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ANIMAL NUTRITION |
* University of Illinois, Department of Animal Sciences, Urbana 61801;
and
Agri-King Inc., Fulton, IL 61252; and
Prince Agri Products, Quincy, IL 62306
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Abstract |
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 Top Abstract INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Key Words: digestibility • enzyme • growing pig • nutrient
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INTRODUCTION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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showed that commercial products of β-glucanase varied in their effects on digestibility of DM, energy, and CP. It seems the most likely application would be in weanling pig diets in which pancreatic enzyme production may be limiting (Lindemann et al., 1986). Exogenous enzymes seem less effective in older pigs (Thacker et al., 1988, 1989, 1992; Baas and Thacker, 1996; Nyachoti et al., 2006), because older pigs are more able to digest fiber than younger pigs. Exogenous enzymes were also more effective in chick diets based on poorly digested cereals (Classen et al., 1995; Scott et al., 1998a,b), which contain more fiber (Fernandez and Jørgenson, 1986; Noblet and Bach-Knudsen, 1997; Le Goff et al., 2002).
Previous studies (Thacker et al., 1990; Li et al., 1996; Zijlstra et al., 2004) on supplying carbohydrases in pig diets were mainly focused on barley, wheat, or rye-based diets. Gaining an advantage from the use of exogenous enzymes has been more challenging for corn-soybean meal (SBM) diets due to decreased fiber concentrations and high nutrient digestibility. Corn and SBM are known to contain 10 and 22% of nonstarch polysaccharides, respectively (CVB, 1998). In conjunction with the variation in nutrient composition of corn (Sullivan et al., 1989; Burgoon et al., 1992; Summers, 2001) and SBM (Grieshop et al., 2003), there may be potential to improve digestibility of corn-SBM diets with exogenous enzymes. A novel β-glucanase-protease enzyme blend product (EBP) has been developed to improve performance of grower-finisher pigs fed a corn-SBM-based diet, in which exogenous enzymes have not often been shown to be effective. The objective of this experiment was to investigate the effect of EBP on digestibility of energy and nutrients in the small intestine, hindgut, and overall gastrointestinal tract.
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MATERIALS AND METHODS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Experimental procedures were approved by the Institutional Animal Care and Use Committee of the University of Illinois.
Fifteen growing barrows (22.5 ± 0.5 kg of BW, PIC-337 sire x Camborough-22, PIC dam) were housed in galvanized metal metabolism crates (0.86 x 1.60 m) with mesh floors in a power ventilated, environmentally controlled room. Pigs were able to move about freely, turn around, and lie down in the crates. Each crate was equipped with a low-pressure drinking nipple, a feeder, and a screen for collecting feces. Room lights were illuminated for 24 h/d, and the room temperature was controlled to approximately 22°C.
Surgical Procedures and Animal Care
The pigs had 7 d to adapt to their surroundings before the surgery. Pigs had ad libitum access to feed during this period. Feed was withheld from each animal 12 h before surgery, but the pigs had continuous access to water. Pigs were surgically fitted with a simple T-cannula at approximately 10 cm cranial to the ileo-cecal junction, according to procedures adapted from Sauer et al. (1983).
The cannulation site was cleaned daily with soft detergent and warm water during the entire experiment. Also, a zinc oxide-lanolin-based cream (Desitin, Pfizer Inc., New York, NY) was applied daily to the flank area to minimize irritation and to maintain skin integrity during the entire experiment. The pigs were fed 2 meals daily until full feeding was achieved 4 d after surgery. A period of 10 d was allowed for surgical recovery before the beginning of the experiment.
Experimental Design, Enzyme Product, and Diets
Twelve of the 15 cannulated pigs (38.2 ± 1.7 kg of BW at the beginning of period 1) were blocked by previous feed intake into 3 groups with 4 pigs each. Three extra pigs were available to be used in the event that pigs were removed from the study, but none was used. Within each group, pigs were randomly assigned to 1 of 4 treatments in a 4 x 4 Latin square design, with pigs and periods as the main factors. The 4 treatments were basal diet (Basal), Basal + 0.05% of EBP (0.05% EBP), Basal + 0.10% of EBP (0.10% EBP), and a hydrolyzed casein diet (Casein). The Casein contained hydrolyzed casein at a 10% level (as-fed basis) as the sole protein source and was included to enable the calculation of standardized ileal digestibility of CP and amino acids. The Basal consisted mainly of corn and SBM and was formulated to provide 3.36 Mcal of ME/kg and 1.1% total lysine (Table 1) to meet or exceed the nutrient requirements (NRC, 1998). The EBP (Agri-King Inc., Fulton, IL) contained β-glucanase activity (guaranteed not less than 660,000 β-glucanase units/kg) and protease activity (guaranteed not less than 22,000 hemoglobin units/kg). One β-glucanase unit liberates 1 mM reducing sugar (glucose equivalence) per minute. One hemoglobin unit of protease produces, in 1 min, a hydrolysate, in which absorbance at 275 nm is equal to that of a solution containing 1.1 mg/mL of tyrosine in 0.006 N hydrochloric acid. The manufacturer’s suggested usage of the EBP in growing pig diets is 0.05%. The 0.05% EBP and 0.10% EBP were made by mixing 0.05 and 0.10% of the EBP into the Basal, respectively, on an as-fed basis. The 4 chromic oxide diets were made by mixing 0.35% of chromic oxide (as-fed basis) into the 4 regular diets (Basal, 0.05% EBP, 0.10% EBP, and Casein). Both the regular and chromic oxide diets were sampled at the feed mill when the diets were mixed and at the feeders during period 4 of the experiment. Because the analyzed nutrient contents of the diets sampled at both the feed mill and the feeder were similar, the nutrient contents of the diets sampled at the feed mill were used in calculation of digestibility. The analyzed nutrient contents of the 4 regular diets sampled at the feed mill are listed in Table 2.
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The daily feed intake of pigs allowed ad libitum consumption before the beginning of the experiment ranged from 1,725 to 2,400 g/d. To ensure that all pigs could consume the feed allowance, the feed intake of all 4 pigs within each square during the first period was restricted to 85% of the previous intake of the pig with the lowest intake (i.e., 1,460, 1,820, and 1,980 g/ d, respectively). The feeding level was increased by 100 g/d in each subsequent period to accommodate the increased nutrient needs due to growth of the pigs. The feeding levels were 3.3 to 4.3 times the maintenance energy requirement (106 kcal of ME per kg of BW0.75; NRC, 1998) in the first period and 2.7 to 3.1 times maintenance in the last period. Pigs were fed 2 equal meals daily, at 12-h intervals. Water was available free choice. Spilled feed was collected and, unless it was heavily contaminated with feces, was returned to the feeder.
There were 4 periods in the experiment, and each pig received different treatment diets in each period. Each period was 14 d in duration. The first 4 d of each period were the diet adaptation phase. The chromic oxide diets were fed at the beginning and end of fecal collection to identify when to begin and end the collection of feces. Pigs were fed the diets containing chromic oxide in the morning meal of d 5. After that, the pigs were still fed the regular diets. Approximately 24 to 36 h later, the chromic oxide appeared in the feces, after which the feces were collected twice daily at 12-h intervals. Fecal collections were conducted from d 6 to 11. In the morning meal on d 10 of each period (120 h after the first feeding of chromic oxide), pigs were fed the diets containing chromic oxide until the end of the period. Again, approximately 24 to 36 h later, chromic oxide appeared in the feces, and collection of feces was stopped. The feces of the 5-d collection were pooled by pig. Fresh feces were frozen at –20°C.
The collection phase of ileal digesta was conducted on d 13 and 14. Ileal effluents were collected continuously for the same 12-h interval. Ileal digesta were collected by attaching polyethylene tubing (5 x 25 cm; Rand Materials Handling Equipment Co. Inc., Pawtucket, RI) to the cannula barrel with a cable tie. There was no chemical preservative in the tubing. The tubing was changed at least once every hour. As soon as ileal digesta was dumped into a container, it was placed in a freezer at –20°C for storage. Ileal digesta samples were pooled by pig and frozen at –20°C until the end of each collection period.
Chemical Analyses
Fresh feces of each pig were weighed. All fecal and ileal samples were thawed and mixed for homogeneity. A subsample was taken for chemical analyses. Fecal, ileal, and feed samples were dried at 60°C and ground to an 800-µm particle size before analyses. The contents of DM, ADF, NDF, total dietary fiber, CP, ether extract, ash, Ca, P, Mg, K, Na, Fe, Cu, Zn, Mn, S, Cl, and pH were determined by the methods of the AOAC (2002). Hemicellulose was calculated as the difference between NDF and ADF. Soluble dietary fiber and insoluble dietary fiber were analyzed according to the method described by Prosky et al. (1992). Amino acids were determined by the method of the AOAC (2002), but using an Adsorbosphere Opa HPLC precolumn (Alltech, 2000). Tryptophan was determined by an HPLC method using 7.5 N NaOH at 103 kPa for 16 h rather than barium hydroxide. Energy was measured using a bomb calorimeter (Model 1108, Parr Instrument Company, Moline, IL). Starch was measured by the method of Trinder (1969). Acid-hydrolyzed fat was determined by the method of Oser (1965). Chromium was analyzed by the method developed by Williams and Iismaa (1962) and Binnerts et al. (1968). Ammonia-nitrogen was determined by the method of Peters et al. (2003). The VFA were analyzed by capillary electrophoresis using a method adapted from that of Cancalon (1993).
Calculations of Digestibility
The fecal digestibility was determined by the equation described by Adeola (2001). The apparent ileal digestibility, endogenous amino acid loss, and standardized ileal digestibility were determined by the equations described by Smiricky et al. (2002). The endogenous amino acid loss used to calculate the standardized ileal digestibility in a square (group) was the average of the 4 pigs in the square.
The digestibility of nutrients in the hindgut was calculated by using the following equations:
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where FW = the dried fecal weight (kg of DM); NCf = the nutrient content in feces (% of DM); DMI = the DM intake (kg); NCF = the nutrient content in feed (% of DM); and AID = apparent ileal digestibility (%).
Statistical Analyses
Data were analyzed by the GLM and UNIVARIATE procedures (SAS Inst. Inc., Cary, NC) to test for homogeneity, normality, and outliers. Data were then analyzed by the MIXED procedure of SAS with groups, pigs, periods, and diets in the model. Means were reported as least squares means. Treatment comparisons were made using the following orthogonal contrasts: 1) effect of EBP [(0.05% EBP + 0.10% EBP)/2 vs. Basal] and 2) effect of level of EBP (0.05 vs. 0.10% EBP), with an 
level of 0.05 used to determine statistical significance.
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RESULTS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The calculated β-glucanase activities were 330 and 660 β-glucanase units/kg of diet, and the calculated protease activities were 11 and 22 hemoglobin units/ kg of diet for 0.05% EBP and 0.10% EBP, respectively. The contents of DM, GE, CP, total dietary fiber, and indispensable amino acids among Basal, 0.05% EBP, and 0.10% EBP were similar after manufacturing (Table 2
), indicating that the EBP did not change the main nutrient composition of the diets.
Periods
The fecal digestibility and apparent ileal digestibility of DM, CP, and energy, as well as apparent ileal digestibility of amino acids, were decreased (P < 0.001) in period 1 compared with periods 2, 3, and 4 (data not shown). Although pigs were allowed 10 d for recovery after surgery with 4 additional days for dietary adaptation, this may not have been sufficient time for recovery from surgery. However, digestibility values did not vary among periods 2, 3, and 4, indicating the effect of age of grower-finisher pigs on nutrient digestibility was limited.
Feed Intake and Fecal Characteristics
All pigs consumed the allotted feed, gained weight, had no diarrhea, and appeared healthy during the experiment. Feed intake did not differ among the treatments (Table 3). Dried fecal weight was decreased (P < 0.001) in the pigs fed the EBP than in the pigs fed the Basal. Fecal pH and concentrations of DM, ammonia, propionic acid, butyric acid, and valeric acid did not differ between the pigs fed the EBP and the Basal. Acetic acid was greater (P < 0.001) in feces from the pigs fed the EBP than from those fed the Basal.
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The fecal digestibility of DM, OM, energy, CP, and ash was greater (P < 0.05) for the pigs fed the EBP than the pigs fed the Basal (Table 4), whereas the fecal digestibility of these items did not differ between the pigs fed 0.05% EBP and 0.10% EBP. Pigs fed the EBP had greater fecal digestibility (P < 0.05) of total dietary fiber and acid-hydrolyzed fat than pigs fed the Basal. Within total dietary fiber, the fecal digestibility of insoluble dietary fiber was increased (P < 0.05) by the EBP, whereas the fecal digestibility of soluble dietary fiber was not affected by the EBP. Almost all starch was digested over the whole digestive tract, and there was no difference in fecal digestibility of starch among treatments. The fecal digestibility of Ca, P, Mg, S, Fe, and Cu also was increased (P < 0.05) when pigs were fed the EBP.
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The apparent ileal digestibility of DM, energy, starch, and nonfiber carbohydrate did not differ among treatments (Table 5). The pigs fed EBP had decreased (P < 0.05) apparent ileal digestibility of CP than the pigs fed the Basal. The apparent ileal digestibility of NDF and hemicellulose of the pigs fed the EBP was greater (P < 0.05) compared with that of the pigs fed the Basal. The apparent ileal digestibility of acid-hydrolyzed fat in the pigs fed the EBP tended to be greater (P = 0.051) than in those fed the Basal. Ileal digestibility of most amino acids was not affected by treatment, but the EBP reduced the apparent (Table 5) and standardized (Table 6) digestibility of methionine, alanine, and serine (P < 0.05) and arginine, glycine, threonine, and valine (P < 0.05 for apparent digestibility only).
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There was no difference among treatments in the difference between fecal and ileal digestibility except for hemicellulose, which was decreased (P < 0.05) for the pigs fed the EBP compared with pigs fed the Basal (Table 7). The difference between fecal and ileal digestibility of NDF for the pigs fed the EBP tended to be decreased (P = 0.050) compared with the pigs fed the Basal. For the digestibility based on the nutrients entering the hindgut (Table 8), the pigs fed the EBP had greater values (P < 0.05) for CP and DM digestibility compared with pigs fed the Basal treatment.
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DISCUSSION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The carbohydrate of cereal grains includes sugars, starch, and cell wall polysaccharides. The endosperm cell walls of barley, oat, wheat, rye, sorghum, corn, and triticale are composed mainly of arabinoxylans and β-glucans (Henry, 1985; Bedford, 1995; de Lange, 2000). The current results showed that the fecal digestibility of total dietary fiber and insoluble dietary fiber was improved by supplying the EBP. The total dietary fiber consists of soluble materials such as β-glucans, arabinoxylans, pectins, and gums, and the insoluble galacto-and glucomannans, cellulose and lignin (Asp et al., 1983; Bedford, 1995; Souffrant, 2001). It is unclear why the EBP increased digestibility of insoluble rather than soluble dietary fiber, because the substrate of the β-glucanase is soluble.
The increase in ileal digestibility of NDF and hemicellulose when the EBP was added to the diet, with no change in fecal digestibility, indicates that the enzyme product may have shifted some of the digestion of these nutrients from hindgut fermentation to small intestinal enzymatic digestion. Such a change would avoid the fermentative loss of energy as heat and methane and presumably increase the energetic efficiency of fiber use.
The cell walls enclose starch, protein, and lipids. Supplying enzymes to enhance breakdown of the components of these cell walls can increase exposure of the nutrients in cells to the digestive enzymes. However, because of the difference of ingredients, age of pigs, and enzyme products, the effectiveness of supplying enzyme in pig diets may be different. Some studies showed that supplying β-glucanase increased ileal digestibility of DM, energy, CP or fibers (Taverner and Campbell, 1988; Inborr and van der Meulen, 1993; Yin et al., 2001a,b), and fecal digestibility of fibers (Yin et al., 2001a), whereas other studies showed supplying β-glucanase did not improve ileal digestibility of DM, energy, or CP (Graham et al., 1989; Nyachoti et al., 2006) and fecal digestibility of DM, energy, CP, or fiber (Taverner and Campbell, 1988; Graham et al., 1989; Nyachoti et al., 2006). These previous studies focused on the enzyme effect on diets based on fibrous cereals such as barley and wheat. The effect of enzyme on corn-SBM-based diet has not previously been well addressed. The current results showed that apparent ileal digestibility of NDF and hemicellulose and the fecal digestibility of DM, energy, CP, and total dietary fiber were improved by the EBP. This indicates that even under the difficult conditions of corn-SBM diet and pigs several months old, supplying exogenous enzymes still have the potential to improve digestibility of DM, energy, and fibers. However, because the capacity of degrading fiber is different for the enzymes obtained from different microbes (Nevins et al., 1978), the effectiveness of various commercial enzyme products was different (Baas and Thacker, 1996). The enzyme product used in the current study was an effective fiber-degrading enzyme product.
The effectiveness of an enzyme may be affected by many factors, such as the activity of the enzyme, the type and concentration of substrate, particle size, pH, temperature, and time of the feed staying in the small intestine. The calculated β-glucanase activities were 330 and 660 units/kg of diet for 0.05% EBP and 0.10% EBP, respectively. The levels of β-glucanase in previous studies varied from 10 (Graham et al., 1986) and 100 (Graham et al., 1989) units per kilogram of diet to 600 β-glucanase units/kg of diet (Yin et al., 2001a,b). In this case, the lower level of EBP tested (0.05%) seemed to be sufficient to achieve maximum effect.
The apparent ileal digestibility of acid-hydrolyzed fat was increased by supplying EBP, whereas Graham et al. (1989) reported no effect. The increase in fecal digestibility of acid-hydrolyzed fat by supplying EBP was caused by increased digestibility in the small intestine. Because the fecal digestibility and apparent ileal digestibility of ether extract did not differ, the increase of apparent ileal digestibility of acid-hydrolyzed fat may be because EBP digested cell wall materials and released the lipids from the cell membranes, which constitute most of the difference between ether extract and acid-hydrolyzed fat. The fecal digestibility of minerals including Ca, P, Mg, S, Cl, Fe, Cu, and Mn was improved by the EBP and may result from the EBP releasing minerals bound to or in the cell wall.
The EBP did not increase ileal digestibility of amino acids. We cannot explain why the standardized ileal digestibility of methionine, alanine, and serine was reduced by the EBP. In previous studies, the effect of supplying exogenous enzymes on ileal digestibility of protein was contradictory in pigs. Protease treatment of SBM had no effect on ileal digestibility of CP and amino acids in newly weaned pigs (Caine et al., 1997a,b). Li et al. (1996) demonstrated that β-glucanase supplementation increased the ileal digestibility of β-glucans, CP, and the majority of amino acids in a hulless barley-SBM diet. Yin et al. (2001b) showed that supplying β-glucanase and xylanase in weaned pig diets increased the apparent ileal digestibility of lysine, glycine, threonine, histidine, and valine. Yin et al. (2001a) obtained similar results from another study, in which β-glucanase improved apparent ileal digestibility of CP and of most amino acids. The current study showed that the supplementation of protease in the corn-SBM-based growing pig diets was ineffective. It may be because the protease activities in the EBP (11 and 22 hemoglobin units/kg of diet for 0.05% EBP and 0.10% EBP, respectively) were decreased compared with that in a previous study (500 units/kg in pig diets; Yin et al., 2001a).
The feeding levels were 3.3, 4.1, and 4.3 times maintenance in the first period and 2.7, 3.0, and 3.1 times maintenance in the fourth period. The degree of restriction increased as the pigs grew. The feeding levels did not affect the fecal digestibility or apparent ileal digestibility of DM, CP, or energy, as well as apparent ileal digestibility of all amino acids, which agrees with the previous results of Haydon et al. (1984), Albin et al. (2001), Gómez et al. (2002), and Moter and Stein (2004). Other studies showed an inverse relationship between feeding levels and fecal digestibility of DM and CP (Rao and McCracken, 1991) and ileal digestibility of CP and most amino acids (Moter and Stein, 2004). Because the comparisons were at different levels of restriction rather than restriction to ad libitum, it is possible the response to the EBP may be greater in ad libitum pigs.
In conclusion, the fecal digestibility of DM, OM, energy, CP, total dietary fiber, acid-hydrolyzed fat, Ca, and P and the apparent ileal digestibility of hemicellulose and NDF were improved, but standardized ileal digestibility of amino acids was not increased by the EBP in corn-SBM-based growing pig diets. The EBP used in the study was effective. Supplying 0.05% was as effective as 0.10% EBP in this study with limit-fed pigs.
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Footnotes |
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2 Corresponding author: jepettig{at}uiuc.edu
Received for publication May 10, 2007. Accepted for publication March 3, 2008.
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LITERATURE CITED |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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