熊本牡蛎中国群体与美国群体杂交效应及杂交三倍体优势分析

为了评估熊本牡蛎(Crassostreasikamea)中国群体与美国群体的杂交效应与杂交三倍体优势,构建了杂交群体和自交群体,并使用细胞松弛素B诱导了杂交三倍体,比较分析了幼虫、稚贝与成贝的生长与存活优势。自交群体和杂交群体的幼虫、稚贝与成贝的培养条件均相同,室内培育幼虫,培育密度为4～5个/mL,自然海区养殖稚贝与成贝,养殖密度为40～45个/吊。结果表明,与自交组相比,杂交二倍体具有较高的卵裂率(13.61%)与D幼率(5.67%)(P0.05),但杂交二倍体幼虫的壳高生长表现杂种劣势(–0.43%),而稚贝、成贝的壳长与壳高的生长表现杂种优势,平均优势率分别为3.96%与6.65%。杂交三倍体诱导组幼虫的壳高生长优势率为3.69%,稚贝及成贝的壳长与壳高平均生长优势率分别为12.69%与13.64%,并且日龄越大生长优势越显著。总体上,杂交三倍体诱导组在3～180日龄具有存活劣势,并且15日龄存活劣势率最大(–48.72%),而在360日龄存活优势率为6.70%。杂交二倍体幼虫与成贝均具有存活优势,平均优势率分别为10.44%与4.59%。本研究表明熊本牡蛎中美地理群体杂交二倍体的生长和存活优于自交二倍体,而杂交三倍体诱导组的生长优势较显著,并且在成贝期具有显著的成活率优势,表明杂交三倍体诱导组的优势来源于三倍体优势和部分杂种优势。     

CB与6-DMAP诱导香港牡蛎三倍体的效果比较

以三倍体率、卵裂率、D幼率、生产成本等为指标,比较了CB、6-DMAP两种化学试剂诱导香港牡蛎三倍体的效果,研究了试剂浓度、诱导时机、诱导持续时间及受精卵密度等4种因素对香港牡蛎三倍体的诱导效应。结果显示,在温度28~30°C、盐度15~25,受精卵密度为2.0×10~8个/L条件下,采用0.5 mg/L的CB在受精后15~18 min处理,诱导持续时间为20 min,可产生100%三倍体;合子的卵裂率为53.16%~63.00%,D形幼虫孵化率为47.32%~53.09%,诱导效率指数为0.47~0.53,生产成本为260元/L。相同条件下,采用浓度为75~100 mg/L的6-DMAP处理,诱导持续时间为20~25 min,受精卵处理密度4.5×10~7个/L,可产生62.52%~72.36%的三倍体;合子的卵裂率为60.00%~66.25%,D形幼虫孵化率为74.43%~90.00%,诱导效率指数为0.47~0.65,生产成本为139~185元/L。综合比较两种方法,6-DMAP诱导方法更加适合用于大规模的香港牡蛎三倍体苗种生产。本研究为香港牡蛎多倍体育种提供了研究数据与实践经验。     

长牡蛎中国群体和美国群体杂交效应与三倍体的优势

将长牡蛎中国群体二倍体分别与美国群体二倍体和四倍体进行杂交,实验共设置4组,分别为杂交二倍体组、杂交三倍体组、中国二倍体组和美国二倍体组,比较了各实验组卵裂率、D幼率、D形幼虫大小及幼虫期、稚贝期的壳高生长、存活率等生物学指标,并估算杂交二倍体的杂种优势率和杂交三倍体的三倍体优势率。结果表明,杂交二倍体幼虫壳高生长的杂种优势率不明显,平均杂种优势率为1.21%,幼虫的存活率及稚贝的壳高生长表现出明显的杂种优势,平均杂种优势率分别为34.47%和20.39%。杂交三倍体的D形幼虫大小、幼虫和稚贝的壳高生长、存活率均表现出三倍体优势,D形幼虫大小三倍体优势率为5.19%,幼虫期壳高生长和存活的平均三倍体优势率分别为4.00%和19.92%,稚贝壳高生长和存活的平均三倍体优势率分别为30.18%和54.43%,200日龄,杂交三倍体鲜体质量的三倍体优势率为202.96%,存活三倍体优势率为73.60%。此外,稚贝期的杂交二倍体生长性状的杂种优势率和杂交三倍体的三倍体优势率均高于幼虫期的优势率。研究表明,中、美两地理群体杂交获得的三倍体长牡蛎子代生长和存活性状都比二倍体优良。杂交三倍体的优良性状主要是三倍体优势,杂交优势的贡献率还有待进一步实验证实。     

三种香港牡蛎三倍体幼虫诱导方法的效果比较

以卵裂率、D形幼虫发生率、三倍体率等为指标,比较了高盐、低温及咖啡因诱导香港牡蛎(Crassostrea hongkongensis)三倍体幼虫的效果,同时也分析了诱导因子强度(或浓度)、持续时间、受精卵密度及受精后开始处理时间对诱导效果的影响。结果表明,高盐诱导三倍体的最适条件组合是在卵子密度1.0×107个/L的情况下受精后15 min,用盐度40的海水持续处理10 min,此时卵裂率为(39.60±2.14)%, D形幼虫发生率为(31.46±1.06)%,三倍体率可以达到(59.53±5.90)%;低温诱导三倍体的最适条件组合是密度为2.0×10~7个/L的卵子在受精后15 min,用10℃海水持续处理10 min,此时卵裂率为(21.00±4.90)%, D形幼虫发生率为(12.68±1.21)%,三倍体率为(51.09±2.67)%;咖啡因诱导三倍体的最适条件组合是卵子密度为1.0×10~8个/L,用咖啡因终浓度2.0g/L的海水在受精后15min,持续处理20 min,卵裂率为(85.46±4.78)%, D形幼虫发生率为(71.79±3.92)%,三倍体率可以达到(56.36±2.07)%。最适诱导条件下,咖啡因的诱导效率指数0.405高于高盐处理0.187、低温处理0.065。高盐及低温处理方法诱导出的幼虫三倍体率随生长降低极快且存活率低于咖啡因诱导方法,说明3种方法中咖啡因可能更适合用于香港牡蛎三倍体幼虫的诱导。本研究为香港牡蛎三倍体育种提供了基础数据和实践经验。     

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，     

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%。     

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.     

杂交鲍三倍体诱导技术研究

为研究杂交鲍的三倍体诱导技术,通过预试验,观察和了解23.5 ℃下杂交鲍受精卵第一极体和第二极体释放的时间。采用细胞松弛素B抑制第二极体释放的方法,分别在不同的诱导起始时间、诱导剂浓度及诱导持续时间下抑制第二极体的释放,测定鲍的三倍体率,进行了杂交鲍三倍体的诱导试验。试验结果表明:杂交鲍在水温23.5 ℃下,处理起始时间为20 min,诱导剂浓度为0.70 mg·L-1,诱导的持续时间为10 min时,表观三倍体率最高,达75 %以上。检查稚鲍的倍性,三倍体率达到66 %。     

6-二甲基氨基嘌呤诱导扇贝异源三倍体

用 6 二甲基氨基嘌呤处理栉孔扇贝♀×虾夷扇贝♂受精卵 ,抑制第二极体释放诱导异源三倍体。水温 17℃下 ,分别进行了不同起始处理时间 (受精后 2 0～ 4 0min)、不同药物处理浓度 (4 0～ 80mg/L)和不同持续处理时间 (5～ 2 0min)的实验。起始处理时间实验以受精后 35min开始处理效果最好 ,三倍体率为 5 1 8% ,D形幼虫成活率可达 78% ;药物处理浓度和持续处理时间最佳组合为 6 0～ 70mg/L 6 DMAP处理 2 0min ,三倍体率可达 5 9 6 5 % ,D形幼虫成活率为 2 2 0 5 %。综合考虑 ,诱导扇贝异源三倍体的适宜参数为 :当 5 0 %受精卵排出第一极体时 ,以 6 0～ 70mg/L 6 DMAP处理 2 0min。     

壳金长牡蛎自交和杂交家系生长与存活比较

选择具有生长优势的长牡蛎(Crassostrea gigas)G2(A)家系及具有明显生长和存活优势的G19(B)和G28(C)家系进行完全双列杂交,得到包括3个自交组(AA、BB、CC)和6个杂交组(AB、AC、BA、BC、CA、CB)共9个壳金长牡蛎实验组,分析各实验组在幼虫期及稚贝期不同日龄的各生长指标和存活性能,并评估了杂交组的杂种优势。结果表明,在整个培育阶段,大多数杂交组在不同生长时期均表现出较高的生长和存活性能,其中,浮游幼虫期5日龄,所有杂交组的壳高和壳长均显著大于自交组(P0.05);10日龄后,杂交组CB、BC和AC的壳高显著大于相应的自交组(P0.05),杂种优势明显;稚贝期85和130日龄,杂交组CB的壳高性能大于其他实验组。浮游幼虫期10日龄,CB组存活率显著大于AA、CA组(P0.05);稚贝期85日龄后,杂交组AC、CA和CB的存活率杂种优势逐渐增大;190日龄,杂交组AC、BC与自交组AA、CC之间存活率差异显著(P0.05)。研究结果为壳金长牡蛎新品种的培育及杂种优势的充分利用奠定了重要基础。     

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.     

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.     

Comparative studies of the growth,survival, and reproduction of diploid and triploid Kumamoto oyster,Crassostrea sikamea

Triploid oysters have been used for farming to improve growth but have not been created in the Kumamoto oyster, Crassostrea sikamea, which is one of the crucial aquaculture species on the southern coast of China. In the present study, triploids were created using cytochalasin B to inhibit polar body II release in C. sikamea, with the untreated oysters as controls. Triploidy rates of 87 and 57.67%, on average, were obtained in larvae and adults, respectively. Larval growth and survival of the triploid were significantly lower than that of the controls (p < 0.05). In contrast, the triploid postlarvae and adults had a significant growth advantage over the controls (p < 0.05) during the period of 180 (December) to 450 days (September of the next year). Moreover, the triploids clearly exhibited significant sterility in the reproductive season. The glycogen and triglyceride contents in the gonad, adductor muscle, mantle, and gill were higher in triploids than in controls from 180 to 450 days. As a result, high physiological energy supply was strongly correlated with superior growth and reduced reproduction in triploid C. sikamea. The triploid C. sikamea is an excellent oyster species and can be used to improve growth for C. sikamea farming.     

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     

Triploid induction by hyperosmotic shock in the Yesso scallop,Patinopecten yessoensis

The triploid technology is a new frontier in shellfish aquaculture and has shown encouraging results in numerous shellfish species. We induced triploid larvae in the Yesso scallop, Patinopecten yessoensis, using hyperosmotic shock for the first time in this study. Different induction parameters, including salinity strength, treatment starting point, and the duration, were tested. The highest triploid ratio of D‐shaped larvae (72.12%) was obtained by 60 ppt salinity treatment for 20 min at the first appearance of zygote showing polar body II (PB2). A significantly faster growth rate was observed during the swimming larvae stage despite a decrease in the hatching and survival ratios. The triploid ratio decreased to 46.67%, and approximately 1.76 million triploid juveniles were harvested after 90 days of cultivation. The treatment parameters can be further optimized to improve the yield of Yesso scallop triploids.     

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.     

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.     

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.