金鱼生殖细胞多倍化现象分析

通过精巢染色体制片观察金鱼的生殖细胞染色体数,结合流式细胞仪测定成熟精子DNA含量,探讨了金鱼生殖细胞发生过程中染色体特征及产生的精子的倍性。结果表明,金鱼初级精母细胞大部分行正常减数分裂,形成50个二价体,另外还发现17.0%分裂相的二价体数呈现不同程度的加倍现象,观察到了二价体数为100、150、200、250、300,甚至远远超过300的分裂相,与鲫鲤远缘杂交导致生殖细胞染色体数加倍一致,不一样的是金鱼稳定产生DNA含量减半的精子。结果表明,近缘杂交和远缘杂交都可以导致生殖细胞染色体数加倍,但是否能产生染色体数加倍的功能性配子与杂交亲本亲缘关系的远近有关。     

泥蚶受精过程的细胞学荧光显微观察

采用荧光染色剂ＤＡＰＩ已固定的泥蚶成熟卵和受精卵进色染色的方法，在显微镜下观察泥蚶受精细胞学过程。通过ＤＡＰＩ的染色，在荧光显微镜下可清楚观察到，泥蚶的成熟未受精卵处于第一次成熟分裂的中期，精子入卵后分裂再次启动，释放出第一和第二极体，完成成熟分裂，部分受精卵的第一极体有极明显的分裂现象，精子入卵的部位是随机的，且自精子入卵至成熟分裂完成，入卵的精子在形态等方面将发生一系列变化，形成雄性原核。雌雄     

菲律宾蛤仔受精及早期胚胎发育过程的细胞学观察

利用HOECHST 33258荧光染料对已固定的菲律宾蛤仔受精卵及早期胚胎样品进行染色的方法,连续观察记录了菲律宾蛤仔受精及早期胚胎发育的细胞生物学过程。结果表明,菲律宾蛤仔的成熟未受精卵子处于第一次成熟分裂中期;精卵混合后精子迅速结合于卵子周围并开始进行顶体反应;精子人卵后去致密并且激发卵子恢复两次成熟分裂,排放出第一及第二极体,完成成熟分裂;受精卵完全排放出第二极体的同时早期的精卵原核形成,随后二者相互靠拢,体积扩散膨大,在细胞中央位置形成各自的染色体组后完成联合;菲律宾蛤仔早期胚胎发育速度进程较快;在受精过程中存在极少数的多精受精现象。     

中国对虾受精过程的细胞学研究

通过光镜和电镜切片技术，对中国对虾的受挤过程进行了观察。结果表明，中国对虾成熟卵子处于第1次成熟分裂前期，卵子激活后产生胶质层，精子在卵子的诱导下产生顶体反应后进入卵子。中国对虾为多精入卵、单精受精。精子入卵后，卵子完成成熟分裂并举起围卵膜，但精子的存在并非此过程所必需。在卵子进行第1次成熟分裂时，其纺锤体旋转90°。卵子完成成熟分裂后，精卵原核原核化并向卵子中央移动，最后，精卵原核结合形成合子核，卵子完成受挤过程。     

栉孔扇贝（♀）×虾夷扇贝（♂）精子入卵过程的电镜观察

采用扫描电镜和透射电镜技术对栉孔扇贝(Chlamys farreri,)×虾夷扇贝(Patinopecten yessoensis,)的精子入卵过程进行观察。结果表明,这2种扇贝杂交与亲本自交的精子入卵过程没有本质的区别。成熟的精卵相遇时,相互激活,产生一系列胞间反应,卵子的激活集中表现为皮层反应、受精膜举起、成熟分裂重新启动;精子激活集中表现为顶体反应和受精锥形成。在16～18℃条件下,精子入卵的时间集中在精卵混合后的第6 min。8 min后,精子的线粒体随精核一起进入卵子中。精子入卵后精核略微膨胀,卵细胞中的线粒体在精核附近出现聚集现象。杂交中有多精入卵现象。本研究目的旨为优化扇贝种间杂交技术及探讨种间受精生物学过程提供基础依据。     

缢蛏受精和早期卵裂过程的细胞学变化观察

采用普通光镜、荧光显微镜和石蜡切片技术,对缢蛏受精和早期卵裂过程中的精子入卵、减数分裂、雌雄原核形成与结合、早期卵裂以及多精入卵等细胞学事件进行了显微观察。结果表明,缢蛏成熟未受精卵多呈圆球形或卵圆形,少数呈梨形,卵径为82～88μm,核相处于第一次成熟分裂中期;精子为典型的原生型,全长55～58μm,头部呈保龄球形,顶体前端的顶体杆呈特别细长的花丝状;在水温21～22℃、盐度10的条件下进行人工授精,精、卵混合后,精子迅速附着于卵子表面,启动卵子发育;受精后4～6 min,精子的头部已进入卵内并明显膨胀,卵子外形变圆,卵外附着的精子量明显减少;在受精后12～15 min、20～25 min,受精卵先后排出第一极体、第二极体,完成两次成熟分裂;第二次成熟分裂结束以后,精、卵核体积迅速膨胀,雄原核的膨胀早于雌原核,核膜重建,在受精后约30 min形成雌、雄原核;雌、雄原核均向卵子中央移动,雄原核旁边的精子星光清晰可见,随后二者在卵子中央以染色体联合的方式结合,联合核的染色体共同排列在纺锤体的赤道板上,形成第一次有丝分裂的中期分裂相;受精后40 min左右,受精卵进行第一次卵裂,染色体在纺锤丝的牵引下向两极移动,结果形成2个大小不等的卵裂球;受精后45～50 min,卵子进行第二次卵裂,核相变化与第一次卵裂相同,在与第一次卵裂垂直的纵轴方向上发生不等全裂,最终形成3小1大4个卵裂球;受精后60～70 min,胚胎进行第三次卵裂,仍为不等全裂,但自此次卵裂起已开始进行螺旋分裂。此外,对实验中发现的缢蛏极少量多精入卵、多极分离等异常细胞学现象进行了分析,并探讨了海洋贝类卵子阻止多精入卵发生的机制。     

单环刺螠受精过程的细胞学观察

利用组织学和荧光显微镜对单环刺螠(Urechis unicinctus)受精过程中精子入卵以及核相变化进行了观察.结果表明:单环刺螠成熟卵呈卵圆形,核相处于生发泡尚未破裂的第1次成熟分裂前期.在(21±1)℃时,精卵混合后约5 min,精子由卵偏向植物极一侧入卵,受精膜开举起,继而精核发生第1次膨胀.直至受精后约25 min,受精膜完全举起；受精后10～50 min,卵核相继完成二次成熟分裂,分别形成第一极体和第二极体；受精后50～70 min,精核卵核分别膨胀,形成雄原核和雌原核,进而相向迁移,最后在卵的中央联合形成合子核.受精后70～90 min,受精卵完成第1次卵裂,形成2个大小相等的卵裂球.单环刺螠受精全过程历时约70 min,略长于多数海洋无脊椎动物的受精持续时间.本研究旨在为今后单环刺螠人工育苗和新品种的培育提供基础资料.     

刺参受精及早期胚胎发育过程的细胞学观察

采用HOECHST 33258染色荧光显微方法,对刺参成熟未受精卵以及受精过程中精子入卵、极体排放、雌雄原核的形成与结合、早期卵裂以及多精入卵等细胞学进行了研究。结果显示,刚产出的刺参成熟未受精卵呈圆形,核相处于第一次成熟分裂中期;在水温22~23 ℃、盐度29条件下进行受精,受精后12 min,完成第一次成熟分裂,释放第一极体;受精后20 min,大部分受精卵完成第二次成熟分裂,放出第二极体。受精后35 min,雌、雄原核开始在卵中央发生染色体联合;受精后80 min,部分受精卵完成第一次卵裂,受精后100 min,部分受精卵完成第二次卵裂。刺参在受精过程中存在极少数的多精入卵现象。     

大菱鲆成熟精子、卵子及精子入卵早期过程的电镜观察

通过人工授精和电镜技术,观察和描述了大菱鲆Scophthalmus maximus成熟精卵的形态、精子入卵的早期过程及精子入卵过程发生的一系列形态结构变化。大菱鲆卵子表面均匀地布满纵横交错的网纹,并整齐地分布许多微小孔,在动物极有一个受精孔。大菱鲆精子为无顶体类精子,由头部、中片和尾部三部分组成,中片有9～14个圆形线粒体。大菱鲆精子入卵速度非常快,授精后0～5s已经有精子通过受精孔(Micropyle)进入卵子。精子入卵过程还伴随着其他一些结构的变化,如精孔管管壁边缘由锯齿状变为平滑的环状等。     

瘤背石磺两性腺周年发育规律的研究

研究了江、浙、沪地区瘤背石磺（Onchidium struma）两性腺周年发育规律。结果显示,瘤背石磺雌雄同体,雄性先熟,生物学最小型为体长2.81cm,体质量1.81g。其两性腺发育为1年1个周期,发育经历增殖期、精子发生期、精卵同步发生期、排放期和休止期。其中排放期又分精子排放、精卵排放和卵子排放,体现了精卵发生的异时性。精子的发生包括精原细胞、初级精母细胞、次级精母细胞、精子细胞和精子;卵子的发生包括卵原细胞期、无卵黄期、卵黄形成期、卵黄形成后期和成熟期。成熟系数周年呈明显单峰分布,峰值出现在7～9月间;蛋白腺系数、卵黄腺系数与成熟系数具有一定的相关性,蛋白腺系数基本随着成熟系数变化而变化,卵黄腺系数随着成熟系数变化的过程中出现3个峰值,分别在6、8和12月。     

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.     

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.     

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.     

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).