Group Selection for Adaptation to Multiple-Hen Cages: Hematology and Adrenal Function1,2

BREEDING AND GENETICS Group Selection for Adaptation to Multiple-Hen Cages: Hematology and Adrenal Function1,2 PATRICIA Y. HESTER, W. M. MUIR, J. V. CRAIG, and J. L. ALBRIGHT Department of Animal Sciences, Purdue University, West Lafayette, Indiana 47907 a decrease in packed cell volume, appeared to adapt more quickly to the new waterer system of multiple-hen cages than did the control and commercial lines. At 33 wk of age, the control and commercial lines in multiplehen cages experienced heterophilia and increased heterophil to lymphocyte ratios, whereas the selected line did not, when compared with these same lines in single-hen cages. This leucocytic response could be interpreted to mean that the selected line of chickens adapted better to social competition than either the control or commercial lines; however, a similar leucocytic response was not observed at 18 or 44 wk of age. In conclusion, the physiological characterization of the selected line of Leghorns showed evidence of improved adaptation to multiple-hen cages when compared to the other stocks. In some cases, the selected line responded less intensely to stress; however, trends were not always consistent.

(Key words: selection, adaptation, multiple-hen cages, hematology, adrenal function) 1996 Poultry Science 75:1295-1307

INTRODUCTION Animal welfare groups and consumers in general have criticized the housing of laying hens in cages. Such groups suggest that the bird's well-being is compromised because cages induce social stress and cause behavioral aberrations such as cannibalism, feather picking, vacuum nest-building activity, and stereotypic pacing. Such concerns have caused Sweden and Switzerland to gradually phase out the use of cages for housing laying hens. Alternative housing systems have been designed with perches, nests, and sand baths in an attempt to improve the well-being of laying hens. These designs offered advantages such as improved bone strength and in some cases reduced hysteria, but there were adverse effects such as fecal contamination and the laying of eggs in non-nest areas (for a review, see Craig and Swanson, 1994).

Received for publication November 6, 1995. Accepted for publication July 1, 1996. journal Article Number 14,655 of the Purdue University Agricultural Research Programs, West Lafayette, IN 47907. financial support for this study was provided by USDA Award Number 58-3602-3118.

Cage systems offer several advantages over alternative housing systems, including improved feed efficiency, reduced labor, cleaner eggs, elimination of internal parasites, and waste management control (North and Bell, 1990). It is because of these economic advantages that the United States egg industry produces approximately 98% of its table eggs from hens kept in cages, according to a 1991 survey (Bell, 1993). Because of high investment costs, single-bird cages are seldom used for housing commercial egg layers in the United States. Instead, hens are placed in multiple-bird cages that vary in size to accommodate 2 to 30 birds per cage, with the most common colony size ranging from 3 to 10 hens (North and Bell, 1990). To improve animal well-being, another option, besides designing an alternative housing system, would be to alter the genetics of the birds so as to make them more adaptable to multiple-hen cages. A selected line of White Leghorns developed by Muir (1996) has shown improved survivability and reduced feather loss in multiple-hen cages (Craig and Muir, 1996). Group selection, denned as selection of birds based on group rather than individual performance, was based on family averages for egg production and survival (Muir, 1996). A randombred control line of Leghorns, the North

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ABSTRACT A selected line of White Leghorns that has shown improved survivability and productivity and reduced feather loss in multiple-hen cages was evaluated for hematological and adrenal responses under both stressed and unstressed conditions. It was hypothesized that hens selected for adaptation to multiple-bird cages would react less intensely to stressors. Three lines of chickens (selected, control, and commercial) were housed in either single-hen (1 hen) or multiple-hen cages (12 hens, social competition) at 16.7 or 17.1 wk of age. They were subsequently subjected to cold exposure at 33 wk of age and heat exposure at 44 wk of age. Genetic stock as a main effect, and the interaction of genetic stock with either a cold or heated environment or with cage size, had no effect on plasma levels of cholesterol and corticosterone. At the time of transfer to laying cages, the selected line of pullets, as indicated by

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HESTER ET Ah.

MATERIALS AND METHODS Three genetic stocks of White Leghorns were compared. A line selected over seven generations for survival and hen-day egg production in multiple-bird cages was derived from and compared to an unselected control line. The third stock was a commercial line of layers. The three strains of chicks were hatched on June 16, 1993 at the Purdue University Hatchery and reared in groups of 16, with others of their own strain and sex, in 61 x 91 cm wire cages. During the entire experimental period, feed and water were provided for ad libitum consumption. Beak trimming was not performed. More details on the origin of the genetic stocks, the management of the birds, and the assignment of the three genetic lines of pullets with respect to room, cage row within the room, and cage size (single- vs multiple-hen) are described by Craig and Muir (1996).

Experiment 1 Chickens of the three genetic lines were placed in either single- (1 pullet per cage providing 1,085 cm 2 per bird) or multiple-hen (12 pullets per cage, which provided 362 cm 2 per bird) cages in one of four independently heated, ventilated, and lighted rooms of the Purdue University Layer Research Unit. Any observed effects of single- vs

multiple-bird cage environments would be due to both the absence (1 bird) or presence (12 birds) of social competition as well as differences due to bird density (1,085 vs 362 cm 2 per bird, respectively). One-half of the birds were transferred from the pullet rearing facility to two rooms of the layer facility at 16.7 wk (117 d) of age. The remaining one-half of the pullets were transferred to the other two rooms of the layer facility 3 d later at 17.1 wk of age. Water was provided through drip nipples with a single-caged hen having access to two drip nipples and the 12 hens of a multiple-bird cage having access to five drip nipples (Craig and Muir, 1996). Each room contained eight rows of cages, in a fourdeck, modified stair step arrangement (North and Bell, 1990). Experimental units, consisting of either four consecutive single-bird cages or one multiple-hen cage, were repeated twice for each of the three genetic stocks within a cage row using a restricted randomization scheme (Craig and Muir, 1996). Only the pullets housed in the two middle rows per side (Rows 2, 3, 6, and 7) were used in Experiment 1. When the pullets were 18 wk of age, one bird per experimental unit was selected at random for blood sampling. Blood samples were collected from 192 pullets [2 d of transfer from pullet to laying cages (at 16.7 and 17.1 wk of age) by two rooms per day of transfer by four rows per room by two experimental units per cage row by three genetic lines by two cage sizes (single- vs multiple-hen)].

Experiment 2 When the hens were 33 wk of age, the second experiment dealing with a cold environmental temperature was initiated on January 31. The temperature of two of the four rooms was decreased to a mean temperature of 0 C for 72 h with a range of - 5 to 7 C. Recording thermographs and humidigraphs monitored the environment of each of the four rooms continuously throughout the experiment. It took 1.5 h to lower the temperature from control levels. Relative humidity for the two colder rooms varied from 50 to 74%. The other two rooms remained at their control temperature (x = 21 C; range of 18 to 24 C) with relative humidity varying from 35 to 44%. Although each room (18.9 x 68.9 m) had its own environmental controls, they were designed to imitate commercial layer facilities with in-room conditions influenced by outside environmental conditions. Water provided to the hens via drip nipples remained unfrozen by allowing it to flow continuously through the polyvinyl chloride pipes. Blood (4.5 mL per bird) was collected from birds of paired cold and control rooms on two occasions. Different hens were bled on each occasion following the initiation of the colder temperatures. Hens of Rows 2 and 6 (second deck level from the top) of each of the two rooms were bled 4 to 6 h after a temperature of 0 C was achieved on Day 1 of the experimental period. Samples were collected by two teams of personnel between 1830 and 2230 h. Blood samples were first collected from hens of Row 2 of each of

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Central Randombred Control, was maintained without selection to enable comparison with the selection results. The competitive environment was produced by housing the selected birds from about 17 to 71 wk of age in multiple-bird cages of 9 or 12 hens each. The noncompetitive environment was produced by placing the control line in individual cages (Muir, 1996). It was hypothesized that hens genetically selected for adaptation to a multiple-bird environment should react less intensely to social competition, and perhaps to all stressors in general, than the unselected controls. Brown and Nestor (1973) showed that excitability of turkeys was correlated with selection for high and low levels of plasma corticosterone. Birds selected for low levels of corticosterone were observed to be less excitable than those selected for high concentrations of corticosterone. Turkeys selected for low corticosterone levels also had better growth, egg production, and lower mortality due to natural causes and induced stress responses to cold (Brown and Nestor, 1973, 1974). The objective of the present study was to physiologically characterize a line of chickens that has shown marked improvements in survival and productivity and reduced feather loss in multiple-hen cages (Craig and Muir, 1996; Muir, 1996). Hematological indicators of stress (Siegel, 1971, 1995; Maxwell, 1993) and adrenal function of the selected line were compared to an unselected control line derived from the same foundation stock, and a commercial line of Leghorns under both stressed and unstressed conditions.

PHYSIOLOGY OF HENS ADAPTED TO MULTIPLE-HEN CAGES the two control rooms beginning at 1830 h. Hens of Rows 2 and 6 of the cold environmental rooms were bled between 1930 and 2130 h (4 to 6 h after achieving a 0 C temperature). Hens of Row 6 of the two control rooms were bled between 2130 and 2230 h. Additional hens of Rows 3 and 7 (third deck level from the top) were bled 4 to 6 h following the end of the 72-h treatment of 0 C on Day 3 using the same schedule as described for the first sampling period. Hens that were bled in Experiment 2 were palpated to confirm the presence of a plumped egg. Only two bled hens failed to meet this criterion and had no egg in the uterus. None of the hens of Experiment 2 had ever been bled before. One bird was bled per experimental unit, resulting in a total of 192 hens (1 hen per experimental unit by two experimental units per row by two rows per room by two rooms per environmental temperature by two sampling periods by three genetic lines by two cage sizes by two environmental temperatures).

The same hens that were exposed to a 0 C temperature at 33 wk of age were subjected to a mean environmental temperature of 38 C (range of 32.5 to 41.5 C) for 3 h at 44 wk of age. By raising the thermostat and using space heaters in the hallways, it took 0.5 h to raise the temperature from control levels. Relative humidity averaged 36% during the 3-h heating episode. The same two control rooms used in Experiment 2 were also used as controls in Experiment 3. Ambient temperature of the two control rooms was maintained at 30 C with a relative humidity of 39%. Hens of Rows 2 and 6 (second deck level from the top) of each of the two heated rooms were bled 1 to 3 h after a mean temperature of 38 C was achieved on Day 1 of the experimental period. Samples were collected by two teams of personnel between 1700 and 2100 h. Blood samples were first collected from hens of Row 2 of each of the control rooms beginning at 1700 h. Hens of Rows 2 and 6 of the heated rooms were bled between 1800 and 2000 h (1 to 3 h after achieving a 38 C temperature). Hens of Row 6 of the two control rooms were bled between 2000 and 2100 h. Hens previously subjected to 38 C for 3 h were exposed to a second heating episode 24 h later on Day 2 of the experimental period. Using the same schedule as described for the first heating episode, hens of two rooms were subjected to a mean temperature of 38 C (range of 35:5 to 40 C) for 3 h. Relative humidity of the two rooms averaged 32%. The two control rooms were the same as used in the first heating episode. Mean temperature of the two control rooms was 28 C with a relative humidity of 38%. Blood samples were first collected from hens of Row 3 of each of the control rooms beginning at 1700 h. Hens of Rows 3 and 7 of the heated rooms were bied between 1800 and 2000 h (1 to 3 h after achieving a mean temperature of 38 C). Hens of Row 7 of the two control rooms were bled between 2000 and 2100 h. Blood samples were obtained

from hens not previously bled, as described for the first heating episode. As in Experiment 2, one hen per experimental unit was bled for a total of 192 hens. Of this total, 11 hens (5.7%) did not have an egg in the uterus; 4 of the 11 hens were from the control environment.

Experiment 4 When the hens were 62 wk of age, adrenal glands were excised from two hens per experimental unit of Rows 2 and 6 of each of the four rooms. All four rooms were maintained at control temperatures. The two hens of each experimental unit (four consecutive single-hen cages or a multiple-hen cage) were selected randomly, killed via cervical dislocation, weighed, and the right adrenal excised and weighed. The right adrenal gland was retrieved from a total of 192 hens (2 hens per experimental unit by two experimental units per row by two rows per room by four rooms by three genetic lines by two cage sizes).

Blood and Plasma Sampling A 4.5-mL blood sample was collected from the brachial vein of 192 birds for each of the first three experiments by two teams of personnel. Because Craig and Craig (1985) had shown that handling time u p to 120 s in an unselected line of Leghorn hens and u p to 180 s in a selected line of White Leghorns had no effect on basal concentrations of corticosterone, blood samples were collected from each bird within 2 min of removing it from the cage, as verified by a timer. Duplicate packed cell volumes (PCV) were determined after spinning microhematocrits for 15 min. Blood smears for a differential leucocyte count were collected from each bird. Slides were stained with Wright's stain for the differential count. Two hundred leucocytes were counted per slide in Experiments 1 and 2. In Experiment 3, leucocyte numbers were lowered in birds of the heated rooms; therefore, only 100 leucocytes per slide were differentiated. Individual body weights were obtained following blood sampling. To prevent reusing the same bird in later experiments, a colored ring was placed on one of the legs before returning the pullet to its cage. Individual heparinized blood samples were centrifuged at 400 x g for 15 min at 4 C. Aliquots of plasma were frozen for later analysis of cholesterol (Rudel and Morris, 1973) and corticosterone by radioimmunoassay (Pierson et al., 1981; Klingensmith et a\., 1984).

Statistical Analysis The design for the four experiments was a completely randomized block with the four rooms serving as blocks. Data were subjected to an analysis of variance with cage size and genetic stock as fixed effects. Rows within rooms of housing; experimental units per cage row; and samples within experimental units (Experiment 4 only) were considered random effects. Newman-Keuls' sequential range test was used to partition differences among means (Steel and Torrie, 1980).

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Experiment 3

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HESTER ET AL.

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RESULTS Experiment 1 Although the differential leucocyte count of the control and selected lines of pullets did not differ from each other, the hematology of these two lines differed markedly from

the commercial line (Table 1). The percentages of heterophils and eosinophils were significantly smaller, whereas the percentage of lymphocytes was significantly greater, in the selected and control lines of pullets than in the commercial stock. As a result of the shift in the differential leucocyte count, the heterophil to lymphocyte (H:L) ratio was significantly lower in the selected and control lines of chickens than the commercial line. Monocytes, basophils, and PCV did not differ among genetic stocks. Heterophils, lymphocytes, eosinophils, and PCV of 18-wk-old pullets did not differ between single- or multiple-hen cages and were also unaffected by length of time in the laying cages prior to blood sampling (4 vs 7 d). The percentages of basophils and monocytes were affected by day of housing, but not by cage size (data not shown in tabular form). Seven days in laying cages prior to blood sampling resulted in an increase percentage of basophils and a decrease in the percentage of monocytes when compared to 4 d of housing in laying cages. The interactions of genetic stock with cage size or days following housing were not significant for the differential leucocyte count. For PCV, a three-way interaction occurred for cage size by genetic stock by days of housing (P < 0.03, Figure 1). Seven days following transfer from pullet to laying cages, PCV did not differ among the genetic lines placed in either single- or multiple-hen cages. However, a significant and opposing trend occurred with PCV at 4 d posthousing among the three lines of chickens due to cage size. Hemodilution, as indicated by a decrease in PCV, was evident for the selected line housed in multiple-hen cages and the commercial pullets of single-bird cages as compared to their counterparts in the opposing cage size. The PCV of the control line of chickens did not differ between single- vs multiple-hen cages at 4 d posthousing. Plasma cholesterol and corticosterone concentrations did not differ among genetic lines, cage size, or days following transfer from pullet to laying cages (data not presented in tabular form). Neither were any of the

TABLE 1. Packed cell volume (PCV) and differential leucocyte count of three lines of genetic stock in singleand multiple-hen cages at 18 wk of age, Experiment 1 Treatment

PCV

Heterophils (H)

Lymphocytes (L)

H:L Ratio x 100

Monocytes

Eosinophils

Basophils

C%1 Single-hen cages Selected line Control line Commercial line Multiple-hen cages Selected line Control line Commercial line SEM x x x x x

Single-hen cages Multiple-hen cages Selected line Control line Commercial line ab

32.4 32.8 31.2

12.4 12.7 16.8

81.3 80.7 77.1

16.4 17.8 24.6

4.1 3.8 3.1

0.7 0.9 1.5

1.4 2.0 1.5

32.1 33.0 32.2 0.5 32.2 32.4 32.3 32.9 31.7

11.6 12.9 18.5 1.3 14.0 14.3 12.0b 12.8b 17.6a

82.6 81.2 74.7 1.4 79.7 79.5 81.9a 80.9" 75.9b

14.7 16.9 26.1 2.4

3.4 3.3 4.0 0.4 3.7 3.5 3.7 3.5 3.6

0.8 1.0 1.4

1.7 1.6 1.3 0.2

19.6 19.2 15.6b 17.4b 25.3a

' Means within a column with no common superscript differ significantly (P < 0.05).

0.1 1.1 1.1 0.8b 0.9b 1.4a

1.6 1.5 1.5 1.8 1.4

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For Experiment 1, two rooms each were assigned to either 4 or 7 d of housing in laying cages. For Experiments 2 and 3, two rooms were assigned to one of two environmental temperatures. For all three experiments, the two rooms within days of housing (Experiment 1) or environmental temperature (Experiments 2 and 3) were considered to be random effects and served as the respective error term for days of housing or environmental temperature. For Experiments 2 and 3, the completely randomized block design also included a split plot in time. For Experiment 2, the two times represented the period when cold exposure was administered and the recovery phase following treatment. Time represented the two heating episodes in Experiment 3. Bleeding time was considered a fixed effect. For Experiment 4, rows within rooms were random and served as the error term for rooms. Plasma cholesterol and corticosterone concentrations were analyzed as whole blood concentrations using the following formula: y = x[100 - (0.96z)]/100, where y = whole blood concentrations; x = plasma concentrations; and z = the perentage PCV (Guyton, 1986); however, as the statistical trends were the same for both plasma and whole blood concentrations, only the plasma levels of cholesterol and corticosterone will be presented. Percentage data for the differential leucocyte count were transformed to arc sine and reanalyzed (Steel and Torrie, 1980). Because statistical trends were similar for both transformed and untransformed data, the untransformed results will be presented.

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PHYSIOLOGY OF HENS ADAPTED TO MULTIPLE-HEN CAGES Four Days of Housing

Four Days of Housing

Genetic Stock

Selected

Control Genetic Stock

Seven Days of Housing Seven Days of Housing

• Multiple-Hen Cages

5 4.8 4.6 4.4 4.2 -

Genetic Stock

FIGURE 1. The packed cell volume of 18-wk-old chickens of three genetic lines (selected, control, and commercial) housed in either singleor multiple-hen cages at 4 and 7 d following the transfer from pullet to laying cages (Experiment 1). ** Values significantly different from the mean in the opposing cage size (genetic stock by cage size by days of housing interaction, P < 0.03). SEM = 0.5.

interactions significant with the exception of a three-way interaction of genetic stock by cage size by day of housing for plasma corticosterone (P < 0.02, Figure 2). With one exception, the plasma corticosterone levels of pullets were relatively consistent among genetic stocks or between cage sizes at 4 and 7 d following transfer to laying cages. The one exception was commercial chickens in single-bird cages at 4 d following transfer to laying cages. Significantly higher levels of plasma corticosterone were measured in this group than in the remaining groups of genetic stock housed in single- or multiple-hen cages at either 4 or 7 d following transfer to laying cages.

Experiment 2. Cold Environmental Temperatures Cold environmental temperature had little effect on the hematology of the 33-wk-old hens with two exceptions for

4

-

1,

11

Control

Commercial

Genetic Stock

FIGURE 2. Plasma corticosterone concentrations of 18-wk-old chickens of three genetic lines (selected, control, and commercial) housed in either single- or multiple-hen cages at 4 and 7 d following the transfer from pullet to laying cages (Experiment 1). * Value significantly different from the mean in the opposing cage size (genetic stock by cage size by days of housing interaction, (P < 0.02). SEM = 0.1.

PCV and the H:L ratio. Birds in the cold environment experienced hemoconcentration as indicated by an increase in PCV during the time of cold exposure (Time 1, Figure 3). Four to 5 h following the end of the cold treatment (Time 2), hens previously exposed to the cold environment had recovered, because PCV were similar to the controls. With respect to the H:L ratios, hens exposed to the cold environment had higher ratios than those of the control environment (x of 39.5 vs 37.6, SEM = 0.3, P < 0.04). During the time that two of the four rooms were in the cold (Time 1), the hens had a greater percentage of heterophils and monocytes (x of 26.6 and 4.5, respectively) than those in the recovery phase following cold exposure (Time 2, x of 23.1 and 3.6, respectively). Genetic stock as a main effect as well as its interaction with environmental temperature had no effect on plasma levels of cholesterol and corticosterone (Table 2) and had

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D Single-Hen Cages 5.2

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HESTER ET AL. TABLE 2. Plasma cholesterol and corticosterone concentrations of three genetic lines in single- or multiple-hen cages and exposed to either a cold (0 C) or control (21 C) environment, Experiment 2 Cholesterol Treatment

Time l i

Corticosterone Time 1

Time 2

(mg/100 mL)

— (ng/mL)

99 102

119 126

3.13 2.71

2.51 2.31

128 105

111 107

3.29 2.37

2.54 2.32

97 105

101 121

3.12 2.36

2.56 2.04

7

0.18

113 109

120 119

3.08a 2.51b

2.59a 2.13b

105 100

100 117

3.28* 2.46b

2.48* 2.32 ab

2

0.04

a b

- Means within a column with no common superscript differ significantly (P < 0.05). Time 1 represents 4 to 5 h into the cold exposure treatment, which lasted 72 h. Time 2 represents the recovery period, 4 to 5 h following the end of cold exposure. x

little effect on the hematology of the birds (Table 3). The only exception was that the control line had higher PCV than the commercial hens (Table 3). The PCV of the selected line of hens did not differ from either the control or commercial lines of hens. Heterophils did not differ among genetic lines or between hens housed in single- vs multiple-bird cages, although the main effect of cage size approached significance (Table 3, P < 0.07). The interaction of genetic stock with cage size (P < 0.02) was due to a significantly higher percentage of heterophils among the colony-caged commercial line of hens than among the other two genetic

lines in the same cage size. Although the heterophils were consistently lower for all three genetic lines in single-hen cages than for hens in multiple-hen cages, percentage heterophils were similar among the genetic lines housed in single-hen cages. The control and commercial lines of chickens housed in multiple-bird cages, as compared to single-hen cages, exhibited heterophilia, whereas the percentage of heterophils of the selected line of chickens did not differ between cage sizes. The statistical effects described above for heterophils also occurred with the H: L ratio (Table 3).

TABLE 3. Packed cell volume (PCV) and differential leucocyte count of three lines of genetic stock in either single- or multiple-hen cages at 33 wk of age, Experiment 2 Treatment

PCV

Heterophils (H)

Lymphocytes (L)

H:L Ratio x 100

Monocytes

Eosinophils

Basophils

cd

(%) Single-hen cages Selected line Control line Commercial line Multiple-hen cages Selected line Control line Commercial line SEM x Single-hen cages x Multiple-hen cages x Selected line x Control line x Commercial line 1_d

28.9 29.6 28.0

cd

23.9 21.3d 23.6 d

68.5 71.5 69.2

37.3 30.8d 35.2d

4.2 3.6 3.8

1.6 1.9 1.6

1.9 1.7 1.9

28.9 30.0 28.6 0.7

24.7bc 25.8b 29.3 a 0.5 22.9 26.6 24.3 23.5 26.4

66.5 70.0 63.5 1.0 69.7 65.7 67.5 69.2 66.4

39.2^ 40.3b 48.2 a 1.5 34.4 42.6 38.2 35.6 41.7

4.9 3.8 3.9 0.7

1.9 1.7 1.7 0.2 1.7 1.8 1.8 1.8 1.6

1.8 1.8 1.6 0.3 1.8 1.7 1.9 1.8 1.8

28.8 29.2 28.9 ab 29.8" 28.3b

Means within a column with no common superscript differ significantly (P < 0.05).

3.8 4.2 4.6 3.7 3.8

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Selected line Cold environment Control environment Control line Cold environment Control environment Commercial line Cold environment Control environment SEM Single-hen cages Cold environment Control environment Multiple-hen cages Cold environment Control environment SEM

Time 2

PHYSIOLOGY OF HENS ADAPTED TO MULTIPLE-HEN CAGES

1301

TABLE 4. Packed cell volume (PCV) and differential leucocyte count of three lines of genetic stock in singleand multiple-hen cages subjected to heated and control environments at 44 wk of age, Experiment 3 Treatment Selected line Heated environment Control environment Control line Heated environment Control environment Commercial line Heated environment Control environment Single-hen cages Heated environment Control environment Multiple-hen cages Heated environment Control environment SEM a_d

PCV

Heterophils (H)

Lymphocytes (L)

H:L Ratio x 100

Monocytes

Eosinophils

Basophils

(01 \ (,il)

30.0 31.2

28.6" 21.1=

57.1* 68.3"

61.7* 32.5b

8.3 6.7

1.9 1.7

4.01 2.11

30.4 31.4

28.9" 22.8=

56.8^ 68.5*

59.5" 36.4b

8.2 5.7

2.2 1.5

3.91 2.03

29.7 29.8

25.8b 25.8 b

60.4= 64.6b

50.3" 42.5 ab

8.1 6.2

2.5 1.6

3.24 1.81

29.3 29.9

27.1 21.9

59.4 69.1

54.1 33.7

7.0 5.7

2.5" 1.4b

3.94" 1.81d

30.8 31.7 0.2

28.4 24.6 1.3

56.8 65.1 0.9

60.2 40.6 4.9

9.4 6.8 0.4

1.9b 1.9b 0.1

3.50b 2.15= 0.04

After 4 to 5 h of cold exposure, both single- and colonycaged hens exposed to 0 C had significantly higher levels of plasma corticosterone than hens of the control environment (Time 1, Table 2). During the recovery period, i.e., 4 to 5 h following the end of the 72-h cold treatment (Time 2), the single-caged hens previously exposed to cold continued to exhibit significantly higher levels of plasma corticosterone than the hens of the control environment. In contrast, plasma corticosterone of hens of multiple-bird cages previously exposed to cold had returned to normal levels and did not differ from the hens of the control environment (Time 2, environmental temperature by cage size by time interaction, P < 0.04). A cold environmental temperature did not affect plasma concentrations of cholesterol (Table 2).

was more pronounced in the selected and control lines than in the commercial line under high temperature conditions. The percentage of monocytes were significantly increased in colony-caged hens as compared to those of single-hen cages (x of 8.1 vs 6.4%, respectively, SEM = 0.3, P < 0.05). Significant interactions between environmental temperature and cage size occurred with eosinophils (P < 0.03) and basophils (P < 0.01; Table 4). Eosinophilia

a

D Cold environment • Control environment

Experiment 3. High Environmental Temperatures ab

Hens of single-bird cages as compared to multiple-bird cages experienced hemodilution, as indicated by a decrease in PCV (x of 29.6 vs 31.3%, respectively; P < 0.01). Packed cell volume did not differ among genetic lines nor was it affected by environmental temperature. Interactions for PCV were not significant (Table 4). Significant interactions between environmental temperature and genetic stock occurred with heterophils (P < 0.003), lymphocytes (P < 0.003), and the ratio of H:L (P < 0.03, Table 4). The percentage of heterophils and the ratio of H:L increased in the selected and control line of chickens subjected to high environmental temperatures, whereas the percentage of heterophils and the H:L ratio were unaffected by heat exposure in the commercial line of laying hens. High temperature induced lymphopenia in all three genetic lines of chickens when compared to the control temperature regimen; however, the lymphopenia

^^^^H

Time 1 (Treatment period)

Time 2 (Recovery period)

FIGURE 3. Packed cell volume of hens exposed to a cold environment (0 C) as compared to hens of the control environment (21 C). Time 1 represents 4 to 5 h into the cold treatment, which lasted 72 h. Time 2 represents the recovery period, 4 to 5 h following the end of cold exposure. Values with no common superscripts differ at a P < 0.05 (environmental temperature by time interaction, SEM = 0.3).

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Means within a column with no common superscript differ significantly (P < 0.05).

HESTER ET AL.

1302 Heated Environment

QHeated Environment • Control Environment

% Basophils

s

Control Gsn«tJc Stock

First Heating Episode

Second Heating Episode

Control Environment

I r l r l fbl Control

Genetic Stock

FIGURE 4. Percentage basophils of 44-wk-old chickens of three genetic lines (selected, control, and commercial) housed in either singleor multiple-hen cages and exposed to either a heated (38 C) or control (28 to 30 C) environment (Experiment 3). * Values significantly different from the mean in the opposing cage size (environmental temperature by cage size by genetic stock interaction, P < 0.02). SEM = 0.3.

occurred in heat-exposed single-caged hens, whereas eosinophils of colony-caged hens were unaffected by environmental temperature. Heat exposure induced basophilia in both single- and colony-caged hens. The three-way interaction of environmental temperature by cage size by genetic stock for basophils further delineates the effect (P < 0.02, Figure 4). Within a heated environment, the basophilic responses of the three genetic lines differed between cage sizes. Specifically, the selected and control lines of chickens housed in single-hen cages experienced a greater increase in the percentage of basophils than the same lines of multiple-hen cages. The basophils of the commercial line of chickens showed the opposite trend in that basophilia occurred in multiple-hen cages more often than in single-hen cages. Within a control temperature regimen, the basophilic responses of the three genetic lines were similar and did not differ between single- vs multiple-hen cages. The hematological profiles of the hens were consistent between heating episodes except for basophils, in which

interactions with environmental temperature and cage size occurred. Hens of the first heating episode had a higher basophil percentage than hens of the second heating episode (x of 3.6 vs 2.1, respectively, SEM = 0.2; P < 0.04). It was specifically the hens of the heated environment of the first heating episode that experienced basophilia, as indicated by the significant interaction of environmental temperature with time or heating episode (P < 0.03, Figure 5). Hens subjected to a high environmental temperature regimen, as compared to those of a control temperature, experienced basophilia during the first heating episode but not during the second heating episode. An opposing basophilic response of colony-caged hens between heating episodes resulted in a significant interaction between cage size and heating episode (P < 0.01, Figure 6). Colony-caged hens of the first heating episode had fewer basophils than single-caged hens with the opposite trend occurring during the second heating episode. The three-way interaction of environmental temperature by cage size by heating episode (P < 0.004, Figure 7) further identifies the effect of high temperature exposure during the first heating episode on basophilia. Although both single- and colony-caged hens of the heated environment of the first heating episode experienced dramatic basophilia, it was the single-caged hens that showed the largest increase in basophils. Hens in both single- and multiple-bird cages of the heated environment in the second heating episode had percentage of basophils similar to that of hens of the control environment; however, percentage basophils were significantly greater for colony-caged hens than for single-caged hens. The same trend for percentage basophils occurred with hens of the control environment during the first heating episode, in that colony-caged hens had higher percentage of basophils than single-caged hens. The

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FIGURE 5. Percentage basophils of 44-wk-old chickens exposed to two heating episodes of 38 C as compared to hens of the control environment (28 to 30 C, Experiment 3). * Values significantly different from the other means (environmental temperature by heating episode interaction, P < 0.03). SEM = 0.3.

• Single-Hen Cages • Multiple-Hen Cages

PHYSIOLOGY OF HENS ADAPTED TO MULTIPLE-HEN CAGES

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TABLE 5. Plasma cholesterol and corticosterone concentrations of three genetic lines in single- or multiple-hen cages and exposed to either a heated (38 C) or control (28 to 30 C) environment, Experiment 3 Cholesterol First heating episode

Treatmer it

Corticosterone

Second heating episode

First heating episode

(ng/mL)

(mg/100 mL)

i

119 112

119 111

4.7 3.9

4.5 4.6

123 114

118 121

4.7 3.9

4.0 3.7

124 113

123 125

5.6 4.2

5.6 4.1

10

0.5

129 120

140 134

5.4 4.0

4.8 4.1

114 106

102 103

4.6 4.0

4.6 4.1

14

0.8

second heating episode did not affect the basophilic response of the single- vs colony-caged hens of the control environment. Plasma concentrations of cholesterol and corticosterone were unaffected by a high environmental temperature, genetic stock, or cage size (Table 5). None of the interactions were significant.

relative right adrenal weights of the selected line were significantly heavier than adrenals of the control and commercial lines (P < 0.01). Hens of the control line had heavier relative right adrenal weights than those of the commercial line. The room effect and the interaction of genetic stock with cage size were not significant for body and adrenal weights.

Experiment 4. Adrenal Weights

DISCUSSION

Lighter body weights and heavier relative right adrenal weights occurred with hens in colony cages as compared to single-hen cages (P < 0.03, Table 6). Body weights were similar among the three genetic lines. The absolute and

Our hypothesis was that hens selected for adaptation to a multiple-bird environment should react less intensely to stressors than would the unselected controls. Turkeys selected for high and low adrenal responses

TABLE 6. Body and adrenal weights of three genetic lines in singleand multiple-hen cages at 62 wk of age, Experiment 4

Treatment Single-hen cages Selected line Control line Commercial line Multiple-hen cages Selected line Control line Commercial line SEM x Single-hen cages x Multiple-hen cages x Selected line x Control line x Commercial line a_c

Body weight

Absolute right adrenal

Relative right adrenal

(kg)

(mg)

(mg/100 g)

1.68 1.72 1.76

74.6 67.0 63.2

4.5 3.9 3.6

1.61 1.58 1.63 .03 1.72a 1.60b

77.2 74.2 69.2 2.7

4.8 4.8 4.4 0.1 4.0 b 4.6a 4.6a 4.3b 4.0=

1.64 1.65 1.69

68.3 73.5 75.9* 70.6b 66.2b

Means within a column with no common superscript differ significantly (P < 0.05).

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Selected line Heated environment Control environment Control line Heated environment Control environment Commercial line Heated environment Control environment SEM Single-hen cages Heated environment Control environment Multiple-•hen cages Heated environment Control environment SEM

Second heating episode

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HESTER ET AL. Heated Environment • Single-Hen Cages • Multiple-Hen Cages

.

D Single-Hen Cages

a

• Multiple-Hen Cages

** b

**

c

1

•

First Heating Episode

| First Heating Episode

Control Environment

Second Heating Episode

f j Single-Hen Cages • Multiple-Hen Cages

First Healing Episode

Second Heating Episode

FIGURE 7. Percentage basophils of hens in either single- or multiplebird cages exposed to two heating episodes of 38 C as compared to hens of the control environment (28 to 30 C, Experiment 3). ** Values significantly different from the mean in the opposing cage size (environmental temperature by cage size by heating episode interaction, P < 0.004). SEM = 0.08.

1996), more similarities would be expected between these two lines than the commercial line. Because water consumption was not measured, PCV 4 d following the transfer of pullets to laying cages provided indirect evidence of improved adaptation of the selected line to multiple-hen cages. Using hemodilution as a criterion, it appeared that the selected line in multiple-hen cages adapted more quickly to the new waterer system than the unselected and commercial lines. At 4 d following housing, the selected line of Leghorns experienced decreased PCV or hemodilution in a competitive environment of 12 hens as compared to the same genetic line in single-hen cages. Even though there were fewer drip nipples per hen in multiple cages than in single cages, the selected line of chickens, as a group of 12 hens, may have taught each other how to use the waterers more quickly than the singly caged hen of the same genetic line. In contrast, the commercial line, whose parents and grandparents were housed and selected in single-hen cages, appeared to adapt better to

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FIGURE 6. Percentage basophils of hens in either single- or multiplehen cages exposed to two heating episodes (cage size by time interaction, Experiment 3). Values with no common superscripts differ at P < 0.01. SEM = 0.06.

during stress support this hypothesis in that the low corticosterone lines were less excitable and had improved production traits (Brown and Nestor, 1973, 1974). Likewise, chickens selected for a high response to adrenocorticotropin had a shorter survival time of 12 min than the low response line when subjected to acute heat stress of 45 C (Edens and Siegel, 1975; Siegel, 1981). The selected line of Leghorns of the current study has shown evidence of responded less intensely to stress than do the other genetic stocks. Mortality during an acute heating episode provided the strongest evidence that the selected line responded less intensely to stress. Specifically, the selected line of chickens in multiple-hen cages showed an increased resistance to heat exposure, as indicated by lower mortality, when compared to the control and commercial lines housed in multiple-hen cages (Hester et al., 1996). A slower rate of decline in egg production in multiple-bird cages as a result of handling and temperature extremes (Hester et al., 1996), a reduction in beak-inflicted injuries in multiple-bird cages, and improved feathering of the selected line (Craig and Muir, 1996) provided additional support that the selected line of chickens was either more resistant to stress or better adapted to multiple-bird cages than were the control and commercial lines. The current study dealing with the physiological response of three genetic lines to stress provided some additional proof that the selected line was better adapted to multiple-bird cages; however, trends were not always consistent. The selected and control lines often showed similar physiological responses that were indistinguishable from each other, but distinct from those of the commercial line (Table 1). Because the selected line was derived from the control line (Muir,

Second Heating Episode

PHYSIOLOGY OF HENS ADAPTED TO MULTIPLE-HEN CAGES

of frostbitten combs (Hester et al, 1996) and the lack of return of circulating levels of corticosterone during the recovery phase following cold exposure (Experiment 2, Table 2). Because of the presence of cage mates and the transfer of heat through conduction, hens of colony cages were more likely to maintain a higher core body temperature during exposure to the cold than singlecaged hens. In addition to adrenal hypertrophy, the differential leucocyte responses during heating (Experiment 3) could be interpreted to represent a more intensive stress reaction by the selected line; however, the same argument used above for the basophilic response under heating conditions can also apply to the heterophil and lymphocyte responses. Although heat exposure induced heterophilia, caused pronounced lymphopenia, and increased the H:L ratio in the selected line as compared to the commercial line, the unselected controls showed the same response as the selected line (Table 4). The lack of an effect of high temperature on circulating levels of corticosterone in the current study was not anticipated, but has been reported previously in 10- to 17-d-old chicks exposed to additive stressors including heat stress (McFarlane and Curtis, 1989). Siegel (1995) suggested that the young age of the chicks used in the study of McFarlane and Curtis (1989) may have contributed to the absence of a glucocorticoid response, because adrenals from chicks of this age are relatively insensitive to adrenocorticotropic hormone or stressors (Siegel, 1962a,b). Other researchers have shown that birds exhibit an increase in plasma corticosterone during temperature exposure (Siegel, 1971; El-Halawani et al, 1973; Edens and Siegel, 1975; Beuving and Vonder, 1978). For the current study, blood samples were collected 4 to 6 h and 1 to 3 h after the initiation of cold and heat exposure, respectively. These time periods were chosen because in broilers, plasma corticosterone peaked 70 to 100 min following the initiation of acute heat exposure and remained at higher levels than that of the controls when the experiment was terminated 2.3 h later (Edens and Siegel, 1975). Likewise, White Leghorns showed an increase in plasma corticosterone at 45 min, 1.5 h, and 3 h, but not at 5 h following the initiation of a 37 C temperature regimen (Beuving and Vonder, 1978). Increased levels of plasma corticosterone were still prevalent in male turkeys 1 or 2 wk following the initiation of either a 7 or a 32 C ambient temperatures, respectively (El-Halawani et al., 1973). Based on these previous reports, the times chosen for blood collection in the current study were not inappropriate for detecting a rise in circulating levels of corticosterone. The lack of a corticosterone response to heat exposure in the present study could be due to acclimation. Siegel (1995) discussed evidence that suggest that chronic or repeated exposures to stress could cause subsequent declines in adrenal responsiveness and thus corticosterone output (Freeman et al., 1979; Gross and Siegel, 1979; Siegel and Gould, 1982). Previous exposures to stressors

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the drip nipples of single-hen cages as opposed to multiple-hen cages, as indicated by hemodilution or decreased PCV (genetic stock by cage size by days of housing interaction, P < 0.03, Figure 1). The H:L ratios of Experiment 2 provided additional support of improved adaptation of the selected line to multiple-hen cages. It has been reported that circulating heterophils and H:L ratios increase with environmental stress (Wolford and Ringer, 1962; Gross and Siegel, 1983; McFarlane and Curtis, 1989; Maxwell, 1993). When the hens of the current study were 33 wk of age, the control and commercial lines in multiple-hen cages experienced heterophilia and increased H:L ratios when compared to these same two lines in single-hen cages. In contrast, the selected line of chickens housed in multiple-hen cages did not experience heterophilia or increased H:L ratios when compared to their single-caged counterparts (genetic stock by cage size interaction, P < 0.02, Table 3). This leucocytic response could be interpreted to mean that the selected line of chickens adapted better to social competition than either the control or commercial lines. However, a similar interaction for the leucocytic response did not occur in Experiment 3 when the hens were 44 wk of age nor was the effect apparent in the pullets of Experiment 1. Basophilia is another indicator of stress (Maxwell, 1993). In the current study, high temperature caused an increase in basophils in both single-caged and in colonycaged hens (Table 4 and Figure 4); however, using the basophilic response as an indicator that the selected line of chickens is better adapted to multiple-hen cages cannot apply here because both the selected and control lines had reduced basophils in colony cages as comp a r e d to the commercial line, w h i c h experienced basophilia in colony cages (temperature by cage size by genetic stock interaction, P < 0.02, Figure 4). Evidence that the selected line reacted more intensely to stressors is limited. The greater adrenal weights of the selected line as compared to the control and commercial lines can be interpreted as a more intensive response to stress. Adrenal hypertrophy could also be indicative of greater adaptation to stress (Siegel, 1971). At the time that the adrenals were excised, half of the birds had not been subjected to high temperature for 15 wk. Although adrenal weights were not measured during cold and heat exposures, a difference in the plasma corticosterone response among genetic lines due to environmental temperature was not evident (Tables 2 and 5). Social competition in multiple-hen cages was persistent throughout the study and resulted in adrenal hypertrophy when compared with single-caged hens (Table 6). Social competition induced by grouping young chickens into pens for 4 h / d for 22 d resulted in adrenal hypertrophy when compared to controls that were not moved (Siegel and Siegel, 1961). Single-caged hens exposed to a low ambient temperature of 0 C for 72 h were more adversely affected by the cold than hens of multiple bird cages as indicated by the higher incidence

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HESTER ET AL.

Beuving and Vonder (1977) reported that the laying hen's circadian rhythm of corticosterone peaks immediately prior to oviposition and is at its lowest level at the beginning of the dark period. Stressed hens of the current study, most of which had a plumped egg in the uterus, were bled 1 to 4 h prior to the end of the dark period and at a time when the ovipositional-related peak in corticosterone would not occur. Hens were bled prior to scotophase because there is some evidence that animals will not respond to further increases in adrenal steroid output when they are already at peak levels, such as prior to oviposition (El-Halawani et al., 1973; Perry, 1973). Individual hens differ genetically in how they perceive stress, which can result in a highly variable response to the same stressor (Gross and Siegel, 1993). For example, Beuving and Vonder (1978) reported that when some hens were immobilized, they responded with a large increase in plasma corticosterone (20 n g / mL), whereas other immobilized hens showed no response (2 ng/mL). Due to the variability in plasma corticosterone, lines of birds with high and low levels of circulating corticosterone have been selected for successfully (Brown and Nestor, 1973; Edens and Siegel, 1975; Siegel, 1979). Hens of the current study also had highly variable plasma corticosterone responses to temperature exposure, with values ranging from 1.1 to 17.7 n g / m L . Control hens had a range of 0.7 to 7.9 n g / m L . In conclusion, the selected line in multiple-hen cages as compared to the unselected control and commercial

lines of chickens reacted less intensely to stressors as indicated by hemodilution (decreased PCV) at the time of transfer to laying cages and the lack of a leucocytic response due to cage size at 33 wk of age. However, trends were not always consistent because the selected line experienced adrenal hypertrophy at 64 wk of age and did not show a similar leucocytic response due to cage size at 18 or 44 wk of age as it did at 33 wk of age.

ACKNOWLEDGMENTS Technical assistance from Marisue Freed, Julie Ladd, Jean Craig, Brent Ladd, Deena Liggett, Debbie Miles, Mollie McComb, Jamie Carrigan, and Kim Berry was greatly appreciated. Gratitude is also expressed to Ken Wolber for the managerial care of the birds and to Mark Einstein for statistically analyzing the data. Hatching eggs of the commercial strain were kindly donated by DeKalb® Poultry Research, Inc., DeKalb, IL 60115.

REFERENCES Bell, D., 1993. The egg industry of California and the USA in the 1990s: a survey of systems. World's Poult. Sci. J. 49: 58-64. Beuving G., and G.M.A. Vonder, 1977. Daily rhythm of corticosterone in laying hens and the influence of egg laying. J. Reprod. Fertil. 51:169-173. Beuving, G., and G.M.A. Vonder, 1978. Effect of stressing factors on corticosterone levels in the plasma of laying hens. Gen. Comp. Endocrinol. 35:153-159. Brown, K. I., and K. E. Nestor, 1973. Some physiological responses of turkeys selected for high and low adrenal response to cold stress. Poultry Sci. 52:1948-1954. Brown, K. I., and K. E. Nestor, 1974. Interrelationships of cellular physiology and endocrinology with genetics 2. Implications of selection for high and low adrenal response to stress. Poultry Sci. 53:1297-1306. Craig, J. V., and J. A. Craig, 1985. Corticosteroid levels in White Leghorn hens as affected by handling, laying-house environment, and genetic stock. Poultry Sci. 64:809-816. Craig, J. V., and W. M. Muir, 1996. Group selection for adaptation to multiple-hen cages: Beak-related mortality, feathering, and body weight responses. Poultry Sci. 75: 294-302. Craig, J. V., and J. C. Swanson, 1994. Review: Welfare perspectives on hens kept for egg production. Poultry Sci. 73:921-938. Davidson, J. M., L. E. Jones, and S. Levine, 1968. Feedback regulation of adrenocorticotropin secretion in "basal" and "stress" conditions: Acute and chronic effects of intrahypothalamic corticoid implantation. Endocrinology 82: 655-663. Edens, F. W., and H. S. Siegel, 1975. Adrenal responses in high and low ACTH response lines of chickens during acute heat stress. Gen. Comp. Endocrinol. 25:64-73. El-Halawani, M. E., P. E. Waibel, J. R. Appel, and A. L. Good, 1973. Effects of temperature stress on catecholamines and corticosterone of male turkeys. Am. J. Physiol. 224: 384-388. Freeman, B. M., A.C.C. Manning, and I. H. Flack, 1979. Habituation by the immature fowl in response to repeated injections of corticotrophin. Br. Poult. Sci. 20:391-399.

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could increase the binding of corticosterone to receptors in the hypothalamus, thus inhibiting the release of corticotropin-releasing factor (Davidson et al., 1968; Kobayashi and Wada, 1973). The colony-caged hens of the current study were exposed to continuous social interaction beginning at 16.7 to 17 wk of age. Behavioral observations, including fear-related behaviors, were conducted on the birds throughout the study. Half of the birds were subjected to a low ambient temperature at 33 wk of age in which higher circulating levels of corticosterone were observed due to cold exposure (Table 2, Experiment 2). Because there was no mortality, the cold exposure was considered to be mild. Finally, the same hens that had been exposed to a low ambient temperature at 33 wk of age were again subjected to a stressor at 44 wk of age, this time two heating episodes each of 3 h duration. Under conditions of repeated stress, which prevailed in this study, Siegel (1995) suggested that end-organ responses such as H:L ratios may be better indicators of physiological stress and that plasma hormone levels such as corticosterone may be better indicators of acute or nonrepeated stress. Plasma corticosterone levels of hens of the current study exposed to a high environmental temperature were unaffected (Table 5). The additive stressors applied to juvenile chicks in the study of McFarlane and Curtis (1989) may have also resulted in acclimation offering an explanation for the observed increases in heterophils and the H:L ratio with no effect on corticosterone.

PHYSIOLOGY OF HENS ADAPTED TO MULTIPLE-HEN CAGES

Pierson, F. W., P. Y. Hester, and E. K. Wilson, 1981. The effect of caponization and dietary 17 a-methyltestosterone on the incidence of leg abnormalities in turkeys. Poultry Sci. 60: 2144-2149. Rudel, L. L., and M. D. Morris, 1973. Determination of cholesterol using o-phthalaldehyde. J. Lipid Res. 14: 364-366. Siegel, H. S., 1962a. Age and sex modification of responses to adrenocorticotropin in young chickens. II. Changes in adrenal cholesterol and blood constituent levels. Poultry Sci. 41:321-334. Siegel, H. S, 1962b. Critical ages for responses to ACTH in chicks. Gen. Comp. Endocrinol. 2:385-388. Siegel, H. S., 1971. Adrenals, stress and the environment. World's Poult. Sci. J. 27:327-349. Siegel, H. S., 1981. Adaptation of poultry to modern production practices. Pages 57-66 in: World Poultry Production: Where and How? C. W. Scheele and C. W. VeerKamp, ed. Spelderholt Institute for Poultry Research, Beekbergen, The Netherlands. Siegel, H. S., 1995. Stress, strains and resistance. Br. Poult. Sci. 36:3-22. Siegel, H. S., and N. R. Gould, 1982. Corticosteroid binding to lymphocytes of various tissues in growth birds subjected to high temperatures. Gen. Comp. Endocrinol. 48:348-354. Siegel, H. S., and P. B. Siegel, 1961. The relationship of social competition with endocrine weights and activity in male chickens. Anim. Behav. 9:151-158. Siegel, P. B., 1979. Behavior genetics in chickens: a review. World's Poult. Sci. J. 35:9-19. Steel, R.G.D., and J. H. Torrie, 1980. Principles and Procedures of Statistics. A Biometrical Approach. 2nd ed. McGrawHill Book Co., Inc., New York, NY. Wolford, J. H., and R. R. Ringer, 1962. Adrenal weight, adrenal ascorbic acid, adrenal cholesterol and differential leucocyte counts as physiological indicators of "stressor" agents in laying hens. Poultry Sci. 41:1521-1529.

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Gross, W. B., and H. S. Siegel, 1983. Evaluation of the heterophil /lymphocyte ratio as a measure of stress in chickens. Avian Dis. 27:972-979. Gross, W. B., and P. B. Siegel, 1979. Adaptation of chickens to their handler, and experimental results. Avian Dis. 23: 708-714. Gross, W. B., and P. B. Siegel, 1993. General principles of stress and welfare. Pages 21-34 in: Livestock Handling and Transport. T. Grandin, ed. CAB International, Wallingford, U.K. Guyton, A. C , 1986. The body fluids: osmotic equilibria between extracellular and intracellular fluids. Page 384 in: Textbook of Medical Physiology. 7th ed. W. B. Saunders, Co., Philadelphia, PA. Hester, P. Y., W. M. Muir, J. V. Craig, and J. L. Albright, 1996. Group selection for adaptation to multiple-hen cages: Production traits during heat and cold exposures. Poultry Sci. 75:1308-1314. Klingensmith, P. M., P. Y. Hester, and E. K. Wilson, 1984. Relationship of plasma corticosterone and adrenal cholesterol and corticosterone to the production of softshelled and shell-less eggs. Poultry Sci. 63:1841-1845. Kobayashi, H., and M. Wada, 1973. Neuroendocrinology in birds. Pages 287-347 in: Avian Biology. Vol. 3. D. S. Farner and J. R. King, ed. Academic Press, New York, NY. Maxwell, M. H., 1993. Avian blood leucocyte responses to stress. World's Poult. Sci. J. 49:34-43. McFarlane, J. M., and S. E. Curtis, 1989. Multiple concurrent stressors in chicks. 3. Effects on plasma corticosterone and the heterophiklymphocyte ratio. Poultry Sci. 68:522-527. Muir, W. M., 1996. Group selection for adaptation to multiplehen cages: Selection program and direct responses. Poultry Sci. 75:447-458North, M. O., and D. D. Bell, 1990. Cage management. Pages 297-370 in: Commercial Chicken Production Manual. 4th ed. Van Nostrand Reinhold, New York, NY. Perry, G., 1973. Can the physiologist measure stress? New Scientist, 18th October, pp. 175-177.

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