Estimation of digestible sulphur amino acids requirements for growth performance and immune responses to Newcastle disease and avian influenza vaccination in broilers

Figures & data

Table 1. Ingredients and nutrients composition of the basal diet^a.

Items	Grower (d11–24)
Ingredient, g/kg as-fed basis
Corn (ME = 3498 kcal/kg, CP = 7.8%)	551.90
Soybean meal (ME = 2800, CP = 45.22%)	376.50
Soybean oil (ME = 8820 kcal/kg)	29.00
Limestone	10.60
Dicalcium phosphate	16.60
Sodium chloride	4.40
Vitamin premix^b	2.50
Mineral premix^c	2.50
L-Lysine-HCL	1.20
L-Threonine	0.80
Filler (corn starch)	4.00
Determined nutrients composition^d, as-fed basis
Metabolisable energy, kcal/kg	3100
Crude protein, %	21.50
Calcium, %	0.87
Available phosphorus, %	0.43
Sodium, %	0.20
Digestible lysine, %	1.15
Digestible methionine, %	0.30
Digestible sulphur amino acids, %	0.59
Digestible threonine, %	0.77

^a The diets were provided in a way that a batch of basal diet (without methionine supplementation) was made and then divided into six equal portions, the DL-methionine supplement ‘as a feed grade (ME = 5020 kcal/kg, CP = 58.1% and Met = 99%, Evonik Degussa GmbH, Hanau‐Wolfgang, Germany)’, was added at the rate of 0, 0.7, 1.4, 2.1, 2.8 and 3.5 g/kg to the top of each portion at the expense of filler (corn starch) and mixed to provide six diets with 0.59, 0.66, 0.73, 0.80, 0.87, and 0.94% digestible sulphur amino acids levels.

^b Vitamin premix supplied the followings per kilogram of diet: vitamin A (all-trans-retinol), 12000 IU; vitamin D3 (cholecalciferol), 5000 IU; vitamin E (α-tocopherol), 18 IU; vitamin K3 (menadione), 2.65 mg; vitamin B1 (thiamin), 2.97 mg; vitamin B2 (riboflavin), 8.0 mg; vitamin B3 (niacin), 57.42 mg; vitamin B5 (pantothenic acid), 17.86 mg; vitamin B6 (pyridoxine), 4.45 mg; vitamin B9 (folic acid), 1.9 mg; vitamin B12 (cyanocobalamin), 0.02 mg; vitamin H2 (biotin), 0.18 mg; choline chloride, 487.5 mg, and antioxidant 1.0 mg

^c Mineral premix supplied the followings per kilogram of diet: Zn (zinc sulfate), 110 mg; Mn (manganese sulfate), 120.6; Fe (iron sulfate), 40.5; Cu (copper sulfate), 16.1; I (calcium iodate), 1.26; Se (Sodium Selenite), 0.31; choline chloride, 474.0.

^d The determined ingredient analysis was used to calculate nutrient composition (crude protein, calcium and sodium were measured by the AOAC (2002) methods; metabolisable energy, digestible amino acids and available phosphorus were measured by NIR analysis.

Abbreviation: ME: metabolisable energy; CP, crude protein.

Table 2. DL-methionine supplementation and determined nutrients composition in experimental diets^a.

DL-methionine supplementation, g/kg	Determined nutrient composition, as-fed basis						Dig SAAs/Dig Lys, %
	ME, kcal/kg	CP, %	Dig Lys, %	Dig Met, %	Dig SAAs, %	Dig Thr, %
0.0	3100	21.50	1.15	0.30	0.59	0.77	51
0.7	3101	21.54	1.15	0.37	0.66	0.77	57
1.4	3103	21.58	1.15	0.44	0.73	0.77	63
2.1	3104	21.62	1.15	0.51	0.80	0.77	70
2.8	3105	21.66	1.15	0.58	0.87	0.77	76
3.5	3107	21.70	1.15	0.65	0.94	0.77	82

^a The determined ingredient analysis was used to calculate nutrients composition; crude protein was measured by the AOAC (2002) method, and ME, digestible amino acids were measured by NIR analysis.

Abbreviation: ME: metabolisable energy; CP: crude protein; Dig Lys: digestible lysine; Dig Met: digestible methionine; Dig SAAs: digestible sulphur amino acids; Dig Thr: digestible threonine.

Table 3. Effect of dietary digestible sulphur amino acids (DSAAs) level on growth performance, and daily DSAAs intake in male Ross-308 broilers during grower period (d11-24 of age)^a.

Traits	DSAAs level, %						SEM	p-Value
	0.59	0.66	0.73	0.80	0.87	0.94		Linear	Quadratic
Live body weight d11, g	243	246	260	254	251	252	4.28	0.86	0.96
Live body weight d24, g	809	860	985	892	903	929	34.36	0.03	0.08
Daily feed intake, g/d	68.02	81.31	71.38	75.36	80.13	75.98	3.40	0.18	0.38
Daily weight gain, g/d	38.58	45.32	47.00	46.49	46.72	48.50	2.78	0.03	0.24
Feed efficiency, g WG/100 g FI	56.46	55.63	65.63	61.50	58.32	63.74	2.29	0.04	0.36
Daily DSAAs intake, mg/d	401	537	521	603	697	714	26.21	0.01	0.52

^a The values are means of six replicates.

Table 4. Effect of dietary digestible sulphur amino acids (DSAAs) level on carcass and cut relative weight (g/100 g of live body weight) in male Ross-308 broilers on day 24^a.

Traits	DSAAs level, %						SEM	p-Value
	0.59	0.66	0.73	0.80	0.87	0.94		Linear	Quadratic
Carcass^b, %	58.57	59.60	61.75	59.56	61.49	59.61	0.81	.03	.04
Breast, %	19.52	20.40	23.69	23.26	25.52	22.27	0.82	.01	.01
Legs (Thigh + Drum), %	18.64	18.04	18.47	18.30	17.57	17.40	0.41	.60	.50
Frame^c, %	20.41	21.15	19.59	18.01	18.41	19.93	0.77	.08	.10
Liver, %	2.78	2.50	2.73	2.56	2.75	2.82	0.14	.21	.19
Bursa of Fabricius, %	0.24	0.23	0.20	0.21	0.20	0.16	0.02	.01	.71
Heart, %	0.67	0.69	0.68	0.65	0.64	0.72	0.04	.80	.48
GIT^d, %	10.44	10.54	9.81	9.95	9.93	9.82	0.50	.25	.69
Cover (skin + feathers), %	8.45	6.96	6.93	9.11	7.62	7.75	0.70	.91	.72

^a The values are means of six replicates.

^b Carcass obtained by feathers, head, feet, skin and viscera removed.

^c Frame is the carcass without the breast and legs.

^d GIT, gastrointestinal tract is the whole digestive organs (crop, proventriculus, gizzard, small and large intestine).

Table 5. Effect of dietary digestible sulphur amino acids (DSAAs) level on blood metabolites and immune responses (antibody titre) to Newcastle disease, and avian influenza vaccination in male Ross-308 broilers on day 24^a.

DSAAs level, %	TG	Chol	HDL-C	LDL-C	UA	Cr	TP	Alb	ALT	AST	ALP	Immune responses, log2
	(mg/dl)						(g/dl)		(U/l)			NDV	AIV
0.59	94	209	79	95	5.45	0.50	3.30	1.50	3.00	278	15438	4.75	2.50
0.66	65	147	74	63	5.38	0.30	2.88	1.30	4.25	256	10588	6.00	4.25
0.73	63	172	76	70	4.73	0.20	3.98	1.80	1.50	211	9611	6.75	3.25
0.80	75	150	74	65	4.33	0.33	3.23	1.65	2.75	251	11971	6.00	3.00
0.87	61	147	69	67	4.45	0.28	3.65	1.68	2.50	251	10189	5.50	4.25
0.94	66	163	70	72	4.73	0.33	3.35	1.33	2.75	248	10998	6.25	4.50
SEM	4.82	12.63	3.65	5.48	0.48	0.06	0.23	0.12	0.91	14.80	989.19	0.51	0.28
p-Value
Linear	0.01	0.03	0.07	0.04	0.10	0.03	0.41	0.92	0.45	0.07	0.03	0.01	0.01
Quadratic	0.02	0.04	0.98	0.01	0.27	0.04	0.38	0.02	0.59	0.08	0.02	0.01	0.01

^a The values are means of six replicates.

Abbreviation: TG: triglyceride; Chol: whole cholesterol; HDL-C: high‐density lipoprotein cholesterol; LDL-C: low‐density lipoprotein cholesterol; UA: uric acid; Cr: creatinine; TP: total protein; Alb: albumin; ALT: alanine aminotransferase; AST: aspartate aminotransferase; ALP: alkaline phosphatase; NDV: Newcastle disease vaccination; AIV: avian influenza virus vaccination.

Table 6. Partial correlation coefficients (above) and probability values (below) between growth performance traits with blood serum constituent of male Ross-308 broilers on day 24.

Growth performance traits	Blood metabolites
	TG	Chol	HDL-C	LDL-C	TP	Alb	UA	Cr	ALT	AST	ALP
Live body weight	−0.41	−0.19	−0.12	−0.34	0.22	0.31	−0.22	−0.25	−0.50	−0.42	−0.51
	0.01	0.27	0.48	0.04	0.19	0.07	0.19	0.14	0.01	0.01	0.01
Feed efficiency	−0.30	−0.35	−0.27	−0.40	0.07	0.09	−0.39	−0.08	−0.23	−0.02	−0.32
	0.08	0.04	0.11	0.02	0.67	0.60	0.02	0.64	0.18	0.90	0.06
Relative breast weight	−0.24	−0.04	−0.02	−0.13	0.32	0.34	−0.27	−0.04	−0.43	−0.23	−0.32
	0.16	0.83	0.89	0.43	0.06	0.04	0.11	0.81	0.01	0.17	0.05

Abbreviation: TG: triglyceride; Chol; whole cholesterol; HDL-C: high density lipoprotein cholesterol; LDL-C: low density lipoprotein cholesterol; TP: total protein; Alb: albumin, UA: uric acid; Cr: creatinine; ALT: alanine aminotransferase; AST: aspartate aminotransferase; ALP: alkaline phosphatase.

Figure 1. Live body weight at 24 d of age (Y, in g/b) as a function of dietary digestible sulphur amino acids level (X, in % of diet) fed from 11 to 24 d of age. Linear broken-line, Y = 900–764(0.73-X), p < .005, R²=0.29. The break point occurred at 0.73 ± 0.09.

Figure 2. Daily weight gain (Y, in g/b) as a function of dietary digestible sulphur amino acids level (X, in % of diet) fed from 11 to 24 d of age. Linear broken-line, Y = 45.71–123.2(0.69-X), p < .001, R²=0.47. The break point occurred at 0.69 ± 0.03.

Figure 3. Feed efficiency (Y, g WG/100 g FI) as a function of dietary digestible sulphur amino acids level (X, in % of diet) fed from 11 to 24 d of age. Quadratic broken-line, Y = 62.30–236(0.78–X)², p<.006, R²=0.33. The break point occurred at 0.78 ± 0.09.

Figure 4. Breast relative weight at 24 day of age (Y, in % of live body weight) as a function of dietary digestible sulphur amino acids level (X, in % of diet) fed from 11 to 24 d of age. Quadratic broken-line, Y = 23.95-67.93(0.86-X)², p < .001, R²=0.50. The break point occurred at 0.86 ± 0.09.

Figure 5. Serum antibody titre in response to Newcastle disease vaccination (Y, in log₂) as a function of dietary digestible sulphur amino acids level (X, in % of diet) fed from 11 to 24 d of age. Quadratic broken-line, Y = 5.56–1.2(0.73-X)², p < .003, R²=0.41. The break point occurred at 0.73 ± 0.07.

Figure 6. Serum antibody titre in response to avian influenza vaccination (Y, in log₂) as a function of dietary digestible sulphur amino acids level (X, in % of diet) fed from 11 to 24 d of age. Quadratic broken-line, Y = 5.31–136(0.72-X)², p < .001, R²=0.76. The break point occurred at 0.72 ± 0.03.

Table 7. Estimated digestible sulphur amino acids (DSAAs) requirement of male Ross-308 broilers during grower period (d11-24 of age) for optimisation of growth performance, and immune responses (antibody titre log₂) to Newcastle disease and avian influenza vaccination by broken‐line regression fit models analysis^a.

Items	Estimated dig SAAs requirement	Lower 95% confidence levels	Upper 95% confidence levels	p-Value	R^2	Adj. R^2	RMSE	AIC
Linear broken-line regression analysis
Live body weight d24	0.73 ± 0.09	0.53	0.92	.005	0.29	0.27	69.89	293
Body weight gain	0.69 ± 0.03	0.63	0.74	.001	0.47	0.45	4.70	109
Feed efficiency	0.73 ± 0.08	0.56	0.89	.001	0.27	0.24	6.10	135
RBW	0.74 ± 0.03	0.67	0.81	.001	0.49	0.47	1.77	39
Immune response to vaccination
ND	0.68 ± 0.03	0.61	0.75	.003	0.39	0.37	0.89	5
AI	0.68 ± 0.01	0.65	0.71	.001	0.74	0.73	0.47	35
Quadratic broken-line regression analysis
Live body weight d24	0.78 ± 0.10	0.57	0.99	.006	0.28	0.26	70.41	294
Daily weight gain	0.81 ± 0.08	0.66	0.96	.001	0.48	0.46	4.70	113
Feed efficiency	0.78 ± 0.09	0.57	0.98	.006	0.33	0.31	5.19	123
RBW	0.86 ± 0.09	0.67	1.04	.001	0.50	0.49	1.73	37
Immune response to vaccination
ND	0.73 ± 0.07	0.57	0.88	.003	0.41	0.39	0.85	3
AI	0.72 ± 0.03	0.64	0.78	.001	0.76	0.72	0.45	32

^a Responses did not fit in the models to optimised dietary dig SAAs level for feed intake.

Abbreviation: R²: coefficient of determination; Adj. R²: adjusted coefficient of determination, RMSE: root means square error; AIC: Akaike’s information criterion; RBW: relative breast weight; ND: Newcastle disease; AV: avian influenza.

Table 8. A comparison of the digestible sulphur amino acids (DSAAs) requirements of male broiler chicks determined with different parameters.

Breed	Age, d	Parameters	Diet form	Dig Met, %	Dig SAAs, %	Reference
Cobb	1–7	Body weight gain	–	–	0.87	Goulart et al. 2011
Ross-308	1–10	–	–	0.51	0.95	Aviagen 2019
Ross-308	1–10	Body weight gain	Mash	–	0.81	Ghavi et al. 2020
Ross-308	1–10	Feed efficiency & Breast weight	Mash	–	0.93	Ghavi et al. 2020
Cobb-500	1–14	Body weight gain	–	–	0.73	Faridi et al. 2016
Cobb-500	1–14	Feed conversion ratio	–	–	0.78	Faridi et al. 2016
Ross-708	1–14	Feed conversion ratio	–	–	0.88	Dozier and Mercier 2013
Hubbard	1–14	Feed conversion ratio	–	–	0.86	Dozier and Mercier 2013
Cobb	8–21	Body weight gain	–	–	0.71	Goulart et al. 2011
Cobb	8–21	Feed conversion ratio	–	–	0.76	Goulart et al. 2011
NS	11–21	Growth performance and immunity	Pellet	–	0.8–1.0	Ren et al. 2020
Ross-308	11–24	–	–	0.47	0.87	Aviagen 2019
Ross-308	11–28	Growth performance	Pellet	–	0.78	Agostini et al. 2015
Ross	14–34	Feed conversion ratio and breast meat yield	Pellet	–	0.78	Schutte and Pack 1995
Cobb-500	15–28	Body weight gain	–	–	0.68	Faridi et al. 2016
Cobb-500	15–28	Feed conversion ratio	–	–	0.72	Faridi et al. 2016
Ross-308	25–42	–	–	0.43	0.80	Aviagen 2019
Cobb-500	29–42	Body weight gain	–	–	0.62	Faridi et al. 2016
Cobb	22–35	Body weight gain	–	–	0.75	Goulart et al. 2011
Cobb	22–35	Feed conversion ratio	–	–	0.76	Goulart et al. 2011
Cobb	28–42	Body weight gain	Mash	–	0.67	Pontin et al. 2018
Cobb	28–42	Feed conversion ratio	Mash	–	0.82	Pontin et al. 2018
Cobb	28–42	Breast meat yield	Mash	–	0.83	Pontin et al. 2018
Cobb-500	29–42	Feed conversion ratio	–	–	0.66	Faridi et al. 2016
Cobb	36–42	Body weight gain	–	–	0.66	Goulart et al. 2011
Cobb	36–42	Feed conversion ratio	–	–	0.67	Goulart et al. 2011

–: Not specified.

Abbreviation: Dig Met: digestible methionine; Dig SAAs: digestible sulphur amino acids.

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