Nuclear Replacement of In Vitro‐Matured Porcine Oocytes by a Serial Centrifugation and Fusion Method |
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| Authors: | N Maedomari K Kikuchi T Nagai M Fahrudin H Kaneko J Noguchi M Nakai M Ozawa T Somfai LV Nguyen J Ito N Kashiwazaki |
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| Institution: | 1. Division of Animal Sciences, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki, Japan;2. Laboratory of Animal Reproduction, School of Veterinary Medicine, Azabu University, Sagamihara, Kanagawa, Japan;3. Present address: Institute of Laboratory Animals, Graduate School of Medicine, Kyoto University, Kyoto, Kyoto 606‐8501, Japan.;4. National Institute of Livestock and Grassland Science, National Agricultural Research Organization, Tsukuba, Ibaraki, Japan;5. Department of Anatomy, Physiology and Pharmacology, Faculty of Veterinary Medicine, Bogor Agricultural University, Bogor, Indonesia;6. Institute of Biotechnology, Vietnamese Academy of Science and Technology, Hanoi, Vietnam |
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| Abstract: | The objective of the present study was to establish a method for nuclear replacement in metaphase‐II (M‐II) stage porcine oocytes. Karyoplasts containing M‐II chromosomes (K) and cytoplasts without chromosomes (C) were produced from in vitro‐matured oocytes by a serial centrifugation method. The oocytes were then reconstructed by fusion of one karyoplast with 1, 2, 3 or 4 cytoplasts (K + 1C, K + 2C, K + 3C and K + 4C, respectively). Reconstructed oocytes, karyoplasts without fusion of any cytoplast (K) and zona‐free M‐II oocytes (control) were used for experiments. The rates of female pronucleus formation after parthenogenetic activation in all groups of reconstructed oocytes (58.2–77.4%) were not different from those of the K and control groups (58.2% and 66.0%, respectively). In vitro fertilization was carried out to assay the fertilization ability and subsequent embryonic development of the reconstructed oocytes. The cytoplast : karyoplast ratio did not affect the fertilization status (penetration and male pronuclear formation rates) of the oocytes. A significantly high monospermy rate was found in K oocytes (p < 0.05, 61.6%) compared with the other groups (18.2–32.8%). Blastocyst formation rates increased significantly as the number of the cytoplasts fused with karyoplasts increased (p < 0.05, 0.0–15.3%). The blastocyst rate in the K + 4C group (15.3%) was comparable with that of the control (17.8%). Total cell numbers in both the K + 3C and K + 4C groups (16.0 and 15.3 cells, respectively) were comparable with that of the control (26.2 cells). Our results demonstrate that a serial centrifugation and fusion (Centri‐Fusion) is an effective method for producing M‐II chromosome transferred oocytes with normal fertilization ability and in vitro development. It is suggested that the number of cytoplasts fused with a karyoplast plays a critical role in embryonic development. |
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