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。     

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。     

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.     

6_二甲基氨基嘌呤诱导太平洋牡蛎三倍体_抑制受精卵第一极体释放

于 1996～ 1997年 ,用 6-二甲基氨基嘌呤 ( 6-DMAP)抑制受精卵第一极体的释放 ,诱导太平洋牡蛎产生三倍体。选用L16( 45)设计 ,进行三因素四水平的正交试验 :6-DMAP浓度 ,设15 0、30 0、45 0和 60 0μmol/L ;诱导时机 (即精卵混合后的时间 ) ,设 10、15、2 0和 2 5min ;诱导持续时间 ,设 10、15、2 0和 2 5min。试验平行重复二次。结果为最高三倍体诱导率为 ( 71.3± 1.2 ) % ,该实验组胚胎孵化率为 ( 5 5 .5± 3.1) % ,D形幼虫畸形率为 ( 10 .7± 1.6) %。根据直观分析结果 ,得出诱导太平洋牡蛎三倍体各因素的最优水平组合 :水温 2 5～ 2 5 .5℃ ,精卵混合 10min时 ,将受精卵浸泡在含 6-DMAP 60 0μmol/L的海水中 15min ;决定三倍体产生的三因素的主次顺序 :6-DMAP浓度→诱导时机→诱导持续时间。 6-DMAP浓度对三倍体诱导率影响显著 ,但诱导时机和诱导持续时间不显著。     

6-二甲基氨基嘌呤诱导泥蚶三倍体实验

于2001年5月,用6-DMAP抑制泥蚶受精卵第二极体的释放诱导三倍体的产生。实验设三个6-DMAP浓度梯度,分别为300μmol/L、450μmol/L、600μmol/L,处理时机为受精卵第一极排放体30％,处理持续时间为10min。结果:三倍体诱导率300μmol/L时为35.5％,450μmol/L时为48.6％,600μmol/L时约为58.5％,另外本实验还就6-DMAP浓度对D形幼虫孵化率和畸形率的影响以及在染色体制片过程中的有关问题进行了初步探讨。     

低渗诱导太平洋牡蛎三倍体以及与其他诱导方法的比较

通过低渗方法抑制受精卵第二极体(PB2)的释放,诱导太平洋牡蛎(Crassostrea gigas)三倍体.在水温为23～25℃的条件下,分别采用不同低渗海水(盐度分别为4、6、8、10、12、14、16),在不同处理时机,即第1个第一极体(PB1)出现、30%的PB1出现、40%的PB1出现、50%的PB1出现以及第1个第二极体(PB2)出现时,对太平洋牡蛎受精卵进行10 min、15 min、20 min、25 min持续处理.处理后,于正常盐度海水中孵化,收集D形幼虫,通过流式细胞仪进行倍性测定,确定最佳诱导条件.结果表明,当太平洋牡蛎受精卵PB1出现40%时,在盐度为8的低渗海水中,持续处理15 min,得到最高三倍体诱导率为(89.16±1.39)%.与对照组相比,低渗诱导获得的三倍体幼虫表现出明显的生长优势.将低渗方法同高温(32℃)、低温(2℃)和6-DMAP(450μmo1/L)处理等多倍体诱导方法进行比较,结果显示,低渗组的卵裂率和孵化率显著高于高温、低温和6-DMAP诱导的太平洋牡蛎三倍体(P＜0.05).6-DMAP诱导的三倍体率虽略高于低渗诱导,但差异不显著(P＞0.05).通过综合评价指数(Ie)比较,认为低渗诱导多倍体的方法比高温、低温和6-DMAP诱导具有明显的优势.     

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%。     

人工诱导栉孔扇贝雌核发育二倍体的初步研究

2004年5~6月,用紫外线照射法使精子遗传物质失活,结合用6-二甲基氨基嘌呤(6-dime-thylaminopurine,6-DMAP)处理受精卵抑制第2极体释放,诱导栉孔扇贝雌核发育二倍体。采用中心波长254nm、光照强度800μw/cm2·s的紫外线照射栉孔扇贝精子50s,然后立即与正常卵子授精,在20℃水温下,受精后35~40min光镜下观察到20%~30%卵子排出第1极体时,分别以40、50、60、70、80mg/L的6-DMAP持续处理受精卵10、15、20min,染色体计数法检测各处理组二倍体率,从而筛选诱导栉孔扇贝雌核发育二倍体两因素的最佳组合。结果表明,6-DMAP的浓度和持续处理时间对二倍体诱导率和D形幼虫率影响较显著,6-DMAP浓度以60、70mg/L较为适宜,最佳处理时间为20min,综合考虑,60mg/L6-DMAP处理20min组得到了最佳效果,二倍体率为57.6%、D形幼虫率22.25%。随后对筛选出的最佳条件组担轮幼虫进行了大量的染色体统计和流式细胞仪分析,结果基本一致,二倍体率分别为53.7%和57.3%。     

扇贝异源三倍体诱导

荧光显微观察表明,20℃水温下,栉孔扇贝(Chlamysfarreri)的卵与海湾扇贝(Argopecten irradians)的精子可以正常受精和发育,具备人工诱导三倍体的可行性.亲贝充分促熟后,分开催产,以20:1的精卵比授精;在50%的受精卵排出第1极体时,以60mg/L 6-二甲基氨基嘌呤(6-DMAP)处理受精卵10～25 min,可诱导75.23%～92.14%的三倍体;6-DMAP处理15 min综合诱导效果最好,三倍体诱导率可达88.56%,孵化率可达53.52%.得到的三倍体幼虫经基因组原位杂交(Genomicin situ hybridization,GISH)验证,为含有2套栉孔扇贝染色体组和1套海湾扇贝染色体组的异源三倍体.孵化后诱导组与杂交对照组(未经6-DMAP处理)幼虫生长越来越缓慢,受精后14 d其幼虫存活率分别下降到0.000 67%和0.002 24%,没有幼虫度过附着变态期.GISH分析显示,栉孔扇贝与海湾扇贝不同倍性的杂种后代早在担轮幼虫阶段就出现母本偏向性染色体转变.     

三倍体九孔鲍的育种和养成技术研究

当 5 0 %九孔鲍受精卵出现第一极体后 ,以 6 -DMAP为诱导剂 ,用不同的浓度和不同的持续时间抑制第二极体的释放诱导三倍体 ;并进行了生产性苗种培育和养成试验。结果表明 ,6 -DMAP的诱导浓度为30 0μmol·dm-3 、诱导持续时间为 10min时 ,其胚胎的三倍体率为 90 %以上 ,并且胚胎的孵化率高达 85 % ,幼体的畸形率较低 ,为 5 0 %～ 5 5 % ;经过 7个月培育 ,试验组养成鲍的三倍体率为 6 5 %以上 ;试验组养成鲍的平均壳长比对照组增长 10 % ,平均体重比对照组增重 30 %。与二倍体鲍相比较 ,三倍体鲍在壳长增长和体重增重等方面 ,显示了显著的优势 [t>t0 .0 1( 58) ]。     

Association between the interannual variation in the oceanic environment and catch rates of bigeye tuna (Thunnus obesus) in the Atlantic Ocean

The environmental processes associated with variability in the catch rates of bigeye tuna in the Atlantic Ocean are largely unexplored. This study used generalized additive models (GAMs) fitted to Taiwanese longline fishery data from 1990 to 2009 and investigated the association between environmental variables and catch rates to identify the processes influencing bigeye tuna distribution in the Atlantic Ocean. The present findings reveal that the year (temporal factor), latitude and longitude (spatial factors), and major regular longline target species of albacore catches are significant for the standardization of bigeye tuna catch rates in the Atlantic Ocean. The standardized catch rates and distribution of bigeye tuna were found to be related to environmental and climatic variation. The model selection processes showed that the selected GAMs explained 70% of the cumulative deviance in the entire Atlantic Ocean. Regarding environmental factors, the depth of the 20 degree isotherm (D20) substantially contributed to the explained deviance; other important factors were sea surface temperature (SST) and sea surface height deviation (SSHD). The potential fishing grounds were observed with SSTs of 22–28°C, a D20 shallower than 150 m and negative SSHDs in the Atlantic Ocean. The higher predicted catch rates were increased in the positive northern tropical Atlantic and negative North Atlantic Oscillation events with a higher SST and shallow D20, suggesting that climatic oscillations affect the population abundance and distribution of bigeye tuna.     

Rare serotype occurrence and PFGE genotypic diversity of Streptococcus agalactiae isolated from tilapia in China

Previously, we reported 10 PEGE types of 85 tilapia Streptococcus agalactiae(GBS), which shifted from Streptococcus iniae in China, by using PEGE method. Presently, larger and more representative tilapia GBS were isolated, for the ?rst time in China, to characterize their serotypes and genetic diversities more precisely than had done before. 168 GBS strains were distributed in ?ve provinces of China, in which Guangdong, Guangxi and Hainan were the major ones, holding36.9%(62/168), 37.5%(63/168) and 19.6%(33/168), respectively. Serotypes, Ia, Ib and III, were observed in these strains and the most predominant one was Ia(95.2%), which mainly distributed in Guangdong, Guangxi and Hainan. Ia initially occurred in 2009, it shoot up to 32.1% in 2010,but decreased to 16.1% in 2011 before went up to 45.2% in 2012. Ib sporadically occurred during2007–2011, III onlyoccurred in 2012. 14 different PFGE types, including 4 new types(N, O,P and Q), were observed, in which B, D, F and G were the predominant types, holding 83.9%(141/168) of the total GBS strains. Ia corresponded to 11 PFGE types(A–H, N–P), in which type D predominated(51%). Ib represented 3 genotypes(I, J and Q) and III harbored only 2genotypes(N and F). Type N and Fsynchronously presented in Ia and III. In summary, the genetic diversity of tilapia GBS varied by serotypes and changed with geographical locations and years.Although Iastillpredominated, new rareserotypeIII alreadyoccurred in China.     

Evaluation of a Fast Method Based on the Presence of Two Restriction Sites in the Mitochondrial ND5 (mt ND5) Gene for the Identification of Scomber Species

The purpose of this work was to evaluate the suitability of a method based on the presence of two restriction sites (for Hae III and Hindf I) in the mitochondrial NADH dehydrogenase subunit 5 (mt ND5) gene to identify Scomber species. The evaluation was performed on 144 reference and market samples by sequencing of the entire 505-bp fragment of the mt ND5 gene and of a 464-bp fragment of the Kocher fragment of the cytochrome b gene (mt Cytb). Sequence analysis of any of the two fragments allows the identification of each of the four Scomber species, but S. japonicus and S. colias had the same restriction sites at the ND5 amplicon and would not have been differentiated by this analysis. Similarly, loss of the Hae III site in some S. scombrus individuals would have misidentified them as not being Scomber. All the market products were correctly labeled except one acquired in Spain labeled as originating in the Atlantic and containing S. japonicus.     

Growth performance,digestive enzyme activity and immune response of Japanese sea bass,Lateolabrax japonicus fed with fructooligosaccharide

In this experiment, a feeding trial was performed to determine the effects of fructooligosaccharide (FOS) on growth performance, digestive enzyme activity and immune response of Japanese sea bass, Lateolabrax japonicus juveniles (initial weight 38.3 ± 0.5 g), and the fish were examined following feeding with six levels of FOS (0, 0.5, 1, 2, 4 and 6 g/kg) for 28 days. Significant enhancement of weight gain (WG) and specific growth rate (SGR) was found in fish fed 1 g/kg FOS incorporated diets (p < .05), while the feed conversion ratio (FCR) in the 1, 2 g/kg FOS groups reduced significantly compared with the control (p < .05). Besides, the crude lipid in the 4, 6 g/kg FOS groups increased significantly compared with the control (p < .05). On the other hand, the erepsin and lipase activities significantly elevated in intestine of fish fed 2 g/kg FOS (p < .05) and the lysozyme activity in serum of fish fed 2 g/kg FOS were significantly higher than that in the control (p < .05). Moreover, the alkaline phosphatase activities in serum of fish fed 0.5, 1, 2 g/kg FOS were significantly higher than in control (p < .05). Regression analysis showed that the relationships between dietary FOS levels and either SGR, FCR, erepsin or lysozyme activities were best expressed by regression equations, and the optimal inclusion levels are 1.37, 1.80, 3.06, 3.11, 1.93 and 1.80 g/kg for SGR, FCR, erepsin, lipase, lysozyme and total superoxide dismutase activities, respectively. Overall, this study revealed that FOS incorporated diets could beneficial for L. japonicus culture in terms of increasing the growth, digestion and immune activities. Under the present experimental condition, the optimal supplementary level of FOS in the diet of L. japonicus is 1–3 g/kg.     

Growth hormone (GH) and reproduction: a review

Interaction between growth and reproduction occurs in many vertebrates and is particularly obvious at certain stages of the life cycle in fish. Endocrine interactions between the gonadotropic axis and the somatotropic axis are described, the potential role of GH being emphasised. A comparative analysis of these phenomena in mammals, amphibians and fish, suggests a specific role of GH in the physiology of puberty, gametogenesis and fertility. It also shows the original contribution made by studies on the fish model in this field of investigations.     

Purification and partial characterization of GtHs (cfLH and cfFSH) from Indian walking catfish (Clarias batrachus) (L.) and development of a homologous ELISA for cfLH

Two gonadotropins (GtH; Qa and Qb) were purified by gel filtration and ion exchange chromatography from the pituitaries of Indian walking catfish (Clarias batrachus). The presence of GtH during purification was assessed by in vitro oocyte maturation and in vivo steroidogenic activity, and their identities were determined by elution profiles, molecular weight, biological activities and yield. The molecular weights of Qa and Qb were 37 and 42 kDa, respectively, and composed of distinct subunits (Qa: 20 and 14 kDa and Qb: 26 and 18 kDa). Polyclonal antibodies raised against Qa immunostained Qa, Qb and pituitary GtH cells. A competitive Qa‐ELISA was developed whose sensitivity was 6.25 ng mL^?1 (1.25 ng well^?1) with intra‐ (3.5%) and inter‐ (12.4%) assay coefficients of variation. Displacement curves parallel to the standard were obtained with plasma and pituitary extracts of catfish, Qb and carp GtHII. The assay was validated by measuring the plasma Qa levels after LHRH treatment and in relation to ovarian growth in the female catfish during different reproductive phases. Based on the results, Qa and Qb corresponded to fish LH and FSH respectively. The findings will increase the knowledge of the mechanisms controlling fish reproduction and identification of sensitive phases in fish in captivity for hormonal manipulation.     

Controlled infection of Poecilia reticulata Peters (guppy) with Tetrahymena by immersion and intraperitoneal injection

Tetrahymena is a protozoan parasite, which infects guppy, Poecilia reticulata Peters, and causes substantial economical losses in commercial farms worldwide. Studies of guppy infected by Tetrahymena require standardized infection protocols. The LD50 for Tetrahymena infection of guppies by intraperitoneal (IP) injection was calibrated, and the level obtained was 946 parasites per fish. Guppy infection with Tetrahymena by immersion, imitating the natural route of infection via the integument, was studied under normal or stress conditions. Exposure to cold and netting (CNI) and to cold only (CI) followed by immersion exposure to 10 000 Tetrahymena per mL resulted in 22.5% and 19.2% mortality, respectively, as compared to 14.2% and 10% in groups that were netted only (NI) or non‐stressed (I). Histopathology revealed that immersion infection resulted in a systemic infection. Lysozyme levels, measured 3 weeks after infection, were significantly higher in the CNI group (288 μg per mg protein) compared with CI‐, NI‐ and I‐treated groups (94.5, 64 and 62.3 μg mg^?1, respectively). There was no evident parasite immobilization activity in body homogenates, suggesting no development of acquired immunity. Re‐infection by IP injection revealed no increase in protection in any of the treatment groups, mortality range of 56.3–75%, higher than in the non‐exposed control (40.6% mortality).