Rainbow Trout Eggs Research Articles

Chilled storage of unfertilized and fertilized rainbow trout (Oncorhynchus mykiss) eggs in sealed polyethylene bags at different temperatures

Three experiments were conducted to study the effect of chilling temperature on unfertilized and fertilized rainbow trout eggs sealed in polyethylene (PE) bags. Experiment 1 showed that after 18days of storage at 2°C fertilization capacity of eggs was undiminished. At 7°C, unimpaired fertilization capacity was maintained for 12days and at 10°C for 6days. This leads to the conclusion that for storage up to approximately 1week an ordinary household refrigerator will suffice; for more extended storage periods lower temperatures are advised. If PE bags filled with unfertilized eggs were submerged in water during chilling, similar results were obtained, though at a slightly lower level. The second experiment showed that fluctuations in chilling temperature between 2°C and 10°C were tolerated by unfertilized eggs as long as sufficient adaptation time was granted. One or two temperature changes within a 12day period had no impact on fertilization capacity, whereas repeated changes at 2day intervals caused a reduction in fertilization capacity. The attempt to apply the method of chilled storage of unfertilized rainbow trout eggs in PE bags to fertilized eggs (Experiment 3) indicated that early eyed eggs (14days incubation at 10°C) may be stored at 2°C for 10days without loss in hatching rate. Reasonable hatching rates may be achieved at 7°C for about one week and at 10°C for about 4days. Storage of fertilized eggs of a more advanced stage of development (21days incubation at 10°C) calls for low temperature (2°C) and storage periods of no >4days.

Bisphenol A in eggs causes development-specific liver molecular reprogramming in two generations of rainbow trout

Bisphenol A (BPA) is widely used in the manufacture of plastics and epoxy resins and is prevalent in the aquatic environment. BPA disrupts endocrine pathways in fish, but the long-term developmental implications are unknown. We demonstrate that BPA deposition in the eggs of rainbow trout (Oncorhynchus mykiss), an ecologically and economically important species of fish, reprograms liver metabolism in the offspring and alters the developmental growth trajectory in two generations. Specifically, BPA reduces growth during early development, followed by a catch-up growth post-juveniles. More importantly, we observed a developmental shift in the liver transcriptome, including an increased propensity for protein breakdown during early life stages to lipid and cholesterol synthesis post- juveniles. The liver molecular responses corresponded with the transient growth phenotypes observed in the F1 generation, and this was also evident in the F2 generation. Altogether, maternal and/or ancestral embryonic exposure to BPA affects liver metabolism leading to development-distinct effects on growth, underscoring the need for novel risk assessment strategies for this chemical in the aquatic environment. This is particularly applicable to migratory species, such as salmon, where distinct temporal changes in growth and physiology during development are critical for their spawning success.

HPLC Quantification of astaxanthin and canthaxanthin in Salmonidae eggs.

Astaxanthin and canthaxanthin are naturally occurring antioxidants referred to as xanthophylls. They are used as food additives in fish farms to improve the organoleptic qualities of salmonid products and to prevent reproductive diseases. This study reports the development and single-laboratory validation of a rapid method for quantification of astaxanthin and canthaxanthin in eggs of rainbow trout (Oncorhynchus mykiss) and brook trout (Salvelinus fontinalis М.). An advantage of the proposed method is the perfect combination of selective extraction of the xanthophylls and analysis of the extract by high-performance liquid chromatography and photodiode array detection. The method validation was carried out in terms of linearity, accuracy, precision, recovery and limits of detection and quantification. The method was applied for simultaneous quantification of the two xanthophylls in eggs of rainbow trout and brook trout after their selective extraction. The results show that astaxanthin accumulations in salmonid fish eggs are larger than those of canthaxanthin. As the levels of these two xanthophylls affect fish fertility, this method can be used to improve the nutritional quality and to minimize the occurrence of the M74 syndrome in fish populations.

Assessment of genetic differentiation and genetic assignment of commercial rainbow trout strains using a SNP panel

Rainbow trout (Oncorhynchus mykiss) is the most widely cultured cold freshwater fish in the world. Troutlodge, Inc., one of the largest commercial rainbow trout egg producers in the world, has eight breeding populations of rainbow trout. Assessment of population genetic differentiation is critical for selective breeding, and genetic assignment is often required to address production issues arising on fish farms. Previously, we developed a SNP (single nucleotide polymorphism) panel for parentage assignment in rainbow trout. The objectives of this study were: (1) to characterize the genetic differentiation of the eight Troutlodge breeding populations; and (2) to evaluate the accuracy of genetic assignment to identify Troutlodge fish using the same SNP panel previously developed for parentage assignment. A total of 1732 breeders of the eight Troutlodge breeding populations were genotyped with the SNP panel. The global FST over all SNPs was 0.13 and the pairwise FST between any two breeding populations ranged from 0.056 to 0.195. Both phylogenetic tree and structure analyses revealed that the odd year and even year populations for the same strain were closely related to each other. Also, the eight populations were clustered into two groups. Based on the results of self-assignment, 97.1% of the Troutlodge breeders were correctly assigned to the population of origin. To further evaluate the accuracy of genetic assignment, we also genotyped 280 egg production fish from three Troutlodge populations of known origin, 49 fish from a Canadian farm, 70 fish from a farm in Idaho and 188 fish from four known non-Troutlodge strains. Among the 280 production fish, 98.2% fish were correctly assigned to the Troutlodge strain of origin. Consistent with the purchasing records, the fish from the two farms were also correctly assigned to two Troutlodge strains. Based on the SNP genotypes, 185 out of 188 fish (98.4%) from the four non-Troutlodge strains could be excluded as Troutlodge fish. In conclusion, our previously developed SNP panel for parentage assignment is also useful to characterize genetic differentiation and has sufficient power for genetic assignment of commercial rainbow trout strains used in this study. Statement of relevanceIdentification of fish strains using a SNP panel.

Identification of Mitochondrial Genome-Encoded Small RNAs Related to Egg Deterioration Caused by Postovulatory Aging in Rainbow Trout.

Many factors have been reported to affect rainbow trout egg quality, among which, postovulatory aging is one of the most significant causes as reared rainbow trout do not usually volitionally oviposit the ovulated eggs. In order to uncover the genetic regulation underling egg deterioration caused by postovulatory aging in rainbow trout, mitochondrial genome-encoded small RNA (mitosRNAs) were analyzed from unfertilized eggs on Days 1, 7, and 14 postovulation with fertilization rates of 91.8, 73.4, and less than 50%, respectively. A total of 248 mitosRNAs were identified from Illumina high-throughput sequencing of the small RNA libraries derived from the eggs of ten females. Ninety-eight of the small RNAs exhibited more than a threefold difference in expression between eggs from females exhibiting high fertilization rates at Day 1 and low fertilization rates at Day 14. The differentially expressed mitosRNAs were predominantly derived from mitochondrial D-loop, tRNA, rRNA, COII, and Cytb gene regions. Real-time quantitative PCR analysis was carried out for 14 differentially expressed mitosRNAs, of which, 12 were confirmed to be consistent with the sequencing reads. Further characterization of the differentially expressed mitosRNAs may lead to the development of new biomarkers for egg quality in rainbow trout.

Persistent Organohalogens in Paired Fish Fillet and Eggs: Implications for Fish Consumption Advisories.

Fish consumption is associated with both health benefits from high-quality proteins, minerals, vitamins, and fatty acids and risks from contaminants in fish. Fish consumption advisories are issued by many government agencies to keep exposure to contaminants at a safe level. Such advisories are typically based on fillets and neglect consumption of other fish parts such as eggs by certain subpopulations. To evaluate potential for dietary exposure to toxic organic chemicals via fish eggs, we analyzed polybrominated diphenyl ethers (PBDEs), polychlorinated naphthalenes (PCNs), dioxin-like polychlorinated biphenyls (dlPCBs), and polychlorinated dibenzodioxins/furans (PCDD/Fs) in paired fillet and eggs of fish from a tributary to Lake Ontario, one of the North American Great Lakes. All wet weight based concentrations in fish eggs were statistically higher than in the paired fillet samples. In fish eggs, concentrations of Σ14PBDEs, Σ14PCNs, and Σ12dlPCBs were 41-118, 0.3-1.7, and 30-128 ng/g wet weight (ww), respectively; Σ3PCDD/Fs and total (dlPCB+ PCDD/Fs) toxic equivalents (TEQs) were 4-22 and 9-54 pg/g ww, respectively. In fillet samples, Σ14PBDEs, Σ14PCNs, and Σ12dlPCBs were 4-116, 0.05-0.66, and 6-85 ng/g, respectively; Σ3PCDD/Fs and TEQs were 2-10 and 3.4-31 pg/g ww, respectively. In contrast, the fillets had higher lipid normalized concentrations than the paired egg samples, suggesting that these chemicals did not reach equilibrium between the fillets and eggs. Accordingly, measured concentrations in eggs or empirical relationship with fillet rather than prediction from equilibrium partitioning model should be used to evaluate contaminant exposure via consumption of fish eggs. For fatty fish from the lower Great Lakes area, we suggest one fillet meal be reduced from the advised fish consumption frequency for consumptions of 207 ± 37, 39 ± 2, 105 ± 51, and 119 ± 9 g fish eggs of brown trout, Chinook salmon, Coho salmon, and rainbow trout, respectively.

Production of germ cell‐deficient salmonids by dead end gene knockdown, and their use as recipients for germ cell transplantation

We previously established a spermatogonial transplantation model in fish using triploid recipients. Although triploid salmonids are sterile, they carry a limited number of immature triploid germ cells that potentially compete with the donor-derived germ cells for their niche. We therefore assessed the biological characteristics of germ cell-deficient gonads in rainbow trout for their suitability as recipients for germ cell transplantation in this study. Antisense morpholino oligonucleotides against the dead end gene were microinjected into the fertilized eggs of rainbow trout to eliminate endogenous germ cells, leaving only their supporting cells. Unlike similar approaches performed in zebrafish and medaka, these germ cell-deficient rainbow trout did not show a male-biased sex ratio. Approximately 30,000 spermatogonia were then transplanted into the body cavities of both germ cell-deficient and control recipients. The donor-derived germ cells showed significantly higher proliferation in the gonads of germ cell-deficient recipients than those in the gonads of the control recipients. Finally, the applicability of the germ cell-deficient recipients for xenogeneic transplantation was evaluated by transplanting rainbow trout spermatogonia into germ cell-deficient masu salmon recipients. The resulting recipient salmon matured normally and produced trout gametes, and early survival of the resulting trout offspring was as high as that of the control offspring. Thus, dead end-knockdown salmonids appear to be ideal recipients for the intraperitoneal transplantation of spermatogonia.

Viability and DNA fragmentation of rainbow trout embryos (Oncorhynchus mykiss) obtained from eggs stored at 4 °C

In vitro storage of salmonid eggs leads to aging of the cells causing a decline in quality and reducing their capacity to develop and produce embryos. The quality of salmonid embryos is assessed by morphologic analyses; however, data on the application of biomarkers to determine the cell viability and DNA integrity of embryos in these species are limited. The aim of this study was to evaluate the effect on embryo development, viability and DNA fragmentation in the embryonic cells of in vitro storage time at 4 °C of rainbow trout (Oncorhynchus mykiss) eggs. The embryos were obtained by IVF from eggs stored for 0 (control), 48, and 96 hours at 4 °C. At 72 hours after fertilization, dechorionated embryos were examined to determine percentages of developed embryos (embryos with normal cell division morphology), viability (LIVE/DEAD sperm viability kit), and DNA integrity (terminal deoxynucleotidyl transferase [TdT] dUTP nick-end labeling assay). The percentage of developing embryos decreased (P < 0.05) with storage time of the eggs (95.10 ± 2.55; 88.14 ± 4.50; 79.99 ± 6.60 for 0, 48, and 96 hours, respectively). Similarly, cell viability decreased (P < 0.05; 96.07 ± 7.15; 80.42 ± 8.55; 77.47 ± 7.88 for 0, 48, and 96 hours, respectively), and an increase (P < 0.05) in DNA fragmentation in the embryos was observed at 96-hour storage. A positive correlation was found between cell DNA fragmentation and storage time (r = 0.8173; P < 0.0001). The results revealed that terminal deoxynucleotidyl transferase [TdT] dUTP nick-end labeling assay technique is reliable mean to assess the state of the DNA in salmonid embryos and that in vitro eggs storage for 96h reduces embryo development and cell DNA integrity. DNA integrity evaluation constitutes a biomarker of the quality of the ova and resulting embryos so as to predict their capacity to produce good-quality embryos in salmonids, particularly under culture conditions.

Egg Cortisol Exposure Enhances Fearfulness in Larvae and Juvenile Rainbow Trout

AbstractWe investigated the effects of an early boost of cortisol exposure in rainbow trout (Oncorhynchus mykiss) eggs during fertilisation on subsequent behavioural responses when exposed to a sudden stimulus in larvae and juveniles. At 55 d post‐fertilisation (dpf), treatment had no effect on high accelerations occurring after a sudden event. At 146 dpf, these high accelerations were more frequent in cortisol‐treated fish than in controls. At 146 dpf also, swimming activity was increased in cortisol‐treated fish both before and after the sudden stimulus. This study underlines the important behavioural modifications in both larvae and juveniles, linked to a change in the surrounding environment of the embryo. Indeed, fish exposed to cortisol as eggs showed a higher level of fearfulness later in life. Our findings are of major interest for stress management in an aquaculture context and also allow for a better understanding of the long‐lasting effects of a permanent and/or acute stress – mediated by cortisol – that could be encountered by females, affecting population's life history trajectory.

Application of The UV-Irradiated Homologous and Heterologous Sperm for Activa Tion of the Rainbow Trout (Oncorhynchus Mykiss) Eggs and Production of the Gynogenetic Stocks

Abstract Meiotic gynogenetic development of the rainbow trout (Oncorhynchus mykiss, Walbaum 1792) was induced in the course of egg activation performed by the UV-irradiated homologous and heterologous (European grayling Thymallus thymallus Linnaeus, 1758) spermatozoa. To recover diploidy in the gynogenetic zygotes, activated eggs were subjected to the high pressure shock in order to inhibit extrusion of the second polar body. Gynogenetic rainbow trout progeny hatched from the eggs activated by the irradiated rainbow trout and grayling milt with similar hatching rates of 28.19% and 29.22%, respectively. However, gynogenetic rainbow trout produced with grayling semen had shown lower survival than gynogenotes provided with the homologous spermatozoa during two years of rearing. Viable hybrids are not produced between rainbow trout and grayling which ensured that fish obtained in this experiment were true gynogenetic progenies. A Robertsonian polymorphism characteristic for the rainbow trout from the studied strain was also observed among the gynogenetic specimens that exhibited diploid chromosome number ranging from 58 to 62 and stable chromosome arm number (FN= 104). No radiation-induced fragments of the paternal chromosomes were observed in the gynogenetic individuals. Fish produced in both experimental variants were genotypic (XX) and phenotypic (gonads) females. The results confirmed that the gynogenetic protocol used in the present research is an efficient means of producing all-female gynogenetic rainbow trout stocks.

Application of nanosilver-coated zeolite as water filter media for fungal disinfection of rainbow trout (Oncorhynchus mykiss) eggs

This study investigated the indirect use of silver nanoparticles (AgNPs) for reduction of fungal infections during incubation period of fertilized rainbow trout eggs. Different concentrations of nanosilver-coated zeolite (0.5, 1, and 1.5 % AgNPs) were compared with unmodified zeolite as water filter media in semi-recirculation systems. For testing the effect of AgNPs on reduction of fungal infection, fertilized eggs were transferred in incubators receiving water from filters coated with nanosilver. The eggs in each incubator were inoculated with Saprolegnia-infected trout eggs. Any dead or infected eggs and embryos were periodically removed, while the performance of the filters was assessed by calculating the survival rates from fertilization up to completion of the yolk–sac absorption stage. The results showed that the filters containing 0.5 % AgNPs increased the survival rate by 4.56 % from fertilization to the swim up stage compared to the control (p < 0.05). Also, the additional application of activated carbon (as absorbent media) along with AgNP-coated media in filters caused an increase of about 11.24 % in the survival rate for the larval stage (p < 0.05). In contrast to the control group with about 6 % fungal infection, no infections were observed during the incubation period in the incubators containing nanosilver-coated filters. Therefore, the final results confirmed that the indirect use of AgNPs in the aforementioned filters were significantly effective for preventing fungal infections in semi-recirculation systems for rainbow trout, making them a candidate for replacing the chemical fungicides currently used during egg incubation in hatchery systems.

MicroRNA expression profiles from eggs of different qualities associated with post-ovulatory ageing in rainbow trout (Oncorhynchus mykiss).

BackgroundEgg quality is an important aspect in rainbow trout farming. Post-ovulatory aging is one of the most important factors affecting egg quality. MicroRNAs (miRNAs) are the major regulators in various biological processes and their expression profiles could serve as reliable biomarkers for various pathological and physiological conditions. The objective of this study was to identify miRNAs that are associated with egg qualities in rainbow trout using post-ovulatory aged eggs.ResultsEgg samples from females on day 1, day 7, and day 14 post-ovulation (D1PO, D7PO and D14PO), which had the fertilization rates of 91.8%, 73.4% and less than 50%, respectively, were collected and small RNAs isolated from these samples were subjected to deep sequencing using the Illumina platform. The massive sequencing produced 27,342,477, 26,910,438 and 29,185,371 reads from the libraries of D1PO, D7PO and D14PO eggs, respectively. A three-way comparison of the miRNAs indicated that the egg samples shared 392 known and 236 novel miRNAs, and a total of 414, 481, and 470 known and 243, 298, and 296 novel miRNAs were identified from D1PO, D7PO and D14PO eggs, respectively. Four known miRNAs (omy-miR-193b-3p, omy-miR-203c-3p, omy-miR-499-5p and omy-miR-7550-3p) and two novel miRNAs (omy-miR-nov-95-5p and omy-miR-nov-112-5p) showed significantly higher expression in D1PO eggs relative to D14PO eggs as revealed by both deep sequencing and real time quantitative PCR analysis. GO analysis of the predicted target genes of these differentially expressed miRNAs revealed significantly enriched GO terms that are related to stress response, cell death, DNA damage, ATP generation, signal transduction and transcription regulation.ConclusionsResults indicate that post-ovulatory ageing affects miRNA expression profiles in rainbow trout eggs, which can in turn impact egg quality. Further characterization of the differentially expressed miRNAs and their target genes may provide valuable information on the role of these miRNAs in controlling egg quality, and ultimately lead to the development of biomarkers for prediction of egg quality in rainbow trout.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1400-0) contains supplementary material, which is available to authorized users.

The Impact of Egg Ozonation on Hatching Success, Larval Growth, and Survival of Atlantic Cod, Atlantic Salmon, and Rainbow Trout.

The direct exposure of fish eggs to ozonated water has generated interest as a means of ensuring pathogen-free eggs without the use of harsh chemicals. However, there are numerous knowledge gaps, including safe contact times, exposure levels, and potential long-term effects on aquaculture species in both freshwater and seawater. The effect of different ozone (O3) doses (0.5-1.0, 1.5-2.0, and 2.5-3.0 mg of O3/L for 90 s) on recently fertilized eggs of Atlantic Cod Gadus morhua and eyed eggs of Atlantic Salmon Salmo salar and Rainbow Trout Oncorhynchus mykiss was evaluated in comparison with the effects of two commercial disinfectants: Perosan (0.004 mg/L) and Ovadine (100 mg/L). The impact of ozone application was evaluated based on hatching success, larval nucleic acid concentration, larval growth, and survival. Overall, results indicated that ozonation of Atlantic Cod eggs at a dose less than 3.0 mg/L for 90 s produced no negative effect on the larvae up to 30 d posthatch. Furthermore, ozonation of Atlantic Salmon and Rainbow Trout eggs generated no negative effect on the larvae, based on monitoring until 85% yolk sac re-absorption (16 d posthatch).

English

The authors investigated the growth of hydromycobiota on the eggs of rainbow trout (Oncorhynchus mykiss) in waters of different trophicity. Of the O. mykiss eggs that were investigated, 16.6% were found to be infected by mycotal species. Thirty mycotal species were found on eggs obtained from adult female representatives. Achlya polyandra, A. radiosa, Aphanomyces laevis, Leptomitus lacteus, Saprolegnia ferax and Saprolegnia parasitica belonged to the species that were most commonly encountered. Aphanomyces frigidophilus, Candida albicans and Zoopage phanera were rarely found in salmonid fishes. Amino acid, carbohydrate and urease tests were used, and all analyses of species from the Achlya, Aphanomyces, Leptolegnia, Pythium and Saprolegnia genera showed that they assimilate glucose and starch. However, they did not assimilate glycine, leucine, lysine, ornithine, and arabinose. Urease was assimilated only by species from the Leptolegnia, Pythium and Saprolegnia genera. Key words: Oncorhynchus mykiss, rainbow trout, eggs, mycotal species, infection, hydrochemistry.

Kpna7 interacts with egg-specific nuclear factors in the rainbow trout (Oncorhynchus mykiss)

Nuclear proteins are required for the initiation of transcription in early embryos before embryonic genome activation. The regulated transport of nuclear proteins is mediated by factors known as importins (karyopherins). Kpna7, a newly discovered member of the importin α family, is critical for early development in mammals. In this study, we characterize rainbow trout Kpna7. The cDNA for rainbow trout Kpna7 encodes a 519 amino acid protein that contains a conserved importin β binding (IBB) domain and seven armadillo/beta-catenin-like repeat (ARM) motifs. Reverse-transcriptase PCR and Western blot analyses revealed that Kpna7 is specifically expressed in eggs/ovary. Real-time PCR analysis demonstrated that expression of Kpna7 mRNA is high in unfertilized eggs, gradually decreases in early-stage embryos until 3 days post-fertilization, and declines sharply thereafter, reaching a level that is barely detectable in 4-day-old embryos. Using a yeast two-hybrid screening system, we identified two Kpna7-interacting proteins from a rainbow trout egg cDNA library: Stl3 (rhamnose-binding lectin 3) and an uncharacterized protein. Both genes appear to be expressed specifically in eggs/testis. Co-immunoprecipitation assays confirmed the interaction between Kpna7 and Stl3, and co-transfection experiments using EGFP-tagged Stl3 showed that Kpna7 facilitates the nuclear transport of Stl3 through an interaction with the predicted nuclear-localization signal cluster at the carboxy-terminus of Stl3. Our data suggest that Kpna7 may function in early embryonic development as a unique nuclear transporter for egg-specific proteins.

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

Since formalin is widely used in prevention of Sapro- legnia infections in salmonid fish hatcheries, there is a need for more environmentally safe treatment methods. Therefore, we screened 360 bacterial iso- lates against their ability to antagonize the growth of Saprolegnia parasitica hyphae in vitro, and best strains were selected according to their antagonistic proper- ties and colonization capability on rainbow trout egg surface. Protective bacterial cultures of Pseudomonas sp. M162, Pseudomonas sp. M174 and Janthinobacte- rium M169 were tested for prevention of Saprolegnia sp. infections during incubation trials of rainbow trout (Oncorhynchus mykiss )e ggs with UV irradiated (400 mWs cm � 2 ) and non-treated inlet water. UV irradiation of inlet water significantly decreased mor- tality during the incubation. Lowest mortalities were observed in protective culture treated groups incu- bated with UV-irradiated inlet water. UV irradiation increased the dominance of the main bacterial colo- nizers and variation in the bacterial species diversity between the experimental units.

Whole Egg and Alevin Changes of Cortisol and Interrenal Tissue Differences in Rainbow TroutOncorhynchus mykissExposed to Different Stocking Densities During Early Development

Cortisol level changes in response to stocking density in the early stages of rainbow trout were measured. Eggs were exposed to low, normal, and high (2.55, 5.10 and 7.65 eggs cm(-2)) densities during the incubation period. Cortisol of maternal origin was found in pre-fertilized eggs (5.09 ± 0.12 ng g(-1)) of rainbow trout. In newly fertilized eggs, resting Cortisol levels (3.68 ± 0.14 ng g(-1)) decreased to 0.58 ± 0.08, 0.60 ± 0.12, and 0.57 ± 0.16 ng g(-1) at low, normal and high densities by day 10 (organo-genesis), respectively. Resting Cortisol levels remained constant until the eyed stage (day 18). Then, Cortisol showed an increase at hatching to 1.16 ± 0.11, 1.20 ± 0.12, and 1.21 ± 0.14 ng g(-1) at low, normal, and high densities, respectively. The pattern of change in Cortisol level was similar in all three densities. Interrenal cells were observed in 1-day old alevins at all three densities. Hematopoietic tissue, renal tubules and nucleated red blood cells were clarified through the head part of kidney. Higher numbers and larger interrenal cells were observed at high-density groups. Chronic density stress test conducted on embryonic stages of rainbow trout revealed no differences in Cortisol levels, but had an effect on the abundance and size of the interrenal cells. Densities were equaled after hatching (200 alevins per replicate) to investigate the different densities of eggs on stress indices in rainbow trout alevins. An acute stress (air exposure of eggs for five minutes) was applied in three treatments two weeks after hatching, and samples were taken at 0, 1, 3, 6, and 24 hps (hours post stress). Cortisol content increased under low density in 1 hps and reached from 5.21 ± 0.13 ng g(-1) to 6.01 ± 0.18 ng g(-1) (P < 0.05). Cortisol levels increased under normal density in 1 hps from 6.03 ± 0.28 ng g(-1) to 10.84 ± 0.18 ng g(-1) (P < 0.05). In high density also Cortisol increased from 6.83 ± 0.23 ng g(-1) to 8.86 ± 0.26 ng g(-1) (P < 0.05). At 3 hps;, Cortisol level was returned to basal level under low (P > 0.05) and normal (P > 0.05) densities, but significantly decreased (P < 0.05) under high density. Results revealed that the Cortisol biosynthesis was observed in rainbow trout between eyeing to the hatching stage. An increase in the density of eggs until 7.65 egg cm(-2) impaired Cortisol secretion and feedback system in alevins. However, more studies are needed to identify the exact time for Cortisol synthesis ability from eyeing to hatching in this species.

In vitroandin vivoantifungal activity ofSatureja cuneifoliaten essential oil onSaprolegnia parasiticastrains isolated from rainbow trout (Oncorhynchus mykiss, Walbaum) eggs

In the present study, the chemical composition and the antifungal properties against Saprolegnia parasitica (in vitro and in vivo) of the essential oils of thyme (Satureja cuneifolia) from Mediterranean region of Turkey were evaluated for the first time. The composition of oils was analysed using gas chromatography/mass spectrometry (GC/MS). The major constituents of oil of S. cuneifolia were cavracrol (46,84%) and cymene (16.90%). Antifungal effects of S. cuneifolia essential oil against S. parasitica strains (A1 and E1) were detected by disc diffusion and tube dilution assays. The antifungal effect of S. cuneifolia was determined to be stronger against S. parasitica E1 isolate (MIC 50 μL mL−1, MLC 250 μL mL−1) compared with S. parasitica A1 isolate (MIC 50 μL mL−1, MLC 500 μL mL−1). Following in vitro assays, effective doses of S. cuneifolia for disease control in rainbow trout eggs experimentally infected with S. parasitica were investigated. For this aim, infected eggs were treated with the essential oil (0, 5, 10, 20 and 50 ppm) during incubation period (21 days) after fertilization. Formalin (5 mL L−1) was used as positive control. Hatching rate of eggs at the end of incubation period were calculated. The highest hatching rates were recorded in S. parasitica E1 strain at 5 and 10 ppm concentrations of S. cuneifolia and in S. parasitica A1 strain at 10 and 20 ppm (P < 0.05).

Heritable Targeted Inactivation of the Rainbow Trout (Oncorhynchus mykiss) Master Sex-Determining Gene Using Zinc-Finger Nucleases

Gene targeting is a powerful tool for analyzing gene function. Recently, new technology for gene targeting using engineered zinc-finger nucleases (ZFNs) has been described in fish species. However, it has not yet been widely used for cold water and slow developing species, such as Salmonidae. Here, we present the results of successful ZFN-mediated disruption of the sex-determining gene sdY (sexually dimorphic on the Y chromosome) in rainbow trout (Oncorhynchus mykiss). Three pairs of ZFN mRNA targeted to different regions of the sdY gene were injected into fertilized rainbow trout eggs. Sperm from 1-year-old male founders (parental generation one or P1) carrying a ZFN-induced mutation in their germline were then used to produce F1 non-mosaic animals. In these F1 populations, we characterized 14 different mutations in the sdY gene, including one mutation leading to the deletion of leucine 43 (L43) and 13 mutations at other target sites that had different effects on the SdY protein, i.e., amino acid insertions, deletions, and frameshift mutations producing premature stop codons in the mRNA. The gonadal phenotype analysis of the F1-mutated animals revealed that the single L43 amino acid deletion did not lead to a male-to-female sex reversal, but all other mutations induced a clear ovarian phenotype. These results show that targeted gene disruption using ZFN is efficient in rainbow trout but depends on the ZFN design. We also characterized new sdY mutations resulting in male-to-female sex reversal, and we conclude that L43 seems dispensable for SdY function.

Toxicity of Copper Sulfate to Flavobacterium psychrophilum and Rainbow Trout Eggs

Tests were conducted to determine the concentrations of copper sulfate needed to kill Flavobacterium psychrophilum, the cause of bacterial coldwater disease, either in vitro or on Rainbow Trout Oncorhynchus mykiss eggs. For the in vitro test, a plastic strip dipped in a solution of F. psychrophilum was exposed for 15 min to copper sulfate solutions of 0, 1, 5, 10, 20, 35, 50, 75, or 100 mg/L. Bacteria were "too numerous to count" at concentrations ≤10 mg/L CuSO4; significant reductions in prevalence relative to untreated controls were noted for concentrations ≥35 mg/L. However, CFUs were still observed at 50 and 75 mg/L (20% of plates with tryptone yeast extract salts media). No yellow-pigmented CFUs typical of F. psychrophilum were observed at 100 mg/L CuSO4. For the in vivo test, eggs were exposed for 15 min to 100, 300, 500, and 700 mg/L CuSO4 or 100 mg/L iodine (control). Survival to hatch was significantly lower at 500 (44.3 ± 15.2%, mean ± SD) or 700 mg/L CuSO4 (1.7 ± 0.8%) than for controls treated with 100 mg/L iodine (93.6 ± 0.9%) or at copper sulfate concentrations ≤300 mg/L. The 15-min LD50 and LD10 for copper sulfate were 461 mg/L (95% confidence interval: 457-466 mg/L) and 259 mg/L (251-266 mg/L). The prevalence of yellow CFUs at 100 mg/L CuSO4 (40.0%) was significantly higher than in untreated controls. Significant reductions in yellow CFUs were achieved using 300, 500, or 700 mg/L CuSO4 (7.5, 2.5, or 0.0% of plates with CFUs, respectively) or 100 mg/L iodine (2.5%), relative to untreated control eggs. Overall, since the concentrations of copper sulfate required to eliminate F. psychrophilum were toxic to the eggs, copper sulfate is not recommended for coldwater disease control in Rainbow Trout eggs based on conditions and parameters in this study.