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Hokkaido, Japan

Kageyama S.,Animal Biotechnology Group | Hirayama H.,Animal Biotechnology Group
Journal of Mammalian Ova Research | Year: 2012

Loop-mediated isothermal amplification (LAMP) is a novel DNA amplification method that amplifies a target sequence specifically under isothermal conditions. The LAMP product is detected by the turbidity of the reaction mixture without electrophoresis. We have developed a rapid sexing method for bovine preimplantation embryos using LAMP. Sexing is performed utilizing two LAMP reactions, male-specific and male-female common reactions, after DNA extraction. The time needed for sexing is <1 h. Sixty-one fresh sexed embryos, of which 23 and 38 had been judged as male and female, respectively, were transferred to recipient animals (one embryo per animal). The pregnancy rate was 57.4% (35/61) and all calves born were of the predicted sex. Therefore, LAMP-based embryo sexing accurately determined gender and was proven to be suitable for field application. In this review, we describe the procedure for detecting male-specific DNA sequence by LAMP for the rapid identification of freemartins. In addition, we also describe an efficient procedure for embryo sexing of water buffalo (Bubalus bubalis) with LAMP. © 2012 Japanese Society of Mammalian Ova Research. Source


Kobayashi Y.,Okayama University | Yamamoto Y.,Okayama University | Kageyama S.,Animal Biotechnology Group | Hirayama H.,Animal Biotechnology Group | And 3 more authors.
Reproduction | Year: 2016

Nitric oxide (NO) is a regulator of sperm motility, oocyte/embryo survival, and waves of contraction/relaxation in mammalian oviducts. As follicles control oviductal functions by two routes at least, (1) a systemic way via blood vessels before ovulation, (2) a direct way by entering of follicular fluid through fimbria at ovulation, we hypothesized that NO synthesis in the bovine oviduct is regulated by follicular steroids and prostaglandins (PGs). Quantification of mRNA expressions in the ampullary tissues showed that inducible NO synthase (NOS2) mRNA expression was highest on the day of ovulation (day 0). By contrast, NOS2 mRNA expression in the isthmus was highest on days 5-6 and lowest on days 19-21. Endothelial NOS (NOS3) mRNA expressions in either the ampulla or the isthmus did not change during the estrous cycle. PGE2 and PGF2α increased NOS2 mRNA expressions in cultured ampullary oviductal epithelial cells after 1-h incubation. These increases were suppressed by an antagonist of E-prostanoid receptor type 2, one of the PGE2 receptor. Estradiol-17β decreased the expression of NOS2 mRNA expression in cultured isthmic epithelial cells 24 h after treatment. This effect was suppressed by an antagonist of estrogen receptor α (ESR1). Expression of ESR1 was highest on days 19-21 in the isthmic tissues. The overall findings indicate region-specific difference of NO synthesis in the oviduct. PGs flowed from ruptured follicle may up-regulate NO synthesis in the oviductal epithelium, whereas circulating E2 seems to inhibit NO synthesis via ESR1 in the isthmus at the follicular stage. © 2016 Society for Reproduction and Fertility. Source


Hirayama H.,Animal Biotechnology Group | Ushizawa K.,Japan National Institute of Agrobiological Science | Takahashi T.,Japan National Institute of Agrobiological Science | Sawai K.,Iwate University | And 8 more authors.
Journal of Reproduction and Development | Year: 2012

We conducted this study to analyze apoptotic changes in the bovine placentome at spontaneous and induced parturition. Cows delivered i) after the administration of dexamethasone followed by prostaglandin F2α and estriol, ii) after the administration of prostaglandin F2α and estriol or iii) spontaneously. Prepartum changes in plasma progesterone and estradiol- 17β concentrations were similar between spontaneous and induced parturition. Messenger RNA of BCL2-related protein A1 (BCL2A1), an antiapoptotic gene, was expressed by trophoblast binucleate cells and caruncular epithelial cells. Quantitative RT-PCR showed that the expression of BCL2A1 mRNA in cotyledonary and caruncular portions was significantly lower in spontaneous parturition than induced parturition. The expression of BCL2-associated X protein (BAX) mRNA, a proapoptotic gene, was significantly higher in cotyledons at spontaneous parturition than parturition induced without dexamethasone. Caspase-3 (CASP3) mRNA and pre-activated CASP3 protein were predominantly detected in caruncular epithelial cells regardless of how parturition proceeded. Activated CASP3 protein was found in trophoblast uninucleate cells and binucleate cells rather than caruncular epithelial cells. In spontaneous parturition, intense staining of activated CASP3 was detected in caruncular epithelial cells. Spontaneous and dexamethasone-induced parturition increased apoptotic cells in the placentome compared with parturition induced without dexamethasone. The number of binucleate cells was significantly decreased in spontaneous parturition. The present results suggest that although the clinical dose of dexamethasone induces apoptosis in the placentome at term, neither dexamethasone nor prostaglandin F2α evoke normal physiological changes in the placentome during delivery such as a change in the balance of apoptosis-related genes and disappearance of binucleate cells. © 2012 by the Society for Reproduction and Development. Source


Hirayama H.,Animal Biotechnology Group | Kageyama S.,Animal Biotechnology Group | Moriyasu S.,Animal Biotechnology Group | Sawai K.,Iwate University | Minamihashi A.,Animal Biotechnology Group
Journal of Reproduction and Development | Year: 2013

In domestic animals of the family Bovidae, sex preselection of offspring has been demanded for convenience of milk/beef production and animal breeding. Development of the nonsurgical embryo transfer technique and sexing methods of preimplantation embryos made it possible. Sexing based on detection of Y chromosome-specific DNA sequences is considered the most reliable method to date. PCR enables amplification of a target sequence from a small number of blastomeres. However, it requires technical skill and is time consuming. Furthermore, PCR has the risk of false positives because of DNA contamination during handling of the PCR products in duplicate PCR procedures and/or electrophoresis. Therefore, for embryo sexing to become widely used in the cattle embryo transfer industry, a simple, rapid and precise sexing method needs to be developed. Loop-mediated isothermal amplification (LAMP) is a novel DNA amplification method, and the reaction is carried out under isothermal conditions (range, 60 to 65 C) using DNA polymerase with strand displacement activity. When the target DNA is amplified by LAMP, a white precipitate derived from magnesium pyrophosphate (a by-product of the LAMP reaction) is observed. It is noteworthy that LAMP does not need special reagents or electrophoresis to detect the amplified DNA. This review describes the development and application of an embryo sexing method using LAMP in cattle and water buffaloes. © 2013 by the Society for Reproduction and Development. Source


Hirayama H.,Animal Biotechnology Group | Moriyasu S.,Animal Biotechnology Group | Kageyama S.,Animal Biotechnology Group | Sawai K.,Iwate University | And 8 more authors.
Theriogenology | Year: 2014

This study was performed to elucidate the changes in IFNT messenger RNA (mRNA) levels in invivo-fertilized and parthenogenetic bovine embryos and their interferon-τ (IFNT) secretion amounts during the elongation phase. We assessed the induction capability of maternal recognition of pregnancy by parthenogenetic embryos and attempted cotransfer of invivo-fertilized and parthenogenetic embryos. The expression level of IFNT mRNA in invivo-fertilized embryos peaked on Day 18 after estrus, and the highest amount of uterine IFNT was observed on Day 20. Transfer of 10 parthenogenetic embryos produced a detectable amount of uterine IFNT. Transfer of one or three parthenogenetic embryos inhibited luteolysis. An increase in ISG15 mRNA levels in peripheral granulocytes was induced by the transfer of three parthenogenetic embryos. Cotransfer of three parthenogenetic embryos significantly improved the pregnancy rate on Day 40 in code 3 invivo-fertilized embryos compared with single transfer without parthenogenetic embryos (65% vs. 35%). However, the pregnancy rate on Day 90 (35%) in cotransfer of code 3 invivo-fertilized embryos did not differ from that upon single transfer (29%), because the cotransfer group had a higher incidence of pregnancy loss than with single transfer (47% vs. 17%) after Day 40. Cotransfer did not affect the pregnancy rate of code 2 invivo-fertilized embryos. The incidence of pregnancy loss was higher in cotransfer of code 2 invivo-fertilized embryos than in single transfer (30% vs. 7%). In conclusion, parthenogenetic embryos in the elongation phase secreted IFNT, enabling induction of maternal recognition of pregnancy. The present study revealed that enhancement of the maternal recognition of pregnancy using parthenogenetic embryos promoted the viability of poor-quality embryos until Day 40 of gestation. However, the incidence of pregnancy loss increased after Day 40 in the cotransfer of parthenogenetic embryos. A technique for promoting the full-term survival of poor-quality embryos is needed. © 2014 Elsevier Inc. Source

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