Effect of digestible threonine on performance, egg quality, blood metabolites, and immune responses in laying hens fed a wheat-based diet in the second cycle

Figures & data

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

Ingredient, g/kg as fed basis	Basal diet
Wheat (CP = 11.37 %, ME = 3213 kcal/kg)	638.3
Soybean meal (CP = 44.47 %, ME = 2230 kcal/kg	143.6
Corn gluten meal (CP = 53.86 %, ME= 3740 kcal/kg)	35.0
Vegetable oil (ME = 8820 kcal/kg)	29.7
Limestone	122.6
Dicalcium phosphate (Phosphorus = 18.7 %)	17.6
Common salt	3.5
Vitamin premix^b	2.5
Mineral premix^c	2.5
DL-Methionine	1.4
L-Lysine-HCL	2.3
Corn starch	1.0
Determined nutrient composition^d, as-fed basis
Metabolisable energy (ME), kcal/kg	2780
Crude protein (CP), %	15.81
Calcium, %	5.12
Phosphorus (available), %	0.43
Sodium, %	0.19
Digestible lysine, %	0.73
Digestible methionine, %	0.35
Digestible methionine + cystine, %	0.60
Digestible threonine, %	0.44
Digestible threonine/ digestible lysine, %	60

^aThe diets were provided in a way that a batch of basal diet (lowest digestible threonine concentration) was made and then divided into 6 equal portions; the L- threonine (ME = 3570 kcal/kg, CP = 72.4, Threonine = 98%, Evonik Degussa GmbH, Hanau-Wolfgang, Germany) was added at the rate of 0.00, 0.21, 0.41, 0.61, 0.82 and 1.02 g/kg to the top of each portion at the expense of filler (corn starch) and mixed to provide six diets with 0.44, 0.46, 0.48, 0.50, 0.52 and 0.54% digestible threonine levels so as to form ratios of 60, 63, 66, 69, 72 and 75% of dig Thr/dig Lys.

^bVitamin premix supplied the following per kilogram of diet. vitamin A (all-trans-retinol), 4400 IU; vitamin D3 (cholecalciferol), 1000 IU; vitamin E (α-tocopherol), 11 IU; vitamin K3 (menadione), 2.33 mg; vitamin B1 (thiamin), 2.97 mg; vitamin B2 (riboflavin), 4.4 mg; vitamin B3 (niacin), 22 mg; vitamin B5 (pantothenic acid), 10 mg; vitamin B6 (pyridoxine), 4.45 mg; vitamin B9 (folic acid), 1.9 mg; vitamin B12 (cyanocobalamin), 0.011 mg; vitamin H2 (biotin), 0.18 mg; choline chloride, 487.5 mg, antioxidant 1.0 mg.

^cMineral premix supplied the following per kilogram of diet. Zn (zinc oxide), 75 mg; Mn (manganese oxide), 75; Fe (iron sulfate), 75; Cu (copper sulfate), 5; I (ethylene diamine dihydroiodide), 0.76; Se (Sodium Selenite), 0.1; choline chloride, 474.0.

^dThe 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.

ME: metabolisable energy; CP: crude protein.

Table 2. Effect of dietary digestible threonine level on feed and nutrient intake of laying hens during the second cycle (105–116 wk of age)^a.

Items	Feed consumption, g/b/d				ME intake, kcal/b/d				CP intake, g/b/d				Digestible threonine intake, mg/b/d
	105–108 wk	109–112 wk	113–116 wk	Overall	105–108 wk	109–112 wk	113–116 wk	Overall	105–108 wk	109–112 wk	113–116 wk	Overall	105–108 wk	109–112 wk	113–116 wk	Overall
Digestible threonine level,%
0.44	108.67	107.22	106.25	107.38	302	298	295	299	16.96	16.74	16.59	16.76	478	472	467	472
0.46	110.15	110.57	106.38	109.03	306	307	296	303	17.19	17.26	16.61	17.02	507	509	489	502
0.48	106.69	108.99	105.51	107.06	297	303	293	298	16.65	17.01	16.47	16.71	512	523	506	514
0.50	106.87	106.89	101.72	105.16	297	297	283	292	16.68	16.68	15.88	16.42	534	534	509	526
0.52	107.60	107.35	103.43	106.13	299	298	288	295	16.80	16.76	16.15	16.57	560	558	538	552
0.54	107.92	107.61	104.29	106.60	300	299	290	296	16.85	16.80	16.28	16.64	583	581	563	576
SEM	0.997	0.784	1.619	0.667	2.77	2.17	4.52	1.85	0.155	0.122	0.253	0.104	4.91	3.71	7.57	3.17
Regression analysis
Linear	0.268	0.114	0.325	0.125	0.267	0.114	0.327	0.124	0.269	0.114	0.325	0.125	0.001	0.001	0.001	0.001
Quadratic	0.283	0.112	0.349	0.125	0.281	0.112	0.351	0.125	0.285	0.112	0.349	0.126	0.297	0.387	0.346	0.188

^aData are means of 8 replications of 8 hens each.

ME: metabolisable energy; CP: crude protein; SEM: Standard error of the means.

Table 3. Effect of dietary digestible threonine level on productive performance of laying hens during the second cycle (105–116 wk of age)^a.

Items	Egg production, %				Egg mass, g/b/d				Feed conversion ratio, g FI/ g EM				Egg weight, g/egg
	105–108 wk	109–112 wk	113–116 wk	Overall	105–108 wk	109–112 wk	113–116 wk	Overall	105–108 wk	109–112 wk	113–116 wk	Overall	105–108 wk	109–112 wk	113–116 wk	Overall
Digestible threonine level,%
0.44	78.33	77.05	72.10	75.83	51.12	49.38	47.16	49.22	2.137	2.225	2.259	2.207	65.29	63.88	65.40	64.86
0.46	83.04	80.90	75.36	79.77	53.75	52.74	49.90	52.13	2.051	2.103	2.137	2.097	64.73	65.18	66.25	65.39
0.48	83.60	82.07	76.41	80.69	53.54	53.65	50.79	52.66	1.994	2.034	2.081	2.036	64.04	65.38	66.48	65.30
0.50	81.59	78.82	73.89	78.10	53.14	50.70	48.18	50.67	2.014	2.115	2.119	2.083	65.13	64.32	65.22	64.89
0.52	79.47	78.77	74.79	77.67	52.16	52.09	49.53	51.26	2.069	2.069	2.092	2.077	65.64	66.13	66.20	65.99
0.54	80.65	77.99	72.01	76.88	52.79	52.39	48.92	51.37	2.048	2.058	2.132	2.079	65.45	67.16	67.94	66.85
SEM	1.296	1.690	1.221	1.033	1.003	1.430	0.815	0.774	0.039	0.068	0.047	0.038	0.604	0.742	0.591	0.388
Regression analysis
Linear	0.036	0.093	0.009	0.005	0.063	0.061	0.068	0.053	0.031	0.030	0.031	0.022	0.256	0.005	0.037	0.002
Quadratic	0.035	0.091	0.008	0.006	0.066	0.072	0.070	0.056	0.034	0.031	0.035	0.025	0.245	0.569	0.274	0.136

^aData are means of 8 replications of 8 hens each.

FI: feed intake; EM: egg mass; SEM: Standard error of the means.

Figure 1. Fitted plots of egg weight (Y, in g) vs. daily digestible threonine consumption (X, in mg/bird per day) of Hy-line-W36 laying hens fed from 100 to 112 weeks of age.

Figure 2. Fitted plots of hen-day egg production (Y, in %) vs. daily digestible threonine consumption (X, in mg/bird per day) of Hy-line-W36 laying hens fed from 100 to 112 weeks of age. Equations: Y = 78.41–0.0043(507-X)² × I, I = 1 (if X < 507 or I = 0 (if X > 507), p <.0001, adj. R² = 0.54. The break point occurred at 507 ± 8.8.

Figure 3. Fitted plots of egg mass (Y, in g/b/d) vs. daily digestible threonine consumption (X, in mg/bird per day) of Hy-line-W36 laying hens fed from 100 to 112 weeks of age. Equations: Y = 51.50 – 0.0022(514-X)² × I, I = 1 (if X < 514 or I = 0 (if X > 514), p <.0001, adj. R² = 0.59. The break point occurred at 514 ± 9.1.

Figure 4. Fitted plots of feed conversion ratio (Y, in g feed intake/ g egg mass) vs. daily digestible threonine consumption (X, in mg/bird per day) of Hy-line-W36 laying hens fed from 100 to 112 weeks of age. Equations: Y = 2.08 + 0.000074(520 – X)² × I, I = 1 (if X < 520 or I = 0 (if X > 520), p <.0001, adj. R² = 0.45. The break point occurred at 520 ± 13.3.

Table 4. Estimated digestible threonine requirements (mg/bird per day) for optimisation egg production parameters in laying hens during the second cycle (105–116 wk of age) by quadratic broken-line methodology^a.

Parameters	Requirement^b	Approximate confidence limits		P-Value	R^2	Adj. R^2	Predicted value^c	RMSE	AIC	Equation^d
		Lower 95%	Upper 95%
Egg production, %
105–108 wk	492	478	506	0.0001	0.36	0.35	81.09	3.62	136	Y = 81.09 − 0.0183(R-X)^2 × I
109–112 wk	517	459	574	0.0023	0.24	0.22	79.84	4.23	148	Y = 79.84 − 0.00248(R-X)^2 × I
113–116 wk	512	465	558	0.0001	0.37	0.36	74.24	3.56	135	Y = 74.24 − 0.00164(R-X)^2 × I
Overall	507	489	525	0.0001	0.55	0.54	78.41	2.29	107	Y = 78.41 − 0.0043(R-X)^2 × I
Egg mass, g/b/d
105–108 wk	492	477	209	0.0002	0.31	0.29	52.71	2.49	111	Y = 52.71 − 0.0105(R-X)^2 × I
109–112 wk	527	462	592	0.0059	0.20	0.19	52.37	3.42	132	Y = 52.37 − 0.0013(R-X)^2 × I
113–116 wk	523	478	567	0.0001	0.44	0.43	49.49	2.42	111	Y = 49.49 − 0.001(R-X)^2 × I
Overall	514	496	532	0.0001	0.60	0.59	51.50	1.33	86	Y = 51.50 − 0.0022(R-X)^2 × I
Feed conversion ratio
105–108 wk	490	472	507	0.0010	0.27	0.25	2.058	0.09	67	Y = 2.0577 + 0.000514 (R-X)^2 × I
109–112 wk	540	455	625	0.0268	0.15	0.13	2.073	0.15	67	Y = 2.073 + 0.000033(R-X)^2 × I
113–116 wk	546	436	655	0.0192	0.16	0.14	2.128	0.13	67	Y = 2.128 + 0.000017(R-X)^2 × I
Overall	520	493	547	0.0001	0.46	0.45	2.080	0.07	67	Y = 2.08 + 0.000074(R-X)^2 × I

^aResponses did not fit in the models to estimate the digestible threonine requirements for egg weight

^bExpressed as mg/bird per day.

^cPredicted values by models to obtain optimum egg production (%); optimum egg mass (g/bird per day); and optimum feed conversion ratio.

^dI = 1 (if X < R or I = 0 (if X > R).

R: requirement; R²: coefficient of determination; Adj. R²: adjusted coefficient of determination; RMSE: root means square error; AIC: Akaike’s information criterion; RBW.

Table 5. Effect of dietary digestible threonine level on egg qualitative traits of laying hens during the second cycle (105–116 wk of age)^a.

Items	Albumen, %				Yolk, %				Shell, %
	108 wk	112 wk	116 wk	Overall	108 wk	112 wk	116 wk	Overall	108 wk	112 wk	116 wk	Overall
Digestible threonine level, %
0.44	64.04	63.02	62.84	63.30	25.80	27.28	27.10	26.73	10.15	9.70	10.06	9.97
0.46	63.56	63.41	63.25	63.41	26.51	26.75	26.58	26.61	9.93	9.84	10.17	9.98
0.48	63.84	63.85	63.13	63.61	26.73	26.51	26.85	26.70	9.42	9.64	10.02	9.69
0.50	63.07	64.05	62.69	63.27	26.29	26.31	27.06	26.55	10.64	9.63	10.25	10.17
0.52	63.75	64.26	63.85	63.95	26.10	26.27	26.10	26.16	10.15	9.47	10.05	9.89
0.54	63.41	64.01	62.76	63.39	26.89	26.64	26.95	26.82	9.70	9.35	10.30	9.78
SEM	0.555	0.592	0.541	0.359	0.437	0.564	0.442	0.288	0.217	0.225	0.251	0.159
Regression analysis
Linear	0.461	0.098	0.845	0.553	0.318	0.272	0.632	0.667	0.848	0.109	0.646	0.581
Quadratic	0.643	0.414	0.651	0.660	0.757	0.277	0.667	0.458	0.648	0.560	0.834	0.704

^aData are means of 8 replications of 6 eggs each.

SEM: Standard error of the means.

Table 6. Effect of dietary digestible threonine level on egg qualitative traits of of laying hens during the second cycle (105–116 wk of age)^a.

Items	Egg shape index (%)				Haugh unit^2				Specific gravity (g/cm^3)				Shell thickness (mm)
	108 wk	112 wk	116 wk	Overall	108 wk	112 wk	116 wk	Overall	108 wk	112 wk	116 wk	Overall	108 wk	112 wk	116 wk	Overall
Digestible threonine level,%
0.44	77.47	77.21	75.96	76.88	70.90	82.82	75.12	76.28	1.082	1.078	1.077	1.079	338	332	344	338
0.46	77.67	75.60	77.16	76.81	77.38	79.58	75.51	77.49	1.082	1.080	1.077	1.080	334	339	359	344
0.48	76.47	75.85	77.04	76.45	82.58	78.23	76.51	79.11	1.076	1.078	1.073	1.076	321	336	330	329
0.50	77.07	77.11	77.29	77.16	84.27	81.56	74.94	80.26	1.084	1.078	1.078	1.080	348	329	359	345
0.52	77.92	77.15	77.87	77.65	82.26	80.32	74.47	79.02	1.081	1.077	1.078	1.079	338	333	354	342
0.54	77.10	77.13	77.27	77.17	81.56	79.66	74.16	78.46	1.078	1.076	1.076	1.077	322	328	349	333
SEM	0.697	0.529	0.622	0.423	3.563	2.498	1.537	1.479	0.002	0.002	0.001	0.001	9.127	8.519	7.608	5.225
Regression analysis
Linear	0.934	0.238	0.084	0.303	0.504	0.999	0.507	0.421	0.191	0.102	0.743	0.293	0.636	0.499	0.932	0.751
Quadratic	0.641	0.165	0.267	0.735	0.043	0.923	0.641	0.155	0.630	0.452	0.466	0.981	0.591	0.669	0.945	0.582

^aData are means of 8 replications of 6 eggs each.

SEM: Standard error of the means.

Table 7. Effect of dietary digestible threonine level on blood metabolites of laying hens determined at the end of the experiment (116 wk of age)^a.

Items	Calcium, mg/dL	Phosphorous, mg/dL	Uric acid, mg/dL	Total protein, g/dL	ALP, U/L
Digestible threonine level,%
0.44	15.94	6.04	6.92	6.02	358.48
0.46	16.38	5.62	7.52	5.74	410.40
0.48	16.06	5.60	6.92	5.74	544.80
0.50	17.06	6.04	7.50	5.60	362.60
0.52	16.94	5.78	7.34	6.02	327.80
0.54	15.64	5.72	6.60	5.72	443.20
SEM	0.531	0.246	0.238	0.206	75.286
Regression analysis
Linear	0.788	0.730	0.558	0.638	0.992
Quadratic	0.109	0.697	0.098	0.399	0.608

^aData are means of 8 replications of 2 blood samples, each.

ALP: alkaline phosphatase; SEM: Standard error of the means.

Table 8. Effect of dietary digestible threonine levels on antibody titres responses to sheep red blood cell (SRBC) inoculation, cutaneous basophil hypersensitivity (CBH) responses elicited by phytohemagglutinin-P (PHA-M) intradermal injection, of laying hens determined 115–116 wk of age^a.

Items	Antibody concentration responses to SRBC inoculation at 115 and 116 wk of age (log^2)						CBH-responses^b (µm)
	7d after the first injection			7d after the second injection			Hours Post-injection
	Total Antibody^c	IgY^c	IgM^c	Total Antibody^c	IgY^c	IgM^c	6	12	18	24
Dietary digestible threonine level, %
0.44	7.20	6.60	0.60	9.20	8.40	0.80	1.08	0.65	0.71	0.72
0.46	7.60	7.20	0.40	8.80	7.60	1.20	1.24	0.85	0.96	0.86
0.48	6.60	5.60	1.00	8.60	7.60	1.00	1.33	1.02	0.97	0.81
0.50	7.00	6.60	0.40	8.00	7.40	0.60	1.11	1.05	1.08	1.03
0.52	7.00	6.20	0.80	9.20	8.00	1.20	1.64	1.03	1.15	1.02
0.54	6.60	6.20	0.40	8.40	7.60	0.80	1.19	0.98	0.89	0.83
SEM	0.584	0.495	0.217	0.383	0.467	0.268	0.158	0.103	0.123	0.110
Regression analysis
Linear	0.360	0.357	0.837	0.322	0.407	0.865	0.284	0.012	0.150	0.181
Quadratic	0.995	0.703	0.399	0.338	0.269	0.757	0.401	0.084	0.087	0.223

^aThe values are means of 8 replicates of 2 samples each.

^bCBH = (skin thickness, PHA-injected foot) – (skin thickness, PBS-injected foot).

^cData represent means of log2 of the reciprocal of the last dilution exhibiting agglutination.

SEM: Standard error of the means.

AOAC. 2002. Official methods of analysis. 15th ed. Arlington (VA): Association of Official Analytical Chemists.

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