Meiotic Gynogenesis Induction in Muskellunge Esox masquinongy by Thermal and Pressure Shocks

Mass production of fast‐growing, all‐female muskellunge Esox rnasquinongy by gynogenesis requires optimized techniques of preventing second polar body extrusion. Heat, cold, and pressure shocks were evaluated for their efficiency of doubling the maternal genome. Muskellunge eggs (20–40 g) were activated with 1 mL ultraviolet (UV)‐irradiated (1,248 J/ m²) heterologous sperm of yellow perch Perca flavescens. Survival and ploidy (by flow cytometry) were determined during the eyed‐stage. Cold shocks of 1.3 × 1 C were applied at 5 or 20 min after gamete activation with water (time of initiation, TI) for a duration of 150 min and pressure shocks of 48,263 or 55,158 kPa (7,000 or 8,000 psi, respectively) at a TI of 4 rnin for 12 min. These shock treatments resulted in 43.7–95.0% diploid gynogens with corresponding yield of diploid gynogen (percent diploid gynogens × total percent survival) of 2.6–11.I%. Cold shocks applied at TI of 5 or 20 min after activation resulted in statistically similar percent survival, percent diploid gynogens, and yield of diploid gynogens. Heat shocks of 31 × 0.1C applied at a TI of 5 to 15 min for a duration of 5 min resulted in 4.8–21.1% diploid gynogens with yields of 0.1–0.4%. Cold and pressure shocks have better potential than heat shock for preventing the second polar body extrusion. Muskellunge eggs activated with UV‐irradiated yellow perch sperm, but not exposed to shock, resulted in 100% haploids with survival of 2.3–5.8%. No viable embryos were produced from the hybrid cross between muskellunge and yellow perch, thus, all diploids produced after the shock treatments were unambiguous meiotic gynogens. Muskellunge eggs fertilized with fresh muskellunge sperm (controls) showed 60.4–64.0% survival to the eyedstage and 100% diploidy. Considering that the sex‐determining mechanism in muskellunge follows the WZ female, ZZ male system, future efforts should be directed to test the efficiency of cold and pressure shocks for mass‐producing gynogenetic super female (WW) muskellunge.     

Induce triploidy by heat shock in Eurasian perch,Perca fluviatilis

In Eurasian perch (Perca fluviatilis), females grow significantly faster than males. Moreover, gonadal development has a significant negative impact on somatic growth and fillet yield. In order to induce sterility, triploidy induction was attempted by subjecting fertilised eggs to heat shocks. Different combinations of temperature (28, 30, 34, 35 and 36 °C), duration (2, 5, 10 and 25 min) and time of shock initiation (TI = 3, 5 and 7 min post-fertilisation) were tested. Flow cytometry analysis was used to assess ploidy level of control and heat-shocked larvae. Low intensity (28–30 °C) and long duration (10 and 25 min) shocks lead to significantly higher survival (44 ± 26%) and triploidisation (71 ± 26%) rates than high intensity (34–36 °C) and short duration (2 and 5 min) shocks (17 ± 19% and 21 ± 26%, respectively). The most effective conditions for efficient triploidy induction were low intensity shock of 30 °C, applied 5 min post-fertilisation for 25 min. This treatment led to the production of all-triploid populations (100%) with up to 43% survival rate.     

Induction of triploidy in the turbot (Scophthalmus maximus): II. Effects of cold shock timing and induction of triploidy in a large volume of eggs

Triploidy was induced in the turbot (Scophthalmus maximus, L.) by applying cold shocks shortly after fertilization. The combined effects of the timing of cold shock commencement after fertilization, cold shock duration and cold shock temperature were investigated. Ploidy was assessed by counting the number of nucleoli per nucleus (NOR) in larvae and also by measuring erythrocyte size in juveniles. A clear peak in triploidy induction was obtained when shocks were started between 6 and 7 min after fertilization at a pre-shock temperature of 13–14°C. With this timing, shocks of 20-min duration at 0°C gave >90% triploidy, with survival about 80% of the untreated controls. In order to ensure both high triploidy rates and high survival, it was necessary to carefully maintain the water temperature just below 0°C. Experiments with small and large volumes of eggs were performed in order to determine how changes in the relative volumes of eggs and chilled water could affect survival and triploidy induction. The best combination to induce triploidy in the turbot was as follows: shock commencement 6.5 min after fertilization, shock duration 25 min, and shock temperature between 0 and −1°C. With this combination, 100% triploidy could consistently be induced with survival 60% of the untreated control. This was successfully applied to a large volume of eggs (300 ml; 1 ml 800 eggs) in order to mass-produce triploid turbot. Triploids had lower survival rate than diploids at hatching but similar thereafter, with the ability to complete the different stages of larval rearing, indicating the viability to produce triploid turbot under farming conditions.     

Triploidy induction by hydrostatic pressure in the pearl oyster, Pinctada martensii Dunker

Triploidy of the pearl oyster, Pinctada martensii Dunker, was induced with hydrostatic pressure shock. The efficiency of inductions changed with the time of initiation of treatment, the duration of treatment and the intensity of pressure. The most effective procedure for inducing triploidy was the treatment of eggs at 200–250 kg/cm² 5–7 min (for the first polar body retention) and 17–19 min (for the second polar body retention) after insemination for 10 min duration, which resulted in 76% triploid embryos. Under this optimal treatment, high hatchability was also obtained.     

Induction of Triploidy and Tetraploidy in Nile Tilapia, Oreochromis niloticus (L.)

Abstract.— Induction of triploidy and tetraploidy in Nile tilapia, Oreochromis niloticus , was investigated by heat shock, cold shock, hydrostatic pressure, and/or chemicals (cytochalasin A, B, and D). Additionally, efficacy of combined protocols was determined. Heat shock 10 min after fertilization induced triploidy when incubation temperature was 24 C but not when incubation temperature was 31 C. Heat shock of 40–41 C at 4–6 min after fertilization was effective in inducing up to 100% triploidy with hatchability similar to controls. Cold shock at 13 C for 45 min five min after fertilization induced 85–100% triploids. Heat shock and multiple heat shocking were the most effective treatments for the induction of tetraploidy. Two heat treatments of 41 C applied at 65 and 80 min after fertilization for 5 min each produced approximately 80% tetraploidy in hatched fry. Immersion of fertilized eggs in cytochalasin A, B, or D at concentrations up to 10 μg/L applied at various times and durations was ineffective in inducing triploidy or tetraploidy.     

Gynogenesis in the African catfish, Clarias gariepinus (Burchell, 1822). Optimizing the induction of polar-body gynogenesis with combined pressure and temperature shocks

The effect of a simultaneous temperature and hydrostatic pressure shock applied a few minutes after activation of the egg was evaluated by monitoring the short-term survival rate of polar-body gynogenetic embryos of African catfish, Clarias gariepinus (Burchell, 1822). The aim was to evaluate the interdependence of temperature and pressure in retaining the second polar body. The temperature shock varied between 4°C and 44°C in increments of 4°C, while the pressure shock varied between 0.1 and 68.8 MPa in increments of 13.8 MPa. Highest survival rates were attained at a pressure between 40 and 55 MPa, regardless of the temperature at which the shocks were applied. Optimal survival rates of shocks applied at lower temperatures required longer durations. If reliability to induce retention of the polar body is of prime concern, hydrostatic pressure shocks of 55 MPa at room temperature are recommended.     

Thermal shock induction of triploidy in Atlantic cod (Gadus morhua L.)

The effects of thermal treatments on induction of triploidy in Atlantic cod have been investigated. Cold shock [−1.7±0.1°C at 20 min post fertilization (PF) for 2 h] was based on a previously developed protocol, and heat shocks, below the lethal threshold of 24°C, were at 16, 18 or 20°C applied 20, 30 or 40 min PF for 20 min. Cold shock did not affect larval survival and was ineffective for producing triploids (range 0–4%). A heat shock of 20°C at 20 min PF generated the highest percentages (range 66–100%) of triploid larvae at hatching, with survival ranging from 10% to 20% relative to the controls. Lower heat shock temperatures or delayed shocks increased survival but decreased the number of triploids, providing no net gain in triploid yield (range 1–9%). Heat shocks applied later than 20 min PF produced 2–4% tetraploid larvae at hatching. A thermal shock of 20°C initiated at 20 min PF and lasting 20 min proved to be the most generally efficient treatment for induction of triploidy in Atlantic cod.     

Induced triploidy in grass carp, Ctenopharyngodon idella Val

Induction of triploidy in grass carp was accomplished by means of thermal shocks to eggs shortly after fertilization. Triploidy occurred most often with cold shocks at 5–7°C and at durations of 25–30 min starting 2.0–4.5 min after fertilization. Estimated percent triploid ranged from 50 to 100% on five occasions. With one exception, cold shocks of 5–7°C for less than 25 min did not induce triploidy, and cold shock durations of 30 min or longer generally resulted in 100% mortality. A heat shock of 40°C for 1 min, 4.75 min after activation, was the only heat treatment which produced triploidy (8%) with 81% surviving to the blastula stage. Fertilized eggs immersed in a solution of cytochalasin B (10 mg/l, 0.1% DMSO) for 10 min, 12 min after activation, resulted in 54% of the eggs surviving to the blastula stage with none found to be triploid.     

Optimization of triploidy induction in the blacklip abalone, Haliotis rubra (Leach, 1814), using 6-dimethylaminopurine

Optimal conditions of 6‐dimethylaminopurine (6‐DMAP) for triploidy induction in the blacklip abalone Haliotis rubra (Leach, 1814) were investigated, targeting inhibition of second polar body (PB2) formation. Two experiments were conducted at a water temperature of 17.5–18.5°C where factorial combination of (1) four dosages (DSs) of 100, 150, 200 and 250 μM 6‐DMAP, four starting times (STs) of 15, 20, 25 and 30 min post fertilization, and two treatment durations (TDs) of 20 and 30 min and (2) three DSs of 50, 100 and 150 μM 6‐DMAP, three STs of 15, 20 and 25 min post fertilization, and three TDs of 10, 20 and 30 min, were applied respectively. Day 3 larvae were sampled for triploidy and survival. Percent triploidy was verified using flow cytometry (FCM). Results show that optimal inductions that combine both high rates of triploidy and reasonable survival were those treatments commenced 15 or 20 min post fertilization and continued for 20 or 30 min, using 100 μM 6‐DMAP. These conditions induced rates of triploidy and relative survival of 80.5–93.3% and 36.5–40.2%, respectively, in the first experiment, and corresponding rates were 79.1–93.6% and 20.7–43.0% in the second experiment. High percent triploidy were also obtained in a number of treatments using 150 μM 6‐DMAP, but with overall survival rates generally lower than those using 100 μM 6‐DMAP.     

Long-term effects and interactions of dietary vitamins C and E on growth and reproduction of yellow perch, Perca flavescens

This study was conducted to examine the effects of dietary vitamin C and/or E and their interaction on growth and reproductive performance in yellow perch. Yellow perch juveniles were divided into four treatments with triplicate groups and fed with one of four semi-purified diets for 32 weeks. The experimental diets were formulated to contain no or high vitamin E levels (160 mg/kg) without or with vitamin C supplementation (250 mg/kg) designated as diets −C−E, −C+E, +C−E, or +C+E, respectively. The growth rates and survival of yellow perch fed with +C−E and +C+E diets were significantly higher than the fish fed with −C−E diet. Total- and dehydro-ascorbate concentrations in liver and testis increased in response to its dietary supplementation. The -tocopherol concentration in sperm was significantly affected by dietary supplementation of vitamin C. Gonadosomatic index of males was lower in the fish fed with −C−E diet compared to that of fish fed with other diets. Hatching rates of embryos obtained from testing sperm viability was significantly improved in fish fed with the diets sufficient in vitamin C (82±7%) compared to the fish fed with the −C−E diet (59±7). This study indicates that supplementation of dietary vitamin C and E increase growth rate, and semen quality can be improved by dietary vitamin C supplementation within one maturation cycle, and vitamin C may spare sperm vitamin E depending on vitamin E stores in tissues.     

Induced triploidy and tetraploidy in the European flat oyster, Ostrea edulis L.

Eggs of the European flat oyster, Ostrea edulis, were treated with cytochalasin B (1 mg/l, 20°C, 20 min), at different time intervals after in vitro fertilization. Ploidy levels were assessed by chromosome counting on 1- and 54-day-old specimens. Evidence for bimodal distribution was found to separate meiotic I and meiotic II triploids. Peaks were located at 30–35 min and 90–100 min post-fertilization, triploid rates reaching 70% and 68% respectively. Tetraploid embryos were induced in two major groups. The effective shocks were those applied at 5–25 min and 260–280 min after fertilization (respectively 40% and 53% tetraploid metaphases). Karyological examinations of embryos and spat, carried out 20 h and 54 days after fertilization, showed a differential mortality among triploids and diploids in all treated groups and no tetraploids among the spat.     

Induction of triploidy in spotted sand bass (Paralabrax maculatofasciatus Steindachner, 1868) by cold shock

Conditions for the induction of triploidy with cold shock of fertilized eggs of the spotted sand bass Paralabrax maculatofasciatus (Steindachner) were investigated. Different temperatures (12, 8 and 4 °C), timing of cold shock application (5, 10 and 15 min after fertilization) and duration of the shock (5, 10, 15 and 20 min) were tested. Triploidy was determined using flow cytometry at 12 h after larvae hatched. Triploids were produced only when the cold shock treatment was applied 5 min after fertilization. No significant difference was observed in the percentage of triploidy between temperature and the shock duration. At 8 and 4 °C, 100% triploidy was obtained at different durations of cold shock. Survival was significantly lower at 12 or 4 °C than at 8 °C. No significant difference was observed for shock duration at the temperature of 8 or 12 °C; however, at 4 °C, survival was significantly lower at longer durations. We recommend induction of triploidy by applying cold shock at 8 °C for a duration of 15–20 min starting at 5 min after fertilization, in the spotted sand bass.     

Pressure Induced Triploidy in Hybrid Lepomis

Worldwide expansion of aquaculture will require identification of species more adapted to localized conditions and less likely to affect genetic diversity of natural populations in the event of escape. Hybrids of Lepomis sunfish have the potential as an alternative for aquaculture in regions of North America where the growing season is unsuitable for production of traditional coldwater and warm-water species. Triploidy was induced in hybrids of green sunfish Lepomis cyanellus males and bluegill L. macrochirus females using hydrostatic pressure shocks. A total of 27 combinations of pressure (41,369, 48,264, and 55,158 kPa), duration (2, 3, 4 min), and post-fertilization time of shock initiation (2, 3, 4 min) were tested to identify those which were most efficient for inducing triploidy. Several of the shock regimes produced 100% triploid hybrid sunfish with at least 90% survival relative to unshocked controls. The two shock treatments deemed best overall were: 1) 48,264 kPa for 4 min begun 2 min after fertilization; and 2) 41,369 kPa for 2 min begun 3 min after fertilization.     

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.     

Evaluation of cytochalasin B (CB) treatments for triploidy induction in the blacklip abalone, Haliotis rubra (Leach, 1814)

The effectiveness of cytochalasin B (CB) treatments for inducing triploidy was evaluated in the blacklip abalone Haliotis rubra (Leach, 1814) in two orthogonal design experiments. The first experiment employed three dosages (DSs) of 0.25, 0.5 and 1.0 mg CB L^?1, three starting times (STs) of 5, 15 and25 min post fertilization and three treatment durations (TDs) of 10, 20 and 40 min, for a total of 27 treatments. The second experiment comprised of two DSs of 0.25 and 0.5 mg CB L^?1, five STs of 5, 15, 20, 25and 30 min post fertilization, and three TDs of 10, 20 and 40 min, for a total of 30 treatments. Water temperature was held at 17.5–18.5°C. Day 3 larvae were sampled for triploidy using flow cytometry (FCM) and survival. Optimal inductions were treatments starting at 15 or 20 min post fertilization and continuing for 40 min, and those initiated 25 or 30 min post fertilization for 20 or 40 min, using 0.5 mg CB L^?1. These treatments were all targeted at inhibition of the second polar body (PB2) formation and yielded triploidy rates of 84.8–89.5% coupled with (relative) survival rates of 20.1–52.1% in the first experiment, and corresponding rates of 86.5–96.5% and 33.0–74.1%, respectively, in the second experiment. A common and essential feature of these optimal conditions is that treatment must fully span the period of time for most of the eggs to extrude PB2. Treatments that resulted in suppression of the first polar body (PB1) formation induced triploidy levels below 71.5% and 57.6% in experiments 1 and 2 respectively. Treatments that had overlapping effects on both PB1 and PB2 extrusion led to triploidy rates above 80% but very low survival rates of 1.8% and 5.4% in experiments 1 and 2 respectively.     

Triploid Induction of Green Tiger Shrimp,Penaeus semisulcatus (De Haan, 1844) Using Temperature and Chemical Shock

Triploidy in fertilized eggs of Penaeus semisulcatus was induced by temperature and chemical shocks. The eggs, which were obtained from the shrimp broodstock maintained at 29 C, were exposed to cold temperature (8, 10, 12, and 14 C) and 6‐dimetiloaminopurine (6‐DMAP) concentrations (100, 150, 200, and 250 μM) for different durations (4, 6, and 8 min) 9 min after spawning was detected. While the highest triploidy rate of 49.7 ± 4.5% was obtained with a 200 μM 6‐DMAP concentration for a duration of 8 min, the best mean triploidy rate of 45.5 ± 2.8% for cold shock was obtained at a temperature of 10 C for a duration of 8 min. Temperature and 6‐DMAP concentration did not have significant effect on triploidy rate (P > 0.05) but shock duration had significant effect on triploidy rate for individual cold temperature shock or 6‐DMAP chemical shock (P < 0.05). Although longer durations of shock agent increased the rates of triploid induction, they generally had an adverse effect on hatching rates in the study.     

Induction of heterozygous and homozygous diploid gynogenesis in Betta splendens (Regan) using hydrostatic pressure

Abstract Optimum conditions for hydrostatic pressure treatment for duplication of chromosome set in gynogenetically activated fighting fish, Betta splendens (Regan), eggs were identified. Maximum survival of heterozygous gynogens was 50%, when 2·5-min-old eggs, after insemination with UV irradiated tilapia sperm, were pressure shocked at 7000psi for 6 min. The frequency (21%) of homozygous gynogenetic fry was high, when the 34min (post-insemination) old eggs, which were inseminated with tilapia sperm, were pressure shocked for 5 min. Sex ratio of gynogenetic progeny suggested that the mechanism of sex determination in this fish is homogametic female and heterogametic male.     

Effects of storage temperature and duration on the setting and post-set spat survival of the tropical oyster, Crassostrea iredalei (Faustino)

The effects of six storage temperatures (5°C, 10°C, 15°C, 20°C, 25°C and room temperature) and six storage durations (6, 12, 24, 48, 72 and 96 h) on pre-settlement larvae of Crassostrea iredalei indicated that settlement rate deteriorated with time for all temperatures. The highest settlement rate (40.1%) was attained at a storage temperature of 20°C for 6 h. This was followed by 10°C and 15°C for 6 h, with mean percent sets of 35.4% and 33.5%, respectively. An above-average set of 29.5% was obtained for the control larvae (larvae directly from the rearing tanks) compared to larvae stored between 10°C and 20°C for 12 to 24 h (21.1–28.2%). Average sets obtained for storage between 10°C and 20°C was 16.6–19.7% for up to 48 h, and sets for room temperature (ca. 30°C) and 5°C for 12 h were 11.9% and 16.9%, respectively; whereas at 25°C the set rate was 10.7% for 6 h. Storage at all other levels of temperature and duration resulted in poor set rates of less than 8%. All successfully set larvae from this experiment were further kept in the hatchery for three weeks to observe their short-term post-settlement survival. Survival rates were closely related to the setting rates, whereby higher sets contributed to better survival rates. The highest survival rates, 61.3–84.8%, were recorded for larvae set at temperatures ranging from 10°C to 20°C with a storage time of up to 48 h. These levels were comparable to the control (68.0%) and 5°C for up to 12 h (68.9%). Storage at 72 h resulted in total mortality at all temperatures, except for those stored at of 10°C (51.5% survival) and 20°C (14.7%).     

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。     

Optimal conditions for cold-shock induction of triploidy in red tilapia

Production of sterile triploid red tilapia [Oreochromis mossambicus (Mozambique tilapia); Peters, 1852 × Oreochromis niloticus (Nile tilapia); Linnaeus, 1758] is an effective strategy to overcome their prolific breeding. Optimal conditions for cold-shock induction of triploidy in red tilapia were investigated by experimentally examining two variables: appropriate temperature of the shock and duration of shock treatment. A constant time after insemination of 4 min was used to determine the best combination of temperature (6, 7, 8, 9, 11, 13, 15 °C) with different durations of shock (10, 20, 30, 40, 50 min) with resultant ploidy level verified karyotypically. Shock duration for 30 min at a temperature of 9 °C was found most effective in producing maximum triploidy (98.7 %) with higher rates of hatching (63.2 %) and survival up to yolk-sac stage (75.8 %). The chromosome count confirmed that triploid percentages were higher when cold shock was used for longer durations at each temperature; however, hatching rates were generally decreased. The maximum triploid yield (82.1 %) obtained was higher than the yield obtained using heat shock (72.7 %) in red tilapia previously. The application of the results of this study has the potential to greatly improve the production of triploid red tilapia in commercial aquaculture.