Embryo collection from pigs post‐pseudopregnancy induced by estradiol dipropionate

We aimed to define whether embryo collection carried out after pseudopregnancy was of similar outcome and quality as after artificial abortion. To induce pseudopregnancy, 30 gilts or sows were given 20 mg intramuscular estradiol dipropionate (EDP) 10–11 days after the onset of estrus. Ten additional pigs were inseminated artificially at natural estrus as a control group. Prostaglandin F_2α (PGF_2α) was administered twice with a 24 hr interval beginning 15, 20, or 25 days after EDP‐treatment (n = 10 per group) or between 23 and 39 days after artificial insemination in control pigs. Following this, all pigs were given 1,000 IU equine chorionic gonadotropin and 500 IU human chorionic gonadotropin (hCG) and then inseminated. Embryos were recovered 6 or 7 days after hCG treatment and outcome was recorded. There was no significant difference in the number of normal embryos collected from the pigs with PGF_2α initiated at different time points or from the control group. Embryonic developmental stages 7 days after hCG treatment also did not differ among groups. These results indicate that the use of EDP to induce pseudopregnancy, followed by PGF_2α administration to synchronize estrus for subsequent embryo harvest, is a suitable alternative to the artificial abortion method.     

Molecular changes associated with adaptation of equine influenza H3N8 virus in embryonated chicken eggs

Embryonated chicken eggs (ECEs) are routinely used to isolate equine influenza virus. Propagation of the virus in ECEs results in selection of variants. In the present study, we determined nucleotide sequences of entire coding regions of parent A/equine/Tottori/1/07 (H3N8) and its derivatives that have different passage histories in ECE. After 12 passages, nucleotide sequence analysis predicted 3 amino acid substitutions in hemagglutinin (HA; 2 in HA1 and 1 in HA2). The two amino acid substitutions in HA1 were located in the vicinity of the cell receptor-binding site. Three other amino acid substitutions were predicted in internal proteins, 1 in the M1, 1 in the NP and 1 in the PA. This is the first report showing mutations in the internal protein genes of equine influenza virus associated with adaptation to ECE.     

National-scale estimation of methane emission from paddy fields in Japan: Database construction and upscaling using a process-based biogeochemistry model

We have estimated methane (CH₄) emission from total rice (Oryza sativa L.) paddies in Japan by means of a process-based biogeochemistry model, DeNitrification-DeComposition(DNDC)-Rice, combined with a geographic information system (GIS) database of climate, soil and farming practices. In the GIS database, 2 million ha of rice paddies were divided into 17,408 units according to 136 climate areas, 16 soil types, four classes of drainage rate and two classes of groundwater level, to simulate CH₄ flux from each of the units applying the DNDC-Rice model. As a result, the national-scale CH₄ emission in 1990 was estimated to be 216 Gg carbon (C), 13% lower than a previous inventory estimated by the Tier 2 method. By our Tier 3 approach, a relatively higher CH₄ flux was estimated from eastern regions than from western regions of Japan, presumably due to the differences in climate and water management. Sensitivity analysis and uncertainty assessment indicated that it is important to account for the heterogeneity in soil properties such as field water capacity, iron (Fe) concentration and drainage rate, in order to reduce the uncertainty in regional estimates.     

Increase in Iron and Zinc Concentrations in Rice Grains Via the Introduction of Barley Genes Involved in Phytosiderophore Synthesis

Increasing the iron (Fe) and zinc (Zn) concentrations of staple foods, such as rice, could solve Fe and Zn deficiencies, which are two of the most serious nutritional problems affecting humans. Mugineic acid family phytosiderophores (MAs) play a very important role in the uptake of Fe from the soil and Fe transport within the plant in graminaceous plants. To explore the possibility of MAs increasing the Fe concentration in grains, we cultivated three transgenic rice lines possessing barley genome fragments containing genes for MAs synthesis (i.e., HvNAS1, HvNAS1, and HvNAAT-A and HvNAAT-B or IDS3) in a paddy field with Andosol soils. Polished rice seeds with IDS3 inserts had up to 1.40 and 1.35 times higher Fe and Zn concentrations, respectively, compared to non-transgenic rice seeds. Enhanced MAs production due to the introduced barley genes is suggested to be effective for increasing Fe and Zn concentrations in rice grains.     

Generation of PDX‐1 mutant porcine blastocysts by introducing CRISPR/Cas9‐system into porcine zygotes via electroporation

Recently, we established the GEEP (“gene editing by electroporation of Cas9 protein”) method, in which the CRISPR/Cas9 system, consisting of a Cas9 protein and single guide RNA (sgRNA), is introduced into pig zygotes by electroporation and thus induces highly efficient targeted gene disruption. In this study, we examined the effects of sgRNA on the blastocyst formation of porcine embryos and evaluated their genome‐editing efficiency. To produce an animal model for diabetes, we targeted PDX‐1 (pancreas duodenum homeobox 1), a gene that is crucial for pancreas development during the fetal period and whose monoallelic disruption impairs insulin secretion. First, Cas9 protein with different sgRNAs that targeted distinct sites in the PDX‐1 exon 1 was introduced into in vitro‐fertilized zygotes by the GEEP method. Of the six sgRNAs tested, three sgRNAs (sgRNA1, 2, and 3) successfully modified PDX‐1 gene. The blastocyst formation rate of zygotes edited with sgRNA3 was significantly (p < 0.05) lower than that of control zygotes without the electroporation treatment. Our study indicates that the GEEP method can be successfully used to generate PDX‐1 mutant blastocysts, but the development and the efficiency of editing the genome of zygotes may be affected by the sgRNA used for CRISPR/Cas9 system.     

Prevalence of canine coronavirus (CCoV) in dog in Japan: detection of CCoV RNA and retrospective serological analysis

We collected rectal swabs from dogs in Japan during 2011 to 2014, and canine coronavirus (CCoV) nucleocapsid gene was detected by RT-PCR. The relationship between CCoV infection and the manifestation of diarrhea symptoms was investigated, and a correlation was noted (df=1, χ²=8.90, P<0.005). The types of CCoV detected in samples from CCoV-infected dogs were CCoV-I in 88.9% and CCoV-II in 7.4%, respectively. We retrospectively investigated the seroprevalence of CCoV-I in dogs in Japan during 1998 to 2006. The sera were tested with a neutralizing antibody test. In the absence of CCoV-I laboratory strain, we used feline coronavirus (FCoV)-I that shares high sequence homology in the S protein with CCoV-I. 77.7% of the sera were positive for neutralizing anti-FCoV-I antibodies.     

Protrudin induces neurite formation by directional membrane trafficking

Guanosine triphosphatases of the Rab family are key regulators of membrane trafficking, with Rab11 playing a specific role in membrane recycling. We identified a mammalian protein, protrudin, that promoted neurite formation through interaction with the guanosine diphosphate (GDP)-bound form of Rab11. Phosphorylation of protrudin by extracellular signal-regulated kinase (ERK) in response to nerve growth factor promoted protrudin association with Rab11-GDP. Down-regulation of protrudin by RNA interference induced membrane extension in all directions and inhibited neurite formation. Thus, protrudin regulates Rab11-dependent membrane recycling to promote the directional membrane trafficking required for neurite formation.     

Healing characteristics of deep digital flexor tenorrhaphy within the digital sheath of horses

OBJECTIVE: To describe the healing characteristics of deep digital flexor tenorrhaphy within the digital sheath. STUDY DESIGN: Experimental study. ANIMALS: Five mature horses. METHODS: Right thoracic limb, deep digital flexor tenorrhaphy was performed within the digital sheath. Limbs were cast in partial flexion using a short limb cast for 6 weeks. Next, extended heel shoes were used for limb support for 14 weeks. Healing was evaluated by sequential ultrasonographic examinations, and limb use was evaluated by force plate analysis. At 26 weeks, mechanical strength and morphologic characteristics of the repair site were evaluated. RESULTS: Gap (mean, 0.93 cm.) formation was evident in unloaded limbs at 3 weeks. This increased markedly by 6 weeks and was 5 cm at 26 weeks. Demarcation between the deep and superficial flexor tendons decreased as the transected ends adhered to the dorsal surface of the superficial flexor tendon. The intrathecal space was reduced by fibrous tissue. Mean maximum load to failure of the repair tissue was 4,616 +/- 3,556 N, with a mean stress of 12.99 +/- 2.78 MPa. The repair consistently failed at the adhesion between the transected tendon and the superficial flexor tendon. CONCLUSIONS: Intrathecal tenorrhaphy with external coaptation (in partial limb flexion) for 6 weeks resulted in gap healing, fibrous adhesion between the deep and superficial flexor tendons, fibrous tissue reduction of the intrathecal space, and a pasture-sound horse at 26 weeks. CLINICAL RELEVANCE: Without improved methods for immobilizing the deep digital flexor tendon, intrathecal tenorrhaphy is unlikely to result in first intention tendon healing.