Interaction between dietary levels of iron and vitamin C on growth, hematology, immune response and resistance of channel catfish (Ictalurus punctatus) to Edwardsiella ictaluri challenge

Aquaculture 185 Ž2000. 313–327 www.elsevier.nlrlocateraqua-online

Interaction between dietary levels of iron and vitamin C on growth, hematology, immune response and resistance of channel catfish žIctalurus punctatus/ to Edwardsiella ictaluri challenge Chhorn Lim a,) , Phillip H. Klesius a , Meng H. Li b, Edwin H. Robinson b a

Aquatic Animal Health Research Laboratory, USDA-ARS, MSA, P.O. Box 952, Auburn, AL 36830 USA b Thad Cochran National Warmwater Aquaculture Center, Mississippi State UniÕersity, StoneÕille, MS 38776 USA Accepted 21 October 1999

Abstract Nine egg-white-based diets supplemented with three levels of vitamin C from L-ascorbyl-2polyphosphate Ž0, 50 and 3000 mgrkg. for each of the three levels of iron from iron methionine Ž0, 30 and 300 mgrkg. were each fed to juvenile channel catfish in triplicate aquaria twice daily to apparent satiation for 14 weeks. Fish fed with iron-deficient diets had decreased weight gain, feed conversion and survival. Supplementation of ascorbic acid to the iron-deficient diets further decreased weight gain and survival. Feed conversion was not significantly affected by dietary level of vitamin C or iron and vitamin C interaction. No gross signs of vitamin C deficiency were observed. Total cell count ŽTCC., red blood cell count ŽRBC., hematocrit ŽHCT. and hemoglobin ŽHb. were significantly lower for fish fed the iron deficient diet. TCC and RBC significantly increased when 3000 mg of vitamin Crkg was added. However, in the absence of dietary iron, supplementation of ascorbic acid resulted in significant decrease in HCT and Hb values. Hepatic iron concentration increased with increasing dietary level of iron. Supplementation of high level of ascorbic acid to the diet containing 300 mgrkg of supplemental iron significantly increased the liver iron content. Liver ascorbate increased with increasing dietary level of ascorbic acid. Dietary level of iron and the interaction between iron and vitamin C had no effect on liver content of vitamin C. Mean macrophage migration in the absence or presence of Edwardsiella ictaluri exoantigen was significantly higher for fish fed the iron supplemented diets. The significant effect

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Corresponding author. Tel.: q1-334-887-3741; fax: q1-334-887-2983. E-mail address: [email protected] ŽC. Lim..

0044-8486r00r$ - see front matter q 2000 Published by Elsevie Science B.V. PII: S 0 0 4 4 - 8 4 8 6 Ž 9 9 . 0 0 3 5 2 - X

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of vitamin C was obtained only when high level Ž3000 mgrkg. was used. Neither dietary levels of iron nor vitamin C or their interaction influenced survival of juvenile channel catfish against E. ictaluri 14-day post challenge. However, the onset of mortality was earlier for fish fed the iron-deficient diet. q 2000 Published by Elsevier Science B.V. Keywords: Iron; Ascorbic acid; Hematology; Immune response; Disease resistance

1. Introduction Iron is an essential element for the functioning of organs and tissues of higher animals, including fish, because of its important role in oxygen transport and cellular respiration. Iron is also one of the most important micronutrients in terms of its effect on immune system functions and host defense against infections ŽBeisel, 1982; Bhaskaram, 1988.. Fish can absorb soluble iron from the water across the gill membrane and intestinal mucosa ŽRoedar and Roedar, 1968.. However, feed is considered the major source of iron for fish due to low concentrations of soluble iron in natural waters ŽNRC, 1993.. The total dietary iron requirement for optimum growth, feed efficiency, hematological values and immune response of juvenile channel catfish has been determined to be about 30 mgrkg diet ŽGatlin and Wilson, 1986; Lim et al., 1996; Sealey et al., 1997.. Chemotactic response of peritoneal macrophages to Edwardsiella ictaluri exoantigen was depressed in iron-deficient channel catfish ŽLim and Klesius, 1997; Sealey et al., 1997. but this abnormality was remedied by feeding an iron-replete diet for 4 weeks ŽLim and Klesius, 1997.. The effect of dietary iron on resistance of channel catfish to bacterial pathogens is unclear, although Sealey et al. Ž1997. suggested that high level of dietary iron Ž180 mgrkg. may lead to increased susceptibility. Ascorbic acid or vitamin C is essential for most fishes, including channel catfish ŽNRC, 1993.. The requirement varies depending on the metabolic function and form of vitamin C used. Using ascorbic acid as the dietary source, a level of 30 mgrdiet was adequate for optimum growth, but 60 mgrkg was needed for prevention of all deficiency signs ŽAndrews and Murai, 1975; Lim and Lovell, 1978; Li and Lovell, 1985.. When a stable form of vitamin C ŽL-ascorbyl-2-polyphosphate. was used, 11 to 45 mgrkg diet was adequate for growth and prevention of deficiency signs ŽEl Naggar and Lovell, 1991; Robinson, 1992.. Evidence of the role of ascorbic acid in fish immunity and disease resistance is not consistent, although numerous studies have shown that feeding fish ascorbic acid at levels higher than those required for growth and prevention of deficiency signs enhanced their immune response and resistance to bacterial challenges. Ascorbic acid is known to be involved in the metabolism of iron in animals including fish ŽHarper, 1975; Monsen, 1982; Hilton, 1989; NRC, 1993.. Ascorbic acid enhances the absorption of iron from the intestine by reducing ferric iron to the ferrous state, a more soluble form that is readily absorbed ŽEl-Alwary et al., 1975; Harper, 1975; Monsen, 1982.. Ascorbic acid is also involved with adenosine triphosphate ŽATP. in the release and reduction of the ferric iron from ferritin and its subsequent incorporation with iron-binding protein, apoferritin and transferrin, into tissue ferritin ŽMazur et al., 1960; Harper, 1975..

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No studies have been conducted on the combined influence of dietary levels of iron and ascorbic acid on the response of channel catfish. Thus, this study was conducted to evaluate the interaction between dietary levels of iron and ascorbic acid on growth, hematology, immune response and resistance of channel catfish to E. ictaluri challenge.

2. Materials and methods 2.1. Experimental diets The egg-white-based diet used in this study ŽTable 1. was modified from Gatlin and Wilson Ž1986.. The basal diet was supplemented with three levels of iron Ž0, 30 and 300 mgrkg. from iron methionine ŽZinpro, Chaska, MI. and three levels of vitamin C Ž0, 50 and 3000 mgrkg. from L-ascorbyl-2-polyphosphate ŽHoffmann-La Roche, Paramus, NJ. at each iron level at the expense of cellulose Ž3 = 3 factorial experiment.. The intermediate levels of iron and vitamin C represented optimum concentrations necessary for good growth and prevention of deficiency signs ŽLim and Lovell, 1978; Lim et al., 1996; Gatlin and Wilson, 1986; Robinson, 1992.. The diets were formulated to contain approximately 34% crude protein and 3.1 kcal of digestible energyrg based on feedstuff values reported in by NRC Ž1993.. Diets were prepared as 3-mm diameter, semi-moist Žapproximately 25% moisture. pellets as described by Lim et al. Ž1996.. Pellets were broken into small pieces and stored at y188C until needed. Iron content of the basal diet without iron supplementation was determined to be 9.2 mg ironrkg by an inductivelycoupled argon plasma ŽICAP. spectrophotometer according to the method of Campbell and Plank Ž1992..

Table 1 Composition of basal diet Ingredient

grkg Diet Ždry matter basis.

Egg white Corn starch Cellulose Cod liver oil Corn oil Iron-free mineral mix a Vitamin C-free vitamin mix b Ethoxyquin

399.0 475.8 5.0 32.5 32.5 40.0 15.0 0.2

a

Contains Žas grkg of premix.: calcium carbonate, 300.0; potassium phosphate, dibasic, anhydrous, 319.0; sodium phosphate, monobasic, 200.34; magnesium sulfate heptahydrate, 132.0; zinc sulfate heptahydrate, 3.00; sodium chloride, 43.50; cobalt chloride, 1.00; manganous sulfate monohydrate, 0.80; cuprous chloride, 0.20; potassium iodide, 0.15; sodium selenite, 0.011. b Contains Žas grkg of premix.: retinyl acetate, 1.20; cholecalciferol, 0.17; menadione, 3.33; DL-alpha tocopheryl acetate, 4.00; inositol, 10.00; choline chloride, 150.0; niacin, 9.00; riboflavin, 2.00; pyridoxine hydrochloride, 2.00; thiamin hydrochloride, 2.00; D-calcium pantothenate, 6.00; biotin, 0.31; folic acid, 0.18; cyanocobalamin, 0.0027; celufil, 809.807.

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2.2. Fish and feeding USDA-ARS strain 103 channel catfish Ž Ictalurus punctatus . fingerlings from a single spawn which had been maintained at the USDA Aquatic Animal Health Research Laboratory on commercial diets to an average weight of 10.6 " 0.1 g were randomly stocked into twenty-seven 110-l aquaria at a density of 100 fish per aquarium. Aquaria were supplied with flow-through dechlorinated tap water at a rate of 0.6–1.2 lrmin. Water flow rates were checked and adjusted daily to insure proper water exchange rate. Water temperature was maintained by a centralized heater at 26 " 28C. Water was continuously aerated and photoperiod was maintained at 12:12 h light:dark schedule. The water contained less than 0.5 mg ironrl. Fish in triplicate aquaria were randomly assigned to each of the nine experimental diets and fed their respective diets twice daily Žbetween 0730–0800 and 1430–1500 h. to satiation for a period of 14 weeks. During each feeding, feed was offered by hand three to four times until satiation was reached. The quantity of feed consumed was recorded daily by calculating the differences in weights of feeds prior to the first and after the last feeding. All aquaria were cleaned once weekly by scrubbing and siphoning accumulated wastes. On cleaning days fish were fed only once in the afternoon. Fish in each aquarium were counted and weighed collectively at biweekly intervals. No feeding was done on sampling days. 2.3. Measurement of liÕer contents of iron and ascorbic acid At the end of 12 weeks, five fish from each of the triplicate tanks were randomly taken and sacrificed for measurement of liver concentrations of iron and ascorbic acid. Livers from each of the five fish from the same aquarium were pooled to obtain one composite sample and stored at y808C. Iron content of the liver was determined by an ICAP spectrophotometer according to the method of Campbell and Plank Ž1992.. Liver levels of total vitamin C were analyzed using reverse-phase high performance liquid chromatography with electrochemical detection following the procedures described by Wang et al. Ž1988.. 2.4. Hematological assay Blood samples were obtained from fish at the end of week 14. Two to five fishrtank for the treatment fed the diet containing 0 mg iron and 3000 mg vitamin Crkg, and five fish from each tank of the other treatments were randomly chosen and anesthetized with tricaine methanesulfonate ŽMS-222; Argent Chemical, Redmond, WA. at 125 mgrl. Blood samples were collected from the caudal vein with heparinized 27-gauge needles and tuberculin syringe Ž20 unitsrml. for determination of hematocrit ŽHCT., total cell ŽTCC. and red blood cell counts ŽRBC., and hemoglobin ŽHb.. HTC was determined by the microhematocrit method described by Brown Ž1988.. TCC cell and RBC were determined by diluting whole blood and enumeration in a hemacytometer. Hemoglobin was determined by the total Hb kit ŽSigma Diagnostics; Sigma, St. Louis, MO. which is a standardized procedure of the cyanomethemoglobin method. Hb values were adjusted

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by the cyanomethemoglobin correction factor for channel catfish described by Larsen Ž1964.. 2.5. Collection of peritoneal exudate cells Collection and isolation of peritoneal exudate cells followed the procedure of Klesius and Sealey Ž1996.. At the end of week 14, three fishrtank for fish fed the diet containing 0 mg iron and 3000 mg vitamin Crkg, and five fish from each tank of other treatment were randomly chosen, injected intraperitoneally Ži.p.. with 0.25 ml of squalene ŽSigma. and transferred into 57-l aquaria where they continued to be fed the various experimental diets. Five to seven days later, fish were anesthetized with MS-222 and injected Ži.p.. with 15-ml sterile, cold phosphate-buffered saline ŽPBS.. Then, PBS was removed along with the squalene-elicited exudate cells using a 20-gauge needle attached to a three-way valve into a 50-ml centrifuge tube. The peritoneal fluids of five fish from the same tank were combined and centrifuged at 300 = g for 10 min. The supernatant was discarded, and the cells suspended in calcium- and magnesium-free Hank’s Balance Salt Solution ŽHBSS. without phenol red ŽGibco, Grand Island, NY. for chemotaxis assay. Cell counts and viability were established following enumeration with a hemocytometer in 5% Trypan blue counting solution. 2.6. Chemotaxis assay Chemotaxis was determined by a modification of the lower-surface method of Boyden Ž1962. as described by Klesius and Sealey Ž1996.. Assays were performed in triplicate using blind well chemotactic chambers ŽCorning CoStar, Cambridge, MA. and 8-mm pore diameter polycarbonate membrane filters ŽNucleopore, Pleasonton, CA. pre-soaked for 5 min in RPMI-1640 ŽGibco. containing 1% horse serum. In the bottom of the chambers 20 ml of either E. ictaluri exoantigen Ž6.10 mg proteinrml. ŽKlesius, 1993. was added together with 180 ml RPMI-1640q 1% horse serum. In the bottom of the control chamber only RPMIq 1% horse serum was added. Peritoneal macrophages were added to the upper compartment of the chamber Žseparated from the bottom chamber by a filter. at a concentration of approximately 5 = 10 5 cellsrchamber. The chambers were incubated on a horizontal platform shaker Ž100 rpm. for 100 min at 258C. Following incubation, filters were removed, inverted, placed on a slide, attached with clear finger nail polish and stained with Leukostat. The number of macrophages on the surface of the filter were counted in five fields of triplicate filters at 100 = . 2.7. E. ictaluri challenge E. ictaluri ŽAL-75-94. from a virulent outbreak of ESC was grown in brain–heart infusion ŽBHI. broth for 24 h and used for bacterial challenge ŽKlesius, 1992.. At the end of week 14, 20 fish from each of three replicate tanks per diet were randomly selected, placed in perforated 5-gallon plastic buckets and immersed for 1 h in static, aerated aquaria containing 1.7–2.0 = 10 7 cellsrml of E. ictaluri. Each group of fish was then returned to their respective aquarium with flowing water adjusted to 0.5–0.6

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lrmin. Water flow and feeding were discontinued for the first 24 h after challenge. Mortality was monitored and recorded twice daily before feeding for 14 days. 2.8. Statistical analysis Data were analyzed by a two-way analysis of variance using the general linear model procedure ŽSAS Institute, Carey, NC, 1993.. Duncan’s multiple-range test was used to determine significant differences due to dietary iron, vitamin C, and iron and vitamin C interactions. When a main effect was found significant but without interaction effect, the differences between treatment means were determine by Duncan’s multiple range test. If a significant interaction was observed, the differences between simple effects were determined by Student’s t-test. Differences were considered significant at the 0.05 probability level.

3. Results Mean final weight gain, feed conversion ratio ŽFCR. and survival are given in Table 2. Fish fed diets without iron supplementation had significantly lower overall weight gain. There were no significant differences among the weight gain of fish fed the 30 or 300 mg supplemental ironrkg diet. Weight gain was also significantly affected by dietary levels of vitamin C. Fish fed diets without vitamin C supplementation and diets supplemented with 50 mg vitamin Crkg had similar mean weight gain. When vitamin C

Table 2 Mean final weight gain per fish1 , survival and feed conversion ratio of channel catfish fed diets containing various levels of iron and vitamin C 2 Iron added Žmgrkg. 0

Vitamin C added Žmgrkg.

0 50 3000 30 0 50 3000 300 0 50 3000 Statistical analysis Ž P level. Iron Vitamin C Iron=vitamin C 1

Weight gain Žg.

Feed conversion ratio 3

Survival Ž%.

35.8"1.5 b 33.3"3.3 bc 22.2"2.9 c 49.2"6.4 a 56.2"5.9 a 48.9"5.3 a 49.7"1.2 a 56.7"4.5a 46.3"6.1ab

1.4"0.09 b 1.6"0.07 a 1.6"0.08 a 1.4"0.07 b 1.4"0.04 b 1.3"0.05 b 1.4"0.01b 1.4"0.02 b 1.4"0.05 b

97.8"1.3 a 88.2"2.7 a 56.9"11.7 b 98.5"1.5a 94.8"3.2 a 98.5"1.5a 100.0"0.0 a 94.8"4.1a 97.8"2.2 a

0.0001 0.05 NS

0.005 NS 4 NS

0.0038 0.0003 0.0008

Initial weights10.6"0.1 g. Column means Ž"S.E.. having the same superscript are not significantly different Ž P ) 0.05.. 3 Feed conversions Dry feed fed Žg.rwet weight gain Žg.. 4 NSs Not significant. 2

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was added at the excessive level Ž3000 mgrkg., a significant reduction of weight gain was observed. There were no interactions between dietary levels of iron and vitamin C for weight gain. Mean FCR was poorest for iron-deficient diets but significantly improved when the diets were supplemented with iron. The addition of vitamin C had no effect on FCR. Likewise, there were no interactions between dietary levels of iron and vitamin C for FCR. Survival was significantly affected by dietary levels of iron, vitamin C and their interaction. Fish fed the iron-deficient diets had significantly lower survival than those fed the 30 mg and 300 mg iron diets. There were no significant differences among the survival of fish fed the 0 and 50 mg vitamin C diets. However, increasing vitamin C to 3000 mgrkg diet significantly decreased survival. Survival was lowest for fish fed the iron-deficient diet supplemented with 3000 mg of vitamin C. No gross signs of vitamin C deficiency were observed in catfish fed vitamin C-deficient diets with or without iron supplementation. Mean hematological values of channel catfish fed diets containing various levels of dietary iron and vitamin C are presented in Table 3. The treatment without iron supplementation had the lowest TCC and RBC. These values significantly increased at each incremental level of iron. The effect of vitamin C on these parameters was significant only when an excessive level was added. When fish were fed iron-deficient diets, TCC and RBC significantly increased as dietary vitamin C level increased. However, in the presence of dietary iron, significant increases of TCC and RBC were obtained only when 3000 mg vitamin Crkg was used. Vitamin C level had no effect on HTC and Hb values. These values were lowest for fish fed iron-deficient diets but significantly increased when dietary iron was added. In the absence of dietary iron, supplementation of vitamin C significantly reduced HTC and Hb. When dietary iron was added, Hb and HTC values were not affected by the addition of vitamin C.

Table 3 Mean total cell count, red blood cell count, hematocrit and hemoglobin of channel catfish fed diets containing various levels of iron and vitamin C for 14 weeks1 Iron added Žmgrkg. 0

Vitamin C added Žmgrkg.

0 50 3000 30 0 50 3000 300 0 50 3000 Statistical analysis Ž P level. Iron Vitamin C Iron=vitamin C 1

Total cell count Ž10 6 rml.

Red blood cell Ž10 6 rml.

Hematocrit Ž%.

Hemoglobin Žgrdl.

1.70"0.04 c 1.90"0.02 b 2.33"0.04 a 1.96"0.03 b 1.75"0.05 c 2.39"0.04 a 1.97"0.01b 1.95"0.02 b 2.34"0.03 a

1.55"0.03 c 1.75"0.02 b 2.15"0.04 a 1.80"0.04 b 1.57"0.05 c 2.22"0.03 a 1.80"0.02 b 1.82"0.02 b 2.16"0.03 a

23.9"0.9 c 15.5"0.8 d 14.7"1.3 d 34.4"2.4 b 36.3"1.3 b 32.5"3.0 b 35.7"1.8 b 37.7"1.4 ab 42.4"2.6 a

5.88"0.37 c 3.02"0.34 d 3.57"0.37 d 10.74"0.54 b 12.50"0.54 a 11.58"0.45ab 10.23"0.41b 12.21"0.59 a 12.58"0.47 a

0.0006 0.0001 0.0001

0.0004 0.0001 0.0001

0.0001 NS 0.0001

0.0001 NS 0.03

Values represent the means of ns9–15 determinationsrtreatment. Column means Ž"S.E.. having the same superscript are not significantly different Ž P ) 0.05..

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Table 4 Mean iron and vitamin C content of livers of channel catfish after 12 weeks of feeding diets containing various levels of iron and vitamin C 1 Iron added Žmgrkg.

Vitamin C added Žmgrkg.

0

0 50 3000 30 0 50 3000 300 0 50 3000 Statistical analysis Ž P level. Iron Vitamin C Iron=vitamin C

Iron content Žmgrg.

Vitamin C content Žmgrg.

41.3"3.8 c 31.2"7.5 c 36.8"7.9 c 64.7"4.9 c 63.6"6.3 c 65.9"2.5 c 288.6"6.7 b 321.6"43.7 b 492.4"31.2 a

5.7"1.3 d 71.3"28.6 c 187.7"13.0 a 12.0"7.4 dc 53.8"9.8 c 137.0"8.9 b 3.2"0.4 d 52.1"9.3 c 167.8"22.5ab

0.0001 0.002 0.006

NS 0.0001 NS

1 Values represent means of ns 2–3 determinationsrtreatment. Column means having the same superscript are not significantly different Ž P ) 0.05..

Liver iron content was significantly affected by dietary levels of iron, vitamin C and their interactions ŽTable 4.. Fish fed iron-deficient diets had the lowest liver iron content but was not significantly different from the group fed diets containing 30 mg of ironrkg.

Table 5 Mean macrophage migration and chemotaxis ratio of channel catfish fed diets containing various levels of iron and vitamin C for 14 weeks1 Iron added Žmgrkg. 0

Vitamin C added Žmgrkg.

0 50 3000 30 0 50 3000 300 0 50 3000 Statistical analysis Ž P level. Iron Vitamin C Iron=vitamin C 1

0 ug Exoantigen

Mean macrophage migration 50 ug Exoantigen

Macrophage chemotaxis ratio 2

0.60"0.07 cd 0.71"0.10 cd 1.00"0.12 c 0.63"0.12 cd 0.78"0.12 cd 3.09"0.12 a 0.78"0.04 cd 0.56"0.09 d 1.87"0.33 b

3.00"17 d 2.71"0.18 d 5.09"0.20 c 4.30"0.49 cd 2.91"0.15d 14.42"1.06 a 3.20"0.23 d 3.16"0.14 d 11.91"1.41b

0.83"0.02 0.80"0.02 0.84"0.01 0.88"0.02 0.80"0.02 0.82"0.01 0.80"0.01 0.78"0.07 0.85"0.03

0.0001 0.0001 0.0001

0.0001 0.0001 0.0001

NS NS NS

Values represent means of ns6–9 determinationsrtreatments. Column means having the same superscript are not significantly different Ž P ) 0.05.. 2 Macrophage chemotactic ratio represents the number of migrating cells with E. ictaluri exoantigen divided by the sum of the number of migrating cells without Žcontrol. and with E. ictaluri exoantigen.

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Table 6 Mean days of first death and cumulative mortality of channel catfish fed diets containing various levels of iron and vitamin C at 14 days post-immersion challenge with E. ictaluri 1 Iron added Žmgrkg. 0

Vitamin C added Žmgrkg.

0 50 3000 30 0 50 3000 300 0 50 3000 Statistical analysis Ž P level. Iron Vitamin C Iron=vitamin C

Days to first mortality

Cumulative mortality Ž%.

4.3"1.2 abc 2.0"1.5 c 3.5"1.5 bc 5.7"0.3 ab 6.0"1.0 ab 5.6"0.3 ab 6.0q0.6 ab 7.3"1.3 a 7.0"0.6 a

66.7"12.0 85.0"12.6 72.5"2.5 66.7"16.9 50.0"21.8 98.3"1.7 81.7"10.1 33.3"13.0 65.0"17.6

0.0021 NS NS

NS NS NS

1 Values represent means of ns 2–3 determinationsrtreatment. Column means having the same superscript are not significantly different Ž P ) 0.05..

These values, however, were significantly lower than those of fish fed diets with 300 mg ironrkg. Also, for the group of fish fed the 300 mg-iron diets, increasing dietary vitamin C level increased the liver iron content but the effect became significant only when 3000 mgrkg was used. Liver vitamin C content was not affected by dietary iron level or the interaction between dietary levels of iron and vitamin C. However, increasing dietary level of vitamin C significantly increased liver content of vitamin C. Mean macrophage migration in the absence or presence of E. ictaluri exoantigen was significantly affected by dietary levels of iron, vitamin C and their interactions ŽTable 5.. Significantly higher mean macrophage migration without or with the presence of exoantigen was obtained in fish fed the 30-mg iron diets. The significant effect of vitamin C on these parameters was observed only when the vitamin was added at 3000 mgrkg. However, the effect of dietary iron, vitamin C or their interaction was not significant when the values were expressed in terms of chemotaxis ratio. The average number of days at which the first mortality occurred after E. ictaluri exposure was significantly earlier for fish fed the iron-deficient diets but did not differ for fish fed diets containing supplemental iron ŽTable 6.. This parameter was not affected by vitamin C or the interaction between vitamin C and iron. Cumulative mortality of fish at day 14 following E. ictaluri challenge was not affected by either dietary iron, vitamin C or their interaction ŽTable 6.. 4. Discussion Fish fed the diets without iron supplementation had significantly lower weight gain, poorer feed conversion, and reduced hematological values Žtotal blood cell count, RBC, HTC and Hb. as previously reported for channel catfish by Gatlin and Wilson Ž1986.,

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Lim et al. Ž1996. and Lim and Klesius Ž1997.. Lim et al. Ž1996., however, did not detect the adverse effect of iron deficiency on feed conversion. The significant decrease in survival observed in this study has also been reported by Lim and Klesius Ž1997. when catfish were fed the iron-deficient diet for more than 13 weeks. Gatlin and Wilson Ž1986. and Lim et al. Ž1996., however, indicated that dietary iron had no effect on the mortality of channel catfish. Ascorbic acid has been reported to be involved in the metabolism of iron in fish ŽHilton, 1989; Maage et al., 1990; Andersen et al., 1998.. In our study, however, there was no interaction between dietary levels of vitamin C and iron on weight gain and feed conversion, indicating that the growth response of channel catfish to varying levels of iron from iron methionine was not affected by the presence of ascorbic acid. No gross signs of vitamin C deficiency were observed in catfish fed the vitamin C-deficient diets for 14 weeks. This is in contrast to earlier studies which showed that channel catfish fed vitamin C-deficient diets exhibited growth retardation, poor feed conversion ŽLi et al., 1993. and developed characteristic signs of scurvy ŽLovell, 1973; Wilson and Poe, 1973; Andrews and Murai, 1975; Lim and Lovell, 1978; Lovell and Lim, 1978; Robinson, 1992.. Significant interactions between dietary levels of ascorbic acid and iron were observed for the survival and hematological values. At each level of iron supplementation, TCC and RBC significantly increased when vitamin C was added at 3000 mgrkg diet. This increase in the number of red blood cells was probably the direct effect of ascorbic acid on erythropoiesis as has been suggested by Dinning Ž1962. and Cox Ž1968.. The development of macrocytic anemia has been reported for channel catfish ŽLim and Lovell, 1978., rainbow trout ŽHilton et al., 1978., snakehead ŽAgrawal and Mahajan, 1980. and hybrid tilapia ŽShiau and Jan, 1992. fed ascorbic acid deficient diets. In contrast, we observed that supplementation of ascorbic acid to iron-deficient diets resulted in decreased HTC, Hb and survival. Anemic conditions were not observed in fish fed the iron supplemented diets with or without addition of vitamin C. Thus, high levels of dietary vitamin C in the absence of dietary iron may accelerate iron deficiency and increase the severity of microcytic anemia in channel catfish. Depletion of stored iron in fish may occur sooner for fish fed the iron-deficient diets supplemented with vitamin C since this vitamin plays an important role in the release of ferritin-bound iron from the livers as well as in the transport of plasma iron to the liver and its subsequent incorporation into tissue ferritin ŽMazur et al., 1960.. A hepatic iron concentration of 30–40 mgrg found in fish fed the iron-deficient diets may be unavailable for utilization and probably be indicative of microcytic anemia in channel catfish. Andersen et al. Ž1996. showed that a hepatic iron concentration of 30 mgrg was insufficient to maintain erythropoiesis and prevent the reduction of blood Hb concentration in Atlantic salmon. Andersen et al. Ž1998. reported that addition of iron exceeding the requirement did not increase hepatic iron in Atlantic salmon. Baker et al. Ž1997. showed that liver iron as well as muscle and serum iron concentrations of African catfish were unaffected by feeding diets containing 663.5 or 6354.4 mg ironrkg, even though fish fed the higher iron diet had significantly reduced growth. In the present study, hepatic iron concentrations increased with increasing dietary iron, although a significant increase was noted when this mineral was supplemented at 300 mgrkg diet Ž10 times the requirement

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level.. The values for fish fed the 30 mg iron diets, although not statistically different from those of fish fed the iron unsupplemented diets, were 53% to more than 100% higher. Liver iron levels were also significantly affected by increasing dietary levels of ascorbic acid. However, significant increase in hepatic iron levels was observed only in fish fed the diet supplemented with 300 mg of iron and 3000 mg of ascorbic acid. With rainbow trout, Hilton et al. Ž1978. found an increase in hepatic iron but a decrease in spleen iron levels with increasing dietary levels of vitamin C. For the same species, Dabrowski and Kock Ž1989. reported that ascorbic acid increased the absorption of ferrous iron, while Lanno et al. Ž1985. found that vitamin C did not appear to enhance the absorption of this form of iron. The lack of a positive effect of increased level of ascorbic acid on the uptake of the ferrous iron by salmonids was because the reduced form of iron ŽFeq2 . is readily available for salmonids ŽHilton, 1989; Andersen et al., 1998.. In the present study, even though iron methionine has been shown to have bioavailability equal to or higher than the ferrous iron from iron sulfate for channel catfish ŽLim et al., 1996; Paripatananont and Lovell, 1997., there was a significant interaction between dietary levels of iron and ascorbic acid. Thus, further studies to evaluate the effect of ascorbic acid on iron metabolism are needed. Hepatic ascorbic acid concentrations increased as dietary ascorbic acid levels increased as have been shown in previous studies with channel catfish ŽLim and Lovell, 1978; Durve and Lovell, 1982; Robinson, 1992; Li and Robinson, 1994; Li et al., 1993, 1998.. A liver vitamin C level below 30 mgrg has been suggested as an indication of vitamin C deficiency ŽLim and Lovell, 1978.. In the present study, although no ascorbic acid deficiency signs were observed, fish fed the diets without vitamin C supplementation had liver vitamin C content as low as 3.2–2.0 mgrg. Robinson Ž1992. indicated that liver ascorbate levels may be indicative of vitamin C status of channel catfish but the exact concentration is difficult to recommend. He found that catfish fed diets containing 45–60 mg of vitamin Crkg which is adequate to prevent deficiency signs had liver ascorbate levels of 16.5 and 18.2 mgrg, respectively. Dietary iron levels had no effect on hepatic vitamin C concentration which is in agreement with previous studies in Atlantic salmon ŽAndersen et al., 1997, 1998.. In rainbow trout, Desjardins Ž1985. observed a decrease in hepatic ascorbic acid concentration with increasing dietary iron levels. However, it was suggested that this apparent interaction was probably due to the effect of iron supplementation on diet rancidity and destruction of ascorbic acid during feed processing and storage. In the present study, diet rancidity and destruction of ascorbic acid were minimal because ethoxyquin was added, the diets were cold-pelleted and stored frozen until fed, and the stable form of vitamin C, L-ascorbyl-2-polyphosphate, was used. Iron deficiency or excess suppressed macrophage chemotactic response, although the effect was more pronounced in fish fed the iron-deficient diets. This is in agreement with Berger Ž1996. who reported that either a deficiency or an excess of iron could compromise the immune system. It has also been reported that iron-deficiency had no affect on the specific antibody response ŽSealey et al., 1997. but decreased macrophage chemotaxis in channel catfish ŽLim and Klesius, 1997; Sealey et al., 1997.. The suppression of macrophage chemotaxis, however, was reversed when the iron-deficient fish were fed the iron-replete diet for 4 weeks ŽLim and Klesius, 1997..

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The mean number of macrophage migration decreased for fish fed the vitamin C-deficient or replete diets but significantly increased when a high level of vitamin C Ž3000 mgrkg diet. was added. The stimulating effect of high dietary level of ascorbic acid on macrophage chemotaxis was observed at all levels of dietary iron supplementation. Li and Lovell Ž1985. found that megadose feeding of ascorbic acid Ž3000 mgrkg. had no effect on phagocytosis but significantly enhanced antibody production and serum complement activity. Li et al. Ž1998., however, found no difference in post-infection antibody levels among catfish fed diets containing 3–257 mg vitamin Crkg. Likewise, Johnson and Ainsworth Ž1991. found no difference in percentage phagocytosis, phagocytic index of anterior kidney neutrophils of channel catfish fed diets containing 100 or 1000 mg ascorbic acidrkg. In guinea pigs, Ganguley et al. Ž1976. observed that macrophages from vitamin C-deficient animals had both normal phagocytic and bactericidal capabilities. However, the migratory ability of the peritoneal exudate cells from deficient animals on a glass surface was significantly impaired as compared to controls. Dietary levels of iron had no effect on cumulative mortality of channel catfish 14 days after exposure to E. ictaluri but iron-deficient fish were more susceptible to infection than those with adequate iron as can be seen by the low number of day to first death. This agrees with the results of a recent study by Lim and Klesius Ž1997., which showed that dietary iron did not protect against mortality of channel catfish from E. ictaluri, but the onset of mortality of iron-deficient fish was earlier than those fed the iron-replete diet. Sealey et al. Ž1997., however, observed increased mortality of iron-deficient channel catfish due to ESC. With Atlantic salmon, it has been reported that fish fed a low-iron diet had some protection against Vibrio anguillarum ŽS.P. Lall, Institute of Marine Biosciences, NRC, Halifax, Canada, personal communication.. Ravndal et al. Ž1994. observed a significant association between high concentrations of serum iron and mortality of Atlantic salmon infected with V. anguillarum. Sherman Ž1992. suggested that a delicate balance exists between the need for iron for host defense mechanisms and the need for iron to sustain microbial growth. The results of previous studies on the effect of ascorbic acid on disease resistance in channel catfish are not consistent. Earlier studies with channel catfish showed that high levels of ascorbic acid increased their resistance against E. ictaluri ŽLi and Lovell, 1985; Liu et al., 1989. and E. tarda ŽDurve and Lovell, 1982.. Li et al. Ž1993. found higher mortality for channel catfish fed vitamin C-free diet but increasing the vitamin C concentration higher than the level required for growth did not improve disease resistance. In a more recent study, however, Li et al. Ž1998. showed that cumulative mortality of catfish 21 days post-challenge with E. ictaluri was lower in fish fed the basal diet Ž3 mg vitamin Crkg. than those fed diets containing supplemental vitamin C. In salmonids, information on the effects of ascorbic acid on disease resistance is also contradictory. High levels of vitamin C have been shown to improve resistance of rainbow trout against V. anguillarum ŽNavarre and Halver, 1989. and Atlantic salmon against Aeromonas salmonicida ŽHardie et al., 1991; Waagbo et al., 1993.. Other studies, however, found no significant effect of high dietary vitamin C level on the resistance of Atlantic salmon infected with A. salmonicida or V. anguillarum ŽLall et al., 1989; Erdal et al., 1991; Hardie et al., 1991.. In our study, dietary levels of vitamin C had no significant effect on the onset of mortality after exposure to E. ictaluri. The

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differences between the results of these studies may be related to differences in species, strain, size and nutritional status of the fish used, feeding management and duration, pathogenicity of the bacteria, and methods and dose of challenge. Results of the present study indicate that iron deficiency resulted in microcytic anemia as has been previously reported. In the absence of dietary iron, supplementation of ascorbic acid may accelerate iron deficiency as can be seen by the significant decrease in weight gain, survival, HTC and Hb values. Hepatic iron content was directly related to the dietary level of iron. At high level of dietary iron Ž300 mgrkg., supplementation of high level of vitamin C Ž3000 mgrkg. further increased liver iron content. Ascorbic acid at 3000 mgrkg were needed to stimulate macrophage chemotaxis. However, neither dietary iron nor ascorbic acid protected juvenile channel catfish from E. ictaluri infection, although iron deficiency accelerate the onset of mortality. In the absence of clear cut information and because nutrient deficiency predisposes the animals to infection due to synergistic effects of nutrient deficiency and infection, adequate levels of iron and vitamin C to meet the requirements for growth and prevent deficiency signs should be used. However, more studies are needed to better understand the interrelationships between dietary levels of iron and ascorbic acid on iron metabolism, immune response and disease resistance.

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