Improvement of preservation method for ammonia-oxidizing bacteria by freeze-drying

Long-term preservation of ammonia-oxidizing bacteria had been achieved until now by using the silica gel-freezing method. Recently, the freeze-drying method for the preservation of microorganisms has been widely used, and has been applied to the preservation of ammonia-oxidizing bacteria. The 10% skim milk-l% pyruvate or glutamate ratio was optimum as a dispersion-medium, and the preservation temperature of the freeze-dried cells was effective at -80°C. This method of preservation was found to be considerably more effective than the silica gel-freezing method.     

Clinical evaluation of growth hormone secretion in cattle using insulin tolerance test

Growth hormone secretion was evaluated in cattle. Clinically healthy bovine growth hormone (bGH) concentrations were 10.7 +/- 1.6 ng/ml in Holstein and 7.8 +/- 3.9 ng/ml in Japanese black cattle. The bGH concentration alternated at three-hour intervals, and tended to be higher at midnight and lower in the morning and before feeding. Insulin tolerance test (ITT) at an insulin dosage of 0.25 U/kg showed a significant increase of bGH concentration to 331 +/- 153% at 60 to 90 min after injection. In ITT applied to five under-growth calves of Japanese black cattle, the basal bGH concentrations were lower and peak values after insulin injection were shown to be significantly low. The ITT is useful for the clinical examination of bGH secretion in cattle.     

The W‐ and Z‐linked EE0.6 sequences used for molecular sexing of captive Japanese crested ibis on Sado Island

The Japanese crested ibis Nipponia nippon is a critically threatened bird. Accurate sexing is necessary to perform effective management of captive breeding toward a national project for a tentative release of the Japanese crested ibis on Sado Island. A PCR‐based sexing method targeting a 0.6 kb EcoRI fragment (EE0.6) sequence on W chromosome with AWS03 and USP3 primers has been developed for the Japanese crested ibis. However, the primers were selected from the EE0.6 sequences from bird species other than the Japanese crested ibis. In this study, we determined the W‐ and Z‐linked EE0.6 sequences in the Japanese crested ibis, and clarified Japanese crested ibis sequence mismatch in the binding sites of the primers. Further, we found no polymorphism in the primer binding sites among five founder birds for the Sado captive Japanese crested ibis population. These findings validated the PCR‐based sexing method with the AWS03 and USP3 as accurate molecular sexing methods of captive Japanese crested ibis on the Sado Island. Additionally, we designed a primer set for a novel PCR‐based sexing, based on the EE0.6 sequences obtained in this study. This novel sexing method may be useful for future ecological research following the release of Japanese crested ibis on Sado Island. This is the first report to show the EE0.6 sequences in Japanese crested ibis.     

The soil seed bank of the threatened plant <Emphasis Type="Italic">Magnolia stellata</Emphasis> is subordinate to the emergence of current-year seedlings

We studied seed bank formation of the threatened star magnolia, Magnolia stellata, to examine the early stage of regeneration. Forty-five seedling plots (2 × 2 m), each including a soil-sampling quadrat (40 × 40 cm), were established randomly under or around the crowns of mature M. stellata trees. Seeds of M. stellata were collected from each quadrat to a depth of 5 cm. Only four seeds of M. stellata were found (0.56 seeds/m²) and all were located under mature crowns. Current-year seedlings were abundant in water channels, on moss, or under mature crowns, suggesting that the seeds may require wet soil conditions for germination. Magnolia stellata seeds show considerable germination below the crowns of mature trees in the year following masting, while some seeds remain dormant in the soil. Considering the soil seed bank and the current-year seedling bank of M. stellata, a frequent supply of seed is essential for the regeneration of this species. Thus, it is important to maintain mature trees in addition to promoting seed production.     

Genotypes and allele frequencies of buried SNPs in a bovine single‐nucleotide polymorphism array in Japanese Black cattle

Single nucleotide polymorphism (SNP) arrays are widely used for genetic and genomic analyses in cattle breeding; thus, data derived from SNP arrays have accumulated on a large scale nationwide. Commercial SNP arrays contain a considerable number of unassigned SNPs on the chromosome/position on the genome; these SNPs are excluded in subsequent analyses. Notably, the position‐unassigned SNPs, or “buried SNPs” include some of the markers associated with genetic disease. In this study, we identified the position of buried SNPs using the Basic Local Alignment Search Tool against the surrounding sequences and characterized the relationship between SNPs and genetic diseases in Online Mendelian Inheritance in Animals based on the genomic position. We determined the position of 285 buried SNPs on the genome and surveyed the genotype and allele frequencies of these SNPs in 5,955 individual Japanese Black cattle. Eleven SNPs associated with genetic disease, which contained five buried SNPs, were found in the population with the risk allele frequency ranging from 0.00008396 to 0.46. These results indicate that buried SNPs in the bovine SNP array can be utilized to identify associations with genetic disorders from large scale accumulated SNP genotype data in Japanese Black cattle.     

Occurrence of ateliosis in Japanese Black calves and their secretory function of growth hormone

Unthrifty calves occurred sporadically in Japanese Black (beef cattle) in an area in northeastern Japan. The states of unthrifty development, pedigree, clinico-biochemistry and the secretory function of bovine growth hormone (bGH) in pituitary were investigated. The total cholesterol concentration and CK, AST and LDH activities in the serum showed higher values than those of control calves. Basal bGH concentrations in the serum and bGH secretory reactivity in the insulin tolerance test (ITT) were showed to be significantly lower than those of the control calves. Furthermore, sperm donated from a specific bull had been used for these unthrifty calves. This study suggested that the present occurrence of unthrifty calves represented ateliosis possibly caused by congenital hypopituitarism which decreased of bGH secretory function.     

Ontogenetic and diel variation in the vertical distribution of larvae of jack mackerel <Emphasis Type="Italic">Trachurus japonicus</Emphasis> in the East China sea

Larvae of jack mackerel Trachurus japonicus were sampled in seven depth layers (0–10 m, 10–20 m, 20–30 m, 30–40 m, 40–50 m, 50–70 m, and 70–100 m), with a closing-type frame net, on 26th–27th February, 2003, at Sta. V (27°50′N, 126°00′E) continental shelf edge in the southern part of East China Sea, to examine their vertical distribution. Most of the larvae (>90%) occurred in the upper 50 m layer during both day and night. In the daytime, larvae of length from 3.0 mm to 4.5 mm were most abundant in the 10–20 m layer, those from 5.0 to 6.0 mm were most abundant in the 20–30 m layer, those from 6.0 to 8.0 mm occurred at greater depth, in the 30–40 m layer, and those more than 9.0 mm were found in the 0–10 m layer and performed ontogenetic vertical migrations. Larvae from 6 mm to 8 mm length performed diurnal vertical migration, for the percentage (16.7%) of larval number in the 0–20 m layer in the daytime increased to 42.1% at night and the percentage (78.1%) in the 20–50 m layer in the daytime decreased to 49.0% at night.     

Assessment of bovine X- and Y-bearing spermatozoa in fractions by discontinuous percoll gradients with rapid fluorescence in situ hybridization

This study was designed to apply the method of discontinuous Percoll gradients for sex preselection in bovine semen by using a current developed molecular technique, fluorescence in situ hybridization (FISH). In addition, we attempted to amplify the level of enrichment of X- or Y-bearing spermatozoa by treating for activating sperm motility performance with 10 mM caffeine. Bovine spermatozoa were fractionated on Percoll gradients into two major subpopulations of motile spermatozoa (bottom fraction) and weak motile spermatozoa (top fraction). The percentage of Y-bearing spermatozoa in the top fraction (52.9%) slightly exceeded and that in the bottom fraction (44.3%) decreased significantly (P<0.001) compared with the theoretical ratio (50:50). Washing sperm with BO medium affected a deviation between the two sex populations, whereas semen activated with caffeine showed no difference in the percentage of X- and Y-bearing spermatozoa in both fractions compared with the theoretical ratio (50:50). These results show that the proportion of X- and Y-bearing bovine spermatozoa can deviate after discontinuous Percoll gradients, although the proportion of X- and Y-bearing bovine spermatozoa was affected by sperm motility of the sample applied.     

Expression of vascular endothelial growth factor (VEGF) associated with histopathological changes in rodent models of osteoarthritis

Vascular endothelial growth factor (VEGF) and its receptors have recently reported to be expressed in human osteoarthritis (OA), suggesting that VEGF could be implicated in the pathogenesis of this disease. In the present study, expression of VEGF in the articular cartilage was determined in three different OA models: medial meniscectomy and monoiodoacetate (MIA) injection in rats and age-associated spontaneous joint cartilage destruction in guinea pigs. VEGF was detected by immunohistochemical analysis in the regenerative and hypertrophic chondrocytes, perichondrium and osteophyte areas and chondrocyte clones. Stain intensity of VEGF immunoreactivity increased simultaneously with the degree of cartilage destruction and reparation. These results suggest that VEGF is a key factor in the articular cartilage in human OA and animal OA models.