METABOLISM AND NUTRITION

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1 METABOLISM AND NUTRITION Effects of trypsin inhibitor and particle size of expeller-extracted soybean meal on broiler live performance and weight of gizzard and pancreas 1 W. J. Pacheco, C. R. Stark, 2 P. R. Ferket, and J. Brake 3 Prestage Department of Poultry Science, North Carolina State University, Raleigh ABSTRACT Soybean meal (SBM) is a co-product that remains after the oil is removed from whole soybeans by either solvent extraction or mechanically pressing soybeans using an expeller. Expeller-extracted soybean meal (ESBM) contains more fat and less protein than solvent-extracted soybean meal (SSBM), but has higher trypsin inhibitors (TI) that limit its inclusion in poultry diets. We hypothesized that increasing the particle size of ESBM could enhance chick tolerance to dietary TI. The experiment consisted of a 2 6 factorial arrangement of 2 ESBM particle sizes (coarse, 1,300 µm, and fine, 530 µm) and 6 TI levels (6, 9, 12, 15, 18, and 21 TIU/mg). A total of 672 male broiler chicks was randomly assigned among the 12 treatment combinations with 8 replicates per combination. The INTRODUCTION Soybean meal (SBM) has been routinely obtained from whole soybeans either as a co-product of oil extraction using solvent or mechanically using extrusionexpeller technology. The extrusion-expeller process has become an alternative as it offers advantages such as low capital investment and low operational costs (Crowe et al., 2001) compared with the traditional solvent extraction process. The best approach to improve SBM quality has been heat treatment, which has been reported to improve SBM nutritional value by denaturing the native protein structure and destroying protease inhibitors (Purushotham et al., 2007). However, the extrusion-expeller process has not provided sufficient heat treatment, which has been shown to modify the birds were provided with 1 kg/bird of starter diet in a crumble form. The pancreas and gizzard were excised and weighed at 16 d of age and organ weights relative to BW calculated. Feed consumption, BW, and feed conversion ratio (FCR) were determined at 7 and 14 d of age. Increasing TI level produced a quadratic effect (P < 0.01) on BW and FCR at 14 d of age. The best performance was obtained when chicks were fed diets containing ESBM at intermediate TI levels. Increasing the particle size of the ESBM from 530 to 1,300 µm improved BW and feed intake (P < 0.01) without affecting FCR. The relative weight of the pancreas increased linearly as TI level increased (P < 0.001). The results of this study indicated that chicks performed better when fed coarse ESBM at an intermediate TI level. Key words: broiler, gizzard, pancreas, particle size, trypsin inhibitor 2014 Poultry Science 93 : /ps Poultry Science Association Inc. Received February 20, Accepted June 1, The use of trade names in this publication does not imply endorsement of the products mentioned nor criticism of similar products not mentioned. 2 Present address: Department of Grain Science and Industry, Kansas State University, Manhattan Corresponding author: jbrake@ncsu.edu structure of the proteins, improve nutrient digestibility, and deactivate trypsin inhibitors commonly present in raw soybeans (Perić et al., 2011). Because soybean processors that have used the extrusion-expeller process have not developed the technology required to apply the proper amount of heat or retain the heated SBM for sufficient time to deactivate the majority of the trypsin inhibitors, other alternatives needed to be explored. One alternative was to increase the particle size of the expeller-extracted soybean meal (ESBM) to modulate the digestive process of the bird or alter the rate of access of digestive processes to the ESBM. Previous studies with cereal grains demonstrated that particle size influenced growth performance, feed passage rate, and gizzard development (Nir et al., 1994). A well-developed gizzard has been associated with increased grinding activity, enhanced reverse peristalsis, and greater absorption of nutrients (Amerah et al., 2007). Pacheco et al. (2013) conducted an experiment using 2 SBM sources [solvent-extracted soybean meal (SSBM) and ESBM], which contained 3.8 and 22.1 TIU/mg, respectively, and 2 particle sizes (coarse, 971 µm, and fine, 465 µm) and reported an interaction between SBM source and particle size on BW at 49 d of age. When birds were fed coarse SBM there were no 1

2 2 Pacheco et al. differences in BW between sources, whereas birds fed diets that contained fine ESBM exhibited lower BW than birds fed diets that contained fine SSBM. The author suggested that increasing the particle size of the ESBM ameliorated the negative effects of trypsin inhibitors. This study was designed to further elucidate the effects of the particle size of ESBM in combination with different levels of trypsin inhibitors on broiler live performance. The hypothesis was that increasing the particle size of ESBM would ameliorate the adverse effects of trypsin inhibitors and improve growth performance, especially at high levels of trypsin inhibitors. MATERIALS AND METHODS SBM, Diets, Husbandry, and Experimental Design Soybean cake was obtained from a facility in Pantego, North Carolina (East Carolina Soy Processors, LLC). Initially, whole soybeans were dried to 10% moisture and passed through a roller mill that cracked them into small pieces and then passed through aspirators where the hulls were removed. Dehulled soybeans were processed using dry extrusion without steam injection and continuous expeller pressing. The shear, friction, and pressure generated during the extrusion process have been reported to disrupt the cell wall structure of the soybeans and release the oil from spherosomes (Wang et al., 2004). The temperature in the extruder was 157 C with a retention time of 1 to 2 min depending on the production rate. According to Nelson et al. (1987), the use of dry extrusion and continuous expeller pressing has accomplished the recovery of 70% of the oil and the inactivation of approximately 90% of the trypsin inhibitors. After the removal of the oil, the soybean cake was transported to the feed mill at North Carolina State University for further processing. The soybean cake containing approximately 21 trypsin inhibitor units (TIU)/mg [1 TIU/mg = 1.9 trypsin inhibitor activity (TIA) mg/g] was ground using a double-pair roller mill (RMS, Harrisburg, SD) to obtain the coarse ESBM at 1,300 µm (the top set of rolls was 100% open and the bottom set 37% open). One-half of the coarse ESBM was placed in 42 cm 25 cm pans at a depth of 6 cm and heated in an autoclave at 100 C for 30 min to reduce the trypsin inhibitor level to approximately 6 TIU/mg. The heating time was assumed to start when the internal temperature of the autoclave reached 100 C. After autoclaving, half of the autoclaved and half of the nonautoclaved coarse ESBM were reground using a roller mill with both sets of rolls completely closed to obtain the fine ESBM at 530 µm. Laboratory Analyses The nutritive value of the ESBM was analyzed for ether extract after HCl hydrolysis (method ), CP (N 6.25) by the combustion method (990.03) using Leco equipment (Leco Corporation, St. Joseph, MI), and crude fiber (method ; AOAC, 2006) before feed formulation, and the main results are reported in Table 1. Expeller-extracted soybean meal samples were sent to the laboratory of Mouriscade (Pontevedra, Spain) to determine trypsin inhibitor levels, urease activity, protein dispersibility index (PDI), and solubility in KOH. Trypsin inhibitor activity, expressed in milligrams of trypsin inhibited per gram of ESBM (mg/g), was determined according to Kakade et al. (1974) as modified by Hamerstrand et al. (1981). A sample (1 g) of ESBM was ground using a laboratory grinder provided with a 0.2-mm screen. After grinding, the ESBM sample was defatted using ether. The trypsin inhibitors were extracted with 50 ml of 0.01 N NaOH at a ph between 8.4 and 10.0 using a magnetic stirrer for 3 h at ambient temperature. The extract was measured for TIA using N-α-benzoyl-dl-arginine-p-nitroanilide as specific substrate. Trypsin inhibitor activity was calculated after measuring the absorbance at 410 nm against a blank reagent, using an UV spectrophotometer. The TIA (mg/g) was converted to TIU/mg using (TIU/mg = TIA 1.9). Residual urease activity was determined in terms of an increase in ph after the ESBM sample was incubated with a buffered urea solution (AACC, 1995). Protein dispersibility index was measured according to the method Ba 10a-05 (AOCS, 2004) and protein solubility in KOH was measured as indicated by Araba and Dale (1990). Dry sieving was used to determine average particle size and particle size distribution of the coarse and fine ESBM according to the ASAE method S319.3 (ASABE, 2007) with the addition of sieve agitators and 0.5% silicon dioxide as dispersing agent per 100 g of sample (Figure 1). Diets Diets were formulated to meet or exceed NRC requirements (NRC, 1994) and were composed principally of corn and ESBM as reported in Table 2. Dietary amino acids were formulated on a digestible amino acid basis and contained 1.14% digestible lysine. A total of 4 basal diets were manufactured using coarse or fine ESBM at 6 or 21 TIU/mg. Dry ingredients were blended in a double ribbon mixer (Hayes and Stolz, Forth Worth, TX), conditioned at 85 C for 45 s, and then pelleted using a pellet mill (CPM California Pellet Mill, Crawfordsville, IN) equipped with a 4.4 mm 35 mm die. Pellets were cooled using a counter-flow cooler (Geelen Counterflow, Orlando, FL) and then crumbled (CPM Roskcamp Champion, Waterloo, IA). The crumbled diets containing coarse and fine ESBM with high (21 TIU/mg) and low (6 TIU/mg) levels of trypsin inhibitors were blended to create 4 diets with intermediate levels of trypsin inhibitors (9, 12, 15, and 18 TIU/mg). A total of 12 diets consisting of 6 levels of trypsin inhibitors (6, 9, 12, 15, 18, and 21 TIU/mg)

3 Table 1. Determined and calculated chemical composition of the expeller-extracted soybean meal (%, as-fed basis, unless stated otherwise) as received from the supplier and before autoclaving Item and 2 particle sizes (coarse, 1,300 µm, and fine, 530 µm) were manufactured and fed from 1 to 16 d of age. Bird Husbandry Value Determined chemical composition 1 DM 95.1 CP 47.6 Ether extract 8.9 Crude fiber 3.8 Sucrose 7.0 Calcium 0.33 Total phosphorus 0.64 CP quality trait Protein dispersibility index, % 18.4 KOH solubility, % 85.0 Urease activity, mg of N/g 0.28 Trypsin inhibitor activity, mg/g 11.4 Trypsin inhibitor units, TIU/mg 21.7 Calculated chemical composition AME n, kcal/kg 3,179 Digestible amino acid Lysine 2.49 Methionine 0.58 TSAA 1.01 Threonine 1.59 Tryptophan 0.55 Arginine In duplicate. Figure 1. Particle size distribution of the coarse and fine expellerextracted soybean meal (SBM). TRYPSIN INHIBITOR AND PARTICLE SIZE The care of the chicks used in this trial conformed to the Guide for the Care and Use of Animals in Agricultural Research and Teaching (FASS, 2010). A total of 672 one-day-old male Ross 708 broiler chicks (Aviagen, Huntsville, AL) were weighed, neck tagged, and placed in 2 environmentally controlled rooms. Each room contained 4 Petersime batteries that had 12 cages spread over 6 decks, electric brooders, and wire mesh floor. Chicks were randomly assigned to 1 of 12 dietary treatments combinations with 8 replicate cages per combination and 7 chicks per cage. Chicks had free access to feed and water throughout the trial. Feed spills were collected from the bottom of each cage and added back to the feeder. The lighting program provided 23 h of artificial light per day throughout the 16-d experimental period. Room temperature was maintained at 32 C for the first 3 d of the experiment and gradually reduced until reaching 29 C at 16 d of age. All chicks were fed 1 kg of starter feed in crumble form from 1 to 16 d of age. Growth Performance Body weight of the chicks and feed consumption were recorded on a cage basis at 1, 7, and 14 d of age. Only 6 birds died during the experimental period. Chicks that died were removed, weighed, and the data were used to calculate and adjust feed conversion ratio (FCR). From these data, feed intake and FCR were calculated. Gizzard and pancreas were collected at 16 d of age. The gizzard was excised, and external fat and internal contents were removed, rinsed, and blotted dry. The Table 2. Ingredient composition and chemical analyses (%, asfed basis, unless stated otherwise) of the experimental diets Item, % unless stated otherwise Value Ingredient Corn Expeller-extracted soybean meal, 47.6% Dicalcium phosphate, 18.5% P 2.03 Limestone 1.14 Poultry fat 0.50 Salt 0.49 dl-methionine, 99% 0.32 l-lysine-hcl, 78% 0.11 Vitamin premix Choline chloride, 60% 0.10 Trace mineral premix Selenium premix Coccidiostat Calculated chemical analyses ME, kcal/g 3.15 CP Crude fat 5.97 Calcium 1.00 Available phosphorus 0.50 Digestible lysine 1.14 Digestible arginine 1.47 Digestible TSAA 0.87 Digestible threonine 0.77 Digestible tryptophan 0.25 Analyzed chemical analysis Gross energy, kcal/g 4.50 CP Ether extract Vitamin premix provided the following per kilogram of diet: vitamin A, 6,600 IU; cholecalciferol, 1,980 IU; niacin, 55 mg; α-tocopherol, 33 mg; pantothenic acid 11 mg; riboflavin, 6.6 mg; pyridoxine, 4 mg; menadione, 2 mg; folic acid, 1.1 mg; thiamine, 2 mg; vitamin B 12, 0.02 mg; and biotin, 0.13 mg. 2 Mineral premix provided the following per kilogram of diet: Zn, 120 mg; Mn, 120 mg; Fe, 80 mg; Cu, 10 mg; I, 2.5 mg; Co, 1.0 mg. 3 Selenium premix provided 0.2 mg of Se/kg. 4 Monensin was included at 99 mg/kg (Coban 90, Elanco Animal Health, Indianapolis, IN). 3

4 4 Pacheco et al. pancreas was dissected away from the duodenal loop and weighed. The gizzard and pancreas weights were expressed relative to BW. Statistical Analysis The experiment was analyzed as a 2 6 factorial randomized complete block design to identify main effects and interactions and consisted of 6 levels of trypsin inhibitors (6, 9, 12, 15, 18, and 21 TIU/mg) and 2 particle sizes (coarse, 1,300 µm, and fine, 530 µm). Cage served as the experimental unit to derive the performance data, which was analyzed using PROC GLM (SAS, 2006). Differences between means were separated by least significant difference test. When appropriate, linear and quadratic responses were evaluated using single degree of freedom orthogonal contrasts with coefficients for evenly spaced treatments. Differences between treatments were considered statistically significant at P < 0.05 unless otherwise specified. RESULTS AND DISCUSSION Quality Variables The results of the laboratory analyses of ESBM are presented in Table 3. The analyzed TIU of the ESBM used in the diets were 6.8, 9.6, 12.6, 16.2, 18.9, and 21.7 TIU/mg, which were in close agreement with values expected at the beginning of the trial. Kratzer et al. (1990) reported a decrease in TIU from 38 to 0 TIU/mg when SSBM was autoclaved for 121 C for 120 min. In a more recent study, Batal et al. (2000) reported a reduction in TIU from 44.2 to 2.6 TIU/mg when soybean flakes were autoclaved at 121 C for 36 min. The urease activity test has been used to evaluate processing time and technique, but has not been found to be important for poultry production (McNaughton et al., 1981). The corresponding results of the urease index test were a 0.05, 0.09, 0.16, 0.20, 0.27, and 0.28 rise in ph ( ph), respectively. The American Feed Manufacturers Association (AFMA) recommended values between 0.05 and 0.20 ph (AFMA, 1979). Whereas urease levels between 0.05 and 0.20 have been considered optimum, levels below 0.05 do not necessarily mean that SBM has been overheated (Waldroup et al., 1985; Araba and Dale, 1990). According to Araba and Dale (1990), there were 2 problems with the urease activity test; low levels were not correlated with good chick performance and urease activity of 0.00 ph was not always an indication of overheating. Nevertheless, the urease test has been one of the most popular and inexpensive methods used to assess the adequacy of heat treatment of SBM as urease has been reported to be destroyed at a similar rate as trypsin inhibitors (Caskey and Knapp, 1944; Wright, 1981). Indeed, when performing trypsin inhibitors analyses, several laboratories have used the urease test to determine the appropriate dilution of the 2 ml of soy sample extract to inhibit 40 to 60% of the trypsin used for the analysis (Hamerstrand et al., 1981). The results of the PDI were 9, 11, 13, 15, 17, and 18%, correspondingly. The National Soybean Processor Association has recommended PDI values between 15 and 30% (Balloun, 1980). The respective results of the KOH solubility were 59, 63, 69, 74, 81, and 85%. Although KOH solubility values have not been established, values below 74% have been considered as an indication of excessive heat treatment and values above 85% have been associated with insufficient heat treatment (Dozier and Hess, 2011). Based on these standards and the PDI and KOH solubility data of the present study, the 3 diets with the lowest solubility containing 6.8, 9.6, and 12.6 TIU/mg were likely overheated. The direct measurement of trypsin inhibitors has been the most desirable assay to evaluate SBM quality and processing conditions (Dozier and Hess, 2011). Nevertheless, the analyses of trypsin inhibitors have been time consuming, have required expensive equipment, have produced inconsistent results probably due to differences in methodologies between laboratories (Rackis et al., 1974), and have been impractical for day-to-day monitoring of SBM quality and adequacy of heat treatment. Therefore, urease activity, PDI, and KOH solubility analyses have become standard methods to measure SBM quality. Table 3. Laboratory analyses of expeller extracted soybean meal Treatment TI activity, 1 mg/g TI units, 1 TIU/mg Urease activity, 2 mg of N 2 /g per min; 30 C PDI, 3 % KOH solubility, 4 % Recommended trypsin inhibitor (TI) levels not defined. 2 American Feed Industry Association recommended levels between 0.05 to 0.20 ph. 3 The National Soybean Processor Association recommended levels between 15 to 30%. PDI = protein dispersibility index. 4 Araba and Dale (1990) recommended levels between 70 to 80%.

5 TRYPSIN INHIBITOR AND PARTICLE SIZE 5 Figure 2. Effect of coarse and fine expeller-extracted soybean meal with different levels of trypsin inhibitor units on broiler BW at 14 d of age. Broiler Live Performance There were no significant interactions between TIU and particle size of the ESBM on live performance in these young broilers. Coarse ESBM generally increased BW and feed intake regardless of the trypsin inhibitor level (Figures 2 and 3) with no differences in FCR (Figure 4). Overall, there was a decrease in feed intake when the TIU were greater than 18.9 TIU/mg (Table 4). Hathcock and Rader (1994) and Shahidi (1997) reported that high levels of naturally occurring protease inhibitors present in feed ingredients reduced feed intake. Chicks fed coarse ESBM had a greater overall feed intake (P < 0.05) than chicks fed fine ESBM, which resulted in higher BW (P < 0.01) at 14 d of age. Although the chicks that consumed the coarse ESBM had a higher feed intake and therefore higher intake of trypsin inhibitors, no differences were observed in FCR. It was possible that coarse ESBM increased the reverse peristalsis between the gizzard and proventriculus, which caused acid denaturation of trypsin inhibitors and brought more bile into the gizzard. Trypsin inhibitor activity toward pancreatic-trypsin can be reduced with alkali and acid denaturation, as well as with Figure 4. Effect of coarse and fine expeller-extracted soybean meal with different levels of trypsin inhibitor units on broiler feed conversion ratio (FCR) at 14 d of age. heat (Liener and Kakade, 1980; Liardon and Friedman, 1987). Additionally, the trypsin inhibitors were probably released more slowly or consistently from the coarse ESBM than from the more finely ground ESBM and hence provided the chick with the opportunity to adapt. There was a quadratic effect (P < 0.01) of trypsin inhibitors on BW at 7 and 14 d of age. The greatest BW was obtained when chicks were fed diets that contained ESBM with intermediate TIU between 12.6 and 16.2 TIU/mg. The decrease in BW observed at TIU lower than 12.6 TIU/mg was likely due to amino acid damage that occurred during autoclaving. According to Sibbald (1980), excessive heat treatment decreased the availability of some amino acids, with lysine being the most sensitive. The decreased BW observed when trypsin inhibitors were greater than 18.9 TIU/mg was likely due to decreased feed intake as well as an inferior FCR. Additionally, trypsin inhibitors have been shown to negatively affect protein digestibility and amino acid availability (de Coca-Sinova et al., 2008) by forming indigestible complexes with pancreatic-produced trypsin (Liener, 1994). Figure 3. Effect of coarse and fine expeller-extracted soybean meal with different levels of trypsin inhibitor units on broiler feed intake at 14 d of age. Figure 5. Effect of trypsin inhibitor on relative pancreas weight of broilers at 16 d of age.

6 6 Pacheco et al. Table 4. Effect of trypsin inhibitor (TI) and particle size of expeller-extracted soybean meal on broiler BW, feed intake, and adjusted feed conversion ratio (FCR) of chicks from 1 to 14 d of age Item Particle size 1 BW, g Feed intake, g FCR, 2 g:g 7 d 14 d 1 7 d 1 14 d 1 7 d 1 14 d TI units, TIU/mg 6 Coarse Coarse Coarse Coarse Coarse Coarse Fine Fine Fine Fine Fine Fine Main effect b 527 bc a 1.09 a 1.19 a b 528 bc ab 1.05 ab 1.17 bc a 542 a a 1.04 b 1.15 c a 537 ab ab 1.06 ab 1.15 c b 523 c b 1.06 ab 1.16 bc b 516 c b 1.10 a 1.18 ab SEM Coarse a a Fine b b SEM Source of variation (P-value) TI level particle size TI level, TIU/mg Particle size, µm Regression analysis (P-value) Linear Quadratic a-c Means with different superscripts in the same column differ significantly (P < 0.05). 1 Treatments consisted of expeller-extracted soybean meal at 6, 9, 12, 15, 18, and 21 TIU/mg and 2 particle sizes: coarse (1,300 µm) and fine (520 µm). 2 FCR = feed intake per cage/total cage BW gain, including BW of mortality that occurred during each time period. 3 Standard error of the means for TI level (n = 16). 4 Standard error of the means for particle size (n = 48). Table 5. Effect of trypsin inhibitor (TI) and particle size of expeller-extracted soybean meal on the relative weight of pancreas and gizzard of broiler chicks at 16 d of age Item Particle size 1 Relative organ weight, g/100 g of BW Pancreas Gizzard TI level 1 Main effect c b b b a a 1.91 SEM Coarse Fine SEM Source of variation (P-value) TI level (TIU/mg) Particle size (µm) Regression analysis (P-value) Linear Quadratic a c Means with different superscripts in the same column differ significantly (P < 0.05). 1 Treatments consisted of expeller-extracted soybean meal at 6, 9, 12, 15, 18, and 21 TI units (TIU)/mg and 2 particle sizes: coarse (1,300 µm) and fine (520 µm). 3 Standard error of the means for TI level (n = 16). 4 Standard error of the means for particle size (n = 48).

7 Similar to the BW results, the level of trypsin inhibitors units in the ESBM produced a quadratic effect on FCR at 14 d of age. Chicks fed diets that contained ESBM with 12.6 and 16.2 TIU/mg exhibited the best FCR. Chicks fed diets that contained ESBM with TIU lower than 9.6 TIU/mg had poorer FCR. Aburto et al. (1998) reported that birds fed overheated SBM exhibited significantly reduced BW and poorer feed efficiency when compared with properly heated SBM, but the addition of 0.08% crystalline lysine restored performance. In general, the growth of the chicks in all the treatments was quite good likely because diets were formulated on a digestible amino acid basis. Pancreas and Gizzard Development The particle size of the ESBM did not affect gizzard relative weight in this study (Table 5). Nir et al. (1994) reported a greater gizzard development at 7 d of age when chicks were fed diets that contained medium and coarse particles. There had been a greater positive effect on gizzard development when diets were fed in a mash versus a pelleted or crumbled form likely due to changes in particle size distribution after pelleting (Amerah et al., 2007). Pelleting and crumbling of the diets used in this trial likely diminished the positive effect of particle size on gizzard development. The relative weight of the pancreas increased linearly as TIU in the ESBM were increased (P < 0.01; Figure 5). Veltman et al. (1986) did not find significant differences in performance and pancreas weight when chicks were fed low trypsin inhibitor levels of 2.9, 4.4, 5.2, and 6.5 TIU/mg, which suggested that a certain level of trypsin inhibitors must be fed to cause pancreatic hypertrophy. The best performance was obtained when chicks were fed coarse ESBM with intermediate TIU between 12.6 and 16.2 TIU/mg. Coarse ESBM increased feed intake and BW in diets that contained more than 9.6 TIU/ mg. 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