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Effects of dietary carnitine on growth rates and
ž
body composition of hybrid striped bass Morone
/
saxatilis male x M. chrysops female
Ronald G. Twibell, Paul B. Brown
)Department of Forestry and Natural Resources, Purdue UniÕersity, West Lafayette, IN 47907-1159 USA
Accepted 29 November 1999
Abstract
The effects of carnitine were evaluated in diets fed to hybrid striped bass. The basal diet contained 34.6% crude protein supplied by casein, gelatin and crystalline L-amino acids and 6.0% lipid supplied by menhaden oil. Four dietary treatments contained L-carnitine concentrations of either 2.1, 41.0, 212.0 or 369.7 mgrkg diet. Dietary treatments were fed to apparent satiation twice daily to triplicate groups of hybrid striped bass initially weighing 13.5 grfish. At the end of the 8-week feeding trial, feed intake and weight gain of fish fed 369.7 mg carnitinerkg diet were significantly increased compared to fish fed the basal diet containing 2.1 mg carnitinerkg diet. However, there were no significant differences in feed intake or weight gain among fish fed 2.1, 41.0 or 212.0 mg carnitinerkg diet. Feed efficiency, total liver lipid concentration, intraperitoneal
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fat ratio IPF and proximate composition of muscle and carcass were not significantly affected by dietary carnitine concentration. Serum concentrations of total and free carnitine and carnitine esters were not significantly different among fish fed any of the diets, although values tended to increase with increasing dietary carnitine. Results of this study indicate that growth rate, but not body composition, of hybrid striped bass can be improved with relatively low concentrations of dietary carnitine.q2000 Elsevier Science B.V. All rights reserved.
Keywords: Carnitine; Fish nutrition; Hybrid striped bass; Lipid
1. Introduction
The ability of dietary carnitine to increase growth rates and reduce tissue lipid concentrations has been evaluated in several species of fish, with mixed results.
)Corresponding author. Tel.:q1-765-494-4968; fax:q1-765-496-2422.
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E-mail address: pb@fnr.purdue.edu P.B. Brown .
0044-8486r00r$ - see front matterq2000 Elsevier Science B.V. All rights reserved. Ž .
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
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concentrations in rainbow trout fed 230 mg carnitinerkg diet Rodehutscord, 1995 or
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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
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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 ŽSantulli 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
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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
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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
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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
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Juvenile hybrid striped bass male Morone saxatilis x female M. chrysops were
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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
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and Use Committee PACUC No. 89-060-98, ‘‘Nutritional Studies with Aquatic Ani-.
mals,’’ Principal Investigator Qualification No. BRO-249 .
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
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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.
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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
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Ingredient Amount grkg dry mixture
Casein 90.0
Amino acid mixture was formulated so that diets contained g amino acidrkg dry diet : arginine, 15.5;
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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 PO2 3 3 2 4PH O, 200;2 KH PO , 200; MgSO2 4 4P7H O, 10; MnSO2 4PH O, 2; CuCl2 2P2H O, 1; ZnSO2 4P7H O, 2; FeSO2 4P7H O, 2;2 NaCl, 12; KI, 0.1; CoCl2P6H O, 0.1; Na MoO2 2 4P2H O, 0.5; AlCl2 3P6H O, 1; and KF, 1.2
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
ŽGriffin et al., 1994b , 1.40% lysine Griffin et al., 1992 and 0.73% total sulfur amino. Ž .
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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
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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 Ždextrin . The energy:protein ratio E: P of the basal diet was calculated as 35.6 kJ. Ž . rg 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
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near optimum for growth of hybrid striped bass Nematipour et al., 1992 .
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Vitamins with the exception of ascorbic acid and choline chloride and minerals
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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,
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Reedville,VA and Sigma, St. Louis, MO, respectively . Vitamins with the exception of .
ascorbic acid , casein, gelatin, dextrin, carboxymethylcellulose, crystalline L-amino
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acids and cellulose were acquired from U.S. Biochemical Cleveland, OH . Roche ŽNutley, NJ supplied ascorbic acid, as L-ascorbyl 2-polyphosphate. L-carnitine was.
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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
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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.
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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
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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 aty208C until needed. 2.3. Analytical procedures
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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
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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
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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
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Table 2
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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-carnitinea
b
Dietary carnitine Initial weight Weight gain Feed efficiency Feed intake Total liver lipids Survival
Žmgrkg. Žgrfish. Žgr100 g Žg gainrg Žg dry Žgr100 g Ž .%
. . . .
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 aty208C for subsequent determination of total lipids
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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-carnitinea
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Dietary Intraperitoneal Carcass gr100 g dry weight Muscle gr100 g dry weight b
carnitine fat ratio Moisture Protein Fat Ash Moisture Protein Fat Ash
Žmgrkg. Žgr100 g
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 .
Table 4
Serum carnitine concentrations of hybrid striped bass fed various levels of dietary L-carnitinea
Ž . Ž .
Dietary carnitine mgrkg Serum L-carnitine umolrl
Total Free Esters
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
No significant differences were detected in feed intake or weight gain of hybrid
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striped bass fed 2.1, 41.0 or 212.0 mg carnitinerkg diet Table 2 . However, feed intake and weight gain of fish fed 369.7 mg carnitinerkg diet were significantly increased compared to those of fish fed the basal diet. There were no significant differences in feed efficiency, total liver lipid concentration or survival among fish fed any of the
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dietary treatments Table 2 . Similarly, dietary carnitine had no significant effect on
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proximate composition of carcass or muscle of hybrid striped bass Table 3 . Serum concentrations of total and free carnitine and carnitine esters were not significantly
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effected by dietary carnitine Table 4 . However, those values tended to increase with increasing dietary carnitine.
4. Discussion
A significant increase in weight gain was observed in juvenile hybrid striped bass fed 369.7 mg carnitinerkg diet. Dietary carnitine has also been shown to increase growth
Ž . Ž
rates of rohu Keshavanath and Renuka, 1998 , red sea bream, Chatzifotis et al., 1995,
. Ž . Ž
1996 , tilapia Jayaprakas et al., 1996 , European sea bass Santulli and D’Amelio,
. Ž .
1986a and African catfish Torreele et al., 1993 . In contrast, dietary carnitine did not
Ž . Ž
effect weight gain of channel catfish Burtle and Liu, 1994 , rainbow trout
Rodehuts-. Ž .
cord, 1995 or Atlantic salmon Ji et al., 1996 . It does not appear that low carnitine concentrations impacted those results. For example, carnitine concentrations as low as
Ž .
150 mgrkg diet increased weight gain of tilapia Jayaprakas et al., 1996 , while concentrations of 3700, 1000 and 230 mg carnitinerkg diet had no significant effect on
Ž . Ž .
growth rates of Atlantic salmon Ji et al., 1996 , channel catfish Burtle and Liu, 1994
Ž .
fat and significant increases in protein were observed in both channel catfish and Ž
Atlantic salmon despite the lack of response in weight gain Burtle and Liu, 1994; Ji et .
al., 1996 . In the study with Atlantic salmon, increased palmitate oxidation was detected Ž
in liver tissue and isolated hepatocytes from fish fed supplemental carnitine Ji et al., .
1996 . Thus, a carnitine-induced increase in lipid utilization will not necessarily result in an improvement in growth rates.
A significant increase in feed intake was also observed in hybrid striped bass fed 369.7 mg carnitinerkg diet. Feed consumption data are not available from all studies as fish were often fed a fixed amount based on body weight. Researchers have speculated that increased growth rates of fish fed supplemental carnitine were due to improved feed conversion via increased fatty acid oxidation and increased utilization of dietary energy. Results of the current study indicate that growth rates of hybrid striped bass were increased due to increased feed consumption and not through improved feed utilization, as there were no significant differences in feed efficiency across dietary treatments. Furthermore, increased fatty acid oxidation was observed in liver tissues of Atlantic salmon fed carnitine without a significant improvement in weight gain or feed
conver-Ž . Ž .
sion Ji et al., 1996 . Similar to our results, Chatzifotis et al. 1996 reported significant increases in feed consumption and growth rates in red sea bream fed carnitine with no significant improvement in feed efficiency.
No significant effect of dietary carnitine was detected in tissue composition of hybrid striped bass. The effects of dietary carnitine on proximate composition of fish tissues have been inconsistent. Similar to our findings, dietary carnitine did not alter tissue
Ž . Ž
composition of rainbow trout Rodehutscord, 1995 or hybrid tilapia Becker et al., .
1999 . In contrast, dietary carnitine reduced tissue lipid concentrations in rohu ŽKeshavanath and Renuka, 1998 , tilapia Jayaprakas et al., 1996 , channel catfish. Ž . ŽBurtle and Liu, 1994 , European sea bass Santulli and D’Amelio, 1986a and Atlantic. Ž .
Ž .
salmon Ji et al., 1996 . Dietary carnitine increased lipid concentrations in liver and
Ž .
muscle of red sea bream Chatzifotis et al., 1995 .
The energy:protein ratio of the diets used in the current study was 35.6 kJrg protein, which is close to the optimum of 33.5 kJrg protein determined for this hybrid ŽNematipour et al., 1992 . In that experiment, E: P ratios higher than 33.5 kJ. rg protein resulted in reduced weight gain and accumulation of lipid in the visceral cavity and muscle. That dietary carnitine had no significant effect on tissue lipid concentrations in the current study may be due to the fact that the diets were formulated to provide the optimum E: P ratio, especially since that estimate was based, in part, on reduced tissue lipid concentrations.
Carnitine was detected in serum of hybrid striped bass fed all diets, including the control diet. However, those values were not significantly different among fish fed any of the dietary treatments. In contrast to hybrid striped bass, carnitine was not detected in blood of Atlantic salmon fed a similar purified diet with no supplemental carnitine, but
Ž
was detected in blood and tissues of salmon fed 3700 mg carnitinerkg diet Ji et al., .
1996 . These findings may indicate that relatively high dietary carnitine concentrations are necessary to increase tissue concentrations significantly in hybrid striped bass.
characteristics of hybrid striped bass, it is unlikely that dietary concentrations higher than 400 mgrkg diet would prove economical given the current price of carnitine, which is approximately US$25rkg active ingredient. Much higher levels have been evaluated in previous studies with fish and were shown to increase weight gain and decrease tissue lipid concentrations in some species, but such high concentrations are unlikely to be used in commercial fish feeds. In the current study, an average increase in weight gain of 19% was observed in hybrid striped bass fed 370 mg carnitinerkg diet compared to control fish and may warrant supplementation of carnitine in practical feeds, although a formal evaluation of the economics should be conducted.
Acknowledgements
This research was supported by Lonza and Purdue University Agricultural Research Programs. This paper is technical contribution number 16155, Purdue Agricultural Research Programs.
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