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Animal Science Journal (2016) 87, 284–292

doi: 10.1111/asj.12411

ORIGINAL ARTICLE The effects of environmental enrichment and transport stress on the weights of lymphoid organs, cell-mediated immune response, heterophil functions and antibody production in laying hens Erdal MATUR,1 İbrahim AKYAZI,1 Evren ERASLAN,1 Elif ERGUL EKIZ,1 Hüseyin ESECELI,2 Mehmet KETEN,1 Kemal METINER3 and Deniz AKTARAN BALA1 1

Department of Physiology, Faculty of Veterinary Medicine, University of Istanbul, Avcilar, Istanbul, 2Balikesir University, Bandirma Vocational High School, Bandirma, and 3Department of Microbiology, Faculty of Veterinary Medicine, University of Istanbul, Avcilar, Istanbul, Turkey

ABSTRACT The effects of environmental enrichment and transport stress on the immune system were investigated in laying hens. A total of 48 1-day-old chickens were used, half of the chickens were reared in conventional cages (RCC) and the rest in enriched cages (REC). Transport stress was applied in the 17th week. Liver weight decreased, spleen and bursa of Fabricius weights, white blood cell count, CD4+ and CD8+ cell proportions increased due to the transport. Environmental enrichment significantly increased antibody production and tended to increase monocyte percentage and CD8+ cell proportion. The effect of transport on, heterophil (H) and lymphocyte (L) percentages was not significant in RCC chickens. While heterophil percentage and H:L ratio increased, lymphocyte percentage decreased in REC chickens subjected to transport. Transport stress increased heterophil functions both in REC and RCC chickens, but the increase was higher in REC hens than in RCC hens. In conclusion, although environmental enrichment did not neutralize the effect of transport on lymphoid organs, it activated the non-specific immune system, cellular and the humoral branches of the specific immune system by increasing heterophil functions, CD8+ cells and antibody production, respectively. Therefore, environmental enrichment suggested for improving animal welfare may also be beneficial to improve the immune system of birds exposed to stress.

Key words: environmental enrichment, heterophil functions, poultry, T cell, transport stress.

INTRODUCTION Stress is an important problem in the poultry industry (Cheng et al. 2002) which decreases production performance and reduces resistance to diseases. There are different types of stressors, such as heat, noise, high stocking density, cold, poor ventilation or social stress (Rosales 1994). Transport is also an important stress factor in laying hens. Furthermore laying hens experience more episodes of transport than other types of chickens (eg. broiler chickens) by first transferring from the hatchery to the growing pens, afterwards to the laying pens, and to the slaughterhouse at the end of the laying period (Mitchell & Kettlewell 2004). Catching and loading of animals, variable environmental temperatures during transportation, fast movement, vibration, loud noise, social disconnectedness, and deprivation of food and water are the main factors contributing to transport stress (Nicol & Scott 1990). Colonization of pathogens in © 2015 Japanese Society of Animal Science

the intestinal tract during transportation (Weedman et al. 2011) leads to penetration to the tissues (Zucker & Krüger 1998) and this causes a reduction in resistance to diseases (Huff et al. 2007). Furthermore, stress has important and considerable effects on the neuroendocrine system, leukocytes and immune mediators regulating stress response (Shini et al. 2008). Different approaches are considered to minimize the effects of stress and to support the immune system in poultry. Feed additives (Wang et al. 2000) or antibiotics (Zulkifli et al. 2000) are used for this purpose. However, these products are considered to be suspicious as some

Correspondence: Erdal Matur, Department of Physiology, Faculty of Veterinary Medicine, University of Istanbul, Avcilar, Istanbul, 34320, Turkey. (Email: [email protected]) Received 22 July 2014; accepted for publication 10 February 2015.

ENVIRONMENTAL ENRICHMENT AND TRANSPORT STRESS

residues remain in animal products. Therefore, new treatments are in need, including natural procedures. Environmental enrichment is defined as using various objects to improve life quality and normal behavior expression of animals which are kept in cages or in a limited space (Belz et al. 2003). The use of environmental enrichment applications both in the laboratory (Belz et al. 2003) and farm animals (Hocking & Jones 2006) is spreading due to their various positive effects (Würbel 2001). Environmental enrichment positively affects immune response (Kingston & Hoffman-Goetz 1996; Arranz et al. 2010), natural killer cell activity (Benaroya-Milshtein et al. 2004), plasma corticosterone concentration and other physiological responses to stress (Meijer et al. 2007) in laboratory animals. Furthermore, environmental enrichment has been reported to cause an increase in motor activity, and sensory, cognitive and social abilities in laboratory animals (Reynolds et al. 2010). Environmental enrichment also increases animal welfare and production performance by decreasing aggression, feather pecking and fear in poultry (Jones et al. 2000). However, the effects of environmental enrichment on the defense system in poultry have not been extensively investigated. Considering that environmental enrichment positively affects stress response in laboratory animals, it can be suggested that it may also positively affect the immune system in poultry. The aim of our study was to investigate the effects of environmental enrichment and transport stress on some lymphoid organs, cellular and humoral immune response and heterophil functions in laying hens. In addition, we examined whether environmental enrichment affects immune system response to stress.

MATERIALS AND METHODS Birds and housing The experimental protocol was approved by the Local Ethic Committee of Istanbul University. A total of 48 the Lohmann Selected Leghorn (LSL, white layer strain) chickens were used. Birds were purchased from a commercial poultry company at the age of 1 day, and placed in the pen (SEN Agricultural Company, Bandirma, Turkey) where the experiment was conducted. The chickens were placed into the cages when they were 3 weeks old. Standard commercial diet and tap water were provided ad libitum throughout the experiment, except the transportation period (no water and food were available during transport). The protein, mineral and energy content of diets were calculated considering the requirements in corresponding stages of development of chickens according to Lohmann Management Guide for Laying Hens. A standard vaccination program was carried out. The environmental conditions (e.g. light, temperature and ventilation) were maintained according to the laying hens breeding standards. Artificial light was provided 13 h/day until the end of the study. All chickens were housed in commercial stainless steel batteries, consisting of wire mesh cages (width = 60 cm, length = 122 cm, height = 45 cm) which were equipped with feeder and nipple drinkers. A total of 1220 cm2 space was provided per chicken, including nest box and sand box area. Animal Science Journal (2016) 87, 284–292

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Environmental enrichment The enriched cages were equipped with a nest box, a sand box and a roost. Colored polypropylene string bunches were hung in the cages for pecking (Jones et al. 2002). The floors of the cages were coated with green artificial turf (Guinebretière et al. 2013). The nest boxes were built of water-resistant plywood with dimensions of 30 cm × 45 cm × 17 cm (width × length × height). An opening with appropriate dimensions to allow a comfortable entrance for chickens was positioned at the rear side of the nest box (close to the backside of the cage). The nest boxes were installed on the left side of the cage and the longest walls of the nest box and cage were perpendicular to each other. The top of the nest box was turned into a sand box using 6 cm height laths mounted on the margins of the roof (Wall et al. 2008a). Sand was used as dust bathing material. A 90 cm-long round wooden stick was installed 20 cm away from the rear wall and 10 cm above the floor of the cage as a perch (Barnett et al. 2009). Approximately 15 cm roosting space was provided per chicken. A sample drawing of the enriched cages is presented (Fig. 1). The sand for dust bathing and the polypropylene string bunches were changed if necessary.

Experimental procedures Chickens were randomly assigned to two groups so that half of the chickens were reared in conventional cages (RCC) and the other half in enriched cages (REC). Both of these groups were replicated four times with six birds per cage. Chickens were subjected to the transport stress when they were 17 weeks old. Four animals from each replicate group were selected randomly to receive transport stress. The animals which were not exposed to transport stress were left in their original cages. Antigenic stimulation was applied to two of the four animals that were transported. Antigenically stimulated animals were marked by attaching a plastic ring to the leg. At the end of transportation, all of the animals were sacrificed except the antigenically stimulated animals. Blood samples of antigenically stimulated hens were collected before and 1 week after transport.

Antigenic stimulation and antibody determination Anticoagulated blood samples collected from sheep were mixed with phosphate-buffered saline (PBS; 1:2 v/v) and centrifuged at 700 × g. Erythrocytes were washed three times using this protocol. Washed pellets of erythrocytes were diluted with PBS and a 7% suspension of sheep red blood cells (SRBC) was obtained; 0.1 mL of the SRBC suspension was intravenously injected into hens. Serum samples were collected 7 days later to determine antibody production against SRBC by microhemagglutination test described by Wegmann and Smithies (1966). The agglutination titer was expressed as the log2 of the highest titer.

Transport Poultry transport crates were used during the transportation of animals (width = 56 cm, length = 95 cm, height = 24 cm). A separate crate was used for each replicate group (total of eight crates). Thus, cage mate birds were transported in the same crate. The hens were transported by a poultry transport truck. The transport procedure, including loading and unloading took 8 h (approximately 1.5 h for loading and unloading, and 6.5 h for transportation). Status of the animals was checked at regular intervals during transportation. Food and water was not © 2015 Japanese Society of Animal Science

286 E. MATUR et al.

Figure 1

A sample drawing of the enriched cage (front view).

supplied to the animals during transportation. Loading and unloading of animals was performed by experienced staff. During transportation, the weather was partly cloudy, and the temperature, humidity and air pressure were recorded as 21°C, 52.5 %, and 30 mb, respectively.

Body and organ weight After the measurement of body weights, chickens were sacrificed by cervical dislocation. Immediately after slaughter, the liver, spleen, thymus and bursa of Fabricius were removed and weighed. Relative organ weights were calculated (organ weight (g) / body weight (BW: g) × 1000). Data were expressed as g/kg of BW.

Total and differential leukocyte count White blood cell (WBC) count was determined using NattHerrick’s solution. A differential leukocyte count was carried out on blood smears stained with May Grunwald-Giemsa stain, and the percentages of heterophil, lymphocyte, eosinophil, monocyte and basophil were determined (Campbell 1995). Heterophil: lymphocyte (H:L) ratio was calculated.

Flow cytometric analysis Heterophil and lymphocyte isolation Lymphocytes and heterophiles were isolated using the methods of Kogut et al. (2002). Briefly, an ethylenediaminetetraacetic acide (EDTA) blood sample was mixed with 2% methylcellulose (Sigma-Aldrich, St. Louis, MO, USA) dissolved in PBS (25 Centipoise) with a 1:1.5 (v/v) ratio. The mixture was centrifuged at 25 × g for 30 min at room temperature. The supernatant liquid and the buffy coat layer were collected with a pipette and transferred into numbered test-tubes and Hanks Balanced Salt Solution was added in a 1:1 (v/v) ratio. The obtained suspension was carefully layered on a Histopaque® (1.077/1.119) gradient (Sigma Chemical Company, St. Louis, MO, USA). The gradient was centrifuged at 250 × g for 60 min. After centrifugation the surface layer of the lower 1.199 band and the upper 1.077 band were carefully collected with a pipette to obtain heterophiles and mononuclear cells, respectively. The collected samples were washed in Roswell Park Memorial Institute (RPMI) 1640 with a 1:1 (v/v) ratio by 10 min centrifugation at 500 × g. The final pellet was resuspended in 1 mL PBS with 1% bovine serum albumin (BSA: Sigma-Aldrich, © 2015 Japanese Society of Animal Science

St Louis, MO, USA). Cell numbers were calculated and adjusted to 1 × 106cell/mL. Cell viability was determined by rhodamine 123 (Robinson et al. 1997). Cell viability was greater than 95%.

Monoclonal antibodies For the determination of CD3+, CD4+ and CD8+ T-cells, chicken-specific monoclonal antibodies were used. Mouse anti-chicken CD3-FITC (clone CT-3), mouse anti-chicken CD4-R-PE (clone CT-4), mouse anti-chicken CD8α-R-PE (clone CT-8) antibodies were used for the determination of CD3+, CD4+ and CD8+ cells, respectively. Besides, fluorescein isothiocyanate (FITC) conjugate mouse IgG1K, (clone 15H6) and R-phycoerythrin (R-PE) conjugate mouse IgG1K, (clone 15H6) were used as isotopic controls to determine the nonspecific bindings. All monoclonal antibodies were purchased from Southern Biotechnology Associates Inc. (Birmingham, AL, USA).

Lymphocyte staining and flow cytometry Monoclonal antibodies were titrated for the determination of the appropriate amount before using. One undred microliters of lymphocyte suspension (1 × 106 cell/mL) were pipetted into the numbered test-tubes (two test-tubes for each sample). One microliter of CD3 FITC (0.5 μg/106 cells) and 1 μL CD4 R-PE (0.1 μg/106 cells) were added for the staining of CD4+ cells. One microliter of CD3 FITC (0.5 μg/106 cells) and 1 μL CD8 R-PE (0.1 μg/106 cells) were added for the staining of CD8+ cells. After 30-min incubation at 4°C in the dark, cells were washed with 2 mL PBS by centrifugation at 300 × g. Subsequently, the supernatant was removed and cell pellets were resuspended with 500 μL 1/10 diluted CellFIX (BD Bioscienses, San Jose, CA, USA). Flow cytometric analyses were performed in 24 h on a FACScalibur™ flow cytometry equipped with Cellquest software (BD Bioscienses, San Jose, CA, USA). A total of 20 000 events were counted.

Heterophil function tests Heterophil functions were evaluated by measuring the density of the fluorescence emission, caused by the transformation of dihydrorhodamine-123 (DHR-123) to rhodamine-123 by activated heterophiles (Bilgic et al. 2007). Three different stimulants, Escherichia coli (109/mL) (stimulation of phagocytic activity), porbol myristate acetate (PMA) (stimulation of chemotaxis) and n-formyl-methionine-leucine-phenylalanine Animal Science Journal (2016) 87, 284–292


(stimulation of oxidative burst), were used to activate the heterophiles. Three test-tubes were prepared for each sample and 1 mL PBS, 20 μL heterophil suspensions and 10 μL DHR-123 were added to each tube, respectively. After 5-min incubation of the prepared heterophiles in a water bath at 38°C, stimulants were pipetted into the test-tubes. Immediately after the pipetting of stimulants (0 min), mean fluorescent intensity was measured using the FL1 detector of the flow cytometer. After the first measurement the heterophils were incubated for 20 min in the water bath and afterwards, the mean fluorescent intensity was measured again. The heterophil functions were calculated using the ratio of the results of these two measurements (Bilgic et al. 2007).

Statistical analysis The experiment was a completely randomized design with treatments factorially arranged. The statistical model included the main effects of environmental enrichment (environmental enrichment exists or not), transport stress (exists or not) and the interactions. Effects of treatments were analyzed as a 2 × 2 factorial arrangement by two-way analysis of variance (ANOVA). Tukey’s Hohestly Significant Difference test was used for multiple comparisons when a significant interaction was detected. Independent sample Student’s t-test was used to compare the result between RCC and REC hens for antibody production. All statements of significance were based on P < 0.05 and tendencies were indicated if the P-value was between 0.05 and 0.10. All statistical analyses of obtained data were performed using the SPSS software package (ver. 11.5.2.1; SPSS Inc., Chicago, IL, USA).

RESULTS The main effect of environmental enrichment did not affect body (P = 0.100), liver (P = 0.326), spleen (P = 0.599), thymus (P = 0.923) and bursa of Fabricius (P = 0.536) weights. Transport did not affect body (P = 0.222) and thymus (P = 0.524) weights. On the other hand, liver weight (P = 0.001) decreased, spleen (P = 0.001) and bursa of Fabricius (P = 0.008) weights increased due to transport. There was no significant interaction between environmental enrichment and transport on body and organ weights (Table 1). Table 1

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The main effect of environmental enrichment did not affect WBC (P = 0.241), heterophil (P = 0.976), lymphocyte (P = 0.490), eosinophil (P = 0.309), basophil (P = 0.268) percentages and the heterophil: lymphocyte (H:L) ratio (P = 0.407). On the other hand, monocyte percentage tended to increase in enriched hens (P = 0.059). The main effect of transport on WBC count was significant (P = 0.001). WBC count increased with transport. There was a significant interaction between environmental enrichment and transport on the H:L ratio, and heterophil and lymphocyte percentages (P = 0.001). The effect of transport on H:L ratio, heterophil and lymphocyte percentages was not significant in RCC chickens; however, heterophil percentage and H:L ratio increased, but lymphocyte percentage decreased in REC chickens exposed to transport. The effect of treatment was not significant on the monocyte, eosinophil and basophil percentages (Table 2). The interaction between environmental enrichment and transport was significant on chemotaxic activity and oxidative burst (P = 0.003 and P = 0.011, respectively). Transport increased chemotaxic activity and oxidative burst both in REC and RCC hens, but the increase was higher in REC hens than in RCC hens. In addition, the increase in phagocytic activity of heterophils tended to be higher (P = 0.082) in REC hens compared to that of RCC hens (Table 3). The main effect of environmental enrichment was that CD8+ cell proportion tended to increase (P = 0.089) in REC hens. In addition, CD4+ cell proportion was numerically higher in REC hens than in RCC hens. The main effect of transport on CD4+ and CD8+ cells was significant. The proportion of these cells in peripheral blood increased due to transport (P = 0.001). On the other hand, CD4:CD8 cell ratio was not affected by either treatment (Table 3). Although there was no significant difference between RCC and REC hens considering antibody levels prior to antigenic stimulation, SRBC-induced antibody production was higher in REC hens than in RCC hens (P < 0.05) (Fig. 2).

Effects of environmental enrichment and transport stress on body weight and some lymphoid organ weights†

RCC hens‡ REC hens¶

Environmental enrichment

Transport stress

Body weight (BW) (g)

Liver (g/kg BW)

Spleen (g/kg BW)

Thymus (g/kg BW)

Bursa of Fabricius (g/kg BW)

+ +

+ + SEM

1318.7 1317.7 1325.8 1264.3 18.8

33.5 18.6 31.3 18.5 1.14 P-values 0.362 0.001 0.326

1.21 1.67 1.32 1.67 0.09

1.54 1.88 1.71 1.76 0.30

1.06 1.70 0.91 1.57 0.23

0.599 0.001 0.547

0.923 0.524 0.645

0.536 0.008 0.955

ANOVA Environmental enrichment§ Transport stress* E. enrichment × Transport**

0.100 0.222 0.111

†Relative organ weight = organ weight (g) / body weight (g) × 1000. ‡RCC hens: reared in conventional cages. ¶REC hens: reared in enriched cages. §Environmental enrichment: main effect of environmental enrichment in rearing period. *Transport stress: main effect of transport stress. **E. enrichment × Transport: interaction between environmental enrichment and transport stress. SEM: standard error of the mean, (n: 8).


© 2015 Japanese Society of Animal Science

288 E. MATUR et al. Table 2

Effects of environmental enrichment and transport stress on total and differential leukocyte count

RCC hens† REC hens‡


Transport stress

WBC (×103/μL)

Heterophil (%)

Lymphocyte (%)

Monocyte (%)

Eosinophil (%)

Basophil (%)

H:L Ratio

+ +

+ + SEM

27.0 34.1 26.8 28.6 1.06

12.8b 17.6b 4.0c 26.3a 2.01

83.5b 79.0b 91.4a 68.2c 1.93 P values 0.490 0.001 0.001

0.62 0.75 2.29 1.87 0.69

2.0 2.3 0.7 2.5 0.5

1.00 0.25 1.57 1.00 0.34

0.154bc 0.229b 0.046c 0.403a 0.038

0.059 0.841 0.707

0.309 0.164 0.218

0.268 0.168 0.799

0.407 0.001 0.001

ANOVA Environmental enrichment¶ Transport stress§ E. enrichment × Transport*

0.241 0.001 0.314

0.976 0.001 0.001

†RCC hens: reared in conventional cages. ‡REC hens: reared in enriched cages. ¶Environmental enrichment: main effect of environmental enrichment in reared period. §Transport stress: main effect of transport stress. *E. enrichment × Transport: interaction between environmental enrichment and transport stress. SEM: standard error of the mean, (n: 8), a , b , c: Means within a column with no common superscript differ significantly.

Table 3

Effects of environmental enrichment and transport stress on CD4+, CD8+ cell proportions and heterophil functions

RCC Hens† REC Hens‡


Transport stress

Phagocytic activity

Chemotaxic activity

Oxidative burst

+ +

+ + SEM

1.78 2.71 1.60 3.48 0.26

1.17c 3.89b 1.48c 5.92a 0.26 P values 0.001 0.001 0.003

1.37c 2.51b 1.71cb 3.77a 0.29

ANOVA Environmental enrichment¶ Transport stress§ E. enrichment × Transport*

0.283 0.001 0.082

0.125 0.001 0.011

CD4+ cell (%) 3.30 28.40 6.63 31.10 3.4 0.391 0.001 0.928

CD8+ cell (%) 1.19 10.23 2.75 12.82 1.1 0.089 0.001 0.664

CD4:CD8 ratio 3.26 3.21 2.63 2.56 0.6 0.347 0.936 0.990

†RCC hens: reared in conventional cages. ‡REC hens: reared in enriched cages. ¶Environmental enrichment: main effect of environmental enrichment in reared period. §Transport stress: main effect of transport stress. *E. enrichment × Transport: interaction between environmental enrichment and transport stress. SEM: standard error mean, (n: 8), a , b , c: Means within a column with no common superscript differ significantly.

Antibody Level (Log2)

6,000 a

5,000

b

4,000 3,000 c

2,000

c

1,000 0 REC Hens, before SRBC injection

RCC Hens, before SRBC injection

REC Hens, 1 week RCC Hens, 1 week after SRBC injection after SRBC injection

Figure 2 The effects of environmental enrichment on sheep red blood cell (SRBC)-induced antibody production in laying hens. (Blood samples were collected before and 1 week after SRBC injection).

DISCUSSION The effects of environmental enrichment and transport stress on the immune system and on immune systemrelated organs were investigated in laying hens. Our results clearly showed that environmental enrichment did not affect body and organ weights, but transport stress had an impact on liver, spleen and bursa of © 2015 Japanese Society of Animal Science

Fabricius weights. It has been previously reported that various stressors change weights of lymphoid organs (Puvadolpirod & Thaxton 2000; Post et al. 2003; Vahdatpour et al. 2009; Quinteiro-Filho et al. 2010). Therefore, the effects of transport stress on the abovementioned organs could be expected. However, whether or not environmental enrichment affects lymphoid organs has not been thoroughly studied. Similar to our results, Huff et al. (2003) reported that environmental enrichment applied to turkeys did not have any positive or negative effects on organs. In contrast, Altan et al. (2013) reported that bursa of Fabricius weight increased in environmentally enriched broiler chicks. Tsai et al. (2002) suggested that the results regarding the effects of environmental enrichment on lymphoid organs are inconsistent in mammals, such as in poultry. The variation in the results may be due to the type or duration of environmental enrichment. In the present study, we specifically investigated whether environmental enrichment affects the response of the immune system to stress. We observed that liver weight decreased, spleen and bursa of Fabricius weights increased not only in RCC but also in REC hens exposed to transport stress. Therefore, our results revealed that environmental Animal Science Journal (2016) 87, 284–292


enrichment applied during the rearing period did not change the effect of stress on immune system-related organs. In the present study, while transport stress increased WBC count, environmental enrichment increased monocyte percentages. Similarly, WBC count was found to increase in turkeys exposed to transport stress (Huff et al. 2005). The increase in the total WBC count may be a result of the transition of leukocytes from the marginal pool to peripheral circulation during stress. Supporting this, it has been reported that leukocytes in the marginal pool mobilize to peripheral circulation during stress in mammals (Duncan & Prasse 1987). It has been reported that physical exercise decreases migration of monocytes from blood vessels to tissues by inhibiting production of serum chemoacttractant protein-1 (SCP-1), and therefore, monocytes accumulate in blood vessels (Singhal et al. 2014). It is known that physical activity increases due to behavioral stimulation in environmentally enriched animals (Ventura et al. 2012). Therefore, the increase of peripheral blood monocyte percentages in REC hens may be due to a possible decrease in SCP-1 secretion. The REC hens that were not exposed to transport stress had a lower heterophil percentage and a higher lymphocyte percentage. All this indicates that environmental enrichment applied in the current study affected leukocytes except monocytes. Nazar and Marin (2011a) found similar results regarding heterophiles and lymphocytes in Japanese quails exposed to environmental enrichment. Thus, results obtained in the current study can be expected. Furthermore, stress response of REC hens was different from those of RCC hens. It had been previously reported that stress response of Japanese quails reared in enriched cages were similar to our report considering leukocyte percentages Nazar and Marin (2011b). The relevant cause of this pattern cannot be completely clarified. However, it has been suggested that birds reared in enriched cages have more interaction with environmental stimuli and therefore, may show a more efficient stress responses (Nazar & Marin 2011a). Stress response of REC hens in our study may be related to this argument. Heterophil functions were examined to reveal the effects of the applications on the non-specific immune system. Our results show that heterophil functions increased in both groups due to transport stress. However, the increase in heterophil functions was more pronounced in REC hens exposed to stress. There may be more than one explanation for this result. In the present study, CD4+ and CD8+ proportions are a little higher in REC hens exposed to transport stress. It has been reported that interleukin 2 (IL-2) expressed from CD4+ and CD8+ cells (Hwang et al. 2005) was effective on the stimulation of phagocytic activity of heterophils (Kogut et al. 2002). Therefore, the increase in CD4+ and CD8+ cells during transportation in the present study may be one of the reasons for an increase in Animal Science Journal (2016) 87, 284–292

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heterophil functions. Moreover, higher exposure to bacterial contamination in enriched hens (Wall et al. 2008b) may be another significant factor, because microorganisms were reported to lead to an increase in phagocytic activity (Harmon 1998) and oxidative burst (Farnell et al. 2003). Another reason for an increase in heterophil functions in REC hens may be related to cytokines produced by CD4+ and CD8+ cells. As part of the inflammatory response, heterophils are activated by cytokines to increase their phagocytic and bactericidal activity (Andreasen et al. 1991; Kogut et al. 2002). However, further studies are needed to clarify exactly which is influential among the above-mentioned mechanisms. Chemotaxic activity has a critical significance on leukocyte migration toward the site of inflammation (Redwine et al. 2003) and phagocytic activity and oxidative burst are effective mechanisms with which heterophils kill bacteria (Farnell et al. 2006). Considering that chickens may have been exposed to various pathogens during transport (Zucker & Krüger 1998), an increase in heterophil functions would be favorable to birds. In the present study, T-cell (CD4+ and CD8+ cells) proportion in peripheral blood was measured to determine effects of the treatments on the specific immune system. Transport stress significantly increased the cells under investigation. Similarly, an increase in CD4+ and CD8+ cells has also been reported in poultry (Han et al. 2010), mice (Satoh et al. 2006), pigs (Qiongxia et al. 2009) and humans (Atanackovic et al. 2013) exposed to stress. Therefore, an increase in these cells due to transport stress was expected. It has been suggested that environmental enrichment did not change (Olsson et al. 2010), improved (Gurfein et al. 2014), or suppressed (Hutchinson et al. 2005) CD4+ and CD8+ cell proportions in lymphoid tissues of rodents. It has also been reported that environmental enrichment effectively improved the immune system in laying hens (Moe et al. 2010) and Japanese quails (Nazar & Marin 2011b), but it has not been examined whether enrichment affected CD4+ and CD8+ cells in these studies. Therefore, the increase in CD8+ cells due to environmental enrichment can be considered as a novel and favorable result in our study. These cells play a prominent role in the adaptive immune system. They help and regulate the activity of other immune cells by releasing cytokines such as IL-2, interferon-.gamma, tumor necrosis factorα and granulocyte-macrophage colony-stimulating factor (Mosmann & Sad 1996). They are also essential in antibody production from B-cells and phagocytic activity of macrophages. In the present study, SRBC-induced antibody production has been examined to determine whether humoral immune response changes due to transport stress applied to hens housed in conventional or enriched cages. Antibody production was higher in REC hens than in RCC hens. It had been suggested that rearing conditions significantly affect antibody production in © 2015 Japanese Society of Animal Science

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SRBC-immunized laying hens (Moe et al. 2010). Similar to our results, it was previously reported that environmental enrichment increased antibody production in laying hens (El-Lethey et al. 2000). In addition, it has been demonstrated that anti-idiotype antibody levels increased in idiotype-vaccine immunized mice after environmental enrichment (Benaroya-Milshtein et al. 2007). Increased antibody production due to environmental enrichment might be related to increased CD4+ cells. It is known that CD4+ cells are key players as T-helper cells in antibody production in poultry (Gore & Qureshi 1997). CD4+ cells stimulate the antigen-specific antibody production of B-cells by secreting various cytokines (Arstila et al. 1994). However, our results indicate only a numerical increase in CD4+ cell count in hens kept in enriched cages. Whatever the underlying mechanism, increased antibody production of B-cells can be seen as a favorable effect of environmental enrichment on the immune system, since the antibody-mediated immune system is a prominent part of the immune system due to certain features, such as agglutination or precipitation of the antigen, activation of the classical complement pathway, and because it forms a bridge between killer cells and the target cells (Abbas et al. 2000).

Conclusion Differential leukocyte count is usually used as an indicator of stress in poultry. In the present study, stress response of RCC hens was different from those of REC hens considering the differential leukocyte count. Because hens reared in enriched cages were more exposed to various environmental stimuli, they more effectively responded to stressors. The effects of transport stress and environmental enrichment on lymphoid organs and the immune system have also been investigated in the present study. In conclusion, although the applied environmental enrichment could not neutralize the effects of stress on lymphoid organs, it did not have a negative effect. Furthermore, environmental enrichment activated the non-specific immune system, cellular and humoral branches of the specific immune system by increasing heterophil functions, CD8+ cells and antibody production, respectively. Stimulation of the immune system by environmental enrichment in hens under stress would increase the resistance to pathogens they may be exposed to during stress. Therefore, we conclude that environmental enrichment used for improving animal welfare may also be beneficial to improve immune systems of birds exposed to stress.

ACKNOWLEDGMENTS We thank Pinar Ertor Akyazi, Nalan Sezer, Mukaddes Ozcan, Murat Arslan, Turgay Çakmak, Dilara Yilmaz, Ezgi Vatansever and Selin Arli for their assistance, and we also would like to thank SEN Agriculture Company © 2015 Japanese Society of Animal Science

for their contributions to this research. This work was supported by Research Fund of the Istanbul University (Project number: BAP 3096).

REFERENCES Abbas AK, Lichtman AH, Pober JS. 2000. Cellular and Molecular Immunology, 4th edn. WB Saunders, Philadelphia, PA. Altan O, Seremet C, Bayraktar H. 2013. The effects of early environmental enrichment on performance, fear and physiological responses to acute stress of broiler. Archiv für Geflügelkunde 77, 23–28. Andreasen CB, Latimer KS, Harmon BG, Glisson JR, Golden JM, Brown J. 1991. Heterophil function in healthy chickens and in chickens with experimentally induced staphylococcal tenosynovitis. Veterinary Pathology 28, 419–427. Arranz L, De Castro NM, Baeza I, Maté I, Viveros MP, De la Fuente M. 2010. Environmental enrichment improves agerelated immune system impairment: long-term exposure since adulthood increases life span in mice. Rejuvenation Research 13, 415–428. Arstila TP, Vainio O, Lassila O. 1994. Central role of CD4+ T cells in avian immune response. Poultry Science 73, 1019–1026. Atanackovic D, Nowottne U, Freier E, Weber CS, Meyer S, Bartels K, et al. 2013. Acute psychological stress increases peripheral blood CD3 + CD56+ natural killer T cells in healthy men: possible implications for the development and treatment of allergic and autoimmune disorders. Stress 16, 421–428. Barnett JL, Tauson R, Downing JA, Janardhana V, Lowenthal JW, Butler KL, Cronin GM. 2009. The effects of a perch, dust bath, and nest box, either alone or in combination as used in furnished cages, on the welfare of laying hens. Poultry Science 88, 456–470. Belz EE, Kennell JS, Czambel RK, Rubin RT, Rhodes ME. 2003. Environmental enrichment lowers stress-responsive hormones in singly housed male and female rats. Pharmacology Biochemistry and Behavior 76, 481–486. Benaroya-Milshtein N, Apter A, Yaniv I, Kukulansky T, Raz N, Haberman Y, et al. 2007. Environmental enrichment augments the efficacy of idiotype vaccination for B-cell lymphoma. Journal of Immunotherapy 30, 517–522. Benaroya-Milshtein N, Hollander N, Apter A, Kukulansky T, Raz N, Wilf A, et al. 2004. Environmental enrichment in mice decreases anxiety attenuates stress responses and enhances natural killer cell activity. European Journal of Neuroscience 20, 1341–1347. Bilgic S, Aktas E, Salman F, Ersahin G, Erten G, Yilmaz MT, Deniz G. 2007. Intracytoplasmic cytokine levels and neutrophil functions in early clinical stage of type-I diabetes. Diabetes Research and Clinical Practice 7, 931–936. Campbell TW. 1995. Avian Hematology and Cytology, 2nd edn. Iowa State University Press, Ames, Iowa. Cheng HW, Singleton P, Muir WM. 2002. Social stress in laying hens: Differential dopamine and corticosterone responses after intermingling different genetic strains of chickens. Poultry Science 81, 1265–1272. Duncan JR, Prasse KW. 1987. Veterinary Laboratory Medicine Clinical Pathology, 2nd edn. Iowa State University Press, Ames, Iowa. El-Lethey H, Aerni V, Jungi TW, Wechsler B. 2000. Stress and feather pecking in laying hens in relation to housing conditions. British Poultry Science 41, 22–28. Farnell MB, Crippen TL, He H, Swaggerty CL, Kogut MH. 2003. Oxidative burst mediated by toll like receptors (TLR) and Animal Science Journal (2016) 87, 284–292


CD14 on avian heterophils stimulated with bacterial toll agonists. Developmental and Comparative Immunology 27, 423–429. Farnell MB, Donoghue AM, De Los Santos FS, Blore PJ, Hargis BM, Tellez G, Donoghue DJ. 2006. Upregulation of oxidative burst and degranulation in chicken heterophils stimulated with probiotic bacteria. Poultry Science 85, 1900–1906. Gore AB, Qureshi MA. 1997. Enhancement of humoral and cellular immunity by vitamin E after embryonic exposure. Poultry Science 76, 984–991. Guinebretière M, Huneau-Salaün A, Huonnic D, Michel V. 2013. Plumage condition, body weight, mortality, and zootechnical performances: the effects of linings and litter provision in furnished cages for laying hens. Poultry Science 92, 51–59. Gurfein BT, Davidenko O, Premenko-Lanier M, Milush JM, Acree M, Dallman MF, et al. 2014. Environmental enrichment alters splenic immune cell composition and enhances secondary influenza vaccineresponses in mice. Molecular Medicine 22, 179–190. Han AY, Zhang MH, Zuo XL, Zheng SS, Zhao CF, Feng JH, Cheng C. 2010. Effect of acute heat stress on calcium concentration, proliferation, cell cycle, and interleukin-2 production in splenic lymphocytes from broiler chickens. Poultry Science 89, 2063–2070. Harmon BG. 1998. Avian heterophils in inflammation and disease resistance. Poultry Science 77, 972–977. Hocking PM, Jones EKM. 2006. On-farm assessment of environmental enrichment for broiler breeders. British Poultry Science 47, 418–425. Huff GR, Huff WE, Balog JM, Rath NC. 2003. The effects of behavior and environmental enrichment on disease resistance of turkeys. Brain, Behavior, and Immunity 17, 339–349. Huff GR, Huff WE, Balog JM, Rath NC, Anthony NB, Nestor KE. 2005. Stress response differences and disease susceptibility reflected by heterophil to lymphocyte ratio in turkeys selected for increased body weight. Poultry Science 84, 709–717. Huff GR, Huff WE, Rath NC, Donoghue A, Anthony N, Nestor K. 2007. Differential effects of sex and genetics on behavior and stress response of turkeys. Poultry Science 86, 1294–1303. Hutchinson E, Avery A, Vandewoude S. 2005. Environmental enrichment for laboratory rodent. Institute for Laboratory Animal Research Journal 46, 148–161. Hwang ES, Hong JH, Glimcher LH. 2005. IL-2 production in developing Th1 cells is regulated by heterodimerization of RelA and T-bet and requires T-bet serine residue 508. Journal of Experimental Medicine 7, 1289–1300. Jones RB, Carmichael NL, Rayner E. 2000. Pecking preferences and pre-dispositions in domestic chicks: implications for the development of environmental enrichment devices. Applied Animal Behaviour Science 25, 291–312. Jones RB, Mcadie TM, Mccorquodale C, Keeling LJ. 2002. Pecking at other birds and at string enrichment devices by adult laying hens. British Poultry Science 43, 337–343. Kingston SG, Hoffman-Goetz L. 1996. Effect of environmental enrichment and housing density on immune system reactivity to acute exercise stress. Physiology and Behavior 60, 145–150. Kogut M, Rothwell L, Kaiser P. 2002. Differential effects of age on chicken heterophil functional activation by recombinant chicken interleukin-2. Developmental and Comparative Immunology 26, 817–830. Meijer MK, Sommer R, Spruijt BM, van Zutphen LF, Baumans V. 2007. Influence of environmental enrichment and


291

handling on the acute stress response in individually housed mice. Laboratory Animals 41, 161–173. Mitchell MA, Kettlewell PJ. 2004. Transport of chicken, pullets and spent hens. In: Perry GC (ed.), Welfare of the laying hen (poultry science symposium series), pp. 361–373. CABI Publishing, Massachusetts, USA. Moe RO, Guémené D, Bakken M, Larsen HJ, Shini S, Lervik S, et al. 2010. Effects of housing conditions during the rearing and laying period on adrenal reactivity, immune response heterophils to lymphocyte (H/L) ratios in laying hens. Animal 4, 1709–1715. Mosmann TR, Sad S. 1996. The expanding universe of T-cell subsets: Th1, Th2 and more. Immunology Today 17, 138–146. Nazar FN, Marin RH. 2011a. Chronic stress and environmental enrichment as opposite factors affecting the immune response in Japanese quail (Coturnix coturnix japonica). Stress 14, 166–173. Nazar FN, Marin RH. 2011b. Effect of stress and early environmental on cellular immunity of juvenile Japanese quail. Revista Argentina de Production Animal 31, 63–69. Nicol CJ, Scott GB. 1990. Pre-slaughter handling and transport of broiler chickens. Applied Animal Behaviour Science 28, 57–73. Olsson IA, Costa A, Nobrega C, Roque S, Correia-Neves M. 2010. Environmental enrichment doesnot compromise the immune response in mice chronically infected with mycobacterium avium. Scandinavian Journal of Immunology 71, 249–257. Post J, Rebel JM, Ter Huurne AA. 2003. Physiological effects of elevated plasma corticosterone concentrations in broiler chickens. An alternative means by which to assess the physiological effects of stress. Poultry Science 82, 1313–1318. Puvadolpirod S, Thaxton JP. 2000. Model of physiological stress in chickens 1. Response parameters. Poultry Science 79, 363–369. Qiongxia L, Shuxia Z, Ruqian Z. 2009. Effects of transportation stress on immune function and regulation of cytokines in pigs. Scientia Agricultura Sinica 42, 2579–2585. Quinteiro-Filho WM, Ribeiro A, Ferraz-de-Paula V, Pinheiro ML, Sakai M, Sá LR, et al. 2010. Heat stress impairs performance parameters, induces intestinal injury, and decreases macrophage activity in broiler chickens. Poultry Science 89, 1905–1914. Redwine L, Snow S, Mills P, Irwin M. 2003. Acute psychological stress: effects on chemotaxis and cellular adhesion molecule expression. Psychosomatic Medicine 65, 598–603. Reynolds S, Lane SJ, Richards L. 2010. Using animal models of enriched environments to inform research on sensory integration intervention for the rehabilitation of neurodevelopmental disorders. Journal of Neurodevelopmental Disorders 2, 120–132. Robinson P, Darzynkiewicz Z, Dean PN, Dressler LG, Rabinovitch PS, Stewart CC, et al. 1997. Assessment of cell viability, unit 9,2. In: Current Protocols in Cytometry. John Wiley & Sons, Inc., New York. Rosales AG. 1994. Managing stress in broiler breeders: a review. The Journal of Applied Poultry Research 3, 199–207. Satoh E, Edamatsu H, Omata Y. 2006. Acute restraint stress enhances calcium mobilization and proliferative response in splenic lymphocytes from mice. Stress 9, 223–230. Shini S, Kaiser P, Shini A, Bryden WL. 2008. Biological response of chickens (Gallus gallus domesticus) induced by corticosterone and a bacterial endotoxin. Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology 149, 324–333.


292 E. MATUR et al.

Singhal G, Jaehne EJ, Corrigan F, Baune BT. 2014. Cellular and molecular mechanisms of immunomodulation in the brain through environmental enrichment. Frontiers in Cellular Neuroscience. doi:10.3389/fncel.2014.00097 Tsai PP, Pachowsky U, Stelzer HD, Hackbarth H. 2002. Impact of environmental enrichment in mice. 1: Effect of housing conditions on body weight, organ weights and haematology in different strains. Laboratory Animals 36, 411–419. Vahdatpour T, Adi K, Nezhad Y, Sis N, Riyazi S, Vahdatpour S. 2009. Effects of corticosterone intake as stress-alternative hormone on broiler chickens: Performance and blood parameters. Journal of Animal and Veterinary Advances 4, 16–21. Ventura BA, Siewerdt F, Estevez I. 2012. Access to barrier perches improves behavior repertoire in broilers. PLoS One 7 (1), e29826, doi:10.1371/journal.pone.0029826 Wall H, Tauson R, Elwinger K. 2008a. Effects of litter substrate and genotype on layers’ use of litter, exterior appearance, and heterophil:lymphocyte ratios in furnished cages. Poultry Science 87, 2458–2465. Wall H, Tauson R, Sørgjerd S. 2008b. Bacterial contamination of eggshells in furnished and conventional cages. The Journal of Applied Poultry Research 1, 11–16.


Wang YW, Field CJ, Sim JS. 2000. Dietary polyunsaturated fatty acids alter lymphocyte subset proportion and proliferation, serum immunoglobulin concentration, and immune tissue development in chicks. Poultry Science 79, 1741–1748. Weedman SM, Rostagno MH, Patterson JA, Yoon I, Fitzner G, Eicher SD. 2011. Animal production-health and well-being yeast culture supplement during nursing and transport affects immunity and intestinal microbial ecology of weanling pigs. Journal of Animal Science 89, 1908–1921. Wegmann TG, Smithies OA. 1966. Simple hemagglutination system requiring small amounts of red cells and antibodies. Transfusion 6, 67–73. Würbel H. 2001. Ideal homes? Housing effects on rodent brain and behaviour. Trends in Neurosciences 24, 207–211. Zucker BA, Krüger M. 1998. Effect of transport stress on the content of endotoxin in blood of slaughter pigs. Berliner und Münchener Tierärztliche Wochenschrift 111, 208–210. Zulkifli I, Abdullah N, Azrin NM, Ho YW. 2000. Growth performance and immune response of two commercial broiler strains fed diets containing Lactobacillus cultures and oxytetracycline under heat stress conditions. British Poultry Science 41, 593–597.
