The Effects of Gonadal Hormones on Hyperossification in the Domestic Fowl1

The Effects of Gonadal Hormones on Hyperossification in the Domestic Fowl1 GEORGE J. MTJLKEY AND EDWARD F. GODFREY Department of Poultry Science, University 0} Maryland, College Park, Maryland 20742 (Received for publication March 5, 1969)

I

T HAS been adequately demonstrated that estrogens promote medullary proliferation of bone. Martin et al. (1949) demonstrated that natural or synthetic estrogens given repeatedly to adult pigeons produces osteosclerosis of the femur. Keyes and Potter (1934) noted that female pigeons have solid bones and males have marrow-filled bones. They reported that the cyclic ossification of the red marrow of the long bones of the leg of female pigeons was coincident with the maturation of ovarian follicles and suggested that the production of ovaiian estrogen accounts for the sex difference. Benoit et al. (1942) demonstrated a calcification of the marrow and pneumatic cavities of long bones in pigeons and ducks using estradiol dipropionate. Quist et al. (1950) by administering 2.0 mg. doses of estradiol benzoate increased the formation of endosteal bone in mice. Sutro (1940) demonstrated osteosclerosis of the lower end of the femur and the upper end of the tibia using estradiol benzoate on immature mice. Rubinstein et al. (1939) administered 1.0 mg. testosterone daily from the 26th to the 80th day of life of rats. The results obtained indicated a decrease in body weight and length in rats. Gardner (1945) reported that steroid hormones sped calcification of the epiphysis and produced increased formation and 1

Scientific Article No. A-1504 Contribution No. 4154 of the Maryland Agricultural Experiment Station. Department of Poultry Science.

strength of medullary bone in mice. Adequate or prolonged administration of FSH or perhaps LH of the anterior pituitary resulted in an increase of serum calcium in normal pigeons (Riddle and Dotti, 1936). Landauer et al. (1941) found that the administration of estradiol benzoate to Mallard and Pekin ducks produced an extensive rise in serum calcium along with hyperossification of the long bones. Avery et al. (1940) by injecting estrone into immature pullets increased blood calcium level by 64 percent. It seems that both estrogens and androgens are necessary for maximum hyperossification of bone in domestic fowl (Godfrey and Jaap, 1951). The following work was designed to investigate the possibility of a synergistic relationship between diethylstilbestrol and testosterone propionate on growth inhibition of the tarsometatarsus. These hormones were chosen because previous research has indicated that these particular substances are quite effective in producing changes in bone ossification. EXPERIMENTAL

In this study a mating between Rhode Island Red males X Barred Plymouth Rock females produced the autosexing experimental birds. The inherent advantage of this crossbred is that the sex of the chick can be determined at one day of age with 100 percent accuracy. The birds were weighed at three weeks of age and paired on the basis of weight

1414

1415

GONADAL HORMONES ON HYPEROSSIFICATION

and sex; thus, each bird to be treated had an individual control. This procedure was followed for every trial except number four in which a factorial design was used with the birds being assigned at random to a treatment at three weeks of age. Paired observation analysis was used in trials one, two, three, and five while an analysis of variance was used for the factorial design of trial four. Three treatments were used in trial one, with the treatments being testosterone propionate (TP), diethylstilbestrol (DES) and DES plus TP. Each treatment was applied to females and males; therefore, six groups were needed. In subsequent trials, male subjects were excluded because more promising results were obtained from the female subjects. In trial two the treatments remained the same as in trial one. In trial three DES and DESTP were the two treatments employed. Trial four, a factorial design, had four treatments; DES, TP, DES-TP and a control group. Each was fed two levels of vitamin D 3 in the diet (330 I.C.U./kg. of feed and 130 I.C.U./kg. of feed). The composition of the ration fed in all trials, with the exception of trial four in which the level of vitamin D3 was altered, is presented in Table 1. In trial five the combined treatment of DES and TP was the only treatment studied. The estrogen treatment was administered as a single dose at three weeks of age in the form of a pellet containing 12 mg. of diethylstilbestrol. The pellet was implanted beneath the skin at the base of the skull with a pellet gun. The androgen, testosterone propionate, was dissolved in corn oil and was injected subcutaneously in the breast region weekly from week three to week seven in trials three, four and five. The birds were given 1.0 mg. of TP/week for weeks three and four in all experiments. The dose level was

TABLE 1.—Composition of diet Ingredients Ground yellow corn Stabilized fat Fish meal, 60% protein Soybean meal, 50% protein Alfalfa meal, 17% protein Iodized salt Delamix with zinc DL methionine Dicalcium phosphate Limestone1 Antibiotic B.H.T., 25% Vitamin mix2 Vitamin B12, 10 mg./454 gm. Vitamin A, 10,000 A/gm. Vitamin D 3 , 3,000 D 3 /gm.

% of Ration 57.675 3.000 2.500 31.000 2.500 0.500 0.050 0.025 2.000 0.500 0.050 0.050 0.075 0.015 0.035 0.025

1 To contain 10 gm. aureomycin or terramycin or 5 gms. of penicillin in combination with 15 gms. streptomycin. 2 To contain 2 gm. riboflavin, 4 gm. pantothenic acid, 9 gm. niacin, 100-110 gm. choline chloride/454 gms.

raised to 1.5 mg. of TP/week for the remaining weeks of the experimental period. The dose level of TP was raised in the latter part of the experimental period because of the increase in body weight of the subjects. Body weight and the length of the tarsometatarsus were recorded at nine weeks of age in trials one and two and at eight weeks of age in trials three through five. Ether extracts of dry carcass were determined for the diethylstilbestrol treated females of trial two (Association of Official Agricultural Chemists, 1965). Serum values for calcium and inorganic phosphorous (orthophosphate) were recorded in trial five for weeks four through eight. The laboratory analysis of calcium and phosphorous was carried out on an Auto Analyzer (Technicon Instruments Corporation). RESULTS AND DISCUSSION

Trial 1. The DES treatment produced a significant increase in body weight in the male group ( P = <.05), but not in the

1416

G. J . MULKEY AND E . F . GODFREY

female group. (Table 2). The average body weight for the DES treated males was 1107.8 grams compared with 1000.7 for the controls. The TP treatment did not produce any significant change in body weight for either sex. The combined treatment of DES plus TP increased the body weight of the males ( P = < . 0 5 ) , but not of the females. The increase in body weight noted above was probably due to the increased deposition of body fat. Lorenz (1945) reported that estrogen increases the deposits of abdominal and liver fat with lesser increases in other organs. Two of the six treatments produced appreciable changes in length of the tarsometatarsus (shank) (Table 2). The DES-TP treatment produced a highly significant decrease ( P = < . 0 1 ) in shank

length of the female birds. The average length for the treated group was 8.49 centimeters and it was 8.82 centimeters for the control group. The DES treatment produced a significant decrease (P = <.05) in shank length of the male birds with the average being 9.37 centimeters for the treated group compared with 9.78 centimeters for the control group. Trial 2. In trial two the DES treatment resulted in a highly significant ( P = <.01) increase in body weight. The average body weight for the treated and control groups was 1088.9 grams and 992.7 grams respectively, but an extreme amount of variability was observed in the control group. No significant change in body weight was found in the TP or DES-TP group. Ether extract values expressed as a per-

TABLE 2.—Body weights and tarsomelatarsus lengths of trials 1 and 2 Length of tarsometa tarsus (cms.)

No pairs

9 wk. wt. (gms.)

F

15

857.8 819.9

>

10

8.73 8.94

<

20

TP Control

F

14

842.4 799.5

>

10

8.73 8.91

>

10

DES-TP Control

F

14

818.7 792.2

<

10

8.49 8.82

<

01

DES Control

M

12

1,107.8 1,000.7

<

05

9.37 9.78

< .05

TP Control

M

13

988.4 1,001.5

<

40

9.71 9.65

> .30

DES-TP Control

M

11

1,054.3 982.9

< .05

9.06 9.56

< .10

DES Control

F

2

18

1,088.9 992.7

< .01

9.38 9.64

< .01

TP Control

F

2

18

1,052.2 1,048.8

< .50

9.68 9.60

> .40

DES-TP Control

F

2

18

1,076.9 1,069.1

< .50

9.17 9.76

< .01

Treatment

Sex

DES Control

Trial

P = Probability. DES = Diethylstilbestrol. TP=Testosterone Propionate.

P

P

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GONADAL H O R M O N E S ON HYPEROSSIFICATION

TABLE 3.—Body weights and tarsometatarsus lengths of trials 3 and 5 Length of tarsometatarsus (cms.)

Treatment

Sex

Trial

No pairs

! wk. wt. (gms.)

DES Control

F

3

18

900.3 769.3

<.01

8.85 9.05

<.05

DES-TP Control

F

3

18

867.6 802.5

<.05

8.72 9.24

<.01

DES-TP Control

F

5

47

803.4 838.5

<.05

8.43 8.81

<.01

centage extract of dry samples of whole, ground carcasses were recorded for the D E S treated birds. T h e average value obtained for the D E S treated birds was 34.35 percent as compared with 22.19 percent for the controls. On a wet basis, the respective values were 14.54 and 8.49 percent. T h e percent moisture for the D E S treated birds was 57.59 percent and it was 61.27 percent for the controls. T h e combined value of moisture and ether extract for the treated group was 72.16 percent while the value was 69.76 percent for the control group. I t appeared t h a t the increased fat deposition was a t the expense of moisture. Bird (1946) reported t h a t estrogen tends to decrease the metabolism of chickens which in part explains the fattening effect. I n trial two significant decreases in shank length were found to be produced b y both the D E S treatment and the combined t r e a t m e n t of D E S - T P . T h e average value for the D E S treated birds was 9.38 centimeters as compared with 9.64 centimeters for the controls. For the combined treatment the average value was 9.17 centimeters compared with 9.76 centimeters for their controls. I n this trial the combined t r e a t m e n t was more effective t h a n the D E S t r e a t m e n t in the reduction of shank length. T h e average decrease in shank length of the D E S treated birds over the control group was 0.26 centi-

meters and a comparative value for the D E S - T P treated birds was 0.59 centimeters. T h e above results indicate t h a t a definite synergistic relationship exists between D E S and T P as far as reduction of shank length is concerned. Trial 3. I n trial three only two treatments were used, namely D E S and D E S plus T P . Both treatments produced significant increases in body weight with the D E S treatment producing a greater inincrease than the combined t r e a t m e n t (Table 3). Significant differences in shank length were also found for both groups with t h e D E S - T P treatment producing a greater decrease t h a n the D E S treatment. T h e combined treatment of D E S - T P had an average value of 8.72 centimeters as compared with 9.24 centimeters for the control group and the D E S treated birds had an average value of 8.85 centimeters as compared with 9.05 centimeters for the controls. Trial 4. A factorial arrangement of treatments (4X2) was employed in trial four to determine the effect of four hormone treatments over two levels of vitamin D3 on growth of the tarsometatarsus and on b o d y weight. An analysis of variance indicated t h a t there were no differences in body weight or shank length between levels of vitamin T>%. A significant difference (P = < .01) in shank length

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G. J . MUXKEY AND E . F . GODFREY

TABLE 4.—-Treatment means for body weig

and tarsometatarsus lengths of trial 4 Treatment

Sex No.

DES+330 I.C.U. Ds/Kg. DES + 130 I.C.U. Ds/Kg. TP+330I.C.U. Ds/Kg. TP+130 I.C.U. Ds/Kg. DES-TP+330 I.C.U. Ds/Kg. DES-TP+130 I.C.U. Ds/Kg. Cont+330 I.C.U. Ds/Kg. Cont+130 I.C.U. Ds/Kg.

F F F V F F F F

8 8 8 8 8 8 8 8

Length of 8wk. wt. tarsometatarsus (gms.) (cms.) 928 a 902 a 854 a 860 a 869 a 879 a 858 a 912 a

8.93 8.99 8.94 8.86 8.49 8.65 9.02 9.01

a a a a b a,b a a

Means in any vertical column not followed by the same letter are significantly different (P=<-05) according to Duncan's multiple range test.

was found for hormone treatments, but the hormone treatments did not produce any significant changes in body weight. A Duncan's multiple range test (1955) indicated that the difference in shank length was due to the hormone treatment of DES-TP (Table 4). Trial 5. In trial five, only one treatment, DES plus TP; was employed. As in previous trials, the combined treatment produced a highly significant decrease (P = <.01) in shank length. Also, the DES-TP treatment produced a significantly lower (P = < .05) body weight than did the control group (Table 3). Serum calcium and inorganic phosphorus (orthophosphate) were measured for weeks four through eight. At week four, the serum calcium level of the treated birds was greater than that of the control birds, but the difference had dissipated by week five. The serum calcium

level, seven days following the implantation of the DES pellet and the first injection of TP, was 18.87 mg./lOO ml. of serum and the control group had a value of 8.21 mg./lOO ml. of serum. The calcium levels for weeks five through eight were similar for both the treated and the control groups with the values ranging from 8.63 to 9.58 mg./lOO ml. of serum (Table 5) In trial five the DES-TP treatment produced significant decreases in inorganic phosphorus at weeks four, six and seven (Table 5). At week four the DES-TP birds had an average value of 4.97 mg./ 100 ml. of serum as compared with a value of 6.51 mg./lOO ml. of serum for the controls. Similar comparisons for weeks five through eight can be found in Table 5. It appeared that the control group reached a maximum level of inorganic phosphorus at week six. The level remained constant for weeks six, seven and eight with values of 7.90, 7.53 and 7.51 mg./lOO ml. of serum respectively. A definite synergistic relationship was found between DES and TP for reduction of shank length. It has been indicated that estrogens increase ossification of bones, particularly endosteal bone of domestic fowl. Landauer and Zondek (1944) suggested that estrogens may produce localized vascular reactions in the interior of long bones. The synergistic relationship between DES and TP on

TABLE 5.—Serum calcium (Ca) and inorganic phosphorus (P) levels for weeks five through eight of female birds treated with DES-TP in trial 5 Weeks

T, . treatment

No ^

Element ( m g . / l 0 0 ml.)

DES-TP Control

26

Ca

18.87** 8.21

9.58 8.92

DES-TP Control

26

P

4 97** 6^51

6.68 6.90

** = P < . 0 1 . * =P<.05.

4

5

5

7

8

8.73 8.63

9.10 8.63

9.20 9.14

6.89* 7.90

7.05* 7.53

6.95 7.51

GONADAL HORMONES ON HYPEROSSIFICATION

bone growth can probably be attributed to the physiological changes in mineral metabolism produced by the androgen, TP. The results, obtained in these trials, indicated that the maximum decrease in length of the shank could only be produced when both hormones, DES and TP, were present. The immediate rise in serum calcium of the DES-TP treated birds of trial five was due to the DES pellet. In most cases an increase in serum calcium precedes hyperossification, but hyperossification has been produced in pigeons (Bloom et ah, 1942) without elevating the calcium levels. It would seem logical to suspect an increased mobilization of the elements which are directly concerned with formation of new endosteal bone and these changes may be depicted in blood levels of these elements; chiefly calcium and phosphorous. SUMMARY

A combined treatment of diethylstilbestrol (DES) and testosterone propionate (TP) produced significant decreases in shank length for the five trials conducted. The DES treatment also produced some significant decreases in shank length, but the combined treatment was about twice as effective as the DES treatment in reducing the shank length. A definite synergistic relationship was found between DES and TP in causing reduction of shank length. The reduction in bone length occurred in most trials without appreciable changes in body weight. Serum calcium and phosphorus (orthophosphate) studies were included in trial five for the DES-TP treated birds to suggest a basis for the decrease in shank length from the standpoint of serum levels of these elements. Two prominent changes were observed; an increase in calcium level after the first week of treatment and

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a decrease in inorganic phosphorus which continued through the experimental period. The rise in calcium was attributed to the DES pellet which was administered only once, but it was difficult to delineate the basic reasons for the decrease in inorganic phosphorus. The lag in decrease of calcium after an initial increase and the reduction of inorganic phosphorus appeared to be predictors of increased endosteal bone formation in birds treated with DES-TP The increases in fat deposition produced by the DES pellet were in agreement with the work of many researchers who have reported the fattening effect of estrogenic compounds. REFERENCES Association of Official Agricultural Chemists, 1965. Methods of Analysis, Assoc. Off. Agr. Chem., Washington, D. C , p. 346. Avery, T. B., H. M. Scott and R. M. Conrad, 1940. Blood calcium levels of the fowl following injections of theelin. Endocrinology, 27: 83-86. Benoit, J., J. Clavert and R. Grangaud, 1942. Action de la folliculine sur le metabolisme du calcium chez les oiseaux. 1. Ossification folliculine. Variations quantitatives des principaux constituants chimiques de l'os provoguees chez le canard et le pigeon par l'hormone sexuelle femelle. Bull. Soc. Chim. Biol. 24: 1311-1322. Bird, S., 1946. The influence of injested estrogens on feed intake, metabolic, rate and lipemia in male fowl. Endocrinology, 39: 149-154. Bloom, M. A., F. C. McLean and W. Bloom, 1942. Calcification and ossification. The formation of medullary bone in male and castrate pigeons under the influence of sex hormones. Anat. Rec. 83:99-120. Duncan, B. D., 1955. Multiple range and multiple F tests. Biometrics, 11: 1-42. Gardner, W. U., 1945. Influence on sex hormones on bone. Recent research on osseous tissues. Yale Sci. Mag. 20(3): 7-8, 24, 26, 28. Godfrey, E. F., and R. G. Jaap, 1951. Some effects of sex hormones on bone development and growth. Poultry Sci. 30: 915. Keyes, P., and T. S. Potter, 1934. Physiological marrow ossification in female pigeons. Anat. Rec. 60: 377-379.

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Landauer, W. C , C. A. Pfeiffer, W. U. Gardner and J. C. Shaw, 1941. Blood serum and skeletal changes in two breeds of ducks receiving estrogens. Endocrinology, 28: 458-464. Landauer, W. C , and B. Zondek, 1944. Observations on the structure of bone in estrogen treated cocks and drakes. Amer. J. Path. 20: 179-209. Lorenz, F. W., 1945. The influence of diethylstilbestrol on fat deposition and meat quality in chickens. Poultry Sci. 24: 128-134. Martin, E., C. Pfister and G. Riolten, 1949. Oestrogens et os. Bull. Schwerz. Akad. Med. Wiss. 5(4) 233-243.

Quist, M. R., A. M. Budy and F. C. McLean, 1950. Endosteal bone formation in estrogen treated mice. J. Bone Joint Surgery, 30A(1): 143-162. Riddle, O., and L. B. Dotti, 1936. Blood calcium in relation to anterior pituitary and sex hormones. Science, 84: 557-559. Rubinstein, H. S., A. A. Kurland and M. Goodwin, 1939. The somatic growth depressing effect of testosterone propionate. Endocrinology, 25: 724728. Sutro, C. J., 1940. Effects of subcutaneous injection of estrogen upon skeleton in immature mice. Proc. Soc. Exp. Biol. Med., 44: 151-154.

Effects of Prednisolone on Growth and Nucleic Acid Metabolism of Pekin Ducks G. D. BOTTOMS, M. D. MCCRACKEN AND W. W. CARLTON Department of Veterinary Physiology and Pharmacology, School of Veterinary Science and Medicine, Purdue University, Lafayette, Indiana 47907 (Received for publication March 7, 1969)

INTRODUCTION

T

HE adrenal corticosteroids are being used extensively for many therapeutic purposes. Although many gross effects are well known, their primary site or sites of action have not been elucidated. The results of numerous in vivo and in vitro experiments in recent years indicate rapid changes in nucleic acid metabolism after corticosteroid treatment. It has been established that corticosteroids stimulate protein synthesis within the liver (Weber et al., 1965), but depress protein synthesis and increase protein catabolism in extrahepatic tissues (Bethell et al., 1965). Effects of Cortisol treatment on increased RNA synthesis in rat liver was observed within 2-3 hours, followed 4-10 hours by increased protein synthesis (Lang and Sekeris, 1964). In Published as Paper No. 3407, Purdue University, Agricultural Experiment Station, Lafayette, Indiana 47907.

tissues known to involute in response to glucocorticoids, e.g., the spleen and thymus, the synthesis of RNA was depressed (Feigelson, 1964). The rate of DNA synthesis was depressed by glucocorticoids to a greater degree than RNA synthesis in mouse fibroblast growing in vitro (Pratt and Aronow, 1966). Because adrenal physiology of birds resembles in certain of its aspects that of mammals and as chicks are more sensitive than rats to the growth inhibition effect of Cortisol (Bellamy and Leonard, 1965), it seemed appropriate to examine more closely glucocorticoid actions on growth inhibition and the rate of nucleotide incorporation into nucleic acids in different tissues of the Pekin duck. The following studies were made: (1) the effects of prednisolone (11/3, 17a, 21-trihydroxy-A 1,4 pregnadien-3,20 dione) in the diet on weight gains; and (2) the effects of single and repeated injections of prednisolone on the incorporation of labeled nucleotides