Effect of Moringa oleifera (Lam.) pods as feed additive on egg antioxidants, chemical composition and performance of commercial layers

The present study was designed to investigate the influence of Moringa oleifera (Lam.) pod meal (MPM) on production, immunity, and functional food index of chicken eggs. Two hundred HyLine W36 layer birds aged 50 weeks, 1469 ± 46.63 g, were assigned to four treatments in a completely randomized design, with five replicates and ten birds each. Diets A, B, C, and D were formulated with the same caloric and protein levels, but with differing MPM dose levels of 0, 5, 10, and 15g MPM/kg finisher diet, respectively. Data for production performance, quality, and chemical composition of eggs were analysed by one-way ANOVA, and means were compared with Duncan’s multiple range test. As a result of this study, feed conversion ratio (FCR) and egg mass (EM) were significantly decreased and recorded lowest in Group B, which was offered 5 g/kg above the basal diet. Bioactives such as β-carotene, quercetin, and selenium levels were increased (540, 121, & 72.21μg/100g of yolk, respectively), whereas cholesterol levels in egg yolk and serum were decreased significantly, that is, 201.87 mg/100g and 8.47 mg/dl, respectively. Serum biochemical indicators, including serum glutamic-pyruvic transaminase (SGPT), glucose, creatinine and cholesterol levels, were lowered significantly. Proximate analysis of egg yolk showed that moisture and ether extract were decreased, whereas crude protein (CP), ash and minerals, sodium (Na), potassium (K), calcium (Ca), magnesium (Mg), and phosphorus (P) contents were increased. The outcomes of this study showed that MPM supplementation affects EM, serum biochemistry and bioactive compounds of the egg yolk positively. ______________________________________________________________________________________


Introduction
Conventional synthetic feed additives such as antibiotic growth promoters, antioxidants, anti-parasitic agents, and anti-fungal agents have been used in poultry feed for decades.However, they created multiple complications, such as traceability in animal products and resistance to antibiotics in the consumer, which became public health issues (Wallace et al., 2010;Ao et al., 2011;Kirkpinar et al., 2011;Embuscado, 2015;).On these grounds, the use of all kinds of antibiotic growth promoters was banned in animal feed in Europe (Nkukwana et al., 2014a).Revolutions in animal feed production gave rise to the idea of phytogenic feed additives (Grashorn, 2010).Plants and their metabolites, known as bioactive compounds, play a key role because of their feed additive attributes.These bioactive compounds, such as carotenoids, flavonoids, and essential oils, help to maintain animal health and productivity, and to produce safe and healthy chicken eggs (Windisch et al., 2008;Wallace et al., 2010;Ao et al., 2011;Embuscado, 2015).The primary mode of action of these active ingredients is inhibition of pathogenic microbes and endotoxins in the gut and enhanced pancreatic activity, resulting in better nutrient metabolism and utilization (Windisch et al., 2008;Grashorn, 2010).
Few studies are available on the use of Moringa pods as a phytogenic feed additive, and on its impact on bioactive compound enrichment in animal products.This study was planned in view of the challenges to animal feed producers regarding animal health, productivity, consumer health, traceability issues in animal products, and environment safety.Its objective was to investigate the effects of essential nutrients and antioxidant-enriched Moringa pods on production performance, immunity, and bioactive compounds of chicken eggs with various levels of supplementation.

Materials and Methods
Moringa oleifera pods were collected and stored in polythene bags after shade drying and grinding for further analysis and addition to feed (Banjo, 2012).The pod meal was analysed for chemical composition (Table 1) in the Department of Animal Nutrition laboratory, University of Veterinary and Animal Sciences (UVAS) and Pharmacy Department, University of Punjab, according to standard procedures (Mehta et al., 2003;AOAC, 2005).Selenium and bioactive compounds (β-carotene and quercetin) were analysed with an atomic absorption spectrophotometer and high-performance liquid chromatography (HPLC), respectively, in Quality Operations Laboratory (QOL), UVAS (AOAC, 2005;Farida et al., 2008;Saini et al., 2014a).Two hundred (50 weeks old) commercial layer birds, with a production percentage of 65-66, were assigned to four treatments and five replicates with ten birds each in a completely randomized design.Four levels (0, 5, 10, and 15g MPM/kg finished diet) of MPM were added to the four isocaloric and isonitrogenous diets: Diets A (ML 0%), B (MLM 0.5%), C (MLM 1.0%), and D (MLM 1.5%), with 160 g/kg crude protein and 2725 kcal/kg metabolizable energy (Table 2).
Table 2 Ingredients and chemical composition of layer experimental diets Note: DCP: di-calcium phosphate; CF: crude fibre, Se: selenium; A, B, C, and D: MPM dose levels of 0, 5, 10, and 15g respectively Cholesterol estimation in the egg yolk samples was performed by the standard method using a UVvisible spectrophotometer (AOAC, 2005).Equal quantities of acetone and egg yolk were taken and shaken vigorously for two minutes.After centrifugation, the supernatant was removed.This procedure was repeated three times and the pooled supernatant was evaporated to remove acetone, and kept for cholesterol analysis.Before quantification of cholesterol, it was de-esterified.The supernatant was dissolved in a few ml isopropanol and vortexed.In another tube, 1 ml sample was separated and 5 ml isopropanol was added and again vortexed.Reagents were added in properly labelled cuvettes almost simultaneously.The samples were allowed to rest for 10 minutes and the absorbance read at 500nm.
Data were analysed through one-way ANOVA (Steel et al., 1997) using PROC GLM in SAS software (SAS Inc. 9.4).Significant means were separated through Duncan's multiple range test (Duncan, 1955).The following mathematical model was used: Where: Y ij = observation of dependent variable recorded on i th treatment µ = population mean Ʈ i = effect of i th level (1, 2, 3, 4) Ԑ ij = residual effect of j th observation on i th treatment NID ~ 0, σ 2 A daily feed allowance of 100g per bird was offered.Feed offered, feed refused, feed intake, and mortality were recorded daily and tabulated cumulatively for FCR every week.Daily egg production was recorded from each experimental unit separately to calculate various parameters, including EM, egg weight, feed per dozen eggs, and feed per kg eggs.Egg shape index, surface area, volume, shell thickness, yolk index, and Haugh unit score were measured at the start of the experiment and then every two weeks throughout the experimental period.Three eggs were picked at random from each unit and subjected to egg geometry measurement.
Bird handling and collection of samples were performed according to the procedure approved by Advance Studies and Research Board (ASRB), UVAS.Blood samples were taken with sterile syringes containing anticoagulant from the wing web on days 28 and 42 of the experiment and stored.Blood samples were centrifuged and the serum was separated and stored for analysis of parameters, including glucose, serum glutamic-pyruvic transaminase (SGPT), alanine transaminase (ALT), creatinine, and cholesterol, with protocols for every parameter using a commercial kit from Merck Microlab-300, in WTO Laboratory, University of Veterinary and Animal Sciences, Lahore Pakistan.The serum samples were analysed for antibody titers against Newcastle Disease by haemagglutination (HA) and haemagglutination inhibition (HI) techniques.
Egg yolk and feed samples were analysed to estimate moisture, crude protein, crude fibre, ether extract, and ash (AOAC, 2005).Ash samples were used for mineral analysis.The wet digestion procedure was used for selenium analysis (AOAC, 2005).
HPLC techniques were used to estimate carotenoids (β-carotene) and flavonoids (quercetin) in the yolk samples (Tokusoglu et al., 2003;Saini et al., 2014b).Yolk samples were analysed for cholesterol level on a UV-visible spectrophotometric with the method described by AOAC (2005).
Quantification of carotenoids was done with the HPLC technique, and yolk samples were prepared as described by Saini et al. (2014a).In an HPLC tube 1 g yolk sample was weighed with 8 ml methanol and 2 ml 1N HCL.The yolk sample was vortexed for five minutes, and this process was repeated three times.The sample was centrifuged at 4000 rpm for 15 minutes.The supernatant was removed and dried in a water bath.When the sample was dried, 1 ml mobile phase was added with acetonitrile, dichloromethane and methanol in the ratio of 70:20:10 v/v/v.The sample was vortexed and filtered with Whatman filter paper and passed to the HPLC to quantify β-carotene.For this purpose, a C18 reverse phase column was used with inner dimensions of 250 mm × 4.6 mm, and a flow rate of 1 ml min − 1.A standard solution was prepared with 0.01 to 0.08 mg/L serial dilutions.
Egg yolk samples were analysed for quercetin levels using the HPLC technique with slight modifications to the standard method (Tokusoglu et al., 2003).For sample preparation, a measured 1 g weight of egg yolk was taken in HPLC tubes and acidified methanol was added.After lowering the temperature, the sample was centrifuged at 1500 g and 5000 rpm for 15 minutes and repeated three times.The supernatant was filtered and shifted to HPLC vials for quercetin analysis.The sample volume injected was 20 µl and the column used was reverse phase (C18) with dimensions (250 × 4.6 mm; 5 µm particle size).Two solvents, namely 3% trifluoro acetic acid and 80:20 v/v acetonitrile and methanol, were used as mobile phase in equal proportions and with a flow rate of 1.0 mL min _1 at 30 ℃.

Results and Discussion
The results of the present study showed that supplementation of MPM to the layer diet affected EM and FCR per kg EM significantly (P ≤0.05).The results showed significant effect of MPM supplementation on EM and FCR per kg EM (Table 3).The best EM and FCR values were observed in Group B, which was supplemented with 5g MPM/kg feed (P ≤ 0.05) (Table 3).All other parameters, including feed intake, egg weight, production percentage and FCR per dozen eggs, remained the same with the supplementation of MPM (Table 3).Improved EM and FCR may be attributed to the essential nutrients added by supplementation, especially amino acids (lysine, methionine) and vitamins, along with extra protein (0 g, 0.10, 0.19, & 0.28 g) per bird daily, in addition to total daily protein intake (16 g).Extra protein resulted in an increase in essential amino acids, especially methionine and lysine, which might affect EM positively (Rezaei et al., 2004).Higher fibre and anti-nutritional compounds resulted in bulkiness and decreased digestibility, which affected some parameters of production and egg quality negatively.Because the digestive system of poultry lacks fibre degrading enzymes, a quadratic response was observed with the higher dose level of MPM, which resulted in slightly poorer FCR and decreased EM compared with Diet B with a 0.5% dose rate of MPM (Abou-Elezz et al., 2011;El-Sheikh et al., 2015).The lowest FCR recorded was 1.67 in Diet B, with the supplementation of 5 g MPM/kg.Because feed intake was not significantly affected, there was no change in egg weight and production during the experimental period.Feed intake, egg weight, and egg production have positive correlations.Quercetin, a flavonoid that affects EM positively, improves FCR per kg EM (Mohammed et al., 2012;Liu et al., 2014).Similar results have been reported in other studies that showed the positive effect of Moringa on EM (Olugbemi, 2010).Contrary to these results, Paguia et al. (2014) used low levels (0% to 0.5%) of Moringa leaf meal and reported that feed consumption, FCR and egg production remained unchanged.
Table 3 Mean (± SE) production performance and egg characteristics of commercial layers fed on various levels of Moringa oleifera pod meal for 6 weeks (55-61 weeks old) Superscripts on different means within row show significant difference (P ≤0.05) FI: feed Intake (kg); EM: egg mass (kg); egg weight (g); FCRdz: feed conversion ratio/dozen eggs; FCRem: feed conversion ratio /kg EM; surface area (cm 2 ); volume (cm 3 ); HU: Haugh unit; ST: shell thickness (mm); A, B, C, and D: MPM dose levels of 0, 5, 10, and 15g respectively MPM supplementation had a significant (P ≤0.05) effect on Haugh unit and egg shell thickness.Significant decreases in these parameters were observed with the increasing dose rate (Table 3).Egg shape index, surface area, volume, and egg yolk index remained unchanged during the experimental period.Highest values of Haugh unit and shell thickness were observed in the control group, which had no supplementation of MPM (Table 3).Quercetin in smaller quantities potentiates phytoestrogens, but in higher quantities may act as antagonists and block receptors and finally result in lowered antioxidant activity and Haugh unit of eggs yolk (Liu et al., 2014).Low egg shell thickness in birds supplemented with Diet D could be attributed to the antinutritional factors present in Moringa pods that disturb the calcium metabolism (Olugbemi et al. 2010;Paguia et al., 2014).Similar results were reported by Tesfaye et al. (2014), who showed that egg quality parameters such as Haugh unit and shell thickness did not change with increasing levels of Moringa in the diet, whereas feed efficiency increased.As reported by Gakuya et al. (2014), egg quality parameters remained unchanged in the treatment groups throughout the supplementation period.Contrary to the current results, other experiments reported that plants containing bioactive compounds, such as essential oils, flavonoids, and carotenoids, affect Haugh unit and shell thickness positively (Nobakht & Moghaddam, 2013;Abbas, 2013) (Gakuya et al., 2014;Liu et al., 2014).This type of response may be attributed to higher levels of β-carotene and quercetin, 2.7-3.10 mg and 80-150 mg per 100g pods, respectively (Lako et al., 2007;Tesfaye et al., 2014).Similarly, other researchers stated that βcarotene is deposited in egg yolk when supplemented with coloured carrots at 70g/bird/day in the basal diet (Okonkwo, 2009;Hammershoj et al., 2010).Most of the β-carotene is enriched in egg yolk, whereas quercetin is enriched in egg albumin in chelation with amino acids.Similar results were reported in other studies in which nutritionists used canthacol, M. oleifera leaf meal, tomato peel, coloured carrots, and apple skin to enrich egg yolk with β-carotene and quercetin (Olson et al., 2008;Gakuya et al., 2014;Liu et al., 2014).Similarly, various studies showed the positive impact of M. oleifera on antioxidant compound deposition in chicken egg yolks (Mbikay, 2012).
Egg yolk selenium values in commercial layers showed a significant linear increase as the dose rate was increased (P ≤0.05).The group supplemented with maximum levels showed the highest values of selenium, whereas the control group showed the lowest values (Table 4).This higher level of enrichment is based on the form of selenium (seleno-methionine, seleno-cystine), because organic selenium has better bioavailability and retention in tissues (Delezie et al., 2014).Moringa oleifera leaves and pods have 2.88 mg and 25.7 mg/100 g Se per 100g dried leaves, respectively (Table 1).Eggs can be enriched easily with selenium, a key trace mineral that is involved in multiple metabolic, antioxidant, immune stimulator, and antiageing processes.Because M. oleifera is enriched with organic selenium, its supplementation may affect egg selenium content (Delezie et al., 2014).
In a study conducted by Farrell ( 2013), it was concluded that supplementation of layer diets with organic selenium @ 0.4 mg/kg of feed, the deposition in eggs would reach up to 20-60 µg/100 g edible egg.
Selenium and methionine have a close relationship in metabolism.Birds supplemented only with methionine (3.2, 4.0, and 5.4g/kg) without selenium showed 0.48, 0.63, and 0.62 µg/g of deposited selenium in egg yolk, whereas this deposition was increased with an increment of selenium (Wang et al., 2010;Gravena et al., 2011).An organic form of selenium can easily enrich eggs, as was shown in a study conducted by Bennett and Cheng, (2010), in which they used sodium selenite (0.3 µg Se/g of feed) along with selenium yeast SSAF-600 ® (Diamond V Mills, Cedar Rapids, IA, USA), an organic source with the concentrations 1.0, 2.4, and 5.1 µg Se /g of diet.They reported that by supplementing 0.3-0.5 µg/g feed the eggs were enriched up to 10-29 µg Se.From these studies, it can be concluded that the higher the bioavailability and concentration of the selenium source in the supplemented diet, the higher the assimilation rate in the egg yolk.Similarly, other studies reported that selenium yeast, seleno-methionine and sodium selenite enrich the egg yolk (Wang et al., 2010;Delezie et al., 2014).
Dietary supplementation of MPM manipulated the lipid profile of egg yolk, resulting in significant (P ≤0.05) decrease in total cholesterol (Table 4).The control diet showed the highest level of cholesterol.The lowest levels were recorded in the Group D supplemented with the maximum dose (Table 4).Plants are enriched with phytosterols, which decrease the cholesterol levels of eggs, and serum (Hussain et al., 2014).MPM is enriched with a sterol (β-sitosterol), which is responsible for decreasing cholesterol content (Hussain et al., 2014).Cholesterol levels of eggs are also influenced by antioxidants (flavonoids and carotenoid) in the diet.Antioxidants potentiate the production of bile salts, which results in emulsification of fats and decrease in the absorption of lipids so decreasing the levels of cholesterol (Srinivasan, 2005;Nobakht & Moghaddam, 2013).Plant sterols and antioxidants decrease the absorption process and expedite the faecal drain of cholesterol, resulting in a hypocholesterolemic effect (Benakmoum et al., 2013).Reduction in egg cholesterol level could be due to fibre contents of M. oleifera and cassava peel, which play a significant role in binding and excreting cholesterol (Ghasi et al., 2000;Oladun-joye et al., 2010;Olugbemi et al., 2010).
Table 4 Mean (±SE) bioactive compounds and selenium in egg yolk and diet samples of commercial layers fed with different levels of Moringa oleifera pod meal Superscripts on different means within row show significant difference (P ≤0.05); β-carotene, quercetin and selenium in diet sample: mg/kg; β-Carotene, quercetin and selenium in yolk sample: µg/100 g, cholesterol: mg/100 g, DPPH: (1, 1diphenyl -2-picrylhydrazyl) %, A, B, C, and D: MPM dose levels of 0, 5, 10, and 15g respectively The nutrient profile of egg yolk was significantly affected (P ≤0.05) by the supplementation of MPM in commercial layers.Moisture, crude protein, ash and ether extract were recorded during the trial (Table 5).The highest moisture and ether extract levels were recorded in the control group, and were lowest in MPM 1.5% group D. Maximum values of crude protein and ash were recorded in Group D whereas minimum values were reported in the control group (Table 5).The nutrient profiles of egg yolk and albumen may be altered with manipulation of the feed (Naber, 1979;Nimalarante & Wu, 2015).Antioxidants, flavonoids, carotenoids, amino acids, additional protein and energy levels that result in lowered moisture may be the reason for the improved nutrient density of egg yolk (Nkukwana et al., 2014a;Nimalarante & Wu, 2015).
Table 5 Mean (± SE) nutrients (g/100g) and mineral profile (mg/100g) of egg yolk in commercial layers fed on different levels of Moringa oleifera pod meal Superscripts on different means within row show significant difference (P ≤0.05) 1 Parameters for proximate analysis were expressed in g/100 g 2 Parameters for mineral profile were expressed in mg/100 g A, B, C, and D: MPM dose levels of 0, 5, 10, and 15g respectively Treatment groups recorded mineral profiles that were significantly different (P ≤0.05) from the control during the experimental period.The lowest values of sodium, potassium, calcium, magnesium, and phosphorus were observed in the group offered the basal diet, and highest in the groups supplemented with  5).Moreover, all these values remained significantly the same in the 1.0 and 1.5% supplementation groups.The mineral profile of egg yolk samples was significantly improved (P ≤0.05) and can be attributed to the higher ash content of MPM, which eventually results in higher levels of all minerals in the egg yolk (Surai & Sparks, 2001).Similar findings were reported in previous studies (Nkukwana et al., 2016;Qwele et al., 2013).Similar results were reported in the estimation of calcium in tibia bone and ash content with M. oleifera supplementation (Nkukwana et al., 2014b;Gravena et al., 2011).Feed supplemented with MPM significantly affected serum biochemistry (P ≤0.05).Serum SGPT, glucose, creatinine and cholesterol levels were highest in the control group and lowest in the group supplemented 1.0% MPM (Table 6).However, all other biomarkers, expressed a quadratic trend as the supplementation level was increased.Antibody titers against Newcastle disease remained the same during the whole experimental period.MPM did not affect the immune status of the birds (Table 6).Bioactive compound flavonoids (quercetin) and carotenoids (β-Carotene) positively affected and reduced the levels of SGPT, creatinine, glucose and cholesterol levels in the serum, which showed improved liver performance, whereas lowered creatinine levels indicated better kidney functionality in MLM-supplemented groups (Melesse et al., 2013;Elkloub et al., 2015).Lowered cholesterol levels in the serum show the hypocholesterolemic effect of MLM, which might be attributed to β-sitosterol-rich plant material, which has same structure as cholesterol and lowers uptake from the intestine (Ghasi et al., 2000).These antioxidants (β-Carotene and quercetin) and phytosterols (β-Sitosterol) affect the functionality of liver, kidneys and heart, resulting in improved metabolism, as indicated in biochemical parameters and antibody titers (Ghasi et al., 2000).Essential oils in plants affect antibody titers positively (Ozek et al., 2011;Goudarzi et al., 2016).Contrary to the current study, Bardzardi et al. (2014) reported that no change in the immune response of birds when their diet was supplemented with bioactive compounds of plant origin.

Conclusion
The results of the present study reveal that MPM could affect egg production and immunity positively, and might alter the bioactive compound index of egg yolk with variable results at different dose levels.The best results were recorded in the group supplemented with 1.5% MPM in the basal diet.Additionally, it was shown that M. oleifera pods could be used as alternative growth promoters, which improves antioxidant content, and the performance of layer birds.

Table 1
Chemical composition of Moringa oleifera pod meal *MPM: Moringa oleifera pod meal . Dietary supplementation of MPM in commercial layers may enrich egg yolks with β-carotene and quercetin.The results of the present study revealed that the highest values(539.93 & 121.47) of β-carotene and quercetin were recorded in Group D, supplemented with 1.5% of MPM, whereas the lowest values were control group.A similar trend was observed in the β-carotene and quercetin content of feed samples.Moringa pods are enriched with carotenoids and flavonoids, which are strong natural antioxidants that can modify egg yolk β-carotene and quercetin levels