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Effects of density and perch availability on the immune status of broilers
IMMUNOLOGY AND MOLECULAR BIOLOGY Effects of Density and Perch Availability on the Immune Status of Broilers R. A. Heckert,*,1 I. Estevez,† E. Russek-Cohen,† and R. Pettit-Riley† *Department of Veterinary Medicine and †Department of Animal and Avian Sciences, University of Maryland at College Park, College Park, Maryland 20742
(Key words: housing density, immune status, heat stress, perch) 2002 Poultry Science 81:451–457
nize that stress alters the immune system, the ability to assess the immune status of commercial broilers is difficult and no single assay is available to evaluate immunocompetence. Measures of immunity that have been commonly used and assessed in poultry are lymphoid organ weights, (Pope, 1991) antibody response to foreign antigens, (Gross and Siegel, 1980,1990; Montgomery et al., 1991; Patterson and Siegel, 1998; Scott et al., 1994) heterophil:lymphocyte (H:L) ratios (Gross and Siegel, 1983; Cravener et al., 1992; Patterson and Siegel, 1998; Al-Murrani et al., 1997) and lymphocyte blastogenesis assays (Nagano and Lee, 1978; Barta;et al., 1992; Cunnick et al., 1994; Talebi et al., 1995; Gogal et al., 1997). Lymphoid organ weights are easily measured and reflect the body’s ability to provide lymphoid cells during an immune response. Humoral immunity, or the ability to produce an antibody response, is another commonly employed method of assessing immunocompetence. The most commonly used antigens in this assessment have been SRBC (T-dependent) and Brucella abortus (T-independent). In Leghorns, housing density has been shown to have no effect on antibody responses to either of these two antigens (Montgomery et al., 1991; Patterson and Siegel, 1998). To our knowledge, the assessment of lymphoid organ weights or the immune response to antigens has not been examined in commer-
INTRODUCTION In modern poultry rearing operations, stress can occur due to a variety of factors (e.g., elevated rearing densities or high summer temperatures). These unfavorable or stressful environmental conditions can negatively affect the animal’s immune system, compromising their performance and ability to overcome viral and bacterial infections. It is well documented that the nervous and immune systems are integrated in their response to stress (Besedovsky et al., 1983; Trout and Mashaly, 1994). Much of the research into neuroendocrine-immune interactions has focused on the relationship between the hypothalamo-pituitary-adrenal axis and the immune system. In animals treated with adrenocorticotropic hormone or subjected to chronic stress the weights of both primary (Davison et al., 1988; Donker and Beuving, 1989) and secondary (Donker and Beuving, 1989) lymphoid organs decreased. Profiles of circulating leukocytes were also affected when treated with adrenocorticotropic hormones or exposed to stress; lymphocyte and monocyte numbers decrease and heterophil numbers increase (Glick, 1958; Onsrud and Thorsby, 1981; Gross and Siegel, 1983; Stevenson and Taylor, 1988). Although we recog-
2002 Poultry Science Association, Inc. Received for publication November 6, 2000. Accepted for publication November 30,2001. 1 1To whom correspondence should be umail.umd.edu.
addressed:
Abbreviation Key: H:L = heterophil:lymphocyte ratio; SI = stimulation index.
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of perches to the pens also significantly decreased the bursa weights and bursa/body weight index (P < 0.01). No other significant effects were observed for the flock performance, morphometric data, or immunological tests between treatments. We concluded that under the conditions of this study, which tried to simulate commercial conditions, the bursal weight was the best indicator of stress that was related to housing density. Addition of perches appeared to increase this level of stress because the birds used the perches infrequently, and therefore there might have been a further reduction in the availability of floor space to the birds.
ABSTRACT This study attempted to evaluate the effect of various housing densities and perch availability on the immune status of commercial broilers. Birds were raised from hatch to 42 d of age with 10, 15, and 20 birds/m2 in pens, with and without the availability of horizontal perches. The immune parameters that were assessed were lymphoid organ weights, antibody response to SRBC in the last 10 d of growth, heterophil:lymphocyte ratios at 32 and 42 d of age, and lymphocyte blastogenesis of peripheral blood lymphocytes collected at 32 and 42 d of age. As density increased, bursa weight and bursa/body weight ratios decreased significantly (P < 0.05). Addition
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commercial conditions are the most crowded and show the greatest degree of health related problems.
MATERIALS AND METHODS Birds and General Management Practices This study was designed as a sub-study within a large trial aimed at investigating the effects of different perch designs and housing density on broiler behavior and performance (Pettit-Riley and Estevez, 2001). Two thousand four hundred twenty-four, 1-d-old sexed broiler chickens (Avian × Avian) were obtained from a commercial hatchery and reared in a broiler house at the Applied Poultry Research Facility, University of Maryland. The broiler house was divided into 36 identical 4.46 m2 pens (18 pens used in the substudy), and the floor was covered with 5 cm of wood shavings litter. The birds, raised under commercial management practices, were fed a commercial two-phase broiler diet and provided water from nipple drinkers ad libitum throughout the rearing period. The diet consisted of a starter crumble with amprolium for the first 21 d (21.85% protein; 3,102 kcal/kg energy), followed by a grower/finisher pellet with amprolium from Day 22 to the end of the growing period (19.23% protein; 3,190 kcal/kg energy). A withdrawal diet was not required with this feeding system. The amount of feeding and drinking space available per bird was kept constant across density by blocking a proportion of the feeding area and drinking nipples within the pen. Each pen contained one feeding bin and seven drinking nipples, of which all seven were available in the high density pens, five were available in the moderate density pens, and three were available in the low density pens. A combination of natural and artificial lighting was used. Artificial light was provided continuously for the first 3 d and from 0600 to 1800 h for the remainder of the growing period. Temperatures were maintained between 32 and 34 C at the beginning of the growing period, and gradually decreased every 2 to 3 d to reach 22 C ± 1 at the end of the growing period. The temperature was controlled using electric brooders and by opening and closing the automatic temperature regulated wall curtains in the house. Positive pressure ventilation was used to provide 10 total air changes for each room per hour during the brooding period and 12 total air changes during the growout period. Although every effort was made to maintain a temperature of 22 C during the study, unusually high temperatures (reaching up to 33 C with high relative humidity values some days) occurred at the end of the growing period. Therefore, as ambient temperatures occasionally exceeded the optimum range toward the end of the rearing period the temperature inside the house could not be maintained low enough to remain within the avian thermoneutral zone. This heat stress situation was unexpected and unplanned in this study.
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cial broiler strains for ability to identify potential stress problems due to increase housing densities, or for the potential effects of perch availability. Although the H:L ratio has been commonly used as a measure of stress in poultry, there have been mixed reports on its usefulness as an indicator of stress due to housing density. One study conducted in seven-week-old broilers showed an increased H:L ratio with increasing density (Cravener et al., 1992), although in caged leghorns, increased cage density had no effect on the H:L ratio (Patterson and Siegel, 1998). On the other hand, little work has been done using the lymphocyte blastogenesis assay in assessing stress in broilers under field conditions. This assay has been used mainly on lymphocytes from inbred populations of birds under laboratory conditions (Nagano and Lee, 1978; Barta et al., 1992; Cunnick et al., 1994; Talebi et al., 1995; Gogal et al., 1997). Numerous studies have been conducted to characterize the effects of density on broiler performance (Hansen and Becker, 1960; Deaton et al., 1968; Bolton et al., 1972; Proudfoot et al., 1979; Shanawany, 1988; Cravener et al., 1992; Estevez et al., 1997). In all studies, increasing density led to a reduction in performance. Although birds at higher densities appeared to be stressed (Proudfoot et al., 1979; Cravener et al., 1992) no comprehensive evaluation of the immune status of the birds was done. Although it is well recognized that stress can negatively impact the immune system, there have been very few controlled studies under field conditions to determine the impact of rearing density on the immune status of birds with industry standard genetics. High rearing densities in broilers are associated with an increased incidence of leg problems (Sørensen et al., 2000; Sanotra et al., 2001), which may be related to the reduced level of activity observed with increasing housing densities (Estevez, et al., 1997). It has been suggested that providing broilers access to perches, under high housing density conditions, may be an effective means of increasing bird activity, therefore potentially reducing the incidence of leg problems, while at the same time increasing air flow among the birds (Hughes and Elson, 1977; LeVan Fiscus et al., 2000; Pettit-Riley and Estevez, 2001). Therefore, perch availability, as a form of environmental enrichment for poultry, has the potential to reduce some of the stressors related to high housing densities (Newberry, 1995). We hypothesized that increasing housing density would result in increased levels of stress and consequently decreased immunocompetence in the birds, that could be in part mitigated by availability of perches (by increasing space availability in the third dimension with better air flow). Therefore, the goals of this study were to determine the immunological status of commercial broilers under increasing housing densities (below, at and above current industry standards) and to determine if any immunological indicators of stress could be mitigated by adding environmental enrichment in the form of horizontal perches. The final 2 wk of the growing period were targeted for study because this is when birds under
DENSITY, PERCHES, AND THE IMMUNE STATUS
Experimental Design
Immunological Assays Several parameters were evaluated to assess the immunological status of the 90 birds at 32 and 42 d of age: 1) humoral immune response to SRBC, 2) lymphocyte blastogenesis assay, 3) H:L ratios, and 4) lymphoid organ weights and organ to body weight ratios. The same birds were caught and tested at 32 and 42 d of age unless they had died during the interval. To assess the ability of the birds under different density and perch availability to mount an immune response
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Gibco-BRL P.O. Box 9418, Gaithersburg, MD 20898. Sigma Chemical Company, 3050 Spruce St., St. Louis, MO 63103. 4 ICN Biomedicals Inc., 3300 Hyland Ave., Costa Mesa, CA 92626. 5 Harvester 96, Tomtec, Inc., 607 Harbor View Rd., CT 06477. 6 Wallac 1450, MicroBeta Trilux, 549 Albany St., Boston, MA 02118. 3
against a foreign antigen, the birds were exposed to SRBC, as described (Gross and Siegel, 1980). Briefly, the chickens were inoculated IV at 32 d of age with 0.1 mL of a 0.25% suspension of SRBC. Plasma was collected from the same birds (where available) by brachial venipuncture before SRBC inoculation at 32 d of age and again 10 d later. The plasma samples were assessed for hemagglutination titers (the last dilution to give a positive result) by a standard microtiter method as described by Gross and Siegel (1980). To assess the cellular immune responses of the birds a mitogen lymphocyte blastogenesis assay was performed essentially as described by Talebi et al.(1995) with some modifications. Briefly, 2 mL of whole blood was collected into heparinized vacutainer tubes at 32 and 42 d of age by brachial venipuncture. The blood was diluted 1/50 in serum-free RPMI 1640 media2 and 200 µL/well cultured in triplicate, with and without mitogen in 96-well flatbottom tissue culture microplates. Concanavalin A3 was added to half of the wells, at 20 µl/well of a 20 µg/mL stock solution, to attain a final concentration of 0.4 µg of mitogen/well. After 90 h of incubation in a humidified chamber at 40 C the cultures were pulsed with 0.2 uCi of [3H]-thymidine4/well. After a further 18 h of incubation the cultures were harvested onto filter mats using a cell harvester.5 The filter mats were then saturated with scintillation fluid in a plastic bag and the disintegrations per minute (DPM) measured in a scintillation analyzer.6 The stimulation index (SI) for each sample was calculated according to the following formula: SI =
mean DPM of stimulated cultures mean DPM of unstimulated cultures
The H:L ratio was also used to determine the degree of a chicken’s stress. The H:L ratios were determined using the stained-slide method, as described by Gross and Siegel (1983). Immunosuppressed or stressed birds classically have smaller lymphoid organs (Pope, 1991). Therefore, at the termination of the study (42 d of age) the bursa, spleen and body weights were determined and the spleen/body and bursa/body weight ratios calculated.
Statistical Analysis A mixed model analysis of variance was used to determine the effects of density (10, 15, 20 birds/m2), perch availability (control versus horizontal), and gender. Although we originally analyzed the data as a randomized block study, block effects were often estimated as zero and in no case were close to significant. Pens were treated as a random effect nested within density by perch treatment combinations. Gender was considered as a subplot factor as gender was not always even within a pen. The mixed model procedure in SAS software (version 6.12) (Littell et al., 1996) was used to analyze the data and calculate least-squares means. Density was analyzed using linear and quadratic orthogonal polynomial contrasts.
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A total of five experimental birds per pen were randomly selected at 1 d of age from each of 18 pens belonging to one of the following treatments: control (no perches) or horizontal (three 0° perches), in a nested design with one of three bird densities: 10 birds/m2, 15 birds/m2, (standard commercial density) and 20 birds/ m2. Birds per pen were 45, 67, and 90, respectively. Each treatment/density combination had a 1:1 sex ratio and was replicated three times. The five chicks for this study were tagged at Day 1 for individual identification. In addition to these treatments, the main experiment consisted of 36 pens and included another two perch treatments that were not included in this experiment. Horizontal perches were constructed of 2.6 cm outside diameter PVC pipe held together by PVC connectors. Perching areas were covered with 3M rubber texturizing tape, to provide a more stable perching surface to the birds. The perches were placed at a height of 8.5 cm and consisted of a main bar (91 cm), with five evenly spaced horizontal cross bars (28 cm). The perch design provided 693 cm of linear perching space for the three perches in each treatment pen, as described (Pettit-Riley and Estevez, 2001). Perch placement resulted in the theoretical unavailability of approximately 7,644 square cm of floor space (PettitRiley and Estevez, 2001). Calculations of unavailable floor area were made by using the dimensions of the perches and the Pythagorean theorem. The length of the floor area covered by the particular perch and the width of the crossbars were multiplied to calculate a total area occupied by that perch. However, this calculation is an overestimation of ‘unavailable floor space,’ as the birds frequently use the space between the crossbars to rest (Pettit-Riley and Estevez, 2001). Bird mortality and culls were recorded daily for each pen, as was the tentative cause of death or reason for culling. Sex of the birds was determined by necropsy. Final body weights were taken at the end of the rearing period (42 d of age).
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HECKERT ET AL. TABLE 1. Morphometric measurements of various lymphoid organs at three different housing densities, with or without perches: treatment by density least square means and 95% confidence intervals
Density (m2) 10
Treatment C5 H6
15
C H
20
C H
Body1,2 weight (g)
Spleen1,2 weight (g)
Bursa3 weight (g)
Spleen weight 1,2 × 100 body weight
Bursa weight 1,4 × 100 body weight
1,847.39 1,715.45–1,989.50 1,856.99 1,685.28–2,046.19 1,744.48 1,613.92–1,885.61 1,756.66 1,636.24–1,885.95 1,955.24 1,802.46–2,120.9.6 1,719.14 1,571.26–1,880.93
2.408 2.04–2.85 2.055 1.697–2.487 2.074 1.732–2.482 2.024 1.725–2.376 1.923 1.602–2.308 1.826 1.495–2.230
3.636a 3.070–4.202 2.735bc 2.090–3.381 3.552ac 2.943–4.161 2.665b 2.100–3.230 2.764bc 2.145–3.383 2.202b 1.529–2.875
0.130 0.109–0.156 0.113 0.088–0.144 0.118 0.097–0.144 0.115 0.097–0.137 0.098 0.081–0.120 0.110 0.086–0.132
0.184a 0.149–0.226 0.139ab 0.105–0.185 0.198a 0.158–0.248 0.145ab 0.118–0.178 0.133b 0.106–0.167 0.120b 0.093–0.155
Means within a column lacking a common superscript differ (P < 0.05) using a least significant difference procedure (Sokal and Rohlf, 1995). Analyzed using log10. First line is estimate, back transformed. Second line is 95% CI (Sokal and Rohlf, 1995). All least square means were adjusted for gender. 2 Using ANOVA, no significant differences (P > 0.1) were found between the means; see text. Lack of superscripts indicate no significant differences between means. 3 As density increased, bursa weight decreased significantly (P < 0.05); however, most of the effect was due to a decline in bursa weight at high density. Addition of horizontal perches significantly (P < 0.01) lowered bursa weight at all densities. No other significant (P > 0.1) effects were found. 4 As density increased, the bursa/body weight ratio decreased marginally (P < 0.1) and the addition of horizontal perches significantly (P < 0.02) lowered the ratio. No other significant effects were found. 5 Control—no perch treatment. 6 Horizontal perch treatment. a–c 1
RESULTS Flock Performance There were no significant differences (P > 0.1) among the final body weights of the birds at the different rearing densities or perch treatments in this sub-study (Table 1). In addition, there were no significant differences in final body weights or feed conversion between any group or perch treatment in the main study involving 2,424 birds
(Pettit-Riley and Estevez, 2001). In our substudy males were heavier than females, but no significant effects of the main factors in the study or their interactions were found (P > 0.1). During the last 2 wk of the growing period, there were two episodes of high environmental temperatures (Week 5: June 7, 1999, and June 8, 1999, 31 and 33 C, respectively; Week 6: June 14, 1999, 28.3 C), which accounted for a substantial proportion of the total mortality in the study, which ranged from 9.0 to 18.9%. Heat stress mortality showed a significant increase with increasing density (Pettit-Riley and Estevez, 2001).
Immunology The body weights at 42 d of age, lymphoid organ weights and the organ to body weight ratios are shown in Table 1. Treatments did not significantly affect the spleen weights; however, there was a tendency to smaller spleen weights with increasing density and a tendency to smaller spleen weights for horizontal perch treatments within any density. A similar effect was found for the bursa weight, which was significantly lighter with increasing density (P < 0.05) mainly due to a large decrease in bursa weight at the highest density. In addition, bursa weight was also significantly smaller for horizontal perch treatments than that of the controls (P < 0.01; overall, but not necessarily between densities). There were no significant differences between density or perch treatment in the spleen/body weight ratios (P > 0.5). However, there was a marginal reduction in bursa/ body weight ratios with increasing density (P < 0.1) and within horizontal perch treatments over that of the control
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A test of density by treatment interactions was done similarly. Prior to analysis, homogeneity of variance was examined. Spleen weight, bursa weight, and the ratios of spleen to body weight and bursa weight to body weight were log-base 10 transformed prior to analysis. For these latter variables, a 95% confidence interval was computed using the log data and subsequently back-transformed to generate an estimate of the median and corresponding confidence interval for that variable on the original scale (Sokal and Rohlf, 1995). Similarly, SI and antibody titer (hemagglutination) values were also log transformed prior to analysis. Because of missing values due to mortality between the two sampling times, we analyzed H:L and SI separately at 32 d and at 42 d as well as the 42 minus 32d difference on an individual bird level before calculating the mean. We also asked if the average difference (between time points) was different from zero for the H:L ratio by using a t-test based on least-squares means. The least-squares means are for treatment by density and are therefore adjusted for differences in gender composition among the pens.
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DENSITY, PERCHES, AND THE IMMUNE STATUS TABLE 2. Least square means for measures of immune status of birds raised at three different densities, with or without perches Density (m2) 10
Treatment1 C7 H
15
8
C H
20
C H
H:L2,3 at 32 d
H:L2,3 at 42 d
H:L difference2,4 42 d–32 d)
SI3,5 at 32 d
SI3,5 at 42 d
HA3,6 titer
0.271 0.140–0.596 0.368 0.017–0.475 0.246 0.153–0.595 0.374 0.006–0.454 0.230 0.069–0.547 0.308 0.119–0.398
0.439 0.290–0.588 0.441 0.292–0.590 0.341 0.176–0.505 0.393 0.252–0.535 0.56 0.335–0.738 0.359 0.189–0.528
0.174 –0.106–0.453 0.052 –0.208–0.311 0.140 –0.142–0.422 0.013 –0.240–0.265 0.299 –0.033–0.631 0.054 –0.233–0.342
23.387 8.684–62.989 19.418 7.304–51.628 12.111 4.393–33.392 25.572 9.992–65.451 10.279 4.016–26.309 9.713 3.445–27.389
2.574 1.138–5.821 4.471 1.977–10.113 2.847 1.158–6.999 1.756 0.809–3.814 4.813 1.574–14.714 3.322 1.293–8.535
347.97 31.387–3857.75 2300 234.739–22,539.95 347.97 31.387–3857.75 95.59 15.511–589.13 744.06 48.632–11,384.02 156.66 14.131–1736.84
1
groups (P < 0.02), again mainly due to a large decrease in bursa weight at the highest density. The interaction of density and perch treatment was not significant (P > 0.1) for any of the variables in this study. All of the immunological parameters tested, their means and the 95% confidence intervals for each treatment are shown in Table 2. There were no significant differences seen between different densities, perch treatment or the interaction of both factors in the H:L values, the SI values, their 42 minus 32-d differences, and the antibody titers induced to SRBC. Although, there was too much individual bird variability to make any conclusions from the H:L or SI values between treatments, it was clear that from 32 to 42 d there was a change in both of these parameters. The SI values decreased dramatically from 32 to 42 d, but because these values were not standardized between the two time points this decline may or may not be entirely due to a decrease in lymphocyte blastogenesis. During this same time period, the H:L ratios tended to increase (not significantly P > 0.1) across the treatment groups.
DISCUSSION Although it has been shown that there is increasing profitability with increasing housing density, the effects on the immunological competence and welfare of broilers are unknown. In this study, many of the measures of immunocompetence did not show significant differences between treatment groups because of large bird-to-bird variability. Despite this a number of interesting trends
and in some cases significant differences were found. Of all the immunological parameters assessed, the morphometric measures that examined bursa weight and the bursa/body weight ratio gave the most consistent and reliable indication of stress. This is a commonly assessed measure in studying the immune system of the bird and is easily accessible at slaughter (Pope, 1991). This parameter clearly showed decreasing organ weight with increasing housing density, indicating a higher degree of stress (Table 1). In addition, it was noted that the availability of perches in the pen also decreased the bursa and bursa/ body weight ratios, indicating that availability of horizontal perches within the pen in this experiment did not reduce stress levels, but seemed to exacerbate the problems. This effect was probably related to the fact that perches were occupying space (therefore increasing the effects of relative density) and their level of use was very low with an average of 2.6% (± 0.15) (Pettit-Riley and Estevez, 2001). Of all of the immunological variables assessed, none showed a significant effect of changing housing density or perch availability. This was primarily due to a great degree of variability seen between birds when applying these immunological measures. Talebi et al. (1995) also showed that there was a great deal of individual bird variability in the lymphocyte blastogenesis assay under even more controlled conditions than used in this study. Although there were changes in the SI and H:L values towards the end of the study (both indicating an increasing degree of stress) none were different between the groups or treatments. These changes were most likely
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Each treatment group at the beginning of the study consisted of 15 birds in three replicate pens (five birds per pen) for a total of 90 birds in the study. Each bird was individually identified and samples taken at 32 and 42 d were matched to bird identity. Due to mortality during the study, the group size at the sampling times varied from 13 to 15 birds. 2 H:L = heterophil/lymphocyte ratio. 3 Using ANOVA, no significant differences (P > 0.1) were found among means within a column; see text. Lack of superscripts indicate no significant differences between means. All least square means are adjusted for gender. 4 H:L = heterophil/lymphocyte ratio difference (samples taken from the same bird on different days and then averaged). None of these means were significantly different from zero, even though all were positive. Second line is 95% confidence interval. 5 SI = stimulation index (the mean disintergrations per minute or the stimulated cultures divided by the disintergrations per minute of the unstimulated cultures). The background disintergrations per minute in the unstimulated cultures ranged from hundreds to thousands. These data were analyzed using log10 values. The first line is an estimate, back transformed. The second line is a 95% confidence interval. Difference computed on log scale corresponds to log (ratio). 6 HA = Hemagglutination. HA was analyzed using log10 values. Back-transformed values in table correspond to geometric mean titers (Sokal and Rohlf, 1995). Titer is the last dilution of serum to produce a positive HA result. 7 Control—no perch treatment. 8 Horizontal perch treatment.
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standard deviation on a log base 10 scale was 0.54 and the difference we observed was about 0.5 (see Table 2). Therefore, to be able to show any significant differences, we needed a sample size of 19 birds at each time period. While more realistic than for HI values, this is probably still too large to be economical as a standard tool for monitoring flocks. We conclude, that increasing housing density may produce immunosuppression in broilers, which can be most easily and reliably assessed at slaughter by measuring the bursa weights and or the bursa to body weight ratios. These measures, although retrospective, may give the producer an indication of the immune status of their flock. Furthermore, we found that perches, although used by young broilers, may add to immunosuppression in older birds towards the end of the rearing period when housed at high densities if perches are not commonly used (as occurs with high ambient temperatures). We should emphasize however, that the potential effect of perch availability on the stress levels of laying hens could be very different than the results reported in this study for broilers.
ACKNOWLEDGMENTS We thank N. Tablante, S. Elankumaran, S. Kennedy, M. Murtagh, R. Samson, S. McGary and T. L. Cornetto for technical assistance in the data collection and analysis. We also thank the Maryland Agricultural Experiment Station for supporting Pettit-Riley with salary and for the funding for this experiment (Grant No. AASC-99-13).
REFERENCES Al-Murrani, W. K., A. Kassab, H. Z. Al-Sam, and A. M. AlAthari. 1997. Heterophil/lymphocyte ratio as a selection criterion for heat resistance in domestic fowls. Br. Poult. Sci. 38:159–163. Barta, O., V. Barta, and F. W. Pierson. 1992. Optimum conditions for the chicken lymphocyte transformation test. Avian Dis. 36:945–955. Besedovsky, H. E., A.E. del Rey, and E. Sorkin. 1983. What do the immune system and the brain know about each other. Immunology 4:342–346. Bolton, W., W. A. Dewar, and R. Morley Jones. 1972. Effect of stocking density on performance of broiler chicks. Br. Poult. Sci. 13:157–162. Cravener, T. L., W. B. Roush, and M. M. Mashaly. 1992. Broiler production under varying population densities. Poult. Sci. 71:427–433. Cunnick, J. E., L. D. Kojic, and R. A. Hughes. 1994. Stressinduced changes in immune function are associated with increased production of an interleukin-1-like factor in young domestic fowl. Brain Behav. Immun. 8:123–136. Davison, T. F., B. H. Misson, R. A. Williamson, and J. Rea. 1988. Effect of increased circulating corticosterone in the immature fowl on the blastogenic responses of peripheral blood lymphocytes. Dev. Comp. Immunol. 12:131–144. Deaton, J. W., F. N. Reece, and T. H. Vardaman. 1968. The effect of temperature and density on broiler performance. Poult. Sci. 47:203–300. Donker, R. A., and G. Beuving. 1989. Effect of corticosterone infusion on plasma corticosterone concentration, antibody production, circulating leukocytes and growth in chicken
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due to the combined effects of decreasing space availability and the severe heat stress experienced in the final two wk of grow out. Although this heat stress experienced was severe, this degree of thermal stress is common in many of the poultry rearing areas of the US. Due to the heat stress and this being an out-bred population of birds, many of the immunological tests may have had more variability than is commonly seen in these assays when applied to defined genetic lines of birds under controlled experimental conditions. This variability made it difficult to form strong conclusions regarding the effect of housing density or perch use on immunological status. However, several indicators suggested that there was an increasing amount of immunological impairment with the addition of perches, which would indicate that instead of adding useable space to the pen and decreasing stress levels of the birds, the addition of perches, when perch use is low, may have actually resulted in the opposite effect. This result may not be surprising as perching frequency in broilers, particularly in the last 2 wk of the growing period was very low (Pettit-Riley and Estevez, 2001), in contrast with the high frequency of perch use observed in laying hens (Newberry et al., 2001). This low level of perch use in broilers was consistent with the results reported by LeVan et al. (2000) and is especially noticeable when ambient temperatures are high. Several findings were made in this study, which have previously been unreported and will help investigators perform these types of studies in the future. First, we note that many of the common laboratory techniques used to assess immunocompetence, while useful under controlled laboratory conditions in inbred populations of birds, can have a high degree of variability when applied to out-bred populations of chickens under field conditions. This variability may be further increased when environmental parameters are beyond the thermotolerance level of the birds. We tried to minimize this variability by sampling the same bird over time for several of the assays (SI and H:L). However, in a practical field setting, this will be difficult and a better approach would be to use large sample sizes to overcome inherent variability in assay parameters. In an attempt to assess the utility of these measures in a production setting, we assumed the variability within a pen is an estimate for variability within a flock. We also looked at the magnitude of change we observed between 32 and 42 d of age for the control treatment with density of 15 birds /square m. With such values, we used a sample size calculation (Samuels and Witmer, 1999) needed to achieve 80% power assuming a two-sample t-statistic at alpha = 0.05. Under these conditions, we would have needed about 100 birds at each time period to detect a change comparable to what we observed using H:L values. This is a considerably larger sample size than what is realistically manageable for field use. Of course, the magnitude of change observed in H:L values was very small (0.1, see Table 2), perhaps too small to be of meaningful consequence. For the SI values, the within pen
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Onsrud, M., and E. Thorsby. 1981. Influence of in vivo hydrocortisone on some human blood lymphocyte subpopulations. I. Effect on natural killer cell activity. Scand. J. Immunol. 13:573–579. Patterson, P. H., and H. S. Siegel. 1998. Impact of cage density on pullet performance and blood parameters of stress. Poult. Sci. 77:32–40. Pettit-Riley, R., and I. Estevez. 2001. Effects of density on perching behavior of broiler chickens. Appl. Anim. Behav. Sci. 71:127–140 Pope, C. R. 1991. Pathology of lymphoid organs with emphasis on immunosuppression. Vet. Immunol. Immunopathol. 30:31–44. Proudfoot, F. G., H. W. Hulan, and D. R. Ramey. 1979. The effect of four stocking densities on broiler carcass grade, the incidence of breast blisters, and other performance traits. Poult. Sci. 58:791–793. Samuels, M., and J. Witmer. 1999. Statistics for the Life Sciences. 2nd ed. Prentice-Hall, Upper Saddle River, NJ. Sanotra S. G., L. G. Lawson, K. S. Vestergaard, and M. G. Thomsen. 2001. Influence of stocking density on tonic immobility, lameness, and tibial dischondroplasia in broilers. J. Appl. Anim. Welfare Sci. 4:71–87. Scott, T. R., E. A. Dunnington, and P. B. Siegel. 1994. Brucella abortus antibody response of white leghorn chickens selected for high and low antibody responsiveness to sheep erythrocytes. Poult. Sci. 73:346–349. Shanawany, M. M. 1988. Broiler performance under high stocking densities. Br. Poult. Sci. 29:43–52. Sokal, R. R., and Rohlf. 1995. Biometry. 3rd ed. W. H. Freeman and Sons, New York. Sørensen, P., G. Su and S. C. Kestin. 2000. Effects of age and stocking density on leg weakness in broiler chickens. Poult. Sci. 79:864–870. Stevenson, J. R., and R. Taylor. 1988. Effects of glucocorticoid and antiglucocorticoid hormones on leukocyte numbers and function. Int. J. Immunopharmacol. 10:1–6. Talebi, A., P. R. Torgerson, and G. Mulcahy. 1995. Optimal conditions for measurement of blastogenic responses of chickens to concanavalin a in whole blood assays. Vet. Immunol. Immunopathol. 46:293–301. Trout, J. M., and M. M Mashaly. 1994. The effects of adrenocorticotropic hormone and heat stress on the distribution of lymphocyte populations in immature male chickens. Poult. Sci. 73:1694–1698.
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lines selected for humoral immune responsiveness. Br. Poult. Sci. 30:361–369. Estevez, I., R. C. Newberry, and de Reyna L.A. 1997. Broiler chickens: A tolerant social system? Etologia 5:19–29. Glick, B. 1958. The effect of cortisone acetate on the leukocytes of young chickens. Poult. Sci. 37:1446–1452. Gogal, R. M. J., S. A. Ahmed, and C. T. Larsen. 1997. Analysis of avian lymphocyte proliferation by a new, simple, nonradioactive assay (Lympho-Pro). Avian Dis. 41:714–725. 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. 1980. Effects of early environmental stresses on chicken body weight, antibody response to RBC antigens, feed efficiency, and response to fasting. Avian Dis. 24:569–579. Gross, W. B., and P. B. Siegel. 1990. Genetic-environmental interactions and antibody response in chickens to two antigens. Avian Dis. 34:843–847. Hansen, R. S., and W. A. Becker. 1960. Feeding space, population density and growth of young chickens. Poult. Sci. 39:654–661. Hughes, B. O., and H. A. Elson. 1977. The use of perches by broilers in floor pens. Br. Poult. Sci. 18:715–722. LeVan Fiscus, N., I. Estevez, and R. W. Stricklin. 2000. Use of horizontal and angled perches by broiler chickens. Appl. Anim. Behav. Sci. 65:349–365. Littell, R. C., G. A. Milliken, W. W. Stroup, and R. D. Wolfinger. 1996. SAS System for Mixed Models. SAS Institute Inc., Cary NC. Montgomery, R. D., C. R. Boyle, and W. R. Maslin. 1991. Influence of antigen concentration, inoculation interval, number of exposures, type of housing, and placement concentration on the tear antibody response to brucella abortus in chickens. Avian Dis. 35:606–614. Nagano, Y., and T. Lee. 1978. Different types of virus inhibitory factors in interferon produced by different organs. CR Seances. Soc. Biol. Fil. 172:805–808. Newberry, R. C. 1995. Environmental enrichment: increasing the biological relevance of captive environments. Appl. Anim. Behav. Sci. 44:229–243. Newberry, R. C., I. Estevez, and L.J. Keeling. 2001. Group size and perching behavior in young domestic fowl. Appl. Anim. Behav. Sci. 73:117–129.
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(Key words: housing density, immune status, heat stress, perch) 2002 Poultry Science 81:451–457
nize that stress alters the immune system, the ability to assess the immune status of commercial broilers is difficult and no single assay is available to evaluate immunocompetence. Measures of immunity that have been commonly used and assessed in poultry are lymphoid organ weights, (Pope, 1991) antibody response to foreign antigens, (Gross and Siegel, 1980,1990; Montgomery et al., 1991; Patterson and Siegel, 1998; Scott et al., 1994) heterophil:lymphocyte (H:L) ratios (Gross and Siegel, 1983; Cravener et al., 1992; Patterson and Siegel, 1998; Al-Murrani et al., 1997) and lymphocyte blastogenesis assays (Nagano and Lee, 1978; Barta;et al., 1992; Cunnick et al., 1994; Talebi et al., 1995; Gogal et al., 1997). Lymphoid organ weights are easily measured and reflect the body’s ability to provide lymphoid cells during an immune response. Humoral immunity, or the ability to produce an antibody response, is another commonly employed method of assessing immunocompetence. The most commonly used antigens in this assessment have been SRBC (T-dependent) and Brucella abortus (T-independent). In Leghorns, housing density has been shown to have no effect on antibody responses to either of these two antigens (Montgomery et al., 1991; Patterson and Siegel, 1998). To our knowledge, the assessment of lymphoid organ weights or the immune response to antigens has not been examined in commer-
INTRODUCTION In modern poultry rearing operations, stress can occur due to a variety of factors (e.g., elevated rearing densities or high summer temperatures). These unfavorable or stressful environmental conditions can negatively affect the animal’s immune system, compromising their performance and ability to overcome viral and bacterial infections. It is well documented that the nervous and immune systems are integrated in their response to stress (Besedovsky et al., 1983; Trout and Mashaly, 1994). Much of the research into neuroendocrine-immune interactions has focused on the relationship between the hypothalamo-pituitary-adrenal axis and the immune system. In animals treated with adrenocorticotropic hormone or subjected to chronic stress the weights of both primary (Davison et al., 1988; Donker and Beuving, 1989) and secondary (Donker and Beuving, 1989) lymphoid organs decreased. Profiles of circulating leukocytes were also affected when treated with adrenocorticotropic hormones or exposed to stress; lymphocyte and monocyte numbers decrease and heterophil numbers increase (Glick, 1958; Onsrud and Thorsby, 1981; Gross and Siegel, 1983; Stevenson and Taylor, 1988). Although we recog-
2002 Poultry Science Association, Inc. Received for publication November 6, 2000. Accepted for publication November 30,2001. 1 1To whom correspondence should be umail.umd.edu.
addressed:
Abbreviation Key: H:L = heterophil:lymphocyte ratio; SI = stimulation index.
rh175@
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of perches to the pens also significantly decreased the bursa weights and bursa/body weight index (P < 0.01). No other significant effects were observed for the flock performance, morphometric data, or immunological tests between treatments. We concluded that under the conditions of this study, which tried to simulate commercial conditions, the bursal weight was the best indicator of stress that was related to housing density. Addition of perches appeared to increase this level of stress because the birds used the perches infrequently, and therefore there might have been a further reduction in the availability of floor space to the birds.
ABSTRACT This study attempted to evaluate the effect of various housing densities and perch availability on the immune status of commercial broilers. Birds were raised from hatch to 42 d of age with 10, 15, and 20 birds/m2 in pens, with and without the availability of horizontal perches. The immune parameters that were assessed were lymphoid organ weights, antibody response to SRBC in the last 10 d of growth, heterophil:lymphocyte ratios at 32 and 42 d of age, and lymphocyte blastogenesis of peripheral blood lymphocytes collected at 32 and 42 d of age. As density increased, bursa weight and bursa/body weight ratios decreased significantly (P < 0.05). Addition
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commercial conditions are the most crowded and show the greatest degree of health related problems.
MATERIALS AND METHODS Birds and General Management Practices This study was designed as a sub-study within a large trial aimed at investigating the effects of different perch designs and housing density on broiler behavior and performance (Pettit-Riley and Estevez, 2001). Two thousand four hundred twenty-four, 1-d-old sexed broiler chickens (Avian × Avian) were obtained from a commercial hatchery and reared in a broiler house at the Applied Poultry Research Facility, University of Maryland. The broiler house was divided into 36 identical 4.46 m2 pens (18 pens used in the substudy), and the floor was covered with 5 cm of wood shavings litter. The birds, raised under commercial management practices, were fed a commercial two-phase broiler diet and provided water from nipple drinkers ad libitum throughout the rearing period. The diet consisted of a starter crumble with amprolium for the first 21 d (21.85% protein; 3,102 kcal/kg energy), followed by a grower/finisher pellet with amprolium from Day 22 to the end of the growing period (19.23% protein; 3,190 kcal/kg energy). A withdrawal diet was not required with this feeding system. The amount of feeding and drinking space available per bird was kept constant across density by blocking a proportion of the feeding area and drinking nipples within the pen. Each pen contained one feeding bin and seven drinking nipples, of which all seven were available in the high density pens, five were available in the moderate density pens, and three were available in the low density pens. A combination of natural and artificial lighting was used. Artificial light was provided continuously for the first 3 d and from 0600 to 1800 h for the remainder of the growing period. Temperatures were maintained between 32 and 34 C at the beginning of the growing period, and gradually decreased every 2 to 3 d to reach 22 C ± 1 at the end of the growing period. The temperature was controlled using electric brooders and by opening and closing the automatic temperature regulated wall curtains in the house. Positive pressure ventilation was used to provide 10 total air changes for each room per hour during the brooding period and 12 total air changes during the growout period. Although every effort was made to maintain a temperature of 22 C during the study, unusually high temperatures (reaching up to 33 C with high relative humidity values some days) occurred at the end of the growing period. Therefore, as ambient temperatures occasionally exceeded the optimum range toward the end of the rearing period the temperature inside the house could not be maintained low enough to remain within the avian thermoneutral zone. This heat stress situation was unexpected and unplanned in this study.
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cial broiler strains for ability to identify potential stress problems due to increase housing densities, or for the potential effects of perch availability. Although the H:L ratio has been commonly used as a measure of stress in poultry, there have been mixed reports on its usefulness as an indicator of stress due to housing density. One study conducted in seven-week-old broilers showed an increased H:L ratio with increasing density (Cravener et al., 1992), although in caged leghorns, increased cage density had no effect on the H:L ratio (Patterson and Siegel, 1998). On the other hand, little work has been done using the lymphocyte blastogenesis assay in assessing stress in broilers under field conditions. This assay has been used mainly on lymphocytes from inbred populations of birds under laboratory conditions (Nagano and Lee, 1978; Barta et al., 1992; Cunnick et al., 1994; Talebi et al., 1995; Gogal et al., 1997). Numerous studies have been conducted to characterize the effects of density on broiler performance (Hansen and Becker, 1960; Deaton et al., 1968; Bolton et al., 1972; Proudfoot et al., 1979; Shanawany, 1988; Cravener et al., 1992; Estevez et al., 1997). In all studies, increasing density led to a reduction in performance. Although birds at higher densities appeared to be stressed (Proudfoot et al., 1979; Cravener et al., 1992) no comprehensive evaluation of the immune status of the birds was done. Although it is well recognized that stress can negatively impact the immune system, there have been very few controlled studies under field conditions to determine the impact of rearing density on the immune status of birds with industry standard genetics. High rearing densities in broilers are associated with an increased incidence of leg problems (Sørensen et al., 2000; Sanotra et al., 2001), which may be related to the reduced level of activity observed with increasing housing densities (Estevez, et al., 1997). It has been suggested that providing broilers access to perches, under high housing density conditions, may be an effective means of increasing bird activity, therefore potentially reducing the incidence of leg problems, while at the same time increasing air flow among the birds (Hughes and Elson, 1977; LeVan Fiscus et al., 2000; Pettit-Riley and Estevez, 2001). Therefore, perch availability, as a form of environmental enrichment for poultry, has the potential to reduce some of the stressors related to high housing densities (Newberry, 1995). We hypothesized that increasing housing density would result in increased levels of stress and consequently decreased immunocompetence in the birds, that could be in part mitigated by availability of perches (by increasing space availability in the third dimension with better air flow). Therefore, the goals of this study were to determine the immunological status of commercial broilers under increasing housing densities (below, at and above current industry standards) and to determine if any immunological indicators of stress could be mitigated by adding environmental enrichment in the form of horizontal perches. The final 2 wk of the growing period were targeted for study because this is when birds under
DENSITY, PERCHES, AND THE IMMUNE STATUS
Experimental Design
Immunological Assays Several parameters were evaluated to assess the immunological status of the 90 birds at 32 and 42 d of age: 1) humoral immune response to SRBC, 2) lymphocyte blastogenesis assay, 3) H:L ratios, and 4) lymphoid organ weights and organ to body weight ratios. The same birds were caught and tested at 32 and 42 d of age unless they had died during the interval. To assess the ability of the birds under different density and perch availability to mount an immune response
2
Gibco-BRL P.O. Box 9418, Gaithersburg, MD 20898. Sigma Chemical Company, 3050 Spruce St., St. Louis, MO 63103. 4 ICN Biomedicals Inc., 3300 Hyland Ave., Costa Mesa, CA 92626. 5 Harvester 96, Tomtec, Inc., 607 Harbor View Rd., CT 06477. 6 Wallac 1450, MicroBeta Trilux, 549 Albany St., Boston, MA 02118. 3
against a foreign antigen, the birds were exposed to SRBC, as described (Gross and Siegel, 1980). Briefly, the chickens were inoculated IV at 32 d of age with 0.1 mL of a 0.25% suspension of SRBC. Plasma was collected from the same birds (where available) by brachial venipuncture before SRBC inoculation at 32 d of age and again 10 d later. The plasma samples were assessed for hemagglutination titers (the last dilution to give a positive result) by a standard microtiter method as described by Gross and Siegel (1980). To assess the cellular immune responses of the birds a mitogen lymphocyte blastogenesis assay was performed essentially as described by Talebi et al.(1995) with some modifications. Briefly, 2 mL of whole blood was collected into heparinized vacutainer tubes at 32 and 42 d of age by brachial venipuncture. The blood was diluted 1/50 in serum-free RPMI 1640 media2 and 200 µL/well cultured in triplicate, with and without mitogen in 96-well flatbottom tissue culture microplates. Concanavalin A3 was added to half of the wells, at 20 µl/well of a 20 µg/mL stock solution, to attain a final concentration of 0.4 µg of mitogen/well. After 90 h of incubation in a humidified chamber at 40 C the cultures were pulsed with 0.2 uCi of [3H]-thymidine4/well. After a further 18 h of incubation the cultures were harvested onto filter mats using a cell harvester.5 The filter mats were then saturated with scintillation fluid in a plastic bag and the disintegrations per minute (DPM) measured in a scintillation analyzer.6 The stimulation index (SI) for each sample was calculated according to the following formula: SI =
mean DPM of stimulated cultures mean DPM of unstimulated cultures
The H:L ratio was also used to determine the degree of a chicken’s stress. The H:L ratios were determined using the stained-slide method, as described by Gross and Siegel (1983). Immunosuppressed or stressed birds classically have smaller lymphoid organs (Pope, 1991). Therefore, at the termination of the study (42 d of age) the bursa, spleen and body weights were determined and the spleen/body and bursa/body weight ratios calculated.
Statistical Analysis A mixed model analysis of variance was used to determine the effects of density (10, 15, 20 birds/m2), perch availability (control versus horizontal), and gender. Although we originally analyzed the data as a randomized block study, block effects were often estimated as zero and in no case were close to significant. Pens were treated as a random effect nested within density by perch treatment combinations. Gender was considered as a subplot factor as gender was not always even within a pen. The mixed model procedure in SAS software (version 6.12) (Littell et al., 1996) was used to analyze the data and calculate least-squares means. Density was analyzed using linear and quadratic orthogonal polynomial contrasts.
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A total of five experimental birds per pen were randomly selected at 1 d of age from each of 18 pens belonging to one of the following treatments: control (no perches) or horizontal (three 0° perches), in a nested design with one of three bird densities: 10 birds/m2, 15 birds/m2, (standard commercial density) and 20 birds/ m2. Birds per pen were 45, 67, and 90, respectively. Each treatment/density combination had a 1:1 sex ratio and was replicated three times. The five chicks for this study were tagged at Day 1 for individual identification. In addition to these treatments, the main experiment consisted of 36 pens and included another two perch treatments that were not included in this experiment. Horizontal perches were constructed of 2.6 cm outside diameter PVC pipe held together by PVC connectors. Perching areas were covered with 3M rubber texturizing tape, to provide a more stable perching surface to the birds. The perches were placed at a height of 8.5 cm and consisted of a main bar (91 cm), with five evenly spaced horizontal cross bars (28 cm). The perch design provided 693 cm of linear perching space for the three perches in each treatment pen, as described (Pettit-Riley and Estevez, 2001). Perch placement resulted in the theoretical unavailability of approximately 7,644 square cm of floor space (PettitRiley and Estevez, 2001). Calculations of unavailable floor area were made by using the dimensions of the perches and the Pythagorean theorem. The length of the floor area covered by the particular perch and the width of the crossbars were multiplied to calculate a total area occupied by that perch. However, this calculation is an overestimation of ‘unavailable floor space,’ as the birds frequently use the space between the crossbars to rest (Pettit-Riley and Estevez, 2001). Bird mortality and culls were recorded daily for each pen, as was the tentative cause of death or reason for culling. Sex of the birds was determined by necropsy. Final body weights were taken at the end of the rearing period (42 d of age).
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HECKERT ET AL. TABLE 1. Morphometric measurements of various lymphoid organs at three different housing densities, with or without perches: treatment by density least square means and 95% confidence intervals
Density (m2) 10
Treatment C5 H6
15
C H
20
C H
Body1,2 weight (g)
Spleen1,2 weight (g)
Bursa3 weight (g)
Spleen weight 1,2 × 100 body weight
Bursa weight 1,4 × 100 body weight
1,847.39 1,715.45–1,989.50 1,856.99 1,685.28–2,046.19 1,744.48 1,613.92–1,885.61 1,756.66 1,636.24–1,885.95 1,955.24 1,802.46–2,120.9.6 1,719.14 1,571.26–1,880.93
2.408 2.04–2.85 2.055 1.697–2.487 2.074 1.732–2.482 2.024 1.725–2.376 1.923 1.602–2.308 1.826 1.495–2.230
3.636a 3.070–4.202 2.735bc 2.090–3.381 3.552ac 2.943–4.161 2.665b 2.100–3.230 2.764bc 2.145–3.383 2.202b 1.529–2.875
0.130 0.109–0.156 0.113 0.088–0.144 0.118 0.097–0.144 0.115 0.097–0.137 0.098 0.081–0.120 0.110 0.086–0.132
0.184a 0.149–0.226 0.139ab 0.105–0.185 0.198a 0.158–0.248 0.145ab 0.118–0.178 0.133b 0.106–0.167 0.120b 0.093–0.155
Means within a column lacking a common superscript differ (P < 0.05) using a least significant difference procedure (Sokal and Rohlf, 1995). Analyzed using log10. First line is estimate, back transformed. Second line is 95% CI (Sokal and Rohlf, 1995). All least square means were adjusted for gender. 2 Using ANOVA, no significant differences (P > 0.1) were found between the means; see text. Lack of superscripts indicate no significant differences between means. 3 As density increased, bursa weight decreased significantly (P < 0.05); however, most of the effect was due to a decline in bursa weight at high density. Addition of horizontal perches significantly (P < 0.01) lowered bursa weight at all densities. No other significant (P > 0.1) effects were found. 4 As density increased, the bursa/body weight ratio decreased marginally (P < 0.1) and the addition of horizontal perches significantly (P < 0.02) lowered the ratio. No other significant effects were found. 5 Control—no perch treatment. 6 Horizontal perch treatment. a–c 1
RESULTS Flock Performance There were no significant differences (P > 0.1) among the final body weights of the birds at the different rearing densities or perch treatments in this sub-study (Table 1). In addition, there were no significant differences in final body weights or feed conversion between any group or perch treatment in the main study involving 2,424 birds
(Pettit-Riley and Estevez, 2001). In our substudy males were heavier than females, but no significant effects of the main factors in the study or their interactions were found (P > 0.1). During the last 2 wk of the growing period, there were two episodes of high environmental temperatures (Week 5: June 7, 1999, and June 8, 1999, 31 and 33 C, respectively; Week 6: June 14, 1999, 28.3 C), which accounted for a substantial proportion of the total mortality in the study, which ranged from 9.0 to 18.9%. Heat stress mortality showed a significant increase with increasing density (Pettit-Riley and Estevez, 2001).
Immunology The body weights at 42 d of age, lymphoid organ weights and the organ to body weight ratios are shown in Table 1. Treatments did not significantly affect the spleen weights; however, there was a tendency to smaller spleen weights with increasing density and a tendency to smaller spleen weights for horizontal perch treatments within any density. A similar effect was found for the bursa weight, which was significantly lighter with increasing density (P < 0.05) mainly due to a large decrease in bursa weight at the highest density. In addition, bursa weight was also significantly smaller for horizontal perch treatments than that of the controls (P < 0.01; overall, but not necessarily between densities). There were no significant differences between density or perch treatment in the spleen/body weight ratios (P > 0.5). However, there was a marginal reduction in bursa/ body weight ratios with increasing density (P < 0.1) and within horizontal perch treatments over that of the control
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A test of density by treatment interactions was done similarly. Prior to analysis, homogeneity of variance was examined. Spleen weight, bursa weight, and the ratios of spleen to body weight and bursa weight to body weight were log-base 10 transformed prior to analysis. For these latter variables, a 95% confidence interval was computed using the log data and subsequently back-transformed to generate an estimate of the median and corresponding confidence interval for that variable on the original scale (Sokal and Rohlf, 1995). Similarly, SI and antibody titer (hemagglutination) values were also log transformed prior to analysis. Because of missing values due to mortality between the two sampling times, we analyzed H:L and SI separately at 32 d and at 42 d as well as the 42 minus 32d difference on an individual bird level before calculating the mean. We also asked if the average difference (between time points) was different from zero for the H:L ratio by using a t-test based on least-squares means. The least-squares means are for treatment by density and are therefore adjusted for differences in gender composition among the pens.
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DENSITY, PERCHES, AND THE IMMUNE STATUS TABLE 2. Least square means for measures of immune status of birds raised at three different densities, with or without perches Density (m2) 10
Treatment1 C7 H
15
8
C H
20
C H
H:L2,3 at 32 d
H:L2,3 at 42 d
H:L difference2,4 42 d–32 d)
SI3,5 at 32 d
SI3,5 at 42 d
HA3,6 titer
0.271 0.140–0.596 0.368 0.017–0.475 0.246 0.153–0.595 0.374 0.006–0.454 0.230 0.069–0.547 0.308 0.119–0.398
0.439 0.290–0.588 0.441 0.292–0.590 0.341 0.176–0.505 0.393 0.252–0.535 0.56 0.335–0.738 0.359 0.189–0.528
0.174 –0.106–0.453 0.052 –0.208–0.311 0.140 –0.142–0.422 0.013 –0.240–0.265 0.299 –0.033–0.631 0.054 –0.233–0.342
23.387 8.684–62.989 19.418 7.304–51.628 12.111 4.393–33.392 25.572 9.992–65.451 10.279 4.016–26.309 9.713 3.445–27.389
2.574 1.138–5.821 4.471 1.977–10.113 2.847 1.158–6.999 1.756 0.809–3.814 4.813 1.574–14.714 3.322 1.293–8.535
347.97 31.387–3857.75 2300 234.739–22,539.95 347.97 31.387–3857.75 95.59 15.511–589.13 744.06 48.632–11,384.02 156.66 14.131–1736.84
1
groups (P < 0.02), again mainly due to a large decrease in bursa weight at the highest density. The interaction of density and perch treatment was not significant (P > 0.1) for any of the variables in this study. All of the immunological parameters tested, their means and the 95% confidence intervals for each treatment are shown in Table 2. There were no significant differences seen between different densities, perch treatment or the interaction of both factors in the H:L values, the SI values, their 42 minus 32-d differences, and the antibody titers induced to SRBC. Although, there was too much individual bird variability to make any conclusions from the H:L or SI values between treatments, it was clear that from 32 to 42 d there was a change in both of these parameters. The SI values decreased dramatically from 32 to 42 d, but because these values were not standardized between the two time points this decline may or may not be entirely due to a decrease in lymphocyte blastogenesis. During this same time period, the H:L ratios tended to increase (not significantly P > 0.1) across the treatment groups.
DISCUSSION Although it has been shown that there is increasing profitability with increasing housing density, the effects on the immunological competence and welfare of broilers are unknown. In this study, many of the measures of immunocompetence did not show significant differences between treatment groups because of large bird-to-bird variability. Despite this a number of interesting trends
and in some cases significant differences were found. Of all the immunological parameters assessed, the morphometric measures that examined bursa weight and the bursa/body weight ratio gave the most consistent and reliable indication of stress. This is a commonly assessed measure in studying the immune system of the bird and is easily accessible at slaughter (Pope, 1991). This parameter clearly showed decreasing organ weight with increasing housing density, indicating a higher degree of stress (Table 1). In addition, it was noted that the availability of perches in the pen also decreased the bursa and bursa/ body weight ratios, indicating that availability of horizontal perches within the pen in this experiment did not reduce stress levels, but seemed to exacerbate the problems. This effect was probably related to the fact that perches were occupying space (therefore increasing the effects of relative density) and their level of use was very low with an average of 2.6% (± 0.15) (Pettit-Riley and Estevez, 2001). Of all of the immunological variables assessed, none showed a significant effect of changing housing density or perch availability. This was primarily due to a great degree of variability seen between birds when applying these immunological measures. Talebi et al. (1995) also showed that there was a great deal of individual bird variability in the lymphocyte blastogenesis assay under even more controlled conditions than used in this study. Although there were changes in the SI and H:L values towards the end of the study (both indicating an increasing degree of stress) none were different between the groups or treatments. These changes were most likely
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Each treatment group at the beginning of the study consisted of 15 birds in three replicate pens (five birds per pen) for a total of 90 birds in the study. Each bird was individually identified and samples taken at 32 and 42 d were matched to bird identity. Due to mortality during the study, the group size at the sampling times varied from 13 to 15 birds. 2 H:L = heterophil/lymphocyte ratio. 3 Using ANOVA, no significant differences (P > 0.1) were found among means within a column; see text. Lack of superscripts indicate no significant differences between means. All least square means are adjusted for gender. 4 H:L = heterophil/lymphocyte ratio difference (samples taken from the same bird on different days and then averaged). None of these means were significantly different from zero, even though all were positive. Second line is 95% confidence interval. 5 SI = stimulation index (the mean disintergrations per minute or the stimulated cultures divided by the disintergrations per minute of the unstimulated cultures). The background disintergrations per minute in the unstimulated cultures ranged from hundreds to thousands. These data were analyzed using log10 values. The first line is an estimate, back transformed. The second line is a 95% confidence interval. Difference computed on log scale corresponds to log (ratio). 6 HA = Hemagglutination. HA was analyzed using log10 values. Back-transformed values in table correspond to geometric mean titers (Sokal and Rohlf, 1995). Titer is the last dilution of serum to produce a positive HA result. 7 Control—no perch treatment. 8 Horizontal perch treatment.
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standard deviation on a log base 10 scale was 0.54 and the difference we observed was about 0.5 (see Table 2). Therefore, to be able to show any significant differences, we needed a sample size of 19 birds at each time period. While more realistic than for HI values, this is probably still too large to be economical as a standard tool for monitoring flocks. We conclude, that increasing housing density may produce immunosuppression in broilers, which can be most easily and reliably assessed at slaughter by measuring the bursa weights and or the bursa to body weight ratios. These measures, although retrospective, may give the producer an indication of the immune status of their flock. Furthermore, we found that perches, although used by young broilers, may add to immunosuppression in older birds towards the end of the rearing period when housed at high densities if perches are not commonly used (as occurs with high ambient temperatures). We should emphasize however, that the potential effect of perch availability on the stress levels of laying hens could be very different than the results reported in this study for broilers.
ACKNOWLEDGMENTS We thank N. Tablante, S. Elankumaran, S. Kennedy, M. Murtagh, R. Samson, S. McGary and T. L. Cornetto for technical assistance in the data collection and analysis. We also thank the Maryland Agricultural Experiment Station for supporting Pettit-Riley with salary and for the funding for this experiment (Grant No. AASC-99-13).
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due to the combined effects of decreasing space availability and the severe heat stress experienced in the final two wk of grow out. Although this heat stress experienced was severe, this degree of thermal stress is common in many of the poultry rearing areas of the US. Due to the heat stress and this being an out-bred population of birds, many of the immunological tests may have had more variability than is commonly seen in these assays when applied to defined genetic lines of birds under controlled experimental conditions. This variability made it difficult to form strong conclusions regarding the effect of housing density or perch use on immunological status. However, several indicators suggested that there was an increasing amount of immunological impairment with the addition of perches, which would indicate that instead of adding useable space to the pen and decreasing stress levels of the birds, the addition of perches, when perch use is low, may have actually resulted in the opposite effect. This result may not be surprising as perching frequency in broilers, particularly in the last 2 wk of the growing period was very low (Pettit-Riley and Estevez, 2001), in contrast with the high frequency of perch use observed in laying hens (Newberry et al., 2001). This low level of perch use in broilers was consistent with the results reported by LeVan et al. (2000) and is especially noticeable when ambient temperatures are high. Several findings were made in this study, which have previously been unreported and will help investigators perform these types of studies in the future. First, we note that many of the common laboratory techniques used to assess immunocompetence, while useful under controlled laboratory conditions in inbred populations of birds, can have a high degree of variability when applied to out-bred populations of chickens under field conditions. This variability may be further increased when environmental parameters are beyond the thermotolerance level of the birds. We tried to minimize this variability by sampling the same bird over time for several of the assays (SI and H:L). However, in a practical field setting, this will be difficult and a better approach would be to use large sample sizes to overcome inherent variability in assay parameters. In an attempt to assess the utility of these measures in a production setting, we assumed the variability within a pen is an estimate for variability within a flock. We also looked at the magnitude of change we observed between 32 and 42 d of age for the control treatment with density of 15 birds /square m. With such values, we used a sample size calculation (Samuels and Witmer, 1999) needed to achieve 80% power assuming a two-sample t-statistic at alpha = 0.05. Under these conditions, we would have needed about 100 birds at each time period to detect a change comparable to what we observed using H:L values. This is a considerably larger sample size than what is realistically manageable for field use. Of course, the magnitude of change observed in H:L values was very small (0.1, see Table 2), perhaps too small to be of meaningful consequence. For the SI values, the within pen
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