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Supplemental boric acid does not prevent the development of footpad dermatitis in broilers subjected to high stocking density
Supplemental boric acid does not prevent the development of footpad dermatitis in broilers subjected to high stocking density ¨ oksal,∗ Onur Tatlı,∗ Eren Kuter,∗ Umair Ahsan,∗ G¨ ul¸sen G¨ uven,† Ozcan Cengiz,∗,1 Bekir Hakan K¨ ∗ ‡ ∗ ¨ ¨ okhan Onol Omer Sevim, Sacit F. Bilgili, and Ahmet G¨ ∗
Department of Animal Nutrition and Nutritional Diseases, Faculty of Veterinary Medicine, Adnan Menderes University, I¸sıklı, Aydın 09016, Turkey; † Department of Chemistry, Faculty of Art and Science, Adnan Menderes University, Aydın 09100, Turkey; and ‡ Department of Poultry Sciences, College of Agriculture, Auburn University, Auburn, AL 36849-5416, USA to 21 days only. Dietary boric acid had no effect on the growth performance of broiler chickens. Litter pH, moisture, and NH3 volatilization were higher in broiler chickens subjected to HSD (P < 0.05). Thus, the incidence and severity of FPD increased in response to HSD (P < 0.05). Dietary boric acid reduced the litter pH and NH3 volatilization on day 42 of experiment (P < 0.05). However, dietary boric acid supplementation had no effect on the incidence and severity of FPD. Boric acid supplementation in broiler diets increased the serum and litter boron levels at day 42 in broiler chickens subjected to NSD or HSD (P < 0.05). In conclusion, HSD resulted in poor growth performance, litter quality, and greater incidence and severity of FPD in broiler chickens. Dietary boric acid was ineffective against FPD in broiler chickens although it improved the litter quality by lowering the litter pH and NH3 volatilization.
ABSTRACT The present study was conducted to evaluate the protective effect of dietary boric acid supplementation on the development of incidence and severity of footpad dermatitis (FPD) in broiler chickens subjected to normal or high stocking densities (NSD or HSD). A total of 576 1-day-old ROSS 308 broiler chickens were randomly allocated to 4 treatments (8 replicate pens per treatment) in a 2 × 2 factorial arrangement of dietary boric acid (0 and 60 mg/kg) and stocking density (NSD 14 birds/m2 and HSD 22 birds/m2 ). Basal diets were formulated for starter, grower, and finisher phases. Growth performance, litter quality (litter pH, moisture, temperature, and NH3 volatilization), serum and litter boron levels, and incidence and severity of FPD were recorded. The HSD affected the body weight gain and feed intake of broiler chickens during all phases and 0 to 42 (P < 0.05), whereas feed conversion ratio (FCR) was poor at 0
Key words: boric acid, broiler, footpad dermatitis, litter quality, stocking density 2018 Poultry Science 0:1–9 http://dx.doi.org/10.3382/ps/pey337
INTRODUCTION
However, HSD causes severe deterioration of litter quality in poultry houses that affects leg health resulting in FPD in poultry. In the past, different strategies have been adopted to prevent and treat the FPD in broilers. These strategies involve interventions of dietary levels of methionine (Chavez and Kratzer, 1972), biotin (Mayne et al., 2007a; Cengiz et al., 2012a), sodium (Cengiz et al., 2012b), zinc (Cengiz et al., 2014), protein source (Cengiz et al., 2013), bedding type (Cengiz et al., 2011), and the supplementation of exogenous enzymes (Cengiz et al., 2012c, d). However, these manipulations have been unsuccessful to completely ameliorate or prevent the broilers from occurrence of FPD. Consequently, researchers have necessitated developing alternative solutions in order to overcome this problem. It is well known that increased litter ammonia (NH3 ) increases litter pH that makes the litter caustic in nature, therefore, associated with poor foot health and FPD in poultry (Mayne et al., 2007b). Litter amendments can be helpful in the prevention of FPD in
High stocking density (HSD) has become a point of concern from broiler welfare perspective in recent years. The HSD is one of the predisposing factors involved in the development of footpad dermatitis (FPD) in poultry (Shepherd and Fairchild, 2010) which is not only important from economic point of concern, but has become animal welfare, food safety, and product quality issue as well. Increasing the stocking density results in beneficial economic impact provided it does not go beyond an optimum range of 34 to 40 kg/m2 assuming 2.5 kg final BW on an average (Dozier et al., 2005, 2006; Estevez, 2007). However, going beyond this range worsens the health and performance of broilers. C 2018 Poultry Science Association Inc. Received October 2, 2017. Accepted July 10, 2018. 1 Corresponding author: E-mail: [email protected]
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CENGIZ ET AL. Table 1. Composition and chemical analysis of basal diets for starter, grower, and finisher (g/kg as fed basis). Experimental diets Items Corn Soybean meal Vegetable oil Limestone Dicalcium phosphate Salt DL-Methionine L-Lysine Vitamin-mineral premix1 Calculated nutrient composition Metabolizable energy (MJ/kg) CP Ca Available P Methionine + Cysteine Lysine Sodium Analyzed nutrient composition Dry matter CP Ether extract Nitrogen free extract NDF Ca Total P Boron (ppm), (basal/boric acid diet)
Starter (0 to 10 d)
Grower (11 to 24 d)
Finisher (25 to 42 d)
556.3 375.0 25.0 8.9 23.0 3.5 3.7 2.1 2.5
561.5 360.0 41.5 8.5 20.0 3.5 2.5 2.5
590.0 335.0 42.5 8.0 17.3 3.5 1.2 2.5
12.7 230 10.0 5.0 10.9 14.2 1.6 884 227.8 61.9 511.1 84.6 11.8 8.6 13.15/20.68
13.2 220 9.1 4.5 9.5 12.1 1.6 883 218.0 78.0 507.6 79.7 10.9 7.9 12.56/23.45
13.4 210 8.0 4.0 8.0 11.5 1.6 887 208.5 80.8 524.2 75.4 9.9 7.3 13.08/24.15
1 Supplied per kilogram of diet: vitamin A, 12,000 IU; vitamin D3, 1,500 IU; vitamin E, 30 mg; vitamin K3, 5 mg; vitamin B1, 3 mg; vitamin B2, 6 mg; vitamin B6, 5 mg; vitamin B12, 0.03 mg; niacin, 40 mg; calcium D-pantothenate, 10 mg; folic acid, 0.75 mg; D-Biotin, 0.075 mg; choline chloride, 375 mg; Mn, 80 mg; Fe, 40 mg; Zn, 60 mg; Cu, 5 mg; I, 0.4 mg; Co, 0.1 mg; Se, 0.15 mg; and antioxidant, 10 mg.
broilers. Boric acid possesses the ability to absorb NH3 (Siemer, 1986), thereby it can reduce NH3 volatilization from litter in poultry houses. Conversely, dietary boric acid can reduce the litter pH, which, in turn, can help to prevent the incidence and reduce the severity of FPD in broilers. Therefore, an experiment was conducted to evaluate the litter quality improvement and reduction of incidence and severity of FPD by dietary supplementation of boric acid in broilers subjected to normal stocking density (NSD) or HSD. To the best of our knowledge, no such report is available in literature that describes the use of supplemental dietary boric acid to improve the litter quality, and footpad health in broilers. The aim of the present study was to investigate the effect of dietary boric acid on growth performance, litter quality, serum and litter boron levels, and incidence and severity of FPD in broilers subjected to NSD or HSD.
MATERIALS AND METHODS The study was conducted at Poultry Research Unit of Faculty of Veterinary Medicine, Adnan Menderes University, Aydın, Turkey, with prior approval from the animal care and use committee of the university (approval letter no. 050.04/2012/007).
Study Design and Experimental Groups The study was conducted as a completely randomized design in a 2 × 2 factorial arrangement of dietary boric acid supplementation (0 and 60 mg/kg of diet) and stocking density (14 and 22 birds/m2 ). A total of 576 1-day-old male broiler chicks of Ross 308 strain were randomly allocated to 4 experimental groups, each comprising of 8 replicate pens adjustable at the front. Sixteen replicate pens were subjected to HSD, whereas remaining replicate pens were subjected to NSD. Corn/soybean meal-based basal diets were formulated for starter (days 0 to 10), grower (days 11 to 24), and finisher (days 25 to 42) phases of broilers in accordance with the recommendations of NRC (1994) to meet or exceed the nutrient requirements of broilers (Table 1). Two experimental groups subjected to NSD and HSD were fed basal diets. Other experimental groups (NSD and HSD) were fed basal diets supplemented with boric acid (containing 17.5% boron) at 60 mg per kg of diet. The experimental groups have been presented in Table 2.
Management of Birds A total of 32 replicate pens were established in the experimental rooms. Initially, each replicate pen sized
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BORIC ACID AND FOOTPAD DERMATITIS IN BROILERS Table 2. Description of experimental groups. Serial No.
Description
1 2 3 4
NSD HSD NSD HSD
+ + + +
0 mg/kg boric acid 0 mg/kg boric acid 60 mg/kg boric acid 60 mg/kg boric acid
NSD, normal stocking density; HSD, high stocking density.
1.1 m × 1.5 m with adjustable pane at the front. The replicate pens meant for HSD provided floor space of 0.045 m2 /bird. The replicate pens subjected to NSD provided the floor space 0.071 m2 /bird. Floor space per bird was exclusive of the space occupied by feeders and drinkers. Approximately 7 to 8 cm deep layer of soft pine wood shavings was laid as a litter in each pen. Temperature of experimental rooms was maintained at 32 to 33◦ C before the arrival of chicks. Oneday-old male broilers were purchased from a commercial hatchery (Egetav Tavuk¸culuk San. ve Tic. A.S ¸ ., ˙ Izmir, Turkey). The groups subjected to HSD received 22 chicks per replicate pen, whereas those subjected to NSD received 14 chicks per replicate pen. The experiment lasted for 42 d. Birds had ad libitum access to feed and water throughout the experiment. Metal feeders (50 × 8 cm; 0.04 m2 ) were used during the starter phase (day 0 to 10) and tube feeders (r = 40 cm; 0.1256 m2 ) were used afterwards (day 11 to 42). Each pen was provided with one feeder. Water was supplied to each pen using nipple drinkers in each pen. The number of feeders and drinkers per pen remained unchanged throughout the experiment. Fluorescence lights were used to lit the experimental rooms during the dark periods of the day (which provided light period of 23L:1D). A constant temperature of 33 ◦ C was maintained at days 0 to 7 of the experiment. Later, 3◦ C was reduced per week until a constant temperature of 24◦ C was achieved that was maintained to the end of the experiment.
Chemical Analysis of Feed Feed samples were oven-dried at 105◦ C for 8 h to estimate the dry matter content of basal diets. Kjeldahl method was used for the estimation of nitrogen (N) content, and crude protein was calculated as N × 6.25. Soxhlet extraction technique was used for ether extract estimation. Van Soest method was employed to determine the neutral detergent fiber content of basal diets (Van Soest et al., 1991). Feed samples were burnt in muffle furnace at 600◦ C for crude ash estimation. Nitrogen-free extract was calculated by subtracting crude protein, ether extract, neutral detergent fiber, and crude ash from dry matter content of the diet. Calcium (Ca) content of basal diets was determined following the wet ash digestion method using nitric and perchloric acids (AOAC,
3
2000; Method 935.13). Phosphorus (P) content of diets was estimated according to the alkalimetric ammonium molybdophosphate method (AOAC, 2000; Method 964.06).
Growth Performance Birds were weighed weekly on per pen basis. The weight of dead bird was also recorded. Feed intake was recorded weekly by the difference between amount of feed offered and amount of feed not consumed. Body weight (BW) gain and feed intake per pen were calculated for the periods 0 to 21, 21 to 42, and 0 to 42 days. BW gain was calculated by adjusting the weight of dead birds (if any) in the pen weight of that pen during the specific phase. Feed conversion ratio (FCR) was calculated using the formula of ratio between feed intake and BW gained.
Litter Quality Litter quality was measured in terms of litter quality variables such as pH, moisture, temperature, NH3 volatilization. For this purpose, approximately 100 g litter samples were collected in plastic bags from each pen at the time of commencement of experiment (initial) and at the end of experiment (final). Each sample was collected from different localities within a pen e.g., near the drinkers, around the feeder, and the corners. The sample was mixed thoroughly to homogenize in order to avoid variability. Litter pH was measured by mixing 100 mL of distilled water in 10 g of homogenized sample. The mixture was agitated for 5 min and suspended for 30 min. A portable waterproof pH meter (Model: HI 9124, Hanna Instruments Inc., RI) was inserted into the agitated mixture. Reading was taken upon the stability of pH value on the meter. Litter moisture was determined by drying the 80 g of homogenized sample at 105◦ C for 24 h in a hot air oven. The samples were weighed after drying. The difference between initial and final samples was taken as litter moisture and presented as a percentage. Litter temperature was measured in each pen at 3 different sites (near feeder, around drinker, and near the bird’s resting place within the pen) with the help of a digital probe thermometer (TFA Dostmann GmbH + Co. KG, Germany). Probe of the thermometer was inserted 2 to 3 cm deep in to the litter of each pen. The reading was taken when the temperature became stable. The NH3 volatilization was measured above 20 cm from the surface level of litter in each pen. Dr¨ ager NH3 meter (Dr¨ager Pac 7000, Dr¨ ager Safety AG and Co. KGaA, Luebeck, Germany) was used with the chips sensitivity ranging from 0.2 to 5 ppm, 2 to 50 ppm, and 10 to 150 ppm NH3 level.
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CENGIZ ET AL.
Feed, Serum, and Litter Boron Levels Boron content of the diets was determined using inductively coupled plasma optical emission spectrometry (ICP-OES). Briefly, 0.3 g feed sample was subjected to wet digestion with 8 mL 65% nitric acid and 2 mL 37% hydrochloric acid in a sealing vessel at 180◦ C in a specialized microwave for 20 min. Blood samples (10 mL) were collected from caudal tibial vein of one bird from each pen on 28 and 42 d of the experiment by inserting sterile syringe (needle 22-gauge, 1-inch length) at a shallow angle. Blood samples were processed for serum separation in a centrifuge (NF 200 Bench-Top ¨ Centrifuge, NUVE, Ankara, Turkey), immediately after clotting of blood samples. Serum boron level was measured using ICP-OES. A known quantity of serum (2 mL) was mixed with 5 mL 65% nitric acid and 2 mL 35% hydrogen peroxide in a sealing vessel that was placed in a specialized microwave (MARS 6 One Touch Microwave Reaction System, CEM Corporation, Matthews, NC) at 200◦ C for digestion for 15 min. At the end of experiment, litter samples for boron analysis collected from each pen were dried at 60◦ C for 72 h and 0.2 g dried litter sample was mixed with 10 mL 65% nitric acid in a sealing vessel that was placed in a specialized microwave at 180◦ C for digestion for 20 min. The digested samples of feed, serum, and litter were cooled, filtered in flask, and diluted to 25 mL by adding deionized water for analysis in ICP-OES spectrometer.
FPD Scoring On 14, 28, and 42 days of experiment, all birds were subjected to 3-point FPD scoring according to the method previously described by Bilgili et al. (2006). Briefly, footpads without any lesion were scored as 0, footpads with lesion size ≤7.5 mm were scored as 1, and those with lesion size >7.5 mm were scored as 2. Average FPD score was calculated for each score and presented as percentage. The following mathematical equation was applied for average FPD score: Average FPD score (Ni ) = Number of birds showing score 0, 1, or 2 (ni ) /Total number of birds scored
Statistical Analysis The data were analyzed using a statistical software package SPSS (version 22.0 Armonk, NY) to assess the effect of dietary boric acid on performance, litter quality, serum and litter boron levels, and incidence and severity of FPD in broilers subjected to NSD or HSD. Non-normalized parameters were tested with the help of Kruskal–Wallis test and transformed using logarithmic or square root transformation method. The percentage data (FPD score) were transformed to arcsine
values before analysis, however, presented in percentage in the results. Two-way analysis of variance was applied in GLM procedures of SPSS. Confidence interval of 95% (P-value < 0.05) was considered as significant for interactions and main effects. The interaction means were differentiated using Tukey’s HSD as post hoc test in case of significant interactions.
RESULTS The study revealed that there was no interaction between stocking density and dietary boric acid supplementation for growth performance of broilers (Table 3). HSD reduced (P < 0.05) the BW gain and feed intake at 0 to 21, 21 to 42, and 0 to 42 days in broilers compared to those subjected to NSD. FCR of broilers subjected to HSD improved at 0 to 21 days as compared to those reared under NSD (P < 0.05). There was a numerical difference that approached significance in FCR of broilers at 21 to 42 days in response to HSD compared to NSD (P = 0.06). Dietary boric acid supplementation had no significant effect (P > 0.05) on BW gain, feed intake, and FCR of broilers in comparison with those fed diets without boric acid. Although the interaction was not significant between stocking density and dietary boric acid supplementation, dietary boric acid supplementation lowered (P = 0.057) NH3 volatilization in broilers subjected to NSD or HSD (Table 4). There was no difference in litter pH, moisture, and temperature at day 0 in either group subjected to stocking density (HSD or NSD) or fed dietary boric acid supplementation (0 or 60 mg/kg). However, litter pH and moisture increased at the end of experiment (day 42) in groups subjected to HSD compared to NSD (P < 0.05). An increase in NH3 volatilization was observed at 28 and 42 days of experiment in groups reared under HSD in comparison with those under NSD (P < 0.05). Dietary boric acid supplementation reduced the litter pH and NH3 volatilization on day 42 of experiment in comparison with those fed diets without boric acid supplementation (P < 0.05). Litter temperature remained unaffected irrespective of the treatments. No interaction was noted between stocking density and dietary boric acid supplementation for serum boron levels at day 28 of the experiment (Table 5). However, significant interaction was observed for serum and litter boron levels (P < 0.05) at the end of the experiment (day 42). While dietary boric acid supplementation increased serum boron levels in birds placed at NSD (P < 0.05), it had no effect on those placed at HSD. Similarly, broilers subjected to NSD and HSD fed diets supplemented with boric acid had higher (P < 0.05) litter boron levels in comparison with those fed diets without supplementation placed at NSD or HSD. There was no interaction between stocking density and dietary boric acid supplementation for incidence and severity of FPD in broilers (Table 6). Broilers
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BORIC ACID AND FOOTPAD DERMATITIS IN BROILERS
Table 3. Body weight gain (BWG), feed intake (FI), and feed conversion ratio (FCR) of broilers subjected to normal or high stocking density fed dietary boric acid. BWG (g) Item NSD 0 mg/kg boric acid 60 mg/kg boric acid HSD 0 mg/kg boric acid 60 mg/kg boric acid Pooled SEM Stocking Density NSD HSD Pooled SEM Boric Acid 0 mg/kg 60 mg/kg Pooled SEM P-values Stocking density Boric acid Stocking density × boric acid
FI (g)
FCR
0 to 21 d
21 to 42 d
0 to 42 d
0 to 21 d
21 to 42 d
0 to 42 d
0 to 21 d
21 to 42 d
0 to 42 d
937 923
2059 2067
2996 2990
1322 1321
3670 3722
4992 5043
1.41 1.43
1.78 1.80
1.67 1.69
888 919 10.20
1802 1734 37.51
2690 2653 36.21
1211 1217 22.97
3337 3276 78.46
4549 4493 81.49
1.36 1.32 0.02
1.85 1.89 0.04
1.69 1.70 0.02
990a 903b 7.21
2,063a 1,768b 26.52
2,993a 2,671b 25.60
1,321a 1,214b 16.25
3,696a 3,307b 55.48
5,018a 4,521b 57.62
1.42a 1.34b 0.02
1.79 1.87 0.03
1.68 1.69 0.02
913 921 7.21
1930 1901 26.52
2843 2821 25.60
1267 1269 16.25
3504 3499 55.48
4770 4768 57.62
1.39 1.38 0.02
1.82 1.85 0.03
1.68 1.69 0.02
0.015 0.426 0.122
< 0.001 0.434 0.320
< 0.001 0.557 0.674
< 0.001 0.922 0.872
< 0.001 0.957 0.473
< 0.001 0.981 0.518
0.001 0.674 0.141
0.058 0.451 0.793
0.536 0.595 0.756
a, b : Means bearing different superscripts within the same column are statistically significant (P < 0.05). NSD: Normal stocking density (14 birds/m2 ). HSD: High stocking density (22 birds/m2 ). n: Number of replicates (n = 8).
Table 4. Litter pH, moisture (%), temperature (◦ C), and NH3 volatilization (ppm) from litter of broilers subjected to normal or high stocking density fed dietary boric acid. pH Item NSD 0 mg/kg boric acid 60 mg/kg boric acid HSD 0 mg/kg boric acid 60 mg/kg boric acid Pooled SEM Stocking density NSD HSD Pooled SEM Boric acid 0 mg/kg 60 mg/kg Pooled SEM P-values Stocking density Boric acid Stocking density × boric acid
Moisture
Temperature
NH3 volatilization
day 0
day 42
day 0
day 42
day 0
day 42
day 28
day 42
5.98 6.03
8.76 8.48
8.97 8.61
21.39 21.50
20.13 20.88
22.50 22.82
1.64 1.82
2.98 2.70
6.22 5.82 0.12
9.33 8.49 0.14
9.13 8.94 0.23
34.33 32.99 0.73
19.88 20.12 0.42
23.19 22.93 0.29
2.76 2.59 0.15
4.15 3.39 0.12
6.01 6.02 0.09
8.62b 9.09a 0.10
8.79 9.04 0.17
21.44b 33.66a 0.52
20.50 20.00 0.30
22.66 23.06 0.21
1.73b 2.68a 0.10
2.84b 3.77a 0.08
6.09 5.92 0.09
9.04a 8.67b 0.10
9.04 8.78 0.17
27.86 27.24 0.52
20.00 20.50 0.30
22.84 22.88 0.21
2.21 2.21 0.10
3.56a 3.05b 0.08
0.602 0.403 0.117
0.003 0.013 0.480
0.296 0.262 0.700
< 0.001 0.401 0.326
0.241 0.241 0.554
0.186 0.918 0.356
< 0.001 0.983 0.243
< 0.001 < 0.001 0.057
a, b : Means bearing different superscripts within the same column are statistically significant (P < 0.05). NSD: Normal stocking density (14 birds/m2 ). HSD: High stocking density (22 birds/m2 ). n: Number of replicates (n = 8).
subjected to HSD had greater incidence and severity of FPD at days 14 (P = 0.05 for score 0, P = 0.06 for score 1, and P < 0.05 for total score), 28, and 42 (P < 0.05) in comparison with those placed at NSD. Dietary boric acid supplementation had no effect on the incidence and severity of FPD in broilers compared to those fed diets without boric acid supplementation.
DISCUSSION Growth Performance Several studies have reported that HSD is associated with lower growth performance of broilers in terms of BW gain, feed intake, and FCR (Puron et al., 1995; Feddes et al., 2002; Dozier et al., 2005; Abudabos et al., 2013; Cengiz et al., 2015). These results are in
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CENGIZ ET AL.
and drinkers compared to those placed at NSD that leads to lowered feed and water intake. This may result in poorer performance of broilers although it might not be the case in the starter phase attributed to less space requirements and relatively smaller size of the chicks (Cengiz et al., 2015). However, HSD may affect the behavior of broilers toward feed intake in the starter phase as the age progresses and increasing physiological stress. Feddes et al. (2002) reported that poor growth performance is attributed to the difficulty in exchange of heat and gases within the microclimate of broilers subjected to HSD. Puron et al. (1995) demonstrated an inverse relationship between growth performance of broilers and HSD beyond a threshold. Therefore, growth performance was negatively affected in broilers subjected to HSD in comparison with those reared under NSD. Other possible explanations in support of declined growth performance in broilers subjected to HSD may be the disruption in gut microbiota that helps in digestion, absorption of nutrients, and improvement of intestinal microarchitecture by reducing the pathogenic bacterial populations (Biswas et al., 1999; Bedford and Apajalathi, 2001; L´ azaro et al., 2003; J´ ozefiak et al., 2004; Guardia et al., 2011). In addition, HSD may be associated with the surge in airborne pathogens (Sauter et al., 1981) probably due to reduced removal of dust (Banhazi et al., 2008), a possible cause of poor growth performance of broilers reared under HSD. There are contrasting results reported by different researchers in the past. In the present study, dietary boric acid supplementation had no effect on growth performance of broilers. These results are in agreement
Table 5. Serum and litter boron levels of broilers subjected to normal or high stocking density fed dietary boric acid. Serum boron levels (mg/L) Item NSD 0 mg/kg boric acid 60 mg/kg boric acid HSD 0 mg/kg boric acid 60 mg/kg boric acid Pooled SEM Stocking density NSD HSD Pooled SEM Boric acid 0 mg/kg 60 mg/kg Pooled SEM P-values Stocking density Boric acid Stocking density × boric acid
Litter boron levels (mg/L)
day 28
day 42
day 42
7.20 14.20
8.30c 15.95a
0.58c 2.71a
5.74 13.61 0.66
10.27b,c 11.97b 0.73
0.77c 1.96b 0.14
10.70 9.67 0.47
12.12 11.12 0.52
1.64 1.37 0.10
6.47b 13.90a 0.47
9.29 13.90 0.52
0.68 2.33 0.10
0.134 < 0.001 0.520
0.183 < 0.001 < 0.001
0.055 < 0.001 0.002
a–c : Means bearing different superscripts within the same column are statistically significant (P < 0.05). NSD: Normal stocking density (14 birds/m2 ). HSD: High stocking density (22 birds/m2 ). n: Number of replicates (n = 8).
conformity with those observed in the present study. HSD creates hindrance, not letting the broilers to move freely, therefore, confining the mobility of birds to a constrained area of the pen. Consequently, the birds subjected to HSD may not have easier access to feeders
Table 6. Incidence and severity of FPD in broilers subjected to normal or high stocking density fed dietary boric acid. Incidence and severity of FPD (%) Day 14 Item NSD 0 mg/kg boric acid 60 mg/kg boric acid HSD 0 mg/kg boric acid 60 mg/kg boric acid Pooled SEM Stocking density NSD HSD Pooled SEM Boric acid 0 mg/kg 60 mg/kg Pooled SEM P-values Stocking density Boric acid Stocking density × boric acid
0 100 100
1
Day 28 2
T
0
0 0
0 0
0 0
94 95
98 94 1.92
1 6 1.82
1 0 0.75
2 6 2.09
100 96 1.36
0 3 1.29
0 1 0.53
99 97 1.36
0 3 1.29 0.060 0.267 0.267
0.050 0.387 0.387
1
Day 42 2
T
2 5
4 0
6 5
40 45 5.40
30 29 4.21
30 26 6.21
60 55 5.40
0b 4a 1.48
94a 42b 3.82
4b 30a 2.98
2b 28a 4.39
1 0 0.53
1 3 1.48
67 70 3.82
16 17 2.98
0.326 0.326 0.326
0.048 0.535 0.535
< 0.001 0.560 0.689
< 0.001 0.826 0.671
0
2
T
6 6
1 0
7 6
42 45 4.16
45 43 3.37
13 12 2.92
58 55 4.17
6b 58a 3.82
94a 43b 2.94
5b 44a 2.38
1b 13a 2.07
6b 57a 2.95
17 13 4.39
33 30 3.82
67 70 2.94
26 24 2.38
7 6 2.07
33 30 2.95
< 0.001 0.518 0.960
< 0.001 0.560 0.689
< 0.001 0.582 0.777
< 0.001 0.797 0.686
< 0.001 0.657 0.983
< 0.001 0.604 0.755
a, b : Means bearing different superscripts within the same column are statistically significant (P < 0.05). NSD: Normal stocking density (14 birds/m2 ). HSD: High stocking density (22 birds/m2 ). 0: No lesion; 1: Mild lesion (< 7.5 mm); 2: Severe lesion (> 7.5 mm). n: Number of replicates (n = 8).
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93 94
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BORIC ACID AND FOOTPAD DERMATITIS IN BROILERS
with those of Yıldız et al. (2013) and Eliot and Edwards (1992), who reported no effect of dietary boric acid on growth performance of broilers. However, other studies have reported an increase in BW of broilers following the inclusion dietary boron (Kurtoglu et al., 2001; Fassani et al., 2004; C ¸ ınar et al., 2015). In contrast, some studies have reported that dietary boric acid supplementation enhanced the growth performance of broilers (Bai and Hunt, 1996; Bozkurt et al., 2012; K¨ uc¸u ¨kyilmaz et al., 2017). The evaluation of difference in results is difficult. It might be attributed to the differences in research protocol, breed, age, composition of diet, and nutritive value of feeds.
Litter Quality
7
seems that reduced litter NH3 volatilization in broilers fed supplemental dietary boric acid might be attributed to reduced litter pH. In addition, boric acid is a good absorbent of NH3 (Siemer, 1986) that has its application in animal nutrition in Kjeldahl method for crude protein estimation in feeds to trap NH3 . This may further reduce NH3 volatilization from the litter of broiler chickens fed diets supplemented with boric acid.
Serum and Litter Boron Levels Dietary boric acid supplementation increased the serum and litter boron levels in broilers subjected to NSD or HSD on day 42. No literature is available describing the effect of dietary boric acid supplementation on serum and litter boron levels of broilers under NSD or HSD. As expected, dietary boron levels increased in broilers subjected to NSD or HSD compared to those fed unsupplemented diets and subjected to NSD or HSD. Serum and litter boron levels were highest in broiler chickens fed boric acid supplemented diets and subjected to NSD followed by those subjected to HSD. Eren et al. (2012) reported a dose-dependent increase in serum boron levels in broilers following the consumption of diets supplemented with 0, 500, 750, and 1,000 mg/kg boric acid. The exact mechanism by which dietary boric acid increased the serum and litter boron levels is not known. This might be attributed to increased boron metabolism that increased the bioavailability of boron in broiler chickens fed boric acid supplemented diets either under NSD or HSD compared to those fed diets without boric acid. Similarly, the increased litter boron levels might be due to the excretion of indigested and excessive boron, boric acid, or its salts through feces in broilers fed boric acid supplemented diets. At present, it is not clear in what form boron is excreted; however, it is assumed that boron salts might be excreted by broilers into the litter. However, broilers subjected to HSD had comparatively lower serum and litter boron levels in comparison with the corresponding groups. The exact mechanism of reduced litter boron levels in broilers subjected to HSD is not known. Apparently, it seems that lowered feed intake in broilers subjected to HSD might have reduced the bioavailability and excretion of boron. In addition, it may be assumed that HSD poses severe physiological stress in broiler chickens that might have affected the metabolism and excretion of boron resulting in lowered serum and litter boron levels.
It is known that HSD quickly deteriorates the quality of litter by increasing the litter moisture, pH, and NH3 volatilization (Sorensen et al., 2000; Dozier et al., 2006; Ravindran et al., 2006; Estevez, 2007; Petek et al., 2014; Kang et al., 2016). High litter moisture helps the microbial growth in the litter and increased microbial activity in the moist litter is a substantial factor in NH3 volatilization (Al-Homidan et al., 2003) thereby increasing the litter pH. It is also evident from litter pH and NH3 volatilization results of our study. In contrast, a recent study reported that gradual increase in stocking density from 16 to 22 birds/m2 had no effect on litter moisture, pH, and NH3 volatilization (Farhadi et al., 2016). Similarly, Zhang et al. (2011) reported no difference in litter pH of broilers subjected to gradual increase in stocking density from 12 to 20 birds/m2 (12, 16, and 20 birds/m2 ). This is the first report describing the effect of dietary boric acid supplementation on litter quality of broilers subjected to NSD or HSD. As anticipated, the supplementation of boric acid in broiler diets reduced the litter pH and NH3 volatilization at day 42 of experiment. In addition, it had no effect on litter moisture. The exact mechanism by which dietary boric acid supplementation reduced the litter pH is not known yet. Most probably, this might be due to the acidic nature of boric acid or boron excreted by the broilers in groups fed dietary boric acid at 60 mg/kg that reduced the litter pH. The explained notion is evident from the increased litter boron levels of broilers fed diets supplemented with boric acid. Moreover, dietary boric acid reduced NH3 volatilization in broilers subjected to NSD or HSD. The NH3 volatilization usually occurs due to microbial decomposition of nitrogen containing compounds (uric acid and urates) in poultry litter. This NH3 is available Incidence and Severity of FPD either as free NH3 or as ammonium ion (NH4 + ) that deThe present study revealed that HSD increased the pends on litter pH. Alkaline pH (pH > 7) favors the decomposition of uric acid. At a litter pH increasing above incidence and severity of FPD in broilers. Many studies 8.5, ureolytic bacteria proliferate at a substantial rate have reported that HSD plays an important role in and release of uricase enzyme (an enzyme that catalyzes the incidence of FPD in poultry in comparison with the decomposition of uric acid) rises, which is associated comparatively lower stocking densities (McIlroy et al., with greater NH3 liberation. The activity of uricase en1987; Ekstrand et al., 1997; Sorensen et al., 2000; Dozier et al., 2005, 2006; Haslam et al., 2007; Meluzzi zyme reaches to its maximum at a pH of 9. Therefore, it Downloaded from https://academic.oup.com/ps/advance-article-abstract/doi/10.3382/ps/pey337/5074500 by University of the Western Cape user on 18 August 2018
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CENGIZ ET AL.
et al., 2008b). However, there are contrasting views of scientists regarding the effective stocking density for the development of FPD. According to Dawkins et al. (2004), leg health issues arise at or beyond the stocking density of 42 kg/m2 . On the other hand, Buijs et al. (2009) reported that development of FPD started at a stocking density of 56 kg/m2 . In contrast, some other studies have demonstrated little or no association between HSD and FPD development in poultry (Martenchar et al., 2002; Sirri et al., 2007; Meluzzi et al., 2008a). The development of FPD occurs due to sudden onset of deterioration in the quality of litter. Higher stocking densities increase the water intake per bird that results in watery feces thereby increasing the litter moisture (Feddes et al., 2002; Bessei, 2006). Mayne et al. (2007b) reported that litter moisture level <30% is optimal for maintenance of footpad health in broilers. Apparently, it seems to happen in the present study as HSD increased the litter moisture (>30%) along with litter pH and NH3 volatilization. Consequently, the incidence and severity of FPD greatly increased in broilers in response to HSD. No literature is available describing the effect of dietary boric acid supplementation on the incidence and severity of FPD in broilers. Despite the fact that dietary boric acid supplementation reduced litter pH and NH3 volatilization, it had no effect on the incidence and severity of FPD in the present study. This might be due to the absence of any difference in litter moisture of broilers fed diets with or without boric acid supplementation. Mayne et al. (2007b) reported that litter moisture alone is sufficient to initiate the development of FPD to a significant extent provided the litter moisture is more than 30%. In the present study, the litter moisture remained lower than 30% and there was no difference in the litter moisture between the groups fed diets with and without boric acid supplementation which explains the possible ineffectiveness of dietary boric acid against the development of FPD in the present study.
CONCLUSIONS The present study showed that dietary boric acid supplementation in broilers subjected to NSD or HSD had no effect on growth performance, litter quality, and incidence and severity of FPD. However, it increased the serum and litter boron levels in broilers subjected to either stocking densities i.e., NSD or HSD. As expected, HSD resulted in poor growth performance, litter quality, and higher incidence and severity of FPD in broilers. Moreover, dietary boric acid reduced the litter pH and NH3 volatilization to a significant extent along with no interactions between boric acid supplementation and stocking density. Therefore, it appears that higher boric acid inclusion levels may be required to further improve the litter quality in terms of pH and NH3 volatilization. Nonetheless, higher inclusion levels may be practiced with the caution of boron toxicity
that requires further investigation. Alternatively, application of boric acid directly to the litter may be taken into consideration as a litter amendment technique to reduce the incidence and severity of FPD in poultry that is only possible after further research.
ACKNOWLEDGMENTS The project was funded by the Scientific and Tech¨ ITAK) ˙ nological Research Council of Turkey (TUB vide project No. 112O389. The funding agency had no role in the study design, execution, analysis, and preparation of this manuscript. The authors are grateful ¨ ITAK ˙ to TUB for the award of doctoral fellowship to ˙ Umair Ahsan under the BIDEB-2215 program for international students.
REFERENCES Abudabos, A. M., E. M. Samara, E. O. S. Hussein, M. Q. Al-Ghadi, and R. M. Al-Atiyat. 2013. Impacts of stocking density on the performance and welfare of broiler chickens. Ital. J. Anim. Sci. 12:66–71. Al-Homidan, A., J. F. Robertson, and A. M. Petchey. 2003. Review of the effect of ammonia and dust concentrations on broiler performance. World’s Poult. Sci. J. 59:340–349. AOAC International. 2000. Official Methods of Analysis, 17th ed. Assoc. Off. Anal. Chem. International, Gaithersburg, MD, US. Bai, Y., and C. D. Hunt. 1996. Dietary boron enhances efficacy of cholecalciferol in broiler chicks. J. Trace Elem. Exp. Med. 9:117– 132. Banhazi, T. M., J. Seedorf, M. Laffrique, and D. L. Rutley. 2008. Identification of the risk factors for high airborne particle concentrations in broiler buildings using statistical modelling. Biosyst. Eng. 101:100–110. Bedford, M. R., and J. Apajalathi. 2001. Microbial interactions in the response to exogenous enzyme utilization. Pages 299–314 in Enzymes in Farm Animal Nutrition. M. R. Bedford, and G. G. Partridge, eds. CABI Publishing, London, UK. Bessei, W. 2006. Welfare of broilers: a review. World’s Poult. Sci. J. 62:455–466. Bilgili, S. F., M. A. Alley, J. B. Hess, and M. Nagaraj. 2006. Influence of age and sex on footpad quality and yield in broiler chickens reared on low and high density diets. J. Appl. Poult. Res. 15:433– 441. Biswas, T., L. Mandal, and S. K. Sarker. 1999. Studies of enzymes supplementation and herbal preparation at different levels of energy on the performance of broilers. J. Interacad. 3:53–58. Bozkurt, M., K. K¨ uc¸u ¨kyilmaz, A. U. C ¸ atlı, and M. C ¸ ınar. 2012. Effects of Boron supplementation to diets deficient in Calcium and Phosphorus on performance with some serum, bone and fecal characteristics of broiler chickens. Asian-Australas. J. Anim. Sci. 25:248–255. Buijs, S., E. Van Poucke, F. A. M. Tuyttens, L. Keeling, and S. Rettenbacher. 2009. Stocking density effects on broiler welfare: Identifying sensitive ranges for different indicators. Poult. Sci. 88:1536–1543. ¨ J. B. Hess, and S. F. Bilgili. 2011. Effect of bedding type Cengiz, O., and transient wetness on footpad dermatitis in broiler chickens. J. Appl. Poult. Res. 20:554–560. ¨ J. B. Hess, and S. F. Bilgili. 2012a. Dietary biotin supCengiz, O., plementation does not alleviate the development of footpad dermatitis in broiler chickens. J. Appl. Poult. Res. 21:764–769. ¨ J. B. Hess, and S. F. Bilgili. 2012b. Influence of graded Cengiz, O., levels of dietary sodium on the development of footpad dermatitis in broiler chickens. J. Appl. Poult. Res. 21:770–775. ¨ J. B. Hess, and S. F. Bilgili. 2012c. Feed enzyme supCengiz, O., plementation does not ameliorate foot pad dermatitis in broiler chickens fed on a corn-soyabean diet. Br. Poult. Sci. 53:401–407.
Downloaded from https://academic.oup.com/ps/advance-article-abstract/doi/10.3382/ps/pey337/5074500 by University of the Western Cape user on 18 August 2018
BORIC ACID AND FOOTPAD DERMATITIS IN BROILERS ¨ B. H. K¨ ¨ ¨ Sevim, H. Avcı, Cengiz, O., oksal, A. G. Onol, O. Tatlı, O. and S. F. Bilgili. 2012d. Influence of dietary enzyme supplementation of barley-based diets on growth performance and footpad dermatitis in broiler chickens exposed to early high-moisture litter. J. Appl. Poult. Res. 21:117–125. ¨ J. B. Hess, and S. F. Bilgili. 2013. Effect of protein Cengiz, O., source on the development of footpad dermatitis in broiler chickens reared on different flooring types. Arch. fur Geflugelk. 77:166– 170. ¨ B. H. K¨ ¨ ¨ Sevim, and S. F. Bilgili. Cengiz, O., oksal, A. G. Onol, O. 2014. Dietary zinc supplementation does not ameliorate footpad dermatitis in broilers reared under heat stress. Proc. XXVI Intl. Poult. Symp. pp. 25. ( Abstr.) ¨ B. H. K¨ ¨ Sevim, U. Ahsan, A. G. Uner, ¨ Cengiz, O., oksal, O. Tatlı, O. P. A. Uluta¸s, D. Beyaz, S. B¨ uy¨ uky¨ or¨ uk, A. Yakan, and A. G. ¨ Onol. 2015. Effect of dietary probiotic and high stocking density on the performance, carcass yield, gut microflora, and stress indicators of broilers. Poult. Sci. 94:2395–2403. Chavez, E., and F. H. Kratzer. 1972. Prevention of foot pad dermatitis in poults with methionine. Poult. Sci. 51:1545–1548. C ¸ inar, M., K. K¨ uc¸u ¨kyilmaz, M. Bozkurt, A. U. C ¸ atli, E. Binta¸s, H. Ak¸sit, R. Konak, C ¸ . Yamaner, and K. Seyrek. 2015. Effects of dietary boron and phytase supplementation on growth performance and mineral profile of broiler chickens fed on diets adequate or deficient in calcium and phosphorus. Br. Poult. Sci. 56:576–589. Dawkins, M. S., C. A. Donnelly, and T. A. Jones. 2004. Chicken welfare is influenced more by housing conditions than by stocking density. Nature 427:342–344. Dozier, W. A., J. P. Thaxton, S. L. Branton, G. W. Morgan, D. M. Miles, W. B. Roush, B. D. Lott, and Y. Vizzier-Thaxton. 2005. Stocking density effects on growth performance and processing yields of heavy broilers. Poult. Sci. 84:1332–1338. Dozier, W. A., III, J. P. Thaxton, J. L. Purswell, H. A. Olanrewaju, S. L. Branton, and W. B. Roush. 2006. Stocking density effects on male broilers grown to 1.8 kilograms of body weight. Poult. Sci. 85:344–351. Ekstrand, C., B. Algers, and J. Svedberg. 1997. Rearing conditions and foot-pad dermatitis in Swedish broiler chickens. Prev. Vet. Med. 31:167–174. Eliot, M. A., and H. M. Edwards. 1992. Studies to determine whether an interaction exists among boron, calcium, and cholecalciferol on the skeletal development of broiler chickens. Poult. Sci. 71:677– 690. Eren, M., F. Uyanik, B. K. Guclu, and A. Atasever. 2012. The influence of dietary Boron supplementation on performance, some biochemical parameters and organs in broilers. Asian J. Anim. Vet. Adv. 7:1079–1089. Estevez, I. 2007. Density allowances for broilers: where to set the limits? Poult. Sci. 86:1265–1272. Farhadi, D., S. M. Hosseini, and B. T. Dezfuli. 2016. Effect of house type on growth performance, litter quality and incidence of foot lesions in broiler chickens reared in varying stocking density. J. Biosci. Biotech. 1:69–78. Fassani, E. J., A. G. Bertechini, J. A. G. Brito, R. K. Kato, E. T. Fialho, and A. Geraldo. 2004. Boron supplementation in broiler diets. Rev. Bras. Cienc. Avic. 6:213–217. Feddes, J. J. R., E. J. Emmanuel, and M. J. Zuidhof. 2002. Broiler performance, body weight variance, feed and water intake, and carcass quality at different stocking densities. Poult. Sci. 81:774– 779. Guardia, S., B. Konsak, S. Combes, F. Levenez, L. Cauquil, J. F. Guillot, C. Moreau-Vauzelle, M. Lessire, H. Juin, and I. Gabriel. 2011. Effects of stocking density on the growth performance and digestive microbiota of broiler chickens. Poult. Sci. 90:1878–1889. Haslam, S. M., T. G. Knowles, S. N. Brown, L. J. Wilkins, S. C. Kestin, P. D. Warriss, and C. J. Nicol. 2007. Factors affecting the prevalence of foot pad dermatitis, hock burn and breast burn in broiler chicken. Br. Poult. Sci. 48:264–275. J´ ozefiak, D., A. Rutkowski, M. Fratczak, and D. Boros. 2004. The effect of dietary fibre fractions from different cereals and microbial enzymes supplementation on performance, ileal viscosity and short-chain fatty acids concentration in caeca of broiler chickens. J. Anim. Feed Sci. 13:487–496.
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Kang, H. K., S. B. Park, S. H. Kim, and C. H. Kim. 2016. Effects of stock density on the laying performance, blood parameter, corticosterone, litter quality, gas emission and bone mineral density of laying hens in floor pens. Poult. Sci. 95:2764–2770. K¨ uc¸u ¨kyilmaz, K., M. Bozkurt, M. C ¸ ınar, and A. E. T¨ uz¨ un. 2017. Evaluation of the Boron and phytase, alone or in combination, in broiler diets. Jpn. Poult. Sci. 54:26–33. Kurtoglu, V., F. Kurtoglu, and B. Co¸skun. 2001. Effects of boron supplementation of adequate and inadequate vitamin D3containing diet on performance and serum biochemical characters of broiler chickens. Res. Vet. Sci. 71:183–187. L´azaro, R., M. Garcia, P. Medel, and G. G. Mateos. 2003. Influence of enzymes on performance and digestive parameters of broilers fed rye-based diets. Poult. Sci. 82:132–140. Martenchar, A., E. Boilletot, D. Huonnic, and F. Pol. 2002. Risk factors for foot-pad dermatitis in chicken and turkey broilers in France. Prev. Vet. Med. 52:213–226. Mayne, R. K., R. W. Else, and P. M. Hocking. 2007a. High dietary concentrations of biotin did not prevent foot pad dermatitis in growing turkeys and external scores were poor indicators of histopathological lesions. Br. Poult. Sci. 48:291–298. Mayne, R. K., R. W. Else, and P. M. Hocking. 2007b. High litter moisture alone is sufficient to cause footpad dermatitis in growing turkeys. Br. Poult. Sci. 48:538–545. McIlroy, S. G., E. A. Goodall, and C. H. McMurray. 1987. A contact dermatitis of broilers – epidemiological findings. Avian Pathol. 16:93–105. Meluzzi, A., C. Fabbri, E. Folegatti, and F. Sirri. 2008a. Survey of chicken rearing conditions in Italy: effects of litter quality and stocking density on productivity, foot dermatitis and carcase injuries. Br. Poult. Sci. 49.257–264. Meluzzi, A., F. Sirri, E. Folegatti, and C. Fabbri. 2008b. Effect of less intensive rearing conditions on litter characteristics, growth performance, carcase injuries and meat quality of broilers. Br. Poult. Sci. 49:509–515. National Research Council. 1994. Nutrient Requirements of Poultry. 9th rev. ed. Natl. Acad. Press, Washington, DC. ¨ uner, and D. Ye¸silba˘ Petek, M., H. Ust¨ g. 2014. Effects of stocking density and litter type on litter quality and growth performance of broiler chicken. Kafkas Univ. Vet. Fak. Derg. 20:743–748. Puron, D., R. Santamaria, J. C. Segaura, and J. L. Alamilla. 1995. Broiler performance at different stocking densities. J. Appl. Poult. Res. 4:55–60. Ravindran, V., D. V. Thomas, D. G. Thomas, and P. C. H. Morel. 2006. Performance and welfare of broilers as affected by stocking density and zinc bacitracin supplementation. Anim. Sci. J. 77:110–116. Sauter, E. A., C. F. Petersen, E. E. Steele, J. F. Parkinson, J. E. Dixon, and R. C. Stroh. 1981. The airborne microflora of poultry houses. Poult. Sci. 60:569–574. Shepherd, E. M., and B. D. Fairchild. 2010. Footpad dermatitis in poultry. Poult. Sci. 89:2043–2051. Siemer, D. D. 1986. Use of solid boric acid as an ammonia absorbent in the determination of nitrogen. Analyst 111:1013–1015. Sirri, F., G. Minelli, E. Folegatti, S. Lolli, and A. Meluzzi. 2007. Foot dermatitis and productive traits in broiler chickens kept with different stocking densities, litter types and light regimen. Ital. J. Anim. Sci. 6:734–736. Sorensen, P., G. Su, and S. C. Kestin. 2000. Effects of age and stocking density on leg weakness in broiler chickens. Poult. Sci. 79:864– 870. Van Soest, P. J., J. B. Robertson, and B. A. Lewis. 1991. Methods for dietary fiber, neutral detergent fiber, and nonstarch polysaccharides in relation to animal nutrition. J. Dairy Sci. 74:3583– 3597. Yildiz, G., B. H. Koksal, and O. Sizmaz. 2013. Effects of dietary boric acid addition on growth performance, cholesterolemia, some carcass and tibia characteristics in different rearing periods in broiler chickens. Rev. M´ed. V´et. 164:219–224. Zhang, H. F., H. C. Jiao, Z. G. Song, and G. Lin. 2011. Effect of alum-amended litter and stocking density on ammonia release and footpad and hock dermatitis of broilers. Agric. Sci. China 10:777–785.
Downloaded from https://academic.oup.com/ps/advance-article-abstract/doi/10.3382/ps/pey337/5074500 by University of the Western Cape user on 18 August 2018
Department of Animal Nutrition and Nutritional Diseases, Faculty of Veterinary Medicine, Adnan Menderes University, I¸sıklı, Aydın 09016, Turkey; † Department of Chemistry, Faculty of Art and Science, Adnan Menderes University, Aydın 09100, Turkey; and ‡ Department of Poultry Sciences, College of Agriculture, Auburn University, Auburn, AL 36849-5416, USA to 21 days only. Dietary boric acid had no effect on the growth performance of broiler chickens. Litter pH, moisture, and NH3 volatilization were higher in broiler chickens subjected to HSD (P < 0.05). Thus, the incidence and severity of FPD increased in response to HSD (P < 0.05). Dietary boric acid reduced the litter pH and NH3 volatilization on day 42 of experiment (P < 0.05). However, dietary boric acid supplementation had no effect on the incidence and severity of FPD. Boric acid supplementation in broiler diets increased the serum and litter boron levels at day 42 in broiler chickens subjected to NSD or HSD (P < 0.05). In conclusion, HSD resulted in poor growth performance, litter quality, and greater incidence and severity of FPD in broiler chickens. Dietary boric acid was ineffective against FPD in broiler chickens although it improved the litter quality by lowering the litter pH and NH3 volatilization.
ABSTRACT The present study was conducted to evaluate the protective effect of dietary boric acid supplementation on the development of incidence and severity of footpad dermatitis (FPD) in broiler chickens subjected to normal or high stocking densities (NSD or HSD). A total of 576 1-day-old ROSS 308 broiler chickens were randomly allocated to 4 treatments (8 replicate pens per treatment) in a 2 × 2 factorial arrangement of dietary boric acid (0 and 60 mg/kg) and stocking density (NSD 14 birds/m2 and HSD 22 birds/m2 ). Basal diets were formulated for starter, grower, and finisher phases. Growth performance, litter quality (litter pH, moisture, temperature, and NH3 volatilization), serum and litter boron levels, and incidence and severity of FPD were recorded. The HSD affected the body weight gain and feed intake of broiler chickens during all phases and 0 to 42 (P < 0.05), whereas feed conversion ratio (FCR) was poor at 0
Key words: boric acid, broiler, footpad dermatitis, litter quality, stocking density 2018 Poultry Science 0:1–9 http://dx.doi.org/10.3382/ps/pey337
INTRODUCTION
However, HSD causes severe deterioration of litter quality in poultry houses that affects leg health resulting in FPD in poultry. In the past, different strategies have been adopted to prevent and treat the FPD in broilers. These strategies involve interventions of dietary levels of methionine (Chavez and Kratzer, 1972), biotin (Mayne et al., 2007a; Cengiz et al., 2012a), sodium (Cengiz et al., 2012b), zinc (Cengiz et al., 2014), protein source (Cengiz et al., 2013), bedding type (Cengiz et al., 2011), and the supplementation of exogenous enzymes (Cengiz et al., 2012c, d). However, these manipulations have been unsuccessful to completely ameliorate or prevent the broilers from occurrence of FPD. Consequently, researchers have necessitated developing alternative solutions in order to overcome this problem. It is well known that increased litter ammonia (NH3 ) increases litter pH that makes the litter caustic in nature, therefore, associated with poor foot health and FPD in poultry (Mayne et al., 2007b). Litter amendments can be helpful in the prevention of FPD in
High stocking density (HSD) has become a point of concern from broiler welfare perspective in recent years. The HSD is one of the predisposing factors involved in the development of footpad dermatitis (FPD) in poultry (Shepherd and Fairchild, 2010) which is not only important from economic point of concern, but has become animal welfare, food safety, and product quality issue as well. Increasing the stocking density results in beneficial economic impact provided it does not go beyond an optimum range of 34 to 40 kg/m2 assuming 2.5 kg final BW on an average (Dozier et al., 2005, 2006; Estevez, 2007). However, going beyond this range worsens the health and performance of broilers. C 2018 Poultry Science Association Inc. Received October 2, 2017. Accepted July 10, 2018. 1 Corresponding author: E-mail: [email protected]
1 Downloaded from https://academic.oup.com/ps/advance-article-abstract/doi/10.3382/ps/pey337/5074500 by University of the Western Cape user on 18 August 2018
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CENGIZ ET AL. Table 1. Composition and chemical analysis of basal diets for starter, grower, and finisher (g/kg as fed basis). Experimental diets Items Corn Soybean meal Vegetable oil Limestone Dicalcium phosphate Salt DL-Methionine L-Lysine Vitamin-mineral premix1 Calculated nutrient composition Metabolizable energy (MJ/kg) CP Ca Available P Methionine + Cysteine Lysine Sodium Analyzed nutrient composition Dry matter CP Ether extract Nitrogen free extract NDF Ca Total P Boron (ppm), (basal/boric acid diet)
Starter (0 to 10 d)
Grower (11 to 24 d)
Finisher (25 to 42 d)
556.3 375.0 25.0 8.9 23.0 3.5 3.7 2.1 2.5
561.5 360.0 41.5 8.5 20.0 3.5 2.5 2.5
590.0 335.0 42.5 8.0 17.3 3.5 1.2 2.5
12.7 230 10.0 5.0 10.9 14.2 1.6 884 227.8 61.9 511.1 84.6 11.8 8.6 13.15/20.68
13.2 220 9.1 4.5 9.5 12.1 1.6 883 218.0 78.0 507.6 79.7 10.9 7.9 12.56/23.45
13.4 210 8.0 4.0 8.0 11.5 1.6 887 208.5 80.8 524.2 75.4 9.9 7.3 13.08/24.15
1 Supplied per kilogram of diet: vitamin A, 12,000 IU; vitamin D3, 1,500 IU; vitamin E, 30 mg; vitamin K3, 5 mg; vitamin B1, 3 mg; vitamin B2, 6 mg; vitamin B6, 5 mg; vitamin B12, 0.03 mg; niacin, 40 mg; calcium D-pantothenate, 10 mg; folic acid, 0.75 mg; D-Biotin, 0.075 mg; choline chloride, 375 mg; Mn, 80 mg; Fe, 40 mg; Zn, 60 mg; Cu, 5 mg; I, 0.4 mg; Co, 0.1 mg; Se, 0.15 mg; and antioxidant, 10 mg.
broilers. Boric acid possesses the ability to absorb NH3 (Siemer, 1986), thereby it can reduce NH3 volatilization from litter in poultry houses. Conversely, dietary boric acid can reduce the litter pH, which, in turn, can help to prevent the incidence and reduce the severity of FPD in broilers. Therefore, an experiment was conducted to evaluate the litter quality improvement and reduction of incidence and severity of FPD by dietary supplementation of boric acid in broilers subjected to normal stocking density (NSD) or HSD. To the best of our knowledge, no such report is available in literature that describes the use of supplemental dietary boric acid to improve the litter quality, and footpad health in broilers. The aim of the present study was to investigate the effect of dietary boric acid on growth performance, litter quality, serum and litter boron levels, and incidence and severity of FPD in broilers subjected to NSD or HSD.
MATERIALS AND METHODS The study was conducted at Poultry Research Unit of Faculty of Veterinary Medicine, Adnan Menderes University, Aydın, Turkey, with prior approval from the animal care and use committee of the university (approval letter no. 050.04/2012/007).
Study Design and Experimental Groups The study was conducted as a completely randomized design in a 2 × 2 factorial arrangement of dietary boric acid supplementation (0 and 60 mg/kg of diet) and stocking density (14 and 22 birds/m2 ). A total of 576 1-day-old male broiler chicks of Ross 308 strain were randomly allocated to 4 experimental groups, each comprising of 8 replicate pens adjustable at the front. Sixteen replicate pens were subjected to HSD, whereas remaining replicate pens were subjected to NSD. Corn/soybean meal-based basal diets were formulated for starter (days 0 to 10), grower (days 11 to 24), and finisher (days 25 to 42) phases of broilers in accordance with the recommendations of NRC (1994) to meet or exceed the nutrient requirements of broilers (Table 1). Two experimental groups subjected to NSD and HSD were fed basal diets. Other experimental groups (NSD and HSD) were fed basal diets supplemented with boric acid (containing 17.5% boron) at 60 mg per kg of diet. The experimental groups have been presented in Table 2.
Management of Birds A total of 32 replicate pens were established in the experimental rooms. Initially, each replicate pen sized
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BORIC ACID AND FOOTPAD DERMATITIS IN BROILERS Table 2. Description of experimental groups. Serial No.
Description
1 2 3 4
NSD HSD NSD HSD
+ + + +
0 mg/kg boric acid 0 mg/kg boric acid 60 mg/kg boric acid 60 mg/kg boric acid
NSD, normal stocking density; HSD, high stocking density.
1.1 m × 1.5 m with adjustable pane at the front. The replicate pens meant for HSD provided floor space of 0.045 m2 /bird. The replicate pens subjected to NSD provided the floor space 0.071 m2 /bird. Floor space per bird was exclusive of the space occupied by feeders and drinkers. Approximately 7 to 8 cm deep layer of soft pine wood shavings was laid as a litter in each pen. Temperature of experimental rooms was maintained at 32 to 33◦ C before the arrival of chicks. Oneday-old male broilers were purchased from a commercial hatchery (Egetav Tavuk¸culuk San. ve Tic. A.S ¸ ., ˙ Izmir, Turkey). The groups subjected to HSD received 22 chicks per replicate pen, whereas those subjected to NSD received 14 chicks per replicate pen. The experiment lasted for 42 d. Birds had ad libitum access to feed and water throughout the experiment. Metal feeders (50 × 8 cm; 0.04 m2 ) were used during the starter phase (day 0 to 10) and tube feeders (r = 40 cm; 0.1256 m2 ) were used afterwards (day 11 to 42). Each pen was provided with one feeder. Water was supplied to each pen using nipple drinkers in each pen. The number of feeders and drinkers per pen remained unchanged throughout the experiment. Fluorescence lights were used to lit the experimental rooms during the dark periods of the day (which provided light period of 23L:1D). A constant temperature of 33 ◦ C was maintained at days 0 to 7 of the experiment. Later, 3◦ C was reduced per week until a constant temperature of 24◦ C was achieved that was maintained to the end of the experiment.
Chemical Analysis of Feed Feed samples were oven-dried at 105◦ C for 8 h to estimate the dry matter content of basal diets. Kjeldahl method was used for the estimation of nitrogen (N) content, and crude protein was calculated as N × 6.25. Soxhlet extraction technique was used for ether extract estimation. Van Soest method was employed to determine the neutral detergent fiber content of basal diets (Van Soest et al., 1991). Feed samples were burnt in muffle furnace at 600◦ C for crude ash estimation. Nitrogen-free extract was calculated by subtracting crude protein, ether extract, neutral detergent fiber, and crude ash from dry matter content of the diet. Calcium (Ca) content of basal diets was determined following the wet ash digestion method using nitric and perchloric acids (AOAC,
3
2000; Method 935.13). Phosphorus (P) content of diets was estimated according to the alkalimetric ammonium molybdophosphate method (AOAC, 2000; Method 964.06).
Growth Performance Birds were weighed weekly on per pen basis. The weight of dead bird was also recorded. Feed intake was recorded weekly by the difference between amount of feed offered and amount of feed not consumed. Body weight (BW) gain and feed intake per pen were calculated for the periods 0 to 21, 21 to 42, and 0 to 42 days. BW gain was calculated by adjusting the weight of dead birds (if any) in the pen weight of that pen during the specific phase. Feed conversion ratio (FCR) was calculated using the formula of ratio between feed intake and BW gained.
Litter Quality Litter quality was measured in terms of litter quality variables such as pH, moisture, temperature, NH3 volatilization. For this purpose, approximately 100 g litter samples were collected in plastic bags from each pen at the time of commencement of experiment (initial) and at the end of experiment (final). Each sample was collected from different localities within a pen e.g., near the drinkers, around the feeder, and the corners. The sample was mixed thoroughly to homogenize in order to avoid variability. Litter pH was measured by mixing 100 mL of distilled water in 10 g of homogenized sample. The mixture was agitated for 5 min and suspended for 30 min. A portable waterproof pH meter (Model: HI 9124, Hanna Instruments Inc., RI) was inserted into the agitated mixture. Reading was taken upon the stability of pH value on the meter. Litter moisture was determined by drying the 80 g of homogenized sample at 105◦ C for 24 h in a hot air oven. The samples were weighed after drying. The difference between initial and final samples was taken as litter moisture and presented as a percentage. Litter temperature was measured in each pen at 3 different sites (near feeder, around drinker, and near the bird’s resting place within the pen) with the help of a digital probe thermometer (TFA Dostmann GmbH + Co. KG, Germany). Probe of the thermometer was inserted 2 to 3 cm deep in to the litter of each pen. The reading was taken when the temperature became stable. The NH3 volatilization was measured above 20 cm from the surface level of litter in each pen. Dr¨ ager NH3 meter (Dr¨ager Pac 7000, Dr¨ ager Safety AG and Co. KGaA, Luebeck, Germany) was used with the chips sensitivity ranging from 0.2 to 5 ppm, 2 to 50 ppm, and 10 to 150 ppm NH3 level.
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CENGIZ ET AL.
Feed, Serum, and Litter Boron Levels Boron content of the diets was determined using inductively coupled plasma optical emission spectrometry (ICP-OES). Briefly, 0.3 g feed sample was subjected to wet digestion with 8 mL 65% nitric acid and 2 mL 37% hydrochloric acid in a sealing vessel at 180◦ C in a specialized microwave for 20 min. Blood samples (10 mL) were collected from caudal tibial vein of one bird from each pen on 28 and 42 d of the experiment by inserting sterile syringe (needle 22-gauge, 1-inch length) at a shallow angle. Blood samples were processed for serum separation in a centrifuge (NF 200 Bench-Top ¨ Centrifuge, NUVE, Ankara, Turkey), immediately after clotting of blood samples. Serum boron level was measured using ICP-OES. A known quantity of serum (2 mL) was mixed with 5 mL 65% nitric acid and 2 mL 35% hydrogen peroxide in a sealing vessel that was placed in a specialized microwave (MARS 6 One Touch Microwave Reaction System, CEM Corporation, Matthews, NC) at 200◦ C for digestion for 15 min. At the end of experiment, litter samples for boron analysis collected from each pen were dried at 60◦ C for 72 h and 0.2 g dried litter sample was mixed with 10 mL 65% nitric acid in a sealing vessel that was placed in a specialized microwave at 180◦ C for digestion for 20 min. The digested samples of feed, serum, and litter were cooled, filtered in flask, and diluted to 25 mL by adding deionized water for analysis in ICP-OES spectrometer.
FPD Scoring On 14, 28, and 42 days of experiment, all birds were subjected to 3-point FPD scoring according to the method previously described by Bilgili et al. (2006). Briefly, footpads without any lesion were scored as 0, footpads with lesion size ≤7.5 mm were scored as 1, and those with lesion size >7.5 mm were scored as 2. Average FPD score was calculated for each score and presented as percentage. The following mathematical equation was applied for average FPD score: Average FPD score (Ni ) = Number of birds showing score 0, 1, or 2 (ni ) /Total number of birds scored
Statistical Analysis The data were analyzed using a statistical software package SPSS (version 22.0 Armonk, NY) to assess the effect of dietary boric acid on performance, litter quality, serum and litter boron levels, and incidence and severity of FPD in broilers subjected to NSD or HSD. Non-normalized parameters were tested with the help of Kruskal–Wallis test and transformed using logarithmic or square root transformation method. The percentage data (FPD score) were transformed to arcsine
values before analysis, however, presented in percentage in the results. Two-way analysis of variance was applied in GLM procedures of SPSS. Confidence interval of 95% (P-value < 0.05) was considered as significant for interactions and main effects. The interaction means were differentiated using Tukey’s HSD as post hoc test in case of significant interactions.
RESULTS The study revealed that there was no interaction between stocking density and dietary boric acid supplementation for growth performance of broilers (Table 3). HSD reduced (P < 0.05) the BW gain and feed intake at 0 to 21, 21 to 42, and 0 to 42 days in broilers compared to those subjected to NSD. FCR of broilers subjected to HSD improved at 0 to 21 days as compared to those reared under NSD (P < 0.05). There was a numerical difference that approached significance in FCR of broilers at 21 to 42 days in response to HSD compared to NSD (P = 0.06). Dietary boric acid supplementation had no significant effect (P > 0.05) on BW gain, feed intake, and FCR of broilers in comparison with those fed diets without boric acid. Although the interaction was not significant between stocking density and dietary boric acid supplementation, dietary boric acid supplementation lowered (P = 0.057) NH3 volatilization in broilers subjected to NSD or HSD (Table 4). There was no difference in litter pH, moisture, and temperature at day 0 in either group subjected to stocking density (HSD or NSD) or fed dietary boric acid supplementation (0 or 60 mg/kg). However, litter pH and moisture increased at the end of experiment (day 42) in groups subjected to HSD compared to NSD (P < 0.05). An increase in NH3 volatilization was observed at 28 and 42 days of experiment in groups reared under HSD in comparison with those under NSD (P < 0.05). Dietary boric acid supplementation reduced the litter pH and NH3 volatilization on day 42 of experiment in comparison with those fed diets without boric acid supplementation (P < 0.05). Litter temperature remained unaffected irrespective of the treatments. No interaction was noted between stocking density and dietary boric acid supplementation for serum boron levels at day 28 of the experiment (Table 5). However, significant interaction was observed for serum and litter boron levels (P < 0.05) at the end of the experiment (day 42). While dietary boric acid supplementation increased serum boron levels in birds placed at NSD (P < 0.05), it had no effect on those placed at HSD. Similarly, broilers subjected to NSD and HSD fed diets supplemented with boric acid had higher (P < 0.05) litter boron levels in comparison with those fed diets without supplementation placed at NSD or HSD. There was no interaction between stocking density and dietary boric acid supplementation for incidence and severity of FPD in broilers (Table 6). Broilers
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BORIC ACID AND FOOTPAD DERMATITIS IN BROILERS
Table 3. Body weight gain (BWG), feed intake (FI), and feed conversion ratio (FCR) of broilers subjected to normal or high stocking density fed dietary boric acid. BWG (g) Item NSD 0 mg/kg boric acid 60 mg/kg boric acid HSD 0 mg/kg boric acid 60 mg/kg boric acid Pooled SEM Stocking Density NSD HSD Pooled SEM Boric Acid 0 mg/kg 60 mg/kg Pooled SEM P-values Stocking density Boric acid Stocking density × boric acid
FI (g)
FCR
0 to 21 d
21 to 42 d
0 to 42 d
0 to 21 d
21 to 42 d
0 to 42 d
0 to 21 d
21 to 42 d
0 to 42 d
937 923
2059 2067
2996 2990
1322 1321
3670 3722
4992 5043
1.41 1.43
1.78 1.80
1.67 1.69
888 919 10.20
1802 1734 37.51
2690 2653 36.21
1211 1217 22.97
3337 3276 78.46
4549 4493 81.49
1.36 1.32 0.02
1.85 1.89 0.04
1.69 1.70 0.02
990a 903b 7.21
2,063a 1,768b 26.52
2,993a 2,671b 25.60
1,321a 1,214b 16.25
3,696a 3,307b 55.48
5,018a 4,521b 57.62
1.42a 1.34b 0.02
1.79 1.87 0.03
1.68 1.69 0.02
913 921 7.21
1930 1901 26.52
2843 2821 25.60
1267 1269 16.25
3504 3499 55.48
4770 4768 57.62
1.39 1.38 0.02
1.82 1.85 0.03
1.68 1.69 0.02
0.015 0.426 0.122
< 0.001 0.434 0.320
< 0.001 0.557 0.674
< 0.001 0.922 0.872
< 0.001 0.957 0.473
< 0.001 0.981 0.518
0.001 0.674 0.141
0.058 0.451 0.793
0.536 0.595 0.756
a, b : Means bearing different superscripts within the same column are statistically significant (P < 0.05). NSD: Normal stocking density (14 birds/m2 ). HSD: High stocking density (22 birds/m2 ). n: Number of replicates (n = 8).
Table 4. Litter pH, moisture (%), temperature (◦ C), and NH3 volatilization (ppm) from litter of broilers subjected to normal or high stocking density fed dietary boric acid. pH Item NSD 0 mg/kg boric acid 60 mg/kg boric acid HSD 0 mg/kg boric acid 60 mg/kg boric acid Pooled SEM Stocking density NSD HSD Pooled SEM Boric acid 0 mg/kg 60 mg/kg Pooled SEM P-values Stocking density Boric acid Stocking density × boric acid
Moisture
Temperature
NH3 volatilization
day 0
day 42
day 0
day 42
day 0
day 42
day 28
day 42
5.98 6.03
8.76 8.48
8.97 8.61
21.39 21.50
20.13 20.88
22.50 22.82
1.64 1.82
2.98 2.70
6.22 5.82 0.12
9.33 8.49 0.14
9.13 8.94 0.23
34.33 32.99 0.73
19.88 20.12 0.42
23.19 22.93 0.29
2.76 2.59 0.15
4.15 3.39 0.12
6.01 6.02 0.09
8.62b 9.09a 0.10
8.79 9.04 0.17
21.44b 33.66a 0.52
20.50 20.00 0.30
22.66 23.06 0.21
1.73b 2.68a 0.10
2.84b 3.77a 0.08
6.09 5.92 0.09
9.04a 8.67b 0.10
9.04 8.78 0.17
27.86 27.24 0.52
20.00 20.50 0.30
22.84 22.88 0.21
2.21 2.21 0.10
3.56a 3.05b 0.08
0.602 0.403 0.117
0.003 0.013 0.480
0.296 0.262 0.700
< 0.001 0.401 0.326
0.241 0.241 0.554
0.186 0.918 0.356
< 0.001 0.983 0.243
< 0.001 < 0.001 0.057
a, b : Means bearing different superscripts within the same column are statistically significant (P < 0.05). NSD: Normal stocking density (14 birds/m2 ). HSD: High stocking density (22 birds/m2 ). n: Number of replicates (n = 8).
subjected to HSD had greater incidence and severity of FPD at days 14 (P = 0.05 for score 0, P = 0.06 for score 1, and P < 0.05 for total score), 28, and 42 (P < 0.05) in comparison with those placed at NSD. Dietary boric acid supplementation had no effect on the incidence and severity of FPD in broilers compared to those fed diets without boric acid supplementation.
DISCUSSION Growth Performance Several studies have reported that HSD is associated with lower growth performance of broilers in terms of BW gain, feed intake, and FCR (Puron et al., 1995; Feddes et al., 2002; Dozier et al., 2005; Abudabos et al., 2013; Cengiz et al., 2015). These results are in
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CENGIZ ET AL.
and drinkers compared to those placed at NSD that leads to lowered feed and water intake. This may result in poorer performance of broilers although it might not be the case in the starter phase attributed to less space requirements and relatively smaller size of the chicks (Cengiz et al., 2015). However, HSD may affect the behavior of broilers toward feed intake in the starter phase as the age progresses and increasing physiological stress. Feddes et al. (2002) reported that poor growth performance is attributed to the difficulty in exchange of heat and gases within the microclimate of broilers subjected to HSD. Puron et al. (1995) demonstrated an inverse relationship between growth performance of broilers and HSD beyond a threshold. Therefore, growth performance was negatively affected in broilers subjected to HSD in comparison with those reared under NSD. Other possible explanations in support of declined growth performance in broilers subjected to HSD may be the disruption in gut microbiota that helps in digestion, absorption of nutrients, and improvement of intestinal microarchitecture by reducing the pathogenic bacterial populations (Biswas et al., 1999; Bedford and Apajalathi, 2001; L´ azaro et al., 2003; J´ ozefiak et al., 2004; Guardia et al., 2011). In addition, HSD may be associated with the surge in airborne pathogens (Sauter et al., 1981) probably due to reduced removal of dust (Banhazi et al., 2008), a possible cause of poor growth performance of broilers reared under HSD. There are contrasting results reported by different researchers in the past. In the present study, dietary boric acid supplementation had no effect on growth performance of broilers. These results are in agreement
Table 5. Serum and litter boron levels of broilers subjected to normal or high stocking density fed dietary boric acid. Serum boron levels (mg/L) Item NSD 0 mg/kg boric acid 60 mg/kg boric acid HSD 0 mg/kg boric acid 60 mg/kg boric acid Pooled SEM Stocking density NSD HSD Pooled SEM Boric acid 0 mg/kg 60 mg/kg Pooled SEM P-values Stocking density Boric acid Stocking density × boric acid
Litter boron levels (mg/L)
day 28
day 42
day 42
7.20 14.20
8.30c 15.95a
0.58c 2.71a
5.74 13.61 0.66
10.27b,c 11.97b 0.73
0.77c 1.96b 0.14
10.70 9.67 0.47
12.12 11.12 0.52
1.64 1.37 0.10
6.47b 13.90a 0.47
9.29 13.90 0.52
0.68 2.33 0.10
0.134 < 0.001 0.520
0.183 < 0.001 < 0.001
0.055 < 0.001 0.002
a–c : Means bearing different superscripts within the same column are statistically significant (P < 0.05). NSD: Normal stocking density (14 birds/m2 ). HSD: High stocking density (22 birds/m2 ). n: Number of replicates (n = 8).
conformity with those observed in the present study. HSD creates hindrance, not letting the broilers to move freely, therefore, confining the mobility of birds to a constrained area of the pen. Consequently, the birds subjected to HSD may not have easier access to feeders
Table 6. Incidence and severity of FPD in broilers subjected to normal or high stocking density fed dietary boric acid. Incidence and severity of FPD (%) Day 14 Item NSD 0 mg/kg boric acid 60 mg/kg boric acid HSD 0 mg/kg boric acid 60 mg/kg boric acid Pooled SEM Stocking density NSD HSD Pooled SEM Boric acid 0 mg/kg 60 mg/kg Pooled SEM P-values Stocking density Boric acid Stocking density × boric acid
0 100 100
1
Day 28 2
T
0
0 0
0 0
0 0
94 95
98 94 1.92
1 6 1.82
1 0 0.75
2 6 2.09
100 96 1.36
0 3 1.29
0 1 0.53
99 97 1.36
0 3 1.29 0.060 0.267 0.267
0.050 0.387 0.387
1
Day 42 2
T
2 5
4 0
6 5
40 45 5.40
30 29 4.21
30 26 6.21
60 55 5.40
0b 4a 1.48
94a 42b 3.82
4b 30a 2.98
2b 28a 4.39
1 0 0.53
1 3 1.48
67 70 3.82
16 17 2.98
0.326 0.326 0.326
0.048 0.535 0.535
< 0.001 0.560 0.689
< 0.001 0.826 0.671
0
2
T
6 6
1 0
7 6
42 45 4.16
45 43 3.37
13 12 2.92
58 55 4.17
6b 58a 3.82
94a 43b 2.94
5b 44a 2.38
1b 13a 2.07
6b 57a 2.95
17 13 4.39
33 30 3.82
67 70 2.94
26 24 2.38
7 6 2.07
33 30 2.95
< 0.001 0.518 0.960
< 0.001 0.560 0.689
< 0.001 0.582 0.777
< 0.001 0.797 0.686
< 0.001 0.657 0.983
< 0.001 0.604 0.755
a, b : Means bearing different superscripts within the same column are statistically significant (P < 0.05). NSD: Normal stocking density (14 birds/m2 ). HSD: High stocking density (22 birds/m2 ). 0: No lesion; 1: Mild lesion (< 7.5 mm); 2: Severe lesion (> 7.5 mm). n: Number of replicates (n = 8).
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93 94
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BORIC ACID AND FOOTPAD DERMATITIS IN BROILERS
with those of Yıldız et al. (2013) and Eliot and Edwards (1992), who reported no effect of dietary boric acid on growth performance of broilers. However, other studies have reported an increase in BW of broilers following the inclusion dietary boron (Kurtoglu et al., 2001; Fassani et al., 2004; C ¸ ınar et al., 2015). In contrast, some studies have reported that dietary boric acid supplementation enhanced the growth performance of broilers (Bai and Hunt, 1996; Bozkurt et al., 2012; K¨ uc¸u ¨kyilmaz et al., 2017). The evaluation of difference in results is difficult. It might be attributed to the differences in research protocol, breed, age, composition of diet, and nutritive value of feeds.
Litter Quality
7
seems that reduced litter NH3 volatilization in broilers fed supplemental dietary boric acid might be attributed to reduced litter pH. In addition, boric acid is a good absorbent of NH3 (Siemer, 1986) that has its application in animal nutrition in Kjeldahl method for crude protein estimation in feeds to trap NH3 . This may further reduce NH3 volatilization from the litter of broiler chickens fed diets supplemented with boric acid.
Serum and Litter Boron Levels Dietary boric acid supplementation increased the serum and litter boron levels in broilers subjected to NSD or HSD on day 42. No literature is available describing the effect of dietary boric acid supplementation on serum and litter boron levels of broilers under NSD or HSD. As expected, dietary boron levels increased in broilers subjected to NSD or HSD compared to those fed unsupplemented diets and subjected to NSD or HSD. Serum and litter boron levels were highest in broiler chickens fed boric acid supplemented diets and subjected to NSD followed by those subjected to HSD. Eren et al. (2012) reported a dose-dependent increase in serum boron levels in broilers following the consumption of diets supplemented with 0, 500, 750, and 1,000 mg/kg boric acid. The exact mechanism by which dietary boric acid increased the serum and litter boron levels is not known. This might be attributed to increased boron metabolism that increased the bioavailability of boron in broiler chickens fed boric acid supplemented diets either under NSD or HSD compared to those fed diets without boric acid. Similarly, the increased litter boron levels might be due to the excretion of indigested and excessive boron, boric acid, or its salts through feces in broilers fed boric acid supplemented diets. At present, it is not clear in what form boron is excreted; however, it is assumed that boron salts might be excreted by broilers into the litter. However, broilers subjected to HSD had comparatively lower serum and litter boron levels in comparison with the corresponding groups. The exact mechanism of reduced litter boron levels in broilers subjected to HSD is not known. Apparently, it seems that lowered feed intake in broilers subjected to HSD might have reduced the bioavailability and excretion of boron. In addition, it may be assumed that HSD poses severe physiological stress in broiler chickens that might have affected the metabolism and excretion of boron resulting in lowered serum and litter boron levels.
It is known that HSD quickly deteriorates the quality of litter by increasing the litter moisture, pH, and NH3 volatilization (Sorensen et al., 2000; Dozier et al., 2006; Ravindran et al., 2006; Estevez, 2007; Petek et al., 2014; Kang et al., 2016). High litter moisture helps the microbial growth in the litter and increased microbial activity in the moist litter is a substantial factor in NH3 volatilization (Al-Homidan et al., 2003) thereby increasing the litter pH. It is also evident from litter pH and NH3 volatilization results of our study. In contrast, a recent study reported that gradual increase in stocking density from 16 to 22 birds/m2 had no effect on litter moisture, pH, and NH3 volatilization (Farhadi et al., 2016). Similarly, Zhang et al. (2011) reported no difference in litter pH of broilers subjected to gradual increase in stocking density from 12 to 20 birds/m2 (12, 16, and 20 birds/m2 ). This is the first report describing the effect of dietary boric acid supplementation on litter quality of broilers subjected to NSD or HSD. As anticipated, the supplementation of boric acid in broiler diets reduced the litter pH and NH3 volatilization at day 42 of experiment. In addition, it had no effect on litter moisture. The exact mechanism by which dietary boric acid supplementation reduced the litter pH is not known yet. Most probably, this might be due to the acidic nature of boric acid or boron excreted by the broilers in groups fed dietary boric acid at 60 mg/kg that reduced the litter pH. The explained notion is evident from the increased litter boron levels of broilers fed diets supplemented with boric acid. Moreover, dietary boric acid reduced NH3 volatilization in broilers subjected to NSD or HSD. The NH3 volatilization usually occurs due to microbial decomposition of nitrogen containing compounds (uric acid and urates) in poultry litter. This NH3 is available Incidence and Severity of FPD either as free NH3 or as ammonium ion (NH4 + ) that deThe present study revealed that HSD increased the pends on litter pH. Alkaline pH (pH > 7) favors the decomposition of uric acid. At a litter pH increasing above incidence and severity of FPD in broilers. Many studies 8.5, ureolytic bacteria proliferate at a substantial rate have reported that HSD plays an important role in and release of uricase enzyme (an enzyme that catalyzes the incidence of FPD in poultry in comparison with the decomposition of uric acid) rises, which is associated comparatively lower stocking densities (McIlroy et al., with greater NH3 liberation. The activity of uricase en1987; Ekstrand et al., 1997; Sorensen et al., 2000; Dozier et al., 2005, 2006; Haslam et al., 2007; Meluzzi zyme reaches to its maximum at a pH of 9. Therefore, it Downloaded from https://academic.oup.com/ps/advance-article-abstract/doi/10.3382/ps/pey337/5074500 by University of the Western Cape user on 18 August 2018
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CENGIZ ET AL.
et al., 2008b). However, there are contrasting views of scientists regarding the effective stocking density for the development of FPD. According to Dawkins et al. (2004), leg health issues arise at or beyond the stocking density of 42 kg/m2 . On the other hand, Buijs et al. (2009) reported that development of FPD started at a stocking density of 56 kg/m2 . In contrast, some other studies have demonstrated little or no association between HSD and FPD development in poultry (Martenchar et al., 2002; Sirri et al., 2007; Meluzzi et al., 2008a). The development of FPD occurs due to sudden onset of deterioration in the quality of litter. Higher stocking densities increase the water intake per bird that results in watery feces thereby increasing the litter moisture (Feddes et al., 2002; Bessei, 2006). Mayne et al. (2007b) reported that litter moisture level <30% is optimal for maintenance of footpad health in broilers. Apparently, it seems to happen in the present study as HSD increased the litter moisture (>30%) along with litter pH and NH3 volatilization. Consequently, the incidence and severity of FPD greatly increased in broilers in response to HSD. No literature is available describing the effect of dietary boric acid supplementation on the incidence and severity of FPD in broilers. Despite the fact that dietary boric acid supplementation reduced litter pH and NH3 volatilization, it had no effect on the incidence and severity of FPD in the present study. This might be due to the absence of any difference in litter moisture of broilers fed diets with or without boric acid supplementation. Mayne et al. (2007b) reported that litter moisture alone is sufficient to initiate the development of FPD to a significant extent provided the litter moisture is more than 30%. In the present study, the litter moisture remained lower than 30% and there was no difference in the litter moisture between the groups fed diets with and without boric acid supplementation which explains the possible ineffectiveness of dietary boric acid against the development of FPD in the present study.
CONCLUSIONS The present study showed that dietary boric acid supplementation in broilers subjected to NSD or HSD had no effect on growth performance, litter quality, and incidence and severity of FPD. However, it increased the serum and litter boron levels in broilers subjected to either stocking densities i.e., NSD or HSD. As expected, HSD resulted in poor growth performance, litter quality, and higher incidence and severity of FPD in broilers. Moreover, dietary boric acid reduced the litter pH and NH3 volatilization to a significant extent along with no interactions between boric acid supplementation and stocking density. Therefore, it appears that higher boric acid inclusion levels may be required to further improve the litter quality in terms of pH and NH3 volatilization. Nonetheless, higher inclusion levels may be practiced with the caution of boron toxicity
that requires further investigation. Alternatively, application of boric acid directly to the litter may be taken into consideration as a litter amendment technique to reduce the incidence and severity of FPD in poultry that is only possible after further research.
ACKNOWLEDGMENTS The project was funded by the Scientific and Tech¨ ITAK) ˙ nological Research Council of Turkey (TUB vide project No. 112O389. The funding agency had no role in the study design, execution, analysis, and preparation of this manuscript. The authors are grateful ¨ ITAK ˙ to TUB for the award of doctoral fellowship to ˙ Umair Ahsan under the BIDEB-2215 program for international students.
REFERENCES Abudabos, A. M., E. M. Samara, E. O. S. Hussein, M. Q. Al-Ghadi, and R. M. Al-Atiyat. 2013. Impacts of stocking density on the performance and welfare of broiler chickens. Ital. J. Anim. Sci. 12:66–71. Al-Homidan, A., J. F. Robertson, and A. M. Petchey. 2003. Review of the effect of ammonia and dust concentrations on broiler performance. World’s Poult. Sci. J. 59:340–349. AOAC International. 2000. Official Methods of Analysis, 17th ed. Assoc. Off. Anal. Chem. International, Gaithersburg, MD, US. Bai, Y., and C. D. Hunt. 1996. Dietary boron enhances efficacy of cholecalciferol in broiler chicks. J. Trace Elem. Exp. Med. 9:117– 132. Banhazi, T. M., J. Seedorf, M. Laffrique, and D. L. Rutley. 2008. Identification of the risk factors for high airborne particle concentrations in broiler buildings using statistical modelling. Biosyst. Eng. 101:100–110. Bedford, M. R., and J. Apajalathi. 2001. Microbial interactions in the response to exogenous enzyme utilization. Pages 299–314 in Enzymes in Farm Animal Nutrition. M. R. Bedford, and G. G. Partridge, eds. CABI Publishing, London, UK. Bessei, W. 2006. Welfare of broilers: a review. World’s Poult. Sci. J. 62:455–466. Bilgili, S. F., M. A. Alley, J. B. Hess, and M. Nagaraj. 2006. Influence of age and sex on footpad quality and yield in broiler chickens reared on low and high density diets. J. Appl. Poult. Res. 15:433– 441. Biswas, T., L. Mandal, and S. K. Sarker. 1999. Studies of enzymes supplementation and herbal preparation at different levels of energy on the performance of broilers. J. Interacad. 3:53–58. Bozkurt, M., K. K¨ uc¸u ¨kyilmaz, A. U. C ¸ atlı, and M. C ¸ ınar. 2012. Effects of Boron supplementation to diets deficient in Calcium and Phosphorus on performance with some serum, bone and fecal characteristics of broiler chickens. Asian-Australas. J. Anim. Sci. 25:248–255. Buijs, S., E. Van Poucke, F. A. M. Tuyttens, L. Keeling, and S. Rettenbacher. 2009. Stocking density effects on broiler welfare: Identifying sensitive ranges for different indicators. Poult. Sci. 88:1536–1543. ¨ J. B. Hess, and S. F. Bilgili. 2011. Effect of bedding type Cengiz, O., and transient wetness on footpad dermatitis in broiler chickens. J. Appl. Poult. Res. 20:554–560. ¨ J. B. Hess, and S. F. Bilgili. 2012a. Dietary biotin supCengiz, O., plementation does not alleviate the development of footpad dermatitis in broiler chickens. J. Appl. Poult. Res. 21:764–769. ¨ J. B. Hess, and S. F. Bilgili. 2012b. Influence of graded Cengiz, O., levels of dietary sodium on the development of footpad dermatitis in broiler chickens. J. Appl. Poult. Res. 21:770–775. ¨ J. B. Hess, and S. F. Bilgili. 2012c. Feed enzyme supCengiz, O., plementation does not ameliorate foot pad dermatitis in broiler chickens fed on a corn-soyabean diet. Br. Poult. Sci. 53:401–407.
Downloaded from https://academic.oup.com/ps/advance-article-abstract/doi/10.3382/ps/pey337/5074500 by University of the Western Cape user on 18 August 2018
BORIC ACID AND FOOTPAD DERMATITIS IN BROILERS ¨ B. H. K¨ ¨ ¨ Sevim, H. Avcı, Cengiz, O., oksal, A. G. Onol, O. Tatlı, O. and S. F. Bilgili. 2012d. Influence of dietary enzyme supplementation of barley-based diets on growth performance and footpad dermatitis in broiler chickens exposed to early high-moisture litter. J. Appl. Poult. Res. 21:117–125. ¨ J. B. Hess, and S. F. Bilgili. 2013. Effect of protein Cengiz, O., source on the development of footpad dermatitis in broiler chickens reared on different flooring types. Arch. fur Geflugelk. 77:166– 170. ¨ B. H. K¨ ¨ ¨ Sevim, and S. F. Bilgili. Cengiz, O., oksal, A. G. Onol, O. 2014. Dietary zinc supplementation does not ameliorate footpad dermatitis in broilers reared under heat stress. Proc. XXVI Intl. Poult. Symp. pp. 25. ( Abstr.) ¨ B. H. K¨ ¨ Sevim, U. Ahsan, A. G. Uner, ¨ Cengiz, O., oksal, O. Tatlı, O. P. A. Uluta¸s, D. Beyaz, S. B¨ uy¨ uky¨ or¨ uk, A. Yakan, and A. G. ¨ Onol. 2015. Effect of dietary probiotic and high stocking density on the performance, carcass yield, gut microflora, and stress indicators of broilers. Poult. Sci. 94:2395–2403. Chavez, E., and F. H. Kratzer. 1972. Prevention of foot pad dermatitis in poults with methionine. Poult. Sci. 51:1545–1548. C ¸ inar, M., K. K¨ uc¸u ¨kyilmaz, M. Bozkurt, A. U. C ¸ atli, E. Binta¸s, H. Ak¸sit, R. Konak, C ¸ . Yamaner, and K. Seyrek. 2015. Effects of dietary boron and phytase supplementation on growth performance and mineral profile of broiler chickens fed on diets adequate or deficient in calcium and phosphorus. Br. Poult. Sci. 56:576–589. Dawkins, M. S., C. A. Donnelly, and T. A. Jones. 2004. Chicken welfare is influenced more by housing conditions than by stocking density. Nature 427:342–344. Dozier, W. A., J. P. Thaxton, S. L. Branton, G. W. Morgan, D. M. Miles, W. B. Roush, B. D. Lott, and Y. Vizzier-Thaxton. 2005. Stocking density effects on growth performance and processing yields of heavy broilers. Poult. Sci. 84:1332–1338. Dozier, W. A., III, J. P. Thaxton, J. L. Purswell, H. A. Olanrewaju, S. L. Branton, and W. B. Roush. 2006. Stocking density effects on male broilers grown to 1.8 kilograms of body weight. Poult. Sci. 85:344–351. Ekstrand, C., B. Algers, and J. Svedberg. 1997. Rearing conditions and foot-pad dermatitis in Swedish broiler chickens. Prev. Vet. Med. 31:167–174. Eliot, M. A., and H. M. Edwards. 1992. Studies to determine whether an interaction exists among boron, calcium, and cholecalciferol on the skeletal development of broiler chickens. Poult. Sci. 71:677– 690. Eren, M., F. Uyanik, B. K. Guclu, and A. Atasever. 2012. The influence of dietary Boron supplementation on performance, some biochemical parameters and organs in broilers. Asian J. Anim. Vet. Adv. 7:1079–1089. Estevez, I. 2007. Density allowances for broilers: where to set the limits? Poult. Sci. 86:1265–1272. Farhadi, D., S. M. Hosseini, and B. T. Dezfuli. 2016. Effect of house type on growth performance, litter quality and incidence of foot lesions in broiler chickens reared in varying stocking density. J. Biosci. Biotech. 1:69–78. Fassani, E. J., A. G. Bertechini, J. A. G. Brito, R. K. Kato, E. T. Fialho, and A. Geraldo. 2004. Boron supplementation in broiler diets. Rev. Bras. Cienc. Avic. 6:213–217. Feddes, J. J. R., E. J. Emmanuel, and M. J. Zuidhof. 2002. Broiler performance, body weight variance, feed and water intake, and carcass quality at different stocking densities. Poult. Sci. 81:774– 779. Guardia, S., B. Konsak, S. Combes, F. Levenez, L. Cauquil, J. F. Guillot, C. Moreau-Vauzelle, M. Lessire, H. Juin, and I. Gabriel. 2011. Effects of stocking density on the growth performance and digestive microbiota of broiler chickens. Poult. Sci. 90:1878–1889. Haslam, S. M., T. G. Knowles, S. N. Brown, L. J. Wilkins, S. C. Kestin, P. D. Warriss, and C. J. Nicol. 2007. Factors affecting the prevalence of foot pad dermatitis, hock burn and breast burn in broiler chicken. Br. Poult. Sci. 48:264–275. J´ ozefiak, D., A. Rutkowski, M. Fratczak, and D. Boros. 2004. The effect of dietary fibre fractions from different cereals and microbial enzymes supplementation on performance, ileal viscosity and short-chain fatty acids concentration in caeca of broiler chickens. J. Anim. Feed Sci. 13:487–496.
9
Kang, H. K., S. B. Park, S. H. Kim, and C. H. Kim. 2016. Effects of stock density on the laying performance, blood parameter, corticosterone, litter quality, gas emission and bone mineral density of laying hens in floor pens. Poult. Sci. 95:2764–2770. K¨ uc¸u ¨kyilmaz, K., M. Bozkurt, M. C ¸ ınar, and A. E. T¨ uz¨ un. 2017. Evaluation of the Boron and phytase, alone or in combination, in broiler diets. Jpn. Poult. Sci. 54:26–33. Kurtoglu, V., F. Kurtoglu, and B. Co¸skun. 2001. Effects of boron supplementation of adequate and inadequate vitamin D3containing diet on performance and serum biochemical characters of broiler chickens. Res. Vet. Sci. 71:183–187. L´azaro, R., M. Garcia, P. Medel, and G. G. Mateos. 2003. Influence of enzymes on performance and digestive parameters of broilers fed rye-based diets. Poult. Sci. 82:132–140. Martenchar, A., E. Boilletot, D. Huonnic, and F. Pol. 2002. Risk factors for foot-pad dermatitis in chicken and turkey broilers in France. Prev. Vet. Med. 52:213–226. Mayne, R. K., R. W. Else, and P. M. Hocking. 2007a. High dietary concentrations of biotin did not prevent foot pad dermatitis in growing turkeys and external scores were poor indicators of histopathological lesions. Br. Poult. Sci. 48:291–298. Mayne, R. K., R. W. Else, and P. M. Hocking. 2007b. High litter moisture alone is sufficient to cause footpad dermatitis in growing turkeys. Br. Poult. Sci. 48:538–545. McIlroy, S. G., E. A. Goodall, and C. H. McMurray. 1987. A contact dermatitis of broilers – epidemiological findings. Avian Pathol. 16:93–105. Meluzzi, A., C. Fabbri, E. Folegatti, and F. Sirri. 2008a. Survey of chicken rearing conditions in Italy: effects of litter quality and stocking density on productivity, foot dermatitis and carcase injuries. Br. Poult. Sci. 49.257–264. Meluzzi, A., F. Sirri, E. Folegatti, and C. Fabbri. 2008b. Effect of less intensive rearing conditions on litter characteristics, growth performance, carcase injuries and meat quality of broilers. Br. Poult. Sci. 49:509–515. National Research Council. 1994. Nutrient Requirements of Poultry. 9th rev. ed. Natl. Acad. Press, Washington, DC. ¨ uner, and D. Ye¸silba˘ Petek, M., H. Ust¨ g. 2014. Effects of stocking density and litter type on litter quality and growth performance of broiler chicken. Kafkas Univ. Vet. Fak. Derg. 20:743–748. Puron, D., R. Santamaria, J. C. Segaura, and J. L. Alamilla. 1995. Broiler performance at different stocking densities. J. Appl. Poult. Res. 4:55–60. Ravindran, V., D. V. Thomas, D. G. Thomas, and P. C. H. Morel. 2006. Performance and welfare of broilers as affected by stocking density and zinc bacitracin supplementation. Anim. Sci. J. 77:110–116. Sauter, E. A., C. F. Petersen, E. E. Steele, J. F. Parkinson, J. E. Dixon, and R. C. Stroh. 1981. The airborne microflora of poultry houses. Poult. Sci. 60:569–574. Shepherd, E. M., and B. D. Fairchild. 2010. Footpad dermatitis in poultry. Poult. Sci. 89:2043–2051. Siemer, D. D. 1986. Use of solid boric acid as an ammonia absorbent in the determination of nitrogen. Analyst 111:1013–1015. Sirri, F., G. Minelli, E. Folegatti, S. Lolli, and A. Meluzzi. 2007. Foot dermatitis and productive traits in broiler chickens kept with different stocking densities, litter types and light regimen. Ital. J. Anim. Sci. 6:734–736. Sorensen, P., G. Su, and S. C. Kestin. 2000. Effects of age and stocking density on leg weakness in broiler chickens. Poult. Sci. 79:864– 870. Van Soest, P. J., J. B. Robertson, and B. A. Lewis. 1991. Methods for dietary fiber, neutral detergent fiber, and nonstarch polysaccharides in relation to animal nutrition. J. Dairy Sci. 74:3583– 3597. Yildiz, G., B. H. Koksal, and O. Sizmaz. 2013. Effects of dietary boric acid addition on growth performance, cholesterolemia, some carcass and tibia characteristics in different rearing periods in broiler chickens. Rev. M´ed. V´et. 164:219–224. Zhang, H. F., H. C. Jiao, Z. G. Song, and G. Lin. 2011. Effect of alum-amended litter and stocking density on ammonia release and footpad and hock dermatitis of broilers. Agric. Sci. China 10:777–785.
Downloaded from https://academic.oup.com/ps/advance-article-abstract/doi/10.3382/ps/pey337/5074500 by University of the Western Cape user on 18 August 2018