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The effect of benzoic acid concentration on nitrogen metabolism, manure ammonia and odour emissions in finishing pigs
Animal Feed Science and Technology 163 (2011) 194–199
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Animal Feed Science and Technology journal homepage: www.elsevier.com/locate/anifeedsci
The effect of benzoic acid concentration on nitrogen metabolism, manure ammonia and odour emissions in finishing pigs D.P. Murphy, J.V. O’Doherty, T.M. Boland, C.J. O’Shea, J.J. Callan, K.M. Pierce, M.B. Lynch ∗ School of Agriculture, Food Science and Veterinary Medicine, University College Dublin, Lyons Research Farm, Newcastle, Co. Dublin, Ireland
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Article history: Received 26 January 2010 Received in revised form 18 October 2010 Accepted 19 October 2010
Keywords: Ammonia Benzoic acid Odour Pigs
a b s t r a c t A complete randomised block design experiment was conducted to investigate the effects of benzoic acid inclusion level on nitrogen (N) metabolism, and manure ammonia (NH3 ) and odour emissions in finishing pigs. Sixteen boars (64 kg live weight ± 1.5 kg) were assigned to one of four dietary treatments (T) varying in benzoic acid concentration: (T1) 0 g benzoic acid/kg (as fed); (T2) 10 g benzoic acid/kg; (T3) 20 g benzoic acid/kg; (T4) 30 g benzoic acid/kg. Animals were housed in individual metabolism crates and feed was provided ad libitum. All diets were formulated to have similar concentration of digestible energy and ileal digestible lysine with benzoic acid replacing wheat in the diet. There was a linear decrease in NH3 emission (P<0.001), as the dietary benzoic acid concentration increased (141.4 mg/g versus 40.5 mg/g N intake (S.E.M. 12.1) over the 240-h storage period). However, there was no effect (P>0.05) of benzoic acid on odour concentration. Urinary nitrogen (N) excretion, total N excretion and the urinary:faecal N ratio were linearly reduced (P<0.05) with increasing benzoic acid inclusion. Furthermore, N retention increased linearly (P<0.05) as benzoic acid concentration increased from 0 g/kg to 30 g/kg in the diet. In conclusion, the inclusion of benzoic acid in the diet of finishing pigs has the potential to reduce total and urinary N excretion and the urinary to faecal N ratio. This was mirrored by reductions in manure NH3 emissions in the benzoic acid supplemented treatments. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Emissions from animal waste constitute the largest single source of total annual national ammonia (NH3 ) emissions in Ireland, with emissions from the pig industry estimated to be between 8.4 and 11% of overall national emissions (Hyde et al., 2003). Odour nuisance from pig production facilities is also a growing concern due to the increasing complaints of neighbours of these facilities. Reduction in odour nuisance plays an important role for strategies concerning where to permit pig production facilities to be located and determines the maximum size of the facilities (Le et al., 2008). NH3 and odour are derived from chemical compounds which are intermediate or end products of microbial turnover of unutilized nutrients in faeces and slurry. Recent studies have shown that it is possible to mitigate the production of NH3 and odour by altering the conditions in the slurry through dietary intervention (Lynch et al., 2007, 2008; O’Shea et al., 2009). Benzoic acid increases the level of biodiversity of gastrointestinal (GIT) microbiota compared with conventional pig diets (Halas et al., 2010; Kluge et al., 2006), an effect which is mainly credited to a reduced GIT pH (Sauer et al., 2009). Reducing
Abbreviations: N, nitrogen; NH3 , ammonia; T, treatment; TAN, total ammoniacal nitrogen; TKN, total Kjeldahl nitrogen. ∗ Corresponding author. Tel.: +353 1 7167769; fax: +353 1 7161103. E-mail address: [email protected] (M.B. Lynch). 0377-8401/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.anifeedsci.2010.10.009
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Table 1 Composition and analysis of experimental diets (as fed g/kg). Benzoic acid inclusion level 0 g/kg
10 g/kg
20 g/kg
30 g/kg
Composition (g/kg) Wheat Barley Soya bean meal Maize Tallow Limestone Salt Minerals & vitaminsa Lysine HCl
382 250 170 150 18 12.5 5 2.5 2.3
372 250 170 150 18 12.5 5 2.5 2.3
362 250 170 150 18 12.5 5 2.5 2.3
352 250 170 150 18 12.5 5 2.5 2.3
Analysed composition (g/kg) Dry matter Crude protein Gross energy Ash Phosphorus Calcium Neutral-detergent fibre
867 164 160 44 4.1 7.1 124
861 164 161 42 4.2 7.4 128
862 160 163 41 3.5 7.1 142
862 155 162 42 3.3 6.3 132
8.0 4.6 4.9 1.5
7.9 4.7 4.9 1.5
7.8 4.9 4.9 1.4
7.9 5.2 5.1 1.3
Calculated composition (g/kg) Digestible lysineb Methionine & cysteineb Threonineb Tryptophanb
a Premix provided per kg of complete diet: 3 mg retinol, 0.05 mg cholecalciferol, 40 mg alpha-tocopherol, 90 mg copper as copper II sulphate, 100 mg zinc as zinc oxide, 0.3 mg selenium as sodium selenite, 25 mg manganese as manganous oxide and 0.2 mg iodine as calcium iodate on a calcium sulphate/calcium carbonate carrier. b Sauvant et al. (2004).
odour from pig production facilities by altering the (GIT) microbiota through dietary intervention may be of interest, because it could alter odour at the source of production, whereas other odour abatement techniques, for example, bioscrubbers, bio-filters, chemical and biological additives, masking agents and treatment of wastes are end-of-pipeline interventions and generally costly and/or prone to malfunction. In addition, benzoic acid is absorbed in the small intestine, where it is conjugated with glycine to produce hippuric acid, which is subsequently excreted in the urine (Bridges et al., 1970). The presence of hippuric acid in the urine lowers urinary pH and thus the activity of urease which is pH dependent (Buhler et al., 2006). A reduced urinary pH can be effective in reducing emissions as it is one of the most important factors governing NH3 emission from manure, and consequently in reducing environmental pollution from pig production (Buhler et al., 2006). However, there is limited information in the literature concerning the effects of benzoic acid on odour and NH3 emissions from manure storage. Therefore, the hypothesis of this study was that the addition of benzoic acid as a feed additive in pig diets would reduce manure NH3 and odour emissions from finishing pigs. 2. Materials and methods All procedures described in this experiment were conducted under experimental licence from the Irish Department of Health in accordance with the Cruelty to Animals Act (1876) and the European Communities (Amendments of the Cruelty to Animals Act, 1976) Regulations (1994). 2.1. Experimental design and diets The experiment was designed as a complete randomised design comprising of four dietary treatments. All diets were formulated to have similar concentration of digestible energy (DE) (14.0 MJ/kg as fed) and ileal digestible lysine (8.5 g/kg as fed). The amino acid requirements were met relative to lysine according to the ideal protein concept (Close, 1994). The dietary composition and analysis are presented in Table 1. The experimental treatments were as follows: (T1) 0 g benzoic acid/kg; (T2) 10 g benzoic acid/kg; (T3) 20 g benzoic acid/kg; and (T4) 30 g benzoic acid/kg. All diets were fed in meal form. 2.2. Animals and management Twenty-four finishing boars (progeny of meat line boars × (Large White × Landrace sow)) with an initial live weight of 64 kg (standard deviation 1.5 kg) were used in this experiment. The pigs were blocked on the basis of live weight and were
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randomly allocated to one of four dietary treatments. The pigs were allowed a 14-day dietary adaptation period after which time they were weighed and 16 pigs were selected according to a uniform weight and transferred to individual metabolism crates (n = 4). The pigs were given a further 5 days to adapt to the metabolism crates before collections begun. The collection period was subdivided into three parts to facilitate studies on NH3 emission (days 1–2), odour emission (days 3–5) and N balance (days 6–10). The daily feed allowance (DE intake (MJ/day) = 3.44 × (live weight)0.54 (Close, 1994), was divided over two meals 50/50. Water was provided with meals in a 1:1 ratio. Between meals, fresh water was provided ad libitum from a designated container in the meal trough. The metabolism crates were located in a temperature-controlled room, maintained at a constant temperature of 22 ◦ C. 2.3. NH3 emission study Faeces and urine were collected according to the method described by Lynch et al. (2008). NH3 emission from the manure was measured over 240 h from the first container, in a laboratory scale set-up according to the method of Derikx and Aarnink (1993). The concentration of ammonia-nitrogen (NH3 -N) in the impingers was determined by the micro-diffusion technique of Conway (1957). NH3 production (mg) from manure was compared between the different dietary treatments using the quantity volatilised from 0 to 240 h/g of N intake. The pH of the manure and urine was recorded using a Mettler Toledo MP 220 pH meter, which was calibrated with certified pH 4 and pH 7 buffer solutions. 2.4. Air sample collection and odour concentration measurement Manure and air samples were collected according to the method described by Lynch et al. (2008). Odour concentration was determined using an Ecoma T08 dynamic olfactometer (ECOMA, Honigsee, Germany) as described by Hayes et al. (2004). 2.5. N metabolism study During collections from day 6 to 10, urine was collected in a plastic container, via a funnel below the crate, containing 20 ml of sulphuric acid (25%). To avoid N volatilisation, the funnel was sprayed four times daily with a weak solution of sulphuric acid (20 g/kg). The urine volume was recorded daily at 08.00 and a 50-ml sample was collected and frozen for laboratory analysis. Total faeces weight was recorded daily at 08.00 and all faeces were oven dried at 100 ◦ C. A sample of freshly voided faeces was collected twice daily and frozen for N analysis. At the end of the collection period, the faeces samples were pooled, homogenised and a subsample was retained for laboratory analysis. Feed intake was estimated through feed disappearance each day. Feed samples were collected each day, pooled and a subsample was retained for chemical analysis. Digestibility values and N balance data were calculated as follows: • Total tract N digestibility = ((N in feed g/kg × feed intake kg/day) − (N in faeces g/kg × faecal output kg/day)/(N in feed g/kg × feed intake kg/day)). • Total N excretion (g/day) = (faecal N excretion (g/day) + urinary N excretion (g/day)). • N retention (g/day) = (N intake − total N excretion). • N retention as a proportion of intake = (N retention/N intake). 2.6. Laboratory analysis of samples Proximate analysis of diets for dry matter (DM) (method 934.01) and ash (method 942.05) was carried out according to Association of Official Analytical Chemists (AOAC, 1995). The DM of the food and faeces was determined after drying for 24 h at 103 ◦ C. Gross energy of diets was determined using an adiabatic bomb calorimeter (Parr Instruments, IL, USA). Ash was determined after ignition of a weighed sample in a muffle furnace (Nabertherm, Bremen, Germany) at 550 ◦ C for 6 h. The ash was then digested in aqua regia (HCl/HNO3 mixture). This solution was used for P and Ca determination. Ca concentration was determined using a Varian ‘50’ atomic absorption spectrophotometer (Ramakrishna et al., 1968). The concentration of P was determined spectrophotometrically using the method of Cavell (1995). The neutral-detergent fibre (NDF) content was determined using a Fibertec extraction unit (Tecator, Hoganas, Sweden) according to the method of Van Soest et al. (1991). The crude protein content of the feed was determined as N × 6.25 using the LECO FP 528 instrument (Leco Instruments, UK Ltd., Stockport, Cheshire, UK). The N content of fresh faeces was analysed by the macro-Kjeldahl technique using a Buchi distillation apparatus. 2.7. Statistical analysis Linear and quadratic effects of dietary benzoic acid concentration were tested using the general linear model procedure of Statistical Analysis Institute (SAS, 1985). Metabolic live weight (live weight0.75 ) was included as a covariate in the model. The individual pig served as the experimental unit. The probability value that denotes significance is P<0.05. The data was
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Table 2 Effect of dietary benzoic acid inclusion level on manure composition (LSM ± S.E.M.). Treatment
N Benzoic acid (g/kg as fed) Manure volume (kg/day) Ammonia (mg/g N intake DM) 0–96 h 96–240 h 0–240 h Odour OuE /m3 (72 h) TAN (g/kg) 0h 240 h TKN (g/kg) 0h 240 h TAN:TKN 0 h TAN:TKN 240 h Slurry pH 0h 240 h Urine pH
S.E.M.
1
2
3
4
4 0 3.85
4 10 3.69
4 20 3.74
4 30 4.53
60.71 80.64 141.36 22,090
38.81 60.29 99.11 27,620
27.54 56.29 83.83 35,462
2.73 7.38
2.12 6.40
10.70 10.63 0.26 0.70 8.16 8.97 8.52
Significance Linear
Quadratic
0.441
ns
ns
14.30 26.16 40.47 32,627
5.98 8.73 12.06 5,709
***
ns ns ns ns
2.45 6.66
2.69 3.30
0.212 0.923
9.38 9.70 0.23 0.64
9.31 9.54 0.28 0.68
8.50 7.57 0.32 0.44
0.868 0.819 0.035 0.041
7.32 8.46 7.48
6.51 8.09 6.00
6.40 7.40 5.77
0.207 0.335 0.221
*** ***
ns ns *
ns *
ns **
***
ns ns ns ns ns ns ns
** ***
ns
ns, not significant (P > 0.05). * P < 0.05. ** P < 0.01. *** P < 0.001.
checked for normality using Proc Univariate and the data in the tables are presented as least-square means (LSM) ± standard error of the mean. 3. Results 3.1. Manure composition and NH3 and odour emissions There was a linear reduction (P<0.001) in manure NH3 emissions as benzoic acid increased in the diet during 0–96, 96–240 and 0–240 h of storage (Table 2). NH3 losses during storage (0–240 h) in the present study were reduced by 30, 41 and 72% as the dietary benzoic acid concentration increased up to 10, 20 and 30 g/kg respectively. There was a linear decrease in total ammoniacal nitrogen (TAN) (P<0.05) and total Kjeldahl nitrogen (TKN) (P<0.05) concentrations in the manure at 240 h as the level of dietary benzoic acid concentration increased. There was a linear decrease in the TAN:TKN at 240 h as the dietary benzoic acid inclusion level increased (P<0.01). Additionally, there was a linear reduction in slurry pH at 0 h (P<0.001) and 240 h (P<0.01) as the benzoic acid concentration increased in the diet. A linear decrease in urinary pH was also reported as the level of dietary benzoic acid increased (P<0.001). However, there was no effect (P>0.05) of benzoic acid inclusion on odour concentration. 3.2. N metabolism study Increasing the dietary benzoic acid concentration resulted in a linear reduction in urinary N excretion (P<0.05), total N excretion (P<0.05) and the urinary:faecal N ratio (P<0.05) (Table 3). This culminated in a linear increase in N retained/intake as the concentration of benzoic acid in the diet increased (P<0.05). 4. Discussion Due to the environmental impact of NH3 emission, national emission limits have been set within each European country. Ireland has been set a national target of 116 kt of NH3 by the year 2010. Because of the significant contribution made by pig production to national NH3 emissions (Hyde et al., 2003), methods to abate NH3 emission are required by the pig industry. The reduction of urinary pH and subsequent slurry pH through the addition of organic acids to the diet has demonstrated significant potential. The main findings from the current study would suggest that dietary benzoic acid can reduce manure NH3 emissions in storage greater than has been previously reported through dietary intervention (Mroz et al., 2000a,b; Lynch et al., 2008). The substantial reductions in NH3 losses during storage are in agreement with the work of Hansen et al. (2007) who reported the addition of 30 g/kg of benzoic acid to the feed of finisher pigs reduced NH3 emission in the exhaust air by 57%. This reduction in NH3 loss is mainly attributed to the decrease in slurry pH in the benzoic acid dietary treatments.
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Table 3 Effect of dietry benzoic acid inclusion level on the coefficient of total tract apparent digestibility (CTTAD) of N and and N balance (LSM ± S.E.M.). Treatment
N Benzoic acid (g/kg as fed) DM intake (kg/day DM) CTTAD of N Faeces dry matter (g/kg DM) Fresh faeces output (kg/day) N intake (g/day DM) Faecal N (g/day) Urinary N (g/day) Total N excretion (g/day) N retention (g/day) N retained/intake Urinary:faecal N
S.E.M.
1
2
3
4
4 0 2.29 0.824 329.0 1.19 69.4 12.2 25.4 37.6 31.8 0.46 2.08
4 10 2.20 0.820 321.7 1.19 66.9 12.1 21.6 33.7 33.3 0.49 1.83
4 20 2.29 0.837 322.0 1.13 68.2 11.1 23.6 34.7 33.5 0.49 2.14
4 30 2.25 0.827 304.3 1.11 64.8 11.2 19.2 30.4 34.5 0.53 1.78
0.074 0.013 17.45 0.066 2.24 0.95 1.67 1.99 2.19 0.026 0.213
Significance Linear
Quadratic
ns ns ns ns ns ns
ns ns ns ns ns ns ns ns ns ns ns
* *
ns 0.050 0.050
ns, not significant (P > 0.05). * P < 0.05.
Additionally, the 20% reduction in total nitrogen excretion as the dietary benzoic acid inclusion rate increased from 0 g/kg to 30 g/kg indicates that there is less ammoniacal N available for volatilisation in T2–T4 dietary treatments. Ammoniacal N together with other factors such as the dietary electrolyte balance and temperature also have an impact on NH3 emission (Canh et al., 1998). The decrease in both slurry and urine pH in the current study is in agreement with the findings of Buhler et al. (2006), who documented that dietary benzoic acid significantly lowered urinary pH in the grower and finisher periods. Benzoic acid is initially absorbed in the small intestine, it is then metabolized in the liver to form hippuric acid, which is excreted rapidly by the urinary pathway (Bridges et al., 1970). The concentration of hippuric acid in the urine consequently lowers urinary pH and thus the pH of the slurry. NH3 is in equilibrium with ammonium in manure, and the rate of NH3 emission is therefore dependant on this equilibrium. Slurry pH is one of the most significant factors influencing NH3 emission, with NH3 volatilisation increasing with an increasing manure pH (Le et al., 2005). At a solution pH of 9.4, NH3 occurs equally in the form of NH4 + and NH3 . Below a pH of 7, NH3 is almost exclusively present as NH4 + , thereby reducing volatilisation as NH3 gas (Le et al., 2005). In spite of this profound reduction in NH3 emission and slurry pH, there were no significant differences in odour emission in any of the experimental treatments. Previous studies have shown there is little correlation between swine manure NH3 and odour emissions (Leek et al., 2007). Manure odour originates from the decomposition of proteins, whereas NH3 production is not a result of the same degradation kinetics as the major part of NH3 derives from urea hydrolysis (Zhu, 2000). As the dietary benzoic acid concentration increased from 0 g/kg to 30 g/kg, there was a 38% reduction in the TAN:TKN ratio at 240 h. Total ammoniacal nitrogen is considered as the sum of NH4 + and NH3 (Blachier et al., 2006). NH3 present in manure largely arises from the breakdown of urea, with NH3 of faecal origin in the form of bacterial protein only having a minimal involvement (Béline et al., 1998). Urea is formed in the liver as the end product of protein degradation in the pig and is excreted by the kidneys in the form of urine. The urea is then quickly hydrolysed by the enzyme urease present in the faeces and converted into NH3 (Le et al., 2005). Therefore, if the nitrogen excretion pathway was to be altered through reducing urinary nitrogen excretion in favour of faecal nitrogen excretion, as indicated by the significant linear decrease in the urinary:faecal nitrogen ratio in the current study, this would consequently result in a reduced TAN:TKN ratio as the benzoic acid concentration increased in the diet. The increase in N retained/intake as the dietary benzoic acid concentration increased from 0 g/kg to 30 g/kg concurs with the findings of Kluge et al. (2006), who documented that pigs supplemented with benzoic acid at 5 and 10 g/kg had marginally higher nitrogen absorption than control pigs offered no benzoic acid. Kluge et al. (2006) attributed this increase in nitrogen absorption to a reduction in the total aerobic bacteria in the ileum, thus increasing ileal digestibility. Additionally, Sauer et al. (2009) documented that acidification of the diet with benzoic acid may increase absorption and retention, either directly by lowering the pH in the gastric contents or indirectly by reducing the rate of gastric emptying.
5. Conclusion The inclusion of benzoic acid in the diet of finishing pigs has the potential to reduce total and urinary nitrogen excretion and the urinary to faecal nitrogen ratio. This was mirrored in reductions in manure NH3 emissions in the benzoic acid supplemented treatments. However, there was no effect of benzoic acid on odour emissions.
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Animal Feed Science and Technology journal homepage: www.elsevier.com/locate/anifeedsci
The effect of benzoic acid concentration on nitrogen metabolism, manure ammonia and odour emissions in finishing pigs D.P. Murphy, J.V. O’Doherty, T.M. Boland, C.J. O’Shea, J.J. Callan, K.M. Pierce, M.B. Lynch ∗ School of Agriculture, Food Science and Veterinary Medicine, University College Dublin, Lyons Research Farm, Newcastle, Co. Dublin, Ireland
a r t i c l e
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Article history: Received 26 January 2010 Received in revised form 18 October 2010 Accepted 19 October 2010
Keywords: Ammonia Benzoic acid Odour Pigs
a b s t r a c t A complete randomised block design experiment was conducted to investigate the effects of benzoic acid inclusion level on nitrogen (N) metabolism, and manure ammonia (NH3 ) and odour emissions in finishing pigs. Sixteen boars (64 kg live weight ± 1.5 kg) were assigned to one of four dietary treatments (T) varying in benzoic acid concentration: (T1) 0 g benzoic acid/kg (as fed); (T2) 10 g benzoic acid/kg; (T3) 20 g benzoic acid/kg; (T4) 30 g benzoic acid/kg. Animals were housed in individual metabolism crates and feed was provided ad libitum. All diets were formulated to have similar concentration of digestible energy and ileal digestible lysine with benzoic acid replacing wheat in the diet. There was a linear decrease in NH3 emission (P<0.001), as the dietary benzoic acid concentration increased (141.4 mg/g versus 40.5 mg/g N intake (S.E.M. 12.1) over the 240-h storage period). However, there was no effect (P>0.05) of benzoic acid on odour concentration. Urinary nitrogen (N) excretion, total N excretion and the urinary:faecal N ratio were linearly reduced (P<0.05) with increasing benzoic acid inclusion. Furthermore, N retention increased linearly (P<0.05) as benzoic acid concentration increased from 0 g/kg to 30 g/kg in the diet. In conclusion, the inclusion of benzoic acid in the diet of finishing pigs has the potential to reduce total and urinary N excretion and the urinary to faecal N ratio. This was mirrored by reductions in manure NH3 emissions in the benzoic acid supplemented treatments. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Emissions from animal waste constitute the largest single source of total annual national ammonia (NH3 ) emissions in Ireland, with emissions from the pig industry estimated to be between 8.4 and 11% of overall national emissions (Hyde et al., 2003). Odour nuisance from pig production facilities is also a growing concern due to the increasing complaints of neighbours of these facilities. Reduction in odour nuisance plays an important role for strategies concerning where to permit pig production facilities to be located and determines the maximum size of the facilities (Le et al., 2008). NH3 and odour are derived from chemical compounds which are intermediate or end products of microbial turnover of unutilized nutrients in faeces and slurry. Recent studies have shown that it is possible to mitigate the production of NH3 and odour by altering the conditions in the slurry through dietary intervention (Lynch et al., 2007, 2008; O’Shea et al., 2009). Benzoic acid increases the level of biodiversity of gastrointestinal (GIT) microbiota compared with conventional pig diets (Halas et al., 2010; Kluge et al., 2006), an effect which is mainly credited to a reduced GIT pH (Sauer et al., 2009). Reducing
Abbreviations: N, nitrogen; NH3 , ammonia; T, treatment; TAN, total ammoniacal nitrogen; TKN, total Kjeldahl nitrogen. ∗ Corresponding author. Tel.: +353 1 7167769; fax: +353 1 7161103. E-mail address: [email protected] (M.B. Lynch). 0377-8401/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.anifeedsci.2010.10.009
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Table 1 Composition and analysis of experimental diets (as fed g/kg). Benzoic acid inclusion level 0 g/kg
10 g/kg
20 g/kg
30 g/kg
Composition (g/kg) Wheat Barley Soya bean meal Maize Tallow Limestone Salt Minerals & vitaminsa Lysine HCl
382 250 170 150 18 12.5 5 2.5 2.3
372 250 170 150 18 12.5 5 2.5 2.3
362 250 170 150 18 12.5 5 2.5 2.3
352 250 170 150 18 12.5 5 2.5 2.3
Analysed composition (g/kg) Dry matter Crude protein Gross energy Ash Phosphorus Calcium Neutral-detergent fibre
867 164 160 44 4.1 7.1 124
861 164 161 42 4.2 7.4 128
862 160 163 41 3.5 7.1 142
862 155 162 42 3.3 6.3 132
8.0 4.6 4.9 1.5
7.9 4.7 4.9 1.5
7.8 4.9 4.9 1.4
7.9 5.2 5.1 1.3
Calculated composition (g/kg) Digestible lysineb Methionine & cysteineb Threonineb Tryptophanb
a Premix provided per kg of complete diet: 3 mg retinol, 0.05 mg cholecalciferol, 40 mg alpha-tocopherol, 90 mg copper as copper II sulphate, 100 mg zinc as zinc oxide, 0.3 mg selenium as sodium selenite, 25 mg manganese as manganous oxide and 0.2 mg iodine as calcium iodate on a calcium sulphate/calcium carbonate carrier. b Sauvant et al. (2004).
odour from pig production facilities by altering the (GIT) microbiota through dietary intervention may be of interest, because it could alter odour at the source of production, whereas other odour abatement techniques, for example, bioscrubbers, bio-filters, chemical and biological additives, masking agents and treatment of wastes are end-of-pipeline interventions and generally costly and/or prone to malfunction. In addition, benzoic acid is absorbed in the small intestine, where it is conjugated with glycine to produce hippuric acid, which is subsequently excreted in the urine (Bridges et al., 1970). The presence of hippuric acid in the urine lowers urinary pH and thus the activity of urease which is pH dependent (Buhler et al., 2006). A reduced urinary pH can be effective in reducing emissions as it is one of the most important factors governing NH3 emission from manure, and consequently in reducing environmental pollution from pig production (Buhler et al., 2006). However, there is limited information in the literature concerning the effects of benzoic acid on odour and NH3 emissions from manure storage. Therefore, the hypothesis of this study was that the addition of benzoic acid as a feed additive in pig diets would reduce manure NH3 and odour emissions from finishing pigs. 2. Materials and methods All procedures described in this experiment were conducted under experimental licence from the Irish Department of Health in accordance with the Cruelty to Animals Act (1876) and the European Communities (Amendments of the Cruelty to Animals Act, 1976) Regulations (1994). 2.1. Experimental design and diets The experiment was designed as a complete randomised design comprising of four dietary treatments. All diets were formulated to have similar concentration of digestible energy (DE) (14.0 MJ/kg as fed) and ileal digestible lysine (8.5 g/kg as fed). The amino acid requirements were met relative to lysine according to the ideal protein concept (Close, 1994). The dietary composition and analysis are presented in Table 1. The experimental treatments were as follows: (T1) 0 g benzoic acid/kg; (T2) 10 g benzoic acid/kg; (T3) 20 g benzoic acid/kg; and (T4) 30 g benzoic acid/kg. All diets were fed in meal form. 2.2. Animals and management Twenty-four finishing boars (progeny of meat line boars × (Large White × Landrace sow)) with an initial live weight of 64 kg (standard deviation 1.5 kg) were used in this experiment. The pigs were blocked on the basis of live weight and were
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randomly allocated to one of four dietary treatments. The pigs were allowed a 14-day dietary adaptation period after which time they were weighed and 16 pigs were selected according to a uniform weight and transferred to individual metabolism crates (n = 4). The pigs were given a further 5 days to adapt to the metabolism crates before collections begun. The collection period was subdivided into three parts to facilitate studies on NH3 emission (days 1–2), odour emission (days 3–5) and N balance (days 6–10). The daily feed allowance (DE intake (MJ/day) = 3.44 × (live weight)0.54 (Close, 1994), was divided over two meals 50/50. Water was provided with meals in a 1:1 ratio. Between meals, fresh water was provided ad libitum from a designated container in the meal trough. The metabolism crates were located in a temperature-controlled room, maintained at a constant temperature of 22 ◦ C. 2.3. NH3 emission study Faeces and urine were collected according to the method described by Lynch et al. (2008). NH3 emission from the manure was measured over 240 h from the first container, in a laboratory scale set-up according to the method of Derikx and Aarnink (1993). The concentration of ammonia-nitrogen (NH3 -N) in the impingers was determined by the micro-diffusion technique of Conway (1957). NH3 production (mg) from manure was compared between the different dietary treatments using the quantity volatilised from 0 to 240 h/g of N intake. The pH of the manure and urine was recorded using a Mettler Toledo MP 220 pH meter, which was calibrated with certified pH 4 and pH 7 buffer solutions. 2.4. Air sample collection and odour concentration measurement Manure and air samples were collected according to the method described by Lynch et al. (2008). Odour concentration was determined using an Ecoma T08 dynamic olfactometer (ECOMA, Honigsee, Germany) as described by Hayes et al. (2004). 2.5. N metabolism study During collections from day 6 to 10, urine was collected in a plastic container, via a funnel below the crate, containing 20 ml of sulphuric acid (25%). To avoid N volatilisation, the funnel was sprayed four times daily with a weak solution of sulphuric acid (20 g/kg). The urine volume was recorded daily at 08.00 and a 50-ml sample was collected and frozen for laboratory analysis. Total faeces weight was recorded daily at 08.00 and all faeces were oven dried at 100 ◦ C. A sample of freshly voided faeces was collected twice daily and frozen for N analysis. At the end of the collection period, the faeces samples were pooled, homogenised and a subsample was retained for laboratory analysis. Feed intake was estimated through feed disappearance each day. Feed samples were collected each day, pooled and a subsample was retained for chemical analysis. Digestibility values and N balance data were calculated as follows: • Total tract N digestibility = ((N in feed g/kg × feed intake kg/day) − (N in faeces g/kg × faecal output kg/day)/(N in feed g/kg × feed intake kg/day)). • Total N excretion (g/day) = (faecal N excretion (g/day) + urinary N excretion (g/day)). • N retention (g/day) = (N intake − total N excretion). • N retention as a proportion of intake = (N retention/N intake). 2.6. Laboratory analysis of samples Proximate analysis of diets for dry matter (DM) (method 934.01) and ash (method 942.05) was carried out according to Association of Official Analytical Chemists (AOAC, 1995). The DM of the food and faeces was determined after drying for 24 h at 103 ◦ C. Gross energy of diets was determined using an adiabatic bomb calorimeter (Parr Instruments, IL, USA). Ash was determined after ignition of a weighed sample in a muffle furnace (Nabertherm, Bremen, Germany) at 550 ◦ C for 6 h. The ash was then digested in aqua regia (HCl/HNO3 mixture). This solution was used for P and Ca determination. Ca concentration was determined using a Varian ‘50’ atomic absorption spectrophotometer (Ramakrishna et al., 1968). The concentration of P was determined spectrophotometrically using the method of Cavell (1995). The neutral-detergent fibre (NDF) content was determined using a Fibertec extraction unit (Tecator, Hoganas, Sweden) according to the method of Van Soest et al. (1991). The crude protein content of the feed was determined as N × 6.25 using the LECO FP 528 instrument (Leco Instruments, UK Ltd., Stockport, Cheshire, UK). The N content of fresh faeces was analysed by the macro-Kjeldahl technique using a Buchi distillation apparatus. 2.7. Statistical analysis Linear and quadratic effects of dietary benzoic acid concentration were tested using the general linear model procedure of Statistical Analysis Institute (SAS, 1985). Metabolic live weight (live weight0.75 ) was included as a covariate in the model. The individual pig served as the experimental unit. The probability value that denotes significance is P<0.05. The data was
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Table 2 Effect of dietary benzoic acid inclusion level on manure composition (LSM ± S.E.M.). Treatment
N Benzoic acid (g/kg as fed) Manure volume (kg/day) Ammonia (mg/g N intake DM) 0–96 h 96–240 h 0–240 h Odour OuE /m3 (72 h) TAN (g/kg) 0h 240 h TKN (g/kg) 0h 240 h TAN:TKN 0 h TAN:TKN 240 h Slurry pH 0h 240 h Urine pH
S.E.M.
1
2
3
4
4 0 3.85
4 10 3.69
4 20 3.74
4 30 4.53
60.71 80.64 141.36 22,090
38.81 60.29 99.11 27,620
27.54 56.29 83.83 35,462
2.73 7.38
2.12 6.40
10.70 10.63 0.26 0.70 8.16 8.97 8.52
Significance Linear
Quadratic
0.441
ns
ns
14.30 26.16 40.47 32,627
5.98 8.73 12.06 5,709
***
ns ns ns ns
2.45 6.66
2.69 3.30
0.212 0.923
9.38 9.70 0.23 0.64
9.31 9.54 0.28 0.68
8.50 7.57 0.32 0.44
0.868 0.819 0.035 0.041
7.32 8.46 7.48
6.51 8.09 6.00
6.40 7.40 5.77
0.207 0.335 0.221
*** ***
ns ns *
ns *
ns **
***
ns ns ns ns ns ns ns
** ***
ns
ns, not significant (P > 0.05). * P < 0.05. ** P < 0.01. *** P < 0.001.
checked for normality using Proc Univariate and the data in the tables are presented as least-square means (LSM) ± standard error of the mean. 3. Results 3.1. Manure composition and NH3 and odour emissions There was a linear reduction (P<0.001) in manure NH3 emissions as benzoic acid increased in the diet during 0–96, 96–240 and 0–240 h of storage (Table 2). NH3 losses during storage (0–240 h) in the present study were reduced by 30, 41 and 72% as the dietary benzoic acid concentration increased up to 10, 20 and 30 g/kg respectively. There was a linear decrease in total ammoniacal nitrogen (TAN) (P<0.05) and total Kjeldahl nitrogen (TKN) (P<0.05) concentrations in the manure at 240 h as the level of dietary benzoic acid concentration increased. There was a linear decrease in the TAN:TKN at 240 h as the dietary benzoic acid inclusion level increased (P<0.01). Additionally, there was a linear reduction in slurry pH at 0 h (P<0.001) and 240 h (P<0.01) as the benzoic acid concentration increased in the diet. A linear decrease in urinary pH was also reported as the level of dietary benzoic acid increased (P<0.001). However, there was no effect (P>0.05) of benzoic acid inclusion on odour concentration. 3.2. N metabolism study Increasing the dietary benzoic acid concentration resulted in a linear reduction in urinary N excretion (P<0.05), total N excretion (P<0.05) and the urinary:faecal N ratio (P<0.05) (Table 3). This culminated in a linear increase in N retained/intake as the concentration of benzoic acid in the diet increased (P<0.05). 4. Discussion Due to the environmental impact of NH3 emission, national emission limits have been set within each European country. Ireland has been set a national target of 116 kt of NH3 by the year 2010. Because of the significant contribution made by pig production to national NH3 emissions (Hyde et al., 2003), methods to abate NH3 emission are required by the pig industry. The reduction of urinary pH and subsequent slurry pH through the addition of organic acids to the diet has demonstrated significant potential. The main findings from the current study would suggest that dietary benzoic acid can reduce manure NH3 emissions in storage greater than has been previously reported through dietary intervention (Mroz et al., 2000a,b; Lynch et al., 2008). The substantial reductions in NH3 losses during storage are in agreement with the work of Hansen et al. (2007) who reported the addition of 30 g/kg of benzoic acid to the feed of finisher pigs reduced NH3 emission in the exhaust air by 57%. This reduction in NH3 loss is mainly attributed to the decrease in slurry pH in the benzoic acid dietary treatments.
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Table 3 Effect of dietry benzoic acid inclusion level on the coefficient of total tract apparent digestibility (CTTAD) of N and and N balance (LSM ± S.E.M.). Treatment
N Benzoic acid (g/kg as fed) DM intake (kg/day DM) CTTAD of N Faeces dry matter (g/kg DM) Fresh faeces output (kg/day) N intake (g/day DM) Faecal N (g/day) Urinary N (g/day) Total N excretion (g/day) N retention (g/day) N retained/intake Urinary:faecal N
S.E.M.
1
2
3
4
4 0 2.29 0.824 329.0 1.19 69.4 12.2 25.4 37.6 31.8 0.46 2.08
4 10 2.20 0.820 321.7 1.19 66.9 12.1 21.6 33.7 33.3 0.49 1.83
4 20 2.29 0.837 322.0 1.13 68.2 11.1 23.6 34.7 33.5 0.49 2.14
4 30 2.25 0.827 304.3 1.11 64.8 11.2 19.2 30.4 34.5 0.53 1.78
0.074 0.013 17.45 0.066 2.24 0.95 1.67 1.99 2.19 0.026 0.213
Significance Linear
Quadratic
ns ns ns ns ns ns
ns ns ns ns ns ns ns ns ns ns ns
* *
ns 0.050 0.050
ns, not significant (P > 0.05). * P < 0.05.
Additionally, the 20% reduction in total nitrogen excretion as the dietary benzoic acid inclusion rate increased from 0 g/kg to 30 g/kg indicates that there is less ammoniacal N available for volatilisation in T2–T4 dietary treatments. Ammoniacal N together with other factors such as the dietary electrolyte balance and temperature also have an impact on NH3 emission (Canh et al., 1998). The decrease in both slurry and urine pH in the current study is in agreement with the findings of Buhler et al. (2006), who documented that dietary benzoic acid significantly lowered urinary pH in the grower and finisher periods. Benzoic acid is initially absorbed in the small intestine, it is then metabolized in the liver to form hippuric acid, which is excreted rapidly by the urinary pathway (Bridges et al., 1970). The concentration of hippuric acid in the urine consequently lowers urinary pH and thus the pH of the slurry. NH3 is in equilibrium with ammonium in manure, and the rate of NH3 emission is therefore dependant on this equilibrium. Slurry pH is one of the most significant factors influencing NH3 emission, with NH3 volatilisation increasing with an increasing manure pH (Le et al., 2005). At a solution pH of 9.4, NH3 occurs equally in the form of NH4 + and NH3 . Below a pH of 7, NH3 is almost exclusively present as NH4 + , thereby reducing volatilisation as NH3 gas (Le et al., 2005). In spite of this profound reduction in NH3 emission and slurry pH, there were no significant differences in odour emission in any of the experimental treatments. Previous studies have shown there is little correlation between swine manure NH3 and odour emissions (Leek et al., 2007). Manure odour originates from the decomposition of proteins, whereas NH3 production is not a result of the same degradation kinetics as the major part of NH3 derives from urea hydrolysis (Zhu, 2000). As the dietary benzoic acid concentration increased from 0 g/kg to 30 g/kg, there was a 38% reduction in the TAN:TKN ratio at 240 h. Total ammoniacal nitrogen is considered as the sum of NH4 + and NH3 (Blachier et al., 2006). NH3 present in manure largely arises from the breakdown of urea, with NH3 of faecal origin in the form of bacterial protein only having a minimal involvement (Béline et al., 1998). Urea is formed in the liver as the end product of protein degradation in the pig and is excreted by the kidneys in the form of urine. The urea is then quickly hydrolysed by the enzyme urease present in the faeces and converted into NH3 (Le et al., 2005). Therefore, if the nitrogen excretion pathway was to be altered through reducing urinary nitrogen excretion in favour of faecal nitrogen excretion, as indicated by the significant linear decrease in the urinary:faecal nitrogen ratio in the current study, this would consequently result in a reduced TAN:TKN ratio as the benzoic acid concentration increased in the diet. The increase in N retained/intake as the dietary benzoic acid concentration increased from 0 g/kg to 30 g/kg concurs with the findings of Kluge et al. (2006), who documented that pigs supplemented with benzoic acid at 5 and 10 g/kg had marginally higher nitrogen absorption than control pigs offered no benzoic acid. Kluge et al. (2006) attributed this increase in nitrogen absorption to a reduction in the total aerobic bacteria in the ileum, thus increasing ileal digestibility. Additionally, Sauer et al. (2009) documented that acidification of the diet with benzoic acid may increase absorption and retention, either directly by lowering the pH in the gastric contents or indirectly by reducing the rate of gastric emptying.
5. Conclusion The inclusion of benzoic acid in the diet of finishing pigs has the potential to reduce total and urinary nitrogen excretion and the urinary to faecal nitrogen ratio. This was mirrored in reductions in manure NH3 emissions in the benzoic acid supplemented treatments. However, there was no effect of benzoic acid on odour emissions.
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