Alpha-tocopherol acetate and alpha lipoic acid may mitigate the development of wooden breast myopathy in broilers at an early age

Figures & data

Table 1. Feed ingredients and calculated nutritional composition of starter diets^1.

Item	Control	ATA	ALA	ATA and ALA
Ingredients, g/kg
Corn	514.0	513.9	513.5	513.4
Soybean meal	336.2	336.2	336.2	336.2
Poultry byproduct meal	75.0	75.0	75.0	75.0
Sodium chloride	2.2	2.2	2.2	2.2
Limestone	11.0	11.0	11.0	11.0
Dicalcium phosphate	4.6	4.6	4.6	4.6
Premix^2	3.5	3.5	3.5	3.5
L-Lys HCL	1.5	1.5	1.5	1.5
DL-Met	3.4	3.4	3.4	3.4
L-Thr	1.1	1.1	1.1	1.1
NaHCO3	1.0	1.0	1.0	1.0
Selenium^3	1.0	1.0	1.0	1.0
Amprolium	10.0	10.0	10.0	10.0
Dl-alpha-tocopherol acetate	-	0.16	-	0.16
Soy oil	35.5	35.5	35.5	35.5
ALA	-	-	0.5	0.5
Calculated Nutrients and energy
AME, kcal/kg	3000	3000	3000	3000
Protein, g/kg	237.3	237.3	237.3	237.2
Calcium, g/kg	9.6	9.6	9.6	9.6
Available phosphorus, g/kg	4.8	4.8	4.8	4.8
Digestible Lys^4, g/kg	12.8	12.8	12.8	12.8
Digestible Met+Cys, g/kg	9.5	9.5	9.5	9.5
Dl-alpha-tocopherol acetate, mg/kg	53.0	133.0	53.0	133.0

¹Broilers in the control group were fed corn-soybean meal basal diet. Alpha-tocopherol acetate (ATA; 160 mg/kg) and alpha lipoic acid (ALA; 500 mg/kg) were supplemented independently and in combination in the dietary treatments during the starter phase (0 to 7 d). ²The premix provides the following per kg of diet: retinol, 5 mg; cholecalciferol, 0.15 mg; alpha-tocopherol, 11 mg; cyanocobalamin, 11 µg; folic acid, 1.5 mg; biotin, 150 µg; calcium carbonate, 25 mg; Mn, 60 mg; Zn, 40 mg; I, 0.33 mg; Fe, 80 mg; Cu, 8 mg; Se, 0.15 mg; and ethoxyquin, 150 mg (Provimi North America, Brookville, OH). ³Selenium provided 0.2 mg selenium (as Na₂SeO₃) per kg of diet. Total selenium content in feed was 0.2 mg/kg. ⁴The calculated digestible Arg/Lys was 1.13, Ile/Lys was 0.68, Met/Lys was 0.52, and Thr/Lys was 0.67.

Table 2. Feed ingredients and calculated nutritional composition of grower diets^1.

Item	Control	ATA	ALA	ATA and ALA
Ingredients, g/kg
Corn	565.6	565.4	565.1	564.9
Soybean meal	280.6	280.6	280.6	280.6
Poultry byproduct meal	75.0	75.0	75.0	75.0
Sodium chloride	2.3	2.3	2.3	2.3
Limestone	10.2	10.2	10.2	10.2
Dicalcium phosphate	2.9	2.9	2.9	2.9
Premix^2	3.5	3.5	3.5	3.5
L-Lys HCL	1.5	1.5	1.5	1.5
DL-Met	3.0	3.0	3.0	3.0
L-Thr	0.9	0.9	0.9	0.9
NaHCO3	1.0	1.0	1.0	1.0
Selenium^3	1.0	1.0	1.0	1.0
Amprolium	10.0	10.0	10.0	10.0
Dl-alpha-tocopherol acetate	-	0.16	-	0.16
Soy oil	42.5	4.25	4.25	4.25
ALA	-	-	0.5	0.5
Calculated Nutrients and energy
AME, kcal/kg	3102	3102	3100	3100
Protein, g/kg	215.6	215.6	215.6	215.6
Calcium, g/kg	8.7	8.7	8.7	8.7
Available phosphorus, g/kg	4.4	4.4	4.4	4.4
Digestible Lys^4, g/kg	11.5	11.5	11.5	11.5
Digestible Met+Cys, g/kg	8.7	8.7	8.7	8.7
Dl-alpha-tocopherol acetate, mg/kg	54.1	134.1	54.1	134.1

¹Broilers in the control group were fed corn-soybean meal basal diet. Alpha-tocopherol acetate (ATA; 160 mg/kg) and alpha lipoic acid (ALA; 500 mg/kg) were supplemented independently and in combination in the dietary treatments during the grower phase (8 to 21 d). ²The premix provides the following per kg of diet: retinol, 5 mg; cholecalciferol, 0.15 mg; alpha-tocopherol, 11 mg; cyanocobalamin, 11 µg; folic acid, 1.5 mg; biotin, 150 µg; calcium carbonate, 25 mg; Mn, 60 mg; Zn, 40 mg; I, 0.33 mg; Fe, 80 mg; Cu, 8 mg; Se, 0.15 mg; and ethoxyquin, 150 mg (Provimi North America, Brookville, OH). ³Selenium provided 0.2 mg selenium (as Na₂SeO₃) per kg of diet. Total selenium content in feed was 0.2 mg/kg. ⁴The calculated digestible Arg/Lys was 1.12, Ile/Lys was 0.68, Met/Lys was 0.53, and Thr/Lys was 0.67.

Table 3. Primer sequences for real-time quantitative PCR

Gene	Accession number	Forward primer	Reverse primer	Amplicon (bp) size
Muscle formation and growth
MYOD1^1	AY641567.1	5’--GACGGCATGATGGAGTACAG-3’	5’-AGCTTCAGCTGGAGGCAGTA-3’	234
MYOG ^2	AY560111.3	5’-CCTTTCCCACTCCTCTCCAAA-3’	5’-GACCTTGGTCGAAGAGCAACT-3’	201
Adipogenesis
PPARγ^3	NM_001001460.1	5’-CCACTGCAGGAACAGAACAA-3’	5’-CTCCCGTGTCATGAATCCTT-3′	249
CEBPα^4	NM_001031459.1	5’-CAGTGGACAAGAACAGCAACGA-3′	5’-CCTTCACCAGCGAGCTTTCG-3′	227
Extracellular matrix
SDC4^5	NM_001007869.1	5’-CCAACAGCAGCATCTTTGAA-3’	5’-GATGGGTTTCTTCCCAAGGT-3’	155
Oxidative stress and inflammation
SELE^6	XM_025153162	5’-CAACAGAGAGCAGTCGCTGA-3’	5’-GTAAGCCCGTAAGCAAGAAGAG-3’	157
Housekeeping gene
GAPDH^7	U94327.1	5’-GAGGGTAGTGAAGGCTGCTG-3’	5’-CCACAACACGGTTGCTGTAT-3’	175

^aMYOD1 = Myogenic determination factor 1. ²MYOG = Myogenin. ³PPARγ = Peroxisome proliferator-activated receptor gamma. ⁴CEBPα = CCAAT/enhancer-binding protein alpha. ⁵SDC4 = Syndecan-4. ⁶SELE = Selectin E. ⁷GAPDH = Glyceraldehyde-3-phosphate dehydrogenase

Table 4. Effect of alpha-tocopherol acetate and alpha lipoic acid on broiler growth performance from 1 to 3 weeks post-hatch

Item	Age (weeks)	Treatments^1				SEM^2	P-value^3 (ATA)	P-value (ALA)	P-value (ATA and ALA)	P-value(Treatment)
		Control	ATA	ALA	ATA and ALA
AWG^4 (g)	1	94.62	94.81	93.52	93.51	1.969	0.99	0.98	0.98	0.95
	2	178.30	178.83	180.2	179.11	4.527	0.99	0.99	0.99	0.99
	3	207.10	205.45	208.87	213.01	5.607	0.99	0.99	0.89	0.82
FI^5 (g)	1	101.45	103.10	98.85	103.08	1.221	0.85	0.46	0.79	0.06
	2	272.47	276.01	268.96	276.19	2.434	0.78	0.75	0.72	0.13
	3	367.61^a	349.99^b	352.40^b	370.53^a	3.161	< 0.01	0.01	0.92	< 0.01
FCR^6	1	1.06	1.10	1.06	1.11	0.026	0.65	0.99	0.27	0.21
	2	1.53	1.58	1.53	1.57	0.044	0.87	0.99	0.93	0.79
	3	1.80	1.72	1.73	1.78	0.042	0.60	0.66	0.99	0.52
Final body weight (g)	1	143.03	138.96	136.51	147.09	4.043	0.48	0.26	0.48	0.28
	2	338.56	362.76	346.71	342.61	11.754	0.15	0.64	0.81	0.50
	3	507.72	498.7	517.11	507.19	12.993	0.64	0.62	0.98	0.79
P. major^7 weight (g)	1	10.30	9.70	8.56	10.14	0.501	0.45	0.07	0.84	0.09
	2	27.86	31.98	31.14	30.46	1.942	0.15	0.25	0.36	0.51
	3	62.22	57.76	61.18	62.66	2.359	0.19	0.76	0.90	0.46

^a,bValues within a row without a common letter are significantly different (P ≤ 0.05). ¹Broilers in the control group were fed corn-soybean meal basal diet. Alpha-tocopherol acetate (ATA; 160 mg/kg) and alpha lipoic acid (ALA; 500 mg/kg) were supplemented independently and in combination in the dietary treatments during the entire study (1 to 3 weeks). ²SEM = Standard error of means. ³Effects of ATA, ALA, or combination of ATA and ALA were determined by setting up orthogonal contrasts using data from each treatment to test the responses of the three treatments against the control group. ⁴AWG = Average weekly gain per bird. ⁵FI = Feed intake per week per bird.

⁶FCR = Feed conversion ratio per bird. ⁷P. major = Pectoralis major muscle per bird.

Table 5. Effect of alpha-tocopherol acetate and alpha lipoic acid on microscopic wooden breast (WB) score of broiler Pectoralis major muscle

		Treatments^1, n (%)^2
Age (weeks)	WB score^3	Control	ATA	ALA	ATA and ALA	P-value^4 (ATA)	P-value (ALA)	P-value (ATA and ALA)	P-value (Treatment)
1	0	5 (50)	6 (60)	5 (50)	6 (60)	0.65	-	0.65	0.94
	1	5 (50)	4 (40)	5 (50)	4 (40)
	2	0	0	0	0
	3	0	0	0	0
2	0	2 (20)	5 (50)	5 (50)	7 (70)	0.19	0.19	0.05	0.14
	1	6 (60)	5 (50)	5 (50)	3 (30)
	2	2 (20)	0	0	0
	3	0	0	0	0
3	0	1 (10)	6 (60)	5 (50)	6 (60)	0.05	0.11	0.05	0.18
	1	8 (80)	4 (40)	5 (50)	4 (40)
	2	1 (10)	0	0	0
	3	0	0	0	0

^aBroilers in the control group were fed corn-soybean meal basal diet. Alpha-tocopherol acetate (ATA; 160 mg/kg) and alpha lipoic acid (ALA; 500 mg/kg) were supplemented independently and in combination in the dietary treatments during the entire study (1 to 3 wk).

^bThe number (percentage) of the broilers with different WB score at each age within each dietary treatment. ³The WB scores were based on the degree of fibrosis, necrosis, and immune cell infiltration. Score zero = none WB, one = mild WB, two = moderate WB, three = severe WB. ⁴Effects of ATA, ALA, or combination of ATA and ALA were determined by setting up orthogonal contrasts using data from each treatment to test the responses of the three treatments against the control group.

Figure 1. Representative photomicrographs of wooden breast affected (a,c,e,g) or not affected (b,d,f,h) Pectoralis major muscle samples in the four treatments at 3 weeks of age. Broilers in the control group (a,b) were fed corn-soybean meal basal diet. Alpha-tocopherol acetate (ATA; 160 mg/kg) was supplemented in the ATA group (c,d) and alpha lipoic acid (ALA; 500 mg/kg) was supplemented in the ALA group (e,f). Combination of ATA and ALA was supplemented in the ATA and ALA group (g,h). C = Collagen; F = Fat cell; H = Hypercontracted myofiber; P = Perimysial connective tissue; and E = Endomysial connective tissue. Scale bar = 100 μm

Table 6. Effect of alpha-tocopherol acetate and alpha lipoic acid on fibre width, perimysial and endomysial connective tissue spacing, and morphology score of broiler Pectoralis major muscle

		Treatments^1
Item	Age (weeks)	Control	ATA	ALA	ATA and ALA	SEM^2	P-value^3 (ATA)	P-value (ALA)	P-value (ATA and ALA)	P-value (Treatment)
Fibre width (μm)	1	5.39	5.29	5.14	5.05	0.164	0.71	0.33	0.20	0.55
	2	8.35	8.98	9.07	9.58	0.364	0.23	0.17	0.08	0.15
	3	9.72	10.09	10.61	10.39	0.378	0.82	0.35	0.60	0.73
Perimysium (μm)	1	9.07	9.17	8.99	8.86	0.300	0.85	0.87	0.65	0.92
	2	8.48	8.74	8.57	8.65	0.423	0.68	0.89	0.78	0.98
	3	7.51	7.47	7.49	7.77	0.344	0.79	0.76	0.38	0.84
Endomysium (μm)	1	2.39	2.48	2.30	2.62	0.128	0.67	0.66	0.26	0.36
	2	2.31	2.20	2.28	2.04	0.089	0.44	0.80	0.06	0.20
	3	2.68	2.60	2.65	2.65	0.142	0.53	0.71	0.71	0.94
Morphology score^4	1	1.87	2.29	2.05	2.27	0.132	0.09	0.44	0.11	0.14
	2	2.14	2.48	2.51	2.23	0.141	0.16	0.13	0.72	0.18
	3	2.65	2.90	3.04	2.61	0.179	0.36	0.27	0.74	0.51

¹Broilers in the control group were fed corn-soybean meal basal diet. Alpha-tocopherol acetate (ATA; 160 mg/kg) and alpha lipoic acid (ALA; 500 mg/kg) were supplemented independently and in combination in the dietary treatments during the entire study (1 to 3 weeks). ²SEM = Standard error of means. ³Effects of ATA, ALA, or combination of ATA and ALA were determined by setting up orthogonal contrasts using data from each treatment to test the responses of the three treatments against the control group. ⁴Morphology score was evaluated using a one to five scale by three trained panelists. A score of one was given to samples with limited or no perimysial or endomysial connective tissue spacing, and excessive myofiber degradation and necrosis. A score of five was given to samples with well-structured muscle fibre bundles and myofibers with ample perimysial and endomysial connective tissue spacing. Scores of two to four were intermediate.

Table 7. Effect of alpha-tocopherol acetate and alpha lipoic acid on gene expression in broiler Pectoralis major muscle

		Treatments^1
Item (arbitrary unit)	Age (weeks)	Control	ATA	ALA	ATA and ALA	SEM^2	P-value^3 (ATA)	P-value (ALA)	P-value (ATA and ALA)	P-value (Treatment)
Muscle formation and growth
MYOD1^4	1	16.30	15.02	16.24	16.51	0.608	0.37	0.21	0.16	0.29
	2	15.80	14.38	15.12	14.39	0.582	0.14	0.64	0.62	0.45
	3	11.26^a	10.56^b	10.33^bc	9.91 ^c	0.320	0.01	0.01	0.51	< 0.01
MYOG ^5	1	0.79	0.66	0.73	0.79	0.069	0.71	0.56	0.16	0.47
	2	2.21	2.12	2.02	2.17	0.099	0.81	0.51	0.30	0.66
	3	1.42^a	1.26^ab	1.03^bc	0.86 ^c	0.092	0.12	0.01	0.98	< 0.01
Adipogenesis
PPARγ^6	1	0.049	0.039	0.051	0.048	0.0058	0.26	0.25	0.44	0.36
	2	0.047^a	0.029^b	0.036^b	0.031^b	0.0029	<0.01	0.12	0.03	< 0.01
	3	0.027^a	0.018^b	0.007 ^c	0.013^bc	0.0021	0.29	<0.01	0.01	< 0.01
CEBPα^7	1	0.030	0.025	0.030	0.025	0.0025	0.06	0.98	0.94	0.31
	2	0.030^a	0.027^b	0.028^b	0.029^a	0.0005	0.05	0.64	<0.01	< 0.01
	3	0.013^a	0.014^a	0.010^b	0.010^b	0.0008	0.04	<0.01	0.06	< 0.01
Extracellular matrix
SDC4^8	1	0.039	0.036	0.028	0.043	0.0094	0.51	0.90	0.34	0.71
	2	0.098	0.084	0.095	0.110	0.0130	0.84	0.52	0.36	0.72
	3	0.180	0.130	0.180	0.130	0.0320	0.11	0.97	0.98	0.43
Oxidative stress and inflammation
SELE^9	1	0.048	0.053	0.042	0.039	0.0072	0.87	0.07	0.50	0.29
	2	0.061^a	0.064^a	0.054^b	0.058^ab	0.0018	0.17	0.01	0.85	0.02
	3	0.009^a	0.009^a	0.005^b	0.010^a	0.0014	< 0.01	0.01	< 0.01	< 0.01

^a-cMeans within a row without a common letter are significantly different (P ≤ 0.05). ¹Broilers in the control group were fed corn-soybean meal basal diet. Alpha-tocopherol acetate (ATA; 160 mg/kg) and alpha lipoic acid (ALA; 500 mg/kg) were supplemented independently and in combination in the dietary treatments during the entire study (1 to 3 weeks). ²SEM = Standard error of means. ³Effects of ATA, ALA, or combination of ATA and ALA were determined by setting up orthogonal contrasts using data from each treatment to test the responses of the three treatments against the control group. ⁴MYOD1 = Myogenic determination factor 1. ⁵MYOG = Myogenin. ⁶PPARγ = Peroxisome proliferator-activated receptor gamma. ⁷CEBPα = CCAAT/enhancer-binding protein alpha. ⁸SDC4 = Syndecan-4. ⁹SELE = Selectin E.