A fish feed composition to reduce mortality and/or improve growth in juvenile salmon upon transfer to seawater. The fish feed includes L-carnitine in admixture with juvenile salmon feed compositions sufficient to improve gluconeogenesis and/or glycogen reserves during smoltification and adaption to seawater.

FIELD OF THE INVENTION 
The invention relates to the effects of L-carnitine on the growth, 
metabolism and body composition of juvenile Atlantic salmon (Salmo salar) 
during smoltification. 
BACKGROUND AND BRIEF SUMMARY OF THE INVENTION 
Sea-type salmon such as Atlantic salmon (Salmo salar) and coho salmon 
(Oncorhynchus kisutch) return from salt water to fresh water rivers to 
spawn, thereby generating fresh water fry. Within generally a two-year 
period or so, the salmon pass through the parr development stage and 
become smolt, in which state they can return to the sea for salt water 
maturation. The time period during which the fish remain smoltified is, 
however, quite limited, usually just several weeks for the Atlantic 
salmon. 
When this process is simulated in hatcheries, the same limited period of 
smoltification exists. If the smolts are not seasonably introduced to sea 
water they revert back to parr characteristics, unsuited for salt water 
survival and growth. In nature, another year of freshwater life as parr is 
required before smolting again occurs. However, in the hatchery operation, 
they do not have occasion to revert back to parr and in the seawater they 
either die or their growth is stunted. 
Outward manifestations of the conversions from the parr to the smolt are 
the loss of parr markings (black bars on the lateral surface), the turning 
silver of the skin, the osmotic adaptations of gills and kidneys, and the 
stretching or lengthening and thinning of the fish into a streamlined form 
with pointed heads and tails. For a limited time, in smoltified condition, 
the fresh-water-grown fish is ready for the stress and shock of 
introduction into salt water and subsequent life therein. If introduction 
is delayed the fish reverts back to its parr characteristics, regaining 
spots or stripes, losing the silver and becoming dull or lead-color and 
darker, losing scales, losing the streamlining, and fattening out with 
rounding of its head and tail. 
The harvesting of salmon via aquaculture is currently practiced and various 
methods have been described, see for example, U.S. Pat. No. 3,777,709, 
4,385,589 and 4,509,458. When smolts are transferred to seawater or 
seawater pens there is often a low survival rate, usually owing to the 
transfer outside of the narrow window of peak smoltification. 
In contrast to mammals, fish have the unusual ability to maintain liver 
glycogen (animal starch). An exception to this is salmon during 
smoltification. During smoltification, energy metabolism is accelerated 
and liver glycogen is virtually depleted, despite heavy feeding. Smaller 
molecules, such as lactate (produced from muscle contraction) and amino 
acids (generated by digestion of a high protein diet) are reassembled to 
create sugar and glycogen (starch). This enzymatic sequence of about a 
dozen reactions is called gluconeogenesis. As with other carnivores, 
salmon are largely dependent upon gluconeogenesis to provide blood sugar 
(glucose) and glycogen stores. 
Glycogen in liver and muscle is an important source of energy, as is blood 
sugar. There are some published reports on changes in blood sugar with 
smoltification but these reports are contradictory. In recent studies, 
seasonal changes in liver glycogen in juvenile Atlantic salmon were 
measured and it was found the amount declined steadily from January to a 
minimum value at or near peak smoltification in April/May (unpublished 
results, FIG. 1). A key regulatory enzyme controlling gluconeogenesis is 
pyruvate carboxylase (PC). Season changes in PC activity followed closely 
seasonal changes in liver glycogen (unpublished results, FIG. 2). 
Accordingly, work was undertaken to determine whether impaired 
gluconeogenesis might be responsible for the reduced levels of blood 
glucose and liver glycogen observed during smoltification. Since lactate 
is a superior gluconeogenic substrate in Atlantic salmon, lactate was used 
as the gluconeogenic precursor in the measurements. Since PC is widely 
viewed as the rate-controlling enzyme in lactate-dependent 
gluconeogenesis, PC activity was assayed in the same samples of liver. 
Reduced blood sugar and depletion of liver glycogen during smoltification 
was found to be associated with a 43-54% reduction in the capacity of the 
liver for lactate-dependent gluconeogenesis. In addition, reduced capacity 
for gluconeogenesis, was found to be associated with a 64-75% decline in 
PC activity (unpublished results, Table I). 
TABLE 1 
__________________________________________________________________________ 
Biochemical measurements in parr and smolts 
Measure Parr Smolt Post smolt 
__________________________________________________________________________ 
Gill Na.sup.+ /K.sup.+ -ATPase 
6.8 .+-. 1.0 
41.1 .+-. 6.5* 
10.0 .+-. 3.2.dagger. 
(.mu.mol Pi/mg protein/hr) 
(Jan.; n = 4) 
(n = 4) 
(n = 4) 
Plasma glucose 2.0 .+-. 0.3 
-- 1.04 .+-. 0.16* 
(mg/ml) (age-matched; n = 6) 
(n = 6) 
Liverglycogen 95.3 .+-. 26.8 
-- 31.2 .+-. 16.4* 
(mg/g liver) (Jan.; n = 6) (n = 6) 
Pyruvate carboxylase activity 
67.8 .+-. 44 
16.7 .+-. 4.5* 
24.5 .+-. 4.7*.dagger. 
(nmol product/mg protein/min) 
(Jan.; n = 6) 
(n = 6) 
(n = 6) 
90.5 .+-. 4.7 
(age-matched; n = 6) 
Lactate-dependent gluconeogenesis 
3.98 .+-. 0.54 
2.26 .+-. 0.44* 
1.83 .+-. 0.31*.dagger. 
(.mu.mol/g liver/hr) 
(Jan.; n = 4) 
(n = 6) 
(n = 3) 
3.45 .+-. 0.62 
(age-matched; n = 3) 
__________________________________________________________________________ 
*Values are different from the corresponding value shown for parr (P &lt; 
0.05). 
.dagger.Values are different from the corresponding value shown for smolt 
(P &lt; 0.05). 
In the above experiments, parr were assayed 3-4 months before 
smoltification, or age-matched cohorts that failed to undergo 
smoltification. Values shown are means .+-.SD (number of samples assayed). 
Glucose and glycogen were assayed by enzymatic methods. Oubain-sensitive 
release of Pi from ATP by homogenates of gill filaments in detergent 
provided values for Na.sup.+ /K.sup.+ -ATPase. Pyruvate-dependent 
oxidation of NADH by mitochondrial lysates incubated with excess malate 
dehydrogenase and substrates for both enzymes gave values for pyruvate 
carboxylase activity. Incorporation of radiolabeled lactate into glucose 
by suspensions of liver cubes provided the measure of gluconeogenesis. 
The invention generally relates to diet supplements, particularly those 
including carnitine, for juvenile salmon for preventing blood sugar and 
liver glycogen depletion during smoltification. The use of a carnitine 
feed supplement for preventing stress in catfish grown via aquaculture is 
described in U.S. Pat. No. 5,030,657. The use of L-carnitine as a diet 
supplement for fish and shellfish is also known to enhance the health of 
the fish and/or increase resistance to pathogens, see Japanese Application 
55-24343, Feb. 27, 1989 and U.S. Pat. Nos. 4,960,795; 5,401,797; and 
Santulli, A. and D'Amelio, V. D., The Effects of Carnitine on the Growth 
of Sea Bass, J. Fish Biol., 28:81-86, 1986; Santulli, A.; Madica, A.; 
Curatolo, A.; and D'Amelio, V. D.; Carnitine Administration to Sea Bass, 
Aquaculture, 68:345-351, 1989. However, the ability of carnitine to 
enhance gluconeogenesis has not been described. 
It has been observed that smolt liver exhibits about half the gluconeogenic 
capacity of that exhibited by parr liver and concluded that reduced levels 
of blood glucose and liver glycogen during smoltification may reflect 
impaired gluconeogenesis. 
To determine why gluconeogenesis might be impaired in smolts, the activity 
of the primary regulatory enzyme in the gluconeogenic pathway (pyruvate 
carboxylase, or PC) was measured and found to be reduced some 75% during 
smoltification. This determination suggested that genetic expression of PC 
is suppressed and that if the activity of PC could be increased, capacity 
for gluconeogenesis might be restored and the blood glucose and liver 
glycogen increased. 
It has now been discovered that parr fed a diet supplemented with carnitine 
exhibit more than triple the PC activity observed in parr fed 
carnitine-free diet. This increased PC activity was associated with more 
than a doubling in the capacity for gluconeogenesis and a 5.4.times. rise 
in muscle glycogen. If prevention of the reduction in gluconeogenesis and 
resultant loss of liver glycogen and blood glucose can similarly be 
achieved in smolts, the increase in energy reserves could improve the 
yield, i.e., greater survival and/or growth of the smolt upon transfer to 
seawater.

DETAILED DESCRIPTION OF THE INVENTION 
The suggestion that dietary carnitine might improve the capacity for fatty 
acid oxidation was supported by evidence that palmitate oxidation by 
salmon mitochondria is carnitine-dependent, and that hepatocytes isolated 
from carnitine-fed salmon oxidized palmitate at a rate 2.5.times. greater 
than hepatocytes from carnitine-deprived salmon (see Table 3 below). 
The suggestion that increased fatty acid oxidation may stimulate PC flux 
was supported by evidence showing an 81% increase in PC flux in 
mitochondria isolated from carnitine-fed fish. Assays of particle-free 
extracts revealed mitochondria from carnitine-fed fish to contain from 
3.3.times. more PC, suggesting enzyme induction, or reduction in enzyme 
turnover, in the mechanism. 
The suggestion that increased PC flux might stimulate lactate-dependent 
gluconeogenesis was supported by evidence obtained in assays with liver 
cubes and isolated hepatocytes. Results showed a 120% to 210% increase in 
the incorporation of lactate into glucose in liver from carnitine-fed 
fish. 
Based on the foregoing findings, additional data, which follows was 
gathered. Based on the following data, one skilled in the art can 
reasonably conclude that feeding juvenile salmon a diet supplemented with 
carnitine during the smoltification process will increase the yield of the 
salmon by reducing mortality and/or improving the growth of the juvenile 
salmon upon transfer to seawater. 
Materials and Methods 
Preparation of diets. The composition of the carnitine-free (control) diet 
is shown in Table 2. Dry ingredients except gelatin were mixed by 
triturating equal amounts in ascending order, by geometric progression, 
with a mortar and pestle. The dry mixture was then mixed with fortified 
oil and choline-Vitamin B.sub.12 solution using a KitchenAid Mixer Model 
K5SS (KitchenAid Inc., St. Joseph, Mich.). In a separate beaker, gelatin 
was added to 259 mL water and dissolved with stirring while heating to 
85.degree. C. in a water bath. Where indicated, L-carnitine, Sigma 
Chemical Co., St. Louis, Mo. (Product No. C0158), was added in place of an 
equal weight of sucrose by dissolving the carnitine in a small aliquot of 
the water used to prepare the gelatin solution. The hot suspension of 
gelatin was immediately blended into the other ingredients in the mixer. 
After cooling for 10 minutes, the warm diets were extruded through a thin 
spaghetti extrusion plate (KitchenAid, Model SN/FGA). The strands of diets 
were cooled, broken into small pellets with the mixer, and stored at 
-20.degree. C. until use (within 8 weeks). 
Animals and feeding. Atlantic salmon fry (Penobscot-strain) obtained from 
the North Attleboro National Fish Hatchery, North Attleboro, Mass., were 
reared at the East Farm Aquaculture Center of the University of Rhode 
Island. They were fed a commercial fish feed (Nelson's Sterling Silver 
Cup, Murray Elevators, Murray, Utah) until experimental use. At the start 
of the experimental feeding schedule juveniles hatched the previous March 
were transferred to 450 L tanks to give a rearing density of 1 g/L. Each 
tank was supplied with supplemental aeration and single-pass, ambient 
(10.degree.-14.degree. C.) freshwater at a flow rate of 1 to 1.5 L/min. 
The fish were reared under simulated natural photoperiod, provided by 
fluorescent lighting and a 24 h timer. After all fish were acclimated to 
the carnitine-free (control) diet for 2 weeks, they were fed one of 2 
diets for 9 weeks: carnitine-free (control) and carnitine-supplemented, at 
23 mmol (3.7 g)/kg wet weight of finished diet. Feed was dispensed at 1.5 
h intervals from 0800 to 1700 hours, using automatic feeders (Model SF7, 
Aquatic Eco-Systems, Apopka, Fla.) to provide a dally ration at 3% of the 
body weight. 
Isolation of liver mitochondria. Liver mitochondria were isolated from 
juvenile salmon according to the method described for rainbow trout by 
Suarez, R. K. and Hochachka, P. W., Preparation and Properties of Rainbow 
Trout Liver Mitochondria, J. Comp. Physiol, 145B:2175-279, 1981. 
Isolation of hepatocytes. Hepatocytes were isolated from 2-year old salmon 
weighing 300 to 500 g by collagenase digestion according to the method of 
French, C. J.; Mommsen, T. P. and Hochachka, P. W., Amino Acid Utilisation 
in Isolated Hepatocytes from Rainbow Trout, Eur. J. Biochem., 113:311-317, 
1981. Fish denied food overnight were anesthetized in MS-222 solution 
(0.19 mmol/L), the liver was exposed, the portal vein cannulated and the 
heart punctured. Refrigerated (10.degree.-15.degree. C.) Medium A 
(Modified Hanks Balanced Salt Solution, Hanks, J. H. and Wallace, R. E., 
Relation of Oxygen and Temperature in the Preparation of Tissues by 
Refrigeration, Proc. Soc. Exp. Biol. Med., 71:196-210, 1949, from which 
Ca.sup.2+ and glucose were omitted, bicarbonate was reduced to 10 mmol/L, 
and HEPES buffer (pH 7.4) was added at 10 mmol/L), equilibrated with 
O.sub.2 :CO.sub.2 (99:1), was immediately pumped through the liver via the 
portal vein at 2 mL/(min.multidot.g) until the liver was cleared of blood. 
The liver was then transferred to a gauze sponge secured over a funnel and 
perfused for an additional 15-20 min at 10.degree.-15.degree. C. with 50 
mL Medium B (Medium A supplemented with CaCl.sub.2 (1 mmol/L) and 
collagenase (EC 3. 4. 24. 3, Sigma Product No. C 5138; 276 units/mL), 
recycled through the liver at 2 mL/(min.multidot.g). The perfusion medium 
was gassed with O.sub.2 :CO.sub.2 (99:1) throughout. When the liver became 
soft and swollen, it was minced in Medium B, on ice, with gentle stirring 
to facilitate separation of hepatocytes, then filtered through gauze and 
the filtrate centrifuged at 4.degree. C. at 100.times.g for 1.5 min. The 
pellet of hepatocytes was washed by resuspension in 40 mL Medium A made 1 
mmol/L in CaCl.sub.2 and centrifuged again. The hepatocytes were washed 
with fresh medium twice more and resuspended at a density to give 10-15 mg 
protein/mL. Protein content was determined by the biuret method. 
Viability, as assessed by exclusion of Trypan Blue, was routinely higher 
than 95%. 
Preparation of liver cubes. Juvenile Atlantic salmon were killed by a sharp 
blow to the head. The liver was excised immediately and transferred to 10 
mL of ice-cold Medium A, in a chilled glass petri dish set on ice. The 
livers from 2 to 3 fish (0.5-1 g tissue) were blotted on filter paper, 
weighed, then put back into the petri dish and minced with a razor blade 
into small cubes (.about.1-2 mm.sup.3). The medium was discarded by 
aspiration, and the cubes were washed twice more by repeated suspension in 
10 mL of fresh Medium A and aspiration. Washed cubes were resuspended in 2 
mL of Medium A made 1 mmol/L in CaCl.sub.2. 
Tissue glycogen content. After fish were killed by a blow on the head, 
liver and a piece of muscle adjacent to the dorsal fin were excised and 
stored frozen at -70.degree. C. for less than 2 weeks. Glycogen content 
was assayed by determining glucose released after amyloglucosidase (EC 3. 
2. 1. 3) digestion of the neutralized acid-soluble fraction of a 17% 
homogenate in 0.6 mmol/L HClO.sub.4, according to Keppler, D. and Decker, 
K. Glycogen In: Methods of Enzymatic Analysis (Bergmeyer, H. U., ed.), 3rd 
Edition, Vol. VI, pp. 11-18, Verlag Chemie, Deerfield Beach, Fla., 1983. 
Glucose was determined enzymatically by coupling of glucose oxidase with 
horse-radish peroxidase, using Sigma Diagnostic Kit No. 510-A. Bovine 
liver glycogen served as a standard for comparison, and could be fully 
accounted for as released glucose in the range of glycogen found in salmon 
liver. 
Fatty acid oxidation in isolated mitochondria. Oxidation of fatty acids was 
assayed in 20 mL scintillation vials containing 1.9 mL of reaction mixture 
of the following composition: KH.sub.2 PO.sub.4, 24 mmol/L; Tris buffer, 
9.5 mmol/L, pH 7.4; MgCl.sub.2, 5 mmol/L; KHCO.sub.3, 3 mmol/L; KCl, 85 
mmol/L; malic acid, 0.1 mmol/L; ATP, 1 mmol/L; 1-.sup.14 C! sodium 
palmitate, 0.1 mmol/L, 7.4 kBq and bovine serum albumin (fatty acid free, 
Sigma, A-6003), 1%. The reaction mixture (excluding ATP) was stirred 
overnight at 4.degree. C. to allow albumin and palmitate to form a 
complex. The assay was initiated by addition of ATP and 0.1 mL of 
mitochondrial suspension (4-6 mg protein). Each vial was immediately 
gassed with O.sub.2 :CO.sub.2 (99:1), sealed with a rubber stopper to 
which a plastic center well holding a piece of fluted filter paper was 
attached, and incubated at 15.degree. C. with shaking. After 40 min, 0.3 
mL of 20% KOH was injected through the stopper into the center well and 
the reaction terminated by injection of 0.5 mL of 1.5 mol/L HCl into the 
main chamber. Shaking was continued for another hour to distill .sup.14 
C!CO.sub.2 into the center well. Unreacted palmitic acid precipitated with 
protein to form a white ring on the vial wall. The center well was 
transferred to another scintillation vial and rinsed with 2 mL of water. 
The well and the rinse were mixed with 6.5 mL of Aquasol and counted for 
radioactivity with a TM Analytic Model 6895 Beta Trac Liquid Scintillation 
System (TM Analytic, Inc., Elk Grove, Ill.). The acidified reaction 
mixture was centrifuged at 10,000.times.g for 5 min and 2 mL of resultant 
clear supernatant fluid was transferred to a scintillation vial and mixed 
with Aquasol to determine radioactivity in the acid-soluble fraction. The 
sum of radioactivity from CO.sub.2 trapped in the center well and 
radioactivity in the acid-soluble fraction of the reaction mixture was 
used as a measure of the oxidation of palmitate. 
Pyruvate carboxylase (PC) flux in isolated mitochondria. PC flux was 
assayed by measuring pyruvate-dependent incorporation of .sup.14 
C!KHCO.sub.3 into acid-stable radiolabeled products during 15 min of 
incubation at 15.degree. C., as described for rainbow trout by Suarez, R. 
K. and Hockachka, P. W., The Pyruvate Branch Point in Fish Liver 
Mitochondria: Effects of Acylcarnitine Oxidation on Pyruvate Dehydrogenase 
and Pyruvate Carboxylase Activities, J. Comp. Physiol., 143B:275-279, 
1981. Reaction mixtures, held in 20 mL scintillation vials, were of the 
following composition: potassium phosphate buffer, pH 7.4, 25 mmol/L; Tris 
buffer, pH 7.4, 10 mmol/L; KCl, 72 mmol/L; MgCl.sub.2, 5 mmol/L; sodium 
pyruvate, 5 mmol/L; .sup.14 C!KHCO.sub.3 (37 kBq), 12 mmol/L; 
mitochondria, 4-6 mg protein. Reactions were terminated by the addition of 
0.5 mL of 1.5 mol/L HCl, and the acidified reaction mixtures were baked to 
dryness over a steam bath. The residue so obtained was extracted with 2 mL 
of water and the contents of the vial (extract and remaining residue) were 
diluted with 6.5 mL of Aquasol for determination of acid-stable 
radiolabeled products. 
PC activity in particle-free extracts of mitochondria. Particle-free 
extracts were prepared by homogenizing liver mitochondria (.about.20 mg 
protein) in 1 mL of detergent solution containing: deoxycholate (0.1%), 
Tris buffer (0.1 mol/L, pH 7.2) and glutathione (1 mmol/L). Residual 
insoluble matter was removed by centrifugation at 10,000.times.g at 
4.degree. C. for 5 min, and the clear supernatant fluid was used as the 
source of PC. Protein content was determined by the biuret method. 
Enzymatic activity was assayed by coupling with excess malate 
dehydrogenase and spectrophotometric measurement of the resultant 
pyruvate-dependent oxidation of NADH at 339 nm during 5 min incubation at 
25.degree. C., Suarez, R. K. and Hochachka, P. W., Pyruvate Carboxylase 
from Rainbow Trout Liver, J. Comp. Physiol., 143B:281-288, 1981; Ji, H., 
Bradley, T. M.; and Tremblay, G. C., Characterization and Tissue 
Distribution of Pyruvate Carboxylase in Atlantic Salmon (Salmo Salar), 
Comp. Biochem. Physiol., 106B:587-593, 1993. Correction for potential 
interference by other dehydrogenases was achieved by routinely subtracting 
rates observed when ATP and acetyl CoA were omitted from the reaction 
mixture. These rates were typically &lt;20% of the rates observed with the 
complete reaction mixture. 
Fatty acid oxidation in suspensions of isolated hepatocytes. Palmitate 
oxidation by hepatocytes was assayed in a 50 mL Erlenmeyer flask 
containing 8 mL Medium A, made 1 mmol/L in CaCl.sub.2, 1.25% in bovine 
albumin and 0.31 mmol/L in 1-.sup.14 C! sodium palmitate (9.25 kBq). This 
reaction mixture was stirred overnight at 4.degree. C. to allow the 
formation of albumin-palmitate complex. The assay was started by addition 
of 2 mL of hepatocyte suspension (20-30 mg protein). The flask was sealed 
and gassed in the same way as described for the assay of palmitate 
oxidation by mitochondria (see above). After 1 h incubation at 17.degree. 
C. with shaking, 0.3 mL of 20% KOH was injected into the center well 
followed by of 0.67 mL of 6 mol/L HClO.sub.4 injected into the main 
chamber. The center well and the acidified reaction mixture were treated 
the same way as in the mitochondrial assay of palmitate oxidation (see 
above). The sum of the radioactivity in the center well and the 
acid-soluble fraction of the reaction mixture was taken as a measure of 
palmitate oxidation. 
Gluconeogenesis in isolated hepatocytes. Lactate-dependent gluconeogenesis 
was assayed by measuring the incorporation of radiolabelled lactate into 
glucose according to Mommsen, T. P.; Walsh, P. J. and Moon, T. W., 
Gluconeogenesis in Hepatocytes and Kidney of Atlantic Salmon, Mol. 
Physiol., 8:89-100, 1985 and Walton, M. J. and Cowey, C. B., 
Gluconeogenesis by Isolated Hepatocytes from Rainbow Trout Salmo 
Gairdneri, Comp. Biochem. Physiol., 62B:75-79, 1979. The assay was 
initiated by mixing 2 mL of hepatocyte suspension (20-30 mg protein) with 
8 mL of Medium A made 1 mmol/L in CaCl.sub.2, 1.25% in bovine albumin and 
12.5 mmol/L in 1-.sup.14 C! lithium lactate (74 kBq). Each flask was 
sealed with a rubber stopper, gassed with O.sub.2 :CO.sub.2 (99:1) and 
incubated at 17.degree. C. At 1 h and 2 h of incubation, an aliquot of 1 
mL of reaction mixture, including hepatocytes, was withdrawn and mixed 
with 0.2 mL of 1.8 mol/L trichloroacetic acid (TCA). Precipitated protein 
was removed by centrifugation at 10,000.times.g for 5 min, 1 mL of the 
supernatant fluid was neutralized with 43.8 .mu.L of 6 mol/L K.sub.2 
CO.sub.3. An aliquot (1 mL) of neutralized extract was mixed with 4 mL of 
1 mol/L glucose and 3 g of Amberlite MB-3 resin and shaken for 1 h to 
extract unreacted lactate. The resin was removed by centrifugation and the 
difference in the amount of radioactivity remaining in the supernatant 
fluid between 1 and 2 h of incubation was used as a measure of the rate of 
lactate-dependent gluconeogenesis. 
Assays with liver cubes. Palmitate oxidation was measured in suspensions of 
liver cubes (0.5-1 g tissue in 2 mL) under the same conditions described 
for the corresponding measurement with isolated hepatocytes (see above) 
except that the reaction mixtures were homogenized after acidification. 
For gluconeogenesis, 1 mL of the reaction medium was withdrawn (avoiding 
cubes) after 1 h and 2 h incubation to isolate radiolabeled extracellular 
glucose. 
Statistical analysis. Statistical analysis was accomplished with the 
StatMost computer program from DataMost Corp., Salt Lake City, Utah. 
Statistical significance of differences was determined by the Student 
t-test. 
EXAMPLE I 
The composition of the carnitine-free (control) diet is shown in Table 2. 
Protein, carbohydrate, and fat constituted 505, 242 and 91 g/kg, 
respectively, of the weight of dry ingredients plus oil, or 370, 178 and 
67 g per kg, respectively, of the wet weight of the finished diet. Where 
indicated, L-carnitine replaced an equal weight of sucrose during 
preparation of the carnitine-supplemented diets, in amounts up to 3.7 g or 
23 mmol carnitine/kg wet weight of finished diet. Since carnitine replaced 
an equal weight of sucrose, the carnitine-supplemented diets had a 
slightly lower (.ltoreq.0.5%) caloric content. 
TABLE 2 
______________________________________ 
Composition of diet.sup.1 
Ingredients Amount in Defined Contro1 Diet (g) 
______________________________________ 
Casein 296 
Gelatin 74.1 
Dextrin 88.9 
Sucrose 88.9 
Mineral mix.sup.2 
29.6 
Vitamin mix.sup.3 
29.6 
.alpha.-cellulose 
59.3 
Fortified oil.sup.4 
66.7 
Choline-Vitamin B-12 solution.sup.5 
7.4 
Water 259 
______________________________________ 
.sup.1 Where indicated in the text, carnitine was added in place of an 
equal weight of sucrose. 
.sup.2 Each kg of mineral mix contained: AlCl.sub.3, 0.15 g; ZnCO.sub.3, 
3.75 g; CuSO.sub.4.5H.sub.2 O, 0.75 g; KI, 0.15 g; MgSO.sub.4.H.sub.2 O, 
4.0 g; CoCl.sub.2.6H.sub.2 O, 0.35 g; Na.sub.2 MoO.sub.4 2H.sub.2 O, 0.2 
g; Na.sub.2 SeO.sub.3, 0.075 g; U.S.P. XIII No. 2 salt mixture, 990.575 g 
.sup.3 Each kg of vitamin mix contained: thiamine.HCl, 1.25 g; riboflavin 
1.25 g; pyridoxine, 0.63 g; niacinamide, 7.5 g; Dcalcium pantothenate, 
6.25 g; inositol, 25 g; biotin, 0.13 g; folic acid, 0.38 g; ascorbic acid 
25 g; alphacel (nonnutritive bulk), 932.61 g. 
.sup.4 Each kg of fortified oil contained: retinyl palmitate, 0.111 g; 
cholecalciferol, 0.065 g; DL.alpha.-tocopherol acetate, 6.67 g in 3.5 ml 
ethanol; menadione, 0.276 g, ethoxyquin (antioxidant), 1.663 g; menhaden 
oil, 988.453 g. 
.sup.5 Choline chloride, 300 g and vitamin B12, 0.030 g in 1 L of aqueous 
solution. 
The carnitine content of the diets was confirmed by analysis. The 
carnitine-free defined diet contained no detectable carnitine upon assay 
(&lt;0.01 mmol/kg wet wt). In the description which follows, the indicated 
carnitine content refers to the amount of carnitine contained in 1 kg wet 
wt of finished diet. 
It is postulated that, through its catalytic role in .beta.-oxidation, 
carnitine might stimulate flux through pyruvate carboxylase (PC). Fixation 
of CO.sub.2 by PC generates oxaloacetate, which can replenish 
intermediates of the tricarboxylic acid cycle drawn off for biosynthetic 
purposes. Tests of this working hypothesis were begun by determining 
whether fatty acid oxidation in salmon mitochondria is indeed 
carnitine-dependent, and whether liver cells from carnitine-fed salmon 
exhibit greater rates of lactate-dependent gluconeogenesis, which is 
limited by PC. 
Mitochondria isolated from the liver of juvenile Atlantic salmon oxidized 
palmitate at a 10-fold greater rate when carnitine was added to the 
reaction mixture, whether the mitochondria were isolated from 
carnitine-fed or carnitine-deprived fish (FIG. 3). Furthermore, in the 
absence of added carnitine, hepatocytes isolated from carnitine-fed fish 
oxidized palmitate at 2.5.times. the rate observed with cells from 
carnitine-deprived fish (Table 3). Liver cubes from juvenile salmon fed 
carnitine also oxidized palmitate at a greater rate than cubes from 
carnitine-deprived juveniles. 
TABLE 3 
______________________________________ 
Effect of dietary carnitine on palmitate oxidation in isolated hepato- 
cytes from 2-yr old salmon and liver cubes of juvenile salmon fed carni- 
tine-free or carnitine supplemented diets for 9 weeks.sup.1,2 
Dietary carnitine 
Liver cubes 
Isolated hepatocytes 
(mmol/kg diet) 
(mnol/(g .multidot. h)) 
(nmol/(mg protein .multidot. h)) 
______________________________________ 
0 (Control) 8.96 .+-. 1.41 
0.43 .+-. 0.26 
23 13.24 .+-. 0.45* 
1.08 .+-. 0.15* 
______________________________________ 
.sup.1 Palmitate oxidation was measured as generation of radiolabeled 
CO.sub.2 and acidsoluble products from 1.sup.14 C! palmitate as describe 
in Materials and Methods. 
.sup.2 Values are means .+-. SD (n = 4 for liver cubes and n = 3 for 
hepatocytes). 
*Significantly different (P &lt; 0.05) from values obtained with control 
diet. 
The data in Table 3 (and FIG. 3) clearly show that, in salmon, L-carnitine 
promotes fat oxidation. 
EXAMPLE III 
Evidence was sought to determine whether changes in flux through PC did, in 
fact, take place with carnitine intake. Flux measurements with suspensions 
of mitochondria isolated from the livers of carnitine-fed juvenile 
Atlantic salmon revealed an 81% greater rate of PC flux (FIG. 4). Whether 
the increase in PC flux was sufficient to alter cell metabolism was tested 
by measuring lactate-dependent gluconeogenesis in suspensions of 
hepatocytes and liver cubes. The rate of lactate-dependent gluconeogenesis 
in surviving liver cubes from juvenile salmon and isolated hepatocytes 
from 2-year old salmon was 2.2.times. and 3.1.times.greater, respectively, 
with liver from carnitine-fed fish (FIG. 5). Carnitine-fed juvenile salmon 
also exhibited a 5.4.times. increase in muscle glycogen, but little change 
in liver glycogen, consistent with the primary function of gluconeogenesis 
to maintain extrahepatic glycogen in carnivores (Table 4). These 
observations indicate that the increase in PC flux observed with isolated 
mitochondria alters intermediary metabolism in the organism. They also 
support the hypothesis that other metabolic changes observed in response 
to increased dietary carnitine are coupled to a rise in PC flux. 
TABLE 4 
______________________________________ 
Effect of dietary carnitine on tissue concentration of glycogen in 
juvenile salmon fed carnitine-free or carnitine supplemented diets for 
9 weeks.sup.1,2 
Dietary carnitine 
Liver glycogen (mg 
Muscle glycogen 
(mmol/kg wet wt of diet) 
glycogen/g liver) 
(mg glycogen/g muscle) 
______________________________________ 
0 (Control) 118.3 .+-. 20.1 
1.25 .+-. 0.99 
23 145.2 .+-. 35.1 
6.72 .+-. 2.25* 
______________________________________ 
.sup.1 Glycogen was assayed as glucose released from amyloglucosidase 
digestion as described in Materials and Methods. 
.sup.2 Values are means .+-. SD (n = 6). 
*Significantly different (P &lt; 0.001) from values obtained with fish fed 
the control diet. 
EXAMPLE IV 
PC activity in particle-free extracts was assayed to determine whether 
liver from carnitine-fed fish contained more enzyme. Results show the 
amount of PC to be 3.3.times. greater in mitochondria from carnitine-fed 
salmon (FIG. 6). 
Since the reduced energy reserve in salmon undergoing smoltification may 
contribute to mortality or stunted growth upon transfer to seawater, 
improvement in energy reserved through the addition of carnitine to the 
feed would be expected to reduce mortality and/or improve growth. With the 
onset of smoltification, the juvenile salmon will be fed a commercial feed 
(for example, Nelsons Sterling Silvercup, Murray Elevators, Murray, Utah) 
supplemented with carnitine to satiation daily. The amount of carnitine to 
be added to the feed will vary between 0.01 to 0.5% of the wet weight of 
the juvenile diet. The feed preferably will be distributed in the form of 
pellets. 
The beneficial effect of the L-carnitine may be achieved by administering 
the L-carnitine as an additive to the feed or by any other means known in 
the art. Assuming daily feed consumption at 3% of body weight, generally 
from an average 3 to 150 mg of carnitine per kg body weight of fish would 
be fed per day, preferably, from 10 to 120 mg/day. The feeding of the 
carnitine may be from January (peak PC) to July (about two months after 
transfer to seawater, though beneficial effects may be achieved by feeding 
for more limited periods, e.g. during 3 weeks around peak smoltification). 
All references set forth herein are incorporated by reference into this 
disclosure in their entirety. 
The foregoing description has been limited to a specific embodiment of the 
invention. It will be apparent, however, that variations and modifications 
can be made to the invention, with the attainment of some or all of the 
advantages of the invention. Therefore, it is the object of the appended 
claims to cover all such variations and modifications as come within the 
true spirit and scope of the invention.