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Artificial insemination with cryopreserved semen is a well-developed technique commonly used for controlled reproduction in cattle. However, despite current technical advances, cryopreservation continues to damage bull spermatozoa, resulting in a loss of approximately 30 to 50% of viable spermatozoa post thawing. To further improve the efficiency of cryopreservation of bull spermatozoa, understanding the molecular mechanisms underlying the cryobiological properties that affect cryoinjuries during cryopreservation process of bull spermatozoa is required. In this study, we examined the expression and localization of aquaporin (AQP) 3 and AQP7 in fresh, cooled, and frozen-thawed bull spermatozoa. Furthermore, we investigated the relevance of AQP3 and AQP7 to motility and to membrane integrity in frozen-thawed bull spermatozoa. Western blotting against AQP3 and AQP7 in bull spermatozoa revealed bands with molecular weights of approximately 42 kDa and 53 kDa, respectively. In immunocytochemistry analyses, immunostaining of AQP3 was clearly observed in the principal piece of the sperm tail. Two immunostaining patterns were observed for AQP7 -pattern 1: diffuse staining in head and entire tail, and pattern 2: diffuse staining in head and clear staining in mid-piece. Cooling and freeze-thawing did not affect the localization pattern of AQP7 and the relative abundances of AQP3 and AQP7 evaluated by Western blotting. Furthermore, we demonstrated that the relative abundances of AQP3 and AQP7 varied among ejaculates, and they were positively related to sperm motility, particularly sperm velocity, post freeze-thawing. Our findings suggest that AQP3 and AQP7 are possibly involved in the tolerance to freeze-thawing in bull spermatozoa, particularly in the sperm's tail.

In mouse development, differentiation of the inner cell mass (ICM) and trophectoderm (TE) during the transition from the morula to blastocyst stage is regulated by the Hippo pathway; however, the functions of the Hippo pathway in porcine embryogenesis have not been investigated. In the present study, we examined the gene expression patterns of the Hippo pathway members yes-associated protein 1 (YAP1) and large tumor suppressor 2 (LATS2) and the functions of these genes during porcine preimplantation development using RNA interference. Both YAP1 and LATS2 mRNA levels were shown high in the in vitro matured oocytes and 1-cell stage embryos and fell progressively with development. YAP1 nuclear localization was detected at the morula and blastocyst stages. Downregulation of either YAP1 or LATS2 inhibited porcine preimplantation development and affected the expression levels of POU class 5 homeobox 1 (OCT-4) and SRY-related HMG-box gene 2 (SOX2), transcription factors necessary for the ICM/TE differentiation. Taken together, YAP1 and LATS2 are essential for porcine preimplantation development, and it is possible that the Hippo pathway has important roles in porcine ICM/TE segregation.

The functions of POU class 5 transcription factor 1 (Oct-4) and caudal-type homeobox 2 (Cdx2) in the differentiation of the murine inner cell mass (ICM) and trophectoderm (TE) have been described in detail. However, little is known about the roles of OCT-4 and CDX2 in preimplantation bovine embryos. To elucidate their functions during early development in bovine embryos, we performed OCT-4 and CDX2 downregulation using RNA interference. We injected OCT-4- or CDX2-specific short interfering RNAs (siRNAs) into bovine zygotes. The rate of blastocyst development of OCT-4-downregulated embryos was lower compared with uninjected or control siRNA-injected embryos. Gene expression analysis revealed decreased CDX2 and fibroblast growth factor 4 expression in OCT-4-downregulated embryos. CDX2-downregulated embryos developed to the blastocyst stage; however, in most cases, blastocoel formation was delayed. Gene expression analysis revealed decreased GATA3 expression and elevated NANOG expression in CDX2-downregulated embryos. In conclusion, OCT-4 and CDX2 are essential for early development and gene expression involved in differentiation of ICM and TE lineages in bovine embryos.

The aim of the present study was to clarify the effect of extracerebral dopamine (DA) on salsolinol (SAL)-induced prolactin (PRL) secretion in goats. An intravenous injection of SAL or thyrotropin-releasing hormone (TRH) was given to female goats before and after treatment with an extracerebral DA receptor antagonist, domperidone (DOM), and the PRL-releasing response to SAL was compared with that to TRH. DOM alone increased plasma PRL concentrations and the PRL-releasing response to DOM alone was greater than that to either SAL alone or TRH alone. The PRL-releasing response to DOM plus SAL was similar to that to DOM alone, and no additive effect of DOM and SAL on the secretion of PRL was observed. In contrast, the PRL-releasing response to DOM plus TRH was greater than that to either TRH alone or DOM alone and DOM synergistically increased TRH-induced PRL secretion. The present results demonstrate that the mechanism involved in PRL secretion by SAL differs from that by TRH, and suggest that the extracerebral DA might be associated in part with the modulation of SAL-induced PRL secretion in goats.

Oct-4, a member of the POU family of transcription factors, is a key factor that regulates the segregation of the inner cell mass (ICM) and the trophectoderm (TE) during the transition from morula to blastocyst in mice. However, little is known about its role in porcine early embryogenesis. To determine the function of OCT-4 in the ICM and TE segregation of porcine embryos, we studied the developmental morphology of porcine embryos using RNA interference technology. Our experiments demonstrated that when 1-cell stage embryos were co-injected with the small interfering RNA (siRNA)for targeted knockdown of OCT-4 (OCT-4-siRNA) and tetramethylrhodamine isothiocyanate (TRITC)-dextran conjugate (Dx), they failed to form blastocysts. Therefore, in this study, we constructed chimeric embryos comprising blastomeres that either expressed OCT-4 normally or showed downregulated OCT-4 expression by co-injection of OCT-4-siRNA and Dx into one blastomere in 2- to 4-cell stage embryos. In control embryos, which were co-injected with control siRNA and Dx, Dx-positive cells contributed to the TE lineage in almost all the blastocysts examined. In contrast, Dx-positive cells derived from a blastomere co-injected with OCT-4-siRNA and Dx were degenerated in almost half the blastocysts. This was probably due to the inability of these cells to differentiate into the TE lineage. Real-time RT-PCR analysis revealed no difference in the levels of SOX2, TEAD4, FGF4 and FGFR1-IIIc, all of which are known to be regulated by OCT-4, between the OCT-4-siRNA-injected morulae and the control ones. However, the level of CDX2, a molecule specifically expressed in the TE lineage, was significantly higher in the former than in the latter. Our results indicate that continuous expression of OCT-4 in blastomeres is essential for TE formation of porcine embryos.

Introduction Mechanisms of detachment of fetal membrane after parturition in cattle are poorly understood. Glucocorticoids trigger the initiation of parturition and may facilitate the placental maturation. We compared the disappearance of trophoblast binucleate cells (BNCs) and expression of transforming growth factor-β (TGFB) in term placentomes between spontaneous and induced parturition to investigate the influences of glucocorticoids on the placental maturity. Methods Cows were delivered spontaneously (SP group) or after the administration of prostaglandin (PG) F 2 α (PG group); dexamethasone, PGF 2 α, and estriol (DEX group); and triamcinolone acetonide, PGF 2 α, and betamethasone (BET group) and placentomes were collected immediately after parturition. The number of BNCs in hematoxylin and eosin stained section was examined. Protein localization and mRNA levels of TGFB and its receptor (TGFBR) were analyzed using immunohistochemistry and qRT-PCR, respectively. Results TGFB1 is characteristically localized in the maternal septum in caruncle in contrast to TGFB2 and TGFB3, which are mainly found in cotyledonary villi and maternal epithelial cells. TGFBR1 and TGFBR2 colocalized in BNCs. The number of BNCs was lower in the SP group than in PG and DEX groups. mRNA levels of TGFB1 , TGFBR1 and TGFBR2 in the SP group differed from PG and DEX groups. There was no difference between SP and BET groups in all analyses. Discussion These results indicate that parturition inductions using PGF 2 α or dexamethasone were not able to induce disappearance of BNCs and change of TGFB signaling. Results in the BET group suggest that investigation into types, dose, and dosage schedule of glucocorticoids may facilitate placental maturation.

Kruppel-like protein Gli-similar 1 (GLIS1) is known as a direct reprogramming factor for the generation of induced pluripotent stem cells. The objective of this study was to investigate the role of GLIS1 in the preimplantation development of bovine embryos. GLIS1 transcripts in in vitro-matured oocytes and 1-cell to 4-cell stage embryos were detected, but they were either absent or at trace levels at the 8-cell to blastocyst stages. We attempted GLIS1 downregulation of bovine early embryos by RNA interference and evaluated developmental competency and gene transcripts, which are involved in zygotic gene activation (ZGA) in GLIS1-downregulated embryos. Injection of specific siRNA resulted in a distinct decrease in GLIS1 transcript in bovine embryos at the 4-cell stage. Although the bovine embryos injected with GLIS1-siRNA could develop to the 16-cell stage, these embryos had difficulty in developing beyond the 32-cell stage. Gene transcripts of PDHA1 and HSPA8, which are transcribed after ZGA, showed lower level in GLIS1 downregulated embryos. It is possible that GLIS1-downregulated embryos fail to initiate ZGA. Our results indicated that GLIS1 is an important factor for the preimplantation development of bovine embryos.

This study was performed to elucidate the changes in IFNT messenger RNA (mRNA) levels in in vivo-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 in vivo-fertilized and parthenogenetic embryos. The expression level of IFNT mRNA in in vivo-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 in vivo-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 in vivo-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 in vivo-fertilized embryos. The incidence of pregnancy loss was higher in cotransfer of code 2 in vivo-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.

The aim of the present study was to clarify the effects of hypothalamic dopamine (DA) on salsolinol (SAL)-induced prolactin (PRL) release in goats. The PRL-releasing response to an intravenous (i.v.) injection of SAL was examined after treatment with augmentation of central DA using carbidopa (carbi) and L-dopa in male goats under 8-h (8 h light, 16 h dark) or 16-h (16 h light, 8 h dark) photoperiod conditions. The carbi and L-dopa treatments reduced basal PRL concentrations in the 16-h photoperiod group (P

The aim of the present study was to clarify the effect of photoperiod on secretory patterns of growth hormone (GH) in male goats. Adult male goats were kept at 20°C with an 8-h or 16-h light photoperiod, and secretory patterns of GH secretion were compared. In addition, plasma profiles of prolactin (PRL), insulin-like growth factor-I (IGF-I) and testosterone (T) were also examined to characterize GH secretion. GH was secreted in a pulsatile manner. There was no significant difference in pulse frequency between the 8-h and 16-h photoperiods. However, GH pulse amplitude tended to be greater in the group with the 16-h photoperiod (P = 0.1), and mean GH concentrations were significantly greater in the 16-h photoperiod (P < 0.05). The GH-releasing response to GH releasing hormone was greater in the 16-h than 8-h photoperiod (P < 0.05). Plasma PRL and IGF-I levels were higher in the 16-h than 8-h photoperiod (P < 0.05). In contrast, plasma T levels were lower in the 16-h photoperiod (P < 0.05). These results show that a long light photoperiod enhances the secretion of GH as well as PRL and IGF-I, but reduces plasma T concentrations in male goats.

The aim of the present study was to clarify the relationship between hypothalamic dopamine (DA) and salsolinol (SAL) for the secretion of prolactin (PRL) in goats. SAL or thyrotropin-releasing hormone (TRH) was intravenously injected into female goats treated with or without the D2 DA receptor antagonist haloperidol (Hal), which crosses the blood-brain barrier, and the PRL-releasing response to SAL was compared with that to TRH. PRL-releasing responses to SAL, Hal, and Hal plus SAL were also examined after a pretreatment to augment central DA using carbidopa (Carbi) and L-dopa. The PRL-releasing response to Hal alone was greater than that to SAL or TRH alone. The PRL-releasing response to Hal plus SAL was similar to that of Hal alone. In contrast, the PRL-releasing response to Hal plus TRH was greater than that to TRH or Hal alone. The treatment with Carbi plus L-dopa inhibited SAL- and Hal-induced PRL secretion. The inhibition of the PRL-releasing response to SAL disappeared when SAL was injected with Hal. These results indicate that the mechanisms underlying the SAL-induced PRL response differ from those of TRH, and suggest that hypothalamic DA and its synthesis is associated in part with SAL-induced PRL secretion in goats.

Exogenous growth factors may increase the efficiency of embryo development in vitro . The aim of the present study was to examine the effects of insulin-like growth factor (IGF)-I on porcine embryo development. Porcine embryos obtained by in vitro fertilization were cultured for seven days in the presence of IGF-I (50, 100 or 150 ng/ml). Subsequently, relative transcript abundance (RA) of IGF-related genes ( IGFR1 , IGFBP2 , and IGFBP3 ), glucose transporter genes ( SLC2A4 and SLC2A8 ), and apoptosis-related genes ( BAX and BCL-XL ) was analyzed. No differences were observed in the cleavage rate on day 2 post insemination (pi) and blastocysts rate on day 7 pi between IGF-treated and control embryos. IGF-I treatment did not affect RA of IGFR1 , IGFBP3 , and SLC2A4 genes, but decreased RA of IGFBP2 and SLC2A8 genes. The percentage of TUNEL-positive nuclei in blastocysts did not differ between the experimental groups. However, RA of BAX and BCL-XL genes decreased in response to all IGF-I concentrations, whereas the BCL-XL / BAX RA ratio was enhanced when embryos were cultured in medium containing 150 ng/ml of IGF-I. These results indicate that IGF-I did not stimulate in vitro development of porcine embryos through the IGF signaling system, nor did IGF-I stimulate RA of glucose transporter genes. However, IGF-I at the highest dose was able to increase the BCL-XL/BAX transcript expression ratio. This may indicate that the primary role of IGF-I during the first days of embryo development in the pig is associated with anti-apoptotic actions rather than with growth stimulation.

The objective of this study was to investigate the role of the POU family transcription factor, Oct-4, in the early development of porcine embryos. We attempted Oct-4 downregulation of porcine early embryos by RNA interference, and evaluated Oct-4 suppression of developmental competencies and gene transcripts in porcine embryos. Injection of specific siRNA resulted in a distinct decrease in Oct-4 mRNA and protein expression in porcine embryos until at least the morula stage. Although the porcine embryos injected with Oct-4 siRNA were able to develop to the morula stage, these embryos failed to form blastocysts. Gene transcripts of caudal-like transcription factor (Cdx2) and fibroblast growth factor 4 (Fgf4), which were involved in segregation of the trophectderm and functionalization of the inner cell mass, were unchanged by Oct-4 siRNA injection. Our results indicated that Oct-4 is an important factor for porcine embryos and, in particular, for the regulation of porcine blastocyst formation.

In mouse embryos, segregation of the inner cell mass (ICM) and trophectoderm (TE) lineages is regulated by genes, such as OCT-4, CDX2 and TEAD4. However, the molecular mechanisms that regulate the segregation of the ICM and TE lineages in porcine embryos remain unknown. To obtain insights regarding the segregation of the ICM and TE lineages in porcine embryos, we examined the mRNA expression patterns of candidate genes, OCT-4, CDX2, TEAD4, GATA3, NANOG, FGF4, FGFR1-IIIc and FGFR2-IIIc, in blastocyst and elongated stage embryos. In blastocyst embryos, the expression levels of OCT-4, FGF4 and FGFR1-IIIc were significantly higher in the ICM than in the TE, while the CDX2, TEAD4 and GATA3 levels did not differ between the ICM and TE. The expression ratio of CDX2 to OCT-4 (CDX2/OCT-4) also did not differ between the ICM and TE at the blastocyst stage. In elongated embryos, OCT-4, NANOG, FGF4 and FGFR1-IIIc were abundantly expressed in the embryo disc (ED; ICM lineage), but their expression levels were very low in the TE. In contrast, the CDX2, TEAD4 and GATA3 levels were significantly higher in the TE than in the ED. In addition, the CDX2/OCT-4 ratio was markedly higher in the TE than in the ED. We demonstrated that differences in the expression levels of OCT-4, CDX2, TEAD4, GATA3, NANOG, FGF4, FGFR1-IIIc and FGFR2-IIIc genes between ICM and TE lineages cells become more clear during development from porcine blastocyst to elongated embryos, which indicates the possibility that in porcine embryos, functions of ICM and TE lineage cells depend on these gene expressions proceed as transition from blastocyst to elongated stage.

Transcription factor TEA domain family transcription factor 4 (Tead4) is one of the key factors involved in the differentiation of the trophectoderm (TE) in murine embryos. However, knowledge on the roles of TEAD4 in preimplantation development during bovine embryos is currently limited. This study examined the transcript and protein expression patterns of TEAD4 and attempted to elucidate the functions of TEAD4 during bovine preimplantation development using RNA interference. TEAD4 mRNA was found to be upregulated between the 16-cell and morula stages, and nuclear localization of the TEAD4 protein was detected at the morula stage, as well as in subsequent developmental stages. TEAD4 downregulation did not affect embryonic development until the blastocyst stage, and TEAD4-downregulated embryos were capable of forming the TE under both 5% and 21% O2 conditions. Results of gene expression analysis showed that TEAD4 downregulation did not affect the expression levels of POU class 5 transcription factor 1 (OCT-4), NANOG, caudal-type homeobox 2 (CDX2), GATA binding protein 3 (GATA3), and interferon-tau (IFNT). In conclusion, TEAD4 might be dispensable for development until the blastocyst stage and TE differentiation in bovine embryos.

The aim of the present study was to clarify the effect of melatonin (MEL) on the salsolinol (SAL)-induced release of prolactin (PRL) in goats. Female goats were kept at 20°C with 16 h of light, 8 h of darkness, and orally administered saline or MEL for 5 weeks. A single intravenous (i.v.) injection of saline (controls), SAL, thyrotropin-releasing hormone (TRH) or a dopamine receptor antagonist, sulpiride, was given to the goats 3 weeks after the first oral administrations of saline or MEL, and the responses were compared. The mean basal plasma PRL concentrations in the control group were higher for the saline treatments than MEL treatments (P

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 F(2α) and estriol, ii) after the administration of prostaglandin F(2α) 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 F(2α) 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.

DNA methylation is an important factor for the regulation of gene expression in early embryos. It is well known that the satellite I sequence is more heavily methylated in bovine somatic cell nuclear transfer (NT-SC) embryos than in embryos derived from in vitro fertilization (IVF). However, the methylation status of bovine embryos obtained by other procedures is not well known. To clarify DNA methylation levels of bovine embryos obtained from various procedures, we examined satellite I sequences in bovine blastocyst (BC) embryos derived from NT-SC, NT using embryonic blastomeres (NT-EM), in vivo (Vivo), IVF and parthenogenetic treatment (PA). Furthermore, in order to evaluate the efficacy of DNA demethylation by the NT procedure, we determined the DNA methylation levels in bovine embryos in which NT was recapitulated (Re-NT). Although the DNA methylation levels in the NT-SC embryos were higher than those in the other embryos, the NT-EM embryos exhibited lower DNA methylation levels. The satellite I sequence in the NT-SC embryos was more demethylated than that in the donor cells. Although the DNA methylation level in the individual NT-SC embryos showed variation, the full-term developmental efficacy of these embryos were not different. These findings suggest that the methylation level of the satellite I sequence at the BC stage is not related to the abnormalities of bovine embryos produced by NT-SC. There was no difference in methylation levels between Re-NT and NT-SC embryos. Our results indicated that the DNA methylation status differed among embryos produced by various methods and that at least some of the demethylation of the donor cell genome occurred in the recipient cytoplast after NT-SC, but the demethylation ability of the NT procedure was noted in the first NT but not in the second NT.

For the successful production of cloned animals by somatic cell nuclear transfer (NT), the epigenetic status of the differentiated donor cell is reversed to an embryonic totipotent status. However, in NT embryos, this process is aberrant, with genomic hypermethylation consistently observed. Here, we investigated the effects of silencing DNA methyltransferase 1 (DNMT1) mRNA by small interfering RNA (siRNA) on the DNA methylation status of the satellite I region and in vitro development of bovine NT embryos. First, the levels of DNMT1 expression were analyzed at 0, 24, 48, 72, 120 and 192 h after in vitro culture. Real-time PCR and western blotting analyses detected a significant decrease in DNMT1 mRNA in the siRNA-injected NT (siRNA-NT) group up to 72 h after in vitro culture. Next, the levels of DNA methylation of the satellite I region were analyzed at several time points after in vitro culture. The level of DNA methylation detected in siRNA-NT embryos was significantly less than those in NT embryos throughout in vitro development. Moreover, the developmental rate of embryos to blastocysts in the siRNA-NT group was significantly higher than that of NT embryos. Our data suggest that knockdown of DNMT1 mRNA in NT embryos can induce DNA demethylation, which may enhance reprogramming efficiency.

The aims of the present study were to clarify the effect of kisspeptin10 (Kp10) on the secretion of growth hormone (GH) from bovine anterior pituitary (AP) cells, and evaluate the ability of sex steroid hormones to enhance the sensitivity of somatotrophic cells to Kp10. AP cells prepared from 8-11-month-old castrated calves were incubated for 12 h with estradiol (E(2), 10(-8) mol/L),progesterone (P(4), 10(-8) mol/L), testosterone (T, 10(-8) mol/L), or vehicle only (control), and then for 2 h with Kp10. The amount of GH released in the medium was measured by a time-resolved fluoroimmunoassay. Kp10 (10(-6) or 10(-5) mol/L) significantly stimulated the secretion of GH from the AP cells regardless of steroid treatments (P < 0.05), and E(2), P(4), and T had no effect on this response. The GH-releasing response to growth hormone-releasing hormone (GHRH, 10(-8) mol/L) was significantly greater than that to Kp10 (P < 0.05). The present results suggest that Kp10 directly stimulates the release of GH from somatotrophic cells and sex steroid hormones do not enhance the sensitivity of these cells to Kp10. Furthermore, they suggest that the GH-releasing effect of Kp10 is less potent than that of GHRH.

The aims of the present study were to clarify the effect of kisspeptin-10 (Kp10) on the secretion of luteinizing hormone (LH), follicle stimulating hormone (FSH), growth hormone (GH) and prolactin (PRL) in goats, and compare the characteristics of any response with those of the response to gonadotropin-releasing hormone (GnRH). The experiments were performed using four female goats (4-5 years old) in the luteal phase of estrous cycle. A single intravenous (i.v.) injection of 1, 5 and 10 microg/kg b.w. (0.77, 3.85 and 7.69 nmol/kg b.w.) of Kp10 stimulated the release of LH. Maximum values were observed 20-30 min after the injection. On the other hand, Kp10 did not alter plasma GH and PRL concentrations significantly. Three consecutive i.v. injections of Kp10 (5 microg/kg b.w.) or GnRH (5 microg/kg b.w.: 4.23 nmol/kg b.w.) at 2-h intervals increased both plasma LH and FSH levels after each injection (P

Cloning Futures Cloning mammals by somatic cell nuclear transfer is a technique with many potential applications in regenerative medicine, agriculture, and pharmaceutics; however, it is inefficient because of the incidence of aberrant genomic reprogramming. Inoue et al. (p. 496 , published online 16 September) found that the gene product of Xist , which normally inactivates one of the two X chromosomes in females, was unexpectedly expressed ectopically from active X chromosomes in cloned mice. When Xist was deleted from the mice, gene expression returned to normal and the efficiency of somatic cell nuclear transfer increased about ninefold, offering promise for future nuclear transfer technology.

Negative plate lugs of flooded lead-acid battery were corroded during partial state-of-charge (PSoC) pattern cycle life tests simulated from stop and go vehicle driving. Potential step was applied to Pb–Ca–Sn alloy electrode at various potential and time regimes, and the electrode surface was observed by in situ electrochemical atomic force microscope (EC-AFM) to investigate the corrosion mechanisms during the potential step cycles. It was found out that the severe corrosion occurs when the oxidation of Pb to PbSO4 and partial reduction of passive layer of PbSO4 take turns many times. It was also found out that the periodic full charge, the optimization of the alloy composition, addition of the material that may make the reaction mechanism change to electrolyte were effective to suppress the corrosion rate.

The secretion of prolactin (PRL) is stimulated by thyrotropin-releasing hormone (TRH), and inhibited by dopamine (DA). However, we have recently demonstrated that salsolinol (SAL), a DA-derived endogenous compound, is able to stimulate the release of PRL in ruminants. The aims of the present study were to compare the characteristics of the PRL-releasing response to SAL and TRH, and examine the relation between the effects that SAL and DA exert on the secretion of PRL in ruminants in vivo and in vitro. Three consecutive intravenous (i.v.) injections of SAL (5mg/kg body weight (b.w.): 19.2micromol/kgb.w.) or TRH (1microg/kgb.w.: 2.8nmol/kgb.w.) at 2-h intervals increased plasma PRL levels after each injection in goats (P

The aims of the present study were to clarify the effect of kisspeptin-10 (Kp10) on the secretion of luteinizing hormone (LH), follicle-stimulating hormone (FSH) and growth hormone (GH) in prepubertal male and female cattle. The experiments were performed from May to June using five male (4-6 months old) and five female (5-6 months old) Japanese Black calves. A single intravenous (iv) injection of Kp10 (5 microg/kg body weight (b.w.): 3.85 nmol/ kg b.w.) significantly stimulated the release of LH and FSH in male and female calves (P

The low efficiency of animal production using somatic cell nuclear transfer (NT-SC) procedures is considered to be the result of an incomplete reprogramming of donor cell nucleus, which leads to abnormal expression of developmentally important genes. We analyzed the abundance of gene transcripts of Insulin-like growth factor (IGF)- related genes in single embryos derived from NT-SC, and determined changes in the transcription of IGF-related genes in blastocyst and elongated stage embryos produced by NT-SC. The present results of an analysis of mRNA transcripts at two different stages of development demonstrate that bovine NT-SC embryos show deviations in their expression patterns with respect to IGF-related genes. Changes in the expression of the IGF family may be responsible for the altered growth characteristics seen in fetuses and offspring originating from bovine embryos obtained using the NT-SC procedure.

We conducted this study to elucidate a factor causing a poor sign of parturition and prolonged gestation, which is frequently observed in cows carrying somatic clone fetuses. Pre-partum rises in concentrations of plasma estrone and estradiol-17β in the recipient cows pregnant with clones were subtle. By contrast, the plasma concentration of estrone sulfate in clone pregnancies increased gradually from pre-initiation of parturition induction whereas control cows that receivedin vivo-derived embryos showed a significant increase at parturition. Therefore, in clone pregnancies, the ratio of estrone/estrone sulfate was low during the pre-partum period compared with control. Messenger RNA expression of estrogen sulfotransferase (SULT1E1) in the placenta at parturition was significantly higher in clone pregnancies than control pregnancies and was localized in binucleate cells (BNC).SULT1E1mRNA abundance was negatively and positively correlated with concentrations of maternal estrone and estrone sulfate at parturition respectively. Messenger RNA expressions of estrogen sulfatase (STS) and aromatase (CYP19) were similar between clone and control pregnancies and were localized in BNC and caruncular epithelial cells.STSandCYP19mRNA abundances showed positive correlations with maternal estradiol-17β concentration. The population of BNC in the placenta did not differ between clone and control pregnancies. Plasma cortisol concentration of vaginally delivered newborn clone calves was comparable with those of control, although cesarean section delivered clone calves showed a low concentration. These results suggest that excess estrogen sulfoconjugation is the reason for the perturbed low ratio of active to inactive estrogens and the resulting hormonal imbalance contributes to the lack of overt signs of readiness for parturition in cows pregnant with clones.

Pb–Ca foil laminated on rolled sheet for positive grid of lead-acid battery is proposed to prevent premature capacity loss (PCL) during charge–discharge cycling. Batteries with Pb–Ca foil laminated on positive grid had longer life during charge–discharge cycle than conventional battery, which failed early by PCL. PCL is a phenomenon due to the increase of the interfacial resistance between the positive grid and the positive active mass (PAM) during discharging by PbSO 4 formation in the corrosion layer. Positive plates suffered from PCL when the compression between the grid and the PAM was poor, H 2 SO 4 concentration at the interface was high or the corrosion layer mainly consisted of β-PbO 2 . Adhesion between the PAM and Pb–5%Sb alloy or Pb–1%Ca alloy was firmer than that between the PAM and Pb–0.06%Ca–1.5%Sn. Corrosion layer formed at the interface between grid material and the PAM during cycle included more α-PbO 2 on Pb–5%Sb and on Pb–1%Ca than on Pb–0.06%Ca–1.5%Sn. It was found out that excellent cycle life performance with Pb–1%Ca foil against PCL is due to firm adhesion between the PAM and grid material, and that α-PbO 2 is formed at the interface as a result of firm adhesion of the PAM and Pb–1%Ca grid.

Potential step was applied to Pb–Ca–Sn alloy electrode at various potential and time regimes. No severe corrosion was observed during potential step cycle with cathodic potential under −140 mV or over −40 mV versus Pb/PbSO4 (3.39 M H2SO4), or at constant potential without stepping. On the other hand, the Pb–Ca–Sn alloy was severely corroded during potential step with cathodic potential from −120 mV to −60 mV and with anodic potential of +40 mV or more positive. The corrosion could not be decreased with periodical rest at 0 mV, while it could be decreased with periodical reduction at high polarization of, e.g. −160 mV. It was found out that the severe corrosion occurs when the oxidation of Pb to PbSO4 and partial reduction of passive film of PbSO4 take turns many times.

The results of road tests on valve-regulated lead-acid (VRLA) batteries in an idling-stop (stop and go) vehicle are reported. Idling-stop systems are simple systems to improve fuel economy of automobiles. They are expected to spread widely from an environmental perspective. Performances of a conventional flooded battery, a conventional VRLA battery, and an improved VRLA battery were compared in road tests with an idling-stop vehicle. It was found that the improved VRLA battery was suited to idling-stop applications because it had a smaller capacity loss than the conventional flooded battery during partial-state-of-charge (PSoC) operation. The positive grid was corroded in layers, unlike the usual grain boundary corrosion of SLI battery grid. It is because the corrosion proceeded mainly under PSoC conditions. The corrosion rate could be controlled by potential control of positive plates.

The aim of the present study was to clarify the effect of extracerebral dopamine (DA) on salsolinol (SAL)-induced prolactin (PRL) secretion in goats. An intravenous injection of SAL or thyrotropin-releasing hormone (TRH) was given to female goats before and after treatment with an extracerebral DA receptor antagonist, domperidone (DOM), and the PRL-releasing response to SAL was compared with that to TRH. DOM alone increased plasma PRL concentrations and the PRL-releasing response to DOM alone was greater than that to either SAL alone or TRH alone. The PRL-releasing response to DOM plus SAL was similar to that to DOM alone, and no additive effect of DOM and SAL on the secretion of PRL was observed. In contrast, the PRL-releasing response to DOM plus TRH was greater than that to either TRH alone or DOM alone and DOM synergistically increased TRH-induced PRL secretion. The present results demonstrate that the mechanism involved in PRL secretion by SAL differs from that by TRH, and suggest that the extracerebral DA might be associated in part with the modulation of SAL-induced PRL secretion in goats.

Additives in the negative active-material of lead–acid batteries were examined to determine whether they could prevent progressive accumulation of lead sulfate (PbSO 4 ) in negative plates during high-rate partial-state-of-charge (HRPSoC) operation. This phenomenon is caused by progressive growth of PbSO 4 particles and a lack of conductive paths near these PbSO 4 particles. Barium sulfate (BaSO 4 ) particles in various sizes and synthetic lignin were added to the negative active-material to control PbSO 4 particle size during HRPSoC cycle-life. Some types of carbon fibres were also added to form conductive paths around the PbSO 4 particles. Synthetic lignin was found to be the most effective additive for improving battery life in HRPSoC cycle-life tests, whereas the other factors such as BaSO 4 size or carbon fibre extended less influence. The growth rate of PbSO 4 particles per cycle was much lower in a cell with synthetic lignin than in a cell with natural lignin.

This paper discusses battery temperature limits as a challenge to be answered when using valve-regulated lead-acid (VRLA) batteries in motor vehicles, and then describes the results obtained in road tests on VRLA batteries used in an idling-stop (stop and go) vehicle. In general, using lead-acid batteries at high-temperature increases grid corrosion and water loss, and accelerates deterioration. VRLA batteries are more susceptible to the effects of temperature than flooded batteries, but that is largely due to their structure. Water loss is fatal to VRLA batteries because water replenishment is impossible. At high temperature not only does the electrochemical decomposition of water increase considerably, but a substantial amount of water also evaporates due to the increased vapor pressure. This requires control to keep batteries from exceeding their maximum temperature. The low-temperature limit of lead-acid batteries is at least −50 to −60 °C, and that temperature is higher at a low SOC. This is dependent on change in the solidification point of the sulfuric acid electrolyte. From an environmental perspective there are expectations that idling-stop systems will find wide use as simple systems to improve fuel economy. We studied the performance of a conventional flooded battery, a conventional VRLA battery, and an improved VRLA battery in road tests with an idling-stop vehicle, and found that the improved VRLA battery is suited to idling-stop applications because it had a smaller capacity loss than the conventional flooded battery.

Band 3 deficiency with hereditary spherocytosis and hemolytic anemia in Japanese black cattle, band 3(Bov.Yamagata), is caused by a total lack of band 3 protein with an autosomal dominant inheritance. Genotyping for band 3 deficiency and sexing were successfully achieved in biopsied embryo cells with efficiencies of 98.4% and 97.4%, respectively. Transfer of the embryo that was determined as homozygous for the mutant allele into a recipient cow resulted in the production of a fetus exhibiting the genotype and red cell phenotypes characteristic of band 3(Bov.Yamagata). These results demonstrate that our procedure is reliable and applicable to produce animals free from or homozygous for the mutant allele by breeding carrier animals.

The objective of this study was to investigate the developmental ability of the whale embryos with interspecies somatic cell nuclear transfer (SCNT) in comparison with bovine or porcine SCNT embryos and parthenotes. There were no significant differences among the proportions of pseud-pronucleus (PPN) formation of whale SCNT oocytes, intraspecies SCNT oocytes and parethenotes. Furthermore, no significant difference was found in the cleavage rates of whale SCNT embryos between 6-dimethylaminopurin (DMAP) and cycloheximide (CHX) as secondary activation treatments. In effects of different donor cell types (viable or non-viable cells) on whale SCNT embryos, there was no significant difference in the cleavage rates of whale SCNT embryos between the two cell types. Two- to four-cell stages of whale SCNT embryos were obtained, but none of the embryos reached the blastocyst stage. The cleaved whale embryos were confirmed to have whale genomic DNA. From the results, it was shown that both bovine and porcine oocyte ...

Loop-mediated isothermal amplification (LAMP) is a novel DNA amplification method that amplifies a target sequence specifically under isothermal conditions. The product of LAMP is detected by the turbidity of the reaction mixture without electrophoresis. The objective of this study was to develop a rapid sexing method for bovine preimplantation embryos using LAMP. The first experiment was conducted to optimize the DNA extraction method for LAMP-based embryo sexing. The DNA of single blastomeres was extracted using three methods: heat, NaOH, and proteinase K-Tween 20 (PK-TW) treatments. Sexing was performed with two LAMP reactions, male-specific and male-female common reaction, after DNA extraction. The rates of correct determination of sex were 88.9-94.4%, with no difference among methods. The sensitivity and accuracy of LAMP-based embryo sexing were evaluated in the next experiment. The proportion of samples in which the sex was correctly determined was 75-100% for one to five biopsied cells. Lastly, in vivo-derived embryos were examined to verify the usefulness of LAMP-based embryo sexing, and some of these fresh, sexed embryos were transferred into recipient animals. The time needed for sexing was

Renal tubular dysplasia is an autosomal recessively inherited disorder in Japanese black cattle that is due to deletion mutations in the claudin-16 gene and causes chronic renal failure and death of affected animals. Here, we report a multiplex-PCR procedure to determine the genotype for claudin-16 deficiency in preimplantation embryos. The presence or absence of the wild-type and mutant allele(s) was precisely detected with the multiplex-PCR using as little as 5 pg of genomic DNA from leukocytes. When biopsied embryo cells were examined for claudin-16 deficiency, 97.2% of genotypes were consistent with the PCR results obtained for the corresponding embryos. In addition, sexing of embryos by PCR was performed using an aliquot of DNA extracted from biopsied embryo cells, and determination of claudin-16 genotype and sex was successfully achieved with an efficiency of 91.7% for claudin-16 genotyping and 83.3% for sexing. The production of a 100-day fetus that was male and homozygous for claudin-16 deficiency, as expected from the analysis of biopsied embryo cells, gave evidence of the reliability and applicability of this procedure for preventing the transmission of this disease and for enabling advances in animal breeding.

The objective of this study was to investigate the relationship between the plasma concentrations of vitamin A (VA), vitamin E (VE) and β-carotene (BC) during embryo collection in Japanese Black cows that had undergone superovulation treatment and the embryo collection results. Following superovulation treatment in 116 Japanese Black cows, we collected 1317 embryos by nonsurgical means seven days after artificial insemination. The collected embryos were classified into transferable embryos, unfertilized oocytes and degenerated embryos. After embryo collection, we collected blood samples from the cows and measured the plasma concentrations of VA, VE and BC. The cows were then divided into 2 groups depending on the plasma concentration of VA (L and H: < 80 IU/dl and ≥ 80 IU/dl), VE (L and H: < 150 µg/dl and ≥ 150 µg/dl) and BC (L and H: < 150 µg/dl and ≥ 150 µg/dl). As a result, the number of collected embryos in the H group of VE was significantly (P

Embryonic stem (ES) cells are pluripotent cells with the potential capacity to generate any type of cell. We describe here the isolation of pluripotent ES-like cells from equine blastocysts that have been frozen and thawed. Our two lines of ES-like cells (E-1 and E-2) appear to maintain a normal diploid karyotype indefinitely in culture in vitro and to express markers that are characteristic of ES cells from mice, namely, alkaline phosphatase, stage-specific embryonic antigen-1, STAT-3 and Oct 4. After culture of equine ES-like cells in vitro for more than 17 passages, some ES-like cells differentiated to neural precursor cells in the presence of basic fibroblast growth factor (bFGF), epidermal growth factor and platelet-derived growth factor. We also developed a protocol that resulted in the differentiation of ES-like cells in vitro to hematopoietic and endothelial cell lineages in response to bFGF, stem cell factor and oncostatin M. Our observations set the stage for future developments that may allow the use of equine ES-like cells for the treatment of neurological and hematopoietic disorders.

The present study was conducted to examine the ability of porcine oocytes to achieve male pronuclear (MPN) formation when they are matured and penetrated in vitro under various culture conditions. When cumulus-enclosed oocytes were cultured for 24-48 h in modified Whitten's medium (pH 7.4) supplemented with 10% porcine follicular fluid, 10 IU eCG/ml, and 10 IU hCG/ml (designated mWM-FG), nuclear maturation of oocytes reaching metaphase II was completed by 36 h after the start of culture. However, there were no differences in the proportions (94-95%) of oocytes penetrated in vitro by cryopreserved ejaculated spermatozoa or in the rates (35-45%) of MPN formation between oocytes cultured for 36 and 48 h. When cumulus-enclosed oocytes were cultured for 36 h in mWM-FG supplemented with 2% (v:v) minimal essential medium (MEM) essential amino acids (EAA) with the addition of 0.1 mM glutamine and/or 1% (v:v) MEM nonessential amino acids (NEAA) and inseminated in vitro, 93-97% of oocytes were penetrated regardless of the presence of amino acids during maturation, but the rates of MPN formation were higher in the presence (79-84%) than in the absence (51%) of any amino acids. The addition of EAA+NEAA and/or 0.57 mM cysteine to mWM-FG also did not affect sperm penetration in vitro, while it promoted MPN formation (76-83%) in penetrated oocytes as compared with those matured in the absence of amino acids and cysteine (53%). When oocytes were freed from cumulus cells after culture in mWM-FG, sperm penetration rates were not different between cumulus-enclosed (100%) and cumulus-free (92%) oocytes, but the rate of MPN formation was higher in cumulus-enclosed (53%) than in cumulus-free (28%) oocytes. When EAA+NEAA+cysteine was added to mWM-FG, MPN formation was not improved in cumulus-free oocytes but was much improved (78%) in cumulus-enclosed oocytes. These results indicate that MPN formation in porcine oocytes is promoted by the addition of amino acids and/or cysteine in simple maturation medium and by the presence of cumulus cells at fertilization in vitro.

The present study was designed to clarify the duration of maturation of porcine oocytes when cysteine promotes male pronuclear (MPN) formation through oocyte glutathione (GSH) synthesis. When cumulus-oocyte complexes (COCs) were cultured in a serum-free maturation medium supplemented or not supplemented with 0.57 mM cysteine, about 90% of oocytes reached metaphase I (M-I) to metaphase II (M-II) and M-II at 36 and 48 h of culture, respectively. When cysteine was added to medium at 0, 12, 24, and 36 h of culture and COCs were cultured for a total of 48 h, oocyte GSH concentrations at the end of culture and the incidence of MPN formation after sperm penetration in vitro were both higher than the values in oocytes cultured for 48 h without cysteine. In contrast, the GSH concentration at 48 h and the incidence of MPN formation were not increased when cysteine was present only during the first 24 h of maturation culture. When cysteine was added to medium every 3 h from 36 h of culture on, a higher incidence of MPN formation was obtained in oocytes cultured in the presence of cysteine from 36 to 42 h than from 36 to 45 h of culture, although GSH concentrations were higher in oocytes cultured with cysteine from 36 to 42 h than from 39 to 42 h of culture. These results suggest that the presence of cysteine in maturation medium is critical only between 42 and 48 h of culture when porcine oocytes are in the late M-I to M-II stage of development. At that time cysteine is utilized for GSH synthesis, which is subsequently instrumental in the formation of MPN after sperm penetration in vitro.

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.