INTRODUCTION. DIVYA JARONI,*, SHEILA E. SCHEIDELER,*,1 MARY BECK,* and CRAIG WYATT

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1 The Effect of Dietary Wheat Middlings and Enzyme Supplementation. 1. Late Egg Production Efficiency, Egg Yields, and Egg Composition in Two Strains of Leghorn Hens DIVYA JARONI,*, SHEILA E. SCHEIDELER,*,1 MARY BECK,* and CRAIG WYATT *Animal Science, University of Nebraska, Lincoln, Nebraska and Finnfeeds International, Inc., Marlborough Wiltshire SNSAAA, United Kingdom ABSTRACT A study was conducted to test the effects of wheat middlings (WM) with and without enzyme (xylanase and protease) supplementation on late egg production (EP), egg yields, and egg composition in two strains of Leghorn hens (DeKalb Delta and Hisex White). Six diets were randomly assigned to 300 birds (150 birds per strain) from 42 to 60 wk of age. Diet 1 was a cornsoybean meal control, Diets 2 and 3 had 8 and 16% WM, respectively, Diet 4 had 8% WM with 0.1% enzyme, and Diets 5 and 6 had 16% WM with 0.1 and 0.2% enzyme, respectively. Each dietary treatment was replicated five times per strain. Egg production, feed consumption (FC), feed efficiency (FE), percentage dirty eggs, specific gravity (SP), egg weight (EW), and egg mass (EM) were measured on a weekly basis. Egg components (EC) were measured biweekly and birds were weighed every 4 wk. Egg production was not significantly affected by diet, with averages of 83.7, 85.7, 84.1, 84.2, 82.3, and 84.0% for Diets, 1, 2, 3, 4, 5, and 6, respectively. Strain had a marked effect on FC, with Hisex having higher FC than DeKalb hens. Feed consumption also showed a significant diet by strain effect, being lower for Hisex hens fed diets with enzyme compared to diets without enzyme. DeKalb hens had better FE than Hisex hens, and addition of enzyme to 16% WM improved it further. Strain and diet had a significant effect on EC with higher percentages of albumen and yolk for diets with enzyme and for DeKalb hens. Percentage shell was lower for DeKalb hens than for Hisex hens but was not affected by diet. There was also a significant increase in EW for Diets 4 (63.1 g), 5 (63.8 g), and 6 (63.2 g) compared to Diets 1 (62.1 g), 2 (62.4 g), and 3 (63.0 g), with DeKalb hens showing an increase with Diet 5 compared to Hisex hens. Egg mass improved with the higher rate of enzyme in 16% WM diet compared to the lower enzyme level. Specific gravity declined with enzyme supplementation in WM diets. Eggs from DeKalb hens had lower SP than those from Hisex hens for all diets. Percentage dirty eggs did not differ significantly among treatments but was higher for the Hisex strain than for the DeKalb strain. There were no differences in BW between the two strains of hens or among diets. Adding protease and xylanase to diets rich in fiber can improve the egg output without affecting production parameters. (Key words: layer, wheat middlings, protease, xylanase, egg components) 1999 Poultry Science 78: INTRODUCTION High levels of production and efficient feed conversion are characteristic of the modern poultry industry. Improvements in the efficiency of production must rely on obtaining maximum nutrient utilization from feedstuffs, which would also enable the use of a wide range of ingredients currently considered inferior. Corn has been used consistently as a major ingredient for poultry rations because of its high energy content and low cost. However, as corn markets tighten and corn supplies go to nonagricultural uses such as ethanol production, there appears to be a need for alternative grain sources for pullet and layer rations. Wheat makes an excellent replacement for corn in poultry feeds, but dietary modifications need to be made because of its anti-nutritive fraction nonstarch polysaccharide. Wheat contains relatively high levels of nonstarch polysaccharide as a structural carbohydrate in the aleurone and endosperm walls (Annison, 1990; Ward, 1995). Cellulose is a small proportion of the grain cell wall; the majority of the carbohydrate fraction is derived from b-glucan and arabinoxylan (Annison and Choct, 1991; Classen and Bedford, 1991). Arabinoxylans make Received for publication August 5, Accepted for publication February 25, To whom correspondence should be addressed: ses@unlinfo. unl.edu Abbreviation Key: D = DeKalb Delta strain; EC = egg components; EM = egg mass; EP = egg production; EW = egg weights; FC = feed consumption; FE = feed efficiency; H = Hisex White strain; SP = specific gravity; WM = wheat middlings. 841

2 842 up the largest portion, mainly in the form of arabinose and xylose with a small amount of mixed linked b- glucans (Ward, 1995). After ingestion, arabinoxylans become soluble, resulting in increased digesta viscosity (Burnett, 1966; Antoniou and Marquardt, 1982; White et al., 1983; Salih et al., 1991; Teitge et al., 1991). The viscous nature of intestinal digesta seems to be responsible for negative effects exhibited by wheat pentosans. Increased intestinal viscosity reduces the passage rate of digesta (Antoniou et al., 1981; Antoniou and Marquardt, 1983). With a reduction in digesta passage rate, the overall feed consumption (FC) would be less and could contribute to a decrease in live performance. As the viscosity increases, the rate of nutrient absorption decreases (Fengler and Marquardt, 1988), which, in turn, could reduce nutrient assimilation rate due to reduced enzyme:substrate reactions in the intestine (Ikeda and Kusano, 1983). High viscosity digesta also lead to increased incidence of sticky droppings, increasing the occurrence of dirty eggs (Classen and Bedford, 1991). Exogenous enzymes can improve feed efficiency (FE), reduce pollution associated with poultry manure, and increase the use of low cost ingredients. In recent years, research has led to a much improved understanding of the ways in which exogenous enzymes can improve the nutritional value of wheat in poultry diets. One of the most commonly used enzymes for wheat-based diets is xylanase, which mainly acts on the arabinoxylan fraction of the wheat (Classen and Bedford, 1991), thereby releasing the nutrients encapsulated within the cell walls and making them more available to the endogenous enzymes. However, as protein appears to be a potential bonding point for arabinoxylan, enzymes capable of breaking these bonds might enhance pentosan release (Classen and Bedford, 1991). The inclusion of protease has therefore been suggested. Surprisingly little information is available regarding the feeding of wheat middlings (WM) to commercial laying hens. A product of milling, WM consist of fine particles of bran, germ, and aleurone layer (Patrick and Shaible, 1981). Summers et al. (1968) and Moran et al. (1970) evaluated wheat samples and milling fractions for energy utilization and protein quality in young growing chicks; however, WM were not included in their evaluation. Patterson et al. (1988) studied the effects of WM supplemented by cellulase as an alternate feedstuff for laying hens. The purpose of the present study was to compare different dietary levels of WM, with and without xylanase-protease supplementation, with a conventional 2DeKalb Poultry Research, Inc., DeKalb, IL Euribrid B. V., 5830 AA Boxmeer, The Netherlands. 4Finnfeeds International Ltd., Marlborough, Wiltshire, SN8 1AA, U.K. 5Gooch Milling and Elevator Co., Division of ADM Milling Co., Lincoln, NE JARONI ET AL. corn-soybean meal diet, with respect to laying hen performance, egg yields, and egg composition, in two strains of Leghorn hens, and to study the effect of xylanase and protease enzyme addition to WM diets. MATERIALS AND METHODS Experimental Design Two strains of Single Comb White Leghorn hens, DeKalb Delta2 (D) and Hisex White3 (H) (150 birds per strain), 42 wk of age, were housed in layer cages at a density of five birds per pen (412 cm2 per bird). An experimental unit consisted of one pen supplied with individual feed pan and nipple drinker. Factorial treatments (two strains by six diets) were randomly assigned to experimental units in five blocks; i.e., each replicate experimental unit appeared once in each row of cages, and each treatment was represented within each block. The birds had ad libitum access to water and were given 16 h of light. Diets The diets were formulated to be isocaloric (2.9 kcal ME/g) and isonitrogenous (17% CP) and to meet or exceed NRC (1994) nutrient requirements for laying hens. Data from diet analysis, according to the Association of Official Analytical Chemists (AOAC, 1990) procedure, showed slight variations in the protein content. Results are shown in Tables 1 and 2. Based on age and rate of production, the birds were fed the Phase II diet from 42 to 54 wk of age (Table 1) and the Phase III diet from 54 to 60 wk of age (Table 2) (NRC, 1994). The control diet was a standard corn-soybean meal diet. The other five diets had varying levels of standard WM added to the basal diet with or without the supplementation of enzymes. Diets 2 and 3 had 8 and 16% WM, respectively, without enzyme supplementation; Diet 4 had 8% WM with 0.1% enzyme; and Diets 5 and 6 had 16% WM with 0.1 and 0.2% enzyme supplementation, respectively. Laboratory analysis of the WM indicated a protein content of 19.0% for Phase II and 17.4 to 17.6% for Phase III; other nutritional values were taken from the NRC (1994) nutrient composition table. The enzyme added was a commercial preparation (Avizyme 1300) of xylanase and protease, provided by Finnfeeds International.4 The crude enzyme preparation was derived from the fermentation of a selected strain of Trichoderma viride and Bacillus subtilis, and the enzyme activity determined by the manufacturer was 2,500 units for xylanase and 800 units for protease. The wheatmiddlings used were provided by Gooch Milling Company5 and were added to the basal diet at the University Feed Mill (Mead, NE). The enzyme mix was added later at the Animal Science Department.

3 WHEAT MIDDLINGS AND ENZYME SUPPLEMENTATION 843 Ingredients TABLE 1. Composition of the basal diets (Phase II) Control (Diet 1) 16% 8% Middlings 2 Middlings 3 (Diet 2 Diet 3, 5, and 4) and 6) (%) Corn Soybean meal Oats Limestone Tallow Oyster shell Dicalcium phosphate Salt Corn oil Poultry premix Methionine Wheat middlings Lysine Calcium carbonate Nutrient composition ME, kcal/kg 2,820 2,820 2,825 Protein, calculated Protein, analyzed Fiber Ca Available P Methionine TSAA Lysine Poultry Premix provided per kilogram of diet Mn, 88 mg; Cu, 6.6 mg; Fe, 8.5 mg; Zn, 88 mg; Se, 0.30 mg; vitamin A, 6,600 IU; cholecalciferol, 2,805 IU; vitamin E, 10 IU; vitamin K, 2.0 mg; riboflavin, 4.4 mg; pantothenic acid, 6.6 mg; niacin, 24.2 mg, choline, 110 mg; vitamin B 12, 8.8 mg; ethoxyquin, 1.1 mg/kg. 2Diet 4 had 0.1% enzyme added to the basal diet. 3Diet 5 had 0.1% and diet 6 had 0.2% enzyme added to the basal diet. Measurements Hen Parameters. Hens were weighed individually every 4 wk and average pen weight was calculated to determine BW change. Eggs were collected daily and EP was calculated on a hen-day basis. Egg mass (EM) was calculated as a factor of egg weight (EW) and EP. Eggs with any adhering manure were classed as dirty, and the percentage calculated. Feed consumption was recorded on a daily basis and calculated per hen per day. Feed efficiency was calculated as grams of feed:grams of egg. Egg Parameters. Eggs were weighed every week for 1 d of production and the eggs were examined for shell quality by specific gravity (SP). The components of egg (albumen, yolk, and shell) were measured by biweekly breakouts on two eggs per replicate pen and expressed as percentage of EW. Statistical Analysis Analysis of variance was performed on the data as a randomized complete block design using the Repeated Measures Analysis of SAS software (Proc Mixed; SAS Institute, 1995), with the following model: Y ijkl = m +r k + a i + b j + ab ij + abr ijk +t l + abt ijl +e ijkl where Y ijkl = variable measured; m = overall mean; r k = block (treated as random effect); a i = diets (treated as fixed effect); b j = strain (treated as fixed effect); ab ij = test for an interaction between a and b; abr ijk = test for interaction between a i, b j and r k ;t l = time (in weeks); abt ijl = test for interaction between a i, b j and t l ;e ijkl = error component. Differences among means were determined by the following orthogonal contrasts, where main and interactions effects were taken into account: 1) Diet 1 vs 2 and 3; 2) Diet 1 vs 4, 5 and 6; 3) Diets 2 and 3 vs 4, 5 and 6; 4) Diet 5 vs 6; 5) Diet 1 vs 2 and 3 * Strain D vs H; 6) Diet 1 vs 4, 5 and 6 * Strain D vs H; 7) Diets 2 and 3 vs 4, 5 and 6 * Strain D vs H and 8) Diet 5 vs 6 * Strain D vs H. TABLE 2. Composition of the basal diets (Phase III) Ingredients RESULTS Hen Weights and Egg Production Egg production and hen BW change (Table 3) were not different between hens fed the control diet or those fed WM at 8 and 16% with or without enzyme supplementation. Average EP was 83.7, 85.7, 84.1, 84.2, 82.3, and 84.0% and average hen weights were 1.56, 1.59, 1.59, 1.60, 1.61, and 1.58 g for Diets 1, 2, 3, 4, 5, and 6, respectively. There were also no significant strain or diet by strain effects on EP and hen weights. Control (Diet 1) 16% 8% Middlings 2 Middlings 3 (Diet 2 (Diet 3, 5, and 4) and 6) (%) Corn Soybean meal Oats Limestone Tallow Oyster shell Dicalcium phosphate Salt Corn oil Poultry premix Methionine Wheat middlings Lysine Calculated nutrient composition ME, kcal/kg 2,820 2,820 2,820 Protein, calculated Protein, analyzed Fiber Ca Available P Methionine TSAA Lysine Poultry premix provided per kilogram of diet Mn, 88 mg; Cu, 6.6 mg; Fe, 8.5 mg; Zn, 88 mg; 0.30 mg; vitamin A, 6,600 IU, cholecalciferol, 2,805 IU; vitamin E, 10 IU; vitamin K, 2.0 mg; riboflavin, 4.4 mg; pantothenic acid, 6.6 mg; niacin, 24.2 mg, choline, 110 mg; vitamin B 12, 8.8 mg; ethoxyquin, 1.1 mg/kg. 2Diet 4 had 0.1% enzyme added to the basal diet. 3Diet 5 had 0.1% and diet 6 had 0.2% enzyme added to the basal diet.

4 844 JARONI ET AL. TABLE 3. Effects of dietary wheat middlings and enzyme supplementation on weight gain and hen parameters Dietary treatment Wheat Egg Body Feed Feed Strain 1 Enzyme middlings Diet production weight consumption efficiency (%) (kg) (g) (g feed:g egg) Main effects D H Interaction effects D H D H D H D H D H D H SEM D = DeKalb Delta and H = Hisex White. Feed Intake and Feed Efficiency A strain by diet interaction occurred for FC (P < 0.05), such that Strain D increased consumption of WM and enzyme when compared to Strain H for the same diets. Strain H already had a greater FC when fed the control diet and did not have a marked increase with WM. Strain H showed an improvement in FC in 8% WM diet supplemented with enzyme compared to 8% WM without enzyme supplementation. Diet by strain interactions were also observed for FE. Strain D had a better FE (1.93) than Strain H (2.11) when fed the control diet (P < 0.05) (Tables 3 and 4). Egg Weight, Egg Mass, and Specific Gravity Diets supplemented with enzyme improved EW significantly (P < 0.05) when compared with control and WM diets without enzyme. Average EW were 62.1, 62.4, 63.0, TABLE 4. Effects of dietary wheat middlings and enzyme supplementation on weight gain and hen parameters (statistical probabilities) Egg Body Feed Feed Source of variation production weight consumption efficiency ANOVA Diet NS NS NS NS Strain NS NS Diet * Strain NS NS NS NS Probability Contrasts 1 vs 2 & 3 NS NS NS NS 1 vs 4, 5 & 6 NS NS NS NS 2 & 3 vs 4, 5 & 6 NS NS NS NS 5 vs 6 NS NS NS NS Interaction effects (Diet Strain) 1 vs 2 & 3 D vs H NS NS vs 4, 5 & 6 D & H NS 0.01 NS NS 2 & 3 vs 4, 5 & 6 D vs H NS NS NS NS 5 vs 6 D vs H NS NS NS NS

5 1D = DeKalb and H = Hisex. WHEAT MIDDLINGS AND ENZYME SUPPLEMENTATION 845 TABLE 5. Effects of dietary wheat middlings and enzyme supplementation on egg parameters Dietary treatment Wheat Egg Egg Specific Dirty Strain 1 Enzyme middlings Diet weight mass gravity Albumen Yolk Shell eggs (%) (g) (g/hen/d) (%) Main effects D H Interaction effects D H D H D H D H D H D H SEM , 63.8, and 63.2 g for Diets 1, 2, 3, 4, 5, and 6, respectively. There was also a significant strain by diet effect (P < 0.05) with Strain D showing a greater increase in EW for 16% WM with 0.1% enzyme supplementation when compared to Strain H fed the same diet (Table 5 and 6). Egg mass showed a similar response, being greater in hens fed the enzyme-supplemented diet (53.6, 52.5, and 53.3 g for Diets 4, 5, and 6, respectively) when compared with the control diet (51.7 g). Egg mass was also significantly improved (P < 0.06) by the higher rate of enzyme supplementation (0.2%) in the 16% WM diet compared to the lower (0.1%) enzyme level. Strain of hen had a significant effect (P < 0.10) on EM, which was greater for Strain H (53.6 g) than for Strain D (52.4 g). Specific gravity declined significantly for WM diets with enzyme supplementation (P < 0.01) compared to those without. This decline was particularly noticeable in the H strain, which started at a SP of and declined to with supplementation of enzyme (Table 5). The SP of eggs from the control diet fed hens did not differ significantly from WM diets supplemented with enzyme. Overall, Strain D showed lower SP than Strain H for all the experimental diets. Egg Composition Percentage albumen was significantly higher (P < 0.05) for diets supplemented with enzyme when compared TABLE 6. Effect of dietary wheat middlings and enzyme supplementation on egg parameters (statistical probabilities) Egg Egg Specific Dirty Source of variation weight mass gravity Albumen Yolk Shell eggs ANOVA Diet NS NS NS NS Strain NS NS NS Strain * Diet 0.03 NS NS NS NS NS NS Probability Contrasts 1 vs 2 & NS NS NS NS 1 vs 4, 5 & NS NS NS 2 & 3 vs 4, 5 & NS NS NS 5 vs 6 NS 0.06 NS NS NS NS NS Interaction effects (Diet Strain) 1 vs 2 & 3 D vs H NS NS NS NS NS NS NS 1 vs 4, 5 & 6 D vs H NS NS NS NS NS NS NS 2 & 3 vs 4, 5 & 6 D vs H NS NS NS NS NS NS NS 5 vs 6 D vs H 0.04 NS NS NS NS NS NS

6 846 with control or WM diets without enzyme. Average percentage albumen was 60.5, 60.7, 61.0, 60.8, 60.7, and 60.8% for Diets 1, 2, 3, 4, 5, and 6, respectively. There were no strain or strain by diet effects on percentage albumen. Percentage yolk was also increased for WM diets supplemented with enzyme. Average percentage yolk was 27.0, 26.9, 26.8, 27.3, 27.2, and 27.3 for Diets 1, 2, 3, 4, 5, and 6, respectively. There was also a diet by strain effect (P < 0.05), in which Strain D showed an increase in percentage yolk for enzyme-supplemented diets in comparison to Strain H (Table 6). Percentage shell was affected by the strain of the hen. It was lower for Strain D (10.86) than for Strain H (11.2) throughout the experiment, for all the diets (P < 0.01). Percentage shell was not affected by dietary treatments. Dirty Eggs Percentage dirty eggs did not differ significantly among various dietary treatments. However, there was a highly significant (P < 0.01) strain effect, with Strain H having a higher percentage of dirty eggs (5.68) than Strain D (3.93). DISCUSSION Results of the present study have shown similar EP for hens fed control and WM diets with or without enzyme supplementation. Similar results were obtained by Brenes et al. (1993), who reported that enzyme addition to a laying diet containing wheat did not affect EP. Patterson et al. (1988) also found no effect of WM on EP. Feed consumption and FE where not significantly affected by diet, which is in contrast to other studies that have reported improved feed utilization due to enzyme addition (Pawlik et al., 1990; Brenes et al., 1993). Strain did, however, have a dramatic effect on FC. The fact that Strain D increased FC with high WM diets compared to Strain H, which did not show any increase in FC with WM diets, could be due to the type of diet the two strains are fed in different regions of the world. The H strain is bred in The Netherlands, where the characteristics of the breeding stock are selected in birds on a much more diverse diet that is higher in fiber. On the other hand, the D strain, bred in Illinois, is fed a simpler cornsoybean meal diet. The significant increase in EW and EM for hens fed enzyme-supplemented diets appears to be associated with increased albumen, which was greater for WM diets with enzyme than for control diets or WM diets without enzyme. The increase in albumen could have been the result of improved protein digestibility, and, thus, amino acid availability, through protease supplemented in the diet. Increase in EM with enzymesupplemented barley diets has been reported to be the result of increased amino acid availability (Wyatt, 1992). Egg mass was also shown to improve by 5% with JARONI ET AL. enzyme addition to barley diets in a study by Graham et al. (1993). Results obtained with barley can be correlated very well with wheat-based diets, as both grains contain the same anti-nutritional factor, nonstarch polysaccharide. Egg yolk also contributed toward increased EW for WM diets supplemented with enzyme in this study. The decline in SP with enzyme supplementation could have been a result of increased albumen and yolk relative to shell. This relationship is suggested by the fact that the H strain had increased egg albumen and yolk with increased enzyme supplementation and showed a corresponding decline in SP at a highly significant level. Studies in the past, on other cerealbased diets, have also reported that enzyme supplementation can have a negative effect on shell quality (Aimonen and Uusi Rawa, 1991). However, the overall SP values of to 1.086, obtained in the present experiment, are quite acceptable for optimal shell quality for this age of hens (42 to 60 wk) in Phases II and III of production. Strain differences observed for SP are in accordance with reports by Bowman and Challender (1963) and Al-Batshan et al. (1994), who showed that SP can vary with strain. Enzyme supplementation did not reduce the number of dirty eggs. The fact that Strain H produced more dirty eggs than Strain D could be due to the genetic differences between the two strains and the higher rate of FC by Strain H. The results of the present study show that WM with enzyme supplementation is promising as an alternative feed source for laying hens. Eggs were larger and heavier with WM-enzyme diets, most of which was attributable to increases in percentage albumen and yolk rather than shell. Even though EP per se was not increased by WM-enzyme diets in this study the increase in total product in the form of EW and EM would be economically advantageous. Further investigation is needed to sort out strain response differences to WM-enzyme diets as well as to determine the optimum levels of both WM and enzyme. ACKNOWLEDGMENTS The authors would like to thank Minnie Stephens, Lyle Robeson, and Tommi Jones for their technical assistance and Finnfeeds International for their financial support. REFERENCES Aimonen, E.M.J., and E. Uusi-Rawa, Replacement of barley by oats and enzyme supplementation in diets for laying hens. 1. Performance and balance trial results. Acta Agric. Scand. 41: Al-Batshan, A. H., S. E. Scheideler, B. L. Black, J. D. Garlich, and K. E. Anderson, Duodenal calcium uptake, femur ash and eggshell quality decline with age and increase following molt. Poultry Sci. 73:1590. Annison, G., Polysaccharide composition of Australian wheats and the digestibility of their starches in broiler chicken diets. Aus. J. Expt. Agri. 30:

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