The effect of artificial insemination prior to transfer of a limited number of vitrified and warmed porcine embryos by open pulled straw (OPS) method on their survival ability for farrowing

Embryo transfer (ET) of 20 porcine expanded blastocysts (ExBs) vitrified and warmed (VW) by open pulled straw (OPS) to a recipient allows stable piglet production. The efficiency of artificial insemination (AI) prior to ET of 10 VW ExBs for piglet production was investigated. For one trial, 10–15 VW ExBs from single donor were assigned, 10 were used for ET and the remains were assessed for their in vitro viability. In the non‐AI/ET group, 10 were transferred to each of five recipients. As AI/ET group, 10 were transferred to each of five recipients after AI. In AI/non‐ET group, only AI was performed to seven gilts. In the non‐AI/ET group, the pregnancy rate was 40%, but none of them farrowed. In the AI/ET group, all recipients produced piglets. Four (80.0%) delivered piglets from transferred VW ExBs. The survival rate of VW ExBs to term was 20.0% (10/50). In the AI/non‐ET group, six of the seven gilts farrowed. There was no difference in in vitro viability between the non‐AI/ET and AI/ ET groups (62.5% and 68.3%, respectively). AI prior to ET can be an appropriate way to maintain pregnancy and assist the development of a low number of VW ExBs to term.     

The Dnmt3b splice variant is specifically expressed in in vitro-manipulated blastocysts and their derivative ES cells

Manipulation of preimplantation embryos in vitro, such as in vitro fertilization (IVF), in vitro culture (IVC), intracytoplasmic sperm injection (ICSI), somatic cell nuclear transfer (SCNT) and other assisted reproduction technologies (ART), has contributed to the development of infertility treatment and new animal reproduction methods. However, such embryos often exhibit abnormal DNA methylation patterns in imprinted genes and centromeric satellite repeats. These DNA methylation patterns are established and maintained by three DNA methyltransferases: Dnmt1, Dnmt3a and Dnmt3b. Dnmt3b is responsible for the creation of methylation patterns during the early stage of embryogenesis and consists of many alternative splice variants that affect methylation activity; nevertheless, the roles of these variants have not yet been identified. In this study, we found an alternatively spliced variant of Dnmt3b lacking exon 6 (Dnmt3bΔ6) that is specific to mouse IVC embryos. Dnmt3bΔ6 also showed prominent expression in embryonic stem (ES) cells derived from in vitro manipulated embryos. Interestingly, IVC blastocysts were hypomethylated in centromeric satellite repeat regions that could be susceptible to methylation by Dnmt3b. In vitro methylation activity assays showed that Dnmt3bΔ6 had lower activity than normal Dnmt3b. Our findings suggest that Dnmt3bΔ6 could induce a hypomethylation status especially in in vitro manipulated embryos.     

The use of multi-detector row computed tomography (MDCT) as an alternative to specimen preparation for anatomical instruction

The purpose of the study reported here was to establish a method of teaching veterinary anatomy, including radiologic anatomy, for clinical practice using computer-aided diagnosis (CAD). Two clinically healthy dogs and three cats were scanned using multi-detector row computed tomography (MDCT). Images were made by means of imaging processing software. At the workstation, by observing the transverse, dorsal-plane, or sagittal sections and three-dimensional (3D) images simultaneously, it is much easier to understand the 3D anatomical structure. With this educational support system, anatomical figures can be explained using living animals instead of specimens. In addition, clinical representative examples can be used to show anatomical disorders to students. Veterinary students (N = 62) who filled out a questionnaire evaluating how the method aided their understanding of both experimental study and clinical examples gave it a score of 88.2 +/- 20.6 (Mean +/- SD) on a visual analog scale. This system can enhance veterinary students' understanding and interest in anatomy and can enable us to offer them a quality veterinary medical education. We concluded that CAD is a useful new option not only for clinical service but also for veterinary education.     

Effect of soft X-ray irradiation on the migratory ability of primordial germ cells in chickens

1. The present study was conducted to elucidate the effect of soft X-ray irradiation on the migratory ability of primordial germ cells (PGCs) to the germinal ridges of chicken embryos. 2. PGCs (Barred Plymouth Rock, BPR) were isolated from embryonic blood and irradiated with soft X-rays for 1-10 min. Then, the PGCs were transfected in vitro with GFP gene by lipofection. The manipulated PGCs were transferred to recipient embryos (White Leghorn, WL) and migration to the germinal ridges was analysed by examining GFP gene expression in the gonads of recipient embryos under UV light at x40 magnifications. The expression of GFP gene was detected in all the gonads of recipient embryos examined up to 10.5 d of culture. 3. Migration of PGCs irradiated with soft X-rays to the germinal ridges was also confirmed by detecting a single nucleotide polymorphism in the D-loop region of the mitochondrial DNA of BPR and WL chickens. Freshly collected PGCs (BPR) were transferred to the bloodstream of recipient embryos (WL). The fate of the transferred donor PGCs was traced by detecting the single nucleotide polymorphism in the D-loop region of the mitochondrial DNA in BPR and WL used in this study. Transferred donor PGC-derived cells were detected in all the gonads of 17-d cultured embryos by PCR. 4. The results suggest that PGCs irradiated with soft X-rays still retain the ability to migrate to the germinal ridges of recipient embryos.     

Characterization of local rhizobia in Thailand and distribution of malic enzymes

TWenty-six isolates were obtained from nodules of various legume plants (Glycine max, Vigna sinensis, Arachis hypogaea, Desmanthus virgatus, Acacia mangium, Centrosema pascuorum, Pterocarpus indicus, Xylia xylocarpa, and Sesbania rostrata) in Thailand. After confirming their nodulation and nitrogen-fixing abilities, they were identified by 16S rRNA gene analysis as Bradyrhizobium japonicum, Bradyrhizobium elkanii, Rhizobium leguminosarum, Rhizobium gallicum, and Rhizobium galegae. Using these local isolates, the distribution of the activities of both NAD⁺-dependent (DME: EC 1.1.1.39) and NADP⁺-dependent (TME: EC 1.1.1.40) malic enzymes was surveyed. The malic enzyme activities were present in all the isolated rhizobia and in other 17 local Bradyrhizobium strains in Thailand. In almost all the rhizobia, the DME activity predominated whereas the TME activity predominated only in the Rhizobium gallicum strains that were major symbionts of Sesbania rostrata. Southern hybridization analysis was performed to survey the distribution of the malic enzyme genes among the local rhizobia, which are similar to those of B. japonicum. DNA probes (ME1 for DME and ME2 for TME) were prepared by polymerase chain reaction (PCR) using degenerated primers from conserved regions of the protein sequences of bacterial malic enzymes. Southern blot analysis with ME1 as a probe showed a single band in about half of the isolates, especially in B. japonicum and R. leguminosarum strains, suggesting the wide distribution of such DME genes among local rhizobia. In contrast, Southern blot analysis with ME2 as a probe detected a single band only in five B. japonicum strains, suggesting that the TME genes, which are similar to those of B. japonicum, would be unique in a group of B. japonicum.     

Mild drying of sandy soil can physically limit the uptake of phosphorus by rainfed lowland rice in northeast Thailand

ABSTRACT

Poor response of rice to phosphorus (P) fertilization and low phytoavailability of soil P have been reported in sandy rainfed fields in northeast Thailand. In order to evaluate the effects of mild soil drying on the uptake of P by rainfed lowland rice, we carried out nutrient omission trials for nitrogen (N) and P at Ubon Ratchathani Rice Research Center under rainfed and flooded conditions. The surface soil was classified as sandy loam. To avoid severe soil drying and drought stress in the rainfed field, soil water potential at a depth of 20 cm was maintained at the field capacity (> ?20 kPa) by flush irrigation. The effects of flooding and drying on the soil properties were also evaluated in the laboratory using soils with diverse textures in and around the center. In the field experiments, the above-ground biomass of rice plants (RD6) did not respond significantly to P fertilization in the rainfed field, although it responded positively to N fertilization. Root length in the surface 10 cm under the rainfed condition was significantly smaller than that under the flooded condition due partly to the increased soil hardness upon drying, but this could not quantitatively explain the large discrepancy of P uptake observed between the rainfed and flooded conditions. Under the rainfed condition, the P uptake did not increase significantly, even when the concentration of soil Bray P was tripled by transferring the surface soil from the flooded to the rainfed field. From the laboratory experiments, it was further suggested that soil P was supplied mainly by diffusion and that the effective diffusion coefficient for P can become less than one-tenth of the value in the flooded field when the sandy soil with clay at around 10% dried to ?100 kPa. Our results suggest that the uptake of P by the rainfed lowland rice grown in sandy soil can be limited physically by mild soil drying that reduces the supply of P to roots by diffusion rather than the chemical extractability of soil P.     

Engineering of a tyrosol-producing pathway, utilizing simple sugar and the central metabolic tyrosine, in Escherichia coli

Metabolic engineering was applied to the development of Escherichia coli capable of synthesizing tyrosol (2-(4-hydroxyphenyl)ethanol), an attractive phenolic compound with great industrial value, from glucose, a renewable carbon source. In this strain, tyrosine, which was supplied not only from the culture medium but also from the central metabolism, was converted into tyrosol via three steps: decarboxylation, amine oxidation, and reduction. The engineered strain synthesized both tyrosol and 4-hydroxyphenylacetate (4HPA), but disruption of the endogenous phenylacetaldehyde dehydrogenase gene shut off 4HPA production and improved the production of tyrosol as a sole product. The engineered mutant strain was capable of producing 0.5 mM tyrosol from 1% (w/v) glucose during a 48 h shake flask cultivation.