Citation of this paper

Effects of green tea powder supplementation on growth performance, immune response and gut health in broiler chickens

Phan Thi Hong Phuc¹, Tran Thi Kim Ngan² and Nguyen Duy Hoan¹

¹Thai Nguyen University of Agriculture and Forestry – Quyet Thang Ward, Thai Nguyen Province, Vietnam
²Vinh University – 182 Le Duan Street, Vinh City, Nghe An Province, Vietnam
nganttk@vinhuni.edu.vn

Abstract

This study evaluated the effects of dietary green tea powder (GTP) on growth performance, immune response and gut health in Mia × Luong Phuong broilers. A total of 250 one-day-old chicks were randomly assigned to five treatments with GTP supplementation at 0, 0.2, 0.4, 0.6 and 0.8% over 56 days. Growth performance was not significantly affected (p > 0.05), although body weight and feed efficiency showed an improving trend. Carcass yield increased significantly (p < 0.05), with the highest value observed at 0.8% GTP. Immune organ indices (spleen, bursa, thymus) and serum immunoglobulins (IgA, IgG, IgM) were significantly enhanced (p < 0.05), particularly at 0.4–0.6% GTP. Additionally, GTP improved leukocyte profiles by increasing lymphocyte proportions and reducing heterophils, indicating reduced physiological stress. Antibody titers against Newcastle disease were also elevated, with peak responses at 0.4% GTP. Gut health was improved through reduced intestinal pH, decreased pathogenic bacteria (E. coli, Salmonella) and increased beneficial microbes (Lactobacillus, Bifidobacterium), along with enhanced intestinal villi morphology. Overall, GTP supplementation at 0.4–0.6% is recommended to optimise immune function, gut health and production efficiency in broilers.

Keywords: green tea powder, Mia x Luong Phuong chickens, growth performance, immunity, gut health

Introduction

In the context of poultry production shifting toward enhanced biosecurity and sustainability, the use of locally available feed resources is considered a promising and appropriate strategy, as it can reduce production costs, minimise environmental impacts and facilitate the gradual replacement of antibiotics in line with global antimicrobial stewardship trends. Previous studies have reported positive effects of green tea powder or extract on improving body weight, survival rate and alleviating heat stress in broilers (Qiu et al 2025; Nguyen Duy Hoan et al 2021; Aziz-Aliabadi 2024). However, existing studies remain fragmented and have not comprehensively evaluated key biological indicators that reflect internal physiological changes in poultry supplemented with green tea products. Therefore, this study addresses these gaps by investigating parameters related to metabolism, immunity and gut health, thereby contributing to a more robust scientific basis for the application of phytogenic feed additives in sustainable poultry production.

Vietnam ranks fifth globally in tea cultivation area (approximately 128,000 ha) and seventh in production, with an annual output of about 1.02 million tons of fresh tea leaves. In addition to processed tea, surplus fresh leaves that cannot be utilised are estimated at 1.5–1.7 million tons per year (General Statistics Office, 2023). This represents a substantial and underutilised resource with strong potential for use in animal nutrition.

Materials and methods

Animals, location and experimental design

The experiment was conducted using 250 one-day-old crossbred broilers (Mia × Luong Phuong), supplied by CP Vietnam Livestock Corporation (Vinh City, Nghe An Province). The trial was carried out from July to September 2025 at the experimental farm of Vinh University. A completely randomised design was applied with five dietary treatments, each including five replicates of 10 birds. The treatments corresponded to dietary supplementation levels of green tea powder (GTP) at 0% (control), 0.2%, 0.4%, 0.6% and 0.8%. The GTP used was 100% pure and sourced from Nhat Thuc Tea Cooperative (Thai Nguyen Province, Vietnam). The green tea powder used in this experiment was derived from the leaves of the green tea plant (Camellia sinensis). These materials consisted of older leaves, pest- or disease-damaged leaves and leaves that did not meet quality standards for human consumption, typically accounting for approximately 20–30% of the total harvested biomass.The processing of tea leaves into animal feed involved three main stages: (1) collection of mature tea leaves unsuitable for human use; (2) withering and drying to reduce the moisture content to below 10%, thereby preserving bioactive compounds; and (3) fine grinding (particle size of approximately 0.5–1 mm) to produce green tea powder for inclusion in animal diets. The processed green tea powder was sent to a feed manufacturing facility for incorporation into the diet at predetermined inclusion levels. A low-temperature pelleting method (below 70°C) was applied to prevent the degradation of bioactive compounds.

Housing conditions and diet

Birds were reared on a litter-based housing system with biological bedding, following VietGAP biosecurity standards. The poultry house was naturally ventilated, with ambient temperature maintained at 24–28 °C and a lighting regime of 20–24 h/day at approximately 3 lux. Feed and water were provided ad libitum throughout the experimental period. All experimental diets were formulated using locally available feed ingredients (Table 1). The feed was manufactured in pellet form by CP Vietnam Livestock Corporation upon request of the research team

Table 1. Ingredient composition and calculated nutrient contents of local diets
Iterm/ Ingredients (%)	Starter (0–21 d)	Grower (22–56 d)

Maize	50.0	55.0
Rice bran	10.0	15.0
Soybean meal (home-processed)	20.0	15.0
Roasted whole soybean	10.0	8.0
Cassava meal	3.0	3.0
Green forage meal	3.0	2.0
Snail/clam meal	3.0	1.5
Eggshell powder	0.8	0.8
Wood ash	0.7	0.7
Salt	0.5	0.5
Total (%)	100.0	100.0
Calculated nutrient contents
Metabolizable energy (kcal/kg)	2850	2950
Crude protein (%)	19.0	17.0
Calcium (%)	1.00	0.90
Available phosphorus (%)	0.40	0.35
Crude fiber (%)	4.5	5.0

Measured parameters and analytical methods

The study comprehensively evaluated growth performance, immune response and gut health parameters. Growth performance indicators, including survival rate, body weight, feed conversion ratio and carcass yield, were determined using standard procedures. Immune parameters included immune organ indices, leukocyte profiles, serum total protein, immunoglobulins (IgA, IgG, IgM), and antibody titers against Newcastle disease virus. Blood samples were collected at 21 and 56 days of age (five birds per treatment), centrifuged to obtain serum for haematological analysis using an automated analyser and immunoglobulin concentrations were measured by ELISA. Newcastle antibody titers were determined using HI and ELISA methods (positive threshold ≥ 3 log₂). At the end of the experiment, five birds per treatment were randomly selected for slaughter to evaluate immune organ development. Gut health was assessed based on intestinal pH, microbial populations and intestinal villi morphology. Small intestine samples (duodenum, jejunum, ileum) were fixed in 10% neutral buffered formalin, processed histologically, stained with H&E, and analysed microscopically using image analysis software. Faecal samples were collected and cultured on selective media to quantify E. coli, Salmonella, Lactobacillus spp. and Bifidobacterium spp. using the colony counting method. Data were analysed using analysis of variance (ANOVA) in SPSS Statistics 27 (IBM Corp., USA) and differences among treatments were considered significant at p < 0.05.

Results and discussion

Effects of dietary green tea powder supplementation on growth performance

Table 2. Effects of GTP on growth performance and carcass traits of broilers
Parameters	GTP levels (%)					SEM	p
Parameters	0.0	0.2	0.4	0.6	0.8	SEM	p

Growth performance (n = 10)
Body weight (g)	1117	1131	1148	1142	1152	18.9	0.64
Average daily gain (g/day)	19.9	20.2	20.5	20.4	20.6	0.63	0.58
Feed conversion ratio	3.04	2.93	2.87	2.92	2.84	0.09	0.51
Survival rate (%)	100.00	100.00	100.00	100.00	100.00	–	–
Carcass traits, % (n = 5)
Carcass yield	67.2ᵃ	68.4ᵃ	69.6ᵃ	69.7ᵃᵇ	70.0ᵇ	1.23	0.04
Thigh muscle	10.5	11.9	11.42	11.77	10.91	0.52	0.40
Breast muscle	7.87	9.11	9.36	9.54	9.02	0.41	0.09
Abdominal fat	0.45	0.92	0.70	0.42	0.47	0.12	0.29
Means within a row with different superscripts differ significantly (p < 0.05)

Data presented in Table 2 indicate that Dietary supplementation of green tea powder (GTP) at 0–0.8% did not significantly affect growth performance ( p > 0.05), although a consistent improvement trend was observed. Body weight increased from 1117 to 1152 g, while feed conversion ratio decreased from 3.04 to 2.84, indicating enhanced feed efficiency without statistical significance. Survival rate remained at 100% across all treatments, confirming the safety of GTP inclusion. These findings are in agreement with Biswas et al. and Uuganbayar et al.. In contrast, carcass yield was significantly improved (67.2–70.0%; p = 0.046), with the highest value observed at 0.8% GTP, suggesting enhanced lean tissue deposition. Breast muscle percentage increased from 7.8 to 9.5%, whereas abdominal fat showed a decreasing trend (0.92 to 0.42%) at higher inclusion levels, although not statistically significant. These results align with those of Sarker et al (2010) and Tang et al (2012), indicating improved carcass traits and modulation of lipid metabolism.

Effects of dietary green tea powder supplementation on selected immune parameters

Table 3. Effects of GTP on immune organ indices, % (n = 5)
Parameters	GTP levels (%)					SEM	p
Parameters	0.0	0.2	0.4	0.6	0.8	SEM	p

Spleen	0.20ᶜ	0.22ᵇ	0.24ᵃ	0.23ᵃᵇ	0.22ᵇ	0.02	0.03
Bursa	0.30ᶜ	0.33ᵇ	0.36ᵃ	0.34ᵃᵇ	0.32ᵇ	0.02	0.01
Thymus	0.66ᶜ	0.71ᵇ	0.75ᵃ	0.73ᵃᵇ	0.70ᵇ	0.03	0.02
Means within a row with different superscripts differ significantly (p < 0.05)

Dietary supplementation of green tea powder (GTP) significantly affected immune organ indices (p< 0.05). Spleen, bursa and thymus indices increased from 0.20, 0.30 and 0.66% in the control to peak values of 0.24, 0.36 and 0.75% at 0.4% GTP, respectively, then slightly declined at 0.6–0.8%. This quadratic response suggests that moderate GTP levels enhance immune organ development, whereas higher levels may reduce efficacy. The 0.4% improvement is likely due to the antioxidant and immunomodulatory effects of tea polyphenols. This trend is consistent with growth performance results, where 0.4–0.6% GTP also showed better feed efficiency and body weight, indicating a close relationship between improved immune function and growth performance in broilers. The results are consistent with those of Aziz-Aliabadi et al (2024); Luo et al (2023) and Qiu et al (2025), indicating that green tea helps maintain and support immune responses in poultry.

Table 4. Effects of dietary GTP on leukocyte parameters
Parameter	Age (days)	GTP levels (%)					SEM	p
Parameter	Age (days)	0.0	0.2	0.4	0.6	0.8	SEM	p

Total WBC (×10⁹/L)	21	32.8ᵃ	27.6ᵇ	28.0ᵇ	29.2ᵇ	29.6ᵇ	2.16	0.03
Total WBC (×10⁹/L)	56	18.2	16.3	15.1	16.5	16.4	1.02	0.08

Lymphocytes (%)	21	56.6ᶜ	57.5ᶜ	61.7ᵇ	62.8ᵃᵇ	63.1ᵃ	3.45	0.01
Lymphocytes (%)	56	50.7ᶜ	53.3ᵇ	55.7ᵃᵇ	56.8ᵃ	55.4ᵃᵇ	2.82	0.03

Heterophils (%)	21	31.4ᵃ	30.1ᵃᵇ	28.4ᵇ	27.2ᵇᶜ	26.5ᶜ	1.17	0.01
Heterophils (%)	56	32.8ᵃ	31.4ᵃᵇ	29.9ᵇ	28.7ᵇᶜ	28.2ᶜ	1.15	0.02

Monocytes (%)	21	7.40ᵃ	6.48ᵇ	6.75ᵃᵇ	6.70ᵃᵇ	6.94ᵃᵇ	0.24	0.04
Monocytes (%)	56	4.14ᵃ	3.75ᵃᵇ	3.38ᵇ	3.83ᵃᵇ	3.86ᵃᵇ	0.43	0.04

Eosinophils (%)	21	0.18ᶜ	0.25ᵇ	0.28ᵃᵇ	0.29ᵃᵇ	0.31ᵃ	0.02	0.03
Eosinophils (%)	56	0.14ᶜ	0.16ᶜ	0.21ᵇ	0.25ᵃᵇ	0.26ᵃ	0.03	0.02

Basophils (%)	21	2.89ᵃ	2.34ᵇ	2.11ᶜ	2.25ᵇᶜ	2.13ᶜ	0.12	0.01
Basophils (%)	56	2.34	2.37	2.27	2.29	2.36	0.17	0.61

Table 4. indicates that GTP supplementation positively modulated the immune response in chickens. At 21 days, WBC was significantly affected (P = 0.038), with a slight decrease at low levels and stabilisation at higher levels. Lymphocytes increased (p = 0.01) while heterophils decreased (p = 0.009), improving the H/L ratio, a key stress indicator. These findings agree with Zhao et al (2021) and Liu et al (2020), who reported that green tea polyphenols and phytogenic compounds enhance immunity and regulate heterophil–lymphocyte balance. Other immune cells (monocytes, eosinophils, basophils) were also significantly affected ( p < 0.05), indicating a broad impact on innate immunity. At 56 days, the effect on WBC was no longer significant (p = 0.08), but lymphocytes, heterophils, monocytes and eosinophils remained significantly influenced (p < 0.05), confirming sustained immunomodulatory effects, while basophils remained unchanged (p = 0.61).

Table 5. Effects of GTP on serum immunoglobulins (n = 5)
Parameter	Age (days)	GTP levels (%)					SEM	p
Parameter	Age (days)	0.0	0.2	0.4	0.6	0.8	SEM	p

IgG (g/L)	21	4.33ᶜ	4.41ᶜ	4.77ᵃ	4.70ᵃᵇ	4.72ᵃᵇ	0.18	0.032
IgG (g/L)	56	8.77ᶜ	8.82ᶜ	10.4ᵃ	10.5ᵃ	10.4ᵃ	0.62	0.018

IgM (g/L)	21	0.23ᶜ	0.28ᶜ	0.48ᵃ	0.50ᵃ	0.51ᵃ	0.02	<0.001
IgM (g/L)	56	0.42ᶜ	0.44ᶜ	0.68ᵃ	0.70ᵃ	0.69ᵃ	0.03	<0.001

IgA (g/L)	21	0.47ᶜ	0.48ᶜ	0.66ᵃ	0.67ᵃ	0.68ᵃ	0.015	<0.001
IgA (g/L)	56	0.66ᶜ	0.62ᶜ	0.77ᵃ	0.79ᵃ	0.81ᵃ	0.04	<0.001
Means within a row with different superscripts differ significantly (p < 0.05)

Data presented in Table 5 indicate that GTP supplementation significantly improved serum immunoglobulin levels in a dose-dependent manner. At both 21 and 56 days, birds receiving 0.4–0.8% GTP showed higher IgG, IgM and IgA compared with the control (p < 0.05), indicating enhanced humoral immune response. The most pronounced effect was observed at 0.4–0.6%, suggesting a plateau at higher inclusion levels. These findings are consistent with reports that green tea polyphenols enhance antioxidant status and immune modulation in poultry (Wang et al 2019; Li et al 2020). Similar improvements in immunoglobulin production were also described by Zhang et al (2021).

Table 6. Effects of GTP on antibody titers against Newcastle disease virus (HI test) (n = 5)
Age (days)	Parameter	GTP levels (%)
Age (days)	Parameter	0.0	0.2	0.4	0.6	0.8

14	log₂GMT	5.50	6.00	6.50	6.25	6.00
14	GMT	45.3	64.0	90.5	76.1	64.0

28	log₂GMT	6.00	6.50	7.00	6.75	6.50
28	GMT	64.0	90.5	128.0	108.0	90.5

42	log₂GMT	5.50	6.00	6.25	6.00	5.75
42	GMT	45.3	64.0	76.1	64.0	54.6
GMT (Geometric Mean Titer) : the geometric mean of antibody titers, commonly used to summarise skewed immunological data. GMT was calculated as: GMT = 2^(log₂GMT)

Table 6 demonstrates that GTP supplementation significantly influenced antibody titers against Newcastle disease virus over time and across inclusion levels. At 14 days of age, the 0.4% GTP group showed a higher immune response than the control, indicating early immunostimulatory effects. By 28 days, antibody titers increased markedly and peaked at 0.4% GTP (log₂GMT = 7.00; GMT = 128), suggesting an optimal post-vaccination immune response. At 42 days, titers declined slightly but remained higher than the control at 0.2–0.4% levels. These findings are consistent with Abd El-Hack et al (2020), who reported improved Newcastle antibody responses with phytogenic additives. Similarly, Gheisar and Kim (2018) highlighted the role of green tea polyphenols in enhancing humoral immunity via oxidative stress regulation and lymphocyte activation. Zhao et al (2021) also confirmed improved vaccine responses with flavonoid-rich plant extracts in poultry.

Effects of dietary green tea powder supplementation on gut health in broilers

Table 7. Effects of GTP on intestinal pH and gut microbiota (n = 5)
Parameter	GTP levels, %					SEM	p
Parameter	0.0	0.2	0.4	0.6	0.8

Intestinal pH
Duodenum	6.22ᵃ	6.18ᵃ	6.12ᵇ	6.08ᵇ	6.10ᵇ	0.04	0.02
Jejunum	6.46ᵃ	6.40ᵃᵇ	6.33ᵇ	6.27ᵇᶜ	6.25ᶜ	0.05	0.01
Ileum	6.80ᵃ	6.73ᵃᵇ	6.66ᵇ	6.61ᵇᶜ	6.58ᶜ	0.05	0.01
Cecum	6.72ᵃ	6.58ᵇ	6.46ᵇᶜ	6.34ᶜ	6.36ᶜ	0.06	<0.01
Gut microbiota (log10 CFU/g)
E. coli	5.18ᵃ	4.95ᵇ	4.72ᶜ	4.60ᶜ	4.68ᶜ	0.07	0.04
Salmonella	1.20	1.05	0.98	0.92	0.95	0.05	0.06 (trend)
Total aerobic bacteria	7.18	7.05	6.92	6.88	6.90	0.06	0.21
Lactobacillus	5.10ᶜ	5.25ᵇᶜ	5.42ᵃᵇ	5.38ᵃᵇ	5.30ᵇᶜ	0.05	0.03
Bifidobacterium	5.50ᶜ	5.85ᵇ	6.18ᵃ	6.05ᵃᵇ	5.62ᵇᶜ	0.09	0.01
Means within a row with different superscripts differ significantly (p < 0.05)

Dietary GTP supplementation markedly improved intestinal health and modulated gut microbiota in broilers (Table 7). Intestinal pH decreased significantly across all gut segments, with the strongest reduction observed in the cecum (p < 0.01), indicating enhanced intestinal acidification. This environment likely suppresses pathogenic bacteria while supporting beneficial microbes. Correspondingly, Escherichia coli counts were significantly reduced in a dose-dependent manner, whereas Lactobacillus and Bifidobacterium populations increased significantly (p < 0.05). Salmonella showed a decreasing trend (p = 0.06), although not statistically significant. Total aerobic bacteria were unaffected by GTP supplementation. Overall, the 0.4–0.6% GTP groups showed the most favorable microbial balance. These results suggest that GTP enhances gut microbial homeostasis by reducing pathogenic bacteria, promoting beneficial bacteria and lowering intestinal pH, thereby contributing to improved intestinal health and potentially supporting immune function in broilers.

Across the duodenum, jejunum and ileum, villus morphology showed a relatively consistent trend across GTP supplementation levels. In the control group (0%), villi were generally shorter, sparse and unevenly arranged. At 0.2–0.4%, particularly 0.4%, villi became longer, more slender, denser and more uniformly distributed, increasing the absorptive surface area. However, at higher levels (0.6–0.8%), villus height and uniformity tended to decline. This pattern may be explained by the antioxidant effects of polyphenols, their role in modulating gut microbiota and enhancing mucosal immunity. In contrast, higher tannin levels at excessive inclusion rates may impair digestion and inhibit intestinal epithelial regeneration.

Conclusions

GTP supplementation (0.0–0.8%) did not significantly affect growth but improved feed efficiency and carcass yield, particularly at 0.4–0.8%. It enhanced immune organ development and humoral immunity (IgG, IgM, IgA, Newcastle antibody titers), while modulating leukocyte profiles (↓heterophils, ↑lymphocytes), indicating reduced physiological stress. GTP also improved gut health by lowering intestinal pH, suppressing pathogenic bacteria and promoting beneficial microbiota (Lactobacillus, Bifidobacterium). Overall, 0.4–0.6% GTP is optimal for improving health, immunity and production efficiency in broilers.

Acknowledgments

This study was funded by the National Research Foundation of Korea (NRF), supported by the Ministry of Education (MOE) (Grant No. NRF-2023H1A7A2A02000078).

The authors would like to thank Vinh University, Hue University of Agriculture and Forestry and the Key Laboratory of Veterinary Biotechnology, Vietnam National University of Agriculture, for their support in conducting experiments and sample analysis.

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