145 COMPARISON OF DEVELOPMENT AND QUALITY OF PORCINE EMBRYOS CULTURED IN DIFFERENT OXYGEN CONCENTRATIONS
S.-A. Ock A , J.-G. Kim A , L.-Y. Shi A , H.-F. Jin A , B. Mohana Kumar A , S.-Y. Choe A and G.-J. Rho AACollege of Veterinary Medicine, Gyeongsang National University, Chinju, 660-701, Republic of Korea. Email: jinrho@nongae.gsnu.ac.kr
Reproduction, Fertility and Development 17(2) 223-223 https://doi.org/10.1071/RDv17n2Ab145
Submitted: 1 August 2004 Accepted: 1 October 2004 Published: 1 January 2005
Abstract
In spite of valuable uses of pig IVP embryos, including IVF and cloned embryos for production of valuable offspring that provide recombinant protein and xenotransplantation, several problems limit the success. In cattle embryo culture, lowering the oxygen concentration resulted in increased development, quality, and cell number, and reduced apoptosis incidence. The high O2 concentration in in vitro culture caused overproduction of reactive oxygen specias (ROS), causing damage to the cell membrane or DNA. The present study, therefore, evaluated the developmental ability of porcine embryos by culturing in either low or high oxygen concentration on the rates of cleavage, development, cell number, and apoptosis. These values were then compared to parthenote embryos, which were activated with electric stimulation by 2 DC pulses of 2.0 kV/cm, 30 μs in 0.3 M mannitol containing 100 μM CaCl2 and 100 μM MgCl2 and then transferred into NCSU23 medium supplemented with 7.5 μg/mL cytochalasin B for 6 h. Cumulus-oocyte complexes from slaughterhouse ovaries were matured in TCM 199 medium supplemented with 10 ng/mL EGF, 0.5 μg/mL FSH, 0.5 μg/mL LH, 0.57 mM cysteine, and 0.91 mM Na pyruvate for 24 h and further cultured in the same medium without FSH and LH for 20 h at 38.5°C, 5% CO2 in air. IVF was carried out in mTBM for 5 h with 1 × 105 sperm/mL by following the previously reported protocols (2004 Methods Mol. Biol. 253, 227–234) with minor modifications. Zygotes were allocated two O2 concentrations; zygotes were cultured in NCSU23 medium supplemented with 0.17 mM Na pyruvate, 2.73 mM Na lactate, and 0.4% BSA for 54 h and subsequently cultured in the same medium supplemented with 5.55 mM glucose for 90 h at 38.5°C, 5% CO2, 5% O2 and 90% N2 (Treatment 1) or 5% CO2, 20% O2 in air (Treatment 2). Statistical analysis was performed with one-way ANOVA by SPSS 10.0 (P < 0.05). Most oocytes (>82%) cleaved and the rates did not differ between groups. However, the rates of blastocyst development from oocytes used as parthenote controls (5% O2, 40.0 ± 13.0 (70/162); 20% O2, 34.5 ± 11.2 (56/161)) were significantly (P < 0.05) higher than those in IVF embryos (5% O2, 27.9 ± 8.0 (70/247); 20% O2, 27.1 ± 7.2 (68/249)). Similarly cell number and apoptosis index by TUNEL staining of blastocysts in parthenote (5% O2, 42 and 15%; 20% O2, 39 and 20%) were significantly (P < 0.05) higher than those in IVF (5% O2, 32 and 13%; 20% O2, 27 and 14%, all respectively). In conclusion, no differences between low and high oxygen concentrations in culture of porcine IVF and parthenote embryos were observed on the rates of cleavages and development into blastocyst cell number and apoptosis incidence. Further research should be carried out to develop a reliable IVP system.
This work was supported by grant No. R05-2004-000-10702-0 from Ministry of Science & Technology, Republic of Korea.
