Nuclear transfer in loach (Paramisgurnus dabryanus Sauvage) by cell-to-cell electrofusion

To study nuclear transfer in the loach (Paramisgurnus dabryanus Sauvage), blastula and gastrula cells were fused with UV-inactivated oocytes by cell-to-cell electrofusion. To facilitate nuclear transfer, blastula and gastrula cells were cultured or incubated at 4 °C in different solutions. TC-199 medium supplemented with 20% calf serum was the best culture solution, and effectively retained the totipotence of blastula or gastrula cells for up to 10 days. It was found that gastrula cells incubated at 4 °C had the same totipotence as blastula cells. The optimal UV dosage for inactivation of the oocyte chromatin was 180–240 mJ cm⁻². Electrofusion was carried out in a cone-shaped fusion chamber, which permitted the recipient oocyte and the donor blastula cell to contact one another. The electrofusion procedure resulted in a 10% success rate of normal-appearing fish. Genetic analysis indicated that the nuclear material originated from the donor cell (blastomere) and the oocyte pronucleus did not take part in development.     

Stages of embryonic development and changes in enzyme activities in embryogenesis of turbot (Scophthalmus maximus L.)

The early development of turbot (Scophthalmus maximus) from fertilization to hatching was described. Hatching occurred at 108 h post-fertilization (hpf) in 14 °C. Yolk syncytial layer and blastocoel formed at morula stage and low stage, respectively. Neural rod derived from the ectoderm appeared and the first somite formed in the middle of the embryonic body at 90 % epiboly stage, and notochord primordium formed at complete epiboly stage. Kupffer’s vesicle appeared at 59 h 35 min hpf and degenerated at 89 hpf. At 72 hpf, the digestive tract formed in the ventral side of the embryonic body, and the posterior digestive tract of embryo was ciliated at 89 hpf. Enzymes play a key role in the catabolism of yolk during embryogenesis of fishes. In this study, the main enzymes alkaline phosphatase (AP), leucine aminopeptidase N (LAP), pepsin, trypsin and Leucine-alanine peptidase (Leu-ala) were all observed in unfertilized eggs and embryo of S. maximus, but amylase was not detected, speculating that amino acids appear to be the main energy substrate during embryonic development of S. maximus, while carbohydrates is less essential. AP reached the lowest value at the gastrula stage and then increased rapidly reaching the highest value at hatching. LAP showed the highest value in unfertilized eggs and kept on decreasing until the blastula stage with the lowest value and then increased at the gastrula stage, followed by a gradual decline thereafter. Trypsin reached the lowest value at the blastula stage and then fluctuated with the maximal value at hatching. Pepsin reached the highest and the lowest values at the unfertilized eggs and the cleavage stage, respectively, but disappeared at hatching. Leu-ala had the maximum activity at the blastula stage and then declined to the minimum at the gastrula stage followed by a gradual increase thereafter.     

单环刺螠Vasa基因的早期发育表达图式

Vasa基因编码DEAD-box家族成员中一种ATP依赖的RNA解旋酶,是决定生殖系发育的重要调控因子之一。采用同源克隆策略及SMART-RACE技术,克隆了海洋经济螠虫动物单环刺螠Vasa基因的全长cDNA,该序列长4 080 bp,开放阅读框2 322 bp,编码733个氨基酸,具有DEAD-box蛋白家族共有的全部9个保守域。整体原位杂交和半定量RT-PCR结果显示,Vasa基因在未受精卵、受精卵、2～8细胞的早期胚胎中均有明显的表达,显示其母源性提供的特点;从囊胚开始,表达量明显降低,在原肠胚表达信号主要集中在内中胚层细胞中;至担轮幼虫,表达信号进一步集中在消化道处;当发育至体节幼虫时,阳性细胞分布于消化道和体节的隔膜处,进入蠕虫状幼虫,信号仅在头部的腹刚毛附近以及后肠周围的细胞中表达。实验结果为探知刺螠动物生殖系的起源和分化以及低等型生殖腺的发生提供重要的数据。     

Oxygen deficiency during somitogenesis causes centrum defects in red sea bream, Pagrus major (Temminck et Schlegel)

Vertebral deformities in red sea bream, Pagrus major, remain serious obstacles to the improvement of seedling quality for its aquaculture. However, the causalities of the deformities remain unclear and prevention methods have not yet been established. In this paper, oxygen deficiency during somitogenesis was demonstrated to cause centrum defects (formerly called fused vertebrae in many cases), which are the major vertebral deformity in cultured red sea bream. An induction experiment of centrum defects was conducted by placing fertilized red sea bream eggs under low dissolved oxygen conditions (10.3–16.6%). The low oxygen treatment was carried out for five different developmental stages of embryo: two‐cell stage to blastula stage; gastrula stage; three to 10 somites stage; 11–17 somites stage and 18–24 somites stage. Oxygen deficiency during somitogenesis induced a high incidence of centrum defects. In contrast, it hardly induced centrum defects during the other stages. The dissolved oxygen concentration in the rearing water should be carefully regulated for fertilized eggs, especially during somitogenesis to reduce the incidence of vertebral deformities in the red sea bream.     

Effects of cell-cycle arrest agents on cleavage and development of loach (Misgurnus anguillicaudatus) embryos

The aim of this study was to determine the lowest concentration of nocodazole and colchicine to arrest blastomere division during the cleavage stage of loach embryos and to assess the reversibility and toxicity of the treatments in the treated embryos. Eight-cell loach embryos were incubated for 4, 8, 12, or 16 h in 1/10× Holtfreter supplemented with either nocodazole, an inhibitor of tubulin polymerization, or colchicine, an inhibitor of tubulin assembly. Complete arrest of cell cycle was observed, at a colchicine concentration of 0.996 mM and at a nocodazole concentration of 0.275 μM, respectively (the lowest effective concentration). No major morphological alteration in chromatin was observed. Reversibility and toxicity of both agents were dose and exposure period dependent. For both agents, prolonging cleavage arrest for more than 4 h (at the effective concentrations) is detrimental to development of embryos. Nocodazole treatment was less cytotoxic, whereas the concentrations of colchicine which induce cleavage arrest were detrimental to development beyond the blastula stage. Toxic effects beyond the blastula stage could be minimized for both agents by reducing the period of treatment and concentration. This revised version was published online in August 2006 with corrections to the Cover Date.     

Cloning,sequence analysis,and expression of tyrp1a and tyrp2 genes related to body colour in different developmental stages and tissues of rainbow trout Oncorhynchus mykiss

Tyrosinase-related protein 1 (tyrp1) and tyrosinase-related protein 2 (tyrp2) genes are important downstream regulatory factors for body colour formation in animals. The present study aimed to explore the relationship between tyrp1a and tyrp2 expression and body colour variation between wild-type (WT) and yellow mutant (YM) rainbow trout. The full-length cDNA sequences of trout tyrp1a and tyrp2 were obtained using rapid amplification of cDNA ends, and the bioinformatic analysis was performed. Quantitative real-time PCR (qRT-PCR) was used to investigate the different expression levels of tyrp1a and tyrp2 in different developmental stages and tissues. The full-length cDNAs of tyrp1a and tyrp2 were 2409 bp and 2219 bp encoding predicted proteins of 522 and 529 amino acid residues, respectively. Both proteins possess six conserved domains, including a signal peptide, an EGF-motif, a Pfam tyrosinase motif, a transmembrane structure, and two copper binding sites (CuA and CuB). Amino acid sequence comparisons showed higher conservation of Tyrp1a and Tyrp2 proteins amongst fishes than amongst other vertebrates, which was further confirmed by phylogenetic analysis. qRT-PCR analysis revealed that tyrp1a and tyrp2 expression levels in WT rainbow trout, and tyrpla in YM rainbow trout, were detected from the fertilised stage to 12 months post hatching (12 M), while tyrp1a did not expressed until the blastula in YM rainbow trout. In addition, the expression levels of both of the genes post hatching were significantly higher than those in the embryonic stages, which showed extremely low expression levels. Additionally, there were extremely significant differences (P < 0.01) in expression at the same stages between WT and YM rainbow trout, e.g., at the blastula, gastrula, 7 days post hatching (7 dph), 10 dph, 2 M, 3 M, 6 M, and 12 M stages for tyrp1a; and at the 4-cell, 16-cell, multicell, blastula, somites, heartbeating, 1 dph, 5 dph, 7 dph, 3 M, 6 M, and 12 M stages for tyrp2. Besides, variable expression levels of tyrp1a and tyrp2 were detected in all tissues; significantly high expression was detected in the dorsal skin and eye compared with that in the other tissues in WT and YM rainbow trout (P < 0.05). These results suggested that the expression level changes of tyrp1a and tyrp2 might be closely related to the variation of rainbow trout body colour, which will enrich our knowledge of the molecular mechanism of skin colour variation in rainbow trout and provide data for research into fish skin colour inheritance and improvement.

     

Biochemical characterization of growth hormone receptor in rainbow trout (Oncorhynchus mykiss) before and after purification

The aim of this work was to verify if, compared to mammals, the lower molecular weight of GH-R previously reported in salmonid is real or due to the experimental process. For this purpose, we compared the apparent molecular weight of GH-R, obtained by SDS-PAGE after cross-linking with ⁵⁰ I-rtGH, obtained from rainbow trout crude liver membrane preparation, incubated in different buffers with those obtained after purification with affinity chromatography.Using crude liver membrane preparation, two specific bands of ⁵⁰ I-rtGH-protein complex were observed: the major one corresponds to a MW of 70 kDa and the minor one to 45 kDa. However, the pattern of electrophoresis varied according to the different incubation buffers tested. Digestion of the cross-linked complex with -galactosidase and phospholipase did not significantly modify the position of bands, whilst N-glycosidase F induced a large smear including 4 more pronounced bands (50, 65, 97 and > 130 kDa), the heavier band corresponding to the most intensive signal.GH receptors were purified using solubilisation and affinity chromatography. The yield of the liver GH-R from crude liver membrane preparation by the solubilization technique was optimized (48%) using Triton 1% for 1 h (12°C ). Specific binding sites in the solubilized membrane proteins were saturable when incubated with increasing ⁵⁰ I-rtGH concentrations, and revealed a high affinity constant (Ka=0.7×10⁹ M^–1). After affinity chromatography, specific binding activity was increased 64,000 fold. However, the purity of the preparation was partial and the purification yield was very low (about 0.3%). This enriched fraction, analysed by SDS-PAGE after cross-linking, showed a very intense band (about 63 kDa) which disappeared with an excess of cold rtGH.These results suggest that the lower molecular weight observed in salmonid (41 kDa), compared to mamals, is not due to the experimental process. The significance of GH-R size difference between salmonids and mammals is discussed.     

黑龙江茴鱼胚胎的发育及仔、稚、幼鱼的生长

采用Bouin氏液固定、剥离卵膜的方法对黑龙江茴鱼(Thymallus arcticus grubei Dybowski)的胚胎发育及孵出后仔鱼卵黄囊的吸收情况进行了观察,同时测定65日龄内的仔、稚、幼鱼生长状况。结果显示:黑龙江茴鱼卵是典型的端黄卵,其卵裂方式为盘状卵裂。胚胎发育可分为22个期,包括受精卵、2细胞期、4细胞期、8细胞期、16细胞期、32细胞期、64细胞期、多细胞期、囊胚早期、囊胚中期、囊胚晚期、原肠早期、原肠中期、原肠晚期、神经胚期、胚孔封闭期、眼囊出现期、胸鳍芽出现期、晶体出现期、眼色素沉积期、循环期和出膜期,至发眼(晶体出现期)需75.84℃.d,至破膜需117℃.d;受精卵膜较鲑科鱼类薄而柔软,胚体头尾绕卵黄囊超过一周,晶体出现在眼色素沉积期之前;仔鱼卵黄囊的体积随着日龄的增加而逐渐减小,4日龄前吸收较快,此后吸收相对较慢;仔鱼在240℃.d(11日龄)左右时出现鳔并开始上浮,260℃.d(16日龄)左右时开口;积温达到346.9℃.d(20日龄)时卵黄囊完全被吸收,仔鱼期结束,进入稚鱼期;稚鱼在36日龄之前生长相对较缓慢,在36日龄后增长较快;在积温达到546.3℃.d(38日龄)时,稚鱼奇鳍褶开始退化,进入幼鱼期。     

Age-related changes in 2-[125I]-iodomelatonin binding sites in the brain of sea breams (Sparus aurata,L.)

The pineal organ of fish, through its 24h rhythmic release of melatonin, acts as a transducer of the photoperiod, influencing different physiological functions (e.g., reproduction, growth). The target sites for melatonin are poorly known in fish, especially marine species. A radioligand study was undertaken using the gilthead sea bream (Sparus aurata) maintained under natural temperature and photoperiod (at 28°N latitude). This species exhibits the property of changing sex during growth. Brains of one year-old males were collected at 16:00h and brains of three year-old females at 03:00, 10:00, 16:00 and 23:00h. Membrane homogenate receptor assays were run using 2-[¹²⁵I]iodomelatonin as a ligand. Binding sites were detected in brains of young and old fish. In the younger, the exhibited a B_max between 3.52 and 4.29 fmol mg protein^–1 and a K_D between 358–380 pmol l^–1. In the older fish, the K_D varied according to a daily pattern: values were three times higher at 03:00 and 10:00h (500–600 pmol l^–1) than at 16:00 and 23:00h (150–300 pmol l^–1). The number of sites also were higher at 03:00 and 10:00h (180–200 fmol mg protein^–1) than at 16:00 and 23:00h (95–110 fmol mg protein^–1). Melatonin and iodomelatonin displaced 2-[¹²⁵I]iodomelatonin binding in a dose dependent manner, the second being more potent than the first. Binding was also inhibited by GTP. The results provide the first evidence for the presence of membrane melatonin binding sites in the brain of an exclusively marine fish. They suggest that their number and affinity varies during growth and throughout a light/dark cycle. Future experiments will aim to precise the anatomical location and role of these binding sites.     

云纹石斑鱼(♀)×七带石斑鱼(♂)杂交子一代胚胎发育及仔稚幼鱼形态学观察

以云纹石斑鱼(Epinephelus moara)♀为母本,七带石斑鱼(E.septemfasciatus)♂为父本进行种间远缘杂交,系统观察了杂交子一代胚胎及仔稚幼鱼的生长发育及形态特征。对杂交子一代的胚胎发育按卵裂期、囊胚期、原肠胚期、神经胚期和器官形成期各阶段进行观察,在水温21～22.5℃、盐度29、pH 8～8.1的海水中,整个胚胎发育过程经35 h 20min完成。根据在卵黄囊期、第二背鳍棘和腹鳍棘生长消退过程、鳞片生长、体表色素变化的不同,观察比较了胚后发育的仔鱼期、稚鱼期、幼鱼期形态特征。鱼苗培育至58 d后完成了稚鱼至幼鱼的变态:初孵仔鱼4 d以前为内源性营养(卵黄囊仔鱼);4 d后开口摄食,为外源性营养;(42±2)d基本完成变态进入稚鱼阶段,(58±4)d全身被鳞,进入幼鱼期。同时,用同一母本(云纹石斑鱼)同批卵与同种雄鱼采集的精子授精后获得的受精卵作为对照组,进行孵化和仔稚幼鱼培育,与杂交子一代进行比较,结果发现杂交子一代在仔稚幼鱼阶段生长速度快于云纹石斑鱼,与七带石斑鱼相近。     

Relationship between dietary phospholipid classes and neutral lipid absorption in newly-weaned turbot, shape Scophthalmus maximus

A 28-day feeding trial was conducted for comparing the effect of different dietary phospholipid (PL) classes on the growth of post-larval turbot and on the incorporation of dietary neutral lipid fatty acids into their body lipids. Prior to the experiment the turbot were weaned for one week on a PL-free diet. The nine experimental diets were isolipidic and contained an equal amount of highly unsaturated fatty acids in the form of ethyl esters. They differed by their PL content (0, 1 or 2%) and by the PL class composition of the added soybean PL fractions.Compared to the PL-free diet, diets enriched with phosphatidylcholine (PC) resulted in a better growth, a higher triglyceride content (% body dry matter) and increased levels of docosahexaenoic acid (% total fatty acids) in each of the examined body lipid classes (neutral lipid, phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol). The effects of the other soybean PL fractions were less explicit than those noted for soybean PC.The results support the idea that dietary PC plays a role in the intestinal absorption of neutral lipid fatty acids. This might, at least partially, explain the superiority of PC for enhancing growth. Abbreviations: DHA – docosahexaenoic acid (22:6n-3); EPA – eicosapentaenoic acid (20:5n-3); HUFA – highly unsaturated fatty acid; PA – phosphatidic acid; PC – phosphatidylcholine; PE – phosphatidylethanolamine; PI – phosphatidylinositol; PL – phospholipid; PS – phosphatidylserine; PUFA – polyunsaturated fatty acid.     

Acute Toxicity of Synthetic Pyrethroid Cypermethrin on the Common Carp (<Emphasis Type="Italic">Cyprinus carpio</Emphasis> L.) Embryos and Larvae

In this study, the toxic effects on the embryos and larvae of the common carp were used as a model to investigate the synthetic pyrethroid pesticide, cypermethrin, which contaminates aquatic ecosystems. Data obtained from the cypermethrin acute toxicity tests were evaluated using the Probit Analysis Statistical Method. The control and eight test experiments were repeated five times. The number of dead embryos significantly increased in response to cypermethrin concentrations 0.0001, 0.001, 0.01, 0.1, 1, 2, 4 and 8 μg l⁻¹ (p<0.05 for each case). The 48 h LC₅₀ value (with 95% confidence limits) of cypermethrin for common carp embryos was estimated at 0.909 (0.256–5.074) μg l⁻¹. Dose–response decreases in hatching success were recorded as 87.4, 85.0, 80.2, 71.4, 56.3, 48.6, 38.8 and 23.5%, respectively. The lowest concentration of cypermethrin (0.0001 μg l⁻¹) produced a significant increase in the number of dead larvae compared to the control group (p<0.05). The number of dead larvae significantly increased with increasing cypermethrin concentrations exposed for 1–96 h (p<0.05). The highest concentration (8 μg l⁻¹) showed the highest larvae mortality. The 96 h LC₅₀ value (with 95% confidence limits) of cypermethrin for common carp larvae was estimated at 0.809 (0.530–1.308) μg l⁻¹. The results of the study suggest that low levels of cypermethrin in the aquatic environment may have a significant effect on the reproduction and development of carp.     

温度对四川华鳊胚胎发育的影响

为探索温度对四川华鳊胚胎发育的影响,笔者观察16、19、22、25、28、31℃6个温度条件下四川华鳊的胚胎发育过程,描述总结(25±0.5)℃下胚胎发育各个阶段的形态特征。研究结果显示,四川华鳊胚胎发育过程可划分为受精卵、胚盘形成、卵裂、囊胚、原肠胚、神经胚、器官分化和出膜等8个连续发育阶段;在(25±0.5)℃下孵化总历时44.83 h;在温度为16℃时,胚胎发育至原肠胚晚期全部死亡;19℃时孵化率为30.00%,其中畸形致死占87.19%,显著高于其余4个温度组(P<0.05);温度由19℃升至31℃时,胚胎发育所需时间变短,各温度间差异显著(P<0.05),其中器官分化阶段均用时最长,占整个胚胎孵化历时的72.53%~77.07%;胚胎畸形率随着温度升高而上升,31℃的胚胎畸形率约为28℃的2倍;在22~28℃时,胚胎的受精率和孵化率、成活率最高,畸形率最低。研究结果表明,水温在(19±1)℃,卵径(y)与胚胎孵化时间(x)的关系为y=49.56-44.36x+47.92x^2(r^2=0.996);水温22~28℃为适宜孵化温度,最佳水温约为25℃;胚胎发育的生物学零度为12.69℃,有效积温为522.35~595.11℃·h。     

Growth and production of the Amazonian tambaqui in fixed cages under different feeding regimes

Experimental culture of the native Amazonian fish tambaqui, Colossoma macropomum, in fixed cages was carried out over a period of 8 months, in Lake Urubu (Rio Grande do Norte, Brazil), to assess the viability of fixed cage culture of tambaqui and to test the influence of diet on growth rates. Nine synthetic net cages (1 m³) were each stocked with 45-day-old fish (mean weight 3 g; mean total body length 51 mm) at a density of 34 fry m^–3. During the first 2 months of culture, fish were fed a balanced formulated feed on an as-fed basis at the rate of 5% body weight day^–1. During months 3–8 this continued for fish in treatment 1 while those in treatment 2 were fed tropical regional fruits, on a wet weight basis at the rate of 5% body wt day^–1. Fish in treatment 3 were given no supplementary feed. Monthly biometric measurements were made on all fish. Fixed cage fish culture was shown to be a viable and simple technique. Survival in all treatments was 100%. With balanced supplementary feed, production was 14.4 kg m^–3, compared with 4.9 kg m^–3 and 2.1 kg m^–3, respectively, in the treatments where fish were fed with fruits and were not given any supplementary feed.     

Preliminary investigation to determine the toxicity of various cryoprotectants on striped gourami (Trichogaster fasciata) embryos

As a study of cryoprotectant toxicity is an essential prerequisite for the development of a cryopreservation protocol, this study focuses on determining the toxicity of four permeable cryoprotectants: dimethyl sulfoxide (DMSO), propylene glycol (PG), methanol (MeOH), and acetamide (Ac). In cryoprotectant toxicity experiments, striped gourami (Trichogaster fasciata) embryos at three different developmental stages (multi cell, 100% epiboly, and proliferation of somites) were exposed to cryoprotectant solutions with concentrations from 1 to 4 M for a period of 5 and 15 min. Following these treatments, the embryos were incubated until the evaluation of hatching rate. Embryos were tolerant to low concentrations of all cryoprotectants tested in the range of 1 to 2 M for all developmental stages. Early stage embryos were more vulnerable to high concentration (3 and 4 M) than late stage embryos. Results also showed that as concentration and duration of exposure increased, the hatching rate significantly decrease (P < 0.05). On a molar-equivalent basis, DMSO appeared to be less toxic to PG, MeOH, and Ac in general. Exposure to cryoprotectants revealed a stage-dependent sensitivity. Toxicity increased in the order of MeOH < DMSO < PG < Ac in multi cell stage and DMSO < MeOH < PG < Ac in 100% epiboly and proliferation of somites stages. The proliferation of somites stage embryos was less sensitive to exposure to cryoprotectants than multi cell and 100% epiboly stages. These findings could be important for designing cryopreservation protocols for this demanding ornamental species.

     

Growth and Chemical Composition of Spirulina Maxima and Spirulina Platensis Biomass at Different Temperatures

The influence of temperature on growth and biomass composition of two species of Spirulina, S. maxima and S. platensis used for food was studied. A 4L fermenter with temperature and agitation control was used to cultivate both species. Under continuous light, maximum cell production of 2.4 g l^–1 was verified for both cultures studied at temperatures above 25 °C: S. maxima (30 °C and 35 °C) and S. platensis (25 °C and 30 °C). An accentuated lag phase was observed for all cultures at lower temperatures (15–20 °C), and a maximum biomass production of 1.5 g l^–1 was achieved. It was also observed that an increase of temperature caused a marked decrease in protein content, while carbohydrate synthesis was stimulated. The concentration of -linolenic acid varied from 11–16% for S. maxima and from 12–14% for S. platensis, at the optimum growth temperatures. Greater culture volumes were also studied in order to compare the performance of glass and plastic containers. At optimum growth temperature, S. maxima produced the same cell growth and similar final biomass composition.     

Diel Rhythms of Serum Metabolites and Thyroid Hormones in Red Porgy Held in Different Photoperiod Regimes

Diel rhythms in serum glucose, lactate, cholesterol, triglycerides and thyroid hormones were studied in red porgy, Pagrus pagrus, held under different photoperiod regimes (constant darkness – DD, 8L:16D,12L:12D), at a constant temperature (17.1–18.7 °C) and fed with commercial pellets, by means of a self-feeder. A clear diurnal rhythm in feeding activity, regardless of the photoperiod regime was demonstrated. All serum components showed significant diel rhythms, although they were not necessarily consistent or circadian in periodicity. As well as this, differences in the average values among the varying treatments were observed. Fish held under the 12L:12D protocol displayed significantly higher average T₄, T₃ and lactate levels during the day rather than at night. Maximum glucose values occurred 8–12 h after dawn and maximum lactate concentrations at 06:00 or 14:00 h. Diel variations in cholesterol were evident only in the DD group with peak values inversely correlated with the rhythm of food intake. Triglycerides displayed a similar pattern of changes. Significant diel fluctuations in T₄ serum levels were observed only in fish exposed to the 12L:12D protocol, with peak values at dawn. A clear diurnal peak (10:00 h) in T₃ concentrations was observed in fish subjected to the 12L:12D regime, while fish exposed to the 8L:16D protocol showed two peaks: one in the photophase (10:00 h) and another in the scotophase (02:00 h). The light–dark alternation and the general activity rhythm of fish seem to be the main synchronizers of the diel rhythms observed in this study.     

Nile tilapia broodfish fed high‐protein diets: Digestive enzymes in eggs and larvae

The aim of this study was to assess the activity of gastric, pancreatic and intestinal digestive enzymes during the embryonic and larval development of Nile tilapia (Oreochromis niloticus) GIFT strain Aqua America^® 1 obtained from a broodstock fed two levels of crude protein (CP). A total of 72 females and 24 males, 10 hapas, two CP levels (32% and 38%) and six phases of embryonic (cleavage, blastula, gastrula) and larval (hatching, 7 and 10 days post hatch, dph) stages were used. The eggs were collected in cleavage, blastula and gastrula stages, 300 mg was collected, and kept in cryogenic tubes in liquid nitrogen. For the samples at larval stage, the remaining eggs were separated according to crude protein level and kept in hatcheries and samples were collected on 7 and 10 dph the same way as before. A total of 48 samples were collected: at each protein level (32% and 38% CP), four samples were collected in each phase of embryonic and larval development. Statistical differences were not observed during embryonic development for acid proteases, trypsin, amylase and lipase activity at both levels of crude protein (32 and 38% CP). Differences for acid proteases were noticed on 7 dph; trypsin and amylase on 7 dph and 10 dph. Significant differences on blastula and 7 dph for protease; as for aminopeptidase, there was significant difference on 7 dph. The data indicated early appearance of digestive enzymes in Nile tilapia broodfish receiving 32% CP taking into account the rapid growth and development of this species.     

虹鳟胚胎受精率适宜发育期的研究

用鉴别液J1、J2与Bouin’s液固定剥膜比较方法,测定了虹鳟(Oncorhynchus mykiss)不同发育期胚胎的受精率。结果表明:用鉴别液J1处理后测得的二细胞期、四细胞期、八细胞期、桑葚期、高囊胚期、原肠中期胚胎的受精率分别为53.21%、77.89%、82.00%、88.2%、87.71%和81.43%;用鉴别液J2处理测得的受精率分别为51.64%、77.86%、81.43%、89.07%、87.50%和81.86%。二者与Bouin’s液固定剥膜后测定的受精率差异不显著(P>0.05),说明这两种鉴别液均可用于生产中测定受精率。在不同的发育时期,鉴别的准确性有所不同,细胞分裂期的准确性远低于桑葚期至囊胚期,这可能是由于胚胎发育不同步和早期胚胎细胞较少所致,因此,鉴别虹鳟鱼卵的受精率最好在桑葚期和囊胚期之间(12～24℃.d)的时间点。