Effects of dietary chitosan oligosaccharides supplementation on meat quality, chemical composition and anti-oxidant capacity in frizzled chickens

Abstract

The aim of this study was to evaluate dietary chitosan oligosaccharides (COS) supplementation on meat quality, chemical composition and oxidative stability in frizzled chickens. A total of 360 one-day-old female frizzled chickens with an average body weight of 34.38 ± 1.23 g were randomly allocated to four groups with six replications (10 chickens/replication) in this 84-day experiment. The chickens in the control group (CON) fed the basal diet and the other three experimental diets were based on the basal diet with 300, 600 and 900 mg/kg COS supplementation, respectively. The results indicated that dietary COS increased growth performance, eviscerated and breast muscle yield while decreased abdominal fat yield, accompanied with increasing pH_45min, pH_24h, Lightness, drip loss and cooking loss in breast and thigh muscle. The scavenging activity of 2,2-diphenyl-1-picrylhydrazyl, superoxide radical, hydroxyl radical and 2,2-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt, the activity of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and catalase (CAT) in breast and thigh muscle were improved by COS while malondialdehyde content was decreased. COS up-regulated the gene expression of nuclear factor erythroid 2-related factor 2, heme oxygenase-1, NAD(P)H quinone dehydrogenase 1, SOD, CAT and GSH-Px in breast and thigh muscle. In conclusion, dietary COS supplementation improved meat quality by improving pH, colour, water holding capacity and anti-oxidant capacity, accompanied with decreased lipid metabolism in frizzled chickens. Dietary 600 mg/kg COS was recommended in frizzled chickens’ diet to improve meat quality.

Highlights

• COS improves growth performance of frizzled chickens.

• COS improves meat quality of frizzled chickens.

• COS enhances anti-oxidant capacity of frizzled chickens’ meat.

Keywords:

• Meat quality
• chemical composition
• anti-oxidant capacity
• frizzled chickens
• chitosan oligosaccharides

Introduction

China was the second largest producer of chicken meat in the world, and almost half was from Chinese yellow-feathered chickens, which satisfied consumers’ preferences due to its desirable flavour and nutritional profiles (Gou et al. 2016). With the improving living standards, consumers, producers and scientists paid more and more attention to meat quality, and the potential influence on consumers’ health. Lipid oxidation was one of the major threats to meat quality and nutritional characteristics, which led to health concerns, consumer acceptance and economic loss (Mielnik et al. 2006; Estévez 2011; Xiao et al. 2011; Skřivan et al. 2012; Estévez 2015). Meanwhile, poultry meat was sensitive to lipid oxidation due to high polyunsaturated fatty acid content (Liu et al. 2009a). Currently, anti-oxidants supplementation to poultry diets was an effective strategy to alleviate lipid oxidation, which diminished lipid oxidation in meat prior to and after slaughter (Jung et al. 2010; Zhang et al. 2015; Chang et al. 2020; Xu et al. 2020). Along these lines, the natural anti-oxidants attracted widespread attention, due to safe consideration, palatability, stability, application potential for consumers’ acceptability, and shelf-life of meat products (Naveena et al. 2008; Park and Kim 2008; Jung et al. 2010).

Among various natural anti-oxidant sources, chitosan oligosaccharides (COS), the degraded products of chiton or chitin derived from the shells of crustaceans, had received wide attention due to excellent anti-oxidant capacity (Naveed et al. 2019; Zhou et al. 2021). COS performed excellent anti-oxidant activity in vitro and in vivo by exhibiting strong free radical scavenging capacity, as well as regulating the anti-oxidant enzyme activities and reduces lipid peroxidation (Chang et al. 2020; Lan et al. 2020a, 2021). The precise radical scavenging mechanism of COS was not clear, but related to the reaction with amino and hydroxyl groups at the C-2, C-3 and C-6 positions of the pyranose ring (Zou et al. 2016; Naveed et al. 2019; Zhou et al. 2021). Nuclear factor erythroid 2-related factor 2 (Nrf2)/anti-oxidant response element (ARE) signalling pathway played a vital role in regulating oxidative homeostasis by regulating phase II detoxification enzymes related genes, including heme oxygenase-1 (HO-1), NAD(P)H quinone dehydrogenase 1 (NQO1), glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and catalase (CAT) (Jaiswal 2004; Jeong et al. 2016). Former literature demonstrated that COS prevented reactive oxidative reaction and the related pathways of cell damage caused by oxidative stress by activation of the Nrf2/ARE signalling pathway. Chang et al. (2022) reported that COS could alleviate heat stress-induced oxidative damage in breast muscle of yellow-feathered chickens by activating Nrf2 pathway and up-regulation of the expression of HO-1 and GSH-Px. Thus, COS had the potential to improve the anti-oxidant capacity of poultry meat via regulating the Nrf2–ARE signalling pathway. However, few studies were conducted to examine the effects of COS supplementation on meat quality, chemical composition and anti-oxidant capacity of breast and thigh muscle in frizzled chickens. Therefore, changes of meat quality, chemical composition and anti-oxidant capacity with COS supplementation were evaluated.

Materials and methods

Animals and experimental design

A total of 360 one-day-old female frizzled chickens with an average body weight (BW) of 34.38 ± 1.23 g were randomly allocated to four groups (60 chickens/group) with six replications (10 chickens/replication) in this 84-day experiment. The chickens were placed in a three-level battery cages and environmentally controlled room. The temperature of the room was maintained at 33 ± 1 °C for the first 3-week. From day 22, the temperature was gradually reduced by 0.5 °C per day until maintained at 24 °C. Artificial light was provided 23 h/d by fluorescent lights. The chickens in the control group (CON) fed the basal diet (Supporting information, Table S1), and the other three experimental diets were based on the basal diet with 300, 600 and 900 mg/kg COS supplementation, respectively. The basal diets were formulated to meet or exceed the nutrient requirement of the Feeding Standard of Chicken, China (NY/T 3645-2020). COS was purchased from Jiangsu Xinrui Biotechnology Co., Ltd., China (HPLC purity 95%, deacetylation degree over 95% and average molecular weight below 3200 Da). The chickens’ administration followed the China Standard of Feeding management regulations of yellow-feathered chicken (NY/T 1871-2010).

Growth performance

On day 1, 28, 56 and 84, chickens were weighed on cage basis; feed consumption was recorded throughout the experiment. Average daily gain (ADG), average daily feed intake (ADFI) and feed conversion ratio (FCR) were calculated.

Carcass characteristics and sample collection

At the end of the experiment, chickens were deprived of feed overnight, free access to water, then six chickens from each replication (1/cage) were randomly selected, weighted individually, and sacrificed. The carcass characteristics date was measured following the method of China Standard (NY/T823-200). The BW, carcass weight (CW), half-eviscerated weight (HEW), eviscerated weight (EW), left breast muscle weight (LBMW), left thigh muscle weight (LTMW) and abdominal fat weight (AFW) were recorded. The carcass characteristics were calculated following the formulas: carcass yield % = CW/BW × 100%; half-eviscerated yield % = HEW/BW × 100%; eviscerated yield % = EW/BW × 100%; breast muscle yield % = 2*LBMW/EW × 100%; thigh muscle yield % = 2*LTMW/EW × 100%; abdominal fat yield % = AFW/BW × 100%. Then, the left breast and thigh muscle were stored at 4 °C for 24 h for the following meat quality measurement. The right breast and thigh muscle were used for the remaining biochemical assays. Triplicate samples (approximately 1 g) were immediately collected into RNase free tube, frozen in liquid nitrogen, then stored at −80 °C for analysis of anti-oxidant related gene expression. Triplicate samples (approximately 1 g) were collected and stored at −20 °C for anti-oxidant enzyme activity analyse, as well as the scavenging activity of superoxide radical (O²–) and hydroxyl radical (OH·). Triplicate samples (approximately 1 g) were stored at −20 °C for the analysis of the scavenging activity of 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS⁺·), before analysis, the samples were thawed and dried at 60 °C for 72 h, then finely ground to a size that could pass through a 600-μm screen. The rest of the samples were collected to determine chemical composition.

Meat quality and chemical composition

After breast and thigh muscle samples were stored at 4 °C for 24 h, meat quality was estimated following the methods described in our former study (Chang et al. 2020). Briefly, duplicate pH was measured using a pH meter (AZ8694, Taiwan Hengxin) at post-mortem 45 min (pH_45min) and 24 h (pH_24h). The Lightness (L*), Redness (a*) and Yellowness (b*) of meat colour were measured using a model CR-10 Plus Chroma meter (Konica Minolta Sensing Inc., Osaka, Japan). The colorimeter was standardised with a white tile (L* = 99.04, a* = –0.12 and b* = 1.03). About 4 g sample (W_i) was weighed for drip loss; the samples were packaged in a plastic bag and stored at 4 °C for 24 h, then the excess moisture was wiped out and weighed (W_f). Dripping loss was calculated following the equation, dripping loss % = (W_i – W_f)/W_i × 100%. About 4 g sample (W₁) was weighed for cooking loss determination, placed in a plastic bag and cooked to an internal temperature of 75 °C in water bath, then the cooked samples were cooled for 30 min, blotted dry and weighed (W₂). Cooking loss was calculated following the equation, cooking loss % = (W₁ – W₂)/W₁ × 100%. Share force was measured after cooking loss, using a texture analyser (Bulader-TS100, Beijing Bulader Co., Ltd., Beijing, China), the cooked samples were cut perpendicular to the fibre orientation.

About 50 g fresh breast and thigh muscle sample from each chicken were weighed, sliced up, placed in a ceramic plate, then dried at 60 °C for 48 h in a freeze dryer (Labconco Corp., Kansas City, MO) and reweighed. The moisture was determined by calculating the difference between the initial and dried sample weights. The dried muscle sample was ground into powder and subsequently analysed for crude protein (CP) content and intramuscular fat content, namely ether extract (EE). The CP (991.36) and EE (991.36) contents of breast and thigh muscle samples were analysed following the methods described by the AOAC (2010).

Anti-oxidant stability

The scavenging activity of DPPH (Art. BC4755), O²– (Art. BC1415), OH· (Art. BC1325) and ABTS⁺· (Art. BC4775), the content of MDA (Art. BC0025), the activities of GSH-Px (Art. BC1195), SOD (Art. BC0175) and CAT (Art. BC0205) were determined following the introduction of the commercial kits purchased from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). Briefly, for DPPH and ABTS⁺· scavenging activity measurement, 0.05 g sample was added to 1 mL extract buffer provided in the kits, respectively, and kept at 40 °C for 30 min, then centrifuged at 10,000 × g for 10 min at 4 °C, the supernatant was stored at −80 °C for further analysis. The percentage inhibition of DPPH and ABTS⁺· generation was calculated following the formula: DPPH/ABTS⁺· scavenging ability % = [[A_blank – (A_sample – A_control)] ÷ A_blank] × 100%. The other parameters measurement: 0.1 g sample was homogenised in ice-cold 1 mL extract buffer provided in the kits, respectively, 8000 × g for 10 min at 4 °C, then the supernatant was stored at −80 °C for further analysis of MDA, GSH-Px, SOD, CAT, O²– and OH·. The protein concentrations of the 10% breast and muscle homogenates were determined by using the bicinchoninic acid assay (BCA, Art. PC0020).

Gene expression

Total RNA extraction, cDNA synthesis and real-time quantitative polymerase chain reaction (RT-PCR) were followed by our described methods (Lan et al. 2014). The primer sequences are listed in Table S2. β-actin was used as an internal control gene. The relative gene expression of each target gene was calculated using the 2^–ΔΔCt method (Livak and Schmittgen 2001).

Statistical analysis

The cage was used as the experiment unit and all data were analysed with SAS 2003 (v. 9.1, SAS Institute Inc., Cary, NC) by using one-way analysis of variance. Orthogonal comparison was conducted using polynomial regression to measure the linear and quadratic effects of increasing level of COS. p < .05 was considered as statistically significantly different.

Results

Growth performance

As shown in Table 1, there was a linear improvement (p < .05) in BW on day 28, 56 and 84, associated with the increasing level of COS, and dietary 600 mg/kg COS had higher (p < .05) BW than the other groups on day 28. On day 56 and 84, dietary 300, 600 and 900 mg/kg COS had better (p < .05) BW compared with the CON group, and dietary 600 mg/kg COS had the highest BW. During day 1 to 28, there was a linear and quadratic improvement (p < .05) in ADG, a linear improvement (p < .05) in ADFI and FCR was associated with the increasing level of COS, and dietary 600 mg/kg COS had better (p < .05) ADG, ADFI and FCR than the other groups. During day 29–56 and day 1–84, there was a linear improvement (p < .05) in ADG and FCR associated with the increasing level of COS. Dietary 300, 600 and 900 mg/kg COS had better (p < .05) ADG, dietary 600 and 900 mg/kg COS had better (p < .05) FCR compared with the CON group. During day 57–84, no significant differences were observed in ADG, ADFI or FCR associated with the increasing level of COS.

Table 1. Dietary chitosan oligosaccharides (COS) supplementation on growth performance of frizzled chickens.

Item^1	COS supplementation level, mg/kg				SEM^2	p Value^3
	0	300	600	900		Linear	Quadratic
Body weight, g
Day 1	34.15	34.75	34.55	34.05	0.5232	.5948	.5395
Day 28	319.49^a	332.09^a	372.89^b	329.85^a	5.2798	<.0001	.0413
Day 56	839.49^a	887.92^b	973.82^c	887.85^b	8.2328	<.0001	.0779
Day 84	1308.15^a	1365.59^b	1453.82^c	1357.85^b	10.9417	<.0001	.2640
Day 1–28
ADG, g	10.19^a	10.62^a	12.09^b	10.57^a	0.1922	<.0001	.0390
ADFI, g	20.18^a	20.50^a	22.03^b	0.42^a	0.2974	.0003	.1137
FCR	1.98^a	1.93^a	1.82^b	1.94^a	0.0264	.0004	.3904
Day 29–56
ADG, g	18.57^a	19.85^b	21.46^c	19.93^b	0.2656	<.0001	.6175
ADFI, g	55.30^ab	57.54^a	54.81^b	54.68^b	0.8383	.6818	.0251
FCR	2.98^a	2.90^a	2.56^b	2.75^c	0.0423	<.0001	.0194
Day 57–84
ADG, g	16.74	17.06	17.14	16.79	0.3990	.4812	.8098
ADFI, g	65.19	65.43	65.16	64.81	0.6393	.9666	.7473
FCR	3.90	3.85	3.81	3.88	0.0972	.5202	1.0000
Day 1–84
ADG, g	11.51^a	12.12^b	13.19^c	12.11^b	0.1452	<.0001	.2121
ADFI, g	46.89	47.83	47.33	46.64	0.4561	.4998	.2152
FCR	4.08^a	3.95^ab	3.59^c	3.86^b	0.0530	<.0001	.0831

^a,b,cMean values with no same superscript in a row were significantly different (p < .05).

¹ ADG: average daily gain; ADFI: average daily feed intake; FCR: feed conversion ratio.

² SEM: standard error of mean.

³ Linear and quadratic effects with increasing level of chitosan oligosaccharides (COS) conducted by orthogonal polynomial contrast.

Carcass characteristics

As shown in Table 2, with increasing level of COS, there was a linear increase (p < .05) in eviscerated yield and breast muscle yield, while a linear decrease (p < .05) in abdominal fat yield. Dietary 600 mg/kg COS had higher (p < .05) eviscerated yield than other groups. Dietary 300, 600 and 900 mg/kg COS had higher (p < .05) breast muscle yield, while lower (p < .05) abdominal fat yield compared with the CON group.

Table 2. Dietary chitosan oligosaccharides (COS) supplementation on carcass characteristics of frizzled chickens.

Item, %	COS supplementation level, mg/kg				SEM^1	p Value^2
	0	300	600	900		Linear	Quadratic
Carcass yield	90.64	90.07	91.39	92.12	0.6453	.4178	.2470
Half-eviscerated yield	82.14	82.06	82.00	81.26	0.6118	.8761	.9947
Eviscerated yield	65.81^a	67.46^a	69.64^b	67.17^a	0.6481	.0005	.7443
Breast muscle yield	15.86^a	19.35^b	19.94^b	19.32^b	0.3916	<.0001	.0068
Leg muscle yield	21.53	22.27	23.91	23.30	0.9239	.0839	.6972
Abdominal fat yield	2.37^a	2.15^b	1.96^b	2.14^b	0.0622	.0001	.8041

^a,bMean values with no same superscript in a row were significantly different (p < .05).

¹ SEM: standard error of mean.

² Linear and quadratic effects with increasing level of chitosan oligosaccharides (COS) conducted by orthogonal polynomial contrast.

Meat quality and chemical composition

As shown in Table 3, L* was linearly and quadratically decreased, and pH_45min was quadratically reduced with the increasing level of COS in breast muscle. Dietary 300 and 600 mg/kg COS had higher (p < .05) pH_45min, dietary 900 mg/kg COS had higher (p < .05) pH_24h, dietary 300 mg/kg COS had lower (p < .05) drip loss, dietary 300, 600 and 900 mg/kg COS had lower (p < .05) L* and cooking loss compared with the CON group. In thigh muscle, the pH_45min was linearly and quadratically increased while L* was decreased, and pH_24h was quadratically increased with increasing level of COS. Dietary 600 and 900 mg/kg COS had higher (p < .05) pH_45min, dietary 300 and 600 mg/kg COS had higher (p < .05) pH_24h while lower (p < .05) cooking loss, dietary 300, 600 and 900 mg/kg COS had lower (p < .05) L*, dietary 300 mg/kg COS had lower (p < .05) drip loss compared with the CON group. No significant differences were observed in chemical composition of breast and thigh muscle with COS supplementation.

Table 3. Dietary chitosan oligosaccharides (COS) supplementation on meat quality and nutrient composition of frizzled chickens.

Item	COS supplementation level, mg/kg				SEM^1	p Value^2
	0	300	600	900		Linear	Quadratic
Breast muscle
pH45min	6.30^b	6.51^a	6.39^ab	6.49^a	0.0562	.2709	.0264
pH24h	5.86^a	5.91^ab	5.92^ab	5.98^b	0.0453	.3872	.4795
Lightness, L*	52.71^a	50.90^b	51.29^b	51.26^b	0.3785	.0151	.0272
Redness, a*	2.40	2.43	2.42	2.60	0.1582	.9180	.8986
Yellowness, b*	9.11	9.33	9.14	9.14	0.1337	.8484	.2325
Drip loss, %	1.25^a	1.07^b	1.14^ab	1.24^ab	0.5366	.1689	.0809
Cooking loss, %	17.09^a	16.20^b	16.23^b	16.22^b	0.3240	.0742	.2584
Share force, N	23.15	22.18	23.08	25.00	1.4233	.9726	.5963
Moisture, %	73.60	73.48	74.19	73.65	0.7208	.5649	.6461
Crude protein, %	23.96	23.76	23.83	23.80	0.5060	.8558	.8361
Intramuscular fat, %	3.71	3.68	3.91	3.72	0.4772	.7681	.8263
Thigh muscle
pH45min	6.53^b	6.56^b	6.69^a	6.76^a	0.0378	.0083	.0041
pH24h	6.03^b	6.18^a	6.19^a	6.13^ab	0.0399	.0117	.2100
Lightness, L*	52.71^a	50.90^b	51.29^b	51.26^b	0.3785	.0151	.0272
Redness, a*	2.40	2.44	2.42	2.60	0.1582	.9180	.8986
Yellowness, b*	9.11	9.33	9.14	9.14	0.1337	.8482	.2325
Drip loss, %	1.59^a	1.32^b	1.42^ab	1.58^ab	0.0878	.1990	.0966
Cooking loss, %	18.45^a	17.13^b	17.26^b	17.40^ab	0.4335	.0658	.1863
Share force, N	25.38	24.41	25.31	27.22	1.4228	.9719	.5965
Moisture, %	69.27	69.15	69.86	69.32	0.7208	.5657	.6461
Crude protein, %	21.74	21.54	21.61	21.58	0.5060	.8558	.8361
Intramuscular fat, %	6.26	6.40	6.80	6.27	0.4412	.4011	.8124

^a,bMean values with no same superscript in a row were significantly different (p < .05).

¹ SEM, standard error of mean.

² Linear and quadratic effects with increasing level of chitosan oligosaccharides (COS) conducted by orthogonal polynomial contrast.

Free radical scavenging activity

Dietary COS supplementation linearly and quadratically increased (p < .05) the scavenging activity of DPPH (Figure 1(A1)), O²– (Figure 1(B1)), OH· (Figure 1(C1)) and ABTS⁺· (Figure 1(D1)) in breast muscle, as well as DPPH (Figure 1(A2)) and O²– (Figure 1(B2)) in thigh muscle (Figure 1). The OH· (Figure 1(C2)) and ABTS⁺· (Figure 1(D2)) scavenging activity of thigh muscle was also linearly increased (p < .05). Dietary 300, 600 and 900 mg/kg COS had higher (p < .05) DPPH, O²–, OH· and ABTS⁺·scavenging activity in breast and thigh muscle, and dietary 600 mg/kg had the highest.

Figure 1. Effects of chitosan oligosaccharides (COS) supplementation on free radical scavenging activity in breast and thigh muscle of frizzled chickens. ^a,b,cMean values with no same superscript in a row were significantly different (p < .05); DPPH: 2,2-diphenyl-1-picrylhydrazyl; O²–: superoxide radical; OH·: hydroxyl radical; ABTS⁺·: 2,2-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt; free radical scavenging activity was calculated on the basis of the protein content in muscle. Linear and quadratic effects with increasing level of chitosan oligosaccharides (COS) conducted by orthogonal polynomial contrast.

Anti-oxidant capacity

Dietary COS supplementation linearly decreased (p < .05) the MDA content in breast and thigh muscle (Figure 2(A1,A2)), while linearly increased (p < .05) the activity of SOD (Figure 2(C1,C2)) and CAT (Figure 2(D1,D2)). The GSH-Px activity was linearly increased (p < .05) in breast muscle (Figure 2(B1)), and linearly and quadratic increased in thigh muscle (Figure 2(B2)). Dietary 600 mg/kg COS increased (p < .05) GSH-Px, SOD and CAT activity, while decreased MDA content in breast muscle when compared with the CON group. In thigh muscle, dietary 300, 600 and 900 mg/kg COS increased (p < .05) GSH-Px, SOD and CAT activity, while lower MDA content, and dietary 600 mg/kg had the lowest MDA content, and highest GSH-Px, SOD and CAT activity.

Figure 2. Effects of chitosan oligosaccharides (COS) supplementation on anti-oxidant capacity in breast and thigh muscle of frizzled chickens. ^a,b,cMean values with no same superscript in a row were significantly different (p < .05); MDA: malondialdehyde; GSH-Px: glutathione peroxidase; SOD: superoxide dismutase; CAT: catalase; linear and quadratic effects with increasing level of chitosan oligosaccharides (COS) conducted by orthogonal polynomial contrast.

Anti-oxidant gene expression

Dietary COS supplementation linearly and quadratically increased (p < .05) the relative gene expression of NQO1 (Figure 3(C1)) and GSH-Px (Figure 3(F1)) in breast muscle, as well as GSH-Px (Figure 3(F2)) in thigh muscle. The relative gene expression of Nrf2 (Figure 3(A2)), HO-1 (Figure 3(B2)) and SOD (Figure 3(D2)) was linearly increased (p < .05) in thigh muscle, quadratically increased in relative gene expression of SOD (Figure 3(D1)) and CAT (Figure 3(E1)) in breast muscle, and NQO1 (Figure 3(C2)) in thigh muscle. Compared to the CON group, dietary 300 mg/kg COS increased (p < .05) relative gene expression of Nrf2 and CAT in breast muscle, as well as NQO1 in thigh muscle. Dietary 600 mg/kg COS increased (p < .05) relative gene expression of NQO1 in breast muscle, as well as Nrf2 and HO-1 in thigh muscle. Dietary 900 mg/kg COS increased (p < .05) relative gene expression of SOD in breast muscle and thigh muscle. Dietary 600 and 900 mg/kg COS increased (p < .05) relative gene expression of GSH-Px in breast muscle and thigh muscle, as well as dietary 300 mg/kg COS increased (p < .05) in breast muscle.

Figure 3. Effects of chitosan oligosaccharides (COS) supplementation on relative gene expression of anti-oxidant related genes in breast and thigh muscle of frizzled chickens. ^a,b,cMean values with no same superscript in a row were significantly different (p < .05); Nrf2: nuclear factor erythroid 2-related factor 2; HO-1: heme oxygenase-1; NQO1: NAD(P)H quinone dehydrogenase 1; SOD: superoxide dismutase; CAT: catalase; GSH-Px: glutathione peroxidase; linear and quadratic effects with increasing level of chitosan oligosaccharides (COS) conducted by orthogonal polynomial contrast.

Discussion

Growth performance

Positive effects on ADG, ADFI and FCR of frizzled chickens were observed from day 1 to 28, day 29 to 56, and the overall period (day 1–84) with the supplementation of COS. Consistent with our results, Li et al. (2019) and Wang et al. (2022) reported that dietary COS had lower FCR in broilers and yellow-feathered chickens, Zhou et al. (2009) reported that dietary COS had a beneficial effects on BW gain and feed intake, Li et al. (2007) reported that dietary COS exerted better ADG, ADFI and FCR. Likewise, Li et al. (2016) reported that dietary COS had no effects on ADG, ADFI or FCR in frizzled chickens aged day 50–92. Growth performance was highly related to gut health, which played important role in nutrient digestion and absorption (Sobolewska et al. 2017). Growth hormones (GH) and insulin-like growth factor-1 (IGF-1) level also related to the growth rate (Scanes 2009). Therefore, several possible reasons could attribute to the beneficial effect of COS on growth performance, including the improvement of intestinal function such as intestinal morphology, gut bacterial flora, digestion, absorption and barrier function, immunity and anti-oxidant capacity (Li et al. 2007; Wan et al. 2017; Nuengjamnong and Angkanaporn 2018; Ibitoye et al. 2019; Li et al. 2019; Lan et al. 2021; Elnesr et al. 2022; Tao et al. 2022), as well as the increasing level of GH and IGF-1 (Li et al. 2007; Chang et al. 2020; Ahmed et al. 2021).

Carcass characteristics

The eviscerated and breast muscle yield were linearly increased, while linearly decreased abdominal fat yield, without affecting carcass, half-eviscerated or thigh muscle yield with dietary COS. Similar results were also reported by former literature, which indicated that dietary COS had no effects on carcass yield, breast or thigh muscle yield, while decreased the abdominal fat yield (Zhou et al. 2009; Arslan and Tufan 2018; Wang et al. 2022). Abdominal fat deposition in broilers was considered as a waste in poultry industry because it reduced feed efficiency, carcass production, and increasing the cost and spoiling the environment for processing waste adipose tissue (Liu et al. 2019). The decreasing abdominal fat deposition suggested that COS had inhibitory effects on body fat deposition, meanwhile, several studies had demonstrated that COS was effective in lipid metabolism, had positive effects in alleviating lipid accumulation (Zhou et al. 2009; Li et al. 2016; Tao et al. 2019; Wang et al. 2022). Dietary COS supplementation could decrease the abdominal fat yield mainly because COS supplementation decreased liver lipogenesis and suppressed triglyceride accumulation in adipose tissue (Zhou et al. 2009; Li et al. 2016; Wang et al. 2022).

Meat quality and chemical composition

pH was an important indicator of meat quality as it was related to water-holding capacity, colour, juiciness, tenderness and shelf life (Mir et al. 2017). Drip loss and cooking loss were the important indicators of water-holding capacity and reflected the juiciness of meat. Meat colour was the most important quality attribute of cooked or raw poultry meat because consumers associated it with the product’s freshness, attractiveness and acceptability at purchase (Mir et al. 2017). The results of the present study showed higher pH_45min and pH_24h of breast and thigh muscle, which resulted in better water-holding capacity and colour. The decreased L* indicated less pale meat (Jiang et al. 2014). The better water-holding capacity may be due to the improvement of anti-oxidant of meat, the drip loss and cooking loss were negatively correlated with the anti-oxidant capacity of meat (Zhu et al. 2019). In consistent with our results, Wang et al. (2022) reported that COS supplementation decreased cooking loss in yellow-feathered chickens. Chang et al. (2020) reported that dietary COS had lower pH_24h and cooking loss in heat-stressed yellow-feathered chickens. The results indicated that dietary COS had positive effects on meat quality of breast and thigh muscle of frizzled chickens.

No significant differences in moisture, CP or intramuscular fat content in breast and thigh muscle with COS supplementation. These results may be associated with the experimental period, former study indicated that significant changes were observed in chemical composition of broilers aged from day 1 to 35, or over day 90 (Mahmood et al. 2005). Meanwhile, some literature also indicated that dietary anti-oxidative additives, such as curcumin and resveratrol, had no effects on meat chemical composition of broilers or ducks (Galli et al. 2020; Wang et al. 2020; Jin et al. 2021; Xu et al. 2021).

Anti-oxidative capacity

Meat quality was highly related to anti-oxidant capacity of muscle (Pan et al. 2018; Zhang et al. 2019). MDA was the end product of lipid peroxidation, was a process in which carbon-carbon double bonds were attacked by free radicals, and reflected the degree of lipid peroxidation. SOD, GSH-Px and CAT were the main components of enzymatic anti-oxidant defence system in scavenging free radicals (Cheng et al. 2019). The free radicals could be eliminated by the combined action of the available anti-oxidant defence. Former literature indicated that COS performed excellent anti-oxidant activity in vitro and in vivo by exerting free radical scavenging activity, reducing lipid peroxidation and regulating the anti-oxidant enzyme activities by improving the activity of GSH-Px, SOD and CAT (Liu et al. 2009b; Zou et al. 2016; Yang et al. 2017; Naveed et al. 2019). In the present study, dietary COS increased the scavenging activity of DPPH, O²–, OH· and ABTS⁺·, decreased MDA content, as well as increased the activity of GSH-Px, SOD and CAT in breast and thigh muscle, indicating that COS could improve anti-oxidant capacity of meat, and the improvement meat quality was related to the improvement of anti-oxidant capacity. In consistent with our results, former literature indicated that COS supplementation improved the scavenging activity of DPPH, O²– and hydrogen peroxide (H₂O₂), increased the activity of GSH-Px and SOD, while decreased the MDA content in the breast muscle of heat-stressed yellow-feathered chickens (Chang et al. 2020, 2022). Lan et al. (2020a, 2020b) reported that dietary COS significantly decreased the MDA content, while increased the GSH-Px, SOD activity in small intestine, liver and spleen of heat-stressed yellow-feathered chickens. In addition, dietary COS also improved the scavenging activity of DPPH, O²–, OH· and ABTS⁺· in jejunum of H₂O₂ challenged rats, decreased MDA content, and increased GSH-Px activity in small intestine (Lan et al. 2021). Although the precise radical scavenging mechanism of COS was not clear, we speculated it mainly depended on the abstraction of a proton from free radicals by an amino group in the C2 position and hydroxy groups in the C3 and C6 positions of the pyranose ring (Adhikari et al. 2021). In addition, the enhancement of GSH-Px, SOD and CAT activity, and the decreased MDA content also indicated the improvement anti-oxidant capacity in breast and thigh muscle with COS supplementation. The enhancement of the free radical scavenging activity may partly be related to the increasing anti-oxidant enzyme activities.

The Nrf2–ARE signalling pathway was a promising pathway to regulate the anti-oxidant capacity and lipid metabolism in muscle by regulating the gene expression of phase anti-oxidant enzymes, such as HO-1, GSH-Px, SOD and CAT in various cell types (Sahin et al. 2012; Song et al. 2018; Xu et al. 2018; Zhang et al. 2018; Hu et al. 2020). Former studies indicated that some feed additives could improve meat quality and muscle anti-oxidant capacity by regulating Nrf2-related signalling gene expression (Zhao et al. 2021). Hu et al. (2020) reported that dietary glutamine increased the protein level of Nrf2, as well as the mRNA expression level of the GSH-Px, SOD and CAT in the thigh muscle. In line with previous literature, in the present study, dietary COS up-regulated the mRNA expression of Nrf2, NQO-1, SOD, CAT and GSH-Px in breast muscle, as well as Nrf2, HO-1, NQO-1, SOD and GSH-Px in thigh muscle. Similarly, Chang et al. (2022) reported that dietary COS up-regulated the mRNA gene expression of HO-1 and GSH-Px in breast muscle of heat-stressed yellow-feathered chickens, as well as improved the meat quality. These results may explain the positive effects of COS on anti-oxidative capacity in meat of frizzled chickens.

Conclusions

It was concluded that dietary COS had beneficial effect on growth performance and meat quality. The effects of COS supplementation on meat qualitywas acheived by improving pH, colour, water holding capacity, as well as improving anti-oxidative capacity by improving free radical scavenging activity, anti-oxidative enzyme activities, and activating the Nrf2-mediated gene expression of Nrf2, HO-1, GSH-Px and SOD in breast and thigh muscle. Dietary COS supplementation had beneficial effects on meat quality of breast and thigh muscle of frizzled chickens, mainly due to an improvement in the anti-oxidant capacity of meat via activating the Nrf2–ARE pathway. Based on the results, dietary supplementation with 600 mg/kg COS seemed to be the optimised dose for improving meat quality of frizzled chickens.

Ethical approval

The experimental protocol used in this study was approved by the Animal Care and Use Committee of Guangdong Ocean University (SYXK-2018-0147).

Disclosure statement

No conflict of interest was reported by the authors.

Data availability statement

The original data of the paper are available upon request from the corresponding author.

Additional information

Funding

This work was supported by the Program for Scientific Research Start-Up Funds of Guangdong Ocean University [101402/R18005].