
==== Front
Poult Sci
Poult Sci
Poultry Science
0032-5791
1525-3171
Elsevier

S0032-5791(24)00755-7
10.1016/j.psj.2024.104176
104176
MANAGEMENT AND PRODUCTION
Evaluation of the growth performance, meat quality, and gut microbiota of broilers fed diets containing walnut green husk extract
Wu Ying *
Zhai Shaohua †
Fang Meiyan ⁎
Zhang Huiling *
Chen Yong cy@xjau.edu.cn
*1
⁎ College of Animal Science, Xinjiang Agricultural University, Urumqi, Xinjiang, 830052, China
† College of Veterinary Medicine, Xinjiang Agricultural University, Urumqi, Xinjiang, 830052, China
1 Corresponding author: cy@xjau.edu.cn
06 8 2024
11 2024
06 8 2024
103 11 10417629 5 2024
1 8 2024
© 2024 The Authors
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
This study was conducted to evaluate the effects of walnut green husk extract (WGHe) on the growth performance, meat quality, antioxidative status, gut morphology, and microbiota diversity of broilers. A total of 216 one-day-old broilers were divided into 4 groups, each consisting of 9 replicates (6 birds per replicate) as follows: 1) control group, basal diet; 2) antibiotic group, basal diet supplemented with enduracidin and colistin sulfate; 3) low-dose group, basal diet supplemented with 5.0 g/kg WGHe; and 4) high-dose group, basal diet supplemented with 10.0 g/kg WGHe. The results revealed that the percentage of abdominal fat decreased, and the ratio of the duodenal villus length to crypt depth (V/C), as well as the α-diversity of the ileal microbiota, increased with 10.0 g/kg WGHe supplementation (P < 0.05). The shear force of the breast muscle and plasma malondialdehyde (MDA) concentration decreased, whereas the plasma peroxidase (POD) activity, Trolox equivalent antioxidant capacity (TEAC), and jejunal villus length increased in response to WGHe supplementation (P < 0.05). Compared with the antibiotic diet, the addition of 5.0 g/kg WGHe resulted in a significant increase in the relative abundances of Candidatus Arthromitus, Eubacterium coprostanoligenes, and Ruminococcaceae UCG-014 (P < 0.01). Furthermore, the addition of 10.0 g/kg WGHe increased the relative abundances of Candidatus Arthromitus and Lachnoclostridium, whereas the relative abundance of unidentified Chloroplast decreased (P < 0.05). In conclusion, dietary supplementation with 10.0 g/kg WGHe is advantageous for intestinal health, meat quality, and antioxidant status in broilers, suggesting its potential as a functional additive in poultry production.

Key words

walnut green husk extract
meat tenderness
antioxidant
microbiota
broiler
==== Body
pmcINTRODUCTION

The use of antimicrobial agents (AMA) as growth-promoting agents (antimicrobial growth promoters [AGP]) became popular in livestock production after their discovery (Samantha, 2018). However, problems of overuse and abuse of AMA are attracting increasing attention because of the potential development of antibiotic-resistant bacteria in human populations and the residues of AMA in animal products, which pose potential harm to human health. Therefore, the use of AMA in animal production has been banned by multiple countries (Plata et al., 2022). The complete removal of AGP has led to a significant increase in pathogen-induced infections, which has negatively affected the health and performance of livestock (Urban et al., 2024). In recent decades, much attention has been focused on identifying potential alternatives with the same positive effects as AGP but without potential harm effects. Probiotics, plant extracts, enzymes, antimicrobial peptides, organic acids, bacteriophages, and nanoparticles have shown potential in replacing AMA and combating multidrug-resistant bacteria (Mwafy et al., 2023; Wickramasuriya et al., 2024). Among these, plant extracts are considered one of the most promising alternatives because they contain a diverse range of active components, including flavonoids, alkaloids, phenols, and quinones, which exhibit good antibacterial properties, as presented in recent studies (Li et al., 2022; Ramaiyulis et al., 2023).

Plant extracts, also called phytobiotics, have attracted much attention for their potential to replace AGPs because they are natural compounds that are safe, highly efficient, and eco-friendly. In poultry production, plant extracts are believed to possess significant antibacterial activity against undesirable pathogenic bacteria, to improve intestinal health, to regulate the composition of the gut microbiota, and to promote growth performance. Essential oils (EO) derived from thyme (Thymus vulgaris) and oregano (Origanum vulgare) exhibit significant inhibitory effects against Escherichia coli and Salmonella spp. (Boskovic et al., 2015). Pomegranate (Punica granatum) leaf and aloe extracts show promising antibacterial activity against Salmonella gallinarum isolated from chickens (Mkangara and Mpenda, 2022). Plant extracts play crucial roles in regulating the gut microbiota, which is essential for maintaining normal health in animals. The inclusion of thyme and cinnamon in broiler chicken feed has been shown to reduce the total bacterial count and the number of coliform bacteria in the gastrointestinal tract (Al-Kassie, 2010). Allicin, an active compound in garlic (Allium sativum), improves intestinal epithelial barrier function and prevents barrier damage induced by lipopolysaccharides in pig intestinal epithelial cells (IPEC-J2) (Gao et al., 2022). It also improves the composition of the gut microbiota by reducing the abundances of gut pathogenic bacteria such as Enterobacter, Erysipelatoclostridium, and Colidextribacter, while enriching beneficial bacteria such as Bifidobacterium, Lactobacillus, Clostridium, and Akkermansia (Zhang et al., 2020; Li et al., 2023). The antimicrobial properties of plant extracts, when used as feed additives, contribute to enhanced feed conversion and growth performance in food animals. For example, the addition of oregano aqueous extract to the feed of broiler chickens from 22 to 42 d of age significantly enhances body weight, average daily gain, and feed conversion rates (Zhang et al., 2023). Moreover, plant extracts are thought to bolster the immune functions of the organism. Herbs and spices, along with their extracts, have the capacity to increase the activity of lymphocytes, macrophages, and natural killer cells; stimulate the process of phagocytosis; and modulate the synthesis of interferon (Urban et al., 2024). Additionally, plant extracts also exhibit significant antioxidant activity. Phytobiotics containing cinnamon oil reduce blood concentrations of malondialdehyde and lipid hydroperoxide, increase catalase (CAT) activity, and improve the ferric reducing ability of plasma (Krauze et al. 2021). Furthermore, plant extracts are utilized for antiparasitic purposes. Essential oil derived from star anise (Illicium verum), and extracts of Carica papaya, Morus Macroura and Lepidium Sativum have been found to be effective in controlling Eimeria spp. in broiler chickens and rabbits (Al-Hoshani et al., 2023; Saleh et al., 2023; Abbas and Alkheraije, 2023).

Walnut (Juglans regia) green husk (WGH), which is a traditional Chinese medicinal material, is widely used to treat pruritus and analgesia in China. WGH contains a variety of active ingredients, including naphthoquinones, polyphenols, flavonoids, terpenoids, and polysaccharides (Cosmulescu et al., 2010; Wang et al., 2023a). WGH extracts have shown beneficial bacteriostatic effects on S. aureus, S. epidermis and E. coli in vitro (Fernández-Agulló et al., 2013; Cheraghali et al., 2019; Wang et al., 2023b). WGH extracts not only demonstrate reliable antibacterial activity but also exhibit favorable ferric reducing antioxidant power (FRAP), oxygen radical absorbance capacity (ORAC), and scavenging abilities against 2,2-diphenyl-1picrylhydrazyl (DPPH), hydroxyl radical (•OH), and 2,2-azino-bis-3-ethylbenzothiazoline-6-sulfonate (ABTS) (Soto-Madrid et al., 2021; Xi et al., 2023). Therefore, WGH could be a valuable source of natural compounds with antioxidant and antimicrobial properties, and WGH extracts may represent a potential alternative to AGPs. However, whether WGH extracts can improve poultry performance and meat quality remains unclear.

In our previous study, we reported that a 25% hydroethanol extract of WGH (WGHe) exhibited high total phenolic and flavonoid contents, as well as strong antimicrobial activity (Wu et al., 2017). Therefore, this study was conducted to evaluate the effects of WGHe on growth performance, nutrient digestibility, carcass traits, meat quality, the immune organ index, plasma antioxidative properties, and the gut morphology and bacterial community of broilers to provide a reference for the development of AGP alternatives in poultry production.

MATERIALS AND METHODS

All experimental procedures involving animals in this study were approved (protocol No. 2016003) by the Animal Care and Use Committee of Xinjiang Agricultural University, Urumqi, Xinjiang, China.

Preparation of WGHe

The extraction of WGH was carried out according to the method reported by Wei et al. (2024). In brief, naturally dried WGHs were crushed through an 80-mesh sieve using a crusher (DFY-1000, Dingli Medical Equipment Co., Ltd., Wenzhou, China) and extracted with a 25% ethanol aqueous solution in a magnetic stirring tank (Zhejiang Jhenten Machinery Co., Ltd., Wenzhou, China) at 37°C for 8 h. The resulting mixture was filtered through a bag filter (Φ = 5 μm), and the filtrate was concentrated by rotary evaporation in a rotary evaporator (RE-5210A, Ya-Rong Biochemical Instruments Plant, Shanghai, China) and then dried at 50°C until a constant weight was achieved to obtain the WGHe. The analysis revealed that WGHe contained 3.59% crude protein (CP), 27.84% polysaccharides, 6.01% total phenols, and 2.99% flavonoids and had a DPPH free radical scavenging rate of 69.16%.

Birds, Diets, and Feeding Management

A total of 216 one-day-old male Chinese yellow-feathered broilers were selected, reared on the ground to 8 d of age, and then divided into 4 groups, each with 9 replicates and 6 birds per replicate cage. The 4 groups were as follows: 1) basal diet (control group); 2) basal diet supplemented with 8 mg/kg diet of enramycin and colistin sulfate from 9 to 28 d and then with 6 mg/kg from 29− to 70 d (antibiotic group); 3) basal diet supplemented with 5.0 g/kg diet of WGHe (low-dose group); and 4) basal diet supplemented with 10.0 g/kg diet of WGHe (high-dose group). From 1− to 8 d, all the birds were fed the basic diet, and from 9 d of age, the animals were fed the corresponding diet. The basal diet was formulated according to the Feeding Standard of Chicken (MOA, 2004). The formulation and nutrient composition of the basal diet are shown in Table 1.Table 1 Formulation and nutrient composition of the basal diet (air-dry basis).

Table 1Ingredient (g/kg)	1 to 28 d of age	29 to 70 d of age	Nutrient level2	1 to 28 d of age	29 to 70 d of age	
Corn	607.8	647.0	Dry matter (%)	95.89	95.81	
Extruded soybean	104.9	58.8	Organic matter (%)	92.82	92.67	
Soybean meal	152.0	127.5	Crude protein (%)	20.75	18.61	
Cottonseed meal	78.4	98.0	Gross energy (MJ/kg)	15.28	16.12	
Soybean oil	9.8	23.5	Metabolizable energy (MJ/kg)	12.30	12.80	
NaCl	2.9	2.9	Calcium (%)	0.86	0.78	
CaHPO4	17.6	15.7	Phosphorus (%)	0.73	0.66	
Limestone	11.8	11.3	Lysine (%)	1.20	1.10	
Zeolite	4.8	5.3	Methionine (%)	0.45	0.45	
Premix1	10.0	10.0				
1 The premix provided the following per kg of diet: VA 10,000 IU, VD3 2 500 IU, VE 30.0 IU, VK3 2.00 mg, thiamin 2.20 mg, riboflavin 8.00 mg, niacin 30.0 mg, choline 400 mg, pantothenic acid 10.0 mg, pyridoxine 3.00 mg, biotin 0.15 mg, folic acid 1.00 mg, VB12 0.013 mg, Fe (as ferrous sulfate) 80.0 mg, Cu (as copper sulfate) 8.00 mg, Mn (as manganese sulfate) 110 mg, Zn (as zinc sulfate) 65.0 mg, I (as potassium iodide) 1.50 mg, Se (as sodium selenite) 0.30 mg.

2 Nutrient level were measured values except for metabolizable energy, lysine and methionine.

Before the experiment, the chicken housing, surrounding environment, and equipment were thoroughly disinfected. From 9 d of age, the chickens in all the groups were maintained on wire floors. During the first week, the temperature was controlled at 35 to 38°C; the temperature was then decreased by 2 to 3°C every week until a temperature of 20 to 22°C was reached. On the first day of the experiment, artificial light was provided for 20 h, after which it was decreased by 1 h every week until it was shortened to 18 h/d, after which it was maintained until the end of the experiment. During the experiment, all chicks had ad libitum access to the diet and water. The body weight (BW) of each bird was measured weekly, and the feed efficiency was calculated. The whole experiment was performed according to the yellow-feathered chicken feeding management standard (MOA, 2010), and the feeding experiment ended at 70 d of age.

Growth Performance

The fasting BW of each bird and the voluntary feed intake of each cage were recorded weekly, and the daily weight gain, feed intake, and feed conversion ratio (FCR) were calculated.

Sampling and Chemical Analyses

A metabolism experiment was conducted during the last 5 d (65− to 70 d), and the total excreta collection method was adopted to determine nutrient digestibility. Complete excreta from each replicate were collected daily. After the feathers, feed, dandruff, and other impurities were carefully removed, the excreta were weighed and thoroughly mixed. A portion of 200 g was sampled daily, and 5 mL of 10% (v/v) hydrochloric acid was added to prevent loss of ammonia nitrogen. The samples were stored at -20°C. Five days of samples of excreta from the same replicate were pooled and dried at 65°C until a constant weight was reached. The samples of excreta and feed were milled to pass through a 100-mesh (0.15 mm) screen and used to measure dry matter (DM), CP, ash, calcium (Ca), and phosphorus (P) (AOAC, 2005) to calculate the apparent nutrient digestibility. Gross energy (GE) was determined via an automatic rapid calorimeter (OR2014, Shanghai Ourui Instrument and Equipment Co., Ltd., China).

Slaughtering Performance

At d 70, nine birds (one bird per replicate) per treatment with a live BW closest to the average BW of the cage were selected for slaughter. The birds were euthanized, bled, scalded, plucked, and eviscerated. The dressed percentage and half-eviscerated and eviscerated yields were expressed as percentages of the live BW. The breast muscle, leg muscle, and abdominal fat were separated and weighed, the percentages of muscle mass to eviscerated carcass weight were calculated, and the percentages of fat mass to the weights of eviscerated carcass and abdominal fat were calculated (Chen et al., 2019).

Meat Quality

The left breast muscle was collected to determine the pH, meat color, drip loss, shear force, and cooking loss. The pH values at 1 h (pH1) and 24 h (pH24) after slaughter were measured using a meat pH meter (pH-STAR, Matthaus Company, Germany) following the methods of Berri et al. (2008). The meat color at 45 min (OPTO-Star1) and 24 h (OPTO-Star24) after slaughter was measured using a meat colorimeter (OPTO-STAR, Matthaus Company, Germany) as reported by Chai et al. (2010). Drip loss was measured as described by Jiang et al. (2018). The shear force was measured with a meat tenderness determinator (C-LM3B, Northeast Agricultural University, China) as described by Shao et al. (2018). Cooking loss was measured as described by An et al. (2015).

Immune Organ Index

Immune organs, including the liver, bursa of Fabricius, thymus, and spleen, were separated carefully and weighed. Organ indices are expressed as percentages of live BW.

Plasma Antioxidant Properties

Before slaughter, 5 mL of blood was sampled from the wing vein. The plasma was separated by centrifugation (TD5A-WS, Hunan Xiangyi Centrifuge Instrument Co., Ltd., China) at room temperature after anticoagulation with heparin sodium and stored immediately at -80°C for subsequent use. The plasma antioxidant properties, including peroxidase (POD) and glutathione peroxidase (GSH-Px) activities; Trolox equivalent antioxidant capacity (TEAC); and the concentrations of glutathione (GSH), lipid peroxide (LPO) and malondialdehyde (MDA), were assayed using kits from Jiancheng Bioengineering Research Institute Co., Ltd. (Nanjing, China) according to the manufacturer's protocols. One unit of POD activity was expressed as 1 μg of tetraguaiacol formed per minute per mL of plasma at 37°C. One unit (U) of GSH-Px activity was expressed as the amount in 1 mL of plasma that led to a decrease of 1 μmol/L GSH per minute at 37°C.

Intestinal Morphology

After slaughter, the intestine was quickly stripped, and 1 cm segments of the middle part of the duodenum, jejunum, and ileum were fixed in 10% formalin and stored in the dark for 48 h for slicing. The fixed tissues were embedded in paraffin, and 5-μm-thick sections were cut with a microtome (YD-335Ⅲ, Jinhua Yidi Medical Equipment Co., Ltd., Zhejiang, China) and stained with hematoxylin and eosin. The sections were observed and photographed under a light microscope (ECLIPSE Ci-L, Nikon Corporation, Tokyo, Japan), and the villus height (from the tip of the villus to the villus‒crypt junction) and crypt depth (from the villus‒crypt junction to the lower limit of the crypt) were measured with NIS-Elements D imaging software (Nikon Corporation, Tokyo, Japan), and ratio of villus height/crypt depth (V/C) was calculated.

The Gut Microbiota

Total DNA from 1.0 g of digesta in the middle part of the ileum was extracted using a QIAamp DNA Stool Mini Kit (Qiagen, Hilden, Germany). The qualified DNA was used as a template to amplify the V3-V4 hypervariable region of the bacterial 16S rRNA gene with the barcoded primer pair 341F (5′-cctaygggrbgcascag-3′) and 806R (5′-ggactacnngggtatctaat-3′) (Yang et al., 2022). Purified PCR amplicons were used to construct sequencing libraries and were subjected to paired-end sequencing on the Illumina MiSeq platform (Illumina Inc., San Diego, CA) according to the standard protocols provided by Novogene Bioinformatics Technology (Biejing, China). The UCHIME algorithm software (https://www.drive5.com/usearch/manual/uchime_algo.html, accessed on 3 Nov. 2023) was used for quality control of the raw sequence to generate clean reads. All the clean reads were then clustered using Uparse software (v 7.0, https://drive5.com/uparse/). Sequences sharing 97% similarity were clustered into an operational taxonomic unit (OTU), and a single representative sequence was chosen from each OTU for taxonomic annotation using the Mothur algorithm with the SILVA rRNA database (https://www.arb-silva.de/, accessed on 19 Nov. 2023). The α-diversity indices of the ileal microbiota, including the OTUs and the Shannon, Simpson and Chao1 indices, were calculated using QIIME 2 (https://qiime2.org/, accessed on 20 Nov. 2023). The β-diversity index and principal coordinate analysis (PCoA) results were evaluated according to the unweighted UniFrac distances. Venn diagrams of groups based on OTUs were generated at https://magic.novogene.com (accessed on 22 Nov. 2023).

Statistical Analysis

The general linear model procedure of SPSS (v. 18.0) was used to analyze the data on growth, digestibility, carcass traits, meat quality, plasma antioxidant properties, and gut morphology. When the main effect of treatment was significant (P ≤ 0.05), Duncan's multiple range test was used to analyze the significance of differences between groups. The Kruskal‒Wallis nonparametric test in GraphPad Prism (v. 9.0) was used to evaluate the differences in the α- and β-diversity indices. Metastats were used to analyze the relative abundance of taxa at the phylum and genus levels (White et al., 2009). The significance level was set at P ≤ 0.05.

RESULTS

Growth Performance and Nutrient Digestibility

The growth performance data are presented in Table 2. WGHe supplementation had no significant effect on BW, weight gain, feed intake, or FCR (P > 0.05). The effects of WGHe supplementation on the apparent digestibility of nutrients are shown in Table 3. Compared with the control treatment, 10 g/kg WGHe supplementation significantly increased the apparent digestibility of P (P = 0.036). No significant differences were found among the groups in terms of the apparent digestibility of DM, OM, CP, GE, or Ca (P > 0.05).Table 2 Effects of WGHe supplementation on growth performance in broilers.

Table 2Parameters	Antibiotics	WGHe, g/kg diet	SEM	P-value	
0	5.0	10.0	
9 to 28 d of age							
BW at 9 d of age (g)	105.04	105.04	104.92	105.05	0.058	0.333	
BW at 28 d of age (g)	682.79	688.59	661.89	665.52	10.856	0.251	
Weight gain (g/d)	30.41	30.71	29.31	29.50	0.571	0.254	
Feed intake (g/d)	50.08	49.97	48.57	48.73	1.051	0.634	
FCR	1.65	1.63	1.66	1.65	0.013	0.429	
29 to 49 d of age							
BW at 49 d of age (g)	1588	1608	1597	1629	27.8	0.748	
Weight gain (g/d)	43.09	43.80	44.53	45.87	1.074	0.316	
Feed intake (g/d)	98.84	100.99	100.62	101.03	1.924	0.831	
F/G	2.30	2.31	2.26	2.20	0.034	0.114	
50 to 70 d of age							
BW at 70 d of age (g)	2353	2352	2367	2377	39.3	0.967	
Weight gain (g/d)	36.47	35.43	36.65	35.62	1.227	0.865	
Feed intake (g/d)	113.23	112.48	113.80	113.71	2.157	0.971	
FCR	3.09	3.14	3.09	3.17	0.101	0.922	
9 to 70 d of age							
Weight gain (g/d)	36.86	36.84	37.08	37.24	0.645	0.967	
Feed intake (g/d)	88.61	89.05	88.94	89.11	1.337	0.994	
FCR	2.41	2.42	2.40	2.39	0.030	0.924	
Abbreviations: BW, fasting body weight; ADG, average daily gain; ADFI, average daily feed intake; F/G, ratio of ADFI to ADG.

a-bMean values without a common superscript letter within a row are significantly different (P < 0.05).

Table 3 Effects of WGHe supplementation on apparent nutrient digestibility in broilers.

Table 3Nutrients	Antibiotics	WGHe, g/kg diet	SEM	P-value	
0	5.0	10.0	
Dry matter (%)	76.61	78.20	77.40	76.97	0.725	0.458	
Organic matter (%)	77.86	79.91	79.41	78.79	0.700	0.210	
Crude protein (%)	52.33	54.20	54.11	54.66	1.645	0.763	
Gross energy (%)	79.96	79.28	80.16	79.38	0.686	0.757	
Calcium (%)	38.86	35.85	40.56	43.13	2.171	0.137	
Phosphorus (%)	41.02ab	38.52b	42.38ab	46.24a	1.804	0.036	
a-b Mean values without a common superscript letter within a row are significantly different (P < 0.05).

Slaughtering Performance and Meat Quality

Table 4 summarizes the effects of WGHe on the slaughtering performance and meat quality of broilers. No significant differences were observed in slaughter weight, dressed weight, dressed percentage, half-eviscerated yield, eviscerated yield, or mass of breast muscle or leg muscle among the 4 groups (P > 0.05). The abdominal fat content (%) of chickens fed the diet supplemented with 10.0 g/kg WGHe was significantly lower than that of birds fed the basal diet or antibiotic diet (P < 0.05).Table 4 Effects of WGHe supplementation on slaughter performance and meat quality of broilers.

Table 4Items	Antibiotics	WGHe, g/kg diet	SEM	P-value	
0	5.0	10.0	
Slaughter performance							
Slaughter weight (g)	2402	2435	2435	2433	50.2	0.956	
Dressed weight (g)	2186	2223	2225	2217	46.3	0.926	
Dressed percentage (%)	91.00	91.27	91.38	91.12	0.337	0.872	
Half-eviscerated yield (%)	86.21	86.08	86.26	85.93	0.320	0.877	
Eviscerated yield (%)	76.26	75.80	76.01	75.61	0.386	0.678	
Breast muscle (%)	16.62	18.02	17.00	17.03	0.664	0.497	
Leg muscle (%)	21.72	23.48	22.65	21.53	0.769	0.270	
Abdominal fat (%)	2.62a	2.72a	2.48ab	2.34b	0.084	0.019	
Meat quality							
OPTO-Star1	81.10	83.36	79.82	84.35	1.464	0.136	
OPTO-Star24	78.05	77.54	75.75	75.67	2.046	0.784	
pH1	6.35	6.07	6.20	6.21	0.162	0.670	
pH24	5.84	5.71	5.82	5.84	0.073	0.525	
Drip loss (%)	1.61	1.24	1.55	1.22	0.137	0.115	
Shear force (N)	61.52a	56.80ab	51.12b	50.90b	2.711	0.026	
Cooking loss (%)	6.37	6.66	6.61	7.52	0.794	0.747	
a-b Mean values without a common superscript letter within a row are significantly different (P < 0.05).

Dietary addition of WGHe had no significant effect on meat color, pH, drip loss, or cooking loss (P > 0.05). Compared with those in the antibiotic group, the shear force of the breast muscle in the WGHe supplementation groups was significantly lower (P < 0.05).

Immune Organ Indices

Table 5 shows that there were no significant differences in the liver index, thymus index, spleen index, or bursa index among the 4 groups (P > 0.05).Table 5 Effects of WGHe supplementation on immune organ indices in broilers.

Table 5Index	Antibiotics	WGHe, g/kg diet	SEM	P-value	
0	5.0	10.0	
Liver (%)	1.40	1.33	1.41	1.41	0.045	0.542	
Thymus (%)	0.52	0.46	0.52	0.43	0.043	0.348	
Spleen (%)	0.13	0.11	0.14	0.12	0.012	0.240	
Bursa of fabricius (%)	0.24	0.22	0.25	0.29	0.030	0.477	

Plasma Antioxidant Properties

The effects of WGHe on the plasma antioxidant properties of broilers are illustrated in Table 6. Compared with the antibiotic group, the WGHe supplementation groups presented significantly greater plasma POD activity (P = 0.025) and TEAC (P = 0.017). Compared with the control group, the antibiotic group and the 2 WGHe supplementation groups presented a decreased plasma MAD concentrations (P < 0.001). In addition, WGHe supplementation tended to decrease LPO in plasma (P = 0.08).Table 6 Effects of WGHe supplementation on plasma antioxidant properties in broilers.

Table 6Parameters	Antibiotics	WGHe, g/kg diet	SEM	P-value	
0	5.0	10.0	
POD (U/mL)	1.43b	1.66ab	1.71a	1.84a	0.088	0.025	
GSH-PX (U/mL)	8.24	8.82	8.53	8.38	0.697	0.942	
GSH (μmol/L)	8.83	8.29	8.61	8.99	0.398	0.638	
TEAC (mmol/L)	0.20b	0.25ab	0.29a	0.29a	0.021	0.017	
LPO (μmol/L)	3.95	3.40	3.33	2.85	0.291	0.080	
MDA (μmol/L)	3.02b	3.95a	2.97b	2.98b	0.136	<0.001	
Abbreviations: POD, peroxidase; GSH-Px, glutathione peroxidase; GSH, glutathione; TEAC, trolox equivalent antioxidant capacity; LPO, lipid peroxide; MDA, malondialdehyde.

a-b Mean values without a common superscript letter within a row are significantly different (P < 0.05).

Intestinal Morphology

The effects of WGHe supplementation on the intestinal morphology of broilers are presented in Table 7. Compared with that in the antibiotic group, the jejunal villus length in the 2 groups receiving WGHe supplementation was significantly increased (P < 0.05). Furthermore, compared with the control diet, dietary supplementation with 10 g/kg WGHe resulted in significant increases in the duodenal V/C ratio and jejunal villus length (P < 0.05).Table 7 Effects of WGHe supplementation on intestinal morphology of broilers.

Table 7Parameters	Antibiotics	WGHe, g/kg diet	SEM	P-value	
0	5.0	10.0	
Duodenum							
Villus length	1233.3	1270.4	1343.8	1323.2	40.59	0.225	
Crypt depth	131.88	154.89	147.93	129.15	8.495	0.114	
V/C ratio	9.48ab	8.25b	9.39ab	10.68a	0.571	0.043	
Jejunum							
Villus length	947.4c	988.64bc	1103.8ab	1155.6a	46.87	0.012	
Crypt depth	119.71	118.49	123.72	115.27	7.028	0.863	
V/C ratio	8.15	8.66	9.01	10.24	0.606	0.113	
Ileum							
Villus length	684.7	807.1	801.6	817.7	59.69	0.366	
Crypt depth	87.00	102.68	115.35	101.54	9.067	0.201	
V/C ratio	7.98	8.06	7.08	8.43	0.505	0.292	
V/C, ratio of villus height to crypt depth.

a-c Mean values without a common superscript letter within a row are significantly different (P < 0.05).

The Gut Microbiota

After high-throughput sequencing and quality control, more than 70 thousand clean reads of bacterial 16S rDNA were obtained from each sample, with an average length of 416 bp per read. All the raw high-throughput sequencing data of the 16S rDNA amplicons are available at https://www.ncbi.nlm.nih.gov/sra/SRR25178040-25178043, 25178045-25178054, 25178056-25178065, and 25178067-25178074. The effects of WGHe on the microbial ɑ-diversity indices of chicken ileal bacteria are shown in Figure 1. The number of OTUs was significantly greater in the high-dose group than in the control group (P < 0.05). Moreover, the Chao1 index in the high-dose group was greater than that in both the control group (P < 0.01) and the antibiotic group (P < 0.05).Figure 1 Effects of WGHe on the microbial ɑ-diversity indices of chicken ileal bacteria. (A), Operational taxonomic units (OTUs); (B), Shannon index; (C), Simpson index; (D), Chao1 index. C, the control group; A, the antibiotic group; L, the low-dose group; H, the high-dose group. *, P < 0.05; **, P < 0.01.

Figure 1

The results of the analysis of the ileal bacterial OTUs and β-diversity are shown in Figure 2. A total of 702 OTUs were shared by the 4 groups, while 50, 110, 114, and 529 unique OTUs were found in the control, low-dose, high-dose, and antibiotic groups, respectively (Figure 2A).Figure 2 Analysis of ileal bacterial OTUs and β-diversity. (A), Venn diagram of OTUs of ileal bacteria with different treatments; (B), comparison of the β-diversity index calculated on the basis of the unweighted UniFrac distance; (C), principal coordinate analysis (PCoA) of the ileal bacterial community on the basis of the unweighted UniFrac distance at the OTU level. C, the control group; A, the antibiotic group; L, the low-dose group; H, the high-dose group. **, P < 0.01; ***, P < 0.001.

Figure 2

Based on the unweighted UniFrac distance, the β-diversity index was significantly greater in the antibiotic group than in the control (P < 0.01), low-dose (P < 0.001), and high-dose groups (P < 0.01) (Figure 2B). The PCoA at the OTU level revealed that PC1 was 27.15%, and PC2 was 9.43% for bacteria (Figure 2C).

The top 10 phyla and 25 genera are highlighted in Figures 3A and 3B. At the phylum level, the dominant bacteria (relative abundance ≥ 5.0%) in the control group were Firmicutes. The supplementation of antibiotics or WGHe changed the ileal microbial community. In the WGHe supplementation groups, Firmicutes and Proteobacteria were the dominant bacteria, whereas in the antibiotic group, Firmicutes, Proteobacteria, and Cyanobacteria were the dominant taxa (Figure 3A). The relative abundance of Firmicutes was significantly increased in the control group compared with the antibiotic group (P < 0.05) (Figure 3C), whereas the relative abundance of Proteobacteria in the control group was significantly decreased (P < 0.05) (Figure 3D). At the genus level, Lactobacillus, Candidatus_Arthromitus, and Romboutsia were the most dominant bacteria (Figure 3B). In the high-dose group, Lactobacillus, Candidatus Arthromitus, and Escherichia-Shigella were the dominant bacteria.Figure 3 Relative abundances of broiler ileal bacteria and MetaStats analysis of the differences at the phylum and genus levels. (A–B), Relative abundances of the ileal bacterial community at the phylum and genus levels; (C–D), relative abundances of the phyla Firmicutes and Proteobacteria; (E–N) relative abundances of the genera Candidatus Arthromitus, Eubacterium coprostanoligenes, Ruminococcaceae UCG-014, Ruminococcaceae UCG-005, Lachnoclostridium, Romboutsia, Halomonas, Thauera, Shewanella, and unidentified Chloroplast, respectively. C, the control group; A, the antibiotic group; L, the low-dose group; H, the high-dose group. *, P < 0.05; **, P < 0.01.

Figure 3

The results of the MetaStats analysis revealed significant differences in the relative abundances of 253 genera between the groups. The top 10 genera (average relative abundance ≥ 1.0%) are shown in Figure 3. Compared with those in the antibiotic group, the relative abundances of Candidatus Arthromitus, Eubacterium coprostanoligenes, Ruminococcaceae UCG-014, Ruminococcaceae UCG-005, and Lachnoclostridium increased in the WGHe-supplemented groups (Figures 3E, 3F, 3G, 3H, and 3I). As the dosage increased, WGHe seemed to decrease the abundance of the 4 aforementioned genera and to increase the abundance of Lachnoclostridium. Moreover, the abundances of Romboutsia, Halomonas, Thauera, Shewanella, and unidentified Chloroplast were significantly increased in the antibiotic group (P < 0.05) (Figures 3J, 3K, 3L, 3M, and 3N).

DISCUSSION

In this study, for the first time, WGHe supplementation was shown to reduce abdominal fat deposition in broilers. Flavonoids and polyphenols may be important components of WGH extracts that contribute to reduced fat deposition. Walnut polyphenols have hypolipidemic effects on obese mice (Shi et al., 2014). The extract of walnut shells strongly inhibited triglyceride accumulation in 3T3-L1 preadipocytes (Yang et al., 2014). Walnut meal extracts can reduce or inhibit adipogenesis by inhibiting the differentiation of 3T3-L1 cells and decreasing triglyceride accumulation (Liang et al., 2017). Polyphenols and flavonoids efficiently suppress preadipocyte differentiation, inhibit adipogenesis, and induce adipocyte apoptosis. Ellagic acid, the main bioactive constituent in WGH, has shown strong inhibitory activity against fatty acid synthase (Wu et al., 2013). Caffeic acid, a polyphenol in WGH, downregulates lipogenesis gene expression, decreases cholesterol and triglyceride production, and increases the phosphorylation of enzymes related to lipid metabolism (Liao et al., 2014). Myricetin, a multifunctional flavonol in WGH, downregulates the mRNA and protein levels of major adipogenic transcription factors and reduces the mRNA levels of other adipogenesis-related transcription factors (Wang et al., 2015a). Since these enzymes and factors play important roles in the biosynthesis pathways of fatty acids and lipolysis, these findings suggest that WGHe may reduce fat production and triglyceride accumulation in adipocytes, thereby inhibiting fat deposition and promoting lipolysis in vivo. More research is needed to determine the exact mechanisms of action of WGHe.

Previous research has shown that dietary marigold and alfalfa extracts reduce shear force (Dong et al., 2011; An et al., 2015; Wang et al., 2017). In this study, muscle shear force was decreased by WGHe supplementation, indicating that WGHe increased muscle tenderness. Studies have shown that some plant extracts can increase the hydrolysis of sarcoplasmic reticulum, collagen, and myofibrillar proteins; change the structure of muscle fibers; increase the density of muscle fibers and the length of sarcomeres; and reduce the diameter of muscle fibers, thereby improving muscle tenderness (He et al., 2015).

The extracts of walnut byproducts show favorable antioxidant properties in vitro (Wang et al., 2015b; Cheraghali et al., 2019). WGH extracts obtained with water, alcohol, or a combination can effectively scavenge DPPH free radicals and have strong reduction ability (Oliveira et al., 2008; Fernández-Agulló et al., 2013). Polysaccharides extracted from WGH can effectively scavenge DPPH free radicals and exhibit excellent effects on O2·− and HO· due to their role as hydrogen donors, which alleviate the oxidative stress caused by free radicals (He et al., 2020). To date, studies on the antioxidant properties of WGH extracts have focused mainly on in vitro experiments. Whether WGH extracts initiate the same antioxidant effects in vivo is not clear. In the present work, the addition of WGHe improved POD activity and TEAC. Polyphenols and flavonoids are important antioxidants found in walnut and their byproducts. Polyphenols enhance the reducing power and free radical scavenging ability of WGH extracts in a concentration-dependent manner (Oliveira et al., 2008). After consumption of walnut meal, plasma γ-tocopherol and hydrophilic and lipophilic ORAC increased, and MDA and oxidized LDL decreased, in healthy adults (Haddad et al., 2014). These findings indicated that WGHe had antioxidant activity in vivo.

MDA is a biomarker that reflects oxidative stress and lipid peroxidation levels. Moreover, plant polyphenols and phenolic acids inhibit lipid peroxidation and reduce MDA formation (Caruso et al., 2022). Furthermore, juglone also has positive effects on antioxidation. This phytochemical has been found to increase the contents of lysozyme and glutathione in the thymus and to reduce the levels of MDA and NO in the blood of mice (Li et al., 2015). In this study, the plasma MDA concentrations were reduced under supplementation with WGHe, suggesting decreased oxidative injury; this decrease may have been due to increased oxygen free radical scavenging through increased POD activity and TEAC.

Dietary addition of polyphenols increases the villus height of the duodenum in broilers, as well as the V/C ratio of the duodenum and jejunum (Prihambodo et al., 2021). Wang and colleagues reported that the 50% hydroethanolic extract of WGH is rich in polyphenols such as catechin, rutin, and caffeic acid (Wang et al., 2021a). Addition of 100 to 200 mg/kg of the extract to a high-fat diet not only reduced the mRNA levels of inflammatory cytokines in rat colonic tissue but also increased the activity of antioxidant enzymes and the expression of intestinal tight junction proteins, indicating that WGH extract enhanced the physical barrier function of the intestine and prevented damage to intestinal tissue caused by a high-fat diet (Wang et al., 2021a). Furthermore, polysaccharides in WGH have been found to have similar effects (Wang et al., 2021b). After the addition of WGHe in this study, there were significant changes in the villus height of the jejunum, which are closely related to the high content of polyphenols and polysaccharides in the extract.

The intestinal microbiota serves as another barrier in the body. Many factors, particularly diet, have the potential to disrupt the homeostasis of the intestinal microbiota. The addition of WGH extracts or polysaccharides derived from WGH to the diet reduces the abundance of some potentially harmful bacteria, such as Lachnospiraceae and Christensenellaceae, while concurrently increasing the abundance of potentially beneficial bacteria, including Muribaculaceae, Ruminococcaceae, Lactobacillus, and Akkermansia (Wang et al., 2021a,b; Fang et al., 2022; Yang et al., 2023). In this study, supplementation with a high dose of WGHe increased the microbial diversity in the ileum. Compared with those in the antibiotic group, the relative abundances of Candidatus Arthromitus, Eubacterium coprostanoligenes, Ruminococcaceae UCG-014, and Ruminococcaceae UCG-005 in the WGHe-supplemented groups were increased in this study. Candidatus Arthromitus is crucial for the development of a well-functioning immune system in the host, as it tightly anchors to the epithelial cells of the ileal mucosa and specifically modulates the immune response of the host (Thompson et al., 2013). The epithelial-binding activity of Candidatus Arthromitus promotes a healthier early digestive system in turkeys, thus serving as an immunostimulatory probiotic (Danzeisen et al., 2013). The Eubacterium coprostanoligenes group harbors the gene encoding IsmA, a key enzyme involved in cholesterol metabolism. In synergy with Oscillibacter, it converts cholesterol into coprostanol, a compound that cannot be absorbed by the intestine, ultimately leading to a decrease in blood cholesterol levels (Li et al., 2024). Previous studies have shown that the Eubacterium coprostanoligenes group is negatively associated with triglycerides, total cholesterol and low-density lipoprotein cholesterol (LDL-C) (Wei et al., 2021; Deng et al., 2022). Additionally, the Eubacterium coprostanoligenes group is a butyrate-producing bacterium that helps maintain the integrity of colonic tissue and a healthy mucosal layer while alleviating intestinal inflammation (Fang et al., 2024). Ruminococcaceae UCG-014 and Ruminococcaceae UCG-005 utilize cellulose and starch to produce short-chain fatty acids (SCFA), including acetate, propionate, and butyrate. SCFAs not only are crucial energy sources for intestinal epithelial cells but also play roles in regulating immune responses, repairing the gut barrier, and enhancing fatty acid oxidation in liver and muscle tissues while inhibiting de novo lipogenesis and lipolysis (Lange et al., 2023). Consequently, these bacteria are considered a part of a healthy microbiome in the animal gut. On the basis of these findings, we speculate that in the WGHe-supplemented group, Candidatus Arthromitus and Ruminococcaceae work synergistically to maintain intestinal health, whereas Eubacterium coprostanoligenes and Ruminococcaceae cooperate to reduce blood lipid levels and decrease fat deposition in broiler chickens. Notably, high-dose WGHe apparently inhibited Candidatus Arthromitus, the Eubacterium coprostanoligenes group, Ruminococcaceae UCG-014, and Ruminococcaceae UCG-005. This phenomenon may be attributed to the broad-spectrum antibacterial properties of the WGH extract (Wang et al., 2023b).

CONCLUSIONS

WGHe is rich in polysaccharides, flavonoids, and phenolic compounds and has favorable antioxidant properties. Dietary WGHe supplementation does not increase the growth performance, feed conversion, slaughter traits, or immune organ indices of broiler chickens. However, WGHe supplementation decreases abdominal fat deposition; improves meat tenderness, antioxidant status, and intestinal villus integrity; and increases intestinal microbiota diversity. Genera such as Candidatus Arthromitus, Ruminococcaceae UCG-014, Ruminococcaceae UCG-005, and the Eubacterium coprostanoligenes group may play positive roles in maintaining intestinal health and decreasing lipid deposition. The recommended inclusion level of WGHe in broiler chicken feed is 10.0 g/kg.

DISCLOSURES

The authors declare no conflicts of interest.

ACKNOWLEDGMENTS

This work was funded by the National Natural Science Foundation of China (No.31560653 ); the Special Project of the Central Government Guidance on Local Science and Technology Development (ZYYD2023B09 ).
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