Dietary broccoli stem-and-leaf meal in laying hens: Effects on production performance, egg quality, antioxidant status, immune function, and gut microbiota composition

Abstract

This study examined the potential of broccoli stems and leaves meal (BSLM) as a functional feed ingredient for aged laying hens, focusing on its effects on production performance, egg quality, antioxidant status, gut immunity, and cecal microbiota during the late-laying phase. A 12-week trial involved 270 Hy-Line Brown hens (66 weeks old), divided into three dietary groups: control (basal diet), 0.5 % BSLM, and 1 % BSLM, with six replicates of 15 hens each. Production and egg quality parameters were presented across three age-defined phases: 66–69 weeks (Weeks 1–4), 70–73 weeks (Weeks 5–8), and 74–77 weeks (Weeks 9–12), as well as over the entire 12-week experimental period. Results showed that dietary BSLM increased egg production rates (P < 0.05) at all time points, with the 1 % BSLM group showing the highest values, while egg weight remained significantly higher in the control group during weeks 9-12 (P < 0.05). Feed intake remained unchanged, whereas the overall effect showed that both BSLM inclusion levels improved the feed-to-egg ratio compared to the control (P < 0.05). Eggshell thickness was greater in the 0.5 % BSLM group, while the 1 % BSLM group demonstrated superior albumen height, Haugh unit, and yolk colour (P < 0.05). Serum analysis indicated significant dietary effects (P < 0.05); 0.5 % BSLM reduced glucose and alkaline phosphatase levels, whereas 1 % BSLM lowered malondialdehyde levels, a marker of oxidative stress. Jejunal mucosal cytokines suggested a shift towards anti-inflammatory profiles in BSLM-fed hens, with the 1 % BSLM group exhibiting increased IL-2 and decreased IL-1β, IL-6, and IL-10 compared to the control (P < 0.05). Cecal microbiota analysis via 16S rRNA sequencing revealed that BSLM selectively enriched short-chain fatty acid-producing genera (Ruminococcaceae, Lachnospiraceae) and Parabacteroides, which correlated with increased IL-10 levels, indicating microbiota-driven anti-inflammatory effects. Overall, BSLM supplementation at 1 % inclusion level significantly improved production performance, egg quality, antioxidant capacity, and gut immune function, while positively modulating the cecal microbiota in aged laying hens. These findings suggest BSLM as a promising dietary strategy to enhance sustainability and productivity in poultry systems.

Introduction

Feed formulation remains a vital factor for productivity in poultry systems, with dietary ingredients markedly influencing nutrient intake, physiological functions, and product quality (Moss et al., 2021). As the nutritional landscape of poultry feeding continues to develop, non-traditional plant-derived ingredients have gained importance due to their rich supply of bioactive compounds capable of enhancing egg quality, regulating oxidative balance, and supporting immune function (Dansou et al., 2023; Obianwuna et al., 2024; Herranz et al., 2024). Recent research shows that these functional ingredients can improve laying performance (Tian et al., 2022; Liu et al., 2023), egg quality (Abdel-Hack et al., 2023; Esenbuga and Ekinci, 2023), immune and antioxidant responses (Mahfuz et al., 2021; Wang et al., 2024), and gut microbiota composition (Hu et al., 2024; Dedousi et al., 2024). Overall, these findings offer a foundation for investigating specific plant-derived by-products with both nutritional and functional benefits, thereby encouraging further exploration of broccoli residues.

Broccoli (Brassica oleracea L. var. italica) is increasingly recognised as a feed ingredient for poultry and other monogastric animals due to its rich nutrient profile and bioactive compounds. It provides vitamin C, dietary fibre, carotenoids, and glucosinolates with anticancer properties (Rao et al., 2021), along with fermentable oligosaccharides that improve hindgut fermentation in birds (Paturi et al., 2010). Fresh broccoli has high moisture content (about 86 %), moderate protein levels (around 5 %), 5–6 % carbohydrates, very low fat (<0.5 %), and valuable minerals such as calcium, phosphorus, iron, and zinc, highlighting its nutritional density (Wadmare et al., 2019). Global production has increased by 32 % over the past decade, reaching 37.2 million tonnes in 2018, mainly driven by China and India (De Evan et al., 2020). However, only the florets (∼25 %) are typically consumed, while stems and leaves (∼75 %) are discarded (Drabinska et al., 2022; Guddino et al., 2024), raising environmental concerns due to their perishability. Therefore, redirecting broccoli residues into poultry diets requires careful nutritional assessment to determine their suitability as functional feedstuffs.

Nutritional assessments show that broccoli residues are a nutrient-rich biomass, with their chemical composition strongly influenced by the leaf-to-stem ratio. Leaves typically contain higher levels of protein, minerals, and bioactive compounds than stems, which explains the variation reported across studies. For example, broccoli leaves contain approximately 12.1 % crude protein (CP) and 12.8 % crude fibre (CF), whereas stems provide 8.8 % CP and 15.0 % CF (Campas-Baypoli et al., 2009). When leaves and stems are combined, CP and CF values range from 24.86 % CP and 12.95 % CF (Mahmoud and Kholif, 2022) to 22.53 % CP and 10.97 % CF (Peña et al., 2025), though earlier work reported even higher CP (27.13 %) and lower CF (8.85 %) (Hu et al., 2012). Protein concentrations in residual leaves alone may reach 90–300 g/kg DM (Prade et al., 2021), and vitamins and minerals are present at two- to fourfold higher levels in leaves and stems compared with florets (Ying et al., 1992). Beyond their nutritive composition, broccoli by-products also supply substantial amounts of bioactive compounds. Peña et al. (2025) reported notably high carotenoid and xanthophyll concentrations in broccoli stem-and-leaf meal (BSLM). Leaf extracts exhibit strong antioxidant capacity attributed to phenolic compounds (Hou et al., 2023; Drabinska et al., 2018), whereas stem extracts possess antimicrobial activity linked to fatty-acid derivatives (Gudiño et al., 2024). These residues also contain appreciable quantities of fibre, protein, ash, essential fatty acids (linoleic, palmitic, linolenic), and amino acids such as aspartate, tyrosine, proline, glutamate, and valine (Meneses et al., 2020). Furthermore, glucosinolates, polyphenolics, peroxidases, flavonoids, and carotenoids confer additional functional properties, including anti-allergic, anticancer, and anti-obesity activities (Thomas et al., 2018; Pedroza et al., 2018). Collectively, these compositional and functional characteristics highlight the substantial potential of broccoli residues as a nutrient-dense, bioactive-rich feed ingredient that can enhance the nutritional and functional value of diets formulated for laying hens.

Evidence from animal feeding trials demonstrates that broccoli residues can serve as effective functional feed additives for laying hens, improving egg quality and yolk nutritional value. Graded inclusion of broccoli stem-and-leaf meal (BSLM; 30–90 g/kg) has been shown to maintain normal egg production while significantly improving yolk pigmentation and reducing yolk cholesterol in a dose-dependent manner (Hu et al., 2011). In older hens, supplementation with broccoli stems and leaves similarly enhanced egg production and increased yolk xanthophyll deposition, demonstrating sustained efficacy even in late-production birds (Peña et al., 2025). Dried broccoli floret residues further improved yolk quality by increasing yolk α-tocopherol concentrations and colour scores without adverse effects on laying rate, feed intake, or efficiency (Mustafa and Baurhoo, 2018). Additional evidence indicates that broccoli meal enriches the nutritional profile of eggs and deepens yolk pigmentation while maintaining overall productivity and egg-quality traits (Pedroza et al., 2018). Collectively, these studies highlight the potential of broccoli by-products as functional feed ingredients that can enhance yolk nutrient value and pigmentation without compromising hen performance.

A key limitation of including broccoli residues in poultry diets is their glucosinolate content, which, upon enzymatic breakdown, forms isothiocyanates capable of inhibiting iodine absorption, impairing thyroid function, and reducing performance when consumed in excess (Tripathi and Mishra, 2007). Although poultry can tolerate glucosinolate levels up to 5.4 μmol/g, these antinutritional factors require careful optimisation of dietary inclusion rates to prevent adverse effects. Such safety concerns likely contribute to the inconsistent responses in performance and egg quality reported across previous feeding trials, where moderate to high inclusion rates increased the anti-nutritional load (Hu et al., 2011; Mustafa and Baurhoo, 2018; Peña et al., 2025). These limitations underline the importance of dose–response assessments to establish safe and effective inclusion levels for broccoli by-products in layer diets. Accordingly, while broccoli residues possess considerable nutritional and functional potential, their practical use depends on evidence-based determination of suitable dietary levels. To address these considerations, the present study evaluated broccoli stem-and-leaf meal (BSLM) at low inclusion levels designed to minimise glucosinolate exposure while retaining the potential functional benefits of its phenolic and antioxidant constituents. For this reason, 0.5 % and 1 % BSLM were selected as biologically relevant, safety-conscious supplementation levels for aged laying hens.

Despite growing interest in utilizing broccoli by-products as functional feed ingredients, comprehensive evaluations of their effects in layer diets remain limited. However, previous studies have not simultaneously examined these by-products at low, safety-focused inclusion levels while assessing productive performance, serum antioxidant and immune responses, jejunal cytokine activity, and cecal microbiota in older laying hens. To address this gap, the current study investigates the functional potential of BSLM through a comprehensive evaluation of productivity, physiological markers, and gut microbiome changes. This multi-dimensional approach contributes new insights to the literature, as previous research has not integrated these endpoints to develop a detailed, mechanistic understanding of BSLM’s effects. The analysis of functional and biological outcomes of BSLM supplementation aims to promote the valorisation of plant-based agricultural residues as sustainable feed sources and to offer practical guidance for designing functional diets for modern laying hens.

Materials and methods

Animal ethics statement

All experimental protocols were approved by the Animal Care and Use Committee of the Institute of Feed Research, Chinese Academy of Agricultural Sciences (ACE-CAAS-20180704), and all animal experiments were conducted following the ARRIVE guidelines (Kilkenny et al., 2010).

Birds, diets and study design

A total of 270 healthy Hy-Line Brown laying hens, aged 66 weeks, were used for the experiment. The selected birds for the experiments have similar body weight and laying rate, the birds were acclimatized for a period of two weeks, before commencement of 12-week trial. The hens were randomly assigned to 3 experimental groups, each containing 90 hens (six replicates of 15 hens each). The groups were designated as follows: Control (basal diet without BSLM), basal diets supplemented with BSLM at 0.5 and 1 % respectively. The basal diets were formulated, to meet Chinese Feeding Standard of Chicken, Ministry of Agriculture of the People’s Republic of China (2004) and National Research Council, and Subcommittee on Poultry Nutrition (1994) requirements (Table 1). The experiment period lasted for 12 weeks (age of birds: 66 weeks old to 78-week-old). The hens were fed ad libitum, housed in three-tier battery cages (40 cm × 40 cm × 35 cm), with three birds per cage, corresponding to a stocking density of approximately 533 cm²/bird (0.053 m²/bird; 18.8 birds/m²). The environmental conditions (temperature range of 22-24°C and a relative humidity of 60-70 %) were maintained throughout the feeding trial. The animals were healthy throughout the feeding trial.

Table 1.

Composition and nutrient level of the experimental diet.

Ingredients	CON	0.5 % BSLM	1 % BSLM
Corn	64.00	64.00	64.00
Soybean meal	23.45	23.45	23.45
Coarse stone grains	7.00	7.00	7.00
Fine stone grains	2.00	2.00	2.00
Soybean oil	1.00	1.00	1.00
Calcium phosphate	0.96	0.96	0.96
Sodium Chloride	0.25	0.25	0.25
DL-Methionine	0.19	0.19	0.19
Premix	0.15	0.15	0.15
Wheat bran	1.00	0.50	0.00
BSLM	0.00	0.50	1.00
Total	100.00	100.00	100.00
Nutrient Content %
Crude protein	16.50(16.61)	16.50(16.72)	16.50(16.68)
ME MJ/Kg	11.32	11.32	11.32
Calcium	3.50(3.53)	3.50(3.49)	3.50(3.51)
Available Phosphorus (NPP)	0.28	0.28	0.28
Total Phosphorus	0.49(0.48)	0.49(0.49)	0.49(0.48)
SID Methionine+Cysteine	0.65	0.65	0.65
SID Lysine	0.75	0.75	0.75

BSL: Broccoli stem-leaf meal, 1 Premix supplied per kilogram of diet: vitamin A, 12,500 IU; vitamin D3, 4,125 IU; vitamin E, 15 IU; vitamin K3, 2 mg; thiamine, 1 mg; riboflavin, 8.5 mg; pyridoxine 8 mg vitamin B12, 0.04 mg; biotin, 0.1 mg; folic acid, 1.25 mg; Ca-pantothenate, 50 mg; niacin, 32.5 mg; Cu, 8 mg; Zn, 65 mg; Fe, 60 mg; Mn, 65 mg; I, 1 mg; Choline chloride, 500 mg; phytase, 200 mg. 2 The values in parenthesis indicate analyzed values. Others are calculated values. 3 CP (GB/T6432-2018), Ca (GB/T6436-2018), and TP (GB/T6437-2018) were measured values, while the other nutrient levels were calculated values referred to NY/T33-2004. Nutritional value of BSLM (Crude protein: 23.51 %, Crude fat: 4.27 %, Crude fiber: 7.13 %, Crude ash: 11.89 %, DM 90.04 %, ME. 13.62 MJ/kg)

Sample collection and analytical determination

Laying performance

Daily egg production and egg weight were monitored on a replicate basis. Laying rate was expressed as average hen-day production, calculated by dividing the total number of eggs by the total number of bird-days and multiplying by 100. Daily egg mass (DEM) was determined by dividing the total weight of normal eggs by the number of bird-days and multiplying by 100. Average egg weight (AEW) was calculated from total egg weight in grams per number of eggs produced. Feed intake (FI) was recorded weekly on a replicate basis and expressed as average daily feed intake (ADFI). Feed conversion ratio (FCR) was calculated as the feed-to-egg ratio, expressed as grams of feed consumed per gram of egg produced. The production performance indices obtained were presented at four-week intervals throughout the feeding trial (week 1 to 4, week 5 to 8, and week 9 to 12).

Egg quality assessment

Egg quality was determined at the interval of 4 weeks. The collection of eggs (30 eggs per treatment: 5 eggs per replicate), with a weight of a similar range, was done on the last day of every 4 weeks. The eggs were collected when freshly laid, and egg quality was determined within 12 h of collection. All sample eggs for egg quality evaluation were kept at room temperature before egg quality assessment. The eggs were each weighed with a sensitive electronic scale, and before starting of experimental trial and after the end of the trial, eggshell strength of all the eggs was measured with an Egg Force Reader (ORKA Technology Ltd., Ramat HaSharon, Israel). The eggs were then broken, and albumen height, yolk color score, and Haugh unit value were measured with an Automatic Egg Analyzer (ORKA Food Technology Ltd., Ramat HaSharon, Israel). The albumen and yolk were then separated using an egg separator, and each component was weighed. The eggshell was weighed after air-drying for 48 h. The percentage proportion of each component was obtained by subtracting the weight from the whole egg weight and multiplying by 100 (Sarlak et al., 2021). The thick and thin albumen were separated with a sieve (40-mesh), and the weight of each fraction was measured, following procedures described in the study by Zhou et al. (2021).

Serum biochemical indices and tissue collection

At the end of the 12-week trial and 77 weeks of age, 36 birds (1 bird per replicate, 6 birds per treatment) were randomly selected. The birds were fasted for 10 h before slaughtering, and then approximately 5 mL of blood was collected from the wing veins of the selected animals. The collected blood samples in the micro-anticoagulant tubes were kept in a fixed slant position for 30 min and then centrifuged (at 3000 g for 15 min). The separated serum was collected in Eppendorf tubes and stored at 20 °C until analyzed. Birds were euthanized immediately after blood collection. The jejunum was excised, opened longitudinally, rinsed with cold sterile saline, and the mucosal layer was gently scraped with a sterile glass slide. Mucosal samples were collected into pre-labeled cryotubes, snap-frozen in liquid nitrogen, and stored at –80 °C until biochemical and immunological analyses. Cecal contents were aseptically collected following the same procedure, snap-frozen, and stored at –80 °C until microbiota sequencing.

Before the analysis of the serum indices, the serum was thawed and maintained at 4 °C to avoid activation of enzymes. The concentrations of Alkaline phosphatase (ALP), Total protein (TP), albumin (ALB), GLB, alanine aminotransferase (ALT), Total cholesterol (TC), high density lipoprotein cholesterol (HDL-C), Triglyceride (TG), glucose (GLU), uric acid (UA), and ALP, levels in serum were determined by serum biochemistry test kit (Shanghai Kehua Bioengineering Co., Ltd., Shanghai, China). The concentrations of total superoxide dismutase (T-SOD), catalase (CAT), glutathione peroxidase (GSH-Px), malondialdehyde (MDA), and total antioxidant capacity (T-AOC) in the serum were analyzed with corresponding ELISA kits and spectrophotometrically measured (Shanghai Kehua Experimental System Co., Shanghai, China, Instrument model: Excellence 310, 2012). All procedures adopted were according to the manufacturer's instructions.

16S rRNA sequencing of cecal microbiota

Total genomic DNA was extracted from frozen cecal content samples using the E.Z.N.A.® Soil DNA Kit (Omega Bio-tek, Norcross, GA, USA), following the manufacturer’s protocol. DNA integrity was verified via gel electrophoresis. The V3-V4 hypervariable regions of the bacterial 16S rRNA gene were amplified using primer pair 338F/806R (5’-ACT CCT ACG GGA GGC AGC AG-3’ and 5’ -GGA CTA CHV GGG TWT CTA AT-3’). PCR amplification was performed with an initial denaturation at 95°C for 3 min, followed by 30 cycles of 95°C for 30 s, 55°C for 30 s, and 72 °C for 45 s, ending with a final extension at 72°C for 10 min. Amplicons were excised from 2 % agarose gels and purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA) in accordance with the kit instructions. The products were quantified, pooled, and subjected to paired-end sequencing on the Illumina MiSeq PE300 platform (Illumina, San Diego, USA), conducted by Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China). Microbial data analysis was performed using the free online Majorbio Cloud Platform (www.majorbio.com), provided by Shanghai Majorbio Bio-Pharm Technology Co., Ltd. Sequencing data were processed based on amplicon sequence variants (ASVs). Alpha diversity indices, including ACE, Chao1, and Shannon, were applied to evaluate species richness and community evenness. Beta diversity was assessed through Principal Coordinate Analysis (PCoA) based on Bray–Curtis dissimilarity. Group-wise differences were further examined using the Kruskal Wallis H test bar Plot on genus level.

Statistical analysis

“All data, excluding cecal microbiota, were analyzed using SPSS software (IBM SPSS Statistics, Version 27, 2020). Prior to analysis, datasets were evaluated for normality and homogeneity of variances. One-way ANOVA was used to assess the effects of dietary treatments, and when significant differences were detected, Tukey’s HSD test was applied to separate group means. Orthogonal polynomial contrasts were additionally performed to assess linear and quadratic responses to increasing BSLM inclusion. Statistical significance was set at P < 0.05. All results are presented as means ± pooled standard error of the mean (SEM).

Results

Effect of dietary BSLM on laying performance

The effects of dietary BSLM supplementation on laying performance are presented in Table 2. Egg production (EP) was significantly affected at all measurement points (P < 0.05) and over the entire period, with the 1 % BSLM group consistently showing the highest EP. Daily egg mass (DEM) was also significantly improved by BSLM inclusion, whereas egg weight differed only during Weeks 9–12, where the control group recorded higher values (P < 0.05). Feed conversion ratio (FCR; feed to egg ratio) improved significantly when the overall period was considered (P < 0.05), while feed intake was not influenced by dietary treatment at any time point (P > 0.05). Analysis of dose–response patterns showed clear linear increases in EP, DEM, and overall FCR as BSLM inclusion dose from 0 % to 1 %. Egg weight exhibited a weak quadratic effect during the final phase, reflecting higher values in the control group but no consistent pattern earlier in the study. Feed intake did not display linear or quadratic responses.

Table 2.

Effect of dietary BSLM on production performance of laying hens.

Items	Groups			SEM	P-value
	CON	0.5 % BSLM	1 % BSLM		ANOVA	Linear	Quadratic
66 to 69 wk old
EP, %	89.64b	90.75ab	92.67a	0.51	0.049	0.016	0.704
DEM, g	60.85	61.93	62.61	0.32	0.079	0.026	0.763
AEW, g	67.93	68.27	67.70	0.20	0.507	0.643	0.286
ADFI, g	121.70	117.03	117.72	1.36	0.331	0.241	0.359
FCR	2.00	1.89	1.89	0.03	0.138	0.077	0.357
70 to 73 wk old
EP, %	87.61b	89.08ab	91.41a	0.59	0.026	0.008	0.716
DEM, g	59.89	61.16	62.03	0.39	0.082	0.027	0.812
AEW, g	68.51	68.72	68.14	0.18	0.432	0.409	0.318
ADFI, g	117.75	116.93	115.89	0.94	0.734	0.436	0.956
F/E	1.97	1.91	1.87	0.02	0.194	0.074	0.844
74 to 77 wk old
EP, %	85.60b	88.46a	89.21a	0.54	0.015	0.006	0.342
DEM, g	58.30	60.14	59.87	0.40	0.132	0.112	0.215
AEW, g	67.25a	66.85ab	65.32b	0.33	0.039	0.016	0.404
ADFI, g	120.34	116.40	117.54	1.49	0.553	0.453	0.433
F/E	2.07	1.94	1.94	0.03	0.082	0.046	0.298
66 to 77 wk
EP, %	87.57c	89.40b	91.12a	0.32	<0.001	<0.001	0.939
DEM, g	59.60ab	61.07ab	61.58a	0.22	<0.001	<0.001	0.314
AEW, g	68.20ab	68.40a	67.80b	0.10	0.046	0.105	0.060
ADFI, g	119.93	116.79	117.05	0.74	0.158	0.112	0.277
F/E	2.01a	1.92b	1.90b	0.01	0.002	0.001	0.190

Abbreviations: EP, egg production; DEM, daily egg mass; AEW, average egg weight; ADFI, average daily feed intake; F/E, feed-to-egg ratio. n = 6 replicates per treatment (15 birds of each replicate). ^a-c Within a row, means with no common superscript differ significantly (P < 0.05).

Effect of dietary BSLM on egg quality

As presented in Table 3, supplementation of BSLM influenced egg quality traits including egg shape index, albumen height, Haugh unit, egg yolk proportion, eggshell thickness and yolk color (P < 0.05). At the end of 12 weeks, dietary effects were significant for egg shape index, albumen height, yolk color and Haugh unit (P < 0.05). Whereas, eggshell thickness showed variations across all time measurements (P < 0.05), with 0.5 % BSLM showing the highest value at the end of week 12. Nevertheless, there was no dietary effect on egg weight, eggshell strength, and the proportions of whole egg (shell, albumen and yolk) (P > 0.05).

Table 3.

Effect of dietary BSLM on egg quality of laying hens.

Items	Groups			SEM	P-value
	CON	0.5 % BSLM	1 % BSLM		ANOVA	Linear	Quadratic
69 wk old (wk 4)
EW (g)	67.82	65.74	66.25	0.44	0.128	0.140	0.162
Eggshell Prop (%)	10.54	10.63	10.34	0.11	0.534	0.450	0.409
Albumen Prop (%)	62.71	61.35	61.80	0.26	0.097	0.155	0.102
Yolk Prop (%)	26.75	28.02	27.85	0.24	0.056	0.054	0.145
Eggshell index	1.32	1.30	1.32	0.01	0.381	1.000	0.166
Eggshell strength	41.30	40.03	39.37	0.78	0.597	0.319	0.857
Eggshell Thickness	0.35	0.36	0.36	0.00	0.298	0.147	0.575
Albumen Height	6.60	6.49	6.57	0.19	0.975	0.961	0.825
Haugh Unit	78.06	77.41	74.46	1.52	0.592	0.338	0.723
Yolk color L	60.54	60.01	60.73	0.16	0.147	0.613	0.059
Yolk color a	1.85c	2.00ab	2.24a	0.08	0.112	0.039	0.773
Yolk color b	58.24	59.24	59.52	0.25	0.087	0.036	0.493
73 wk old (wk 8)
EW (g)	67.90	67.71	67.70	0.51	0.984	0.873	0.936
Eggshell Prop (%)	9.74	9.68	9.77	0.11	0.943	0.902	0.749
Albumen Prop (%)	64.57	64.07	64.72	0.25	0.532	0.805	0.274
Yolk Prop (%)	25.73	26.26	25.51	0.20	0.302	0.650	0.140
Eggshell index	1.33	1.32	1.32	0.01	0.968	0.852	0.862
Eggshell strength	35.54	37.52	36.28	0.64	0.449	0.639	0.241
Eggshell Thickness	0.37a	0.36ab	0.35b	0.002	<0.001	<0.001	0.955
Albumen Height	6.46	6.30	6.36	0.27	0.971	0.881	0.849
Haugh Unit	72.56	71.03	73.04	2.18	0.927	0.928	0.706
Yolk color L	62.38	64.25	64.13	0.67	0.451	0.292	0.488
Yolk color a	0.36	-0.24	0.16	0.14	0.190	0.538	0.086
Yolk color b	57.25	55.56	57.08	0.43	0.213	0.870	0.081
77 wk old (wk 12)
EW (g)	67.96	66.79	67.37	0.46	0.590	0.607	0.375
Eggshell Prop (%)	10.41	10.53	10.21	0.13	0.580	0.521	0.411
Albumen Prop (%)	63.34	62.19	63.12	0.31	0.278	0.765	0.117
Yolk Prop (%)	26.25	27.29	26.68	0.26	0.262	0.500	0.136
Eggshell index	1.37a	1.32b	1.33b	0.01	0.003	0.015	0.015
Eggshell strength	36.11	34.23	35.19	0.69	0.542	0.590	0.334
Eggshell Thickness	0.36	0.36	0.37	0.002	0.077	0.053	0.231
Albumen Height	5.93b	6.43ab	7.02a	0.18	0.045	0.013	0.904
Haugh Unit	69.85b	76.70ab	80.45a	1.70	0.034	0.011	0.660
Yolk color L	61.38	61.40	61.45	0.185	0.988	0.886	0.955
Yolk color a	1.64b	1.82ab	2.13a	0.082	0.046	0.014	0.703
Yolk color b	40.75	40.54	40.21	0.304	0.769	0.474	0.921

n = 6 replicates per treatment. (5 eggs per replicate). ^a-b Within a row, means with no common superscript differ significantly (P < 0.05).

Linear and quadratic contrast analyses highlighted specific dose–response patterns. At 69 weeks, yolk redness (a) and yellowness (b) increased linearly with BSLM inclusion (P = 0.039 and P = 0.036, respectively), despite the absence of a significant overall ANOVA effect. At 73 weeks, eggshell thickness increased linearly (P < 0.001) as BSLM inclusion rose from 0 % to 1 %, with no evidence of a quadratic response. At 77 weeks, eggshell index showed both linear and quadratic effects (P = 0.015 for each), reflecting the higher value in the control group and similar lower values in both BSLM groups, while albumen height, Haugh unit, and yolk a displayed clear linear increase with increasing BSLM inclusion (P = 0.013, 0.011, and 0.014, respectively). No linear or quadratic trends were detected for the remaining traits.

Effect of dietary BSLM on serum biochemical parameters

The effect of dietary BSLM supplementation on serum biochemical indices in are presented in Table 4. There were significant treatment effects on serum glucose levels (P < 0.05), with BSLM-fed hens showing lower values, and the 0.5 % group recording the lowest level. However, other serum biochemical indices (TP, ALB, GB, AST, UA, ALP, TC, HDLC, and TG), were not influenced by dietary treatments (P > 0.05). Polynomial contrasts further clarified these responses. Serum glucose displayed a significant quadratic effect (P = 0.015), reflecting the sharp reduction at 0.5 % BSLM followed by a return toward control levels at 1 % inclusion, whereas no linear trend was detected (P = 0.806). HDL-C also exhibited a significant quadratic response (P = 0.036), despite the absence of a main treatment effect, indicating a non-linear shift across diets. No other serum variables showed significant linear or quadratic patterns.

Table 4.

Effect of dietary BSLM on serum biochemical indices of laying hens.

Items	Groups			SEM	P-value
	CON	0.5 % BSLM	1 % BSLM		ANOVA	Linear	Quadratic
TP, g/L	71.55	70.09	70.96	0.743	0.758	0.756	0.507
ALB, g/L	25.21	25.26	25.05	0.280	0.963	0.845	0.853
GLB, g/L	46.35	45.30	45.91	0.644	0.829	0.793	0.588
AST, U/L	242.00	209.17	222.67	6.403	0.117	0.208	0.081
UA, μmol/L	399.00	328.00	374.17	17.000	0.234	0.546	0.113
ALP, U/L	230.00	157.50	214.75	14.905	0.194	0.630	0.086
GLU, mmol/L	4.00a	1.88b	3.78a	0.405	0.048	0.806	0.015
TC, mmol/L	2.44	2.93	3.05	0.150	0.229	0.109	0.559
HDL-C, mmol/L	15.35	17.82	14.67	0.614	0.094	0.616	0.036
TG, mmol/L	47.02	42.86	43.78	2.324	0.799	0.598	0.654

Abbreviations: TP, total protein; ALB, albumin; GLB, globulin; AST, aspartate transaminase; UA, uric acid; ALP, alkaline phosphatase; GLU, glucose; TC, total cholesterol; HDLC, High-Density Lipoprotein Cholesterol; TG, triglyceride. The data were expressed as Standard Error Mean (SEM), n = 6. Means annotated within a row, different superscript letters are significantly different (P < 0.05).

The effect dietary BSLM supplementation on antioxidant status of laying hens is presented in Table 5. There was a marked significant effect of BSLM on the levels of MDA, an oxidative marker (P < 0.05), the serum concentrations were significantly lower in the BSLM groups than the control. However, there was no effect of dietary treatments on other antioxidant enzymes; SOD, CAT, GSH-Px, and T-AOC (P > 0.05). Polynomial contrasts supported this observation. MDA showed a significant linear decrease with increasing BSLM inclusion (P = 0.009), indicating a dose-dependent reduction in lipid peroxidation, while the quadratic effect was not significant (P = 0.449). GSH displayed a trend toward a linear increase (P = 0.086), although this did not reach statistical significance. No linear or quadratic patterns were detected for SOD, CAT, or T-AOC.

Table 5.

Effect of dietary BSLM on the antioxidant status of laying hens.

Items	Groups			SEM	P-value
	CON	0.5 % BSLM	1 % BSLM		ANOVA	Linear	Quadratic
SOD, U/ml	79.21	82.28	79.58	2.195	0.894	0.947	0.652
CAT, U/ml	122.27	100.91	117.33	6.167	0.342	0.755	0.172
GSH, U/ml	213.50	229.52	243.97	7.531	0.205	0.086	0.965
T-AOC, U/ml	0.93	0.97	1.20	0.128	0.689	0.735	0.735
MDA, nmol/ml	4.15a	3.00ab	2.43b	0.262	0.026	0.009	0.449

Abbreviations: SOD, superoxide dismutase; CAT, catalase; GSH, glutathione Peroxidase; T-AOC, total antioxidant capacity; MDA, malondialdehyde. The data were expressed as Standard Error Mean (SEM), n = 6. Means annotated within a row, different superscript letters are significantly different (p <0.05).

Effect of dietary BSLM on jejunal mucosal immunity

The influence of dietary BSLM on jejunal mucosal immunity of laying hens is presented in Table 6. There was a marked influence of dietary BSLM supplementation on the jejunal mucosal immunity, significant variations among treatments were notable (P < 0.05): The immune indices including IL-1β, IL-6, and IL-10 concentrations were markedly higher in the control group compared with BSLM-fed hens (P < 0.05). In contrast, IL-2 was significantly elevated in the BSLM groups, with the highest values at 1 % inclusion, while the control group recorded the lowest levels (P < 0.05).

Table 6.

Effect of dietary BSLM on jejunal mucosal immunity.

Items	Groups			SEM	P-value
	CON	0.5 % BSLM	1 % BSLM		ANOVA	Linear	Quadratic
IL-1β, g/L	219.79a	176.69b	182.55b	7.082	0.004	0.01	0.026
IL-2, g/L	110.69c	131.06b	149.79a	4.253	<0.001	<0.001	0.745
IL-6, g/L	19.91a	17.15ab	16.66b	0.583	0.047	0.021	0.280
IL-10, g/L	34.91a	29.97b	28.25b	1.030	0.012	0.005	0.305

Abbreviations: IL-1β (Interleukin-1 beta), IL-interleukin 2, 6 and 10. The data were expressed as Standard Error Mean (SEM), n = 6. Means annotated within a row, different superscript letters are significantly different (p <0.05).

The polynomial contrast analysis showed a dose-response relationships; IL-1β exhibited both a significant linear effect (P = 0.010) and a significant quadratic effect (P = 0.026), reflecting the marked reduction from the control to BSLM-fed groups. IL-2 showed a strong linear increase (P < 0.001) with no quadratic component, confirming a consistent upward response to increasing BSLM inclusion. IL-6 demonstrated a significant linear reduction (P = 0.021), whereas its quadratic effect was not significant (P = 0.280). Similarly, IL-10 declined in a significant linear pattern (P = 0.005) without evidence of a quadratic response. Depicting dietary dose-dependent immunomodulatory effect, characterized by suppression of pro-inflammatory cytokines and enhancement of mucosal immune competence.

Cecal microbiota composition

16S rRNA sequencing showed that dietary BSLM did not affect α-diversity indices (Simpson, Chao1; P > 0.05) or global community structure, as PCoA/PCA revealed overlapping clusters with non-significant PERMANOVA values (R = 0.015–0.078, P > 0.18). Thus, overall microbial richness and diversity were preserved across treatments, as illustrated in Fig. 1. Despite this stability, genus-level analysis (Kruskal–Wallis, P < 0.05) identified selective taxonomic shifts. BSLM at 1 % enriched norank_f__Ruminococcaceae, Lachnospiraceae_UCG-, and norank_f__Erysipelotrichaceae, whereas BSLM at 2 % increased Faecalicoccus and Family_XIII_UCG-001. These taxa are saccharolytic and SCFA-associated, suggesting functional enhancement of gut fermentation, as illustrated in Fig. 2. Core genera (Bacteroides, Rikenellaceae_RC9_gut_group, Prevotellaceae) remained dominant in all groups, indicating that BSLM supplementation did not disrupt the basal community. Correlation analysis linked microbial shifts with host physiology, as illustrated in Fig. 3. Deferribacterota correlated positively with MDA, IL-1β, and IL-6 (P < 0.05), implicating this phylum in oxidative and inflammatory stress. Conversely, Parabacteroides correlated positively with IL-10 (P < 0.05), highlighting an immune-regulatory role. SCFA-producing genera (Faecalibacterium, Mediterraneibacter, Phascolarctobacterium) showed negative trends with MDA and IL-6, supporting their anti-inflammatory potential.

Fig. 1.

Effect of dietary BSLM on the microbiota diversity of laying hens. (A-C), alpha diversity indices: Simpson Index, Ace Index and Chao Index. (D-E) Beta diversity: principal coordinate analysis (PCoA). Abbreviations: CON, control; BSL, Broccoli Stem-Leaf meal: BSL1(0.5 %) and BSL2 (1 %), respectively.

Fig. 2.

Effect of dietary BSLM on the cecal bacterial composition of laying hens. (A) Effect of dietary BSLM supplementation on cecal bacterial differences at genus level in laying hens. (B-C) Microbial composition in layers’ cecum at the phylum and genus levels. Abbreviations: CON, control; BSL, Broccoli Stem-Leaf meal: BSL1(0.5 %) and BSL2 (1 %), respectively.

Fig. 3.

Heatmap of Pearson’s correlation between cecal microbiota and serum oxidative biomarker and jejunal mucosal immunity indices. morphological indices. The intensity of the colors ranging from red to blue represented the magnitude of correlation. Significant correlation was noted by * 0.01 ≤ P < 0.05.

Discussion

Our findings demonstrate that BSLM affects various physiological aspects in late-cycle hens, supporting previous research on plant by-products as regulators of productivity and gut–immune health. At a 1 % inclusion level, BSLM was linked to improved performance and specific enhancements in egg quality, along with biochemical and immunological changes indicative of reduced oxidative stress and a more balanced mucosal immune environment. These responses occurred alongside targeted shifts in SCFA-producing microbiota, suggesting that BSLM’s effects stem from coordinated modulation of metabolic, immune, and microbial pathways. This integrated pattern provides a basis for understanding the specific outcomes that follow.

Production performance

Laying performance in ageing hens generally declines due to reduced ovarian activity and decreased metabolic efficiency, both of which hinder the digestion, absorption, and utilisation of nutrients. In the present study, BSLM at a 1 % inclusion level improved laying rate, daily egg mass, and feed efficiency without changing feed intake, indicating that the enhanced performance was due to better utilisation of consumed nutrients rather than increased intake. Because feed intake did not differ among treatments, the higher productivity of BSLM-fed hens reflects more efficient absorption and metabolic use of nutrients, a pattern consistent with reports that phytogenic bioactives can enhance digestive function, antioxidant status, and nutrient partitioning in poultry (Mahfuz et al., 2021; Darmawan et al., 2022). These findings also align with earlier work showing that broccoli residues improve nutrient utilisation and egg production in layers without influencing feed intake (Hu et al., 2011). Similarly, Peña et al. (2025) reported improved performance in aged hens consuming broccoli by-products, although their diets contained slightly higher crude protein, suggesting that both improved nutrient quality and the activity of broccoli phytochemicals may contribute to the positive response. Evidence from quails supports this interpretation, with performance benefits at 10 g/kg but not 5 g/kg of broccoli floret powder (Tüzün et al., 2024), indicating that broccoli-derived additives exert their effects within an optimal inclusion range.

These improvements are likely linked to the phytochemical composition of broccoli by-products, which supply phenolics, glucosinolates, carotenoids, fibre, and vitamins that support gut integrity, stabilise redox balance, and enhance metabolic processing of dietary nutrients (Paturi et al., 2010; Andrés et al., 2025). Through these actions, BSLM likely enables a greater proportion of available nutrients to be channelled into egg production, consistent with improvements reported for other phytogenic additives that enhance digestive and metabolic efficiency in ageing hens (Mahfuz et al., 2021; Darmawan et al., 2022). Another factor contributing to the improved laying performance may be the enhanced antioxidant status observed in BSLM-fed hens, as indicated by reduced MDA levels. Broccoli by-products naturally contain glucosinolates, phenolics, carotenoids, and vitamins that help regulate oxidative balance and modulate inflammatory pathways, processes directly related to metabolic productivity and ovarian longevity in older birds (Andrés et al., 2025). Greater antioxidant capacity mitigates oxidative damage and supports mitochondrial efficiency, thereby allowing nutrients to be used more effectively for egg production rather than cellular repair. Comparable antioxidant-driven improvements in laying performance have been recorded with spirulina, ginger extract, green tea, and other phytogenic additives (Savary et al., 2017; Tufarelli et al., 2021; Luo et al., 2024; Derese et al., 2025).

Conversely, inconsistent findings in studies using much higher inclusion levels of broccoli by-products (30–120 g/kg) are likely related to increased intake of anti-nutritional compounds such as phytate, protease inhibitors, and oxalates, which impair digestibility and reduce metabolisable energy (Choudhury and Khaled, 2014; Hughes et al., 2009). Similar performance depressions have been reported for other high-inclusion vegetable residues, such as dried tomato pulp (Jafari et al., 2006). In contrast, the low inclusion level of BSLM used here appears to maximise the beneficial metabolic and antioxidant effects of broccoli phytochemicals while minimising anti-nutritional impacts. Together, these results indicate that BSLM supports laying persistence in aged hens by improving nutrient utilisation and enhancing antioxidant protection, both of which help sustain productive performance during the late-laying phase.

Egg quality

Egg quality is a crucial factor affecting the economic value of laying hens and generally declines with age as albumen quality, shell strength, and pigment deposition decrease due to reproductive and metabolic senescence. In this study, BSLM supplementation provided targeted yet significant improvements in albumen height, Haugh unit, and eggshell thickness, with varying effects on yolk colour, depending on the inclusion level. The lack of changes in eggshell strength and component proportions suggests that BSLM influenced specific quality traits rather than producing a uniform effect across all egg characteristics.

The increase in albumen height and Haugh unit at 1 % BSLM indicates that albumen quality in ageing hens is sensitive to oxidative and inflammatory stress. Albumen viscosity depends on the integrity of ovomucin and the functional state of the magnum, both of which decline as oxidative stress rises with age. Phytogenic antioxidants are known for preserving magnum epithelial structure, stabilising albumen proteins, and stimulating protein synthesis, thus providing a scientifically sound basis for the improvements observed here. For example, Obianwuna et al. (2022) demonstrated that plant-derived additives enhanced albumen quality by modulating protein metabolism and protecting oviductal tissues, while Spirulina platensis improved Haugh unit through increased protein synthesis (Salahuddin et al., 2024). Tea polyphenols likewise increased albumen height in aged hens by stabilising ovomucin and improving magnum morphology (Wang et al., 2018), and ginger extract or theabrownin elevated albumen indices by enhancing antioxidant enzyme activity and reducing oxidative markers (Wen et al., 2019; Zhang et al., 2022). These studies collectively highlight a common mechanism by which phytogenic antioxidants reduce oxidative damage, maintain epithelial integrity, and preserve albumen protein structure. The current findings support this pathway: hens fed 1 % BSLM showed higher albumen height and Haugh unit, along with decreased MDA and lower IL-1β and IL-6, indicating that improved redox balance and mucosal protection likely promoted more efficient albumen secretion.

However, inconsistent results have also been reported. A study by Hu et al. (2011) found that feeding 30–90 g/kg BSLM did not affect albumen traits or shell quality, whereas Mustafa and Baurhoo (2018) reported no changes in Haugh unit, albumen height, or shell strength with dried broccoli floret residues. Similarly, Peña et al. (2025) reported that broccoli leaves and stems did not affect Haugh unit or eggshell strength in aged hens. In quails, Tüzün et al. (2024) also found no improvement in albumen or shell parameters with broccoli floret powder. These discrepancies likely reflect differences in the botanical fraction used, nutrient density, and especially the inclusion level. Higher inclusion rates introduce greater amounts of anti-nutritional factors such as glucosinolate derivatives, fibre-bound phenolics, and oxalates, which can hinder nutrient absorption and impair both oviduct and shell gland function, thereby enveloping any phytochemical benefits. The positive albumen responses observed at the lower BSLM level in the present study suggest that improvements in egg quality occur within an inclusion window where the antioxidant and metabolic benefits of broccoli phytochemicals outweigh potential anti-nutritional effects.

Yolk pigmentation, a key consumer-driven quality attribute, is determined almost entirely by the hen’s dietary carotenoid intake, particularly xanthophylls such as lutein and zeaxanthin. These pigments are absorbed in the intestine via micelle-mediated lipid assimilation, incorporated into circulating lipoproteins primarily very-low-density lipoproteins (VLDL) and vitellogenin and subsequently transported to the ovary for selective deposition into the developing yolk (Karadas et al., 2006). Because broccoli stems and leaves contain abundant carotenoids and xanthophylls, their inclusion in the diet provides a direct substrate for this physiological pathway. Thus, the improvements in yolk colour observed in BSLM-fed hens are biologically consistent with known mechanisms of pigment absorption, transport, and yolk deposition. The carotenoid-rich nature of broccoli by-products has been clearly established.

The study by Peña et al. (2025) documented markedly higher xanthophyll levels in broccoli leaves and stems than in florets, leading to significant increases in yolk colour and yolk xanthophyll content when included in layer diets. Similarly, Mustafa and Baurhoo (2018), showed that carotenoid-rich broccoli florets increased yolk lutein and α-tocopherol concentrations in a linear manner, confirming efficient transfer of broccoli-derived pigments into yolk tissue. Earlier work by Hu et al. (2011) also reported dose-dependent improvements in yolk colour with BSLM, reinforcing the consistency of this response across broccoli fractions and inclusion rates. Studies in quails further support this mechanistic interpretation, with Tüzün et al. (2024) demonstrating that 10 g/kg broccoli floret powder enhanced yolk pigmentation through increased dietary pigment intake. Comparable findings from other carotenoid-rich feed ingredients underline the centrality of this mechanism. Microalgae such as Chlorella vulgaris and Spirulina platensis, among the richest natural sources of xanthophylls, produce robust improvements in yolk pigmentation (Panaite et al., 2023; Khan et al., 2021; Tufarelli et al., 2021; Salahuddin et al., 2024), primarily due to their high content of lutein, zeaxanthin, and β-carotene (El-Sabrout et al., 2022). These studies collectively demonstrate that when hens consume carotenoid-dense ingredients, the pigments are reliably absorbed, packaged into lipoproteins, and deposited in the yolk through a well-characterized physiological mechanism. Although pigment levels were not directly quantified in the present study, the enhancement of yolk colour in BSLM-fed hens aligns with established mechanistic pathways and the known pigment profile of broccoli by-products. These findings suggest that BSLM effectively supplies bioavailable carotenoids that enter the intestinal-lipoprotein-ovarian axis and improve yolk pigmentation. This mechanism is particularly relevant in aged layers, where digestive efficiency and pigment deposition may decline, meaning that carotenoid-rich feedstuffs such as BSLM can help sustain both functional and visual egg quality during late lay.

Eggshell quality in ageing hens is particularly vulnerable to oxidative stress and disturbances in calcium metabolism, because prolonged laying reduces shell-gland (uterine) efficiency, weakens antioxidant defenses, and impairs the expression and activity of calcium-transport proteins required for shell mineralisation. Consequently, eggshell formation becomes highly responsive to dietary phytochemicals capable of restoring redox balance and supporting uterine calcium transport. This mechanistic sensitivity is well-documented in aged birds: quercetin-based treatments improved eggshell thickness and strength by modulating eggshell-gland morphology, regulating immune responses, and altering the serum metabolome in late-laying hens (Wei et al., 2025). Similarly, quercetin supplementation (400 mg/kg) enhanced eggshell thickness and strength and improved albumen quality in older hens, effects linked to enhanced antioxidant status and adjustments in lipid metabolism (Liu et al., 2023).

In the present study, BSLM supplementation improved eggshell thickness, suggesting that the phenolic-rich matrix of broccoli by-products may have counteracted age-related declines in shell deposition by stabilising redox homeostasis within the shell gland. Evidence from other phytogenic interventions further supports this interpretation. Mulberry-leaf flavonoids increased eggshell thickness and strength by enhancing antioxidant capacity, promoting calcium deposition, and upregulating uterine calcium-transport genes such as CALB1 and PMCA1 (Huang et al., 2022). Likewise, polyphenol- and saponin-rich Quillaja and Yucca extracts improved shell mechanical properties by enhancing mineralisation efficiency in the uterine fluid (Souza et al., 2023). Quercetin, a key phenolic also present in broccoli leaves has been shown to improve shell quality in aged hens by suppressing oxidative stress, preventing lipid peroxidation in uterine tissues, and facilitating more efficient utilisation of dietary calcium (Fu et al., 2024). Given that broccoli by-products naturally contain quercetin, ferulic acid, and other potent phytogenic antioxidants, it is biologically plausible that BSLM exerted similar effects by reducing oxidative pressure in the shell-gland epithelium, improving mitochondrial function, and supporting the coordinated activity of calcium-binding and calcium-transport proteins essential for eggshell calcification.

The lack of improvements in earlier studies using broccoli residues (Peña et al., 2025; Hu et al., 2011; Mustafa and Baurhoo, 2018; Tüzün et al., 2024) may reflect the use of higher inclusion levels, different botanical fractions, or diets with a greater glucosinolate burden, which can depress calcium utilisation and offset potential phytochemical benefits. In contrast, the low-inclusion strategy used in the present study likely maximized the antioxidant and mineralisation-supportive properties of broccoli-derived phenolics while minimising anti-nutritional constraints. Overall, the improvement in eggshell thickness observed here indicates that BSLM can enhance shell quality in aged hens by supporting uterine redox stability and calcium deposition pathways at biologically appropriate inclusion levels.

In summary, BSLM supplementation was associated with improvements in albumen quality, yolk pigmentation, and eggshell thickness in aged hens. While the precise mechanisms were not directly measured in this study, our results are consistent with literature attributing such effects to antioxidant-mediated preservation of protein metabolism (albumen), dietary carotenoid deposition (yolk pigmentation), and enhanced calcium utilization and shell gland function (eggshell quality). These inferred pathways highlight the potential of broccoli by-products to sustain both the functional and visual qualities of eggs in aged layers. Beyond these improvements in egg quality traits, BSLM also influenced systemic metabolic status, as reflected in serum biochemical indices, providing additional insight into its physiological benefits.

Serum biochemical indices

Serum biochemical indices provide essential insights into metabolic and hepatic function in laying hens. In this study, broccoli stem–leaf meal (BSLM) influenced two key markers—serum glucose and alkaline phosphatase (ALP), indicating that broccoli-derived phytochemicals can modulate metabolic and liver status in aged layers.

Improved glucose regulation is particularly important in aged layers because it supports hepatic energy supply, mitigates age-related metabolic stress, and helps maintain egg production and egg quality during late lay (Noetzold and Zuidhof, 2025). In this study, serum glucose levels decreased significantly at 0.5 % BSLM, whereas the 1 % level produced values comparable to the control. This response suggests more efficient glucose utilisation and enhanced metabolic stability in older hens, which are typically more susceptible to reduced insulin sensitivity. Although the specific bioactive compounds in the BSLM used here were not analysed, the glucose-lowering effect aligns with established metabolic actions of broccoli-derived phytochemicals. Broccoli stems and leaves contain glucosinolates and phenolic compounds such as sulforaphane and quercetin (Wang et al., 2025). Sulforaphane, in particular, is known to reduce hepatic gluconeogenesis and improve insulin sensitivity through Nrf2- and AMPK-mediated pathways, with supporting evidence from prediabetic adults (Dwibedi et al., 2025) and obese mice supplemented with broccoli leaf extracts (Ranaweera et al., 2022). The stronger effect observed at 0.5 % is also consistent with the hormetic dose–response characteristic of isothiocyanates (Wang et al., 2024), where moderate inclusion levels elicit the most favourable physiological outcomes. Collectively, these findings suggest that 0.5 % BSLM may enhance glucose regulation in aged hens in a manner consistent with known metabolic activities of broccoli-derived phytochemicals, despite the absence of direct compound quantification in this study.

Alkaline phosphatase (ALP) is a key indicator of hepatic and biliary function, with elevated activity often reflecting hepatocellular stress, impaired bile flow, or disruptions in mineral metabolism (Righi et al., 2021). In this study, hens receiving 0.5 % BSLM exhibited significantly lower serum ALP activity than both the control and 1 % BSLM groups, indicating a more favourable hepatic status at the moderate inclusion level. This pattern aligns with the hormetic dose–response commonly observed with phytogenic compounds, in which moderate levels produce maximal hepatoprotective effects, while higher doses offer limited additional benefit (Barreiro-Sisto et al., 2024). The response is significant in aged layers, whose continuous egg production imposes heavy metabolic and oxidative demands on the liver (Alessandro et al., 2024). Although bioactive constituents of the BSLM were not quantified here, broccoli stems and leaves are known sources of phenolics and flavonoids, including ferulic acid, quercetin, and kaempferol: compounds associated with enhanced hepatic antioxidant capacity in poultry (Duan et al., 2021; Rodríguez-García et al., 2022). Comparable reductions in ALP have been reported in birds supplemented with phytogenic additives such as clove buds (Rahman et al., 2017), olive–laurel–rosemary leaf mixtures (Alessandro et al., 2024), spirulina (Salahuddin et al., 2024), and phenolic-rich compounds including quercetin (Cao et al., 2024) and ferulic acid (Shu et al., 2022). A broader synthesis by Righi et al. (2021) similarly highlights consistent reductions in hepatic enzyme leakage following supplementation with plant-derived additives. Broccoli by-products share this phytogenic profile. Ferulic acid is the predominant phenolic constituent in broccoli residues (Rodríguez-García et al., 2022), and broccoli leaves contain markedly higher total phenolics than stems (0.70 vs. 0.17 g GAE/100 g dry weight), along with notable quantities of quercetin and kaempferol (Duan et al., 2021). These compounds plausibly contributed to the lower ALP observed at 0.5 % BSLM by stabilising hepatocytes, reducing oxidative stress, and supporting nutrient metabolism, mirroring hepatoprotective effects reported for other phytogenic feed additives.

Serum lipid fractions, including triglycerides (TG) and high-density lipoprotein (HDL) cholesterol, serve as key indicators of systemic lipid transport and metabolic status in laying hens. In this study, dietary BSLM did not significantly affect these circulating lipid parameters, suggesting that its metabolic influence did not extend to systemic lipid mobilisation under the conditions tested. This outcome is consistent with the tightly regulated lipid homeostasis characteristic of aged layers, which must continually synthesise yolk precursors while preventing excessive hepatic lipid accumulation. Previous studies, however, have reported lipid-related effects of broccoli residues at the yolk rather than serum level. Hu et al. (2011) documented reduced yolk cholesterol in hens fed BSLM, associated with altered hepatic HMG-CoA reductase activity and increased cecal short-chain fatty acids, while Mustafa and Baurhoo (2018) observed enhanced lutein and α-tocopherol deposition along with lower yolk cholesterol when hens were supplemented with broccoli florets. Comparable improvements in lipid and protein metabolism have also been noted in rabbits receiving broccoli by-products (Mahmoud and Kholif, 2022), indicating that these residues can modulate nutrient utilisation in a tissue-specific manner. Collectively, these findings suggest that broccoli-derived residues may preferentially influence yolk lipid deposition pathways without necessarily altering circulating lipid fractions, which aligns with the absence of serum lipid changes observed in the present study.

Overall, the serum biochemical profile indicates that moderate inclusion of BSLM (0.5 %) exerts beneficial metabolic and hepatic effects in aged laying hens, reflected in reduced serum glucose and lower ALP activity. These responses align with established actions of broccoli-derived phytochemicals and support the concept of a hormetic dose–response, where moderate levels optimise physiological benefits. The absence of changes in serum lipid fractions suggests that BSLM does not disrupt systemic lipid homeostasis, despite evidence of yolk-level effects in previous studies. Collectively, the findings suggest 0.5 % BSLM as a potentially adequate inclusion level to support metabolic regulation and hepatic health in late-lay hens.

Jejunal mucosal immunity and antioxidant status

Maintaining vigorous jejunal immunity and antioxidant function is crucial in aged laying hens because the intestine is the primary site of nutrient absorption and is highly vulnerable to inflammation and oxidative stress, both of which impair epithelial integrity and overall productivity. Strengthening mucosal defence in older birds, therefore, has direct implications for feed efficiency, nutrient utilisation, and sustained egg production.

Cytokines play a central role in regulating intestinal immunity and maintaining mucosal homeostasis (Salemi et al., 2022). In poultry, elevated interleukin-1β (IL-1β) and interleukin-6 (IL-6) indicate epithelial stress or inflammation, whereas reductions imply improved mucosal integrity (Adedokun and Adeola, 2016). Interleukin-2 (IL-2) reflects T-cell activation and mucosal immune competence (Shouse et al., 2024), while interleukin-10 (IL-10) acts as an anti-inflammatory regulator. Although protective, increased IL-10 can also represent compensatory responses to intestinal stress (Wu et al., 2016). In the present study, dietary BSLM reduced jejunal IL-1β and IL-6 and elevated IL-2, indicating suppression of mucosal inflammation and enhancement of T-cell–mediated immune activity. The concurrent reduction in IL-10 further suggests that mucosal immune signalling was stabilised rather than hyper-suppressed, reflecting a shift toward balanced immune homeostasis. Such reductions in inflammatory load lower the metabolic cost of immune activation, allowing more nutrients to be allocated to productive processes such as egg formation and albumen synthesis. Importantly, the cytokine pattern observed in this study is consistent with the hormetic behavior of natural bioactive antioxidants, which exert their strongest immunomodulatory effects at low to moderate doses. Barreiro-Sisto et al. (2024) emphasized that many phytogenic compounds, including phenolics, flavonoids, and glucosinolate derivatives, modulate redox and inflammatory signalling through non-linear, dose-dependent pathways. This framework supports the interpretation that the low BSLM inclusion levels used here (0.5–1 %) fell within an optimal functional range, enabling effective suppression of pro-inflammatory cytokines while preserving or enhancing beneficial immune activity.

These immunomodulatory patterns are consistent with the known activities of phytogenic antioxidants. Several studies demonstrate that natural phenolics attenuate intestinal inflammation by reducing pro-inflammatory cytokine expression and strengthening epithelial defense. Quercetin and vitamin E reduced jejunal IL-1β and IL-6 while enhancing mucosal antioxidant capacity and secretory IgA in aged breeder hens (Amevor et al., 2022). Similarly, quercetin supplementation ameliorated LPS-induced intestinal inflammation and stabilized gut microflora in broilers (Sun et al., 2022), while lutein downregulated IL-1β, IL-6, and IFN-γ in chicken epithelial cells (Lin et al., 2023). Feng et al. (2023) also confirmed that quercetin reduced jejunal inflammatory cytokines in stressed hens, preserving mucosal structure. Broccoli stems and leaves are rich sources of quercetin and lutein (Duan et al., 2021), and the reductions in IL-1β and IL-6 observed in BSLM-fed hens are consistent with the well-established anti-inflammatory actions of these bioactives. Supporting this interpretation, lutein supplementation in aged hens reduced hepatic MDA, IL-6, IL-1β, and TNF-α while enhancing antioxidant enzymes (Dansou et al., 2024), paralleling the cytokine and oxidative patterns observed in this study. Collectively, these findings indicate that BSLM does not merely act as a generic phytogenic additive but provides bioactives with documented immunoregulatory and antioxidant properties. This mechanistic alignment helps explain why hens receiving BSLM displayed improved physiological resilience, enabling better utilization of dietary nutrients and contributing to measurable improvements in performance traits.

The improvement in antioxidant status further strengthens this interpretation; BSLM supplementation reduced serum malondialdehyde (MDA), indicating reduced lipid peroxidation and lower systemic oxidative stress. This is relevant to intestinal health, as oxidative injury compromises epithelial integrity and amplifies pro-inflammatory signalling. Phytogenic bioactives, including quercetin, lutein, and other phenolics abundant in broccoli leaves, are known to enhance endogenous antioxidant defences such as superoxide dismutase (SOD) and total antioxidant capacity (T-AOC), thereby protecting epithelial cells from reactive oxygen species (Duan et al., 2021). Quercetin acts as a potent ROS scavenger and suppresses NF-κB activation, while lutein stabilises lipid membranes against oxidative damage (Dansou et al., 2024; Feng et al., 2023). The combined presence of these compounds likely explains the dual improvement observed in this study: reduced IL-1β and IL-6, normalised IL-10, and enhanced IL-2 expression. Together, these effects reflect an integrated redox–immune stabilisation that preserves mucosal function, improves nutrient assimilation, and contributes to the superior feed efficiency and albumen quality observed in BSLM-fed hens. Improved mucosal health reduces nutrient losses to inflammation and enhances digestive efficiency, thereby supporting sustained productivity in late-lay periods.

Overall, BSLM supplementation promoted a more balanced mucosal cytokine profile and reduced oxidative stress, as evidenced by lower MDA levels and modulation of pro- and anti-inflammatory cytokines. These findings indicate improved mucosal immune competence and epithelial homeostasis in aged hens. Given the well-established interplay between mucosal immunity, oxidative balance, and microbial composition, these physiological improvements likely contributed to the favourable shifts in cecal microbiota and the enhanced productivity and egg quality observed in this study. Thus, the intestinal immunological and antioxidant benefits of BSLM provide a plausible biological basis for the improved production outcomes recorded in BSLM-fed hens.

Microbiota composition

Plant-derived by-products are increasingly recognised for their capacity to modulate the gut microbiota of aged laying hens beneficially. For example, fermented blueberry pomace improved barrier integrity and reshaped cecal microbial structure in late-phase hens (Qin et al., 2024), while fermented soybean meal enhanced performance, egg quality, nutrient digestibility, and antioxidant and immune status through microbiota-mediated effects (Obianwuna et al., 2024). These observations support the overarching concept that phytogenic residues can remodel microbial communities to enhance host physiology.

In the present study, BSLM supplementation selectively altered the cecal microbiota, enriching several key saccharolytic taxa, including Ruminococcaceae, Lachnospiraceae_UCG, Faecalicoccus, and Family_XIII_UCG-001, without affecting alpha diversity. These bacterial groups are consistently associated with fibre fermentation and short-chain fatty acid (SCFA) production, suggesting a shift toward a more fermentative microbial profile. Short-chain fatty acid production, particularly butyrate, is essential for maintaining intestinal health. Butyrate-producing taxa primarily within Ruminococcaceae and Lachnospiraceae play central roles in fermenting complex carbohydrates such as cellulose and resistant starch. Butyrate enhances epithelial barrier function, stimulates mucin production, and mitigates inflammation by suppressing NF-κB signalling and activating regulatory immune pathways (Rychlik et al., 2020; Ducatelle et al., 2023; Yue et al., 2024). These families are highly oxygen-sensitive and often decline under intestinal inflammation, making their enrichment in BSLM-fed hens indicative of a more favourable, less inflammatory gut environment.

Additional enriched taxa also support this interpretation. Faecalicoccus, an Erysipelotrichaceae member originally isolated from the chicken cecum (De Maesschalck et al., 2014), ferments carbohydrates into butyric, lactic, and formic acids. Its abundance increases in response to ferulic acid supplementation (Liu et al., 2021), suggesting that phenolic compounds in broccoli residues, particularly ferulic acid, may have contributed to its proliferation. Similarly, Family_XIII_UCG-001, a Clostridiales-affiliated group, is frequently co-enriched with Ruminococcaceae and Lachnospiraceae in chicken cecal networks (Farkas et al., 2022), implying functional complementarity in saccharolytic fermentation. Evidence from pigs associates the related Family XIII AD3011 with enhanced acetate, propionate, and valerate production (Wan et al., 2024), and poultry metabolomics link Family_XIII_UCG-001 to aromatic metabolite variation (Yang et al., 2024), further supporting its involvement in SCFA and aromatic compound turnover. Beyond fermentative metabolism, BSLM supplementation was associated with a notable correlation between Parabacteroides abundance and IL-10 levels. Parabacteroides distasonis stimulates IL-10 production in human PBMCs (Chamarande et al., 2022), and P. goldsteinii promotes immune normalization in inflamed piglets (Deng et al., 2025). Although direct evidence in poultry remains limited, the positive association observed in this study suggests that Parabacteroides may participate in immunoregulatory processes that contribute to the balanced cytokine profiles observed in BSLM-fed hens.

Taken together, the enrichment of SCFA-associated taxa and the immune-related correlation with Parabacteroides indicate that BSLM fosters a cecal environment conducive to fermentative metabolism, epithelial protection, and balanced immune responses. Although SCFAs were not directly quantified, their well-established roles in barrier reinforcement, nutrient absorption, and inflammation control provide a biologically plausible mechanism linking microbial remodelling with the improved feed efficiency, laying performance, and egg quality observed in this study. These findings suggest that BSLM supplementation shifts the gut microbiota toward a more fermentative, anti-inflammatory configuration, reinforcing the systemic antioxidant and immunomodulatory benefits described earlier.

In summary, the improvements observed across performance, egg quality, serum biochemistry, antioxidant status, and mucosal immunity support a unified mechanism in which BSLM enhances redox balance and immune stability while remodelling the cecal microbiota toward SCFA-producing taxa. Together, these physiological and microbial adaptations create the metabolic and immunological conditions necessary for sustained productivity and improved egg quality in aged laying hens.

Conclusion

This study shows that broccoli stem–leaf meal (BSLM) can be used effectively at low inclusion levels (0.5–1 %) to enhance productivity and physiological function in aged laying hens. The 1 % level produced the strongest improvements in laying rate, daily egg mass, feed efficiency, albumen height, Haugh unit, yolk pigmentation, and antioxidant status, indicating a clear dose-responsive benefit. Both 0.5 % and 1 % improved eggshell thickness, though the response was more consistent at the higher inclusion. Metabolic effects varied by dose, with 0.5 % producing the greatest reduction in serum glucose, whereas 1 % more strongly supported hepatocellular stability, as indicated by lower ALP. Immunologically, pro-inflammatory cytokines (IL-1β, IL-6) were reduced at both levels, but IL-2 enhancement, a marker of mucosal immune competence was greatest at 1 %. Microbiota shifts also favored the higher inclusion, as 1 % BSLM enriched key saccharolytic, SCFA-producing taxa, supporting improved gut function. Overall, 0.5 % BSLM offered moderate metabolic and anti-inflammatory benefits, while 1 % generated a broader and more pronounced set of improvements across performance, egg quality, immunity, antioxidant status, and cecal microbial composition. These findings highlight BSLM as a sustainable functional feed ingredient that enhances late-lay performance while contributing to the valorisation of vegetable by-products in circular agriculture.

CRediT authorship contribution statement

Ghazanfar Hussain: Writing – original draft, Methodology, Investigation. Uchechukwu Edna Obianwuna: Writing – review & editing. Congrui Wen: Investigation. Haihua Zhang: Writing – review & editing. Habtamu Ayalew: Data curation. Haijun Zhang: Investigation. Shugeng Wu: Resources, Project administration, Funding acquisition. Kai Qiu: Resources, Project administration, Funding acquisition.

Disclosures

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.