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The current body of work on rearing larval/juvenile zebrafish is based on (1) utilization of freshwater and (2) diurnal light/dark cycle, (3) provision of live feed at modest density, and (4) culture in high visibility environment. We challenged these rearing approaches by maintaining zebrafish under constant light for 46-48 days (days postfertilization [dpf]), while securing continuous feeding in high turbidity and saline (1.8-2.1 parts per thousand) environment for the experiment's duration, allowing 24 h feeding/growth of fish from first exogenous feeding to maturation. There was no evidence of negative effects on zebrafish larvae behavior, growth, survival, and life cycle duration at constant illumination when food was continuously available. Zebrafish were stocked at high initial density (100 larvae/L) in a static system and fed high densities of rotifers (Brachionus plicatilis) (200-400/mL) from 6 to 12 dpf. Fish density was then reduced by 50% and two diet treatments, live rotifers or brine shrimp (Artemia) nauplii (10/mL), followed. Fish were reared on these two diets until first maturation. Performance of adult zebrafish fed live rotifer followed by Artemia nauplii diet was the highest recorded in the literature after 42 dpf, 250 ± 29 (males) and 430 ± 5 mg (females). Use of these rearing conditions, during the entire life cycle, until reproduction, resulted in the shortest ever recorded generation time (from egg to egg) of 43-45 dpf and fertilization rate (1 dpf) of 80.3%-94%.

We have shown previously that diets in which the nitrogen portion is based on synthetic dipeptides (PP) resulted in weight gain by rainbow trout alevins when free amino acid (FAA) based diets did not. However, the protein-based diet used as a control in the previous study resulted in a significantly better performance of fish than peptide- or FAA based diet fed fish. Therefore, the objectives of our study were (i) to test how stomachless fish respond to peptide-based diets, (ii) to evaluate PP and protein-PP mixture diets and (iii) to examine if post-prandial response to FAA concentrations in the fish body can be used as an indicator of the availability of dietary amino acid sources. The first experiment was conducted with a 4-day old Koi carp (Cyprinus carpio) larvae and included groups fed a casein-gelatin (CG) based diet, a commercial diet and live Artemia nauplii. In the second experiment, fish fed live Artemia for 3 weeks (69 +/- 12 mg) were placed in glass aquaria in triplicate per dietary treatment. Four diets provided equivalent amounts of nitrogen in the form of a CG, a PP, a 50% PP and 50% CG (PP50) mix and as FAA. Fish were fed at 1 and 3 h intervals, and the survival and growth were monitored during the second and third weeks of experiments 1 and 2 respectively. Following the completion of feeding, juvenile carp were sampled prior to feeding and 3 h after a meal. Whole body FAA analyses were carried out. We observed marginal suitability of the CG diet for larval Koi carp, as earlier indicated in common carp. Juvenile Koi carp fed the CG diet achieved 236 +/- 19 mg, whereas PP50, PP and FAA diet fed fish grew to 140 +/- 37, 70 +/- 8 and 73 +/- 5 mg respectively. Free amino acids in the fish body, and in particular indispensable amino acids (IDAA), were excellent indicators of dietary availability. The present experiment shows that a dietary 1:1 ratio of protein to synthetic PPs results in better growth, survival and whole body IDAA concentrations in relation to FAA- and PP-based diets confirming earlier reports on the use of synthetic PP-based diets, although this diet does not match the performance of complete protein, CG-based diet.

Studies on larval rearing of silver bream (Vimba vimba), a migratory cyprinid fish have addressed on limited scale larval and juvenile rearing using commercial and semipurified diets along with live feeds, such as brine shrimp Artemia nauplii. The objectives of the present study were (1) to determine whether experimental, protein-, peptide-, free amino acid-based diets are adequate for larval silver bream, a stomachless fish, (2) to evaluate whether commercial and purified diets are comparable as the first/exclusive feed for growth and survival of silver bream, and (3) to examine whether free amino acid concentrations in fish body are potential indicators of availability of amino acid sources. We report here the differences in diets acceptance, fish growth and diet utilization in silver bream in comparison to other cyprinid fishes. We specifically address the response in free amino acids in the body to dietary treatments. Experimental diets included: a commercial Aglo Norse feed, casein-gelatin based diet (CG), free amino acid mixture diet (FAA), dipeptide (PP), dipeptide-protein (PP50) based diet, and dipeptide diet without arginine (NoArg). In addition, live Artemia were offered to 3 groups and "fasting" control treatment was included during 3 week long trial. Fish offered Artemia overperformed those offered formulated diets both in terms of mass (80.7+/-26.3 mg) and survival (97.2%). We also indicate that commercial and purified diets are comparable as the first/exclusive feed for growth and survival of silver bream. Our experiment also showed that the whole body free amino acid concentrations of 9 indispensable amino acids (IDAA) out of 10 (His, Thr, Arg, Val, Met, Ile, Leu, Trp, Lys) in the PP50 group was the highest among 7 diet treatments and the totalfree amino acid concentration, total dispensable amino acids (DAA) and total IDAA of the PP50 diet fed fish showed the same trend. This may indicate that diets based on 50% of dipeptides and 50% of protein are adequate for silver bream larvae as they result in similar growth, survival and whole body IDAA concentrations as in fish fed CG (protein-based) diet fed fish.

The premise that free amino acid or dipeptide based diets will resolve the nutritional inadequacy of formulated feeds for larval and juvenile fish and improve utilization of nitrogen in comparison to protein-based diets was tested in stomachless fish, common carp (Cyprinus carpio L.) larvae. We examined the postprandial whole body free amino acid (FAA) pool in fish that were offered a FAA mixture based diet for the duration of 2 or 4 weeks. We found that the total amount and all indispensable amino acids concentrations in the whole body decreased after a meal. We then fed juvenile carp with dietary amino acids provided in the FAA, dipeptide (PP), or protein (live feed organisms; brine shrimp Artemia salina nauplii, AS) forms. Histidine concentrations in the whole fish body increased in all dietary groups after feeding whereas all other indispensable amino acids decreased in FAA and PP groups in comparison to the AS group. Taurine appears to be the major osmotic pressure balancing free amino acid in larval freshwater fish which may indicate a conditional requirement. We present the first evidence in larval fish that in response to synthetic FAA and PP diets, the whole body indispensable free AA concentrations decreased after feeding. This study shows that amino acids given entirely as FAA or PP cannot sustain stomachless larval fish growth, and may result in depletion of body indispensable AA and most of dispensable AA. The understanding of these responses will determine necessary changes in diet formulations that prevent accelerated excretion of amino acids without protein synthesis.

The premise that a dietary dipeptide approach will improve the understanding of amino acid utilization in the fastest-growing vertebrate, the teleost fish, was tested by examining the muscle free amino acid (FAA) pool and enzyme activities, in concert with growth response, when dietary amino acids were provided in free, dipeptide or protein molecular forms. We present the first evidence in fish that, in response to a synthetic dipeptide diet, muscle FAA varies as a result of both growth rate and amino acid availability of specific peptides. We demonstrate significantly diminished muscle indispensable FAA (3-10-fold) in rainbow trout alevins fed a dipeptide-based diet compared with a protein-based diet. The dipeptide-based diet did not contain proline, resulting in 10-27-fold less muscle free proline and hydroxyproline in alevins. The response of alevins fed FAA-based or peptide-based diets can be indicative of collagen turnover (Hyp/Pro ratio) and showed significant differences between dietary treatments. Pyrroline-5-carboxylate (P5C) reductase activity was detected, suggesting that P5C may ameliorate proline deficiency, but synthesis from glutamate could not maintain free proline levels in muscle. This finding will provide an impetus to test whether proline is conditionally indispensable in young fish, as in mammals and birds. This study shows that amino acids given entirely as dipeptides can sustain fish growth, result in muscle FAA and enzyme responses in line with dietary levels and identify growth-limiting amino acids. The understanding of these factors necessitates a diet formulation that will improve the accuracy of determining amino acid requirements in the early life stages of vertebrates.

Most studies on the antioxidants, lipoic acid (LA) and ascorbic acid (AA), focused on species that, unlike teleost fish, are not scurvy-prone, and are able to synthesize AA. The antioxidant properties of LA may make it useful in aquaculture nutrition, but several effects must first be investigated, and we address here plasma free amino acids (FAA). In mammals, LA and AA in high doses were claimed to alter plasma FAA profile; to our knowledge, however, no data are available in fish. We therefore studied the effects of dietary LA and AA on plasma FAA in the South American teleost fish pacu, which is being used increasingly in aquaculture. LA treatment decreased concentrations of 18 of 23 individual FAA; specifically, dispensable and total FAA were significantly affected. Ornithine was elevated (+26%) in LA-treated fish and significantly decreased ratios of plasma [Arg]/[Orn] and other individual [FAA]/[Orn] were observed. LA and AA both affected sulfur FAA concentrations. Plasma cystine levels were significantly increased in the LA-supplemented groups. AA had little effect on most amino acids, and no interaction with LA was detected. AA supplementation did, however, significantly lower taurine (-42%) and cystathionine (-31%) levels in plasma. No effect on the branched chain:aromatic amino acid ratios was observed. The data indicate that at the dietary level studied, LA and AA independently affect selected plasma FAA in pacu, and suggest that any use of LA in particular as a dietary supplement should take into account an altered plasma FAA profile.

Gossypol is an antifertilizing agent in males and females. However, gossypol and its metabolite, gossypolone, have also gained interest because of their anticarcinogenic activities. This paper examines for the first time both enantiomers of tissue gossypol and gossypolone in mature rainbow trout fed two diets containing low (15%) and high (60%) levels of cottonseed meal (CM) for 9 months. The gossypol concentration was highest in liver followed by kidney, intestine, testis, blood plasma, stomach, and muscle. Gossypol was detected in muscles of fish fed low- and high-CM diets (0.31 +/- 0.03 and 1.95 +/- 0.59 microg of total gossypol/g, wet basis, respectively). The (+)-gossypol enantiomer was predominantly retained in all tissues. The ratio of (-)- to total gossypol ranged from 30 to 44% in fish fed the high-CM diet and from 23 to 30% in fish fed the low-CM diet except for muscle tissue (44%). Higher gossypolone concentrations were found in intestine than in liver. Gossypolone, however, was not detected in blood plasma, muscle, and testis of fish fed the low-CM diet. The ratio of gossypolone to gossypol was highest in muscle (1.75), followed by intestine (1.59), stomach (1.50), kidney (0.43), liver (0.34), testis (0.28), and blood plasma (0.27). This study indicated that the retention of the (-)-gossypol enantiomer is dependent on dietary concentrations and that the oxidative conversion of gossypol to gossypolone occurs more actively in the digestive tract and muscle than in other tissues in rainbow trout.

In a preceding study, complete substitution of fish meal protein with cottonseed meal (CM) protein did not affect the survival or growth rate of adult rainbow trout over a 6-mo period. Gossypol, a naturally occurring compound in cottonseeds, has an antifertility effect in terrestrial animals, but information regarding salmonid fish is lacking. Female rainbow trout in this experiment were fed diets with either 0, 25, 50, 75, or 100% (diets 1 to 5) of the fish meal protein replaced with CM protein until first maturation and spawning to study long-term effects on growth and reproduction. Feeding diets containing CM over a total period of 10 mo did not result in differences in growth and mortality compared to the control group (P > 0.05). Increased CM incorporation levels resulted in decreased (P < 0.05) blood hemoglobin (10.6 +/- 1.3, 8.4 +/- 1.8, 7.3 +/- 1.1, 6.9 +/- 0.8, and 5.6 +/- 1.4 g/dL) and hematocrit (49.6 +/- 3.9, 38.5 +/- 9.3, 34.4 +/- 3.7, 34.8 +/- 4.9 and 28.0 +/- 6.8%) levels in diets 1 to 5, respectively. The CM incorporation level had no effect (P > 0.05) on the number of eggs produced per female but led to a reduction (P < 0.05) in egg weight. Eyed stage survival of embryos was low in all dietary groups and did not show differences (P > 0.05). However, an increasing CM incorporation level led to a linear increase (P < 0.05) in the number of females that produced no viable embryos (23.1, 37.5, 42.9, 60.0, and 71.4%). Gossypol in the diet was absorbed by the female trout and transferred to the eggs (0, 2.2 +/- 0.5, 6.7 +/- 1.6, 10.6 +/- 4.2, and 20.0 +/- 2.6 micrograms/g in diets 1 to 5, respectively). A high concentration of gossypol remained in the juveniles at the swim-up stage (endogenous yolk-absorbed) (0.6 +/- 0.3, 2.4 +/- 0.3, 3.4 +/- 0.0, and 4.7 +/- 1.0 micrograms/g, diets 2 to 5, respectively). The findings suggest that replacement of the dietary fish meal protein with CM protein has no effect on fish growth and mortality but may lead to a reduction in reproductive performance in female rainbow trout.

In scurvy-prone animals ascorbic acid uptake is regulated by the number of intestinal "brush-border" carriers available for transport. The number of carriers is negatively affected by an increasing dietary ascorbic acid intake, thus limiting the maximum attainable uptake. We tested a new approach towards manipulating the accumulation of ascorbic acid in rainbow trout by repeatedly feeding a 10 day ascorbic acid allowance in a 1 or 3 day "pulse" followed by withdrawal, rather than spread out over the entire 10 day period. Pulse dosing led to initially increased mortalities compared to continuous dosing, but this was reversed after 55 days. Although individual weight and cumulative mortality were not significantly different after 88 days, total liver ascorbic acid levels were significantly higher in the 1-day pulse group (16.3 +/- 3.9 micrograms/g) than in the 3-day pulse (9.8 +/- 0.9 micrograms/g) or continuous (9.3 +/- 0.9 micrograms/g) groups. This led us to conclude that after an initial adaptation phase a feeding profile with intermittent ascorbic acid withdrawal results in a compensatory increase in uptake of ascorbic acid from the diet and/or a better conservation of the body pool of ascorbic acid, opening interesting new avenues for ascorbic acid dosing and therapy.

Ascorbic acid is an essential nutrient in rainbow trout diets and has been shown to play an important role in fish reproduction. Recommended dietary levels are based on immature fish, and the specific requirements for brood stock are unknown. To establish the optimum dietary level for mature rainbow trout, six graded levels of ascorbyl-2-monophosphate were fed to groups of female fish over a period of 10 mo until spawning. Increasing dietary levels of ascorbyl monophosphate resulted in significantly increased ascorbic acid concentrations in liver, kidney, ovaries, and ovulated eggs. Liver and egg concentrations were saturable at 109.3 and 266.6 micrograms ascorbic acid/g tissue, respectively. Tissue saturation levels of 83.7% and 91.2%, respectively, were reached at the highest dietary level (870 mg ascorbyl monophosphate/kg diet) tested. Both fecundity and embryo survival increased significantly with dietary ascorbyl monophosphate levels. The results indicated that the present National Research Council recommended dietary level of 50 mg ascorbic acid/kg diet for rainbow trout is inadequate for brood stock fish. An amount 8 times higher is necessary to optimize tissue ascorbic acid levels and achieve maximum reproductive success.

High concentrations of ascorbic acid occur in scurvy-prone, teleost fish plasma. We quantified seasonal relationships between 1) dietary level of vitamin C and level of seminal plasma ascorbic acid, and 2) seminal plasma ascorbic acid concentration and sperm quality, in rainbow trout (Oncorhynchus mykiss). We maintained six groups of 2-yr-old rainbow trout on diets supplemented with 0, 30, 110, 220, 440, and 870 ppm ascorbyl monophosphate beginning in May 1992. Sperm were produced during the end of October 1992 through April 1993; we collected milt 13 times from 3-8 fish per treatment. We quantified ascorbic acid concentration in seminal plasma, and sperm concentration, motility, and weight. Seminal plasma ascorbic acid concentrations were affected directly by the ascorbyl monophosphate level in the diet. Seminal plasma ascorbic acid concentrations also were affected by season. Ascorbic acid deficiency did not influence semen quality (sperm concentration and motility) at the beginning of the spawning season. However, sperm concentration and motility in a group fed an ascorbic acid-free diet declined during the period of study. Ascorbic acid deficiency reduced both sperm concentration and motility, and thus fertility, of rainbow trout. These results indicate that vitamin C is important for male fish reproduction; the dietary requirement for seminal plasma ascorbic acid saturation exceeds that for optimum growth.

The high cost of fish meal in tilapia diets warrants the potential use of cottonseed meal (CSM) as an alternative source of high quality protein. The effects of varying levels of CSM (0, 25, 50, 75 and 100%) as fish meal protein replacement on growth, blood parameters, spleen characteristics, free and bound gossypol in blood plasma, haemoglobin and haematocrit were determined in tilapia. Gossypol (C30H30O8) is a polyphenolic substance found in cottonseed that has known toxic effects in fish. Tilapias ( n = 219, average weight = 11.3 ± 3.9 g) were randomly distributed into 15, 32-L glass aquaria, representing five dietary treatments and three replicates per treatment. Each aquarium containing 13–16 fish was supplied with thermoregulated, recirculating water (27 ± 1 °C) at 1 L min−1 flow rate and photoperiod was constant (12 h L/12 h D). Fish fed 25–50% CSM protein replacement showed similar body weights and total lengths as the controls at the completion of the 16-week trial. Fish fed 75 and 100% CSM protein replacement showed a significant decline in body weight and total length. Fish fed 25–100% CSM protein replacement had significantly lower haematocrit and haemoglobin (ANOVA/LSD, P < 0.05) compared with levels in controls. The decline was most prominent in groups fed diets with 50–100% CSM protein replacement. Total and free gossypol concentrations of blood plasma significantly increased with increasing levels of CSM replacement ( P < 0.05). No gossypol was found in blood plasma of fish from the control group. The occurrence of immature and abnormal erythrocytes was significantly greater among fish fed 75 and 100% CSM diets compared with fish fed 0–50% CSM diets. Spleen-somatic index (spleen weight/body weight × 100) did not differ between control fish and fish fed 50–100% CSM diets. Spleen abnormalities, such as large depositions of haemosiderin and melanin pigments and proliferation of melano-macrophage centres, lymphocytic depletion of the white pulp areas (hypocellularity), and presence of vacuoles and necrotic areas were observed among fish fed 50–100% CSM protein diets. In general, the pathological effects of gossypol in tilapia (low haemoglobin and haematocrit levels, abundance of immature red blood cells or polychromatocytes, abnormal spleen morphology) were similar to the effects of vitamin E and/or vitamin C deficiencies observed in other studies. [ABSTRACT FROM AUTHOR]

Rainbow trout Oncorhynchus mykiss broodstocks were fed five experimental diets in which fish meal protein was gradually replaced with cottonseed meal (CS) protein (0%, 25%, 50%, 75%, and 100%; diets 1–5, respectively) during a 22-month period. The effect of increasing dietary levels of CS on reproductive performance was gender specific. Sperm fertilizing ability significantly decreased when CS exceeded 50% protein replacement (72.6±2.0%, 73.6±2.0%, 69.0±3.3%, 43.3±5.1%, and 36.8±4.7%, for diets 1–5, respectively). In contrast, in females, the viability of embryos was only significantly affected at 25% and 50% replacement levels (56.7±15.6%, 20.5±24.9%, 11.4±18.6%, 48.8±25.9%, and 39.6±31.1%, for diets 1–5, respectively). Progenies from multiple parents per dietary treatment were combined and reared on a commercial diet over a 2- or 3-month period fed at a rate of 4% of their body weight. The paternal origin (fresh sperm, experiment 1) had a highly significant effect on growth performance of progenies, and progenies from males fed with 25%, 50%, and 75% CS grew significantly (P<0.05) faster than progenies from males fed with 0% and 100% CS. Growth performance of progenies produced using cryopreserved sperm (experiment 2) was not affected regardless of the CS levels fed to male rainbow trout. Progenies from females (experiment 3) fed a diet containing 50% CS grew significantly (P<0.05) slower than the other groups. Sex ratio was examined histologically after completion of feeding experiments with progenies. Regardless of maternal or paternal origin, males dominated among the progenies. Thus, we postulated that other substances such as flavonoids, present in the CS and possibly transferred to yolk sac reserves, might affect the sex ratio in favor of males. [Copyright &y& Elsevier]

The leaching loss of free amino acids (FAA) from an experimental protein-walled microencapsulated diet (MC) for larval fish after immersion in water was measured and compared with leaching loss from a gelatin microbound diet (MB) containing the same dietary ingredients. The loss of FAA (as a percentage of total dietary FAA) was significantly higher in the MB diet compared to the MC diet. After 5 min of re-hydration, 22% of FAA were leached from the MB diet compared with 8% from the MC diet. After 60 min, 85% of FAA from the MB diet were leached into the water compared to 17% from the MC diet. The addition of free lysine to protein-walled microcapsules (MC-L) was also investigated to determine the suitability of MC for delivering specific FAA. Lysine was incorporated into the particles with an efficiency of 7.5% and accounted for approximately 60% of the total FAA in the diet. The loss of this amino acid from MC-L after 60 min of immersion was 1.4%. The loss of specific FAA from MB and MC diets was found to be negatively and positively correlated to the hydrophobic character (hydropathy index) of each FAA, respectively. These results support the use of protein-walled microcapsules as a vector for specific dietary amino acids using a macronutrient-balanced diet. [Copyright &y& Elsevier]