Chronic intravenous infusion of chicken growth hormone increases body fat content of young broiler chickens

Camp. Biochem. Physiol. Vol. I IOA. No. I, pp. 47-56. 1995 Copyright c 1995 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0300-9629/95 $9.50 + 0.00

Pergamon 0300-%29(94)00151-O

Chronic intravenous infusion of chicken growth hormone increases body fat content of young broiler chickens Robert F. Moellers and Larry A. Cogburn Department of Animal Science and Agricultural Biochemistry, College Sciences, University of Delaware, Newark, DE 19717-1303, U.S.A.

of Agricultural

The purpose of this study was to determine the effects of programmed intravenous infusion of chicken growth hormone (cGH) on growth and metabolism of young broiler chickens (4-7 weeks of age). Four-week-old broiler cockerels, fitted with indwelling jugular catheters, were randomly assigned to three treatment groups (6 birds/group): pulsatile infusion of buffer (phosphate buffer, pH 7.4)[PB-Pj at 3 hr intervals, pulsatile infusion of cGH (15 pg/kg at 3 hr intervals)[GH-Pj, or continuous infusion of cGH (120 ag/kg-day)(GH-Cj. Birds were bled 5 min before (0-min) and 5 min post-infusion (relative to the pulses of PB and cGH) at 5, 6, and 7 weeks of age. Pulsatile infusion of cGH increased (P < 0.05) feed consumption by 24% and reduced (P < 0.05) feed efficiency by 14% without affecting body weight (BW) gain. The relative weights (%BW) of liver, abdominal fat, and bursa of Fabricius were not affected by the pattern of cGH infusion. However, the body fat content of cGH-infused chickens was increased (P < 0.05) by 13% (GH-C) and 17% (GH-P), while body protein and water contents were slightly reduced. Body ash content was not affected by pattern of cGH infusion. When compared with the PB-P controls, the GH-P treatment depressed (P < 0.05) hepatic GH-binding activity by 52% without affecting plasma insulin-like growth factor-I (IGF-I) levels. Continuous infusion of cGH increased (P < 0.05) plasma IGF-I by 16%, thyroxine (T.,) by 31%, and glucagon levels by 55%, although plasma GH levels were only 47% higher than those of the PB-P group. However, the GH-P treatment was only half as effective as the GH-C pattern in elevating plasma levels of T4 and glucagon. This study shows that programmed intravenous infusion of cGH increases deposition of body fat in young rapidly-growing broiler chickens. Key words: Growth rate; Feed efficiency; Body composition; hormones; 5’-Monodeiodinase activity; Hepatic GH-binding Comp. Biochem.

Physiol.

IlOA, 47-56,

Intravenous GH infustion; activity; Chicken.

Plasma

199.5.

Introduction The pronounced episodic pattern of GH secretion found in young broiler chickens quickly subsides to maintenance of low basal GH levels in maturing chickens (Johnson et al., 1987;

Vasilatos-Younken and Zarkower, 1987; Johnson, 1988). The high frequency and amplitude of endogenous GH pulses, found at 3-4 weeks of age, are reflected as a developmental peak in the mean plasma GH level of broiler chickens (Johnson et al., 1987; Goddard et al., 1988; McGuinness and Cogburn, 1990). A recent study shows a good correlation between the ontogeny of pituitary GH mRNA content and plasma GH levels which reach peak levels at 4 weeks of age in the chicken (McCannLevorse et al., 1993). The administration of

to: L. A. Cogburn, Department of Animal Science and Agricultural Biochemistry, Room 040 Townsend Hall, University of Delaware, Newark, DE 19717-1303, U.S.A. *Published as Journal Paper No. 1389 from the Delaware Agricultural Experiment Station. Received 29 January 1994; revised 8 August 1994; accepted 16 August 1994.

Correspondence

47

48

Robert

F. Moellers

chicken GH (cGH) by single daily injection or continuous subcutaneous infusion during the period in which basal GH levels are falling (i.e. after 4 weeks of age) does not stimulate growth rate and usually increases the accumulation of body fat (Leung et al., 1986; Burke et al., 1987; Cogburn et al., 1989; Cogburn, 1991). In 8week-old broiler pullets which have very low basal GH levels, pulsatile intravenous infusion of cGH, designed to mimic episodic GH secretory pattern of young chickens, improves growth efficiency and body composition (Vasilatos-Younken et al., 1988). However, the continuous infusion of cGH in a separate experiment did not stimulate growth or reduce accumulation of body fat. Unfortunately, it is not possible to draw any conclusions about the pattern of intravenous cGH infusion in broiler chickens because the pattern of cGH administration was confounded by the two separate experiments and two different preparations of pituitary-derived cGH. Continuous subcutaneous infusion of cGH from 12 to 15 weeks of age increases the growth rate of male broiler chickens, although there is a disproportionate increase in abdominal fat weight @canes et al., 1990). It is possible that the positive response of older broiler chickens to exogenous cGH (Vasilatos-Younken et al., 1988; Scanes et al., 1990) is simply due to greater abundance of GH receptors in target tissue. This idea is supported by the fact that GH binding to hepatic receptors is barely detectable by radioreceptor assay until chickens reach sexual maturity (Leung et al., 1987; Krishnan et al., 1989; Cogburn, 1991; Burnside and Cogburn, 1992). We have recently shown that expression of hepatic GH receptor mRNA is very low at hatching and progressively increases as the broiler chicken reaches sexual maturity (Burnside and Cogburn, 1992). These observations suggest that the stage of development and/or the pattern of cGH administration could determine the responsiveness of the broiler chicken to exogenous cGH. In the hypophysectomized rat, pulsatile (or intermittent) administration of GH is more effective than continuous infusion in restoring growth rate (Clark et a/., 1985; Maiter et al., 1988; Bick et al., 1992) elevating plasma IGF-I levels (Maiter et al., 1988, 1992) and increasing the abundance of IGF-I mRNA in liver (Maiter et al., 1992). skeletal muscle and cartilage (Isgaard et al., 1988). The importance of pattern of intravenous cGH infusion has not been established for young broiler chickens. The purpose of the present study was to compare, in a single study, the effects of pulsatile versus continuous infusion of biosynthetic cGH in young broiler chickens during the period in

and Larry

A. Cogburn

which endogenous GH levels are rapidly declining (i.e. 4-7 weeks of age). This study shows that, regardless of the infusion pattern, exogenous cGH increases the accumulation of body fat in young rapidly growing broiler chickens. A brief portion of this work was presented earlier (Moellers and Cogburn, 1991).

Materials and Methods Animals One-day-old broiler cockerels (Ross x Arbor Acre strain), obtained from a commercial hatchery (Perdue Hatcheries, Salisbury, MD), were housed in a heated brooder-unit (Petersime Incubator Co., Gettysburg, OH) for the first three weeks. A commercial starter ration (Pennfield Corp., Lancaster, PA) and water were provided ad libitum during the brooding period. At 3 weeks of age, 18 chicks of uniform body weight (830-880 g range) were selected for cannulation and randomly assigned to individual stainless steel wire cages (46 x 36 x 36 cm). The birds were maintained in a controlled-environment room held at 21 ‘C and under a 20L : 4D light/dark cycle. From 3 to 7 weeks of age, the chickens were provided with a pelleted broiler-finisher ration (Pennfield Corp., Lancaster, PA) and water ad libitwn. Programmed

intravenous

injiuion

yf cGH

At 24 days of age, the chickens were surgically fitted with indwelling jugular catheters (Silastic medical grade tubing, 0.025 in i.d. x 0.047 in o.d.; Dow Corning, Midland, MI) and a swiveltether-infusion system (Alice King Chatman Medical Arts, Los Angeles, CA) as previously described (Moellers and Cogburn, 1994). Before cannulation, the intravascular end of the Silastic cannula was treated for 1 min with a heparin solution (TDMAC-heparinate, Polysciences Inc., Warrington, PA) to maintain catheter patency. After surgery, catheters were flushed with a sterile saline solution (0.9% NaCI) containing heparin (100 Uiml). An intramuscular (i.m.) injection of procaine-G penicillin ( 100,000 U) was given to each bird on the day before, and immediately after surgery. This was followed by a daily i.m. injection of gentamicin (15 mg/bird) for 2 days following surgery. All birds were given continuous (0.08 ml/hr) infusion of sterile saline (0.9?& NaCl) until cGH treatments began. The 4-week-old broiler cockerels, fitted with an indwelling jugular cannula, were randomly assigned to three treatment groups (six birds/group): pulsatile infusion of buffer (0.05 M phosphate buffer, pH 7.4)[PB-P] at 3 hr intervals, pulsatile infusion of GH (15 Llg/kg at 3 hr intervals or 120 pg/kg-day)[GH-P], or con-

Chronic

cGH

infusion

tinuous infusion of GH (120 pgg/kg-day)[GH-C]. The birds were given these treatments for 3 weeks (i.e. 4-7 weeks of age). Twelve infusion pumps (Auto Syringe Model AS20A, Travenol Laboratories, Inc., Hookset, NH) were programmed for pulsatile delivery of either PB (N = 6) or cGH (N = 6) (0.3 ml over a 10 min period every 3 hr or 2.4 ml/day) starting at 09.00 hr. The remaining six pumps were programmed for continuous infusion of cGH at 0.1 ml/hr (2.4 ml/day). Biosynthetic cGH (Genentech, Inc., South San Francisco, CA) was prepared for infusion by diluting the stock cGH solution in filtersterilized (0.2 pm filter unit, Nalgene) phosphate buffer (pH 7.4). The biological properties of this commercial preparation of biosynthetic cGH were described earlier (Burke et al., 1987). All solutions used for infusion were loaded into sterile syringes under aseptic conditions. Spent syringes were replaced each day with a new syringe containing a fresh solution of cGH or buffer prior to the 09.00 hr pulse. Birds were weighed at 2-day intervals to adjust the cGH dosage for body weight gain. Feed consumption and body weight gain were determined at weekly intervals for each bird. Blood samples (4 ml) were taken 5 min before (0-min) and 5 min post-infusion relative to the midday (12.00 hr) PB-P and GH-P pulses at 5, 6, and 7 weeks of age. The 5 min post-infusion sampling time was selected from a preliminary experiment which showed a 6-fold elevation in plasma GH levels at 5 min post-infusion of cGH pulses (15 pg/kg at 3 hr intervals for 5 days; data not shown). Due to the physical restraint of the back harness, blood samples were taken from each bird via the contralateral leg vein. Plasma was obtained by centrifugation (1500 g for 30 min at 4°C) and stored at - 20°C until the hormone/metabolite assay. At the end of the treatment period (i.e. 7 weeks of age), birds were killed by cervical dislocation for determination of the weights of the liver, abdominal fat pad, and bursa of Fabricius. A liver sample (20 g) was frozen in liquid nitrogen for measurement 5’-monodeiodinase (5’-MDI) activity $ogb urn et al., 1989) and specific GH binding activity (Krishnan et al., 1989). Liver membranes were washed with 3.4 M MgCl, to remove endogenous ligand bound to the hepatic GH receptors (Kelly et al., 1979). The GH binding assay was conducted in 1.5 ml microcentrifuge tubes containing 2 mg of liver membrane and ‘~SI-labelled 50,000 cpm of biosynthetic cGH (Burnside et al., 1992). The fractional occupancy of total GH receptors was determined by the formula described by Maiter et al., 1988): (total binding sites - free binding sites)/total binding sites x 100. Each carcass,

in broiler

chickens

49

including the liver and fat pad, was frozen at -20°C for proximate analysis. The entire frozen carcass of each bird was ground twice in a meat grinder fitted with a 3.5mm dye (Biro Model 5, Biro, Marblehead, OH). Body composition (i.e. moisture, protein, fat, and ash content) was determined in duplicate ground samples by standard analytical procedures (Horowitz, 1980). Plasma

hormone

and metabolite

assays

Plasma hormone levels were measured by specific radioimmunoassay (RIA). Double-antibody RIA procedures were used to quantitate plasma levels of cGH (Proudman, 1984) and IGF-1 (McGuinness and Cogburn, 1990). A mixture of ethanol : acetone : acetic acid (60:30:10 by vol.) (Enright et al., 1989) was used to extract IGF-1 from chicken plasma to eliminate interference of the IGF-1 binding proteins in the RIA. A non-equilibrium doubleantibody RIA was used to measure plasma insulin levels (McMurtry et al., 1983). Plasma levels of T,, T,, and glucagon were determined by using commercial RIA kits (ICN Biomedicals, Inc., Costa Mesa, CA). Calorimetric assay kits were used to determine plasma levels of glucose (Sigma Chemical, St. Louis, MO) and non-esterified fatty acids (NEFA-C, Wako Chemical, Dallas, TX). All plasma samples were assayed in a single hormone or metabolite assay to avoid inter-assay variability. The intra-assay coefficients of variation were 5.2% for GH, 2.4% for IGF-I, 5.0% for T,, 7.1% for Tqr 2.1% for insulin, and 5.4% for glucagon. Statistical

anulysis

Treatments were assigned to cages according to a randomized-complete-block design with cage height (or level) from the floor (level 1 = 32 cm; level 2 = 125 cm) as the blocking factor, Measures of growth efficiency such as average daily gain (ADG, g/bird), average daily feed consumption (ADFC, g/bird), and the feed-to-gain ratio (FTG, kg feed/kg weight gain) were analyzed, with treatment and week as the main effects. The final body weight, liver weight, bursal weight, hepatic 5’-MD1 activity, and body composition were analyzed with treatment as the main effect. Analysis of variance was used to determine significant (P < 0.05) differences in plasma hormone levels due to the main effects of treatment, time of infusion (prevs. post-infusion), week of age (5,6 and 7 weeks) and their interactions. Fisher’s least significant difference test was used to determine significant differences (P < 0.05) due to cGH treatment. For convenience, plasma hormone data were averaged across weeks of age and are presented

50

Robert Table

l. Final

Treatment PB-P GH-P GH-C

body weight programmed

F. Moellers

and Larry

A. Cogburn

(FBW) and growth performance of broiler infusion of cGH from 4 to 7 weeks of age

Body weight (kg)

ADG

3.10 & 0.04 3.30 * 0.05 3.19 * 0.10

77.2 + 2.9 83.8 + 2.6 80.4 k 3.6

cockerels

ADFC

given

FTG

130.6 + 3.1b 161.6 k 6.4” 140.4 * 7.3b

1.70* 0.03b 1.93 + 0.02” I .74 + 0.04b

PB-P = pulsed infusion of phosphate buffer, GH-P = pulsed infusion of cGH (15 p g/kg BW at 3 hr intervals), GH-C = continuous infusion of cGH (120 pg/kg-day). Average daily gain (ADG) and average daily feed consumption (ADFC) are given in g/bird. Feed-to-gain ratio (FTG) is expressed as kg feed/kg weight gain. Each value represents the mean If: SEM of six birds. Means within a eiven column oossessing a different superscript are significantly (P c 0.05) different.

as the main infusion.

effects

of treatment

and

time of

Results Growth, feed,

Qiciency

and body composition

Chronic infusion of cGH did not stimulate growth rate of broiler cockerels (Table 1). Pulsed infusion of cGH increased (P < 0.05) feed consumption by 24% when compared with that of the other treatments; in particular, feed intake was 10% higher during the first week of infusion and increased to 34% above that of the PB-P group during the last 2 weeks of infusion. This disproportionate increase in feed intake of GH-P birds, without improving body weight gain, caused a 14% reduction (P < 0.05) in overall feed efficiency when compared with that of the PB-P birds. Hepatic 5’-MDT activity in the GH-C birds was slightly lower (14%), but not significantly different from that of the other groups (Table 2). There were no significant differences in the relative weights of liver, abdominal fat, or bursa among treatment groups. Body fat content was increased (P < 0.05) by 13% with continuous cGH infusion and by 17% with pulsatile infusion of cGH (Table 3). Body moisture and protein contents were reduced (P < 0.05) by both patterns of cGH infusion, although ash content was not affected by either treatment.

Plasma hormone and metabolite levels Pulsed infusion of cGH for 21 days provided a 6-fold elevation in plasma GH at 5 min postinfusion when compared with the pre-infusion level of this treatment group (Fig. 1A). Basal or pre-infusion GH levels for the GH-P and GH-C groups did not differ significantly from those of the PB-P group. However, the basal (preinfusion) GH level of the pulsatile GH-infusion group was lower than the plasma GH level maintained by continuous GH infusion. At 5 min post-infusion, GH levels for the GH-P group (52.4ng/ml) were 3.3- and 5-fold higher (P < 0.05) than those in GH-C and PB-P respectively. Although plasma GH groups, levels in the GH-C group were consistently maintained about 47% higher than those of the PB-P group, this difference was not significant. There was also a main effect of week of age on plasma GH levels, since the average GH level (across treatment and time of infusion) at 5 weeks of age was 50% higher (P < 0.05) than that at 7 weeks of age. Plasma IGF-I levels were 16% higher (P i 0.05) in birds given continuous cGH infusion when compared with those of the GH-P and PB-P groups (Fig. 1B). Pulsatile infusion of cGH depressed (P < 0.05) GH binding to MgClz-treated liver membranes by 52’/u when compared with that of the PB-P group (Fig. 2). The binding of GH to free (unoccupied) binding sites on hepatic membranes was 23% lower in birds given either

Table 2. Hepatic 5’-monodeiodinase activity (5’-MDI) and relative weights (%BW) of abdominal fat, and the bursa of Fabricius in broiler cockerels after programmed infusion of cGH from 4 to 7 weeks of age % BW Treatment PB-P GH-P GH-C

5’-MD1

Liver

3.82 2 0.33 3.91 & 0.28 3.30 + 0.12

1.83 F0.16 2.14_+0.15 2.04 + 0.03

Abdominal 2.14iO.17 2.20+0.17 1.91 kO.19

fat

Bursa 0.19~0.01 0.19+0.03 0.17 * 0.02

PB-P = pulsed infusion of phosphate buffer, GH-P = pulsed infusion of cGH (15 pg/kg BW @ 3 hr intervals), GH-C = continuous infusion of cGH (120 pg/kg-day). Hepatic 5’-MD1 is expressed as ng T, generated per mg protein per hour (ng T,/mg protein-hr). Each value represents the mean i SEM of six birds.

Chronic Table 3. Body composition

cGH

infusion

in broiler

chickens

of broiler cockerels after programmed from 4 to 7 weeks of aee

51 infusion

of cGH

% BW Treatment

Moisture

PB-P GH-P GH-C

65.6 * 0.4” 63.8 k 0.5b 64.2 + 0.5b

Protein 18.5 + 0.2” 17.9 f 0.2b 18.0 + 0.3b

Fat

Ash

12.9 f OZ? 15.1 & 0.5” 14.6 + 0.8’

2.13&0.04 2.19 & 0.08 2.27 + 0.05

PB-P = pulsed infusion of phosphate buffer, GH-P = pulsed infusion of cGH (15 pg/kg BW Q 3 hr intervals), GH-C =continuous infusion of cGH (120 pg/kg-day). Each value represents the mean -+ SEM of six birds. Means a different superscript are significantly within a given column possessing (P < 0.05) different.

pattern of GH infusion, although this difference was not statistically different from that of the PB-P group. Pre-treatment of liver membranes with MgCl, to remove endogenous ligand increased specific GH-binding by 2.5-fold. The fractional occupancy of total GH receptors was 34% lower in liver membranes from GH-P birds (43% occupancy) when compared with that of the PB-P (63% occupancy) and GH-C (67% occupancy) groups. There was a main effect of time on plasma T, levefs, since post-infusion T, levels were 7% lower (P < 0.05) than pre-infusion levels for all

PRE-INFUSION POST-INFUSION

0 m0 60,A

a

treatment groups (Fig. 3A). There was a main effect (P < 0.05) of cGH infusion on plasma T, levels (Fig, 3B). In particular, the average T, level for the GH-C group (7.5 ng/ml) was 3 1% higher (P < 0.05) than that of the PB-P group, while the average plasma T4 level of the GH-P group was only 16% higher (P < 0.05) than that of the PB-P controls. There was a treatment by week interaction (P < 0.05) on plasma T, levels because plasma T, levels in the PB-P and GH-P birds were maintained at about 2.8 ng/mf throughout the 3-week infusion period. In contrast, T, levels in the GH-C group were initially depressed at 5 weeks of age, but increased steadily at 6 and 7 weeks of age. Thus, the two-way interaction occurred during the last week of treatment, since T, levels in the GH-C group were higher than those of the GH-P and PB-P groups. The response of pancreatic hormones to chronic cGH infusion is shown in Fig. 4. There was a main effect of treatment on plasma

PE-P PB-P

GH-P

GH-P

GH-C

GH-C

Fig. I. Plasma levels of GH (A) and IGF-I (9) in broiler cockerels given different patterns of cGH infusion from 4 to 7 weeks of age. Each bar represents the mean + SEM of six birds bled 5 min before (0 min) and 5 min post-infusion (relative to the PB and cGH pulses) and averaged across 5, 6, and 7 weeks of age (N = 18). Means within a given time (pre- versus post-infusion) possessing a different superscript are significantly (P < 0.05) different.

Fig. 2. Specific hepatic GH-binding activity of broiler cockerels given different patterns of cGH infusion from 4 to 7 weeks of age. The open bar represents binding of “‘I-cGH to unoccupied (non-treated) membrane receptors, while the striped bar denotes GH-binding activity after pre-treatment of membranes with 3.4 M MgClz to remove endogenous ligand. Each bar represents the mean i SEM of six birds. Means possessing a different superscript are significantly (P < 0.05) different.

Robert

52

F. Moellers

a PRE--INFUSION kXIl POST-INFUSION

and Larry

A. Cogburn

main effect of time on glucose level, since the average post-infusion glucose level was 10% higher (P < 0.05) than the pre-infusion glucose level. There was also a main effect of week on plasma NEFA levels, since the average NEFA level at 7 weeks of age was 10% lower (P < 0.05) than that at 5 weeks of age.

Discussion

b

P&P

GH-P

GH-C

Fig. 3. Plasma levels of T, (A) and T, (B) in broiler cockerels given different patterns of cGH infusion from 4 to 7 weeks of age. Each value represents the mean thyroid hormone level of six birds bled 5 min before (pre-infusion) and 5 min post-infusion (relative to the PB and cGH pulses) and averaged across 5, 6, and 7 weeks of age (N = 18). Treatment means possessing a different superscript are significantly (P < 0.05) different.

glucagon levels, since the average glucagon level for the GH-C group was 55% higher (P < 0.05) than that of the PB-P group (Fig. 4B). The average glucagon level for the GH-P group was 26% higher (P < 0.05) when compared with that of the PB-P treatment. There was a treatment by week interaction (P < 0.05) on plasma insulin levels due to elevated (P < 0.05) insulin levels at 6 weeks of age in the GH-C group. After 2 weeks of infusion, plasma insulin levels in the GH-C group were 38% higher (P < 0.05) than those in the GH-P group and 50% higher than those in the PB-P group. This elevation was only temporary, since insulin levels for the GH-C group fell to control levels by the end of the treatment period (i.e. 7 weeks of age). No effect of cGH treatment or interactions between treatment and time of infusion was found on plasma metabolite levels (Fig. 5). However, there was a main effect (P < 0.05) of the week on plasma glucose levels, since glucose levels decreased by 8% between 5 and 7 weeks of age (Fig. 5A). This effect was mainly due to glucose levels for the GH-P group being 9% higher (P < 0.05) than those in the PB-P group after the first week of treatment. There was a

This study clearly shows that chronic intravenous infusion of cGH in young rapidly growing broiler chickens does not improve growth rate, feed efficiency, or body composition. Instead, both patterns of cGH infusion increased the deposition of body fat, despite major differences in the magnitude of hormonal responses between the continuous and pulsatile GH-infusion patterns. Pulsatile infusion of cGH appears to reduce fat deposition in &week-old broiler pullets that are treated with cGH for either 1 week (Rosebrough ef nl., 1991) or 3 weeks (Vasilatos-Younken et al., 1988). The present study supports earlier reports of a lack of growth response and higher accumulation of body fat in young broiler chickens given a single daily injection of cGH (Leung er nl.. 1986; 0 PRE-INFUSION &5I POST-INFUSION 2.0 T A

g

0.6

5 3 t3

0.4

%

0.2

Y a

0.0 PB-P

GH-P

GH-C

Fig. 4. Plasma levels of insulin (A) and glucagon (B) in broiler cockerels given different patterns of cGH infusion from 4 to 7 weeks of age. Each value represents the mean plasma level of six birds bled 5 min before (pre-infusion) and 5 min post-infusion (relative to the PB and cGH pulses) and averaged across 5, 6, and 7 weeks of age (N = 18). Means possessing a different superscript are significantly (P < 0.05) different.

Chronic

cGH

infusion

0 PRE-INFUSION mS POST-INFUSION 300

2 &

250 200

3 8

150

3 0

100

$ 3

50

a

5.

A

-

0I 1.0

%

0.0

E

0.6

B

iz ii 4 5 4 a_

0.4

0.2

P&P

GH-P

GH-C

Fig. 5. Plasma glucose (A) and NEFA (B) levels in broiler cockerels given different patterns of cGH infusion from 4 to 7 weeks of age. Each value represents the mean metabohte level of six birds bled 5 min before (pre-infusion) and 5 min post-infusion (relative to the PB and cGH pulses) and averaged across 5, 6 and 7 weeks of age (N = 18).

Burke et al.. 1987; Cogburn et al., 1989; Cogburn, 1991). There appears to be some redistribution of fat in GH-infused chickens since the percentage of body fat was increased without affecting abdominal fat weight. In broiler pullets, continuous cGH infusion did not affect abdominal fat weight, although the percentage of fat in breast muscle was increased (Vasilatos-Younken et al., 1988). Our study shows that continuous cGH infusion had no effect on the growth rate or feed efficiency of broiler chickens, whereas pulsatile infusion of cGH increased feed intake and reduced feed efficiency. In contrast, we have recently found that pulsatile intravenous infusion of GRF depresses feed intake and growth rate of young broiler chickens (Moellers and Cogburn, 1994). Daily injection of broiler chickens with recombinant bovine GH (rbGH) for 14 days (4-6 weeks of age) increased the feed intake, although this effect was attenuated during the second week of treatment by development of antibodies to the heterologous GH (Buonomo and Baile, 1988). Vasilatos-Younken et al. (1988) indicated that the feed efficiency of older (8-1 I-week-old) broiler pullets was reduced by continuous cGH infusion, whereas pulsatile cGH infusion improved feed efficiency. They also reported

in broiler

chickens

53

that the positive effect of pulsatile cGH infusion on the feed efficiency of broiler pullets waned with each week of treatment. Collectively, these findings indicate that exogenous cGH per se has very little potential for improving the growth rate, feed efficiency, or body composition of young rapidly-growing broiler chickens. Since the dose and route of administration were similar between the current study and that of Vasilatos-Younken et al. (1988) the positive response of older broiler chickens to exogenous cGH (Vasilatos-Younken et al., 1988; Rosebrough et al., 1991; Scanes et al., 1990) could be simply due to greater expression of the GH receptor in target tissue (Burnside and Cogburn, 1992). In young broiler chickens, circulating GH reaches the highest basal levels at 334 weeks of age (Johnson et al., 1987; Goddard et a1.,1988; McGuinness and Cogburn, 1990). In contrast, hepatic GH-binding activity (Leung et al., 1984; Krishnan et al., 1989; Cogburn, 1991) and expression of GH receptor mRNA (Burnside and Cogburn, 1992) are very low in young chickens, which have the highest plasma GH levels, and extremely high in mature chickens which have the lowest basal GH levels. This inverse relationship between circulating GH levels and expression of the GH receptor in young broiler chickens appears to limit their responsiveness to exogenous cGH. Furthermore, a single daily injection of chickens with cGH causes down-regulation of hepatic GHbinding activity (Leung et al., 1984; Cogburn, 1991). The hepatic GH-binding activity of birds in the present study was depressed by 52% with pulsatile infusion of cGH, whereas continuous cGH infusion did not affect GH binding. In contrast, continuous GH infusion in the hypophysectomized rat increases hepatic GH binding (Maiter et al., 1988; Bick et al., 1992; Maiter et al., 1992) without increasing the abundance of GH receptor mRNA (Maiter et al., 1992). The fact that hepatic GH-receptor binding activity (Leung et al., 1984, 1987; Krishnan et al., 1989) and expression of hepatic GH receptor mRNA (Burnside and Cogburn, 1992) progressively increase with age suggests that older chickens should be more responsive to cGH infusion (Vasilatos-Younken et al., 1988; Rosebrough et al., 1991; Scanes et al., 1990). Thus, it appears that the age-dependent expression of the GH receptor in target tissue, rather than the pattern of cGH administration, constitutes the major factor which controls the responsiveness of young broiler chickens to exogenous cGH. In mammals, the anabolic activity of GH (i.e. increased protein accretion and skeletal growth) is mediated through its stimulatory effects on IGF-I synthesis (Hart and Johnsson, 1986). Although IGF-I levels were elevated by

54

Robert

F. Moellers

continuous infusion of cGH in young broiler chickens (the present study) and older broiler pullets (Vasilatos-Younken et al., 1988; Rosebrough et al., 199 l), no improvements were made in protein accretion or ash content (i.e. skeletal growth). Similarly, chronic treatment of young broiler chickens with biosynthetic human IGF-I does not improve body composition (McGuinness and Cogburn, 1991). On the other hand, pulsatile infusion of cGH did not increase plasma IGF-I levels of young broiler chickens, despite the ability of this treatment to provide 6-fold elevations in plasma GH levels eight times per day for 21 days. This observation supports data from a previous study (Cogburn et al., 1989) in which a daily S.C. injection of cGH (100-200 pg/kg) for 14 days (from 3 to 5 weeks of age) failed to increase IGF-I levels of broiler chickens. The present study shows that continuous infusion of cGH elevates IGF-I levels, albeit basal GH levels are only slightly higher. This observation is consistent with reports of enhanced IGF-I levels in older broiler pullets continuously infused with similar doses of cGH (Vasilatos-Younken et al., 1988; Rosebrough et al., 1991). Thus, continuous infusion of cGH appears to be more effective than pulsatile infusion of cGH in increasing plasma levels of IGF-I in the broiler chicken. In the present study, continuous cGH infusion depressed thyroid activity which was indicated by reduced plasma T, levels, slightly depressed hepatic 5’-MD1 activity, and elevated T, levels. A single injection of ovine GH was found to increase hepatic 5’-MD1 activity in adult chickens (Kuhn et al., 1987). In support of this finding, Vasilatos-Younken et al. (1988) showed an increase in T, levels with a concurrent decrease in T, levels of older broiler pullets (8-l 1 weeks of age) given pulsatile infusion of cGH. In a more recent study, a single injection of cGH into chicken embryos stimulated hepatic 5’-MD1 activity, although the ability of cGH to stimulate T, to T, conversion vanished after hatching (Darras ef al., 1990). More recent work suggests that endogenous GH regulates plasma T, levels in the chicken by depressing T,-degrading (Type III) deiodinase activity (Darras et al., 1993). In young rapidly-growing broiler chickens, plasma T, levels were not affected by daily injection of GH from 3 to 5 weeks of age (Cogburn et al.,1989) or 2-24 days of age (Burke ef al., 1987), despite significant elevations of plasma GH. Our finding that 5’-MD1 activity was slightly depressed in birds given continuous, but not pulsatile, cGH infusion suggests that the effects of cGH on 5’-MD1 activity are determined primarily by basal GH levels rather than the peak amplitude achieved by pulsatile cGH infusion. We have consistently

and Larry

A. Cogburn

found that exogenous cGH does not stimulate peripheral conversion of T, into T, in young broiler chickens (Cogburn, 199 1). The transient increase in plasma insulin levels in chickens after 2 weeks of continuous cGH infusion was similar to the effect of this treatment on plasma T, levels. In fact, there are some remarkable similarities between plasma profiles of insulin and T, in young broiler chickens infused with cGH. Pancreatic hormones (insulin and glucagon) have been reported to affect thyroid activity in the adult chicken (Mitchell and Raza, 1986). However, we maintain that the thyroid hormones (particularly T,) are important regulators of pancreatic hormone secretion in young chickens (Cogburn et al., 1986; Cogburn, 1991). The thyroid hormones were not affected in older (8-l 1-week-old) broiler pullets given continuous cGH infusion (VasilatosYounken et al., 1988) or young (3-5 weeks of age) broiler chickens given daily S.C. injections of cGH (Cogburn et al., 1989) despite elevated insulin levels. However, Cogburn et a/. (1986) showed that plasma insulin levels were increased in chickens by dietary T, treatment from 3 to 6 weeks of age. Our present data suggest that continuous infusion of cGH increases insulin levels by depressing thyroid activity. Since glucose levels were not elevated in chickens given continuous infusion of cGH, it is unlikely that cGH caused insulin resistance which has been reported in older chickens given continuous GH infusion (Vasilatos-Younken et al., 1988). In our study, plasma glucagon levels were increased in response to both pulsatile and continuous infusion of cGH, although this effect was greater in birds given continuous cGH. This suggests that cGH has a direct effect on glucagon secretion in chickens; however, glucagon levels in chickens were not affected by a daily S.C. injection of cGH for 14 days (Cogburn et al., 1989). Since glucagon and insulin levels in chickens are known to fluctuate regularly to maintain metabolic homeostasis (Hazelwood, 1984), it is possible that glucagon levels in cGH-treated birds in the present study were elevated to compensate for slightly higher insulin levels. Another possibility is that cGH infusion reduces the sensitivity of adipose tissue to glucagon, because plasma NEFA levels were not affected despite higher plasma glucagon levels in cGH-infused chickens. Although plasma glucose and NEFA levels were not altered by continuous infusion of cGH, we found a transient increase in plasma glucose levels after 1 week of pulsatile cGH infusion. Mitchell and Raza (1986) indicated that glucagon was a potent stimulus for glucose release in the chicken. However, our data

Chronic

cGH

infusion I in broiler

suggest that the glycolytic effect of glucagon could be secondary to that of cGH. This idea is supported by our observation that continuous infusion of cGH failed to elevate glucose levels despite higher glucagon levels, whereas a significant increase in glucose levels was found in birds given pulsatile cGH infusion. The hyperglycemic response of chickens to pulsatile cGH treatment is consistent with the finding that cGH is glycolytic in young broiler chickens (Hall et al., 1987). Our data confirm an earlier observation that the glucose response to an injection of cGH is blunted in chickens chronically treated with GH (Hall et al., 1987). Since continuous cGH infusion was not glycolytic, this suggests that chickens became refractory with constant exposure to cGH. It is also possible that glucose levels increased in response to continuous cGH treatment, but were returned to normal levels by a compensatory increase in insulin levels. Although there has been some suggestion that exogenous GH enhances immune function of chickens (Scanes and Lauterio, 1984), this notion is based largely on studies in which chickens were treated with heterologous (i.e. bovine or ovine) GH preparations (Marsh ef al., 1984; Buonomo and Baile, 1988). Scanes et al. (1990) reported that continuous infusion of cGH into 12-week-old broiler chickens increased the weight of the bursa of Fabricius, but not thymus weight. However, stimulation of bursal growth in their study could be due to the addition of 1% bovine serum albumin (BSA), a potent antigen, to the cGH infusate rather than a direct effect of cGH. In the present study, infusion of cGH into young broiler chickens for 3 weeks had no effect on the weight of the bursa of Fabricus at 7 weeks of age. In conclusion, our study clearly shows that both pulsatile and continuous intravenous infusion of cGH increase deposition of body fat in young broiler chickens. Pulsatile cGH treatment progressively increased feed intake without increasing growth rate. Hepatic GH-binding activity was dramatically reduced by pulsatile infusion of cGH. Continuous cGH infusion increased plasma IGF-I levels; however, pulsatile infusion of cGH had no effect on plasma IGF-I levels. Infusion of young chickens with cGH appears to depress thyroid activity which leads to higher plasma insulin levels. Regardless of major differences in the magnitude of hormonal responses to continuous or pulsatile infusion patterns, chronic intravenous infusion of cGH promotes deposition of body fat in young rapidly-growing broiler chickens. are grateful to Dr Cheng Zhong with the hepatic GH-binding assays.

Acknowledgemen/-We

his assistance

for

chickens

55

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Buonomo F. C. and Baile C. A. (1988) Recombinant bovine somatotropin stimulates short term increases in growth rate and insulin-like growth factor-I (IGF-I) in chickens. Dorm Anim. Endocr. 5, 219-229.

Burke W. H., Moore J. A., Ogez J. R. and Builder S. E. (1987) The properties of recombinant chicken growth hormone and its effects on growth, body composition, feed efficiency, and other factors in broiler chickens. Endocrinology

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an Id Larry

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McGuinness M. C. and Cogburn L. A. (1991) Response of young broiler chickens to chronic injection of recombinant-derived human insulin-like growth factor-l. Dom Anim. Endow. 8, 61 l-620. McMurtry J. P., Rosebrough R. W. and Steele N. C. (1983) A homologous radioimmunoassay for chicken insulin. Poult. Sci. 62, 697-701. Maiter D., Underwood L. E.. Maes M., Davenport M. L. and Ketelslegers J. M. (1988) Different effects of intermittent and continuous growth hormone (GH) administration on serum somatomedin-C/insulin-like growth factor-l and liver GH receptors in hypophysectomized rats. Endocrinology 123, 1053-1059. Maiter D., Walker J. L., Adam E., Moatsstaats B., Mulumba N.. Ketelslegers J. M. and Underwood L. E. (1992) Differential regulation by growth hormone (GH) of insulin-like growth factor-1 and GH receptor binding protein gene expression in rat liver. Endocrinology 130, 3251-3264. Marsh J. A.. Cause W. C., Sandhu S. and Scanes C. G. (1984) Enhanced growth and immune development in dwarf chickens treated with mammalian growth hormone and thyroxine. Proc. Sot. e.~p. Biol. Med. 175, 351-360. Mitchell M. A. and Raza A. (1986) The effects of glucagon and insulin on plasma thyroid hormone levels in fed and fasted domestic fowls. Camp. Biochcm. Physiol. 2, 217-223. Moellers R. M. and Cogburn L. A. (1991) Growth and metabolism of young broiler chickens given pulsatile or continuous infusion of chicken growth hormone (cGH). Poult. Sci. 70 (Suppl. l), 84 (Abstract). Moellers R. M. and Cogburn L. A. (1994) Pulsatile infusion of growth hormone-releasing factor depresses growth of young broiler chickens. Conip. Biochem. Physiol. 107A, 665-672. Proudman J. A. (1984) Recombinant-derived chicken growth hormone used for radioimmunoassay. Proc. Sot. e.xp. Biol. Med. 175, 417-419. Rosebrough R. W., McMurtry J. P. and Vasilatos-Younken R. (1991) Effect of pulsatile or continuous administration of pituitary-derived chicken growth hormone (p-cGH) on lipid metabolism in broiler pullets. Camp. Biochem. Physiol. 99A, 2077214. Scanes C. G. and Lauterio T. J. (1984) Growth hormone: Its physiology and control. J. esp. 2001. 232, 443.-452. Scanes C. G., Peterla T. A., Kantor S. and Ricks C. A. (1990) In ciro effects of biosynthetic chicken growth hormone in broiler-strain chickens. Growth Derl. Aging 54,95slOl. Vasilatos-Younken R.. Cravener T. L.. Cogburn L. A.. Mast M. G. and Wellenreiter R. H. (1988) Effect of pattern of administration on the response to exogenous, pituitary-derived chicken growth hormone by broilerstrain pullets. Gen. camp. Endow. 71, 268-283 Vasilatos-Younken R. and Zarkower P. G. (1987) Agerelated changes in plasma immunoreactive growth hormone secretory patterns in broiler pullets. Gron~th 51, 171-180.