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Effect of micronized pea and enzyme supplementation on nutrient utilization and manure output in growing pigs¹

C. M. Nyachoti^*,2, S. D. Arntfield, W. Guenter^*, S. Cenkowski and F. O. Opapeju^*

^* Departments of Animal Science, and Food Science, and and Biosystems Engineering, University of Manitoba, Winnipeg, Manitoba, Canada R3T 2N2

	Abstract

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An experiment was done to determine manure output, N and P excretion, and apparent digestibilities of AA, CP, P, and DM in growing pigs fed barley-based diets containing micronized or raw peas with or without supplementation with enzyme containing primarily ß-glucanase and phytase (Biogal S⁺). Eight barrows (21.5 ± 1.2 kg of initial BW) fitted with T-cannulas at the distal ileum were used in a 40-d trial and housed in metabolism cages. Pigs were assigned in a replicated 4 x 4 Latin square design to 4 experimental diets: 1) barley-raw peas control (BRP), 2) barley-micronized peas (BMP), 3) BRP plus enzyme, and 4) BMP plus enzyme (BMP+E). Pigs received 2.6 times maintenance energy requirements based on BW at the beginning of each experimental period. During each experimental period, pigs were acclimatized to their respective diets for 5 d followed by a 3-d period of total fecal and urine collection and another 2-d period of ileal digesta collection. Samples were analyzed for DM, AA (diets and digesta only), N, and P. Wet fecal output of BRP plus enzyme-fed pigs tended to be lower (P = 0.07) than the amount produced by BMP-fed pigs. The amounts of dry feces and urine produced were not different among treatments (P > 0.10). Supplementing the BRP and BMP diet with enzyme increased (P = 0.002) the daily P retained per pig. Pigs fed the enzyme-supplemented diets tended to have lower (P = 0.06) fecal P excretion and greater urinary P excretion (P = 0.001) compared with pigs fed the nonsupplemented diets, but total P excretion was not influenced by diet (P > 0.10). Pigs fed the BMP+E diet retained more (P = 0.006) N per day than pigs fed the BMP diet. However, N excretion was not influenced by dietary treatment (P > 0.10), although BMP+E-fed pigs excreted 13.2% less N in the feces compared with those fed the nonenzyme supplemented controls. Inclusion of micronized peas with or without enzyme supplementation did not affect urinary or fecal N excretion (P > 0.10) compared with the BRP. Dietary treatment had no effect (P > 0.10) on ileal or fecal DM or CP digestibilities. Apparent ileal digestibilities of AA were usually lower (P < 0.05) in the BRP diet compared with the other diets. Enzyme supplementation improved P digestibility at the ileal and fecal level. The current results indicate that utilizing micronized peas in barley-based pig grower diets enhances P retention.

Key Words: enzyme supplementation • manure output • micronization • nutrient utilization • pig

	INTRODUCTION

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Environmental issues associated with intensive livestock production have attracted a lot of research interest in recent years. The majority of the research thus far has focused on minimizing the amount of N and P excreted in livestock manure because high concentrations of these nutrients limit its application onto agricultural land in intensive swine production areas (Cromwell and Coffey, 1991). Nutritional management strategies offer the most cost-effective means to reduce the negative environmental impact associated with swine production (Leneman et al., 1993).

Supplementing swine diets with exogenous phytase or phytase in combination with carbohydrase enzymes has been shown to improve phytate P utilization with reductions in manure P content (Jongbloed and Lenis, 1998; Grandhi, 2001; Zhang et al., 2003). Also, ingredient processing to inactivate antinutritional factors, reducing particle size to improve DM and nutrient digestibilities, or disruption of cell wall components to make them more susceptible to enzymatic digestion may be used to reduce N and P content in swine manure (van Kempen, 2000).

Additionally, decreased manure output will reduce the environmental impact of swine production. Using ingredients with low fiber content in place of high fiber ingredients has been shown to reduce manure volume (Grandhi, 2001). Heat processing procedures such as micronization have been reported to disrupt cell wall components and increase DM and nutrient digestibilities in pigs (Lawrence, 1973) and poultry (Igbasan and Guenter, 1996). However, the potential use of micronized feedstuffs and or supplemental enzymes to reduce nutrient excretion and swine manure output has not been examined extensively (Zhang et al., 2003).

Therefore, the objective of this study was to evaluate the inclusion of micronized peas with or without enzyme supplementation in a barley-based diet fed to growing pigs on N and P excretion, manure output, and nutrient digestibilities.

	MATERIALS AND METHODS

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Animals and Housing
The use of animals in the current study was reviewed and approved by the Animal Care Committee of the University of Manitoba, and pigs were cared for according to the guidelines of the Canadian Council on Animal Care (CCAC, 1993).

Eight unrelated Cotswold barrows with an initial BW of 21.5 ± 1.2 kg were obtained from Glenlea Swine Research Farm of the University of Manitoba for use in this experiment. Pigs were housed individually in adjustable metabolism crates (0.91 x 1.52 m) with smooth, transparent plastic sides and plastic-covered, woven metal flooring in a temperature-controlled (20 to 22°C) room. After a 7-d adaptation period, pigs were surgically fitted with a simple T-cannula at the terminal ileum following the procedures described by Sauer et al. (1983). The design of the cannulas was modified according to de Lange et al. (1989). After surgery, the pigs were immediately returned to the metabolism pens and allowed a 14-d recovery period. During this period they were fed increasing amounts of a corn and soybean meal-based pig starter diet twice daily and had unlimited access to water from low-pressure nipples. After the study, the pigs were killed to determine whether cannulation had caused any intestinal abnormalities.

Preparation of Experimental Diets
Micronized peas (Pisum sativum) were prepared as described by Arntfield et al. (2004). Briefly, pea (cv. Croma) samples were tempered to a 25% moisture content and then micronized at a temperature range of 110 to 115°C for 90 s in a gas-fired micronizer unit (Micronizer Ltd. Co., Suffolk, UK). Micronized peas as well as raw peas were dried at room temperature for 4 d to reduce the moisture level to 12% and then ground to pass through a 4.76-mm screen for diet formulation. The 4 experimental diets (Table 1) were 1) barley-raw peas control (BRP), 2) barley-micronized peas (BMP), 3) BRP plus enzyme (BRP+E), and 4) BMP plus enzyme (BMP+E). The enzyme used was a multienzyme blend (Biogal S⁺) providing 500 units of ß-glucanase and 300 units of phytase per kilogram of diet plus a broad spectrum of other enzyme activities including protease, amylase, cellulase, and pectinase (Canadian Biosystems Inc., Calgary, Alberta, Canada). Vitamins and minerals were supplemented to meet or exceed NRC (1998) recommendations, except for Ca and P. Chromic oxide (0.3%) was included as an indigestible marker for determining apparent nutrient digestibilities. All diets were fed in a mash form.

During each experimental period, pigs were allowed to acclimatize to their respective experimental diets for 5 d. Feces and urine were then collected quantitatively over a 3-d period. Each metabolism crate had a collection tray for urine and a fine-mesh plastic net just above the tray for fecal collection. In addition, glass wool was placed in the funnel of the collection trays to trap any feces not retained by the net. The sample collection procedures were similar to those described by Grandhi (2001) and Zhang et al. (2003). Urine was collected into plastic pails containing 50 mL of 5% H₂SO₄. After measuring the daily volume, 100-mL aliquots were taken for each 24-h period and kept frozen until analyzed. Feces were collected frequently (at least every 2 h) and stored in sealed plastic bags to minimize N loss as ammonia. Feces were weighed daily and stored at –20°C until analyzed. Ileal digesta was collected continuously for a 12-h period on d 9 and 10 to determine apparent ileal AA digestibilities. Digesta were collected into plastic bags that were attached to the barrel of the T-cannulas by a hose clamp. Collection bags contained 10 mL of 10% (vol/vol) formic acid to minimize bacterial activity. Every 1 to 2 h, the collected digesta were removed and immediately frozen at –20°C until processed.

Sample Preparation and Chemical Analysis
All analyses were performed in duplicate. Digesta and fecal samples were pooled per pig and experimental period. Digesta and fecal samples were freeze-dried and, along with diet samples, were ground to pass through a 1-mm screen, then thoroughly mixed before taking samples for analysis. Samples were analyzed for DM according to AOAC procedures (1990). Nitrogen was determined using a Leco NS 2000 Nitrogen analyzer (LECO Corporation, St. Joseph, MI). Total P (%) was determined according to the AOAC (1990) procedure. Briefly, 1 g of sample was ashed for 12 h in a muffle furnace at 600°C. To the sample was added 10 mL of a solution containing 5 N HCl and HNO₃ (1% vol/vol), and the mixture was heated in a sonicator water bath at 65°C for 1 h. The mixture was allowed to settle overnight. Standards with P concentration ranging from 0 to 15 µg/mL were prepared from a stock of KH₂PO₄ (2 mg of P/mL) to generate the standard curve. The absorbance of samples was read against distilled water at 400 nm using a Pharmacia Ultrospec 2000 spectrophotometer (Pharmacia Biotech, Cambridge, UK). After reacting with ammonium molybdate to form an ammonium phosphomolybdate complex, as described by Fiske and Subbarow (1925), urinary P content was determined colorimetrically using a Vitros PHOS Slides (Ortho Clinical Diagnostics, Mississauga, ON). Chromic oxide was analyzed, after the samples were ashed for 12 h in a muffle furnace, using an atomic absorption spectrophotometer (Instrumentation Laboratory Inc., Wilmington, MA) according to the procedure described by Williams et al. (1962).

Diet and digesta samples were analyzed for AA content. A 100-mg sample was weighed for acid hydrolysis according to AOAC (1984), and as modified by Mills et al. (1989) for AA analysis. Briefly, samples were digested in 4 mL of 6 N HCl for 24 h at 110°C, followed by neutralization with 4 mL of 25% (wt/vol) NaOH and cooling to room temperature. The mixture was then made to 50 mL volume with sodium citrate buffer (pH 2.2) and analyzed using an LKB 4151 Alpha analyzer (LKB Biochrom, Cambridge, UK). Amino acids were quantified with the internal standard method by measuring the absorption of reaction products with ninhydrin at 570 nm. The sulphur-containing AA and Trp were not determined.

Calculations and Data Analysis
Analyzed dietary N and P concentrations and feed intake were used to calculate the amount consumed. The concentrations determined in the feces and urine together with the quantities of feces and urine voided were used to calculate N and P excretion. Retention of each of these nutrients was determined from the difference between their respective intake and excretion values. Apparent ileal DM, AA, CP, and P digestibilities were calculated using the chromic oxide concentration in the diets and digesta as reported previously (Nyachoti et al., 2002).

Data were subjected to ANOVA using the GLM procedures of SAS (SAS Inst. Inc., Cary, NC). Diet, experimental period, pig, square, and BW (covariate) were included as sources of variation. When a significant F-value for treatment (P < 0.05) was observed in the ANOVA, treatment means were compared using Fisher’s protected least significant difference test.

	RESULTS AND DISCUSSION

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The pigs seemed healthy, readily consumed their daily feed allowance, and grew normally during the experimental periods. The initial and final BW were 21.5 (SD = 1.2) and 43.2 (SD = 2.4) kg, respectively. The average daily DMI was not different (P = 0.30) among treatments because daily feed allowance was provided at a fixed rate based on BW, and it averaged 1,091 g/d. A postmortem examination revealed no adverse effects of cannulation. The analyzed CP and lysine composition of the experimental diets were close to calculated values (Table 1). However, analyzed dietary P concentrations (Table 1) were greater than formulated values (NRC, 1998).

The amount of feces and urine produced on a daily basis is shown in Table 2. The amount of wet or dry feces and urine volume were not different (P > 0.10) among diets. Zhang et al. (2003) reported that including micronized peas in barley-based pig grower diets resulted in significant reductions in fecal volume.

The increased proportion of P excreted via the urine suggests that enzyme supplementation increased P absorption beyond the P requirements for the pigs used in the study. Supplemental phytase has been reported to increase the amount of P excreted in urine (Kemme et al., 1997; Zhang et al., 2003). This is an important observation considering that there is a lot of interest in understanding the effect of diet manipulation, including enzyme supplementation, on P forms and solubility in livestock manure and manure-amended soils (Baxter et al., 2003; Gollany et al., 2003; Wienhold and Miller, 2004).

Nitrogen intake tended (P = 0.08) to differ among dietary treatments. Pigs fed the BMP+E diet retained more N on a daily basis compared with pigs fed the nonsupplemented diets (P = 0.006). When expressed as percentage of intake, no differences (P > 0.10) were observed in N retention. The amounts of N in the feces, urine, and manure (feces + urine) were not different among the dietary treatments (Table 2). The current N utilization data does not support previous studies reporting improved N utilization in pigs fed diets containing micronized hulless barley (Huang et al., 1998; Thacker, 1999), and peas (Zhang et al., 2003).

Nutrient Digestibilities
Apparent ileal DM and CP digestibilities were similar among diets (P > 0.10), and they averaged 89.3 and 80.7%, respectively (Table 3). Similarly, there were no differences (P > 0.10) among diets in the apparent total tract digestibilities of DM and CP, which averaged 87.9 and 87.6%, respectively. The effect of micronization on the apparent ileal digestibility of CP in pigs has been reported in only one previous study (Owusu-Asiedu et al., 2002), in which micronization was also found to have no effect. However, significant improvements in pea protein digestibility due to micronization have been reported with broiler chickens (Igbasan and Guenter, 1996). Furthermore, micronization of hulless barley has been shown to improve apparent ileal and total tract DM and CP digestibilities in starter (Huang et al., 1998) and growing pigs (Thacker, 1999). The lack of effect of micronization on DM and CP digestibility in the current study might have been due to the fact that micronization does not have consistent effects on different feed-stuffs. For example, in the study by Thacker (1999) micronization had only a minor effect on these responses in hulled barley, whereas significant improvements were observed for hulless barley.

The apparent ileal digestibilities for AA are presented in Table 3. Compared with the BRP, apparent ileal digestibilities of all AA except Arg, Ser, and Pro were greater (P < 0.05) in the BMP diet. No differences (P > 0.10) were seen in the apparent ileal AA digestibilities between the BMP diet and diets supplemented with enzymes (BRP+E and BMP+E). The current results are in close agreement with our previous study in which micronization of peas was shown to significantly improve apparent ileal AA digestibilities in young pigs (Owusu-Asiedu et al., 2002). Improved ileal AA digestibility implies that micronization led to an increase in AA bioavailability, which may partly explain the improvements in pig growth performance observed in previous studies (Huang et al., 1997; Thacker, 1999; Zhang et al., 2003). Micronization, which involves infrared heating, may lead to conformational changes in the matrix of storage proteins, thus rendering them more susceptible to enzymatic attack (van der Poel, 1990; Owusu-Ansah and McCurdy, 1991).

Micronization of peas did not influence (P > 0.10) apparent ileal (Table 3) and total tract P digestibility. Total tract digestibility of P in BRP and BMP averaged 58%. There are no reports in the literature on the effect of micronization on P digestibility. The lack of effect of micronization on P digestibility is consistent with P retention and P excretion in feces and urine results. However, in a previous study, we showed significant improvements in P utilization in growing pigs fed similar diets as in the current study (Zhang et al., 2003). It was expected that micronization would improve P digestibility due to its ability to disrupt cell wall components. Therefore, the effect of micronization on nutrient utilization is contradictory and requires further evaluation. Supplementing BRP and BMP diets with a multi-enzyme blend improved (P < 0.05) apparent ileal P digestibility by 12 and 21%, respectively (Table 3). Likewise, enzyme supplementation improved (P < 0.05) apparent total tract digestibility of P in BRP diet (66 vs. 57%) and BMP diet (67 vs. 69%). The enzyme blend used in the current study was similar to that used in the study by Zhang et al. (2003). Thus, the current data are consistent with those of Zhang et al. (2003), who reported a reduction in P excretion when pigs are fed micronized peas with or without enzyme supplementation.

	IMPLICATIONS

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The results of this project show that formulating pig grower diets with micronized peas improved amino acid digestibility, whereas enzyme supplementation regardless of micronization improved phosphorus retention. The results support the proposition that careful dietary manipulations can be used effectively to minimize the impact of pork production on the environment.

	Footnotes

 
¹ Manitoba Livestock Manure Management Initiative, Manitoba Pulse Growers Association, and Agri-Food Research and Development Initiative provided funding for this research. We are grateful to B. A. Slominski and Canadian Biosystems Inc. for providing the enzyme cocktail used in the current project, M. Zhang, R. Stuski, O. F. Omogbenigun, and S. Kawadza for helping with animal care and sample collection, and J. Chalmers, Veterinary Laboratory, Manitoba Agriculture and Food, for assistance with urine phosphorus analysis.

² Corresponding author: martin_nyachoti{at}umanitoba.ca

Received for publication September 2, 2004. Accepted for publication April 24, 2006.

	LITERATURE CITED

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