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.     

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.     

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.     

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.     

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.     

Triploidization of European catfish, Silurus glanis L., by heat shock

Heat shock was applied to fertilized eggs of European catfish, Silurus glanis L., for the induction of triploidy. A heat shock of 40. 5°C lasting 1 min and starting 9 min after gamete activation gave the best results with 88.93% of hatched viable fry (92.41% in control group). Yields of hatched viable triploid fry reached 63.14% or 50.60%, when expressed as percentage of the absolute number of viable fry or number of living eggs in eye-bud stage, respectively (P < 0.05).     

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.     

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.     

Manipulation of ploidy in yellow perch (Perca flavescens) by heat shock, hydrostatic pressure shock, and spermatozoa inactivation

Heat shocks, hydrostatic pressure shocks, and ultraviolet radiation were evaluated for their efficacy as methods of manipulating ploidy in yellow perch (Perca flavescens). The most effective methods of inducing triploidy were heat shocks of 28–30°C applied at a time of initiation (TI) of 5 min postfertilization for durations of 10 or 25 min, and hydrostatic pressure shocks of 9000 or 11 000 psi applied at a TI of 5 min for a duration of 12 min. These treatments resulted in triploidy induction rates that ranged from 54–100%, and embryonic survival rates of 16–80%. Cold shocks of 0°C had no effect on the ploidy or survival of embryos. For perch, hydrostatic pressure shock offered several advantages over heat shock as a method of manipulating ploidy. The most effective methods of inducing tetraploidy were hydrostatic pressure shocks of 9000 psi applied at a TI of 192 min for durations of 16 or 24 min. Ultraviolet radiation of perch sperm with doses of 3240–6480 ergs/mm² resulted in 100% inactivation of paternal chromosomes, and perch eggs fertilized with inactivated sperm had survival rates of > 50%, thereby establishing methods for producing gynogenetic perch. Studies comparing the growth and performance of diploid vs. triploid perch are underway. Tetraploid perch are being reared to sexual maturity to evaluate their potential as brood fish.     

Tetraploid induction in tropical oysters,Crassostrea belcheri (Sowerby) and Crassostrea iredalei (Faustino)

Tetraploid induction has been conducted on temperate oysters but not on tropical oysters. In this study, different heat shocks (32, 35 and 38°C) and cold shocks (1, 4 and 7°C) were used to induce tetraploidy in two tropical oyster species, Crassostrea belcheri and Crassostrea iredalei, through meiosis I inhibition. Temperature shocks were applied on the newly fertilized eggs at 8–10 min post fertilization and terminated when second polar bodies began to form in the control eggs. The ploidy of the larvae and spat was determined via direct chromosome count. The percentage of larval survival until Day 20 was low (between 0.4% and 42.9%) for both temperature shocks and oyster species. No surviving larva was recorded for induction at 1, 4 and 38°C. Tetraploid spat was only recorded in C. iredalei but the percentage is low through heat shock induction of 32 and 35°C. This study shows that the tetraploid induction success rate was slightly higher in C. iredalei compared to C. belcheri. No surviving tetraploid spat were recorded for both oyster species through the cold shock method. This study shows that heat shock can be used to inhibit meiosis for the production of tetraploids but more experiments need to be conducted to determine the optimum temperature when dealing with tropical oysters.     

Effects of ultra-violet irradiation on sperm motility and diploid gynogenesis induction in large yellow croaker (<Emphasis Type="Italic">Pseudosciaena crocea</Emphasis>) undergoing cold shock

Large yellow croaker, Pseudosciaena crocea, exhibit sexually dimorphic growth, with females growing faster and reaching larger adult sizes than males. Thus, development of techniques for preferentially producing females is necessary to optimize production of these species. We have established a protocol to produce all-female croaker P. crocea through induction of meiotic gynogenesis with homologous sperm. The first set of experiments investigated the ultra-violet (UV) irradiation on sperm motility and duration of sperm activity to determine the optimal UV dosage for genetic inactivation of sperm, yet retaining adequate motility for activation of eggs. Milt from several males was diluted 1:100 with Ringer’s solution and UV irradiated with doses ranging from 0–150 J cm⁻². The results indicated that motility and duration of activity generally decreased with increased UV doses. At UV doses greater than 105 J cm⁻², after fertilization, motility was <10% and fertilization rates were significantly lower. Highest hatching rate was obtained at 75 J cm⁻². A second set of experiments was carried out to determine appropriate conditions of cold shock for retention of the 2nd polar body in P. crocea eggs after fertilization with UV-inactivated sperm by altering the timing, temperature and duration of shock. At 20°C, shock applied at 3 min after fertilization resulted in higher survival rate of larvae at 6 h after hatching. Results of different combinations of three shock temperatures (2°C, 3°C or 4°C) and five shock durations (4 min, 8 min, 12 min, 16 min or 20 min) at 3 min after fertilization demonstrated that shocks of 12 min gave highest production of diploid gynogens. Statistical analysis revealed that maximum production of diploid gynogens (44.55 ± 2.99%) were obtained at 3°C. The results of this study indicate that the use of UV-irradiated homologous sperm for activation of P. crocea eggs and cold shock for polar body retention is an effective method for producing gynogenetic offspring.     

Induction of triploidy in the zebrafish, Brachydanio rerio (Hamilton)

Abstract. Triploidy was induced in the zebrafish, Brachydanio rerio (Hamilton), by varying all possible combinations of the time after fertilization (AF) (1-3min after insemination), temperature (36-42°C) and shock duration (1-7min). A thermal shock of 41°C for 4min, 2.5min AF ensured 100% triploidy and maximum (51%) survival. Induction of triploidy was confirmed by measurement of erythrocyte nuclear volume and chromosome counting. There was no significant difference in the growth rate of triploid and diploid fishes. All surviving triploids developed into males, and produced a few spermatozoa unable to fertilize normal eggs. A study on thermal and other characteristics required to ensure 100% triploidy rate in fish indicates that these characteristics are species specific.     

Induction of triploidy and tetraploidy in stinging catfish, Heteropneustes fossilis (Bloch), using heat shock

Induction of triploidy and tetraploidy was attempted in Heteropneustes fossilis using heat shock. The optimal age of zygote, temperature level and duration of thermal shock required for effective induction of triploidy and tetraploidy was investigated in a series of experiments. A maximum of 82±7% triploids (3n=87) were obtained when fertilized eggs (2.5‐min old) were heat shocked at 40°C for 4‐min duration. A maximum of 40±8% tetraploids (4n=116) was obtained when the fertilized eggs (30‐min old) were heat shocked at 40°C for 4‐min duration. The triploid and tetraploid red blood cells (RBCs) nucleus volumes were 1.4 and 2.1 times greater, respectively, than that of the diploid RBC nucleus.     

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.     

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.     

Triploid Induction in the Yellowtail Tetra,Astyanax altiparanae,Using Temperature Shock: Tools for Conservation and Aquaculture

Triploidization is an interesting tool to produce sterile fish. In the yellowtail tetra, Astyanax altiparanae, this can be applied for aquaculture and surrogate technologies. In this study, we compared the efficacy of cold (2 C) or heat shock (38 C, 40 C, and 42 C) on triploid induction in the yellowtail tetra. The eggs were treated with cold or heat shock, 2 min postfertilization (30 min in cold shock or 2 min in heat shock). Intact embryos served as the control group. Ploidy status was confirmed by karyotyping, flow cytometry, and nuclear diameter of erythrocytes. The hatching rate decreased after cold shock (12.69 ± 15.76%) and heat shock at 42 C (0.35 ± 0.69%) in comparison with the control group (63.19 ± 16.82%). At 38 C and 40 C, hatching rates (61.29 ± 17.73% and 61.75 ± 22.1%, respectively) were not decreased. Only one triploid arose at 38 C (1/80). At 40 C, a high number of triploids arose (72/78). At 42 C, very few embryos developed into the hatching stage. A large number of haploid individuals arose after cold shock (61/75), with only one triploid. Our results indicate that heat shocking of embryos at 40 C is optimum for triploid production in the yellowtail tetra.     

Preliminary study on performance of triploid Thai silver barb, Puntius gonionotus

The triploid chromosome condition was induced in Thai silver barb (Puntius gonionotus) by application of cold shock (2°C) to eggs at time intervals after activation of 0.5 min with a duration of 10 min which resulted in mean triploidy yield of 72.5% at 9 months of age. Growth rate of the 2–9-month-old, cold shock group (0.1–6.2 g/month) did not differ from that of the control (0.1–5.7 g/month). Gonadal somatic indices (GSI) of presumed triploid males and females were lower than that of control (GSI values of the presumed triploids were 35.0–60.2% and 28.7–75.9% of control males and females, respectively). Spermatogenesis and oogenesis were retarded in triploids. However, all stages of spermatogenic cells were observed in triploid males, including few spermatozoa. Oocytes of triploid females did not undergo vitellogenesis while normal oogenesis was observed in diploids. Nuclear volume of red blood cells (RBCs) of triploid fish was 1.63 times larger than that of diploids.     

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。     

Induction of triploidy in Melicertus kerathurus (Forskal, 1775) with temperature shock

Triploidy in fertilized eggs of Melicertus kerathurus was induced by cold (8, 10, 12°C) and heat (34, 36, 38°C) shock for different duration times (2, 4 and 8 min) after 10 min of post spawning. The best individual treatment produced 64.5% triploid nauplii in cold shock application at a temperature of 10°C for a duration of 8 min. Temperature did not have significant effect (P > 0.05) on triploid rate but duration time had a significant effect (P < 0.05) for individual cold or heat shock. This study demonstrates that because of a wide variety of effective parameters, it is essential to optimize shock conditions for each species strain at each location.     

Induction of diploid gynogenesis in turbot <Emphasis Type="Italic">Scophthalmus maximus</Emphasis> with left-eyed flounder <Emphasis Type="Italic">Paralichthys olivaceus</Emphasis> sperm

Turbot Scophthalmus maximus exhibits sexually dimorphic growth, with females growing faster and reaching larger adult sizes than males. Thus, development of techniques for preferentially producing females is necessary to optimize production of these species. In this paper, gynogenetic diploids of turbot were induced by activating egg development with ultraviolet (UV)-irradiated left-eyed flounder Paralichthys olivaceus sperm combined with cold shock to prevent extrusion of the second polar body. The results of UV irradiation experiments showed that survival, motility, and duration of activity of P. olivaceus sperm generally decreased with increase in UV dose. The typical Hertwig’s effect was observed after fertilized turbot eggs with UV-irradiated P. olivaceus sperm and the optimal UV dose for gynogenetic haploid production was 36,000 erg mm⁻². At 15°C, appropriate timing of cold shock for retention of the second polar body in turbot eggs was at 6 min after fertilization. Results of different combinations of two shock temperatures (1 or 3°C) and four shock durations (15, 25, 35 or 45 min) at 6 min after fertilization demonstrated that shock of 25 min at 1°C gave the highest production of diploid gynogens (39.58% relative to its diploid control). The results of this study reveal that the use of UV-irradiated P. olivaceus sperm for activation of turbot eggs and cold shock for polar body retention is an effective method to produce gynogenetic offspring.