Utilizing original XPC™ in feed to reduce stress susceptibility of broilers

Utilizing original XPC™ in feed to reduce stress susceptibility of broilers Abstract Reduction of stress is an important factor in improving poultry welfare, especially during periods of heat stress. A study was conducted to evaluate the effects of feeding the functional metabolites of Diamond V Original XPCTM to broilers reared under ambient or heat stress temperatures. Dietary treatments included: control feed (CON) and Original XPC fed continuously at 1.25 kg/MT (XPC). Half the birds in each dietary treatment were subjected to either no heat stress (24°C constant) or heat stress (35°C:24°C for 18:6 h daily) from 28 to 42 d. At the end of the heat stress period, blood was collected from 40 birds/treatment. Blood was analyzed for plasma corticosterone (CORT), plasma HSP70 (HSP70), and heterophil/lymphocyte ratios (H/L). At 42 d, bilateral metatarsal traits were also measured in 40 birds/treatment to assess physical asymmetry. Birds fed XPC had significantly lower CORT levels than CON (P < 0.001; 5,129 ± 617 vs. 8,433 ± 730, respectively). Physical asymmetry scores were also significantly higher in CON compared to XPC fed broilers (P < 0.001; 1.50 ± 0.13 vs. 0.54 ± 0.05, respectively). H/L ratios were significantly greater in CON than for XPC birds (P = 0.01; 0.81 ± 0.05 mm vs. 0.62 ± 0.05 mm, respectively). No differences were observed between CON and XPC fed broilers in HSP70. However, heat stress did increase (P < 0.0001) HSP70 compared to no heat stress birds (5.65 ± 0.12 vs. 4.78 ± 0.11 pg/mL, for heat stress and no heat stress, respectively). Feeding XPC to broiler chickens improved animal welfare via reduced stress indicators while under heat stress or no heat stress conditions. These results indicate that feeding XPC may improve poultry welfare by reducing heat stress susceptibility. INTRODUCTION As the broiler industry strives to provide the most efficient and highest-quality meat products to consumer markets, special attention has been given to stocking density. The average floor space in North America for commercial broilers is around 0.07 m2/bird (NCC, 2015). While commercial production environments are designed to provide adequate air flow, humidity, access to feed/water, and constant temperature of around 26°C (Aviagen, 2014), episodic conditions may still be considered as stressful to birds. The modern consumer has become increasingly conscious of the perceived plight of food production animals in confinement and a market category has begun to focus on even more humane treatment of these animals. There is an opportunity to focus on food and water ingestion as a key area for controlling stress. One goal in producing food animals is to increase their average daily gain (ADG) to meet market demands, making ingestion of quality feed paramount. Selection for fast-growing, high-yielding broilers has been shown to have detrimental effects on the ability of a bird to cope with heat stress (Berrong and Washburn, 1998; Tan et al., 2010; Soleimani et al., 2011). Syafwan et al. (2011) showed that heat stress in commercial broiler strains can lead to cellular oxidative stress, which increases susceptibility to infectious diseases. However, the fact that broilers ingest a large amount of feed and water allows for the addition of stress-reducing agents to that feed and water. Previous studies have shown that functional metabolites of Diamond V Original XPCTM (DiamondV Mills, Cedar Rapids, IA) can help balance the immune response and stress hormone levels in production poultry (Firman et al., 2013). Gao et al. (2008, 2009) showed increased secretory IgA and intestinal IgM in birds fed Original XPC, and Al-Mansour et al. (2011) showed that Original XPC significantly decreased heterophil/lymphocyte (H/L) ratios in broilers. Original XPC has also shown to have a positive impact on weight gain, feed conversion, and mortality in broilers (Gao et al., 2008, 2009), even in broilers that were challenged with ingestion of used litter during a heat-stress period (Teeter, 1993). A new study was undertaken to measure the effects of stress on broilers fed Original XPC during long-term heat stress and non-heat stress. It is hypothesized that supplementing broiler feed with Original XPC will decrease the stress susceptibility of the broilers subjected to heat stress or routine management stressors during rearing. MATERIALS AND METHODS Animals and Husbandry A total of 320 Ross 708 broilers were used in this experiment. Birds were equally housed at 20 birds per pen across a total of 16 pens (0.91 m × 2.74 m). Birds were randomly assigned to each pen; however, initial pen weights were equalized. Each pen was lined with clean pine shavings and constructed of solid black plastic on all but the front side, which was made of mesh wire. Each pen was equipped with one bell feeder and nipple drinking system. A 2 × 2 factorial design was used in this experiment in which pens were assigned at random to one of 2 dietary treatments: control diet (CON) or control diet with 1.25 kg/MT of Original XPC (DiamondV Mills, Cedar Rapids, IA) added (XPC) and one of 22 environmental treatments (heat stress or non-heat stress). Table 1 describes the experimental design. Half of the birds were subjected to 2 wk of heat stress (35°C for at least 18 h daily and the remaining 6 h at 24°C to allow for feeding) between 28 and 42 d of age. Dietary treatments were equally represented between rooms that were environmentally controlled. Each room was equipped with fluorescent lighting, oscillating fans for additional airflow, and a single-pass central ventilation system equipped with high-efficiency particulate air (HEPA) filters per U.S. Department of Agriculture (USDA) Biosecurity Level 2 protocols. Birds were fed a 3-phase diet consisting of a starter (D 0–14, crumble), grower (d 14 to 28, pellet), and finisher (d 28 to 42, pellet) presented in Table 2. Birds were allowed ad libitum access to feed and water. A photoperiod of 24L:0D was maintained to 10 d of age and then reduced to 20L:4D for the remainder of the study. The birds were managed according to the guidelines set forth in the Guide for the Care and Use of Agricultural Animals in Research and Teaching (FASS, 2010) and all procedures were approved by the USDA-ARS animal care committee (ACUC #2,015,005). Table 1. Experimental design for commercial broilers fed a diet with and without Diamond V Original XPCTM and exposed to either a non-heat stress or a heat stress environment.   Treatment  Variable  Control (CON)  Original XPC (XPC)1  Product Inclusion Rate  N/A  1.25 kg/MT  Heat Stress Pens  4  4  Non-Heat Stress Pens  4  4  Total Number of Birds  160  160    Treatment  Variable  Control (CON)  Original XPC (XPC)1  Product Inclusion Rate  N/A  1.25 kg/MT  Heat Stress Pens  4  4  Non-Heat Stress Pens  4  4  Total Number of Birds  160  160  1Diamond V Original XPCTM, Cedar Rapids, IA, USA View Large Table 2. Experimental diets and calculated nutrient content for the starter, grower, and finisher phases. Nutrient Name  Units  Starter  Grower  Finisher  Corn  %  58.40  63.10  68.95  Soybean  %  34.40  29.85  24.10  DL-methionine  %  0.32  0.28  0.24  Lysine  %  0.20  0.21  0.20  L-threonine  %  0.07  0.08  0.08  Animal fat  %  2.85  2.85  3.00  Limestone  %  1.44  1.36  1.25  Biophosphate  %  1.56  1.45  1.30  Salt  %  0.44  0.37  0.24  Sodium bicarbonate  %  0.00  0.12  0.32  Trace minerals  %  0.05  0.05  0.05  Trace vitamins  %  0.25  0.25  0.25  Salinomycin  %  0.05  0.05  0.05  Dry matter  %  90.05  89.98  89.91  Moisture  %  9.96  10.02  10.09  Protein  %  22.07  20.26  17.94  Crude fat  %  5.40  5.53  5.84  Crude fiber  %  2.63  2.55  2.46  Calcium  %  0.90  0.84  0.76  Phosphorus  %  0.70  0.67  0.61  Av phosphate  %  0.45  0.42  0.38  ME POULTRY kcal/kg  kcal/kg  3056.74  3102.72  3169.19  Cystine  %  0.35  0.33  0.30  Methionine  %  0.65  0.58  0.52  Tsaa  %  1.01  0.92  0.82  Lysine  %  1.32  1.21  1.05  Tryptophan  %  0.26  0.24  0.20  Threonine  %  0.89  0.82  0.73  Arginine  %  1.45  1.31  1.13  Histidine  %  0.58  0.53  0.47  Valine  %  1.00  0.92  0.81  Leucine  %  1.87  1.75  1.59  Isoleucine  %  0.90  0.82  0.71  Phenylalanine  %  1.03  0.94  0.83  Glycine  %  0.90  0.82  0.72  Phe + Tyr  %  2.19  1.98  1.74  Vitamin D3 IU/kg  IU/kg  7715.09  7715.28  7715.47  Vitamin K mg/kg  mg/kg  3.06  3.07  3.08  Vitamin A KIU/kg  kIU/kg  22.63  22.67  22.73  Vitamin E IU/kg  mg/kg  105.82  106.72  107.85  Thiamin  mg/kg  9.02  9.04  9.06  Riboflavin  mg/kg  13.52  13.43  13.33  Pantothenic acid  mg/kg  47.91  47.42  46.79  Niacin  mg/kg  113.43  113.56  113.70  Choline  mg/kg  1562.50  1467.43  1346.71  Biotin  mg/kg  1.25  1.23  1.22  Folic acid  mg/kg  4.18  4.14  4.08  Vitamin B12 mg/kg  mg/kg  32.50  32.50  32.50  Vitamin b6  mg/kg  20.14  20.24  20.36  Linoleic acid  %  2.01  2.09  2.23  Electrolytes  mEq/kg  205.18  200.08  199.74  Sodium  %  0.19  0.20  0.20  Potassium  %  0.86  0.78  0.69  Chloride  %  0.34  0.31  0.22  Magnesium  %  0.18  0.18  0.16  Sulfur  %  0.22  0.20  0.18  (Na+K)-(Cl+S)  mEq/kg  7.03  7.61  8.97  Iron  mg/kg  144.74  139.13  131.99  Copper  mg/kg  13.91  13.37  12.68  Zinc  mg/kg  89.42  87.77  85.66  Manganese  mg/kg  78.87  77.25  75.18  Iodine  mg/kg  0.40  0.40  0.40  Selenium  mg/kg  0.05  0.05  0.05  Nutrient Name  Units  Starter  Grower  Finisher  Corn  %  58.40  63.10  68.95  Soybean  %  34.40  29.85  24.10  DL-methionine  %  0.32  0.28  0.24  Lysine  %  0.20  0.21  0.20  L-threonine  %  0.07  0.08  0.08  Animal fat  %  2.85  2.85  3.00  Limestone  %  1.44  1.36  1.25  Biophosphate  %  1.56  1.45  1.30  Salt  %  0.44  0.37  0.24  Sodium bicarbonate  %  0.00  0.12  0.32  Trace minerals  %  0.05  0.05  0.05  Trace vitamins  %  0.25  0.25  0.25  Salinomycin  %  0.05  0.05  0.05  Dry matter  %  90.05  89.98  89.91  Moisture  %  9.96  10.02  10.09  Protein  %  22.07  20.26  17.94  Crude fat  %  5.40  5.53  5.84  Crude fiber  %  2.63  2.55  2.46  Calcium  %  0.90  0.84  0.76  Phosphorus  %  0.70  0.67  0.61  Av phosphate  %  0.45  0.42  0.38  ME POULTRY kcal/kg  kcal/kg  3056.74  3102.72  3169.19  Cystine  %  0.35  0.33  0.30  Methionine  %  0.65  0.58  0.52  Tsaa  %  1.01  0.92  0.82  Lysine  %  1.32  1.21  1.05  Tryptophan  %  0.26  0.24  0.20  Threonine  %  0.89  0.82  0.73  Arginine  %  1.45  1.31  1.13  Histidine  %  0.58  0.53  0.47  Valine  %  1.00  0.92  0.81  Leucine  %  1.87  1.75  1.59  Isoleucine  %  0.90  0.82  0.71  Phenylalanine  %  1.03  0.94  0.83  Glycine  %  0.90  0.82  0.72  Phe + Tyr  %  2.19  1.98  1.74  Vitamin D3 IU/kg  IU/kg  7715.09  7715.28  7715.47  Vitamin K mg/kg  mg/kg  3.06  3.07  3.08  Vitamin A KIU/kg  kIU/kg  22.63  22.67  22.73  Vitamin E IU/kg  mg/kg  105.82  106.72  107.85  Thiamin  mg/kg  9.02  9.04  9.06  Riboflavin  mg/kg  13.52  13.43  13.33  Pantothenic acid  mg/kg  47.91  47.42  46.79  Niacin  mg/kg  113.43  113.56  113.70  Choline  mg/kg  1562.50  1467.43  1346.71  Biotin  mg/kg  1.25  1.23  1.22  Folic acid  mg/kg  4.18  4.14  4.08  Vitamin B12 mg/kg  mg/kg  32.50  32.50  32.50  Vitamin b6  mg/kg  20.14  20.24  20.36  Linoleic acid  %  2.01  2.09  2.23  Electrolytes  mEq/kg  205.18  200.08  199.74  Sodium  %  0.19  0.20  0.20  Potassium  %  0.86  0.78  0.69  Chloride  %  0.34  0.31  0.22  Magnesium  %  0.18  0.18  0.16  Sulfur  %  0.22  0.20  0.18  (Na+K)-(Cl+S)  mEq/kg  7.03  7.61  8.97  Iron  mg/kg  144.74  139.13  131.99  Copper  mg/kg  13.91  13.37  12.68  Zinc  mg/kg  89.42  87.77  85.66  Manganese  mg/kg  78.87  77.25  75.18  Iodine  mg/kg  0.40  0.40  0.40  Selenium  mg/kg  0.05  0.05  0.05  View Large Stress Analysis On D 40 to 42, blood samples were collected from 10 birds per pen. The area around the jugular vein was sanitized with 70% alcohol, and in preparation, the inside of a 10 mL syringe was lined with a small amount of heparin. Between 2 to 3 mL of blood was collected from each bird, and a drop was used to prepare a blood-smear slide. The remaining blood was injected into a vacutainer (BD 368,056, BD, Franklin Lakes, NJ). Vacutainers were temporarily stored in an ice bath prior to transport to the lab for further processing. Once all samples were collected, the vacutainers were spun down in a Beckman GS-6R centrifuge (Beckman Coulter, Brea, CA) for 15 min at 4,000 rpm to separate the cells from the plasma. The plasma was drawn into 2 mL micro-centrifuge tubes and stored at –19°C until further analysis. The blood-smear slides were stained using a hematology staining kit (Cat# 25,034, Polysciences Inc., Warrington, PA), then air-dried. Plasma corticosterone concentrations were measured using a commercially available enzyme-linked immunosorbent assay (ELISA) kit (Enzo Life Sciences, ADI-901–097, Farmingdale, NY). Plasma HSP70 concentrations were measured using a commercially available ELISA kit (Enzo Life Sciences, EKS-715, Farmingdale, NY). Inter and Intra assay %CV were less than 5%. White blood cell differentials were measured by taking the blood smear slides and observing them under 1,000× magnification (10× eyepiece, 100× oil emersion lens) using an Omax DCE-2 microscope (Kent, WA). H/L ratio were measured by taking an area of the slide that had moderate cell density (no overlapping cells) and counting cells under 1,000× magnification, and the numbers of both heterophils and lymphocytes were counted until the total observed number reaches 100 (Campo et al., 2008). A keystroke counter was used to accurately keep track of the number of cells observed. Physical asymmetry was measured on 8 birds per pen, following the protocol outlined in Archer and Mench (2013). Using a calibrated Craftsman IP54 Digital Caliper (Sears Holdings, Hoffman Estates, IL), the middle toe length, metatarsal length, and metatarsal width were measured for both the right and left legs. The composite asymmetry score was calculated by taking the sum of the absolute value of left minus right of each trait, then dividing by the total number of traits. Thus, the formula for this trial would be (|L-R|MTL+|L-R|ML+|L-R|MW)/3 = composite asymmetry score. Statistical Analysis Stress measures were all analyzed using the GLM procedure in Minitab 17.1.0. The model tested for the effects of diet (CON and XPC), environment (heat stress and non-heat stress) and the 2-way interaction between diet and environment. Significant differences were considered at P < 0.05. Mean separation was performed using the Least Significant Difference (LSD) post hoc procedure. RESULTS White blood cell populations and total WBC count are presented in Table 3. No significant 2-way interactions between diet and environment were observed for these measured parameters. The application of a heat stress did not have a significant effect on heterophil or eosinophil populations. However, heat stress did significantly decrease (P = 0.002) lymphocyte populations (51.2 ± 1.6 vs. 58.4 ± 1.7, respectively) compared to no heat stress. No significant diet effects were observed between CON and XPC birds for monocyte, eosinophil or basophil populations. XPC significantly reduced (P = 0.004) heterophil populations compared to CON (30.1 ± 1.6 vs. 36.1 ± 1.4, respectively) while significantly increasing (P = 0.02) lymphocyte populations (57.5 ± 1.7 vs. 52.0 ± 1.6, respectively). Total WBC counts were also significantly decreased (P = 0.05) for XPC compared to CON (32,417 ± 1,304 vs. 36,235 ± 1,402, respectively). Table 3. Percentage of white blood cell populations and total white blood cell count (mean ± SE) for commercial broilers fed a diet with and without Diamond V Original XPCTM and exposed to either a non-heat stress or a heat stress environment. Variable  Heterophil  Lymphocyte  Monocyte  Eosinophil  Basophil  Total White Blood Cell Count  Diet Main Effect  CON  36.1 ± 1.4a  52.0 ± 1.6b  6.0 ± 0.4  2.5 ± 0.4  2.5 ± 0.4  36,235 ± 1402a  XPC  30.1 ± 1.6b  57.5 ± 1.7a  6.5 ± 0.7  2.7 ± 0.4  2.7 ± 0.4  32,417 ± 1304b  Environment Main Effect  No Heat Stress  31.4 ± 1.6  58.4 ± 1.7a  4.4 ± 0.4b  2.9 ± 0.4  3.0 ± 0.3b  32,910 ± 1437  Heat Stress  34.8 ± 1.4  51.2 ± 1.6b  8.1 ± 0.7a  2.3 ± 0.4  3.9 ± 0.3a  35,742 ± 1282  P-value  Diet Effect  0.004  0.02  0.55  0.68  0.47  0.05  Environment Effect  0.10  0.002  <0.0001  0.36  0.05  0.14  Interaction  0.46  0.94  0.12  0.71  0.32  0.60  Variable  Heterophil  Lymphocyte  Monocyte  Eosinophil  Basophil  Total White Blood Cell Count  Diet Main Effect  CON  36.1 ± 1.4a  52.0 ± 1.6b  6.0 ± 0.4  2.5 ± 0.4  2.5 ± 0.4  36,235 ± 1402a  XPC  30.1 ± 1.6b  57.5 ± 1.7a  6.5 ± 0.7  2.7 ± 0.4  2.7 ± 0.4  32,417 ± 1304b  Environment Main Effect  No Heat Stress  31.4 ± 1.6  58.4 ± 1.7a  4.4 ± 0.4b  2.9 ± 0.4  3.0 ± 0.3b  32,910 ± 1437  Heat Stress  34.8 ± 1.4  51.2 ± 1.6b  8.1 ± 0.7a  2.3 ± 0.4  3.9 ± 0.3a  35,742 ± 1282  P-value  Diet Effect  0.004  0.02  0.55  0.68  0.47  0.05  Environment Effect  0.10  0.002  <0.0001  0.36  0.05  0.14  Interaction  0.46  0.94  0.12  0.71  0.32  0.60  a,bDifferent letters within column and measure indicate significant differences P < 0.05. View Large Heat stress significantly increased both monocyte (P < 0.0001; 8.1 ± 0.7 vs. 4.4 ± 0.4, respectively) and basophil (P = 0.05; 3.9 ± 0.3 vs. 3.0 ± 0.3, respectively) populations compared to no heat stress. Heat stress did not significantly change total WBC count. Heterophil/lymphoctye ratio, plasma corticosterone concentration, composite asymmetry score, and plasma HSP 70 concentration are presented in Table 4. No significant 2-way interactions between diet and environment were observed for these measured parameters. The application of the heat stress also resulted in significantly greater (P = 0.02) H/L ratios (heat stress, 0.80 ± 0.06 vs. no heat stress, 0.63 ± 0.05). The H/L ratio was significantly greater (P = 0.01) in the CON birds (0.81 ± 0.05) when compared to those fed XPC (0.62 ± 0.05). Table 4. Heterophil/lymphocyte ratio, plasma corticosterone concentration (pg/mL), composite asymmetry score, and plasma heat-shock protein 70 (pg/mL) for commercial broilers fed a diet with and without Diamond V Original XPCTM and exposed to either a non-heat stress or a heat stress environment. Variable  Heterophil/Lymphocyte Ratio  Plasma Corticosterone Concentration  Composite Asymmetry Score  Plasma HSP 70 Concentration  Diet Main Effect  CON  0.81 ± 0.05a  8433 ± 730a  1.50 ± 0.09a  5.06 ± 0.16  XPC  0.62 ± 0.05b  5129 ± 617b  0.54 ± 0.03b  5.29 ± 0.17  Environment Main Effect  No Heat Stress  0.63 ± 0.05a  5105 ± 444b  0.98 ± 0.10  4.75 ± 0.14b  Heat Stress  0.80 ± 0.06b  8456 ± 846a  1.05 ± 0.08  5.62 ± 0.17a  P-value  Diet Effect  0.01  <0.0001  <0.0001  0.35  Environment Effect  0.02  <0.0001  0.58  <0.0001  Interaction  0.72  0.95  0.88  0.61  Variable  Heterophil/Lymphocyte Ratio  Plasma Corticosterone Concentration  Composite Asymmetry Score  Plasma HSP 70 Concentration  Diet Main Effect  CON  0.81 ± 0.05a  8433 ± 730a  1.50 ± 0.09a  5.06 ± 0.16  XPC  0.62 ± 0.05b  5129 ± 617b  0.54 ± 0.03b  5.29 ± 0.17  Environment Main Effect  No Heat Stress  0.63 ± 0.05a  5105 ± 444b  0.98 ± 0.10  4.75 ± 0.14b  Heat Stress  0.80 ± 0.06b  8456 ± 846a  1.05 ± 0.08  5.62 ± 0.17a  P-value  Diet Effect  0.01  <0.0001  <0.0001  0.35  Environment Effect  0.02  <0.0001  0.58  <0.0001  Interaction  0.72  0.95  0.88  0.61  a,bDifferent letters within column and measure indicate significant differences P < 0.05. View Large Heat stress significantly increased (P < 0.0001) plasma corticosterone concentration (8456 ± 846 vs. 5105 ± 444, respectively) compared to no heat stress. No significant effects of heat stress were observed on composite asymmetry score; however, heat stress did significantly increase the plasma HSP70 concentrations compared to no heat stress (P < 0.0001, heat stress, 5.62 ± 0.17 pg/mL vs. no heat stress, 4.75 ± 0.14 pg/mL). XPC fed birds had a significantly lower stress response as indicated by both plasma corticosterone concentrations (P < 0.0001; 5129 ± 617 vs. 8433 ± 730, respectively) and composite physical asymmetry scores (P < 0.0001; 0.54 ± 0.03 vs. 1.50 ± 0.09, respectively). No significant differences were observed between XPC and CON birds for HSP70 concentrations; DISCUSSION To determine the impact of XPC as an effective dietary additive to help mitigate stress in broilers, it was first necessary to ensure that the model used did, in fact, elicit a stress response from the birds. The parameters measured in this study included: HSP70, CORT, H/L and composite asymmetry and are associated with stress in broilers, as well as bird welfare (Gross and Siegel, 1983; Zulkifli et al., 1999; Campo and Da’vila, 2000; Al-Aqil et al., 2009). In this study, the expression of HSP70 measured in blood plasma as well as plasma corticosterone was increased in birds exposed to heat stress regardless of dietary treatment. Additionally, H/L ratios were also increased in birds exposed to heat stress regardless of dietary treatment. Increases in all of these targeted stress measurements illustrate that the heat stress model used in this study was perceived as a stress in the birds. Therefore, the model used provided an acceptable means to determine the potential use of XPC in reducing stress and improving animal welfare measures. The inclusion of XPC in the treatment diet showed measurable effects on the stress parameters collected during this current study. The H/L ratios were decreased in XPC birds (at 1.25 kg/MT) in this current study. These data agree with the report by Al-Mansour et al. (2011) where reduced H/L ratios were observed after feeding XPC to broilers. Increased H/L ratios indicate increased stress in poultry (Gross and Siegel, 1983; McFarlane and Curtis, 1989) and these results indicate that feeding XPC decreased the stress susceptibility of broilers either during heat stress or during normal rearing conditions. The total WBC population was also reduced by feeding XPC. This further indicates that XPC is playing a role in immune modulation, which has been demonstrated previously by Chou et al. (2017), who observed supplementation with XPC resulted in improved immunocompetence. Birds fed XPC also exhibited a significant reduction in plasma corticosterone in both heat stress and non-heat stress environments, an indication that feeding XPC can reduce stress susceptibility during times of heat stress or during normal rearing conditions. The reduction in corticosterone by feeding XPC is consistent with research in turkeys (Bartz et al., 2015, 2016) which saw similar effects. While corticosterone was reduced by XPC treatment in this current study, HSP70 was not affected. Bensi et al. (1990) and Beutler and Cerami (1989) have shown that HSP70 can actually inhibit the expression of cytokines interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-α). XPC has been shown to up-regulate IL-1, IL-2, and TNF-α in gene extraction studies (Park, 2014). This may further indicate the importance of HSP70 as a protective mechanism in environmental heat stress conditions, but further research is needed to identify the possible relationship between feeding XPC and HSP70 gene expression. It is possible as well that feeding XPC may also affect stress-related gene regulation, though this needs further investigation. Composite asymmetry scoring was improved in birds fed XPC compared to CON; this was observed in both heat stress and non-heat stress environments. The reduction in composite asymmetry further indicates that feeding XPC can reduce stress susceptibility during heat stress or during normal rearing conditions, as asymmetry is an indicator of current stress levels (Kellner and Alford, 2003). The reduction of stress susceptibility when faced with a heat stress or solely the stressors of production (growth, stocking density, etc.) demonstrates that XPC is having a substantial impact on the overall health and well-being. Further research is needed to determine better understand the mechanisms affecting not only the stress response, but also the immune system. The reduction in stress susceptibility observed in this current study and the immune modulation observed by (Chou et al., 2017) may be why others have observed increased growth and feed conversion (Gao et al., 2008, 2009) in birds feed XPC. Overall, the inclusion of XPC in the diet of broiler chickens reduced stress susceptibility in all 3 independent evaluations in this study: H/L ratio, plasma corticosterone, and physical asymmetry, without changes to HSP70. The mechanism for stress reduction is unknown; it may be related to gene expression, as there is evidence that the immune system is modulated in this way by XPC consumption. These data, however, support the use of XPC as a useful management tool for improving the welfare of poultry during times of extreme stress, e.g., elevated temperatures, or the routine management stress associated with rearing. ACKNOWLEDGEMENTS We would like to thank all the undergraduates who helped with this project as well as Denise Caldwell for all her assistance. REFERENCES Al-Aqil A., Zulkifli I., Sazili A. Q., Omar A. R., Rajion M. A.. 2009. The effects of the hot, humid tropical climate and early age feed restriction on stress and fear responses, and performance in broiler chickens. Asian-Australas. J. Anim. Sci.  22: 1581– 1586. Google Scholar CrossRef Search ADS   Al-Mansour S., Al-Khalf A., Al-Homidan I., Fathi M. M.. 2011. Feed efficiency and blood hematology of broiler chicks given a diet supplemented with yeast culture. Int. J. Poult. Sci.  10: 603– 607. Google Scholar CrossRef Search ADS   Archer G. S., Mench J. A.. 2013. The effects of light stimulation during incubation on indicators of stress susceptibility in broilers. Poult. Sci.  92: 3103– 3108. Google Scholar CrossRef Search ADS PubMed  Aviagen, 2014. Ross Broiler Management Handbook . Aviagen Group, Huntsville, AL. Bartz B., Grimes J., Black S., Barasch I., McIntyre D.. 2015. Effect of induced stress on turkey hen performance provided with and without AviCare and Original XPC. Poult. Sci.  94 (Suppl. 1): 18. (Abstr.). Bartz B., Grimes J., Black S., Barasch I., McIntyre D.. 2016. Effects of heat and crowding stress on commercial turkey hen performance supplemented with dietary XPC™ or combined with AviCare™ in water. 2016 Int. Poult. Sci. Forum, Atlanta, Georgia. (Abstr.). Bensi G., Mora M., Raugei G., Buonmassa D., Rossini M., Melli M.. 1990. An inducible enhancer controls the expression of the human interleukin-1β gene. Cell Growth Diff.  1: 491– 497. Berrong S. L., Washburn K. W.. 1998. Effects of genetic variation on total plasma protein, body weight gains, and body temperature responses to heat stress. Poult. Sci.  773: 379– 385. Google Scholar CrossRef Search ADS   Beutler B., Cerami A.. 1989. The biology of cachectin/TNF: a primary mediator of the host response. Annu. Rev. Immun.  7: 625– 655. Google Scholar CrossRef Search ADS   Campo J. L., Dávila S. G.. 2000. Changes in heterophil to lymphocyte ratios of heat-stressed chickens in response to dietary supplementation of several related stress agents. Arch. Geflügelk.  66: 80– 84. Campo J. L., Prieto M. T., Davila S. G.. 2008. Effects of housing system and cold stress on heterophil-to-lymphocyte ratio, fluctuating asymmetry, and tonic immobility duration of chickens. Poult. Sci.  87: 621– 626. Google Scholar CrossRef Search ADS PubMed  Chou W. K., Park J., Carey J. B., McIntyre D. R., Berghman L. R.. 2017. Immunomodulatory effects of saccharomyces cerevisiae fermentation product supplementation on immune gene expression and lymphocyte distribution in immune organs in broilers. Front Vet. Sci.  4: 37. Google Scholar CrossRef Search ADS PubMed  Federation of Animal Science Societies (FASS). 2010. Guide for the Care and Use of Agricultural Animals in Research and Teaching . Third Edition. Federation of Animal Science Societies, Champaign, IL. Firman J. D., Moore D., Broomhead J., McIntyre D.. 2013. Effects of dietary inclusion of a Saccharomyces cerevisiae fermentation product on performance and gut characteristics of male turkeys to market weight. Int. J. Poult. Sci.  12: 141– 143. Google Scholar CrossRef Search ADS   Gao J., Zhang H. J., Yu S. H., Wu S. G., Yoon I., Quigley J., Gao Y. P., Qi G. H.. 2008. Effects of yeast culture in broiler diets on performance and immunomodulatory functions. Poult. Sci.  87: 1377– 1384. Google Scholar CrossRef Search ADS PubMed  Gao J., Zhang H. J., Wu S. G., Yu S. H., Yoon I., Moore D., Gao Y. P., Yan H. J., Qi G. H.. 2009. Effect of Saccharomyces cerevisiae fermentation product on immune functions of broilers challenged with Eimeria tenella. Poult Sci.  88: 2141– 2151. Google Scholar CrossRef Search ADS PubMed  Gross W. B., Siegel H. S.. 1983. Evaluation of the heterophil lymphocyte ratio as a measure of stress in chickens. Avian Dis.  27: 972– 979. Google Scholar CrossRef Search ADS PubMed  Kellner J. R., Alford R. A.. 2003. The ontogeny of fluctuating asymmetry. Am. Nat.  161: 931– 947. Google Scholar CrossRef Search ADS PubMed  McFarlane J. M., Curtis S. E.. 1989. Multiple concurrent stressors in chicks. 3. Effects on plasma corticosterone and the heterophil: lymphocyte ratio. Poult. Sci.  68: 522– 527. Google Scholar CrossRef Search ADS PubMed  NCC. 2015. National Chicken Council Animal Welfare Guidelines and Audit Checklist for Broilers . National Chicken Council, Washington, DC. Park J. W. 2014. Effects of yeast product on modulating the adaptive immune function in broilers. PhD Diss. Texas A&M University, College Station, TX. Soleimani A. F., Zulkifli I., Hair-Bejo M., Omar A. R., Rahal A. R.. 2011. The role of heat shock protein 70 in resistance to Salmonella Enteritidis in broiler chickens subjected to neonatal feed restriction and thermal stress. Poult. Sci.  91: 340– 345. Google Scholar CrossRef Search ADS   Syafwan S. K., Kwakkel R. P., Verstegen M. W. A.. 2011. Heat stress and feeding strategies in meat-type chickens. World. Poult. Sci. J.  67: 653– 673. Google Scholar CrossRef Search ADS   Tan G. Y, Yang L., Fu Y. Q., Feng J. H., Zhang M. H.. 2010. Effects of different acute high ambient temperatures on function of hepatic mitochondrial respiration, antioxidative enzymes, and oxidative injury in broiler chickens. Poult. Sci.  891: 115– 122. Google Scholar CrossRef Search ADS   Teeter R. 1993. Effect of yeast culture in broilers under heat stress and nonspecific antigen challenge. Yeast culture poultry research report 2. Department of Animal Science, Oklahoma State University, Stillwater, OK. Zulkifli I., Dass R. T., Che Norma M. T.. 1999. Acute heat-stress effects on physiology and fear-related behaviour in red jungle fowl and domestic fowl. Can. J. Anim. Sci.  79: 165– 170. Google Scholar CrossRef Search ADS   © 2017 Poultry Science Association Inc. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Poultry Science Oxford University Press

Utilizing original XPC™ in feed to reduce stress susceptibility of broilers

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Oxford University Press
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© 2017 Poultry Science Association Inc.
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0032-5791
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1525-3171
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10.3382/ps/pex386
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Abstract

Abstract Reduction of stress is an important factor in improving poultry welfare, especially during periods of heat stress. A study was conducted to evaluate the effects of feeding the functional metabolites of Diamond V Original XPCTM to broilers reared under ambient or heat stress temperatures. Dietary treatments included: control feed (CON) and Original XPC fed continuously at 1.25 kg/MT (XPC). Half the birds in each dietary treatment were subjected to either no heat stress (24°C constant) or heat stress (35°C:24°C for 18:6 h daily) from 28 to 42 d. At the end of the heat stress period, blood was collected from 40 birds/treatment. Blood was analyzed for plasma corticosterone (CORT), plasma HSP70 (HSP70), and heterophil/lymphocyte ratios (H/L). At 42 d, bilateral metatarsal traits were also measured in 40 birds/treatment to assess physical asymmetry. Birds fed XPC had significantly lower CORT levels than CON (P < 0.001; 5,129 ± 617 vs. 8,433 ± 730, respectively). Physical asymmetry scores were also significantly higher in CON compared to XPC fed broilers (P < 0.001; 1.50 ± 0.13 vs. 0.54 ± 0.05, respectively). H/L ratios were significantly greater in CON than for XPC birds (P = 0.01; 0.81 ± 0.05 mm vs. 0.62 ± 0.05 mm, respectively). No differences were observed between CON and XPC fed broilers in HSP70. However, heat stress did increase (P < 0.0001) HSP70 compared to no heat stress birds (5.65 ± 0.12 vs. 4.78 ± 0.11 pg/mL, for heat stress and no heat stress, respectively). Feeding XPC to broiler chickens improved animal welfare via reduced stress indicators while under heat stress or no heat stress conditions. These results indicate that feeding XPC may improve poultry welfare by reducing heat stress susceptibility. INTRODUCTION As the broiler industry strives to provide the most efficient and highest-quality meat products to consumer markets, special attention has been given to stocking density. The average floor space in North America for commercial broilers is around 0.07 m2/bird (NCC, 2015). While commercial production environments are designed to provide adequate air flow, humidity, access to feed/water, and constant temperature of around 26°C (Aviagen, 2014), episodic conditions may still be considered as stressful to birds. The modern consumer has become increasingly conscious of the perceived plight of food production animals in confinement and a market category has begun to focus on even more humane treatment of these animals. There is an opportunity to focus on food and water ingestion as a key area for controlling stress. One goal in producing food animals is to increase their average daily gain (ADG) to meet market demands, making ingestion of quality feed paramount. Selection for fast-growing, high-yielding broilers has been shown to have detrimental effects on the ability of a bird to cope with heat stress (Berrong and Washburn, 1998; Tan et al., 2010; Soleimani et al., 2011). Syafwan et al. (2011) showed that heat stress in commercial broiler strains can lead to cellular oxidative stress, which increases susceptibility to infectious diseases. However, the fact that broilers ingest a large amount of feed and water allows for the addition of stress-reducing agents to that feed and water. Previous studies have shown that functional metabolites of Diamond V Original XPCTM (DiamondV Mills, Cedar Rapids, IA) can help balance the immune response and stress hormone levels in production poultry (Firman et al., 2013). Gao et al. (2008, 2009) showed increased secretory IgA and intestinal IgM in birds fed Original XPC, and Al-Mansour et al. (2011) showed that Original XPC significantly decreased heterophil/lymphocyte (H/L) ratios in broilers. Original XPC has also shown to have a positive impact on weight gain, feed conversion, and mortality in broilers (Gao et al., 2008, 2009), even in broilers that were challenged with ingestion of used litter during a heat-stress period (Teeter, 1993). A new study was undertaken to measure the effects of stress on broilers fed Original XPC during long-term heat stress and non-heat stress. It is hypothesized that supplementing broiler feed with Original XPC will decrease the stress susceptibility of the broilers subjected to heat stress or routine management stressors during rearing. MATERIALS AND METHODS Animals and Husbandry A total of 320 Ross 708 broilers were used in this experiment. Birds were equally housed at 20 birds per pen across a total of 16 pens (0.91 m × 2.74 m). Birds were randomly assigned to each pen; however, initial pen weights were equalized. Each pen was lined with clean pine shavings and constructed of solid black plastic on all but the front side, which was made of mesh wire. Each pen was equipped with one bell feeder and nipple drinking system. A 2 × 2 factorial design was used in this experiment in which pens were assigned at random to one of 2 dietary treatments: control diet (CON) or control diet with 1.25 kg/MT of Original XPC (DiamondV Mills, Cedar Rapids, IA) added (XPC) and one of 22 environmental treatments (heat stress or non-heat stress). Table 1 describes the experimental design. Half of the birds were subjected to 2 wk of heat stress (35°C for at least 18 h daily and the remaining 6 h at 24°C to allow for feeding) between 28 and 42 d of age. Dietary treatments were equally represented between rooms that were environmentally controlled. Each room was equipped with fluorescent lighting, oscillating fans for additional airflow, and a single-pass central ventilation system equipped with high-efficiency particulate air (HEPA) filters per U.S. Department of Agriculture (USDA) Biosecurity Level 2 protocols. Birds were fed a 3-phase diet consisting of a starter (D 0–14, crumble), grower (d 14 to 28, pellet), and finisher (d 28 to 42, pellet) presented in Table 2. Birds were allowed ad libitum access to feed and water. A photoperiod of 24L:0D was maintained to 10 d of age and then reduced to 20L:4D for the remainder of the study. The birds were managed according to the guidelines set forth in the Guide for the Care and Use of Agricultural Animals in Research and Teaching (FASS, 2010) and all procedures were approved by the USDA-ARS animal care committee (ACUC #2,015,005). Table 1. Experimental design for commercial broilers fed a diet with and without Diamond V Original XPCTM and exposed to either a non-heat stress or a heat stress environment.   Treatment  Variable  Control (CON)  Original XPC (XPC)1  Product Inclusion Rate  N/A  1.25 kg/MT  Heat Stress Pens  4  4  Non-Heat Stress Pens  4  4  Total Number of Birds  160  160    Treatment  Variable  Control (CON)  Original XPC (XPC)1  Product Inclusion Rate  N/A  1.25 kg/MT  Heat Stress Pens  4  4  Non-Heat Stress Pens  4  4  Total Number of Birds  160  160  1Diamond V Original XPCTM, Cedar Rapids, IA, USA View Large Table 2. Experimental diets and calculated nutrient content for the starter, grower, and finisher phases. Nutrient Name  Units  Starter  Grower  Finisher  Corn  %  58.40  63.10  68.95  Soybean  %  34.40  29.85  24.10  DL-methionine  %  0.32  0.28  0.24  Lysine  %  0.20  0.21  0.20  L-threonine  %  0.07  0.08  0.08  Animal fat  %  2.85  2.85  3.00  Limestone  %  1.44  1.36  1.25  Biophosphate  %  1.56  1.45  1.30  Salt  %  0.44  0.37  0.24  Sodium bicarbonate  %  0.00  0.12  0.32  Trace minerals  %  0.05  0.05  0.05  Trace vitamins  %  0.25  0.25  0.25  Salinomycin  %  0.05  0.05  0.05  Dry matter  %  90.05  89.98  89.91  Moisture  %  9.96  10.02  10.09  Protein  %  22.07  20.26  17.94  Crude fat  %  5.40  5.53  5.84  Crude fiber  %  2.63  2.55  2.46  Calcium  %  0.90  0.84  0.76  Phosphorus  %  0.70  0.67  0.61  Av phosphate  %  0.45  0.42  0.38  ME POULTRY kcal/kg  kcal/kg  3056.74  3102.72  3169.19  Cystine  %  0.35  0.33  0.30  Methionine  %  0.65  0.58  0.52  Tsaa  %  1.01  0.92  0.82  Lysine  %  1.32  1.21  1.05  Tryptophan  %  0.26  0.24  0.20  Threonine  %  0.89  0.82  0.73  Arginine  %  1.45  1.31  1.13  Histidine  %  0.58  0.53  0.47  Valine  %  1.00  0.92  0.81  Leucine  %  1.87  1.75  1.59  Isoleucine  %  0.90  0.82  0.71  Phenylalanine  %  1.03  0.94  0.83  Glycine  %  0.90  0.82  0.72  Phe + Tyr  %  2.19  1.98  1.74  Vitamin D3 IU/kg  IU/kg  7715.09  7715.28  7715.47  Vitamin K mg/kg  mg/kg  3.06  3.07  3.08  Vitamin A KIU/kg  kIU/kg  22.63  22.67  22.73  Vitamin E IU/kg  mg/kg  105.82  106.72  107.85  Thiamin  mg/kg  9.02  9.04  9.06  Riboflavin  mg/kg  13.52  13.43  13.33  Pantothenic acid  mg/kg  47.91  47.42  46.79  Niacin  mg/kg  113.43  113.56  113.70  Choline  mg/kg  1562.50  1467.43  1346.71  Biotin  mg/kg  1.25  1.23  1.22  Folic acid  mg/kg  4.18  4.14  4.08  Vitamin B12 mg/kg  mg/kg  32.50  32.50  32.50  Vitamin b6  mg/kg  20.14  20.24  20.36  Linoleic acid  %  2.01  2.09  2.23  Electrolytes  mEq/kg  205.18  200.08  199.74  Sodium  %  0.19  0.20  0.20  Potassium  %  0.86  0.78  0.69  Chloride  %  0.34  0.31  0.22  Magnesium  %  0.18  0.18  0.16  Sulfur  %  0.22  0.20  0.18  (Na+K)-(Cl+S)  mEq/kg  7.03  7.61  8.97  Iron  mg/kg  144.74  139.13  131.99  Copper  mg/kg  13.91  13.37  12.68  Zinc  mg/kg  89.42  87.77  85.66  Manganese  mg/kg  78.87  77.25  75.18  Iodine  mg/kg  0.40  0.40  0.40  Selenium  mg/kg  0.05  0.05  0.05  Nutrient Name  Units  Starter  Grower  Finisher  Corn  %  58.40  63.10  68.95  Soybean  %  34.40  29.85  24.10  DL-methionine  %  0.32  0.28  0.24  Lysine  %  0.20  0.21  0.20  L-threonine  %  0.07  0.08  0.08  Animal fat  %  2.85  2.85  3.00  Limestone  %  1.44  1.36  1.25  Biophosphate  %  1.56  1.45  1.30  Salt  %  0.44  0.37  0.24  Sodium bicarbonate  %  0.00  0.12  0.32  Trace minerals  %  0.05  0.05  0.05  Trace vitamins  %  0.25  0.25  0.25  Salinomycin  %  0.05  0.05  0.05  Dry matter  %  90.05  89.98  89.91  Moisture  %  9.96  10.02  10.09  Protein  %  22.07  20.26  17.94  Crude fat  %  5.40  5.53  5.84  Crude fiber  %  2.63  2.55  2.46  Calcium  %  0.90  0.84  0.76  Phosphorus  %  0.70  0.67  0.61  Av phosphate  %  0.45  0.42  0.38  ME POULTRY kcal/kg  kcal/kg  3056.74  3102.72  3169.19  Cystine  %  0.35  0.33  0.30  Methionine  %  0.65  0.58  0.52  Tsaa  %  1.01  0.92  0.82  Lysine  %  1.32  1.21  1.05  Tryptophan  %  0.26  0.24  0.20  Threonine  %  0.89  0.82  0.73  Arginine  %  1.45  1.31  1.13  Histidine  %  0.58  0.53  0.47  Valine  %  1.00  0.92  0.81  Leucine  %  1.87  1.75  1.59  Isoleucine  %  0.90  0.82  0.71  Phenylalanine  %  1.03  0.94  0.83  Glycine  %  0.90  0.82  0.72  Phe + Tyr  %  2.19  1.98  1.74  Vitamin D3 IU/kg  IU/kg  7715.09  7715.28  7715.47  Vitamin K mg/kg  mg/kg  3.06  3.07  3.08  Vitamin A KIU/kg  kIU/kg  22.63  22.67  22.73  Vitamin E IU/kg  mg/kg  105.82  106.72  107.85  Thiamin  mg/kg  9.02  9.04  9.06  Riboflavin  mg/kg  13.52  13.43  13.33  Pantothenic acid  mg/kg  47.91  47.42  46.79  Niacin  mg/kg  113.43  113.56  113.70  Choline  mg/kg  1562.50  1467.43  1346.71  Biotin  mg/kg  1.25  1.23  1.22  Folic acid  mg/kg  4.18  4.14  4.08  Vitamin B12 mg/kg  mg/kg  32.50  32.50  32.50  Vitamin b6  mg/kg  20.14  20.24  20.36  Linoleic acid  %  2.01  2.09  2.23  Electrolytes  mEq/kg  205.18  200.08  199.74  Sodium  %  0.19  0.20  0.20  Potassium  %  0.86  0.78  0.69  Chloride  %  0.34  0.31  0.22  Magnesium  %  0.18  0.18  0.16  Sulfur  %  0.22  0.20  0.18  (Na+K)-(Cl+S)  mEq/kg  7.03  7.61  8.97  Iron  mg/kg  144.74  139.13  131.99  Copper  mg/kg  13.91  13.37  12.68  Zinc  mg/kg  89.42  87.77  85.66  Manganese  mg/kg  78.87  77.25  75.18  Iodine  mg/kg  0.40  0.40  0.40  Selenium  mg/kg  0.05  0.05  0.05  View Large Stress Analysis On D 40 to 42, blood samples were collected from 10 birds per pen. The area around the jugular vein was sanitized with 70% alcohol, and in preparation, the inside of a 10 mL syringe was lined with a small amount of heparin. Between 2 to 3 mL of blood was collected from each bird, and a drop was used to prepare a blood-smear slide. The remaining blood was injected into a vacutainer (BD 368,056, BD, Franklin Lakes, NJ). Vacutainers were temporarily stored in an ice bath prior to transport to the lab for further processing. Once all samples were collected, the vacutainers were spun down in a Beckman GS-6R centrifuge (Beckman Coulter, Brea, CA) for 15 min at 4,000 rpm to separate the cells from the plasma. The plasma was drawn into 2 mL micro-centrifuge tubes and stored at –19°C until further analysis. The blood-smear slides were stained using a hematology staining kit (Cat# 25,034, Polysciences Inc., Warrington, PA), then air-dried. Plasma corticosterone concentrations were measured using a commercially available enzyme-linked immunosorbent assay (ELISA) kit (Enzo Life Sciences, ADI-901–097, Farmingdale, NY). Plasma HSP70 concentrations were measured using a commercially available ELISA kit (Enzo Life Sciences, EKS-715, Farmingdale, NY). Inter and Intra assay %CV were less than 5%. White blood cell differentials were measured by taking the blood smear slides and observing them under 1,000× magnification (10× eyepiece, 100× oil emersion lens) using an Omax DCE-2 microscope (Kent, WA). H/L ratio were measured by taking an area of the slide that had moderate cell density (no overlapping cells) and counting cells under 1,000× magnification, and the numbers of both heterophils and lymphocytes were counted until the total observed number reaches 100 (Campo et al., 2008). A keystroke counter was used to accurately keep track of the number of cells observed. Physical asymmetry was measured on 8 birds per pen, following the protocol outlined in Archer and Mench (2013). Using a calibrated Craftsman IP54 Digital Caliper (Sears Holdings, Hoffman Estates, IL), the middle toe length, metatarsal length, and metatarsal width were measured for both the right and left legs. The composite asymmetry score was calculated by taking the sum of the absolute value of left minus right of each trait, then dividing by the total number of traits. Thus, the formula for this trial would be (|L-R|MTL+|L-R|ML+|L-R|MW)/3 = composite asymmetry score. Statistical Analysis Stress measures were all analyzed using the GLM procedure in Minitab 17.1.0. The model tested for the effects of diet (CON and XPC), environment (heat stress and non-heat stress) and the 2-way interaction between diet and environment. Significant differences were considered at P < 0.05. Mean separation was performed using the Least Significant Difference (LSD) post hoc procedure. RESULTS White blood cell populations and total WBC count are presented in Table 3. No significant 2-way interactions between diet and environment were observed for these measured parameters. The application of a heat stress did not have a significant effect on heterophil or eosinophil populations. However, heat stress did significantly decrease (P = 0.002) lymphocyte populations (51.2 ± 1.6 vs. 58.4 ± 1.7, respectively) compared to no heat stress. No significant diet effects were observed between CON and XPC birds for monocyte, eosinophil or basophil populations. XPC significantly reduced (P = 0.004) heterophil populations compared to CON (30.1 ± 1.6 vs. 36.1 ± 1.4, respectively) while significantly increasing (P = 0.02) lymphocyte populations (57.5 ± 1.7 vs. 52.0 ± 1.6, respectively). Total WBC counts were also significantly decreased (P = 0.05) for XPC compared to CON (32,417 ± 1,304 vs. 36,235 ± 1,402, respectively). Table 3. Percentage of white blood cell populations and total white blood cell count (mean ± SE) for commercial broilers fed a diet with and without Diamond V Original XPCTM and exposed to either a non-heat stress or a heat stress environment. Variable  Heterophil  Lymphocyte  Monocyte  Eosinophil  Basophil  Total White Blood Cell Count  Diet Main Effect  CON  36.1 ± 1.4a  52.0 ± 1.6b  6.0 ± 0.4  2.5 ± 0.4  2.5 ± 0.4  36,235 ± 1402a  XPC  30.1 ± 1.6b  57.5 ± 1.7a  6.5 ± 0.7  2.7 ± 0.4  2.7 ± 0.4  32,417 ± 1304b  Environment Main Effect  No Heat Stress  31.4 ± 1.6  58.4 ± 1.7a  4.4 ± 0.4b  2.9 ± 0.4  3.0 ± 0.3b  32,910 ± 1437  Heat Stress  34.8 ± 1.4  51.2 ± 1.6b  8.1 ± 0.7a  2.3 ± 0.4  3.9 ± 0.3a  35,742 ± 1282  P-value  Diet Effect  0.004  0.02  0.55  0.68  0.47  0.05  Environment Effect  0.10  0.002  <0.0001  0.36  0.05  0.14  Interaction  0.46  0.94  0.12  0.71  0.32  0.60  Variable  Heterophil  Lymphocyte  Monocyte  Eosinophil  Basophil  Total White Blood Cell Count  Diet Main Effect  CON  36.1 ± 1.4a  52.0 ± 1.6b  6.0 ± 0.4  2.5 ± 0.4  2.5 ± 0.4  36,235 ± 1402a  XPC  30.1 ± 1.6b  57.5 ± 1.7a  6.5 ± 0.7  2.7 ± 0.4  2.7 ± 0.4  32,417 ± 1304b  Environment Main Effect  No Heat Stress  31.4 ± 1.6  58.4 ± 1.7a  4.4 ± 0.4b  2.9 ± 0.4  3.0 ± 0.3b  32,910 ± 1437  Heat Stress  34.8 ± 1.4  51.2 ± 1.6b  8.1 ± 0.7a  2.3 ± 0.4  3.9 ± 0.3a  35,742 ± 1282  P-value  Diet Effect  0.004  0.02  0.55  0.68  0.47  0.05  Environment Effect  0.10  0.002  <0.0001  0.36  0.05  0.14  Interaction  0.46  0.94  0.12  0.71  0.32  0.60  a,bDifferent letters within column and measure indicate significant differences P < 0.05. View Large Heat stress significantly increased both monocyte (P < 0.0001; 8.1 ± 0.7 vs. 4.4 ± 0.4, respectively) and basophil (P = 0.05; 3.9 ± 0.3 vs. 3.0 ± 0.3, respectively) populations compared to no heat stress. Heat stress did not significantly change total WBC count. Heterophil/lymphoctye ratio, plasma corticosterone concentration, composite asymmetry score, and plasma HSP 70 concentration are presented in Table 4. No significant 2-way interactions between diet and environment were observed for these measured parameters. The application of the heat stress also resulted in significantly greater (P = 0.02) H/L ratios (heat stress, 0.80 ± 0.06 vs. no heat stress, 0.63 ± 0.05). The H/L ratio was significantly greater (P = 0.01) in the CON birds (0.81 ± 0.05) when compared to those fed XPC (0.62 ± 0.05). Table 4. Heterophil/lymphocyte ratio, plasma corticosterone concentration (pg/mL), composite asymmetry score, and plasma heat-shock protein 70 (pg/mL) for commercial broilers fed a diet with and without Diamond V Original XPCTM and exposed to either a non-heat stress or a heat stress environment. Variable  Heterophil/Lymphocyte Ratio  Plasma Corticosterone Concentration  Composite Asymmetry Score  Plasma HSP 70 Concentration  Diet Main Effect  CON  0.81 ± 0.05a  8433 ± 730a  1.50 ± 0.09a  5.06 ± 0.16  XPC  0.62 ± 0.05b  5129 ± 617b  0.54 ± 0.03b  5.29 ± 0.17  Environment Main Effect  No Heat Stress  0.63 ± 0.05a  5105 ± 444b  0.98 ± 0.10  4.75 ± 0.14b  Heat Stress  0.80 ± 0.06b  8456 ± 846a  1.05 ± 0.08  5.62 ± 0.17a  P-value  Diet Effect  0.01  <0.0001  <0.0001  0.35  Environment Effect  0.02  <0.0001  0.58  <0.0001  Interaction  0.72  0.95  0.88  0.61  Variable  Heterophil/Lymphocyte Ratio  Plasma Corticosterone Concentration  Composite Asymmetry Score  Plasma HSP 70 Concentration  Diet Main Effect  CON  0.81 ± 0.05a  8433 ± 730a  1.50 ± 0.09a  5.06 ± 0.16  XPC  0.62 ± 0.05b  5129 ± 617b  0.54 ± 0.03b  5.29 ± 0.17  Environment Main Effect  No Heat Stress  0.63 ± 0.05a  5105 ± 444b  0.98 ± 0.10  4.75 ± 0.14b  Heat Stress  0.80 ± 0.06b  8456 ± 846a  1.05 ± 0.08  5.62 ± 0.17a  P-value  Diet Effect  0.01  <0.0001  <0.0001  0.35  Environment Effect  0.02  <0.0001  0.58  <0.0001  Interaction  0.72  0.95  0.88  0.61  a,bDifferent letters within column and measure indicate significant differences P < 0.05. View Large Heat stress significantly increased (P < 0.0001) plasma corticosterone concentration (8456 ± 846 vs. 5105 ± 444, respectively) compared to no heat stress. No significant effects of heat stress were observed on composite asymmetry score; however, heat stress did significantly increase the plasma HSP70 concentrations compared to no heat stress (P < 0.0001, heat stress, 5.62 ± 0.17 pg/mL vs. no heat stress, 4.75 ± 0.14 pg/mL). XPC fed birds had a significantly lower stress response as indicated by both plasma corticosterone concentrations (P < 0.0001; 5129 ± 617 vs. 8433 ± 730, respectively) and composite physical asymmetry scores (P < 0.0001; 0.54 ± 0.03 vs. 1.50 ± 0.09, respectively). No significant differences were observed between XPC and CON birds for HSP70 concentrations; DISCUSSION To determine the impact of XPC as an effective dietary additive to help mitigate stress in broilers, it was first necessary to ensure that the model used did, in fact, elicit a stress response from the birds. The parameters measured in this study included: HSP70, CORT, H/L and composite asymmetry and are associated with stress in broilers, as well as bird welfare (Gross and Siegel, 1983; Zulkifli et al., 1999; Campo and Da’vila, 2000; Al-Aqil et al., 2009). In this study, the expression of HSP70 measured in blood plasma as well as plasma corticosterone was increased in birds exposed to heat stress regardless of dietary treatment. Additionally, H/L ratios were also increased in birds exposed to heat stress regardless of dietary treatment. Increases in all of these targeted stress measurements illustrate that the heat stress model used in this study was perceived as a stress in the birds. Therefore, the model used provided an acceptable means to determine the potential use of XPC in reducing stress and improving animal welfare measures. The inclusion of XPC in the treatment diet showed measurable effects on the stress parameters collected during this current study. The H/L ratios were decreased in XPC birds (at 1.25 kg/MT) in this current study. These data agree with the report by Al-Mansour et al. (2011) where reduced H/L ratios were observed after feeding XPC to broilers. Increased H/L ratios indicate increased stress in poultry (Gross and Siegel, 1983; McFarlane and Curtis, 1989) and these results indicate that feeding XPC decreased the stress susceptibility of broilers either during heat stress or during normal rearing conditions. The total WBC population was also reduced by feeding XPC. This further indicates that XPC is playing a role in immune modulation, which has been demonstrated previously by Chou et al. (2017), who observed supplementation with XPC resulted in improved immunocompetence. Birds fed XPC also exhibited a significant reduction in plasma corticosterone in both heat stress and non-heat stress environments, an indication that feeding XPC can reduce stress susceptibility during times of heat stress or during normal rearing conditions. The reduction in corticosterone by feeding XPC is consistent with research in turkeys (Bartz et al., 2015, 2016) which saw similar effects. While corticosterone was reduced by XPC treatment in this current study, HSP70 was not affected. Bensi et al. (1990) and Beutler and Cerami (1989) have shown that HSP70 can actually inhibit the expression of cytokines interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-α). XPC has been shown to up-regulate IL-1, IL-2, and TNF-α in gene extraction studies (Park, 2014). This may further indicate the importance of HSP70 as a protective mechanism in environmental heat stress conditions, but further research is needed to identify the possible relationship between feeding XPC and HSP70 gene expression. It is possible as well that feeding XPC may also affect stress-related gene regulation, though this needs further investigation. Composite asymmetry scoring was improved in birds fed XPC compared to CON; this was observed in both heat stress and non-heat stress environments. The reduction in composite asymmetry further indicates that feeding XPC can reduce stress susceptibility during heat stress or during normal rearing conditions, as asymmetry is an indicator of current stress levels (Kellner and Alford, 2003). The reduction of stress susceptibility when faced with a heat stress or solely the stressors of production (growth, stocking density, etc.) demonstrates that XPC is having a substantial impact on the overall health and well-being. Further research is needed to determine better understand the mechanisms affecting not only the stress response, but also the immune system. The reduction in stress susceptibility observed in this current study and the immune modulation observed by (Chou et al., 2017) may be why others have observed increased growth and feed conversion (Gao et al., 2008, 2009) in birds feed XPC. Overall, the inclusion of XPC in the diet of broiler chickens reduced stress susceptibility in all 3 independent evaluations in this study: H/L ratio, plasma corticosterone, and physical asymmetry, without changes to HSP70. The mechanism for stress reduction is unknown; it may be related to gene expression, as there is evidence that the immune system is modulated in this way by XPC consumption. These data, however, support the use of XPC as a useful management tool for improving the welfare of poultry during times of extreme stress, e.g., elevated temperatures, or the routine management stress associated with rearing. ACKNOWLEDGEMENTS We would like to thank all the undergraduates who helped with this project as well as Denise Caldwell for all her assistance. REFERENCES Al-Aqil A., Zulkifli I., Sazili A. Q., Omar A. R., Rajion M. A.. 2009. The effects of the hot, humid tropical climate and early age feed restriction on stress and fear responses, and performance in broiler chickens. Asian-Australas. J. Anim. Sci.  22: 1581– 1586. Google Scholar CrossRef Search ADS   Al-Mansour S., Al-Khalf A., Al-Homidan I., Fathi M. M.. 2011. Feed efficiency and blood hematology of broiler chicks given a diet supplemented with yeast culture. Int. J. Poult. Sci.  10: 603– 607. Google Scholar CrossRef Search ADS   Archer G. S., Mench J. A.. 2013. 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Poultry ScienceOxford University Press

Published: Mar 1, 2018

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