Polypolar spindle formation during first cell cycle in rainbow trout <Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis> embryos after heat-shock treatment

ABSTRACT:   Heat shock has been used to inhibit cleavage for the induction of monogenic diploids or tetraploids in animals, but usually the success rate is low. Heat-shocked rainbow trout Oncorhynchus mykiss embryos were used in this histological study to clarify the causes of this low success rate. Embryos treated with hydrostatic pressure were used for comparison. After heat shock had disorganized the spindles, polypolar (tripolar or tetrapolar) spindles in addition to bipolar spindles were often reassembled soon after treatment. The embryos then completed tripolar or tetrapolar division at the first mitosis, and directly turned into three- or four-cell embryos as a result of the first cleavage. During the second mitosis, a monopolar spindle was formed in each blastomere of four-cell embryos and approximately 60% of three-cell embryos. In the remaining three-cell embryos, two of the three blastomeres formed a monopolar spindle, and the third one formed a bipolar spindle. The formation of polypoles is assumed to be caused by insufficient disorganization of daughter centrioles and splitting from the mother centriole by heat shock. Polypolar division is considered to be the cause of aneuploidy and the low success rate of chromosome set doubling. In the case of hydrostatic pressure treatment, the regenerated spindles were bipolar in almost all embryos.     

Effects of organic acids on growth and phosphorus utilization in rainbow trout <Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis>

ABSTRACT:   An experiment was conducted to investigate the effect of various organic acid (OA) supplementation on phosphorus (P) and nitrogen (N) retention by rainbow trout fed low fishmeal-based diets. Six experimental diets were formulated, and diet 0.5P was arranged as a positive control diet with 0.5% calcium phosphate and 0P as a negative control without additional P. Diets CA and LA were supplemented with 1% citric (CA), and lactic (LA) acids, respectively, and diets MHA and LTE were supplemented with 1% methionine hydroxy analog (MHA) and 1% liquid trace elements (LTE), respectively. Fish fed CA and LTE showed similar growth to that of the positive control diet. P retention was lowest in the 0.5P group and was elevated with CA and LTE diets. Therefore, in this study it is suggested that it might not be necessary to supplement the low fishmeal-based diet of rainbow trout with P if certain organic acids such as CA are added.     

Thermal tolerance of a rainbow trout <Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis> strain selected by high-temperature breeding

ABSTRACT:   Thermal tolerance was studied in a rainbow trout strain successively selected through high temperature breeding at 20–27°C since 1966 in Miyazaki Prefecture, Japan. The hatching rate and fry mortality at high temperatures were examined in the selected strain along with normal strains cultured at water temperature of 9–17°C. The hatching rate of embryos fertilized at either 10 or 14°C and subsequently subjected to high temperatures in the blastula or neurula stage of the selected strain, was marginally higher than that of the normal strain counterparts. The upper 50% lethal temperatures (LT₅₀) for embryos in the early segmentation, blastula and neurula stages of the selected strain were also higher than those of the normal strain counterparts. Death temperatures and LT₅₀ of fries acclimated to 20°C of the selected strain were significantly higher than those of the normal strains. However, no difference in the critical thermal maximum was detected between the different strains. These results suggest that the selected strain of rainbow trout established by selecting successively for many generations at high temperatures acquired a degree of thermal tolerance.     

Effect of light intensity on self-feeding of rainbow trout <Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis> reared individually

The self-feeding abilities of individually reared rainbow trout Oncorhynchus mykiss were investigated under four different light intensities (7×10⁻², 1×10°, 5×10¹, and 7×10² lx). There was no significant effect of light intensity on self-feeder learning ability and food wastage (which did not exceed 0.5%). However, there was high individual variability in the ability irrespective of light intensity. The total number of trigger actuations was significantly higher at 1×10° lx than at 7×10⁻² and 5×10¹ lx. Feeding patterns during the light phase of a day were classified as early or late in relation to the timing of significant peaks in fish feeding, or non-steady. The last pattern was defined when significant peaks were not observed. Under 7×10⁻², 1×10°, or 7×10² lx, a non-steady feeding pattern was the most common. Early feeding appeared only under the 1×10° lx regime with the random pattern, while the late pattern was observed under 7×10⁻², 5×10¹, and 7×10² lx. Under 5×10¹ lx regime half of the fish fed in the afternoon. These results suggest that light intensity can affect the timing of feeding judged from the frequency of pulling bite tags, but does not affect waste or learning period.     

Effects of slaughter methods on physical,biochemical and microbiological quality of rainbow trout <Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis> and mirror carp <Emphasis Type="Italic">Cyprinus carpio</Emphasis> filleted in pre-, in- or post-rigor periods

Rainbow trout Oncorhynchus mykiss and mirror carp Cyprinus carpio were slaughtered by either percussion or asphyxiation. For determination of the physical (texture value) and biochemical quality attributes [pH, lactic acid, total volatile basic nitrogen (TVB-N), and malondialdehyde (MDA) values] of trout and carp, the fish were filleted immediately (within 2 h), 12 h after harvest (pre-rigor), 24 and 36 h after harvest (in-rigor), and finally 48 and 60 h after harvest (post-rigor). For microbiological quality attribute (mesophilic and psychrophilic bacteria count) analyses, the fish were filleted immediately (within 2 h) after harvest (pre-rigor), 24 h after harvest (in-rigor) and 48 and 72 h after harvest (post-rigor). By percussion slaughtering followed by pre-rigor filleting, it was possible to process rainbow trout and mirror carp without inflicting excessive handling stress. Percussion slaughter delayed onset of rigor and the percussion-slaughtered fish exhibited a long pre-rigor period (≈24 h), giving plenty time for pre-rigor processing. Percussion slaughtering and pre-rigor filleting of fish was considered to be superior to the traditional asphyxiation slaughtering with respect to texture, TVB-N and MDA attributes.     

Effect of different permeable and non-permeable cryoprotectants on the hatching rate of rainbow trout (<Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis>) embryos

The purpose of the present study was to investigate the toxicity of cryoprotectants on the hatching rate of rainbow trout (Oncorhynchus mykiss) embryos. Epiboly and first eye pigmentation stage embryos were immersed in six permeable cryoprotectants, dimethyl sulfoxide (DMSO), glycerol (Gly), methanol (MeOH), propylene glycol (PG), ethylene glycol (EG), and acetamide (Ac), in concentrations of 1–5 M for 5 or 10 min and two non-permeable cryoprotectants, sucrose (Suc) (10%, 15%, 20%) and polyvinyl pyrrolidone (PVP) (5%, 10%, 15%) for 5 min. The embryos were then washed and incubated until hatching occurred. The toxicity of the cryoprotectant was assessed by the hatching rate. The results illustrated that permeable cryoprotectant toxicity for rainbow trout embryos increased in the order of PG < DMSO < MeOH < Gly < EG < Ac. The hatching rate of the embryos treated with permeable cryoprotectants decreased (P < 0.05) with increased concentration and duration of exposure. There were no significant decreases in hatching rate of embryos treated with sucrose and PVP with the increase of concentration; sucrose had higher hatching rates than PVP. Rainbow trout embryos at first eye pigmentation stage exhibited greater tolerance to cryoprotectants than embryos at epiboly stage.     

Comparison of fatty acid contents of wild and cultured rainbow trout <Emphasis Type="Italic">Onchorhynchus mykiss</Emphasis> in Turkey

Abstract:   The total lipid content and fatty acid composition were determined in the flesh and skin of wild and cultured rainbow trout in Turkey. The effect of diet content was also investigated on cultured trout. Gas chromatography-mass spectrometry (GC-MS) was used for fatty acid analyses. Total lipid content of skin was higher than flesh in both types and when compared appreciably higher in cultured fish. The predominant fatty acid was palmitic acid (C16:0) in saturated fatty acids and oleic acid (C18:1n-9) in monounsaturated fatty acids. The amount of eicosapentaenoic acid was double in wild and docosahexaenoic acid (DHA) 1.5 times higher in cultured fish flesh. The n-3/n-6 ratio was higher in cultured fish than wild fish. The levels of palmitic, oleic, linoleic (C18:2n-6) and palmitoleic (C16:1n-7) acids were high in skin. The level of EPA was the same in skin of wild fish but 5.5 times higher in cultured fish, whereas the proportion of DHA in skin was lower for wild and 3.5 times higher in cultured fish. Wild fish had a high level of linoleic, arachidonic (C20:4n-6) and linolenic (C18:3n-3) acids. The total amount of n-3 and n-6 polyunsaturated fatty acids was higher in flesh of wild fish than cultured fish, contrary to skin of cultured fish. The data obtained demonstrated that fatty acid composition of cultured fish did not depend on that of feed.     

Motility Parameters of Rainbow Trout (<Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis>) Spermatozoa in Relation to Sequential Collection of Milt,Time of <Emphasis Type="Italic">Post-mortem</Emphasis> Storage and Anesthesia

The present study investigated the effects of sequential collection of milt, time of post-mortem storage and anesthesia on rainbow trout (Oncorhynchus mykiss) sperm motility parameters (using computer-assisted sperm analysis – CASA) as well as seminal plasma osmolality and sperm concentration. The post-mortem storage and time of anesthesia altered motility characteristics of rainbow trout sperm to different extents. The moderate impact of time of anesthesia was manifested in a shortened duration of sperm motility after 10 min exposure of fish to anesthetic. The prolonged post-mortem storage (≥40–60 min), in addition to lowering sperm motility duration, also significantly influenced sperm motility parameters, such as sperm velocities, percentage of motile sperm and sperm trajectory parameters. These results clearly demonstrate that when milt from sacrificed fish is used for sperm motility studies, the time of post-mortem storage significantly alters sperm motility characteristics. Since sperm motility rate and swimming velocity could predict fertilizing ability, detrimental effects of prolonged post-mortem storage may lead to reduced fertilization success. Sperm concentration and seminal plasma osmolality were lower in the first fractions and increased with successive collections of milt. It suggests the presence of urine contamination of the first milt fractions which were collected by stripping. Therefore, testing of sperm concentration and/or seminal plasma osmolality should be mandatory while handling stored milt.     

Probiotic bacteria <Emphasis Type="Italic">Lactobacillus rhamnosus</Emphasis> influences the blood profile in rainbow trout <Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis> (Walbaum)

This paper reports the effect of feeding probiotic diets on blood profiles in rainbow trout. Two experiments were performed: in the first, fish of average weight 75 g were offered either a commercial feed or the same incorporated with 10⁹ CFU g⁻¹ of lactic acid bacteria Lactobacillus rhamnosus for 30 days; in the second study performed for a similar duration, fish of average weight 126 g were offered formulated diets that either contained the same bacteria in heat-killed or freeze-dried form (nearly 10¹¹ CFU g⁻¹), or the basal diet without the bacteria. Blood samples were collected at different times after commencement of probiotic feeding to determine the total cholesterol, triglyceride contents, the plasma alkaline phosphatase activity, plasma protein and hematocrit value. The plasma cholesterol significantly increased upon probiotic feeding in the first experiment. A significant elevation (P < 0.05) of plasma cholesterol and triglyceride and alkaline phosphatase activity level was found in the freeze-dried probiotic fed groups at 20 and 30 days postfeeding. This was concomitant with the increased plasma protein and hematocrit values in FD group at 20 and 30 days. Likewise, the heat-killed probiotic fed group registered significantly high values of triglycerides, alkaline phosphatase activity, and plasma protein compared to the control diet fed groups after 20 days of feeding. Thus, alterations in the blood profiles could serve as supplementary information when examining the benefits of probiotics for fish.     

Adverse and beneficial effects of long-term high-concentration ascorbic acid supplementation in rainbow trout <Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis>

The present study examined the effects of high-concentration ascorbic acid (AsA) supplementation over a long period on growth, plasma components, nonspecific immune responses, and thermal tolerance in rainbow trout Oncorhynchus mykiss. Four commercial diets supplemented with 0, 100, 1000, and 5000 mg AsA per kg of diet (designated AsA0, AsA100, AsA1000, and AsA5000, respectively) were fed to rainbow trout (initial weight 1.85 g) for 100 days. AsA contents in liver increased with increasing dietary AsA levels. Feeding period, growth performance, and plasma components did not differ significantly between the AsA groups. On the other hand, the phagocytic assay [nitro-blue tetrazolium (NBT) assay] was significantly higher in fish fed AsA1000 and AsA5000 than those fed a diet without AsA (AsA0) at the end of feeding trial. Superoxide dismutase (SOD) activity in the intestines of fish fed AsA5000 was also significantly higher than that of fish fed AsA0. In the thermal tolerance test, fish fed AsA5000 only showed significantly lower cumulative mortality compared with the AsA0 group. In conclusion, high-concentration AsA supplementation such as AsA5000 over a long period does not induce any adverse effects and can enhance disease and stress resistance in rainbow trout.     

Molecular characterization of PRR13 and its tissue-specific expression in rainbow trout (<Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis>)

The proline-rich protein 13 (PRR13) is reported to be a key regulator of the resistance to cytostatica by decreasing the copy number of the proapoptotic gene thrombospondin-1. We isolated and characterized the complete PRR13 gene sequence of rainbow trout (Oncorhynchus mykiss). The gene comprises four exons and three introns, the latter of comparatively short lengths (100–811 bp). The full-length PRR13 cDNA consists of 1,101 nucleotides, including an open reading frame of 563 bp, which is predicted to encode a 187 amino acid protein with a molecular mass of 18.8 kDa. A continuous stretch of ten serine residues at the C-terminus is highly conserved and characteristic for vertebrate PRR13, but not for other known proline-rich proteins. Phylogenetic analyses suggest a clear separation of teleostean PRR13 proteins and those from mammalian and reptilian species. Comparison of the tissue-specific PRR13 mRNA abundance in two strains of the rainbow trout coastal form (TCO Steelhead II-WA vs. BORN Steelhead II-Germany) revealed an increased expression in the BORN trout in nearly all examined tissues. The major expression differences were detected in gill (2.29-fold) and in liver tissue (2.16-fold). Hence, the increased PRR13 expression in BORN trout might cause improved protection from natural cytostatica and therefore support our assumption that PRR13 is a candidate gene possibly involved in the varying ability of the two rainbow trout strains to handle environmental stress under local conditions of the Southern Baltic.     

Impact of microwave-assisted enzymatic hydrolysis on functional and antioxidant properties of rainbow trout <Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis> by-products

Fishery by-products can be better utilized following enzymatic hydrolysis treatment to produce fish protein hydrolysates (FPH) with potentially enhanced interface-stabilizing properties (e.g. functionality). The production of FPH could be accelerated through the application of rapid heating methods [e.g. microwave-assisted heating (MW)] rather than slower conventional heating (CH) treatments. The objective of this study was to investigate the effects of microwave heating during enzymatic hydrolysis on the functionality and antioxidant properties of FPH. Trout by-products were hydrolyzed with Alcalase at an enzyme substrate ratio (E:S) of 0.5, 1.7, and 3.0% (w/v), respectively, for 3, 5 and 15 min using a microwave system (1200 W, 20% power with 50% duty cycle at 50–55 °C) and a conventional heating method (water bath at 50 °C). The degree of hydrolysis and protein solubility was higher (P < 0.05) for the MW-FPH than for the CH-FPH. MW-FPH at 5 min (0.5% E:S) demonstrated higher (P < 0.05) emulsifying activity and emulsion stability than CH-FPH with the same treatment. Foam capacity and stability were also greater (P < 0.05) for MW-FPH samples that were treated 15 min by microwave-assisted heating (0.5% E:S) when compared to CH. Overall, MW-FPH exhibited higher (P < 0.05) 2,2-diphenyl-1-picryhydrazyl and ferric ion reducing capacity than CH-FPH. We therefore conclude that microwave-assisted hydrolysis is an alternative method to produce FPH with improved solubility, emulsifying activity, foaming properties and antioxidant activity.     

Effects of chronic high stocking density on liver proteome of rainbow trout (<Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis>)

The main aim of the present study was to assess the effects of chronic high stocking density on liver proteome of rainbow trout. Rainbow trout juveniles (42.6 ± 2.3 g average body weight) were randomly distributed into six tanks at two stocking densities (low stocking density (LD) = 20 kg m^?3 and high stocking density (HD) = 80 kg m^?3). Both treatments were performed in triplicate tanks for a period of 60 days. High stocking density caused a reduction in the growth performance compared with LD fish. Lysozyme activity increased with stocking density, while serum complement activity presented the opposite pattern. Serum cortisol and total protein levels did not show significant differences (P > 0.05) between experimental groups. The fish reared at high stocking density showed significantly lower osmolality and globulin values but higher albumin level. The HD group had significantly higher activities of catalase, glutathione peroxidase and superoxide dismutase, and malondialdehyde content in the liver when compared to the LD group. Comparative proteomics was used to determine the proteomic responses in livers of rainbow trout reared at high stocking density for 60 days. Out of nine protein spots showing altered abundance (>1.5-folds, P < 0.05), eight spots were successfully identified. Two proteins including apolipoprotein A-I-2 precursor and mitochondrial stress-70 protein were found to increase in HD group. The spots found to decrease in the HD group were identified as follows: 2-peptidylprolyl isomerase A, two isoforms of glyceraldehydes-3-phosphate dehydrogenase, an unnamed protein product similar to fructose-bisphosphate aldolase, 78 kDa glucose-regulated protein, and serum albumin 1 protein.     

Medicinal plant extracts modulate respiratory burst and proliferation activity of rainbow trout (<Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis>) leukocytes

The present study was designed to investigate the immunomodulatory effects of Aloe vera, Curcuma longa, Echinacea purpurea, Lavandula officinalis, Origanum vulgare, Panax ginseng, and Rheum officinale extracts on leukocytes purified from rainbow trout (Oncorhynchus mykiss) head kidney. The cells were cultured in a medium containing increasing doses of extracts; afterwards, they were tested for reactive oxygen species production after stimulation with phorbol myristate acetate (PMA) and proliferation in the presence or absence of phytohemagglutinin from Phaseolus vulgaris (PHA-P). After a 2-h exposure, the extracts of L. officinalis, O. vulgare, and R. officinale strongly reduced the oxidative burst activity of PMA-stimulated leukocytes, in a dose-dependent manner (P ≤ 0.05). A. vera, C. longa, E. purpurea, and P. ginseng extracts reduced this response with lower efficacy and especially at lower concentrations. On the contrary, the highest concentration of ginseng extract stimulated the respiratory burst of leukocytes compared to untreated control cells. After a 72-h exposure, the extracts of L. officinalis, R. officinale, C. longa, E. purpurea, and P. ginseng had a clear dose-dependent stimulatory effect on leukocyte proliferation (P ≤ 0.05). The results suggest that these medicinal plants can be considered as reliable sources of new antioxidants or immunostimulants to be used in aquaculture.     

Effects of long-term silymarin oral supplementation on the blood biochemical profile of rainbow trout (<Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis>)

Silymarin, an extract from “milk thistle” (Silybum marianum) plant is traditionally used as herbal medicine. The present study was conducted to investigate the clinical effects and possible side effects of silymarin on biochemical blood parameters of rainbow trout (Oncorhynchus mykiss). Fishes were treated with 0 (control), 100, 400, and 800 mg of silymarin per kg of food during 4 weeks. Plasma alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), alkaline phosphatase (ALP), creatine kinase (CK), glucose, total protein, creatinine, triglyceride, cholesterol, urea, uric acid and liver cellular total antioxidant, and protein content were measured after 7, 14, and 28 days of silymarin treatment. The results showed that oral administration of silymarin in fish significantly reduced plasma glucose and cholesterol levels and relatively increased plasma total protein and globulin concentrations (P < 0.05). Increasing plasma albumin levels indicate the important role of albumin in drug transportation in circulatory system of fish. Silymarin also stabilized cellular membrane structure and regulated the levels of AST, ALT, ALP, CK, and LDH activity. In conclusion, on the basis of these results, oral administration of silymarin up to 400 mg per 1 kg of food has no side effect on blood biochemical and clinical parameters of fishes. However, oral administration of 800 mg/kg- of silymarin caused cytotoxicity and modifications in blood biochemical parameters of fish.     

Stress response of juvenile rainbow trout (<Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis>)to chemical cues released from stressed conspecifics

The objective of this study was to determine whether exposure of rainbow trout (Oncorhynchus mykiss) to water containing a stressed trout or skin extract from stressed and non-stressed trout would elicit a stress response in conspecifics. Juvenile rainbow trout were exposed for 1 hour to water containing a stressed fish, homogenized skin extracts from a non-stressed fish, skin extract from a stressed fish and water with none of these factors. The stress response was measured over a 24-h period (1, 6, 12, 24 h after exposure). Plasma cortisol levels increased at 12 h in fish exposed to water from a stressed fish and skin extract from a stressed fish. Plasma glucose and hepatic hsp70 levels were not affected by treatments. The results suggest that rainbow trout elicit a stress response when exposed to stress-related alarm cues released from conspecifics.     

Availability of genetically modified feed ingredient: investigations of ingested foreign DNA in rainbow trout <Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis>

ABSTRACT:   Foreign DNA fragments from genetically modified defatted soybean meal (GM SBM) in rainbow trout was traced by nested polymerase chain reaction (PCR) and located by in situ hybridization. Either a GM or non-GM SBM formulated diet (42% protein) was fed to fish (average weight 50.5 g) for 2 weeks. The degradation results showed that the cauliflower mosaic virus 35S promoter (220 bp) fragment was detected in the contents of digestive system only in fish fed the GM SBM diet, and it was not detected on the third day after changing the diet to the non-GM SBM diet. For the possible transferal results, the promoter fragment was detected in the leukocyte, head kidney and muscle only of fish fed the GM SBM diet; it was not detected on the fifth day after changing the diet to the non-GM SBM diet. These results suggest that a foreign DNA fragment was not completely degraded and might be taken up into organs through the gastrointestinal tract. However, foreign DNA was not detected after the withdrawal period. Thus, the data show that uptake of DNA from GM SBM might not remain in the tissues of fish fed GM SBM diet.