Liveweight Gains, Blood Levels of Metabolites, Proteins and Hormones Following Implantation of Anabolic Agents in Steers

Br. vet. J. (1977), 133, 62

LIVEWEIGHT GAINS, BLOOD LEVELS OF METABOLITES, PROTEINS AND HORMONES FOLLOWING IMPLANTATION OF ANABOLIC AGENTS IN STEERS By R. J.

HEITZMAN*,

K.

H. CHAN* AND

I.

C. HARTt

*Agricultural Research Council, Institute for Research on Animal Diseases, Compton, Newbury, Berkshire and t National Institute for Research in Dairying, Shinfield, Reading, Berkshire SUMMARY

Eleven steers aged 12- I 4- months were divided into groups of three based on age, breed and liveweight. One animal in each group was implanted with trenbolone acetate, another with trenbolone acetate + oestradiol- I 7fl, and the third animal served as control. The mean liveweight gains throughout -a 9 week period were 54-·3 kg, 64-·3 kg and 77.2 kg for control, trenbolone acetate and trenbolone acetate + oestradiol treated groups respectively. The mean values for weight gains of the treated animals were significantly higher (P < 0 ·05) than the control values. The improvement in feed conversion in the treated animals was significant (P < 0 ·00 I) in the animals receiving trenbolone acetate + oestradiol. No changes were seen in the plasma concentrations of growth hormone, prolactin and insulin. There was, however, a significant decrease in the concentration of total thyroxine in the plasma of the treated animals. The decrease was greatest in steers implanted with trenbolone acetate + oestradiol-I7fl. The levels of serum urea and albumin were decreased in the steroid treated animals, whereas there were no alterations in the concentrations of plasma glucose, or serum total protein, magnesium, calcium, inorganic phosphorus, iron, copper, sodium and potassium. Some modifications of behaviour were noted in steers implanted with the combined steroid preparation. INTRODUCTION

The growth rate of bulls, castrate males and heifers fed on a uniform diet at a standard rate per kg liveweight confirms that food conversion efficiency and weight gain is greatest in the bull and lowest in the female. These differences may be attributable to differing hormonal status between the sexes; the par-

ANABOLIC AGENTS IN STEERS

ticular hormonal profile of the bull conferring the advantage of higher feed conversion efficiency and weight gain. Nevertheless, Grandadam et al. (1973) have shown that the weight gain of bulls can be further improved by steroid administration. The aim of this study was to investigate the effects in steers of the administration of an anabolic steroid, alone or in combination with an oestrogen, in an attempt to determine a hormonal status associated with improved growth rate. Anabolic agents are usually either androgens, which have properties and molecular structure related to testosterone, or oestrogenic substances. This paper demonstrates that administration of either androgenic steroids or combined preparations of androgens and oestrogens results in significant alteration in rates of growth. The results emphasize that the choice of appropriate anabolic agents is related to the sex hormone status of the treated animal. H eitzman & Chan (1974) reported that following administration of an androgenic steroid to heifers there was an improvement in weight gain and feed conversion. The levels of urea and albumin in plasma were decreased during the second month after implantation. At the same time no changes were observed in the concentrations of glucose, insulin, several minerals and total protein. A further aim of the present study was to determine the concentration of these metabolites in plasma or serum in steers following implantation of an androgenic steroid alone or in combination with an oestrogen. MATERIALS AND METHODS

Eleven steers were randomly allocated to three groups on the basis of age, breed and weight. The ages ranged from 12-14 months. The groups and steroid treatment are shown in Table I. Each animal was tied in an individual stall and fed a measured ration of concentrate and hay. A single batch of concentrate was made up, sufficient for the whole experiment. It contained the same constituents as the ration for previous experiments. (Chan, Heitzman & Kitchenham, 1975)' Analysis showed a crude protein content of II'5% of wet weight matter. Similarly a single load of hay was purchased and this contained between 7'1 and 8'2% crude protein in wet weight matter. The steers were fed either 2 '70 kg concentrate or in the case of the two smallest steers (G68I and G690) 2 '27 kg concentrate and sufficient hay to provide the estimated protein requirement for an expected growth of 0'5 kg/day (National Research Council's table, U.S.A., 1970). The hay and concentrate diet was fed for at least eight weeks before implantation. Water was given ad libitum. The weights of the animals were recorded weekly for two weeks before implantation, 20 h before implantation and at weekly intervals for a further nine weeks after implantation. Blood samples were taken at weekly intervals from one week before implantation until nine weeks after implantation. The samples were taken from the jugular vein and always between 09 '00 hand IO '00 h. The concentration in plasma of glucose and in serum of urea-N, protein, albumin, calcium, magnesium, copper, iron, sodium, potassium and inorganic phosphorus were 3

BRITISH VETERINARY JOURNAL, 133, I

64-

TABLE I GROUPING, TREATMENT AND LIVEWEIGHTS OF STEERS BEFORE AND AFTER ADMINisTRATION OF ANABOLIC AGENTS LiveUieight (kg )

44 {G6 G646 G3 29 G66 { G6684 G665 8 { G9 G88 G359 0 { 06 9 0681

Breed

Treatment

At implantation

After 63 days

F F x F F x F F F x x

Control 'fA + OE 2 TA Control TA + OE 2 TA Control TA + OE 2 TA Control TA + OE 2

3°4. 8 30 7 .9 3 14 .3 263.5 263.5 260·3 355 .6 332 .4 34 2 .9 206·4 233 ·1

355.6 384. 1 377.8 323.8 339 ·7 3 2 7.0 408 .6 4 17. 2 405"4 259·3 305" 1

F

F

F F

H

H

H H

Weight gain (kg) 63 days after implantation

5° ·8 76 .2 63·5 60·3 76 .2 66·7 53.0 84 .8 62·5 52 .9 72·0

Each matched group is enclosed by parentheses. TA + OE 2 - trenbolone acetate (140 mg) + oestradiol-17.B (20 mg) (Revalor; RousselUelaf, France). TA-trenbolone acetate (300 mg) (Finaplix; Hoechst, U.K.) . The steroids were implanted subcutaneously as pellets at the base of the ear. F-Friesian F x H-Friesian x Hereford.

measured using auto-analytical methods described by Payne et al. (1970) and Manston & R owlands (1973). The radioimmunoassays for prolactin (Hart, 1973) and growth hormone (GH) (Hart, Flux, Andrews & McNeilly, 1975) have been described previously. Insulin was assayed by a double antibody technique using reagents purchased from Wellcome Reagents Ltd. and total thyroxine (T 4) was measured using antisera kindly supplied by Dr C. W. Burke (Radcliffe Infirmary, Oxford) and Dr T. C. Merrett and Miss L. Nye (St. Bartholomews Hospital, London). The behavioural traits of the individual steers were independently assessed by an experienced stockman at nine weeks after implantation. The data on growth rate and feed conversion was analyzed for significance using the t test. Other data was analyzed for significance using analysis of variance. Because of variation in concentrations among the groups prior to the experiment, the concentrations in each animal in each week after treatment were adjusted by subtracting its average concentration prior to treatment. Analysis of variance was performed on the adjusted data. RESULTS

The growth rates of the steers are shown in Fig. I. The animals receiving the combined implant of trenbolone acetate plus oestradiol-I 7,8 (R evalor) gained weight much faster than the control steers. At the end of the nine week period following implantation the mean increased weight gain was 22 ·9 kg, whereas in the steers implanted with the trenbolone acetate alone (Finaplix) the in-

ANABOLIC AGENTS IN STEERS

I 350

C' .x

..... .c:

325

C'

00; ~

~

300

:::i

275

Weeks from Finaplix impl anta ti on

Weeks from Revolor implantation

Fig, I, Growth curves for steers implanted with anabolic steroids, Each value represents the m ean liveweight for animals in each treatment group (Table I ), Key - -.- control, ---.--- 140 mg trenbolone acetate + 20 mg oestradiol-17,B, --- A--- 300 mg trenbolone acetate, The animals were implanted at time 0 weeks,

creased weight gain was 9.6 kg only. There were several periods in the growth curves where the control animals gained weight faster than the steers implanted with androgen alone. The steers receiving the combined implant were more efficient in terms of feed conversion than steers treated with androgen alone (Table II). The mean values of concentrations for glucose in the plasma, and urea-N, albumin and protein in the serum are shown in Fig. 2. There was a significant reduction (P < 0 '05) in the mean levels of urea and albumin in the serum of TA BL E II DAIL Y LI VE WEIGHT GAIN AND FEE D CONVERSION RA T IOS

(FCR )

IN STEERS

AND HEIFERS IMPLANT E D WITH STEROID PREPARATIONS

Animal Steer Steer Steer H eifer H eifer

Treatment

Mean daily live weight gain kg/day ± SEM

Control TA TA + OE 2 Control TA

0 ,862 ± 0 '033 1'021 ± 0'043" 1·225 ± 0 '020 b o'654 d 1'1 I I d

+

FeR

± SEM

± o·g 9'2 ± 0·7 c 7'4 ± O ' 2 b 13'gd 8 '5d

10'2

TA + OE 2 - trenbolone acetate (140 mg) oestradiol-17 ,8 (20 mg) (Revalor ; RousselUelaf, France), TA-trenbolone acetate (300 mg) , (Finaplix; Hoechst, U,K,), The steroids were implanted subcutaneously as pellets at the base of the ear. a,b Values significantly differe nt from control- a = P < 0'05, b = P < 0 '001, c Is not significant. d Values from H eitzman & Chan (1974) , SEM- standard error of the m ean,

66

BRITISH VETERINARY JOURNAL, 133, 1 Al bum i n

• ~30E

. I ~ 25

~4:~ . .....

65 . . ....•

'"

&.,

I

I I

1

I

I 5 Weeks 1

I 7

1

:3

I

1

1

I

.If,.'

..

'

I

1

70r t pr~te i n -~ ~~ . ~.'Il\.~

Glucose

4 -I

1

'~

•

'b~.o

·1 \ ......./ • 60

I 9

1

I

-I

..&

I

1

I

1

I

I

3 5 Weeks

I

I

7

9

Fig. 2. Changes in concentration of plasma/serum components following implantation of anabolic steroids in steers. Each value represents the mean of values for 3 or 4 animals in each group (Table I). ---.&--- trenbolone acetate (300 mg), -.- trenbolone acetate (140 mg) + oestradiol-17.B (20 mg), 0 - control. Glucose was measured in plasma, urea, albumin and protein were measured in serum.

,

40

E

...... Cl

Q.

80

GH

30

70

20

60

10

50

.~

T4

~

"-/ -",,/

'/"\ .~

40

0 E

.

1&

j"''' \ / I~

12 E

...... ::)

:I-


8 4

QI Q.

Cl

c

90 60

t

Prolactin . ..... 6

i

30

Insulin

0

0 -1

3

5

Weeks

7

9

-1

3

5

7

9

WEEKS

Fig. 3. Changes in the concentrations of hormones in plasma of steers treated with anabolic steroids. The key to the animals and treatments is the same as for Fig. 2 .

ANABOLIC AGENTS IN STEERS

steers given either the combined or androgenic implant. The individual values for urea were significantly lower (P < 0 '05) for both treatment groups compared with controls at weeks I to 7 and for albumin at weeks 5, 8 and 9. No significant differences were observed between treatments in the concentrations of glucose, total protein, magnesium, calcium, inorganic phosphorus, iron, copper, sodium and potassium. The average concentrations of plasma GH, prolactin, insulin and total T4 are shown (Fig. 3) for the three groups of steers before and after implantation. No significant (P > o' I) changes in plasma GH or insulin were observed either after implantation or between the three different treatments. A significantly (P < 0 '02) higher concentration of prolactin was found in the trenbolone acetate treated animals, but the level of this hormone was also high before implantation. Implantation resulted in a substantial fall in circulating total T4 (P < 0'01). Further analysis of the data showed that the combined implant caused a greater reduction in total T 4 (P < 0'0 I) than trenbolone acetate alone (P < 0 '05). No differences were observed in patterns of behaviour between control animals and those implanted with, the androgen. However, the animals implanted with the combined preparation showed some differences in behaviour, so that nine weeks after implantation they had raised tail heads and protruding eyes. When tied they tended to have a more nervous disposition and shied at being approached or touched. There was a distinct tendency to aggressiveness with lowering of the head. When they were led outside, they were more difficult to handle and showed increased aggressiveness adopting a 'bullish' attitude with head down and a tendency to butt. They had an unnatural nervous and jumpy gait, rather than normal forward progress and they carried their tails in the air. DISCUSSION

The influence of sex hormones on growth in cattle The growth rate of cattle appears to be related to the sexual status of the animal. Bulls grow faster than steers, and steers grow faster than heifers. Because bulls have the highest circulating levels of testosterone, it was thought that raising the circulating level of androgens in steers and cows might enhance their growth. In part this was true and the administration of androgens as anabolic steroids greatly increased the weight gain of h eifers (Best, 1972; H eitzman & Chan, 1974; Table II) and cull cows (Beranger & Malterre, 1968). However, the growth response of steers administered androgenic steroids alone has been variable. In some early studies Andrews et al. ( 1950, 1954) reported that steers implanted with testosterone or testosterone propionate failed to grow faster than controls. Again in a study where testosterone was injected daily, steers grew slightly faster than controls but heifers showed marked increases in liveweight gain (Burris et al., 1952). Some recent studies have recorded that trenbolone acetate did not increase the liveweight gain of bulls or cryptorchid bulls compared with controls (MacLeod, Macdearmid & White, 1975). Similar

68

BRITISH VETERINARY JOURNAL, 133,

I

observations were made in the present study i.e. androgens when administered alone to steers did not elicit the maximum growth response. This poses the question why did androgens result in higher growth rates in heifers and cows compared with steers? A possible reason was that both androgenic and oestrogenic steroids have important roles in the control of growth so that androgens may potentiate the action of oestrogens or vice versa. The concentration of plasma oestrogens in the female (Glencross et at., 1973; Dobson & Dean, 1974) was higher than that of steers (Mongkonpunya et at., 1974), while the level in bulls does not seem to have been accurately determined. On the other hand th e concentration of testosterone was found to be higher in steers than heifers but several times lower than the level found in bull plasma (Gortsema et at., 1974; Shemesh & Hansel, 1974). Because of the relatively high endogenous concentrations of oestrogens in plasma of heifers and cows, the administration of an androgenic steroid probably created a favourable hormonal profile associated with increased growth. The response in steers following androgen administration alone may be limited by the low endogenous level of oestrogen. Thus growth in steers which possess low endogenous oestrogen levels is best improved by addition of exogenous oestrogen. Several other studies have shown that growth rate in steers was greatly increased following oestrogen treatment (Perry et at., 1955; O'Mary et at., 1956; Kilkenny & Sutherland, 1970; Hale & Ray, 1973). The administration of an oestrogen plus an androgen was probably sufficient to create a hormonal status similar to that of a bull. Thus as in previous studies (Kilkenny & Sutherland, 1970; Grandadam et at., 1973; Koers et at., 1974) combined implants of an androgen and oestrogen were shown to be effective in increasing liveweight gain.

Hormonal interrelationships Although a significant amount of work has been carried out to investigate the effects of other growth promoting agents (e.g. diethyl stilboestrol, DES; melengesterol acetate, MGA) on circulation levels of various other hormones (Hafs, Purchas, & Pearson, 197 I) little or no work has been carried out using anabolic steroids which are androgens. Our results with steers indicated that the anabolic effect of trenbolone acetate, either alone or in combination with oestradiol- I 7,8 is not mediated by changes in the blood concentrations of GH, prolactin or insulin. The findings for insulin are similar to those for heifers (Heitzman & Chan, 1974) and contrary to the situation resulting from DES treatment where a positive correlation between increased insulin and increased weight gain has been found (H afs, Purchas & Pearson, 1971 ). Because of the wide daily variation in concentrations of prolactin and growth hormone in the bovine any alterations in concentrations following anabolic steroids may well be obscured (Hart, Bines, Balch & Cowie, 1975). The findings for total T 4 are of much interest as a considerable amount of work has been carried out in ruminants to determine the effect of thyroid depressant drugs on bodyweight (BIaxter, Reineke, Crompton & Petersen, 1949; Draper, Haynes, Falconer & Lamming, 1969). Karg ( 1966) reported for example that thyroid depressing drugs resulted in increases in liveweight

ANABOLIC AGENTS IN STEERS

69

gain. Interpretation of the T 4 data in this experiment is hampered by the fact that the radioimmunoassay measures both free T 4 and that T 4 which was originally bound to plasma proteins (e.g, thyroxine-binding globulin, TBG; thyroxine-binding prealbumin, TBPA). Concentrations of both of these binding proteins have been shown to alter following the administration of androgens (Braverman et al., 1968; Barbosa, Seal & Doe, 1971; Kley et at., 1973) and oestrogens (Musa, Doe & Seal, 1967). Because of the problems encountered in measuring the unbound hormone (Burke & Eastman, 1974) little is known of the effect of anabolic steroids on free T 4 in plasma. Since it is generally thought that thyroid hormone action is mediated by the non-protein bound moiety in blood the results must still be regarded with some caution. Thus if free T 4 is the only form in which the hormone can exert a physiological action it seems unlikely that the anabolic effects of trenbolone acetate, either alone or in combination with oestradiol-17,B, are mediated by changes in circulating T 4.

Metabolites The alterations in concentrations of metabolites in blood of steers following administration of the androgen and the combined implant were similar to the results reported for heifers treated with the androgen alone (Heitzman & Chan, 1974). The conoentration of urea was decreased in both treated groups compared with control. This suggested that anabolic agents change nitrogen metabolism, usually with an observed increase in nitrogen retention. Anabolic steroids have widely differing effects on plasma albumin a~d globulins (Kruskemper, 1968) and probably the lower albumin concentrations observed following implantation were of only minor importance for growth. The concentrations of several other biochemically important blood substances remained unchanged. Residues The steers were slaughtered at nine weeks after implantation and samples of tissues analyzed for residues of the anabolic steroid. The concentration of trenbolone in the treated steers wa.s 0 '~-0'3 parts/ l 0 9 in liver, muscle, fat and blood and < 0' 1 parts/l0 9 in kidney. (Heitzman & Harwood, unpublished data) . The plasma level of oe.stradiol.r7,B in steers receiving Revalor was < 0 '03 parts/l0 9 at slaughter. Clearly residues were present at the time of slaughter but they were extremely low. Alterations in behaviour patterns of steers No alterations in behaviour had been seen in heifers treated with androgen and similar re.sults were observed in the androgen implanted steers. There were. however, some changes in the behaviour of the steers implanted with the combined agents. Both male and female characteristics were accentuated, e.g. aggressiveness and rai.sed tail head. However, the animals did not present a management problem, and at no time could the steers be classed as dangerous.

BRITISH VETERINARY JOURNAL, 133, I ACKNOWLEDGEMENTS

The authors wish to thank Mr N. Elcock for his experienced help with the comments on behaviour of the animals. They also thank Mrs M. Wheeler and Mr F. Matthews for looking after the animals, Mr R. Manston and Mr M. Vagg for carrying out the measurement of certain metabolites in the blood samples and Mrs K. A. M. Darby and Mr A. D . Simmonds for technical assistance with the hormone assays. The implants of Finaplix and Revalor were kindly donated by Hoechst UK, and Roussel-Uelaf, France respectively. REFERENCES

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December 1975)