Genetic Variation in Responses to Repeated Administrations of ACTH and Hydrocortisone in Immature Chickens

Genetic Variation in Responses to Repeated Administrations of A C T H and Hydrocortisone in Immature Chickens H. S. SIEGEL1 AND P. B. SIEGEL Virginia Polytechnic Institute, Blacksburg (Received for publication January 14, 1966)

B

1

Present address: Southeast Poultry Research Laboratory, ARS, U.S.D.A., 934 College Station Road, Athens, Georgia.

were investigated for several criteria of response to ACTH and hydrocortisone. EXPERIMENTAL METHODS Two experiments were completed. In experiment 1, 60 male chicks from four genetic lines maintained at the Virginia Agricultural Experiment Station were randomized into a 3 X 4 factorial by treatments and lines, respectively. In experiment 2, 180 males of the same stocks were divided into a 3 X 4 X 3 factorial by treatments, lines and ages, respectively. There were thus five chicks per subgroup in each experiment. Treatments consisted of: (1) ACTH2—One U.S.P. unit per 100 g. body weight per day for five days given intramuscularly in the pectoral area in a 15% gelatin vehicle. (2) Hydrocortisone3—Five-tenths mg. per 100 g. body weight per day as a suspension in 0.85% saline administered, as above. (3) Gel-control—15% gelatin administered as above. Concentrations were adjusted so that volume did not exceed one ml. per injection. The four genetic lines used were as follows: (1) High weight (HW) and low weight (LW) lines in the seventh generation of 2 ACTH — Adrenocorticotropin — Depo-ACTH, The Upjohn Company. 3 Hydrocortisone—A4—Pregnene—lip, 17a, 21triol-3-20-dione) -Cortef—The Upjohn Company.

901

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EFORE one considers the relationships between responses to exogenous ACTH or glucocorticoid administration and responses to environmental stimuli, it is important to consider whether birds from varying genetic backgrounds respond similarly to these exogenous materials. Thiessen and Nealy (1962), using eosinophil count and adrenal weight as response criteria to handling stress, reported that the pattern of response for one of five inbred strains of mice could not be used to predict the pattern for the others. Highly significant differences in changes of plasma corticosterone levels resulting from electric shock treatments were found among four inbred mouse strains by Levine and Treiman (1964). Genetic differences in thiouracil-induced hypothyroidism (El-Ibiary and Shaffner, 1951; Shaklee and Shaffner, 1955), and in assay sensitivity to gonadotropins (Siegel and Siegel, 1964a, b) have been observed; however, little has been reported concerning genetic influences upon the response of the avian pituitary-adrenal cortical system to exogenous hormone stimulation. Heritability estimates are relatively high for adrenal weights of White Rock males (Siegel and Siegel, 1960), which indicates that a large proportion of the total variation is genetic. In this study, the magnitude of the genotype X environment interactions, or more specifically, line X treatment interactions

902

H . S. SlEGEL AND P . B . SlEGEL

TABLE 1.—Effect of five-day injections of A CTH and hydrocortisone on body weight of cockerels of four lines, Experiment 1 Change in body weight, gm. Line

x+ SD

Gel

ACTH

Hydrocort.

HW

77.6

47.6

-27.8

32.59 ±17.1

LW

33.0

13.4

-20.8

8.5b + 14.9

HM

44.2

34.6

-37.0

13.9b + 24.1

Line

The chicks were brooded and reared in metal batteries in a room maintained at 20°C. (±5°C.) under continuous artificial illumination. A practical corn-soya starter diet was fed ad lib. until 12-18 hours prior to autopsy when it was removed. Water was provided ad lib. RESULTS

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Experiment 1. Changes in body weight LM 45.4 18.6 -35.2 9.6b ±17.3 which occurred during the injection period Treat. 50.0c 28.5b -30.2a from the 26th through the 30th day of age x+ SD + 10.0 +35.0 +11.6 are shown in Table 1. Gains by the ACTHtreated group were significantly lower than Line or treatment means with the same superscripts are not significantly different (p<0.05). those of the gel-injected birds while there selection; source-White Plymouth Rocks was a significant loss in weight by the hydrocortisone groups over the five-day peri(P. Siegel, 1962). (2) High mating frequency (HM) and od. As expected, body weights of the high low mating frequency (LM) lines in the weight (HW) line birds were significantly sixth generation of selection; source-Ath- greater than those of the other lines. ens-Canadian Randombreds (Siegel and Table 2 presents the absolute and relaSiegel, 1962). tive weights of the adrenals and bursae of In experiment 1, injections were begun Fabricius for the four lines. Because body at 26 days and completed at 30 days of weights were obviously influenced by the age. In the second experiment injections hormone injection, relative gland weights were commenced at 3, 11 and 28 days and were based on body weight prior to treatwere completed at 7, IS and 32 days of ment. ACTH did not significantly increase age. Body weights were recorded prior to absolute adrenal weights or those relative the initiation of treatment and before au- to body weight. Hydrocortisone did reduce topsy which was approximately 18 hours them significantly, however, The effects of after the last injection. After weighing, 10 line were only significant when the adrenal ml. of blood was drawn, by cardiac punc- weights were adjusted for body weight. ture, using heparinized syringes, then Relative weights of the adrenals of the LW cooled and centrifuged. The plasma was line were significantly greater than those of stored at — 20°C. for future analysis. The the HW and HM lines, while those of the birds were killed by exsanguination and the LM line were intermediate. adrenals and bursae of Fabricius were reBoth absolute and relative bursa weights moved and weighed. In experiment 2 adre- were appreciably reduced by ACTH and nals were stored at — 20°C. for future further reduced by hydrocortisone. Bursae study. Plasma and adrenal corticosterone of the LW line were lighter than those of concentrations were determined fiuoromet- the other lines, but this difference disaprically as described by Guillemin et al. peared when the weights were adjusted to (1959). Plasma total cholesterol was ob- body weight. tained by the Zlatkis et al. (1953) method Plasma corticosterone levels were not and adrenal cholesterol by the Knobil et al. significantly influenced by treatment or line (1954) procedure. (Table 3). Variation was quite high, how-

VARIATION IN

ACTH

903

AND HYDROCORTISONE RESPONSES

Q ever, which might have been due to plasma fluorescence (Moncloa et al., 1959). Plas>> +1 M +l o>+| ma cholesterol levels were significantly ina ti 3 creased by ACTH and further elevated by o o hydrocortisone. No appreciable differences o ^ O o were noted among lines. ~ % X ^ a The absence of line X treatment interac- o x ^ 3 tions in these criteria of response indicated o that there was the same relative magnitude o < 5 o of response to the individual hormones by "9 ain s the four lines. a +1 The sources of variance heterogeneity Ex •X o o E-< fl +1 +1 are shown in Table 4. Plasma corticoste- u bb ^ rone and cholesterol were not included "o> s 3 since Bartlett's test did not demonstrate 1 +S o MS O heterogeneity. The theoretical error in the X o 2 Analysis of Variance for s was used to test .a «0 x >o the treatment X line interaction (X2, DF = cs 3 < •* 1), which in turn was used to test the re"3 ^4" maining variances. The line variance was H o Q further subdivided into orthogonal comparw A isons according to line source (White Rock •3 £ •a>> +1 * c o * ^ ^ H < vs. Randombred) and direction of selection 1 8 .ao 0 ™+l " +l for the selected trait (high or low). "S> bb 13 8 >-< Treatment variances were heterogeneous >o s* o for changes in body weight and bursa • is *""* X weight. In the former, the ACTH treated > ~£ 0 groups were higher than the others (Table x MS " -a H 3 1). In the latter, variances appeared to be < o -* < »o related to the size of the means, with the a o ~* § gel group the largest and the hydrocor- 1 a tisone group the smallest. Overall, line-vari+1 4 ances for bursa weight were homogeneous; ~§ 10 +1 •*+! however, when broken-down orthogonally, s 0 significantly greater variation in bursa a 3 weight was associated with the lines obtained from the Randombreds (HM and % O -* X LM). < X S8 H Experiment 2. Additional information was «

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sought on the inter-relationship of age and genetic background in response to the two hormones under study in this experiment. The data in Table 5 show the changes in body weight by treatment, line and age. The results were essentially the same as

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904

H . S. SlEGEL AND P . B . SlEGEL TABLE 3.—Effect

of ACTH and hydrocortisone on plasma corticosterone and cholesterol levels of cockerels from four lines, Experiment 1

Corticosterone, jug.% Line

Cholesterol, mg. % Linex±SD

Gel

ACTH Hydrocort.

Line x + SD Gel

ACTH Hydrocort.

19.4

40.8

24.4

28.2" ±19.1

376

401

523

433" 110

LW

18.2

18.7

30.8

22.6" ±18.1

281

382

484

382" 85

HM

26.1

29.8

31.0

29.0" ±18.9

328

395

523

415" 50

LM

20.8

14.1

21.3

20.4" ±16.2

339

384

476"

400" 56

21.1" 14.1

27.2" 22.2

26.9" 15.9

331" 75

390b 70

502' 84

Treat, x + SD

Line or treatment means with the same superscript are not significantly different (p<0.05).

those of experiment 1, i.e., gains by the ACTH treated groups were less than those of the gel-controls and those of the hydrocortisone treated groups were further depressed or a weight loss was observed. But the effect was not the same at all ages. ACTH did not significantly depress gains at 7 or IS days of age, although hydrocortisone did. When injected at 32 days, however, ACTH did significantly reduce gains and hydrocortisone caused significant losses in weight. Analysis of these data revealed a highly significant age X treatment interac-

TABLE 4

tion which appeared to be due primarily to the ACTH effect. As observed in the previous experiment, ACTH had no significant effect on adrenal weights, while a reduction resulting from hydrocortisone injection was observed (Table 6). This was generally true, irrespective of age. Adrenals of the LW line were significantly smaller than those of the other lines before adjustment for body weight. When adjusted for body weight, significant differences in adrenal weight were not observed before the 3 2-day autop-

—Analyses of variance of the log variances for Experiment 1 Mean squares

Source of variation Total Treatment (T) Line (L) Line Source Hi vs. Lo Residual TXL Error 3 1

D.F.

Body wt. change

—

11 2 3 1 1 1 6

—

1.37** .07 .02 .01 .16 .12 .02

Adr. wt., mg.

Adr. wt.1 mg./lOO g.

—

—

.25 .27 .02 .80 .01 .15 .02

.44 .31 .16 .36 .40 .17 .02

Bursa wt., mg.

Bursa wt.1,2 mg./lOO g.

— 1.60** .25 .72** .03 .00 .06 .02

Relative gland weights based on 100 grams of body weight before initiation of treatment. X10" 1 Theoretical error for ANOVA of s 2 = (logi 0 e) 2 /2(n-l) (Bartlett and Kendall, 1946). * p<0.05, **p<0.01

2 3

1.13** .27 74** .01 .06 .08 .02

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HW

VARIATION IN

ACTH

905

AND HYDROCORTISONE RESPONSES

TABLE 5.—Changes in body weights during injection period, Experiment 2

Age, days

Change in body wt. g. Line

Gel

ACTH

H-Cort.

Line x ±SD

HW

21

20

13

18b ±5

LW

14

9

7

10s ±4

HM

15

15

4

LM

21

15

8

11" ±7 14a ±5

18b +6

15b ±4

8" ±5

HW

35

36

14

27b ±8

LW

11

22

2

12a ±8

HM

33

28

12

LM

33

33

15

24b ±7 27b +8

HW

28b ±12 39

30 b ±5 0

11" ±6 -19

LW

21

-5

-23

-7a ±14

HM

31

3

-31

3a ±19

LM

42

-1

-31

5a ±10

32-day f+SD

33° + 12

-lb ±21

-26a ±27

Grand x+SD

26b + 10

14»b ±10

7

7-day x+SD 15

15-day S+SD 32

7a ±28

6s ±13

Line or treatment means with the same superscript are not significantly different (p<0.05).

produced significant increases. As observed previously, there were no significant differences due to line. Adrenal corticosterone values of the 32-day old chicks were considerably lower than at the two preceding ages (Table 9) Although ACTH did not significantly reduce corticosterone values, hydrocortisone did cause significant reductions at the 15and 3 2-day autopsies. Significant differences were found among lines during the first two periods, but it was difficult to ascertain a consistent pattern. ACTH significantly reduced cholesterol

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sy when values for both the LW and HM lines were significantly higher than those of the HW line. A significant age X treatment interaction was not found. Bursa responses were essentially the same as in experiment 1 (Table 7), in which absolute and relative atrophy was found as a result of ACTH and hydrocortisone administration. The age X treatment interaction, associated essentially with ACTH, was evident. At 7 and 14 days ACTH did not reduce bursa weights significantly. At 32 days of age significant bursa atrophy was found. Hydrocortisone reduced bursa weights at all three autopsy periods. As in the first experiment, absolute bursa weights of the LW line were significantly lower than those of the other lines; this was true at all three ages. It was also noted that lines derived from the White Rock source generally had significantly smaller bursae, relative to body weight, than lines derived from the Randombreds. This trend was also apparent but not significant in the first experiment. The values for plasma corticosterone shown in Table 8 are lower than those in experiment 1. The variation, also considerably lower, was probably due to corrections for plasma fluorescence (Moncloa et al., 1959). Although the values of the hydrocortisone treated groups were generally higher than the others, within age groups this was only true at 15 days. The HW line had higher levels of the circulating hormone at seven days of age, but the differences were not significant subsequently. Thus the analysis of variance indicated a significant age X line interaction. ACTH significantly increased plasma cholesterol levels (Table 8) and hydrocortisone increased them still further. The age X treatment interaction was again apparent. At 7 and 15 days, only hydrocortisone caused substantial increases in cholesterol levels, but at 32 days ACTH also

906

H . S. SlEGEL AND P . B . SlEGEL TABLE 6.—A bsolute and relative1 adrenal weights after five day injections with A CTH or hydrocortisone, Experiment 2 Age days

7

Adrenal wt., mg Gel

Line x

16.6

15.0

13.6

15.1" ±2.8

LW

15.1

14.7

11.5

HM

17.4

18.5

LM

17.3

b

Gel

ACTH H-Cort. Line x 29.8

28.2

30.5" ±5.1

13.7" ±3.5

37.7

35.9

28.6

33.9" ±7.8

11.8

15.8»b + 3.1

27.2

39.2

22.6

32.4" ±7.5

19.4

14.6

17.l b ±2.4

34.1

38.6

31.1

34.6" ±6.1

16.6 b ±3.2

16.8 b ±3.9

12.8" ±4.0

35.3" ±7.0

35.4 b ±7.5

27.6" ±6.2

HW

25.6

23.8

20.6

23.1" ±3.9

26.2

23.0

22.8

24.0" ±5.3

LW

16.8

21.1

15.8

17.9" ±2.5

25.2

29.1

21.6

25.3" + 3.6

HM

25.3

25.9

18.2

23. l b ±3.1

25.0

28.0

19.9

24.3" ±3.7

LM

27.2

25.8

21.3

24.8 b ±5.5

27.4

25.3

21.3

24.7" ±4.3

23.7 b ±4.9

24.2 b ±3.1

19.0" + 3.4

26.0 b ±3.3

26.5 b ±3.6

21.5" ±5.7

HW

73.0

70.2

55.3

66.2 b ±8.3

13.4

11.8

12.9

12.7" ±2.7

LW

61.3

57.0

55.5

57.9" ±11.7

15.4

15.6

14.3

15. l b ±3.0

HM

69.4

69.7

56.7

65.3 b ±7.5

17.2

15.8

13.9

15.6 b ±4.1

LM

66.6

70.8

55.5

64.9 b ±9.5

13.6

14.8

12.6

13.6" b ±1.4

32-day x ± S D

67.6 b 66.9 b ±8.7 ±11.1

55.8" ±8.4

14.9" ±7.8

14.5" ±2.2

13.4" ±3.8

Grand x ± S D

35.9 b ±5.6

28.9" ±5.3

21.5 b ±6.0

25.3 b ±4.4

20.8" ±5.3

36.5 b ±6.0

1 Relative gland weights were taken as mg./lOO g. body weight prior to initiation of treatment. Line or treatment means with same superscripts are not significantly different (p<0.05).

levels in the adrenal at all three ages (Table 9) which was consistent with the results of experiment 1. Hydrocortisone, by contrast, had no significant effect. Again, significant differences among lines at 7 and IS days of age appeared to follow no consistent pattern, although the LW line was

lowest in both cases at 7 days and highest at IS days of age. It should be pointed out again that in no criterion tested was there a significant line X treatment interaction. The sources of the variance heterogeneities are shown in the analyses in Table

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33.0

15-day x ± S D 32

ACTH H-Cort.

HW

7-day x ± S D 15

Adrenal wt., mg./lOO g body

Line

VARIATION IN

ACTH

907

AND HYDROCORTISONE RESPONSES

TABLE 7.—A bsolute and relative1 bursa weights after five-day injections with A CTH or hydrocortisone, Experiment 2 Age days 7

Gel

ACTH H-Cort. Line x

Gel

ACTH H-Cort. Line x

140

142

81

125" ±36

276

279

167

248"" ±54

LW

103

71

64

80" ±19

257

173

157

197" ±40

HM

130

110

92

112" ±48

264

235

178

228"" ±82

LM

183

158

83

127" ±63

363

252

178

137" ±55

123" ±49

80* ±10

285" + 98

254" ±98

170" ±44

HW

303

283

157

239" ±75

304

281

157

LW

148

208

87

148" ±50

212

284

119

205" ±50

HM

472

342

177

330" ±96

460

372

195

342" ±103

LM

401

476

253

377= ±134

392

464

248

368" ±103

331" + 41

330" ±73

168' + 66

342" ±126

354" + 82

179" ±57

15-day x ± S D

277" ±119

239" ±70

HW

1,855

1,238

588

1,227" ±329

333

213

122

223" ±43

LW

1,168

809

595

857" ±175

291

222

151

221" ±42

HM

1,777

1,072

783

1,211" ±503

392

243

173

269" ±77

LM

1,837

1,231

876

1,325" ±460

375

263

191

282" + 91

32-day x + SD

1,659" ±542

1,087" + 246

702" + 306

348= ±94

235" ±58

158* ±56

Grand x ± S D

709= ±212

530" ±123

311" ±124

325= ±106

281" ±79

169" ±52

32

1 Relative gland weights were taken as mg./lOO g. body weight prior to initiation of treatment. Line or treatment means with same superscripts are not significantly different (p<0.05).

10. As in experiment 1, the heterogeneous treatment variance for bursa weight appeared to be directly associated with the magnitude of the means (Table 6). The orthogonal comparison again indicated that differences in bursa variance among lines was primarily dependent on the source of

the stock, with lines derived from the White Rock gene pool having lower variation than those derived from the Randombreds. A significant difference in the variance associated with body weight change appears to be determined, essentially, by

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HW

7-day x ± S D 15

Bursa wt., mg./lOO g . body

Bursa wt., mg. Line

908

H . S. SlEGEL AND P . B . SlEGEL TABLE 8.—Plasma corticosterone and cholesterol levels by ages and lines after five-day injections oj A CTH or hydrocortisone, Experiment 2 Age days

7

Plas. cortico., fig. Line Gel

Plas. cholest., mg.

Line x

Gel

ACTH H-Cort

% Line x

b

317

324

434

351" + 51

17.3

12.6

23.8

17.2 ±9.0

LW

12.3

15.3

11.6

12.7" ±6.2

302

355

416

358" ±78

HM

14.5

12.2

13.3

13.4" b + 4.6

308

300

380

331" ±64

LM

12.0

15.8

17.1

13.3" b ±4.7

308

288

425

341" ±55

14.2" ±7.8

12.5" ±4.0

16.0" ±7.0

308" ±49

314" ±51

413 b ±79

HW

10.7

9.5

13.4

11.5" ±5.0

317

356

413

366" ±51

LW

7.5

11.2

13.7

10.8" ±3.8

313

355

369

346" ±64

HM

10.2

10.7

15.2

12.0" ±3.8

332

372

433

379" ±60

LM

9.3

13.2

17.6

11.8" ±4.2

327

306

398

344" ±70

322" ±54

347" + 61

404b + 74

9.4" ±3.8

15-day X±SD

11.3" b 13.7 b + 4.2 + 4.2

HW

10.0

17.1

13.3

13.5" ±6.2

353

344

515

404" ±45

LW

14.5

14.3

15.2

14.7" ±4.8

344

374

502

407" ±67

HM

17.0

16.8

16.4

16.7" ±3.9

327

406

460

398" ±63

LM

12.3

13.3

15.7

13.6" ±3.5

324

426

444

395" ±71

32-day x ± S D

13.5" ±5.3

15.3" + 5.5

15.2" ±8.9

337" ±66

338 b ±74

482= + 57

Grand x ± S D

12.3" ±5.6

12.9" ±4.6

±6.7

15.2>>

322" ±56

351 b ±62

432° ±70

32

Line or treatment means with same superscripts are not significantly different (p<0.05).

whether selections were made high or low for the particular selected trait (body weight or mating frequency), especially at 32 days of age (Table 5). Except relative bursa weight, a significant difference in variation due to age was found for every response criterion. Examination of the

standard deviations indicated that variation increased with the magnitude of the age means. DISCUSSION Reduced growth or actual weight loss in chickens due to glucocorticoid administra-

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HW

7-day x ± S D 15

ACTH H-Cort

Jo

VARIATION IN

ACTH

909

AND HYDROCORTISONE RESPONSES

TABLE 9.—Adrenal corticosterone and cholesterol concentrations by ages and lines after five-day injections of A CTH or hydrocortisone, Experiment 2 Age days 7

Gel.

Gel.

ACTH H-Cort. Line x

6.95

6.61

6.87"" ±2.43

44.6

40.2

57.3

46.2"" ±13.1

LW

3.79

4.50

6.68

5.02" ±2.86

52.8

24.5

41.0

39.5" ±2.0

HM

6.97

5.97

8.84

7.33" ±3.86

47.4

34.2

61.9

49.7" ±10.1

LM

7.72

5.66

9.89

7.76" ±2.93

43.4

36.3

46.1

41.5"" ±16.3

51.5" 46.9"" 34.6" ± 9 . 9 + 16.3 ± 1 2 . 0

8.08" 5.89" 6.39" ± 3 . 4 8 + 3.20 ± 4 . 0 0 HW

6.67

7.62

5.50

6.45"" ±2.99

42.0

33.8

39.6

38.6" ±10.8

LW

9.07

7.65

4.32

7.01" ±2.32

53.1

31.9

54.8

45.8" ±8.4

HM

4.64

4.19

4.02

4.28" ±3,50

42.5

31.9

51.7

42.0"" + 7.1

LM

7.12

4.75

4.58

5.48"" ±2.99

42.6

28.2

47.9

40.4"" ±7.7

44.8" ±8.8

47.8" 31.5" + 6.6 ± 1 2 . 0

5.97"" 4.64" 6.87" ±3.68 ±3.42 ±2.28 HW

3.31

2.62

1.89

2.61" + .80

32.6

25.3

34.3

30.5" ±5.26

LW

2.13

3.57

1.51

2.40" ±1.67

32.4

21.9

35.9

30.1" ±3.28

HM

2.87

2.53

1.64

2.35" + 1.34

33.4

24.1

37.8

31.8" ±6.88

LM

2.41

2.57

1.54

2.22" ±.82

31.7

23.5

36.9

30.8" ±5.29

36.2" ±7.4

32-day x ± S D

2.82" 1.65" 2.68" ± 1 . 2 7 ± 1 . 5 5 ±0.89

32.5" ±3.5

23.8" ±5.4

Grand x ± S D

4.86" 5.33" 4.79" ±2.81 ±2.72 ±2.39

41.7" ±7.4

45.2" 29.3" + 9.4 + 10.5

Line or treatment means with same superscripts are not significantly different (p<0.05).

tion has been observed by several authors (Kudzia and Champion, 1953; Dulin, 1956; Glenn et al., 1961). Nagra and Meyer (1963) have reported that glucocorticoids alter in vivo disposition of glucose carbon into more lipid and less protein and carbohydrate. Net synthesis of DNA and

RNA is inhibited, with a reduction in protein content of skeletal muscle and a change in major muscle cation balance. Loss of potassium concentration is not balanced by the increase in sodium (Bellamy and Leonard, 1965). In vitro studies indicate that 11-oxygenated steroids stimulate

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6.98

15-day x ± SD

&

ACTH H-Cort. Line x

HW

7-day x ± S D 15

Adr. cholest., /ig./mg.

Adr cortico. Mg./100 mg. Line

910

H. S. SIEGEL AND P. B. SIEGEL TABLE 1C .—Analyses

of variance of the log variances for Experiment 2 Mean squares

Source of variation

35 2 3 1 1 1 2 6 4 6 12

Adr. wt. 1 mg./lOO g.

Body wt. change

Adr. wt., mg.

—

—

.03 .43* .02 1.26** .01 1.92** .12 .62 .26 .09

.12 .02 .00 .04 .00 3.20** .12 .11 .23* .07

.19 .16 .00 .10 .39 2.67** .31 .18 .20 .14

Bursa wt. mg.

— 1.40** 1.16** 2.20** .11 1.16 11.36** .24 .17 .09 .10

Bursa Wt. 1 ' 2

mg./lOO g.

Adr. cortico.

Adr. cholest.

.—

—

—

.92* .91* 2 42** .00 .29 .18 .14 .11 .03 .20

.17 .18 .01 .48 .06 4.61** .48 .28 .25 .21

.37 .17 .07 .03 .39 1.45** .24 .25 .22 .17

Relative gland weights based on 100 grams of body weight before initiation of treatment. XIO" 1 * p<0.05, ** p<0.01 :

glucose formation from amino acids (Haynes, 1962) probably as a result of an increase in alpha-ketoglutarate transamination (Chan and Cohen, 1964). Thus despite adequate feed intake, reductions in proteinaceous tissue may occur. The significant age X treatment interactions observed in Experiment 2 for body growth, bursa weights and plasma cholesterol confirm previous observations with ACTH (Siegel, 1961; H. Siegel, 1962). In general, responses to ACTH were not evident until the last autopsy at 32 days of age. A major portion of the activity of ACTH is mediated through the adrenal, thus lowered glandular activity or low sensitivity of target tissues at early ages seems possible. Insensitivity of the target tissues to adrenal secretion appears improbable because a direct effect on these tissues by hydrocortisone was found. Lack of sensitivity of the adrenal to ACTH stimulation during the early ages is unlikely since ACTH caused cholesterol depletion at all three ages and significant, although variable tissue responses were previously observed at three days of age (H. Siegel, 1962). Functional insufficiency of adrenal cortical tissue, in which synthesis of gluco-

corticoids may be reduced due to lack of precursor materials, appears more likely. The presence of yolk has been previously found necessary for maximum bursa response at 24 days of age and plasma corticosterone levels have been found to be higher in ACTH-treated chicks with yolk intact (Siegel, 1964). On the other hand, the reduction in concentration of adrenal corticosterone at the 32-day autopsy suggests that the total amount of adrenal tissue may be important, especially since it was only at this age that hydrocortisone significantly reduced corticosterone concentrations. It is of interest that hydrocortisone did not cause adrenal cholesterol depletion at any age, which suggests that the action is not directly on the adrenal, but rather by a feedback mechanism. Plasma corticosterone values of the second experiment were similar to those reported for peripheral plasma by Nagra et al. (1960). Although corticosterone levels in the adrenal effluent are raised by ACTH (Nagra et al., 1960), it is probable that increases resulting from the dosages of ACTH used in these experiments would be metabolized by the liver and would not be apparent peripherally.

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Total Treatment (T) Line (L) Line Source Hi vs. Lo Residual Age (A) TXL TXA LXA TXLXA

Df

VARIATION IN

ACTH

AND HYDROCORTISONE RESPONSES

The heterogeneity in variance experienced in these experiments, especially with regard to age, points out that tests for heterogeneity and possible transformation may be required when data from widely divergent ages or stocks are considered. SUMMARY

Two experiments, involving 240 male chicks from four genetic lines, were conducted to evaluate line X treatment interactions in response to ACTH and hydrocortisone administration. The influence of age on such interactions was also observed. Effects on body, adrenal and bursa weight, adrenal cholesterol and corticosterone concentrations and plasma cholesterol and corticosterone levels were used as criteria of response. Lack of significant line X treatment interactions for any of the criteria indicated that responses by the four lines were essentially parallel. However, significantly greater variance associated with bursa weight in two of the lines sug-

gested that individuals within lines may be more or less responsive. Heterogeneity in variance with regard to age and line effects indicates the necessity of data transformation when widely divergent ages or stocks are evaluated. Significant age X treatment interactions involving responses to ACTH but not hydrocortisone were observed and the possibility of functional insufficiency of adrenal cortical tissue during early post-hatch life was discussed. ACKNOWLEDGMENTS

The authors wish to thank Mrs. Mary Coleman and Mrs. Mary McLaughlin for technical aid and the Upjohn Company, Kalamazoo, Michigan, for kindly providing ACTH and Hydrocortisone. REFERENCES Bartlett, M. S., and D. G. Kendall, 1946. The statistical analysis of variance-heterogeneity and the logarithmic transformation. J. Royal Statistical Soc. Supl. 8: 128-138. Bellamy, D., and R. A. Leonard, 196S. Effect of Cortisol on the growth of chicks. Gen. Comp. Endocrinol. 5: 402-410. Chan, S., and P. P. Cohen, 1964. A comparative study of the effect of hydrocortisone injection on tyrosine transaminase activity of different vertebrates. Arch. Biochem. Biophys. 104: 335337. Dulin, W. E., 1956. Effects of corticosterone, cortisone, and hydrocortisone on fat metabolism in the chick. Proc. Soc. Exp. Biol. Med. 92: 253-255. El-Ibiary, H. M., and C. S. Shaffner, 1951. The effects of induced hypothyroidism on the genetics of growth in the chicken. Poultry Sci. 30: 435-444. Glenn, E. M., B. J. Bowman, R. B. Bayer and C. E. Meyer, 1961. Hydrocortisone and some of its effects on intermediary metabolism. Endocrinology, 68: 386-410. Guillemin, R., G. W. Clayton, H. S. Lipscomb and J. O. Smith, 1959. Fluorometric measurement of rat plasma and adrenal corticosterone concentration. J. Lab. Clin. Med. 53: 830-832. Haynes, R. C , 1962. Studies of an in vitro effect of glucocorticoids on gluconeogenesis. Endocrinology, 7 1 : 399-406. Knobil, E., M. C. Hagney, E. I. Wilder and F. N.

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The major reason for undertaking this research was to determine the magnitude and extent of genetic X treatment interactions for responses to two hormones which are considered important in the bird's adaptive processes. The lack of significant line X treatment interactions in both experiments indicates that the four lines used here responded in a parallel manner to the dosages of ACTH and hydrocortisone employed. However, a broad interpretation that all chicks will respond to these hormones to a similar degree requires caution. The greater variation experienced for bursa weight by two of the lines indicates the possibility that certain individuals within the lines may be more or less responsive. Age of dam was not a factor (Temple and Jaap, 1961), since the chicks were all from dams of the same age. Certainly additional stocks need to be evaluated before definite conclusions can be drawn.

911

912

H. S. SIEGEL AND P. B. SIEGEL Siegel, H. S., 1964. Yolk influence on responses to ACTH in the chick. Virginia J. Sci. 15: 299. Siegel, H. S., and P. B. Siegel, 1964b. Genetic variation in chick bioassays for gonadotropins II. Histological and histochemical responses Virginia J. Sci. IS: 204-217. Siegel, P. B., 1962. Selection for body weight at eight weeks of age. 1. Short term response and heritabilities. Poultry Sci. 4 1 : 954-962. Siegel, P. B., and H. S. Siegel, 1960. Genetic parameters of gland and body weights in White Rock cockerels. J. Hered. 51: 59-62. Siegel, P. B., and H. S. Siegel, 1962. The quantitative inheritance of mating ability in chickens. Amer. Zool. 2 : 558. Siegel, P. B., and H. S. Siegel, 1964a. Genetic variation in chick bioassays for gonadotropins I. Testes weight and responses. Virginia J. Sci. 15: 187-203. Snedecor, G. W., 1962. Statistical Methods. 5th Edition, Iowa State University Press, Ames, Iowa. Temple, R. W., and R. G. Jaap, 1961. Age of dam and response to selection for increased weight of the bursa of Fabricious in day-old chicks. Poultry Sci. 40: 1355-1358. Thiessen, D. D., and V. G. Nealy, 1962. Adrenocortical activity, stress response and behavioral reactivity of five inbred mouse strains. Endocrinology, 71: 267-270. Zlatkis, A., B. Zak and A. J. Boyle, 1953. A new method for direct determination of serum cholesterol. J. Lab. Clin. Med. 4 1 : 486-492.

NEWS AND NOTES (Continued from page 900) Problem; and Disposal through Mechanical Opof a two-session symposium on Food with the erations. Section on Chemistry. Organizations cosponsoring this Symposium on KANSAS NOTES Pollution are the American Society of Agronomy, the American Society of Animal Science, the AmerThe new poultry farm of Kansas State Uniican Society of Plant Physiologists, the Poultry versity was dedicated as the "T. B. Avery ReScience Association, the Society of American Forestsearch Center" as the concluding event of the ers, and the Soil Conservation Society of America. first annual Kansas Poultry Industry Conference Section O itself is a cosponsor of the Symon March 24th. posium on "Migration to the Arid Lands of the Dr. Glenn H. Beck, Vice-President for AgriculUnited States," sponsored by the Committee on ture, Kansas State University, paid tribute to Arid Lands; it is a joint sponsor of a symposium Avery. Although only 54 at the time of his death, with the Section on Statistics, and a joint sponsor Avery already had won an international reputa(Continued on page 945)

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Briggs, 1954. Simplified method for determination of total adrenal cholesterol. Proc. Soc. Exp. Biol. Med. 87: 48-50. Kudzia, J. J., and L. R. Champion, 1953. Investigations concerning the effects of cortisone on domestic fowl. Poultry Sci. 32: 476-481. Levine, S., and D. M. Treiman, 1964. Differential plasma corticosterone response to stress in four inbred strains of mice. Endocrinology, 75:142-144. Moncloa, F., F. G. Peron and R. I. Dorfman, 1959. The fluorometric determination of corticosterone in rat adrenal tissue and plasma: Effect of administering ACTH subcutaneously. Endocrinology, 65: 717-724. Nagra, C. L., G. J. Baum and R. K. Meyer, 1960. Corticosterone levels in adrenal effluent blood of gallinaceous birds. Proc. Soc. Exp. Biol. Med. 105: 68-70. Nagra, C. L., and R. K. Meyer, 1963. Influence of corticosterone on the metabolism of palmitate and glucose in cockerels. Gen. Comp. Endocrinol. 3 : 131-138. Shaklee, W. E., and C. S. Shaffner, 1955. Some effects of selecting for high and low thyroidal response to thiouracil feeding in New Hampshire chickens. Poultry Sci. 34: 572-577. Siegel, H. S., 1961. Age and sex modification of responses to adrenocorticotropin in young chickens. 1. Changes in adrenal and lymphatic gland weights. Poultry Sci. 40: 1263-1274. Siegel, H. S., 1962. Age and sex modification to adrenocorticotropin in young chickens 2. Change in adrenal cholesterol and blood constituent levels. Poultry Sci. 4 1 : 321-334.