Individual and Combined Effects of Moringa and Neem Leaves on Immune Response and Gut Microflora in Japanese Quails

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INTRODUCTION
I n an attempt, to augment the animal protein for human use quails were introduced. They are very prolific, have a short generation interval, and are disease resistant. Quails are a popular protein source because of their fast growth rates, early sexual maturity, short generation intervals, and resistance to a number of bird diseases (Mulaudzi et al., 2019). Quail farming is a relatively new industry that complements the production of low-cost animal proteins such as chicken, turkey, and ostrich. As a result, they helped to bridge the gap between the availability of other animal products on the table for human use. Traditionally used synthetic feed additives like antimicrobial growth stimulants have recently banned in commercial poultry farming (including quails' farmin), prompting further research into alternative growth promoters for animal production (Kamboh et al., 2015;Shahin et al., 2020). Many alternatives to these growth boosters, such as organic acids and medicinal herbs for chicken feed, have been proposed to enhance the performance of the birds (El-Saadany et al., 2022;Saki et al., 2012). Several plants like moringa, neem, ginger etc., were rported in recent literature for their significant potential to replace antibiotics in the poultry industry (Ashour et al., 2020;Untari et al., 2022).
Moringa oleifera has been shown to contain natural antioxidants like flavonoids, flavones and other phenolic compounds (Siddhuraju and Becker, 2003). Moringa leaves are rich in protein, vitamins (A, B, and C), iron, phosphorus, and calcium (Murro et al., 2003). Furthermore,

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heavy metals that might be harmful, such as mercury, arsenic, and cadmium, are not present in M. oleifera leaves, making its use in poultry diets safe (Donkor et al., 2013). Furthermore, polyphenols, anthocyanin, tannins, thiocarbamates, and glycosides, which activate antioxidant enzymes, remove free radicals, and inhibit oxidases, contributed to the antioxidant activity of moringa leaves (Luqmans et al., 2012) that ultimately effects immunity and modulate it towards the positive direction (Eladia and Ampode, 2021). According to a recent study conducted by our research group, moringa supplementation showed significant immunomodulatory effects in broilers (Afzal et al., 2020). Neem (Azadirachta indica) is a fast-growing evergreen tree that has the potential to provide medical and nutritional benefits to broilers. Extracts of neem leaves have been demonstrated in the literature to have antibacterial activity against bacteria like Streptococcus spp., Pseudomonas spp., Staphylococcus spp., and Escherichia coli, as well as against certain fungal species (Valarmathy et al., 2010). In addition, neem supplementation has been found to have immunomodulatory effects in farm animals (Landy et al., 2011) and aquaculture (Kaur et al., 2019).
Keeping in view the biological activities of moringa and neem, current study planned to investigate the individual and combined immunomodulatory and gut microflora modulatory effects of Moringa oleifera and Azadirachta indica leaf powder in Japanese quails.

Experimental plan and housing
The experimental design and procedures were approved by the Directorate of Advanced Studies, Sindh Agriculture University Tandojam (No. DAS/1425/of 2021) and were carried out according to prescribed ethical standard. Total eighty unsex day-old Japanese quail chicks were purchased from a commercial hatchery in Karachi and were raised in cage system available in the Department of Animal Nutrition, SAU, Tandojam. After arrival, chicks were first weighed and equally divided into four groups i.e., T0, T1, T2, and T3. Each treatment was consists of four replicates with 5 chicks in each replicate. T0 was fed control diet (basal diet), whereas T1 was supplemented 5% moringa leaf meal (MLM) in basal diet, T2 was supplemented 1% neem leaf meal (NLM) and T3 was supplemented with mixtures of 5% MLM and 1% NLM in basal diet respectively. Both the doses of neem and moringa (1 and 5%, respectively) were chosen based on a pilot study conducted on broilers (unpublished data). The experiment was lasted until five weeks of age.
Chicks were kept in cages during whole experimental period. Using disinfectant and fresh water, the cages were fully cleaned before transfer chicks to them. Rice husk was utilised as litter up to a depth of 2-4 inches for each treatment of birds. To minimise gas generation, litter was turned on a regular basis. Paper sheets were utilised to shield the litter during the first week of brooding. The temperature was kept at 90-95 o F for the first week, then reduced by 5 o F each week until the temperature was kept at 70 o F. It was customary to feed the birds twice a day. The birds have unlimited access to fresh and clean water. The feed was prepared according to the instructions of National Research Council (NRC, 1994) and its ingredients and chemical composition is shown in Table I. While the nutritive value of MLM and NLM is presented in Table II.

Determination of relative weight of lymphoid organs
Eight birds from each group were weighed and killed via islamic slaughtering method of neck cutting at 35 days of age, and the carcass weight as well as the weight of lymphoid organs (thymus, spleen, and bursa) was determined using a digital balance. The relative weight of lymphoid organs was calculated using organ and live bird weight (Memon et al., 2019).

Complete blood count
At 35 day, eight birds from each group were slaughtered and a two milliliter of blood sample was collected in tubes from the both treated and control group. The sample were used for hematological analysis using an automatic hematology analyser (SINNOWA Medical Science and Technology Co., Ltd., Nanjing, China) in order to investigate the supplemental effects of moringa and neem on hematological indices including RBC (red blood cell) count, MCH (mean corpuscular haemoglobin), MCV (mean corpuscular volume), PCV (packed cell volume), MCHC (mean corpuscular haemoglobin concentration), PDW (platelet distribution width), PCT (plateletcrit), MPV (mean platelet volume), and WBC (white blood cell) count. The hematology profile also illuminated the heterophil to lymphocytes ratio (H/L index), which is a vital indicator of immunity.

Quantification of cecal microflora
To count the cecal microflora in each group, duplicate cecal contents were collected aseptically from the intestines of slaughtered Japanese quail birds at 35 days into sterile Eppendorf tubes. These were well combined and kept in the refrigerator at 4°C for future examination. The method of Memon et al. (2019) with slight modifications was used for overall bacterial counts and enumeration of particular species such as E. coli bacteria and lactobacilli. In brief, a total of five dilution tubes holding 9 mL of sterile normal saline solution were used. One mL of the sample was used to prepare a 10-fold dilution. A 0.1 ml of every tube were then cultured on nutrient agar (Oxoid, UK) plates and stored for 24 h at 37°C. After incubation, colonies were counted using a colony counter and CFU/g results were calculated using following formula: CFU/g = (colony no. x dilution factor)/ volume plated All bacterial isolates were recognized based on the standardized cultural, staining and biochemical properties following the Bergey's Manual of Systematic Bacteriology (Whitman et al., 2012).

Statistical analysis
The results obtained in the work were analyzed using one-way analysis of variance (ANOVA). The JMP statistical package software (version 5.0.1a; SAS Institute, 2000) was used for calculating the differences between different supplementation levels and p< 0.05 was considered as limit for significance.

Live body weight, carcass weight and relative weight of immune organs
Effect of supplementation of MLM and NLM powder in Japanese quail diet on live weight, carcass weight and relative weight of immune organs was shown in Table III. The results exhibited that live body weight, carcass weight and relative immune organs weight (g) of Japanese quail was improved (p<0.05) by the supplementation of MLM and NLM powder. T3 (171.28 g) exhibited the highest raise in live body weight which was significantly (p<0.05) higher as compared to T2 (136.43 g), while non significantly higher (p > 0.05) than T1 (157.67 g) and T0 (156.01 g) groups. Carcass weight was also recorded highest in T3 (94.34 g) which was significantly (p<0.05) higher than control (T0, 71.2 g) and other supplemented groups. Relative weight of spleen was found higher (p<0.05) in T2 and T3 groups (0.17 g) as compared to T0 (0.14 g) and T1 (0.13 g) groups. Relative weight of thymus was recorded higher (p<0.05) in T3 (0.25 g) as compared to control (0.18 g) and other treated groups. Relative weight of bursa was

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higher (p<0.05) in T1 and T3 groups (0.22 g) as compared to T0 (control, 0.16 g) and T2 (0.14 g) groups.
All treated groups exhibited significantly high (p < 0.01) total bacterial count as compared to control group (T0). Lactobacillus count and Bifidobacterium count was also recorded highest in T3 (4.363 and 4.264 log cfu/g) followed by T2 (4.214 and 4.195 log cfu/g), T1 (4.190and 4.178 log cfu/g) and T0 (4.167 and 4.097 log cfu/g), respectively. All supplemented groups exhibited high (p < 0.01) Lactobacillus and Bifidobacterium count as compared to control group (T0). However, E. coli and Salmonella count was found highest in T0 (4.380 and 4.365 log cfu/g) followed by T1 (4.264 and 4.243 log cfu/g), T2 (4.130 and 4.195 log cfu/g) and T3 (4.056 and 4.096 log cfu/g), respectively. All treated groups exhibited significantly lower (p < 0.01) E. coli and Salmonella count as compared to control group (T0).

DISCUSSION
There has been a lot of research done on the impact of moringa and neem supplementation in chicken to modulate immunity, digestibility, and gut microbiota in a favourable circumstances (Eladia and Ampode, 2021;Malik et al., 2019). However, there is lack of literature on the individual and combined effects of these medicinal plants on immunity and gut microflora in quails. Therefore, MLM and NLM were tested in different doses in broilers in our laboratory (unpublished data) for immune and gut microflora modulatory effects and best doses (1% neem and 5% moringa) were again investigated in current research in Japanese quail to see how they affect growth, immunological responses, and gut microbiota in these birds.
The results obtained from this study exhibited that combined supplementation of MLM and NLM has significantly improved weight gain and carcass weight. This study confirms previous findings indicated that moringa leaf meal promoted growth and productivity in poultry which is attributed to its nutrients and phytochemicals that had antimicrobial properties and significantly may reduce the microbial load of birds and improve the feed utilization that ultimately enhance weight gain (Fahey et al., 2001;Kakengi et al., 2007). Also, the improvement in final body weight and body weight gain could be credited to best digestibility potential of moringa plant as it has natural enzymes which could enhance nutrients absorption (Backer, 1995). These findings are in agreement with the finding of Elkloub et al. (2015) who reported that the supplementation of moringa leaves meal with 0.2, 0.4 and 0.6% levels in the feed of the growing Japanese quails significantly (p<0.05) improved their final body weight and body weight gain compared with control group. Teteh et al. (2013) reported that 1 to 2% moringa leaf meal have positive effect (p<0.05) on daily weight gain and FCR, however it has no effect (p>0.05) on daily feed intake of Ross broilers.
The addition of MLM and NLM powder in combined form enhanced the relative immune organ weight ( Japanese quail (p<0.05). However, positive effects of individual treatments were also evident as compared to control group, but these effects were little when compared with combined supplementation. In agreement to our findings, the study of Ansari et al. (2012) reported a little (p<0.05) improvement in the relative weight of immune organs in neem supplemented chickens. In another study, broilers were supplemented with 1 and 2% of moringa leaves in diet and it was reported that moringa leaves dose-dependently improved (p <0.05) the weight of bursa, spleen and thymus as compared to control group (Teteh et al., 2013). In Japanese quails, MLM and NLM powder modulated the intestinal microbiota towards a positive direction that results significant increase in total viable bacteria, Lactobacillus and Bifidobacterium count, while reduced the count of E. coli and Salmonella. The results support the findings of Djakalia et al. (2011) who reported that moringa leaf extracts exhibit antibacterial properties. They stop growth of harmful bacteria (like Staphylococcus aureus) from growing in animal intestines and feed. The intestinal microbiota was substantially decreased with MOL powder. The study of Assunção et al. (2019) also reported the marvelous decline in the intestinal population of E. coli in neem supplemented broilers. The study of Nantapo (2018) indicated that broilers supplemented with 5% moringa leaf powder showed significantly lower E. coli and Clostridium perfringens and higher Lactobacillus count in duodenum and jejunum as compared to those fed 1% moringa leaf powder. These results are also in agreement with the study of Mandal et al. (2014), those further reported that 2% moringa in diet of broilers is also beneficial in reducing the microbial load of pathogenic bacteria in the meat. The presence of various photochemical and essential oils in moringa leaves may have role in decreasing the microbial load and coliform count at all levels of MOL feeding compared to control and even antibiotic fed groups (Yang et al., 2006).
The results of haematology and serum analysis are often utilised to determine an animal's health condition. Haematological and serum variables have been recognized as an excellent markers of an animal's physiological state, and variations in these parameters are essential in determining the animal's reaction to various physiological circumstances (Khan and Zafar, 2005). Our results demonstrated that all haematological values were within the normal range (Aiello and Mays, 1998). The significant increase in haemoglobin and RBC count in MLM and NLM supplemented groups indicates that these botanicals have potential to prevent animals from anemia (Ansari et al., 2012). Moreover, significant raise of haemoglobin in MLM (T1) and MLM+NLM (T3) groups indicates the hepatoprotective effects of both supplements, as liver is known to regulate the synthesis of haemoglobin in the bone morrow via release of erythropoietic factors (Browman et al., 1976). Similarly, significant increase in WBC count in MLM and NLM combined supplemented groups reflects that this combinatorial treatment has immune-stimulating potential, because WBCs are the soldiers of body defense system that known to eliminate invading pathogens by several means like neutralization, opsonization and phagocytosis (Lewis Marffy and McCarthy, 2020).
It is well established that raised H/L ratio in birds is a suitable indictor of environmental stress conditions (Landy et al., 2011). In our study, all moringa and neem supplemented groups significantly improved (p < 0.05) the lymphocyte %, while decreased (p < 0.05) the heterophil % and H/L ratio as compared to control group (T0). These results affirmed that like other flavonoid-rich botanicals, MLM and NLM (which are rich in flavonoids) have potential antioxidant effects thus may reduce the stress at cellular level which is evident by decreased H/L ratio (Silas et al., 2014;Landy et al., 2011) heterophil count which is also recognized as a biomarker of antioxidants against environmental stresses (Oyagbemi and Adejinmi, 2012). These results are parallel with the study of Khan et al. (2021) who reported a significant decline in H:L in broilers supplemented moringa extract in drinking water. Similarly, the study of Landy et al. (2011) reported a decreased H/L ratio in broilers fed neem fruit powder in diet.
In current investigation, combined effect of MLM and NLM was observed superior as compared to their individual effects, which might be due to tendency of both botanicals to form synergism. In previous research, it is well documented that plant compounds may produce additive, synergistic or antagonistic effects when combined with each other (Kamboh and Zhu, 2014). However, further investigation is recommended to illuminate this phenomenon using specially designed in vitro or in vivo studies.

CONCLUSIONS
The study concluded that moringa and neem have significant potential to modulate cecal microflora and immunity in quails, particularly when supplemented jointly. Thus, both plant leaves could be used in commercial quail farming (in a dose of 5% moringa + 1% neem) for better production and to overcome immunity-related issues. Meanwhile, further challenge studies are recommended using infections agents and/or their products to estimate the actual immunomodulatory potential of moringa and neem leaf meal powder.