The comparative effects of testosterone propionate and testosterone suspension on the combs and testes of young chicks with and without sunlight

254 45 4MB

English Pages 107

Report DMCA / Copyright

DOWNLOAD FILE

Polecaj historie

The comparative effects of testosterone propionate and testosterone suspension on the combs and testes of young chicks with and without sunlight

Citation preview

THE COMPARATIVE EFFECTS OF TESTOSTERONE PROPIONATE AND TESTOSTERONE SUSPENSION ON THE COMBS AND TESTES OF YOUNG CHICKS WITH AND WITHOUT SUNLIGHT

A Dissertation Presented to the Faculty of the Department of Zoology University of Southern California

In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy

by Harry Yuen Chee Wong August 1950

UMI Number: DP32343

All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion.

Dissertation Publishing

UMI DP32343 Published by ProQuest LLC (2014). Copyright in the Dissertation held by the Author. Microform Edition © ProQuest LLC. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code

ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48 10 6 - 1346

T h i s dissertation, w r it t e n by

............... u n d e r the g u id a n c e o f h . da.. F a c u l t y C o m m itte e on S tu d ie s , a n d a p p r o v e d by a l l its m e m b e rs , has been p re sen ted to a n d accep ted by the C o u n c i l on G ra d u a te S tu d y a n d R e se arch , in p a r t i a l f u l ­ f i l l m e n t o f re q u ire m e n ts f o r the degree o f DOCTOR

OF

P H IL O S O P H Y

^ D a te

C o m m itte e on S tu dies

.......

ACKNOWLEDGEMENTS The author wishes to express his deep appreciation and gratitude for the helpful suggestions and criticisms of Dr. Francis M. Baldwin, under whose supervision this experiment was conducted.

He wishes to extend his thanks

also to other members of his Committee, Dr. Bruce M. Harrison, Dr. Irving Rehman, Dr. Tilden Roberts, and Dr. William Mayer for their encouragement and assistance in the preparation of this dissertation. Special mention should be given to Abbott Laboratories of North Chicago, Illinois, and Schering Corporation in Bloomfield, New Jersey, for the supply of hormones necessary for this experiment.

TABLE OF CONTENTS CHAPTER

PAGE

ACKNOWLEDGEMENT .................................. I.

INTRODUCTION

..............................

Review of the literature

ii 1

................

5

Effect on capons

......................

6

Effect on chicks

......................

8

Effect on the f e m a l e ..................

14

Methods of bioassays of androgens on b i r d s ................................

18

Measurement of combs i z e ...............

20

..................

20

Planimetric method......................

21

Weight of drawing

...................

23

W e i g h t ................................

24

V o l u m e ................................

25

Length of the c o m b ....................

26

Length and h e i g h t ......................

26

Length + h e i g h t ........................

27

l/2 length + h e i g h t ....................

28

Length x h e i g h t ........................

28

Sum of height, length, and width

29

Photoelectric method

....

V L x H mm.

29

Height x l e n g t h ........................ 2

30

iv CHAPTER

PAGE

II.

MATERIALS AND M E T H O D S ......................

31

III.

R E S U L T S ....................................

35

Body w e i g h t ............................

35

Comb i n c r e a s e ..........................

38

Chicks injected with testosterone s u s p e n s i o n .......................... Effects of testosterone propionate

42

...

43

Effects of hormones on testes ..........

55

D i s c u s s i o n ..............................

67

Summary and conclusions..................

72

Conclusions............................

75

B I B L I O G R A P H Y ....................................

76

LIST OP TABLES TABLE I.

PAGE Body Weight of Different Series of Chicks Injected with Male Sex Hormones from the 5th to the 9th Day at the End of the 5th, 10th, 15th, and 20th Day With and Without S u n ..............................

II.

36

Average Comb Factor for 5 Day Old Chicks Injected With Testosterone Aqueous and Testosterone Propionate from the 5th to the 9th Day at Different Intervals with S u n l i g h t ................................

III.

40

Average Comb Factor of 5 Day Old Chicks Injected with Testosterone Propionate and Testosterone Suspension from the 5th to the 9th Day at Different Periods Without S u n l i g h t ................................

IV.

4l

Average Testes Weight of Chicks Injected With Testosterone Suspension and Testosterone Propionate at the End of the Different Intervals With and Without S u n ......................................

V.

56

Mean Testis Weights Expressed as Percentages of Mean Body Weights in 5, 10, 15, and 20 Day Old C h i c k s ..........................

63

LIST OF FIGURES FIGURE 1.

PAGE Difference in Comb Increase of 5* 10* 15* and 20 Day Old Chicks Injected Daily from the 5th to the 9th Day With Testosterone Suspension With and Without Sunlight ........................

2.

44

Difference in Comb Growth of 5* 10* 15* and 20 day old Chicks Injected Daily From the 5th to the 9th Day With Testosterone Propionate With and Without Sunlight . . .

3.

46

Representative Chicks of the Series Without Sunlight Injected With Testosterone Propionate and Testosterone Suspension at the End of the 10th D a y .............. a.

50

Chicks Injected with Testosterone Propionate on the 10th Day Without S u n l i g h t................................

b.

Representative Chicks of the Same Series on the 15th D a y ........................

4.

a.

51

51

Chicks Injected With Testosterone Suspension in an Aqueous Solution on the 10th Day Without S u n l i g h t ..............

b.

52

Representative Chicks of the Same Series on the 15th D a y ........................

52

vii FIGURE 5.

PAGE a.

Chicks Injected With Testosterone Propionate as Shown on the 10th Day With Sunlight ................................

b.

Representative Chicks of the Same Series on the 15th Day..........................

6.

a.

53

53

Chicks Injected With Testosterone Suspension as Shown on the 10th Day in the Sun l i g h t ............................

b.

54

Representative Chicks of the Same Series on the 15th D a y ........................

7 . Testis

54

Weights Expressed as Percentage of

Body Weights in Chicks Injected with Testosterone Suspension With and Without Sunlight at the End of the 10th, 15th, and 20th D a y ............................ 8 . Testis

60

Weights Expressed as Percentage of

Body Weights in Chicks Injected With Testosterone Propionate With and Without Sunlight at the End of the 10th, 15th, and 20th D a y ............................

6l

CHAPTER I INTRODUCTION Of the various tests that exist for the hioassay of the male sex hormone, the comb growth response of both the castrated and uncastrated chick is generally recognized as the test being the most satisfactory.

One of the other

tests that have been utilized is the effect of androgens on atrophied organs of the castrated rat.

However, Koch (1937)

states that due to variation of rats this test is not reliable.

According to Miescher, Wettstein, and Tschopp

(1936), the comb test is the only method for bioassay.

Of

the two, the chief criteria for the bioassay of male sex hormone is the quantitative test developed by Gallagher and Koch (1930). It has been found that the combs and wattles of poulards, capons, and young cockerels can be made to develop more rapidly by the administration of androgenic hormones by subcutaneous injections, inunctions, or directly injected into the combs.

However, it has been reported by many

investigators that sunlight has an antagonistic action on the comb growth response property of the androgens, parti­ cularly that of testosterone propionate.

Many experiments

have been undertaken to study the effect of difference of

2 light and temperature on the combs of young male and female chicks as well as on capons. It has been observed by Benoit (1927) that the growth of the comb of the Leghorn fowls, hatched ea.rly in the year, showed a remarkable depression toward the end of summer and the beginning of autumn.

This phenomena was not ob­

served in this investigation or if present it was only so very slight as to be overlooked in birds reared during the latter part of the year.

Domm (1930) found that the combs

of ovariectomized birds increased greatly in size during the winter months, which he attributed to the lack of sun­ light.

According to Womack, Koch, Domm, and Juhn (1931)

Brown Leghorn capons which had access to sunlight had combs that responded less to injections of male sex hormones. Buckner, Insko, and Martin (1932-3*0 observed that White Leg­ horn cockerels had larger testes and smaller combs when th^r were exposed to sunlight.

Those without sunlight were

found to have relatively smaller testes, but they had very large and less erect combs.

Furthermore, removal of the

combs and wattles resulted in the chicks having smaller testes.

However, Zawadowsky (1935) reported that the comb

may act as an antagonist physiologically and confirmed the report made by Buckner that larger testes result in cock­ erels when the comb was removed.

According to Nikolaiczuk

and Maw (1941) sunlight has an insignificant effect upon

3 the final mature body weight, but it does induce a reduction in comb and wattle size.

David (1938) found that injections

of androsterone into chicks for a period of four days caused a maximum growth of the comb late in the fall and during the winter months; it is rapidly reduced in the spring and reaches its minimum growth during the summer months.

He

also mentioned that chicks kept in the dark had greater comb growth than those in the sun.

Starkey, Grauer, and Saier

(1940) stated that the amount of light affected the response of the combs.

They found that the comb weights were great­

est when the birds were exposed to natural light and de­ creased as the light exposure was diminished.

It was noted

by Hain (1938) that not only is there a variation in the time of the year but that the variation in temperature had some effects on the growth of the comb.

This was confirmed

by Lamoreaux (1943) who stated that difference in light and temperature had an effect upon the size of the combs of White Leghorn cockerels.

The most effective method of

inhibiting the comb growth of chicks was to maintain a low temperature.

In an unpublished report, Wong (1947) has

shown that White Leghorn cockerels when injected with testosterone propionate had smaller combs when kept in the sunlight as compared to those without sunlight. Though the comb reaction is used extensively in the bioassay of male sex hormones, there sire certain features

4 that are of disconcerting nature such as the action of sun­ light on the comb. The purpose of this investigation was twofold: first, to determine the physiological effects of testosterone propionate in sesame oil in contrast to that of testosterone suspension in an aqueous medium; and secondly, to note weight changes that may occur in the testes due to the ac­ tion of sunlight or the absence of it in the various dosages of testosterone suspension and testosterone propionate with and without sunlight. It is evident that two important variable factors influence the growth of the combs in the chicks; namely, the dosage of the male sex hormone employed, and whether the chicks were exposed to sunlight.

Thus, these experiments

were undertaken to study the importance of exposing the birds, in experimental and control groups, to an adequate and constant source of light as compared to those without sunlight when injected with various dosages of the male sex hormone.

This is especially true of testosterone suspension

in an aqueous solution which has not been used by previous investigators.

In addition, there is here included a study

of the comparative and quantitative response of the comb growth of young cockerels injected with different dosages kept in sunlight and without sunlight.

5 REVIEW OF THE LITERATURE It has been found that birds possess a great number of sexual characteristics which make them excellent mater­ ial for the study of hormones.

The importance of the

sexual organs upon the secondary sexual characters, as well as upon the other parts of the body, has been investigated intensively in recent years in the vertebrate series, but more particularly in the birds.

Birds, possessing a great

variety of dependent somatic sexual characters, have been employed extensively as useful indicators of the activity of the sex hormones.

Due to these useful indicators of the

activity of hormonal response possessed by the many species of birds, including those that are domesticated, birds have been used frequently for experiments.

The most important

and superficially obvious among these indicators are the combs and wattles of the capons, cockerels, and hens.

The

differential growth of the combs and wattles under differ­ ent experimental procedures have not been surpassed as indicators of hormonal activity.

Thus, records of hormonal

influences may be recorded and observed as the experiments progress.

It has been adequately demonstrated that the

injections of certain dosages of the male sex hormones result in an increase of the surface of the comb which can be measured on the comb of young capons, cockerels, pullets or hens.

6 Effect on capons.

John Hunter (179^) was first to

demonstrate the hormonal action of the testes when he proved that the rudimentary spur of a hen developed into a wellformed masculine spur when transplanted into the leg of a cock.

However, when the spur of the cock was implanted

into the leg of a hen, no similar development took place. The hormonal function of the testes remained obscure until Berthold (18^9) transplanted the testes of a castrated rooster into its abdominal cavity.

It showed that the

testes did produce a hormone, which in the absence of ex­ ternal secretion, maintained the secondary sexual character­ istics of the bird.

Pezard (1911) found that he could pro­

duce comb growth in black Orpington capons by the twice weekly injections of five to ten cubic centimeters of an extract from one-tenth of a cryptorchid hog testes for a period of over three months.

It resulted in growth of the

atrophied combs and wattles and induced other secondary sexual characteristics such as crowing and aggressiveness. However, as soon as the injections ceased, the birds resumed the behavior of a typical capon.

Loewy (1903) found that

young capons injected repeatedly with a testicular substance showed an increase in development of the skeleton and the comb.

Oslund (1928) demonstrated that three caponized

cockerels injected with fresh semen from the vas deferens resulted in positive growth of the comb, thus proving to

7 himself that the testicular hormone was produced by the germinal epithelium.

Funk and Harrow (1929) and Funk,

Harrow, and Lejwa (1930) reported that comb growth of capons was increased when urine of young men was injected into the birds with a regression taking place after the cessation of the injections.

Dessau (1937) found that only

1/150 as much urinary extract was necessary for a positive reaction when smeared on the comb of the capon as when the same extract was injected intramuscularly.

Domm and Van

Dyke (1932) noticed a pronounced growth of the comb when Brown Leghorn cockerels received daily subcutaneous injec­ tions of hebin.

This growth could be definitely noted in

several cockerels as early as 48 hours after the injections began.

In addition to comb growth, precocious sexual

behavior was observed.

Young cockerels were found to crow

when 9 days old and treading was noticed on the 13th day. Unilateral and bilateral castrate Leghorns which received daily injections of hebin were reported by Domm (1933)*

It

was noticed that the unilateral castrate ones responded similarly to the normal chicks when injected with hebin daily.

However, the capon showed no such response. When androgens are administered by the intramuscular

implantation of solid crystals or tablets of the pure hormone, a very high effectiveness and prolongation of the action can be obtained in capons.

This was the basis for

8 the experiment of Schoeller and Gehrke (1938) who found that implantation of 2 mg. of testosterone produced a maximum response of 480$ increase in the comb 16-21 days later, whereas an equal amount injected in an oil solution gave a comparatively small response of only 15$ soon after the injection.

The same method, by implantation, was used by

Emmens (1938) in investigating the maximum growth rate of the capon comb.

By implantation of 15-16 mg. of testosterone

propionate per bird he was able to obtain a maximum growth of the comb at the end of the 40-50 days, having increased the comb size 700-1000$ in length times the height over that of the normal cock.

Hamilton and Dorfman (1939) found

that the implantation of crystals of testosterone propionate in one injection of 20 mg. into day old chicks caused a comb growth which lasted for 71 days. Effect on chicks.

The use of the chick as a test

object was first mentioned by Ruzicka (1935) •

Ptissganger

(1934) utilized the chick as a test object by applying androgen externally to the crest region.

He claimed that

1/50 of a capon unit gave a response as great as 1 capon unit when given intramuscularly to the capon.

However,

Dessau (1937) claims that if the male hormone was applied percutaneously to the back, the effect was only 1/150 as potent as when applied to the comb.

Ruzicka’s report was

confirmed by Burrows, Byerly and Evans (1936) that baby

9 chicks will grow combs when injected with testosterone or androsterone.

It was demonstrated by Juhn, Gustavson, and

Gallagher (1932) that marked response of the combs of Immature chicks occxirred when injected with male sex hor­ mone . In a study to determine the quantitative response of the chick’s comb to androgen, Duff and Darby (19^1) and McCullagh and Guillet (19^1) described experiments of comb— response to androgen by inunction and found that the comb increased in size.

Frank and Klempner (1937) applied

various androgens in oil to the base of the comb by in­ unction and they also claimed a quantitative response.

The

latter reported no curve of response in their original report.

By applying the hormones to the comb by inunction,

Deansely and Parkes (1937) were able to produce an effec­ tive comb increase of 200 times.

Dorfman and Greulich (1937)

presented a response curve when known quantity of androgen was injected into fowls.

Frank and Klempner gave some

measure of comb-growth for the various doses of androsterone applied to the comb of the chick, but no typical curve of response was presented.

Gallagher and Koch (1935) proved

that length of the comb rather than the height is a fairly constant index of comb size in their experiments.

By the

injection of 0.5 mg. of testosterone propionate in White Leghorn chicks at 2 days of age for 27 days, Hamilton (1938)

10 was able to induce precocious sex behavior (crowing and aggressiveness) in the male.

Crowing of the birds was

noted 180 hours after the initial injection.

Cock-like

fighting and stunting of the body growth were observed afterward. ments.

Noble and Zitrin (1942) reported similar experi­

These authors found that all the sexual behavior of

the adult cock can be exhibited in male chicks when injected with testosterone propionate in sesame oil.

They observed

that crowing of the birds began as early as the 4th day of age and treading was noted on the 15th.

The birds were

found to have the same copulatory pattern as seen in sex­ ually mature birds.

After the cessation of injection,

growth of the androgenized males was retarded with a re­ gression of the stimulated comb to subnormal level.

David

(1938) reported that the injection of testosterone and androsterone into chicks caused maximum growth of the comb during the fall and winter months and the smallest in the summer months.

In addition, he noted that chicks kept in

the sunlight had smaller comb growth than the birds which were confined indoors.

It was reported by Breneman (1939)

that 5 day old male chicks injected with hormones crowed at the end of the 11th day with the growth of secondary sexual characteristics after the injection of either dihydroandrosterone-benzoate or testosterone propionate.

The

effects of androstanediol benzoate and testosterone on the

11 comb and testes of White Leghorn chicks were reported by Breneman (1937)*

Subcutaneous injections of 0.1 mg. per

day of either substance for 5 days into 5 or 10 day old chicks led to immediate cessation of testicul ar growth which lasted only during the period of injection.

Month-

old chicks injected with male sex hormone from the 5th to the 10th day had an average gonad weight 90^ heavier than the controls.

Androstanediol benzoate injected with

pituitary hormone was found to be effective in maintaining testicular growth, showing that the former acted by depress­ ing the gonadotropic activity of the pituitary gland. Considerable comb-growth occurred in the experimental birds. Breneman (1938a-) has shown that androstanediol benzoate has a greater action than testosterone propionate in the combgrowth of intact chicks, and that divided doses of the latter are more effective than a single dose.

After the cessation

of injections, the combs continued to grow more rapidly than normally for several weeks, and Breneman believes this is due to increased secretion of androgenic substance by the bird, since such long continued growth did not occur in injected capon chicks and is not due to the slow absorption of the retained hormone.

No crowing was observed in the

treated capons but it occurred in all treated intact chicks. By the inunction of 12.5 nig. of androsterone in 10 daily doses, Morato - Manaro, Albrieux, and Buno (1938) were able

12 to produce comb-growth of 99*3$ (measured by area) in a three months old cockerel.

Upon examination of this bird,

the testes were found to be greatly atrophied and damaged, showing no spermatogenesis and tubules with only a few rows of cells containing mostly dead cells in their lumens. Chu (19^0) also observed that injections of testosterone in pigeons resulted in damage to the testis, although the same treatment given to hypophysectomized birds produced some degree of testicular maintenance.

When testosterone is

injected into small chicks, it causes growth of the genital eminence in both sexes according to Kosin (1940).

It was

also reported by Kosin (1942) that the sex eminence of young chicks of both sexes was very sensitive to testosterone propionate injected intramuscularly, whereas the inunction method was less efficient.

Chicks of both sexes treated

with hormones resulted in a ’’puffing" of the cloacal region. Crowing was observed in the 72 hour chicks of both sexes 24 hours after the first injection.

Daily intramuscular

administration of androsterone in dosages of 2.5 nig. rising to 5 nig. was found by Callow and Parkes (1935) to produce a comb-growth in a Leghorn capon.

Very rapid and extensive

growth of the comb of a Leghorn capon was produced by Emmen (1938) by intramuscular implantation of a compressed tablet of crystalline testosterone propionate.

A daily dose of 0.3

- 1.7 mg. of testosterone propionate is imperative to

13 maintain comb size in castrated cocks according to Caridroit and Regnier (1938).

Turner (1948) reported that adding 20

mg. of methyl testosterone per kilogram of feed produced a growth of the comb of either the male or female White Plymouth Rock chick up to the 4th week of age.

Rakoff,

Paschkis, and Cantarow (1944) found three day old chicks very satisfactory for androgen assay purposes.

Snedecor

(1949) made a study of the injections of hormones consist­ ing of stilbestrol, dienestrol, pregnant mare serum, testosterone propionate, and dihydroandrosterone benzoate into the albumen of hen's eggs and noted its effect on the sex characteristics.

Androgen is found to have a marked

effect on the comb growth of the unhatched chicks.

There­

fore, it indicates that a comb growth factor may be pro­ duced by the normal male before hatching. Caridriot (1941) was unable to produce comb growth in chicks with one injection of 25 gammas of testosterone propionate but with the addition of Vitamin E to testos­ terone propionate, comb growth was produced.

Bernstorf

(1948) found that chicks of one to three weeks of age, each caponized and given an intrasplenic testis graft, developed larger combs than did the capons, but smaller combs than the controls.

The highly significant difference between

the combs of the grafted and control chicks demonstrated partial hepatic removal of the androgen, while in comparison

14 with the control capons it showed the extent of incomplete androgen removal. As found by the earlier investigators with impure extracts, the production of

abnormal

adult cocks proved difficult.

comb growth in the

Greenwood and Blyth (1935)

reported that intra-comb injections of 100 gammas per day for 5 days failed to cause additional growth of the comb of the sexually active male fowl.

A tablet of testosterone

propionate (6 mg.) implanted into the breast muscle resulted in the growth of the comb of a normal Silver-Black Rock cock beyond the normal genetic limit of this breed. Effect on the female.

The comb of the hen, which is

much larger and more vascular than that of the capon, has been used as a test for androgen.

It is reduced by ovariec­

tomy until the right gonad is regenerated, thus showing that comb-promoting substances are produced by the ovary of the hen, probably a 17 ketosteroid, in addition to the female sex hormone, estrogen. The first to produce comb growth in normal young hens was Walker (1908) who injected one-half cubic centimeter quantities of normal saline extract made from fresh, macerated cockerel's testes into young hens.

Injections

were made subcutaneously daily for several months in two hens and resulted in a growth of the comb and the wattles. However, as soon as the injections ceased, the combs and

15 wattles regressed to their original size.

Callow and

Parkes (1935) injected a normal Brown Leghorn hen with 2.5 mg. of androsterone daily and obtained considerable combgrowth.

Emmens (193$) administered testosterone which was

given subcutaneously to a normal hen and found that this hormone produced a growth of the comb.

Pullets differ from

cockerels in several important features such as the body, comb, gonad, and the anterior pituitary, as reported by Breneman (1946).

Breneman (1940a) showed that pullets had

a greater comb increment when subjected to a restricted diet in comparison to the normal dieted chicks.

Burrows,

Byerly, and Evans (1936) reported that as a test animal the pullet was superior to that of the cockerel.

However, it

was reported by Danby (1936) that she was unable to detect any differences between the two.

Breneman (1940b) observed

that pullets injected from either the first day to the fourth day or those that were injected from the fifth to the ninth day after hatching did not have as marked response to testosterone propionate and dihydroandrosterone-benzoate as cockerels of the same age.

In addition it was noticed

that the combs after cessation of injections grew much slower than those of cockerels receiving the same treatment. Herrick and Lockhart (1940) demonstrated that young female chicks receiving small doses of testosterone propionate totaling 2.5 mg. had ovaries slightly smaller than their

16 controls. The Isolation of the male sex hormone by McGee (1927), and McGee, Juhn and Domm (1928) extracted from bull’s testes was injected into two bilaterally castrated hens at the rate of 2 cc. daily for six weeks and resulted in the growth of the comb.

However, if this material was administered orally

1 cc. twice daily to a capon, negative results were obtained. Domm (1927) studied the influence of the male sex hormones on the sexual behavior of domestic fowls.

He first ex­

perimented on the effects of androgen on bilaterally ovariectomized poulards.

The poulards resemble capons in

that they are neutral in behavior.

With the injection of

androgen, the poulards became aggressive and fought violent­ ly.

The hens first crowed on the l6th day of injection and

within three weeks ”waltzed11 at the introduced female or dummy (female).

The poulards were observed to peck the head

of the female and the dummy viciously.

Experiments on the

effects of androgen in sinistrally ovariectomized poulards were continued by the same investigator.

The effects were

similar to the above experiment with the exception that testosterone on sinistrally ovariectomized poulards caused more aggressiveness.

The only case on record of a sinis­

trally ovariectomized poulard performing the male copulatory act was reported by Domm (1927).

Juhn, D*Amour and Womack

(1930) have demonstrated the effect of simultaneous

17 injections of male and female sex hormone on capons and females over a period of time.

They found that birds re­

ceiving simultaneous injections developed female plumage and male head furnishings.

Funk, Harrow, and Lejwa (1930)

injected urine of young men into female chickens and found no effects on comb growth and the wattles.

It was shown by

Allee, Collias and Lutherman (1939) that the social order of hens may be modified with the injections of testosterone propionate.

Birds injected with the male hormone produced

a comb growth and other head furnishings immediately.

In

addition, each adult hen injected with the hormone showed a rise in social status and eventually occupied the top posi­ tion in each flock.

In young pullets, the investigators

showed that the injected birds dominated each flock.

Other

effects mentioned which are of interest in addition to that of comb growth are initiation of crowing and with three hens, courtship.

Dorfman and Greulich (1937) used day old female

chicks which were injected with androgen and found that they were satisfactory for assay purposes.

The same workers

used 4 hour old chicks as a test and found that there was a wide variation since it was impossible to obtain a homogenous group of the same age.

According to Hamilton (1938) injec­

tion of male hormone into females will cause a pronounced growth in the combs and wattles and in body weight changes.

18 Methods of bioassays of androgens on birds.

It was

first suggested by Gallagher and Koch (1929) that by measuring the growth of the capon comb a quantitative method for the assaying of androgenic extracts could be determined. They defined a "capon” unit in terms of the average amount of growth in a group of at least five Brown Leghorn capons injected daily for five days, which yielded an average of 5 mm. increase in length and height of the combs.

The same

type of "characteristic curve" in comb-growth response simi­ lar for androsterone, transdehydrosterone, androstenedione, and androstanediol has been reported by Koch (1937)•

The

dose-response "curve" has been interpreted as a straight line when the dose is plotted logarithmically by Greenwood, Blyth, and Callow (1935) and Hall and Dryden (1939) or doses plotted in ordinary units in a straight line as shown by Emmens (1939)•

It has been reported by Womack and Koch

(1930), Womack, Koch, Domm, and Juhn (1931)* Gallagher and Koch (1929) that the comb-growth response value is affected by the initial length of the comb and by the intensity of the light to which the birds may be exposed as well as the solvent used in the preparation of the hormone.

Domm (1930)

observed that the combs of normal cocks and poulards vary greatly in size with the seasons, being smaller in the spring and summer and larger in the fall and winter.

He

believes that light and possible exercise are the factors

19 which cause these variations.

He has observed since begin­

ning his experiments on birds, that cocks and poulards, confined in small cages with outside pens where they have access to sunlight, have considerably smaller combs than those birds kept away from sunlight.

Buckner, Insko, and

Martin (l93^b) report that light plays a very important role in determining the size of gonads and combs.

They observed

that when White Leghorn cockerels are raised without sun­ light, exceptionally large and less erect combs were developed, but relatively small testes.

However, when the

cockerels received sunlight, their combs were smaller and more erect, but the testes were larger than normal.

Further­

more, removal of the comb and wattle resulted in cockerels having small testes, whereas in pullets no such effect was observed in ovarian weight.

However, Starkey, Grauer, and

Saier (19^0) report that exposure to various degrees of light influenced the weight response of the combs of the male chicks to which androsterone had been applied by inunction.

They claimed that birds receiving normal sunlight

as compared to those that are kept in the dark or inadequate light had a greater weight response to androgenic stimulation. Lamoreaux (19^3) undertook the study of the effects of ultra­ violet light, short and long daily periods of light, and the differences in environmental temperature upon the comb-growth of White Leghorn cockerels.

He found that ultraviolet light

20 irradiation has little direct effect on the growth of the comb; exposure to artificial light during the short periods of time daily does not induce greater comb-growth than long daily exposure to similar light; and confinement of cockerels indoors at low temperature levels result in effective inhibition of growth of the comb. Measurement of comb size.

In reviewing the litera­

ture, several methods have been employed to determine the increase of the size of the combs.

An attempt has been made

to consider in this report all the more important methods that have been utilized thus far to express comb size. Photoelectric method.

This was used by Gradstein

(1935) as a means of determining the increase of comb size by photoelectric cells.

This method requires the use of

special equipment and is so complicated that it is not prac­ tical for routine bioassay.

The results obtained are

similar to the results produced by the planimetric method which will be discussed further as being severely criticized as a method of comb determination by Gallagher and Koch (1935)*

From Gradstein’s illustrations, it is apparent that

his method of determination is confined to combs of small birds, since it is necessary to arbitrarily determine the junction of the comb on the skull by drawing a straight base-line between two arbitrary points on the margins of

21 the comb that has distinguishing points.

As the comb grows,

it follows the curvature of the head-furnishing so that a certain portion of the head is invariably included when measured by this type of apparatus-

Since the comb tends to

follow the contour of the head and neck, considerable error is made by the omission of the posterior portion of the comb. Planimetric method.

Freud, de Fremery, and Laqueur

(1932) devised a method of determining comb growth known as the planimetric method.

Their method was to photograph the

shadows cast by the comb before and after injection and determined the change in area of the shadows by the planimeter, thus measuring the comb-growth response.

Their

definition of a unit was the minimum daily dose, which, administered in two injections daily for four days, causes an increase of 15 per cent in area in over 50 per cent of the capons on the 5th day.

To complete an accurate check

by this method may necessitate four to five weeks due to the fact that the minimum dose must be determined directly and not calculated from a characteristic curve.

Buckner,

Insko, and Martin (1933) also used this method for deter­ mining the surface area of the comb.

However, the authors

do not mention whether the measurements were taken from the photographs of the combs or from the combs themselves.

They

have also given the weights of the combs and wattles; the surface area was determined by means of a planimeter.

22 Gallagher and Koch (1935) have supplied evidence that the determination of the area by means of the planimeter from photographs is subject to considerable error.

These

investigators employed the planimetric method in determining the area of comb response by photographs.

Due to the fact

that they felt it was impossible to properly control a common base line for the base of the comb during the course of their experiment, they did not use the shadow photograph. In order to eliminate this difficulty they photographed the heads of the capons and then determined the areas on the image when projected to the original size.

The head of the

capon was held at a fixed distance on a special board, the comb flattened against a glass plate, the beak was painted white to enable one to determine accurately the anterior end of the comb, and the posterior juncture of the comb with the head was indicated by a black line on a white marker held at that point.

A straight line from the posterior

juncture of the comb to the beginning of the comb was taken as the base line.

In order to determine the accuracy of

this method, seven photographs each were taken of twenty capons, and the negative was projected to actual size on a ground glass plate.

By means of a planimeter, the area of

each photograph was determined at least three times.

The

authors found that there was a variation ranging from three to thirty-two per cent for the twenty combs which were each

23 photographed seven times.

An average variation in area of

15 per cent was attributed to photography, while variations ranging from 1 to 10 per cent were due to measurements of area on the same photograph.

However, if precaution is

taken in establishing the base line and in photographing the comb, there is no reason to believe that the shadow photographs as suggested by the various authors would be more accurate.

As it has been proven by Gallagher and Koch

(1935) the inaccuracy of this method is due to the diffi­ culty of establishing a base line for the comb.

Variations

in area due to photography as well as in the measurements of the area of the same photograph indicate that this method is of no practical value. Gallagher and Koch (1935) mention that two qualified workers made direct measurements of the length and height of the comb after the birds were photographed.

They sum­

marize from a comparison of their data with the planimetric method that it was easier, simpler, as well as more rapid when the direct measurement method was employed. Ogata and Ito (1933), and Ogata, Hirano, and Tanaka (193^) also used the planimetric method and based their results on an increase in area. Weight of drawing.

Morato-Manaro and Albrieux (1939)

presented a similar method of measuring comb increase as in the planimetric method.

They impressed the outline of the

24 comb on photographic paper and then had this retraced on a paper of uniform thickness, cut out and weighed on a scale. By comparing the weights of the same comb before and after injection of hormone, they were able to calculate the per­ centage increase and decrease in size.

This is a time-

consuming method, and the results show only the relative sizes of the combs and give very slight indication of the actual size and changes. Weight. Many research workers have attempted to determine the size of the comb by weighing.

This method has

been reported by Burrows, Byerly, and Evans (1936), Frank and Klempner (1937), Frank, Klempner, and Hollander (193$), Breneman (1940a, 1941a), Duff and Darby (1941), Dorfman (1948), and by many others for determining the response of the comb of immature chicks to male and female sex hormones. The determination of the size of the combs by the weight method is excellent, but it is subjected to errors and variations due to the method of removal and error in weighing and vascularization.

Previous to the report of

Frank, Klempner, Hollander and Kriss (1942a), the removal of the comb had been made by clipping the comb as close to the skull as possible.

Their method of determining the size

of the comb by the weight method was to make two parallel longitudinal incisions with a sharp scalpel along the base of the comb vertically down to the skull.

The entire comb

25 is removed by undercutting directly on the skull, starting at the posterior extremity of the comb base and moving the scalpel forward to the anterior part of the beak.

Thus the

removal of the comb is made by three simple, clearly defined incisions.

The comb is grasped lightly with a pair of

tweezers and its base is lightly touched to a filter paper or towel in order to remove any blood from the cut surface and then weighed on a balance. Reasonable care in weighing reduces that source of error to minimum.

Weighing has an advantage in adjusting

for any change in the thickness of the comb which is not possible to account for by any method of linear measurement. However, the very nature of some experiments concern­ ing the increase in comb size make this type of measurement impossible. Volume.

This method might be a better measure of

comb size than weight if the specific gravity of combs were subject to great variation. Greenwood and Crew (1927) were the first to measure the size of comb by volume.

This method is likely sensitive

enough to enable one to distinguish relatively small changes in the size of the comb.

Their method for the determination

of the volume of the comb was made by taking an impression of the comb with dental wax and from these impressions permanent plaster casts were made.

The difference in comb

26

volume could readily be determined by means of these casts. Due to the complexity of this method, it is of little value in bio-assays of comb response other than experimental work on small numbers of birds. The possibility of determining the volume of the comb by measuring the amount of water it displaces was investi­ gated by Engel (1939)*

This method may have been used by

Marlow and Payne (1940) in determining the volume of the comb.

As they have not reported their procedure, it is

probable that this method of determining the volume

of the

comb was used since they obtained weights of the combs as well. There is little to be gained by using this method in determining comb size since it is possible to use the weight as a measure of comb size. Length of the comb.

This method was first employed

by Pezard (1911, 1920) in determining the size of the comb. Others who have used this method are Benoit (1929), Funk and Harrow (1929), and Funk, Harrow, and Lejwa (1930).

Funk

and his colleagues (1929, 1930) expressed the comb response to injections of male sex hormones in terms of the percentage increase in length when injections were begun over the length of the comb. Length and height.

Some workers believe that this

27 method is much more accurate in the measurement of comb growth than the length of the comb alone.

This method, to

determine the increment of the comb has been used by Pezard (1918), Finlay (19?5), .Tnhn, D'Amour, and Womack (1930), Domm (1931), Domm and Van Dyke (1932), and Asmundson and Wolfe (1935). Length + height.

The sum of length and height

has been employed as a means of measuring the comb size. This is actually a slight variation of length and height. The increment of the size of the comb is calculated by determining the increase in the length + height of the comb during the experimental period.

This method is advantageous

in that it expresses the amount of increase of the comb in one figure rather than in two.

Thus, this method is simpler

in comparing the size of the comb before and after injections of hormones into birds.

Also, it would be just as accurate

if there were a respective variation in the length and height of the comb, i.e., if the growth in one is supplemented by a growth in the other.

Several workers have used this method

for determining comb increase.

They are Gallagher and Koch

(1930), Greenwood and Blyth (1935), Hain (1938a), Hoskins and Koch (1939), and McCullagh and Cuyler (1939)*

However,

there has been some disagreement among the workers as to whether or not this method is satisfactory.

According to

Asmundson, Gunn, and Klose (1937) this method is not an

28 accurate measure of actual difference in weight.

They

found that a 37 mm. comb (length + height) weighed about 0.3 gram, whereas a 127 mm. comb weighed 12.1 grams. 1/2 length + height.

Only two groups of investi­

gators have used this method but they failed to give their reasons for dividing the length plus the height by two. They are Womack, Koch, Domm, and Juhn (1931) and Bates, Lahr, and Riddle (1935)• Length x height.

This method has been used by McGee,

Juhn, and Domm (1928), Blyth, Dodds, and Gallimore (1931), Preud, de Premery, and Laqueur (1932), Deansley and Parkes (1937), and Emmens (1938) to reveal the size of the comb. In such a method it would seem that these investigators were trying to acquire a measurement of comb surface area. Assuming that it is slightly rectangular in shape, such a measurement would result in the surface area of one side of the comb. The total surface area would then be twice the sum of the length times the height method.

According to Freud,

de Fremery, and Laqueur (1932) they consider this a better measurement of the size of the comb than length plus height. By this method they feel that the comb resembles a parallelo­ gram and that the product of the sides of a parallelogram is more characteristic of the area than the sum of the two

29 sides.

Hill and Parkes (1934) suggest that the best

measurement of comb size is by volume, but they discovered that this was not satisfactory as it failed to account for the variation in the thickness of the comb.

As a result

they have relied on the length times the height method for comb measurement but remain skeptical of this procedure. They claim that this does not give the changes which are three dimensional, though it gives a fair indication of surface area.

Callow and Parkes (1935) hold the same

opinion regarding this method.

They also feel this method

does not give the actual changes in the appearance of the comb although the length and height are a convenient basis for assay work. Sum of height, length, and width.

This method was

the basis of the measurement of comb development in both male and female chicks by Noble and Zitrin (1942).

They

give no reason for using the sum of the height, length and width. V l x H mm.

A new method of expressing comb increase

was introduced by Breneman (1937) which he has continued to use in all his reports.

He expresses comb size as the

square root of length times height in millimeters.

Breneman

states that the usual dilemma was encountered as regards the proper method of establishing a factor for comb size.

After

30 measurement of the size of the combs the figure of V L x H ram.

appeared to be the most reliable in the mea­

surement of comb size.

This comb factor is used to indi­

cate the differences in si ze, resulting from experimental treatments, in terms of the length of one side of a square having a comparable area.

This figure aids in the visuali­

zation of actual changes in the dimensions of the comb. Jones and Lamoreaux (19^3) reported their study of five different measurements of comb size to determine which was most closely related with the actual weight of the comb. The measurements of the comb size undertaken in this study were (a) length,

(b) height, (c) the sum of the length and

height, (d) the product of length and height, and (e) the square root of the product of length and height.

They found

the product of length and height, the sum of length and height and the square root of the product of length and height were closely related but the results of either the length or the height were somewhat lower. Height x length. The latest method for the estima2 tion of comb size was reported by Bernstorf (19^8) as height times the length in millimeters divided by two.

He fails to

give any reason for using this figure in the measurement of comb growth other than stating it was a reliable indication.

CHAPTER II MATERIALS AND METHODS A total of 872 single comb White Leghorn cockerels were used in all the experiments.

These all were secured

from one source, Birkey’s Hatchery in South Bend, Indiana, within 24 hours after hatching. the hatchery as being cockerels.^

All chicks were sexed by The synthetic male sex

hormone, testosterone propionate and testosterone in an aqueous solution, was used in all experiments.

The tes­

tosterone propionate dissolved in oil was supplied by Schering Corporation.

0

The 50 mg* per 1 cc. solution was

diluted with sesame oil to 2.5 mg., 3*5 mg., and 5*0 mg. per 1 cc., respectively.

The testosterone suspension in an

aqueous solution was supplied by Abbott Laboratories. was in an ampoule containing 60 mg. per 1 cc.

This

This was

diluted with 0.9^ physiological saline solution to the

chick.

1 Autopsy was taken to determine the sex of each They were found to be 98$ cockerels.

p

The author is indebted to Dr. W. H. Stoner of the Schering Corporation, Bloomfield, New Jersey, for the ’’Oreton1 used in this research. 3 The author wishes to acknowledge the kindness of Dr.George Hazel of Abbott Laboratories, North Chicago, Illinois, for the hormone used in these experiments.

32 corresponding values of 2.5 nig., 3-5 nig., and 5-0 nig. per 1 cc.

One series of the experimental chicks was injected

with 0.1 cc. of the aqueous solution and the other received the oil hormone.

Injections were given on alternate thighs

intramuscularly with a 1 cc. tuberculin syringe graduated into 1/100 cc. the injections.

A Yale B-D needle size 25 was used in all The five day old chicks were injected daily

for 5 days at 24-hour intervals.

Chicks younger than 5 days

old have been reported by Frank and Klempner (1937) to have a very high mortality rate when injected as described above. The temperature of the cages without sunlight fluctuated between

96

- 100 degrees Fahrenheit which was regulated by

thermostatic control.

The temperature of the cages of the

chicks kept in the sunlight ranged from 68 degrees to 83 degrees Fahrenheit with an average of 75 degrees during the experiment.

Sanitary cages 48" x 36” x 12" were used

with 1/4 inch wire mesh flooring.

Plenty of chick’s starter

mash and water were available at all times except at weigh­ ing time when they were fasted for the previous 24 hour interval.

Water, however, was taken ad lib. during this

interval. For convenience in maintenance and handling, eight groups of chicks were used for the experiments.

Four groups

of chicks were kept in the sunlight, while the remaining groups were kept in a room where sunlight was totally

33 excluded.

These were further subdivided into groups re­

ceiving different dosages of both the testosterone pro­ pionate in sesame oil and testosterone suspension in an aqueous solution. One group, consisting of some kept in the sunlight and some without sunlight, received a total of 1.25 mg. of testosterone propionate and testosterone suspension of 0.5 cc. in sesame oil and physiological saline solution res­ pectively in 5 days; the second group received a total of 1.75 m g • of testosterone propionate and testosterone sus­ pension in 0.5 cc. of oil and aqueous solution in 5 days; the third group received a total of 2.5 mg. of testosterone propionate and testosterone suspension in 0.5 cc. of oil and aqueous solution in 5 days; and the fourth group, the controls, received a total of 0.5 cc. of oil and physio­ logical saline solution in 5 days. One set of chicks was sacrificed at the end of the 10th day after hatching; another at the end of the 15th day, and the last set at the end of the 20th day. Prior to the sacrificing of the birds of each group at the termination of different periods--5, 10, 15, and 20 days--the combs were measured by a caliper graduated in millimeters.

The measurements were taken as the greatest

posterior-anterior length as well as the height which was measured from the base of the comb to the highest point of

34 the comb.

In order to reduce the amount of error in the

taking of the combs, only one person took the comb measure­ ments during the entire experiment. Measurements of the combs were taken daily for the first five days and every five days thereafter. The body weights of the chicks were taken on the 5th day with the initial application of the hormone.

As men­

tioned previously, all chicks were fasted the previous 24 hours before weighing to insure that the weights were uniform.

This was also done on the 10th, 15th, and 20th

days. In order to avoid confusion, the chicks were tagged prior to the 5th day with different colored bands and metallic tags which were numbered for the different series with and without sunlight. The chicks were sacrificed, five at a time, by placing them in a can which contained a wad of cotton saturated with ether.

A laparotomy was done immediately in 0

order to determine the sex of the chick by inspection of the gonads.

The testes were removed immediately and

weighed to the nearest milligram on an analytical balance.

CHAPTER III RESULTS Body weight.

The daily dose of testosterone pro­

pionate in sesame oil and testosterone suspension in an aqueous solution was 0.25 mg., 0.35 nig., and 0.5 mg. in the different series of chicks with and without sunlight. Despite this difference in dosage, the average body weight of the 5* 10, and 15 day old chicks showed a remarkable uniformity in the averages.

However, on the 20th day, the

controls of series D exceeded the body weights of the ex­ perimental of that series.

In series H, which was kept in

the sunlight, the experimentals were heavier than the controls.

The body weight of the controls and the experi­

mental results of the different series on the 5th, 10th, 15th, and 20th days with and without sunlight are shown on Table I. These results show clearly that sunlight does not affect the body weight of the cockerels greatly during the period of injections with testosterone propionate or testosterone suspension.

In addition, the body weight of

the chicks to the 15th day is fairly constant in relation­ ship to each other, whether with or without sunlight. The physiological effects of radiation on somatic growth of chicks were reported by Bovie (1925).

He showed

36 TABLE I BODY WEIGHT OP DIFFERENT SERIES OF CHICKS INJECTED WITH MALE SEX HORMONES FROM THE 5TH TO THE 9TH DAY AT THE END OF THE 5TH, 10TH, 15TH AND 20TH DAY WITH AND WITHOUT SUN Without sunlight Treatraent Series Control Control 1.25 mg. 1.75 rag. 2.5 rag. Control 1.25 mg. 1.75 rag. 2.5 mg. Control 1.25 mg. 1.75 mg. 2.5 mg.

Age Testosterone in N o .of aqueous ave. N o .of days birds birds in grams

A B

5 10

C

15

D

20

11 71 75 81 33 31 25 29 4-0 11 7 7 6

total -• 321

Control Control 1.25 mg. 1.75 mg. 2.5 mg. Control 1.25 mg. 1.75 mg. 2.5 mg. Control 1.25 m g . 1.75 mg. 2.5 mg.

E F

G

H

11 40.3 47.6 70 46.8 65 46.9 59 47.1 59 30 58.7 60.2 27 62.2 25 62.9 23 80.1 8 72.0 9 6 68.2 6 71.3 total -■ 264

With sunlight 11 11 40.5 36 32 45.1 47.2 32 31 45.8 4-0 30 47.2 34 30 18 15 17 53.5 16 16 57.9 60.6 21 15 59.6 17 15 20 6 70.7 7 6 5 74.3 8 83.7 5 6 76.0 6 total -• 152 total- 135 5 10 10

Testosterone propionate in grams 40.3 45.9 46.7 48.4 46.1 56.9 56.6 63.8 60.4 73-5 74.3 73-1 68.3

40.5 47.2 48.1 45.7 46.0 61.2 63.8 63.6 56.5 77.0 82.0 83.2 80.0

37 that chicks kept in the open sunlight and those exposed to rays of quartz mercury vapor lamp for 20 minutes a day were much larger and heavier than those which received sunlight through the glass roof of a greenhouse.

This was used

clinically in children and similar results occurred. According to Hamilton (1938) male chicks injected with sex hormones up to the age of 7 days do not differ significantly, but as the birds grow older there is considerable difference in the body weights.

He states the controls are heavier

than the experimentals.

This, he believes, is due to the

fact that crowding, feed, and other factors may influence this change.

Breneman (1939) using smaller doses than were

used by Hamilton found that the weight was essentially the same in the control and experimental chicks until after the end of the treatment at which time the controls were heavier than the experimentals.

However, Noble and Zitrin (1942)

reported that male chicks injected with testosterone pro­ pionate did not weigh as much as the controls.

Buckner,

Insko, and Martin (1933) observed that the weight of cockerels at the age of 24 weeks had made no marked increase in body weight as a result of irradiation from the sun. Similar results were reported by Nikolaiczuk and Maw (l94l) showing that somatic differences were very slight when chicks were kept in the sunlight.

According to Taber (19^9)

male chicks injected with pregnant mare serum on the first

38 day of hatching and for periods from one to 33 days there­ after showed no difference in tody weight between the con­ trol and treated birds. Small doses of testosterone propionate were adminis­ tered to young rats by Rubenstein and Kurland (19^1) and they found no significant differences between the body weights of any groups either before or after treatment for 10 or 20 days. The results, herein studied, show that the male sex hormones have little influence in somatic development during the period of injection of chicks without sunlight and con­ firm the results of Breneman (1939).

However, the data show

testosterone propionate and testosterone suspension have some effect on body weight when the birds were kept in the sunlight.

The experimentals are heavier than the controls

at the end of the 20th day. Comb increase.

The chicks in all the series except

the controls, injected with testosterone propionate and testosterone suspension, had received a total of 1.23 nig., 1.75 nig., and 2.5 nig. respectively, at the end of the 5th day of injection, the 9th day after hatching.

The controls

of both series were injected with sesame oil and normal physiological saline solution (0.9$) respectively.

They

received a total of 0.5 cc. during the five days of injec­ tions beginning on the 5th to the 9th day.

39 The earliest indication of comb stimulation became apparent 48 hours after the initial injection of testoster­ one propionate and testosterone suspension at which time the combs appeared turgid, redder, and much thicker than those of the controls.

Daily measurement of the comb for

the first five days showed that there is a regression of the comb 24 hours after the first injection and that the comb begins to grow after the second day of injection. This confirms the report of Domm and Van Dyke (1932) and Breneman (1938b) that there is a “lag" in the development of the combs of the chicks and that an increase in size does not take place until 48 or 72 hours after the initial injection.

Breneman states that there is needed a "priming"

period prior to the increase in the size of the comb.

It

was observed that the greatest increment in height and length of the comb did not occur until the last 24 hours prior to the 10th day. The development of the comb is taken as the square root of the product of the comb length and height as ex­ pressed by Breneman (1937).

This figure aids in the

visualization of actual changes in the dimensions of the comb. It is obvious that injections of testosterone pro­ pionate and testosterone suspension into normal immature cockerels gave varied results.

Tables II and III summarize

40 TABLE II AVERAGE COMB FACTOR FOR 5 DAY WITH TESTOSTERONE AQUEOUS PROPIONATE FROM THE 5TH AT DIFFERENT INTERVALS Age of bird in days injected with Testosterone aqueous 5

OLD CHICKS INJECTED AND TESTOSTERONE TO THE 9TH DAY WITH SUNLIGHT

Comb factor V l x H mm ---------------------------------Dosage in milligrams ---------------------------------Control 1.25 nig. 1.75 mg.

36n

40

31 5.0

5-1

2.5 mg.

3^

5.0

5-1

10

18* 15 6.0(17.6)P 7.2(44)

19 7.4(48)

17 7.8(53)

15

12 8 .1 (58.8 )

10 8.7(74)

13 8.9(78)

11 9-5(86.2)

20

6 8.6(68.6)

6 10.0(100)

8 10.5(110)

6 10.8(111)

Testosterone propionate

32

30

30

32

4.9

5.1

5.4

5.1

10

15 6.1(24.5)

16 7.0(37.2)

15 7.6(40.7)

!5 v 7.6(49)

15

10 7.8(57.1)

11 9.7(90)

10 10.2(88.8)

9 10.3(101)

20

7 8.5(73)

5 11.2(111)

5 11.3(109)

6 11.5(125.

5

n - Number of birds at beginning of experiment * - Number of birds sacrificed p - Percentage increase of comb

4l TABLE III AVERAGE COMB FACTOR OF 5 DAY OLD CHICKS INJECTED WITH TESTOSTERONE PROPIONATE AND TESTOSTERONE SUSPENSION FROM THE 5TH TO THE 9TH DAY AT DIFFERENT PERIODS WITHOUT SUNLIGHT Age of birds in days in­ jected with Testosterone aqueous 5

Comb factor = V

l

x H nun

Dosage in milligrams Control

1.25 mg*

71n 4.7

4.8

10

40* 5.5(17)P

75

1.75 mg.

2.5 mg.

81

83

4.8

4.9

50 6.7(39.6)

52 6.8(41.6)

43 7-3(49)

15

20 6.5(38.3)

18 8.5(80.4)

22 8 .9 (85.4 )

34 9.1(87.7)

20

11 9.0(91.4)

7 10.(108)

7 10.1(110.4)

6 10.5(114.2)

Testosterone propionate 5 10 15 20

70n

65

59

59

5.0

4.9

4.9

4.9

40* 5-5(10)P

38 7.2(47)

34 7-3(49)

36 7.6(57.1)

22 6.1(22) 8 8.4(68)

18 9.1(85.7)

19 9.8(100) 6 9 11.2 (128.5 ) 11.4(132.6)

n - Number of birds at start of experiments * - Number of birds sacrificed p - Percentage of increase of comb

17 10.2 (108.1 ) 6 11.7(138.7)

42 the average amount of increase in the comb factor and the increase by percentage of the birds injected with male sex hormones at the end of the 10, 15, and 20 days. Chicks injected with testosterone suspension.

The

injections of 0.25 mg*, 0.35 mg., and 0.5 mg. from the 5th to the 9th day produced an increase of the comb factor which was much larger for the series in the sunlight in comparison to those without sunlight. The series of chicks in the sun injected with the different dosages showed an average of 17*6$ for the con­ trol, 44$ for the 1.25 mg., 48$ for the 1.75 mg., and 53$ for the series injected with 2.5 mg. at the end of the 10th day.

This is significantly higher than the series in no

sun injected with the same amount of hormone at the end of the same interval and having an increase of 17$, 39*8$, 4l.6$ and 49$.

The series injected with 1.75 mg. in the sunlight

produced an increment of the comb almost as large as that produced by the 2.5 mg. series without sunlight. The average increase in the size of the comb at the end of the 10th day for the series injected with 1.25 mg. of testosterone suspension and testosterone propionate with and without sunlight is 39*6$ and 44$, respectively, more than the controls.

This indicates that the difference in

the comb size is due to the influence of the hormonal action on the comb.

^3 The net increase of the comb factor in per cent shows at the end of the 10th day a progressive change as the dosage was increased accordingly in both series.

However, the in­

crease in the series injected with testosterone suspension in the sunlight was much larger than in those kept without light for the different dosages.

This is of great signifi­

cance inasmuch as all previous experiments have shown that sunlight inhibits the comb response of birds injected with testosterone propionate.

This confirms the preliminary

report made by Wong (19^8 ) that sunlight has little effect on the comb development of birds injected with testosterone suspension in an aqueous medium. No significant change in the comb size of the control for the first 8 days was observed.

Thereafter the combs

increased greatly in size for both series with a smaller slope for the chicks in the sunlight than those without sunlight from the 15-20 days. cally by comparing

Figure 1 shows this graphi­

the percentage of increase of the comb

as a result of the effect of testosterone suspension in both series, with and without sun, at the end of different intervals. Effects of testosterone propionate.

The injection

of testosterone propionate in the same amount was given in the different series of chicks, with and without sunlight.

Figure 1. Difference in comb increase of 5, 10, 15, and 20 day old chicks injected daily from the 5th to the 9th day with testosterone suspension with and without sunlight. Straight line represents chicks in the sunlight; broken line indicates those without sun. Color scheme represents different doses: black, control; green, 1.25 mg.; blue, 1.75 mg.; and red, 2.5 mg.

02

6T

8T" AT

§T

ST

TT

£T

2T

IT OX

6

8

A

9

45 The average increase of the comb factor and the increase in percentage of the different series is shown in Tables II and III. The series of chicks injected with testosterone pro­ pionate

that were kept in the sunlight show that the comb

averages on the 10th day are much smaller than those in the sunlight.

The average increase of the combs of the chicks

at the end of the 10th day in which sunlight is excluded was 10$, 47$, 49$, and 53$, in comparison to those kept in the sun.

The latter had an average increase of 24.5$,

37.2$, 40.7$, and 49$. As noted clearly in Tables II and III, the combgrowth response of the experimental series with and without sunlight exceeds that of the controls.

Figure 2 shows

graphically the difference in the increase of the comb by percentage as a result of the effect of testosterone pro­ pionate in various dosages on the development of the comb at the end of the 10th, 15th, and 20th day with and without sunlight.

Chicks without sunlight responded with an in­

crease of the comb as the dosage of the hormone injected into them was increased.

But the line of response of the

series receiving the same dosage which was kept in the sun­ light resulted in a smaller increase when the amount of the hormone was increased when compared to the series kept in the sunlight.

This confirms the reports of Domm (1930)*

Figure 2. Difference in comb growth of 5, 10, 15, and 20 day old chicks injected daily from the 5th to the 9th day with testosterone propionate with and with­ out sunlight. Straight line represents chicks in the sun; broken line indicates those without sunlight. Color scheme represents different doses: black, control green, 1.25 mg.; blue, 1.75 mg.; and red, 2.5 mg.

ffirfH

1 T i- n

F+Tm

47 Womack and Koch (1930), Womack, Koch, Domm, and Juhn (1931), Gallagher and Koch (1929), Buckner, Insko, and Martin (1934), Lamoreaux (1943), and Wong (1947) that sunlight does have a physiological effect on the development of the comb in chicks when kept in the sun. The results observed in this experiment substantiate the report of Severinghaus and Sikkema (1946) that testos­ terone propionate produced a more prolonged result than testosterone suspension.

It is contrary to the report made

by Abbott Laboratories (1947) that testosterone suspension in an aqueous solution had a prolonged effect in comparison to testosterone propionate.

At the end of the 20th day the

comb factor as well as the percentage of increase of the increment of the comb is greater in all series injected with testosterone propionate in comparison to testesterone suspension. Figures 1 and 2 show that injection of male sex hormones into chicks with and without sunlight resulted in a comb increase as the dosage was increased.

The increase

from the 5th to the 10th day is considered to be due to hormone injected into the birds, but after the 10th day, the comb continued to increase in the experimental series. Two variable factors may influence comb growth during this period: one, comb growth is proportional to the body weight; two, increase in the size of the comb may be due to

48 hormonal action.

This continuation of the comb growth for

several days after cessation of the injections has been observed by Breneman (1938) to continue for several weeks. He believes that this is due to increased secretion of androgenic substances by the bird since such long continued action was not observed in injected capon chicks and is not due to the slow absorption of the retained hormone.

He

explains the slow growth of the early control chicks as being due to: first, the failure of the comb to increase in size according to the hormone secreted at that time; or secondly, to lack of hormone being secreted by the testes. If secretion is taking place only very small amounts are being liberated. Domm (1934) has been reported to have produced larger combs in Brown Leghorn male chicks than those produced with testosterone propionate or testosterone suspension by the injection of gonadotropic hormone over a period from the 2nd to the l8th day.

However, with the cessation of the

injections, there occurred a decrease in size of the comb and normal appearance was reached shortly.

The comb factor

obtained in these experiments in comparison to that of Breneman (1938a, 1942a) is much lower than that obtained by him.

This may be due to the difference in season, as it

has been shown by Domm (1930), Benoit (1927), David (1938), Hain (1938) and Turner (1948) that comb development of

49 chicks varies with the time of the year. The photographs in Figures 3, 3&, 3b* 4a, 4b, 5a, 5b, 6a, 6b, and 7 illustrate some of the characteristic differences between the controls and the experimental chicks at the end of the 10th and 15th day with and without sun­ light.

The birds illustrated on the right of Figure 3

show that there is an increase in the size of the comb in comparison to the control on the left when they are injected with either testosterone propionate or testosterone suspen­ sion and confined indoors without sunlight.

Since the

difference in the development of the comb of both series is slight, it was possible to represent these birds with one photograph. Some of the other secondary sexual characteristics are of interest.

There is a development of the wattles of

the chicks when injected with either hormone in comparison to the control where no growth was observed during the experiments.

The largest wattle developed was found in the

series injected with 2.5 nig. with either testosterone pro­ pionate or testosterone suspension. Another interesting secondary sexual characteristic which was noticed was the crowing of the birds, which occurred on the 11th day after hatching.

This was observed

in the series injected with testosterone propionate. earliest crowing observed in the series injected with

The

Figure 3 Representative chicks of the series without sunlight injected with testosterone propionate and testosterone suspension at the end of the 10th day. Chicks reading from left to right are: control, 1.25 mg., 1.75 mg., and 2.5 mg.

50

FIGORE 3

Plate 1:

Explanation of Figures Figure 3a

Chicks injected with testosterone propionate on the 10th day without sun­ light. Reading from left to right; control, 1.25 mg., 1.75 mg., and 2.5 mg.

Figure 3b Representative chicks of the same series on the 15th day.

51

FIGOEB 3b

Plate 2:

Explanation of Figures Figure 4a

Chicks injected with testosterone suspension in an aqueous solution on the 10th day without sunlight. Reading from left to right: control, 1.25 nig., 1.75 m g ., and 2.5 nig. Figure 4b Representative chicks of the same series on the 15th day.

52

FIGURE 4a

FIGURE 4b

Plate 3:

Explanation of Figures Figure

Chicks injected with testosterone propionate as shown on the 10th day with sunlight. Reading from left to right: control, 1.23 m g ., I .75 mg., and 2.5 nig. Figure 5h Representative chicks of the same series on the 15th day. Chicks in reverse order of above.

53

FIGURE 5a

FIGURE 5b

Plate 4:

Explanation of Figures Figure 6a

Chicks injected with testosterone suspension as shown on the 10th day in the sunlight. Reading left to right: control, 1.25 mg., 1-75 mg., and 2.5 mg. Figure 6b Representative chicks of the same series on the 15th day.

54

FIGURE 6b

55 testosterone suspension was on the 15th day after hatching. At the end of the 20th day most of the chicks injected were found to be crowing.

Domm (1934) observed that crowing

occurred on the ninth day in his experimental chicks.

It

was reported by Hamilton (1938) that injection of White Leghorn chicks with 0.5 mg. of testosterone propionate on the second day and for twenty-seven days thereafter, re­ sulted in crowing at the end of 180 hours after the initial injection.

Zitrin and Noble (1942) observed crowing as

early as the 4th day in birds injected with the same hormone. Hamilton and Dorfman (1939) caused male chicks to crow 60 hours after hatching by one injection of 20 mg. of crystal­ line testosterone propionate.

Crowing in young cockerels

at the age of 9 days was observed by Domra and Van Dyke (1932) when birds were injected with pituitary hebin. These authors noticed that no effect occurred on similarly treated young pullets even though sufficient androgen was produced. Effects of hormones on testes.

Results concerning

the testes of the experimental chicks and controls are presented in Table IV.

These results show clearly that

injection of testosterone suspension and testosterone pro­ pionate into chicks under these experimental conditions was accompanied by an almost immediate cessation of growth of

56 TABLE IV

AVERAGE TESTES WEIGHT OP CHICKS INJECTED WITH TESTOSTERONE SUSPENSION AND TESTOSTERONE PROPIONATE AT THE END OP THE DIFFERENT INTERVALS WITH AND WITHOUT SUN

Treat­ ment

Series

Age in days

Testosterone suspension Ave.wt.of No. of testes in birds milligrams

Testosterone propionate Ave.wt.of No. of testes in birds milligrams

Without sunlight Control Control 1.25 mg* 1.75 mg. 2.5 mg. Control 1.25 mg. 1.75 mg. 2.5 mg. Control 1.25 mg. 1.75 mg. 2.5 mg.

Control Control 1.25 mg. 1.75 mg. 2.5 mg. Control 1.25 mg. 1.75 mg. 2.5 mg. Control 1.25 mg. 1.75 mg. 2.5 mg.

A B

5 10

C

15

D

20

E F

5 10

G

15

H

20

11 4-0 50 52 ^3 20 18 22 34 11 7 7 6

9*74 10.32 6.55 6.87 7.28 11.95 12.01 12.10 12.80 22.4 18.0 19.6 16.0

11 40 38 34 36 22 18 19 17 8 9 6 6

9.74 9.84 7.78 7.63 7.23 11.17 9.91 9.47 9.20 20.1 16.0 19.2 15.5

With sunlight 8.6 11 18 9.01 15 7.9 6.87 19 6.43 17 12 19.1 10 II.36 13.14 13 15 13.1 6 21.0 6 17.6 8 19.0 6 16.6

11 15 16 16 15 10 11 10 9 7 5 6 6

8.6 9.2 6.84 5.6 5.29 17.0 12.5 11.7 11.4 19.0 19.O 18.0 16.3

57 the testes of the birds and that during this period of injections an inhibition occurred.

However, both series F

of the experimental chicks which were kept in the sunlight and injected with either hormone and series B without light and injected with testosterone propionate showed a decrease in the average weight of the testes as the dosage of the hormone was increased accordingly at the end of the 10th day.

Series B, without sun, injected with testosterone

suspension, showed a decrease in testes weight in compari­ son to the control, but the greatest decrease occurred in the series injected with 1.25 nig. of hormone.

As the

dosage was increased there was an increment of weight accordingly for this series.

The fact that this series did

not demonstrate the same phenomena as the others indicates a different reaction to the hormone.

However, since a

sufficient number of chicks were used in this series, this result may be of definite importance. In order to investigate this interesting problem of whether injections of male hormones would inhibit the gonads during the period of injections and after the cessation of the injections, the chicks were sacrificed at intervals of from 5 days to the 20th day. Some variability was indicated by the occasional greater injury to the testes of one male than to another receiving identical treatment.

On the whole, however, a

58 great uniformity was found when equivalent dosages were compared.

In Table IV is presented a summary of the find­

ings of the number of males sacrificed on the 5th, 10th, 15th, and 20th day which were injected with hormones as compared to the controls.

The removal of the testes pre­

vious to injections on the 5th and on subsequent days gives an accurate indication of the effects noted during the period of injections and following periods.

In almost every case,

the testes of birds removed prior to the commencement of the injections were found to be normal. An analysis of Table IV and Figures 7 and 8 shows that the chicks in no sunlight injected with 1.25 mg., 1*75 mg., and 2.5 nig. of testosterone suspension had a decrease in testis weight of 33$, 30$, and 2^.3$, respectively, in comparison to the series which was placed in the sunlight and which had an average decrease of 8$, 21$, and 26$, with similar doses.

It is interesting to note that the

experimental chicks of series C in no sunlight, injected with testosterone suspension, at the end of the 15th day were much heavier than the controls.

All other series

whether injected with testosterone propionate or suspension at the end of the 15th day showed the experimental chicks to be lighter than the controls. On the other hand, injections of testosterone pro­ pionate into chicks indicated that a greater reduction in

59 testis weight occurred in the series which were placed in the sun.

The percentage of decrease as the dosage of the

hormone was increased from 1.25 m g • to 2.5 mg. was 21$, 35$, and 39*0$, respectively, in comparison to the series which received no sunlight and had a decrease of 21$, 22$, and 26$ when injected with the same dose at the end of the 10th day after hatching.

Therefore, this indicates that

testosterone suspension in no sunlight and testosterone propionate in the sunlight had the greatest effect on the testis weight.

The least testicular development in both

series injected with either hormone was found in the series that received sun in the suspension group and in no sun in the propionate group. The experimental data as well as the graphs in Figures 7 and 8 indicate that during the period of injec­ tions from the 5th to the 9th day, the sex hormones inhibit the pituitary gland so that an insufficient amount of gonadotropic hormone is liberated by the gland, and as a result, the testes remain much smaller than in the controls. However, as soon as the injections cease, a larger quantity of gonadotropic hormone is secreted by the pituitary gland which results in an increase of the testes weight on the 15th day in both series of the experimental, (with the exception of the series injected with testosterone propionate which received no sunlight) showing a slow recovery from the

Figure 7 Testis weights expressed as percentage of body weights in chicks injected with testosterone suspension with and without sunlight at the end of the 10th, 15th, and 20th day. Straight line represents series in the sunlight; broken line indicates chicks in no sun. Color scheme represents different doses: black, control; green, 1.25 mg.; blue, 1.75 nig.; and red, 2.5 nig.

Days

L

Figure 8 Testis weights expressed as percentage of body weights in chicks injected with testosterone propionate with and without sunlight at the end of the 10th, 15th, and 20th day. Straight line represents series in the sun; broken line indicates chicks in no sunlight. Color scheme repre­ sents different doses: black, control; green, I.25.; blue, 1.75 and red, 2.5 nig.

02 6t 81 LT

9t ST

CT 2T TT 0T 6

8

L

9

01rTO*

7

62

10th to the 15th day. Prom the 15th to the 20th day, all series of the experimentals showed a rapid increase in testis weight indicating that no damage was done to the testis with the exception of delaying development of the testis during the period of injections. In analyzing the graphs in Figures 7 and 8, the controls of both series, with and without sunlight, injected with testosterone suspension and testosterone propionate show a slight reduction of testes weight when calculated on the mean testis weight as a percentage of the body weight of the chick (Table V).

This decrease on the 10th day of

the control may be due to the fact that androgen is not being secreted at this period since it has been mentioned that secretion takes place after the 10th day by Breneman (1938) and if it is secreted before this time, the amount is probably in very small quantity.

However, during the

period of the 5th to the 10th day, there is a great change in the weight and when the mean testis weight is divided by the body weight, there will a smaller increase. Since Moore and Price (1930) first studied the effects of male hormone on the testicles, there has been a great controversy as to the effect of androgens on the testes.

They found that gonadal hormones suppress the

hypophysis in 30 day old rats and leads to an inhibition of

63

TABLE V MEAN TESTIS WEIGHTS EXPRESSED AS PERCENTAGES OF MEAN BODY WEIGHTS IN 5 , 1 0 , 15 AND 20 DAY OLD CHICKS

Treat­ ment Control Control 1 .2 5 mg. 1 .7 5 mg. 2 .5 mg. Control 1 .2 5 mg. 1 .7 5 mg. 2 .5 mg. Control 1.25 mg. 1 .7 5 mg. 2 .5 mg.

Bird 5 10

15

20

Testosterone aqueous No Sun Sun 0 .2 47 0 .2 1 7 0.1 4 0 0.149 0 .1 5 4 0 .2 0 4 0 .1 9 9 0 .1 9 4 0 .2 1 0 0 .2 7 9 0 .2 5 0 0 .2 87 0 .2 2 4

0.2 1 2 0 .1 99 0 .1 6 8

0.150 0 .1 3 6 0 .3 5 6 0 .1 97 0 .2 15 0 .2 2 0 0 .2 9 6 0 .2 3 6 0 .2 2 7 0 .2 1 8

Testosterone propionate No Sun Sun 0 .2 4 7 0 .2 1 4 0.166 0 .1 5 8 0 .1 5 6 0 .2 20 0.175 0 .1 49 0.152 0 .2 7 4 0 .2 1 5 0 .2 6 2 0 .2 26

0 .2 1 2 0 .1 9 7 0.1 41 0 .1 2 2 0 .1 1 6 0 .2 7 8 0 .1 96 0 .1 8 4 0 .2 02 0 .2 4 6 0.2 3 1 0 .2 1 6 0 .2 0 4

64 the testis with subsequent degeneration.

Such an inhibitory

effect was assumed to be due to lack of hormones secreted by the pituitary gland, resulting in a decrease in weight of the testis.

However, this may be prevented if an occasional

fresh pituitary implant be administered the rat.

Using

synthetically prepared male hormone, the same investigators were able to demonstrate that in normal rats treated with androsterone, the testes of the experimentals were lighter than those in the controls.

A similar condition was also

observed by Greene and Burrill (1940) when they administered androgenic extract of bull testes to immature rats and found that it had an inhibitory effect.

Similar results occurred

when crystalline androsterone, testosterone, and testosterone propionate was injected into the animals.

However, this

inhibition was only temporary as the testes recovered to normal if given sufficient time.

Others who have observed

this reduction in testis weight are Selye and Friedman (1941), Mark and Biskind (1941), and Shay, Gershon-Cohen, Paschkis, and Fels (1941).

According to Rubenstein and

Kurland (1941) the testes of animals treated with small doses of testosterone propionate for 10 days were found to be 17$ heavier than in the untreated rats.

Shay, Gershon-

Cohen, Paschkis, and Fels observed that if they gave large doses of male hormone to rats, there was an increase in testis weight rather than a decrease.

Therefore, they felt

65 that androgenic hormone in large doses stimulates the pituitary gland rather than depressing its activity. However, Greene and Burrill (19^1) deny that the testis may be stimulated by an increase in dosage. It has been previously observed by Domm (1931) on older cockerels that homeoplastic hypophyseal implants caused a hypertrophy of the testes.

The experimental

animals invariably weighed much more than the controls.

In

two groups he mentioned that there was an increase in excess of 800$ In favor of the experimental animals.

Breneman

(1937) reported that chicks Injected with androgenic hormones manifested a decrease in testes weight.

This

observation was also noticed in chicks that were placed on a limited diet.

The testes weight of the dieted chicks was

lighter in comparison to that of the controls.

However,

injection of a male hormone into normal dieted chicks caused a reduction in the weight of the testes.

Morato-Manaro and

Albrieux (1939) reported that application of androsterone locally on the comb of adult cocks caused a marked comb growth but led to a definite decrease of the testis weight and an injury to the organ.

Testis weight Increasing 170$

from the 5th to the 23rd day was reported by Breneman (19^5&) when chicks were injected with gonadotropic hormones. In similar experiments conducted by the same investigator (19^2b) he noticed that chicks administered estrone had a

66 substantially lowered weight of the testes at the end of the 15th day, however, prolactin did not inhibit the testes as greatly as estrone.

Breneman (1941b) also mentioned

that White Leghorn chicks injected with estrone and stilbestrol caused a loss of testes weight as much as 35$ by estrone and 60$ by stilbestrol.

According to Bouvier,

Sartori, and Torres (19^9) implantation of 0.015 mg. of stilbestrol under the skin of the neck of four White Leghorn cockerels caused a complete atrophy of the testes within two and a half months. Injuries to the testes with subsequent reduction in weight has been reported to be observed in other species when androgenic hormones were administered.

Chu (1940)

noticed that injections of 2 mg. daily for 30 days of testosterone in pigeons resulted in loss of testis weight. According to Bottomley and Folley (1938) injections of hormones into young guinea pigs resulted in an injury to the testes.

Wells (1943) observed that injection of

testosterone and testosterone propionate into young ground squirrels with a daily dose of 0.05 to 20.0 mg. for 18-20 days resulted in the inhibition of the testes in the major­ ity of the cases.

Lahr and Riddle (194-4-) noticed that ten

daily injections of different steroid hormones into mature ring doves resulted in the reduction of testis weight by as much as 14-22$.

67 Another interesting observation noted during the course of the experiment was the larger size of the left testis than the right at the time of autopsy at the end of the 5th, 10th, 15th, and 20th day of all series.

This

tendency was noted in embryonic chicks by several authors. Firket (1914) reported that the left testis on the seventh day of incubation was much smaller than the right in the chick.

According to Swift (1916) the left embryonic testis

was much larger than the right one in the five day old chick and the difference of the testes remained in favor of the left testis in the six and nine day chicks.

Riddle

(1916) found no essential difference occurred between the left and right testes in common fowl. mention the age of the birds.

However, he does not

It was reported by Domm and

Juhn (1927) that the left testes were larger than the right in 50 cases of control cockerels up to the age of 24 weeks, and at 32 weeks the left testes were larger in two out of every three pairs.

DISCUSSION Considering the data obtained from these experiments, it is evident that injections of testosterone propionate and testosterone suspension in chicks with different doses — 1.25 mg., 1.75 nig., and 2.5 nig. — growth in the size of the comb.

caused considerable

As the dosage was increased

68 an increment of the comb occurred.

However, the combs of

chicks injected with testosterone propionate placed in the sun had a smaller comb factor than similarly treated birds that were kept where sunlight was excluded.

This is in

accordance with the results of the majority of investigators who found that sunlight inhibited the comb response of capons and cockerels. In the literature, only one postulation of the mechanism which explains the hormonal effect of the combgrowth stimulation between lack of sunlight and sunlight has been made, and that by Womack, Koch, Domm, and Juhn (1931) who believe that the hormone in vivo is destroyed by the light.

On the other hand, Lamoreaux (19^3) states

that ultra violet rays and artificial light had no differ­ ent effects on the comb size.

In his experiments, he did

not use any male hormones. The data indicate

the effects of testosterone

suspension demonstrate that the opposite result occurred in birds with and without sunlight.

The chicks in the sun­

light had greater comb enlargement in comparison to those without sunlight.

This suggests that the theory of Womack,

Koch, Domm, and Juhn may satisfactorily account for the male hormone, testosterone propionate, in that sunlight may inactivate the hormone.

Chicks injected with testosterone

suspension, on the other hand, showed an increase of the

69 comb regardless of sunlight and this is probably due to the crystallization of the suspension at the point of injection so that this method is similar to the implantation method as used by several investigators. Hardesty (1931) reported her studies of the tissue of the comb from a systematic microscopic and macroscopic study of the development of the comb from the embryo to the adult male and female and showed the gradual differentia­ tion of comb tissue under the influence of the gonadal hormone of a sexually maturing bird.

It is concluded that

the hormone causes the secretion of large quantities of mucoid by the cells of the intermediate layer, and that the presence of this mucoid is primarily responsible for the other changes which appear.

Comparison of the comb of lay­

ing and non-laying hens suggests that the female differs only quantitatively from the male in hormonal effect upon the comb.

Autoplastic comb grafts have been made.

Castra­

tion and injection bring the same histological results in the grafts and stumps as in normal combs.

The stumps re­

maining after amputation do not regenerate. Kochakian (193$) made a careful study of the effectiveness of various compounds of testosterone on the castrated rat.

His results indicate there are specific

differences between the hydrolysis of some of the compounds in birds and in the rat.

70 The results show that there is a relationship between the comb and the testes.

As the dosage of the hormone was

increased there was a corresponding increase in the comb, but a decrease of the testes weight occurred.

However, in

one series injected with testosterone suspension in the sunlight, there was a decrease in testis weight in compari­ son with the controls.

Nevertheless, as the dosage was

increased, the testis weight also was slightly increased. Hoskins and Koch (1939b) believed this interrela­ tionship was expressed by three organs as follows: Pituitary s = = = s . Testis Comb Zawadowsky (1935) explains this reciprocal relation­ ship as based as follows: Pituitary

Testis

.x. Comb

He suggested that the appendages of the head elicited a substance which inhibits the development of the testes.

He

also mentioned that the removal of the furnishings of the head resulted in a loss of heat producing area, thus caus­ ing a change in body temperature which, in turn, is reflected in testes enlargement. never been corroborated.

This supposition has

71 Landauer (1942) confirmed the report that removal of the comb causes a testicular development.

Buckner, Insko,

and Martin (1933) reported that removal of the comb resulted in an increase of the wattles as compared to those of the controls.

They interpret this enlargement of the wattles

as being caused by a compensatory mechanism of the comb. Nikolaiczuk and Maw (1941) however, deny that such a rela­ tionship exists.

According to Landauer (1942) the changes

caused by the removal of the comb are due to trauma. Marlow and Payne (1940) reported that the body weight of chickens is increased in birds in which the head appendages had been removed and removal of the comb resulted in the testes weight being doubled.

This increase in body weight

of the cockerels that have been dubbed was confirmed by Nikolaiczuk and Maw (1941). According to Marlow (1948) the removal of the combs of three-day-old chicks resulted in a significant increase in the size of the testes, but the increase in the wattles was not very great.

Also he found that topical application

to the comb of a chick of a purified alcoholic extract of rooster combs resulted in an enlargement of the testes and combs over that in the controls.

Andrew and Erbs (1943)

claim that testicular weight of White Leghorn chicks injected with gonadotropic hormone for ten days, commencing on the 2nd day, was more than three times that of the controls,

72 while testicular weight of birds receiving a similar number of injections on the 29th day was less than twice that of the controls. In a study of the effect of prolactin on broodiness and on the cock's testes, Nalbandov and his associates (19^5 ) found this hormone inhibits the development of the gonads by shutting off the hypophyseal follicle stimulat­ ing hormone.

There is a reduction of testes weight, and

accompanying this is a decrease in the androgenic activity which, in turn, is marked by the absence of crowing, tread­ ing, and the lack of comb growth. Fiske (1939) observed the effects of artificial light and darkness on the activity of the pituitary of rats. Male rats which were kept in the light had heavier testes than the males which received no light.

According to

Burger (19^) light does not have an effect on the testes directly.

However, he found that injection of testosterone

propionate in sparrows produced within two weeks an in­ crease in the size of the testes which was followed by a return to the original size or smaller.

SUMMARY AND CONCLUSIONS The effects of testosterone propionate and testoster­ one suspension injected in several groups of 5 day old chicks

73 in doses totaling 1.25 mg., 1.75 mg.., and 2.5 mg. over a period of 5 days showed that: 1.

The chicks in the dark with sunlight excluded

demonstrated a greater increment of the comb than those that were placed in the sunlight when injected with testos­ terone propionate.

At the end of the 10th day, the series

of chicks which were in the sun and injected with testos­ terone suspension had a comb increment of 17.6$ for the control, 44$ for the 1.25 mg., 48$ for the 1.75 mg., and 53$ for the series injected with 2.5 mg. as compared to the series that were in no sunlight and had similar dosages had an average comb increase of 17$, 39*6$, 41.6$ and 49$.

In

comparison, the comb increase was larger than in the series injected with testosterone propionate in the sunlight. However, the series with no sunlight had a smaller comb factor than the testosterone propionate series.

The series

placed in the sun and injected with propionate had an average increase of 24.5$, 37*2$, 40.7$ and 49$ when injected with 0.5 cc. of sesame oil, 1.25 mg., 1.75 mg., and 2.5 mg. of hormone, respectively, in comparison to the series in no sunlight which had an increase of 10$, 47$, 49$ and 53$ at the end of the 10th day.

As the dosage was

increased there was a greater development of the combs in comparison to the controls.

This was also observed for the

15 and 20 day series of chicks.

74 2.

With the commencement of injections on the 5th

day, there was an immediate interruption of the development of the testes of the chicks which received hormones and this delay of the growth of the organ remained during the period of injections.

As soon as the injections ceased,

there was an immediate increase in size of the testes which continued to develop.

In chicks that received no sunlight

and which were injected with testosterone suspension in the different doses —

1.25 nig., 1.75 mg., and 2.5 mg. -- there

was a greater decrease in testis weight which averaged 33$, 30$, and 24.3$, respectively, in comparison to the series that were placed in the sun which had an average decrease of 8$, 21$, and 26$, respectively, with similar doses.

It

was interesting to note that the series of chicks receiving no sunlight injected with testosterone suspension had testes that were heavier than the controls at the end of the 15th day.

All other series were lighter than the controls. However, chicks injected with testosterone pro­

pionate had a greater reduction in the weight of the testis in the series that were placed in the sunlight.

The per­

centage of decrease in weight as the dosage was increased from 1.25 mg. to 2.5 mg. was 21$, 35$, and 39$, respectively. In comparison, at the end of the 10th day, the series which received no sunlight had a decrease of 21$, 22$, and 26$ when injected with the same dose.

75 3.

With the cessation of injections, the combs of

the cockerels continued to develop.

This indicated that

the androgenic substance necessary for the growth of the comb was produced by the testes of the chicks. 4.

There was a marked development of the wattles in

the series with and without sunlight when injected with either hormone as compared to the controls which had no development. 5.

Weight gained in the group of chicks in the

absence of sunlight was not significantly different in the experimentals and controls in the sun.

However, after the

cessation of the injections, it appears that the controls weighed slightly more than those that were injected. Conclusions.

In conclusion, the foregoing experi­

ments showed that light played an important role in the bio-assay of the male sex hormone, testosterone propionate. It appears that sunlight inhibits the comb growth of chicks kept in the sun when compared to those without sun­ light.

On the other hand, sunlight does not have any

influence on the combs of chicks in the bio-assay of testosterone suspension in an aqueous solution.

B I B L I O G R A P H Y

76 BIBLIOGRAPHY Allee, W.C., N.E. Collias, and C.Z. Lutherman 1939 "Modification of the social order in flocks of hens by the injection of testosterone propionate," Physiological Zoology, 12:412-440. Andrews, P.M., and R. E. Erb 1943 "Ascorbic acidgonadotropic hormone relationship in the chick," Endocrinology, 32:140-144. Anonymous 19^7 MProlonging the effect of sex hormone preparations," What1s New published by Abbott Labora­ tories, North Chicago, Illinois, Number 115. Asmundson, V. S., and M. J. Wolfe 1935 "Effect of pregnant mare's serum on the immature fowl,” Proceedings of the Society for Experimental Biology and Medicine, 32: 1107-1109. _______ , C. A. Dunn, and A. A. Klose 1937 "Some response of the immature female fowls to injections of mare gonadotropic hormones and oestrin," Poultry Science, 16:194-206. Bates, R. W., E. L. Lahr,and 0. Riddle 1935 "The gross action of prolactin and follicle stimulating hormone on mature ovary and sex accessories of the fowl, " American Journal of Physiology, 111:361-368. Benoit, J. 1927 "La croissance conditioned de la crete chez des poulets de race leghorn etudiee a diverses epoques de 1'anee," Comptes Rendus de la Societe de Biologie, 97:279-282. _______ , 1929 nLe determinsme des caracteres sexuels secondaire du coq domestique," Archives de Zoologie Experimentale et Generate, Paris, 69:1-49. Berthold, C. 1849 "Transplantation der hoden," Archiv fur Anatomie und Physiologie, S . 42. Bernstorf, E. C. 1948 "Hepatic inactivation of testicu­ lar androgen in White Leghorn cockerels," Proceedings of the Society for Experimental Biology and Medicine,

77 Blyth, J. S. S. 1927 "Some observation on the relation between comb growth and gonadic structure on the male of the domestic fowl," Proceeding of the Royal Physical Society, 21:173-163. _______ , E. C. Dodds, and E. I. Gallimore 1931 "Observa­ tions on the assay of comb growth promoting hormone," Journal of Physiology, 73:136-140. Bottomley, A. C., and S. J. Folley 1936 "The effects of high doses of androgenic substances on the weights of the testes, accessory reproductive organs, and endo­ crine glands of young male guinea pigs," ibid., 94:26-39. Bovie, W. T. 1925 11The physiological effects of radiation -- the electromagnetic spectrim," The Boston Medical and Surgical Journal, 192:1035-1037. Bouvier, G., D. Sartori, and F. A. Torres 1949 "Ensaios de castracao quimica de frangos," Biologico, San Paulo, 15:31-34. Breneman, W. R. 1935 "The effect of fractionated and unfractionated gonadotropic preparations on testes and comb growth in the chick," Anatomical Record, 64: Supplement 1. _______ , 1937 "Male hormone and the testis-comb relation­ ship in the chick," Endocrinology, 21:503-510. _______ , 1938a "Relative effectiveness of testosterone propionate and dihydroandrosterone-benzoate in the chick as indicated by comb growth," ibid.,23:44-52. _______ , 1938b "Some aspects of male hormone secretion in White Leghorn chicks," Indiana University Publication, Science Series, 7 . _______ , 1939 "Variations in the reaction of chicks to different methods of administering androgens," Endocrinology, 24:55-62. _______ , 1940a "Limitation of food consumption as a factor influencing endocrine reactions in the chick," ibid., 26 :1091-1098 . _______ , 1940b"Response of pullets to androgen," Poultry Science, 19:147-153.

78 Breneman, ¥. R. 194la "The effectiveness of androgens during inanition in the chicks," Endocrinology, 28 :222-228 . _______ , 194 lb "Comparative action of estrone and stil­ bestrol on testes and comb growth in the chick," Anatomical Record, 84: Supplement 30_______ , 1942a "The chick comb for androgen assay," Endocrinology, 30:277-285. _______ , 1942b "Action of prolactin and estrone on weights of reproductive organs and viscera of the cockerels," ibid., 30:609“6l5. _______ , 1945a "The gonadotropic activity of the anterior pituitary of cockerels," ibid., 38:190-199. _______ , 1945b "Response of chicks to pituitary gonado­ tropins and pregnant mare serum," Proceedings of the Indiana Academy of Science, 5^: 207-213Buckner, G. D., W. M. Insko, Jr., and J. H. Martin 1932 "The effects of confinement on growth of chicken combs and testes," American Journal of Physiology, 102:271275. _______ , _______, _______ 1933 "Abnormal growth of wattles and testes of cockerels after removal of combs," ibid., 103:647-650. _______ , _______, _______ 1934a "Sex characters in battery brooder and colony raised White Leghorns," Poultry Science, 71:3^5 _______ , J. H. Martin, and W. M. Insko, Jr. 1934b "The effect of confinement brooding on growth and egg production," Bulletin Number 346, Kentucky Agricultural Experimental Station. Burrows, W. H., T. C. Byerly, and E. I. Evans 1938 "Sensitivity of the baby comb to male sex hormone," Proceedings of the Society for Experimental Biology and Medicine, 35:50-52. Callow, R. K . , and A. S. Parkes 1935 "Growth and main­ tenance of the fowl’s comb by administration of androsterone," Biochemical Journal, 29:l4l4-l423.

79 Caridroit, F. 1942 ”La vitamine E abaisse le seuil de response de la crete du chapon a 1'hormone male,” La Revue Scientifique, 80:31* _______ , and V. Regnier 193$ "Determination du seuil de conditionment total de la crete du coq domestique," Comptes Rendus de la Societe de Biologie, 129:445-447Ghu, J. P. 1940 "Effects of estrone and testosterone and of pituitary extracts on the gonads of hypophysectomized pigeons," Journal of Endocrinology, 2:21-37* Danby, M. 1938 "The assay of male hormones by the chicken test," Acta Brevia Neerlandica, 8:90. David, K. 1938 "Uber de einfluss der saison auf die empfindlic-hkect das hahnenkamnes," ibid., 8 :133-136. Deanesly, R., and A. S. Parkes 1937 "Factors influencing the effectiveness of administered hormone," Proceedings of the Royal Society, London, B, 134:279-297* Dessau, F. 1937 "Wertere untersuchungen uber die wirksamkeit kammwachstumstoffen irn schmierversuh," Acta Brevia N eerlandica, 7:1-4. Domm, L. V. 1927 "New experiments on ovariotomy and the problem of sex inversion in the fowl," Journal of Experimental Zoology, 48:31-174. , 1930 "A factor modifying growth of head furnish­ ings in Leghorn fowls," Anatomical Record, 47:314. _______ , and M. Juhn 1927 "Compensatory hypertrophy of the testes in Brown Leghorns," Biological Bulletin, 52:458-473* _______ , 1931 "Precocious development of sexual characters in the fowl by homeoplastic hypophyseal implants. I. the male," Proceedings of the Society for Experimental Biology and Medicine, 29:308-309. _______ , 1934 "The precocious development of sexual characters in the male chick by daily injections of hebin," Anatomical Record, 58: Supplement 6. _______, 1937 "Observations concerning anterior pituitary gonadal interrelations in the fowl," Cold Spring Harbor Symposia on Quantitative Biology, 5:241-257*

80 Domm, L. V., and H. B. Van Dyke 1932 "Precocious ment of sexual characters in the fowl by daily tions of hebin, the male hormone," Proceedings Society for Experimental Biology and Medicine, 349-350.

develop­ injec­ of the 30:

_______ , _______ 1933 "The precocious development of sexual characters in the fowl by daily injections of hebin," Science. 77:456-457. Dorfman, R. X. 1947 “Studies on the bioassay of hormones: The relative potencies of testosterone propionate and testosterone-17-B-diethyl amino ethyl carbonate hydro­ chloride," Endocrinology, 41:451-455. _______ , 1948 "Studies on the bioassay of hormones. The relative potency of the combs of various breeds of chicks to androgens," ibid., 42:7-14. _______ , and W. W. Greulich 1937 "Response of the chick's comb to naturally occurring androgens and estrogens," Yale Journal of Biology and Medicine, 10:79-88. Duff, P. A., and H. H. Darby 1941 "The chick comb response to androgens," Endocrinology, 28:643-647Emmens, C. W. 1938 "Maximum growth rate of capon comb," Journal of Physiology, 93:413-415_______ , 1939 "Reports on biological standards," Medical Research Council (British), Special Reprint Series, 2WEngel, P. 1939 "Untersuchungen uber den hahnenkammtest zum nachweise des mannlichen hormon," Archives Internationales de Pharmacodynamie et de Therapie, 61:354-359. Finlay, G. F. 1925 "Studies in sex difference in fowls," British Journal of Experimental Zoology, 2:439-^68. Firket, J. 1914 "Recherches sur I'organenese des glandes sexuelles chez les oiseaux," Archives de Biologie, 29:201-352. Fiske, V. M. 1939 "The effects of light and darkness on the activity of the pituitary of rats," Proceedings of the Society for Experimental Biology and Medicine,

407189-191.

81 Prank, R. T., and E. Klempner 1937 "The comb of the baby chick as a test for the male sex hormone," ibid., 36:763-765. _______ , _______ , and P. Hollander 1938 "The comb of the baby chick as a test for the male sex hormone," ibid., 38:853-856. _______ , _______ , _______ and B. Kriss 1942a "Detailed description of technique for androgen assay by the chick comb method," Endocrinology, 31s63-70. _______ , _______ , _______ , _______ 1942b "Report on assays of known quantities of androsterone by the chick method," Endocrinology, 31:71-73. Freud, J., 1930 "On the biological tests for the male hormone," Proceedings of the Second International Congress for Sex Research, 3047 _______ , P. de Premery, and E. Laqueur 1932 "Eichung des mannlichen hormones mit hilfe der kammwachstumsreaktion," Pflugger's Archiv fur dies Gesmate Physiologie, 239:763-7867 Funk, C., and B. Harrow 1929 "The male hormone," Proceedings of the Society for Experimental Biology and Medicine, 267325-326. _______ , _______ , and A. Lejwa 1930 "The male hormone," American Journal of Physiology, 92:440-449. Fussganger, R. 1934 "Ein Beitrag zum wirkunamechanismus des mannlichen sexualhormons," Medizin und Chemie, 2:94-204. Gallagher, T. F., and F. C. Koch 1929 "Testicular hormone," Journal of Biological Chemistry, 84:495-500. _______ , ________ 1930 "The quantitative assay for the testicular hormone by the comb growth reaction," Journal of Pharmacology and Experimental Therapy, 40:327-339• _______ , ________ 1935 "The quantitative assay for the testicular hormone by the comb growth reaction," ibid., 55:97-117.

82 Gradstein, S. 1935 "A photo-electric method of measuring the comb of capons in the assay of male hormone," Archives Internationales de Pharamcodynamie et de Therapie, 51:113Greene, R. R., and M. W. Burrill 1940 "Recovery of testes after androgen induced inhibition," Endocrinology, 26:516-518. _______ , ________ 1941 "Effects of large amounts of androgen on testes of prepuberal rats," ibid., 29: 64-72. Greenwood, A. W., and F. A. E. Grew 1927 "On the quanti­ tative relation of comb size and gonadic activity in the fowl," Proceedings of the Royal Society of Edinburg, 47:190-1987 , and J. the site of response in Physiology,

S. S. Blyth 1935 "On the relation between injection of androsterone and the comb the fowl," Quarterly Journal of Experimental 25:267-277-

_______ , _______ , and R. K. Callow 1935 "Quantitative studies on the response of the capon’s comb to andro­ sterone," Biochemical Journal, 29:1400-1413. Hain, A. M. 1938a "The effect of temperature on the response of Bantam capons to androsterone," Quarterly Journal of Experimental Physiology, 28:353-355, 1938b "Comb atrophy after adult castration (Bantam Cocks)," ibid., 28:349-352. Hall, S. R., and L. P. Dryden 1939 "Dose-response rela­ tionship of androsterone by direct application to the capon’s comb," Proceedings of the Society for Experi­ mental Biology and Medicine, 41:378-382. Hamilton, J. B. 1938 "Precocious masculine behavior following administration of synthetic male hormone substance," Endocrinology, 23:53~57_______ , and R. I. Dorfman 1939 "Influence of the vehicle upon the length and strength of the action of the male hormone substance, testosterone propionate," ibid., 24:711-719-

83 Hardesty, M. 1931 "The structural basis for the response of the comb of the Brown Leghorn fowl to sex hormones,” American Journal of Anatomy, 47:277-323* Herrick, E. H., and C. H. Lockhart 1940 "The effect of male sex hormone on the developing ovaries of young fowls,” Endocrinology, 26:508-510. Hill, R. T., and A. S. Parkes 1934 "Hypophysectomy of birds. III. Effect on gonads, accessory organs, and head furnishings,” Proceedings of the Royal Society, London, B, 116:221-236. Hoskins, W. F., and F. C. Koch 1939a "Inhibition of comb growth in cockerels and capons by estrone,” Endocrin­ ology, 25:266-274. _______ , _______ , 1939b "Reciprocal nature of the testis-comb relationship,” ibid., 25:257-265. , G. W. Beach, J. R. Coffman, and F. C. Koch "The baby cockerel test for androgens," ibid., 28:651-653.

1941

Imig. C. J., J. D. Thompson, and H. M. Hines 1948 Testicular degeneration as a result of microwave irradiation,” Proceedings of the Society for Experimental Biology and Medicine, 69:3B2~-386. Itho, M., and T. Kon 1935 "Action de l'hormone male sur les organes genitaux de jeunes chiens males,” Comptes Rendus de la Societe de Biologie. 120:678-681. Jones, D. G., and W. F. Lamoreaux 1943 "Estimation of the size of comb on live fowl,” Endocrinology, 32:356-360. Juhn, M., F. D ‘Amour, and E. B. Womack 1930 "The effects of simultaneous injections of the female and male hormones in capons,” American Journal of Physiology, 95:641-649. _______ , R. G. Gustavson, and T. F. Gallagher 1932 "The factor of age with reference to reactivity to sex hormones in fowls," Journal of Experimental Zoology, 64:133-188. Koch, F. C. 1937 "The male sex hormones,” Physiological Reviews, 17:153-238.

84 Kochakian, C. D. 1938 "The comparative efficacy of various androgens as determined by the rat assay method," ibid., 22:181-192. Kosin, I. L., 1942 "Observations on effect of esterified androgen on sex eminence of the chick," Endocrinology, 30:767-772. _______ , and S. S. Munro 1942 "Effect of sex hormones separably and combined on the proliferation and hydration of combs and cloacae of male chicks," ibid., 30 :102-106 . Lahr, E. L., and 0. Riddle 1944 "The action of steroid hormones on the mature dove testis," ibid., 35: 261-266 . Lamoreaux, ¥. P., and D. G. Jones 1942 "The effects of dubbing White Leghorn males," Poultry Science, 21:437-444. _______ , 1943 "Effect of differences in light and tempera­ ture upon the size of combs of White Leghorns," Endocrinology, 32:497-504. Landauer, W. .1942 "Experiments concerning supposed influence of cock's head appendage on size of testes," ibid., 31:78-83. Loewy, A. 1903 "Neure untersuchungen zur physiologie der geschlechtsorgane," Ergebnisse der Physiologie, 2:130-158. Mark, J., and G. R. Biskind 1941 "The effect of long term stimulation on male and female rats with estrone, estradiol-benzoate, and testosterone propionate administered in pellet form," Endocrinology, 28:465-477. Marlow, H. W., and L. P. Payne 1940 "A hormone study of dubbed and normal cockerels," Poultry Science, 19:401-402. , 1948 53771-77.

"Comb-testis relationship," Endocrinology,

McCullagh, D. R., and W. K. Cuyler 1939 "Response of the capon's comb to androsterone," Journal of Pharmacology and Experimental Therapy, 66:379-388.

85 McCullagh, D. R., and R. Guillet 1941 "The use of the chicks for assays of androgens," Endocrinology, 28:648-650. McGee, L. C. 1927 "The effects of the injections of a lipoid fraction of hull testicles in capons," Proceedings of the Institute of Medicine, Chicago, 2:242. _______ , M. Juhn, and L. V. Domm 1928 "The development of secondary sex characters in capons by Injections of extracts of bull testes," American Journal of Physiology,

87 : 406- 435 Miescher, K., A. Wettstein, and E. Tschopp 1936 "The activation of male hormone," Biochemical Journal, 30:1977-1990. Morato-Manaro, J., and A. Albrieux 1939 "The effects of sex hormones in combs of castrated normal cocks," Endocrinology, 24:518-522. _______ , _______ , and W. Buno 193$ "Wirkung der sexualhormone auf den hahnenkamm," Klinische Wochenschrift, 17:784-785. Moore, C. R., and D. Price 1930 "The question of sex hormone antagonism," Proceedings of the Society for Experimental Biology and Medicine, 28:38-40. Nalbandov, A. V., M. Hochhauser, and M. Dugas 1945 "A study of the effect of prolactin on broodiness and on cock testes," Endocrinology, 36:251-258. Nikolaiczuk, N., and W. A. Maw 1941 "A preliminary study of the effect of sunlight, dubbing, and fractionated anterior pituitary extract upon growth, endocrine glands, and sexual capacity of single comb White Leghorn cockerels," Poultry Science, 21:483-496. Noble, G. K., and A. Zitrin 1942 "Induction of mating behavior in male and female chicks following the injec­ tion of sex hormones including notations on body weight and comb growth," Endocrinology, 30:327-334. Ogata, A., S. Hirano, and S. Tanaka 1934 "Uber auswertung des mannlichen sexualhormons nach der hahnenkamm methode," Journal of Pharmaceutical Society, Japan, 54: 49. Cited by R. C. Gustavson in Sex and Internal Secretion, Baltimore: Williams and Wilkins Company, 1939, p. 879.

86 Ogata, A., and Y. Ito 1933 "Eine neue methode zur ausmessug den hahnenkamm wachstum-koeffizienten,11 Ibid., 53:39* Cited by R. C. Gustavson in Sex and Internal Secretion, Baltimore: Williams and Wilkins Company, 1939~, 879* Oestring, R. B., and B. Webster 1938 nThe sex hormone excretion of children/’ Endo cr in o1ogy, 22:307* Oslund, R. M. 1928 ’’Source of the testicular hormones,” Proceedings of the Society for Experimental Biology and Medicine, 25:8^5r8¥57 1

'



— p.*T—

A

1 "7**1

11 —

i i1mami ana

i i■

■■ip i■i■ ■ ■■ ■



mium

■ ■ ■

■! ■ «■

Parkes, A. S. 1937 ’’Androgenic activity of ovarian extracts,” Nature, 139:965* Parker, J. E., F. F. McKenzie, and H. L. Klempster 1942 ’’Development of the testes and combs of White Leghorn and New Hampshire cockerels,” Poultry Science, 21:35-44. Pezard, A. 1911 ”Sur la determination des caracteres sexuels secondaires chez les gallinaces,” Comptes Rendus de la Societe de Biologie, 153:1027* _______ , 1918 ”Le conditionment physiologique des caracteres sexuels secondaires chez les oiseaux,” Bulletin Biologie, Paris, 52:76. _______ , 1920 ’’Secondary sexual characteristics and endocrinology,” Endocrinology, 4:527-540. Rakoff, A. E., K. E. Paschkis, and A. Cantarow 1944 ’’Androgen assay on three day old male chicks,” Proceed­ ings of the Society for Experimental Biology and Medicine, 55:125-127. Riddle, 0. 1916 "Size and length relation of the right ard left testis of pigeons in health and disease,” Anatomical Record, 11:87-102. Rubenstein, H. S., and A. A. Kurland 1940 "Response of the testis to small doses of testosterone propionate," Endocrinology, 27:461-462. _______ , _______ , 1941 "The effect of testosterone pro­ pionate on the rat testis,” ibid,, 28:495-505. Ruzicka, L. 1935 "L*hormone masculine et sa preparation artificielle au laboratorie," Bulletin de la Societe Chimique de France, 5:1497-1512.

87 Ruzicka, L. 1937 20:69-79.

“The male sex hormones," Chemical Review,

Sehoeller, W., and M. Gehrke 1938 Tierphysiologische versuche uber die wirkung mannlicher keimdrusenhormone," Klinische Woehensehrift, 17:694-699* Selye, H., and S. Friedman 1941 "The action of various steroid hormones on the testis," Endocrinology, 28: 129-140. Severinghaus, E. L., and S. Sikkema 1946 "Therapy with aqueous suspensions of testosterone," Journal of Clinical Endocrinology, 6:415-420. Shockaert, J. A. 1933 "Difference between anterior pituitary sex hormones and pregnancy urine substances as tested on the male mammal and bird," American Journal of Physiology, 115:497-507. Simpson, M. E., C. H. Li, and H. E. Evans 1942 "Comparison of methods for standardization of pituitary interstitialcell-stimulating hormone (ICSH)," Endocrinology, 30: 977-984. Snedecor, J. G. 1949 "A study of some effects of sex hormones on the embryonic reproductive system and comb of the White Leghorn chick," Journal of Experimental Zoology, 110:205-246. Souba, A. J. 1923 "Variations and correlations of the organs of single comb White Leghorn cockerels," Anatomical Record, 26:291-297. Swift, C. H. 1916 "Origin of the sex cords and the definitive spermatagonia in the male chick," American Journal of Anatomy, 20:373-^10. Taber, E. 1949 "The sources and effects of androgen in the male chicks treated with gonadotropins," American Journal of Anatomy, 85:231-252. Turner, C. W. 1948 "Influence of season on comb develop­ ment of chicks following oral administration of methyl testosterone," Poultry Science, 27:553-557. Walker, C. E. 1908 "Influence of the testis upon the secondary sexual characteristics of fowls," Proceedings of the Royal Society of Medicine and Pathology, Section 1, 153*

88 Wells, L. J. 1943 "Effects of large doses of androgen on the testis in the ground squirrel, Citellus trldecelineatus," Endocrinology, 32:455-562. Womack, E. B., and P. C. Koch 1930 "Biochemical studies on the yield of testicular hormone from tissues and body fluids and on factors affecting the comb growth responses in Brown Leghorn capons," Proceedings of the Second International Congress for Sex Research, 329* _______ , P. C. Koch, L. V. Domm, and M. Juhn 1931 "Some factors affecting comb-growth response in Brown Leghorn capons," Journal of Pharmacology and Experimental Therapy, fl:173-175'. Wong, H. Y. C. 1947 "The effects of testosterone propionate in various dosages on comb-growth of young chicks with and without sunlight," Unpublished material, University of Southern California, Department of Zoology. _______ , 1948 "The effects of testosterone suspension in various dosages in young immature chicks with and without sunlight, ibid. Zawadowsky, M. M. 1935 "The influence of secondary sexual characters on the gonads," Animal Breeding Abstracts, 3 (4): 434. Zitrin, A. 1943 "Induction of male copulatory behavior in a hen following administration of male hormone," Endocrinology, 31:690.

ot S o u t h e r n Caiifemta life*.-