Comparison of artificial spawning effectiveness of hCG, CPH and GnRHa in combination with dopamine inhibitors in a wild strain of ide Leuciscus idus (L.) in hatchery conditions.

Optimization of artificial reproduction, by increasing the survival rate of embryos and hatching rate, is of major importance for reducing genetic diversity, especially in fish captured from their natural habitat that subsequently spawn in hatcheries. The artificial reproduction of ide, Leuciscus idus (L.) was conducted in controlled conditions. The spawning agents included: different doses of human chorionic gonadotropin (hCG) that was compared with common carp pituitary homogenate (CPH), Ovopel - a commercial agent for induction of spawning that contains mammalian gonadotropin releasing hormone analogue (mGnRHa) with dopamine antagonists (DA): metoclopramide (MET) and Ovaprim - a commercial agent containing salmon gonadotropin releasing hormone analogue (sGnRHa) and a dopamine antagonists (DA): domperidone (DOM). There were no ovulations in females from control groups. There were no differences between the ovulation rates (90 %) or embryo survival (> 92 %) and hatching rates (> 91 %) when there was administration of hCG doses between 500 and 1000 IU/kg. When there was comparison of different spawning agents, the ovulation rate was 100 % for all treated groups. There were the shortest and longest latency times to the time of ovulation after administration of CPH (26 h) and hCG (79 h), respectively. The greatest embryo survival (> 93 %) and hatching (> 91 %) rates occurred as a result of hCG administration with these values being slightly greater than when there was treatment with Ovaprim. The association between latency time and hatching rate indicated that when there was a slower final oocyte maturation (FOM) there were greater hatching rates.

Expression analysis of the heat shock protein genes and cellular reaction in dojo loach (Misgurnus anguillicaudatus) under the different pathogenic invasion.

As molecular chaperones, heat shock proteins (HSPs) play essential roles in cells in response to stress conditions. Recent studies about immune functions of HSPs in fish have also been reported. In this study, based on the reported cDNA sequences of the four HSP genes, HSP70, HSC70, HSP90α and HSP90ß, the temporal expression patterns of the four genes during embryonic development of dojo loach(Misgurnus anguillicaudatus) was assayed with qRT-PCR. All of the four genes were ubiquitously expressed in all detected embryonic developmental stages. Among of them, HSP70, HSC70 and HSP90ß were highly expressed in the organ formation stage, while HSP90α was the highest expressed in myotome formation stage. Further, the immune responses of the four HSP genes were assayed when loach were infected with three different pathogens, bacterium (Flavobacterium cloumnare G4), parasite (Ichthyophthirius multifiliis) and fungus (Saprolegnia). All of the four genes were differentially expressed in four tissues such as skin, gills, spleen and kidney in response to the pathogenic invasion, but both HSP70 and HSP90α expressions were dramatically up-regulated. Further, the cellular responses of the loach skinand gill tissues were observed, in which the number of the skin goblet cells were significantly increased, and the gill lamellae became shorter and wider after infected. Thus, our work indicated that the HSPs may directly or indirectly involved in immune defense in fish, at least in the loach.

Outcomes of in vitro fertilization with frozen-thawed sperm: An analysis of post-thaw recovery of sperm, embryogenesis, offspring morphology, and skeletogenesis for a cyprinid fish.

BACKGROUND: Gamete cryopreservation causes cellular damage and death. This study develops cryopreservation techniques for Levantine scraper, and deciphers how early offspring development is affected when eggs are sired with fresh and frozen-thawed sperm. RESULTS: Cryopreserved sperm did not affect embryogenesis at two- and four-cell stages, but impaired embryonic development at eight-cell stage. Embryonic viability decreased at organogenesis, where only 34-49% of embryos showed viability with frozen-thawed sperm. Hatching success and percentage of normal hatched embryos declined when fertilized with frozen-thawed sperm. Considering only frozen-thawed cells the dimethyl sulfoxide (DMSO)-5%, methanol (METH)-5%, and METH-10% treatments yielded highest hatch, while METH-5% and propylene glycol-5% yielded the most normal hatched embryos. Larval spinal torsion was higher for fresh than frozen-thawed sperm, where larvae with spinal torsion showed vertebral fusion and shape alterations during exogenous feeding. Both fresh and cryopreserved treatments showed abnormalities in caudal skeleton, while rates of defective yolk-sacs were higher for cryopreserved sperm, where larvae with defective yolks showed oversized yolk extension. Percentage of larvae with defective heads/eyes were also higher for cryopreserved sperm. CONCLUSIONS: Results show how frozen-thawed sperm impairs embryonic/larvae development and identifies frequency and position of abnormalities. Future studies should investigate how sperm DNA damage may have caused these alterations.

Intra-ooplasmic injection of a multiple number of sperm to induce androgenesis and polyploidy in the dojo loach Misgurnus anguillicaudatus (Teleostei: Cobitidae).

SummaryPolyspermy was initiated by microinjecting a multiple number of sperm into the activated and dechorionated eggs of dojo loach Misgurnus anguillicaudatus (Teleostei: Cobitidae). A 10-nl sperm suspension from an albino (recessive trait) male (105, 106, 107 or 108 sperm ml -1) was microinjected into eggs from a wild-type female. Although the rates of embryos developing into the blastula stage in the injection group at the highest sperm concentration were similar to that of the control group, the hatching rates of the injection group were much lower. A large proportion of embryos that developed from the injected eggs was haploid and were mosaics containing haploid cells. Most of the haploid and mosaic embryos inherited only paternally derived alleles in the microsatellite markers (i.e. androgenesis was initiated by injecting multiple sperm). In contrast, some haploid embryos contained both paternal and maternal alleles despite haploidy, suggesting that they were mosaics consisting of cells with either paternal or maternal inheritance. The injected eggs displayed diploid, hypotriploid and triploid cells, all of which included both maternally and paternally derived alleles. One albino tetraploid with only paternal alleles was also observed from the injected eggs. These results suggested that part of the sperm microinjected into the ooplasm should form a male pronucleus/pronuclei, which could develop by androgenesis or could fuse with the female pronucleus/pronuclei. Therefore, microinjection of multiple sperm should be considered a potential technique to induce androgenesis and polyploidy.

Molecular cloning, characterization, and expression profiles of the Sox3 gene in Chinese loach Paramisgurnus dabryanus.

A number of studies have established that in vertebrates, Sox3 is involved in a wide range of developmental processes, including sex differentiation and neurogenesis. However, the exact functions of the Sox3 gene have not been documented so far in teleosts. Here, we cloned the full length cDNA of Sox3 from the teleost fish, Paramisgurnus dabryanus, which we designated PdSox3. Sequence analysis revealed that PdSox3 encodes a hydrophilic protein, and shares high homology with Sox3 in other species, ranging from mammals to fishes. Quantitative real-time reverse transcription PCR, and in situ hybridization showed that PdSox3 is consistently expressed during embryogenesis, mainly localized in the developing central nervous system. Tissue distribution analyses revealed that PdSox3 is abundant in the adult brain, especially in particle cell layer. Furthermore, PdSox3 expression was higher in gonads, in primary spermatocyte cells, primary oocytes, and previtellogenic oocyte cells. All of these results suggest that PdSox3 plays an important role in early embryonic development, in particular the formation and development of the nervous system, and gonad development, similarly to other vertebrates. This is the first report describing Sox3 gene expression from this species, and the results are necessary to provide fundamental information on both the functional and evolutionary role of Sox3 across different species.

The yolk syncytial layer of loach, Misgurnus fossilis (Teleostei) during early development.

The yolk syncytial layer (YSL) of Teleostei is a dynamic multifunctional temporary system. This paper describes the YSL structure of Misgurnus fossilis (Cobitidae) during its early developmental stages, studied using histological methods. YSL formation is prolonged. From the late blastula stage, the basal surface of the YSL is uneven and has protuberances, but becomes smoother during development. There are syncytial 'islands' with 1-2 yolk syncytial nuclei in the yolk mass. During epiboly, gastrulation and early segmentation, loach YSL is of different thickness in different regions along the dorso-ventral and antero-posterior axes of an embryo. The YSL is thickened in the dorsal region of gastrulae compared with the ventral region. Although the development of M. fossilis is similar to the development of zebrafish, there are important differences in YSL formation and organization that await further study and analysis. The study of YSL organization contributes to our knowledge of teleost developmental diversity and to the biology of temporary structures.

[Dynamics of the Incubation Medium Redox Potential during Early Development Stages of Misgurnus fossilis Loach].

The analysis of kinetic curve of redox potential changes (ORP) of aqueous medium in the process of loach embryo population development at 17°C was conducted. The system of critical points defining periodization of the major stages of embryo development was identified on the ORP curve in the time interval up to τ(0) using novel methods for processing of nonlinear signals. It was shown that τ(0) was a natural periodization parameter of main morphological changes in an embryo known in the developmental biology.

[Variability of Quantitative Morphogenetic Parameters during Early Morphogenesis of the Loach, Missgurnus fossilis L].

Analysis of normal variation in quantitative morphological characters during the early embryonic development of the loach, based on fixed material and individual developmental trajectories of living embryos, shows that the dorsoventral differentiation of the blastoderm proceeds in two stages. Initially, at the onset of epiboly, the sagittal (short) and transverse (long) blastoderm meridians are marked off, and only then, upon germ ring (GR) formation, differentiation between the opposite poles of the sagittal meridian takes place. The embryonic shield (ES) usually appears in the segment of the blastoderm where the radius of its external curvature reaches a maximum and, therefore, the active surface tension at the blastoderm boundary with the YSL (peri-blast) and yolk is the highest. In this case, the convergence of inner cells toward the future dorsal segment (leading to ES formation) is a mechanical consequence of surface tension anisotropy. The normal course of epiboly is associated with periodic changes in the curvature of the blastoderm external surface, with new structures (the dorsal segment, GR, and ES) are marked off only when the surface curvature becomes maximally uniform. Although the ES in most embryos appears within the initial dorsal segment, individual developmental trajectories have been traced where the GR starts to form at the dorsal pole of the blastoderm but the ES develops on its opposite site, at the point of GR closure. In both cases, GR formation is initiated at the point of convergence of centrifugal cell migration flows that arise in the marginal zone of the blastoderm upon GR initiation or closure.

Early Development and Post Embryonic Skeletal Morphology of the Progeny of Spined Loach Cobitis taenia L. (Teleostei, Cobitidae) and its Naturally Occurring Allotriploids.

Polyploid fishes of the genus Cobitis represent a valuable model system to study the origin and consequences of hybridization and polyploidization within vertebrates. These naturally accessible polyploids are an excellent subject to determine the advantages or disadvantages of polyploidy. We investigated the embryonic and larval development with skeletal morphology of diploid and polyploid Cobitis progeny, obtained from crosses between females and males of Cobitis taenia and between allotriploid Cobitis females and C. taenia males. Observations were made during first fourteen days post fertilization. The pattern of development of all investigated individuals was the same. However the diploids developed synchronically, achieving successive stages faster than the polyploid ones; hatching was observed at 50 and 63 hours post fertilization, respectively. Statistically significant differences in hatching success and survival rate between diploid and polyploid progeny were not observed. All newly hatched larvae were characterized by a large amount of yolk, forty myomeres, body pigmentation and four external gills. Skeletal elements of the chondrocranium in the first days post hatching consisted of the otic capsule, ethmoid plate, trabeculae cranii and Meckel's cartilage. In contrast to the diploids, the polyploid larvae were characterized by a higher number of deformities. This study gives new comparative data on the features of early development of diploid and polyploid Cobitis progeny.

[The influence of low-intensity laser irradiation on the loach embryo cells].

The ultrastructural organization of loach embryo cells (Misgurnus fossilis L) at the stage of the first and the tenth embryo divisions was investigated in the control and under the influence of low intensity helium-neon laser irradiation of 5 min exposure. The effect of laser irradiation led to ultrastructural changes in cell organelles, increasing the number and size of mitochondria, and as a result their shape changes. Under the influence of laser irradiation, the activation of cellular digestion processes took place, the number of vacuoles and lysosomes increased. The results explain the possible mechanism laser irradiation impact at the cellular level.

Homologue of Sox10 in Misgurnus anguillicaudatus: sequence, expression pattern during early embryogenesis.

A number of genetic studies have established that Sox10 is a transcription factor associated with neurogenesis in vertebrates. We have isolated a homologue of Sox10 gene from the brain of Misgurnus anguillicaudatus by using homologous cloning and RACE method, designated as MaSox10b. The full-length cDNA of MaSox10b contained a 311 bp 5'UTR, a 312 bp 3'UTR and an ORF encoding a putative protein of 490 amino acids with a characteristic HMG-box DNA-binding domain of 79 amino acids (aa: 105-183). Phylogenetic tree shows that the MaSOX10b fits within the Sox10 clade and clusters firmly into Sox10b branches. During embryogenesis, MaSox10b was first detected in gastrulae stage. From somitogenesis stage and thereafter, distinct expression was observed in the medial neural tube, extending from the hindbrain through the posterior trunk. Taken together, these preliminary findings suggested that MaSox10b is highly conserved during vertebrate evolution and involved in a wide range of developmental processes including embryogenesis and neurogenesis.

[Discovery and its significance of spawning grounds of Jinshaiasinensis from upper and middle Jinshajiang River].

During April, July to August and November of 2012,three surveys were conducted to estimate the early-stage fish resources in the area of Liyuan power station on Jinshajiang River of Yunnan Province. During the survey conducted at the crossing of Daju Town, Yulong County, Yunnan Province, from July to August, 2012, two egg-tides of Jinshaia sinensis were detected. One was from 8:00 of August 1(st) to 10:00 of August 4(th) and the other one was from 23:00 of August 9(th) to 6:00 of August 13(th). The average egg diameter was 3.89 mm, ranging from 3.37 to 4.41 mm. Natural fertilization and hatching rate as well as abnormality rate in mimic wild environment were 91.5%, 97.1% and 2.2%, respectively. Meanwhile, three spawning grounds had been conjectured, which were Longpan Town, Liming Town and Judian Town of Yulong County, Yunnan Province. Moreover, the total number of eggs from those three spawning grounds during the two egg-tides was approximately 1.49×10(7) ind.. The discovery of the three spawing grounds is of importance in fullfilling the study of Jinshaia sinensis, and in protecting fish resources under the influences of power stations.

Development of the mandibular, hyoid arch and gill arch skeleton in the Chinese barb Puntius semifasciolatus: comparisons of ossification sequences among Cypriniformes.

The morphogenesis and sequence of ossification and chondrification of skeletal elements of the jaws, and hyoid arch and gill arches of Puntius semifasciolatus are described. These data provide a baseline for further studies and enable comparisons with other described cypriniforms. Some general patterns of ossification in the hyoid arch and branchial arches in cypriniforms were notable. First, the overall development is from anterior to posterior, with the exception of the fifth ceratobranchial bone, which ossifies first. Second, where ossification of iterated elements is sequential, it tends to proceed from posterior to anterior, even when more posterior chondrifications are the smallest in the series. Ossification of the ceratobranchial, epibranchial and pharyngobranchial bones tends to proceed from ventral to dorsal. The comparisons revealed small sets of skeletal elements whose ossification sequence appears to be relatively conserved across cyprinid cypriniforms. Several potentially key timing changes in the ossification sequence of the jaws, hyoid arch and gill arches were identified, such as the accelerated timing of ossification of the fifth ceratobranchial bone, which may be unique to cypriniforms.

[Morphogenetic consequences of partial removal of blastomere cytoplasm during early embryonic development of the loach, Misgurnus fossilis L].

The development of loach embryos is successfully regulated (normalized) after partial removal of the cytoplasm from one blastomere at the two- or four-cell stage or complete removal of one or two blastomeres at the stage of 8-16 cells. Using time-lapse video imaging and morphometric analysis, it has been shown that this regulation is a two-stage process. At the first stage, the ratio between the volumes of the blastodisk and yolk sac is rapidly (within one or two cell cycles) restored almost to the initial level; at the second stage, morphogenesis of the embryo is modified according to its new structural features acquired after the operation. After several rounds of cytokinesis, the cytoplasm remaining in the operated blastomere fuses with the marginal yolk syncytium (periblast),which at the blastula stage forms a distinct extension at the operation site. This extension marks the site of embryonic shield formation. The results of morphometric analysis show that restoration of the initial blastoderm volume in operated embryos leads to a reduction of active tension at the blastoderm--yolk boundary and an increase in the ratio of blastoderm surface to its volume at the moment of epiboly initiation. As a result, the convergence of blastoderm cells to the operation site and the embryonic shield formation begin at a lesser degree of epiboly, compared to the control.

Morphogenesis of active shells.

We consider the active shell as a single-cell or epithelial sheet surface that, sharing basic properties of stretched elastic shells, is capable of active planar movement owing to recruiting of the new surface elements. As model examples of their morphogenesis, we consider the growth and differentiation of single-cell hairs (trichomes) in plants of the genus Draba, and the epiboly and formation of the dorsoventral polarity in loach. The essential feature of the active shell behavior at both cellular and supracellular levels is regular deviating from the spatially homogeneous form, which is a primary cause of originating of the active mechanical stresses inside the shell in addition to its passive stretching by the intrinsic forces. Analyzing the quantitative morphological data, we derive the equations in which the temporal self-oscillations and spatial differentiation are distinguishable only at the parametric level depending on the proportion of active to passive stresses. In contrast to the ordinary activator-inhibitor systems, the self-oscillation dynamics is principally non-local and, consequently, one-parametric, the shell surface curvature being an analog of the inhibitor, while its spatial variance being an analog of the activator of shaping. Analyzing variability and evolution of the hair cell branching, we argue that the linear ontogeny (succession of the developmental stages) is a secondary evolutionary phenomenon originating from cyclic self-organizing algorithms of the active shell shaping.

Developmental potential of embryonic cells in a nucleocytoplasmic hybrid formed using a goldfish haploid nucleus and loach egg cytoplasm.

In teleosts, viable nucleocytoplasmic hybrids, formed by combining a nucleus from one species with the egg cytoplasm of another, have been used as one of the methods for breed improvement in aquaculture, but have been little exploited for developmental biology studies. Here, we used an artificial androgenesis technique to form nucleocytoplasmic hybrids comprising a goldfish haploid nucleus and loach egg cytoplasm. These hybrids were used to investigate interactions between the nucleus and cytoplasm during embryonic development. Additionally, the developmental characteristics of embryonic cells of nucleocytoplasmic hybrids were examined in chimeras produced by transplantation of blastomeres into recipient loach or goldfish embryos. We found that the nucleocytoplasmic hybrids arrested at the dome stage of embryonic development and did not form any gastrula structures. The goosecoid (gsc) and no tail (ntl) genes were expressed normally before gastrulation in nucleocytoplasmic hybrids, similar to diploid loach. However, expression of the gsc and ntl genes was not maintained in nucleocytoplasmic hybrids. In chimeric embryos, blastomeres derived from nucleocytoplasmic hybrids were found to mix with the cells of recipient loach embryos at the gastrula stage. The transplanted blastomeres formed small clusters at the somitogenesis stage and, finally, small spots at the hatching stage. In contrast, when the blastomeres were transplanted into goldfish embryos, the transplanted blastomeres aggregated in the chimeric embryos. Thus, embryonic cells from nucleocytoplasmic hybrids that arrest before gastrulation could survive beyond the somitogenesis stage depending on the cytoplasmic environment in the recipient embryos.

[The influence of potassium 4-toluenethiosulfonate on membrane potential and ATPase activity of plasmatic membranes of loach embryos].

The influence of novel biologically active substance potassium 4-toluenethiosulfonate in concentration of 4 x 10(-5) M on the changes of electrophysiological parameters of embryonic cells in early development of fish (Misgurnus fossilis L.) was investigated including the changes of membrane potential (TMP) and enzyme activity of plasmatic membranes of loach embryos during the period of synchronous division of blastomers in the early period of development. The evaluation of influence of these matters was studied and aperiodic changes of the level of TMP were shown. The diminishing of amplitude in every period by 7/12 mV and diminishing of growth of maximal values of vibrations of TMP by 39 mV in comparison to control was noticed. It was related to inhibition of some biosynthetic processes and results in the decline of activity of membrane enzyme (Na+, K(+)-ATPase) by 75.5 +/- 4.1% and 78.4 +/- 10.4% both at the action of high (4 x 10(-3) M) and low (4 x 10(-9) M) concentrations, accordingly, at first hours of development with subsequent renewal of its activity to the level of control only for the actions of low concentration.

[Stage-specific changes in membrane microviscosity in Misgurnus fossilis embryos].

Natural and probe fluorescence as well as membrane microviscosity was studied in eggs and embryos of Misgurnus fossilis by fluorescence microscopy. The lateral mobility of the probe (pyrene) increased in loach embryos from early to late blastula, which indicates a decrease in plasma membrane microviscosity. At the later stage of mid-gastrula, the microviscosity remained largely invariant. Considering that the embryo exposure to different temperatures changes the quantum yield of fluorescence and the degree of pyrene excimerization, one can gain information about both the temperature-induced structural changes and changes in membrane microviscosity in the embryos. Natural and probe fluorescence of embryonic membranes is proposed as at tool to study morphogenetic mechanisms.