst Stage ◽  
Vitro Fertilization ◽  
Pronuclear Formation ◽  
Bovine Oocytes

This study was conducted to examine the effects of carbohydrates and amino acids on the maturation and fertilization of bovine oocytes. To evaluate the effect of each treatment without any unpredictable interference, oocytes were cultured in a simply defined medium (modified Tyrode’s medium; mT) without the addition of hormones and proteins. In Experiment 1, oocyte maturation to the metaphase-II stage was significantly (P<0.0001) enhanced after the addition of glucose (5.6 mМ), lactate (10 mМ) and/or pyruvate (0.5 mМ) to mT (37–74%) than after no addition (0%). In mT supplemented with glucose, the addition of 19 essential and non-essential amino acids (aa; 0, 0.01, 0.1, 1, 5 or 10%) did not further improve in vitro maturation (Experiment 2) or in vitro fertilization (Experiment 3) of oocytes. However, more (P<0.05) pronuclear formation after in vitro-insemination was found in oocytes matured in mT with 1% aa and glucose than in oocytes matured in mT with glucose alone (56% vs. 35%). Penetration of spermatozoa into the ooplasm was initiated at 3 h after insemination and pronuclear formation from 8 h (Experiment 4). When cultured inseminated oocytes were examined up to 192 h post insemination, a significant (P<0.05) increase in the number of 2-cell (18 v. 38%) and 8-cell embryos, (7 v. 20%) and morulae (0 v. 8%) was found after the addition of 1% aa to mT with glucose than after no addition (Experiment 5). A limited number of oocytes matured in mT with aa and glucose developed to the blastocyst stage (6%). These results indicate that exogenous carbohydrates and amino acids are prerequisites for the maturation and fertilization of bovine oocytes in vitro. Glucose alone promotes the nuclear maturation of oocytes, whereas amino acids aid the pronuclear formation of fertilized oocytes.

scholarly journals 318EFFECT OF PIG FOLLICLE FLUID AND FETAL CALF SERUM ON PORCINE OOCYTE MATURATION AND SUBSEQUENT DEVELOPMENT AFTER ACTIVATION AND SOMATIC CELL NUCLEAR TRANSFER

2004 ◽  
Vol 16 (2) ◽  
pp. 278
Author(s):  
Z. Liu ◽  
L. Lai ◽  
G. Im ◽  
M. Samuel ◽  
D. Wax ◽  
...  
Keyword(s):  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
In Vitro Maturation ◽  
Fetal Calf Serum ◽  
Calf Serum ◽  
Defined Medium ◽  
Chemically Defined Medium ◽  
Maturation Rate ◽  
Blastocyst Rate

In vitro maturation of porcine oocytes is very important for understanding porcine somatic cell nuclear transfer (SCNT). In order to develop an in vitro maturation system that can provide more high quality oocytes, the effect of porcine follicle fluid (pFF) (gathered from 3–5-mm porcine follicles) and fetal calf serum (FCS: Sigma, St. Louis, MO, USA), as an important additional component of a chemically-defined medium was studied. Cumulus-oocyte complexes (COC) derived from follicles 3–5mm in diameter were cultured in three different media: a chemically-defined medium (CDM: TCM-199 with 0.1mgmL−1 cysteine, 10ngmL−1 EGF, 0.5μgmL−1 LH and 0.5μgmL−1 FSH); CDM with 10% pFF (CDM+p); and CDM with 10% FCS (CDM+F). After 42–44h of maturation, oocytes with a clear polar body were classified as matured oocytes. Matured oocytes stimulated by electric pulse (120v, 30μs, 2 pulse), or enucleated and fused with fibroblasts to construct SCNT embryos by using the same electrical parameters. All of these parthenogenetic and SCNT embryos were cultured in Porcine Zygote Medium-3. The blastocyst rate was assessed under a stereomicroscope on Day 6, and the number of nuclei in the blastocysts was counted under a fluorescent microscope after staining with 5μgmL−1 of Hoechst 33342. All data were subjected to a Generalized Linear Model Procedure (PROC-GLM) of Statistical Analysis System (SAS). The maturation rates of porcine oocytes in CDM and CDM+p were 53.2±3.8% (539/1050) and 69.7±3.8% (587/847), respectively;; in CDM and CDM+F, 61.1±3.1% (471/776) and 70.2±3.7% (577/844), respectively. Oocytes matured in CDM+p and CDM+F showed a higher (P&lt;0.05) maturation rate than those in CDM. The percentages of parthenogenetic blastocysts of oocytes matured in CDM and CDM+p were 13.9±2.1% (35/250) and 20.2±5.3% (64/300), and the numbers of nuclei in these blastocysts were 25.8±2.3 and 25.8±1.4, respectively. The blastocyst rate from CDM- and CDM+F-matured oocytes were 20.1±2.0% (53/272) and 22.2±4.7%(71/298), and the numbers of nuclei in these blastocysts were 24.7±1.5 and 25.3±1.5, respectively. There were no significant (P&gt;0.05) differences in the percentages of parthenogenetic blastocysts and nuclei numbers between CDM and CDM+p, or CDM and CDM+F. The percentages of blastocysts in SCNT embryos derived from CDM and CDM+p were 8.1±1.5% (14/192) and 12.3±1.9% (24/192), while the nuclei numbers in these blastocysts were 26.6±1.2 and 34.5±2.2, respectively. The percentages of blastocysts after SCNT from oocytes matured in CDM and CDM+F were 24.3±4.9% (35/139) and 27.1±5.5% (45/176), while the numbers of nuclei were 29.8±2.5 and 32.2±1.9, respectively. There were no significant (P&gt;0.05) differences between CDM and CDM+p, or CDM and CDM+F in SCNT embryo blastocyst rate, but the SCNT embryos derived from CDM+p showed a higher (P&lt;0.05) nuclear number. In conclusion, these results indicate that 10% pFF or FCS in CDM can promote a higher maturation rate of porcine oocytes. As recipient cytoplasm for SCNT, oocytes matured in CDM+p can support development of blastocysts that contain more nuclei than those matured in CDM alone. Supported in part by Food for the 21st Century and RR13438.

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