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
Ž .
Mean final weight gain, feed conversion ratio FCR 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 fish
1
, survival and feed conversion ratio of channel catfish fed diets containing various levels of iron and vitamin C
2
Iron added Vitamin C
Weight gain Feed conversion
Survival
3
Ž .
Ž .
Ž . Ž .
mgrkg added mgrkg
g ratio
b b
a
35.81.5 1.40.09
97.81.3
bc a
a
50 33.33.3
1.60.07 88.22.7
c a
b
3000 22.22.9
1.60.08 56.911.7
a b
a
30 49.26.4
1.40.07 98.51.5
a b
a
50 56.25.9
1.40.04 94.83.2
a b
a
3000 48.95.3
1.30.05 98.51.5
a b
a
300 49.71.2
1.40.01 100.00.0
a b
a
50 56.74.5
1.40.02 94.84.1
ab b
a
3000 46.36.1
1.40.05 97.82.2
Ž .
Statistical analysis P level Iron
0.0001 0.005
0.0038
4
Vitamin C 0.05
NS 0.0003
Iron=vitamin C NS
NS 0.0008
1
Initial weight s10.60.1 g.
2
Ž .
Ž .
Column means S.E. having the same superscript are not significantly different P 0.05 .
3
Ž . Ž .
Feed conversions Dry feed fed g rwet weight gain g .
4
NSs Not significant.
Ž .
was added at the excessive level 3000 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 weeks
1
Iron added Vitamin C
Total cell count Red blood cell
Hematocrit Hemoglobin
6 6
Ž .
Ž .
Ž .
Ž .
Ž . Ž
. mgrkg
added mgrkg 10 rml
10 rml grdl
c c
c c
1.700.04 1.550.03
23.90.9 5.880.37
b b
d d
50 1.900.02
1.750.02 15.50.8
3.020.34
a a
d d
3000 2.330.04
2.150.04 14.71.3
3.570.37
b b
b b
30 1.960.03
1.800.04 34.42.4
10.740.54
c c
b a
50 1.750.05
1.570.05 36.31.3
12.500.54
a a
b ab
3000 2.390.04
2.220.03 32.53.0
11.580.45
b b
b b
300 1.970.01
1.800.02 35.71.8
10.230.41
b b
ab a
50 1.950.02
1.820.02 37.71.4
12.210.59
a a
a a
3000 2.340.03
2.160.03 42.42.6
12.580.47 Ž
. Statistical analysis P level
Iron 0.0006
0.0004 0.0001
0.0001 Vitamin C
0.0001 0.0001
NS NS
Iron=vitamin C 0.0001
0.0001 0.0001
0.03
1
Ž .
Values represent the means of ns9–15 determinationsrtreatment. Column means S.E. having the Ž
. same superscript are not significantly different P 0.05 .
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 Vitamin C
Iron content Vitamin C content
Ž .
Ž .
Ž .
Ž .
mgrkg added mgrkg
mgrg mgrg
c d
41.33.8 5.71.3
c c
50 31.27.5
71.328.6
c a
3000 36.87.9
187.713.0
c dc
30 64.74.9
12.07.4
c c
50 63.66.3
53.89.8
c b
3000 65.92.5
137.08.9
b d
300 288.66.7
3.20.4
b c
50 321.643.7
52.19.3
a ab
3000 492.431.2
167.822.5 Ž
. Statistical analysis P level
Iron 0.0001
NS Vitamin C
0.002 0.0001
Iron=vitamin C 0.006
NS
1
Values represent means of ns 2–3 determinationsrtreatment. Column means having the same super- Ž
. script are not significantly different P 0.05 .
Liver iron content was significantly affected by dietary levels of iron, vitamin C and Ž
. their interactions Table 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 weeks
1
Iron added Vitamin C
Mean macrophage migration Macrophage
2
Ž .
Ž .
mgrkg added mgrkg
chemotaxis ratio 0 ug Exoantigen
50 ug Exoantigen
cd d
0.600.07 3.0017
0.830.02
cd d
50 0.710.10
2.710.18 0.800.02
c c
3000 1.000.12
5.090.20 0.840.01
cd cd
30 0.630.12
4.300.49 0.880.02
cd d
50 0.780.12
2.910.15 0.800.02
a a
3000 3.090.12
14.421.06 0.820.01
cd d
300 0.780.04
3.200.23 0.800.01
d d
50 0.560.09
3.160.14 0.780.07
b b
3000 1.870.33
11.911.41 0.850.03
Ž .
Statistical analysis P level Iron
0.0001 0.0001
NS Vitamin C
0.0001 0.0001
NS Iron=vitamin C
0.0001 0.0001
NS
1
Values represent means of ns6–9 determinationsrtreatments. Column means having the same super- Ž
. script are not significantly different P 0.05 .
2
Macrophage chemotactic ratio represents the number of migrating cells with E. ictaluri exoantigen Ž
. divided by the sum of the number of migrating cells without control and with E. ictaluri exoantigen.
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 Vitamin C
Days to first Cumulative
Ž .
Ž .
Ž . mgrkg
added mgrkg mortality
mortality
abc
4.31.2 66.712.0
c
50 2.01.5
85.012.6
bc
3000 3.51.5
72.52.5
ab
30 5.70.3
66.716.9
ab
50 6.01.0
50.021.8
ab
3000 5.60.3
98.31.7
ab
300 6.0q0.6
81.710.1
a
50 7.31.3
33.313.0
a
3000 7.00.6
65.017.6 Ž
. Statistical analysis P level
Iron 0.0021
NS Vitamin C
NS NS
Iron=vitamin C NS
NS
1
Values represent means of ns 2–3 determinationsrtreatment. Column means having the same super- Ž
. script are not significantly different 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 Ž
significantly affected by dietary levels of iron, vitamin C and their interactions Table .
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
Ž .
for fish fed diets containing supplemental iron Table 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 Ž
. dietary iron, vitamin C or their interaction Table 6 .
4. Discussion
