Spindle formation and microtubule organization during first division in reconstructed rat embryos produced by somatic cell nuclear transfer

The present study was conducted to demonstrate the spindle formation and behavior of chromosomes and microtubules during first division in reconstructed rat embryos produced by somatic cell nuclear transfer (SCNT) with cumulus cell nuclei. To demonstrate the effect of oocyte aging after ovulation on the cleavage of SCNT embryos, micromanipulation was carried out 11, 15 and 18 h after injection of hCG. SCNT oocytes were activated by incubation in culture medium supplemented with 5 microM ionomycin for 5 min followed by treatment with 2 mM 6-dimethylaminopurine (6-DMAP) in mR1ECM for 2-3 h. For immunocytochemical observation, the SCNT embryos were incubated with monoclonal anti-alpha-tubulin antibody and then fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG. Cleavage rates were significantly higher for oocytes collected after 15 and 18 h rather than for those collected 11 h after injection of hCG (56 and 53%, respectively vs. 28%; P<0.05). Premature chromosome condensation occurred before activation of the SCNT oocytes, but adequate spindle formation was only rarely observed. The distribution of microtubules in SCNT embryos after activation was different from those of fertilized and parthenogenic oocytes, i.e., a dense microtubule organization shaped like a ring was observed. Eighteen to 20 h post-activation, most SCNT embryos were in the 2-cell stage, but no nucleoli were clearly visible, which was quite different from the fertilized oocytes. In addition, first division with and without small cellular bodies containing DNA was observed in the rat SCNT embryos in some cases. The present study suggests that reorganization of transferred nuclei in rat SCNT embryos may be inadequate in terms of formation of the mitotic assembly and nucleolar reorganization.     

Phylogenetic characterization of rabies virus isolates from Carnivora in Brazil

The incidence of canine rabies has been widely reported in Brazil, and new rabies virus (RV) variants, genetically similar to canine RV, have recently been isolated from foxes. In order to derive the epidemiological characteristics of Brazilian Carnivora RV, Brazilian RVs isolated from dogs, cats, and foxes were genetically analyzed. Brazilian Carnivora RV isolates were divided into 2 main lineages. The predominant lineage was found in dogs and cats, which included the Argentinean and Bolivian Carnivora RV isolates, and was extensively distributed throughout Brazil and surrounding countries. The other lineage consisted of three sublineages containing Brazilian dog and fox RV isolates, with the dog sublineages located on an internal branch of 2 fox sublineages, suggesting that RV transmission events might have occurred between foxes and dogs in the past. These results suggest that contact between dogs and wildlife has the potential to generate new rabies variants and that it is important to control RV infection cycles in both dogs and wildlife to prevent spread of rabies infection.     

A case of hyperplastic dermatosis of the West Highland White Terrier controlled by recombinant canine interferon-gamma therapy

A 3.5-year-old, male West Highland White Terrier was diagnosed as having hyperplastic dermatosis by clinical and histopathological findings. Controlling of Malassezia overgrowth by antifungal drugs provided a temporal improvement of the skin lesions, but the disease was deteriorated within the next 2 months despite the negative demonstration of the yeasts. Induction of recombinant canine interferon-gamma (rCaIFN-gamma) therapy resulted in almost complete cure of the skin lesions within 2 months after the initiation of the therapy. No adverse effects were detected during the therapy. Our results suggested that the rCaIFN-gamma therapy is potential to be a novel therapeutic option for controlling the breed-specific hyperplastic skin disease.     

Rapid brain MRI protocols result in comparable differential diagnoses versus a full brain protocol in most canine and feline cases

Evaluation of brain disease in veterinary patients uses a wide variety of MRI sequences. A shortened protocol that maintains consistency of interpretation would reduce radiologist reporting time, patient anesthetic time, and client cost. The aims of this retrospective, methods comparison, observer agreement study were to evaluate whether abbreviated MRI protocols alter differential diagnoses and recommendations compared to our institution's standard protocol; evaluate interobserver agreement on standard brain MRIs; and assess whether differential diagnoses change after postcontrast images. Normal and pathologic canine and feline brain MRIs were retrieved from hospital archives. Three protocols were created from each: a 5-sequence noncontrast enhanced Fast Brain Protocol 1 (FBP1); a 6-sequence contrast-enhanced Fast Brain Protocol 2 (FBP2); and an 11-sequence standard brain protocol (SBP). Three blinded veterinary radiologists interpreted FBP images for 98 cases (1 reader/case) and SBP images for 20 cases (3 readers/case). A fourth observer compared these interpretations to the original MRI reports (OMR). Overall agreement between FBPs and OMR was good (k = 0.75) and comparable to interobserver agreement for multiple reviews of SBP cases. Postcontrast images substantially altered conclusions in 17/97 cases (17.5%), as well as improved interobserver agreement compared to noncontrast studies. The conclusions reached with shortened brain protocols were comparable to those of a full brain study. The findings supported the use of a 6-sequence brain MRI protocol (sagittal T2-weighted [T2w] TSE; transverse T2w turbo spin echo fluid-attenuated inversion recovery, T2*-weighted gradient recalled echo, T1-weighted spin echo, and diffusion weighted imaging/apparent diffusion coefficient; and postcontrast transverse T1-weighted spin echo) for dogs and cats with suspected intracranial disease.     

Cryopreservation of shoot apices of hawthorn in vitro cultures originating from East Asia

The objective of this study was to establish a cryopreservation protocol for hawthorn shoot apices (Crataegus pinnatifida Bge.). Cryopreservation was carried out via encapsulation–dehydration, vitrification, and encapsulation–vitrification on shoot apices excised from in vitro cultures. We began by showing that cold-acclimation enhanced the regrowth of cryopreserved apices from 10.0 to 65.5% in encapsulation–dehydration. We then decided that the encapsulation–dehydration method was an optimal cryopreservation method for hawthorn shoot apices in terms of its high recovery after cryopreservation as well as its ease of use compared with vitrification and encapsulation–vitrification. In encapsulation–dehydration, the protocol leading to optimal regrowth was as follows: after cold-acclimation at 5 °C in the dark for 2 weeks, excised shoot tips were pretreated for 24 h at 25 °C on hormone-free Murashige and Skoog [Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol. Plant. 15, 473–497] (MS) basal medium with 0.4 mol/L sucrose, then encapsulated and precultured in liquid MS medium with 0.8 mol/L sucrose for 16 h at 25 °C. Precultured beads were dehydrated for 6 h at 25 °C in the dessicator containing 50 g silica gel to a moisture content of 15.3% (fresh-weight basis) before cryostorage for 1 h. In addition, we examined the effect of adding glycerol to both the alginate beads and loading solution to enhance regrowth after cryopreservation in encapsulation–dehydration. In the present study, it was shown that adding 0.5 mol/L glycerol resulted in high regrowth percentages (82.5–90.0%) in four Crataegus species.     

Effects of instream structures on Kirikuchi charr, southernmost population of genus Salvelinus, in a small mountain stream of Japan

ABSTRACT:   The changes in physical habitats and responses of fish, e.g. the Kirikuchi charr Salvelinus leucomaenis japonicus , were examined by placing instream structures in a stream on the upper drainage of the Totsu River system of the Kii Peninsula, Japan, over a two-year period. Instream structures created pools below the structures, and these pools have maintained their functions as fish habitats over two years. In two treatment sections, sections A and B (some red-spotted masu salmon were removed in advance in section B), mean water depth increased soon after placing the structures, but decreased over two years. Mean water velocity slowed after placing the structures in both sections. Total fish abundance has been relatively high at the two treatment sections compared with a control section after placing the structures. Age 1 Kirikuchi charr, however, increased only in section B. These results indicate that instream structures can serve as habitat enhancement for fish, including Kirikuchi charr, in the study area if other appropriate habitat factors for respective species are taken into consideration.     

Reproductive season and female maturity size of coconut crab <Emphasis Type="Italic">Birgus latro</Emphasis> on Hatoma Island,southern Japan

Information about reproductive season, size at sexual maturity and size-dependent reproductive potentials is important for efficient resource management. Such information is necessary to determine seasonal closures and minimum legal size limits. To conserve resources of coconut crab Birgus latro, which are in danger of extinction due to excessive exploitation, their reproductive season, female size at functional maturity and relationship between female size and reproductive potentials were investigated. The reproductive season started in early June and ended late August, and females with higher reproductive potentials showed a tendency to spawn early in the reproductive season. The prohibition of exploiting the resources during the reproductive season, especially early in the season, would be appropriate for sustainability of high reproductive outputs. Fifty percent of investigated females matured functionally at 24.5-mm thoracic length (TL). However, the size at which all females reached functional maturity was 32.3 mm TL, and there was a wide variation in the size of functional maturity among individuals. We must determine minimum legal size by taking the variation into account to allow all individuals to mate at least once before they are caught, which may prevent serious depression in reproductive rate and genetic diversity.     

Influence of light intensity on feeding, growth, and early survival of leopard coral grouper (Plectropomus leopardus) larvae under mass-scale rearing conditions

This study investigated the effect of different light intensities on feeding, growth and survival of early stage leopard coral grouper Plectropomus leopardus larvae. Four different light intensities (0, 500, 1000 and 3000 lx) were used and larvae were kept under constant light conditions from 0 day after hatching (DAH) to 5 DAH. The larvae were fed a small S-type of Thai strain rotifers at a density of 20 individuals/mL from 2 DAH. The number of rotifers in larval digestive organ and total length of larvae were examined at 3 h intervals between 04:00 and 22:00 h on 3 DAH, and thereafter at 6 h intervals until the end of the experiment (5 DAH). Four experimental trials of the larval rearing were repeated using by 60 kL mass-scale rearing tanks. The results indicate that coral grouper larvae are visual feeders and their food intake increases with increasing light intensity. Food intake of larvae reared at 3000 lx was significantly higher than those reared at 0–1000 lx on 3 DAH despite being the first-feeding day (P < 0.01). On 4 DAH, total length of larvae reared at 3000 lx was significantly larger than those reared at the lower light intensities (0, 500 and 1000 lx), and thereafter light intensity significantly influenced larval feeding and growth until the end of the experiment. Survival on 5 DAH did not show a significant difference between light intensities, but survival rate at 3000 lx and 1000 lx had a tendency to be higher than those reared at the lower light intensities (0 and 500 lx). In contrast, larvae reared at 0 lx exhibited stagnant and/or negative growth. These results indicate that light intensity is significantly the factor affecting larval feeding, growth, and survival in coral grouper larvae under the rearing conditions.     

Mixed cropping with ice plant alleviates the damage and the growth of cowpea under consecutive NaCl treatment and after the recovery from high salinity

We investigated the alleviative effects of mixed cropping using ice plant, which is one of the salt-accumulating halophytes, on the damage and growth inhibition of cowpea, which is not tolerant to high salinity. Three cropping patterns (mono cropping of cowpea and ice plant and their combination) were tested. The plants were treated with 0, 100, 200 and 300 mM NaCl for 14 days (consecutive NaCl). The plants were also treated with NaCl for 3 days, followed by 2 weeks (short-term recovery) and 1 month (long-term recovery) recovery. Salinity levels for short-term recovery were similar to those of the consecutive experiment, while the concentration of long-term recovery was 250 mM. The alleviative effects of mixed cropping in the consecutive NaCl experiment were observed at 200 and 300 mM NaCl. Mixed cropping significantly reduced the Na content in the cowpea leaves at 200 and 300 mM NaCl compared with mono cropping. In addition, the Na content in the soil of mix-cropped cowpea at 200 and 300 mM NaCl was statistically lower than that of the mono cropping. Mixed cropping was effective to recover from high concentration of NaCl in the experiments of short- and long-term recovery. These results indicate that mixed cropping with a halophyte could be effective in mitigating the damage and growth inhibition of a glycophyte not only under salinity but also under recovery periods.