fuminori_tanihara/published_papers/52266324 Megumi Nagahara Takeshige Otoi Momo Nakagawa Kazuki Yoshida Shigeki Morikawa Kohei Torii Oky Setyo Widodo Mao Matsushita Yuichiro Nakayama Fuminori Tanihara Development of a Portable Device-Based Method for Predicting Follicle Number Using Vulvar Blood Flow Volume Measurement in Cows. Embryo recovery and transfer techniques efficiently assist superior cow selection for beef and milk production. In this study, we evaluated the use of vulvar blood flow volume (BFV) measurements obtained with a portable blood flow meter in predicting the follicle number. We explored the relationship between vulvar BFV and follicular hormone dynamics in cows. Eleven Japanese Black cows underwent superovulation treatment, after which vulvar BFV, serum estradiol (E2) and progesterone levels, and follicle number were measured. All measurements were repeated in the luteal phase (7 days before estrus), 1 and 2 days before estrus, and 2 and 7 days after estrus. The vulvar BFV was significantly elevated 1 day before estrus compared with that during the luteal phase. Furthermore, the vulvar BFV dynamics mirrored the follicle number and serum E2 level, both of which were significantly elevated 1 day before estrus compared with those during the luteal phase. In addition, positive correlations were observed between vulvar BFV and follicle number, between serum E2 level and follicle number, and between vulvar BFV and E2 level. Thus, vulvar BFV measurements could serve as a practical and non-invasive strategy for predicting follicle number and may provide a valuable tool for farmers. Animal science journal = Nihon chikusan Gakkaiho 97 1 e70146 null 20260000 10.1111/asj.70146 41492866 fuminori_tanihara/published_papers/52266323 Qingyi Lin Takeshige Otoi Oky Setyo Widodo Theerawat Tharasanit Kaywalee Chatdarong Zhao Namula Maki Hirata Aya Nakai Yuichiro Nakayama Megumi Nagahara Fuminori Tanihara Synergistic enhancement of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 -mediated gene editing in porcine zygotes through combined lipofection and electroporation of cationic lipid-packaged ribonucleoproteins. BACKGROUND AND AIM: Genetically engineered pigs are invaluable biomedical models for xenotransplantation and the study of human diseases. Although electroporation (EP) and lipofection are individually effective for clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) ribonucleoprotein (RNP) delivery, their combined application in porcine embryos has not been systematically evaluated. This study aimed to determine whether packaging Cas9-guided RNA complexes in cationic lipids enhances EP-mediated gene editing efficiency without compromising embryonic development. MATERIALS AND METHODS: Porcine zygotes with their zona pellucida removed were edited using RNPs targeting beta-1,4-N-acetyl-galactosaminyl transferase 2 (B4GALNT2) or growth hormone receptor (GHR) genes. Four treatment groups were tested: (1) EP with RNPs (EP), (2) EP with lipofectamine-packaged RNPs (EPL), (3) transfection with lipofectamine-packaged RNPs before EP (TL + EPL), and (4) EP followed by lipofection (EPL + TL). Blastocyst formation was evaluated morphologically, and mutation rates were assessed by Sanger sequencing followed by tracking of indels by decomposition (TIDE) analysis. RESULTS: Blastocyst formation rates were comparable across all treatments, indicating that lipofectamine packaging and EP caused no detectable cytotoxicity. For B4GALNT2, no mutations were induced by EP alone, whereas TL + EPL treatment significantly increased total and mosaic mutation rates (p < 0.05). For GHR, the total mutation and mosaic mutation rates were likewise higher in TL + EPL compared with EP, although mutation efficiency (indel percentage per edited embryo) remained unchanged. These results suggest that pre-EP lipofection promotes RNP uptake by facilitating lipid-membrane interactions that are potentiated by subsequent membrane destabilization through EP. CONCLUSION: Packaging RNPs in cationic lipids and applying sequential lipofection followed by EP significantly enhances CRISPR/Cas9-mediated mutagenesis in porcine zygotes without affecting developmental competence. This dual-delivery approach provides a simple, reproducible, and low-toxicity workflow for generating gene-edited embryos, with potential applicability to large-animal biomedical models. Veterinary world 18 12 3806 3814 20251200 10.14202/vetworld.2025.3806-3814 41716181 fuminori_tanihara/published_papers/52266326 Maki Hirata Zhao Namula Qingyi Lin Megumi Nagahara Shangquan Gan Xianghong Ju Koki Takebayashi Aya Nakai Manita Wittayarat Takeshige Otoi Fuminori Tanihara IL2RG deficient X-SCID pigs generated via electroporation-mediated gene editing using CRISPR/Cas9. Interleukin 2 receptor gamma (IL2RG) knockout (KO) is frequently used to generate X-linked severe combined immunodeficiency (X-SCID) models. Humans and pigs have anatomical and physiological similarities; hence, X-SCID pigs are being actively developed. IL2RG KO X-SCID pigs are typically achieved through somatic cell nuclear transfer using genetically modified somatic cells as nuclear donors. Therefore, we investigated the possibility of generating IL2RG KO pigs through electroporation-mediated zygotic gene editing using CRISPR/Cas9. First, we designed and selected an efficient guide RNA targeting the porcine IL2RG gene. Subsequently, we obtained one mosaic mutant male piglet carrying a frame-shift mutation (-19 bp) in IL2RG without off-target events, after the transfer of gene-edited zygotes. We also confirmed that F1 female pigs successfully inherited the -19bp mutation (XKOX). Subsequently, we obtained 30 offspring, including 10 IL2RG-KO male pigs (XKOY), from three F1 XKOX litters. The XKOY pigs showed thymic tissue hypoplasia, and flow cytometry analysis revealed absence of cytotoxic T lymphocytes, helper T, and natural killer cells. Typically, X-linked disorders predominantly affect males. Therefore, female carriers pigs are usually maintained and mated with WT males, generating animal models of the disease. In this study, we successfully generated IL2RG-modified F0 male hemizygous pigs rescued by mosaic genotype via electroporation-mediated zygotic gene editing and subsequently generated IL2RG KO pigs by mating F0 pigs with WT females. Our results demonstrated the feasibility of using zygote electroporation to generate IL2RG-deficient pigs. Furthermore, while obtaining a fertile IL2RG mosaic male pig capable of transmitting the mutation was serendipitous, it presents a potential alternative approach for generating X-linked immunodeficiency models. Journal of animal science null null 20251118 10.1093/jas/skaf350 41252434 fuminori_tanihara/published_papers/52266325 Zhao Namula Takeshige Otoi Theerawat Tharasanit Kaywalee Chatdarong Megumi Nagahara Oky Setyo Widodo Aya Nakai Suong Thi Nguyen Yuichiro Nakayama Maki Hirata Fuminori Tanihara Establishment of a Single-Oocyte Culture System for Pigs and Its Validation Using Curcumin as a Model Antioxidant for Oocyte Maturation. Since individual embryos cannot be evaluated in group culture, establishing a single culture from in vitro maturation to in vitro culture may provide new insights into oocyte and embryo quality. This study aimed to develop a single culture system for individual oocytes, from in vitro maturation through fertilization to embryo development. The effects of curcumin supplementation during in vitro maturation on oocyte maturation, embryo development, and embryo quality were examined in single and group culture systems. Porcine oocytes were cultured individually in 20 µL microdroplets, with one oocyte per droplet, or in groups of 50 oocytes per 500 µL. The maturation medium contained curcumin at concentrations of 20 µM or less. Supplementation with 10 µM curcumin increased oocyte maturation in both systems compared to the controls. The fertilization rates and oocyte/embryo quality did not differ among the treatment groups. Oocytes matured with 10 µM curcumin in a single culture showed a higher blastocyst formation rate (7.0%) than the control (2.3%). In the group culture, 10 µM curcumin increased cleavage rates compared to the control (75.2% vs. 63.0%), but blastocyst formation rates did not differ. Blastocyst formation rates were similar between single and group cultures under control (2.3% and 4.3%, respectively) or 10 µM curcumin (7.0% and 11.4%, respectively) conditions. Therefore, porcine oocytes can develop to the blastocyst stage in a single culture system. Incorporating antioxidants during in vitro maturation may be an effective condition for in vitro embryo culture that can be implemented in a single oocyte. Animals : an open access journal from MDPI 15 22 null null 20251114 10.3390/ani15223295 41302003 fuminori_tanihara/published_papers/52266327 Yuichiro Nakayama Takeshige Otoi Namula Zhao Suong Thi Nguyen Nanaka Torigoe Qyingi Lin Liu Bin Oky Setyo Widodo Theerawat Tharasanit Kaywalee Chatdarong Maki Hirata Megumi Nagahara Fuminori Tanihara Indole-3-propionic acid supplementation during in vitro maturation improves in vitro production of porcine embryos. BACKGROUND: Oxidative stress is a critical factor affecting the maturation and developmental competence of porcine oocytes during in vitro culture, reducing the efficiency of blastocyst formation. Antioxidant supplementation in the culture medium has been proposed to mitigate oxidative stress and improve developmental outcomes. This study aimed to evaluate the effects of indole-3-propionic acid (IPA), a potent antioxidant, on porcine oocyte maturation, fertilization, and subsequent blastocyst development under in vitro conditions. METHODS: Porcine cumulus-oocyte complexes (COCs) were matured for 44 h in maturation medium supplemented with IPA at various concentrations (0.5, 1, 5, and 10 µM). As a control, COCs were cultured in maturation medium containing the IPA dilution vehicle (ethanol) without IPA. After maturation culture, in vitro fertilization and embryo culture were performed to evaluate fertilization rate and developmental competence. Meiotic competence, fertilization status, blastocyst formation, and DNA integrity of oocytes and blastocysts were evaluated. To further investigate the protective effect of IPA against oxidative stress, COCs were exposed to 1 mM H2O2 in a maturation medium supplemented with or without IPA (0.5 µM) during maturation culture. After maturation, intracellular reactive oxygen species (ROS) and glutathione (GSH) levels in oocytes were assessed. RESULTS: Supplementation with 0.5 μM IPA in the in vitro maturation medium significantly enhanced the rates of oocyte maturation, fertilization, and blastocyst formation compared with the control group (p < 0.05). In addition, IPA supplementation at concentrations ranging from 0.5 to 5 µM significantly reduced DNA fragmentation in both matured oocytes and blastocysts. Furthermore, IPA supplementation effectively reduced the intracellular ROS levels elevated by H2O2 while significantly increasing intracellular GSH levels (p < 0.05). These findings indicate that IPA supplementation protects porcine oocytes from oxidative stress during in vitro maturation by reducing ROS generation and enhancing GSH levels. Consequently, IPA improves oocyte quality, fertilization potential, and developmental competence of embryos. CONCLUSIONS: This study demonstrates the potential of IPA as a beneficial supplement to in vitro maturation medium for improving the efficiency of porcine embryo production by enhancing oocyte quality and protecting against oxidative damage. BMC veterinary research 21 1 642 642 20251103 10.1186/s12917-025-05049-4 41184843 fuminori_tanihara/published_papers/51300253 Suong T Nguyen Takeshige Otoi Zhao Namula Oky Setyo Widodo Theerawat Tharasanit Kaywalee Chatdarong Yuichiro Nakayama Megumi Nagahara Aya Nakai Maki Hirata Fuminori Tanihara Mitochondrial-Targeted Protective Potential of Elamipretide for the In Vitro Production of Porcine Embryos. Mitochondrial-targeted antioxidant supplementation is promising for in vitro culture of mammalian embryos. Elamipretide (SS-31) is a synthetic tetrapeptide that binds to the inner mitochondrial membrane, contributing to the prevention of oxidative stress. In this study, the effects of SS-31 supplementation in maturation medium on the developmental competence of porcine oocytes and its protective function were evaluated. Porcine oocytes were matured in maturation medium with SS-31 at 0.001, 0.01, 0.1, 0.5, 1, 1.5, 2.5, and 5 µM, with 0 µM and DMSO-treated groups established as controls. In vitro fertilization and embryo culture were performed to analyze developmental potential. Oocytes cultured in medium with 1 µM SS-31 exhibited higher maturation and blastocyst formation rates than the control (0 µM) (78.3 ± 3.8% vs. 55.2 ± 4.1% and 7.6 ± 1.6% vs. 2.8 ± 1.8%, respectively). Oocytes treated with 1 µM SS-31 showed significantly lower reactive oxygen species, higher glutathione content, and improved mitochondrial membrane potential than those without treatment. DNA fragmentation in oocytes after in vitro maturation was significantly lower in the 1 µM SS-31 supplemented group than in the control. This study demonstrates that SS-31 exerts beneficial effects on the in vitro production of porcine embryos via the enhancement of the mitochondrial antioxidant capacity. Animals : an open access journal from MDPI 15 17 null null 20250825 10.3390/ani15172497 40941292 fuminori_tanihara/published_papers/51300254 Bin Liu Takeshige Otoi Zhao Namula Oky Setyo Widodo Maki Hirata Aya Nakai Qingyi Lin Yuichiro Nakayama Megumi Nagahara Fuminori Tanihara Oxidative stress index of porcine follicular fluid influences meiotic maturation and embryo development during in vitro culture. BACKGROUND AND AIM: Porcine follicular fluid (pFF) is frequently used to mimic the follicular microenvironment during in vitro maturation (IVM) of oocytes. However, the influence of oxidative stress levels within pFF on oocyte quality and embryo development remains unclear. This study aimed to investigate how varying oxidative stress index (OSI) of pFF affect porcine oocyte meiotic progression, fertilization, and embryonic development during IVM. MATERIALS AND METHODS: Oocytes were matured in IVM media supplemented with 30% pFF classified into low (OSI 19), medium (OSI 22), and high (OSI 25) oxidative stress groups, based on the ratio of diacron-reactive oxygen metabolites to biological antioxidant potential. Post-IVM, oocytes were assessed for meiotic stage, DNA fragmentation, reactive oxygen species (ROS), and glutathione (GSH) levels. Fertilization and embryo development outcomes were monitored following in vitro fertilization and culture. RESULTS: The OSI 19 group showed significantly higher maturation to the metaphase II stage and improved fertilization and blastocyst formation rates compared to OSI 22 and OSI 25 groups (p < 0.05). ROS and GSH levels were also significantly elevated in OSI 19 oocytes, without an increase in DNA fragmentation. Blastocysts from the OSI 25 group exhibited significantly higher DNA fragmentation index than those from the OSI 19 group (p < 0.05). CONCLUSION: The OSI of pFF modulates porcine oocyte competence and embryonic outcomes. Lower OSI is associated with enhanced antioxidant balance, meiotic maturation, and embryo quality. Monitoring pFF oxidative status may improve assisted reproductive outcomes in swine. Veterinary world 18 7 2078 2086 20250700 10.14202/vetworld.2025.2078-2086 40926853 fuminori_tanihara/published_papers/51300256 Manita Wittayarat Kimika Kawanishi Haruka Ohata Megumi Nagahara Rentsenkhand Sambuu Otgonjargal Sambuu Maki Hirata Fuminori Tanihara Masayasu Taniguchi Takeshige Otoi Yoko Sato Aberrant Expression Levels of Androgen Receptor and SRD5A2 in Epididymal Epithelial Cells of Crossbred Infertile Cattle-Yak. Although yaks and cattle belong to the same Bovinae subfamily and have the same number of chromosomes, hybrid males are sterile because of the inactivation or abnormality of gene expression related to the production of healthy normal sperm. Recently, the analysis of gene expression not only in the testis but also in the epididymis has offered hints about the mechanism of infertility, because the epididymis supports the maturation of sperm in acquiring the capacity of fertilisation. Sperm maturation processes have been thought to be androgen-dependent, and the androgen receptor (AR) can be activated by dihydrotestosterone converted from plasma testosterone by the 5α-reductase isoform 2 (SRD5A2) in epididymal cells. In the present study, we investigated the immuno-expression levels of the AR and SRD5A2 in the epithelial cells of the hybrid cattle-yak epididymal caput in comparison with yak samples using image analysis. Epididymal tissues from yaks (1-3 years of age) and hybrid cattle-yaks (2 years of age) were used in this study. In yaks, AR signal intensity did not show any changes in epididymal epithelial cells during maturation. However, in 2-year-old hybrid cattle-yaks, AR signal intensity was significantly higher in the principal cells of the epididymis compared to that of yaks of the same age, indicating that hybrid sterility is not likely related to AR deficiency in the epididymal epithelium. On the other hand, SRD5A2 signal intensity was stable during maturation in the epithelial cells of the yak epididymis. However, the epididymal SRD5A2 signal intensity in the epithelial cells of the hybrid cattle-yak was lower than that of the yak. This suggests that a deficiency in SRD5A2 production in the epididymis may result in hybrid infertility, as it can subsequently cause incomplete AR signal transduction and altered spermatozoa physiology. Animals : an open access journal from MDPI 15 5 null null 20250224 10.3390/ani15050660 40075943 fuminori_tanihara/published_papers/51300795 M. Nagahara M. Hirata Q. Lin K. Takebayashi A. Nakai T. Otoi F. Tanihara Analysis of myofiber composition in myostatin monoallelic mutant pigs Journal of Livestock Science Journal of Livestock Science 2277-6214 16 2 168 172 20250212 10.33259/jlivestsci.2024.168-172 fuminori_tanihara/published_papers/49413011 Nanaka Torigoe Qingyi Lin Bin Liu Yuichiro Nakayama Aya Nakai Megumi Nagahara Fuminori Tanihara Maki Hirata Takeshige Otoi Effects of Electroporation Timing and Cumulus Cell Attachment on In Vitro Development and Genome Editing of Porcine Embryos. Pig genome editing using the oviductal nucleic acid delivery (GONAD) method with electroporation would allow the efficient obtention of genetically modified pigs. However, oocytes and zygotes at various stages after ovulation must be targeted, and cumulus cell attachment and mosaic mutations are major obstacles. Therefore, we investigated whether two parameters (electroporation timing and the cumulus cell attachment) influence the effectiveness of multiplex genome editing by electroporation in porcine oocytes or zygotes. Three gRNAs targeting either GGTA1, CMAH or B4GALNT2 were introduced individually into oocytes and zygotes with and without cumulus cells at three different time points, 0 h before in vitro fertilisation (IVF) and 5 h and 10 h after IVF initiation. The introduction of gRNAs into oocytes and zygotes did not significantly affect the rates of blastocyst formation and total mutation of the resulting blastocysts irrespective of cumulus cell attachment and electroporation timing. In conclusion, the electroporation timing and the cumulus cell attachment did not interfere with the efficient delivery of the CRISPR/Cas9 system to the oocytes/zygotes, indicating that porcine genome editing in the oviduct using GONAD method may be possible. Reproduction in domestic animals = Zuchthygiene 60 2 e70011 null 20250200 10.1111/rda.70011 39901848 fuminori_tanihara/published_papers/51300255 Supitcha Kaewma Takeshige Otoi Oky Setyo Widodo Megumi Nagahara Aya Nakai Suong T Nguyen Yuichiro Nakayama Theerawat Tharasanit Maki Hirata Fuminori Tanihara Zinc Chloride Supplementation During In Vitro Fertilization Reduces Polyspermic Fertilization of Porcine Oocytes. As zinc is important for the fertilization competency of sperms and oocytes, we investigated the effects of zinc chloride (ZnCl2) supplementation during in vitro fertilization (IVF) on porcine oocyte fertilization and development. We evaluated the effects of ZnCl2 concentration (0, 1, 10, and 20 μg/mL) on the quality of frozen-thawed boar spermatozoa cultured for 5 h and on oocyte fertilization and embryo development after IVF. Spermatozoa from three different boars were additionally tested. ZnCl2 supplementation effects along with metal chelators, calcium ethylenediaminetetraacetic acid (Ca-EDTA) and zinc EDTA (Zn-EDTA), were also examined. ZnCl2 supplementation did not affect the quality of frozen-thawed spermatozoa after culture for 5 h. Supplementation with 1-μg/mL ZnCl2 decreased the percentage of polyspermic fertilization compared to that at 0- and 20-μg/mL ZnCl2. Moreover, it increased blastocyst formation rate compared to that in the other supplementation groups. In different boar spermatozoa, ZnCl2 supplementation (1 μg/mL) decreased polyspermic fertilization but did not improve embryo development. Co-incubation with Ca-EDTA did not reduce polyspermic fertilization, but Zn-EDTA co-incubation reduced polyspermic fertilization similar to that with ZnCl2 alone. In conclusion, 1-μg/mL ZnCl2 supplementation during IVF reduces polyspermic fertilization but may not improve embryo development. Animal science journal = Nihon chikusan Gakkaiho 96 1 e70095 null 20250000 10.1111/asj.70095 40859724 fuminori_tanihara/published_papers/49413015 Qingyi Lin Koki Takebayashi Nanaka Torigoe Bin Liu Zhao Namula Maki Hirata Fuminori Tanihara Megumi Nagahara Takeshige Otoi Genome editing of porcine zygotes via lipofection of two guide RNAs using a CRISPR/Cas9 system. CRISPR/Cas9-based multiplex genome editing via electroporation is relatively efficient; however, lipofection is versatile because of its ease of use and low cost. Here, we aimed to determine the efficiency of lipofection in CRISPR/Cas9-based multiplex genome editing using growth hormone receptor (GHR) and glycoprotein alpha-galactosyltransferase 1 (GGTA1)-targeting guide RNAs (gRNAs) in pig zygotes. Zona pellucida-free zygotes were collected 10 h after in vitro fertilization and incubated with Cas9, gRNAs, and Lipofectamine 2000 (LP2000) for 5 h. In Experiment 1, we evaluated the mutation efficiency of gRNAs targeting either GHR or GGTA1 in zygotes transfected using LP2000 and cultured in 4-well plates. In Experiment 2, we examined the effects of the culture method on the development, mutation rate, and mutation efficiency of zygotes with simultaneously double-edited GHR and GGTA1, cultured using 4-well (group culture) and 25-well plates (individual culture). In Experiment 3, we assessed the effect of additional GHR-targeted lipofection before and after simultaneous double gRNA-targeted lipofection on the mutation efficiency of edited embryos cultured in 25-well plates. No significant differences in mutation rates were observed between the zygotes edited with either gRNA. Moreover, the formation rate of blastocysts derived from GHR and GGTA1 double-edited zygotes was significantly increased in the 25-well plate culture compared to that in the 4-well plate culture. However, mutations were only observed in GGTA1 when zygotes were transfected with both gRNAs, irrespective of the culture method used. GHR mutations were detected only in blastocysts derived from zygotes subjected to GHR-targeted lipofection before simultaneous double gRNA-targeted lipofection. Overall, our results suggest that additional lipofection before simultaneous double gRNA-targeted lipofection induces additional mutations in the zygotes. The Journal of reproduction and development 70 6 356 361 20241213 10.1262/jrd.2024-054 39218670 fuminori_tanihara/published_papers/49413012 Qingyi Lin Nanaka Torigoe Bin Liu Yuichiro Nakayama Aya Nakai Zhao Namula Megumi Nagahara Fuminori Tanihara Maki Hirata Takeshige Otoi Efficient gene editing of pig embryos by combining electroporation and lipofection. BACKGROUND AND AIM: Mosaicism, which is characterized by the presence of wild-type and more than one mutant allele, poses a serious problem in zygotic gene modification through the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 system. Therefore, we used pig embryos to compare the gene editing efficiencies achieved by combining electroporation and lipofection using different aminopeptidase N (APN)-targeting guide RNA (gRNA) sequences. MATERIALS AND METHODS: Six gRNAs (gRNA1-6) with different target sequences were designed to target APN. Zona pellucida (ZP)-intact zygotes collected 10 h after the start of in vitro fertilization (IVF) were electroporated with each gRNA to compare their gene editing efficiency. The gRNA sequences that achieved the lowest and highest mutation rates (gRNA4 and gRNA6, respectively) were selected for additional lipofection to assess gene editing efficiency following combined treatment. As ZP removal is essential for lipofection, ZP-free zygotes were electroporated with gRNA4 or gRNA6 10 h after IVF initiation, followed by lipofection with the same gRNAs 24 or 29 h after IVF initiation. The electroporated ZP-intact and ZP-free zygotes were used as controls. RESULTS: gRNA4 and gRNA6 exhibited the lowest and highest mutation rates, respectively. gRNA4-targeted ZP-free embryos subjected to additional lipofection 29 h after IVF initiation exhibited significantly higher total and biallelic mutation rates than ZP-intact embryos that received only electroporation. Additional lipofection of gRNA6-targeted embryos had no obvious effect on mutation rates. CONCLUSION: Electroporation combined with lipofection using gRNAs with low mutation rates may improve gene editing efficiency in pig embryos. However, the effects may vary based on the timing of gene editing. Veterinary world 17 11 2701 2707 20241100 10.14202/vetworld.2024.2701-2707 39829671 fuminori_tanihara/published_papers/49413013 Koki Takebayashi Manita Wittayarat Maki Hirata Qingyi Lin Zhao Namula Nanaka Torigoe Bin Liu Megumi Nagahara Aya Nakai Takeshige Otoi Fuminori Tanihara Optimization of embryonic stage for aggregation to generate chimeric pigs using gene-edited blastomeres. In vitro cellular & developmental biology. Animal null null 20241024 10.1007/s11626-024-00987-z 39446279 fuminori_tanihara/published_papers/49413019 Thanh-Van Nguyen Koki Takebayashi Lanh Thi Kim Do Zhao Namula Manita Wittayarat Megumi Nagahara Maki Hirata Takeshige Otoi Fuminori Tanihara Generation of allogenic chimera carrying mutations in PDX1 and TP53 genes via phytohemagglutinin-mediated blastomere aggregation in pigs. The generation of genetically engineered pig models that develop pancreas-specific tumors has the potential to advance studies and our understanding of pancreatic cancer in humans. TP53 mutation causes organ-nonspecific cancers, and PDX1-knockout results in the loss of pancreas development. The aim of the present study was to generate a PDX1-knockout pig chimera carrying pancreas complemented by TP53 mutant cells via phytohemagglutinin (PHA)-mediated blastomere aggregation using PDX1 and TP53 mutant blastomeres, as a pig model for developing tumors in the pancreas with high frequency. First, the concentration and exposure time to PHA to achieve efficient blastomere aggregation were optimized. The results showed that using 300 µg/mL PHA for 10 min yielded the highest rates of chimeric blastocyst formation. Genotyping analysis of chimeric blastocysts derived from aggregated embryos using PDX1- and TP53-edited blastomere indicated that approximately 28.6% carried mutations in both target regions, while 14.3-21.4% carried mutations in one target. After the transfer of the chimeric blastocysts into one recipient, the recipient became pregnant with three fetuses. Deep sequencing analysis of the PDX1 and TP53 regions using ear and pancreas samples showed that one fetus carried mutations in both target genes, suggesting that the fetus was a chimera derived from embryo-aggregated PDX1 and TP53 mutant blastomeres. Two out of three fetuses carried only the PDX1 mutation, indicating that the fetuses developed from embryos not carrying TP53-edited blastomeres. The results of the present study could facilitate the further improvement and design of high-frequency developing pancreatic tumor models in pigs. In vitro cellular & developmental biology. Animal 60 7 708 715 20240800 10.1007/s11626-024-00870-x 38379097 fuminori_tanihara/published_papers/49413018 Thanh-Van Nguyen Lanh Thi Kim Do Qingyi Lin Megumi Nagahara Zhao Namula Manita Wittayarat Maki Hirata Takeshige Otoi Fuminori Tanihara Programmed cell death-1-modified pig developed using electroporation-mediated gene editing for in vitro fertilized zygotes. Programmed cell death-1 (PD-1) is an immunoinhibitory receptor required to suppress inappropriate immune responses such as autoimmunity. Immune checkpoint antibodies that augment the PD-1 pathway lead to immune-related adverse events (irAEs), organ non-specific side effects due to autoimmune activation in humans. In this study, we generated a PD-1 mutant pig using electroporation-mediated introduction of the CRISPR/Cas9 system into porcine zygotes to evaluate the PD-1 gene deficiency phenotype. We optimized the efficient guide RNAs (gRNAs) targeting PD-1 in zygotes and transferred electroporated embryos with the optimized gRNAs and Cas9 into recipient gilts. One recipient gilt became pregnant and gave birth to two piglets. Sequencing analysis revealed that both piglets were biallelic mutants. At 18 mo of age, one pig showed non-purulent arthritis of the left elbow/knee joint and oligozoospermia, presumably related to PD-1 modification. Although this study has a limitation because of the small number of cases, our phenotypic analysis of PD-1 modification in pigs will provide significant insight into human medicine and PD-1-deficient pigs can be beneficial models for studying human irAEs. In vitro cellular & developmental biology. Animal 60 7 716 724 20240800 10.1007/s11626-024-00869-4 38485817 fuminori_tanihara/published_papers/49413017 Qingyi Lin Koki Takebayashi Nanaka Torigoe Bin Liu Zhao Namula Maki Hirata Megumi Nagahara Fuminori Tanihara Takeshige Otoi Evaluation of culture methods and chemical reagent combinations on CRISPR/Cas9 gene editing systems by lipofection in pig zygotes. The delivery of CRISPR/Cas ribonucleoprotein (RNP) complexes is gaining attention owing to its high cleavage efficiency and reduced off-target effects. Although RNPs can be delivered into porcine zygotes via electroporation with relatively high efficiency, lipofection-mediated transfection appears to be versatile because of its ease of use, low cost, and adaptation to high-throughput systems. However, this system requires improvements in terms of embryo development and mutation rates. Therefore, this study elucidated the effects of culture methods and reagent combinations on the CRISPR/Cas9 gene editing systems by using three lipofection reagents: Lipofectamine™ CRISPRMAX™ Cas9 Transfection Reagent (CM), Lipofectamine™ 2000 Transfection Reagent (LP), and jetCRISPR™ RNP Transfection Reagent (Jet). Porcine zona pellucida-free zygotes were incubated for 5 h with Cas9, a guide RNA targeting CD163, and the above lipofection reagents. When examining the effect of culture methods using 4-well (multiple embryo culture) and 25-well plates (single embryo culture) on the efficiency of CM-mediated zygote transfection, the culture of embryos in 25-well plates significantly increased the blastocyst formation rate; however, there was no difference in mutation rates between the 4-well and 25-well plates. When assessing the effects of individual or combined reagents on the efficiency of zygote transfection, the mutation rate was significantly lower for individual LP compared to individual CM- and Jet-mediated transfections. Moreover, combinations of lipofection transfection reagents did not significantly increase the mutation rate or mutation efficiency. In vitro cellular & developmental biology. Animal 60 7 725 731 20240800 10.1007/s11626-024-00908-0 38664280 fuminori_tanihara/published_papers/49413016 Bin Liu Manita Wittayarat Koki Takebayashi Qingyi Lin Nanaka Torigoe Zhao Namula Maki Hirata Megumi Nagahara Fuminori Tanihara Takeshige Otoi Effects of centrifugation treatment before electroporation on gene editing in pig embryos. Genetic mosaicism, characterized by multiple genotypes within an individual, is considered an obstacle to CRISPR/Cas9 genome editing in animal models. Despite the various strategies for minimizing mosaic mutations, no definitive methods exist to eliminate them. This study aimed to enhance gene editing efficiency in porcine zygotes using CRISPR/Cas9, which targets specific genes through centrifugation and zona pellucida removal before electroporation. Centrifugation at 2000 × g did not adversely affect blastocyst formation rates in zygotes electroporated with gRNA targeting the GGTA1 gene; instead, it led to increased total and monoallelic mutation rates compared with control zygotes without centrifugation. However, the groups had no significant differences in biallelic mutation rates. In zygotes electroporated with gRNA targeting the CMAH gene, centrifugation treatments exceeding 1000 × g significantly increased both biallelic mutation rates and mutation efficiency. The combination of centrifugation and zona pellucida removal did not have a detrimental effect on blastocyst formation rates. It led to a higher rate of double biallelic mutations in embryos targeting both GGTA1 and CMAH compared to embryos without centrifugation treatment. In summary, our results demonstrate that pre-electroporation treatments, including centrifugation and zona pellucida removal, positively influenced the reduction of mosaic mutations, with the effectiveness of centrifugation depending on the specific gRNA used. In vitro cellular & developmental biology. Animal 60 7 732 739 20240800 10.1007/s11626-024-00926-y 38833208 fuminori_tanihara/published_papers/49413014 Megumi Nagahara Zhao Namula Qingyi Lin Koki Takebayashi Nanaka Torigoe Bin Liu Fuminori Tanihara Takeshige Otoi Maki Hirata Effects of ergothioneine supplementation on meiotic competence and porcine oocyte development. BACKGROUND AND AIM: The antioxidant effects of ergothioneine (EGT) on in vitro culture of porcine zygote are not well established. The study investigated the impact of EGT supplementation on meiotic competence and development of porcine oocytes. MATERIALS AND METHODS: The impact of EGT concentrations (0, 5, 10, 25, 50, and 100 μM) during in vitro maturation (IVM) on the progression of meiotic maturation, fertilization, and blastocyst formation in porcine oocytes was assessed. The DNA fragmentation level was evaluated to determine oocyte and blastocyst quality. RESULTS: The proportion of metaphase II oocytes was significantly greater (p < 0.05) in EGT-cultured oocytes than in control oocytes, regardless of the EGT concentration, and those oocytes with 10 μM or more EGT had fewer DNA-fragmented nuclei (p < 0.05). Blastocysts derived from oocytes cultured with 10 μM EGT had the highest proportion (p < 0.05), while those from control oocytes or oocytes cultured with 50 μM or less EGT had significantly higher proportions. Despite EGT supplementation, there were no noticeable differences in total cell numbers and DNA fragmentation indices in the derived blastocysts. CONCLUSION: Supplementing with EGT during IVM leads to better oocyte maturation, quality, and embryonic development due to decreased DNA fragmentation. The present study failed to elucidate the mechanism of DNA fragmentation reduction by EGT. More research needs to be conducted to explore the antioxidant mechanism of EGT. Veterinary world 17 8 1748 1752 20240800 10.14202/vetworld.2024.1748-1752 39328438 fuminori_tanihara/published_papers/42267787 Fuminori Tanihara Maki Hirata Zhao Namula Manita Wittayarat Lanh Thi Kim Do Qingyi Lin Koki Takebayashi Hiromasa Hara Megumi Nagahara Takeshige Otoi GHR-mutant pig derived from domestic pig and microminipig hybrid zygotes using CRISPR/Cas9 system. BACKGROUND: Pigs are excellent large animal models with several similarities to humans. They provide valuable insights into biomedical research that are otherwise difficult to obtain from rodent models. However, even if miniature pig strains are used, their large stature compared with other experimental animals requires a specific maintenance facility which greatly limits their usage as animal models. Deficiency of growth hormone receptor (GHR) function causes small stature phenotypes. The establishment of miniature pig strains via GHR modification will enhance their usage as animal models. Microminipig is an incredibly small miniature pig strain developed in Japan. In this study, we generated a GHR mutant pig using electroporation-mediated introduction of the CRISPR/Cas9 system into porcine zygotes derived from domestic porcine oocytes and microminipig spermatozoa. METHODS AND RESULTS: First, we optimized the efficiency of five guide RNAs (gRNAs) designed to target GHR in zygotes. Embryos that had been electroporated with the optimized gRNAs and Cas9 were then transferred into recipient gilts. After embryo transfer, 10 piglets were delivered, and one carried a biallelic mutation in the GHR target region. The GHR biallelic mutant showed a remarkable growth-retardation phenotype. Furthermore, we obtained F1 pigs derived from the mating of GHR biallelic mutant with wild-type microminipig, and GHR biallelic mutant F2 pigs through sib-mating of F1 pigs. CONCLUSIONS: We have successfully demonstrated the generation of biallelic GHR-mutant small-stature pigs. Backcrossing of GHR-deficient pig with microminipig will establish the smallest pig strain which can contribute significantly to the field of biomedical research. Molecular biology reports 50 6 5049 5057 20230600 10.1007/s11033-023-08388-3 37101010 fuminori_tanihara/published_papers/42267791 Thanh Van Nguyen Lanh Thi Kim Do Zhao Namula Qingyi Lin Nanaka Torigoe Megumi Nagahara Maki Hirata Fuminori Tanihara Takeshige Otoi DEVELOPMENT AND GENOME MUTATION OF BOVINE ZYGOTES VITRIFIED BEFORE AND AFTER GENOME EDITING VIA ELECTROPORATION Cryo-Letters 0143-2044 44 2 118 122 20230300 10.54680/fr23210110612 2-s2.0-85152276879 fuminori_tanihara/published_papers/49413020 Qingyi Lin Koki Takebayashi Nanaka Torigoe Bin Liu Zhao Namula Maki Hirata Fuminori Tanihara Megumi Nagahara Takeshige Otoi Comparison of chemically mediated CRISPR/Cas9 gene editing systems using different nonviral vectors in porcine embryos. The transfection efficiency of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas ribonucleoprotein complexes was compared using three nonviral vector transfection reagents: nonliposomal polymeric (TransIT-X2), lipid nanoparticle delivery (CRISPRMAX), and peptide (ProteoCarry) systems. Porcine zona pellucida-free zygotes and embryos were incubated for 5 h with CRISPR-associated protein 9 (Cas9), guide RNA (gRNA) targeting GGTA1, and one of the reagents. In Experiment 1, optimization of Cas9 protein to gRNA molar ratios of 1:2, 2:2, and 4:2, along with single or double doses of reagents, was performed on zygotes at 10 h post-in vitro fertilization. In Experiment 2, optimization of timing was performed at 10 or 29 h post-in vitro fertilization, using optimal molar ratios and reagent doses. Blastocyst formation, mutation rates, and mutation efficiency were measured in each experiment. For each reagent, a 4:2 Cas9:gRNA molar ratio and addition of a double reagent dose exhibited a higher mutation rate; however, blastocyst rate tended to decrease compared with that of control. Moreover, the optimal transfection time varied depending on the reagent, and the proportions of blastocysts carrying mutations were <34%. In conclusion, the above three transfectants allowed gene editing of porcine zygotes and embryos; however, this newly established chemistry-based technology needs further improvement, especially regarding editing efficiency and embryo development. Animal science journal = Nihon chikusan Gakkaiho 94 1 e13878 null 20230000 10.1111/asj.13878 37818780 fuminori_tanihara/published_papers/47639740 Chommanart Thongkittidilok Maki Hirata Qingyi Lin Nanaka Torigoe Bin Liu Yoko Sato Megumi Nagahara Fuminori Tanihara Takeshige Otoi Mosaic TP53 Mutation on Tumour Development in Pigs: A Case Study. Pigs rarely develop cancer; however, tumour protein p53 (TP53)-modified pigs may have an increased incidence of cancer. In this study, two pigs with mosaic mutations induced by gene editing were compared to determine the role of the wild-type TP53 sequence in tumorigenesis and to speculate how amino acid changes in TP53 sequences are related to tumorigenesis. The pig without tumours had a wild-type TP53 sequence and a 1-bp deletion in the TP53 sequence that resulted in a premature stop codon. In contrast, the pig with nephroblastoma had 6- and 7-bp deletions in the TP53 sequence, resulting in the absence of two amino acids and a premature stop codon, respectively. Our results indicated that TP53 mutations with truncated amino acids may be related to tumour formation. Veterinary medicine international 2023 7000858 7000858 20230000 10.1155/2023/7000858 37609627 fuminori_tanihara/published_papers/42551928 Fuminori Tanihara Maki Hirata Zhao Namula Lanh Thi Kim Do Naoaki Yoshimura Qingyi Lin Koki Takebayashi Tetsushi Sakuma Takashi Yamamoto Takeshige Otoi Pigs with an INS point mutation derived from zygotes electroporated with CRISPR/Cas9 and ssODN. Just one amino acid at the carboxy-terminus of the B chain distinguishes human insulin from porcine insulin. By introducing a precise point mutation into the porcine insulin (INS) gene, we were able to generate genetically modified pigs that secreted human insulin; these pigs may be suitable donors for islet xenotransplantation. The electroporation of the CRISPR/Cas9 gene-editing system into zygotes is frequently used to establish genetically modified rodents, as it requires less time and no micromanipulation. However, electroporation has not been used to generate point-mutated pigs yet. In the present study, we introduced a point mutation into porcine zygotes via electroporation using the CRISPR/Cas9 system to generate INS point-mutated pigs as suitable islet donors. We first optimized the efficiency of introducing point mutations by evaluating the effect of Scr7 and the homology arm length of ssODN on improving homology-directed repair-mediated gene modification. Subsequently, we prepared electroporated zygotes under optimized conditions and transferred them to recipient gilts. Two recipients became pregnant and delivered five piglets. Three of the five piglets carried only the biallelic frame-shift mutation in the INS gene, whereas the other two successfully carried the desired point mutation. One of the two pigs mated with a WT boar, and this desired point mutation was successfully inherited in the next F1 generation. In conclusion, we successfully established genetically engineered pigs with the desired point mutation via electroporation-mediated introduction of the CRISPR/Cas9 system into zygotes, thereby avoiding the time-consuming and complicated micromanipulation method. Frontiers in cell and developmental biology 11 884340 null 20230000 10.3389/fcell.2023.884340 36711037 fuminori_tanihara/published_papers/42551927 Fuminori Tanihara Maki Hirata Takeshige Otoi GEEP Method: An Optimized Electroporation-Mediated Gene Editing Approach for Establishment of Knockout Pig Lines. Pigs are excellent large animal models owing to their several physiological and anatomical similarities to humans. Somatic cell nuclear transfer using gene-modified cells is the mainstream approach for generating genetically modified pigs. Recent advances in improving gene editors such as the CRISPR/Cas9 system have enabled direct gene modification in zygotes/embryos. Here, we describe the gene editing by electroporation of Cas9 protein (GEEP) method, an optimized electroporation-mediated method for the introduction of CRISPR/Cas9 into porcine zygotes/embryos. The simplicity and micromanipulation-free procedures are the major advantages of this method. Methods in molecular biology (Clifton, N.J.) 2637 293 300 20230000 10.1007/978-1-0716-3016-7_22 36773155 fuminori_tanihara/published_papers/42551926 Koki Takebayashi Manita Wittayarat Qingyi Lin Nanaka Torigoe Bin Liu Maki Hirata Megumi Nagahara Fuminori Tanihara Takeshige Otoi Efficiency of genetic modification in gene-knockout sperm-derived zygotes followed by electroporation of guide RNA targeting the same gene. Genetic mosaicism is considered one of the main limitations of the electroporation method used to transfer CRISPR-Cas9/guide RNA (gRNA) into porcine zygotes. We hypothesized that fertilization of oocytes with sperm from gene-deficient boars, in combination with electroporation (EP) to target the same region of the gene in subsequent zygotes, would increase the gene modification efficiency. As myostatin (MSTN) and α1,3-galactosyltransferase (GGTA1) have beneficial effects on agricultural production and xenotransplantation, respectively, we used these two genes to test our hypothesis. Spermatozoa from gene-knockout boars were used for oocyte fertilization in combination with EP to transfer gRNAs targeting the same gene region to zygotes. No significant differences in the rates of cleavage and blastocyst formation as well as in the mutation rates of blastocysts were observed between the wild-type and gene-deficient spermatozoa groups, irrespective of the targeted gene. In conclusion, the combination of fertilization with gene-deficient spermatozoa and gene editing of the same targeted gene region using EP had no beneficial effects on embryo genetic modification, indicating that EP alone is a sufficient tool for genome modification. Animal science journal = Nihon chikusan Gakkaiho 94 1 e13842 null 20230000 10.1111/asj.13842 37218074 fuminori_tanihara/published_papers/42045306 Koki Takebayashi Manita Wittayarat Qingyi Lin Maki Hirata Naoaki Yoshimura Nanaka Torigoe Megumi Nagahara Lanh Thi Kim Do Fuminori Tanihara Takeshige Otoi Gene editing in porcine embryos using a combination of electroporation and transfection methods. CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9) technology is growing rapidly and has been greatly influencing the efficiency and effectiveness of genetic modifications in different applications. One aspect of research gaining importance in the development of the CRISPR/Cas9 system is the introduction of CRISPR materials into target organisms. Although we previously demonstrated the efficacy of electroporation- and lipofection-mediated CRISPR/Cas9 gene disruption in porcine zygotes, we still believe that the efficiency of this system could be improved by combining these two methods. The present study was thus conducted to clarify the effects of a combination of electroporation and lipofection for delivering CRISPR/Cas9 components into zona pellucida (ZP)-intact and -free zygotes. The results revealed that electroporation alone significantly increased the biallelic mutation rates in the resulting blastocysts compared to lipofection alone, irrespective of the presence of ZP. None of ZP-intact zygotes treated by lipofectamine alone had any mutations, suggesting that removal of the ZP is necessary for enabling CRISPR/Cas9-based genome editing via lipofection treatment in the zygotes. Additional lipofectamine treatment after electroporation did not improve the rates of total and biallelic mutations in the resulting blastocysts derived from either ZP-intact or -free zygotes. Reproduction in domestic animals = Zuchthygiene 57 10 1136 1142 20221000 10.1111/rda.14184 35699358 fuminori_tanihara/published_papers/42551929 Quynh Anh Le Manita Wittayarat Zhao Namula Qingyi Lin Koki Takebayashi Maki Hirata Fuminori Tanihara Lanh Thi Kim Do Takeshige Otoi Multiple gene editing in porcine embryos using a combination of microinjection, electroporation, and transfection methods. BACKGROUND AND AIM: Mosaicism - the presence of both wild-type and mutant alleles - is a serious problem for zygotic gene modification through gene editing using the Clustered regularly interspaced short palindromic repeats-Cas9 (CRISPR/Cas9) system. Different delivery methods, such as microinjection (MI), electroporation (EP), and transfection (TF), can be used to transfer CRISPR/Cas9 components into porcine zygotes. This study aimed to develop a method that combines MI, EP, and TF to improve mutation efficiency mediated through the CRISPR/Cas9 system for a triple-gene knockout in pigs. MATERIALS AND METHODS: The study consisted of three groups: The MI group with three simultaneously microinjected guide RNAs (gRNAs) targeting α-1,3-galactosyltransferase (GGTA1), cytidine 32 monophosphate-N-acetylneuraminic acid hydroxylase (CMAH), and β-1,4-N-acetyl-galactosaminyltransferase 2 (B4GALNT2); the MI + EP group with two gRNAs targeting GGTA1 and B4GALNT2 genes delivered into zygotes through MI, followed by EP of gRNA targeting the CMAH 1 h later; and the MI + EP + TF group with MI of gRNA targeting GGTA1 gene into zygotes, followed by EP of gRNA targeting CMAH 1 h later, and then TF of gRNA targeting the B4GALNT2 gene into zona-free zygotes after another hour. RESULTS: The rate of blastocysts carrying mutations in one or two gene(s) was significantly higher in the MI + EP + TF group than in the MI group. However, the blastocyst formation rate of zygotes in the MI + EP + TF group was lower than that of the zygotes in the other treatment groups. CONCLUSION: The combination of CRISPR/Cas9 delivery methods may improve the mutation efficiency of triple-gene edited porcine blastocysts. Veterinary world 15 9 2210 2216 20220900 10.14202/vetworld.2022.2210-2216 36341066 fuminori_tanihara/published_papers/37061999 Zhao Namula Manita Wittayarat Lanh Thi Kim Do Thanh Van Nguyen Qingyi Lin Koki Takebayashi Maki Hirata Fuminori Tanihara Takeshige Otoi Effects of the timing of electroporation during in vitro maturation on triple gene editing in porcine embryos using CRISPR/Cas9 system. Mosaicism, including alleles comprising both wild-type and mutant, is a serious problem for gene modification by gene editing using electroporation. One-step generation of F0 pigs with completely desired gene modifications saves cost and time, but the major obstacles have been mosaic mutations. We hypothesized that the timing of electroporation prior to in vitro fertilization (IVF) can increase the rates of biallelic mutation for multiple gene knockout as the permeability of mature oocytes is greater than that of zygotes. Hence, we determined whether the timing of electroporation during in vitro maturation (IVM) culture enhances triple gene editing in the resulting blastocysts. Three gRNAs targeting KDR, PDX1, and SALL1 were simultaneously introduced into the oocytes that had been incubated for 40, 42, and 44 h from the start of the IVM culture. Electroporation with three gRNAs at 40 h and 42 h during IVM culture decreased the blastocyst formation rates and did not improve the mutation rates and target number of biallelic mutations in the resulting blastocysts. The blastocyst formation rate, mutation rates, and target numbers in the resulting blastocysts from oocytes treated by electroporation at 44 h of IVM culture were similar to those of control zygotes electroporated at 13 h after the initiation of IVF. In conclusion, multiple gene editing efficiency in the resulting blastocysts was comparable between oocytes electroporated before and after the fertilization, indicating that oocytes with completed maturation time may allow better functioning of materials accepting gene editing application. Veterinary and animal science 16 100241 100241 20220600 10.1016/j.vas.2022.100241 35265771 fuminori_tanihara/published_papers/37061995 Megumi Shimazaki Manita Wittayarat Rentsenkhand Sambuu Asami Sugita Masaki Kawaguchi Maki Hirata Fuminori Tanihara Mitsuhiro Takagi Masayasu Taniguchi Takeshige Otoi Yoko Sato Disruption of cell proliferation and apoptosis balance in the testes of crossbred cattle-yaks affects spermatogenic cell fate and sterility. The balance between proliferation, differentiation, and apoptosis is well-coordinated in spermatogenesis for the timely production of appropriate numbers of sperm in animals. Disruption or decrease in sperm production is due to many conditions, including changes in testicular cell fate balance. Interspecies hybridisation of domestic yaks and cattle results in sterility in males because of spermatogenic arrest; however, the underlying mechanisms involved in sterility are still unclear. In the present study, we investigated the proliferation and apoptosis status during the development of yaks and crossbred cattle-yaks using immunohistochemistry of proliferating cell nuclear antigen and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assays. Testicular tissues from yaks (immature: 1 year old, mature: 2-3 years old) and backcrossed hybrids (2 year old) were collected and used to investigate the expression of each parameter in testicular cells. During the maturation of yak testes, proliferation and apoptosis became active only in spermatogenic cells, and not in other somatic cells, such as Sertoli cells, myoid cells, and Leydig cells. Furthermore, hybrid cattle-yak testes maintained proliferation ability but less apoptotic ability in spermatogenic cells when compared to yaks of the same age, suggesting that normal spermatogenic cell fate control is disrupted by changes in the balance between proliferation and apoptosis. In addition, Leydig cell proliferation rate was higher than apoptosis rate in the cattle-yak testes, indicating an increased number of Leydig cells, which may affect spermatogenesis through changes in steroidogenesis. Although epigenetic changes may be involved in cattle-yak testes, further studies are needed to clarify the modulation of proliferation and apoptosis to elucidate the mechanisms of infertility in hybrid cattle-yak males. Reproduction in domestic animals = Zuchthygiene 57 9 999 1006 20220525 10.1111/rda.14166 35614560 fuminori_tanihara/published_papers/37062000 Qingyi Lin Quynh Anh Le Koki Takebayashi Maki Hirata Fuminori Tanihara Chommanart Thongkittidilok Osamu Sawamoto Takeshi Kikuchi Takeshige Otoi Viability and developmental potential of porcine blastocysts preserved for short term in a chemically defined medium at ambient temperature. This study developed an efficient method for liquid storage of in vitro-derived porcine blastocysts at ambient temperature for 24 hr. We evaluated the effects of new chemically defined media (cell wash and preservation solution, Cellstor® -W [Cell-W] and cell suspension and preservation solution, Cellstor® -S [Cell-S]) for short-term storage. In the first experiment, in vitro-derived blastocyst were stored at 25ºC for 24 hr in Cell-W solution, Cell-S solution and pig embryo culture (PBM) medium. There were no differences in the rates of survival and development of stored blastocysts between the Cell-S and Cell-W solutions, but the total cell number of embryos that survived after storage in Cell-S solution was significantly higher (p < .05) than that in the Cell-W solution. In the second experiment, Cell-S solution was used to store the in vitro-derived blastocysts at 20°C, 25°C and 30°C. Storage at 20°C resulted in a significant decrease in the rates of survival and development of stored blastocysts compared to storage at 25°C or 30°C. No differences in survival and development rates were observed between storage at 25°C and 30°C, but the damage to the embryo quality after storage and culture was significantly lower at 25°C than at 30°C. In the third experiment, Cell-S solution was supplemented with β-mercaptoethanol and curcumin, either alone or in combination, as antioxidant agents. Although the supplementation with curcumin did not improve survival, it significantly increased the development rate of stored blastocysts compared with the control blastocysts stored without antioxidants. In conclusion, when porcine blastocysts were stored at 25°C for 24 hr, a Cell-S solution may be effective for maintaining the survival and development of in vitro embryos. Reproduction in domestic animals = Zuchthygiene 57 5 556 563 20220500 10.1111/rda.14095 35137478 fuminori_tanihara/published_papers/37061997 Asami Okada Misuzu Yamada-Yamashita Yukari Tominaga Kyoka Jo Hiroyasu Mori Reiko Suzuki Masashi Ishizu Motoyuki Tamaki Yuko Akehi Yuichi Takashi Daisuke Koga Eisuke Shimokita Fuminori Tanihara Kiyoe Kurahashi Sumiko Yoshida Yukari Mitsui Shiho Masuda Itsuro Endo Ken-Ichi Aihara Shoji Kagami Masahiro Abe Kevin Ferreri Yoshio Fujitani Munehide Matsuhisa Akio Kuroda Novel method utilizing bisulfite conversion with dual amplification-refractory mutation system polymerase chain reaction to detect circulating pancreatic β-cell cfDNA. AIMS/INTRODUCTION: Several research groups have reported methods for quantifying pancreatic beta cell (β-cell) injury by measuring β-cell-specific CpG unmethylation of the insulin gene in circulation using digital droplet PCR or next-generation sequencing. However, these methods have certain disadvantages, such as the need to consider the background signal owing to the small number of target CpG sites and the need for unique equipment. MATERIALS AND METHODS: We established a novel method for detecting four CpG unmethylations of the insulin gene using two-step amplification refractory mutation system PCR. We applied it to type 1 diabetes (T1D) patients with a wide range of disease durations and to healthy adults. RESULTS: The assay showed high linearity and could detect a single copy of unmethylated insulin DNA in experiments using methylated and unmethylated plasmid DNA. The unmethylated insulin DNA level in the type 1 diabetes group, whose β-cell mass was considerably reduced, was similar to that of healthy adults. An inverse correlation was observed between copy number and disease duration in patients with unmethylated insulin DNA-positive type 1 diabetes. CONCLUSIONS: We developed a novel method for detecting unmethylated insulin DNA in circulation that can be performed using a conventional real-time PCR system. This method would be useful for analyzing dynamic profiles of β-cells in human disease such as type 1 diabetes. Journal of diabetes investigation 13 7 1140 1148 20220409 10.1111/jdi.13806 35396829 fuminori_tanihara/published_papers/37062003 Praopilas Phakdeedindan Manita Wittayarat Theerawat Tharasanit Mongkol Techakumphu Megumi Shimazaki Rentsenkhand Sambuu Maki Hirata Fuminori Tanihara Masayasu Taniguchi Takeshige Otoi Yoko Sato Aberrant levels of DNA methylation and H3K9 acetylation in the testicular cells of crossbred cattle-yak showing infertility. Although the interspecies hybridization of bovids, such as cattle-yak (Bos taurus × Bos grunniens), has heterosis benefits, the infertility of hybrid males affects the maintenance of dominant traits in subsequent generations. To achieve reproductive capacity, male germ cell development requires coordinated changes in gene expression, including DNA methylation and generalized histone modifications. Although gene expression-related mechanisms underlying hybrid male sterility have been investigated recently, information on the cell types and stage-specific controls remains limited. Here, we used immunohistochemistry and image analyses to evaluate the 5-methylcytosine (5MC) and acetyl-histone H3 Lys9 (AcK9) expression in all spermatogonia and testicular somatic cell types to determine their roles in cattle-yak spermatogenesis. Testicular tissues from yak (1-3 years old) and backcrossed hybrids (2 years old) were used. In yak, the AcK9 expression levels increased in all cell types during maturation, but the 5MC expression levels did not change until reaching 3 years when they increased in all testicular cell types, except spermatogonia. Cattle-yak hybrids showed higher 5MC expression levels and different AcK9 expression levels in all cell types compared to the same-aged yak. These results suggested that both gene modulation by AcK9 and constant levels of DNA methylation are required for spermatogenesis during maturation in yak. Therefore, inappropriate expression levels of both AcK9 and DNA methylation might be the major factors for disruption of normal germ cell development in cattle-yak. Additionally, various modulations occurred depending on the cell type. Further experiments are needed to identify the stage-specific gene expression modulations in each cell type in yak and cattle-yak to potentially solve the infertility issue in crossbreeding. Reproduction in domestic animals = Zuchthygiene 57 3 304 313 20220300 10.1111/rda.14061 34854139 fuminori_tanihara/published_papers/37062002 Chommanart Thongkittidilok Quynh Anh Le Qingyi Lin Koki Takebayashi Thi Kim Lanh Do Zhao Namula Maki Hirata Fuminori Tanihara Takeshige Otoi Effects of individual or in-combination antioxidant supplementation during in vitro maturation culture on the developmental competence and quality of porcine embryos. The oocyte maturation process requires a high supply of energy, which generates reactive oxygen species (ROS), adversely affecting oocyte and embryo development. Balancing ROS by antioxidant supplementation is essential for maintaining oocyte maturation and embryonic quality in vitro. This study aimed to evaluate the impact of four antioxidants: β-mercaptoethanol (β-ME), chlorogenic acid (CGA), curcumin and sericin, when applied individually or in combinations, during oocyte maturation on development of porcine oocytes. Cumulus-oocyte complexes were collected, cultured in maturation medium supplemented with antioxidants for 44 hr and subsequently subjected to in vitro fertilization (IVF) and culture for 7 days. Combining all four (β-ME + CGA + curcumin + sericin) or three (β-ME + CGA + curcumin) antioxidants increased blastocyst formation rates. However, sericin supplementation alone, or in combination with β-ME or CGA, failed to improve blastocyst formation rates. The total cell numbers of blastocysts from the group supplemented with three antioxidants (β-ME + CGA + curcumin) were significantly higher than those from the other groups, except for the curcumin-supplement group. There were no differences in the maturation rates and proportions of oocytes with fragmented DNA between the antioxidant-supplemented and the non-supplemented control groups. In conclusion, supplementation with three antioxidants (β-ME + CGA + curcumin) during the maturation culture enhanced blastocyst formation and improved blastocyst quality. Reproduction in domestic animals = Zuchthygiene 57 3 314 320 20220300 10.1111/rda.14063 34862995 fuminori_tanihara/published_papers/37061996 Zhao Namula Quynh Anh Le Manita Wittayarat Qingyi Lin Koki Takebayashi Maki Hirata Lanh Thi Kim Do Fuminori Tanihara Takeshige Otoi Triple gene editing in porcine embryos using electroporation alone or in combination with microinjection. Background and Aim: We previously developed the gene-editing by electroporation (EP) of Cas9 protein method, in which the CRISPR/Cas9 system was introduced into porcine in vitro fertilized (IVF) zygotes through EP to disrupt a target gene. This method should be further developed, and a combination of EP and MI methods should be evaluated in pigs. This study aimed to determine that a combination of microinjection (MI) and EP of CRISPR/Cas9 system could increase the rates of biallelic mutation for triple-gene knockout in porcine blastocysts. We targeted the pancreatic and duodenal homeobox1 (PDX1) gene using cytoplasmic MI 1 h before or after EP, which was used to edit alpha-1,3-galactosyltransferase (GGTA1) and cytidine 32 monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) genes in porcine zygotes. Materials and Methods: We introduced guide RNAs targeting PDX1, GGTA1, and CMAH with the Cas9 protein into IVF zygotes (one-cell stage) through EP 10 h after the start of IVF (IVF; EP group) or in combination with MI (1 h before, MI-EP group, or after EP treatment EP-MI group) and evaluated the blastocyst formation rate and efficiency of target mutations in the resulting blastocysts. Results: Our results revealed a significant reduction in the rate of blastocyst formation in the two groups that underwent MI before and after EP (MI-EP and EP-MI group), compared with that in the groups treated with EP alone (EP group) (p=0.0224 and p<0.0001, respectively) and control (p=0.0029 and p<0.0001, respectively). There was no significant difference in the total mutation rates among the treatment groups in the resulting blastocysts. As an only positive effect of additional MI treatment, the rate of blastocysts carrying biallelic mutations in at least one target gene was higher in the MI-EP group than in the EP group. However, there was no difference in the rates of embryos carrying biallelic mutations in more than 2 target genes. Conclusion: These results indicate that although a combination of MI and EP does not improve the mutation efficiency or biallelic mutation for triple-gene knockout, MI treatment before EP is better to reduce mortality in porcine zygotic gene editing through a combination of MI and EP. Veterinary world 15 2 496 501 20220200 10.14202/vetworld.2022.496-501 35400948 fuminori_tanihara/published_papers/42551930 Naoaki Yoshimura Yasuhiro Morita Mitsuo Yamamoto Chika Higashine Koki Takebayashi Taichi Kumegawa Yoshimichi Higashiyama Masatoshi Niimi Fuminori Tanihara Takeshige Otoi Relationship between GnRH-induced LH increase profiles in the serum and vaginal mucus of Japanese Black beef cows. This study aimed to investigate the relationship between increases in the luteinizing hormone (LH) profiles in the serum and vaginal mucus of cows induced by gonadotropin-releasing hormone (GnRH). Samples for LH determination were collected from Japanese Black beef cows during estrus, which was induced with a controlled internal progesterone-releasing device and the administration of cloprostenol immediately before GnRH administration and every 30 min from the start of GnRH administration until 6.5 h. The peak serum LH concentration was clearly identified at 2.5 h post-GnRH administration, with serum concentrations returning to near-pre-GnRH-administration values after 6.5 h, whereas the peak vaginal mucus LH concentration was identified 4.5 h after GnRH administration. These results indicate that the LH secretion peak in vaginal mucus appeared about 2 h after peak LH secretion in the serum. Archives animal breeding 65 3 353 356 20220000 10.5194/aab-65-353-2022 36267479 fuminori_tanihara/published_papers/37062005 Zhao Namula Maki Hirata Quynh Anh Le Qingyi Lin Koki Takebayashi Naoaki Yoshimura Fuminori Tanihara Chommanart Thongkittidilok Takeshige Otoi Zona pellucida treatment before CRISPR/Cas9-mediated genome editing of porcine zygotes. BACKGROUND: Increasing the permeability of the zona pellucida (ZP) of oocytes before CRISPR/Cas9 electroporation may improve the efficiency of gene editing; however, the effects of this approach on subsequent developmental processes are unclear. In this study, the effects of ZP treatment before electroporation on embryonic development and gene editing in porcine embryos were evaluated. METHODS: The ZP of zygotes was weakened or removed by exposure to 0.5% actinase E, followed by electroporation of the Cas9 protein with guide RNA targeting GGTA1. RESULTS: The blastocyst formation rate of ZP-free zygotes after electroporation was significantly lower (p < 0.05) than that of ZP-intact zygotes. The mutation rate in blastocysts from ZP-weakened zygotes was similar to that in ZP-intact zygotes, whereas ZP removal increased the mutation rate. The mutation efficiency in blastocysts from electroporated zygotes did not differ among ZP treatment groups. CONCLUSIONS: Our results indicate that weakening the ZP does not affect the developmental competence, mutation rate, or mutation efficiency of electroporated zygotes, whereas ZP removal has a detrimental effect on embryonic development but may increase the mutation rate. Veterinary medicine and science 8 1 164 169 20220100 10.1002/vms3.659 34674375 fuminori_tanihara/published_papers/37061998 Qingyi Lin Quynh Anh Le Koki Takebayashi Maki Hirata Fuminori Tanihara Osamu Sawamoto Takeshi Kikuchi Takeshige Otoi Short-term preservation of porcine zygotes at ambient temperature using a chemically defined medium. We aimed to develop a simple method for the short-term preservation of in vitro-produced porcine zygotes at 25°C for up to 2 days. Firstly, we evaluated the efficiency of three storage solutions to preserve porcine zygotes at 25°C for 24 h. Two of these were commercially available defined media for cell storage (Cell-W and Cell-S), and the third was fetal bovine serum (FBS). Thereafter, we examined the effects of storing the zygotes in the Cell-W solution for 24-72 h at 25°C. The Cell-W solution was the most efficient for 24 h storage of porcine zygotes at 25°C, with no apparent effects on blastocyst quality. However, short-term storage of porcine zygotes for 24 h reduced the blastocyst formation rate compared with the fresh control group. As storage duration increased from 24 to 48 or 72 h, blastocyst formation rates were significantly decreased from 11.3% to 4.4% and 0%, respectively. In conclusion, during zygote storage, the developmental competence to the blastocyst stage decreased with time. Storage of zygotes in chemically defined media did not affect blastocyst quality, but the storage in 100% serum had an adverse effect on developing embryos causing apoptosis. Animal science journal = Nihon chikusan Gakkaiho 93 1 e13711 null 20220100 10.1111/asj.13711 35373427 fuminori_tanihara/published_papers/37062001 Maki Hirata Manita Wittayarat Zhao Namula Quynh Anh Le Qingyi Lin Koki Takebayashi Chommanart Thongkittidilok Taro Mito Sayuri Tomonari Fuminori Tanihara Takeshige Otoi Generation of mutant pigs by lipofection-mediated genome editing in embryos. The specificity and efficiency of CRISPR/Cas9 gene-editing systems are determined by several factors, including the mode of delivery, when applied to mammalian embryos. Given the limited time window for delivery, faster and more reliable methods to introduce Cas9-gRNA ribonucleoprotein complexes (RNPs) into target embryos are needed. In pigs, somatic cell nuclear transfer using gene-modified somatic cells and the direct introduction of gene editors into the cytoplasm of zygotes/embryos by microinjection or electroporation have been used to generate gene-edited embryos; however, these strategies require expensive equipment and sophisticated techniques. In this study, we developed a novel lipofection-mediated RNP transfection technique that does not require specialized equipment for the generation of gene-edited pigs and produced no detectable off-target events. In particular, we determined the concentration of lipofection reagent for efficient RNP delivery into embryos and successfully generated MSTN gene-edited pigs (with mutations in 7 of 9 piglets) after blastocyst transfer to a recipient gilt. This newly established lipofection-based technique is still in its early stages and requires improvements, particularly in terms of editing efficiency. Nonetheless, this practical method for rapid and large-scale lipofection-mediated gene editing in pigs has important agricultural and biomedical applications. Scientific reports 11 1 23806 23806 20211213 10.1038/s41598-021-03325-5 34903813 fuminori_tanihara/published_papers/37062004 Takeshi Kikuchi Masuhiro Nishimura Maki Hirata Fuminori Tanihara Natsuki Komori Makoto Tanaka Osamu Sawamoto Takeshige Otoi Shinichi Matsumoto Development and characterization of Gal KO porcine bone marrow-derived mesenchymal stem cells. BACKGROUND: We demonstrated that neonatal porcine bone marrow-derived mesenchymal stem cell (npBM-MSCs) could improve a critical ischemic limb disease in rat model more efficiently compared with human MSCs. However, since porcine MSC presents galactosyl-alpha 1,3-galactose antigen (Gal antigen), MSC could be eliminated by the xenogeneic rejection. Recently, we established Gal knockout (KO) pigs by a technique of the electroporation of the CRISPR/Cas9 system into vitro-fertilized zygotes. In this study, we hypothesized that MSC from the established Gal KO pigs could further improve the efficacy. Before examining the hypothesis, in this study, we have established and characterized bone marrow-derived MSC from the Gal KO adult pigs (apBM-MSCs). METHODS: Mononuclear cells (MNCs) were isolated from bone marrow cells of both Gal KO adult pigs and wild-type (WT) adult pigs. MNCs were further manipulated to create Gal KO apBM-MSCs and WT apBM-MSCs. Both MSCs were assessed by their surface markers, the capability of differentiation into adipocytes, osteocytes and chondrocytes, grow speed and colony-forming assay. To assess the efficacy of Gal KO apBM-MSCs, angiogenesis-related genes and immunosuppression-related genes were assessed by cytokine stimulation. RESULTS: Gal KO apBM-MSC showed no Gal antigen on their cell surfaces. Both Gal KO apBM-MSCs and WT apBM-MSCs, presented little or no negative surface markers of MSCs, while they presented positive surface markers of MSCs. Furthermore, Gal KO apBM-MSCs were able to differentiate into adipocytes, osteocytes, and chondrocytes as well as WT apBM-MSCs. There was no difference in doubling time between Gal KO apBM-MSCs and WT apBM-MSCs. Interestingly, the colony-forming efficiency of Gal KO apBM-MSCs was about half that of WT apBM-MSC. However, angiogenesis and immunosuppression-related genes were equally upregulated in both Gal KO apBM-MSCs and WT apBM-MSCs by cytokine stimulation. CONCLUSION: We created and characterized Gal KO apBM-MSCs which showed similar characteristics and cytokine-induced gene upregulation to the WT apBM-MSCs. Xenotransplantation 28 6 e12717 null 20211100 10.1111/xen.12717 34730861 fuminori_tanihara/published_papers/35198891 Qingyi Lin Quynh Anh Le Koki Takebayashi Chommanart Thongkittidilok Manita Wittayarat Maki Hirata Fuminori Tanihara Takeshige Otoi Timing and duration of lipofection-mediated CRISPR/Cas9 delivery into porcine zygotes affect gene-editing events. OBJECTIVE: Lipofection-mediated introduction of the CRISPR/Cas9 system in porcine zygotes provides a simple method for gene editing, without requiring micromanipulation. However, the gene editing efficiency is inadequate. The aim of this study was to improve the lipofection-mediated gene editing efficiency by optimizing the timing and duration of lipofection. RESULTS: Zona pellucida (ZP)-free zygotes collected at 5, 10, and 15 h from the start of in vitro fertilization (IVF) were incubated with lipofection reagent, guide RNA (gRNA) targeting GGTA1, and Cas9 for 5 h. Lipofection of zygotes collected at 10 and 15 h from the start of IVF yielded mutant blastocysts. Next, ZP-free zygotes collected at 10 h from the start of IVF were incubated with lipofection reagent, gRNA, and Cas9 for 2.5, 5, 10, or 20 h. The blastocyst formation rate of zygotes treated for 20 h was significantly lower (p < 0.05) than those of the other groups, and no mutant blastocysts were obtained. Moreover, the mutation rates of the resulting blastocysts decreased as the incubation time increased. In conclusion, a lipofection-mediated gene editing system using the CRISPR/Cas9 system in ZP-zygotes is feasible; however, further improvements in the gene editing efficiency are required. BMC research notes 14 1 389 389 20211009 10.1186/s13104-021-05800-8 34627381 fuminori_tanihara/published_papers/37062006 Fuminori Tanihara Maki Hirata Takeshige Otoi Current status of the application of gene editing in pigs. Genetically modified animals, especially rodents, are widely used in biomedical research. However, non-rodent models are required for efficient translational medicine and preclinical studies. Owing to the similarity in the physiological traits of pigs and humans, genetically modified pigs may be a valuable resource for biomedical research. Somatic cell nuclear transfer (SCNT) using genetically modified somatic cells has been the primary method for the generation of genetically modified pigs. However, site-specific gene modification in porcine cells is inefficient and requires laborious and time-consuming processes. Recent improvements in gene-editing systems, such as zinc finger nucleases, transcription activator-like effector nucleases, and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (CRISPR/Cas) system, represent major advances. The efficient introduction of site-specific modifications into cells via gene editors dramatically reduces the effort and time required to generate genetically modified pigs. Furthermore, gene editors enable direct gene modification during embryogenesis, bypassing the SCNT procedure. The application of gene editors has progressively expanded, and a range of strategies is now available for porcine gene engineering. This review provides an overview of approaches for the generation of genetically modified pigs using gene editors, and highlights the current trends, as well as the limitations, of gene editing in pigs. The Journal of reproduction and development 67 3 177 187 20210621 10.1262/jrd.2021-025 33840678 fuminori_tanihara/published_papers/34863968 Naoaki Yoshimura Masayasu Taniguchi Tsukasa Terazono Tetsushi Ono Mitsuhiro Takagi Yoko Sato Maki Hirata Fuminori Tanihara Takeshige Otoi Vaginal stimulation enhances ovulation of queen ovaries treated using a combination of eCG and hCG Wiley Veterinary Medicine and Science 2053-1095 7 5 1569 1574 20210616 10.1002/vms3.552 fuminori_tanihara/published_papers/42185597 L. T. K. Do M. Wittayarat Y. Sato K. Chatdarong T. Tharasanit M. Techakumphu M. Hirata F. Tanihara M. Taniguchi T. Otoi Comparison of Blastocyst Development between Cat-Cow and Cat-Pig Interspecies Somatic Cell Nuclear Transfer Embryos Treated with Trichostatin A Pleiades Publishing Ltd Biology Bulletin 1608-3059 48 2 107 117 20210300 10.1134/s1062359021020035 fuminori_tanihara/published_papers/31974886 Fuminori Tanihara Maki Hirata Nhien Thi Nguyen Osamu Sawamoto Takeshi Kikuchi Takeshige Otoi One-Step Generation of Multiple Gene-Edited Pigs by Electroporation of the CRISPR/Cas9 System into Zygotes to Reduce Xenoantigen Biosynthesis. 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. International journal of molecular sciences 22 5 null null 20210224 10.3390/ijms22052249 33668187 fuminori_tanihara/published_papers/31974885 Maki Hirata Manita Wittayarat Zhao Namula Quynh Anh Le Qingyi Lin Koki Takebayashi Chommanart Thongkittidilok Fuminori Tanihara Takeshige Otoi Lipofection-Mediated Introduction of CRISPR/Cas9 System into Porcine Oocytes and Embryos. 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. Animals : an open access journal from MDPI 11 2 null null 20210223 10.3390/ani11020578 33672168 fuminori_tanihara/published_papers/31974888 Quynh Anh Le Fuminori Tanihara Manita Wittayarat Zhao Namula Yoko Sato Qingyi Lin Koki Takebayashi Maki Hirata Takeshige Otoi Comparison of the effects of introducing the CRISPR/Cas9 system by microinjection and electroporation into porcine embryos at different stages. 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. BMC research notes 14 1 7 7 20210106 10.1186/s13104-020-05412-8 33407863 fuminori_tanihara/published_papers/35198895 Zhao Namula Yasuhiro Isumi Yoko Sato Quynh Anh Le Qingyi Lin Koki Takebayashi Maki Hirata Fuminori Tanihara Chommanart Thongkittidilok Takeshige Otoi Improvement of the in vitro fertilization and embryo development using frozen-thawed spermatozoa of microminipigs. This study aimed to compare the quality and the penetration ability of frozen-thawed spermatozoa from three microminipigs and Large White boars and to evaluate the effects of caffeine and heparin as well as the sperm-oocyte co-incubation length on the fertilization and embryonic development in vitro. Results showed that the fertilization rates of spermatozoa from three microminipig boars were significantly lower than those of a Large White boar. In the post-thaw spermatozoa from one of three microminipig boars, the sperm quality, penetration ability, and the oocyte development after in vitro fertilization were significantly lower than those of the spermatozoa from other boars. The caffeine supplementation in the fertilization media increased the rates of fertilization and blastocyst formation for the microminipig spermatozoa with low sperm quality. In addition to caffeine supplementation, the rates of fertilization and blastocyst formation after using microminipig spermatozoa were significantly higher with a 10 h sperm-oocyte co-incubation than with 3 h of co-incubation length. Our results indicate that the differences between the males and the breed influence the quality and fertility of frozen-thawed boar spermatozoa. In conclusion, the presence of caffeine in the in vitro fertilization (IVF) medium and adequate length of sperm-oocyte co-incubation may have beneficial effects for improving IVF results when using microminipig spermatozoa with low quality. Archives animal breeding 64 1 265 271 20210000 10.5194/aab-64-265-2021 34189254 fuminori_tanihara/published_papers/31974887 Manita Wittayarat Maki Hirata Zhao Namula Yoko Sato Nhien T Nguyen Quynh A Le Qingyi Lin Koki Takebayashi Fuminori Tanihara Takeshige Otoi Introduction of a point mutation in the KRAS gene of in vitro fertilized porcine zygotes via electroporation of the CRISPR/Cas9 system with single-stranded oligodeoxynucleotides. 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. Animal science journal = Nihon chikusan Gakkaiho 92 1 e13534 null 20210100 10.1111/asj.13534 33638256 fuminori_tanihara/published_papers/31012092 Zhao Namula Yoko Sato Manita Wittayarat Quynh Anh Le Nhien Thi Nguyen Qingyi Lin Maki Hirata Fuminori Tanihara Takeshige Otoi Curcumin supplementation in the maturation medium improves the maturation, fertilisation and developmental competence of porcine oocytes. 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. Acta veterinaria Hungarica null null 20201120 10.1556/004.2020.00041 33221737 fuminori_tanihara/published_papers/31012093 Maki Hirata Manita Wittayarat Fuminori Tanihara Yoko Sato Zhao Namula Quynh Anh Le Qingyi Lin Koki Takebayashi Takeshige Otoi One-step genome editing of porcine zygotes through the electroporation of a CRISPR/Cas9 system with two guide RNAs. 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. In vitro cellular & developmental biology. Animal 56 8 614 621 20200900 10.1007/s11626-020-00507-9 32978715 fuminori_tanihara/published_papers/31012094 Fuminori Tanihara Maki Hirata Nhien Thi Nguyen Osamu Sawamoto Takeshi Kikuchi Masako Doi Takeshige Otoi Efficient generation of GGTA1-deficient pigs by electroporation of the CRISPR/Cas9 system into in vitro-fertilized zygotes. 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. BMC biotechnology 20 1 40 40 20200818 10.1186/s12896-020-00638-7 32811500 fuminori_tanihara/published_papers/31012095 Maki Hirata Manita Wittayarat Zhao Namula Quynh Anh Le Qingyi Lin Nhien Thi Nguyen Koki Takebayashi Yoko Sato Fuminori Tanihara Takeshige Otoi Evaluation of multiple gene targeting in porcine embryos by the CRISPR/Cas9 system using electroporation. 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. Molecular biology reports 47 7 5073 5079 20200700 10.1007/s11033-020-05576-3 32519310 fuminori_tanihara/published_papers/31012194 Quynh A. Le Maki Hirata Nhien T. Nguyen Koki Takebayashi Manita Wittayarat Yoko Sato Zhao Namula Masahiro Nii Fuminori Tanihara Takeshige Otoi Effects of electroporation treatment using different concentrations of Cas9 protein with gRNA targeting Myostatin ( MSTN ) genes on the development and gene editing of porcine zygotes Wiley Animal Science Journal 1740-0929 91 1 e13386 null 20200600 10.1111/asj.13386 fuminori_tanihara/published_papers/31012097 Fuminori Tanihara Maki Hirata Nhien Thi Nguyen Quynh Anh Le Takayuki Hirano Takeshige Otoi Generation of viable PDX1 gene-edited founder pigs as providers of nonmosaics. 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. Molecular reproduction and development 87 4 471 481 20200400 10.1002/mrd.23335 32166879 fuminori_tanihara/published_papers/31012096 Takayuki Hirano Maki Hirata Shigeyuki Fujimoto Nhien Thi Nguyen Quynh Anh Le Fuminori Tanihara Takeshige Otoi Comparative analysis of bilirubin glucuronidation activity in 2D- and 3D-cultured human hepatocellular carcinoma HepG2 cells. In vitro cellular & developmental biology. Animal 56 4 277 280 20200400 10.1007/s11626-020-00451-8 32394241 fuminori_tanihara/published_papers/47123284 N. T. Nguyen M. Hirata F. Tanihara Y. Sato Z. Namula Q. A. Le M. Wittayarat M. Fahrudin T. Otti In vitro Development of Zona Pellucida-free Porcine Zygotes Cultured Individually after Vitrification NLM (Medline) Cryo letters 0143-2044 41 2 86 91 20200301 33988658 2-s2.0-85106552666 fuminori_tanihara/published_papers/27285734 Sato Y Kuriwaki R Hagino S Shimazaki M Sambuu R Hirata M Tanihara F Takagi M Taniguchi M Otoi T Abnormal functions of Leydig cells in crossbred cattle-yak showing infertility. Reproduction in domestic animals = Zuchthygiene 1439-0531 55 2 209 216 20191200 10.1111/rda.13609 31858644 fuminori_tanihara/published_papers/27285719 Tanihara F Hirata M Morikawa S Nguyen NT LE QA Hirano T Fukumi Y Abe T Otoi T The effects of electroporation on viability and quality of in vivo-derived bovine blastocysts. The introduction of exogenous molecules into embryos is required for analyses of molecular dynamics and specific gene functions during early embryonic development. Electroporation is an effective method to transport exogenous molecules into cells, but is rarely used in bovine embryos. First, we evaluated the viability of in vivo-derived bovine blastocysts after electroporation with fluorescein (FAM) labeled-oligonucleotides with varying pulse numbers (3, 5, 7, and 10), while keeping the pulse duration at 1 msec and the electric field of 20 V/mm. Next, we examined the effects of zona pellucida status on blastocyst quality after electroporation, by comparing the average diameter of blastocysts before and after electroporation using blastocysts with intact zona pellucida and hatching/hatched blastocysts. Electroporation successfully introduced exogenous molecules into in vivo-derived bovine blastocysts without loss of viability. Moreover, the status of the zona pellucida may be associated with the quality of blastocysts after electroporation. The Journal of reproduction and development 0916-8818 65 5 475 479 20191000 10.1262/jrd.2019-049 31178553 fuminori_tanihara/published_papers/27285718 Namula Z Wittayarat M Hirata M Hirano T Nguyen NT Le QA Fahrudin M Tanihara F Otoi T Genome mutation after the introduction of the gene editing by electroporation of Cas9 protein (GEEP) system into bovine putative zygotes. The present study was designed to investigate the effects of voltage strength on embryonic developmental rate and mutation efficiency in bovine putative zygotes during electroporation with the CRISPR/Cas9 system to target the MSTN gene at different time points after insemination. Results showed that there was no significant interaction between electroporation time and voltage strength on the embryonic cleavage and blastocyst formation rates. However, increasing the voltage strength to 20 V/mm to electroporate the zygotes at 10 h after the start of insemination yielded significantly lower blastocyst formation rates (P < 0.05) than those of the 10-V/mm electroporated zygotes. Mutation efficiency was then assessed in individual blastocysts by DNA sequence analysis of the target sites in the MSTN gene. A positive correlation between mutation rate and voltage strength was observed. The mutation efficiency in mutant blastocysts was significantly higher in the zygotes electroporated with 20 V/mm at 10 h after the start of insemination (P < 0.05) than in the zygotes electroporated at 15 h, irrespective of the voltage strength. We also noted that a certain number of blastocysts from zygotes that were electroporated with more than 15 V/mm at 10 h (4.8-16.7%) and 20 V/mm at 15 h (4.8%) were biallelic mutants. Our results suggest that the voltage strength during electroporation as well as electroporation time certainly have effects on the embryonic developmental rate and mutation efficiency in bovine putative zygotes. In vitro cellular & developmental biology. Animal 1071-2690 55 8 598 603 20190900 10.1007/s11626-019-00385-w 31297696 fuminori_tanihara/published_papers/27285716 Tanihara F Hirata M Nguyen NT Le QA Wittayarat M Fahrudin M Hirano T Otoi T Generation of <i>CD163-</i>edited pig via electroporation of the CRISPR/Cas9 system into porcine <i>in vitro-</i>fertilized zygotes. CD163 is a putative fusion receptor for virus of porcine reproductive and respiratory syndrome (PRRS). In this study, we introduced a CRISPR/Cas9 system [guide RNAs (gRNAs) with Cas9 protein] targeting the CD163 gene into in vitro-fertilized porcine zygotes by electroporation to generate CD163-modified pigs. First, we designed four types of gRNAs that targeted distinct sites in exon 7 of the CD163 gene. Cas9 protein with different gRNAs was introduced into in vitro-fertilized zygotes by electroporation. When the electroporated zygotes were allowed to develop to blastocysts in vitro and the genome editing efficiency was evaluated using these blastocysts, three (gRNA1, 2, and 4) of the four gRNAs tested successfully edited the CD163 gene. To generate CD163-knockout pigs, a total of 200 electroporated zygotes using these three gRNAs were transferred into the oviducts of oestrous-synchronized surrogate and the surrogate gave birth to eight piglets. Subsequent sequence analysis revealed that one of the piglets carried no wild-type sequence in CD163 gene. The other seven piglets carried only wild-type sequence. Thus, we successfully generated a CD163-edited pig by electroporation of the CRISPR/Cas9 system into in vitro-fertilized zygotes, although further improvement is required to generate genetically modified pigs with high efficiency. Animal biotechnology 1049-5398 1 8 20190900 10.1080/10495398.2019.1668801 31558095 fuminori_tanihara/published_papers/27285717 Hirata M Wittayarat M Hirano T Nguyen NT Le QA Namula Z Fahrudin M Tanihara F Otoi T The Relationship between Embryonic Development and the Efficiency of Target Mutations in Porcine Endogenous Retroviruses (PERVs) <i>Pol</i> Genes in Porcine Embryos. Porcine endogenous retrovirus (PERV) is a provirus found in the pig genome that may act as an infectious pathogen in humans who receive pig organ xenotransplantation. Inactivation of the PERV pol gene in porcine cells reportedly affects cell growth. Therefore, the mutation of PERV pol gene in porcine embryos using genome editing may affect the embryonic development. The present study was carried out to investigate the relationship between the mutation of the PERV pol gene in porcine embryos and their development. We introduced, either alone or in combination, three different gRNAs (gRNA1, 2, and 3) into porcine zygotes by genome editing using electroporation of the Cas9 protein (GEEP) system. All three gRNAs targeted the PERV pol gene, and we assessed their effects on porcine embryonic development. Our results showed that the blastocyst formation rates of zygotes electroporated with gRNA3-alone and in combination-were significantly lower (p < 0.05) than those of zygotes electroporated with gRNA1. The mutation rates assessed by the PERV pol gene target site sequencing in individual blastocysts and pooled embryos at the 2-to-8-cell stage did not differ among the three gRNAs. However, the frequency of indel mutations in mutant embryos at the 2-to-8-cell stage trended higher in the embryos electroporated with gRNA3 alone and in combination. Embryonic development may be affected by gRNAs that induce high-frequency indel mutations. Animals : an open access journal from MDPI 9 9 null null 20190800 10.3390/ani9090593 31443357 fuminori_tanihara/published_papers/27285709 Tanihara F Hirata M Iizuka S Sairiki S Nii M Nguyen NT Le QA Hirano T Otoi T Relationship among ovarian follicular status, developmental competence of oocytes, and anti-Müllerian hormone levels: A comparative study in Japanese wild boar crossbred gilts and Large White gilts. The aim of this study was to investigate the ovarian follicular development, developmental competence of oocytes, and plasma anti-Müllerian hormone (AMH) levels of Japanese wild boar crossbred (wild hybrid) gilts, whose litter size is inferior to that of European breeds. Ovary and plasma samples were collected from two different breeds of gilts (wild hybrid and Large White breeds). The ovaries from the wild hybrid gilts had a lower average numbers of secondary follicles and vesicular follicles in ovarian cross-sections and of good quality oocytes collected from ovarian follicles as compared with those from Large White gilts (p < 0.05). The development rate to the blastocyst stage of good quality oocytes after in vitro maturation, fertilization and culture was also lower (p < 0.05) in wild hybrid gilts than in Large White gilts. Plasma AMH levels with >0.16 ng/ml were detected in 8.3% of the examined wild hybrid gilts and 33% of the Large White gilts. These results indicate that the low reproductive performance of wild hybrid breed may result in part from low numbers of vesicular follicles and good quality oocytes, and low developmental competence of oocytes. Moreover, plasma AMH levels may support low number of vesicular follicles in ovaries of wild hybrid gilts. Animal science journal = Nihon chikusan Gakkaiho 1344-3941 90 6 712 718 20190600 10.1111/asj.13200 30977253 fuminori_tanihara/published_papers/27285712 Thi Nguyen N Hirata M Tanihara F Hirano T Le QA Nii M Otoi T Hypothermic storage of porcine zygotes in serum supplemented with chlorogenic acid. The current study was conducted to investigate the effects of 100% foetal bovine serum (FBS) and 100% porcine follicular fluid (pFF) as a storage medium on the developmental competence of porcine zygotes stored at 25°C for 24 hr. Moreover, we evaluated the additive effects of chlorogenic acid (CGA) in the storage medium. When in vitro-produced zygotes were stored at 25°C for 24 hr in tubes containing either tissue culture medium (TCM) 199 supplemented with 1 mg/ml bovine serum albumin (BSA), 100% of FBS or 100% of pFF, the rate of blastocyst formation was significantly higher in 100% of FBS than in BSA-containing TCM 199. When the effects of CGA supplementation in 100% of FBS on the development of zygotes stored at 25°C for 24 hr was evaluated, more zygotes stored with 50 µM CGA developed to blastocysts compared with the other concentrations of CGA. When the formation date and quality of blastocysts derived from zygotes stored in 100% of FBS supplemented with 50 µM CGA were investigated, the highest ratio of blastocysts formation in the storage group appeared 1 day later than in the non-stored control group. However, a higher proportion of blastocysts with apoptotic nuclei was observed in the stored group as compared to the non-stored group. In conclusion, 100% of FBS is available for a short storage medium of porcine zygotes. The supplementation of 50 µM CGA into the storage medium improves the rates of blastocyst formation of zygotes after storage, but the quality of embryos from the stored zygotes remains to be improved. Reproduction in domestic animals = Zuchthygiene 0936-6768 54 5 750 755 20190500 10.1111/rda.13417 30788874 fuminori_tanihara/published_papers/27285711 Hirata M Tanihara F Wittayarat M Hirano T Nguyen NT Le QA Namula Z Nii M Otoi T Genome mutation after introduction of the gene editing by electroporation of Cas9 protein (GEEP) system in matured oocytes and putative zygotes. The application of CRISPR/Cas9 strategy promises to rapidly increase the production of genetically engineered animals since it yields stably integrated transgenes. In the present study, we investigated the efficiency of target mutations after electroporation with the CRISPR/Cas9 system using sgRNAs to target the MSTN or FGF10 genes in porcine-matured oocytes and putative zygotes. Effects of pulse number (3-7 pulse repetitions) during electroporation on the embryonic development and mutation efficiency were also investigated. Our results showed that the cleavage rate of matured oocytes with electroporation treatment significantly decreased as compared with electroporated putative zygotes (p < 0.05). Moreover, the rates of blastocyst formation from oocytes/zygotes electroporated with more than 5 pulses decreased. Mutation efficiency was then assessed after sequencing the target sites in individual blastocysts derived from oocytes/zygotes electroporated by 3 and 5 pulses. No bi-allelic mutations in all examined blastocysts were observed in this study. There were no differences in the mutation rates (50-60%) between blastocysts derived from matured oocytes electroporated by 3 and 5 pulses, irrespective of targeting gene. In the targeting MSTN gene, however, the mutation rate (12.5%) of blastocysts derived from putative zygotes electroporated by 3 pulses tended to be lower than that (60%) from 5-pulsed electroporated putative zygotes. These data indicate that the type of eggs may influence not only their development after electroporation treatment but also the mutation rate in the resulting blastocysts. In vitro cellular & developmental biology. Animal 1071-2690 55 4 237 242 20190400 10.1007/s11626-019-00338-3 30820813 fuminori_tanihara/published_papers/27285710 Namula Z Tanihara F Wittayarat M Hirata M Nguyen NT Hirano T Le QA Nii M Otoi T Effects of Tris (hydroxymethyl) aminomethane on the quality of frozen-thawed boar spermatozoa. Tris (hydroxymethyl) aminomethane (Tris) has been used as a pH regulator for buffering the pH of dilution extenders for boar semen, such as the Modena extender. The purpose of the present study was to assess the effects of Tris supplementation at different concentrations (0, 8, 24 and 72 μM) into the freezing extender on the quality and fertilising capacity of frozen-thawed boar spermatozoa. The results showed that the supplementation of 24 μM of Tris gave significantly higher percentages of sperm viability and plasma membrane integrity than those of the control group at any time point of assessment (0 h and 3 h post-thawing) (P < 0.05). However, there were no significant differences in the acrosome integrity parameter among the groups. Higher percentages of sperm motility were observed in the spermatozoa cryopreserved with 24 μM of Tris compared to the control groups when the samples were analysed 0 h after thawing (P < 0.05). However, an increase of the Tris concentration to 72 μM did not enhance the sperm motility parameters. The total numbers of fertilised oocytes and blastocysts obtained with spermatozoa frozen with 24 μM Tris were significantly higher than those of the control group without Tris (P < 0.05). In conclusion, the supplementation of 24 μM Tris into the freezing extender contributes to a better boar sperm quality and fertilising capacity after the process of freezing and thawing. Acta veterinaria Hungarica 0236-6290 67 1 106 114 20190300 10.1556/004.2019.012 30922097 fuminori_tanihara/published_papers/27285713 Tanihara F Hirata M Nguyen NT LE QA Hirano T Otoi T Effects of concentration of CRISPR/Cas9 components on genetic mosaicism in cytoplasmic microinjected porcine embryos. Cytoplasmic microinjection (CI) of the CRISPR/Cas9 system enabled the induction of site-specific mutations in porcine zygotes and resulting pigs. However, mosaicism is a serious problem for genetically modified pigs. In the present study, we investigated suitable timing and concentration of CRISPR/Cas9 components for introduction into oocytes/zygotes by CI, to reduce mosaicism in the resulting blastocysts. First, we introduced 20 ng/μl of Cas9 protein and guide RNA (gRNA), targeting the α-1,3-galactosyltransferase (GalT) gene in oocytes before in vitro fertilization (IVF), in zygotes after IVF, or in oocytes/zygotes before and after IVF, twice. CI treatment had no detrimental effects on blastocyst formation rates. The highest value of the rate of mutant blastocysts was observed in zygotes injected after IVF. Next, we injected Cas9 protein and gRNA into zygotes after IVF at a concentration of 20 ng/μl each (20 ng/μl group) or 100 ng/μl each (100 ng/μl group). The ratio of the number of blastocysts that carried mutations to the total number of blastocysts examined in the 100 ng/μl group was significantly higher (P < 0.05) than that in the 20 ng/μl group. Although no blastocysts from the 20 ng/μl group carried a biallelic mutation, 16.7% of blastocysts from the 100 ng/μl group carried a biallelic mutation. In conclusion, increasing the concentration of Cas9 protein and gRNA is effective in generating biallelic mutant blastocysts. To reduce mosaicism, however, further optimization of the timing of CI, and the concentration of CRISPR/Cas9 components, is needed. The Journal of reproduction and development 0916-8818 65 3 209 214 20190200 10.1262/jrd.2018-116 30726783 fuminori_tanihara/published_papers/7252502 Hirata M Tanihara F Taniguchi M Takagi M Terazono T Otoi T Follicular development of canine ovaries stimulated by a combination treatment of eCG and hCG. Veterinary medicine and science 2053-1095 4 4 333 340 20181000 10.1002/vms3.121 30273973 fuminori_tanihara/published_papers/14024040 Tanihara F Hirata M Nguyen NT Le QA Hirano T Takemoto T Nakai M Fuchimoto DI Otoi T Generation of PDX-1 mutant porcine blastocysts by introducing CRISPR/Cas9-system into porcine zygotes via electroporation. Recently, we established the GEEP ("gene editing by electroporation of Cas9 protein") method, in which the CRISPR/Cas9 system, consisting of a Cas9 protein and single guide RNA (sgRNA), is introduced into pig zygotes by electroporation and thus induces highly efficient targeted gene disruption. In this study, we examined the effects of sgRNA on the blastocyst formation of porcine embryos and evaluated their genome-editing efficiency. To produce an animal model for diabetes, we targeted PDX-1 (pancreas duodenum homeobox 1), a gene that is crucial for pancreas development during the fetal period and whose monoallelic disruption impairs insulin secretion. First, Cas9 protein with different sgRNAs that targeted distinct sites in the PDX-1 exon 1 was introduced into in vitro-fertilized zygotes by the GEEP method. Of the six sgRNAs tested, three sgRNAs (sgRNA1, 2, and 3) successfully modified PDX-1 gene. The blastocyst formation rate of zygotes edited with sgRNA3 was significantly (p < 0.05) lower than that of control zygotes without the electroporation treatment. Our study indicates that the GEEP method can be successfully used to generate PDX-1 mutant blastocysts, but the development and the efficiency of editing the genome of zygotes may be affected by the sgRNA used for CRISPR/Cas9 system. Animal science journal = Nihon chikusan Gakkaiho 1344-3941 90 1 55 61 20181000 10.1111/asj.13129 30368976 fuminori_tanihara/published_papers/7252505 Nguyen TV Wittayarat M Do LTK Nguyen TV Nii M Namula Z Kunihara T Tanihara F Hirata M Otoi T Effects of chlorogenic acid (CGA) supplementation during in vitro maturation culture on the development and quality of porcine embryos with electroporation treatment after in vitro fertilization. Electroporation is the technique of choice to introduce an exogenous gene into embryos for transgenic animal production. Although this technique is practical and effective, embryonic damage caused by electroporation treatment remains a major problem. This study was conducted to evaluate the optimal culture system for electroporation-treated porcine embryos by supplementation of chlorogenic acid (CGA), a potent antioxidant, during in vitro oocyte maturation. The oocytes were treated with various concentrations of CGA (0, 10, 50, and 100 μmol/L) through the duration of maturation for 44 hr. The treated oocytes were then fertilized, electroporated at 30 V/mm with five 1 msec unipolar pulses, and subsequently cultured in vitro until development into the blastocyst stage. Without electroporation, the treatment with 50 μmol/L CGA had useful effects on the maturation rate of oocytes, the total cell number, and the apoptotic nucleus indices of blastocysts. When the oocytes were electroporated after in vitro fertilization, the treatment with 50 μmol/L CGA supplementation significantly improved the rate of oocytes that developed into blastocysts and reduced the apoptotic nucleus indices (4.7% and 7.6, respectively) compared with those of the untreated group (1.4% and 13.0, respectively). These results suggested that supplementation with 50 μmol/L CGA during maturation improves porcine embryonic development and quality of electroporation-treated embryos. Animal science journal = Nihon chikusan Gakkaiho 1344-3941 89 8 1207 1213 20180800 10.1111/asj.13049 29806122 fuminori_tanihara/published_papers/7252503 Namula Z Hirata M Wittayarat M Tanihara F Thi Nguyen N Hirano T Nii M Otoi T Effects of chlorogenic acid and caffeic acid on the quality of frozen-thawed boar sperm. Chlorogenic acid (CGA) and caffeic acid (CA) are potent antioxidants that are mostly found in coffee beans. This study aimed to investigate the effects of CGA and CA supplementation during semen freezing on the quality of frozen-thawed boar spermatozoa. The antioxidants CGA and CA were added to a semen extender to achieve final concentrations of 50, 100, 200 and 400 µM. Supplementation of 100 µM CGA and CA yielded a significantly higher percentage of sperm viability (increased by 8%-10%) and plasma membrane integrity (increased by 4%-6%) than the control groups without the antioxidants at 0 and 3 hr after thawing (p < 0.05). At a concentration of 100 µM, CGA and CA also yielded beneficial effects on total and progressive sperm motility. Increases of CGA and CA concentrations to more than 200 µM did not enhance any sperm quality parameters. When the sperm penetrability and oocyte development by spermatozoa frozen with CGA and CA were evaluated, CGA and CA supplementations had no positive effects on the percentages of total fertilization, monospermic fertilization, cleavage and blastocyst formation. In conclusion, the supplementation of 100 µM CGA and CA during sperm freezing improved certain sperm parameters including motility, viability and plasma membrane integrity. Reproduction in domestic animals = Zuchthygiene 0936-6768 53 6 1600 1604 20180700 10.1111/rda.13288 30053311 fuminori_tanihara/published_papers/7252506 Tanihara F Hirata M Nhien NT Hirano T Kunihara T Otoi T Effect of ferulic acid supplementation on the developmental competence of porcine embryos during in vitro maturation. The value of laboratory and genetically-modified pigs is becoming increasingly clear; however, their in vitro development remains inefficient. Trans-ferulic acid (trans-FA) is an aromatic compound that is abundant in plant cell walls, and which exhibits antioxidant effects in vitro. Trans-FA is known to improve sperm viability and motility; however, its effects on porcine oocytes are unknown. Our aim was to investigate the effects of trans-FA supplementation during in vitro maturation on the meiotic and developmental competence of porcine oocytes. Oocytes were matured either without (control) or with trans-FA (10, 100 and 1,000 µM), fertilized, and cultured in vitro for 7 days. The maturation rate of oocytes cultured with 10 µM trans-FA (81.6%) was significantly higher than that of controls (65.0%; P<0.05). The fertilization rate of oocytes matured with 10 µM trans-FA (57.4%) was also significantly higher than that of controls (32.7%) and oocytes cultured with other concentrations (33.1% and 22.7% for 100 and 1,000 µM, respectively; P<0.05). Moreover, the blastocyst formation rate of oocytes matured with 10 µM trans-FA (6.9%) was significantly higher than that of controls (2.3%; P<0.05). Our results suggest that in vitro maturation with 10 µM trans-FA is beneficial for the in vitro production of porcine embryos and has the potential to improve production system. The Journal of veterinary medical science 0916-7250 80 6 1007 1011 20180600 10.1292/jvms.18-0131 29769451 fuminori_tanihara/published_papers/14024020 K. Nishio F. Tanihara T. V. Nguyen T. Kunihara M. Nii M. Hirata T. Takemoto T. Otoi Effects of voltage strength during electroporation on the development and quality of in vitro-produced porcine embryos Blackwell Publishing Ltd Reproduction in Domestic Animals 1439-0531 53 2 313 318 20180401 10.1111/rda.13106 29135047 2-s2.0-85033793022 fuminori_tanihara/published_papers/14024021 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 Effects of antifreeze protein supplementation on the development of porcine morulae stored at hypothermic temperatures Cryo-Letters 0143-2044 39 2 131 136 20180301 29734422 2-s2.0-85050717199 fuminori_tanihara/published_papers/7252504 Ono T Takagi M Kawashima C Wijayagunawardane MPB Vos PLAM Taniguchi M Tanihara F Otoi T Comparative Effects of Different Dosages of hCG on Follicular Development in Postpartum Dairy Cows With Cystic Ovarian Follicles. Frontiers in veterinary science 2297-1769 5 130 null 20180000 10.3389/fvets.2018.00130 30009161 fuminori_tanihara/published_papers/34145565 Maki Hirata Fuminori Tanihara Masayasu Taniguchi Mitsuhiro Takagi Tsukasa Terazono Takeshige Otoi Viability of Canine Ovaries Autografted to Different Peripheral Sites INTERNATIONAL JOURNAL OF APPLIED RESEARCH IN VETERINARY MEDICINE 1559-470X 16 2 140 148 20180000 fuminori_tanihara/published_papers/34145557 Megumi Shimazaki Saki Urasoko Masako Tanaka Yoko Sato Fuminori Tanihara Maki Hirata Masayasu Taniguchi Mitsuhiro Takagi Takeshige Otoi Effects of Orvus Es Paste (OEP) on the Viability of Bull Spermatozoa After Double Freezing and Thawing INTERNATIONAL JOURNAL OF APPLIED RESEARCH IN VETERINARY MEDICINE 1559-470X 16 1 33 39 20180000 fuminori_tanihara/published_papers/14024039 Tanihara F Hirata M Nguyen NT Le QA Hirano T Takemoto T Nakai M Fuchimoto DI Otoi T Generation of a TP53-modified porcine cancer model by CRISPR/Cas9-mediated gene modification in porcine zygotes via electroporation. TP53 (which encodes p53) is one of the most frequently mutated genes in cancers. In this study, we generated TP53-mutant pigs by gene editing via electroporation of the Cas9 protein (GEEP), a process that involves introducing the Cas9 protein and single-guide RNA (sgRNA) targeting exon 3 and intron 4 of TP53 into in vitro-fertilized zygotes. Zygotes modified by the sgRNAs were transferred to recipients, two of which gave birth to a total of 11 piglets. Of those 11 piglets, 9 survived. Molecular genetic analysis confirmed that 6 of 9 live piglets carried mutations in TP53, including 2 piglets with no wild-type (WT) sequences and 4 genetically mosaic piglets with WT sequences. One mosaic piglet had 142 and 151 bp deletions caused by a combination of the two sgRNAs. These piglets were continually monitored for 16 months and three of the genome-edited pigs (50%) exhibited various tumor phenotypes that we presumed were caused by TP53 mutations. Two mutant pigs with no WT sequences developed mandibular osteosarcoma and nephroblastoma. The mosaic pig with a deletion between targeting sites of two sgRNAs exhibited malignant fibrous histiocytoma. Tumor phenotypes of TP53 mosaic mutant pigs have not been previously reported. Our results indicated that the mutations caused by gene editing successfully induced tumor phenotypes in both TP53 mosaic- and bi-allelic mutant pigs. PloS one 13 10 e0206360 null 20180000 10.1371/journal.pone.0206360 30352075 fuminori_tanihara/published_papers/14024022 T-V Nguyen F. Tanihara L. T. K. Do Y. Sato M. Taniguchi M. Takagi T. Van Nguyen T. Otoi Chlorogenic acid supplementation during in vitro maturation improves maturation, fertilization and developmental competence of porcine oocytes REPRODUCTION IN DOMESTIC ANIMALS 1439-0531 52 6 969 975 20171200 10.1111/rda.13005 2-s2.0-85021411742 fuminori_tanihara/published_papers/7252507 Katsutoshi Nishio Mado Yamazaki Masayasu Taniguchi Kazuhiko Besshi Fumio Morita Toshiki Kunihara Fuminori Tanihara Tatsuya Takemoto Takeshige Otoi SENSITIVITY OF THE MEIOTIC STAGE TO HYPERTHERMIA DURING IN VITRO MATURATION OF PORCINE OOCYTES ACTA VETERINARIA HUNGARICA 1588-2705 65 1 115 123 20170300 10.1556/004.2017.012 28244334 fuminori_tanihara/published_papers/14024023 L. T. K. Do M. Wittayarat T. Terazono Y. Sato M. Taniguchi F. Tanihara T. Takemoto Y. Kazuki K. Kazuki M. Oshimura T. Otoi Effects of duration of electric pulse on in vitro development of cloned cat embryos with human artificial chromosome vector REPRODUCTION IN DOMESTIC ANIMALS 1439-0531 51 6 1039 1043 20161200 10.1111/rda.12766 27568550 2-s2.0-84983472668 fuminori_tanihara/published_papers/7252509 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 Somatic cell reprogramming-free generation of genetically modified pigs SCIENCE ADVANCES 2375-2548 2 9 e1600803 null 20160900 10.1126/sciadv.1600803 27652340 fuminori_tanihara/published_papers/7252508 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 CHARACTERISTICS AND FERTILITY OF SUMATRAN TIGER SPERMATOZOA CRYOPRESERVED WITH DIFFERENT SUGARS CRYOLETTERS 1742-0644 37 4 264 271 20160700 27925009 fuminori_tanihara/published_papers/7252510 Yasuhiro Morita Masayasu Taniguchi Fuminori Tanihara Aya Ito Zhao Namula Lanh Thi Kim Do Mitsuhiro Takagi Tatsuya Takemoto Takeshige Otoi The optimal period of Ca-EDTA treatment for parthenogenetic activation of porcine oocytes during maturation culture JOURNAL OF VETERINARY MEDICAL SCIENCE 1347-7439 78 6 1019 1023 20160600 10.1292/jvms.15-0658 26947170 fuminori_tanihara/published_papers/14024024 M. Nakai J. Ito N. Kashiwazaki N. T. Men F. Tanihara J. Noguchi H. Kaneko A. Onishi K. Kikuchi Treatment with protein kinase C activator is effective for improvement of male pronucleus formation and further embryonic development of sperm-injected oocytes in pigs THERIOGENOLOGY 1879-3231 85 4 703 708 20160300 10.1016/j.theriogenology.2015.10.011 26559470 2-s2.0-84960431389 fuminori_tanihara/published_papers/28126842 T. Ono T. Isobe Y. Morita L. T. K. Do F. Tanihara M. Taniguchi M. Takagi T. Otoi Effects of parity and season on pregnancy rates after the transfer of embryos to repeat-breeder Japanese Black beef cattle ARCHIVES ANIMAL BREEDING 2363-9822 59 1 45 49 20160000 10.5194/aab-59-45-2016 fuminori_tanihara/published_papers/7252511 Tetsushi Ono Asako Takaoka Yasuhiro Morita Lanh Thi Kim Do Fuminori Tanihara Masayasu Taniguchi Takeshige Otoi EFFECTS OF DIBUTYRYL CYCLIC ADENOSINE MONOPHOSPHATE AND HUMAN CHORIONIC GONADOTROPIN ON THE FORMATION OF ANTRAL FOLLICLE-LIKE STRUCTURES BY BOVINE CUMULUS-OOCYTE COMPLEXES ACTA VETERINARIA HUNGARICA 1588-2705 63 4 485 498 20151200 10.1556/004.2015.045 26599095 fuminori_tanihara/published_papers/14024026 L. T. K. Do Y. Shibata M. Taniguchi M. Nii T. V. Nguyen F. Tanihara M. Takagi T. Otoi Melatonin Supplementation During In Vitro Maturation and Development Supports the Development of Porcine Embryos REPRODUCTION IN DOMESTIC ANIMALS 1439-0531 50 6 1054 1058 20151200 10.1111/rda.12607 26392209 2-s2.0-84947036615 fuminori_tanihara/published_papers/7252522 Tomohiro Isobe Yoshihisa Ikebata Lanh Thi Kim Do Fuminori Tanihara Masayasu Taniguchi Takeshige Otoi In vitro development of OPU-derived bovine embryos cultured either individually or in groups with the silk protein sericin and the viability of frozen-thawed embryos after transfer ANIMAL SCIENCE JOURNAL 1740-0929 86 7 661 665 20150700 10.1111/asj.12341 25488699 fuminori_tanihara/published_papers/14024025 Megumi Shimazaki Rentsenkhand Sambuu Yoko Sato Lanh Thi Kim Do Fuminori Tanihara Masayasu Taniguchi Takeshige Otoi EFFECTS OF ORVUS ES PASTE ON THE MOTILITY AND VIABILITY OF YAK (BOS GRUNNIENS) EPIDIDYMAL AND EJACULATED SPERMATOZOA AFTER FREEZING AND THAWING CRYOLETTERS 1742-0644 36 4 264 269 20150700 26576001 2-s2.0-84956578451 fuminori_tanihara/published_papers/7252521 Yasuhiro Morita Ni Wayan Kurniani Karja Lanh Thi Kim Do Fuminori Tanihara Masayasu Taniguchi Takeshige Otoi Formation of an Antral Follicle-Like Structure by Bovine Cumulus-Oocyte Complexes Embedded with Fragmin/Protamine Microparticles ANIMAL BIOTECHNOLOGY 1532-2378 26 4 273 275 20150000 10.1080/10495398.2015.1015681 26158458 fuminori_tanihara/published_papers/7252524 Hiroyuki Kaneko Kazuhiro Kikuchi Fuminori Tanihara Junko Noguchi Michiko Nakai Junya Ito Naomi Kashiwazaki Normal reproductive development of pigs produced using sperm retrieved from immature testicular tissue cryopreserved and grafted into nude mice THERIOGENOLOGY 1879-3231 82 2 325 331 20140700 10.1016/j.theriogenology.2014.04.012 24835638 fuminori_tanihara/published_papers/7252527 Manita Wittayarat Akira Fujiwara Kaywalee Chatdarong Mongkol Techakumphu Yoko Sato Fuminori Tanihara Yasuhiro Morita Masayasu Taniguchi Takeshige Otoi CELL CYCLE ANALYSIS AND INTERSPECIES NUCLEAR TRANSFER OF CAT CELLS TREATED WITH CHEMICAL INHIBITORS ACTA VETERINARIA HUNGARICA 1588-2705 62 2 233 242 20140600 10.1556/AVet.2013.050 24334073 fuminori_tanihara/published_papers/7252523 Tamas Somfai Koji Yoshioka Fuminori Tanihara Hiroyuki Kaneko Junko Noguchi Naomi Kashiwazaki Takashi Nagai Kazuhiro Kikuchi Generation of Live Piglets from Cryopreserved Oocytes for the First Time Using a Defined System for In Vitro Embryo Production PLOS ONE 1932-6203 9 5 e97731 null 20140500 10.1371/journal.pone.0097731 24844283 fuminori_tanihara/published_papers/7252525 Fuminori Tanihara Michiko Nakai Nguyen Thi Men Noriko Kato Hiroyuki Kaneko Junko Noguchi Takeshige Otoi Kazuhiro Kikuchi Roles of the zona pellucida and functional exposure of the sperm-egg fusion factor 'IZUMO' during in vitro fertilization in pigs ANIMAL SCIENCE JOURNAL 1740-0929 85 4 395 404 20140400 10.1111/asj.12164 24450993 fuminori_tanihara/published_papers/7252526 Zhao Namula Namula Z Risa Kodama Kodama R Fuminori Tanihara Tanihara F Yasuhiro Morita Morita Y Yoko Sato Sato Y Manita Wittayarat Wittayarat M Masayasu Taniguchi Taniguchi M Takeshige Otoi Otoi T EFFECTS OF SKIM-MILK SUPPLEMENTATION ON THE QUALITY AND PENETRATING ABILITY OF BOAR SEMEN AFTER LONG-TERM PRESERVATION AT 15 degrees C Effects of skim-milk supplementation on the quality and penetrating ability of boar semen after long-term preservation at 15 °C. ACTA VETERINARIA HUNGARICA Acta veterinaria Hungarica 1588-2705 62 1 106 116 20140300 10.1556/AVet.2013.046 24334075 fuminori_tanihara/published_papers/7252529 Istvan Egerszegi Tamas Somfai Michiko Nakai Fuminori Tanihara Junko Noguchi Hiroyuki Kaneko Takashi Nagai Jozsef Ratky Kazuhiro Kikuchi Comparison of cytoskeletal integrity, fertilization and developmental competence of oocytes vitrified before or after in vitro maturation in a porcine model CRYOBIOLOGY 1090-2392 67 3 287 292 20131200 10.1016/j.cryobiol.2013.08.009 23993921 fuminori_tanihara/published_papers/7252528 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 Effect of trehalose on DNA integrity of freeze-dried boar sperm, fertilization, and embryo development after intracytoplasmic sperm injection THERIOGENOLOGY 1879-3231 80 9 1033 1044 20131200 10.1016/j.theriogenology.2013.08.001 24041826 fuminori_tanihara/published_papers/7252513 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 Fertilization Ability of Porcine Oocytes Reconstructed from Ooplasmic Fragments Produced and Characterized after Serial Centrifugations JOURNAL OF REPRODUCTION AND DEVELOPMENT 0916-8818 59 6 549 556 20131200 10.1262/jrd.2013-042 23965685 fuminori_tanihara/published_papers/7252512 Fuminori Tanihara Yukine Kaedei Zhao Namula Vien Viet Luu Yoko Sato Manita Wittayarat Masayasu Taniguchi Takeshige Otoi COMPARISON OF ACTIVATION ABILITY BETWEEN FELINE AND BOVINE OOCYTES ACTA VETERINARIA HUNGARICA 1588-2705 61 4 491 494 20131200 10.1556/AVet.2013.032 23974933 fuminori_tanihara/published_papers/7252530 Hiroyuki Kaneko Michiko Nakai Fuminori Tanihara Junko Noguchi Kazuhiro Kikuchi Improved developmental ability of porcine oocytes grown in nude mice after fusion with cytoplasmic fragments prepared by centrifugation: A model for utilization of primordial oocytes THERIOGENOLOGY 1879-3231 80 8 887 892 20131100 10.1016/j.theriogenology.2013.07.014 23981298 fuminori_tanihara/published_papers/7252518 Zhao Namula Namula Z Yoko Sato Sato Y Risa Kodama Kodama R Kouta Morinaga Morinaga K Vien Viet Luu Luu VV Masayasu Taniguchi Taniguchi M Michiko Nakai Nakai M Fuminori Tanihara Tanihara F Kazuhiro Kikuchi Kikuchi K Takashi Nagai Nagai T Takeshige Otoi Otoi T Motility and fertility of boar semen after liquid preservation at 5 degrees C for more than 2 weeks Motility and fertility of boar semen after liquid preservation at 5°C for more than 2 weeks. ANIMAL SCIENCE JOURNAL Animal science journal = Nihon chikusan Gakkaiho 1344-3941 84 8 600 606 20130800 10.1111/asj.12049 23607795 fuminori_tanihara/published_papers/7252516 Fuminori Tanihara Michiko Nakai Hiroyuki Kaneko Junko Noguchi Takeshige Otoi Kazuhiro Kikuchi Evaluation of Zona Pellucida Function for Sperm Penetration During In Vitro Fertilization in Pigs JOURNAL OF REPRODUCTION AND DEVELOPMENT 0916-8818 59 4 385 392 20130800 10.1262/jrd.2013-021 23666494 fuminori_tanihara/published_papers/7252514 Hiroyuki Kaneko Kazuhiro Kikuchi Michiko Nakai Tamas Somfai Junko Noguchi Fuminori Tanihara Junya Ito Naomi Kashiwazaki Generation of Live Piglets for the First Time Using Sperm Retrieved from Immature Testicular Tissue Cryopreserved and Grafted into Nude Mice PLOS ONE 1932-6203 8 7 e70989 null 20130700 10.1371/journal.pone.0070989 23923039 fuminori_tanihara/published_papers/7252515 Vien Viet Luu Luu VV Keisuke Hanatate Hanatate K Fuminori Tanihara Tanihara F Yoko Sato Sato Y Lanh Thi Kim Do Do LT Masayasu Taniguchi Taniguchi M Takeshige Otoi Otoi T The effect of relaxin supplementation of in vitro maturation medium on the development of cat oocytes obtained from ovaries stored at 4 degrees C The effect of relaxin supplementation of in vitro maturation medium on the development of cat oocytes obtained from ovaries stored at 4°C. REPRODUCTIVE BIOLOGY Reproductive biology 1642-431X 13 2 122 126 20130600 10.1016/j.repbio.2013.04.002 23719116 fuminori_tanihara/published_papers/7252517 Tamás Somfai Michiko Nakai Fuminori Tanihara Junko Noguchi Hiroyuki Kaneko Naomi Kashiwazaki István Egerszegi Takashi Nagai Kazuhiro Kikuchi Comparison of ethylene glycol and propylene glycol for the vitrification of immature porcine oocytes Journal of Reproduction and Development 0916-8818 59 4 378 384 20130000 10.1262/jrd.2013-015 23666455 2-s2.0-84879550324 fuminori_tanihara/published_papers/14024027 Y. Kaedei M. Naito H. Naoi Y. Sato M. Taniguchi F. Tanihara K. Kikuchi T. Nagai T. Otoi Effects of (-)-Epigallocatechin Gallate on the Motility and Penetrability of Frozen-Thawed Boar Spermatozoa Incubated in the Fertilization Medium REPRODUCTION IN DOMESTIC ANIMALS 0936-6768 47 6 880 886 20121200 10.1111/j.1439-0531.2012.01984.x 22299777 2-s2.0-84868685306 fuminori_tanihara/published_papers/7252519 Hiroyuki Kaneko Kazuhiro Kikuchi Michiko Nakai Fuminori Tanihara Junko Noguchi Michiko Noguchi Junya Ito Naomi Kashiwazaki Normal reproductive development of offspring derived by intracytoplasmic injection of porcine sperm grown in host mice THERIOGENOLOGY 0093-691X 78 4 898 906 20120900 10.1016/j.theriogenology.2012.04.004 22626781 fuminori_tanihara/published_papers/14024028 T. Terazono Y. Kaedei F. Tanihara Z. Namula V. L. Viet M. Takagi M. Inoue Y. Sato M. Taniguchi T. Otoi Follicle Formation in the Canine Ovary After Autografting to a Peripheral Site REPRODUCTION IN DOMESTIC ANIMALS 0936-6768 47 2 e16 e21 20120400 10.1111/j.1439-0531.2011.01880.x 21883513 2-s2.0-84858439180 fuminori_tanihara/published_papers/14024029 T. Terazono M. Inoue Y. Kaedei F. Tanihara Z. Namula V. L. Viet Y. Taura M. Takagi T. Takuma T. Otoi Assessment of canine ovaries autografted to various body sites THERIOGENOLOGY 0093-691X 77 1 131 138 20120100 10.1016/j.theriogenology.2011.07.026 21872320 2-s2.0-83055194532 fuminori_tanihara/published_papers/7252520 Masao Murakami Ya Juan Dong Tatsuyuki Suzuki Masayasu Taniguchi Yukine Kaedei Yoko Sato Fuminori Tanihara Takeshige Otoi Development and subsequent cryotolerance of domestic cat embryos cultured in serum-free and serum-containing media CRYOBIOLOGY 0011-2240 63 3 170 174 20111200 10.1016/j.cryobiol.2011.06.002 21893054 fuminori_tanihara/published_papers/14024030 Masayasu Taniguchi Rie Arikawa Yukine Kaedei Fuminori Tanihara Zhao Namula Vien Luu Viet Yoko Sato Takeshige Otoi EFFECTS OF CRYOPROTECTANT AGENTS AND EQUILIBRATION METHODS ON DEVELOPMENTAL COMPETENCE OF PORCINE OOCYTES CRYOLETTERS 1742-0644 32 5 410 414 20110900 22020463 2-s2.0-81255147116 fuminori_tanihara/published_papers/14024031 E. V. Abakushina Y. Morita Y. Kaedei F. Tanihara Z. Namula V. L. Viet T. Otoi Formation of an Antral Follicle-like Structure of Bovine Cumulus-Oocyte Complexes Embedded Individually or in Groups in Collagen Gels REPRODUCTION IN DOMESTIC ANIMALS 0936-6768 46 3 423 427 20110600 10.1111/j.1439-0531.2010.01684.x 20723136 2-s2.0-79955648322 fuminori_tanihara/published_papers/14024034 A. Fujii Y. Kaedei F. Tanihara A. Ito K. Hanatate K. Kikuchi T. Nagai T. Otoi In Vitro Maturation and Development of Porcine Oocytes Cultured in a Straw or Dish Using a Portable Incubator with a CO2 Chamber REPRODUCTION IN DOMESTIC ANIMALS 0936-6768 45 4 619 624 20100800 10.1111/j.1439-0531.2008.01316.x 19144033 2-s2.0-77956131585 fuminori_tanihara/published_papers/14024035 Morteza Yavari Hideaki Naoi Yukine Kaedei Fuminori Tanihara Zhao Namula Vien Luu Viet Takeshige Otoi Effects of epigallocatechin-3-gallate on the developmental competence of parthenogenetic embryos in the pig ITALIAN JOURNAL OF ANIMAL SCIENCE 1594-4077 9 4 386 389 20100000 10.4081/ijas.2010.e73 2-s2.0-78449305529 fuminori_tanihara/published_papers/14024033 Yukine Kaedei Akira Fujiwara Fuminori Tanihara Zhao Namula Vien Luu Viet Takeshige Otoi IN VITRO DEVELOPMENT OF CAT INTERSPECIES NUCLEAR TRANSFER USING PIG&apos;S AND COW&apos;S CYTOPLASM BULLETIN OF THE VETERINARY INSTITUTE IN PULAWY 0042-4870 54 3 405 408 20100000 2-s2.0-77957945739 fuminori_tanihara/published_papers/14024032 Yukine Kaedei Akira Fujiwara Aya Ito Fuminori Tanihara Yasuhiro Morita Keisuke Hanatate Vien Luu Viet Zhao Namula Takeshige Otoi Effect of Roscovitine Pretreatment on the Meiotic Maturation of Bovine Oocytes and their Subsequent Development after Somatic Cell Nuclear Transfer JOURNAL OF ANIMAL AND VETERINARY ADVANCES 1680-5593 9 22 2848 2853 20100000 10.3923/javaa.2010.2848.2853 2-s2.0-78649803917 fuminori_tanihara/published_papers/14024036 Budiyanto Agung Farid Barati Yukine Kaedei Fuminori Tanihara Takeshige Otoi Meiotic Competence and DNA Damage of Porcine Oocytes from Ovaries Exposed to an Elevated Temperature Journal of Animal and Veterinary Advances 1680-5593 7 10 1179 1183 20081000 2-s2.0-53949123120 fuminori_tanihara/misc/28648474 K. Kikuchi N. V. Linh M. Nakai F. Tanihara J. Noguchi H. Kaneko T. Q. Dang-Nguyen N. T. Men N. Van Hanh T. Somfai B. X. Nguyen T. Nagai N. Manabe Fusion of cytoplasmic fragments to porcine oocytes improves the ability for embryonic development REPRODUCTION IN DOMESTIC ANIMALS 0936-6768 47 451 451 20120800 fuminori_tanihara/misc/28618265 F. Tanihara M. Nakai H. Kaneko J. Noguchi T. Otoi K. Kikuchi Effects of the zona pellucida on in vitro sperm penetration into porcine oocytes REPRODUCTION IN DOMESTIC ANIMALS 0936-6768 47 513 514 20120800 fuminori_tanihara/misc/28633592 V. V. Luu Z. Namula Y. Kaedei F. Tanihara T. Otoi EFFECTS OF OVARY STORAGE TIME ON THE QUALITY AND MEIOTIC COMPETENCE OF CAT OOCYTES REPRODUCTION FERTILITY AND DEVELOPMENT 1031-3613 24 1 205 206 20120000 fuminori_tanihara/misc/28329316 Z. Namula R. Kodama Y. Kaedei F. Tanihara V. L. Vien T. Otoi EFFECTS OF SKIM MILK ON THE QUALITY AND FERTILITY OF BOAR SEMEN FOLLOWING LIQUID PRESERVATION AT 5 degrees C AND 15 degrees C REPRODUCTION FERTILITY AND DEVELOPMENT 1031-3613 23 1 115 116 20110000 fuminori_tanihara/misc/28327045 Y. Kaedei A. Fujiwara F. Tanihara Z. Namula V. L. Vien T. Otoi IN VITRO DEVELOPMENT OF CANINE EMBRYOS PRODUCED BY INTERSPECIES SOMATIC CELL NUCLEAR TRANSFER USING ENUCLEATED BOVINE OOCYTES REPRODUCTION FERTILITY AND DEVELOPMENT 1031-3613 23 1 126 126 20110000 fuminori_tanihara/misc/28269167 T. Terazono Y. Kaedei Z. Namula V. L. Vien F. Tanihara T. Otoi VIABILITY OF OOCYTES FROM CANINE OVARIES GRAFTED IN THE PROXIMAL PORTION OF THE BODY SURFACE REPRODUCTION FERTILITY AND DEVELOPMENT 1031-3613 23 1 233 234 20110000 fuminori_tanihara/misc/52357286 谷原史倫 谷原史倫 菊地和弘 菊地和弘 中井美智子 野口純子 金子浩之 鈴木千恵 吉岡耕治 永井卓 大豆由来レシチンを用いたブタ凍結精液の受精・発生能 Journal of Reproduction and Development 0916-8818 56 Suppl Japanese Issue null null 20100000