Aquaculture 236 (2004) 583 – 592 www.elsevier.com/locate/aqua-online Correlation of plasma IGF-I concentrations and growth rate in aquacultured finfish: a tool for assessing the potential of new diets Anthony R. Dyer a,*, Christopher G. Barlow b, Matthew P. Bransden c, Chris G. Carter c, Brett D. Glencross d, Neil Richardson e,f, Philip M. Thomas g, Kevin C. Williams h, John F. Carragher g a TGR Biosciences, PO Box 185, Hindmarsh, SA 5007, Australia Queensland Department of Primary Industries, Freshwater Fisheries and Aquaculture Centre, Walkamin, QLD 4872, Australia c School of Aquaculture, Tasmanian Aquaculture and Fisheries Institute, University of Tasmania, Locked Bag 1-370, Launceston, TAS 7250, Australia d Western Australia, PO Box 20, North Beach, WA 6020, Australia e School of Life Sciences, Queensland University of Technology, GPO Box 2434, Brisbane, QLD 4001, Australia f Aquatic Sciences, South Australian Research and Development Institute, PO Box 20, Henley Beach, SA 5020, Australia g School of Biological Sciences, Flinders University, GPO Box 2100, Adelaide, SA 5001, Australia h CSIRO Marine Research, PO Box 120, Cleveland, Qld. 4163, Australia b Received 24 November 2003; received in revised form 18 December 2003; accepted 30 December 2003 Abstract A recently developed radioimmunoassay (RIA) for measuring insulin-like growth factor (IGF-I) in a variety of fish species was used to investigate the correlation between growth rate and circulating IGF-I concentrations of barramundi (Lates calcarifer), Atlantic salmon (Salmo salar) and Southern Bluefin tuna (Thunnus maccoyii). Plasma IGF-I concentration significantly increased with increasing ration size in barramundi and IGF-I concentration was positively correlated to growth rates obtained in Atlantic salmon (r2=0.67) and barramundi (r2=0.65) when fed a variety of diet formulations. IGF-I was also positively correlated to protein concentration (r2=0.59). This evidence suggested that measuring IGF-I concentration may provide a useful tool for monitoring fish growth rate and also as a method to rapidly assess different aquaculture diets. However, no such correlation was demonstrated in the tuna study probably due to seasonal cooling of sea surface temperature shortly before blood was sampled. Thus, some recommendations for the * Corresponding author. Tel.: +61-8-8354-6186; fax: +61-8-8354-6188. E-mail address: Anthony.Dyer@tgr-biosciences.com.au (A.R. Dyer). 0044-8486/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2003.12.025 584 A.R. Dyer et al. / Aquaculture 236 (2004) 583–592 design and sampling strategy of nutritional trials where IGF-I concentrations are measured are discussed. D 2004 Elsevier B.V. All rights reserved. Keywords: IGF-I; Nutrition; Growth; Fish 1. Introduction The important role of hormones in controlling growth and development has been extensively documented (Jones and Clemmons, 1995). One group of hormones fundamentally involved in growth regulation is the insulin-like growth factors (IGFs). In mammals, it appears that IGF-I is the major growth-promoting agent (Daughaday et al., 1987), and the concentration found circulating in the plasma is regulated by a complex interaction between receptors and various binding proteins. It has also been shown that plasma IGF-I concentration is largely regulated by nutritional status (Ketelslegers et al., 1995). In fish, circulating IGF-I levels seem to play an important role in growth regulation and has also been shown to be nutritionally regulated (Duan and Hirano, 1992; Duan and Plisetskaya, 1993; Niu et al., 1993; Moriyama et al., 1994; Perez-Sanchez et al., 1995; Silverstein et al., 1998, 2000). If IGF-I concentration could be used as a reproducible marker of growth performance, this could impact significantly on fish farm management practices. Indeed, the hypothesis that the GH-IGF-I axis could be used as a marker of growth performance and nutritional status in aquaculture has already been suggested (Perez-Sanchez and Le Bail, 1999). Our research group has recently developed a radioimmunoassay for detection of IGF-I in a wide variety of fish species (Dyer et al., 2004). Thus, in this paper, we present data from several studies that examine the correlation between growth rates of fish fed different diets and their plasma IGF-I concentrations with the aim of demonstrating this principle. 2. Materials and methods 2.1. Effect of ration size on IGF-I and growth in barramundi (Lates calcarifer) Juvenile barramundi (12.0F0.3 g, MeanFS.E., n=15) were maintained in glass aquaria (160 l) with recirculating filtered freshwater on a commercial pellet diet (Ridley Agriproducts, Brisbane) at rations of 2%, 4% and 10% body weight per day for 7 weeks. Fish were netted and weighed at weekly intervals. At the termination of the trial, barramundi were euthanased by immersion in MS222 and blood samples obtained. 2.2. Effect of diet formulation on IGF-I and growth in barramundi Barramundi (80.0F1.1 g, n=15) were maintained in recirculating fresh water in four replicate tanks (180 l) and were fed once daily to satiation on one of 12 different diets. These diets were formulated to contain three lipid concentrations of 13%, 18% and 23% A.R. Dyer et al. / Aquaculture 236 (2004) 583–592 585 and were combined with four crude protein concentrations (CP), which varied serially between 49% and 65%, 48% and 61% and 44% and 60%, respectively, as described by Williams et al. (2003). Lipid and CP concentrations were manipulated by substituting fish and soy oil (for lipid) and casein (for CP) for starch and diatomaceous earth. Barramundi were maintained on these diets for 6 weeks. At the end of the experiment, four fish from each tank were anaesthetised, weighed and blood sampled and IGF-I levels determined. 2.3. Effect of diet formulation on IGF-I and growth in Atlantic salmon (Salmo salar) Six dietary treatments, including three formulations of moist pellet that differed in nutrient composition (CRC-A, CRC-B and CRC-C), one commercial northern bluefin tuna extruded dry pellet (Com.NBT), a bait-fish (pilchards) diet, and a commercial salmon feed (Com.Sal; Pivot Aquaculture, Tasmania, Australia) were fed to Atlantic salmon during a 6week growth trial. The composition of these diets is described by Bransden et al. (2001). Salmon (mean weight 161.4 gF14.0 S.E.) were randomly allocated to one of twelve 300l tanks until a total of 18 fish were in each tank. Fish were fed twice daily at 2% tank biomass using automatic belt feeders. At the conclusion of the trial, five fish from each tank were anaesthetised, weighed and blood sampled for IGF-I analysis. 2.4. Effect of diet formulation on IGF-I and growth in southern bluefin tuna (Thunnus maccoyii) The southern bluefin tuna aquaculture industry in South Australia relies upon the capture of wild tuna schools by purse seining, followed by a grow-out period in which tuna are fed a diet of pilchards and other baitfish. While effective, this practice may not necessarily be the most economical or promote the highest growth rates. For these reasons, the use of artificial diets has been investigated as an alternative to baitfish (Glencross et al., 1999). Wild captured southern bluefin tuna (mean weight 16.95 kgF4.23 S.E.) were divided between 10 floating sea-cages (12 m in diameter), with two cages per treatment. Tuna were fed twice daily to satiation with five of the same diets that were fed to Atlantic salmon (refer to Section 2.3), with the exception of the Salmon pellets. The diets were fed for 16 weeks until July (i.e. midwinter), whereupon the fish were harvested, weighed and 10 fish randomly chosen from each sea-cage were blood sampled for IGF analysis. 2.5. Blood sampling and processing Blood samples were obtained using heparinised (60 IU heparin) needle and syringes and centrifuged at approximately 4000g for 2 min to separate blood plasma. Plasma was then snap frozen for later analysis. For IGF-I analysis, plasma samples were acid/ethanol extracted (Shimizu et al., 2000). 2.6. IGF-I radioimmunoassay Full details and validation of the IGF-I radioimmunoassay (RIA) are described by Dyer et al. (2004). However, briefly, plasma IGF-I was determined by RIA using commercially 586 A.R. Dyer et al. / Aquaculture 236 (2004) 583–592 available reagents—recombinant tuna IGF-I standards, tuna IGF-I tracer and rabbit antibarramundi IGF-I polyclonal antiserum (GroPep, Adelaide, Australia). Separation was achieved using sheep anti-rabbit gamma-globulin and polyethylene glycol. The minimum detectable limit of the assay was 0.15 ng/ml. Inter-assay variation was 16% and intra-assay variation was 3%. Samples were assayed in duplicate. 2.7. Statistical analysis One-way analysis of variance (ANOVA; SPSS software) was used to compare group means. Differences were determined to be significant if P<0.05. Correlations were indicated by regression analysis and bivariate correlations used to determine the significance of the relationship. Values are meansFS.E. except where indicated. 3. Results 3.1. Effect of ration size on IGF-I and growth in barramundi Juvenile barramundi fed a commercial pellet diet at 2%, 4% and 10% body weight per day grew at significantly different rates, with average daily growth (ADG) observed to be 0.08, 0.32, 0.67 g/day, respectively ( P<0.01) (Fig. 1). Circulating IGF-I concentration also increased significantly with increasing ration size, with IGF-I levels of 30.1, 50.9 and 55.0 Fig. 1. Effect of increasing feed ration size on plasma IGF-I concentration and growth in juvenile barramundi. Plasma IGF-I concentration increased with increasing ration sizes of 2% (n), 4% (o) and 10% (.) body weight/ day. Values are meanFS.E. A.R. Dyer et al. / Aquaculture 236 (2004) 583–592 587 ng/ml ( P<0.01). Although there was a large difference (63%) in bodyweight between 4% and 10% ration groups at the end of the experiment, the differences in IGF-I concentrations were substantially lower (only 10%). 3.2. Effect of diet formulation on IGF-I and growth in barramundi Barramundi were fed 12 diets containing different levels of crude protein (43.8 – 64.7%). Food conversion ratios (FCRs) for all diets ranged between 0.70 and 0.95 and demonstrated that all formulations were close to optimal for this species under these conditions. Plasma IGF-I showed a strong correlation (r2=0.65) to weight gain during this trial (Fig. 2) and also to the %protein composition of each diet (r2=0.5905, Fig. 3). 3.3. Effect of diet formulation on IGF-I and growth in southern bluefin tuna IGF-I concentrations were measured at the termination of a 16-week feed trial in which four experimental pellet diets and a baitfish reference diet were fed to Southern bluefin tuna. Fish fed on four of the diets performed well, growing by 70– 80 g/day over the 16 weeks. Fish fed the extruded dry pellet diet performed less well (20 g/day) due to poor diet acceptability. There was no difference between the plasma IGF-I concentrations measured in the groups of fish fed the diets despite the obvious growth rate differences that they supported (Fig. 4). Fig. 2. Effect of different diets on IGF-I concentration and growth in barramundi. Barramundi maintained on 12 different diets containing from 43.8% to 64.7% crude protein demonstrated a strong correlation between IGF-I concentration and growth rate at the end of the 6-week trial. Values are meanFS.E. Vertical error bars refer to the y-axis and horizontal error bars to the x-axis. 588 A.R. Dyer et al. / Aquaculture 236 (2004) 583–592 Fig. 3. Effect of protein composition on IGF-I concentration in barramundi. In barramundi maintained on 12 different diets, circulating IGF-I levels were positively correlated to the %crude protein of the diet at the end of the 6-week trial. Values are meanFS.E. Fig. 4. Effect of different diets on IGF-I concentration and weight gain in Southern bluefin tuna. In tuna maintained on five different diets for 16 weeks, including three formulations of moist pellet that differed in nutrient composition (CRC-A (.), CRC-B (o) and CRC-C (n), one commercial northern bluefin tuna extruded dry pellet (E) and a bait-fish (pilchards) diet (D), IGF-I concentration did not show a correlation to weight gain at the termination of the trial. Values are meanFS.E. Vertical error bars refer to the y-axis and horizontal error bars to the x-axis. A.R. Dyer et al. / Aquaculture 236 (2004) 583–592 589 Fig. 5. Effect of different diets on IGF-I concentration and growth in Atlantic salmon. In salmon maintained on six different diets, including three formulations of moist pellet that differed in nutrient composition (CRC-A (.), CRC-B (o) and CRC-C (n), one commercial northern bluefin tuna extruded dry pellet (E), a bait-fish (pilchards) diet ( D), and a commercial salmon feed (5), mean IGF-I concentration was positively correlated to growth rate. Values are meanFS.E. Vertical error bars refer to the y-axis and horizontal error bars to the x-axis. 3.4. Effect of diet formulation on IGF-I and growth in Atlantic salmon To minimise the influence of external factors on growth and/or IGF-I concentrations, Atlantic salmon kept in a recirculating-water system were used in the subsequent experiment. In addition to the five diets used in the Southern bluefin tuna trial, a commercial salmon feed was used as a further control. After 9 weeks, mean IGF-I concentration was positively correlated to weight gain (r2=0.67, Fig. 5) and provided an indication of the growth performance obtained with each diet. The dry extruded pellets provided the highest growth rates and also the highest IGF-I levels. The experimental diets performed less well with a corresponding decrease in IGF-I concentrations and a diet of pilchards resulted in the lowest growth rate and IGF-I levels. 4. Discussion The fish IGF-I assay used in these studies has been shown to reliably and accurately measure IGF-I in several species of aquacultured finfish including salmonids, barramundi, southern bluefin tuna, silver perch, red sea-bream and tilapia (Dyer et al., 2004). In this paper, we measured IGF-I in fish sampled from several nutritional trials and identified positive correlations between IGF-I concentrations and the growth rates obtained using different diets or feeding regimes. In the trial with juvenile barramundi fed 2%, 4% and 10% rations, both IGF-I concentrations and weight gain increased when more food was available for consump- 590 A.R. Dyer et al. / Aquaculture 236 (2004) 583–592 tion. Interestingly, there was a relatively small difference in IGF-I concentrations in the fish fed 4% and 10% body weight at the end of the trial. This was mirrored by similar growth rates in these two groups in the last week of the experiment and may suggest that plasma IGF-I concentration is a better indicator of recent growth performance rather than growth measured over a longer period of time. This may also account for the finding in the nutritional trial conducted with Southern bluefin tuna in which fish sampled at the conclusion of an extensive grow-out period showed no correlation between weight gained and IGF-I concentration. Recent studies have identified that growth of the captive tuna declines to minimal levels towards the later stages of the production season, concomitant with a decline in water temperature (Glencross et al., 2002). It is possible then that when the samples were collected for this study, growth may have effectively already ceased in the animals. We suspect that this decline in growth, to essentially maintenance metabolism, may have had an effect on circulating IGF-I concentrations at this time. This hypothesis is supported by the previous studies showing IGF-I concentrations changing with environmental cues such as season and photoperiod (Beckman et al., 1998, Dickhoff et al., 1997). These findings suggest that if IGF-I concentration is to be measured, and perhaps used as an indicator of growth rate, then studies should be designed such that blood sampling occurs when environmental and/or nutritional conditions are stable and representative of the duration of the experiment. The benefits of these recommendations for experimental design can be observed in the two shorter-term barramundi and salmon studies conducted in recirculation systems. In the first of these studies, IGF-I concentrations in barramundi fed 12 highly developed diets were indicative of fish growth rate, with fish growing at faster rates having the highest IGF-I levels. In addition, IGF-I level was well correlated to the %protein found in the diet (a finding also demonstrated by Perez-Sanchez et al., 1995) and suggested that measuring IGF-I concentration might be a useful tool for evaluating the performance and composition of aquaculture diets. This hypothesis was supported in a similar trial with Atlantic salmon. IGF-I concentrations in salmon fed six different diets correlated well to weight gain and again were indicative of diet performance, with the established diets of dry extruded pellets resulting in the highest growth rates and IGF-I levels and a poorly accepted diet of pilchards resulting in the lowest. It is well documented that IGF-I is one of the key factors in controlling growth in most vertebrate species, and that IGF-I is nutritionally regulated. Thus, it seems reasonable to conclude that a change in diet composition resulting in detectable growth rate differences could also be identified by a change in IGF-I level (assuming the assay is sufficiently sensitive). Indeed, the results described here support this hypothesis, with IGF-I concentrations in Altantic salmon and barramundi reflecting the growth rates obtained with different diets. In turn, this suggests that measuring IGF-I could be a useful tool to detect, and possibly predict, subtle changes in growth, and provide an alternative method to traditional techniques for optimising diet formulation. The benefit of this type of analysis over conventional methods is from the speed at which diets may be evaluated. Shortening the length of time required for any grow out trial provides significant financial savings to what is often a lengthy and labour-intensive exercise. Although the time frame required for an accurate IGF-I prediction of diet performance has not been defined, Pierce et al., 2001, A.R. Dyer et al. / Aquaculture 236 (2004) 583–592 591 recently demonstrated that a change in IGF-I level in response to a change in nutritional status could be detected in 2– 4 weeks in salmonids, suggesting it may be possible to assess a new diet in as little as 14 days. References Beckman, B.R., Larsen, D.A., Moriyama, S., Lee-Pawlak, B., Dickhoff, W.W., 1998. Insulin-like growth factor-I and environmental modulation of growth during smoltification of spring Chinook salmon (Oncorhynchus tshawytscha). Gen. Comp. Endocrinol. 109, 325 – 335. Bransden, M.P., Carter, C.G., Nowak, B.F., 2001. Effects of dietary protein source on growth, immune function, blood chemistry and disease resistance of Atlantic salmon (Salmo salar) parr. Animal Sci. 73, 105 – 114. Daughaday, W.H., Hall, K., Salmon Jr., J.L., Van den Brande, J.L., Van Wyk, J.J. 1987. On the nomenclature of the somatomedins and insulin-like growth factors. J. Clin. Endocrinol. Metab. 65, 1075 – 1076. Dickhoff, W.W., Beckman, D.A., Larsen, D.A., Duan, C., Moriyama, S., 1997. The role of growth in endocrine regulation of salmon smoltification. Fish Physiol. Biochem. 17, 231 – 236. Duan, C., Hirano, T., 1992. Effects of insulin-like growth factor I and insulin on the in vitro uptake of sulphate by eel branchial cartilage: evidence for the presence of independent hepatic and pancreatic sulphation factors. J. Endocrinol. 133, 211 – 219. Duan, C., Plisetskaya, E.M., 1993. Nutritional regulation of insulin-like growth factor-I mRNA expression in salmon tissues. J. Endocrinol. 139, 243 – 252. Dyer, A.R., Upton, Z., Stone, D., Thomas, P.M., Higgs, N., Quinn, K., Carragher, J.F., 2004. Development and validation of a radioimmunoassay for fish insulin-like growth factor I (IGF-I) and the effect of aquaculture related stressors on circulating IGF-I levels. Gen. Comp. Endocrinol. 135, 268 – 275. Glencross, B.D., van Barneveld, R.J., Carter, C.G., Clarke, S.M., 1999. On the path to a manufactured feed for farmed southern bluefin tuna. World Aquac. Mag. 30, 42 – 46. Glencross, B.D., Carter, C.G., Gunn, J., van Barneveld, R.J., Rough, K.M., Clarke, S.M., 2002. Southern bluefin tuna. In: Webster, C.D., Lim, C.E. (Eds.), Nutrient requirements and Feeding of Finfish for Aquaculture CABI Publishing, Wallingford, UK, pp. 159 – 172. Jones, J.I., Clemmons, D.R., 1995. Insulin-like growth factors and their binding proteins: biological actions. Endocr. Rev. 16, 3 – 34. Ketelslegers, J.M., Maiter, D., Maes, M., Underwood, L.E., Thissen, J.P., 1995. Nutritional regulation of insulin like growth factor I. Metab. Clin. Exp. 44, 50 – 57. Moriyama, S., Swanson, P., Nishii, M., Takahashi, A., Kawauchi, H., Dickhoff, W.W., Plisetskaya, E.M., 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 protein assay for teleost insulin-like growth factor (IGF)-like: relationships between growth hormone (GH) and IGF-like in the blood of rainbow trout (Oncorhynchus mykiss). Fish Physiol. Biochem. 11, 1 – 6. 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. J. Nutr. 125, 546 – 552. Pierce, A.L., Beckman, B.R., Shearer, K.D., Larsen, D.A., Dickhoff, W.W., 2001. Effects of ration on somatotropic hormones and growth in coho salmon. Comp. Biochem. Physiol. 128B, 255 – 264. 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. Effects of growth and fatness on sexual development of Chinook salmon (Oncorhynchus tshawytscha) parr. Can. J. Fish. Aquat. Sci. 55, 2376 – 2382. 592 A.R. Dyer et al. / Aquaculture 236 (2004) 583–592 Silverstein, J.T., Wolters, W.R., Shimizu, M., Dickhoff, W.W., 2000. Bovine growth hormone treatment of channel catfish: strain and temperature effects on growth, plasma IGF-I levels, feed intake and efficiency and body composition. Aquaculture 190, 77 – 88. Williams, K.C., Barlow, C.G., Rodgers, L., Hockings, I., Agcopra, C., Ruscoe, I., 2003. Asian seabass Lates calcarifer (Bloch) perform well when fed pelleted diets high in protein and lipid. Aquaculture 225, 191 – 206.