Changes of serum 3-methylhistidine concentration and energy-associated metabolites in dairy cows with ketosis

The present study examined the Serum 3-methylhistidine concentrations and energy-associated variables of 5 healthy Holstein cows and 5 Holstein cows with ketosis. The serum total cholesterol and apolipoprotein B-100 concentrations and lecithin-cholesterol acyltransferase (LCAT) activity of the ketotic cows were lower than those of the healthy cows 14 days before parturition. The serum non-esterified fatty acid (NEFA) concentration on the day of parturition and 3-methylhistidine concentration 14 days after parturition were higher in the ketotic cows. The serum 3-methylhistidine concentration 14 days after parturition was negatively correlated with the serum LCAT activity 14 days before parturition and was positively correlated with the serum NEFA concentration on the day of parturition. Insufficiency of cholesterol metabolism and acceleration of body fat degradation occur before parturition in cows with ketosis, and these characteristics are correlated with acceleration of protein degradation after parturition.     

Association between embryonic loss and damage to the zona pellucida by invasive micromanipulation during oviductal transfer of early-stage embryos in pigs

The aim of this study was to examine the impact of zona pellucida damage, which might arise during somatic cell nuclear transfer (SCNT), on the development and survival of transferred embryos. The zonae pellucidae of in vitro matured oocytes were either punctured with 8- to 10-microm square-ended nuclear injection pipettes and piezo pulses or slit with 35- to 40-microm enucleation pipettes. Intact oocytes were used as controls. These oocytes were electroactivated to induce parthenogenesis and transferred to the oviducts of estrus-synchronized recipient gilts. After 5 to 7 days, the recipient uteri were flushed to collect embryos, and embryonic development (morula-blastocyst stage embryos/collected embryos) and survival (viable embryos/collected embryos) were determined. In total, 221 zona-punctured, 129 zona-slitted and 57 intact embryos were transplanted into four, two and two gilts, respectively. The efficiency of embryo recovery was similar in all groups (64.3 to 79.1%). However, the zona-penetrated and incised embryos exhibited unstable development and survival compared with the controls; development and survival of the control embryos were 94.7 and 87.7%, whereas those of the zona-punctured embryos were 69.0 and 47.9% (P<0.01) and those of the zona-slit embryos were 64.7 and 50.0% (P<0.01). Cells with large foci that appeared to be macrophage giant cells were observed at the surface or inside the degenerated zona-damaged embryos. These results indicate that the recipient's immune response to damage to the zona pellucida may impair embryonic development after transplantation to the oviduct. This may be one of the factors causing the reduced efficiency of live progeny production by SCNT.     

Generation of α‐1,3‐Galactosyltransferase‐Deficient Porcine Embryonic Fibroblasts by CRISPR/Cas9‐Mediated Knock‐in of a Small Mutated Sequence and a Targeted Toxin‐Based Selection System

The CRISPR/Cas9 system has enabled the editing of mammalian genomes; however, its applicability and efficiency in the pig genome has not been studied in depth. The α‐gal epitope synthesized by α‐1,3‐galactosyltransferase gene (GGTA1) is known as a xenoantigen obtained upon pig‐to‐human xenotransplantation. We here employed the CRISPR/Cas9 system‐mediated knock‐in of endogenous GGTA1 via targeted homologous recombination (HR). Linearized donors with ~800‐bp homology flanking the CRISPR/Cas9 target site [exon 4 (containing ATG) of GGTA1] served as a template for gene targeting by HR. Using a targeted toxin strategy to select clones lacking α‐gal epitope expression, we successfully obtained several knock‐in clones within 3 weeks of initial transfection. These results suggest that the use of CRISPR/Cas9‐mediated HR to knock‐in a mutated fragment at defined loci represents an efficient strategy to achieve the rapid modulation of genes of interest in swine cells and is a promising tool for the creation of KO piglets.     

EFFECT OF BIODEGRADABLE CHELATING LIGAND ON IRON BIOAVAILABILITY AND RADISH GROWTH

The effect of chelating ligands on iron (Fe) uptake and growth of radish (Raphanus sativus L.) was investigated. The ethylenediaminetetraacetic acid (EDTA) increased ⁵⁵Fe uptake in roots of radish though its subsequent translocation from roots to shoots and leaves did not increase. About 70%—80% of the total ⁵⁵Fe was distributed in the roots while about 5%—15% and 11%—17% were in shoots and leaves, respectively. The EDTA increased iron uptake into the roots of radish, but not in the above ground parts of the plant. The growth of radish (Raphanus sativus L.) decreased drastically in alkaline condition (pH > 9), even though the concentration of iron was sufficient in the growth medium. The growth of radish was enhanced successfully by the addition of hydroxyiminodisuccinic acid (HIDS) and EDTA. This might be because HIDS and EDTA solubilize iron from its precipitation with hydroxides at higher pH, and increase iron bioavailability. The influence of EDTA and HIDS on radish growth was comparable. Increase of radish growth by ethylenediaminedisuccinic acid (EDDS) and methylglicinediacetic acid (MGDA) was less than those by EDTA and HIDS. Considering the reproducibility of the radish growth (biomass production) at pH 10, HIDS is supposed to be more effective compared to EDTA.     

Self-EcoTILLING to identify single-nucleotide mutations in multigene family

TILLING (Targeting Induced Local Lesions IN Genomes) is a low-cost, high-throughput reverse genetic technique that employs a mismatch-specific endonuclease CEL-1 to discover induced point mutations in the genes of interest. The use of the TILLING technique to survey natural variation in genes is called EcoTILLING. Here, we report a modified EcoTILLING method for the discovery of mutations in multigene family, which we coin “Self-EcoTILLING” by using an allotetraploid Monochoria vaginalis ALS multigene family as an example. The mutations could be detected by TILLING of PCR products resulting from the primers specific to both Als1 and Als3 without involving the experimental step of mixture of reference and query DNA. Either of the two co-amplified loci could serve as reference DNA to the other. We demonstrate with this example that Self-EcoTILLING is a fast, reliable and economical technique of detecting single-nucleotide mutations in polyploid plants containing multigene family.     

Nitrogen flow associated with food production and consumption and its effect on water quality in Japan from 1961 to 2005

Using statistical data, we evaluated food supply and consumption in terms of nitrogen flow to clarify its effects on water quality from 1961 to 2005 in Japan. We revised a nitrogen flow model to incorporate food production, trade and consumption as well as nitrogen mineralization of crop residues and livestock manure and denitrification. Food consumption increased steadily from 1961 to the mid 1980s and has been almost stable since then. There was a notable increase in the consumption of livestock products. By 1996, consumption had risen to fivefold the 1961 level of consumption, but it has been stable since then. We concluded that the demand for food reached a maximum in the 1990s. The increasing demand for animal feed was filled by an increase in the imports of coarse cereals until the 1980s and of oil cakes until now. As the consumption of food and animal feed increased until the mid 1980s, the nitrogen load to the environment also gradually increased during this period, after which it tended to decrease. We estimated the nitrogen concentration in river water for 8 km × 8 km grid cells from 1961 to 2005 and compared these estimated values with measurements taken at more than 4000 points from public bodies of water in 1998. The spatial distribution and prefectural average of the estimated nitrogen concentration roughly corresponded with the measured values, and both indicate that food supply and consumption have had considerable effects on water quality in Japan.     

Molecular pathogenesis of bovine paratuberculosis and human inflammatory bowel diseases

Paratuberculosis (Ptb), caused by Mycobacterium avium subsp. paratuberculosis (Map), is a chronic enteritis that affects many ruminants and other wild animals worldwide. Ptb is a great concern in animal health and in etiology of human Crohn's disease (CD). In the present study, we detected Map-specific insertion sequence IS900 of DNA in tissue sections surgically removed from lesions of patients with CD (29 samples), ulcerative colitis (UC) (17 samples), and non-inflammatory bowel disease (IBD) (20 samples). We then compared the histopathological findings of 29 CD and 17 UC cases with those of 35 cases of bovine Ptb, since few comparative pathological studies of human IBD and Ptb have been conducted. The QPCR examination indicated positive results in 13.37% of CD cases, 3.57% of UC cases, and 10% of non-IBD cases. Human CD tissues typically exhibited destructive full thickness enteritis with severe lympho-plasma infiltration and scattered additional granulomas; UC lesions exhibited much less inflammation than CD lesions. Non-IBD control samples did not exhibit pathological changes. Human CD and UC lesions were very different from Ptb lesions that are characterized by predominant granuloma formation. Immunohistochemistry for Map antigen and acid-fast staining were negative in all human IBD cases but were always positive in Ptb cases. Our present comparative study strongly suggests that we reconsider the previous hypothesis that "Map infection" causes CD, even though human intestines were considered to have been exposed to the Map antigen containing the DNA.     

Extractives of muirapiranga (<Emphasis Type="Italic">Brosimun</Emphasis> sp.) and its effects on the vibrational properties of wood

The potential of muirapiranga (Brosimun sp.) as a substitute material for violin bows was estimated in terms of vibrational properties, and the influence of extractives on the vibrational properties was examined. The loss tangent of muirapiranga was somewhat higher and the specific dynamic Young’s modulus was rather lower than the respective values for pernambuco, which is regarded as the best material for violin bows. Therefore, muirapiranga is a poorer bow material in terms of vibrational properties. Impregnation of crude extractives from muirapiranga decreased the loss tangent of other wood specimens. The main compounds of the extractives were identified as xanthyletin and luvangetin. Impregnations of isolated xanthyletin and commercially available methoxsalen, which was tested as an analogue of luvangetin, markedly decreased the loss tangent of other wood specimens. Methoxsalen and xanthyletin differ from conventional loss tangent-decreasing substances, namely protosappanin B and hematoxylin, in terms of water insolubility and the absence of hydroxyl groups. From the similarity in molecular characteristics of loss tangent-decreasing substances found so far, restriction of molecular motion due to an impregnated substance in the wood matrix is suggested as one loss tangent-decreasing mechanism.     

Effects of smolt size and timing of migration on recovery rate of wild masu salmon <Emphasis Type="Italic">Oncorhynchus masou</Emphasis>

Little is known about the survival rate of wild masu salmon Oncorhynchus masou. To examine the effects of smolt length and migration timing on the recovery rate of wild masu salmon, we reanalyzed past tagging and recovery data (1993–1994). The tagging study was conducted in the Shokanbetsu River, northern Japan; 863 wild masu salmon smolts were captured, tagged, and released in a downstream site, and a total of 19 fish were recovered in coastal fisheries and in the natal river the following year. The data were analyzed by a logistic regression analysis with recapture as a response variable and tagging date and smolt length as explanatory variables; the tagging date had a significant effect on the recapture rate, whereas the effect of smolt size was not significant. Despite the small number of recaptures, this study indicates that migration timing is a factor affecting the marine survival of wild masu salmon smolts, although this conclusion has been repeatedly documented for other species of Pacific salmon Oncorhynchus spp.     

Dynamic left ventricular outflow tract obstruction secondary to hypovolemia in a German Shepard dog with splenic hemangiosarcoma

Dynamic left ventricular outflow tract obstruction (DLVOTO) is a common condition in cats and humans. In this case report, a dog is described with DLVOTO secondary to severe intra-abdominal hemorrhage caused by a hemangiosarcoma. The dog was a 9-year-old, 35.7-kg, spayed female German Shepard dog that presented with a history of tachypnea and collapse. A Levine II/VI systolic murmur was present at the heart base. Abdominal ultrasonography revealed a splenic mass and a large amount of ascites. Echocardiography showed a reduced left ventricular diameter and an increased aortic velocity caused by systolic anterior motion (SAM) of the mitral valve apparatus. The heart murmur and the SAM were resolved after treatment including a splenectomy and a blood transfusion.