MATERIALS AND METHODS

Birds and experimental design: This study was applied in Experimental Poultry Unit, Veterinary Medicine College, Damanhur University within October and November, 2015. About 120 Cobb 500 chicks and 120 Ross 308 chicks were obtained from Arab poultry breeders Company Ommat. Chicks were brooded under gas brooder supplied 33°C at the first week reduced 3°C per week till reaching 24°C. Light supplied for 24 h during the first 48 h of life, then lighting duration reduced to 18 h day^–1 according to Schwean-Lardner¹⁹. Chicks were fed with starter ration (23% CP) during first 3 weeks, followed by starter ration (21% CP) from 2 till 6 weeks of age. Experiment initiated at 2 weeks of age where the chicks of each strain were distributed into 3 treatments (40 birds treatment^–1) including (1) Birds drink water supplied with 2% M. oleifera leaves aqueous extract (MW 2%), (2) Birds drink water supplied with 3% M. oleifera leaves aqueous extract (MW 3%) and (3) Birds drink water without any supplementation (control); moreover chicks of each treatment were distributed into 4 replicates (10 birds replicate^–1). All chicks were vaccinated with HB1+H120 at 8 days of age; IBD at 12 days and La Sota at 18 days of age and all vaccines were applied through drinking water after following all precautions.

Moringa source and preparation: Moringa oleifera leaves used in our experiment were obtained as a powder product from the farm of Moringa friends at Sadat city where the plant was analyzed in the Desert Development Center, The American University in Cairo, Research Station in Sadat City, Soil Testing Laboratory according to AOAC²⁰, the main results were illustrated in Table 1. Moringa oleifera leaves aqueous extract was prepared by soaking the leaves powder in distilled water for 24 h using 1:2 ratios (weight/volume), the preparation was then filtered to separate the debris and filtrate, the filtrate placed in a sealed clean container and kept in refrigerator at 4°C until used²¹ (Table 1).

Performance traits: During the experiment many performance traits were estimated including weekly body weight to the nearest gram using sensitive scale, weekly body weight gain, feed intake per bird per week, water intake per bird per week, mortality per treatment per week and feed conversion which was estimated according to Lambert et al.²².

Carcass traits: At 6 weeks of age 12 birds treatment^–1 (3 birds replicate^–1) were slaughtered after starvation for 12 h with continued supplying with water. The birds were weighed before slaughtering then weighed again after evisceration to calculate dressing percentage. Carcass weight, abdominal fat weight (including fat around gizzard) and internal organs weights (including intestine, liver, gizzard and heart) were estimated to the nearest gram using sensitive scale.

Table 1:	Moringa oleifera leaves analysis

Carcasses were divided and the weight of thigh, shoulder and left breast were measured.

Total antioxidant capacity estimation: Blood samples (12 samples per group) were collected from wing vein at end of experiment (42 days), serum were separated through centrifugation at 3000 rpm for 15 min and preserved in a deep freezer at -20°C until the time of analysis. Total Antioxidant Capacity (TAC) was measured according to Koracevic et al.²³.

Lactobacillus count and haemagglutination inhibition test (HI): Lactobacillus count was done using Rogosa agar as a selective medium used for the isolation of lactobacilli according to Rogosa et al.²⁴. The NDV antigen, la Sota strain, was used to test serum samples collected at 42 days of age (12 samples per group) for antibody titers against NDV as described by Allan et al.²⁵. The HI titer was expressed as the reciprocal of the highest dilution that causes inhibition of agglutination and Geometric Mean Titer (GMT) was calculated.

Statistical analysis: Body weight data were analyzed three way analysis of co-variance for 2 weeks b.wt., data, however other productive and carcass traits absolute weight data were analyzed two ways analysis of variance by SAS²⁶, Proc GLM.

RESULTS

Productive performance: Ross breed had significantly higher weekly body weight and weight gain than Cobb breed (p<0.05) as illustrated in Table 2 and 3. With respect to effect of M. oleifera water supplementation on body weight and weight gain addition of M. oleifera leaves aqueous extract to water at 3% concentration did not differ significantly from control treatment. However, MW 2% decreased weekly body weights significantly (p<0.05) compared to the two other treatments except with final body weight (W6) where there were no significant differences between the three treatments (1857.47, 1803.47 and 1900.68 g for control; MW 2 and 3%, respectively). Moreover, MW 3% increased WG2 (weight gain from 3-4 weeks) significantly than MW 2% and control treatment (420.62, 364.71 and 390.665 g, respectively p<0.05).

Feed conversion ratio: Cobb breed recorded significantly higher FCR from 2-3 weeks than Ross breed (2.45 and 2.09 for Cobb band Ross breeds, respectively p<0.05) and this may be attributed to its significantly lower weight gain thereafter the differences between the two breeds will be disappeared till the end of the experiment. Moringa oleifera leaves water supplementation either at 2 or 3% decreased FCR than control treatments, however the difference was significant only at FCR1 (2.42 vs 2.20 for control and M. oleifera supplied treatments, respectively). Moreover, the response of different breeds to M. oleifera supplementation had the same trend of general M. oleifera effect as with the two breeds the control treatments recorded higher FCR compared to the two M. oleifera treatments Table 3.

Feed intake and water consumption: Ross breed recorded higher average feed intake per bird per week (F/B/W) and water consumption per bird per week (W/B/W) compared to Cobb breed (841.67 vs 695.83 g F/B/W and 1835.83 vs 1370 mL W/B/W for Ross and Cobb, respectively p<0.05).

Table 2:	Weekly body weights of broilers in relation to breed and M. oleifera concentration
Means within the same column under the same category carry different superscripts are significantly different

Table 3:	Weight gain and feed conversion ratios of broilers in relation to breed and M. oleifera concentration
Means within the same column under the same category carry different superscripts are significantly different. WG1: Weight gain from 2-3 weeks, WG2: Weight gain from 3-4 weeks, WG3: Weight gain from 4-5 weeks, WG4: Weight gain from 6-5 weeks, FCR1: Feed conversion from 2-3 weeks, FCR2: Feed conversion from 3-4 weeks, FCR3: Feed conversion from 4-5 weeks, FCR4: Feed conversion from 5-6 weeks

Table 4:	Feed intake per bird per week (F/B/W), water intake per bird per week (W/B/W) and mortality (%) per week (M/W) of broilers in relation to breed and M. oleifera concentration
Means within the same column under the same category carry different superscripts are significantly different

On the other hand mortality percentage per week (M/W) was significantly higher with Cobb than Ross breed (1.33 vs 0.25% for Cobb and Ross, respectively), moreover the Total Feed Intake (TFI), Total Water Consumption (TWC) and total mortality (TM) percentage take the same trend of weekly average (Table 4). Moringa oleifera water supplementation decreased F/B/W significantly than control treatment with no significant differences between MW 2 and 3% (797.5 vs 745 and 763.75 g for control; MW 2 and 3%, respectively p<0.05). However, W/B/W and weekly mortality (%) did not affect significantly with M. oleifera supplementation (Table 5). With respect to the effect of breed on feed intake under different M. oleifera treatments, researchers observed that Cobb breed was more sensitive to M. oleifera supplementation than Ross breed where their weekly feed intake decreased significantly with M. oleifera supplementation than control treatment, however Ross breed weekly average feed intake did not affect with M. oleifera supplementation this mean that Ross breed is more adapted breed to new management than Cobb breed. Table 4 shows the highest F/B/W, W/B/W and mortality (%) were recorded for W5 (Table 4, 5).

Carcass traits: Ross breed achieved higher carcass weight and carcass cuts weights compared to Cobb breed (Table 6), however the differences were significant only with carcass and shoulder weights (1524.44 and 84.44 vs 1305.56 and 68.89 g for Ross and Cobb carcass and shoulder weights, respectively p<0.05).

Table 5:	Means and standard errors of Total Feed Intake (TFI), Total Weight Gain (TWG), Total Feed Conversion Ratio (TFCR), Total Water Consumption (TWC) and Total Mortality (TM%) of broilers in relation to breed and M. oleifera concentration
Means within the same column under the same category carry different superscripts are significantly different

Table 6:	Means and standard errors of the effect of breed, M. oleifera supplementation and their interactions on carcass weight, dressing (%), thigh, breast and shoulder weights of broilers
Means within the same column under the same category carry different superscripts are significantly different. N = 12 birds per treatment per 3 replicate

Internal organs weight did not differ significantly between the two breeds except for intestine and heart weights which were significantly higher in Ross than Cobb breed (Table 7). The significantly higher carcass, intestine and heart weights of Ross breed than Cobb breed may be resulted from the significantly higher body weight and feed intake of this breed. Although the differences between treatments in carcass weight, carcass cuts and organs weights were not significant MW 3% treatments recorded higher values than control and MW 2% (Table 6, 7).

Total Antioxidant Capacity (TAC): Ross breed recorded significantly higher TAC compared to Cobb breed (0.09 vs 0.05 p<0.05) which ensure its higher benefit by M. oleifera component than Cobb breed. However, differences between control treatments and M. oleifera treatments were not significant Table 8.

Lactobacillus count and HI test: The effect of M. oleifera on immune response, indicated that Ross 308 breed showed a higher beneficial bacterial count in the intestinal tract and also, increased immunity against NDV than Cobb 500 breed (Table 9, 10, Fig. 1).

DISCUSSION

Response of both breeds to MW 3% was better than MW 2%, however the differences in final body weight and weight gain between different treatments were non-significant. These results may be attributed to the high level of substances absorption through water¹ so high M. oleifera level (3%) leads to higher absorption of M. oleifera benefit contents from water that compensate low feed intake resulted from its bitter taste and improve weight gain, moreover M. oleifera positive effect may be obtained at early ages rather than oldest ages.

Table 7:	Effect of breed, Moringa oleifera supplementation and their interactions on internal organs weight of broilers
Means within the same column under the same category carry different superscripts are significantly different. N = 12 birds per treatment per 3 replicate

Table 8:	Total Antioxidant Capacity (TAC μmol mL–1) in all treated chicken groups
N = 12 birds per treatment per 3 replicate

Table 9:	Lactobacillus count of intestinal samples at 42 days of age

Table 10:	Serological response for NDV vaccine by HI titers of serum samples collected at 42 days of age (12 samples per chicken group

Fig. 1:	Lactobacillus colonies in M. oleifera 3% treated Ross birds

Moringa oleifera received groups recorded lower FCR than control groups and this is a good indicator where under M. oleifera treatments birds consumed lower amount of ration per each kilogram meat. Similar results were obtained by Portugaliza and Fernandez²⁷ where they studied the effect of water supplementation of various levels of M. oleifera leaf aqueous extract (30, 60 and 90 mL L^–1) on broiler performance, it is found that water supplementation of 30, 60 and 90 mL concentrations of M. oleifera aqueous leaf extract had improved the actual live weight, Feed Conversion Ratio (FCR) and return of investment (ROI) of Cobb broilers. However, Banjo²⁸ added M. oleifera leaf meal to broiler rations with metabolizable energy levels ranging from 2800-2900 kcal kg^–1 at 0, 1, 2 and 3% levels where the birds fed on these diets for 4 weeks, thereafter it is concluded that addition of M. oleifera 2% significantly (p<0.05) enhanced weight gain however, Moringa supplementation did not significantly enhanced feed intake and feed conversion. These conflicting results may be attributed to the use of M. oleifera leaf meal as a powder in ration not as an aqueous extract in drinking water as in this experiment.

With respect to breed effect on growth performance there are many studies ensure the significant effect of breed on performance, carcass weight, feed conversion and carcass composition^11,12,15,16. However, the preference between Cobb 500 and Ross 308 breed was differ between studies where researchers^29-31 take the same trend of this study and recommended response of Ross over Cobb breed for new managements. However, Hristakieva et al.³² concluded opposite results this disagreement may be attributed to the different sources of chicks.

Moringa oleifera water supplementation decreased F/B/W significantly than control treatment with no significant differences between MW 2 and 3%. Similar results were obtained by Onunkwo and George³³ where they attributed their results to the bitter taste of M. oleifera leaves meals which resulted in reduced palatability and thus reduce feed intake of the broiler diets.

Carcass cuts weights and internal organs weights were not significantly affected with M. oleifera aqueous extract water supplementation and similarly it is concluded that supplying weaned rabbits a diet containing M. oleifera leaf meal significantly (p<0.05) increased daily weight gain¹⁰ however, it had no significant (p>0.05) effect on carcass characteristics. The researchers attributed the improvement of rabbit growth to the higher level of vitamin A in M. oleifera leaf meal as reported by Grubben and Denton³⁴.

The results of TAC in this study did not indicated by Verma et al.³⁵ where it is found that the whole plant extract of M. oleifera inhibits the lipid peroxidation in chicken liver homogenates which indicating their antioxidant effect in preserving chicken meat. The cause of disagreement between results may be attributed to the used part of plant as they used whole plant extract, however we used plant leaf aqueous extract only.

The clinical data indicated the better weight gain and FCR in Ross 308 chickens treated with M. oleifera (3%) as it has a significant increase in Lactobacillus count inducing better feed digestion, absorption, increased digestive enzymes as well as reducing the bad effect of harmful bacteria in the intestinal tract. As some previous data³⁶ indicated the positive effect of M. oleifera (3% dried leaves) on enhancement of duodenum traits, reduced E. coli and increased Lactobacillus counts in ileum improving the intestinal health of broilers which helped in increasing the production of digestive secretions and nutrient absorption.

The effect of M. oleifera on immune response, indicated that Ross 308 breed showed an increased immunity (HI titers) against NDV vaccine than Cobb 500 breed and these data were a confirmation to Eze et al.³⁷, who reported that M. oleifera extract increased ND HI titers in the vaccinated chicken groups with NDV vaccines.

CONCLUSION AND FUTURE RECOMMENDATIONS

Ross breed responded better than Cobb breed to new managements and that water supplementation of M. oleifera leaves aqueous extract improved weight gain, feed conversion, carcass characteristics, slightly increased the immune response to NDV vaccine and increased Lactobacillus count as beneficial bacteria in the intestinal tract, however with concentration not less than 3%. Further studies on water supplementation of M. oleifera leaf aqueous extract regarding the antibacterial effect, immunomodulatory effect in the intestinal tract, growth performance and antioxidant effect are required.

SIGNIFICANCE STATEMENTS

The effect of water supplementation Moringa oleifera (M. oleifera) leaves aqueous extract on productive performance, carcass characters, blood antioxidants level, immune response to Newcastle Disease Virus (NDV) vaccine and Lactobacillus count of different broiler breeds were studied. All procedures were carried out in accordance with the recommendations in the guide for the care and use of laboratory animals of the national institutes of health.