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 Ž
. The egg-white-based diet used in this study Table 1 was modified from Gatlin and
Ž .
Ž Wilson 1986 . The basal diet was supplemented with three levels of iron 0, 30 and 300
. Ž
. Ž
mgrkg from iron methionine Zinpro, Chaska, MI and three levels of vitamin C 0, 50 .
Ž .
and 3000 mgrkg from
L
-ascorbyl-2-polyphosphate Hoffmann-La Roche, Paramus, NJ Ž
. at each iron level at the expense of cellulose
3 = 3 factorial experiment . The intermediate levels of iron and vitamin C represented optimum concentrations necessary
Ž for good growth and prevention of deficiency signs Lim 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 Ž
. values reported in by NRC 1993 . Diets were prepared as 3-mm diameter, semi-moist
Ž .
Ž .
approximately 25 moisture pellets as described by Lim et al. 1996 . 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 inductively- Ž
. coupled argon plasma ICAP spectrophotometer according to the method of Campbell
Ž .
and Plank 1992 .
Table 1 Composition of basal diet
Ingredient grkg Diet
Ž .
dry matter basis Egg white
399.0 Corn starch
475.8 Cellulose
5.0 Cod liver oil
32.5 Corn oil
32.5
a
Iron-free mineral mix 40.0
b
Vitamin C-free vitamin mix 15.0
Ethoxyquin 0.2
a
Ž .
Contains as 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
Ž .
Contains as 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.
2.2. Fish and feeding Ž
. USDA-ARS strain 103 channel catfish 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 Ž
. diets and fed their respective diets twice daily between 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 Ž
. ICAP spectrophotometer according to the method of Campbell and Plank 1992 . Liver
levels of total vitamin C were analyzed using reverse-phase high performance liquid chromatography with electrochemical detection following the procedures described by
Ž .
Wang et al. 1988 . 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 Ž
. tricaine methanesulfonate MS-222; Argent Chemical, Redmond, WA at 125 mgrl.
Blood samples were collected from the caudal vein with heparinized 27-gauge needles Ž
. Ž
. and tuberculin syringe 20 unitsrml for determination of hematocrit HCT , total cell
Ž .
Ž .
Ž .
TCC and red blood cell counts RBC , and hemoglobin Hb . HTC was determined by Ž
. the microhematocrit method described by Brown 1988 . TCC cell and RBC were
determined by diluting whole blood and enumeration in a hemacytometer. Hemoglobin Ž
. was determined by the total Hb kit Sigma Diagnostics; Sigma, St. Louis, MO which is
a standardized procedure of the cyanomethemoglobin method. Hb values were adjusted
by the cyanomethemoglobin correction factor for channel catfish described by Larsen Ž
. 1964 .
2.5. Collection of peritoneal exudate cells Collection and isolation of peritoneal exudate cells followed the procedure of Klesius
Ž .
and Sealey 1996 . 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
Ž .
treatment were randomly chosen, injected intraperitoneally i.p.
with 0.25 ml of Ž
. squalene Sigma 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 Ž
. Ž
. and injected i.p. with 15-ml sterile, cold phosphate-buffered saline PBS . 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
Ž .
Ž .
Hank’s Balance Salt Solution HBSS without phenol red Gibco, 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
Ž .
Ž .
Boyden 1962 as described by Klesius and Sealey 1996 . Assays were performed in Ž
. triplicate using blind well chemotactic chambers Corning CoStar, Cambridge, MA and
Ž .
8-mm pore diameter polycarbonate membrane filters Nucleopore, Pleasonton, CA Ž
. pre-soaked for 5 min in RPMI-1640 Gibco containing 1 horse serum. In the bottom
Ž . Ž
of the chambers 20 ml of either E. ictaluri exoantigen 6.10 mg proteinrml Klesius,
. 1993 was added together with 180 ml RPMI-1640 q 1 horse serum. In the bottom of
the control chamber only RPMI q 1 horse serum was added. Peritoneal macrophages Ž
were added to the upper compartment of the chamber separated from the bottom .
5
chamber by a filter at a concentration of approximately 5 = 10 cellsrchamber. The Ž
. chambers were incubated on a horizontal platform shaker 100 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 Ž
. E. ictaluri AL-75-94 from a virulent outbreak of ESC was grown in brain–heart
Ž .
Ž .
infusion BHI broth for 24 h and used for bacterial challenge Klesius, 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
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
Ž .
procedure SAS 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
