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Silver crucian carp Carassius auratus langsdorfii comprises a diploid-polyploid complex in wild Japanese populations. Bisexually reproducing diploids are sympatrically distributed with gynogenetically developing triploids and tetraploids. Triploid and tetraploid males are very rare among Japanese silver crucian carp due to their gynogenetic reproduction. We examined the genetic characteristics of progeny that arose in a tank by natural spawning of a tetraploid silver crucian carp pair. The ploidy status of 120 samples randomly collected from these progeny was determined to be tetraploid by DNA content flow cytometry. DNA fingerprints from a random amplified polymorphic DNA assay indicated that almost all the progeny examined had genotypes identical to the maternal tetraploid female with no paternally derived fragments. Selected specimens’ cytogenetic analyses revealed that the progeny examined had tetraploid chromosome numbers, categorized into 40 metacentric, 80 submetacentric, and 80 subtelocentric or telocentric chromosomes, which were arranged into quartets and six supernumerary microchromosomes. Fluorescence in situ hybridization signals were detected in four homologous chromosomes in all analyzed metaphases prepared from diploid goldfish specimens. Contrary, tetraploid silver crucian carp gave eight rDNA signals. These results suggest that gynogenetic development in eggs spawned by tetraploid females should be triggered by tetraploid males’ homospecific sperm. [ABSTRACT FROM AUTHOR]

To develop a suitable cryopreservation diluent for spermatozoa of the endangered Sakhalin taimenHucho perryi, all possible combinations of cryoprotectants (glycerol, dimethyl sulfoxide[DMSO], methanol) and extenders (fetal bovine serum[FBS], 300 mM glucose solution[GS], artificial seminal plasma for masu salmon) were examined by observing sperm motility 10 s after thawing. Spermatozoa cryopreserved with diluents such as mixtures of 10% glycerol plus 90% FBS, 10% DMSO plus 90% FBS, and 10% methanol plus 90% GS showed the highest motility. The maximal post-thaw motility was observed at 10% among all concentrations (0, 5, 10, 15 and 20%) of these three cryoprotectants. No significant difference among three diluents was observed in motility at 10 s. Mixtures of 10% glycerol plus 90% FBS, 10% DMSO plus 90% FBS, and 10% methanol plus 90% GS are suitable cryopreservation diluents for Sakhalin taimen spermatozoa. [ABSTRACT FROM AUTHOR]

Gynogenesis is a phenomenon in which only the maternal genome contributes to offspring development. Artificial gynogenesis induced using genetically inactivated sperm is a useful technique for sex control and rapid production of inbred lines in aquaculture and breeding. For gynogenesis induction, eggs require fertilization stimulus from the sperm; however, the effects of sperm characteristics on gynogenetic success remain unexplored. In this study, we investigated the induction of gynogenesis in medaka embryos by sperm from distant species and the correlation of sperm head size relative to the micropyle with success in gynogenesis. Results revealed that sperm heads of Nile tilapia and rainbow trout had smaller areas than those of the medaka micropyle, and thus, the sperm successfully activated medaka eggs for development. Furthermore, gynogenetic haploid embryos could be induced using genetically inactivated sperm from these species, which were viable after diploidization using heat shock treatment. In contrast, goldfish and zebrafish sperm, with larger head areas than that of medaka micropyle, could not activate medaka eggs for development, suggesting that sperm entry into medaka micropyle was inhibited. Therefore, we propose that the relative physical features of sperm and eggs influence the success of gynogenesis. The findings provide new insights into the application of gynogenesis in aquacultural fish. We developed an automatic method for measuring sperm head size, which can facilitate the recording of physical characteristics of sperm and predicting gynogenesis success. • Sperm collected from distant species are suitable for gynogenesis in fish. • Sperm head size relative to micropyle size can indicate gynogenetic success. • A method for the automatic measurement of the sperm head size was developed. [ABSTRACT FROM AUTHOR]

Ginbuna (Carassius auratus langsdorfii (Teleostei: Cyprinidae)) occur in diploid, triploid, and tetraploid forms in wild populations. Diploid females reproduce bisexually, whereas polyploid (triploid and tetraploid) females reproduce gynogenetically with no contribution from sperm nuclei. However, tetraploid males produce diploid sperm. The mechanism responsible for the differences in egg and sperm ploidy has not been elucidated as tetraploid males are rare in wild populations. Here, we aimed to characterize the types of sperm and elucidate the mechanism of spermatogenesis in ginbuna. In the present study, we artificially produced tetraploid males by crossbreeding triploid ginbuna females with diploid goldfish (Carassius auratus auratus) males via accidental incorporation of sperm nuclei. We then examined spermatogenesis to reveal the process by which reduced diploid sperm are generated from tetraploid germ cells. DNA fingerprinting by random amplified polymorphic DNA (RAPD)-PCR indicated that the tetraploid progeny had a paternally derived genome. For the tetraploid male sperm, there were narrow (N-type) and broad (B-type) flow cytometrical histograms. The N-type were determined to be diploid with a low coefficient of variation (CV) by flow cytometry. The B-type were found to be aneuploid (hypodiploid to hexaploid) with a high CV. The head sizes of B-type sperm were variable, whereas those of the N-type sperm were uniform. Computer-assisted sperm analysis (CASA) revealed that both the haploid and diploid B-type sperm were weakly motile compared with the haploid sperm of goldfish and the diploid N-type sperm of tetraploid males. Bivalents and various multivalents were observed in the meiotic configurations of diploid spermatogenesis. In aneuploid spermatogenesis, most of the chromosomes were unpaired univalents and there were very few bivalents. Our findings provide empirical evidence for two different types of spermatogenesis in tetraploid C. a. langsdorfii males. Meiotic synapses might explain the observed differences in the ploidy status of the two sperm types. • Two types of sperm, diploid and aneuploid, are found among tetraploid ginbuna. • Incorporation of the paternal genome into triploid eggs produced tetraploids. • Aneuploid sperm was less motile than haploid and diploid sperm. • Spermatocytes with multivalent configurations generate diploid sperm. • The occurrence of synapsis at meiosis may cause ploidy differences in sperm. [ABSTRACT FROM AUTHOR]

The aim of this study was to evaluate the cryopreservation of masu salmon (Oncorhynchus masou) semen, using 0.18 M glucose in 9% methanol as an extender, and to characterize semen parameters (sperm motility; sperm concentration; and seminal plasma parameters such as pH, electric conductivity, DNA concentration, and protein concentration), with respect to post-thaw sperm motility. Semen samples were collected from fishes in the middle of the spawning season, at Toya Lake station, Hokkaido University, and the end of the spawning season, at the Nanae freshwater station, Hokkaido University. The semen was diluted with the extender at a 1:5 ratio (semen:extender) and then equilibrated for 15 min. The final concentration was 0.15 M and 7.5% for glucose and methanol, respectively. Sperm motility was assessed for fresh, fresh-diluted, and post-thaw semen using computer-assisted sperm analysis. The post-thaw sperm showed lower motility, compared with those of fresh and fresh-diluted sperm, which ranged from 33.9% to 82.9% and 1.5% to 27.1% in the semen collected in the middle and end the spawning season, respectively. Sperm concentration, pH, and electric conductivity of the semen collected at the end of the spawning season were lower than those of semen collected in the middle of the spawning season. Moreover, remarkably high DNA concentrations were detected in the semen at the end of the spawning season, although no difference was observed in protein concentration. Finally, semen cryopreserved using the aforementioned method could be used for artificial fertilization to produce offspring. These results indicate that the glucose-methanol extender is useful for sperm cryopreservation in masu salmon and that the suitability of semen for cryopreservation is affected by the condition of the parental fish. • Glucose-methanol extender could be used for sperm cryopreservation in masu salmon. • Sperm cryopreservation success was affected by spawning season. • DNA concentration in seminal plasma is a robust biomarker for sperm quality. [ABSTRACT FROM AUTHOR]

Silver crucian carp Carassius auratus langsdorfii comprises a diploid-polyploid complex in wild Japanese populations. Bisexually reproducing diploids are sympatrically distributed with gynogenetically developing triploids and tetraploids. Triploid and tetraploid males are very rare among Japanese silver crucian carp due to their gynogenetic reproduction. We examined the genetic characteristics of progeny that arose in a tank by natural spawning of a tetraploid silver crucian carp pair. The ploidy status of 120 samples randomly collected from these progeny was determined to be tetraploid by DNA content flow cytometry. DNA fingerprints from a random amplified polymorphic DNA assay indicated that almost all the progeny examined had genotypes identical to the maternal tetraploid female with no paternally derived fragments. Selected specimens’ cytogenetic analyses revealed that the progeny examined had tetraploid chromosome numbers, categorized into 40 metacentric, 80 submetacentric, and 80 subtelocentric or telocentric chromosomes, which were arranged into quartets and six supernumerary microchromosomes. Fluorescence in situ hybridization signals were detected in four homologous chromosomes in all analyzed metaphases prepared from diploid goldfish specimens. Contrary, tetraploid silver crucian carp gave eight rDNA signals. These results suggest that gynogenetic development in eggs spawned by tetraploid females should be triggered by tetraploid males’ homospecific sperm. [ABSTRACT FROM AUTHOR]

We induced season independent smolts in masu salmon (Oncorhynchus masou) by rearing juveniles under an advanced photoperiod (AP) in which LD15:9 was interrupted by LD9:15 from June for 4 months. AP fish showed higher degree of skin slivering from November than fish reared under a simulated natural photoperiod (SNP) and became larger in length and lower in condition factor in January. Gill Na+,K+-ATPase in AP fish started increasing in November and peaked in December to typical spring smolt levels while it remained low in SNP fish. In addition, AP fish had higher hypo-osmoregulatory ability than SNP fish, as judged by the lower serum sodium and chloride concentrations 48 h after seawater transfer. Serum levels of insulin-like growth factor (IGF)-I and IGF-binding protein (IGFBP)-1b, components of the somatotropic axis, in AP fish peaked in November and January, respectively. Serum IGFBP-1b levels were increased 48 h after transfer to 70% seawater in both groups, suggesting that it is not a quantitative index of hypo-osmoregulatory ability in the short-term but a marker of osmotic stress. The present study showed that off-season smolts could be induced in underyearling masu salmon by a square-wave photoperiod, and off-season smolts showed morphological and physiological changes comparable to yearling spring smolts. Increased levels of circulating IGF-I and IGFBP-1b in smolting fish suggest that these endocrine parameters are involved in the smoltification process. • Underyearling masu salmon smolts can be induced by a square-wave photoperiod. • Serum IGF-I and IGFBP-1b are involved in the smoltification process. • Gill NKA activity in off-season smolt is comparable to that in spring smolt. [ABSTRACT FROM AUTHOR]

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.

The induction of germ-line chimerism in fish is a strategy for the reproduction of endangered or genetically valuable fish species. Chimeras can be created by transplanting a single primordial germ cell or multiple blastomeres from a donor into a sterile host embryo. When the host reaches sexual maturity, it will produce donor-originating gametes throughout its reproductive life span. This technique provides unique experimental conditions for basic biology research in model fish species like zebrafish. The success of cell transplantation relies on the effective sterilization of host embryos, the correct identification of developing germ cells, and the synchronization of migratory cues between the host and the transplanted cells. Developments in non-transgenic methods of germ cell ablation and identification have made germ cell transplantation more applicable to use in conservation and aquaculture. In this chapter, we provide a protocol for germ cell labeling by injection of chimeric RNA or FITC-dextran, the sterilization of host embryos using an antisense morpholino oligonucleotide, and two methods for producing germ-line chimeras in zebrafish: single primordial germ cell transplant and blastomere transplant.

Microinjection is a powerful tool for studying embryonic development and analyzing gene functions in fish. This technique was first applied to model species of fish such as zebrafish and medaka whose egg chorions could be removed or softened before microinjection. Recent progress in genome editing using TALEN and CRISPR has opened the opportunity to analyze gene functions in a much wider range of fish including those important to marine aquaculture. Therefore, application of the microinjection technique is also required in these species. However, the characteristics of fish eggs vary widely between species and several technical difficulties need to be overcome in order to use microinjection in a wider range of species. To obtain consistent results with microinjection, an optimal method has to be developed for each target species. In this chapter, we describe the physical characteristics of the eggs of fish species that have been used in microinjection experiments in our laboratory and detail the microinjection system we developed for fish eggs with a hard chorion, such as those of marine species.