2014 Poultry Science Association, Inc. Effects of xylanase supplementation of corn-soybean meal-dried distiller s grain diets on performance, metabolizable energy, and body composition when fed to first-cycle laying hens E. A. Bobeck,* N. A. Nachtrieb,* A. B. Batal, and M. E. Persia * 1 * Department of Animal Science, Iowa State University, Ames 50011; and Department of Poultry Science, University of Georgia, Athens 30602 Primary Audience: Industry Nutritionists, Researchers SUMMARY The increased cost of energy for laying hen diets has resulted in the use of enzymes, such as xylanase, to increase energy digestibility and thereby reduce the need for dietary energy. A 24--long experiment was conducted using 432 twenty-week-old Hy-line W36 first-cycle laying hens to determine the effects of energy concentrations and xylanase supplementation (Hostazym X 100) on hen performance, ME, and body composition. Three concentrations of dietary energy were fed with and without xylanase supplementation in a 3 2 factorial arrangement of treatments. Diets were reformulated twice over the experiment ( 30 and 40) due to increased feed intake or changes in egg production. Dietary energy main effect treatments included a control (C), C 77 kcal/kg, and C 154 kcal/kg diets. Egg production, feed intake, BW, egg weight, egg mass, and FE data were collected during the experiment. Nitrogen-corrected AME was determined at 32 and 44 of age, and featherless body composition (crude fat, CP, and ash) was determined at 44 of age. Hen-day egg production was increased in hens fed dietary xylanase during 31 to 40 and 20 to 44. Feed intake was increased with reduced dietary energy during all periods evaluated. Egg mass and FE were improved with xylanase supplementation during 31 to 40 and 20 to 44. Hen BW was not different among treatments and egg weight was only significantly different at select time periods with no consistent responses over time. Main effects at hen 32 showed that reduced energy resulted in reduced AME n and xylanase increased AME n. In contrast, xylanase treatment resulted in reduced AME n when measured at the end of 44. overall, supplementation of xylanse to laying hens increased egg production, egg mass, and FE, although AME n results were inconsistent and body composition data were dependent on dietary energy. Key words: laying hen, xylanase, production, energy, body composition 2014 J. Appl. Poult. Res. 23 :174 180 http://dx.doi.org/ 10.3382/japr.2013-00841 DESCRIPTION OF PROBLEM Ethanol and biofuel production diverts corn and oil from animal agriculture, resulting in an increased cost of dietary energy for laying hens [1]. Dietary enzymes have been developed to increase energy and nutrient utilization for poultry and other species [2, 3]. Xylanase enzymes have 1 Corresponding author: mpersia@iastate.edu
Bobeck et al.: XYLANASE SUPPLEMENTATION 175 been suggested to increase the production of soluble, fermentable oligomers, which results in enhanced cecal fermentation and improved utilization by poultry of grains such as barley and wheat, which contain a high content of soluble nonstarch polysaccharides (NSP) [4]. Xylanase preparations were first demonstrated as efficacious in high-soluble-nsp diets by reducing viscosity and improving nutrient utilization, digestion, and performance [5]. However, xylanase effectiveness in corn-soybean meal (SBM) diets has taken longer to demonstrate due to differences in constituent NSP [3]. Xylanase, in combination with a protease, increased egg weight and improved feed conversion in laying hens fed corn-oat-wheat middling-based diets [6]. A 12- experiment with second-cycle layers showed xylanase increased hen BW and egg weight inconsistently over the experimental period [7]. The objective of the present experiment was to determine the effects of dietary energy concentration and xylanase supplementation on laying hen production, ME, and body composition of hens fed corn-sbm-dried distillers grains with solubles (DDGS)-based diets. MATERIALS AND METHODS The experiment was approved by the Institutional Animal Care and Use Committee at Iowa State University and was conducted at the Iowa State University Poultry Research and Teaching Unit. A total of 432 seventeen-week-old Hy-Line W36 hens [8] were fed a standard laying hen diet over a 2- transition period, after which they were provided ad libitum access to 1 of 6 experimental diets and water. Initial photostimulation was 12L:12D upon receiving pullets, and the photoperiod was increased 0.5 h of light/ until 16L:8D was achieved and maintained. Hens were monitored twice daily, with mortalities removed from the cages and recorded as they occurred. Each experimental unit (EU) was defined as 3 adjacent cages of 3 hens (439 cm 2 /hen), resulting in 8 EU for each of the 6 treatment groups (72 hens per dietary treatment). Experimental diets were corn-sbm- DDGS-based and were arranged as a 3 2 factorial by reducing energy (soybean oil) from the control diet to provide 3 levels of energy: control (C), C 77 kcal/kg (C-77), and C 154 kcal/ kg (C-154; Table 1) with or without the addition of 0.01% Hostazym X 100 (target xylanase activity of 1,500 endo-pentosanase units/kg of diet) [9]. All 6 diets were mixed using an overall basal diet to generate 3 sub-basal diets before experimental diets were mixed. Experimental diets were generated by either mixing the nonenzyme diet alone or by the addition of enzyme (added on top of diet) mixed into approximately 2 to 3 kg of basal diet before mixing with the remainder of the experimental diet. Diets were reformulated over time due to increasing feed intake and changes in egg production, resulting in 3 dietary phases fed during 20 to 30, 31 to 40, and 41 to 44 of age, respectively. Eggs were collected daily at approximately 1100 h and egg production data were recorded by EU. Feed intake was determined weekly by calculating feed offered subtracting the amount of feed refused. Hens were weighed by cage and pooled to determine average hen weight per EU at the initiation of the experiment and every 4 until the completion of the experiment. Egg weight and egg mass were determined for a 1- period every 4 by combining 5 d of egg production [7]. Feed efficiency was calculated as the ratio of egg mass to feed intake and calculated for a 1- period every 4. Excreta samples were collected over the last 48 h of hen 32 and 44 for AME n determination [10]. On the last day of the experiment, 3 hens per EU (1 hen randomly selected per cage) were euthanized using carbon dioxide asphyxiation. Hens were defeathered before being ground for wet chemistry body composition analysis [11]. Data were analyzed using SAS statistical software [12]. RESULTS AND DISCUSSION Hen Performance Hen-day egg production was increased by 2.24 and 2.16% with xylanase supplementation over the 31- to 40- and 20- to 44- periods, respectively (Table 2; P 0.05). Dietary energy had no effect on hen-day egg production, although an interaction resulted at 41 to 44 because xylanase supplementation increased egg production when supplemented to the control diet but had no effect on egg production
176 JAPR: Research Report Table 1. Formulation of dietary treatments and nutrient composition of laying hen diets: control (C), C 77 kcal/kg (C-77), and C 154 kcal/kg (C-154), with or without 0.01% xylanase Item, % (unless otherwise noted) Wk Wk Wk C C-77 C-154 C C-77 C-154 C C-77 C-154 Ingredient Corn 43.11 44.70 46.30 50.51 52.11 53.70 48.37 49.96 50.90 Soybean meal (47.5% CP) 25.55 25.40 25.25 17.74 17.59 17.44 16.34 16.19 16.64 DDGS 1 10.00 10.00 10.00 10.00 10.00 10.00 15.00 15.00 15.00 Bakery meal 5.00 5.00 5.00 5.00 5.00 5.00 5.00 5.00 5.00 Soy oil 4.26 2.82 1.39 3.44 2.00 0.56 2.98 1.55 0.20 Salt 0.24 0.24 0.24 0.19 0.19 0.19 0.16 0.16 0.16 dl-met 2 0.19 0.19 0.19 0.12 0.12 0.11 0.10 0.10 0.09 l-lys 3 0.06 0.06 0.06 0.09 0.09 0.10 0.11 0.12 0.09 Limestone, small 4 4.89 4.89 4.89 4.90 4.90 4.90 4.93 4.93 4.93 Limestone, large 5 4.89 4.89 4.89 4.90 4.90 4.90 4.93 4.93 4.93 Dicalcium phosphate 1.21 1.20 1.20 1.26 1.25 1.25 1.16 1.15 1.15 Choline chloride 60% 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 Vitamin-mineral premix 6 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 Phytase 7 0.006 0.006 0.006 0.006 0.006 0.006 0.006 0.006 0.006 Celite 1.00 1.00 1.00 Titanium dioxide 0.25 0.25 0.25 0.25 0.25 0.25 Calculated nutrient profile CP 19.29 19.33 19.37 16.12 16.16 16.19 16.62 16.66 16.91 Determined CP 19.05 19.92 18.60 16.10 15.92 16.37 17.03 17.50 18.61 Digestible sulfur amino acids 0.74 0.74 0.74 0.60 0.60 0.60 0.60 0.60 0.60 Digestible Lys 0.89 0.89 0.89 0.71 0.71 0.71 0.71 0.71 0.71 Digestible Thr 0.66 0.67 0.67 0.55 0.55 0.55 0.56 0.56 0.57 ME, kcal/kg 2,900 2,823 2,746 2,875 2,798 2,721 2,850 2,773 2,696 Determined crude fat 6.51 5.21 3.88 5.38 4.51 3.08 6.68 8 5.37 8 4.13 8 Linoleic acid 2.75 2.21 1.67 2.54 2.00 1.46 2.42 1.88 1.36 Calcium 4.10 4.10 4.10 4.10 4.10 4.10 4.10 4.10 4.10 Nonphytate P 0.35 0.35 0.35 0.35 0.35 0.35 0.35 0.35 0.35 Determined ash 13.63 14.33 14.20 14.95 14.46 13.94 ND 8 ND ND 1 Dried distiller s grains with solubles produced from corn grain. 2 dl-methionine (Evonik Industries, Kennesaw, GA) contains 99% dl-met. 3 Bio-Lys (Evonik Industries) contains 50.7% of l-lys in the form of l-lys sulfate. 4 Unical-P (ILC Resources, Alden, IA) contains a finely ground calcium carbonate. 5 Bone and Shell Builder (ILC Resources) contains a coarse particulate calcium carbonate. 6 Vitamin and mineral premix contained the following per kilogram of diet: vitamin A, 6,605 IU; vitamin E, 14.31 IU; cholecalciferol, 2,200 IU; menadione, 880 µg; vitamin B 12, 9.3 µg; biotin, 33.0 µg ; choline, 357 mg; folic acid, 1,100 µg; niacin, 33.0 mg; pantothenic acid, 8.81 mg; pyridoxine, 0.88 mg; riboflavin, 4.4 mg; thiamine, 1.1 mg; iron, 102.5 mg; magnesium, 10 mg; manganese, 100 mg; zinc, 100 mg; copper, 10 mg; iodine, 0.7 μg; and selenium, 200 µg. 7 Escherichia coli phytase was added at 300 phytase units per kilogram of diet. 8 Not determined or the value was calculated. when supplemented to the C-77 and C-154 diets. This is inconsistent with overall egg production and might be related to elevated environmental temperature over the 41- to 44- period. Feed intake was significantly increased in hens fed low-energy diets versus control-fed birds over all periods measured (Table 2; P 0.05) and enzyme supplementation had no effect on feed intake. It is interesting to note that W36 laying hens fed reduced dietary energy were able to increase feed intake over the experiment; this is in contrast to previous reports [13, 14]. Although feeding second-cycle W36 laying hens diets containing 4 concentrations of energy resulted in decreased feed consumption as energy increased, these were second-cycle hens that had increased BW (and the ability to consume more feed) in relationship to the smaller first-cycle hens in this experiment [7]. Similar data in other strains of laying hens have
Bobeck et al.: XYLANASE SUPPLEMENTATION 177 also been reported. Bovans White and Dekalb White first-cycle laying hens were able to adjust dietary intake to maintain energy intake [15]. No significant main effects or interactions for egg weight were observed (data not shown), as egg weights increased from 54.6 to 61.4 g/egg over the experimental period. Dietary energy had no significant effect on egg mass or FE throughout the experiment and no interactions were noted between dietary energy and enzyme supplementation. Xylanase treatment increased egg mass during the 31- to 40- and 20- to 44- periods and FE during the 31- to 40- period (P 0.05; Table 3). Previous work with xylanase supplementation in hens was shown to increase egg weight and mass [6]. In the current experiment, egg mass was increased due to increased egg production and not increased egg weight. Regardless of treatment, egg mass was reduced during 44 of age; again, this decrease in egg mass corresponds to elevated environmental temperature. The SEM for both egg mass and FE from 41 to 44 is elevated in comparison to the earlier time points and suggests an adverse response to the elevated environmental temperature. As only one interaction was seen between enzyme and dietary energy for egg production ( ; during a time of elevated environmental temperature), and none for feed intake, egg mass, or FE, it appeared that effects of xylanase on laying hen performance were independent of dietary energy. No treatment differences in hen BW were noted for the duration of the experiment. Hen BW were 1.32, 1.44, 1.48, 1.48, 1.50, 1.51, and 1.51 kg for 20, 24, 28, 32, 36, 40, and 44, respectively. In total, 5 mortalities were reported for the experiment; 3 in the control diet without xylanase, and 1 each in the reduced energy diets without xylanase. AME n Results for AME n data were inconsistent between 32 and 44. At 32, reducing dietary energy significantly reduced AME n, whereas the addition of xylanase resulted in a significant increase in AME n (main effects of both energy and enzyme on AME n, P 0.05; Table 4). At 44, xylanase reduced AME n (P 0.05) and no significant effects due to energy were observed. The opposite results between 32 and 44 are perplexing, although the inconsistent AME n results coincide with increased variability in Table 2. Hen-day egg production and feed intake (by age) of hens fed 3 dietary energy concentrations with and without xylanase 1 Hen-day egg production, % Feed intake, g/hen per day Energy Enzyme Control (C) 82.5 94.0 88.8 88.3 90.3 b 104.5 b 102.9 b 95.5 b C 77 kcal/kg 82.0 95.7 91.3 89.2 92.1 ab 106.8 a 104.1 b 97.4 a C 154 kcal/kg 81.2 94.8 92.2 88.6 93.1 a 107.4 a 107.3 a 98.3 a SEM 1.25 0.82 0.86 0.78 0.71 0.63 0.84 0.66 P-value 0.78 0.35 0.02 0.72 0.03 0.01 0.01 0.02 None 80.9 93.8 b 90.3 87.8 b 91.7 106.2 105.3 97.0 Xylanase 82.9 95.9 a 91.1 89.7 a 92.0 106.2 104.2 97.1 SEM 1.02 0.67 0.70 0.64 0.58 0.51 0.69 0.54 P-value 0.17 0.03 0.42 0.04 0.71 0.96 0.24 0.82 C None 82.0 92.0 86.7 b 87.0 89.2 103.6 102.8 94.6 Xylanase 82.9 95.9 90.9 a 89.6 91.4 105.4 102.9 96.4 C 77 kcal/kg None 80.4 94.6 92.1 a 88.2 92.3 106.6 104.4 97.5 Xylanase 83.6 96.7 90.6 a 90.2 91.8 106.9 103.8 97.3 C 154 kcal/kg None 80.2 94.6 92.3 a 88.2 93.5 108.3 108.7 98.8 Xylanase 82.2 95.1 91.9 a 89.1 92.8 106.4 105.9 97.7 SEM 1.77 1.16 1.22 1.11 1.01 0.89 1.19 0.93 P-value 0.81 0.36 0.05 0.74 0.27 0.11 0.45 0.28 a,b Columns without similar superscript letters denote significant difference (P 0.05). 1 n = 16 for dietary energy, n = 24 for enzyme comparison, and n = 8 for the interaction.
178 JAPR: Research Report Table 3. Egg mass and FE (by age) of hens fed 3 dietary energy concentrations with or without xylanase 1 Egg mass, g/hen per day FE, g of egg/kg of feed intake Energy Enzyme Control (C) 52.4 55.9 52.4 54.2 557 534 508 538 C 77 kcal/kg 52.4 57.0 55.0 55.1 546 536 505 534 C 154 kcal/kg 52.6 56.1 55.5 54.8 541 523 491 524 SEM 0.75 0.46 1.01 0.51 7.5 5.9 18.8 6.4 P-value 0.98 0.20 0.08 0.43 0.31 0.26 0.78 0.30 None 51.7 55.4 b 53.8 53.9 b 542 523 b 499 526 Xylanase 53.2 57.2 a 54.8 55.5 a 555 539 a 504 539 SEM 0.61 0.38 0.83 0.42 6.0 4.8 15.3 5.3 P-value 0.09 0.01 0.40 0.01 0.12 0.02 0.83 0.09 C None 51.7 54.2 50.4 52.8 557 525 517 535 Xylanase 53.2 57.5 54.4 55.6 558 544 500 542 C 77 kcal/kg None 51.3 56.0 56.0 54.4 532 526 524 527 Xylanase 53.4 58.0 54.0 55.8 561 546 487 541 C 154 kcal/kg None 52.2 56.0 55.0 54.5 536 519 457 515 Xylanase 53.0 56.2 56.0 55.1 547 527 525 533 SEM 1.06 0.65 1.42 0.72 10.5 8.3 26.1 9.1 P-value 0.83 0.06 0.13 0.32 0.45 0.73 0.13 0.82 a,b Columns without similar superscript letters denote significant difference (P 0.05). 1 n = 16 for dietary energy, n = 24 for enzyme comparison, and n = 8 for the interaction. FE and egg mass during the time of increased environmental temperature that could have adversely affected AME n measured at 44. Previous research has demonstrated that heat stress can alter energy digestibility in 4--old broiler chickens fed a nutrient-dense summer diet and might explain some of the inconsistency in the data collected at 44 of age during elevated environmental temperatures [16]. The data reported in 32 agree with previous reports, as xylanase significantly improved AME n and metabolizability coefficients of amino acids in laying hens fed wheat, rye, or SBM diets [17], albeit these diets have a greater level of soluble NSP. Body Composition Wet chemistry analysis of hen body composition revealed no differences in carcass protein or ash content. However, an interaction between dietary energy and enzyme supplementation was noted, as xylanase treatment increased carcass fat in birds fed the C diet, decreased carcass fat in hens fed the C-77 diet, and had no effect on birds fed the C-154 diet (Table 5). The high-energy (control) diet with the xylanase treatment could be expected to result in higher carcass fat if excess energy is stored after productive energy requirements are met, but the higher carcass fat from the C-77 diet without xylanase is unexplained. Although few data have been pub- Table 4. Nitrogen-corrected AME (kcal/kg) of first-cycle laying hens fed various dietary energy concentrations with or without xylanase at 32 and 44 of age 1 Energy Enzyme Wk 32 Wk 44 Control (C) 3,042 a 3,112 C 77 kcal/kg 3,002 a 3,057 C 154 kcal/kg 2,945 b 3,123 SEM 19.2 21.1 P-value 0.01 0.07 None 2,953 b 3,123 a Xylanase 3,039 a 3,071 b SEM 15.7 17.2 P-value 0.01 0.04 C None 3,004 3,130 Xylanase 3,080 3,094 C 77 kcal/kg None 2,956 3,120 Xylanase 3,049 2,994 C 154 kcal/kg None 2,900 3,120 Xylanase 2,990 3,126 SEM 27.1 29.8 P-value 0.95 0.09 a,b Columns without similar superscript letters denote significant difference (P < 0.05). 1 n = 16 for dietary energy, n = 24 for enzyme comparison, and n = 8 for the interaction.
Bobeck et al.: XYLANASE SUPPLEMENTATION 179 Table 5. Body composition of defeathered hens fed 3 dietary energy concentrations with or without xylanase 1 Energy, % Enzyme Carcass protein Carcass fat Carcass ash Control (C) 43.8 41.0 9.25 C 77 kcal/kg 43.9 39.6 9.29 C 154 kcal/kg 45.1 37.3 9.42 SEM 0.77 0.72 0.279 P-value 0.43 0.01 0.90 None 44.5 39.3 9.24 Xylanase 44.0 39.3 9.40 SEM 0.63 0.59 0.228 P-value 0.51 0.95 0.64 C None 44.8 39.1 bc 9.31 Xylanase 42.9 42.9 a 9.19 C 77 kcal/kg None 44.2 41.4 ab 8.90 Xylanase 43.7 37.9 c 9.68 C 154 kcal/kg None 44.8 37.5 c 9.52 Xylanase 45.4 37.0 c 9.32 SEM 1.09 1.03 0.394 P-value 0.52 0.01 0.39 a c Columns without similar superscript letters denote significant difference (P < 0.05). 1 n = 16 for dietary energy, n = 24 for enzyme comparison, and n = 8 for the interaction. lished regarding the effects of dietary energy on hen body composition, it appears that carcass fat content may be a more sensitive indicator of dietary energy status than egg production. These data are in agreement with previous research showing hens reduced energy storage in the abdominal fat pad before reducing egg production, when limit-fed diets with a 90-kcal/kg reduction in dietary energy [13]. CONCLUSIONS AND APPLICATIONS 1. The addition of xylanase (Hostazym X100) increased egg production, egg mass, and FE of first-cycle Hy-Line W36 laying hens ( of age) regardless of dietary energy. 2. Hy-Line W36 laying hens were sensitive to dietary energy, as both 77 and 154 kcal/kg reductions in dietary energy content resulted in increased feed intake over the experiment. 3. At 32, AME n decreased as dietary energy decreased, whereas xylanase supplementation increased AME n ; at 44, under elevated environmental temperatures, dietary energy did not affect AME n and xylanase supplementation decreased AME n. High-energy xylanase diets increased carcass fat, suggesting that this variable might be a more sensitive indicator of dietary energy than egg production. REFERENCES AND NOTES 1. Pardue, S. L. 2010. Food, energy, and the environment. Poult. Sci. 89:797 802. 2. Bedford, M. R., and H. Schulze. 1998. Exogenous enzymes for pigs and poultry. Nutr. Res. Rev. 11:91 114. 3. Slominski, B. A. 2011. Recent advances in research on enzymes for poultry diets. Poult. Sci. 90:2013 2023. 4. Choct, M., R. J. Hughes, J. Wang, M. R. Bedford, A. J. Morgan, and G. Annison. 1996. Increased small intestinal fermentation is partly responsible for the anti-nutritive activity of non-starch polysaccharides in chickens. Br. Poult. Sci. 37:609 621. 5. Mathlouthi, N., M. A. Mohamed, and M. Larbier. 2003. Effect of enzyme preparation containing xylanase and beta-glucanase on performance of laying hens fed wheat/ barley- or maize/soybean meal-based diets. Br. Poult. Sci. 44:60 66. 6. Jaroni, D., S. E. Scheideler, M. Beck, and C. Wyatt. 1999. 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. Poult. Sci. 78:841 847. 7. Gunawardana, P., D. A. Roland, and M. M. Bryant. 2009. Effect of dietary energy, protein, and a versatile enzyme on hen performance, egg solids, egg composition, and egg quality of Hy-Line W-36 hens during second cycle, phase two. J. Appl. Poult. Res. 18:43 53. 8. Hy-Line International, Dallas Center, IA. 9. Enzymes was added at manufacturer s recommendations (0.1% of the diet) and contained a minimum of 15,000
180 JAPR: Research Report endo-pentosanase units of xylanase activity per gram of premix derived from Trichoderma longibrachiatum (Huvepharma, Sofia, Bulgaria). Diet samples were analyzed for enzyme activity at Eurofins Scientific Inc. (Des Moines, IA) using the manufacturer s proprietary method. Enzyme analysis for diets 1 to 6 was <100, <100, 180, 1,430, 1,550, and 1,380 endo-pentosanase units/kg of diet, respectively. 10. The collected excreta samples were frozen at 20 C before they were oven-dried at 65 C for 3 d to determine DM. Feed samples were corrected to a DM basis by measuring 5.0 g of each diet, drying them in an oven at 100 C for 24 hr, and calculating the ratio between the dry weight and predry weight. The excreta samples were then ground through a 1-mm screen and the feed samples were ground using a 0.5-mm screen (Brinkmann Instruments Company, Westbury, NY). Feed and excreta samples were assayed for the AME n by determining the gross energy (GE) content using an adiabatic oxygen bomb calorimeter and nitrogen content by the micro-kjeldahl method using a Kjeltech 1028 distilling unit (U.S. Tecator Inc., Herndon, PA) [18]. Titanium dioxide was determined in the excreta and feed samples to calculate the AME n as AME n = Diet GE [Excreta GE Diet Ti/Excreta Ti 8.22 (Diet N Excreta N Diet Ti/ Excreta Ti)] [19, 20]. 11. Ground carcass samples were freeze-dried before being analyzed for CP (LECO Tru-Mac N, St. Joseph, MI), ash [21], and crude fat [22]. After euthanasia, the hen carcass was submerged in water heated between 60 and 71 C for approximately 15 to 20 s before feather removal via a counter-top poultry plucker drum style with one-third horsepower electric motor. Feather removal was stopped after a visual inspection for completeness although hair and pinfeathers were not removed with any sort of flame treatment. Hens were defeathered to reduce variance due to incomplete feather grinding resulting in differences in ground particulate size and a nonhomogenous sample. 12. All data, with the exception of feed intake and egg production, were subjected to a two-way ANOVA using the GLM procedures of SAS (SAS Institute, Cary, NC) as a 2 3 factorial, and if ANOVA was significant means were separated using the least significance procedure. Feed intake and egg production data were subjected to a two-way ANOVA using repeated measures within the mixed procedures of SAS as a 2 3 factorial, and if ANOVA was significant means were separated using Tukey s method. All data were presented as means with pooled SEM estimates and were considered significantly different if P 0.05. Three consecutive cages within a dietary treatment were considered an experimental unit with a randomized complete block design. Hens were assigned to treatment groups using blocks within house and treatments were randomized within block. Measurements for BW, egg weight, egg mass, and FE were used as subsamples within each feeding period for statistical analysis. The 3 birds collected for body mass determination were pooled within each experimental unit. For all parameters measured n = 24 for enzyme comparison, n = 16 for dietary energy, and n = 8 for the interaction. 13. Murugesan, G. R., and M. E. Persia. 2013. Validation of the effects of small differences in dietary ME and feed restriction in first-cycle laying hens. Poult. Sci. 92:1238 1243. 14. Harms, R. H., V. Olivero, and G. B. Russell. 2000. A comparison of performance and energy intake of commercial layers based on body weight or egg weight. J. Appl. Poult. Res. 9:179 184. 15. Wu, G., M. M. Bryant, R. A. Voitle, and D. A. Roland Sr.. 2005. Effect of dietary energy on performance and egg composition of Bovans White and Dekalb White hens during phase I. Poult. Sci. 84:1610 1615. 16. Bonnet, S., P. A. Geraert, M. Lessire, B. Carre, and S. Guillaumin. 1997. Effect of high ambient temperature on feed digestibility in broilers. Poult. Sci. 76:857 863. 17. Pirgozliev, V., M. R. Bedford, and T. Acamovic. 2010. Effect of dietary xylanase on energy, amino acid and mineral metabolism, and egg production and quality in laying hens. Br. Poult. Sci. 51:639 647. 18. AOAC. 2006. Official Method of Analysis 984.13. Total Nitrogen or Crude Protein using Kjeldahl. AOAC International, Gaithersburg, MD. 19. Leone, J. L. 1973. Collaborative study of quantitative-determination of titanium-dioxide in cheese. J. Assoc. Off. Anal. Chem. 56:535 537. 20. Scott, M. L., M. C. Nesheim, and R. J. Young. 1982. Nutrition of the Chicken. 3rd ed. Scott and Associates, Ithaca, NY. 21. AOAC. 1980. Official Method of Analysis 7.003. Determination of Moisture. AOAC International, Gaithersburg, MD. 22. AOAC. 2005. Official Method of Analysis 960.39, 39.1.05, 18th ed. Determination of Fat (Crude) or Ether Extract in Meat. AOAC International, Gaithersburg, MD.