Effect of storage time and dietary enzyme on the metabolizable energy and digesta viscosity of barley-based diets for poultry

Effect of Storage Time and Dietary Enzyme on the Metabolizable Energy and Digesta Viscosity of Barley-Based Diets for Poultry1 J. M. FUENTE,* P. PEREZ DE AYALA,* A. FLORES,* and M. J. VILLAMIDE†,2 Trouw Nutrition Espan˜a S.A. Ronda de Poniente 9, 28760 Tres Cantos, Madrid, Spain, and de Produccio´n Animal, E.T.S.I. Agro´nomos, Universidad Polite´cnica, 28040 Madrid, Spain

†Departamento

respectively, P < 0.001). Interactions of enzyme and age by barley storage time (P < 0.02 and P < 0.001, respectively) were also detected. These data indicate that the minimum time of barley storage before its inclusion in broiler feed depends on the animals’ age (more than 6 wk for 10-d-old chickens and 3 wk for 30-d-old chickens), and that the use of enzymes allowed a reduction in the time of barley storage. Digesta viscosity decreased with barley storage time (P < 0.001), and with enzyme addition (P < 0.001), an interaction of storage time by enzyme addition was shown (P < 0.007). Digesta viscosity was also negatively related to the dietary AMEn content (r = –0.68, P < 0.01). In vitro barley viscosity explained 53 and 90% of the variation in gut viscosity produced by unsupplemented and enzyme supplemented diets, respectively. In Experiment 2, the same diets as Experiment 1 and the barley grain were intubated into 120 adult roosters (Hy-Line) to determine TMEn. Dietary and barley TMEn values were not affected by barley storage time or enzyme addition (3,237 and 3,037 kcal TMEn/kg DM for diets and barley, respectively).

(Key words: nitrogen-corrected apparent metabolizable energy, gut viscosity, barley storage, enzymes, poultry age) 1998 Poultry Science 77:90–97

chickens, led to the development and use of commercial b-glucanases, which were able to alleviate many of these problems (McNab and Smithard, 1992). The seasonal production of barley, as of other crops, leads to its storage for varying periods of time. During storage, some changes in the chemical composition of barley occur, the best known being changes in DM content, although variations in b-glucan content and barley viscosity have been also detected with anaerobic storage treatment (Hesselman et al., 1981). Similarly, Brufau et al. (1993) observed a decrease in total b-glucan content and barley viscosity with barley storage time at room temperature. These changes seemed not to affect barley energy value, as determined in chicks from 18 to

INTRODUCTION Barley is being used more frequently today as a feed component for poultry, because of the better knowledge of its chemical composition and the remarkable progress in biotechnological production of commercial enzymes (Jeroch and Da¨nicke, 1995). The identification of bglucans as the main antinutritive factors in barley, responsible for increasing gut viscosity (White et al., 1983) and sticky droppings (Gohl et al., 1978), as well as impairing nutrient digestibility (Salih et al., 1991) in

Received for publication March 31, 1997. Accepted for publication July 31, 1997. 1Financial support provided by Centro para el Desarrollo Tecnolo´gico e Industrial. Project Number 910050. 2To whom correspondence should be addressed: [email protected]

Abbreviation Key: NSP = nonstarch polysaccharide.

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ABSTRACT The effect of barley storage time and dietary enzyme addition on the energy value of barleybased broiler diets was studied in two experiments. A two-rowed winter barley (Beka cultivar) was stored at room temperature for 0, 3, 6, 16, and 32 wk after harvesting. At these dates, diets were formulated using 50% barley with and without the addition of a commercial b-glucanase-based enzyme product. In Experiment 1, 320 Arbor Acres chickens (eight replicates of three 10-d-old birds and eight replicates of one 30-d-old bird, per treatment) were fed the experimental diets to determine the AMEn following a 2 × 2 × 5 (age by enzyme by barley storage time) factorial design. At the end of the metabolism trial, viscosity of the intestinal contents was determined in 30-d-old broilers. Total bglucan, nonstarch polysaccharides (NSP), in vitro viscosity, and endogenous enzyme activity of barley grain decreased with increasing storage time. Dietary AMEn increased with barley storage time (from 2,755 to 2,939 kcal/kg DM, P < 0.001, for 0 and 32 wk of storage, respectively), with enzyme addition (2,861 vs 2,919 kcal/ kg DM, P < 0.003), and with the age of animals (2,826 and 2,958 kcal/kg DM for 10- and 30-d-old chickens,

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STORAGE TIME AND ENZYME ADDITION ON BARLEY ENERGY VALUE

MATERIALS AND METHODS

Barley, Enzyme, and Diets A two-rowed winter barley (Beka cultivar grown in 1993) was stored in cloth bags holding 200 kg for 0, 3, 6, 16, or 32 wk after harvesting in the storage section of a mill, at a room temperature (15 to 25 C). At the designated times, a representative sample from each storage period was collected for proximate analysis, nonstarch polysaccharides (NSP), b-glucans, in vitro viscosity, and b-glucanase activity. A commercial multienzyme complex obtained from Trichoderma longibrachiatum, Trichoderma viridae, and Aspergillus niger3 containing 300 U/g of b-glucanase (EC 3.2.1.6) and 300 U/g of xylanase (EC 3.2.1.8) activities was used at 1 g/kg dose to study the effect of enzyme addition. A basal diet containing 50% barley was formulated to meet the National Research Council (1984) requirements (Table 1). The basal diet with and without enzyme addition was prepared five times, one for each barley storage time; the only difference among diets was the storage time of barley. Feedstuffs were ground to pass through a 2.5-mm sieve screen. After mixing, diets were pelleted at 70 C. The resulting pellets were 3 mm in diameter and 6 mm long. Chickens and adult cockerels were randomly assigned to dietary treatments. The two experiments described below were conducted at the Experimental Farm of Trouw Ibe´rica S.A. (Spain). The animals were held in environmentally controlled

3Avizyme

1100, Finnfeeds International Ltd., Marlborough, U.K.

TABLE 1. Composition of basal diet Ingredients and analysis Barley Soybean meal Full-fat soybean Sunflower seed meal Lard Dicalcium phosphate Calcium carbonate Sodium chloride Vitamin-mineral premix1 Methionine2 Lysine2 Calculated analysis Crude protein Starch Ash Metabolizable energy, kcal/kg

Percentage 50.0 28.6 10.9 1.0 5.0 1.6 0.9 0.25 0.8 0.8 0.2 22.7 31.8 6.0 2,920

1Supplied per kilogram of diet: selenium, 0.15 mg; iodine, 2 mg; cobalt, 0.2 mg; copper, 6 mg; iron, 30 mg; zinc, 50 mg; manganese, 80 mg; retinyl acetate, 7,500 IU; cholecalciferol, 1,500 IU; dl-a-tocopheryl acetate, 7.5 IU; riboflavin, 5.3 mg; pantothenic acid, 8 mg; pyridoxine, 1.8 mg; folic acid, 0.5 mg; menadione sodium bisulfite, 2 mg; vitamin B12, 12.5 mg; niacin, 24 mg; choline, 350 mg. 2Commercial supplement of methionine and lysine with 20% of DLmethionine and L-lysine, respectively.

rooms, with temperatures ranging from 23 to 27 C. The lighting program for chicks and roosters, during the experiments was 16 h:8 h light:dark cycle. Throughout the experiments, animals were handled according to the principles for the care of animals in experimentation established by the Royal Decree 223/88 of Spain (1988). For each barley storage time, the two AMEn trials (with chickens of 10 and 30 d of age, respectively) of Experiment 1 and the TMEn trial of Experiment 2 were performed simultaneously.

Experiment 1 Three hundred and twenty Arbor Acres chickens (eight replicates of three 10-d-old birds and eight replicates of one 30-d-old bird, per treatment) were used in this experiment. Chickens were fed a commercial broiler starter diet until the beginning of the experiment. For the trials run with 10-d-old chickens, the birds were transferred to metabolic cages when they were 3-d-old. After a 7-d adaptation period to experimental diets, the birds were deprived of feed for 16 h, then allowed ad libitum consumption for 4 d, and deprived of feed again for 16 h prior to the end of the collection period. Total excreta voided during the balance period (4 d) was collected twice (at 48 and 96 h) and frozen at –20 C. Excreta samples were dried in a forced draught oven at 70 C for 48 h, and then ground for gross energy and nitrogen analyses in order to determine dietary AMEn. Correction to zero nitrogen retention was made using 8.22 kcal/g of retained nitrogen (Hill and Anderson, 1958). For the trials run with 30-d-old chickens, dietary AMEn was determined as described above, with the

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25 d of age (Brufau et al., 1993), but, nevertheless, an interaction of storage time by enzyme addition on dietary AMEn was detected. Traditionally, barley is stored for a minimum time period, varying from 2 to 8 wk before its use in animal nutrition because of its impairment of animal performance. However, changes in the nutritive value of barley during the 1st wk of storage have not been previously studied, and therefore, the minimum storage time required has not been determined. The susceptibility of birds to b-glucans depends on their age (Almirall et al., 1993), and hence, the response to enzyme addition in performance (Petterson et al., 1991) and nutrient digestibility (Salih et al., 1991) varies with age. Thus, the effect of storage time could be different due to the age of the birds. The aims of the current work were 1) to study the effect of barley storage time, enzyme addition and age of chickens on dietary AMEn; 2) to establish the effect of barley storage time and enzyme addition on gut viscosity content of 30-d-old chickens; and 3) to establish the effect of barley storage time and enzyme addition on dietary and barley TMEn as determined in adult roosters.

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FUENTE ET AL. TABLE 2. Evolution of physicochemical parameters of barley1 with storage time (DM basis) Barley storage time Content

0 wk

3 wk

6 wk

16 wk

32 wk

Non starch polysaccharides Arabinose Galactose Glucose Mannose Rhamnose Xylose Total b-glucan Cellulose2 b-glucanase activity, U/g In vitro viscosity, cSt

14.94 1.91 0.23 9.20 0.21 0.05 3.34 6.23 2.97 57 5.04

15.66 2.00 0.23 9.49 0.19 0.06 3.69 6.60 2.89 24 4.51

(%) 14.85 2.18 0.24 8.46 0.22 <0.01 3.74 6.77 1.69 19 4.65

12.10 1.81 0.20 6.59 0.21 0.18 3.11 5.26 1.33 <17 4.15

12.75 2.01 0.23 6.60 0.21 0.16 3.54 4.73 1.87 <14 4.08

1Chemical composition of barley not affected by storage time (percentage DM): ash (2.25), crude protein (13.7), crude fiber (3.8), ether extract (2.2), and starch (60.2). 2Calculated as difference between glucose and total b-glucan content.

Experiment 2 In this experiment, TMEn of the same diets used in Experiment 1 (unsupplemented and enzymesupplemented) and of barley grain was determined according to Sibbald (1986). A total of 160 adult roosters (Hy-Line, 1-yr-old) were used. After a feed deprivation period of 24 h, experimental animals (eight roosters per diet) were intubated with 30 g of ground feed, and deprived of feed for 48 h. Eight additional roosters were deprived of feed for 72 h to estimate the endogenous energy losses. At the end of these periods, total excreta voided were collected and frozen at –20 C. Excreta samples were analyzed as described for Experiment 1.

Analyses Dry matter, crude protein, crude fiber, ether extract, ash, and starch were analyzed according to AOAC (1984) methods. The NSP content of barley was determined by gas liquid chromatography4 (English and Cummings, 1984). Gross energy was measured with an adiabatic bomb calorimeter.5 Endogenous b-glucanase activity was

4Waters Model 150, Waters (Division of Millipore), Milford, MA 01757. 5IKA-4000, Shotch Ibe ´ rica, Spain. 6Kapillar Viskosimeter number 516-10. Schott-Gerate GmbH. D 6238 Hofheim a. Ts. Germany. 7LVDV II+ CP number 28754. Brookfield Engineering Laboratories Inc., Stoughton, MA 02072.

determined following the McCleary and Schemeer (1987) methodology, and total mixed-linked b-glucan content was calculated as described by Aman and Graham (1987). In vitro barley viscosity was determined at pH = 5.2 using as Ostwald viscometer6 according to Henry (1984), and chicken digesta viscosity measured with a Brookfield DVII + viscometer7 as described by Bedford and Classen (1993). Statistical analyses were performed using the General Linear Models procedure of SAS Institute (1985). A 2 × 2 × 5 factorial ANOVA (age by enzyme by barley storage time) was carried out to study the main effects and interactions on dietary AMEn in Experiment 1. Orthogonal contrasts were performed to evaluate the time from which there was no differences in AMEn for each age and type of diet. Dietary TMEn in Experiment 2 and digesta viscosity in Experiment 1 were analyzed using a 2 × 5 factorial ANOVA (enzyme by barley storage time). Correlation and regression analyses were performed among chemical parameters and biological determinations.

RESULTS Nonstarch polysaccharide content, total b-glucan, in vitro viscosity and b-glucanase activity of barley (Table 2) were negatively correlated to barley storage time (P < 0.05). Nonstarch polysaccharide content remained constant until 6 wk of storage (average 15%) and then decreased to 12.5%, mainly due to the concomitant decrease in glucose residues, which explained 95% (P < 0.01) of the NSP variation. Total mixed-linked b-glucan content, the main NSP component (with a correlation of 0.89), showed a similar tendency, remaining constant (6.5%) from 0 to 6 wk, and decreasing to 5.3 and 4.7% for 16 and 32 wk, respectively). Glucose residue content decreased quadratically with barley storage time (P < 0.07). In contrast, other NSP sugars did not vary with barley storage time. Endogenous b-glucanase activity

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adaptation period beginning when the birds were 23-d-old. At the end of the metabolic trial, chickens were euthanatized by cervical dislocation. The small intestine was removed from the end of the duodenal loop to the ileo-ceco-colic junction, and the digesta collected from the jejunum to measure digesta supernatant viscosity (see below). The intestinal tract (duodenum, jejunum, and ileum) was weighed empty and full.

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STORAGE TIME AND ENZYME ADDITION ON BARLEY ENERGY VALUE TABLE 3. Apparent MEn of 10- and 30-d-old broiler chickens fed barley based diets with and without enzyme supplementation. Barley was stored during different time periods1 Main effect means

Storage time Treatment

0 wk

3 wk

6 wk

16 wk

32 wk

x

Age

2,800 2,855

2,827

Enzyme2

(kcal/kg DM) 10 d of age Barley Barley + enzyme 30 d of age Barley Barley + enzyme Mean ± SEM

(7) 2,671 (7) 2,785

(8) 2,655 (7) 2,667

(7) 2,706 (8) 2,915

(7) 3,000 (7) 3,001

(8) 2,966 (5) 2,951

(5) 2,741 (5) 2,944 2,776 ± 80.0c

(8) 3,012 (7) 2,979 2,823 ± 75.4c

(7) 2,963 (7) 3,062 2,912 ± 62.0b

(8) 2,946 (8) 3,015 2,990 ± 34.3a

(5) 2,921 2,931 (8) 2,915 2,984 2,939 ± 43.9ab Probability

2,958 2,919 (+)

<0.001 0.003 <0.001 0.02 <0.001 0.933 0.59

a–cMeans

with no common superscript differ significantly (P < 0.05). 1Determined from (n) chicks. 2(–) without enzyme; (+) with enzyme.

and in vitro barley viscosity decreased exponentially (P < 0.01) with time. In both cases, the greatest decrease occurred from harvesting to 3 wk of storage (57 to 24 U/ g, and 5.04 to 4.51 cSt for endogenous b-glucanase activity and barley viscosity, respectively). Dietary energy value determined in chickens (Experiment 1) was affected by barley storage time (P < 0.001), enzyme addition (P < 0.003), and age of animals (P < 0.001) (Table 3). Apparent MEn increased from 2,776 kcal/kg DM just after harvesting to 2,823 kcal/kg DM when barley was stored for 3 wk and to 2,939 kcal/kg DM, from 6 to 32 wk of storage. Enzyme addition increased (P < 0.003) the dietary AMEn from 2,862 to 2,919 kcal/kg DM. The highest energy values were obtained (P < 0.001) with older chickens (2,958 vs 2,827 kcal AMEn/kg DM for 30- and 10-d-old chickens, respectively). The effect of enzyme addition and age on the AMEn of the chickens was different depending on the barley storage time. This difference resulted in enzyme by storage time (P < 0.02) and age by storage time (P < 0.001) interactions. Thus, orthogonal contrasts of AMEn values for broilers of different ages were made between adjacent storage times to determine the minimum time of storage for barley with and without enzymes (Table 4). Digesta viscosity decreased with barley storage time (P < 0.001), and with enzyme addition (P < 0.001) (Table 5); however, the decrease in unsupplemented diets was greater in the first stages of storage than in enzyme supplemented diets. This effect resulted in a storage time by enzyme interaction (P < 0.007). Enzyme addition reduced digesta viscosity (7.0 cps as an average) as

compared with unsupplemented diets (14.1 cps) and the standard deviation of this parameter was three times lower for enzyme-supplemented than for unsupplemented diets. Empty weight of the intestinal tract of 30-d-old chickens (data not shown) was not affected by barley storage time, but was reduced by 5% (P = 0.12) by enzyme addition (56.87 and 53.62 g, respectively). The empty weight of the intestinal tract was linearly related to digesta viscosity (r = 0.64); this relationship was higher in broilers fed diets without enzymes (r = 0.9; P < 0.09). Dietary TMEn determined with adult cockerels (Table 6, Experiment 2) was not affected by barley storage time or enzyme addition (3,237 kcal TMEn/kg DM, on average). Similarly, barley TMEn (mean value, 3,307 kcal/kg DM) was not affected by storage time.

TABLE 4. Orthogonal contrast of AMEn to evaluate the minimum time of storage in barley (Beka cultivar) before its use as broiler feed (P > F) 10 d of age

30 d of age

Orthogonal contrast weeks of storage

Without enzyme

With enzyme

Without enzyme

With enzyme

(wk) 0 vs 3, 6, 16, 32 3 vs 6, 16, 32 6 vs 16, 32 16 vs 32

0.002 0.001 0.001 0.575

0.1271 0.001 0.371 0.564

0.002 0.232 0.642 0.739

0.369 0.708 0.070 0.080

1Contrast

0 vs 6, 16, and 32 wk of storage (P < 0.001).

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Source of variation Storage time Enzyme Age Storage time by enzyme Storage time by age Enzyme by age Storage time by enzyme by age

2,862 (–)

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FUENTE ET AL. TABLE 5. Digesta viscosity content of 30-d-old chickens fed diets with 50% barley with or without enzyme supplementation. Barley was stored during different time periods1 Barley storage time

Treatment

0 wk

3 wk

6 wk

Barley Barley + enzyme Mean ± SEM

(7) 23.4 (7) 11.4 17.4 ± 4.7a

(7) 23.0 (7) 8.0 15.5 ± 3.4a

(cps)2 (5) 8.0 (6) 6.6 (7) 6.6 (6) 4.2 7.2 ± 0.5ab 5.4 ± 0.9b Probability

Source of variation Storage time Enzyme Storage time by enzyme

16 wk

32 wk

x

SEM

(6) 5.4 (6) 4.1 4.8 ± 0.4b

14.1 7.0

3.3 1.0

0.001 0.001 0.007

a–cMeans

with no common superscript differ significantly (P < 0.05). from (n) chicks. 2cps = centipoise. 1Determined

Barley storage time did not affect proximate components of barley. However, cellulose and mixed-linked bglucan content decreased with barley storage time in a similar pattern as NSP glucose content. Brufau et al. (1993), working with a two-rowed barley sample stored for 1 yr between 10 and 30 C, observed no differences in proximate components. Total mixed-linked b-glucan content decreased during storage time, whereas insoluble b-glucan increased at the expense of soluble b-glucan during the same period. Similarly, Hesselman et al. (1981) observed a marked decrease in barley viscosity with a minimal decrease in total b-glucan content during 7 wk of anaerobic storage, and concluded that there were structural changes of the b-glucans with time. Barley, viscosity, caused by soluble components, and endogenous b-glucanase activity decreased with storage time, both parameters being positively related (r = 0.86). The endogenous enzyme content of barley grain was shown by Burnett (1962) to be the main reason for the

TABLE 6. True MEn determined in adult cockerels fed diets with 50% barley with or without enzyme supplementation, or barley grain. Barley was stored during different time periods1 Barley storage time Treatment

0 wk

3 wk

6 wk

16 wk

32 wk

x

SEM

(8) 3,251 (8) 3,208 3,230 ± 39.2

3,241 3,324

57.0 49.5

(7) 3,348

3,308

41.21 NS

(kcal/kg DM) Diets Barley Barley + enzyme Mean ± SEM Source of variation Storage time Enzyme Storage time by enzyme Grain Barley 1Determined

from (n) roosters.

(6) 3,224 (8) 3,209 3,216 ± 52.9

(5) 3,279 (6) 3,326 3,305 ± 75.9

(6) 3,241 (8) 3,222 (6) 3,257 (8) 3,198 3,249 ± 64.5 3,210 ± 40.3 Probability 0.490 0.816 0.938

(6) 3,253

(6) 3,352

(7) 3,292

(7) 3,299

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differences found in the nutritive value of U.S. barleys. Preece and McKenzie (1952) had previously identified endogenous enzyme systems in barley grain, responsible for the decrease in viscosity without an increment in reduced sugars content; that is, these enzymes had the capacity to modify the structure but not the content of bglucans. As long as barley grain has endogenous enzyme activity, there could be a transformation from soluble to insoluble b-glucans (with a concomitant decrease in viscosity). Thus, the existence of endo 1-3 b-glucanase activity in barley is possible. Corroborating these results, Aman et al. (1990) found a decrease in b-glucan solubility with increasing barley storage time. These changes in the physicochemical characteristics of barley with storage time affected barley energy value in Experiment 1 (Table 3) as determined with chickens (AMEn) at 10 and 30 d of age. Apparent MEn of unsupplemented diets, determined with 10-d-old chickens, was negatively related to total mixed-linked bglucan content (r = –0.91; P < 0.05) and barley viscosity

DISCUSSION

STORAGE TIME AND ENZYME ADDITION ON BARLEY ENERGY VALUE

to the diets, the minimum storage time was between 6 and 16 wk (Table 4); but the addition of enzymes decreased this period to less than 6 wk. When barley was supplied to 30-d-old chickens and no enzyme was used, the minimum storage time was about 3 wk, whereas the use of dietary enzymes allowed barley use without any storage time. Thus, at both ages studied, the use of enzymes allowed a reduction in the time of barley storage. Digesta viscosity values in 30-d-old chickens fed barley-based diets were affected by barley storage time, enzyme addition, and their interaction (Table 5, Experiment 1). The effect was opposite to that observed for AMEn. In fact, gut viscosity explained 38% of the decrease in AMEn as shown in the following equation (n = 10); 30 d old: (r2 = 0.38; P < 0.01; SEM = 48.48): AMEn (kcal/kg DM) = 3,172 (± 54) – 15.4 (± 5.4) digesta viscosity (cps) Thus, for each centipoise unit of increase in digesta viscosity, the AMEn declined 15.4 kcal. This value agrees with that found in a previous study using increasing levels of barley without enzyme addition (–14.1 kcal/ cps; Fuente et al., 1995). Rotter et al. (1990) have shown that reduction in gut viscosity is the most important factor in the performance improvement in broiler chickens fed high viscosity cereals, and that enzyme effectiveness is related to its capacity to decrease digesta viscosity. Almirall et al. (1993) reported a significant decrease in endogenous digestive enzyme activities (amylase, trypsin, and lipase) in the intestine as well as a decrease in nutrient ileal digestibility caused by the intestinal viscosity. Digesta viscosity (centipoise) of intestinal contents was positively related to in vitro barley viscosity (cst) (r = 0.73, P = 0.159, and r = 0.95, P < 0.01 for unsupplemented and enzyme-supplemented diets, respectively). These results agree with those obtained in a previous study (Villamide et al., 1997), in which a positive relationship between barley viscosity and the increment in AMEn due to enzyme addition was found (r = 0.69). High digesta viscosity of chickens fed barley-based diets (with or without enzyme addition) produced an increase in empty weight of intestinal tract, as shown by the relationship found between these parameters (r = 0.64; P < 0.02). This relationship was higher in broilers fed unsupplemented diets (r = 0.9; P < 0.09; n = 5) than in broilers fed enzyme-supplemented diets. The best fit equation relating the empty weight of the digestive tract (grams) to digesta viscosity (centipoise) was (n = 10, r2 = 0.41; P < 0.02): Empty digestive tract weight (g) = 50.28 (± 2.1) + 0.69 (± 0.2) Digesta viscosity (cps) This relationship might be of interest because field observations indicate that when animals are fed diets

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(r = –0.85; P < 0.1), and that determined with 30-d-old chickens was negatively related to endogenous b-glucanase activity (r = –0.85; P < 0.1). This relationship between b-glucan content and AMEn had been previously reported by Choct and Annison (1990). These authors found a very high negative correlation (r = –0.98) between barley energy value (measured at 24 d of age), and b-glucan and pentosan content. Previously, Thomke (1972) had shown that the improvement in the nutritive value of barley during grain maturation was due to the decrease in its viscosity. In the present experiment, no relationship was found between AMEn of enzyme-supplemented diets or TMEn values (Experiment 2) and variables summarized in Table 1. Enzyme addition increased dietary energy values (AMEn) by 2% on average (P < 0.003, Table 3). This increase could be due to hydrolysis of b-glucans, allowing digestive enzymes access to substrates such as protein and starch with a consequent improvement in the digestibility of nutrients (from 2 to 23%, Salih et al., 1991), and in the AMEn (from 0 to 12%, Rotter et al., 1990; Fuente et al., 1995). The age of animals also affected (P < 0.001) the dietary energy value; the AMEn determined in 30-d-old chickens was 4.6% higher than that of 10-d-old chicks. Salih et al. (1991) also showed that negative effects of high barley levels in broiler diets was dependent on the animals’ age and decreased in older chicks. Brenes (1992) explained that, during their 1st d after hatch, due to the immaturity of their digestive tract, chickens are more sensitive to the negative effects of antinutritional factors in cereals and other raw materials. The interaction between time of storage and enzyme addition (P < 0.002) in the present work showed that enzymes had different effects on barley energy value depending on the barley storage time. Thus, dietary AMEn increased markedly due to enzyme addition, just after harvesting or storage for 6 wk (157 kcal/kg DM on average, which implies 314 kcal/kg DM in barley AMEn), whereas no further improvement was observed for barley stored for 3, 16, and 32 wk. These results could be explained by the higher b-glucan and barley viscosity found for the earlier storage times. However, no statistical relationship was found between this AMEn increment and barley analytical parameters, possibly due to the unexpected lack of enzyme effect at 3 wk of storage. In contrast, Brufau et al. (1993) detected an interaction between storage time and enzyme addition in one of the two cultivars studied, by which the effect of the enzyme was greater as the storage time increased. In the present trial, the interaction between time of storage and age (P < 0.001) showed that the effect of barley storage time on energy value was more important for 10- than for 30-d-old chickens. Thus, the minimum barley storage time required is a function of the bird’s age and of the enzyme addition. When barley was supplied to 10-d-old chickens and no enzyme was added

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ACKNOWLEDGMENTS The authors gratefully acknowledge the valuable comments of Marı´a Fernanda Soto-Salanova and Hadden Graham during the preparation of this paper.

REFERENCES Almirall, M., J. Brufau, and E. Esteve-Garcı´a, 1993. Effects of intestinal viscosity on digestive activities of intestinal content and ileal digestibilities of poultry fed barley diets at different ages supplemented with b-glucanases. Pages 69–72 in: Proceedings of the 1st Symposium of Enzymes in Animal Nutrition. Kartause Ittingen, Switzerland. Aman, P., and H. Graham, 1987. Analysis of total and insoluble mixed-linked (1-3), (1-4) b-D-glucan in barley and oats. J. Agric. Food Chem. 35:704–709. Aman, P., D. Pettersson, and H. Graham, 1990. Chemical and nutritional evaluation of airtight storage of high-moisture barley and high moisture barley treated with lactobacilli or lactobacilli and yeast. Anim. Feed Sci. Technol. 29: 223–235. Association of Official Analytical Chemists, 1984. Official Methods of Analysis. 14th ed. Association of Official Analytical Chemists, Washington, DC.

Bedford, M. R., and H. L. Classen, 1993. An in vitro assay for prediction of broiler intestinal viscosity and growth when fed rye-based diets in the presence of exogenous enzymes. Poultry Sci. 72:137–143. Brenes, A., 1992. Influencia de la adicio´n de enzimas sobre el valor nutritivo de las raciones en la alimentacio ´ n aviar. Pages 139–148 in: Proceedings of the XXIX Symposium de la Seccio´n Espan˜ola de la World’s Poultry Science Association, Salamanca, Spain. Brenes, A., M. Smith, W. Guenter, and R. R. Marquardt, 1993. Effect of enzyme supplementation on the performance and digestive tract size of broiler chickens fed wheat and barley based diets. Poultry Sci. 72:1731–1739. Brufau, J., M. Francesch, A. M. Pe´rez-Vendrell, and E. EsteveGarcı´a, 1993. Effects of post-harvest storage on nutritive value of barley in broilers. Pages 125–128 in: Proceedings of the 1st Symposium of Enzymes in Animal Nutrition. Kartause Ittingen, Switzerland. Burnett, G. S., 1962. The effect of damaged starch, amylolytic enzymes, and proteolytic enzymes on the utilization of cereals by chickens. Br. Poult. Sci. 3:89–103. Choct, M., and G. Annison, 1990. Antinutritive activity of wheat pentosans in broiler diets. Br. Poult. Sci. 31:811–821. English, N., and J. Cummings, 1984. Simplified method for the measurement of total non starch polysaccharides by gas liquid chromatography of constituent sugars as alditol acetates. Analyst 109:937–942. Fuente, J. M., P. Pe´rez de Ayala, and M. J. Villamide, 1995. Effect of dietary enzyme on metabolizable energy of diets with increasing levels of barley fed to broilers at different ages. Anim. Feed Sci. Technol. 56:45–53. Gohl, B., S. Alde´n, K. Elwinger, and S. Thomke, 1978. Influence of b-glucanase on feeding value of barley for poultry and moisture content of excreta. Br. Poult. Sci. 19:41–47. Henry, R. J., 1984. A simplified enzymatic method for the determination of 1-3, 1-4 betaglucan in barley. J. Inst. Brew. 90:178–180. Hesselman, K., K. Elwinger, M. Nilsson, and S. Thomke, 1981. The effect of b-glucanase supplementation, stage of ripeness and storage treatment of barley in diets fed to broiler chickens. Poultry Sci. 60:2664–2671. Hill, F. W., and D. L. Anderson, 1958. Comparison of metabolizable energy and productive energy determinations with growing chicks. J. Nutr. 64:587–603. Jeroch, H., and S. Da¨nicke, 1995. Barley in poultry feeding: a review. World’s Poult. Sci. J. 51:271–291. McCleary, B. V., and J. Schemeer, 1987. Assay of malt bglucanase using azo-barley glucan: an improved precipitant. J. Inst. Brew. 93:285–297. McNab, J. M., and R. R. Smithard, 1992. Barley B-glucan: an antinutritional factor in poultry feeding. Nutr. Res. Rev. 5: 45–60. National Research Council, 1984. Nutrient Requirement of Poultry. 8th rev. ed. National Academy Press, Washington, DC. Pettersson, D., H. Graham, and P. Aman, 1991. The nutritive value for broiler chickens of pelleting and enzyme supplementation of a diet containing barley, wheat and rye. Anim. Feed Sci. Technol. 33:1–14. Preece, I. A., and K. G. McKenzie, 1952. Non starch polysaccharides of cereal grains. Fraction of the barley gums. J. Inst. Brewing. 58:353–362. Rotter, B. A., O. D. Friesen, W. Guenter, and R. R. Marquardt, 1990. Influence of enzyme supplementation on the bioavailable energy of barley. Poultry Sci. 69:1174–1181.

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that produce an increased viscosity of intestinal contents, the weight of their digestive tract increase, and thus, the percentage carcass yield at processing decreases. A similar effect was shown by Brenes et al. (1993) and Viveros et al. (1994), who found that enzyme addition decreased the digestive tract weight by 20% when expressed as a percentage of live weight. Dietary and barley energy values in adult cockerels (TMEn) were not influenced by barley storage time, and enzyme addition did not affect these parameters (Experiment 2, Table 6). Thus, the chemical parameters that change with barley storage time (NSP, b-glucan, barley viscosity), or the changes in digesta viscosity produced by enzyme addition did not affect barley energy values (TMEn) of adult cockerels. These results agree with those found by Rotter et al. (1990), showing that energy improvement due to enzyme addition was high in broiler chickens (AMEn determinations), but it did not exist in adult cockerels (TMEn determinations). The authors explained this lack of effect was due to the age of animals, although because of the experimental design, the effects of age and methodology were confounded. Likewise, Almirall et al. (1993) reported no differences in digestive enzyme activity or ileal digestibilities when high barley diets (60%) were enzyme-supplemented and fed to cockerels. A reduction in the intestinal viscosity was observed, suggesting that intestinal viscosity was not a limiting factor in Leghorn cockerels, as in young birds. Nevertheless, the current work shows the low susceptibility of adult birds to barley b-glucan and the lack of response to enzyme addition in terms of energy value, although part of these results could be due to the methodology used (force feeding).

STORAGE TIME AND ENZYME ADDITION ON BARLEY ENERGY VALUE Royal Decree 223/88 of Spain, 1988. Sobre proteccio ´ n de los animales utilizados para experimentacio ´ n y otros fines cientı´ficos. Pages 8509–8511 in: Boletı´n Oficial del Estado. nu´mero 67, Madrid, Spain. Salih, M. E., H. L. Classen, and G. L. Campbell, 1991. Response of chickens fed on hull-less barley to dietary b-glucanase at different ages. Anim. Feed Sci. Technol. 33:139–149. SAS Institute, 1985. SAS User’s Guide: Statistics. Version 5 Edition. SAS Institute Inc., Cary, NC. Sibbald, I. R., 1986. The TME system of feed evaluation: methodology, feed composition data, and bibliography. Technical Bulletin 1986 4E, Agriculture Canada. Ottawa, ON, Canada. Thomke, S., 1972. Deterioration processes in newly harvested grains. V. Experiment with barley deteriorated under

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controlled conditions and fed to broiler chickens. Swedish J. Agric. Res. 2:27–37. Villamide, M. J., J. M. Fuente, P. Pe´rez de Ayala, and A. Flores, 1997. Energy evaluation of eight barley cultivars for poultry: Effect of dietary enzyme addition. Poultry Sci. 76: 834–840. Viveros, A., A. Brenes, M. Pizarro, and M. Castan˜o, 1994. Effect of enzyme supplementation of a diet based on barley, and autoclave treatment, on apparent digestibility, growth performance and gut morphology of broiler diets. Anim. Feed Sci. Technol. 48:237–251. White, W. B., H. R. Bird, M. L. Sunde, J. A. Marlett, N. A. Prentice, and W. C. Burger, 1983. Viscosity of b-D-glucan as a factor in the enzymatic improvement of barley for chicks. Poultry Sci. 62:853–862.

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