Effect of Enzyme Addition on the Performance and Gastrointestinal Tract Size of Chicks Fed Lupin Seed and Their Fractions A. Brenes,*,1 R. R. Marquardt,† W. Guenter,† and A. Viveros‡ *Instituto de Nutricio´n y Bromatologı´a, Facultad de Farmacia, Ciudad Universitaria, 28040 Madrid, Spain; †Department of Animal Science, University of Manitoba, Winnipeg, Manitoba, Canada, R3T 2N2; and ‡Departamento de Produccio´n Animal, Facultad de Veterinaria, Ciudad Universitaria, 28040 Madrid, Spain effects were partially counteracted by the action of enzymes. In the third experiment, increasing concentration of whole lupins (15, 35, and 45%) in broiler chicken diets caused a depression in the performance of birds fed 35 and 45% whole lupins as compared to those fed the wheat-soy diet. In contrast, 15% lupins improved weight gains compared to that obtained with the nonlupin diets. The lower content of lupins in the diet also had no or little effect on other performance values compared to the control group, whereas 35 and 45% dietary lupins tended to have negative effects. Likewise, increasing lupin content in the diet produced an enlargement in the relative size of several sections of the gastrointestinal tract. Enzyme supplementation of lupin diets improved weight gain (5.5%) and feed consumption (3.8%) with the values being similar to those obtained with the wheat-soy diet. Moreover, the enzymes also reduced the relative size of digestive organs from 5.3% for pancreas to 22.2% for crop. In summary, lupins appear to contain fibrous components that reduce the performance of the birds and increase the size of the gastrointestinal tract. The addition of enzymes counteracted these negative effects in birds fed whole, dehulled lupins and lupin hull diets.
ABSTRACT Three experiments were conducted to study the effects of adding a crude enzyme preparation to diets containing whole, dehulled lupins and lupin hulls on performance, dry matter retention (DMR), AME, apparent protein digestibility (APD), and size of gastrointestinal tract of Leghorn and broiler chicks. In the first experiment, Leghorn chicks fed diets containing up to 70% whole lupins showed a depression in the performance. Progressive decreases in DMR (up to 30.2%), AME (up to 6.5%), and APD (up to 6.5%) and an increase in the relative gizzard weight (18.8%) were observed with increasing concentration of lupins (23.1, 46.9, and 70%) in the diet. Enzyme supplementation of diets containing lupins significantly improved the performance of the chicks. DMR and AME were improved by 4.2 and 3.1, respectively, and gizzard size was reduced (7.1%) by addition of the enzymes. In the second experiment, addition of 11.2 and 22.4% of lupin hulls to a dehulled lupin diet resulted in a dramatic depression in chick performance, with values ranging from 6.3% for feed consumption to 60.5% for fed to gain ratio, and an increase in the relative organ weight (up to 29.9%) and length (35.6%). These
(Key words: lupin, dehulling, hull, enzyme, chicken) 2002 Poultry Science 81:670–678
sions up to 30%; however, these levels increase the viscosity of intestinal content, increase litter moisture content, alter the profile of intestinal microflora, and definitely produce growth depression (Bedford and Schulze, 1998; Rubio et al., 1998). The fiber content of lupin seed may also contribute to the growth depression observed in poultry. Van Barneveld (1997) and Bach Knudsen (1997) reported that sweet white lupin seeds contained 24.1% hull and that consisted mainly of hemicellulose, cellulose, and lignin. Carre et al. (1985) and Evans et al. (1993) showed that lupin kernels contain high amounts of nonstarch polysaccharides (NSP), with the major polysaccha-
INTRODUCTION Low-alkaloid lupin seeds are a potential source of good quality protein supplements for monogastric animals such as poultry and pigs, and they are also free of other antinutritional compounds such as trypsin and hemagglutinins (Schoeneberger et al., 1983). In raw form, these seeds can be tolerated as 15 to 25% of the diet without detrimental effects on growth or other production parameters (Centeno et al., 1990; Roth Maier and Kirchgessner, 1994a). Other studies have indicated even greater inclu-
2002 Poultry Science Association, Inc. Received for publication July 25, 2001. Accepted for publication November 30, 2001. 1 To whom correspondence should be addressed:
[email protected].
Abbreviation Key: APD = apparent protein digestibility; DL = dehulled lupin diet; DMR = dry matter retention; E = enzyme preparation; NSP = nonstarch polysaccharides; WL = wheat-lupin; WS = wheat-soy.
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ENZYME ADDITION TO LUPINS IN CHICKEN DIETS
ride being β (1-4)-galactan (a mixture of D-galactose, Larabinose, L-rhamnose, and galacturonic acid). These NSP are thought to have adverse effects on diet intake and digestibility of nutrients (Carre et al., 1985; Ferraz de Oliveira, 1998; Naveed et al., 1999). Lupin seeds also contain significant levels of oligosaccharides of the raffinose family (Brenes et al., 1989; Saini, 1989; Van Kempen and Jansman, 1994). These oligosaccharides appear to be indigestible in the small intestine of monograstric animals due to the lack of α-galactosidase in the intestinal mucosa (Carre et al., 1985). Total β-galactosides in different lupin species range from 7 to 12% (Trugo and Almeida, 1988; Muzquiz et al., 1989). Stachyose is reported as the main sugar in lupin seeds, accounting for up to 50% of the total sugars (Van Kempen and Jansman, 1994). Positive production responses from the addition of supplementary enzymes to poultry diets containing lupin have been reported by Brenes et al. (1993a), Annison et al. (1996), and Ferraz de Oliveira (1998). However, no effect or a negative effect has been reported by Alloui et al. (1994), Roth Maier and Kirchgessner (1995), and Eder et al. (1996). Preliminary, short time studies by Marquardt et al. (1996) demonstrated that the inclusion of enzymes and limited amounts of lupins in the diet of poultry increases chick performance over that obtained with a conventional cereal-soybean meal based diet. They did not, however, establish the mechanism of the beneficial effects of both enzymes and lupins and only limited information was provided on the beneficial effects of adding limited amounts of lupins to conventional diets. The objectives of these experiments were to more conclusively establish the effects on chick performance of crude enzymes when added to diets containing different concentrations of whole, dehulled lupins and lupin hulls. In addition, the effects of the diets on nutrient digestibilities and gastrointestinal tract size were determined.
MATERIALS AND METHODS General Experimental Procedures The lupin (Lupinus albus) cultivar, Amiga, used in the experiments was grown in the province of Saskatchewan2 and did not contain any of the lupin alkaloids (Brenes et al., 1993a). Dehulling was carried out by a commercial pea splitter. The process involved the cracking of the seed with a plate grinder followed by mechanical removal of the hulls from the cotyledon fraction (dehulled lupin). Hulls were further purified by air classification. The dehulled lupins and the hulls were estimated to be more than 95% pure. The crude enzyme preparations used were: Energex威 (multiple-enzyme complex for a broad range of carbohydrates), Bio-Feed Pro威 (proteolytic en-
2
Seedtec Ltd., Qu Appelle, SK, S0G 4A0 Canada. Novo Nordisk A/S, Bagsvaerd, Denmark. Parr Instrument Co., Moline, IL 61265.
3 4
671
zyme), and Novozyme威 SP-230威 (high in β-galactosidase activity).3 The enzyme activities per gram crude product as determined by the manufacturers were as follows: Energex威, 75 fungal β-glucanase units, 150,000 hemicellulase units, 10,000 pectinase units, and 400 endoglucanase units; Bio-Feed Pro威, 150,000 protease units; Novozyme威 SP-230威, 500 α-galactosidase units. The mixed crude enzyme preparation (E) consisted of Energex, Bio-Feed Pro, and Novozyme at 0.10% each and was used in all three experiments. This enzyme mixture has been shown to produce a beneficial response when added to diets containing a high content of lupin seeds (Brenes et al., 1993a). All diets were given in mash form and the birds had free access to water and feed throughout the entire experiment. The experimental diets were formulated to meet or exceed the minimum National Research Council (1994) requirements for Leghorn and broiler chickens. Chick performance was measured in terms of feed consumption, weight gain, and feed to gain ratio. Seeds were analyzed for alkaloids according to the procedure of Mu´zquiz et al. (1989).
Experiment 1 The objective of this study was to determine the optimal amount of lupin and enzymes that could be used in a diet to achieve maximal performance and their correspondingly effect on nutrient digestibility and gizzard and crop size. All chicks were fed commercial chick starter diets during the 7 d pre-experimental period. At 7 d of age, the birds were randomly distributed in Petersime batteries brooder with six birds per pen and six pens per treatment. The experimental diets are shown in Table 1. The 288 male Leghorn chicks were randomly distributed among the following eight treatments: 1) wheat-lupin (WL) + wheat-soy (WS) (0 + 100); 2) WL + WS (0 + 100) plus E; 3) WL + WS (67:33 mixture); 4) WL + WS (67:33) plus E; 5) WL + WS (33:67); 6) WL + WS (33:67) plus E; 7) WL + WS (100 + 0), and 8) WL + WS plus E. The actual lupin concentration in Diets 1 and 2, 3 and 4, 5 and 6, and 7 and 8 were 0, 23.1, 46.9, and 70%, respectively. The experimental design was a completely randomized block with a factorial arrangement of treatment: 4 (four diets with different concentrations of lupins) × 2 (two enzymes doses, 0 and 0.3%). The experimental diets were fed to birds from 7 to 21 d of age. The chicks were killed (12 randomly selected birds) at 22 d, and the crop and the gizzard were removed. Feed and excreta samples were collected 3 d before the last day of the experiment for determinations of dry matter retention (DMR), AME, and apparent protein digestibility (APD). Chromic oxide was analyzed by the procedure of Williams et al. (1962). Dry matter was determined as described by AOAC (1984). DMR was calculated as described by Marquardt et al. (1979). Gross energy was determined using a Parr adiabatic oxygen bomb calorimeter.4 APD was determined as described by Rotter et al. (1989).
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BRENES ET AL. TABLE 1. Composition of diets used in Experiments 1 and 2 Experiment 1 Ingredients Wheat1 Soybean meal (47.1% CP) Lupin (31.5% CP) Dehulled lupin (39.5% CP) Starch Hulls2 Sunflower oil Dicalcium phosphate Calcium carbonate DL-Methionine Vitamins3 Minerals4 Chromic oxide Enzyme mix5 Calculated analysis Crude protein (N × 6.25) ME, kcal/kg Lysine,% Methionine + cysteine,% Calcium,% Available phosphorus,%
Wheat-soy
Experiment 2
Wheat-lupin
0% Hulls
11.2% Hulls
22.4% Hulls
63.32 30.00 – – – – 2.00 1.24 1.79 – 1.00 0.35 0.30 −/+
17.84 – 70.00 – – – 8.00 1.14 1.34 0.03 1.00 0.35 0.30 −/+
(%) – – – 65.00 29.59 – 1.00 1.17 1.42 0.17 1.00 0.35 0.30 −/+
– – – 62.20 18.39 14.00 1.00 1.17 1.42 0.17 1.00 0.35 0.30 −/+
– – – 59.40 7.19 28.00 1.00 1.17 1.42 0.17 1.00 0.35 0.30 −/+
23.71 2,896 0.95 0.70 1.17 0.40
24.77 2,922 1.19 0.60 0.84 0.42
24.50 2,978 0.98 0.60 0.80 0.40
24.50 2,500 0.98 0.60 0.88 0.40
24.50 2,160 0.98 0.60 0.95 0.40
Katepwa variety, protein content 15.4% (N × 6.25). Hulls contained some dehulled lupin. The 14 and 28% hull fractions contained approximately 11.2 and 22.4% hulls and 2.8 and 5.6% dehulled lupin, respectively. Therefore, amount of dehulled lupin added to each diet was 65%.The estimated percentage of hulls in lupin was approximately 16%. 3 Vitamin supplied the following per kilogram of diet: vitamin A, 8,250 IU; cholecalciferol, 1,000 IU; vitamin E, 11 IU; vitamin K, 1.1 mg; vitamin B12, 11.5 µg; riboflavin, 5.5 mg; Ca panthotenate, 11 mg; niacin, 53.3 mg; choline chloride, 1,020 mg; folic acid, 0.75 mg; biotin, 0,25 mg; delaquin, 125 mg; DL-methionine, 500 mg; amprol, 1 g. 4 Mineral mix supplied the following per kilogram of diet: Mn, 55 mg; Zn, 50 mg; Fe, 80 mg; Cu, 5 mg; Se, 0.1 mg; I, 0.18 mg; NaCl, 2,500 mg. 5 Enzyme added to the diets were 0.1% each of Energex威 + Bio-Feed Pro威 + Novozyme威 (Novo Nordisk A/ S, Bagsvaerd, Denmark). 1 2
Experiment 2 The objective of Experiment 2 was to determine the influence of lupin hulls and enzymes on chick performance. The experimental procedure was as outlined for Experiment 1, and the diets are shown in Table 1. Two hundred sixteen Leghorn chicks (216) were randomly distributed among the following six treatments: 1) dehulled lupin diet (DL); 2) DL plus E; 3) DL plus hulls (11.2%); 4) DL plus hulls (11.2%) plus E; 5) DL plus hulls (22.4%), and 6) DL plus hulls (22.4%) plus E. In the diets, the lupin hulls were partially substituted for by starch, maintaining the protein concentration constant and incorporating the same level of fat (1%). The experimental design was a completely randomized block with a factorial arrangement of treatments: 3 (three lupin diets) × 2 (two enzymes doses, 0, 0.3%).
Experiment 3 The objectives of this study were to determine if a similar pattern of response to the inclusion of different concentrations of lupin and enzymes in the diet could be obtained if broiler chicks were fed the diets over the entire productive cycle rather than for only a 14-d-period as carried out in Experiments 1 and 2.
Day-old broiler chicks (1,440), which had been vaccinated (Marek’s), were purchased from a commercial hatchery. Each treatment was replicated three times, and 60 birds were randomly assigned to each pen. Pens were located in an environmentally controlled house with lowintensity, continuous light. Starter (0 to 3 wk) and growerfinisher (3 to 6 wk) broiler diets of different inclusion levels of whole lupin (0, 15, 35, and 45%) were fed for 6 wk. The treatments were as follows: 1) WS; 2) WS plus E; 3) wheat-lupin 15% diet (WL 150); 4, WL 150 plus E; 5, wheat-lupin 35% diet (WL 350); 6, WL 350 plus E; 7, wheat-lupin 45% diet (WL 450) and 8, WL 450 plus E. The composition of the lupin starter and grower-finisher diets are shown in Table 2. Weight gain and feed consumption were determined at 5 wk of age due to a problem with the supply of additional lupin. For that reason, 20 chicks were randomly separated per treatment at 5 wk of age. These chicks were killed (14 per treatment) at 6 wk of age after 18 h of feed withdrawal, and the entire gastrointestinal tract and abdominal fat were removed and weighed. Residual digesta was removed from the digestive tract prior to weighing. The experimental design was a completely randomized block with a factorial arrangement of treatments: 4 (four lupin diets) × 2 (2 enzymes doses, 0, 0.3%).
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ENZYME ADDITION TO LUPINS IN CHICKEN DIETS TABLE 2. Composition of experimental starter and grower-finisher diets, Experiment 3 Starter diets Ingredients Wheat1 Lupin (31.5% CP) Soybean meal (47.1% CP) Fishmeal (60% CP) Sunflower oil L-Lysine DL-Methionine Dicalcium phosphate Calcium carbonate Vitamins2 Minerals3 Calculated analysis Crude protein (N × 6.25) ME, kcal/kg Lysine,% Methionine + cysteine,% Calcium,% Available phosphorus,%
0% Lupin
15% Lupin
35% Lupin
Grower-finisher diets 45% Lupin
0% Lupin
15% Lupin
35% Lupin
45% Lupin
(%) 62.18 – 27.00 1.30 5.00 0.03 0.15 1.29 1.70 1.00 0.35
56.21 15.00 17.50 2.00 5.00 0.07 0.20 1.15 1.52 1.00 0.35
44.15 35.00 8.00 2.00 6.50 0.08 0.27 1.13 1.52 1.00 0.35
38.67 45.00 3.00 2.00 7.00 0.08 0.30 1.08 1.52 1.00 0.35
72.55 – 18.00 – 5.00 0.04 0.14 1.34 1.58 1.00 0.35
64.06 15.00 11.00 – 5.50 0.05 0.18 1.27 1.59 1.00 0.35
53.01 35.00 1.00 – 6.50 0.09 0.25 1.22 1.58 1.00 0.35
43.67 45.00 – – 7.00 – 0.24 1.18 1.56 1.00 0.35
22.91 3,087 1.20 0.85 1.00 0.45
22.71 3,042 1.20 0.85 1.00 0.45
22.73 3,038 1.20 0.85 1.00 0.45
22.70 3,023 1.20 0.85 1.00 0.45
19.55 3,158 0.90 0.72 0.90 0.40
19.70 3,119 0.90 0.72 0.90 0.40
19.64 3,090 0.90 0.72 0.90 0.40
20.88 3,053 0.90 0.72 0.90 0.40
Katepwa variety, protein content 15.4% (N × 6.25). Vitamin supplied the following per kilogram of diet: vitamin A, 8,250 IU; cholecalciferol, 1,000 IU; vitamin E, 11 IU; vitamin K, 1.1 mg; vitamin B12, 11.5 µg; riboflavin, 5.5 mg; Ca panthotenate, 11 mg; niacin, 53.3 mg; choline chloride, 1,020 mg; folic acid, 0.75 mg; biotin, 0.25 mg; delaquin, 125 mg; DL-methionine, 500 mg; amprol, 1 g. 3 Mineral mix supplied the following per kilogram of diet: Mn, 55 mg; Zn, 50 mg; Fe, 80 mg; Cu, 5 mg; Se, 0.1 mg; I, 0.18 mg; NaCl, 2,500 mg. 1 2
Statistical Analyses The data (pen means) were subjected to analysis of variance using the general linear models procedure of SAS software (SAS Institute, 1986). Experiments 1, 2, and 3 were analyzed by ANOVA in 4 (lupin concentration) × 2 (enzyme concentration), 3 × 2, and 4 × 2 factorial arrangements of treatments, respectively, and single df contrasts were used to separate treatment means in the factorial experiments. The means of the data for the major effect of diet were subjected to analysis with linear, quadratic, and cubic polynomials.
RESULTS Experiment 1 Analysis of variance of the data demonstrated that in most comparisons there were significant main effects for lupin concentration in the diet and enzyme addition (Table 3). The percentage lupin in the diet had a slight effect on chick performance (Table 3). The weight gain achieved with 23.1% lupin (Diet 3) was superior (9%; P < 0.05) to that obtained with 70% lupins (Diet 4). Likewise, feed to gain ratio was increased (6%) in the chicks fed 70% lupins in comparison to 23.1 and 46.9% lupins. In addition, there was a progressive and dramatic decrease (P < 0.001) in DMR (up to 30.2%), AME (up to 6.5%), and APD (up to 6.5%) and an increase in the relative gizzard weight (18.8%) with increasing concentration of lupins in the diet (Table 3). In general, enzymes had a dramatic effect on the diets as they increased chick performance by 6 to 13% (Table 3). They also improved DMR and AME by 4.2 and 3.1%,
respectively, and reduced gizzard size 7.1%. Other effects were not significant (P > 0.05). The statistical analysis also showed three significant interactions for each of feed consumption, gizzard size, and AME. The individual values for these interactions are presented in the footnotes of Table 3. However, according to the procedure of Little (1981), these interactions are not emphasized because the percentage of total treatment sum of square for the lupin diet × E interactions were 16, 7 and 11% for feed consumption, gizzard relative weight, and AME, respectively. Therefore, interpretation of the data was relative to their main effects. Likewise, quadratic and cubic increases for relative weights of gizzard (up to 18.8%) and linear, quadratic, and cubic decreases for DMR (up to 30.2%), AME (up to 6.5%), and APD (6.5%) were observed in response to increasing lupin concentration in the diets.
Experiment 2 The results demonstrated that the partial substitution of 14 and 28% lupin hulls for starch in the diet resulted in dramatic decreases in weight gains (12.9 and 33.6%, respectively, P < 0.001) and increases in feed to gain ratios (22 and 60.5%, respectively, P < 0.001) compared to those obtained when chicks consumed a diet containing 65% dehulled lupin (Table 4). Feed consumption partially corrected for the effect of hulls in the feed (6.3% increase, P < 0.001). Also, the chicks partially adapted to increased concentrations of hulls in the diet by increasing size or length of all sections of the gastrointestinal tract with the effect being much greater at the higher hull inclusion rate. The values for increased organ size ranged from 5.6% for the gizzard to 20% for the jejunum and ileum, when the
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TABLE 3. Performance, crop and gizzard relative weight, dry matter retention (DMR), AME, and apparent protein digestibility (APD) of Leghorn chicks (7 to 21 d) fed enzymes supplemented diets containing lupin seeds, Experiment 1
Main effects
Weight gain
Feed consumption (g)
Diets1 WS + WL (100:0) WS + WL (67:33) WS + WL (33:67) WS + WL (0:100) Enzyme3 No enzyme Enzyme Pooled SEM Source of variation4 Lupin in diet (LD) Enzyme (E) LD × E Type of response due to amount of lupin in diet Linear Quadratic Cubic
Feed to gain ratio (g/g)
Relative weight Crop
Gizzard
(g/100 g body weight)
DMR
AME
APD
(%)
(kcal/kg)
(%)
116ab 123a (+6.0)2 121ab (+4.3) 113b (−2.6)
246 252 (+2.4) 251 (+2.0) 246 (0.0)
2.13b 2.07b (−2.8) 2.06b (−3.3) 2.19a (+2.8)
0.42 0.43 (+2.3) 0.47 (+11.9) 0.45 (+7.1)
2.82c 2.86c (+1.4) 3.03b (+7.4) 3.35a (+18.8)
74.64a 65.58b (−12.1) 58.76c (−21.3) 52.09d (−30.2)
3,073a 2,970b (−3.4) 2,925bc (−4.8) 2,874c (−6.5)
88.97a 86.87b (−2.4) 84.80c (−4.7) 83.17c (−6.5)
111b 125a (+12.6) 8.65
241b 256a (+6.2) 10.43
2.18a 2.04b (−6.4) 0.09
0.43 0.45 (+4.7) 0.10
3.12a 2.90b (−7.1) 0.27
61.49b 64.05a (+4.2) 1.27
2,915b 3,006a (+3.1) 48.16
85.36 86.54 (+1.4) 1.46
Probabilities 0.044 0.001 NS
NS 0.001 0.016
0.011 0.001 NS
NS NS NS
0.001 0.001 0.001
0.001 0.001 NS
0.001 0.001 0.032
0.001 NS NS
NS NS NS
NS NS NS
NS NS NS
NS NS NS
NS 0.001 0.001
0.001 0.001 0.001
0.020 0.021 0.005
0.005 0.008 0.001
Means within each column for diet or enzyme without a common letter are significantly different (P < 0.05). WS = Wheat-soy; WL = Wheat-lupin. The wheat-lupin diet contained 80% lupin, whereas the wheat-soy diet contained 0% lupin. 2 Values in brackets represent percentage increase or decrease relative to chicks fed diets containing WS + WL (100:0) and diet without enzyme. 3 Enzyme added to the diets were 0.1% each of Energex威 + Bio-Feed Pro威 + Novozyme威. 4 Individual values for significant LD × E interaction for feed consumption for Diets 1, 2, 3 and 4 without and with enzymes were as follows: 244 and 247, 240 and 251, 236 and 268, and 245 and 256, respectively. Corresponding values for gizzard relative weight were: 2.85 and 2.79, 3.57 and 3.13, 2.86 and 2.85, and 3.22 and 2.82. Values for AME were as follows: 3,009 and 3,138, 2,832 and 2,917, 2,976 and 2,964, and 2,843 and 3,007. The percentages of total treatment sum of square for the LD × E interaction were 16% for feed consumption, 7% for gizzard relative weight, and 11% for AME. a-d 1
diets contained 11.2% added hulls, and from 29.9% for the gizzard to 35.6% for the ileum when the diet contained 22.4% hulls compared to the diet with no hulls (P < 0.001). The statistical analysis did not show a significant interaction among the many main effects. Addition of enzymes also had a considerable effect on chick performance and digestive organ size or length. Improvements in weight gain, feed consumption, and feed to gain ratios with enzyme addition were dramatic at 22.7, 6.9, and 13.2%, respectively. The size of all intestinal organs appeared to decrease with enzyme treatment with the effects being observed for the gizzard (16.7%; P < 0.001), jejunum (6.5%; P < 0.012), ileum (5.8%; P < 0.05), and cecum (7.2%; P < 0.009).
Experiment 3 Analysis of variance of the data demonstrated that there were several significant main effects (P < 0.05) for enzyme treatment and lupin content of the diet (Table 5). In contrast, only the interaction with feed consumption was significant (P < 0.032) and accounted for only 22% of the total variance when the sums of squares were partitioned. The responses in weight gain and feed consumption to lupin content of the diet were also greatest for diets containing 15% lupins compared to those containing no lupin, 35 and 45% lupin (P < 0.001). The responses to lupin content of the diet were linear (P < 0.001) and quadratic (P < 0.038 to 0.040). The results further confirm that the
response to lupin content in the diet was not directly proportional to its content. The feed to gain ratio was affected by the inclusion of 15% lupin in the diet (P < 0.013) and increased when the inclusion rate was 30 and 45%. The feed to gain ratio was also affected linearly (P < 0.003) with increasing lupin content in the diets. In general, the relative size of different sections of the gastrointestinal tract increased when the lupin inclusion level increased in the diet (P < 0.05). The organs that were most affected by inclusion of 45 or 30% lupin in the diet were the crop (50.0 and 17.6%, increase), proventriculus (12.1 and 12.1% increase), gizzard (30.1 and 19.9%, increase), and the duodenum (12.0 and 12.0%, increase). Relative liver, pancreas, and abdominal fat content were not affected by lupin content of the diet (P > 0.05). The addition of an enzyme mixture to the different lupin diets increased feed intake (3.8%, P < 0.003) and weight gain (5.5%, P < 0.001) but did not affect the feed to gain ratio. The failure to obtain an interaction or interactions suggest that the response to enzyme treatment was essentially independent of the lupin diet. Enzyme treatment, also decreased (P < 0.05) the relative size of all sections of the intestinal tract except for that of the colon. The magnitude of decreases ranged from 22.2% for the crop to 6.9% for the gizzard. The relative sizes of the liver and pancreas also declined (P < 0.05) with enzyme addition to the diets; average values were 5.5 and 5.3%, respectively. In contrast, abdominal fat was increased by 9.5% (P < 0.05) by enzyme addition to the diet.
0.001 0.009 NS 0.001 0.050 NS 1
a-c
0.001 0.001 NS Source of variation Diet Enzyme Diet × enzyme
Means within each column for diet or enzyme without a common letter are significantly different (P < 0.05). The dehulled lupin diet contained 65% dehulled lupin. 2 Values in brackets represent percentage increase or decrease relative to chicks fed either dehulled lupin diet or diet without enzyme. 3 Enzymes added to the diets were 0.1% each of Energex威 + Bio-Feed Pro威 + Novozyme威 (Novo Nordisk A/S, Bagsvaerd, Denmark).
0.001 0.012 NS 0.001 NS NS 0.001 0.001 NS 0.002 NS NS 0.001 0.001 NS 0.001 0.001 NS
10.63a 9.92b (−7.2) 1.12 21.45a 20.21b (−5.8) 2.72 19.59a 18.40b (−6.5) 2.04 9.52 9.17 (−3.7) 1.04 3.95a 3.29b (−16.7) 0.37 Probabilities 3.35 3.27 (−2.4) 0.26 233b 249a (+6.9) 9.05 88b 108a (+22.7) 5.92
2.72a 2.36b (−13.2) 0.18
17.60c 21.06b (+19.7) 23.87a (+35.6) 16.20c 19.46b (+20.1) 21.32a (+31.6) 3.33a 3.19b (−4.2) 3.43a (+3.0) 2.00c 2.44b (+22.0) 3.21a (+60.5) 231b 246a (+6.5) 245a (+6.1)
Diets Dehulled lupins (DL)1 DL + Hulls (11.2%) DL + Hulls (22.4%) Enzyme3 No enzyme Enzyme Pooled SEM
Main effects
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DISCUSSION
(g) 116a 101b (−12.9)2 77c (−33.6)
(g/100 g body weight) (g/g)
3.24b 3.42b (+5.6) 4.21a (+29.9)
8.17c 9.17b (+12.2) 10.66a (+30.5)
(cm/100 g body weight)
Ileum Jejunum Liver
Gizzard
Duodenum
Relative length Relative weight
Feed to gain ratio Feed consumption Weight gain
TABLE 4. Performance, relative gizzard and liver weights, and intestinal length of Leghorn chicks (7 to 21 d) fed enzyme-supplemented diets containing dehulled lupin and hulls, Experiment 2
Cecum
8.87c 10.10b (+13.9) 11.83a (+33.3)
ENZYME ADDITION TO LUPINS IN CHICKEN DIETS
The ability of lupins to improve weight gain and feed efficiency may be related to an appetite-enhancing factor that increased feed intake. However, the decrease in the digestibility of nutrients may be in part attributed to the content of hulls or nondigestible fiber that limits maximum feed intake. As a result, birds on diets that contained 23.1% lupins had improved performance, whereas those on diets with higher concentrations had decreased performance. It has been reported that a crude fiber content above 4.63% may be a limiting factor in the ability of young birds to consume enough feed for maximum gain (Halvorson et al., 1988). The detrimental effects of fiber on growth performance were further demonstrated by adding hulls (11.2 and 22.4%) to a dehulled lupin diet. Sweet lupin hulls contain 76 to 79% NSP with cellulose being (51%) the major constituent of hull structural polysaccharides (Brillouet and Riochet, 1983; Brenes et al., 1993a). So far, there is limited information on the physiological effects of legume NSP in monogastric animals. Considerable amounts of lupin NSP become soluble when ingested by the chicken, as shown by the increase in the ilealsoluble NSP levels (Annison et al., 1996) and the ileal viscosity. This water-soluble NSP fraction can physically complex with intestinal enzymes, keeping them from reacting with substrates. Lupin seeds also contain significant levels of oligosaccharides of the raffinose family that appear to be indigestible in the stomach and the small intestine of monogastric animals due to the lack of αgalactosidase. However, oligosaccharides in lupins actually appear to contribute to metabolizable energy content and should not be regarded as having antinutritive effects on poultry diets (Hughes et al., 1998; Kocher et al., 1999). Maximum performance in chicks fed a lupin diet is achieved by the removal of hulls from lupins. Because dehulled seeds contain less NSP, their nutritive value is higher than that of whole seeds. Brenes et al. (1993a), Smulikowska et al. (1995), Flis et al. (1997) and Gdala et al. (1998) reported that the crude protein content of dehulled lupin seeds increased approximately 20% and that of crude fiber decreased by 70%. As a consequence of these changes in nutrient proportions, the nutritive value of dehulled seeds produced an increase in the digestibility of energy (18%) and that of protein by 7% (Brenes et al., 1993a). The positive effect of dehulling have also been demonstrated by Jansman and Mieczkowska (1998) in diets for roosters, increasing the ileal digestibility of the dry matter. The presence of lupin hulls and lupin seeds in the diets also produced an effect on the different organs of the digestive tract. An increase in dietary fiber caused significant enlargement in the gastrointestinal tract of birds (Hollands et al., 1965; Kondra et al., 1974; McKay, 1980). Changes in the upper tract could be related to an increased muscular development to cope with higher levels of nondigestible material (in the case of lupin, NSP fraction). Increases in the lower part could be caused by an
0.024 0.003 0.032
NS 0.040 NS
0.001 0.038 NS
2,421b 2,514a (+3.8) 65
1,406b 1,484a (+5.5) 27
0.001 0.001 NS
2,459ab 2,524a (+2.6) 2,490a (+1.3) 2,397b (−2.5)
1,473b 1,506a (+2.2) 1,420c (−3.6) 1,380d (−6.3)
(g)
Feed consumption
0.003 NS NS
0.013 NS NS
1.72 1.69 (−1.7) 0.05
1.66b 1.68b (+1.2) 1.75a (+5.4) 1.74a (+4.8)
(g/g)
Feed to gain ratio
0.005 NS NS
0.015 0.021 NS
0.45a 0.35b (−22.2) 0.19
0.34b 0.36b (+5.9) 0.40ab (+17.6) 0.51a (+50.0)
Crop
0.012 NS NS
0.012 0.012 NS
0.37a 0.34b (−8.1) 0.05
0.33b 0.37a (+12.1) 0.37a (+12.1) 0.37a (+12.1)
Proventriculus
0.001 NS NS
0.001 0.021 NS
1.73a 1.61b (−6.9) 0.26
1.46c 1.56c (+6.8) 1.75b (+19.9) 1.90a (+30.1)
Gizzard
0.002 NS NS
0.004 0.010 NS
0.55a 0.51b (−7.3) 0.08 Probabilities
0.50b 0.49b (−2.0) 0.56a (+12.0) 0.56a (+12.0)
Duodenum
Ileum
NS NS NS
NS 0.001 NS
1.02a 0.92b (−9.8) 0.13
0.95 0.97 (+2.1) 0.97 (+2.1) 0.98 (+3.2)
NS NS NS
NS 0.001 NS
0.83a 0.74b (−10.8) 0.12
0.75 0.80 (+6.7) 0.81 (+8.0) 0.80 (+6.7)
(g/100 g body weight)
Jejunum
Relative weight
NS 0.001 NS
0.001 NS NS
0.16 0.15 (−6.3) 0.03
0.14b 0.18a (+28.6) 0.17a (+21.4) 0.14b (0.0)
Colon
NS NS NS
NS 0.033 NS
1.82a 1.72b (−5.5) 0.21
1.72 1.75 (+1.7) 1.81 (+5.2) 1.81 (+5.2)
Liver
0.035 NS NS
NS 0.003 NS
0.19a 0.18b (−5.3) 0.03
0.19 0.19 (0.0) 0.18 (−5.3) 0.17 (−10.5)
Pancreas
NS NS NS
NS NS NS
1.68 1.84 (+9.5) 0.46
1.80 1.81 (+0.6) 1.86 (+3.3) 1.58 (−12.2)
Abdominal
1
a-c
Means within each column for lupin content or enzyme without a common letter are significantly different (P < 0.05). Values in brackets represent percentage increase or decrease relative to chicks fed diets containing 0% lupin or diets without enzyme. 2 Enzyme added to the diets were 0.1% each of Energex威 + Bio-Feed Pro威 + Novozyme威 (Novo Nordisk A/S, Bagsvaerd, Denmark). 3 Individual values for the significant LC × E interaction for feed consumption for the 0, 15, 30 and 45% lupin diets with and without enzymes were as follows: 2,429 and 2,489, 2,542 and 2,505, 2,403 and 2,576, and 2,309 and 2,485, respectively. The percentage of total treatment sum of square for the LC × E interaction was 22% for feed consumption.
Source of variation Lupin content (LC) Enzyme (E) LC × E3 Type of response due to percentage lupin in diet Linear Quadratic Cubic
Lupin content1 0% 15% 30% 45% Enzyme2 0% 0.3% Pooled SEM
Main effects
Weight gain
TABLE 5. Performance of broiler chicks (0 to 5 weeks) and relative organ weights and abdominal fat of broiler chicks (6 wk) fed enzyme-supplemented diets containing lupin seeds, Experiment 3
676 BRENES ET AL.
ENZYME ADDITION TO LUPINS IN CHICKEN DIETS
increased muscle thickness to assist expulsion of bulky feces and increased microbial activity to stimulate intestinal growth (Topping and Visek, 1977; Rubio et al., 1998). The increase in the intestinal viscosity in the birds fed lupin diets (Annison et al., 1996) could also complement this effect, similar to that produced by the viscous βglucan in barley (Brenes et al., 1993b; Viveros et al., 1994). The results of the current studies also demonstrated that the addition of a crude enzyme mixture to diets containing lupin hulls and lupin seeds generally improved weight gain, feed consumption and feed to gain ratio of chicks. In a preliminary study, Brenes et al. (1993a) showed that enzyme addition to a lupin diet (50 and 70% of inclusion) had a beneficial effect on the performance of birds. This effect has been confirmed in our experiments and demonstrated by other authors (Bryden et al., 1994; Roth Maier and Kirchgessner, 1994ab, 1995; Annison et al., 1996; Ferraz de Oliveira, 1998; Naveed et al., 1999). Kocher et al. (2000) also showed that the addition of a commercial enzyme preparation to a lupin-based diet resulted in a significant increase in the ileal digestibility of NSP, which was related mainly to the digestibility of glucose, xylose and arabinose, and polymers present predominantly in hull NSPs. The response to enzymes was positive when the concentration of lupin in the diets was as low as 15%, although, in part, this result may be attributed to an effect of enzyme on other dietary components. As xylanase activity was almost absent from the enzymes used, the effect seen is that of the enzymes on the lupin fraction of the diet rather than on the wheat present. The present study also showed that the addition of enzyme to lupin-based diets was associated with a reduction in the size of certain sections of the gastrointestinal tract and pancreas but not liver. Changes in gastrointestinal morphology have also been demonstrated in birds fed a high level of lupin (70%) in a corn-soy diet (Brenes et al., 1993a). Annison et al. (1996), Kocher et al. (2000) and Hughes et al. (2000) showed that an enzymatic preparation was able to depolymerize the lupin NSPs to some extent as the viscosity of the ileal contents was reduced, resulting probably in an improvement in fermentability in the hindgut. The modifications of the intestinal microflora in lupin-fed birds (Rubio et al., 1998) could also influence the changes produced in the weight and length of the gastrointestinal organs. In conclusion, lupins, when added in high concentrations to a wheat-soy diet, result in reduced chick performance and increased size of digestive organs. These effects may be attributed mainly to the high content of hulls in lupin diets and less to the high nonstarch polysaccharides content of the cotyledon portion of the seed. Lower concentrations of whole lupins (23.1 and 15% lupins in Experiments 1 and 3, respectively) in the diet, in contrast to the high concentration, enhanced feed intake and weight gains when added to wheat-soy diets. The corresponding magnitude of improvement in weight gains for the two experiments were 5.2 and 2.2%. Enzyme addition to lupin containing diets also improved chick perfor-
677
mance and reduced the size of different sections of the gastrointestinal tract. These data demonstrate that the inclusion of moderate concentrations of lupin and enzymes in a chick diet will result in weight gains that are superior to those obtained with a soy-control diet, whereas an opposite effect is obtained when high levels of whole or hulls lupins with or without enzymes are added to a wheat-soy diet.
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