Effects of FBS(Fetal Bovine Serum) and pFF(Porcine Follicular Fluid) on In Vitro Maturation and Development of Porcine Parthenogenetic and Nuclear Transfer Embryos원문보기
Moon, Hyo-Jin
(Division of Animal Biotechnology, National Institute of Animal Science, RDA)
,
Shim, Joo-Hyun
(Division of Animal Biotechnology, National Institute of Animal Science, RDA)
,
Hwang, In-Sun
(Division of Animal Biotechnology, National Institute of Animal Science, RDA)
,
Park, Mi-Rung
(Division of Animal Biotechnology, National Institute of Animal Science, RDA)
,
Kim, Dong-Hoon
(Division of Animal Biotechnology, National Institute of Animal Science, RDA)
,
Ko, Yeoung-Gyu
(Division of Animal Biotechnology, National Institute of Animal Science, RDA)
,
Park, Choon-Keun
(College of Animal Science, Kangwon National University)
,
Im, Gi-Sun
(Division of Animal Biotechnology, National Institute of Animal Science, RDA)
In this study, in vitro maturation system using fetal bovine serum (FBS) or porcine follicular fluid (pFF) was investigated to produce comparable oocytes to those derived from in vivo. Control group of oocytes was cultured in TCM 199 supplemented with 0.1% polyvinyl alcohol (PVA). Other three groups...
In this study, in vitro maturation system using fetal bovine serum (FBS) or porcine follicular fluid (pFF) was investigated to produce comparable oocytes to those derived from in vivo. Control group of oocytes was cultured in TCM 199 supplemented with 0.1% polyvinyl alcohol (PVA). Other three groups of oocytes were cultured in TCM 199 supplemented with 10% FBS, 10% pFF or 5% FBS + 5% pFF, respectively. After 44 h maturation, oocytes with the first polar body were activated with two electric pulses (DC) of 1.2 kv/cm for 30 ${\mu}sec$. Also, matured oocytes of four groups were reconstructed and fused. Reconstructed embryos were cultured in PZM-3 under 5% $CO_2$ in air at $38.5^{\circ}C$ for 6 days. The oocytes matured in the medium supplemented with FBS or/and pFF showed significantly higher maturation rates (64.0 vs. 73.9 to 85.2%). In PA embryos, cleavage rates (89.7 vs. 77.1 to 86.6%) and blastocysts rates (30.0 vs. 16.2 to 26.2%) were significantly higher in pFF group (p<0.05). In NT embryos, there was no difference among treatments in cleavage rate, but the blastocyst rates (28.5 vs. 15.5 to 24.6%) were significantly higher in pFF group (p<0.05). The apoptosis rate was significantly higher (p<0.05) in the control than other groups (10.8 vs. 4.9 to 8.2% for PA, 3.1 vs. 0.5 to 1.3% for NT). In order to select the comparable oocyte to in vivo oocytes, each group of oocytes was stained with Brilliant cresyl blue (BCB) after 42h maturation. The matured oocytes were separated according to color of cytoplasm; stained group (BCB+) and unstained group (BCB-). The oocytes matured in the presence of FBS or/and pFF showed significantly higher staining rates (70.3 to 72.7 vs. 35.1%) (p<0.05). To verify the fact that the supplementation of FBS or/and pFF can increase the maturation rates, cdc2 kinase activity, the catalytic subunit of MPF, was determined. The cdc2 kinase activity of the oocytes matured in the medium supplemented with FBS or/and pFF was significantly higher than control group (6.7 to 9.3 vs. 3.8). In conclusion, the supplementation of FBS or/and pFF can support in vitro maturation rate of porcine oocytes through the increment of cdc2 kinase activity level in the cytoplasm.
In this study, in vitro maturation system using fetal bovine serum (FBS) or porcine follicular fluid (pFF) was investigated to produce comparable oocytes to those derived from in vivo. Control group of oocytes was cultured in TCM 199 supplemented with 0.1% polyvinyl alcohol (PVA). Other three groups of oocytes were cultured in TCM 199 supplemented with 10% FBS, 10% pFF or 5% FBS + 5% pFF, respectively. After 44 h maturation, oocytes with the first polar body were activated with two electric pulses (DC) of 1.2 kv/cm for 30 ${\mu}sec$. Also, matured oocytes of four groups were reconstructed and fused. Reconstructed embryos were cultured in PZM-3 under 5% $CO_2$ in air at $38.5^{\circ}C$ for 6 days. The oocytes matured in the medium supplemented with FBS or/and pFF showed significantly higher maturation rates (64.0 vs. 73.9 to 85.2%). In PA embryos, cleavage rates (89.7 vs. 77.1 to 86.6%) and blastocysts rates (30.0 vs. 16.2 to 26.2%) were significantly higher in pFF group (p<0.05). In NT embryos, there was no difference among treatments in cleavage rate, but the blastocyst rates (28.5 vs. 15.5 to 24.6%) were significantly higher in pFF group (p<0.05). The apoptosis rate was significantly higher (p<0.05) in the control than other groups (10.8 vs. 4.9 to 8.2% for PA, 3.1 vs. 0.5 to 1.3% for NT). In order to select the comparable oocyte to in vivo oocytes, each group of oocytes was stained with Brilliant cresyl blue (BCB) after 42h maturation. The matured oocytes were separated according to color of cytoplasm; stained group (BCB+) and unstained group (BCB-). The oocytes matured in the presence of FBS or/and pFF showed significantly higher staining rates (70.3 to 72.7 vs. 35.1%) (p<0.05). To verify the fact that the supplementation of FBS or/and pFF can increase the maturation rates, cdc2 kinase activity, the catalytic subunit of MPF, was determined. The cdc2 kinase activity of the oocytes matured in the medium supplemented with FBS or/and pFF was significantly higher than control group (6.7 to 9.3 vs. 3.8). In conclusion, the supplementation of FBS or/and pFF can support in vitro maturation rate of porcine oocytes through the increment of cdc2 kinase activity level in the cytoplasm.
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문제 정의
This study investigated the effect of FBS and pFF on maturation and in vitro development of porcine PA and NT embryos. In this study, pFF supported better maturation and in vitro developmental rate.
가설 설정
Data are expressed as relative percentage of the level of p34cdc2 activity in porcine oocytes.a, b : Bars with different letters are significantly different (p<0.05).
제안 방법
Following BCB exposure, the COCs were transferred to mDPBS and washed twice. After washing, the COCs were examined under a stereomicroscope and divided into two groups according to their cytoplasm coloration: oocytes with any degree of blue coloration in the cytoplasm (BCB+) and oocytes without blue cytoplasm (BCB-).
(SAS Institute, Cary, NC, USA). Differences among treatment means were determined by using the Dun can's multiple range tests. All data were expressed as Least Square (LS) mean±SEM (Standard Error of the sample Mean).
In order to select the comparable oocytes to in vivo oocytes, BCB staining was conducted. It has been demonstrated that brilliant cresyl blue (BCB) can be us ed for the selection of competent oocytes of prepuber tal pigs, goats and cattle (Rodriguezef al.
To carry out the BCB test, the compact COCs were washed three times in Dulbecco's PBS modified by the addition of 0.4% BSA (A-7888; mDPBS). Then the COCs were exposed to 26 yM of BCB (B-5388) diluted in mPBS for 90 min at 38.
데이터처리
Data were subjected to a Generalized Linear Model procedure (PROC-GLM) of the Statistical Analysis Sys tem (SAS Institute, Cary, NC, USA). Differences among treatment means were determined by using the Dun can's multiple range tests.
성능/효과
In conclusion, the oocytes matured in TCM 199 supplemented with FBS or pFF showed significantly higher maturation rates. However, the developmental rate to blastocyst stage was higher in the oocytes ma tured in the presence of pFF.
However, the developmental rate to blastocyst stage was higher in the oocytes ma tured in the presence of pFF. These results indicate that FBS or pFF can support the increment of cdc2 ki nase activity level in the cytoplasm of oocytes resulting in the in vitro developmental ability.
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