翘嘴鳜人工雌核发育的初步研究

采用紫外线照射灭活精子,经冷休克诱导染色体加倍的方法对翘嘴鳜(Siniperca chuatsi)进行人工诱导雌核发育育种。灭活翘嘴鳜精子,紫外线照射剂量80 mJ/cm2是最佳的辐射剂量。冷休克结果表明,在0～3℃冰水下,冷休克起始时间为授精后3～7 min均有效,以5 min为最适;冷休克持续时间10-50 min均有效,以30 min最佳。综合以上两项因素,授精5 min后,休克时间持续30 min组的原肠胚存活率和孵化率均为最高,与其他处理组差异显著(P<0.05),此时雌核发育率最高,达到4.72%。     

黄姑鱼雌核发育诱导及鉴定

为建立黄姑鱼雌核发育诱导方法,实验利用紫外线照射使黄姑鱼精子遗传失活,与卵子授精后再通过冷休克抑制极体排放进行倍性恢复,成功诱导出黄姑鱼雌核发育二倍体。精子经强度为3 800μW/(cm²·s)的紫外线照射10～100 s,受精卵孵化率呈现明显的Hertwig效应;当照射时长达到60 s以上,各实验组全部孵出仔鱼均呈现单倍体综合症。精子的紫外线照射时间、受精卵冷休克起始和持续时间等三因素三水平正交实验结果表明,精子经紫外线照射60 s,受精2 min后卵子在3～4 ℃海水中持续冷休克10 min为最佳诱导条件组合,可以获得最高的孵化率(16%)。最佳组合仔鱼形态和细胞相对DNA含量与正常二倍体一致,经微卫星标记检验证明全部不含有父本基因,为雌核发育二倍体。实验报道了成功诱导黄姑鱼雌核发育二倍体的条件,为进一步开展黄姑鱼良种选育和性别控制工作奠定了基础。     

人工诱导泥鳅雌核发育试验

用经UV灭活的野鲤精子激活泥鳅卵,以冷休克与热休克二种方式诱导激活卵的染色体加倍。冷休克组进行休克起始时间、持续时间二参数组合试验,休克起始时间分别为3min、4min、5min,持续时间为20min、25min,休克处理温度0℃-3℃。热休克组休克起始时间为3min35s,持续时间为2min,休克处理温度为39℃-41℃。结果:冷休克组各组合均得到雌核发育二倍体泥鳅苗,以休克起始时间为3min、持续时间为25min组合雌核发育二倍体的诱导率最高,达28.8%-34.1%。热休克组雌核发育二倍体诱导率52.1%-56.3%,与冷休克各组合间均存在极显著差异(P0.01),诱导效果显著好于冷休克组。泥鳅苗经12d培育,冷休克组成活率28.6%,全长0.7-1.6cm;热休克组成活率43%,全长0.5-1.4cm;对照组成活率70.5%,规格与冷休克组相当。     

真鲷精子诱导牙鲆减数分裂雌核发育

采用真鲷精子激活牙鲆卵子,经(0±0.5)℃冷休克处理,诱导了牙鲆减数分裂雌核发育。灭活真鲷精子,紫外线照射剂量3.4mJ/cm2时孵化率最低;随着照射剂量的增加,孵化率恢复,照射剂量73mJ/cm2时,孵化率最高,呈现典型的Hertwig效应。用流式细胞仪检测倍性,未经冷休克处理的胚胎全部为单倍体,经冷休克处理的均为二倍体,表明精子灭活有效。冷休克实验结果表明,冷休克起始时间2～5min均有效,以3min为最好;冷休克持续时间30～90min均有效,以45min为最好。综合两项因素,授精3min后,休克时间持续45min组的授精率和孵化率均为最高,与其他处理组差异显著(P<0.05)。RAPD分析结果显示,异源精子的遗传信息未在雌核发育二倍体的电泳图中出现,表明其遗传信息没有传递给子代。     

冷、热休克法诱导黄颡鱼三倍体的比较研究

分别采用冷、热休克抑制第二极体释放的方法诱导黄颡鱼三倍体。结果表明,在卵受精后2min,5℃处理20min,胚胎时期的三倍体率达70%左右,孵化率50%左右,幼鱼时期三倍体(含嵌合体)的检出率为25%,此条件为冷休克处理的优化参数;在卵受精后2min,40℃处理2min,胚胎时期的三倍体诱导率达58%,孵化率为39%,幼鱼时期三倍体(含嵌合体)的检出率为40%,此条件为热休克处理的优化参数。正交分析得出,冷休克条件下起始休克时间是原肠期三倍化率和孵化率的重要影响因子,温度对畸形个体的产生有重要影响;热休克条件下,参考三倍体率、畸形率、孵化期相对存活率三者而言,休克温度均是重要因素。比较观察到冷休克处理组的胚胎受损情况严重,后期的成活率较热休克处理组要低,总体诱导效果逊于热休克处理组。     

真鲷精子诱导漠斑牙鲆减数雌核发育

采用紫外线(UV)灭活的冷冻真鲷(Pagrosomus major)精子激发漠斑牙鲆(Paralichthys lethostigma)卵子发育.冷冻真鲷精子适宜的UV灭活剂量为72 mJ/cm2,随着处理时间延长,胚胎的孵化率呈现哈特维希效应,其中40 s灭活组胚胎孵化率达到最高[(29士1.9)％],单倍体率100％.利用冷休克法抑制卵子第二极体排出,成功获得雌核发育二倍体仔鱼.冷休克处理温度为0～2℃,处理起始时间3 min、持续时间45 min时,雌核发育二倍体胚胎的孵化率最高,达(9.4士0.71)％,获得批量雌核发育仔鱼.雌核发育仔鱼经流式细胞仪和染色体倍性鉴定,均为二倍体(2n=48),未发现单倍体和非整倍体现象.雌核发育二倍体组和单倍体组与正常二倍体组组孵化时间差异显著,分别历时64 h 59 min和55 h 49 min.雌核发育二倍体仔鱼与正常二倍体仔鱼形态特征差异不显著(P＞0.05),单倍体仔鱼明显畸形.研究表明,采用适宜的紫外线剂量灭活的冷冻真鲷精子可成功诱导漠斑牙鲆雌核发育,获得雌核发育二倍体后代,研究结果可为漠斑牙鲆全雌化苗种生产提供技术和理论依据.     

黑鲷精子诱导漠斑牙鲆雌核发育研究

采用紫外线遗传失活的黑鲷精子与漠斑牙鲆卵子授精,获得了漠斑牙鲆雌核发育单倍体受精卵,经冷休克处理后诱导出漠斑牙鲆雌核发育二倍体.试验结果表明,黑鲷精子经紫外线照射后(0～180 J/cm2)与漠斑牙鲆卵子受精,胚胎孵化率呈现明显的Hertwig效应.黑鲷精子遗传失活的最适紫外线照射剂量为120 J/cm2.18℃的水温条件下,冷休克处理的最适时刻为受精后4 min.处理持续时间和处理温度组合试验表明,在相同处理持续时间条件下,3℃休克水温处理效果均优于对应1℃休克水温各组,且45 min处理持续时间诱导效果最佳.综合试验结果,黑鲷精子诱导漠斑牙鲆雌核发育的适合条件为:水温18℃,将黑鲷精子经120 J/cm2紫外线照射后与漠斑牙鲆卵子受精,受精后4min,用3℃冷海水处理45min,可获得46.59％的雌核发育漠斑牙鲆二倍体初孵仔鱼.     

施氏鲟精子诱导匙吻鲟雌核发育

采用照射强度为10 188 μW/(cm2·s),照射距离为15 cm的紫外线对施氏鲟(Acipenser schrenckii)精子进行4～7 min的照射,用照射后的精子对匙吻鲟(Polyodon spathula)卵进行人工授精,以探索精子的最佳紫外线照射时间.设计热休克起始时间、持续时间和休克温度3因子、3水平的正交试验,以探索人工诱导匙吻鲟雌核发育的理想热休克条件.结果表明:用紫外线照射5 min处理的施氏鲟精子,与匙吻鲟卵受精所得胚胎经染色体观察均为单倍体(n=60),表明照射精子遗传失活的有效性和可靠性;若热休克处理前受精卵保持在(18±013)℃,在一定的休克温度(35～39℃)条件下,于不同时间(受精后16～20 min)起始热休克,持续处理2 min均能诱导受精卵染色体加倍.对热休克处理条件的优化结果表明,将受精卵于受精后18 min,在37℃的水中热休克处理2 min,二倍体诱导率最高,达18.8%.染色体鉴定显示,该条件下处理的胚胎均为二倍体(2n=120),未发现单倍体和非整倍体.本研究为进一步分析异源精子诱导匙吻鲟雌核发育机制以及匙吻鲟性别决定的分子机制奠定了基础.     

紫外辐射灭活大黄鱼和黄姑鱼精子及其激活大黄鱼卵子发育为胚胎的效果

为探讨大黄鱼(Larimichthys crocea)和黄姑鱼(Nibea albiflora)精子的紫外辐射灭活适宜剂量及其激活大黄鱼卵子发育为胚胎的效果,以Ringer氏液为稀释液,按1:30稀释大黄鱼和黄姑鱼精子,采用自制紫外灭活装置[紫外辐射强度2200μW/(cm~2·s),紫外波长254 nm]对这2种鱼的精子进行紫外照射处理及活力测定,然后与正常大黄鱼卵子进行人工授精,授精后一部分卵未作冷休克处理,另一部分卵进行了冷休克处理(受精2 min 30 s,3℃海水,冷休克10 min),并进行了早期胚胎成活率和仔鱼孵化率的测定与比较。结果显示:1)大黄鱼和黄姑鱼精子的激活率与紫外照射处理时间呈负相关,精子的快速运动时间变化呈典型的Hertwig效应。2)未冷休克组中大黄鱼和黄姑鱼诱导的早期胚胎成活率与精子的紫外照射时间总体呈负相关,而仔鱼孵化率呈Hertwig效应。3)冷休克组中大黄鱼和黄姑鱼精子诱导的早期胚胎成活率和仔鱼孵化率随紫外照射时间的增加呈Hertwig效应,分别于2 min 20 s和1min 30 s时达相对峰值,此时,大黄鱼早期胚胎成活率和仔鱼孵化率分别为(38.3±4.3)%和(66.5±5.1)%,黄姑鱼早期胚胎成活率和仔鱼孵化率分别为(43.3±3.3)%和(67.7±6.3)%。分析认为,大黄鱼和黄姑鱼精子遗传失活的紫外辐射剂量分别以308 m J/cm~2和198 m J/cm~2为佳。本研究旨在为大黄鱼雌核发育技术的改进提供依据。     

3种染色体组加倍方法诱导黄河鲇雌核发育的比较

用经紫外线照射后遗传失活的黄河鲇精子、刺激性成熟的黄河鲇卵子,进行雌核发育,采用冷休克、热休克和秋水仙素浸泡等3种染色体组加倍方法诱导黄河鲇卵进行二倍体雌核发育.实验结果表明:在人工诱导黄河鲇二倍体雌核发育过程中,以冷休克处理组的效果最好,胚胎孵化率达到8.1%;其次为秋水仙素处理组,胚胎孵化率达到6.3%;热休克处理组诱导效果较差,胚胎孵化率仅为4.0%.     

Protein and amino acid composition from the mantle of juvenile O ctopus vulgaris exposed to prolonged starvation

Protein and amino acid composition of the mantle of juvenile O ctopus vulgaris (Cuvier, 1797) during fasting for 27 days were determined. Average protein content of octopus mantle was of 711.19 ± 46.80 g kg^?1 DW, and it decreased with increasing fasting days. The non‐essential amino acids content was higher (486.18 ± 11.08 g kg^?1 protein) than essential amino acids (425.82 ± 9.15 g kg^?1 protein) at the start of the experiment (unstarved animals). The results suggest that the amino acid profile of the mantle where the most abundant amino acids are Arg, His, Lys, Gly, Leu and Pro could indicate a prolonged fasting condition (>20 days) or poor nutrition of O . vulgaris. This study supports the idea of using mantle for metabolic needs of starved O . vulgaris suggesting that the degradation pathway of amino acids to pyruvate and tricarboxylic acid cycle intermediates was favoured contrary to the degradation pathway of ketogenic amino acids. Special considerations should be taken concerning Thr, Ile, Ser, Ala, Asx (Asp, Asn), Glx (Glu, Gln) (because of their fast intake) and Lys and His (due to their stable contents) during a prolonged period of fasting.     

美人鱼发光杆菌对凡纳滨对虾非特异性 免疫功能及抗病力的影响

在基础饲料中分别添加0.1%和1%美人鱼发光杆菌灭活菌、0.1%美人鱼发光杆菌活菌配制成3种免疫实验饲料,以基础饲料为空白对照组饲料,每组设3个平行样。对个体质量为(4.83±0.36)g的凡纳滨对虾进行为期20 d的饲养实验,分别在0、5、10、15和20d进行取样,以血清中的酚氧化酶(PO)、酸性磷酸酶(ACP)、碱性磷酸酶(AKP)、超氧化物歧化酶(SOD)和溶菌酶(UL)活性为免疫指标,探讨了美人鱼发光杆菌作为免疫制剂对凡纳滨对虾非特异性免疫效应的影响;在投喂免疫饲料后的第22天,按0.004 2 kg/kg体重的剂量,直接投喂对虾白斑综合征病毒(WSSV)病料,并记录累积死亡率。结果表明,美人鱼发光杆菌免疫实验组对凡纳滨对虾血清中PO、ACP、AKP、UL和SOD活性影响明显高于对照组,并且在饲料中添加美人鱼发光杆菌后,明显提高了对虾抵御WSSV感染的能力。其中0.1%美人鱼发光杆菌活菌实验组的抗病毒感染能力最强,WSSV感染14d内累计死亡率为63.3%±5.8%;而对照组为96.7%±3.3%。研究表明,美人鱼发光杆菌添加在对虾饲料中能提高凡纳滨对虾非特异性免疫水平,增强抵抗疾病的能力,将其作为对虾免疫增强剂具有良好的应用前景。     

Effects of Spirulina platensis as a feed additive on growth and coloration of blue dolphin cichlids (Cyrtocara moorii Boulunger, 1902)

This study was carried out to investigate the effects of replacing fish meal with dietary Spirulina as a feed supplement on the growth performance and coloration of blue dolphin cichlids (Cyrtocara moorii). Five isonitrogenous (47% crude protein) and isocaloric (17.36 kJ/g digestible energy) diets were for formulated to replace FM with 0, 5, 10, 15 and 20% Spirulina (designated as Control, SP5, SP10, SP15 and SP20 respectively) and fed to the fish (initial body weight, 3.15 ± 0.01 g). Fish were randomly distributed into fifteen 120 L aquariums (26.5 ± 1.00°C), 15 fish per aquarium. The diets were tested in triplicate for 12 weeks. Experimental groups were fed twice daily (09:00 and 17:00) by hand to satiation. At the end of the feeding trial, significantly (p < 0.05) higher weight gain (WG), specific growth rate (SGR), protein efficiency ratio (PER) and lower feed conversion ratio (FCR) were observed in fish fed the SP10 diet when compared to the SP20 diet. There was no significant difference in these parameters between the other groups. The skin coloration of blue dolphin cichlid fed a diet containing Spirulina meal was enhanced. The best coloration was observed in the SP15 group. These impressions were objectively validated by chemical determinations of carotenoids extracted from fish skins and passed statistical tests of significance. The study findings show that Spirulina meal does not diminish growth rates except at very high levels.     

A Minchinia mercenariae‐like parasite infects cockles Cerastoderma edule in Galicia (NW Spain)

The cockle Cerastoderma edule fishery has traditionally been the most important shellfish species in terms of biomass in Galicia (NW Spain). In the course of a survey of the histopathological conditions affecting this species in the Ria of Arousa, a haplosporidan parasite that had not been observed in Galicia was detected in one of the most productive cockle beds of Galicia. Uni‐ and binucleate cells and multinucleate plasmodia were observed in the connective tissue mainly in the digestive area, gills and gonad. The parasite showed low prevalence, and it was not associated with abnormal cockle mortality. Molecular identification showed that this parasite was closely related to the haplosporidan Minchinia mercenariae that had been reported infecting hard clams Mercenaria mercenaria from the Atlantic coast of the United States. The molecular characterization of its SSU rDNA region allowed obtaining a fragment of 1,796 bp showing 98% homology with M. mercenariae parasite. Phylogenetic analysis supported this identification as this parasite was clustered in the same clade as M. mercenariae from the United States and other M. mercenariae‐like sequences from the UK, with bootstrap value of 99%. The occurrence of M. mercenariae‐like parasites infecting molluscs outside the United States is confirmed.     

Dietary thiamin and pyridoxine requirements of fingerling Indian major carp,Cirrhinus mrigala (Hamilton)

Two experiments were conducted to quantify the dietary thiamin (experiment I) and pyridoxine (experiment II) requirements of fingerling Cirrhinus mrigala for 16 weeks. In experiment I, dietary thiamin requirement was determined by feeding seven casein–gelatin‐based diets (400 g kg^?1 CP; 18.69 kJ g^?1 GE) with graded levels of thiamin (0, 0.5, 1, 2, 4, 8 and 16 mg kg^?1 diet) to triplicate groups of fish (6.15 ± 0.37 cm; 1.89 ± 0.12 g). Fish fed diet with 2 mg kg^?1 thiamin had highest specific growth rate (SGR), protein retention (PR), RNA/DNA ratio, haemoglobin (Hb), haematocrit (Hct), RBCs and best feed conversion ratio (FCR). However, highest liver thiamin concentration was recorded in fish fed 4 mg thiamin kg^?1 diet. Broken‐line analysis of SGR, PR and liver thiamin concentrations exhibited the thiamin requirement in the range of 1.79–3.34 mg kg^?1 diet (0.096–0.179 μg thiamin kJ^?1 gross energy). In experiment II, six casein–gelatin‐based diets (400 g kg^?1 CP; 18.69 kJ g^?1 GE) containing graded levels of pyridoxine (0, 2, 4, 6, 8 and 10 mg kg^?1 diet) were fed to triplicate groups of fish (6.35 ± 0.37 cm; 1.97 ± 0.12 g). Fish fed diet containing 6 mg kg^?1 pyridoxine showed best SGR, FCR, PR, RNA/DNA ratio, Hb, Hct and RBCs, whereas maximum liver pyridoxine concentration was recorded in fish fed 8 mg kg^?1 dietary pyridoxine. Broken‐line analysis of SGR, PR and liver pyridoxine concentrations reflected the pyridoxine requirement from 5.63 to 8.61 mg kg^?1 diet. Data generated during this study would be useful in formulating thiamin‐ and pyridoxine‐balanced feeds for the intensive culture of this fish.     

Sea bream culture in Egypt; status, constraints and potential

Marine fish farming in Egypt began in 1976 with the culture of gilthead sea bream, (Sparus aurata) as this fish was notably adaptable to brackish and marine pond conditions. Today, marine fish and shrimp farms amount to about 19,000 ha, out of which 42% is already in production while the rest, i.e., 58% is still under construction. In 1997, cultured gilthead sea bream production of 2,250 tons made up 3% of the 75,000 tons total aquaculture catch. In polyculture, usually with the grey mullet and sea bass, gilthead sea bream contributed 440 kg ha^–1 to the total yield of 1,700 kg ha^–1 (26%) over a period of 16 months. For the same period, the yield of monoculture ponds averaged 100 kg ha^–1, while in marine cages, yields ranged from 4–10 kg m³. In 1996–1997, fry of 0.25–1 g and fingerlings 1–10 g with a total of 3 million, were collected from the wild and 1 million fry were produced in the three marine hatcheries out of the four existing ones. The development of sea bream culture in Egypt is now severely inhibited by the shortage of seeds and adequate feeds. Exports of both sea bream and sea bass, during 1994–1996 averaged 1,300 tons per year.     

Fertilization by refrigerator stored sperm of the Indian major carp, Labeo calbasu (Hamilton, 1822)

This study reports on the spermatological properties, and on the development of a protocol for refrigerator storage (4°C) of Labeo calbasu (Hamilton, 1822) sperm for artificial breeding. Volume, motility, concentration and pH of the freshly collected sperm were 2.21 ± 0.53 (μL g^?1 of fish weight) (mean ± SD), 95 ± 1 (%), 1.93 ± 0.44 × 10⁹ (cells mL^?1) and 7.56 ± 0.17 respectively. Sperm activation was evaluated at different osmolalities of NaCl solution, and motility ceased completely when osmolality of the extender was ≥287 mOsmol kg^?1. Sperm retained motility for 24, 72 and 108 h, after refrigerator storage when sperm were undiluted, suspended in Alsever's solution and suspended in Alsever's solution containing 5% methanol respectively. Fertilization rate of fresh eggs with sperm stored at 4°C in Alsever's solution and Alsever's solution containing 5% methanol was 77% and 60% with a hatching rate of 60% and 43% respectively. The fertilization and hatching success of the stored sperm suggests potential to use refrigeration for transporting genetic material to hatcheries for artificial breeding of L. calbasu in Bangladesh.     

A novel approach to denitrification processes in a zero-discharge recirculating system for small-scale urban aquaculture

This paper presents an innovative process to solve the nitrate build-up problem in recirculating aquaculture systems (RAS). The novel aspects of the process lie in a denitrification bioreactor system that uses solid cotton wool as the primary carbon source and a unique degassing chamber. In the latter, the water is physically stripped of dissolved gaseous O₂ (by means of a Venturi vacuum tube), and the subsequent denitrification becomes more efficient due to elimination of the problems of oxygen inhibition of denitrification and aerobic consumption of cotton wool. The cotton wool medium also serves as a physical barrier that traps organic particles, which, in turn, act as an additional carbon source for denitrification. Operation in the proposed system gives an extremely low C/N ratio of 0.82 g of cotton wool/g of nitrate N, which contributes to a significant reduction of biofilter volume. The additional advantage of using solid cotton wool as the carbon source is that it does not release organic residuals into the liquid to be recycled. Operation of the system over a long period consistently produced effluents with low nitrate levels (below 10 mg N/l), and there was only a very small need to replace system water. The overall treatment scheme, also incorporating an aerobic nitrification biofilter and a granular filtration device, produced water of excellent quality, i.e., with near-zero levels of nitrite and ammonia, a sufficiently high pH for aquaculture, and low turbidity. The proposed system thus provides a solution for sustainable small-scale, urban aquaculture operation with a very high recovery of water (over 99%) and minimal waste disposal.     

Effects of metomidate anaesthesia or transfer to pur sea water on plasma parameters in ammonia-exposed Atlantic salmon (Salmo salar L) in sea water

Atlantic salmon (Salmo salar L) postsmolts weighing 150 ± 53 g were exposed to 14–15 mg l^–1 TA-N (total ammonia-N) in sea water in 1 m³ tanks for 24h. Blood samples were then taken A) immediately after the fish were netted from the exposure tanks and stunned by a blow to the head; B) 2–20 min after the fish were transferred to 15 l of an anaesthetic solution of metomidate in ammonia-free sea water; or C) 2–20 min after the fish were transferred to 15 l of ammonia-free sea water. Plasma TA-N level was 18% lower in the anaesthetised fish compared to in the fish sampled directly from the exposure tanks (p 0.05), and accordingly 16% lower in the fish transferred to pure sea water although this difference was not significant (p = 0.07). Plasma glucose level was higher in the fish transferred to pure sea water than in the fish receiving the two other treatments (p 0.05), but plasma urea, osmolality, Na^{+, Cl–, Ca2+ or Mg2+} levels did not vary significantly between the different treatments. Plasma TA-N level increased with time in the fish in the metomidate solution (p 0.02).