Transgenic, high-protein sorghums display promise in poultry diets in an initial comparison

Abstract

This study aimed to compare the inclusion of transgenic sorghums against commercially available sorghums on growth performance in broiler chickens. Isonitrogenous and isoenergetic diets were offered to a total 288 male Ross 308 broiler chickens from 14 to 35 d posthatch. Three dietary treatments were diets based on transgenic sorghums with a mean protein content of 154.7 g/kg and 5 treatments were based on commercially available sorghum hybrids with a mean protein content of 90.6 g/kg. Soybean meal inclusions in the commercial sorghum diets averaged 215 g/kg, which was reduced to 171 g/kg in the transgenic sorghum diets because of the higher protein contents. Overall growth performance was highly satisfactory, and commercial sorghums supported 2.55% (2,330 vs. 2,272 g/bird; P = 0.010) more weight gains and 2.74% (2,929 vs. 2,851 g/bird; P = 0.012) higher feed intakes; however, the transgenic sorghums supported a fractionally better FCR (1.255 vs 1.257; P = 0.826). There were no statistical differences in apparent jejunal and ileal starch and protein (N) digestibility coefficients between treatments. The transgenic sorghum diets generated slightly, but significantly, higher AME:GE ratios and AMEn, but the commercial sorghum diets generated 6.33% (235 vs. 221 g/kg; P < 0.001) greater breast meat yields. Apparent ileal digestibility coefficients of 16 amino acids averaged 0.839 and 0.832 for transgenic and commercial sorghum-based diets, respectively, without any significant differences in individual amino acids. This outcome suggests amino acid digestibilities of the transgenic sorghums may be inherently higher than commercial hybrid sorghums as the 25.7% higher average soybean meal inclusions would have advantaged amino acid digestibilities in commercial sorghum diets. The possibility that the digestibilities of amino acids in the kafirin component of transgenic sorghums was enhanced by modifications to the structure of kafirin protein bodies is discussed. In conclusion, transgenic sorghums with higher protein concentrations led to 20.5% reduction of soybean meal inclusions in broiler diets, and this change did not compromise feed conversion efficiency compared to standard commercial hybrid sorghums.

INTRODUCTION

Wheat and, to a lesser extent, sorghum are the 2 feed grains of choice for chicken-meat production in Australia. However, sorghum is a drought tolerant crop (Mutava et al., 2011), which is advantageous given the challenges to crop production presented by climate change (Lobell et al., 2011). Generally, wheat has a higher crude protein (CP) content than sorghum. In one local survey (Bryden et al. 2009), 27 wheat samples had an average CP content of 116 g/kg (range: 88–162 g/kg) as opposed to an average CP content of 102 g/kg (range: 71–118 g/kg) in 17 sorghum samples. The lower CP sorghum content typically results in higher soybean meal inclusions in least-cost formulated broiler diets to meet amino acid specifications than in wheat-based diets; however, imported soybean meal is an expensive feedstuff. This tends to inflate feed costs for sorghum-based diets and disadvantages sorghum relative to wheat. Therefore, 3 transgenic, high-protein sorghums (mean: 154.7 g/kg CP) were compared with 5 commercial hybrids (mean: 90.6 g/kg CP) as the feed grain basis in broiler chicken diets. The total amino acid concentrations or “true protein” contents of the transgenic and commercial sorghums averaged 149.2 and 83.3 g/kg, respectively.

RNA silencing (RNAi) approaches were used to produce these transgenic sorghums, targeting the downregulation (not knockout) of 2 genes. GS3 encodes a G-protein gamma-subunit (Mason and Botella, 2000; Botella, 2012), known to be a negative regulator of grain size in sorghum. Allelic variation at this locus plays a major role in grain size and seed protein content (Tao et al., 2020, 2021). The other gene, designated PD, encodes a protein foldase gene involved in the correct folding of kafirin proteins into discrete protein bodies during grain fill. Genetic mechanisms influencing sorghum protein digestibility have been identified (Edmondson and Busche, 2015) and mutations involving kafirin protein bodies have been shown to enhance nutritional traits in sorghum (Wu et al., 2013).

Amino acid, crude protein, kafirin, and starch concentrations in the 8 sorghums were determined as were the amino acid profiles of kafirin per se. The parameters evaluated in broiler chickens included growth performance, relative abdominal fat-pad weights, carcass traits, nutrient utilization, jejunal and ileal starch and protein (N) digestibility coefficients, disappearance rates and amino acid digestibility coefficients in distal jejunum and distal ileum. The objective of the feeding study was to complete an initial evaluation of transgenic sorghums to assess the feasibility of pursuing their development. The average soybean meal inclusion in commercial hybrid sorghum diets was 255 g/kg in this study, as opposed to 171 g/kg in transgenic sorghum diets. Therefore, it was hypothesized that the utilization of transgenic sorghum could reduce soybean meal inclusion in broiler diets without compromising growth performance.

MATERIALS AND METHODS

This study fully complied with the specific guidelines (2020/1776) approved by the Research Integrity and Ethics Administration of The University of Sydney.

Experimental Design

The experimental design consisted of 8 dietary treatments, which were offered to 288 off-sex (parent line) male Ross 308 broiler chickens from 14 to 35 d posthatch. Three treatments were diets based on high-protein, transgenic sorghums and 5 treatments were diets based on commercially available sorghum hybrids. Each dietary treatment was offered to 6 replicate cages (6 birds per cage) or a total of 36 birds per treatment. The 8 sorghums genotypes were analyzed for gross energy and concentrations of starch, crude protein, kafirin, and 16 proteinogenic amino acids (Table 1, Table 2). Concentrations of amino acids in kafirin per se were determined (Table 3).

Table 1.

Nutrient composition of 8 sorghum genotypes.

	Transgenics			Commercial hybrids
Item (g/kg)	C1R5	PD12	PD14	Bazley	Buster	Cracka	Blend	Liberty
Dry matter	942	950	936	895	892	900	862	902
Gross energy (MJ/kg)	18.01	17.93	18.03	17.02	16.86	16.66	16.02	16.64
Starch	552	573	521	638	632	613	669	665
Crude protein	157	155	152	100	92	87	81	93
Kafirin	98.1	94.7	102.0	47.1	56.2	46.5	47.4	58.3
Kafirin proportion (%)	62.5	61.1	67.1	47.1	61.1	53.4	58.5	62.7
Arginine	5.50	4.41	4.24	3.06	3.14	3.71	2.78	2.81
Histidine	3.65	3.54	3.49	2.35	2.40	2.21	2.23	2.26
Isoleucine	6.56	6.67	6.42	3.84	3.70	3.25	3.37	3.96
Leucine	21.50	22.80	22.00	12.70	12.20	9.51	11.00	13.20
Lysine	3.23	2.54	2.39	1.90	1.97	2.51	1.78	1.68
Methionine	1.85	1.63	1.57	1.10	1.19	1.06	1.03	1.34
Phenylalanine	8.51	8.77	8.35	4.95	4.75	4.05	4.28	5.04
Threonine	4.93	4.69	4.58	2.87	2.82	2.70	2.54	2.92
Valine	7.87	7.70	7.48	4.67	4.59	4.22	4.17	4.66
Alanine	14.50	15.00	14.40	8.38	8.12	6.64	7.15	8.57
Aspartic acid	11.00	10.20	9.92	5.96	5.87	5.86	5.31	5.84
Glutamic acid	33.80	35.20	33.90	19.80	19.10	15.10	17.10	20.30
Glycine	4.67	4.06	3.86	2.90	2.93	3.15	2.71	2.71
Proline	13.40	13.80	13.30	7.83	7.50	6.05	6.79	8.07
Serine	6.98	6.91	6.56	4.17	4.11	3.71	3.70	4.12
Tyrosine	3.93	2.68	2.60	1.37	1.59	1.82	1.32	1.99
Total amino acids	151.9	150.6	145.1	87.9	86.0	75.6	77.3	89.5

Table 2.

Average amino acid profiles of transgenic and commercial hybrid grain sorghums.

	Transgenics		Commercial hybrids
Amino acid	Concentration (g/kg)	Proportion (%)	Concentration (g/kg)	Proportion (%)
Arginine	4.72	3.16	3.10	3.73
Histidine	3.56	2.39	2.29	2.75
Isoleucine	6.55	4.39	3.62	4.35
Leucine	22.10	14.81	11.72	14.09
Lysine	2.72	1.82	1.97	2.36
Methionine	1.68	1.13	1.14	1.37
Phenylalanine	8.54	5.73	4.61	5.54
Threonine	4.73	3.17	2.77	3.33
Valine	7.68	5.15	4.46	5.36
Alanine	14.63	9.81	7.77	9.34
Aspartic acid	10.37	6.95	5.77	6.93
Glutamic acid	34.30	22.99	18.28	21.97
Glycine	4.20	2.81	2.88	3.46
Proline	13.50	9.05	7.25	8.71
Serine	6.82	4.57	3.96	4.76
Tyrosine	3.07	2.06	1.62	1.94
Total	149.18	100.0	83.22	100.0

Table 3.

Amino acid concentrations in kafirin across 8 sorghum genotypes.

Amino acid (g/kg)	Transgenics			Commercial hybrids
	C1R5	PD12	PD14	Bazley	Buster	Cracka	Blend	Liberty
Arginine	2.53	2.49	2.51	1.29	1.55	1.42	1.39	1.50
Histidine	2.01	1.94	2.14	0.98	1.21	1.00	1.06	1.18
Isoleucine	4.28	4.12	4.46	2.07	2.43	2.07	2.07	2.58
Leucine	15.4	14.8	16.2	7.42	8.80	7.09	7.22	9.28
Lysine	1.02	1.02	0.96	0.53	0.62	0.71	0.68	0.64
Methionine	0.69	0.69	0.75	0.29	0.43	0.25	0.41	0.41
Phenylalanine	5.88	5.70	6.19	2.83	3.30	2.74	2.75	3.56
Threonine	2.15	2.07	2.22	0.96	1.15	0.94	0.98	1.25
Valine	4.72	4.52	4.93	2.33	2.76	2.37	2.38	2.85
Alanine	9.72	9.33	10.2	4.56	5.48	4.46	4.46	5.64
Aspartic acid	6.77	6.49	6.90	3.01	3.64	3.26	3.20	3.56
Glutamic acid	23.8	23.0	25.1	11.4	13.7	11.0	11.2	14.3
Glycine	1.98	1.97	1.98	1.16	1.33	1.25	1.25	1.31
Proline	9.14	8.89	9.53	4.44	5.16	4.28	4.28	5.42
Serine	3.45	3.37	3.57	1.46	1.82	1.43	1.62	1.92
Tyrosine	4.55	4.38	4.80	2.33	2.77	2.25	2.43	2.88
Total	98.1	94.7	102.0	47.1	56.2	46.5	47.4	58.3

Transgenic Sorghums

Transgenic sorghums were produced with RNAi gene silencing methods (Eamens et al., 2008) following the method of Liu and Godwin (2012). Replicated field trials were performed at the University of Queensland Gatton campus field site (27.5°S, 152.3 ^oE) of 27 different transgenic lines in summer 2017/2018 under Office of the Gene Technology Regulator Licence DIR153 (Liu et al., 2019). Further selections were made in subsequent years, with the plants grown for this experimental grain in the summer of 2020/21. Plants were grown in 75 cm rows, on soil with a pre-sowing application of 100 kg/ha N and adequate irrigation. Harvested grain was dried to 10% moisture then transported to the Poultry Research Foundation at the Camden Campus of the University of Sydney in mid-2021.

Diet Preparation

The dietary treatments were formulated to be isonitrogenous and isoenergetic based on the above analyses and digestible amino acids predicted by near-infrared (NIR) spectroscopy of the sorghums and soybean meal using the AMINOIR Advanced program (Evonik Nutrition & Care GmbH, Hanau, Germany). The diets based on each tested sorghum were formulated on a least-cost basis to form practical broiler diet under commercial scenario. In order to reduce the variation of dietary compositions in each diet, all diets were then combined to form a Blend, the final experimental diet for each tested sorghum contained 50% of the original test-sorghum-based diet and 50% of the Blend. The final dietary composition and nutrient specifications of the experimental diets are shown in Table 4, Table 5, respectively. The analyzed concentrations of gross energy, starch, CP and amino acids are displayed in Table 6. All 8 diets were formulated to contain 210.0 g/kg CP, 11.0 g/kg digestible lysine, 13.0 MJ/kg metabolizable energy with a constant dietary electrolyte balance (DEB) of 230 mEq/kg. To facilitate the formulation of isonitrogenous diets, the sorghum component of each diet contained predominantly the designated sorghum but also a blend of the balance of sorghums. The transgenic sorghum diets contained on average 515 g/kg of the designated sorghum or 71.0% of the sorghum total of 725 g/kg. The corresponding values in the standard sorghum diets were 428 g/kg of the designated sorghum or 66.6% of the sorghum total of 643 g/kg. The reciprocal adjustment in soybean meal inclusions saw a decline from 253 g/kg in standard sorghum diets to 171 g/kg in transgenic sorghum diets. A phytate-degrading feed enzyme was included across all dietary treatments. The analyzed concentrations of the 8 dietary treatments are shown in Table 7. The sorghums were mediumly ground (4.0 mm hammer-mill screen) prior to being blended into the complete diets which were steam-pelleted through a Palmer PP330 pellet press (Palmer Milling Engineering, Griffith, NSW, Australia) at a conditioning temperature of 80°C with a conditioner residence time of 14 s and were then cooled.

Table 4.

Composition (g/kg) of 8 dietary treatments.

Feed ingredient	C1R5	PD12	PD14	Bazley	Buster	Cracka	Blend	Liberty
C1R5	516
PD12		517
PD14			512
Bazley				435
Baxter					435
Cracka						418
Blend							425
Liberty								427
Sorghum blend	210	210	211	217	207	217	218	217
Soybean meal	171	169	173	245	255	261	263	253
d,l-methionine	3.8	3.9	3.9	3.6	3.5	3.5	3.5	3.5
Glycine	3.3	3.6	3.6	2.3	2.0	1.8	1.8	2.1
l-arginine	3.5	3.9	3.8	1.8	1.5	1.1	1.3	1.6
l-histidine	0.2	0.2	0.2	0.1	0.1	0.5	0.1	0.1
l-isoleucine	1.0	1.0	1.0	0.5	0.4	0.4	0.3	0.4
l-lysine HCl	6.0	6.4	6.3	4.2	3.8	3.5	3.6	4.0
l-threonine	2.8	2.9	2.9	2.3	2.2	2.1	2.1	2.2
l-tryptophan	0.1	0.1	0.1	0.0	0.0	0.0	0.0	0.0
l-valine	1.5	1.6	1.6	1.1	1.0	1.0	0.9	1.0
Soy oil	30.3	30.1	30.6	40.1	41.6	42.6	33.3	41.3
Limestone	14.1	14.1	14.1	13.8	13.8	13.8	13.8	13.8
Dicalcium phosphate	6.7	6.7	6.7	6.1	6.0	6.0	5.9	6.0
Potassium carbonate	2.3	2.3	2.2	0.5	0.5	0.5	0.5	0.5
Sodium chloride	0.6	0.6	0.6	1.3	1.7	1.8	2.0	1.6
Sodium bicarbonate	3.5	3.5	3.5	2.4	2.0	1.7	1.5	2.1
Vitamin-mineral premix1	2.2	2.2	2.2	2.2	2.2	2.2	2.2	2.2
Choline Cl	1.0	1.0	1.0	1.0	1.0	1.0	1.0	1.0
Phytase	0.1	0.1	0.1	0.1	0.1	0.1	0.1	0.1
Celite	20.0	20.0	20.0	20.0	20.0	20.0	20.0	20.0
Nonbound amino acids	21.4	27.8	22.6	15.4	14.0	13.5	13.1	14.4

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.

Table 5.

Nutrient specifications (g/kg) of 8 dietary treatments.

Item	C1R5	PD12	PD14	Bazley	Buster	Cracker	Blend	Liberty
Metabolizable energy (MJ/kg)	13.0	13.0	13.0	13.0	13.0	13.0	13.0	13.0
Crude protein	210	210	210	210	210	210	210	210
Lysine1	11.0	11.0	11.0	11.0	11.0	11.0	11.0	11.0
Methionine	5.60	5.70	5.70	5.60	5.60	5.60	5.60	5.70
Methionine + cysteine	8.10	8.10	8.10	8.10	8.10	8.10	8.10	8.10
Threonine	7.70	7.70	7.70	7.70	7.70	7.70	7.70	7.70
Valine	8.70	8.70	8.70	8.70	8.70	8.70	8.70	8.70
Isoleucine	7.60	7.60	7.60	7.60	7.60	7.60	7.60	7.60
Leucine	16.2	16.6	16.4	15.4	15.5	15.0	15.5	15.7
Arginine	12.1	12.1	12.1	12.1	12.1	12.1	12.1	12.1
Glycine equivalents	13.2	13.2	13.2	13.2	13.2	13.2	13.2	13.2
Histidine	3.90	3.90	3.90	4.10	4.20	4.70	4.30	4.20
Tryptophan	1.96	1.96	1.96	2.25	2.30	2.33	2.35	2.46
Phenylalanine + tyrosine	14.4	14.4	14.3	14.8	15.0	14.9	15.1	14.4
Fat	59.4	59.1	59.6	67.3	68.6	69.4	66.7	72.0
Fiber	17.7	17.7	17.7	18.0	18.0	18.1	18.2	18.1
Calcium	8.70	8.70	8.70	8.70	8.70	8.70	8.70	8.70
Total phosphorus	4.94	4.94	4.95	5.07	5.09	5.10	5.12	5.13
Available phosphorus	4.35	4.35	4.35	4.35	4.35	4.35	4.35	4.35
Choline (mg)	1,497	1,491	1,502	1,694	1,722	1,739	1,752	1,758
Electrolyte balance (mEq/kg)	230	230	230	230	230	230	230	230

¹Amino acid concentrations are reported as standardized ileal digestible basis.

Table 6.

Analyzed concentrations in 8 dietary treatments.

Item (g/kg)	C1R5	PD12	PD14	Bazley	Buster	Cracker	Blend	Liberty
Gross energy (MJ/kg)	17.1	17.0	17.1	17.1	17.0	17.1	16.7	17.0
Crude protein	209	204	208	209	207	213	210	207
Starch	460	491	448	412	439	410	438	394
Histidine	4.70	4.56	4.79	4.93	5.04	5.41	5.05	4.86
Serine	9.11	8.78	9.17	9.52	9.75	9.87	9.68	9.41
Arginine	12.6	12.0	12.9	12.6	12.1	12.2	12.4	12.4
Glycine	9.77	9.46	10.2	9.56	9.31	9.35	9.52	9.55
Aspartic acid	17.1	16.3	17.4	18.9	19.3	19.7	19.5	18.7
Glutamic acid	38.7	38.1	38.9	38.3	39.3	39.3	38.3	37.7
Threonine	9.13	8.94	9.42	9.20	9.06	9.23	9.28	9.20
Alanine	13.1	13.1	13.1	11.7	12.0	11.9	11.4	11.4
Proline	13.3	13.1	13.3	12.3	12.6	12.5	12.1	12.1
Lysine	12.6	11.5	12.6	12.4	12.0	12.1	12.2	12.3
Tyrosine	4.74	3.51	4.08	4.37	3.74	4.00	3.91	4.00
Methionine	5.08	5.08	5.08	4.90	4.65	4.60	4.80	5.01
Valine	10.7	10.6	11.1	10.7	10.8	10.7	10.8	10.4
Isoleucine	9.33	9.07	9.42	9.33	9.38	9.42	9.19	9.06
Leucine	21.1	21.0	21.1	19.4	19.9	19.7	18.9	18.9
Phenylalanine	10.2	10.0	10.2	10.3	10.5	10.5	10.3	10.1
Total amino acids	201	195	202	198	199	201	197	195

Table 7.

Effect of dietary treatment on growth performance and relative abdominal fat-pad weights from 14 to 35 d posthatch.

Dietary treatment	Growth performance				Relative fat-pad weights (g/kg)
	Weight gain (g/bird)	Feed intake (g/bird)	FCR (g/g)	Mortality (%)
C1R5	2,291	2,887	1.261	3.33	8.01
PD12	2,238	2,819	1.261	0.00	9.69
PD14	2,289	2,847	1.244	6.67	7.71
Bazley	2,318	2,942	1.269	0.00	8.20
Buster	2,294	2,893	1.261	0.00	7.35
Cracka	2,312	2,908	1.258	0.00	7.46
Blend	2,363	2,963	1.254	0.00	7.29
Liberty	2,362	2,937	1.244	3.33	7.32
Main effect
Transgenic (n = 3)	2,272a	2,851a	1.255	3.33	8.47b
Commercial (n = 5)	2,330b	2,929b	1.257	0.67	7.72a
SEM	14.54	20.17	0.0061	1.121	0.220
Significance (P =)	0.010	0.012	0.8260	0.110	0.005

^a,bMeans within columns not sharing a common suffix are significant different at the 5% level of probability.

Bird Management

A total of 288 male, off-sex Ross 308 chicks were procured from a commercial hatchery and were initially offered a standard starter diet. At 14 d posthatch birds were individually identified (wing-tags) and allocated into bioassay cages (floor area: 750 × 750 mm; height: 500 mm) by bodyweight so that mean weights and variations within cages were statistically identical. Each of 8 dietary treatments were offered to 6 replicate cages (6 birds per cage) from 14 to 35 d posthatch. Birds had unrestricted access to feed and water in an environmentally controlled facility, which remained illuminated for 18 h daily. 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 fifth week.

Data and Sample Collection, Chemical Analyses, Calculations

Growth performance (weight again, feed intake, FCR, mortality/cull rates) and relative abdominal fat-pad-weights was determined from 14 to 35 d posthatch. Birds were weighed at d 14 and 35 and feed intakes were monitored over this interval, the bodyweights of any dead or culled birds were recorded daily to correct feed intakes on a per cage basis and adjust FCR calculations. Total excreta outputs and feed intakes were monitored from 27 to 29 d posthatch to determine parameters of nutrient utilization. These parameters included apparent metabolizable energy (AME), apparent metabolizable to gross energy ratios (AME:GE), nitrogen (N) retention and N-corrected AME (AMEn). Excreta were weighed before and after drying in a forced-air oven at 80◦C for 24 h to determine excreta dry matter and the gross energy (GE) of excreta and diets were determined using a Parr 1,281 adiabatic bomb calorimeter (Parr Instruments Co., Moline, IL). The AME values of the diets on a dry matter basis were calculated from the following equation:

$$AME=\frac{\left(Feedintake\times GEDiet\right)-\left(Excreta\times GEexcreta\right)}{Feedintake}$$

AME: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:

$$Nretention=\frac{\left(Feedintake\times NinDiet\right)-\left(Excreta\times NinExcreta\right)}{Feedintake\times NinDiet}\times 100\%$$

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). At 35 d posthatch, birds were euthanized by an intravenous injection of sodium pentobarbitone, the abdominal cavities opened and abdominal fat-pads dissected out and weighed. The small intestine was removed and digesta was gently expressed in its entirety from the distal half of the jejunum and ileum and pooled by cage, homogenized, freeze dried and weighed to determine the apparent digestibility coefficients of starch, protein (N) and amino acids. Also, Pectoralis major, Pectoralis minor, and thigh muscles were removed from the carcass straight after slaughter without chilling and recorded against final body weights to calculate relative weights of carcass traits.

Starch concentrations were determined by a procedure based on dimethyl sulfoxide, α-amylase and amyloglucosidase, as described in Mahasukhonthachat et al. (2010). Celite (Celite Corporation. Lompoc, CA) was included in diets at 20 g/kg to provide an acid insoluble ash (AIA) dietary marker and AIA and protein (N) concentrations were determined by methods described in Siriwan et al. (1993).

Kafirin concentrations were quantified by procedures developed by the Australian Proteome Analytical Facility (Macquarie University) and a detailed description is provided in Truong et al. (2015). In essence, kafirin was extracted from sorghum following the methodologies of Wallace et al. (1990) and Hamaker et al. (1995). The methodology for analyses of amino acids in kafirin, sorghum, diets, and digesta were based on Cohen (2001), but the amino acids were analyzed by ultra-performance liquid chromatography (Boogers et al., 2008). Apparent digestibility coefficients (ADC) of starch, protein and amino acids in the jejunum and ileum were calculated using the following equation:

$$ADC=\frac{(nutrient/AIAdiet)-(nutrient/AIAdigesta)}{nutrient/AIAdiet}$$

Starch and protein disappearance rates (DR) were calculated using the following equation:

$$DR=Feedintake(g/kg)\times Dietarynutrient(g/kg)\times ADC$$

Statistical Analysis

The experimental data were subject to analyses of variance using the JMP Pro 16.0 software package (SAS Institute Inc., JMP Software, Cary, NC) to compare transgenic and standard sorghums. Pearson correlations were established when deemed suitable. Experimental units were cage means (6 replicate cages of 6 birds per dietary treatment) and a probability level of less than 5 % was taken as statistically significant.

RESULTS

Sorghum Characteristics

The nutrient composition of 8 sorghum genotypes is shown in Table 1. On average, the gross energy content of the transgenic sorghums was higher (17.99 vs. 16.64 MJ/kg) than the commercial varieties as was the CP content (155 vs. 91 g/kg). The mean kafirin concentration was 98.3 g/kg (63.4% of CP) in the transgenic sorghums as opposed to 51.1 g/kg kafirin (56.2% of CP) in the commercial sorghums. Predictably, the commercial sorghum hybrids had higher average starch concentrations (643 vs. 549 g/kg). The average amino acid profiles of transgenic and commercial grain sorghums are shown in Table 2. The transgenic sorghums have a mean total concentration of 149.18 g/kg of the 16 amino acids assessed, which was 79.3% higher than the 83.22 g/kg total concentration in the commercial sorghums. The amino acid concentrations in kafirin per se across the 8 sorghum varieties are shown in Table 3. On a proportional basis, the amino acid profiles of the transgenic and commercial sorghums are quite similar, although kafirin in the transgenic sorghums contained slightly more serine and threonine and slightly less tyrosine, arginine, glycine and lysine than the commercial sorghums. However, it is interesting that the shape of the kafirin protein bodies in the transgenic sorghums is more irregular than standard as detected by Transmission Electron Microscopy, as shown in Figure 1, which implies their structure is modified. In Figure 2, Scanning Electron Microscopy shows the structure of the peripheral endosperm in the transgenic sorghum consisting of tightly packed kafirin protein bodies.

Figure 1.

Transmission electron microscopy of transgenic sorghum displays irregular shapes kafirin protein bodies.

Figure 2.

Scanning electron microscope shows the structure of the peripheral endosperm in the transgenic sorghum consisting of tightly packed kafirin protein bodies.

Experimental Results

Growth Performance

The effects of dietary treatments on growth performance and relative fat-pad weights from 14 to 35 d posthatch are shown in Table 7. Weight gain and feed intake in birds offered diets based on commercial sorghums were higher by 2.46% (2,328 vs. 2,272 g/bird; P = 0.006) and by 2.49% (2,922 vs. 2,851 g/bird; P = 0.010), respectively, than their transgenic sorghum counterparts. However, transgenic- and commercial sorghum-based diets supported effectively identical feed conversion ratios (1.255 vs. 1.257). Relative abdominal fat-pad weights were heavier in birds offered transgenic, sorghum-based diets by 9.72% (8.47 vs. 7.72 g/kg; P = 0.023) than commercial sorghum diets. The overall mortality rate was an acceptable 1.33%, although the mortality rate was numerically higher in birds offered transgenic, sorghum-based diets (3.33 vs. 0.67%; P = 0.110).

Nutrient Utilization

Effects of dietary treatments on parameters of nutrient utilization and N concentrations in excreta from 27 to 29 d posthatch are shown in Table 8. Birds offered transgenic, sorghum-based diets had better energy utilization monitored as AME:GE ratios by 1.51% (0.809 vs 0.797; P = 0.005) and higher AMEn by 0.20 MJ (13.99 vs. 13.79; P = 0.015) than their commercial sorghum-based counterparts. In the same comparison, AME was numerically higher (15.35 vs 15.25 MJ/kg) and N retention was numerically lower (69.0 vs. 69.4%). Excreta N concentrations were higher (4.74 vs. 4.54 g/kg; P < 0.001) in birds offered transgenic, sorghum-based diets.

Table 8.

Effect of dietary treatment on parameters of nutrient utilization and N concentrations in excreta from 27 to 29 d posthatch.

Dietary treatment	AME (MJ/kg DM)	AME:GE ratio	N retention (%)	AMEn (MJ/kg DM)	Excreta N (g/kg)
C1R5	15.20	0.802	68.4	13.82	4.72
PD12	15.46	0.817	69.5	14.10	4.80
PD14	15.38	0.809	69.2	14.04	4.70
Bazley	15.38	0.798	70.0	13.88	4.35
Buster	15.36	0.800	70.3	13.91	4.37
Cracka	15.11	0.789	67.4	13.68	4.63
Blend	15.06	0.793	68.7	13.60	4.51
Liberty	15.35	0.805	70.6	13.89	4.41
Main effect
Transgenic (n = 3)	15.35	0.809b	69.0	13.99b	4.74b
Commercial (n = 5)	15.25	0.797a	69.4	13.79a	4.54a
SEM	0.056	0.0028	0.454	0.053	0.049
Significance (P =)	0.251	0.005	0.553	0.015	< 0.001

^a,bMeans within columns not sharing a common suffix are significant different at the 5% level of probability.

Relative Weights of Carcass Traits

Treatment effects on carcass traits at 35 d posthatch is displayed in Table 9. Commercial sorghums supported greater total breast meat yields than transgenic sorghums by 6.33% (235 vs. 221 g/kg; P < 0.001). There were not any differences in thigh yields (P = 0.728).

Table 9.

Effect of dietary treatment on carcass traits at 35 d posthatch.

Dietary treatment	Pectoralis major (g/kg)	Pectoralis minor (g/kg)	Total breast (g/kg)	Thigh (g/kg)
C1R5	193	33.0	226	209
PD12	188	32.5	221	200
PD14	183	32.8	216	200
Bazley	197	37.3	234	217
Buster	192	33.8	226	202
Cracka	203	35.8	239	205
Blend	204	35.2	239	204
Liberty	203	34.3	238	194
Main effect
Transgenic (n = 3)	188a	32.8	221a	203
Commercial (n = 5)	200b	35.3	235b	204
SEM	1.499	0.622	1.771	2.629
Significance (P =)	< 0.001	0.088	< 0.001	0.728

^a,bMeans within columns not sharing a common suffix are significant different at the 5% level of probability.

Starch and Protein Digestibility Coefficients in Distal Jejunum

Treatment effects on apparent starch and protein (N) digestibility coefficients, disappearance rates and starch:protein disappearance rate ratios in the distal jejunum are shown in Table 10. The mean distal jejunum starch digestibility coefficient was 0.956, without any difference (P = 0.851) between transgenic and commercial-based sorghum diets. The distal jejunum protein (N) digestibility coefficient was numerically higher in birds offered commercial sorghum-based diets by 4.16% (0.676 vs. 0.649; P = 0.139). The distal jejunum starch disappearance rate was higher in birds offered transgenic sorghum-based diets by 8.52% (60.60 versus 55.84 g/bird/d; P < 0.001), but the protein (N) disappearance rate was higher in birds offered commercial sorghum-based diets by 8.11% (19.72 vs. 18.24 g/bird/d; P = 0.012). The starch:protein disappearance rate ratio was higher in birds offered transgenic sorghum-based diets (3.34 vs. 2.82; P < 0.001) than their commercial sorghum counterparts.

Table 10.

Effect of dietary treatment on apparent distal jejunal starch and protein (N) digestibility coefficients, disappearance rates (g/bird/d) and starch:protein disappearance rate ratios at 35 d posthatch.

	Starch		Protein (N)		Starch:Protein ratio
Dietary treatment	Digestibility	Disappearance	Digestibility	Disappearance
C1R5	0.962	60.82	0.658	18.91	3.22
PD12	0.963	63.52	0.679	18.59	3.44
PD14	0.946	57.47	0.610	17.23	3.34
Bazley	0.954	55.06	0.661	19.33	2.87
Buster	0.968	58.50	0.702	20.01	2.95
Cracka	0.958	54.43	0.685	20.21	2.70
Blend	0.953	58.98	0.684	20.31	2.93
Liberty	0.947	52.25	0.647	18.74	2.80
Main effect
Transgenic (n = 3)	0.957	60.60b	0.649	18.24a	3.34b
Commercial (n = 5)	0.956	55.84a	0.676	19.72b	2.85a
SEM	0.0027	0.659	0.0129	0.384	0.0496
Significance (P =)	0.851	< 0.001	0.139	0.012	< 0.001

^a,bMeans within columns not sharing a common suffix are significant different at the 5% level of probability.

Starch and Protein Digestibility Coefficients in Distal Ileum

Treatment effects on the same parameters in the distal ileum are shown in Table 11. The mean distal ileal starch digestibility coefficient was 0.996, without any difference (P = 0.324) between the 2 sorghum categories. Similarly, the mean distal ileal protein (N) digestibility coefficient was 0.808, without any difference (P = 0.460) between the 2 sorghum categories; nor was there any difference (P = 0.077) in protein (N) disappearance rates. The distal ileal starch disappearance rate was higher in birds offered transgenic sorghum-based diets by 8.60% (63.11 vs. 58.11 g/bird/d; P < 0.001). The starch:protein disappearance rate ratio in the distal ileum was higher in birds offered transgenic sorghum-based diets (2.78 vs. 2.46; P < 0.001) than their commercial sorghum counterparts.

Table 11.

Effect of dietary treatment on apparent distal ileal starch and protein (N) digestibility coefficients, disappearance rates (g/bird/d) and starch:protein disappearance rate ratios at 35 d posthatch.

	Starch		Protein (N)		Starch:Protein ratio
Dietary treatment	Digestibility	Disappearance	Digestibility	Disappearance
C1R5	0.996	63.02	0.826	23.73	2.66
PD12	0.997	65.78	0.808	21.12	2.98
PD14	0.996	60.52	0.790	22.28	2.72
Bazley	0.995	57.45	0.803	23.49	2.45
Buster	0.990	59.89	0.801	22.24	2.71
Cracka	0.997	56.64	0.800	23.62	2.40
Blend	0.996	61.59	0.807	23.96	2.58
Liberty	0.997	54.96	0.831	24.04	2.29
Main effect
Transgenic (n = 3)	0.997	63.11b	0.839	22.71	2.78b
Commercial (n = 5)	0.995	58.11a	0.831	23.47	2.46a
SEM	0.0010	0.627	0.0068	0.2881	0.037
Significance (P =)	0.324	< 0.001	0.460	0.077	< 0.001

^a,bMeans within columns not sharing a common suffix are significant different at the 5% level of probability.

Amino Acid Digestibility Coefficients

Effects of dietary treatment on distal jejunal apparent amino acid digestibility coefficients are shown in Table 12. Amino acid digestibilities were significantly higher in birds offered commercial sorghum-based diets for 5 amino acids, including histidine (6.05%), phenylalanine (5.80%), aspartate (6.73%), glutamate (5.06%) and serine (9.34%), where the percentage increases are shown in paratheses. The corresponding data in the distal ileum is displayed in Table 13. However, not any significant differences in digestibilities were observed for the 16 assessed amino acids.

Table 12.

Effect of dietary treatment on apparent amino acid digestibility coefficients in distal jejunum at 35 d posthatch.

Dietary treatment	Arginine	Histidine	Isoleucine	Leucine	Lysine	Methionine	Phenylalanine	Threonine
Transgenic	0.802	0.678a	0.669	0.646	0.815	0.850	0.655a	0.691
Commercial	0.804	0.719b	0.693	0.670	0.816	0.859	0.693b	0.707
SEM	0.0082	0.0096	0.0103	0.0103	0.0092	0.0059	0.0097	0.0096
Significance (P =)	0.936	0.007	0.131	0.131	0.968	0.313	0.014	0.269
Dietary treatment	Valine	Alanine	Aspartate	Glutamate	Glycine	Proline	Serine	Tyrosine
Transgenic	0.676	0.637	0.654a	0.692a	0.733	0.663	0.621a	0.529
Commercial	0.696	0.657	0.698b	0.727b	0.736	0.683	0.679b	0.564
SEM	0.0100	0.0107	0.0103	0.0086	0.0092	0.0089	0.0124	0.0152
Significance (P =)	0.203	0.225	0.008	0.011	0.834	0.155	0.005	0.140

^a,bMeans within columns not sharing a common suffix are significant different at the 5% level of probability

Table 13.

Effect of dietary treatment on apparent amino acid digestibility coefficients in distal ileum at 35 d posthatch.

Dietary treatment	Arginine	Histidine	Isoleucine	Leucine	Lysine	Methionine	Phenylalanine	Threonine
Transgenic	0.912	0.829	0.834	0.825	0.900	0.933	0.834	0.819
Commercial	0.901	0.831	0.826	0.816	0.888	0.926	0.834	0.807
SEM	0.0051	0.0071	0.0075	0.0082	0.0054	0.0038	0.0073	0.0070
Significance (P =)	0.155	0.813	0.535	0.477	0.176	0.228	0.993	0.282
Dietary treatment	Valine	Alanine	Aspartate	Glutamate	Glycine	Proline	Serine	Tyrosine
Transgenic	0.828	0.803	0.819	0.845	0.847	0.810	0.810	0.773
Commercial	0.819	0.791	0.822	0.848	0.830	0.801	0.811	0.766
SEM	0.0073	0.0090	0.0070	0.0067	0.0062	0.0075	0.0079	0.0127
Significance (P =)	0.445	0.396	0.769	0.762	0.074	0.440	0.929	0.709

^a,bMeans within columns not sharing a common suffix are significant different at the 5% level of probability.

DISCUSSION

The overall growth performance recorded in this study, given that the diets were sorghum-based, was highly acceptable. The birds on trial exceeded 2022 Aviagen growth performance objectives by 21.5% (2,308 vs. 1,900 g/bird) in weight gain, by 1.68% (2,900 vs. 2,852 g/bird) in feed intake and by 16.3% (1.257 vs. 1.501) in FCR. Moreover, transgenic (1.255) and commercial sorghum-based diets (1.257) supported almost identical, highly efficient feed conversion ratios from 14 to 35 d posthatch. The promise of transgenic sorghums is reflected in the 32.9% (141 vs. 255 g/kg) reduction in average soybean meal inclusions, which was permitted in isonitrogenous diets with their replacement of commercial sorghums hybrids. This would have the potential to reduce total feed costs by in the order of 7.5% at current feed ingredient prices.

The growth performance of broiler chickens offered sorghum-based diets tends to be variable and, at times, inferior (Hughes and Brooke, 2005). The prime reason for the inclusion of sorghum in broiler diets is the provision of energy, which is principally derived from starch. However, Truong et al. (2016) reported a distal ileal starch digestibility coefficient of 0.883 from a review of 7 studies in which birds were offered sorghum-based diets, which was lower than the digestibility coefficients of 0.916 for wheat (9 studies) and 0.950 for maize (11 studies). Nutritionally equivalent diets based on 2 varieties of maize, wheat and sorghum were compared in broiler chickens by Moss et al. (2020). Distal ileal starch digestibility coefficients in sorghum diets (0.804) were clearly inferior to wheat (0.926) and maize (0.913) diets. This was also the case for efficiency of energy utilization (AME:GE ratios) where the ranking was 0.785 for maize, 0.765 for wheat and 0.713 for sorghum.

In contrast, in the present study, overall starch digestibility was of a high order in the jejunum and ileum with average digestibility coefficients of 0.956 and 0.996, respectively. Moreover, transgenic sorghums supported slightly, but significantly, better energy utilization expressed as AME:GE ratios (0.809 vs. 0.797) or AMEn (13.99 vs. 13.79 MJ/kg) than commercial hybrid sorghum diets. Sub-optimal energy utilization in birds offered sorghum-based diets is seen as a distinct problem where kafirin and “non-tannin” phenolic compounds are inherent, and effectively unique, factors in sorghum that almost certainly compromise starch digestion (Selle et al., 2021). Kafirin protein bodies and starch granules are both embedded in the glutelin protein matrix of sorghum endosperm and this proximity facilitates starch-protein interactions (Rooney and Pflugfelder, 1986). These interactions include the formation of disulphide cross-linkages between the cysteine-rich peripheral layers (β-kafirin and γ- kafirin) of kafirin protein bodies and starch granule-associated proteins (Taylor and Emmambux, 2010).

In a complete diet, the sorghum component provides about 30% of dietary protein; however, there is a greater range of amino acid digestibility values in sorghum protein than protein in maize or wheat (Salunkhe et al., 1977). On the basis of 2 reports, sorghum protein consists of kafirin (54.7%), glutelin (30.8%), albumin (7.5%) and globulin (7.0%) in 2 studies, where the proportions shown in parentheses (Selle et al., 2010). Also, from the data of Taylor et al. (1984), it does appear that as the protein content of sorghum increases, the proportion of kafirin escalates at the expense of glutelin (Selle, 2011). Essentially kafirin contains 3 fractions: α-kafirin (67.3%), β-kafirin (22.7%) and γ-kafirin (10.0%) as recorded by Oria et al. (1995); however, quite different proportions have also been reported. In addition, the amino acid profiles of the 3 kafirin fractions differ as there is less cysteine in α-kafirin (1.1 mole %) than in β-kafirin (4.9 mole %) and γ-kafirin (6.9 mole %) based on the data of Shull et al. (1992). The digestibility of protein/amino acids in sorghum is decreased by “wet-cooking” or hydrothermal treatments. Duodu et al. (2003) proposed that disulphide crosslinking may be the most single important factor negatively influencing sorghum protein digestibility. If follows that disulphide crosslinking between the cysteine rich γ-kafirin and β-kafirin fractions in the periphery of kafirin protein bodies impede the digestion of α-kafirin, the centrally located major storage protein fraction in sorghum (Belton et al., 2006).

Instructively, Truong et al. (2015) compared sorghum-based diets in which the 2 sorghums contained either 50.7 or 61.5 g/kg kafirin and the corresponding isonitrogenous diets contained either 29.0 or 39.6 g/kg kafirin. The diets based on the lower kafirin sorghum generated improvements in weight gains of 3.75% (1,302 vs. 1,255 g/bird) and FCR of 3.56% (1.524 vs. 1.601) from 7 to 28 d posthatch. The same diets enhanced AME by 1.06 MJ (13.61 vs. 12.55 MJ/kg), ME:GE ratios by 4.81% (0.806 vs. 0.769), N retention by 5.6 percentage units (63.6 vs. 58.0%) and AMEn by 1.03 MJ (12.38 vs. 11.35 MJ/kg). These performance advantages were partially attributed to the 26.8% lower kafirin concentration in the relevant diets. This outcome is entirely at odds with the present study where the transgenic sorghum-based diets contained an average of 65.1 g/kg kafirin as opposed to 36.7 g/kg kafirin in the commercial sorghum hybrid-based diets and yet both sets of diets supported comparable weight gains and feed intakes and identical FCR.

While there were significant differences for the digestibility of 5 amino acids in the distal jejunum, in favor of the commercial sorghum-based diets, this was not the case in the distal ileum where not any significant differences were observed. Indeed, the mean apparent digestibilities of 16 amino acids for the transgenic and commercial sorghum-based diets were nearly identical at 0.839 and 0.832, respectively. The likelihood is that about 58% of dietary protein was derived from soybean meal in commercial hybrid sorghum diets as opposed to 39% in the transgenic sorghum diets. However, amino acid digestibilities in soybean meal are usually superior to sorghum in broiler chickens. The ileal digestibility of 14 amino acids in diets where either soybean meal or sorghum were the only protein sources was reported by Ravindran et al. (1999). In diets supplemented with 1200 FTU/kg phytase, mean apparent ileal amino acid digestibility coefficients were 7.46% higher (0.850 vs. 0.791) for soybean meal than sorghum. Therefore, that there were not any significant differences in ileal amino acid digestibility coefficients between transgenic and commercial sorghum-based diets in the present study implies that the ileal amino acid digestibilities of the transgenic sorghums were possibly superior to the commercial hybrid sorghums.

The high-protein, transgenic sorghums did display promise in this preliminary evaluation as they supported acceptable growth performance while permitting reduced soybean meal dietary inclusions. It appears that the transgenic sorghums generated more favorable starch digestibility, energy utilization and amino acid digestibilities than the commercial hybrids. The genesis of these favourable trends may be the modifications to the shape and structure of the kafirin protein bodies. Similarly, Hamaker and Bugusu (2003) reported higher protein digestibility in sorghum following the modification of kafirin protein bodies. In standard sorghums, kafirin protein bodies are spherical, with a 1 to 3 mm diameter, with a central core of α-kafirin with peripheral layers of β- and γ-kafirin, which are relatively rich in cysteine. Thus, there is the potential for the formation of disulphide cross-linkages, which is exacerbated by hydrothermal processes (McCuistion et al., 2019). A highly digestible sorghum mutant cultivar in which kafirin protein bodies had a unique folded structure was identified by Oria et al. (2000). The kafirin protein bodies were irregular in shape with numerous invaginations. Also, transgenic sorghums with altered kafirin synthesis were shown to be advantageous probably resulting from less disulphide-bonded kafirin polymerisation by Da Silva et al. (2011). We propose that the change in the protein-starch matrix, and in particular the changes in the physico-chemical structure of the kafirin protein bodies attenuated the anti-nutritive properties of kafirin in the transgenic sorghums.

Future Directions

The sorghums tested were genetically modified organisms (GMO) and hence would be unlikely to be commercialized due to the costs of the regulatory process in most or all jurisdictions. Gene edited crops, particularly those with simple edits, are not regulated as GMOs in many target markets such as the United States, Brazil, Argentina and Australia (Keiper et al., 2023; Smyth, 2022). Hence our current focus is to deliver the same phenotypes using gene editing approaches (Massel et al., 2021).

The emphasis on the properties of these gene edited sorghums will remain focused on the kafirin protein bodies. However, there are several parameters that should prove instructive as it is possible to determine the concentrations of both free sulfhydryl groups, disulphide bonds and protein solubility in sorghum (Selle et al., 2013) and the formation of disulphide cross-linkages has negative impacts on protein and energy utilization. Phenolic compounds have the capacity to form starch-phenolic complexes (Tomasik and Schilling, 1998) and Thompson et al. (1984) found negative relationships between polyphenol intakes and blood-glucose responses in humans. It is likely that “non-tannin” phenolic compounds have the potential to depress starch digestion and glucose absorption and, in turn, energy utilization in sorghum-based diets. Therefore, concentrations of the various phenolic compounds in the selected sorghums (Khoddami et al., 2015) and their CIELAB color scores (Hughes et al., 2020) merit consideration, which also applies to RVA starch pasting profiles (Truong et al., 2017). It is then noteworthy that the transgenic sorghums were of a light dun color which may be indicative of relatively low concentrations of phenolic compounds, which should be advantageous. Furthermore, the transgenic sorghums exhibited larger dimensions compared to the control line T×430 (Figure 3).

Figure 3.

The color and size of the transgenic sorghum grains/C1R5 (bottom row) used in the present study in comparison to T×430 control line (top row).