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Preliminary study on the effects of dry powder and water extract of Neolamarckia cadamba on growth performance, immunity, and intestinal health of yellow-feathered broilers

BMC Veterinary Research volume 21, Article number: 233 (2025) Cite this article

Abstract

Background

In recent years, the livestock industry has shown increasing concern regarding the need to find effective alternatives to antibiotic products while also striving to produce high-quality livestock and poultry products. Woody feed sources exhibit wide distribution and variety, containing a variety of bioactive substances such as flavonoids and alkaloids. Among these woody plants, Neolamarckia cadamba (Nc) has the characteristics of high leaf yield, fast growth rate, and rich nutritional value, which has great development potential. However, whether Nc supplementation can improve growth performance, immunity, and gut health of yellow-feathered broilers remains to be explored. In the present study, we aimed to investigate the effects of diet supplemented with dry powder or water extract of Nc on the growth performance, immunity, and intestinal health of yellow-feathered broilers.

Results

The results showed that, (1) There was no significant difference in Body weight (BW), Average daily gain (ADG), Average daily feed intake (ADFI), and Feed conversion ratio (FCR) between the Nc experimental groups and the control group (P > 0.05); (2) Compared to the control group, the thymus index at 63 days was significantly increased in the 1% Nc dry powder group (P < 0.05). Conversely, the bursa index of Fabricius was significantly decreased (P < 0.05) in the same group; (3) Compared to the control group and antibiotic group, there were no significant differences in the serum levels of albumin (ALB), globulin (GLB), total protein (TP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and triglycerides (TG) in each experimental group (P > 0.05); (4) Compared to the control group, serum IgG levels were significantly increased in both the 1% Nc dry powder group and the 0.05% water extract group (P < 0.05); (5) Compared to the control group, 0.05% Nc water extract could significantly increase the mRNA expression levels of ZO-1 and TGF-β4 in the jejunum, as well as claudin-1, ZO-1, and TGF-β4 mRNA in the ileum(P < 0.05). Furthermore, the use of 1% Nc dry powder also resulted in a significant increase in the mRNA expression levels of TGF-β4 in the jejunum and ZO-1 mRNA in the ileum (P < 0.05).

Conclusions

These results suggest that the dietary supplementation with Nc can enhance immune function, improve small intestinal barrier integrity, and reduce intestinal inflammation to a certain extent. Among the four doses (1% dry powder, 2% dry powder, 0.05% water extract and 0.1% water extract), 1% dry powder and 0.05% water extract were more effective. These findings underscore the importance of exploring alternative feed additives in livestock production, as they may offer a viable strategy for promoting animal health and performance in the absence of traditional antibiotics.

Peer Review reports

Background

With the gradual development of intensive poultry production, it has become critical to enhance poultry growth performance while improve the immune function to improve production efficiency and product quality. For many years, antibiotics have been widely used to promote growth and enhance the health of poultry [1]. However, the emergence of antibiotic resistance has raised significant concerns in recent times. Prolonged use of antibiotics can lead to the development of drug-resistant bacteria, the accumulation of drug residues, and various associated issues, all of which pose substantial threats to food safety and public health [2,3,4]. Consequently, there is an urgent need to identify healthy, safe, and effective alternatives to antibiotics in poultry farming. Addressing these challenges is essential not only for the sustainability of poultry production but also for ensuring the health of consumers and the integrity of food systems.

Woody plants, such as Morus alba, Moringa oleifera and Neolamarckia cadamba (Nc), exhibit a wide distribution, notable variety, and considerable nutritional richness, indicating their great development potential [5,6,7]. Importantly, extracts from these woody plants often contain a wealth of bioactive components, such as flavonoids, alkaloids, and polyphenols, which are recognized for their antibacterial, antioxidant, and immunomodulatory effects [8,9,10]. Consequently, these extracts are considered promising alternatives to traditional antibiotics [11,12,13]. Studies have shown that the addition of Morus alba and Moringa oleifera could improve humoral immune response and performance of broiler chickens [14, 15]. The other one notable example is Nc, a semi-deciduous, evergreen tree belonging to the Rubiaceae family, which is widely distributed across tropical and subtropical regions. It is known as the “miracle tree” in Southeast Asia because of its high leaf yield, fast growth rate, rich nutritional value, and antibacterial properties [16,17,18]. Studies have shown that Nc contains various bioactive compounds, including flavonoids, phenols, terpenoids, steroidal saponins, and other active ingredients, which exhibit diverse effects such as anti-inflammatory, antioxidant, anti-diarrheal, and anti-tumor activities [19,20,21]. Given these beneficial properties, Nc and its extracts hold substantial potential for development and utilization in fields such as biomedicine [22,23,24]. In addition, studies have shown that Nc extracts can enhance the immune function of rats [25]. And dietary supplementation with Nc extract improves broiler meat quality by enhancing antioxidant capacity and regulating metabolites [26]. However, there is a scarcity of research focusing on the use of Nc in the immune function and intestinal health of yellow-feathered broilers. Therefore, this study chose two easily prepared methods (dry powder and water extract) to investigate the effects of different dosages of Nc on the growth performance, immune function, and intestinal health of yellow-feathered broilers. By incorporating varying doses of Nc into a basic diet that excludes antibiotics, this research aims to provide data to support the application of Nc in broiler production. Furthermore, the findings aim to serve as a reference for the potential development of Nc as a feed additive, thereby contributing to more sustainable poultry farming practices.

Therefore, two easily prepared methods (dry powder and water extract) were chosen in this study to investigate the effects of different doses of Nc (in both dry powder and water extract forms) on the growth performance, immune function, and intestinal health of yellow-feathered broilers. By adding different doses of Nc to the basal diet, excluding antibiotics, this study aims to provide data support for the use of Nc in broiler production and other animal systems. Additionally, the results are intended to inform the potential development of Nc as a feed additive, thereby promoting more sustainable poultry farming practices.

Results

Performance of production

There was no significant difference in Body weight (BW), Average daily gain (ADG), Average daily feed intake (ADFI), and Feed conversion ratio (FCR) between the Nc experimental groups and the control group (P > 0.05). However, compared to the antibiotic group, BW at 21, 35, and 63 days, and the ADG from 1 to 21 days and 1 to 63 days was significantly decreased (P < 0.05), and the FCR of 1 to 21 days was significantly increased in 2% Nc dry powder group (P < 0.05). The BW at 21 and 63 days, the ADG from 1 to 21 days, 36 to 63 days, and 1 to 63 days, and ADFI at 22–35 and 1–63 days were significantly decreased in 0.1% water extract group (P < 0.05). (Table 1). All the above results indicated that both the dry powder and water extract of Nc did not affect the growth performance of yellow-feathered broilers.

Table 1 Effects of Nc dry powder and water extract on growth performance of yellow-feathered broilers (n = 6)
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Effects of Nc dry powder and water extract on immune organ index

We further explored the effect of Nc on immune organ indices in yellow-feathered broilers. Compared to the control group, the thymus index at 63 days was significantly increased in the 1% Nc dry powder group (P < 0.05), while the bursa of Fabricius index showed a significant decrease (P < 0.05). Furthermore, there were no significant differences noted in the thymus index, spleen index, or bursa of Fabricius index on other sampling dates (P > 0.05). Notably, the spleen index was significantly decrease on day 21, and the thymus index was significantly decrease on day 35 in the 0.1% water extract group compared to the antibiotic group (P < 0.05) (Table 2). Based on these results, we speculate that both the 1% Nc dry powder and the 0.05% water extract may affect the growth and development of immune organs to a certain extent, thereby potentially enhancing the immune function of yellow-feathered broilers.

Table 2 Effects of Nc dry powder and water extract on immune organ index of yellow-feathered broilers (n = 6)
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Effects of Nc dry powder and water extract on serum biochemical indexes

We further evaluated the effects of Nc dry powder and water extract on serum biochemical indexes in yellow-feathered broilers on day 63. Compared to the control group and antibiotic group, there were no significant differences in the serum levels of albumin (ALB), globulin (GLB), total protein (TP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and triglycerides (TG) in each experimental group (P > 0.05). Notably, among these variables, the serum AST content of the 2% Nc dry powder group was the highest, significantly exceeding that of the 0.05% water extract group (P < 0.05). (Table 3). This suggests that addition of both dry powder and water extract of Nc did not significantly affect the body health of yellow-feathered broilers during production.

Table 3 Effects of Nc dry powder and water extract on serum biochemical indexes of yellow-feathered broilers on day 63 (n = 6)
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Effects of Nc dry powder and water extract on serum immune indexes

Based on the above results, we found that both the 1% Nc dry powder and the 0.05% water extract had the potential to enhance the immune function of yellow-feathered broilers without adversely affecting their overall health. To further investigate this potential, we evaluated the serum immune indices of yellow-feathered broilers treated with 1% Nc dry powder and 0.05% water extract. The results showed that, compared to the control group, serum Immunoglobulin G (IgG) levels were significantly increased in the 1% Nc dry powder group and the 0.05% water extract group (P < 0.05), and 1% Nc dry powder group increased by about 30%, and 0.05% water extract group increased by about 50% (Fig. 1a). In comparison to the antibiotic group, the serum IgG level was increased in the 0.05% water extract group (P = 0.056) (Fig. 1a). Otherwise, there were no significant differences in the serum levels of Tumor Necrosis Factor-alpha (TNF-α), Interleukin-2 (IL-2), and Interleukin-6 (IL-6) among the experimental groups (P > 0.05) (Fig. 1b, c and d). In summary, the addition of 1% Nc dry powder and 0.05% water extract resulted in an increased IgG content in the serum of yellow-feathered broilers, suggesting a potential enhancement of immune function.

Fig. 1
figure 1

Effects of Nc dry powder and water extract on immune indexes in serum of yellow-feathered broilers (n = 6). (a) Serum IgG content. (b) Serum TNF-α content. (c) Serum IL-2 content. (d) Serum IL-6 content. Abbreviations: IgG Immunoglobulin G, TNF-α Tumor Necrosis Factor-alpha, IL-2 Interleukin-2, IL-6 Interleukin-6. a−b Within a column, values with different letter superscripts mean significant difference (P < 0.05)

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Effects of Nc dry powder and water extract on the expression of intestinal barrier and inflammation-related genes

mRNA expression levels of intestinal barrier and inflammation-related genes in the jejunum

In the present experiment, we examined the expression of intestinal barrier and immune-related genes in yellow-feathered broilers on day 63. The results showed that, compared with the control group, the mRNA expression level of ZO-1 in the jejunum of the 0.05% water extract group was significantly increased (P < 0.05) (Fig. 2c). Additionally, the mRNA expression level of transforming growth factor β4 (TGF-β4) in the jejunum was significantly increased in both the1% Nc dry powder group and the 0.05% water extract group (P < 0.05) (Fig. 2g). However, the mRNA expression levels of Interferon-gamma (IFN-γ) in the 1% Nc dry powder group and 0.05% water extract group were significantly lower than those in the antibiotic group (P < 0.05) (Fig. 2d). Notably, when compared to the control and antibiotic groups, there were no significant differences in the mRNA expression of Claudin-1, Occludin, interleukin-8 (IL-8), and interleukin-10 (IL-10) in jejunum among the Nc groups (P > 0.05) (Fig. 2a, b, e and f). Overall, the 0.05% water extract could significantly increase the expression of the intestinal barrier related gene ZO-1, while both the 1% dry powder and the 0.05% water extract could promote the expression of the anti-inflammatory factor TGF-β4. These findings suggest that supplementing the both dosage of Nc in the daily ration of yellow-feathered broilers could enhance intestinal barrier function and regulate the inflammatory response.

Fig. 2
figure 2

Effects of Nc on the mRNA expression of intestinal barrier and inflammation-related genes in the jejunum (n = 6). (a-c) Expression of intestinal barrier related genes Claudin-1, Occludin, and ZO-1. (d-g) Expression of inflammation related genes IFN-γ, IL-8, IL-10, and TGF-β4. Abbreviations: IFN-γ Interferon-gamma, IL-8 Interleukin-8, IL-10 Interleukin-10, TGF-β4 Transforming Growth Factor beta 4. a−b Within a column, values with different letter superscripts mean significant difference (P < 0.05)

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mRNA expression levels of intestinal barrier and inflammation-related genes in the ileum

In addition to measuring the expression of barrier and inflammation-related genes in the jejunum, we further examined these indices in the ileum. The results showed that, compared to the control group, the mRNA expression levels of claudin-1, and ZO-1 were significantly increased in the 0.05% water extract group in the ileum (P < 0.05) (Fig. 3a and c). And the mRNA expression of ZO-1 was significantly increased in the 1% dry powder group (P < 0.05) (Fig. 3c), the mRNA expression level of pro-inflammatory factor IL-8 was significantly decreased in the 1% dry powder group (P < 0.05) (Fig. 3e). Compared to the antibiotic group, the mRNA expression levels of anti-inflammatory factor IL-10 and TGF-β4 were significantly increased in the 0.05% water extract group (P < 0.05) (Fig. 3f and g), and TGF-β4 was significantly increased in the 1% dry powder group (P < 0.05) (Fig. 3g). However, when compared to the control and antibiotic groups, there were no significant differences in the mRNA expression of Occludin and IFN-γ in ileum among the Nc groups (P > 0.05) (Fig. 3b and d). In conclusion, the addition or supplementation of 0.05% Nc water extract and 1% Nc dry powder to the diet of yellow-feathered broilers enhanced the intestinal barrier function and regulated the inflammatory response in ileum.

Fig. 3
figure 3

Effects of Nc on the mRNA expression of intestinal barrier and inflammation-related genes in the ileum (n = 6). (a-c) Expression of intestinal barrier related genes Claudin-1, Occludin, and ZO-1. (d-g) Expression of inflammation related genes IFN-γ, IL-8, IL-10, and TGF-β4. Abbreviations: IFN-γ Interferon-gamma, IL-8 Interleukin-8, IL-10 Interleukin-10, TGF-β4 Transforming Growth Factor beta 4. a−b Within a column, values with different letter superscripts mean significant difference (P < 0.05)

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Discussion

As effective alternatives to antibiotics, plant extracts have garnered increasing public attention worldwide due to their performance and safety [27]. These extracts contain a variety of bioactive components, such as polysaccharides, alkaloids, phenols, and flavonoids, which are widely utilized in animal husbandry to promote growth and enhance immune function in livestock [28,29,30]. Among these, Nc is particularly rich in alkaloids, phenols, flavonoids, and other bioactive substances [31,32,33,34]. Flavonoids are known to promote the proliferation and differentiation of porcine muscle stem cells and chicken primordial germ cells [35, 36]. Meanwhile, phenols compounds have been shown to enhance growth and gastrointestinal barrier function in broilers [37] and possess the potential to inhibit Staphylococcus aureus [38]. Additionally, the supplementation of alkaloids can improve the growth performance of broiler chickens, particularly under heat stress conditions [39]. In this study, neither 1% nor 2% dry powder, nor the 0.05% or the 0.1% water extract, had any significant effect on the growth performance of yellow-feathered broilers. However, it is worth noting that other dosages may need to be explored in future studies to fully evaluate the potential effects of the contained active substances.

Immune organs such as thymus, spleen, and bursa of Fabricius are essential components of the immune system in poultry, and they are commonly used to evaluate the immune function of these animals [40]. Study have shown that Nc extract significantly increased splenocyte proliferation and regulated the networking of pro-inflammatory cytokines (TNF-α) and anti-inflammatory cytokines (IL-10) to maintain the body’s homeostasis, thereby regulating immune function [25]. Various bioactive components, including polysaccharides, flavonoids, terpenoids, and alkaloids, have been shown to regulate the immune function in animals [41,42,43]. For instance, flavonoids are known to increase IgG content in the serum of piglets [44]. Dietary daidzein can increase the IgG content and improve the immune function of laying hens [45]. While, studies have shown that Nc contains a variety of bioactive substances such as polysaccharides, flavonoids, terpenoids, and alkaloids [46, 47]. In this study, 1% Nc dry powder and the 0.05% water extract may affect the growth and development of immune organs to a certain extent, and the supplementation of 1% Nc dry powder and 0.05% water extract was found to increase the IgG content in the serum of yellow-feathered broilers. We speculate that this improvement may be attributed to the effects of polysaccharides, flavonoids, terpenoids, and alkaloids, with flavonoids likely playing a pivotal role in increasing serum IgG levels.

In terms of regulating intestinal function, studies have demonstrated that naturally derived alkaloids and phenols hold significant potential for treating intestinal diseases [48,49,50]. For instance, plant-derived isoquinoline alkaloids have been shown to enhance intestinal barrier function and inhibit the expression of intestinal inflammatory factors such as IL-6 and TNF-α in chickens under heat stress [39]. Additionally, early-life supplementation of grape polyphenol extract has been found to promote polyphenol absorption and increase the mRNA expression of key intestinal barrier genes [51]. Furthermore, phenol-enriched olive oils can positively regulate intestinal immune function [52]. In this study, the 0.05% water extract was observed to significantly increase the expression of the intestinal barrier related gene ZO-1, while the 1% dry powder and 0.05% water extract could promote the expression of the anti-inflammatory factor TGF-β4. While, alkaloids and phenols were found in Nc [46, 53]. We speculate that the regulation of intestinal function and immune responses may be linked to the alkaloids and phenols present in Nc.

Conclusions

In summary, the inclusion of Nc in the diet of yellow-feathered broilers has been shown to promote immune function. Specifically, among the four doses explored here (1% dry powder, 2% dry powder, 0.05% water extract, and 0.1% water extract), 1% dry powder and 0.05% water extract increased serum IgG levels in yellow-feathered broilers, improve the barrier function of the intestine, and have the potential to mitigate intestinal inflammatory responses. Consequently, Nc can be considered a beneficial dietary additive for improving the overall health of yellow-feathered broilers, exhibiting potential as an alternative to antibiotics to some degree. However, further exploration is warranted to determine the optimal dosage for these additions in order to maximize their beneficial effects.

Methods

Raw materials preparation

Fresh leaves and twigs of Nc were collected from the experimental farm of South China Agricultural University, with the species identified by Professor Xiaoyang Chen. Following collection, the plant material was dried in an oven at 65 °C for 24 h. The dried material was then pulverized and passed through a 40-mesh sieve to obtain the dry powder, which was subsequently sealed and stored at room temperature. To prepare the water extract, the dry powder of Nc was mixed with water at a ratio of 1:30, and a concentrated solution was obtained through cyclic extraction, and then the water-extracted solid powder was obtained by spray drying.

Experimental design and diets

The experiment was approved by the Animal Care Institution and Ethics Committee of South China Agriculture University. All animal experiments were adhered to the animal experiment policy of South China Agriculture University, China. (SYXK2014-0136). A total of 432 yellow-feathered broilers (half male and half female) aged 1 day were randomly divided into 6 groups: control group, antibiotic group (50 mg/kg chlortetracycline + 2.5 mg/kg nosiheptide + 20 mg/kg virginiamycin), 1% Nc dry powder group, 2% Nc dry powder group, 0.05% water extract group, and 0.1% water extract group. Each group had 6 replicates, with each replicate consisting of 12 chickens. Throughout the experiment, food and water were provided ad libitum, and lighting was maintained throughout the day. The broilers were raised in three stages: from 1 to 21 days old, from 22 to 35 days old, and from 36 to 63 days of age. The duration of the experiment was 63 days. The composition and nutrient levels of diets at different stages were see in Table 4.

Table 4 Composition and nutrient levels of experimental diets (air-dry basis, %)
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Sample collection

During the experiment, the number of deaths and the health status of the chickens were recorded. When the chickens reached the age of 21, 35, and 63 days, six broilers were randomly selected from each group. The broilers were fasted for 12 h, the chickens were euthanized by cervical dislocation after blood samples collection. Blood samples were centrifuged (2,500 r/min at 4 °C for 10 min). And samples of the small intestine, thymus, spleen, and bursa of Fabricius were collected. Serum was stored at -20 °C and small intestine at -80 °C for further analysis.

Performance of growth

At day 21, day 35 and day 63, after 12 h of fasting, all broilers were weighed, and feed intake was measured. ADG, ADFI, and FCR were calculated.

Immune organ index

The thymus, spleen, and bursa of Fabricius were isolated at 21, 35, and 63 days, and calculated according to the following formula.

Immune organ index = organ weight/body weight before slaughter (g/kg).

Serum biochemical indicators

TG, TP, ALB, GLB, ALT, and AST were measured by Guangzhou Lanji Biotechnology Co. LTD (Guangdong, China).

Serum immune indicators

The serum concentrations of IgG, TNF-α, IL-2, IL-6 were analyzed with ELISA kits (Shanghai Enzyme-linked, Shanghai, China) as described by the manufacturer’s instructions.

RNA extraction and RT-qPCR analysis

Total RNA was extracted from intestinal tissue using Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol, and a reverse transcription kit (EZB A0010CG) was used for reverse transcription. RT-qPCR was performed according to the instructions of RealStar Fast SYBR (A301-10) to determine the expression of mRNA. The sequences of the primers utilized in RT-qPCR assays are presented in Table 5, and the relative mRNA expression of the above target genes was calculated by the 2−ΔΔCt method usingβ-actin as the reference gene.

Table 5 Primer sequences for RT-qPCR
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Data analysis

Office Excel software 2016 was used to sort out the preliminary data, and SPSS 20.0 Statistical Package for the Social Sciences was used for One-way ANOVA statistical analysis. Duncan was used for post hoc multiple comparisons, and plots were performed using software Graphpad 8.4.2. A value of P < 0.05 was considered statistically significant.

Data availability

No datasets were generated or analysed during the current study.

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Acknowledgements

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Funding

This work was supported by the National key research and development program (2022YFD1300904); National Natural Science Foundation of China (32072814, 32072812); and Natural Science Foundation of Guangdong Province (2021A1515011310, 2023A1515012127); The National broiler industry technology system project (CARS-41-Z12).

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Author notes

  1. Yuxuan Wang, Guoping Chen and Changsong Liu contributed equally to this work.

Authors and Affiliations

  1. College of Animal Science, Guangdong Province Key Laboratory of Animal Nutritional Regulation, National Engineering Research Center for Breeding Swine Industry, State Key Laboratory of Livestock and Poultry Breeding, South China Agricultural University, Guangzhou, 510642, Guangdong, China

    Yuxuan Wang, Guoping Chen, Yunxin Liao, Hailong Wang, Junyi Luo, Jiajie Sun, Qianyun Xi, Yongliang Zhang & Ting Chen

  2. Zhangzhou Yiyuan Bio-technology Co., Ltd., Longhai, Fujian, 363100, China

    Changsong Liu

  3. Hainan Key Laboratory of Tropical Animal Breeding and Epidemic Research, Institute of Animal Husbandry and Veterinary Research, Hainan Academy of Agricultural Sciences, Haikou, 571100, Hainan, China

    Limin Wei

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  1. Yuxuan Wang
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Contributions

Conceptualization, T.C and Y.L.Z.; Methodology, G.P.C, Y.X.W, C.S.L, Y.X.L, and H.L.W.; Software, Y.X.W and G.P.C.; Validation, T.C and Y.L.Z.; Formal Analysis, G.P.C.; Investigation, J.Y.L, J.J.S, Q.Y.X, T.C, and Y.L.Z.; Resources, L.M.W, Q.Y.X, T.C, and Y.L.Z.; Data Curation, G.P.C.; Writing – Original Draft Preparation, Y.X.W and G.P.C.; Writing – Review & Editing, Y.X.W, T.C, Y.X.L, and H.L.W; Visualization, Y.X.W and G.P.C.; Supervision, T.C and Y.L.Z.; Project Administration, J.Y.L, J.J.S, Q.Y.X, T.C, and Y.L.Z.; Funding Acquisition, L.M.W, Q.Y.X, T.C, and Y.L.Z. All authors read and approved the final manuscript.

Corresponding authors

Correspondence to Yongliang Zhang or Ting Chen.

Ethics declarations

Ethics approval and consent to participate

The experiment was approved by the Animal Care Institution and Ethics Committee of South China Agriculture University. All animal experiments were adhered to the animal experiment policy of South China Agriculture University, China. (SYXK2014-0136). Farm managers gave permission for their animal samples to be used in this study.

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The authors declare no competing interests.

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Wang, Y., Chen, G., Liu, C. et al. Preliminary study on the effects of dry powder and water extract of Neolamarckia cadamba on growth performance, immunity, and intestinal health of yellow-feathered broilers. BMC Vet Res 21, 233 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12917-025-04481-w

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BMC Veterinary Research

ISSN: 1746-6148

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