A Study on the Interaction of Xylanase and Phytase Enzymes in Wheat-Based Diets Fed to Commercial White and Brown Egg Laying Hens 1

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1 A Study on the Interaction of Xylanase and hytase Enzymes in Wheat-Based s Fed to Commercial White and Brown Egg Laying Hens 1 F. G. Silversides,* 2 T. A. Scott,* 3 D. R. Korver, M. Afsharmanesh, and M. Hruby *Agassiz Research Centre, Agassiz, British Columbia, Canada V0M 1A0; Department of Agricultural, Food, and Nutritional Science, University of Alberta, Edmonton, Canada T6G 25; Department of oultry Science, Shahid Bahonar University, Kerman, Iran ; and Danisco Animal Nutrition, Marlborough, Wiltshire, United Kingdom SN8 1XN ABSTRACT A trial was conducted to investigate potential had lower albumen height; higher egg, shell, and albumen interactions between phytase and xylanase enzymes in wheat-based laying diets. Hens (480 ISA-White and 480 ISA-Brown) were distributed into 160 experimental units and fed one of 10 diets containing 75 to 77% wheat from 33 to 64 wk of age with a diet change at 49 wk. Two diets were adequate in content (0.3 and 0.25% available in the 2 phases) with or without xylanase (0 or 2,000 U/kg; Avizyme 2300, Danisco Animal Nutrition, Marlborough, Wiltshire, United Kingdom), and 8 diets had reduced (0.2 and 0.15% available ) with or without xylanase and phytase (0, 300, 500, and 700 ppu/kg; hyzyme 5000G, Danisco Animal Nutrition). Egg production was higher for ISA-Brown than for ISA-White hens, and ISA-Brown hens were larger. Eggs from ISA-Brown hens weights; and lower yolk weight than those from ISA- White hens. Egg production was not affected by the diet. In -reduced diets without xylanase, phytase significantly increased BW gain in the first period with no change in feed intake or feed efficiency. In -adequate diets, xylanase increased egg and albumen weight and albumen height. In -reduced diets with xylanase, increasing levels of phytase increased egg and albumen weight. This trial demonstrated no negative interactions between these enzymes for production traits and no interactions between the diet and strain of hen. These data suggest that poultry producers can use these enzymes individually or together in feed for the Brown and White egg layers used in this study without concern for the strain of hen. Key words: laying hen, xylanase, phytase, egg production, egg quality 2006 oultry Science 85: INTRODUCTION The poultry feed industry has greatly increased its use of exogenous enzymes over the past 15 yr. Adding appropriate exogenous enzymes to the feed can improve the extraction of nutrients from the feed, thereby decreasing feed costs, improving bird performance, and decreasing the environmental impact of manure application to land (Bedford and Schulze, 1998). To date, the industry has concentrated on enzymes that break down soluble nonstarch polysaccharides (NS), which are found in some cereals, and those that cause the release of from phytates, which are the primary storage form of in plants. Xylans are the principal NS of wheat, and high levels of wheat in poultry diets can increase the viscosity of the gut contents, which impedes the circulation and absorption 2006 oultry Science Association, Inc. Received July 18, Accepted October 1, Agriculture and Agri-Food Canada Contribution Number Corresponding author: silversidesf@agr.gc.ca 3 Current address: University of Sydney, rivate Mail Bag 3, Camden, New South Wales 2570, Australia. of nutrients, causing reduced feed intake, BW gain, and feed efficiency (Annison and Choct, 1991). Xylanase is used extensively in wheat-based diets to counteract the effects of NS in broiler (Bedford and Schulze, 1998) and layer diets (an et al., 1998; Scott et al., 1999a). The use of phytase in poultry diets has become widespread because it reduces the content of manure and can replace inorganic in feed, thereby reducing excretion and feed costs. Research on the effects of phytase in broiler diets has been extensive (Nelson et al., 1971; Broz et al., 1994; Silversides et al., 2004); research on the effects of phytase in layer diets has been less so (Van der Klis et al., 1997; Scott et al., 1999b, 2000, 2001; Keshavarz, 2003; Lim et al., 2003). Leske and Coon (1999) reported that commercial phytase increases the release of phytate from various feedstuffs fed to layers and broilers from between 23 and 34.9% to between 46.8 and 72.4%. Research has shown that xylanase increases digestibility of nutrients and that phytase increases retention; the effects of both are beneficial. However, some xylanases may increase the viscosity of gut contents when added at low levels because they can increase the release of NS from the cell walls (M. R. Bedford, Wiltshire, UK, personal communication), and phytase affects availability of many 297

2 298 SILVERSIDES ET AL. nutrients in addition to, including Ca, Mg, Fe, Cu, Mn, protein, and energy (Ravindran et al., 1995; Liu et al., 1998; Namkung and Leeson, 1999; Ravindran et al., 2000). Both xylanase and phytase cause the release of Ca and and could therefore disrupt the balance of these minerals, which is important to layers (Roland and Gordon, 1996), which need both minerals to ensure adequate eggshell quality and bone strength. Whereas adequate dietary Ca and are needed, an excess of Ca can interfere with the availability of other minerals (National Research Council, 1994) and an excess of can negatively affect eggshell quality (National Research Council, 1994; Scott et al., 1999b). Interactions have been found between xylanase and phytase used in broiler diets (Ravindran et al., 1999; Żyła et al., 2000; Juanpere et al., 2005), with phytase affecting the intestinal viscosity and xylanase affecting availability. The aim of this experiment was to evaluate the effects of including xylanase and phytase enzymes together in wheat-based laying diets fed to 2 strains of laying hens. MATERIALS AND METHODS At 18 wk of age, 480 ISA-White and 480 ISA-Brown pullets were housed in a cage facility with a day length of 14 h, which was increased gradually to a constant 16 h by 28 wk. Each cage held 3 pullets of the same strain, and adjacent cages formed experimental units. This provided 160 experimental units, with the strains randomly distributed within 8 blocks. Both feed and water were available ad libitum, and birds were cared for following Canadian Council of Animal Care (1993) guide. Experimental diets (Table 1) were fed from 33 to 64 wk in a phase feeding program with a diet change at 49 wk. Feed was formulated to provide adequate levels of all nutrients according to the National Research Council (1994) and management guides (ISA, 2000), and the calculated nutrient composition was the same for all diets except for enzyme additions and available levels. One dietary treatment was formulated to contain an adequate level of available and was fed without enzyme. The remaining diets were formulated to contain low levels of available and were fed with one of 2 levels of xylanase enzyme (0 and 2,000 U/kg; Avizyme 2300, Danisco Animal Nutrition, Marlborough, Wiltshire, UK) and 1 of 4 levels of phytase enzyme (0, 300, 500, and 700 ppu/kg; hyzyme 5000G, Danisco Animal Nutrition). All diets included 0.5% Celite (Celite Corp., Lompar, CA), an acid-insoluble ash, to allow determination of nutrient retention. The wheat was pelleted and reground before incorporation into the feed to destroy endogenous phytase (Bedford et al., 2000). Feed intake was based on measurements of 2-cage units. The birds were weighed at the start and end of the trial and at the diet change. Throughout the trial period, egg production was recorded for each cage for 5 d/wk and extrapolated to 7-d production. All eggs produced on 1 d/ wk were weighed. Data on feed intake were combined with those for egg production and egg weight to calculate efficiency of feed used for egg mass. When the hens were 38, 49, and 62 wk of age, all eggs produced on one day by the hens in one-half of the experimental units were collected and weighed and then broken for determination of albumen height and yolk weight. Shells were washed, dried, and weighed, and albumen weight was determined by difference. At 37, 54, and 62 wk, a 6-h collection of excreta was made from one-half of the units in each treatment group to allow calculation of AME. At the end of the trial, a blood sample was taken from the brachial vein of 8 hens of each strain per diet and allowed to coagulate. Serum was frozen and sent for analysis of Ca,, and Mg levels (British Columbia Agriculture, Food, and Fisheries, Abbotsford, British Columbia, Canada). These hens were euthanized by cervical dislocation. The right wing was removed, and the radius and humerus were cleaned of flesh and stored at 20 C. After thawing, the area and density of the cortical bone and the bone in the trabecular space of the radius and humerus were measured by quantitative computed tomography (QCT) following techniques described by Korver et al. (2004). Using this technique, medullary and trabecular bone are not distinguishable, and bone in the trabecular space at the midpoint of the bone is thought to be largely medullary. Finally, the breaking strength and bending stress (load corrected for cross-sectional area as determined by QCT with the assumption that the bone is circular) were determined (modified from Knott et al., 1995). Two measurements of cortical area of the humerus were deleted from the data set because their values were approximately twice the mean. All data were subjected to an ANOVA using the GLM program of SAS (Littell et al., 1991). The model used for all data except the egg quality data included the effects of strain of hen (2), diet (10), and the interaction between them. For some measures, an ANOVA was performed on data from only the -reduced diets, using the main effects of strain (2), xylanase (2), and phytase (4), along with interactions among these effects. The model used for analysis of egg quality data included the strain, diet, week, and the strain by week interaction. Other interactions were not significant and were removed from the model. The eggs produced from a single cage at different times likely represented different hens and so were not treated as repeated measures. Differences were considered to be significant when < RESULTS The ISA-Brown hens weighed more than the ISA-White hens at the start of the experiment, but gained less in both periods (Table 2). The ISA-Brown hens ate less and produced eggs more efficiently in both periods, but feed wastage was a problem for the ISA-White hens, and the data for these hens do not reflect their true consumption. The hens assigned to dietary treatments did not differ in BW at the start of the experiment. In the first phase (34 to 49 wk), addition of phytase had a significant effect on BW when diets had reduced and contained no supplemental xylanase, but phytase had no effect on BW when these diets

3 XYLANASE AND HYTASE FOR LAYERS 299 Table 1. Composition of diets fed to ISA-Brown and ISA-White layers from 34 to 64 wk -adequate diet -reduced diet Ingredient 1 34 to 49 wk 50 to 64 wk 34 to 49 wk 50 to 64 wk Wheat, hard red Soybean meal (47.5% C) Canola meal Limestone, ground Oyster shells, ground Corn dent gluten meal Vegetable oil Celite Ca dibasic (18% Ca, 21% ) NaCl Layer premix Lysine (98%) DL-Methionine Calculated composition ME, kcal/kg 2,776 2,776 2,776 2,776 C, % Available, % Ca, % One kilogram of Avizyme 2300 (Danisco Animal Nutrition, Marlborough, Wiltshire, United Kingdom) and 60, 100, or 140 g of hyzyme 5000G (Danisco Animal Nutrition) were added per ton to complete the diets. 2 Celite Corp., Lompar, CA. 3 The premix contained (/kg of diet): vitamin A, 9,600 IU; cholecalciferol, 3,120 IU; vitamin E, 36 IU; menadione, 2.4 mg; vitamin B12, mg; riboflavin, 7.2 mg; pantothenic acid, 14.4 mg; niacin, 60 mg; thiamine, 1.2 mg; pyridoxine, 2.4 mg; folic acid, 0.72 mg; biotin, 0.10 mg; Zn, 100 mg; Fe, 80 mg; Mn, 100 mg; Cu, 12 mg; I, 1 mg; and Se, 0.3 mg. (%) were supplemented with xylanase. In -adequate diets, the level of xylanase had no effect on BW gain. The differences in BW change associated with dietary treatment were not reflected in feed intake or feed efficiency. There was no significant effect of diet on BW change between 50 and 64 wk. by diet interactions for BW change, feed intake, or feed efficiency were not significant. The ISA-Brown hens produced significantly more eggs than ISA-White hens from 34 to 49 wk but not from 50 to 64 wk (Table 3). As expected, the eggs from ISA-Brown hens were heavier than those from ISA-White hens. There was no effect of diet on egg production, but the effect of diet was significant for egg weight late in production. An ANOVA (not shown) including only the -reduced diets with strain, xylanase, and phytase along with the 2- and 3-way interactions demonstrated a significant interaction between xylanase and phytase for egg weight from 50 to 64 wk. Reference to Table 3 shows that without xylanase, phytase level had little effect. Without either enzyme, egg weight appeared to be lower, but this was reversed with higher levels of phytase addition. The weights of the eggs that were broken for interior egg quality measures (Table 4) confirmed results shown in Table 3. As expected, age and strain of the hen had a significant effect on albumen height, egg weight, and weights of the principal components. The ISA-Brown hens laid eggs with a lower albumen height and greater shell and albumen weight, but slightly smaller yolks than those from ISA-White hens. As the hens aged, the albumen height decreased, and yolk weight increased. Egg and albumen weights increased with age of the hen, but those at 49 and 62 wk were not significantly different, and shell weight was highest at 49 wk. The effect of diet was significant for all measures. Eggs from hens fed the -adequate diets were larger and had greater albumen height and weight if the diets were supplemented with xylanase. Egg data from hens fed -reduced diets were also analyzed as a factorial (xylanase, phytase, strain, and diet; analysis not shown), which demonstrated an interaction between the enzymes for albumen height and yolk weight. As shown in Table 4, eggs from hens fed -reduced diets without xylanase had greater albumen height with addition of phytase, and yolk weight was lowest with intermediate levels of phytase. In -reduced diets with xylanase, phytase level had no significant effect on albumen height or yolk weight. The AME of the 10 diets was measured at 3 times over the course of the trial (Table 5) and varied between 2,710 and 3,280 kcal/kg. of hen did not significantly affect the AME, but the effect of diet was significant. In the - adequate diet, xylanase supplementation decreased AME significantly at 37 wk, whereas at 54 wk, the difference between xylanase supplemented and unsupplemented diets was not significant. At 62 wk, the AME of the 2 diets was identical. A separate ANOVA including only the - reduced diets with levels of xylanase (2) and levels of phytase (4) as main effects showed that the interaction between enzymes was significant at 37 and 54 wk and that each of the enzymes was significant at 64 wk, although the interaction was not. In the -reduced diet at 37 wk, the effect of phytase addition was inconsistent, but in opposite directions with and without xylanase. At 54 wk with - reduced diets and no xylanase, phytase supplementation increased AME, but with xylanase the lowest level of AME was observed with the highest level of phytase. At 62

4 300 SILVERSIDES ET AL. Table 2. Body weight change, feed intake, and feed efficiency of ISA-Brown and ISA-White hens fed one of 10 diets differing in level and xylanase and phytase supplementation 1 BW change Daily feed intake Feed conversion BW at 34 to 50 to 34 to 50 to 34 to 50 to Xylanase hytase 2 33 wk 49 wk 64 wk 49 wk 64 wk 49 wk 64 wk (U/kg) (ppu/kg) (g) (g of feed: g of egg) ISA-Brown 1,965 a 89 b 54 b 119 b 114 b 2.14 b 2.18 b ISA-White 1,655 b 115 a 80 a 122 a 121 a 2.32 a 2.34 a SEM Adequate 0 0 1, bc , , c Deficient 0 0 1, c , abc , abc , a , , abc , , ab , , abc , , abc SEM <0.01 <0.05 <0.05 <0.01 <0.01 <0.01 <0.01 NS <0.05 NS NS 0.07 NS NS diet NS NS NS NS NS NS NS a c Means within main effects without a common letter differ ( < 0.05). 1 Total number of observations was 160. wk, phytase supplementation of -reduced diets decreased AME both with and without xylanase. Serum Ca and Mg (Table 5) were both affected by the strain of hen; ISA-Brown hens had higher levels of both. The diet did not affect either mineral. When all diets were considered, serum was not significantly affected by either strain or diet, but when only -reduced diets were considered, phytase supplementation increased serum level Table 3. Egg production and weight of eggs produced by ISA-Brown and ISA-White hens fed 1 of 10 diets differing in phosphorus level and xylanase and phytase supplementation 1 Hen day egg production Egg weight 34 to 50 to 34 to 50 to Xylanase hytase 2 49 wk 64 wk 49 wk 64 wk (U/kg) (ppu/kg) (%) (g) ISA-Brown 91.6 a a 62.4 a ISA-White 89.1 b b 61.2 b SEM Adequate abc 2, a Reduced abc abc c ab 2, bc 2, bc 2, abc 2, abc SEM <0.01 NS <0.01 <0.01 NS NS NS <0.05 diet NS NS NS NS a c Means within main effects without a common letter differ ( < 0.05). 1 Total number of observations was 160 for each measure; each observation represents egg production by 6 hens.

5 XYLANASE AND HYTASE FOR LAYERS 301 Table 4. Albumen height, egg weight, and weight of the principal components of eggs produced by ISA-Brown and ISA-White laying hens fed 1 of 10 diets differing in phosphorus level and xylanase and phytase supplementation 1 Albumen Egg Shell Yolk Albumen Xylanase hytase 2 height weight weight weight weight (U/kg) (ppu/kg) (mm) (g) ISA-Brown 6.96 b 61.4 a 5.92 a 16.4 b 39.0 a ISA-White 7.65 a 59.9 b 5.84 b 16.7 a 37.4 b SEM Adequate c 59.9 cd 5.83 bc 16.4 bc 37.6 b 2, a 61.3 ab 5.97 ab 16.5 bc 38.8 a Reduced c 61.1 abc 6.01 a 16.8 ab 38.3 ab abc 60.5 abcd 5.90 abc 16.4 c 38.3 ab ab 60.0 bcd 5.80 c 16.4 bc 37.9 ab a 61.8 a 5.95 abc 16.9 a 38.9 a 2, abc 59.7 d 5.80 c 16.6 abc 37.3 b 2, a 59.7 d 5.84 abc 16.5 bc 37.3 b 2, ab 60.9 abcd 5.79 c 16.8 ab 38.3 ab 2, abc 61.4 a 5.91 abc 16.6 abc 38.9 a SEM Age 38 wk 7.65 a 59.2 b 5.81 b 15.8 c 37.6 b 49 wk 7.25 b 61.3 a 5.97 a 16.8 b 38.5 a 62 wk 6.95 c 61.7 a 5.87 b 17.2 a 38.6 a SEM <0.01 < <0.01 <0.01 <0.05 <0.01 <0.05 <0.05 <0.01 Age <0.01 <0.01 <0.01 <0.01 <0.01 age NS NS NS <0.01 NS a d Means within main effects without a common letter differ ( < 0.05). 1 Total number of observations for each measurement varied from 1,174 to 1,180. from 5.17 mg/dl with no phytase to 6.3 mg/dl with 700 ppu/kg of phytase. The density and area of the bone in the trabecular space of the radius were both affected by the strain of hen with ISA-White hens have greater density and ISA-Brown hens having greater area (Table 6). Cortical density of the radius was not different between strains, and cortical area was higher for ISA-Brown hens. The breaking strength and bending stress of the radius were also higher for ISA-Brown hens than for ISA-White hens. Of measures of the radius, only the trabecular area was affected by diet. Measures from hens on the -reduced diet and no enzymes were highest, and those from hens on the -reduced diet with xylanase at 500 ppu/kg were lowest. The humerus of only 4 hens contained trabecular bone (Table 7), and density of the bone in the trabecular space was not analyzed statistically. The area of the trabecular space of the humerus was greater for ISA-Brown hens than for ISA-White hens. Density of cortical bone in the humerus was slightly higher for ISA-White hens. by diet interactions were significant for cortical bone area, breaking strength, and bending stress and were investigated further as shown in Table 8. Among hens fed -reduced diets, phytase appears to have a different effect for area of cortical bone and breaking strength of the humerus for ISA-Brown hens than for ISA-White hens. The significant interaction between strain and diet for bending strength shown in Table 7 appears to be because the -adequate diet without enzyme produced the highest value (1.411 kg/mm 2 ) of all diets for ISA-Brown hens and the second lowest value for ISA-White hens (1.283 kg/mm 2 ). DISCUSSION The principal aim of this study was to investigate the interaction between xylanase and phytase enzymes in laying diets. Two strains of commercial layers were used because of the possibility that their digestion or mineral metabolism differed. roduction characteristics of the 2 strains confirmed previously reported results and are in line with breeder expectations. Body and egg weights of ISA-Brown hens were greater than those of ISA-White hens, but changed less over the production cycle, confirming results of Scott et al. (2001). Corresponding changes in egg and BW are expected because the traits are correlated (Emsley et al., 1977). Egg production was also higher for ISA-Brown hens, as was reported by Scott et al. (2001), and as shown previously (Scott and Silversides, 2000; Silversides and Scott, 2001), the height of the albumen and the yolk weight from ISA- Brown eggs were lower than those from ISA-White eggs, and albumen and shell weights were higher. Feed wastage by the ISA-White hens reduced the utility of data on feed intake and feed efficiency. Overall, the diet had only minor effects on production measures, possibly because the diet might not have been

6 302 SILVERSIDES ET AL. Table 5. Apparent metabolizable energy (DM basis) and serum Ca,, and Mg at 64 wk of ISA-Brown and ISA- White hens fed 10 diets differing in level and xylanase and phytase supplementation 1 AME Xylanase hytase 2 37 wk 54 wk 62 wk Ca Mg (U/kg) (ppu/kg) (kcal/kg of DM) (mg/dl) ISA-Brown 3,000 2,940 2, a a ISA-White 3,010 2,960 2, b b SEM Adequate 0 0 3,160 b 2,870 cd 2,980 ab , ,800 d 2,790 d 2,980 ab Reduced 0 0 2,940 c 2,930 bc 2,980 ab ,200 ab 2,940 bc 2,930 b ,710 d 3,050 a 2,850 c ,130 b 3,100 a 2,860 c , ,120 b 2,930 bc 3,030 a , ,760 b 3,060 a 2,940 b , ,280 a 3,020 ab 2,910 bc , ,950 c 2,810 d 2,940 b SEM NS NS 0.05 <0.01 NS <0.05 <0.01 <0.01 <0.01 NS 0.06 NS diet NS NS NS NS NS NS a d Means within main effects without a common letter differ ( < 0.05). 1 Total number of observations was 80 at each age (79 at 54 wk) for AME and 160 for each measure for serum Ca,, and Mg. deficient in. Without xylanase in the -reduced diet, phytase increased BW, as would be expected because low levels of reduce BW gain over the laying cycle (Scott et al., 2000, 2001), but phytase had no effect when xylanase was added, suggesting that the xylanase caused the release of enough to allow maximum BW gain. Keshavarz (2003) and Francesch et al. (2005) found that low levels decreased egg production and that phytase reversed the effect, but both studies used lower levels of than were used in this trial, where no effect of diet was observed. Table 6. Measures on the radius of 64 wk old ISA-Brown and ISA-White hens fed 1 of 10 diets differing in phosphorus level and xylanase and phytase supplementation 1 Trabecular Cortical Bending Breaking Xylanase hytase 2 Density Area Density Area strength stress (U/kg) (ppu/kg) (mg/cm 3 ) (mm 3 ) (mg/cm 3 ) (kg) (mm 3 ) (kg/mm 2 ) ISA-Brown 199 b 2.99 a a a 4.76 a ISA-White 233 a (79) 1.70 b (79) b b 3.66 b SEM Adequate ab , bc Reduced a ab bc ab , ab , ab , c , (15) 2.50 ab (15) SEM <0.01 <0.01 NS <0.01 <0.01 <0.01 NS <0.05 NS NS 0.09 NS diet NS NS NS NS NS 0.10 a c Means within main effects without a common letter differ ( < 0.05). 1 Total number of observations was 80 for each strain and 16 for each diet, unless indicated in parentheses following the mean.

7 XYLANASE AND HYTASE FOR LAYERS 303 Table 7. Measures on the humerus of ISA-Brown and ISA-White hens from 10 diets differing in phosphorus level and xylanase and phytase supplementation 1 Trabecular Cortical Breaking Bending Xylanase hytase 2 Density Area Density Area strength stress (U/kg) (ppu/kg) (mg/cm 3 ) (mm 3 ) (mg/cm 3 ) (mm 3 ) (kg) (kg/mm 2 ) ISA-Brown 127 (3) a 1,000 b (78) (79) (79) ISA-White 84 (1) b 1,024 a (79) SEM Adequate (2) , , , Reduced , , (2) , (14) (15) (15) , , , , , , (15) 2, , SEM <0.01 <0.01 <0.05 <0.05 <0.01 NS 0.08 NS NS NS diet NS <0.05 <0.05 <0.05 a,b Means within main effects without a common letter differ ( < 0.05). 1 Total number of observations was 80 for each strain and 16 for each diet, unless indicated in parentheses following the mean. Egg quality was affected by the diet. Egg weight late in production was increased by phytase in -reduced diets with xylanase but not in the diets without xylanase, suggesting an interaction between the enzymes that increased egg weight slightly. Several studies (Keshevarz, 2003; Scott et al., 2001; Francesch et al., 2005) have found that phytase increases egg weight, at least for some strains and diets, but the effects described were small. Data shown here also showed that the effect on egg weight was due to a difference in albumen weight rather than shell or yolk weight. Scott et al. (2001) also found that phytase increased albumen height, although they described this for corn-based diets but not wheat-based diets. Used in -adequate diets, xylanase increased albumen height. Used in -reduced diets without xylanase, phytase increased albumen height, whereas with xylanase, phytase had no effect. Factors contributing to albumen height are not well understood (Silversides and Scott, 2001), and these data suggest that may be involved without providing information on mechanisms. The AME extracted from the feed was very similar for the 2 strains, as could be expected because, although AME is affected by strain (March and Biely, 1971), both of these strains are high-producing laying strains and likely have a similar digestive physiology. In the -adequate diet, xylanase did not increase AME, which was not surprising because enzymes that reduce intestinal viscosity have their greatest effect in young birds (Jeroch and Dänicke, 1995). Alternately, it is possible that the level of NS in the wheat used was sufficiently low that there was little room for improvement with the addition of enzymes. In contrast to these results, an et al. (1998) found that a commercial crude enzyme preparation did increase the AME of wheatbased diets. The effects of enzymes in -reduced diets were inconsistent. At 54 wk, phytase increased AME, especially without xylanase, but at 62 wk, the opposite happened. At 37 wk, no pattern was apparent. Serum Ca and Mg levels were higher for ISA-Brown hens than for ISA-White hens. Higher serum Ca level could be related to differences in egg production or eggshell deposition because plasma Ca level changes with the ovulation cycle (Johnson, 1986). Serum levels were not affected by the strain of hen. had little effect on serum minerals, except that serum was increased by phytase when used in -reduced diets. This could be expected because phytase increases the release of (Leske and Coon, 1999) and because the serum levels in hens fed the - reduced diets without enzyme were low. The radius, which normally contains medullary bone, had lower bone density in the trabecular space of ISA- Brown hens than ISA-White hens. This may be related to the higher serum Ca and differences in egg laying because Ca is metabolized from the bone and transported to the oviduct for deposition on the shell (Simkiss, 1967). The greater trabecular area for the radius of ISA-Brown hens may relate to increased mobilization of cortical bone to support greater egg production and larger eggs from this strain. Endocortical thinning of bones is associated with the development of osteoporosis in laying hens (Whitehead et al., 2003). The density of cortical bone of the radius was not affected by the strain, but the increase in the area of cortical bone resulted in a greater breaking strength for ISA- Brown hens. An increase in cortical area, with no change in cortical density, would be expected to increase breaking

8 304 SILVERSIDES ET AL. Table 8. Cortical bone area, and bone breaking strength and bending stress of humeri of ISA-Brown and ISA- White hens fed -reduced diets differing in the addition of xylanase and phytase 1,2 Cortical area Breaking strength Bending stress ISA-Brown ISA-White ISA-Brown ISA-White ISA-Brown ISA-White (mm 3 ) (kg) (kg/mm 2 ) Xylanase, U/kg (31) , (31) SEM hytase, ppu/kg b b a a a (15) ab (15) b ab SEM Xylanase NS NS NS NS hytase NS <0.01 NS <0.05 NS NS Xylanase phytase NS NS NS NS NS NS a,b Means within main effects without a common letter differ ( < 0.05). 1 Total number of observations was 32 for each level of xylanase and 16 for each level of phytase, unless indicated in parentheses following the mean. strength. Despite this, the bending stress was not different between strains. This trial found that 4 humeri of the 160 measured contained bone in the trabecular space (which is confounded with medullary bone in the QCT measurements; Korver et al., 2004), confirming reports (Fleming et al., 1994; McCoy et al., 1996) that the humerus rarely contains medullary bone. The greater trabecular area for the humerus of ISA- Brown hens may relate to increased mobilization of bone Ca in hens of this strain to support higher egg production from 34 to 49 wk and greater egg size throughout the trial. The greater trabecular area may be a result of greater endocortical mobilization of bone, resulting in a decreased cortical area and an increase in trabecular space (Whitehead et al., 2003). The higher density and area of the cortical bone of the humeri of ISA-White hens is in contrast to findings on the radius, but is consistent with the higher breaking strength observed. As was found for the radius, bending stress was not affected by the strain of hen, suggesting a biological compensation for differences in body size. Budgell and Silversides (2004) found that ISA-Brown and Babcock 300 hens had similar levels of bone breakage, supporting this suggestion. Conversely, Riczu et al. (2004) reported that a brown-egg layer strain (Shaver 579) had greater humerus cortical area and breaking strength than a white-egg strain (Shaver 2000). The diet had little effect on measures of bone. The trabecular area of the radius of hens fed -reduced diets was highest without either enzyme, but was lowest when xylanase and 500 ppu of phytase/kg were added. This effect on the radius was observed despite the fact that the diet had no effect on production. The enzyme-free, -reduced diet might have caused the birds to mobilize more structural bone to support eggshell formation, in effect adding to the trabecular area. The addition of enzymes might have made up for at least part of the deficit. Rennie et al. (1997) reported that hens fed dietary total as low as 0.45% had trabecular and medullary bone amounts in the proximal tarsometatarsus and free thoracic vertebra that were not different from those of hens fed 0.6% total. The present study might have provided sufficient to avoid defects in bone mineralization. by strain interactions for several of the bone measurements suggested that the 2 strains may differ in their metabolism, and these interactions deserve further investigation. In this trial, egg production of ISA-Brown hens was as high as or higher than that of ISA-White hens. In addition, ISA-Brown hens weighed more and laid larger eggs, but body and egg weight of ISA-Brown hens increased less over the course of the trial than did those of ISA-White hens. Only minor interactions were observed between the xylanase and phytase enzymes when used in -reduced diets, and none appeared to be negative. There were no strain by diet interactions for any measures of production, and although these results apply only to the 2 strains tested, they can likely be extrapolated to other strains. ACKNOWLEDGMENTS The authors acknowledge Agriculture and Agri-Food Canada and Danisco Animal Nutrition for their support. We also recognize the dedication and care for the birds provided by the poultry staff of the acific Agri-Food Research Centre, and we thank Kerry Nadeau for QCT analysis and Wendy Clark for helpful discussions. REFERENCES Annison, G., and M. Choct Anti-nutritive activities of cereal non-starch polysaccharides in broiler diets and strategies minimizing their effects. World s oult. Sci. J. 47: Bedford, M. R., and H. Schulze Exogenous enzymes for pigs and poultry. Nutr. Res. Rev. 11:

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