Ascorbic acid turnover in rainbow trout, Oncorhynchus mykiss: Is there a vitamin enrichment effect during embryonic period on the juvenile fish “sensitivity” to deficiency?

Aquaculture 320 (2011) 99–105

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Ascorbic acid turnover in rainbow trout, Oncorhynchus mykiss: Is there a vitamin enrichment effect during embryonic period on the juvenile fish “sensitivity” to deficiency? Bahram Falahatkar a, 1, Konrad Dabrowski a,⁎, Murat Arslan a, b a b

The School of Environment and Natural Resources, The Ohio State University, Columbus, OH 43210, USA Department of Fisheries and Aquaculture, Ispir Hamza Polat Vocational School, Ataturk University, Ispir, Erzurum 25900, Turkey

a r t i c l e

i n f o

Article history: Received 29 November 2010 Received in revised form 7 August 2011 Accepted 9 August 2011 Available online 17 August 2011 Keywords: Ascorbic acid Metabolism Rainbow trout Feeding

a b s t r a c t Proper feeding results in the transfer of nutrients from broodstocks to eggs and alevins and consequently in better growth and survival throughout the early ontogeny. In the present study, however, following fertilization, rainbow trout eggs had been exposed to different forms and levels of vitamin C enrichment that included 0, 1000 mg L− 1 ascorbic acid (AA) neutralized (N) with NaOH, 1000 and 2000 mg L− 1 ascorbyl phosphate (AP). Fertilized eggs were immersed in water containing ascorbate at above concentrations and forms. After hatching and yolk absorption, feeding trials were carried out on offspring with different vitamin exposure history in two separate experiments of 6 week duration each. Two casein-gelatin based experimental diets were formulated. The first diet contained 500 mg/kg ascorbyl phosphate (AP) (+C) and the second diet was the negative control with no ascorbic acid (−C). In the first experiment (first 6-week period), dietary treatments did not have a significant effect on growth performance and survival of fish. However, the fish fed +C diet had significantly higher ascorbic acid (TAA) levels in the whole body and viscera (P b 0.05) than those on −C diet. After exposure to hypoxia, survival and oxygen consumption did not differ significantly between dietary treatments. In the second experiment (the second 6-week period), all experimental fish were pooled into two groups (+C and −C) based on the dietary treatments they were previously allocated regardless of the vitamin C exposure at the beginning of embryonic development. At the end of experiment 2, fish fed −C diet had a lower final mean weight (2.08 ± 1.12 g) than those fed +C diet (2.37 ± 0.11 g). Survival was significantly lower in fish fed −C diet (91.6 ± 6.8%) than those fed +C diet (100%) (P b 0.05). Liver TAA concentration was significantly higher in fish fed +C diet than those on −C diet (P b 0.05). Deficiency symptoms, including exophthalmia, anorexia and lethargy, lordosis, and scoliosis were observed. Our results suggest that dietary ascorbic acid supplementation plays more important role than enrichment of rainbow trout eggs with ascorbic acid through immersion bath and it is unlikely that “rebound-scurvy” might occur in alevins and juveniles. No signs of enhanced ascorbic acid degradation (turnover) were observed based on body ascorbate depletion following ascorbate-free diets. Published by Elsevier B.V.

1. Introduction It has been postulated that in rainbow trout the dietary essentiality of AA is due to the absence of the enzyme L-gulonolactone oxidase which catalyzes the conversion of L-gulonolactone to AA in comparison to more ancient non-teleost species of fish, including sturgeon (Moreau and Dabrowski, 2000). The vitamin C requirement for different teleost fish has been well documented (Dabrowski, 2001). This requirement may also vary in fish ontogeny, for instance during gonad maturation or larval metamorphosis (Ciereszko and Dabrowski, 1995). Tissues vary considerably in their concentration of ascorbic acid, and gonads (ovaries ⁎ Corresponding author. Tel.: + 1 614 292 4555; fax: + 1 614 292 7432. E-mail address: [email protected] (K. Dabrowski). 1 Present address: Fisheries Department, Faculty of Natural Resources, The University of Guilan, Sowmeh Sara, P.O. Box 1144, Iran. 0044-8486/$ – see front matter. Published by Elsevier B.V. doi:10.1016/j.aquaculture.2011.08.012

and seminal plasma) represent one of the highest levels, several-fold higher than in blood plasma (Blom and Dabrowski, 1995a; Ciereszko and Dabrowski, 1995). Very limited evidence exists regarding the relation between the maternal status of ascorbic acid in rainbow trout broodstock and egg ascorbic acid concentration (Blom and Dabrowski, 1995b; Dabrowski and Ciereszko, 2001). Also of interest is whether high maternal ascorbate levels convey a benefit to the offspring. An initial experiment by Dabrowski and Blom (1994) on 2-year-old fish during their first reproductive cycle found that, despite a significant difference in ascorbate concentration in eggs [82 versus 316 μg g − 1 in fish fed AA-free and ascorbyl phosphate (AP)-supplemented diet groups, respectively], there was no significant effect of vitamin C depletion on the embryos' survival. Determination of factors that affect eggs and larval quality remains difficult as good criteria are lacking (Kjorsvik et al., 1990), but it has

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been demonstrated that the nutritional status of broodstock can affect offspring quality. The accumulation of essential nutrients in eggs is dependent on the nutrient reserves in the female fish and, therefore, on the dietary intake of broodstock in the period preceding and during gonadogenesis (Bell et al., 1997; Blom and Dabrowski, 1996). In this regard, broodstock nutrition deserves special attention to guarantee optimal survival and development of the larvae/alevins. During the period of endogenous feeding and start of exogenous feeding the uptake of essential nutrients is critical (Lavens et al., 1999). The possible means of enrichment with vitamins in early stages of fish includes immersion in a bath with a high concentration of water-soluble compounds (Falahatkar et al., 2006). Also, enrichment of live prey is used to deliver vitamins to fish (Fernandez et al., 2008). The question of vitamin C dependency, or so called ‘rebound scurvy’ (Olsen and Hodges, 1987) has not been addressed in teleost fish. By examining the depletion of tissue ascorbate in endogenously feeding fish, the effect of enrichment by treatment or by maternal vitamin C status, may be extended to offspring. Fish accumulate high levels of AA in tissues when fed adequate dietary amount, and it provides additional protection against oxidative damage caused by environmental stress. In the earlier studies with golden shiner (Notemigonus crysoleucas) it was demonstrated that dietary ascorbic acid supplementation significantly reduced mortality caused by stress factors (Chen et al., 2004). The present study was designed to examine the effects of dietary ascorbic acid and ascorbate immersion enrichment of early embryos on growth performance, susceptibility to hypoxia, and ascorbic acid retention in rainbow trout alevins hatched from eggs exposed after fertilization to different concentrations and forms of ascorbic acid. 2. Materials and methods 2.1. Collection and transport of gametes Unfertilized gametes of rainbow trout were obtained from J. Perry Egan State Fish Hatchery, Utah Division of Wildlife Resources, Bicknell, UT. Gametes were delivered by overnight shipment to our laboratory in Columbus, OH. Gametes were obtained from 4 females and 4 males. Ova were hand-stripped from each female. Approximately 100 g (~2500 oocytes) were placed into a Ziploc container and placed on crushed ice (0–1 °C) in an isotherm box for transportation. Sperm was collected from each male, and transferred to 0.5 L Ziploc bag container and placed directly on crushed ice. The temperature inside the isotherm box was always lower than 4 °C, and the temperature of ovarian fluid was 1.9 °C upon arrival.

The water was drained after 3 min through a net and fertilized egg were transferred to the ascorbic acid treatments (Falahatkar et al., 2006). Each container was then placed into the cooling system (7.9 °C) for 3 h. After hardening, eggs were rinsed with hatchery water, put in small baskets with a nylon screen bottom and placed into a vertical incubator with flow-through water system (Californiatype hatching trays, Flex-a-Lite Consolidated, Inc., Tacoma, WA, USA). 2.4. Experiment 1 2.4.1. Fish, experimental design and diets Formulations of two casein-gelatin based experimental diets are shown in Table 1. The first diet contained 500 mg/kg L-ascorbyl monophosphate (Showa Denko K. K. NY, USA) (+C) and the second was a negative control with no ascorbic acid supplementation (−C). The dry ingredients of the diets were mixed, oil and water were added, and the diets were pelleted with a laboratory pelleting machine (Hobart, Troy, OH, USA). Pellets were freeze-dried, crushed into 0.5–1 mm particles, and stored at −20 °C until use. Rainbow trout (Oncorhynchus mykiss) alevins (0.09 ± 0.007 g initial average weight) from the eggs formerly exposed to varying forms and concentrations of ascorbic acid (0, 1000 N, 1000 AP, and 2000 AP) were used in the present study. Each fish group with different immersion history was offered +C and −C diets separately. Forty liter aquaria were used and 25 fish per unit were stocked in triplicates. The aquaria were provided with a continuous water flow of dechlorinated, aerated city water at an ambient temperature of 18.1 ± 0.4 °C in a semi-closed recirculation system. Prior to onset of the experiment, five fish were sacrificed and kept at −80 °C for total ascorbic acid (TAA) and dehydroascorbic acid (DHAA) analyses. Fish were fed 6 times per day on the basis of 10% of body weight at the beginning and 3.5% toward the end of 12 weeks of feeding. This feeding rate approached apparent satiation throughout the trial.

Table 1 Composition (%) of the experimental diets fed to rainbow trout. Ingredients 1

Casein Gelatin2 Dextrin3 CPSP4 Lecithin5 Vitamin mixture6 Mineral mixture7 Phosphitan8 CMC9 L-Arginine10 L-Methionine2 L-Lysine11 Choline chloride2

2.2. Experimental stock solutions Four different concentrations of ascorbic acid including 0 (control), 1000 mg L − 1 L-ascorbic acid (Sigma, St Louis, MO, USA) neutralized (N) by 1 M NaOH, 1000 mg L − 1 and 2000 mg L − 1 L-ascorbyl monophosphate (AP) (Phospitan® C, Showa Denko K. K. NY, USA) (50% ascorbic acid) were used (Falahatkar et al., 2006). pH of the solutions was measured using Beckman Φ 72 pH meter (Fullerton, CA, USA). 2.3. Fertilization and incubation Fertilization was carried out as described earlier (Falahatkar et al., 2006). In summary, ova from the four females were pooled together before exposing to the ascorbic acid concentrations, and 10 μL of semen per 4 g (~100 ova) was used for each fertilization. This amounted to approximately 1.5 × 10 9 spermatozoa per egg. Ova were placed into a 100 mL plastic container and sperm was added (on the eggs or just near the eggs). Then 10 mL of dechlorinated city water was added for sperm/ova activation.

1

+C

−C

40.0 8.0 21.25 5.0 14.0 4.0 3.0 0.05 2.0 0.5 0.4 0.8 1.0

40.0 8.0 21.3 5.0 14.0 4.0 3.0 0.0 2.0 0.5 0.4 0.8 1.0

Casein Vitamin Free, MP Biomedicals LLC, OH. MP Biomedicals LLC, OH. 80% water soluble, Dextrin Commercial Grade Type II, MP Biomedicals LLC, OH. 4 Fish hydrolysate C.P.S.P 90, (crude protein 82–84%; crude lipid 9–13% WW) Sopropeche S.A. Cedex, France. 5 Lecithin Soy Refined, MP Biomedicals LLC, OH. 6 Roche Performance Premix (L-Hoffman and Roche, Inc., Nutley, NJ), composition per gram of the vitamin mixture: vitamin A acetate, 2645.50 IU; vitamin D3, 220.46 IU; vitamin E, 44.09 IU; vitamin B12, 13 μg; riboflavin, 13.23 mg; niacin, 61.73 mg; thiamin, 7.95 mg; D-biotin, 0.31 mg. 7 MP Biomedicals LLC, OH, composition (g/100 g): calcium carbonate, 2.1; calcium phosphate dibasic, 73.5; citric acid, 0.227; cupric acid, 0.046; ferric acid citrate (16 to 17% Fe), 0.558; magnesium oxide, 2.5; magnesium citrate, 0.835; potassium iodide, 0.001; potassium phosphate dibasic, 8.1; potassium oxide, 6.8; sodium chloride, 3.06; sodium phosphate, 2.14; and zinc citrate, 0.133. Five mg of Se in the form of sodium selenate was added per kg of the salt mixture. 8 (Mg-L-ascorbyl-2-phosphate), Showa Denko, K.K, Japan. 9 Carboxymethylcellulose, MP Biomedicals LLC, OH. 10 L-Arginine-free base, MP Biomedicals LLC, OH. 11 L-Lysine Monohydrochloride, MP Biomedicals LLC, OH. 2 3

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2.4.2. Hypoxia stress test and oxygen consumption At the end of the first feeding experiment (week 6), 1 L beaker with hatchery water was used to set up hypoxic condition. Oxygen was removed by using a system where O2 was stripped with N2 (Blom et al., 1993) to reach less than 0.5 mg L− 1. Dissolved oxygen levels were monitored to maintain a level of oxygen around b0.5 mg L− 1 for hypoxia and N6.8 mg L− 1 for normoxia (at 18–19 °C water temperature). Oxygen was recorded with an YSI Probe (Model 1100, NexSens Technology). Five fish from each aquarium were removed and kept in the beaker for 30 min. After hypoxia exposure period, 3 fish were placed in closed containers for 15 min to determine oxygen consumption. Oxygen in the container was measured before and after this period. Also in each experiment, a container without fish was used to determine the oxygen consumption of bacteria and other likely organisms that consume oxygen in order to subtract it from the total oxygen consumption in the containers where fish were exposed. 2.5. Ascorbic acid analysis Visceral tissues (digestive tract, liver) or whole fish were homogenized in 50 g L− 1 trichloroacetic acid (TCA) in 250 mmol L− 1 HClO4 containing 0.8 g L − 1 ethylenediaminetetraacetic acid (EDTA). An Omni 5000 spin homogenizer (Omni International, Waterbury, CN, USA) was used, and supernatant was centrifuged at 29,000 g for 30 min in a J2-21 Beckmann centrifuge at 4 °C. Supernatants were tested for ascorbic acid level using the dinitrophenyllhydrazine (DNPH) method modified by Dabrowski and Hinterleitner (1989). 2.6. Experiment 2 Because ascorbic acid exposure history did not affect fish growth and tissue TAA and DHAA concentrations at the end of the first experiment, all fish were pooled into two groups regardless of the forms or concentrations of ascorbic acid exposure after fertilization. All fish with different enrichment history were offered the same diet as was offered during the first experiment. The same rearing system and conditions used in the first experiment were also applied, and 10 fish per unit were stocked in 6 replicates. Fish were fed +C and − C diets over a six-week period. At the end of the experiment, growth performance, feed conversion ratio, and survival were calculated as in experiment 1. Feed intake (% body weight) was calculated following ad libitum feeding between weeks 4 and 6. Four fish were sampled from each experimental unit and the livers from these fish were combined and considered a replicate. Livers were then analyzed for TAA and DHAA. 2.7. Statistical analysis In experiment 1, a two-way ANOVA was used to test the effects of embryonic vitamin exposure and dietary vitamin C on growth performance, oxygen consumption rate and vitamin C levels in the whole body and viscera following validation of the normal distribution of the data. In experiment 2, a one-way ANOVA was used to test the effect of dietary vitamin C on growth, food consumption rate and vitamin C levels in the liver. When differences were found, Duncan's multiple comparison test was used by the SPSS statistical package (version 13.0, SPSS, Chicago, IL). Percentages data were arcsin transformed prior to the analysis. Differences were considered significant at P b 0.05.

3. Results 3.1. Vitamin C analysis prior to the experiment At the beginning of the experiment, TAA and DHAA in fish body did not differ significantly among fish exposed to different ascorbic acid treatments (P N 0.05). It varied from 68.3 ± 5.6 (TAA) and 29.5 ± 5.2 μg g − 1 (DHAA) in control group to 78.2 ± 13.2 (TAA) and 30.6 ± 5.8 μg g − 1 (DHAA) in fish exposed to 2000 AP, respectively (Fig. 1). 3.2. Experiment 1 3.2.1. Growth Six weeks after the first feeding, fish fed +C and − C diets did not have significant differences in growth performance. However, fish increased their body weight up to 12 fold through this period (Table 2). Cumulative mortality was not significantly affected by dietary treatments averaging 4.0%. FCR ranged from 0.66 to 0.69 with no significant difference among dietary treatments. 3.2.2. Tissue ascorbic acid concentration Four weeks after the first feeding, all groups on + C diet had significantly higher (P b 0.05) TAA and DHAA levels in the whole body than those fed − C diet regardless of initial ascorbic acid exposure type or concentration (Fig. 2). Also, differences were significant among − C groups and + C groups with different immersion treatment history at the initiation of embryonic development (P b 0.05). Among fish fed + C diet, those from eggs initially exposed to AP at 2000 mg L − 1 had the highest level of ascorbic acid in their bodies. Among the groups fed − C diet, fish from eggs with no ascorbic acid exposure had the highest TAA level in their bodies. However, at week 6, no significant differences were observed among different groups fed the same diet (P N 0.05). At the end of the feeding trial, there was 40-fold more TAA in the fish body fed +C diet than in those fed −C diet (Table 3). However, DHAA remained at 44.3–50% of TAA in fish when fed +C diet in comparison to −C diet fed fish where DHAA level amounted to 75–87.5% of TAA (Fig. 2). The highest ratio of DHAA/TAA was observed in fish from 1000 N treatment and the lowest in the control fish. TAA results in the viscera showed that amounts in groups fed the −C diet were severely depleted while in groups fed the +C diet a 41–51% increase was observed. However, no significant differences were observed among different immersion groups (Fig. 3). At week 4, the fish fed −C diet of 2000 AP treatment showed a 93.3% decrease in body TAA while fish in 0 (−C) group showed a 91.1% decline. At week 6, 1000 N (−C) and 0 (−C) groups showed 97.8% and 96.9% reduction in TAA, respectively, in comparison to the start of the experiment. The comparison of TAA between week 4 and 6

Ascorbic acid (µg/g)

Feeding coefficients (FCR; dry feed/wet weight gain) were measured. Mortality was recorded daily. Biomass was measured to the nearest ± 0.01 g at 2-week intervals. At week four, two fish per aquarium were sampled to measure TAA, and at week six, four fish per aquarium were sacrificed for TAA analysis; two for the whole body and two for viscera.

101

100 90 80 70 60 50 40 30 20 10 0 0

1000 N

1000 AP

2000 AP

Level of vitamin C (mg/L) Fig. 1. Total ascorbic acid (whole column, TAA) and oxidized ascorbic acid (black column, DHAA) concentrations in whole body of control rainbow trout and those enriched with vitamin C at the beginning of the feeding trial (n = 5). N: neutralized AA; AP: ascorbyl phosphate.

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Table 2 Effects of the experimental diets with (+ C) or without (− C) ascorbic acid on growth performance and survival of rainbow trout with different ascorbate yolk sac exposure history (6 week period; experiment 1).

Treatments 0 (− C) 0 (+C) 1000 N (− C) 1000 N (+C) 1000 AP (− C) 1000 AP (+C) 2000 AP (− C) 2000 AP (+C)

Final weight (g)

Weight gain1 (%)

SGR2 (%)

Survival (%)

FCR3

1.11 ± 0.05 1.15 ± 0.02 1.14 ± 0.00 1.12 ± 0.11 1.09 ± 0.03 1.12 ± 0.05 1.14 ± 0.04 1.10 ± 0.06

1134 ± 55 1172 ± 26 1167 ± 10 1144 ± 127 1106 ± 41 1138 ± 66 1168 ± 46 1115 ± 68

2.5 ± 0.0 2.4 ± 0.0 2.5 ± 0.0 2.5 ± 0.1 2.5 ± 0.0 2.5 ± 0.0 2.5 ± 0.1 2.5 ± 0.0

96.0 ± 4.0 94.6 ± 6.1 96.0 ± 4.0 94.6 ± 4.6 97.3 ± 2.3 96.0 ± 6.9 97.3 ± 2.3 96.0 ± 4.0

0.66 ± 0.01 0.66 ± 0.01 0.63 ± 0.04 0.65 ± 0.07 0.66 ± 0.02 0.67 ± 0.02 0.66 ± 0.02 0.69 ± 0.03

NS

NS

NS

NS

NS

NS

NS

NS

NS

NS

NS

NS

Two-way ANOVA4 Embryonic NS exposure Dietary NS treatment Interaction NS

Level of AA (μg/g) 0

1000 N

1000 AP

2000 AP

Treatments −C +C

1.18 ± 0.36b 48.6 ± 6.6a

0.96 ± 0.40b 49.0 ± 5.0a

1.18 ± 0.44b 49.9 ± 4.7a

1.19 ± 0.31b 49.8 ± 9.6a

Two-way ANOVA1 Embryonic exposure Dietary treatment Interaction

NS S NS

NS S NS

NS S NS

NS S NS

N: neutralized. AP: ascorbyl phosphate. 1 NS = not significant, S = significant.

N: neutralized. AP: ascorbyl phosphate. Values are means from triplicate groups of fish ± standard deviation (SD). 1 WG (weight gain): [(final weight − initial weight) × 100] / initial weight. 2 SGR (specific growth rate): [(loge final weight − loge initial weight) × 100] / duration of the experiment in days. 3 FCR (feed conversion ratio): dry feed intake/wet weight gain. 4 NS = not significant.

indicated a 48.5% decrease in 1000 AP (− C) group and a 41.7% increase in 1000 AP (+ C) group (Fig. 3).

3.2.3. Hypoxia test and oxygen consumption After hypoxia tests at the end of the feeding trial, no significant differences were observed in mortality of fish among the dietary treatments. Among the fish with different embryonic vitamin exposure and different nutritional history, oxygen consumption did not change significantly (Fig. 4).

45

Table 3 Total AA concentration in viscera of rainbow trout fed with or without vitamin C diets after 6 weeks in experiment 1 (n = 6). Means with different superscript letters in a column are significantly different (P b 0.05). No significant differences in growth and survival at this point were recorded.

3.3. Experiment 2 3.3.1. Growth Growth did not differ significantly between dietary treatments until week 4 (Fig. 5). Following this period, growth parameters (final weight, weight gain and SGR) were significantly lower in fish fed − C diet (2.08 ± 1.12 g, 69.5 ± 8.6%, and 1.25 ± 0.12% day − 1, respectively) than those fed + C (2.37 ± 0.11 g, 92.7 ± 6.8%, and 1.56 ± 0.08% day − 1, respectively). Survival was significantly higher (100 vs. 91.6 ± 6.8%) and FCR was significantly better (1.1 ± 0.1 vs.1.9 ± 0.5) in fish fed + C diet. Symptoms caused by the deficiency of vitamin C (lordosis) began to be observed during the second experiment (Fig. 6). The incidence of lordotic fish was around 15%. Feed intake was significantly higher (2 fold) in fish fed + C diet at both week 4 and 6 following an acute ad libitum feeding test (Table 4).

3.3.2. Liver ascorbic acid concentration At the end of the feeding trial (12 weeks), fish fed + C diet had 78.9 ± 6.5 and 18.1 ± 2.6 μg g − 1 TAA and DHAA, respectively, in their livers while only trace amounts of TAA and DHAA were detected in the liver of fish fed −C diet (Table 5).

A Experimental period

a

35

ab b

0 –4 weeks

60

0 –6 weeks

15 5 45

40 y

x

x

x

B

35 25 15 5 0 (-C)

0 (+C)

1000 N (-C)

1000 N 1000 AP 1000 AP 2000 AP 2000 AP (+C) (-C) (+C) (-C) (+C)

Level of vitamin C (mg/L) Fig. 2. Total ascorbic acid (whole column, TAA) and oxidized ascorbic acid (black column, DHAA) concentrations in whole body of rainbow trout fed diets with or without vitamin C after 4 (A) and 6 weeks (B) (n = 6). Different letters indicate significant difference between treatments for TAA (P b 0.05). N: neutralized AA; AP: ascorbyl phosphate.

Loss/gain of TAA (%)

Ascorbic acid (µg/g)

b

25

4 –6 weeks

20 0 -20 -40 -60 -80 -100

0 (-C)

0 (+C)

1000 N (-C)

1000 N 1000 AP 1000 AP 2000 AP 2000 AP (+C) (+C) (+C) (-C) (-C)

Level of vitamin C (mg/L) in immersion treatments Fig. 3. Changes in total ascorbic acid during first experiment from start of feeding to 4, 6 and between 4 and 6 week periods. N: neutralized AA; AP: ascorbyl phosphate.

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103

Oxygen consumption (mg/g fish/15 min)

0.4

A 0.3

0.2

0.1

B

0.0 0 (-C)

0 (+C)

1000N (-C)

1000N 1000AP 1000AP 2000AP 2000AP (+C) (-C) (+C) (-C) (+C)

Fig. 6. Appearance of fish fed diets with (A) or without (B) vitamin C at week 10.

Level of vitamin C (mg/L) Fig. 4. Oxygen consumption by rainbow trout with different early life history after exposure to the hypoxia. Asterisk indicates significant difference between 0 (+ C) group and others (P b 0.05) N: neutralized AA; AP: ascorbyl phosphate.

4. Discussion Saturation of ovarian ascorbic acid levels in broodstock rainbow trout resulted in an increase in egg quantity and superior egg quality (Blom and Dabrowski, 1995b). The effect of vitamin C deficiency extends to early life history. Ascorbic acid deficiency in larval fish has been associated with hyperplasia of collagen and cartilage, scoliosis, lordosis, internal hemorrhages, resorbed opercules, and abnormal support cartilage in gills, spine, and fins with deformation of the jaw and snout (Halver, 2002). We observed that 6 weeks into the first experiment, rainbow trout juveniles, exposed to different levels and forms of ascorbic acid during the early stage of embryonic development (according to the method of Falahatkar et al., 2006), whether fed diets with or without vitamin C, showed no differences in growth parameters (Table 2). Moreover, there were no deficiency symptoms in fish fed vitamin C devoided diet. The comparable growth performance in fish fed diet without vitamin C might have been secured by the low rate of ascorbate degradation and relatively high level at the beginning of the experiment. It seems that the depletion rate of this vitamin was not sufficient to cause a decrease in growth rate and overt deficiency

3.0 Yolk sac history effect

Dietary effect

Individual weight (g)

2.5

a

a (+) vitamin C (- vitamin C (-)

b

2.0

b

1.5

symptoms for the period of 6 weeks and over a 10-fold body weight increase. However, subsequent rearing of fish for another 6 weeks in the second experiment led to vitamin C deficiency symptoms (Fig. 6). Significant decrease in survival and growth performance was clearly demonstrated. In the present study, both the deficiency symptoms and the depression in growth rate were first seen 10 weeks after the initiation of feeding. It seems that in 10 weeks the ascorbic acid depletion in tissues reached a point where metabolic needs were not being met. Similar to our first 8 week results, rainbow trout juveniles fed an ascorbic aciddeficient diet for a prolonged period did not show any decrease in growth rate, gross deficiency signs, or increased mortality (Waagbø et al., 1989). In another study, two groups of rainbow trout were maintained on either 0 or 450 mg kg− 1 dietary vitamin C for 4 months. While the average weight increased from 13.1± 0.1 to 107 ±4.9 g, no effects on growth or mortality were observed, nor were external signs of scurvy visible. At the end of this period, liver vitamin C levels were significantly higher in fish fed the vitamin supplemented diet 93.9 ±17.2 μg g− 1 vs. the vitamin C-free diet 1.9 ±1.1 μg g− 1 (Blom et al., 1999). These results might be caused by a lower vitamin C requirement for biochemical functions with increasing age/size in salmonids, more efficient endogenous reuse, or an increased storage capacity (Hilton et al., 1978; Sato et al., 1978; Waagbø et al., 1989). Blom and Dabrowski (1996) demonstrated that mortality of high ascorbic acid offspring was not affected by the dietary ascorbic acid level until after 18 weeks from initial exogenous feeding in rainbow trout. However, fish fed the marginal ascorbate level (20 mg kg − 1) diet achieved a significantly lower individual weight starting at week 7. Ascorbic acid deficient offspring fed a marginal ascorbic acid diet showed continuously high mortalities, reaching 92.3% after 15 weeks. Feeding a high ascorbic acid diet to ascorbic acid deficient offspring of tilapia led to the rapid improvement of growth and survival, comparable to offspring rich in ascorbic acid, and fed a high amount of this nutrient (Soliman et al., 1986). Apparently, a severe deficiency in

1.0 Table 4 Effects of the experimental diets with or without vitamin C on feed intake of rainbow trout with different early life history in ad libitum feeding test at different times (experiment 2). Feed intake was expressed as % body weight. Means with different superscript letters in a row are significantly different (P b 0.05).

0.5 0.0 0

2

4

6

8

10

12

Time (weeks) Fig. 5. Effect of early life history and dietary treatment on the growth of rainbow trout fed diets with or without vitamin C over 12 weeks. Different letters indicate significant difference between treatments (P b 0.05).

Time of ad libitum feeding

At 4 weeks At 6 weeks

Feed intake (% body weight) −C

+C

1.6 ± 0.5b 1.4 ± 1.1b

2.5 ± 0.3a 3.1 ± 0.3a

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Table 5 Total (TAA) and oxidized (DHAA) ascorbic acid concentrations in liver of rainbow trout juveniles fed diets with and without vitamin C over 12 weeks (n = 6; each experimental unit was considered a replicate; each replicate is the combined sample of 4 livers). Means with different superscript letters in a row are significantly different (P b 0.05). Ascorbic acid form

TAA DHAA

Level of AA (μg/g) −C

+C

0.9 ± 0.2b 0.5 ± 0.1b

78.9 ± 6.5a 18.1 ± 2.6a

ascorbic acid during embryonic development and yolk-sac absorption did not result in an irreversible damage to most of the fish, and those which survived the initial recovery phase were capable of further normal growth. We must point out, however, that the high ascorbic acid offspring diet used in the above study contained 10 times as high ascorbic acid as the level recommended for optimum growth in tilapia by the NRC (1993). Diets containing less than 500 mg kg − 1 ascorbic acid may result in decreased growth and survival of fish from eggs containing less than 5 μg g − 1 ascorbic acid (Blom and Dabrowski, 1995a). Blom and Dabrowski (1996) indicated that in rainbow trout, there was no indication of any long term maternal effect on the half-life of ascorbic acid in their offspring. The long half-life of ascorbic acid in rainbow trout, 30–40 days in the whole body in that study or 17–142 days in individual tissues (Tucker and Halver, 1986), as compared to guinea pigs (3 days, Banay and Dimant, 1962) may protect fish juveniles against more severe “rebound scurvy” effects as reported for guinea pig pups (Basu, 1985; Norkus and Rosso, 1975). After the first six-week period of the present study, the depletion of vitamin C reached the level of detection in fish fed − C diet. The decrease in concentrations in fish fed + C was about 65–70% at week 4 and 45–50% of the initial amount at week 6. At the end of the second experiment, we found high amounts of ascorbic acid in the liver of fish fed + C diet, whereas there was below 2 μg/g of ascorbic acid in the liver of fish fed − C. This finding shows the efficient storage of vitamin C in juvenile rainbow trout. There was an apparent increase (25–40%) in the level of vitamin C from week four through week six (Fig. 3). Fish may start to conserve vitamin C when the level of this vitamin decreases to a certain threshold level (Dabrowski and Ciereszko, 1993). The ontogenetic trend of ascorbate concentrations has been quantified in several species of freshwater and marine fish larvae and juveniles. It is accepted that the total ascorbate (reduced and oxidized) declines in fish ontogeny from levels as high as 150–800 μg g − 1 in the whole body to 5–10 μg g − 1 in severalmonth-old juveniles. These findings may become criteria when dietary levels of ascorbic acid in larval and juvenile fish feeds used in intensive culture are recommended (Dabrowski et al., 1996). Whole body ascorbic acid levels were low after 6 weeks of feeding without ascorbic acid (Fig. 2). This supports earlier findings on the ontogenetic trend in ascorbic acid concentrations in rainbow trout (Dabrowski, 1992). As a result, however, the measured tissue ascorbic acid concentrations were close to the detection limit, thus lacked sufficient accuracy to unequivocally show significant effects of the dietary treatment on tissue storage of the vitamin. Matusiewicz et al. (1994) demonstrated that during the depletion phase of the experiment, fish fed a diet supplemented with ascorbyl monophosphate, the equivalent of 240 mg kg − 1 of ascorbate, had a 100% chance of surviving for about 30 days while only 50% survived after 15 days on a diet supplemented with a two-fold higher level of free ascorbic acid. Survival during the depletion phase was dependent on the supplementation dose during the saturation phase. Blom and Dabrowski (1997) demonstrated that after an initial adaptation phase to 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 ascorbic acid body pool, opening interesting new avenues for ascorbic acid dosing and therapy. Hypoxic and hyperoxic conditions are common occurrences in aquaculture systems and both conditions severely affect fish mortality by impairing energy metabolism in the brain, liver, and muscle tissue (van Raaij et al., 1994). Acute (hours) hypoxia and hyperoxia in trout result in severe physiological changes compared to fish maintained under normoxic conditions (van Raaij et al., 1996). Fish under normoxic conditions acclimated to a confinement challenge, whereas ability of hypoxic or hyperoxic fish to tolerate confinement did not improve (Caldwell and Hinshaw, 1994). Increased oxygen demand imposed by the stress was met by an increased cellular recruitment of red blood cells from the spleen in normoxic fish, and by cellular swelling in hypoxic and hyperoxic fish as compensatory strategies. In the present study, among the fish exposed to hypoxia, there was no significant difference in oxygen consumption. Earlier studies on the relationship between hypoxia and ascorbic acid status in fishes pointed to a protective role of this vitamin. Channel catfish fed without AA supplementation died at higher residual oxygen levels than when they were fed a diet with 78 or 390 mg kg − 1 supplementation of this vitamin (Mazik et al., 1987). Ishibashi et al. (1992) observed that intermittent hypoxia negatively affected the growth and survival of parrot fish fed no-ascorbate diet, whereas 750 mg AA kg − 1 diet fed fish were barely affected. Dabrowski et al. (2004) showed that higher levels of dietary AA have beneficial effects on growth in the hypoxia and normoxia conditions and there seems to be a trend in an increased rate of tissue AA degradation in hyperoxia. The liver ascorbate concentrations were significantly affected by both dietary ascorbate and dissolved oxygen levels by the 12th week, but not by oxygen levels at the final 18th week, in this study. However, the trend of a decrease in tissue ascorbate in fish kept in hyperoxia was observed. Similar to our results, rainbow trout under hypoxic conditions did not show increased oxygen consumption or enhanced swimming activity, indicating that the oxygen transport system in trout is limited, i.e., it has a limited adaptation capacity to the reduced oxygen availability (Bushnell et al., 1984). First, as ascorbic acid is known in hypoxic conditions for its protective role in mitochondrial injury and consequently in maintaining membrane fluidity (integrity), this may result in reduced mitochondrial function (energy supply). We hypothesized that a supplement of vitamin C above “the requirement level for optimum growth” would be necessary to demonstrate its preventive role in hypoxia. Second, hyperoxia through the generation of free radicals and other prooxidants may result in damage to lipids, proteins, and DNA (Arrigoni and DeTullio, 2002). Whole body percentage of the reduced ascorbic acid was decreased to 1.0–1.7% in vitamin C-depleted fish indicating that most of the ascorbate was present as dehydroascorbic acid, an oxidized form. These levels were associated with higher mortality or reduced weight gain in golden shiners fed unsupplemented practical diets (Chen et al., 2004). However, no vertebral deformities (scoliosis and lordosis) or other characteristic signs of ascorbic acid deficiency appeared in golden shiners fed the ascorbate-unsupplemented diet.

5. Conclusions The present experiment established, for the first time, the criteria to be used for evaluating the state of ascorbic acid in the rainbow trout ontogeny, and in salmonids in general, that are related to immersion treatments in ascorbic acid. In conclusion, this study showed that dietary ascorbic acid supplementation played more important role than immersion treatment in growth and ascorbic acid retention. Fish do not appear to develop ascorbate-dependency due to an enhanced provision of vitamin C, and that “rebound scurvy” is a very unlikely event in teleost fish.

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