Researchers Database

tanihara fuminori

    Center for Development of Advanced Medical Technology,Animal Resource Laboratory Associate Professor
Last Updated :2021/10/17

Researcher Information

Research funding number

  • 90754680

J-Global ID

Research Interests

  • oocyte   gene editing   in vitro fertilization   pig   

Research Areas

  • Life sciences / Zoological sciences

Academic & Professional Experience

  • 2020/10 - Today  Jichi Medical University先端医療技術開発センター准教授

Association Memberships

  • THE JAPANESE SOCIETY FOR REGENERATIVE MEDICINE   SOCIETY FOR REPRODUCTION AND DEVELOPMENT   International Society for Transgenic Technologies   日本異種移植研究会   日本ゲノム編集学会   日本獣医学会   

Published Papers

  • Fuminori Tanihara, Maki Hirata, Nhien Thi Nguyen, Osamu Sawamoto, Takeshi Kikuchi, Takeshige Otoi
    International journal of molecular sciences 22 (5) 2021/02 [Refereed][Not invited]
     
    Xenoantigens cause hyperacute rejection and limit the success of interspecific xenografts. Therefore, genes involved in xenoantigen biosynthesis, such as GGTA1, CMAH, and B4GALNT2, are key targets to improve the outcomes of xenotransplantation. In this study, we introduced a CRISPR/Cas9 system simultaneously targeting GGTA1, CMAH, and B4GALNT2 into in vitro-fertilized zygotes using electroporation for the one-step generation of multiple gene-edited pigs without xenoantigens. First, we optimized the combination of guide RNAs (gRNAs) targeting GGTA1 and CMAH with respect to gene editing efficiency in zygotes, and transferred electroporated embryos with the optimized gRNAs and Cas9 into recipient gilts. Next, we optimized the Cas9 protein concentration with respect to the gene editing efficiency when GGTA1, CMAH, and B4GALNT2 were targeted simultaneously, and generated gene-edited pigs using the optimized conditions. We achieved the one-step generation of GGTA1/CMAH double-edited pigs and GGTA1/CMAH/B4GALNT2 triple-edited pigs. Immunohistological analyses demonstrated the downregulation of xenoantigens; however, these multiple gene-edited pigs were genetic mosaics that failed to knock out some xenoantigens. Although mosaicism should be resolved, the electroporation technique could become a primary method for the one-step generation of multiple gene modifications in pigs aimed at improving pig-to-human xenotransplantation.
  • Maki Hirata, Manita Wittayarat, Zhao Namula, Quynh Anh Le, Qingyi Lin, Koki Takebayashi, Chommanart Thongkittidilok, Fuminori Tanihara, Takeshige Otoi
    Animals : an open access journal from MDPI 11 (2) 2021/02 [Refereed][Not invited]
     
    Liposome-mediated gene transfer has become an alternative method for establishing a gene targeting framework, and the production of mutant animals may be feasible even in laboratories without specialized equipment. However, how this system functions in mammalian oocytes and embryos remains unclear. The present study was conducted to clarify whether blastocyst genome editing can be performed by treatment with lipofection reagent, guide RNA, and Cas9 for 5 h without using electroporation or microinjection. A mosaic mutation was observed in blastocysts derived from zona pellucida (ZP)-free oocytes following lipofection treatment, regardless of the target genes. When lipofection treatment was performed after in vitro fertilization (IVF), no significant differences in the mutation rates or mutation efficiency were found between blastocysts derived from embryos treated at 24 and 29 h from the start of IVF. Only blastocysts from embryos exposed to lipofection treatment at 29 h after IVF contained biallelic mutant. Furthermore, there were no significant differences in the mutation rates or mutation efficiency between blastocysts derived from embryos at the 2- and 4-cell stages. This suggests that lipofection-mediated gene editing can be performed in ZP-free oocytes and ZP-free embryos; however, other factors affecting the system efficiency should be further investigated.
  • Quynh Anh Le, Fuminori Tanihara, Manita Wittayarat, Zhao Namula, Yoko Sato, Qingyi Lin, Koki Takebayashi, Maki Hirata, Takeshige Otoi
    BMC research notes 14 (1) 7 - 7 2021/01 [Refereed][Not invited]
     
    OBJECTIVE: Cytoplasmic microinjection and electroporation of the CRISPR/Cas9 system into zygotes are used for generating genetically modified pigs. However, these methods create mosaic mutations in embryos. In this study, we evaluated whether the gene editing method and embryonic stage for gene editing affect the gene editing efficiency of porcine embryos. RESULTS: First, we designed five guide RNAs (gRNAs) targeting the B4GALNT2 gene and evaluated mutation efficiency by introducing each gRNA with Cas9 protein into zygotes by electroporation. Next, the optimized gRNA with Cas9 protein was introduced into 1-cell and 2-cell stage embryos by either microinjection or electroporation. The sequence of gRNA affected the bi-allelic mutation rate and mutation efficiency of blastocysts derived from electroporated embryos. Microinjection significantly decreased the cleavage rates in each embryonic stage and blastocyst formation rates in 2-cell stage embryos compared with electroporation (p < 0.05). However, the bi-allelic mutation rate and mutation efficiency of blastocysts from the 1-cell stage embryos edited using microinjection were significantly higher (p < 0.05) than those of blastocysts from the 2-cell stage embryos edited by both methods. These results indicate that the gene editing method and embryonic stage for gene editing may affect the genotype and mutation efficiency of the resulting embryos.
  • Manita Wittayarat, Maki Hirata, Zhao Namula, Yoko Sato, Nhien T Nguyen, Quynh A Le, Qingyi Lin, Koki Takebayashi, Fuminori Tanihara, Takeshige Otoi
    Animal science journal = Nihon chikusan Gakkaiho 92 (1) e13534  2021/01 [Refereed][Not invited]
     
    This study aimed to investigate the efficiency of KRAS gene editing via CRISPR/Cas9 delivery by electroporation and analyzed the effects of the non-homologous end-joining pathway inhibitor Scr7 and single-stranded oligodeoxynucleotide (ssODN) homology arm length on introducing a point mutation in KRAS. Various concentrations (0-2 µM) of Scr7 were evaluated; all concentrations of Scr7 including 0 µM resulted in the generation of blastocysts with a point mutation and the wild-type sequence or indels. No significant differences in the blastocyst formation rates of electroporated zygotes were observed among ssODN homology arm lengths, irrespective of the gRNA (gRNA1 and gRNA2). The proportion of blastocysts carrying a point mutation with or without the wild-type sequence and indels was significantly higher in the ssODN20 group (i.e., the group with a ssODN homology arm of 20 bp) than in the ssODN60 group (gRNA1: 25.7% vs. 5.4% and gRNA2: 45.5% vs. 5.9%, p < .05). In conclusion, the CRISPR/Cas9 delivery with ssODN via electroporation is feasible for the generation of point mutations in porcine embryos. Further studies are required to improve the efficiency and accuracy of the homology-directed repair.
  • Zhao Namula, Yoko Sato, Manita Wittayarat, Quynh Anh Le, Nhien Thi Nguyen, Qingyi Lin, Maki Hirata, Fuminori Tanihara, Takeshige Otoi
    Acta veterinaria Hungarica 2020/11 [Refereed]
     
    This study was conducted to determine the effects of supplementing the maturation medium with the antioxidant curcumin on the in vitro maturation (IVM), fertilisation and development of porcine oocytes. Curcumin supplementation was performed at concentrations of 0, 5, 10, 20, and 40 µM. At concentrations of 5-20 µM, curcumin had significant positive effects (P < 0.05) on maturation and fertilisation rates compared to the non-treated group. Of the groups cultured with 5-20 µM curcumin, the number of oocytes with DNA-fragmented nuclei after IVM was significantly lower than in groups matured without curcumin. Moreover, curcumin supplementation at 10 µM also gave a significantly higher rate of blastocyst formation compared with oocytes matured without curcumin. Increasing the curcumin concentration to 40 µM yielded negative effects on fertilisation and embryonic development compared with the groups treated with lower concentrations of curcumin. Supplementation with 10 µM curcumin had beneficial effects on the oocyte maturation rate and DNA fragmentation index compared to the non-treated group both in the presence and absence of hydrogen peroxide. These results indicate that curcumin supplementation at a suitable concentration (10 µM) is potentially useful for porcine oocyte culture systems, in terms of protecting oocytes from various forms of oxidative stress.
  • Maki Hirata, Manita Wittayarat, Fuminori Tanihara, Yoko Sato, Zhao Namula, Quynh Anh Le, Qingyi Lin, Koki Takebayashi, Takeshige Otoi
    In vitro cellular & developmental biology. Animal 56 (8) 614 - 621 2020/09 [Refereed]
     
    In the present study, we investigated whether electroporation could be used for one-step multiplex CRISPR/Cas9-based genome editing, targeting IL2RG and GHR in porcine embryos. First, we evaluated and selected guide RNAs (gRNAs) by analyzing blastocyst formation rates and genome editing efficiency. This was performed in embryos electroporated with one of three different gRNAs targeting IL2RG or one of two gRNAs targeting GHR. No significant differences in embryo development rates were found between control embryos and those subjected to electroporation, irrespective of the target gene. Two gRNAs targeting IL2RG (nos. 2 and 3) contributed to an increased biallelic mutation rate in porcine blastocysts compared with gRNA no. 1. There were no significant differences in the mutation rates between the two gRNAs targeting GHR. In our next experiment, the mutation efficiency and the development of embryos simultaneously electroporated with gRNAs targeting IL2RG and GHR were investigated. Similar embryo development rates were observed between embryos electroporated with two gRNAs and control embryos. When IL2RG-targeting gRNA no. 2 was used with GHR-targeting gRNAs no. 1 or no. 2, a significantly higher double biallelic mutation rate was observed than with IL2RG-targeting gRNA no. 3. In conclusion, we demonstrate the feasibility of using electroporation to transfer multiple gRNAs and Cas9 into porcine zygotes, enabling the double biallelic mutation of multiple genes with favorable embryo survival.
  • Fuminori Tanihara, Maki Hirata, Nhien Thi Nguyen, Osamu Sawamoto, Takeshi Kikuchi, Masako Doi, Takeshige Otoi
    BMC biotechnology 20 (1) 40 - 40 2020/08 [Refereed]
     
    BACKGROUND: Xenoantigens are a major source of concern with regard to the success of interspecific xenografts. GGTA1 encodes α1,3-galactosyltransferase, which is essential for the biosynthesis of galactosyl-alpha 1,3-galactose, the major xenoantigen causing hyperacute rejection. GGTA1-modified pigs, therefore, are promising donors for pig-to-human xenotransplantation. In this study, we developed a method for the introduction of the CRISPR/Cas9 system into in vitro-fertilized porcine zygotes via electroporation to generate GGTA1-modified pigs. RESULTS: We designed five guide RNAs (gRNAs) targeting distinct sites in GGTA1. After the introduction of the Cas9 protein with each gRNA via electroporation, the gene editing efficiency in blastocysts developed from zygotes was evaluated. The gRNA with the highest gene editing efficiency was used to generate GGTA1-edited pigs. Six piglets were delivered from two recipient gilts after the transfer of electroporated zygotes with the Cas9/gRNA complex. Deep sequencing analysis revealed that five out of six piglets carried a biallelic mutation in the targeted region of GGTA1, with no off-target events. Furthermore, staining with isolectin B4 confirmed deficient GGTA1 function in GGTA1 biallelic mutant piglets. CONCLUSIONS: We established GGTA1-modified pigs with high efficiency by introducing a CRISPR/Cas9 system into zygotes via electroporation. Multiple gene modifications, including knock-ins of human genes, in porcine zygotes via electroporation may further improve the application of the technique in pig-to-human xenotransplantation.
  • Maki Hirata, Manita Wittayarat, Zhao Namula, Quynh Anh Le, Qingyi Lin, Nhien Thi Nguyen, Koki Takebayashi, Yoko Sato, Fuminori Tanihara, Takeshige Otoi
    Molecular biology reports 47 (7) 5073 - 5079 2020/07 [Refereed]
     
    The CRISPR/Cas9 system now allows for unprecedented possibilities of genome editing. However, there are some limitations, including achieving efficient one-step multiple genome targeting to save costs, time, and ensure high quality. In the present study, we investigated the efficiency of one-step multiple gene modification by electroporation in porcine zygotes using pooled guide RNAs (gRNAs) targeting CMAH, GHR, GGTA1, and PDX1. We first selected the best-performing gRNA from three different designs for each gene based on the effect on embryo development and mutation efficiency. The three gRNAs showed equivalent effects on the rates of blastocyst formation in each targeted gene; however, gRNAs CMAH #2, GHR #3, GGTA1 #3, and PDX1 #3 showed the highest biallelic mutation rate, although the total mutation rate of PDX1 #3 was significantly lower than that of PDX1 #1. Therefore, CMAH #2, GHR #3, GGTA1 #3, and PDX1 #1 were used as a mixture in electroporation to further clarify whether multiple genes can be targeted simultaneously. Individual sequencing of 43 blastocysts at the target sites of each gene showed mutations in one and two target genes in twenty-four (55.8%) and nine (20.9%) blastocysts, respectively. No mutation was detected in any target gene in ten (23.3%) blastocysts and no blastocysts had a mutation in three or more target genes. These results indicate that electroporation could effectively deliver multiple gRNAs and Cas9 protein into porcine zygotes to target multiple genes in a one-step process. However, the technique requires further development to increase the success rate of multiple gene modification.
  • Quynh A. Le, Maki Hirata, Nhien T. Nguyen, Koki Takebayashi, Manita Wittayarat, Yoko Sato, Zhao Namula, Masahiro Nii, Fuminori Tanihara, Takeshige Otoi
    Animal Science Journal 91 (1) e13386  1344-3941 2020/06 [Refereed]
  • Fuminori Tanihara, Maki Hirata, Nhien Thi Nguyen, Quynh Anh Le, Takayuki Hirano, Takeshige Otoi
    Molecular reproduction and development 87 (4) 471 - 481 2020/04 [Refereed]
     
    Pancreatic duodenal homeobox 1 (PDX1) is a crucial gene for pancreas development during the fetal period. PDX1-modified pigs have the potential to be used as a model of diabetes mellitus. However, the severe health problems caused by the PDX1 mutation limit phenotypic studies of PDX1-modified pigs as diabetes models. In this study, we generated PDX1-modified pigs by the CRISPR/Cas9 system introduced into zygotes via electroporation and investigated the mosaicism, phenotypes, and inheritance of the resulting pigs. After the embryo transfer of PDX1-modified zygotes, nine mutant piglets were delivered. Two piglets were apancreatic biallelic mutants. For the other seven piglets, the ratio of mutant alleles to total alleles was 17.5-79.7%. Two mutant piglets with high mutation rates (67.7% and 79.7%) exhibited hypoplasia of the pancreas, whereas the other five piglets were healthy. One of the male mutant piglets was further analyzed. The ejaculated semen from the pig contained PDX1-mutant spermatozoa and the pig showed normal reproductive ability. In conclusion, the frequency of the PDX1 mutation is presumed to relate to pancreas formation, and PDX1 mutant founder pigs generated from zygotes introduced to the CRISPR/Cas9 system can serve as providers of nonmosaics to contribute to medical research on diabetes mellitus.
  • Takayuki Hirano, Maki Hirata, Shigeyuki Fujimoto, Nhien Thi Nguyen, Quynh Anh Le, Fuminori Tanihara, Takeshige Otoi
    In vitro cellular & developmental biology. Animal 56 (4) 277 - 280 2020/04 [Refereed]
  • Sato Y, Kuriwaki R, Hagino S, Shimazaki M, Sambuu R, Hirata M, Tanihara F, Takagi M, Taniguchi M, Otoi T
    Reproduction in domestic animals = Zuchthygiene 0936-6768 2019/12 [Refereed][Not invited]
  • Tanihara F, Hirata M, Morikawa S, Nguyen NT, LE QA, Hirano T, Fukumi Y, Abe T, Otoi T
    The Journal of reproduction and development 65 (5) 475 - 479 0916-8818 2019/10 [Refereed][Not invited]
  • Namula Z, Wittayarat M, Hirata M, Hirano T, Nguyen NT, Le QA, Fahrudin M, Tanihara F, Otoi T
    In vitro cellular & developmental biology. Animal 55 (8) 598 - 603 1071-2690 2019/09 [Refereed][Not invited]
  • Tanihara F, Hirata M, Nguyen NT, Le QA, Wittayarat M, Fahrudin M, Hirano T, Otoi T
    Animal biotechnology 1 - 8 1049-5398 2019/09 [Refereed][Not invited]
  • Hirata M, Wittayarat M, Hirano T, Nguyen NT, Le QA, Namula Z, Fahrudin M, Tanihara F, Otoi T
    Animals : an open access journal from MDPI 9 (9) 2019/08 [Refereed][Not invited]
  • Tanihara F, Hirata M, Iizuka S, Sairiki S, Nii M, Nguyen NT, Le QA, Hirano T, Otoi T
    Animal science journal = Nihon chikusan Gakkaiho 90 (6) 712 - 718 1344-3941 2019/06 [Refereed][Not invited]
  • Thi Nguyen N, Hirata M, Tanihara F, Hirano T, Le QA, Nii M, Otoi T
    Reproduction in domestic animals = Zuchthygiene 54 (5) 750 - 755 0936-6768 2019/05 [Refereed][Not invited]
  • Hirata M, Tanihara F, Wittayarat M, Hirano T, Nguyen NT, Le QA, Namula Z, Nii M, Otoi T
    In vitro cellular & developmental biology. Animal 55 (4) 237 - 242 1071-2690 2019/04 [Refereed][Not invited]
  • Namula Z, Tanihara F, Wittayarat M, Hirata M, Nguyen NT, Hirano T, Le QA, Nii M, Otoi T
    Acta veterinaria Hungarica 67 (1) 106 - 114 0236-6290 2019/03 [Refereed][Not invited]
  • Tanihara F, Hirata M, Nguyen NT, LE QA, Hirano T, Otoi T
    The Journal of reproduction and development 0916-8818 2019/02 [Refereed][Not invited]
  • Hirata M, Tanihara F, Taniguchi M, Takagi M, Terazono T, Otoi T
    Veterinary medicine and science 2018/10 [Refereed][Not invited]
  • Tanihara F, Hirata M, Nguyen NT, Le QA, Hirano T, Takemoto T, Nakai M, Fuchimoto DI, Otoi T
    Animal science journal = Nihon chikusan Gakkaiho 90 (1) 55 - 61 1344-3941 2018/10 [Refereed][Not invited]
  • Nguyen TV, Wittayarat M, Do LTK, Nguyen TV, Nii M, Namula Z, Kunihara T, Tanihara F, Hirata M, Otoi T
    Animal science journal = Nihon chikusan Gakkaiho 89 (8) 1207 - 1213 1344-3941 2018/08 [Refereed][Not invited]
  • Namula Z, Hirata M, Wittayarat M, Tanihara F, Thi Nguyen N, Hirano T, Nii M, Otoi T
    Reproduction in domestic animals = Zuchthygiene 0936-6768 2018/07 [Refereed][Not invited]
  • Tanihara F, Hirata M, Nhien NT, Hirano T, Kunihara T, Otoi T
    The Journal of veterinary medical science 80 (6) 1007 - 1011 0916-7250 2018/06 [Refereed][Not invited]
  • K. Nishio, F. Tanihara, T. V. Nguyen, T. Kunihara, M. Nii, M. Hirata, T. Takemoto, T. Otoi
    Reproduction in Domestic Animals 53 (2) 313 - 318 1439-0531 2018/04 [Refereed][Not invited]
     
    This study was conducted to determine suitable conditions for an experimental method in which the CRISPR/Cas9 system is introduced into in vitro-produced porcine zygotes by electroporation. In the first experiment, when putative zygotes derived from in vitro fertilization (IVF) were electroporated by either unipolar or bipolar pulses, keeping the voltage, pulse duration and pulse number fixed at 30 V/mm, 1 msec and five repeats, respectively, the rate of blastocyst formation from zygotes electroporated by bipolar pulses decreased compared to zygotes electroporated by unipolar pulses. In the second experiment, the putative zygotes were electroporated by electroporation voltages ranging from 20 V/mm–40 V/mm with five 1-msec unipolar pulses. The rate of cleavage and blastocyst formation of zygotes electroporated at 40 V/mm was significantly lower (p < .05) than that of zygotes electroporated at less than 30 V/mm. Moreover, the apoptotic nuclei indices of blastocysts derived from zygotes electroporated by voltages greater than 30 V/mm significantly increased compared with those from zygotes electroporated by voltages less than 25 V/mm (p < .05). When zygotes were electroporated with Cas9 mRNA and single-guide RNA (sgRNA) targeting site in the FGF10 exon 3, the proportions of blastocysts with targeted genomic sequences were 7.7% (2/26) and 3.6% (1/28) in the embryos derived from zygotes electroporated at 25 V/mm and 30 V/mm, respectively. Our results indicate that electroporation at 25 V/mm may be an acceptable condition for introducing Cas9 mRNA and sgRNA into pig IVF zygotes under which the viability of the embryos is not significantly affected.
  • Thanh Van Nguyen, Fuminori Tanihara, Maki Hirata, Takayuki Hirano, Katsutoshi Nishio, Lanh Thi Kim Do, Lanh Thi Kim Do, Thanh Van Nguyen, Masahiro Nii, Takeshige Otoi
    Cryo-Letters 39 131 - 136 0143-2044 2018/03 [Refereed][Not invited]
     
    © CryoLetters. BACKGROUND: Short-term storage is valuable method to reuse manipulated embryos. OBJECTIVE: The present study evaluated the effects of antifreeze protein (AFP) supplementation on the quality and development of in vitro-produced porcine morulae after short-term storage (24 h). MATERIALS AND METHODS: The morulae were stored with various concentrations of AFP type III for 24 h at 5, 15 and 25 C. RESULTS: Supplementation of AFP type III (1.0 µg/ml) improved the developmental competence of embryos stored at 25°C. The proportions of DNA-fragmented nuclei in the blastocysts did not differ between the embryos stored at 25°C and the control embryos without storage treatment. However, the developmental competence of embryos stored at hypothermic temperatures decreased relative to that of the control embryos. CONCLUSION: Supplementation of AFP type III (1.0 µg/ml) maintained the quality of embryos stored at 25°C, but did not have beneficial effects on the development of embryos stored at hypothermic temperatures.
  • Ono T, Takagi M, Kawashima C, Wijayagunawardane MPB, Vos PLAM, Taniguchi M, Tanihara F, Otoi T
    Frontiers in veterinary science 5 130  2018 [Refereed][Not invited]
  • Tanihara F, Hirata M, Nguyen NT, Le QA, Hirano T, Takemoto T, Nakai M, Fuchimoto DI, Otoi T
    PloS one 13 (10) e0206360  2018 [Refereed][Not invited]
  • T-V Nguyen, F. Tanihara, L. T. K. Do, Y. Sato, M. Taniguchi, M. Takagi, T. Van Nguyen, T. Otoi
    REPRODUCTION IN DOMESTIC ANIMALS 52 (6) 969 - 975 0936-6768 2017/12 [Refereed][Not invited]
     
    Chlorogenic acid (CGA) is a quinic acid conjugate of caffeic acid, and a phytochemical found in many fruits and beverages that acts as an antioxidant. The present study investigated the effects of CGA supplementation during in vitro maturation (IVM), on in vitro development of porcine oocytes, to improve the porcine in vitro production (IVP) system. Oocytes were matured either without (control) or with CGA (10, 50, 100 and 200 mu M). Subsequently, the matured oocytes were fertilized and cultured in vitro for 7 day. The rates of maturation, fertilization and blastocyst formation of oocytes matured with 50 mu M CGA were significantly (p < .05) higher than those of the control oocytes. Hydrogen peroxide (H2O2) is one of the reactive oxygen species and induces DNA damage in porcine oocytes. When oocytes were matured with 1 mM H2O2 to assess the protective effect of CGA, 50 mu M CGA supplementation improved the maturation rate and the proportion of DNA--fragmented nuclei in oocytes compared with control oocytes matured without CGA. Moreover, when oocytes were matured with either 50 mu M CGA (control) or caffeic acid (10, 50 and 100 mu M), the rates of maturation, fertilization and the blastocyst formation of oocytes matured with 50 mu M CGA were similar to those of oocytes matured with 10 and 50 mu M caffeic acid. Our results suggest that CGA has comparable effects to caffeic acid, and IVM with 50 mu M CGA is particularly beneficial to IVP of porcine embryos and protects oocytes from DNA damage induced by oxidative stress. Supplementation of CGA to the maturation medium has a potential to improve porcine IVP system.
  • Katsutoshi Nishio, Mado Yamazaki, Masayasu Taniguchi, Kazuhiko Besshi, Fumio Morita, Toshiki Kunihara, Fuminori Tanihara, Tatsuya Takemoto, Takeshige Otoi
    ACTA VETERINARIA HUNGARICA 65 (1) 115 - 123 0236-6290 2017/03 [Refereed][Not invited]
     
    The present study was conducted to clarify whether the meiotic stage of porcine oocytes has the highest sensitivity to hyperthermia during in vitro maturation by evaluating meiotic competence and DNA damage. Oocytes were exposed to 41 degrees C for 12 h at various intervals during 48 h of maturation culture. When the oocytes were exposed to 41 degrees C from 12 to 24 h of the maturation culture, the proportion of oocytes reaching metaphase II (MII) decreased as compared to the control oocytes cultured at 38.5 degrees C (P < 0.05). Moreover, the proportions of DNA fragmentation in all oocytes exposed to 41 degrees C in each culture period after 12 h from the start of maturation culture were significantly higher (P < 0.05) than for the control oocytes. When the meiotic stage of oocytes cultured at 38.5 degrees C between 12 and 24 h was examined, the majority of oocytes remained at the germinal vesicle (GV) stage at 12 h and approximately half of the oocytes reached metaphase I (MI) at 24 h. These results indicate that the meiotic stage of porcine oocytes having the highest sensitivity to hyperthermia during in vitro maturation is a transition period from the GV stage to the MI stage.
  • L. T. K. Do, M. Wittayarat, T. Terazono, Y. Sato, M. Taniguchi, F. Tanihara, T. Takemoto, Y. Kazuki, K. Kazuki, M. Oshimura, T. Otoi
    REPRODUCTION IN DOMESTIC ANIMALS 51 (6) 1039 - 1043 0936-6768 2016/12 [Refereed][Not invited]
     
    The current applications for cat cloning include production of models for the study of human and animal diseases. This study was conducted to investigate the optimal fusion protocol on in vitro development of transgenic cloned cat embryos by comparing duration of electric pulse. Cat fibroblast cells containing a human artificial chromosome (HAC) vector were used as genetically modified nuclear donor cells. Couplets were fused and activated simultaneously with a single DC pulse of 3.0 kV/cm for either 30 or 60 mu s. Low rates of fusion and embryo development to the blastocyst stage were observed in the reconstructed HAC-transchromosomic embryos, when the duration of fusion was prolonged to 60 mu s. In contrast, the prolongation of electric pulse duration improved the embryo development and quality in the reconstructed control embryos without HAC vector. Our results suggested that the optimal parameters of electric pulses for fusion in cat somatic cell nuclear transfer vary among the types used for donor cells.
  • Fuminori Tanihara, Tatsuya Takemoto, Eri Kitagawa, Shengbin Rao, Lanh Thi Kim Do, Akira Onishi, Yukiko Yamashita, Chisato Kosugi, Hitomi Suzuki, Shoichiro Sembon, Shunichi Suzuki, Michiko Nakai, Masakazu Hashimoto, Akihiro Yasue, Munehide Matsuhisa, Sumihare Noji, Tatsuya Fujimura, Dai-ichiro Fuchimoto, Takeshige Otoi
    SCIENCE ADVANCES 2 (9) e1600803  2375-2548 2016/09 [Refereed][Not invited]
     
    Genetically modified pigs for biomedical applications have been mainly generated using the somatic cell nuclear transfer technique; however, this approach requires complex micromanipulation techniques and sometimes increases the risks of both prenatal and postnatal death by faulty epigenetic reprogramming of a donor somatic cell nucleus. As a result, the production of genetically modified pigs has not been widely applied. We provide a simple method for CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 gene editing in pigs that involves the introduction of Cas9 protein and single-guide RNA into in vitro fertilized zygotes by electroporation. The use of gene editing by electroporation of Cas9 protein (GEEP) resulted in highly efficient targeted gene disruption andwas validated by the efficient production of Myostatin mutant pigs. Because GEEP does not require the complex methods associated with micromanipulation for somatic reprogramming, it has the potential for facilitating the genetic modification of pigs.
  • Ni Wayan Kurniani Karja, Mokhamad Fahrudin, Mohamad Agus Setiadi, Ligaya I. T. A. Tumbelaka, Retno Sudarwati, Yohana Tri Hastuti, Bongot Huaso Mulia, Ardyta Widianti, Keni Sultan, Tsukasa Terazono, Zhao Namula, Masayasu Taniguchi, Fuminori Tanihara, Tatsuya Takemoto, Kazuhiro Kikuchi, Yoko Sato, Takeshige Otoi
    CRYOLETTERS 37 (4) 264 - 271 0143-2044 2016/07 [Refereed][Not invited]
     
    BACKGROUND: Cryopreservation of semen is one of the most important methods for the preservation of endangered tigers. OBJECTIVE: This study evaluated the effects of sugar supplementation on the cryosurvival of spermatozoa from Sumatran tigers (Panthera tigris sumatrae). MATERIALS AND METHODS: The post-thaw characteristics and fertility of spermatozoa cryopreserved with different sugars (glucose, lactose, and trehalose) were evaluated using heterologous in-vitro fertilisation with cat oocytes. RESULTS: All parameters of post-thaw spermatozoa significantly decreased as compared with those of fresh spermatozoa. The index of sperm motility for semen cryopreserved with lactose was significantly higher than that for semen cryopreserved with trehalose. The percentage of total fertilisation for tiger spermatozoa cryopreserved with trehalose was significantly lower than that for control cat spermatozoa. CONCLUSION: Our findings indicated that supplementation with lactose or glycerol as the main sugar in the egg yolk extender resulted in a better motility and fertility potential for post-thawed spermatozoa.
  • Yasuhiro Morita, Masayasu Taniguchi, Fuminori Tanihara, Aya Ito, Zhao Namula, Lanh Thi Kim Do, Mitsuhiro Takagi, Tatsuya Takemoto, Takeshige Otoi
    JOURNAL OF VETERINARY MEDICAL SCIENCE 78 (6) 1019 - 1023 0916-7250 2016/06 [Refereed][Not invited]
     
    The changes triggered by sperm-induced activation of oocytes, which are required for normal oocyte development, can be mediated by other agents, thereby inducing the parthenogenesis. In this study, we exposed porcine oocytes to 1 mM Ca-EDTA, a metal-ion chelator, at various intervals during 48 hr of in vitro maturation to determine the optimum period of Ca-EDTA treatment for parthenogenetic activation. When the oocytes were cultured with or without Ca-EDTA from 36 hr (post-12), 24 hr (post-24), 12 hr (post-36) and 0 hr (post-48) after the start of maturation culture, the blastocyst formation rates were significantly higher (P<0.05) in the post-24, post-36 and post-48 groups (3.3%, 4.0% and 2.6%, respectively) than those in the control group without treatment (0%). Furthermore, when the oocytes were cultured with Ca-EDTA for 0 hr (control), 12 hr (pre-12), 24 hr (pre-24), 36 hr (pre-36) and 48 hr (pre-48) from the start of maturation culture, the oocytes formed blastocysts only in the pre-36 and pre-48 groups (0.4% or 0.8%, respectively). Pronuclei (<66.7%) were observed only when the periods of Ca-EDTA treatment were more than 12 hr during maturation culture. In the control group, no pronuclei were detected. Our findings demonstrate that porcine immature oocytes can be parthenogenetically activated by Ca-EDTA treatment for at least 24 hr to 36 hr during maturation culture, leading to pronucleus formation followed by the formation of blastocysts.
  • M. Nakai, J. Ito, N. Kashiwazaki, N. T. Men, F. Tanihara, J. Noguchi, H. Kaneko, A. Onishi, K. Kikuchi
    THERIOGENOLOGY 85 (4) 703 - 708 0093-691X 2016/03 [Refereed][Not invited]
     
    To assist the process of oocyte activation, which is essential for promotion of fertilization events, i.e., resumption of meiosis, extrusion of the second polar body and formation of the pronucleus (PN), artificial stimuli such as an electrical pulse have been applied to porcine oocytes after injection of sperm. However, the efficiency of fertilization and embryonic development remains low. It is well known that in vertebrates, inactivation of mitogen-activated protein (MAP) kinase is required for oocyte activation. We have hypothesized that even after electrical stimulation of sperm-injected oocytes, MAP kinase may not be inactivated. As it has been reported that MAP kinase activity is regulated by protein kinase C, we examined the effectiveness of phorbol 12-myristate 13-acetate (PMA), a protein kinase C activator, for improvement of fertilization and embryonic development of sperm injected porcine oocytes. First, we examined the concentrations (0, 0.01, 0.1, 1, and 10 mu M) and durations (0, 1, 3, 5 hours) of PMA treatment that were efficient for the extrusion of two polar bodies and formation of two PNs (2PB+2PN) and embryonic development. When the sperm-injected oocytes were treated with 0.01-mu M PMA for 3 hours after electrical stimulation, the rates of 2PB+2PN and embryonic development were higher than those in the other treatment groups. We then examined the effect of PMA treatment (0.01 mu M, 3 hours) on MAP kinase activity. Unexpectedly, after electrical stimulation, the activity remained low until PN formation, irrespective of whether or not the oocytes had been treated with PMA. On the other hand, transformation of the injected sperm nucleus into the male PN was accelerated after the PMA treatment. Our present results suggest that the low efficiency of fertilization and embryonic development in sperm-injected oocytes is not due to high activity of MAP kinase but due to poor transformation of the injected sperm nucleus into the male PN. Furthermore, a combination of electrical stimulation and PMA is a fairly effective artificial protocol for promoting 2PB+2PN and embryonic development in sperm-injected porcine oocytes. (C) 2016 Elsevier Inc. All rights reserved.
  • T. Ono, T. Isobe, Y. Morita, L. T. K. Do, F. Tanihara, M. Taniguchi, M. Takagi, T. Otoi
    ARCHIVES ANIMAL BREEDING 59 (1) 45 - 49 0003-9438 2016 [Refereed][Not invited]
     
    Repeat-breeder (RB) cows are a major source of economic waste due to their decreased fertility. Embryo transfer (ET) is an alternative tool to improve the fertility of RB cows. The aims of the present study were to evaluate the effects of recipient parity and the season on pregnancy rates following ET in RB Japanese Black beef cattle. Embryos were transferred nonsurgically to recipients, consisting of 155 heifers (< 2 years old) and 172 cows (< 8 years old), which were defined as RB cattle. Of the recipients that were presented for ET, 57 recipients received a fresh embryo and 270 recipients received a frozen embryo. There were no differences in the pregnancy rates between cattle that received fresh embryos or frozen embryos. The rates of recipients with pregnancy, abortion, stillbirth, and normal calving were similar between heifers and cows. In cows, the pregnancy rates were lower (P < 0.05) in summer (June to August) than in spring (March to May) and winter (December to February). In heifers, however, there were no differences in the pregnancy rates among the seasons. Our findings indicate that in RB Japanese Black beef cattle, the parity of the recipients does not have an effect on the pregnancy rates following the transfer of fresh and frozen embryos. However, heat stress may affect reproductive performance in RB Japanese Black cows.
  • Tetsushi Ono, Asako Takaoka, Yasuhiro Morita, Lanh Thi Kim Do, Fuminori Tanihara, Masayasu Taniguchi, Takeshige Otoi
    ACTA VETERINARIA HUNGARICA 63 (4) 485 - 498 0236-6290 2015/12 [Refereed][Not invited]
     
    This study evaluated the effect of dibutyryl cyclic adenosine monophosphate (dbcAMP) and human chorionic gonadotropin (hCG) on the formation of antral follicle-like structures (AFLSs) and on the meiotic status of bovine cumulus-oocyte complexes (COCs) embedded in collagen gel. Supplementation with dbcAMP increased the mean diameter of AFLSs during days 4-8 of culture compared with that of control COCs, irrespective of the concentration of dbcAMP used (0.5-2.0 mM). When the embedded COCs were cultured for 8 days with hCG, the diameters of AFLSs after 4 days of culture tended to be lower in the supplemented COCs than in the control COCs without hCG, irrespective of the concentration used (1-100 IU/mL). Supplementation with 10 IU/mL hCG increased the concentrations of anti-Mullerian hormone but not progesterone and oestradiol in the culture medium after 4 days of culture. Almost all oocytes collected from AFLSs had resumed meiosis by the end of culture, irrespective of supplementation of dbcAMP and hCG. These results indicate that although dbcAMP had a positive effect on AFLS formation and development, supplementation with hCG was detrimental. Moreover, hCG supplementation did not influence the luteinisation of granulosa cells in the AFLS for 4 days after the start of culture.
  • L. T. K. Do, Y. Shibata, M. Taniguchi, M. Nii, T. V. Nguyen, F. Tanihara, M. Takagi, T. Otoi
    REPRODUCTION IN DOMESTIC ANIMALS 50 (6) 1054 - 1058 0936-6768 2015/12 [Refereed][Not invited]
     
    Melatonin has been reported to improve the in vitro development of embryos in some species. This study was conducted to investigate the effect of melatonin supplementation during in vitro maturation (IVM) and development culture on the development and quality of porcine embryos. In the first experiment, when the in vitro fertilized embryos were cultured with different concentrations of melatonin (0, 10, 25 and 50 ng/ml) for 8 days, the blastocyst formation rate of embryos cultured with 25 ng/ml melatonin (10.7%) was significantly increased (p < 0.05) compared to the control embryos cultured without melatonin (4.2%). The proportion of DNA-fragmented nuclei in blastocysts derived from embryos cultured with 50 ng/ml melatonin was significantly lower (p < 0.05) than that of embryos cultured without melatonin (2.1% vs 7.2%). In the second experiment, when oocytes were cultured in the maturation medium supplemented with different concentrations of melatonin (0, 10, 25 and 50 ng/ml), fertilized and then cultured with 25 ng/ml melatonin for 8 days, there were no significant differences in the rates of cleavage and blastocyst formation among the groups. However, the proportions (2.7-5.4%) of DNA-fragmented nuclei in blastocysts derived from oocytes matured with melatonin were significantly decreased (p < 0.05) compared to those (8.9%) from oocytes matured without melatonin, irrespective of the concentration of melatonin. Our results suggest that supplementation of the culture media with melatonin (25 ng/ml) during IVM and development has beneficial effects on the developmental competence and quality of porcine embryos.
  • Tomohiro Isobe, Yoshihisa Ikebata, Lanh Thi Kim Do, Fuminori Tanihara, Masayasu Taniguchi, Takeshige Otoi
    ANIMAL SCIENCE JOURNAL 86 (7) 661 - 665 1344-3941 2015/07 [Refereed][Not invited]
     
    The optimization of single-embryo culture conditions is very important, particularly in the in vitro production of bovine embryos using the ovum pick-up (OPU) procedure. The purpose of this study was to examine the development of embryos derived from oocytes obtained by OPU that were cultured either individually or in groups in medium supplemented with or without sericin and to investigate the viability of the frozen-thawed embryos after a direct transfer. When two-cell-stage embryos were cultured either individually or in groups for 7 days in CR1aa medium supplemented with or without 0.5% sericin, the rates of development to blastocysts and freezable blastocysts were significantly lower for the embryos cultured individually without sericin than for the embryos cultured in groups with or without sericin. Moreover, the rate of development to freezable blastocysts of the embryos cultured individually with sericin was significantly higher than that of the embryos cultured without sericin. When the frozen-thawed embryos were transferred directly to recipients, the rates of pregnancy, abortion, stillbirth and normal calving in the recipients were similar among the groups, irrespective of the culture conditions and sericin supplementation. Our findings indicate that supplementation with sericin during embryo culture improves the quality of the embryos cultured individually but not the viability of the frozen-thawed embryos after transfer to recipients.
  • Megumi Shimazaki, Rentsenkhand Sambuu, Yoko Sato, Lanh Thi Kim Do, Fuminori Tanihara, Masayasu Taniguchi, Takeshige Otoi
    CRYOLETTERS 36 (4) 264 - 269 0143-2044 2015/07 [Refereed][Not invited]
     
    BACKGROUND: The addition of the detergent Orvus ES Paste (OEP) to semen freezing extenders has been observed to improve the post-thaw survival and longevity of spermatozoa from various species but has never been evaluated for yak spermatozoa. OBJECTIVE: This study evaluated the effects of OEP on the post-thaw motility and viability of epididymal and ejaculated yak spermatozoa. MATERIALS AND METHODS: Semen samples were frozen and thawed in semen freezing extender supplemented with 0%, 0.375%, 0.75% or 1.5% OEP. The motility and viability of frozen-thawed spermatozoa were evaluated before and after 3 h of incubation. RESULTS: The addition of 0.75% OEP to the freezing extender significantly improved the mean motility and viability values of both the epididymal and ejaculated spermatozoa immediately after thawing, but the beneficial effects on motility disappeared after 3 h of incubation. CONCLUSION: Our findings indicate that the addition of 0.75% OEP is effective for the preservation of yak spermatozoa.
  • Yasuhiro Morita, Ni Wayan Kurniani Karja, Lanh Thi Kim Do, Fuminori Tanihara, Masayasu Taniguchi, Takeshige Otoi
    ANIMAL BIOTECHNOLOGY 26 (4) 273 - 275 1049-5398 2015 [Refereed][Not invited]
     
    Fragmin/protamine microparticles (F/P MPs) approximately 0.5-1 mu M in diameter were prepared by the simple mixing of fragmin with protamine. This study investigated the effects of F/P MP-containing collagen gels as a hormone carrier on the formation of antral follicle-like structures and on the development of growing bovine oocytes. The supplementation of F/P MPs in collagen gels contributed to the beneficial effects of follicle stimulating hormone (FSH) on the formation and size of antral follicle-like structures. The F/P MPs may serve as potential hormone carriers for the growth of cultured bovine oocytes from early antral follicles.
  • Hiroyuki Kaneko, Kazuhiro Kikuchi, Fuminori Tanihara, Junko Noguchi, Michiko Nakai, Junya Ito, Naomi Kashiwazaki
    THERIOGENOLOGY 82 (2) 325 - 331 0093-691X 2014/07 [Refereed][Not invited]
     
    Xenografting of immature testicular tissue combined with cryopreservation can preserve and use genetic information of prepubertal animals. For establishment of this new approach, it is essential to clarify whether offspring derived from sperm grown in host mice harboring cryopreserved xenografts show normal reproductive development. This study examined serum profiles of gonadal hormones during sexual maturation in pigs generated by intracytoplasmic sperm injection using sperm derived from cryopreserved xenografts (CryoXeno pigs; three males and three females). We also assessed the reproductive abilities of the male CryoXeno pigs by mating them with conventionally produced (conventional) pigs, and by examining the in vitro fertilizing ability of their sperm. For female CryoXeno pigs, reproductive ability was evaluated by artificial insemination with semen from a conventional boar. During the growth of male CryoXeno pigs, the serum concentrations of inhibin and testosterone showed similar changes (P > 0.17) to those in conventional pigs (n = 4). Histologic analyses of the testes revealed no differences (P > 0.2) in the growth and differentiation of seminiferous tubules between CryoXeno and conventional pigs. Three conventional sows delivered 13.0 +/- 1.0 (mean standard error of the mean) live piglets after being mated with the three CryoXeno males. Sperm obtained from all CryoXeno pigs had the ability to penetrate oocytes, and these fertilized oocytes reached the blastocyst stage in vitro. During the growth of female CryoXeno pigs, the serum inhibin profile was similar (P > 0.17) to that observed in conventional pigs (n = 5). The first rise in serum progesterone concentration to more than 2 ng/mL was noted at 32.0 +/- 2.3 weeks of age in the CryoXeno pigs and at 32.0 +/- 3.3 weeks in the conventional pigs, suggesting that both pigs reached puberty at a similar age. After puberty, female CryoXeno pigs farrowed 8.3 +/- 1.7 (mean +/- standard error of the mean; n = 3) live piglets after artificial insemination with semen from a conventional boar. In conclusion, these findings demonstrate that both male and female CryoXeno pigs have normal reproductive abilities. (C) 2014 Elsevier Inc. All rights reserved.
  • Manita Wittayarat, Akira Fujiwara, Kaywalee Chatdarong, Mongkol Techakumphu, Yoko Sato, Fuminori Tanihara, Yasuhiro Morita, Masayasu Taniguchi, Takeshige Otoi
    ACTA VETERINARIA HUNGARICA 62 (2) 233 - 242 0236-6290 2014/06 [Refereed][Not invited]
     
    This study investigated the effect of chemical inhibitors on the cell-cycle synchronisation in cat fibroblast cells and evaluated the development of interspecies embryos reconstructed from cat donor cells and enucleated bovine oocytes. Cat fibroblast cells were treated with 15 mu g/mL roscovitine or 0.05 mu g/mL demecolcine prior to cell cycle analysis and nuclear transfer. The percentage of cat fibroblast cells arrested at the G0/G1 phase in the roscovitine group was similar to that in the control group without any treatment. The percentage of cells arrested at the G2/M phase was significantly higher in the demecolcine group than in the control group. The fusion rate of interspecies couplets was significantly greater in the roscovitine group than in the control group. Most embryos stopped the development at the 2- or 4-cell stage, and none developed into blastocysts. Chemical inhibitor-induced donor cell cycle synchronisation did not overcome developmental arrest in interspecies cloned embryos.
  • Tamas Somfai, Koji Yoshioka, Fuminori Tanihara, Hiroyuki Kaneko, Junko Noguchi, Naomi Kashiwazaki, Takashi Nagai, Kazuhiro Kikuchi
    PLOS ONE 9 (5) e97731  1932-6203 2014/05 [Refereed][Not invited]
     
    We report the successful piglet production from cryopreserved oocytes for the first time by using a simple, high capacity vitrification protocol for preservation and a defined system for in vitro embryo production. Immature cumulus-oocyte complexes (COCs) from prepubertal gilts were vitrified in microdrops and stored in liquid nitrogen. After warming, COCs were subjected to in vitro maturation (IVM), fertilization (IVF), and subsequent culture (IVC). Adjusting warmplate temperature to 42 degrees C during warming prevented temperature drops in a medium below 34.0 degrees C and significantly increased the percentage of oocyte survival and thus blastocyst yields obtained from total vitrified oocytes compared with that of warming at 38 degrees C (87.1% vs 66.9% and 4.4% vs 2.7%, respectively). Nuclear maturation and fertilization of oocytes were not affected by vitrification and warming temperature. Blastocyst development on day 7 (day 0 = IVF) of the surviving oocytes after warming at 38 degrees C and 42 degrees C was not different but lower (P<0.05) than those of non-vitrified control oocytes (4.6%, 5.2% and 17.9%, respectively). However, blastocyst cell numbers in the control and vitrified groups were similar irrespective of warming temperature. Omitting porcine follicular fluid (pFF) from IVM medium (POM) did not affect maturation, fertilization and embryo development of vitrified-warmed oocytes. Transfer of blastocysts obtained on day 5 from vitrified oocytes matured either with or without pFF into 4 recipients (2 for each group) resulted in 4 pregnancies and the delivery of a total of 18 piglets. In conclusion, optimization of warming temperature was a key factor for achieving high survival rates, and surviving oocytes could be utilized in vitro using defined media. Using these modifications, live piglets could be obtained from cryopreserved oocytes for the first time.
  • Fuminori Tanihara, Michiko Nakai, Nguyen Thi Men, Noriko Kato, Hiroyuki Kaneko, Junko Noguchi, Takeshige Otoi, Kazuhiro Kikuchi
    ANIMAL SCIENCE JOURNAL 85 (4) 395 - 404 1344-3941 2014/04 [Refereed][Not invited]
     
    The zona pellucida (ZP) is considered to play important roles in the prevention of polyspermy in mammalian oocytes. However, in pigs we have shown that the presence of the ZP accelerates sperm penetration into the ooplasm during in vitro fertilization (IVF). In the present study, we investigated the effects of the ZP on sperm binding, acrosomal status, and functional exposure of IZUMO, a critical factor involved in sperm-egg fusion, during IVF in pigs. We evaluated the numbers and acrosomal statuses of sperm binding to the ZP and oolemma, and being present in the ZP and perivitelline space (PVS) using ZP-intact and ZP-free oocytes. More sperm bound to the ZP than to the oolemma. The average number of sperm present in the PVS was 0.44-0.51 per oocyte, and all sperm had lost their acrosomes. The proportion of sperm that were immunopositive for anti-IZUMO antibody was significantly higher after they were passing or had passed through the ZP. Furthermore, addition of anti-IZUMO antibody to the fertilization medium significantly inhibited the penetration of sperm into ZP-free oocytes. These results suggest that, in pigs, the ZP induces the acrosome reaction, which is associated with the functional exposure of IZUMO, resulting in completion of fertilization.
  • Zhao Namula, Risa Kodama, Fuminori Tanihara, Yasuhiro Morita, Yoko Sato, Manita Wittayarat, Masayasu Taniguchi, Takeshige Otoi
    ACTA VETERINARIA HUNGARICA 62 (1) 106 - 116 0236-6290 2014/03 [Refereed][Not invited]
     
    This study investigated the effects of skim-milk supplementation on the quality and penetrating ability of boar semen preserved at 15 degrees C. When boar semen samples were preserved in Modified Modena extender supplemented with various concentrations (0, 7.5, 15, 30 and 50 mg/mL) of skim milk powder at 15 degrees C for 4 weeks, higher sperm motility and viability were observed in the case of 7.5 mg/mL skim-milk supplementation compared with the control group (0 mg/mL) during the preservation (P < 0.05). When in vitro matured oocytes were co-incubated with boar sperm that had been preserved in Modified Modena extender with three different concentrations (0, 7.5 or 15 mg/mL) of skim milk powder at 15 degrees C for two weeks, there were no apparent effects of skim-milk supplementation on the rates of fertilisation and development to blastocysts of oocytes after co-incubation. However, the monospermic fertilisation rate of sperm preserved with 15 mg/mL skim milk powder was higher (P < 0.05) than that of fresh non-preserved sperm, but did not differ among the preservation groups. The results indicate that the supplementation of Modified Modena extender with 7.5 mg/mL skim milk powder improves the motility and viability, but not the penetrating ability, of sperm after liquid preservation for at least two weeks.
  • Istvan Egerszegi, Tamas Somfai, Michiko Nakai, Fuminori Tanihara, Junko Noguchi, Hiroyuki Kaneko, Takashi Nagai, Jozsef Ratky, Kazuhiro Kikuchi
    CRYOBIOLOGY 67 (3) 287 - 292 0011-2240 2013/12 [Refereed][Not invited]
     
    Aim of the study was to investigate the effect of vitrification on viability, cytoskeletal integrity and in vitro developmental competence after in vitro fertilization (IVF) of oocytes vitrified before or after in vitro maturation (IVM) using a pig model. Oocytes from abattoir-derived porcine ovaries were vitrified at either the germinal vesicle (GV) or metaphase II (MII) stage by modified solid surface vitrification (SSV). Oocyte viability was evaluated by stereomicroscopic observation whereas their nuclear stage and morphology of microtubules and F-actin were observed by confocal microscopy after immunostaining. Fertilization was assessed by orcein staining. The survival rate after vitrification was higher for MII-stage than for GV-stage oocytes. However, the ability of surviving oocytes to reach the MII stage after vitrification at the GV stage (GV-vitrified oocytes) was similar to that of control oocytes. Furthermore, after IVM, GV-vitrified oocytes had better spindle and F-actin integrity than oocytes vitrified at the NI stage (MII-vitrified oocytes). In accordance with this result, GV-vitrified oocytes had better ability to extrude the second polar body and support male pronucleus formation after in vitro fertilization (IVF), in comparison to MII-vitrified oocytes. Fertilization rates did not differ among groups. Finally, the ability of GV-vitrified oocytes to develop into embryos was superior to that of MII-vitrified oocytes. However, both vitrified groups showed reduced blastocyst development compared with the control group. In conclusion vitrification of porcine oocytes at the GV stage is advantageous in conferring better cytoskeletal organization and competence to develop to the blastocyst stage in comparison with vitrification at the MII stage. (C) 2013 Elsevier Inc. All rights reserved.
  • Nguyen Thi Men, Kazuhiro Kikuchi, Michiko Nakai, Atsunori Fukuda, Fuminori Tanihara, Junko Noguchi, Hiroyuki Kaneko, Nguyen Viet Linh, Bui Xuan Nguyen, Takashi Nagai, Atsushi Tajima
    THERIOGENOLOGY 80 (9) 1033 - 1044 0093-691X 2013/12 [Refereed][Not invited]
     
    Freeze-drying (FD) medium containing ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) is reported to be beneficial for maintenance of sperm DNA integrity after FD. Recently, trehalose has also been reported to have notable ability to stabilize the protein structure and biomembranes of sperm in a dry state. In this study, we examined the effect of a combination of EGTA and different concentrations of trehalose in FD medium on sperm DNA integrity and the in vitro development of IVM porcine oocytes after intracytoplasmic sperm injection (ICSI) using freeze-dried boar sperm. Ejaculated sperm from a boar were suspended in basic FD medium supplemented with 0, 3.75, 7.5, 15, 30, 60, or 90 mM trehalose and freeze-dried. After rehydration, the sperm in all groups were subjected to DNA damage detection using a Halomax kit. It was found that the level of DNA damage in 15-mM group was significantly lower than that in 0-mM group, and no difference was observed between the 15-, 7.5-, and 3.75-mM groups. Moreover, there were no significant differences in the DNA damage level among 0, 3.75 mM, and other groups treated with trehalose. When freeze-dried sperm were used for ICSI, the fertilization rates and blastocyst formation rates (observed at 10 hours and 6 days of IVC after ICSI, respectively) in the 7.5- and 15-mM groups were not different from those in 0-mM group. These results suggest that FD medium supplemented with trehalose at appropriate concentrations improves sperm DNA integrity, but does not improve fertilization and preimplantation embryo development of IVM oocytes following ICSI. (C) 2013 Elsevier Inc. All rights reserved.
  • Nguyen Viet Linh, Kazuhiro Kikuchi, Michiko Nakai, Fuminori Tanihara, Junko Noguchi, Hiroyuki Kaneko, Thanh Quang Dang-Nguyen, Nguyen Thi Men, Nguyen Van Hanh, Tamas Somfai, Bui Xuan Nguyen, Takashi Nagai, Noboru Manabe
    JOURNAL OF REPRODUCTION AND DEVELOPMENT 59 (6) 549 - 556 0916-8818 2013/12 [Refereed][Not invited]
     
    Mitochondria are reported to be critical in in vitro maturation of oocytes and subsequent embryo development after fertilization, but their contribution for fertilization has not been investigated in detail. In the present study, we investigate the contribution of mitochondria to fertilization using reconstructed porcine oocytes by fusion of ooplasmic fragments produced by serial centrifugations (centri-fusion). Firstly, we evaluated the characteristics of ooplasmic fragments. Three types of fragments were obtained by centrifugation of porcine oocytes matured in vitro for 46 h: brownish (B), transparent (T) and large (L) fragments containing both B and T parts in a fragment. The production efficiencies of these types of fragments were 71.7, 91.0 and 17.8 fragments/100 oocytes, respectively. In experiments, L fragments were excluded because they contained both brownish and transparent components that were apparently intermediate between B and T fragments. Observations by confocal microscopy after staining with MitoTracker Red CMXRos (R) and transmission electron microscopy revealed highly condensed active mitochondria in B fragments in contrast to T fragments that contained only sparse organelles. We reconstructed oocytes by fusion of a karyoplast and two cytoplasts from B and T fragments (B and T oocytes, respectively). The B oocytes showed higher sperm penetration (95.8%) and male pronuclear formation rates (94.2%) by in vitro fertilization than T oocytes (66.7% and 50.0%, respectively). These results suggest that the active mitochondria in oocytes may be related to their ability for fertilization.
  • Fuminori Tanihara, Yukine Kaedei, Zhao Namula, Vien Viet Luu, Yoko Sato, Manita Wittayarat, Masayasu Taniguchi, Takeshige Otoi
    ACTA VETERINARIA HUNGARICA 61 (4) 491 - 494 0236-6290 2013/12 [Refereed][Not invited]
     
    Research comparing the activation sensitivity of oocytes to chemical treatment among mammalian species remains limited. We compared the activation ability of oocytes from bovine and feline ovaries when treated with ethanol alone, with ethanol and cycloheximide, and without any chemical treatment; the oocytes were then cultured for 72 h. After in vitro maturation (IVM), 5% of feline oocytes were activated and 1% were cleaved, whereas there were no prematurely activated bovine oocytes. Activation rates with ethanol and ethanol/cycloheximide were significantly higher (P < 0.01) in bovine oocytes than in feline oocytes (74.2% vs. 34.1% and 86.3% vs. 52.5%, respectively). Thus, our findings indicate that feline oocytes can be prematurely activated by the end of IVM, and that bovine oocytes may have a higher sensitivity of parthenogenetic activation to chemical treatment than do feline oocytes.
  • Hiroyuki Kaneko, Michiko Nakai, Fuminori Tanihara, Junko Noguchi, Kazuhiro Kikuchi
    THERIOGENOLOGY 80 (8) 887 - 892 0093-691X 2013/11 [Refereed][Not invited]
     
    Primordial oocytes are a potential resource for medical and zoological application, but those of large animals have not yet been reported to show efficient embryonic development. In the present study, we established a pig model for production of blastocysts from primordial oocytes that had been grafted into nude mice and matured in vitro, in combination with fusion of cytoplasmic fragments. Neonatal porcine ovaries in which most follicles are at the primordial stage were minced and grafted into nude mice (Crlj:CD1-Foxn1(nu)). About 60 days after detection of vaginal opening, the mice were given 62.5 U/mL porcine FSH for 2 weeks by infusion to enhance follicular development. Developmentally competent oocytes collected from porcine ovaries (conventional oocytes) were matured in vitro and subjected to serial centrifugation to prepare cytoplasmic fragments without a metaphase plate (cytoplasts). Three cytoplasts were fused by electro-stimulation to an oocyte retrieved from a host mouse (xenogeneic oocyte) and matured in vitro. Then these fused oocytes were fertilized and subsequently cultured in vitro. No blastocysts were generated from xenogeneic oocytes without fusion of cytoplasm. When xenogeneic oocytes had been fused with three cytoplasts, the blastocyst rate increased significantly to 143%, comparable to that for untreated conventional oocytes (20.0%). The numbers of cells in blastocysts for these fused oocytes (37.2 cells/blastocyst) were not significantly different from those for conventional oocytes (25.4 cells/blastocyst). Our findings show that it is possible to use primordial oocytes of large mammals in combination with xenografting of ovarian tissue and also ooplasmic fusion. (C) 2013 Elsevier Inc. All rights reserved.
  • Zhao Namula, Yoko Sato, Risa Kodama, Kouta Morinaga, Vien Viet Luu, Masayasu Taniguchi, Michiko Nakai, Fuminori Tanihara, Kazuhiro Kikuchi, Takashi Nagai, Takeshige Otoi
    ANIMAL SCIENCE JOURNAL 84 (8) 600 - 606 1344-3941 2013/08 [Refereed][Not invited]
     
    This study investigated the effects of skim milk on the quality and fertility of boar spermatozoa under long-term chilled preservation. Semen samples were stored in Modena solution supplemented with 0 (control) to 50 mg/mL skim milk at 5 degrees C for 4 weeks; spermatozoa stored with 7.5 and 15 mg/mL of skim milk (7.5-SM and 15-SM groups, respectively) exhibited significantly higher motility indices than those of the control group up to 3 weeks (P < 0.05), and the 7.5-SM group showed improved motility indices even after 4 weeks (P < 0.05). In vitro fertilization using spermatozoa in the 7.5-SM and 15-SM groups stored at 5 degrees C for 2 weeks showed significantly higher fertilization rates of spermatozoa and the development rates to blastocyst than the control group (P < 0.05), and the 7.5-SM group showed similar rates of fertilization and blastocyst formation in the fresh non-stored spermatozoa group. After artificial insemination using spermatozoa stored for 2 weeks in the 7.5-SM group, healthy piglets were obtained. Boar spermatozoa can be stored at 5 degrees C in a Modena solution containing skim milk. Supplementation of 7.5 mg/mL skim milk improves boar spermatozoa motility and fertility even after liquid preservation at 5 degrees C for 2 weeks.
  • Fuminori Tanihara, Michiko Nakai, Hiroyuki Kaneko, Junko Noguchi, Takeshige Otoi, Kazuhiro Kikuchi
    JOURNAL OF REPRODUCTION AND DEVELOPMENT 59 (4) 385 - 392 0916-8818 2013/08 [Refereed][Not invited]
     
    In porcine oocytes, the function of the zona pellucida (ZP) with regard to sperm penetration or prevention of polyspermy is not well understood. In the present study, we investigated the effects of the ZP on sperm penetration during in vitro fertilization (IVF). We collected in vitro-matured oocytes with a first polar body (ZP+ oocytes). Some of them were freed from the ZP (ZP- oocytes) by two treatments (pronase and mechanical pipetting), and the effects of these treatments on sperm penetration parameters (sperm penetration rate and numbers of penetrated sperm per oocyte) were evaluated. There was no evident difference in the parameters between the two groups. Secondly, we compared the sperm penetration parameters of ZP+ and ZP- oocytes using frozen-thawed epididymal spermatozoa from four boars. Sperm penetration into ZP+ oocytes was found to be accelerated relative to ZP- oocytes. Thirdly, we evaluated the sperm penetration of ZP+ and ZP- oocytes at 1-10 h after IVF (3 h gamete co-incubation). The proportions of oocytes penetrated by sperm increased significantly with time in both groups; however, the number of penetrated sperm per oocyte did not increase in ZP- oocytes. Finally, we performed IVF using ZP- oocytes divided into control (3 h) and prolonged gamete co-incubation (5 h) groups. Greater numbers of sperm penetrated in the 5 h group than in the control group. These results suggest that the ZP and oolemma are not competent factors for prevention of polyspermy in our present porcine IVF system. However, it appears that ZP removal is one of the possibilities for reducing polyspermic penetration in vitro in pigs.
  • Hiroyuki Kaneko, Kazuhiro Kikuchi, Michiko Nakai, Tamas Somfai, Junko Noguchi, Fuminori Tanihara, Junya Ito, Naomi Kashiwazaki
    PLOS ONE 8 (7) e70989  1932-6203 2013/07 [Refereed][Not invited]
     
    Cryopreservation of immature testicular tissues is essential for increasing the possibilities of offspring generation by testicular xenografting for agricultural or medical purposes. However, successful production of offspring from the sperm involved has never been reported previously. In the present study, therefore, using intracytoplasmic sperm injection (ICSI), we examined whether xenogeneic sperm obtained from immature pig testicular tissue after cryopreservation would have the capacity to produce live piglets. Testicular fragments from 9- to 11-day-old piglets were vitrified after 10- or 20-min immersion in vitrification solution containing ethylene glycol (EG), polyvinyl pyrrolidone (PVP) and trehalose as cryoprotectants, and then stored in liquid nitrogen for more than 140 days. Thirty nude mice were assigned to each immersion-time group. Testicular fragments were transplanted under the back skin of castrated mice immediately after warming and removal of the cryoprotectants. Blood and testicular grafts were then recovered from the recipient mice on days 60, 120, 180 and 230-350 (day 0 = grafting). Histological assessment of the testicular grafts and analyses of inhibin and testosterone production revealed no significant differences between the two immersion-time groups, indicating equal growth activity of the cryopreserved tissues. A single sperm obtained from a mouse in each group on day 230-350 was injected into an in vitro-matured porcine oocyte, and then the ICSI oocytes were transferred to the oviducts of estrus-synchronized recipient gilts. One out of 4 gilts that had received oocytes fertilized using sperm from the 10-min immersion group delivered 2 live piglets, and one of another 4 gilts from the 20-min group delivered 4 live piglets. Thus, we have successfully generated porcine offspring utilizing sperm from immature testicular tissues after cryopreservation and transplantation into nude mice. The present model using pigs will be applicable to many large animals, since pigs are phylogenetically distant from the murine recipients.
  • Vien Viet Luu, Keisuke Hanatate, Fuminori Tanihara, Yoko Sato, Lanh Thi Kim Do, Masayasu Taniguchi, Takeshige Otoi
    REPRODUCTIVE BIOLOGY 13 (2) 122 - 126 1642-431X 2013/06 [Refereed][Not invited]
     
    Relaxin is a member of the insulin-like family of hormones that promotes growth in a number of reproductive tissues, including the granulosa and theca cells. Cat oocytes collected from cold-stored ovaries remain capable of maturing in vitro, but the developmental ability of the oocytes decreases after 24 h of cold storage. To improve the developmental ability of cat oocytes from cold-stored ovaries, we investigated the effect of relaxin supplementation of maturation medium on their meiotic ability and subsequent development. Cat oocytes were collected from ovaries stored at 4 degrees C for one day and cultured in maturation medium supplemented with different concentrations (0, 10, 20, and 40 ng/ml) of relaxin for 24 h. They were then fertilized in vitro for 12 h with frozen-thawed spermatozoa. After in vitro fertilization, the zygotes were cultured in synthetic oviduct fluid medium for 8 days. There were no significant differences in the maturation rates and glutathione contents of oocytes among the groups, irrespective of relaxin supplementation. The rate of blastocyst formation from oocytes matured with 10 ng/ml relaxin (16.0%) was higher (p<0.05) than that from oocytes matured without relaxin (5.9%). Our findings indicate that supplementation of 10 ng/ml rela)tin into maturation medium may improve blastocyst formation of cat oocytes after in vitro fertilization. (C) 2013 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.
  • Tamás Somfai, Michiko Nakai, Fuminori Tanihara, Junko Noguchi, Hiroyuki Kaneko, Naomi Kashiwazaki, István Egerszegi, Takashi Nagai, Kazuhiro Kikuchi
    Journal of Reproduction and Development 59 (4) 378 - 384 0916-8818 2013 [Refereed][Not invited]
     
    Our aim was to optimize a cryoprotectant treatment for vitrification of immature porcine cumulus-oocyte complexes (COCs). Immature COCs were vitrified either in 35% ethylene glycol (EG), 35% propylene glycol (PG) or a combination of 17.5% EG and 17.5% PG. After warming, the COCs were in vitro matured (IVM), and surviving oocytes were in vitro fertilized (IVF) and cultured. The mean survival rate of vitrified oocytes in 35% PG (73.9%) was higher (P< 0.05) than that in 35% EG (27.8%). Oocyte maturation rates did not differ among vitrified and non-vitrified control groups. Blastocyst formation in the vitrified EG group (10.8%) was higher (P< 0.05) than that in the vitrified PG group (2.0%) but was lower than that in the control group (25.0%). Treatment of oocytes with 35% of each cryoprotectant without vitrification revealed a higher toxicity of PG on subsequent blastocyst development compared with EG. The combination of EG and PG resulted in 42.6% survival after vitrification. The maturation and fertilization rates of the surviving oocytes were similar in the vitrified, control and toxicity control (TC treated with EG+PG combination without cooling) groups. Blastocyst development in the vitrified group was lower (P< 0.05) than that in the control and TC groups, which in turn had similar development rates (10.7%, 18.1% and 23.3%, respectively). In conclusion, 35% PG enabled a higher oocyte survival rate after vitrification compared with 35% EG. However, PG was greatly toxic to oocytes. The combination of 17.5% EG and 17.5% PG yielded reasonable survival rates without toxic effects on embryo development. © 2013 by the Society for Reproduction and Development.
  • Y. Kaedei, M. Naito, H. Naoi, Y. Sato, M. Taniguchi, F. Tanihara, K. Kikuchi, T. Nagai, T. Otoi
    REPRODUCTION IN DOMESTIC ANIMALS 47 (6) 880 - 886 0936-6768 2012/12 [Refereed][Not invited]
     
    Epigallocatechin gallate (EGCG) is the major polyphenol in green tea (Camellia sinensis) and is known for its antioxidant effects. The objective of the present study was to examine the effects of EGCG during in vitro fertilization (IVF) on the sperm quality and penetrability into oocytes. In the first experiment, the effects of concentration and incubation period of EGCG on the motility and penetrability of spermatozoa were examined. When frozenthawed spermatozoa were incubated in IVF medium supplemented with 0 (control), 1, 50 and 100 mu M EGCG for 1, 3 and 5 h, supplementation with 50 and 100 mu M EGCG improved motility of the spermatozoa (p < 0.05), but not viability, as compared with the control group. When frozenthawed spermatozoa were co-incubated with in vitro-matured (IVM) oocytes in IVF medium supplemented with 50 and 100 mu M EGCG for 5 h, supplementation of EGCG had positive effects on sperm penetration rates. In the second experiment, the effects of supplementation of EGCG in IVF medium on penetrability of sperm from different boars and development of fertilized oocytes were evaluated. When frozenthawed spermatozoa from six boars were co-incubated with IVM oocytes in IVF medium supplemented with 50 mu M EGCG, the effect of EGCG on sperm penetration and development of oocytes after fertilization was found to vary with individual boar. Our results indicate that motility and penetrability of boar spermatozoa are improved by co-incubation with 50 mu M EGCG, but the effects vary with individual boars.
  • Hiroyuki Kaneko, Kazuhiro Kikuchi, Michiko Nakai, Fuminori Tanihara, Junko Noguchi, Michiko Noguchi, Junya Ito, Naomi Kashiwazaki
    THERIOGENOLOGY 78 (4) 898 - 906 0093-691X 2012/09 [Refereed][Not invited]
     
    For establishment of gonadal xenografting, it is essential to clarify whether offspring derived from gametes grown in host mice harboring xenografts have normal reproductive development. This study examined the secretory profiles of gonadal hormones in relation to sexual maturation or ovarian cyclicity in pigs generated by intracytoplasmic sperm injection using xenogeneic sperm (Xeno-ICSI pigs, four males and one female). We also assessed the developmental activity of gametes obtained from these pigs using in vitro culture systems, or by mating with conventionally produced (conventional) pigs. During the growth of male Xeno-ICSI pigs, serum inhibin and testosterone concentrations were generally within ranges for those hormones in conventional pigs. Histologically, there were no differences in the growth and differentiation of seminiferous tubules between Xeno-ICSI and conventional pigs. Parameters of semen quality, including volume, pH, sperm concentration, and the percentage of motile sperm were not different from those in conventional pigs. Among the Xeno-ICSI pigs, individual differences were noted in the ability of sperm to penetrate oocytes and to produce blastocysts. However, oocytes after in vitro fertilization using these sperm developed into blastocysts containing more than 31 cells. One conventional sow delivered 12 piglets after being mated with a male Xeno-ICSI pig. During growth of the female Xeno-ICSI pig, serum progesterone concentrations had a sudden increase at 41 wk of age, suggesting CL formation. After puberty, this animal showed cyclic changes in the serum concentrations of progesterone and inhibin, and delivered 10 piglets after AI using fresh sperm obtained from a conventional boar. In conclusion, these findings demonstrated that both male and female Xeno-ICSI pigs had normal reproductive abilities. (C) 2012 Elsevier Inc. All rights reserved.
  • T. Terazono, Y. Kaedei, F. Tanihara, Z. Namula, V. L. Viet, M. Takagi, M. Inoue, Y. Sato, M. Taniguchi, T. Otoi
    REPRODUCTION IN DOMESTIC ANIMALS 47 (2) e16 - e21 0936-6768 2012/04 [Refereed][Not invited]
     
    This study reports about follicular development on the surface of canine ovarian tissue after autografting under the fascia of the thoracolumbar muscle and about meiotic resumption of follicle-derived oocyte after maturation culture. After ovarian excision from a bitch, each ovary of the pairs was cut approximately into half. The hemi-ovaries were transplanted into the bitch of origin at three different body sites (under the fascia of the quadriceps femoris muscle and the thoracolumbar muscle, and in the deltoid muscle in the scapular region). All grafted ovaries were recovered from the bitch at 35 days post-transplantation. A visible antral follicle was observed on the surface of the ovary grafted under the thoracolumbar fascia. Histological examination revealed viable follicles at different stages of development irrespective of graft site. Most granulosa cells in the follicles at different stages of development expressed proliferating cell nuclear antigen (PCNA). A total of three oocytes were collected from an ovary grafted under the fascia of the thoracolumbar muscle, wherein an oocyte reached metaphase I after maturation culture. This is the first report to demonstrate follicular development and meiotic resumption of oocytes recovered from autografted canine ovarian tissues.
  • T. Terazono, M. Inoue, Y. Kaedei, F. Tanihara, Z. Namula, V. L. Viet, Y. Taura, M. Takagi, T. Takuma, T. Otoi
    THERIOGENOLOGY 77 (1) 131 - 138 0093-691X 2012/01 [Refereed][Not invited]
     
    The influence of graft site on the survival of canine follicles and oocytes after autografting was investigated. Hemi-ovaries were autografted to three locations (quadriceps femoris muscle fascia, kidney capsule, and gastrosplenic ligament), and grafted ovaries were recovered (under anesthesia) 28 to 31 d after transplantation. The grafted hemi-ovaries were bisected: one-quarter ovary was used for histological assessment and another quarter for evaluation of oocyte viability. As controls, the remaining fresh hemi-ovaries were used to assess the viability of follicles and oocytes in non-transplanted ovaries. Most follicles in the histological sections of the grafts were classified as primordial or primary follicles. Antral follicles were not observed in the grafts, irrespective of the graft site. The percentages of viable follicles in the sections from control ovaries, and the ovaries grafted to the kidney capsule, the quadriceps femoris muscle fascia, and the gastrosplenic ligament were 17.4, 22.9, 18.3, and 32.4%, respectively. A total of 12 oocytes was recovered from the 15 hemi-ovaries grafted in five bitches, of which five (41.7%) oocytes from the ovaries grafted to the quadriceps femoris muscle fascia and the kidney capsule were cultured for assessment of meiotic competence. Three oocytes were viable but remained in the germinal vesicle stage after 72 h of maturation culture. The quadriceps femoris muscle fascia might be useful for grafting like the kidney capsule, but improvement of follicle survival and meiotic competence of oocytes in the grafts is necessary. (C) 2012 Elsevier Inc. All rights reserved.
  • Masao Murakami, Ya Juan Dong, Tatsuyuki Suzuki, Masayasu Taniguchi, Yukine Kaedei, Yoko Sato, Fuminori Tanihara, Takeshige Otoi
    CRYOBIOLOGY 63 (3) 170 - 174 0011-2240 2011/12 [Refereed][Not invited]
     
    The objectives of this study were to examine the effects of the presence or absence of serum during the in vitro culturing period of domestic cat embryos on their developmental potential into blastocysts as well as their tolerance to cryopreservation using a slow-freezing method. In vitro-fertilized cat oocytes were incubated in a modified synthetic oviduct fluid (mSOF) containing 4 mg/mL bovine serum albumin (BSA) throughout culturing (BSA group) or in mSOF containing 4 mg/mL BSA for the first 3 days followed by mSOF containing 5% fetal bovine serum (FBS group). The developmental potential of the embryos to the blastocyst and expanded blastocyst stages was evaluated 7 days after in vitro fertilization. The blastocysts were frozen-thawed by the slow-freezing method and cultured for 3 days to examine their viability in vitro. There were no differences in the formation rates of blastocysts or expanded blastocysts, or number of cells in the embryos between the two groups. After cryopreservation, the hatching rates of the expanded blastocysts in the BSA group were significantly higher (P < 0.05) than those of the FBS group. The postthaw viability of blastocysts was lower than that of expanded blastocysts irrespective of culture medium. These results indicate that the developmental potential of cat embryos cultured in serum-free medium is comparable to those cultured in serum-containing medium. Furthermore, expanded blastocysts produced without serum exhibit better postthaw viability than those produced with serum. (C) 2011 Elsevier Inc. All rights reserved.
  • Masayasu Taniguchi, Rie Arikawa, Yukine Kaedei, Fuminori Tanihara, Zhao Namula, Vien Luu Viet, Yoko Sato, Takeshige Otoi
    CRYOLETTERS 32 (5) 410 - 414 0143-2044 2011/09 [Refereed][Not invited]
     
    Chemical toxicity of cryoprotectants to in vitro developmental competence of porcine oocytes was examined. In vitro-matured oocytes were exposed to 40% ethylene glycol (EG), glycerol (GLY), or 1,2-propanediol (PD), fertilized with spermatozoa, and cultured for 8 d. Compared to treatment with other cryoprotectants, exposure to EG resulted in the development of significantly more blastocysts, but the rate was significantly lower than that of non-exposed control oocytes. In vitro-matured oocytes were also equilibrated in 40% EG by 3 multi-step methods, after which their developmental competence was evaluated. The rate of blastocyst development was higher in the 4-step method than in the 2- and 3-step methods of equilibrium. These results indicate that cryoprotectants and equilibration methods affect the developmental competence of porcine oocytes and that EG may be a superior cryoprotectant for vitrification of these cells.
  • E. V. Abakushina, Y. Morita, Y. Kaedei, F. Tanihara, Z. Namula, V. L. Viet, T. Otoi
    REPRODUCTION IN DOMESTIC ANIMALS 46 (3) 423 - 427 0936-6768 2011/06 [Refereed][Not invited]
     
    Contents Culture techniques of antral follicle-like structure (AFLS) derived from cumulus-oocyte complexes (COCs) might provide important insights into follicular development and oocyte maturation. This study was undertaken to investigate the effects of embedding bovine COCs individually (one COC) or in groups (4-5 COCs) in collagen gels on the formation of AFLS and the meiotic status of oocytes. The observations of AFLS formation were performed every second day for 14 days. The AFLS was formed at Day 2 or 4 after the start of culture (Day = 0), irrespective of the culture methods. The mean diameters of AFLS during Days 4-14 using the individual culture method were significantly higher (p < 0.05) than those using the group culture method. However, the AFLS formation rate in the individual culture method was significantly lower compared to that in the group culture method (26.1% vs 62.7%, p < 0.01). Almost all oocytes had undergone the germinal vesicle breakdown stage, irrespective of the culture method or AFLS formation. In conclusion, comparison with the individual culture method revealed that the mean diameters of AFLS in the group culture method were smaller, but more COCs formed AFLS. The group culture method might be useful for evaluating the various hypotheses of follicular formation and interfollicular communication. However, improvement of the group culture system is necessary to prevent the meiotic resumption of oocytes, because the AFLS formation is dependent on the cumulus/granulosa cells surrounding oocytes.
  • A. Fujii, Y. Kaedei, F. Tanihara, A. Ito, K. Hanatate, K. Kikuchi, T. Nagai, T. Otoi
    REPRODUCTION IN DOMESTIC ANIMALS 45 (4) 619 - 624 0936-6768 2010/08 [Refereed][Not invited]
     
    We investigated the effects of a portable incubator with a CO2 chamber on the viability and development of porcine oocytes/embryos for their transportation and examined the operational suitability of a straw or dish as a container for culturing the oocytes or embryos in the portable incubator. In the first experiment, the cumulus-oocyte complexes (COCs) were placed either in a dish or straw; and they were then cultured for 44 h in a standard CO2 incubator, in the CO2 chamber in an incubator, or in the CO2 chamber in a portable incubator. The matured oocytes were fertilized with frozen-thawed spermatozoa and then cultured in a dish in the standard CO2 incubator for 8 days. There were no differences in the proportions of oocytes reaching metaphase II stage among the groups. However, the proportions of cleavage and development to blastocysts derived from oocytes matured in a straw were lower than those from oocytes matured in a dish, irrespective of the type of incubator used. In the second experiment, the COCs were matured in a dish in the standard CO2 incubator, and the matured oocytes were fertilized and then placed either in a dish or straw. These were then cultured for 8 days in the standard CO2 incubator or portable incubator. Some zygotes cultured in the portable incubator developed to the blastocyst stage. The proportions of cleavage and development to blastocysts were significantly lower for putative zygotes cultured in straw than for those cultured in dish, irrespective of the type of incubator used. Our results indicate that a portable incubator with a CO2 chamber can maintain the viability and development of oocytes/embryos, but the straw is not a suitable system for in vitro culture of the oocytes/embryos during transportation.
  • Morteza Yavari, Hideaki Naoi, Yukine Kaedei, Fuminori Tanihara, Zhao Namula, Vien Luu Viet, Takeshige Otoi
    ITALIAN JOURNAL OF ANIMAL SCIENCE 9 (4) 386 - 389 1594-4077 2010 [Not refereed][Not invited]
     
    This study was conducted to examine the effects of (-)-epigallocatechin gallate (EGCG) supplementation on the developmental competence and quality of parthenogenetic porcine embryos during culture. Parthenogenetic embryos derived from in vitro matured oocytes were cultured for eight days in a modified North Carolina State University (NCSU)-37 solution supplemented with EGCG at different concentrations (0, 1, 5, 10 and 50 mu M). Supplementation of 1 and 5 mu M EGCG during in vitro culture of embryos showed no significant influence on the rate of cleavage or that of blastocyst formation or on the total cell number and DNA fragmentation indices of blastocysts when compared to those of a control group. However, when 10 and 50 mu M EGCG were supplemented into the culture medium, the cleavage rates were significantly lower than those of the other groups. No embryo developed to the blastocyst stage. Results suggest that treatment with low EGCG during in vitro culture has no influence on the developmental competence of porcine embryos but the presence of high concentrations of EGCG is apparently harmful for in vitro development of porcine parthenotes.
  • Yukine Kaedei, Akira Fujiwara, Fuminori Tanihara, Zhao Namula, Vien Luu Viet, Takeshige Otoi
    BULLETIN OF THE VETERINARY INSTITUTE IN PULAWY 54 (3) 405 - 408 0042-4870 2010 [Not refereed][Not invited]
     
    This study was conducted to investigate the influence of recipient cytoplasm on the development of somatic cell nuclear transfer (SCNT) embryos, using recipient oocytes and donor cells that were obtained from cats, cows, and pigs. Bovine and porcine oocytes were collected from ovaries obtained at a slaughterhouse, and cat oocytes were collected from ovaries obtained at local veterinary clinics following ovariohysterectomy. Cumulus cells from oocytes of each species were used as donors. When cat cumulus cells were transferred into cow, pig, and cat oocytes, the percentages of fusion and cleavage in the cow-cat and pig-cat interspecies groups were similar to those in the cat-cat intraspecies group. There were no significant differences in the percentages of fusion and cleavage between the interspecies (cow-cat and pig-cat groups) and intraspecies SCNT embryos (cow-cow and pig-pig groups) in each recipient oocyte species. However, none of the interspecies SCNT embryos developed to the morula and blastocyst stage. The percentages of fusion and cleavage were significantly higher (p<0.05) in cow-cat SCNT embryos than in pig-cat SCNT embryos. In conclusion, bovine and porcine cytoplasm can be used to support the early embryonic development of interspecies SCNT with cat donor nucleus. However, the interspecies SCNT embryos could not develop to the late embryonic stage.
  • Yukine Kaedei, Akira Fujiwara, Aya Ito, Fuminori Tanihara, Yasuhiro Morita, Keisuke Hanatate, Vien Luu Viet, Zhao Namula, Takeshige Otoi
    JOURNAL OF ANIMAL AND VETERINARY ADVANCES 9 (22) 2848 - 2853 1680-5593 2010 [Not refereed][Not invited]
     
    Roscovitine, a specific inhibitor of M-phase promoting factor kinase activity was used to inhibit the completion of meiotic maturation of bovine oocytes. The objectives of this study were to evaluate the nuclear maturation of bovine oocytes pre-cultured with various concentrations (0, 50, 100 and 200 mu M) of roscovitine before in vitro Maturation (IVM) and to examine the development of Somatic Cell Nuclear Transfer (SCNT) embryos derived from the oocytes pre-cultured with roscovitine. Before IVM, 72% of oocytes that were cultured without roscovitine (control) had reached the Metaphase II (MII) stage whereas culture with roscovitine decreased the rates of oocytes reaching MII (11-27%). After IVM, the maturation rate of oocytes pre-cultured with 200 mu M roscovitine was significantly higher than that of control oocytes (79 vs. 58%). Moreover, significantly more oocytes extruded the first polar body in the 50 mu M roscovitine group than in the control group (64 vs. 51%). The rate of blastocyst formation of reconstructed embryos derived from oocytes pre-cultured with 50 mu M roscovitine was significantly higher than that from the control oocytes (14 vs. 6%). In this study, the addition of roscovitine to culture medium delays the completion of meiotic maturation of bovine oocytes and the cytoplasm derived from oocytes pre-cultured under meiotic inhibition can support the development of SCNT embryos.
  • Budiyanto Agung, Farid Barati, Yukine Kaedei, Fuminori Tanihara, Takeshige Otoi
    Journal of Animal and Veterinary Advances 7 (10) 1179 - 1183 1680-5593 2008/10 [Not refereed][Not invited]
     
    The present study was conducted to investigate the effects of the exposure length of ovaries to an elevated temperature (41 degrees C) on the meiotic competence and DNA damage of oocytes. Ovaries were stored in physiological saline at 41 degrees C for 0 h (control), 0.5, 1.0 and 1.5 h. After exposure of ovaries to the elevated temperature, oocytes were collected and then cultured for 44 h. The length of exposure of ovaries to 41 degrees C had no effect on the proportions of total oocytes with DNA-fragmented nuclei before maturation culture, but it did influence the proportions at the end of maturation culture. The proportion of oocytes reaching metaphase II (MII) significantly decreased with increasing exposure time. In addition, significantly more oocytes from ovaries exposed to 41 degrees C for 1.5 h had DNA-fragmented nuclei compared with control oocytes. These results indicate that the meiotic competence and DNA damage of porcine oocytes are dependent on the duration of exposure of ovaries to the elevated temperature. Moreover, the occurrence of DNA damage in oocytes becomes more apparent after maturation culture than before the culture.

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