Evaluation of dietary crude protein concentrations, fishmeal, and sorghum inclusions in broiler chickens offered wheat-based diet via Box-Behnken response surface design.

The objective of this study was to investigate the impacts of dietary crude protein (CP), fishmeal and sorghum on nutrient utilisation, digestibility coefficients and disappearance rates of starch and protein, amino acid concentrations in systemic plasma and their relevance to growth performance of broiler chickens using the Box-Behnken response surface design. The design consisted of three factors at three levels including dietary CP (190, 210, 230 g/kg), fishmeal (0, 50, 100 g/kg), and sorghum (0, 150, 300 g/kg). A total of 390 male, off-sex Ross 308 chicks were offered experimental diets from 14 to 35 days post-hatch. Growth performance, nutrient utilisation, starch and protein digestibilities and plasma free amino acids were determined. Dietary CP had a negative linear impact on weight gain where the transition from 230 to 190 g/kg CP increased weight gain by 9.43% (1835 versus 2008 g/bird, P = 0.006). Moreover, dietary CP linearly depressed feed intake (r = -0.486. P < 0.001). Fishmeal inclusions had negative linear impacts on weight gain (r = -0.751, P < 0.001) and feed intake (r = -0.495, P < 0.001). There was an interaction between dietary CP and fishmeal for FCR. However, growth performance was not influenced by dietary inclusions of sorghum. Total plasma amino acid concentrations were negatively related to weight gain (r = -0.519, P < 0.0001). The dietary transition from 0 to 100 g/kg fishmeal increased total amino acid concentrations in systemic plasma by 35% (771 versus 1037 μg/mL, P < 0.001). It may be deduced that optimal weight gain (2157 g/bird), optimal feed intake (3330 g/bird) and minimal FCR (1.544) were found in birds offered 190 g/kg CP diets without fishmeal inclusion, irrespective of sorghum inclusions. Both fishmeal and sorghum inclusions did not alter protein and starch digestion rate in broiler chickens; however, moderate reductions in dietary CP could advantage broiler growth performance.

Introduction

Developing reduced crude protein (CP) diets where soybean meal partially replaced with supplementary amino acids is a promising nutritional strategy to achieve sustainable chicken meat production with reduced nitrogen excretion and improved bird welfare. Dietary CP reduction from 220 to 160 g/kg decreased nitrogen excretion by 35% and dietary CP reduction from 198 to 169 g/kg reduced foot-pad lesion scores by 59% in broiler chickens as reviewed by Greenhalgh et al. [1]. Moreover, a dietary CP reduction from 222 to 165 g/kg reduced soybean meal inclusions by 74% in Chrystal et al. [2]. However, broiler chickens responded to reduced CP diets inconsistently; for instance, broilers offered maize-based, reduced-CP diets improved growth performance in comparison to standard CP diets; whereas, broilers offered wheat-based, reduced-CP diets displayed inferior growth performance [2].

Starch and protein digestive dynamics in broiler chickens are important for optimising growth performance in broiler chickens offered reduced-CP diets because dietary CP reduction increases non-bound amino acid (NBAA) inclusions and starch content [3]. Both glucose and monomeric amino acids are co-absorbed with Na+ via their respective Na+-dependent transport systems; whereas, di- and tri- peptides are absorbed via PepT-1 transporter [4, 5]. Glucose and monomeric amino acids may compete for intestinal uptakes [6] and it is possible that such competitions are more pronounced in reduced-CP diets. Liu et al. [7] investigated the influence of diets based on feedstuffs with predetermined starch and protein digestion rate on broiler growth performance from 7 to 35 days post-hatch, where retarding starch digestion rates and/or accelerating protein digestion rates improved FCR and condensed starch:protein digestion rate ratios quadratically (r = 0.648; P < 0.001) improved FCR. In Australia, sorghum is the main alternative feed grain to wheat; starch digestions rates in sorghum are considered slower than wheat under in vitro conditions [8]. Moreover, sorghum starch digestion rates were slower than wheat starch by 56% (0.075 versus 0.117 min -1) as determined in broiler chickens in Selle et al. [9]. Fishmeal inclusions at 175 g/kg in sorghum-soybean-meal–based diets significantly increased protein disappearance rates in proximal and distal ileum which indicates that fishmeal can be a more rapidly digestible protein source than soybean meal [10]. Previous studies reported the relevance of protein digestion rate in broiler diet using casein [11] and whey protein concentrate [12]. It is intended to apply a more practical feed ingredient in the present study to test the relevance of starch and protein digestive dynamics. Therefore, the objective of the present study was to evaluate the relevance of starch and protein digestive dynamics in reduced CP diets by varying inclusion levels of fishmeal and sorghum in a wheat-soybean-meal based diets.

Box-Behnken design (BBD) is a multivariate optimization design with the advantage of testing multiple nutrients simultaneously with less number of treatments [13]. It was previously used to optimise digestive dynamics and compare relative importance of dietary factors [14, 15]. Therefore, the experimental diets in the present study contained three levels of dietary CP (190, 210, 230 g/kg), three levels of fishmeal (0, 50, 100 g/kg), and three levels of sorghum (0, 150, 300 g/kg) and response surface was plotted to visualise experimental results. The hypothesis was both starch and protein digestive dynamics, which was reflected by variable dietary fishmeal and sorghum inclusions, would influence growth performance and nutrient utilisation; moreover, it was expected the impact of starch and protein digestive dynamics was more pronounced in diets containing lower CP and higher NBAA.

Materials and methods

This feeding study was conducted in compliance with the guidelines of the Animal Ethics Committee of The University of Sydney (Project number 2019/1516).

Experimental design

A three factor, three level Box-Behnken response surface experiment with 13 dietary treatments was used to investigate the impact of three dietary CP levels (190, 210, 230 g/kg) with three inclusion levels of fishmeal (0, 50, 100 g/kg) and sorghum (0, 150, 300 g/kg) on growth performance, nutrient utilisation, starch protein digestibility coefficients and plasma amino acid concentrations from 14 to 35 days post-hatch. The factorial arrangement and their respective levels across 13 dietary treatments are shown in Table 1. The central points were 210 g/kg dietary CP level, 50 g/kg fish meal inclusions, and 150 g/kg sorghum inclusions as described for treatment 13M.

Table 1

Factor levels for Box-Behnken design in present study.

Diet	Sorghum (g/kg)	Fishmeal (g/kg)	Dietary crude protein (g/kg)
1A	300	100	210
2B	300	0	210
3C	0	100	210
4D	0	0	210
5E	150	100	230
6F	150	100	190
7G	150	0	230
8H	150	0	190
9I	300	50	230
10J	300	50	190
11K	0	50	230
12L	0	50	190
13M	150	50	210

Diet preparation

Dietary composition and nutrient specifications are shown in Tables 2 and 3. The nutritionally equivalent diets were formulated based on near-infrared spectroscopy (NIR) of wheat, sorghum and soybean meal using the AMINONir® Advanced program (Evonik Nutrition & Care GmbH, Hanau, Germany). Experimental diets were based on wheat, soybean meal, canola meal with or without sorghum and fishmeal. Varying levels of maize starch ranged from 7.50 to 150 g/kg were added to Diets 1A, 3C, 6F, 8H, 10J, and 12L in order to make all diets iso-energetic (13.0 MJ/kg). Experimental diets were formulated based on digestible amino acids with 10 g/kg of lysine across all dietary treatments. NBAA including lysine, methionine, threonine, tryptophan, valine, arginine, isoleucine, leucine, and glycine were supplemented to maintain similar ideal protein ratios for TSAA, Thr, Trp, Ile, Val, Arg and Gly-equivalent. A commercial starter diet based on wheat and soybean meal with 12.13 MJ/kg energy and 220 g/kg crude protein, was offered to broiler chickens from 1 to 13 days post-hatch. Acid insoluble ash (AIA; CeliteTM World Minerals, Lompoc, CA, USA) was included at 20 g/kg in diets as an inert marker in order to determine nutrient digestibility coefficients in two small intestinal sites. Sorghum and wheat were mediumly ground (4.0 mm hammer-mill screen) prior to being blended into the complete diets. All diets were cold-pelleted and contained xylanase and offered to broiler chickens from 14 to 35 days post-hatch.

Table 2

Composition of experimental diets.

Ingredients (g/kg)	Experimental diets
	1A	2B	3C	4D	5E	6F	7G	8H	9I	10J	11K	12L	13M
Wheat	392	301	692	608	492	328	392	525	272	276	578	569	502
Sorghum	300	300	-	-	150	150	150	150	300	300	-	-	150
Maize starch	8.69	-	14.9	-	-	150	-	7.50	-	117	-	128	-
Soybean meal	36.1	172	30.7	163	119	20.1	253	91.5	171	59.1	163	52.4	92.9
Canola meal	83.8	100	81.7	100	56.8	100	72.4	100	100	100	100	100	100
Fishmeal	100	-	100	-	100	100	-	-	50	50	50	50	50
Soybean oil	33.6	62.0	36.0	64.7	42.2	52.9	70.0	51.8	58.8	35.2	61.8	37.1	50.2
l-lysine HCl	2.47	3.08	2.48	3.13	0.48	3.30	1.19	5.27	0.73	4.28	0.76	4.32	2.82
d,l-methionine	1.52	2.05	1.25	1.76	1.00	2.25	1.58	2.43	1.27	2.48	0.97	2.23	1.59
l-threonine	1.13	1.06	1.23	1.16	0.33	1.84	0.32	2.06	0.20	1.91	0.29	2.02	1.14
l-tryptophan	-	-	0.02	-	-	0.28	-	0.11	-	0.19	-	0.22	-
l-valine	0.09	0.14	0.27	0.32	-	1.24	-	1.37	-	1.23	-	1.43	0.19
l-arginine	2.66	5.55	2.35	5.16	3.17	3.23	6.11	5.12	4.22	4.36	3.83	4.04	3.75
l-isoleucine	0.25	0.11	0.39	0.26	-	1.27	-	1.31	-	1.23	-	1.40	0.27
l-leucine	-	-	-	-	-	-	-	-	-	-	-	0.79	-
Glycine	4.33	0.47	3.90	0.04	2.48	5.88	-	2.03	0.74	4.19	0.28	3.80	2.18
Salt	-	0.79	-	0.77	0.73	-	1.51	-	1.14	-	1.12	-	0.37
NaHCO3	4.77	4.90	4.68	4.82	3.80	4.78	3.96	5.99	3.73	5.44	3.64	5.34	4.78
Limestone	4.94	1.74	5.04	11.8	4.89	4.78	11.7	12.1	8.01	8.39	8.10	8.47	8.34
MDCP2	-	12.4	-	12.3	-	0.36	12.5	13.0	5.44	6.71	5.38	6.68	6.00
Xylanase	0.10	0.10	0.10	0.10	0.10	0.10	0.10	0.10	0.10	0.10	0.10	0.10	0.10
Choline chloride	0.90	0.90	0.90	0.90	0.90	0.90	0.90	0.90	0.90	0.90	0.90	0.90	0.90
Celites	20.0	20.0	20.0	20.0	20.0	20.0	20.0	20.0	20.0	20.0	20.0	20.0	20.0
Sand	-	-	-	-	-	46.8	-	-	-	-	-	-	-
Vit min premix1	2.00	2.00	2.00	2.00	2.00	2.00	2.00	2.00	2.00	2.00	2.00	2.00	2.00

1Vitamin-trace mineral premix supplied per tonne of feed; [million international units, MIU] retinol 12, cholecalciferol 5, [g] tocopherol 50, menadione3, 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.

2MDCP, Mono Dicalcium Phosphate.

Table 3

Nutrient specification of experimental diets.

Nutrient (g/kg)	Diet 1A	Diet 2B	Diet 3C	Diet 4D	Diet 5E	Diet 6F	Diet 7G	Diet 8H	Diet 9I	Diet 10J	Diet 11K	Diet 12L	Diet 13M
Calculated values
ME (MJ/kg)	13.0	13.0	13.0	13.0	13.0	13.0	13.0	13.0	13.0	13.0	13.0	13.0	13.0
Crude protein	210	210	210	210	230	190	230	190	230	190	230	190	210
Starch	440	377	440	377	400	428	340	427	360	462	358	463	406
Starch: protein	2.10	1.80	2.10	1.80	1.74	2.25	1.48	2.25	1.57	2.43	1.57	2.44	1.93
Calcium	8.25	8.25	8.25	8.25	8.25	8.25	8.25	8.25	8.25	8.25	8.25	8.25	8.25
Phosphorous available	4.13	4.13	4.13	4.13	4.13	4.13	4.13	4.13	4.13	4.13	4.13	4.13	4.13
Crude fat	67.2	87.2	61.9	82.3	71.4	75.9	89.7	73.7	88.2	64.0	83.6	57.0	76.1
Crude fibre	22.5	25.7	21.3	24.6	20.8	20.4	23.2	24.2	25.8	23.1	24.6	21.5	24.3
Digestible amino acids
Lysine	10.0	10.0	10.0	10.0	10.0	10.0	10.0	10.0	10.0	10.0	10.0	10.0	10.0
TSAA	7.40	7.40	7.40	7.40	7.40	7.40	7.40	7.40	7.40	7.40	7.40	7.40	7.40
Threonine	6.70	6.70	6.70	6.70	6.70	6.70	6.70	6.70	6.70	6.70	6.70	6.70	6.70
Tryptophan	1.90	2.14	1.90	2.11	2.20	1.90	2.42	1.90	2.34	1.90	2.32	1.90	2.01
Isoleucine	7.00	7.00	7.00	7.00	7.75	7.00	7.82	7.00	7.85	7.00	7.72	7.00	7.00
Leucine	13.2	13.5	11.6	11.9	14.1	10.7	14.3	10.8	15.0	11.6	13.5	10.7	12.5
Arginine	10.4	10.4	10.4	10.4	10.4	10.4	10.4	10.4	10.4	10.4	10.4	10.4	10.4
Valine	8.00	8.00	8.00	8.00	8.00	8.84	8.71	8.00	8.95	8.00	8.79	8.00	8.00
Histidine	3.92	4.13	3.97	4.17	4.52	3.41	4.68	3.51	4.63	3.43	4.68	3.46	4.05
Phenylalanine	5.53	8.13	5.35	7.95	6.66	4.25	9.19	6.80	7.96	5.61	7.79	5.39	6.72
Tyrosine	5.43	5.19	5.29	5.03	6.39	4.68	6.11	4.10	6.23	4.46	6.09	4.29	5.19
Proline	9.78	11.8	10.8	12.9	11.1	7.58	13.0	11.6	11.5	9.08	12.5	10.0	11.4
Aspartic acid	3.63	9.26	4.92	10.4	7.88	2.58	1.34	6.81	9.08	4.07	10.3	5.27	6.75
Glutamic acid	17.4	24.0	28.2	34.9	27.3	13.8	33.5	26.5	22.8	14.7	33.7	25.2	25.7
Glycine	8.57	6.83	8.51	6.78	7.74	9.38	7.29	7.62	6.95	8.53	6.89	8.51	7.68
Serine	5.23	7.66	5.30	7.73	6.39	4.08	8.74	6.55	7.49	5.27	7.57	5.31	6.46
Gly- equivalent	12.3	12.3	12.3	12.3	12.3	12.3	12.3	12.3	12.3	12.3	12.3	12.3	12.3
Alanine	1.97	3.98	2.91	4.88	3.73	1.47	5.69	3.38	3.86	1.96	4.78	2.85	3.32
Sodium	1.90	1.90	1.90	1.90	1.90	1.90	1.90	1.90	1.90	1.90	1.90	1.90	1.90
Potassium	4.98	7.33	4.63	6.95	6.32	4.14	8.61	5.59	7.59	5.06	7.22	4.69	5.92
Chloride	1.80	1.80	1.80	1.80	1.80	1.80	1.80	1.80	1.80	1.80	1.80	1.80	1.80
DEB	159	219	150	209	193	135	251	174	226	159	216	150	183
Analysed values
GE (MJ/kg)	16.5	17.1	16.6	17.1	16.7	16.0	17.3	16.8	17.1	16.5	17.2	16.5	17.0
Crude protein	192	201	195	202	219	179	222	183	216	182	223	184	197
Starch	392	351	405	343	369	386	297	360	359	422	322	429	400
Starch: protein	2.04	1.75	2.08	1.70	1.68	2.16	1.34	1.97	1.66	2.32	1.44	2.33	2.03
Total NBAA	12.5	12.5	11.9	11.8	7.47	19.3	9.20	19.7	7.15	19.9	6.13	20.3	11.9

Bird management

A total of 390 off-sex 14-days old male broilers (Ross 308) were randomly distributed into 65 battery cages each of 6 birds (13 treatments × 5 replicates). The variance of average initial body weight was maintained at 1.02% between cages. The dimensions of the cages were 750 mm in width and depth and 500 mm in height. An environmentally controlled housing facility was used and birds had ad-libitum access to feed and water. An initial room temperature of 32 ± 1°C was maintained for the first week, which was gradually decreased to 22 ± 1°C by the end of the third week and maintained at this temperature with a ‘18-hours-on-6-hours-off’ lighting regime for the duration of the feeding study. Initial and final body weights were recorded to determine weight gain. FCR was calculated from feed intake divided by weight gain for the experiment period and any culled/dead bird’s body weights were recorded to adjust feed intakes and FCR calculations.

Sample collection and chemical analysis

Total excreta were collected from 27 to 29 days post-hatch and feed intake during this period was measured separately to determine apparent metabolizable energy (AME), metabolisable energy to gross energy ratio (ME:GE), N retention and N-corrected apparent metabolisable energy (AMEn). Excreta were dried in a forced-air oven at 80°C for 24 hours and the gross energy (GE) of excreta and diets were determined using an adiabatic bomb calorimeter (Parr 1281 bomb calorimeter, Parr Instruments Co., Moline, IL, USA). The AME values (MJ/kg) of the diets were calculated on a dry matter basis from the following equation: 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-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 [16].

At day 34, three birds at random were selected from each cage and blood samples were taken from the brachial vein to determine the concentrations of 20 amino acids in systemic plasma. Collected blood samples were then centrifuged and the decanted plasma samples were then kept at −80˚C before analysis. Amino acids concentration in systemic plasma was quantified by 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC; Waters™ AccQTag Ultra; www.waters.com) followed by separation of the derivatives and quantification by reversed phase ultra-performance liquid chromatography [17]. All amino acids were detected by UV absorbance.

On day 35, all birds were euthanized by intravenous injection of sodium pentobarbitone and digesta samples were collected in their entirety from the distal jejunum and distal ileum to determine apparent digestibility coefficients protein (N) and starch in the distal jejunum and ileum. Small intestines were removed from euthanised birds and samples of digesta were gently expressed from the distal jejunum (below the mid-point between duodenal loop and Meckel’s diverticulum) and distal ileum (below the mid-point between Meckel’s diverticulum and the ileo-caecal junction) in their entirety and pooled for each cage. The digesta samples were then freeze-dried. Starch concentrations in diets and digesta were determined by a procedure based on dimethyl sulphoxide, α-amylase and amyloglucosidase as described by Mahasukhonthachat et al. [18]. N concentrations were determined as already stated and AIA concentrations were determined by the method of Siriwan et al. [19]. The apparent digestibility coefficients for starch and protein (N) in two small intestinal sites were calculated from the following equation:

Starch and protein (N) disappearance rates (g/bird/day) were deduced from the following equation:

Statistical analyses

The experimental units were replicate cage means (6 birds per cage) and statistical procedures included model prediction and linear regression analysis. The surface plots of 3-factor, 3-level Box-Behnken design were obtained by R 3.5.3 software. Best fitted models for response surface designs were predicted by combinations of first- and second-degree polynomial regressions. In model prediction, non-significant coefficients were omitted and the equations were re-predicted with only significant coefficients for each response variable. When more than one models were fitted and significantly different, “Akaike Information Criterion” was used for model comparison and selection. Solver function in Microsoft excel was used to calculate best dietary CP levels with optimal inclusions of fishmeal and sorghum based on response surface design equations.

Results

The influence of dietary treatments on weight gain, feed intake, FCR, and relative fat pad weights are shown in Table 4. The overall average weight gain and feed intake for all treatments from 14 to 35 days post-hatch were higher than 2019 Ross performance objectives by 3.41% (1912 versus 1849 g/bird) and 4.48% (3052 versus 2921 g/bird), respectively and FCR was comparable (1.601 versus 1.579). The mortality rate in the present study was 1.28% which was not influenced by the dietary treatments (P = 0.77).

Table 4

Effect of dietary treatments on growth performance, and relative abdominal fat-pad weights in broiler chickens from 14 to 35 days post-hatch.

Diet	Weight gain (g/bird)	Feed intake (g/bird)	FCR (g/g)	Relative fat-pad weights (g/kg)
1A	1764	2883	1.642	13.3
2B	2053	3160	1.540	12.7
3C	1682	2783	1.655	12.7
4D	2063	3144	1.525	9.74
5E	1678	2889	1.723	12.7
6F	1875	3158	1.685	16.0
7G	2002	3128	1.563	10.3
8H	2140	3270	1.529	13.0
9I	1800	2934	1.630	11.4
10J	1953	3129	1.602	15.8
11K	1857	2901	1.561	11.0
12L	2064	3267	1.583	14.6
13M	1925	3024	1.571	12.9
Crude protein (g/kg)
190	2008	3206	1.600	14.9
210	1897	2999	1.587	12.3
230	1835	2963	1.619	11.3
Linear relationship (r =)	-0.414	-0.486	0.094	-0.620
P-value	0.006	<0.0001	0.457	<0.0001
Sorghum (g/kg)
0	1917	3024	1.581	12.0
150	1924	3094	1.614	13.0
300	1893	3027	1.603	13.3
Linear relationship (r =)	-0.058	0.005	0.108	0.228
P-value	0.648	0.967	0.393	0.068
Fishmeal (g/kg)
0	2065	3176	1.539	11.4
50	1920	3051	1.589	13.2
100	1750	2928	1.676	13.7
Linear relationship (r =)	-0.751	-0.495	0.660	0.395
P-value	<0.0001	<0.0001	<0.001	0.0011

The response surface and contour plot for weight gain is illustrated in Fig 1 and there was no interaction between dietary factors. However, increasing dietary CP and fishmeal inclusions linearly reduced the weight gain regardless of the sorghum inclusion. According to the coefficient of the equation, the negative impact of dietary CP was greater than fishmeal inclusion, as described by the following equation, Based on the above equation, the optimal weight gain of 2157 g/bird was predicted with 190 g/kg dietary CP and no fishmeal regardless of sorghum inclusions.

Fig 1

Response surface and contour plot describing influence of fishmeal inclusion and dietary crude protein level on weight gain in broiler chickens from 14–35 days post-hatch.

The response surface for feed intake is illustrated in Fig 2. Only fishmeal inclusion and dietary CP level influenced feed intake where increasing fishmeal inclusion depressed feed intake and dietary CP had quadratic relationship with feed intake. The predicted equation for the feed intake is,

Fig 2

Response surface and contour plot describing influence of fishmeal inclusion and dietary crude protein level on feed intake in broiler chickens from 14–35 days post-hatch.

The response surface and contour plot for FCR is illustrated in Fig 3. There was a CP and fishmeal interaction on FCR. That negative impact of fish meal inclusions on FCR was more pronounced in diets containing 230 g/kg CP than diets containing 190 g/kg CP. The response of FCR was described by the following equation,

Fig 3

Response surface and contour plot describing influence of fishmeal inclusion and dietary crude protein level on FCR in broiler chickens from 14–35 days post-hatch.

Fig 4 illustrates response surface and contour plots describing the relationship between fish meal inclusions, sorghum inclusions and dietary CP with relative fat pad weights. The surface design shows that increasing fish meal and sorghum inclusions increased the relative fat pad weights whereas dietary CP had negative impact. The predicted equation for relative fat pad weight is as follows,

Fig 4

The response surface designs and contour plots describing the impact of dietary crude protein level together with sorghum and fishmeal inclusions on relative fat pad weights in broiler chickens at 35 days post-hatch (left–slice at 210 g/kg of CP; middle–slice at 5 g/kg of FM; right–slice at 150 g/kg of sorghum).

The predicted lowest relative fat pad weight of 8.67 g/kg was obtained at 230 g/kg dietary CP with no inclusion of fish meal and sorghum.

The impact of dietary treatments on nutrient utilization including AME, ME:GE, nitrogen retention and N corrected apparent metabolisable energy in broiler chickens from 27–29 post-hatch is reported in Table 5. The response surface and contour plots for ME:GE ratio is illustrated in Fig 5. Increasing fishmeal and sorghum inclusion and decreasing dietary CP resulted higher ME:GE ratio. There is an interaction between fishmeal and CP on ME:GE ratio where higher inclusion of fishmeal to reduced CP diets increased ME:GE to larger extent than under high CP diets (Fig 5 right). The predicted equation for response surface design for ME:GE ratio as follows,

Table 5

Effect of dietary treatments on nutrient utilisation (AME: Apparent metabolisable energy, ME:GE: Metabolisable to gross energy ratio, N: Nitrogen retention, AMEn: N-corrected AME) in broiler chicken from 27 to 29 days post-hatch.

Diet	AME (MJ/kg)	ME:GE ratio	N retention (%)	AMEn (MJ/kg)
1A	15.49	0.82	65.52	14.38
2B	15.46	0.79	66.81	14.14
3C	15.53	0.82	65.93	14.45
4D	15.04	0.77	61.78	13.81
5E	15.26	0.80	63.73	14.04
6F	15.45	0.85	71.58	14.22
7G	15.32	0.77	62.21	13.99
8H	15.13	0.78	7076	13.86
9I	15.23	0.77	60.93	14.05
10J	15.60	0.83	69.62	14.44
11K	14.99	0.77	61.09	13.79
12L	15.20	0.81	68.78	13.92
13M	15.35	0.80	64.10	14.21
Crude protein (g/kg)
190	15.34	0.82	70.18	13.97
210	15.37	0.80	64.83	14.20
230	15.20	0.78	61.99	13.96
Linear relationship (r =)	-0.185	-0.554	-0.709	-0.160
P-value	0.140	<0.0001	<0.0001	0.203
Sorghum (g/kg)
0	15.19	0.79	64.40	13.99
150	15.30	0.80	66.48	14.06
300	15.44	0.80	65.72	14.26
Linear relationship (r =)	0.319	0.126	0.115	0.303
P-value	0.009	0.310	0.364	0.014
Fishmeal (g/kg)
0	15.23	0.78	65.39	13.95
50	15.27	0.82	64.90	14.08
100	15.43	0.80	66.69	14.27
Linear relationship (r =)	0.247	0.598	0.113	0.368
P-value	0.047	<0.001	0.372	0.003

Fig 5

The response surface designs and contour plots describing the impact of dietary crude protein level with sorghum and fishmeal inclusions on AME:GE ratio in broiler chickens in 27–29 days post-hatch (left–slice at 210 g/kg of CP; middle–slice at 5 g/kg of FM; right–slice at 150 g/kg of sorghum).

The highest predicted ME:GE ratio of 0.85 was estimated at 190 g/kg of dietary CP level without fish meal and sorghum inclusions.

The effect of dietary treatments on apparent digestibility coefficients and disappearance rates of starch in distal jejunum and distal ileum at 35 days post-hatch is reported in Table 6. Average starch digestibility coefficients in distal jejunum and distal ileum are 0.966 and 0.995 respectively. Crude protein concentrations linearly decreased starch disappearance rate in the distal jejunum (r = -0.800, P < 0.001) and distal ileum (r = -0.812, P < 0.001). Sorghum inclusions had no impact on starch digestibility and disappearance rate. Fishmeal inclusions slightly increased starch digestibility in distal ileum from 0.995 to 0.998 (r = 0.334, P = 0.009). The results of Apparent digestibility coefficients, disappearance rates of protein and starch: protein disappearance rate ratios in distal jejunum and distal ileum are shown in Table 7. Dietary crude protein did not influence apparent protein digestibility coefficients in the distal jejunum and ileum; but it linearly reduced starch and protein disappearance rate ratios in the distal jejunum (r = -0.639, P < 0.001) and ileum (r = -0.779, P < 0.001). Sorghum inclusions had no impact on apparent jejunal protein digestibilities but increasing sorghum inclusion linearly decreased ileal protein digestibility (r = -0.260, P = 0.036). Fishmeal inclusion also had no impact on apparent protein digestibility coefficients in the distal jejunum and ileum. However, increasing fishmeal inclusion increased starch and protein disappearance rate ratios in the distal jejunum (r = 0.342, P = 0.005) and distal ileum (r = 0.375, P = 0.005).

Table 6

Effect of dietary treatments on apparent starch digestibility coefficients and disappearance rates in distal jejunum and distal ileum at 35 days post-hatch.

Diet	Digestibility coefficients		Disappearance rates (g/bird/day)
	Distal jejunum	Distal ileum	Distal jejunum	Distal ileum
1A	0.969	0.999	60.0	62.2
2B	0.970	0.997	58.4	60.5
3C	0.966	0.999	59.4	61.4
4D	0.968	0.996	56.8	59.5
5E	0.964	0.996	56.2	58.1
6F	0.968	0.998	64.2	67.4
7G	0.968	0.970	48.9	50.0
8H	0.951	0.997	61.1	64.0
9I	0.970	0.997	56.0	57.5
10J	0.971	0.998	69.9	71.9
11K	0.964	0.997	48.6	49.2
12L	0.965	0.999	73.6	76.1
13M	0.964	0.997	63.0	65.2
Crude protein (g/kg)
190	0.964	0.998	67.2	70.0
210	0.967	0.998	59.6	61.8
230	0.967	0.995	52.4	59.9
Linear relationship (r =)	0.094	-0.284	-0.800	-0.812
P-value	0.455	0.027	<0.0001	<0.0001
Sorghum (g/kg)
0	0.966	0.998	59.6	62.4
150	0.963	0.995	58.7	60.1
300	0.970	0.998	61.2	63.1
Linear relationship (r =)	0.129	0.004	0.086	0.071
P-value	0.306	0.978	0.496	0.589
Fishmeal (g/kg)
0	0.964	0.995	56.4	58.4
50	0.967	0.998	62.2	64.6
100	0.967	0.998	59.9	62.0
Linear relationship (r =)	0.071	0.334	0.190	0.190
P-value	0.577	0.009	0.130	0.143

Table 7

Effect of dietary treatments on apparent crude protein digestibility coefficients, disappearance rates and starch: Protein disappearance rate ratios in distal jejunum and distal ileum at 35 days post-hatch.

Diet	Digestibility coefficients		Disappearance rates (g/bird/day)		Starch: protein disappearance rate ratio
	Distal jejunum	Distal ileum	Distal jejunum	Distal ileum	Distal jejunum	Distal ileum
1A	0.618	0.725	18.69	22.01	3.27	2.80
2B	0.598	0.723	20.84	25.14	2.93	2.41
3C	0.623	0.736	18.39	21.76	3.27	2.82
4D	0.610	0.706	21.15	24.53	2.73	2.30
5E	0.586	0.738	20.23	25.51	2.79	2.28
6F	0.528	0.727	16.31	22.30	4.15	2.96
7G	0.578	0.671	21.87	25.37	2.31	1.99
8H	0.619	0.749	20.21	24.50	3.05	2.62
9I	0.607	0.697	21.05	24.20	2.69	2.39
10J	0.583	0.703	18.14	21.83	3.90	3.30
11K	0.649	0.774	22.60	26.96	2.16	1.89.
12L	0.660	0.766	21.57	25.02	3.43	3.05
13M	0.618	0.752	19.93	24.12	3.20	2.70
Crude protein (g/kg)
190	0.598	0.736	19.06	23.41	3.63	2.98
210	0.613	0.729	19.80	23.53	3.08	2.61
230	0.604	0.720	21.44	25.51	2.49	2.15
Linear relationship (r =)	0.033	-0.128	0.292	0.354	-0.639	-0.779
P-value	0.796	0.321	0.019	0.004	<0.0001	<0.0001
Sorghum (g/kg)
0	0.636	0.746	20.93	24.57	2.90	2.56
150	0.586	0.728	19.71	24.38	3.10	2.49
300	0.602	0.712	19.68	23.29	3.20	2.72
Linear relationship (r =)	-0.160	-0.260	-0.153	-0.214	0.167	0.169
P-value	0.203	0.036	0.223	0.087	0.184	0.226
Fishmeal (g/kg)
0	0.601	0.713	21.01	24.89	2.76	2.33
50	0.623	0.738	20.66	24.44	3.08	2.70
100	0.589	0.732	18.41	22.89	3.37	2.70
Linear relationship (r =)	-0.058	0.147	-0.321	-0.336	0.342	0.375
P-value	0.643	0.241	0.009	0.006	0.005	0.005

The impact of dietary treatments on free amino acids concentrations in the systemic plasma is shown in Tables 8 and 9. Dietary crude protein linearly increased plasma concentrations of Arg, His, Ile, Leu, Phe, Asn+Asp and Tyr. Dietary crude protein linearly decreased plasma concentrations of Met, Thr, Glu+Gln and Gly. Sorghum inclusions linearly decreased plasma concentrations of Leu, Val and Tyr. Fishmeal inclusions linearly increased plasma concentrations of His, Ile, Leu, Met, Phe, Thr, Val, Ala, Cys, Glu+Gln, Gly, Pro and Ser; but decreased plasma concentrations of Arg, Lys and Asn + Asp.

Table 8

Effects of dietary treatments on free essential amino acid systemic plasma concentrations (μg/mL) at 34 days post-hatch.

Diet	Arg	His	Ile	Leu	Lys	Met	Phe	Thr	Trp	Val
1A	77.5	20.5	11.4	29.0	14.5	15.5	22.4	109.3	4.6	23.0
2B	119.2	8.1	9.8	22.5	17.8	10.8	19.0	58.6	4.9	18.4
3C	66.9	21.8	14.2	23.7	13.8	14.5	22.7	106.1	5.0	28.9
4D	110.6	8.7	11.2	18.2	22.0	10.0	17.0	64.3	4.8	21.2
5E	106.5	26.2	16.1	31.7	15.5	12.6	24.0	92.3	5.4	31.9
6F	58.8	5.5	13.9	20.3	15.2	17.6	17.0	114.2	5.4	30.8
7G	106.5	13.8	12.4	23.1	17.2	8.3	20.0	54.5	5.3	22.7
8H	100.6	5.0	12.1	15.8	21.9	14.2	16.2	72.5	5.1	24.6
9I	116.2	21.4	14.8	31.9	16.7	12.0	23.5	73.2	5.6	29.1
10J	87.8	9.6	12.1	22.3	17.4	17.3	19.8	100.6	4.9	25.8
11K	120.5	22.1	15.5	26.2	17.5	10.8	22.1	74.3	5.5	30.2
12L	76.9	5.2	13.9	17.9	15.6	15.1	18.0	102.7	5.3	31.1
13M	100.6	13.9	11.1	23.1	14.2	12.0	20.4	80.5	5.00	21.9
Linear relationships
Crude protein (g/kg)
190	81.0	6.3	13.0	19.1	17.5	16.1	17.8	97.5	5.2	28.1
210	95.0	14.6	11.5	23.3	16.5	12.5	20.3b	83.8	4.9	22.6
230	112.4	20.9	14.7	28.2	16.7	10.9	22.4	73.6	5.4	28.5
r =	0.453	0.726	0.267	0.661	-0.066	-0.598	0.614	-0.424	0.177	0.029
P =	<0.0001	<0.001	0.032	<0.001	0.602	<0.0001	<0.001	<0.001	0.160	0.821
Sorghum (g/kg)
0	93.7	14.5	13.7	21.5	17.2	12.6	20.0	86.8	5.1	27.8
150	94.6	12.9	13.1	22.8	16.8	13.0	19.5	82.8	5.2	26.4
300	100.2	14.9	12.0	26.4	16.6	13.9	21.2	85.4	5.0	24.1
r =	0.093	0.022	-0.258	0.357	-0.047	0.154	0.159	-0.025	-0.081	-0.275
P =	0.460	0.864	0.038	0.004	0.711	0.221	0.206	0.842	0.521	0.026
Fishmeal (g/kg)
0	109.2	8.9	11.4	19.9	19.7	10.8	18.1	62.5	5.0	21.7
50	100.4	14.5	13.5	24.3	16.3	13.4	20.7	86.3	5.3	27.6
100	77.4	18.5	13.9	26.2	14.7	15.1	21.5	105.5	5.1	28.6
r =	-0.459	0.477	0.393	0.450	-0.397	0.492	0.460	0.761	0.055	0.504
P =	<0.001	<0.001	0.001	0.0002	0.001	<0.001	0.001	<0.001	0.665	<0.001

Table 9

Effects of dietary treatments on free non-essential and total amino acid systemic plasma concentrations (μg/mL) at 34 days post-hatch.

Diet	Ala	Asn+Asp	Cys	Glu+Gln	Gly	Pro	Ser	Tyr	Total
1A	78.9	94.7	16.0	242.2	138.9	72.4	83.2	40.7	1036.0
2B	65.2	137.1	15.9	190.8	47.7	41.7	54.7	41.7	775.2
3C	72.8	79.9	18.5	277.0	127.3	81.8	76.5	37.1	1042.2
4D	59.8	130.4	15.1	196.4	43.8	45.6	54.4	34.2	763.4
5E	77.7	122.4	17.4	223.1	105.4	78.7	80.4	43.0	1025.2
6F	78.7	73.9	16.7	274.8	165.4	57.7	89.6	29.7	1044.9
7G	58.7	123.1	14.8	181.0	46.9	45.0	54.3	38.3	751.8
8H	68.5	114.5	14.7	221.4	48.9	48.3	49.6	28.6	793.0
9I	77.8	134.7	15.8	202.8	76.0	60.1	58.1	47.3	919.3
10J	74.5	105.5	15.6	243.7	110.2	57.8	72.5	30.9	954.8
11K	71.2	137.4	16.9	202.3	71.4	66.3	64.8	39.7	912.2
12L	72.6	97.3	17.0	283.5	100.5	59.4	72.3	26.8	963.8
13M	68.3	116.5	15.7	208.2	81.3	59.2	61.1	37.5	864.6
Linear relationships
Crude protein (g/kg)
190	73.6	97.8	16.0	255.9	106.3	55.8	71.0	29.0	939.1
210	69.0	111.7	16.4	222.9	87.8	60.1	66.0	38.4	896.4
230	71.4	129.4	16.2	202.3	74.9	62.5	64.4	42.1	902.1
r =	-0.076	0.448	0.054	-0.434	-0.301	0.175	-0.187	0.736	-0.105
P =	0.135	0.0002	0.671	0.0003	0.015	0.164	0.135	<0.001	0.405
Sorghum (g/kg)
0	69.1	111.3	16.8	239.8	85.7	62.3	67.0	34.4	920.5
150	70.4	110.1	15.9	221.7	89.6	57.8	67.0	35.4	895.9
300	74.1	118.0	16.0	219.9	93.2	58.0	67.1	40.2	921.3
r =	0.171	0.095	-0.197	-0.162	-0.197	-0.137	0.003	0.323	0.002
P =	0.173	0.450	0.116	0.198	0.572	0.276	0.982	0.009	0.985
Fishmeal (g/kg)
0	63.0	126.3	15.1	197.4	46.8	45.1	53.3	35.7	770.8
50	72.9	118.3	16.2	228.1	87.9	60.6	65.8	36.5	922.9
100	77.0	92.7	17.4	254.3	134.3	72.7	82.4	37.6	1037.2
r =	0.479	-0.478	0.554	0.461	0.839	0.715	0.830	0.110	0.756
P =	<0.001	<0.0001	<0.001	0.0001	<0.001	<0.000	<0.001	0.384	<0.001

Discussion

In the present study, dietary CP reductions from 230 to 210 and 190 g/kg linearly increased weight gain (r = 0.414, P < 0.01) and feed intake (r = 0.486, P < 0.001) without influencing FCR. Accordingly, birds offered 190 g/kg CP diets had 9.43% (2008 versus 1835 g/bird, P < 0.05) higher weight gain and 8.20% (3206 versus 2963 g/bird, P < 0.05) higher feed intake compared to birds offered 230 g/kg CP diets. However, the 190 g/kg CP diet represents a modest reduction in dietary CP for birds from 14 to 35 days post-hatch. Chrystal et al. [20] reported modest reduction in dietary CP increased weight gain by 8.22% (2396 versus 2214 g/bird) and reduced FCR 2.62% (1.453 versus 1.415) from 7–35 days post-hatch. Thus, birds have the potential to perform satisfactorily when offered moderately reduced-CP diets which was consistent with the present study. However, more tangible reductions in dietary CP usually compromise growth performance with an increase in carcass fat deposition [21, 22]. In the present study, on average, transition of dietary CP from 230 to 190 g/kg reduced soybean meal inclusions (56 versus 177 g/kg) whilst increasing NBAA inclusions (7.49 versus 19.8 g/kg). This outcome supports the rationale in Selle et al. [23] that replacing soybean meal with NBAA in reduced-CP diets could be a promising strategy to reduce the chicken-meat industry’s dependence on soybean meal. However, further reductions of dietary CP requires higher inclusions of NBAA as evident in Chrystal et al. [2]. This may generate amino acid imbalances at sites of protein synthesis; if so, increased deamination would generate ammonia and may compromise broiler growth performance [24].

Fishmeal is usually recognised as a prolific source of digestible amino acids in broiler chickens [25] and pigs [26] and was included in the present experiment to diversify the rate of protein digestion in order to investigate the influence of protein and starch digestive dynamics in diets containing different CP levels. However, it was not anticipated that the 100 g/kg fish meal inclusion would significantly compromise growth performance in the present study. This is in complete contrast to the positive growth performance responses generated by 175 g/kg fishmeal inclusions in sorghum-based broiler diets observed by Sydenham et al. [10]. Nevertheless, the unexpected impact of fishmeal is not without precedent. It was reported decades ago [27] that overheating fishmeal during the rendering process depressed the availability of amino acids in broiler chickens. Lysine and aspartic acid, in particular, appear to be vulnerable to improper processing in both fishmeal and other protein meals [28-30]. In the present study, fishmeal inclusion negatively influenced plasma concentrations of Lys (r = -0.397, P = 0.001) and Asn + Asp (r = -0.478, P < 0.001). Unfortunately, due to the lack of variable impact of sorghum and fishmeal inclusions on distal jejunal starch and protein digestibilities, respectively, the relevant importance of starch and protein digestive dynamics in diets containing different CP was not tested as originally planned. However, attention needs to be drawn on inconsistent growth performance in broiler chickens when offered diets containing sorghum and fishmeal.

Sorghum has been associated with sub-optimal growth performance in broiler chickens, consequently, its inclusion in practical diets is often limited. However, in the present study, the substitution of wheat with sorghum did not influence broiler performance, which implies the two feed grains used in the present study were nutritionally equivalent where broilers offered diets with 0 and 300 g/kg sorghum generated comparable starch digestibility coefficients in distal jejunum (0.966 versus 0.970). This suggests that the starch digestibility/energy utilization of sorghum used in the present study was of an unusually high order. Wheat is commonly recognised as a better feed grain under Australian conditions, but this is not necessarily always the case. The performance of broilers offered maize-, sorghum- and wheat-based diets, without and with exogenous phytase, was compared from 1 to 27 days post-hatch in Liu et al. [31]. As main effects, sorghum supported significantly better weight gain by 6.70% (1338 versus 1254 g/bird) and FCR by 3.60% (1.471 versus 1.526) than wheat-based diets. Alternatively, Moss et al. [32] compared two sorghum-based diets with two wheat-based diets offered to broilers from 1 to 35 days post-hatch. While average weight gains were nearly identical (2670 versus 2676 g/bird), birds offered wheat-based diets enjoyed an advantage of 3.41% (1.415 versus 1.465) in FCR. Thus, the relative nutritional values of the two feed grains are sufficiently variable that the outcome from any one comparison cannot be predicted with any accuracy. The comparable quality of sorghum starch to wheat starch in the present study failed to generate different starch digestion rates.

The Box-Behnken design is challenged in the present study because there are additional variations in dietary compositions to the three factors being evaluated. Concentrations of soybean meal ranged from 20.1 to 253 g/kg and NBAA from 6.13 to 19.90 g/kg across the 13 dietary treatments (Table 2). Interestingly, significant multiple linear regressions were detected for feed intakes (r = 0.746; P < 0.0001) and weight gains (r = 0.860; P < 0.0001) when soybean meal and NBAA were considered in addition to fishmeal. The equations are as follows: Thus, in both instances, inclusions of soybean meal and NBAA promoted feed intakes and weight gains; whereas, fishmeal inclusions had negative impacts on growth performance.

Conclusions

Growth performance were compromised by higher fishmeal inclusion and was not influenced by sorghum substitution. Both fishmeal and sorghum inclusions did not alter protein and starch digestion rate in broiler chickens; however, it is evident that moderate reductions in dietary CP could advantage broiler growth performance.

(XLSX)

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6 Oct 2021

PONE-D-21-26429Box-Behnken evaluation of dietary crude protein concentrations, fishmeal, and sorghum inclusions in wheat-based diets in broiler chickens from 14 to 35 days post-hatchPLOS ONE

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Reviewer #3: Yes

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5. Review Comments to the Author

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Reviewer #1: The paper is interesting. I am not familiar to Box-Behnke design and I think you should write more of this design. How were the number of the treatments decided? I was also wodering was the amino acid balance similar in all the diets. Do you have any information of the processing conditions of fish meal. It is surprised be that fish meal lowered the weight gain.

Reviewer #2: The manuscript titled ‘Box-Behnken evaluation of dietary crude protein concentrations, fishmeal, and sorghum inclusions in wheat-based diets in broiler chickens from 14 to 35 days post-hatch’ has been designed well to elucidate the effect of providing high and low levels of crude protein, sorghum, and fish meal compared with frequently used levels in the diet on the performance and nutrient utilization in growing-finishing broilers. Overall, the authors have done nice work, but there are a few important pieces of feedback for the improvement of the conclusiveness of this study. The addressing of these comments should be considered before the manuscript would be deemed acceptable for publication.

1. It would be better to present digestible amino acids value for at least major limiting amino acids as well in the nutrient composition. Since crystalline (non-bound) amino acids are used to balance the essential amino acids in the diet, it would change the level of digestible amino acids in the formulated feed. The declaration of the digestible amino acids in the diets used for this study is vital because it is a complex design and shows the predicted optimum composition of feed based on the best-fit regression model. This study is not only evaluating different ingredients but is also adding crude protein in the model and due to the variation in the digestibility of amino acids of the ingredients used in this study, it could make a certain conclusion about the effect of crude protein in the diet on growth performance which may not necessarily be true. Some previous studies have already shown that broilers grown on a low crude protein diet can have better performance. However, the information about the digestible value will prove that even high protein in the diet after a certain inclusion level may not improve the performance of broilers.

2. The second question is about the digestible amino acid value of the fish meal. The authors could provide the digestible crude protein and amino acids value of the fish meal used in this study as the digestible profile of this product can be substantially variable depending on source and processing methods.

3. Should make it Box-Behnken design or experimental model in the title. The phrase 'Box-Behnken evaluation' doesn’t sound appropriate.

4. Line 41-43: Negative opinion for soybean production should not be put forward without it being the theme of discussion. Securing sources of protein could also lead to overfishing, excess petroleum extraction etc.

5. Line 47: Should be footpad lesion scores.

6. Table 2 and rest of tables: better mention choline chloride. A common name would be better in the ingredient list. OR define in the caption below the table. Also, provide more information in the description caption of tables such as what the treatments from 1A to 13 M mean. The tables and figures should be standalone.

7. Line 123: Change ‘-6-hour-off’ to ‘-6-hours-off’.

8. Line 196: Change ‘weigh’ to ‘weight’ in the equation.

9. Line 305: Change ‘compromised’ to ‘compromise’.

Reviewer #3: Dear Authors,

I have a series of questions that I consider necessary to address in the present manuscript (Discussion) and that may be useful to you for future studies as well.

Main comments:

- Fishmeal: the justification to study “fishmeal” as main factor seems not totally well justified. It does not appear to be a more sustainable protein source compared to SBM and so helping to have a more sustainable meat chicken meat production as stated in L41-43.

o Moreover, with 100 g/kg fishmeal, canola meal was also partially replaced, so that would apparently induce an even higher environmental impact.

o Was fishmeal quality measured, and compared to previous studies (L301-311).

- The interaction between starch and protein digestive dynamics has been previously studied by your group. The Box-Behnken design studying these three factors (CP level, fish meal level and sorghum level) is not well justified, and hypothesis for their potential interaction are not well described in the introduction and discussion sections.

- Conclusion. Form L341 and 350 it is not a conclusion but a results summary. The only conclusion written is in L351-352.

Study design:

- Why was the study conducted from day 14 onwards and not from day 0 until the end of growing period? Why is your design more relevant than having different feeding phases.

- Why was phytase not used in the experimental diets!?

- Were test feedstuffs analysed before diet reformulation and production? I would expect that at least wheat, sorghum, SBM and fishmeal were analysed. These results should be presented here as well.

- Why only 5 replicates were used per treatment? This number seems very small.

- Why was excreta collected from 27-29 days and intestinal contents on day 34? The age difference could have make the comparison of these parameters not possible.

Additional comments:

- L144: 20 amino acids. Why Asn+Asp and Glu+Gln are presented together as a sum?

- L149-150: reference method?

- L151: what vein?

- L151: Why were (390!) birds (individually) sacrificed by intravenous injection instead of by using CO2 per pen? The individual sacrifice should have cost a large amount of time and seems not to be that convenient.

- L152: Why was ileal content collected from distal jejunum + distal ileum to determine ileal digestibility? This is a rather large section at this bird age.

- L154-158: repetititve.

- L157: it seems that the contents of both distal jejunum and distal ileuam were pooled.

- L161: it is okay to repeat reference method.

- L163: What 4 sites??? In Table 6 and 7 there are only 2 sites presented.

- L168: average Feed intake 14-35d?

- L203-206: no interaction?

- L207: R2: exactly the same as for BWG?

- L214: P-value?

- L222: interactions?

- L238: Why is ME:GE ratio a relevant parameter?

- L260-263: Why not explanation of main factors?

- L270-274: Not presented? What is the relevance of these results?

Table 2:

- Maize starch was used as an ingredient. So, why not targeting the same starch content in all diets? Sand was used as a filler (only in diet 6F?).

- Glycine: was it L-glycine?

- C5H12ClNO => Choline chloride

- I would suggest to merge tables 2 and 3. If that does not happen, I would suggest to present “Total NBAA”, “Starch specs” and “Starch analysed” in Table 3; “Starch analysed” is currently presented in both Table 2 and 3.

Table 3:

- Present (calculated and analysed) C Fat and CFibre levels.

- It would be more interesting to present digestible AA contents rather than total.

- dEB: why was it not corrected by using K-carbonate or another K source? Discuss the effect of dEB on your study.

Table 5:

- There is a redundant row Under “Sorghum” main effect.

Table 7: P-values of CP factor?

Figures: add units of the response parameters. e.g. %, g, use the same units as on the Tables. Image quality is poor.

Figure 2 is the same as in Figure 1 for BWG!!!! The figure of FI is not shown!!!

**********

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24 Oct 2021

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Updated

Additional Editor Comments:

This manuscript deals with an interesting and important topic in poultry nutrition. Nevertheless there are still some points of concern from the reviewers, before the manuscript can be accepted for publication. Please focus on animal material and method, Tables and figures. As there are some points unclear to me regarding the trial execution, sampling and statements in the manuscript, I recommend major revision.

Thank you, we have address all reviewers’ comments.

Review Comments to the Author

Reviewer #1: The paper is interesting. I am not familiar to Box-Behnken design and I think you should write more of this design. How were the number of the treatments decided? I was also wondering was the amino acid balance similar in all the diets. Do you have any information of the processing conditions of fish meal. It is surprised be that fish meal lowered the weight gain.

Thank you, the below is included in the Introduction,

Box-Behnken design (BBD) is a multivariate optimization design with the advantage of testing multiple nutrients simultaneously with less number of treatments (De Leon et al., 2010). It was previously used to optimise digestive dynamics and compare relative importance of dietary factors (Liu et al., 2016; 2019). Therefore, the experimental diets in the present study contained three levels of dietary CP (190, 210, 230 g/kg), three levels of fishmeal (0, 50, 100 g/kg), and three levels of sorghum (0, 150, 300 g/kg) and response surface was plotted to visualise experimental results.

BBD is a classic way of optimisation. Once experimental factors are confirmed to be influential by factorial design, BBD can help to find the best numerical combinations of these variables. It is widely used in engineering. Unfortunately, we do not have processing and source information of fishmeal which we regret. It is certainly a surprise fishmeal depressed performance here.

Reviewer #2: The manuscript titled ‘Box-Behnken evaluation of dietary crude protein concentrations, fishmeal, and sorghum inclusions in wheat-based diets in broiler chickens from 14 to 35 days post-hatch’ has been designed well to elucidate the effect of providing high and low levels of crude protein, sorghum, and fish meal compared with frequently used levels in the diet on the performance and nutrient utilization in growing-finishing broilers. Overall, the authors have done nice work, but there are a few important pieces of feedback for the improvement of the conclusiveness of this study. The addressing of these comments should be considered before the manuscript would be deemed acceptable for publication.

1. It would be better to present digestible amino acids value for at least major limiting amino acids as well in the nutrient composition. Since crystalline (non-bound) amino acids are used to balance the essential amino acids in the diet, it would change the level of digestible amino acids in the formulated feed. The declaration of the digestible amino acids in the diets used for this study is vital because it is a complex design and shows the predicted optimum composition of feed based on the best-fit regression model. This study is not only evaluating different ingredients but is also adding crude protein in the model and due to the variation in the digestibility of amino acids of the ingredients used in this study, it could make a certain conclusion about the effect of crude protein in the diet on growth performance which may not necessarily be true. Some previous studies have already shown that broilers grown on a low crude protein diet can have better performance. However, the information about the digestible value will prove that even high protein in the diet after a certain inclusion level may not improve the performance of broilers.

Totally agreed, we apologise for not pointing this out in the original submission. All experimental diets were formulated to digestible AA basis, Table 3 has been updated to reflect this. The amino acids formulated to constant levels (digestible basis) include TSAA, Thr, Arg and Gly-equivalent. The amino acids formulated to minimal levels (digestible basis) include Trp, Ile and Val.

2. The second question is about the digestible amino acid value of the fish meal. The authors could provide the digestible crude protein and amino acids value of the fish meal used in this study as the digestible profile of this product can be substantially variable depending on source and processing methods.

We regret the fishmeal we used in the present study was not analysed by NIR for dig AAs as fishmeal is not routinely used in broiler diets. It is surprising in the present study fishmeal did not perform as well as our previous study in 2016. The below was used for formulation based on our historical wet chemistry data in the 2016 experiment.

% % % % % %

Ingredients Nitrogen Protein Factor Starch Ca P Na

Fish meal 10.175 63.594 6.250 0.105 6.807 3.604 0.655

Liu, SY, Sydenham, CJ, Selle, PH (2016) Feed access to, and inclusions of fishmeal and corn starch in, sorghum-based broiler diets influence growth performance and nutrient utilisation as assessed by the Box-Behnken response surface design. Animal Feed Science and Technology 220, 46-56.

3. Should make it Box-Behnken design or experimental model in the title. The phrase 'Box-Behnken evaluation' doesn’t sound appropriate.

This title is updated to “Evaluation of dietary crude protein concentrations, fishmeal, and sorghum inclusions in broiler chickens offered wheat-based diet via Box-Behnken response surface design”

4. Line 41-43: Negative opinion for soybean production should not be put forward without it being the theme of discussion. Securing sources of protein could also lead to overfishing, excess petroleum extraction etc.

Deleted

5. Line 47: Should be footpad lesion scores.

Corrected

6. Table 2 and rest of tables: better mention choline chloride. A common name would be better in the ingredient list. OR define in the caption below the table. Also, provide more information in the description caption of tables such as what the treatments from 1A to 13 M mean. The tables and figures should be standalone.

Table 2 is updated; however, we are not sure whether it is practical to include footnote describing 13 treatments because this is different from factorial design. This is the reason table 1 was included.

7. Line 123: Change ‘-6-hour-off’ to ‘-6-hours-off’.

Corrected

8. Line 196: Change ‘weigh’ to ‘weight’ in the equation.

Corrected

9. Line 305: Change ‘compromised’ to ‘compromise’.

Corrected

Reviewer #3: Dear Authors,

I have a series of questions that I consider necessary to address in the present manuscript (Discussion) and that may be useful to you for future studies as well.

Main comments:

- Fishmeal: the justification to study “fishmeal” as main factor seems not totally well justified. It does not appear to be a more sustainable protein source compared to SBM and so helping to have a more sustainable meat chicken meat production as stated in L41-43. Moreover, with 100 g/kg fishmeal, canola meal was also partially replaced, so that would apparently induce an even higher environmental impact.

Totally agree L41-43 is deleted and the first paragraph of Introduction is included as below,

“Developing reduced crude protein (CP) diets where soybean meal particularly replaced with supplementary amino acids is a promising nutritional strategy to achieve sustainable chicken meat production with reduced nitrogen excretion and improved bird welfare. Dietary CP reduction from 220 to 160 g/kg reduced nitrogen excretion by 35% and dietary CP reduction from 198 to 169 g/kg reduced foot-pad lesion scores by 59% in broiler chickens as reviewed by Greenhalgh et al. [1]. Moreover, a dietary CP reduction from 222 to 165 g/kg reduced soybean meal inclusions by 74% in Chrystal et al. [3]. However, broiler chickens responded to reduced CP diets inconsistently; for instance, broilers offered maize-based, reduced-CP diets improved growth performance in comparison to standard CP diets; whereas, broilers offered wheat-based, reduced-CP diets displayed inferior growth performance [3].”

“Previous studies reported the relevance of protein digestion rate in broiler diet using casein and whey protein concentrate. It is intended to apply a more practical feed ingredient in the present study to test the relevance of starch and protein digestive dynamics.”

The intention of this study is not to use fishmeal to replace SBM, it is preliminary test the relevance of starch and protein digestive dynamics in reduced CP diets. We previously reported the relevance by using whey-protein, and we gradually moved to a semi-practical ingredient in this study. Unfortunately, fishmeal in the present study did not perform as a rapidly digestible protein source as we expected, which raised another interesting question of consistency of fishmeal quality.

Was fishmeal quality measured, and compared to previous studies (L301-311).

Unfortunately and regrettably not because of the challenge of sourcing fishmeal on time. Fishmeal is not routinely used in broiler diets presently.

- The interaction between starch and protein digestive dynamics has been previously studied by your group. The Box-Behnken design studying these three factors (CP level, fish meal level and sorghum level) is not well justified, and hypothesis for their potential interaction are not well described in the introduction and discussion sections.

The below is included in Introduction:

“Previous studies reported the relevance of protein digestion rate in broiler diet using casein and whey protein concentrate. It is intended to apply a more practical feed ingredient in the present study to test the relevance of starch and protein digestive dynamics. The hypothesis was both starch and protein digestive dynamics, which was reflected by variable dietary fishmeal and sorghum inclusions, would influence growth performance and nutrient utilisation; moreover, it was expected the impact of starch and protein digestive dynamics was more pronounced in diets containing lower CP and higher NBAA.”

- Conclusion. Form L341 and 350 it is not a conclusion but a results summary. The only conclusion written is in L351-352.

Conclusion is updated as below

“Growth performance were compromised by higher fishmeal inclusion and was not influenced by sorghum substitution. Both fishmeal and sorghum inclusions did not alter protein and starch digestion rate in broiler chickens; however, it is evident that moderate reductions in dietary CP could advantage broiler growth performance.”

Study design:

- Why was the study conducted from day 14 onwards and not from day 0 until the end of growing period? Why is your design more relevant than having different feeding phases.

It is for the below reasons:

(1) Performance in younger chicks (starter phase) could be confounded by breeder flock and hatchery conditions.

(2) Significant number of additional treatments would be required to truly test phase effect unless only cumulative response was determined.

(3) This one is a practical/commercial concern. For reducing dietary crude protein, it is more profitable and less risk in grower and finisher phases, because they have much higher feed consumption and more resilient digestive and immune system.

- Why was phytase not used in the experimental diets!?

It is not used because fishmeal was included at variable levels. Fishmeal contains high levels of mineral, the below is a historical (2016) wet chemistry analysis, we do not want to confound the study by using phytase with mineral matrix and worrying about inconsistent activity recovery and variation of dietary phytate levels.

% % % % % %

Ingredients Nitrogen Protein Factor Starch Ca P Na

Fish meal 10.175 63.594 6.250 0.105 6.807 3.604 0.655

- Were test feedstuffs analysed before diet reformulation and production? I would expect that at least wheat, sorghum, SBM and fishmeal were analysed. These results should be presented here as well.

Wheat, sorghum and SBM were tested before formulation and unfortunately, fishmeal was not tested due to the challenge of sourcing it. The results were not included because dig AAs used for formulation is analysed by NIR which is industry standard but we were suggested previously by other reviewers not to report it.

However, the below is included in M&M,

“The nutritionally equivalent diets were formulated based on near-infrared spectroscopy (NIR) of wheat, sorghum and soybean meal using the AMINONir® Advanced program (Evonik Nutrition & Care GmbH, Hanau, Germany).”

- Why only 5 replicates were used per treatment? This number seems very small.

This is typical for BBD which emphasises the number of treatments (dots on the surface) is more critical than number of replications for each dot.

- Why was excreta collected from 27-29 days and intestinal contents on day 34? The age difference could have make the comparison of these parameters not possible.

We agree that age would influence AME and N retention results, and this is why we conduct collection between 27-29 days. In the literature, this is the age nutrient utilisation was reported and most matrix value used in formulation is quantified around 28 days post-hatch. The collection of intestinal content is for quantification of digestibility.

Additional comments:

- L144: 20 amino acids. Why Asn+Asp and Glu+Gln are presented together as a sum?

This is to do with amino acid analysis method as they are not separated by ultra-performance liquid chromatography.

- L149-150: reference method?

Included

Cohen, SA (2001) Amino acid analysis using precolumn derivatisation with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate. Methods in Molecular Biology 159, 39-47.

- L151: what vein?

Updated to “brachial vein”

- L151: Why were (390!) birds (individually) sacrificed by intravenous injection instead of by using CO2 per pen? The individual sacrifice should have cost a large amount of time and seems not to be that convenient.

This is the routine method approved by our Ethic Committee

- L152: Why was ileal content collected from distal jejunum + distal ileum to determine ileal digestibility? This is a rather large section at this bird age.

Corrected to “digesta samples were collected in their entirety from the distal jejunum and distal ileum to determine apparent digestibility coefficients protein (N) and starch in the distal jejunum and ileum.”

- L154-158: repetititve.

Corrected

- L157: it seems that the contents of both distal jejunum and distal ileuam were pooled.

Yes, jejunal samples from the same cage were pooled and ileal samples from the same cage were pooled.

- L161: it is okay to repeat reference method.

Okay

- L163: What 4 sites??? In Table 6 and 7 there are only 2 sites presented.

Corrected

- L168: average Feed intake 14-35d?

Updated to “…The overall average weight gain and feed intake for all treatments from 14 to 35 days post-hatch …”

- L203-206: no interaction?

No interaction here, non-significant term is excluded in the equation.

- L207: R2: exactly the same as for BWG?

Updated

- L214: P-value?

Included

- L222: interactions?

No interaction here, non-significant term is excluded in the equation

- L238: Why is ME:GE ratio a relevant parameter?

We have seen in the past better correlation between performance and ME:GE than with AME per se. This original idea is from

Black, JL, Hughes, RJ, Nielsen, SG, Tredrea, AM, MacAlpine, R, Van Barneveld, RJ (2005) The energy value of cereal grains, particularly wheat and sorghum, for poultry. Proceedings of Australian Poultry Science Symposium 17, 21-29.

- L260-263: Why not explanation of main factors?

Good suggestion, thank you and now they are included as shown in the track-change version

- L270-274: Not presented? What is the relevance of these results?

Deleted and updated as

“The impact of dietary treatments on free amino acids concentrations in the systemic plasma is shown in Table 8A and 8B. Dietary crude protein linearly increased plasma concentrations of Arg, His, Ile, Leu, Phe, Asn + Asp and Tyr. Dietary crude protein linearly decreased plasma concentrations of Met, Thr, Glu + Gln and Gly. Sorghum inclusions linearly decreased plasma concentrations of Leu, Val and Tyr. Fishmeal inclusions linearly increased plasma concentrations of His, Ile, Leu, Met, Phe, Thr, Val, Ala, Cys, Glu + Gln, Gly, Pro and Ser; but decreased plasma concentrations of Arg, Lys and Asn + Asp.”

Table 2:

- Maize starch was used as an ingredient. So, why not targeting the same starch content in all diets? Sand was used as a filler (only in diet 6F?).

The crude protein levels also changed, if starch content is fixed. Low CP diet will draw fat to formulate iso-energetic diets. Consequently, these diets will have much higher level of filler (> 10%) which is impossible to pellet.

- Glycine: was it L-glycine?

There is no L, D-form of Glycine

- C5H12ClNO => Choline chloride

Corrected

- I would suggest to merge tables 2 and 3. If that does not happen, I would suggest to present “Total NBAA”, “Starch specs” and “Starch analysed” in Table 3; “Starch analysed” is currently presented in both Table 2 and 3.

Only showing Starch and Total NBAA in Table 3.

Table 3:

- Present (calculated and analysed) C Fat and CFibre levels.

Calculated crude fat and fibre values are included in Table 3

- It would be more interesting to present digestible AA contents rather than total.

Corrected

- dEB: why was it not corrected by using K-carbonate or another K source? Discuss the effect of dEB on your study.

The impact of DEB on performance of reduced CP diets were reported in Chrystal et al. (2020) where dropping DEB from 230 to 156 mEq/kg in reduced CP diet did not influence growth performance, energy utilisation and protein digestibilities.

Chrystal, PV, Moss, AF, Khoddami, A, Naranjo, VD, Selle , PH, Liu, SY (2020) Effects of reduced crude protein levels, dietary electrolyte balance, and energy density on the performance of broiler chickens offered maize-based diets with evaluations of starch, protein, and amino acid metabolism. Poultry Science 99, 1421-1431.

Table 5:

- There is a redundant row Under “Sorghum” main effect.

Deleted

Table 7: P-values of CP factor?

Included

Figures: add units of the response parameters. e.g. %, g, use the same units as on the Tables. Image quality is poor.

All updated

Figure 2 is the same as in Figure 1 for BWG!!!! The figure of FI is not shown!!!

Corrected

8 Nov 2021

Evaluation of dietary crude protein concentrations, fishmeal, and sorghum inclusions in broiler chickens offered wheat-based diet via Box-Behnken response surface design

PONE-D-21-26429R1

Dear Dr. Liu,

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Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

**********

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Reviewer #3: Yes

**********

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Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #2: Yes

Reviewer #3: Yes

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PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #3: (No Response)

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Reviewer #2: Yes: Amit Kumar Singh

Reviewer #3: No

10 Nov 2021

PONE-D-21-26429R1

Evaluation of dietary crude protein concentrations, fishmeal, and sorghum inclusions in broiler chickens offered wheat-based diet via Box-Behnken response surface design

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