Effect of different levels of green tea (Camellia sinensis) and mulberry (Morus alba) leaves powder on performance, carcass characteristics, immune response and intestinal morphology of broiler chickens

Abstract Background In recent years, the use of medicinal plants as an alternative to antibiotics has expanded. Plants containing medicines and antioxidants can improve the performance of poultry. Objectives The purpose of this study was to achieve the appropriate levels of green tea leaf powder (GTP) and mulberry leaf powder (MLP) in the diet, which positively affects broilers’ performance. Methods 648 one‐day‐old Ross 308 broiler chickens were allocated to nine dietary treatments with six replicates and each replicate containing 12 birds based on a completely randomised design (CRD) in a factorial arrangement of 3 × 3 with three levels of GTP, and three levels of MLP for 42 days. Treatments included: (1) no GTP + no MLP (control), (2) 1% GTP + no MLP, (3) 2% GTP + no MLP, (4) no GTP + 1% MLP, (5) 1% GTP + 1% MLP, (6) 2% GTP + 1% MLP, (7) no GTP + 2% MLP, (8) 1% GTP + 2% MLP and (9) 2% GTP + 2% MLP. Results The results revealed that the effect of added powders (2%) was significant and increased daily weight gain (DWG) and reduced feed conversion ratio (FCR) compared to the control group during the grower and whole phases (p < 0.05). On 35 days, the control and 2% GTP + 2% MLP treatment had the lowest and highest antibodies titre (total and immunoglobulin G [IgG]), respectively (p < 0.05). The groups fed with 1% GTP + 1% MLP showed higher villus height (VH) compared to the control, 2% GTP + 1% MLP, 1% GTP + 2% MLP, and 2% GTP + 2% MLP groups (p < 0.05). The ratio of the villus height to crypt depth (VH: CD) in treatments 1% GTP + no MLP, 2% GTP + no MLP and 1% GTP + 1% MLP was significantly higher than the control treatment (p < 0.05). Conclusions It was concluded that the addition of 2% GTP or MLP could improve humoral immune response and performance, and the addition of 1% GTP without MLP increased VH: CD in broilers.


INTRODUCTION
Nowadays, to increase the performance in the poultry industry as well as the production of chemical-free meat, the use of natural additives has been considered by broiler farmers. Herbal supplements containing antioxidants can improve growth performance and immune response in chickens (Romeo et al., 2009). In order to improve production performance in livestock, leaves of medicinal plants (powder or extract), spices, and other related products are used as a substitute for antibiotics. These compounds can to activate digestive enzymes and improve performance (Brenes & Roura, 2010).
Tea (Camellia sinensis L.) is an evergreen plant with white flowers and green fruits with two to three seeds (Khan, 2014). Green tea is nutritionally valuable, encompasses urgent nutrients, including amino acids, of which L-theanine accounts for more than half of the total amino acids (Khan, 2014), and has polyphenol catechin (Angga et al., 2018). Green tea is very healthy and non-toxic nutritious; it has been found that green tea contains large amounts of beneficial antioxidants (Wolfram, 2007). White berry (Morus alba) is the name of a species of berry genus; mulberry leaves are rich in protein (15-35%), minerals (calcium: 2.42-4.71%; phosphorus: 0.23-0.97%), energy (1.130-2.240 kcal/kg) and very few (negligible) anti-nutritional factors (Sarita et al., 2006). Plants rich in flavonoids, such as green tea, mulberry leaves and thyme with, have antibacterial effects that improve the performance and immune system (Cook & Samman, 1999).
Although carcass efficiency is significantly affected by age and genetics, carcass composition is also greatly affected by diet (Esmail, 1999). Yang et al. (2003) Compared different levels of green tea by-products (0.5%, 1% and 2%) with antibiotics and obtained significant results regarding body weight gain. Shomal et al. (2012) stated that performance and health of broilers improved when diets containing green tea were consumed. These advantageous effects can be attributed to the presence of bioactive compounds, such as catechins, flavonoids and flavonols in green tea (Khan, 2014). The attendance of phenolic acids such as tannins in the green tea leaf could intervene with the utilisation of protein and other nutrients, and potentially damaging the performance of animals (Mahlak et al., 2021). Hence, it is momentous that a maximum inclusion level of green tea in broilers' diets must be identified so as not to compromise their performance. On the other hand, because of the great amount of crude fibre in mulberry leaves and the presence of anti-nutritional factors such as tannin, the excessive addition of mulberry leaves could affect the performance and health of poultry, which, to a certain extent, restricts it is too much use in animal production (Srivastava et al., 2006). Thus, this study aimed to achieve the appropriate levels of green tea leaf powder (GTP) and mulberry leaf powder (MLP) in the diet, which positively affects broilers' performance.

Preparation of green tea and mulberry leaves powder
Dry green tea and mulberry leaves were purchased from an herbal pharmacy in Rasht-Iran. Samples were ground using a Grinder (Kinematica AG, Malters, Switzerland) to a particle size of 2 mm. The green tea and mulberry leaf powders were analysed to determine the amount of dry matter (DM), crude protein (CP), crude fibre (CF) and ash (AOAC, 2005). The results of the approximate analysis are shown in Table 1. GTP + 2% MLP.

Birds, diets and housing
The nutrient composition of feed ingredients published (NRC, 1994) was utilised for feed formulation. Ingredients and nutrient composition of diets are shown in Table 2. All diets were formulated based on the Ross 308 nutrition guidelines (Aviagen, 2014a

Growth performance
The chickens in each pen were weighed collectively at 11, 24 and 42 days of age. The daily weight gain (DWG) and daily feed intake (DFI) were determined for each pen. Feed intake was measured by subtracting the remaining feed from the offered feed in each pen during each study period. Feed conversion ratio (FCR) was corrected for mortality and represented as grams of feed consumed by all chickens in each pen divided by grams of body weight gain. Mortality for each pen was recorded daily. anti-SRBC titres. Antibody titres were represented as log 2 of the reciprocal of the highest dilution giving visible agglutination (Dietert, 2009).

Intestinal morphology
Two birds from each pen were haphazardly selected and euthanised by cervical dislocation at 42 days of age. The entire intestinal tract was pick up, and pieces of approximately 1 cm were taken from the middle part of the jejunum. The pieces were fixed in 10% neutral buffered formalin solution and infixed in paraffin wax later. All histological morphometric studies are accomplished on 5 µm sections and stained with haematoxylin and eosin. To study the morphology of the tissue samples, a computer-connected optical microscope (Olympus model BX51 microscope; magnification 100) was used to obtain images of the samples, and consequently, the parameters including VH, villus width (VW), crypt depth (CD), and the thickness of muscle layer were measured using the relevant software (Garcia et al., 2007).

Statistical analyses
This trial was performed based on a completely randomised design (CRD) in a factorial arrangement of 3 × 3 with three levels of GTP and three levels of MLP with six replicates by the GLM 1 procedure of SAS 9.4 (2012) software. Results are showed as means ± standard error of the mean (SEM). All data were normalised by the Shapiro-Wilk test.
The significance of treatment differences was analysed using ANOVA.

Growth performance
The results of DWG, DFI and FCR for the starter, grower, and finisher phases are indicated in Table 3. Different levels of GTP and MLP did not significantly affect DFI (p > 0.05). However, the main effects of GTP and MLP were significant (p < 0.05). Thereby, the groups receiving 2% GTP or MLP showed more DWG compared to the control group (p < 0.05). However, no significant difference was observed between the groups receiving 1% and 2% GTP or MLP (during the grower and whole phases) (p > 0.05). Regarding the main effects of GTP and MLP on FCR, the treatments receiving 2% GTP or MLP showed a better FCR compared to the control treatment (during the grower and whole phases) (p < 0.05). The interactions were not significant (p > 0.05).

Carcass characteristics
The results of carcass characteristics percentage at 42 days of age are presented in Table 4. Carcass characteristics were not affected by experimental treatments (p > 0.05).

Immune response
The data related to the main and interaction effects of GTP and MLP on the total Anti-SRBC, IgG, and IgM on days 21, 28, 35 and 42 are shown in

Intestinal morphology
The main and interaction effects of GTP and MLP on intestinal morphology are given in Table 6. About the main effects of GTP, the groups fed with 2% GTP had lower VH and VH: CD than the control or groups receiving 1% GTP (p < 0.05). Regarding the main effects of MLP, the groups fed with 2% MLP had lower VH than the groups receiving 1% MLP (p < 0.05). Concerning the interaction effects of GTP and MLP, the highest and lowest VH were observed in the treatments containing 1% GTP + 1% MLP, and 2% GTP + 2% MLP, respectively, which were significantly different from the control group (p < 0.05). Treatments: 1% GTP + 1% MLP, 1% GTP + no MLP and 2% GTP + no MLP showed the highest VH: CD, which were significantly higher than the control treatment (p < 0.05).

DISCUSSION
Phytochemicals or bioactive ingredients in herbs, if used in sufficient amounts, will be beneficial for animal health (Adegbeye et al., 2020).
Digestive enzymes and immune system function are stimulated by plant leaves (powdered or herbal extract), herbs, spices, and other plant-based products (as a substitute for antibiotics) (Brenes & Roura, 2010). Former surveys have found that adding 10% and 20% fermented and unfermented mulberry leaves in the broilers' diet considerably decreased the final body weight and digestibility of dry matter and crude protein (Has et al., 2013). One study showed that birds receiving different levels of MLP (0.1%, 0.2% and 0.3%) had better performance compared to the control group, especially at a concentration of 0.3% (Saenthaweesuk et al., 2010). In the current study, although there was no significant difference between the treatments in terms of DFI, the consumption of different levels of GTP and MLP increased the DWG, and these changes improved the FCR, so that the treatments receiving 2% GTP or MLP showed a better FCR compared to the control treatment during the grower and whole phases. The active compounds in herbals play a role in diminishing the population of harmful bacteria and increasing the population of beneficial bacteria in the digestive tract, and stimulating immunity. These beneficial bacteria have a significant role in improving the digestion of birds through the production of many essential enzymes in the digestive tract (Seidavi & Simoes, 2015). It is proposed that dietary supplementation of MLP boosts the broilers' growth performance via improving intestinal tissue structure, digestive enzyme activities and nutrient digestibility (Ding et al., 2021). In the present study, GTP and MLP have stimulated humoral immunity and increased the VH: CD. As a result, performance improvement can be attributed to strengthening immunity, and enhancing digestion. A study showed that the green tea supplementation at 0.5%, 1% and 1.5% in the form of powder in the broilers' diet has no effect on feed consumption (Hrncar & Bujko, 2017). Saraee et al. (2015) observed that the supplementation of fish oil (0.00%, 1.50% and 2.00%), GTP (0.00%, Means within a column without a common superscript significantly differ (p < 0.05).

TA B L E 4
Effects of different levels of green tea (GTP) and mulberry leaf powder (MLP) on carcass characteristics (%) of broilers at 42 days of age. reactive oxygen species or cell damage by free radicals that cause oxidative stress (Surai, 2019). The reasons for the difference in bird performance could be the source and composition of green tea (total, hydrolysable, and condensed polyphenols), the age and species of birds and the measured performance parameters. Hrncar and Bujko (2017) showed that the various amounts of green tea did not affect carcass parameters, carcass products and most internal organs in broilers. In agreement with their study, we did not observe any significant difference between the treatments. Biswas and Wakita (2001) reported that the dressing percentage was not affected by the GTP added in four levels (0.5%, 0.75%, 1% and 1.5%). On the other hand, Olmo et al. (2012) found that the substitution of mulberry leaf meal at 10%, 20% and 30% decreases the weight of carcass, breast, thigh, leg and abdominal fat. Ding et al. (2021) reported that the sup- One study checked the effect of green tea against coccidiosis in chickens and observed that there was increased cellular, and humoral immunity against coccidiosis, due to improvement in immunogenic response and immunoglobulin titres (total, IgG and IgM) (Abbas et al., 2017). The titre of antibodies in broilers reverberates the potency of humoral immunity. The superior the antibody titre, the more powerful the resistance of birds to infectious illness, and the less the chance of infectious diseases in birds (Gheorghiu et al., 1985). Immunoglobulins G, M and A are the primary immunoglobulins generated by activated B-lymphocytes, indicating the humoral immune conditions. Amongst them, IgG is the principal member of antibodies in the serum and body fluids. It mostly takes part in the body's re-humoral immune response (Lebacqverheyden et al., 1974). IgM is the first immunoglobulin is generated by activated B cells stimulated by antigen (Dekruyff et al., 1985). This type of immunoglobulin has a potent antigen-binding TA B L E 5 Effects of different levels of green tea (GTP) and mulberry leaf powder (MLP) on total antibody titres, IgG and IgM (log2). Means within a column without a common superscript significantly differ (p < 0.05).

TA B L E 6
Effects of different levels of green tea (GTP) and mulberry leaf powder (MLP) on jejunum morphology at 42 days of age. of green tea on the intestinal tissue, and it is maybe the effect of green tea on apoptosis that leads to these differences in the morphology of the intestine. It has been shown that dietary antioxidants significantly protect the gut epithelial cells from stress, and improve the growth of epithelial cells (Miller et al., 2001). Previous studies revealed that as the VH increases, the intestinal digestive and absorptive functions elevate, therefore enhancing absorptive surface area, brush border enzymes' expression, systems of nutrient transport, and body weight (Awad et al., 2009;Mohamed et al., 2014 and powder (1, 2 and 3 g/kg) in the broilers' diet and reported that green tea increased VH and VW in the different intestinal parts, compared with control. When studying the effect of orange essential oil on the histology of the intestine of broiler chickens, Erhan and Bolukbasi (2017) found that using 3 mL/kg of oil in the diets increased the VH.

Experimental treatments
They related the increase in the VH to the antioxidant properties of the studied essential oil. It is known that green tea contains a high level of polyphenols, (Wu et al., 2016) and the main ingredient of polyphenols is catechins (Pasrija et al., 2015), which can efficaciously prevent the proliferation of pathogenic bacteria, and ameliorate the animal intestinal health (Hara, 2000).

CONCLUSIONS
The results showed that 2% green tea leaf powder or mulberry leaf powder in the broilers' diet during the grower and whole phases caused a significant increase and decrease in daily weight gain and feed conversion ratio, respectively. Regarding the immune response, the use of 2% green tea leaf powder and mulberry leaf powder showed a higher titre for total anti-sheep red blood cells and IgG. Meanwhile, the highest villus height to crypt depth ratio was observed in the groups fed with 1% green tea leaf powder + 1% mulberry leaf powder. In general, higher levels (2%) of green tea leaf powder and mulberry leaf powder are likely to improve the performance and immune response in broilers, while achieving the best villus height to crypt depth ratio, using a level of 1% green tea leaf powder without mulberry leaf powder seems to be sufficient.

AUTHOR CONTRIBUTIONS
Fatemeh investigation;

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.

ETHICS STATEMENT
All procedures were approved by the Animal Care and Use Committee of the Ferdowsi University of Mashhad, Mashhad, Iran.