Triploidy induction in Australian greenlip abalone Haliotis laevigata (Donovan) with cytochalasin B

Triploid induction in Australian greenlip abalone, Haliotis laevigata (Donovan), was conducted by blocking the formation of the second polar body using cytochalasin B (CB). Twenty minutes after fertilization, the zygotes of greenlip abalone were treated with four CB concentrations (0, 0.25, 0.5 and 0.75 mg L⁻¹) for 10, 15 and 20 min. The ploidy of resultant larvae was determined using flow cytometry at 72-h post fertilization. Our study showed that fertilization, hatching, survival and induced triploidy of abalone larvae were significantly affected by the CB concentration and treatment duration. The effective range of CB concentration for triploid induction on greenlip abalone was 0.5–0.75 mg L⁻¹ with an induction duration of 10–15 min. The results indicate that the most effective treatment combination for triploid induction in greenlip abalone is 0.5 mg CB L⁻¹ for 15 min starting at 20-min post fertilization.     

Hybridization between Haliotis rufescens and Haliotis discus hannai: evaluation of fertilization,larval development,growth and thermal tolerance

Two introduced abalone species are currently produced in Chile, red abalone Haliotis rufescens and Japanese abalone Haliotis discus hannai. However, red abalone accounts for 99% of total production, while the Japanese abalone has not adapted well to Chilean coastal waters. This study reports the hatching, growth and thermal tolerance performance in interspecific hybrids produced between red (R) and Japanese (J) abalone. Our results show that egg age and sperm concentration were critical factors to produce hybrids. The cross R♀ × J♂ showed a fertilization rate of 55.3 ± 3.5% using 20‐min‐old eggs and sperm concentrations of 14 × 10⁶ cells mL^?1, while the reciprocal cross (J♀ × R♂) was not successful. Further, larval development stages were similar in RR, JJ and RJ hybrid abalones. Among the experimental trials, settlement rate varied from 12.3% to 18.6% and final survival from 20.1% to 31.7%, being the RJ hybrid rates intermediate between parental species. The final shell lengths were similar between RR and RJ hybrids, but significantly higher in JJ abalones. In addition, thermal tolerance was ascertained due its pivotal role for the abalone physiology. Thus, RJ hybrids showed the highest HSP70 gene expression and offers new possibilities to expand Chilean abalone production in warm waters zones.     

Experimental production of triploid brown trout, Salmo trutta L., using heat shock

Abstract. This study was designed to investigate the potential of heat shock to produce triploidy in brown trout, Salmo trutta L., and to develop a methodof routinely identifying triploids in this species. Triploids were produced in all heat-shocked batches and were identified by the size of their erythrocyte nucleus, which had a volume ratio of 1:1-57 relative to diploid controls. Cytogenetic and flow cytometric analyses confirmed that trout with the larger nuclei were triploid. Heat shock of 28°Cof 10 min duration initiated 5-15 min post-insemination produced high rates of triploidy in experimental batches (88-2-100%), later shocks at 20-25 min producing lower rates (down to 60%). Reproducibilicy of tripioid rates was generally good, a maximum difference between replicates of 21.9% being observed, the majority of differences being considerably less. The highest triploid yield was produced with a heat shock of 28°C for 10 min initiated at 10 or 20 min post-insemination, the difference between replicates being due to variability in survival to hatch. Survival to hatch was generally lower in groups having higher rates of tripioidy.     

Juvenile growth and survival of heat-shocked triploid hybrid saugeyes, Stizostedion vitreum × S. canadense

Triploid hybrid saugeyes may be a desirable stocking alternative because diploid saugeyes are fertile and can compromise the genetic integrity of parental stocks. Four groups of saugeye eggs were heat-shocked 5 min after fertilization for 15 min at 31 °C, which resulted in an 86.7 ± 9.4% triploidy rate and 57.8 ± 24.3% survival to the eyed-stage. Heat shocked and control saugeyes were stocked in two ponds for 40 days at 395 000 ind. ha^–1. Mean length and weight of triploid and heat-shocked diploid saugeyes were greater ( P  < 0.01) than unshocked diploid saugeyes. Survival of heat-shocked saugeyes (22.4%) was lower than unshocked saugeyes (94.7%). Heat-shocked fish ( n =25 851) harvested from ponds were stocked for 153 days in O'Shaughnessy Reservoir, Ohio, USA. Triploids were 34 mm shorter and weighed 41 g less than heat-shocked diploids ( P  < 0.01). Mean length and weight of triploids were comparable with unshocked diploids elsewhere in Ohio.     

Early survival rates and subsequent morphological abnormalities in landlocked,anadromous and hybrid (landlocked × anadromous) diploid and triploid Atlantic salmon

Upwards of 95% triploidy was induced in landlocked, anadromous and hybrid (landlocked female × anadromous male) Atlantic salmon using heat shocks (5 min at 32°C, 20 min after fertilization and incubation at 10°C). On average, 23% of the treated eggs died within 24 h of fertilization. Most of the subsequent mortality occurred prior to hatching. In all cases, triploid hybrids had better survival rates than triploids of either pure form; triploid hybrids had survival rates comparable to those of diploid hybrids, whereas the survival rates of triploid pure forms were substantially lower than their respective diploid pure forms.Two types of morphological abnormalities were apparent in underyearling parr derived from control and heat shocked groups. Short gill covers were present in both diploid and triploid groups, and were not associated with the heat shock. Protruding lower jaws, however, were found almost exclusively in triploid fish, but were not present in all triploid fish within any particular treated group. No diploid fish originating from previously heat shocked eggs had protruding jaws, so the predisposition to this abnormality seems to depend on the triploid condition and not the heat shock.     

An investigation into the reproducibility of triploid brown trout, Salmo trutta L., production using heat shock

A single heat shock of 28°C for 10 min, applied 15 min after water activation to five different batches of brown trout, Salmo trutta L., ova and replicated within one batch produced triploid rates ranging from 22% to 100% and variable survival to hatch ranging from 9.4% to 47.9%. Variability was greater among batches than among replicates. In general, the treatment reduced survival to hatch by 50%. The range of triploid yields obtained from a single heat shock regime is discussed in terms of potential advantages to aquaculture.     

Induction of triploidy in mud loach (Misgurnus mizolepis) and its effect on gonad development and growth

Triploidy was induced in mud loach (Misgurnus mizolepis) by cold shocking fertilized eggs 5 min post-fertilization at 2°C for 15 to 60 min. Best results were obtained when eggs were shocked for 60 min; 98% of fish examined in that treatment were triploids. Triploidy was confirmed by erythrocyte measurements and chromosome counts. Diploids had 48 chromosomes, while triploids had 72. Histological analysis of 9-month-old triploid ovaries showed an appreciable number of oocytes at the chromatin nucleolus stage with considerable interstitial tissue. However, diploids had well developed oocytes. Diploid testes from diploid males exhibited normal spermatids and spermatozoa, while a few were seen in triploid males. Growth rate was evaluated over a 9-month growth trial. Although male and female triploids were slightly heavier than their diploid counterparts from the third to the ninth month, their growth rates were not significantly different compared to their diploid controls.     

二倍体和三倍体皱纹盘鲍精子发生过程的超微结构

比较了二倍体和三倍体皱纹盘鲍精子发生过程中细胞和细胞器的超微结构变化。结果表明,二倍体皱纹盘鲍的精子发生经历了精原细胞、初级精母细胞、次级精母细胞、精子细胞和精子5个阶段。其形态结构发生了一系列变化,主要包括：核染色质浓缩、线粒体的发达与融合、顶体形成和胞质的减少。三倍体皱纹盘鲍各种生精细胞的直径和核径均大于二倍体,精原细胞结构与二倍体相似;初级、次级精母细胞的胞质中,除溶酶体外,线粒体、内质网等细胞器少于二倍体,线粒体大小与二倍体没有差别,但形态不典型,层状嵴不发达;三倍体皱纹盘鲍的精子发生停滞在精子细胞阶段,表现出各种畸形状态,很多趋于解体,没有发现成熟精子。     

Gynogenesis induction and sex determination in the Eurasian perch,Perca fluviatilis

In the present study, we used meiotic gynogenesis, widely used in studies on sex determination, to confirm female homogamety in Eurasian perch, Perca fluviatilis. Sperm irradiated with UV for 400 s was used to artificially fertilized eggs. The diploid of the resulting embryos was restored by a heat shock (30 °C) applied to the eggs 5 min postfertilization, for 25 min. Fertilization (ranging between 45% and 75%) and survival rates at hatching (ranging between 3.4% and 46.6%) were not significantly different (P>0.05) between the diploid control and gynogenetics. The diploid controls and two batches of gynogenetics contained 100% diploid larvae, whereas two other batches of gynogenetics contained 6.7% and 10.0% triploid larvae. The sex ratios of the diploid controls were not significantly different from 1:1, whereas all gynogenetic families were 100% female. These results confirm female homogamety in Eurasian perch, demonstrated by the use of hormonally mascilinized breeders in a previous study.     

Triploidization in rohu × mrigal hybrid and comparison of growth performance of triploid hybrid

The present study was aimed at the identification of treatment optima to induce triploidy in ‘Labeo rohita (rohu) × Cirrhinus cirrhosus (mrigal)’ hybrid using heat shock treatment. The eggs were exposed at four different temperature regimes viz., 38, 39, 40 and 41°C for 1–3 min, applied 3–5 min after fertilization. After 4 min of fertilization, heat shock treatments for 1 and 1.5 min durations were found the best inducing triploidy up to 100% and 96% respectively. Survival rates upto yolk sac absorption were found to be 73% and 71% in rohu and mrigal, 68% and 67% in the reciprocal diploid hybrids and 61% and 60% in the reciprocal triploid hybrids (RTH). Triploidy was confirmed by chromosome counting that revealed the diploid chromosome number of rohu and mrigal at 2n = 50 and in their triploid hybrid chromosome number was found to be 3n = 75. Growth rate of the RTH showed a significant difference (P < 0.05) from the single species and the diploid hybrids. Triploids also showed higher survival rate over the diploids.     

Growth and feeding in juvenile triploid and diploid blacklip abalone, Haliotis rubra (Leach, 1814), at two temperatures

Growth and feeding of juvenile triploid and diploid blacklip abalone Haliotis rubra (Leach, 1814) were investigated at two temperatures of 17 and 21 °C over a 50‐day period. There were no differences in growth between triploid and diploid abalone as measured by shell length and body weight. Both triploid and diploid abalone increased in length but not in weight at 21 °C. Condition indices were similar for triploid abalone maintained at both temperatures; however, those for diploid abalone were significantly higher at 17 °C than at 21 °C. Food intake was significantly greater yet feed conversion efficiency was significantly lower in triploid than in diploid abalone. Both the feeding variables were independent of temperature. On average, diploid abalone were able to convert 1 g of dry food ingested to 0.58 g of body weight, but triploid abalone only 0.44 g.     

Optimization of triploid induction by the use of 6-DMAP for the oyster Crassostrea gigas (Thunberg)

Abstract. A novel method using 6-dimethylaminopurine (6-DMAP) has recently been demonstrated to be a good triploid inductor for bivalve molluscs. The effects of 6-DMAP concentration, timing of treatment after fertilization, and treatment duration on survival and triploid induction of Crassostrea gigas (Thunberg) embryos were examined. An influence of sperm concentration was also detected and discussed. Survival to D-stage was inversely related to 6-DMAP concentration and the percentage of triploids was shown to be 6-DMAP dose dependent. Two optimal moments for treatment application were determined, corresponding to extrusions of the first and second polar bodies. Optimal treatment of 450μmol/1 6-DMAP beginning 15min after fertilization over a 10min period yielded mean survival to D-stage of 64% with a mean of 85% triploid production. As a comparison, a sample treated with 1 mg/1 of cytochalasin B (CB), 20 min after fertilization over a 15 min period, yielded a mean survival to D-stage of only 36% with a mean of 95% triploid production. The advantages of 6-DMAP over CB are clearly identified: this chemical is not carcinogenic, cheaper than cytochalasin B and much easier to use as it is water soluble.     

Induced triploidy in the European clam, Ruditapes decussates (L.), and performance of triploid larvae

Abstract. For the first time, effective treatments using cytochalasin B were developed to induce triploidy in the European clam, Ruditapes decussatus (L.). The percentage of triploid embryos was assessed by karyological or image analysis. Two treatments (0·5 or 1 mg of cytochatasin B (CB) per ml of dimethyisulfoxide in 1 litre of sea water) were applied at different times after fertilization (10 to 25min), for two different periods (15 and 20min). Best results were obtained for a CB concentration of 1 mg/1. When treatment was applied 15min after fertilization for a 20min period, 94% and 95% of triploid embyros were obtained in two repeated experiments. At metamorphosis, the treated larvae appeared to be no smaller than the control larvae in all experiments. However, in general, significantly higher mortalities for CB-treated batches were found when compared with the untreated batch.     

Reproductive ability of triploid ornamental (koi) carp (Cyprinus carpio L.) × goldfish (Carassius auratus L.) hybrids and characteristics of their offspring

The purpose of this study was to investigate reproductive ability of backcross triploid koi (Cyprinus carpio L.) × goldfish (Carassius auratus L.) hybrids. These triploids have been obtained by crossing of F₁ hybrid females producing diploid eggs with males of parental species. Triploid hybrid females, when crossed with goldfish or koi males, produced mostly aneuploid fish with ploidy range from approximately 2.2n–3.2n with a mean value 2.5n; some fish in crosses of triploid females with koi males were tetraploid (4.0n). Since analysed fish had in their genomes one haploid set from parental males, the data indicate that triploid hybrid females mostly produced aneuploid eggs with ploidy range from approximately 1.2n–2.2n and a mean ploidy around 1.5n while some eggs were triploid. Triploid hybrid males were completely sterile and have not released any sperm after hormonal injection. Despite their low viability, some aneuploid fish obtained from triploid hybrid females were raised in indoor recirculating systems until the age of 2 years and their reproductive ability has been evaluated. One aneuploid female with ploidy 2.1n produced larvae with ploidy range from 2.9n to 3.4n with a mean ploidy of 3.1n when crossed with a koi male; about 60% of obtained larvae had ploidy from 3.0n to 3.2n. These data indicate that this female produced mostly eggs with unreduced ploidy level.     

Growth and reproductive performance of triploid and diploid blacklip abalone, Haliotis rubra (Leach, 1814)

Growth and reproduction of triploid and diploid blacklip abalone Haliotis rubra (Leach, 1814) were compared in a 30-month study. Triploidy was induced by inhibition of the second polar body formation using 6-dimethylaminopurine (6-DMAP) or cytochalasin B (CB). There were no significant differences in growth and survivorship between triploid and diploid abalone. However, triploid abalone had a more elongated shell and greater foot muscles than diploid abalone. A slightly curvilinear growth in shell length was conformed to all treatments. While diploid abalone had reached sexual maturity and spawned during the study, gonadal development and gamete maturation were abnormal in triploids. Female triploids lacked an apparent gonad at the macroscopic level but microscopic examination revealed that they had a thin layer of oogonia development. In contrast, male triploids were able to form similar-sized gonads to diploids during most of the reproductive period, but with brown-yellow discolouration and stalled gametogenesis at spermatocyte formation. Sex ratio of triploid abalone did not deviate from 1:1. With the onset of sexual maturation, growth and gonadal maturation occurred concurrently in diploid abalone, and there was no indication that growth of (diploid) abalone was reduced.     

Community and trophic structures of abalone <Emphasis Type="Italic">Haliotis diversicolor</Emphasis> habitat in Sagami Bay,Japan

Abstract:   The community and trophic structures of the subtidal habitat of the abalone Haliotis diversicolor were examined in Nagai, Sagami Bay, Japan. Conventional community indices showed no significant differences among three sampling stations. The overall continuum of stable isotope ratios was structured into three different trophic linkages: (i) brown algae-dependent benthic food chain; (ii) red algae-dependent benthic food chain; and (iii) planktonic food chain. Brown algae and red algae likely play different roles with respect to carbon sources in the habitat. Conventional fractionation values indicated that the abalone H. diversicolor (δ¹³C = −12.4 ± 1.0‰, δ¹⁵N = 9.3 ± 0.5‰) feeds on the lamina of Undaria pinnatifida during juvenile and adult stages (8.0–65.6 mm shell length). Stable isotope signatures suggested that the juveniles of other abalone species as well as some amphipods and a sea cucumber Holothuria decorata are competitors of H. diversicolor , whereas some Muricidae gastropods such as Thais bronni and Ergalatax contractus are predators. The isotopic differences among macroalgal species and the subsequent transfer to consumers indicate that stable isotopic analysis is an effective means of studying food webs in an open rocky shore community with little influence from external primary production such as terrestrial vegetation.     

静水压休克法诱导三倍体鲶鱼（silurus asotus L）的研究

采用静水压休克法诱导三倍体鲶鱼。通过对受精时间、静水压力及持续施压处理时间三方面进行筛选试验的结果表明，鲶鱼卵受精4－5min，用600－649kg/cm^2的静水压力处理3min，可以获得100％的三倍体鲶鱼，而且胚胎存活率也较高，孵化率达对照组的90％以上，是静水压休克法诱导三倍体鲶鱼的最佳条件。三倍体鲶鱼的倍性用细胞遗传学方法验证。     

Ovaprim treatment promotes oocyte development and milt fertilization rate in diploid and triploid African catfish (<Emphasis Type="Italic">Clarias gariepinus)</Emphasis>

Triploid fish are increasingly used in aquaculture because they are generally unable to reproduce successfully. Energy is channeled into somatic growth rather than gonadal development, and in the event of escape, the animals are unlikely to breed successfully among themselves or with wild conspecifics. This study tested the ability of recently matured triploid African catfish (Clarias gariepinus) to produce and fertilize eggs with and without ovaprim treatment. Triploid females did not show the increase in ovary size observed in diploid members of the same cohort between 8 and 9 months of age, or the coincident decrease in visceral fat deposits, and this was unaffected by up to 5 weekly i.m. injections of 0.5 ml kg⁻¹ Ovaprim. However, we observed advanced vitellogenin (Vtg) sequestration in oocytes of triploid females, albeit to a lesser degree and with lesser cortical alveoli, compared to oocytes from diploid cohort members. Histological sections revealed a positive trend of oocyte development up to the third weekly ovaprim injection followed by a negative gonadal development in weeks four and five. Milt from triploid males injected 9–12 h earlier with 0.25 ml kg⁻¹ ovaprim i.m. fertilized more diploid eggs than milt from untreated triploid males (30 vs. 20%), but none of the developing embryos of triploid paternity survived to hatch. In contrast, milt of diploid males fertilized 49% of eggs, and 20% of the developing embryos hatched successfully. These rates were improved in ovaprim-injected diploid males to 70% fertilization and 33% hatch. This study demonstrates potential of overcoming non-viability of eggs from triploid female African catfish, and enhancing the ability of triploid milt to fertilize eggs.     

Preventive efficacy of sodium hypochlorite against water mold infection on eggs of chum salmon Oncorhynchus keta

ABSTRACT:   To protect chum salmon eggs from water mold infection during incubation, the eggs were treated daily with sodium hypochlorite solution (NaOCl) at 10 mg/L residual chlorine concentration for 15 min during their developmental period from fertilization to eyed stages. The number of infected eggs and number of eyed eggs were observed on day 23 of incubation. The percentage of infected eggs to total eggs was significantly lower with NaOCl treatment (1.8 : 33.4%) than in the control (11.3 : 59.3%, P  < 0.01). The percentage of eyed eggs to total eggs was significantly higher with NaOCl (85.9 : 98.6%) than in the control (66.1 : 97.5%, P  < 0.01). The antifungal activity of NaOCl resulted in improving egg survival. Accordingly, NaOCl is a useful antifungal agent against water mold infection on chum salmon eggs.     

Survival, growth and gonad development of triploid red sea bream, Pagrus major (Temminck et Schlegel): use of allozyme markers for ploidy and family identification

Abstract. Triploidy was induced in three full-sib families of sea bream, Pagrus major (Temminck & Schlegel), by subjecting the fertilized eggs to cold-shock treatment at 0°C for a 12 min duration, starting 3 min after insemination. Results showed that this treatment gave 100% triploid fish with reasonable survival rates of more than 61% up to first feeding, Triploidy induction was proved by the use of allozyme markers at three ( ADH *, EST * and 6PGD *) loci and red blood cell size analysis.
At 2 months of age, the survival of the triploid group was lower than that of the diploids but it was comparable at 10 months. No differences in growth rate between diploid and triploid full-sibs were observed up to the age of 10 months. However, differences in body weight between families were noticed both in diploid and triploid groups when cultured in the same net cage. This possibly suggests a high heritability for the trait examined in the broodstock. The gonads of the triploid fish were significantly smaller than those of the diploids, Histological analysis of the gonads revealed sterility in triploid fish.