Protection of crucian carp (Carassius auratus Gibelio) against septicaemia caused by Aeromonas hydrophila using specific egg yolk immunoglobulins

Egg yolk immunoglobulins (IgY) were obtained from laying hens immunized with inactivated Aeromonas hydrophila. The purified IgY was shown to inhibit the growth of A. hydrophila in vitro and the optimum concentration for inhibition of A. hydrophila‐specific IgY was 75 mg mL^?1. In a subsequent challenge trial, 100 carp (200~250 g) were assigned to one of ten tanks with ten carp per tank. The fish in one tank were unchallenged whereas the remaining 90 fish were injected intraperitoneally with 100 μL of A. hydrophila at a concentration of 10⁸ cfu mL^?1. For the next 21 days, all fish were moved in their respective groups to a clean tank for 20 min day^?1. The fish in four tanks (one unchallenged tank and three challenged tanks) received no treatment whereas the fish in the remaining six tanks were immersed in either 0.5 g L^?1 aqueous nonspecific IgY (N = 3) or 0.5 g L^?1 aqueous specific IgY (N = 3). Haemoglobin concentrations, white and red blood cell numbers as well as the mortality of specific IgY‐treated fish were significantly different from those of the control. These results suggest that passive immunization by immersion with pathogen‐specific IgY may provide a valuable treatment for A. hydrophila infection in carp.     

Effects of monensin dose and treatment time on xenoma reduction in microsporidial gill disease in rainbow trout, Oncorhynchus mykiss (Walbaum)

The objectives of the study were (1) to determine an acceptable dose of dietary monensin against microsporidial gill disease (MGD) of salmon caused by Loma salmonae and (2) to determine the life stage of L. salmonae at which the action of monensin was most effective at reducing clinical disease. Rainbow trout (RBT) were fed monensin diets at concentrations of 0, 10, 100, 1000 and 5000 ppm starting 1 week before per os exposure to L. salmonae infected gill material. Lethal sampling occurred at weeks 6, 7, 8 and 9 post‐exposure (PE). The acceptable dose of dietary monensin was determined to be 1000 ppm because it was the lowest concentration that produced a significant and constant reduction in xenoma formation. RBT were fed monensin at 1000 ppm beginning at 0, 1, 2 and 3 weeks before exposure and at 1, 2 and 3 weeks after exposure and continuing until week 9 PE. Fish in which treatment was initiated at the time of exposure or 1 week before, showed the greatest reduction in xenoma production when compared with the untreated RBT.     

Loma salmonae-associated growth rate suppression in rainbow trout, Oncorhynchus mykiss (Walbaum), occurs during early onset xenoma dissolution as determined by in situ hybridization and immunohistochemistry

Experimental infection of rainbow trout, Oncorhynchus mykiss (Walbaum), juveniles with Loma salmonae at a water temperature of 15 °C yielded detectable parasite DNA within the gills by week 2 post-exposure (PE) and detectable spore-wall antigen within developing xenomas by week 3 PE, as determined by in situ hybridization and monoclonal antibody (Mab) based immunohistochemistry, respectively. The microsporidian was most commonly located within endothelial cells of lamellar basal channels. Whereas the onset of xenoma formation appeared to be relatively synchronous, as expected from previous studies, xenoma dissolution followed an unexpected biphasic pattern with peaks at weeks 4 and 9 PE. The onset of significant growth rate suppression, at week 4 PE in exposed fish, was temporally associated with the appearance of gill lesions which, in turn, were centred about sites of premature xenoma dissolution. The latter was determined by the detection of spore-wall antigen within lesions. Co-habitant control fish began developing xenomas by week 10, indicating the infective potential of those spores released from the principal fish during early xenoma dissolution. Although infection with L. salmonae significantly affects fish growth rates, the time-course of this suppression is limited, and as an unexpected finding, growth rate recovery commences prior to the infection’s resolution.     

拟态弧菌的绿色荧光蛋白标记及其在感染草鱼体内的动态分布

为了示踪研究拟态弧菌感染草鱼的动态过程,将增强型绿色荧光蛋白编码基因EGFP克隆至质粒pBAD24,并转化到拟态弧菌04-14菌株构建荧光标记重组菌.重组菌经阿拉伯糖诱导后,能高效表达EGFP蛋白;荧光显微镜观察和流式细胞仪检测均发现重组菌能够发出明显的绿色荧光信号,且传至30代后质粒稳定率仍为100％;生物学特性检测结果显示,与野生株相比,重组菌的形态、生长特性和细胞黏附性均未发生明显改变.用标记重组菌浸泡感染草鱼,定点采集鳃、肠道、肌肉、头肾、脾脏和肝脏,借助荧光信号检测4d内细菌在不同组织脏器中的动态分布.结果发现感染4h后即可在肠道和鳃中检测到绿色荧光信号,标记菌检出量分别为3.60×108和2.36×106 CFU/g,直至10 h,其含量无明显变化,12 h后含菌量逐渐下降,但持续存在直至鱼死亡.标记菌在肌肉、头肾、脾脏和肝脏中呈现相似的动力学,感染24 h后才检测到荧光信号,24～ 85 h时间段含菌量呈现先增加后下降的变化,48 h达到峰值,检出量分别为9.58×104(肌肉)、8.75×104(头肾)、1.50×104(脾脏)和4.50×104 CFU/g(肝脏),但均低于肠道中的检出量,结果表明肠道是拟态弧菌黏附定植与繁殖的主要靶器官.     

Loma salmonae-associated xenoma onset and clearance in rainbow trout, Oncorhynchus mykiss (Walbaum): comparisons of per os and cohabitation exposure using survival analysis

Survival analysis techniques were used to compare experimental exposure methods of Loma salmonae (Microspora) in rainbow trout (RBT) by measuring xenoma onset and clearance time. Twenty‐eight naive RBT were exposed per os (fed L. salmonae spores) and 28 RBT were exposed by cohabitation with 28 L. salmonae‐infected RBT. Exposed fish were examined once every week (7 days) post exposure (PE). For xenoma onset, the median survival time, the time to first appearance of branchial xenomas, was 6 weeks PE for both per os and cohabitation exposure. For xenoma clearance, the median survival time, the time to total clearance of branchial xenomas, was 10 weeks for per os exposure and 12 weeks for cohabitation exposure. There was a significant difference between the survival curves of per os fish compared with cohabited fish for both xenoma onset and xenoma clearance. The incidence rate of xenoma development was greater for per os exposure (0.1745 cases per fish‐week) compared with cohabitation exposure (0.1342 cases per fish‐week). Similarly, the rate of xenoma clearance was greater for per os exposure (0.1028 cases per fish‐week) compared with cohabitation exposure (0.0885 cases per fish‐week). Differences between exposure methods were attributed to differences in the frequency and duration of exposure to spores. Survival analysis was useful for examining the onset and clearance of L. salmonae and may have applications for understanding disease dynamics in aquaculture.     

肖四海湖五种渔具的鳜渔获结构特征及其对鳜资源的影响

为评估不同渔具对鳜资源的影响,于2007年5月和12月对长江中游浅水湖泊肖四海湖刺网、延绳钓、网簖、电拖网和电捕仪5种渔具捕获的鳜渔获物结构特征进行了调查分析.结果发现,共采集鳜样本359尾,全长分布范围为92 ～600 mm,优势全长集中于251～350 mm;体质量分布范围为10～3 380 g,优势体质量集中于300 ～500 g.种群由5个年龄组构成,2～3龄为优势龄组,占总数的74.6％.刺网、延绳钓和网簖对鳜有较强的捕捞选择性,网目大小为80 mm和100 mm刺网的鳜渔获物中2龄及以上成熟个体占总数的93.3％,个体平均体质量466 g,“标鳜”(0.4 ～0.75 kg)个体占总数量的57.0％;延绳钓捕获的鳜渔获物中2龄及以上成熟个体占总数的86.9％,“标鳜”个体占总数量的43.5％;网簖捕获的鳜渔获物中90％以上为1龄的未成熟个体.电拖网和电捕仪捕获的鳜全长范围明显较大,其渔获物以1龄和2龄个体为主.综合分析表明,刺网适于作为鳜捕捞的主要渔具,延绳钓可以作为一种鳜捕捞的辅助渔具.网簖对鳜补充群体有较大危害,不适于作为鳜的捕捞网具.电拖网和电捕仪均属于违法渔具,对鳜资源危害巨大,应该加大监管力度,严禁使用.     

Enhanced production of the sea urchin Paracentrotus lividus in integrated open-water cultivation with Atlantic salmon Salmo salar

Survivorship and performance was investigated for two size classes of the sea urchin Paracentrotus lividus grown adjacent to open-water Atlantic salmon (Salmo salar) cultivation on the north-west coast of Scotland. Juvenile P. lividus were maintained for 12 months at 0 m, 50 m and 2.5 km from the mariculture activity. The sea urchins at the 0 m station showed higher survivorship than at the 50 m and 2.5 km stations and had significantly greater test diameter than at the 50 m station. Only urchins at 0 m developed gonads and, although small, these were of acceptable or excellent colouration in terms of their marketability. Adult P. lividus were maintained for 3 months at 0 m and 15 m from the mariculture activity, either with or without additional macroalgae Laminaria spp. Sea urchins at the 0 m station and fed additional macroalgae showed higher gonadal growth than sea urchins at the 15 m station held without additional food. Gonad colouration in the adult urchins, irrespective of the treatment, was acceptable or excellent. The 0 m station also received significantly greater quantities of particulate organic matter (POM) than the other stations in both the juvenile and adult experiments. At the 0 m station, the presence of the fatty acid 22:1n-11 and elevated levels of the long chain n-3 fatty acid DHA 22:6n-3 in the gonadal tissue of the urchins confirmed the consumption of fish farm derived POM and highlighted the potential human health benefits from consuming sea urchin roe.The results show that P. lividus can thrive in the salmon culture environment and suggests that the integration of P. lividus with Atlantic salmon can provide a viable means to culture this species, even at this northerly latitude. Salmon-sea urchin co-culture would enable fish farms to diversify into producing a second highly valuable product and would reduce the increasing worldwide pressure of sea urchin fisheries on wild stocks.     

Amoebic gill disease (AGD)-affected Atlantic salmon, Salmo salar L., are resistant to subsequent AGD challenge

There is inconsistent evidence of resistance of Atlantic salmon, Salmo salar L., to amoebic gill disease (AGD). Here, evidence is presented that demonstrates that Atlantic salmon exposed and subsequently challenged with AGD are more resistant than naïve control fish. Seventy‐three per cent of Atlantic salmon previously exposed to AGD survived to day 35 post‐challenge compared with 26% exposed to Neoparamoeba sp. for the first time, yet the gill pathology of surviving naïve control or previously exposed fish was not significantly different. Development of resistance to AGD is associated with anti‐Neoparamoeba sp. antibodies that were detectable in serum of 50% of surviving Atlantic salmon previously exposed to AGD. However, anti‐Neoparamoeba sp. antibodies were not detectable in cutaneous mucus of resistant fish. Increased resistance of Atlantic salmon after secondary Neoparamoeba sp. infection and detection of specific serum antibodies provides support for the development of a vaccine for AGD.     

肉桂活性成分肉桂醛杀灭离体多子小瓜虫效果

研究从肉桂中分离纯化出具有抑杀多子小瓜虫活性的化合物。以乙醇为提取剂,用索氏超声提取法从肉桂中提取浸膏,再用石油醚、乙酸乙酯、甲醇为萃取剂,萃取不同有效组分,以不同浓度分别进行杀灭离体多子小瓜虫实验,发现石油醚萃取物的杀虫效果最优。然后对石油醚萃取物采用硅胶层析柱和制备型高效液相色谱进行分离纯化,利用质谱和核磁波谱分析,最终鉴定其杀虫活性成分为肉桂醛;将肉桂醛溶于二甲基亚砜并用二倍梯度稀释法配成不同浓度的药液测试其对离体小瓜虫的杀灭活性。结果表明,100%杀灭滋养体和感染性幼虫的剂量分别为50和8 mg/L,半数有效浓度分别为13.9和1.8 mg/L;使用剂量在50 mg/L可完全抑制小瓜虫包囊孵化。     

In situ studies on the bioaccumulation of microcystins in the phytoplanktivorous silver carp (Hypophthalmichthys molitrix) stocked in Lake Taihu with dense toxic Microcystis blooms

The phytoplanktivorous silver carp is an important biomanipulation fish to control cyanobacterial blooms and is also a food fish with the greatest production in China. The accumulation of the hepatotoxic microcystins (MCs) determined by LC-MS in various organs of silver carp was studied monthly in Lake Taihu dominated by toxic Microcystis aeruginosa. Average recoveries of spiked fish samples were 78% for MC-RR and 81% for MC-LR. The highest content of MCs was found in the intestine (97.48 μg g^− 1 DW), followed by liver (6.84 μg g^− 1 DW), kidney (4.88 μg g^− 1 DW) and blood (1.54 μg g^− 1 DW), and the annual mean MC content was in the order of intestine > liver > kidney > blood > muscle > spleen > gallbladder > gill. Silver carp could effectively ingest toxic Microcystis cells (up to 84.4% of total phytoplankton in gut contents), but showed fast growth (from 141 g to 1759 g in 1 year in mean weight). Silver carp accumulated less microcystins in liver than other animals in the same site or other fish from different water bodies at similar level of toxin ingestion. There was possible inhibition of the transportation of the most toxic MC-LR across the gutwall. Muscle of silver carp in Lake Taihu should not be consumed during period of dense Microcystis blooms while viscera were risky for consumption in more months.     

Susceptibility by amberjack (Seriola dumerili), yellowtail (S. quinqueradiata) and Japanese flounder (Paralichthys olivaceus) to Neobenedenia girellae (Monogenea) infection and their acquired protection

This study investigated: 1) susceptibility differences to infection by Neobenedenia girellae (Capsalidae) between amberjack Seriola dumerili (Carangidae), yellowtail S. quinqueradiata and Japanese flounder Paralichthys olivaceus (Paralichthyidae); 2) growth and egg production of N. girellae on each fish species; 3) acquired protection of each fish species against this parasite. The number of N. girellae on S. dumerili was significantly higher than on S. quinqueradiata and P. olivaceus when these fishes were exposed to oncomiracidia in the same aquarium. Neobenedenia girellae growth on S. dumerili was fastest and, thus the number of eggs laid by parasites on S. dumerili was greater than on the other two species. Seriola dumerili and P. olivaceus, which were previously infected with N. girellae and treated by freshwater bath, acquired partial protection against re-infection by N. girellae. The relative re-infection of three S. dumerili individuals out of eleven individuals was markedly low compared with the initial infection, and the relative initial infection and re-infection on two P. olivaceus out of eleven individuals was markedly low. The results of this study could be useful to control N. girellae infections when cultivating S. dumerili, S. quinqueradiata and P. olivaceus.     

The induction of laboratory-based amoebic gill disease revisited

Previous work in our laboratory defined a method of inducing laboratory‐based amoebic gill disease (AGD) in Atlantic salmon, Salmo salar L. Gills of AGD‐affected fish were scraped and the debris placed into fish‐holding systems, eliciting AGD in naïve Atlantic salmon. While this method is consistently successful in inducing AGD, variability in the kinetics and severity of infections has been observed. It is believed that the infections are influenced by inherently variable viability of post‐harvest amoeba trophozoites. Here, a new method of experimental induction of AGD is presented that redefines the infection model including the minimum infective dose. Amoebae were partially purified from the gills of AGD‐affected Atlantic salmon. Trophozoites were characterized by light microscopy and immunocytochemistry and designated Neoparamoeba sp., possibly Neoparamoeba pemaquidensis. Cells were placed into experimental infection systems ranging in concentration from 0 to 500 cells L^?1. AGD was detected by gross and histological examination in fish held in all systems inoculated with amoebae. The number of gross and histological AGD lesions per gill was proportional to the inoculating concentration of amoebae indicating that the severity of disease is a function of amoeba density in the water column. The implications of these observations are discussed in the context of the existing AGD literature base as well as Atlantic salmon farming in south‐eastern Tasmania.     

Recruitment and effects of Discocotyle sagittata (Monogenea) infection on farmed trout

Farmed rainbow trout Oncorhynchus mykiss and brown trout Salmo trutta were monitored over 3 years for infection with the blood-feeding gill fluke Discocotyle sagittata. Parasite transmission is seasonal: new infections take place during summer/autumn, and transmission is generally negligible during winter/spring. There are 2 sources of infection for naïve fish-of-the-year: limited invasion when fish are in the raceways by riverborne larvae originating external to the farm; and internally, within the farm, when 0+ fish are transferred to ponds previously occupied by older cohorts of infected fish. Thereafter, infection levels continue to increase in rainbow trout primarily through transmission within the farm. Prevalence rose to 100% in 1+ fish by the end of their second summer. In O. mykiss, mean abundance reached 194 worms/host for 1+ fish (up to 489 worms/host) and 160 worms/host for 2+ fish. By contrast, in S. trutta, parasite prevalence never exceeded 85% and, after the first year's invasions, infection levels decreased over time: in 1+ and 2+ brown trout, parasite mean abundance was < 4 (maximum 15) worms/host. We present evidence of the detrimental effects of D. sagittata on the host: high burdens are associated with pale gills, decreased body condition and host mortality. Parasite burdens become overdispersed during the warmer part of the year, as prevalence and mean abundance increase. However, the degree of parasite overdispersion decreases over winter; we cannot distinguish whether decreased aggregation is due to parasite losses from infected fish (including immune-mediated parasite mortality) or parasite-induced host mortality.     

Distribution of a fish pathogen Listonella anguillarum in the Japanese flounder Paralichthys olivaceus hatchery

The present study was undertaken to investigate the distribution of Listonella anguillarum in a Japanese flounder (Paralichthys olivaceus) hatchery. A total of 2704 isolates were obtained from the developing fish, live diets and artificial feeds of Japanese flounder and their rearing water, 439 of which were identified as L. anguillarum by the combining incubation on thiosulfate-citrate-bile salt-sucrose (TCBS) agar at 35 °C overnight with polymerase chain reaction (PCR) detection for the VAH1 hemolysin gene. L. anguillarum was detected in all seven rotifer samples, with densities of 2.5 × 10³ to 4.6 × 10⁶ colony forming units (CFU) g^− 1. Both the analyzed samples of Nannochloropsis oculata contained this bacterium at densities of 1.6 × 10⁴ to 1.4 × 10⁵ CFU g^− 1. L. anguillarum was detected in only one of four samples of Artemia nauplii with a density of 4.8 × 10⁵ CFU g^− 1 (35%) and it was not detected in the two analyzed artificial feed samples. L. anguillarum was detected in 11 of 18 specimens of larval and juvenile Japanese flounder at densities of 5.0 × 10¹ to 7.4 × 10⁵ CFU g^− 1, while it was not detected in the two analyzed egg specimens of Japanese flounder. These results indicate that L. anguillarum associated with the developing Japanese flounder is likely derived from rearing water and live diets such as rotifers. Further, it is strongly suggested that L. anguillarum is a transient bacterium of the intestinal microflora for the Japanese flounder but is a permanently indigenous one for the Japanese flounder hatcheries.     

Effect of Dietary Inclusion of N-Acetyl Cysteine on Mucus Viscosity and Susceptibility of Rainbow Trout, Oncorhynchus mykiss, and Atlantic Salmon, Salmo salar, to Amoebic Gill Disease

The treatment of amoebic gill disease (AGD) in cultured Atlantic salmon, Salmo salar L., using mucolytic agents has been previously reported. The agent L‐cysteine ethyl ester reduces salmonid mucus viscosity and potentially increases the flushing of the gill. In the present study, the effects of the mucolytic agent N‐acetyl cysteine (NAC) were assessed. Cutaneous mucus from rainbow trout, Oncorhynchus mykiss Walbaum, and Atlantic salmon was shown to have reduced viscosity when mixed in vitro with 100 or 200 μg/mL NAC. Saltwater‐acclimated rainbow trout and Atlantic salmon were fed an oil‐incorporated, NAC‐medicated diet (8 g NAC/kg diet) for up to 24 d and challenged with inoculation of 300 cells/L Neoparamoeba spp., the etiological agent of AGD. Control fish were fed normal oil‐coated pellets and received no NAC. NAC medication failed to reduce the severity of gill lesions associated with AGD even though the mucus viscosity from medicated fish was less than that of controls. Oral NAC medication does not appear to be an effective method for controlling AGD in salmonids despite reducing cutaneous mucus viscosity.     

Evaluation of three seaweeds Gracilaria bursa-pastoris, Ulva rigida and Gracilaria cornea as dietary ingredients in European sea bass (Dicentrarchus labrax) juveniles

The aim of this study was to evaluate the inclusion of three seaweeds Gracilaria bursa-pastoris (GP), Ulva rigida (UR) and Gracilaria cornea (GC) as dietary ingredients on the performance, nutrient utilisation and body composition of European sea bass juveniles. Six experimental diets were formulated to replace 5% (GP-5, UR-5, and GC-5 Diets) and 10% (GP-10, UR-10 and GC-10 Diets) fish protein hydrolysate (CPSP) by each of the three seaweeds. A control diet was used, without inclusion of any seaweed. Diets were fed to duplicate groups of 25 European sea bass (Dicentrarchus labrax) juveniles (IBW = 4.7 g) for 10 weeks. Growth performance was only significantly reduced (P < 0.05) in fish fed the GC-10 diet, whereas the feed conversion ratio increased significantly in those fish. The apparent digestibility coefficients of dry matter and lipid were significantly lower in fish fed diet GC-10 relative to those fed the control diet. Carcass composition was similar among treatments, although fish fed GC-10 exhibited significantly higher ash content.The results obtained in this study suggest that the inclusion of G. bursa-pastoris (GP) and U. rigida (UR), up to 10%, can be considered as very interesting ingredients in diets for sea bass juveniles, as no negative consequences on growth performance, nutrient utilization or body composition were observed. On the other hand, the inclusion of G. cornea (GC) should be limited to 5% of the diet.     

Sarcoma of the swim bladder of Atlantic salmon (Salmo salar L.)

A neoplastic disease of the swim bladder of farmed Atlantic salmon (Salmo salar L.) was investigated. The tumour was classified as a leiomyosarcoma and had a detected prevalence of 4.6% of a population of 500 second-year sea fish.     

副溶血弧菌对斑节对虾非特异性免疫酶活性的影响

为了研究副溶血弧菌对斑节对虾非特异性免疫酶活性的影响,分别对斑节对虾注射生理盐水和副溶血弧菌,于不同时间点测定肝胰腺和鳃中的总抗氧化能力(T-AOC)、酸性磷酸酶(ACP)、碱性磷酸酶(ALP)和溶菌酶(LSZ)的活性变化。结果显示,与对照组相比,感染副溶血弧菌后,肝胰腺和鳃中T-AOC活性分别于12和6 h达到最大值(P0.05),随后逐渐降低;肝胰腺中ACP活性于3 h显著升高,随后逐渐降低,并于24 h达到最小值(P0.05),而鳃中ACP活性于12 h达到最大值,随后逐渐降低,并于48 h显著低于对照组(P0.05);肝胰腺和鳃中ALP活性整体均呈现出先升高后下降的趋势,但未出现显著低于对照组的现象;肝胰腺和鳃中LSZ活性均于3 h显著升高至最大值(P0.05),随后逐渐恢复至初始水平。研究表明,副溶血弧菌感染对斑节对虾非特异性免疫酶活有显著影响,对其机体免疫防御系统有明显的破坏作用。     

Cryoprotectant microinjection toxicity and chilling sensitivity in gilthead seabream (Sparus aurata) embryos

Cryopreservation of fish embryos requires an optimal distribution of cryoprotectants inside all embryo compartments. Traditional techniques for the incorporation of cryoprotectants (CPAs) have failed to protect all fish compartments, especially the yolk sac which has been considered the principal point of embryo chilling sensitivity. In the present study, microinjection was used to incorporate cryoprotectants into the yolk sac of gilthead seabream (Sparus aurata) embryos at tail bud stage. The effect of microinjection viability, cryoprotectant toxicity and chilling resistance was evaluated through the hatching rate. Larval survival at first feeding was also determined in microinjection viability and cryoprotectant toxicity studies. Permeabilized seabream embryos were microinjected with 2.35 nl dimethyl sulfoxide (Me₂SO), methanol (MeOH), ethylene glycol (EG) (5 M, 10 M and pure) or sucrose (10% and 15%). In a second experiment, 29.5 nl and 154.0 nl of the highest concentration of each cryoprotectant were used in the same embryo stage. To test the effect of microinjected cryoprotectants on embryo chilling resistance, 29.5 nl of pure Me₂SO or 15% sucrose was microinjected into the yolk sac of tail bud stage embryos and then at a later stage, (tail-bud-free), were exposed to 3 M Me₂SO solution at − 10 °C for 30 min. Our results showed that microinjection technique did not affect the viability of tail bud stage embryos as is shown by the high hatching and survival rates. Hatching and larval survival rate at first feeding were not affected with any of the CPAs tested, showing percentages higher than 75% and 90%, respectively, when embryos were microinjected with a smaller quantity of cryoprotectant. Sucrose was the cryoprotectant better tolerated at higher concentration and volume. Cryoprotectant concentration inside the yolk higher than 1.18 M for Me₂SO, 1.5 M for EG and 2 M for methanol decreased the hatching rate. Microinjection allowed the delivery of high concentrations of CPAs into the yolk sac without deleterious effects on the embryo, but did not provide a significant level of protection for the whole embryo against chilling injury.     

Importance of river migration to the development of seawater tolerance in Columbia River anadromous salmonids

Gradually increasing levels of gill Na⁺K⁺ ATPase activity were observed in juvenile chinook, Oncorhynchus tshawytscha, and coho, Oncorhynchus kisutch, salmon and steelhead trout, Salmo gairdneri, undergoing parr-smolt transformation in artificial rearing facilities on the Columbia River. Portions of the same populations released to migrate seaward, however, generally showed much greater increases in enzyme activity with time and distance from the release point. After migrating 714 km to the Columbia River estuary, spring chinook salmon had a mean gill Na⁺K⁺ ATPase activity 2.5 times greater than fish retained at the hatchery and 1.9 times greater than fish adapted to 28 ppt seawater for 208 days. Similar observations were made on coho salmon.