Effects of reducing dietary crude protein and whole grain feeding on performance and amino acid metabolism in broiler chickens offered wheat-based diets

Animal Feed Science and Technology 260 (2020) 114386

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Effects of reducing dietary crude protein and whole grain feeding on performance and amino acid metabolism in broiler chickens offered wheat-based diets

T

Dafei Yina,b, Peter V. Chrystalb,c, Amy F. Mossb,d, Sonia Yun Liub,e, Jianmin Yuana, Peter H. Selleb,* a

State Key Laboratory of Animal Nutrition, College of Animal Science and Technology, China Agricultural University, Beijing 100193, China Poultry Research Foundation Within The University of Sydney, Camden 2570, NSW, Australia c Baiada Poultry Pty Limited, Pendle Hill 2145 NSW, Australia d School of Environmental and Rural Science, University of New England, Armidale 2351 NSW, Australia e School of Life and Environmental Sciences, Faculty of Science, The University of Sydney, Camperdown 2050 NSW, Australia b

A R T IC LE I N F O

ABS TRA CT

Keywords: Amino acids Broiler chickens Growth performance Reduced-CP diets Whole grain feeding

A total of 336 off-sex, male Ross 308 chicks were offered seven dietary treatments from 7 to 35 days post-hatch; each treatment was offered to eight replicate cages with six birds per cage. Three wheat-based diets were formulated to declining crude protein (CP) levels of 215, 190 and 165 g/ kg but with a constant energy density (12.70 MJ/kg), electrolyte balance (250 mEq/kg) and digestible lysine level (11.00 g/kg). In a 2 × 3 factorial arrangement birds were offered either 215 or 165 g/kg CP diets to which 0%, 12.5% and 25.0% whole gain was incorporated postpelleting. In addition, a ground grain, 190 g/kg CP diet served as a seventh treatment. The assessed parameters included growth performance, relative gizzard, pancreas and abdominal fatpad weights, nutrient utilisation, concentrations of free amino acid in portal (anterior mesenteric vein) and systemic (brachial vein) plasma and apparent jejunal and ileal amino acid digestibility coefficients and disappearance rates. The CP reduction from 215 to 165 g/kg compromised FCR by 5.99% (1.576 versus 1.487; P < 0.005) and increased relative fat-pad weights by 12.2% (8.02 versus 7.15 g/kg; P < 0.025). Whole grain feeding (25.0%) significantly decreased relative fat-pad weights by 14.6% (6.91 versus 8.09 g/kg) but did not otherwise improve the performance of birds offered reduced-CP diets. Whole grain feeding enhanced energy utilisation (AME, ME:GE ratios, AMEn) but the CP reduction significantly compromised AME and AMEn. The transition from 215 to 165 g/kg CP in ground grain diets significantly depressed ileal digestibilities of histidine, alanine, aspartic acid, glycine, serine and tyrosine and generally depressed jejunal and ileal amino acid disappearance rates. However, methionine disappearance rates were increased by 28.6% (0.485 versus 0.377 g/bird/day; P < 0.005) and 22.8% (0.539 versus 0.439 g/bird/ day; P < 0.001), respectively. The effects of 215, 190 and 165 g/kg CP diets on portal free amino acid concentrations were diverse as lysine was linearly increased but histidine, leucine, tryptophan, asparagine, glycine, serine and tyrosine were decreased. The effects of reduced CP diets and whole grain feeding on systemic free amino acid concentrations were also diverse as significant treatment interactions were observed for isoleucine, leucine, lysine, valine, glutamine

Abbreviations: AIA, acid insoluble ash; AME, apparent metabolisable energy; AMEn, nitrogen corrected apparent metabolisable energy; CP, crude protein; DEB, dietary electrolyte balance; DI, distal ileum; DJ, distal jejunum; FCR, feed conversion ratio; FI, feed intake; ME:GE ratio, metabolisable to gross energy ratio; MJ, megajoules; mEq, milliequivalents; N, nitrogen; pGI, predicted glycaemic index ⁎ Corresponding author at: Poultry Research Foundation within The University of Sydney, Camden, NSW, Australia. E-mail address: [email protected] (P.H. Selle). https://doi.org/10.1016/j.anifeedsci.2019.114386 Received 6 May 2019; Received in revised form 16 October 2019; Accepted 30 December 2019 0377-8401/ © 2019 Published by Elsevier B.V.

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and proline. Reducing dietary CP compromised feed conversion efficiency which was not attenuated by whole grain feeding although whole grain feeding did reduce relative abdominal fatpad weights.

1. Introduction There is an intense global and local focus on the successful development of reduced-CP diets where feed grain contents are elevated at the expense of soybean meal and an increasing range of synthetic or unbound amino acids are included to meet amino acid requirements. The potential benefits of reduced-CP diets are very real and range from environmental advantages in respect of less nitrogen pollution and ammonia emissions to reduced feed costs. However, the successful development of tangibly reduced-CP diets is not straightforward. For example, Fancher and Jensen (1989b) reduced dietary CP from 215 to 160 g/kg in maize-based diets offered to male broiler chickens from 21 to 42 days post-hatch where the four low protein diets were supplemented with synthetic amino acids or potassium. Pursuant to this transition, FCR was compromised by an average of 12.1% (2.084 versus 1.859) and relative abdominal fat-pad weights were increased by 39.6% (21.5 versus 15.4 g/kg). These outcomes illustrate the challenge of developing reduced-CP diets, which is epitomised by compromised FCR and increased fat deposition. Whole grain feeding is a well-accepted practice in countries where wheat is the dominant feed grain for chicken-meat production. In Australia, for example, probably all broiler chickens are offered some whole grain usually as a blend with a pelleted concentrate where a proportion of 15–20% whole grain is commonly applied. Whole grain feeding is thought to promote efficiency of energy utilisation, improve FCR, enhance gut integrity, advantage litter quality and, in addition, reduce feed-milling costs (Liu et al., 2015; Moss et al., 2019). The hallmark response to whole grain feeding is increased relative weights of gizzards but the ramifications of heavier and, presumably, more functional gizzards on digestive processes in broiler chickens still require clarification (Moss et al., 2019). However, the gizzard has been described at the ‘pace-maker’ of gut motility and it is likely that whole grain feeding will promote episodes of reverse peristalsis (Duke, 1982; Ferket, 2000). In theory, both the gastric and small intestinal refluxes could accelerate the digestion of ‘intact’ protein by increasing the exposure of dietary proteins to pepsin and pancreatic enzymes in the gizzard. Given that synthetic or unbound amino acids do not require digestion and are rapidly absorbed (Wu, 2009) it then follows that if whole grain feeding accelerates the digestion of intact protein this would promote more synchronous intestinal uptakes of unbound and protein-bound amino acids (Liu and Selle, 2017). In the quest to develop reduced-CP diets insufficient attention appears to have been paid to jejunal and ileal amino acid digestibility coefficients and concentrations of free amino acids in portal and systemic plasma. The catabolism of digested or absorbed amino acids in the gut mucosa for energy provision (Wu, 1998) will influence their transition into the portal and systemic circulations and glutamate, glutamine and glucose are the prime energy substrates in avian enterocytes (Watford et al., 1979) However, a reduced-CP diet will contain more starch, and potentially glucose, but less amino acids than a conventional diet. This raises the possibility that this differential will alter the pattern of catabolism of glucose versus amino acids in the gut mucosa; an aspect that has been investigated in pigs by Van der Schoor et al. (2001). Therefore, apparent amino acid digestibility coefficients and disappearance rates in jejunum and ileum and their free concentrations in portal plasma in birds offered 215, 190 and 165 g/kg CP diets were determined. Thus, the objective of the present study was to evaluate reduced-CP diets in the context of whole grain feeding with an emphasis on the metabolism of amino acids and their post-enteral availability.

2. Materials and methods 2.1. Experimental design The experimental design consisted of seven dietary treatments as shown in Table 1 where three diets were formulated to contain protein levels of 215, 190 and 165 g/kg. Six of these diets comprised a 2 × 3 factorial array of treatments with two levels of protein (215 and 165 g/kg) and three levels of whole grain (0, 12.5 and 25%). Also, the linear effects of 215, 190 and 165 g/kg CP diets based on ground wheat were assessed. Table 1 Schedule of dietary treatments. Designation

Description

1A 2B 3C 4D 5E 6F 7G

215 g/kg 190 g/kg 165 g/kg 215 g/kg 215 g/kg 165 g/kg 165 g/kg

2

crude crude crude crude crude crude crude

protein protein protein protein protein protein protein

plus plus plus plus

125 g/kg 250 g/kg 125 g/kg 250 g/kg

whole whole whole whole

grain gain grain grain

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2.2. Diet preparation Three basal wheat-based diets were formulated to declining crude protein levels of 215, 190 and 165 g/kg but their energy density (12.70 MJ/kg), dietary electrolyte balance (250 mEq/kg) and and digestible lysine levels (11.00 g/kg) remained constant as shown in Tables 2 and 3. The 215 g/kg protein diet contained synthetic lysine methionine and threonine; whereas, the 165 g/kg protein diet contained a total of nine synthetic or unbound essential amino acids designed to meet requirements. The analysed crude protein and amino acid concentrations of the experimental diets are shown in Table 4. Wheat was ground (3.2 mm hammer-mill screen) prior to being blended into the complete diets which were steam-pelleted at a temperature of 80 °C with a conditioner residence time of 14 s. When appropriate, 12.5 or 25% whole wheat was blended with the balancing concentrate post-pelleting. 2.3. Bird management This study fully complied with the guidelines (2017/1252) specifically approved by the Research Integrity and Ethics Administration of The University of Sydney. A total of 336 off-sex, male Ross 308 chicks were procured from a commercial hatchery and were initially offered a standard starter diet. At 14 days post-hatch birds were individually identified (wing-tags) and allocated into bioassay cages on the basis of body-weights so that mean weights and variations within cages were nearly identical. Each of seven dietary treatments was offered to eight replicate cages (six birds per cage) from 14 to 35 days post-hatch. Birds had unrestricted access to feed and water in environmentally controlled facility, which remained illuminated for 16 h per day. An initial room temperature of 32 °C was maintained for the first week, which was gradually decreased to 22 °C by the end of the fourth week and maintained throughout the fifth week. 2.4. Data and sample collection, chemical analyses, calculations Growth performance (weight again, feed intake, FCR) was determined from 14 to 35 days post-hatch. Bird weights were determined at 14 and 35 days and feed intakes were monitored over the experimental period. The body-weights of any dead or culled birds were recorded on a daily basis in order to correct feed intakes for relevant cages and to adjust FCR calculations accordingly. Total excreta outputs and feed intakes were recorded from 25 to 27 days post-hatch to determine parameters of nutrient utilisation [apparent metabolisable energy (AME), metabolisable to gross energy ratio (ME:GE), nitrogen (N) retention and N-corrected AME (AMEn)] by the classical approach. Excreta were dried in a forced-air oven at 80 °C for 24 h and the gross energy (GE) of excreta and Table 2 Composition of the three basal experimental diets. Feed ingredient (g/kg)

Diet 1A

Diet 2B

Diet 3C

Wheat Soybean meal Canola meal Vegetable oil Lysine HCl (780 g/kg) d,l-Methionine (980 g/kg) Threonine (980 g/kg) Tryptophan (980 g/kg) Valine (980 g/kg) Arginine (980 g/kg) Isoleucine (980 g/kg) Leucine (980 g/kg) Histidine (980 g/kg) Sodium chloride Sodium bicarbonate Potassium carbonate Limestone Dicalcium phosphate Xylanase2 Phytase3 Choline chloride (600 g/kg) Vitamin-trace mineral premix1 Celite

551 247 100 52.2 1.74 1.84 0.74 – – – – – – 1.81 2.13 – 8.46 9.77 0.05 0.10 0.90 2.00 20.0

649 147 100 36.8 4.63 2.57 2.02 0.12 1.53 2.32 1.52 1.47 0.30 0.91 3.44 3.69 8.58 10.82 0.05 0.10 0.90 2.00 20.0

747 47.2 100 21.4 7.51 3.31 3.30 0.24 3.05 4.64 3.04 2.93 0.60 – 4.76 7.37 8.70 11.87 0.05 0.10 0.90 2.00 20.0

1Vitamin-trace mineral premix supplies in MIU/kg or mg/kg of diet: [MIU] retinol 12, cholecalciferol 5, [mg] tocopherol 50, menadione 3, thiamine 3, riboflavin 9, pyridoxine 5, cobalamin 0.025, niacin 50, pantothenate 18, folate 2, biotin 0.2, copper 20, iron 40, manganese 110, cobalt 0.25, iodine 1, molybdenum 2, zinc 90, selenium 0.3. 2Xylanase is a preparation of endo-1,4-ß-xylanase produced by Trichoderma reesei (Danisco® Xylanase, DuPont Animal Nutrition, Leiden, The Netherlands). Minimum activity of 40,000 U/g endo-1,4 beta-xylanase. 3Phytase used was derived from Buttiauxella sp. expressed in Trichoderma reesei (Axtra® PHY, DuPont Animal Nutrition, Leiden, The Netherlands). Minimum phytase activity of 10,000 FTU/g. 3

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Table 3 Nutritional specifications of the three basal experimental diets. Item (g/kg)

Diet 1A

Diet 2B

Diet 3C

Metabolisable energy (MJ/kg) Crude protein Digestible amino acids Lysine Methionine Cysteine Threonine Tryptophan Isoleucine Leucine Arginine Valine Histidine Phenylalanine Proline Glutamic acid Glycine Serine Starch Fat Fibre Calcium Total phosphorus Available phosphorus Sodium Potassium Chloride Dietary electrolyte balance (mEq/kg)

12.70 215

12.70 190

12.70 165

11.00 4.79 3.35 7.37 2.50 7.82 13.92 12.28 8.88 4.79 8.96 12.25 18.51 7.51 8.63 341 72.7 30.4 8.25 5.61 4.13 1.80 8.96 2.03 250

11.00 5.09 3.05 7.37 2.16 7.76 12.85 11.86 8.84 4.21 7.26 11.10 11.02 6.16 6.98 401 57.8 28.8 8.25 5.37 4.13 1.80 9.04 2.11 250

11.00 5.39 2.75 7.37 1.82 7.70 11.77 11.44 8.80 3.63 5.56 9.94 3.53 4.81 5.33 460 42.9 27.1 8.25 5.14 4.13 1.80 9.12 2.18 250

Table 4 Analysed crude protein (N) and amino acid concentrations (g/kg) in experimental diets. Item (g/kg)

1A

2B

3C

4D

5E

6F

7G

Crude protein Arginine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Threonine Valine Alanine Aspartic acid Glutamic acid Glycine Proline Serine Tyrosine Total

224 12.1 5.3 8.7 15.0 11.3 3.4 9.7 8.1 9.7 8.1 17.4 42.5 8.9 13.6 9.7 4.3 187.9

202 11.3 4.6 8.2 13.4 10.9 3.6 8.1 7.7 9.3 6.9 13.9 38.5 7.7 12.5 8.1 3.6 168.0

185 11.3 4.0 8.0 12.1 10.8 3.9 6.8 7.5 9.0 5.7 10.6 34.3 6.6 11.6 6.8 2.9 151.9

217 12.1 5.3 8.7 14.9 11.3 3.4 9.6 8.1 9.8 8.0 17.2 42.6 8.9 13.6 9.6 4.3 187.2

210 11.4 5.0 8.2 14.3 10.5 3.33 9.3 7.7 9.2 7.7 16.1 40.9 8.6 13.2 9.2 4.1 178.6

182 10.8 4.0 8.0 11.1 11.4 3.8 6.9 7.5 9.0 5.8 10.8 35.0 6.7 11.8 6.9 3.0 152.4

178 10.5 3.9 7.5 10.3 10.9 3.6 6.7 7.1 8.6 5.5 9.9 33.9 6.5 11.6 6.7 2.8 146.2

diets were determined using an adiabatic bomb calorimeter. The AME values of the diets on a dry matter basis were calculated from the following equation: AMEdiet (MJ/kg DM) = (feed intake x GEdiet) – (excreta output x GEexcreta) feed intake ME:GE ratios were calculated by dividing AME by the gross energy (GE) of the appropriate diets. N contents of diets and excreta were determined using a nitrogen determinator (Leco Corporation, St Joseph, MI) and N retentions calculated from the following equation: N retention (%) = (feed intake x Ndiet) – (excreta output x Nexcreta) x 100 (feed intake x Ndiet) N-corrected AME (AMEn MJ/kg DM) values were calculated by correcting N retention to zero using the factor of 36.54 kJ/g N retained in the body (Hill and Anderson, 1958). 4

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At 34 days post-hatch, blood samples were taken from the brachial vein from 3 birds per replicate cage to determine concentrations of free amino acids in the systemic circulation. The birds sampled were those offered 215 g/kg protein diets with either 0 or 25% whole grain or 165 g/kg with either 0 or 25% whole grain. Blood samples were centrifuged and decanted plasma samples were then kept at ―80 °C prior to analysis. Concentrations of twenty proteinogenic amino acids in plasma taken from the brachial vein were determined using precolumn derivatisation amino acid analysis with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC; Waters™ AccQTag Ultra; Waters Australia PL; www.waters.com) followed by separation of the derivatives and quantification by reversed phase ultra-performance liquid chromatography (RP-UPLC). All amino acids were detected by UV absorbance using analytical standards and this procedure is fully described in Selle et al. (2016). At day 35, birds were euthanased by an intravenous injection of sodium pentobarbitone, the abdominal cavity opened, and pH of digesta within the gizzard was determined in situ with a pH probe. The weights of full and emptied gizzards and abdominal fat pad weights were recorded. In the case of birds offered 215, 190 and 165 g/kg protein diets without whole grain, blood samples were taken from the anterior mesenteric vein from three birds per cage to determine concentrations of free amino acids in the portal circulation as already outlined. The small intestine was removed from all birds and digesta gently expressed in its entirety from the distal half of the jejunum and ileum and pooled by cage, homogenised, freeze-dried and weighed to determine the apparent digestibility coefficients of protein (N) and amino acids. Acid insoluble ash (AIA) and protein (N) concentrations were determined by methods described in Siriwan et al. (1993). Amino acid concentrations of diets and digesta were determined via 24 h liquid hydrolysis at 110◦C in 6 M HCl followed by analysis of 16 amino acids using the Walters AccQTag Ultra chemistry on a Waters Acquity UPLC. Apparent digestibility coefficients were calculated by the following equation: Digestibility coefficient = (Nutrient/AIA)diet− (Nutrient/AIA)digesta (Nutrient/AIA)diet Disappearance rates (g/bird/day) of amino acids and protein (N) were calculated from the product of dietary concentrations of nutrient (g/kg), daily feed intake (g/day) from 14 to 35 days post-hatch and the relevant apparent ileal digestibility coefficient. 2.5. Statistical analysis The experimental data were analysed as a 2 × 3 factorial array of treatments or one-way analyses of variance using the Statistics 24 program (SPSS, 2016) and adopting standard statistical procedures. Experimental units were cage means (8 replicate cages of 6 birds per dietary treatment) and a probability level of less than 5% was considered statistically significant. 3. Results The effects of reduced-crude protein diets and whole grain feeding on growth performance are shown in Table 5. The transition from 215 to 165 g/kg CP diets compromised FCR by 5.65% (1.576 versus 1.487; P < 0.005) and tended to increase feed intake by 3.36% (3197 versus 3093 g/bird; P = 0.058). Weight gain and mortality rate was not influenced by treatment and whole grain feeding did not influence growth performance. The effects of dietary treatments on gizzard, pancreas and fat-pads are shown in Table 6. The reduced-CP diet increased relative Table 5 Effects of reduced-crude protein diets and whole grain feeding on growth performance and mortality rates from 14 to 35 days post-hatch. Treatment Crude protein (g/kg)

Whole grain (g/kg)

215

0 125 250 0 125 250

165

SEM Main effects: Protein 215 165 Whole grain 0 12.5 25.0 Significance (P =) Crude protein (CP) Whole grain (WG) CP x WG interaction

Weight gain (g/bird)

Feed intake (g/bird)

FCRa (g/g)

Mortality/cull rate (%)

2096 2136 2022 2106 2002 1994 46.0

3097 3123 3060 3255 3151 3186 65.5

1.476 1.464 1.519 1.546 1.583 1.600 0.0335

10.42 4.17 4.17 12.50 10.42 6.25 4.277

2084 2034

3093 3197

1.487a 1.576b

6.25 9.72

2101 2069 2008

3176 3139 3123

1.511 1.523 1.560

11.46 7.29 5.21

0.190 0.132 0.281

0.058 0.709 0.591

0.002 0.338 0.756

0.326 0.340 0.854

ab

Means within columns not sharing a common suffix are significantly different at the 5% level of probability. Mean values from 8 replicates of 6 birds per replicate cage per treatment. a feed conversion ratio. 5

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Table 6 Effects of reduced-crude protein diets and whole grain feeding on relative gizzard weights, relative gizzard contents, relative pancreas weights, relative abdominal fat-pad weights and pH of gizzard digesta at 35 days post-hatch. Treatment Crude protein (g/ kg)

Whole grain (g/ kg)

215

0 125 250 0 125 250

165

SEM Main effects: Protein 215 165 Whole grain 0 12.5 25.0 Significance (P =) Crude protein (CP) Whole grain (WG) CP x WG interaction

Relative gizzard weight (g/kg)

Relative gizzard content (g/kg)

Relative pancreas weight (g/kg)

Relative fat-pad weight (g/kg)

Gizzard digesta pH

8.33 11.47 13.37 9.11 12.33 13.45 0.355

1.81 5.39 5.92 3.30 6.46 5.95 0.444

1.89 1.65 1.78 1.92 1.68 1.72 0.102

7.32 7.48 6.66 8.87 803 7.17 0.418

3.84 2.90 2.81 3.39 2.94 2.71 0.116

11.06 11.63

4.37a 5.24b

1.771 1.771

7.15a 8.02b

3.19 3.01

8.72a 11.90b 13.41c

2.56a 5.92b 5.94b

1.90 1.66 1.75

8.09b 7.76b 6.91a

3.62b 2.92a 2.76a

0.054 < 0.001 0.496

0.022 < 0.001 0.247

0.996 0.070 0.871

0.015 0.021 0.381

0.074 < 0.001 0.109

abc Means within columns not sharing a common suffix are significantly different at the 5% level of probability. Mean values from 8 replicates of 6 birds per replicate cage per treatment.

fat-pad weights by 12.2% (8.02 versus 7.15 g/kg; P < 0.025), increased gizzard contents by 19.9% (5.24 versus 4.37 g/kg P < 0.025) and tended to increase relative gizzard weights and depressed gizzard digesta pH. Whole grain feeding influenced relative gizzard weights, contents and pH of digesta to highly significant (P < 0.001) extents. The transition from 0% to 25% whole grain diet increased gizzard weights by 53.8% (13.41 versus 8.72 g/kg), gizzard contents by 1.2% (5.94 versus 2.56 g/kg) and depressed gizzard digesta pH from 3.62 to 2.76. Whole grain feeding significantly (P < 0.025) influenced relative fat-pad weights; the transition from 0% to 25% whole grain decreased fat pad weights by 14.6% (6.91 versus 8.09 g/kg). Table 7 records the effects of reduced-crude protein diets and whole grain feeding on parameters of nutrient utilisation. Reducing dietary protein enhanced N retention by 6.02% (0.616 versus 0.581; P < 0.005) but compromised AME by 0.43 MJ (11.98 versus 12.41 MJ/kg; P < 0.005) and AMEn by (10.87 versus 11.28 MJ/kg; P < 0.05). Whole grain feeding significantly (P = 0.005Table 7 Effects of reduced-crude protein diets and whole grain feeding on parameters of nutrient utilisation [AME: apparent metabolisable energy, ME:GE: metabolisable to gross energy ratio, N retention: nitrogen retention, AMEn: nitrogen-corrected apparent metabolisable energy]. Treatment Crude protein (g/kg)

Whole grain (g/kg)

215

0 125 250 0 125 250

165

SEM Main effects: Protein 215 165 Whole grain 0 12.5 25.0 Significance (P =) Crude protein (CP) Whole grain (WG) CP x WG interaction

AME (MJ/kg DM)

ME:GE ratio

N retention

AMEn (MJ/kg DM)

11.77 12.76 12.68 11.52 12.13 12.29 0.162

0.691 0.746 0.743 0.700 0.737 0.742 0.0098

0.547 0.616 0.580 0.602 0.623 0.623 0.0129

10.71 11.55 11.58 10.36 11.22 11.03 0.219

12.41b 11.98a

0.727 0.726

0.581a 0.616b

11.28b 10.87a

11.65a 12.45b 12.48b

0.696a 0.741b 0.742b

0.575a 0.619b 0.601b

10.53a 11.38b 11.30b

0.002 < 0.001 0.502

0.954 < 0.001 0.653

0.002 0.005 0.176

0.027 < 0.001 0.863

ab

Means within columns not sharing a common suffix are significantly different at the 5% level of probability. Mean values from 8 replicates of 6 birds per replicate cage per treatment. 6

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< 0.001) improved all four parameters as a main effect. The transition from 0% to 25% whole grain increased AME by 0.83 MJ (12.48 versus 11.65 MJ/kg), ME:GE ratios by 6.61% (0.742 versus 0.696), N retention by 4.52% (0.601 versus 0.575) and AMEn by 0.77 MJ (11.30 versus 10.53 MJ/kg). The effects of reduced-crude protein diets and whole grain feeding on concentrations of free amino acids in systemic plasma are shown in Tables 8 and 9. Significant treatment interactions were observed for isoleucine (P < 0.001), leucine (P < 0.005), lysine (P < 0.001), valine (P < 0.001), glutamine (P < 0.05), and proline (P < 0.05). In the majority of instances the genesis of these interactions was that the dietary combination of 165 g/kg crude protein and 25% whole grain generated the highest plasma concentrations; the exception was leucine where the same dietary combination generated the lowest plasma concentration. As a main effect, the lower crude protein level reduced concentrations of arginine by 31.8% (39.8 versus 58.4 μg/ml; P < 0.001), histidine by 53.8% (3.6 versus 7.8 μg/ml; P < 0.001), phenylalanine by 24.3% (13.1 versus 17.3 μg/ml; P < 0.001), glycine by 27.8% (29.9 versus 41.4 μg/ml; P < 0.001)), serine by 15.9% (42.3 versus 50.3 μg/ml; P < 0.005) and tyrosine by 26.2% (16.9 versus 22.9 μg/ ml; P < 0.001). Alternatively, the lower crude protein level increased concentrations of methionine by 40.5% (11.8 versus 8.4 μg/ ml; P < 0.001) and threonine by 32.1% (62.9 versus 47.6 μg/ml; P = 0.001). Whole grain feeding increased concentrations of alanine by 19.8% (55.6 versus 46.4 μg/ml; P < 0.025), asparagine by 42.3% (19.5 versus 13.7 μg/ml; P < 0.005) and cysteine by 7.30% (14.7 versus 13.7 μg/ml; P < 0.05) as a main effect. Systemic concentrations of tryptophan, aspartic acid and glutamic acid were not influenced by treatments. The effects of 215, 190 and 165 g/kg CP diets, based on ground grain only, on growth performance and selected parameters are shown in Table 10. It is evident that feed intake was numerically increased by 5.10% and FCR compromised by 4.74% in birds offered the 165 g/kg CP diet in comparison to the 215 g/kg CP diet, although statistical differences were not observed. Similarly, relative fatpad weights were numerically heavier by 21.2% and the linear regression with dietary CP approached significance (r =−0.389; P = 0.060). Gizzard pH was significantly decreased from pH 3.84 to 3.39 by reducing dietary CP and N retention was significantly increased from 0.547 to 0.602 with the reduction in dietary CP. The mortality rate was not related to treatment (P > 0.80). The effects of reducing dietary CP on the jejunal and ileal digestibility coefficients of essential amino acids are shown in Table 11. Essentially, there were no tangible effects of treatment as the only significant difference observed was a 6.21% (0.695 versus 0.741) decline in ileal histidine digestibility. The effects of reducing dietary CP on amino acid disappearance rates in jejunum and ileum of essential amino acids are shown in Table 12. In the jejunum, the disappearance rate of methionine significantly increased by 28.6% (0.485 versus 0.377 g/bird/day) when the 165 and 215 g/kg CP diets are compared. In contrast, in the same comparison, arginine decreased by 14.1% (1.356 versus 1.578 g/bird/day), histidine by 24.0% (0.355 versus 0.467 g/bird/day) and phenylalanine by 27.9% (0.618 versus 0.857 g/bird/day). Again, in the ileum, the disappearance rate of methionine significantly increased by 15.3% (0.539 versus 0.439 g/bird/day); whereas, there were decreases of 25.2% (0.431 versus 0.576 g/bird/day) for histidine and 50.0% (0.539 versus 1.087 g/bird/day) for phenylalanine. The effects of reducing dietary CP on the jejunal and ileal digestibility coefficients of non-essential amino acids are shown in Table 13, where no significant treatment effects were observed in the jejunum. However, the ileal digestibility of alanine (13.2%), aspartic acid (14.8%), glycine (8.42%), serine (9.96%) and tyrosine (11.0%) were significantly depressed by the reduction in dietary Table 8 Effects of reduced-crude protein diets and whole grain feeding on concentrations (μg/ml) of free essential amino acids in systemic plasma taken from the brachial vein at 34 days post-hatch. Treatment Protein

Whole grain

215 g/kg

0 250 g/kg 0 250 g/kg

165 g/kg

SEM Main effects: protein 215 g/kg 165 g/kg Whole grain 0% 25% Significance (P =) Protein (CP) Whole grain (WG) CP x WG interaction

Arg

His

Ile

Leu

Lys

Met

Phe

Thr

Trp

Val

60.8 56.0 40.1 39.5 3.57

7.4 8.3 3.3 3.9 0.63

11.1a 11.9a 11.4a 18.4b 0.75

17.4c 18.1c 13.5b 9.5a 0.76

30.4ab 20.8a 39.0b 73.6c 3.89

8.9 8.0 11.8 11.9 0.78

16.9 17.6 12.6 13.5 0.62

45.9 49.4 60.1 65.8 3.93

5.4 5.0 5.0 4.8 0.26

21.4a 23.3a 23.9a 37.8b 1.52

58.4 b 39.8 a

7.8b 3.6a

11.5 14.9

17.8 11.5

25.6 56.3

8.4a 11.8 b

22.3 30.8

11.3 15.1

15.4 13.8

34.7 47.2

47.6 a 62.9 b

5.2 4.9

5.3 6.1

17.3 b 13.1 a

5.2 4.9

22.6 30.5

< 0.00 1 0.24 1 0.843

< 0.00 1 < 0.001 < 0.001

< 0.00 1 0.04 2 0.004

< 0.001 0.003 < 0.001

14.8 15.6

53.0 57.6

< 0.00 1 0.197 0.920

0.00 1 0.256 0.789

0.24 3 0.24 3 0.813

< 0.00 1 < 0.001 < 0.001

50.4 47.8 < 0.00 1 0.458 0.568

ab

10.3 9.9 < 0.00 1 0.636 0.528

Means within columns not sharing a common suffix are significantly different at the 5% level of probability. Mean values from 8 replicates of 3 birds per replicate cage sampled per treatment. 7

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Table 9 Effects of reduced-crude protein diets and whole grain feeding on concentrations (μg/ml) of free non-essential amino acids in systemic plasma taken from the brachial vein at 34 days post-hatch. Treatment Protein

Whole grain

215 g/kg

0 250 g/kg 0 250 g/kg

165 g/kg

SEM Main effects: protein 215 g/kg 165 g/kg Whole grain 0% 25% Significance (P =) Protein (CP) Whole grain (WG) CP x WG interaction

Ala

Asp

Asn

Cys

Glu

Gln

Gly

Pro

Ser

Tyr

46.6 48.5 46.1 62.6 3.85

9.1 6.8 7.6 8.0 1.37

15.0ab 20.3c 12.4a 18.8bc 1.61

14.1 14.9 13.3 14.5 0.46

17.3 16.6 16.3 17.8 0.79

139.9a 144.1a 171.9b 216.3c 9.58

40.2 42.5 28.5 31.3 1.72

38.9a 41.0ab 34.4a 47.9b 2.73

51.6 48.9 39.5 45.0 2.42

22.0 23.8 15.3 18.6 1.35

47.6 54.4

7.9 7.8

17.6 15.6

14.5 13.9

16.9 17.0

142.0 194.1

39.9 41.1

46.4 a 55.6 b

8.4 7.7

13.7a

13.7 a 14.7 b

16.8 17.2

155.9 180.2

41.4 b 29.9 a

50.3 b 42.3 a

22.9 b 16.9 a

0.93 8 0.585 0.190

< 0.001 0.017 0.045

45.6 46.9

18.6 21.2

0.003 0.574 0.099

< 0.001 0.068 0.552

19.5b 0.92 8 0.470 0.323

0.088 0.024 0.068

0.211 0.001 0.729

0.084 0.038 0.591

36.6 44.4

34.4 36.9

0.667

< 0.001 0.156 0.885

0.008 0.046

abc Means within columns not sharing a common suffix are significantly different at the 5% level of probability. Mean values from 8 replicates of 3 birds per replicate cage sampled per treatment. Table 10 Effects of reduced-crude protein diets on growth performance, mortality rates and selected parameters from 14 to 35 days post-hatch. Treatment

Weight gain (g/ bird)

Feed intake (g/ bird)

FCR (g/g)

Mortality/cull rate (%)

Relative fat pad weights (g/kg)

Gizzard digesta pH

N retention

215 g/kg CP 190 g/kg CP 165 g/kg CP SEM Significance (P =) LSD (P < 0.05)

2096 2058 2106 41.1 0.680 –

3097 3077 3255 88.0 0.315 –

1.476 1.497 1.546 0.0316 0.286 –

10.42 8.34 12.50 5.073 0.844 –

7.32 7.76 8.87 0.562 0.159 –

3.84b 3.70ab 3.39a 0.123 0.048 0.351

0.547 0.585 0.602 0.0128 0.020 0.0375

ab

Means within columns not sharing a common suffix are significantly different at the 5% level of probability. Mean values from 8 replicates of 6 birds per replicate cage per treatment.

Table 11 Effects of reduced-crude protein diets on apparent jejunal and ileal digestibility coefficients of essential amino acids at 35 days post-hatch. Treatment Distal jejunum 215 g/kg CP 190 g/kg CP 165 g/kg CP SEM Significance (P =) LSD (P < 0.05) Distal ileum 215 g/kg CP 190 g/kg CP 165 g/kg CP SEM Significance (P =) LSD (P < 0.05)

Arg

His

Ile

Leu

Lys

Met

Phe

Thr

Val

0.643 0.677 0.706 0.0228 0.180 –

0.599 0.602 0.573 0.0283 0.712 –

0.581 0.627 0.652 0.0284 0.234 –

0.585 0.608 0.609 0.0305 0.824 –

0.622 0.659 0.676 0.0295 0.439 –

0.756 0.792 0.803 0.0214 0.316 –

0.601 0.600 0.586 0.0323 0.934 –

0.526 0.551 0.577 0.0302 0.497 –

0.557 0.599 0.617 0.0280 0.340 –

0.803 0.807 0.809 0.0060 0.757 –

0.741b 0.724b 0.695a 0.0078 0.002 0.0231

0.752 0.779 0.768 0.0198 0.640 –

0.744 0.743 0.736 0.0104 0.640 –

0.766 0.766 0.775 0.0097 0.764 –

0.881 0.887 0.892 0.0085 0.699 –

0.764 0.750 0.680 0.0343 0.190 –

0.675 0.681 0.687 0.0103 0.7154 –

0.713 0.725 0.726 0.0092 0.569 –

ab

Means within columns not sharing a common suffix are significantly different at the 5% level of probability. Mean values from 8 replicates of 6 birds per replicate cage per treatment.

CP and the percentage reductions when the 215 to the 165 g/kg CP diets are compared are shown in parentheses. The disappearance rates of non-essential amino acids in the jejunum and ileum are shown in Table 14. The reductions in dietary CP significantly depressed the disappearance rates of alanine, aspartic acid, glycine, serine and tyrosine in the jejunum. In the ileum, reductions in 8

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Table 12 Effects of reduced-crude protein diets on apparent disappearance rates (g/bird/day) of essential amino acids in the distal jejunum and distal ileum at 35 days post-hatch. Treatment Distal jejunum 215 g/kg CP 190 g/kg CP 165 g/kg CP SEM Significance (P =) LSD (P < 0.05) Distal ileum 215 g/kg CP 190 g/kg CP 165 g/kg CP SEM Significance (P =) LSD (P < 0.05)

Arg

His

Ile

Leu

Lys

Met

Phe

Thr

Val

1.578b 1.328a 1.356a 0.0521 0.006 0.1536

0.467b 0.407ab 0.355a 0.0235 0.013 0.0671

0.742 0.755 0.809 0.0442 0.533 –

1.288 1.196 1.141 0.0731 0.388 –

1.030 1.054 1.133 0.0615 0.474 –

0.377a 0.418a 0.485b 0.0182 0.002 0.0536

0.857b 0.713a 0.618a 0.0442 0.004 0.1305

0.625 0.623 0.670 0.0412 0.653 –

0.793 0.818 0.861 0.0498 0.630 –

1.425 1.339 1.417 0.0442 0.333 –

0.576c 0.489b 0.431a 0.0164 < 0.001 0.0482

0.960 0.940 0.951 0.0412 0.946 –

1.637b 1.462a 1.379a 0.0523 0.008 0.1544

1.270 1.225 1.297 0.0427 0.479 –

0.439a 0.469a 0.539b 0.0152 < 0.001 0.0448

1.087a 0.892a 0.539b 0.0471 < 0.001 0.1389

0.802 0.770 0.798 0.0280 0.662 –

1.015 0.989 1.012 0.0343 0.837 –

abc Means within columns not sharing a common suffix are significantly different at the 5% level of probability. Mean values from 8 replicates of 6 birds per replicate cage per treatment. Table 13 Effects of reduced-crude protein diets on apparent jejunal and ileal digestibility coefficients of non-essential amino acids at 35 days post-hatch. Treatment Distal jejunum 215 g/kg CP 190 g/kg CP 165 g/kg CP SEM Significance (P =) LSD (P < 0.05) Distal ileum 215 g/kg CP 190 g/kg CP 165 g/kg CP SEM Significance (P =) LSD (P < 0.05)

Ala

Asp

Glu

Gly

Pro

Ser

Tyr

0.536 0.514 0.456 0.0412 0.377 –

0.513 0.489 0.431 0.0396 0.333 –

0.674 0.700 0.693 0.0240 0.737 –

0.525 0.509 0.469 0.0343 0.496 –

0.635 0.654 0.655 0.0228 0.797 –

0.550 0.527 0.490 0.0343 0.462 –

0.504 0.494 0.439 0.0412 0.487 –

0.687b 0.660b 0.596a 0.0139 < 0.001 0.0410

0.697b 0.559ab 0.594a 0.0133 < 0.001 0.0393

0.813 0.818 0.810 0.0080 0.779 –

0.689b 0.679b 0.631a 0.0097 0.001 0.0285

0.773 0.780 0.769 0.0077 0.591 –

0.723b 0.694b 0.651a 0.0110 0.001 0.0325

0.711b 0.689b 0.633a 0.0126 0.001 0.0372

ab

Means within columns not sharing a common suffix are significantly different at the 5% level of probability. Mean values from 8 replicates of 6 birds per replicate cage per treatment.

Table 14 Effects of reduced-crude protein diets on apparent disappearance rates (g/bird/day) of non-essential amino acids in the distal jejunum and distal ileum at 35 days post-hatch. Treatment Distal jejunum 215 g/kg CP 190 g/kg CP 165 g/kg CP SEM Significance (P =) LSD (P < 0.05) Distal ileum 215 g/kg CP 190 g/kg CP 165 g/kg CP SEM Significance (P =) LSD (P < 0.05)

Ala

Asp

Glu

Gly

Pro

Ser

Tyr

0.637b 0.520ab 0.403a 0.0427 0.004 0.1261

1.309c 0.998b 0.708a 0.0799 < 0.001 0.2358

4.201 3.957 3.683 0.0182 0.163 –

0.685b 0.575ab 0.479a 0.0412 0.009 0.1215

1.268 1.200 1.175 0.0168 0.518 –

0783b 0.626a 0.516a 0.0442 0.002 0.1305

0.318b 0.162ab 0.197a 0.0228 0.004 0.0674

0.816c 0.668b 0.526a 0.0255 < 0.001 0.0753

1.780c 1.34b 0.974a 0.0523 < 0.001 0.1544

5.067b 4.619a 4.298a 0.0143 0.005 0.4208

0.899c 0.766b 0.644a 0.0255 < 0.001 0.0753

1.542 1.430 1.380 0.0442 0.058 –

1.028c 0.825b 0.984a 0.0280 < 0.001 0.0825

0.448c 0.364b 0.284a 0.0132 < 0.001 0.0391

abc Means within columns not sharing a common suffix are significantly different at the 5% level of probability. Mean values from 8 replicates of 6 birds per replicate cage per treatment.

9

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dietary CP significantly depressed the disappearance rates of alanine (28.6%), aspartic acid (45.3%), glutamic acid (15.2%), glycine (28.4%), serine (4.28%) and tyrosine (36.6%). The effects of reduced-crude protein diets on concentrations of free essential amino acids in the portal circulation are shown in Table 15. Reducing dietary protein levels linearly decreased concentrations of histidine (r = 0.606; P < 0.005), leucine (r = 0.668; P < 0.001), phenylalanine (r = 0.637; P < 0.005) and tryptophan (r = 0.453; P < 0.05). Conversely, reducing protein levels linearly increased lysine (r = -0.430; P < 0.05) concentrations and tended to increase concentrations of methionine (r = -0.368; P = 0.077) and threonine (r = -0.373; P = 0.072). The effects of dietary treatments on concentrations of non-essential amino acids are shown in Table 16. Reducing dietary protein linearly decreased concentrations of asparagine (r = 0.419; P < 0.05), glycine (r = 0.637; P < 0.005), serine (r = 0.494; P < 0.025) and tyrosine (r = 0.760; P < 0.001) and tended to decrease glutamine concentrations (r = 0.394; P = 0.057).

4. Discussion The overall growth performance of broiler chickens in this study comfortably exceeded Ross 308 2014 performance objectives for weight gain (2059 versus 1795 g/bird), feed intake (3135 versus 2965 g/bird) and FCR (1.526 versus 1.652). The overall mortality rate of 8.04% was not influence by treatment (P > 0.70) and many of these losses were attributed to Ascites during the final week of the study. As shown in Tables 5 and 6, the 50 g/kg reduction in dietary CP from 215 to 165 g/kg as a main effect compromised FCR by 5.99% and increased fat-pad weights by 12.2%, which illustrates the challenge in developing reduced-CP diets. Instructively, 25% whole grain feeding decreased relative fat-pad weights by 14.6%, which is an important observation given that reduced-CP diets invariably increase fat deposition. For example, Belloir et al. (2017) reported a 20.8% (26.1 versus 21.6 g/kg) increase in abdominal fat with the transition from a 190 to a 160 g/kg CP diet which was associated with a 3.05% (1.69 versus 1.64) inferior FCR in birds at 35 days post-hatch. The present study suggests that whole grain feeding may hold some promise to attenuate increased fat deposition in birds offered reduced-CP diets. Nevertheless, the addition of 25% whole grain to the 215 g/kg CP diet numerically decreased weight gain by 3.53% and increased FCR by 2.91%; whereas, 25% whole grain in the 165 g/kg CP diet decreased weight gain by 5.32% and increased FCR by 3.49% (Table 5). While increases in FCR in response to whole grain feeding are not typical, the outcomes do not support the contention that whole grain feeding will enhance growth performance of birds offered reduced-CP diets. It is not feasible to determine amino acid digestibility coefficients in birds offered whole grain diets with post-pelleting whole grain incorporation into rations as was the case in the present study. This is because the dietary marker can only be included in the pelleted concentrate and not the entire diet, so for this reason jejunal and ileal amino acid digestibility coefficients were determined for the three conventional, ground grain diets with CP contents of 215, 190 and 165 g/kg. The reduction in dietary CP linearly depressed (P < 0.001) the ileal digestibility of histidine, alanine, aspartic acid, glycine, serine and tyrosine. From a comparison of the 215 to 165 g/kg CP wheat-based diets, ileal digestibilities of histidine, alanine, aspartic acid, glycine, serine and tyrosine were significantly decreased by from 8.42% up to 14.8%. Indeed, the mean ileal digestibility coefficient of 16 amino acids was decreased by 4.02% (0.716 versus 0.746) in this comparison. In complete contrast, Chrystal et al. (2019) reported that a transition from 210 to 165 g/kg CP diets significantly increased the mean ileal digestibility coefficient of 17 amino acids by 6.18% (0.790 versus 0.744) in maize-based diets. Moreover, the jejunal digestibility of all amino acids assessed were linearly increased by the reduction in dietary CP study such that the mean jejunal digestibility coefficient increased by 29.4% (0.594 versus 0.459). Again, in contrast, the mean jejunal digestibility coefficients were not influenced (0.590 versus 0.588) by the reduction from 215 to 165 g/kg CP diets in the present study. The diverse responses in broiler chickens offered maize- versus wheat-based, reduced-CP diets may be partially due to the capacity of wheat pentosans to exacerbate endogenous amino acid losses in chickens as demonstrated by Angkanaporn et al. (1994). Increased quantities of endogenous amino acids in the ileum will depress apparent amino acid digestibility coefficients. According to Ravindran et al. (2004), glutamic acid, aspartic acid, threonine and glycine are the predominant amino acids in avian ileal digesta and the dietary presence of proteins and peptides will increase endogenous amino acid flows. On this basis, reducing dietary CP of maize-based would tend to decrease endogenous amino acid flows and enhance apparent amino acid digestibility; Table 15 Effects of reduced-crude protein diets on concentrations (μg/ml) of free essential amino acids in portal plasma taken from the anterior mesenteric vein at 35 days post-hatch. Protein (g/kg)

Arg

His

Ile

Leu

Lys

Met

Phe

Thr

Trp

Val

215 190 165 SEM Significance (P =) LSD (P < 0.05) Linear effect

72.9 62.3 59.8 5.61 0.237 – r = 0.337 P = 0.107

12.8b 9.0a 7.5a 1.05 0.006 3.07 r = 0.606 P = 0.002

20.6 17.4 18.1 1.21 0.162 – r = 0.293 P = 0.164

32.3b 24.9a 22.9a 1.54 0.001 4.52 r = 0.668 P < 0.001

38.6 42.3 51.6 4.19 0.100 – r = -0.430 P = 0.036

11.5 11.3 14.4 1.08 0.099 – r = -0.368 P = 0.077

25.9b 20.5a 19.0a 1.21 0.002 3.57 r = 0.637 P = 0.001

56.0 56.8 70.0 9.93 0.130 – r = -0.373 P = 0.072

6.4 6.1 5.6 0.23 0.081 – r = 0.453 P = 0.026

33.3 30.5 33.0 1.85 0.520 – r = 0.020 P = 0.925

ab

Means within columns not sharing a common suffix are significantly different at the 5% level of probability. Mean values from 8 replicates of 3 birds per replicate cage sampled per treatment. 10

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Table 16 Effects of reduced-crude protein diets on concentrations (μg/ml) of free non-essential amino acids in portal plasma taken from the anterior mesenteric vein at 35 days post-hatch. Protein (g/kg)

Ala

Asp

Asn

Cys

Glu

Gln

Gly

Pro

Ser

Tyr

215 190 165 SEM Significance (P =) LSD (P < 0.05) Linear effect

76.0 58.0 62.4 5.60 0.083 –

13.3 14.9 12.1 1.39 0.389 –

23.5b 13.6a 15.8a 2.36 0.018 6.93

17.0 16.6 16.5 1.04 0.940 –

40.1 38.0 34.5 2.78 0.368 –

154.1 160.8 192.8 13.71 0.128 –

53.6b 41.9a 38.8a 3.43 0.014 10.1

57.6 52.5 55.1 5.30 0.793 –

59.5b 46.6a 46.8a 3.27 0.015 9.61

32.3c 24.5b 19.5a 1.68 < 0.001 4.94

r = 0.334 P = 0.111

r = 0.119 P = 0.579

r = 0.419 P = 0.041

r = 0.074 P = 0.731

r = 0.298 P = 0.157

r = 0.394 P = 0.057

r = 0.637 P = 0.001

r = 0.072 P = 0.738

r = 0.494 P = 0.014

r = 0.760 P < 0.001

abc Means within columns not sharing a common suffix are significantly different at the 5% level of probability. Mean values from 8 replicates of 3 birds per replicate cage sampled per treatment.

alternatively, endogenous amino acid flows may be increased by soluble pentosans in wheat-based diets to the detriment of amino acid digestibility. Free amino acid concentrations in plasma taken from the anterior mesenteric vein were determined in the same birds offered the 215, 190 and 165 g/kg CP diets. Any interpretations of the outcomes of free amino acid concentrations in the portal circulation are difficult because the origin of the amino acids may be from either the gut lumen or the arterial circulation. The likelihood is that more amino acids enter enterocytes from the bloodstream than from the gut lumen (Newsholme and Carrié, 1994). Reducing dietary protein levels linearly decreased concentrations of histidine, leucine, phenylalanine, tryptophan, asparagine, glycine, serine and tyrosine in the portal circulation. In contrast, concentrations of lysine (P = 0.036) linearly increased and methionine (P = 0.077) and threonine (P = 0.072) tended to be increased. It is then relevant that the proportions of unbound lysine, methionine and threonine were substantial in the low-protein diet; whereas, phenylalanine, asparagine, glycine, serine and tyrosine were present only as protein-bound amino acids. Wu (1998) suggested that branched-chain amino acids, arginine, proline and perhaps certain other amino acids are catabolised in the gut mucosa in addition to glutamate, arterial glutamine and aspartate. This outcome suggests that unbound or synthetic amino acids may be less prone to catabolism in the gut mucosa than protein-bound amino acids. This could stem from the fact that unbound amino acids are absorbed more proximally and rapidly in the small intestine where more glucose is available as an alternative energy substrate for enterocytes. The effects of reduced-crude protein diets and whole grain feeding on free amino acid concentrations in systemic plasma are of interest. As a main effect, the lower dietary crude protein level reduced concentrations of arginine, histidine, phenylalanine, glycine, serine and tyrosine but increased systemic concentrations of methionine and threonine. However, there is a precedent for these variable responses as Fancher and Jensen (1989a) reported that the transition from 183 to 159 g/kg CP maize-soy diets increased systemic plasma concentrations of methionine by 55% (216 versus 139 nmol/mL) and threonine by 87% (1635 versus 879 nmol/mL) in female broilers at 42 days post-hatch. One possible explanation is that methionine and threonine concentrations in the systemic circulation were in excess of requirements for protein accretion relative to the balance of amino acids. Whole grain feeding increased concentrations of alanine, asparagine and cysteine as a main effect. However, significant treatment interactions were observed for isoleucine, leucine, lysine, valine, glutamine and proline. The majority of these interactions stemmed from the observation that the combination of 165 g/kg crude protein and 25% whole grain generated the highest plasma concentrations. However, leucine was the noticeable exception across the interactions because the high whole grain/low-CP diet generated the lowest plasma concentration. Moreover, it is interesting that the pattern of interactions for isoleucine and valine on one hand, and leucine on the other, were totally opposite. This possibly reflects antagonism between branched-chain amino acids which has been shown to exist in poultry (Smith and Austic, 1978; Calvert et al., 1982). Only free concentrations of tryptophan, aspartic acid and glutamic acid in systemic plasma were not influenced by treatment. Concentrations of free amino acids in the portal and systemic circulations were determined in birds offered the 215 g/kg CP diet without whole grain. Total concentrations of 837 μg/ml in the portal circulation declined to 620 μg/ml in the systemic circulation and the concentrations of all amino acids declined to varying extents. Earlier, Truong et al. (2017) reported a similar uniform decline in free amino acid concentrations from 933 μg/ml in portal plasma to 820 μg/ml in systemic plasma in broiler chickens. The dietary inclusion of 25% whole grain increased gizzard weights by 53.8% relative to diets containing only ground grain which is a robust response. Whole grain feeding generated tangible responses in AME, ME:GE ratios, N retention and AMEn, which is in accordance with the reviews of Liu et al. (2015) and Moss et al. (2019). However, the reduction in dietary CP of wheat-based diets significantly reduced AME and AMEn by 0.43 MJ and 0.41 MJ, respectively. This was not anticipated give the contrasting findings reported by both Moss et al. (2018) and Chrystal et al. (2019) for maize-based diets. In both studies, tangible improvements in energy utilisation were reported in response to reduced-CP diets. Thus, there appear to be different response patterns in birds offered either maize- or wheat-based, reduced-CP diets and it is worth noting that any improvements if protein digestibility will have a positive impact on energy utilisation. In addition, the properties of starch in wheat or maize are quite different as reported by Giuberti et al. (2012). These researchers compared 14 maize and 12 wheat samples in an in vitro study and found that wheat contained more rapidly digestible starch but less resistant starch than maize. Also, the rate of wheat starch digestion was more rapid and the predicted glycaemic index of wheat (pGI 65.8) was greater than maize (pGI 39.5). Alternatively, the extent of ileal wheat starch digestibility in 11

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broiler chickens is less than that of maize (Truong et al., 2016). Additionally, there is the real possibility that there is competition between glucose and amino acids for intestinal uptakes via Na+-dependent transport systems in birds offered reduced-CP diets (Moss et al., 2018), which well may be influenced by the starch properties of the dietary feed gain. The 165 g/kg CP diet compromised FCR by 5.65% relative to the positive control diet; however, somewhat surprisingly, whole grain feeding numerically disadvantaged FCR. It is common for reduced-CP diets to compromise FCR and whole grain feeding has been shown to improve FCR (Liu et al., 2015; Moss et al., 2019) but this was not the case in the present study. Reducing dietary CP from 215 to 190 and 165 g/kg (Diets 1A, 2B, 3C, where whole grain was not involved) numerically compromised FCR and increased relative fat-pad weights. The linear effects of reduced-CP diets in relation to FCR (r = -0.326; P = 0.120) and fat-pad weights (r = -0.389; P = 0.060) approached significance. Reducing dietary CP did not influence parameters of energy utilisation (AME, ME:GE ratios, AMEn) and, again, this was not an anticipated outcome. Alternatively, reducing dietary crude protein linearly increased N retention (r = -0.546; P = 0.006) and birds offered the low protein diet had 5.5 percentage units greater N retention (60.2% versus 54.7%) than their high protein counterparts. 5. Conclusion Reductions in dietary crude protein and whole grain feeding had differing impacts on the post-enteral availability of amino acids as assessed by their free concentrations in portal and systemic plasma and the proportion of unbound relative to protein-bound amino acids appeared to be influential. Thus, it is possible that reductions in dietary crude protein are generating imbalances in the postenteral availability of amino acids that are compromising growth performance. Also, there is the indication, when the outcomes of this wheat-based study are compared with the maize-based studies completed at this Institution that there are differences between these two feed grains when they form the basis of reduced-crude protein diets. The genesis of these differences may stem from inherent differences in protein contents and starch characteristics between the two feed grains giving rise to variations in the digestibility and post-enteral availability of amino acids. Declaration of Competing Interest The authors declare that there are not any conflicts of interest. Acknowledgements The authors wish to acknowledge AgriFutures Australia for their financial support of the project (PRJ-010623: Utilisation of synthetic amino acids by poultry) of which the present study forms a component part. The authors would also like to thank Mr Bernie McInerney and Dr Leon McQuade and their colleagues at Australian Proteome Analytical Facility within Macquarie University and the indefatigable Ms Joy Gill and her team in the Poultry Research Foundation for their indispensable inputs. References Angkanaporn, K., Choct, M., Bryden, W.L., Annison, E.F., Annison, G., 1994. Effects of wheat pentosans on endogenous amino acid losses in chickens. J. Sci. Food Agric. 66, 399–404. Belloir, P., Médal, B., Lambert, W., Corrent, E., Juin, H., Lessire, M., Tesseraud, S., 2017. Reducing the CP content in broiler feeds: impact on animal performance, meat quality and nitrogen utilization. Animal 11, 1881–1889. Calvert, C.C., Klasing, K.C., Austic, R.E., 1982. 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