Effects of Chicken GH on Body Weight Gain in Chickens

Large quantities of chicken pituitary GH were purified to examine its effect on growth (Leung et al., 1986b). The purified chicken GH (cGH), which was biologically active in the rat tibia bioassay, gave a dose-dependent response parallel to that of the bovine GH standard. The amino acid composition of cGH was similar to that of mammalian GH, and particle-sequencing analysis of cGH showed 79 percent homology with bovine GH. Four-week-old Hubbard × Hubbard broiler cockerels were used in all experiments. Thirty-six birds were individually caged in a temperature- and light-controlled room; they were randomly divided into four treatment groups of nine birds each, with food and water available ad libitum.

The purified cGH was dissolved in physiological saline and given daily by intravenous injection via the brachial vein at concentrations of 5, 10, and 50 µg/bird in 100-µl volumes. Body weight and feed consumption were recorded twice weekly for 2 weeks. At the end of the experiment, birds were killed, defeathered, and ground in a meat grinder. Tissues were analyzed by New Jersey Feed Laboratory, Inc. (Plains-field, N.J.), for moisture, protein, and fat content, according to the procedure recommended by the Association of Official Analytical Chemists.

Birds that received 5 µg of cGH daily showed significant weight gains (20.6 and 13.5 percent over control birds) on days 3 and 6, respectively. Birds that received 10 µg of cGH also showed significant weight gains over control birds after 3 and 6 days of treatment (19.6 and 11.3 percent, respectively). Birds that received 50 µg of cGH showed an improvement in weight gain over control birds, but the increase was not statistically significant. Overall, the increase in body weight gain seemed to be transient, so that the stimulating effect of cGH was diminished by the end of the experiment. There was no difference in the effect of feed conversion efficiency on carcass composition between cGH-treated and control birds.

Effects of Human Pancreatic GRF and TRH on Body Weight Gain in Chickens

Chicken hypothalamic GRF has not yet been isolated and purified, but a synthetic human pancreatic GRF (hpGRF) has been shown to be active in stimulating cGH release in chickens both in vivo and in vitro (Leung and Taylor, 1983; Scanes et al., 1984). In addition, TRH, which is a hypo-thalamic peptide, has been shown to stimulate cGH release in vivo. The objective of the studies described below was to determine the effect of hypothalamic peptides on growth in chickens.

Four-week-old Hubbard × Hubbard broiler cockerels were used in all experiments. In the hpGRF experiment, birds were individually caged and randomly distributed into four treatment groups of nine birds each. In the TRH experiment, birds were individually caged and randomly divided into four treatment groups of 8 to 10 birds. All birds were housed in a temperature- and light-controlled room (25ºC; 14 hours of light, 10 hours of darkness) and provided with food and water ad libitum. Food consumption and weight were recorded twice weekly for 2 weeks. At the end of the experiment, birds were killed and defeathered, and carcass composition was analyzed as described in the previous section. The hpGRF₄₄ (Bachem, Torrance, Calif.) and TRH (Beckman, Palo Alto, Calif.) were dissolved in physiological saline and injected via the brachial vein at concentrations of 0.1, 1.0, or 10.0 µg/bird in a 100-µl volume. Control birds received 100 µl of a saline solution.

Birds that received 0.1 µg of hpGRF daily showed a significant increase in body weight gain early on, but that soon diminished. The similarly transient stimulating effect of cGH and hpGRF on body weight gain suggests that hpGRF is also mediated through pituitary GH.

Birds that received 1.0 or 10.0 µg of TRH daily showed significant increases in body weight compared to controls. In contrast to the effect of hpGRF, the growth response to TRH injections was not transient (Leung et al., 1984c). The difference between the effects of the two hormones is probably due to the additional stimulation of thyroid hormone by TRH. Thyroid hormones (triiodothyronine [T₃] and thyroxine [T₄]) have been shown to influence body weight gain in chickens (Leung et al., 1985).

Somatomedin-C

The growth activity of GH is believed to be mediated by SM-C growth factor, generated mainly in the liver. Somatomedin-C is GH-dependent, and purified SM-C has been shown to stimulate body weight gain in both hypophysectomized and intact rats (Hizuka et al., 1986; Schoenle et al., 1982). Since chicken SM-C has not been isolated and purified, a human SM-C radioimmunoassay (RIA) was used to measure serum immunoreactive SM-C when purified cGH was injected into 4-week-old cockerels (Leung et al., 1986b). Purified cGH did not affect weight or incorporation of ³H-proline or ³⁵SO₄ in 9- to 10-day-old chicken embryo cartilage cultured in vitro, but purified human SM-C had a significant effect (Burch et al., 1985). Thus, it seems that the growth promotion axis of hypothalamic GRF-pituitary GH-hepatic SM-C in chickens is similar to that in mammals, but investigation of the biological effects of purified chicken GRF and chicken SM-C is needed to validate this hypothesis.

Growth Hormone Receptor

Hormone-receptor interaction is the first step in hormone action, but receptor physiology has only recently been given attention. Many human diseases are known to result from receptor defects, but the biological significance of the receptor is only beginning to be recognized. For example, analysis of the amino acid and nucleotide sequences of purified epidermal growth factor receptor (EGF-R) has enabled scientists to link the structure-function relationships of oncogenes (v-erbB) and EGF-R (Downward et al., 1984). Although there is no structural analysis (amino acid response) for the GH receptor as yet, its eventual determination will lead to an understanding of the molecular basis of GH action.

Leung et al. (1984a) demonstrated a specific hepatic GH receptor in chickens and observed paradoxically high blood concentrations of GH, as measured by a homologous cGH RIA (Leung et al., 1984b), in sex-linked dwarf chickens (Lilburn et al., 1986). These chickens grew to less than half the size of normal chickens, leading Leung et al. (1984a) to examine GH receptor binding in the same strain. There was a significant decrease in hepatic receptor binding at 6, 8, and 20 weeks of age compared to that of normal, fast-growing broiler chickens (Leung et al., 1987). Huybrechts et al. (1985) reported that sex-linked dwarf chickens also had significantly lower circulating immunoreactive SM-C concentrations compared to those of normal birds. And Leung et al. (1984a) observed that sex-linked dwarf chickens had significantly higher hepatic (IGF-I) receptor binding.

These observations may provide evidence that dwarfism is sex-linked and may be due to a defect in the GH receptor. Based on preliminary results, we believe that GH receptors may be the limiting factor in the growth promoter axis in chickens. For example, normal Leghorn chickens, which grow at a much slower rate than broiler chickens, possess significantly fewer GH receptors than broiler chickens (Leung et al., 1987). However, that hypothesis does not agree with data reported for mammalian species. Growth hormone has been shown to maintain its own receptors in rat adipocytes and to up-regulate its hepatic receptors (Baxter and Zaltsman, 1984). Recently, Chung and Etherton (1986) reported that the number of hepatic GH receptors is increased in pigs that have received GH injections. The method of regulating GH receptors in other agricultural animals is not known. However, if GH up-regulates its receptors at the target tissue, it is logical to assume that an increase in circulating GH would result in an amplified biological response to GH.

Gene Insertion

The technology for introducing foreign genes into mammalian embryos forms the basis of a powerful approach for studying gene regulation and the genetic basis of development (Palmiter and Brinster, 1985). A dramatic growth increase in transgenic mice from eggs that were microinjected with a metallothionein GH foreign gene suggests that this technology could be valuable for agricultural applications. Indeed, Hammer et al. (1985) successfully introduced foreign genes into the genes of rabbits, sheep, and pigs by microinjecting eggs, using mouse metallothionein-human GH recombinant DNA. The foreign DNA was integrated and expressed in transgenic rabbits and pigs. Thomas E. Wagner (Ohio University, personal communication, 1986) also successfully introduced foreign genes in pigs by microinjection. Leung and coworkers have attempted to directly inject foreign DNA into the blastoderm of freshly laid eggs with recombinant DNA technology (unpublished data). And Souza et al. (1984) used the retroviral approach in introducing foreign genes into chickens.

Kopchick et al. (1985) constructed a recombinant DNA (pbGH-4) that is an avian retroviral long-terminal repeat (LTR), ligated to the structural bovine GH (bGH) gene. This recombinant DNA is biologically active in a transient eukaryotic expression assay system. When this recombinant DNA was totally integrated into a mouse fibroblast cell line, mature bGH was expressed and secreted into the culture medium. Leung et al. (1986a) purified and characterized the recombinant bGH from culture medium and showed that the recombinant bGH possesses the same physiochemical and physical properties as native pituitary bGH. This recombinant bGH DNA was then introduced into the germinal disk of the freshly laid egg by opening a window in the egg and injecting various amounts of DNA in circular or linear form with a micropipette. Only seven of the chicks that hatched from the 3,000 injected eggs had measurable circulating immunoreactive bGH. When serum samples were measured with both a homologous cGH RIA and a bGH RIA, the cross-reactivity of purified cGH and bGH in the RIA was less than 5 percent. The expression of bGH was transient; no detectable immunoreactive bGH was present after 10 weeks of age. All the chickens were killed or crossed after sexual maturity. Tissue DNA was analyzed by dot blot and Southern gel assays. No measurable immunoreactive bGH was detected by RIA from seven samples collected from first-generation offspring. It appears, therefore, that this method is inefficient. In addition, since the germinal disk in freshly laid eggs consists of at least 500 to 1,000 cells, even if the foreign DNA is integrated in the host cell genome it is unlikely that the foreign DNA will enter the germ line.

Use of a retroviral vector to introduce foreign genes into chicken genes provides an alternative experimental approach. Indeed, Souza et al. (1984) generated a recombinant retrovirus by cloning chicken GH cDNA into a modified Rous sarcoma virus Sehmiedt-Ruspin A genome in which the sac gene was entirely deleted. Recombinant infectious virus that expresses cGH was generated to infect 9-day-old chick embryos. Subsequently born chicks expressed circulating concentrations of cGH that were two- to threefold higher than those of normal birds. In addition, the birds were uremic. Salter et al. (1986) obtained similar results using a different retroviral vector. These results suggest that the retro-viral approach may be more effective than direct injection of foreign DNA in introducing foreign genes into the germ line of chickens.