Effect of specific sound frequency on production performance, egg quality and physiological characteristics of laying hens

Figures & data

Table 1. Composition and calculated analysis (% as fed) of the diet for laying hens.

Ingredients^a	%
Corn	57.0
Soybean meal, CP 44%	21.6
WB	0
FWBMP	0
Phytase, unit	0
Fish meal, CP 65%	3.0
DDGS	4.7
Full fat soybean meal	1.2
Soybean oil	2.0
Limestone	7.3
Dicalcium phosphate	2.6
dl-Methionine	0.13
Salt	0.15
Choline chloride	0.07
Vitamin premix^b	0.125
Mineral premix^b	0.125
Total	100.0
Calculated nutrient value
ME, kcal/kg	2850.0
Dry matter, %	88.5
Crude protein, %	18.0
Crude fat, %	5.58
Calcium, %	3.56
Total phosphorus, %	0.86
Available phosphorus, %	0.68
Lysine, %	0.93
Methionine, %	0.46
Cysteine, %	0.28
Analysed nutrient value
Dry matter, %	89.2
Crude protein, %	18.1
Total phosphorus, %	0.81

^a All groups were fed the basal diet (corn–soybean meal).

^b Diet composition per kilogram of feed: vitamin A, 8000 IU; vitamin D, 1500 IU; riboflavin, 4 mg; cobalamin, 10 μg; vitamin E, 15 mg; vitamin K, 2 mg; choline, 500 mg; niacin, 25 mg; manganese, 60 mg; zinc, 50 mg; iron, 50 mg; copper, 3 mg; selenium, 0.26 mg.

CP: Crude protein.

Table 2. Characteristics and performance data of the primers used for qPCR analysis.

Gene name	Primer sequence^a	Tm^b	Size^c	GenBank no.
β-actin	F: 5′-CTGGCACCTAGCACAATGAA-3′	56.4	464	X00182.1
	R: 5′-ACATCTGCTGGAAGGTGGAC-3′
NF-κB	F: 5′-GAAGGAATCGTACCGGGAACA-3′	58.5	131	D13719.1
	R: 5′-CTCAGAGGGCCTTGTGACAGTAA-3′
iNOS	F: 5′-AGCATAACTCCCGTGTTCCA-3′	56.4	239	NM_204961.2
	R: 5′-GATTTCCCAGTCTCGGTTGC-3′
TNF-α	F: 5′-TGTGTATGTGCAGCAACCCGTAGT-3′	57.7	229	NM_204267.2
	R: 5′-GGCATTGCAATTTGGACAGAAGT-3′
ZO-1	F: 5′-AGGTGAAGTGTTTCGGGTTG-3′	56.4	171	XM_015278975.4
	R: 5′-CCTCCTGCTGTCTTTGGAAG-3′
Occludin	F: 5′-GTCTGTGGGTTCCTCATCGT-3′	57.4	156	NM_205128.1
	R: 5′-GTTCTTCACCCACTCCTCCA-3′

NF-κB: nuclear factor kappa-light-chain-enhancer of activated B cells p50; iNOS: inducible nitric oxide synthase; TNF-α: tumour necrosis factor-alpha; ZO-1: zonula occludens-1.

^a For each gene, the primer sequence for forward and reverse 5′ to 3′.

^b Tm, the melting temperature in °C.

^c The product size (size, bp).

Table 3. Effects of specific sound frequency on the production performance of laying.

Items	Treatments^1				SEM	p Values
	Control	Violin	Bass	Mozart
1–12 weeks
Feed intake, g/bird/day	219.5	219.0	217. 8	219.6	1.01	.592
Laying rate, %	82.8	86.8	84.7	79.3	1.84	.072
Egg mass, g/egg	51.9^a	52.9^a	55.4^a	45.5^b	1.36	.002
FCR^2	2.4^ab	2.3^b	2.3^b	2.6^a	0.06	.037

Each value represents the mean with standard error of mean (n = 4). Means within the same row with different letters (a, b) are significantly different (p < .05).

¹ Control: background sound; Violin: 3969.03 Hz; Bass: 41.39 Hz; Mozart: K. 448.

² FCR: feed conversion rate.

Table 4. Effects of specific sound frequency on the egg quality of laying hens.

Items	Treatments^1				SEM	p Values
	Control	Violin	Bass	Mozart
1–12 weeks
Egg weight, g	62.6	61.1	62.0	61.5	0.77	.614
Eggshell strength, kg/cm^2	3.9	3.9	3.9	3.9	0.15	.991
Shell thickness, mm	0.4	0.4	0.4	0.4	0.010	.443
Thick albumin height, mm	7.1^b	7.8^a	7.3^ab	7.5^ab	0.17	.049
Haugh unit, HU	83.0^b	87.6^a	84.9^b	86.1^a	0.77	.008
Yolk colour score	6.7	6.6	6.5	6.7	0.08	.495
Yolk height, mm	17.3	17.6	17.4	17.5	0.12	.514
Yolk width, mm	41.8	41.5	41.5	42.0	0.23	.376
Yolk index	0.405^c	0.438^a	0.427^b	0.426^b	0.003	< .001

Each value represents the mean with standard error of mean (n = 4). Means within the same row with different letters (^a–c) are significantly different (p < .05).

¹ Control: background sound; Violin: 3969.03 Hz; Bass: 41.39 Hz; Mozart: K. 448.

Table 5. Effects of specific sound frequency on blood biochemistry of laying hens.

Items	Treatments^1				SEM	p Values
	Control	Violin	Bass	Mozart
GLU, mg/dL	243	245.7	236.7	243.5	7.02	.822
BUN, mg/dL	2.0	1.8	1.8	1.8	0.14	.801
CREA, mg/dL	0.16	0.18	0.16	0.15	0.01	.597
UA, mg/dL	3.9	4.7	4.1	4.3	0.32	.430
SGOT, U/L	157.3	147.7	173.2	154.5	6.89	.093
T-P, g/dL	5.3	4.8	5.0	5.1	0.18	.436
ALB, g/dL	2.1	2.0	2.1	2.3	0.59	.104
GLO, g/dL	3.2	2.8	2.9	2.9	0.17	.521
Alk-P, IU/L	492.2	452.7	416.5	402.7	80.49	.862
CHOL, mg/dL	99.8	84.0	89.5	100.8	12	.711
TG, mg/dL	1039.0	763.0	867.8	1286.2	182.94	.230
HDL-C, mg/dL	24.0	29.2	31.0	27.8	1.71	.054
LDL-C, mg/dL	12.5	12.2	12.8	9.2	1.1	.104

GLU: glucose; BUN: blood urea nitrogen; UA: urea; SGOT: serum glutamic-oxaloacetic transaminase; SGPT: serum glutamic-pyruvic transaminase; T-P: total protein; ALB: albumin; GLO: globulin; AlkP: alkaline phosphatase; CHOL: cholesterol; HDL-C: high-density lipoprotein cholesterol; LDL-C: low-density lipoprotein cholesterol.

Each value represents the mean with standard error of mean (n = 6).

¹ Control: background sound; Violin: 3969.03 Hz; Bass: 41.39 Hz; Mozart: K. 448.

Figure 1. The effects of specific sound frequency on the mRNA expression levels of antioxidant-regulated genes in laying hens were investigated. The mRNA expression levels of NF-κB (A), iNOS (B), TNF-α (C), Occludin (D) and ZO-1 (E) were examined and exhibited. ^a–cMeans within the same row with different letters are significantly different (p < .05).

Table 6. Effects of specific sound frequency on jejunum–ileum’s and serum’s antioxidant activity of laying hens.

Items	Treatments^1				SEM	p Values
	Control	Violin	Bass	Mozart
Jejunum–ileum
MDA, µM	105.0	79.8	89.8	82.1	6.85	.089
SOD, U/mg protein	17.4	39.2	45.1	32.7	5.91	.050
CAT, nmol/min/mg protein	119.5	139.9	144.4	105.5	21.40	.568
GSH-Px, nmol/min/mg protein	14.7^b	44.1^a	38.5^a	34.7^a	4.73	.005
Serum
MDA, µM	32.2	23.7	39.4	25.9	4.56	.100
SOD, U/mg protein	8.9^b	18.4^a	12.9^ab	17.7^a	2.41	.039
CAT, nmol/min/mg protein	0.50	0.34	0.38	0.42	0.10	.698
GSH-Px, nmol/min/mg protein	4.4^b	6.9^a	7.5^a	7.6^a	0.60	.004

Each value represents the mean with standard error of mean (n = 4). Means within the same row with different letters (a, b) are significantly different (p < .05). MDA, malondialdehyde; SOD, superoxide dismutase; CAT, catalase; GSH-Px, glutathione peroxidase.

¹ Control: background sound; Violin: 3969.03 Hz; Bass: 41.39 Hz; Mozart: K. 448.

Table 7. Effects of specific sound frequency on jejunum–ileum’s immunity of laying hens.

Items	Treatments^1				SEM	p Values
	Control	Violin	Bass	Mozart
TNF-α, pg/mg protein	2.5	1.8	2.3	2.4	0.80	.927
IL-1β, pg/mg protein	1762.1^a	1163.9^bc	1052.5^c	1545.0^ab	135.95	.020
IL-6, pg/mg protein	3751.3^a	2505.9^bc	2388.9^c	3083.0^b	199.50	.005
IL-10, pg/mg protein	690.2	533.1	461.9	626.1	63.24	.130

Each value represents the mean with standard error of mean (n = 4). Means within the same row with different letters (a–c) are significantly different (p < .05). TNF-α, tumor necrosis factor alpha; IL-1β, interleukin-1β; IL-6, interleukin-6; IL-10, interleukin-10.

¹ Control: background sound; Violin: 3969.03 Hz; Bass: 41.39 Hz; Mozart: K. 448.

Table 8. Effects of specific sound frequency on jejunum–ileum’s transcription factors of laying hens.

Items	Treatments^a				SEM	p Values
	Control	Violin	Bass	Mozart
Nrf2 (ADU)^b	0.062	0.050	0.052	0.058	0.0066	.547
NF-κB (ADU)	0.015	0.016	0.015	0.016	0.0009	.799

Each value represents the mean with standard error of mean (n = 4). Nrf2, nuclear factor erythroid 2-related factor 2; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells.

^a Control: background sound; Violin: 3969.03 Hz; Bass: 41.39 Hz; Mozart: K. 448.

^b ADU: arbitrary densitometry units.

Figure 2. The jejunum–ileum intestinal morphology of laying hens were examined, with presentations of the control group (A), Violin group (B), Bass group (C) and Mozart group (D) jejunum–ileum intestines.

Table 9. Effect of specific sound frequency on jejunum-ileum morphology of laying hens.

Items	Treatments^a				SEM	p Values
	Control	Violin	Bass	Mozart
Villus height, µm	337.0	316.8	343.9	359.7	12.79	.158
Crypt depth, µm	55.9	60.8	59.0	50.9	4.76	.490
Villus height/crypt depth	6.2	5.2	6.0	7.6	0.59	.064
Muscle layer, µm	134.3	134.4	135.5	134.1	11.17	1.000

Each value represents the mean with standard error of mean (n = 4).

^a Control: background sound; Violin: 3969.03 Hz; Bass: 41.39 Hz; Mozart: K. 448.

Figure 3. (A) The distribution of amplicon sequence variants (ASVs) across both the control group and the Violin group were analysed. (B) Boxplots illustrating the alpha-diversity indices of both the control group and the Violin group were generated. The Shannon (a) and Simpson (b) indices are used to measure microbial diversity within samples, the observed features (c) represent the actual total species count within samples, the Pielou (d) index estimates species evenness within samples, and the Menhinick (e) and Margalef (f) indices estimate community richness based on the total number of species and total sample size.

Figure 4. (A) Community composition of the gut microbiota among the control group and the Violin group of laying hens were analysed at the family (a), genus (b) and species (c) levels, respectively. (B) Welch’s test indicated significant differences at the genus (a) and species (b) levels. (C) MetagenomeSeq results exhibited significant differences at the genus (a) and species (b) level.

Figure 5. Two functions were detected existing significant difference in the control group and the Violin group by using PICRUSt.

Data availability statement

The data presented in this study are available on request from the corresponding author upon reasonable request.