Effects of a growth hormone-releasing factor analogue and an estradiol-trenbolone acetate implant on somatotropin, insulin-like growth factor I, and metabolite profiles in growing Hereford steers.

Effects of a Growth Hormone-Releasing Factor Analogue and an Estradiol-Trenbolone Acetate Implant on Somatotropin, Insulin-Like Growth Factor I, and Metabolite Profiles in Growing Hereford Steers't2

Department of Animal Science, University of Minnesota, St. Paul 55108

ABSTRACT: Hereford steers (290 k 6 kg of BW) were implanted (n = 4) with 140 mg of trenbolone acetate (TBA) and 28 mg of estradiol-17p (E&) or nonimplanted (controls, n = 4). In Trial 1, effects of a single i.v. injection of 0, 20, 40, or 80 pg of a growth hormone-releasing factor (1-29 NH2) analogue (GRFa) on release of endogenous somatotropin (ST) were evaluated in a double 4 x 4 Latin square design. Plasma samples (n = 21) were obtained from -20 to 240 min after GRFa injection. Area under the ST response curve (AUC) increased ( P = .009)in a dose-dependent manner (2, 2.6, 3.6,4.3 rng.min-l .mL-', respectively). Mean ST concentration was not affected ( P = .238) by implant but AUC was greater ( P = .009) in

implanted than in control steers. There was no interaction ( P = .4601 between dose of GRFa and presence of implant. In Trial 2, 80 pg of GRFa was administered at 12-h intervals to the same eight steers. Response of ST (AUC) to the first and last (13th) i.v. injection of GRFa was similar and not affected by implant. Before GRFa administration, plasma insulin-like growth factor I (IGF-I) concentrations were greater ( P = .039) in implanted than in control steers (272 vs 164 ng/mL). Administration of GRFa increased plasma IGF-I ( P = .0001), decreased plasma urea N (PUN) ( P = .00011, and did not alter plasma glucose ( P = .447) in both control and implanted steers. Data indicate that effects of GRFa and TBA/E2P on plasma IGF-I and PUN concentrations were additive in this study.

Key Words: Somatotropin, Anabolic Steroids, Hypothalamic Releasing Hormones, IGF-I, Steers J. Anim. Sci. 1992. 70:1439-1448

Introduction

'Published as paper no. 18,704 of the scientific journal series of the Minnesota Agric. Exp. Sta. on research conducted under Minnesota Agric. Exp. Sta. Project No. 16-045 and 16-075 and supported in part by the Minnesota Beef Council. 2Authors express their appreciation to the NIDDK and NHPP, Univ. of Maryland School of Med., for growth hormone RIA preparations, to L. E. Underwood, J. J. Van Wyk, and the NHPP for IGF-I antiserum, to R. M. Campbell, T. F. Mowles, and Hoffmann-LaRoche, Inc. for the GRF, and to R. J. Grant and Hoechst-Roussel --Vet Co. for the trenbolone acetate-estradiol implants. 3To whom correspondence should be addressed: 130 Haecker Hall, 1364 Eckles Ave. Received July 12, 1991. Accepted December 5, 1991.

Administration of growth hormone-releasing factor (GRF) or one of its analogues (GRFal to farm animals increases blood concentrations of somatotropin (ST)and insulin-like growth factor-I (IGF-I) (Gluckman et al., 1987; Enright et al., 1989). Treatment of swine (Dubreuil et al., 1990; Pommier et al., 19901, sheep (Beermann et al., 1990; Godfredson et al., 19901, and calves (Lapierre et al., 1991) with GRF increased ADG, decreased lipid deposition, and increased lean tissue accretion. Additional studies are needed to examine the ability of GRF to alter IGF-I and to define treatment regimens that stimulate growth of farm animals. The ability of steroid implants to improve

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D. D. Hongerholt, B. A. Crooker3, J. E. Wheaton, K. M. Carlson, and D. M. Jorgenson

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HONGERHOLT ET AL.

Materials and Methods Animals, Housing, and Diet. Eight Hereford steers of similar genetic background were purchased from two Minnesota farms for the two trials in this study. Upon receipt, steers were treated for coccidiosis and dewormed. Steers were vaccinated with killed viruses for Haernophilus sornnus, infectious bovine rhinotracheitis (IBR), bovine viral diarrhea (BVD),parainfluenza-3 (PI3], leptospirosis (5-way Leptol, and Clostridia (7-way Clostridium). Booster vaccinations were administered 14 d later. Steers were housed in an enclosed, ventilated barn maintained at 13"C and were allowed 1 mo to become accustomed to environmental conditions and management practices before the initiation of Trial 1. A total mixed

Table 1. Composition of total mixed diet ~~

Ingredients and composition Ingredients, YO of DM Corn silage Corn Protein supplement' Composition Crude protein, O/O of DM Acid detergent fiber, YO of DM Calcium, YO of DM Phosphorus, % of DM Magnesium, % of DM Potassium, 46 of DM Net energy for maintenanceb, Mcal/kg Net energy for gainb, Mcal/kg

Diet contribution 58

38 4

11.7 14.1 .38 .38 .18

1.07 2.03 1.37

'Protein supplement contained 59.34% soybean meal ( 4 4 % CPJ,31.43% corn, 4.53% limestone,3.2% tracemineralsalt, 1.19% dicalcium phosphate, YO SeaQ trace mineral, . I % Mg, .03% vitamins A and E, and .O8% vitamin A (3,000 IU/gl. bCalculated from ADF content.

diet consisting of 58% corn silage, 38% corn grain, and 4 % soybean meal with a vitamin-mineral supplement (DM basis) was fed to all steers throughout the study (Table 1). Fresh water was available at all times. Steers were weighed weekly. Steers were fed as a group during Trial 1 and on a n individual basis during Trial 2. During both trials, steers were fed quantities of feed sufficient to maintain a n ADG of 1.0 kg (NRC, 19841. Feed samples were obtained weekly and composited every 4 wk throughout both trials. Treatments. Twenty-five days after arrival, steers were assigned on a BW basis to an implant (258 k 7 kg) or control (264 k 8 kg) group. Implanted steers received 140 mg of TBA and 28 mg of E2P in a single pellet (Revalor@,Hoechst-Roussel Agri Vet, Somerville, NJ1 placed at the base of an ear. Control steers received a sham implantation. Implantation occurred 16 d before the initiation of Trial 1. In both trials, control and implanted steers received i.v. injections of GRFa. This analogue ([DesNH2Tyr1,D-Ala2,Ala151hGRF(1-291NH2;Compound RO 23-7863, Hoffman-LaRoche, Nutley, NJI is more resistant to biodegradation and has a greater biopotency than its native counterpart (Felix et al., 19881. Before the start of each trial, GRFa was dissolved in sterile saline (.9%), divided into aliquots of individual injection volumes, and stored at -20" C until it was used. Administration of GRFa was through indwelling jugular catheters. The entire dose was always administered within 10 s , and the residue remaining in the catheter was immediately flushed into the jugular vein with 5 mL of sterile saline. Blood Sampling and Preparation. Steers could move freely within their pen except during the 4-h period associated with GRFa challenges. During this time, steers were haltered, tied to the pen fence, and permitted to lie down, but they were not allowed to eat or drink. Blood samples were obtained via jugular catheters (1.02 mm i.d. x 1.78 mm 0.d.; Tygon microbore tubing, Norton Plastics and Synthetics Division, Akron, OH), inserted 1 d before the first GRFa injection in each trial. An intermittent infusion plug (Argyle, St. Louis, MO) was attached to each catheter. Catheters were routinely filled with 1,000 units (U)of heparin per milliliter of saline except during intensive sampling periods when catheters were filled with sterile saline (successive samples obtained at < 10-min intervals) or sterile saline containing 200 U of heparin/mL (successive samples obtained at 10- to 60-min intervals). Blood samples (approximately 6 mL1 were mixed with 100 pL of sterile saline and heparin (25 U/tube) in 16-m x 100-mm test tubes and placed in an ice-water bath until they were centrifuged (1,240 x g for 10 mid.


efficiency of growth, feed conversion, and carcass composition of beef steers is well established (Galbraith and Topps, 19811, but the mechanism(s1 responsible for these improvements remains unknown. Some studies suggest that steroid implants function indirectly through increased plasma ST concentrations (Galbraith and Watson, 1978; Peters et al., 1984; Grigsby and Trenkle, 1986; Gluckman et al., 19871. The objective of this study was to evaluate the potential for steroid implants and GRF to function together to enhance animal performance. Specifically, we determined effects of a trenbolone acetate/estradiol- 17p (TBA/E$I implant on acute response of plasma ST to dose of a GRF analogue in Trial 1 and response of plasma ST, IGF-I, urea nitrogen (PUN), and glucose to chronic (6 dl GRFa administration in Trial 2.

SOMATOTROPIN AND IGF-I LEVELS IN STEERS

were obtained at 12-h intervals. These samples were used to determine concentrations of ST,IGFI, PUN, and glucose before, during, and after the 6-d GRFa treatment period. In addition, for the first and last injection of GRFa, blood samples were obtained as described in Trial 1 to monitor ST response (AUC, peak height, time to peak, and preinjection mean concentration of ST). Whenever time of blood sampling and GRFa administration coincided, sampling occurred first. Assays. Unless noted otherwise, chemicals were obtained from Sigma Chemical (St. Louis, M01. Nutrient content of diet composites was determined by wet chemical methods (Dairy Herd Improvement Forage Testing Laboratory, Ithaca, NY). Bovine ST concentrations in plasma were measured using a heterologous double-antibody procedure (Wheaton et al., 19861. Assay components were USDA-I-1 bGH for radioiodination and AFP-5340-B for bovine standards, Sensitivity of four assays averaged 2.1 ng/mL. Intra- and interassay CV averaged 5.4 and 8.0%, respectively. A modified heterologous, double-antibody procedure was used to measure IGF-I in plasma (Godfredson et al., 1990).Dissociation and removal of binding proteins from IGF-I occurred before analysis. Equal volumes of plasma and .2 M glycylglycine HCl pH 2.0 were incubated in polystyrene tubes for 48 h at 37°C (Plaut et al., 1991) in a shaking water bath (Model 3575, Lab-line Instruments, Melrose Park, ILI. A 200-pL aliquot of this sample preparation was placed on a Sephadex G50 column (.7 cm x 30 cm) and washed into the column with 1 mL of eluent (1 M acetic acid). Binding proteins were contained in the first 5 mL and IGF-I in the second 5 mL eluting from the columns. The IGF-I fraction was collected in a siliconized graduated cylinder containing 10 mL of assay buffer (.02% protamine sulfate grade X, .4 1% sodium phosphate [monobasicl, 25% BSA 199% pure], .02% sodium azide, and .37% EDTA, pH 7.51. This mixture of IGF-I and assay buffer was quantitatively transferred to siliconized, 100-mL round-bottomed flasks, frozen in cold ethanol, and lyophilized (Model 10-lOOV, Virtus, Gardiner, NY1 overnight. Lyophilized samples were reconstituted in 2 mL of assay buffer. Reconstituted samples were placed in 12-mm x 75-mm polystyrene tubes and stored at -20°C until they were assayed. All samples were processed before IGF-I analysis. Immediately before analysis, reconstituted Samples were thawed and a 100-pLaliquot was diluted 1 : l O in assay buffer. A 100-pL aliquot of the diluted sample was assayed for IGF-I content (Godfredson et al., 1990). Recovery of IGF-I added to serum before glycyl-glycine incubation (83.4%) agreed with that reported by Plaut et al. (19911 when similar procedures were employed. Sensitivity


Plasma was removed, placed in 12-mm x 75-mm glass tubes, and stored at -20" C until it was assayed. Trial I. The objective of Trial 1 was to determine the effect of dose of GRFa on ST release in control and implanted steers. Steers (290 f 6 kg of BW) were housed in a single pen (11.43 m x 3.89 m) within the barn and were group-fed daily at 1200. Challenges (0,20, 40, or 80 pg of GRFa in 2 mL of saline) were administered (at 0800) before feeding as recommended by Moseley et al. (1987a1. Preliminary results indicated that the dose of 80 pg of GRFa would provide a plateau in pituitary response (ST release). Control steers were assigned to the first square and implanted steers were assigned to the second square of a double 4 x 4 Latin square. Within each square, steers were assigned randomly to a sequence of GRFa doses. Each period of the trial consisted of a single GRFa = challenge, and plasma samples (n 21 .steer-' .dose-l) were obtained at -20, -15, -10, -5, 0, 5, 10, 15, 20, 25, 30, 40, 50, 60, 75, 90, 105, 120, 150, 180, and 240 min relative to injection of GRFa. Successive periods (challenges) were separated by at least 1 d and the trial was completed within 8 d. Effects of TBA/E2P on the ability of GRFa to induce a release of pituitary ST were assessed by area under the ST response curve (AUC),peak height, time to peak, and mean ST concentration before GRFa administration. Trial 2. The objective of Trial 2 was to determine the effects of prolonged (6 d) administration of GRFa on plasma ST, IGF-I, PUN, and glucose in control and TBA/E2P implanted steers. The pen space used in Trial 1 was enlarged and divided into eight individual adjacent pens (each 3.05 m x 3.89 ml so that individual intakes could be determined. Other management practices were as described for Trial 1. Mean initial BW was 306 f 7 and 324 f 8 kg for control and implanted steers, respectively. All steers received i.v. injections (total n = 13)of GRFa (80 pg/injection) a t 12-h intervals (0800 and 2000). Single blood samples (12 mL stored as two equal aliquots of plasma) from each steer were obtained 24 and 0 h before the first GRFa injection. During the first 48 h of the GRFa treatment period, blood samples were obtained at 6-h intervals to monitor potential shifts in mean concentration of hormones (ST, IGF-I) and metabolites (PUN, g l u cose). We assumed adjustments in mean concentration would be minimal after this point and sampled a t 12-h intervals during the final 96 h of the GRFa treatment period. After the last injection of GRFa, blood samples were again obtained at 6-h intervals for the first 48 h to monitor readjustments in mean concentrations due to removal of GRFa. During the subsequent 24 h, blood samples

1441

1442

HONGERHOLT ET AL.

-"-I

I I

1

I

ADG (kg)

Table 2. Effect of implant on mean somatotropin concentrations before administration of growth hormone-releasing factor analogue Treatment ~~~

Trial lb 2c

~

~

~

~

Control

Implanta

SE

P

5.2 6.2

8.5

1.3 .9

.24 .20

8.1

&Steers were implanted with a combination of trenbolone acetate (140 mg) and estradiol-17P (28 mgl. bLeast squares means (nanograms/milliliter) determined from plasma samples (n = 5) obtained from each steer (n = 4 per treatment) during the 20 min before administration of each dose (n = 4) of a growth hormone-releasing factor analogue (GRFal (total n = 80 samples per treatment group). Administration of GRFa occurred 16 to 23 d postimplantation. CLeast squares means (nanograms/milliliter) determined from plasma samples obtained from each steer 24 h before first injection of GRFa (n = 1 per steer), from samples obtained during the 20 min before the 1st and 13th injection of GRFa (n = 4 per steer per injection), and from samples obtained at 0200, 0800, 1400, and 2000 (n = 28 per steer) throughout the trial (total n = 148 samples per treatment group). Administration of GRFa occurred 44 to 51 d postimplantation.

was Yijkl = p. + Ii + Sj(Ii) + Dk(IiI + Ti + ITil, where Yijkl = the observation made for the dependent variable, p. = the overall mean, Ii = the implant (or square), Si = the steer, Dk = the day of treatment, and T1 = GRFa treatment. Effects of prolonged administration of GRFa on plasma ST response (AUC) after the 1st and 13th GRFa injection in control and implanted steers (Trial 2) were evaluated as a completely randomized block design using the model Yijk = p. + Ii + Sj(Ii) + Dk + IDik, where abbreviations are as previously defined. Effects of prolonged (6 dl administration of GRFa on plasma components (IGF-I, urea nitrogen, and glucose) in control and implanted steers were assessed using repeated measures analyses. The GLM procedure of SAS for personal computers (1988) was used to analyze all data. Comparisons were considered significantly different if P c .05.

Results

-

-

Trial 1

Trial 2

200

0

1

2

3

4

5

6

7

8

9 1 0 1 1 1 2 1 3 1 4

Relative Time (weeks)

Figure 1. Mean body weights (n = 4) of control (m) and trenbolone acetatelestradiol-l7P-implanted ( 0 ) steers. Average daily gain was calculated from time of implantation to 5 d after the end of trial 2 (57 d). Vertical bars represent SEM.

Control and implanted steers had similar BW before implantation with TBA and E& (Figure 1). From implantation to the end of Trial 2, implanted steers had a greater ( P = .0001) ADG than controls. During Trial 2, average DMI were 0.95 and 6.85 kg/d for implanted and control steers, respectively. Although mean plasma concentration of ST before GRFa administration (Table 2) tended to be greater in implanted than in control steers in both trials, the differences were not significant. Individ-


averaged 12.5 ng/mL. Intraassay, intra-, and intercolumn CV averaged 3.4, 21.1, and 22.O%, respectively. Measurement of PUN was accomplished with a diagnostic kit (Procedure 640, Sigma Chemical) using 15 pL of plasma or standard solution. Absorbance (Gilford Response 11, Ciba-Corning Diagnostics, Medfield, MA) was determined at 570 nm. Intra- and interassay CV were 3.0 and 3.3%, respectively. Plasma glucose was assayed with a diagnostic kit (Procedure 5 10, Sigma Chemical) using 20 pL of plasma or standard and 2.0 mL of combined enzyme-color reagent. Absorbance was determined a t 450 nm. Intra- and interassay CV were 2.3 and 5.8%, respectively. Statistical AnuZyses and CuZcuZutions. For individual steers, ADG was calculated as the slope of the regression line describing the relationship between body weight and day of study (calculated over a n 8-wk period from 2 d postimplantation to 5 d after the end of Trial 2). Effect of TBA/E2P implant on ADG was assessed by Student's t-test. Calculation of AUC was by trapezoid summation of the area between successive pairs of concentration and time coordinates after subtracting the mean ST concentration before administration of GRFa (preinjection ST mean). In addition, preinjection ST mean, peak ST response (greatest ST concentration above preinjection ST mean within 30 min postadministration of GRFal, and time to ST peak (time of peak minus time of GRFa injection) were compared between control and implanted steers. The model used to evaluate treatment effects in the two concurrent 4 x 4 Latin squares of Trial 1

1443


100

Dose

0

80

-

I

A

E

\

2 0 pg Dose

80

0

C

Y

0'

-30

L

1

0

30

I

I

60

90

I

I

,

I

120 150 180 210 240 270

-30

0

30

Relative Time (min.)

z \

D

C

Y

I-

60

t

90

120 150 180 210 240 270

Relative Time (rnin.)

100 I h

60

I

4 0 pg Dose

40 u)

20

0 -30

I

0 ' 0

30

60

90

120 150 180 210 2 4 0 270


-30

o

30

60

go

120 150 180 210 240 2 7 0


Figure 2. Plasma concentrations of somatotropin (ST) in control (- - -) and trenbolone acetate/estradiolsteers relative to time (0)of i.v. injection of 0, 20, 40, or 80 vg of a growth hormone-releasing 17P-implanted )-( factor analogue per steer. Average SEM (n = 4) for control and implanted ST concentrations at individual time points were 1.0 and 4.9, 3.7 and 4.8,3.9 and 6.6,and 3.2 and 9.0 for 0-, 20-, 40-, and 80-1.18 dose, respectively.


Implanted steers had a greater (P = .009)ST response (AUC) than controls (Table 31. Responses of ST to 20-, 40-,and 80-pg doses of GRFa were greater (P = .009)than responses of ST to the 0-pg dose, but there were no differences in AUC among non-zero doses. The numerical difference between AUC of control and implanted steers decreased as dose of GRFa increased (Table 31,but there was no interaction ( P = .741) between dose of GRFa and presence of implant. Responses of control and implanted steers were most similar when 80 pg of GRFa was administered. An objective of Trial 2 was to examine effects of steroid implants on plasma IGF-I response to GRFa administration. To minimize potential interference caused by differences in plasma ST content, the 80-pg dose of GRFa was used in Trial 2. Trial 2. One control steer (#7)did not respond to the first injection of GRFa, but, based on the

ual responses were variable (see below), but both control and implanted steers responded to GRFa by increasing plasma ST concentration within 5 to 15 min (Figures 2 and 31. Trial 1. One implanted steer (#3)did not respond to the dose of 40 pg of GRFa until 30 min after the dose was administered, and data from this steer were omitted from the analyses. Response of all other steers to all non-zero doses of GRFa occurred within 15 min. Time required to reach peak plasma ST concentration was similar for control and implanted steers (18 min) and was not affected ( P = .3881 by dose of GRFa. Administration of GRFa to implanted steers tended (P = .lo21 to increase peak height above that of control steers (Table 3). Compared with saline (dose of 0 p g of GRFa), non-zero doses of GRFa resulted in greater ( P = .011) peak height but there were no differences among the non-zero doses (Table 31.

1444

HONGERHOLT ET AL.

chronic administration of 80 pg of GRFa. Peak height was not affected by presence of implant [35.1 vs 42.7 ng/mL for control and implanted steers, respectively; P = .432) but tended ( P = .090) to increase with chronic administration of GRFa [31.4 vs 46.3 ng/mL for the first and last injection). Mean preinjection ST concentrations (Table 21 were not affected by presence of implant ( P = .198) or the chronic administration of GRFa ( P = .369). There was no interaction between time (1st vs 13th injection) of GRFa administration and presence of implant on peak height (P = .8421 or mean ST concentrations (P = .404). Plasma IGF-I concentrations were greater ( P = .039) in implanted than in control steers (272 v s 164

100

100

Controls

60

Injection 1

I

I

r

AUC 5155

0 '

10

0 ' -30

I 0

30

60

90

120

160

180

210

240

c

"

"

'

I

"

.

'

a

P-.o6

40

**

PI.01

*

270

T

c v)

fm

Implants

6o

Injection

1 13

t

AUC 4567 4955

= I I

I

E

&

c I

LL

p

0 ' -30

I

I

0

30

60

90

120

150

180

210

240

270

Relative l i m e (min)

Figure 3. Plasma concentrations of somatotropin (ST) in control and trenbolone acetatelestradiol-17fl-implanted steers relative to time ( 0 ) of i.v. injection of 80 pg of a growth hormone-releasing factor analogue. Area under the ST response curve (AUC) for the first 1-( and 13th (- -) injection (12-h injection interval) were similar for both control and implanted steers. Average SEM (n = 4) for ST concentration at individual time points of 1st and 13th injection were 7.0 and 4.0, and 7.8 and 7.3 for control and implanted steers, respectively-

450 400

r

* **

T

380

a

-

--

,.y--*---r 35 2 0 00 1

I

200

I

50 0 -

-..

--*

PC.06 PS.01

*---I

0

a

a

a*

*-

*a

*

. I

v v v v v v v v v v v v v

1

0

1

2

3

4

5

Relative Time

6

7

8

9

1

0

(days)

Figure 4. Plasma concentrations of insulin-like growth factor I [IGF-I), urea nitrogen (PUN), and glucose relative to time of initiation of treatment with 80 pg of a growth hormone-releasing factor analogue (GRFa) at 12-h intervals for control (m) and trenbolone acetate/estradiol-l7P-implanted( 0 ) steers. The GRFa treatment is noted by V; associated asterisks indicate effect of GRFa on daily mean of plasma components for both groups of steers relative to d 0. Asterisks associated with vertical standard error bars indicate differences between control and implanted steers. Data points represent means of 2 to 4 samples.steer-' .d-'.


increase in plasma IGF-I, did respond to subsequent injections of GRFa. Data from this steer were included in the analyses. One implanted steer (#5) responded more slowly (20 to 25 min) to both the 1st and 13th injection of GRFa than the other steers (5 min). Administration of 80 pg of GRFa caused similar increases in plasma ST concentrations in control and implanted steers (Figure 3). This response was not altered by prolonged (13 treatments a t 12-h intervals) administration of GRFa (Figure 3). Time required to reach peak plasma ST concentration was similar for control and implanted steers (18 vs 19 min, respectively) and was similar to that observed in Trial 1. Time to peak was not affected (P = .320) by


1445

Table 3. Effect of dose of growth hormone-releasing factor analogue (GRFa) on somatotropin (ST) response and peak height in control and implanted steers Peak heighta DoseC 0 20 40 80

Meadh

Control 6.4 25.2 37.1 31.1 24.9

Implantd 21.0 53.7 44.7 48.4 41.9

AUCb Meane

SE

Control

Implant

Meanf

SE

13.7 39.5 40.9 39.7 33.4

5.2 4.4 5.2 4.4 3.3

335 1,129 2,618 3,496 1.894

44 4.070 4,690 5,041 3.461

189 2,599 3,654 4,268 2.678

704 704 813 704 518

ng/mL) before GRFa administration (Figure 4). Plasma IGF-I in control steers increased ( P = .010) after the first two GRFa injections but the response was delayed in implanted steers. After this initial difference, prolonged administration of GRFa increased IGF-I concentrations a t a similar rate (13.9 ng.mL-l.d-l) in both groups of steers (Figure 4).Concentration of IGF-I seemed to reach a plateau in control and implanted steers after 4 to 5 d of GRFa administration. The GRFa-induced increase in plasma IGF-I (plasma concentration of IGF-I at plateau minus that before GRFa administration) tended to be greater in control than in implanted steers (120.3vs. 67.8 k 21.1 ng/mL; P = .1281. Within 3 d after GRFa administration ceased, IGF-I concentrations had returned to preinjection concentrations in implanted steers but remained elevated (42.5%) in control steers. Presence of implant decreased ( P = .003)PUN concentrations from 6.7 to 4.7 mg/dL (Figure 41. After treatment with GRFa was initiated, PUN in control and implanted steers decreased ( P = .OOOll by 1.8 and 1.3 mg/dL, respectively. After GRFa administration ceased, PUN in both groups returned to preadministration concentrations within 3 d. Neither presence of implant nor chronic administration of GRFa affected plasma glucose concentrations.

Discussion Our results indicate that ADG of implanted steers was increased by 47.2% compared with ADG of control steers. These results agree with other reports of the effects of TBA/E2P implants on ADG. In a review of effects of TBA and E2P on growth performance of beef steers, Anderson (19901 reported that ADG, feed efficiency, and carcass lean

were improved 24, 15, and 5% over controls, respectively. When steers received implants containing 140 mg of TBA and 28 mg of E2P during wk 0, 10,and 18 of a long-term study, ADG was 32.4% greater than that of control steers (Henricks et al., 1988). Growing Hereford x Friesian beef steers implanted with 140 mg of TBA and 20 mg of E2P exhibited a 59% improvement in ADG compared with sham-implanted steers (Lobley et al., 1985). Mean ST concentrations before GRFa administration were not altered by presence of implant in our study. This is in agreement with other studies of steers implanted with TBA/E2P (Heitzman et al., 1977; Henricks et al., 1988; Hunt et al., 19911, of bulls implanted with TBA/E2P (Hunt et al., 19911, and of bulls implanted with 300 mg of TBA 21 d after receiving 45 mg of hexoestrol (Galbraith, 1982). In contrast, Galbraith and Watson (1978) determined that steers implanted with 60 mg of a n estrogen analogue (hexoestrol) or with 300 mg of TBA and 60 mg of hexoestrol had greater serum ST concentrations than controls or steers implanted with 300 mg of TBA. Several investigations besides our own have examined the possibility that steroids stimulate growth in steers and sheep by directly affecting pituitary release of ST (Gluckman et al., 19871,but results have been inconsistent. The high variability of ST concentrations within treatment groups precluded detection of differences in the study of Henricks et al. (1988)and in our study and may partially explain inconsistencies in the literature about effects of steroids on blood ST concentrations. Our results indicate that a minimum of 30 steers per implant group would be required to detect a difference ( P c .lo1 of 1.7 ng of ST/mL (Table 21. Differences in age and physiological status (finishing vs growing) of the steers, dose and choice of implant (300 mg of TBA/6O mg of


&Highestplasma ST concentration within 30 min after GRFa administration minus preadministration ST mean. bArea under curve as nanograms. minute-’ ‘milliliter-’. CThe GRFa (micrograms .steer-’ .d-’) was administered 18 to 23 d postimplantation. dSteers implanted with a combination of trenbolone acetate (140 mgl and estradiol-17P (28 mg). eNon-zero and zero dose effects differ (P = .011). fNon-zero and zero dose effects differ UJ = ,0091. gImplant effect on peak height (P = ,1021. hImplant effect on area under curve (P = .OOgl.

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HONGERHOLT ET AL.

effects caused by increases in mean ST concentrations. However, Hunt et al. (1991) observed similar IGF-I concentrations in serum from control and TBA/E2P-implanted steers. Alternatively, the nonsignificant, but consistent, numerical difference between mean ST (8.1 vs 6.2 ng/mL, for implants and controls, respectively) may have been sufficient to elicit the increase in plasma IGF-I concentrations in the implanted steers. An increased number of ST receptors, a n increased affinity of receptors for ST, or a postreceptor modification could also cause the increase in plasma IGF-I content in implanted steers without altering plasma ST content (Gluckman et al., 19871. Armstrong et al. (1990) detected increased (37%) IGF-I in plasma from steers treated with GRF compared with that from control steers. Similar effects of GRF administration have been demonstrated in lactating dairy cows (Enright et al., 1989; Abribat et al., 19901. In our study, plasma IGF-I concentration increased ( P = .0001) in both control and implanted steers when GRFa was administered. However, the response tended (P = ,128) to be lower in implanted steers. These results indicate that effects of GRFa and TBA/E2P on IGF-I are additive and agree with the additive effects of GRF and E2P reported by Enright et al. (19901. In contrast, Wagner and colleagues (1988a) compared IGF-I concentrations in plasma from control steers with those from steers treated with 24 mg of E#, 960 mg of ST/14 d, or both E# and ST and concluded that the effects of E2p and ST on IGF-I were not additive (195, 291, 495, and 511 ng of IGFI/mL, respectively). However, the effects of E2P and ST on ADG and feed efficiency were additive (Wagner et al., 1988a). Although these results may be interpreted to suggest that these metabolic modifiers function through different mechanisms, an alternative conclusion also is possible. The smaller IGF-I response to GRFa by implanted steers could result from a smaller difference between basal IGF-I and maximal IGF-I response possible in implanted steers compared with control steers. Although the maximum attainable IGF-I response in steers is not known, implanted steers had greater circulating IGF-I concentrations than control steers. If this greater IGF-I concentration were close to some upper limit, then the opportunity for GRFa to elicit an IGF-I response would be reduced in implanted steers. The metabolic modifiers could be functioning by either the same or different mechanisms in this situation. Regardless of the mechanism involved, the two metabolic modifiers induced a n additive response in IGF-I. Both control and implanted steers had low concentrations of PUN before GRFa administration. Byers and Moxon (1980) observed similar


hexoestrol vs 140 mg of TBA/28 mg of EzP), and sampling period (70 vs 56 d) may explain why Galbraith and Watson (1978) observed an increase in mean ST concentration, whereas we did not. In our study, all non-zero doses of GRFa increased ( P = .009)ST AUC in both groups of steers (Trial 1). Although there was a n overall effect of implant on AUC IP = .009), response by control and implanted steers to individual doses of GRFa was not different (Table 3). The large degree of variation in ST response by steers contributed to the lack of a n implant effect for individual doses. Response of ST in control and implanted steers was most similar when 80 pg of GRFa was administered. Our results indicate that a minimum of 10 steers per implant group would be required to detect a difference ( P e .lo) of 2,000 ng.min-l .mL-l (Table 3) at any particular dose of GRFa. An objective of Trial 2 was to examine effects of steroid implants on plasma IGF-I response to GRFa administration. To minimize potential interference caused by differences in plasma ST content, the 80-pg dose of GRFa was used in Trial 2. The lack of a n implant effect on ST response in Trial 2 is consistent with results obtained in Trial 1 for the dose of 80 pg of GRFa. Data describing effects of TBA/E2P implants and administration of GRF on plasma IGF-I and ST concentrations in growing steers are very limited. Hunt et al. (1991) observed no difference between mean serum ST concentrations from control and TBA/E@ implanted steers after a single injection of GRF. To our knowledge, our study is the first to examine the interaction of TBA/E2P and chronic administration of GRF. Chronic administration (5 or 20 dl of GRF or its analogues to steers and bulls (Moseley et al., 1985, 1987b1 has not decreased responsiveness of the pituitary to GRF. In our study, the first and last (13th) injection of GRFa yielded similar ST responses (AUC) and response was not affected by presence of the TBA/EzP implant (Figure 3). These results agree with the concept that pituitary response is not affected by chronic administration of GRF or its analogues and indicate that the presence of TBA/E2P implants does not alter this sustained response. Administration of ST to growing (Wagner et al., 1988; Lema1 et al., 1989; Crooker et al., 1990) or lactating (Cohick et al., 1989) cattle increases plasma IGF-I concentrations, Although mean ST concentrations and AUC were not different between control and implanted steers, mean IGF-I concentrations were greater ( P = .039) in implanted steers before administration of GRFa. This suggests that plasma IGF-I may be directly affected by steroid implants in addition to indirect


Implications Results of this study demonstrate that chronic (6 dl administration of a growth hormone-releasing analogue and a n estradiol-trenbolone acetate implant alone and in combination can increase plasma somatotropin and insulin-like growth factor I and decrease plasma urea nitrogen concentrations. These results suggest that effects of these metabolic modifiers are additive and that they

could be used in combination to increase efficiency of production in cattle.

Literature Cited Abribat, T., H. Lapierre, P. Dubreuil, G. Pelletier, P. Gaudreau, P. Brazeau, and D. Petitclerc. 1990. Insulin-like growth factor-1 concentration in Holstein female cattle: variations with age, stage of lactation and growth hormone-releasing factor administration. Domest. h i m . Endocrinol. 7:93. Anderson, P. T. 1990.Strategies for use of trenbolone acetate in growing/finishing beef cattle systems. Minnesota Nutr. Conf. 51:132. Armstrong, J. D., R. W. Harvey, J. W. Spears, J. G. Ross, R. Campbell, and T. Mowles. 1990. Concentrations of growth hormone and insulin-like growth factor 1 and performance following chronic administration of an analog of growth hormone releasing factor in finishing steers. J. Anim. Sci. 68(suppl. 1):15 IAbstr.1. Beermann, D. H., D. E. Hogue, V. K. Fishell, S . Aronica, H. W. Dickson, and B. R. Schricker. 1990. Exogenous growth hormone-releasing factor and ovine somatotropin improve growth performance and composition of gain in lambs. J. h i m . Sci. 68:4122. Byers, F. M., and A. L. Moxon. 1980. Protein and selenium levels for growing and finishing beef cattle. J. Anim. Sci. 50:1136. Cohick, W. S.,K. Plaut, S . J. Sechen, and D. E. Bauman. 1989. Temporal pattern of insulin-like growth factor-1 response to exogenous bovine somatotropin in lactating cows. Domest. Anim.Endocrinol. 6:263. Coxam, V., M. J. Davicco, F. A. Opmeer, J. P. Ravault, and J. P. Barlet. 1987. Plasma somatotropin and somatomedin C concentrations following GRF on TRH injections in newborn calves. Reprod. Nutr. Dev. 27(2B):533. Crooker. B. A,, M. A. McGuire, W. S . Cohick, M. Harkins, D. E. Bauman, and K. Sejrsen. 1990. Effect of dose of bovine somatotropin on nutrient utilization in growing dairy heifers. J. Nutr. 120:1256. Dubreuil, P., D. Petitclerc, G. Pelletier, P. Gaudreau, C. Farmer, T. F. Mowles, and P. Brazeau. 1990. Effect of dose and frequency of administration of a potent analog of human growth hormone-releasing factor on hormone secretion and growth in pigs. J. Anim. Sci. 68:1254. Enright, W. J., L. T. Chapin, W. M. Moseley, S . A. Zinn, M. B. Kamdar, L. F. Krabill, and H. A. Tucker. 1989. Effects of infusions of various doses of bovine growth hormone-releasing factor on blood hormones and metabolites in lactating Holstein cows. J. Endocrinol. 122:671. Enright, W. J., J. F. Quirke, P. D. Gluckman, B. H. Breier. L. G. Kennedy, I. C. Hart, J. F. Roche, A. Coert, and P. Allen. 1990. Effects of long term administration of pituitary-derived bovine growth hormone and estradiol on growth in steers. J. Anim. Sci. 68:2345. Felix, A. M., E. P. Heimer, C.-T. Wang, T. J. Lambros, A. Fournier, T. F. Mowles, S . Maines, R. M. Campbell, B. B. Wegrzynski, V. Toome, D. Fry, and V. S . Madison. 1988. Synthesis, biological activity and conformational analysis of cyclic GRF analogs. Int. J. Peptide Protein Res. 32:441. Galbraith, H. 1982. Growth, hormonal and metabolic response of post-pubertal entire male cattle to trenbolone acetate and hexoestrol. Anim. Prod. 35:269. Galbraith, H., and J. H. Topps. 1981. Effect of hormones on the growth and body composition of animals. Nutr. Abstr. Rev. Series B 51:521. Galbraith, H., and H. B. Watson. 1978. Performance, blood and carcass characteristics of finishing steers treated with trenbolone acetate and hexoestrol. Vet. Rec. 103:28. Gluckman, P. D., B. H. Breier, and S . R. Davis. 1987. Physiology


PUN values (7.8 mg of N/loO mL) when beef cattle were fed diets containing 11.6% CP. Presence of implant reduced (300/0)PUN concentration (Figure 31, in agreement with results of Galbraith and Watson, (19781, Galbraith (19821, and Henricks et al. (19881. Administration of GRFa caused PUN to decrease further in both groups (Figure 31. Plouzek and colleagues (19881reported that administration of .067 pg of rat hypothalamic Nle27 GRF (1-29)NH2/kg of BW to bull calves every 4 h for 10 d decreased PUN 6% below that in controls. The smaller PUN response observed by Plouzek et al. (1988) could be due to variations in amount and potency of GRF and possibly to differences in time of blood sampling relative to time of feeding. The reduced PUN concentrations associated with separate and combined administration of GRFa and TBA/E2P support the ability of these metabolic modifiers to improve nitrogen utilization. In addition, reductions in PUN concentrations were associated with increased circulating IGF-I concentrations. These results are in agreement with the relationship between nitrogen retention and IGF-I described by Crooker et al. (19901 in growing Holstein heifers treated with varying doses of ST and are consistent with a possible role of IGF-I in nitrogen utilization. That administration of GRFa was able to further reduce PUN concentrations in implanted steers indicates that, compared with their separate use, greater improvements in nitrogen utilization may occur when the two metabolic modifiers are used in conjunction. Glucose concentrations were not altered by presence of implant or GRFa administration (Figure 3) and were within the normal range (40 to 80 mg/dL1 for cattle (Swenson, 1984). These results agree with those from other studies that have investigated the effects of E2p, TBA, and TBA/E2P (Heitzman et al., 1977; Galbraith and Watson, 1978; Wagner et al., 1988b) and GRF (Coxam et al., 1987; Plouzek et al., 1988) on blood glucose. This lack of alteration in blood glucose suggests that administration of TBA/E2P and GRF does not adversely affect the mechanisms that regulate glucose homeostasis.

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of the somatotrophic axis with particular reference to the ruminant. J. Dairy Sci. 70:442. Godfredson, J. A,, J. E. Wheaton, B. A. Crooker, E. A. Wong, R. M. Campbell, and T. F. Mowles. 1990. Growth performance and carcass composition of lambs infused for 28 days with a growth hormone-releasing factor analogue. J. Anim. Sci. 6833624.

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SAS. 1988. SAS User’s Guide: Statistics. SAS Inst. Inc., Cary, NC. Swenson, M. J. 1984. Physiological properties and cellular and chemical constituents of blood; In: Dukes’ Physiology of Domestic Animals, (10th Ed.). p 34. Cornel1 University Press, Ithaca, NY. Wagner, J. F., T. Cain, D. B. Anderson, P. Johnson, and D. Mowrey. 1988a. Effect of growth hormone (GH) and estradiol IE.$I alone and in combination on beef steer growth performance, carcass and plasma constituents. J. Anim. Sci. 66(suppl. 11:283 Ubstr.). Wagner, J. F., R. E. Paxton, D. B. Anderson, E. L. Potter, and D. Mowrey. 1988b. The effect of bovine growth hormone (bGH1 and estradiol (E2pl alone and in combination on urinary nitrogen excretion in beef steers. J. Anim. Sci. 66(suppl. 1): 253 LAbstr.). Wheaton, J. E., S. N. Al-Raheem, Y. S. Massri, and J. M. Marcek. 1986. Twentyfour-how growth hormone profiles in Angus steers. J. Anim. Sci. 62:1267.


Grigsby, M. E., and A. Trenkle. 1986. Plasma growth hormone, insulin, glucocorticords and thyroid hormones in large, medium and small breeds of steers with and without an estradiol implant. Domest. Anim. Endocrinol. 3:261. Heitzman, R. J., K. H. Chan, and I. C. Hart. 1977. Liveweight gains, blood levels of metabolites, proteins and hormones following implantation of anabolic agents in steers. Br. Vet. J. 13332. Henricks, D. M., T. Gimenez, T. W. Gettys, and B. D. Schanbacher. 1988. Effect of castration and an anabolic implant on growth and serum hormones in cattle. Anim. Prod. 46:35. Hunt, D. W., D. M. Henricks, G. C. Skelley, and L. W. Grimes. 1991. Use of trenbolone acetate and estradiol in intact and castrate male cattle Effects on growth, serum hormones, and carcass characteristics. J. h i m . Sci. 69:2452. Lapierre, H., G . Pelletier, D. Petitclerc, P. Dubreuil, J. Morisset, P. Gaudreau, Y. Couture, and P. Brazeau. 1991. Effect of human growth hormone-releasing factor and(or1 thyrotropin-releasing factor on growth, carcass composition, diet digestibility, nutrient balance, and plasma constituents in dairy calves. J. Anim. Sci. 69:587. Lemal, D., R. Renaville, V. Claes, L. Ruelle, J. Fabry, A. Burny, L. E. Underwood, and J.-M. Ketelslegers. 1989. Effect of pituitary somatotropin injections on plasma insulin-like growth factor I and somatotropin profiies in growing heifers. J. Anim. Sci. 67:2715. Lobley, G . E., A. Connell, G . S . Mollison, A. Brewer, C . I. Harris, V. Buchan, and H. Galbraith. 1885. The effects of a combined implant of trenbolone acetate and oestradiol- 176 on protein and energy metabolism in growing beef steers. Br. J. Nutr. 543381. Moseley, W. M., G. R. Naniz, W. H. Claflin, and L. F. Krabill. 1987a. Food intake alters the serum GH response to bGRF in meal-fed Holstein steers. J. Anim. Sci. 65(suppl. 11247 LAbstr.1. Moseley, W. M., J. Huisman, and E. J. VanWeerden. l987b. Serum growth hormone and nitrogen metabolism responses in young bull calves infused with growth hor-

mone-releasing factor for 20 days. Domest. Anim. Endocrinol. 4:51. Moseley, W. M., L. F. Krabill, A. R. Friedman, and R. F. Olsen. 1985. Administration of synthetic human pancreatic growth hormone-releasing factor for five days sustains raised serum concentrations of growth hormone in steers. J. Endocrinol. 104:433. NRC. 1984. Nutrient Requirements of Beef Cattle (6th Ed.) National Academy Press, Washington, DC. Peters, A. R., D. G. Evans, D. J. Read, J. M. Beeby, and W. Haresign. 1984. Effects of trenbolone acetate and hexoestrol on live-weight gain, and serum hormone and metabolite concentrations in steers. Anim. Prod. 38:385. Plaut, K., W. S. Cohick, D. E. Bauman, and R. C. Baxter. 1991. Evaluation of interference by insulin-like growth factor I (IGF-I)binding proteins in a radioimmunoassay for IGF-I in serum from dairy cows. Domest. Anim. Endocrinol. 8:393. Plouzek, C. A., W. Vale, J. Rivier, L. L. Anderson, and A. Trenkle. 1988. Growth hormone-releasing factor on growth hormone secretion in prepubertal calves. Proc. SOC.Exp. Biol. Med. 188:198. Pommier, S. A,, P. Dubreuil, G. Pelletier, P. Gaudreau, T. F. Mowles, and P. Brazeau. 1990. Effect of a potent analog of human growth hormone-releasing factor on carcass composition and quality of crossbred market pigs. J. Anim.Sci.

Secretion of insulinlike growth factor I and insulinlike growth factor-binding proteins by murine bone marrow stromal cells.

Effect of puberty on insulinlike growth factor I and HbA1 in type I diabetes.

Effect of growth hormone-releasing factor infusion on somatotropin, prolactin, thyroxine, insulin, insulin-like growth factor I and blood metabolites in control and somatostatin-immunized growing pigs.

A unique receptor-independent mechanism by which insulinlike growth factor I regulates the availability of insulinlike growth factor binding proteins in normal and transformed human fibroblasts.

Effect of tamoxifen on serum insulinlike growth factor I levels in stage I breast cancer patients.

Growth hormone and insulin-like growth factor-I responses in steers actively immunized against somatostatin or growth hormone-releasing factor.

Influence of nutritional deprivation on insulin-like growth factor I, somatotropin, and metabolic hormones in swine.

Relationships between the thyroid and somatotropic axes in steers. I: Effects of propylthiouracil-induced hypothyroidism on growth hormone, thyroid stimulating hormone and insulin-like growth factor I.

Effects of partial hepatectomy on hepatic insulinlike growth factor binding protein-1 expression.

Truncated and native insulinlike growth factor I enhance mucosal adaptation after jejunoileal resection.

Insulinlike growth factor I: a potent mitogen for human osteogenic sarcoma.

Effect of active immunization against growth hormone releasing factor on concentrations of somatotropin and insulin-like growth factor I in lactating beef cows.

Effect of prepartum administration of growth hormone-releasing factor on somatotropin, insulin-like growth factor I, milk production, and postpartum return to ovarian activity in primiparous beef heifers.

In vitro effects of epidermal growth factor, insulin-like growth factor-I, fibroblast growth factor, and follicle-stimulating hormone on hamster follicular deoxyribonucleic acid synthesis and steroidogenesis.

Overexpression of the human insulinlike growth factor I receptor promotes ligand-dependent neoplastic transformation.

Recombinant bovine somatotropin improves growth performance in finishing beef steers.

Mitogenic response of canine fundic epithelial cells in short-term culture to transforming growth factor alpha and insulinlike growth factor I.

Growth hormone increases insulin-like growth factor-I (IGF-I) and decreases IGF-II in plasma of growing pigs.

Effects of growth hormone and insulin-like growth factor I on rabbit proximal convoluted tubule transport.

Urinary growth hormone and insulin-like growth factor I. Effects of growth-hormone injection schedule.

Characterization of trenbolone acetate and estradiol metabolite excretion profiles in implanted steers.

Destripeptide insulin-like growth factor-I in milk from bovine somatotropin-treated cows.

Comparative effects of epidermal growth factor, an insulin-glucagon combination, and a hepatocyte growth factor preparation on epidermal growth factor receptors.

Influence of growth hormone and insulin-like growth factor-I on kidney function and kidney growth.