Corticosterone in Commercial Broilers1

2005 Poultry Science Association, Inc.

Corticosterone in Commercial Broilers1 J. P. Thaxton,*,2 P. Stayer,† M. Ewing,† and J. Rice† *Department of Poultry Science, Box 9665, Mississippi State University, Mississippi State, Mississippi 39762-9665; and †Sanderson Farms, Inc., Laurel, Mississippi 39440

SUMMARY The welfare of commercially produced broilers is of concern to producers and consumers. When birds are reared under stressful conditions they often exhibit several adaptive responses. The major response is secretion of the stress hormone, corticosterone (CS), by the adrenal glands. The objective of this study was to assess welfare of commercially reared broilers by determining plasma CS concentrations at various times during grow out. Results show that CS concentrations of all birds tested remained in the nonstress range. The conclusion of this study is that commercial production of broilers in tunnel-ventilated houses that maintained temperatures in the comfort zone and light at low intensities did not cause stress as measured by circulating blood levels of CS. Key words: welfare, stress, corticosterone, broiler production 2005 J. Appl. Poult. Res. 14:745–749

DESCRIPTION OF PROBLEM Today much concern is being expressed about stocking density as it relates to the wellbeing and welfare of broilers. Several grocery, and fast food, and restaurant chains, as well as public advocates concerned with animal welfare, are acutely concerned with this important issue [1]. Integrated broiler companies share this concern about the welfare of their birds [2]. Presentday production practices are designed and implemented to maximize production and minimize stressful conditions. Research has shown that the concentration of blood CS is the most sensitive indicator of stress in broilers [3, 4, 5, 6, 7]. Many studies of chickens reared under research 1

condition have indicated that when the wellbeing of chickens is compromised by stressful conditions, blood concentrations of CS will be elevated [8, 9, 10, 11]. However, there are no reports concerning blood CS levels in broilers reared by contract growers on commercial broiler farms. The specific objective of this study was to determine CS blood concentration in commercially reared broilers.

METHODS AND MATERIALS Husbandry Eight contract growers were selected by management personnel of Sanderson Farms, Inc. at Laurel, MS, to grow and care for broilers in this study. The broilers were placed in a single

Journal article number J-10714 from the Mississippi Agricultural and Forestry Experiment Station, Mississippi State, MS. To whom correspondence should be addressed: [email protected].

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Primary Audience: Researchers; Production, Processing, and Sales Personnel; Poultry Health Specialists

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746 TABLE 1. Density and slaughter age of broilers in this study Grower 1 2 3 4 5 6 7 8

House type1

Birds placed (n)

Floor space (ft2)

Density (ft2/bird)

Slaughter age (d)

BC BC BC BC BC BS BS CC

27,500 24,400 24,400 18,000 25,000 24,400 24,400 16,000

22,000 22,000 22,000 14,400 20,000 22,000 22,000 14,400

0.8 0.9 0.9 0.8 0.8 0.9 0.9 0.9

53 61 52 59 49 53 59 61

house on each of the 8 farms during the first week of April 2004. Three different housing conditions were incorporated in this study. Table 1 summarizes housing type, space allotments, bird numbers, and stocking densities of the grow out houses. Six of the 8 houses had curtain sidewalls, whereas the other 2 had solid sidewalls. Of the 6 curtain-sided houses, 5 were fitted with black curtains and the other had clear curtains. This means that 7 of the 8 houses were blackout houses and 1 essentially had natural lighting. In the 7 blackout houses, the lighting schedule and light intensity measured in footcandles (fc) were as follows: 0 to 7 d = 24L:0D at 1.50 fc, 8 to 14 d = 19L:5D at 1.00 fc, 15 to 22 d = 20L:4D at 0.50 fc, and d 22 until processing = 22L:2D at 0.50 fc. Company recommended house temperature profiles were adhered to in all houses. These temperatures were regulated by tunnel ventilation systems. Litter temperatures in the houses for d 1, 2, and 3 were 88, 86, and 84°F, respectively. After the first 3 d, air temperatures were measured at 2 in. above the litter. The recommended ambient temperatures are 84°F from 4 to 10 d, 83°F from 11 to 12 d, 82°F from 13 to 14 d, 81°F from 15 to 16 d, 80°F from 17 to 18 d, 78°F from 19 to 20 d, 77°F from 21 to 22 d, 76°F from 23 to 24 d, 75°F from 25 to 26 d, and 70 from 74°F on d 27 until processing. Bird density was 0.8 or 0.9 ft2/bird, and the birds were processed at 47 to 61 d of age. Birds were fed starter, grower, finisher, and withdrawal diets. Protein contents of the diets were 21, 20, 16, 15.5%, respectively, and caloric contents were 1,405, 1,430, 1,440, 1,460 kcal/lb, respectively.

Blood Collection Procedure At designated times during growing, broilers in 1 house on each of the 8 farms were bled by service personnel. These company employees collected blood samples into heparinized test tubes from 12 chickens. Each chicken was selected at random and quickly bled by cardiac stab. Each service person understood that it was critical to catch the chicken and draw the blood sample in less than 45 s. The designated times of collection were when the chicks were at the following ages: 17 to 18, 35 to 39, 43 to 47, 49 to 53, and 59 to 61 d. Blood samples were capped, placed on ice, and delivered to the Sanderson Farms Laboratory as soon as possible after collection had been completed. Laboratory personnel centrifuged the samples (3,120 rpm for 10 min), decanted each plasma sample into a vial, capped each vial, and stored the vials at −4°F. Within 1 wk after freezing, a group of samples that had been collected at the same bleeding interval were removed from the freezer, packed in dry ice in a cryoprotective container, and shipped by overnight carrier to the Poultry Science Department at Mississippi State University. As soon as received, samples were transferred to a freezer (−4°F) until CS determinations were performed. Measurements Plasma CS concentrations were determined by an enzyme-linked immunoassay (EIA) [12]. This EIA was validated for chickens by adding known amounts of CS to chicken plasma that had been stripped of steroids using activated charcoal. The mean intra- and interassay coeffi-

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BC = blackout, black curtain sidewalls, solid walls; BS = blackout, solid walls; CC = natural lighting, clear curtain sidewalls.

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TABLE 2. Corticosterone (CS, pg/mL) in commercial broilers

Grower 1 2 3 4 5 6 7 8

Bleeding age (d)

House type1

17–19

35–39

43–47

49–53

BC BC BC BC BC BS BS CC

723 629 762 746 647 568 537 705

559 500 538 578 548 493 539 542

518 563 543 563 541 509 558 597

525 517 541 539 534 548 566 566

59–61 600 604

596 713 466

Mean ± SEM2 581 572 598 606 568 543 583 575

± ± ± ± ± ± ± ±

11 7 10 14 15 8 12 14

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cients of variation were 4.5 and 3.1%, respectively. Validation of this assay against the accepted RIA has been completed. Results indicate that accuracy and precision for the EIA for CS are greater than the RIA procedure. The EIA uses sheep polyclonal antibody against CS. To assay, 10 ␮L of each plasma sample was pipetted into an Eppendorf tube, and 10 ␮L of steroid inhibitor buffer was added and then vortexed. All samples were diluted to a 1:400 concentration with Tris buffer solution containing sodium azide. Each sample (100 ␮L) was pipetted into microtiter plate wells (coated with donkey-anti-sheep IgG) in duplicate. In addition, 50 ␮L of alkaline phosphatase conjugated with CS and 50 ␮L of sheep polyclonal antibody were added into each well, respectively. After 2 h of incubation on a shaker (500 rpm) at room temperature (72°F), plates were washed 3 times in Tris buffer solution containing detergents and sodium azide. A solution of p-nitrophenyl phosphate (200 ␮L) was added into each well, and the plate was incubated for a further 1 h (without shaking) at room temperature. To stop the reaction, 50 ␮L of trisodium phosphate solution was added, and absorbance was read at 405 nm using a microplate spectrophotometer [13]. Standard curves and sample concentrations were calculated using KC Junior software [13]. This software package utilizes a 4-parameter logistic curve-fitting program for standard and unknown samples. Statistical Procedures Data were analyzed by one-way ANOVA. The main effects were age of bird at time of

collection of blood samples and grower. Means were separated by Bonferonni test. All statistical procedures were conducted using Statistix 8 Analytical Software [14].

RESULTS AND DISCUSSION Results of this study are summarized in Table 2. In this study, blood CS concentrations were determined on 192 broilers. The highest single CS value recorded was 1,070 pg/mL, and the lowest recorded individual CS value was 340 pg/mL. The mean CS concentration for all 192 birds was 607 with an SEM of 10. Mean CS concentrations for the 8 growers, over all time periods, did not differ at P ≤ 0.05. These means ranged from 543 ± 8 to 606 ± 14. The CS levels did not appear to be affected by housing type. Mean CS levels were numerically lowest in solid wall houses (CS = 555 ± 6 pg/mL) as compared with blackout houses with dark curtains (CS = 573 ± 5 pg/mL) or the house with clear curtains (CS = 588 ± 14 pg/ mL). Additional experimentation, however, is required to statistically compare CS levels in broilers as related to housing types, especially in regards to light intensities. A model to evaluate stress in chickens has been described [3, 4, 5, 6, 7]. Osmotic pumps loaded with ACTH were implanted under the skin of broiler chicks. These pumps released ACTH at a dosage of 8 IU/kg of BW per d for 7 consecutive days. The ACTH is a hormone secreted by the pituitary gland that causes the adrenal glands to synthesize and secrete CS [15]. In this stress model, ACTH caused a series of physiological responses. In fact, 42 different re-

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BC = blackout, black curtain sidewalls; BS = solid walls; CC = natural lighting, clear curtain sidewalls; Mean CS ± SEM of houses: BC = 573 ± 5, BS = 555 ± 6, CC = 588 ± 14 did not differ at critical value of comparison of 18. Means CS of growers did not differ (P ≤ 0.05) at critical value for comparison of 67.

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sponses were studied, and blood CS concentration was shown to be the most predictable indicator of the stress condition in broilers. In this stress model, nonstressed broilers were found to have a mean blood CS concentration of 624 pg/mL. These workers also demonstrated that maximum CS concentration in stressed broilers exceeded 15,000 pg/mL. Blood levels of CS have been shown to be elevated when broilers are simply held by a human in excess of 45 s [16]. It also has been demonstrated that at the onset of stress in chickens, blood concentrations of CS rise to levels between 1,500 to 3,000 pg/mL [5, 10]. Based on the previous findings [3, 4, 5, 6, 7, 8, 9, 10, 11], not a single bird in this study was found to exhibit a concentration of blood CS indicative of the stress condition. It is also

interesting to note that there was a decrease in blood CS concentration over time. Specifically, from 2 to 7 wk of age CS concentrations of all 192 birds decreased consistently as shown in the scatter plot of Figure 1. The R2 value for CS was 0.1058 (P < 0.01). Apparently, physiological adaptation by broilers to their environment is enhanced as the birds approach market age [17]. The commercial production practices used in the present study are representative of industry norm, and these practices did not cause a stress condition, based on blood concentrations of CS. Apparently, when broilers are maintained within the thermoneutral zone at preferred target comfort zone temperatures in a dim light environment with minimal human intervention as the bird grows to market age, physiological fitness is improved as indicated by the age-related decrease in CS concentration.

CONCLUSIONS AND APPLICATIONS 1. Broilers reared under state-of-the-art commercial production practices did not exhibit a stress condition based on normal plasma concentrations of CS. 2. Broilers exhibited enhanced physiological adaptation to their environment as they neared processing age as evidenced by an age-related decrease in plasma CS.

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FIGURE 1. Simple regression of corticosterone (CS in pg/mL) and age in days; the regression equation for CS = 658.82 to 2.0880; R2 = 0.1058; P ≤ 0.01.

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10. Downing, J. A., and W. L. Bryden. 1999. Stress, Hen Husbandry and Welfare: Literature Review of Stress in Poultry. RIRDC/ EIRDC, Barton, ACT, Australia. 11. Thaxton, J. P. 2004. Stress and the welfare of laying hens. Pages 81–95 in Welfare of the Laying Hen. G. C. Perry, ed., CAB Int., London. 12. Assay Designs, Inc., Ann Arbor, MI. 13. ␮Quant Model Spectrophotometer and K Junior Software, Bio-Tek Instruments, Inc., Vinooski, VT. 14. Statistix Analytical Software, Inc., Tallahassee, FL. 15. Frankel, A. I. 1970. Neurohumoral control of the avian adrenal: A review. Poult. Sci. 49:869–921. 16. Beuving, G., R. B. Jones, and H. J. Blokhuis. 1989. Adrenocortical and heterophil/lymphocyte responses to challenge in hens showing short or long tonic immobility reactions. Br. Poult. Sci. 30:175–184. 17. Murchison, C. 1936. The experimental measurement of a social hierarchy in Gallus domesticus. VI. Preliminary identification of social law. J. Gen. Psychol. 13:227–247.

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5. Puvadolpirod, S., and J. P. Thaxton. 2000. Model of physiological stress in chickens. 3. Temporal patterns of response. Poult. Sci. 79:377–382.

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