Suppression of Saprolegnia infections in rainbow trout (Oncorhynchus mykiss) eggs using protective bacteria and ultraviolet irradiation of the hatchery water

Since formalin is widely used in prevention of Saprolegnia infections in salmonid fish hatcheries, there is a need for more environmentally safe treatment methods. Therefore, we screened 360 bacterial isolates against their ability to antagonize the growth of Saprolegnia parasitica hyphae in vitro, and best strains were selected according to their antagonistic properties and colonization capability on rainbow trout egg surface. Protective bacterial cultures of Pseudomonas sp. M162, Pseudomonas sp. M174 and Janthinobacterium M169 were tested for prevention of Saprolegnia sp. infections during incubation trials of rainbow trout (Oncorhynchus mykiss) eggs with UV irradiated (400 mWs cm^?2) and non‐treated inlet water. UV irradiation of inlet water significantly decreased mortality during the incubation. Lowest mortalities were observed in protective culture treated groups incubated with UV‐irradiated inlet water. UV irradiation increased the dominance of the main bacterial colonizers and variation in the bacterial species diversity between the experimental units.     

The bacterial flora of rainbow trout, Salmo gairdneri Richardson, and brown trout, Salmo trutta L., eggs and its relationship to developmental success

Abstract. The bacterial flora of rainbow, Salmo gairdneri Richardson, and brown trout, Salmo trutta L., eggs was studied during incubation. Few bacteria were considered to exist internally, although Pseudomonas species and Aeromonas hydrophila were isolated. During incubation, large numbers of bacteria approaching 500 colony forming units/mm² accumulated around egg surfaces. Both rainbow and brown trout eggs placed in a through-flow system were colonized mainly by Pseudomonas sp. and A. hydrophila , whilst rainbow trout eggs in a separate recycling system were dominated by a Cytophaga species. Glass beads were also incubated (as an inert surface) and found to support significantly lower numbers of bacteria. Egg mortalities were recorded daily and found to differ significantly between both fish species and water conditions. Statistical analysis indicated a potentially significant correlation between hatching success and numbers of surface bacteria.     

用显微注射技术导入外源DNA对鲤鱼和虹鳟鱼受精卵孵化率的影响

本报道了利用显微注射技术导入外源基因分别对鲤鱼和虹鳟鱼授精卵孵化率影响的研究。从1993年－1996年4年中，我们用显微注射方法，将全鱼生长激素基因导入虹鳟鱼授精卵（包括囊胚期卵）5，676粒，孵出鱼苗364尾，孵化率为6.4%(4年平均，虹鳟鱼正常孵化率为80％）；而用这种方法，将全鱼生长激素基因导入鲤鱼授精卵12，196粒，孵出鱼苗4，290，孵化率为34．0％（4年平均，鲤鱼正常孵化率为70％左右）。实验结果：显微注射技术在虹鳟鱼和鲤鱼两种受精卵上的效果明显不同，斑点杂交和SouthernBlot杂交结果相近。     

Mortality of marine cultured rainbow trout, Oncorhynchus my kiss (Walbaum), during the summer in the Netherlands

Abstract. In northern Europe, rearing of rainbow trout, Oncorhynchus mykiss (Walbaum), in sea cages is a substantial part of fish culture. One of the major constraints for the development of this system in other parts of Europe is the unexplained mortality of fish during the summer. To investigate environmental aspects of this mortality, rainbow trout, cultured in cages in sea water, were monitored from May to October for three successive years. No evidence of bacterial involvement could be found, while water temperature and salinity strongly influence the mortality. This supports the suggestion that the mortality has been caused by patho-physiological changes due to higher metabolic requirements for osmoregulation at elevated water temperature in the saline environment which might result in a dissipation of O₂ by CO₂ on the hemoglobin.     

The effect of short-term starvation on ovarian development and egg viability in rainbow trout (Salmo gairdneri)

Adult female rainbow trout (Salmo gairdneri) were fed a commercial trout production diet until approximately 40 days prior to spawning at which time the fish were divided in two groups. One group continued to be fed while the other group was starved until spawning. Egg size, egg number, ovary weight, and egg proximate composition were examined periodically starting 5 months before spawning. Muscle tissues of the fish were sampled at spawning to measure somatic depletion of proximate consituents in the two groups. Egg hatchability was determined by incubating test lots from each spawning female. No significant differences (P > 0.05) were found in egg hatchability between the fed and starved groups. Changes in proximate composition were observed in the eggs during the months prior to spawning, but no differences were detected between the eggs of the starved and the fed groups. Short-term starvation did not significantly alter the proximate composition of muscle tissue of the spawned female trout.     

Are environmental conditions in Finnish streams limiting to early life-history survival in the nonnative rainbow trout?

The nonnative rainbow trout Oncorhynchus mykiss has been an unsuccessful invader in North European streams, although it has been widely introduced. Here we studied whether early life history stages (egg incubation and hatching, first overwintering) act as filters for the establishment of hatchery rainbow trout. Survival of hatched alevins was approximately 80%, whereas only 47% of the embryos survived. However, the latter value was impacted by the high number of unfertilized eggs. Correlation coefficients with embryo survival rate and environmental variables (pH and temperature) were statistically insignificant. In the overwintering experiments, the survival of rainbow trout was 93%. The growth was generally slowed during the winter, but in the spring the growth of rainbow trout exceeded that of the native brown trout. Our data demonstrated that the survival and growth of rainbow trout during early life-history stages were relatively high and comparable to those of the native brown trout. Based on the variables considered in our study, our results suggest that environmental conditions during early life-history stages are not detrimental for rainbow trout in the study streams.     

Egg Quality Determinants in Finfish The Role of Overripening with Special Reference to the Timing of Stripping in the Atlantic Halibut Hippoglossus hippoglossus

Egg quality, those characteristics of the egg that determine its capacity to survive, is a significant problem for many species of fish currently being farmed. Little is known about the determinants of egg quality and there is little agreement regarding reliable methods for its assessment. To be of practical benefit, assessments should be simple to perform and should be carried out as early in development as is possible. Fertilization rates are often used as measures of quality. For the Atlantic halibut Hippoglossus hippoglossus, fertilization rate and assessments of cell symmetry at early cleavage stages provide reasonable indicators of quality. Regardless of assessment method, it is strongly recommended that performance data from all batches of each broodfish be examined when surveying the overall quality of a stock. The misleading effects of pooling such information are demonstrated for a rainbow trout Oncorhynchus mykiss broodstock. Although a large number of factors have been implicated as possible determinants of egg quality, only 1) bacterial colonization of the eggs, 2) nutritional status of the broodfish and, 3) overripening, the process of aging that occurs when eggs are retained within the broodfish after ovulation, have been clearly shown to affect egg quality. The effect of overripening on fish egg quality is discussed in detail. Species-specific differences in the time scale of overripening are pointed out and related to spawning strategy and water temperature. Rainbow trout eggs and those of other salmonids should be fertilized within approximately one week of ovulation. Overripening proceeds much more rapidly in warm water species, e.g., tilapia eggs must be fertilized within an hour or so of ovulation. Egg viability also decreases rapidly for batch spawning species. Fertilization data are presented for Atlantic halibut demonstrating that egg quality decreases 4–6 h after ovulation. The rate of overripening was comparable for eggs held in vitro in ovarian fluid to those retained within the ovarian lumen. These halibut data, combined with information from other marine and freshwater fish, indicate that overripening is a significant determinant of egg quality for many if not all fish.     

Individual variation in progeny size and quality in rainbow trout, Oncorhynchus mykiss (Walbaum)

Early life history traits of fish are very variable as a result of both genetic and environmental factors. In this study, we examine individual variation in early life history traits in progeny of seven females crossed with one male rainbow trout, Oncorhynchus mykiss (Walbaum). Individual eggs were followed from fertilization through hatching until death of the larvae by starvation. Larvae and yolksac size (size was shown to be a good indicator of energy content) increased with increasing egg size, but there were still differences between families after variation in egg size was accounted for. Incubation time was not correlated with egg size, and did not differ between families. The progeny from the different families utilized the available energy differently, as both longevity and growth-rate without food was independent of yolk-sac size, but strongly dependent on family. The observed between-family differences in early life history traits in rainbow trout were mainly caused by genetically based effects; egg size and thus probably egg quality, which differed strongly bet ween females, could not adequately explain these differences.     

Control of Listeria Monocytogenes and Escherichia coli O157:H7 Inoculated on Fish Fillets Using Alginate Coating Containing Lactoperoxidase System and Zataria multiflora Boiss Essential Oil

The present study was conducted to investigate the inhibitory effect of alginate coating incorporated with Zataria multiflora Boiss essential oil (ZEO) and lactoperoxidase system (LPOS), individually and in combination, in order to control inoculated Listeria monocytogenes and Escherichia coli O₁₅₇:H₇ in rainbow trout fillets during 16days of storage at 4°C. The antibacterial activity of ZEO was evaluated against Listeria monocytogenes and E. coli O₁₅₇:H₇ through determination of minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC). In vivo study was performed by inoculating four strains of L. monocytogenes as culture cocktail and one strain of E. coli O₁₅₇:H₇ on trout fillets for their survival evaluation during the 16-day storage. Results indicated that ZEO and LPOS, when used in combination in alginate solution, had a stronger effect on the control of mentioned bacteria in trout fillets; however, their individual use could significantly inhibit their growth, when compared to the control. Moreover, it has been implicated that alginate coating, when used with no antimicrobial agent, had a supportive effect on the growth of these pathogenic bacteria. Therefore, the application of alginate coating containing ZEO and LPOS is recommended in foods, especially fish and fish products.     

Preservation of trout sperm in liquid or frozen stateFlüssig- und gefrierkonservierung von regenbogenforellensperma

A series of experiments to study the preservation of rainbow trout semen in the liquid and frozen state was carried out.For the preservation of liquid semen both undiluted and diluted semen was stored in total darkness at 20° or 4°C under a number of different gasses (air, O₂, O₂ + N₂ (1:1), N₂, CO₂, N₂ + H₂ + CO₂ (8:1:1)). The best results were obtained when storing semen in an undiluted state at 4°C under O₂ (hatching rate after 15 days was 80.6%, controls were 98.2%). Good results were also obtained under air (after 9 days 94.7% vs 94.9% for the controls). Anaerobic conditions, dilution or an increase in temperature all rendered less favourable results.For the purpose of cryopreservation a diluent was developed on the basis of the composition of rainbow trout seminal plasma. The semen was mixed with the diluent immediately before freezing at a ratio of 1:1 or 1:3. The pellet-technique (Nagase, 1964) was found to be more convenient and successful than freezing in straws. The pellets were thawed in a 1% NaHCO₃-solution. The hatching results after storage periods of 1–4 months were very variable (2.6–80.3%).     

Hydrogen peroxide oxygenation and disinfection capacity in recirculating aquaculture systems

Hydrogen peroxide (H₂O₂) treatment is an alternative for disinfection in aquaculture, which may be advantageous as it dissociates and disinfects while increasing water oxygen concentration. Yet, accurate dosing remains undeveloped in Recirculating Aquaculture Systems (RAS). Dosage requirements can depend on organic burden, stocking density, feeding frequency, salinity, temperature and biofilter performance. The present case study investigated the dual effect of H₂O₂ application for oxygen enrichment and disinfection when continuously applied to a RAS rearing European seabass. H₂O₂ addition equivalent to 2.4 and 15.8 H₂O₂ mg L⁻¹ were applied for 4 h per day in three 5-days experiments. H₂O₂ was injected at the inlet of protein skimmer and/or the rearing tanks in or without combination with traditional disinfection methods. Water microbial load and oxygen saturation were determined, along with stress markers glucose and cortisol in blood plasma of fish. Doses of 15.8 mg L⁻¹ H₂O₂ steadily increased oxygen levels in holding tank water from ∼50 % to over 100 % saturation while reducing microbial load (from 604.4 CFU ml⁻¹ in the rearing tanks before dosing to 159.8 CFU ml⁻¹ after application), achieving suitable conditions for commercial fish densities in RAS. The doses used had negligible impact on biofilter performance and did not affect the fish in terms of stress. Overall results indicate H₂O₂ is effective for disinfection and oxygenation of RAS systems when applied at appropriate dosage and we recommend the protein skimmer as the safest position in order to protect the bacterial community of the biofilters and the reared fish.     

Cryopreservation of rainbow trout spermatozoa: The influence of sperm quality,egg quality and extender composition on post-thaw fertility

The presence of 5% to 20% hen's egg yolk in a sucrose-based extender significantly improved post-thaw fertility of cryopreserved rainbow trout (Salmo gairdneri) spermatozoa compared to when the extender without hen's egg yolk was used. However, the degree of increased cryoprotection associated with hen's egg yolk was affected by the quality of the milt. Considerable variation was detected in the performance of various batches of trout eggs used to test post-thaw fertility and the composition of the extender was shown to affect fertilizations differentially with some of the eggs. Despite this variation, the extender containing 10% hen's egg yolk consistently gave high post-thaw fertility in samples of cryopreserved milt (67.3±3.0% S.E.M.) in thirty replicated trials. As such, the method described is reliable for cryopreserving rainbow trout milt and fertilizing small quantities of eggs.     

Studies on cryopreservation of eggs from rainbow trout (Salmo gairdneri) and coho salmon (Oncorhynchus kisutch)

The effect of pre-freezing treatments as well as freezing of inseminated, not water-activated eggs from rainbow trout Salmo gairdneri, and coho salmon, Oncorhynchus kisutch, was investigated in relation to survival and further development.Effects above freezing temperatures included: the temperature at insemination, viability of inseminated and unactivated eggs after storage, suitability of an incubation medium and the tolerance of eggs to various levels of the cryoprotectant dimethylsulfoxide (DMSO). Freezing experiments included: investigating the action of DMSO (0, 1, 2 mole) and the tolerance of coho eggs to temperatures between ?4.6 to ?30°C. Insemination temperatures between 0.5°C and 9.8°C (coho eggs) as well as incubation in an artifical medium (1-0°C) for 80 min (rainbow trout eggs) and 170 min (coho eggs) did not influence subsequent fertility. Storage of inseminated and unactivated rainbow trout eggs for 135 min and beyond reduced egg fertility. DMSO at 2 and 4 mole was detrimental to coho eggs (1-0°C). One mole DMSO had no (coho) or reduced (rainbow trout) influence on egg fertility when it was added gradually.In the presence of 1 mole DMSO most eggs remained unfrozen (67–89%) when kept for 10 min in frozen artificial medium (?4.6%) and 27–32% subsequently reached the eyed stage (control = 100). Further cooling (0.3°C/min) to ?10°C was still tolerated (62% unfrozen, 22% eyed eggs) but not to ?20°C (6% unfrozen, no development) and ?30°C (no survival). Use of 2 mole DMSO did not improve the results.     

Accumulation of aluminum on atlantic salmon eggs during the incubation period

Groups of rainbow trout Onchorhyncus mykiss eggs were incubated in duplicate trays with PVC screens and the original aluminum screens. During the incubation period, aluminum bound to sediments was deposited on the eggs and in the whole hatching system.

The pH of the inlet water to the hatching system with PVC screen was experimentally lowered by adding sulphuric acid. Buffering of the outlet water was observed at pH lower than 5, and at pH 4·5 and lower, an increased aluminum concentration in the outlet water was recorded.

These experiments show that aluminum accumulated on eggs and in the hatching system during periods with neutral water represents a serious threat to the survival of eggs and fry of salmonids if pH drops below 5·0, even for a few hours. In sensitive lakes and rivers, pH drops below 5·0 are common during heavy rain or during the snow melting period.     

Fecundity, egg size and total egg volume differences in 12 stocks of rainbow trout, Oncorhynchus mykiss Richardson

Abstract. Measurements of egg number, egg size and total egg volume in relation to post-stripped weight, made on the eggs stripped from 12 separately maintained farm stocks of 4-year-old rainbow trout. Oncorhynchus mykiss Richardson, revealed highly significant differences between populations when analysed by regression and covariance statistical techniques. All of the stocks showed increases in fecundity with increasing fish size, with 61% (coefficient of determination or r²) of the variation in fecundity of the 12 stocks explained by the common regression on parental fish size. Comparison of the regressions of the different stocks revealed that the rate of increase of fecundity with increasing fish weight was constant throughout the weight range (i.e. the regressions had similar slopes) although there were highly significant differences in elevation, with some stocks producing almost twice as many eggs as some of the others. By contrast, the relationship of egg size with fish weight was much more variable with three of the stocks showing no significant regression of egg size on fish weight and a further two stocks with data poorly fitted to the regressions. Of the remaining seven stocks only one showed a significant difference in slope of the regression with modest increases in fish weight being associated with large increases in egg size. Only four of the remaining stocks of fish showed significant differences in the elevation of their regressions, i.e. the egg sizes were significantly different after the potentially conflicting influence of fish size was partitioned using covariance techniques. Assessments of total egg volume produced far improved regressions on post-strip weight with 75% (r²) of the variation in the pooled data of the 12 stocks being described by the line of the common regression. In general, there appeared to be a “trade-off” between egg number and size with the most and least fecund fish producing somewhat smaller and larger eggs respectively. All 12 regressions of totai egg volume on fish weight showed a common slope. However, there were significant differences in elevation of the regressions, with some of the stocks of trout producing up to a 55% greater volume of eggs. There appeared to be no relationship between the differences in egg number, size or total egg volume and the type or geograpfiical location of the farms on which the stocks were held. Although it remains to be determined to what extent these differences in reproductive performance are Inheritable, it is recommended that these characteristics should be taken into account by hatcheries in tbe design of long-term broodstock selection programmes for rainbow trout.     

Evaluating the Rearing condition of Rainbow Trout (Oncorhynchus Mykiss) Using Fuzzy Inference System

Rainbow trout (Oncorhynchus mykiss) is one of the most popular aquacultured species in the world. Sustainable production of this fish at commercial scale is very important but requires maintaining good water quality throughout the total rearing period. The present study aimed to develop a rainbow trout production index in order to raise awareness about the conditions of the rearing environment, enhance production, and reduce losses. For this purpose, an intensive rainbow trout production system was selected as the study system. In this system, there were seven stations including (a) 3000 5-g fish, (b) 3000 25-g fish, (c) 3000 50-g fish, (d) 3000 100-g fish, (e) 3000 220-g fish, (f) 2000 350-g fish, and (g) 2000 830-g fish. The fuzzy inference system was used to develop the target rearing index. Water quality parameters involved in the variation in the rainbow trout rearing conditions were classified into three groups including un-ionized ammonia, nitrite, and nitrate, Alkalinity and phosphate, along with dissolved oxygen and linear velocity. For each group and condition of rearing, a separate fuzzy inference system was defined and the output of each fuzzy system was named I₁, I₂, I₃. Finally, I₁, I₂, and I₃ were considered as the inputs to a fuzzy system in order to evaluate their effects on the index of general rearing conditions (I). The results indicated that un-ionized ammonia, nitrite, nitrate, and phosphate had negative effects while dissolved oxygen, linear velocity, and alkalinity positively affected water quality and rearing index. Most of the decline in the rainbow trout rearing index was related to the effect of un-ionized ammonia, nitrite, and nitrate due to food decomposition. Therefore, intelligence feeding based on fish appetite through reducing food conversion rate and water pollution can improve rainbow trout production in this system. The index of rainbow trout production conditions reflects the type, amount, and effect of water quality pollutants on rearing conditions. Producers can use this information to reduce the negative environmental effects and improve the product quality.     

A genetic analysis of egg,alevin and fry mortality in salmon (Salmo salar), sea trout (Salmo trutta) and rainbow trout (Salmo gairdneri)

Heritability of mortality in eggs, alevins and fry were estimated using data from salmon, sea trout and rainbow trout. The highest value (average for all species and both estimation methods) based on the sire component was obtained for the eyed egg stage, h² = 0.08, followed by the alevin stage, h² = 0.05, while estimates for fry mortality did not differ significantly from zero.The maternal effect was rather large for uneyed eggs and eyed eggs, while it was low for alevin mortality. Maternal effects were also found to have a significant influence on the mortality of salmon fry. It was, however, not possible to separate maternal effects from tank effects.     

Comparison of iodine and glutaraldehyde as surface disinfectants for red porgy (Pagrus pagrus) and white sea bream (Diplodus sargus sargus) eggs

The efficacy of iodine and glutaraldehyde as fish egg surface disinfectants were assessed in red porgy (Pagrus pagrus) and white sea bream (Diplodus sargus sargus) eggs, two species of interest for Mediterranean aquaculture. Iodine was effective in reducing the bacterial load of the 1-day-old eggs when applied at 50 mg L⁻¹ for 5 min. The same concentration did not cause any significant change in hatching success or survival of the larvae for the first 5 days. Glutaraldehyde failed to reduce the bacterial load of the fish eggs at concentrations that were safe for the eggs (100 mg L⁻¹ for 5 min), as it had a significant effect in preventing hatching of the developed embryo. Disinfecting 0-day-old eggs with iodine resulted in a significant reduction of hatching percentage, while larval survival thereafter was unaffected. The results of the present study suggest that iodine may be an appropriate egg disinfectant for both red porgy and white sea bream.     

Egg quality and egg pigment content in salmonid fishes

The widespread belief in the superior quality of highly pigmented eggs is examined by bringing together evidence concerning the effect of the carotenoid content of salmonid eggs on their viability. It is concluded that, in rainbow trout, there is not a simple linear relation between these two quantities. There is some evidence that a critical level exists (ca. 1–3 μg carotenoid/g eggs) above which hatching percentages of more than 80% may be expected but below which hatching percentages of less than 50% tend to occur. However, the evidence is not conclusive and suitable experiments under controlled conditions have not been performed. Eggs of wild fish are frequently of higher quality than eggs of farmed fish but this difference is not necessarily caused by the large difference in egg carotenoid content but rather by the generally superior nutrition of the wild fish. The effects of egg carotenoid composition, including the artificial pigmentation of eggs with dietary canthaxanthin, on egg viability are considered, and the possible functions of egg carotenoids during embryonic development are discussed. Only one such function has been conclusively demonstrated, namely that they act as a source of pigment for the chromatophores of the alevin. Analogy with other animals suggests that β-carotene, when present, may act as a precursor of vitamin A. There is growing evidence that carotenoids may perform some undefined function in respiration of egg or alevin when oxygen is deficient.     

Water recirculation and good management: potential methods to avoid disease outbreaks with Flavobacterium psychrophilum

Flavobacterium psychrophilum infections cause high mortality among rainbow trout, Oncorhynchus mykiss, fry in Danish fish farms and hatcheries. Hatcheries based entirely on bore‐hole water recirculation systems have been suggested as a possibility for eliminating F. psychrophilum or at least keeping the amount of this bacterium low. The occurrence of the bacterium in a bore‐hole water recirculation system was compared with a combined bore‐hole water and stream water flow‐through system in a hatchery where outbreaks of rainbow trout fry syndrome caused by F. psychrophilum often occurred. Broodfish, unfertilized and fertilized eggs, eyed eggs and fry, as well as water samples from the tanks/troughs with broodfish/fry, were examined. Suspect yellow bacterial colonies were either confirmed or rejected as F. psychrophilum by growth characteristics and by PCR. As both virulent and less virulent F. psychrophilum isolates are known, isolates were characterized. The isolates were ribotyped and grouped according to ribotyping patterns. Representatives of the groups were serotyped. Fry isolates were very homogeneous whereas isolates from broodfish were heterogeneous, whether the isolates originated from external surfaces of the fish (mucus from skin and gills, haemorrhages and ulcers) or internal organs. Flavobacterium psychrophilum was isolated from broodfish in both water systems; 56% of investigated broodfish from the borehole/flowthrough system and 36% from the recirculation facility harboured the bacterium. In the recirculation system, the bacterium was isolated from fish (ulcers, milt, liver, abdominal cavity) kept in the system for 11 months. Flavobacterium psychrophilum was found in milt and ovarian fluid as well as on the surface of fertilized eggs, but not inside the eggs. Fry also harboured F. psychrophilum, but in the water recirculation system the bacterium was first isolated from the fry after they had been graded. Flavobacterium psychrophilum was found regularly in other parts of the hatchery (outside the recirculation facility), including at the time of grading, suggesting that the occurrence of F. psychrophilum in the fry recirculation facility was due to contamination from the borehole/flowthrough hatchery. It is suggested that the combination of bore‐hole water recirculation systems and good management procedures (including egg disinfection) is a possible method for hatcheries to avoid disease outbreaks due to F. psychrophilum.