Physiological Stress in Laying Hens

Physiological Stress in Laying Hens1 J. Odihambo Mumma,*2 J. P. Thaxton,*3 Y. Vizzier-Thaxton,* and W. L. Dodson† *Department of Poultry Science, and †School of Human Sciences, Mississippi State University, Mississippi State 39762 ABSTRACT Stress responses in laying hens were mediated by continuous infusion of adrenocorticotropin (ACTH) via osmotic pumps. The ACTH was dissolved in saline solution (0.85%), and each pump delivered 8 IU of ACTH per kilogram of BW per day at the rate of 1 ␮L/h for 7 d. Control hens received pumps loaded with saline. Measurements were made at 6 d postpump implantation, unless otherwise indicated. The ACTH-treatment increased BW and total carcass, rear half of carcass, intestinal, and liver weights. Proximate analyses of liver showed increases in dry weight, moisture, protein, fat, carbohydrate, and ash content. Weights of the front half of the carcass, as well as weights of the abdominal fat pad, heart, head, feet, and skin were unaffected by ACTHtreatment. Plasma corticosterone, glucose, cholesterol, and high-density lipoproteins were increased by ACTH,

whereas triglycerides were decreased. Feed and water intake, total excreta, and excretory DM were all increased in ACTH-treated hens. The ACTH decreased carbohydrate in excreta, whereas ash, protein, fiber, and gross energy of excreta were unaffected. The ACTH did not affect digestibility of dry matter, proteins, carbohydrates, fats, or gross energy; however, absorption of ash, protein, carbohydrates, and gross energy were increased by ACTH. Antibody levels to sheep red blood cells, cellmediated immunity (wattle index to phytohemagglutinin-phosphate), and relative spleen weight were reduced by ACTH, whereas heterophil:lymphocyte ratio was increased. Reproduction in hens was negatively affected by ACTH treatment, as measured by cessation of laying on the third day of treatment, atretic follicles, and decreased oviduct weight.

Key words: layer, stress, adrenocorticotropin 2006 Poultry Science 85:761–769

blood metabolites, digestion and metabolism, and immunity. The model of Puvadolpirod and Thaxton (2000a,b,c,d) has not been extended to adult birds, and there is limited information on ACTH-mediated stress responses in laying hens. Most reports have been aimed at understanding the effects of specific environmental factors on a limited number of physiological responses (Thaxton, 2004). A comprehensive evaluation of adaptive responses known or suspected to occur in stressed adult fowl, as well as the temporal relationships of these responses, does not exist. The purpose of the present study was to investigate multiple stress responses of laying hens mediated by continuous infusion of ACTH.

INTRODUCTION Stress occurs when an animal experiences changes in the environment that stimulate body responses aimed at reestablishing the homeostatic condition. Stress and its physiological implications in domestic fowl, especially in juvenile birds, have been reviewed (Freeman, 1971, 1976, 1985; Siegel, 1971, 1980, 1985, 1995; Maxwell, 1993; Jones, 1996; Downing and Bryden, 1999). Puvadolpirod and Thaxton (2000a,b,c,d) and Thaxton and Puvadolpirod (2000) proposed a model to study stress in domestic fowl. This model involves continuous infusion of adrenocorticotropin (ACTH). Post et al. (2003) offered an alternative stress model utilizing dietary corticosterone (CS). In both models, only broilers were studied. Puvadolpirod and Thaxton (2000a,b,c,d) studied 42 different parameters and assigned each of them to 1 of 4 stress response categories: morphology, endocrine and

MATERIALS AND METHODS Animal Husbandry Nine separate trials were conducted during this study. Single Comb White Leghorn hens were used in all trials, and hens in each trial were selected at random from a single flock of hens that were the same age; however, age of hens in different trials varied from 36 to 65 wk of age. In all cases, hens were palpated to select hens in lay at the onset of each trial. Hens were maintained on the

2006 Poultry Science Association, Inc. Received October 10, 2005. Accepted November 17, 2005. 1 Paper number J-10808 of the journal series of the Mississippi Agriculture and Forestry Experiment Station, Mississippi State, MS 39762. 2 Present address: Emory University School of Medicine, Department of Human Genetics, Atlanta, GA. 3 Corresponding author: [email protected]

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Poultry Research Farm of the Mississippi Agriculture and Forestry Experiment Station. A layer ration formulated to meet or exceed all known nutritional requirements of laying hens (NRC, 1994) and water were available on an ad libitum basis. The lighting schedule consisted of 17L:7D with lights on from 0500 to 2200 h.

tein (AOAC 39.1.19), fat (AOAC 39.1.05), carbohydrate (AOAC 39.1.06), and ash (AOAC 39.1.01) contents. All values were recorded as percentage of sample. The relative value of each muscle and liver sample was determined. These calculations were made by multiplying percentage of each liver sample by total CW and each breast and thigh sample by the weight of the FH or RH of the carcass, respectively.

ACTH Treatment All procedures conducted in this study were in accordance with Mississippi State University’s Animal Care Committee. In all trials, 2 treatments were administered using mini-osmotic pumps (Model 2001, Durect Corp., Cupertino, CA) loaded either with ACTH (Sigma Aldrich Fine Chemicals, St. Louis, MO) dissolved in 0.85% saline solution (ACTH treatment) or 0.85% saline (control treatment). The ACTH-treated hens received 8 IU of ACTH per kilogram of BW per day for 7 d, and control hens received a like amount of saline. Delivery rate of the pumps was 1 ␮L/h. Pumps were implanted according to the procedure of Puvadolpirod and Thaxton (2000a). All parameters were measured on d 6 after pump installation. Digestion and metabolism parameters were also measured on d 2 and 4.

Morphology and Reproduction Trials 1 and 2 In trial 1, 8 control and 8 ACTH hens, were weighed and killed. Hearts, spleens, livers, and oviducts were removed and weighed. Livers were sealed in Ziploc bags (Ziploc, S. C. Johnson and Son Inc., Racine, WI) and immediately placed on ice for later proximate analysis (AOAC, 1995). Before removing the oviduct from each hen, the left ovary was inspected. The number of preovulatory follicles exhibiting rapid development (>2 cm) were counted, as well as follicles that were exhibiting atresia (Johnson, 1986). In trial 2, 10 control and 10 ACTH hens were weighed, bled by cardiac puncture, and killed. Body weight, as well as abdominal fat pad, heart, spleen, liver, oviduct, and intestinal tract (intestines plus attached organs, i.e., proventriculus, gizzard, and pancreas) weights were determined. Additionally, the head, feet, and skin plus feathers (residual) were removed and weighed. The whole carcass (CW) was dissected into 2 parts, the front half (FH) and rear half (RH), and weighed. The back was severed laterally between the last thoracic vertebrae and the first lumbar vertebrae, (i.e., immediately proximal to the dorsal aspect of the synsacrum). Samples of breast (pectoralis major) and thigh (biceps femoris) muscle tissue, as well as livers, were dissected, sealed in Ziploc bags, and placed on ice. Samples were then frozen (−20°C) for later proximate analysis (AOAC, 1995). As in trial 1, numbers of ovarian follicles, as well as numbers of atretic follicles, were determined. Separate 5-g samples of livers from trials 1 and 2, along with breast and thigh meat from trial 2, were thawed, ground, and analyzed for moisture (AOAC 39.1.02), pro-

Endocrine and Blood Metabolites: Trials 3 and 4 Sixteen control and a like number of ACTH hens in trial 3 and trial 4 were bled by puncture of the ulnar vein. Sampling time never exceeded 45 s. Beuving et al. (1989) demonstrated that plasma corticosterone (CS) level in laying hens was elevated after only 45 s of restraint. Blood was collected between 1200 and 1400 h to avoid interfering with the egg-laying pattern. Blood samples were collected into Luer monovettes (Sarstedt Inc., Newton, NC) containing lithium heparin as an anticoagulant and immediately placed in an ice bath. Samples were centrifuged at 5,000 × g for 10 min. The resulting plasma samples were decanted, stored frozen at −20°C, and thawed before analysis for levels of CS, glucose (GLU), cholesterol, highdensity lipoprotein (HDL), and triglycerides (TRI). Concentrations of GLU, cholesterol (CHOL), HDL, and TRI were determined using an autoanalyzer (Etkachem Model DT 60 analyzer, Eastman Kodak Co., Rochester, NY). This analyzer used colorimetric detection procedures described by Elliot (1984). Plasma CS concentrations were determined by enzymelinked immunoassay (EIA; Correlate-EIA for CS, Assay Designs, Inc., Ann Arbor, MI). A sheep polyclonal antibody was the primary reactive agent in this assay. The mean intra- and interassay coefficients of variation (n = 32) were 4.5 and 3.1%, respectively. This EIA for chicken CS was compared with an accepted radioimmunoassay. Chicken plasma samples (n = 8) were assayed for CS by both EIA and radioimmunoassay methods, and means ± SEM were 12,940 ± 293 and 14,720 ± 356 ng/mL, respectively. Precision and accuracy of CS measurement did not differ between the 2 methods. To assay CS, 10 ␮L of steroid inhibitor buffer was added to each sample and then vortexed. All samples were diluted 1:40 using Tris buffer solution with sodium azide (25 ␮L of buffer containing 28 ␮mol of Na azide as a preservative). Duplicate 100 ␮L aliquots of each sample were pipetted into wells of a microtiter plate (coated with donkey-anti-sheep IgG). In addition, 50 ␮L of alkaline phosphatase conjugated with CS and sheep polyclonal antibody was added into the wells. After 2 h of incubation on a shaker (500 rpm) at room temperature, plates were washed 3 times with a Tris buffer solution (400 ␮L/well) containing ∼30 ␮mol of detergent and sodium azide. Trisodium phosphate solution was added, and absorbance was read spectrophotometrically (Correlate-EIA for CS, Assay Designs, Inc., Ann Arbor, MI) at 405 nm. Standard curves and sample concentrations were calculated using KC Junior software

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(␮Quant Microtiterplate Spectrophotometer, Bio-Tek Instruments, Inc., Winooski, VT).

Table 1. Effects of continuous infusion of adrenocorticotropin (ACTH) for 6 d on morphological characteristics of laying hens1 Treatment1

Immunology: Trials 5, 6, and 7 In trial 5, the ulnar vein of each of 10 control and 10 ACTH-treated hens was pricked with a needle, and 1 drop of whole blood was used to make a thin smear on a clean microscope slide. These slides were air-dried and stained with Wright’s stain. One hundred leukocytes, including both granular and agranular cells, were counted microscopically (Cook, 1959). The heterophil:lymphocyte (H:L) ratios were determined by dividing the percentage of heterophils by the percentage of lymphocytes. In trial 6, 10 control and 10 ACTH-treated hens received an IV injection of 1 mL of a 10% suspension of sheep red blood cells (SRBC) in 0.9% saline immediately after pumps were implanted. On d 6 after SRBC challenge, each hen was bled by venipuncture, and 2 mL of whole blood was collected into a clean tube that did not contain anticoagulant. Clotted blood samples were incubated (∼37 C) for 2 h, then serum was decanted into clean tubes and frozen at −20°C for later analysis of antiSRBC antibody levels by microhemagglutination procedure (Witlin, 1967). The highest dilution that exhibited hemaggglutination was recorded as the titer of the serum sample. The titer of each sample was converted to the appropriate log2 value (Thaxton and Siegel, 1972). In trial 7, each of 10 control and 10 ACTH-treated hens was given a single intradermal injection of 100 ␮L of 0.85% saline containing 100 ␮g of phytohemagglutinphosphate (PHA-P) into the subcutaneous layer of lower wall of the right wattle. An equivalent volume of avian saline was injected into the lower wall of the left wattle. Each hen served as its own control. These injections were made immediately after pumps were implanted. Left and right wattle thicknesses were determined 24 h after the intradermal injections using a metric constant tensionmicrometer. Wattle thickness indices were calculated by dividing the thickness of the right wattle by the thickness of the left wattle.

Digestion and Metabolism: Trials 8 and 9 Trials 8 and 9 were replicates using sister hens. Trial 8 was conducted when hens were 62 wk of age, and trial 9 was conducted using sister hens that were 65 wk of age. Hens (i.e., 30 control and 30 ACTH-treated hens in trial 8 and 16 control and 16 ACTH-treated hens in trial 9) were transferred from cages (35 length × 25 width × 30 height cm) in the layer house to battery cages (60 length × 60 width × 30 height cm) 4 wk before each trial was conducted. Feed and water intakes, as well as total excreta output, were calculated on a daily basis. Mean values of each parameter in both control and ACTH-treated hens are reported. Proximate analyses by AOAC (1995) methodology were used to determine gross energy, protein, ash, and lipid content of both feed and excreta. Carbohy-

Parameter2 (g) Body weight (1 ± 2) Carcass weight Front half weight Rear half weight Head, feet, skin weight Liver weight (1 ± 2) Liver analysis: Dry weight Moisture Protein Fat Carbohydrate Ash Abd. fat pad weight Intestinal tract weight Heart weight (1 ± 2)

Control

ACTH

1,673 825 431 394 359 47

± ± ± ± ± ±

27 9 8 6 6 2

1,807 890 432 432 348 69

± ± ± ± ± ±

36* 9* 5* 5* 5 4

13.2 29.58 7.03 5.41 1.39 0.51 73 128 7.4

± ± ± ± ± ± ± ± ±

0.84 1.46 0.25 0.52 0.31 0.02 5 3 0.4

20.11 43.96 9.54 9.55 3.92 0.71 77 164 8.2

± ± ± ± ± ± ± ± ±

0.86* 1.48* 0.22* 0.51* 0.33* 0.02* 6 5* 0.4

1 Means ± SEM of control and ACTH-trated hens are presented. Control hens received 1 ␮L of 0.85% saline/kg of BW/d for 7 d, and ACTH hens received 8 IU of ACTH/kg of BW/d for 7 d. 2 Parameters followed by (1 ± 2) represent combined results of trials 1 ± 2, whereas parameters lacking this designation are results of only trial 2. *Each ACTH mean with this symbol differs (P ≤ 0.05) from the corresponding control mean.

drate content was calculated by subtracting protein, fat, and ash contents from DM content. Nutrient digestibility (%) was calculated by dividing nutrient intake per day by nutrient excreted per day, multiplying the quotient by 100, and dividing the outcome by nutrient intake per day. Nutrient absorption (grams or kilocalories) was calculated by subtracting weight of nutrient excreted per day (grams or kilocalories) from weight of nutrient per day (grams or kilocalories). All parameters of digestion and metabolism are calculated and reported at 2, 4, and 6 d postpump installation.

Statistics All data were subjected to ANOVA using the GLM procedure of Stat-8 (Statistix, 2004). Means were compared using the least significant difference. Statements of significance are based on P ≤ 0.05.

RESULTS Morphology Effects of continuous ACTH infusion on morphological parameters of laying hens are presented in Table 1. The ACTH treatment caused increases in BW, CW, and RH weights, as well as liver and intestinal weights, when compared with appropriate control hen responses. However, FH weight, residue of feet, head and skin weight, heart weight, and abdominal fat pad weight were not affected by ACTH treatment. Proximate analysis of livers showed that dry weight, moisture, protein, fat, carbohydrate, and ash contents were all increased by ACTH.

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Table 2. Effects of continuous infusion of adrenocorticotropin (ACTH) for 6 d on breast and thigh muscle in laying hens Treatment1 Parameter (g)

Muscle

Control

Moisture

Breast Thigh Breast Thigh Muscle Thigh Breast Thigh Breast Thigh

317 276 108 83 3 34 1 1 5 4

Protein Fat Carbohydrate Ash

± ± ± ± ± ± ± ± ± ±

6 6 2 2 0.3 4 0.1 0.1 0.1 0.6

ACTH 338 301 115 115 6 45 0 4 5 4

± ± ± ± ± ± ± ± ± ±

8 5* 3 3 0.2* 3 0 1 0.1 0.4

1 Means ± SEM of control and ACTH-treated hens are presented. Control hens received 1 ␮L of 0.85% saline/kg of BW/d for 7 d, and ACTH hens received 8 IU of ACTH/kg of BW/d for 7 d. *Each ACTH mean with this symbol differs (P ≤ 0.05) from the corresponding control mean.

Results of proximate analyses of breast and thigh meat samples collected from the FH and RH of carcasses, respectively, from hens of trial 2 are presented in Table 2. The ACTH treatment had 2 effects: increased moisture in thigh samples and increased fat in breast samples. All other meat parameters in both breast and thigh samples were unaffected by ACTH treatment.

Endocrine and Blood Metabolites Effects of ACTH on endocrine and metabolic parameters are presented in Table 3. The ACTH treatment caused elevations in blood levels of CS, GLU, CHOL, and HDL, whereas TRI levels were reduced by ACTH treatment. Effects of the ACTH treatment on immune parameters are presented in Table 4. AntiSRBC antibody levels, wattle index in response to PHA-P, and relative spleen weight were all decreased by ACTH treatment. Additionally, the H:L ratio was increased in ACTH-treated hens, as compared with controls.

Table 3. Effects of continuous infusion of adrenocorticotropin (ACTH) for 6 d on plasma corticosterone (CS), glucose (GLU), cholesterol (CHOL), high density lipoprotein (HDL), and triglycerides (TRI) in laying hens of trials 1 + 2 Treatment1 Parameter CS (ng/mL) GLU (mg/dL) CHOL (mg/dL) HDL (mg/dL) TRI (mg/dL)

Control 4,470 202 121 3 1,842

± ± ± ± ±

420 3 2 1 139

ACTH 10,280 554 202 38 1,565

± ± ± ± ±

460* 28* 3* 9* 142*

1 Means of control and ACTH-treated hens are presented. Control hens received 1 ␮L of 0.85 saline/kg of BW/d for 7 d, and ACTH hens received 8 IU of ACTH/kg of BW/d for 7 d. *Each ACTH mean with this symbol differs (P ≤ 0.05) from the corresponding control mean.

Digestion and Metabolism Effects of ACTH on digestive and metabolic parameters are summarized in Table 5. ACTH caused increased intake of both feed and water and total feces and excretory DM at all times of measurement. Proximate analysis of fecal matter showed that carbohydrate content on a percentage basis in feces from ACTH-treated hens was reduced on d 4, as compared with control hens. However, percentages of carbohydrate, as well as percentages of ash, protein, fat, fiber, and gross energy (kilocalories per gram) were not affected by ACTH treatment at any of the measurement times. Digestibility and absorption profiles were determined on fecal samples. Digestibility of none of the nutrients was affected by ACTH treatment at any of the times of measurement. However, absorption was affected by ACTH treatment. Specifically, ACTH treatment increased absorption of ash, protein, and gross energy on d 2 and 6; increased absorption of carbohydrate on d 2, 4, and 6; and increased absorption of fat on d 6.

Reproduction In Table 6, results of ACTH on reproductive parameters of hens are summarized. The number of maturing follicles present on the ovary was not affected by ACTH; however, all of the follicles of ACTH-treated hens appeared to be experiencing atresia. Additionally, weight of the oviduct was reduced in ACTH-treated hens, as compared with control hens. All ACTH hens ceased laying by d 3 after implantation of pumps.

DISCUSSION Results of this study indicate that hens exhibited increases in BW when subjected to continuous infusion of ACTH. This effect contrasts results reported in broilers. For example, continuous infusion of ACTH and addition of CS to the drinking water caused cessation of growth in 5-wk-old broilers, and this effect persisted until the end of the grow-out period (Puvadolpirod and Thaxton, 2000a; Post et al., 2003). Cessation of growth in broilers in these reports was explained as a result of adrenocortical-mediated gluconeogenesis; specifically, protein reserves were catabolized for energy purposes. In another study, Tankson et al. (2001) demonstrated that ACTH infusion decreased BW and CW in broilers. Chemical analyses showed that moisture, protein, and caloric content of meat samples from ACTH-treated broilers were decreased. These results support the contention that elevated CS increases catabolism of proteins. Klasing and Jarrell (1985) injected 16-d-old Leghorn chicks with cortisol, CS, or dexamethasone. These glucocorticoids resulted in cessation of growth due to inhibition of protein accretion. The rate of protein catabolism was not altered after any of the hormonal treatments. Results in the present study using adult laying hens agree with those of Klasing and Jarrell (1985) in 18-d-old pullet chicks

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STRESS IN LAYERS Table 4. Effects of continuous infusion of adrenocorticotropin (ACTH) for 7 d on immune parameters of laying hens Treatment1 Parameter2

Control

AntiSRBC antibody level (log2) Wattle index to PHA-P Relative spleen weight (mg/100 g) Heterophil:lymphocyte

6.37 3.14 9.24 0.34

± ± ± ±

ACTH

0.21 0.13 1.23 0.02

4.46 1.65 5.46 1.35

± ± ± ±

0.35* 0.12* 1.21* 0.12*

1 Means of control and ACTH-treated hens are presented. Control hens received 1 ␮L of 0.85% saline/kg of BW/d for 7 d and ACTH hens received 8 IU of ACTH/kg of BW/d for 7 d. 2 Sheep red blood cell (SRBC) challenge was 1 mL of 10% suspension of SRBC in saline immediately after pump placement and serum collection for anti-SRBC antibodies was made at 7 d postpump installation. Phytohemagglutin-phosphate (PHA-P) challenge (100 ␮g in 100 ␮L of saline) was made immediately postpump placement, and wattle thicknesses were made 24 h later. *Each ACTH mean with this symbol differs (P ≤ 0.05) from the corresponding control mean.

that elevated CS levels did not affect protein catabolism in muscle and liver samples. Broilers are noted for their ability to assimilate skeletal muscle mass, especially that associated with the breast, at a rate far greater than that of Leghorns. Dupont et al. (1999) reported that CS altered insulin signaling in chicken muscle and liver. Additionally, Tesseraud et al. (2003) showed in broilers that insulin-like growth factor (IGF)-I, but not IGF-II, controls lean tissue growth. The role of insulin and IGF-I and IGF-II in stress in chickens has not been completely elucidated; however, cessation of growth in broilers is probably caused by downregula-

tion of receptors for IGF-I (Dupont et al., 2004). The finding in the present study of lack of effect of ACTH treatment on protein content in skeletal muscles is expected because IGF-I is known to stimulate accretion of lean tissue mass in growing animals, whereas it is largely ineffective in adults that have completed growth (LeRoith et al., 2003; Dupont et al., 2004). Present results indicate that BW, as well as RH, liver, and intestinal weights, were increased in laying hens by ACTH treatment. The findings of increased liver weight, as well as increases in liver moisture, protein, fat, carbohydrate, and ash levels, are indicative of stress. During glu-

Table 5. Effects of continuous infusion of adrenocorticotropin (ACTH) for 6 d on digestion and metabolism in laying hens Treatment1 Day 2 Parameter Feed itnake (g/d) Water intake (mL/d) Total excreta (g/d) Excreta dry matter (g/d) Analysis of excreta Ash (%) Protein (%) Fat (%) Carbohydrate (%) Fiber (%) Gross energy (kcal/g) Nutrient digestibility2 Dry matter (%) Protein (%) Carbohydrate (%) Fat (%) Gross energy (%) Nutrient absorption3 Ash (g/d) Protein (g/d) Carbohydrate (g/d) Fat (g/d) Gross energy (kcal/d)

Control

Day 4 ACTH

Control

Day 6 ACTH

Control

ACTH

104 167 124 24

± ± ± ±

5 9 5 2

128 308 175 30

± ± ± ±

5* 27* 14* 2*

104 159 122 26

± ± ± ±

6 7 4 2

128 298 194 34

± ± ± ±

6* 34* 15* 2*

112 209 101 25

± ± ± ±

6 16 5 1

152 391 161 32

± ± ± ±

7* 40* 8* 3*

26 36 7 31 65 3.3

± ± ± ± ± ±

1 2 0.2 2 0.6 0.1

28 33 7 32 63 3.2

± ± ± ± ± ±

1 1 0.6 2 0.6 0.1

28 34 6 32 63 3.2

± ± ± ± ± ±

1 0.5 0.2 1 0.4 0.1

31 37 5 26 62 3.0

± ± ± ± ± ±

1 2 0.3 2* 0.5 0.1

31 34 4 30 63 2.8

± ± ± ± ± ±

2 2 0.1 1 1 0.1

33 35 5 26 62 2.9

± ± ± ± ± ±

1 2 0.4 2 1 0.1

79 48 88 50 76

± ± ± ± ±

1 4 1 2 1

80 53 88 50 76

± ± ± ± ±

1 2 1 3 1

75 40 86 49 72

± ± ± ± ±

2 4 1 5 2

76 36 89 51 75

± ± ± ± ±

1 4 1 4 1

83 59 91 72 83

± ± ± ± ±

1 3 1 2 1

84 59 92 70 83

± ± ± ± ±

1 3 1 2 1

11 8 57 1.6 252

± ± ± ± ±

0.8 0.6 3 0.1 12

14 11 75 2.1 334

± ± ± ± ±

0.4* 0.5* 3* 0.2 13*

9 6 52 1.5 225

± ± ± ± ±

0.9 1 3 0.2 16

11 7 69 2.0 295

± ± ± ± ±

1 1 2* 0.2 11*

14 12 74 2.9 347

± ± ± ± ±

1 1 4 0.2 19

19 16 100 3.8 461

± ± ± ± ±

0.1* 1* 4* 0.2* 15*

1 Means ± SEM of control and ACTH-treated hens are presented. Control hens received 1 ␮L of 0.85% saline/ kg of BW/d for 7 d, and ACTH hens received 8 IU of ACTH/kg of BW/d for 7 d. 2 Digestibility was calculated by dividing nutrient intake per day by nutrient excreted per day. 3 Absorption was calculated by substracting weight of nutrient excreted per day (grams or kilocalories) from weight of nutrient intake per day (grams or kilocalories). *Each ACTH mean with this symbol differs (P ≤ 0.05) from the corresponding control mean.

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Table 6. Effects of continuous infusion of adrenocorticotropin (ACTH) for 6 d on reproductive parameters of hens of trials 1 and 2 Treatment1 Parameter Preovulatory follicles (no.) Atretic follicles (%) Oviduct weight (g)

Control

ACTH

6.25 ± 1.21 0 164 ± 5

5.5 ± 0.14 100 128 ± 3*

1 Means of control and ACTH-treated hens are presented. Control hens received 1 ␮L of 0.85% saline/kg of BW/d for 7 d, and ACTH hens received 8 IU of ACTH/kg of BW/d for 7 d. *Each ACTH mean possessing this symbol differs (P ≤ 0.05) from the corresponding control mean.

coneogenesis, lipids accumulate in liver because amino acids are being preferentially catabolized. The liver is essential to gluconeogenesis via the Cori cycle (Cori and Cori, 1931). Therefore, hypertrophy of liver, along with increased levels of water, protein, fat, carbohydrate, and ash, are expected (Groen et al., 1986). The average increase in BW caused by ACTH treatment was 134 g. The increases in CW and the intestinal tract account for 63 and 33 g, respectively, of the increase in BW. Analyses of muscle samples from both breast and thigh muscle samples of each hen showed that ACTH did not cause a decrease in protein content. However, there was an ACTH-mediated increase in water content in thigh muscle but not in breast muscle. The mechanism of the ACTH-mediated increase in water in thigh muscle is not known, but changes in cations, hydrophobisity, and pH all are known to affect water-holding capacity of meat (Goldberg, 1969; Hedrick et al., 1994; Raymer et al., 1997). Additionally, ACTH-treated hens drank on average 145 mL more water than controls, and fecal water content was increased by 54 mL/d in ACTH-treated hens vs. controls. Increased body water content probably accounted for most of the increase in BW in ACTHtreated hens. Endocrine and biochemical changes are definitive responses in most species, including adult and juvenile fowl. Clearly, CHOL, GLU, HDL, and TRI levels are indicators of stress in all fowl. Corticosterone, however, is not accepted by all as a stress response in laying hens. Mashaly et al. (1984) showed that stocking density elevated plasma CS in hens. Craig et al. (1986) reported that increased stocking density increased total plasma corticosteroids in some experiments but was without effect in others. Conversely, Davis et al. (2000) found that increasing stocking density did not affect CS levels in hens. Downing and Bryden (1999) and Fitko et al. (1993) showed that handling hens elevated plasma CS. In the report of Downing and Bryden (1999), CS levels were elevated within 15 min after handling the hens; however, these levels returned to baseline within 12 h. Chen et al. (2002) showed that intermingling visitor hens with residents resulted in elevated CS levels in visitors. Davis et al. (1994) showed that beak trimming resulted in elevated CS levels in hens; however, Compton et al. (1980) found that declawing hens did not affect blood CS levels.

In the present study, the magnitude of stress responses in laying hens, as indicated by ACTH-mediated elevations in plasma levels of CS, GLU, and CHOL, mimicked elevations recorded in broilers (Puvadolpirod and Thaxton, 2000a,b,c,d). Increased circulating glucocorticoids induce gluconeogenesis, a process that leads to elevation of plasma GLU (Nagra and Meyer, 1963). In addition, glucocorticoids can inhibit GLU uptake into peripheral tissues by antagonizing the effect of insulin (Buren et al., 2002). Glucocorticoids released during stress initially mobilize lipids in laying hens, especially TRI (Sahin and Kucuk, 2001), from adipose tissue. Glucocorticoids also mobilize structural proteins (Nagra and Meyer, 1963). Immediately after lipids are mobilized, they are broken down into nonesterified fatty acids and moved to the liver to be converted into TRI by phosphatidyl phosphodihydrolase (Brindley, 1981). Very-low density lipoprotein (VLDL) transports TRI from liver to tissues such as muscle and adipose, where TRI is hydrolyzed by lipoprotein lipase (Assmann and Jerzy-Roch, 2003). Hydrolysis and removal of TRI from VLDL by the action of lipoprotein lipase converts VLDL to CHOL ester-rich low-density lipoprotein, whereas some of the VLDL surface lipids are transformed into HDL (Walzem, 1996). During stress, plasma CHOL levels are increased, as are HDL levels, because the function of HDL is to transport CHOL to liver from body tissues. Plasma TRI levels were decreased in ACTH-treated hens in this study and in heat-stressed hens (Sahin and Kucuk, 2001). These results are in contrast to those observed in broilers (Latour et al., 1996; Puvadolpirod and Thaxton, 2000a-d), in which ACTH caused elevation in TRI level. Continuous infusion of ACTH affected immune parameters of the laying hen in a similar manner as has been reported in broilers (Puvadolpirod and Thaxton, 2000a,b,c,d). Specifically, the primary humoral immune response to SRBC in hens was suppressed to the same degree as that caused by a single IM injection of ACTH in broilers (Thaxton and Siegel, 1973). Cell-mediated immunity, as indicated by cutaneous basophil hypersensitivity to PHA-P (measured by determining wattle index), was suppressed to approximately the same degree as reported by single depot injections of ACTH in broilers (Murray et al., 1987). Relative spleen weight was decreased, and H:L was increased by continuous infusion of ACTH to similar degrees in both hens in the present study and that of Mashaly et al. (1993) and in broilers (Puvadolpirod and Thaxton, 2000a,b,c). Plasma CS levels of laying hens increase when exposed to different stressors including tonic immobility (Beuving et al., 1989; Jones et al., 2005), increased stocking density in cages (Edens et al., 1982), and feed restriction (Beuving and Vonder, 1978). Lymphocytes have high affinity receptors for CS in their cytoplasm, and these receptors increase in number in response to immune stimulation (Freier and Fuchs, 1994). Free steroids pass through the cell membranes and bind to these receptors. The steroid-receptor complex then enters the nucleus and binds to specific

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DNA sequences that induce synthesis of mRMA, which in turn triggers synthesis of protein that inhibits intracellular glucose transport and lipid synthesis (Lewis and Jacobs, 2002). As a result, glucose uptake and protein synthesis of the cells are suppressed (Rushakoff and Kalkhoff, 1983; Lillehoj et al., 1992). Injections of ACTH will increase the amount of endogenously produced CS that is bound to cytoplasm and nucleus of lymphatic tissue cells of chickens (Gould and Siegel, 1980). Increased CS concentrations have also been shown to cause a decline in monocyte accumulation at inflammatory sites and reduce responses to macrophage inhibition factor (Webster et al., 2002). This CS-mediated reduction in macrophage numbers is also attributed to inhibition of enzymes necessary for phagocytosis, as well as inhibition of secretion of interleukin-2 (Eskay et al., 1990), the cytokine needed for proliferation and maturation of lymphocytes in chickens (Lillehoj et al., 1992). The effects of ACTH-mediated stress on digestion and metabolism in mature hens are different from that of broilers. The ACTH caused a remarkable increase in nutrient absorption in laying hens. Gross energy and carbohydrate absorption were increased throughout the stress period, whereas protein and ash absorption were increased on d 2 and 6, as was fat absorption on d 6. Digestion of nutrients, however, was not affected by ACTH treatment in laying hens. In broilers, ACTH caused a general decrease in digestion, whereas all nutrients were absorbed at rates comparable with control birds, with the exception of protein, which was decreased (Puvadolpirod and Thaxton, 2000d). A unifying physiological explanation for the discrepancies in digestion and metabolism of mature hens and juvenile broilers is not known. In fact, we are not aware of previous reports that describe these differences. It is known that under nonstressful circumstances, juvenile birds expend much of their ME on growth (Kuenzel and Kuenzel, 1977), whereas adult hens expend a considerable amount of ME on reproduction, specifically, daily egg production (Polin and Wolford, 1977). However, when broilers were acutely stressed by ACTH infusion, growth immediately ceased (Puvadolpirod and Thaxton, 2000d), as did egg production in the hens of the present study. Therefore, mounting the necessary adaptive responses seemed to be paramount to both adult hens and juvenile broilers. It is well documented that during stress, the need for energy increases markedly as the animal tries to adapt to the stressor (Siegel and Van Kampen, 1984). The cessation of growth observed in broiler birds was the direct result of redirecting energy normally used for accretion of body mass to adaptive responses (Nagra and Meyer, 1963; Siegel and Van Kampen, 1984). Because hens increased BW during the stress period and because egg production ceased by d 3, it is possible that adults share broilers’ proclivity for redirecting energy to adaptive responses. Reproduction in laying hens appeared to be severely negatively affected by ACTH infusion. The limited repro-

ductive results collected in this study, as well as a large body of evidence that various environmental factors limit egg production and quality of eggs (Thaxton, 2004), support a conclusion that physiological stress can hamper reproduction. It is obvious that in-depth studies are needed to understand the underlying mechanism(s) responsible for a normal number of ovarian follicles concomitant with extensive atresia. The present results suggest that follicular integrity and ovulation were compromised by ACTH treatment. It is not known if these ACTHmediated effects on egg laying were temporary or permanent. Equally, it is not known if broilers would have exhibited impairment in egg production if reared to adulthood. Studies aimed at evaluating reproduction in ACTH-treated layers and broiler-breeders promise to yield interesting results. In conclusion, adaptive responses in laying hens, when mediated by continuous infusion of ACTH, can be categorized into 5 response categories: morphology, endocrine secretions and blood metabolites, digestion and metabolism, immunity, and reproduction. The major differences between stress responses in layers and broilers occurred in morphology, digestion and metabolism, and reproduction. Specifically, BW in stressed layers increased, whereas growth was terminated in broilers. Reduced digestion of nutrients seemed to be the major consequence of stress in broilers, whereas reduction in nutrient absorption occurred in layers. Egg laying ceased in layers, and this was apparently due to death of follicles. Responses in levels of CS, blood metabolites, and all immune parameters were very similar in layers and broilers. The only notable difference was that ACTH-treated layers exhibited a reduction in TRI levels, whereas their broiler counterparts exhibited increased TRI levels.

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