Elevated incubation temperature improves later-life swimming endurance in juvenile Chinook salmon, Oncorhynchus tshawytscha.

The effect of incubation and rearing temperature on muscle development and swimming endurance under a high-intensity swimming test was investigated in juvenile Chinook salmon (Oncorhynchus tshawytscha) in a hatchery experiment. After controlling for the effects of fork length (LF ) and parental identity, times to fatigue of fish were higher when fish were incubated or reared at warmer temperatures. Significant differences among combinations of pre- and post-emergence temperatures conformed to 15-15°C > 15-9°C > 9-9°C > 7-9°C > 7-7°C in 2011 when swimming tests were conducted at 300 accumulated temperature units post-emergence and 15-9°C > (7-9°C = 7-7°C) in 2012 when swimming tests were conducted at an LF of c. 40 mm. The combination of pre- and post-emergence temperatures also affected the number and size of muscle fibres, with differences among temperature treatments in mean fibre cross-sectional area persisting after controlling for LF and parental effects. Nonetheless, neither fibre number nor fibre size accounted for significant variation in swimming endurance. Thus, thermal carryover effects on swimming endurance were not mediated by thermal imprinting of muscle structure. This is the first study to test how temperature, body size and muscle structure interact to affect swimming endurance during early development in salmon.

Carnitine palmitoyl transferase activity and whole muscle oxidation rates vary with fatty acid substrate in avian flight muscles.

Birds primarily fuel migratory flights with fat, and the composition of that fat has the potential to affect overall lipid oxidation rates. We measured the whole muscle lipid oxidation rates in extensor digitorum communis muscles from white-throated sparrows (Zonotrichia albicollis Gmelin) incubated for 20 min at 20°C with radiolabeled stearate (18:0), oleate (18:1ω9), or linoleate (18:2ω6). Lipid oxidation rates were ~40% higher with linoleate than oleate (oleate: 36 ± 8.54 µmol CO(2) g(-1) h(-1)), and ~75% lower with stearate compared with oleate, indicating that maximal lipid oxidation rates can indeed be affected by the type of fatty acid supplied to the muscle. Additionally, we investigated the activity of the mitochondrial fatty acid transport-associated enzyme carnitine palmitoyl transferase (CPT) in pectoralis muscles of 5 bird species (Zonotrichia albicollis, Philomachus pugnax, Sturnus vulgaris, Taeniopygia guttata, Passer domesticus). Activity was measured in homogenized samples using various fatty acyl-CoA substrates (16:0, 16:1, 18:0, 18:1ω9, 18:2ω6, 18:3ω3, 18:3ω6, 20:0, 20:4ω6, 22:6ω3) in a spectrophotometric assay. CPT activity increased with the degree of unsaturation and decreased with chain length. CPT activity did not differ between ω3 and ω6 isomers of 18:3, nor was the pattern of CPT substrate preference different between captive white-throated sparrows in a migratory (i.e., displaying Zugunruhe) or non-migratory state. These findings can explain previously observed differences in peak performance induced by dietary fat composition and suggest that lipid supply is limiting to maximal exercise performance in birds.

Fuel use during glycogenesis in rainbow trout (Oncorhynchus mykiss Walbaum) white muscle studied in vitro.

The purpose of this study was to examine fuel used during muscle glycogenesis in rainbow trout Oncorhynchus mykiss using an in vitro muscle slice preparation to test the hypothesis that intracellular lactate is the major glycogenic substrate and the muscle relies upon extracellular substrates for oxidation. Fish were exhaustively exercised to reduce muscle glycogen content, muscle slices were taken from exhausted fish and incubated for 1 h in medium containing various substrates at physiological concentrations. 14C-labeled lactate, glycerol or palmitate was added and 14C incorporation into muscle glycogen and/or CO2 was measured. Lactate clearance in the absence of net glycogenesis suggests that when suitable oxidizable extracellular substrates were lacking, intracellular lactate was oxidized. Only muscle incubated in lactate, glycerol or palmitate synthesized glycogen, with the greatest synthesis in muscle incubated in lactate plus glycerol. The major fate of these extracellular substrates was oxidative, with lactate oxidized at rates 10 times that of palmitate and 100 times that of glycerol. Neither extracellular lactate nor glycerol contributed significantly to glycogenesis, with lactate carbon contributing less than 0.1% of the total glycogen synthesized, and glycerol less than 0.01%. There was 100 times more extracellular lactate-carbon incorporated into CO2 than into glycogen. In the presence of extracellular lactate, palmitate or glycerol, intracellular lactate was spared an oxidative fate, allowing it to serve as the primary substrate for in situ glycogenesis, with oxidation of extracellular substrates driving ATP synthesis. The primary fate of extracellular lactate is clearly oxidative, while that of intracellular, glycolytically derived lactate is glycogenic, which suggests intracellular compartmentation of lactate metabolism.

Hormonal regulation of glycogen metabolism in white muscle slices from rainbow trout (Oncorhynchus mykiss Walbaum).

To test the hypothesis that cortisol and epinephrine have direct regulatory roles in muscle glycogen metabolism and to determine what those roles might be, we developed an in vitro white muscle slice preparation from rainbow trout (Oncorhynchus mykiss Walbaum). In the absence of hormones, glycogen-depleted muscle slices obtained from exercised trout were capable of significant glycogen synthesis, and the amount of glycogen synthesized was inversely correlated with the initial postexercise glycogen content. When postexercise glycogen levels were <5 micromol/g, about 4.3 micromol/g of glycogen were synthesized, but when postexercise glycogen levels were >5 micromol/g, only about 1.7 micromol/g of glycogen was synthesized. This difference in the amount of glycogen synthesized was reflected in the degree of activation of glycogen synthase. Postexercise glycogen content also influenced the response of the muscle to 10(-8) M epinephrine and 10(-8) M dexamethasone (a glucocorticoid analog). At high glycogen levels (>5 micromol/g), epinephrine and dexamethasone stimulated glycogen phosphorylase activity and net glycogenolysis, whereas at low (<5 micromol/g) glycogen levels, glycogenesis and activation of glycogen synthase activity prevailed. These data clearly indicate not only is trout muscle capable of in situ glycogenesis, but the amount of glycogen synthesized is a function of initial glycogen content. Furthermore, whereas dexamethasone and epinephrine directly stimulate muscle glycogen metabolism, the net effect is dependent on initial glycogen content.

A regulatory role for cortisol in muscle glycogen metabolism in rainbow trout Oncorhynchus mykiss Walbaum.

To test the hypothesis that cortisol has a regulatory role in fish muscle glycogenesis post-exercise, rainbow trout were treated 1 h prior to exercise with either saline (control) or metyrapone (2-methyl-1, 2-di-3-pyridyl-1-propanone) to block cortisol synthesis. Following exercise (time 0), half of the metyrapone-treated fish received a single injection of cortisol, to mimic the post-exercise rise usually observed. Muscle glycogen and the relative activities of glycogen phosphorylase a (Phos a) and glycogen synthase I (GSase I), regulatory enzymes for glycogen resynthesis, were monitored 4 h post-exercise. Metyrapone treatment succeeded in blocking the post-exercise rise in plasma cortisol (17+/-2 vs 118+/-13 ng ml(-1) in controls at time 0), and cortisol injection resulted in a larger and more prolonged cortisol increase than in controls (159+/-22 vs 121+/-14 ng ml(-1) in controls at 1 h). Muscle glycogen was completely restored in the metyrapone-treated fish within 2 h after exercise (8.3+/-0.6 vs 8+/-0.7 micromol g(-1) pre-exercise), only partially restored in control fish at 4 h (5.4+/-01.4 vs 8.8+/-1.3 micromol g(-1) pre-exercise), and not at all in cortisol-treated fish (1.0+/-0.5 micromol g(-1) at 4 h). The rapid glycogen resynthesis in the metyrapone-treated fish was associated with a more rapid inactivation of Phos a and stimulation of GSase I compared to controls. In cortisol-treated fish, Phos a activity persisted throughout 4 h post-exercise; there was also a significant stimulation of GSase I activity. As a consequence of dual activation of Phos a and GSase I, glycogen cycling probably occurred, thus preventing net synthesis. This explains why the post-exercise elevation of cortisol inhibits net glycogen synthesis in trout muscle.

Lactate efflux from sarcolemmal vesicles isolated from rainbow trout Oncorhynchus mykiss white muscle is via simple diffusion.

Lactic acid is produced as an end product of glycolysis in rainbow trout white muscle following exhaustive exercise. The metabolically produced lactic acid causes an intramuscular acidosis that must be cleared, either via net transport out of the muscle or by conversion to glycogen, thereby replenishing the muscle energy store. Trout muscle has been shown to retain lactate and utilise it as a substrate for in situ glycogen resynthesis. The giant sarcolemmal vesicle preparation was used to characterise the potential for lactate loss from white muscle of rainbow trout. Minimal lactate loss was expected due to the requirement within the intramuscular compartment of lactate for glycogen resynthesis. The sarcolemma was found to be highly resistant to lactate loss, with efflux rates approximately 500-fold lower than influx rates [0.049+/-0.006 nmol mg(-1) min(-1) (N=21) versus 26.4+/-6.3 nmol mg(-1) min(-1) (N=5), respectively, at 25 mmol l(-1) lactate concentration]. Lactate efflux was linear over the range 10-250 mmol l(-1) lactate, and greatest under conditions when intravesicular pH was lower than extravesicular pH, but was unaffected by alpha-cyano-4-hydroxycinnamate, a known inhibitor of lactate transport. These results suggest that lactate is relatively impermeant to the trout white muscle membrane and any lactate loss occurs via passive diffusion. This resistance to lactate diffusion can explain why trout muscle retains lactate post-exercise, despite transmembrane gradients that should favour net efflux.