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Effects of ration on somatotropic hormones and growth in coho salmon
Comparative Biochemistry and Physiology Part B 128 Ž2001. 255᎐264
Effects of ration on somatotropic hormones and growth in coho salmon Andrew L. Pierce a,U , Brian R. Beckmana,b, Karl D. Shearer b, Donald A. Larsen b, Walton W. Dickhoff a,b b
a School of Fisheries, Uni¨ ersity of Washington, Seattle, WA 98195, USA Integrati¨ e Fish Biology Program, Northwest Fisheries Science Center, National Marine Fisheries Ser¨ ice, 2725 Montlake Boule¨ ard E., Seattle, WA 98112, USA
Received 7 June 2000; received in revised form 5 October 2000; accepted 16 October 2000
Abstract We examined the response of growth hormone ŽGH., total plasma insulin-like growth-factor I ŽIGF-I., and growth rate to a change in ration in coho salmon. Tanks of individually tagged fish were placed on high, medium, or low ration, and sampled every 2 weeks for 8 weeks to create a range of growth rates. Some fish received non-lethal blood draws, while others were sampled terminally. Plasma IGF-I levels were higher in high ration fish than in low ration fish from 4 weeks after the beginning of experimental diets to the end of the experiment. GH levels were low and similar in all fish after changing rations, except for the fish in the low ration group at week 2. IGF-I was strongly correlated with specific growth rate in weight in terminally sampled fish after 4 weeks. GH did not correlate with growth rate or IGF-I levels. Growth parameters Žlength, weight, specific growth rates in weight and length, and condition factor. responded to ration. Serial sampling reduced growth rates and hematocrit, but did not change hormone levels. This study shows that IGF-I responds to changed rations within 2᎐4 weeks in salmonids. Published by Elsevier Science Inc. Keywords: Insulin-like growth factor-I ŽIGF-I.; Growth hormone ŽGH.; Growth; Nutrition; Coho salmon Ž Oncorhynchus kisutch.; Somatotrophic; Hatchery; Time course
1. Introduction The somatotropic endocrine axis regulates growth in all vertebrates. The axis consists of the peptide hormone growth hormone ŽGH. secreted
Corresponding author. Tel.: q1-206-860-3248; fax: q1-206860-3267. E-mail address: [email protected] ŽA.L. Pierce.. 1096-4959r01r$ - see front matter Published by Elsevier Science Inc. PII: S 1 0 9 6 - 4 9 5 9 Ž 0 0 . 0 0 3 2 4 - 9
by the pituitary gland, insulin-like growth factor-I ŽIGF-I. produced in most tissues, and their receptors and binding proteins. According to the widely accepted somatomedin hypothesis, many of the growth promoting effects of GH are mediated by circulating IGF-I, which is produced by the liver in response to GH ŽDaughaday and Rotwein, 1989.. In mammals, circulating IGF-I levels are often strongly correlated with growth rate ŽStraus, 1994..
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Nutritional status regulates the somatotropic axis. In all species studied to date, IGF-I declines during fasting, whereas GH increases in nearly all species ŽStraus, 1994; McMurtry et al., 1997; Duan, 1998.. Hepatic GH sensitivity is important in nutritional regulation of IGF-I. Changes in metabolic hormones Ži.e. insulin, glucagon, thyroid hormones, and glucocorticoids. and nutrient levels during fasting or feed restriction cause decreases in hepatic GH receptor mRNA and protein, which results in decreased IGF-I production ŽThissen et al., 1999.. The subsequent decrease in circulating IGF-I level may increase GH secretion via relaxation of negative feedback ŽThissen et al., 1999.. All components of the somatotropic axis exist in fish, and the basic operation of the axis appears to be similar in fish and mammals ŽDuan, 1998.. Furthermore, nutritional regulation of the somatotropic axis in fish appears to fit the mammalian model. Nutritional status positively regulates IGF-I, and negatively regulates GH ŽDuan, 1998.. Studies in the marine teleost gilthead sea bream Ž Sparus aurata. have shown that ration and dietary protein level regulate hepatic GH receptor levels, plasma IGF-I, and plasma GH ŽPerezSanchez et al., 1994, 1995; Company et al., 1999.. In salmonids, hepatic GH receptor level is regulated by nutrition ŽGray et al., 1992.. Negative feedback of IGF-I on GH secretion has been demonstrated in salmonids ŽPerez-Sanchez et al., 1992; Blaise et al., 1995.. Of the studies on nutrition and endocrine growth regulation in teleosts, only two have measured both plasma GH and IGF-I levels ŽNiu et al., 1993; Perez-Sanchez et al., 1995.. In these studies, the fish were not sampled until 4 ŽNiu et al., 1993. and 7 ŽPerez-Sanchez et al., 1995. weeks after beginning feeding treatments. Changes in growth occur much earlier than this, and one would expect plasma GH and IGF-I levels to respond earlier as well. Curiously, the single study examining the time course of the hepatic IGF-I mRNA response to fasting did not find a reduction until 28 days after fasting began ŽDuan and Plisetskaya, 1993.. We sought to document the time course of changes in plasma GH, IGF-I, and growth rate in response to a change in ration, sampling every 2 weeks for 8 weeks. Previous investigators have looked at the average growth rates and hormone levels of tanks of fish. By using tagged fish, we were able to assess the
relationship between growth rates and hormone levels of individuals.
2. Materials and methods The experiment used 1994 brood University of Washington strain coho salmon Ž Oncorhyncus kisutch., obtained in April 1996 from the Sandpoint hatchery of the Biological Resources Division, United States Geological Survey ŽSeattle, WA.. Fish were housed in a 1.3-m cylindrical tank, under simulated natural photoperiod, in a recirculating freshwater hatchery at the Northwest Fisheries Science Center ŽSeattle, WA.. The fish were fed commercial pelleted feed ŽBiodiet Grower, Bioproducts, Inc.,1 wet weight composition 43% protein, 10.5% carbohydrate, 14.5% lipid., 5 daysrweek by belt feeder at an overall feeding rate of 1.4% body weightrday. On 28 June 1996 Žweek y2., 180 fish w100.4" 2.8 g, mean " standard error ŽS.E..x were implanted intraperitoneally with passive integrated transponder tags ŽPIT tags, DestronrIDI., weighed, measured, and divided into three tanks. Based on appearance, fish were post-smolts at this time. Rations of 1.4% body weightrday were continued for 2 weeks. On 10 July 1996 Žweek 0., the tagged fish were switched onto experimental rations. The high ration was calculated to produce maximal growth, and the low ration to maintain current weight without permitting growth. The medium ration was the average of high and low rations. Ration calculations were performed according to Cho Ž1992., adjusted daily for temperature and weight of fish in each tank, with an estimated thermalunit growth coefficient of 0.0023 g 1r3⬚Cy1 dayy1 . The fish were fed Biodiet pellets 7 daysrweek. Average rations were high 1.6, medium 1.1, and low 0.6% body weightrday. High and medium rations were fed by belt feeder, whereas the low ration was fed by hand, to allow all fish a chance at the food. The fish in the low ration tank were seen to eat all of the food supplied, and no uneaten food was ever observed in the high or medium ration tanks. The fish were fed experimental rations for 8 weeks, during which time the
1 Use of trade names does not imply endorsement of NOAA, U.S. Department of Commerce.
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water temperature ranged from 11 to 15⬚C, average 12.5⬚C, without seasonal trends. Samples were taken at week 0 and every 2 weeks thereafter Žweek 2: 24 July 1996; week 4: 7 August; week 6: 21 August; week 8: 4 September.. All fish were removed from each treatment tank. The fish were then individually anesthetized in a 0.05 ᎐ 0.1-grl buffered solution of tricane methanesulfonate ŽMS-222., and weight, fork length, and PIT tag number were recorded with an automated system ŽBiomark Inc., Boise, ID.. Pre-determined fish, identified by a PIT tag number, either received non-lethal blood draws Žserial samples., or were killed Žterminal samples.. Fish not scheduled for serial or terminal sampling were returned to the treatment tank. Sixteen fish per tank were serially sampled at weeks 0, 2, 4, and 6. Four fish per tank were terminally sampled at week 0, and 10 fish per tank were terminally sampled at weeks 2, 4 and 6. All remaining fish were terminally sampled at week 8. Serial samples were taken from the same fish at each time point. On each sampling date, tanks were sampled from 08.00 h to approximately 13.00 h in the same order: medium, low, high. For serial sampling, the caudal artery was punctured with a sterile 22-gauge hypodermic needle and ; 200 l of blood was drawn into a heparinized syringe, after which the fish was returned to the tank. For terminal sampling, the caudal peduncle was transected and blood from the caudal artery was collected into heparinized test tubes. Blood was centrifuged for 5 min at 3000 = g, hematocrit level noted, and plasma frozen at y80⬚C for later radioimmunoassays. Individual fish specific growth rates in length ŽSGRL. were calculated by the method of Ricker Ž1979. SGRL s 100U wlnŽlength 2. y lnŽlength 1.xrdays, where ‘length 2’ and ‘length 1’ were fish lengths at the end and beginning of the growth period, respectively, and ‘days’ was the duration of the growth period. Specific growth rates in weight ŽSGRW. were calculated similarly. The condition factor ŽCF . was calculated as 100U weight Žg.rlength Žcm. 3. Plasma GH was assayed according to Swanson Ž1994.. Samples were run in two assays with intraassay coefficients of variation Ž%CV. of 17.6 and 6.2% and an interassay %CV of 8.0%. Values from the second assay were adjusted to the first by a linear correction factor. Plasma IGF-I was assayed after acid᎐ethanol extraction accord-
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ing to Shimizu et al. Ž2000.. Briefly, 25-l plasma samples were incubated with 100-l of an acid᎐ethanol solution Ž87.5% ethanol, 12.5% 2 N HCl. for 30 min at room temperature, neutralized with 80 l 0.855 M Tris, and centrifuged for 30 min at 1800 = g. The supernatent was assayed in duplicate for IGF-I using recombinant salmon IGF-I tracer and a standard, and anti-recombinant barramundi IGF-I antibody Žcomponents purchased from GroPep Pty Ltd., Adelaide, Australia.. An appropriate amount of acid᎐ethanol q Tris solution was added to standard curves. Samples were run in two assays with intraassay %CVs of 9.40 and 8.73%, including the extraction step and an interassay %CV of 0.77%. IGF-I was not measured in all samples due to insufficient plasma. Measurements were performed for 144 out of 150 terminal samples and 137 out of 237 serial samples. Plasma from the last two sampling points Žweeks 6 and 8. was inadvertently thawed and kept in a refrigerator Ž4⬚C. for 2 weeks before the IGF-I assay. The effects of ration and serial sampling on growth, hormone levels, condition factor, and hematocrit were examined at the level of tanks of fish as a matter of convenience, since each tank was fed a different ration. Multiple-way analyses of variance ŽANOVAs. with date, ration, and serial vs. terminal fish as effects were performed for each dependent variable. Significant effects were further examined by one-way ANOVA followed by Fisher’s protected least significant difference ŽZar, 1996.. Relations between growth and hormone levels were investigated by linear regression. Relations between growth rate rankings of individual fish over different periods were examined using the Spearman rank correlation test. Results were considered statistically significant at P- 0.05. The results described here are statistically significant unless otherwise stated.
3. Results 3.1. Growth Ration, date, serial sampling, and the interaction of date and ration affected fish size. High and medium ration fish increased in size over the course of the experiment, and this increase was influenced by ration level, with higher ration fish growing more ŽFig. 1a,b.. Fish on low rations did
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Fig. 1. Ža. Weight, Žb. length, Žc. condition factor, Žd. specific growth rate in weight ŽSGRW., and Že. length ŽSGRL. through the experiment. High ration: ᎏ`ᎏ ; medium ration: ---I---; low ration: --^--. Points sharing a common superscript or without superscripts at each sampling date do not differ significantly. Asterisks indicate significant differences vs. week 0 Žpanels a, b, and c. or week y2᎐0 Žpanels d and e..
not gain significant weight or length through the experiment, and were lighter and shorter than medium and high ration fish beginning at week 4. There was a significant difference in weight between medium and high ration fish only at week 6. Differences in condition factor ŽCF., a measure of fish body shape, developed between ration treatments ŽFig. 1c.. Differences among fish in all three tanks became significant at the week 4 sampling and were maintained throughout the rest of the experiment, with high and medium ration CF increasing through the experiment and low ration CF unchanged. Ration and date affected weight growth rate
ŽSGRW. ŽFig. 1d.; significant differences among all treatments appeared at week 0᎐2, and were maintained up to week 6᎐8, when the difference between high and medium ration became non-significant. The SGRW of fish on the high ration increased from week y2᎐0 to all later intervals, although SGRW declined from week 2᎐4 to week 4᎐6. Medium ration SGRW increased from week y2᎐0 to 0᎐2, but not later intervals. There were no further significant changes in medium ration SGRW. Low ration SGRW decreased after the switch onto experimental rations. A subsequent increase from week 0᎐2 to 2᎐4 approached significance Ž Ps 0.0580..
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and CF were not significantly affected by repeated blood draws. SGRW was depressed in serially sampled fish relative to terminals at weeks 4, 6 and 8, and SGRL at weeks 6 and 8. From week 0 to week 8, serial fish hematocrit declined from 40.1" 0.5 to 30.9" 1.2 Žmean " S.E. of the mean., with the greatest decline from week 0 to 2. Hematocrit levels of terminally sampled fish did not vary significantly through the experiment. Plasma IGF-I and GH levels did not differ significantly between serial and terminal fish, and there were no trends toward any such difference. 3.3. Ration effects on GH and IGF-I
Fig. 2. Relationship between SGRW for weeks 0᎐4 and weeks 4᎐8 in individual fish terminally sampled at week 8. High ration: `, Spearman rank correlation Rhos 0.625, Ps 0.0194; medium ration: I, Rhos 0.564, Ps 0.0241; low ration: ^, Rhos 0.600, Ps 0.0248.
Ration and date affected length growth rate ŽSGRL. ŽFig. 1e.. After the switch to experimental diets, fish on the high ration had a higher SGRL than those on low ration over every interval. Fish on the high ration also had a higher SGRL than those on the medium ration for weeks 0᎐2, 2᎐4 and 4᎐6, and fish on the medium ration had a higher SGRL than those on the low ration for weeks 2᎐4 and 6᎐8. In high and low ration fish, SGRL increased from week y2᎐0 to all later intervals. Also in high and low ration fish, SGRL decreased from week 2᎐4 to week 6᎐8. In medium ration fish, SGRL increased from week y2᎐0 to week 2᎐4 only. The SGRWs of individual fish identified by PIT tag number showed consistent ranking over time, both overall and within each tank ŽFig. 2.. Excluding the 2-week period prior to beginning experimental diets Žweek y2᎐0., all possible comparisons of SGRW of individual fish over different 2-week periods showed significant consistency of SGRW rank over time by the Spearman rank correlation. 3.2. Serial sampling Serially sampled fish had lower hematocrit, SGRW, and SGRL than terminally sampled fish Ždata not shown.. In contrast, fish length, weight,
Date and ration affected the plasma levels of both GH and IGF-I. GH peaked in fish in the low ration treatment at week 2, at which point it was higher in this treatment than in either of the others ŽFig. 3a.. GH levels did not vary signifi-
Fig. 3. Ža. GH and Žb. IGF-I levels through the experiment. High ration: ᎏ`ᎏ; medium ration: ---I---; low ration: --^--. Points sharing a common superscript or without superscripts at each sampling date do not differ significantly. Asterisks show significant differences vs. week 0. No line connects points from weeks 4 and 6 to indicate that weeks 6 and 8 should not be compared to earlier time points. Plasma from weeks 6 and 8 was inadvertently thawed and kept in a refrigerator for 2 weeks before IGF-I was assayed.
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cantly between treatments at any point other than week 2. The GH level at week 2 in low ration fish was higher than GH levels in low ration fish at weeks 0, 4, 6 and 8. In fish the high ration treatment, GH declined from week 0 to all later time points. A similar, but non-significant, decline occurred in fish in the medium ration treatment. In a two-way ANOVA with date and ration as effects, IGF-I increased with increasing ration. IGF-I levels were similar at week 0. A non-significant trend toward ranking of treatments appeared at week 2, with high ration having highest IGF-I, medium ration intermediate, and low ration having lowest IGF-I. At weeks 4 and 6, high ration fish had higher IGF-I than low or medium ration fish, whereas at week 8, high and medium ration fish had higher IGF-I than low ration fish. In the two-way ANOVA, a decline in IGF-I level occurred from weeks 2 and 4 to weeks 6 and 8, i.e. IGF-I was lower in samples that were accidentally left in a refrigerator for 2 weeks. Changes over time were not significant within tanks. 3.4. Growth and hormone le¨ el correlations Plasma IGF-I levels at week 4 were positively correlated with SGRW for the previous 2 weeks in both terminal and serial fish ŽFig. 4.. The strongest correlation was found in terminal fish ŽFig. 4a, P- 0.0001, r 2 s 0.623., whereas that in serial fish tended to be weaker ŽFig. 4b, Ps 0.001, r 2 s 0.257., except in low ration fish, where a high correlation was found Ž Ps 0.0005, r 2 s 0.687.. Week 4 IGF-I was also positively correlated with SGRL for the previous 2 weeks in both terminal Ž Ps 0.0003, r 2 s 0.389. and serial Ž Ps 0.0017, r 2 s 0.236. fish. Regression analysis of possible relations between GH at each sampling point and IGF-I level, SGRW, SGRL, and condition factor did not show any significant relations.
4. Discussion This study provides the first characterization of the time course of the combined GH and IGF-I response to a change in ration in a teleost. IGF-I responded to changed ration and correlated with
Fig. 4. Relationship between week 4 IGF-I and SGRW for weeks 2᎐4 in Ža. terminally sampled and Žb. serially sampled fish. High ration: `; medium ration: I; low ration: ^. Regression lines shown for all fish combined. Regression statistics: Ža. all fish: P - 0.0001, r 2 s 0.623; high ration: P s 0.118; medium ration: Ps 0.0026, r 2 s 0.699; low ration: P s 0.109; Žb. all fish: Ps 0.001, r 2 s 0.257; high ration: Ps 0.707; medium ration: Ps 0.097; low ration: Ps 0.0005, r 2 s 0.687.
growth rate within 4 weeks. GH increased transiently at the lowest ration, but did not show a consistent relationship to growth rate. A trend toward IGF-I ranking by ration appeared at week 2, with higher ration fish showing higher IGF-I, and the high vs. low ration difference became significant at week 4. IGF-I ranking by ration continued up to week 8. The ranking of IGF-I by ration agrees with models of growth regulation in which IGF-I is under nutritional control. Positive relations of IGF-I with ration
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are found in birds ŽMcMurtry et al., 1997. mammals ŽThissen et al., 1994. and fish ŽDuan, 1998; Perez-Sanchez and Le Bail, 1999.. Previous studies in fish have found effects of feeding level on plasma IGF-I level. Reduced plasma IGF-I was found after fasting for 25 days in coho salmon smolts ŽMoriyama et al., 1994. and after fasting for 6 weeks in rainbow trout ŽFoucher et al., 1992.. Combined temperature and ration Ž1.4 and 1.8% body weightrday. treatments of chinook salmon resulted in the separation of IGF-I levels after approximately 1 month ŽBeckman et al., 1998.. IGF-I ranked by ration after 7 weeks of feeding treatment in gilthead sea bream ŽPerezSanchez et al., 1995.. Hepatic IGF-I mRNA levels were decreased after 4 weeks of fasting and restored by 2 weeks of refeeding in coho salmon ŽDuan and Plisetskaya, 1993.. IGF-I like immunoreactivity was reduced by 4 weeks of fasting in rainbow trout ŽNiu et al., 1993.. In barramundi, 21 days of fasting reduced hepatic IGF-I mRNA ŽMatthews et al., 1997.. This study confirms and extends these findings by showing that, in coho salmon under our rearing conditions, plasma IGF-I responds to relatively mild changes in ration in 2᎐4 weeks. This is considerably longer than the IGF-I response to fasting in rats, which takes 2᎐3 days Že.g. Frystyk et al., 1999.. This difference may be due in part to the less severe nutritional restriction employed in this study, and species differences associated with poikilothermy and adaptation to prolonged fasting in salmonids ŽDuan, 1998.. Plasma IGF-I at week 4 correlated strongly Ž r 2 s 0.623. with SGRW for the previous 2 weeks in terminally sampled fish. Some previous studies have not found significant or strong correlations between individual IGF-I levels and growth rates Žunpublished studies reviewed in Plisetskaya, 1998; Silverstein et al., 1998.. However, tank average IGF-I levels and monthly specific growth rates in length correlated strongly Ž r 2 s 0.50 and 0.65. in two studies on chinook salmon ŽBeckman et al., 1998, 1999., and forthcoming data show strong correlations Ž r 2 s 0.5᎐0.8. between individual chinook salmon IGF-I levels and monthly specific growth rates ŽB. Beckman, unpublished data.. Studies in juvenile mammals have found strong correlations of IGF-I with various measures of growth, with r 2 values in the 0.7᎐0.8 range ŽStraus, 1994.. Plisetskaya Ž1998. speculates that the correlation between plasma IGF-I and growth
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rate in fish may vary seasonally and with developmental stage, and that these factors and the characteristics of IGF-I assays and IGFBP extraction techniques used may explain the lack of correlation in some studies. IGF-I probably both reflects past growth and predicts future growth. Further work is necessary to determine the time period and growth measurement which gives the strongest correlation. In this study, correlations between IGF-I at week 4 and SGRW for the previous 2 weeks were weaker in high and medium ration serially sampled fish than in high and medium ration terminally sampled fish. Serially sampled fish had had two previous blood draws at this point. In contrast, low ration serially sampled fish showed a comparably strong correlation Ž r 2 s 0.687.. The reasons for these differences are not clear, but they may be related to the stress of the repeated blood draws, andror differences in the suite of IGF binding proteins between different ration and sampling groups. Plasma GH levels peaked in low ration fish 2 weeks after the feed was reduced. Fasting and decreased rations increase plasma GH in salmonids, within a period of 5 days to 4 weeks ŽDuan and Plisetskaya, 1993; Bjornsson, 1997.. An inverse relation between ration and GH was also found in birds ŽMcMurtry et al., 1997., and mammals ŽKetelslegers et al., 1996. except rats, where fasting was associated with decreased plasma GH Že.g. Frystyk et al., 1999.. However, the graded inverse relation at intermediate ration levels Ž0᎐2 or 3% body weightrday. found in gilthead sea bream ŽPerez-Sanchez et al., 1995; Company et al., 1999. and tilapia ŽToguyeni et al., 1996. has not, to our knowledge, been demonstrated in salmonids and was not found in the present study. Lower rations Žless than 0.6% body weightrday. may be required to elicit sustained increases in GH in salmonids than in sea bream or tilapia. The return to baseline GH levels at 4 weeks after the ration switch is interesting. Studies of the effects of fasting or low rations on GH in fish have shown continuously increasing plasma levels. The present study is the first we are aware of that shows a transitory increase in GH following decreased rations. The return to baseline values may represent an accommodation of the somatotropic axis to the change in nutrition. It is interesting to note that a reduction in SGRW Žweek 0᎐2. coincided with the GH peak in low ration fish at week 2. Low ration SGRW for
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weeks 2᎐4 was higher when GH had returned to a baseline level Ž Ps 0.058.. Reductions in growth rate and feed efficiency under conditions of high GH have been found in sea bream ŽPerez-Sanchez et al., 1995; Company et al., 1999.. No correlation was found in this study between GH and growth rate, or between circulating GH and IGF-I levels. Thus, plasma IGF-I levels were more strongly and directly related to feeding level and growth rate than plasma GH levels. Bjornsson et al. Ž1995. found a non-linear positive correlation between GH and growth rate in smolting Atlantic salmon, in which plasma GH remained relatively constant Ž0᎐2 ngrml. for SGRW up to 0.6% body weightrday, but increased to higher levels Ž2᎐7 ngrml. for SGRW above 0.6% body weightrday. In contrast, no relation between plasma GH and growth rate over comparable ranges was observed in Atlantic salmon in seawater ŽHandeland et al., 2000.. This suggests a permissive role for GH in growth. As previously discussed, however, GH increases during fasting in salmonids and other fish species. Exogenous GH stimulates increases in growth ŽMcLean and Donaldson, 1993. hepatic IGF-I mRNA ŽCao et al., 1989. and plasma IGF-I ŽMoriyama et al., 1994.. Niu et al. Ž1993. reported a positive correlation between endogenous GH and IGF-I measured 1.5 h later in rainbow trout terminally sampled throughout the day. The lack of such a correlation in the present study may be due to the absence of an appropriate delay between samples for GH and IGF-I measurement. Alternatively, circulating IGF-I levels may not reflect the level of GH under our experimental conditions. In spite of the clear importance of GH, the regulation of IGF-I is complex, involving multiple hormones and nutritional factors ŽDuan, 1998; Perez-Sanchez and Le Bail, 1999.. In addition, GH secretion is episodic in salmonids ŽGomez et al., 1996., as in carp ŽZhang et al., 1994. and, thus, the single GH measure per fish at each sampling point in this study may not be representative of GH status. Growth rates increased with increasing ration level ŽFig. 1., consistent with energetic constraints ŽBrett, 1979.. Fish on the three ration levels showed overlapping ranges of growth rates. Individual SGRW rankings were consistent over time in all tanks ŽFig. 2.. This could be due to consistent individual differences in feed intake, digestive processes, metabolic rate, growth efficiency,
or a combination thereof. The food intake of individual rainbow trout was consistently ranked over time in tanks of fish fed restricted rations by belt feeders ŽJobling and Koskela, 1996.. This was attributed to dominance hierarchies operating within tanks. Dominance hierarchies may also explain the consistent ranking found in the present study. Alternatively, differences in voluntary feed intake may be responsible. Fish subjected to repeated blood draws Žserial sampling. showed reduced hematocrit and growth rates. The reduction in hematocrit indicates that fish were not able to completely replace lost red blood cells between samplings. The reduction in growth may be due to the stress of frequent blood draws ŽBarton et al., 1987.. The use of a single tank at each ration level does not permit the exclusion of tank effects. However, the responses seen in size, shape, and growth rate fit reasonable expectations, and the hormonal responses are consistent with previous studies on growth regulation in fish and mammals. Thus, these differences are likely to result from the different rations fed to fish in the tanks. Based on the results of this study, the nutritional regulation of plasma IGF-I appears to be similar in salmonids, other teleosts, and mammals. IGF-I responded to a change in ration over 2᎐4 weeks. IGF-I correlated strongly with growth rate after 4 weeks. In contrast, GH responded only transiently to the lowest ration, and was not correlated with growth rate. Further work is necessary to determine the course of events in the somatotropic axis response to changes in nutritional status in greater detail.
Acknowledgements This study was funded by fellowships from the H. Mason Keeler Endowment and the John E. Halver Endowment at the University of Washington School of Fisheries ŽALP., and grants from the US Department of Agriculture and the Bonneville Power Administration Žproject no. 199202202. ŽWWD.. We would like to thank Dr. Penny Swanson for assistance and advice on radioimmunoassays, and Beeda Lee-Pawlak for sampling help. This publication was supported by the Joint Institute for the Study of the Atmosphere and Ocean ŽJISAO. under NOAA
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Cooperative Agreement no. NA67RJ0155, Contribution no. 775. References Barton, B.A., Schreck, C.B., Barton, L.D., 1987. Effects of chronic cortisol administration and daily acute stress on growth, physiological conditions, and stress responses in juvenile rainbow trout. Dis. Aquat. Org. 2, 173᎐185. Beckman, B.R., Dickhoff, W.W., Zaugg, W.S. et al., 1999. Growth, smoltification, and smolt-to-adult return of spring chinook salmon from hatcheries on the Deschutes river. Oregon. Trans. Am. Fish. Soc. 128, 1125᎐1150. Beckman, B.R., Larsen, D.A., Moriyama, S., LeePawlak, B., Dickhoff, W.W., 1998. Insulin-like growth factor-I and environmental modulation of growth during smoltification of spring chinook salmon Ž Oncorhynchus tshawytscha.. Gen. Comp. Endocrinol. 109, 325᎐335. Bjornsson, B.T., 1997. The biology of salmon growth hormone: from daylight to dominance. Fish Physiol. Biochem. 17, 9᎐24. Bjornsson, B.T., Stefansson, S.O., Hansen, T., 1995. Photoperiod regulation of plasma growth hormone levels during parr᎐smolt transformation of Atlantic salmon: implications for hypoosmoregulatory ability and growth. Gen. Comp. Endocrinol. 100, 73᎐82. Blaise, O., Weil, C., Le Bail, P.Y., 1995. Role of IGF-I in the control of GH secretion in rainbow trout Ž Oncorhynchus mykiss.. Growth. Regul. 5, 142᎐150. Brett, J.R., 1979. Environmental factors and growth. In: Hoar, W.S., Randall, D.J., Brett, J.R. ŽEds.., Fish Physiology, Vol. 8. Bioenergetics and growth. Academic Press, New York, pp. 599᎐675. Cao, Q.P., Duguay, S.J., Plisetskaya, E., Steiner, D.F., Chan, S.J., 1989. Nucleotide sequence and growth hormone-regulated expression of salmon insulin-like growth factor I mRNA. Mol. Endocrinol. 3, 2005᎐2010. Cho, C.Y., 1992. Feeding systems for rainbow trout and other salmonids with reference to current estimates of energy and protein requirements. Aquaculture 100, 107᎐123. Company, R., Calduch-Giner, J.A., Kaushik, S., PerezSanchez, J., 1999. Growth performance and adiposity in gilthead sea bream Ž Sparus aurata.: risks and benefits of high energy diets. Aquaculture 171, 279᎐292. Daughaday, W.H., Rotwein, P., 1989. Insulin-like growth factors I and II. Peptide, messenger ribonucleic acid and gene structures, serum, and tissue concentrations. Endocr. Rev. 10, 68᎐91. Duan, C., 1998. Nutritional and developmental regula-
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tion of insulin-like growth factors in fish. J. Nutr. 128, 306S᎐314. Duan, C., Plisetskaya, E.M., 1993. Nutritional regulation of insulin-like growth factor-I mRNA expression in salmon tissues. J. Endocrinol. 139, 243᎐252. Foucher, J.L., Le Bail, P.Y., Le Gac, F., 1992. Influence of hypophysectomy, castration, fasting, and spermiation on SBP concentration in male rainbow trout Ž Oncorhyncus mykiss.. Gen. Comp. Endocrinol. 85, 101᎐110. Frystyk, J., Delhanty, P.J., Skjaerbaek, C., Baxter, R.C., 1999. Changes in the circulating IGF system during short-term fasting and refeeding in rats. Am. J. Physiol. 277, E245᎐252. Gomez, J.M., Boujard, T., Fostier, A., Le Bail, P.Y., 1996. Characterization of growth hormone nycthemeral plasma profiles in catheterized rainbow trout Ž Oncorhyncus mykiss.. J. Exp. Zool. 274, 171᎐180. Gray, E.S., Kelley, K.M., Law, S., Tsai, R., Young, G., Bern, H.A., 1992. Regulation of hepatic growth hormone receptors in coho salmon Ž Oncorhyncus kisutch.. Gen. Comp. Endocrinol. 88, 243᎐252. Handeland, S.O., Berge, A., Bjornsson, B.T., Lie, O., Stefansson, S.O., 2000. Seawater adaptation by outof-season Atlantic salmon Ž Salmo salar L. smolts at different temperatures. Aquaculture 181, 377᎐396. Jobling, M., Koskela, J., 1996. Interindividual variations in feeding and growth in rainbow trout during restricted feeding and in a subsequent period of compensatory growth. J. Fish Biol. 49, 658᎐667. Ketelslegers, J.M., Maiter, D., Maes, M., Underwood, L.E., Thissen, J.P., 1996. Nutritional regulation of the growth hormone and insulin-like growth factorbinding proteins. Horm. Res. 45, 252᎐257. Matthews, S.J., Kinhult, A.K., Hoeben, P., Sara, V.R., Anderson, T.A., 1997. Nutritional regulation of insulin-like growth factor-I mRNA expression in barramundi, Lates calcarifer. J. Mol. Endocrinol. 18, 273᎐276. McLean, E., Donaldson, E.M., 1993. The role of growth hormone in the growth of poikilotherms. In: Schreibman, M.P., Scanes, C.G., Pang, P.K.T. ŽEds.., The Endocrinology of Growth, Development and Metabolism in Vertebrates. Academic Press, San Diego, pp. 43᎐71. McMurtry, J.P., Francis, G.L., Upton, Z., 1997. Insulin-like growth factors in poultry. Domest. Anim. Endocrinol. 14, 199᎐229. Moriyama, S., Swanson, P., Nishii, M. et al., 1994. Development of a homologous radioimmunoassay for coho salmon insulin- like growth factor-I. Gen. Comp. Endocrinol. 96, 149᎐161. Niu, P.D., Perez-Sanchez, J., Le Bail, P.Y., 1993. Development of a protein binding assay for teleost
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insulin-like growth factor ŽIGF.-like: Relationships between growth hormone ŽGH. and IGF-like in the blood of rainbow trout Ž Oncorhyncus mykiss.. Fish Physiol. Biochem. 11, 381᎐391. Perez-Sanchez, J., Le Bail, P.-Y., 1999. Growth hormone axis as marker of nutritional status and growth performance in fish. Aquaculture 177, 117᎐128. Perez-Sanchez, J., Marti-Palanca, H., Kaushik, S.J., 1995. Ration size and protein intake affect circulating growth hormone concentration, hepatic growth hormone binding and plasma insulin-like growth factor-I immunoreactivity in a marine teleost, the gilthead sea bream Ž Sparus aurata.. J. Nutr. 125, 546᎐552. Perez-Sanchez, J., Marti-Palanca, H., Le Bail, P.Y., 1994. Homologous growth hormone ŽGH. binding in gilthead sea bream Ž Sparus aurata.. Effect of fasting and refeeding on hepatic GH-binding and plasma somatomedin-like immunoreactivity. J. Fish Biol. 44, 287᎐301. Perez-Sanchez, J., Weil, C., Le Bail, P.Y., 1992. Effects of human insulin-like growth factor-I on release of growth hormone by rainbow trout Ž Oncorhynchus mykiss. pituitary cells. J. Exp. Zool. 262, 287᎐290. Plisetskaya, E.M., 1998. Some of my not so favorite things about insulin and insulin-like growth factors in fish. Comp. Biochem. Physiol. 121B, 3᎐11. Ricker, W.E., 1979. Growth rates and models. In: Ricker, W.E., Hoar, W.S., Randall, D.J., Brett, J.R. ŽEds.., Fish Physiology, vol. 8. Bioenergetics and Growth. Academic Press, New York, pp. 738᎐743. Shimizu, M., Swanson, P., Fukada, H., Hara, A., Dickhoff, W.W., 2000. Comparison of extraction methods and assay validation for salmon insulin-like growth
factor-I using commercially available components. Gen. Comp. Endocrinol. 119, 26᎐36. Silverstein, J.T., Shearer, K.D., Dickhoff, W.W., Plisetskaya, E.M., 1998. The effects of growth and fatness on sexual development of chinook salmon Ž Oncorhynchus tshawytscha. parr. Can. J. Fish. Aquat. Sci. 55, 2376᎐2382. Straus, D.S., 1994. Nutritional regulation of hormones and growth factors that control mammalian growth. Faseb J. 8, 6᎐12. Swanson, P., 1994. Radioimmunoassay of fish growth hormone, prolactin, and somatolactin. In: Hochachka, P.W., Mommsen, T.P. ŽEds.., Biochemistry and Molecular Biology of Fishes, vol. 3. Analytical Techniques. Elsevier Science, Amsterdam, pp. 545᎐565. Thissen, J.P., Ketelslegers, J.M., Underwood, L.E., 1994. Nutritional regulation of the insulin-like growth factors. Endocr. Rev. 15, 80᎐101. Thissen, J.P., Underwood, L.E., Ketelslegers, J.M., 1999. Regulation of insulin-like growth factor-I in starvation and injury. Nutr. Rev. 57, 167᎐176. Toguyeni, A., Baroiller, J.F., Fostier, A. et al., 1996. Consequences of food restriction on short-term growth variation and on plasma circulating hormones in Oreochromis niloticus in relation to sex. Gen. Comp. Endocrinol. 103, 167᎐175. Zar, J.H., 1996. Biostatistical Analysis, Third Edition Prentice Hall, Upper Saddle River, New Jersey. Zhang, W.M., Lin, H.R., Peter, R.E., 1994. Episodic growth hormone secretion in the grass carp, Ctenopharyngodon idellus ŽC. & V... Gen. Comp. Endocrinol. 95, 337᎐341.
Effects of ration on somatotropic hormones and growth in coho salmon Andrew L. Pierce a,U , Brian R. Beckmana,b, Karl D. Shearer b, Donald A. Larsen b, Walton W. Dickhoff a,b b
a School of Fisheries, Uni¨ ersity of Washington, Seattle, WA 98195, USA Integrati¨ e Fish Biology Program, Northwest Fisheries Science Center, National Marine Fisheries Ser¨ ice, 2725 Montlake Boule¨ ard E., Seattle, WA 98112, USA
Received 7 June 2000; received in revised form 5 October 2000; accepted 16 October 2000
Abstract We examined the response of growth hormone ŽGH., total plasma insulin-like growth-factor I ŽIGF-I., and growth rate to a change in ration in coho salmon. Tanks of individually tagged fish were placed on high, medium, or low ration, and sampled every 2 weeks for 8 weeks to create a range of growth rates. Some fish received non-lethal blood draws, while others were sampled terminally. Plasma IGF-I levels were higher in high ration fish than in low ration fish from 4 weeks after the beginning of experimental diets to the end of the experiment. GH levels were low and similar in all fish after changing rations, except for the fish in the low ration group at week 2. IGF-I was strongly correlated with specific growth rate in weight in terminally sampled fish after 4 weeks. GH did not correlate with growth rate or IGF-I levels. Growth parameters Žlength, weight, specific growth rates in weight and length, and condition factor. responded to ration. Serial sampling reduced growth rates and hematocrit, but did not change hormone levels. This study shows that IGF-I responds to changed rations within 2᎐4 weeks in salmonids. Published by Elsevier Science Inc. Keywords: Insulin-like growth factor-I ŽIGF-I.; Growth hormone ŽGH.; Growth; Nutrition; Coho salmon Ž Oncorhynchus kisutch.; Somatotrophic; Hatchery; Time course
1. Introduction The somatotropic endocrine axis regulates growth in all vertebrates. The axis consists of the peptide hormone growth hormone ŽGH. secreted
Corresponding author. Tel.: q1-206-860-3248; fax: q1-206860-3267. E-mail address: [email protected] ŽA.L. Pierce.. 1096-4959r01r$ - see front matter Published by Elsevier Science Inc. PII: S 1 0 9 6 - 4 9 5 9 Ž 0 0 . 0 0 3 2 4 - 9
by the pituitary gland, insulin-like growth factor-I ŽIGF-I. produced in most tissues, and their receptors and binding proteins. According to the widely accepted somatomedin hypothesis, many of the growth promoting effects of GH are mediated by circulating IGF-I, which is produced by the liver in response to GH ŽDaughaday and Rotwein, 1989.. In mammals, circulating IGF-I levels are often strongly correlated with growth rate ŽStraus, 1994..
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Nutritional status regulates the somatotropic axis. In all species studied to date, IGF-I declines during fasting, whereas GH increases in nearly all species ŽStraus, 1994; McMurtry et al., 1997; Duan, 1998.. Hepatic GH sensitivity is important in nutritional regulation of IGF-I. Changes in metabolic hormones Ži.e. insulin, glucagon, thyroid hormones, and glucocorticoids. and nutrient levels during fasting or feed restriction cause decreases in hepatic GH receptor mRNA and protein, which results in decreased IGF-I production ŽThissen et al., 1999.. The subsequent decrease in circulating IGF-I level may increase GH secretion via relaxation of negative feedback ŽThissen et al., 1999.. All components of the somatotropic axis exist in fish, and the basic operation of the axis appears to be similar in fish and mammals ŽDuan, 1998.. Furthermore, nutritional regulation of the somatotropic axis in fish appears to fit the mammalian model. Nutritional status positively regulates IGF-I, and negatively regulates GH ŽDuan, 1998.. Studies in the marine teleost gilthead sea bream Ž Sparus aurata. have shown that ration and dietary protein level regulate hepatic GH receptor levels, plasma IGF-I, and plasma GH ŽPerezSanchez et al., 1994, 1995; Company et al., 1999.. In salmonids, hepatic GH receptor level is regulated by nutrition ŽGray et al., 1992.. Negative feedback of IGF-I on GH secretion has been demonstrated in salmonids ŽPerez-Sanchez et al., 1992; Blaise et al., 1995.. Of the studies on nutrition and endocrine growth regulation in teleosts, only two have measured both plasma GH and IGF-I levels ŽNiu et al., 1993; Perez-Sanchez et al., 1995.. In these studies, the fish were not sampled until 4 ŽNiu et al., 1993. and 7 ŽPerez-Sanchez et al., 1995. weeks after beginning feeding treatments. Changes in growth occur much earlier than this, and one would expect plasma GH and IGF-I levels to respond earlier as well. Curiously, the single study examining the time course of the hepatic IGF-I mRNA response to fasting did not find a reduction until 28 days after fasting began ŽDuan and Plisetskaya, 1993.. We sought to document the time course of changes in plasma GH, IGF-I, and growth rate in response to a change in ration, sampling every 2 weeks for 8 weeks. Previous investigators have looked at the average growth rates and hormone levels of tanks of fish. By using tagged fish, we were able to assess the
relationship between growth rates and hormone levels of individuals.
2. Materials and methods The experiment used 1994 brood University of Washington strain coho salmon Ž Oncorhyncus kisutch., obtained in April 1996 from the Sandpoint hatchery of the Biological Resources Division, United States Geological Survey ŽSeattle, WA.. Fish were housed in a 1.3-m cylindrical tank, under simulated natural photoperiod, in a recirculating freshwater hatchery at the Northwest Fisheries Science Center ŽSeattle, WA.. The fish were fed commercial pelleted feed ŽBiodiet Grower, Bioproducts, Inc.,1 wet weight composition 43% protein, 10.5% carbohydrate, 14.5% lipid., 5 daysrweek by belt feeder at an overall feeding rate of 1.4% body weightrday. On 28 June 1996 Žweek y2., 180 fish w100.4" 2.8 g, mean " standard error ŽS.E..x were implanted intraperitoneally with passive integrated transponder tags ŽPIT tags, DestronrIDI., weighed, measured, and divided into three tanks. Based on appearance, fish were post-smolts at this time. Rations of 1.4% body weightrday were continued for 2 weeks. On 10 July 1996 Žweek 0., the tagged fish were switched onto experimental rations. The high ration was calculated to produce maximal growth, and the low ration to maintain current weight without permitting growth. The medium ration was the average of high and low rations. Ration calculations were performed according to Cho Ž1992., adjusted daily for temperature and weight of fish in each tank, with an estimated thermalunit growth coefficient of 0.0023 g 1r3⬚Cy1 dayy1 . The fish were fed Biodiet pellets 7 daysrweek. Average rations were high 1.6, medium 1.1, and low 0.6% body weightrday. High and medium rations were fed by belt feeder, whereas the low ration was fed by hand, to allow all fish a chance at the food. The fish in the low ration tank were seen to eat all of the food supplied, and no uneaten food was ever observed in the high or medium ration tanks. The fish were fed experimental rations for 8 weeks, during which time the
1 Use of trade names does not imply endorsement of NOAA, U.S. Department of Commerce.
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water temperature ranged from 11 to 15⬚C, average 12.5⬚C, without seasonal trends. Samples were taken at week 0 and every 2 weeks thereafter Žweek 2: 24 July 1996; week 4: 7 August; week 6: 21 August; week 8: 4 September.. All fish were removed from each treatment tank. The fish were then individually anesthetized in a 0.05 ᎐ 0.1-grl buffered solution of tricane methanesulfonate ŽMS-222., and weight, fork length, and PIT tag number were recorded with an automated system ŽBiomark Inc., Boise, ID.. Pre-determined fish, identified by a PIT tag number, either received non-lethal blood draws Žserial samples., or were killed Žterminal samples.. Fish not scheduled for serial or terminal sampling were returned to the treatment tank. Sixteen fish per tank were serially sampled at weeks 0, 2, 4, and 6. Four fish per tank were terminally sampled at week 0, and 10 fish per tank were terminally sampled at weeks 2, 4 and 6. All remaining fish were terminally sampled at week 8. Serial samples were taken from the same fish at each time point. On each sampling date, tanks were sampled from 08.00 h to approximately 13.00 h in the same order: medium, low, high. For serial sampling, the caudal artery was punctured with a sterile 22-gauge hypodermic needle and ; 200 l of blood was drawn into a heparinized syringe, after which the fish was returned to the tank. For terminal sampling, the caudal peduncle was transected and blood from the caudal artery was collected into heparinized test tubes. Blood was centrifuged for 5 min at 3000 = g, hematocrit level noted, and plasma frozen at y80⬚C for later radioimmunoassays. Individual fish specific growth rates in length ŽSGRL. were calculated by the method of Ricker Ž1979. SGRL s 100U wlnŽlength 2. y lnŽlength 1.xrdays, where ‘length 2’ and ‘length 1’ were fish lengths at the end and beginning of the growth period, respectively, and ‘days’ was the duration of the growth period. Specific growth rates in weight ŽSGRW. were calculated similarly. The condition factor ŽCF . was calculated as 100U weight Žg.rlength Žcm. 3. Plasma GH was assayed according to Swanson Ž1994.. Samples were run in two assays with intraassay coefficients of variation Ž%CV. of 17.6 and 6.2% and an interassay %CV of 8.0%. Values from the second assay were adjusted to the first by a linear correction factor. Plasma IGF-I was assayed after acid᎐ethanol extraction accord-
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ing to Shimizu et al. Ž2000.. Briefly, 25-l plasma samples were incubated with 100-l of an acid᎐ethanol solution Ž87.5% ethanol, 12.5% 2 N HCl. for 30 min at room temperature, neutralized with 80 l 0.855 M Tris, and centrifuged for 30 min at 1800 = g. The supernatent was assayed in duplicate for IGF-I using recombinant salmon IGF-I tracer and a standard, and anti-recombinant barramundi IGF-I antibody Žcomponents purchased from GroPep Pty Ltd., Adelaide, Australia.. An appropriate amount of acid᎐ethanol q Tris solution was added to standard curves. Samples were run in two assays with intraassay %CVs of 9.40 and 8.73%, including the extraction step and an interassay %CV of 0.77%. IGF-I was not measured in all samples due to insufficient plasma. Measurements were performed for 144 out of 150 terminal samples and 137 out of 237 serial samples. Plasma from the last two sampling points Žweeks 6 and 8. was inadvertently thawed and kept in a refrigerator Ž4⬚C. for 2 weeks before the IGF-I assay. The effects of ration and serial sampling on growth, hormone levels, condition factor, and hematocrit were examined at the level of tanks of fish as a matter of convenience, since each tank was fed a different ration. Multiple-way analyses of variance ŽANOVAs. with date, ration, and serial vs. terminal fish as effects were performed for each dependent variable. Significant effects were further examined by one-way ANOVA followed by Fisher’s protected least significant difference ŽZar, 1996.. Relations between growth and hormone levels were investigated by linear regression. Relations between growth rate rankings of individual fish over different periods were examined using the Spearman rank correlation test. Results were considered statistically significant at P- 0.05. The results described here are statistically significant unless otherwise stated.
3. Results 3.1. Growth Ration, date, serial sampling, and the interaction of date and ration affected fish size. High and medium ration fish increased in size over the course of the experiment, and this increase was influenced by ration level, with higher ration fish growing more ŽFig. 1a,b.. Fish on low rations did
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Fig. 1. Ža. Weight, Žb. length, Žc. condition factor, Žd. specific growth rate in weight ŽSGRW., and Že. length ŽSGRL. through the experiment. High ration: ᎏ`ᎏ ; medium ration: ---I---; low ration: --^--. Points sharing a common superscript or without superscripts at each sampling date do not differ significantly. Asterisks indicate significant differences vs. week 0 Žpanels a, b, and c. or week y2᎐0 Žpanels d and e..
not gain significant weight or length through the experiment, and were lighter and shorter than medium and high ration fish beginning at week 4. There was a significant difference in weight between medium and high ration fish only at week 6. Differences in condition factor ŽCF., a measure of fish body shape, developed between ration treatments ŽFig. 1c.. Differences among fish in all three tanks became significant at the week 4 sampling and were maintained throughout the rest of the experiment, with high and medium ration CF increasing through the experiment and low ration CF unchanged. Ration and date affected weight growth rate
ŽSGRW. ŽFig. 1d.; significant differences among all treatments appeared at week 0᎐2, and were maintained up to week 6᎐8, when the difference between high and medium ration became non-significant. The SGRW of fish on the high ration increased from week y2᎐0 to all later intervals, although SGRW declined from week 2᎐4 to week 4᎐6. Medium ration SGRW increased from week y2᎐0 to 0᎐2, but not later intervals. There were no further significant changes in medium ration SGRW. Low ration SGRW decreased after the switch onto experimental rations. A subsequent increase from week 0᎐2 to 2᎐4 approached significance Ž Ps 0.0580..
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and CF were not significantly affected by repeated blood draws. SGRW was depressed in serially sampled fish relative to terminals at weeks 4, 6 and 8, and SGRL at weeks 6 and 8. From week 0 to week 8, serial fish hematocrit declined from 40.1" 0.5 to 30.9" 1.2 Žmean " S.E. of the mean., with the greatest decline from week 0 to 2. Hematocrit levels of terminally sampled fish did not vary significantly through the experiment. Plasma IGF-I and GH levels did not differ significantly between serial and terminal fish, and there were no trends toward any such difference. 3.3. Ration effects on GH and IGF-I
Fig. 2. Relationship between SGRW for weeks 0᎐4 and weeks 4᎐8 in individual fish terminally sampled at week 8. High ration: `, Spearman rank correlation Rhos 0.625, Ps 0.0194; medium ration: I, Rhos 0.564, Ps 0.0241; low ration: ^, Rhos 0.600, Ps 0.0248.
Ration and date affected length growth rate ŽSGRL. ŽFig. 1e.. After the switch to experimental diets, fish on the high ration had a higher SGRL than those on low ration over every interval. Fish on the high ration also had a higher SGRL than those on the medium ration for weeks 0᎐2, 2᎐4 and 4᎐6, and fish on the medium ration had a higher SGRL than those on the low ration for weeks 2᎐4 and 6᎐8. In high and low ration fish, SGRL increased from week y2᎐0 to all later intervals. Also in high and low ration fish, SGRL decreased from week 2᎐4 to week 6᎐8. In medium ration fish, SGRL increased from week y2᎐0 to week 2᎐4 only. The SGRWs of individual fish identified by PIT tag number showed consistent ranking over time, both overall and within each tank ŽFig. 2.. Excluding the 2-week period prior to beginning experimental diets Žweek y2᎐0., all possible comparisons of SGRW of individual fish over different 2-week periods showed significant consistency of SGRW rank over time by the Spearman rank correlation. 3.2. Serial sampling Serially sampled fish had lower hematocrit, SGRW, and SGRL than terminally sampled fish Ždata not shown.. In contrast, fish length, weight,
Date and ration affected the plasma levels of both GH and IGF-I. GH peaked in fish in the low ration treatment at week 2, at which point it was higher in this treatment than in either of the others ŽFig. 3a.. GH levels did not vary signifi-
Fig. 3. Ža. GH and Žb. IGF-I levels through the experiment. High ration: ᎏ`ᎏ; medium ration: ---I---; low ration: --^--. Points sharing a common superscript or without superscripts at each sampling date do not differ significantly. Asterisks show significant differences vs. week 0. No line connects points from weeks 4 and 6 to indicate that weeks 6 and 8 should not be compared to earlier time points. Plasma from weeks 6 and 8 was inadvertently thawed and kept in a refrigerator for 2 weeks before IGF-I was assayed.
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cantly between treatments at any point other than week 2. The GH level at week 2 in low ration fish was higher than GH levels in low ration fish at weeks 0, 4, 6 and 8. In fish the high ration treatment, GH declined from week 0 to all later time points. A similar, but non-significant, decline occurred in fish in the medium ration treatment. In a two-way ANOVA with date and ration as effects, IGF-I increased with increasing ration. IGF-I levels were similar at week 0. A non-significant trend toward ranking of treatments appeared at week 2, with high ration having highest IGF-I, medium ration intermediate, and low ration having lowest IGF-I. At weeks 4 and 6, high ration fish had higher IGF-I than low or medium ration fish, whereas at week 8, high and medium ration fish had higher IGF-I than low ration fish. In the two-way ANOVA, a decline in IGF-I level occurred from weeks 2 and 4 to weeks 6 and 8, i.e. IGF-I was lower in samples that were accidentally left in a refrigerator for 2 weeks. Changes over time were not significant within tanks. 3.4. Growth and hormone le¨ el correlations Plasma IGF-I levels at week 4 were positively correlated with SGRW for the previous 2 weeks in both terminal and serial fish ŽFig. 4.. The strongest correlation was found in terminal fish ŽFig. 4a, P- 0.0001, r 2 s 0.623., whereas that in serial fish tended to be weaker ŽFig. 4b, Ps 0.001, r 2 s 0.257., except in low ration fish, where a high correlation was found Ž Ps 0.0005, r 2 s 0.687.. Week 4 IGF-I was also positively correlated with SGRL for the previous 2 weeks in both terminal Ž Ps 0.0003, r 2 s 0.389. and serial Ž Ps 0.0017, r 2 s 0.236. fish. Regression analysis of possible relations between GH at each sampling point and IGF-I level, SGRW, SGRL, and condition factor did not show any significant relations.
4. Discussion This study provides the first characterization of the time course of the combined GH and IGF-I response to a change in ration in a teleost. IGF-I responded to changed ration and correlated with
Fig. 4. Relationship between week 4 IGF-I and SGRW for weeks 2᎐4 in Ža. terminally sampled and Žb. serially sampled fish. High ration: `; medium ration: I; low ration: ^. Regression lines shown for all fish combined. Regression statistics: Ža. all fish: P - 0.0001, r 2 s 0.623; high ration: P s 0.118; medium ration: Ps 0.0026, r 2 s 0.699; low ration: P s 0.109; Žb. all fish: Ps 0.001, r 2 s 0.257; high ration: Ps 0.707; medium ration: Ps 0.097; low ration: Ps 0.0005, r 2 s 0.687.
growth rate within 4 weeks. GH increased transiently at the lowest ration, but did not show a consistent relationship to growth rate. A trend toward IGF-I ranking by ration appeared at week 2, with higher ration fish showing higher IGF-I, and the high vs. low ration difference became significant at week 4. IGF-I ranking by ration continued up to week 8. The ranking of IGF-I by ration agrees with models of growth regulation in which IGF-I is under nutritional control. Positive relations of IGF-I with ration
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are found in birds ŽMcMurtry et al., 1997. mammals ŽThissen et al., 1994. and fish ŽDuan, 1998; Perez-Sanchez and Le Bail, 1999.. Previous studies in fish have found effects of feeding level on plasma IGF-I level. Reduced plasma IGF-I was found after fasting for 25 days in coho salmon smolts ŽMoriyama et al., 1994. and after fasting for 6 weeks in rainbow trout ŽFoucher et al., 1992.. Combined temperature and ration Ž1.4 and 1.8% body weightrday. treatments of chinook salmon resulted in the separation of IGF-I levels after approximately 1 month ŽBeckman et al., 1998.. IGF-I ranked by ration after 7 weeks of feeding treatment in gilthead sea bream ŽPerezSanchez et al., 1995.. Hepatic IGF-I mRNA levels were decreased after 4 weeks of fasting and restored by 2 weeks of refeeding in coho salmon ŽDuan and Plisetskaya, 1993.. IGF-I like immunoreactivity was reduced by 4 weeks of fasting in rainbow trout ŽNiu et al., 1993.. In barramundi, 21 days of fasting reduced hepatic IGF-I mRNA ŽMatthews et al., 1997.. This study confirms and extends these findings by showing that, in coho salmon under our rearing conditions, plasma IGF-I responds to relatively mild changes in ration in 2᎐4 weeks. This is considerably longer than the IGF-I response to fasting in rats, which takes 2᎐3 days Že.g. Frystyk et al., 1999.. This difference may be due in part to the less severe nutritional restriction employed in this study, and species differences associated with poikilothermy and adaptation to prolonged fasting in salmonids ŽDuan, 1998.. Plasma IGF-I at week 4 correlated strongly Ž r 2 s 0.623. with SGRW for the previous 2 weeks in terminally sampled fish. Some previous studies have not found significant or strong correlations between individual IGF-I levels and growth rates Žunpublished studies reviewed in Plisetskaya, 1998; Silverstein et al., 1998.. However, tank average IGF-I levels and monthly specific growth rates in length correlated strongly Ž r 2 s 0.50 and 0.65. in two studies on chinook salmon ŽBeckman et al., 1998, 1999., and forthcoming data show strong correlations Ž r 2 s 0.5᎐0.8. between individual chinook salmon IGF-I levels and monthly specific growth rates ŽB. Beckman, unpublished data.. Studies in juvenile mammals have found strong correlations of IGF-I with various measures of growth, with r 2 values in the 0.7᎐0.8 range ŽStraus, 1994.. Plisetskaya Ž1998. speculates that the correlation between plasma IGF-I and growth
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rate in fish may vary seasonally and with developmental stage, and that these factors and the characteristics of IGF-I assays and IGFBP extraction techniques used may explain the lack of correlation in some studies. IGF-I probably both reflects past growth and predicts future growth. Further work is necessary to determine the time period and growth measurement which gives the strongest correlation. In this study, correlations between IGF-I at week 4 and SGRW for the previous 2 weeks were weaker in high and medium ration serially sampled fish than in high and medium ration terminally sampled fish. Serially sampled fish had had two previous blood draws at this point. In contrast, low ration serially sampled fish showed a comparably strong correlation Ž r 2 s 0.687.. The reasons for these differences are not clear, but they may be related to the stress of the repeated blood draws, andror differences in the suite of IGF binding proteins between different ration and sampling groups. Plasma GH levels peaked in low ration fish 2 weeks after the feed was reduced. Fasting and decreased rations increase plasma GH in salmonids, within a period of 5 days to 4 weeks ŽDuan and Plisetskaya, 1993; Bjornsson, 1997.. An inverse relation between ration and GH was also found in birds ŽMcMurtry et al., 1997., and mammals ŽKetelslegers et al., 1996. except rats, where fasting was associated with decreased plasma GH Že.g. Frystyk et al., 1999.. However, the graded inverse relation at intermediate ration levels Ž0᎐2 or 3% body weightrday. found in gilthead sea bream ŽPerez-Sanchez et al., 1995; Company et al., 1999. and tilapia ŽToguyeni et al., 1996. has not, to our knowledge, been demonstrated in salmonids and was not found in the present study. Lower rations Žless than 0.6% body weightrday. may be required to elicit sustained increases in GH in salmonids than in sea bream or tilapia. The return to baseline GH levels at 4 weeks after the ration switch is interesting. Studies of the effects of fasting or low rations on GH in fish have shown continuously increasing plasma levels. The present study is the first we are aware of that shows a transitory increase in GH following decreased rations. The return to baseline values may represent an accommodation of the somatotropic axis to the change in nutrition. It is interesting to note that a reduction in SGRW Žweek 0᎐2. coincided with the GH peak in low ration fish at week 2. Low ration SGRW for
262
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weeks 2᎐4 was higher when GH had returned to a baseline level Ž Ps 0.058.. Reductions in growth rate and feed efficiency under conditions of high GH have been found in sea bream ŽPerez-Sanchez et al., 1995; Company et al., 1999.. No correlation was found in this study between GH and growth rate, or between circulating GH and IGF-I levels. Thus, plasma IGF-I levels were more strongly and directly related to feeding level and growth rate than plasma GH levels. Bjornsson et al. Ž1995. found a non-linear positive correlation between GH and growth rate in smolting Atlantic salmon, in which plasma GH remained relatively constant Ž0᎐2 ngrml. for SGRW up to 0.6% body weightrday, but increased to higher levels Ž2᎐7 ngrml. for SGRW above 0.6% body weightrday. In contrast, no relation between plasma GH and growth rate over comparable ranges was observed in Atlantic salmon in seawater ŽHandeland et al., 2000.. This suggests a permissive role for GH in growth. As previously discussed, however, GH increases during fasting in salmonids and other fish species. Exogenous GH stimulates increases in growth ŽMcLean and Donaldson, 1993. hepatic IGF-I mRNA ŽCao et al., 1989. and plasma IGF-I ŽMoriyama et al., 1994.. Niu et al. Ž1993. reported a positive correlation between endogenous GH and IGF-I measured 1.5 h later in rainbow trout terminally sampled throughout the day. The lack of such a correlation in the present study may be due to the absence of an appropriate delay between samples for GH and IGF-I measurement. Alternatively, circulating IGF-I levels may not reflect the level of GH under our experimental conditions. In spite of the clear importance of GH, the regulation of IGF-I is complex, involving multiple hormones and nutritional factors ŽDuan, 1998; Perez-Sanchez and Le Bail, 1999.. In addition, GH secretion is episodic in salmonids ŽGomez et al., 1996., as in carp ŽZhang et al., 1994. and, thus, the single GH measure per fish at each sampling point in this study may not be representative of GH status. Growth rates increased with increasing ration level ŽFig. 1., consistent with energetic constraints ŽBrett, 1979.. Fish on the three ration levels showed overlapping ranges of growth rates. Individual SGRW rankings were consistent over time in all tanks ŽFig. 2.. This could be due to consistent individual differences in feed intake, digestive processes, metabolic rate, growth efficiency,
or a combination thereof. The food intake of individual rainbow trout was consistently ranked over time in tanks of fish fed restricted rations by belt feeders ŽJobling and Koskela, 1996.. This was attributed to dominance hierarchies operating within tanks. Dominance hierarchies may also explain the consistent ranking found in the present study. Alternatively, differences in voluntary feed intake may be responsible. Fish subjected to repeated blood draws Žserial sampling. showed reduced hematocrit and growth rates. The reduction in hematocrit indicates that fish were not able to completely replace lost red blood cells between samplings. The reduction in growth may be due to the stress of frequent blood draws ŽBarton et al., 1987.. The use of a single tank at each ration level does not permit the exclusion of tank effects. However, the responses seen in size, shape, and growth rate fit reasonable expectations, and the hormonal responses are consistent with previous studies on growth regulation in fish and mammals. Thus, these differences are likely to result from the different rations fed to fish in the tanks. Based on the results of this study, the nutritional regulation of plasma IGF-I appears to be similar in salmonids, other teleosts, and mammals. IGF-I responded to a change in ration over 2᎐4 weeks. IGF-I correlated strongly with growth rate after 4 weeks. In contrast, GH responded only transiently to the lowest ration, and was not correlated with growth rate. Further work is necessary to determine the course of events in the somatotropic axis response to changes in nutritional status in greater detail.
Acknowledgements This study was funded by fellowships from the H. Mason Keeler Endowment and the John E. Halver Endowment at the University of Washington School of Fisheries ŽALP., and grants from the US Department of Agriculture and the Bonneville Power Administration Žproject no. 199202202. ŽWWD.. We would like to thank Dr. Penny Swanson for assistance and advice on radioimmunoassays, and Beeda Lee-Pawlak for sampling help. This publication was supported by the Joint Institute for the Study of the Atmosphere and Ocean ŽJISAO. under NOAA
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