3 results
Glucose in a maturation medium with reduced NaCl improves oocyte maturation and embryonic development after somatic cell nuclear transfer and in vitro fertilization in pigs
- Yongjin Lee, Hanna Lee, Joohyeong Lee, Seung Tae Lee, Geun-Shik Lee, Eunsong Lee
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This study was conducted to examine whether glucose in maturation medium containing reduced NaCl could improve oocyte maturation and embryonic development in pigs. The base medium was bovine serum albumin-free porcine zygote medium (PZM)-3 containing 10% (v/v) pig follicular fluid (FPZM) or 0.1% (w/v) polyvinyl alcohol (PPZM). Using each medium, the effects of NaCl concentrations (108 and 61.6 mM) and 5.56 mM glucose supplementation (designated as PZM108N, PZM108G, PZM61N, and PZM61G, respectively) were examined using a 2 × 2 factorial arrangement. When oocytes were matured in FPZM, glucose supplementation improved nuclear maturation compared with no supplementation, regardless of the NaCl concentrations. FPZM61G showed a higher blastocyst formation compared with FPZM108N and FPZM108G after parthenogenesis (PA). Blastocyst formations of somatic cell nuclear transfer (SCNT) embryos derived from FPZM61N and FPZM61G were higher compared with those of oocytes from FPZM108N. When oocytes were matured in PPZM, glucose added to PPZM108 and PPZM61 increased nuclear maturation compared with no supplementation. However, glucose added to PPZM108 did not alter embryonic development after PA. Additionally, oocytes matured in PPZM61G showed a higher blastocyst formation compared with those from PPZM61N. In SCNT, blastocyst formation was not influenced by glucose supplementation of PPZM108, but was increased by maturation in glucose-supplemented PPZM61. In embryonic development of in vitro fertilization (IVF), oocytes matured in medium with reduced NaCl and glucose showed significantly higher blastocyst formation compared with those matured in PPZM108G. Our results demonstrated that glucose in maturation medium containing 61.6 mM NaCl increased oocyte maturation and embryonic development after PA, SCNT, and IVF.
Blastocyst production by in vitro maturation and development of porcine oocytes in defined media following intracytoplasmic sperm injection
- M. Kobayashi, S. Asakuma, Y. Fukui
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The present study was carried out to establish porcine defined IVP. In Experiments 1 and 2, we investigated the efficacy of additional 0.6 mM cystine and/or 100 µM cysteamine (Cys) to a defined TCM199 maturation medium with regard to the intracellular glutathione (GSH) concentration and the developmental competence of in vitro matured porcine oocytes following intracytoplasmic sperm injection (ICSI). The control medium was a modified TCM199 containing 0.05% (w/v) polyvinyl alcohol (PVA). Cys and/or cystine were added to the control medium. The control group and immature oocytes (presumptive germinal vesicle oocytes; GV) were prepared for GSH assay. In Experiment 3, the efficacy of epidermal growth factor (EGF) addition to a modified porcine zygote medium (mPZM) for in vitro culture (IVC) medium was investigated on embryonic development and the mean cell number of blastocysts following ICSI. As a positive or negative control, 0.3% BSA (mPZM-3) or 0.3% PVA (mPZM-4), respectively, was added to the base medium. The defined IVC medium was supplemented with 5 or 10 ng/ml EGF. In Experiment 1, no significant difference was found in the rates of cleavage (31.4–64.3%) and blastocyst formation (6.5–22.9%) among the treatment and control groups. The mean cell numbers per blastocyst ranged from 30 to 48 among the groups without significant differences. However, in Experiment 2, the intracellular GSH concentrations in the oocytes cultured in the medium supplemented with 100 µM Cys (9.6 pmol/oocyte) or Cys + cystine (9.9 pmol/oocyte) were significantly (p < 0.05) higher than the control (2.5 pmol/oocyte) and 0.6 mM cystine (6.5 pmol/oocyte) groups, but not different from the GV group (9.0 pmol/oocyte). The GSH concentration in the cystine group was also significantly (p < 0.05) higher than that in the control group, but not different from the GV group. In Experiment 3, the rates of cleavage and blastocyst formation and the mean cell numbers of blastocysts were not significantly different among the groups. However, the addition of 5 ng/ml EGF into the mPZM-4 resulted in a significantly (p < 0.05) higher blastocyst rate per cleaved embryo than the other two defined groups (mPZM-4 + 5 ng/ml: 48.6%, mPZM-4 and mPZM-4 +10 ng/ml: 23.4% and 23.1%, respectively).
The present results indicate that the addition of Cys to a defined medium for in vitro maturation (IVM) of porcine oocytes increases intracellular GSH concentration. Further addition of cystine into the IVM medium containing 100 µM Cys is not necessary and TCM199 plus Cys (100 µM) could be used as a defined IVM medium for porcine oocytes. The addition of 5 ng/ml EGF to a defined IVC medium has enhanced subsequent development after ICSI. This study shows that porcine blastocysts can be produced by defined media throughout the steps of IVP (IVM, ICSI and IVC).
Additional effect of epidermal growth factor during in vitro maturation for individual bovine oocytes using a chemically defined medium
- Takahiro Oyamada, Hiroshi Iwayama, Yutaka Fukui
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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.