Fuminori Tanihara, Maki Hirata and Takeshige Otoi : GEEP Method: An Optimized Electroporation-Mediated Gene Editing Approach for Establishment of Knockout Pig Lines., Methods Mol Biol., 2023.
(Summary)
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.
Thanh-Van Nguyen, Kim Lanh Thi Do, Qingyi Lin, Megumi Nagahara, Zhao Namula, Manita Wittayarat, Maki Hirata, Takeshige Otoi and Fuminori Tanihara : Programmed cell death-1-modified pig developed using electroporation-mediated gene editing for in vitro fertilized zygotes., In Vitro Cellular & Developmental Biology. Animal, 2024.
(Summary)
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.
Thanh-Van Nguyen, Koki Takebayashi, Kim Lanh Thi Do, Zhao Namula, Manita Wittayarat, Megumi Nagahara, Maki Hirata, Takeshige Otoi and Fuminori Tanihara : Generation of allogenic chimera carrying mutations in PDX1 and TP53 genes via phytohemagglutinin-mediated blastomere aggregation in pigs., In Vitro Cellular & Developmental Biology. Animal, 2024.
(Summary)
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.
Supitcha Kaewma, Zhao Namula, Thi Suong Nguyen, Qingyi Lin, Nanaka Torigoe, Bin Liu, Megumi Nagahara, Masahiro Nii, Masayasu Taniguchi and Takeshige Otoi : Effects of ergothioneine supplementation on the quality of liquid-preserved and frozen-thawed boar semen., Acta Veterinaria Hungarica, Vol.71, No.3-4, 219-222, 2024.
(Summary)
This study examined the effects of ergothioneine (EGT) supplementation as an antioxidant on the quality of boar spermatozoa when using liquid and frozen preservation methods. In the first experiment, boar semen was preserved in an extender supplemented with 0, 50, 100 and 200 µM EGT, at 15 °C, part of the samples for one and another part for three weeks. In comparison with the control (without EGT), EGT supplementation at 100 µM significantly increased the percentage of total motility of spermatozoa that were preserved as a liquid both for one and three weeks (P < 0.05). EGT supplementation did not affect the quality of preserved spermatozoa, irrespective of the EGT concentration. In the second experiment, semen was frozen and thawed in the freezing extender supplemented with 0, 50, 100 and 200 µM EGT. In comparison with the control, the 100 µM EGT supplementation significantly increased the percentages of total and progressive motility of frozen-thawed spermatozoa (P < 0.05). EGT (100 µM) supplementation did not affect the viability, the plasma membrane integrity, or the acrosomal integrity of frozen-thawed spermatozoa. These findings indicate that supplementing extenders with 100 µM EGT may improve the motility of boar sperm in both liquid and freezing preservation methods.
T Suong Nguyen, Ayane Edo, Megumi Nagahara, Takeshige Otoi, Masayasu Taniguchi and Mitsuhiro Takagi : Selection of spermatozoa with high motility and quality from bovine frozen-thawed semen using the centrifuge-free device., Animal Reproduction Science, Vol.260, No.260, 2024.
(Summary)
This study aimed to assess the potential of the centrifuge-free commercial device (MIGLIS®) in selecting functional frozen-thawed bovine sperm by migration-sedimentation, its effect on embryo development, and compare the potential with that of centrifugation-based techniques, including washing and Percoll density gradient centrifugation (DGC). In experiment 1, different dilutions (1.5×, 2×, and 3×) of frozen-thawed spermatozoa were assessed to identify the adequate one for the MIGLIS method. In experiment 2, the recovery rates, quality, and reactive oxygen species (ROS) concentrations of the spermatozoa selected using MIGLIS, washing, and Percoll DGC were compared. In experiment 3, the resultant in vitro fertilised embryos from spermatozoa selected using the three methods were evaluated for blastocyst formation rates and intracellular ROS concentrations at the 2-4 cell stage. The intracellular ROS concentrations were investigated using 2', 7'-dichlorodihydrofluorescein diacetate staining. Using the MIGLIS device, significantly more spermatozoa were recovered at 2× dilution compared with the other dilution ratio, but the motility was not affected by the dilution ratio. On the selection of spermatozoa using the three methods, employing MIGLIS decreased the recovery rates. However, the MIGLIS method increased motility, viability, and acrosome integrity rates compared to those in spermatozoa from the other methods. The ROS concentration of spermatozoa in the MIGLIS method was significantly lower than that in the washing method. Nevertheless, blastocyst formation rates were similar among the three methods, but the ROS concentration of early-stage embryos produced using MIGLIS was significantly lower than those produced using Percoll DGC. In conclusion, the MIGLIS method has the potential to select functional, high-quality frozen-thawed bovine spermatozoa.
Nanaka Torigoe, Megumi Nagahara, Thi Suong Nguyen, Qingyi Lin, Koki Takebayashi, Bin Liu, Mutsumi Aihara, Masayasu Taniguchi and Takeshige Otoi : Development of porcine embryos cultured in media irradiated with ultraviolet-C., Reproduction in Domestic Animals = Zuchthygiene, Vol.59, No.1, e14520, 2024.
(Summary)
Sterilization of the culture medium using ultraviolet (UV)-C reduces the potential adverse effects of microorganisms and allows for long-term use. In the present study, we investigated the effects of a medium directly irradiated with UV-C prior to in vitro culture on the development and quality of porcine in vitro-fertilized embryos and the free amino acid composition of the culture media. The culture media (porcine zygote medium [PZM-5] and porcine blastocyst medium [PBM]) were irradiated with UV-C at 228 and 260 nm for 1 and 3 days, respectively. Next, the culture media were irradiated with UV-C at 228 nm for 3, 7, or 14 days. After in vitro fertilization, the embryos were cultured in the UV-C-irradiated media for 7 days. Free amino acid levels in culture media irradiated with 228 and 260 nm UV-C for 3 days were analysed. The blastocyst formation rate of embryos cultured in media irradiated with 260 nm UV-C for 3 days was significantly lower than that of embryos cultured in non-irradiated control media. However, 228 nm UV-C irradiation for up to 14 days did not affect blastocyst formation rates and quality in the resulting blastocysts. Moreover, 260 nm UV-C irradiation significantly increased the taurine concentration in both culture media and decreased methionine concentration in the PBM. In conclusion, UV-C irradiation at 228 nm before in vitro culture had no detrimental effects on embryonic development. However, 260 nm UV-C irradiation decreased embryo development and altered the composition of free amino acids in the medium.
Hashimoto Shogo, Taniguchi Masayasu, Edo Ayane, Ono Tetsushi, Siagian Barunawati Tetty, Sekine Hiroaki, Megumi Nagahara, Takeshige Otoi and Takagi Mitsuhiro : Impact of redox status of donor cows before superovulation treatment on in vivo embryo production., Archives Animal Breeding, Vol.66, 433-437, 2023.
8.
Takeshi Kikuchi, Masuhiro Nishimura, Natsuki Komori, Naho Iizuka, Takeshige Otoi and Shinichi Matsumoto : Development and characterization of islet-derived mesenchymal stem cells from clinical grade neonatal porcine cryopreserved islets., Xenotransplantation, e12831, 2023.
(Summary)
The method described herein was successful in of developing clinical grade npISLET-MSCs from cryopreserved islets. Cryopreserved clinical grade porcine islets could be an excellent stable source of MSCs for cell therapy.
Qingyi Lin, Koki Takebayashi, Nanaka Torigoe, Bin Liu, Zhao Namula, Maki Hirata, Fuminori Tanihara, Megumi Nagahara and Takeshige Otoi : Comparison of chemically mediated CRISPR/Cas9 gene editing systems using different nonviral vectors in porcine embryos., Animal Science Journal, Vol.94, No.1, e13878, 2023.
(Summary)
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.
Hisayoshi Omori, Junko Chikamoto, Megumi Nagahara, Maki Hirata and Takeshige Otoi : Evaluating variations in bilirubin glucuronidation activity by protease inhibitors in canine and human primary hepatocytes cultured in a 3D culture system., Toxicology In Vitro, Vol.93, 2023.
(Summary)
Bilirubin is excreted into the bile from hepatocytes, mainly as monoglucuronosyl and bisglucuronosyl conjugates, reflecting bilirubin glucuronidation activity. However, there is limited information on the in vitro evaluation of liver cell lines or primary hepatocytes. This study aimed to investigate variations in the bilirubin metabolic function of canine and human hepatocyte spheroids formed in a three-dimensional (3D) culture system indicated by the formation of bilirubin glucuronides when protease inhibitors such as atazanavir, indinavir, ritonavir, and nelfinavir were treated with bilirubin. The culture supernatant was collected for bilirubin glucuronidation assessment and the cells were used to evaluate viability. On day 8 of culture, both canine and human hepatocyte spheroids showed high albumin secretion and distinct spheroid formation, and their bilirubin glucuronidation activities were evaluated considering cell viability. Treatment with atazanavir and ritonavir remarkably inhibited bilirubin glucuronide formation, wherein atazanavir showed the highest inhibition, particularly in human hepatocyte spheroids. These results may reflect the effects on cellular uptake of bilirubin and its intracellular metabolic function. Thus, primary hepatocytes cultured in a 3D culture system may be a useful in vitro system for the comprehensive evaluation of bilirubin metabolic function and risk assessment in bilirubin metabolic disorders for drug development.
Chommanart Thongkittidilok, Maki Hirata, Qingyi Lin, Nanaka Torigoe, Bin Liu, Yoko Sato, Megumi Nagahara, Fuminori Tanihara and Takeshige Otoi : Mosaic TP53 Mutation on Tumour Development in Pigs: A Case Study., Veterinary Medicine International, Vol.2023, 7000858, 2023.
(Summary)
mutations with truncated amino acids may be related to tumour formation.
Fuminori Tanihara, Maki Hirata, Zhao Namula, Manita Wittayarat, Lanh Thi Kim Do, Qingyi Lin, Koki Takebayashi, Hiromasa Hara, Megumi Nagahara and Takeshige Otoi : GHR-mutant pig derived from domestic pig and microminipig hybrid zygotes using CRISPR/Cas9 system., Molecular Biology Reports, Vol.50, No.6, 5049-5057, 2023.
(Summary)
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. 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. 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.
Megumi Nagahara, Zhao Namula, Qingyi Lin, Koki Takebayashi, Nanaka Torigoe, Bin Liu, Mutsumi Aihara, Masahiro Nii and Takeshige Otoi : Effects of curcumin supplementation on quality of porcine spermatozoa irradiated with ultraviolet-C at 228 nm during liquid preservation., Reproduction in Domestic Animals = Zuchthygiene, 2023.
(Summary)
It is important to prevent microbial contamination during liquid preservation of semen in pigs. We examined the effects of curcumin supplementation on the quality of porcine spermatozoa irradiated with ultraviolet-C (UV-C) at 228 nm. UV-C is used to disinfect gases and solid surfaces. In the first experiment, porcine semen was preserved with 0, 10, 25, 50 or 100 μM curcumin under UV-C irradiation at 228 nm for 7 days at 15°C. The irradiation did not affect the motility and viability of preserved spermatozoa but decreased the percentage of plasma membrane integrity of spermatozoa. Curcumin supplementation at 25 μM significantly improved the plasma membrane and acrosome integrity of irradiated spermatozoa compared with spermatozoa preserved without curcumin (p < .05). In the second experiment, semen was preserved with or without 25 μM curcumin with UV-C irradiation at 228 or 260 nm for 3 days at 15°C. Curcumin supplementation increased the percentages of total motility, sperm viability and plasma membrane integrity of preserved spermatozoa at both irradiation wavelengths (p < .05). All quality parameters of 260 nm irradiated spermatozoa decreased compared to those of the other groups, irrespective of curcumin supplementation. The collective findings indicate that porcine spermatozoa can retain their viability even after continuous long-duration irradiation with 228 nm UV-C. Curcumin supplementation improves the quality of UV-C irradiated spermatozoa during semen preservation.
Van Thanh Nguyen, Kim Lanh Thi Do, Thi Ngoc-Anh Nguyen, Kazuhiro Kikuchi, Tamas Somfai and Takeshige Otoi : Oocyte Maturation System and Chlorogenic Acid Supplementation during Embryo Culture on the Development of Porcine Cloned Embryos Derived from Native Vietnamese Ban Pigs., Veterinary Medicine International, Vol.2023, 2023.
(Summary)
M chlorogenic acid (CGA). Furthermore, this study examined parthenogenetic embryos. The IVM medium and duration of hormone treatment did not affect embryo development. CGA supplementation to the culture medium significantly increased blastocyst formation rates in parthenogenetic embryos but not in SCNT embryos. However, CGA supplementation significantly reduced the apoptotic index in blastocysts regardless of embryo source. In conclusion, the IVM method did not affect SCNT embryo production, while CGA supplementation during embryo culture improved the quality of SCNT embryos in indigenous pig breeds.
Thanh-Van Nguyen, Kim Lanh Thi Do, Zhao Namula, Qingyi Lin, Nanaka Torigoe, Megumi Nagahara, Maki Hirata, Fuminori Tanihara and Takeshige Otoi : Development and Genome Mutation of Bovine Zygotes Vitrified Before and After Genome Editing via Electroporation, Cryo Letters, Vol.44, No.2, 118-122, 2023.
Qingyi Lin, Mutsumi Aihara, Akihiro Shirai, Ami Tanaka, Koki Takebayashi, Naoaki Yoshimura, Nanaka Torigoe, Megumi Nagahara, Takeo Minamikawa and Takeshige Otoi : Porcine embryo development and inactivation of microorganisms after ultraviolet-C irradiation at 228 nm, Theriogenology, Vol.197, 252-258, 2023.
(Summary)
It is important to prevent contamination inside the incubator as a method of preventing microbial infections during the embryo culture. In the present study, we examined the effects of ultraviolet-C (UV-C) irradiation, used for microorganism inactivation, on embryo development and the growth of bacteria, including Escherichia coli and Staphylococcus aureus, and the fungus Cladosporium cladosporioides. In the embryo irradiation experiment, we examined the effects of the plastic lid of the culture dish, irradiation distances (10, 20, and 25 cm), and different irradiation wavelengths (228 and 260 nm) during embryo culture for 7 days on the development and quality of porcine in vitro-fertilized embryos. None of the embryos cultured in dishes without plastic lids developed into blastocysts after irradiation with 228 nm UV-C. When porcine embryos were cultured in a culture dish with lids, the 228 nm UV-C irradiation decreased blastocyst formation rates of the embryos but not their quality, irrespective of the UV-C irradiation distance. Moreover, irradiation with 260 nm UV-C, even with plastic lids, had more detrimental effects on embryo development than irradiation with 228 nm UV-C. Investigation of the inactivating effects of UV-C irradiation at 228 nm and 260 nm on the growth of the bacteria and fungus showed that 260 nm UV-C reduced the viability to a greater extent than 228 nm UV-C. Moreover, the disinfection efficacy for the bacteria increased when the irradiation duration increased and the distance decreased. In conclusion, porcine embryos can develop into blastocysts without loss of quality even after continuous long-duration irradiation (7 days) with 228 nm UV-C, which can inactivate the growth of bacteria and the tested fungus; however, the development rate of the embryo is reduced.
Fuminori Tanihara, Maki Hirata, Zhao Namula, Kim Lanh Thi Do, Naoaki Yoshimura, Qingyi Lin, Koki Takebayashi, Tetsushi Sakuma, Takashi Yamamoto and Takeshige Otoi : Pigs with an INS point mutation derived from zygotes electroporated with CRISPR/Cas9 and ssODN, Frontiers in Cell and Developmental Biology, Vol.11, 2023.
(Summary)
electroporation-mediated introduction of the CRISPR/Cas9 system into zygotes, thereby avoiding the time-consuming and complicated micromanipulation method.
Koki Takebayashi, Manita Wittayarat, Qingyi Lin, Nanaka Torigoe, Bin Liu, Maki Hirata, Megumi Nagahara, Fuminori Tanihara and Takeshige Otoi : Efficiency of genetic modification in gene-knockout sperm-derived zygotes followed by electroporation of guide RNA targeting the same gene., Animal Science Journal, Vol.94, No.1, e13842, 2023.
(Summary)
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.
(Keyword)
Male / Animals / Swine / Gene Editing / Zygote / CRISPR-Cas Systems / Semen / Electroporation / RNA, Guide, CRISPR-Cas Systems
Naoaki Yoshimura, Yasuhiro Morita, Mitsuo Yamamoto, Chika Higashine, Koki Takebayashi, Taichi Kumegawa, Yoshimichi Higashiyama, Masatoshi Niimi, Fuminori Tanihara and Takeshige Otoi : Relationship between GnRH-induced LH increase profiles in the serum and vaginal mucus of Japanese Black beef cows., Archives Animal Breeding, Vol.65, No.3, 353-356, 2022.
(Summary)
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.
Naoaki Yoshimura, Takeshi Tsuka, Takaaki Yoshimura and Takeshige Otoi : Efficacy of Abdominal Ultrasonography for Differentiation of Gastrointestinal Diseases in Calves., Animals : An Open Access Journal from MDPI, Vol.12, No.19, 2022.
(Summary)
This study investigated the clinical efficacy of abdominal ultrasonography for abomasal dilation in three calves, intestinal volvulus in five calves, intussusception in one calf, and internal hernia in one calf. In the abdominal ultrasonograms of the abomasal dilation cases, this disease was commonly characterized by severely extended lumens, including heterogeneously hyperechoic ingesta without intraluminal accumulations of gas. In the animals with intestinal volvulus and intussusception, a to-and-fro flow was observed to be a common ultrasonographic characteristic that led to suspicion of an intestinal obstruction. The use of abdominal ultrasonography for five cases with intestinal volvulus gave no reason to suspect this disease, despite its efficacy in one case, based on an acutely angled narrowing. Although three of five animals with intestinal volvulus had intestinal ruptures, no ultrasonographic evidence could be obtained. When abdominal ultrasonography was used for one case with intussusception, this pathological condition could be strongly suspected, as a "target" sign was observed. This finding supported surgical intervention for this case, followed by treatment with manual reduction, resulting in a favorable outcome. In terms of the differential and definitive diagnosis for various intestinal diseases, abdominal ultrasonography may be poor at providing indicative evidence, but very helpful for confirming intestinal obstruction.
Anh Quynh Le, Manita Wittayarat, Zhao Namula, Qingyi Lin, Koki Takebayashi, Maki Hirata, Fuminori Tanihara, Kim Lanh Thi Do and Takeshige Otoi : Multiple gene editing in porcine embryos using a combination of microinjection, electroporation, and transfection methods., Veterinary World, Vol.15, No.9, 2210-2216, 2022.
(Summary)
The combination of CRISPR/Cas9 delivery methods may improve the mutation efficiency of triple-gene edited porcine blastocysts.
Yasutaka Fujita, Masuhiro Nishimura, Tamaki Wada, Natsuki Komori and Takeshige Otoi : Dimethyl sulfoxide-free cryopreservation solution containing trehalose, dextran 40, and propylene glycol for therapy with human adipose tissue-derived mesenchymal stromal cells., Cytotechnology, Vol.74, No.5, 515-529, 2022.
(Summary)
We evaluated a dimethyl sulfoxide (Me2SO)-free cryopreservation solution to freeze human adipose-derived mesenchymal stromal cells (hADSCs). In the first experiment, we compared the combined effects of 3% trehalose (3 T) and 5% dextran (5D) in lactated Ringer's solution (LR) as a cryopreservation base solution containing 10% propylene glycol (PG). The cell viability of hADSCs immediately after thawing was significantly higher (p < 0.05) in LR supplemented with 3 T (LR-3 T) and with 3 T and 5D (LR-3 T-5D) than in LR. In the second experiment, we compared the cell characteristics of hADSCs freeze-thawed in LR-3 T-5D containing either 10% Me2SO or 10% PG. The cell viability, annexin V-positive ratio, colony-forming capacity, cell proliferation, cell surface antigen positivity, adipogenic differentiation, osteogenic differentiation, and genetic response to cytokine stimulation of hADSCs immediately after thawing were similar between the LR-3 T-5D containing 10% Me2SO and 10% PG. In the third experiment, we examined various concentrations of PG on the cell proliferative capacity of freeze-thawed hADSCs. The cell proliferative capacity of hADSCs frozen with LR-3 T-5D containing 2.5% to 5% PG was significantly higher (p < 0.05) than LR-3 T-5D containing 10% PG. Furthermore, the cell proliferative capacity of hADSCs frozen with LR-3 T-5D containing 4% PG was similar to that of fresh hADSCs. These results indicate that the combination of 3 T-5D in an LR solution as a basic solution is effective for post-thaw cell viability, and that the optimal concentration of PG to maintain the cell characteristics of hADSCs frozen with LR-3 T-5D is 2.5% to 5%, which is promising for cell therapy applications.
Hisayoshi Omori, Junko Chikamoto, Takayuki Hirano, Kazuhiko Besshi, Naoaki Yoshimura, Maki Hirata and Takeshige Otoi : Comparative analysis of bilirubin glucuronidation activity in canine and human primary hepatocytes using a 3D culture system., In Vitro Cellular & Developmental Biology. Animal, Vol.58, No.8, 712-718, 2022.
(Summary)
Species differences in bilirubin glucuronidation activity are observed between humans and dogs through liver microsomes and recombinant UDP-glucuronosyltransferase 1A1. Humans exhibit higher activity than that of dogs. In this study, bilirubin glucuronidation activity was examined in canine and human primary hepatocyte spheroids formed using a 3D culture system. When spheroid development in canine and human primary hepatocytes was evaluated on days 7 and 14 after the start of culture, canine primary hepatocyte spheroids had a more distinct spherical shape than human hepatocyte spheroids, irrespective of the culture period. Furthermore, mono- and di-glucuronide generation detected in spheroids were significantly higher (P < 0.05) in human primary hepatocytes than in canine primary hepatocytes after 24 h of incubation with bilirubin for each culture period. These results suggest that there are species differences in the bilirubin glucuronidation activity of primary hepatocytes with spheroid formation between humans and dogs, with the activity being higher in humans than in dogs.
Koki Takebayashi, Manita Wittayarat, Qingyi Lin, Maki Hirata, Naoaki Yoshimura, Nanaka Torigoe, Megumi Nagahara, Kim Lanh Thi Do, Fuminori Tanihara and Takeshige Otoi : Gene editing in porcine embryos using a combination of electroporation and transfection methods., Reproduction in Domestic Animals = Zuchthygiene, Vol.57, No.10, 1136-1142, 2022.
(Summary)
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.
(Keyword)
Animals / CRISPR-Associated Protein 9 / CRISPR-Cas Systems / Electroporation / Gene Editing / Swine / Transfection / Zygote
Farid Barati, Mobina Ehsani, Takeshige Otoi, A Aziz Fallah and Habibiyan Saied Dehkordi : Reproductive cycle and in vitro maturation of canine oocyte: A meta-analysis approach., Theriogenology, Vol.188, 22-27, 2022.
(Summary)
Different outcomes of canine in vitro oocyte maturation depend on the phases of the donor's estrus cycle. This study aimed to examine this topic using a meta-analysis approach. From 1765 identified records in three major scientific databases, 145 were selected after the screening, 27 were evaluated for eligibility, and, finally, by removing 3 studies with high heterogeneity, 11 studies were used for the meta-analysis. The anestrus (ANE) phase was the reference for evaluating the luteal (LUT) and follicular (FOL) phases. The effect size data were selected as dichotomous types, and publication bias and heterogeneity were used for the quality assessments. The results showed a higher risk ratio (RR) of meiosis resumption in oocytes from the germinal vesicle stage in the LUT and FOL phases (∼30% more than ANE oocytes). The RR of being oocytes at the germinal vesicle breakdown (GVBD) stage was similar in all three phases. When compared to ANE, the RR of oocytes reaching the metaphase I (MI) stage from GVBD were higher in the FOL and LUT phases (30% and 14%, respectively). Moreover, the rate of oocytes reaching metaphase II (MII) was higher in the FOL and LUT phases (85% and 30%, respectively) than in the ANE phase. Being at an active phase (FOL or LUT phases) increased the rates of transition from the GVBD to MI stages (18%) and the MI to MII stages (48%) as compared with the ANE phase. It was assumed that oocytes in the LUT phase can reach GVBD and MI, whereas oocytes in the FOL phase are more capable of reaching MII than oocytes in anestrus.
Megumi Shimazaki, Manita Wittayarat, Rentsenkhand Sambuu, Asami Sugita, Masaki Kawaguchi, Maki Hirata, Fuminori Tanihara, Mitsuhiro Takagi, Masayasu Taniguchi, Takeshige Otoi and Yoko Sato : Disruption of cell proliferation and apoptosis balance in the testes of crossbred cattle-yaks affects spermatogenic cell fate and sterility., Reproduction in Domestic Animals = Zuchthygiene, Vol.57, No.9, 999-1006, 2022.
(Summary)
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 hybridization 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 labelling 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.
Zhao Namula, Manita Wittayarat, Kim Lanh Thi Do, Thanh Nguyen Van, Qingyi Lin, Koki Takebayashi, Maki Hirata, Fuminori Tanihara and Takeshige Otoi : Effects of the timing of electroporation during in vitro maturation on triple gene editing in porcine embryos using CRISPR/Cas9 system, Veterinary and Animal Science, Vol.16, 2022.
Zhao Namula, Anh Quynh Le, Manita Wittayarat, Qingyi Lin, Koki Takebayashi, Maki Hirata, Kim Lanh Thi Do, Fuminori Tanihara and Takeshige Otoi : Triple gene editing in porcine embryos using electroporation alone or in combination with microinjection., Veterinary World, Vol.15, No.2, 496-501, 2022.
(Summary)
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.
Qingyi Lin, Anh Quynh Le, Koki Takebayashi, Maki Hirata, Fuminori Tanihara, Chommanart Thongkittidilok, Osamu Sawamoto, Takeshi Kikuchi and Takeshige Otoi : Viability and developmental potential of porcine blastocysts preserved for short term in a chemically defined medium at ambient temperature., Reproduction in Domestic Animals = Zuchthygiene, Vol.57, No.5, 556-563, 2022.
(Summary)
-W [Cell-W] and cell suspension and preservation solution, Cellstor
(Keyword)
Animals / Blastocyst / Culture Media / Curcumin / Embryo, Mammalian / Fertilization in Vitro / Swine / Temperature
Manita Wittayarat, Saritvich Panyaboriban, Navapol Kupthammasan, Takeshige Otoi and Kaywalee Chatdarong : Effects of green tea polyphenols and α-tocopherol on the quality of chilled cat spermatozoa and sperm IZUMO1 protein expression during long-term preservation., Animal Reproduction Science, Vol.237, 2022.
(Summary)
Sperm IZUMO1 protein was recently found to be a crucial mediator in the interaction and fusion with eggs, indicating an important role in assuring the favourable outcome from long-term preservation of chilled semen. The purpose of this study was to investigate whether supplementation of chilled semen extender with green tea polyphenols together with α-tocopherol would provide synergistic effects to prolong sperm survival and maintain IZUMO1 protein stability in cat spermatozoa. Sperm samples were collected from the cat epididymis before being diluted with semen extender containing various concentrations of α-tocopherol (0, 2.5, 5 and 7.5 µg/ml) and 0.75 mg/ml green tea polyphenols and cooled to 4 °C. One sample without antioxidants served as a control. Sperm characteristics and IZUMO1 protein expression were investigated before and after chilling at 3, 6, 9, 12 and 15 days. Using α-tocopherol at 5 µg/ml together with 0.75 mg/ml green tea polyphenols in the semen extender is the most suitable condition to retain the sperm characteristics up to nine days of preservation. Cat IZUMO1 proteins, 17 kDa, were identified at the equatorial segment of acrosome reacted sperm. Without antioxidant, cold storage can gradually degrade the IZUMO1 protein level. Sperm IZUMO1 protein was markedly conserved by supplementation of 5 µg/ml α-tocopherol together with 0.75 mg/ml green tea polyphenols up to 12 days in cold storage. These findings indicate that green tea polyphenols and α-tocopherol have protective effects on the preservation of sperm characteristics and IZUMO1 protein integrity of cat epididymal sperm during long-term chilling.
Qingyi Lin, Anh Quynh Le, Koki Takebayashi, Maki Hirata, Fuminori Tanihara, Osamu Sawamoto, Takeshi Kikuchi and Takeshige Otoi : Short-term preservation of porcine zygotes at ambient temperature using a chemically defined medium., Animal Science Journal, Vol.93, No.1, 2022.
(Summary)
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.
(Keyword)
Animals / Blastocyst / Culture Media / Fertilization in Vitro / Swine / Temperature / Zygote
Maki Hirata, Manita Wittayarat, Zhao Namula, Anh Quynh Le, Qingyi Lin, Koki Takebayashi, Chommanart Thongkittidilok, Taro Mito, Sayuri Tomonari, Fuminori Tanihara and Takeshige Otoi : Generation of mutant pigs by lipofection-mediated genome editing in embryos., Scientific Reports, Vol.11, No.1, 23806, 2021.
(Summary)
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.
Chommanart Thongkittidilok, Anh Quynh Le, Qingyi Lin, Koki Takebayashi, Lanh Thi Kim Do, Zhao Namula, Maki Hirata, Fuminori Tanihara and Takeshige Otoi : Effects of individual or in-combination antioxidant supplementation during in vitro maturation culture on the developmental competence and quality of porcine embryos., Reproduction in Domestic Animals = Zuchthygiene, Vol.57, No.3, 314-320, 2021.
(Summary)
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.
(Keyword)
Animals / Antioxidants / Blastocyst / Dietary Supplements / Embryonic Development / Fertilization in Vitro / In Vitro Oocyte Maturation Techniques / Oocytes / Swine
Praopilas Phakdeedindan, Manita Wittayarat, Theerawat Tharasanit, Mongkol Techakumphu, Megumi Shimazaki, Rentsenkhand Sambuu, Maki Hirata, Fuminori Tanihara, Masayasu Taniguchi, Takeshige Otoi and Yoko Sato : Aberrant levels of DNA methylation and H3K9 acetylation in the testicular cells of crossbred cattle-yak showing infertility., Reproduction in Domestic Animals = Zuchthygiene, Vol.57, No.3, 304-313, 2021.
(Summary)
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.
(Keyword)
Acetylation / Animals / Cattle / Cattle Diseases / DNA Methylation / Infertility, Male / Male / Spermatogenesis / Testis
Zhao Namula, Maki Hirata, Anh Quynh Le, Qingyi Lin, Koki Takebayashi, Naoaki Yoshimura, Fuminori Tanihara, Chommanart Thongkittidilok and Takeshige Otoi : Zona pellucida treatment before CRISPR/Cas9-mediated genome editing of porcine zygotes., Veterinary Medicine and Science, Vol.8, No.1, 164-169, 2021.
(Summary)
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.
Qingyi Lin, Anh Quynh Le, Koki Takebayashi, Chommanart Thongkittidilok, Manita Wittayarat, Maki Hirata, Fuminori Tanihara and Takeshige Otoi : Timing and duration of lipofection-mediated CRISPR/Cas9 delivery into porcine zygotes affect gene-editing events., BMC Research Notes, Vol.14, No.1, 389, 2021.
(Summary)
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.
Naoaki Yoshimura, Masayasu Taniguchi, Tsukasa Terazono, Tetsushi Ono, Mitsuhiro Takagi, Yoko Sato, Maki Hirata, Fuminori Tanihara and Takeshige Otoi : Vaginal stimulation enhances ovulation of queen ovaries treated using a combination of eCG and hCG., Veterinary Medicine and Science, Vol.7, No.5, 1569-1574, 2021.
(Summary)
Follicular changes throughout the oestrous phase have been poorly documented in queens because of the location and the small size of ovaries. We investigated follicular development in queens treated with a combination of equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG) and evaluated the effects of vaginal stimulation by a tomcat on ovulation induction. A hormonal treatment was administered using a simple crossover design. Four queens were administered 150 IU of eCG (day 1) and 250 IU of hCG on day 5 and 6. Half of the queens were mated with a vasectomised tomcat for 3 days after hCG injection. Ultrasound imaging of the ovaries clamped at a subcutaneous site was performed once a day from day 1 to 7, and on day 13, and the serum concentrations of oestradiol and progesterone were examined on day 1, 5, 7 and 13. The mean number of follicles gradually increased with the eCG treatment and decreased after hCG injection. The ovulation rate of follicles was significantly higher in the vaginal stimulation group (70.0%) than in the control group (42.6%). During the hormonal treatments, the serum concentration of oestradiol and progesterone did not differ between the two groups. Ultrasound imaging of the ovaries clamped at a subcutaneous site showed that eCG and hCG treatment promoted the follicular growth and corpus luteum formation, respectively. The combination of hCG injection with vaginal stimulation by a vasectomised tomcat enhanced the ovulation rate of follicles.
Zhao Namula, Yasuhiro Isumi, Yoko Sato, Anh Quynh Le, Qingyi Lin, Koki Takebayashi, Maki Hirata, Fuminori Tanihara, Chommanart Thongkittidilok and Takeshige Otoi : Improvement of the in vitro fertilization and embryo development using frozen-thawed spermatozoa of microminipigs., Archives Animal Breeding, Vol.64, No.1, 265-271, 2021.
(Summary)
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.
Fuminori Tanihara, Maki Hirata, Thi Nhien Nguyen, Osamu Sawamoto, Takeshi Kikuchi and Takeshige Otoi : One-Step Generation of Multiple Gene-Edited Pigs by Electroporation of the CRISPR/Cas9 System into Zygotes to Reduce Xenoantigen Biosynthesis., International Journal of Molecular Sciences, Vol.22, No.5, 2249, 2021.
(Summary)
triple-edited pigs. Immunohistological analyses demonstrated the downregulation of xenoantigens; however, these multiple gene-edited pigs were genetic mosaics that failed to knock out some xenoantigens. Although mosaicism should be resolved, the electroporation technique could become a primary method for the one-step generation of multiple gene modifications in pigs aimed at improving pig-to-human xenotransplantation.
Maki Hirata, Manita Wittayarat, Zhao Namula, Quynh Le Anh, Qingyi Lin, Koki Takebayashi, Chommanart Thongkittidilok, Fuminori Tanihara and Takeshige Otoi : Lipofection-Mediated Introduction of CRISPR/Cas9 System into Porcine Oocytes and Embryos., Animals : An Open Access Journal from MDPI, Vol.11, No.2, 2021.
(Summary)
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.
Anh Quynh Le, Fuminori Tanihara, Manita Wittayarat, Zhao Namula, Yoko Sato, Qingyi Lin, Koki Takebayashi, Maki Hirata and Takeshige Otoi : Comparison of the effects of introducing the CRISPR/Cas9 system by microinjection and electroporation into porcine embryos at different stages., BMC Research Notes, Vol.14, No.1, 7, 2021.
(Summary)
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.
Kim Thi Lanh Do, Manita Wittayarat, Yoko Sato, Kaywalee Chatdarong, Theerawat Tharasanit, Mongkol Techakumphu, Maki Hirata, Fuminori Tanihara, Masayasu Taniguchi and Takeshige Otoi : Comparison of blastocyst development between cat-cow and cat-pig interspecies somatic cell nuclear transfer embryos under the treatment of Trichostatin A., Biology bulletin of the Russian Academy of Sciences, Vol.48, 107-117, 2021.
Nguyen Nhien Thi, Wittayarat Manita, Zhao Namula, Sato Yoko, Anh Le Quynh, Lin Qingyi, Takebayashi Koki, Fuminori Tanihara, Maki Hirata and Takeshige Otoi : Chlorogenic acid and insulin-transferrin-selenium supplementation during in vitro maturation enhances the developmental competence of interspecies chimera blastocysts following cell injection., Journal of Applied Animal Research, Vol.49, No.1, 486-491, 2021.
Manita Wittayarat, Maki Hirata, Zhao Namula, Yoko Sato, T Nhien Nguyen, A Quynh Le, Qingyi Lin, Koki Takebayashi, Fuminori Tanihara and 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., Animal Science Journal, Vol.92, No.1, e13534, 2021.
(Summary)
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.
Fuminori Tanihara, Maki Hirata, Thi Nhien Nguyen, Le Anh Quynh, Manita Wittayarat, Mokhamad Fahrudin, Takayuki Hirano and Takeshige Otoi : Generation of CD163-edited pig via electroporation of the CRISPR/Cas9 system into porcine in vitro-fertilized zygotes., Animal Biotechnology, Vol.32, 147-154, 2021.
Maki Hirata, Manita Wittayarat, Fuminori Tanihara, Yoko Sato, Zhao Namula, Anh Quynh Le, Qingyi Lin, Koki Takebayashi and Takeshige Otoi : One-step genome editing of porcine zygotes through the electroporation of a CRISPR/Cas9 system with two guide RNAs., In Vitro Cellular & Developmental Biology. Animal, Vol.56, No.8, 614-621, 2020.
(Summary)
In the present study, we investigated whether electroporation could be used for one-step multiplex CRISPR/Cas9-based genome editing, targeting IL2RG and GHR in porcine embryos. First, we evaluated and selected guide RNAs (gRNAs) by analyzing blastocyst formation rates and genome editing efficiency. This was performed in embryos electroporated with one of three different gRNAs targeting IL2RG or one of two gRNAs targeting GHR. No significant differences in embryo development rates were found between control embryos and those subjected to electroporation, irrespective of the target gene. Two gRNAs targeting IL2RG (nos. 2 and 3) contributed to an increased biallelic mutation rate in porcine blastocysts compared with gRNA no. 1. There were no significant differences in the mutation rates between the two gRNAs targeting GHR. In our next experiment, the mutation efficiency and the development of embryos simultaneously electroporated with gRNAs targeting IL2RG and GHR were investigated. Similar embryo development rates were observed between embryos electroporated with two gRNAs and control embryos. When IL2RG-targeting gRNA no. 2 was used with GHR-targeting gRNAs no. 1 or no. 2, a significantly higher double biallelic mutation rate was observed than with IL2RG-targeting gRNA no. 3. In conclusion, we demonstrate the feasibility of using electroporation to transfer multiple gRNAs and Cas9 into porcine zygotes, enabling the double biallelic mutation of multiple genes with favorable embryo survival.
Fuminori Tanihara, Maki Hirata, Thi Nhien Nguyen, Osamu Sawamoto, Takeshi Kikuchi, Masako Doi and Takeshige Otoi : Efficient generation of GGTA1-deficient pigs by electroporation of the CRISPR/Cas9 system into in vitro-fertilized zygotes., BMC Biotechnology, Vol.20, No.1, 40, 2020.
(Summary)
We established GGTA1-modified pigs with high efficiency by introducing a CRISPR/Cas9 system into zygotes via electroporation. Multiple gene modifications, including knock-ins of human genes, in porcine zygotes via electroporation may further improve the application of the technique in pig-to-human xenotransplantation.
Maki Hirata, Manita Wittayarat, Zhao Namula, Anh Quynh Le, Qingyi Lin, Thi Nhien Nguyen, Koki Takebayashi, Yoko Sato, Fuminori Tanihara and Takeshige Otoi : Evaluation of multiple gene targeting in porcine embryos by the CRISPR/Cas9 system using electroporation., Molecular Biology Reports, Vol.47, No.7, 5073-5079, 2020.
(Summary)
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.
Fuminori Tanihara, Maki Hirata, Nhien Nguyen Thi, Le Anh Quynh, Takayuki Hirano and Takeshige Otoi : Generation of viable PDX1 gene-edited founder pigs as providers of nonmosaics., Molecular Reproduction and Development, Vol.87, No.4, 471-481, 2020.
(Summary)
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.
Yoko Sato, Kiyoshi Asahina, Miki Yoshiike, Shiari Nozawa, Takeshige Otoi and Teruaki Iwamoto : A change in the steroid metabolic pathway in human testes showing deteriorated spermatogenesis., Reproductive Biology, Vol.20, No.2, 210-219, 2020.
(Summary)
-pathway. A new insight into a change of metabolites in Low-JS testes will be relevant to understand the mechanism of the deteriorated spermatogenesis under the normal range of testosterone level.
T N Nguyen, Maki Hirata, Fuminori Tanihara, Y Sato, Z Namula, Le Trong Quang, M Wittayarat, M Fahrudin and Takeshige Otoi : In vitro Development of Zona Pellucida-free Porcine Zygotes Cultured Individually after Vitrification., Cryo Letters, Vol.41, No.2, 86-91, 2020.
(Summary)
Our results suggest that the removal of the ZP does not cause detrimental effects to the development of vitrified-warmed zygotes.
In Mongolia, yak (Bos grunniens) are able to live in alpine areas and their products greatly influence the lives of the local people. Increased vigour in hybridized yak and cattle can offer benefits for livestock farmers. However, male hybrids show reproductive defects resulting from spermatogenesis arrest, affecting the conservation and maintenance of dominant traits in the next generation. The underlying mechanisms involved in hybrid cattle-yak infertility have recently been investigated; however, the genetic cause is still unclear. Androgens and androgen receptor (AR) signalling are required for spermatogenesis. We, therefore, evaluated the expression of AR, 3β-hydroxysteroid dehydrogenase (3βHSD) and 5α-reductase 2 (SRD5A2) in Leydig cells to investigate their function in cattle-yak spermatogenesis. Testicular tissues from yaks (1-3 years old) and hybrids (F1-F3, 2 years old) were collected and subjected to immunohistochemistry and image analyses to investigate the expression of each parameter in the Leydig cells. After maturation at 2 years, the expression levels of AR increased and the levels of 3βHSD decreased, but the SRD5A2 levels remained constant in yak. However, the cattle-yak hybrid F2 showed immature testicular development and significantly different expression levels of AR and 3βHSD compared with mature yak. These results suggest that the decreased expression of AR and increased expression of 3βHSD in the Leydig cells of cattle-yak hybrid testes may represent one of the causes of infertility. Our study might help in solving the problem of infertility in crossbreeding.
Namula Zhao, Yoko Sato, Manita Wittayarat, Quynh Le Anh, Thi Nhien Nguyen, Qingyi Lin, Maki Hirata, Fuminori Tanihara and Takeshige Otoi : Curcumin supplementation in maturation medium improves the maturation, fertilisation, and developmental competence of porcine oocytes., Acta Veterinaria Hungarica, Vol.68, No.3, 298-304, 2020.
(Summary)
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.
(Keyword)
Animals / Blastocyst / Cell Proliferation / Curcumin / DNA Fragmentation / Dose-Response Relationship, Drug / Embryo Culture Techniques / Hydrogen Peroxide / In Vitro Oocyte Maturation Techniques / Oocytes / Oxidative Stress / Sus scrofa
A Quynh Le, Maki Hirata, T Nhien Nguyen, Koki Takebayashi, Manita Wittayarat, Yoko Sato, Zhao Namula, Masahiro Nii, Fuminori Tanihara and 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., Animal Science Journal, Vol.91, No.1, e13386, 2020.
(Summary)
This study was conducted to investigate the effect of seven concentrations of Cas9 protein (0, 25, 50, 100, 200, 500, and 1,000 ng/µl) on the development and gene editing of porcine embryos. This included the target editing and off-target effect of embryos developed from zygotes that were edited via electroporation of the Cas9 protein with guide RNA targeting Myostatin genes. We found that the development to blastocysts of electroporated zygotes was not affected by the concentration of Cas9 protein. Although the editing rate, which was defined as the ratio of edited blastocysts to total examined blastocysts, did not differ with Cas9 protein concentration, the editing efficiency, which was defined as the frequency of indel mutations in each edited blastocyst, was significantly decreased in the edited blastocysts from zygotes electroporated with 25 ng/µl of Cas9 protein compared with that of blastocysts from zygotes electroporated with higher Cas9 protein concentrations. Moreover the frequency of indel events at the two possible off-target sites was not significantly different with different concentrations of Cas9 protein. These results indicate that the concentration of Cas9 protein affects gene editing efficiency in embryos but not the embryonic development, gene editing rate, and non-specific cleavage of off-target sites.
Shinya Ishihara, Q Thanh Dang-Nguyen, Kazuhiro Kikuchi, Aisaku Arakawa, Satoshi Mikawa, Makoto Osaki, Takeshige Otoi, Minh Quang Luu, Son Thanh Nguyen and Masaaki Taniguchi : Characteristic features of porcine endogenous retroviruses in Vietnamese native pigs., Animal Science Journal, Vol.91, No.1, e13336, 2020.
(Summary)
We aimed to clarify the genomic characteristics of porcine endogenous retroviruses (PERVs) in Vietnamese native pig (VnP) breeds. First, we investigated genetic polymorphisms in β- and γ-like PERVs, and we then measured the copy numbers of infectious γ-like PERVs (PERV-A, B, and C). We purified genomic DNA from 15 VnP breeds from 12 regions all over the country and three Western pig breeds as controls, and investigated genetic polymorphisms in all known PERVs, including the beta (β)1-4 and gamma (γ)1-5 groups. PERVs of β1, β2, β3, and γ4 were highly polymorphic with VnP-specific haplotypes. We did not identify genetic polymorphisms in β4, γ1, or γ2 PERVs. We then applied a real-time polymerase chain reaction-based method to estimate copy numbers of the gag, pol, and env genes of γ1 PERVs (defined as A, B, and C). VnP breeds showed significantly lower copy number of the PERV genes compared with the Western pig breeds (on average, 16.2 and 35.7 copies, respectively, p < .05). Two VnP breeds showed significantly higher copy number compared with the other VnPs (p < .05). Our results elucidated that VnPs have specific haplotypes and a low copy number of PERV genes.
Ha Minh Ngo, MaríaCruz Arnal, Ryosuke Sumi, Junna Kawasaki, Ariko Miyake, K Chris Grant, Takeshige Otoi, Daniel de Luco Fernández and Kazuo Nishigaki : Tracking the Fate of Endogenous Retrovirus Segregation in Wild and Domestic Cats., Journal of Virology, Vol.93, No.24, 2019.
(Summary)
genes of both FeLV and murine ERV provides a common mechanism shared by endogenous and exogenous retroviruses by which ERVs can be inactivated after endogenization. The antiviral role of Refrex-1 predates cat exposure to feline retroviruses. The existence of two ERV-DC14 phenotypes provides a unique model for understanding both ERV fate and cat domestication.
Thanh-Van Nguyen, Kim Lanh Thi Do, Tamas Somfai, Takeshige Otoi, Masayasu Taniguchi and Kazuhiro Kikuchi : Presence of chlorogenic acid during in vitro maturation protects porcine oocytes from the negative effects of heat stress., Animal Science Journal, Vol.90, No.12, 1530-1536, 2019.
(Summary)
Chlorogenic acid (CGA) is known to protect oocytes from oxidative stress. Here we investigated the effects of CGA on porcine oocyte maturation under heat stress and subsequent embryonic development after parthenogenetic activation. For in vitro maturation (IVM) at 41.0°C (hyperthermic condition), supplementation of the maturation medium with 50 μM CGA significantly improved the percentage of matured oocytes and reduced the rate of apoptosis relative to oocytes matured without CGA (p < .05). CGA treatment of oocytes during IVM under hyperthermia tended to increase (p < .1) percentage of blastocyst formation after parthenogenesis and significantly increased (p < .05) the total cell number per blastocyst relative to oocytes matured without CGA. For IVM at 38.5°C (isothermic condition), CGA significantly improved the rate of blastocyst development compared with oocytes matured without CGA (p < .05), but did not affect oocyte maturation, apoptosis rate or the number of cells per embryo. Omission of all antioxidants from the IVM medium significantly reduced the rate of oocyte maturation, but the rate was restored upon addition of CGA. These results demonstrate that CGA is a potent antioxidant that protects porcine oocytes from the negative effects of heat stress, thus reducing the frequency of apoptosis and improving the quality of embryos.
Maki Hirata, Manita Wittayarat, Takayuki Hirano, Thi Nhien Nguyen, Le Anh Quynh, Zhao Namula, Mokhamad Fahrudin, Fuminori Tanihara and Takeshige Otoi : The relationship between embryonic development and the efficiency of target mutations in porcine endogenous retroviruses (PERVs) pol genes in porcine embryos., Animals : An Open Access Journal from MDPI, Vol.9, No.9, E593, 2019.
Namula Zhao, Manita Wittayarat, Maki Hirata, Hirano Takayuki, Nguyen Thi Nhien, Le Anh Quynh, Fahrudin Mokhamad, Fuminori Tanihara and Takeshige Otoi : Genome mutation after the introduction of the gene editing by electroporation of Cas9 protein (GEEP) system into bovine putative zygotes, In Vitro Cellular & Developmental Biology. Animal, Vol.55, No.8, 598-603, 2019.
(Summary)
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.
Fuminori Tanihara, Maki Hirata, Morikawa Shigeki, Thi Nhien Nguyen, Le Anh Quynh, Hirano Takayuki, Fukumi Yoshiyuki, Abe Toshiaki and Takeshige Otoi : The effects of electroporation on viability and quality of in vivo-derived bovine blastocysts, The Journal of Reproduction and Development, Vol.65, No.5, 475-479, 2019.
(Summary)
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.
Fuminori Tanihara, Maki Hirata, Satoru Iizuka, Shinya Sairiki, Masahiro Nii, Nhien Thi Nguyen, Quynh Anh Le, Takayuki Hirano and Takeshige Otoi : Relationship among ovarian follicular status, developmental competence of oocytes, and anti-M llerian hormone levels: A comparative study in Japanese wild boar crossbred gilts and Large White gilts., Animal Science Journal, Vol.90, No.6, 712-718, 2019.
(Summary)
The aim of this study was to investigate the ovarian follicular development, developmental competence of oocytes, and plasma anti-M llerian 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.
Nhien Nguyen Thi, Maki Hirata, Fuminori Tanihara, Takayuki Hirano, Quynh Anh Le, Masahiro Nii and Takeshige Otoi : Hypothermic storage of porcine zygotes in serum supplemented with chlorogenic acid., Reproduction in Domestic Animals = Zuchthygiene, Vol.54, No.5, 750-755, 2019.
(Summary)
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 M 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 M 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 M 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.
Zhao Namula, Fuminori Tanihara, Manita Wittayarat, Maki Hirata, Nhien Thi Nguyen, Takayuki Hirano, Quynh Anh Le, Masahiro Nii and Takeshige Otoi : Effects of Tris (hydroxymethyl) aminomethane on the quality of frozen-thawed boar spermatozoa., Acta Veterinaria Hungarica, Vol.67, No.1, 106-114, 2019.
(Summary)
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 M) into the freezing extender on the quality and fertilising capacity of frozen-thawed boar spermatozoa. The results showed that the supplementation of 24 M 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 M 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 M did not enhance the sperm motility parameters. The total numbers of fertilised oocytes and blastocysts obtained with spermatozoa frozen with 24 M Tris were significantly higher than those of the control group without Tris (P < 0.05). In conclusion, the supplementation of 24 M Tris into the freezing extender contributes to a better boar sperm quality and fertilising capacity after the process of freezing and thawing.
Maki Hirata, Fuminori Tanihara, Manita Wittayarat, Takayuki Hirano, Nhien Thi Nguyen, Quynh Anh Le, Zhao Namula, Masahiro Nii and Takeshige Otoi : Genome mutation after introduction of the gene editing by electroporation of Cas9 protein (GEEP) system in matured oocytes and putative zygotes., In Vitro Cellular & Developmental Biology. Animal, Vol.55, No.4, 237-242, 2019.
(Summary)
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.
Fuminori Tanihara, Maki Hirata, Nhien Thi Nguyen, Quynh Anh LE, Takayuki Hirano and Takeshige Otoi : Effects of concentration of CRISPR/Cas9 components on genetic mosaicism in cytoplasmic microinjected porcine embryos., The Journal of Reproduction and Development, Vol.65, No.3, 209-214, 2019.
(Summary)
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/ l of Cas9 protein and guide RNA (gRNA), targeting the -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/ l each (20 ng/ l group) or 100 ng/ l each (100 ng/ l group). The ratio of the number of blastocysts that carried mutations to the total number of blastocysts examined in the 100 ng/ l group was significantly higher (P < 0.05) than that in the 20 ng/ l group. Although no blastocysts from the 20 ng/ l group carried a biallelic mutation, 16.7% of blastocysts from the 100 ng/ l 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.
Fuminori Tanihara, Maki Hirata, Nhien T. Nguyen, Quynh A. Le, Takayuki Hirano, Tatsuya Takemoto, Michiko Nakai, Dai-Ichiro Fuchimoto and Takeshige Otoi : Generation of PDX-1 mutant porcine blastocysts by introducing CRISPR/Cas9-system into porcine zygotes via electroporation., Animal Science Journal, Vol.90, No.1, 55-61, 2018.
(Summary)
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.
Fuminori Tanihara, Maki Hirata, Nhien Thi Nguyen, Quynh Anh Le, Takayuki Hirano, Tatsuya Takemoto, Michiko Nakai, Dai-Ichiro Fuchimoto and Takeshige Otoi : Generation of a TP53-modified porcine cancer model by CRISPR/Cas9-mediated gene modification in porcine zygotes via electroporation., PLoS ONE, Vol.13, No.10, e0206360, 2018.
(Summary)
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.
Maki Hirata, Fuminori Tanihara, Masayasu Taniguchi, Mitsuhiro Takagi, Tsukasa Terazono and Takeshige Otoi : Follicular development of canine ovaries stimulated by a combination treatment of eCG and hCG., Veterinary Medicine and Science, 2018.
(Summary)
Ovarian follicular dynamics is not well known in dogs. Imaging of ovaries is technically difficult; however, ovaries clamped at a subcutaneous site can more easily be monitored using ultrasound imaging. This study investigated the follicular development of canine ovaries stimulated by hormone treatment using ultrasound imaging of the ovaries clamped at a subcutaneous site. Oestrus was induced using subcutaneous administration of 500 IU equine chorionic gonadotropin (eCG) and 1000 IU human chorionic gonadotropin (hCG) (eCG/hCG). Five bitches were given 1000 IU hCG 11 days after eCG/hCG administration. Examinations with ovarian ultrasonography using a 7.5-MHz sector transducer, vaginal cytology, and assays of serum oestrogen and progesterone were performed daily until 20 days after eCG/hCG administration. Serosanguineous vaginal discharges and vaginal cytology of two of the bitches were observed. New follicular growth (>1.0 mm in diameter) was observed in all bitches from 2 to 8 days after eCG/hCG administration. The mean diameter of follicles and maximum numbers of follicles per ovary ranged from 2.8 to 5.5 mm and 4 to 16, respectively. The elevation in oestrogen concentrations after eCG/hCG administration was observed in all bitches, and elevation in progesterone concentration (>2 ng mL ) was observed in three bitches. However, no follicles ovulated until 9 days after hCG administration. In conclusion, although the number of examined bitches were limited, follicular growth in ovaries clamped at a subcutaneous site can be monitored using ultrasound imaging. Ovarian ultrasonography showed that eCG/hCG administration induced new follicular growth and hCG administration induced increases in oestrogen concentrations but not ovulation by hCG administration.
Maki Hirata, Fuminori Tanihara, Taniguchi Masayasu, Takagi Mitsuhiro, Terazono Tsukasa and Takeshige Otoi : Viability of canine ovaries autografted to different peripheral sites, The International Journal of Applied Research in Veterinary Medicine, Vol.16, No.2, 140-148, 2018.
Zhao Namula, Maki Hirata, Manita Wittayarat, Fuminori Tanihara, Nhien Nguyen Thi, Takayuki Hirano, Masahiro Nii and Takeshige Otoi : Effects of chlorogenic acid and caffeic acid on the quality of frozen-thawed boar sperm., Reproduction in Domestic Animals = Zuchthygiene, 2018.
(Summary)
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.
Tetsushi Ono, Mitsuhiro Takagi, Chiho Kawashima, B Missaka P Wijayagunawardane, M Peter L A Vos, Masayasu Taniguchi, Fuminori Tanihara and Takeshige Otoi : Comparative Effects of Different Dosages of hCG on Follicular Development in Postpartum Dairy Cows With Cystic Ovarian Follicles., Frontiers in Veterinary Science, Vol.5, 2018.
(Summary)
= 0.07) for group hCG-1 compared to group hCG-C on day 5. The preliminary findings of this study suggest that multiple smaller doses of hCG might be equally effective as a single large dose of hCG in modulating ovarian follicular development in dairy cows with COFs.
Thanh-Van Nguyen, Manita Wittayarat, Kim Lanh Thi Do, Van Thanh Nguyen, Masahiro Nii, Zhao Namula, Toshiki Kunihara, Fuminori Tanihara, Maki Hirata and Takeshige Otoi : 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., Animal Science Journal, Vol.89, No.8, 1207-1213, 2018.
(Summary)
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.
Fuminori Tanihara, Maki Hirata, Thi Nguyen Nhien, Takayuki Hirano, Toshiki Kunihara and Takeshige Otoi : Effect of ferulic acid supplementation on the developmental competence of porcine embryos during in vitro maturation., The Journal of Veterinary Medical Science, Vol.80, No.6, 1007-1011, 2018.
(Summary)
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.
V T Nguyen, Fuminori Tanihara, Maki Hirata, Takayuki Hirano, Katsutoshi Nishio, T Do L Kim, V T Nguyen, M Nii and Takeshige Otoi : Effects of Antifreeze Protein Supplementation on the Development of Porcine Morulae Stored at Hypothermic Temperatures., Cryo Letters, Vol.39, No.2, 131-136, 2018.
(Summary)
Supplementation of AFP type III (1.0 microgram per mL) maintained the quality of embryos stored at 25C, but did not have beneficial effects on the development of embryos stored at hypothermic temperatures.
Megumi Shimazaki, Urasoko Saki, Tanaka Masako, Sato Yoko, Fuminori Tanihara, Maki Hirata, Taniguchi Masayasu, Takagi Mitsuhiro and Takeshige Otoi : Effects of Orvus Es Paste (OEP) on the viability of bull spermatozoa after double freezing and thawing., The International Journal of Applied Research in Veterinary Medicine, Vol.16, 32-38, 2018.
Katsutoshi Nishio, Fuminori Tanihara, T-V Nguyen, Toshiki Kunihara, M Nii, Maki Hirata, Tatsuya Takemoto and Takeshige Otoi : Effects of voltage strength during electroporation on the development and quality of in vitro-produced porcine embryos., Reproduction in Domestic Animals = Zuchthygiene, Vol.53, No.2, 313-318, 2017.
(Summary)
This study was conducted to determine suitable conditions for an experimental method in which the CRISPR/Cas9 system is introduced into in vitro-produced porcine zygotes by electroporation. In the first experiment, when putative zygotes derived from in vitro fertilization (IVF) were electroporated by either unipolar or bipolar pulses, keeping the voltage, pulse duration and pulse number fixed at 30 V/mm, 1 msec and five repeats, respectively, the rate of blastocyst formation from zygotes electroporated by bipolar pulses decreased compared to zygotes electroporated by unipolar pulses. In the second experiment, the putative zygotes were electroporated by electroporation voltages ranging from 20 V/mm-40 V/mm with five 1-msec unipolar pulses. The rate of cleavage and blastocyst formation of zygotes electroporated at 40 V/mm was significantly lower (p < .05) than that of zygotes electroporated at less than 30 V/mm. Moreover, the apoptotic nuclei indices of blastocysts derived from zygotes electroporated by voltages greater than 30 V/mm significantly increased compared with those from zygotes electroporated by voltages less than 25 V/mm (p < .05). When zygotes were electroporated with Cas9 mRNA and single-guide RNA (sgRNA) targeting site in the FGF10 exon 3, the proportions of blastocysts with targeted genomic sequences were 7.7% (2/26) and 3.6% (1/28) in the embryos derived from zygotes electroporated at 25 V/mm and 30 V/mm, respectively. Our results indicate that electroporation at 25 V/mm may be an acceptable condition for introducing Cas9 mRNA and sgRNA into pig IVF zygotes under which the viability of the embryos is not significantly affected.
T-V Nguyen, Fuminori Tanihara, Ltk Do, Y Sato, M Taniguchi, M Takagi, T Nguyen Van and Takeshige Otoi : Chlorogenic acid supplementation during in vitro maturation improves maturation, fertilization and developmental competence of porcine oocytes., Reproduction in Domestic Animals = Zuchthygiene, Vol.52, No.6, 969-975, 2017.
(Summary)
Chlorogenic acid (CGA) is a quinic acid conjugate of caffeic acid, and a phytochemical found in many fruits and beverages that acts as an antioxidant. The present study investigated the effects of CGA supplementation during in vitro maturation (IVM), on in vitro development of porcine oocytes, to improve the porcine in vitro production (IVP) system. Oocytes were matured either without (control) or with CGA (10, 50, 100 and 200 μM). Subsequently, the matured oocytes were fertilized and cultured in vitro for 7 day. The rates of maturation, fertilization and blastocyst formation of oocytes matured with 50 μM CGA were significantly (p < .05) higher than those of the control oocytes. Hydrogen peroxide (H2 O2 ) is one of the reactive oxygen species and induces DNA damage in porcine oocytes. When oocytes were matured with 1 mM H2 O2 to assess the protective effect of CGA, 50 μM CGA supplementation improved the maturation rate and the proportion of DNA-fragmented nuclei in oocytes compared with control oocytes matured without CGA. Moreover, when oocytes were matured with either 50 μM CGA (control) or caffeic acid (10, 50 and 100 μM), the rates of maturation, fertilization and the blastocyst formation of oocytes matured with 50 μM CGA were similar to those of oocytes matured with 10 and 50 μM caffeic acid. Our results suggest that CGA has comparable effects to caffeic acid, and IVM with 50 μM CGA is particularly beneficial to IVP of porcine embryos and protects oocytes from DNA damage induced by oxidative stress. Supplementation of CGA to the maturation medium has a potential to improve porcine IVP system.
Katsutoshi Nishio, Mado Yamazaki, Masayasu Taniguchi, Kazuhiko Besshi, Fumio Morita, Toshiki Kunihara, Fuminori Tanihara, Tatsuya Takemoto and Takeshige Otoi : Sensitivity of the meiotic stage to hyperthermia during in vitro maturation of porcine oocytes., Acta Veterinaria Hungarica, Vol.65, No.1, 115-123, 2017.
(Summary)
The present study was conducted to clarify whether the meiotic stage of porcine oocytes has the highest sensitivity to hyperthermia during in vitro maturation by evaluating meiotic competence and DNA damage. Oocytes were exposed to 41 °C for 12 h at various intervals during 48 h of maturation culture. When the oocytes were exposed to 41 °C from 12 to 24 h of the maturation culture, the proportion of oocytes reaching metaphase II (MII) decreased as compared to the control oocytes cultured at 38.5 °C (P < 0.05). Moreover, the proportions of DNA fragmentation in all oocytes exposed to 41 °C in each culture period after 12 h from the start of maturation culture were significantly higher (P < 0.05) than for the control oocytes. When the meiotic stage of oocytes cultured at 38.5 °C between 12 and 24 h was examined, the majority of oocytes remained at the germinal vesicle (GV) stage at 12 h and approximately half of the oocytes reached metaphase I (MI) at 24 h. These results indicate that the meiotic stage of porcine oocytes having the highest sensitivity to hyperthermia during in vitro maturation is a transition period from the GV stage to the MI stage.
(Keyword)
Animals / DNA Damage / Hot Temperature / In Vitro Oocyte Maturation Techniques / Meiosis / Oocytes / Swine
Genetically modified pigs for biomedical applications have been mainly generated using the somatic cell nuclear transfer technique; however, this approach requires complex micromanipulation techniques and sometimes increases the risks of both prenatal and postnatal death by faulty epigenetic reprogramming of a donor somatic cell nucleus. As a result, the production of genetically modified pigs has not been widely applied. We provide a simple method for CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 gene editing in pigs that involves the introduction of Cas9 protein and single-guide RNA into in vitro fertilized zygotes by electroporation. The use of gene editing by electroporation of Cas9 protein (GEEP) resulted in highly efficient targeted gene disruption and was validated by the efficient production of Myostatin mutant pigs. Because GEEP does not require the complex methods associated with micromanipulation for somatic reprogramming, it has the potential for facilitating the genetic modification of pigs.
L Do, M Wittayarat, T Terazono, Y Sato, M Taniguchi, Fuminori Tanihara, Tatsuya Takemoto, Y Kazuki, K Kazuki, M Oshimura and Takeshige Otoi : Effects of duration of electric pulse on in vitro development of cloned cat embryos with human artificial chromosome vector., Reproduction in Domestic Animals = Zuchthygiene, Vol.51, No.6, 1039-1043, 2016.
Manita Wittayarat, Yoko Sato, Kim Lanh Thi Do, Kaywalee Chatdarong, Theerawat Tharasanit, Mongkol Techakumphu, Masayasu Taniguchi and Takeshige Otoi : Epigenetic modulation on cat-cow interspecies somatic cell nuclear transfer embryos by treatment with trichostatin A., Animal Science Journal, 2016.
(Summary)
This study aimed to determine the acetylation at lysine 9/18/23 of histone H3 (H3K9ac/H3K18ac/H3K23ac; H3K9/18/23 ac) and the di-methylation at lysine 9 of histone H3 (H3K9me2) during early embryogenesis among trichostatin A (TSA)-treated interspecies somatic cell nuclear transfer (iSCNT) cat-cow (TSA-iSCNT) embryos, TSA-untreated iSCNT cat-cow control (control) embryos and bovine in vitro fertilization (IVF) embryos, because TSA-iSCNT embryos can develop to blastocysts. Compared to the control embryos, higher expressions of H3K9/18/23 ac were observed in TSA-iSCNT embryos and IVF embryos at most following stages (2 h post-fusion / post-insemination (PF/PI) to eight-cell stage). At 6 h PF/PI the expression of H3K9/23 ac in TSA-iSCNT embryos and IVF embryos were lower than those in control embryos, and the expression of H3K18ac was no difference among the three groups. The expression of H3K9/23 ac increased in TSA-iSCNT embryos and IVF embryos at pronuclear (PN) stages. The expression of H3K9me2 in TSA-iSCNT embryos resembled that of IVF embryos at 2 h PF/PI to PN stages, and these expression levels were greater than those of control embryos. These results suggest that treatment of iSCNT embryos with TSA modifies the patterns of histone modification at certain lysine residues in a manner that is comparable with that seen in IVF during early embryogenesis.
N Kurniani Karja Wayan, M Fahrudin, MA Setiadi, LI Tumbelaka, R Sudarwati, YT Hastuti, BH Mulia, A Widianti, K Sultan, T Terazono, Z Namula, M Taniguchi, Fuminori Tanihara, Tatsuya Takemoto, K Kikuchi, Y Sato and Takeshige Otoi : Characteristics and fertility of sumatran tiger spermatozoa cryopreserved with different sugars., Cryo Letters, Vol.37, No.4, 264-271, 2016.
(Summary)
Cryopreservation of semen is one of the most important methods for the preservation of endangered tigers. This study evaluated the effects of sugar supplementation on the cryosurvival of spermatozoa from Sumatran tigers (Panthera tigris sumatrae). The post-thaw characteristics and fertility of spermatozoa cryopreserved with different sugars (glucose, lactose, and trehalose) were evaluated using heterologous in-vitro fertilisation with cat oocytes. All parameters of post-thaw spermatozoa significantly decreased as compared with those of fresh spermatozoa. The index of sperm motility for semen cryopreserved with lactose was significantly higher than that for semen cryopreserved with trehalose. The percentage of total fertilisation for tiger spermatozoa cryopreserved with trehalose was significantly lower than that for control cat spermatozoa. Our findings indicated that supplementation with lactose or glycerol as the main sugar in the egg yolk extender resulted in a better motility and fertility potential for post-thawed spermatozoa.
Kyohei Kuse, Jumpei Ito, Ariko Miyake, Junna Kawasaki, Shinya Watanabe, Isaac Makundi, Ha Minh Ngo, Takeshige Otoi and Kazuo Nishigaki : Existence of two distinct infectious ERVs in domestic cats and their different strategies for adaptation to transcriptional regulation., Journal of Virology, 2016.
(Summary)
The domestic cat genome contains many endogenous retroviruses, including ERV-DCs. These ERV-DCs have been acquired through germ cell infections with exogenous retroviruses. Some of these ERV-DCs are still capable of producing infectious virions. Hosts must tightly control these ERVs because replication-competent viruses in the genome pose a risk to the host. Here, we investigated how ERV-DCs are adapted by their hosts. Replication-competent viruses with strong promoter activity such as ERV-DC10 and ERV-DC18 were suppressed by promoter methylation in LTRs. On the other hand, replication-competent virus with weak promoter activity such as ERV-DC14 seemed to escape from strict control via promoter methylation by the host. Interestingly, ERV-DCs with weak promoter activity such as ERV-DC14 have expanded in the cat genome significantly more than ERV-DCs with strong promoter activity. Our results improve understanding of the host-virus conflict and how ERVs adapt in their hosts over time.
Morita Yasuhiro, Taniguchi Masayasu, Fuminori Tanihara, Ito Aya, Namula Zhao, DO Thi Kim Lanh, Takagi Mitsuhiro, Tatsuya Takemoto and Takeshige Otoi : The optimal period of Ca-EDTA treatment for parthenogenetic activation of porcine oocytes during maturation culture, The Journal of Veterinary Medical Science, 2016.
(Summary)
The changes triggered by sperm-induced activation of oocytes, which are required for normal oocyte development, can be mediated by other agents, thereby inducing the parthenogenesis. In this study, we exposed porcine oocytes to 1 mM Ca-EDTA, a metal-ion chelator, at various intervals during 48 hr of in vitro maturation to determine the optimum period of Ca-EDTA treatment for parthenogenetic activation. When the oocytes were cultured with or without Ca-EDTA from 36 hr (post-12), 24 hr (post-24), 12 hr (post-36) and 0 hr (post-48) after the start of maturation culture, the blastocyst formation rates were significantly higher (P<0.05) in the post-24, post-36 and post-48 groups (3.3%, 4.0%, and 2.6%, respectively) than those in the control group without treatment (0%). Furthermore, when the oocytes were cultured with Ca-EDTA for 0 hr (control), 12 hr (pre-12), 24 hr (pre-24), 36 hr (pre-36) and 48 hr (pre-48) from the start of maturation culture, the oocytes formed blastocysts only in the pre-36 and pre-48 groups (0.4% or 0.8%, respectively). Pronuclei (<66.7%) were observed only when the periods of Ca-EDTA treatment were more than 12 hr during maturation culture. In the control group, no pronuclei were detected. Our findings demonstrate that porcine immature oocytes can be parthenogenetically activated by Ca-EDTA treatment for at least 24 hr to 36 hr during maturation culture, leading to pronucleus formation followed by the formation of blastocysts.
Ono Tetsushi, Isobe Tomohiro, Morita Yasuhiro, Do Lanh Thi Kim, Fuminori Tanihara, Taniguchi Masayasu, Takagi Mitsuhiro and Takeshige Otoi : Effects of parity and season on pregnancy rates after the transfer of embryos to repeat-breeder Japanese Black beef cattle., Archives Animal Breeding, Vol.59, 45-49, 2016.
(Summary)
<jats:p>Abstract. Repeat-breeder (RB) cows are a major source of economic waste due to their decreased fertility. Embryo transfer (ET) is an alternative tool to improve the fertility of RB cows. The aims of the present study were to evaluate the effects of recipient parity and the season on pregnancy rates following ET in RB Japanese Black beef cattle. Embryos were transferred nonsurgically to recipients, consisting of 155 heifers (< 2 years old) and 172 cows (< 8 years old), which were defined as RB cattle. Of the recipients that were presented for ET, 57 recipients received a fresh embryo and 270 recipients received a frozen embryo. There were no differences in the pregnancy rates between cattle that received fresh embryos or frozen embryos. The rates of recipients with pregnancy, abortion, stillbirth, and normal calving were similar between heifers and cows. In cows, the pregnancy rates were lower (P < 0.05) in summer (June to August) than in spring (March to May) and winter (December to February). In heifers, however, there were no differences in the pregnancy rates among the seasons. Our findings indicate that in RB Japanese Black beef cattle, the parity of the recipients does not have an effect on the pregnancy rates following the transfer of fresh and frozen embryos. However, heat stress may affect reproductive performance in RB Japanese Black cows. </jats:p>
Tetsushi Ono, Asako Takaoka, Yasuhiro Morita, Lanh Do, Fuminori Tanihara, Masayasu Taniguchi and 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, Vol.63, No.4, 485-498, 2015.
(Summary)
This study evaluated the effect of dibutyryl cyclic adenosine monophos-phate (dbcAMP) and human chorionic gonadotropin (hCG) on the formation of antral follicle-like structures (AFLSs) and on the meiotic status of bovine cumulus- oocyte complexes (COCs) embedded in collagen gel. Supplementation with dbcAMP increased the mean diameter of AFLSs during days 4-8 of culture compared with that of control COCs, irrespective of the concentration of dbcAMP used (0.5-2.0 mM). When the embedded COCs were cultured for 8 days with hCG, the diameters of AFLSs after 4 days of culture tended to be lower in the supplemented COCs than in the control COCs without hCG, irrespective of the concentration used (1-100 IU/mL). Supplementation with 10 IU/mL hCG increased the concentrations of anti-Müllerian hormone but not progesterone and oestradiol in the culture medium after 4 days of culture. Almost all oocytes collected from AFLSs had resumed meiosis by the end of culture, irrespective of supplementation of dbcAMP and hCG. These results indicate that although dbcAMP had a positive effect on AFLS formation and development, supplementation with hCG was detrimental. Moreover, hCG supplementation did not influence the luteinisation of granulosa cells in the AFLS for 4 days after the start of culture.
L T. K. Do, Y Shibata, M Taniguchi, M Nii, T V. Nguyen, Fuminori Tanihara, M Takagi and Takeshige Otoi : Melatonin Supplementation During In Vitro Maturation and Development Supports the Development of Porcine Embryos., Reproduction in Domestic Animals = Zuchthygiene, Vol.50, No.6, 1054-1058, 2015.
(Summary)
Melatonin has been reported to improve the in vitro development of embryos in some species. This study was conducted to investigate the effect of melatonin supplementation during in vitro maturation (IVM) and development culture on the development and quality of porcine embryos. In the first experiment, when the in vitro fertilized embryos were cultured with different concentrations of melatonin (0, 10, 25 and 50 ng/ml) for 8 days, the blastocyst formation rate of embryos cultured with 25 ng/ml melatonin (10.7%) was significantly increased (p < 0.05) compared to the control embryos cultured without melatonin (4.2%). The proportion of DNA-fragmented nuclei in blastocysts derived from embryos cultured with 50 ng/ml melatonin was significantly lower (p < 0.05) than that of embryos cultured without melatonin (2.1% vs 7.2%). In the second experiment, when oocytes were cultured in the maturation medium supplemented with different concentrations of melatonin (0, 10, 25 and 50 ng/ml), fertilized and then cultured with 25 ng/ml melatonin for 8 days, there were no significant differences in the rates of cleavage and blastocyst formation among the groups. However, the proportions (2.7-5.4%) of DNA-fragmented nuclei in blastocysts derived from oocytes matured with melatonin were significantly decreased (p < 0.05) compared to those (8.9%) from oocytes matured without melatonin, irrespective of the concentration of melatonin. Our results suggest that supplementation of the culture media with melatonin (25 ng/ml) during IVM and development has beneficial effects on the developmental competence and quality of porcine embryos.
(Keyword)
Animals / Blastocyst / Culture Media / Embryonic Development / Female / Fertilization in Vitro / Melatonin / Oocytes / Swine
Y Fushimi, M Takagi, D Monniaux, S Uno, E Kokushi, U Shinya, C Kawashima, Takeshige Otoi, E Deguchi and J Fink-Gremmels : Effects of Dietary Contamination by Zearalenone and Its Metabolites on Serum Anti-Müllerian Hormone: Impact on the Reproductive Performance of Breeding Cows., Reproduction in Domestic Animals = Zuchthygiene, Vol.50, No.5, 834-839, 2015.
(Summary)
We investigated the effects of in vivo exposure to low zearalenone levels on the anti-Müllerian hormone endocrine levels and the reproductive performance of cattle. Urine and blood samples and reproductive records were collected from two Japanese Black breeding female cattle herds with dietary zearalenone contamination below the threshold levels (<1 ppm) at 30 days after calving. Urinary zearalenone, α-zearalenol and β-zearalenol concentrations were measured by chromatography-tandem mass spectrometry, and serum anti-Müllerian hormone concentrations were determined along with serum biochemical parameters. Urinary concentrations of α-zearalenol were significantly higher (p < 0.05) in cattle in Herd 1 than in cattle in Herd 2, reflecting the different amounts of zearalenone in the diet of the two herds. Although the number of 5-mm and 10-mm follicles of the herds and their fertility after artificial insemination were similar, the serum anti-Müllerian hormone concentrations in herds 1 and 2 were 438.9 ± 48.6 pg/ml and 618.9 ± 80.0 pg/ml, respectively, with a trend towards a significant difference (p = 0.053), which may indicate differences in the antral follicle populations between herds. Thus, zearalenone intake from dietary feed, even when below the threshold zearalenone contamination level permitted in Japan, may affect the ovarian antral follicle populations, but not the fertility, of post-partum cows.
Yasuhiro Morita, Ni Wayan Kurniani Karja, Lanh Thi Kim Do, Fuminori Tanihara, Masayasu Taniguchi and Takeshige Otoi : Formation of an Antral Follicle-Like Structure by Bovine Cumulus-Oocyte Complexes Embedded with Fragmin/Protamine Microparticles., Animal Biotechnology, Vol.26, No.4, 273-275, 2015.
(Summary)
Fragmin/protamine microparticles (F/P MPs) approximately 0.5-1 µM in diameter were prepared by the simple mixing of fragmin with protamine. This study investigated the effects of F/P MP-containing collagen gels as a hormone carrier on the formation of antral follicle-like structures and on the development of growing bovine oocytes. The supplementation of F/P MPs in collagen gels contributed to the beneficial effects of follicle stimulating hormone (FSH) on the formation and size of antral follicle-like structures. The F/P MPs may serve as potential hormone carriers for the growth of cultured bovine oocytes from early antral follicles.
Megumi Shimazaki, Rentsenkhand Sambuu, Yoko Sato, Kim Lanh Thi Do, Fuminori Tanihara, Masayasu Taniguchi and Takeshige Otoi : EFFECTS OF ORVUS ES PASTE ON THE MOTILITY AND VIABILITY OF YAK (BOS GRUNNIENS) EPIDIDYMAL AND EJACULATED SPERMATOZOA AFTER FREEZING AND THAWING., Cryo Letters, Vol.36, No.4, 264-269, 2015.
(Summary)
The addition of the detergent Orvus ES Paste (OEP) to semen freezing extenders has been observed to improve the post-thaw survival and longevity of spermatozoa from various species but has never been evaluated for yak spermatozoa. This study evaluated the effects of OEP on the post-thaw motility and viability of epididymal and ejaculated yak spermatozoa. Semen samples were frozen and thawed in semen freezing extender supplemented with 0 %, 0.375 %, 0.75 % or 1.5 % OEP. The motility and viability of frozen-thawed spermatozoa were evaluated before and after 3 h of incubation. The addition of 0.75 % OEP to the freezing extender significantly improved the mean motility and viability values of both the epididymal and ejaculated spermatozoa immediately after thawing, but the beneficial effects on motility disappeared after 3h of incubation. Our findings indicate that the addition of 0.75 % OEP is effective for the preservation of yak spermatozoa.
Cut Yasmin, Takeshige Otoi, Mohamad Agus Setiadi and Ni Wayan Kurniani Karja : Maturation and fertilisation of sheep oocytes cultured in serum-free medium containing silk protein sericin., Acta Veterinaria Hungarica, Vol.63, No.1, 110-117, 2015.
(Summary)
Sericin is a water-soluble component of silk and has been used as a biomaterial due to its antibacterial and ultraviolet radiation-resistant properties. This study was designed to evaluate the effect of sericin supplementation in a maturation medium on the meiotic competence and fertilisability of sheep oocytes. Cumulus-oocyte complexes (COCs) were cultured in TCM199 supplemented with sericin at various concentrations of 0 (control), 0.1, 0.25 and 0.5%, either with or without bovine serum albumin (BSA). When the COCs were matured without BSA, the supplementation of 0.1% sericin significantly increased the rates of maturation to metaphase II and the total fertilisation of oocytes compared with the other concentrations of sericin. When the COCs were matured with BSA, the beneficial effects of 0.1% sericin supplementation on the maturation and fertilisation of oocytes were not observed. Our findings indicate that supplementation with 0.1% sericin during maturation culture may improve the nuclear maturation and fertilisability of sheep oocytes. Moreover, it may be possible to replace BSA with sericin in chemically defined media without the risk of disease transmission.
Lanh Thi Kim Do, Vien Viet Luu, Yasuhiro Morita, Masayasu Taniguchi, Masahiro Nii, Augustine T. Peter and Takeshige Otoi : Astaxanthin present in the maturation medium reduces negative effects of heat shock on the developmental competence of porcine oocytes., Reproductive Biology, Vol.15, No.2, 86-93, 2015.
(Summary)
Astaxanthin, one of the most common carotenoids, elicits antioxidant effects on cellular viability and embryonic development. This study was conducted to investigate the effects of astaxanthin on maturation, fertilization and development of porcine oocytes matured in vitro under heat stress conditions, and then fertilized and cultured under standard conditions. Porcine oocytes were cultured in maturation medium supplemented with different concentrations of astaxanthin (0, 0.25, 0.5 or 1 ppm) for 46 h at either 38.5 or 41 °C. In comparison to oocytes cultured at 38.5 °C, the exposure of porcine oocytes to 41.0 °C during in vitro maturation (IVM) significantly inhibited maturation and development of fertilized oocytes to the blastocyst stage. Supplementation of maturation medium with astaxanthin (0.5 ppm) significantly improved oocyte maturation, fertilization and development to the blastocysts stage in both oocyte groups. However, the total cell number and apoptosis index of blastocysts did not differ among groups. Moreover, astaxanthin (0.5 ppm) significantly increased the rate of oocytes that reached metaphase II and decreased proportion of apoptotic oocytes exposed to H2O2 (1.0mM) during IVM. In summary, we demonstrated that supplementation of maturation medium with astaxanthin (0.5 ppm) exerted antioxidative effects and improved the ability of maturation, fertilization, and development of porcine oocytes exposed to heat stress.
Tomohiro Isobe, Yoshihisa Ikebata, Lanh Thi Kim Do, Fuminori Tanihara, Masayasu Taniguchi and 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, Vol.86, No.7, 661-665, 2014.
(Summary)
The optimization of single-embryo culture conditions is very important, particularly in the in vitro production of bovine embryos using the ovum pick-up (OPU) procedure. The purpose of this study was to examine the development of embryos derived from oocytes obtained by OPU that were cultured either individually or in groups in medium supplemented with or without sericin and to investigate the viability of the frozen-thawed embryos after a direct transfer. When two-cell-stage embryos were cultured either individually or in groups for 7 days in CR1aa medium supplemented with or without 0.5% sericin, the rates of development to blastocysts and freezable blastocysts were significantly lower for the embryos cultured individually without sericin than for the embryos cultured in groups with or without sericin. Moreover, the rate of development to freezable blastocysts of the embryos cultured individually with sericin was significantly higher than that of the embryos cultured without sericin. When the frozen-thawed embryos were transferred directly to recipients, the rates of pregnancy, abortion, stillbirth and normal calving in the recipients were similar among the groups, irrespective of the culture conditions and sericin supplementation. Our findings indicate that supplementation with sericin during embryo culture improves the quality of the embryos cultured individually but not the viability of the frozen-thawed embryos after transfer to recipients.
Masayasu Taniguchi, Manita Wittayarat, Kota Morinaga, Yoko Sato, Lanh Kim Thi, Kaywalee Chatdarong, Mongkol Techakumphu, Masahiro Nii and Takeshige Otoi : Chelation of trace elements in preservation medium influences the quality of boar spermatozoa during liquid preservation at 5°C for 4 weeks., Cryo Letters, Vol.35, No.4, 336-344, 2014.
(Summary)
The addition of a metal chelator, ethylenediaminetetraacetic acid (EDTA), to semen extender has the purpose of capturing trace element ions. This study was conducted to evaluate the effects of EDTA on the quality and in vitro fertilisability of liquid-preserved boar spermatozoa. In Experiment 1, semen samples were preserved in the semen extender supplemented with 0, 3, 6, or 12 mM of Na-EDTA at 5 degree C for 4 weeks. In Experiment 2, semen samples were preserved in the extender supplemented with 3 mM of Na-EDTA, Ca-EDTA, or Zn-EDTA and without chelator EDTA. When Na-EDTA was used as a chelating substance in the extender, 3 mM was a most suitable concentration for sperm motility and viability after cold preservation. The supplementation of 3 mM Ca-EDTA had advantages regarding sperm motility, viability and plasma membrane integrity. Our findings indicate that 3 mM Ca-EDTA is the most suitable metal-chelating substance for the liquid preservation of boar semen.
(Keyword)
Animals / Cell Survival / Chelating Agents / Culture Media / Edetic Acid / Fertilization in Vitro / Male / Oocytes / Protective Agents / Refrigeration / Semen Preservation / Sperm Motility / Spermatozoa / Swine / Time Factors
(Link to Search Site for Scientific Articles)
● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 25282502
Manita Wittayarat, Akira Fujiwara, Kaywalee Chatdarong, Mongkol Techakumphu, Yoko Sato, Fuminori Tanihara, Yasuhiro Morita, Masayasu Taniguchi and Takeshige Otoi : Cell cycle analysis and interspecies nuclear transfer of cat cells treated with chemical inhibitors., Acta Veterinaria Hungarica, Vol.62, No.2, 233-242, 2014.
(Summary)
This study investigated the effect of chemical inhibitors on the cell-cycle synchronisation in cat fibroblast cells and evaluated the development of interspecies embryos reconstructed from cat donor cells and enucleated bovine oocytes. Cat fibroblast cells were treated with 15 μg/mL roscovitine or 0.05 μg/mL deme-colcine prior to cell cycle analysis and nuclear transfer. The percentage of cat fibroblast cells arrested at the G0/G1 phase in the roscovitine group was similar to that in the control group without any treatment. The percentage of cells arrested at the G2/M phase was significantly higher in the demecolcine group than in the control group. The fusion rate of interspecies couplets was significantly greater in the roscovitine group than in the control group. Most embryos stopped the development at the 2- or 4-cell stage, and none developed into blastocysts. Chemical inhibitor-induced donor cell cycle synchronisation did not overcome developmental arrest in interspecies cloned embryos.
Urara Watanabe, Mitsuhiro Takagi, Osamu Yamato, Takeshige Otoi and Koji Okamoto : Retrospective surveillance of metabolic parameters affecting reproductive performance of Japanese Black breeding cows., Journal of Veterinary Science, Vol.15, No.2, 283-288, 2014.
(Summary)
This retrospective study was conducted to confirm the relationship between pre- and postpartum metabolic parameters and postpartum reproductive performance and to clarify seasonal characteristics of the metabolic parameters by using our metabolic profile test (MPT) database of Japanese Black breeding herds. In evaluation 1, MPT databases of blood samples from multiparous cows collected prepartum and postpartum were divided into two groups according to calving interval, and each MPT parameter was compared. In evaluation 2, the same MPT databases used in evaluation 1 were divided into two groups according to the sampling period. Significant differences were found in the prepartal total protein and postpartal γ-glutamyltransferase in evaluation 1. In evaluation 2, significant differences were found in the prepartal and postpartal total protein, albumin/globulin ratio, and glucose. Clear seasonal differences in MPT results emphasized the usefulness of the MPT in breeding cattle herds fed home-pasture roughage and suggest that unsatisfactory reproductive performance during hot periods reflects inadequate nutritional content of the diet and possible reduced feed intake due to heat stress.
Zhao Namula, Risa Kodama, Fuminori Tanihara, Yasuhiro Morita, Yoko Sato, Manita Wittayarat, Masayasu Taniguchi and Takeshige Otoi : Effects of skim-milk supplementation on the quality and penetrating ability of boar semen after long-term preservation at 15 °C., Acta Veterinaria Hungarica, Vol.62, No.1, 106-116, 2014.
(Summary)
This study investigated the effects of skim-milk supplementation on the quality and penetrating ability of boar semen preserved at 15 °C. When boar semen samples were preserved in Modified Modena extender supplemented with various concentrations (0, 7.5, 15, 30 and 50 mg/mL) of skim milk powder at 15 °C for 4 weeks, higher sperm motility and viability were observed in the case of 7.5 mg/mL skim-milk supplementation compared with the control group (0 mg/mL) during the preservation (P < 0.05). When in vitro matured oocytes were co-incubated with boar sperm that had been preserved in Modified Modena extender with three different concentrations (0, 7.5 or 15 mg/mL) of skim milk powder at 15 °C for two weeks, there were no apparent effects of skim-milk supplementation on the rates of fertilisation and development to blastocysts of oocytes after co-incubation. However, the monospermic fertilisation rate of sperm preserved with 15 mg/mL skim milk powder was higher (P < 0.05) than that of fresh non-preserved sperm, but did not differ among the preservation groups. The results indicate that the supplementation of Modified Modena extender with 7.5 mg/mL skim milk powder improves the motility and viability, but not the penetrating ability, of sperm after liquid preservation for at least two weeks.
L T. K. Do, Z Namula, V V. Luu, Y Sato, M Taniguchi, T Isobe, K Kikuchi and Takeshige Otoi : Effect of sericin supplementation during in vitro maturation on the maturation, fertilization and development of porcine oocytes., Reproduction in Domestic Animals = Zuchthygiene, Vol.49, No.2, e17-20, 2014.
(Summary)
This study aimed to examine the effects of sericin supplementation during in vitro oocyte maturation on the nuclear maturation, fertilization and development of porcine oocytes. Cumulus-oocyte complexes (COCs) were cultured in maturation medium supplemented with 0 (control), 0.1, 0.5, 1.0, 2.5 or 5.0% sericin and were then subjected to in vitro fertilization and embryo culture. More COCs matured with 1.0% sericin underwent germinal vesicle breakdown and reached metaphase II compared with the control COCs matured without sericin (p < 0.01). The proportions of oocytes with DNA-fragmented nuclei did not differ between the groups, regardless of the sericin level. The total fertilization rate of oocytes matured with 1.0% sericin was higher (p < 0.05) than that of oocytes matured with 0.1%, 2.5% and 5.0% sericin. Supplementation with more than 1.0% sericin decreased the DNA fragmentation index of the blastocysts compared with the control group (p < 0.05). However, the supplementation of the maturation medium with sericin had no beneficial effects on the cleavage, development to the blastocyst stage and the total cell number of the embryos. Our findings indicate that supplementation with 1.0% sericin during maturation culture may improve the nuclear maturation and the quality of the embryos but does not affect blastocyst formation.
(Keyword)
Animals / Culture Media / Fertilization in Vitro / In Vitro Oocyte Maturation Techniques / Oocytes / Sericins / Swine
Fuminori Tanihara, Michiko Nakai, Nguyen Thi Men, Noriko Kato, Hiroyuki Kaneko, Junko Noguchi, Takeshige Otoi and 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, Vol.85, No.4, 395-404, 2014.
(Summary)
The zona pellucida (ZP) is considered to play important roles in the prevention of polyspermy in mammalian oocytes. However, in pigs we have shown that the presence of the ZP accelerates sperm penetration into the ooplasm during in vitro fertilization (IVF). In the present study, we investigated the effects of the ZP on sperm binding, acrosomal status, and functional exposure of IZUMO, a critical factor involved in sperm-egg fusion, during IVF in pigs. We evaluated the numbers and acrosomal statuses of sperm binding to the ZP and oolemma, and being present in the ZP and perivitelline space (PVS) using ZP-intact and ZP-free oocytes. More sperm bound to the ZP than to the oolemma. The average number of sperm present in the PVS was 0.44-0.51 per oocyte, and all sperm had lost their acrosomes. The proportion of sperm that were immunopositive for anti-IZUMO antibody was significantly higher after they were passing or had passed through the ZP. Furthermore, addition of anti-IZUMO antibody to the fertilization medium significantly inhibited the penetration of sperm into ZP-free oocytes. These results suggest that, in pigs, the ZP induces the acrosome reaction, which is associated with the functional exposure of IZUMO, resulting in completion of fertilization.
(Keyword)
Acrosome Reaction / Animals / Female / Fertilization / Fertilization in Vitro / Immunoglobulins / In Vitro Techniques / Male / Membrane Proteins / Oocytes / Sperm-Ovum Interactions / Spermatozoa / Swine / Zona Pellucida
Z.X. He, Sato Yoko, J.C. Zhang, B.Z. Huang, Wittayarat Manita, Taniguchi Masayasu, Suzuki Tatsuyuki and Takeshige Otoi : Superovulatory response and pregnancies after interspecies transfer of embryos in semi-wild Dulong (Bos frontalis)., Veterinarski arhiv, Vol.84, 183-188, 2014.
104.
Y. Fushimi, Takagi Mitsuhiro, H. Hasunuma, S. Uno, E. Kokushi, U. Watanabe, J. Liu, M. Marey, A. Miyamoto, Takeshige Otoi, E. Deguchi and J. Fink-Gremmels : Application of mycotoxin adsorbent to cattle feed naturally contaminated with zearalenone: Urinary zearalenone excretion and association with anti-Mullerian hormone level., World Mycotoxin Journal, Vol.7, 367-378, 2014.
105.
Vien Viet Luu, Zhao Namula, Lanh Thi Kim Do, Yoko Sato, Masayasu Taniguchi, Ni Wayan Kurniani Karja and Takeshige Otoi : Nuclear status and DNA fragmentation of oocytes from porcine, bovine and feline ovaries stored at 4 degrees C for 5 days., Cryo Letters, Vol.35, No.1, 48-53, 2014.
(Summary)
The cooling of mammalian oocytes to sub-physiological temperatures is widely known to affect their viability through the induction of various abnormalities at all stages of meiosis. This study was to compare the kinetics of nuclear status and oocyte damage in porcine, bovine and feline ovaries stored at 4 degrees C for 5 days. The nuclear status and oocyte quality during storage were evaluated before and after maturation culture. The cold storage of ovaries decreased the proportions of porcine and bovine oocytes that remained at the germinal vesicle stage before maturation culture. The maturation rates of oocytes decreased with increasing storage time, independent of species. None of the porcine oocytes reached metaphase II (MII) after 1 day of storage. In contrast, bovine and feline oocytes from ovaries that were stored for 2 days and 3 days reached MII. DNA fragmentation in porcine oocytes from ovaries stored for 1 day was significantly higher than that in bovine and feline oocytes. The maturation competency of oocytes after the cold storage of ovaries could be related to the meiotic resumption of oocytes during storage and the occurrence of DNA fragmentation in oocytes during maturation culture.
(Keyword)
Animals / Cats / Cattle / Cell Nucleus / DNA Fragmentation / Female / Meiosis / Metaphase / Oocytes / Organ Preservation / Organ Preservation Solutions / Ovary / Refrigeration / Species Specificity / Swine / Time Factors
(Link to Search Site for Scientific Articles)
● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 24872157
Fuminori Tanihara, Yukine Kaedei, Zhao Namula, Vien Viet Luu, Yoko Sato, Manita Wittayarat, Masayasu Taniguchi and Takeshige Otoi : Comparison of activation ability between feline and bovine oocytes., Acta Veterinaria Hungarica, Vol.61, No.4, 491-494, 2013.
(Summary)
Research comparing the activation sensitivity of oocytes to chemical treatment among mammalian species remains limited. We compared the activation ability of oocytes from bovine and feline ovaries when treated with ethanol alone, with ethanol and cycloheximide, and without any chemical treatment; the oocytes were then cultured for 72 h. After in vitro maturation (IVM), 5% of feline oocytes were activated and 1% were cleaved, whereas there were no prematurely activated bovine oocytes. Activation rates with ethanol and ethanol/cycloheximide were significantly higher (P < 0.01) in bovine oocytes than in feline oocytes (74.2% vs. 34.1% and 86.3% vs. 52.5%, respectively). Thus, our findings indicate that feline oocytes can be prematurely activated by the end of IVM, and that bovine oocytes may have a higher sensitivity of parthenogenetic activation to chemical treatment than do feline oocytes.
Manita Wittayarat, Aya Ito, Taichi Kimura, Zhao Namula, Vien Viet Luu, Lanh Thi Kim Do, Yoko Sato, Masayasu Taniguchi and Takeshige Otoi : Effects of green tea polyphenol on the quality of canine semen after long-term storage at 5°C., Reproductive Biology, Vol.13, No.3, 251-254, 2013.
(Summary)
The aim of the present study was to determine the effects of green tea polyphenol on the quality of canine semen after long-term storage at 5°C. The supplementation of a Tris-egg yolk extender with polyphenol (0.5, 0.75, or 1mg/mL) increased the motility and viability of sperm preserved for four weeks at 5°C.
Tomohiro Isobe, Yoshihisa Ikebata, Takeshi Onitsuka, Lanh Thi Kim Do, Yoko Sato, Masayasu Taniguchi and Takeshige Otoi : Cryopreservation for bovine embryos in serum-free freezing medium containing silk protein sericin., Cryobiology, Vol.67, No.2, 184-187, 2013.
(Summary)
Because the use of serum in the embryo cryopreservation increases the probability of animal health problems such as bovine spongiform encephalopathy (BSE) and viral infections, this study was conducted to examine the effects of sericin supplementation for serum-free freezing medium on the survival and development of bovine embryos after freezing-thawing and direct transfer to recipients. When in vitro-produced bovine embryos were frozen conventionally in the freezing medium supplemented with various concentrations (0.1%, 0.5%, and 1.0%) of sericin, the percentages of damaged zona pellucida, survival, and development of frozen-thawed embryos were similar to those of embryos frozen in freezing medium supplemented with 0.4% bovine serum albumin (BSA) and 20% fetal bovine serum (FBS) (0.4BSA/20F; control). When in vivo-derived embryos were frozen with 0.4BSA/20F (control), 0.5% sericin +20% FBS (0.5S/20F) or 0.5% sericin (0.5S) and were subsequently transferred directly to recipients, the percentages of recipients with pregnancy and normal calving in the 0.5S/20F group were higher than those in the control group (47.3% vs. 40.1% and 94.6% vs. 87.3%, respectively). Moreover, the percentages of recipients with pregnancy and normal calving (42.2% and 92.4%, respectively) in the 0.5S group were similar with those of other groups. In conclusion, these results indicate that serum-free freezing medium supplemented with sericin is available for the cryopreservation of bovine embryos and that it is beneficial for the elimination of a potential source of biological contamination by serum or BSA.
Manita Wittayarat, Yoko Sato, Lanh Thi Kim Do, Yasuhiro Morita, Kaywalee Chatdarong, Mongkol Techakumphu, Masayasu Taniguchi and Takeshige Otoi : Histone deacetylase inhibitor improves the development and acetylation levels of cat-cow interspecies cloned embryos., Cellular Reprogramming, Vol.15, No.4, 301-308, 2013.
(Summary)
Abnormal epigenetic reprogramming, such as histone acetylation, might cause low efficiency of interspecies somatic cell nuclear transfer (iSCNT). This study was conducted to evaluate the effects of trichostatin A (TSA) on the developmental competence and histone acetylation of iSCNT embryos reconstructed from cat somatic cells and bovine cytoplasm. The iSCNT cat and parthenogenetic bovine embryos were treated with various concentrations of TSA (0, 25, 50, or 100 nM) for 24 h, respectively, following fusion and activation. Treatment with 50 nM TSA produced significantly higher rates of cleavage and blastocyst formation (84.3% and 4.6%, respectively) of iSCNT embryos than the rates of non-TSA-treated iSCNT embryos (63.8% and 0%, respectively). Similarly, the treatment of 50 nM TSA increased the blastocyst formation rate of parthenogenetic bovine embryos. The acetylation levels of histone H3 lysine 9 (H3K9) in the iSCNT embryos with the treatment of 50 nM TSA were similar to those of in vitro-fertilized embryos and significantly higher (p<0.05) than those of non-TSA-treated iSCNT embryos (control), irrespective of the embryonic development stage (two-cell, four-cell, and eight-cell stages). These results indicated that the treatment of 50 nM TSA postfusion was beneficial for development to the blastocyst stage of iSCNT cat embryos and correlated with the increasing levels of acetylation at H3K9.
Fuminori Tanihara, Michiko Nakai, Hiroyuki Kaneko, Junko Noguchi, Takeshige Otoi and Kazuhiro Kikuchi : Evaluation of zona pellucida function for sperm penetration during in vitro fertilization in pigs., The Journal of Reproduction and Development, Vol.59, No.4, 385-392, 2013.
(Summary)
In porcine oocytes, the function of the zona pellucida (ZP) with regard to sperm penetration or prevention of polyspermy is not well understood. In the present study, we investigated the effects of the ZP on sperm penetration during in vitro fertilization (IVF). We collected in vitro-matured oocytes with a first polar body (ZP+ oocytes). Some of them were freed from the ZP (ZP- oocytes) by two treatments (pronase and mechanical pipetting), and the effects of these treatments on sperm penetration parameters (sperm penetration rate and numbers of penetrated sperm per oocyte) were evaluated. There was no evident difference in the parameters between the two groups. Secondly, we compared the sperm penetration parameters of ZP+ and ZP- oocytes using frozen-thawed epididymal spermatozoa from four boars. Sperm penetration into ZP+ oocytes was found to be accelerated relative to ZP- oocytes. Thirdly, we evaluated the sperm penetration of ZP+ and ZP- oocytes at 1-10 h after IVF (3 h gamete co-incubation). The proportions of oocytes penetrated by sperm increased significantly with time in both groups; however, the number of penetrated sperm per oocyte did not increase in ZP- oocytes. Finally, we performed IVF using ZP- oocytes divided into control (3 h) and prolonged gamete co-incubation (5 h) groups. Greater numbers of sperm penetrated in the 5 h group than in the control group. These results suggest that the ZP and oolemma are not competent factors for prevention of polyspermy in our present porcine IVF system. However, it appears that ZP removal is one of the possibilities for reducing polyspermic penetration in vitro in pigs.
(Keyword)
Animals / Female / Fertilization in Vitro / Male / Oocytes / Sperm-Ovum Interactions / Swine / Zona Pellucida
Yoko Sato, Kaoru Yoshida, Shiari Nozawa, Miki Yoshiike, Michiko Arai, Takeshige Otoi and Teruaki Iwamoto : Establishment of adult mouse Sertoli cell lines by using the starvation method., Reproduction, Vol.145, No.5, 505-516, 2013.
(Summary)
Sertoli cells were isolated from the testes of 6-week-old mice and stable Sertoli cell lines with higher proliferation rates were subcloned after starvation of primary cultured cells. After two rounds of this subcloning, 33 subcloned lines were selected on the basis of their proliferation rates. In addition, these subclones were screened according to their phagocytic activity and the characteristics of mature Sertoli cells, such as the expression of androgen receptors (ARs) and progesterone receptors, by using western blotting and immunocytochemical analysis, in addition to their morphology and proliferation rates. After the third round of subcloning, 12 subclones were selected for the final selection using RT-PCR for identification of genes specifically expressed by various testicular cells. Three clones were selected that expressed Sertoli-cell-specific genes, i.e., stem cell factor, clusterin, AR, α-inhibin, transferrin, Wilms' tumour-1, Müllerian inhibitory substance, sex-determining region Y-box 9, FSH receptor (Fshr) and occludin; however, these clones did not express globulin transcription factor 1, steroidogenic factor or androgen-binding protein. These clones also expressed growth and differentiation factors that act on germ cells, such as leukaemia inhibitory factor, transforming growth factor β1 and basic fibroblast growth factor 2, but did not express c-kit (specific for germ cells), LH receptor and 3β-hydroxyl-dehydrogenase (specific for Leydig cells). Immunocytochemical data confirmed the expression of clusterin in these clones. Furthermore, the Bromodeoxyuridine incorporation assay confirmed the proliferation activity of these clones through Fshr after treatment with FSH. These clones are considered to be valuable tools for the study of Sertoli cell-specific gene expression and function.
Vien Viet Luu, Keisuke Hanatate, Fuminori Tanihara, Yoko Sato, Lanh Thi Kim Do, Masayasu Taniguchi and Takeshige Otoi : 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, Vol.13, No.2, 122-126, 2013.
(Summary)
Relaxin is a member of the insulin-like family of hormones that promotes growth in a number of reproductive tissues, including the granulosa and theca cells. Cat oocytes collected from cold-stored ovaries remain capable of maturing in vitro, but the developmental ability of the oocytes decreases after 24h of cold storage. To improve the developmental ability of cat oocytes from cold-stored ovaries, we investigated the effect of relaxin supplementation of maturation medium on their meiotic ability and subsequent development. Cat oocytes were collected from ovaries stored at 4°C for one day and cultured in maturation medium supplemented with different concentrations (0, 10, 20, and 40ng/ml) of relaxin for 24h. They were then fertilized in vitro for 12h with frozen-thawed spermatozoa. After in vitro fertilization, the zygotes were cultured in synthetic oviduct fluid medium for 8 days. There were no significant differences in the maturation rates and glutathione contents of oocytes among the groups, irrespective of relaxin supplementation. The rate of blastocyst formation from oocytes matured with 10ng/ml relaxin (16.0%) was higher (p<0.05) than that from oocytes matured without relaxin (5.9%). Our findings indicate that supplementation of 10ng/ml relaxin into maturation medium may improve blastocyst formation of cat oocytes after in vitro fertilization.
(Keyword)
analysis of variance / Animals / Cats / Cryopreservation / Culture Media / Dose-Response Relationship, Drug / Embryo Culture Techniques / Embryonic Development / Female / Oocytes / ovary / Relaxin / Reproductive Techniques, Assisted
Masahito Komuro, Kenichi Suzuki, Makoto Kanebako, Takashi Kawahara, Takeshige Otoi, Kenji Kitazato, Toshio Inagi, Kimiko Makino, Masakazu Toi and Hiroshi Terada : Novel iontophoretic administration method for local therapy of breast cancer., Journal of Controlled Release, Vol.168, No.3, 298-306, 2013.
(Summary)
Ductal drug therapy is a novel therapeutic approach for primary breast cancers, particularly those involving ductal carcinoma in situ lesions. Total or partial mastectomy with or without radiotherapy is the standard local therapy for primary breast cancer. Here, we propose a novel drug administration method for ductal drug therapy based on a drug delivery system (DDS) for primary breast cancer. This DDS was designed to deliver miproxifen phosphate (TAT-59), an antiestrogen drug, to ductal lesions via the milk duct, where carcinomas originate, more efficiently than systemic administration, using an iontophoretic technique applied to the nipple (IP administration). Autoradiography imaging confirmed that TAT-59 was directly delivered to the milk duct using IP administration. The plasma concentrations of TAT-59 and its active metabolite DP-TAT-59 were quite low with IP administration. The area under the curve value of DP-TAT-59 in the mammary tissue was approximately 3 times higher with IP administration than with oral administration, at a 6-fold lower dose, indicating higher availability of the drug delivered via DDS than via systemic administration. The low plasma concentrations would limit adverse effects to minor ones. These characteristics show that this DDS is suitable for the delivery of active DP-TAT-59 to ductal lesions.
(Keyword)
Animals / Antineoplastic Agents / Breast Neoplasms / Dogs / Drug Delivery Systems / Estrogen Antagonists / Female / Humans / Iontophoresis / Mammary Glands, Animal / Rats / Tamoxifen
Zhao Namula, Yoko Sato, Risa Kodama, Kouta Morinaga, Viet Vien Luu, Masayasu Taniguchi, Michiko Nakai, Fuminori Tanihara, Kazuhiro Kikuchi, Takashi Nagai and Takeshige Otoi : Motility and fertility of boar semen after liquid preservation at 5°C for more than 2 weeks., Animal Science Journal, Vol.84, No.8, 600-606, 2013.
(Summary)
This study investigated the effects of skim milk on the quality and fertility of boar spermatozoa under long-term chilled preservation. Semen samples were stored in Modena solution supplemented with 0 (control) to 50 mg/mL skim milk at 5°C for 4 weeks; spermatozoa stored with 7.5 and 15 mg/mL of skim milk (7.5-SM and 15-SM groups, respectively) exhibited significantly higher motility indices than those of the control group up to 3 weeks (P < 0.05), and the 7.5-SM group showed improved motility indices even after 4 weeks (P < 0.05). In vitro fertilization using spermatozoa in the 7.5-SM and 15-SM groups stored at 5°C for 2 weeks showed significantly higher fertilization rates of spermatozoa and the development rates to blastocyst than the control group (P < 0.05), and the 7.5-SM group showed similar rates of fertilization and blastocyst formation in the fresh non-stored spermatozoa group. After artificial insemination using spermatozoa stored for 2 weeks in the 7.5-SM group, healthy piglets were obtained. Boar spermatozoa can be stored at 5°C in a Modena solution containing skim milk. Supplementation of 7.5 mg/mL skim milk improves boar spermatozoa motility and fertility even after liquid preservation at 5°C for 2 weeks.
(Keyword)
Animals / Cold Temperature / Fertilization in Vitro / Male / Semen / Semen Analysis / Semen Preservation / Sperm Motility / Swine / Time Factors
Budiyanto Agung, Takeshige Otoi, Dai-ichiro Fuchimoto, Shoichiro Senbon, Akira Onishi and Takashi Nagai : In vitro fertilization and development of porcine oocytes matured in follicular fluid., The Journal of Reproduction and Development, Vol.59, No.2, 103-106, 2013.
(Summary)
This study was conducted to assess the fertilization and development of porcine oocytes matured in a solo follicular fluid (pFF) using different in vitro culture systems and insemination periods. Cumulus-oocyte complexes (COCs), follicular cells (FCs), and pFF were collected from the follicles of ovaries. The pFF was used as a maturation medium (MpFF) after supplementation with follicle stimulating hormone (FSH) and antibiotics. The COCs were matured in a 15 ml test tube containing 3.5 ml of MpFF with FCs (5.2 × 10(6) cells/ml; rotating culture system) or 2 ml of MpFF without FCs in a 35-mm petri dish (static culture system) for 44 to 48 h. After maturation culture, oocytes were co-incubated with frozen-thawed spermatozoa for 5 h and then cultured for 7 days. The total mean rates of sperm penetration, normal fertilization, male pronucleus (MPN) formation, cleavage, and development to the blastocyst stage of oocytes after insemination were significantly higher (P<0.01) in the rotating culture system than in the static culture system. In conclusion, compared with the static culture system, the rotating culture system is adequate for the production of developmentally competent porcine oocytes when MpFF is used as a maturation medium.
(Keyword)
Animals / Culture Media / Female / Fertilization in Vitro / Follicular Fluid / In Vitro Oocyte Maturation Techniques / Male / Oocytes / Swine
Taniguchi Masayasu, B Agung, Y. Morita, Sato Yoko and Takeshige Otoi : The meiotic competence and DNA damage of porcine immature oocytes following cryoprotectant exposure and vitrification., Asian Journal of Animal and Veterinary Advances, Vol.8, 670-676, 2013.
117.
Mitsuhiro Takagi, S. Shiga, S. Uno, E. Kokushi, Takeshige Otoi, C. Tshering, E. Deguchi and J. Fink-Gremmels : Periodic alteration in urinary zearalenone excretion in a dairy cattle herd., Animal Nutrition and Food Technology, Vol.13, 303-310, 2013.
Takagi Mitsuhiro, T. Hirai, S. Shiga, S. Uno, E. Kokushi, Takeshige Otoi, E. Deguchi, C. Tshering and J. Fink-Gremmels : Relationship between urinary zearalenone concentration and embryo production in superovulated cattle., Archives Animal Breeding, Vol.56, 360-366, 2013.
(Summary)
<jats:p>Abstract. This field study aimed to investigate the relationships between the urinary zearalenone (ZEN) concentration, which reflects dietary ZEN intake, and the numbers of total and transferable embryos in superovulated cattle. A total of 38 cows (Japanese Black, n=16; Holstein, n=22) were superovulated for commercial embryo production. Urine samples were collected from all cows at the time of embryo flushing and the urinary ZEN concentration was measured. The ZEN concentration was corrected for the creatinine (Crea) concentration as follows: ZEN (pg/mL)/Crea (mg/dL); the corrected ZEN concentration was expressed in pg/mg Crea. The cows were divided into two groups according to whether the urinary ZEN level was less than (group 1) or more than (group 2) the mean value for each breed (Japanese Black: 97.4 pg/mg Crea; range 44.5–91.3 pg/mg Crea; Holstein: 155.5 pg/mg Crea; range 32.7–146.9 pg/mg Crea). The embryo flushing results were compared between the two groups within each breed. Overall, the total number of embryos collected and the number of transferable embryos did not differ significantly between the groups. These results suggest that natural ZEN contamination resulting in urine levels below the threshold value (i.e. below the maximal permissible urinary ZEN concentration) does not affect embryo production in Japanese Black and Holstein cows undergoing superovulation. </jats:p>
Urara Watanabe, Koji Okamoto, Akio Miyamoto, Takeshige Otoi, Osamu Yamato, Chenga Tshering and Mitsuhiro Takagi : Japanese Black breeding herd exhibiting low blood urea nitrogen: a metabolic profile study examining the effect on reproductive performance., Animal Science Journal, Vol.84, No.5, 389-394, 2012.
(Summary)
Ten reared cows of a Japanese Black cattle herd in Kagoshima prefecture, Japan, exhibited extremely low blood urea nitrogen (BUN) concentration (2.6 ± 0.6 mg/dL). Examination of dietary feed nutrition and relevant pastureland soil content suggested a correlation with crude protein (CP) deficiency or unbalanced nutritional dietary feeds. Thirteen months after the introduction of a dietary remedial measure (bean cake supplementation), BUN, total cholesterol and albumin concentration from five of the original 10 cows increased significantly compared with their values of before the dietary remedy. The postpartum day open period was significantly lower after the dietary remedial measure than that before it. The abnormally low BUN levels of the cattle herd may be due to inadequate dietary nutritional content, primarily from the imbalance of total digestible nutrient and CP of the feed and far lower han average CP value. In conclusion, routine examination of serum biochemical parameters in Japanese Black breeding cattle may be a useful strategy for determining subclinical metabolic failure of cattle herds, and consequently, its effect on reproductive performance of the herd.
Urara Watanabe, Mitsuhiro Takagi, Osamu Yamato, Takeshige Otoi, Chenga Tshering and Koji Okamoto : Metabolic profile of Japanese Black breeding cattle herds: usefulness in selection for nutrient supplementation to enhance reproductive performance and regional differences., The Journal of Veterinary Medical Science, Vol.75, No.4, 481-487, 2012.
(Summary)
The study aims were (1) to confirm the effects of nutritional improvement in prepartal and postpartal periods, monitored using the serum metabolic profile test (MPT) and reproductive performance, and (2) to clarify regional characteristics of the MPT results within our jurisdiction by using our MPT database. Experiment 1: Among 42 breeding cattle herds in our jurisdiction mainly fed home-pasture roughage, 3 experimental herds showing subnormal blood urea nitrogen (BUN) levels were selected and compared with 1 representative excellent herd. Dietary remedial measures were implemented from feed analysis in each herd. BUN concentration in all 3 herds increased significantly, and open days postpartum in 2 of the herds were significantly reduced, compared with values before dietary supplementation. Experiment 2: Thirty-seven herds within our jurisdiction were grouped into 3 categories (Area 1, 2 and 3) by location and soil condition of the herd pastureland. The MPT and reproductive performance in cows whose blood samples were collected at both prepartum (60-20 days before calving) and postpartum (30-90 days after calving) were compared among the 3 areas. Significant regional differences were found in prepartal albumin, total cholesterol, BUN, and glucose and postpartal BUN, glucose and open days (P<0.05). Overall, the MPT (especially BUN) might be useful for determining the metabolic nutritional status of breeding cattle herds, particularly those fed home-pasture roughage. Additionally, poor/unsatisfactory reproductive performance of beef breeding cattle herds probably reflects inadequate nutritional content of the diet, possibly arising from regional pastureland differences.
M Wittayarat, A Thongphakdee, K Saikhun, K Chatdarong, Takeshige Otoi and M Techakumphu : Cell cycle synchronization of skin fibroblast cells in four species of family Felidae., Reproduction in Domestic Animals = Zuchthygiene, Vol.48, No.2, 305-310, 2012.
(Summary)
This study was examined whether the species of felid affects synchronization accuracy at the G0/G1 stage of the cell cycle and the occurrence of apoptosis by different protocols, such as serum starvation, confluent and roscovitine treatment. Skin fibroblast cells were obtained from the Asian golden cat, marbled cat, leopard and Siamese cat. The cells from each animal were treated with either serum starvation for 1-5 days, cell confluency-contact inhibition for 5 days or roscovitine at various concentrations (7.5-30 μm). Flow cytometric analysis revealed that serum starvation for 3 days provided the highest cell population arrested at the G0/G1 stage, irrespective of the felid species. In all species, 100% confluency gave a significantly higher percentage of cells arrested at the G0/G1 stage compared with the non-treated control cells. The effects of roscovitine treatment and the appropriate concentration on the rates of G0/G1 cells differed among the felid species. Serum starvation for more than 4 days in the marbled cat and Siamese cat and roscovitine treatment with 30 μm in the Asian golden cat and leopard increased the rates of apoptosis. In conclusion, different felid species responded to different methods of cell cycle synchronization. Asian golden cat and Siamese cat fibroblast cells were successfully synchronized to G0/G1 stage using the serum starvation and roscovitine treatment, whereas only confluency-contact inhibition treatment induced cell synchronization in the leopard. Moreover, these three methods did not successfully induce cell synchronization of the marbled cat. These findings may be valuable for preparing their donor cells for somatic cell nuclear transfer in the future.
Rentsenkhand Sambuu, Mitsuhiro Takagi, Zhao Namula, Masahiro Nii, Masayasu Taniguchi, Seiich Uno, Emiko Kokushi, Chenga Tshering, Regiane Santos Rodrigues dos, Johanna Fink-Gremmels and Takeshige Otoi : Effects of long-term in vitro exposure of ejaculated boar sperm to zearalenone and α-zearalenol in sperm liquid storage medium., Animal Science Journal, Vol.84, No.1, 28-34, 2012.
(Summary)
The effects of in vitro exposure of porcine spermatozoa to zearalenone (ZEN) and α-zearalenol (α-ZOL) were studied by evaluating several parameters of an in vitro fertilization (IVF) system. For this purpose, boar spermatozoa cultured with semen storage medium containing 0 (control), 10 and 1000 µg/L of ZEN and α-ZOL for 1 week at 5°C were used for IVF of in vitro matured oocytes. Overall, there were no significant differences in the rates of total penetration, monospermic fertilization, and polyspermic fertilization of oocytes inseminated with spermatozoa from the different groups. Similarly, ZEN and α-ZOL at 10 and 1000 µg/L did not have detrimental effects on the cleavage and development to blastocysts of oocytes after in vitro fertilization. Although the motility, viability, and plasma membrane integrity of spermatozoa significantly decreased after 3 weeks of storage compared to non-stored spermatozoa (P < 0.05), ZEN and α-ZOL at the evaluated concentrations did not exert detrimental effects on the above parameters, even after 3 weeks of storage. These results indicate that prolonged exposure of boar spermatozoa to ZEN and α-ZOL up to 1000 µg/L under reduced metabolic conditions does not affect their in vitro function.
(Keyword)
Animals / Cells, Cultured / Culture Media / Dose-Response Relationship, Drug / Estrogens, Non-Steroidal / Fertilization / Fertilization in Vitro / Male / Sperm Motility / Spermatozoa / Swine / Temperature / Time Factors / Zearalenone / Zeranol
T Isobe, Y Ikebata, T Onitsuka, M Wittayarat, Y Sato, M Taniguchi and Takeshige Otoi : Effect of sericin on preimplantation development of bovine embryos cultured individually., Theriogenology, Vol.78, No.4, 747-752, 2012.
(Summary)
The silk protein sericin has been identified as a potent antioxidant in mammalian cells. This study was conducted to examine the effects of sericin on preimplantation development and quality of bovine embryos cultured individually. When two-cell-stage embryos were cultured individually for 7 days in CR1aa medium supplemented with 0, 0.1, 0.5, or 1% sericin, rates of total blastocyst formation and development to expanded blastocysts from embryos cultured with 0.5% sericin were higher (P < 0.05) than those from embryos cultured with 0 or 1% sericin. When embryos were cultured individually for 7 days in the CR1aa medium supplemented with 0 or 0.5% sericin under two oxidative stress conditions (50 or 100 μm H(2)O(2)), the addition of sericin significantly improved the blastocyst formation rate of embryos exposed to 100 μm H(2)O(2). However, the protective effect of sericin was not observed in development of embryos exposed to 50 μm H(2)O(2). When embryos were exposed to 100 μm H(2)O(2) during culture, the DNA fragmentation index of total blastocysts from embryos cultured with 0.5% sericin was lower than blastocysts derived from embryos cultured without sericin (4.4 vs. 6.8%; P < 0.01). In conclusion, the addition of 0.5% sericin to in vitro culture medium improved preimplantation development and quality of bovine embryos cultured individually by preventing oxidative stress.
(Keyword)
Animals / Blastocyst / Cattle / Cell Survival / Cells, Cultured / Culture Media / Embryo Culture Techniques / Embryonic Development / Fertilization in Vitro / Hydrogen Peroxide / Individuality / Oxidative Stress / Quality Control / Sericins
Y Kaedei, M Naito, H Naoi, Y Sato, M Taniguchi, Fuminori Tanihara, K Kikuchi, T Nagai and Takeshige Otoi : Effects of (-)-epigallocatechin gallate on the motility and penetrability of frozen-thawed boar spermatozoa incubated in the fertilization medium., Reproduction in Domestic Animals = Zuchthygiene, Vol.47, No.6, 880-886, 2012.
(Summary)
Epigallocatechin gallate (EGCG) is the major polyphenol in green tea (Camellia sinensis) and is known for its antioxidant effects. The objective of the present study was to examine the effects of EGCG during in vitro fertilization (IVF) on the sperm quality and penetrability into oocytes. In the first experiment, the effects of concentration and incubation period of EGCG on the motility and penetrability of spermatozoa were examined. When frozen-thawed spermatozoa were incubated in IVF medium supplemented with 0 (control), 1, 50 and 100 m EGCG for 1, 3 and 5 h, supplementation with 50 and 100 m EGCG improved motility of the spermatozoa (p < 0.05), but not viability, as compared with the control group. When frozen-thawed spermatozoa were co-incubated with in vitro-matured (IVM) oocytes in IVF medium supplemented with 50 and 100 m EGCG for 5 h, supplementation of EGCG had positive effects on sperm penetration rates. In the second experiment, the effects of supplementation of EGCG in IVF medium on penetrability of sperm from different boars and development of fertilized oocytes were evaluated. When frozen-thawed spermatozoa from six boars were co-incubated with IVM oocytes in IVF medium supplemented with 50 m EGCG, the effect of EGCG on sperm penetration and development of oocytes after fertilization was found to vary with individual boar. Our results indicate that motility and penetrability of boar spermatozoa are improved by co-incubation with 50 m EGCG, but the effects vary with individual boars.
(Keyword)
Animals / catechin / Cryopreservation / Dose-Response Relationship, Drug / Female / Fertilization in Vitro / Male / Semen Preservation / Sperm Motility / Sperm-Ovum Interactions / Spermatozoa / Swine
Yoko Sato, Shiari Nozawa, Miki Yoshiike, Takeshige Otoi and Teruaki Iwamoto : Glycoconjugates recognized by peanut agglutinin lectin in the inner acellular layer of the lamina propria of seminiferous tubules in human testes showing impaired spermatogenesis., Human Reproduction, Vol.27, No.3, 659-668, 2012.
(Summary)
The aim of this study was to evaluate the histochemical characteristics of the thickened inner acellular layer (IL) of the lamina propria specifically present in the human seminiferous tubules of testes showing impaired spermatogenesis. Eighteen biopsies for the investigation of infertility and 10 orchiectomies for testicular cancer and cryptorchidism were used. Lectin staining [peanut agglutinin (PNA), Maackia amurensis (MAA), Sambuccus nigra (SNA)], PNA lectin staining with sialidase digestion, immunohistochemistry and binding assay of progesterone were performed and analysed quantitatively. The IL of the thickened lamina propria of the seminiferous tubules in the testes showed PNA lectin affinity and binding affinity for progesterone. Both affinities of MAA and SNA were in the IL of only fairly thickened lamina propria. Furthermore, a positive correlation was present between the thickness of the lamina propria and the accumulation of glycoconjugates showing PNA lectin affinity (r = 0.829, P < 0.001) or progesterone (r = 0.629, P < 0.001) in the IL. However, ILs show no immunoreactivities of progesterone receptor, androgen receptor or human serum albumin. Progesterone inhibited the binding affinity of PNA lectin to the IL (P < 0.001), but not the affinity to the spermatogenic cells. In addition, sialidase digestion increased the PNA affinity not in the IL but in the spermatogenic cells (P < 0.001). These results indicate that the IL of the thickened lamina propria always consists of glycoconjugates with PNA lectin affinity and possible binding affinity to progesterone. In addition, the glycoconjugates in the IL may be predictors of abnormal spermatogenesis in the testes of infertile patients.
Manita Wittayarat, Taichi Kimura, Risa Kodama, Zhao Namula, Kaywalee Chatdarong, Mongkol Techakumphu, Yoko Sato, Masayasu Taniguchi and Takeshige Otoi : Long-term preservation of chilled canine semen using vitamin C in combination with green tea polyphenol., Cryo Letters, Vol.33, No.4, 318-326, 2012.
(Summary)
Vitamin C and green tea polyphenol are known to have antioxidant effects. The aim of this study was to evaluate the quality of canine semen after preservation with diluents containing vitamin C and polyphenol at 5 degree C for 4 weeks. In experiment 1, we investigated the effects of vitamin C combined with polyphenol supplementation on chilled semen quality. The addition of vitamin C (0.5 or 1 mM) with 0.75 mg per mL polyphenol to semen extender provided significantly higher percentages of sperm motility and viability during cold storage compared to unsupplemented semen. In experiment 2, we determined the optimal working concentration of vitamin C in the semen extender by comparison of a range of concentrations between 0.1 and 20 mM. Supplementation of 0.5 mM vitamin C plus polyphenol yielded the highest percentages of sperm motility and viability; however, there was no beneficial effect on the plasma membrane and acrosomal integrity of the spermatozoa.
Pimprapar Wongsrikeao, Dyana Saenz, Tommy Rinkoski, Takeshige Otoi and Eric Poeschla : Antiviral restriction factor transgenesis in the domestic cat., Nature Methods, Vol.8, No.10, 853-859, 2011.
(Summary)
Studies of the domestic cat have contributed to many scientific advances, including the present understanding of the mammalian cerebral cortex. A practical capability for cat transgenesis is needed to realize the distinctive potential of research on this neurobehaviorally complex, accessible species for advancing human and feline health. For example, humans and cats are afflicted with pandemic AIDS lentiviruses that are susceptible to species-specific restriction factors. Here we introduced genes encoding such a factor, rhesus macaque TRIMCyp, and eGFP, into the cat germline. The method establishes gamete-targeted transgenesis for the first time in a carnivore. We observed uniformly transgenic outcomes, widespread expression, no mosaicism and no F1 silencing. TRIMCyp transgenic cat lymphocytes resisted feline immunodeficiency virus replication. This capability to experimentally manipulate the genome of an AIDS-susceptible species can be used to test the potential of restriction factors for HIV gene therapy and to build models of other infectious and noninfectious diseases.
Masayasu Taniguchi, Rie Arikawa, Yukine Kaedei, Fuminori Tanihara, Zhao Namula, Vien Luu Viet, Yoko Sato and Takeshige Otoi : Effects of cryoprotectant agents and equilibration methods on developmental competence of porcine oocytes., Cryo Letters, Vol.32, No.5, 410-414, 2011.
(Summary)
Chemical toxicity of cryoprotectants to in vitro developmental competence of porcine oocytes was examined. In vitro-matured oocytes were exposed to 40 percent ethylene glycol (EG), glycerol (GLY), or 1,2-propanediol (PD), fertilized with spermatozoa, and cultured for 8 days. Compared to treatment with other cryoprotectants, exposure to EG resulted in the development of significantly more blastocysts, but the rate was significantly lower than that of non-exposed control oocytes. In vitro-matured oocytes were also equilibrated in 40 percent EG by 3 multi-step methods, after which their developmental competence was evaluated. The rate of blastocyst development was higher in the 4-step method than in the 2- and 3-step methods of equilibrium. These results indicate that cryoprotectants and equilibration methods affect the developmental competence of porcine oocytes and that EG may be a superior cryoprotectant for vitrification of these cells.
T Terazono, Y Kaedei, Fuminori Tanihara, Z Namula, Vl Viet, M Takagi, M Inoue, Y Sato, M Taniguchi and Takeshige Otoi : Follicle formation in the canine ovary after autografting to a peripheral site., Reproduction in Domestic Animals = Zuchthygiene, Vol.47, No.2, e16-21, 2011.
(Summary)
This study reports about follicular development on the surface of canine ovarian tissue after autografting under the fascia of the thoracolumbar muscle and about meiotic resumption of follicle-derived oocyte after maturation culture. After ovarian excision from a bitch, each ovary of the pairs was cut approximately into half. The hemi-ovaries were transplanted into the bitch of origin at three different body sites (under the fascia of the quadriceps femoris muscle and the thoracolumbar muscle, and in the deltoid muscle in the scapular region). All grafted ovaries were recovered from the bitch at 35 days post-transplantation. A visible antral follicle was observed on the surface of the ovary grafted under the thoracolumbar fascia. Histological examination revealed viable follicles at different stages of development irrespective of graft site. Most granulosa cells in the follicles at different stages of development expressed proliferating cell nuclear antigen (PCNA). A total of three oocytes were collected from an ovary grafted under the fascia of the thoracolumbar muscle, wherein an oocyte reached metaphase I after maturation culture. This is the first report to demonstrate follicular development and meiotic resumption of oocytes recovered from autografted canine ovarian tissues.
T Terazono, M Inoue, Y Kaedei, Fuminori Tanihara, Z Namula, L V Viet, Y Taura, M Takagi, T Takuma and Takeshige Otoi : Assessment of canine ovaries autografted to various body sites., Theriogenology, Vol.77, No.1, 131-138, 2011.
(Summary)
The influence of graft site on the survival of canine follicles and oocytes after autografting was investigated. Hemi-ovaries were autografted to three locations (quadriceps femoris muscle fascia, kidney capsule, and gastrosplenic ligament), and grafted ovaries were recovered (under anesthesia) 28 to 31 d after transplantation. The grafted hemi-ovaries were bisected: one-quarter ovary was used for histological assessment and another quarter for evaluation of oocyte viability. As controls, the remaining fresh hemi-ovaries were used to assess the viability of follicles and oocytes in non-transplanted ovaries. Most follicles in the histological sections of the grafts were classified as primordial or primary follicles. Antral follicles were not observed in the grafts, irrespective of the graft site. The percentages of viable follicles in the sections from control ovaries, and the ovaries grafted to the kidney capsule, the quadriceps femoris muscle fascia, and the gastrosplenic ligament were 17.4, 22.9, 18.3, and 32.4%, respectively. A total of 12 oocytes was recovered from the 15 hemi-ovaries grafted in five bitches, of which five (41.7%) oocytes from the ovaries grafted to the quadriceps femoris muscle fascia and the kidney capsule were cultured for assessment of meiotic competence. Three oocytes were viable but remained in the germinal vesicle stage after 72 h of maturation culture. The quadriceps femoris muscle fascia might be useful for grafting like the kidney capsule, but improvement of follicle survival and meiotic competence of oocytes in the grafts is necessary.
Masao Murakami, Juan Ya Dong, Tatsuyuki Suzuki, Masayasu Taniguchi, Yukine Kaedei, Yoko Sato, Fuminori Tanihara and Takeshige Otoi : Development and subsequent cryotolerance of domestic cat embryos cultured in serum-free and serum-containing media., Cryobiology, Vol.63, No.3, 170-174, 2011.
(Summary)
The objectives of this study were to examine the effects of the presence or absence of serum during the in vitro culturing period of domestic cat embryos on their developmental potential into blastocysts as well as their tolerance to cryopreservation using a slow-freezing method. In vitro-fertilized cat oocytes were incubated in a modified synthetic oviduct fluid (mSOF) containing 4 mg/mL bovine serum albumin (BSA) throughout culturing (BSA group) or in mSOF containing 4 mg/mL BSA for the first 3 days followed by mSOF containing 5% fetal bovine serum (FBS group). The developmental potential of the embryos to the blastocyst and expanded blastocyst stages was evaluated 7 days after in vitro fertilization. The blastocysts were frozen-thawed by the slow-freezing method and cultured for 3 days to examine their viability in vitro. There were no differences in the formation rates of blastocysts or expanded blastocysts, or number of cells in the embryos between the two groups. After cryopreservation, the hatching rates of the expanded blastocysts in the BSA group were significantly higher (P<0.05) than those of the FBS group. The postthaw viability of blastocysts was lower than that of expanded blastocysts irrespective of culture medium. These results indicate that the developmental potential of cat embryos cultured in serum-free medium is comparable to those cultured in serum-containing medium. Furthermore, expanded blastocysts produced without serum exhibit better postthaw viability than those produced with serum.
Rentsenkhand Sambuu, Mitsuhiro Takagi, Zhao Namula, Takeshige Otoi, Satoshi Shiga, Regiane Dos Santos Rodrigues and Johanna Fink-Gremmels : Effects of exposure to zearalenone on porcine oocytes and sperm during maturation and fertilization in vitro., The Journal of Reproduction and Development, Vol.57, No.4, 547-550, 2011.
(Summary)
The influence of acute exposure to zearalenone (ZEN) on porcine oocyte maturation, fertilization or sperm penetration ability during both in vitro maturation and fertilization was evaluated. First, oocytes were cultured in ZEN-containing (0-1000 µg/l) maturation medium and then fertilized. The oocytes maturing in vitro without ZEN were then fertilized in ZEN-containing fertilization medium. The maturation rates of oocytes and penetration ability of sperm decreased significantly in the presence of 1000 µg/l of ZEN. However, neither increases in the rates of degeneration and DNA fragmentation of oocytes nor reductions in normal and polyspermic fertilization were observed. ZEN did not affect the sperm penetration rates; however, 1000 µg/l ZEN had positive effects on normal and polyspermic fertilization rates. Therefore, it can be suggested that an acute exposure of porcine oocytes during maturation and of oocytes and sperm during fertilization to ZEN up to 1000 µg/l may not affect the fertility of the oocytes.
Atsushi Taniyama, Yasutaka Watanabe, Youji Nishino, Tetsurou Inoue, Yasuho Taura, Mitsuhiro Takagi, Chikara Kubota and Takeshige Otoi : Assisted hatching of poor-quality bovine embryos increases pregnancy success rate after embryo transfer., The Journal of Reproduction and Development, Vol.57, No.4, 543-546, 2011.
(Summary)
Embryos of good, fair and poor quality were collected from superovulated cows and subjected to zona cutting (ZC) treatment using a needle under either an inverted microscope or a stereomicroscope. One (single transfer) or 2 (twin transfer) embryos with or without prior ZC treatment were transferred nonsurgically to recipients. Without the ZC treatment, lower embryonic quality resulted in lower pregnancy success rates. However, the ZC treatment increased the pregnancy success rate following transfer of poor-quality embryos, but not the pregnancy rate after transfer of good- or fair-quality embryos. No differences were observed between the pregnancy success rates after the transfer of embryos treated under the inverted microscope and those after transfer of embryos treated under the stereomicroscope, and this was the same after single and twin transfer. Moreover, ZC treatment of embryos prior to transfer did not result in an increased abortion rate, irrespective of the number of transferred embryos. In conclusion, ZC treatment improves pregnancy success rates following transfer of poor-quality embryos. Moreover, the results indicate that ZC treatment by using a stereomicroscope is practical for on-farm application.
Pimprapar Wongsrikeao, Shizuyo Sutou, Miho Kunishi, Ya Juan Dong, Xuejin Bai and Takeshige Otoi : Combination of the somatic cell nuclear transfer method and RNAi technology for the production of a prion gene-knockdown calf using plasmid vectors harboring the U6 or tRNA promoter., Prion, Vol.5, No.1, 39-46, 2011.
(Summary)
By combining RNAi technology with SCNT method, we attempted to produce transgenic calves with knocked down bPRNP for technological assessments. The respective utilities of type II (tRNA) and type III (hU6) Pol III promoters in mediating plasmid vector-based RNAi for the production of a bPRNP-knockdown calf were compared. Plasmid harboring DNA for siRNA expression was introduced stably into the genome of primary cultured bovine cells. By inserting the transgenic cell into an enucleated bovine egg, SCNT embryos were produced. The ability for SCNT embryos to develop to blastocysts was higher in hU6 based vector groups (44-53%) than in a tRNA group (32%). In all, 30 hU6-embryos and 12 tRNA-embryos were transferred to 11 recipients. Only tRNA-embryos were able to impregnate recipients (6 out of 11 transfers), resulting in four aborted fetuses, one stillbirth, and one live-born calf. The expression of EGFP, a marker, was detected in all six. The bPRNP transcript levels in the nervous tissues (brain, cerebellum, spinal bulb, and spinal cord) from the calf, which was killed 20 days after birth, were reduced to 35% of those of the control calf on average, as determined by qRT-PCR. The PrPC levels, as estimated by western blot were reduced to 86% on average in the nervous tissues. These findings suggest that SCNT technology remains immature, that the tRNA promoter is useful, and that RNAi can significantly reduce PRNP mRNA levels, but insufficient reduction of PrPC levels exists in cattle under these conditions.
Rentsenkhand Sambuu, Mitsuhiro Takagi, Satoshi Shiga, Seiichi Uno, Emiko Kokushi, Zhao Namula, Takeshige Otoi, Akio Miyamoto, Eisaburo Deguchi and Johanna Fink-Gremmels : Detection of zearalenone and its metabolites in naturally contaminated porcine follicular fluid by using liquid chromatography-tandem mass spectrometry., The Journal of Reproduction and Development, Vol.57, No.2, 303-306, 2010.
(Summary)
Zearalenone (ZEN) and its metabolites are important nonsteroidal estrogenic mycotoxins that cause reproductive disorders in domestic animals, especially pigs. We aimed to simultaneously detect ZEN and its metabolites á-zearalenol (α-ZOL) and β-zearalenol (β-ZOL) in porcine follicular fluid (FF) by liquid chromatography-tandem mass spectrometry. ZEN and α-ZOL, but not β-ZOL, were detected in all pooled FF samples collected from coexisting follicles (diameter ≥ 6 mm) within 10 ovaries. Furthermore, ZEN and α-ZOL were detected in samples pretreated with β-glucuronidase/arylsulfatase, but not in those left untreated, suggesting that the FF samples contained glucuronide-conjugated forms of the mycotoxins that may be less harmful to porcine oocytes due to glucuronidation affecting the receptor binding. Nonetheless, the effects of the glucuronide-conjugated forms should be studied, both in vitro and in vivo.
V E Abakushina, Y Morita, Y Kaedei, Fuminori Tanihara, Z Namula, L V Viet and Takeshige 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 = Zuchthygiene, Vol.46, No.3, 423-427, 2010.
(Summary)
Culture techniques of antral follicle-like structure (AFLS) derived from cumulus-oocyte complexes (COCs) might provide important insights into follicular development and oocyte maturation. This study was undertaken to investigate the effects of embedding bovine COCs individually (one COC) or in groups (4-5 COCs) in collagen gels on the formation of AFLS and the meiotic status of oocytes. The observations of AFLS formation were performed every second day for 14 days. The AFLS was formed at Day 2 or 4 after the start of culture (Day=0), irrespective of the culture methods. The mean diameters of AFLS during Days 4-14 using the individual culture method were significantly higher (p<0.05) than those using the group culture method. However, the AFLS formation rate in the individual culture method was significantly lower compared to that in the group culture method (26.1% vs 62.7%, p<0.01). Almost all oocytes had undergone the germinal vesicle breakdown stage, irrespective of the culture method or AFLS formation. In conclusion, comparison with the individual culture method revealed that the mean diameters of AFLS in the group culture method were smaller, but more COCs formed AFLS. The group culture method might be useful for evaluating the various hypotheses of follicular formation and interfollicular communication. However, improvement of the group culture system is necessary to prevent the meiotic resumption of oocytes, because the AFLS formation is dependent on the cumulus/granulosa cells surrounding oocytes.
Yavari Morteza, Naoi Hideaki, Kaedei Yukine, Fuminori Tanihara, Namula Zhao, Viet Luu Vien and Takeshige Otoi : Effects of epigallocatechin- 3-gallate on the developmental competence of parthenogenetic embryos in the pig, Italian Journal of Animal Science, Vol.9, No.4, 386-389, 2010.
(Summary)
This study was conducted to examine the effects of (-)-epigallocatechin gallate (EGCG) supplementation on the developmental competence and quality of parthenogenetic porcine embryos during culture. Parthenogenetic embryos derived from in vitro matured oocytes were cultured for eight days in a modified North Carolina State University (NCSU)-37 solution supplemented with EGCG at different concentrations (0, 1, 5, 10 and 50 µM). Supplementation of 1 and 5 µM EGCG during in vitro culture of embryos showed no significant influence on the rate of cleavage or that of blastocyst formation or on the total cell number and DNA fragmentation indices of blastocysts when compared to those of a control group. However, when 10 and 50 µM EGCG were supplemented into the culture medium, the cleavage rates were significantly lower than those of the other groups. No embryo developed to the blastocyst stage. Results suggest that treatment with low EGCG during in vitro culture has no influence on the developmental competence of porcine embryos but the presence of high concentrations of EGCG is apparently harmful for in vitro development of porcine parthenotes.
K. Itamoto, K. Hara, K. Tani, M. Nakaichi, M. Okuda, Takeshige Otoi, H. Inokuma and Y. Taura : Influence of volatile anesthetics on muscle relaxant effect of vecuronium in dogs., Journal of Animal Veterinary advances., Vol.9, No.7, 1131-1136, 2010.
139.
K. Itamoto, M. Nakaichi, M. Okuda, Takeshige Otoi, H. Inokuma and Y. Taura : Effects of medetomidine and atipamezole on cerebral perfusion pressure in dogs., Journal of Animal Veterinary advances, Vol.9, No.5, 913-919, 2010.
140.
Makoto Seki, Norio Watanabe, Kenyo Ishii, Yoh-Ichi Kinoshita, Takehiro Aihara, Shuji Takeiri and Takeshige Otoi : Plasma progesterone profiles in Beagle bitches with and without the whelping experience., Acta Veterinaria Hungarica, Vol.58, No.1, 117-124, 2010.
(Summary)
The aim of this study was to investigate differences between the progesterone profiles of Beagle bitches with (multiparous) and without (nulliparous) the whelping experience and to examine whether the selection of bitches by progesterone analyses before the programmed mating improved the whelping rates. In the first experiment, the progesterone profiles of nulliparous and multiparous bitches were evaluated from Days 1 to 13 (onset of prooestrus = Day 0). The mean duration of the elevation in progesterone levels (> 2 ng/mL) after the onset of prooestrus tended to be approximately 1 day shorter in nulliparous bitches (7.7 days) than in multiparous bitches (8.5 days). In the second experiment, progesterone analyses in the bitches were carried out once on Days 4, 5 or 6. Bitches with progesterone levels of > 10 ng/mL were excluded from mating because it was unclear when the progesterone levels reached > 10 ng/mL considering the optimal date for mating. No significant differences were observed in the percentages of bitches excluded from mating and in the whelping rates of the mated bitches between the groups, irrespective of the day of progesterone analysis and the type of bitch. In conclusion, the initial elevation of progesterone levels was of shorter duration in nulliparous bitches. The selection of bitches by the measurement of progesterone levels once before mating was not effective for the programmed mating.
B Agung, Y Piao, D Fuchimoto, S Senbon, A Onishi, Takeshige Otoi and T Nagai : Effects of oxygen tension and follicle cells on maturation and fertilization of porcine oocytes during in vitro culture in follicular fluid., Theriogenology, Vol.73, No.7, 893-899, 2010.
(Summary)
The objective was to investigate the effects of oxygen tension and follicle cells (FCs) during in vitro maturation of porcine oocytes in only porcine (Sus scrofa domesticus) follicular fluid (pFF), using static and non-static (rotating) culture systems, on the nuclear maturation and subsequent in vitro fertilization of the oocytes. In the first experiment, cumulus-oocyte complexes (COCs) were matured for 48 h in pFF supplemented with (+) or without (-) FCs (5.2x10(6) cells/mL), using the static (S) and rotating (R) culture systems (+FC/S, -FC/S, +FC/R, and -FC/R) under 5% or 20% O(2). Co-culture with FCs in the static culture system (+FC/S) had a detrimental effect on the meiotic competence of oocytes, whereas co-culture with FCs in the rotating culture system (+FC/R) increased maturation rates. In both culture systems, oxygen tension had no apparent effects on meiotic competence of oocytes, irrespective of culture system and FC addition. In the second experiment, COCs were matured under 5% or 20% O(2) using the -FC/S or +FC/R culture systems and then fertilized. Oxygen tension had no significant effects on fertilization parameters, irrespective of the culture system. The rotating culture system increased rates of sperm penetration and male pronuclear formation and decreased polyspermic fertilization compared with the static culture system (P < 0.05). In conclusion, both -FC/S and +FC/R culture systems supported meiotic competence, irrespective of oxygen tension. However, the +FC/R culture system may be superior to the -FC/S culture system for promoting fertilization.
Makoto Seki, Norio Watanabe, Kenyo Ishii, Yoh-ichi Kinoshita, Takehiro Aihara, Shuji Takeiri and Takeshige Otoi : Influence of parity and litter size on gestation length in beagle dogs., Canadian Journal of Veterinary Research, Vol.74, No.1, 78-80, 2010.
(Summary)
This study was conducted to investigate the effects of parity and litter size on gestation length in beagle bitches. The mean duration of the initial elevation (>2 ng/mL) in progesterone concentrations after the onset of proestrus was shorter (P < 0.05) in bitches without (nulliparous) whelping experience than in bitches with (multiparous) whelping experience (6.9 d versus 8.0 d). When calculated as the interval between the day of initial elevation in progesterone concentrations and the day of whelping, the gestation length in the nulliparous bitches was noted to be similar to that in the multiparous bitches (64.3 d versus 64.2 d). No significant correlation between gestation length and litter size was observed in any of the bitches. Our results indicate that the gestation length in beagle bitches is not affected by parity or litter size.
Kaedei Yukine, Fujiwara Akira, Ito Aya, Fuminori Tanihara, Morita Yasuhiro, Hanatate Keisuke, Viet Luu Vien, Namula Zhao and 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, Vol.9, No.22, 2848-2853, 2010.
(Summary)
Roscovitine, a specific inhibitor of M-phase promoting factor kinase activity was used to inhibit the completion of meiotic maturation of bovine oocytes. The objectives of this study were to evaluate the nuclear maturation of bovine oocytes pre-cultured with various concentrations (0, 50, 100 and 200 μM) of roscovitine before in vitro Maturation (IVM) and to examine the development of Somatic Cell Nuclear Transfer (SCNT) embryos derived from the oocytes pre-cultured with roscovitine. Before IVM, 72% of oocytes that were cultured without roscovitine (control) had reached the Metaphase II (MII) stage whereas culture with roscovitine decreased the rates of oocytes reaching MII (11-27%). After IVM, the maturation rate of oocytes pre-cultured with 200 μM roscovitine was significantly higher than that of control oocytes (79 vs. 58%). Moreover, significantly more oocytes extruded the first polar body in the 50 μM roscovitine group than in the control group (64 vs. 51%). The rate of blastocyst formation of reconstructed embryos derived from oocytes pre-cultured with 50 μM roscovitine was significantly higher than that from the control oocytes (14 vs. 6%). In this study, the addition of roscovitine to culture medium delays the completion of meiotic maturation of bovine oocytes and the cytoplasm derived from oocytes pre-cultured under meiotic inhibition can support the development of SCNT embryos.
Y Kaedei, A Fujiwara, Fuminori Tanihara, Z Namula, VL Viet and Takeshige Otoi : In vitro development of cat interspecies nuclear transfer using pig's and cow's cytoplasm, Bulletin of the Veterinary Institute in Puławy, Vol.54, No.3, 405-408, 2010.
Tetsuya Takuma, Sayoko Sakai, Daisuke Ezoe, Hitoshi Ichimaru, Takaomi Jinnouchi, Yukine Kaedei, Takashi Nagai and Takeshige Otoi : Effects of season and reproductive phase on the quality, quantity and developmental competence of oocytes aspirated from Japanese black cows., The Journal of Reproduction and Development, Vol.56, No.1, 55-59, 2009.
(Summary)
The aim of the present study was to determine whether the season (hot and cool) and reproductive phase (pregnant and non-pregnant) of the cow affect follicular recruitment and oocyte development. Follicular oocytes were aspirated from Japanese black cows by the ovum pick-up (OPU) method, which was performed 2 to 6 times within 1.5 months in pregnant cows and 2 to 4 times within 2 months in non-pregnant cows, during the hot (July to September) and cool (October to November) seasons. After follicular aspiration, the number and morphology of cumulus-oocyte complexes (COCs) and the developmental competence of oocytes after in vitro maturation (IVM), in vitro fertilization (IVF) and in vitro culture were evaluated. The quality of aspirated COCs did not differ between the hot and cool seasons, irrespective of the reproductive phase of the donor cows. In the pregnant cows, the season did not affect follicular recruitment, early embryonic development or the quality of embryos. In the non-pregnant cows, however, the mean number of aspirated follicles and collected oocytes decreased during the hot season as compared with the cool season. When the data for the 2 seasons were combined to assess the effects of reproductive phase on oocyte development, the total proportions of cleavage, development into blastocysts and freezable embryos were higher for embryos obtained from pregnant cows (P<0.05) than those obtained from non-pregnant cows. In conclusion, the season did not have any apparent effects on the quality of aspirated COCs and the developmental competence of oocytes after IVM-IVF, but it may affect follicular recruitment in non-pregnant cows. Moreover, the reproductive phase may influence the developmental competence of the recovered oocytes.
(Keyword)
Animals / Cattle / Cumulus Cells / Embryo, Mammalian / Estrous Cycle / Female / Fertilization in Vitro / Hot Temperature / Japan / Oocyte Retrieval / Oocytes / Ovarian Follicle / Pregnancy / Seasons
M Hayashi, H Tsuchiya, Takeshige Otoi, B Agung, N Yamamoto and K Tomita : Influence of freezing with liquid nitrogen on whole-knee joint grafts and protection of cartilage from cryoinjury in rabbits., Cryobiology, Vol.59, No.1, 28-35, 2009.
(Summary)
Improving survival rates for sarcoma patients are necessitating more functional and durable methods of reconstruction after tumor resection. Frozen osteoarticular grafts are utilized for joint reconstruction, but the joint may develop osteoarthritic change. We used a frozen autologous whole-rabbit knee joint graft model to investigate the influence of freezing on joint components. Thirty rabbit knee joints that had been directly immersed into liquid nitrogen (L) or saline (C) without use of cryoprotectants were re-implanted. Histological observations were made after 4, 8, and 12 weeks. Both groups had bone healing. In group L, despite restoration of cellularity to the menisci and ligaments, no live chondrocytes were observed and cartilage deterioration progressed over time. It was concluded that cryoinjury of chondrocytes caused osteoarthritic change. Then we tested whether a vitrification method could protect cartilage from cryoinjury. Full-thickness articular cartilage of rabbit knee was immersed into liquid nitrogen with and without vitrification. Histology, ultrastructure, and chondrocyte viability were examined before and after 24h of culture. Vitrified cartilage cell viability was >85% compared with that of fresh cartilage. Transmission electron microscopy revealed preservation of original chondrocyte structure. Our vitrification method was effective for protecting chondrocytes from cryoinjury. Since reconstructing joints with osteoarticular grafts containing living cartilage avert osteoarthritic changes, vitrification method may be useful for storage of living cartilage for allografts or, in Asian countries, for reconstruction with frozen autografts containing tumors.
A Fujii, Y Kaedei, Fuminori Tanihara, A Ito, K Hanatate, K Kikuchi, T Nagai and Takeshige 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 = Zuchthygiene, Vol.45, No.4, 619-624, 2009.
(Summary)
We investigated the effects of a portable incubator with a CO(2) chamber on the viability and development of porcine oocytes/embryos for their transportation and examined the operational suitability of a straw or dish as a container for culturing the oocytes or embryos in the portable incubator. In the first experiment, the cumulus-oocyte complexes (COCs) were placed either in a dish or straw; and they were then cultured for 44 h in a standard CO(2) incubator, in the CO(2) chamber in an incubator, or in the CO(2) chamber in a portable incubator. The matured oocytes were fertilized with frozen-thawed spermatozoa and then cultured in a dish in the standard CO(2) incubator for 8 days. There were no differences in the proportions of oocytes reaching metaphase II stage among the groups. However, the proportions of cleavage and development to blastocysts derived from oocytes matured in a straw were lower than those from oocytes matured in a dish, irrespective of the type of incubator used. In the second experiment, the COCs were matured in a dish in the standard CO(2) incubator, and the matured oocytes were fertilized and then placed either in a dish or straw. These were then cultured for 8 days in the standard CO(2) incubator or portable incubator. Some zygotes cultured in the portable incubator developed to the blastocyst stage. The proportions of cleavage and development to blastocysts were significantly lower for putative zygotes cultured in straw than for those cultured in dish, irrespective of the type of incubator used. Our results indicate that a portable incubator with a CO(2) chamber can maintain the viability and development of oocytes/embryos, but the straw is not a suitable system for in vitro culture of the oocytes/embryos during transportation.
T Takuma, T Otsubo, Y Kurokawa, H Ichimaru and Takeshige Otoi : Effects of twice-weekly follicular punctures of ovaries with or without the corpus luteum on follicular and luteal dynamics., Reproduction in Domestic Animals = Zuchthygiene, Vol.45, No.1, 50-54, 2008.
(Summary)
We investigated the effects of twice-weekly follicular punctures of ovaries with or without corpus luteum (CL) on follicular and luteal dynamics. A cross-over design was used, with each cow (seven Japanese Black beef cows) being assigned to one of the three groups at 2-month intervals. Follicular punctures were performed twice weekly for three consecutive weeks until day 20 (oestrus = day 0). All visible follicles (diameter >3 mm) in the ovaries bearing CL (ipsilateral group) or those in the contralateral ovaries (contralateral group) were aspirated. As a control, all visible follicles in both ovaries were aspirated (bilateral group). Follicular development, CL formation and progesterone concentrations in each cow were monitored from days 0 to 30. Follicular growth profiles in the punctured ovaries during/after puncture treatment were similar, irrespective of the presence of follicles in the unpunctured ovary and the CL in the punctured or unpunctured ovaries. After puncture, two cows (28.6%) each in the ipsilateral and bilateral groups did not exhibit behavioural oestrus until day 30, whereas all cows in the contralateral group exhibited oestrus. CL growth and increase in progesterone concentrations after the last follicular puncture in the bilateral group were delayed when compared with those in the ipsilateral group. Our results indicate that the presence of follicles in the unpunctured ovary and the CL in the punctured or unpunctured ovaries does not significantly influence follicular growth in punctured ovaries during/after puncture treatment. However, follicular puncture in ovaries bearing CL may disturb or delay oestrus occurrence after treatment.
(Keyword)
Animals / Cattle / Corpus Luteum / Female / Ovarian Follicle / Ovary / Progesterone / Punctures / Tissue and Organ Harvesting
Mitsuhiro Takagi, Shuhei Mukai, Toshiyuki Kuriyagawa, Katsuhito Takagaki, Seiichi Uno, Emiko Kokushi, Takeshige Otoi, Agung Budiyanto, Koumei Shirasuna, Akio Miyamoto, Osamu Kawamura, Koji Okamoto and Eisaburo Deguchi : Detection of zearalenone and its metabolites in naturally contaminated follicular fluids by using LC/MS/MS and in vitro effects of zearalenone on oocyte maturation in cattle., Reproductive Toxicology, Vol.26, No.2, 164-169, 2008.
(Summary)
Zearalenone (Zen) and its metabolites are estrogenic and may be important factors involved in reproductive disorders in domestic animals. We aimed to (1) simultaneously detect Zen and its metabolites in bovine follicular fluids (FFs) by liquid chromatography-tandem mass spectrometry and (2) examine the in vitro effects of Zen on bovine oocytes. Zen and its metabolites were detected in 6 of 32 normal follicles and 7 of 20 cystic follicles. Bovine oocytes were cultured in a maturation media containing various Zen concentrations (0 [control], 1, 10, 100, and 1000microg/L), fertilized, and cultured further. Maturation rates decreased dose-dependently. Further, maturation of 62 (50%) of 124 oocytes examined in the 1000-microg/L group was arrested in metaphase I, without affecting the fertilization rate. Blastocyst-formation rates did not significantly differ among the groups. Zen and its metabolites were detectable in bovine FFs. High Zen concentration may adversely affect meiotic competence but not the fertilization and development rates.
(Keyword)
Animals / Cattle / Chromatography, Liquid / Embryonic Development / Estrogens, Non-Steroidal / Female / Follicular Fluid / In Vitro Techniques / Oocytes / Tandem Mass Spectrometry / Zearalenone
H Naoi, B Agung, N W. K. Karja, P Wongsrikeao, R Shimizu, M Taniguchi and Takeshige Otoi : Effects of the reproductive status on morphological oocyte quality and developmental competence of oocytes after in vitro fertilization and somatic cell nuclear transfer in cat., Reproduction in Domestic Animals = Zuchthygiene, Vol.43, No.2, 157-161, 2008.
(Summary)
This study was conducted to examine the effects of the reproductive cycle of donor cat on the quality of oocytes at recovery and developmental competence of oocytes after in vitro fertilization (IVF) and somatic cell nuclear transfer (NT). Based on the presence or absence of follicles and corpora lutea, the ovarian pairs collected were classified into inactive, follicular or luteal stages. After collection of oocytes, the oocytes were classified into four grades according to the morphological condition of oocyte cytoplasm and cumulus cells. A total of 16 558 oocytes were obtained from 198 ovarian pairs. The total mean numbers of oocytes and the mean numbers of oocytes with high quality (grade I) were significantly higher in ovarian pairs at the inactive stage (111.1 and 19.0 oocytes, respectively) than in ovarian pairs at the follicular stage (67.1 and 11.4 oocytes, respectively). A significant difference in the proportions of oocytes with grade I out of the total examined oocytes was observed between the follicular and luteal stages of ovaries (14.9% vs 20.2%, p < 0.05). The proportions of IVF embryos cleaved and developed to blastocysts significantly decreased with decreased quality of oocytes at recovery, irrespective of the reproductive status of ovaries. Moreover, there were no significant differences in the proportions of cleavage and development to the blastocyst stage of IVF and NT embryos among three oestrous stages of ovaries. These results indicate that the reproductive cycle stage of donor cat ovaries has no apparent effects on the in vitro development of oocytes after IVF and NT, but the quality of oocytes at recovery influences the development of IVF embryos.
(Keyword)
Animals / Blastocyst / Cats / Culture Techniques / Female / Fertilization in Vitro / Male / Nuclear Transfer Techniques / Oocytes / Ovarian Follicle
F Barati, B Agung, P Wongsrikeao, M Taniguchi, T Nagai and Takeshige Otoi : Meiotic competence and DNA damage of porcine oocytes exposed to an elevated temperature., Theriogenology, Vol.69, No.6, 767-772, 2008.
(Summary)
The present study was conducted to investigate the effects of the length of exposure to an elevated temperature (41 degrees C) on the meiotic competence and DNA damage of porcine oocytes. Oocytes were recovered from ovaries, loaded into straws, and then exposed at 41.0 or 38.5 degrees C (sham control) for 0, 0.5, 1.0, or 1.5h, followed by culture for 44 h. The proportion of oocytes reaching the metaphase II (MII) stage gradually decreased with increasing exposure time, irrespective of the exposure temperature. A higher proportion of oocytes stored at 38.5 degrees C reached MII (57-63%) than those exposed to 41 degrees C (14-29%; P<0.01). The proportion of total oocytes with DNA fragmentation gradually increased with increasing exposure time, irrespective of the exposure temperature. The proportion of DNA fragmentation in total oocytes exposed to 41 degrees C (37-57%) was higher (P<0.01) than that in total oocytes stored at 38.5 degrees C (14-24%). When the oocytes were stored at 38.5 degrees C for up to 1.5 h, there were no differences in the proportions of MII-stage oocytes, with DNA-fragmented nuclei among all groups (P>0.05). However, a higher proportion of MII-stage oocytes exposed to 41 degrees C for more than 1h exhibited DNA-fragmented nuclei, compared with MII-stage oocytes stored at 38.5 degrees C (P<0.05). In conclusion, exposure of porcine oocytes to an elevated temperature had a detrimental effect on the meiotic competence and quality of oocytes; furthermore, the effect was dependent on the duration of exposure.
(Keyword)
Animals / DNA Damage / DNA Fragmentation / Female / Hot Temperature / Meiosis / Metaphase / Oocytes / Swine / Time Factors
Agung Budiyanto, Barati Farid, Kaedei Yukine, Fuminori Tanihara and Takeshige Otoi : Meiotic Competence and DNA Damage of Porcine Oocytes from Ovaries Exposed to an Elevated Temperature, Journal of Animal and Veterinary Advances, Vol.7, No.10, 1179-1183, 2008.
(Summary)
The present study was conducted to investigate the effects of the exposure length of ovaries to an elevated temperature (41°C) on the meiotic competence and DNA damage of oocytes. Ovaries were stored in physiological saline at 41°C for 0 h (control), 0.5, 1.0 and 1.5 h. After exposure of ovaries to the elevated temperature, oocytes were collected and then cultured for 44 h. The length of exposure of ovaries to 41°C had no effect on the proportions of total oocytes with DNA-fragmented nuclei before maturation culture, but it did influence the proportions at the end of maturation culture. The proportion of oocytes reaching metaphase II (MII) significandy decreased with increasing exposure time. In addition, significantly more oocytes from ovaries exposed to 41°C for 1.5 h had DNA-fragmented nuclei compared with control oocytes. These results indicate that the meiotic competence and DNA damage of porcine oocytes are dependent on the duration of exposure of ovaries to the elevated temperature. Moreover, the occurrence of DNA damage in oocytes becomes more apparent after maturation culture than before the culture.
M. Yavari, A. Fujii, R. Shimizu, A. Ito, Y. Kaedei, Y. Morita, Fuminori Tanihara and Takeshige Otoi : Meiotic competence of porcine oocytes after percoll sedimentation treatment for oocyte selection., Journal of Animal Veterinary advances, Vol.6, No.11, 1333-1336, 2007.
154.
M Takagi, D Yamamoto, S Ogawa, Takeshige Otoi, M Ohtani and A Miyamoto : Messenger RNA expression of angiotensin-converting enzyme, endothelin, cyclooxygenase-2 and prostaglandin synthases in bovine placentomes during gestation and the postpartum period., The Veterinary Journal, Vol.177, No.3, 398-404, 2007.
(Summary)
The bovine placenta contains local vasoactive-related systems, including angiotensin-converting enzyme (ACE), endothelin-1 (ET-1), ET-A receptor (ETAR) and ET-B receptor (ETBR), as well as arachidonic acid (AA) cascade-related enzymes, such as cyclooxygenase-2 (Cox-2), prostaglandin E-synthase (PGES) and prostaglandin F-synthase (PGFS). The mRNA expression of these molecules was examined in bovine placentomes (caruncles and cotyledons) collected immediately (0 h) and 6h after spontaneous parturition from 15 cows with early (fetal membranes released within 6 h of parturition) or late (fetal membranes released 6-12 h after parturition) detachment, as well as from 15 pregnant cows at a slaughterhouse. Significant differences were observed in expression of ET-1, ETAR and ETBR mRNAs between gestation and the postpartum period in both caruncles and cotyledons. Significant differences were also found between 0 and 6 h postpartum in the expression of ETBR mRNA in the early detachment group and PGES mRNA in the early and late detachment groups. Compared to PGFS, both Cox-2 and PGES exhibited opposite mRNA expression patterns during gestation and the postpartum period. The vasoactive-related peptide systems and AA cascade-related enzymes may mediate placental development and fetal membrane detachment after parturition in cattle.
Ken Maeda, Shigeru Yasumoto, Akari Tsuruda, Kiyohiko Andoh, Kazushige Kai, Takeshige Otoi and Tomio Matsumura : Establishment of a novel equine cell line for isolation and propagation of equine herpesviruses., The Journal of Veterinary Medical Science, Vol.69, No.9, 989-991, 2007.
(Summary)
In the present study, an equine-derived cell line was established by transfecting primary fetal horse kidney (FHK) cells with expression plasmid encoding simian virus 40 (SV40) large T antigen and then cloning them by limiting dilution. The cloned cell line, named FHK-Tcl3, grew well and could be propagated over 30 times by splitting them 1:3. Equine herpesvirus (EHV)-1 and EHV-4 replicated well in FHK-Tcl3. EHV-2 and EHV-4 were isolated from samples collected from horses in the field using FHK-Tcl3, and EHV-3 also propagated in FHK-Tcl3. These results indicated that this novel cell line, FHK-Tcl3, can be used for isolation and propagation of equine herpesviruses.
Shizuyo Sutou, Miho Kunishi, Toshiyuki Kudo, Pimprapar Wongsrikeao, Makoto Miyagishi and Takeshige Otoi : Knockdown of the bovine prion gene PRNP by RNA interference (RNAi) technology., BMC Biotechnology, Vol.7, 44, 2007.
(Summary)
Since prion gene-knockout mice do not contract prion diseases and animals in which production of prion protein (PrP) is reduced by half are resistant to the disease, we hypothesized that bovine animals with reduced PrP would be tolerant to BSE. Hence, attempts were made to produce bovine PRNP (bPRNP) that could be knocked down by RNA interference (RNAi) technology. Before an in vivo study, optimal conditions for knocking down bPRNP were determined in cultured mammalian cell systems. Factors examined included siRNA (short interfering RNA) expression plasmid vectors, target sites of PRNP, and lengths of siRNAs. Four siRNA expression plasmid vectors were used: three harboring different cloning sites were driven by the human U6 promoter (hU6), and one by the human tRNAVal promoter. Six target sites of bovine PRNP were designed using an algorithm. From 1 (22 mer) to 9 (19, 20, 21, 22, 23, 24, 25, 27, and 29 mer) siRNA expression vectors were constructed for each target site. As targets of siRNA, the entire bPRNP coding sequence was connected to the reporter gene of the fluorescent EGFP, or of firefly luciferase or Renilla luciferase. Target plasmid DNA was co-transfected with siRNA expression vector DNA into HeLaS3 cells, and fluorescence or luminescence was measured. The activities of siRNAs varied widely depending on the target sites, length of the siRNAs, and vectors used. Longer siRNAs were less effective, and 19 mer or 21 mer was generally optimal. Although 21 mer GGGGAGAACTTCACCGAAACT expressed by a hU6-driven plasmid with a Bsp MI cloning site was best under the present experimental conditions, the corresponding tRNA promoter-driven plasmid was almost equally useful. The effectiveness of this siRNA was confirmed by immunostaining and Western blotting. Four siRNA expression plasmid vectors, six target sites of bPRNP, and various lengths of siRNAs from 19 mer to 29 mer were examined to establish optimal conditions for knocking down of bPRNP in vitro. The most effective siRNA so far tested was 21 mer GGGGAGAACTTCACCGAAACT driven either by a hU6 or tRNA promoter, a finding that provides a basis for further studies in vivo.
Pimprapar Wongsrikeao, Takashi Nagai, Budiyanto Agung, Masayasu Taniguchi, Miho Kunishi, Shizuyo Suto and Takeshige Otoi : Improvement of transgenic cloning efficiencies by culturing recipient oocytes and donor cells with antioxidant vitamins in cattle., Molecular Reproduction and Development, Vol.74, No.6, 694-702, 2007.
(Summary)
The present study was conducted to investigate effects of antioxidants during maturation culture of recipient oocytes and/or culture of gene-transfected donor cells on the meiotic competence of recipient oocytes, and the developmental competence and quality of the reconstructed embryos after nuclear transfer (NT) in cattle. Gene-transfected donor cells had negative effects on the proportions of blastocyst formation, total cell numbers, and DNA fragmentation indices of reconstructed embryos. Supplementation of either vitamin E (alpha-tocopherol: 100 microM) or vitamin C (ascorbic acid: 100 microM) during maturation culture significantly enhanced the cytoplasmic maturation of oocytes and subsequent development of embryos reconstructed with the oocytes and gene-transfected donor cells, but did not have synergistic effects. The supplementation of vitamin E during maturation culture of recipient oocytes increased the proportions of fusion and blastocyst formation of gene-transfected NT embryos, in which the proportions were similar to those of nontransfected NT embryos. When the gene-transfected donor cells that had been cultured with 0, 50, or 100 microM of vitamin E were transferred into recipient oocytes matured with vitamin E (100 microM), 50 microM of vitamin E increased the proportion of blastocyst formation and reduced the index of DNA fragmentation of blastocysts. In conclusion, gene-transfected donor cells have negatively influenced the NT outcome. Supplementation of vitamin E during both recipient oocyte maturation and donor cell culture enhanced the blastocyst formation and efficiently blocked DNA damage in transgenic NT embryos.
Masayasu Taniguchi, Akiko Ikeda, Eri Arikawa, Pimprapar Wongsrikeao, Budiyanto Agung, Hideaki Naoi, Takashi Nagai and Takeshige Otoi : Effect of cryoprotectant composition on in vitro viability of in vitro fertilized and cloned bovine embryos following vitrification and in-straw dilution., The Journal of Reproduction and Development, Vol.53, No.4, 963-969, 2007.
(Summary)
In this study the efficacy of the combination of glycerol (GLY) and ethylene glycol (EG) as cryoprotectants in a vitrification method developed for direct embryo transfer was evaluated by in vitro development of in vitro fertilized (IVF) and somatic cell nuclear transfer (SCNT) embryos after vitrification. The IVF and SCNT blastocysts were vitrified in either 40% GLY, 30% GLY + 10% EG, or 20% GLY + 20% EG using French straws. After warming, the straws were held vertically for 1 min without shaking and were then placed horizontally for 5 min to dilute the cryoprotectants. After washing, the vitrified-warmed embryos were cultured in vitro for 72 h. There were no differences among the vitrification solutions with respect to the rates of vitrified-warmed IVF and SCNT embryos surviving and developing to the hatched blastocyst stage. However, the rates of development to the hatched blastocyst stage of the SCNT embryos vitrified with 40% GLY tended to be higher than those vitrified with 30% GLY + 10% EG or 20% GLY + 20% EG (26% vs. 7-8%, respectively). The development rates to the hatched blastocyst stage of the IVF and SCNT embryos vitrified with solution containing EG were significantly lower (P<0.05) than those of non-vitrified embryos. These results suggest that use of the combination of GLY and EG as cryoprotectants had no beneficial effect on the viability of embryos after in-straw dilution. However, this method is so simple that it can be used for practical direct transfer of vitrified embryos in the field.
Takeshige Otoi, T Shin, D C. Kraemer and M E. Westhusin : Role of cumulus cells on in vitro maturation of canine oocytes., Reproduction in Domestic Animals = Zuchthygiene, Vol.42, No.2, 184-189, 2007.
(Summary)
The objectives of the present study were to investigate the relationship between the morphological status of cumulus cells surrounding canine oocytes after maturation culture and the meiotic stage of the oocytes. In addition, the effect of the removal of cumulus cells from canine cumulus-oocyte complexes (COCs) during maturation culture on their meiotic competence was examined. Canine COCs were collected from bitches at the anoestrous and dioestrous stages and only COCs with >110 microm in vitelline diameter were cultured in medium 199 with 10% canine serum for 72 h. In the first experiment, the relation between the morphological status of cumulus cells surrounding oocytes cultured for 72 h and their meiotic stages was examined. At the end of maturation culture, the proportions of intact, partially nude and completely nude oocytes were 65.2%, 22.9% and 11.9%, respectively. The proportion of maturation to metaphase II of completely nude oocytes was highest among the oocytes with different morphological status of cumulus cells. In the second experiment, the cumulus cells were partially or completely removed from COCs at 48 h after the start of maturation culture and the oocytes were cultured for a further 24 h. The proportion of oocytes reaching metaphase II in the completely denuded oocytes was significantly higher than that in the control oocytes without the removal treatment of cumulus cells. The results indicate that morphological status of cumulus cells surrounding oocytes may be related to the nuclear maturation of canine oocytes, and the removal of cumulus cells from COCs during maturation culture can promote the completion of oocyte meiotic maturation.
Hideaki Naoi, Takeshige Otoi, Takano Shimamura, Ni Wayan Kurniani Karja, Budiyanto Agung, Ryohei Shimizu, Masayasu Taniguchi and Takashi Nagai : Developmental competence of cat oocytes from ovaries stored at various temperature for 24 h., The Journal of Reproduction and Development, Vol.53, No.2, 271-277, 2006.
(Summary)
We conducted two experiments to investigate the effects of storage temperature and period for cat ovaries on the meiotic and developmental competence of oocytes collected from the ovaries. In Experiment 1, ovaries were stored in physiological saline for 24 h at 4 C, 23-25 C, or 38 C (cold, room, and incubator temperature groups, respectively), and then oocytes were collected from the ovaries in each group. Morphologically intact oocytes were then selected and cultured in maturation medium for 24 h. Significantly more oocytes reached metaphase II (MII) in the cold temperature group (53.4%) than in the room and incubator temperature groups (20.0 and 2.4%, respectively). In Experiment 2, ovaries were stored in physiological saline at room temperature for 0, 6, 12 or 18 h, and then they were stored at 4 C (cold storage) until reaching a total storage period of 24 h. After storage of the ovaries, morphologically intact oocytes were matured, fertilized with frozen-thawed spermatozoa, and cultured in vitro. The rates of morphologically intact oocytes obtained from the ovaries stored at room temperature for 0, 6, 12 or 18 h were 35.3, 30.0, 26.4 and 14.7%, respectively, and the rates of intact oocytes that reached MII were 63.2, 36.4, 26.5 and 11.9%, respectively. The results suggested that the numbers of morphologically intact oocytes and intact oocytes that reached MII after in vitro maturation decrease gradually as the period of storage at room temperature before cold storage increases. Only oocytes from ovaries stored for 6 h developed to the blastocyst stage after in vitro maturation and fertilization when ovaries were stored at room temperature before cold storage. These results indicate that 24 h storage of ovaries at high temperatures (>23 C) decreases the meiotic competence of oocytes. The quality and developmental competence of oocytes are influenced by the length of storage at room temperature before cold storage.
(Keyword)
Animals / Cats / Cleavage Stage, Ovum / Female / Fertilization in Vitro / Male / Meiosis / Oocytes / Organ Preservation / Ovary / Temperature / Time Factors
M Taniguchi, A Ikeda, E Arikawa, R Shimizu, M Seki, M Karaki, R Rajamahendran and Takeshige Otoi : Ovarian follicular and corpus luteum changes, progesterone concentrations, estrus and ovulation following estradiol benzoate/progesterone based treatment protocol in cross-bred cows., Animal Reproduction Science, Vol.99, No.3-4, 389-394, 2006.
(Summary)
The objectives of the present study were to investigate the effects of the stage of the estrous cycle at the start of an estradiol benzoate (EB) and progesterone (P) based treatment protocol on new follicular wave emergence, subsequent estrus and ovulation. The experiment was conducted using a crossover design with each cow (five cross-bred cows) being assigned to one of three groups at 3-month intervals within a 1-year period. Estrous cycle stage in individual cows was initially synchronized with prostaglandin F(2)alpha. After detection of estrus, each cow was injected intramuscularly (i.m.) with 2 mg EB and 200 mg P (EB/P) on day 5, 12 or 17 of the estrous cycle (estrus=day 0), followed by 1 mg EB i.m. 12 days after the EB/P treatment. Ovarian ultrasonographic examinations showed that the emergence of a new follicular wave occurred after EB/P treatment in all groups and the mean interval from EB/P treatment to wave emergence did not differ among the groups (3.2-3.8 days). All cows in each group exhibited behavioral estrus and ovulated the newly formed dominant follicle. However, cows in the day-17 group exhibited estrus 1-3 days before the second EB injection. The concentrations of progesterone showed faster reduction, during the treatment period, in the day-12 and -17 groups compared to the day-5 group. These results indicate that the EB/P treatment induces an emergence of a new follicular wave, irrespective of the estrous cycle stage at the start of treatment, but the effect of EB/P protocol on estrous/ovulation synchronization is influenced by the stage of the estrous cycle.
Ni Wayan Kurniani Karja, Takeshige Otoi, Pimprapar Wongsrikeao, Ryohei Shimizu, Masako Murakami, Budiyanto Agung, Mokhamad Fahrudin and Takashi Nagai : Effects of electric field strengths on fusion and in vitro development of domestic cat embryos derived by somatic cell nuclear transfer., Theriogenology, Vol.66, No.5, 1237-1242, 2006.
(Summary)
The present study was conducted to determine the effect of electric field strength on the rate of membrane fusion between the somatic cell and cytoplast and on subsequent in vitro development of reconstructed embryos. Additionally, the in vitro developmental competence of cat oocytes artificially activated after 44 h of maturation culture was examined. An efficient fusion rate (64.2%) was obtained by applying a single pulse of 1.5 kV/cm for 50 micros, and the fusion rate remained almost constant at the higher field intensity (59.8 and 54.9% at 1.7 and 2.0 kV/cm, respectively). Although the cleavage rate of fused embryos increased with an increase of the electric field strength, there were no differences among the groups with respect to the proportion of development to the morula and blastocyst stages. In the additional experiment, oocytes at the metaphase II stage after culture for 44 h were activated by the combination of calcium ionophore (CaI) with cycloheximide (CHX). Some (11.8%) of activated oocytes developed to the blastocyst stage. Results from this study indicated that electric field strength affects the rates of fusion and cleavage but has no significant effects on the development to the blastocyst stage of reconstructed embryos. Prolonged maturation culture of cat oocytes (up to 44 h) decreased their ability to develop to the blastocyst stage.
Takeshige Otoi, R Shimizu, H Naoi, P Wongsrikeao, B Agung and M Taniguchi : Meiotic competence of canine oocytes embedded in collagen gel., Reproduction in Domestic Animals = Zuchthygiene, Vol.41, No.1, 17-21, 2006.
(Summary)
The present study was conducted to examine the meiotic competence of canine oocytes embedded in collagen gel, and to investigate the effects of timed exposure of the oocytes embedded in collagen gel to gonadotrophins during maturation culture, on their nuclear maturation. Cumulus-oocyte complexes (COCs) were collected from bitches at the anoestrous and dioestrous stages of the reproductive cycle. In the first experiment, half of the COCs were embedded in collagen gels. The COCs with or without collagen-gel embedding were cultured in a TCM-199 medium supplemented with 0.1 IU/ml human menopausal gonadotropin (hMG) and 10 IU/ml human chorionic gonadotropin (hCG) for 72 h. In the second experiment, the COCs embedded in collagen gels were cultured in TCM-199 medium with gonadotrophins (hMG and hCG) for various periods (0, 24, 48 and 72 h) and then cultured in the medium without gonadotrophins until reaching total culture period (72 h). The percentage of the oocytes reaching metaphase I and metaphase II (MI/MII) was significantly higher (p < 0.05) in COCs with collagen-gel embedding than in COCs without collagen-gel embedding. The percentage of oocytes that were arrested at the germinal vesicle stage was significantly lower (p < 0.05) in oocytes cultured with gonadotrophins than in oocytes cultured without gonadotrophins. However, there were no significant differences in the percentages of oocytes that reached each stage of meiosis among the groups, irrespective of the duration of exposure to gonadotrophins. These observations indicate that embedding of COCs by collagen gel enhances the meiotic competence of canine oocytes, but removal of hormone supplement from maturation medium does not improve the ability of the oocytes to reach MII stage.
Pimprapar Wongsrikeao, Takeshige Otoi, Hirofumi Yamasaki, Budiyanto Agung, Masayasu Taniguchi, Hideaki Naoi, Ryohei Shimizu and Takashi Nagai : Effects of single and double exposure to brilliant cresyl blue on the selection of porcine oocytes for in vitro production of embryos., Theriogenology, Vol.66, No.2, 366-372, 2006.
(Summary)
The aim of this study was to evaluate the effectiveness and toxicity of single and double application of the brilliant cresyl blue (BCB) test on the selection of porcine oocytes as an indirect measure of oocyte growth for in vitro fertilization (IVF) and nuclear transfer. In the first experiment, oocytes were exposed to BCB before and after maturation culture and classified according to their cytoplasmic coloration: blue coloration and colorless. The classified oocytes were fertilized with spermatozoa and then cultured for 7 days. The percentages of maturation to metaphase II in blue oocytes at the start of maturation culture were higher than those of colorless oocytes (68.7-70.1% versus 0.8-3.6%, P < 0.05). However, double application of BCB test before and after maturation culture had a toxic effect on fertilization and embryonic development. No significant differences in the blastocyst formation were found between blue oocytes without double application of BCB test and control oocytes without any application of BCB test, whereas the total cell number per blastocyst from the blue oocytes was higher than that from the control oocytes (48.0 versus 34.2, P < 0.05). In the second experiment, oocytes were exposed to the BCB test before or after maturation culture, and then the matured oocytes were activated to evaluate the ability of parthenogenetic development. The proportion of blastocyst formation of blue oocytes classified after maturation culture was lower than that of blue oocytes classified before maturation culture (10.0% versus 27.0%, P < 0.05). Therefore, double application of the BCB test before and after maturation culture impaired fertilization and embryonic development, whereas a single application before maturation culture was efficient to select oocytes for IVF and nuclear transfer.
(Keyword)
Animals / Blastocyst / Coloring Agents / Dose-Response Relationship, Drug / Female / Fertilization in Vitro / Oocytes / Oxazines / Sexual Maturation / Swine / Tissue and Organ Harvesting
M Murakami, N W. K. Karja, P Wongsrikeao, B Agung, M Taniguchi, H Naoi and Takeshige Otoi : Development of cat embryos produced by intracytoplasmic injection of spermatozoa stored in alcohol., Reproduction in Domestic Animals = Zuchthygiene, Vol.40, No.6, 511-515, 2005.
(Summary)
This study was conducted to examine whether cat spermatozoa stored in ethanol for 1 month was capable of developing into pronuclei and of supporting normal embryonic development. In vitro matured oocytes were injected with frozen-thawed spermatozoa and ethanol-stored spermatozoa. The status of oocytes and sperm nuclei was examined at 4 and 18 h after injection of spermatozoa, and the presumptive zygotes were cultured for 7 days to assess the development of oocytes injected with each storage sperm. The percentage of enlarged sperm head at 4 h after injection was higher in ethanol-stored spermatozoa than in frozen-thawed spermatozoa, but there was no significant difference in the development of oocytes and sperm nuclei at 18 h after injection between the two groups. The development of oocytes to the blastocyst stage after injection of spermatozoa was observed only in oocytes with frozen-thawed spermatozoa. Of oocytes injected with ethanol-stored spermatozoa, two (2.8%) oocytes developed to the 16-cell stage. These results indicate that cat spermatozoa stored in ethanol can decondense and form male pronuclei after intracytoplasmic injection. However, the oocytes injected with ethanol-stored spermatozoa did not have the ability to develop to the blastocyst stage.
Budiyanto Agung, Takeshige Otoi, Pimprapar Wongsrikeao, Masayasu Taniguchi, Ryohei Shimizu, Hajime Watari and Takashi Nagai : Effect of maturation culture period of oocytes on the sex ratio of in vitro fertilized bovine embryos., The Journal of Reproduction and Development, Vol.52, No.1, 123-127, 2005.
(Summary)
It has been suggested that the maturational stage of oocytes at time of insemination influences the sex ratio of resulting embryos. However, there are very few reports concerning the relationship between the maturation culture period of oocytes and the sex ratio of resulting embryos. The objective of this study was to investigate the effects of in vitro maturation culture period for bovine oocytes on the sex ratio of in vitro produced blastocysts using a novel technique of loop-mediated isothermal amplification (LAMP). Cumulus-oocyte complexes were collected from the ovaries of slaughtered cows, and then matured in vitro for various periods (16, 22, 28, and 34 h). After maturation culture for each period, the oocytes were inseminated with frozen-thawed spermatozoa, and then cultured in vitro. Blastocysts were harvested on Day 7 after insemination, and the sex of the embryos was examined using the LAMP method. The rates of oocytes matured to the metaphase II stage were significantly lower (P < 0.05) in the 16-h maturation group than in the other groups. The proportion of blastocyst formation after insemination was significantly higher (P < 0.05) in the 22-h maturation group than in the other groups. The proportion of male blastocysts increased with the increase in maturation culture period. The proportion of male blastocysts derived from oocytes matured for 34 h was significantly higher (P < 0.05) than from oocytes matured for 16 and 22 h. These results indicate that the sex ratio of in vitro fertilized embryos is apparently influenced by the maturation culture period of the oocytes.
(Keyword)
Animals / Cattle / Embryo Culture Techniques / Embryo, Mammalian / Fertilization in Vitro / Oocytes / Sex Determination Analysis / Sex Ratio / Time Factors
Ni Wayan Kurniani Karja, Takeshige Otoi, Masako Murakami, Pimprapar Wongsrikeao, Agung Budiyanto, Mokhamad Fahrudin and Takashi Nagai : Effect of cycloheximide on in vitro development of electrically activated feline oocytes., The Journal of Reproduction and Development, Vol.51, No.6, 783-786, 2005.
(Summary)
This study was conducted to improve parthenogenetic development in vitro of feline oocytes following a combined activation treatment of electrical stimulation and cycloheximide. In vitro matured (IVM) oocytes were stimulated electrically by a DC electrical pulse of 2 kV/cm for 50 micros. The stimulated oocytes were then incubated in MK-1 medium with or without cycloheximide and subsequently cultured in vitro for 6 days. No significant differences were observed between the two groups with respect to the proportions of cleavage, development to the morula stage, and the cell number of blastocysts. However, exposure of electrically stimulated oocytes to cycloheximide significantly increased the rate of development of the stimulated oocytes into the blastocyst stage compared with oocytes stimulated by electrical stimulation alone (31.0% vs 6.7%). The results from the present study suggested that a single electrical stimulation was insufficient to activate the IVM cat oocytes at 24 h of maturation and that exposure to cycloheximide following electrical stimulation improved the efficacy of the parthenogenetic development of domestic cat oocytes.
(Keyword)
Animals / Blastocyst / Cats / Cells, Cultured / Cycloheximide / Electric Stimulation / Female / In Vitro Techniques / Nuclear Transfer Techniques / Oocytes / Parthenogenesis / Protein Synthesis Inhibitors
F Rajaei, N W. K. Karja, B Agung, P Wongsrikeao, M Taniguchi, M Murakami, R Sambuu, M Nii and Takeshige Otoi : Analysis of DNA fragmentation of porcine embryos exposed to cryoprotectants., Reproduction in Domestic Animals = Zuchthygiene, Vol.40, No.5, 429-432, 2005.
(Summary)
The chemical toxicity of cryoprotectants to porcine embryos was examined by the evaluation of survival and DNA damage after exposure to cryoprotectants. Porcine blastocysts were exposed to 10% of ethylene glycol (EG), 1,2-propanediol (PD) or glycerol (GLY) for 1 h at room temperature (23-25 degrees C) and then cultured in vitro for 24 h. The survival rates of blastocysts exposed to PD and GLY were significantly lower than those of control blastocysts in which the embryos were exposed to carrier solution without cryoprotectants. Significantly more DNA-fragmented nuclei occurred in the cryoprotectant-exposed blastocysts, compared with the control blastocysts. Moreover, the indices of DNA-fragmented nuclei in the blastocysts without blastocoele re-formation after culture were significantly higher than those with blastocoele re-formation, irrespective of the exposure treatment. These results indicate that the exposure of porcine blastocysts to cryoprotectant decreases the survival rates and increases the DNA-fragmented nuclei in embryos.
(Keyword)
Animals / Blastocyst / Cryopreservation / Cryoprotective Agents / DNA Damage / DNA Fragmentation / Embryo, Mammalian / Ethylene Glycol / Glycerol / In Vitro Techniques / Propylene Glycol / Swine / Temperature / Time Factors
Masaomi Mori, Takeshige Otoi, Pimprapar Wongsrikeao, Budiyanto Agung and Takashi Nagai : Effects of beta-mercaptoethanol and cycloheximide on survival and DNA damage of bovine embryos stored at 4 degrees C for 72 h., Theriogenology, Vol.65, No.7, 1322-1332, 2005.
(Summary)
The objectives of this study were to determine the effects of cycloheximide (CHX) and beta-mercaptoethanol (beta-ME) during storage of in vitro-produced (IVP) bovine blastocysts for 72 h at 4 degrees C on their survival, hatching capacity and DNA damage. In Experiment 1, when blastocysts were stored in a medium supplemented with 25, 50 or 100 microg/mL of CHX, or 25, 50 or 100 microM of beta-ME, the blastocysts stored with 25 microg/mL of CHX had a significantly higher survival rate than that of the blastocysts stored without CHX (79.5% versus 54.2%). In contrast, beta-ME had no apparent effects on the survival and hatching capacity of stored embryos. In Experiment 2, to investigate synergistic effects of CHX and beta-ME during storage of blastocysts on their developmental parameters and DNA damage, they were stored in the medium with CHX (25 microg/mL) and beta-ME (50 microM). The combination of CHX and beta-ME had no significant effects on the survival of blastocysts. The proportion (6.8%) of DNA-fragmented cells in the blastocysts stored with CHX was similar to that (5.4%) in the non-stored blastocysts (positive control) and significantly lower than that (9.7%) in the blastocysts stored without CHX and beta-ME (negative control). However, there were no significant differences among the proportions of dead cells of blastocysts in the storage groups. Therefore, the supplementation of CHX in the storage medium had a beneficial effect on the proportions of survival and DNA-fragmented cells in the stored embryos, whereas the beta-ME alone or in combination with CHX had no positive effects on either of these proportions.
(Keyword)
Animals / Blastocyst / Cattle / Cycloheximide / DNA Damage / DNA Fragmentation / Drug Synergism / Embryo, Mammalian / Female / In Situ Nick-End Labeling / Mercaptoethanol / Temperature / Tissue Preservation
Ni Wayan Kurniani Karja, Takeshige Otoi, Pimprapar Wongsrikeao, Masako Murakami, Budiyanto Agung, Mokhamad Fahrudin and Takashi Nagai : In vitro development and post-thaw survival of blastocysts derived from delipidated zygotes from domestic cats., Theriogenology, Vol.65, No.2, 415-423, 2005.
(Summary)
The ability to cryopreserve in vitro-produced feline embryos was investigated. To improve the survival rate of cryopreserved embryos, first the developmental ability of in vitro fertilized feline zygotes (after removal of intracellular lipids) was determined, followed by the post-thaw survival of cryopreserved blastocysts derived from delipidated zygotes. More than 67% of the delipidated zygotes cleaved and 36% of them developed to the morula stage. The developmental ability of delipidated zygotes to the blastocyst stage (26%) was similar to that of sham-operated (30.5%) or control embryos (31.3%). Although the survival rate of delipidated blastocysts (81.8%) after freezing and thawing tended to be higher than that of control embryos without delipidation (60.6%), rates were not significantly different between the both groups. In conclusion, in vitro-produced feline blastocysts were successfully frozen, removal of the cytoplasmic lipid content in feline zygotes did not impair their in vitro developmental competence (up to the blastocyst stage), and reduction of cytoplasmic lipids by aspiration had no apparent effects on the survival of in vitro-derived blastocysts after cryopreservation.
Pimprapar Wongsrikeao, Takeshige Otoi, Masayasu Taniguchi, Ni Wayan Kurniani Karja, Budiyanto Agung, Masaru Nii and Takashi Nagai : Effects of hexoses on in vitro oocyte maturation and embryo development in pigs., Theriogenology, Vol.65, No.2, 332-343, 2005.
(Summary)
The objective was to determine the effects of supplementing hexoses in oocyte maturation and embryo culture medium on in vitro maturation (IVM) and in vitro fertilization (IVF) of porcine oocytes and in vitro development of in vitro produced (IVP) porcine embryos. In the first experiment, oocytes were matured in vitro in modified North Carolina State University (NCSU)-37 medium, supplemented with hexoses (glucose, fructose or galactose) at various concentrations: 0 (control), 2.5, 5.5 and 10 mM. Supplementing the maturation medium with either glucose or fructose (5.5 mM) increased the percentages of oocytes that matured to metaphase II (79.4 and 70.2%, respectively), as compared with the control group (P < 0.05). However, supplementing galactose had no effects on meiotic maturation and fertilization. In the second experiment, cleaved embryos were collected 3 days after IVF of oocytes matured in the maturation medium supplemented with 5.5 mM of glucose; they were cultured for an additional 4 days in modified NCSU-37 medium, supplemented with 5.5mM of glucose, fructose or galactose. The incidence of blastocyst formation was higher (P < 0.05) in the glucose and fructose groups (18.6 and 18.2%, respectively) than in the galactose group and non-supplemented control group (12.9 and 9.2%). Moreover, fructose supplementation increased the total cell number/blastocyst (48.0 versus 37.6) and reduced the index of DNA-fragmented nucleus in the blastocysts (7.6% versus 11.8%), as compared with glucose supplementation (P < 0.05). In conclusion, fructose was a practical alternative to glucose for supporting IVM of porcine oocytes and fructose was superior to glucose for producing high-quality porcine embryos in vitro.
(Keyword)
Animals / Culture Media / Culture Techniques / Embryonic Development / Female / Fertilization in Vitro / Fructose / Galactose / Glucose / Hexoses / Male / Oocytes / Swine
P Wongsrikeao, Y Kaneshige, R Ooki, M Taniguchi, B Agung, M Nii and Takeshige Otoi : Effect of the removal of cumulus cells on the nuclear maturation, fertilization and development of porcine oocytes., Reproduction in Domestic Animals = Zuchthygiene, Vol.40, No.2, 166-170, 2005.
(Summary)
The present study was conducted to investigate the effects of attachment of cumulus cells to porcine oocytes during the process of maturation and fertilization on the nuclear maturation, fertilization and subsequent development after in vitro fertilization (IVF). In the first experiment, the cumulus cells were removed from cumulus-oocyte complexes (COCs) at 0, 24 and 42 h after the onset of maturation culture and were then cultured until reaching 42 h of cultivation. In the second experiment, COCs were denuded as described in the first experiment, then fertilized and cultured for 7 days. As a control, cumulus cells were allowed to maintain attachment to the oocytes until the end of IVF. The proportion of oocytes reaching metaphase II significantly increased with the delay in the removal treatment of cumulus cells. The proportion of normal fertilization gradually increased with delay in the removal treatment of cumulus cells from COCs until the end of IVF. However, no significant difference in the proportion of normal fertilization was found between the 42-h and control groups. The removal treatment of cumulus cells in the 0- and 24-h group significantly (p < 0.05) decreased the proportion of cleaved embryos when compared with the control, and none of them developed to the blastocyst stage. The proportion of development to the blastocyst stage was significantly higher (p < 0.05) in the control group than in the 42-h group (18.1% vs 12.4%; p < 0.05). The present study indicates that the attachment of cumulus cells to the oocyte during maturation and fertilization is important to support oocyte nuclear maturation, fertilization and subsequent embryo development. Particularly, the attachment of cumulus cells to the oocyte during IVF promotes embryonic development.
B Agung, Takeshige Otoi, H Abe, H Hoshi, M Murakami, N W. K. Karja, M K. Murakami, P Wongsrikeao, H Watari and T Suzuki : Relationship between oxygen consumption and sex of bovine in vitro fertilized embryos., Reproduction in Domestic Animals = Zuchthygiene, Vol.40, No.1, 51-56, 2005.
(Summary)
The present study was conducted to examine the relationship between the oxygen consumption rate and sex ratio of bovine in vitro fertilized embryos on each day of blastocyst formation. The quality of blastocysts collected on day 7, 8, and 9 after in vitro fertilization (IVF) were categorized as ranks A and B (excellent and good, respectively) based on microscopic observation of the morphology. The oxygen consumption rate and sex of individual blastocysts were evaluated using two novel techniques: scanning electrochemical microscopy (SECM) and loop-mediated isothermal amplification (LAMP), respectively. The oxygen consumption rates of embryos of rank A were significantly higher (p < 0.05) than those of rank B, irrespective of the day of blastocyst appearance after IVF. Neither did the proportion of male embryos of ranks A and B differ significantly from each other at any of the days examined, nor from the average proportion (53%). The oxygen consumption rate of embryos of rank B collected on day 8 was significantly higher (p < 0.05) in female embryos than in male embryos collected on the same day. However, there were no apparent differences of oxygen consumption rates at each day of blastocyst appearance between male and female embryos of rank A. These results indicate that the oxygen consumption rate of individual embryos reflects their quality but does not correlate with the sex ratio of embryos of excellent quality.
(Keyword)
Animals / Cattle / Culture Techniques / Embryo, Mammalian / Female / Fertilization in Vitro / Male / Microscopy, Electron, Scanning / Oxygen Consumption / Pregnancy / Sex Ratio
Pimprapar Wongsrikeao, Takeshige Otoi, Ni Wayan Kurniani Karja, Budiyanto Agung, Masahiro Nii and Takashi Nagai : Effects of ovary storage time and temperature on DNA fragmentation and development of porcine oocytes., The Journal of Reproduction and Development, Vol.51, No.1, 87-97, 2005.
(Summary)
The present study was conducted to investigate the effects of storage time and temperature of porcine ovaries on the quality and nuclear maturation in vitro of oocytes obtained from stored ovaries and their subsequent development after in vitro fertilization. The ovaries were stored in physiological saline for 0, 3, 6, 9 and 12 h at various temperatures (4, 15, 25 and 35 C). The pH of follicular fluid obtained from the ovaries, DNA fragmentation of the oocyte nucleus and meiotic competence of oocytes were examined. Some oocytes from ovaries stored at 15, 25 and 35 C for 6 h were fertilized in vitro, and then cultured for 7 days to examine the ability of embryos to develop to the blastocyst stage. When the ovaries were stored at 35 C, the pH of follicular fluid decreased and the proportions of oocytes with DNA fragmented nuclei increased as the storage time was prolonged, and the storage of ovaries for 6, 9 and 12 h resulted in lower maturation rates of oocytes. When the ovaries were stored at 4, 15, 25 and 35 C for 6 h, the storage at higher temperatures (> or =15 C) decreased the pH of follicular fluid and induced nucleic DNA fragmentation in higher proportions of oocytes. None of the oocytes from ovaries stored at 4 C reached metaphase II. The storage of ovaries at 15 C reduced the rates of in vitro fertilized oocytes and subsequent embryo development, but there were no significant differences in the rates of fertilization and blastocyst formation between oocytes from ovaries stored at 25 C and 35 C. Our findings indicate that the storage of ovaries at 25-35 C for 6 h is effective for maintaining the developmental competence of porcine oocytes even though the development rates were lower than those of ovaries stored at 35 C for 3 h.
(Keyword)
Animals / Blastocyst / Cell Nucleus / Culture Techniques / DNA Damage / DNA Fragmentation / Embryonic Development / Female / Fertilization in Vitro / Hydrogen-Ion Concentration / In Situ Nick-End Labeling / Male / Oocytes / Ovarian Follicle / Ovary / Specimen Handling / Spermatozoa / Swine / Temperature / Time Factors
Masao Murakami, Takeshige Otoi, Pimprapar Wongsrikeao, Budiyanto Agung, Rentsenkhand Sambuu and Tatsuyuki Suzuki : Development of interspecies cloned embryos in yak and dog., Cloning and Stem Cells, Vol.7, No.2, 77-81, 2005.
(Summary)
Interspecies nuclear transfer (NT) could be an alternative to replicate animals when supply of recipient oocytes is limited or in vitro embryo production systems are incomplete. In the present study, embryonic development was assessed following interspecies NT of donor cumulus cells derived from yak and dog into the recipient ooplasm of domestic cow. The percentages of fusion and subsequent embryo development to the eight-cell stage of interspecies NT embryos were comparable to those of intraspecies NT embryos (cow-cow NT embryos). The percentage of development to blastocysts was significantly lower (p < 0.05) in yak-cow NT embryos than that in cow-cow NT embryos (10.9% vs. 39.8%). In dog-cow NT embryos, only one embryo (0.4%) developed to the blastocyst stage. These results indicate that interspecies NT embryos possess equally developmental competence to the eight-cell stage as intraspecies NT embryos, but the development to blastocysts is very low when dog somatic cells are used as the donor nuclei.
Ni Wayan Kurniani Karja, Pimprapar Wongsrikeao, Masako Murakami, Budiyanto Agung, Mokhamad Fahrudin, Takashi Nagai and Takeshige Otoi : Effects of oxygen tension on the development and quality of porcine in vitro fertilized embryos., Theriogenology, Vol.62, No.9, 1585-1595, 2004.
(Summary)
The present study was conducted to examine the effect of oxygen tension during in vitro culture (IVC) of porcine oocytes/embryos on their development and quality using two different culture systems. Porcine cumulus oocyte complexes (COCs) were matured (IVM) and fertilized (IVF) in vitro, and subsequently cultured for 6 days in a simple and economical portable incubator or a standard CO(2) incubator. While the same temperature (38.5 degrees C) and CO(2) concentration (5%) were used in the both systems, the portable incubator was operated in a negative air pressure (- 300 mmHg) to create an O(2) level at 8-10% (low O(2) concentration), or in a positive air pressure (high O(2) concentration). To compare the two culture systems, IVM and IVF of COCs and subsequent IVC of in vitro produced (IVP) embryos were carried out in the portable incubator with a low O(2) concentration (Group I) or in the standard incubator with a high O(2) concentration (Group II). To assess the effect of O(2) concentration on IVC of IVP embryos, some oocytes that had been cultured in the standard incubator for IVM and IVF were subsequently cultured in the portable incubator with a low O(2) concentration (Group III) or a high O(2) concentration (Group IV). The occurrence of DNA fragmentation in the blastocysts produced under different culture conditions was examined by TUNEL staining to assess embryo quality. The rates of oocytes that reached MII and were penetrated by spermatozoa following IVF did not differ between the two incubation systems. In contrast, the proportions of development to blastocysts and the mean cell number of blastocysts in Group I were higher than those in Group II and Group IV. The index of DNA-fragmented nucleus in the blastocysts of Group I was significantly lower than that in the blastocysts of Group II. Therefore, low oxygen tension during IVM, IVF and IVC enhanced the subsequent development of IVP embryos to the blastocyst stage and improved their quality.
(Keyword)
Animals / Blastocyst / DNA Fragmentation / Embryo, Mammalian / Embryonic Development / Female / Fertilization in Vitro / In Situ Nick-End Labeling / Oxygen / Swine / Tissue Culture Techniques
Ni Wayan Kurniani Karja, Sergey Medvedev, Akira Onishi, Dai-Ichiro Fuchimoto, Masaki Iwamoto, Takeshige Otoi and Takashi Nagai : Effect of replacement of pyruvate/lactate in culture medium with glucose on preimplantation development of porcine embryos in vitro., The Journal of Reproduction and Development, Vol.50, No.5, 587-592, 2004.
(Summary)
The effect of glucose supplementation at different times in in vitro culture on the developmental competence of in vitro produced (IVP) porcine embryos was examined. In Experiment 1, when IVP embryos were cultured in modified NCSU-37 supplemented with pyruvate and lactate (IVC-pyr/lac) for 0 h, 24 h, 48 h, 72 h, 96 h, or 118 h and subsequently in modified NCSU-37 supplemented with glucose (IVC-glu) until Day 6 (Day 0=day of in vitro fertilization), the rates of blastocyst formation were significantly higher in embryos cultured in IVC-pyr/lac for 24 or 48 h (24.4% and 23.0%, respectively) than in embryos cultured in IVC-pyr/lac for the whole culture period (14.5%). However, there were no significant differences between embryos obtained after the energy source replacement and embryos cultured in IVC-glu for the whole culture period on the rates (15.2%-24.4%, and 16.8% respectively). Replacement of pyruvate/lactate with glucose at 58 h of culture in Experiment 2 significantly enhanced the rate (31.3%) compared to those after replacement at 48 h, 53 h and 63 h of culture (20.6%, 20.8%, and 21.1%, respectively). In conclusion, replacement of pyruvate/lactate with glucose as the energy substrate was optimal at 58 h of culture for the development of porcine embryos to the blastocyst stage.
(Keyword)
Animals / Blastocyst / Culture Media / Embryonic Development / Energy Metabolism / Female / Fertilization in Vitro / Glucose / Lactic Acid / Pyruvic Acid / Sus scrofa
Mk Murakami, Takeshige Otoi, N W. K. Karja, P Wongsrikeao, B Agung and T Suzuki : Blastocysts derived from in vitro-fertilized cat oocytes after vitrification and dilution with sucrose., Cryobiology, Vol.48, No.3, 341-348, 2004.
(Summary)
Experiments were conducted to find an optimal incubation period in a sucrose solution during dilution of cryoprotectants for obtaining a higher level of survival and development of cat oocytes cryopreserved by vitrification method. In the first experiment, in vitro-matured fresh oocytes were exposed to 0.5M sucrose solution for 1 or 5 min before in vitro fertilization (IVF). The percentage of development to the blastocyst stage significantly decreased in oocytes exposed for 5 min, compared with oocytes exposed for 1 min and control oocytes without exposure to sucrose (P<0.05). In the second experiment, oocytes that had been vitrified in 40% ethylene glycol and 0.3M sucrose were liquefied and then incubated in 0.5M sucrose for 0.5, 1 or 5 min to dilute the cryoprotectant. The percentage of cleavage (>or=2-cell stage) of vitrified-liquefied oocytes incubated for 0.5 min was significantly higher (P<0.05) than that of other groups. Development of vitrified-liquefied oocytes to the morula and blastocyst stages after IVF was observed only in oocytes incubated in sucrose for 0.5 min. The present study indicates that the oocytes have sensitivity to the toxic effect of sucrose and that the incubation period during dilution of the cryoprotectant is of critical importance for developmental competence of vitrified-liquefied cat oocytes.
A. Kurihara, J. Sekine, M. Hishinuma, Tatsuyuki Suzuki and Takeshige Otoi : Influence of recipient dams of different breeds on performance of Japanese Black calves produced by embryo transfer., Archives Animal Breeding, Vol.47, 431-441, 2004.
Y.J. Dong, X.J. Bai, M.D. Varisanga, N.R. Mtango, Takeshige Otoi, R. Rajamahendran and Suzuki Tatsuyuki : Production of cloned calves by the transfer of somatic cells derived from frozen tissues using simple portable CO2 incubator., Asian-Australasian Journal of Animal Sciences, Vol.17, 168-173, 2004.
181.
Pimprapar Wongsrikeao, Takeshige Otoi, Masako Murakami, Ni Wayan Kurniani Karja, Agung Budiyanto, Masao Murakami, Masaru Nii and Tatsuyuki Suzuki : Relationship between DNA fragmentation and nuclear status of in vitro-matured porcine oocytes: role of cumulus cells., Reproduction, Fertility, and Development, Vol.16, No.8, 773-780, 2004.
(Summary)
The present study was conducted to investigate the effects of the attachment of cumulus cells to oocytes and coculture with cumulus cells during maturation culture on the nuclear status and DNA fragmentation of porcine denuded oocytes (DOs). In the first experiment, cumulus cells were removed from cumulus-oocyte complexes (COCs) at 0, 8, 16, 24 or 32 h after the onset of maturation culture and the DOs were then cultured in their original droplets until 42 h of culture was reached. In the second experiment, all COCs were denuded before the onset of culture and the DOs were cocultured with their removed cumulus cells. The DOs were transferred into fresh medium at 0, 8, 16, 24 or 32 h after the onset of coculture with cumulus cells and then cultured until 42 h of culture was reached. After culture, DNA fragmentation and the nuclear status of oocytes were examined using the terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end-labelling (TUNEL) method. When the DOs were returned to the same droplets after removal of the cumulus cells, the removal of the cumulus cells after 16 h of culture significantly decreased the proportion of oocytes remaining at the germinal vesicle (GV) stage. However, coculture treatment of DOs in the presence of their removed cumulus cells had no significant effects on the GV breakdown (GVBD) of oocytes. There were no significant differences in the proportion maturing to MII oocytes among the groups following removal of cumulus cells after the onset of maturation culture; however, DOs cocultured with cumulus cells until the end of maturation culture exhibited an increased maturation rate compared with DOs cocultured for 8 and 16 h. The total proportion of TUNEL-positive oocytes of oocytes remaining at the GV stage was higher than that of oocytes reaching other stages, irrespective of the removal of cumulus cells and coculture treatments. However, coculture for more than 16 h decreased the total proportion of TUNEL-positive oocytes. Our results indicate that the attachment of cumulus cells to oocytes may have a critical role for oocytes undergoing GVBD and that coculture with cumulus cells promotes the ability of oocytes to complete maturation. Moreover, coculture with cumulus cells may assist the oocyte to avoid undergoing DNA fragmentation.
Takeshige Otoi, Taeyoung Shin, Duane C. Kraemer and Mark E. Westhusin : Influence of maturation culture period on the development of canine oocytes after in vitro maturation and fertilization., Reproduction, Nutrition, Development, Vol.44, No.6, 631-637, 2004.
(Summary)
The objective of this study was to determine an optimum maturation period of canine oocytes for the development in vitro after in vitro fertilization (IVF). Canine oocytes larger than 110 micrometers in diameter, which were collected from ovaries at the follicular phase of the reproductive cycle, were cultured for each time (48, 72 and 96 h) in TCM 199 medium supplemented with 10% canine serum, fertilized, and then cultured in vitro for 8 days. Significantly more oocytes reached metaphase II (MII) in the 72-h culture group than in the 48-h culture group (25.6% vs. 41.0%). The percentages of oocytes that reached MII or beyond after maturation culture did not differ significantly between the 72- and 96-h culture groups, but the percentage of parthenogenetically activated oocytes in the 96-h culture group was significantly higher than that in the 72-h culture group. The percentages of cleaved embryos after IVF were significantly higher in the 48- and 72-h culture groups than in the 96-h culture group. In the 48-h culture group, 3.9% of fertilized oocytes developed to the 16-cell stage or beyond, but none of the cleaved embryos in the 72- and 96-h culture groups developed to the same stage. These results indicate that full nuclear maturation of oocytes collected from ovaries at the follicular phase occurs after 72 h of in vitro culture. However, an optimum maturation period (48 h) for the in vitro development of canine oocytes after IVF may be different from the period necessary to reach the maximal oocyte maturation rate, when based on the developmental stage of the cleaved embryos.
(Keyword)
Animals / Cleavage Stage, Ovum / Culture Media / Dogs / Embryonic Development / Female / Fertilization in Vitro / Male / Oocytes / Time Factors / Tissue and Organ Harvesting
M Yuge, Takeshige Otoi, M Nii, Mk Murakami, N W. K. Karja, F Rajaei, B Agung, P Wongsrikeao, M Murakami and T Suzuki : Effects of cooling ovaries before oocyte aspiration on meiotic competence of porcine oocytes and of exposing in vitro matured oocytes to ambient temperature on in vitro fertilization and development of the oocytes., Cryobiology, Vol.47, No.2, 102-108, 2003.
(Summary)
Experiments were conducted to evaluate the effects of cooling porcine ovaries to low temperature (4 degrees C, 15 degrees C, 20 degrees C, 25 degrees C or 30 degrees C) for 1 h on the meiotic competence of their oocytes. Moreover, it was determined whether or not the exposure of in vitro matured oocytes to ambient temperature (20 degrees C, 25 degrees C or 30 degrees C) for 1 h affects the fertilization and developmental competence of the oocytes. There was no difference between the proportions of oocytes that underwent maturation to metaphase II when isolated from control ovaries held at 35 degrees C and ovaries exposed to 30 degrees C. However, the percentages of oocytes from ovaries exposed to 25 degrees C or less were significantly lower than those of oocytes from ovaries exposed to 30 degrees C and control ovaries. The proportions of total and normal fertilization of oocytes that had been exposed to 20 degrees C before in vitro fertilization (IVF) were significantly lower than those of control oocytes maintained at 38.5 degrees C. However, cooling in vitro matured oocytes had no effects on their cleavage and development to blastocysts after IVF. These data suggest that exposing porcine ovaries to a low temperature of 25 degrees C or less before aspiration of oocytes may adversely affect their subsequent in vitro maturation. It may be necessary to maintain the oocytes at a temperature of more than 25 degrees C during manipulation of oocytes for retaining the fertilizability of the oocytes.
(Keyword)
Animals / Cell Nucleus / Cryopreservation / Female / Fertilization / Fertilization in Vitro / Male / Meiosis / Metaphase / Oocytes / Ovary / Spermatozoa / Swine / Temperature
M. Murakami, S. Ideguchi, M. Fahrudin, Takeshige Otoi, R.A. Godke and Tatsuyuki Suzuki : Influence of the DNA amount per microinjection on the development and EGFP expression in bovine embryos., Archives Animal Breeding, Vol.46, 25-30, 2003.
185.
M Murakami, Takeshige Otoi, N W. K. Karja, A Ooka and T Suzuki : Effects of serum-free culture media on in vitro development of domestic cat embryos following in vitro maturation and fertilization., Reproduction in Domestic Animals = Zuchthygiene, Vol.37, No.6, 352-356, 2002.
(Summary)
This study was conducted to determine the adequate medium for a serum-free culture system of domestic cat embryos produced by in vitro maturation (IVM) and fertilization (IVF). Cumulusoocyte complexes recovered from cat ovaries were matured in vitro for 24 h, and then inseminated in vitro for 12 h. After insemination, the oocytes were cultured in five media [Ham's F10, Waymouth 752/1 (Waymouth), TCM199, modified Earle's balanced salt solution (MK-1) and CR1aa], each of which contained 0.4% bovine serum albumin. There were no significant differences among the rates of fertilization of oocytes cultured in five media following IVF. The rate of oocytes/embryos developed to at least the morula stage was significantly lower (p < 0.05) in Waymouth than in MK-1, TCM199 and CR1aa. Moreover, none of the embryos cultured in Ham's F10 and Waymouth developed to the blastocyst stage. There were no differences among the rates of development to the blastocyst stage of oocytes/embryos cultured in MK-1, TCM199 and CR1aa. These results indicate that the type of serum-free medium has a major impact on in vitro development of domestic cat embryos derived from IVM/IVF, and MK-1, TCM199 and CR1aa media are suitable for in vitro culture of cat embryos in a serum-free culture system.
Takeshige Otoi, L Willingham, T Shin, D C. Kraemer and M Westhusin : Effects of oocyte culture density on meiotic competence of canine oocytes., Reproduction, Vol.124, No.6, 775-781, 2002.
(Summary)
This study was conducted to determine a suitable ratio of oocytes to medium for in vitro maturation (IVM) of cumulus-oocyte complexes (COCs) collected from bitches at anoestrus and dioestrus and to examine the meiotic competence of COCs cultured singly or in different group sizes. In the first experiment, different numbers of COCs (5, 10, 15 and 20 per drop) were cultured for 72 h in 100 microl drops of maturation medium. The meiotic competence of oocytes from ovaries at anoestrus was affected by the number of COCs incubated, whereas at dioestrus, the incubation number of COCs had no effect. In the second experiment, COCs were cultured singly or in different group sizes for 72 h by suitable oocyte density according to the reproductive cycle of the donor. In the anoestrous group, 1, 5 and 10 COCs were cultured in 10, 50 and 100 microl drops of the medium (10 microl per COC), respectively. In the dioestrous group, 1, 5 and 15 COCs were cultured in 7, 35 and 105 microl drops of the medium (7 microl per COC), respectively. There were no differences in the proportions of oocytes reaching metaphase II among the different group sizes in each stage of the reproductive cycle of the donor. The results indicate that the influence of oocyte density on the meiotic competence of oocytes differs according to the stage of the reproductive cycle of the donor. Moreover, the group sizes have no effect on the meiotic competence of oocytes cultured at suitable oocyte density according to the reproductive cycle of the donor.
M Fahrudin, Takeshige Otoi, N W. K. Karja, M Mori, M Murakami and T Suzuki : Analysis of DNA fragmentation in bovine somatic nuclear transfer embryos using TUNEL., Reproduction, Vol.124, No.6, 813-819, 2002.
(Summary)
The production of cloned animals is an inefficient process because of early or late embryonic losses. This study focused on the DNA fragmentation that occurs during embryonic development. The occurrence of DNA fragmentation was examined in bovine embryos produced by in vitro fertilization (IVF) and somatic cell nuclear transfer (NT) using the terminal deoxynucleotidyl transferase (TdT) nick-end labelling (TUNEL). IVF and NT embryos at the two-cell to blastocyst stage were stained by TUNEL for the analysis of DNA-fragmented nuclei and with propidium iodide for determination of the total number of cells. DNA fragmentation was first detected in NT embryos at the four-cell stage, but in IVF embryos at the six- to eight-cell stage. The percentage of embryos with at least one DNA-fragmented nucleus increased with the advance of the developmental stage of embryos in both IVF and NT groups. The DNA-fragmented nucleus index in NT embryos that developed beyond the four-cell stage was significantly higher (P<0.01) than that of IVF embryos at the same stage. In the both IVF and NT groups, TUNEL-labelled cells were detected in almost all blastocysts and were mainly observed in presumptive inner cell mass (ICM) cells of embryos. The DNA-fragmented nucleus index was negatively correlated with the total number of cells in NT blastocysts, but this relationship was not observed in IVF blastocysts. These results suggest that the high occurrence of DNA fragmentation observed in NT embryos may be related to early embryonic loss after transfer.
(Keyword)
Animals / Apoptosis / Cattle / Cell Nucleus / Cells, Cultured / Cloning, Organism / DNA Fragmentation / Embryonic and Fetal Development / Fertilization in Vitro / In Situ Nick-End Labeling / Nuclear Transfer Techniques
M Mori, Takeshige Otoi and T Suzuki : Correlation between the cell number and diameter in bovine embryos produced in vitro., Reproduction in Domestic Animals = Zuchthygiene, Vol.37, No.3, 181-184, 2002.
(Summary)
This study was designed to examine the effects of age and developmental stage of in vitro-produced bovine embryos on the cell number of the embryos and to investigate the correlation between the cell number and diameter in the embryos. The diameter and cell number in blastocysts and expanded blastocysts collected on days 7-9 after in vitro fertilization (IVF) were examined. Although the diameters of the blastocysts collected on days 7 and 8 after IVF were smaller than those of the expanded blastocysts collected on day 9, the cell number in both types of embryos was similar. The cell numbers of the blastocysts and expanded blastocysts decreased with increasing embryo age. There were positive correlations between the cell number and diameter in bovine embryos at each stage collected on each day after IVF. However, the value of the correlation coefficient in the day-9 expanded blastocyst group tended to be higher than that in the other groups. These results indicate that the cell number of in vitro-produced embryos is affected by the embryonic stage and age. The diameter of the embryo may be potentially used for the viability testing of the expanded blastocysts collected on day 9 after IVF.
(Keyword)
Age Factors / Animals / Blastocyst / Cattle / Cell Count / Culture Techniques / Embryonic and Fetal Development / Female / Fertilization in Vitro / Pregnancy
N W. K. Karja, Takeshige Otoi, M Murakami, M Fahrudin and T Suzuki : In vitro maturation, fertilization and development of domestic cat oocytes recovered from ovaries collected at three stages of the reproductive cycle., Theriogenology, Vol.57, No.9, 2289-2298, 2002.
(Summary)
This study was conducted to examine the effect of the donor cat's reproductive cycle stage on in vitro maturation (IVM), in vitro fertilization (IVF), and in vitro development of oocytes recovered from ovaries that were collected and stored at 35 degrees C for a short period (1-6 h). Based on the presence or absence of follicles and corpora lutea, the ovarian pairs collected were classified into inactive, follicular, or luteal stages. Nuclear status of 161 cumulus-oocyte complexes (COCs) were examined immediately after recovery; 91.3% of the oocytes were found to be at the immature germinal vesicle (GV) stage, and 3.7% of the oocytes were at metaphase II (MII) stage. The percentage of the oocytes at the GV stage was significantly lower in the follicular stage than in the inactive stage (P < 0.01). Of the oocytes from the follicular stage, 9.1% were at MII stage. After culture for 24 h, however, the proportions of oocytes that reached metaphase I and MII were not different among the reproductive cycle stages of the ovaries collected (P > 0.05). After co-incubation with sperm, 63.1% of oocytes were fertilized, but there were no significant differences among the reproductive cycle stages of the ovaries with respect to the proportions of normal and polyspermic fertilization. However, the number of oocytes reaching cleavage stage and development to the morula and blastocyst stages from follicular stage ovaries were significantly lower (P < 0.05) than those obtained from inactive and luteal stage ovaries. These results indicate that the reproductive cycle stage of donor cat ovaries, stored at 35 degrees C, has no apparent effects on the frequencies of maturation and fertilization of oocytes, but influences developmental competence of the oocytes following IVM or IVF.
(Keyword)
Animals / Blastocyst / Cats / Culture Techniques / Embryonic and Fetal Development / Female / Fertilization in Vitro / Follicular Phase / Luteal Phase / Meiosis / Morula / Oocytes / Ovary / Tissue and Organ Harvesting
N W. Kurniani Karja, Takeshige Otoi, Masako Murakami, Minori Yuge, Mokhamad Fahrudin and Tatsuyuki Suzuki : Effect of protein supplementation on development to the hatching and hatched blastocyst stages of cat IVF embryos., Reproduction, Fertility, and Development, Vol.14, No.5-6, 291-296, 2002.
(Summary)
The effects of protein supplementation in culture medium on development to the hatching and hatched blastocyst stages of cat in vitro-fertilized embryos were investigated. In the first experiment, presumptive zygotes derived from in vitro maturation and in vitro fertilization (IVF) were cultured in modified Earle's balanced salt solution (MK-1) supplemented with 0.4% bovine serum albumin (BSA) or 5% fetal bovine serum (FBS) for 9 days. There were no significant differences between the BSA and FBS groups with respect to the proportion of cleavage and development to the morula and blastocyst stages of zygotes. However, the presence of FBS in the medium enhanced development to the hatching blastocyst stage of zygotes compared with the BSA group (31.4% v. 7.8%). Moreover, 2.9% of zygotes cultured with FBS developed to the hatched blastocyst stage. The mean cell number of blastocysts derived from zygotes cultured with FBS was significantly higher (P<0.01) than that from zygotes cultured with BSA (136.6 v.101.5). In the second experiment, embryos at the morula orblastocyst stage, which were produced by culturing in MK-1 supplemented with 0.4% BSA after IVF, were subsequently cultured in MK-1 with 0.4% BSA or 5% FBS. Significantly more morulae developed to the blastocyst (P<0.05) and hatching blastocyst stages (P<0.01) in the FBS group than in the BSA group (71.5% and 53.6% v. 44.9% and 6.0%, respectively). Although none of the morulae cultured with BSA developed to the hatched blastocyst stage, 11.5% of morulae cultured with FBS developed to the hatched blastocyst stage. Moreover, the proportion of development to the hatching blastocyst stage of blastocysts was significantly higher (P<0.01) in the FBS group than in the BSA group (68.7% v. 9.8%). None of the blastocysts cultured with BSA developed to the hatched blastocyst stage, whereas 7.3% of blastocysts cultured with FBS developed to the hatched blastocyst stage. The results of the present study indicate that supplementation with FBS at different stages of early embryo development promotes development to the hatching and hatched blastocyst stages of cat IVF embryos.
(Keyword)
Animals / Blastocyst / Cats / Cattle / Culture Media / Culture Techniques / Embryonic and Fetal Development / Female / Fertilization in Vitro / Fetal Blood / Morula / Proteins / Serum Albumin, Bovine / Zygote
Y J. Dong, M D. Varisanga, N R. Mtango, M Aono, Takeshige Otoi and T Suzuki : Improvement of the culture conditions for in vitro production of cattle embryos in a portable CO2 incubator., Reproduction in Domestic Animals = Zuchthygiene, Vol.36, No.6, 313-318, 2001.
(Summary)
The effects of different concentrations of growth hormone (GH) on in vitro maturation (IVM), fertilization (IVF) and culture (IVC) of bovine oocyte/embryos in CR1aa or CR2aa media using a simple CO2 incubator were investigated. The IVM/IVF/IVC of oocytes were carried out in the presence of 0, 50, 100 and 200 ng/ml GH in the medium. The proportion of metaphase II oocytes was significantly higher (p < 0.05) in 200 ng/ml compared with 0 ng/ml GH in CR1aa medium (59 versus 85%, respectively), but this effect was not observed under CR2aa. Higher concentrations of GH yielded lower rates of unfertilized ova and thus superior cleavage rates (36.5 +/- 0.2 and 63.5 +/- 2.0% versus 17.5 +/- 0.2 and 82.5 +/- 1.5% or 40.4 +/- 0.6 and 59.6 +/- 1.4% versus 16.6 +/- 1.2 and 83.4 +/- 6.2% for 0 and 200 ng/ml GH in portable or ordinary incubator, respectively) in CR1aa. This dose-dependent effect was also observed in the percentages of transferable embryos, although not statistically different (17.2 +/- 1.7 versus 27.3 +/- 1.8% and 16.6 +/- 3.1 versus 26.0 +/- 1.4%, for 0 versus 200 ng/ml GH in portable and ordinary incubator, respectively). In contrast to the CR1aa, different concentrations of GH in CR2aa medium did not increase either fertilization or cleavage rates. In fact, higher concentrations of GH in this medium negatively affected the rate of transferable embryos. Hence, percentages of transferable embryos obtained in the portable incubator under 0 or 50 ng/ml GH were higher (p < 0.05) compared with those obtained in 100 or 200 ng/ml GH (35.4 +/- 5.7 or 40.5 +/- 5.4% versus 22.4 +/- 2.4 or 15.5 +/- 2.1%, respectively). There was however, no significant difference in the rate of transferable embryos in an ordinary incubator employing CR2aa medium, but the trend was more or less similar to that observed in the portable incubator. Despite the fact that relatively fewer oocytes were employed for the culture in the ordinary incubator, overall results observed employing the simple portable CO2 incubator were within the range of those obtained in an ordinary incubator: implying that the simple portable incubator can effectively be employed for the in vitro production of bovine embryos under field conditions.
(Keyword)
Animals / Carbon Dioxide / Cattle / Dose-Response Relationship, Drug / Embryo Transfer / Embryonic and Fetal Development / Fertilization / Fertilization in Vitro / Growth Hormone / Incubators / Oocytes
M Fahrudin, Takeshige Otoi and T Suzuki : Developmental competence of bovine embryos reconstructed by the transfer of somatic cells derived from frozen tissues., The Journal of Veterinary Medical Science, Vol.63, No.10, 1151-1154, 2001.
(Summary)
The ability of frozen-thawed fetal skin was examined to generate viable cell lines for nuclear transfer. Fetal skin frozen at -35 degrees C or -80 degrees C in the presence of 5% DMSO were used as tissue explants to generate somatic cells. The resultant confluent cells were then used as donors for nuclear transfer (NT). Of the bovine NT embryos reconstructed from the somatic cells, 78% to 81% showed cleavage, 43% to 48% reached the stage of morula formation and 34% to 35% reached blastocyst formation. There were no significant differences in development (P>0.05) when the NT embryos were compared with those reconstructed from fresh somatic-cell-derived skin tissues (75%, 45%, and 38%, for cleavage, and development to morula and blastocyst stages, respectively). The results indicated that cell lines derived from bovine fetal skin cryopreserved by a simple method could be used as donors in nuclear transfer. The resulting embryos showed similar development in vitro to those reconstructed from unfrozen fetal-skin-derived somatic cells.
(Keyword)
Animals / Cattle / Cryopreservation / Cryoprotective Agents / Culture Techniques / Embryo Transfer / Embryonic and Fetal Development / Female / Fertilization in Vitro / Male / Nuclear Transfer Techniques / Pregnancy / Skin
A S. Abdoon, O M. Kandil, Takeshige Otoi and T Suzuki : Influence of oocyte quality, culture media and gonadotropins on cleavage rate and development of in vitro fertilized buffalo embryos., Animal Reproduction Science, Vol.65, No.3-4, 215-223, 2001.
(Summary)
The present study was designed to examine the influence of oocyte quality, culture media and gonadotropins on cleavage rate and development of in vitro fertilized buffalo embryos. Three experiments were conducted. In experiment 1, oocytes were classified by number of cumulus cell layers and morphology of the ooplasm as good, fair or poor. Oocytes were cultured for IVM, IVF and IVC in CR1aa medium. In experiment 2, good quality oocytes were cultured for maturation in: (1) CR1aa; (2) CR2aa; (3) TCM-199; (4) MEM or (5) RPMI-1640, and then fertilized using frozen thawed buffalo spermatozoa in CR1aa. The oocytes were cultured in the same medium used for maturation after fertilization. In experiment 3, oocytes were classified into three groups: group (1) was without gonadotropin and serve as a control; group (2) in which IVM medium was supplemented with 10microg/ml FSH and group (3) in which IVM medium was supplemented with 10IUml(-1) eCG. In all experiments, oocytes were kept at 38.5 degrees C under 5% CO(2) for IVM, IVF, IVC and examined for cleavage and embryo development rates on days 3 and 8, respectively. Good and fair quality oocytes produced a higher cleavage rate (P<0.01) than poor quality oocytes. Morula production rate was also higher (P<0.01) for good as compared with fair quality oocytes. Embryo development with poor quality oocytes was arrested at the two to sixteen cell stage. In experiment 2, the cleavage rate was higher (P<0.05) in CR1aa than CR2aa, and higher (P<0.01) than TCM-199, MEM and RPMI-1640. The numbers of morulae and blastocysts were higher (P<0.01) for oocytes cultured in CR1aa and CR2aa media than TCM-199 or MEM. In experiment 3, the addition of FSH or eCG to the maturation medium increased (P<0.01) cleavage and developmental rates of buffalo embryo compared with control medium. In conclusion, the IVM of good quality buffalo oocytes in CR1aa or CR2aa medium and the addition of FSH or eCG in maturation medium produced higher cleavage and developmental rates of IVF buffalo embryos.
N R. Mtango, M D. Varisanga, Y J. Dong, Takeshige Otoi and T Suzuki : The effect of prefreezing the diluent portion of the straw in a step-wise vitrification process using ethylene glycol and polyvinylpyrrolidone to preserve bovine blastocysts., Cryobiology, Vol.42, No.2, 135-138, 2001.
(Summary)
A total of 678 bovine blastocysts, which had been produced by in vitro maturation, fertilization, and culture, were placed into plastic straws and were vitrified in various solutions of ethylene glycol (EG) + polyvinylpyrrolidone (PVP). Part of the straw was loaded with TCM199 medium + 0.3 M trehalose as a diluent; the diluent portions of the straw were prefrozen to either -30 or -196 degrees C. Then, the embryos suspended in the vitrification solution were pipetted into the balance of the straw and vitrified by direct immersion into liquid nitrogen. For thawing, the straws were warmed for 3 s in air and 20 s in a water bath at 39 degrees C and then agitated to mix the diluent and cryoprotectant solution for 5 min followed by culture in TCM199 + 10% FCS + 5 + microg/ml insulin + 50 microg/ml gentamycin sulfate for 72 h. Variables that were examined were the time of exposure to EG prior to vitrification, the PVP concentration, and the temperature of exposure to EG + PVP prior to vitrification. Survival and hatching rates of the blastocysts exposed to 40% EG in four steps at 4 degrees C were higher than those of embryos exposed in two steps (81.3 +/- 4.3% and 80.2 +/- 3.4% vs 67.6 +/- 4.5% and 71.5 +/- 4.7%, respectively; P < 0.05). The same indices were superior following vitrification-thawing of the blastocysts in 40% EG + 20% PVP than it was in 40% EG + 10% PVP (76.1 +/- 5.5% vs 63.7 +/- 1.8%; P < 0.05; and 61.6 +/- 6.0% vs 70.5 +/- 4.7%; P < 0.01, respectively). Exposure to the vitrification solution (40% EG + 20% PVP) at higher temperatures (37.5 degrees C vs 4 degrees C) reduced both survival and hatching rates (45.8 +/- 6.9% vs 83.9 +/- 4.4% and 41.5 +/- 1.8% vs 64.0 +/- 4.7%, respectively; P < 0.001). These results indicate that blastocysts vitrified after prefreezing the diluent portions of the straws do favor developmental competence of in vitro produced embryos.
(Keyword)
Animals / Blastocyst / Cattle / Cryopreservation / Cryoprotective Agents / Ethylene Glycol / Female / Fertilization in Vitro / In Vitro Techniques / Male / Povidone / Solutions
Takeshige Otoi, M Murakami, A Ooka, N W. Karja and T Suzuki : Effects of size and storage temperature on meiotic competence of domestic cat oocytes., The Veterinary Record, Vol.148, No.4, 116-118, 2001.
(Keyword)
Animals / Cats / Cell Size / Embryonic and Fetal Development / Female / Fertilization in Vitro / Meiosis / Oocytes / Temperature
(Link to Search Site for Scientific Articles)
● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 11232928
M. Fahrudin, Takeshige Otoi, N.W.K. Karja, M. Murakami and Tatsuyuki Suzuki : The effects of donor cell type and culture medium on in vitro development of domestic cat embryos reconstructed by nuclear transplantation., Asian-Australasian Journal of Animal Sciences, Vol.14, 1057-1061, 2001.
197.
Takeshige Otoi, A Ooka, M Murakami, N W. Karja and T Suzuki : Size distribution and meiotic competence of oocytes obtained from bitch ovaries at various stages of the oestrous cycle., Reproduction, Fertility, and Development, Vol.13, No.2-3, 151-155, 2001.
(Summary)
The present study was conducted to examine the effects of the stage of the oestrous cycle on the meiotic competence of canine oocytes and also to investigate the relationship between the stage of the oestrous cycle and the relative size distribution of oocytes obtained from bitches at three stages of the cycle (anoestrus, follicular phase and dioestrus). Only healthy-looking cumulus-oocyte complexes were used for in vitro maturation and these were divided into three groups based on diameter (< 110, 110 to < 120 and > or = 120 microm). The mean diameter of oocytes from ovaries at anoestrus, the follicular phase and dioestrus was 103.6, 119.2 and 107.7 microm, respectively. The percentage of large oocytes (> or = 120 microm) collected at the follicular phase was higher (P<0.01) than that collected at dioestrus and the percentage of oocytes > or = 120 microm collected from ovaries at dioestrus was higher (P<0.01) than that collected at anoestrus. After culture for 72 h, significantly more oocytes reached metaphase II (MII) in the follicular phase than in the other stages (P<0.01), and more oocytes reached MII in dioestrus than in anoestrus (P<0.05). In the > or = 120 microm group, the frequency of oocytes that resumed meiosis in the follicular phase was higher (P<0.05) than in the other stages. However, in the smaller diameter (< 120 microm) groups, there were no significant differences between ovaries at different stages of the oestrous cycle with respect to the proportion of oocytes reaching each stage of meiosis. Thus, the oestrous cycle stage influences maturation frequency. Moreover, oocytes demonstrated a size-related ability to undergo meiotic maturation, irrespective of the stage of the oestrous cycle. These results suggest that the effects of the stage of the oestrous cycle may result from differences in the distribution of large oocytes.
Takeshige Otoi, M Fujii, M Tanaka, A Ooka and T Suzuki : Canine oocyte diameter in relation to meiotic competence and sperm penetration, Theriogenology, Vol.54, No.4, 535-542, 2000.
(Summary)
This study was conducted to determine the diameter of canine oocytes that are able to attain full meiotic competence and sperm penetration. Oocytes were collected from ovaries of bitches at various stages of the estrous cycle. Only healthy-looking cumulus-oocyte complexes were used for in vitro maturation, and were divided into four groups based on diameter: <100, 100 to <110, 110 to <120 and >120 microm. Following in vitro maturation or fertilization, oocytes were stained to assess nuclear maturation and penetration rates. The mean oocyte diameter was 108.5 +/- 0.4 microm. The oocytes displayed size-related ability to undergo meiotic maturation. After culture for 72 h, the rates of oocytes that remained at the germinal vesicle stage in the <110 microm groups were significantly higher (P<0.01) than in the > or = 110 microm groups. None of the oocytes <110 microm reached metaphase II (MU), but 4.9 and 21.5% of the oocytes that were greater than 110 and 120 microm, respectively, progressed to MII. After in vitro fertilization for 20 h, 10 to 25% of oocytes were penetrated by spermatozoa, but there were no clear relationships between oocyte diameter and penetration rates of the oocyte by sperm. In the <120 microm groups, sperm penetration was mostly found in oocytes arrested at the germinal vesicle stage. However, a total of eight oocytes > or = 120 microm in diameter were penetrated by spermatozoa, of which five oocytes reached MII. These results suggest that there is a clear relationship between oocyte diameter and meiotic competence, but no relationship between oocyte diameter and sperm penetration. Canine oocytes may have acquired meiotic competence once they reach at a diameter of 120 microm, but the oocytes may allow the entry of spermatozoa into the ooplasm irrespective of oocyte diameter.
(Keyword)
Animals / Dogs / Female / Fertilization in Vitro / Male / Meiosis / Oocytes / Sperm-Ovum Interactions / Spermatozoa
Takeshige Otoi, N Koyama, K Yamamoto, N Horikita, S Tachikawa and T Suzuki : Developmental competence of frozen-thawed blastocysts from fair-quality bovine embryos cultured with beta-mercaptoethanol., The Veterinary Journal, Vol.159, No.3, 282-286, 2000.
(Summary)
Two-hundred-and-thirty-one fair-quality embryos at the compacted morula stage collected from 89 superovulated cows were cultured in TCM199 or Brinster's BMOC-3 medium with or without 100 microM beta-mercaptoethanol (beta-ME). After 24 h culture, a total of 142 fair-quality embryos developed to the blastocyst stage, of which 106 were subsequently frozen with 1.8 M ethylene glycol. The mean cell number and development rates of frozen-thawed blastocysts from the fair-quality embryos cultured in TCM199 containing beta-ME were higher than those of the fair-quality embryos directly frozen without culture. The pregnancy rates obtained with frozen blastocysts from fair-quality embryos tended to be lower than those of non-cultured fresh fair-quality embryos and cultured fresh blastocysts. These results indicate that the inclusion of beta-ME in pre-freezing culture media improve the development of frozen-thawed blastocysts from fair-quality embryos, but not the pregnancy rate.
M Fujii, Takeshige Otoi, M Murakami, M Tanaka, S Une and T Suzuki : The quality and maturation of bitch oocytes recovered from ovaries by the slicing method., The Journal of Veterinary Medical Science, Vol.62, No.3, 305-307, 2000.
(Summary)
Oocytes were recovered from bitch ovaries at various stages of the estrous cycle by the slicing method. The proportion of Grade A oocytes (darkly pigmented and surrounded in part, or whole, by dense layers of cumulus cells) were counted. Only Grade A oocytes were cultured in TCM199 supplemented with 5% fetal calf serum for evaluation of meiotic competence. There were no significant differences in the total number of oocytes or the proportion of Grade A oocytes that were recovered from bitches at various stages of the estrous cycle. Only 11% of the oocytes reached metaphase II (MII) at 72 hr after initiation of maturation culture. However, the proportions of oocytes reaching MII did not increase with culturing for up to 120 hr.
(Keyword)
Animals / Dogs / Female / Oocytes / Ovary / Tissue and Organ Harvesting
(Link to Search Site for Scientific Articles)
● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 10770604
Takeshige Otoi, M Murakami, M Fujii, M Tanaka, A Ooka, S Une and T Suzuki : Development of canine oocytes matured and fertilised in vitro., The Veterinary Record, Vol.146, No.2, 52-53, 2000.
Takeshige Otoi, N. Koyama, K. Yamamoto, S. Tachikawa and Tatsuyuki Suzuki : Effect of removal of large follicles after artificial insemination on embryo yield of superovulated beef cows., Canadian Journal of Animal Science, Vol.80, 199-201, 2000.
203.
M. Fahrudin, M.D. Varisanga, M. Murakami, Takeshige Otoi and Tatsuyuki Suzuki : Assessment of developmental competence of nuclei from bovine parthenogenetic embryos., The Journal of Reproduction and Development, Vol.46, 51-56, 2000.
204.
Takeshige Otoi, A SS Abdoon, M TK Omaima, M Tanaka and T Suzuki : Pellet freezing of in vitro produced bovine embryos using dry ice., Cryo Letters, Vol.21, No.1, 31-38, 2000.
(Summary)
In vitro produced bovine embryos were frozen by pellet freezing or vitrification method. In the pellet freezing method, the embryos were cooled on the dry ice and then frozen as pellets. At warming, the pellets were immersed directly into 0.5 M sucrose. The survival rates of blastocysts frozen by the pellet freezing method were higher (P<0.01) in 40% ethylene glycol (EG) than those in the lower concentrations (20 and 30% EG). Higher survival rates of blastocysts frozen by the pellet freezing method were obtained but the development rates did not differ, as compared with those by the vitrification method. There were no significant differences between the pellet freezing and vitrification method in the frequencies of post-thaw survival of hatched blastocysts. These results demonstrate that the pellet freezing method using dry ice can be used successfully for the cryopreservation of blastocysts.
(Link to Search Site for Scientific Articles)
● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 12148062
Takeshige Otoi, K Yamamoto, N Horikita, S Tachikawa and T Suzuki : Relationship between dead cells and DNA fragmentation in bovine embryos produced in vitro and stored at 4 degrees C., Molecular Reproduction and Development, Vol.54, No.4, 342-347, 1999.
(Summary)
DNA fragmentation and its relationship with dead cells were examined in bovine blastocysts produced in vitro and stored at 4 degrees C for 1-5 days. Survival and development to the hatching and hatched blastocyst stage decreased with increasing storage time. Both were significantly lower at 72 hr than at 48 hr. None of the embryos stored for 120 hr developed to the hatching or hatched blastocyst stage. The proportion of dead cells per embryo increased progressively as the time of storage increased, until 69% of embryonic cells were dead after 120 hr of storage. There was no significant difference between the proportions of DNA fragmentation per embryo stored for 0 and 24 hr (12% vs 16%). However, the proportion of DNA fragmentation in embryos stored for longer than 48 hr was significantly greater than that in embryos stored for less than 24 hr. There were no significant differences among those stored for longer than 48 hr (28-33%). These results suggest that the reduced developmental competence of bovine embryos stored at 4 degrees C is characterized by necrotic change rather than apoptotic change.
(Keyword)
Animals / Blastocyst / Cattle / Cell Death / Cell Survival / Cells, Cultured / Cryopreservation / DNA Fragmentation / In Situ Nick-End Labeling / Necrosis
K Yamamoto, Takeshige Otoi, N Koyama, N Horikita, S Tachikawa and T Miyano : Development to live young from bovine small oocytes after growth, maturation and fertilization in vitro., Theriogenology, Vol.52, No.1, 81-89, 1999.
(Summary)
Bovine oocytes (90 to 99 microns in diameter) were isolated from early antral follicles (0.5 to 0.7 mm in diameter). Cumulus-oocyte complexes (COC) with pieces of parietal granulosa were embedded in collagen gels and cultured for 14 d. After in vitro growth culture, oocytes recovered from the collagen gels were further matured, fertilized and cultured in vitro, and then were transferred to recipient cows. After 14 d of growth culture, 37% of the oocytes (203/556) showed normal morphology in the collagen gels. The mean diameter of the oocytes was 110.1 +/- 6.0 microns, significantly larger (P < 0.01) than before growth culture (94.8 +/- 2.7 microns), and 77% were at the germinal vesicle stage while 23% had undergone germinal vesicle breakdown. After 24 h of maturation culture followed by insemination, 27% of in vitro-grown oocytes reached the second metaphase, and 42% of the oocytes were normally fertilized. After insemination, 18.2% of in vitro-grown oocytes cleaved and 3.7% developed to the blastocyst stage. Three blastocysts obtained from in vitro-produced 90- to 99-micron oocytes were transferred to 3 recipients. One recipient subsequently became pregnant and delivered a live calf on Day 277. These results demonstrated for the first time that 90 to 99-micron oocytes from early antral follicles can complete growth and acquire full developmental competence in vitro so that live young can be produced after maturation, fertilization, subsequent culture in vitro, and transfer to recipient cows.
H Abe, Takeshige Otoi, S Tachikawa, S Yamashita, T Satoh and H Hoshi : Fine structure of bovine morulae and blastocysts in vivo and in vitro., Anatomy and Embryology, Vol.199, No.6, 519-527, 1999.
(Summary)
The ultrastructure of bovine morulae and blastocysts developed from in vitro-matured and -fertilized oocytes in a serum-supplemented medium was compared with that of morulae and blastocysts collected non-surgically from superovulated cows. In the in vivo-derived morulae, two characteristic cells types could be identified by the electron-density of their cytoplasm and by their ultrastructural features. One type appeared light in color with low electron-dense cytoplasm. These cells were located in the peripheral layer of the cluster of blastomeres, possessed numerous cellular organelles such as mitochondria and Golgi apparatus and had microvilli projecting into the perivitelline space. The other cell type was distinguished by cytoplasm that stained more densely than that of the lighter-appearing cells. The darker-appearing cells generally possessed fewer organelles than the lighter cells, but many lysosome-like structures were present in the cytoplasm. The in vitro-developed morulae also contained two types of cells similar to those observed in the in vivo morulae. However, most of the in vitro-developed cells possessed numerous lipid droplets and contained fewer lysosome-like structures than the cells of the in vivo-derived morulae. The blastocysts, both in vivo and in vitro, showed a clear differentiation of trophoblast cells and inner cell mass (ICM)-cells. In the in vivo-derived blastocyst, the apical membrane of trophoblast cells was covered with large, numerous microvilli and well-developed junctional complexes were observed. Lipid droplets were present in the cytoplasm of trophoblast and ICM-cells but were not abundant. In vitro-developed blastocysts showed less well-developed junctional complexes between trophoblast cells, less well-developed apical microvilli on the trophoblast cells, and contained large numbers of lipid droplets. This accumulation of lipid droplets was higher in the trophoblast cells than in the ICM-cells. The zonae pellucidae of in vitro-developed embryos were thinner than that of the in vivo-derived embryos. This study demonstrates conspicuous differences in the ultrastructural features between the in vivo-derived and in vitro-developed embryos, suggesting that the ultrastructure may reflect the various physiological anomalies observed in previous studies.
(Keyword)
Animals / Blastocyst / Cattle / Cytoplasm / Female / Fertilization in Vitro / Microscopy, Electron / Morula / Organ Culture Techniques / Superovulation
O.M. Kandil, A.S.S. Abdoon., M. Murakami, Takeshige Otoi and Tatsuyuki Suzuki : New technique, using a portable CO2 incubator, for the production of in vitro fertilized Egyptian buffalo embryos., The Journal of Reproduction and Development, Vol.45, 315-320, 1999.
209.
Takeshige Otoi, M Fujii, M Tanaka, A Ooka and T Suzuki : Effect of serum on the in vitro maturation of canine oocytes., Reproduction, Fertility, and Development, Vol.11, No.7-8, 387-390, 1999.
(Summary)
The meiotic competence of canine oocytes cultured for 72 h in medium supplemented with three different concentrations (5, 10 and 20%) of anoestrous, oestrous or metoestrous bitch serum, or with 0.3% bovine serum albumin (BSA), was examined. The oestrous serum supplement had a positive effect on the resumption of meiosis, compared with the other supplements (P<0.05). The number of oocytes that reached metaphase I (MI) and metaphase II (MII) was significantly higher (P<0.05) with the oestrous serum supplement than with the anoestrous serum supplement. There were no significant differences among the three different concentrations in each serum type with respect to the proportion of oocytes that completed meiosis (MI to MII). The number of oocytes that resumed meiosis in the 10% oestrous serum supplement was significantly higher (P<0.05) than those of each concentration of the anoestrous and metoestrous serum supplements, and of the 0.3% BSA supplement. Moreover, a higher number of oocytes reached MII in the presence of the 10% oestrous serum supplement than with the 10% anoestrous serum supplement. These results suggest that supplementation of the culture medium with 10% oestrous serum is the optimal treatment for in vitro maturation of canine oocytes.
Takeshige Otoi, K Yamamoto, N Koyama, S Tachikawa and T Suzuki : Cryopreservation of mature bovine oocytes by vitrification in straws., Cryobiology, Vol.37, No.1, 77-85, 1998.
(Summary)
Experiments were conducted to determine optimal conditions for vitrification of in vitro matured bovine oocytes in straws. In the first series of experiments, the effects of stepwise addition before exposure of oocytes to vitrification solution consisting of 30% (v/v) ethylene glycol (EG) with 0.35 M sucrose was tested. The rates of morphological survival and cleavage of oocytes vitrified by the three-step addition procedure were higher than those vitrified by the one-step addition procedure. In the second series, the effect of the concentration of the vitrification solution was tested (20, 30, 40, and 50% EG, all with 0.35 M sucrose). The survival rates of oocytes vitrified in 20 and 30% EG were lower than those in 40 and 50% EG. The rates of cleavage and development to blastocysts of oocytes vitrified in 40% EG were the highest among the four groups. In the third series, the effect of duration of exposure of oocytes to 0.5 M sucrose (1, 5, and 10 min) at the first step during the three-step dilution was tested. Although there were no significant differences among the groups with respect to developmental competence of oocytes vitrified in 40% EG, the highest development rate (10%) to blastocysts was observed when oocytes were exposed to sucrose for 5 min. These data demonstrate that improvements to the concentration of cryoprotectant and addition procedures have critical effects on the developmental competence of oocytes vitrified in straws.
Takeshige Otoi, N Koyama, K Yamamoto, S Tachikawa and T Suzuki : Superovulatory responses in Japanese black beef cows following largest follicle aspiration or human chorionic gonadotrophin (hCG) treatment., The Journal of Veterinary Medical Science, Vol.60, No.8, 961-963, 1998.
(Summary)
The effect of largest follicle aspiration or hCG administration before induction of superovulation on the ovarian response of Japanese Black beef cows was investigated using a crossover design in which induction of superovulation was attempted in every cow. The superovulatory response of cows whose largest follicle had been aspirated from the ovaries by ultrasound-guided follicular aspiration 1 day before induction of superovulation, did not differ from the response in non-treated control cows. In contrast, in cows given 5,000 IU of hCG 3 days before induction of superovulation, the proportions of fertilized ova and transferable embryos significantly decreased compared with the other groups.
M Murakami, Takeshige Otoi, C Sumantri and T Suzuki : Effects of centrifugation and lipid removal on the cryopreservation of in vitro produced bovine embryos at the eight-cell stage., Cryobiology, Vol.36, No.3, 206-212, 1998.
(Summary)
The effects of intracellular lipid polarization and lipid removal treatments on the postthawed in vitro development of frozen bovine embryos at the 8-cell stage were studied. As the first step, bovine presumptive zygotes were centrifuged at 16,000 g for 20 min for the cytoplasmic lipid polarization and their lipid layers were removed by micromanipulation in order to examine the influence of these treatments on the developmental capacity of bovine zygotes. As the second step, bovine embryos developed to the 8-cell stage following centrifugation treatment at various forces (8000, 12,000, and 16,000 g) or lipid removal treatment at the 1-cell stage were frozen in 1.8 M ethylene glycol + 0.05 M trehalose supplemented with 5% polyvinylpyrrolidone in a one-step procedure. There were no significant differences among the control (nontreatment), lipid-polarized, and lipid-removed groups with respect to the developmental capacity of fresh nonfrozen zygotes (experiment 1). The rates of survival and development to the blastocyst of frozen-thawed 8-cell embryos increased slightly with increasing force of centrifugation (experiment 2). The rate of development into blastocysts of the frozen-thawed 8-cell embryos was significantly higher in the groups that underwent centrifugation (at 16,000 g for 20 min; P < 0.05) or lipid removal (P < 0.01) treatments than the control (intact) group. However, there were no significant differences among the groups with respect to the rate of development to the expanded/hatched blastocyst stage. In addition, the mean cell numbers of embryos developed into blastocysts (day 8) derived from frozen-thawed 8-cell embryos tended to be low in the centrifugation and lipid removal groups compared to the controls (experiment 3). These results suggest that although the centrifugation with or without lipid removal treatments has no detrimental effects on the developmental capacity of bovine zygotes, the freezing tolerance of bovine 8-cell embryos was not improved by these treatments.
Takeshige Otoi, N Koyama, K Yamamoto and S Tachikawa : Superovulatory response in beef cows following removal of the largest ovarian follicle., The Veterinary Record, Vol.142, No.15, 402-403, 1998.
Takeshige Otoi, K Yamamoto, N Koyama, S Tachikawa and T Suzuki : Bovine oocyte diameter in relation to developmental competence., Theriogenology, Vol.48, No.5, 769-774, 1997.
(Summary)
This study was conducted to determine the diameter of bovine oocytes that were able to attain their full developmental competence to blastocysts. Oocytes were recovered by aspiration of surface-visible follicles (1 to 7 mm in diameter) from slaughterhouse ovaries. Only healthy-looking cumulus-oocyte complexes were used for in vitro maturation, and they were divided into six groups based on diameter: < 110 microm, 110 to < 115 microm, 115 to < 120 microm, 120 to < 125 microm, 125 to < 130 microm and >/= 130 microm. Oocytes were processed through standard procedures for in vitro maturation, fertilization and culture. Following in vitro maturation or fertilization, some oocytes were stained to assess nuclear maturation and penetration rates. The numbers of embryos that cleaved at 42 h post insemination and developed to blastocysts and hatched blastocysts after 8 days of culture were recorded. The mean oocyte diameters were 114.0 +/- 4.8 microm. The oocytes displayed size-related ability to undergo meiotic maturation. The rates of nuclear maturation of oocytes in the greater than 115-microm size range were significantly higher than those of oocytes with diameters < 115 microm. In the < 120 microm diameter groups, the polyspermic fertilization rates of oocytes < 115 microm were significantly higher than those of oocytes 115 to < 120 microm in diameter. The rates of cleavage and development to blastocysts and hatched blastocysts rose as oocyte diameter increased. Among oocytes with a diameter >110 microm, oocytes < 120 microm were found to have significantly lower developmental competence than oocytes 120 to < 130 microm in diameter. These results suggest that bovine oocytes have acquired full meiotic competence at a diameter of 115 microm but not yet attained full developmental competence to blastocysts, and that oocytes have acquired full developmental competence at a diameter of 120 microm.
Takeshige Otoi, K Yamamoto, N Koyama, S Tachikawa, M Murakami, Y Kikkawa and T Suzuki : Cryopreservation of mature bovine oocytes following centrifugation treatment., Cryobiology, Vol.34, No.1, 36-41, 1997.
(Summary)
In vitro matured bovine oocytes were frozen slowly in 1.6 M 1,2-propanediol following centrifugation treatment for polarization of lipid droplets in the cytoplasm. After thawing, the survival of the oocytes was assessed morphologically and also by in vitro fertilization and culture. The polarization of cytoplasmic lipid droplets had a negative effect on the survival of frozen-thawed oocytes. Thus, this treatment did not improve the frequency of normal fertilization and development to blastocysts, compared with that of frozen control oocytes. However, the frequency of polyspermy and activation of lipid-polarized oocytes that survived after freezing-thawing and subsequent in vitro fertilization tended to be less than those of surviving control oocytes. In addition, the effect of centrifugation treatment was to produce a small but significant increase in the cleavage rate of oocytes that survived after freezing-thawing and the development rates to blastocysts of surviving lipid-polarized oocytes tended to increase, compared with those of surviving control oocytes. These results suggest that the freezing tolerance of the spindle and other organelles of in vitro matured bovine oocytes is associated with lipid droplets and may be improved by the polarization of cytoplasmic lipid droplets before cryopreservation.
Takeshige Otoi, K. Yamamoto, N. Koyama and S. Tachikawa : Effects of exposure to room temperature, chilling and freezing of bovine one-cell embryos with centrifugation treatment in the presence of various cryoprotectants on their development to blastocysts., Cryo Letters, Vol.18, 307-316, 1997.
217.
Takeshige Otoi, K Yamamoto, N Koyama and S Tachikawa : Development of oocytes derived from the first dominant follicles of beef cows., The Veterinary Record, Vol.139, No.16, 396, 1996.
Takeshige Otoi, K Yamamoto, N Koyama, S Tachikawa and T Suzuki : A frozen-thawed in vitro-matured bovine oocyte derived calf with normal growth and fertility., The Journal of Veterinary Medical Science, Vol.58, No.8, 811-813, 1996.
(Summary)
The growth and fertility of a female calf obtained from a frozen-thawed bovine oocyte was assessed. The birth weight of the calf was lower than the mean birth weight of calves from in vitro fertilized embryos (IVF-controls) and calves obtained by artificial insemination (AI-controls). The growth rate of the calf up to 6 months was slower than that of the IVF-controls, but similar to that of the AI-controls. When the calf developed into a heifer (200 kg), she was inseminated with frozen semen and 280 days later delivered a male calf. The chromosoms of this cow were normal. These findings suggest that the growth and fertility of the calf derived from the frozen oocyte are normal.
S Saha, Takeshige Otoi, M Takagi, A Boediono, C Sumantri and T Suzuki : Normal calves obtained after direct transfer of vitrified bovine embryos using ethylene glycol, trehalose, and polyvinylpyrrolidone., Cryobiology, Vol.33, No.3, 291-299, 1996.
(Summary)
In the present study, IVF bovine embryos were vitrified using as the cryoprotectants, ethylene glycol plus trehalose plus the polymer, polyvinylpyrrolidone (PVP). In Experiment I, toxicity of the vitrification solution (VS) containing 20% PVP was tested in relation to temperature and exposure time. One hundred percent embryo development was observed with treatment at 5 degrees C for 5 min, whereas only 55.5% embryos were developed when the treatment was carried out at 20 degrees C for 5 min. In Experiment II, embryos were vitrified using one of the three treatments (Treatment A, 40% ethylene glycol (EG); treatment B, 40% EG + 11.3% trehalose; and treatment C, 40% EG + 11.3% trehalose + 20% PVP and rehydrations) was performed directly in mPBS. Highest development (84.1%) and hatching rate (68.2%) were obtained when embryos were vitrified with the vitrification solution used in treatment C. In Experiment III, embryos were vitrified as in Experiment II (treatment C). The development and hatching rates were compared after rehydration in different rehydration solutions. No significant difference was observed among the development and hatching rates when rehydration was carried out in different concentrations of trehalose. Five vitrified-warmed bovine embryos were transferred directly to five recipients and three recipients gave birth to three normal calves.
(Keyword)
Animals / Cattle / Cryopreservation / Cryoprotective Agents / Embryo Transfer / Embryo, Mammalian / Ethylene Glycol / Ethylene Glycols / Evaluation Studies as Topic / Female / Fertilization in Vitro / Povidone / Pregnancy / Solutions / Temperature / Time Factors / Trehalose
Takeshige Otoi, K. Yamamoto, N Koyama, S. Tachikawa and Tatsuyuki Suzuki : Development of in vitro matured bovine oocytes activated with low temperature., Cryo Letters, Vol.17, 131-136, 1996.
221.
S. Saha, Takeshige Otoi and Tatsuyuki Suzuki : The efficiency of ethylene glycol, trehalose and polyvinylpyrrolidone for successful vitrification of IVF bovine embryos., The Journal of Reproduction and Development, Vol.42, 163-169, 1996.
222.
Takeshige Otoi, K Yamamoto, N Koyama and T Suzuki : In vitro fertilization and development of immature and mature bovine oocytes cryopreserved by ethylene glycol with sucrose., Cryobiology, Vol.32, No.5, 455-460, 1995.
(Summary)
Immature and mature bovine oocytes were frozen slowly in 1.8 M ethylene glycol (EG) with different concentrations of sucrose (0, 0.1, and 0.2 M). After thawing and dilution of the cryoprotectant by a one-step procedure, the survival of the oocytes was assessed morphologically and also by in vitro fertilization and culture. Sucrose had no effect on the survival and fertilization rates of frozen-thawed oocytes. However, higher cleavage rates of frozen-thawed immature oocytes were obtained with 0.2 M sucrose used in combination with EG. Moreover, none of the immature oocytes frozen in EG without sucrose developed into blastocysts, but about 1% of immature oocytes frozen with sucrose developed into blastocysts. The cleavage rates of mature oocytes frozen in EG were significantly lower with sucrose than without sucrose. However, no significant difference was observed in the development to blastocysts. Transfer of four blastocysts derived from frozen-thawed immature oocytes into two recipient heifers resulted in one pregnancy.
Takeshige Otoi, K. Yamamoto, N. Koyama, S. Hayashi and Tatsuyuki Suzuki : Developmental capacity of bovine follicular oocytes collected from ovaries of pubertal heifers obtained through a spay device., The Journal of Reproduction and Development, Vol.41, 109-111, 1995.
224.
Takeshige Otoi, K. Yamamoto, N. Koyama, S. Tachikawa and Tatsuyuki Suzuki : The development of immature bovine oocytes cryopreserved by 1,2-propanediol., The Journal of Reproduction and Development, Vol.41, 361-366, 1995.
225.
Takeshige Otoi, S Tachikawa, S Kondo, M Takagi and T Suzuki : Developmental competence of bovine oocytes frozen at different cooling rates., Cryobiology, Vol.31, No.4, 344-348, 1994.
(Summary)
Experiments were conducted to determine the optimal cooling conditions for improving the developmental competence of in vitro-matured bovine oocytes when frozen in 1.6 M 1,2-propanediol. Bovine oocytes were cooled from 0 degree to -5.5 degrees C at 1 degree C/min, seeded, and then cooled to -30 degrees C at different cooling rates (0.3 degree, 0.6 degree or 0.9 degree C/min) and then plunged into LN2. When frozen-thawed oocytes were fertilized in vitro, the proportions of fertilization and developmental competence did not differ among the three groups frozen at different cooling rates. The findings indicated that a cooling rate of less than 1.0 degree C/min in the freezing procedure did not affect the developmental competence of postthaw in vitro-matured bovine oocytes.
(Keyword)
Animals / Cattle / Cryopreservation / Cryoprotective Agents / Evaluation Studies as Topic / Female / Fertilization in Vitro / In Vitro Techniques / Oocytes / Propylene Glycol / Propylene Glycols / Time Factors
M Takagi, L Sakonju, Takeshige Otoi, K Hamana and T Suzuki : Postthaw viability of the inner cell mass of in vitro-matured/in vitro-fertilized bovine embryos frozen in various cryoprotectants., Cryobiology, Vol.31, No.4, 398-405, 1994.
(Summary)
A study was conducted to examine the viability of inner cell mass (ICM) cells of frozen-thawed in vitro-matured (IVM)/in vitro-fertilized (IVF)-derived embryos using various cryoprotectants. Expanded blastocysts were frozen and thawed in 1.4 M glycerol with 0.25 M sucrose (GL), 1.6 M propylene glycol (PG), 1.8 M ethylene glycol (EG), or 1.3 M ethylene glycol monomethyl ether (EME) as cryoprotectants using a one-step method. After thawing, the embryos were cocultured for 24 h with cumulus cells in TCM199. Embryos which were viable after thawing and developed beyond the blastocyst stage were treated by immunosurgery and differential fluorochrome staining for ICM cell counts. Overall, there were no significant differences in the development to blastocyst stage after 24 h culture in each cryoprotectant (P < 0.05, chi 2 analysis). The viability of ICM cells of frozen-thawed embryos with each cryoprotectant was lower (GL, 72.7%; PG, 67.8%; EG, 77.5%; EME, 74.7%) than that of unfrozen embryos (84.4%). In the case of PG as a cryoprotectant, viability of ICM cells was significantly lower than that of unfrozen embryos (P < 0.05, ANOVA analysis). Our results suggest that the viability of ICM cells of frozen-thawed bovine embryos tend to be lower than that of unfrozen embryos irrespective of the cryoprotectants used. PG was significantly more toxic to the ICM cells compared with the other cryoprotectants.
M Takagi, Takeshige Otoi, A Boediono, S Saha and T Suzuki : Viability of frozen-thawed bovine IVM/IVF embryos in relation to aging using various cryoprotectants., Theriogenology, Vol.41, No.4, 915-921, 1994.
(Summary)
Bovine IVF embryos developed on Days 7, 8 and 9 were equilibrated with 1.6 M propylene glycol (PG), 1.8 M ethylene glycol (EG), 1.1 M diethylene glycol (DEG) or 1.3 M ethylene glycol monomethyl ether (EME) for 10 to 20 min in modified phosphate buffered saline. (mPBS) supplemented with 10% superovulated cow serum. The embryos were loaded into 0.25-ml plastic straws and were placed directly into a 0 degrees C alcohol bath chamber and held for 2 min. They were cooled from 0 degrees C to -5.5 degrees C at 1 degrees C/min and then seeded, followed by a 10-min holding period at -5.5 degrees C. The straws were then cooled to -30 degrees C at 0.3 degrees C/min before plunging into liquid nitrogen. Embryos were thawed and placed directly into the culture medium and washed 3 times. The survival rates of the Day-9 embryos based on reappearance of blastocoele, expansion, and hatching after 48 h of post-thaw culture were significantly lower (P<0.01) than those of the Day-7 and 8 embryos, in all of the cryoprotectants tested. On the other hand, while the reappearance of blastocoele and expansion of blastocysts after 48 h of post-thaw culture were not significantly different among each cryoprotectant, the percentage of hatching blastocysts were significantly different between DEG and EME (P<0.05), between DEG and EG (P<0.01) and between PG and EG (P<0.05). These findings demonstrate that the age of the embryo (Day 7 and 8) is very important for the successful freezing of IVF bovine embryos. Also, as to the hatching rates, EME and EG are superior as cryoprotectants than the other 2 cryoprotectants tested.
Takeshige Otoi, S Tachikawa, S Kondo and T Suzuki : Effect of washing, antibiotics and trypsin treatment of bovine embryos on the removal of adhering K99+ Escherichia coli., The Journal of Veterinary Medical Science, Vol.55, No.6, 1053-1055, 1993.
(Summary)
Bovine embryos with the intact zona pellucida were exposed in vitro to K99+ Escherichia coli (K99 E. coli). The recommended procedures for washing and treating embryos were then evaluated for their effectiveness in removing or killing the adherent bacteria. After 10-step washing, bacteria were recovered not only from the embryos exposed to E. coli suspensions but also from those treated with trypsin during washing. On the other hand, no bacteria were recovered from any embryos treated with antibiotics (gentamicin; 50 micrograms/ml) in culture medium before washing, indicating that the recommended washing procedures with appropriate antibiotics assure that zona pellucida-intact bovine embryos are free from E. coli.
M Takagi, Takeshige Otoi and T Suzuki : Survival rate of frozen-thawed bovine IVM/IVF embryos in relation to post-thaw exposure time in two cryoprotectants., Cryobiology, Vol.30, No.5, 466-469, 1993.
(Summary)
The relationship between post-thaw exposure time in cryoprotectants and the viability of in vitro fertilized (IVF)-derived bovine embryos after culture was examined. Good- and excellent-quality Day 7 to Day 8 blastocysts and expanded blastocysts were frozen by the nonstepwise method using 1.6 M propylene glycol (PG) or ethylene glycol (EG) as the cryoprotectant. After the straws were thawed, the embryos were kept in the cryoprotectants for 1, 10, or 30 min. Then the embryos were placed directly into culture medium and washed three times, followed by coculture with cumulus cells. After 48 h of culture, there were no significant differences in survival rate among the various exposure times in PG (79-81%) and EG (71-75%). After 72 h of culture, there were no significant differences among the various exposure times in the fully expanded (PG 76-81%; EG 71-72%), hatching (PG 69-74%; EG 58-71%), and hatched blastocyst rates (PG 60-71%; EG 47-65%). Our findings suggest that different post-thaw exposure times up to 30 min using PG or EG as the cryoprotectant are not harmful to IVF bovine embryos.
Takeshige Otoi, S Tachikawa, S Kondo and T Suzuki : Developmental capacity of bovine oocytes frozen in different cryoprotectants., Theriogenology, Vol.40, No.4, 801-807, 1993.
(Summary)
Iso-osmolar concentrations of 1.6 M of 1,2-propanediol, glycerol and dimethylsulfoxide were compared for their effectiveness as cryoprotectants for in vitro-matured bovine oocytes. Survival, defined as the number of morphologically normal oocytes, after freeze-thawing in 1,2-propanediol (49.8%) was significantly higher (P<0.01) than in glycerol (28.6%) or dimethylsulfoxide (32.6%). After insemination, the fertilization rate of morphologically normal oocytes frozen-thawed in 1,2-propanediol (57.9%) was higher (P<0.05) than that of those frozen in dimethylsulfoxide (38.3%). However, the proportion of surviving oocytes developing to blastocysts was not different among the 3 groups.
Takeshige Otoi, S Tachikawa, S Kondo, F Kono, M Goto, Y Ogano and T Suzuki : Effect of trypsin treatment of in vitro fertilized bovine embryos on their subsequent survival and development., The Journal of Veterinary Medical Science, Vol.55, No.2, 237-239, 1993.
(Summary)
The object of this study was to determine whether several washings with trypsin affected the survival and development of in vitro fertilized (IVF) bovine embryos. The embryos developed to blastocysts 7-8 days after insemination, were washed 12 times in washing medium (modified Dulbecco's phosphate-buffered saline (mPBS) containing 20% fetal bovine serum (FBS): 25 mM Hepes TCM199 with Earle's salts = 1:1), or in a series of three washes in the washing medium, two in mPBS containing 0.3% bovine serum albumin (BSA), two in 0.25% trypsin in Hank's solution (without Ca++ and Mg++) for a total time in the trypsin of 60 to 90 sec and five in the washing medium. After washing, the embryos were either cultured in vitro or cryopreserved. The fresh and frozen-thawed embryos were either cultured for 72 hr in vitro for evaluating development or transferred nonsurgically to recipient cows. Development of fresh and frozen-thawed embryos in vitro in control-washed and trypsin-washed embryos did not differ. Pregnancy rates did not differ (P > 0.05) among recipient cows receiving control-washed or trypsin-washed embryos, and transferring fresh or frozen-thawed embryos. These results indicate that the treatment of bovine IVF embryos with trypsin during washing did not have a positive effect on embryonic development.
(Keyword)
Animals / Blastocyst / Cattle / Cell Survival / Cryopreservation / Embryonic and Fetal Development / Female / Fertilization in Vitro / Organ Culture Techniques / Pregnancy / Pregnancy, Animal / Trypsin
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S Tachikawa, Takeshige Otoi, S Kondo, T Machida and M Kasai : Successful vitrification of bovine blastocysts, derived by in vitro maturation and fertilization., Molecular Reproduction and Development, Vol.34, No.3, 266-271, 1993.
(Summary)
Bovine blastocysts were produced through maturation, fertilization, and development in vitro. For vitrification, solutions designated EFS, GFS, and PFS were prepared; these were 40% ethylene glycol, 40% glycerol, and 40% propylene glycol, respectively, diluted in modified phosphate-buffered saline (PBS) containing 30% Ficoll + 0.5 M sucrose. The embryos were exposed to the solutions in one step at room temperature, kept in the solutions for various times, vitrified in liquid nitrogen, and warmed rapidly. When the embryos were vitrified in EFS solution after 1 or 2 min exposure, the postwarming survival rate, assessed by the reexpansion of the blastocoel, was 74-77%. However, when the exposure time was extended to 3 min or longer, this rate dropped to 7-0%. This reduction was attributed to the toxicity of ethylene glycol. Of the embryos vitrified in GFS solution, 53% survived when they were cooled after 1 min exposure; as the duration of the exposure increased, the survival rate increased, reaching a peak (72%) at 4 min. The rate then decreased gradually with exposure time. In PFS solution, embryos surviving after vitrification were recovered only with 1 min exposure (33%), reflecting the high toxicity of propylene glycol. After vitrification in EFS or GFS solution, two embryos were nonsurgically transferred into each of 14 recipient animals. Of the 14 recipients, ten (71%) became pregnant; two resulted in early stillbirths, four recipients delivered twins (four alive and four stillborn), and two delivered single live calves, demonstrating the effectiveness of this simple vitrification method for the cryopreservation of in-vitro-produced bovine blastocysts.
Takeshige Otoi, S Tachikawa, S Kondo and T Suzuki : Effects of different lots of semen from the same bull on in vitro development of bovine oocytes fertilized in vitro., Theriogenology, Vol.39, No.3, 713-718, 1993.
(Summary)
The effectiveness of 8 different lots of semen from the same bull on the in vitro development of bovine oocytes fertilized in vitro was evaluated. Cleavage and development rates to the blastocyst stage were not significantly different among the 8 lots of semen. However, the cleavage and development rates varied no more than +/-11 and +/-6%, respectively, in overall rates. A maximum of 35.2 and 27.9% difference in cleavage and development rates to the blastocyst stage, respectively, was observed using a single straw from the same semen lot for insemination in 4 trials. This variation tended to be higher than that observed for the cleavage and development rates of oocytes after insemination with double straws from a single lot. These results indicate that the developmental capacity of embryos after insemination are affected by factors associated with a different semen lot from the same bull and with different straws from a single lot.
Takeshige Otoi, S Tachikawa, S Kondo and T Suzuki : Developmental capacity of bovine oocytes cryopreserved after maturation in vitro and of frozen-thawed bovine embryos derived from frozen mature oocytes., Theriogenology, Vol.38, No.4, 711-719, 1992.
(Summary)
The present study was conducted 1) to investigate the post-thaw developmental capacity of in vitro mature bovine oocytes (Metaphase II) frozen by 1.6 M of 1,2-propanediol and 2) to confirm the viability of frozen bovine embryos derived from frozen mature oocytes. The cleavage and developmental rates to the blastocyst stage of frozen-thawed mature oocytes were significantly lower (P<0.01) than that of nonfrozen oocytes. When mature oocytes were treated with hyaluronidase, trypsin, or base solution (solution control) before processing to remove the cumulus cells, the developmental rates to the blastocyst stage of frozen-thawed oocytes were 2.8% (5/180), 3.1% (9/295) and 1.1% (1/89), respectively. The viability and developmental capacity of frozen-thawed bovine embryos derived from frozen mature oocytes were not different from those of frozen-thawed bovine embryos derived from nonfrozen mature oocytes (control). Furthermore, nonfrozen and frozen-thawed embryos derived from frozen-thawed mature oocytes were nonsurgically transferred to recipient cows. One of the four and one of the two recipient cows became pregnant, respectively. The results of this study demonstrated the viability of embryos obtained from frozen-thawed bovine oocytes at Metaphase II followed by in vitro fertilization and culture to the blastocyst stage in vitro.
Takeshige Otoi, S Tachikawa, S Kondo and T Suzuki : Effect of antibiotics treatment of in vitro fertilized bovine embryos to remove adhering bacteria., The Journal of Veterinary Medical Science, Vol.54, No.4, 763-765, 1992.
Fuminori Tanihara, Maki Hirata and Takeshige Otoi : Current status of the application of gene editing in pigs., The Journal of Reproduction and Development, Vol.67, No.3, 177-187, Apr. 2021.
(Summary)
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.
Y. Sato, M. Taniguchi and Takeshige Otoi : Studying spermatogenesis by using in vivo and in vitro models: Advantages and disadvantages of these models for practical use., Journal of Veterinary Science and Technology, Vol.3, No.3, 115, 2012.
4.
Takeshige Otoi : Effects of large follicle aspiration on superovulatory response of beef cows., Archives Animal Breeding, Vol.44, 30-32, Jan. 2001.
5.
T. Miyano, S. Osaki, K. Yamamoto and Takeshige Otoi : In vitro growth of bovine oocytes., 4th Asian Symposium on Animal Biotechnology, Proceedings, 67-74, Jan. 1998.
Proceeding of International Conference:
1.
Lin Qingyi, Maki Hirata, K Takebayashi, N. Torigoe, M. Nagahara and Takeshige Otoi : Comparison of chemically mediated CRISPR/Cas9 gene-editing systems using different transfection mechanisms on the mutation of porcine embryos., The 17th Transgenic Technology Meeting (TT2022), Helsinki, Finland, Sep. 2022.
2.
Maki Hirata, Fuminori Tanihara, Nguyen Thi Nhien, Le Anh Quynh, Hirano Takayuki and Takeshige Otoi : Effects of CRISPR/Cas9-mediated gene targeting of porcine endogenous retrovirus on the developmental competence of porcine embryos, The 15th Transgenic Technology Meeting (TT2019), Kobe, Apr. 2019.
3.
Nguyen Thi Nhien, Fuminori Tanihara, Maki Hirata, Hirano Takayuki, Le Anh Quynh and Takeshige Otoi : Efficiency of gene editing by electroporation of Cas9 protein (GEEP) to generate GGTA1-modified pigs, The 15th Transgenic Technology Meeting (TT2019), Kobe, Apr. 2019.
4.
Le Anh Quynh, Maki Hirata, Fuminori Tanihara, Nguyen Thi Nhien, Hirano Takayuki and Takeshige Otoi : Effect of Cas9 protein levels on genomic mutations using the gene editing by electroporation of Cas9 protein (GEEP) system in putative zygotes, The 15th Transgenic Technology Meeting (TT2019), Kobe, Apr. 2019.
5.
Fuminori Tanihara, Maki Hirata, Nguyen Thi Nhien, Le Anh Quynh, Hirano Takayuki and Takeshige Otoi : Assessment of PDX-1-deficient pigs generated using the CRISPR/Cas9 system introduced into porcine zygotes via electroporation, The 15th Transgenic Technology Meeting (TT2019), Kobe, Apr. 2019.
6.
Katsutoshi Nishio, Fuminori Tanihara, TV Nguyen, Toshiki Kunihara and Takeshige Otoi : Effects of voltage strength on development and quality of electroporated porcine embryos., Transgenic Research, Vol.26, No.1, 29, Utah, USA, Oct. 2017.
7.
Fuminori Tanihara, LTK Do, TV Nguyen, Toshiki Kunihara, Katsutoshi Nishio, Tatsuya Takemoto and Takeshige Otoi : Generation of TP53-modified pigs by GEEP method: CRISPR/Cas9-mediated gene modification introduced into porcine zygotes by electroporation., Transgenic Research, Vol.26, No.1, 38, Utah, USA, Oct. 2017.
8.
Fuminori Tanihara and Takeshige Otoi : A simple-step generation of genetically modified pigs by genome editing by electroporation of Cas9 protein (GEEP) method., Forth World Congress of Reproductive Bioligy (WCRB2017), Okinawa, Sep. 2017.
(Summary)
Pigs are considered as one of the best animals for generating models of human diseases and for providing organs for xenotransplantation. Currently, genetically modified pigs for biomedical applications have been mainly generated using the somatic cell nuclear transfer (SCNT) technique after the generation of engineered donor somatic cells. However, this approach requires complex micromanipulation techniques and a long time for preparing enough number of reconstructed embryos to be transferred to the surrogate pig. Recently, we have established a simple method for CRISPR /Cas9 gene editing that involves the introduction of Cas9 protein and single-guide RNA into in vitro-fertilized zygotes by electroporation. The use of gene editing by electroporation of Cas9 protein (GEEP) resulted in highly efficient targeted gene disruption and was validated by the efficient production of Myostatin mutant pigs. While the genotype of genetically modified pig generated using the SCNT is homogeneous, the introduction of CRISPR/Cas9 system into embryos may induce mosaic genotype. However, GEEP method will be a powerful tool to achieve gene modification in the pig. We are now applying this method to generate various kinds of gene-modified pigs as an intractable disease model. We introduce recent studies and trials for generating genetically modified pigs by GEEP method.
9.
R Kuriwaki, Y Sato, S Hagino, M Shimazaki, R Sambuu, LTK Do, Fuminori Tanihara, M Takagi, M Taniguchi and Takeshige Otoi : Evaluation of the Leydig cell function on crossbreeding yak showing infertility., Forth World Congress of Reproductive Bioligy (WCRB2017), Okinawa, Sep. 2017.
Q Lin, Quynh Anh Le, Manita Wittayarat, Zhao Namula, Koki Takebayashi, Maki Hirata, Fuminori Tanihara, Kim Lanh Thi Do and Takeshige Otoi : Triple gene editing in porcine embryos using a combination of microinjection, electroporation, and transfection methods., 第7回ゲノム編集学会, Jun. 2022.
4.
Lin Qingyi, Chommanart Thongkittidilok, Maki Hirata, QUYNH ANH LE, K. Takebayashi, Fuminori Tanihara and Takeshige Otoi : Lipofection-mediated introduction of CRISPR/Cas9 system into porcine zygotes., 第114回日本繁殖生物学会, Sep. 2021.
5.
TAKEBAYASHI Kohki, Chommanart Thongkittidilok, 林 青怡, LE Anh Quynh, Maki Hirata, Fuminori Tanihara and Takeshige Otoi : ノックアウトブタ由来精子を用いた体外受精胚におけるゲノム編集効率の検討, 第114回日本繁殖生物学会, Sep. 2021.
6.
Fuminori Tanihara, Maki Hirata, Nguyen Thi Nhien, Quynh Anh Le, Takayuki Hirano and Takeshige Otoi : エレクトロポレーションを用いたCRISPR/Cas9システムのブタ体外受精卵への導入によるCD163遺伝子改変ブタの作製, 第162回日本獣医学会学術集会, Sep. 2019.
ブタは生理学的および解剖学的にヒトに近い優れたモデル動物である．これまで遺伝子改変ブタの作製は，遺伝子改変を行った体細胞を用いた核移植によるクローンブタの作製によりなされてきた(体細胞クローン法)．さらに近年Crispr/Cas9システムをはじめとするゲノム編集技術が体細胞の遺伝子改変に活用されている．体細胞クローン法ではbiallelic変異個体を一代で得られるという利点があるが，遺伝子改変細胞株の樹立からクローン胚作製と高度な手技および時間が必要であり，さらに正常産仔の作製効率も未だ低く，実施できる研究者・研究機関は限られている．この問題を解決するため，発表者らはブタ体外受精卵に対しエレクトロポレーションにより CRISPR/Cas9 システムを導入し，簡便かつ高効率に遺伝子改変を行うGEEP法(Genome editing by electroporation of Cas9 protein)を確立した．この手法によりすでに複数のゲノム編集ブタ作製に成功しているが，得られたゲノム編集ブタの解析から，biallelic変異個体の作製に成功しているもののwt配列の残るモザイク個体も存在することがわかり，GEEP法においてモザイク個体率を下げることが課題となっている．今回，ブタ体外受精卵および産仔について，ゲノム編集ブタ作製に使用するガイドRNAとエレクトロポレーションによる導入条件，およびモザイク率の関連を複数の標的遺伝子で解析した．その結果，適切なガイドRNAおよびエレクトロポレーション条件の選択により，biallelic変異個体を一代で得られる可能性が示唆された．
近年，再生医療技術およびゲノム編集技術の進展に伴い，ブタの組織・臓器を用いた異種移植が現実味を帯びてきた．一方，ブタゲノム中にはブタ内在性レトロウイルス(PERV)が多数内在されており，移植後におけるヒトへの感染が懸念されている．ゲノム編集技術により，ブタゲノム中に存在するPERV遺伝子をノックアウトすることにより，PERV感染の制御が可能と考えられるが，内在性レトロウイルスの初期発生との関連性，特に，初期胚発育能に及ぼす影響は未知数である．我々は，これまでにエレクトロポレーションによりCRISPR/Cas9システムをブタの1細胞期体外受精卵に導入することで，ゲノム編集を高効率に行い，かつ胚の生存性を維持できるGEEP法(genome editing by electroporation of Cas9 protein)を確立した．本研究ではブタの体外受精卵において，細胞内でPERV 自身を複製する際に重要なpol 遺伝子を標的にGEEP法によりノックアウトし，ゲノム編集効率と胚発育の関連性を調査した．pol遺伝子を標的とするguideRNA(gRNA)を5種類作製し，それぞれのgRNAをCas9タンパク質とともにブタの体外受精卵に導入した．その結果，一部のgRNA導入群において胚盤胞形成率が非常に低い値を示した．これらの群において4-8細胞期のゲノム編集効率を検討したところ，編集効率は他の群よりも高い値を示し，ゲノム編集効率と胚盤胞形成率に負の関連性が認められた．このことから，PERV遺伝子は胚発生と関連していることが示唆された．
Fuminori Tanihara, Maki Hirata, Nguyen Thi Nhien, Le Anh Quynh, Hirano Takayuki and Takeshige Otoi : CRISPR/Cas9システムによるブタ体外受精卵のINS遺伝子への点変異導入, 第7回日本先進医工学ブタ研究会, Oct. 2019.
3.
Takeshige Otoi, Maki Hirata, Nguyen Thi Nhien, Le Anh Quynh, Hirano Takayuki and Fuminori Tanihara : GEEP法を用いた遺伝子改変ブタの作製と遺伝子改変効率, 第7回日本先進医工学ブタ研究会, Oct. 2019.
4.
Maki Hirata, Fuminori Tanihara, Nguyen Thi Nhien, Le Anh Quynh, Hirano Takayuki and Takeshige Otoi : ブタ体外受精卵におけるCRISPR/Cas9システムを用いた複数遺伝子の同時改変, 第7回日本先進医工学ブタ研究会, Oct. 2019.
5.
Le Anh Quynh, Fuminori Tanihara, Maki Hirata, Nguyen Thi Nhien, Takayuki Hirano and Takeshige Otoi : Concentration of CRISPR/Cas9 components effects on genetic mosaicism of cytoplasmic microinjected porcine embryos, 第6回日本先進医工学ブタ研究会, Oct. 2018.
(Summary)
Cytoplasmic microinjection (CI) of the CRISPR/Cas9 system enabled the induction of site-specific mutations in porcine zygotes and resulting pigs, but mosaicism is a serious problem for genetically modified pigs because of the complicated analysis of phenotypes. In the present study, we examined the suitable timing and concentration of CRISPR/Cas9 for introduction into oocytes/zygotes by CI to reduce mosaicism in the resulting blastocysts. In the first experiment, we introduced 20 ng/μl of Cas9 protein and gRNA, targeting the α-1,3-galactosyltransferase (GalT) gene in oocytes before in vitro fertilization (IVF), in zygotes after IVF, or in oocytes and zygotes before and after IVF twice. CI treatment had no detrimental effects on the blastocyst formation rates, but the number of mutant blastocysts from zygotes injected after IVF tended to increase compared to that from oocytes injected before IVF (p < 0.1). In the second experiment, we injected 20 ng/μl or 100 ng/μl of Cas9 protein and gRNA into zygotes after IVF. Although no blastocysts that were injected with 20 ng/μl of Cas9 protein and gRNA carried a biallelic mutation, 50% of blastocysts that were injected with 100 ng/μl of Cas9 protein and gRNA carried a biallelic mutation. In conclusion, to achieve efficient gene editing in porcine zygotes by CI of Cas9 protein with gRNA, performing CI after IVF is better than performing CI before IVF. Furthermore, 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.
6.
Nguyen Thi Nhien, Fuminori Tanihara, Maki Hirata, Takayuki Hirano, Le Anh Quynh, 新居 雅宏 and Takeshige Otoi : Hypothermic storage of porcine zygotes in serum supplemented with chlorogenic acid, 第6回日本先進医工学ブタ研究会, Oct. 2018.
(Summary)
The current study was conducted to investigate the effects of 100% fetal 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 h. 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 h 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 h 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.
7.
Maki Hirata, Fuminori Tanihara, Takayuki Hirano, Nhien Thi Nguyen, Quynh Anh Le, 新居 雅宏 and Takeshige Otoi : ブタにおける受精前後でのゲノム編集が胚盤胞の変異導入効率に及ぼす影響, 第6回日本先進医工学ブタ研究会, Oct. 2018.
Fuminori Tanihara, Maki Hirata, Nguyen Thi Nhien, Le Anh Quynh, Takayuki Hirano, Tatsuya Takemoto, 中井 美智子, 淵本 大一郎 and Takeshige Otoi : ゲノム編集によるTP53遺伝子改変ブタの作製と表現型の解析, 第6回日本先進医工学ブタ研究会, Oct. 2018.
9.
Fuminori Tanihara, Tatsuya Takemoto, Maki Hirata, N Nguyen Thi, Toshiki Kunihara, R Nishinakamura and Takeshige Otoi : Modification of SALL1 gene via CRISPR/Cas9-mediated gene editing introduced into porcine zygotes by electroporation, KEY Forum: The 3rd International symposium on Stem Cell Traits and Developmental Systems, Jan. 2018.
10.
Fuminori Tanihara and Takeshige Otoi : ゲノム編集技術を用いたブタでの応用例, 第3回日本生物工学会西日本支部講演会「ゲノム編集—多様な生物種への応用研究」, Dec. 2016.
11.
Fuminori Tanihara, Tatsuya Takemoto, Michiko Nakai, Eri Kitagawa, Do Thi Kim Lanh, Akira Onishi, Shunichi Suzuki, Syoichiro Senbon, Daiichiro Fuchimoto and Takeshige Otoi : 新規ゲノム編集技術を用いたPDX-1遺伝子改変ブタの作製, 第4回 日本先進医工学ブタ研究会 要旨集, 21, Oct. 2016.
Report:
1.
Yoshihiko Ueno, Maiko Sawada, Tatsuro Aratake, Ichiro Hashimoto, Takeshi Nikawa, Toshiyuki Yasui, Kenichi Hamada, Yasuhiko Shirayama, Ken-ichi Yamada, Masahide Hojo, Takeshige Otoi, Ray S. Furuya and Yosuke Seki : 平成29年度 徳島大学総合教育センターアドミッション部門 報告書, 平成29年度 徳島大学総合教育センターアドミッション部門 報告書, Mar. 2018.
Development of animal model for kidney-specific spontaneous tumours (Project/Area Number: 22K19896 )
Production of virus infection-resistant animals by a simple genome editing procedure (Project/Area Number: 22H02499 )
Generation of African Swine Fever virus-resistant pigs by applying the concept of gene editing technology (Project/Area Number: 21KK0124 )
Production of immunodeficient micro-mini pig using genome editing technology. (Project/Area Number: 18K12062 )
Production of pigs for safe xenotransplantation by controlling infectious porcine endogenous retrovirus (PERV) (Project/Area Number: 17K19325 )
Production of genetically modified pigs by combined techniques of chromosome engineering and genome editing (Project/Area Number: 17H03938 )
Study of sperm maturation factors on Asian Elephant (Project/Area Number: 15H05259 )
Evaluation of minor histocompatibility antigens by kidney transplant model derived from somatic cell cloned animals (Project/Area Number: 26560212 )
Monitoring for multiple contamination of mycotoxins in feeding environment of cattle herds (Project/Area Number: 26450429 )
Elucidation of male sterility occurred in cattle-yak hybrid and production of calf from the hybrid male in Mongolia (Project/Area Number: 25304039 )
Study of non-coding RNA machinery complex-chromatoid body and dosage compensation. (Project/Area Number: 24570237 )
Monitoring of naturally feed contamination of zearalenone in cattle herds and its preventive measures (Project/Area Number: 23580441 )
Development of transgenic animals using a human artificial chromosome (HAC) vector (Project/Area Number: 22580320 )
Monitoring of natural zearalenone contamination in feeds of cattle herds, with special focus on the effect on reproductive efficacy. (Project/Area Number: 20580355 )
Monitoring of naturally contaminated zearalenone concentrations in feeding environment of cattles.especially the effects for reproductive efficacy (Project/Area Number: 18580318 )
Production of prion gene-knockdown cattle by RNA interference(RNAi)technology (Project/Area Number: 16310140 )
Production of human blood cohesion factor from bovine milk by nuclear transfer following the EGFP gene (Project/Area Number: 12556057 )
Embryo transfer/In vitro fertilization of bovine and buffalo (Project/Area Number: 10044211 )