Some Effects of Growth Hormone Preparations in Pullets and Mature Hens1

EFFECTS OF GROWTH HORMONE

Kratzer, F. H., D. E. Williams, B. Marshall and P. N. Davis, 1954. Some properties of the chick growth inhibitor in linseed oil meal. J. Nutrition, 52: 555-563. McGinnis, J., and H. L. Polis, 1946. Factors affecting the nutritive value of linseed meal for growing chicks. Poultry Sci. 25:408. McGinnis, J., 1948. Toxicity of linseed meal for chicks. Poultry Sci. 27: 141-145. Mani, K. V., N. Nikolaiczuk and W. A. Maw, 1949. Flaxseed mucilage and its effect on the feeding value of linseed oil meal in chick rations. Sci. Ag. 29: 86-90. Slinger, S. J., J. C. Small, I. Motzok and F. M. Marcellus, 1943. Linseed oil meal replacing meat meal in rations for growing chicks. Sci. Ag. 23: 732-740.

Some Effects of Growth Hormone Preparations in Pullets and Mature Hens 1 R. D. CARTER, R. N. RISNER 2 AND H. YACOWITZ Ohio Agricultural Experiment Station, Wooster, Ohio, and The Ohio State University, Columbus, Ohio (Received for publication May 10, 1955)

f~~* ROWTH hormone has been shown to ^ - * have a growth stimulating effect on mature rats and dogs by Evans and Long (1921); Putnam, Benedict and Tell (1929); Li and Evans (1944); Li and Evans (1948); Gaebler (1951). Very little work has been reported on the effects of growth hormone in poultry. Hulbe (1949) injected dwarf chicks daily with an extract of fresh beef pituitaries for a period of 6 months, in monthly increasing doses of 70-230 rat units. The injected birds showed a more prolonged increase in weight than the controls. Hsieh, Wang and Blumenthal (1952) found that growth hormone exerted a diabetogenic and 1 Presented at the 42nd Annual Meeting of the Poultry Science Association, University of British Columbia, Vancouver, B. C. August 1953. 2 Present address: Ohio Department of Agriculture, Division of Plant Industry, Feeds and Fertilizers Section, Reynoldsburg, Ohio.

growth-promoting action when injected into the developing chick embryo. No published reports could be found which describe the effects of purified growth hormone in laying hens. The purpose of this paper is to report the effects of injecting low levels of growth hormone in pullets and mature hens. EXPERIMENTAL Single Comb White Leghorn hens, White Leghorn-New Hampshire cross pullets and White Plymouth Rock pullets were used in the 3 trials conducted. The hens were 15 months of age, and the pullets 7 months of age. The hens were used in the first trial. Pullets were used in the second and third trials. All birds were housed in individual battery cages. The supply of growth hormone was limited, therefore, 4 to 8 birds were used for each treatment. Fresh water,

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Bethke, R. M., G. Bohstedt, H. L. Sassaman, D. C. Kennard and B. H. Edington, 1928. The comparative nutritive value of the proteins of linseed meal and cottonseed meal for different animals. J. Agr. Res. 36: 855-817. Kratzer, F. H., 1946. Treatment of linseed meal to improve its feeding value for chicks. Poultry Sci. 25:541-542. Kratzer, F . H., 1947. Effect of duration of water treatment on the nutritive value of linseed meal. Poultry Sci. 26: 90-91. Kratzer, F. H., 1948. Improvement of linseed meal for chick feeding by the addition of synthetic vitamins. Poultry Sci. 27: 236-238. Kratzer, F. H., 1949. Growth depression of turkey poults caused by linseed oil meal. Poultry Sci. 28:618-620.

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R. D. CARTER, R. N. RISNER AND H. YACOWITZ

RESULTS

Trial 1. Two groups of hens, with 4 hens per group, were used in this experiment. The control group received an inTABLE 1.—Effect

jection of 1 ml. of physiological saline per day for the first 10 days of the injection period and 2 ml. of saline the last 15 days of the injection period. The test group received an injection of 1 mg. of growth hormone for the first 10 days of the injection period. This level was found to be insufficient to produce a growth stimulation in the hens. During the last 15 days of the injection period the level of hormone was therefore increased to 2 mg. per day. The growth hormone preparation contained 40 percent growth hormone and 5 percent thyrotropic hormone (Armour's Growth Hormone Preparation—lot GH3). The hormone was prepared for injection by dissolving in physiological saline so that growth hormone concentration of 1 mg. per ml. of solution was obtained. A very slight gain in body weight was noted in the hormone injected hens during the injection period (Table 1). The average weight increase in the test group was 126 gm., while the control group averaged a gain of 32 gm. during the 25-day injection period. The injection of growth hormone caused

of growth hormone on body weight of pullets and mature hens Average body weight

Injection

Group

Trial 1 Hens Hens Trial 2 Pullets Pullets Pullets

Saline Bovine growth hormone§

10th day preinjection

At start of injections

10th day of injection period*

25th day of injection period f

10th day postinjection

gms.

gms.

gms.

gms.

gms.

1,896 1,864

1,906 1,826

1,890 1,900

1,938 (4-32) t 1,952(4-126)

1,890 1,934

2,230 2,210 2,370

2,261 2,252 2,470

2,326 2,175 2,582

2,329(4-68) 2,275(4-23) 2,727(4-257)

2,374 2,268 2,685

— Saline Bovine growth hormone || Porcine growth hormonelf

* One mg. growth hormone per day for 10 days. f Two mg. growth hormone per day from 10th to 25th day. t Figures in parentheses represent change in weight due to injections. § Lot No. GH-3. || Lot No. R 377237. If Lot No. R 491082 P.

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oyster shells and a 17 percent protein allmash laying ration were provided, ad libitum. Dim all-night lights were used. Individual body weights and egg production data were kept during a pre-injection period prior to the administration of the growth hormone preparations. At the end of the pre-injection period, daily intramuscular injections were started and continued for a period of 25 days for the first two trials and 32 days in the third trial. During the injection period, the birds were weighed daily and egg production recorded. At the end of the 25-day injection period, body weight and egg production records were again collected during a post-injection period. In trials 2 and 3 records were kept of feed consumption. The use of pre-injection and post-injection data in addition to the use of control groups enabled a more reliable measurement of the treatment effects.

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EFFECTS OF GROWTH HORMONE TABLE 2.—Effect

of growth hormone on egg production of pullets and mature hens Average egg production

Group

Injection

Pre-injection (20 days)

Injection period 1-10 days*

daysf

10-25

Post-Injection 1-10 days

10-20 days

Trial 1 Hens Hens

Saline Bovine growth hormone

% 55 54

% 67 48

% 55 38

% 60 28

%

Trial 2 Pullets

Saline

65.5

76.5

70.5

sot

Bovine growth hormone Porcine growth hormone

73. 84

58 73

47 58

53§ 45 45

70t 33§ 63 83

* One mg. growth hormone per day. f Two mg. growth hormone per day. t Average egg production of 4 pullets which continued to receive saline injections. § Average egg production of 4 pullets which were injected with 2 mg. porcine growth hormone per day during this period.

a marked decrease in egg production (Table 2). The decrease was noted 10 days after the injections were started and persisted until 10 days after the injections were stopped. During the second 10 day post-injection period the egg production of the hormone-treated group returned to normal. The control group remained at a relatively stable level of production throughout the experiment. Two of the 4 hens receiving growth hormone began molting during the injection period. One bird started to molt after 12 days of injection and the other after 24 days of injection. None of the control birds molted during the injection period. Trial 2. This experiment was conducted in an attempt to duplicate the results obtained in trial 1. White Leghorn-New Hampshire cross pullets were used. Pullets were included in this trial in order to reduce the possibility of natural molting. Four birds were used in each experimental group except for the control group which contained 8 birds. Two lots of growth hormone were available for this trial; lot R377237, which was extracted from beef pituitary and con-

tained a small amount of thyrotropic hormone as an impurity (0.11 Armour units per mg.), and lot R491082P, which was extracted from swine pituitary and contained a small amount of adrenocorticotropic hormone (ACTH) as an umpurity (0.03 Armour units per mg.). Three groups of pullets were used to test the two preparations. One group was injected with lot R377237 and another with lot R491082P. The control group received injections of saline. The dosage levels and injection periods were the same as those used in trial 1. The growth hormone preparation obtained from beef pituitary was quite soluble in physiological saline and was made up to a concentration of 1 mg. of growth hormone per ml. The growth hormone preparation from swine pituitary would not dissolve in the saline solution. It was necessary to increase the volume and to add sodium hydroxide to raise the pH to 9 before the hormone would go into solution. The final concentration was 1 mg. of growth hormone per 3 ml. of saline. Very little gain in body weight was noted when the bovine growth hormone

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Pullets Pullets

68 63

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R. D. CARTER, R. N. RISNER AND H. YACOWITZ

TABLE 3.—Effect

of growth hormone on feed

consumption of pullets Average feed consumption per kg. of body weight per day Group

Trial 2 Pullets Pullets Pullets

Injection

Saline Bovine growth hormone Porcine growth hormone

Preinjection Injection period period (10 days) (25 days) gms.

gms.

44.8 43.5 46.6

42.5 43.1 50.4

Trial 3. This trial was conducted in an attempt to learn the cause of the broodiness that occurred in trial 2, since broodiness had not been noted in any of the previous experiments in which other growth hormone preparations were used. There were several possible causes of the broodiness: the ACTH present as a contaminant; a combination of ACTH plus growth hormone; or some unknown hormone present in this preparation. The preparation did not contain prolactin, which has been shown to produce broodiness (Riddle, Bates and Lahr, 1935). Three groups of White Plymouth Rock pullets were started on the experiment. The control group, consisting of 7 birds, was injected daily with saline; the other 2 groups, consisting of 5 birds each, were injected with 2 levels of ACTH. One group received 0.08 units of ACTH per day. This was the level of ACTH present as a contaminant in the porcine growth hormone preparation which caused broodiness in trial 2. The third group received 0.32 units of ACTH per day. The ACTH injections were continued for a period of

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preparation was injected (Table 1). The pullets which were injected with bovine growth hormone gained only 23 gm. as compared to a 68 gm. gain in the control group. The pullets injected with the porcine growth hormone preparation showed a small growth response. The hormoneinjected group gained 257 gm. as compared to a 68 gm. gain in the control group. Bovine growth hormone had no effect on feed consumption, however, the pullets injected with porcine growth hormone consumed more feed than the controls (Table 3). The injection of both the bovine and porcine hormone preparations produced a marked decrease in egg production in pullets (Table 2). The decrease persisted for 10 days after the hormone injections were stopped, but egg production returned to normal during the second 10 day postinjection period. This confirmed the results of trial 1. The egg production of the control groups fluctuated somewhat during the trial, but the over-all level of production was higher than the hormoneinjected groups. The pullets which were injected with porcine growth hormone became broody during the trial. Broodiness was evidenced by clucking, ruffling of the feathers and setting. This broody condition was observed in 3 of the 4 pullets, while none of the pullets in the other groups showed

signs of broodiness. Two of the 3 broody pullets continued to lay, although their production was decreased. At the end of the initial 25 day injection period a reversal of treatments was started in order to confirm the observation that the porcine growth hormone preparation caused broodiness. Half of the original control group (4 birds) was injected with porcine growth hormone and the birds previously injected with hormone served as the controls. As in the previous injection period, 3 of the 4 birds injected with porcine growth hormone became broody and their egg production decreased. The injection of saline in the pullets which previously had received porcine growth hormone resulted in normal egg production and disappearance of broodiness.

EFFECTS OF GROWTH HORMONE TABLE 4.—Effect

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of adrenocorticotropic hormone and growth hormone, alone and in combination, on the body weight of pullets Average body weight

Injection

Trial 3 Saline 0.32 units of ACTH per day 0.08 units of ACTH per day

18th day pre-injection

At start of injections

21st day of injection period

gms.

gms.

gms.

2,851 2,746 2,876

2,867 2,770 2,916

2,882 (+15)* 2,764 ( - 6 ) 2,990 ( + 7 4 ) t

53rd day of injection period gms. 2,817 ( - 5 0 ) 2,828 (+58) 2,788 (-128)f

21 days. At the end of this period, 2 mg. of bovine growth hormone were injected along with the low level of ACTH. This combination was made up to duplicate the composition of the porcine growth hormone preparation which caused broodiness in trial 2. The higher level of ACTH was continued for the entire experiment. The two levels of ACTH had no appreciable effect on body weight (Table 4). When the growth hormone was injected in the group already receiving 0.08 units of ACTH, the body weight of the birds dropped rapidly for 3 weeks and then began to increase again. By the end of the experiment, the birds had regained 100 gm. of the weight which had been lost. The feed consumption of all groups, including the controls, decreased during the experimental period (Table 5). The group injected with 0.08 units of ACTH plus 2 mg. of bovine growth hormone showed the greatest drop in feed consumption. Ten days after bovine growth hormone was injected along with the ACTH, 4 of the 5 birds in the group developed watery droppings. These hens stopped eating but drank large quantities of water. By the end of the experiment, these birds had returned to normal feed consumption and the watery droppings had disappeared.

The percent egg production of the control group, when averaged by 10-day periods, fluctuated somewhat from one period to the next (Table 6). During the last week of the trial, 3 of the control birds went out of production. The group receiving 0.32 units of ACTH showed a constant rate of production throughout the trial. At the termination of the trial, all pullets in this group were still laying. In the group injected with 0.08 units of TABLE 5.—Effect of adrenocorticotropic hormone and growth hormone, alone and in combination, on the feed consumption of pullets Average feed consumption per kg. of body weight per day Injection

Trial 3 Saline

Preinjection

During Injection

gms.

gms.

45.4

40.4

0.32 units of ACTH per day

46.9

41.5

0.08 units of ACTH per day plus 2 mg. bovine growth hormone per day*

46.3

35.4

* Growth hormone injections started on 21st day of injection period.

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* Figures in parentheses represent change in body weight in grams when compared with body weight at the start of injection period. f Received 2 mg. of bovine growth hormone per day starting on the 21 st day.

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R. D. CARTER, R. N. RISNER AND H. YACOWITZ TABLE 6.—Effect

of adrenocorticotropic hormone and growth hormone on the egg production of pallets Average egg production Preinjection (18 days)

Injection

Trial 3 Saline 0.32 units of ACTH per day 0.08 units of ACTH per day; plus 2 mg. bovine growth hormone per day starting on 21st day.

TABLE 7.—Effect

0-21 days

22-31 days

32^1 days

%

%

%

%

%

77 79

58 72

53 68

51 72

40 68

77

66

24

12

20

42-53 days

of growth hormone plus 0.08 units of ACTH doubled the weight of the spleen. The oviduct weight was decreased by the combined injection. The decrease in oviduct weight was to be expected in view of the decreased egg production in this group. The ovaries in these birds appeared normal and contained several well developed yolks. There was little effect on the adrenal weight. Bates, Riddle and Miller (1940) developed a chick assay for ACTH in which increased adrenal weight served as the end point. A direct correlation was observed between ACTH dosage and adrenal weight. Since no increase in adrenal weight was observed in the experiment reported herein, the level of ACTH injected was apparently not very high in terms of the normal physiological secretion of birds. The decrease in the thyroid weight of the

of adrenocorticotropic hormone and growth hormone on the organ weight of White Plymouth Rock pullets Organ weights

Injections Spleen

Saline 0.32 units ACTH per day 0.08 units ACTH per day plus 2 mg. bovine growth hormone per day.

Oviduct

Liver

Pancreas

Right adrenal

Thyroid

mg. % body wt.* 88 81

mg.% body wt. 1,734 1,790

mg. % body wt. 1,822 1,565

mg. % body wt. 142 137

mg. % body wt. 3.1 2.9

mg. % body wt. 10.0 5.7

169

1,096

2,078

133

3.4

11.0

* Milligrams of organ per 100 grams of body weight.

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ACTH plus *2 mg. of bovine growth hormone, egg production began to decrease shortly after the growth hormone was injected. Four of the 5 pullets in this group went out of production within a week after the injection of growth hormone was started. One bird in the group continued to lay throughout the experimental period. One of the hens which went out of production began to lay again a week before the end of the experiment. About 13 days after the injection of bovine growth hormone was started, 4 of the 5 pullets started to molt. No molt was observed in the control group or in the group receiving 0.32 units of ACTH. No broodiness was observed in any of the groups during this experiment. The pullets in trial 3 were killed and the weights of various organs were determined (Table 7). The injection of 2 mg.

Injection period

EFFECTS OF GROWTH HORMONE

birds injected with 0.32 units of ACTH per day is puzzling in view of the lack of effect on the adrenal weight. The interpretation of the organ weight data must be considered tentative in view of the small numbers involved. DISCUSSION

The weight loss, watery droppings, and decreased food intake which appeared in trial 3 when a combination of bovine growth hormone and ACTH was injected, may be explained by the report of Reid (1953). Reid found that the diabetogenic activity of the growth hormone in cats was markedly enhanced by ACTH adadministration. ACTH alone had no diabetogenic activity.

The rapid decrease in egg production, which was consistent in all trials and with different hormone preparations, appeared to be due to the growth hormone and not the thyrotropic hormone or ACTH which were present as contaminants. Evans, Simpson and Li (1948) found that the reproductive system of rats was considerably disturbed when purified growth hormone was injected. Only a few small active follicles were present in the ovaries. The observation that the growth hormone caused a decrease in egg production is in agreement with the work of Evans el al. (1948) in rats. The broodiness that occurred in trial 2 when the porcine growth hormone preparation was injected was a definite treatment effect since it was confirmed when the treatments were reversed. The fact the ACTH alone or a combination of bovine growth hormone plus ACTH did not cause broodiness in trial 3 suggests the possibility that an unknown hormone may have been present in the growth hormone preparation used in trial 2. Nalbandov and Card (1945) postulated the existence of an unknown hormone which may interact with prolactin and cause broodiness. Using dairy heifers, Wrenn and Sykes (1952) found that injected growth hormone (containing some thyrotropic hormone) produced more milk secretion than did prolactin. These results indicated that an unknown hormone or a combination of the hormones injected may have caused the broodiness observed in trial 2. Further work will be necessary to establish the cause of the broodiness. SUMMARY

1. Very little growth stimulation was noted in pullets or hens injected with 1 or 2 mg. of the growth hormone preparations used in these trials. 2. The injection of 1 or 2 mg. per day

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In general, the injection of pituitary growth hormone preparations had very little growth stimulating effect on mature hens and pullets at the dosage levels used in these experiments. These same hormone levels, on a body weight basis, have been shown to stimulate growth in rats, Whitney, Bennett, Li and Evans (1948). In one trial, a 10 percent increase in growth was noted when porcine growth hormone was injected in one group of pullets. However, this group was growing rapidly even before the hormone injections were started. The results of these experiments indicate that this small growth response, if not due to chance alone, was most likely due to the growth hormone rather than the ACTH contamination in this preparation since ACTH alone showed no influence on growth in trial 3. Evans, Simpson and Li (1943) found that the growth of normal and gonadectomized male rats was inhibited by the injection of pure ACTH. Thus, it is not likely that the ACTH present in the porcine growth hormone used in this study was responsible for the increased growth.

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NEWS AND NOTES

ACKNOWLEDGMENT

The growth hormone and ACTH preparations used in these experiments were generously supplied by Dr. I. Bunding of Armour and Company. REFERENCES Bates, R. W., O. Riddle and R. A. Miller, 1940. Preparation of adrenotrophic extracts and their assay on two-day chicks. Endocrinology, 27: 781-792. Evans, H. M., and J. A. Long, 1921. The effect of feeding the anterior lobe of the hypophysis on the oestrus cycle of the rat. Proc. Anat. Rec. 21: 62-63. Evans, H. M., M. E. Simpson and C. H. Li, 1943. Inhibiting effect of adrenocorticotropic hormone on the growth of male rats. Endocrinology, 33: 273-238. Evans, H. M., M. E. Simpson and C. H. Li, 1948.

The gigantism produced in normal rats by injection of pituitary growth hormone. I. Body growth and organ changes. Growth, 12: 15-32. Gaebler, O. H., 1951. Remarkable effectiveness of small daily doses of growth hormone in dogs. Am. J. Physiol. 165:486-490. Hsieh, K. M., T. Y. Wang and H. T. Blumenthal, 1952. The diabetogenic and growth-promoting activities of growth hormone (Somatotropin) in the developing chick embryo. Endocrinology, 51:298-301. Hulb6, J., 1949. Langdurige behandeling van dwerghoenders met een groeibevorderend extract uit der voorkwab der runderhypophyse. Natuurwetenschap. Tijdschr. 31: 201-205. Li, C. H., and H. M. Evans, 1944. Isolation of pituitary growth hormone. Science, 99: 183-184. Li, C. H., and H. M. Evans, 1948. Biochemistry of pituitary growth hormone of anterior pituitary. Recent Progr. Hormone Research, 3:3-44. Nalbandov, A. V., and L. E. Card, 1945. Endocrine identification of the broody genotype in cocks. J. Heredity, 36: 35-39. Putnam, T. J., E. B. Benedict and H. M. Tell, 1929. Experimental canine acromegaly produced by injection of anterior lobe pituitary extract. Arch. Surg. 18:1708-1936. Reid, E., 1935. Adrenocorticotrophin in relation to the diabetogenic activity of growth hormone preparations. J. Endocrinology, 9:185-193. Riddle, O., R. W. Bates and E. L. Lahr, 1935. Prolactin induced broodiness in fowl. Am. J. Physiol. 111:352-360. Whitney, J. E., L. L. Bennett, C. H. Li and H. M. Evans, 1948. Effect of growth hormone on the nitrogen excretion and body weight of adult female rats. Endocrinology, 43:329-335. Wrenn, T. R., and J. F. Sykes, 1952. Lactation stimulating effect of prolactin and growth hormone preparation. J. An. Sci. 11: 806.

NEWS AND NOTES {Continued from page 1403) Termohlen, formerly Head of the Poultry Division of the Agricultural Marketing Service, U. S. Department of Agriculture, has served on the Advisory Board of the Library since its inception and was active in raising funds for its endowment. The Library has recently acquired microfilms of all Ph.D. theses, dealing with poultry subjects, written in the United States.

MAINE NOTES F. D. Reed, Extension Service Poultry Specialist at the University of Maine since 1938, has been appointed Extension Poultry Marketing Specialist, succeeding C. R. Eckstrom. Reed is a poultry husbandry graduate of the University of New Hampshire and did graduate work at Pennsylvania State University. He was a Poultry Research Assistant a t

{Continued on page 1440)

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of pituitary growth hormone preparations caused a marked decrease in egg production in both pullets and mature hens. In most birds molting accompanied the decreased egg production. 3. Broodiness was observed in pullets injected with a porcine growth hormone preparation containing a small quantity of ACTH. These results however could not be duplicated with a combination of growth hormone and ACTH formulated to duplicate the original injections. It has been postulated that an unknown hormone or a combination of hormones may have caused the observed broodiness.