Developmental potential of somatic and germ cells of hybrids between Carassius auratus females and Hemigrammocypris rasborella males.

The cause of hybrid sterility and inviability has not been analyzed in the fin-fish hybrid, although large numbers of hybridizations have been carried out. In this study, we produced allo-diploid hybrids by cross-fertilization between female goldfish (Carassius auratus) and male golden venus chub (Hemigrammocypris rasborella). Inviability of these hybrids was due to breakage of the enveloping layer during epiboly or due to malformation with serious cardiac oedema around the hatching stage. Spontaneous allo-triploid hybrids with two sets of the goldfish genome and one set of the golden venus chub genome developed normally and survived beyond the feeding stage. This improved survival was confirmed by generating heat-shock-induced allo-triploid hybrids that possessed an extra goldfish genome. When inviable allo-diploid hybrid cells were transplanted into goldfish host embryos at the blastula stage, these embryos hatched normally, incorporating the allo-diploid cells. These allo-diploid hybrid cells persisted, and were genetically detected in a 6-month-old fish. In contrast, primordial germ cells taken from allo-diploid hybrids and transplanted into goldfish hosts at the blastula stage had disappeared by 10 days post-fertilization, even under chimeric conditions. In allo-triploid hybrid embryos, germ cells proliferated in the gonad, but had disappeared by 10 weeks post-fertilization. These results showed that while hybrid germ cells are inviable even in chimeric conditions, hybrid somatic cells remain viable.

Hybrid between Danio rerio female and Danio nigrofasciatus male produces aneuploid sperm with limited fertilization capacity.

In the Danio species, interspecific hybridization has been conducted in several combinations. Among them, only the hybrid between a zebrafish (D. rerio) female and a spotted danio (D. nigrofasciatus) male was reported to be fertile. However, beyond these investigations, by means of reproductive biology, gametes of the hybrid have also not been investigated genetically. For this study, we induced a hybrid of the D. rerio female and D. nigrofasciatus male in order to study its developmental capacity, reproductive performance and gametic characteristics. Its hybrid nature was genetically verified by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the rhodopsin gene. Almost all the hybrids (36/37) were males, and only one was female. Developing oocytes were observed in the hybrid female, but ovulated eggs have not been obtained thus far. Microscopic observation revealed various head sizes of sperm in the hybrid males. Flow cytometry showed that the hybrid males generated aneuploid sperm with various ploidy levels up to diploidy. In backcrosses between D. rerio females and hybrid males, fertilization rates were significantly lower than the control D. rerio, and most resultant progeny with abnormal appearance exhibited various kinds of aneuploidies ranging from haploidy to triploidy, but only one viable progeny, which survived more than four months, was triploid. This suggested the contribution of fertile diploid sperm of the hybrid male to successful fertilization and development.

Improved Procedure for Induction of the Androgenetic Doubled Haploids in Zebrafish.

Androgenesis is useful for induction of doubled haploids from male genetic resources and contributes to the restoration of individuals from cryopreserved sperm. Here, we determined the suitable conditions for egg in vitro preservation and the suitable dose of UV irradiation for genetic inactivation of the egg nucleus, and established an improved procedure for induction of androgenetic-doubled haploids in zebrafish. The suitable solution for egg preservation was evaluated by the fertilization rate using different types of solutions or conditions. Hank's solution with 0.5% bovine serum albumin (pH8.0) was suitable for the preservation of zebrafish eggs. In addition, we discovered an improvement of fertilization rates by temporal preservation of ovulated eggs in the suitable solution. UV irradiation of eggs at 50-75 mJ/cm2 induced haploid embryos. Microsatellite genotyping using eight loci revealed the paternity and homozygosity of the putative androgenetic doubled haploids. The yield rate of androgenetic doubled haploids, which were induced by UV irradiation and heat shock, ranged from 0.4% to 10.7%.

Treatment evaluation using lung sound analysis in asthmatic children.

BACKGROUND AND OBJECTIVE: Non-invasive assessment of treatment and prediction of attacks in asthmatic children do not yet exist. Lung sound analysis can non-invasively evaluate airway obstruction. We used a recently developed technology for analysing lung sounds using ic700 (index of the chest wall at 700 Hz, sound intensity at 700 Hz) to evaluate response to inhaled corticosteroid (ICS) in asthmatic children. METHOD: Seventy asthmatic children, including infants, underwent lung sound recording in the asymptomatic state prior to and 1, 2, 4, 6 and 8 weeks after ICS treatment, and asthma control was assessed at 10 weeks. The ic700 scores at 4, 6 and 8 weeks were compared with the presence of attack during the following 2 weeks. Subjects were divided into uncontrolled and well-controlled groups. RESULTS: The mean ic700 scores of all subjects significantly reduced after 8 weeks of treatment. The mean scores of the uncontrolled group were significantly higher than those of the well-controlled group at 4, 6 and 8 weeks after starting treatment. The ic700 cut-off value for predicting asthma attacks within 2 weeks following the evaluation was set at 0.0. After 6 weeks of treatment, the area under the curve was 0.92 ± 0.04; the sensitivity, specificity and positive and negative predictive values were 83%, 88% and 88% and 84%, respectively. Similar results were observed at 4 and 8 weeks. CONCLUSION: The ic700 score is useful in assessing the effects of ICS treatment, predicting attack symptoms and identifying asymptomatic asthmatic children at a high risk for asthma attack.

Evaluation of airflow limitation using a new modality of lung sound analysis in asthmatic children.

BACKGROUND: Reliable assessment of not only symptoms but also lung function is essential in asthma management. We developed a new technology for analyzing lung sounds and assessed its clinical usefulness in asthmatic children. METHODS: Forty-four children underwent lung sound recording with simultaneous airflow measurement using a sensor on the upper right anterior chest. We calculated a sound parameter index from the amplitude of inspiratory lung sounds at 700 Hz (ic700). ic700 were compared depending on flow and body size. In addition, 184 asthmatic children and 16 non-asthmatic children underwent lung sound analysis and lung function test in an asymptomatic state. In the asthma group, 135 children received treatment continually. The untreated asthma group included 28 children who had never received treatment continually and 21 children who had not been treated for at least 1 year. The asthmatic children were divided into four classes according to asthma severity. ic700 were compared depending on spirometric parameters and asthma severity classification. RESULTS: The influences of flow and body size were negligible for ic700. ic700 correlated with FEV1%, MMF and FEF50 (r = -0.436, -0.339 and -0.302, respectively). There was a significant difference of ic700 between asthmatic and non-asthmatic children (p < 0.001), and ic700 correlated with the classification of asthma severity (p < 0.001). The ic700 scores of the severe group were higher than those of the intermittent group and non-asthmatic children. CONCLUSIONS: It was possible to evaluate airway dysfunction of asthma using ic700, which was calculated non-invasively by analyzing lung sounds alone, without measuring body size and airflow.