Language：English   Publishing type：Research paper (scientific journal)   Publisher：MDPI AG  

The intergenerational effects from chronic low-dose exposure are matters of concern. It is thus important to elucidate the radiation-induced effects of germ cell maturation, fertilization and embryonic development. It is well known that DNA methylation levels in CpG sites in gametes are reprogrammed in stages during their maturity. Furthermore, the binding of Izumo on the surface of sperm and Juno on the surface of oocytes is essential for fertilization. Thus, there is a possibility that these genes are useful indicators to evaluate fertility in mice after irradiation exposure. Therefore, in this study, we analyzed global DNA methylation patterns in the testes and gene expression of Izumo1 and Izumo1r (Juno) in the gonads of mice after neonatal acute high-dose ionizing radiation (HDR) and chronic low-dose ionizing radiation (LDR). One-week-old male and female mice were irradiated with a total dose of 4 Gy, with acute HDR at 7 days at a dose rate of 30 Gy/h and LDR continuously at a dose rate of 6 mGy/h from 7 to 35 days. Their gonads were subsequently analyzed. The results of global DNA methylation patterns in the testes showed that methylation level increased with age in the control group, the LDR group maintained its DNA methylation level, and the HDR group showed decreased DNA methylation levels with age. In the control group, the gene expression level of Izumo1 in the testis did not show age-related changes, although there was high expression at 100 days of age. However, in the LDR group, the expression level recovered after the end of irradiation, while it remained low regardless of age in the HDR group. Conversely, gene expression of Izumo1r (Izumo1 receptor) in the ovary decreased with age in the control group. Although the gene expression of Izumo1r decreased with age in the LDR group, it remained low in the HDR group. Our results indicate that LDR can induce different DNA methylation patterns, and both high- and low-dose radiation before sexual maturity might affect gametogenesis and fertility.

DOI： 10.3390/biology10121270

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Wiley  

DOI： 10.1111/rda.13887

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Language：English   Publishing type：Research paper (scientific journal)  

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DOI： 10.1080/09553002.2020.1807643

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Authorship：Corresponding author   Publishing type：Research paper (scientific journal)  

© 2020 The aim of this study was to investigate the proliferation and apoptosis of male germ cells during the seasonal reproductive cycle of the large Japanese field mice (Apodemus speciosus). Male mice residing in their natural habitat were captured in Niigata, Japan. Testis sections were stained with haematoxylin and eosin, and mitotic male germ cells were identified using immunofluorescence staining for proliferating cell nuclear antigen (PCNA). Apoptosis was analysed using terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick end labelling (TUNEL) assay. The phases of spermatogenesis during the seasonal reproductive cycle were classified as active, transitional, and inactive based on the diameter of the seminiferous tubules. The number of PCNA-positive germ cells was less during the inactive than other phases. The percentage of TUNEL-positive germ cells per seminiferous tubule was greater during the inactive than active and transitional phases. Spermatogenesis during the seasonal reproductive cycle is controlled by proliferation and apoptosis in male germ cells. This species of undomesticated mice could be used as an animal model to study spermatogenesis as a valuable indicator of the effects of ecological and anthropogenic factors on animal reproduction.

DOI： 10.1016/j.anireprosci.2020.106288

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Authorship：Corresponding author   Publishing type：Part of collection (book)   Publisher：IntechOpen  

DOI： 10.5772/intechopen.84634

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© 2020 Japanese Society of Animal Science. This study was carried out to evaluate the nutritional effects of rice feeding and carnitine addition to a diet for broiler chicks. Thirty-six male 10-day-old broiler chicks were assigned to one of the following four treatment groups: corn-based diet (corn group), rice-based diet (rice group), and each diet with added carnitine (100 ppm). The experimental period was 2 weeks. Rice feeding resulted in significantly higher growth performance (body weight gain and feed efficiency) compared to corn feeding. Carnitine addition also resulted in higher growth performance. Breast muscle and thigh muscle weight (g) were significantly higher in broiler chicks fed rice and those fed diets with added carnitine. Liver mRNA expression of IGF-I was significantly higher in broiler chicks fed rice compared to those fed corn. There was no significant difference in mRNA expression of muscle atrogin-1 or liver CPT-I between broiler chicks fed rice and those fed corn, not between broilers chicks fed diets containing carnitine or not. Overall, these results show that rice feeding and carnitine addition improve the growth performance of broiler chicks by increasing mRNA expression of liver IGF-I. In addition, carnitine action is not affected by different cereals (corn and rice).

DOI： 10.1111/asj.13390

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Authorship：Corresponding author   Publishing type：Research paper (scientific journal)  

© 2019 Blackwell Verlag GmbH Seasonally, bred wild mice provide a unique bioresource, with high genetic diversity that differs from wild-derived mice and laboratory mice. This study aimed to establish an alternative superovulation method using wild large Japanese field mice (Apodemus speciosus) as the model species. Specifically, we investigated how the application of inhibin antiserum and equine chorionic gonadotropin (IASe) during both the reproductive and non-reproductive seasons impact the ovulation rate and competence of embryo development after in vitro fertilization (IVF) with fresh and cryopreserved sperm. When the wild mice were superovulated by injecting eCG followed by human chorionic gonadotropin (hCG), few oocytes were collected during the reproductive and non-reproductive seasons. In comparison, the number of ovulated oocytes was dramatically enhanced by the administration of IASe, followed by isolation of ovulated oocytes 24 hr after 30 IU hCG administration. The IVF oocytes that were in vitro cultured (IVC) with medium containing serum further developed to the 2- and/or 4-cell stage using both fresh and frozen-thawed sperm. In conclusion, we successfully established an alternative protocol for collecting ovulated oocytes from wild large Japanese field mice by administering IASe and hCG during both the reproductive and non-reproductive seasons. This study is the first to develop IVF–IVC wild large Japanese field mice beyond the 2- and/or 4-cell stage in vitro using fresh and cryopreserved sperm. This approach could be used in other species of wild or endangered mice to reduce the number of animals used for experiments, or in maintaining stocks of germ cells or embryos.

DOI： 10.1111/rda.13573

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© 2019 Saito, Kuroda, Suzuki, Kino, Sekine, Shinoda, Yamashiro, Fukuda, Kobayashi, Abe, Nishimura, Urushihara, Yoneyama, Fukumoto and Isogai. In March 2011, an accident at the Fukushima Daiichi Nuclear Power Plant led to major problems, including the release of radionuclides such as Cesium (Cs)-137 into the environment. Ever since this accident, Cs-137 in foods has become a serious problem. In this study, we determined the concentration of Cs-137 in the feces, urine, and ruminal contents of cattle and demonstrated the possibility of its elimination from the body by intestinal bacteria. The results revealed a high Cs-137 concentration in the feces; in fact, this concentration was higher than that in skeletal muscles and other samples from several animals. Furthermore, intestinal bacteria were able to trap Cs-137, showing an uptake ratio within the range of 38-81% in vitro. This uptake appeared to be mediated through the sodium-potassium (Na+-K+) ion pump in the bacterial cell membrane. This inference was drawn based on the fact that the uptake ratio of Cs-137 was decreased in media with high potassium concentration. In addition, it was demonstrated that intestinal bacteria hindered the trapping of Cs-137 by the animal. Cattle feces showed high concentration of Cs-137 and intestinal bacteria trapped Cs-137. This study is the first report showing that intestinal bacteria contribute to the elimination of Cs-137 from the body.

DOI： 10.3389/fvets.2019.00070

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© 2018, The Author(s). Several populations of wild Japanese macaques (Macaca fuscata) inhabit the area around Fukushima Daiichi Nuclear Power Plant (FNPP). To measure and control the size of these populations, macaques are captured annually. Between May 2013 and December 2014, we performed a haematological analysis of Japanese macaques captured within a 40-km radius of FNPP, the location of a nuclear disaster two years post-accident. The dose-rate of radiocaesium was estimated using the ERICA Tool. The median internal dose-rate was 7.6 μGy/day (ranging from 1.8 to 219 μGy/day) and the external dose-rate was 13.9 μGy/day (ranging from 6.7 to 35.1 μGy/day). We performed multiple regression analyses to estimate the dose-rate effects on haematological values in peripheral blood and bone marrow. The white blood cell and platelet counts showed an inverse correlation with the internal dose-rate in mature macaques. Furthermore, the myeloid cell, megakaryocyte, and haematopoietic cell counts were inversely correlated and the occupancy of adipose tissue was positively correlated with internal dose-rate in femoral bone marrow of mature macaques. These relationships suggest that persistent whole body exposure to low-dose-rate radiation affects haematopoiesis in Japanese macaques.

DOI： 10.1038/s41598-018-35104-0

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© 2017 Elsevier Ltd 90Sr specific activity in the teeth of young cattle that were abandoned in Kawauchi village and Okuma town located in the former evacuation areas of the Fukushima-Daiichi Nuclear Power Plant (FNPP) accident were measured. Additionally, specific activity in contaminated surface soils sampled from the same area was measured. (1) All cattle teeth examined were contaminated with 90Sr. The specific activity, however, varied depending on the developmental stage of the teeth during the FNPP accident; teeth that had started development before the accident exhibited comparatively lower values, while teeth developed mainly after the accident showed higher values. (2) Values of 90Sr-specific activity in teeth formed after the FNPP accident were higher than those of the bulk soil but similar to those in the exchangeable fraction (water and CH3COONH4 soluble fractions) of the soil. The findings suggest that 90Sr was incorporated into the teeth during the process of development, and that 90Sr in the soluble and/or leachable fractions of the soil might migrate into teeth and contribute to the amount of 90Sr in the teeth. Thus, the concentration of 90Sr in teeth formed after the FNPP accident might reflect the extent of 90Sr pollution in the environment.

DOI： 10.1016/j.jenvrad.2017.12.005

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Introduction: Impaired diabetic wound healing is an important issue in diabetic complications. The present study aims to evaluate the protective effect on glycemic control against impaired diabetic wound healing using a diabetic rat model. We investigated the wound healing process and effect on the impaired wound repair by glycemic control in the Spontaneously Diabetic Torii (SDT) fatty rat, which is a new animal model of obese type 2 diabetes and may be a good model for study impaired wound healing.Material and methods: Male SDT fatty rats at 15 weeks of age were administered orally with sodium glucose co-transporter (SGLT) 2 inhibitor for 3 weeks. Wounds were induced at 2 weeks after SGLT 2 inhibitor treatment, and the wound areas were periodically examined in morphological and histological analyses.Results: The SDT fatty rats showed a delayed wound healing as compared with the normal rats, but a glycemic control improved the impaired wound healing. In histological analysis in the skin of SDT fatty rats showed severe infiltration of inflammatory cell, hemorrhage and many bacterial masses in the remaining and slight fibrosis of crust on skin tissue. Thought that this results skin performance to be a delay of crust formation and regeneration of epithelium; however, these findings were ameliorated in the SGLT 2 inhibitor treated group.Conclusion: Glycemic control is effective for treatment in diabetic wounds and the SDT fatty rat may be useful to investigate pathophysiological changes in impaired diabetic wound healing.

DOI： 10.5455/medarh.2018.72.4-8

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Authorship：Corresponding author   Publishing type：Research paper (scientific journal)  

© 2017, Sociedade Brasileira de Reproducao Assistida. All rights reserved. Objective: Oocyte-sperm interaction is the essential step in fertilization. Juno, which has been known as Folate receptor 4, is the Izumo1 receptor expressed on the oocyte membrane. This study aims to investigate the location and expression of Juno in mice oocytes during maturation. Methods: To confirm the stage at which Juno expression begins in the mice oocytes and its location pattern, we performed immunostaining methods. Next, we evaluated Juno mRNA expression by a half quantitative RT-PCR. Juno knockdown oocytes were generated by microinjecting siRNA into the germinal vesicle (GV) stage oocytes, and analyzed the maturation rate. Results: Our results showed that Juno was expressed on the surface of the oocyte cytoplasmic membrane at the GV stage and it continues to be expressed at similar levels in the metaphase II (MII) stages of oocytes maturation. Interestingly, Juno is also expressed on the first polar body membrane at the MII stage. Fluorescence showing Juno expression was decreased in the oolemma of siRNA injected oocytes, but it was not completely disappearing in knock down oocytes. MII stage-rates of siRNA injected oocytes were not significantly different from sham controls. Conclusion: Juno was expressed in oocytes at the GV stage and it continues to be expressed at similar levels in later stages of oocytes maturation. Juno accumulation in oolemma during oocyte maturation is essential for fertilization, such as membrane recognition of both gametes.

DOI： 10.5935/1518-0557.20170065

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© 2017 by Radiation Research Society. The Fukushima Daiichi Nuclear Power Plant (FNPP) accident, the largest nuclear incident since the 1986 Chernobyl disaster, occurred when the plant was hit by a tsunami triggered by the Great East Japan Earthquake on March 11, 2011. The subsequent uncontrolled release of radioactive substances resulted in massive evacuations in a 20-km zone. To better understand the biological consequences of the FNPP accident, we have been measuring DNA damage levels in cattle in the evacuation zone. DNA damage was evaluated by assessing the levels of DNA double-strand breaks in peripheral blood lymphocytes by immunocytofluorescence-based quantification of γ-H2AX foci. A greater than two-fold increase in the fraction of damaged lymphocytes was observed in all animal cohorts within the evacuation zone, and the levels of DNA damage decreased slightly over the 700-day sample collection period. While the extent of damage appeared to be independent of the distance from the accident site and the estimated radiation dose from radiocesium, we observed age-dependent accumulation of DNA damage. Thus, this study, which was the first to evaluate the biological impact of the FNPP accident utilizing the γ-H2AX assays, indicated the causal relation between high levels of DNA damage in animals living in the evacuation zone and the FNPP accident.

DOI： 10.1667/RR14630.1

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Authorship：Corresponding author   Publishing type：Research paper (scientific journal)  

© 2017 by Radiation Research Society. In this study we analyzed the effect of chronic and low-dose-rate (LDR) radiation on spermatogenic cells of large Japanese field mice (Apodemus speciosus) after the Fukushima Daiichi Nuclear Power Plant (FNPP) accident. In March 2014, large Japanese field mice were collected from two sites located in, and one site adjacent to, the FNPP ex-evacuation zone: Tanashio, Murohara and Akogi, respectively. Testes from these animals were analyzed histologically. External dose rate from radiocesium (combined 134Cs and 137Cs) in these animals at the sampling sites exhibited 21 μGy/day in Tanashio, 304-365 μGy/day in Murohara and 407-447 μGy/day in Akogi. In the Akogi group, the numbers of spermatogenic cells and proliferating cell nuclear antigen (PCNA)-positive cells per seminiferous tubule were significantly higher compared to the Tanashio and Murohara groups, respectively. TUNEL-positive apoptotic cells tended to be detected at a lower level in the Murohara and Akogi groups compared to the Tanashio group. These results suggest that enhanced spermatogenesis occurred in large Japanese field mice living in and around the FNPP ex-evacuation zone. It remains to be elucidated whether this phenomenon, attributed to chronic exposure to LDR radiation, will benefit or adversely affect large Japanese field mice.

DOI： 10.1667/RR14234.1

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Authorship：Corresponding author   Publishing type：Research paper (scientific journal)  

© Cambridge University Press 2016. The objective of this study was to classify spindle and first polar body (PB1) chromosome images in ovulated mouse oocytes over time to predict the developmental competence of metaphase II (MII) oocytes. Oocytes were collected at 12, 15, 20, and 25 h after human chorionic gonadotropin (hCG) injection, and stained for spindle tubulin, chromosomes, and PB1 chromosomes. MII spindle morphology was classified as tapered type or barrel type and PB1 chromosomes were categorized as aggregated, separated, dot, or collapsed. To determine whether differences in spindle and PB1 images in MII oocytes are associated with fertilization success, we performed in vitro fertilization (IVF) at various times after hCG injection. Barrel-type spindles and aggregate-type PB1 were dominant at 12 h after hCG injection. Oocyte spindles collected 1 h after injection were tapered, and PB1 chromosomes were separated. At 20 and 25 h after treatment, spindle and PB1 images were classified as collapsed. The rate of development to 2-cell embryos after IVF did not differ between the 12 h and 15 h treatments; however, it was significantly lower for the 25 h treatment than for other treatments. The rates of development to blastocysts at 12, 15, 20, and 25 h after hCG injection were 61, 46, 42, and 9%, respectively. MII oocytes with barrel-type spindles and aggregate-type PB1 had high rates of fertilization and blastocyst development, and spindle and PB1 characteristics were correlated with the outcomes of IVF and embryo culture. These results suggested that images of spindles combined with those of PB1 chromosomes enable the prediction of oocytic and/or embryonic quality.

DOI： 10.1017/S096719941600023X

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© 2015 Japanese Society of Animal Science The 2011 earthquake severely damaged the Fukushima Daiichi Nuclear Power Plant (FNPP), resulting in the release of large quantities of radioactive material into the environment. The deposition of these radionuclides in rice straw as livestock feed led to the circulation of contaminated beef in the market. Based on the safety concern of the consumers, a reliable method for estimating concentrations of radioactive cesium in muscle tissue is needed. In this study, we analyzed the concentrations of radioactive cesium in the blood and skeletal muscle of 88 cattle, and detected a linear correlation between them. We then developed software that can be used to estimate radioactive cesium concentrations in muscle tissue from blood samples. Distribution of this software to the livestock production field would allow us to easily identify high-risk cattle, which would be beyond the safety regulation, before shipping out to the market. This software is planned to be released as freeware. This software would contribute to food safety, and aid the recovery of the livestock industry from the damage creacted by the 2011 Tohoku earthquake and tsunami.

DOI： 10.1111/asj.12490

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© 2016 Urushihara et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. The effect of the Fukushima Daiichi Nuclear Power Plant (FNPP) accident on humans and the environment is a global concern. We performed biochemical analyses of plasma from 49 Japanese Black cattle that were euthanized in the ex-evacuation zone set within a 20-km radius of FNPP. Among radionuclides attributable to the FNPP accident, germanium gamma-ray spectrometry detected photopeaks only from 134Cs and 137Cs (radiocesium) commonly in the organs and in soil examined. Radioactivity concentration of radiocesium was the highest in skeletal muscles. Assuming that the animal body was composed of only skeletal muscles, the median of internal dose rate from radiocesium was 12.5 μGy/day (ranging from 1.6 to 33.9 μGy/day). The median of external dose rate calculating from the place the cattle were caught was 18.8 μGy/day (6.0-133.4 μGy/day). The median of internal and external (total) dose rate of the individual cattle was 26.9 μGy/day (9.1-155.1 μGy/day). Plasma levels of malondialdehyde and superoxide dismutase activity were positively and glutathione peroxidase activity was negatively correlated with internal dose rate. Plasma alanine transaminase activity and percent activity of lactate dehydrogenase (LDH)-2, LDH-3 and LDH-4 were positively and LDH-1 was negatively correlated with both internal and total dose rate. These suggest that chronic exposure to low-dose rate of ionizing radiation induces slight stress resulting in modified plasma protein and enzyme levels.

DOI： 10.1371/journal.pone.0155069

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Here we determined the 90Sr concentrations in the teeth of cattle abandoned in the evacuation area of the Fukushima-Daiichi Nuclear Power Plant (FNPP) accident. 90Sr activity concentrations in the teeth varied from 6-831 mBq (g Ca)-1 and exhibited a positive relationship with the degree of radioactive contamination that the cattle experienced. Even within an individual animal, the specific activity of 90Sr (Bq (g Sr)-1) varied depending on the development stage of the teeth during the FNPP accident: teeth that were early in development exhibited high 90Sr specific activities, while teeth that were late in development exhibited low specific activities. These findings demonstrate that 90Sr is incorporated into the teeth during tooth development; thus, tooth 90Sr activity concentrations reflect environmental 90Sr levels during tooth formation. Assessment of 90Sr in teeth could provide useful information about internal exposure to 90Sr radiation and allow for the measurement of time-course changes in the degree of environmental 90Sr pollution.

DOI： 10.1038/srep24077

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© 2015 The Author. It is not an exaggeration to say that, without nuclear accidents or the analysis of radiation therapy, there is no way in which we are able to quantify radiation effects on humans. Therefore, the livestock abandoned in the ex-evacuation zone and euthanized due to the Fukushima Daiichi Nuclear Power Plant (FNPP) accident are extremely valuable for analyzing the environmental pollution, its biodistribution, the metabolism of radionuclides, dose evaluation and the influence of internal exposure. We, therefore, sought to establish an archive system and to open it to researchers for increasing our understanding of radiation biology and improving protection against radiation. The sample bank of animals affected by the FNPP accident consists of frozen tissue samples, formalin-fixed paraffin-embedded specimens, dose of radionuclides deposited, etc., with individual sampling data.

DOI： 10.1093/jrr/rrv069

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© 2016 The Author. We aimed to investigate the effect of chronic radiation exposure associated with the Fukushima Daiichi Nuclear Power Plant (FNPP) accident on the testes of boar and inobuta (a hybrid of Sus scrofa and Sus scrofa domestica). This study examined the contamination levels of radioactive caesium (Cs), especially 134Cs and 137Cs, in the testis of both boar and inobuta during 2012, after the Fukushima accident. Morphological analysis and electron-probe X-ray microanalysis (EPMA) were also undertaken on the testes. The 134Cs and 137Cs levels were 6430 ± 23 and 6820 ± 32 Bq/kg in the boar testes, and 755 ± 13 and 747 ± 17 Bq/kg in the inobuta testes, respectively. The internal and external exposure of total 134Cs and 137Cs in the boar testes were 47.1 mGy and 176.2 mGy, respectively, whereas in the inobuta testes, these levels were 6.09 mGy and 59.8 mGy, respectively. Defective spermatogenesis was not detected by the histochemical analysis of radiation-exposed testes for either animal. In neither animal were Cs molecules detected, using EPMA. In conclusion, we showed that adverse radiation-induced effects were not detected in the examined boar and inobuta testes following the chronic radiation exposure associated with the FNPP accident.

DOI： 10.1093/jrr/rrv070

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This study aimed to determine the seasonality of reproduction throughout the year in Japanese wood mice (Apodemus speciosus). The effect of seasonal changes on testicular morphology and the periodic expression of circadian clock genes in the hypothalamus and testes of male individuals was evaluated. We also examined the morphology of the testes and caudae epididymides of male mice. In addition, RT-PCR analysis was carried out with mRNA extracted from the hypothalamus and testes to evaluate the expression of the circadian clock genes Clock, Bmal1, Per1, and Cry1. The complete induction of testicular activity was detected from February to April and from August to October, with testes weight increasing with the completion of spermatogenesis (reproductive season). From May to early June and from November to early January, testicular weight declined, the seminiferous tubules reduced in size, spermatogenesis was arrested, and sperm were not produced (non-reproductive season). From mid- June to July and mid-January, the re-induction of testicular activity for spermatogenesis was observed in the seminiferous tubules (transitional season). Out of the four examined genes, Cry1 had the highest expression level in both the hypothalamus and testes throughout the year, followed by Bmal1, Per1, and Clock. The expression of Bmal1 was significantly lower in the hypothalamus and testes during the transitional season compared to the reproductive and non-reproductive seasons. Cry1 transcript levels were also significantly lower in the hypothalamus and testes during the transitional season compared to the reproductive season. In conclusion, the results indicating changes in testicular morphology revealed annual reproductive, non-reproductive, and transmission periods in Japanese wood mice. When an increase in testicular activity was observed indicating the onset of the reproductive season, the mean day length was approximately 11–13 h. The expression of the circadian clock genes Bmal1 and Cry1 in the hypothalamus and testes during the reproductive season was significantly higher than that of the same genes during the transitional season. Consequently, completion of spermatogenesis occurred in the seminiferous tubules of Japanese wood mice testes during the reproductive period.

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© 2015 by Bin Tong. Marbling characterized by the amount and distribution of intramuscular fat (IMF) in longissimus muscle (LM) and measured as beef marbling score (BMS), is an economically important trait of beef cattle in Japan. The myosin binding protein C, slow type (MYBPC1) gene, involved in efficient energy metabolism and homeostasis during muscle contraction in slow skeletal muscle, has been previously shown to be expressed at different levels in the LM between high-marbled and lowmarbled steer groups using differential-display PCR (ddPCR). In this study, we found that IMF area (%) in the sacrococcygeus muscle (SM) was positively correlated with BMS in the LM in Japanese Black steers (n=22, r=0.941, P<0.0001). This suggested that the IMF area (%) in the SM tends to equate marbling level in the LM. Furthermore, we showed that the MYBPC1 expression level in SM was significantly higher in the Japanese Black steers (n=5) with high BMS than in the Japanese Black steers (n=5) with low BMS (P<0.001). Moreover, correlation analyses showed that the expression level of the MYBPC1 gene was positively correlated with IMF area (%) (n=22, r=0.858, P<0.0001) and BMS (n=22, r=0.769, P<0.0001), indicating the association of MYBPC1 expression level with marbling trait. These results, together with the previous ddPCR result, suggested that high level of MYBPC1 expression may be associated with the development of marbling in Japanese Black beef cattle.

DOI： 10.1515/aoas-2015-0014

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Publishing type：Research paper (scientific journal)   Publisher：Sciencedomain International  

DOI： 10.9734/bjmmr/2015/17988

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© 2014 Japanese Society of Animal Science. The accident at the Fukushima Daiichi Nuclear Power Plant (FNPP) released a large amount of radioactive substances into the environment. Furthermore, beef contaminated with radioactive cesium above the 500 Bq/kg safety standard was circulated in the food chain in 2011. Japanese consumers remain concerned about the safety of radioactively contaminated food. In our previous study, we detected a linear correlation between radioactive cesium (137Cs) activity in blood and muscle around 500 to 2500 Bq/kg in cattle. However, it was unclear whether the correlation was maintained at a lower radioactivity close to the current safety standard of 100 Bq/kg. In this study, we evaluated 17 cattle in the FNPP evacuation zone that had a 137Cs blood level less than 10 Bq/kg. The results showed a linear correlation between blood 137Cs and muscle 137Cs (Y=28.0X, R2=0.590) at low radioactivity concentration, indicating that cesium radioactivity in the muscle can be estimated from blood radioactivity. This technique would be useful in detecting high-risk cattle before they enter the market, and will contribute to food safety.

DOI： 10.1111/asj.12301

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Copyright © 2014 by the American Association for Laboratory Animal Science. Many attempts have been made to culture germ cells in vitro by mimicking their development in vivo. The objective of this study was to establish an alternative method of xenotransplantation by developing a new approach for the rapid induction of spermatogenesis by using the chorioallantoic membrane of developing chicken embryos. Fertilized chicken eggs were incubated for 7 d, after which a small window was cut into the shell of the egg. We then transplanted testes from 7- to 8-d-old B6D2F1 mice onto the vessels of the chorioallantoic membrane and incubated them at 35.0 °C for 14 d or 37.5 °C for 12 d. After this in ovo CAM (iCAM) culture, the survival rates of the eggs and testes were assessed histologically and immunohistologically. The transplanted testes in the chicken embryos that survived were supported by the CAM, with an associated chronic vascularization response. The testes cultured at 35.0 °C had lower rates of generation and higher rates of death than did those cultured at 37.5 °C. Histologic examination of the testes cultured at 37.5 °C revealed the presence of spermatogonia and primary spermatocyte-like germ cells in the seminiferous tubules. The number of cells positive for synaptonemal complex protein 3 in the seminiferous tubules was significantly higher than that in the noniCAM-cultured testes from control mice. These results suggest that iCAM culturing of neonatal donor testis induces androcyte development. This method could be the foundation for a method that would enable in vitro spermatogenesis.

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DOI： 10.1111/age.12172

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Language：English   Publishing type：Research paper (other academic)  

DOI： 10.5772/56998

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Publishing type：Research paper (scientific journal)   Publisher：Japanese Society of Animal Breeding and Genetics  

DOI： 10.5924/abgri.42.3

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Contents: This study aimed to develop a polymerase chain reaction (PCR)-based sexing and effective semen collection methods for black-headed and straw-necked ibis species. However, most birds are not sexually dimorphic, that is, the sexes appear similar. Therefore, the gender should be determined before semen collection. DNA was extracted from the blood samples of 11 black-headed and 4 straw-necked ibis. The sex was determined after PCR amplification of the EE0.6 region of W-chromosome. The PCR products were separated using gel electrophoresis. A single band indicated the presence of the EE0.6 region and that the individual was a female, while no band indicated that the individual was a male. Further, the single bands from seven specimens were amplified. Semen collection was performed by massage or a combination of massage with electro-ejaculation and was attempted during all four seasons. The semen was successfully collected in March from male straw-necked ibis using the massage method. Limited motility, viability and concentration of straw-necked ibis sperm were observed. The sperm length was 180 μm and that of the nucleus was 30 μm with acrosome located at the tip of the nucleus. Thus, the PCR-based sexing proved to be an accurate molecular sexing method for black-headed and straw-necked ibis. Furthermore, we successfully collected semen and observed the stained sperm nucleus and acrosome of the straw-necked ibis sperm. We propose that the use of this PCR methodology can be applied as a routine method for sex determination and semen collection in ibis species for future ecological research. However, considering our limited success, further studies on semen collection method are required. © 2013 Blackwell Verlag GmbH.

DOI： 10.1111/rda.12200

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Authorship：Lead author,　Corresponding author   Publishing type：Research paper (scientific journal)  

We aimed to investigate the effect of chronic radiation exposure associated with the Fukushima Daiichi Nuclear Plant accident on the testis from 2 bulls. Estimated dose of internal exposure in one bull was 0.7-1.2â.mGy (134 Cs) and 0.4-0.6â.mGy (137 Cs) and external exposure was 2.0â.mGy (134 Cs) and 0.8â.mGy (137 Cs) (196 days). Internal dose in the other was 3.2-6.1â.mGy (134 Cs) and 1.8-3.4â.mGy (137 Cs) and external dose was 1.3â.mGy (134 Cs) and 0.6â.mGy (137 Cs) (315 days). Sperm morphology and spermatogenesis were within normal ranges. 134, 137 Cs radioactivity was detected but Cs was not detectable in the testis by electron probe microanalysis. Thus, adverse radiation-induced effects were not observed in bull testes following chronic exposure to the above levels of radiation for up to 10 months. Since we could analyse a limited number of testes, further investigation on the effects of ionizing radiation on spermatogenesis should be extended to more animals.

DOI： 10.1038/srep02850

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The Fukushima Daiichi Nuclear Power Plant (FNPP) accident released large amounts of radioactive substances into the environment. In order to provide basic information for biokinetics of radionuclides and for dose assessment of internal exposure brought by the FNPP accident, we determined the activity concentration of radionuclides in the organs of 79 cattle within a 20-km radius around the FNPP. In all the specimens examined, deposition of Cesium-134 (134Cs, half-life: 2.065 y) and 137Cs (30.07 y) was observed. Furthermore, organ-specific deposition of radionuclides with relatively short half-lives was detected, such as silver-110m (110mAg, 249.8 d) in the liver and tellurium-129m (129mTe, 33.6 d) in the kidney. Regression analysis showed a linear correlation between the radiocesium activity concentration in whole peripheral blood (PB) and that in each organ. The resulting slopes were organ dependent with the maximum value of 21.3 being obtained for skeletal muscles (R2 = 0.83, standard error (SE) = 0.76). Thus, the activity concentration of 134 Cs and 137Cs in an organ can be estimated from that in PB. The level of radioactive cesium in the organs of fetus and infants were 1.19-fold (R2 = 0.62, SE = 0.12), and 1.51-fold (R2 = 0.70, SE = 0.09) higher than that of the corresponding maternal organ, respectively. Furthermore, radiocesium activity concentration in organs was found to be dependent on the feeding conditions and the geographic location of the cattle. This study is the first to reveal the detailed systemic distribution of radionuclides in cattle attributed to the FNPP accident. © 2013 Fukuda et al.

DOI： 10.1371/journal.pone.0054312

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Publishing type：Research paper (scientific journal)  

We have developed a unique method for mouse transgenesis. The transposase-enhanced pronuclear microinjection (PNI) technique described herein uses the hyperactive piggyBac transposase to insert a large transgene into the mouse genome. This procedure increased transgene integration efficiency by fivefold compared with conventional PNI or intracytoplasmic sperm injection-mediated transgenesis. Our data indicate that the transposase-enhanced PNI technique additionally requires fewer embryos to be microinjected than traditional methods to obtain transgenic animals. This transposase-mediated approach is also very efficient for single-cell embryo cytoplasmic injections, offering an easy-to-implement transgenesis method to the scientific community.

DOI： 10.1073/pnas.1216473109

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Marbling, defined by the am eract with muscle protein including myosin, actin and titin to regulate muscle contraction and to express in slow-twitch oxidative fiber that is observed in high-marbled muscle and involved in development and maintenance of the fiber, possesses expression differences in musculus longissimus muscle between low-marbled Holstein and high-marbled Japanese Black steer groups. In the present study, researchers found that a marker (DIK4787) close to the MYBPC1 was polymorphic between low-marbled Holstein and high-marbled Japanese Black steer groups and exhibited significantly different allelic distribution between Japanese Black sires with extremely high predicted breeding value for marbling and with extremely low one. Further, researchers detected 3 Single Nucleotide Polymorphisms (SNPs) in the promoter region of the MYBPC1 gene between low-marbled Holstein and high-marbled Japanese Black steer groups. A SNP in the upstream of the promoter of the MYBPC1, referred to as g.70014208G>A, exhibited significantly different allelic distribution between animals with extremely high predicted breeding value for marbling and with extremely low one (p = 0.046). The other 2 SNPs seemed not to segregate in Japanese Black beef cattle. The findings suggest that the g.70014208G>A SNP may be related to changes in MYBPC1 gene expression and/or marbling. The g.70014208G>A SNP marker may be useful for effective marker-assisted selection to increase the levels of marbling. © Medwell Journals, 2012.

DOI： 10.3923/javaa.2012.4446.4450

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Growth performance as well as marbling is the main breeding objectives in Japanese Black cattle, the major beef breed in Japan. Researchers have recently reported the existence of Single Nucleotide Polymorphisms (SNPs), g.29850738G>A and g.3834941C>T in promoter regions of the Myosin Heavy chain 1 (MYHl) and the Triadin (TRDN) genes, respectively, in Japanese Black beef cattle population. The MYHl encodes an isoform of myosin heavy chain in type I fiber of skeletal muscle and the TRDN is known to be involved in muscle contraction and the 2 genes have been earlier shown to be located within genomic regions of quantitative trait loci for growth-related carcass traits. Thus, the MYHl and TRDN genes were considered as positional functional candidates for the gene responsible for growth performance. In this study, we analyzed the possible effects of the 2 SNPs on the growth-related carcass traits: Carcass Weight (CWT), Rib Thickness (RT), Rib Eye Area (REA) and Subcutaneous Fat Thickness (SFT) in Japanese Black beef cattle. The g.29850738G>A SNP in the MYHl, exhibited significantly different allelic distribution between Japanese Black sires with extremely high predicted breeding value and the sires with extremely low one for CWT and RT but not for the others. The allelic distribution of the g.3834941C>T SNP in the TRDN was indistinguishable between the sires with extremely high one and with extremely low one for all the traits. These findings suggest possible effect of the g.29850738G>A SNP on the growth-related trait in Japanese Black beef cattle. The MYHl SNP may be useful for effective marker-assisted selection to increase the beef productivity in Japanese Black beef cattle. © Medwell Journals, 2012.

DOI： 10.3923/javaa.2012.4540.4543

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Marbling, defined by the amount and distribution of intramuscular fat is an economically important trait of beef cattle in Japan. Previously, researchers have found that the Myosin Heavy Chain 1 (MYH1) gene coding for an isoform of myosin heavy chain a major contractile protein which converts chemical energy into mechanical energy in muscle which is expressed in slow-twitch oxidative fiber and involved in development of the fiber and Insulin Receptor Substrate 1 (IRS1) gene encoding a signaling adaptor protein which is phosphorylated by insulin receptor and associated with insulin resistance and birth weight, possess expression differences in musculus longissimus muscle between low-marbled and high-marbled steer groups. In the present study, researchers detected Single Nucleotide Polymorphisms (SNPs), g.29850738G>A and g.l 20947716T>C in the promoter regions of the MYH1 and IRS1 genes, respectively between the 2 steer groups. However, the allelic distributions of the 2 SNPs were indistinguishable between Japanese Black sires with extremely high predicted breeding value for marbling and with extremely low one. An unknown SNP in the distal region upstream of ~4kb proximal promoter regions of the MYH1 and IRS 1 genes might be related to changes in gene expression and/or marbling. © Medwell Journals, 2012.

DOI： 10.3923/javaa.2012.4441.4445

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Growth performance as well as marbling is the main breeding objectives in Japanese Black cattle, the major beef breed in Japan. Researchers have recently reported that existence of Single Nucleotide Polymorphisms (SNPs), g.63629097G>C, g.120947716T>C and g.38437771C>T in the promoter regions of the septin 7 (CDC10), the insulin receptor substrate 1 (IRS1) and the mitofusin 2 (MFN2) genes, respectively in Japanese Black beef cattle population. The CDC10 is known to be involved in cellular proliferation and the IRS1 is reported to be associated with insulin resistance and birth weight. The 2 genes have been earlier shown to be located within genomic regions of quantitative trait loci for growth-related traits. Thus, the CDC10 and IRS1 genes were considered as positional functional candidates for the gene responsible for growth performance. In addition, the MFN2 is known to play a role in energy balance through mitochondrial fusion, so the MFN2 gene was considered as functional candidate. In this study, researchers analyzed the possible effects of the 3 SNPs on the growth-related carcass traits: Carcass Weight (CWT), Rib Thickness (RT), Rib Eye Area (REA) and Subcutaneous Fat Thickness (SFT) in Japanese Black beef cattle. The g.63629097G>C and g. 120947716T>C SNPs in the CDC10 and IRS1, respectively, exhibited significantly different allelic distribution between Japanese Black sires with extremely high predicted breeding value and the sires with extremely low one for CWT and RT but not for the others. The g.38437771C>T SNP in the MFN2 had significantly different allelic distribution for RT. These findings suggest possible effects of the g.63629097G>C, g.l 20947716T>C and g.38437771C>T SNP on the growth-related trait in Japanese Black beef cattle. The SNPs in the CDC10, IRS1 and MFN2 genes may be useful for effective marker-assisted selection to increase the beef productivity in Japanese Black beef cattle. © Medwell Journals, 2012.

DOI： 10.3923/javaa.2012.4536.4539

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The objective of this study was to examine the effect of different concentrations of trehalose and raffinose in Tris-citric acid-glucose (TCG) egg yolk extender for the sperm freezability of Red Maasai sheep, another African breed- the Dorper and their crosses. The results showed that Red Maasai, Dorper and their crosses sperm could be cryopreserved by extending with 0.075 to 0.01 M trehalose or raffinose-TCG egg yolk extender. We conclude that the cryopreservation of currently under threat Red Maasai sheep due to uncontrolled exotic breeds sperm makes it possible to establish a gene bank for the storage of animal genetic resources for endangered species, under the management of the International Livestock Research Institute for future genetic options in an uncertain world. © 2011 Academic Journals.

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Efficient integration of functional genes is an essential prerequisite for successful gene delivery such as cell transfection, animal transgenesis, and gene therapy. Gene delivery strategies based on viral vectors are currently the most efficient. However, limited cargo capacity, host immune response, and the risk of insertional mutagenesis are limiting factors and of concern. Recently, several groups have used transposon-based approaches to deliver genes to a variety of cells. The piggyBac (pB) transposase in particular has been shown to be well suited for cell transfection and gene therapy approaches because of its flexibility for molecular modification, large cargo capacity, and high transposition activity. However, safety considerations regarding transposase gene insertions into host genomes have rarely been addressed. Here we report our results on engineering helper-independent pB plasmids. The single-plasmid gene delivery system carries both the piggyBac transposase (pBt) expression cassette as well as the transposon cargo flankedby terminal repeat element sequences. Improvements to the helper-independent structure were achieved by developing new plasmids in which the pBt gene is rendered inactive after excision of the transposon from the plasmid. As a consequence, potentially negative effects that may develop by the persistence of an active pBt gene posttransposition are eliminated. The results presented herein demonstrate that our helper-independent plasmids represent an important step in the development of safe and efficient gene delivery methods that should prove valuable in gene therapy and transgenic approaches.

DOI： 10.1073/pnas.1003674107

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We evaluated the cryosurvival of rat epididymal sperm preserved in raffinose-modified Krebs-Ringer bicarbonate-egg yolk extender supplemented with various energy-yielding substrates (glucose, pyruvate, lactate, and ATP) and assessed the effect on sperm oxygen consumption. The incubation of sperm at 37°C for 10 min in lactate-free extender decreased sperm motility and oxygen consumption before and after thawing compared with those of sperm in glucose- and pyruvate-free mediums. We then focused on the effect of supplementing the extender with lactate (0, 10.79, 21.58, 32.37, and 43.16 mM) and found that sperm frozen and thawed in extender supplemented with 32.37 mM lactate exhibited the highest motility. When we supplemented extender containing 32.37 mM lactate with ATP (0, 0.92, 1.85, 3.70, and 5.55 mM), sperm frozen and thawed in the extender supplemented with 1.85 mM ATP exhibited considerably higher motility and viability than those of sperm frozen and thawed in ATP-free extender. These results provide the first evidence that supplementation of the raffinose-modified Krebs-Ringer bicarbonate-egg yolk extender with 32.37 mM lactate and 1.85 mM ATP increases of number of motile sperm before freezing and enhances the cryosurvival of rat sperm. These supplements to the extender may enhance sperm cryosurvival by improving the metabolic capacity of sperm before freezing. Copyright 2010 by the American Association for Laboratory Animal Science.

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We studied the effects of ATP, ionomycin, and dibutyryl cAMP (dbcAMP) on the motility, freezability, and oxygen consumption of rat epididymal sperm. In vitro fertilization and intrauterine insemination were performed by using frozen-thawed rat sperm. Frozen-thawed sperm diluted in raffinose-modified Krebs-Ringer bicarbonate solution-egg yolk extender containing 1.85 mM ATP and 100 μM dbcAMP exhibited considerably higher motility and viability than sperm diluted in dbcAMP-free extender. Addition of ionomycin and dbcAMP to ATP-containing extenders did not alter the oxygen consumption rate of sperm, suggesting that extracellular ionomycin and dbcAMP are not involved in the mobilization of mitochondrial energy substrates in sperm. Further, high rates of pronucleus formation and progression to the blastocyst stage were observed in embryos produced by the fertilization of oocytes with fresh sperm in an in vitro fertilization medium supplemented with ATP and dbcAMP. Oocytes were not penetrated by frozen-thawed sperm when cocultured with cumulus-oocyte complexes in a medium without ATP and dbcAMP. In contrast, cryopreserved sperm penetrated oocytes when the gametes were cultured in an ATP- and dbcAMP-containing medium, and the resultant embryos formed blastocysts. Our results show that the dilution of rat sperm in raffinose-modified Krebs-Ringer bicarbonate solution-egg yolk extender supplemented with ATP and dbcAMP prior to sperm cryopreservation enhances the freezability of the cryopreserved sperm. Furthermore, the in vitro fertilization medium we developed effectively supports the production of embryos from both fresh and cryopreserved rat sperm. Copyright 2010 by the American Association for Laboratory Animal Science.

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Interspecies somatic cell nucleus transfer (iSCNT) could be a useful bioassay system for assessing the ability of mammalian somatic cells to develop into embryos. To examine this possibility, we performed canine iSCNT using porcine oocytes, allowed to mature in vitro, as recipients. Canine fibroblasts from the tail tips and dewclaws of a female poodle (Fp) and a male poodle (Mp) were used as donors. We demonstrated that the use of porcine oocytes induced blastocyst formation in the iSCNT embryos cultured in porcine zygote medium-3. In Fp and Mp, the rate of blastocyst formation from cleaved embryos (Fp: 6.3% vs. 22.4%; and Mp: 26.1% vs. 52.4%) and the number of cells at the blastocyst stage (Fp: 30.7 vs. 60.0; and Mp: 27.2 vs. 40.1) were higher in the embryos derived from dewclaw cells than in those derived from tail-tip cells (P < 0.05). The use of donor cells of any type in later passages decreased the rate of blastocyst formation. Treatment with trichostatin-A did not improve the rate of blastocyst formation from cleaved dewclaw cell-derived embryos but did so in the embryos derived from the tail-tip cells of Fp. Only blastocysts derived from dewclaw cells of Mp developed outgrowths. However, outgrowth formation was retrieved in the embryos derived from dewclaw cells of Fp by aggregation at the 4-cell stage. We inferred that iSCNT performed using porcine oocytes as recipients could represent a novel bioassay system for evaluating the developmental competence of canine somatic cells. © 2009 Elsevier Inc.

DOI： 10.1016/j.theriogenology.2009.04.011

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Problem statement: Two experiments were conducted to determine the effect of the prostatic fluid from the first and third fractions of the ejaculates on the freezability of poodle dog sperm. Approach: The ejaculates from 2 trained poodle dogs were collected. Effect of prostatic fluid from the first and third fractions of ejaculates on cryosurvival of dog sperm was examined. SDS-PAGE analyses were performed to verify whether there were any modifications in the protein patterns of the first and third fractions. Results: When second fractions of Dog A sperm were combined with the prostatic fluid from the first and third fractions of the ejaculates, sperm motility index was decreased compared to second fractions were collected alone. The post-thaw motility of Dog A sperm was significantly reduced when prostatic fluid from the first and third fractions of the ejaculates was added. The motility of frozen-thawed Dog B sperm did not appear to be altered when it was diluted with either of the prostatic fluids. The major bands present in both prostatic fluids corresponded to proteins with molecular masses of 30 and 75 kDa. The amount of proteins in the prostatic fluid from the third fraction was twice-three times as much protein present compared to the first fraction. Conclusion: Collection of the sperm-rich fraction along with either the prostatic fluid from the 1st and 3rd fractions of the ejaculates during sperm processing has varying detrimental effects on the motility characteristics of frozen-thawed sperm. © 2009 Science Publications.

DOI： 10.3844/ajavsp.2009.14.20

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Progesterone is an important reproductive hormone and measurement of its level by repeated blood samplings is beneficial to monitoring of estrus cycle. However, since toy poodles have a small body size and thin-walled blood vessels, repeated blood samplings cause stress and affect their preparation for mating or artificial insemination (AI). Therefore, a noninvasive method for monitoring progesterone concentration should be developed. Here, we show that time-resolved fluoroimmunoassay (TF-RIA) is a useful noninvasive method for determining the progesterone concentration in serum and fecal samples obtained from toy poodles. Present results demonstrate that progesterone concentrations in the fecal correlated with the serum collected in same time and the sequential changes in progesterone concentrations in the feces are paralleled in the serum. Therefore, this technique may be suitable for monitoring the estrus cycle in toy poodles. © 2008 Science Publications.

DOI： 10.3844/ajavsp.2008.43.46

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The objective of this study was to develop an ideal freezing extender and method for rat epididymal sperm cryopreservation. Epididymal sperm collected from 30 Wistar males was frozen, and experiments were conducted to study its post-thaw characteristics when freezing with raffinose-free buffer or various concentrations of raffinose and egg yolk dissolved in distilled and deionised water, PBS, or modified Krebs-Ringer bicarbonate (mKRB)-based extender. Different concentrations of glycerol, Equex STM, or sodium dodecyl sulfate (SDS) dissolved in either PBS or mKRB containing egg yolk were also tested. Based on the data from these experiments, further experiments tested how different sugars such as raffinose, trehalose, lactose, fructose, and glucose dissolved in mKRB with Equex STM, SDS and egg yolk supplementation affected the post-thaw characteristics of cryopreserved sperm. Cryosurvival of frozen-thawed sperm were judged by microscopic assessment of the sperm motility index (SMI), and acrosome integrity was measured using FITC-PNA staining. Thawed sperm were subjected to 3 h of a thermal resistance test. Beneficial effects on the post-thaw survival of sperm were obtained when 0.1 M raffinose in mKRB was used with 0.75% Equex STM, 0.05% SDS, and 20% egg yolk. Sperm cryopreserved with this treatment exhibited a higher motility index and maintained greater SMI and acrosome integrity throughout incubation when compared to sperm frozen in various concentrations of other cryoprotectants and trehalose, lactose, fructose, glucose. In conclusion, cryopreservation in an extender solution of raffinose dissolved in mKRB containing Equex STM, SDS and egg yolk greatly enhances the freezability of rat epididymal sperm. © 2007 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.cryobiol.2007.08.011

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In an attempt to develop a suitable freezing method for Poodle dog sperm, an experiment was conducted to investigate semen collection methods of digital stimulation and an artificial vagina (AV), using Tris and trehalose-egg yolk extender, on the characteristics and cryopreservation of sperm. Two dogs (dogs A and B) were subjected to semen collection by digital stimulation and AV. The volume, sperm concentration, sperm motility index (SMI) and acrosome status of ejaculates were determined immediately after collection. The remainder was frozen as pellets in Tris and trehalose-egg yolk extender. Sperm motility index was evaluated after thawing and during a thermal resistance test, and acrosome integrity was also assessed. No significant differences regarding sperm concentrations, SMI and acrosome integrity were observed between semen collected by AV and digital stimulation. However, when dog sperm were collected by an AV and frozen in trehalose-egg yolk extender, the motility index of frozen-thawed sperm was significantly improved compared to sperm frozen in Tris-egg yolk extender which were collected by digital stimulation. In conclusion, semen collected by an AV and frozen in trehalose-egg yolk extender was effective in enhancing the freezability of Poodle dog sperm. © 2007 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.anireprosci.2007.02.026

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Background: The objective of this study was to investigate whether the steroid hormone(s) secreted from cumulus-oocyte complexes (COCs) is a prerequisite for bovine oocyte maturation and cumulus expansion using aminoglutethimide (AGT), a P450 cholesterol side-chain cleavage inhibitor. Methods: In experiment 1, COCs were cultured in maturation medium with various concentrations of AGT for 22 h to determine the effective concentration of AGT to inhibit steroid hormone secretion, meiotic maturation and cumulus expansion. In experiment 2, COCs were cultured in conditioned medium (CM) and TCM-199 medium with or without 10 mM AGT to check whether steroid hormones secreted from COCs were responsible for oocyte maturation and cumulus expansion. Experiments 3 and 4 were carried out to determine whether exogenous progesterone or estradiol-17beta was able to overcome the inhibitory effects of AGT on oocytes maturation and cumulus expansion. COCs cultured in 10 mM AGT-containing medium supplemented with various concentrations of progesterone or estradiol-17beta for 22 h were examined for oocyte maturation and cumulus expansion. Results: Experiment 1 showed that a concentration of 10 mM AGT in medium was sufficient to block steroid hormone secretion, oocyte maturation and cumulus expansion, and that these inhibitory effects were fully reversible. In experiment 2, the addition of 10 mM AGT to CM did not significantly prevent oocyte maturation and cumulus expansion, implying that CM contains the steroid hormone(s) secreted from COCs, which are closely associated with oocyte maturation and cumulus expansion. The results in experiments 3 and 4 demonstrated that the addition of any concentration of progesterone or estradiol-17beta in the medium did not reduce the inhibitory effects of AGT on oocyte maturation and cumulus expansion. Conclusion: Our results indicate that bovine oocytes surrounded by cumulus cells are prevented from maturation and cumulus expansion through the inhibition of steroid secretion due to AGT, and that these inhibitory effects of AGT on oocyte maturation and cumulus expansions can not be overcome by the addition of either progesterone or estradiol-17beta in the medium. These observations suggest that some steroid hormone(s) other than P4 and E2 secreted from bovine COCs is essential for their meiotic maturation and cumulus expansion. © 2006 Wang et al; licensee BioMed Central Ltd.

DOI： 10.1186/1477-7827-4-4

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The purpose of this study was to investigate the effects of semen collection into tubes containing extender supplemented with BSA on the cryosurvival of goat spermatozoa. Semen was collected from two goats into empty tubes or tubes containing 10 ml extender supplemented with 0, 0.1, 1, or 5% BSA, and the washed spermatozoa were frozen as pellets in egg yolk-trehalose extender with the addition of 0.04% SDS and 4% glycerol. Sperm motion parameters were evaluated after post-thawing and during a thermal resistance test. The acrosome status of frozen-thawed spermatozoa was also observed using FITC-PNA staining. In frozen semen that was collected into tubes containing extender supplemented with 5% BSA, the post-thawed spermatozoa exhibited a significant improvement in motion parameters and maintained high motility throughout incubation and acrosome integrity, as compared with semen collected into tubes containing extender supplemented with lower concentrations of BSA. In conclusion, semen collection into tubes with a large volume of extender containing high concentrations of BSA dramatically improves the motility and acrosome integrity of frozen-thawed spermatozoa. This suggests that the in vitro functional freezability of spermatozoa is abruptly modified by reducing contact with seminal plasma and by flash contact with BSA at ejaculation.

DOI： 10.1262/jrd.17105

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The objective of this experiment was to investigate whether the motility parameters and acrosome integrity of goat ejaculated spermatozoa are affected by collecting semen into tubes containing an extender, and thereby determine the significance of reducing contact between seminal plasma and the sperm membrane at ejaculation. Semen were collected from three goats into tubes containing 0, 1 or 10 ml extender, or collected into tubes containing 10 ml extender supplemented with 0.1, 1 or 5% BSA. Sperm motion parameters were evaluated immediately after collection, after washing, and during a 3-h thermal resistance test. Acrosome integrity was assessed using FITC-PNA staining. Semen collection into tubes containing 10 ml extender produced higher sperm motility, progressive motility, and acrosome integrity than that using a smaller volume of extender. Furthermore, collection into 5% BSA-containing extender exhibited higher sperm characteristics and maintained high sperm motility and progressive motility throughout incubation. In conclusion, semen collection into tubes with a large volume of extender, especially extender containing higher concentrations of BSA, improved the quality of ejaculated spermatozoa, strongly suggesting that the in vitro functional characteristics of the spermatozoa were abruptly modified by flash sperm contact with accessory sex gland fluid at ejaculation.

DOI： 10.1262/jrd.17104

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Background: The objectives of this study were to develop an easy and rapid method for measuring gene expression in a small number of cells by real-time PCR without RNA extraction and purification, and to use this method to determine more precisely IGF-I gene expression in the cumulus cells surrounding oocytes. Methods: First, after small numbers of cumulus cells were lysed in cell lysis buffer, they were digested with various concentrations of DNase I for different periods at 37 to determine the optimal conditions for digestion of genomic DNA in the lysate. Since nonspecific amplification was liable to occur when the non-purified RT product of the cell lysate was used for real-time PCR with the given primers, the optimal conditions for Mg2+ and annealing temperature were well investigated. Further, to create the same conditions as in the actual sample reaction for measurement by real-time PCR, RT-minus product was added to the reaction mixture of the standard curve, and then the amplification efficiency was assessed. Next, IGF-I gene expression in cumulus cells collected from cumulus oocyte complexes (COCs) every 4h during maturation was determined using the developed method. Results: The optimal conditions for measuring gene expression using the cell lysate from a small number of cells were as follows: incubation of the cell lysate with 0.16 U/ microL DNase I with 10 U/ microL for 30 min, an Mg concentration of 1.5mM for amplification of target gene by real-time PCR using RT-product of the cell lysate. When the RT-minus products added to the reaction mixture for the standard curve, which was prepared for purified 18SrRNA plasmid, the PCR efficiency was similar between the sample and the standard. The IGF-I gene expression in the cumulus cells was elevated up through the first 8h of the culture and then declined gradually by the end of maturation, with the maximal gene expression (778-fold) seen at 8 h. Conclusions: It can be concluded that the method developed here, in which equivalent to cumulus cells collected from 0.03-0.075 COCs were employed per reaction, permits rapid and easy determination of target gene expression in a limited number of cells using real-time PCR without RNA extraction. © 2005 Kumamoto et al., licensee BioMed Central Ltd.

DOI： 10.1186/1477-7827-3-59

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Language：English   Publisher：(一社)日本放射線影響学会  

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Language：Japanese   Publisher：(公社)日本繁殖生物学会  

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Language：English   Publisher：(一社)日本放射線影響学会  

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Language：Japanese   Publisher：(公社)日本繁殖生物学会  

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Language：Japanese   Publisher：(一社)日本卵子学会  

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Language：Japanese   Publisher：(一社)日本放射線影響学会  

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Language：Japanese   Publisher：(一社)日本放射線影響学会  

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Language：Japanese   Publisher：(一社)日本放射線影響学会  

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Language：Japanese   Publisher：北信越畜産学会  

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Language：Japanese  

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Language：Japanese   Publisher：(一社)日本放射線影響学会  

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Language：Japanese   Publisher：(一社)日本放射線影響学会  

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Language：Japanese   Publisher：日本無菌生物ノートバイオロジー学会  

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Language：Japanese   Publisher：日本無菌生物ノートバイオロジー学会  

東京電力福島第一原子力発電所事故により福島県内では多くの野生動物が被災した。我々は福島県内で被災した野生ニホンザルに着目し、2013年より現在までに300頭以上から様々な臓器、筋肉などのサンプリングを行っている。採取した18の主要臓器と末梢血の放射性セシウム濃度を測定した結果、体内における放射性セシウム濃度は骨格筋が最も高く、甲状腺が最も低いことが明らかとなった。筋肉中放射性セシウム濃度と末梢血球像との間に明確な相関はみられなかったが、成獣の骨髄中顆粒球系細胞は筋肉中放射性セシウム濃度と負の相関を示し、脂肪細胞割合は筋肉中放射性セシウム濃度と正の相関を示した。これらの結果は、低線量慢性被ばくによる造血機能の低下を示唆するが、骨髄細胞の解析結果が放射線による影響であるかを結論付けるためには今後も解析を継続して行う必要がある。(著者抄録)

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Language：Japanese   Publisher：(一社)日本卵子学会  

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Language：Japanese   Publisher：(公社)日本畜産学会  

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Language：Japanese   Publisher：北信越畜産学会  

血管新生が盛んであるニワトリ胚漿尿膜(in ovo chorioallantoic membrane;iCAM)を用いて、新生子マウス卵巣の器官培養が可能か否か試みた。7-8日齢のBDF1雌マウスから採取した卵巣を、38.5度で7-9日間孵卵したニワトリ胚の漿尿膜上に静置し、孵卵器で10-12日間培養した。培養後、卵巣のパラフィン切片を作製して、HE染色を施し、卵巣組織および卵胞の発育を観察した。iCAM器官培養後の卵巣には、ニワトリ胚由来の血管が新生されて血液が全体に浸潤したもの、部分的に浸潤したもの、血液が全く浸潤していないものがあった。卵胞の形態を維持したiCAM器官培養後のHE染色した卵巣では、原始卵胞、1次卵胞、2次卵胞および胞状卵胞が観察され、ニワトリ胚由来の血液浸潤による赤血球も見られた。iCAM器官培養後の卵巣の切片1枚あたりの卵胞数は7.29±2.2個であり7-8日齢及び19日齢のBDF1雌マウスの卵巣のそれらと比較して有意に少なかった。また、直径が30μm未満の原始卵胞の割合は、58.9%であり、1次卵胞の21.9%、2次卵胞の17.8%、初期胞状卵胞を含む2次卵胞の2.7%、胞状卵胞の1.4%に比べて高かった。一方、卵胞の形態を維持できなかったiCAM器官培養卵巣では、ニワトリ胚由来の血液浸潤による赤血球が観察されるのみであった。以上、iCAM器官培養した31個の新生子マウス卵巣において、ニワトリ胚由来の血管新生による血液が全体に浸潤したものと部分的に浸潤した24サンプル中、7サンプルの卵巣において卵胞が維持されることが確かめられた。今後は、さらに卵胞を維持できるiCAM器官培養の方法を検討する必要がある。(著者抄録)

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Language：Japanese   Publisher：(公社)日本畜産学会  

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Language：Japanese   Publisher：環境ホルモン学会(日本内分泌撹乱化学物質学会)  

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Language：Japanese   Publisher：環境ホルモン学会(日本内分泌撹乱化学物質学会)  

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Language：Japanese   Publisher：(一社)日本放射線影響学会  

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Language：Japanese   Publisher：(一社)日本放射線影響学会  

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Language：Japanese   Publisher：(一社)日本放射線影響学会  

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Language：Japanese   Publisher：(一社)日本放射線影響学会  

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Language：Japanese  

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Language：Japanese   Publisher：(公社)日本畜産学会  

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Language：Japanese   Publisher：(公社)日本畜産学会  

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Language：Japanese   Publisher：(一社)日本放射線影響学会  

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Language：Japanese   Publisher：(一社)日本放射線影響学会  

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Language：Japanese   Publisher：(一社)日本放射線影響学会  

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Language：Japanese   Publisher：(一社)日本放射線影響学会  

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Language：Japanese   Publisher：日本無菌生物ノートバイオロジー学会  

2011年3月に起きた東京電力福島第一原子力発電所の爆発事故によって大気中に放出された放射性物質は福島県を中心に広範な地域に環境汚染をもたらした。本研究では福島原発20km圏内(警戒区域)で被災家畜の調査を行った。警戒区域内ではガンマースペクトルメーター測定において採取された家畜の筋肉などで134Cs、137Cs、110m Ag、129m Teのピークを認めた。放射性Csは血液などの体液、全身の臓器・組織から検出され、筋肉は最も高いレベルであった。また、血液と筋肉をはじめとする諸臓器との相関を認めた。福島原発の影響を知るためにも被災家畜を通して、人や動物への内部被ばくの影響を明らかにしていく必要がある。(著者抄録)

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Language：Japanese   Publisher：(公社)日本畜産学会  

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Language：Japanese   Publisher：(一社)日本卵子学会  

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Other Link： https://search.jamas.or.jp/index.php?module=Default&action=Link&pub_year=2013&ichushi_jid=J05623&link_issn=&doc_id=20130510150035&doc_link_id=%2Fct6mamma%2F2013%2Fs03002%2F032%2F5032-5032%26dl%3D0&url=https%3A%2F%2Fwww.medicalonline.jp%2Fjamas.php%3FGoodsID%3D%2Fct6mamma%2F2013%2Fs03002%2F032%2F5032-5032%26dl%3D0&type=MedicalOnline&icon=https%3A%2F%2Fjk04.jamas.or.jp%2Ficon%2F00004_2.gif

Language：Japanese  

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Language：Japanese   Publisher：(公社)日本畜産学会  

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Language：Japanese   Publisher：(公社)日本畜産学会  

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Language：Japanese   Publisher：(一社)日本放射線影響学会  

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Language：Japanese   Publisher：(一社)日本放射線影響学会  

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Language：Japanese   Publisher：(一社)日本放射線影響学会  

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Language：Japanese   Publisher：(一社)日本放射線影響学会  

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Language：Japanese   Publisher：(一社)日本放射線影響学会  

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Language：Japanese   Publisher：(公社)日本繁殖生物学会  

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Language：Japanese   Publisher：(公社)日本獣医学会  

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Language：Japanese   Publisher：Japanese Society of Large Animal Clinics / Large Animal Clinic Research Association  

DOI： 10.4190/jjlac.3.102

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Language：Japanese   Publisher：(一社)日本IVF学会  

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Language：Japanese   Publisher：(公社)日本獣医学会  

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Language：Japanese   Publisher：養賢堂  

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Other Link： https://agriknowledge.affrc.go.jp/RN/2010741220

Language：Japanese   Publisher：養賢堂  

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Other Link： https://agriknowledge.affrc.go.jp/RN/2010740797

Language：Japanese   Publisher：養賢堂  

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Other Link： https://agriknowledge.affrc.go.jp/RN/2010732400