Exploring the effects of dietary inulin in rainbow trout fed a high‑starch,100%plant‑based diet

Background High dietary carbohydrates can spare protein in rainbow trout(Oncorhynchus mykiss)but may affect growth and health.Inulin,a prebiotic,could have nutritional and metabolic effects,along with anti-inflammatory properties in teleosts,improving growth and welfare.We tested this hypothesis in rainbow trout by feeding them a 100%plant-based diet,which is a viable alternative to fishmeal and fish oil in aquaculture feeds.In a two-factor design,we examined the impact of inulin(2%)as well as the variation in the carbohydrates(CHO)/plant protein ratio on rainbow trout.We assessed the influence of these factors on zootechnical parameters,plasma metabolites,gut microbiota,production of short-chain fatty acids and lactic acid,as well as the expression of free-fatty acid receptor genes in the mid-intestine,intermediary liver metabolism,and immune markers in a 12-week feeding trial.Results The use of 2%inulin did not significantly change the fish intestinal microbiota,but interestingly,the high CHO/protein ratio group showed a change in intestinal microbiota and in particular the beta diversity,with 21 bacterial genera affected,including Ralstonia,Bacillus,and 11 lactic-acid producing bacteria.There were higher levels of butyric,and valeric acid in groups fed with high CHO/protein diet but not with inulin.The high CHO/protein group showed a decrease in the expression of pro-inflammatory cytokines(il1b,il8,and tnfa)in liver and a lower expression of the genes coding for tight-junction proteins in mid-intestine(tjp1a and tjp3).However,the 2%inulin did not modify the expression of plasma immune markers.Finally,inulin induced a negative effect on rainbow trout growth performance irrespective of the dietary carbohydrates.Conclusions With a 100%plant-based diet,inclusion of high levels of carbohydrates could be a promising way for fish nutrition in aquaculture through a protein sparing effect whereas the supplementation of 2%inulin does not appear to improve the use of CHO when combined with a 100%plant-based diet.

Short-Term Stress Response of Juvenile Rainbow Trout Subjected to Two Different Rearing Densities

Juvenile rainbow trout Oncorhynchus mykiss were subjected to one of four treatments in a two-by-two experimental design: 1) fed at a density of 1.8 g/m3, 2) Fasted at 1.8 g/m3, 3) fed at 30.1 g/m3, and 4) fasted at 30.1 g/m3. Blood glucose and hematocrit were measured at 4, 6, 48, 168, and 336 hours after placement in one of the two rearing densities, with relative fin lengths and organosomatic indices recorded at 336 hours. Glucose levels over time were not significantly different among the density and feeding treatments. Hematocrit levels over time were also not significantly different. Total lengths, weight, hepatosomatic index, viscerosomatic index, and any of the relative fin lengths were not significantly different between the high and low densities. However, the hepatosomatic index was significantly greater in the fed fish compared to those fasted. The splenosomatic index was significantly greater in the higher density treatment. These results likely indicate no short-term stress response to the higher rearing density used in this short-term experiment and no interaction between starvation and density-related stressors.

IMMUNE FUNCTION OF IMMATURE RAINBOW TROUT ( Oncorhynchus mykiss ) RELATED WITH PHOTOPERIOD

Radioimmunoassay,enzyme linked immunospot assay and enzyme immunoassay were used for the determinations of plasma steroid hormone's level,antibody producing cell's counting and IgM level in this study.The decreased number of antibody producing cells and low IgM levels were observed in sexual immature rainbow trout during the spawning season.These fish were reared under almost constant water temperature and natural photoperiod.Moreover,low IgM level was also observed in immature rainbow trout,which were reared under short photoperiod,and IgM level was not changed by treatment of testosterone.The results suggest that photoperoid may cause the changes in immune competence.It is possible that circadian rhythm accompanied with photoperiod may influence physiological function of fish,so that immune competence is changed.

17α,20β-DIHYDROXY-4-PREGNENE-3-ONE SUPPRESSES PLASMA IgM LEVELS IN RAINBOW TROUT (Oncorhynchus mykiss)

The relationships between 17α,20β dihydroxy 4 pregnene 3 one (17α,20β DP) and plasma IgM and total protein levels were investigated in rainbow trout, Oncorhynchus mykiss .IgM and total protein levels decreased in both sexes of mature rainbow trout when 17α,20β DP levels increased during the spawning season,while the elevated 17α,20β DP suppressed IgM levels and reversibly enhanced total protein concentrations in immature trout.This represents the first report of the effect of 17α,20β DP on fish immunity.These data suggest that the increase of 17α,20β DP in spawning season may be related to infectious skin diseases.

Immune response,stress resistance and bacterial challenge in juvenile rainbow trouts Oncorhynchus mykiss fed diets containing chitosan-oligosaccharides

Effects of dietary supplementation of chitosan-oligosaccharides （COS） on the growth performance, immune response, stress resistance, and disease resistance of juvenile rainbow trout Oncorhynchus mykiss were studied. Four experimental diets containing 0, 20, 40, or 60 mg/kg COS （COSO, COS20, COS40, and COS60, respectively） were fed to juvenile rainbow trout （initial weight = 5.2 ± 0.3 g） for 8 weeks. By the end of the feeding trial, representative groups of fish from each dietary treatment were challenged with stressor （30 see air exposure） and pathogen exposure （intraperitoneal injection with Aeromonas hydrophila ）. Results showed that supplementation of COS in diets did not affect production performance and body composition of rainbow trout. However, fish fed the COS40 diet demonstrated improved phagocytic activities, respiratory burst activities and decreased serum cortisol level. Additionally, survival following A. hydrophila challenge was significant higher among fish fed the COS-supplemented feeds, although there was no difference based on the level of supplementation. The present study suggests that COS can be used as an immuno-stimulant in rainbow trout feeds

Effects of full replacement of dietary fishmeal with insect meal from Tenebrio molitor on rainbow trout gut and skin microbiota

Background:Aquaculture must continue to reduce dependence on fishmeal(FM)and fishoil in feeds to ensure sustainable sector growth.Therefore,the use of novel aquaculture feed ingredients is growing.In this regard,insects can represent a new world of sustainable and protein-rich ingredients for farmed fish feeds.Accordingly,we investigated the effects of full replacement of FM with Tenebrio molitor(TM)larvae meal in the diet of rainbow trout(Oncorhynchus mykiss)on fish gut and skin microbiota.Methods:A feeding trial was conducted with 126 trout of about 80 g mean initial weight that were fed for 22 weeks with two isonitrogenous,isolipidic,and isoenergetic extruded experimental diets.Partially defatted TM meal was included in one of the diets to replace 100%(TM 100)of FM,whereas the other diet(TM 0)was without TM.To analyse the microbial communities,the Illumina MiSeq platform for sequencing of 16S rRNA gene and Qiime pipeline were used to identify bacteria in the gut and skin mucosa,and in the diets.Results:The data showed no major effects of full FM substitution with TM meal on bacterial species richness and diversity in both,gut mucosa-and skin mucus-associated microbiome.Skin microbiome was dominated by phylum Proteobacteria and especially by Gammaproteobacteria class that constituted approximately half of the bacterial taxa found.The two dietary fish groups did not display distinctive features,except for a decrease in the relative abundance of Deefgea genus(family Neisseriaceae)in trout fed with insect meal.The metagenomic analysis of the gut mucosa indicated that Tenericutes was the most abundant phylum,regardless of the diet.Specifically,within this phylum,the Mollicutes,mainly represented by Mycoplasmataceae family,were the dominant class.However,we observed only a weak dietary modulation of intestinal bacterial communities.The only changes due to full FM replacement with TM meal were a decreased number of Proteobacteria and a reduced number of taxa assigned to Ruminococcaceae and Neisseriaceae families.Conclusions:The data demonstrated that TM larvae meal is a valid alternative animal protein to replace FM in the aquafeeds.Only slight gut and skin microbiota changes occurred in rainbow trout after total FM replacement with insect meal.The mapping of the trout skin microbiota represents a novel contribution of the present study.Indeed,in contrast to the increasing knowledge on gut microbiota,the skin microbiota of major farmed fish species remains largely unmapped but it deserves thorough consideration.

GFAP expression in the optic nerve and increased Н2S generation in the integration centers of the rainbow trout（Oncorhynchus mykiss） brain after unilateral eye injury

Hydrogen sulfide(H2S)is considered as a protective factor against cardiovascular disorders.However,there are few reports on the effects of H2S in the central nervous system during stress or injury.Previous studies on goldfish have shown that astrocytic response occurs in the damaged and contralateral optic nerves.Glial fibrillary acidic protein(GFAP)concentration in the optic nerves of rainbow trout has not been measured previously.This study further characterized the astrocytic response in the optic nerve and the brain of a rainbow trout(Oncorhynchus mykiss)after unilateral eye injury and estimated the amount of H2S-producing enzyme cystathionineβ-synthase(CBS)in the brain of the rainbow trout.Within 1 week after unilateral eye injury,a protein band corresponding to a molecular weight of 50 kDa was identified in the ipsi-and contralateral optic nerves of the rainbow trout.The concentration of GFAP in the injured optic nerve increased compared to the protein concentration on the contralateral side.The results of a quantitative analysis of GFAP+cell distribution in the contralateral optic nerve showed the largest number of GFAP+cells and fibers in the optic nerve head.In the damaged optic nerve,patterns of GFAP+cell migration and large GFAP+bipolar activated astrocytes were detected at 1 week after unilateral eye injury.The study of H2S-producing system after unilateral eye injury in the rainbow trout was conducted using enzyme-linked immunosorbent assay,western blot analysis,and immunohistochemistry of polyclonal antibodies again st CBS in the integrative centers of the brain:telencephalon,optic tectum,and cerebellum.Enzyme-linked immunosorbent assay results showed a 1.7-fold increase in CBS expression in the rainbow trout brain at 1 week after unilateral eye injury compared with that in intact animals.In the ventricular and subventricular regions of the rainbow trout telencephalon,CBS+radial glia and neuroepithelial cells were identified.After unilateral eye injury,the number of CBS+neuroepithelial cells in the pallial and subpallial periventricular regions of the telencephalon increased.In the optic tectum,unilateral eye injury led to an increase in CBS expression in radial glial cells;simultaneously,the number of CBS+neuroepithelial cells decreased in intact animals.In the cerebellum of the rainbow trout,neuroglial interrelationships were revealed,where H2S was released,apparently,from astrocyte-like cells.The organization of H2S-producing cell complexes suggests that,the amount of glutamate produced in the rainbow trout cerebellum and its reuptake was controlled by astrocyte-like cells,reducing its excitotoxicity.In the dorsal matrix zone and granular eminences of the rainbow trout cerebellum,CBS was expressed in neuroepithelial cells.After unilateral eye injury,the level of CBS activity increased in all parts of the cerebellum.An increase in the number of H2S-producing cells was a response to oxidative stress after unilateral eye injury,and the overproduction of H2S in the cerebellum occurred to neutralize reactive oxygen species,providing the cells of the rainbow trout cerebellum with a protective effect.A structural reorganization in the dorsal matrix zone,associated with the appearance of an additional CBS+apical zone,and a decrease in the enzyme activity in the dorsal matrix zone,was revealed in the zones of constitutive neurogenesis.All experiments were approved by the Commission on Biomedical Ethics,A.V.Zhirmunsky National Scientific Center of Marine Biology(NSCMB),Far Eastern Branch,Russian Academy of Science(FEB RAS)(approval No.1)on July 31,2019.

An Effective Method of Prompting Juvenile Rainbow Trout (Oncorhynchus mykiss) to Cope with Heat Stress

In the present study,juvenile rainbow trout(Oncorhynchus mykiss)were acclimated at 5 temperatures.Fish in the control group(C0)were reared at a constant temperature(16℃);trout in four other treatments(A0,A3,A6,and A9)were acclimated to a high temperature(22℃)for 0,3,6,and 9 d,respectively,and then returned to normal temperature(16℃)for 7d.The temperature was then raised to 20℃and the fish were cultured for 40 d.The results showed that the growth rates of A3,A6,and A9 were higher than that of A0 but lower than that of C0.The growth rate of A9 was the highest among the 4 acclimation groups.The activities of serum transaminase and liver antioxidant enzyme significantly increased during acclimation.At the beginning of growth,the activities of enzymes were lower in A3,A6,and A9 with the lowest in A9.In addition,the transcript abundance of heat shock protein(HSP)60 gene in A9 was not significantly different from that of C0 during growth.HSP70 in A9 significantly increased at the beginning and returned to that of C0 at the end.Our findings indicated that pre-acclimation improved the high temperature tolerance with the best effectiveness observed at 22℃for 9 d.A possible mechanism underlining such phenomenon is the improvement of antioxidant defense system.

Effect of micro-algae Schizochytrium sp.supplementation in plant diet on reproduction of female rainbow trout(Oncorhynchus mykiss):maternal programming impact of progeny

Background:The broodstock diet,and in particular the lipid and fatty acid composition of the diet,is known to play a key role in reproductive efficiency and survival of the progeny in fish.A major problem when replacing both fish meal and fish oil by plant sources is the lack of n-3 long chain polyunsaturated fatty acids,such as eicosapentaenoic acid(EPA)and docosahexaenoic acid(DHA).To address this problem,we studied the effect of the plant-based diet supplemented with Schizochytrium sp.microalgae,source of DHA,compared to a conventional commercial diet rich in fish meal and fish oil on reproductive performance and egg quality and the consequences on progeny,in female rainbow trout broodstock.Results:The results demonstrated that DHA-rich microalgae supplementation in a plant-based diet allowed for the maintenance of reproductive performance and egg quality comparable to a conventional commercial feed rich in fish meal and fish oil and led to an increased significant fry survival after resorption.Moreover,when females were fed a plant-based diet supplemented with micro-algae,the 4-month-old progenies showed a significant higher growth when they were challenged with a similar diet as broodstock during 1 month.We provide evidence for metabolic programming in which the maternal dietary induced significant protracted effects on lipid metabolism of progeny.Conclusions:The present study demonstrates that supplementation of a plant-based diet with DHA-rich microalgae can be an effective alternative to fish meal and fish oil in rainbow trout broodstock aquafeed.

Neurolin expression in the optic nerve and immunoreactivity of Pax6-positive niches in the brain of rainbow trout(Oncorhynchus mykiss) after unilateral eye injury

In contrast to astrocytes in mammals, fish astrocytes promote axon regeneration after brain injury and actively participate in the regeneration process. Neurolin, a regeneration-associated, Zn8-1abeled protein, is involved in the repair of damaged optic nerve in goldfish. At 1 week after unilateral eye injury, the ex- pression of neurolin in the optic nerve and chiasm, and the expression of Pax6 that influences nervous system development in various brain regions in the rainbow trout （Oncorhynchus mykiss） were detected. Immunohistochemical staining revealed that the number of Zn8~ cells in the optic nerve head and intraor- bital segment was obviously increased, and the increase in Zn8~ cells was also observed in the proximal and distal parts of injured optic nerve. This suggests that Zn8＊ astrocytes participate in optic nerve regeneration. ELISA results revealed that Pax6 protein increased obviously at 1 week post-injury. Immunohistochemi- cal staining revealed the appearance of Pax6＋ neurogenic niches and a larger number of neural precursor cells, which are mainly from Pax6＋ radial glia cells, in the nuclei of the diencephalon and optic tectum of rainbow trout （Oncorhynchus mykiss）. Taken together, unilateral eye injury can cause optic nerve reaction, and the formation of neurogenic niches is likely a compensation phenomenon during the repair process of optic nerve injury in rainbow trout （Oncorhynchus mykiss）.

ANNUAL CHANGES IN PLASMA LEVELS OF CORTISOL AND SEX STEROID HORMONES IN MALE RAINBOW TROUT, ONCORHYNCHUS MYKISS

The profiles of cortisol, testosterone, 11 ketotestosterone and 17α, 20β dihydroxy 4 pregnene 3 one in male rainbow trout reared under constant water temperature and natural photoperiod were determined by radioimmunoassay. Gonads of male rainbow trout reached maturity when the fish were two years old. Changes in the plasma levels of both sex steroid hormones and cortisol were closely related to the GSI. Plasma levels of testosterone, 11 ketotestosterone and 17α, 20β dihydroxy 4 pregnene 3 one showed a clear peak in the annual breeding season, when the GSI reached their maxima. Plasma cortisol levels also showed clearly seasonal changes in both two and three year old fish. The results suggest that the elevated plasma levels of cortisol may not just be due to stresses during the breeding season but have certain physiological functions in the reproduction of rainbow trout.

Novel Surgical Technique for Acoustic Transmitter Insertion in Rainbow Trout Reduces the Need for Surgical Training

The implantation of acoustic transmitters into the peritoneal cavity of fish is typically performed by experienced surgeons. This study compared the effectiveness of an experienced and inexperienced surgeon performing two different types of transmitter insertion techniques on rainbow trout (Oncorhynchus mykiss). The methods used were either a traditional sutured ventral incision or a novel unsutured lateral incision. There was no significant difference in surgical duration, tag retention, or fish survival between the experienced and naïve surgeon. The time to complete surgical wound closure was not significantly different between the fish operated on by an experienced or naïve surgeon, and surgeon experience had no significant effect on the duration of inflammation. However, there were significant differences between the two surgical techniques, regardless of surgeon experience. The ventral incision with sutures method took approximately three times longer to complete than the lateral incision-only method. However, complete wound closure was significantly faster in the sutured ventral incision treatment compared to the unsutured lateral treatment. Post-surgery inflammation was over seven times longer in the fish with sutures compared to those only receiving a lateral incision. Tag retention was not significantly different between the two surgical methods. The results of this study indicate that the unsutured lateral surgical technique used in this experiment can be completed with minimal training and surgical experience in contrast to the standard technique requiring sutures. These results also provide additional support to the use of sutureless surgical techniques for acoustic transmitter implantation.

Effects of irradiated Ergosan on the growth performance and mucus biological components of rainbow trout Oncorhynchus mykiss

Effects of irradiated and non-irradiated Ergosan extract(alginic acid) on rainbow trout growth performance and skin mucosal immunity were compared. Ergosan was irradiated at 30 kGy in a cobalt-60 irradiator. A total of 252 fish(128.03±9.4 g) were randomly divided into four equal groups,given the basal diet either unsupplemented with Ergosan(control group) or supplemented with crude Ergosan(5 g/kg),ethanol-extracted Ergosan(0.33 g/kg) or irradiated Ergosan(0.33 g/kg) according to this protocol: basal diet for 15 days,treatment diet for 15 days,basal diet for 10 days and treatment diet for 15 days. Highest growth performance was observed in fish fed irradiated Ergosan(P<0.05). Dietary administration of different Ergosan types did not cause any changes in mucus protein level,but improved alkaline phosphatase level and hemagglutination titer compared with the control(basal diet without Ergosan) on day 55 offeeding trial(P<0.05). Furthermore,the highest value of lysozyme activity was observed in gamma-irradiated Ergosan on day 55. In conclusion,gamma-irradiated Ergosan at 0.33 g/kg was found to improve growth performance and mucus biological components significantly in comparison with the control group(basal diet without Ergosan).

Fatty Acid Composition and Digestive Enzyme Activities of Rainbow Trout in Response to Dietary Docosahexaenoic Acid(DHA)and Eicosapentaenoic Acid(EPA)During Salinity Acclimation

This physiological study aimed to evaluate the effects of dietary docosahexaenoic acid(DHA)and eicosapentaenoic acid(EPA)on the fatty acid composition and digestive enzyme activities of rainbow trout(Oncorhynchus mykiss)during salinity acclimation.Rainbow trout with an average initial weight of 90.61 g±9.25 g were fed diets with the quantities of DHA and EPA equaling to 0.54%,0.95%,1.40%and 1.79%(abbreviated as DE-0.54,DE-0.95,DE-1.40,and DE-1.79,respectively)for eight weeks,after which the gastric and intestinal fatty acids composition were analyzed.Subsequently,the fish underwent salinity acclimation.On days 1,4,7,and 14 after the freshwater was replaced by seawater and at the end of the 8-week period,gastric and intestinal digestive enzyme activities were determined.The results showed that the gastric and intestinal DHA and EPA contents of the fish were positively correlated to their dietary DHA and EPA levels.Low dietary DHA and EPA levels inhibited the protease activity of rainbow trout.Fish in the DE-0.54 group increased the lipase activity to enhance the utilization of lipids maybe due to the inadequate essential fatty acids for fish in this group.Hence,rainbow trout in the DE-0.54 group failed to maintain suitable activities of digestive enzymes after salinity acclimation.Therefore,a diet with minimum 0.95%DHA and EPA levels is necessary for rainbow trout during salinity acclimation.

Behaviour of rainbow trout(Oncorhynchus mykiss) under defensible and indefensible patterns of food delivery

The goal of this study was to investigate the behaviour of rainbow trout(n=30),Oncorhynchus mykiss,in small raceways when either self-feeders(T2) or hand-feeding(t2) were used.The method of food delivery in T2 was defensible while that of t2 was indefensible.Fish in both raceways were subjected to restricted feeding(RF) for 25 days.Food was available in the morning(09:00-10:00) in the downstream area and in the afternoon(16:00-17:00) in the upstream area of the raceways.The results showed that the behaviour of rainbow trout was significantly different under interference competition(T2) for food compared with that under scramble competition(t2).RF in T2 fish limited food availability to meal times when feeding rewards were available while t2 fish only responded to the location of food delivery.The aggressive fish in T2 were dominant,and t2 fish at high densities showed intense social interactions under the indefensible pattern of food distribution;these interactions did not dampen to a minimum level to suppress the development of dominance hierarchies.Further,the stocking density did not break down the dominance hierarchies between the T2 fish.This suggests that decreased efficiency in the search for food or inefficient foraging,induced by interference competition at high densities,affected the behaviour of rainbow trout.

Difference Analysis of Liver Metabolic Response Between Diploid and Triploid Rainbow Trout Oncorhynchus mykiss Under Fishing Stress

To elucidate the effects of fishing stress on the liver glucose metabolism of both diploid and triploid female rainbow trouts as well as differences in their stress response,blood and liver tissues were collected from 0 h to 24 h after the fishing stress.Blood indexes,such as the levels of white blood cells and red blood cells,cortisol,glucose and lactic acid,as well as activities of key enzymes of the glucose metabolism in the liver,were measured.Furthermore,the mRNA expressions of glucocorticoid receptors and enzymes related to energy metabolism were assessed.The results showed that fishing stress exerted significant effects on blood physiological and biochemical indexes and liver glucose metabolism.Differences were found between diploid and triploid female rainbow trouts in the liver glucose metabolism pathway,as well as the level and performance during stress.The basic glucose metabolism intensity of the triploid liver was higher than that of the diploid liver;however,the hepatic glycogen reserve was lower in the triploid liver.After fishing stress,triploid trouts entered the immunosuppressive state earlier than diploid trouts.Triploid trouts also showed an earlier and stronger stress reaction than diploid trouts,while their energy allocation ability and immune recovery ability were weaker than those of diploid trouts.These results showed that the regulatory ability of diploid female rainbow trouts in response to fishing stress was better than that of triploid trouts.

Thermal Tempering Does Not Impact Rainbow Trout and Brown Trout Survival

Hatchery-reared fish are frequently adjusted (tempered) to the higher water temperatures present in the water bodies where they are to be stocked. This study was undertaken to determine the necessity of such tempering practices. This study used rainbow trout (Oncorhynchus mykiss) and brown trout (Salmo trutta) reared at 11.2°C. The first two trials for each species were designed to simultaneously determine the upper incipient lethal temperature and 100% lethal temperature over a 14-day period. The third trial for each species evaluated the effects of an exaggerated 12-hour tempering regime on fish survival after placement in elevated water temperatures. After transfer from a water temperature of 11.2°C, no rainbow trout survived at 26°C, and only 50% survived at 25°C. No brown trout survived at 22°C and only 50% at 20°C. Survival of rainbow trout was not improved by the 12-hour tempering regime where water temperatures were slowly increased from 11.2°C to either 25°C or 26°C. Similarly, tempering did not improve brown trout survival at either 20°C or 22°C. These results suggest that tempering is not needed when hatchery-reared trout are reared and stocked at the water temperatures within the range of those used in this study.

Effects of varying densities on serum reproductive parameters in pen-reared juvenile female rainbow trout Oncorhynchus mykiss farms

The primary goal of this study was to assess the effect of varying densities on serum reproductive parameters of immature rainbow trout Oncorhynchus mykiss. Experimental trout were maintained in intensive, pen-reared farms for 300 days in fresh water reservoirs. Initial densities were 4.6, 6.6, and 8.6 kg/m^3(40, 60, 80 ind./m^3), indicated as SD1, SD2, SD3, and final densities were 31.1, 40.6, 49.3 kg/m^3, respectively. A summary of the ovarian stages were observed by histological examination. Serum E 2(estradiol), T(testosterone) were evaluated by radioimmunoassay and FSH(follicle-stimulatinghormone), LH(luteinizing-hormone), vitellogenin, 17α,20β-P(17α,20βdihydroxy4-pregnen-3-one) were measured by enzyme-linked immunosorbent assay. Our findings demonstrated that ovarian development were retarded(from stage Ⅲ to stage Ⅳ) at highest rearing density(SD3) after 180 days of intensive culture(over 40.6 kg/m^3). In addition, we observed an inverse relationship between serum reproductive parameters and rearing density. Furthermore, compared to serum reproductive parameters of SD1, E 2, T, FSH, vitellogenin, 17α,20β-P, GSI and LH of two higher density groups decreased firstly and significantly at 60(over 15.9 kg/m^3), 180(over 31.7 kg/m^3), 180(over 40.6 kg/m^3), 240(over 36 kg/m^3), 240(over 36 kg/m^3), 240(over 45 kg/m^3) and 300(over 49.3 kg/m^3) days, respectively. Comparing serum reproductive parameters within the same ovarian development stage of rainbow trout from varying densities revealed that higher population density also led to significantly lower overall serum reproductive parameters. Overall, this study presents the reproductive, endocrinological parameters of juvenile female rainbow trout at high rearing densities and indicates the need for rainbow trout(114.44±5.21 g, 19.69±0.31 cm) that are initially stocked at 6.6 or 8.6 kg/m^3 should be classified and subdivided into lower density after 180 days of farming(not over 31.7 kg/m^3).

Sutureless Implantation of Acoustic Transmitters in Rainbow Trout Exceeding 2% Tag-to-Body Ratio

A novel sutureless surgical technique has been successfully used to implant acoustic transmitters in relatively large rainbow trout Oncorhynchus mykiss with a tag-to-body-ratio of 0.88%. This study examined the same technique in smaller rainbow trout in two, 12-week trials comparing both sutured and sutureless surgical techniques. In the first trial using a tag-to-body-ratio of 1.9% ± 0.04%, tag retention was only 16.6% in fish without sutures, which was significantly lower than the 83.3% retention with sutured incisions. Similarly, in the second trial with a tag-to-body-ratio of 3.2% ± 0.03%, tag retention was 55.5% without sutures, which was significantly lower than the 90.0% retention using sutures. Mortality was not significantly different between treatments in either trial. The results of this study indicate that sutures must be used during surgeries to implant acoustic transmitters in relatively smaller fish with tag-to-body-ratios at or above 1.9%.

Correlation between Leukocyte Numbers and Body Size of Rainbow Trout

Immune cells in rainbow trout Oncorhynchus mykiss comprise granulocytes (neutrophils, basophils and eosinophils), macrophages/monocytes and lymphocytes (B- and T-cells). These cellular elements occur early during the ontogenetic development of trout and allow both innate and adaptive responses towards an antigen to be initiated even in fry. The number of leukocytes in individual fish at different developmental stages is likely to influence the capacity of the fish to respond simultaneously to several antigens (pathogens and vaccine components). This parameter may therefore be crucial for both wild and cultured fish and we show that the size of the leukocyte population increase exponentially with body size of rainbow trout. Four groups (5 fish/ group) of naive rainbow trout (Oncorhynchus mykiss) with a mean body weight of 2-4 g (group I), 4-6 g (group II), 25-30 g (group III), and 650-780 g (group IV) were investigated. The number of lymphocytes was generally higher in head kidney compared to blood and spleen but they dominated in all samples (blood, head kidney, and spleen) and their numbers increased exponentially with fish size. Percentages of lymphocytes in relation to neutrophils and macrophages were higher in spleen (98%-99%) compared to blood and head kidney in all groups. Fish fry is therefore equipped to respond specifically against one or a few vaccine antigens but the capacity to raise protective responses against a repertoire of pathogens may be limited until the larger fingerling stage has been reached. The implications for vaccination of early fry are discussed.