The effect of drying method temperature, collection method, and marker type on apparent ileal amino acid digestibility in 21-day-old broilers fed corn-soybean meal-barley based diet

The effect of drying method temperature, collection method, and marker type on apparent ileal... ABSTRACT For accurate estimation of nutrient digestibility, an ideal drying and sampling method is required to preserve the quality of the digesta. A standard corn-soybean meal (corn-SBM) broiler starter diet was fed from d 0 to 10 before birds were placed on the experimental diets until d 21. One hundred and sixty-eight male Cobb 500 broiler chicks were used to evaluate the effect of two drying methods (freeze-dryer vs. forced air-oven) and two drying temperatures (40 vs. 55°C) (Exp 1), while ninety-six chicks were used to evaluate the effect of flushing and squeezing as well as marker types (titanium vs. chromium) on apparent ileal DM, N, Ca, P, and AA digestibility (Exp 2). There were seven (Exp 1) or eight (Exp 2) replicate cages per treatment with 6 birds/cage. Digesta from the distal two thirds of the ileum was obtained from birds following euthanasia on d 21 by squeezing (Exp 1) and squeezing or flushing (Exp 2). Samples collected were stored in the freezer at −20°C until they were either freeze-dried (FD) or oven-dried (OD) at 40 or 55°C. There were no interactions between the drying methods and drying temperatures (Exp 1) on apparent ileal DM, N, and AA digestibility. Met had the highest (92.3%) while Cys had the lowest (73.8%) digestibility value. In Exp 2, no interaction between sampling methods and marker types was observed. The effect of sampling methods was not significant except for Arg and Met where squeezing resulted in higher (P < 0.05) digestibility values. Furthermore, apparent ileal His, Ile, Cys, Ser, and Tyr digestibility tended to be higher (P < 0.1) in squeezed digesta compared to the flushed digesta. Results from these studies showed that OD ileal digesta at 40 or 55°C had no negative effect on apparent ileal AA digestibility. Likewise, marker type did not influence apparent ileal AA digestibility values. INTRODUCTION Nutrient digestibility studies address the importance of accurately estimating how efficiently nutrients in diets are digested and absorbed in the mid-section of the gastrointestinal tract. In avian species, the bio-availability of each amino acid (AA) is critical for evaluating the nutritional value of the feedstuff and for estimating AA requirement of poultry (Poureslami et al., 2012; Kong and Adeola, 2014). Ileal digestibility procedure has been routinely used to estimate AA requirement because excreta AA contents can be influenced by the microorganisms in the hindgut and this microbiota can metabolize some of the undigested AA thus modifying the contents of the excreta (Ravindran et al., 1999; Pig, 2000; Kong and Adeola, 2014). In determining ileal digestibility of nutrients in broilers, several sampling and processing methods have been adopted over the years. Ileal digesta samples are usually collected by gently squeezing (Short et al., 1999; Bandegan et al., 2009; Poureslami et al., 2012) or flushing (Ravindran et al., 1999, 2004; Kluth et al., 2005; Adedokun et al., 2007), and subsequently oven dried (OD) or freeze-dried (FD) prior to chemical analysis. The aforementioned methods have been a bone of contention with no consensus reached as to the standard procedure for preparing samples prior to chemical analyses (Jacobs et al., 2011). Drying is often described as the ratio of percent moisture removed without changing the initial chemical structure of a particular substance (Jindal and Siebenmorgen, 1987). Jindal and Siebenmorgen (1987) suggested that moisture loss using different drying methods may not be the same due to the empirical nature of the methods. Oven drying requires less time than freeze drying which takes longer and subsequently increases the cost and time of sample drying (Hinnant and Kothmann et al., 1988). Research also showed that poultry excreta samples that were OD at 65°C resulted in an average energy loss of about 12% (Manoukas et al., 1964) while nitrogen (N) loss when FD or OD at 60°C resulted in a mean loss of approximately 4.7% (Shannon and Brown, 1969). However, a study conducted by another group indicated that the effect of FD or OD of excreta samples at 60°C on true AA availability, TME, and nitrogen-corrected TME were not significant (Dale et al., 1985). The use of an inert marker in determining the rate of nutrient disappearance along the gastrointestinal tract, its chemical analysis, and calculations have been reported by several authors (Jagger et al., 1992; Short et al., 1996; Adeola, 2001; Myers et al., 2004). The effect of different inert (index) dietary markers in animal digestibility studies is inconclusive with some studies showing higher ileal AA digestibility values when titanium dioxide was used compared to chromic oxide. A higher recovery rate was observed for titanium (Ti) when compared to chromium (Cr) in the ileal digestibility values of N and individual AA (Jagger et al., 1992). Similarly, Olukosi et al. (2012) concluded that apparent ileal AA digestibility values were higher when Ti rather than Cr was the inert marker. Furthermore, a low recovery rate of Cr has been attributed to analytical problems caused by the presence of phosphorus and other minerals (Peddie et al., 1982; Saha and Gilbreath, 1991; Yin et al., 2000), as well as the possibility that Cr may lack uniform distribution in the digesta across the gastrointestinal tract (Oberleas et al., 1990; Sooncharernying and Edwards, 1993). Jacobs et al. (2011) evaluated the effect of drying method on total tract N digestibility in poultry excreta while Poureslami et al. (2012) evaluated the effect of sampling method on apparent ileal AA digestibility in broiler chickens. In this study, we attempted to evaluate the effect of sampling method on ileal AA digestibility of a diet that is expected to produce highly viscous digesta (barley-based diet) as opposed to digesta from birds fed a semi-purified diet (Poureslami et al., 2012). This has the potential to increase digesta effect on the wall of the gastrointestinal tract, especially the mucin covering of the gastrointestinal tract. Hence, the aim of the current study was to evaluate the effect of drying methods, drying temperatures (Exp 1) and sampling method as well as marker types (Exp 2) on apparent ileal AA digestibility in 21 d-old broilers. MATERIALS AND METHODS The experimental procedures for these studies were conducted under protocols approved by the University of Kentucky Institutional Animal Care and Use Committee. General Procedures and Diets In two experiments, Cobb 500 male broiler chicks obtained from a commercial hatchery were weighed and placed in battery cages (6 birds/cage; 0.61 × 0.51 × 0.36 m) with two nipple drinkers per cage in an environmentally controlled room with standard temperature regimes that gradually decreased from 32°C (d 0 to 7) to 24°C (d14 to 21) with appropriate ventilation and lighting (20 h of light and 4 h of dark). Birds were fed a standard corn-soybean meal based diet that was adequate in all nutrients as recommended by National Research Council (1994) for the first 10 d. On d 10, all birds were weighed and randomly allocated to their respective treatment groups in a completely randomized design. The experimental diets were corn-soybean meal-barley based. The diet composition as well as the analyzed nutrient contents of the experimental diets are presented in Tables 1 and 2, respectively. All birds had ad libitum access to experimental diets and water throughout the duration of the studies. Table 1. Dietary composition of the experimental diet (g/kg on as-fed basis). Ingredients, g/kg Experimental diet1 Corn 392 Soybean meal, 48% 360 Soy oil 50 Dicalcium phosphate 19 Limestone 12 Barley 150 Vitamin-mineral premix2 3 L-lysine HCl 2 DL-methionine 1.9 L-threonine 0.7 Salt (NaCl) 4.1 Titanium dioxide3 5 Total 1000 Ingredients, g/kg Experimental diet1 Corn 392 Soybean meal, 48% 360 Soy oil 50 Dicalcium phosphate 19 Limestone 12 Barley 150 Vitamin-mineral premix2 3 L-lysine HCl 2 DL-methionine 1.9 L-threonine 0.7 Salt (NaCl) 4.1 Titanium dioxide3 5 Total 1000 1In addition to the 5 g of titanium dioxide per kg diet already in the diet fed in EXP 1, 5 g of chromic oxide per kg of diet was also added at the expense of corn to EXP 1 diet to produce EXP 2 diet. 2Provided per kilogram of diet: iron, 71.6 mg; copper, 11.0 mg; manganese, 178.7 mg; zinc, 178.7 mg; iodine, 3.0 mg; selenium, 0.4 mg; vitamin A (retinyl acetate), 18,904.3 IU; vitamin D3 (cholecalciferol), 9,480.0 IU; vitamin E (dl-α-tocopheryl acetate), 63.0 IU; vitamin K activity, 6.4 mg; thiamine, 3.2 mg; riboflavin, 9.4 mg; pantothenic acid, 34.7 mg; niacin, 126.0 mg; pyridoxine, 4.7 mg; folic acid, 1.6 mg; biotin, 0.5 mg; vitamin B12, 35.4 μg; choline, 956.9 mg. 3Titanium dioxide (as well as chromic oxide) was added to the diet at the expense of ground corn. View Large Table 1. Dietary composition of the experimental diet (g/kg on as-fed basis). Ingredients, g/kg Experimental diet1 Corn 392 Soybean meal, 48% 360 Soy oil 50 Dicalcium phosphate 19 Limestone 12 Barley 150 Vitamin-mineral premix2 3 L-lysine HCl 2 DL-methionine 1.9 L-threonine 0.7 Salt (NaCl) 4.1 Titanium dioxide3 5 Total 1000 Ingredients, g/kg Experimental diet1 Corn 392 Soybean meal, 48% 360 Soy oil 50 Dicalcium phosphate 19 Limestone 12 Barley 150 Vitamin-mineral premix2 3 L-lysine HCl 2 DL-methionine 1.9 L-threonine 0.7 Salt (NaCl) 4.1 Titanium dioxide3 5 Total 1000 1In addition to the 5 g of titanium dioxide per kg diet already in the diet fed in EXP 1, 5 g of chromic oxide per kg of diet was also added at the expense of corn to EXP 1 diet to produce EXP 2 diet. 2Provided per kilogram of diet: iron, 71.6 mg; copper, 11.0 mg; manganese, 178.7 mg; zinc, 178.7 mg; iodine, 3.0 mg; selenium, 0.4 mg; vitamin A (retinyl acetate), 18,904.3 IU; vitamin D3 (cholecalciferol), 9,480.0 IU; vitamin E (dl-α-tocopheryl acetate), 63.0 IU; vitamin K activity, 6.4 mg; thiamine, 3.2 mg; riboflavin, 9.4 mg; pantothenic acid, 34.7 mg; niacin, 126.0 mg; pyridoxine, 4.7 mg; folic acid, 1.6 mg; biotin, 0.5 mg; vitamin B12, 35.4 μg; choline, 956.9 mg. 3Titanium dioxide (as well as chromic oxide) was added to the diet at the expense of ground corn. View Large Table 2. Analyzed nutrient composition of the experimental diets (%). Experiment 1 diet Experiment 2 diet Indispensable amino acid Arginine 1.46 1.43 Histidine 0.56 0.56 Isoleucine 0.98 0.97 Leucine 1.81 1.79 Lysine 1.43 1.44 Methionine 0.48 0.46 Phenylalanine 1.12 1.12 Threonine 0.87 0.87 Tryptophan 0.27 0.27 Valine 1.08 1.04 Dispensable amino acid Alanine 1.03 1.01 Aspartic acid 2.21 2.19 Cysteine 0.32 0.31 Glutamic acid 3.99 3.98 Glycine 0.91 0.90 Proline 1.32 1.23 Serine 0.92 0.94 Tyrosine 0.76 0.71 Total amino acid 21.75 21.45 Dry matter 90.57 91.17 Nitrogen 3.57 3.57 Calcium ND1 1.23 Phosphorus ND 0.93 Experiment 1 diet Experiment 2 diet Indispensable amino acid Arginine 1.46 1.43 Histidine 0.56 0.56 Isoleucine 0.98 0.97 Leucine 1.81 1.79 Lysine 1.43 1.44 Methionine 0.48 0.46 Phenylalanine 1.12 1.12 Threonine 0.87 0.87 Tryptophan 0.27 0.27 Valine 1.08 1.04 Dispensable amino acid Alanine 1.03 1.01 Aspartic acid 2.21 2.19 Cysteine 0.32 0.31 Glutamic acid 3.99 3.98 Glycine 0.91 0.90 Proline 1.32 1.23 Serine 0.92 0.94 Tyrosine 0.76 0.71 Total amino acid 21.75 21.45 Dry matter 90.57 91.17 Nitrogen 3.57 3.57 Calcium ND1 1.23 Phosphorus ND 0.93 1ND = not determined. View Large Table 2. Analyzed nutrient composition of the experimental diets (%). Experiment 1 diet Experiment 2 diet Indispensable amino acid Arginine 1.46 1.43 Histidine 0.56 0.56 Isoleucine 0.98 0.97 Leucine 1.81 1.79 Lysine 1.43 1.44 Methionine 0.48 0.46 Phenylalanine 1.12 1.12 Threonine 0.87 0.87 Tryptophan 0.27 0.27 Valine 1.08 1.04 Dispensable amino acid Alanine 1.03 1.01 Aspartic acid 2.21 2.19 Cysteine 0.32 0.31 Glutamic acid 3.99 3.98 Glycine 0.91 0.90 Proline 1.32 1.23 Serine 0.92 0.94 Tyrosine 0.76 0.71 Total amino acid 21.75 21.45 Dry matter 90.57 91.17 Nitrogen 3.57 3.57 Calcium ND1 1.23 Phosphorus ND 0.93 Experiment 1 diet Experiment 2 diet Indispensable amino acid Arginine 1.46 1.43 Histidine 0.56 0.56 Isoleucine 0.98 0.97 Leucine 1.81 1.79 Lysine 1.43 1.44 Methionine 0.48 0.46 Phenylalanine 1.12 1.12 Threonine 0.87 0.87 Tryptophan 0.27 0.27 Valine 1.08 1.04 Dispensable amino acid Alanine 1.03 1.01 Aspartic acid 2.21 2.19 Cysteine 0.32 0.31 Glutamic acid 3.99 3.98 Glycine 0.91 0.90 Proline 1.32 1.23 Serine 0.92 0.94 Tyrosine 0.76 0.71 Total amino acid 21.75 21.45 Dry matter 90.57 91.17 Nitrogen 3.57 3.57 Calcium ND1 1.23 Phosphorus ND 0.93 1ND = not determined. View Large Experiment 1 Experiment 1 (Exp 1) was designed to evaluate the effect of two drying methods (freeze-drying vs. forced air-oven drying) and two drying temperatures (40 vs. 55°C) on apparent ileal DM, N, and AA digestibility. The 55°C was selected to reflect temperature close to what is commonly used when samples are dried in forced air oven while 40°C was selected to check if drying at a lower temperature would influence amino acid digestibility values. One hundred and sixty-eight chicks were randomly assigned to treatments in a completely randomized design with a 2 × 2 factorial arrangement of treatment. The design consists of four treatment groups (2 drying methods × 2 drying temperatures) each with seven replicate cages, and each replicate consisted of six birds per cage. Titanium dioxide was included in the experimental diet at 5 g/kg of diet as an inert marker for digestibility calculation. Digesta samples were collected via squeezing. Experiment 2 Experiment 2 (Exp 2) was conducted to evaluate the effect of sampling method (flushing vs. squeezing) as well as marker types (Ti vs. Cr) on apparent ileal DM, N, Ca, P, and AA digestibility. A total of 96 d-old broiler chicks were randomly allocated to two treatments, each having eight replicate cages with six birds per replicate. The experimental diet contained both titanium oxide and chromium oxide which served as the indigestible markers. Each of the marker was added to the diet at 5 g/kg of diet. Sample Collection On d 21, birds were euthanized by argon gas asphyxiation before collection of ileal contents. The body cavity was opened and digesta samples were collected from the distal two thirds of the ileum by squeezing (Exp 1) or by squeezing or flushing (Exp 2) with distilled water. In order to minimize the effect of differences in pressure applied during squeezing, both the flushing and squeezing were done jointly by the same set of people (2 people). The ileal contents from birds within each cage were pooled into a clean and pre-labeled plastic container to allow for sufficient sample for chemical analysis and immediately stored in the freezer at −20°C. Subsequently, digesta samples were either FD or OD at 40 or 55°C (Exp 1) or FD only (Exp 2) at 55°C. The 55°C drying temperature was selected to be close to the temperature that is commonly used for oven drying (forced air oven). We selected 40°C to check if there would be any effect of a reduction in drying temperature on apparent ileal amino acid digestibility. The freeze dryer temperature was raised gradually to avoid a rapid increase in the drying temperature from zero to 40 or 55°C. Diets were ground to pass through a 0.5 mm screen using a mill grinder (Retsch ZM 100, Retsch GmbH and Co., K.G., Haan, Germany) while ileal digesta were ground with a coffee grinder in preparation for chemical analyses. Chemical Analysis Duplicate determinations of percentage dry matter contents of dried ileal digesta and diets was conducted by oven drying samples in duplicates at 105°C for 24 h (AOAC International, 1990; method 925.09). To prevent differences due to analytical errors, FD and OD ileal digesta samples from both experiments as well as their respective diets were subjected to the same analytical techniques at the same time. Titanium, Cr, and AA analyses were conducted at the Agricultural Experiment Station Chemical Laboratories, University of Missouri-Columbia (Columbia, MO). Nitrogen content was determined by the combustion method using a LECO Trumac Nitrogen Analyzer (LECO, St. Joseph, MI; AOAC International, 2000; method 990.03). Amino acid content of the experimental diets and ileal digesta were determined using an amino acid analyzer (Hitachi L-8900, Tokyo, Japan). Prior to analysis, samples were hydrolyzed in 6 N HCl at 110°C for 24 h, and aliquots of the hydrolysates were analyzed and quantified using post-column ninhydrin derivatization [method 982.30 E (a, b, c); AOAC International, 2006]. Samples were oxidized using performic acid prior to estimation of sulfur AA (Met and Cys). Tryptophan content was measured by HPLC after an alkaline hydrolysis in barium hydroxide solution. The concentration of Ti and Cr in the diets and digesta samples were determined. Samples were digested as described by Myers et al. (2004) after which Ti concentration was determined by flame atomic absorption spectroscopy. Cr in the diets and ileal digesta samples were determined as described by Fenton and Fenton (1979) using the Perkin-Elmer Optical Emission Spectrometer (Optima 2000 DV, Waltham, MA). STATISTICAL ANALYSIS Data were subjected to ANOVA using the general linear model procedures of SAS (SAS 9.3, Inc., Cary, NC) that is appropriate for factorial arrangement of treatments. Data were analyzed to determine the significance of main effects (drying temperature and drying method, Exp 1) and their interaction. When interactions were not significant, the interaction term was removed from the code and reanalyzed. For Exp 2, data were analyzed using Proc Mixed procedure of SAS with sampling method (squeezing or flushing) as the main plot and marker type (Ti or Cr) as the subplot. As with Exp 1, the interaction term was removed from the SAS codes whenever the interaction was not significant. Where necessary, differences between treatment means were compared using Tukey. The level of significance was set at P < 0.05. RESULTS The calculated percent moisture contents post drying are reported in Table 3. There was an interaction (P < 0.05) between drying method and drying temperature with OD method resulting in higher (P < 0.05) moisture loss compared to FD method at 40°C. Moisture loss for FD at 55°C was higher (P < 0.05) compared to the OD method at the same temperature. The highest and lowest levels of moisture loss (P < 0.05) were recorded for FD at 55°C and FD at 40°C, respectively (Table 3). There was no interaction between drying method and drying temperature on apparent ileal AA digestibility (Table 4, Exp 1). There was no significant main effect of drying method or drying temperature on apparent ileal AA digestibility. Methionine had the highest (92.3%) while Cys had the lowest (73.8%) ileal digestibility values. In Exp. 2, there was no interaction between sampling methods (flushing vs. squeezing) and marker types (Ti vs. Cr) on apparent ileal DM, N, Ca, P, and AA digestibility (Table 5, Exp 2). Similarly, the samples from the squeezing method had higher (P < 0.05) apparent ileal Arg and Met digestibility while the digestibility values for His, Ile, Cys, Ser, and Tyr tended to be higher (P < 0.1) compared to those from the flushing method (Table 5, Exp 2). Furthermore, the apparent ileal DM, N, Ca, P, and AA digestibility was not affected by the different markers (Table 5). Table 3. Effect of drying method (freeze-drying vs. forced-air oven drying) and temperature (40 vs. 55°C) on moisture contents of ileal digesta of broilers fed corn-soybean meal-barley based diet, (%). Method Temperature oC Freeze 401 Freeze 552 Oven 403 Oven 554 Pooled SEM Method Temperature Method*temperature Moisture 2.2d 8.4a 6.7b 5.6c 0.174 <.0001 <.0001 <.0001 Method Temperature oC Freeze 401 Freeze 552 Oven 403 Oven 554 Pooled SEM Method Temperature Method*temperature Moisture 2.2d 8.4a 6.7b 5.6c 0.174 <.0001 <.0001 <.0001 a-dMeans with different superscripts are different (P < 0.05) 1Freeze-dried samples were dried at −15°C for 40 h, 0°C for 40 h, +5°C for 20 h, +15°C for 20 h, +25°C for 20 h, and +40°C for 40 h. 2Freeze-dried samples were dried at −15°C for 40 h, 0°C for 40 h, +5°C for 20 h, +15°C for 20 h, +25°C for 20 h, and +40°C for 40 h, and +55°C for 40 h. 3Oven dried samples were dried at 40°C for 6 days. 4Oven dried samples were dried at 55°C for 6 days. View Large Table 3. Effect of drying method (freeze-drying vs. forced-air oven drying) and temperature (40 vs. 55°C) on moisture contents of ileal digesta of broilers fed corn-soybean meal-barley based diet, (%). Method Temperature oC Freeze 401 Freeze 552 Oven 403 Oven 554 Pooled SEM Method Temperature Method*temperature Moisture 2.2d 8.4a 6.7b 5.6c 0.174 <.0001 <.0001 <.0001 Method Temperature oC Freeze 401 Freeze 552 Oven 403 Oven 554 Pooled SEM Method Temperature Method*temperature Moisture 2.2d 8.4a 6.7b 5.6c 0.174 <.0001 <.0001 <.0001 a-dMeans with different superscripts are different (P < 0.05) 1Freeze-dried samples were dried at −15°C for 40 h, 0°C for 40 h, +5°C for 20 h, +15°C for 20 h, +25°C for 20 h, and +40°C for 40 h. 2Freeze-dried samples were dried at −15°C for 40 h, 0°C for 40 h, +5°C for 20 h, +15°C for 20 h, +25°C for 20 h, and +40°C for 40 h, and +55°C for 40 h. 3Oven dried samples were dried at 40°C for 6 days. 4Oven dried samples were dried at 55°C for 6 days. View Large Table 4. The effect of drying method (freeze-drying vs. forced-air oven drying) and temperature (40 vs. 55°C) on apparent ileal amino acid digestibility in 21 day-old broilers fed barley based diet (Experiment 1). Drying method Drying temperature (°C) Method Temperature Method* temperature Freeze-dried1 Oven dried2 40 55 SEM Probability Dry matter 67.5 67.7 67.3 67.9 0.73 0.824 0.483 0.227 Nitrogen 81.2 81.5 81.1 81.5 0.48 0.576 0.379 0.701 Indispensable amino acid Arg 88.6 88.9 88.7 88.7 0.46 0.538 0.975 0.131 His 84.9 85.2 84.9 85.2 0.48 0.516 0.498 0.140 Ile 82.8 83.2 83.0 83.0 0.65 0.530 0.931 0.185 Leu 83.8 84.2 84.0 84.1 0.61 0.447 0.853 0.253 Lys 86.4 87.0 86.6 86.8 0.53 0.300 0.701 0.087 Met 92.2 92.5 92.2 92.4 0.37 0.420 0.692 0.165 Phe 84.1 84.5 84.2 84.4 0.57 0.446 0.675 0.198 Thr 78.0 78.4 78.1 78.3 0.62 0.537 0.748 0.206 Trp 87.4 87.6 87.4 87.5 0.46 0.647 0.807 0.218 Val 78.3 79.0 78.6 78.7 0.67 0.375 0.793 0.164 Dispensable amino acid Ala 83.2 83.7 83.3 83.6 0.62 0.454 0.601 0.209 Asp 80.9 81.2 80.9 81.2 0.66 0.662 0.725 0.260 Cys 73.7 73.9 73.5 74.2 0.85 0.855 0.411 0.310 Glu 87.6 87.9 87.7 87.8 0.46 0.476 0.749 0.165 Gly 79.2 79.6 79.1 79.7 0.67 0.599 0.327 0.253 Pro 83.4 83.8 83.6 83.7 0.53 0.447 0.800 0.359 Ser 81.3 81.7 81.3 81.7 0.61 0.444 0.545 0.241 Tyr 84.5 84.9 84.7 84.7 0.62 0.527 0.964 0.116 Total 83.4 83.8 83.5 83.7 0.56 0.474 0.733 0.162 Drying method Drying temperature (°C) Method Temperature Method* temperature Freeze-dried1 Oven dried2 40 55 SEM Probability Dry matter 67.5 67.7 67.3 67.9 0.73 0.824 0.483 0.227 Nitrogen 81.2 81.5 81.1 81.5 0.48 0.576 0.379 0.701 Indispensable amino acid Arg 88.6 88.9 88.7 88.7 0.46 0.538 0.975 0.131 His 84.9 85.2 84.9 85.2 0.48 0.516 0.498 0.140 Ile 82.8 83.2 83.0 83.0 0.65 0.530 0.931 0.185 Leu 83.8 84.2 84.0 84.1 0.61 0.447 0.853 0.253 Lys 86.4 87.0 86.6 86.8 0.53 0.300 0.701 0.087 Met 92.2 92.5 92.2 92.4 0.37 0.420 0.692 0.165 Phe 84.1 84.5 84.2 84.4 0.57 0.446 0.675 0.198 Thr 78.0 78.4 78.1 78.3 0.62 0.537 0.748 0.206 Trp 87.4 87.6 87.4 87.5 0.46 0.647 0.807 0.218 Val 78.3 79.0 78.6 78.7 0.67 0.375 0.793 0.164 Dispensable amino acid Ala 83.2 83.7 83.3 83.6 0.62 0.454 0.601 0.209 Asp 80.9 81.2 80.9 81.2 0.66 0.662 0.725 0.260 Cys 73.7 73.9 73.5 74.2 0.85 0.855 0.411 0.310 Glu 87.6 87.9 87.7 87.8 0.46 0.476 0.749 0.165 Gly 79.2 79.6 79.1 79.7 0.67 0.599 0.327 0.253 Pro 83.4 83.8 83.6 83.7 0.53 0.447 0.800 0.359 Ser 81.3 81.7 81.3 81.7 0.61 0.444 0.545 0.241 Tyr 84.5 84.9 84.7 84.7 0.62 0.527 0.964 0.116 Total 83.4 83.8 83.5 83.7 0.56 0.474 0.733 0.162 1Freeze-dried samples were dried at 40°C: −15°C for 40 h, 0°C for 40 h, +5°C for 20 h, +15°C for 20 h, +25°C for 20 h, and +40°C for 40 h; 55°C: −15°C for 40 h, 0°C for 40 h, +5°C for 20 h, +15°C for 20 h, +25°C for 20 h, and +40°C for 40 h, and +55°C for 40 h. 2Oven dried samples were dried at 40°C for 6 days or at 55°C for 6 days. View Large Table 4. The effect of drying method (freeze-drying vs. forced-air oven drying) and temperature (40 vs. 55°C) on apparent ileal amino acid digestibility in 21 day-old broilers fed barley based diet (Experiment 1). Drying method Drying temperature (°C) Method Temperature Method* temperature Freeze-dried1 Oven dried2 40 55 SEM Probability Dry matter 67.5 67.7 67.3 67.9 0.73 0.824 0.483 0.227 Nitrogen 81.2 81.5 81.1 81.5 0.48 0.576 0.379 0.701 Indispensable amino acid Arg 88.6 88.9 88.7 88.7 0.46 0.538 0.975 0.131 His 84.9 85.2 84.9 85.2 0.48 0.516 0.498 0.140 Ile 82.8 83.2 83.0 83.0 0.65 0.530 0.931 0.185 Leu 83.8 84.2 84.0 84.1 0.61 0.447 0.853 0.253 Lys 86.4 87.0 86.6 86.8 0.53 0.300 0.701 0.087 Met 92.2 92.5 92.2 92.4 0.37 0.420 0.692 0.165 Phe 84.1 84.5 84.2 84.4 0.57 0.446 0.675 0.198 Thr 78.0 78.4 78.1 78.3 0.62 0.537 0.748 0.206 Trp 87.4 87.6 87.4 87.5 0.46 0.647 0.807 0.218 Val 78.3 79.0 78.6 78.7 0.67 0.375 0.793 0.164 Dispensable amino acid Ala 83.2 83.7 83.3 83.6 0.62 0.454 0.601 0.209 Asp 80.9 81.2 80.9 81.2 0.66 0.662 0.725 0.260 Cys 73.7 73.9 73.5 74.2 0.85 0.855 0.411 0.310 Glu 87.6 87.9 87.7 87.8 0.46 0.476 0.749 0.165 Gly 79.2 79.6 79.1 79.7 0.67 0.599 0.327 0.253 Pro 83.4 83.8 83.6 83.7 0.53 0.447 0.800 0.359 Ser 81.3 81.7 81.3 81.7 0.61 0.444 0.545 0.241 Tyr 84.5 84.9 84.7 84.7 0.62 0.527 0.964 0.116 Total 83.4 83.8 83.5 83.7 0.56 0.474 0.733 0.162 Drying method Drying temperature (°C) Method Temperature Method* temperature Freeze-dried1 Oven dried2 40 55 SEM Probability Dry matter 67.5 67.7 67.3 67.9 0.73 0.824 0.483 0.227 Nitrogen 81.2 81.5 81.1 81.5 0.48 0.576 0.379 0.701 Indispensable amino acid Arg 88.6 88.9 88.7 88.7 0.46 0.538 0.975 0.131 His 84.9 85.2 84.9 85.2 0.48 0.516 0.498 0.140 Ile 82.8 83.2 83.0 83.0 0.65 0.530 0.931 0.185 Leu 83.8 84.2 84.0 84.1 0.61 0.447 0.853 0.253 Lys 86.4 87.0 86.6 86.8 0.53 0.300 0.701 0.087 Met 92.2 92.5 92.2 92.4 0.37 0.420 0.692 0.165 Phe 84.1 84.5 84.2 84.4 0.57 0.446 0.675 0.198 Thr 78.0 78.4 78.1 78.3 0.62 0.537 0.748 0.206 Trp 87.4 87.6 87.4 87.5 0.46 0.647 0.807 0.218 Val 78.3 79.0 78.6 78.7 0.67 0.375 0.793 0.164 Dispensable amino acid Ala 83.2 83.7 83.3 83.6 0.62 0.454 0.601 0.209 Asp 80.9 81.2 80.9 81.2 0.66 0.662 0.725 0.260 Cys 73.7 73.9 73.5 74.2 0.85 0.855 0.411 0.310 Glu 87.6 87.9 87.7 87.8 0.46 0.476 0.749 0.165 Gly 79.2 79.6 79.1 79.7 0.67 0.599 0.327 0.253 Pro 83.4 83.8 83.6 83.7 0.53 0.447 0.800 0.359 Ser 81.3 81.7 81.3 81.7 0.61 0.444 0.545 0.241 Tyr 84.5 84.9 84.7 84.7 0.62 0.527 0.964 0.116 Total 83.4 83.8 83.5 83.7 0.56 0.474 0.733 0.162 1Freeze-dried samples were dried at 40°C: −15°C for 40 h, 0°C for 40 h, +5°C for 20 h, +15°C for 20 h, +25°C for 20 h, and +40°C for 40 h; 55°C: −15°C for 40 h, 0°C for 40 h, +5°C for 20 h, +15°C for 20 h, +25°C for 20 h, and +40°C for 40 h, and +55°C for 40 h. 2Oven dried samples were dried at 40°C for 6 days or at 55°C for 6 days. View Large Table 5. The effect of sampling method (flushing vs. squeezing) and marker type (titanium oxide vs. chromium oxide) on apparent ileal dry matter and amino acid digestibility in 21 day-old broilers fed barley-based diet (%, Experiment 2). Method Marker Method Marker Method Marker Method*Marker Flush Squeeze Chromium Titanium SEM Probability Indispensable AA Arg 88.4 89.0 88.8 88.6 0.21 0.21 0.042 0.715 0.971 His 85.4 86.1 85.8 85.7 0.25 0.25 0.070 0.707 0.995 Ile 83.2 84.0 83.7 83.5 0.28 0.28 0.067 0.716 0.979 Leu 84.2 84.6 84.4 84.3 0.31 0.31 0.371 0.738 0.971 Lys 87.0 87.5 87.3 87.2 0.23 0.23 0.113 0.685 0.969 Met 91.8 92.5 92.2 92.1 0.19 0.19 0.009 0.792 0.974 Phe 84.6 85.4 85.1 84.9 0.33 0.33 0.112 0.749 0.998 Thr 78.8 79.3 79.1 79.0 0.33 0.33 0.352 0.689 0.955 Trp 88.7 89.0 88.9 88.8 0.29 0.29 0.390 0.775 0.981 Val 78.5 79.0 78.8 78.6 0.33 0.33 0.324 0.663 0.973 Dispensable AA Ala 83.4 83.7 83.7 83.5 0.31 0.31 0.427 0.704 0.998 Asp 81.3 81.7 81.6 81.4 0.32 0.32 0.329 0.687 0.998 Cys 73.7 75.1 74.5 74.3 0.50 0.50 0.060 0.738 0.990 Glu 88.0 88.4 88.2 88.1 0.22 0.22 0.171 0.745 0.970 Gly 79.5 79.9 79.8 79.6 0.32 0.32 0.430 0.693 0.985 Pro 82.9 83.0 83.1 82.9 0.30 0.30 0.797 0.712 0.982 Ser 82.3 83.2 82.8 82.7 0.31 0.31 0.070 0.706 0.968 Tyr 83.8 84.5 84.2 84.1 0.27 0.27 0.099 0.699 0.983 Total 83.7 84.2 84.0 83.9 0.27 0.27 0.218 0.714 0.976 Dry matter 67.3 67.6 67.6 67.3 0.39 0.39 0.656 0.570 0.988 Nitrogen 81.3 81.8 81.7 81.5 0.28 0.28 0.176 0.642 0.989 Calcium 33.8 32.6 33.6 32.9 1.71 1.71 0.626 0.793 0.981 Phosphorus 45.6 46.8 46.5 46.0 0.84 0.84 0.330 0.677 0.963 Method Marker Method Marker Method Marker Method*Marker Flush Squeeze Chromium Titanium SEM Probability Indispensable AA Arg 88.4 89.0 88.8 88.6 0.21 0.21 0.042 0.715 0.971 His 85.4 86.1 85.8 85.7 0.25 0.25 0.070 0.707 0.995 Ile 83.2 84.0 83.7 83.5 0.28 0.28 0.067 0.716 0.979 Leu 84.2 84.6 84.4 84.3 0.31 0.31 0.371 0.738 0.971 Lys 87.0 87.5 87.3 87.2 0.23 0.23 0.113 0.685 0.969 Met 91.8 92.5 92.2 92.1 0.19 0.19 0.009 0.792 0.974 Phe 84.6 85.4 85.1 84.9 0.33 0.33 0.112 0.749 0.998 Thr 78.8 79.3 79.1 79.0 0.33 0.33 0.352 0.689 0.955 Trp 88.7 89.0 88.9 88.8 0.29 0.29 0.390 0.775 0.981 Val 78.5 79.0 78.8 78.6 0.33 0.33 0.324 0.663 0.973 Dispensable AA Ala 83.4 83.7 83.7 83.5 0.31 0.31 0.427 0.704 0.998 Asp 81.3 81.7 81.6 81.4 0.32 0.32 0.329 0.687 0.998 Cys 73.7 75.1 74.5 74.3 0.50 0.50 0.060 0.738 0.990 Glu 88.0 88.4 88.2 88.1 0.22 0.22 0.171 0.745 0.970 Gly 79.5 79.9 79.8 79.6 0.32 0.32 0.430 0.693 0.985 Pro 82.9 83.0 83.1 82.9 0.30 0.30 0.797 0.712 0.982 Ser 82.3 83.2 82.8 82.7 0.31 0.31 0.070 0.706 0.968 Tyr 83.8 84.5 84.2 84.1 0.27 0.27 0.099 0.699 0.983 Total 83.7 84.2 84.0 83.9 0.27 0.27 0.218 0.714 0.976 Dry matter 67.3 67.6 67.6 67.3 0.39 0.39 0.656 0.570 0.988 Nitrogen 81.3 81.8 81.7 81.5 0.28 0.28 0.176 0.642 0.989 Calcium 33.8 32.6 33.6 32.9 1.71 1.71 0.626 0.793 0.981 Phosphorus 45.6 46.8 46.5 46.0 0.84 0.84 0.330 0.677 0.963 View Large Table 5. The effect of sampling method (flushing vs. squeezing) and marker type (titanium oxide vs. chromium oxide) on apparent ileal dry matter and amino acid digestibility in 21 day-old broilers fed barley-based diet (%, Experiment 2). Method Marker Method Marker Method Marker Method*Marker Flush Squeeze Chromium Titanium SEM Probability Indispensable AA Arg 88.4 89.0 88.8 88.6 0.21 0.21 0.042 0.715 0.971 His 85.4 86.1 85.8 85.7 0.25 0.25 0.070 0.707 0.995 Ile 83.2 84.0 83.7 83.5 0.28 0.28 0.067 0.716 0.979 Leu 84.2 84.6 84.4 84.3 0.31 0.31 0.371 0.738 0.971 Lys 87.0 87.5 87.3 87.2 0.23 0.23 0.113 0.685 0.969 Met 91.8 92.5 92.2 92.1 0.19 0.19 0.009 0.792 0.974 Phe 84.6 85.4 85.1 84.9 0.33 0.33 0.112 0.749 0.998 Thr 78.8 79.3 79.1 79.0 0.33 0.33 0.352 0.689 0.955 Trp 88.7 89.0 88.9 88.8 0.29 0.29 0.390 0.775 0.981 Val 78.5 79.0 78.8 78.6 0.33 0.33 0.324 0.663 0.973 Dispensable AA Ala 83.4 83.7 83.7 83.5 0.31 0.31 0.427 0.704 0.998 Asp 81.3 81.7 81.6 81.4 0.32 0.32 0.329 0.687 0.998 Cys 73.7 75.1 74.5 74.3 0.50 0.50 0.060 0.738 0.990 Glu 88.0 88.4 88.2 88.1 0.22 0.22 0.171 0.745 0.970 Gly 79.5 79.9 79.8 79.6 0.32 0.32 0.430 0.693 0.985 Pro 82.9 83.0 83.1 82.9 0.30 0.30 0.797 0.712 0.982 Ser 82.3 83.2 82.8 82.7 0.31 0.31 0.070 0.706 0.968 Tyr 83.8 84.5 84.2 84.1 0.27 0.27 0.099 0.699 0.983 Total 83.7 84.2 84.0 83.9 0.27 0.27 0.218 0.714 0.976 Dry matter 67.3 67.6 67.6 67.3 0.39 0.39 0.656 0.570 0.988 Nitrogen 81.3 81.8 81.7 81.5 0.28 0.28 0.176 0.642 0.989 Calcium 33.8 32.6 33.6 32.9 1.71 1.71 0.626 0.793 0.981 Phosphorus 45.6 46.8 46.5 46.0 0.84 0.84 0.330 0.677 0.963 Method Marker Method Marker Method Marker Method*Marker Flush Squeeze Chromium Titanium SEM Probability Indispensable AA Arg 88.4 89.0 88.8 88.6 0.21 0.21 0.042 0.715 0.971 His 85.4 86.1 85.8 85.7 0.25 0.25 0.070 0.707 0.995 Ile 83.2 84.0 83.7 83.5 0.28 0.28 0.067 0.716 0.979 Leu 84.2 84.6 84.4 84.3 0.31 0.31 0.371 0.738 0.971 Lys 87.0 87.5 87.3 87.2 0.23 0.23 0.113 0.685 0.969 Met 91.8 92.5 92.2 92.1 0.19 0.19 0.009 0.792 0.974 Phe 84.6 85.4 85.1 84.9 0.33 0.33 0.112 0.749 0.998 Thr 78.8 79.3 79.1 79.0 0.33 0.33 0.352 0.689 0.955 Trp 88.7 89.0 88.9 88.8 0.29 0.29 0.390 0.775 0.981 Val 78.5 79.0 78.8 78.6 0.33 0.33 0.324 0.663 0.973 Dispensable AA Ala 83.4 83.7 83.7 83.5 0.31 0.31 0.427 0.704 0.998 Asp 81.3 81.7 81.6 81.4 0.32 0.32 0.329 0.687 0.998 Cys 73.7 75.1 74.5 74.3 0.50 0.50 0.060 0.738 0.990 Glu 88.0 88.4 88.2 88.1 0.22 0.22 0.171 0.745 0.970 Gly 79.5 79.9 79.8 79.6 0.32 0.32 0.430 0.693 0.985 Pro 82.9 83.0 83.1 82.9 0.30 0.30 0.797 0.712 0.982 Ser 82.3 83.2 82.8 82.7 0.31 0.31 0.070 0.706 0.968 Tyr 83.8 84.5 84.2 84.1 0.27 0.27 0.099 0.699 0.983 Total 83.7 84.2 84.0 83.9 0.27 0.27 0.218 0.714 0.976 Dry matter 67.3 67.6 67.6 67.3 0.39 0.39 0.656 0.570 0.988 Nitrogen 81.3 81.8 81.7 81.5 0.28 0.28 0.176 0.642 0.989 Calcium 33.8 32.6 33.6 32.9 1.71 1.71 0.626 0.793 0.981 Phosphorus 45.6 46.8 46.5 46.0 0.84 0.84 0.330 0.677 0.963 View Large DISCUSSION Accurate nutrient digestibility estimation requires some form of sample preparation including proper drying prior to preparation for laboratory analysis. The drying methods used in this study, oven drying and freeze drying, are used by many laboratories as a routine technique to prepare samples for analysis. After collecting ileal or excreta samples, they were put through the different methods mentioned above until a constant mass was reached after which moisture loss or dry matter contents can be determined by weight difference (Galle, 2001). The per cent moisture obtained in this study shows that the greatest moisture loss was obtained by freeze-drying at 55°C while the least amount of moisture was lost when samples were freeze-dried at 40°C. By increasing the drying temperature from 40 to 55°C, the difference in the moisture contents of ileal digesta was 6.2%-points (FD) compared to 1.1%-points for OD samples. This shows that there may not be any advantage of drying ileal digesta samples beyond 40°C in forced air oven in terms of further moisture removal. Despite this however, freeze drying technique is favored by many industries including soil engineering, food industry because it is believed that freeze-drying protects the primary structure and shape of the sample while decreasing the danger of altering more labile AA (Ray and Kothmann, 1988; Ratti, 2001). Furthermore, ileal AA digestibility studies have often adopted the FD method because of the concern for the quality of AA and N when ileal digesta are dried using the forced air oven. Indeed, in these studies, the ileal digesta samples that were dried in the forced-air oven were extremely hard and were much more difficult to grind compared to samples that were freeze-dried. However, the cost and the need for regular maintenance of a freeze-dryer is challenging. Previous studies showed that OD at 65°C resulted in energy and N loss of 11.4 and 5.2%, respectively, (Manoukas et al., 1964) while FD resulted in energy loss of 1.3% which increased to 5.5% when OD at 60°C (Shannon and Brown, 1969). However, in a different study where the effects of drying method on different tissues were evaluated, it was reported that the amount of moisture lost from tissue when freeze-dried or dried in a desiccator was not different (Sahin et al., 2006). The recoveries of the markers (titanium and chromium) in the experimental diets fed to birds in these studies were >97%. No significant interaction was observed on apparent ileal DM, N, and AA digestibility that could be attributed to both the drying methods and drying temperatures (Exp 1). The digestibility of the AA was similarly not affected by the main factors (drying method and drying temperature) however, Met had the highest digestibility (92.3%) while Cys had the lowest (73.7%) digestibility value. This indicates that the availability and quality of these AA were not affected by the drying method or temperature. In evaluating the effect of the drying procedure on apparent ileal digestibility, the results were different from studies conducted by Dale et al. (1985) which showed that the availability of Ala increased in both the diets and excreta when FD or that of Sibbald (1979) who reported that oven drying of excreta was a better alternative to freeze-drying when estimating true metabolizable energy, however, Hinnant and Kothmann (1988) reported no significant effects of drying methods on N contents of excreta samples (OD, 1.35% vs. FD, 1.38%). In Exp. 2 where two inert dietary markers were added to the diet, ileal digesta samples were either flushed or squeezed to determine the effect of sampling methods (flushing vs. squeezing) and marker type (Ti vs Cr) on apparent ileal DM, N, Ca, P, and AA digestibility. There was no interaction between the sampling methods (flushing vs. squeezing) and marker types (Ti vs Cr). Similarly, the effect of sampling method was not significant except for Arg and Met where squeezing resulted in higher ileal digestibility values. Furthermore, apparent ileal His, Ile, Cys, Ser, and Tyr digestibility showed tendencies to be higher in squeezed digesta. This observation was opposite of what was expected and difficult to explain. However, this observation may be due to the nature of the digesta. Because of the presence of barley in the diet (15%), the digesta was very viscous and relatively difficult to flush compared to squeezing. There is the possibility of an increase adhesion of the digesta to the gastrointestinal wall, which when flushed, sloughed more cells including mucins, dissolving into the distilled water. The contribution of mucin to the ileal digesta can be attributed to increased concentration of amino acids in the digesta (Adedokun et al., 2011). This may explain, in part, the reason for the relatively low apparent ileal AA digestibility in the flushed samples. However, with digesta from less viscous diet, this may not be an issue. This difference in digestibility, even for the two AA that showed significant effect of sampling method, was very low (0.6 and 0.7-percentage points for Arg and Met, respectively). This was significant because of the very low standard error values. It is important to note that any effect or a lack of it may not be attributed to the chemical analysis procedure as digestibility values obtained from Exp 1 and 2 were quite similar. Contrary to the findings reported by Selle et al. (2006) on the meta-analysis of phytase effect on AA digestibility in poultry where the variation in phytase responses was attributed to the choice of inert marker used in the digestibility assays, the outcome from this current study showed no such effect. They (Selle et al., 2006) concluded that AA digestibility responses to phytase were higher when Ti or acid insoluble ash were used compared to Cr. Similarly, Olukosi et al. (2012) concluded that the apparent ileal AA digestibility in broilers and pigs with phytase supplementation determined with Ti as a digestibility marker were considerably higher than the values measured with the Cr marker. Jacobs et al. (2011) reported that there was no significant effect of drying method (FD vs. OD) as well as drying temperature (OD 55 vs. OD 100°C) on the N composition of poultry excreta. Despite the lack of significant difference in N contents of excreta samples that were oven dried at 55 vs. 100°C, the N contents of excreta samples that were dried at 100°C was about 3.8% lower than those dried at 55°C. This lack of significant effect of drying methods is consistent with what was observed for ileal digesta in the current study. Ribeiro et al. (2001) however reported a significant decrease in broiler excreta N contents between excreta samples that were dried at 55 and 100°C. There is a dearth of information on the effect of sampling or collection method on apparent ileal AA digestibility in broiler chickens. Poureslami et al. (2012) observed significant effect of collection method in all the AA they evaluated in their study with flushing having higher ileal AA digestibility compared to squeezing which is different from what we observed in the current study. This may be because of three factors. First, the data from Poureslami et al. (2012) were from 18 d-old broilers compared to 21 d-old broilers that were sampled in the current study. Secondly, the diet tested in the study by Poureslami et al. (2012) was fed for 4 d compared to 12 d in the current study. The difference in the length of feeding of these diets may influence the AA digestibility values as different diets may require different length of time for proper adaptation. The third reason, which may be the most important, is the nature of the diets fed. A semi-purified diet (soybean meal and dextrose-based) was fed to broilers in the study reported by Poureslami et al. (2012) while a complete diet (corn-soybean meal-barley-based) was fed to broilers in the current study. Generally, because of the presence of barley (15%) in the diet fed to broilers in the current study, the digesta from the birds were very viscous and it requires some efforts to squeeze but much more efforts to flush. There is the possibility that flushing may have led to a situation whereby more mucus layer as well as other sources of AA in the gastrointestinal tract (AA of endogenous origin) may have been flushed along with the digesta. This may explain the relatively lower apparent ileal AA digestibility values when ileal digesta were flushed. This may not be a challenge when digesta from birds fed a semi-purified diet or a complete diet with minimal level of non-starch polysaccharides are collected (easy flow). From the results presented in this study, it can be summarized that ileal digesta could be dried using either the freeze-dryer or forced-air oven without jeopardizing the quality of the digesta for AA analysis. Likewise, digesta could be dried at temperature as low as 40 and as high as 55oC without any significant negative effect on digestibility values. Furthermore, the use of Ti or Cr as inert marker did not influence apparent ileal AA digestibility values. The apparent ileal digestibility of two AA, Arg and Met, increased with squeezing while generally, sampling method did not influence AA digestibility in broilers fed corn-soybean meal-barley based diet. Finally, results from Exp 1 showed that in places where access to a freeze-dryer is impossible, forced air oven could be used for ileal digesta samples that were squeezed. If both the freeze-dryer and forced air oven are available, freeze-drying should be the preferred option, especially because freeze-dried samples are much easierer to process (grind) than the oven-dried samples. Footnotes 1 This is publication No. 17-07-098 of the Kentucky Agricultural Experiment Station and is published with the approval of the Director. This work is supported by the National Institute of Food and Agriculture, U.S. Department of Agriculture, Multistate project number KYNE1442 under accession number 1005963. REFERENCES Adedokun S. A. , Parsons C. M. , Lilburn M. S. , Adeola O. , Applegate T. J. . 2007 . Endogenous amino acid flow in broiler chicks is affected by the age of birds and method of estimation . Poult. Sci. 86 : 2590 – 2597 . Google Scholar CrossRef Search ADS PubMed Adedokun S. 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Prod. Sci. 62 : 133 – 141 . Google Scholar CrossRef Search ADS © 2018 Poultry Science Association Inc. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Poultry Science Oxford University Press

The effect of drying method temperature, collection method, and marker type on apparent ileal amino acid digestibility in 21-day-old broilers fed corn-soybean meal-barley based diet

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Abstract

ABSTRACT For accurate estimation of nutrient digestibility, an ideal drying and sampling method is required to preserve the quality of the digesta. A standard corn-soybean meal (corn-SBM) broiler starter diet was fed from d 0 to 10 before birds were placed on the experimental diets until d 21. One hundred and sixty-eight male Cobb 500 broiler chicks were used to evaluate the effect of two drying methods (freeze-dryer vs. forced air-oven) and two drying temperatures (40 vs. 55°C) (Exp 1), while ninety-six chicks were used to evaluate the effect of flushing and squeezing as well as marker types (titanium vs. chromium) on apparent ileal DM, N, Ca, P, and AA digestibility (Exp 2). There were seven (Exp 1) or eight (Exp 2) replicate cages per treatment with 6 birds/cage. Digesta from the distal two thirds of the ileum was obtained from birds following euthanasia on d 21 by squeezing (Exp 1) and squeezing or flushing (Exp 2). Samples collected were stored in the freezer at −20°C until they were either freeze-dried (FD) or oven-dried (OD) at 40 or 55°C. There were no interactions between the drying methods and drying temperatures (Exp 1) on apparent ileal DM, N, and AA digestibility. Met had the highest (92.3%) while Cys had the lowest (73.8%) digestibility value. In Exp 2, no interaction between sampling methods and marker types was observed. The effect of sampling methods was not significant except for Arg and Met where squeezing resulted in higher (P < 0.05) digestibility values. Furthermore, apparent ileal His, Ile, Cys, Ser, and Tyr digestibility tended to be higher (P < 0.1) in squeezed digesta compared to the flushed digesta. Results from these studies showed that OD ileal digesta at 40 or 55°C had no negative effect on apparent ileal AA digestibility. Likewise, marker type did not influence apparent ileal AA digestibility values. INTRODUCTION Nutrient digestibility studies address the importance of accurately estimating how efficiently nutrients in diets are digested and absorbed in the mid-section of the gastrointestinal tract. In avian species, the bio-availability of each amino acid (AA) is critical for evaluating the nutritional value of the feedstuff and for estimating AA requirement of poultry (Poureslami et al., 2012; Kong and Adeola, 2014). Ileal digestibility procedure has been routinely used to estimate AA requirement because excreta AA contents can be influenced by the microorganisms in the hindgut and this microbiota can metabolize some of the undigested AA thus modifying the contents of the excreta (Ravindran et al., 1999; Pig, 2000; Kong and Adeola, 2014). In determining ileal digestibility of nutrients in broilers, several sampling and processing methods have been adopted over the years. Ileal digesta samples are usually collected by gently squeezing (Short et al., 1999; Bandegan et al., 2009; Poureslami et al., 2012) or flushing (Ravindran et al., 1999, 2004; Kluth et al., 2005; Adedokun et al., 2007), and subsequently oven dried (OD) or freeze-dried (FD) prior to chemical analysis. The aforementioned methods have been a bone of contention with no consensus reached as to the standard procedure for preparing samples prior to chemical analyses (Jacobs et al., 2011). Drying is often described as the ratio of percent moisture removed without changing the initial chemical structure of a particular substance (Jindal and Siebenmorgen, 1987). Jindal and Siebenmorgen (1987) suggested that moisture loss using different drying methods may not be the same due to the empirical nature of the methods. Oven drying requires less time than freeze drying which takes longer and subsequently increases the cost and time of sample drying (Hinnant and Kothmann et al., 1988). Research also showed that poultry excreta samples that were OD at 65°C resulted in an average energy loss of about 12% (Manoukas et al., 1964) while nitrogen (N) loss when FD or OD at 60°C resulted in a mean loss of approximately 4.7% (Shannon and Brown, 1969). However, a study conducted by another group indicated that the effect of FD or OD of excreta samples at 60°C on true AA availability, TME, and nitrogen-corrected TME were not significant (Dale et al., 1985). The use of an inert marker in determining the rate of nutrient disappearance along the gastrointestinal tract, its chemical analysis, and calculations have been reported by several authors (Jagger et al., 1992; Short et al., 1996; Adeola, 2001; Myers et al., 2004). The effect of different inert (index) dietary markers in animal digestibility studies is inconclusive with some studies showing higher ileal AA digestibility values when titanium dioxide was used compared to chromic oxide. A higher recovery rate was observed for titanium (Ti) when compared to chromium (Cr) in the ileal digestibility values of N and individual AA (Jagger et al., 1992). Similarly, Olukosi et al. (2012) concluded that apparent ileal AA digestibility values were higher when Ti rather than Cr was the inert marker. Furthermore, a low recovery rate of Cr has been attributed to analytical problems caused by the presence of phosphorus and other minerals (Peddie et al., 1982; Saha and Gilbreath, 1991; Yin et al., 2000), as well as the possibility that Cr may lack uniform distribution in the digesta across the gastrointestinal tract (Oberleas et al., 1990; Sooncharernying and Edwards, 1993). Jacobs et al. (2011) evaluated the effect of drying method on total tract N digestibility in poultry excreta while Poureslami et al. (2012) evaluated the effect of sampling method on apparent ileal AA digestibility in broiler chickens. In this study, we attempted to evaluate the effect of sampling method on ileal AA digestibility of a diet that is expected to produce highly viscous digesta (barley-based diet) as opposed to digesta from birds fed a semi-purified diet (Poureslami et al., 2012). This has the potential to increase digesta effect on the wall of the gastrointestinal tract, especially the mucin covering of the gastrointestinal tract. Hence, the aim of the current study was to evaluate the effect of drying methods, drying temperatures (Exp 1) and sampling method as well as marker types (Exp 2) on apparent ileal AA digestibility in 21 d-old broilers. MATERIALS AND METHODS The experimental procedures for these studies were conducted under protocols approved by the University of Kentucky Institutional Animal Care and Use Committee. General Procedures and Diets In two experiments, Cobb 500 male broiler chicks obtained from a commercial hatchery were weighed and placed in battery cages (6 birds/cage; 0.61 × 0.51 × 0.36 m) with two nipple drinkers per cage in an environmentally controlled room with standard temperature regimes that gradually decreased from 32°C (d 0 to 7) to 24°C (d14 to 21) with appropriate ventilation and lighting (20 h of light and 4 h of dark). Birds were fed a standard corn-soybean meal based diet that was adequate in all nutrients as recommended by National Research Council (1994) for the first 10 d. On d 10, all birds were weighed and randomly allocated to their respective treatment groups in a completely randomized design. The experimental diets were corn-soybean meal-barley based. The diet composition as well as the analyzed nutrient contents of the experimental diets are presented in Tables 1 and 2, respectively. All birds had ad libitum access to experimental diets and water throughout the duration of the studies. Table 1. Dietary composition of the experimental diet (g/kg on as-fed basis). Ingredients, g/kg Experimental diet1 Corn 392 Soybean meal, 48% 360 Soy oil 50 Dicalcium phosphate 19 Limestone 12 Barley 150 Vitamin-mineral premix2 3 L-lysine HCl 2 DL-methionine 1.9 L-threonine 0.7 Salt (NaCl) 4.1 Titanium dioxide3 5 Total 1000 Ingredients, g/kg Experimental diet1 Corn 392 Soybean meal, 48% 360 Soy oil 50 Dicalcium phosphate 19 Limestone 12 Barley 150 Vitamin-mineral premix2 3 L-lysine HCl 2 DL-methionine 1.9 L-threonine 0.7 Salt (NaCl) 4.1 Titanium dioxide3 5 Total 1000 1In addition to the 5 g of titanium dioxide per kg diet already in the diet fed in EXP 1, 5 g of chromic oxide per kg of diet was also added at the expense of corn to EXP 1 diet to produce EXP 2 diet. 2Provided per kilogram of diet: iron, 71.6 mg; copper, 11.0 mg; manganese, 178.7 mg; zinc, 178.7 mg; iodine, 3.0 mg; selenium, 0.4 mg; vitamin A (retinyl acetate), 18,904.3 IU; vitamin D3 (cholecalciferol), 9,480.0 IU; vitamin E (dl-α-tocopheryl acetate), 63.0 IU; vitamin K activity, 6.4 mg; thiamine, 3.2 mg; riboflavin, 9.4 mg; pantothenic acid, 34.7 mg; niacin, 126.0 mg; pyridoxine, 4.7 mg; folic acid, 1.6 mg; biotin, 0.5 mg; vitamin B12, 35.4 μg; choline, 956.9 mg. 3Titanium dioxide (as well as chromic oxide) was added to the diet at the expense of ground corn. View Large Table 1. Dietary composition of the experimental diet (g/kg on as-fed basis). Ingredients, g/kg Experimental diet1 Corn 392 Soybean meal, 48% 360 Soy oil 50 Dicalcium phosphate 19 Limestone 12 Barley 150 Vitamin-mineral premix2 3 L-lysine HCl 2 DL-methionine 1.9 L-threonine 0.7 Salt (NaCl) 4.1 Titanium dioxide3 5 Total 1000 Ingredients, g/kg Experimental diet1 Corn 392 Soybean meal, 48% 360 Soy oil 50 Dicalcium phosphate 19 Limestone 12 Barley 150 Vitamin-mineral premix2 3 L-lysine HCl 2 DL-methionine 1.9 L-threonine 0.7 Salt (NaCl) 4.1 Titanium dioxide3 5 Total 1000 1In addition to the 5 g of titanium dioxide per kg diet already in the diet fed in EXP 1, 5 g of chromic oxide per kg of diet was also added at the expense of corn to EXP 1 diet to produce EXP 2 diet. 2Provided per kilogram of diet: iron, 71.6 mg; copper, 11.0 mg; manganese, 178.7 mg; zinc, 178.7 mg; iodine, 3.0 mg; selenium, 0.4 mg; vitamin A (retinyl acetate), 18,904.3 IU; vitamin D3 (cholecalciferol), 9,480.0 IU; vitamin E (dl-α-tocopheryl acetate), 63.0 IU; vitamin K activity, 6.4 mg; thiamine, 3.2 mg; riboflavin, 9.4 mg; pantothenic acid, 34.7 mg; niacin, 126.0 mg; pyridoxine, 4.7 mg; folic acid, 1.6 mg; biotin, 0.5 mg; vitamin B12, 35.4 μg; choline, 956.9 mg. 3Titanium dioxide (as well as chromic oxide) was added to the diet at the expense of ground corn. View Large Table 2. Analyzed nutrient composition of the experimental diets (%). Experiment 1 diet Experiment 2 diet Indispensable amino acid Arginine 1.46 1.43 Histidine 0.56 0.56 Isoleucine 0.98 0.97 Leucine 1.81 1.79 Lysine 1.43 1.44 Methionine 0.48 0.46 Phenylalanine 1.12 1.12 Threonine 0.87 0.87 Tryptophan 0.27 0.27 Valine 1.08 1.04 Dispensable amino acid Alanine 1.03 1.01 Aspartic acid 2.21 2.19 Cysteine 0.32 0.31 Glutamic acid 3.99 3.98 Glycine 0.91 0.90 Proline 1.32 1.23 Serine 0.92 0.94 Tyrosine 0.76 0.71 Total amino acid 21.75 21.45 Dry matter 90.57 91.17 Nitrogen 3.57 3.57 Calcium ND1 1.23 Phosphorus ND 0.93 Experiment 1 diet Experiment 2 diet Indispensable amino acid Arginine 1.46 1.43 Histidine 0.56 0.56 Isoleucine 0.98 0.97 Leucine 1.81 1.79 Lysine 1.43 1.44 Methionine 0.48 0.46 Phenylalanine 1.12 1.12 Threonine 0.87 0.87 Tryptophan 0.27 0.27 Valine 1.08 1.04 Dispensable amino acid Alanine 1.03 1.01 Aspartic acid 2.21 2.19 Cysteine 0.32 0.31 Glutamic acid 3.99 3.98 Glycine 0.91 0.90 Proline 1.32 1.23 Serine 0.92 0.94 Tyrosine 0.76 0.71 Total amino acid 21.75 21.45 Dry matter 90.57 91.17 Nitrogen 3.57 3.57 Calcium ND1 1.23 Phosphorus ND 0.93 1ND = not determined. View Large Table 2. Analyzed nutrient composition of the experimental diets (%). Experiment 1 diet Experiment 2 diet Indispensable amino acid Arginine 1.46 1.43 Histidine 0.56 0.56 Isoleucine 0.98 0.97 Leucine 1.81 1.79 Lysine 1.43 1.44 Methionine 0.48 0.46 Phenylalanine 1.12 1.12 Threonine 0.87 0.87 Tryptophan 0.27 0.27 Valine 1.08 1.04 Dispensable amino acid Alanine 1.03 1.01 Aspartic acid 2.21 2.19 Cysteine 0.32 0.31 Glutamic acid 3.99 3.98 Glycine 0.91 0.90 Proline 1.32 1.23 Serine 0.92 0.94 Tyrosine 0.76 0.71 Total amino acid 21.75 21.45 Dry matter 90.57 91.17 Nitrogen 3.57 3.57 Calcium ND1 1.23 Phosphorus ND 0.93 Experiment 1 diet Experiment 2 diet Indispensable amino acid Arginine 1.46 1.43 Histidine 0.56 0.56 Isoleucine 0.98 0.97 Leucine 1.81 1.79 Lysine 1.43 1.44 Methionine 0.48 0.46 Phenylalanine 1.12 1.12 Threonine 0.87 0.87 Tryptophan 0.27 0.27 Valine 1.08 1.04 Dispensable amino acid Alanine 1.03 1.01 Aspartic acid 2.21 2.19 Cysteine 0.32 0.31 Glutamic acid 3.99 3.98 Glycine 0.91 0.90 Proline 1.32 1.23 Serine 0.92 0.94 Tyrosine 0.76 0.71 Total amino acid 21.75 21.45 Dry matter 90.57 91.17 Nitrogen 3.57 3.57 Calcium ND1 1.23 Phosphorus ND 0.93 1ND = not determined. View Large Experiment 1 Experiment 1 (Exp 1) was designed to evaluate the effect of two drying methods (freeze-drying vs. forced air-oven drying) and two drying temperatures (40 vs. 55°C) on apparent ileal DM, N, and AA digestibility. The 55°C was selected to reflect temperature close to what is commonly used when samples are dried in forced air oven while 40°C was selected to check if drying at a lower temperature would influence amino acid digestibility values. One hundred and sixty-eight chicks were randomly assigned to treatments in a completely randomized design with a 2 × 2 factorial arrangement of treatment. The design consists of four treatment groups (2 drying methods × 2 drying temperatures) each with seven replicate cages, and each replicate consisted of six birds per cage. Titanium dioxide was included in the experimental diet at 5 g/kg of diet as an inert marker for digestibility calculation. Digesta samples were collected via squeezing. Experiment 2 Experiment 2 (Exp 2) was conducted to evaluate the effect of sampling method (flushing vs. squeezing) as well as marker types (Ti vs. Cr) on apparent ileal DM, N, Ca, P, and AA digestibility. A total of 96 d-old broiler chicks were randomly allocated to two treatments, each having eight replicate cages with six birds per replicate. The experimental diet contained both titanium oxide and chromium oxide which served as the indigestible markers. Each of the marker was added to the diet at 5 g/kg of diet. Sample Collection On d 21, birds were euthanized by argon gas asphyxiation before collection of ileal contents. The body cavity was opened and digesta samples were collected from the distal two thirds of the ileum by squeezing (Exp 1) or by squeezing or flushing (Exp 2) with distilled water. In order to minimize the effect of differences in pressure applied during squeezing, both the flushing and squeezing were done jointly by the same set of people (2 people). The ileal contents from birds within each cage were pooled into a clean and pre-labeled plastic container to allow for sufficient sample for chemical analysis and immediately stored in the freezer at −20°C. Subsequently, digesta samples were either FD or OD at 40 or 55°C (Exp 1) or FD only (Exp 2) at 55°C. The 55°C drying temperature was selected to be close to the temperature that is commonly used for oven drying (forced air oven). We selected 40°C to check if there would be any effect of a reduction in drying temperature on apparent ileal amino acid digestibility. The freeze dryer temperature was raised gradually to avoid a rapid increase in the drying temperature from zero to 40 or 55°C. Diets were ground to pass through a 0.5 mm screen using a mill grinder (Retsch ZM 100, Retsch GmbH and Co., K.G., Haan, Germany) while ileal digesta were ground with a coffee grinder in preparation for chemical analyses. Chemical Analysis Duplicate determinations of percentage dry matter contents of dried ileal digesta and diets was conducted by oven drying samples in duplicates at 105°C for 24 h (AOAC International, 1990; method 925.09). To prevent differences due to analytical errors, FD and OD ileal digesta samples from both experiments as well as their respective diets were subjected to the same analytical techniques at the same time. Titanium, Cr, and AA analyses were conducted at the Agricultural Experiment Station Chemical Laboratories, University of Missouri-Columbia (Columbia, MO). Nitrogen content was determined by the combustion method using a LECO Trumac Nitrogen Analyzer (LECO, St. Joseph, MI; AOAC International, 2000; method 990.03). Amino acid content of the experimental diets and ileal digesta were determined using an amino acid analyzer (Hitachi L-8900, Tokyo, Japan). Prior to analysis, samples were hydrolyzed in 6 N HCl at 110°C for 24 h, and aliquots of the hydrolysates were analyzed and quantified using post-column ninhydrin derivatization [method 982.30 E (a, b, c); AOAC International, 2006]. Samples were oxidized using performic acid prior to estimation of sulfur AA (Met and Cys). Tryptophan content was measured by HPLC after an alkaline hydrolysis in barium hydroxide solution. The concentration of Ti and Cr in the diets and digesta samples were determined. Samples were digested as described by Myers et al. (2004) after which Ti concentration was determined by flame atomic absorption spectroscopy. Cr in the diets and ileal digesta samples were determined as described by Fenton and Fenton (1979) using the Perkin-Elmer Optical Emission Spectrometer (Optima 2000 DV, Waltham, MA). STATISTICAL ANALYSIS Data were subjected to ANOVA using the general linear model procedures of SAS (SAS 9.3, Inc., Cary, NC) that is appropriate for factorial arrangement of treatments. Data were analyzed to determine the significance of main effects (drying temperature and drying method, Exp 1) and their interaction. When interactions were not significant, the interaction term was removed from the code and reanalyzed. For Exp 2, data were analyzed using Proc Mixed procedure of SAS with sampling method (squeezing or flushing) as the main plot and marker type (Ti or Cr) as the subplot. As with Exp 1, the interaction term was removed from the SAS codes whenever the interaction was not significant. Where necessary, differences between treatment means were compared using Tukey. The level of significance was set at P < 0.05. RESULTS The calculated percent moisture contents post drying are reported in Table 3. There was an interaction (P < 0.05) between drying method and drying temperature with OD method resulting in higher (P < 0.05) moisture loss compared to FD method at 40°C. Moisture loss for FD at 55°C was higher (P < 0.05) compared to the OD method at the same temperature. The highest and lowest levels of moisture loss (P < 0.05) were recorded for FD at 55°C and FD at 40°C, respectively (Table 3). There was no interaction between drying method and drying temperature on apparent ileal AA digestibility (Table 4, Exp 1). There was no significant main effect of drying method or drying temperature on apparent ileal AA digestibility. Methionine had the highest (92.3%) while Cys had the lowest (73.8%) ileal digestibility values. In Exp. 2, there was no interaction between sampling methods (flushing vs. squeezing) and marker types (Ti vs. Cr) on apparent ileal DM, N, Ca, P, and AA digestibility (Table 5, Exp 2). Similarly, the samples from the squeezing method had higher (P < 0.05) apparent ileal Arg and Met digestibility while the digestibility values for His, Ile, Cys, Ser, and Tyr tended to be higher (P < 0.1) compared to those from the flushing method (Table 5, Exp 2). Furthermore, the apparent ileal DM, N, Ca, P, and AA digestibility was not affected by the different markers (Table 5). Table 3. Effect of drying method (freeze-drying vs. forced-air oven drying) and temperature (40 vs. 55°C) on moisture contents of ileal digesta of broilers fed corn-soybean meal-barley based diet, (%). Method Temperature oC Freeze 401 Freeze 552 Oven 403 Oven 554 Pooled SEM Method Temperature Method*temperature Moisture 2.2d 8.4a 6.7b 5.6c 0.174 <.0001 <.0001 <.0001 Method Temperature oC Freeze 401 Freeze 552 Oven 403 Oven 554 Pooled SEM Method Temperature Method*temperature Moisture 2.2d 8.4a 6.7b 5.6c 0.174 <.0001 <.0001 <.0001 a-dMeans with different superscripts are different (P < 0.05) 1Freeze-dried samples were dried at −15°C for 40 h, 0°C for 40 h, +5°C for 20 h, +15°C for 20 h, +25°C for 20 h, and +40°C for 40 h. 2Freeze-dried samples were dried at −15°C for 40 h, 0°C for 40 h, +5°C for 20 h, +15°C for 20 h, +25°C for 20 h, and +40°C for 40 h, and +55°C for 40 h. 3Oven dried samples were dried at 40°C for 6 days. 4Oven dried samples were dried at 55°C for 6 days. View Large Table 3. Effect of drying method (freeze-drying vs. forced-air oven drying) and temperature (40 vs. 55°C) on moisture contents of ileal digesta of broilers fed corn-soybean meal-barley based diet, (%). Method Temperature oC Freeze 401 Freeze 552 Oven 403 Oven 554 Pooled SEM Method Temperature Method*temperature Moisture 2.2d 8.4a 6.7b 5.6c 0.174 <.0001 <.0001 <.0001 Method Temperature oC Freeze 401 Freeze 552 Oven 403 Oven 554 Pooled SEM Method Temperature Method*temperature Moisture 2.2d 8.4a 6.7b 5.6c 0.174 <.0001 <.0001 <.0001 a-dMeans with different superscripts are different (P < 0.05) 1Freeze-dried samples were dried at −15°C for 40 h, 0°C for 40 h, +5°C for 20 h, +15°C for 20 h, +25°C for 20 h, and +40°C for 40 h. 2Freeze-dried samples were dried at −15°C for 40 h, 0°C for 40 h, +5°C for 20 h, +15°C for 20 h, +25°C for 20 h, and +40°C for 40 h, and +55°C for 40 h. 3Oven dried samples were dried at 40°C for 6 days. 4Oven dried samples were dried at 55°C for 6 days. View Large Table 4. The effect of drying method (freeze-drying vs. forced-air oven drying) and temperature (40 vs. 55°C) on apparent ileal amino acid digestibility in 21 day-old broilers fed barley based diet (Experiment 1). Drying method Drying temperature (°C) Method Temperature Method* temperature Freeze-dried1 Oven dried2 40 55 SEM Probability Dry matter 67.5 67.7 67.3 67.9 0.73 0.824 0.483 0.227 Nitrogen 81.2 81.5 81.1 81.5 0.48 0.576 0.379 0.701 Indispensable amino acid Arg 88.6 88.9 88.7 88.7 0.46 0.538 0.975 0.131 His 84.9 85.2 84.9 85.2 0.48 0.516 0.498 0.140 Ile 82.8 83.2 83.0 83.0 0.65 0.530 0.931 0.185 Leu 83.8 84.2 84.0 84.1 0.61 0.447 0.853 0.253 Lys 86.4 87.0 86.6 86.8 0.53 0.300 0.701 0.087 Met 92.2 92.5 92.2 92.4 0.37 0.420 0.692 0.165 Phe 84.1 84.5 84.2 84.4 0.57 0.446 0.675 0.198 Thr 78.0 78.4 78.1 78.3 0.62 0.537 0.748 0.206 Trp 87.4 87.6 87.4 87.5 0.46 0.647 0.807 0.218 Val 78.3 79.0 78.6 78.7 0.67 0.375 0.793 0.164 Dispensable amino acid Ala 83.2 83.7 83.3 83.6 0.62 0.454 0.601 0.209 Asp 80.9 81.2 80.9 81.2 0.66 0.662 0.725 0.260 Cys 73.7 73.9 73.5 74.2 0.85 0.855 0.411 0.310 Glu 87.6 87.9 87.7 87.8 0.46 0.476 0.749 0.165 Gly 79.2 79.6 79.1 79.7 0.67 0.599 0.327 0.253 Pro 83.4 83.8 83.6 83.7 0.53 0.447 0.800 0.359 Ser 81.3 81.7 81.3 81.7 0.61 0.444 0.545 0.241 Tyr 84.5 84.9 84.7 84.7 0.62 0.527 0.964 0.116 Total 83.4 83.8 83.5 83.7 0.56 0.474 0.733 0.162 Drying method Drying temperature (°C) Method Temperature Method* temperature Freeze-dried1 Oven dried2 40 55 SEM Probability Dry matter 67.5 67.7 67.3 67.9 0.73 0.824 0.483 0.227 Nitrogen 81.2 81.5 81.1 81.5 0.48 0.576 0.379 0.701 Indispensable amino acid Arg 88.6 88.9 88.7 88.7 0.46 0.538 0.975 0.131 His 84.9 85.2 84.9 85.2 0.48 0.516 0.498 0.140 Ile 82.8 83.2 83.0 83.0 0.65 0.530 0.931 0.185 Leu 83.8 84.2 84.0 84.1 0.61 0.447 0.853 0.253 Lys 86.4 87.0 86.6 86.8 0.53 0.300 0.701 0.087 Met 92.2 92.5 92.2 92.4 0.37 0.420 0.692 0.165 Phe 84.1 84.5 84.2 84.4 0.57 0.446 0.675 0.198 Thr 78.0 78.4 78.1 78.3 0.62 0.537 0.748 0.206 Trp 87.4 87.6 87.4 87.5 0.46 0.647 0.807 0.218 Val 78.3 79.0 78.6 78.7 0.67 0.375 0.793 0.164 Dispensable amino acid Ala 83.2 83.7 83.3 83.6 0.62 0.454 0.601 0.209 Asp 80.9 81.2 80.9 81.2 0.66 0.662 0.725 0.260 Cys 73.7 73.9 73.5 74.2 0.85 0.855 0.411 0.310 Glu 87.6 87.9 87.7 87.8 0.46 0.476 0.749 0.165 Gly 79.2 79.6 79.1 79.7 0.67 0.599 0.327 0.253 Pro 83.4 83.8 83.6 83.7 0.53 0.447 0.800 0.359 Ser 81.3 81.7 81.3 81.7 0.61 0.444 0.545 0.241 Tyr 84.5 84.9 84.7 84.7 0.62 0.527 0.964 0.116 Total 83.4 83.8 83.5 83.7 0.56 0.474 0.733 0.162 1Freeze-dried samples were dried at 40°C: −15°C for 40 h, 0°C for 40 h, +5°C for 20 h, +15°C for 20 h, +25°C for 20 h, and +40°C for 40 h; 55°C: −15°C for 40 h, 0°C for 40 h, +5°C for 20 h, +15°C for 20 h, +25°C for 20 h, and +40°C for 40 h, and +55°C for 40 h. 2Oven dried samples were dried at 40°C for 6 days or at 55°C for 6 days. View Large Table 4. The effect of drying method (freeze-drying vs. forced-air oven drying) and temperature (40 vs. 55°C) on apparent ileal amino acid digestibility in 21 day-old broilers fed barley based diet (Experiment 1). Drying method Drying temperature (°C) Method Temperature Method* temperature Freeze-dried1 Oven dried2 40 55 SEM Probability Dry matter 67.5 67.7 67.3 67.9 0.73 0.824 0.483 0.227 Nitrogen 81.2 81.5 81.1 81.5 0.48 0.576 0.379 0.701 Indispensable amino acid Arg 88.6 88.9 88.7 88.7 0.46 0.538 0.975 0.131 His 84.9 85.2 84.9 85.2 0.48 0.516 0.498 0.140 Ile 82.8 83.2 83.0 83.0 0.65 0.530 0.931 0.185 Leu 83.8 84.2 84.0 84.1 0.61 0.447 0.853 0.253 Lys 86.4 87.0 86.6 86.8 0.53 0.300 0.701 0.087 Met 92.2 92.5 92.2 92.4 0.37 0.420 0.692 0.165 Phe 84.1 84.5 84.2 84.4 0.57 0.446 0.675 0.198 Thr 78.0 78.4 78.1 78.3 0.62 0.537 0.748 0.206 Trp 87.4 87.6 87.4 87.5 0.46 0.647 0.807 0.218 Val 78.3 79.0 78.6 78.7 0.67 0.375 0.793 0.164 Dispensable amino acid Ala 83.2 83.7 83.3 83.6 0.62 0.454 0.601 0.209 Asp 80.9 81.2 80.9 81.2 0.66 0.662 0.725 0.260 Cys 73.7 73.9 73.5 74.2 0.85 0.855 0.411 0.310 Glu 87.6 87.9 87.7 87.8 0.46 0.476 0.749 0.165 Gly 79.2 79.6 79.1 79.7 0.67 0.599 0.327 0.253 Pro 83.4 83.8 83.6 83.7 0.53 0.447 0.800 0.359 Ser 81.3 81.7 81.3 81.7 0.61 0.444 0.545 0.241 Tyr 84.5 84.9 84.7 84.7 0.62 0.527 0.964 0.116 Total 83.4 83.8 83.5 83.7 0.56 0.474 0.733 0.162 Drying method Drying temperature (°C) Method Temperature Method* temperature Freeze-dried1 Oven dried2 40 55 SEM Probability Dry matter 67.5 67.7 67.3 67.9 0.73 0.824 0.483 0.227 Nitrogen 81.2 81.5 81.1 81.5 0.48 0.576 0.379 0.701 Indispensable amino acid Arg 88.6 88.9 88.7 88.7 0.46 0.538 0.975 0.131 His 84.9 85.2 84.9 85.2 0.48 0.516 0.498 0.140 Ile 82.8 83.2 83.0 83.0 0.65 0.530 0.931 0.185 Leu 83.8 84.2 84.0 84.1 0.61 0.447 0.853 0.253 Lys 86.4 87.0 86.6 86.8 0.53 0.300 0.701 0.087 Met 92.2 92.5 92.2 92.4 0.37 0.420 0.692 0.165 Phe 84.1 84.5 84.2 84.4 0.57 0.446 0.675 0.198 Thr 78.0 78.4 78.1 78.3 0.62 0.537 0.748 0.206 Trp 87.4 87.6 87.4 87.5 0.46 0.647 0.807 0.218 Val 78.3 79.0 78.6 78.7 0.67 0.375 0.793 0.164 Dispensable amino acid Ala 83.2 83.7 83.3 83.6 0.62 0.454 0.601 0.209 Asp 80.9 81.2 80.9 81.2 0.66 0.662 0.725 0.260 Cys 73.7 73.9 73.5 74.2 0.85 0.855 0.411 0.310 Glu 87.6 87.9 87.7 87.8 0.46 0.476 0.749 0.165 Gly 79.2 79.6 79.1 79.7 0.67 0.599 0.327 0.253 Pro 83.4 83.8 83.6 83.7 0.53 0.447 0.800 0.359 Ser 81.3 81.7 81.3 81.7 0.61 0.444 0.545 0.241 Tyr 84.5 84.9 84.7 84.7 0.62 0.527 0.964 0.116 Total 83.4 83.8 83.5 83.7 0.56 0.474 0.733 0.162 1Freeze-dried samples were dried at 40°C: −15°C for 40 h, 0°C for 40 h, +5°C for 20 h, +15°C for 20 h, +25°C for 20 h, and +40°C for 40 h; 55°C: −15°C for 40 h, 0°C for 40 h, +5°C for 20 h, +15°C for 20 h, +25°C for 20 h, and +40°C for 40 h, and +55°C for 40 h. 2Oven dried samples were dried at 40°C for 6 days or at 55°C for 6 days. View Large Table 5. The effect of sampling method (flushing vs. squeezing) and marker type (titanium oxide vs. chromium oxide) on apparent ileal dry matter and amino acid digestibility in 21 day-old broilers fed barley-based diet (%, Experiment 2). Method Marker Method Marker Method Marker Method*Marker Flush Squeeze Chromium Titanium SEM Probability Indispensable AA Arg 88.4 89.0 88.8 88.6 0.21 0.21 0.042 0.715 0.971 His 85.4 86.1 85.8 85.7 0.25 0.25 0.070 0.707 0.995 Ile 83.2 84.0 83.7 83.5 0.28 0.28 0.067 0.716 0.979 Leu 84.2 84.6 84.4 84.3 0.31 0.31 0.371 0.738 0.971 Lys 87.0 87.5 87.3 87.2 0.23 0.23 0.113 0.685 0.969 Met 91.8 92.5 92.2 92.1 0.19 0.19 0.009 0.792 0.974 Phe 84.6 85.4 85.1 84.9 0.33 0.33 0.112 0.749 0.998 Thr 78.8 79.3 79.1 79.0 0.33 0.33 0.352 0.689 0.955 Trp 88.7 89.0 88.9 88.8 0.29 0.29 0.390 0.775 0.981 Val 78.5 79.0 78.8 78.6 0.33 0.33 0.324 0.663 0.973 Dispensable AA Ala 83.4 83.7 83.7 83.5 0.31 0.31 0.427 0.704 0.998 Asp 81.3 81.7 81.6 81.4 0.32 0.32 0.329 0.687 0.998 Cys 73.7 75.1 74.5 74.3 0.50 0.50 0.060 0.738 0.990 Glu 88.0 88.4 88.2 88.1 0.22 0.22 0.171 0.745 0.970 Gly 79.5 79.9 79.8 79.6 0.32 0.32 0.430 0.693 0.985 Pro 82.9 83.0 83.1 82.9 0.30 0.30 0.797 0.712 0.982 Ser 82.3 83.2 82.8 82.7 0.31 0.31 0.070 0.706 0.968 Tyr 83.8 84.5 84.2 84.1 0.27 0.27 0.099 0.699 0.983 Total 83.7 84.2 84.0 83.9 0.27 0.27 0.218 0.714 0.976 Dry matter 67.3 67.6 67.6 67.3 0.39 0.39 0.656 0.570 0.988 Nitrogen 81.3 81.8 81.7 81.5 0.28 0.28 0.176 0.642 0.989 Calcium 33.8 32.6 33.6 32.9 1.71 1.71 0.626 0.793 0.981 Phosphorus 45.6 46.8 46.5 46.0 0.84 0.84 0.330 0.677 0.963 Method Marker Method Marker Method Marker Method*Marker Flush Squeeze Chromium Titanium SEM Probability Indispensable AA Arg 88.4 89.0 88.8 88.6 0.21 0.21 0.042 0.715 0.971 His 85.4 86.1 85.8 85.7 0.25 0.25 0.070 0.707 0.995 Ile 83.2 84.0 83.7 83.5 0.28 0.28 0.067 0.716 0.979 Leu 84.2 84.6 84.4 84.3 0.31 0.31 0.371 0.738 0.971 Lys 87.0 87.5 87.3 87.2 0.23 0.23 0.113 0.685 0.969 Met 91.8 92.5 92.2 92.1 0.19 0.19 0.009 0.792 0.974 Phe 84.6 85.4 85.1 84.9 0.33 0.33 0.112 0.749 0.998 Thr 78.8 79.3 79.1 79.0 0.33 0.33 0.352 0.689 0.955 Trp 88.7 89.0 88.9 88.8 0.29 0.29 0.390 0.775 0.981 Val 78.5 79.0 78.8 78.6 0.33 0.33 0.324 0.663 0.973 Dispensable AA Ala 83.4 83.7 83.7 83.5 0.31 0.31 0.427 0.704 0.998 Asp 81.3 81.7 81.6 81.4 0.32 0.32 0.329 0.687 0.998 Cys 73.7 75.1 74.5 74.3 0.50 0.50 0.060 0.738 0.990 Glu 88.0 88.4 88.2 88.1 0.22 0.22 0.171 0.745 0.970 Gly 79.5 79.9 79.8 79.6 0.32 0.32 0.430 0.693 0.985 Pro 82.9 83.0 83.1 82.9 0.30 0.30 0.797 0.712 0.982 Ser 82.3 83.2 82.8 82.7 0.31 0.31 0.070 0.706 0.968 Tyr 83.8 84.5 84.2 84.1 0.27 0.27 0.099 0.699 0.983 Total 83.7 84.2 84.0 83.9 0.27 0.27 0.218 0.714 0.976 Dry matter 67.3 67.6 67.6 67.3 0.39 0.39 0.656 0.570 0.988 Nitrogen 81.3 81.8 81.7 81.5 0.28 0.28 0.176 0.642 0.989 Calcium 33.8 32.6 33.6 32.9 1.71 1.71 0.626 0.793 0.981 Phosphorus 45.6 46.8 46.5 46.0 0.84 0.84 0.330 0.677 0.963 View Large Table 5. The effect of sampling method (flushing vs. squeezing) and marker type (titanium oxide vs. chromium oxide) on apparent ileal dry matter and amino acid digestibility in 21 day-old broilers fed barley-based diet (%, Experiment 2). Method Marker Method Marker Method Marker Method*Marker Flush Squeeze Chromium Titanium SEM Probability Indispensable AA Arg 88.4 89.0 88.8 88.6 0.21 0.21 0.042 0.715 0.971 His 85.4 86.1 85.8 85.7 0.25 0.25 0.070 0.707 0.995 Ile 83.2 84.0 83.7 83.5 0.28 0.28 0.067 0.716 0.979 Leu 84.2 84.6 84.4 84.3 0.31 0.31 0.371 0.738 0.971 Lys 87.0 87.5 87.3 87.2 0.23 0.23 0.113 0.685 0.969 Met 91.8 92.5 92.2 92.1 0.19 0.19 0.009 0.792 0.974 Phe 84.6 85.4 85.1 84.9 0.33 0.33 0.112 0.749 0.998 Thr 78.8 79.3 79.1 79.0 0.33 0.33 0.352 0.689 0.955 Trp 88.7 89.0 88.9 88.8 0.29 0.29 0.390 0.775 0.981 Val 78.5 79.0 78.8 78.6 0.33 0.33 0.324 0.663 0.973 Dispensable AA Ala 83.4 83.7 83.7 83.5 0.31 0.31 0.427 0.704 0.998 Asp 81.3 81.7 81.6 81.4 0.32 0.32 0.329 0.687 0.998 Cys 73.7 75.1 74.5 74.3 0.50 0.50 0.060 0.738 0.990 Glu 88.0 88.4 88.2 88.1 0.22 0.22 0.171 0.745 0.970 Gly 79.5 79.9 79.8 79.6 0.32 0.32 0.430 0.693 0.985 Pro 82.9 83.0 83.1 82.9 0.30 0.30 0.797 0.712 0.982 Ser 82.3 83.2 82.8 82.7 0.31 0.31 0.070 0.706 0.968 Tyr 83.8 84.5 84.2 84.1 0.27 0.27 0.099 0.699 0.983 Total 83.7 84.2 84.0 83.9 0.27 0.27 0.218 0.714 0.976 Dry matter 67.3 67.6 67.6 67.3 0.39 0.39 0.656 0.570 0.988 Nitrogen 81.3 81.8 81.7 81.5 0.28 0.28 0.176 0.642 0.989 Calcium 33.8 32.6 33.6 32.9 1.71 1.71 0.626 0.793 0.981 Phosphorus 45.6 46.8 46.5 46.0 0.84 0.84 0.330 0.677 0.963 Method Marker Method Marker Method Marker Method*Marker Flush Squeeze Chromium Titanium SEM Probability Indispensable AA Arg 88.4 89.0 88.8 88.6 0.21 0.21 0.042 0.715 0.971 His 85.4 86.1 85.8 85.7 0.25 0.25 0.070 0.707 0.995 Ile 83.2 84.0 83.7 83.5 0.28 0.28 0.067 0.716 0.979 Leu 84.2 84.6 84.4 84.3 0.31 0.31 0.371 0.738 0.971 Lys 87.0 87.5 87.3 87.2 0.23 0.23 0.113 0.685 0.969 Met 91.8 92.5 92.2 92.1 0.19 0.19 0.009 0.792 0.974 Phe 84.6 85.4 85.1 84.9 0.33 0.33 0.112 0.749 0.998 Thr 78.8 79.3 79.1 79.0 0.33 0.33 0.352 0.689 0.955 Trp 88.7 89.0 88.9 88.8 0.29 0.29 0.390 0.775 0.981 Val 78.5 79.0 78.8 78.6 0.33 0.33 0.324 0.663 0.973 Dispensable AA Ala 83.4 83.7 83.7 83.5 0.31 0.31 0.427 0.704 0.998 Asp 81.3 81.7 81.6 81.4 0.32 0.32 0.329 0.687 0.998 Cys 73.7 75.1 74.5 74.3 0.50 0.50 0.060 0.738 0.990 Glu 88.0 88.4 88.2 88.1 0.22 0.22 0.171 0.745 0.970 Gly 79.5 79.9 79.8 79.6 0.32 0.32 0.430 0.693 0.985 Pro 82.9 83.0 83.1 82.9 0.30 0.30 0.797 0.712 0.982 Ser 82.3 83.2 82.8 82.7 0.31 0.31 0.070 0.706 0.968 Tyr 83.8 84.5 84.2 84.1 0.27 0.27 0.099 0.699 0.983 Total 83.7 84.2 84.0 83.9 0.27 0.27 0.218 0.714 0.976 Dry matter 67.3 67.6 67.6 67.3 0.39 0.39 0.656 0.570 0.988 Nitrogen 81.3 81.8 81.7 81.5 0.28 0.28 0.176 0.642 0.989 Calcium 33.8 32.6 33.6 32.9 1.71 1.71 0.626 0.793 0.981 Phosphorus 45.6 46.8 46.5 46.0 0.84 0.84 0.330 0.677 0.963 View Large DISCUSSION Accurate nutrient digestibility estimation requires some form of sample preparation including proper drying prior to preparation for laboratory analysis. The drying methods used in this study, oven drying and freeze drying, are used by many laboratories as a routine technique to prepare samples for analysis. After collecting ileal or excreta samples, they were put through the different methods mentioned above until a constant mass was reached after which moisture loss or dry matter contents can be determined by weight difference (Galle, 2001). The per cent moisture obtained in this study shows that the greatest moisture loss was obtained by freeze-drying at 55°C while the least amount of moisture was lost when samples were freeze-dried at 40°C. By increasing the drying temperature from 40 to 55°C, the difference in the moisture contents of ileal digesta was 6.2%-points (FD) compared to 1.1%-points for OD samples. This shows that there may not be any advantage of drying ileal digesta samples beyond 40°C in forced air oven in terms of further moisture removal. Despite this however, freeze drying technique is favored by many industries including soil engineering, food industry because it is believed that freeze-drying protects the primary structure and shape of the sample while decreasing the danger of altering more labile AA (Ray and Kothmann, 1988; Ratti, 2001). Furthermore, ileal AA digestibility studies have often adopted the FD method because of the concern for the quality of AA and N when ileal digesta are dried using the forced air oven. Indeed, in these studies, the ileal digesta samples that were dried in the forced-air oven were extremely hard and were much more difficult to grind compared to samples that were freeze-dried. However, the cost and the need for regular maintenance of a freeze-dryer is challenging. Previous studies showed that OD at 65°C resulted in energy and N loss of 11.4 and 5.2%, respectively, (Manoukas et al., 1964) while FD resulted in energy loss of 1.3% which increased to 5.5% when OD at 60°C (Shannon and Brown, 1969). However, in a different study where the effects of drying method on different tissues were evaluated, it was reported that the amount of moisture lost from tissue when freeze-dried or dried in a desiccator was not different (Sahin et al., 2006). The recoveries of the markers (titanium and chromium) in the experimental diets fed to birds in these studies were >97%. No significant interaction was observed on apparent ileal DM, N, and AA digestibility that could be attributed to both the drying methods and drying temperatures (Exp 1). The digestibility of the AA was similarly not affected by the main factors (drying method and drying temperature) however, Met had the highest digestibility (92.3%) while Cys had the lowest (73.7%) digestibility value. This indicates that the availability and quality of these AA were not affected by the drying method or temperature. In evaluating the effect of the drying procedure on apparent ileal digestibility, the results were different from studies conducted by Dale et al. (1985) which showed that the availability of Ala increased in both the diets and excreta when FD or that of Sibbald (1979) who reported that oven drying of excreta was a better alternative to freeze-drying when estimating true metabolizable energy, however, Hinnant and Kothmann (1988) reported no significant effects of drying methods on N contents of excreta samples (OD, 1.35% vs. FD, 1.38%). In Exp. 2 where two inert dietary markers were added to the diet, ileal digesta samples were either flushed or squeezed to determine the effect of sampling methods (flushing vs. squeezing) and marker type (Ti vs Cr) on apparent ileal DM, N, Ca, P, and AA digestibility. There was no interaction between the sampling methods (flushing vs. squeezing) and marker types (Ti vs Cr). Similarly, the effect of sampling method was not significant except for Arg and Met where squeezing resulted in higher ileal digestibility values. Furthermore, apparent ileal His, Ile, Cys, Ser, and Tyr digestibility showed tendencies to be higher in squeezed digesta. This observation was opposite of what was expected and difficult to explain. However, this observation may be due to the nature of the digesta. Because of the presence of barley in the diet (15%), the digesta was very viscous and relatively difficult to flush compared to squeezing. There is the possibility of an increase adhesion of the digesta to the gastrointestinal wall, which when flushed, sloughed more cells including mucins, dissolving into the distilled water. The contribution of mucin to the ileal digesta can be attributed to increased concentration of amino acids in the digesta (Adedokun et al., 2011). This may explain, in part, the reason for the relatively low apparent ileal AA digestibility in the flushed samples. However, with digesta from less viscous diet, this may not be an issue. This difference in digestibility, even for the two AA that showed significant effect of sampling method, was very low (0.6 and 0.7-percentage points for Arg and Met, respectively). This was significant because of the very low standard error values. It is important to note that any effect or a lack of it may not be attributed to the chemical analysis procedure as digestibility values obtained from Exp 1 and 2 were quite similar. Contrary to the findings reported by Selle et al. (2006) on the meta-analysis of phytase effect on AA digestibility in poultry where the variation in phytase responses was attributed to the choice of inert marker used in the digestibility assays, the outcome from this current study showed no such effect. They (Selle et al., 2006) concluded that AA digestibility responses to phytase were higher when Ti or acid insoluble ash were used compared to Cr. Similarly, Olukosi et al. (2012) concluded that the apparent ileal AA digestibility in broilers and pigs with phytase supplementation determined with Ti as a digestibility marker were considerably higher than the values measured with the Cr marker. Jacobs et al. (2011) reported that there was no significant effect of drying method (FD vs. OD) as well as drying temperature (OD 55 vs. OD 100°C) on the N composition of poultry excreta. Despite the lack of significant difference in N contents of excreta samples that were oven dried at 55 vs. 100°C, the N contents of excreta samples that were dried at 100°C was about 3.8% lower than those dried at 55°C. This lack of significant effect of drying methods is consistent with what was observed for ileal digesta in the current study. Ribeiro et al. (2001) however reported a significant decrease in broiler excreta N contents between excreta samples that were dried at 55 and 100°C. There is a dearth of information on the effect of sampling or collection method on apparent ileal AA digestibility in broiler chickens. Poureslami et al. (2012) observed significant effect of collection method in all the AA they evaluated in their study with flushing having higher ileal AA digestibility compared to squeezing which is different from what we observed in the current study. This may be because of three factors. First, the data from Poureslami et al. (2012) were from 18 d-old broilers compared to 21 d-old broilers that were sampled in the current study. Secondly, the diet tested in the study by Poureslami et al. (2012) was fed for 4 d compared to 12 d in the current study. The difference in the length of feeding of these diets may influence the AA digestibility values as different diets may require different length of time for proper adaptation. The third reason, which may be the most important, is the nature of the diets fed. A semi-purified diet (soybean meal and dextrose-based) was fed to broilers in the study reported by Poureslami et al. (2012) while a complete diet (corn-soybean meal-barley-based) was fed to broilers in the current study. Generally, because of the presence of barley (15%) in the diet fed to broilers in the current study, the digesta from the birds were very viscous and it requires some efforts to squeeze but much more efforts to flush. There is the possibility that flushing may have led to a situation whereby more mucus layer as well as other sources of AA in the gastrointestinal tract (AA of endogenous origin) may have been flushed along with the digesta. This may explain the relatively lower apparent ileal AA digestibility values when ileal digesta were flushed. This may not be a challenge when digesta from birds fed a semi-purified diet or a complete diet with minimal level of non-starch polysaccharides are collected (easy flow). From the results presented in this study, it can be summarized that ileal digesta could be dried using either the freeze-dryer or forced-air oven without jeopardizing the quality of the digesta for AA analysis. Likewise, digesta could be dried at temperature as low as 40 and as high as 55oC without any significant negative effect on digestibility values. Furthermore, the use of Ti or Cr as inert marker did not influence apparent ileal AA digestibility values. The apparent ileal digestibility of two AA, Arg and Met, increased with squeezing while generally, sampling method did not influence AA digestibility in broilers fed corn-soybean meal-barley based diet. 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Poultry ScienceOxford University Press

Published: Mar 5, 2018

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