ABSTRACT Osmotic minipumps containing either sa- line or recombinant human insulin-like growth factor-II. (IGF-II) were implanted into 4-wk-old female broiler.
PHYSIOLOGY AND REPRODUCTION Effect of Recombinant Human Insulin-Like Growth Factor-II on Weight Gain and Body Composition of Broiler Chickens G.S.G. SPENCER,! E. DECUYPERE,2 J. BUYSE, and M. ZEMAN3 Laboratory for Physiology and Immunology of Domestic Animals, Catholic University of Leuven, Kardinaal Mercierlaan 92, B3001 Heverlee, Belgium ABSTRACT Osmotic minipumps containing either saline or recombinant human insulin-like growth factor-II (IGF-II) were implanted into 4-wk-old female broiler chickens such that the treated chickens received 0.5 mg IGF-II/kg body weight per d. At the end of the trial, no differences in body weight gain or bone length were detected between the treated and control groups. Similarly, there were no differences between the two treatments with respect to heart, spleen, liver, or bursa of Fabricius weight. The relative weight of the abdominal fat pads was greater (P < 0.05) in the birds treated with IGF-II than in the controls, whereas the weight of
breast muscle was reduced (P = 0.06) in the birds treated with IGF-II. There was no effect of IGF-II treatment on feed intake or feed conversion efficiency. Plasma growth hormone (GH) levels were acutely depressed by 15 min after IGF-II administration; and also after 2 wk of IGF-II treatment. Plasma triiodothyronine (T3) concentrations were significantly depressed by IGF-II treatment. These results suggest that IGF-II may not stimulate growth in chickens, but can act as a nutrient partitioning agent, either directly or indirectly through altering plasma GH or T3 concentrations.
(Key words: insulin-like growth factor-II, chicken, growth, body composition, fat) 1996 Poultry Science 75:388-392
INTRODUCTION There is considerable circumstantial evidence to suggest that the insulin-like growth factors (IGF) mediate the actions of growth hormone (GH) in stimulating growth in both mammals and birds. However, a number of recent studies have cast doubt on the physiological role of circulating IGF-I on growth in normal birds (Huybrechts et al, 1992; Tixier-Boichard et al, 1992) and mammals (Zapf et al, 1989; Kerr et al, 1990; Spencer et al, 1991a). Insulin-like growth factor-II has been considered to be a fetal somatomedin, and of minor importance in postnatal animals. However, it is also now well established that in mammals other than rodents (Zapf et al, 1981) and in birds (Decuypere et al, 1993) postnatal plasma IGF-II concentrations are at least as high as IGF-I concentrations. There are much fewer data available on the in vivo effects of IGF-II on growth
Received for publication May 25, 1995. Accepted for publication October 26, 1995. Visiting Fellow. Present address: Ruakura Agricultural Centre, Hamilton, New Zealand. 2 To whom correspondence should be addressed. 3 Visiting Fellow. Present address: Institute for Animal Physiology and Genetics, Ivanka pri Dunaji, Slovakia. 4 Krix, Hendrix, Merksem, Belgium. 5 Alzet 2002, Alza Corp., Palo Alto, CA. 6 Ciba-Geigy, Basel, Switzerland.
and body composition. Results from fetal pigs (Spencer, 1986) and rodents (Schoenle et al, 1985; van Buul-Offers et al, 1988; Conlon et al, 1995) suggest that purified IGFII has, at best, only weak growth-promoting activity. There are no reports on the effects of IGF-II administration to chickens, but differences in circulating IGF-II levels between lines of chickens with different growth characteristics have been reported (Scanes et al, 1989; Decuypere et al, 1993). The present paper provides the first report on the effects of continuous infusion of recombinant human IGF-II on growth and body composition in chickens.
MATERIALS AND METHODS Twenty female Ross broiler chickens were used. The birds were housed individually in cages at an ambient temperature of 22 C. They all had ad libitum access to water and to a commercial broiler ration containing 3,200 kcal ME/kg and 23% CP.4 The total feed consumed by each bird throughout the experiment was recorded. At 4 wk of age, the birds were weighed and allocated to one of two groups such that the groups were balanced with regard to body weight. Ten birds were implanted with an osmotic minipump, 5 filled with a solution containing 10 mg of IGF-II such that the birds received approximately 0.5 m g / k g per d of IGF-II (recombinant human IGF-II),6 for 14 d, whereas 10 control birds received saline alone. Another group of 6 388
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INSULIN-LIKE GROWTH FACTOR-II AND GROWTH IN CHICKENS birds was slaughtered at the beginning of the experiment to provide initial carcass energy data. Two weeks after the start of the treatment, blood was collected by brachial vein puncture into heparinized tubes and the birds were then euthanatized by cervical dislocation. The carcasses were immediately weighed and dissected. Liver, spleen, heart, and bursa of Fabricius were removed and weighed. The abdominal fat pads and the breast muscle on one side of the bird were also removed and weighed. One leg was removed, and the shank-toe length (from the proximal end of the tibia to the tip of the third metatarsal) was measured. Liver GH receptors and energy balance, heat production, and energetic efficiency were measured as described elsewhere (Spencer et ah, 1995). Acute hormonal responses to IGF-II administration were measured in heparinized blood collected by brachial vein puncture both before, and at 15-min intervals for 1 h after i.v. administration of IGF-II (100 /xg) or saline, in otherwise untreated birds. In this acute study (which was undertaken to confirm that IGF-II administration was able to alter hormone concentrations) the birds were 7 wk of age and weighed 2.05 ± 0.05 kg (seven chickens per treatment). The blood samples were centrifuged and the plasma frozen at -20 C for later assay of hormone concentrations. Growth hormone was measured by homologous radioimmunoassay using a monoclonal antibody (Berghman et ah, 1987). Thyroxine (T4) was assayed using a commercially available kit, 7 and 3,3',5-triiodothyronine (T3) by radioimmunoassay using a commercially available antiserum, 8 as described elsewhere (Huybrechts et ah, 1992). Plasma IGF-I levels were measured by radioimmunoassay after acid-ethanol extraction (Huybrechts et ah, 1985). Differences between groups were compared statistically by t test (SAS Institute, 1986). The GH data were log transformed before analysis. Hormonal data from the acute study were analyzed by ANOVA for repeated measures (SAS Institute, 1986).
RESULTS Neither body weight nor relative growth rate was affected by IGF-II treatment when compared with the saline-treated controls (Table 1). Similarly, IGF-II administration did not significantly influence the weight of liver, heart, or spleen. The absolute weight of abdominal fat was greater (P = 0.06) in the birds treated with IGF-II, and this was reflected in a significantly (P < 0.05) greater relative amount of abdominal fat when expressed as a percentage of carcass weight. In contrast, the weight of the breast muscle was lower (P = 0.06) in the IGF-IItreated birds. There was no effect of IGF-II treatment on
7
Abbott, Antwerp, Belgium. Mallinckrodt Diagnostica NV, Brussels, Belgium.
8
TABLE 1. Effect of insulin-like growth factor-II (IGF-II) administration for 14 d on body, organ, and tissue growth in female broilers1 Item
Controls
Initial body weight, kg Final body weight, kg Growth velocity, g / d Liver weight, g % carcass weight Heart weight, g % carcass weight Muscle weight, 2 g % carcass weight Spleen weight, g % carcass weight Bursa weight, g % carcass weight Abdominal fat weight, g % carcass weight Leg length, 3 cm
1,141 1,900 54.2 40.6 2.46 10.0 0.60 138 8.25 2.82 0.17 2.22 0.13 30.2 1.79 27.3
IGF-II ± ± ± + ± + + ± ± ± ± ± ± + + ±
30 1,149 42 1,872 2.9 52.6 2.4 38.4 0.18 2.32 0.5 9.68 0.03 0.59 4.9 128 0.20 7.76 0.20 3.18 0.01 0.19 0.27 2.00 0.02 0.12 2.3 37.8 0.11 2.23 0.18 27.0
± ± ± ± + ± ± + ± + + ± ± ± + ±
29 40 3.2 2.2 0.12 0.4 0.02 4.5 0.22+ 0.17 0.01 0.34 0.02 2.9+ 0.16* 0.26
1
Values are means ± SEM (n = 10). Breast muscle (left side). 3 Shank-toe length (from the proximal end of the tibia to the distal end of the third metatarsal). +P = 0.06. *P < 0.05. 2
shank length. Treatment with IGF-II did not affect appetite (Table 2) and also had no significant effect on feed efficiency (body weight gain:weight of feed consumed), energy retention, or heat production; however, energetic efficiency was increased (P < 0.05) with IGF-II treatment. There was an acute fall in plasma GH concentrations in response to IGF-II administration, as indicated by a significant (P < 0.01) treatment by time interaction (Figure 1). There was no significant change in plasma T3, T4, or IGF-I concentrations. In the longer term, plasma GH concentrations of chickens treated with IGF-II were lower than those of control birds (P < 0.05), whereas no difference in hepatic GH receptor populations were observed (Table 3). In addition, those birds that had received IGF-II had lower (P < 0.05) plasma concentrations of T3 than did the controls, but plasma T4 concentrations were similar.
DISCUSSION The results of these studies indicate that administration of recombinant human IGF-II does not stimulate overall somatic growth (either in terms of weight gain or shank length) in broiler chickens; however, administration of IGF-II causes a decrease in plasma GH concentrations. The age and sex of the birds were chosen to maximize the possibility of finding an effect of treatment. Although slower growing female broilers were used rather than males, and although growth rate is slowing at 4 to 6 wk of age, administration of IGF-II did not affect weight gain. Such a lack of effect is similar to that reported for IGF-I administration to normal chickens (McGuinness and Cogburn, 1991; Huybrechts et ah,
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SPENCER ET AL. TABLE 2. Effect of administration for efficiency, total and energetic
insulin-like growth factor-II (IGF-II) 14 d on feed intake, appetite, feed carcass energy, heat production, efficiency in female broilers 1
Item
Controls
Feed intake, g Appetite, g:g BW Feed efficiency (BW:intake) Carcass energy, Meal Energy retention, Meal Heat production, Meal Energetic efficiency
1,253 0.83 0.61 4.11 1.50 3.11 0.26
IGF-II ± ± ± + ± ± ±
40.7 1,206 0.02 0.80 0.62 0.03 0.23 4.49 0.21 1.90 0.21 2.79 0.03 0.36
± ± ± ± ± ± ±
64.4 0.04 0.03 0.21 0.23 0.39 0.03'
TABLE 3. Plasma concentrations of growth hormone (GH), triiodothyronine (T3), thyroxine (T4), and hepatic GH receptor binding in chickens given insulin-like growth factor-II (IGF-II) and control birds given saline 1 Item
Controls
IGF-II
Growth hormone, n g / m L T3, n g / m L T4, ng/mL GH binding, % / m g protein
17.0 0.94 16.3 18.8
9.3 0.57 17.3 16.3
± ± ± +
4.2 0.10 1.8 1.9
± ± ± ±
1.2* 0.09* 1.8 2.1
!Values are means ± SEM (n = 10). *P < 0.05.
'Values are means ± SEM (n = 10). *P < 0.05.
1992; Tixier-Boichard et al, 1992). We are not aware of any report on the in vivo effects of recombinant IGF-II on growth rate in any species, but the results of this experiment indicate that administration of IGF-II (like IGF-I) is unable to stimulate growth in normal chickens. This concurs with the results using partially purified preparations of IGF-II in rodents (Schoenle et al, 1985; van Buul-Offers et al., 1998), and in the fetus (Spencer, 1986). It is possible that, despite 87% homology between chicken and human IGF-II (Spencer et al, 1991b) the recombinant human preparation is not recognized by the IGF Type-I or insulin receptors in birds. However, this seems unlikely in view of the hormonal differences found between treated and control birds in this study. It has been reported that in chicken tissues the cationindependent mannose-6-phosphate receptor does not bind IGF-II (Kasuga et al, 1982; Canfield and Kornfeld, 1989). In such a case, any effect of increased IGF-II would presumably be through the type I receptor (Duclos and Goddard, 1990). Given that administration of IGF-I does not increase growth in chickens (Huybrechts et al, 1992; Tixier-Boichard et al, 1992), it is perhaps not surprising that administration of exogenous IGF-II does not affect overall growth. By contrast with the lack of effect on body size, the present results indicate that IGF-II is able to influence fat deposition with an increase in the proportion of dissectable abdominal fat in the carcass. The carcass energy data confirm the net increase in body fat. For a similar energy intake, less energy was used in heat production and more energy was retained in the carcass of the birds treated with IGF-II than in the controls. The energetic efficiency of the birds treated with IGF-II was calculated to be greater, and a higher energetic efficiency is indicative of increased fat deposition. The enhanced fat deposition of the birds treated with IGF-II is apparently at odds with a lack of effect on feed efficiency; however, it must be recognized that many other factors such as maintenance requirements, metabolizability, and activity, also determine feed efficiency. Particularly, it was observed that the birds treated with IGF-II were much less active than the control chickens. The reduced activity, and hence the inherent reduced "waste" of metabolizable energy,
would counteract the negative effect of an augmented fat deposition on feed efficiency. In support of this finding, the opposite treatment (immunoneutralization of IGF-II) has been found to produce the opposite effect, i.e., a decrease in fatness (Spencer et al, 1994, 1995). These reciprocal effects with the opposite treatments strongly suggest a real effect of IGF-II in regulating fat deposition. It has been shown previously (Decuypere et al, 1987) that T3 is closely associated with abdominal fat deposition in chickens. Moreover, these reduced T3 levels may also explain the lower heat production of the birds treated with IGF-II, as circulating T3 levels are positively correlated with heat production (Buyse et al, 1992). The decreased plasma T3 concentrations associated with IGFII administration in the present study suggest that the changed fat deposition and heat production may be attributed to an indirect effect of IGF-II through thyroid hormone metabolism. Interestingly, it has been shown previously that administration of exogenous IGF-I resulted in increased plasma T3 concentrations and a corresponding decrease in fatness (Huybrechts et al, 1992). These data provide the interesting observation that IGF-I and IGF-II appear to have opposite effects on T3 and body composition in chickens, though apparently working through a single receptor. This does not seem to be a result of compensatory decreases in IGF-I, but there are a number of possible explanations for this result. There may be another, as yet unidentified, receptor for IGF-II in the chicken; evidence for a third type of IGF receptor with preferential binding of IGF-II has been reported in deer antler (Elliott et al, 1993). Another possibility is that the increased amounts of plasma IGF-II, decreases the number of binding sites available for IGF-I to bind to the receptor. The lack of effect of IGF-II administration on heart and liver are consistent with the reported lack of effect of IGF-I on growth of these organs (Skottner et al, 1989; Tixier-Boichard et al, 1992). However, IGF-I administration can increase spleen and thymus weight in hypophysectomized rats (Guler et al, 1988; Skottner et al, 1989; Binz et al, 1990), and mice selected for high plasma concentrations of IGF-I had higher spleen and thymus weights than their counterparts selected for low IGF-I levels (Siddiqui et al, 1992). In the present study, there was a nonsignificant increase in spleen weight and
391
INSULIN-LIKE GROWTH FACTOR-II AND GROWTH IN CHICKENS 2-1
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no effect on the bursa. These results leave yet unresolved the question of whether IGF-II, like IGF-I, can influence the growth of the organs of the immune system. The decrease in plasma GH following administration of IGF-II is consistent with the elevation in plasma GH that has been seen following immunoneutralization of IGF-II in chickens (unpublished data). These data suggest a role for IGF-II in regulating GH-negative feedback; a finding that has also been reported in sheep (Spencer et d.r 1993). In conclusion, administration of recombinant human IGF-II to chickens at a dose of 0.5 m g / k g per d had no effect on growth rate, but was associated with increased carcass fatness and decreased plasma T3 concentrations. Whether a higher dose of IGF-II would stimulate weight gain, or whether IGF-II would be effective in a growthretarded line of chickens, remains to be established.
ACKNOWLEDGMENTS We are very grateful to Drs. Muller and Maerki (CibaGeigy, Basel, Switzerland) for the supply of IGF-II. This
work was made possible thanks to Fellowships from the Catholic University Leuven to GSGS and MZ.
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factors I and II and of growth hormone in hypophysectomized rats. Acta Endocrinol. 108:167-174. Siddiqui, R. A., S. N. McCutcheon, H. T. Blair, D. D. Mackenzie, P. C. Morel, B. H. Breier, and P. D. Gluckman, 1992. Growth allometry of organs, muscles and bones in mice from lines divergently selected on the basis of plasma insulin-like growth factor-I. Growth Dev. Aging 56:53-60. Skottner, A., R. G. Clark, L. Fryklund, and I.C.A.F. Robinson, 1989. Growth responses in a mutant dwarf rat to human growth hormone and recombinant human insulin-like growth factor-1. Endocrinology 124:2519-2526. Spencer, G.S.G., 1986. Hormonal influences on growth of the pig fetus. Pages 1205-1213 in: Swine in Biomedical Research. Vol. 2. M. E. Tumbleson, ed. Plenum Press, New York, NY. Spencer, G.S.G., C. Berry, S. C. Hodgkinson, and J. J. Bass, 1993. On the neuroendocrine role of insulin-like growth factors 1 and 2 in the regulation of growth hormone secretion. Mol. Cell. Neurosci. 4:538-542. Spencer, G.S.G., E. Decuypere, and J. Buyse, 1994. Immunoneutralization of insulin-like growth factors alters fat deposition in chickens. Growth Regl. 4(Suppl. 1):121. Spencer, G.S.G., E. Decuypere, J. Buyse, S. C. Hodgkinson, J. J. Bass, and M. Zeman. 1995. Passive immunisation of insulin-like growth factor (IGF)-l and of IGF-1 and IGF-II in chickens. Comp. Biochem. Physiol. 110C:29-33. Spencer, G.S.G., S. C. Hodgkinson, and J. J. Bass, 1991a. Passive immunisation against insulin-like growth factor-1 does not inhibit growth hormone-stimulated growth of dwarf rats. Endocrinology 128:2103-2109. Spencer, G.S.G., S. C. Hodgkinson, and J. J. Bass, 1991b. Farm animals—the preferred model for IGF research. Pages 71-83 in: Modern Concepts of Insulin-like Growth Factors. E. M. Spencer, ed. Elsevier, New York, NY. Tixier-Boichard, M., L. M. Huybrechts, E. Decuypere, E. R. Kuhn, J.-L. Monvoison, G. Coquerelle, J. Charrier, and J. Simon, 1992. Effects of insulin-like growth factor-I (IGF-I) infusion and dietary tri-iodothyronine (T3) supplementation on growth, body composition and plasma hormone levels in sex-linked dwarf mutant and normal chickens. J. Endocrinol. 133:101-110. Van Buul-Offers, S., C. M. Hoogerbrugge, J. Branger, M. Feijbrief, and J. L. van den Brande, 1988. Growthstimulating effects of somatomedin-/insulin-like peptides in Snell dwarf mice. Horm. Res. 29:229-236. Zapf, J., H. P. Guler, C. Schmid, A. Kurtz, and E. R. Froesch, 1989. In vivo actions of insulin-like growth factor-1. Pages 145-162 in: Advances in Growth Hormone and Growth Factor Research. E. E. Muller, D. Cocchi, and V. Locatelli, ed. Springer Verlag, Berlin, Germany. Zapf, J., H. Walter, and E. R. Froesch, 1981. Radioimmunological determination of insulin-like growth factors I and II in normal subjects and in patients with growth disorders and extrapancreatic tumour hypoglycaemia. J. Clin. Invest. 68: 1321-1330.
