Carnitine, synthesized in vivo from lysine and methionine, is required for transport of long-chain fatty acids into the mitochondria, which is the site of beta oxidation. Early
research with fish indicated that carnitine increased fatty acid oxidation in tissues of Ž
. Ž
. rainbow trout Bilinski and Jonas, 1970 . More recently, Ji et al.
1996 reported increased fatty acid oxidation in liver of Atlantic salmon fed 3700 mg carnitinerkg diet,
which was accompanied by reduced lipid levels in muscle and viscera. Feeding trials with other species of fish have also demonstrated the lipotropic effects of dietary
Ž carnitine Santulli and D’Amelio, 1986a; Santulli et al., 1988; Burtle and Liu, 1994;
. Jayaprakas et al., 1996 . However, carnitine had no significant effect on tissue lipid
Ž .
concentrations in rainbow trout fed 230 mg carnitinerkg diet Rodehutscord, 1995 or Ž
. hybrid tilapia fed 150 or 300 mg carnitinerkg diet
Becker et al., 1999 . Lipid concentrations in muscle and liver of red sea bream increased as dietary carnitine
Ž .
concentration increased from 75 to 2088 mgrkg diet Chatzifotis et al., 1995 . Dietary carnitine has also been shown to increase growth rates of some species of fish
Ž .
Santulli and D’Amelio, 1986a; Torreele et al., 1993; Keshavanath and Renuka, 1998 , Ž
although a positive response has not been observed in all species Rodehutscord, 1995; .
Ji et al., 1996 . Saltwater fish are apparently able to acquire carnitine from the environment, as well as the diet, as growth rates of European sea bass were significantly
Ž increased when carnitine was dissolved in the water of the rearing tanks Santulli and
. D’Amelio, 1986b . The growth promoting effects of dietary carnitine have generally
been explained by increased utilization of dietary energy resulting from increased Ž
. oxidation of fatty acids Becker et al., 1999 . However, increased fatty acid oxidation
was observed in Atlantic salmon fed 3700 mg carnitinerkg diet without a significant Ž
. Ž
. improvement in weight gain or feed efficiency Ji et al., 1996 . Chatzifotis et al. 1996
reported significant improvements in feed consumption and growth rates, but no change in feed conversion, when red sea bream were fed 2088 mg carnitinerkg diet. Thus, it is
Ž . apparent that the mechanism s of growth enhancement of dietary carnitine is not clearly
understood. The objective of this experiment was to evaluate the effects of dietary carnitine on
growth and body composition of hybrid striped bass.
2. Materials and methods
2.1. Fish and animal husbandry Ž
. Juvenile hybrid striped bass male Morone saxatilis x female M. chrysops were
Ž .
obtained from a commercial producer Keo Fish Farms, Keo, AR and transported to the Purdue University Aquaculture Research Facility. All fish were acclimated to laboratory
conditions for 5 weeks prior to initiation of the experiment. Procedures used during transport, quarantine and experimental period were approved by the Purdue Animal Care
Ž and Use Committee PACUC No. 89-060-98, ‘‘Nutritional Studies with Aquatic Ani-
. mals,’’ Principal Investigator Qualification No. BRO-249 .
The closed recirculating system contained 24 individual 38-l aquaria and was equipped with two submerged filtration tanks for solid material removal and denitrifica-
tion of the water. Water was pumped through a sand filter to each aquarium at a rate of ;
1 lrmin and was maintained at 28 18C throughout the experiment. The diurnal light:dark cycle of the aquaculture facility remained at 16 h light:8 h dark throughout the
study. Groups of 20 randomly chosen fish were stocked into each of 12 aquaria. Fish were
acclimated to the experimental system and their respective diets for three weeks prior to the experiment. Following the acclimation period, the number of fish in each tank was
reduced to 12 so that the total weight of fish in each tank was 160.5 5.0 g. Dietary treatments were randomly assigned to triplicate aquaria. Fish were fed to apparent
satiation twice daily during the 8-week experiment. Water quality was monitored daily and was within acceptable limits throughout the experiment. Dissolved oxygen concen-
trations were not below 5.0 mgrl at any point during the experiment. Ammonia–N and nitrite–N concentrations did not exceed 0.25 and 0.20 mgrl, respectively.
2.2. Diet preparation Ž
Our basal diet was the same as used in previous studies with this hybrid Brown et .
Ž .
al., 1993 . The diet was formulated to provide 34.6 crude protein Table 1 . Casein and gelatin served as intact protein sources and provided a total of 10.1 crude protein.
Ž .
An L-amino acid mixture Table 1 supplied the remaining 24.5 crude protein. The L-amino acid mixture was formulated so the basal diet contained 1.55 arginine
Table 1 Composition of basal diet fed to juvenile hybrid striped bass
Ž .
Ingredient Amount grkg dry mixture
Casein 90.0
Gelatin 18.0
a
Amino acid mixture 249.0
b
Mineral premix 80.0
c
Vitamin premix 3.3
d
STAY-C 25 2.0
Dextrin 250.0
Choline chloride 0.7
Carboxymethylcellulose 20.0
Cellulose 227.1
Menhaden oil 60.0
a
Ž .
Amino acid mixture was formulated so that diets contained g amino acidrkg dry diet : arginine, 15.5; Ž .
histidine, 9.5; isoleucine, 19.3; leucine, 31.9; lysine, 14.0; methionine, 4.7; cyst e ine, 2.6; phenylalanine, 21.0; tyrosine, 20.0; threonine, 18.2; tryptophan, 4.9; valine, 23.5; aspartic acid, 21.9; proline, 21.9; glutamic
acid, 21.8; serine, 21.8; glycine, 21.6.
b
Ž .
Mineral premix consisted of grkg of premix : Na SeO , 0.4; CaCO , 350; NaH PO PH O, 200;
2 3
3 2
4 2
KH PO , 200; MgSO P7H O, 10; MnSO PH O, 2; CuCl P2H O, 1; ZnSO P7H O, 2; FeSO P7H O, 2;
2 4
4 2
4 2
2 2
4 2
4 2
NaCl, 12; KI, 0.1; CoCl P6H O, 0.1; Na MoO P2H O, 0.5; AlCl P6H O, 1; and KF, 1.
2 2
2 4
2 3
2 c
Ž .
Vitamin premix supplied the diets with mgrkg dry diet : retinyl acetate, 40; cholecalciferol, 0.1; DL-a-tocopheryl acetate, 80; menadione, 15; niacin, 168 riboflavin, 22; pyridoxine HCl, 40; thiamin
mononitrate, 45; D-Ca pantothenate, 102, biotin, 0.4; folic acid, 10; vitamin B-12, 0.04; and inositol, 450.
d
Contained 25 ascorbic acid equivalents, as L-ascorbyl-2-polyphosphate.
Ž .
Ž .
Griffin et al., 1994b , 1.40 lysine Griffin et al., 1992 and 0.73 total sulfur amino Ž
. acids Griffin et al., 1994a , thus meeting the dietary requirements of hybrid striped bass
for these amino acids. The remaining dietary essential amino acid concentrations met or Ž
. exceeded the highest known requirements for fish
NRC, 1993 . Dietary choline Ž
concentration was maintained at 500 mgrkg diet with choline chloride Griffin et al., .
Ž .
1994c . The basal diet contained 6.0 lipid menhaden oil and 25.0 carbohydrate Ž
. Ž
. dextrin . The energy:protein ratio E: P of the basal diet was calculated as 35.6 kJrg
protein using physiological fuel values of 16.7, 16.7 and 37.7 kJrg for protein, carbohydrate and lipid, respectively. An E: P ratio of 33.5 kJrg protein is considered
Ž .
near optimum for growth of hybrid striped bass Nematipour et al., 1992 . Ž
. Vitamins with the exception of ascorbic acid and choline chloride and minerals
Ž .
were added to the diets as nutritionally complete premixes Table 1 . Menhaden oil and Ž
reagent grade minerals were obtained from commercial suppliers Omega Protein,
. Ž
Reedville,VA and Sigma, St. Louis, MO, respectively . Vitamins with the exception of .
ascorbic acid , casein, gelatin, dextrin, carboxymethylcellulose, crystalline L-amino Ž
. acids and cellulose were acquired from U.S. Biochemical Cleveland, OH . Roche
Ž .
Nutley, NJ supplied ascorbic acid, as L-ascorbyl 2-polyphosphate. L-carnitine was Ž
. provided by Lonza Fairlawn, NJ .
Four dietary treatments were formulated to contain L-carnitine concentrations of either 0, 50, 250 or 500 mg carnitinerkg diet. Dietary carnitine concentrations were
Ž .
analyzed according to the methods of Parvin and Pande 1977 and contained 2.1, 41.0, 212.0 and 369.7 mg carnitinerkg diet, respectively. Carnitine was added to the diets at
the expense of cellulose. Ž
Dry ingredients were thoroughly mixed in a twin-shell V-mixer Patterson-Kelly, .
Ž East Stroudsburg, PA . Diets were then transferred to a Hobart mixer Hobart, Troy,
. OH where lipid and water were added prior to mixing. Diets were adjusted to pH
Ž .
7.0 0.2 with saturated NaOH Wilson et al., 1977 and pelleted. The diets were air-dried for 48 h then stored under air-tight conditions at y208C until needed.
2.3. Analytical procedures Ž
All fish were anesthetized tricaine methanesulfonate, Argent Chemical, Redmond, .
WA and weighed 24 h after the final feeding. Blood was collected with a 22-guage needle and 1-ml syringe from the caudal vein of three fish in each dietary replicate for
determination of serum carnitine concentrations. Blood samples were allowed to clot on ice and then centrifuged at 3000 = g for 20 min for collection of serum. Serum carnitine
Ž .
concentrations were determined according to the methods of Parvin and Pande 1977 . Three randomly chosen fish were collected from each dietary replicate and frozen at
y208C for subsequent carcass proximate analysis. Fillets were obtained from an additional group of three randomly chosen fish and frozen at y208C for subsequent
proximate analysis. Moisture concentration was determined by drying whole fish or muscle for 24 h in a forced-air oven maintained at 1008C. Crude protein was estimated
from whole-body and muscle nitrogen values that were determined in an elemental
Ž .
nitrogen analyzer LECO, St. Joseph, MI . Ash content was determined by incinerating samples at 6008C for 24 h in a muffle furnace. Lipid concentration of carcass and
Ž .
muscle was determined as described by Folch et al. 1957 .
Table 2 Ž
. Mean initial weightrfish, weight gain increase from initial weights , feed efficiency, feed intakerfish, total
liver lipid concentration and survival of hybrid striped bass fed various levels of dietary L-carnitine
a b
Dietary carnitine Initial weight Weight gain Feed efficiency Feed intake Total liver lipids
Survival Ž
. Ž
. Ž
Ž Ž
Ž Ž .
mgrkg grfish
gr100 g g gainrg
g dry gr100 g
. .
. .
initial weight dry feed
feedrfish dry liver
2.1 13.4
320.9b 0.60
71.3b 25.9
97.2 41.0
13.4 331.8a,b
0.61 71.8b
24.8 97.2
212.0 13.7
300.6b 0.61
65.6b 24.4
97.2 369.7
13.3 381.2a
0.62 81.9a
24.2 100
Pooled SEM 0.1650
15.6670 0.0008
2.8132 2.7708
2.3960
c
Probability 0.4892
0.0342 0.3306
0.0207 0.9720
0.8018
a
Ž .
Means in the same column with the same letter designation were not significantly different P - 0.05 .
b
Total liver lipid values are means of nine replications. The remaining values are means of three replications.
c
Ž .
Probability Pr F of treatment differences as determined by ANOVA.
Livers from an additional group of three randomly chosen fish in each dietary replicate were removed and frozen at y208C for subsequent determination of total lipids
Ž .
using the methods of Folch et al. 1957 . Visceral fat was also removed from each fish Ž
. Ž .
for calculation of intraperitoneal fat ratio IPF IPF = 100rbody weight .
2.4. Statistical analyses Data were analyzed in a completely randomized design using each aquarium as an
experimental unit. The data were subjected to one-way ANOVA using the Statistical Ž
. Analysis System SAS Institute 1990 . Analyses were conducted with dietary treatment
as the independent variable. Duncan’s multiple range test separated mean values when
Table 3 Carcass and muscle composition of juvenile hybrid striped bass fed various levels of dietary L-carnitine
a
Ž .
Ž .
Dietary Intraperitoneal Carcass gr100 g dry weight
Muscle gr100 g dry weight
b
carnitine fat ratio
Moisture Protein Fat
Ash Moisture Protein
Fat Ash
Ž .
Ž mgrkg
gr100 g wet body
. weight
2.1 5.8
64.8 51.9
36.0 13.9
75.3 79.5
18.2 5.2
41.0 5.6
65.7 53.8
35.5 13.4
75.5 79.7
16.6 4.8
212.0 5.7
65.6 52.1
34.3 14.0
75.0 77.8
16.9 4.4
369.7 6.4
64.6 52.1
36.9 13.2
74.5 79.1
18.1 4.9
Pooled SEM 0.3404 0.3528
0.9913 1.1886
0.5463 0.3039
1.0738 1.4926 0.3438
c
Probability 0.4172
0.0969 0.5182
0.5232 0.7356
0.1158 0.5931
0.8256 0.5072
a
Values are means of nine replications.
b
Ž .
Ž .
Intraperitoneal fat IPF ratio was calculated as IPF=100rbody weight .
c
Ž .
Probability Pr F of treatment differences as determined by ANOVA.
Table 4 Serum carnitine concentrations of hybrid striped bass fed various levels of dietary L-carnitine
a
Ž .
Ž .
Dietary carnitine mgrkg Serum L-carnitine umolrl
Total Free
Esters 2.1
4.4 1.7
2.7 41.0
4.6 1.7
2.8 212.0
5.3 1.9
3.4 369.7
7.4 2.7
4.8 Pooled SEM
0.9996 0.4295
0.5951
b
Probability 0.1971
0.3822 0.1318
a
Values are means of three replications.
b
Ž .
Probability Pr F of treatment differences as determined by ANOVA.
significant differences were detected by ANOVA. Accepted level of significance was 0.05.
3. Results
