most beneficial approach to increasing nutrient density of diets generally involves excluding ingredients with low protein and energy contents which are often poorly
digested. This strategy was evaluated with red drum and observed to have positive Ž
. effects on water quality of a closed recirculating system Jirsa et al., 1997 .
A previous experiment in our laboratory demonstrated that high dietary energy levels resulted in lower ammonia production by red drum but compromised weight gain and
Ž .
body composition McGoogan and Gatlin, 1999 . Additionally, high dietary energy seemed to have a more beneficial effect at a reduced feeding rate, resulting in favorable
growth rates and reduced nitrogenous waste production. Increasing dietary crude protein Ž
. CP provided greater weight gain but with higher ammonia excretion. Taking these
findings into consideration, the present study was conducted using the protein level Ž
. 45 CP previously determined to support most rapid weight gain and graded energy
levels to determine if an energy level could be ascertained which would provide a reduction in ammonia production without adversely affecting weight gain or body
composition. Because higher dietary energy levels may result in reduced intake, a second experiment was conducted to determine the effects of increasing dietary protein
levels in conjunction with energy while reducing feeding rates to allow adequate dietary protein and energy intake. Although protein and energy are known to have profound
effects on growth and body composition of fish, much less is known about the effects of these manipulations on plasma metabolites and enzyme activities associated with
ammonia production, and therefore, these aspects were also studied.
2. Materials and methods
2.1. Dietary treatments Diets used in the first experiment contained 45 CP and graded levels of energy
Ž ranging from 15.1 to 18.4 kJ estimated digestible energyrg diet Serrano et al., 1992;
. McGoogan and Gatlin, 1998 with alterations in energy accomplished by varying levels
Ž .
of menhaden oil and dextrin Table 1 . In the second experiment, three diets were Ž
. Ž
. formulated Table 1 with increasing levels of digestible protein 33, 40, and 50 and
Ž .
energy 13, 15.5, and 18 kJrg
to meet previously determined requirements for Ž
. maximum growth of red drum McGoogan and Gatlin, 1998 using digestibility determi-
Ž .
nations for low-temperature processed menhaden fish meal Gaylord and Gatlin, 1996 . The diet containing the highest protein and energy levels met the red drum’s require-
ment for digestible protein but was low in energy due to limited space in the formulation. These three diets were fed by hand at decreasing fixed rates of 6, 5, or 4
Ž .
body weight BW rday corresponding to inversely increasing protein and energy levels to provide approximately the same amount of digestible protein and energy each day.
These dietrfeed rate treatments were designated 33r13 at 6, 40r15.5 at 5 and 50r18 at 4. An additional treatment in the second experiment consisted of the diet with intermedi-
ate protein and energy being fed to apparent satiation over a 30-min period, at both daily
Ž .
feedings 40r15.5 at satiate . Procedures for diet preparation and storage were previ- Ž
. ously described McGoogan and Gatlin, 1999 .
B.B. McGoogan,
D.M. Gatlin
III r
Aquaculture
182 2000
271 –
285 274
Table 1 Diet formulations containing various energy levels and different protein and energy levels for red drum in experiments 1 and 2. Values are expressed as gr100 g dry
diet
a
Diet designation Experiment 1
Experiment 2 Diet ingredient
45 CP: 15.1 kJ 45 CP: 15.9 kJ
45 CP: 16.7 kJ 45 CP: 17.6 kJ
45 CP: 18.4 kJ 33r13 at 6
40r17.5 at 5 50r18 at 4
b
Menhaden fish meal 67.44
67.44 67.44
67.44 67.44
57.97 69.56
86.95
c
Menhaden fish oil 9.55
11.48 13.42
15.36 19.13
3.62 4.40
4.05
d
Dextrin 5.81
6.45 7.10
7.74 4.43
3.61 4.35
e
Mineral premix 4.00
4.00 4.00
4.00 4.00
4.00 4.00
4.00
f
Vitamin premix 3.00
3.00 3.00
3.00 3.00
3.00 3.00
3.00
d
Carboxymethyl cellulose 2.00
2.00 2.00
2.00 2.00
2.00 2.00
2.00
d
Cellulose 8.20
5.63 3.04
0.46 0.00
25.80 12.69
f
Formulated to contain Ž
. Energy: protein kJrg
33.5 35.2
37.2 38.9
41.0 39.3
38.9 36.0
Analyzed composition
g
Ž .
dry-matter basis Crude protein
45.0 45.2
45.8 46.0
45.0 37.8
44.2 56.1
Crude lipid 15.7
17.8 21.5
23.8 26.0
10.3 12.5
13.8
a
Ž .
Ž . In experiment 1, each diet contained 45 crude protein CP and graded levels of estimated digestible energy kJ . In experiment 2, each diet contained different
Ž .
digestible protein and energy levels DPrkJ to be fed at a specific percent BWrday.
b
Omega Protein, Houma, LA, USA.
c
Omega Protein, Reedville, VA, USA.
d
United States Biochemical, Cleveland, OH, USA.
e
Ž .
Same as Moon and Gatlin 1991 .
f
Ž .
Diets for experiment 1 were formulated based on CP and estimated digestible energy Serrano et al., 1992 whereas diets for experiment 2 were formulated based on Ž
. DP and energy Gaylord and Gatlin, 1996 .
g
Values represent means of three replicate samples.
2.2. Experimental procedures Ž
. The experiments were conducted in 110-l aquaria containing 80 l of water con-
nected as a recirculating system with salinity maintained at 6‰ by the addition of Ž
. synthetic sea salt Fritz Aquaculture, Dallas, TX . Water quality was maintained within
acceptable ranges for red drum with biological and mechanical filtration and constant aeration. Water temperature in the first and second experiment averaged 26 and 28
8C, respectively. A 12:12 h light:dark cycle were achieved with timed fluorescent lighting.
At the initiation of the first experiment, red drum, Sciaenops ocellatus, weighing approximately 35 g were selected for uniformity of size and sorted into groups of 10
Ž .
weighing 353.6 3.1 grgroup mean SD and placed into each aquarium. For the Ž
. second experiment, 12 fish, averaging 41.7 1.3 grgroup ; 3.5 grfish , were placed
in each aquarium. In both experiments, each dietary treatment was fed to fish in three replicate aquaria.
Fish in the first experiment were initially fed at 4 BWrday and this was lowered equally among all treatments during the course of the experiment to 3 BWrday. Three
Ž treatments of fish in the second experiment were fed at decreasing fixed rates 6 to 4
. BWrday with increasing dietary protein and energy density to allow for similar daily
protein and energy provisions with consumption of the entire ration. All fish fed at fixed rates in both experiments had half their daily ration divided equally between morning
and evening feedings. In the second experiment, fish fed to apparent satiation were given a small quantity of feed every several minutes during a 30-min period in the
morning and evening until active feeding ceased. One day each week, fish within each aquarium were weighed collectively and this weight was used to adjust feed quantities.
The first experiment was conducted for 6 weeks while the second was continued for 8 weeks.
2.3. Weekly and terminal sampling and analysis One day each week in both experiments, ammonia in each aquarium was monitored
Ž .
every 2 h for a total of 16 h postprandial 10 h total in the second experiment . After the last feeding period on the day before ammonia was to be monitored, excess feed or
particulate wastes were removed, and aquarium walls were scrubbed to detach nitrifying bacteria. For the feeding period after which ammonia was to be monitored, fish were
given 30 min to consume their ration and then the recirculating nature of the system was stopped by turning off the pump. Water samples from each aquarium were measured for
ammonia with a Hach spectrophotometer, to indicate initial levels. These initial ammo- nia measurements were subtracted from measurements at each subsequent time point to
determine change in aquarium ammonia. At the conclusion of the ammonia measure- ments, one aquarium from each treatment also had fish removed so that the decline in
ammonia was monitored over time as a measure of nitrification and degassing losses from aquaria. Ammonia measurements were computed as a function of fish biomass in
each aquarium for expression of ammonia produced per kilogram BW.
Red drum in the second experiment were also monitored for consumption of diets one morning and one afternoon each week. After the preceding feeding period, tanks were
cleaned by siphoning fecal and any uneaten feed debris. For consumption measurements, Ž
. fish were fed one-half of their daily ration pre-weighed and allowed 30 min to
consume this quantity before siphoning uneaten feed into containers with a small-diame- ter hose. Feed and water obtained was filtered through pre-weighed coarse filter paper
with the aid of a vacuum pump. Samples were then dried at 60 8C overnight, weighed
and subtracted from the quantity initially provided to quantify feed intake. At the conclusion of the first experiment, one fish from each aquarium at each of five
time points, ranging from 0 to 16 h postprandial, was bled for plasma glucose analysis. Blood was first centrifuged for 5 min after which supernatant was removed with a
transfer pipette. This was then deproteinized with perchloric acid and neutralized with Ž
. potassium carbonate using the method of Brock et al. 1994 . Plasma samples were
Ž stored frozen at y80
8C until glucose was analyzed Sigma Kit a510; Sigma, St. Louis, .
MO . Additional sampling from the first experiment involved muscle and liver tissue Ž
. being excised, wrapped in foil and stored frozen y80
8C until being analyzed for proximate composition. Liver tissue from 2 and 16 h time points was also analyzed for
enzymatic activity as described later. Muscle, liver, and intraperitoneal fat were taken Ž
. from seven fish per aquarium one fish per aquarium from the second experiment for
Ž determination of the following body indices: hepatosomatic index HSI: liver weight =
. Ž
. Ž
. 100rBW , intraperitoneal fat IPF ratio IPF weight = 100rBW , and muscle ratio
Ž .
MR: muscle weight = 100rBW . Ž
. Glutamate dehydrogenase GLDH activity was determined using the procedure of
Ž .
Schmidt and Schmidt 1983 while glutaminase and glutamine synthetase activity were Ž
. analyzed using the procedure of Chamberlin et al. 1992 . All enzyme assays were run at
Ž .
28 8C and activity was expressed per milligram liver protein Lowry et al., 1951 .
Ž .
Differences between means were tested for significance P - 0.05 using the GLM Ž
. procedure
SAS Institute, 1985 . Duncan’s multiple-range test was used to detect significant differences among means.
3. Results
