Effect of green tea (Camellia sinensis L.) as antimicrobial agent on growth performance and ileal histomorphology of broiler chickens

ABSTRACT We investigated the impact of green tea powder (GTP) as an alternative to antibiotics on growth performance and gut health in broilers. A total of 340 one-day-old broilers were assigned to four dietary treatments as control (free of antibiotic), antibiotic, GTP 0.5 g/kg (GTP-1) and GTP 1 g/kg (GTP-2). Overall, the antibiotic group demonstrated significantly (P < 0.05) higher feed intake, weight gain, lower feed conversion ratio and better dressing percentage compared to the GTP groups, and the control group had the lowest values. GTP-1 showed the highest (P < 0.05) villus height (VH), followed by the antibiotic group and GTP-2, while the control group had the lowest villus height. The control group displayed a higher (P < 0.05) crypt depth, followed by GTP-2, while the antibiotic group and GTP-1 had significantly (P < 0.05) lower crypt depth. The villus height to crypt depth ratio was significantly higher in GTP-1, followed by similar values in the antibiotic group and GTP-2, while the control group had the lowest ratio (P < 0.05). From the results of the present study, we concluded that GTP at the level of 0.5 g/kg of feed is an excellent dose vis-à-vis antibiotic in broiler for optimum growth performance and gut health.


Introduction
Antimicrobial agents are extensively utilized as dietary supplements in the rearing of broiler chickens, not only to mitigate and manage diseases, but also due to their potential to enhance broiler performance (Khan et al. 2023).Although they enhance the efficiency of feed utilization, concerns arise regarding residual effects and the emergence of antibioticresistant bacteria, which can render antibiotics ineffective in both poultry and humans upon consumption of poultry products (Khan et al. 2021;Ishaq et al. 2022).Consequently, numerous countries, including the European Union (EU), have enacted legislation prohibiting the incorporation of antibiotics in poultry feed.As a result, there is currently a pervasive quest for substitute measures to replace antibiotics (Ullah et al. 2022;Khan, Tahir, et al. 2022).
The utilization of botanical preparations in the dietary formulation for poultry exhibits the potential to enhance performance (Khan, Fatima, et al. 2022) and reduce mortality rates.Among these preparations, green tea (Camellia sinensis) stands out as a natural and non-toxic product, consisted of a diverse array of bioactive constituents such as polyphenols, alkaloids, volatile oils and polysaccharides (Karori et al. 2007;Khan 2014).Green tea possesses significant nutritional value, containing essential nutrients including amino acids, with L-theanine accounting for over half of the total amino acid content (Khan 2014).Additionally, green tea is rich in polyphenol catechins (Samynathan et al. 2016;Angga et al. 2018).The leaves of green tea encompass approximately 82.4% organic matter, 92.2% dry matter, 8.7% aether extract, 19.3% crude fibre, 18.1% crude protein and 9.8% ash (Abdo et al. 2010).Notably, green tea exhibits various therapeutic properties, including antioxidant, antimicrobial, and immune-modulatory effects (Nishida et al. 2006;Ko and Yang 2008;Erener et al. 2011), making it highly beneficial for poultry.Previous research has focused on aspects like production, immunity, meat quality, faecal microbiota, and egg production in poultry primarily utilized green tea extract or combinations with other feed additives (Ko and Yang 2008;Abdo et al. 2010;Erener et al. 2011;Aziz-Aliabadi et al. 2023).However, there is a limited body of work addressing the impact of green tea powder on intestinal histology in broilers.Furthermore, previous studies have primarily utilized green tea extract or combinations with other feed additives like probiotics, vitamin E and fish oil, mulberry leaves or aqueous extract.This study stands out for its distinctive approach of incorporating green tea powder, shedding light on its effects on both production and intestinal histology in broilers.Therefore, the aims of this investigation encompassed the assessment of growth performance and gut histomorphology in broiler chickens subjected to dietary regimens containing either antibiotics or green tea powder.

Birds grouping and husbandry
A total of 340 one-day-old Arbor Acres male broilers were allocated to 34 pens, accommodating 10 broilers per pen (0.9 × 1.4 m 2 each pen).The floor of the pens was lined with 8-10 cm of wood shavings.Heat provision was accomplished using infrared lamps.Feed and water were made available ad libitum through semi-automatic system.The temperature in the beginning was set at 33°C for the first week, and then gradually reduced to approximately 20-22°C at 28 days of age.The formulation and analysis of the diet are presented in Table 1.The formulated diet was prepared to satisfy the broilers' requirements for nutrition.
In this experiment, twenty-four pens of broilers were randomly allocated into four dietary treatments, with six pens serving as replicates for each treatment.The treatments were assigned as follows: Treatment 1: Control (CON) groupbroilers were fed the basal diet without any antibiotic included in the diet.
Treatment 2: Antibiotic groupbroilers were fed a diet containing 0.4 mg bacillosporin and enduracidin.
Treatment 3: Green tea powder (GTP1) groupbroilers were fed a diet containing 0.5 g/kg green tea powder obtained from a local company.
Each treatment group had six pens, serving as replicates, for a total of twenty-four pens in the study.

Performance traits
During the feeding trial, the broilers' weights were measured at the beginning of the trial and subsequently recorded on a weekly basis.The weights were recorded per pen, representing the collective weight of broilers within each pen.Additionally, the feed intake from each pen was measured on weekly basis.The data of feed intake was utilized to calculate the feed conversion ratio (FCR) of the broilers.

Carcass characteristic and gut sampling
On day 42 of the treatment, a random selection was made of two birds from each pen.The selected birds were subjected to weighing and subsequently euthanized in a manner consistent with humane practices using the cervical dislocation method.Following euthanasia, dress yield was calculated by removing internal organs.Aseptic collection of ileal samples was performed and then transferred into test tubes containing peptone buffer to maintain sterility.Finally, the test tubes were stored at a temperature of −80°C to preserve the integrity of the samples for future analysis.
A 5 cm segment extending from Meckel's diverticulum to the ileocaecocolic junction was obtained for analysis.After rinsing with soluble phosphate-buffered saline, the samples were fixed in 10% buffered formalin for a duration of 2 days, embedded in paraffin, sectioned (3 μm of thickness), and subjected to staining using the haematoxylin-eosin.
For morphometric analysis, 1 cm long segments of the intestine were collected from the central portion of each sample.Three researchers independently conducted measurements on each sample for villous length, crypt depth and their ratio taking into consideration ten villi per section.

Statistical analysis
The collected data was entered into Excel worksheets for further analysis.Statistical analyses were conducted using the general linear model (GLM) method and analysis of variance (ANOVA) procedures in SAS software.To assess differences between means, the LSD (Least Significant Difference) test was employed.The significance level for determining whether the means were statistically significant was set at 0.05.

Results
Table 2 presents the results of feed intake in broilers from the antibiotic and GTP-fed groups.In the second week, significantly higher feed intake was observed in the antibiotic and GTP-1 groups, while the GTP-2 group had the lowest feed intake (P < 0.05).Similarly, during the third week, the antibiotic and GTP-1 groups showed significantly higher feed intake compared to GTP-2 and the control group (P < 0.05).For the fourth week, the antibiotic group exhibited the highest feed intake, followed by GTP-1 and GTP-2, while the control group had the lowest intake (P < 0.05).In the fifth week, GTP-1 showed significantly higher feed intake, whereas the control group had the lowest feed intake (P < 0.05).Overall, the antibiotic group demonstrated significantly higher feed intake compared to the GTP groups, and the control group had the lowest intake (P < 0.05).
The comparison of antibiotic and GTP on weight gain is shown in Table 3.The first week did not observe a significant effect on weight gain.The higher weight gain was found in GTP-1 and antibiotic groups, while the control group had the minimum weight gain (P < 0.05) during the second week.In the third week, significantly (P < 0.05) higher weight gain was observed in antibiotic and GTP-fed groups compared to the control.The antibiotic group also showed significantly higher weight gain compared to the GTP groups, with the control group recording the lowest weight gain at the end of the fourth and fifth weeks (P < 0.05).Overall, the antibiotic group demonstrated the highest weight gain (P < 0.05), followed by the GTP groups, while the control group had the lowest weight gain (P < 0.05).
The effect of antibiotics and GTP positively affected FCR in all weeks except the first week (Table 4).During the second week, GTP-1 exhibited significantly lower FCR, followed by the antibiotic and control groups, while the highest FCR was recorded in GTP-2 (P < 0.05).In the third week, the control group showed significantly higher FCR, while GTP-1, antibiotic, and GTP-2 groups had significantly lower and similar FCR values (P < 0.05).Similarly, during the fourth week, the antibiotic and GTP-2 groups demonstrated significantly lower FCR, followed by GTP-1, while the control group exhibited significantly higher FCR values (P < 0.05).In the fifth week, the antibiotic group had lower FCR (P < 0.05), followed by GTP-1 and GTP-2, while the control group exhibited the highest FCR.Overall, the antibiotic group displayed lower FCR, followed by GTP-1, GTP-2, and the control group had the highest FCR (P < 0.05).
Table 5 presents the effects of antibiotics and GTP on dressing percentage and villus histomorphology in the control and experimental groups of broilers.The results indicate significant (P < 0.05) effects of these factors on the measured parameters.
The antibiotic group exhibited significantly higher dressing percentage compared to GTP-1 and GTP-2, while the control group had the lowest dressing percentage (P < 0.05).GTP-1 showed the highest (P < 0.05) villus height, followed by the antibiotic group and GTP-2, while the control group had the lowest villus height.The control group displayed a higher crypt depth, followed by GTP-2, while the antibiotic group and GTP-1 had significantly lower and similar crypt depths (P < 0.05).The VH: CD was significantly higher in GTP-1, followed by similar values in the antibiotic group and GTP-2, while the control group had the lowest ratio (Figure 1).The histological examination revealed that villus height, width, and crypt depth appeared normal in the control, antibiotic-treated, and GTP-treated groups.There were no signs of toxicity observed in the birds treated with GTP in terms of histological structures.

Discussion
Green tea is recognized for its rich polyphenol content (Wu et al. 2016), with catechins being the primary constituents of these polyphenols (Pasrija and Anandharamakrishnan 2015).Catechins have been shown to possess potent antimicrobial properties, effectively suppressing the growth of pathogenic bacteria (Zhao et al. 2013) and promoting intestinal health in animals (Hara 2000).During the first two weeks of broilers, the intestinal function is not fully developed, and the microflora is not mature enough.Consequently, a relatively higher fibre found in green tea powder, seems to have no significant impact on the feed efficiency of young broilers.This observation aligns with the findings of the present study.The presence of a higher dietary fibre content in the later stages of broiler growth may have Notes: GTP represent green tea powder; 0.5 and 1 g/kg feed for GTP-1 and GTP-2 respectively.a-d values in the similar column not sharing the same superscript vary (P < 0.05).
Table 3.Effect of antibiotics and green tea powder supplementation on weight gain (g) of broilers (Mean ± SE).Notes: GTP represent green tea powder; 0.5 and 1 g/kg feed for GTP-1 and GTP-2 respectively.a-d values in the similar column not sharing the same superscript vary (P < 0.05).Notes: GTP represent green tea powder; 0.5 and 1 g/kg feed for GTP-1 and GTP-2 respectively.a-d values in the similar column not sharing the same superscript vary (P < 0.05).

Groups
potential benefits.It can contribute to the development of a longer and heavier intestine, prolong feed retention, and ultimately enhance nutrient absorption.This suggests that an increased fibre content in the diet may have positive implications for the later stage growth of broilers (Kalmendal et al. 2011).Interestingly, the higher dose of GTP did not improve the growth parameters consistently, this might be due to the increased content of fibre in the feed.These results are similar to the findings of Jelveh et al. (2022).In the current study, FCR was significantly lower in GTP-treated groups.The improvement in FCR observed in the study could potentially be linked to the effect of epigallocatechin gallate, the most abundant catechin found in GTP.Additionally, other components present in green tea have been reported to have antimicrobial properties and enhance nutrient absorption.These combined effects of green tea components contribute to the improvement in FCR (Engelhardt 2010).In the current study, the dressing percentage was also higher in GTP-1 compared to the GTP-2 compared to the antibiotic group.This finding also corresponds to the weight gain in the respective groups.
The VH, CD and VH: CD are crucial parameters for evaluating intestinal capacity for absorption in broilers (Aziz-Aliabadi et al. 2023).These indexes serve as valuable indicators of the structural and functional aspects of the intestinal epithelium, providing insights into the efficiency of nutrient uptake and utilization.In the current study, villus height significantly increased while crypt depth decreased as a result their ratio was also increased in GTP-1 compared to the antibiotic group.The presence of longer villi in the intestinal tract holds significant significance for nutrient absorption, as these structures serve as the primary site for efficient nutrient uptake.Intestinal villi, characterized by their finger-like projections, possess a larger surface area that enables enhanced absorption of nutrients from the ingested food.This increased surface area facilitates the optimal utilization of nutrients, which in turn may contribute to improved growth and overall performance of broilers.The findings of the current study are partially similar to the results of Aziz-Aliabadi et al. (2023) who reported increased improved villus height at the level of 1 and 2% GTP.Previous research findings have demonstrated that an increase in villus height (VH) correlates with enhanced intestinal digestive and absorptive functions.This increase in VH leads to an expansion of the absorptive surface area, upregulation of brush border enzymes' expression, improved systems of nutrient transport, and subsequent positive effects on body weight.These observations have been reported in studies conducted by and Green tea is recognized for its rich polyphenol content, as reported by Wu et al. (2016).Among the polyphenols, catechins are the primary constituents, as highlighted by Pasrija and Anandharamakrishnan (2015).Catechins have been shown to effectively inhibit the growth of pathogenic bacteria and contribute to the improvement of intestinal health in animals, as documented by Hara (2000).

Conclusion
It was concluded that 0.5 g/kg green tea powder improved the growth performance and intestinal histomorphology in broilers compared to the control.Notes: GTP represent green tea powder; 0.5 and 1 g/kg feed for GTP-1 and GTP-2 respectively.a-d values in the similar column not sharing the same superscript vary (P < 0.05).

Table 1 .
Composition of basal feed and chemical composition as fed basis.

Table 2 .
Effect of antibiotic and green tea powder supplementation on feed intake (g) in broilers (Mean ± SE).

Table 4 .
Effect of antibiotics and green tea powder supplementation on FCR of broilers (Mean ± SE).