Effect of dietary synbiotic supplement on behavioral patterns and growth performance of broiler chickens reared under heat stress

Effect of dietary synbiotic supplement on behavioral patterns and growth performance of broiler... Abstract This study examined the effects of a dietary synbiotic supplement on the behavioral patterns and growth performance of broiler chickens exposed to heat stress (HS). Three hundred sixty 1-day-old male Ross 708 broiler chicks were distributed among 24 floor pens (15 chicks per pen); each pen was randomly assigned to one of 3 dietary treatments containing a synbiotic at 0 (control), 0.5 (0.5X) and 1.0 (1.0X) g/kg. From d 15 to 42, birds were exposed to HS at 32°C daily from 08:00 to 17:00. Five broiler chickens were randomly marked in each pen for behavioral observation. Instantaneous scan sampling was used to record the birds’ behavioral patterns. Performance parameters were measured on d 7, 14, 28 and 42. The synbiotic fed birds exhibited more standing, sitting, walking, feeding, preening and less wing spreading and panting behaviors (P < 0.05) compared to birds fed the control diet. The synbiotic group also had higher BW, BW gain and feed intake on d 7, 14 and 42 (P < 0.05), and higher BW, feed intake and feed conversion ratio at d 28 (P < 0.01). There were no treatment effects on drinking behavior, BW gain on d 28 and feed conversion ratio on d 42 (P > 0.05). There were few dose-related differences of the synbiotic on production performance; namely, the 1.0X concentration resulted in the highest BW and feed intake on d 14 and 42 (P < 0.05), while BW gain was higher compared to the control group only on d 42 (P < 0.05). The results suggest that the synbiotic supplement may prove to be an important management tool for the broiler industry to diminish the negative effects of HS, potentially safeguarding the welfare and production of broiler chickens, particularly in areas that experience hot climates. INTRODUCTION Ambient temperature that is elevated beyond the thermoneutral zone can lead to heat stress (HS) which is a common environmental stressor for poultry (Lucas and Rostagno, 2013), due to the increasing proportion of poultry production in tropical and subtropical regions, as well as seasonal effects in moderate climates (Lin et al., 2006). Broiler chickens raised for meat production are particularly susceptible to HS due to the selection for fast growth and feed conversion efficiency (Tan et al., 2010; Soleimani et al., 2011). In broilers, HS is responsible for a reduction in growth rate, immune system impairment, poor meat quality, changes in behavior, and decreased welfare (Sohail et al., 2010; 2012; Mack et al., 2013). Economic analyses suggest an annual loss of $128 million in the poultry industry due to HS (St-Pierre et al., 2003); estimates in today's market are likely higher considering inflation and climate change. One of the biggest contributors to the economic loss associated with HS is the reduction in feed efficiency and growth of broilers. Several studies have quantified the relationship between HS and growth; for example, Ain Baziz et al. (1996) reported that feed consumption was reduced by 3.6% for every 1 °C increase in environmental temperature. Further, Sohail et al. (2012) reported a decrease in BW by 32.6%, with a higher feed conversion ratio (+25.6%) by the end of the production period. To cope with the effects of HS, broiler chickens modify their behavior in an attempt to return their body temperature to thermoneutral conditions. Previous research suggests that walking and standing behaviors, as well as feed intake, are reduced during HS conditions, while sitting, drinking, wing elevation, and panting behaviors are increased in order to dissipate excess heat (Gowe et al., 2008; Mack et al., 2013; Sohail et al., 2013). Lifting wings away from the body (i.e., “wing spreading”) exposes the skin of the apteria (i.e., the area under the wings), which promotes heat transfer to the environment (Gerken et al., 2006), while panting increases water lost through evaporative cooling, and consuming water replenishes this loss (Gowe et al., 2008). These changes in behaviors suggest exposed birds experience discomfort, which provides further evidence that HS causes a reduction in birds’ welfare. In addition to the negative effects on broiler performance and behavioral exhibition, HS activates the hypothalamic-pituitary-adrenal axis (HPA) and alters the microbial composition in the gut of poultry (Burkholder et al., 2008; Yu et al., 2012), causing proliferation of harmful pathogens such as Salmonella and Escherichia coli (Park et al., 2013) and increased susceptibility to disease and mortality (Quinteiro-Filho et al., 2012). Probiotics are live microorganisms that beneficially affect the host by improving its microbial intestinal balance (Fuller, 1989) and improvements in growth performance and feed efficiency in broiler chickens following dietary supplementation with probiotics have been reported in numerous studies (e.g., Cavazzoni et al., 1998; Zulkifli et al., 2000; Al-Fataftah and Abdelqader, 2014; Jahromi et al., 2015), although conflicting results have also been reported (Sandikci et al., 2004; Sohail et al., 2015). Synbiotics are synergistic combinations of prebiotics and probiotics (de Vrese and Schrezenmeir, 2008); prebiotics benefit the host by selectively stimulating the growth and activity of one or more bacteria in the colon (Gibson and Roberfroid, 1995). Together, prebiotics and probiotics work to improve the survival and implantation of beneficial bacteria in the gastrointestinal tract (Awad et al., 2009), and regulate biological functions and behavioral exhibition through both local and systematic pathways, i.e., the microbiota-gut-brain axis (Gareau, 2014) and microbiota-gut-immune axis (Rooks and Garrett, 2016). Therefore, using synbiotics to alter microbial populations and ameliorate the effects of HS may be more beneficial than the application of prebiotics or probiotics alone. Several studies have shown promise in protecting birds against the harmful effects of HS with synbiotics (Sohail et al., 2010; 2011; 2013; Ashraf et al., 2013), although a few studies have reported no effects (Sandikci et al., 2004; Sohail et al., 2015). Differences in the composition or concentration of the synbiotics may be responsible for these conflicting reports. Further, previous studies have not reported changes in behavior or welfare following supplementation with synbiotics. Therefore, the objective of this study was to investigate the effects of dietary supplementation of a synbiotic (a combination of fructo-oligosaccharides and 4 mixed microbial strains) on the behavioral patterns and performance of heat stressed Ross 708 broiler chickens. We hypothesized that the dietary synbiotic supplement would mitigate the effects of HS, resulting in decreased HS-associated behavior, wing spreading and panting, and increased feed intake and BW gain. MATERIALS AND METHODS Synbiotic The commercial synbiotic (PoultryStar® meUS, BIOMIN America Inc., San Antonio, TX) used in this study contained a prebiotic (fructo-oligosaccharides) and a probiotic mixture of 4 microbial strains selected from the different segments of the gastrointestinal tract (Lactobacillus reuteri isolated from the crop; Enterococcus faecium from the jejunum; Bifidobacterium animalis from the ileum; and Pediococcus acidilactici from the cecum). The probiotic mixture was selected for its efficacy to exclude pathogenic bacteria and maintain a healthy gut microbial population, while the prebiotic has been suggested to further modify the activity and growth of beneficial microflora. Its effects and survivability have been tested in previous studies (McReynolds et al., 2009; Murugesan and Persia, 2015; Yan et al., 2015). Animals and Housing All procedures were approved by the Purdue Animal Care and Use Committee prior to the start of the experiment (PACUC Number: 1,111,000,262). Three hundred sixty male broiler chicks (Ross 708 strain; Pine Manor/Miller Poultry, Goshen, IN) were weighed and assigned to 24 floor pens (110 cm × 110 cm per pen) with equal average BW in a temperature controlled room at the Poultry Research Farm of Purdue University. Management of the broilers followed the guidelines of Aviagen (2015). The chicks were maintained at a temperature of approximately 34°C at d 1 with a gradual reduction in temperature to 27°C on d 14. Heat stimulation began on d 15 (i.e., the beginning of the growth phase) at 32°C for 9 h (08:00–17:00) daily until the end of the experiment. Data loggers (HOBO®, Onset Computer Corporation, MA) were placed in the room to record the room temperature and humidity throughout the experiment (Table 1). A thermoneutral control group was not used in this study as we aimed to investigate the effect of synbiotics on heat stressed broiler chickens and 32°C was guaranteed to induce HS based on the results of a previous study (Mahmoud et al., 2015). Narrowing focus to include only HS birds allowed us to reduce animal use by 50%, a key priority of animal welfare scientists (i.e., the 3Rs principal (Russell and Burch, 1959)). Table 1. Temperature and humidity levels throughout the experimental period. Bird age  Temperature  Humidity    Day time (08:00–17:00)  Night time (17:00–08:00)  Day time (08:00–17:00)  Night time (17:00–08:00)  d 15–21  31.87 ± 0.27  25.87 ± 0.36  49.16 ± 1.54  55.12 ± 1.58  d 22–28  31.58 ± 0.27  25.36 ± 0.18  50.94 ± 0.79  52.71 ± 1.50  d 29–35  31.67 ± 0.51  25.45 ± 0.29  60.03 ± 1.72  61.37 ± 1.28  d 36–42  31.57 ± 0.29  25.36 ± 0.12  57.52 ± 1.24  53.65 ± 1.32  Bird age  Temperature  Humidity    Day time (08:00–17:00)  Night time (17:00–08:00)  Day time (08:00–17:00)  Night time (17:00–08:00)  d 15–21  31.87 ± 0.27  25.87 ± 0.36  49.16 ± 1.54  55.12 ± 1.58  d 22–28  31.58 ± 0.27  25.36 ± 0.18  50.94 ± 0.79  52.71 ± 1.50  d 29–35  31.67 ± 0.51  25.45 ± 0.29  60.03 ± 1.72  61.37 ± 1.28  d 36–42  31.57 ± 0.29  25.36 ± 0.12  57.52 ± 1.24  53.65 ± 1.32  View Large Dietary Treatments The 24 pens were randomly assigned to 3 dietary treatments with 8 replicates of 15 broiler chickens each: a regular diet mixed with the synbiotic product at 0 (control), 0.5 (106 cfu/g) (0.5X) and 1.0 (2 × 106 cfu/g) (1.0X) g/kg feed. The PoultryStar® dietary treatments were supplied from d 1 to 42 and made by the step-up procedure as explained in detail by Mahmoud et al. (2015). In brief, the respective amount of PoultryStar® was mixed with a small amount of the regular diet as a small batch, and then incorporated with a larger amount of the diet gradually, until the total amount of each of the particular diets was homogeneously mixed. The base of the diet was formulated according to growth stage requirements (Table 2). Table 2. Components of base diet,1 separated by growth phase. Ingredient, %  Starter  Grower  Finisher    (1–14 d)  (15–28 d)  (29–42 d)  Corn  52.00  52.30  62.80  Soybean meal,48% CP  40.00  39.10  29.70  Soy oil  3.59  4.97  4.11  Sodium chloride  0.51  0.46  0.43  DL Methionine  0.30  0.24  0.23  L-Lysine HCL  0.13  -—-  0.07  Threonine  0.06  -—-  -—-  Limestone  1.29  1.15  1.12  Monocalcium phosphate  1.75  1.48  1.17  Vitamin/mineral premix2  0.35  0.35  0.35  Calculated analyses  Crude protein %  23.40  22.80  19.20  Poultry ME kcal/kg  3050  3151  3200  Calcium %  0.95  0.85  0.75  Available phosphorus %  0.50  0.44  0.36  Methionine %  0.66  0.59  0.53  Methionine+Cystine %  1.04  0.97  0.86  Lysine %  1.42  1.29  1.09  Threonine %  0.97  0.89  0.74  Na %  0.22  0.20  0.19  Ingredient, %  Starter  Grower  Finisher    (1–14 d)  (15–28 d)  (29–42 d)  Corn  52.00  52.30  62.80  Soybean meal,48% CP  40.00  39.10  29.70  Soy oil  3.59  4.97  4.11  Sodium chloride  0.51  0.46  0.43  DL Methionine  0.30  0.24  0.23  L-Lysine HCL  0.13  -—-  0.07  Threonine  0.06  -—-  -—-  Limestone  1.29  1.15  1.12  Monocalcium phosphate  1.75  1.48  1.17  Vitamin/mineral premix2  0.35  0.35  0.35  Calculated analyses  Crude protein %  23.40  22.80  19.20  Poultry ME kcal/kg  3050  3151  3200  Calcium %  0.95  0.85  0.75  Available phosphorus %  0.50  0.44  0.36  Methionine %  0.66  0.59  0.53  Methionine+Cystine %  1.04  0.97  0.86  Lysine %  1.42  1.29  1.09  Threonine %  0.97  0.89  0.74  Na %  0.22  0.20  0.19  1The ration formulation was produced according to Aviagen (2015) 2Provided per kilogram of diet: vitamin A, 13,233 IU; vitamin D3, 6,636 IU; vitamin E, 44.1 IU; vitamin K, 4.5 mg; thiamine, 2.21 mg; riboflavin, 6.6 mg; pantothenic acid, 24.3 mg; niacin, 88.2 mg; pyridoxine, 3.31 mg; folic acid, 1.10 mg; biotin, 0.33 mg; vitamin B12, 24.8 μg; choline, 669.8 mg; iron from ferrous sulfate, 50.1 mg; copper from copper sulfate, 7.7 mg; manganese from manganese oxide, 125.1 mg; zinc from zinc oxide, 125.1 mg; iodine from ethylene diamine dihydroidide, 2.10 mg; selenium from sodium selenite, 0.30 mg. View Large Behavioral Observations Five broiler chickens per pen (40 total per treatment) were randomly selected for observation and marked with livestock spray marker on their backs (Livestock green sharp-mark spray paint marker, Cotran Corporation, Portsmouth, RI). Behavioral observation was performed twice daily from 10:00 to 11:00 and 14:00 to 15:00 3 times per wk (Monday—Wednesday) from d 15 to 42. Behavioral data was collected on alternative days to production data collection to avoid interrupting normal behavior. Behaviors of focal birds were collected according to an ethogram (Table 3) and scored 6 times per observation session using instantaneous scan sampling (Altmann, 1974; Engel, 1996). The focal broiler chickens were observed from outside of the pens at a distance of 1.5 m to avoid disturbance of the normal behavioral repertoire. Data are presented as a proportion of observed behaviors out of the total number possible (Kristensen et al., 2007). Table 3. Ethogram of broiler behaviors collected during heat stress condition. Behavior1  Definition  Standing  The feet are in contact with the ground. No other body part is touching the floor surface. The birds’ body posture is in an upright position.  Sitting  The ventral part of the bird is in contact with the ground. Legs are bent at the knee with the fibula and tibia (i.e., lower part of the leg, under the knee) touching the ground.  Walking  The bird moves at least 2 steps in succession. This may or may not include scratching at the litter with feet.  Feeding  The bird's head is located inside the feeder, presumably consuming feed.  Drinking  The bird is observed pecking at the drinker, presumably consuming water.  Preening  The bird is manipulating its own feathers with beak gently.  Wing Spreading  Wings are extended horizontally from the body such that a space can be seen between the underside of the wing and the surface of the bird's body.  Panting  The bird is breathing with an open beak and respiration rate is abnormally rapid.  Behavior1  Definition  Standing  The feet are in contact with the ground. No other body part is touching the floor surface. The birds’ body posture is in an upright position.  Sitting  The ventral part of the bird is in contact with the ground. Legs are bent at the knee with the fibula and tibia (i.e., lower part of the leg, under the knee) touching the ground.  Walking  The bird moves at least 2 steps in succession. This may or may not include scratching at the litter with feet.  Feeding  The bird's head is located inside the feeder, presumably consuming feed.  Drinking  The bird is observed pecking at the drinker, presumably consuming water.  Preening  The bird is manipulating its own feathers with beak gently.  Wing Spreading  Wings are extended horizontally from the body such that a space can be seen between the underside of the wing and the surface of the bird's body.  Panting  The bird is breathing with an open beak and respiration rate is abnormally rapid.  1All behaviors were mutually exclusive; postures (i.e., standing and sitting) were only counted if the bird performed no other simultaneous behaviors. View Large Growth Performance BW, BW gain, feed intake and feed conversion ratio were recorded on d 7, 14 (the end of starter phase), 28 (the end of grower phase) and 42 (the end of finisher phase). Data collected on d 7 and 14 were used to assess the effect of synbiotics prior to HS, while d 28 and 42 were collected during HS conditions. All birds within a pen were measured at each time point (i.e., 15 birds were sampled in each pen, with 8 replicates per treatment). The BW gain was calculated as the BW of the current time point subtracted the BW of the previous time point. Statistical Analysis The experimental design was conducted in a randomized block design. Pen was considered the experimental unit (n = 8). The overall effects of the synbiotic supplementation on broiler chicken behaviors and performance were analyzed statistically by repeated measures ANOVA. Means of the data were analyzed by using PROC MIXED model with SAS 9.4 software (SAS Institute Inc., Cary, NC). The normality of the data was analyzed by the Shapiro-Wilk test. Transformation of the data was performed for normality when variances were not homogeneous (Steel et al., 1997). BW, BW gain, feed intake, feed conversion ratio, standing, sitting, preening, wing spreading, and panting were log transformed. Statistical trends were similar for both transformed and untransformed data, the untransformed results were presented. Means were compared by Tukey-Kramer test when a significant difference was detected at a probability of α less than 0.05. Expression of the findings is reported as the mean ±  SE. RESULTS Behavioral Patterns The overall relationships between the synbiotic supplementation and behavioral activities are presented in Table 4. The synbiotic fed broiler chickens spent more time standing (F(2, 29.72) = 6.80, P < 0.01), walking (F(2, 36.9) = 139.73, P < 0.001), sitting (F(2, 23.87) = 49.73, P < 0.001), preening (F(2, 26.4) = 16.38, P < 0.001), and feeding (F(2, 23.9) = 11.50, P < 0.001) in comparison to the controls, while wing spreading (F(2, 23.32) = 27.72, P < 0.001) and panting (F(2, 20.56) = 62.30, P < 0.001) were lower in the synbiotic groups than the control groups. There were no differences in drinking behavior (F(2, 25) = 1.32, P = 0.285) between the groups. No statistical differences were found between synbiotic dosage levels, except that the percentage of walking behavior was further increased in the 0.5X group in comparison with the control and 1.0X groups (t(36.9) = 16.17, P < 0.001; t(36.9) = 4.41, P < 0.001, respectively). Table 4. Effect of dietary supplementation of synbiotic (PoultryStar) on behaviors of broiler chickens under heat stress condition. Treatment1  Control  0.5X  1.0X  P-value  Behavior          Standing (%)  3.03b ± 0.23  4.24a ± 0.24  4.01a ± 0.23  0.004  Sitting (%)  11.75b ± 0.97  28.30a ± 1.02  27.42a ± 1.00  0.001  Walking (%)  2.73c ± 0.15  6.16a ± 0.15  5.23b ± 0.15  0.001  Feeding (%)  12.85b ± 0.89  17.25a ± 0.89  18.62a ± 0.89  0.001  Drinking (%)  5.54 ± 0.02  6.38 ± 0.02  6.06 ± 0.02  0.285  Preening (%)  2.86b ± 0.29  5.44a ± 0.31  5.30a ± 0.29  0.001  Wing Spreading (%)  9.17a ± 0.49  4.55b ± 0.52  3.66b ± 0.56  0.001  Panting (%)  50.29a ± 1.58  27.31b ± 1.77  28.07b ± 1.63  0.001  Treatment1  Control  0.5X  1.0X  P-value  Behavior          Standing (%)  3.03b ± 0.23  4.24a ± 0.24  4.01a ± 0.23  0.004  Sitting (%)  11.75b ± 0.97  28.30a ± 1.02  27.42a ± 1.00  0.001  Walking (%)  2.73c ± 0.15  6.16a ± 0.15  5.23b ± 0.15  0.001  Feeding (%)  12.85b ± 0.89  17.25a ± 0.89  18.62a ± 0.89  0.001  Drinking (%)  5.54 ± 0.02  6.38 ± 0.02  6.06 ± 0.02  0.285  Preening (%)  2.86b ± 0.29  5.44a ± 0.31  5.30a ± 0.29  0.001  Wing Spreading (%)  9.17a ± 0.49  4.55b ± 0.52  3.66b ± 0.56  0.001  Panting (%)  50.29a ± 1.58  27.31b ± 1.77  28.07b ± 1.63  0.001  a,b,cMean ± SE with different superscripts in the same row differ (P < 0.01). 1Dietary treatments containing 0 (control), 0.5 (0.5X) and 1.00 (1.0X) gkg−1 synbiotic, n = 8 per treatment (pen was considered the experimental unit, 5 birds per pen were observed, percentage of each behavior was calculated separately using 40 birds per treatment). Heat stimulation began on d 15 at 32°C for 9 h daily until d 42. View Large Growth Performance The relationships between the synbiotic supplementation and growth performance are presented in Table 5. Under thermoneutral condition, the synbiotic fed broilers had higher BW, BW gain, and feed intake, but lower feed conversion ratio in comparison with the controls on d 7 (F(2, 22.99) = 32.04, P < 0.001; F(2,19.37) = 3.80, P < 0.05; F(2, 19.73) = 83.07, P < 0.001; F(2, 20.97) = 15.69, P < 0.001, respectively) and d 14 (F(2, 23.85) = 98.29, P < 0.001; F(2, 24.23) = 96.41, P < 0.001; F(2, 21.01) = 29.53, P < 0.001; F(2, 20.32) = 6.66, P < 0.01, respectively). Under HS condition, compared to broilers fed the control diet, the synbiotic group had higher BW (F(2, 21) = 15.21, P < 0.001), feed intake (F(2, 21) = 19.39, P < 0.001), and feed conversion ratio (F(2, 21) = 7.46, P < 0.01) on d 28, and higher BW (F(2, 20.01) = 18.61, P < 0.001), BW gain (F(2, 19.21) = 3.95, P < 0.05), and feed intake (F(2, 20.36) = 10.41, P < 0.001) on d 42, while BW gain (F(2, 21) = 2.31, P = 0.124) on d 28 and feed conversion ratio (F(2, 21) = 0.78, P = 0.473) on d 42 were not significantly different between treatments. Table 5. Effect of dietary supplementation of synbiotic (PoultryStar) on performance parameters of heat stressed broiler chickens at different growth ages. Treatment1  Control  0.5X  1.0X  P-value  d7          BW (g)  133.69b ± 1.16  145.90a ± 1.16  144.40a ± 1.16  0.001  BW gain (g)  86.69c ± 0.11  94.55a ± 0.11  91.15b ± 0.11  0.041  Feed intake (g)  96.63b ± 0.72  108.73a ± 0.72  107.53a ± 0.72  0.001  Feed conversion ratio (g/g)  0.89a ± 0.01  0.86b ± 0.01  0.84c ± 0.01  0.001  d14          BW (g)  446.33c ± 3.85  501.19b ± 3.85  521.73a ± 3.85  0.001  BW gain (g)  312.62c ± 3.28  355.30b ± 3.28  377.33a ± 3.28  0.001  Feed intake (g)  343.09b ± 7.89  364.10b ± 7.89  424.57a ± 7.89  0.001  Feed conversion ratio (g/g)  1.10a ± 0.02  1.02b ± 0.02  1.12a ± 0.02  0.006  d28          BW (g)  1437.48b ± 13.68  1505.67a ± 13.68  1543.36a ± 13.68  0.001  BW gain (g)  991.15±11.08  1004.48±11.08  1024.53±11.08  0.124  Feed intake (g)  1471.41b ± 13.01  1549.34a ± 13.01  1584.11a ± 13.01  0.001  Feed conversion ratio (g/g)  1.48b ± 0.01  1.54a ± 0.01  1.54a ± 0.01  0.004  d42          BW (g)  2412.50c ± 22.14  2515.86b ± 22.14  2615a ± 22.14  0.001  BW gain (g)  1076.77b ± 21.60  1099.13a,b ± 21.60  1162.77a ± 21.60  0.027  Feed intake (g)  2144.83b ± 27.73  2202.19b ± 27.73  2323.61a ± 27.73  0.001  Feed conversion ratio (g/g)  1.99 ± 0.08  1.88 ± 0.08  1.99 ± 0.08  0.473  Treatment1  Control  0.5X  1.0X  P-value  d7          BW (g)  133.69b ± 1.16  145.90a ± 1.16  144.40a ± 1.16  0.001  BW gain (g)  86.69c ± 0.11  94.55a ± 0.11  91.15b ± 0.11  0.041  Feed intake (g)  96.63b ± 0.72  108.73a ± 0.72  107.53a ± 0.72  0.001  Feed conversion ratio (g/g)  0.89a ± 0.01  0.86b ± 0.01  0.84c ± 0.01  0.001  d14          BW (g)  446.33c ± 3.85  501.19b ± 3.85  521.73a ± 3.85  0.001  BW gain (g)  312.62c ± 3.28  355.30b ± 3.28  377.33a ± 3.28  0.001  Feed intake (g)  343.09b ± 7.89  364.10b ± 7.89  424.57a ± 7.89  0.001  Feed conversion ratio (g/g)  1.10a ± 0.02  1.02b ± 0.02  1.12a ± 0.02  0.006  d28          BW (g)  1437.48b ± 13.68  1505.67a ± 13.68  1543.36a ± 13.68  0.001  BW gain (g)  991.15±11.08  1004.48±11.08  1024.53±11.08  0.124  Feed intake (g)  1471.41b ± 13.01  1549.34a ± 13.01  1584.11a ± 13.01  0.001  Feed conversion ratio (g/g)  1.48b ± 0.01  1.54a ± 0.01  1.54a ± 0.01  0.004  d42          BW (g)  2412.50c ± 22.14  2515.86b ± 22.14  2615a ± 22.14  0.001  BW gain (g)  1076.77b ± 21.60  1099.13a,b ± 21.60  1162.77a ± 21.60  0.027  Feed intake (g)  2144.83b ± 27.73  2202.19b ± 27.73  2323.61a ± 27.73  0.001  Feed conversion ratio (g/g)  1.99 ± 0.08  1.88 ± 0.08  1.99 ± 0.08  0.473  a,b,cMean ± SE with different superscripts in the same row differ significantly (P < 0.05). 1Basal dietary supplemented with 0 (Control), 0.5 (0.5X) and 1.00 (1.0X) gkg−1 synbiotic, n = 8 per treatment (pen was considered the experimental unit, all 15 birds/pen were used to measure performance parameters and averaged for analysis). Heat stimulation began on d 15 at 32°C for 9 h daily until d 42. View Large There were some differences in growth performance results between the 2 concentrations of synbiotics and the control diet. The 1.0X group had the highest BW, BW gain and feed intake on d 14 (thermoneutral conditions) compared to control group (t(23.85) = 13.76, P < 0.001; t(24.23) = 13.81, P < 0.001; t(21.01) = 7.21, P < 0.001, respectively) and 0.5X group (t(23.85) = 3.77, P < 0.001; t(24.23) = 4.75, P < 0.001; t(21.01) = 5.38, P < 0.001, respectively). The 1.0X group also had higher BW and feed intake on d 42 (HS condition) compared to the control group (t(20.01) = 6.10, P < 0.001; t(20.36) = 4.44, P < 0.001, respectively) and the 0.5X group (t(20.01) = 2.99, P < 0.001; t(20.36) = 3.02, P < 0.01, respectively), while BW gain was higher in the 1.0X group on d 42 compared to the control group only (t(19.21) = 2.68, P < 0.05). DISCUSSION Environmental stressors, including HS, are responsible for decreased health, welfare, and production coupled with undesirable meat quality on color, water-holding capacity, tenderness, and/or pale, soft, and exudative breast muscle (Zhang et al., 2012; Fouad et al., 2016) in broiler chickens. Increasing the beneficial bacteria in the gastrointestinal tract has shown promise in increasing growth performance and mitigating the negative effects of HS, thereby safeguarding poultry welfare and economic gains for the industry. When poultry experience stress, including HS, the neuroendocrine system is altered causing dysregulation of the HPA axis and increased plasma corticosterone concentration (Quinterio-Filho et al., 2010). A direct link has been established between microbiota and HPA reactivity; mice lacking a commensal bacterial population (i.e., “germ-free”) had exaggerated corticosterone and adrenocorticotrophin response to stressors (Sudo et al., 2004). Increased corticosterone levels have been associated with numerous behavioral changes indicating heightened states of anxiety, depression, and aggression (Gregus et al., 2005; O’Mahony et al., 2009). Beneficial bacteria, such as probiotics, have been demonstrated to protect or improve the function of the microbiome, leading to improved regulation of the HPA axis and behavior (Cryan and O’Mahony, 2011; Mayer et al., 2015). Under HS conditions, birds reduce their feed intake and activities as a mechanism to decrease the production of body heat (Sohail et al., 2010; 2013). As a result, metabolism is impaired and decreased BW, BW gain, and increased feed conversion ratios are observed (Sohail et al., 2012). Our results suggest that dietary synbiotics can improve production profiles as feed intake, BW and BW gain increased in birds supplemented with synbiotics compared to those consuming a regular diet under both thermoneutral (d 7 and 14) and HS conditions (d 42). Similar to our results, several authors have also reported increased production profiles when supplementing birds with either probiotics or synbiotics under stressful conditions (Fayed and Tony, 2008; Hosseini et al., 2013; Al-Fataftah and Abdelqader, 2014; Jahromi et al., 2015) and normal environmental temperature (Awad et al., 2009; Riad et al., 2010; Saiyed et al., 2015; Sarangi et al., 2016), although others reported no differences in production under stressful conditions (Hassan et al., 2007; Sohail et al., 2012; 2013) and thermoneutral conditions (Salehimanes et al., 2016). Variation in the effects of probiotics and synbiotics in previous studies may be attributed to the differences in strains of bacteria and their concentrations in the diet, as reviewed by Jin et al. (1997). The mechanisms by which bacteria regulate changes in the gastrointestinal tract differ based on the type of prebiotic or probiotic introduced (Patterson and Burkholder, 2003); for example, Sohail et al. (2012, 2013) used mannanoligosaccharides (MOS) as the prebiotic in their synbiotic mixture; unlike the fructo-oligosaccharide prebiotic used in our study, which selectively enriches beneficial bacterial populations, MOS has been suggested to act by binding and removing pathogens from the intestinal tract (Spring et al., 2000). Further research should be conducted to determine the exact mechanisms by which certain combinations of probiotics and prebiotics control growth performance and influence the effects of stress to determine which compositions may be most beneficial. Considering the positive results on production-related measurements in this study the mechanisms and application of this bacterial combination could provide a useful basis for future investigations. In addition to the composition of bacteria, the concentration of the synbiotic may influence the effect on broiler production during stressful conditions. Our study investigated the synbiotic at 2 different concentrations: 0.5X and 1.0X, based on the company's recommendation. A few differences were found between the dosages, namely, at the 1.0X concentration, BW and feed intake were increased compared to the 0.5X and control treatments under thermoneutral (d 14) and HS (d 42) conditions, while BW gain was higher in the 1.0X group compared to the control group only at d 42 (end of the study). Considering the potential for increased production throughout the broiler lifespan, a 1.0X concentration of dietary synbiotics could be utilized. Under HS condition, broiler chickens display a number of behaviors to improve thermoregulation and reduce HS effects, including increased respiratory rate, panting (i.e., open-mouth breathing), and wing spreading (Pereira et al., 2007; Li et al., 2015). The current results showed that, compared to the effects of regular diet, supplementing with synbiotics reduced panting and wing-spreading activities of broiler chickens under an elevated temperature condition. Generally, broiler chickens release body heat through panting and wing spreading due to the lack of sweat glands. Panting is used to dissipate heat through evaporation from the respiratory tract, while wing spreading enhances heat loss by increasing the body surface, exposing more unfeathered areas, and reducing the isolating capacity of the feather cover (Lolli et al., 2010). The beneficial effect of synbiotics on panting and wing spreading activities suggests the birds may have experienced less of an effect of HS. A previous study investigating the effects of probiotics (Lactobacillus spp.) on broiler chickens subjected to HS conditions noted an increase in erythrocyte count, hemoglobin concentration, and hematocrit value; all hematological changes that are associated with improved respiratory ability (Hasan et al., 2015). Although not measured in this study, similar hematological changes may be responsible for reduced HS-associated behaviors in our broilers. Additionally, in our study, synbiotic-fed broilers spent more time standing and walking during HS conditions. The increased movement activities may be related to the improved musculoskeletal health from synbiotic supplementation. A parallel study showed that a probiotic (Bacillus subtilis) improved the bone health of broiler chickens under HS as indicated by higher bone mineral content of the femur and tibia (Yan et al., 2016). Several studies have reported an improvement in bone mineralization due to an increase in bone ash, calcium and phosphorus contents following supplementation with probiotics (Houshmand et al., 2011; Narasimha et al., 2013). However, our results also suggest that sitting behavior increased with supplemental synbiotics. The reasons for the increased sitting behavior in the synbiotic fed birds are unclear but may be related to a decrease in the fear response as demonstrated by several rodent studies; for example, probiotics modulated cognitive processes in stressed mice (Bifidobacteria, Savignac et al., 2015) and reduced fear relapse in rats experiencing maternal separation anxiety (Lactobacillus, Cowan et al., 2016). Alternatively, heavier BW may have increased sitting behavior in the synbiotic groups. Finally, preening activities were increased in the synbiotic supplemented broilers in our study. In support, Fayed and Tony (2008) reported an increase in preening behavior when broiler chickens were fed probiotics during stress experienced by high stocking density. Preening has been considered a comfort-related behavior in poultry (Hinde, 1970) and it is possible that supplementation with synbiotics decreased the discomfort associated with HS. In all, the observed behavioral effects of synbiotics suggest that birds experienced less stress when exposed to high temperatures compared to birds fed a regular diet. However, surprisingly, drinking behavior was not different between treatments. If synbiotics were to decrease water loss under HS conditions, we would expect to observe less drinking behavior. It is possible that synbiotics help dissipate heat loss through the skin (as seen with decreased wing spreading), but are not as effective at controlling overall water loss. Further studies should explore these relationships to determine the degree to which HS effects are controlled by synbiotics. CONCLUSION In the current study, both doses of the synbiotic supplement significantly increased walking, sitting, preening activities while reducing panting and wing spreading (i.e., heat-associated behaviors) in broiler chickens exposed to HS. Overall, our results suggest that supplementing the broiler diet with synbiotics may be useful to improve production profiles, even during HS conditions. Further, supplementing with a 1.0X concentration resulted in a higher increase in feed intake, BW and BW gain at the end of the broiler cycle suggesting the synbiotic may be most effective at this dosage. The poultry industry could consider supplementing the broiler diet with synbiotics as a management strategy, particularly in hot weather or climates, in order to protect broiler production from the negative effects of stress. 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Effect of dietary synbiotic supplement on behavioral patterns and growth performance of broiler chickens reared under heat stress

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Oxford University Press
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Published by Oxford University Press on behalf of Poultry Science Association 2017. This work is written by (a) US Government employee(s) and is in the public domain in the US.
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0032-5791
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Abstract

Abstract This study examined the effects of a dietary synbiotic supplement on the behavioral patterns and growth performance of broiler chickens exposed to heat stress (HS). Three hundred sixty 1-day-old male Ross 708 broiler chicks were distributed among 24 floor pens (15 chicks per pen); each pen was randomly assigned to one of 3 dietary treatments containing a synbiotic at 0 (control), 0.5 (0.5X) and 1.0 (1.0X) g/kg. From d 15 to 42, birds were exposed to HS at 32°C daily from 08:00 to 17:00. Five broiler chickens were randomly marked in each pen for behavioral observation. Instantaneous scan sampling was used to record the birds’ behavioral patterns. Performance parameters were measured on d 7, 14, 28 and 42. The synbiotic fed birds exhibited more standing, sitting, walking, feeding, preening and less wing spreading and panting behaviors (P < 0.05) compared to birds fed the control diet. The synbiotic group also had higher BW, BW gain and feed intake on d 7, 14 and 42 (P < 0.05), and higher BW, feed intake and feed conversion ratio at d 28 (P < 0.01). There were no treatment effects on drinking behavior, BW gain on d 28 and feed conversion ratio on d 42 (P > 0.05). There were few dose-related differences of the synbiotic on production performance; namely, the 1.0X concentration resulted in the highest BW and feed intake on d 14 and 42 (P < 0.05), while BW gain was higher compared to the control group only on d 42 (P < 0.05). The results suggest that the synbiotic supplement may prove to be an important management tool for the broiler industry to diminish the negative effects of HS, potentially safeguarding the welfare and production of broiler chickens, particularly in areas that experience hot climates. INTRODUCTION Ambient temperature that is elevated beyond the thermoneutral zone can lead to heat stress (HS) which is a common environmental stressor for poultry (Lucas and Rostagno, 2013), due to the increasing proportion of poultry production in tropical and subtropical regions, as well as seasonal effects in moderate climates (Lin et al., 2006). Broiler chickens raised for meat production are particularly susceptible to HS due to the selection for fast growth and feed conversion efficiency (Tan et al., 2010; Soleimani et al., 2011). In broilers, HS is responsible for a reduction in growth rate, immune system impairment, poor meat quality, changes in behavior, and decreased welfare (Sohail et al., 2010; 2012; Mack et al., 2013). Economic analyses suggest an annual loss of $128 million in the poultry industry due to HS (St-Pierre et al., 2003); estimates in today's market are likely higher considering inflation and climate change. One of the biggest contributors to the economic loss associated with HS is the reduction in feed efficiency and growth of broilers. Several studies have quantified the relationship between HS and growth; for example, Ain Baziz et al. (1996) reported that feed consumption was reduced by 3.6% for every 1 °C increase in environmental temperature. Further, Sohail et al. (2012) reported a decrease in BW by 32.6%, with a higher feed conversion ratio (+25.6%) by the end of the production period. To cope with the effects of HS, broiler chickens modify their behavior in an attempt to return their body temperature to thermoneutral conditions. Previous research suggests that walking and standing behaviors, as well as feed intake, are reduced during HS conditions, while sitting, drinking, wing elevation, and panting behaviors are increased in order to dissipate excess heat (Gowe et al., 2008; Mack et al., 2013; Sohail et al., 2013). Lifting wings away from the body (i.e., “wing spreading”) exposes the skin of the apteria (i.e., the area under the wings), which promotes heat transfer to the environment (Gerken et al., 2006), while panting increases water lost through evaporative cooling, and consuming water replenishes this loss (Gowe et al., 2008). These changes in behaviors suggest exposed birds experience discomfort, which provides further evidence that HS causes a reduction in birds’ welfare. In addition to the negative effects on broiler performance and behavioral exhibition, HS activates the hypothalamic-pituitary-adrenal axis (HPA) and alters the microbial composition in the gut of poultry (Burkholder et al., 2008; Yu et al., 2012), causing proliferation of harmful pathogens such as Salmonella and Escherichia coli (Park et al., 2013) and increased susceptibility to disease and mortality (Quinteiro-Filho et al., 2012). Probiotics are live microorganisms that beneficially affect the host by improving its microbial intestinal balance (Fuller, 1989) and improvements in growth performance and feed efficiency in broiler chickens following dietary supplementation with probiotics have been reported in numerous studies (e.g., Cavazzoni et al., 1998; Zulkifli et al., 2000; Al-Fataftah and Abdelqader, 2014; Jahromi et al., 2015), although conflicting results have also been reported (Sandikci et al., 2004; Sohail et al., 2015). Synbiotics are synergistic combinations of prebiotics and probiotics (de Vrese and Schrezenmeir, 2008); prebiotics benefit the host by selectively stimulating the growth and activity of one or more bacteria in the colon (Gibson and Roberfroid, 1995). Together, prebiotics and probiotics work to improve the survival and implantation of beneficial bacteria in the gastrointestinal tract (Awad et al., 2009), and regulate biological functions and behavioral exhibition through both local and systematic pathways, i.e., the microbiota-gut-brain axis (Gareau, 2014) and microbiota-gut-immune axis (Rooks and Garrett, 2016). Therefore, using synbiotics to alter microbial populations and ameliorate the effects of HS may be more beneficial than the application of prebiotics or probiotics alone. Several studies have shown promise in protecting birds against the harmful effects of HS with synbiotics (Sohail et al., 2010; 2011; 2013; Ashraf et al., 2013), although a few studies have reported no effects (Sandikci et al., 2004; Sohail et al., 2015). Differences in the composition or concentration of the synbiotics may be responsible for these conflicting reports. Further, previous studies have not reported changes in behavior or welfare following supplementation with synbiotics. Therefore, the objective of this study was to investigate the effects of dietary supplementation of a synbiotic (a combination of fructo-oligosaccharides and 4 mixed microbial strains) on the behavioral patterns and performance of heat stressed Ross 708 broiler chickens. We hypothesized that the dietary synbiotic supplement would mitigate the effects of HS, resulting in decreased HS-associated behavior, wing spreading and panting, and increased feed intake and BW gain. MATERIALS AND METHODS Synbiotic The commercial synbiotic (PoultryStar® meUS, BIOMIN America Inc., San Antonio, TX) used in this study contained a prebiotic (fructo-oligosaccharides) and a probiotic mixture of 4 microbial strains selected from the different segments of the gastrointestinal tract (Lactobacillus reuteri isolated from the crop; Enterococcus faecium from the jejunum; Bifidobacterium animalis from the ileum; and Pediococcus acidilactici from the cecum). The probiotic mixture was selected for its efficacy to exclude pathogenic bacteria and maintain a healthy gut microbial population, while the prebiotic has been suggested to further modify the activity and growth of beneficial microflora. Its effects and survivability have been tested in previous studies (McReynolds et al., 2009; Murugesan and Persia, 2015; Yan et al., 2015). Animals and Housing All procedures were approved by the Purdue Animal Care and Use Committee prior to the start of the experiment (PACUC Number: 1,111,000,262). Three hundred sixty male broiler chicks (Ross 708 strain; Pine Manor/Miller Poultry, Goshen, IN) were weighed and assigned to 24 floor pens (110 cm × 110 cm per pen) with equal average BW in a temperature controlled room at the Poultry Research Farm of Purdue University. Management of the broilers followed the guidelines of Aviagen (2015). The chicks were maintained at a temperature of approximately 34°C at d 1 with a gradual reduction in temperature to 27°C on d 14. Heat stimulation began on d 15 (i.e., the beginning of the growth phase) at 32°C for 9 h (08:00–17:00) daily until the end of the experiment. Data loggers (HOBO®, Onset Computer Corporation, MA) were placed in the room to record the room temperature and humidity throughout the experiment (Table 1). A thermoneutral control group was not used in this study as we aimed to investigate the effect of synbiotics on heat stressed broiler chickens and 32°C was guaranteed to induce HS based on the results of a previous study (Mahmoud et al., 2015). Narrowing focus to include only HS birds allowed us to reduce animal use by 50%, a key priority of animal welfare scientists (i.e., the 3Rs principal (Russell and Burch, 1959)). Table 1. Temperature and humidity levels throughout the experimental period. Bird age  Temperature  Humidity    Day time (08:00–17:00)  Night time (17:00–08:00)  Day time (08:00–17:00)  Night time (17:00–08:00)  d 15–21  31.87 ± 0.27  25.87 ± 0.36  49.16 ± 1.54  55.12 ± 1.58  d 22–28  31.58 ± 0.27  25.36 ± 0.18  50.94 ± 0.79  52.71 ± 1.50  d 29–35  31.67 ± 0.51  25.45 ± 0.29  60.03 ± 1.72  61.37 ± 1.28  d 36–42  31.57 ± 0.29  25.36 ± 0.12  57.52 ± 1.24  53.65 ± 1.32  Bird age  Temperature  Humidity    Day time (08:00–17:00)  Night time (17:00–08:00)  Day time (08:00–17:00)  Night time (17:00–08:00)  d 15–21  31.87 ± 0.27  25.87 ± 0.36  49.16 ± 1.54  55.12 ± 1.58  d 22–28  31.58 ± 0.27  25.36 ± 0.18  50.94 ± 0.79  52.71 ± 1.50  d 29–35  31.67 ± 0.51  25.45 ± 0.29  60.03 ± 1.72  61.37 ± 1.28  d 36–42  31.57 ± 0.29  25.36 ± 0.12  57.52 ± 1.24  53.65 ± 1.32  View Large Dietary Treatments The 24 pens were randomly assigned to 3 dietary treatments with 8 replicates of 15 broiler chickens each: a regular diet mixed with the synbiotic product at 0 (control), 0.5 (106 cfu/g) (0.5X) and 1.0 (2 × 106 cfu/g) (1.0X) g/kg feed. The PoultryStar® dietary treatments were supplied from d 1 to 42 and made by the step-up procedure as explained in detail by Mahmoud et al. (2015). In brief, the respective amount of PoultryStar® was mixed with a small amount of the regular diet as a small batch, and then incorporated with a larger amount of the diet gradually, until the total amount of each of the particular diets was homogeneously mixed. The base of the diet was formulated according to growth stage requirements (Table 2). Table 2. Components of base diet,1 separated by growth phase. Ingredient, %  Starter  Grower  Finisher    (1–14 d)  (15–28 d)  (29–42 d)  Corn  52.00  52.30  62.80  Soybean meal,48% CP  40.00  39.10  29.70  Soy oil  3.59  4.97  4.11  Sodium chloride  0.51  0.46  0.43  DL Methionine  0.30  0.24  0.23  L-Lysine HCL  0.13  -—-  0.07  Threonine  0.06  -—-  -—-  Limestone  1.29  1.15  1.12  Monocalcium phosphate  1.75  1.48  1.17  Vitamin/mineral premix2  0.35  0.35  0.35  Calculated analyses  Crude protein %  23.40  22.80  19.20  Poultry ME kcal/kg  3050  3151  3200  Calcium %  0.95  0.85  0.75  Available phosphorus %  0.50  0.44  0.36  Methionine %  0.66  0.59  0.53  Methionine+Cystine %  1.04  0.97  0.86  Lysine %  1.42  1.29  1.09  Threonine %  0.97  0.89  0.74  Na %  0.22  0.20  0.19  Ingredient, %  Starter  Grower  Finisher    (1–14 d)  (15–28 d)  (29–42 d)  Corn  52.00  52.30  62.80  Soybean meal,48% CP  40.00  39.10  29.70  Soy oil  3.59  4.97  4.11  Sodium chloride  0.51  0.46  0.43  DL Methionine  0.30  0.24  0.23  L-Lysine HCL  0.13  -—-  0.07  Threonine  0.06  -—-  -—-  Limestone  1.29  1.15  1.12  Monocalcium phosphate  1.75  1.48  1.17  Vitamin/mineral premix2  0.35  0.35  0.35  Calculated analyses  Crude protein %  23.40  22.80  19.20  Poultry ME kcal/kg  3050  3151  3200  Calcium %  0.95  0.85  0.75  Available phosphorus %  0.50  0.44  0.36  Methionine %  0.66  0.59  0.53  Methionine+Cystine %  1.04  0.97  0.86  Lysine %  1.42  1.29  1.09  Threonine %  0.97  0.89  0.74  Na %  0.22  0.20  0.19  1The ration formulation was produced according to Aviagen (2015) 2Provided per kilogram of diet: vitamin A, 13,233 IU; vitamin D3, 6,636 IU; vitamin E, 44.1 IU; vitamin K, 4.5 mg; thiamine, 2.21 mg; riboflavin, 6.6 mg; pantothenic acid, 24.3 mg; niacin, 88.2 mg; pyridoxine, 3.31 mg; folic acid, 1.10 mg; biotin, 0.33 mg; vitamin B12, 24.8 μg; choline, 669.8 mg; iron from ferrous sulfate, 50.1 mg; copper from copper sulfate, 7.7 mg; manganese from manganese oxide, 125.1 mg; zinc from zinc oxide, 125.1 mg; iodine from ethylene diamine dihydroidide, 2.10 mg; selenium from sodium selenite, 0.30 mg. View Large Behavioral Observations Five broiler chickens per pen (40 total per treatment) were randomly selected for observation and marked with livestock spray marker on their backs (Livestock green sharp-mark spray paint marker, Cotran Corporation, Portsmouth, RI). Behavioral observation was performed twice daily from 10:00 to 11:00 and 14:00 to 15:00 3 times per wk (Monday—Wednesday) from d 15 to 42. Behavioral data was collected on alternative days to production data collection to avoid interrupting normal behavior. Behaviors of focal birds were collected according to an ethogram (Table 3) and scored 6 times per observation session using instantaneous scan sampling (Altmann, 1974; Engel, 1996). The focal broiler chickens were observed from outside of the pens at a distance of 1.5 m to avoid disturbance of the normal behavioral repertoire. Data are presented as a proportion of observed behaviors out of the total number possible (Kristensen et al., 2007). Table 3. Ethogram of broiler behaviors collected during heat stress condition. Behavior1  Definition  Standing  The feet are in contact with the ground. No other body part is touching the floor surface. The birds’ body posture is in an upright position.  Sitting  The ventral part of the bird is in contact with the ground. Legs are bent at the knee with the fibula and tibia (i.e., lower part of the leg, under the knee) touching the ground.  Walking  The bird moves at least 2 steps in succession. This may or may not include scratching at the litter with feet.  Feeding  The bird's head is located inside the feeder, presumably consuming feed.  Drinking  The bird is observed pecking at the drinker, presumably consuming water.  Preening  The bird is manipulating its own feathers with beak gently.  Wing Spreading  Wings are extended horizontally from the body such that a space can be seen between the underside of the wing and the surface of the bird's body.  Panting  The bird is breathing with an open beak and respiration rate is abnormally rapid.  Behavior1  Definition  Standing  The feet are in contact with the ground. No other body part is touching the floor surface. The birds’ body posture is in an upright position.  Sitting  The ventral part of the bird is in contact with the ground. Legs are bent at the knee with the fibula and tibia (i.e., lower part of the leg, under the knee) touching the ground.  Walking  The bird moves at least 2 steps in succession. This may or may not include scratching at the litter with feet.  Feeding  The bird's head is located inside the feeder, presumably consuming feed.  Drinking  The bird is observed pecking at the drinker, presumably consuming water.  Preening  The bird is manipulating its own feathers with beak gently.  Wing Spreading  Wings are extended horizontally from the body such that a space can be seen between the underside of the wing and the surface of the bird's body.  Panting  The bird is breathing with an open beak and respiration rate is abnormally rapid.  1All behaviors were mutually exclusive; postures (i.e., standing and sitting) were only counted if the bird performed no other simultaneous behaviors. View Large Growth Performance BW, BW gain, feed intake and feed conversion ratio were recorded on d 7, 14 (the end of starter phase), 28 (the end of grower phase) and 42 (the end of finisher phase). Data collected on d 7 and 14 were used to assess the effect of synbiotics prior to HS, while d 28 and 42 were collected during HS conditions. All birds within a pen were measured at each time point (i.e., 15 birds were sampled in each pen, with 8 replicates per treatment). The BW gain was calculated as the BW of the current time point subtracted the BW of the previous time point. Statistical Analysis The experimental design was conducted in a randomized block design. Pen was considered the experimental unit (n = 8). The overall effects of the synbiotic supplementation on broiler chicken behaviors and performance were analyzed statistically by repeated measures ANOVA. Means of the data were analyzed by using PROC MIXED model with SAS 9.4 software (SAS Institute Inc., Cary, NC). The normality of the data was analyzed by the Shapiro-Wilk test. Transformation of the data was performed for normality when variances were not homogeneous (Steel et al., 1997). BW, BW gain, feed intake, feed conversion ratio, standing, sitting, preening, wing spreading, and panting were log transformed. Statistical trends were similar for both transformed and untransformed data, the untransformed results were presented. Means were compared by Tukey-Kramer test when a significant difference was detected at a probability of α less than 0.05. Expression of the findings is reported as the mean ±  SE. RESULTS Behavioral Patterns The overall relationships between the synbiotic supplementation and behavioral activities are presented in Table 4. The synbiotic fed broiler chickens spent more time standing (F(2, 29.72) = 6.80, P < 0.01), walking (F(2, 36.9) = 139.73, P < 0.001), sitting (F(2, 23.87) = 49.73, P < 0.001), preening (F(2, 26.4) = 16.38, P < 0.001), and feeding (F(2, 23.9) = 11.50, P < 0.001) in comparison to the controls, while wing spreading (F(2, 23.32) = 27.72, P < 0.001) and panting (F(2, 20.56) = 62.30, P < 0.001) were lower in the synbiotic groups than the control groups. There were no differences in drinking behavior (F(2, 25) = 1.32, P = 0.285) between the groups. No statistical differences were found between synbiotic dosage levels, except that the percentage of walking behavior was further increased in the 0.5X group in comparison with the control and 1.0X groups (t(36.9) = 16.17, P < 0.001; t(36.9) = 4.41, P < 0.001, respectively). Table 4. Effect of dietary supplementation of synbiotic (PoultryStar) on behaviors of broiler chickens under heat stress condition. Treatment1  Control  0.5X  1.0X  P-value  Behavior          Standing (%)  3.03b ± 0.23  4.24a ± 0.24  4.01a ± 0.23  0.004  Sitting (%)  11.75b ± 0.97  28.30a ± 1.02  27.42a ± 1.00  0.001  Walking (%)  2.73c ± 0.15  6.16a ± 0.15  5.23b ± 0.15  0.001  Feeding (%)  12.85b ± 0.89  17.25a ± 0.89  18.62a ± 0.89  0.001  Drinking (%)  5.54 ± 0.02  6.38 ± 0.02  6.06 ± 0.02  0.285  Preening (%)  2.86b ± 0.29  5.44a ± 0.31  5.30a ± 0.29  0.001  Wing Spreading (%)  9.17a ± 0.49  4.55b ± 0.52  3.66b ± 0.56  0.001  Panting (%)  50.29a ± 1.58  27.31b ± 1.77  28.07b ± 1.63  0.001  Treatment1  Control  0.5X  1.0X  P-value  Behavior          Standing (%)  3.03b ± 0.23  4.24a ± 0.24  4.01a ± 0.23  0.004  Sitting (%)  11.75b ± 0.97  28.30a ± 1.02  27.42a ± 1.00  0.001  Walking (%)  2.73c ± 0.15  6.16a ± 0.15  5.23b ± 0.15  0.001  Feeding (%)  12.85b ± 0.89  17.25a ± 0.89  18.62a ± 0.89  0.001  Drinking (%)  5.54 ± 0.02  6.38 ± 0.02  6.06 ± 0.02  0.285  Preening (%)  2.86b ± 0.29  5.44a ± 0.31  5.30a ± 0.29  0.001  Wing Spreading (%)  9.17a ± 0.49  4.55b ± 0.52  3.66b ± 0.56  0.001  Panting (%)  50.29a ± 1.58  27.31b ± 1.77  28.07b ± 1.63  0.001  a,b,cMean ± SE with different superscripts in the same row differ (P < 0.01). 1Dietary treatments containing 0 (control), 0.5 (0.5X) and 1.00 (1.0X) gkg−1 synbiotic, n = 8 per treatment (pen was considered the experimental unit, 5 birds per pen were observed, percentage of each behavior was calculated separately using 40 birds per treatment). Heat stimulation began on d 15 at 32°C for 9 h daily until d 42. View Large Growth Performance The relationships between the synbiotic supplementation and growth performance are presented in Table 5. Under thermoneutral condition, the synbiotic fed broilers had higher BW, BW gain, and feed intake, but lower feed conversion ratio in comparison with the controls on d 7 (F(2, 22.99) = 32.04, P < 0.001; F(2,19.37) = 3.80, P < 0.05; F(2, 19.73) = 83.07, P < 0.001; F(2, 20.97) = 15.69, P < 0.001, respectively) and d 14 (F(2, 23.85) = 98.29, P < 0.001; F(2, 24.23) = 96.41, P < 0.001; F(2, 21.01) = 29.53, P < 0.001; F(2, 20.32) = 6.66, P < 0.01, respectively). Under HS condition, compared to broilers fed the control diet, the synbiotic group had higher BW (F(2, 21) = 15.21, P < 0.001), feed intake (F(2, 21) = 19.39, P < 0.001), and feed conversion ratio (F(2, 21) = 7.46, P < 0.01) on d 28, and higher BW (F(2, 20.01) = 18.61, P < 0.001), BW gain (F(2, 19.21) = 3.95, P < 0.05), and feed intake (F(2, 20.36) = 10.41, P < 0.001) on d 42, while BW gain (F(2, 21) = 2.31, P = 0.124) on d 28 and feed conversion ratio (F(2, 21) = 0.78, P = 0.473) on d 42 were not significantly different between treatments. Table 5. Effect of dietary supplementation of synbiotic (PoultryStar) on performance parameters of heat stressed broiler chickens at different growth ages. Treatment1  Control  0.5X  1.0X  P-value  d7          BW (g)  133.69b ± 1.16  145.90a ± 1.16  144.40a ± 1.16  0.001  BW gain (g)  86.69c ± 0.11  94.55a ± 0.11  91.15b ± 0.11  0.041  Feed intake (g)  96.63b ± 0.72  108.73a ± 0.72  107.53a ± 0.72  0.001  Feed conversion ratio (g/g)  0.89a ± 0.01  0.86b ± 0.01  0.84c ± 0.01  0.001  d14          BW (g)  446.33c ± 3.85  501.19b ± 3.85  521.73a ± 3.85  0.001  BW gain (g)  312.62c ± 3.28  355.30b ± 3.28  377.33a ± 3.28  0.001  Feed intake (g)  343.09b ± 7.89  364.10b ± 7.89  424.57a ± 7.89  0.001  Feed conversion ratio (g/g)  1.10a ± 0.02  1.02b ± 0.02  1.12a ± 0.02  0.006  d28          BW (g)  1437.48b ± 13.68  1505.67a ± 13.68  1543.36a ± 13.68  0.001  BW gain (g)  991.15±11.08  1004.48±11.08  1024.53±11.08  0.124  Feed intake (g)  1471.41b ± 13.01  1549.34a ± 13.01  1584.11a ± 13.01  0.001  Feed conversion ratio (g/g)  1.48b ± 0.01  1.54a ± 0.01  1.54a ± 0.01  0.004  d42          BW (g)  2412.50c ± 22.14  2515.86b ± 22.14  2615a ± 22.14  0.001  BW gain (g)  1076.77b ± 21.60  1099.13a,b ± 21.60  1162.77a ± 21.60  0.027  Feed intake (g)  2144.83b ± 27.73  2202.19b ± 27.73  2323.61a ± 27.73  0.001  Feed conversion ratio (g/g)  1.99 ± 0.08  1.88 ± 0.08  1.99 ± 0.08  0.473  Treatment1  Control  0.5X  1.0X  P-value  d7          BW (g)  133.69b ± 1.16  145.90a ± 1.16  144.40a ± 1.16  0.001  BW gain (g)  86.69c ± 0.11  94.55a ± 0.11  91.15b ± 0.11  0.041  Feed intake (g)  96.63b ± 0.72  108.73a ± 0.72  107.53a ± 0.72  0.001  Feed conversion ratio (g/g)  0.89a ± 0.01  0.86b ± 0.01  0.84c ± 0.01  0.001  d14          BW (g)  446.33c ± 3.85  501.19b ± 3.85  521.73a ± 3.85  0.001  BW gain (g)  312.62c ± 3.28  355.30b ± 3.28  377.33a ± 3.28  0.001  Feed intake (g)  343.09b ± 7.89  364.10b ± 7.89  424.57a ± 7.89  0.001  Feed conversion ratio (g/g)  1.10a ± 0.02  1.02b ± 0.02  1.12a ± 0.02  0.006  d28          BW (g)  1437.48b ± 13.68  1505.67a ± 13.68  1543.36a ± 13.68  0.001  BW gain (g)  991.15±11.08  1004.48±11.08  1024.53±11.08  0.124  Feed intake (g)  1471.41b ± 13.01  1549.34a ± 13.01  1584.11a ± 13.01  0.001  Feed conversion ratio (g/g)  1.48b ± 0.01  1.54a ± 0.01  1.54a ± 0.01  0.004  d42          BW (g)  2412.50c ± 22.14  2515.86b ± 22.14  2615a ± 22.14  0.001  BW gain (g)  1076.77b ± 21.60  1099.13a,b ± 21.60  1162.77a ± 21.60  0.027  Feed intake (g)  2144.83b ± 27.73  2202.19b ± 27.73  2323.61a ± 27.73  0.001  Feed conversion ratio (g/g)  1.99 ± 0.08  1.88 ± 0.08  1.99 ± 0.08  0.473  a,b,cMean ± SE with different superscripts in the same row differ significantly (P < 0.05). 1Basal dietary supplemented with 0 (Control), 0.5 (0.5X) and 1.00 (1.0X) gkg−1 synbiotic, n = 8 per treatment (pen was considered the experimental unit, all 15 birds/pen were used to measure performance parameters and averaged for analysis). Heat stimulation began on d 15 at 32°C for 9 h daily until d 42. View Large There were some differences in growth performance results between the 2 concentrations of synbiotics and the control diet. The 1.0X group had the highest BW, BW gain and feed intake on d 14 (thermoneutral conditions) compared to control group (t(23.85) = 13.76, P < 0.001; t(24.23) = 13.81, P < 0.001; t(21.01) = 7.21, P < 0.001, respectively) and 0.5X group (t(23.85) = 3.77, P < 0.001; t(24.23) = 4.75, P < 0.001; t(21.01) = 5.38, P < 0.001, respectively). The 1.0X group also had higher BW and feed intake on d 42 (HS condition) compared to the control group (t(20.01) = 6.10, P < 0.001; t(20.36) = 4.44, P < 0.001, respectively) and the 0.5X group (t(20.01) = 2.99, P < 0.001; t(20.36) = 3.02, P < 0.01, respectively), while BW gain was higher in the 1.0X group on d 42 compared to the control group only (t(19.21) = 2.68, P < 0.05). DISCUSSION Environmental stressors, including HS, are responsible for decreased health, welfare, and production coupled with undesirable meat quality on color, water-holding capacity, tenderness, and/or pale, soft, and exudative breast muscle (Zhang et al., 2012; Fouad et al., 2016) in broiler chickens. Increasing the beneficial bacteria in the gastrointestinal tract has shown promise in increasing growth performance and mitigating the negative effects of HS, thereby safeguarding poultry welfare and economic gains for the industry. When poultry experience stress, including HS, the neuroendocrine system is altered causing dysregulation of the HPA axis and increased plasma corticosterone concentration (Quinterio-Filho et al., 2010). A direct link has been established between microbiota and HPA reactivity; mice lacking a commensal bacterial population (i.e., “germ-free”) had exaggerated corticosterone and adrenocorticotrophin response to stressors (Sudo et al., 2004). Increased corticosterone levels have been associated with numerous behavioral changes indicating heightened states of anxiety, depression, and aggression (Gregus et al., 2005; O’Mahony et al., 2009). Beneficial bacteria, such as probiotics, have been demonstrated to protect or improve the function of the microbiome, leading to improved regulation of the HPA axis and behavior (Cryan and O’Mahony, 2011; Mayer et al., 2015). Under HS conditions, birds reduce their feed intake and activities as a mechanism to decrease the production of body heat (Sohail et al., 2010; 2013). As a result, metabolism is impaired and decreased BW, BW gain, and increased feed conversion ratios are observed (Sohail et al., 2012). Our results suggest that dietary synbiotics can improve production profiles as feed intake, BW and BW gain increased in birds supplemented with synbiotics compared to those consuming a regular diet under both thermoneutral (d 7 and 14) and HS conditions (d 42). Similar to our results, several authors have also reported increased production profiles when supplementing birds with either probiotics or synbiotics under stressful conditions (Fayed and Tony, 2008; Hosseini et al., 2013; Al-Fataftah and Abdelqader, 2014; Jahromi et al., 2015) and normal environmental temperature (Awad et al., 2009; Riad et al., 2010; Saiyed et al., 2015; Sarangi et al., 2016), although others reported no differences in production under stressful conditions (Hassan et al., 2007; Sohail et al., 2012; 2013) and thermoneutral conditions (Salehimanes et al., 2016). Variation in the effects of probiotics and synbiotics in previous studies may be attributed to the differences in strains of bacteria and their concentrations in the diet, as reviewed by Jin et al. (1997). The mechanisms by which bacteria regulate changes in the gastrointestinal tract differ based on the type of prebiotic or probiotic introduced (Patterson and Burkholder, 2003); for example, Sohail et al. (2012, 2013) used mannanoligosaccharides (MOS) as the prebiotic in their synbiotic mixture; unlike the fructo-oligosaccharide prebiotic used in our study, which selectively enriches beneficial bacterial populations, MOS has been suggested to act by binding and removing pathogens from the intestinal tract (Spring et al., 2000). Further research should be conducted to determine the exact mechanisms by which certain combinations of probiotics and prebiotics control growth performance and influence the effects of stress to determine which compositions may be most beneficial. Considering the positive results on production-related measurements in this study the mechanisms and application of this bacterial combination could provide a useful basis for future investigations. In addition to the composition of bacteria, the concentration of the synbiotic may influence the effect on broiler production during stressful conditions. Our study investigated the synbiotic at 2 different concentrations: 0.5X and 1.0X, based on the company's recommendation. A few differences were found between the dosages, namely, at the 1.0X concentration, BW and feed intake were increased compared to the 0.5X and control treatments under thermoneutral (d 14) and HS (d 42) conditions, while BW gain was higher in the 1.0X group compared to the control group only at d 42 (end of the study). Considering the potential for increased production throughout the broiler lifespan, a 1.0X concentration of dietary synbiotics could be utilized. Under HS condition, broiler chickens display a number of behaviors to improve thermoregulation and reduce HS effects, including increased respiratory rate, panting (i.e., open-mouth breathing), and wing spreading (Pereira et al., 2007; Li et al., 2015). The current results showed that, compared to the effects of regular diet, supplementing with synbiotics reduced panting and wing-spreading activities of broiler chickens under an elevated temperature condition. Generally, broiler chickens release body heat through panting and wing spreading due to the lack of sweat glands. Panting is used to dissipate heat through evaporation from the respiratory tract, while wing spreading enhances heat loss by increasing the body surface, exposing more unfeathered areas, and reducing the isolating capacity of the feather cover (Lolli et al., 2010). The beneficial effect of synbiotics on panting and wing spreading activities suggests the birds may have experienced less of an effect of HS. A previous study investigating the effects of probiotics (Lactobacillus spp.) on broiler chickens subjected to HS conditions noted an increase in erythrocyte count, hemoglobin concentration, and hematocrit value; all hematological changes that are associated with improved respiratory ability (Hasan et al., 2015). Although not measured in this study, similar hematological changes may be responsible for reduced HS-associated behaviors in our broilers. Additionally, in our study, synbiotic-fed broilers spent more time standing and walking during HS conditions. The increased movement activities may be related to the improved musculoskeletal health from synbiotic supplementation. A parallel study showed that a probiotic (Bacillus subtilis) improved the bone health of broiler chickens under HS as indicated by higher bone mineral content of the femur and tibia (Yan et al., 2016). Several studies have reported an improvement in bone mineralization due to an increase in bone ash, calcium and phosphorus contents following supplementation with probiotics (Houshmand et al., 2011; Narasimha et al., 2013). However, our results also suggest that sitting behavior increased with supplemental synbiotics. The reasons for the increased sitting behavior in the synbiotic fed birds are unclear but may be related to a decrease in the fear response as demonstrated by several rodent studies; for example, probiotics modulated cognitive processes in stressed mice (Bifidobacteria, Savignac et al., 2015) and reduced fear relapse in rats experiencing maternal separation anxiety (Lactobacillus, Cowan et al., 2016). Alternatively, heavier BW may have increased sitting behavior in the synbiotic groups. Finally, preening activities were increased in the synbiotic supplemented broilers in our study. In support, Fayed and Tony (2008) reported an increase in preening behavior when broiler chickens were fed probiotics during stress experienced by high stocking density. Preening has been considered a comfort-related behavior in poultry (Hinde, 1970) and it is possible that supplementation with synbiotics decreased the discomfort associated with HS. In all, the observed behavioral effects of synbiotics suggest that birds experienced less stress when exposed to high temperatures compared to birds fed a regular diet. However, surprisingly, drinking behavior was not different between treatments. If synbiotics were to decrease water loss under HS conditions, we would expect to observe less drinking behavior. It is possible that synbiotics help dissipate heat loss through the skin (as seen with decreased wing spreading), but are not as effective at controlling overall water loss. Further studies should explore these relationships to determine the degree to which HS effects are controlled by synbiotics. CONCLUSION In the current study, both doses of the synbiotic supplement significantly increased walking, sitting, preening activities while reducing panting and wing spreading (i.e., heat-associated behaviors) in broiler chickens exposed to HS. Overall, our results suggest that supplementing the broiler diet with synbiotics may be useful to improve production profiles, even during HS conditions. Further, supplementing with a 1.0X concentration resulted in a higher increase in feed intake, BW and BW gain at the end of the broiler cycle suggesting the synbiotic may be most effective at this dosage. The poultry industry could consider supplementing the broiler diet with synbiotics as a management strategy, particularly in hot weather or climates, in order to protect broiler production from the negative effects of stress. 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Poultry ScienceOxford University Press

Published: Apr 1, 2018

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