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We report for the first time, a comparison of two approaches for artificially induced triploidy in zebrafish (Danio rerio) using cold shock and heat shock treatments. Of the two methods, heat shock treatment proved more effective with a triploid production rate of 100% in particular females. Subsequently, triploid zebrafish larvae were used as recipients for intraperitoneal transplantation of ovarian and testicular cells originating from vas:EGFP strain in order to verify their suitability for surrogate reproduction. Production of donor-derived sperm was achieved in 23% of testicular cell recipients and 16% of ovarian cell recipients, indicating the suitability of triploids as surrogate hosts for germ cell transplantation. Success of the transplantation was confirmed by positive GFP signal detected in gonads of dissected fish and stripped sperm. Germline transmission was confirmed by fertilization tests followed by PCR analysis of embryos with GFP specific primers. Reproductive success of germline chimera triploids evaluated as fertilization rate and progeny development was comparable to control groups. • Zebrafish triploids can be produced by a simple heat shock treatment. • Triploids are convenient candidates for surrogate reproduction in zebrafish. • Donor-derived sperm was produced after germ cell transplantation into triploids. • Fertility performance of triploid germline chimera is similar to control males. [ABSTRACT FROM AUTHOR]

The aim of this study was to evaluate the growth, carcass yield, fatty acid composition, and reproductive parameters of diploid and triploid Astyanax altiparanae . Triploidization was induced by heat shock (40 °C for 2 min) 2 min after fertilization. Fish were reared for 175 days from fertilization. Triploid females showed lower proportion of saturated and poly-unsaturated fatty acids and higher amount of mono-unsaturated fatty acids than diploids. They were sterile, with immature gonads, and showed higher carcass yield than diploid females, which exhibited higher gonadosomatic indices. Triploid and diploid males showed similar gonad morphology, although diploid males produced greater numbers of spermatozoa. All males exhibited the secondary sex characteristic of spines in the pelvic and anal fins. Fatty acid composition and growth were similar in triploid and diploid males. Triploidy did not ensure sterility in males, but triploid females were sterile and had increased carcass yield, and may be used in aquaculture to increase the production of A. altiparanae. Statement of relevance To our knowledge, this is the first study addressing the effect of triploid induction on growth parameters of a neotropical species. The important results for carcass yield and sterility in female triploid will improve the production of A. altiparane , as this species is being used as live-bait for sport fishing and direct consumption. [ABSTRACT FROM AUTHOR]

Androgenesis is a useful manipulation to fix male-specific genetic traits as well as to restore the genotypes of cryopreserved sperm, and it has been induced by the genetic inactivation of the egg nucleus with gamma-, X- or UV-ray irradiation before fertilization. Recently, a technique for cold-shock induced androgenesis was developed in a freshwater species, the loach, Misgurnus anguillicaudatus , in which viable androgenetic diploids and doubled haploids (DHs) were successfully produced by the fertilization of diploid sperm and chromosome doubling by heat-shock treatment, respectively. This technique was immediately applied for cloning in the zebrafish, Danio rerio . Here, we reported the first successful induction of androgenetic development by means of cold-shock treatment (at 0 °C for 15 min) just after the fertilization (within 10 s) of eggs in a marine aquaculture fish species, the Japanese flounder, Paralichthys olivaceus . Then, androgenetic embryos thus generated were subjected to hydrostatic pressure treatment (650 kg/cm 2 , 6 min) after incubation at 17 °C so as to produce DHs by chromosome doubling. The yield rate of putative DHs, which was estimated as the frequency of diploid larvae at the first feeding stage in relation to the total eggs used, ranged from 0.81% to 1.79%. The complete homozygosity of these putative DHs was genetically verified using 36 microsatellite markers that covered 24 linkage groups of Japanese flounder. Thus, an androgenetic clonal line was produced with the sperm of a mito-gynogenetic DH male by using cold-shock and subsequent hydrostatic pressure-shock treatments. Statement of relevance The results of our manuscript confirm the broad applicability of the cold-shock induced androgenesis method in commercially important marine fish species. [ABSTRACT FROM AUTHOR]

Abstract: This study seeks to determine the best method for preventing the second body (PBII) extrusion as a means to induce triploidy in the black tiger shrimp Penaeus monodon. Chemical (cytochalasine-B and 6-dimethylaminopurine) and temperature (heat and cold) shocks were applied to newly fertilized eggs. Cold shock that was administered at 8°C, for 10min at 8min post-spawning, was the best method for PBII triploidy induction, as evidenced by the highest percentage of forming three pronuclei in syngamy. Therefore, cold shock induction was employed to further explore the advantage of triploid over diploid shrimp. After hatching of the cold-shocked eggs, the larvae were allowed to reach juvenile and adult levels of development. The number and amount of chromosomes were determined in juveniles and adult stages, using Fluorescence Activating Cell Sorting methods, by which the shrimp were divided into diploid and triploid groups. At day 150 in culture, the average body weight of the triploid females (35.2±3.2g) and triploid males (31.5±3.5g) was significantly higher (P <0.0001) than that of their diploid counterparts (24.5±0.5g for females and 23.1±3.8g for males), having a ratio of 2 females:1 male for triploid shrimp, and 2 females:3 males for diploid shrimp. These results reveal the advantages of growing triploid over diploid P. monodon, and its feasibility for commercial production. [Copyright &y& Elsevier]

Abstract: Mitotic gynogenetic diploid or doubled haploid Japanese flounder Paralichthys olivaceus were produced by activating eggs with UV irradiated sperm of red sea bream (Pagrus major), followed by hydrostatic pressure treatment to block the first mitotic division. To verify the doubled haploid, we measured the homozygosity of 21 maternally heterozygous microsatellite loci that are located in the telomeric region of chromosomes. These 21 loci have large marker-centromere distances of 43 to 50cM estimated from four meiotic gynogenetic diploid lines, suggesting the location at telomeric regions of chromosomes. These 21 loci cover 16 out of the total 24 linkage groups of Japanese flounder. Microsatellite genotyping of these 21 markers in four mitotic gynogentic diploid lines shows that 75% (58 out of 77) individuals are homozygous at all 21 loci, indicating doubled haploids. The other 25% are heterozygous at 1 to 13 loci, probably representing spontaneously occurring meiotic gynogenetic diploids. These results demonstrate the high efficiency of generating doubled haploids in just one generation by mitotic gynogenesis. We have used these doubled haploids to successfully generate clonal lines via a second round of gynogenesis. [Copyright &y& Elsevier]

Tetraploid fish are an important source of diploid gametes for polyploid production, and they can be induced by inhibition of the first mitotic cell division. Dispite its potential, very few protocols regarding tetraploidization and subsequent application to mass production of triploid fish for aquaculture exist. In the yellowtail tetra Astyanax altiparanae , triploids present sterility and increased carcass yield, suggesting the generation of tetraploid for large-scale production of such triploids. In this study, the efficacy of heat shock treatments (40° for 2 min) at 16, 18, 20, 22, 24, and 26 min post-fertilization (mpf) at an incubation temperature of 22 °C were compared on tetraploid induction of A. altiparanae. Ploidy status was confirmed by flow cytometry, karyotyping, and nuclear diameter of erythrocytes. Afterwards, sperm parameters and fertility capacity of tetraploid males were evaluated. The results showed that heat shock decreased the hatching rate, especially at 22 (19.1 ± 9.6%), 24 (16.0 ± 9.1%), and 26 mpf (10.3 ± 6.2%) in comparison with the control group (62.2 ± 7.7%). The flow cytometric analysis showed that the control group and the treatments with heat shock at 16 and 18 mpf presented only diploid individuals. However, from 20 mpf onwards, tetraploid individuals were observed, and the highest percentage arose at 26 mpf (94.55%). When mature, tetraploid males are capable of producing diploid spermatozoa with fertilization capacity, generating a 100% triploid offspring. The results indicate that the heat shock treatment at 26 mpf (40 °C for 2 min) is optimum for tetraploid induction in the A. altiparanae. Additionally, tetraploid mature males generated diploid spermatozoa and can be used to mass production of triploid fish. The current data are the first report of tetraploids in family Characidae, presenting an application in basic and applied sciences. • Tetraploidization were optimized in Astyanax altiparanae by heat-shock (40 °C; 2 min) at 26 min post-fertilization. • High percentages (94.55%) of tetraploids were obtained. • Tetraploid males are viable and can produce diploid gametes. • 100% triploid offspring were generated with diploid spermatozoa from tetraploid males. • This was the first protocol of induced tetraploids in Neotropical Characins. [ABSTRACT FROM AUTHOR]

Androgenetic doubled haploids (DHs) were induced in the loach Misgurnus anguillicaudatus (Cobitidae) without irradiation of the eggs. The eggs of wild-type females were activated with the intact sperm of an orange-phenotype male, and treated (within 10 s of activation) at 3 ± 0.5 °C for 30 min, to eliminate the female nucleus. The eggs were then incubated in a water bath at 20 ± 0.5 °C for 35 min. Finally, diploidy was restored (65 min after activation) by heat-shock treatment at 42 ± 0.5 °C for 2 min. Under these conditions, the yield rate (mean ± SD) of putative DHs relative to the total number of eggs used was 10.43 ± 1.69%, which was significantly higher ( P < 0.05) than the yield rates obtained under the remaining heat-shock initiation conditions (55 min, 60 min, and 70 min after activation). We analyzed the ploidy status of the putative DH by using flow cytometry. All-male inheritance was confirmed by the expression of the recessive orange body color trait and microsatellite genotypes. We detected no maternally derived alleles or heterozygous genotypes at any of the 28 loci (covering 27 linkage groups) of loach, indicating the exclusively paternal inheritance and homozygosity of the obtained androgenetic DHs. [ABSTRACT FROM AUTHOR]

Abstract: Tetraploid loaches have been discovered amongst specimens recovered from fish markets in Japan. These tetraploids can be used as a source of diploid gametes to assist the further expansion of ploidy manipulation. Here, we produced a first generation neo-tetraploid strain by fertilizing eggs from a normal diploid female with diploid spermatozoa from a natural tetraploid male, followed 5min later by heat shock (42°C, 2min duration) to inhibit release of the second polar body. Diploid spermatozoa from the neo-tetraploid males produced were then used to create androgenetic diploid progeny by fertilizing UV-irradiated eggs from a normal diploid female. Triploid progeny were produced by crossing a normal diploid female with a neo-tetraploid male. Tetraploid progeny were produced by cold shock (1°C, 40min duration), beginning 5min after fertilizing normal eggs with diploid spermatozoa from first generation neo-tetraploid males. Reproductive performance of second generation progeny was also examined. Androgenetic diploid males generated fertile haploid spermatozoa. Triploid males were sterile, but a triploid female laid fertile haploid eggs. Second generation neo-tetraploid males were considered sterile. [ABSTRACT FROM AUTHOR]