Screening for ketosis using multiple logistic regression based on milk yield and composition

Multiple logistic regression was applied to milk yield and composition data for 632 records of healthy cows and 61 records of ketotic cows in Hokkaido, Japan. The purpose was to diagnose ketosis based on milk yield and composition, simultaneously. The cows were divided into two groups: (1) multiparous, including 314 healthy cows and 45 ketotic cows and (2) primiparous, including 318 healthy cows and 16 ketotic cows, since nutritional status, milk yield and composition are affected by parity. Multiple logistic regression was applied to these groups separately. For multiparous cows, milk yield (kg/day/cow) and protein-to-fat (P/F) ratio in milk were significant factors (P<0.05) for the diagnosis of ketosis. For primiparous cows, lactose content (%), solid not fat (SNF) content (%) and milk urea nitrogen (MUN) content (mg/dl) were significantly associated with ketosis (P<0.01). A diagnostic rule was constructed for each group of cows: (1) 9.978 × P/F ratio + 0.085 × milk yield <10 and (2) 2.327 × SNF − 2.703 × lactose + 0.225 × MUN <10. The sensitivity, specificity and the area under the curve (AUC) of the diagnostic rules were (1) 0.800, 0.729 and 0.811; (2) 0.813, 0.730 and 0.787, respectively. The P/F ratio, which is a widely used measure of ketosis, provided the sensitivity, specificity and AUC values of (1) 0.711, 0.726 and 0.781; and (2) 0.678, 0.767 and 0.738, respectively.     

Effects of dietary microalgal species and hormone treatment on the lorica size and reproductivity of heavy-ion beam irradiated rotifers

In aquatic seeding production, the feeding regime of fish larvae consists of rotifers to Artemia, while there is a lack of intermediate-sized feed in the range of 350–600 µm. To solve this problem, in our previous study, the euryhaline rotifer Brachionus plicatilis (L-type, Notojima strain) were irradiated with carbon heavy-ion-beams, and large-sized rotifer mutant lines (>?350 µm lorica length) were established. This study aimed to enhance the mutated morphometric characteristics and reproductivity through the combined use of three microalgal species (Nannochloropsis oculata, Tetraselmis tetrathele, and Chlorella vulgaris), and two hormones [juvenile hormone (JH) and gamma-aminobutyric acid (GABA)]. The combination of T. tetrathele and JH was effective in enhancing the mutated phenotype, and the largest lorica length of 344–425 µm was observed with no significant differences in population growth between the wild-type and mutant strains. The combination of N. oculata and GABA enhanced the population growth of the mutant line, and its growth rate was 2.4 times higher than that of the control group (without GABA). This study indicates that the morphometric characteristics and reproductivity of rotifer mutant lines could be regulated by feed and hormone supplementation. These findings can promote the efficient use of mutant rotifer lines in aquaculture facilities.

     

Characterization of bacterial community composition in rotifer cultures under unexpected growth suppression

Unexpected growth suppression is common in rotifer mass culturing, and bacteria present in culture tanks are believed to be one of the main causes. We used a polymerase chain reaction (PCR)-amplified 16S ribosomal RNA (rRNA) gene finger-printing method to determine the bacterial community composition in water and that in association with rotifers present in tanks when rotifer growth was suppressed. Bacterial 16S rRNA genes representing Nautella sp. and Marinomonas sp. were identified as dominant in control tanks with good rotifer growth. In contrast, the bacterial community composition was more diverse at start-up in tanks where rotifer growth was suppressed. The community composition changed during rotifer cultivation, and bacterial sequences representative of Ruegeria sp. and Hyphomonas sp. were dominant when rotifer growth recovered. These findings indicate that the bacterial community composition varied according to the rotifer growth conditions and suggest that the initial microbial flora present in the culture water may cause rotifer growth suppression.     

Phylogeography based on the nuclear ribosomal DNA internal transcribed spacer region of native Miscanthus sinensis (Poaceae) populations in Japan

Nucleotide sequence variations were investigated with respect to the geographical distribution patterns of Miscanthus sinensis populations that were sampled from 26 Japanese national parks and three populations of the Ryukyu Islands. Twelve homozygous sequences in the nuclear ribosomal DNA internal transcribed spacer region were detected. The populations of M. sinensis in mainland Japan mainly were composed of a monophyletic group with a symapomorphic character, whereas those in the Ryukyu Islands included a polyphyletic group. Only an internal transcribed spacer haplotype with a plesiomorphic character was found in both mainland Japan and the Ryukyu Islands. Thus, no clear geographical isolation was observed in this species. These facts might be caused by the ability of M. sinensis as a pioneer plant to have a high migration potential and high gene flow by outcrossing. On the basis of the results of this study (nuclear ribosomal DNA) and the previous study (chloroplast DNA), a phylogeographical history of M. sinensis was presumed where the ancestral and derived lineages were distributed in the southern and northern regions of Japan, respectively, without strict geographical isolation.     

Design and storage stability of reference materials for microfluidic quantitative PCR-based equine gene doping tests

One method of gene doping in horseracing is administering of exogenous genetic materials, known as transgenes. Several polymerase chain reaction (PCR)-based methods have been developed for detecting transgenes with high sensitivity and specificity. However, novel designs for reference materials (RMs) and/or positive template controls (PTCs) are necessary for simultaneous analysis of multiple transgene targets. In this study, we designed and developed a novel RM for simultaneously detecting multiple targets via microfluidic quantitative PCR (MFQPCR). Twelve equine genes were selected as targets in this study. A sequence region including primers and probes for quantitative PCR was designed, and a 10 bp sequence was inserted to allow the RM to be distinguished from the original transgene sequences. The sequences of individual detection sites were then connected for 12 genes and cloned into a single plasmid vector. We performed fragment size analysis to distinguish between the PCR products of the original transgene sequence and those of the RM, enabling identification of RM contamination. PTCs diluted to 10,000, 1,000, 100, and 10 copies/µl with horse genomic DNA from RM were stably stored at 4°C for 1 year. As digital PCR enabled absolute quantification, the designed substances can serve as an RM. These findings indicate that the RM design and storage conditions were suitable for gene doping tests using MFQPCR.     

Evaluation of immunochromatographic test of Shiga toxin 2e in enrichment cultures of swine edema disease clinical samples

To simplify the diagnosis of swine edema disease, overnight culture supernatants of swine clinical samples were assayed using immunochromatographic test strips we developed previously. Small-intestinal contents, mesenteric lymph nodes, and fecal samples were cultured in casamino acid-yeast extract broth overnight, after which supernatants were loaded onto immunochromatographic test strips to determine whether they could detect Shiga toxin 2e (Stx2e). Among 23 clinical samples in which PCR-identified stx2e-positive E. coli were isolated, samples from seven of ten small-intestinal contents, one of three mesenteric lymph nodes and six of ten fecal samples showed Stx2e-positive reactions in the protein-based immunochromatographic test. Additionally, one small-intestinal content sample, in which stx2e-positive E. coli were not isolated, showed an Stx2e-positive reaction. Furthermore, the immunochromatographic test results of the samples were associated with the toxin concentration determined by sandwich ELISA and cytotoxicity assay results on Vero cells. The toxin concentration range of the samples with positive and negative reactions were 2.1–196.2 ng/ml and 0–12.8 ng/ml, respectively. The sensitivity and specificity of this immunochromatographic test strip calculated from all clinical samples analyzed in this study were 60.9% and 94.4%, respectively. Our immunochromatographic test strip has strong potential for simple and accurate diagnosis for edema disease by detecting toxin expression, complementing the PCR method.     

Effects of Mutant Thermostable α‐Amylases on Rheological Properties of Wheat Dough and Bread

Thermostable mutant α‐amylases (21B, M111, and M77) with various degrees of thermostability were purified from Bacillus amyloliquefaciens F and used as improvers for breadmaking. Test baking with the mutant enzymes was conducted using the long fermentation sponge‐dough method. Addition of an appropriate amount of mutant α‐amylases to the ingredients distinctly increased the specific volume of the bread and improved the softness of breadcrumb as compared with the addition of Novamyl (NM), an exo‐type α‐amylase. M77 was the most effective in retarding the staleness of breadcrumb. The softness of breadcrumb during storage, however, was not correlated with the thermostability. All mutant α‐amylases weakened the mixing property of the dough, whereas they strengthened the property of fermented dough. Especially, M77 and NM had different effects on the dough properties, but their bread qualities were similar to each other. The strong tolerance of M77 dough to the long baking process might be due to the production of hydrolyzed starches, oligosaccharides in the range of maltopentaose to maltohexaose, as compared with NM. Therefore, in the light of present findings, these mutant α‐amylases are possible substitutes for NM as bread improvers.     

A preliminary experiment regarding the natural induction of gonadal development in female Japanese eels without hormone treatment

To test whether gonadal development of female eels could be promoted without any exogenous hormone treatments, we observed the effect of water temperature manipulation. After 3–5 months of water temperature treatments, three silver eels showed higher gonadosomatic indices (GSI). In particular, one eel in the 5–15°C fluctuating temperature treatment group (5°C daytime and 15°C night‐time) had the highest GSI of 8.5 with secondary yolk globule stage oocytes of large diameter (OD; 412 μm) after 3 months, which indicated definite gonadal development compared with those in the initial states (mean GSI, 2.4; OD, 226.7 μm). The 5–15°C fluctuating temperature and constant 5°C groups had low oocyte breakdown (atretic) rates. Because daily temperature fluctuations and cold daytime water are experienced by vertically migrating silver eels in the ocean, these temperature conditions may be key maturation process components that could be useful for hormone‐free artificial maturation.     

Phylogenetic analyses in cetacean species of the family Delphinidae using a short wavelength sensitive opsin gene sequence

The short wavelength sensitive (SWS) opsin gene is expected to contain informative sites for understanding the speciation of the family Delphinidae, because it is not functional in cetaceans. We determined partial SWS gene sequences from 15 delphinid species of 12 genera and from harbor porpoise for comparison. We found a 39-bp insertion that was shared by six species (the insertion group: Delphinus delphis, Delphinus capensis, Stenella longirostris, Stenella coeruleoalba, Lagenodelphis hosei, and Sousa chinensis) and common base substitutions shared by eight species (Stenella frontalis, Tursiops truncatus, and six species of the insertion group). As these insertions and substitutions are not found in the other seven delphinids or in the cloven-hoofed mammals (which are close to cetaceans), it is suggested that these eight species are more closely related to each other than to the other species. This hypothesis is supported by phylogenetic analyses. The eight species with the substitutions formed a clade containing two sister clades, one consisting of the insertion group and the other consisting of the two other species, in both neighbor-joining and Bayes analyses. Phylogenetic analyses also showed that Lissodelphis borealis and Lagenorhynchus obliquidens are closely related and that their common ancestor diverged from the others at an early stage of delphinid evolution.     

Effect of long-term exposure to sulfides on taurine transporter gene expression in the gill of the deep-sea mussel Bathymodiolus platifrons,which harbors a methanotrophic symbiont

Seawater around deep-sea hydrothermal vents and cold seeps contain high levels of hydrogen sulfide, which is toxic to most animals. Invertebrates inhabiting these environments have been reported to accumulate high levels of thiotaurine, a sulfur-containing amino acid. Thiotaurine is likely to play an important role in sulfide detoxification, but its functions in the detoxification process are still unknown. We cloned methane-seep mussel Bathymodiolus platifrons cDNA encoding the taurine transporter (TAUT), which transports thiotaurine and its precursors across the cell membrane. Phylogenetic analyses revealed that the predicted peptide formed a clade with the TAUTs of shallow-water mussel Mytilus galloprovincialis and the hydrothermal vent mussel Bathymodiolus septemdierum that harbors thioautotrophic bacteria. We then reared B. platifrons in the presence or absence of Na₂S and quantified TAUT mRNA using a real-time PCR system. The amount of TAUT mRNA in the gills of B. platifrons increased with rearing in the presence of Na₂S for 69 days, but no change was observed in the absence of sulfide. These results suggest that TAUT plays an important role in sulfide detoxification, even in species that do not harbor thioautotrophic bacteria. The TAUT mRNA level was variable in the mantle and low in the foot throughout the entire rearing period, regardless of the presence/absence of sulfide, suggesting that TAUT gene expression is regulated differentially in each tissue.