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.     

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.     

Induced triploidy in the Pacific oyster,Crassostrea gigas: Optimal treatments with cytochalasin B depend on temperature

Triploidy was induced in Crassostrea gigas using cytochalasin B (CB) (1 mg CB/l) at three temperatures: 18, 20 and 25°C. Between 3 and 5 million eggs/l were treated with CB at 15-min intervals following fertilization.Large differences in survival to straight hinge among mass spawns were observed. These were attributed to variable quality of strip-spawned eggs and treatment with CB. The negative effect of CB treatment was most apparent during critical periods of zygotic development (e.g., fertilization, polar body formation). After 48h, larvae from control and treatment groups had equivalent survival and growth rates.Replicates yielded similar percentages of triploids with standard errors of generally 10% or less. Induction curves were calculated for each temperature; triploid maxima at 18, 20 and 25°C were 52, 76 and 90%, respectively. The highest mean percentages obtained empirically at 18, 20 and 25°C were 62, 74 and 88%, respectively. No evidence for bimodal distributions to separate meiotic I and meiotic II triploids was found. Treatments at lower temperatures delayed triploid maxima which occurred approximately 30, 45 and 50 min after fertilization at 25, 20 and 18°C, respectively. Overall, the optimal treatment for inducing triploidy in the Pacific oyster (C. gigas) appears to be 30–45 min post-fertilization at 25°C, which yielded 88±9% (SE) triploidy over four replicates.     

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.     

不同方法制备的三倍体长牡蛎养殖效果的比较

比较了细胞松弛素B(CB)和6-二甲基氨基嘌呤(6-DMAP)通过抑制受精卵极体释放的方法批量诱导三倍体长牡蛎的养殖效果.长牡蛎卵子在25℃的海水中受精,20～30min后,开始用浓度为0.5mg@L-1的CB处理,持续18～22min,受精卵处理密度为4.0～4.5×107个@L-1,三倍体产率为65.2%～70.1%,面盘幼虫孵化率为12.3%～14.5%,诱导效率指数为0.09.6-DMAP的使用浓度为400～420μmol@L-1,受精卵处理密度为3.0～3.5×107个@L-1,授精水温、处理起始和持续时间等与CB方法相同,三倍体产率为58.7%～65.4%,面盘幼虫孵化率为52.1%～55.4%,诱导效率指数为0.32.两种方法的采苗率基本相同,采苗器为基质较硬的栉孔扇贝贝壳.海区养殖采用浮筏夹苗吊养技术,两种方法诱导的三倍体牡蛎养殖性状没有明显差别.通过比较CB和6-DMAP两种诱导方法及三倍体的养殖效果表明,后者具有更好的应用性.     

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.     

CB诱导熊本牡蛎三倍体及其存活率与倍化率的变化关系

为诱导熊本牡蛎三倍体,研究了细胞松弛素B (CB)浓度、诱导起始时间、诱导持续时间等因素对卵裂率、D幼率、三倍体率的影响,并分析了幼虫、稚贝及成贝的存活率和三倍体率的变化特征。结果显示,CB浓度为0.5~0.6 mg/L,诱导起始时间为40%受精卵释放第一极体,诱导持续时间为20 min时可获得87%的三倍体率。卵裂率、D幼率、三倍体率的最大影响因素分别为CB浓度、诱导持续时间、诱导起始时间与诱导持续时间。三倍体率与卵裂率无显著负相关性,而与D幼率呈显著正相关。因此,减小CB浓度或诱导持续时间,可同时获得较高的三倍体率与幼虫产量。3~15日龄三倍体组与对照组的存活率分别由71.27%与96.09%降低至34.14%与58.80%,成贝期450日龄(9月)三倍体组与对照组的存活率分别为53.62%与44.67%。3~9日龄三倍体率从87%降低至77%,而90~450日龄三倍体率平均值为59.21%±4.99%,表明幼贝与成贝期三倍体率变化较小,三倍体率的维持与存活率无显著相关性。     

合浦珠母贝三倍体的卵诱导四倍体

将合浦珠母贝三倍体的卵与二倍体的精子授精,用０．５μｇ／ｍＬ细胞松弛素Ｂ抑制精卵第一极体的释放诱导四倍体。研究了处理起始时间及持续时间对胚胎孵化率和四倍体诱导率的影响及幼虫的生长及存活。实验结果表明：持续时间与胚胎孵化率呈负相关,而与四倍体诱导率呈正相关,持续时间一般为１５￣１８ｍｉｎ,处理起始时间一般在第一极体出现前３￣５ｍｉｎ。在胚胎期,四倍体诱导率平均为２０％。在幼虫培养阶段,幼虫死亡严重,     

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.     

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.     

Induction of triploidy in the South African abalone using cytochalasin B

An investigation into triploidy induction in the South African abalone, Haliotis midae, was conducted. It was found that 0.5 mg l–1 of Cytochalasin B (CB) in seawater induced triploidy when administered to coincide with the normal timing of the release of either polar body one (PB1) or two (PB2). This concentration of CB produced 70.9% triploid induction in the PB2 treatment and 48.4% induction at PB1. Significant numbers of tetraploid larvae were found in the PB1 treatment. These resulted from the presence of excess sperm (polyspermy) but only when CB was present. Although larval survival after triploid induction was lower than the control animals, it was considered high enough for use in commercial hatcheries. © Rapid Science Ltd. 1998     

Effect of three triploidy induction methods on the growth and survival of larvae and post‐larvae of the Caribbean scallop Argopecten nucleus

Argopecten nucleus is a small scallop from the Caribbean Sea and a relatively new species for aquaculture. One of the key challenges to develop the farming operations for this species from the current pilot scale to commercial level is to improve its harvest size. In this study, we tested three different methods for triploidy induction. Additionally, the effect of these protocols on survival, developmental rate and size of larvae and post‐larvae were assessed. Three different mechanisms to stimulate the inhibition of the release of the second polar body were tested; (1) cold shock (18°C); (2) 6‐dimethylaminopurine (6‐DMAP); (3) cytochalasin B (CB) and (4) dimethylsulphoxide (DMSO). The treatment with 6‐DMAP yielded the highest percentage of triploid larvae (39%). The survival and development rate, however, were higher in non‐treated larvae (control) than in the treatment groups. Interestingly, larvae from CB and the DMSO control groups exhibited lower growth rates in length than those from control and the other two treatments. No influence of the triploidy induction treatments was observed on post‐larvae survival, but the size of post‐larvae was larger for the cold shock treatment and DMSO control group. Our results indicate that the use of 6‐DMAP has the greatest potential to produce triploid larvae of A. nucleus without affecting negatively growth and survival of post‐larvae.     

诱导异源三倍体鲍的初步研究

用细胞松弛素B（CB）处理九孔鲍♂×盘鲍♀受精卵,分别抑制其第一极体和第二极体、以及第二极体释放诱导异源三倍体。水温24℃,九孔鲍♂×盘鲍♀授精后10min,用浓度0.6～1.0mg/L的CB持续处理受精卵20～25min,抑制其第一极体的排放。而九孔鲍♂×盘鲍♀在受精后27min,当40%～50%受精卵排出第一极体时,用浓度0.6～1.0mg/L的CB持续处理受精卵10～15min,分别统计对照组和药物处理组的担轮幼虫率,并用倍体分析仪检测各组稚鲍的倍性。结果表明：对照组和药物处理组担轮幼虫的倍性较复杂,起始处理时间为10min,CB药物处理浓度为0.6mg/L,持续处理时间为25min,其三倍体率可达40.67%,担轮幼虫的孵化率为26.44%。起始处理时间为27min,CB药物处理浓度0.6mg/L,处理持续时间为10min,其三倍体率可达48.11%,担轮幼虫率的孵化率为29.86%。     

Studies on triploid oysters in Australia: Evaluation of cytochalasin B and 6-dimethylaminopurine for triploidy induction in Sydney rock oysters Saccostrea commercialis (Iredale and Roughley)

Naturally spawned Sydney rock oysters Saccostrea commercialis (Iredale and Roughley),were used to determine the appropriate stage of development for inducing triploidy and to compare the effectiveness of cytochalasin B (CB) and 6-dimethylaminopurine (6-DMAP) in dose-optimization trials. Induction should commence at 50% first polar body (PB1) extrusion in eggs (approximately 17-19 min post-fertilization at 25^oC). By day 5 the highest triploidy percentage and yield (number of triploid larvae per 100 fertilized eggs) were achieved in the ranges of 0.75-1.5 mg CB 1^-1 (1.6-3.1 μm CB)or 200-400 μm 6-DMAP (32.6-65.3 mg 6-DMAP l^-1). However, CB treatment resulted in greater survival and triploidy percentage than 6-DMAP in Sydney rock oysters.     

Highly efficient induction of triploid Pacific abalone <Emphasis Type="Italic">Haliotis discus hannai</Emphasis> by caffeine treatment

ABSTRACT:   In order to develop a highly efficient method for mass production of triploid Pacific abalone Haliotis discus hannai , caffeine treatment that is safe and inexpensive was optimized. To suppress the first meiotic division, fertilized eggs were exposed to either a 10- or 15-mM caffeine solution for 24 min beginning at 12 min after fertilization. In most treated batches, the rates of cleaved eggs showed no significant difference from the control batches. However, in most treated batches, the rates of occurrence of normal larvae and the survival rates of the early juveniles were significantly lower than those of the controls. The triploid rates at 6 days to 11 months after settlement in all the treated batches were extremely high (91–100%). There was no significant difference in the mean triploid rates between 10- and 15-mM caffeine treatments. These results suggest that both treatment conditions were conducive to triploid abalone production. One live 2n/3n mosaic specimen was found in the treated batches. However, since the frequency of mosaic was extremely low, the mosaicism would probably not have an adverse effect on the stable production of triploid abalones.     

Tetraploid induction by meiosis inhibition in the dwarf surfclam Mulinia lateralis (Say 1822): effects of cytochalasin B duration

Different durations of a cytochalasin B (CB) treatment were tested for tetraploid induction by meiosis inhibition in the dwarf surfclam Mulinia lateralis Say. Cytochalasin B, 0.67 mg L^?1, was applied to newly fertilized eggs at 8–10‐min post fertilization and removed when in the untreated eggs: (1) Polar body 1 (PB1) was released in 90% of the eggs and polar body 2 (PB2) began to form (T1); (2) Polar body 2 was released in about 25–30% of the eggs (T2); (3) Polar body 2 was released in about 70–75% of the eggs (T3); or (4) eggs began to enter mitosis I or the polar lobe began to form (T4). Three replicates were produced using different sets of parents. The ploidy of resultant larvae and juveniles was determined by flow cytometry. Blocking PB1 alone in T1 groups produced mostly tetraploids, and longer CB treatments in T2 and T3 resulted in increasing numbers of pentaploids. In T4 groups where both PB1 and PB2 were inhibited, larvae were predominantly pentaploids. Pentaploid larvae were arrested at the trochophore stage. The majority of tetraploid larvae died as trochophores, although a small fraction reached D‐stage. Among 478 juvenile clams sampled from a T1 group, three (0.6%) were confirmed as tetraploids. This study shows that tetraploid embryos can be produced at high efficiencies (40–90%) by blocking meiosis I. Tetraploids produced by meiosis inhibition in normal eggs are viable in M. lateralis, but their survival beyond metamorphosis is extremely low.     

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 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.     

热休克诱导马苏大麻哈鱼三倍体的初步研究

为探索马苏大麻哈鱼(Oncorhynchus masou)三倍体育种技术方法,更好地解决其个体小、生长慢和性成熟后死亡率高等问题,采用热休克法进行三倍体诱导实验,设4个诱导温度(24、26、28、30℃),2个起始诱导时间(15 min、20 min)和2个持续诱导时间(15 min、20 min)共分13个实验组和1个对照组。结果显示,13个实验组均能诱导出三倍体个体,而不同温度组的诱导率差异显著,分别为13.3%、31.65%、52.28%和78.81%(P<0.0.5)。随着温度的上升,孵化率呈显著降低的趋势。结果表明,温度是影响诱导成功的关键因素,利用热休克诱导受精卵制备陆封型马苏大麻哈鱼三倍体苗种的方法是可行的,三倍体诱导的最佳条件是:水温28℃,卵子受精后15 min持续处理20 min,发眼率(72.57±0.26)%,孵化率(60.92±0.31)%,三倍体率53.1%,综合诱导效果最佳。     

开都河流域长身高原鳅胚胎发育观察

为了探究长身高原鳅(Triplophysa tenuis)的早期生活史,2018年4月在察汗乌苏鱼类增殖站对长身高原鳅胚胎发育过程进行观察。随机挑取10~15粒受精卵,在XSP-24S生物显微镜下对胚胎发育情况进行连续观察并拍照,记录胚胎发育时序及各个时期的典型特征。结果显示,长身高原鳅成熟卵粒圆形、灰色透明、饱满有弹性、沉性,卵径(1.07±0.10)mm;遇水受精后,迅速出现黏性。水温(20±1)℃时,整个胚胎发育历时86 h 56 min,积温1826℃·h,依次经历胚盘期、卵裂期、囊胚期、原肠胚期、神经胚期、器官形成期和出膜7个连续发育阶段、34个时期。受精后10~15 min完成吸水,吸水后卵径为(1.50±0.09)mm;受精后1 h 22 min进入卵裂期;受精后7 h 40 min进入囊胚期;受精后11 h 17 min进入原肠胚期;受精后15 h 28 min进入神经胚期;受精后19 h 28 min进入器官形成期;受精后77 h 13 min进入出膜前期,受精后86 h 56 min完成出膜,长身高原鳅出膜仔鱼全长(4.81±0.17)mm。