Effects of dietary inclusion of Moringa oleifera methanolic extract on productive performance, humoral immunity and nutrient digestibility in Japanese quails

ABSTRACT A total of 180 unsexed, 1-day-old quail chicks were divided into four treatments (three replicates). The treatment groups were designated as MOLE-0, MOLE-1, MOLE-2 and MOLE-3. In the MOLE-0 group, quails received a basal diet without supplementation. For MOLE-1, quails were provided with 100 mg/L of MOLE in their water, while MOLE-2 received 200 mg/L of water with MOLE supplementation. The fourth treatment, MOLE-3, received a concentration of 300 mg/L of MOLE in their drinking water. The findings revealed that MOLE-3 exhibited significantly higher weight gain and dressing percentage, along with a lower feed conversion ratio (P < 0.05), while the control group (MOLE-0) exhibited the least growth performance. MOLE-3 showed statistically higher antibody titres and immune organ weights, followed by MOLE-2 and MOLE-1. Conversely, the control group (MOLE-0) had a lower antibody titre against ND. Additionally, nutrient digestibility analysis indicated that MOLE-3 had higher digestibility of dry matter and crude protein (P < 0.05), while group MOLE-0 exhibited lower digestibility (P < 0.05). We concluded that supplementing Moringa oleifera leaf extract at a rate of 300 mg/L resulted in improved growth performance, immunity and nutrient digestibility in Japanese quails.


Introduction
Antibiotics are usually added into chicken diet for several purposes such as the treatment of infectious diseases or used subtherapeutically to prevent infections and promote growth performance in broilers (Khan et al. 2022a).However, the widespread use of antibiotics has led to the emergence of antimicrobial resistance.This resistance is linked to humans consuming poultry products containing traces of antibiotics (Khan et al. 2022c).As a reaction, the European Union has prohibited the utilization of antibiotics in feed to reduce their harmful effects (Fatima et al. 2022).Consequently, there is a need to find alternative growth stimulants to replace antibiotics in chicken production.
The prohibition of antibiotics as growth enhancers has spurred comprehensive research into alternatives for poultry production, including probiotics (Khan and Naz 2013), prebiotics (Haq et al. 2020), enzymes (Jabbar et al. 2021), organic acids (Khan et al. 2021), herbs (Kairalla et al. 2022a(Kairalla et al. , 2022b) ) and other compounds (Rahman et al. 2017;Kareem et al. 2018;Chand et al. 2020).Among these alternatives, researchers are exploring phytogenic compounds for use in chickens due to their numerous benefits, such as improved feed conversion ratio, better immunological status and augmented gastrointestinal health (Zamani et al. 2017;Kairalla et al. 2023).M. oleifera, a compact drought-resistant deciduous tree standing 5-12 cm tall (Khan et al. 2021), holds exceptional value for humans and animals alike, providing nutrition, medicinal properties and industrial applications (Mbikay 2012).This plant is rich in beta-carotene, amino acids and vitamins, contributing to its therapeutic potential in treating various ailments (Fuglie 1999).Moreover, it serves as an antioxidant, antimicrobial agent and growth stimulant (Ullah et al. 2022), with antioxidant benefits derived from caffeic acid and cinnamic acid (Khan et al. 2021).With over 92 beneficial components, M. oleifera proves to be a valuable natural resource (Nnam 2009).
Moringa oleifera is commonly used in broiler rations in the form of powder or aqueous extract.However, there have been limited studies investigating the effects of its aqueous extract on broiler performance and health.One such study by Alabi et al. (2020) reported that administering 90 mL of M. oleifera leaf extract (MOLE) reduced feed intake while improving feed to gain ratio in broilers.Similarly, in another study conducted by Paul et al.(2018) the aqueous extract of M. oleifera leaves was found to decrease feed intake and improve feed conversion ratio compared to antibiotics in broilers.In a recent study, Khan et al. (2022b) demonstrated that supplementing broiler chicks with MOLE (120 mL/L) of drinking water led to enhanced carcass yield, improved immunity, blood metabolites, and a reduction in melanodialdehyde.Few studies have documented the methanolic extract of moringa on growth, digestibility of nutrients and immune response of quails.Therefore, the aim of the present study was to find different levels of methanolic extract of growth performance, carcass quality, nutrient digestibility and antibody titre against New Castle disease (ND) virus in Japanese quails.

Preparation of Moringa oleifera MOLE
The leaves of M. oleifera were obtained from the Medicinal Plants Section of Pakistan Forest Institute, Peshawar.The green and fresh leaves of moringa were air dried in a shaded area till the constant weight was achieved.To inhibit fungal growth continuous turning of leaves were done.The dried leaves were properly grinded and were sieved through 0.15 mm sieve.In Soxhlet, 200 grams of leaf powder was soaked overnight in 100 mL of methanol (100 percent methanol).About 50 mL extract were dissolved in dimethyl sulfoxide using clean vessel for 72 h, yielding a compact mass having 10 mg/mL concentration known as Moringa oleifera methanolic extract.

Experimental house and management
The poultry house and equipment's were cleaned using water and disinfected with formalin.Before chick's arrival the house was left empty for 14 days to get microbial free environment.Temperature and humidity were maintained according to age and condition of chicks.

Birds management and diet composition
A total of 180 unsexed, 1-day-old quail chicks were divided into four treatment groups.Each treatment consisted of three replicates, with 15 quails per pen.Throughout the study, all quails had free access to commercial feed (Table 1) and water.The birds were housed in metallic cages measuring 80 × 45 × 45 cm, equipped with feeders and drinkers.The light program initially provided 14 h of light daily, which was later increased to 16 h.The corn-soybean diets were in mash form and were formulated following the National Research Council's guidelines (1994) as shown in Table 1.The birds received vaccinations by a veterinarian at the appropriate age using distilled water.Ambient temperatures were recorded daily with the highest and lowest temperatures ranging from 14 to 23°C.The relative humidity averaged around 60-70%.All quails were raised in wire batteries under consistent management, hygiene, and environmental conditions.The four groups were labelled as MOLE-0, MOLE-1, MOLE-2, and MOLE-3.In the MOLE-0 group, quails received a basal diet without any supplementation.For MOLE-1, quails were provided with MOLE at a rate of 100 mg/L of water, while MOLE-2 received 200 mg/L of water with MOLE supplementation.The fourth treatment, MOLE-3, received MOLE at a concentration of 300 mg/L in their drinking water.

Productive traits
To assess productive response, the following indicators were used: feed intake (FI), weight gain (WG), and feed conversion rate (FCR).At the end of the experiment, 96 male Japanese quails (3 animals in each subgroup, totalling 12 animals in each group) were randomly selected.The cervical dislocation method was used to euthanize the quails, and their skin and internal organs were collected to assess the dressing percentage.

Antibody titre
On day 35 of age, blood samples were collected randomly from five birds per replicate pen.The samples were used to analyse the antibody titre against New Castle disease virus.After collection, the blood samples were centrifuged at room temperature for 10 min at 3000 rpm to obtain serum.

Apparent nutrient digestibility
Upon reaching 35 days of age, three birds from each replicate pen were selected at random and placed into meticulously cleaned cages for the purpose of measuring nutrient digestibility.The cages were equipped with feed troughs and water nipples.The same treatments given during the growth study were administered to the birds.After a 3-day acclimatization period, measurements were taken over 5 days.Excreta collected were meticulously screened to remove any spilled feed and feathers.The collected excreta were then air-dried under ambient room temperature, finely ground, and subsequently employed for proximate analysis.The samples were analysed for crude protein (CP), ether extract (EE), crude fat (CF) and nitrogen free extract (NFE) using the methods of the Association of Official Analytical Chemists ( 2005).The apparent digestibility values for CP, EE, CF, and NFE were then calculated

Statistical analysis
The data was subjected to statistical analysis using a completely randomized design (CRD) to compare means.The least significant difference (LSD) test was applied at a significance level of 5% to determine significant differences between the groups.

Results
The feed intake was non-significant (P < 0.05) during experimental trail from day 0 to day 42 as shown in Table 2. Numerically high feed consumption was recorded during finisher period in MOLE-0 (Control) which were not treated with methanolic extract of M. oleifera as compared to the treated groups (MOLE 1, 2, 3) which were supplemented with Moringa oleifera at the rate of 100, 200 and 300 mg per litre in drinking water.
Similarly, on overall basis, the treated groups showed low feed intake as compare to MOLE-0.The data of weight gain in Japanese quails fed with different levels of methanolic extract of moringa is given in Table 3. Weight gain was substantially (P > 0.05) increased with increasing rate of methanolic extract of M. oleifera in all treated groups.During starter phase, high (P < 0.05) weight gain was recorded in MOLE-3 linearly followed by MOLE-2 and MOLE-1, while low (P < 0.05) weight gain was recorded in MOLE-0.During finisher phase, weight gain was increased (P < 0.05) with increasing level of moringa.Significantly (P < 0.05) high weight gain was recorded in MOLE-3 while the control group experienced the lowest weight gain.Similar pattern of weight gain was recorded for overall.
With increasing levels of methanolic extract of M. oleifera during the course of the trial, FCR was significantly (P < 0.05) enhanced (Table 4).MOLE-0 recorded the greatest (poor) feed FCR during the starter phase.Similarly, higher (poor) FCR was noted in MOLE-0 (control) during the finisher phase, while lower (good) and the same feed conversion ratio were recorded in all treated groups.Overall FCR of MOLE-3 was significantly (P < 0.05) lower and was followed by MOLE-2 and MOLE-1.Overall FCR for the control group was recorded poor.
Immune organs weight was significantly (P < 0.05) improved with increasing level of MOLE (Table 6).Statistically higher relative weight of thymus was found in MOLE-3 followed by MOLE-2, MOLE-1 and MOLE-0 (control).Similarly, high (P < 0.05) and the same relative weight of spleen was found in MOLE-3 and MOLE-2, while lowest (P < 0.05) spleen weight was recorded in the control group.Relative weight of bursa was found to be lowest (P < 0.05) in the control group (MOLE-0) and MOLE-1, while highest (P < 0.05) value was recorded for MOLE-3.The data in Table 7 showed the effect of addition of different MOLE on nutrients digestibility of quails.There was no effect (P > 0.05) in digestibility of ash, EE, CF, and NFE.The digestibility of dry matter and CP was significantly affected by supplementation of MOLE.The digestibility of DM and CP was higher (P < 0.05) in MOLE-3 and lower (P < 0.05) in group MOLE-0 (control group).

Discussion
Our findings showed that supplementing MOLE at a rate of 300 mg/L improved body weight gain and feed conversion ratio (FCR).The leaves of M. oleifera are remarkably rich in protein, containing approximately 25-27%, making them highly suitable for animal consumption.The amino acid profile of M. oleifera leaves and soybean meal exhibited similarity.Moreover, these leaves contain all the essential amino acids needed by animals.Studies have reported that M. oleifera possesses higher iron content four glasses of milk and twice the protein content of milk.The nutritional analysis of M. oleifera leaf meal revealed it contains 22.5% crude fibre, 29.7% crude protein, 4.38% ether extract, 0.26% phosphorus, 27.9% calcium and minimal tannin content.According to a study by Lu et al. (2016) while M. oleifera did not significantly affect feed intake, however, it positively impacted FCR.In comparison to the control group, Riry et al. (2016) observed a reduction in feed consumption among Japanese quails that were fed M. oleifera seed meal.Likewise, Elkloub et al. (2015) discovered that Japanese quails provided M. oleifera leaf meal recorded the lowest feed intake and the most favourable FCR.Kulkarni and Durge (2019) observed an improved FCR in Japanese quails provided with M. oleifera leaf meal.On the other hand, Ahmed and El-Rayes (2019) noted an increased FCR in Japanese quails fed M. oleifera leaf meal in their diet.Nevertheless, there exist inconsistencies in the research outcomes concerning feed intake in birds supplemented with M. oleifera.Castillo et al. (2018) documented a noteworthy rise in feed conversion ratio (FCR) among Japanese quails administered M. oleifera leaf flour, while observing no substantial alteration in feed consumption.Conversely, Alabi et al. (2020) noted that the Moringa meal supplement led to reduced feed consumption, possibly due to increased nutritional satisfaction.
These discrepancies may be attributed to factors such as the dosage, duration, preparation of Moringa, the quail strains used, and variations in experimental design.Adding Moringa leaf meal to broilers' feed has shown to improve weight gain in other studies (Nouman et al. 2014).Likewise, Elkloub et al. (2015) observed that Japanese quails receiving 0.2% Moringa meal exhibited greater weight gain compared to those in the control diet.Talukdar et al. (2020) also identified a notable rise in growth among Japanese quails that were supplemented M. oleifera leaf meal in comparison to the control group that received no supplementation.This indicates that M. oleifera leaf meal holds a promising profile as a viable natural feed additive for boosting the overall performance of Japanese quails.Moreover, Kulkarni and Durge (2019) noted a rise in weight gain among Japanese quails that were supplemented M. oleifera leaf meal, surpassing the weight gain observed in the control group.Similarly, Ahmed and El-Rayes (2019) reported noteworthy increases in weight gain among Japanese quails that were fed M. oleifera leaf meal as part of their diet, in comparison to the control group.In the current study, Mole supplementation at the level of 300 mg/L resulted in significantly higher dressing percentage or carcass weight.M. oleifera leaves are rich in alpha-linoleic acid and various essential amino acids (Khan et al. 2021).The occurrence of dietary antioxidants within M. oleifera is likely accountable for the observed rise in carcass weight (Hekmat et al. 2015).These antioxidants play a role in mitigating stress among birds and enhancing the processes of protein absorption and digestion (Qwele et al. 2013;Saini et al. 2014).Ahmed and El-Rayes (2019) recorded significant rises in dressing weight within the group of Japanese quails that incorporated M. oleifera leaf meal into their diet.The abundance of carbohydrates, protein, and fibre in M. oleifera, along with its low fat content, may offer an explanation for the noticeable increase in carcass weight.Moreover, the substantial presence of pepsin and total soluble protein in M. oleifera leaf meal affirms its appropriateness as a dietary protein source for non-ruminant animals.This, in turn, contributes to the enhancement in carcass weight, as highlighted by Nouman et al. (2014).In the present study, the antibody titre and the weight of immune organs were significantly higher in Japanese quails fed MOLE at the level of 300 mg/L.Similar to our findings, Hassan et al. (2016) also reported that supplementation of Moringa oleifera leaf meal (MOLM) at a rate of 15% resulted in an enhanced antibody titre against New Castle Disease Virus (NDV) in birds.Previous studies, like the one conducted by Du et al. (2007), have also shown that MOLM supplementation in broiler diets can increase antibody titre against diseases.The increased antibody titre observed with MOLM may be attributed to the presence of lectins in Moringa leaves, which are known to enhance the body's defence system (Hassan et al. 2016).As a result, Moringa oleifera is considered an immune-boosting plant that helps the body strengthen its defence against diseases.According to Katanbaf et al. (1989), an increase in relative organ weight is considered an indicator of immunological improvement.The enhanced immunity observed may be attributed to the unique combination of phenolic compounds present in the leaves of M. oleifera, including zeatin, quercetin, kaempferol, and apigenin.
In the current study, the digestibility of dry matter and crude protein was significantly higher in quails supplemented with MOLE 300 mg/L.Nutrient digestibility refers to the proportion of feedstuff absorbed by an animal's body upon consumption.Egbu et al. (2022) conducted a study on Cobb 500 broilers and found that the administration of Moringa oleifera seed extract (MSE) in drinking water had digestion-enhancing qualities similar to other herbal medications.MSE appeared to promote the growth of beneficial bacteria while inhibiting harmful microbes, potentially influencing poultry growth and intestinal microbiota (Akhouri et al. 2013).According to Siti et al. (2019), the active ingredient in MSE works by inhibiting microbial pathogens and endotoxins in the intestine, as well as increasing pancreatic activity, leading to improved nutrient metabolism and utilization.Ossebi (2002) conveyed that the incorporation of Moringa leaves up to a 24% level in feed exhibited no detrimental impacts on nutrient absorption.Conversely, it led to a substantial enhancement in protein digestibility, energy utilization and mineral utilization.

Conclusion
From the results of the present study, it was concluded that supplementation of Moringa oleifera leaves extract at the rate of 300 mg/L resulted in improved growth performance, immunity and nutrient digestibility in Japanese quails.

Table 1 .
Dietary composition of broiler chickens during finisher diet.

Table 2 .
Mean feed intake (g) in broilers fed with different levels of methanolic extract of Moringa oleifera leaves (MOLE).

Table 3 .
Mean weight gain (g) in broilers fed with different levels of methanolic extract of Moringa oleifera leaves (MOLE).
Means in the same column with different superscripts are statistically different (P < 0.05).MOLE represents methanolic extract of Moringa oleifera; 100-300 mg per litre of drinking water.

Table 4 .
Mean feed conversion ratio in broilers fed with different levels of methanolic extract of Moringa oleifera leaves (MOLE).

Table 5 .
Mean dressing percentage and mortality in broilers fed with different levels of Moringa oleifera leaves (MOLE).

Table 6 .
Mean immune organ index in quails fed with different levels of methanolic extract of Moringa oleifera leaves (MOLE).
MOLE represents methanolic extract of Moringa oleifera; 100-300 mg per litre of drinking water.

Table 7 .
Nutrient digestibility in quails fed with different levels of methanolic extract of Moringa oleifera leaves (MOLE).Means in the same column with different superscripts are statistically different (P < 0.05).MOLE represents methanolic extract of Moringa oleifera; 100-300 mg per litre of drinking water.