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

S0032-5791(24)00712-0
10.1016/j.psj.2024.104133
104133
MICROBIOLOGY AND FOOD SAFETY
Effects of dietary rosemary ultrafine powder supplementation on aged hen health and productivity: a randomized controlled trial
Li Wen *
Yang Meixue *
Luo Yuxing *
Liu Wei †
Wang Zhong *
Ning Zhonghua ningzhh@cau.edu.cn
*1
⁎ National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing 100193, China
† Zhuozhou Mufeng Poultry Company Limited, Zhuozhou 072750, China
1 Corresponding author: ningzhh@cau.edu.cn
31 7 2024
11 2024
31 7 2024
103 11 1041332 6 2024
24 7 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/).
Recently, poultry industry has been seeking antibiotic residue-free poultry products and safe nutritious feed additives. Whether rosemary ultrafine powder (RUP) affects productive performance by regulating the intestinal microbiome of aged layers remains unclear. Here, we investigated the effects of dietary RUP supplementation on the production performance, egg quality, antioxidant capacity, intestinal microbial structure, and metabolome of aged hens. The results indicate that RUP had no significant effect on production performance but significantly enhanced Thick albumen height, Haugh unit, yolk color (P < 0.05), daily feed intake, and qualified egg rate. Serum content of non-esterified fatty acids, catalase, and glutathione peroxidase increased significantly (P < 0.05). Furthermore, the liver total protein content was significantly increased (P < 0.05). 16S rRNA sequence analysis revealed that RUP significantly impacted both α- and β-diversity of the caecum microbiota. Linear discriminant analysis of effect size and random forest identified Bacteroides, Muribaculum, Butyricimonas, Odoribacter, and Prevotella as biomarkers in groups A and B. In comparing groups A and C, Barnesiella, Turicibacter, and Acholeplasma were critical bacteria, while comparing groups A and D highlighted Barnesiella and Candidatus Saccharimonas as differential bacteria. FAPROTAX analysis of the caecum microbiota revealed that the functional genes associated with harmful substance biodegradation were significantly increased in the RUP-fed group. Based on Spearman correlation analysis, alterations in microbial genera were associated with divergent metabolites. In summary, dietary RUP can improve egg quality and antioxidant capacity and regulate the intestinal microbiome and metabolome in aged breeders. Therefore, RUP can potentially be used as a feed additive to extend breeder service life at an appropriate level of 1.0 g/kg.

Key words

rosemary ultrafine powder
aged layer
antioxidant capacity
microbiome
metabolome
==== Body
pmcINTRODUCTION

Extending the productive lifespan of laying hens can decrease farming expenses and boost economic performance. However, in their later stages, these hens frequently show reduced productive, reproductive, and immune capabilities (Dai et al., 2020; Kowalczyk et al., 2020). To tackle these challenges, the poultry industry has traditionally used antibiotic growth promoters (AGPs) to maintain gut health, balance microbiota, and enhance growth performance. (Thakur et al., 2019; Mahmood and Guo, 2020; Ma et al., 2021). Studies have shown that the health of the gut microbiota significantly improves feed utilization efficiency, thereby increasing egg production rates. For example, supplementing the diet of laying hens with the probiotic Enterococcus faecalis can help maintain high production levels throughout the laying period (Shi et al., 2024). Additionally, specific gut microbiota such as Lactobacillus and Bacteroides are significantly associated with improved laying performance. Research indicates that dynamic changes in the composition of the gut microbiota during different laying stages directly affect the laying capacity of hens (Wang et al., 2023). Furthermore, research shows that microorganisms influence egg quality by improving physical and chemical properties. For instance, Bacillus subtilis and Rhodobacter capsulatus can enhance egg quality and increase resistance to pathogens such as Salmonella (Peralta-Sánchez et al., 2019).However, The widespread application of antibiotics in poultry farming has led to the rise of various bacterial strains resistant to antibiotics. This not only causes metabolic disorders and weakens the immune system of birds but also decreases intestinal bacterial populations, heightening disease susceptibility and potentially endangering consumer health. (Ashour et al., 2020; Oliveira et al., 2020).

Creating safe and nourishing feed additives that enhance intestinal immune status, boost performance, enhance the beneficial microbiota, and lessen the influence of pathogenic microorganisms has become especially crucial in light of the rising demand for antibiotic-free chicken products worldwide (Suliman et al., 2021). Accordingly, attention has been turned to herbs, spices, and other botanicals to enhance gastric secretion and intestinal health, effectively inhibit pathogen adhesion, exclude competing pathogens, synthesize antimicrobial molecules (Seidavi et al., 2022), and enhance immune defense mechanisms to improve meat and egg production in birds (Abou-Elkhair et al., 2020). Additionally, due to their antioxidant properties, various botanicals can directly enhance immunity (Kim and Lillehoj, 2019) while enhancing the oxidative stability of chicken eggs and meat, extending shelf life (Abd El-Hack et al., 2020; Seidavi et al., 2021; Seidavi, Tavakoli, et al., 2021). Recent research has provided more evidence of the advantages of medicinal plants and their derivatives for enhancing the wellbeing, health, and productivity of chickens, highlighting their significance and potential as feed substitutes for antibiotics (Abd El‐Hack et al., 2021a, b; Al-Sagheer et al., 2019).

For food preservation, rosemary extract (E392) has been certified by the European Union for its safety and effectiveness as a natural antioxidant (Younes et al., 2018). The plant known as rosemary (Rosmarinus officinalis L.) belongs to the genus Rosmarinus and dicotyledonous family Labiatae (Li et al., 2022) that originated in the Mediterranean Sea's coastal regions and is now commonly found in temperate locations, such as Europe and the US (Farouk et al., 2022). Rosemary's primary components include α-pinene, eucalyptin, rosmarinic acid, camphor, and 1,8-cineole (Nematolahi et al., 2018; Sienkiewicz et al., 2013), which are usually extracted from leaves and flowers via supercritical fluid extraction, hydrodistillation, distillation, or maceration techniques (Yao et al., 2023). Primarily composed of phenolic components, diterpenes, and triterpenes, rosemary extract includes phytochemicals such as rosmarinic acid, camphor, caffeic acid, ursolic acid, rhamnolic acid, and rhamnol (Ahmed and Babakir-Mina, 2020). These compounds exhibit antimicrobial (Rashidaie Abandansarie et al., 2019), antioxidant (Ebrahimi et al., 2020), antitumor (Allegra et al., 2020), and anti-inflammatory properties (Rocha et al., 2015; Brindisi et al., 2020; El-Demerdash et al., 2021a). In fact, rosemary's strong antioxidant properties are said to lessen lipid peroxidation and oxidative damage in animals (Yang et al., 2021). Accordingly, rosemary has been used as a natural additive in animal feed (Mohamed et al., 2021), spices, and medicinal supplements (Miladi et al., 2013; El-Demerdash et al., 2021b).

Different forms of rosemary extract at different concentrations are reportedly effective in improving poultry performance. For Romaine Brown laying hens, adding 3.5% and 5.2% rosemary leaf meal (RLM) to the commercial diet enhances feed intake, laying performance, feed conversion ratio, and egg quality (Kedir et al., 2023a). Similarly, laying chickens of all ages produce more eggs and have higher-quality, higher-yielding eggs when 0.6% rosemary powder is added (Alagawany and El-Hack, 2015). Meanwhile, the inclusion of 0.5% rosemary powder in the diet improves both the feed conversion ratio (FCR) and the growth rate of broiler chickens (Nadia et al., 2008), resulting in a 3.72 ± 1.65% increase in growth rate, decreased Escherichia coli abundance and increased Lactobacillus abundance in the intestinal microflora compared to controls (Norouzi et al., 2015). Rosemary supplementation has also been reported to reduce lipid oxidation in chickens, improving carcass characteristics (Loetscher et al., 2013; Rostami et al., 2018). Additionally, Borghei-Rad found that rosemary leaf powder supplementation for aged roosters enhances semen quality, including concentration, total sperm count, motility, and viability, while lowering malondialdehyde (MDA) levels. Furthermore, at 5.0 g/kg supplementation, it improved semen volume, seminal plasma membrane function, sperm penetration with fertilization, and breeding egg hatchability, slowing the decline of reproductive performance in aged roosters (Borghei-Rad et al., 2017). Moreover, rosemary oil mitigates the negative effects elicited by Newcastle disease on poultry growth (Khazaei et al., 2017), potentially due to its ability to enhance blood protein utilization (albumin/globulin ratio) (Rostami et al., 2018). Lastly, Tang et al. found that rosemary extract raises the cellular levels of the cardiac heat stress proteins heat shock protein (HSP)70 and crystallin alpha B (CryAb) as well as enhances antioxidant capacity (Tang et al., 2018), thus improving eggshell quality (Zhang et al., 2021). Research on the effects of supplementing with rosemary ultrafine powder (RUP) on the reproductive and production abilities of older layers is, however, comparatively lacking. Furthermore, it is yet unknown if RUP influences these characteristics by controlling the layers' gut microbiota. To assess the potential benefits of rosemary in laying hens, this study aimed to examine if RUP supplementation could enhance the laying performance and egg quality of older breeder hens by improving their intestinal microbial structure.

MATERIAL AND METHODS

Feed Preparation

RUP was assessed through high-performance liquid chromatography (HPLC), ensuring that the moisture content remained at or below 10%. The primary constituents were β-pinene (35.8%), camphor (23.3%), eucalyptus oleoresin (21.7%), and fennel esters (11.6%); small amounts of alkenes (alcohol compounds) were also detected.

To guarantee that the RUP dosage was met, the meals were prepared every 7 d. Table 1 shows the ingredients and nutrient content of the basal diet, which was comparable to a corn-soybean meal diet. This created three supplement profiles for the diet: low-dose rosemary (basal diet with 0.5 g/kg RUP), medium-dose rosemary (basal diet with 1.0 g/kg RUP), and high-dose rosemary (basal diet with 2.0 g/kg RUP).Table 1 Composition and nutrient levels of basal diets (dry matter basis: %).

Table 1Ingredient	Percent	Nutrient level3	Content	
Corns	61.38	Metabolic energy, MJ/kg	11.13	
Soybean oil	1.00	Crude protein	15.93	
Soybean meal	25.00	Lysine	0.76	
Limestone	10.00	Methionine	0.37	
NaCl	0.35	Methionine + cystine	0.65	
CaHPO4	1.75	Calcium	3.88	
High temperature resistant phytase (104 IU/g)	0.02	Available phosphorus	0.33	
Vitamin premix1	0.03	Total phosphorus	0.52	
Mineral premix2	0.15			
98% DL-Methionine	0.13			
50% Choline chloride	0.14			
Antioxidant	0.05			
Total	100.00			
1 Supplied per kilogram of diet: vitamin A, 13,500 IU; vitamin D3, 4,500 IU; vitamin E, 75 IU; vitamin K3, 3.6 mg; vitamin B1, 3.0 mg; vitamin B2, 9.24 mg; vitaminB6, 6.0 mg; nicotinic acid, 66 mg; pantothenic acid, 16.8 mg; biotin, 0.54 mg; folic acid, 2.10 mg; vitamin B12, 0.03 mg; vitamin C, 135 mg; choline, 675 mg; ethoxyquinoline, 15 mg;

2 Mineral premix provided per kilogram of complete diet: iron, 80 mg; copper, 10 mg; manganese, 100 mg; zinc, 100 mg; iodine, 0.35 mg; selenium, 0.30 mg;

3 ME, CP, and CF were measured values, and the other nutrients were calculated values.

Experimental Design

All animal handling and experimental procedures were approved by the Animal Care and Use Committee of China Agricultural University, Beijing, China (permit number: AW30601202-1-1). The experiment was conducted at Zhuozhou Mufeng Poultry Co., Ltd. (Zhuozhou, Hebei). All experimental chickens were housed in a barn equipped with an automated environmental control system and caged in a 3-layer ladder-type cage, with each replicate cage evenly distributed in the barn. The environmental control system automatically maintained the temperature of the chicken house between 23 and 25°C. Each hen was housed in cages within a chicken house equipped with negative pressure ventilation, receiving 16 h of light per day. The hens had unlimited access to food and water.

The experiment involved 216 Rhode Island Red egg breeders, aged 65 wk, who were in good health and had comparable body weights and output levels. The chickens were randomly divided into 4 groups, each with six replicates of nine birds: Group A received the basal diet; Group B received the basal diet with 0.5 g/kg RUP; Group C received the basal diet with 1.0 g/kg RUP; and Group D received the basal diet with 2.0 g/kg RUP. Following a thorough mixing process, the materials were ground into a powder and pelletized into 2.5 mm-sized granules. Pre-feeding lasted for 2 wk, while the actual experiment lasted for 8 wk out of the ten-week trial period. The formal period was divided into the early phase (65–68 wk) and the late phase (69–72 wk). The hens were fed at 08:30, 13:30, and 17:00 daily, and eggs were collected at 09:00 and 13:00. Manure was cleared once daily, and immunization of the chickens and disinfection of the barns were performed according to routine procedures.

Performance Parameters

Egg weights were recorded daily, as well as the quantity of cracked, abnormal, and deceased eggs laid by the hens. Each replicate cage's feed intake was recorded once a week. The feed-to-egg ratio, average weight of eggs, egg production rate, rate of broken eggs, rate of abnormal eggs, and average daily feed intake (measured at 65–72 wk) were all calculated.

Egg Quality Parameters

In all, 120 eggs were gathered at 68 and 72 wk of age, with 30 eggs (5 per replicate cage) randomly selected from each group. Room temperature testing was used to assess the quality of the eggs. The egg index and eggshell color were assessed using an egg-shaped index tester and a color tester (Konicaminolta CM-2600D), respectively. The eggshell color indices L∗, a∗, and b∗ represent lightness, redness, and yellowness, respectively. The strength of eggshells was tested with a quasi-static compression apparatus (Robotmation, Japan). Using a micrometre screw gauge, the thickness of the eggshell was measured at its bottom, middle, and top ends. The average of these 3 measurements was used to determine the eggshell thickness. An automated device for analyzing egg quality (Robotmation EMT-5200, Japan) was used to assess Thick albumen height, Haugh unit, and yolk color.

Serum and Liver Biochemical Parameters

After the trial was over, a single chicken per replicate cage was chosen at random to have blood drawn from the wing vein (a total of 6 hens/group). After the chicken was killed, liver tissue was also taken and the samples were kept at -80°C. Before analysis, serum was collected and kept at -20°C. Following the manufacturer's instructions, measurements were taken for total cholesterol (TCHO), triglycerides (TG), non-esterified (free) fatty acids (NEFAs), low-density lipoprotein cholesterol (LDL-C), uric acid (UA), high-density lipoprotein cholesterol (HDL-C) and total bile acids (TBAs). Additionally, enzyme markers were used as per the manufacturer's instructions to measure the following parameters: total antioxidant capacity (T-AOC), glutathione (GSH) content, glutathione peroxidase (GSH-Px) activity, malondialdehyde content, total superoxide dismutase (T-SOD) activity, and catalase (CAT) activity in serum and liver. The kits were bought from the Jiancheng Nanjing.

Long-Read 16S rRNA Sequencing Using the MinION Platform

On the final day of the study, one hen each replication (6 hens/group) was chosen at random and put to death with carbon dioxide. Each bird's caecum was removed, and it was quickly frozen in liquid nitrogen to facilitate the extraction of DNA. With the help of a DNeasy PowerSoil Kit (50) (QIAGEN, 47014), microbial genomic DNA was isolated. The purity and concentration of the DNA were ascertained using agarose gel electrophoresis and a NanoDrop One spectrophotometer (NanoDrop Technologies, Wilmington, DE).

The PCR bar-coded primers indicated that the purified DNA was directed towards the V1–V9 region of the 16S rDNA gene (27F: 5′-CAGCACCTGTCTCGTGGGCTCGG-AGAGTTTGATCMTGGCTCAG-3′ and 1492R: 5′-CAGCACCTTCGTCGGCAGCGTC-CGGTTACCTTGTTACGACTT-3′). After the initial denaturation at 94°C, 12 cycles were conducted as follows: 30 s at 94°C, 20 seconds at 60°C, 2 min at 65°C, followed by a final extension step of 10 min at 65°C. The PCR products were cleaned and quantified with AMPure XP beads (Beckman Coulter, Brea, CA) and 1% agarose gel electrophoresis, following the instructions provided by Qubit 3.0 Fluorometer (Life Technologies, Carlsbad, CA) and Nanopore, respectively. Mixed amplification products were pooled according to read number.

Following the manufacturer's instructions, a MinION nanopore sequencer (Oxford Nanopore Technologies, Oxford, UK) fitted with an R10.4 flow cell (FLO-MIN106D) was used to perform 16S rRNA amplicon sequencing. An amplicon library was created using a 16S Barcoding Kit 1–24 (SQK-16S024, Oxford Nanopore Technologies, Oxford, UK). For PCR amplification and barcoding, 0.25 g of template DNA extracted from fecal samples was combined with the LongAmp Taq 2X Master Mix (New England Biolabs, Ipswich, MA). The program MINKNOW (19.12.5) was utilized to collect the data.

Taxonomic Analysis of Nanopore 16S rRNA Sequencing Data

Fast5 files, recognized by GUPPY (https://community.nanoporetech.com/, 5.0.16, barcode kits "SQK-LSK114," require_barcodes_both_ends), were used as the downstream raw data from Nanopore sequencing (De Coster et al., 2018) for base calling and barcoding. NanoFilt v.2.7.1 (De Coster et al., 2018) was utilized to remove sequences with quality values below 10.Primer sequence identification was performed using cutadapt v3.5 (Martin, 2011) software for length filtering and primer sequence identification and removal, resulting in clean sequences devoid of primer sequences. Minimap2 was utilized to identify and eliminate chimera sequences (Li, 2018) and yacrd aligned to the Silva alignment release 123 database (Yilmaz et al., 2014) using LAST software (Kiełbasa et al., 2011). A feature list was created after the sequence with the highest score was determined to be the most ideal comparison outcome. After obtaining the feature list, all samples were smoothed with the minimum number of sequence entries after filtering the low-abundance features using QIIME2 (version 2022.3) software (Bolyen et al., 2019).

R packages were used for downstream analyses (McMurdie and Holmes, 2013; Chong et al., 2020). By utilizing the Chao1 index and the number of detected characteristics, the alpha diversity of gut bacteria was assessed. To assess the significance of the variations in alpha diversity, a paired t-test was employed. Scripts from QIIME 2.0 were employed to analyze the alpha and beta diversities of the chicken gut microbial community. The beta diversity was assessed using the Bray-Curtis dissimilarity method. The R Vennerable and vegan programs were used to create species accumulation curves and Venn diagrams, respectively. The study (Gao et al., 2024) conducted linear discriminant analysis (LDA) of effect size (LEfSe) to identify significant species and taxonomic compositions that underwent considerable alteration due to RUP treatment. For charting and analysis, a linear discriminant analysis value > 3.0 and a factorial Kruskal-Wallis test alpha value with a P-value < 0.05 were used. The projected metagenomic functional content was calculated using FAPROTAX (Louca et al., 2016) software by integrating 16S rRNA data with the 13.5 version of the Greengenes 16S rRNA Gene Database. STAMP (V2.1.3) was used to conduct a pairwise statistical comparison study (Welch's t-test, narrative false discovery rate (FDR) adjustment) of the microbial function (Parks et al., 2014).

Untargeted Metabolomics by Liquid Chromatography-Mass Spectrometry

After gentle thawing at 4°C, a precooled solution consisting of methanol (2 parts), acetonitrile (2 parts), and water (1 part) was added to the fecal samples and carefully mixed. The samples were then centrifuged at 14,000 × g and 4°C for 20 min. Following vacuum drying, the supernatant was redissolved in 100 μL of a 1:1 v/v acetonitrile-water solution, vortexed, and centrifuged at 14,000 × g and 4°C for 15 min. The resulting supernatant was retained for further analysis.

An Agilent 1,290 Infinity LC UHPLC system equipped with an HILIC column was employed to analyze the samples. The flow rate was set to 0.5 mL/min, and the column was maintained at a constant temperature of 25°C. For each sample, 2 μL was injected. The mobile phase consisted of Component A (25 mM ammonium acetate and 25 mM ammonia in water) and Component B (acetonitrile). Samples were stabilized during the study by being stored in an autosampler at 4°C. The order in which the samples were analyzed was random in order to reduce the influence of signal fluctuations and guarantee the accuracy of the findings. Quality control (QC) samples were included in the set to monitor the accuracy of the experimental results and the system's performance. For analysis, an AB Triple TOF 6600 mass spectrometer with an electrospray ionization (ESI) source was used. The mass spectrometry process was conducted in both positive and negative ion modes.

Metabolome Bioinformatics Analysis

Proteo Wizard was the program used to transform raw data. Peak alignment, peak area extraction, and retention time correction were performed using XCMS software. From there, a list of m/z and retention times was acquired for each sample, along with matching intensities for each metabolite. The data were then mapped into the Human Metabolome Database (HMDB, http://www.hmdb.ca/) and the Metabolite and Tandem MS Database (METLIN, https://metlin.scripps.edu/) to identify the metabolites. Both one-dimensional and multidimensional studies were used to identify different metabolites. Metabolites with P ≤ 0.05 and variable importance in projection (VIP) ≥ 1.0 were considered distinct. The normalized data were subjected to principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA). The R tool ggplot2 was used to create volcano graphs. Hierarchical clustering was used for the clustering process, and the metabolic pathway enrichment of differential metabolites was computed using Euclidean distance in the Kyoto Encyclopedia of Genes and Genome (KEGG) database. Metabolic pathway enrichment analysis was conducted for differentially expressed metabolites based on the KEGG database. Based on differential metabolites, metabolic pathways with P < 0.05 were shown to be considerably enriched. Correlations between differential microbiota and metabolite studies were performed using OmicShare tools, a free online data analysis platform (https://www.omicshare.com/tools).

Statistical Analysis

All graphics and data calculations were created using R software (version 4.2.3), Prism8 (GraphPad), and SPSS (version 25.0; SPSS Inc., Chicago, IL). The study utilized ANOVA and Duncan's multiple range test to identify significant mean differences across the treatments. To validate significant differences, the relative abundance (%) of bacteria at the phylum and genus levels within the microbiome was evaluated using the non-parametric Kruskal-Wallis test. Results are shown as the mean and the pooled standard error (SEM). A trend was defined as 0.05 > P < 0.10, and statistical significance was set at P < 0.05.

RESULTS

Laying Performance and Egg Quality

Table 2 displays the laying performance analysis data for the chickens. There were no discernible variations between the groups' egg production, weight, mortality and culling rate (P > 0.05). At 65 to 72 wk of age, groups B, C, and D's feed intake exhibited an increasing trend (P = 0.077) in comparison to Group A. Furthermore, Groups B, C, and D displayed an increasing tendency in the abnormal egg rate from wk 65 to 72 as compared to Group A (P = 0.039).Table 2 Effects of supplemental RUP on growth performance of laying hens in late laying period.

Table 2Item	RUP supplemental level/(mg/kg)	
A	B	C	D	
65 weeks of age	
 Egg production, %	76.85 ± 9.71	76.85 ± 10.78	76.54 ± 6.16	75.62 ± 6.97	
 Egg weight, g	58.44 ± 1.71	58.31 ± 1.50	58.60 ± 1.04	57.75 ± 0.76	
 Abnormal egg, %	1.21 ± 2.14	0.93 ± 1.02	1.05 ± 1.73	0.72 ± 1.12	
65 to 72 weeks of age	
 Egg production, %	73.09 ± 6.97	71.47 ± 7.76	75.10 ± 5.66	71.52 ± 8.08	
 Egg weight, g	58.57 ± 0.67	58.100 ± 1.61	58.37 ± 0.80	57.99 ± 0.90	
 Abnormal egg, %	0.67 ± 0.55c	1.97 ± 1.43ab	2.45 ± 3.21a	1.68 ± 1.45ab	
 Feed intake, g/day/hen	87.64 ± 5.40	87.73 ± 5.78	91.93 ± 6.67	89.13 ± 2.54	
 FCR, g feed/g egg	2.18 ± 0.12	2.30 ± 0.23	2.27 ± 0.22	2.32 ± 0.27	
 Mortality and culling rate, %	1.85 ± 2.87	3.70 ± 6.73	2.78 ± 4.65	0.93 ± 2.27	
FCR, feed conversion ratio.

During the 8-wk trial period, RUP supplementation led to a significant increase in Thick albumen height, Haugh unit, and yolk colour (P < 0.05; Table 3). Groups B and C had considerably lower Eggshell color (ESC-a*) levels at 68 wk (P < 0.05) than the other 2 groups. Compared to Group A, Group D's Eggshell color (ESC-b*) was notably lower (P < 0.05), while Group C's was markedly higher.Table 3 Effects of supplemental rosemary ultrafine power on egg quality of laying hens in late laying period.

Table 3Item	Treatment	
	A	B	C	D	
68 weeks of age					
 ESC-L*	63.62 ± 3.10	65.25 ± 3.35	65.22 ± 3.12	64.61 ± 2.49	
 ESC-a*	15.0 ± 1.59a	14.28 ± 1.32b	14.28 ± 1.27b	15.21 ± 1.24a	
 ESC-b*	18.93 ± 1.25b	19.09 ± 1.14b	20.17 ± 1.08a	17.71 ± 0.81c	
 Shape index	1.27 ± 0.04	1.27 ± 0.03	1.27 ± 0.03	1.27 ± 0.05	
 Egg weight, g	57.78 ± 4.36	57.68 ± 4.78	58.93 ± 3.25	58.374 ± 4.48	
 Eggshell strength, kg/cm2	3.62 ± 0.75	3.96 ± 0.81	3.84 ± 1.03	3.98 ± 0.60	
 Thick albumen height, mm	5.71 ± 1.56b	6.72 ± 0.72a	7.21 ± 1.54a	6.56 ± 1.72a	
 Haugh unit	73.87 ± 12.64b	83.00 ± 5.30a	84.13 ± 9.19a	79.23 ± 14.41ab	
 Yolk colour	5.29 ± 0.60	5.20 ± 0.65	5.39 ± 0.69	5.26 ± 0.53	
 Eggshell thickness, mm	0.39 ± 0.03	0.37 ± 0.03	0.39 ± 0.04	0.39 ± 0.03	
72 weeks of age					
 ESC-L*	62.40 ± 3.59	61.97 ± 2.58	63.75 ± 3.91	63.53 ± 2.98	
 ESC-a*	17.89 ± 1.65	17.04 ± 2.20	18.07 ± 1.54	17.37 ± 1.63	
 ESC-b*	25.47 ± 1.52	24.67 ± 1.92	25.51 ± 1.21	25.44 ± 1.35	
 Shape index	1.27 ± 0.04	1.28 ± 0.04	1.27 ± 0.04	1.28 ± 0.05	
 Egg weight, g	57.49 ± 4.08	57.55 ± 4.37	58.81 ± 3.68	56.71 ± 4.76	
 Eggshell strength, kg/cm2	3.70 ± 0.76	3.62 ± 0.98	3.67 ± 0.86	3.97 ± 0.97	
 Thick albumen height, mm	5.60 ± 1.79	5.72 ± 1.57	6.03 ± 1.41	5.86 ± 1.71	
 Haugh unit	72.66 ± 13.98	74.14 ± 11.96	76.33 ± 10.49	75.17 ± 13.27	
 Yolk colour	4.70 ± 0.61b	5.00 ± 0.92b	5.46 ± 0.69a	4.97 ± 0.82b	
 Eggshell thickness, mm	0.35 ± 0.02	0.34 ± 0.03	0.34 ± 0.02	0.34 ± 0.03	
a,b,c Different superscripts within a row indicate significant differences (P < 0.05).

Serum and Liver Biochemical Parameters

Serum NEFAs and GSH-Px in Groups B and D compared to Group A rose considerably (P < 0.05) as RUP concentration increased (Table 4). Serum CAT, meantime, first went down and then up (P < 0.05). Total protein (TP) rose significantly (P < 0.05) in the liver, while CAT climbed linearly (P = 0.063) and MDA decreased (P = 0.089).Table 4 Effect of supplemental RUP on biochemical parameters of laying hens in late laying period.

Table 4Item1	Treatment	
	A	B	C	D	
Blood biochemical parameters					
 TCHO, mmol/L	2.87 ± 0.72	3.22 ± 1.35	3.85 ± 1.65	3.31 ± 1.46	
 TG, mmol/L	6.82 ± 3.90	9.96 ± 5.72	10.24 ± 5.94	8.73 ± 4.04	
 HDL-C, mmol/L	1.45 ± 0.48	1.94 ± 0.74	1.01 ± 0.43	0.98 ± 1.00	
 LDL-C, mmol/L	5.16 ± 2.57	4.11 ± 1.22	4.79 ± 1.06	5.89 ± 3.68	
 NEFA, mmol/L	0.96 ± 0.28c	1.29 ± 0.43bc	2.12 ± 0.80ab	3.15 ± 1.49a	
 UA, mmol/L	1.79 ± 0.96	2.51 ± 1.01	2.80 ± 1.61	2.20 ± 1.00	
 TBA, μmol/L	16.04 ± 3.67	18.79 ± 10.13	22.55 ± 8.15	22.03 ± 6.97	
 CAT, U/mg Hb	4.51 ± 0.27c	5.19 ± 1.23b	5.20 ± 0.83b	6.73 ± 1.35a	
 MDA, nmol/mg prot	3.58 ± 1.03	2.35 ± 1.20	2.46 ± 2.16	1.56 ± 1.00	
 T-SOD, U/mg prot	328.75 ± 29.89	326.72 ± 15.73	347.98 ± 13.38	325.80 ± 33.49	
 GSH, μmol/g prot	24.35 ± 2.58	23.77 ± 3.85	30.28 ± 12.11	25.85 ± 2.73	
 GSH-Px, U/mg prot	1339.26 ± 281.75b	1365.25 ± 223.63b	1605.57 ± 232.65ab	1673.21 ± 148.03a	
 T-AOC, mM	0.66 ± 0.09	0.70 ± 0.05	0.67 ± 0.08	0.72 ± 0.15	
Liver biochemical parameters					
 TP, μg/mL	7.57 ± 1.26b	8.47 ± 0.89b	10.71 ± 2.46a	9.01 ± 1.24ab	
 CAT, U/mg Hb	46.66 ± 10.45	64.52 ± 10.40	55.32 ± 5.07	59.56 ± 12.28	
 MDA, nmol/mg pro	1.39 ± 0.45	1.10 ± 0.07	0.98 ± 0.06	1.21 ± 0.15	
 T-SOD, U/mg prot	150.43 ± 45.84	187.86 ± 19.21	192.46 ± 43.98	167.04 ± 34.11	
 GSH, μmol/g prot	11.86 ± 5.22	14.65 ± 2.10	13.47 ± 2.90	16.96 ± 7.19	
 GSH-PX, U/mg prot	90.59 ± 2.98	82.04 ± 3.53	64.85 ± 6.82	75.26 ± 4.06	
a,b,c Different superscripts within a row indicate significant differences (P < 0.05).

1 TCHO, total cholesterol; TG, triglycerides; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; NEFA, non-esterified fatty acid; UA, uric acid; TBA, total bile acid; CAT, catalase; MDA, malondialdehyde; T-SOD, total superoxide dismutase; GSH, glutathione; GSH-PX, glutathione peroxidase; T-AOC, total antioxidant capacity; TP, total protein.

Intestinal Bacterial Richness, Diversity, and Similarity

After 16S rRNA sequencing of the 24 cecal coelomic samples from the four treatment groups, 2,189,708 read pairs were obtained. Following splicing, filtering, and chimaera removal, 1,724,904 valid tags were retrieved, providing approximately 71,871 high-quality sequences per sample. The species accumulation and rarefaction curves indicated that the sequencing depth and sample size were adequate for accurately representing the composition of the caecum microbial community (Figures 1A and 1C).Figure 1 Description of the gut microbiota in different groups. (A) Alpha-diversity rarefaction curve of 16S rRNA gene sequence to estimate the rationality of sequencing depth (at 97% similarity). X-axis indicates the number of randomly selected sequence strips, and the Y-axis indicates the number of features obtained based on that number of sequencing strips, each curve represents a sample/grouping and is marked with a different color. (B) Alpha-diversity evaluation of caecum flora richness and evenness. (C) Species accumulation curve is used to estimate the rationality of sequencing sample quantity. X-axis indicates the number of sequencing samples, and Y-axis indicates the number of operational taxonomic unit (OTU) detected. (D) Venn diagram represents the number of OTUs that are unique or common to each group. (E) (PCoA)-2D. (F) (PCoA)-3D.

Figure 1

Alpha diversity was evaluated using the ACE, Chao1 diversity index, Simpson, and Shannon diversity indices (Figure 1B). In comparison to the other 3 groups, Group A's Shannon (P = 0.014) and Simpson (P = 0.015) diversity indices were significantly greater, indicating a more homogenous distribution of gut flora. After analyzing Groups A, B, C, and D, it was discovered that Group A contained 108 distinct operational taxonomic units, while Group B had 38, Group C had 44, and Group D had 60 (Figure 1D).

PCoA, or Principal Coordinate Analysis, was employed to assess beta diversity using Bray–Curtis distances, generating both 2D and 3D plots (Figures 1E and 1F). With principle components PC1, PC2, and PC3 accounting for 38.39, 10.92, and 8.90% of the variation, respectively, groups A, B, C, and D showed good separation (Adonis, R2 = 0.206, P = 0.035). Hence, RUP administration considerably changed the microbial makeup in the hens' caecum.

Caecum Microbial Community Structure

In the caecum of hens, the most prevalent phyla (relative abundance >1%) were Firmicutes, Bacteroidetes, Spirochaetota, Proteobacteria, Fusobacteriota, Desulfobacterota, and Campylobacterota (Figure 2A). The relative abundance of Bacteroidetes (A: 35.41%, B: 45.76%, C: 38.77%, and D: 39.38%) rose with the addition of RUP. Furthermore, Group B had a lower percentage of Spirochaetota (A: 3.72%, B: 1.96%, C: 3.28%, and D: 3.31%).Figure 2 Microbial community structure of caecum. (A) Left: stacked bar chart of microbial composition at the phylum level; Right: ridges chart of the microbiota at the phylum level. (B) Left: stacked bar chart of microbial composition at the genus level; Right: ridges chart of the microbiota at the genus level.

Figure 2

Following Bacteroides, Bact, Helicobacter, Faecalibacterium, and Megamonas as the most common genera, Phascolarctobacterium, Muribaculum, Parabacteroides, and Intestinomonas were found to be the least abundant genera in the caecum microbial composition when analyzed at the genus level. (Figure 2B and Table 5). Notably, in Group B, Bacteroides were significantly enriched (A, 14.19%; B, 29.02%; C, 20.74%; D, 20.57%), whereas Muribaculum was reduced (A, 2.96%; B, 1.86%, C, 2.81%; D, 2.22%).Table 5 Effect of supplemental RUP on Ileum bacteria of laying hens at genus level (%).

Table 5Classification levels of bacteria	Treatment1	SEM2	
Phylum	Genus	A	B	C	D		
Bacteroidetes	Bacteroides	14.19b	29.02a	20.74b	20.57b	3.04	
Bacteroidetes	Bact	8.19	6.52	7.28	7.64	0.35	
Bacteroidetes	Muribaculum	2.96a	1.86c	2.81ab	2.22bc	0.26	
Bacteroidetes	Parabacteroides	2.34	2.62	2.48	2.34	0.07	
Firmicutes	Megamonas	2.64	5.60	4.48	4.08	0.61	
Firmicutes	Faecalibacterium	3.44	4.44	4.98	3.52	0.37	
Firmicutes	Phascolarctobacterium	2.95	3.29	2.97	2.66	0.13	
Firmicutes	Intestinimonas	1.45	2.51	2.05	1.99	0.22	
Campilobacterota	Helicobacter	4.00	1.27	2.41	2.81	0.56	
­	Unclassified	8.87a	6.71b	6.56b	8.05ab	0.55	
-	Other	45.67	34.72	40.79	42.22	2.29	
1 Relative abundance of genus less than 1% are not listed. Values are means, n = 6.

2 SEM, standard error of the mean.

a,b,c Different superscripts within a row indicate significant differences (P < 0.05).

Key Microbial Identification

LEfSe, with LDA scores exceeding 3.0, was utilized to identify the specific taxa responsible for the variations observed among the four groups (Figures 3A–3C). In contrast to Groups A and B, the bacterial phylum Bacteroidetes, order Oscillospirales, and class Negativicutes were significantly enriched in Group B. In the meantime, group A contained a substantially higher concentration of the genera Roseburia, order Acholeplasmatales, family Barnesiellaceae, family Veillonellaceae, family Lachnospiraceae, and order Erysipelotrichales. Compared to Group D, the Barnesiellaceae, Muribaculaceae, Saccharimonadia, and Patescibacteria families were specifically enriched in Group A.Figure 3 Cladogram plot (left) and Histogram of LDA value (right) between the A and B group (A). Cladogram plot (left) and Histogram of LDA values (right) for groups A and C (B). Cladogram plot (left) and Histogram of LDA values (right) for groups A and D (C).

Figure 3

To delve deeper into potentially significant bacteria at the genus level, we utilized a random forest classifier to identify reliable biomarkers of the gut microbial response to RUP (Figures 4A and 4B). The genera Bacteroides, Muribaculum, Butyricimonas, Odoribacter, and Prevotella were identified as biomarkers for distinguishing between Groups A and B; Barnesiella, Turicibacter, and Acholeplasma distinguished Groups A and C; and Barnesiella and Candidatus saccharimonas distinguished Groups A and D.Figure 4 Machine-learning classification based on genera in cecal microbial using random forest algorithms. (A) The top 10 genera according to relative importance by the mean decrease accuracy of “A vs. B,” “A vs. C,” and “A vs. D.” The red circle represents the genera that were confirmed and P < 0.05 simultaneously in “A vs. B.” Green circle represents the genera that were confirmed and P < 0.05 simultaneously in “A vs. C.” Orange circle represents the genera that were confirmed and P < 0.05 simultaneously in “A vs. D.” (B) Heat map of the relative abundance of the genera identified through random forest.

Figure 4

Predicted Functions of Caecum Bacterial Communities

There were notable variations in the gut microbiota between the four groups. Consequently, in order to forecast the possible roles of the gut microbiota, FAPROTAX analysis was carried out. The anticipated metabolic functional categories in Group A were linked to biodegradation and metabolism pathways, such as “respiration of sulphur compounds,” “sulphate respiration,” “aerobic chemoheterotrophy,” and “ureolysis,” after they were filtered for non-bacterial functional pathways. Group A was also enriched in “dark sulphide oxidation” and “aliphatic non-methane hydrocarbon degradation” pathways. Meanwhile, Group B was enriched in “aromatic compound degradation,” “chemoheterotrophy,” and “fermentation,” and Group D was enriched in “xylanolysis” (Figures 5A–5C).Figure 5 Microbial functional analysis performed using FAPROTAX software under different experimental conditions. (A)A vs. B. (B) A vs. C. (C) A vs. D.

Figure 5

Response of Cecum Metabolomic Profiles to RUP Supplementation

All four groups' caecum metabolomes were examined to determine how RUP supplementation affected the caecum chyme. 1,764 metabolites were found using LC-MS. Supervised and unsupervised dimensionality reduction techniques, including PCA and OPLS-DA, were employed to mitigate dimensionality. Both PCA and OPLS-DA showcased discrimination and separation capabilities (Figures 6A and 6B). The OPLS-DA permutation was expected to have strong reliability and predictability with quality parameter values of [R2X (cum) = 0.276, R2Y (cum) = 0.989] and fitness of [Q2 (cum) = 0.415] (Figure 6C). Based on P < 0.05 and | log2FC| > 1 parameters, the volcano plot showed differential metabolites that were up- and down-regulated (Figure 6D).Figure 6 Perturbation of gut metabolites by the addition of RUP (n = 6 hens/group). (A) 3D PCA of caecum metabolome. (B) Orthogonal projections to latent structures-discriminate analysis (OPLS-DA) score plot was performed on caecum in A and C groups. In the permutation validation plot. (C) (7-fold cross-validation) the Y-axis intercepts of R2 and Q2 are 0.989 and 0.415, respectively, indicating that the model is valid. (D) Volcano plots of caecum profiles showing log2(fold-change) and −log10 (P-value) in metabolites levels induced by adding RUP (up-regulated in red and down-regulated in green). The labelled metabolites were of particular interest (OPLS-DA VIP > 1, P < 0.10).

Figure 6

Identification of Differential Metabolites and Critical Metabolic Pathways

Differential metabolites were enriched using KEGG pathway analysis, with a screening threshold set at a P-value < 0.05. The manufacture of plant secondary metabolites, nucleotide metabolism, alkaloids produced from histidine and purines, pyrimidine metabolism, and metabolism were the pathways that were enriched the most (Figure 7A). After annotating the differential metabolites using the HMDB database, the composition was determined as follows: lipids and lipid-like molecules constituted 28.855% of the metabolites in Groups A and C; organic acids and derivatives accounted for 21.429%; organoheterocyclic compounds comprised 12.585%; benzenoids constituted 9.524%; organic oxygen compounds represented 5.782%; phenylpropanoids and polyketides made up 3.175%; and organic nitrogen compounds made up 2.438%.The remaining metabolites accounted for 16.212% (Figure 7B).Figure 7 Metabolome analysis of caecum in the A and C groups. (A) Pathway enrichment of differentially expressed metabolites was analyzed using the KEGG pathway enrichment statistical scatterplot. (B) Classification of differential metabolites in the HMDB database.

Figure 7

Correlations Among Differential Microbiota and Metabolites

To better understand the relationships between metabolites and differential microbiota, a network was established between them. We used a strict threshold criterion (P < 0.01, | FC| > 2, VIP > 4) to find more sensitive and specific metabolite indicators (Figure 8A). We conducted a correlation study with the signature microbiota using the top focus metabolites (LDA > 3, P < 0.05; Figure 8B). Results demonstrated that Acholeplasma, Holdemania, Barnesiella, Roseburia, and Megasphaera were significantly positively and negatively correlated with bisoprolol, gamma-linolenoyl ethanolamide, and N6, N6, N6-Trimethyl-L-lysine, while Flavonifractor exhibited the opposite regulation pattern. Additionally, Megasphaera and Bisoprolol were significantly positive (P < 0.001).Figure 8 Correlation analysis was conducted between the top focus metabolites and the signature microbiota to explore the key factors influencing organismal fitness by adding RUP. (A) Heat map of differential metabolites with a bar graph of VIP values from previous OPLS-DA model. Only if the metabolites were a satisfied condition of VIP > 4 were selected for the following association analysis. (B) Correlation of top focus metabolites and the signature microbiota. Red and blue colors represent positive and negative correlations between microbiomics and metabolomics, *P < 0.05, **P < 0.01, and ***P < 0.001, respectively, and indicate the significant difference between microbiota and metabolites.

Figure 8

DISCUSSION

Since antibiotics are no longer used in poultry feed (Yao et al., 2023), rosemary and its extracts have shown a great deal of promise as feed additives to encourage growth and offer added advantages (Valenzuela-Grijalva et al., 2017). In contrast to earlier research, the current investigation revealed that adding rosemary powder to laying hens' diets had no discernible impact on their performance. In contrast, Group C (1.0 g/kg rosemary powder) had higher egg production, feed intake, and egg-breaking rates than Group A. Garcia discovered that adding 0.2 g/kg of rosemary oil considerably enhanced the 30-week-old Hyland Brown chickens in lay (Herrera-García et al., 2022). These results suggest that variations in laying hen breed and age, rosemary extract form, and the additive amount may impact the outcomes. Additionally, RUP supplementation tended to increase feed intake throughout the study, likely due to its aroma enhancing feed flavor, stimulating appetite, and improving feed quality.

We also found that RUP significantly increased Thick albumen height, Haugh units, and yolk color. Low and medium doses of RUP significantly increased the eggshell color b* value and decreased the ESC-a* value; the opposite was observed in the high-dose group. In line with what we discovered, the addition of rosemary or rosemary extracts dramatically raises Thick albumen height and Haugh units, according to a number of earlier research (Ghanima et al., 2020b; Kedir et al., 2023b; Yesilbag et al., 2011). According to Yesilbag et al. (Yesilbag et al., 2011), fennel and rosemary volatile oils greatly increased the egg Haugh unit when added to diets. Additionally, Abo et al. (Ghanima et al., 2020a) found that supplementing the test group's diet with essential oils of rosemary and cinnamon led to a substantial improvement in protein heights and egg heights when compared to the control group. Laying hens are unable to synthesize yolk pigment and can only ingest it through their diets; therefore, nutrition is the main factor affecting yolk color, with carotenoids (e.g., lutein and zeaxanthin) in the diet the main contributors (Zurak et al., 2022). According to Garcia et al. (Garcia et al., 2019), adding rosemary to one's diet greatly enhanced the color of egg yolks. The presence of bioactive compounds such flavonoids, diterpenoids, triterpenoids, monoterpenoids, sesquiterpenoids, alcohols, esters, ketones, and hydroxycinnamic derivatives may be the reason for rosemary's beneficial influence on egg quality (Santos et al., 2020). We speculate that the carotenoids present in rosemary are absorbed by the laying hens, promoting yolk pigmentation.

Late-laying hens are affected by oxidative stress and ovarian ageing. As the oxidation of lipids and proteins in the body increases, the normal physiological functions of laying hens are impacted (Liu et al., 2018). Antioxidants are important indicators of poultry health (Zhang et al., 2020), and the ability of cells to maintain functional homeostasis depends on enzymatic antioxidants (SOD, GSH-Px, and CAT) and non-enzymatic antioxidants (e.g., GSH) in living cells (Surai et al., 2019). Antioxidant enzymes like GSH-Px and CAT break down oxygen and hydrogen peroxide, which are produced when superoxide anions are converted by SOD into water (Yesilbag et al., 2011). An essential peroxidase called GSH-Px guards against oxides' disruption and destruction of cell membrane structure and function (Luo et al., 2003). Lipid peroxidation, on the other hand, results from oxygen radicals attacking polyunsaturated fatty acids when antioxidant enzyme activity is diminished (Cheng et al., 2020). MDA, as the final byproduct of lipid peroxidation, provides a measure for assessing the extent of lipid peroxidation. This can be accomplished by quantifying the amount of MDA present (Sumida et al., 1989). Serum NEFAs are derived from lipolysis in the adipose tissue and are intermediate products of lipolysis, reflecting the degree of TG mobilization during lipolysis and the body's lipid metabolism (Tienken et al., 2015). The dietary addition of various quantities of rosemary powder in aged laying hens resulted in a considerable increase in antioxidant levels, as seen by increased levels of TP in the liver and serum levels of NEFA, CAT, and GSH-PX activities. Furthermore, a linear decrease in serum and liver MDA levels was observed with increasing rosemary content. At doses ranging from 250 to 1,000 mg/kg, Liu observed a significant increase in serum catalase (CAT) activity following administration of rosemary extract (Liu et al., 2022). According to Alagawany, powdered rosemary leaf significantly increased serum GSH-Px activity in a linear fashion (Alagawany and El-Hack, 2015). Furthermore, Lu reported that by modulating the expression of associated transcription factors like CCAAT/enhancer binding proteins (C/EBPs) and peroxisome proliferator-activated receptors (PPARs), the active chemicals in rosemary can decrease fat accumulation in adipocytes and adipose tissue (Lu et al., 2018). Hence, the hypothesis suggests that rosemary induces increased release of NEFAs into the bloodstream by enhancing lipolysis in adipose tissue. Consequently, adding rosemary to a laying hen's diet can increase its antioxidant capacity in the latter phases of laying without having a negative impact on liver function.

In order to achieve optimal gut health and gut microbiota balance in poultry, it is imperative to comprehend the intricate interactions that exist between the host and its gut microbiome. Thus, by examining the microbiome and metabolome of the caecum following rosemary therapy, we were able to get additional insight into the mechanism underlying the effects of supplementing with varying dosages of rosemary powder on the immune system and production performance of aged laying hens. RUP supplementation considerably reduced the Shannon and Simpson diversity indices, but had no effect on ACE or Chao1. In the meantime, the PCoA demonstrated a distinct difference between the RUP and control groups, suggesting that RUP significantly changed the gut microbiota.

Consistent with earlier research, the most prevalent phyla in the hens' caecum were Firmicutes, Bacteroidetes, Spirochaetota, Proteobacteria, Fusobacteriota, Desulfobacterota, and Campilobacterota (relative abundance > 1%) (Liu et al., 2021). The relative abundances of Bacteroides and Muribaculum and Helicobacter were dramatically reduced and increased, respectively, with the addition of RUP. The ability of Bacteroides to metabolize heparin, heparan sulphate, and chondroitin sulphate is strong (Pan et al., 2021). Furthermore, through their metabolism of chondroitin sulfate in the host and diet, specific endogenous Bacteroides species can alter the relative abundance of gut microbiota. (Dong et al., 2024). One important mucin monosaccharide forager that can prevent Clostridium difficile from colonizing an area is Muribaculum (Pereira et al., 2020). In contrast, Helicobacter spp. are pathogenic bacteria that cause gastritis and gastric carcinoma (Kandpal et al., 2024). Hence, the reduction of Muribaculum and Helicobacter and elevation of Bacteroides suggest that rosemary may inhibit the inflammatory response in the caecum and improve intestinal barrier function in aged laying hens.

LEfSe analysis revealed that Group A was significantly enriched in species belonging to the phylum Patescibacteria and class Bacilli, whereas Groups B, C, and D were enriched in Bacteroidetes. Random forest analysis showed that Bacteroides, Muribaculum, Butyricimonas, Odoribacter, and Prevotella were biomarkers delineating Groups A and B. Furthermore, the increased relative abundance of Bacteroides spp. (A: 15.55%, B: 31.12%, C: 22.10%, and D: 22.30%) in the three RUP-treated groups suggests heightened activity within these groups. According to earlier research, Bacteroides predominate in the caecum and are mostly responsible for breaking down complicated chemicals into simpler ones (Xiao et al., 2017; Kumar et al., 2018). Additionally, it has a positive correlation with growth performance and weight gain, and in chickens, it can suppress Clostridium perfringens through its metabolites (Wrigley, 2004; Chang et al., 2016). Muribaculum, a significant Bacteroidales member that produces bile salt hydrolase (BSH) (Huang et al., 2022) and participates in the deconjugation and oxidation of bile acid (Marion et al., 2020). Gram-negative anaerobic bacteria in the genus Butyricimonas of the family Odoribacteraceae contribute to commensal balance between the host's gut microbiota and has a positive impact on the host's energy metabolism (Lee et al., 2022). It is true that increased Butyricimonas spp. abundance correlates with improved metabolism (Kim et al., 2019; Lee et al., 2018). Animal intestinal microbiota commonly contains the short-chain fatty acid-producing bacterium Odoribacter, which is a member of the Bacteroidales order (Hiippala et al., 2020). Reduced abundance of Odoribacter has been associated with a range of microbiota-related disorders, including inflammatory bowel disease, fatty liver disease, and cystic fibrosis (Morgan et al., 2012; Lewis et al., 2015; Burke et al., 2017; Li et al., 2018; Wang et al., 2018). Because Odoribacter may create short-chain fatty acids (SCFAs), it is advantageous to animals' gut microbiomes when it is present in a balanced and healthy state. Commensal bacteria are responsible for the metabolism of resistant starch and dietary fiber into SCFAs, which are essential sources of energy for intestinal epithelial cells (Koh et al., 2016). First reported by Shar and Collins in 1990 (Tett et al., 2021), Prevotella is a widespread genus of gram-negative anaerobic bacterium that is an adept producer of the SCFA propionate from arabinoxylans and fructooligosaccharides in vitro (Chen et al., 2017). Anti-inflammatory propionate, which is produced by Prevotella, controls the differentiation of anti-inflammatory Treg/Tr1 cells. (Li et al., 2016). It is true that a gut microbiota rich in Prevotella enhances weight loss (Christensen et al., 2018; Hjorth et al., 2005, Hjorth et al., 2005; Ortega-Santos and Whisner, 2019), reduces cholesterol levels (Eriksen et al., 2020), and restricts the bifidogenic response (Chung et al., 2020). The application of rosemary powder to Group B considerably raised the relative abundance of Bacteroides, which at the same time reduced pathogen colonization and preserved intestinal homeostasis.

In the anticipated metagenomes from Group A, microbiological pathways such as “respiration of sulfur compounds,” “sulfate respiration,” “aerobic chemoheterotrophy,” and “ureolysis” were considerably enriched in biodegradation and metabolism, as per the gene function analysis. Group A was also enriched in “dark sulphide oxidation” and “aliphatic non-methane hydrocarbon degradation.” Meanwhile, Group B was associated with “aromatic compound degradation,” “chemoheterotrophy,” and “fermentation.” Due to the raised and interconnected pH, conductivity, and FA levels caused by ureolysis, the makeup of the microbial community was greatly influenced by it and grouped together based on temperature and inoculum (Christiaens et al., 2019). Through increasing pH and directing dissolved inorganic carbon species towards CO32−, several microbial metabolic processes can locally enhance carbonate saturation, thereby indirectly encouraging calcium carbonate precipitation within biofilm microenvironments. These processes comprise ureolysis, photosynthesis, methane oxidation, bicarbonate transport, and the dissimilatory reduction of nitrate to ammonium (Zhu and Dittrich, 2016).

One of the most common and enduring types of pollutants in the environment are aromatic chemicals, and soil and sediment contaminated by petroleum often contain a combination of heterocyclic and polycyclic aromatic hydrocarbons (PAHs) (Seo et al., 2009). Widely dispersed organic pollutants known as PAHs are carcinogenic and genetically damaging, and they have been shown to negatively impact host health and gut flora (Ghosal et al., 2016; Redfern et al., 2021). The toxicity of aromatic compounds is due primarily to their biological activation, generating intermediates like epoxide or catechol derivatives (Phale et al., 2007). T The addition of RUP considerably enhanced the microbial functional genes involved in the breakdown of these dangerous molecules, indicating that Group B's gut microbiota is more adept at breaking down hazardous organic compounds and preserving the environment's homeostasis.

Notably, Group C (1.0 g/kg dose of RUP) displayed relatively better growth performance, egg quality, and antioxidant parameters than Groups B and D; therefore, metabolome sequencing was performed, and the orders Acholeplasmatales, Erysipelotrichales, Barnesiellaceae, Veillonellaceae, Roseburia, and Flavonifractor were significantly enriched in the control Group A. Conversely, LEfSe data revealed a significant increase in the abundance of Favonifractor, particularly F. plautii, within Group C. Using permuted random forest analysis, the taxa Barnesiella, Turicibacter, and Acholeplasma were found to be essential for differentiating between the groups A and C. Despite the fact that a number of studies have indicated a positive correlation between the Flavonifractor genera and increased inflammation and oxidative stress, various species within the same genera have markedly different metabolic profiles (Rodriguez-Castaño et al., 2020). Furthermore, a recent study showed that by blocking interleukin (IL)-17, a greater quantity of F. plautii reduces acute colitis in mice (Mikami et al., 2020). F. plautii partakes in various biological processes, such as converting flavone and catechin into physiologically active metabolites that have the ability to reduce blood pressure, inflammation, and oxidative stress (Sankaranarayanan et al., 2021). In actuality, F. plautii may be a helpful probiotic that supports vascular health (Luo et al., 2023). In the meanwhile, it has been proposed that Barnesiella reduces the inflammatory potential of the gut environment by correlating with many immunoregulatory cells (Weiss et al., 2014). For example, a colon with higher concentrations of Barnesiella is associated with a less inflammatory intestinal environment (Berry and Reinisch, 2013). Butyric acid and isobutyric acid are the primary byproducts of Barnesiella derivation (Sakamoto et al., 2009). Butyrate stimulates epidermal growth factor receptors (EGFR), thereby inhibiting the production of inflammatory proteins like IL-6, IL-1, and NF-B (G. Chen et al., 2018; Clemente et al., 2018). Likewise, a number of research using microbiota profiling have found links between Turicibacter and characteristics of the host's fat metabolism, including dietary lipids and obesity (Dhakal et al., 2020; Jiao et al., 2018; Li et al., 2019; Liu et al., 2016; Petersen et al., 2019; Velázquez et al., 2019). However, these relationships are not all the same (Chen et al., 2020; Golloso-Gubat et al., 2020). One study found that Turicibacter changes host fat mass, lipids, cholesterol, and serum bile acids (Lynch et al., 2023).

Significant enrichment was found in multiple pathways, including metabolic pathways, plant secondary metabolite biosynthesis, nucleotide metabolism, alkaloids derived from histidine and purine biosynthesis, and pyrimidine metabolism, according to the metabolic pathway enrichment analysis between Groups A and C. In some situations, nucleoside supplementation of cysteine-deficient cells restores cell growth, indicating that these changes in nucleotide metabolism can impact cellular fitness. Many of the most significant ferroptosis-induced changes that have been seen are explained by modifications in nucleotide metabolism. For instance, erastin-induced ferroptosis and cysteine availability are associated with nucleotide metabolism and can, under some situations, reduce the rate of cell growth (Allen et al., 2023). One top-focused metabolite, bisoprolol, was found to be abundant in Group A of the current investigation and to have a positive link with the C-signature bacteria Acholeplasma, Holdemania, Barnesiella, Roseburia, and Megasphaera, according to Spearman correlation analysis. Bisoprolol, as a highly selective β1-adrenoceptor antagonist, lacks intrinsic sympathomimetic (partial agonist) or membrane-stabilizing (local anesthetic) properties. Because of their β-blocking and vasodilatory properties, selective β-blockers such as bisoprolol and nebivolol are essential for the management of cardiovascular disorders.

Megasphaera elsdenii is a gram-negative, completely anaerobic bacterium that was initially identified from the rumen in 1953. It is often found in the gastrointestinal system of mammals. Although specifics of its lipopolysaccharide structure are still unknown, Megasphaera is one of the few genera in its phylum having a porous pseudo-outer membrane that causes a negative gram-staining reaction (Sarmikasoglou and Faciola, 2022). Consequently, the gut microbiota is altered by supplementing with medium-dose (1.0 g/kg) rosemary powder, which also improves bacterial interactions to control immune function and metabolism by raising the relative abundance of potentially advantageous bacteria. This is due to the association study between Groups A and C, which demonstrated a high link between changed metabolites and the gut microbiota signature taxa.

Meanwhile, when comparing Groups A and D, Barnesiellaceae, Muribaculaceae, Saccharimonadia, and Patescibacteria spp. were specifically enriched in Group A. In particular, Barnesiella and Candidatus Saccharimonas were differential bacteria. Candidatus Saccharimonas predominance has been linked in the past to inflammatory mucosal disorders (Cruz et al., 2020) and may have a direct impact on inflammatory responses. When comparing Group D to the Control group, the administration of RUP led to a decrease in the prevalence of Candidatus Saccharimonas and Barnesiella. In the adjuvant-induced arthritic rat model, the relative abundance of gut Candidatus Saccharimonas was found to be inversely linked with the expression levels of TLR2, IL-17α, and cadherin11 (Y. Huang et al., 2019), indicating that it plays a negative role in intestinal functions. As a result, the lower concentration of Candidatus Saccharimonas in Group D implies that RUP supplementation may enhance the gut microbiome, prevent the formation of some harmful bacteria, and enhance the well-being of older layers.

Group D's FAPROTAX function prediction suggests that the core microbiota has a significant capacity for xylanolysis. Without affecting the number of interacting bacteria, the addition of RUP greatly boosted the interactions between the gut microbiota, as revealed by an examination of the microbial co-occurrence network patterns. Therefore, bacterial communication was enhanced by RUP supplementation.

There were several restrictions on this study, though. To determine the alterations in the metabolome and gut microbiota brought on by RUP in intestinal cell function, more research is required.

CONCLUSIONS

The gut microbiota of aged laying hens was shown to benefit from an eight-week RUP dosing regimen by lowering the abundance of “harmful” bacteria like Candidatus Saccharimonas and increasing the abundance of “beneficial” organisms like Bacteroides and Barnesiella. Additionally, feeding hens with 1.0 g/kg of dietary RUP supplementation improved antioxidant capacities, reduced inflammation, changed the microbial makeup of their caeces, and aided in growth performance. All of our results point to RUP's potential in chicken diets as an antibiotic replacement.

DISCLOSURES

The authors declare no conflicts of interest.

ACKNOWLEDGMENTS

This work was supported by the China Agriculture Research System (CARS-40). We extend our sincere gratitude to all study participants, as well as the research personnel and students who contributed to this endeavor. We acknowledge the support of the Xihe high-performance computing platform of the National Research Facility for Phenotypic and Genotypic Analysis of Model Animals (Beijing). Additionally, we would like to express our appreciation to Editage (www.editage.cn) for their English language editing assistance.
==== Refs
REFERENCES

Abd El-Hack M.E. Abdelnour S.A. Taha A.E. Khafaga A.F. Arif M. Ayasan T. Swelum A.A. Abukhalil M.H. Alkahtani S. Aleya L. Abdel-Daim M.M Herbs as thermoregulatory agents in poultry: an overview Sci. Total Environ. 703 2020 134399
Abd El-Hack M.E. Alaidaroos B.A. Farsi R.M. Abou-Kassem D.E. El-Saadony M.T. Saad A.M. Shafi M.E. Albaqami N.M. Taha A.E. Ashour E.A Impacts of supplementing broiler diets with biological curcumin, zinc nanoparticles and bacillus licheniformis on growth, carcass traits, blood indices, meat quality and cecal microbial load Animals 11 2021 1878 34202621
Abd El-Hack M.E. El-Saadony M.T. Swelum A.A. Arif M. Ghanima A. M M. Shukry M. Noreldin A. Taha A.E. El-Tarabily K.A Curcumin, the active substance of turmeric: its effects on health and ways to improve its bioavailability J. Sci. Food Agric. 101 2021 5747 5762 34143894
Abo Ghanima M.M. Abd El-Hack M.E. Taha A.E. Tufarelli V. Laudadio V. Naiel M.A.E Assessment of stocking rate and housing system on performance, carcass traits, blood indices, and meat quality of French Pekin ducks Agriculture 10 2020 273
Abo Ghanima M.M. Elsadek M.F. Taha A.E. Abd El-Hack M.E. Alagawany M. Ahmed B.M. Elshafie M.M. El-Sabrout K. Effect of housing system and rosemary and cinnamon essential oils on layers performance, egg quality, haematological traits, blood chemistry, immunity, and antioxidant Animals 10 2020 245 32033082
Abou-Elkhair R. Abdo Basha H. Slouma Hamouda Abd El Naby W. Ajarem J.S. Maodaa S.N. Allam A.A. Naiel M.A.E Effect of a diet supplemented with the moringa oleifera seed powder on the performance, egg quality, and gene expression in japanese laying quail under heat-stress Animals 10 2020 809 32392810
Ahmed H.M. Babakir-Mina M. Investigation of rosemary herbal extracts (Rosmarinus officinalis) and their potential effects on immunity Phytother. Res.: PTR 34 2020 1829 1837 32086980
Alagawany M. Abd El-Hack M. The effect of rosemary herb as a dietary supplement on performance, egg quality, serum biochemical parameters, and oxidative status in laying hens J. Anim. Feed Sci. 24 2015 341 347
Alagawany M. El-Hack M.E.A. The effect of rosemary herb as a dietary supplement on performance, egg quality, serum biochemical parameters, and oxidative status in laying hens J. Anim. Feed Sci. 24 2015 341 347
Allegra A. Tonacci A. Pioggia G. Musolino C. Gangemi S. Anticancer activity of Rosmarinus officinalis L.: mechanisms of action and therapeutic potentials Nutrients. 12 2020 1739 32532056
Allen A.E. Sun Y. Wei F. Reid M.A. Locasale J.W. Nucleotide metabolism is linked to cysteine availability J. Biol. Chem. 299 2023 103039
Al-Sagheer A. Abd El-Hack M. Alagawany M. Naiel M. Mahgoub S. Badr M. Hussein E. Alowaimer A. Swelum A. Paulownia leaves as a new feed resource: chemical composition and effects on growth, carcasses, digestibility, blood biochemistry, and intestinal bacterial populations of growing rabbits Animals 9 2019 95 30889832
Ashour E.A. Abd El-Hack M.E. Swelum A.A. Osman A.O. Taha A.E. Alhimaidi A.R. Ismail I.E Does the dietary graded levels of herbal mixture powder impact growth, carcass traits, blood indices and meat quality of the broilers? Ital. J. Anim. Sci. 19 2020 1228 1237
Berry D. Reinisch W. Intestinal microbiota: A source of novel biomarkers in inflammatory bowel diseases? Best. Pract. Res. Clin. Gastroenterol. 27 2013 47 58 23768552
Bolyen E. Rideout J.R. Dillon M.R. Bokulich N.A. Abnet C.C. Al-Ghalith G.A. Alexander H. Alm E.J. Arumugam M. Asnicar F. Bai Y. Bisanz J.E. Bittinger K. Brejnrod A. Brislawn C.J. Brown C.T. Callahan B.J. Caraballo-Rodríguez A.M. Chase J. Cope E.K. Da Silva R. Diener C. Dorrestein P.C. Douglas G.M. Durall D.M. Duvallet C. Edwardson C.F. Ernst M. Estaki M. Fouquier J. Gauglitz J.M. Gibbons S.M. Gibson D.L. Gonzalez A. Gorlick K. Guo J. Hillmann B. Holmes S. Holste H. Huttenhower C. Huttley G.A. Janssen S. Jarmusch A.K. Jiang L. Kaehler B.D. Kang K.B. Keefe C.R. Keim P. Kelley S.T. Knights D. Koester I. Kosciolek T. Kreps J. Langille M.G.I. Lee J. Ley R. Liu Y.X. Loftfield E. Lozupone C. Maher M. Marotz C. Martin B.D. McDonald D. McIver L.J. Melnik A.V. Metcalf J.L. Morgan S.C. Morton J.T. Naimey A.T. Navas-Molina J.A. Nothias L.F. Orchanian S.B. Pearson T. Peoples S.L. Petras D. Preuss M.L. Pruesse E. Rasmussen L.B. Rivers A. Robeson M.S. 2nd Rosenthal P. Segata N. Shaffer M. Shiffer A. Sinha R. Song S.J. Spear J.R. Swafford A.D. Thompson L.R. Torres P.J. Trinh P. Tripathi A. Turnbaugh P.J. Ul-Hasan S. van der Hooft J.J.J. Vargas F. Vázquez-Baeza Y. Vogtmann E. von Hippel M. Walters W. Wan Y. Wang M. Warren J. Weber K.C. Williamson C.H.D. Willis A.D. Xu Z.Z. Zaneveld J.R. Zhang Y. Zhu Q. Knight R. Caporaso J.G. Reproducible, interactive, scalable and extensible microbiome data science using QIIME 2 Nat. Biotechnol. 37 2019 852 857 31341288
Borghei-Rad S.M. Zeinoaldini S. Zhandi M. Moravej H. Ansari M. Feeding rosemary leaves powder ameliorates rooster age-related subfertility Theriogenology 101 2017 35 43 28708514
Brindisi M. Bouzidi C. Frattaruolo L. Loizzo M.R. Tundis R. Dugay A. Deguin B. Cappello A.R. Cappello M.S. chemical profile, antioxidant, anti-inflammatory, and anti-cancer effects of Italian Salvia rosmarinus Spenn. Methanol leaves extracts Antioxidants 9 2020 826 32899385
Burke D.G. Fouhy F. Harrison M.J. Rea M.C. Cotter P.D. O'Sullivan O. Stanton C. Hill C. Shanahan F. Plant B.J. Ross R.P The altered gut microbiota in adults with cystic fibrosis BMC. Microbiol. 17 2017 58 28279152
Chang C.L.T. Chung C.-Y. Kuo C.-H. Kuo T.-F. Yang C.-W. Yang W.-C. Beneficial effect of Bidens pilosa on body weight gain, food conversion ratio, gut bacteria and coccidiosis in chickens PLoS. One 11 2016 e0146141
Chen G. Ran X. Li B. Li Y. He D. Huang B. Fu S. Liu J. Wang W. Sodium butyrate inhibits inflammation and maintains epithelium barrier integrity in a TNBS-induced inflammatory bowel disease mice model EBioMedicine 30 2018 317 325 29627390
Chen T. Long W. Zhang C. Liu S. Zhao L. Hamaker B.R. Fiber-utilizing capacity varies in Prevotella- versus Bacteroides-dominated gut microbiota Sci. Rep. 7 2017 2594 28572676
Chen X. Sun H. Jiang F. Shen Y. Li X. Hu X. Shen X. Wei P. Alteration of the gut microbiota associated with childhood obesity by 16S rRNA gene sequencing PeerJ. 8 2020 e8317 31976177
Cheng Y. Chen Y. Li J. Qu H. Zhao Y. Wen C. Zhou Y. Dietary β-sitosterol regulates serum lipid level and improves immune function, antioxidant status, and intestinal morphology in broilers Poult. Sci. 99 2020 1400 1408 32111314
Chong J. Liu P. Zhou G. Xia J. Using MicrobiomeAnalyst for comprehensive statistical, functional, and meta-analysis of microbiome data Nat. Protoc. 15 2020 799 821 31942082
Christensen L. Roager H.M. Astrup A. Hjorth M.F. Microbial enterotypes in personalized nutrition and obesity management Am. J. Clin. Nutr. 108 2018 645 651 30239555
Christiaens M.E.R. De Vrieze J. Clinckemaillie L. Ganigué R. Rabaey K. Anaerobic ureolysis of source-separated urine for NH3 recovery enables direct removal of divalent ions at the toilet Water. Res. 148 2019 97 105 30352325
Chung W.S.F. Walker A.W. Bosscher D. Garcia-Campayo V. Wagner J. Parkhill J. Duncan S.H. Flint H.J. Relative abundance of the Prevotella genus within the human gut microbiota of elderly volunteers determines the inter-individual responses to dietary supplementation with wheat bran arabinoxylan-oligosaccharides BMC. Microbiol. 20 2020 283 32928123
Clemente J.C. Manasson J. Scher J.U. The role of the gut microbiome in systemic inflammatory disease BMJ 360 2018 j5145 29311119
Cruz B.C. Conceição d.S. da L.L. Mendes T.A. de O. Ferreira C.L. de L.F. Gonçalves R.V. Peluzio M.d.C.G. Use of the synbiotic VSL#3 and yacon-based concentrate attenuates intestinal damage and reduces the abundance of Candidatus Saccharimonas in a colitis-associated carcinogenesis model Food Res Int. 137 2020 109721
Dai H. Lv Z. Hu C. Shi Z. Wei X. Jin S. Yuan Y. Yu D. Shi F. Alpha-lipoic acid improves the reproduction performance of breeder hens during the late egg-laying period J. Anim. Physiol. Anim. Nutr. (Berl) 104 2020 1788 1797 32881138
De Coster W. D'Hert S. Schultz D.T. Cruts M. Van Broeckhoven C. NanoPack: visualizing and processing long-read sequencing data Bioinformatics. 34 2018 2666 2669 29547981
Dhakal S. McCormack L. Dey M. Association of the gut microbiota with weight-loss response within a retail weight-management program Microorganisms. 8 2020 1246 32824364
Dong J. Cui Y. Qu X. Metabolism mechanism of glycosaminoglycans by the gut microbiota: Bacteroides and lactic acid bacteria: areview Carbohydr. Polym. 332 2024 121905
Ebrahimi E. Haghjou M. Nematollahi A. Goudarzian F. Effects of rosemary essential oil on growth performance and hematological parameters of young great sturgeon (Huso huso) Aquaculture 521 2020 734909
El-Demerdash F.M. El-Sayed R.A. Abdel-Daim M.M Rosmarinus officinalis essential oil modulates renal toxicity and oxidative stress induced by potassium dichromate in rats J. Trace Elements Med. Biol. 67 2021 126791
El-Demerdash F.M. El-Sayed R.A. Abdel-Daim M.M. Hepatoprotective potential of Rosmarinus officinalis essential oil against hexavalent chromium-induced hematotoxicity, biochemical, histological, and immunohistochemical changes in male rats Environ. Sci. Pollution Res. 28 2021 17445 17456
Eriksen A.K. Brunius C. Mazidi M. Hellström P.M. Risérus U. Iversen K.N. Fristedt R. Sun L. Huang Y. Nørskov N.P. Knudsen K.E.B. Kyrø C. Olsen A. Tjønneland A. Dicksved J. Landberg R. Effects of whole-grain wheat, rye, and lignan supplementation on cardiometabolic risk factors in men with metabolic syndrome: a randomized crossover trial Am. J. Clin. Nutr. 111 2020 864 876 32097450
Farouk S.M. Abdel-Rahman H.G. Abdallah O.A. El-Behidy N.G. Comparative immunomodulatory efficacy of rosemary and fenugreek against Escherichia coli infection via suppression of inflammation and oxidative stress in broilers Environ. Sci. Pollut. Res. Int. 29 2022 40053 40067 35112249
Gao Y. Zhang G. Jiang S. Liu Y. Wekemo Bioincloud: a user-friendly platform for meta-omics data analyses Imeta 3 2024 e175 38868508
Garcia E.R.D.M. Chaves N.R.B. Oliveira C.A.L.D. Kiefer C. Melo E.P.D. Performance and egg quality of laying hens fed with mineral sources and rosemary oil Anais Da Academia Brasileira de Ciências 91 2019 e20180516
Ghosal D. Ghosh S. Dutta T.K. Ahn Y. Current state of knowledge in microbial degradation of polycyclic aromatic hydrocarbons (PAHs): a review Front. Microbiol. 7 2016 1369 27630626
Golloso-Gubat M.J. Ducarmon Q.R. Tan R.C.A. Zwittink R.D. Kuijper E.J. Nacis J.S. Santos N.L.C. Gut microbiota and dietary intake of normal-weight and overweight Filipino children Microorganisms. 8 2020 1015 32650516
Herrera-García J.A. Martinez M. Zamora-Tavares P. Vargas-Ponce O. Hernández-Sandoval L. Rodríguez-Zaragoza F.A. Metabarcoding of the phytotelmata of Pseudalcantarea grandis (Bromeliaceae) from an arid zone PeerJ. 10 2022 e12706 35127281
Hiippala K. Barreto G. Burrello C. Diaz-Basabe A. Suutarinen M. Kainulainen V. Bowers J.R. Lemmer D. Engelthaler D.M. Eklund K.K. Facciotti F. Satokari R. Novel odoribacter splanchnicus strain and its outer membrane vesicles exert immunoregulatory effects in vitro Front. Microbiol. 11 2020 575455 33281770
Hjorth M.F. Blædel T. Bendtsen L.Q. Lorenzen J.K. Holm J.B. Kiilerich P. Roager H.M. Kristiansen K. Larsen L.H. Astrup A. Prevotella-to-Bacteroides ratio predicts body weight and fat loss success on 24-week diets varying in macronutrient composition and dietary fiber: results from a post-hoc analysis Int. J. Obes. (Lond) 43 2019 149 157 29777234
Hjorth M.F. Roager H.M. Larsen T.M. Poulsen S.K. Licht T.R. Bahl M.I. Zohar Y. Astrup A. Pre-treatment microbial Prevotella-to-Bacteroides ratio, determines body fat loss success during a 6-month randomized controlled diet intervention Int. J. Obes. (Lond) 42 2018 284 29406520
Huang J. Liu D. Wang Y. Liu L. Li J. Yuan J. Jiang Z. Jiang Z. Hsiao W.W. Liu H. Khan I. Xie Y. Wu J. Xie Y. Zhang Y. Fu Y. Liao J. Wang W. Lai H. Shi A. Cai J. Luo L. Li R. Yao X. Fan X. Wu Q. Liu Z. Yan P. Lu J. Yang M. Wang L. Cao Y. Wei H. Leung E.L. Ginseng polysaccharides alter the gut microbiota and kynurenine/tryptophan ratio, potentiating the antitumour effect of antiprogrammed cell death 1/programmed cell death ligand 1 (anti-PD-1/PD-L1) immunotherapy Gut 71 2022 734 745 34006584
Huang Y. Li M. Zhou L. Xu D. Qian F. Zhang J. Zhou X. Effects of Qingluo Tongbi decoction on gut flora of rats with adjuvant-induced arthritis and the underlying mechanism Evid-Based Comp. Alter. Med.: eCAM 2019 2019 6308021
Jiao N. Baker S.S. Nugent C.A. Tsompana M. Cai L. Wang Y. Buck M.J. Genco R.J. Baker R.D. Zhu R. Zhu L. Gut microbiome may contribute to insulin resistance and systemic inflammation in obese rodents: a meta-analysis Physiol. Genomics. 50 2018 244 254 29373083
Kandpal M. Baral B. Varshney N. Jain A.K. Chatterji D. Meena A.K. Pandey R.K. Jha H.C. Gut-brain axis interplay via STAT3 pathway: Implications of Helicobacter pylori derived secretome on inflammation and Alzheimer's disease Virulence 15 2024 2303853
Kedir S. Tamiru M. Tadese D.A. Takele L. Mulugeta M. Miresa A. Wamatu J. Alkhtib A. Burton E. Effect of rosemary (Rosmarinus officinalis) leaf meal supplementation on production performance and egg quality of laying hens Heliyon. 9 2023 e19124 37649844
Kedir S. Tamiru M. Tadese D.A. Takele L. Mulugeta M. Miresa A. Wamatu J. Alkhtib A. Burton E. Effect of rosemary (Rosmarinus officinalis) leaf meal supplementation on production performance and egg quality of laying hens Heliyon. 9 2023 e19124 37649844
Khazaei R. Esmailzadeh L. Seidavi A. Simões J. Comparison between rosemary and commercial antioxidant blend on performance, caecal coliform flora and immunity in broiler chickens fed with diets containing different levels of poultry fat J. Appl. Anim. Res. 45 2017 263 267
Kiełbasa S.M. Wan R. Sato K. Horton P. Frith M.C. Adaptive seeds tame genomic sequence comparison Genome Res. 21 2011 487 493 21209072
Kim J. Lee H. An J. Song Y. Lee C.-K. Kim K. Kong H. Alterations in gut microbiota by statin therapy and possible intermediate effects on hyperglycemia and hyperlipidemia Front. Microbiol. 10 2019 1947 31551944
Kim W.H. Lillehoj H.S. Immunity, immunomodulation, and antibiotic alternatives to maximize the genetic potential of poultry for growth and disease response Anim. Feed. Sci. Technol. 250 2019 41 50
Koh A. De Vadder F. Kovatcheva-Datchary P. Bäckhed F. From dietary fiber to host physiology: short-chain fatty acids as key bacterial metabolites Cell 165 2016 1332 1345 27259147
Kowalczyk J. Śmiałek M. Tykałowski B. Dziewulska D. Stenzel T. Koncicki A. Research note: effect of age on the distribution of lymphocytes in the oviduct in Turkey breeder hens Poult. Sci. 99 2020 3009 3014 32475436
Kumar S. Chen C. Indugu N. Werlang G.O. Singh M. Kim W.K. Thippareddi H. Effect of antibiotic withdrawal in feed on chicken gut microbial dynamics, immunity, growth performance and prevalence of foodborne pathogens PLoS. One 13 2018 e0192450
Lee H. An J. Kim J. Choi D. Song Y. Lee C.-K. Kong H. Kim S.B. Kim K. A novel bacterium, butyricimonas virosa, preventing HFD-induced diabetes and metabolic disorders in mice via GLP-1 receptor Front. Microbiol. 13 2022 858192
Lee H. Lee Y. Kim J. An J. Lee S. Kong H. Song Y. Lee C.-K. Kim K. Modulation of the gut microbiota by metformin improves metabolic profiles in aged obese mice Gut. Microbes. 9 2018 155 165 29157127
Lewis J.D. Chen E.Z. Baldassano R.N. Otley A.R. Griffiths A.M. Lee D. Bittinger K. Bailey A. Friedman E.S. Hoffmann C. Albenberg L. Sinha R. Compher C. Gilroy E. Nessel L. Grant A. Chehoud C. Li H. Wu G.D. Bushman F.D. Inflammation, antibiotics, and diet as environmental stressors of the gut microbiome in pediatric Crohn's disease Cell Host. Microbe 18 2015 489 500 26468751
Li F. Sun G. Wang Z. Wu W. Guo H. Peng L. Wu L. Guo X. Yang Y. Characteristics of fecal microbiota in non-alcoholic fatty liver disease patients Sci. China. Life Sci. 61 2018 770 778 29948900
Li H. Minimap2: Pairwise alignment for nucleotide sequences Bioinformatics. 34 2018 3094 3100 29750242
Li J. Sung C.Y.J. Lee N. Ni Y. Pihlajamäki J. Panagiotou G. El-Nezami H. Probiotics modulated gut microbiota suppresses hepatocellular carcinoma growth in mice Proc. Natl. Acad. Sci. u S. a 113 2016 E1306 E1315 26884164
Li T.-T. Tong A.-J. Liu Y.-Y. Huang Z.-R. Wan X.-Z. Pan Y.-Y. Jia R.-B. Liu B. Chen X.-H. Zhao C. Polyunsaturated fatty acids from microalgae Spirulina platensis modulates lipid metabolism disorders and gut microbiota in high-fat diet rats Food Chem. Toxicol. 131 2019 110558
Li X. Bi R. Xiao K. Roy A. Zhang Z. Chen X. Peng J. Wang R. Yang R. Shen X. Irwin D.M. Shen Y. Hen raising helps chicks establish gut microbiota in their early life and improve microbiota stability after H9N2 challenge Microbiome 10 2022 14 35074015
Liu W. Crott J.W. Lyu L. Pfalzer A.C. Li J. Choi S.-W. Yang Y. Mason J.B. Liu Z. Diet- and genetically-induced obesity produces alterations in the microbiome, inflammation and Wnt pathway in the intestine of Apc+/1638N mice: comparisons and contrasts J. Cancer 7 2016 1780 1790 27698916
Liu Y. Cheng X. Zhen W. Zeng D. Qu L. Wang Z. Ning Z. Yeast culture improves egg quality and reproductive performance of aged breeder layers by regulating gut microbes Front. Microbiol. 12 2021 633276
Liu Y. Li C. Huang X. Zhang X. Deng P. Jiang G. Dai Q. Dietary rosemary extract modulated gut microbiota and influenced the growth, meat quality, serum biochemistry, antioxidant, and immune capacities of broilers Front. Microbiol. 13 2022 1024682
Liu X. Lin X. Mi Y. Li J. Zhang C. Grape seed proanthocyanidin extract prevents ovarian aging by inhibiting oxidative stress in the hens Oxid. Med. Cell. Longevity 2018 2018 9390810
Loetscher Y. Kreuzer M. Messikommer R.E. Oxidative stability of the meat of broilers supplemented with rosemary leaves, rosehip fruits, chokeberry pomace, and entire nettle, and effects on performance and meat quality Poult. Sci. 92 2013 2938 2948 24135598
Louca S. Parfrey L.W. Doebeli M. Decoupling function and taxonomy in the global ocean microbiome Science 353 2016 1272 1277 27634532
Lu M. Cao Y. Xiao J. Song M. Ho C.-T. Molecular mechanisms of the anti-obesity effect of bioactive ingredients in common spices: a review Food Funct. 9 2018 4569 4581 30168574
Luo G. Ren X. Liu J. Mu Y. Shen J. Towards more efficient glutathione peroxidase mimics: substrate recognition and catalytic group assembly Curr. Med. Chem. 10 2003 1151 1183 12678808
Luo S. Zhao Y. Zhu S. Liu L. Cheng K. Ye B. Han Y. Fan J. Xia M. Flavonifractor plautii protects against elevated arterial stiffness Circ. Res. 132 2023 167 181 36575982
Lynch J.B. Gonzalez E.L. Choy K. Faull K.F. Jewell T. Arellano A. Liang J. Yu K.B. Paramo J. Hsiao E.Y. Gut microbiota Turicibacter strains differentially modify bile acids and host lipids Nat. Commun. 14 2023 3669 37339963
Ma F. Xu S. Tang Z. Li Z. Zhang L. Use of antimicrobials in food animals and impact of transmission of antimicrobial resistance on humans Biosaf. Health 3 2021 32 38
Mahmood T. Guo Y. Dietary fiber and chicken microbiome interaction: where will it lead to? Anim. Nutr. 6 2020 1 8 32211522
Marion S. Desharnais L. Studer N. Dong Y. Notter M.D. Poudel S. Menin L. Janowczyk A. Hettich R.L. Hapfelmeier S. Bernier-Latmani R. Biogeography of microbial bile acid transformations along the murine gut J. Lipid Res. 61 2020 1450 1463 32661017
Martin M. Cutadapt removes adapter sequences from high-throughput sequencing reads EMBnet. J. 17 2011 10
McMurdie P.J. Holmes S. phyloseq: an R package for reproducible interactive analysis and graphics of microbiome census data PLoS. One 8 2013 e61217 23630581
Mikami A. Ogita T. Namai F. Shigemori S. Sato T. Shimosato T. Oral administration of Flavonifractor plautii, a bacteria increased with green tea consumption, promotes recovery from acute colitis in mice via suppression of IL-17 Front. Nutr. 7 2020 610946
Miladi H. Slama R.B. Mili D. Zouari S. Bakhrouf A. Ammar E. Essential oil of thymus vulgarisl. and rosmarinus officinalisl.: Gas chromatography-mass spectrometry analysis, cytotoxicity and antioxidant properties and antibacterial activities against foodborne pathogens Nat. Sci. (Irvine) 5 2013 729 739
Mohamed S.H. Attia A.I. Reda F.M. Abd El-Hack M.E. Ismail I.E Impacts of dietary supplementation of Boswellia serrata on growth, nutrients digestibility, immunity, antioxidant status, carcase traits and caecum microbiota of broilers Ital. J. Anim. Sci. 20 2021 205 214
Morgan X.C. Tickle T.L. Sokol H. Gevers D. Devaney K.L. Ward D.V. Reyes J.A. Shah S.A. LeLeiko N. Snapper S.B. Bousvaros A. Korzenik J. Sands B.E. Xavier R.J. Huttenhower C. Dysfunction of the intestinal microbiome in inflammatory bowel disease and treatment Genome Biol. 13 2012 R79 23013615
Nadia L. Hassan R. R A. Qota E.M. Fayek H.M Effect of natural antioxidant on oxidative stability of eggs and productive and reproductive performance of laying hens Int. J. Poult. Sci. 7 2008 134 150
Nematolahi P. Mehrabani M. Karami-Mohajeri S. Dabaghzadeh F. Effects of Rosmarinus officinalis L. on memory performance, anxiety, depression, and sleep quality in university students: a randomized clinical trial Complement. Ther. Clin. Pract. 30 2018 24 28 29389474
Norouzi B. Qotbi A.A.A. Seidavi A. Schiavone A. Marín A.L.M. Effect of different dietary levels of rosemary (Rosmarinus Officinalis) and yarrow (Achillea Millefolium) on the growth performance, carcass traits and ileal micro-biota of broilers Ital. J. Anim. Sci. 14 2015 3930
Oliveira N.A. Gonçalves B.L. Lee S.H. Caf O. Corassin C.H. Use of antibiotics in animal production and its impact on human health J. Food Chem. Nanotechnol. 06 2020 32 38
Ortega-Santos C.P. Whisner C.M. The key to successful weight loss on a high-fiber diet may be in gut microbiome prevotella abundance J. Nutr. 149 2019 2083 2084 31584088
Pan L. Sun W. Shang Q. Niu Q. Liu C. Li G. Yu G. In vitro fermentation and isolation of heparin-degrading bacteria from human gut microbiota Anaerobe 68 2021 102289
Parks D.H. Tyson G.W. Hugenholtz P. Beiko R.G. STAMP: statistical analysis of taxonomic and functional profiles Bioinformatics. 30 2014 3123 3124 25061070
Peralta-Sánchez J.M. Martín-Platero A.M. Ariza-Romero J.J. Rabelo-Ruiz M. Zurita-González M.J. Baños A. Rodríguez-Ruano S.M. Maqueda M. Valdivia E. Martínez-Bueno M. Egg production in poultry farming is improved by probiotic bacteria Front. Microbiol. 10 2019 1042 31178831
Pereira F.C. Wasmund K. Cobankovic I. Jehmlich N. Herbold C.W. Lee K.S. Sziranyi B. Vesely C. Decker T. Stocker R. Warth B. von Bergen M. Wagner M. Berry D. Rational design of a microbial consortium of mucosal sugar utilizers reduces Clostridiodes difficile colonization Nat. Commun. 11 2020 5104 33037214
Petersen C. Bell R. Klag K.A. Lee S.-H. Soto R. Ghazaryan A. Buhrke K. Ekiz H.A. Ost K.S. Boudina S. O’Connell R.M. Cox J.E. Villanueva C.J. Stephens W.Z. Round J.L. T cell-mediated regulation of the microbiota protects against obesity Science 365 2019 eaat9351 31346040
Phale P.S. Basu A. Majhi P.D. Deveryshetty J. Vamsee-Krishna C. Shrivastava R. Metabolic diversity in bacterial degradation of aromatic compounds OMICS: A J. Integr. Biol. 11 2007 252 279
Rashidaie Abandansarie S.S. Ariaii P. Charmchian Langerodi M. Effects of encapsulated rosemary extract on oxidative and microbiological stability of beef meat during refrigerated storage Food Sci. Nutr. 7 2019 3969 3978 31890175
Redfern L.K. Jayasundara N. Singleton D.R. Di Giulio R.T. Carlson J. Sumner S.J. Gunsch C.K. The role of gut microbial community and metabolomic shifts in adaptive resistance of Atlantic killifish (Fundulus heteroclitus) to polycyclic aromatic hydrocarbons Sci. Total Environ. 776 2021 145955
Rocha J. Eduardo-Figueira M. Barateiro A. Fernandes A. Brites D. Bronze R. Duarte C.M. Serra A.T. Pinto R. Freitas M. Fernandes E. Silva-Lima B. Mota-Filipe H. Sepodes B. Anti-inflammatory effect of rosmarinic acid and an extract of Rosmarinus officinalis in rat models of local and systemic inflammation Basic Clin. Pharmacol. Toxicol. 116 2015 398 413 25287116
Rodriguez-Castaño G.P. Rey F.E. Caro-Quintero A. Acosta-González A. Gut-derived Flavonifractor species variants are differentially enriched during in vitro incubation with quercetin PLoS. One 15 2020 e0227724
Rostami H. Seidavi A. Dadashbeiki M. Asadpour Y. Simões J. Shah A.A. Laudadio V. Losacco C. Perillo A. Tufarelli V. Supplementing dietary rosemary (Rosmarinus officinalis L.) powder and vitamin E in broiler chickens: evaluation of humoral immune response, lymphoid organs, and blood proteins Environ. Sci. Pollut. Res. Int. 25 2018 8836 8842 29330815
Sakamoto M. Takagaki A. Matsumoto K. Kato Y. Goto K. Benno Y. Butyricimonas synergistica gen. Nov., sp. Nov. and Butyricimonas virosa sp. Nov., butyric acid-producing bacteria in the family “Porphyromonadaceae” isolated from rat faeces Int. J. Syst. Evol. Microbiol. 59 2009 1748 1753 19542124
Sankaranarayanan R. Sekhon P.K. Ambat A. Nelson J. Jose D. Bhat G.J. Scaria J. Screening of human gut bacterial culture collection identifies species that biotransform quercetin into metabolites with anticancer properties Int. J. Mol. Sci. 22 2021 7045 34208885
Santos A.R.F.D.C. Cruz J.H.D.A. Guênes G.M.T. Oliveira Filho A.A.D. Alves M.A.S.G. Matricaria chamomilla L: Propriedades farmacológicas Arch. Health Investig. 8 2020 846 852
Sarmikasoglou E. Faciola A.P. Ruminal bacteria lipopolysaccharides: an immunological and microbial outlook J. Anim. Sci. Biotechnol. 13 2022 41 35418112
Seidavi A. Azizi M. Swelum A.A. Abd El-Hack M.E. Naiel M.A.E Practical application of some common agro-processing wastes in poultry diets World's Poult. Sci. J. 77 2021 913 927
Seidavi A. Tavakoli M. Asroosh F. Scanes C.G. Abd El-Hack M.E.. Naiel M.A.E. Taha A.E. Aleya L. El-Tarabily K.A. Swelum A.A Antioxidant and antimicrobial activities of phytonutrients as antibiotic substitutes in poultry feed Environ. Sci. Pollut. Res. Int. 29 2022 5006 5031 34811612
Seidavi A. Tavakoli M. Slozhenkina M. Gorlov I. Hashem N.M. Asroosh F. Taha A.E. Abd El-Hack M.E.. Swelum A.A The use of some plant-derived products as effective alternatives to antibiotic growth promoters in organic poultry production: a review Environ. Sci. Pollut. Res. 28 2021 47856 47868
Seo J.-S. Keum Y.-S. Li Q.X. Bacterial degradation of aromatic compounds Int. J. Environ. Res. Public Health 6 2009 278 309 19440284
Shi K. Liu X. Duan Y. Jiang X. Li N. Du Y. Li D. Feng C. Dynamic changes in intestinal gene expression and microbiota across chicken egg-laying stages Animals 14 2024 1529 38891577
Sienkiewicz M. Łysakowska M. Pastuszka M. Bienias W. Kowalczyk E. The potential of use basil and rosemary essential oils as effective antibacterial agents Molecules. 18 2013 9334 9351 23921795
Suliman G.M. Alowaimer A.N. Al-Mufarrej S.I. Hussein E.O.S. Fazea E.H. Naiel M.A.E. Alhotan R.A. Swelum A.A. The effects of clove seed (Syzygium aromaticum) dietary administration on carcass characteristics, meat quality, and sensory attributes of broiler chickens Poult. Sci. 100 2021 100904
Sumida S. Tanaka K. Kitao H. Nakadomo F. Exercise-induced lipid peroxidation and leakage of enzymes before and after vitamin E supplementation Int. J. Biochem. 21 1989 835 838 2583352
Surai P.F. Kochish I.I. Fisinin V.I. Kidd M.T. Antioxidant defence systems and oxidative stress in poultry biology: an update Antioxidants. (Basel) 8 2019 235 31336672
Tang S. Yin B. Xu J. Bao E. Rosemary Reduces heat stress by inducing CRYAB and HSP70 expression in broiler chickens Oxid. Med. Cell. Longevity 2018 7014126
Tett A. Pasolli E. Masetti G. Ercolini D. Segata N. Prevotella diversity, niches and interactions with the human host Nature Rev. Microbiol. 19 2021 585 599 34050328
Thakur A. Kumar A. Sharma M. Kumar R. Vanita B. Strategies to minimize the impact of antibiotic resistance in livestock production system Int. J. Curr. Microbiol. Appl. Sci. 8 2019 2293 2310
Tienken R. Kersten S. Frahm J. Meyer U. Locher L. Rehage J. Huber K. Kenéz Á. Sauerwein H. Mielenz M. Dänicke S. Effects of an energy-dense diet and nicotinic acid supplementation on production and metabolic variables of primiparous or multiparous cows in periparturient period Arch. Anim. Nutr. 69 2015 319 339 26305382
Valenzuela-Grijalva N.V. Pinelli-Saavedra A. Muhlia-Almazan A. Domínguez-Díaz D. González-Ríos H. Dietary inclusion effects of phytochemicals as growth promoters in animal production J. Anim. Sci. Technol. 59 2017 8 28428891
Velázquez K.T. Enos R.T. Bader J.E. Sougiannis A.T. Carson M.S. Chatzistamou I. Carson J.A. Nagarkatti P.S. Nagarkatti M. Murphy E.A. Prolonged high-fat-diet feeding promotes non-alcoholic fatty liver disease and alters gut microbiota in mice World J. Hepatol. 11 2019 619 637 31528245
Wang X.-Y. Meng J.-X. Ren W.-X. Ma H. Liu G. Liu R. Geng H.-L. Zhao Q. Zhang X.-X. Ni H.-B. Amplicon-based metagenomic association analysis of gut microbiota in relation to egg-laying period and breeds of hens BMC. Microbiol. 23 2023 138 37202719
Wang Y. Gao X. Ghozlane A. Hu H. Li X. Xiao Y. Li D. Yu G. Zhang T. Characteristics of faecal microbiota in paediatric Crohn's disease and their dynamic changes during infliximab therapy J Crohn's Colitis 12 2018 337 346 29194468
Weiss G.A. Chassard C. Hennet T. Selective proliferation of intestinal Barnesiella under fucosyllactose supplementation in mice Br. J. Nutr. 111 2014 1602 1610 24411010
Wrigley D.M. Inhibition of Clostridium perfringens sporulation by Bacteroides fragilis and short-chain fatty acids Anaerobe 10 2004 295 300 16701530
Xiao Y. Xiang Y. Zhou W. Chen J. Li K. Yang H. Microbial community mapping in intestinal tract of broiler chicken Poult. Sci. 96 2017 1387 1393 28339527
Yang M. Yin Y. Wang F. Bao X. Long L. Tan B. Yin Y. Chen J. Effects of dietary rosemary extract supplementation on growth performance, nutrient digestibility, antioxidant capacity, intestinal morphology, and microbiota of weaning pigs J. Anim. Sci. 99 2021 237
Yao Y. Liu Y. Li C. Huang X. Zhang X. Deng P. Jiang G. Dai Q. Effects of rosemary extract supplementation in feed on growth performance, meat quality, serum biochemistry, antioxidant capacity, and immune function of meat ducks Poult. Sci. 102 2023 102357
Yesilbag D. Eren M. Agel H. Kovanlikaya A. Balci F. Effects of dietary rosemary, rosemary volatile oil and vitamin E on broiler performance, meat quality and serum SOD activity Br. Poult. Sci. 52 2011 472 482 21919575
Yilmaz P. Parfrey L.W. Yarza P. Gerken J. Pruesse E. Quast C. Schweer T. Peplies J. Ludwig W. Glöckner F.O. The SILVA and “All-species Living Tree Project (LTP)” taxonomic frameworks Nucleic. Acids. Res. 42 2014 D643 D648 (Database issue) 24293649
Younes M. Aggett P. Aguilar F. Crebelli R. Dusemund B. Filipič M. Frutos M.J. Galtier P. Gott D. Gundert-Remy U. Kuhnle G.G. Lambré C. Lillegaard I.T. Moldeus P. Mortensen A. Oskarsson A. Stankovic I. Waalkens-Berendsen I. Woutersen R.A. Wright M. Boon P. Lindtner O. Tlustos C. Tard A. Leblanc J.C. Refined exposure assessment of extracts of rosemary (E 392) from its use as food additive EFSA J. 2018 16
Zhang L.Y. Peng Q.Y. Liu Y.R. Ma Q.G. Zhang J.Y. Guo Y.P. Xue Z. Zhao L.H. Effects of oregano essential oil as an antibiotic growth promoter alternative on growth performance, antioxidant status, and intestinal health of broilers Poult. Sci. 100 2021 101163
Zhang W. Zou G. Li B. Du X. Sun Z. Sun Y. Jiang X. Fecal microbiota transplantation (FMT) alleviates experimental colitis in mice by gut microbiota regulation J. Microbiol. Biotechnol. 30 2020 1132 1141 32423189
Zhu T. Dittrich M. Carbonate precipitation through microbial activities in natural environment, and their potential in biotechnology: a review Front. Bioeng. Biotechnol. 4 2016 4 26835451
Zurak D. Slovenec P. Janječić Z. Bedeković X.D. Pintar J. Kljak K. Overview on recent findings of nutritional and non-nutritional factors affecting egg yolk pigmentation World's Poult. Sci. J. 78 2022 531 560
