Commercial poultry farming is a major industry in Mexico¹. Poultry feed can include pastes from cottonseed, peanuts, safflower seeds, sesame seeds and coconut as sources of protein and other nutrients, although these are all lysine deficient. Soybean paste, in contrast, is a protein source with an optimal amino acids balance for animal feed². Poultry farming, including growing of backyard chickens, is important in the country’s rural communities since it provides families with meat and eggs as well as surplus birds for sale³. Greater research is needed on protein sources from locally available resources which can be added to the poultry feeds used in rural areas such as corn, food scraps and other materials.

The tree Moringa oleifera has been studied as a protein source for animals^4,5 and humans⁶. Native to India and Pakistan, it was introduced to Mexico⁷. The moringa leaf contains 20 to 30 % protein, 5.0 to 7.5 % fat and 25 to 31 % fiber^8,9. It is also a good source of iron, calcium and vitamin C⁹. Due to the other phytochemicals it contains, moringa leaf has also been recommended for treating gastrointestinal ulcers¹⁰, lowering cholesterol levels and as a source of antioxidants¹¹.

Moringa leaf has been used in feed for rabbits¹², goats⁵, sheep¹³ and cows¹⁴, among other domestic species. Only minimal research has been done on the use of moringa leaf as an ingredient in feed for broilers. Studies have been done evaluating its effect on intestine morphology and the condition of other internal organs^15,16, breast fatty acids content¹⁷, tibia strength and mineral content¹⁸, animal weight, hematology and immune response^4,19. Only two of these studies have evaluated moringa leaf’s effect on weight gain^4,19, and one used Moringa stenopetala⁴, a different species in the same genus.

The present study objective was to characterize nutritional content and vitamin C and condensed tannins levels in Moringa oleifera leaf flour, and evaluate its effect on productive and toxicological parameters when included in diets for broiler chickens.

Leaves were harvested from two-year-old M. oleifera trees in an orchard in the municipality of Gasca, in the state of Guanajuato, Mexico. Immediately after harvest the leaves were washed and disinfected with a 0.1% sodium hypochlorite solution followed by sterile distilled water, and dried in the shade. The dried leaves were milled with a hammer mill (2.0-3.0 mm sieve) and stored at -20 °C until analysis and inclusion in broiler diets. Experimental animals were 180 ROSS-308 line broilers (39 ± 1.5 g initial weight), mixed, which were one-day old at experiment outset.

The animal trial was carried out at the Poultry Unit of the Livestock Area of the Zootechnical Station of the Autonomous University of Aguascalientes, in the municipality of Jesús María, in the state of Aguascalientes. The chickens were randomly selected to form two groups with three repetitions of thirty birds each. They were fed either a conventional feed (T1) or a conventional feed plus 10% moringa leaf flour (MF). Both diets were isoproteic and isoenergetic²⁰ (Table 1). Feed and water were freely available throughout the 42-day experimental period.

Proximal analysis of the moringa flour was done with established methods for protein (960.52), fiber (991.43 G, H), fat (920.85) and ash (923.03)²¹. Carbohydrate content was calculated by the percentage difference compared to the previous analyses. Iron and calcium content were determined by atomic absorption spectrometry²², and vitamin C by high performance liquid chromatography (HPLC)²³. Total phenols²⁴ and condensed tannins²⁵ contents were also determined.

Daily records were kept of feed provided and consumed. At the end of the experiment calculations were done of average weight (AW), daily weight gain (DWG), feed conversion rate (FCR) and the productivity index (PI)²⁶.

After 42 d blood samples were taken to quantify total proteins (TP) and albumin (ALB); as well as blood levels of alanine amino transferase (ALT), aspartate amino transferase (AST), alkaline phosphatase (ALP) and gamma glutamyl transpeptidase (GGT)²⁷.

Blood samples were taken from three chickens selected from each of the three replicates of the two groups originally formed. These were killed by cervical dislocation following the Ethical Management and Humanitarian Sacrifice Guide of the Autonomous University of Aguascalientes. Samples (1 cm³) of liver and kidney were taken for histopathological analysis²⁸. In the liver, evaluations were done of the condition of the histo-architecture of the hepatic parenchyma, presence or absence of inflammation cells, the Möll space, the Disse space, the hepatic sinusoids and the bile ducts. In the hepatocytes evaluations were done of the nucleus-cytoplasm relationship, cell nucleus characteristics (pyknosis, karyolysis and karyorrhexis) and presence or absence of lipid vacuoles in the cytoplasm.

The data were processed with an analysis of variance (ANOVA) and Student t test with repeated measurements and a P<0.05. All statistical analyses were run with the Statistical Analysis System statistical package (SAS, 2001)²⁹.

The moringa leaf flour in the present study had a higher protein content (33.4%) than reported elsewhere for this species (20.0 to 29.0 %)^8,9,30 (Table 2). The higher protein content in the present results was probably because the reported data were from other moringa species and/or local environmental conditions affected protein content⁹. The remaining compounds were within ranges reported in the literature^8,9,30. Moringa is known to be a good source of iron and calcium (Ca= 2.6 g; Fe= 19 mg/100g)⁹, which is consistent with the present results (Table 2)³¹. Moringa may therefore contribute substantial amounts of calcium when added to animal feed. Its high iron content may assist in synthesis when added to broiler diets, and its vitamin C content could promote metabolism³²; indeed, the combination of these compounds could contribute to this phenomenon.

Use of sorghum with high tannin content (1,360 mg/100 g) in broiler diets is reported to reduce weight gain, feed intake and absorption of iron and calcium, among other minerals³³. The moringa leaf flour used in the experiment had a very low tannin content, suggesting it did not have any antinutritional effect in the experimental animals.

Average final weight in the MF treatment was 414 g less than those in the control group (Figure 1A). This weight loss reflects the lower feed intake in the MF treatment (Table 3). Average total feed intake per replicate in each treatment was 123.35 kg in the control group and 107.19 kg in the MF group. This 16.16 kg difference (P<0.05) in the MF treatment can be attributed to higher dietary fiber intake. This contrasts with a study in which broilers were fed diets containing from 5 to 14 % flour from M. stenopetala in which the 5 to 11 % M. stenopetala diets exhibited no weight difference compared to the control⁴. The difference between this study and the present results may be attributed to use of different moringa species in each. The difference in average weight in the present results was also reflected in the DWG results throughout the study (Figure 1B); at no time did DWG in the MF treatment exceed that in the control group.

Asterisks indicate signficant difference (Student t, P<0.05, n=63).

Figure 1 Production parameters in the control and moringa flour treatment: A) Average weight at 42 d (g); B) Daily weight gain (g/d); C) feed conversion index (feed intake/weight); D) productivity index (daily weight gain) (viability)/(FCx10).  

The lower the FCR the more efficiently a feed increases animal weight(²⁶). In the present results the FCR in the two treatments was similar only in the second and third weeks (Figure 1C). In a study conducted with M. stenopetala, no differences in FCR compared to the control were observed in broilers fed diets containing from 5 to 8 % moringa leaf(⁴). However, at higher moringa inclusion levels the FCR increased, with the 14 % moringa leaf diet resulting in the lowest weight. Productivity index (PI) values were 21% higher in the control group than in the MF treatment, probably because average weight, DWG and FCR results for the latter treatment were lower (Figure 2D).

Total protein (TP) content did not differ between the treatments (Figure 2A), although albumin content was higher in the MF treatment (Figure 2B). However, in both cases TP levels were lower than the reported reference values for poultry (3.1 to 5.05 g/dl)(³⁴). The low globulin levels observed in both treatments in the present study may be associated with pathologies in the liver and/or kidney, or intrinsic factors typical of the studied animals.

^ab Different lowercase letters in the same parameter indicate significant difference (P<0.05 n=90).

Figure 2 Blood protein and enzyme activity levels in the control and moringa flour treatment: A) total proteins (g/dl); B) albumin (g/dl); C) alanine amino transferase (IU/L); D) aspartate amino transferase (IU/L); E) alkaline phosphatase (IU/L); and F) gamma glutamyl transferase (IU/L). 

No differences (P>0.05) were observed in ALT and AST activities (Figure 2C and 2D). However, in the MF treatment the ALT level was 35.1 IU/L, near the reference maximum for this enzyme (9.5 to 37.2 IU/L)³⁵. Values above this maximum for ALT suggest hepatic and renal damage³⁶. The lack of a difference in AST is to be expected since chronic liver damage normally only causes subtle cell rupture, which manifests as normal or even decreasing AST levels³⁷, as was observed in the MF treatment (Figure 2D). Values for AST activity above 275 IU/L may be related to liver or muscle disturbances, while values above 800 IU/L strongly suggest severe liver damage³⁸; this was not the case in the present study.

Activities for ALP and GGT differed between the treatments (Figure 2E and 2F). The reference ALP activity value is 600 IU/L⁴, which is higher than occurred in both groups (Control and MF). Increases in ALP activity suggest bone growth, osteomyelitis and neoplasms³⁸, although it is not known if these problems arise at levels higher than the reference. The present results do not allow verification of whether this problem occurred in the MF treatment.

The membrane enzyme GGT is associated with a protein and linked to the level of amino acid metabolism. Increased GGT activity can be caused by inflammation, biliary cholestasis and bile duct hyperplasia³⁹. A 25% increase in GGT activity was observed in the MF treatment (Figure 2F). Normal GGT values are between 0-10 IU/L⁴⁰. Based on the reference values, both groups of birds apparently exhibited active liver lesion⁴¹.

No micromorphological differences were apparent between the groups (Figure 3A and 3B) since the evaluated structures exhibited no congestion, inflammation, scar tissue, liquefaction, lipid vacuoles, pyknosis, karyolysis or karyorrhexis.

A) (a) histological architecture of parenchyma; (b) basophilic cells; (c) Möll space; (d) sinusoids, (e) bile ducts; and (f) hepatocytes.

B) (a) parenchyma; (b) sinusoids; and (c) centrilobular vein.

C) (a) renal glomeruli; (b) proximal tubule; (c) Bowman capsule; (d) glomerular visceral layer.

D) (a) renal glomeruli; (b) reduced urinary space; (c) filtration barrier; (d) apparently normal macula densa; (e) whole proximal tubule.

Figure 3 Histopathologies in the liver of the control (A) and the MF treatment (B), and in the kidneys of the control (C) and the MF treatment (B) 

Histopathological analysis of the kidneys (Figure 3C and D) examined the glomeruli, Bowman’s capsule, the urinary space, podocytes, the vascular and urinary poles, the filtration barrier, the proximal tubules, the Henle loop, the distal tubules, the juxtaglomerular apparatus, the collecting tubules and the renal interstitium. No micromorphological differences were apparent between the two groups since the evaluated structures showed no congestion or inflammation and their structures were complete.

Leaves from Moringa oleifera are a good source of protein and minerals. Addition of 10 % moringa flour to a broiler diet reduced production parameter values during the study period. Differences in protein and enzyme levels were detected although no visible toxic effects were apparent, which was confirmed in the histopathological studies. The higher GGT and ALP activity levels in the moringa flour treatment may be due to certain inflammatory processes in the bile ducts which occurred in both groups and were caused by factors other than consumption of moringa and feed. Establishing the proper inclusion levels of moringa leaf in poultry diets requires further research to compare with the present results.

The research reported here was financially supported by the INIFAP through the project “Potencial agronómico, nutricional y nutracéutico de la moringa (Moringa oleífera) y usos alternativos de la hoja y la pasta de la semilla” (No. Preci 11163719248). MKFE received a master’s scholarship from the CONACYT.