Influence Of L-theanine Dietary Supplementation On Growth Performance,Meat Quality,and Immune Response Of Broiler Chickens

L-theanine is a non-protein water soluble amino acid(AA)mostly found in leaves of Camellia sinensis(green tea).This is a key component of green tea,and is considered as the most abundant i.e.,about 50% of total amino acids in green tea.It has a unique taste constituent of tea,producing an attractive flavor and aroma in tea.It is reported as a safe natural amino acid with a potential of non-toxic phytogenic food additive.It has worth notice biological effects like antioxidant,growth promoter,immune booster,anti-stresser,hepactoprotective,antitumor,anti-aging,antimicrobial,anti-inflammatory and anti-anxiety activities.It could reduce the oxidative damage by reducing the production of reactive oxygen species(ROS),oxidative parameters and lipid damage as well as increasing the activity of antioxidant enzymes.Moreover,L-theanine might improve hepatocyte antioxidative capacity by inhibiting the formation of malondialdehyde(MDA)and restoring the activities of antioxidant enzymes such as catalase(CAT),superoxide dismutase(SOD),and reduced glutathione(GSH)in liver injury disorder.The oral ingestion of L-theanine enhanced γδ Tcell proliferation.Therefore,it is being considered an essential compound of green tea’that has ability to improve immune function.This AA(L-theanine)can be used as a potential ailments for hepatic injury and immune-related liver diseases via the downregulation of the inflammatory response through the induction of nitric oxide(NO)production and GSH synthesis are likely to be critical for the control of hepatic diseases as well as for the improvement of immune function.In addition,it is very effective for treatment of hypercholesterolemia through its vital ability to inhibit lipid peroxidation.After systematically reviewed the literature it had noticed that,mostly studies done on mice,pig,human and butterfly;while dietary supplementation studies of L-theanine in animal and poultry especially among broilers was very limited due to less awareness pertain to this amino acid.So far,according to the authors knowledge most of previous literature was available on human,mice,and pigs;and there was a serious gap of information in literature on the use of L-theanine in farm animal and poultry especially in broiler production.So it was the first time study that conducted on L-theanine which provided a new insight regarding this amino acid that could be used as a best natural feed additive in poultry farming.The study was conducted various experiments discussed as given below.1.Influence of Graded Levels of L-Theanine Dietary Supplementation on Growth Performance,Meat Quality,and Immune Response of Broiler Chickens.This study was conducted,to evaluate the effect of different dietary levels of L-theanine on meat quality,growth performance,immune response,and blood metabolites in broilers.A total of 400 day-old broiler chicks were randomly divided into four treatment groups using a completely randomized design;C-control,basal diet;100LT-basal diet + 100 mg Ltheanine/kg diet;200LT-basal diet + 200 mg L-theanine/kg diet;and 300LT-basal diet + 300 mg L-theanine /kg diet.Results revealed that the intermediate level of L-theanine(200 mg/kg diet)showed better results in terms of body weight gain(BWG),feed consumed(FC),and feed conversion ratio(FCR)as compared with the other supplemented groups and the control.The live weight eviscerated weight and gizzard weight were higher in all L-theanine levels as compared to those of the control group.Increased weight(p ≤ 0.05)of spleen and bursa were found in group 200LT(200 mg L-theanine/kg diet).Concerning meat color parameters,values for yellowness(b*),and redness(a*)were greater in L-theaninesupplemented groups than the control.Supplementing broiler diet with L-theanine reduced(p= 0.02)total serum cholesterol contents while increased HDL.Further analysis revealed lower relative serum cytokines(IL-2 and INF-γ)and reduced m RNA expression of TNF-αand IL-6 in thymus,and IFN-γ and IL-2 in spleen in the treated group.Moreover,supplementation with 200mg/kg of L-theanine improved antioxidant status in blood by increasing SOD,GSH-Px,and relative CAT levels.It is concluded that the optimum supplementation level of L-theanine is 200 mg/kg of diet because it resulted in improved performance parameters in broilers.However,higher levels of L-theanine(300 mg/kg diet)may have deleterious effects on performance and health of broiler chickens.2.16 S rRNA Sequencing Reveals Modulation of Intestinal Microbiome and Immune Response by Dietary L-Theanine Supplementation in Broiler ChickensThe 2nd trail was purely on microbes and immunity of broilers.Gut health is an important component of successful and profitable commercial chicken enterprise.Gut microbiota aids in efficient nutrient uptake and utilization while significantly influencing intestinal functions leading to better growth and health in broiler chicken.Despite the availability of abundant literature on green tea,studies on the use of L-theanine(unique amino acid found only in green tea)as a feed additive in animals,and poultry especially broiler are limited.So,this study was conducted to explore effects of L-theanine on intestinal microbiome and immune response in broiler.A total of 400 day-old chicks were randomly divided into four treatment groups(A,B,C,and D)using a complete randomized design.Treatments were as follows: A;control(basal diet),B;basal diet+100 mg L-theanine / kg diet,C;basal diet+ 200 mg Ltheanine / kg diet,and D;basal diet+ 300 mg L-theanine / kg diet.In our previous study,better growth performance was observed at intermediate level of L-theanine(200mg/kg of diet),so these samples were further used for characterization of intestinal microbiome and immune response.Mucosal samples from ileum and jejunum of broiler chicken were extracted at 21 and 42 days of age.Extraction of genomic DNA was followed by amplification of V3 and V4 hypervariable regions of 16 S r RNA.After Illumina sequencing,comparison of hypervariable region tags was performed with r RNA sequence databases and statistical analyses were performed in mothur software.Additional analyses for predicted metagenomes were performed through Green genes databases.The expressions of tight unction proteins of intestinal ZO-1,Occuldin and Claudin-3 were also determine to evaluate effect of L-theanine by using method of 2-??Ct.Results revealed that treatment with Ltheanine significantly increased population of Lactobacillus in ileum and jejunum as compared to control group but higher population was observed in jejunum at both 21 and 42 days of age.The overall diversity of the jejunum microbiome in treatment group was significantly lower than that of the ileum and control group(P<0.05).Results of this study revealed that m RNA expression of TLRs(TLR-2,TLR-4)and cytokines(TNF-α,IFN-γ and IL-2)was decreased in response to treatment with L-theanine.Moreover,negative correlation of abundance of lactobacillus was observed with expression of IL-2 and IFN-γ in intestine and these effects were highly significant(P<0.01).Overall results revealed that L-theanine exhibited positive influence on intestinal bacteria by favoring beneficial microbes like lactobacillus while decreasing harmful microbes like Clostridium.Moreover,L-theanine showed its ability to modulate intestinal immunity and inflammatory response by regulating expression of cytokines and TLRs.Overall,the dietary L-theanine at a dose of 200 mg/kg significantly increased growth performance,promoted meat quality,and improved immune response in broilers.Thus,Ltheanine can be used as a safe natural additive in the breeding of broilers.