Effects of crystalline amino acid supplementation to the diet on odor from pig manure

M. Ogink and M. W. A. Verstegen P. D. Le, A. J. A. Aarnink, A. W. Jongbloed, C. M. C. van der Peet-Schwering, N. W.

manureEffects of crystalline amino acid supplementation to the diet on odor from pig

published online Dec 4, 2006; J Anim Sci

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Running Head: Amino acids and odor from pig manure1

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Effects of crystalline amino acid supplementation to the diet on odor from pig manure15

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P. D . Le*†, A. J. A. Aarnink*2, A. W. Jongbloed*, C. M. C. van der Peet-Schwering*, N. W.7

M. Ogink* and M. W. A. Verstegen*8

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* Animal Sciences Group, Wageningen-UR, Bornsesteeg 59, 6708 PD Wageningen PO Box 11

17 The Netherlands12

† Department of Animal Sciences, Hue University of Agriculture and Forestry, Vietnam13

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1 Research was supported by the Dutch Ministry of Agriculture, Nature and Food Quality23

2 Corresponding author: [email protected]

Page 1 of 37 Journal of Animal Science

Published Online First on December 4, 2006 as doi:10.2527/jas.2006-644 by on May 14, 2011. jas.fass.orgDownloaded from

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ABSTRACT: The objective of this study was to determine effects of specific crystalline AA 25

supplementation to a diet on odor emission, odor intensity, odor hedonic tone, and ammonia 26

emission from pig manure, and on manure characteristics (pH, ammonium-N, total nitrogen, 27

sulfurous, indolic and phenolic compounds and VFA concentrations). An experiment was 28

conducted with growing pigs (n = 18) in a randomized complete block design with 3 29

treatments in 6 blocks. Treatment groups were 1) 15% CP basal diet with 3 times the 30

requirement of sulfur-containing AA (14.2 g/kg diet, as fed basis); 2) basal diet with 2 times 31

the requirement of Trp, and Phe+Tyr (2.9 and 20.4 g/kg diet, as fed basis, respectively); and 3) 32

basal diet with AA supplementation to levels sufficient for maximum protein gain. Pigs with 33

an initial BW of 41.2 ± 0.8 kg were individually penned in partly slatted floor pens and offered 34

a daily feed allowance of 2.8 x maintenance requirement for NE (293 kJ/BW0.75). Feed was 35

mixed with water, 1/2.5 (w/w). Feces and urine of each pig was allowed to accumulate in 36

separate manure pits under the slatted floor. After an adaptation period of 2 wk, and after 37

cleaning the manure pits, manure was subsequently collected. In the wk 5 of the collection 38

period, separate samples were collected directly from each manure pit for odor, ammonia, and 39

manure composition analysis. Air samples were analyzed for odor concentration, and for 40

hedonic tone and odor intensity above odor detection threshold. Results showed that 41

supplementing crystalline S-containing AA in surplus of the requirement increased odor 42

emission (P < 0.001) and odor intensity (P < 0.05), and reduced odor hedonic tone (P < 0.05) 43

from air above the manure pits. Supplementing crystalline Trp, Tyr, and Phe in surplus of 44

recommended requirements did not affect odor emission, odor intensity, or odor hedonic tone. 45

Regardless of dietary treatment, all pigs had similar performance levels. No differences were 46

observed in ammonia emission from manure of pigs fed different levels of AA 47

supplementation (P = 0.20). In order to reduce odor from pig manure, dietary S-containing AA 48

should be minimized to just meet recommended requirements.49

Key words: crystalline amino acids, diet, growing pig, odor50

Page 2 of 37Journal of Animal Science


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INTRODUCTION51

Odor emission from pig production facilities can cause serious nuisance for residents 52

in the surrounding areas of pig operations. Odor is generated by microbial conversion of feed 53

in the intestinal tract of pigs and by microbial conversion of pig excreta under anaerobic 54

conditions in manure storages. There are a great number of odorous compounds identified in 55

air and manure from animal production facilities (O'Neill and Phillips, 1992; Schiffman et al.,56

2001). Odorants can be classified into 4 main groups: (1) sulfurous compounds, (2) indolic 57

and phenolic compounds, (3) VFA, and (4) ammonia and amines. Many of these compounds 58

are intermediate or end products of AA metabolism. Therefore, AA are important dietary 59

nutrients that should be considered to reduce odor emission. 60

In a literature review Le et al. (2005a) found that previous studies hypothesized that 61

sulfurous, indolic and phenolic compounds are most important for odor nuisance in the air and 62

in manure from pig production facilities. Tryptophan, Phe, and Tyr are main substrates for 63

synthesis of indolic and phenolic compounds. The sulfur-containing AA, Met and Cys, are the 64

main substrates for synthesis of sulfurous compounds such as methanethiol and hydrogen 65

sulfide (Mackie et al., 1998). A change in concentration of these AA in the diet may alter the 66

level of odorous compounds produced in the gut of animals and in the manure.67

Because there are few studies on effects of supplemented crystalline AA types in the 68

diet on odor strength and on odor offensiveness of air from pig manure, our objective was to 69

determine whether AA type influences odor emission, odor strength, odor offensiveness, and 70

ammonia emission from pig manure and manure characteristics (pH, ammonium-N, total 71

nitrogen, sulfurous, indolic and phenolic compounds, and VFA concentrations). In addition 72

this study mimiced odor emission from pig manure in practical situations by collecting odor 73

samples directly from the manure pit.74



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MATERIALS AND METHODS75

Animals, Experimental Design, and Diets 76

All procedures involving animal handling were reviewed and approved by 77

Wageningen University and Research Center Committee on Animal Care and Use. A 78

randomized complete block design with 3 treatments in 6 blocks was used to study effects of 79

AA types in the diet on odor concentration, odor emission, odor intensity, odor hedonic value, 80

and ammonia emission from manure of growing pigs, and on manure characteristics. Three 81

groups of pigs were fed different diets: 1) 15% CP basal diet with 3 times the requirement82

(CVB, 2004) of sulfur-containing AA (SAA) (14.2 g/kg diet, as fed basis); 2) basal diet with 2 83

times the requirement of Trp, and Phe+Tyr (TAA) (2.9 and 20.4 g/kg diet, as fed basis, 84

respectively); and 3) basal diet with AA supplementation to levels sufficient for maximum 85

protein gain (NOAA). In all diets, additional AA were supplemented to the diets in crystalline 86

form. Each treatment was replicated 6 times, 1 replicate in each of 6 blocks, of which a block 87

consisted of samples collected on the same day and animals with similar initial BW.88

In total 18 growing barrows (Great Yorkshire x Dutch Landrace) with an initial BW of 89

41.2 ± 0.8 kg were allocated to 6 blocks, with blocks based on initial BW. Pigs were penned 90

individually in galvanized steel pens (2.1 x 0.96 m), consisting of a slatted floor at the back 91

(0.97 x 0.96 m). There was a separate manure pit under the slatted floor of each pen. The size 92

of the manure pit was 1.35 x 0.91 x 0.36 m (length x width x depth). Pigs were housed in a 93

mechanically ventilated and temperature controlled room. Temperature and relative humidity 94

were recorded every 5 min. The average temperature and relative humidity of the room during 95

the experimental period were 21.0 0C ± 0.84 and 50.0 % ± 5.32 (mean ± SD), respectively.96

Diets were formulated to have similar contents of NE, non-starch polysaccharides 97

(NSP), electrolyte balance, minerals, and vitamins (Tables 1 and 2). The basal diet (NOAA) 98

was formulated to contain 15% CP with AA supplementation to meet the requirement for the 99

pig based on ileal AA digestibility (CVB, 2004). To formulate the SAA diet, additional Met 100



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was added so that sulfur-containing SAA in the diet was 3 times the recommended level. To 101

formulate the TAA diet, additional Trp, Phe, and Tyr were added to provide 2 times the 102

requirements of these AA of Trp, Tyr, and Phe (TAA) based on ileal digestibility. Addition of 103

AA replaced tapioca meal. Analyzed AA composition of the diets are presented in Table 2 104

with concentrations of sulfur-containing AA 0.49, 1.42, and 0.51%; Trp, 0.19, 0.20, and 105

0.29%; and Phe + Tyr, 1.13, 1.15 and 2.04%; in NOAA, SAA, and TAA diets, respectively.106

Experimental diets were analyzed for AA composition, ash, DM, CP, minerals, crude 107

fiber, fat, starch, sugar, and gross energy. The AA (except Met, Cys, and Trp) were assayed by 108

ion-exchange column chromatography after hydrolysis for 23 h in HCl (6 mol/L). Cystine and 109

Met were determined as cysteic acid and methionine sulfone after oxidation with performic 110

acid before hydrolysis (Schram et al., 1954). Tryptophan was determined according to Sato et 111

al. (1984). Starch content was determined enzymatically according to the amyloglucosidase/ 112

hexokinase method (NEN 3574). Sugar was assayed according to the non-starch 113

polysaccharides (NSP) procedure (Gelder et al., 1992). Crude fiber was determined 114

gravimetrically after treatment with sulfuric acid and potassium hydroxide (ISO/DIS 6895). 115

For total fat, samples were hydrolyzed with chloric acid, followed by extraction of fat with 116

petroleum ether. The Ca, P, Mg, Na, K, Cu, and Zn contents were determined using the 117

inductively coupled plasma atomic emission spectrometry (ICP-AES). The Cl content was 118

determined by potentiometric titration of water-diluted solid samples with the chloride specific 119

ion electrode (Jenway Chloride Meter, model PCLM3). For sulfate, samples were extracted 120

with chloric acid. Sulfate was separated with ion chromatography using a water sodium 121

hydroxide gradient and an Ionpac AS 11 (Dionex) as column. Detection takes place by 122

suppressed electric conductivity. Identification and quantification occurs using an external 123

standard solution. The DM was determined gravimetrically after 4 h at 103°C (ISO 6496). 124

Content of ash was determined gravimetrically after ashing at 550 °C (ISO 5984). Nitrogen 125

content was determined by the Kjeldahl method (ISO 5983).126



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https://www.researchgate.net/publication/10382026_Chromatographic_determination_of_cystine_as_cysteic_acid_Biochem?el=1_x_8&enrichId=rgreq-3a13c602-8fc1-418e-8287-0d3d4e6c5251&enrichSource=Y292ZXJQYWdlOzY2NTMwOTE7QVM6MTAxOTI1NjEzNzM1OTM2QDE0MDEzMTIzNjgxNDQ=

Pigs were fed 2.8 times the maintenance NE requirement (293 kJ/BW0.75). Water was 127

restricted by mixing feed with water in the ratio of 1/2.5 (w/w). Apart from water with feed, 128

no additional water was given to the pigs with the aim to excrete the same amount of feed and 129

water intake by the pigs giving almost a similar amount of manure. Pigs were fed twice daily130

day at 0800 and 1500. The amount of feed provided was adjusted each day according to an 131

assumed BW gain of 780 g/d. Feed intake was recorded daily. Pigs were weighed at the 132

beginning and at the end of the experimental period just before the morning feed. Daily gain 133

and G:F were derived from the feed intake and the increase in BW during a period of 46 d. 134

After an adaptation period of 2 wk to allow pigs to acclimatize to the experimental diets and 135

pens, manure pits were cleaned. Subsequently, feces and urine accumulated in the manure pit. 136

In wk 5 of the collection period, odor, ammonia, and manure samples were collected for 137

subsequent analysis.138

Collecting Air Samples and Measuring Odor Concentration, Odor Hedonic Tone, and Odor 139

Intensity140

Collection of Air Samples for Odor Measurement. Air samples were used to measure 141

odor concentration, odor hedonic tone, and odor intensity. Air samples were collected directly 142

from air above the manure in the pit. A schematic view of the odor sampling arrangement is 143

shown in Figure 1. A vessel without a bottom was placed in the middle of the manure pit. The 144

bottom of the vessel touched the bottom of the manure pit. The net surface of the vessel was 145

595 cm2; and the diameter was 28 cm. The vessel was divided into 2 compartments by a lid. 146

The net height of the lower compartment was 40 cm and the net height of the upper 147

compartment was 20 cm. Air entering the upper compartment of the vessel from a pressurized 148

cylinder was odor-free air. Air entered the lower compartment of the vessel via 24 holes of 1149

mm diameter each, located at the edge of the lid. Air was exhausted from the vessel by a hole 150

of 5 mm diameter in the middle of the lid. 151



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The outgoing air from the vessel was split into 2 streams. One stream was used to 152

collect the air sample for odor measurement. It was connected to an odor-sampling bag placed 153

in a rigid container. This container was connected to a critical glass capillary, which had a 154

flow rate of 0.5 L min-1, and then to a vacuum pump. The other stream was used to collect 155

ammonia and was connected to 2 connected impingers. The outgoing air from the impinger 156

was connected to a critical glass capillary, which had a flow rate of 0.5 L m-1, and then to the 157

vacuum pump.158

Air samples were collected according to the European standard (CEN standard 13725, 159

2003). The sampling method for delayed olfactometry was applied using the 'lung principle'. A 160

40 L Nalophaan odor sampling bag was placed in a rigid container. The sample bag had been 161

flushed with compressed and odorless air 3 times before it was placed in a rigid container for 162

collection of the odor sample. The sample bag was used once for each air sample as 163

recommended by European standard (CEN standard 13725, 2003). The air was removed from 164

the container by the vacuum pump. The lower pressure in the container caused the bag to fill 165

with a volume of sample air equal to the volume removed from the container (Figure 1).166

One air sample was collected from each manure pit. During transport and storage, air167

samples were kept at a temperature above the dew point of the sample to avoid condensation. 168

This was achieved by warming the rigid container of the odor bag to about 4 0C above the 169

ambient temperature. The interval between sampling and measuring odor concentration did 170

not exceed 30 h, as recommended by European standard (CEN standard 13725, 2003).171

Measurement of Odor Concentration. Odor concentration was measured by 172

olfactometry according to the European standard (CEN standard 13725, 2003) as described in 173

detail by Le et al. (2005b). Odor concentrations of the examined samples were expressed in 174

European odor units per cubic meter air (ouE m-3). One odor unit is defined as the amount of 175

odor-causing gases which, when diluted in 1 m3 of air, can just be distinguished from clean air 176

by 50% of the members of an odor panel. 177



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Odor emission was defined as the number of odor units emitted from a manure surface 178

per second and it was obtained by multiplying the ventilation rate with the corresponding odor 179

concentration (Equation 1).180

Eodor = (Codor * V * 10,000) / (60 * 1,000 * 595) [1]181

where Eodor = odor emission s-1 m-2 (ouE s-1m-2), Codor = odor concentration (ouE m

-3), and V = 182

ventilation rate (L min-1), 10,000 = cm2 m-2, 60 = sec min-1, 1,000 = liters m-3, and 595 = the 183

cm2 surface area of the manure pit.184

Measuring Odor Hedonic Tone and Odor Intensity. Hedonic tone (H) is used to 185

evaluate the odor offensiveness, which is a measure of the unpleasantness or pleasantness of 186

the perceived odor above the odor detection threshold. Odor intensity (I) refers to the 187

magnitude of the odor sensation and is a measure of the intensiveness of the odor above the 188

odor detection threshold. Odor intensity and hedonic tone were measured at the same time by 189

olfactometry and were determined by the same panel members as for odor concentration. The 190

principle of the measurement is to vary the odor concentration and thus to vary hedonic value 191

and intensity. The odor concentration varied randomly in 5 dilution factors above the detection 192

threshold. At each presentation, each panelist was asked to indicate the perceived hedonic 193

value, using a 9-point hedonic scale ranging from - 4, extremely unpleasant or offensive 194

through 0, neither pleasant nor unpleasant or neutral odor to + 4, extremely pleasant. The 195

panelist was also asked to indicate the perceived odor intensity using a 7-point intensity scale 196

ranging from 1, no odor through 2, very faint odor to 7, overwhelming odor. For each odor 197

sample, the hedonic tone and the odor intensity at each odor concentration level above the 198

detection threshold were calculated as the average of the hedonic tone and the odor intensity 199

perceived by all panelists, and plotted against the logarithm of the odor concentration. From 200

the regression lines obtained, the odor concentration at H = -1 (mildly unpleasant), H = -2 201

(moderately unpleasant), I = 1 (no odor), I = 2 (very faint odor), I = 4 (distinct odor) were 202



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derived. Regression lines of the hedonic tone and the odor intensity were also plotted against 203

the logarithm of the odor concentration for all samples in the same treatment.204

Collecting and Measuring Ammonia Emission 205

Samples for determining ammonia emission were collected during the same period 206

and with the same system as samples for odor measurement (Figure 1). One air sample for 207

ammonia measurement was collected from each manure pit. Ammonia in outgoing air was 208

removed by passing through 2 ammonia traps (impingers), each containing about 20 mL 0.5 M 209

HNO3 solution. The system was run for about 90 min. The ammonia concentration and the 210

volume of the liquid were determined in the first and the second impingers. Ammonia 211

emission per time unit and surface unit was calculated as (Equation 2): 212

MNH3 = (CNH3 * V * 10,000) / (T x 60 * 595) 213

where MNH3 = ammonia emission (mg s-1 m-2), CNH3 = ammonia concentration (mg 214

mL-1 HNO3), V = volume of HNO3 solution (mL), 10,000 = cm2 m-2, T = sampling time 215

(min), 60 = s min-1, and 595 = the cm2 surface area of the manure pit.216

Collection and Measurement of Manure Characteristics 217

Manure samples were analyzed to evaluate the effect of the diets on manure 218

characteristics. These include DM, ash, total N (total-N), ammonium-N, pH, VFA (acetic, 219

propionic, butanoic, pentanoic, iso-butanoic, iso-pentanoic, hexanoic, and heptanoic acid), 220

indolic (indole and 3-methyl indole) and phenolic compounds (phenol, 4-ethyl phenol, and 221

cresols), and sulfurous compounds (carbon disulfide, methyl sulfide, methyl disulfide, and 222

ethanethiol). One manure sample was collected from each manure pit. Immediately after 223

collecting odor samples, manure in each manure pit was mixed thoroughly before a sample of 224

about 1 kg was collected. Manure samples were stored at - 20 0C until analysis. 225

Ammonium was determined spectrophotometrically according to NEN 6472 (Derikx et226

al., 1994). Volatile fatty acids were measured using a Packard 427 gas chromatograph, 227



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equipped with a flame ionization detector. Manure pH was measured by a pH electrode. For 228

determination of indolic and phenolic compounds and sulfurous compounds, 2.5 g fresh 229

manure was extracted with 15 ml 50% methanol for 2 h. The sample was centrifuged and the 230

supernatant was analyzed by the HPLC. The HPLC conditions were a water-methanol gradient 231

as elution solution and Alltima C18 (Alltech) as column. Detection was done by UV-232

absorption at 200 nm. For identification and quantification, an external standard solution was 233

used.234

Statistical Analysis 235

The effect of AA types on ADG, ADFI, G : F, odor emission, odor hedonic value, odor 236

intensity, ammonia emission, and manure characteristics were analyzed using ANOVA 237

appropriate for a randomized complete block design with 6 pigs/treatment. Each treatment was 238

replicated 6 times, 1 replicate in each of 6 blocks, of which a block consisted of samples 239

collected on the same day and animals with similar IBW. The individual pig or the manure pit 240

was the experimental unit. 241

A natural logarithm transformation was applied to odor emission, concentrations of 242

VFA, total-N, ammonium-N, indolic and phenolic compounds, and sulfur-containing 243

compounds since they were skewed and non-normally distributed. Statistical analyses were 244

conducted on the natural logarithm scale. Data were presented as either arithmetic or 245

geometric mean. Geometric mean is the antilog of the arithmetic average of logarithms of data 246

on original scale. In other words, geometric mean is the back-transformed value of data on 247

logarithm scale.248

In each treatment, odor hedonic tone and odor intensity was plotted against the natural 249

logarithm of odor concentration, and odor hedonic tone was plotted against odor intensity. The 250

differences between slopes and between intercepts were tested to decide whether there should 251

be separate regression lines for treatments or a common line for all treatments. The 252

relationship between ammonia emission and odor emission was also determined by linear 253



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regression. Analyses were computed using GenStat statistical package 7th version (GenStat 254

VSN International Ltd., 2004).255

RESULTS256

Effects of Amino Acid Supplementation on Animal Performance 257

To detect whether there are any effects of treatments on production parameters, the 258

effects of AA supplementation to the diet on ADG, ADFI, and G:F are summarized in Table 3. 259

Average daily feed intake, ADG, and G:F were similar among treatments (P > 0.30).260

Effects of Amino Acid Supplementation on Odor Strength and Offensiveness 261

Descriptive statistics and ANOVA of diet effects on odor strength, and offensiveness 262

from pig manure are given in Table 4. Geometric odor concentration and odor emission from 263

pig manure were highest from pigs fed the SAA treatment, 111,302 ouE m-3 and 15.48 ouE s-1 264

m-2, respectively. The NOAA treatment had the lowest odor concentration and odor emission, 265

13,224 ouE m-3 and 1.88 ouE s-1 m-2, respectively. Analyses of variance showed that diets 266

affected odor concentration and odor emission from pig manure (P < 0.001). Further analyses 267

showed that pigs fed the the SAA treatment had a greater odor concentration and odor 268

emission than pigs fed the TAA and NOAA treatments (P < 0.05). No differences were 269

observed in odor concentration and odor emission between pigs fed the TAA and NOAA 270

treatments. At the same level of odor hedonic tone or odor intensity, odor concentration from 271

the pigs fed the SAA treatment was lowest (P < 0.05). No differences in odor concentration at 272

different levels of hedonic tone and odor intensity were found between pigs fed the TAA and 273

NOAA treatments.274

Relationships between odor concentration and odor hedonic tone, between odor 275

concentration and intensity, and between intensity and hedonic tone are shown in Figures 2, 3,276

and 4, respectively. In each figure, a difference in intercepts (P < 0.05) of different regression 277

lines was observed. The intercept of the pigs fed the SAA treatment differed from that of pigs 278



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fed the TAA and NOAA treatments (P < 0.05), but it was similar for pigs fed the TAA and 279

NOAA. The regression lines were similar in slopes. There was a strong linear relationship 280

between hedonic tone and the natural logarithm of odor concentration (R2 = 0.66), between 281

odor intensity and the natural logarithm of odor concentration (R2 = 0.71) and between 282

hedonic tone and intensity (R2 = 0.89). Hedonic tone decreased while odor intensity increased 283

as the odor concentration increased. Hedonic tone decreased as odor intensity increased.284

Effects of AA Supplementation on Manure Characteristics and Ammonia Emission 285

Descriptive statistics and ANOVA of the impacts of AA supplementation on manure 286

characteristics and ammonia emission are shown in Table 5. Volatile fatty acids, carbon 287

disulfide, ethanethiol, phenol, and 3-methyl indole concentrations in the manure of pigs fed 288

diets supplemented with different kinds of AA were similar. Manure from pigs fed SAA diet 289

had the greatest total N (P = 0.04). Ammonium-N concentrations were greater in pigs fed 290

SAA and TAA than in NOAA diet (P < 0.01). Ammonia emission and manure pH were 291

similar between manure of pigs fed different diets. Diets affected cresols (P < 0.01), 4-ethyl 292

phenol (P = 0.03), indole (P < 0.01), and methyl sulfide (P = 0.04) concentrations in the 293

manure. Manure from pigs fed the TAA diet had greater cresols and indole concentrations than 294

manure from pigs fed the SAA and NOAA diets. Manure from pigs fed the SAA diet had 295

greater 4-ethyl phenol concentration than manure from pigs fed the TAA diet. Pigs fed the 296

TAA diet produced manure with greater concentrations of methyl sulfide than pigs fed the 297

SAA and NOAA diets. Methyl disulfide, hexanoic, heptanoic, and pentanoic acids were not 298

detected in the manure of the different treatments. The detection limits of methyl disulfide and 299

the 3 VFA are 0.5 mg/kg and 0.1 g/kg, respectively. The correlation between ammonia 300

emission and odor emission was – 0.3 (P > 0.05).301



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DISCUSSION302

Odor nuisance from pig production facilities is a growing concern for the society and 303

consequently; it needs to be reduced. It is preferable that odor abatement solutions are done at 304

source of production. Diet is the first step in odor generation in animal husbandry (Le et al., 305

2005a) and it is generally accepted that dietary manipulation can influence odor from pig 306

manure. Odor is a complex mixture of various compounds, in which sulfurous compounds, 307

indolic, and phenolic compounds are considered the most important related to odor strength 308

and offensiveness (O'Neill and Phillips, 1992; Mackie et al., 1998). Reducing precursors of 309

these compounds in the diet should reduce odor strength and offensiveness. Therefore, the 310

primary objective of this study was to determine manure odor strength and offensiveness from 311

pigs fed diets with different types of AA supplementation. In addition, ammonia emission and 312

manure characteristics were evaluated.313

We hypothesized that the surplus of sulfur-containing AA in the diet would provide 314

precursors for the production of odorous sulfur compounds, such as hydrogen sulfide and 315

methanethiol, both of which can volatilize from the manure and create odor. As expected, 316

manure of pigs fed the SAA diet had a higher odor concentration, and thus odor emission, than 317

pigs fed the NOAA and TAA diets. 318

In addition to the greater odor concentration and emission, there was a consistently 319

lower odor concentration at different levels of odor hedonic tone and odor intensity of manure 320

from pigs fed the SAA diet, implying that manure from pigs fed the SAA diet produces a 321

strong and offensive odor at lower levels of odor concentration than manure from pigs fed the 322

TAA and NOAA treatments. 323

Literature shows that surplus of sulfur-containing AA in the diet provides precursors 324

for many odorous compounds in manure and in the odorous air such as hydrogen sulfide (Ren, 325

1999; Sutton et al., 1999), methanethiol (Inoue et al., 1995; Yoshimura et al., 2000), dimethyl 326

sulfide (Kelly et al., 1994), dimethyl disulfide (Bonnarme et al., 2001), and dimethyl trisulfide 327



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https://www.researchgate.net/publication/13662441_Biochemical_Identification_and_Biological_Origin_of_Key_Odor_Components_in_Livestock_Waste?el=1_x_8&enrichId=rgreq-3a13c602-8fc1-418e-8287-0d3d4e6c5251&enrichSource=Y292ZXJQYWdlOzY2NTMwOTE7QVM6MTAxOTI1NjEzNzM1OTM2QDE0MDEzMTIzNjgxNDQ=

https://www.researchgate.net/publication/14613178_Structural-analysis_of_the_l-methionine_gamma-lyase_gene_from_Pseudomonas_putida_J_Biochem?el=1_x_8&enrichId=rgreq-3a13c602-8fc1-418e-8287-0d3d4e6c5251&enrichSource=Y292ZXJQYWdlOzY2NTMwOTE7QVM6MTAxOTI1NjEzNzM1OTM2QDE0MDEzMTIzNjgxNDQ=

https://www.researchgate.net/publication/15501562_Biological_production_and_consumption_of_gaseous_organic_sulphur_compounds?el=1_x_8&enrichId=rgreq-3a13c602-8fc1-418e-8287-0d3d4e6c5251&enrichSource=Y292ZXJQYWdlOzY2NTMwOTE7QVM6MTAxOTI1NjEzNzM1OTM2QDE0MDEzMTIzNjgxNDQ=

(Chin and Lindsay, 1994). In addition sulfurous compounds have a lower odor detection 328

threshold than other odorous compounds. Furthermore, concentrations of sulfurous 329

compounds in the odorous air can be relatively high. Finally, the nature of the smell of 330

sulfurous compounds is more offensive than other odorous compounds. This explains why 331

manure from pigs fed the SAA diet had greater odor emission, and odor intensity and lower 332

hedonic tone (more unpleasant) than manure from pigs fed the TAA and NOAA diets.333

It is generally accepted that crystalline sulfur-containing AA are absorbed completely 334

by the time digesta reaches the terminal ileum. The excess sulfur-containing AA are absorbed 335

in the small intestine of animals and end up as pyruvate (from Cys), Succinyl CoA (Met), and 336

SO42-. Sulfates are excreted via urine; in the manure sulfates are quickly converted to sulfur 337

odorous compounds, mainly hydrogen sulfide and methanethiol. According to Spoelstra 338

(1980), sulfate-reducing bacteria produce a trace amount of carbon disulfide, methyl sulfide, 339

and ethanethiol.340

Considering manure characteristics, analyzed sulfurous compounds, carbon disulfide, 341

methyl sulfide, and ethanethiol concentrations in the manure of pigs fed the SAA treatment 342

were not greater than in the other 2 treatments. These 3 compounds may not be important in 343

terms of mass concentration compared to other sulfurous compounds. In this experiment, 344

precursors for sulfurous compounds in manure are mainly sulfates from the urine. Carbon 345

disulfide, methyl sulfide, and ethanethiol are primarily produced from metabolism of sulfur-346

containing AA in the intact protein in the large intestine of animals and in manure. This is 347

supported by the finding of Le et al. (unpublished data) who reported that increased dietary CP 348

levels resulted in greater concentrations of these compounds in the manure. According to 349

Banwart and Bremmer (1975) hydrogen sulfide and methanethiol (methyl mercaptan) 350

represented 70 to 90% of the total S volatilized in the manure while Beard and Guenzi (1983)351

stated that most of the S emitted is in the form of hydrogen sulfide (39%) and methanethiol 352

(34%). In addition, according to O'Neill and Phillips (1992), carbon disulfide and methyl 353



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sulfide are not among the compounds having the lowest odor detection threshold, as 354

methanethiol and hydrogen sulfide were the compounds having the lowest odor detection 355

threshold, 0.0003 and 0.1 µg/m3, respectively. These 2 compounds were not analyzed in this 356

experiment, because they have boiling points (6 and – 60.7 0C, respectively) too low to be 357

captured in manure samples for subsequent analysis. Furthermore, it is difficult to analyze 358

these compounds by a normal gas chromatography. 359

From the results of odor strength and offensiveness and the concentrations of sulfurous 360

compounds in the manure of pigs fed different diets, it is difficult to correlate the 361

concentrations of single odorous compounds in the manure and the odor strength and 362

offensiveness of the odorous air emitting from the manure. Therefore, it is necessary to 363

analyze odorous compounds in the air. We feel the focus should be on very volatile sulfurous 364

compounds because this better reflects the relationship between odor sulfurous compounds 365

and odor strength and offensiveness. Techniques to collect and to analyze compounds in the 366

air are, however, still under development.367

Supplementation with a surplus of Trp and Phe + Tyr to a level twice the requirement 368

estimate did not increase odor concentration, emission, and intensity, nor did it reduce hedonic 369

tone from pig manure. These AA are precursors for phenol (Hammond et al., 1989; Sutton et 370

al., 1999), 4-methylphenol (Hengemuhle and Yokoyama, 1990), 4-ethylphenol (Spoelstra, 371

1977; Hengemuhle and Yokoyama, 1990), indole, and 3-methylindole (Honeyfield and 372

Carlson, 1990; Jensen and Jørgensen, 1994). There are 2 possible reasons for the non-effect of 373

crystalline Trp, Phe, and Tyr supplementation to a diet on odor from pig manure. First, the 374

excess of ileal absorbed Trp, Tyr, and Phe are degraded to a carbon chain and nitrogen, where 375

excess show up only as an increased urea excretion in urine. In our experiment, this led to a 376

greater ammonium concentration of this diet compared to the NOAA diet (Table 5). In 377

general, crystalline AA are absorbed 100% before the digesta reach the ileum. If this is the 378

case, excess Trp, Tyr, and Phe absorbed in the small intestine of animals will not cause much 379



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odor nuisance from the manure. Second, although these compounds are thought to be mainly 380

responsible for the smell in the headspace and ventilation air of pig houses (Schaefer, 1977; 381

Williams, 1984; O'Neill and Phillips, 1992), these phenolic and indolic compounds may not be 382

as important in causing odor nuisance as expected. The hypothesis for the importance of these 383

compounds within previous studies was mainly based on their concentration in the air and/or 384

in manure from pig production facilities and their olfactometry detection threshold. Odor is a 385

complex mixture of various compounds. For example, Schiffman et al. (2001) reported 331 386

compounds, in which the relationship between each individual odor compound or a group of 387

odor compounds and the odor strength and offensiveness of the mixture of the odor air is not 388

yet clear. In a review, Le et al. (2005a) found a large variation in the concentration of an 389

odorous compound and its detection threshold. 390

One may consider the logic using 2 and 3 times the requirements of Trp, Phe + Tyr,391

and S-containing AA for the test diets. This research was designed as a model experiment to 392

verify if an oversupply of specific AA causes much more odor nuisance. In addition, it was a 393

fundamental experiment that provided knowledge on odor precursors and their excretion 394

pathways. Results show that an excess of S-containing AA results in much more odor 395

nuisance, while Trp, Phe + Tyr did not. If a large oversupply does not cause more odor 396

nuisance, then a slight oversupply will not cause odor nuisance, as well.397

From the analyzed concentration of Met, Cys, and sulfate in the diets (Table 2), the 398

concentration of S was calculated. They were 1.55, 3.55, and 1.66 g kg-1 diet as-fed basic, 399

respectively for NOAA, SAA, and TAA diets. Although the SAA diet had 3 times the 400

requirement of S-containing AA, the S concentration was not high compared to other diets.401

For example, diets used in the experiment of Kerr et al. (2006) contained approximately 17 g 402

kg-1. 403

Although the main objective of this study was to determine the effect of AA 404

supplementation to the diet on odor strength and offensiveness, ammonia emission was also 405



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considered because it is a serious environmental problem. Odor abatement strategies are only 406

of interest if they do not increase other environmental problems such as ammonia. Ammonia 407

emission from pig manure is mainly influenced by pH and ammonia concentration. These 2408

factors are mainly influenced by dietary protein content and electrolyte balance (Canh et al., 409

1998a, b). The similar ammonia emission from manure of pigs fed the diets supplemented 410

with different types of AA can be explained by the fact that the pigs fed the NOAA, SAA, and 411

TAA diets had similar CP and electrolyte balance concentrations (Table 2). In addition, 412

although total N and ammonium-N concentrations in the manure from pigs fed the SAA 413

treatment were greater than in manure from pigs fed the NOAA and TAA treatments, 414

differences were small and partly compensated by small differences in pH (Table 6).415

This study showed that the correlation between ammonia emission and odor emission 416

was low and negative (-0.3). Inconsistent findings were found in literature and between our 417

finding and others. Schulte et al. (1985) and Miner (1995) found a high correlation between 418

ammonia and odor emission from pig production facilities. On the other hand Williams 419

(1984), Oldenburg (1989), Liu et al. (1993) and Verdoes and Ogink (1997) found only a low 420

correlation between ammonia and odor emission from pig houses. The inconsistencies in the 421

relationship between ammonia and odor emission likely comes from the fact that ammonia and 422

odor samples were collected from different farms and at different times. Farms are different in 423

animal types, housing design, and dietary composition, especially fermentable carbohydrates 424

which may vary greatly among diets. Different times of sample collection and farms might 425

have different environmental factors. These farms and environmental factors play key roles in 426

influencing odor and ammonia emission (Le et al., 2005a, b) and consequently the relationship 427

between them. In our study, these sources of variation were prevented, because we collected 428

odor and ammonia samples from the manure of the different treatments in the same animal 429

house, at the same time, under the same environmental conditions, and with the same air flow 430

rate.431



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https://www.researchgate.net/publication/245041922_A_Review_of_the_Literature_on_the_Nature_and_Control_of_Odours_from_Pork_Production_Facilities?el=1_x_8&enrichId=rgreq-3a13c602-8fc1-418e-8287-0d3d4e6c5251&enrichSource=Y292ZXJQYWdlOzY2NTMwOTE7QVM6MTAxOTI1NjEzNzM1OTM2QDE0MDEzMTIzNjgxNDQ=

IMPLICATIONS432

This study demonstrates that supplementing crystalline sulfur-containing amino acids 433

to the diet above the requirement for the pig increases odor strength and offensiveness from 434

pig manure. Therefore, to reduce odor from pig manure sulfur-containing amino acids should 435

be formulated very near the requirement for the animal . At the same time, supplementing 436

crystalline phenylalanine, tyrosine, and tryptophan to the diet, above requirement does not 437

increase the odor strength and offensiveness from pig manure. Ammonia emission has a low 438

correlation with odor emission, so strategies that have demonstrated to be successful in 439

reducing ammonia emission may not have a similar impact on odor emission. From this study 440

it is clear that sulfurous compounds contribute significantly to odor nuisance.441

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https://www.researchgate.net/publication/222633678_Indicators_of_piggery_slurry_odour_offensiveness?el=1_x_8&enrichId=rgreq-3a13c602-8fc1-418e-8287-0d3d4e6c5251&enrichSource=Y292ZXJQYWdlOzY2NTMwOTE7QVM6MTAxOTI1NjEzNzM1OTM2QDE0MDEzMTIzNjgxNDQ=




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Table 1. Formulation of experimental diets, as-fed basis

Diet1

Composition, (%)NOAA SAA TAA

Wheat 50.00 50.00 50.00

Tapioca meal (starch 62.5 to 65.7%) 13.13 12.12 11.99

Wheat gluten meal 4.00 4.00 4.00

Potato protein 2.53 2.53 2.53

Wheat middings 15.00 15.00 15.00

Palm kernel expeller (crude fiber < 22%) 6.60 6.60 6.60

Cane molasses, < 47.5% sugar 3.00 3.00 3.00

Soybean oil 1.69 1.69 1.69

K2CO3 0.93 0.93 0.93

CaCO3 1.15 1.15 1.15

Monocalcium phosphate·H2O 0.68 0.68 0.68

Salt 0.48 0.48 0.48

Premix 2 0.20 0.20 0.20

L-Lysine HCl 0.45 0.45 0.45

DL-Methionine 0.06 1.07 0.06

L-Threonine 0.09 0.09 0.09

L-Tryptophan 0.01 0.01 0.15

L-Phenylalanine 0.00 0.00 0.60

L-Tyrosine 0.00 0.00 0.40

1 SAA = 3 times requirement of sulfur-containing AA; TAA = 2 times requirement of Trp, and

Tyr + Phe; NOAA = supplementation of these AA up to requirement.

2 The vitamin-mineral premix supplied per kg feed: 7,000 IU vitamin A, 1,700 IU vitamin D3,



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20 IU vitamin E, 1.5 mg vitamin K, 1.5 mg vitamin B1, 4 mg vitamin B2, 11 mg d-pantothenic

acid, 18 mg niacin, 18 µg vitamin B12, 0.1 mg folic acid, 1.0 mg vitamin B6, 100 mg choline

chloride, 75 mg Fe, 10 mg Cu, 65 mg Zn, 30 mg Mn, 0.15 mg Co, 0.75 mg I, 0.30 mg Se

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Table 2. Nutrient composition of experimental diets, as-fed basis

Diet1

Composition UnitNOAA SAA TAA

Calculated composition

CP % 15.00 15.57 15.60

Digestible CP % 12.18 12.77 12.80

Ileal digestible CP % 12.17 12.74 12.78

NE kcal/kg 2183 2178 2187

NSP2 % 17.99 17.85 17.83

Digestible NSP % 9.00 8.94 8.93

Illeal digestible amino acids

Lys % 0.74 0.74 0.74

Met % 0.27 1.27 0.27

Cys % 0.23 0.23 0.23

Thr % 0.43 0.43 0.43

Trp % 0.14 0.14 0.28

Phe % 0.59 0.59 1.18

Tyr % 0.40 0.40 0.79

Analyzed composition

DM % 86.11 86.68 86.60

CP (N x 6.25) % 14.81 15.36 15.24

Gross energy kcal/kg 3,804 3,901 3,837

Ash % 5.41 5.55 5.59

Total fat % 2.76 3.81 3.88



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Crude fiber % 3.61 3.68 3.86

Starch % 40.14 39.29 39.64

Sugar % 3.89 4.04 4.12

NSP % 19.09 18.63 18.11

Na % 0.19 0.21 0.22

K % 1.15 1.18 1.16

Sulphate % 0.11 0.11 0.11

Cl % 0.45 0.46 0.44

dEB3 meq/kg 251 263 270

dEBS-a4 meq/kg 227 241 246

Amino acids

Ala % 0.56 0.56 0.58

Arg % 0.71 0.74 0.75

Asp % 0.90 0.91 0.92

Cys % 0.25 0.27 0.27

Glu % 3.45 3.48 3.51

Gly % 0.60 0.60 0.61

His % 0.34 0.35 0.36

Ile % 0.51 0.52 0.52

Leu % 0.98 0.99 1.00

Lys % 0.60 0.75 0.76

Met % 0.24 1.15 0.24

Phe % 0.65 0.66 1.20

Pro % 1.19 1.37 1.30

Ser % 0.64 0.64 0.67



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Thr % 0.54 0.54 0.54

Trp % 0.19 0.20 0.29

Tyr % 0.48 0.48 0.84

Val % 0.65 0.66 0.67

1 SAA = 3 times requirement of sulfur-containing AA; TAA = 2 times requirement of

Trp, and Tyr + Phe; NOAA = supplementation of these AA up to requirement.

2 Non-starch polysaccharides (NSP) were calculated as organic matter – (CP + crude fat

+ starch + sugar).

3 dEB was determined as mEq = Na + K – Cl.

4 dEBS-a was determined as mEq = Na + K – Cl – 2S. dEBS-a does not take into

account S present in AA.

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Table 3. Effects of amino acid supplementation to the diet on daily gain, daily feed

intake and feed efficiency

Diet1

ItemNOAA SAA TAA

SEM2 P value

Initial BW, kg 41.4 40.8 41.4 0.4 0.40

Final BW, kg 73.7 73.4 74.4 1.1 0.79

ADFI, kg/ d 1.87 1.87 1.87 0.00 0.30

ADG, g/ d 702 708 716 23 0.91

G:F 0.375 0.379 0.383 0.013 0.90

1 SAA = 3 times requirement of sulfur-containing AA; TAA = 2 times requirement of

Trp, and Tyr + Phe; NOAA = supplementation of these AA up to requirement. Pigs

were fed for 46 d.

2 SEM = Standard errors of means with 6 observations/ treatment.

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Table 4. Effects of amino acid supplementation to the diet on odor strength (concentration and intensity)

and offensiveness (hedonic tone) from pig manure. Both arithmetic and geometric means are given. The

statistical analyses were conducted on natural logarithmic scale

Diet1

NOAA SAA TAAItems

GM2 AM3 GM AM GM AM

SEM4

Concentration5, ouE m-3 13,224a 16,423 111,302b 118,369 16,318a 21,234 0.26

Emission, ouE s-1m-2 1.88a 2.33 15.48b 16.50 2.23a 2.99 0.26

Concentration at H6 = -1, ouE m-3 3.19a 3.83 1.57b 1.86 3.56a 3.83 0.12

Concentration at H = -2, ouE m-3 9.39a 11.58 5.70b 6.25 11.36a 13.06 0.16

Concentration at I7 = 1, ouE m-3 0.75a 0.90 0.29b 0.36 0.83a 0.91 0.20

Concentration at I = 2, ouE m-3 1.72a 2.00 0.84b 0.96 1.92a 2.06 0.14

Concentration at I = 4, ouE m-3 9.03a 10.08 6.89b 7.24 10.27a 11.24 0.10

1 SAA = 3 times requirement of sulfur-containing AA; TAA = 2 times requirement of Trp, and Tyr + Phe;

NOAA = supplementation of these AA up to requirement. Pigs were fed for 46 d.2 GM = geometric mean. Geometric mean is the antilog of the arithmetic mean of the logarithms of a set

of values.3 AM = arithmetic mean.4 SEM = Standard errors of means with 6 observations/ treatment. The analyses were conducted on natural

logarithmic scale and SEM is on natural logarithmic scale.5 Odor concentration: Odor concentration was expressed in European odor units per cubic meter air (ouE

m-3). One odor unit is defined as the amount of odor-causing gases which, when diluted in 1 m3 of neutral

gas, can just be distinguished from clean air by 50% of the members of an odor panel (CEN, 2003). 6 Hedonic tone: odor hedonic tone is a measure of the unpleasantness or pleasantness of the perceived

odor. H = -1 = mildly unpleasant, and H = -2 = moderately unpleasant.7 Intensity: odor intensity refers to the magnitude of the odor sensation. I = 1 = no odor, I = 2 = very faint

odor; and I = 4 = distinct odor.a, b Means within rows missing a common superscript letter are different (P < 0.05)

590



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Table 5. Effects of amino acid supplementation to the diet on manure characteristics and ammonia

emission from pig manure. Both arithmetic and geometric means are given. The statistical analyses

were conducted on natural logarithmic scale

Diet1

ItemsNOAA SAA TAA

GM2 AM3 GM AM GM AMSEM4

DM, g/kg 114.0a 114.1 116.4 a 116.9 110.2 a 110.6 0.03

Ash, g/kg 30.5 a 30.5 33.4 a 33.5 29.3 a 29.4 0.02

Total VFA5, g/kg 6.27 a 6.38 4.86 a 5.00 5.53 a 5.65 0.08

Acetic acid, g/kg 4.09 a 4.15 3.23 a 3.30 3.74 a 3.82 0.07

Propionic acid, g/kg 1.37 a 1.40 1.01 a 1.03 1.10 a 1.13 0.09

Butanoic acid, g/kg 0.37 a 0.42 0.21 a 0.23 0.26 a 0.30 0.23

Iso-butanoic acid, g/kg 0.11 a 0.12 0.11 a 0.12 0.11 a 0.12 0.13

Iso-pentanoic acid, g/kg 0.29 a 0.30 0.27 a 0.28 0.28 a 0.28 0.10

Total N, g/kg 6.0a 6.0 6.6b 6.6 6.1a 6.2 0.02

Ammonium-N g/kg 2.60a 2.61 3.13b 3.14 2.86b 2.87 0.03

pH - 7.75 a - 7.65 a - 7.93 a 0.09

Ammonia emission, mg/ s/m2 0.013 a 0.014 0.011 a 0.012 0.015 a 0.015 0.09

Phenol, mg/kg 16.07 a 16.15 16.93 a 17.15 19.75 a 20.50 0.10

Cresols, mg/kg 40.13a 40.93 44.39a 44.82 65.56b 67.32 0.08

4-ethyl phenol, mg/kg 0.85ab 0.89 1.02b 1.17 0.47a 0.40 0.42

Indole, mg/kg 9.22a 9.85 3.39b 3.52 9.68a 9.90 0.47

3-methyl indole, mg/kg 5.12 a 5.22 4.38 a 4.45 5.41 a 5.50 0.09

Carbon disulfide, mg/kg 0.50 a 0.53 0.39 a 0.40 0.42 a 0.47 0.16

Methyl sulfide, mg/kg 1.84a 1.22 2.89ab 2.36 4.39b 3.78 0.21

Ethanethiol, mg/kg 77.71 a 80.0 97.61 a 99.6 80.88 a 82.3 0.88



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1 SAA = 3 times requirement of sulfur-containing AA; TAA= 2 times requirement of Trp, and Tyr +

Phe; NOAA = supplementation of these AA up to requirement. Pigs were fed for 46 d.2 GM = geometric mean. Geometric mean is the antilog of the arithmetic mean of the logarithms of a

set of values.3 AM = arithmetic mean.4 SEM = Standard errors of means with 6 observations/ treatment. The analyses were conducted on

natural logarithmic scale and SEM is on natural logarithmic scale, except manure pH which was

analyzed on the original scale. 5 Total VFA = acetic acid + propinoic acid + butanoic acid + iso-butanoic acid + iso-pentanoic acid.

a, b Means within rows missing a common superscript letter are different (P < 0.05).



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Figure 1. Schematic view of the odor and ammonia sample collection (1 = odor free air 591

pressurized cylinder, 2 = manure pit, 3 = vessel, 4 = impingers, 5 = critical glass capillary, 6 = 592

rigid plastic container, 7 = odor bag, 8 = vacuum pump)593

594

Figure 2: Hedonic tone (H) as a function of odor concentration with regression lines, SAA = 3 595

times requirement of sulfur-containing AA; TAA = 2 times requirement of Trp and Tyr + Phe; 596

NOAA = supplementation of these AA up to requirement. HSAA = -0.62 (0.12) – 0.78 (0.04) ln 597

(odor concentration), indicated by ………▲; HTAA = -0.10 (0.12) – 0.78 (0.04) ln (odor 598

concentration), indicated by _____■; HNOAA = -0.27 (0.11) – 0.78 (0.004) ln (odor 599

concentration), indicated by ___ ___ ●; R2 = 0.66600

601

Figure 3: Odor intensity (I) as a function of odor concentration with regression lines, SAA = 3 602

times requirement of sulfur-containing AA; TAA = 2 times requirement of Trp, and Tyr + 603

Phe; NOAA = supplementation of these AA up to requirement. ISAA = 2.04 (0.13) + 0.99 604

(0.05) ln (odor concentration), indicated by ………▲; ITAA = 1.66 (0.13) + 0.99 (0.05) ln 605

(odor concentration), indicated by _____■; INOAA = 1.78 (0.13) + 0.99 (0.005) ln (odor 606

concentration), indicated by ___ ___ ●; R2 = 0.71607

608

Figure 4: Hedonic tone (H) as a function of odor intensity (I) with regression lines, SAA = 3 609

times requirement of sulfur-containing AA; TAA = 2 times requirement of Trp, and Tyr + 610

Phe; NOAA = supplementation of these AA up to requirement. HSAA = 0.94 (0.09) – 0.77 611

(0.02) I, indicated by ………▲; HTAA = 1.16 (0.09) – 0.77(0.02) I, indicated by _____■; 612

HNOAA = 1.09 (0.09) – 0.77(0.02) I, indicated by ___ ___ ●; R2 = 0.89613

614

615

616



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Figure 1.617

618

619

620



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Figure 2.621

-4

-3

-2

-1

0

0 1 2 3 4

ln (odor concentration)

Hed

onic

tone SAA

TAA

NOAA

622

623



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Figure 3.624

0

1

2

3

4

5

6

7

0 1 2 3 4ln (odor concentration)

Inte

nsity

SAA

NOAA

TAA

625



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Figure 4.626

-4

-3

-2

-1

0

1 2 3 4 5 6Intensity

Hed

onic

tone

NOAA

TAA

SAA

627

628

629

630



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Effects of crystalline amino acid supplementation to the diet on odor from pig manure

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