SciELO - Scientific Electronic Library Online

 
vol.8 issue3Productivity of Holstein dairy cows without shade at two seasons of the yearBlack tuna meal in chicken diets to increase eicosapentanoic and docosahexaenoic acids author indexsubject indexsearch form
Home Pagealphabetic serial listing  

Services on Demand

Journal

Article

Indicators

Related links

  • Have no similar articlesSimilars in SciELO

Share


Abanico veterinario

On-line version ISSN 2448-6132Print version ISSN 2007-428X

Abanico vet vol.8 n.3 Tepic Sep./Dec. 2018

http://dx.doi.org/10.21929/abavet2018.83.4 

Original papers

Inclusion of the Moringa oleifera leaf on immunological constants in broiler chickens

Mariana Ramírez-Acosta1 

Cecilia Jiménez-Plascencia2 

Carlos Juárez-Woo2 

Jesús Rendón-Guízar1 

Alejandro Ángeles-Espino3 

David Sánchez-Chiprés2  * 

1Inter-institutional Master in Animal Production, Mexico.

2Department of Animal Production, University Center of Biological and Agricultural Sciences, University of Guadalajara. Mexico.

3Department of Agricultural Production University Center of Biological and Agricultural Sciences, University of Guadalajara. Mexico.

ABSTRACT

The national poultry production has acquired great importance for being an activity of high socioeconomic impact and generates high demand for ingredients. Moringa oleifera has acquired great importance as a nutritional supplement and medicinal plant; therefore, it could represent an alternative as an immune stimulant. The effects of the inclusion of the M. oleifera leaf on the immunological constants of broiler chickens were evaluated. 180 chickens (females) Cobb genetic line were used, distributed in 3 treatments: Control, M10, and M20. Intestinal morphometry and the content of IgY were determined at 21 and 42 days. A completely randomized statistical design was used: ANOVA and comparison of Tukey's means were performed. At 42 days, the height of villi of the duodenum of the Control group obtained the lowest values (1485 μm) with respect to M10 and M20 groups. The height of the jejunum villi was significantly higher (P<0.05) in the M10 group, with respect to the M20 and Control groups. In the depth of the jejunum crypts, there was a difference (P<0.05) between the Control and M20, the latter being the least. The IgY content at 21 days was significantly higher (P<0.05) in the M10 and M20 than in the Control. M. oleifera has a positive effect as an immunostimulant and can be used in animal feeding.

Keywords: Moringa; broiler chicken; intestinal morphometry; immunology

INTRODUCTION

The poultry production sectors in developing countries face some problems, such as increased food costs; due to this, alternative sources have been sought in their diet that is available and not expensive. Poultry farming has a high impact in the economic and social spheres; since more than 60 % of the animal protein consumed in Mexico and the world comes from the poultry industry (SAGARPA, 2017).

Moringa oleifera is the genus of a tree belonging to the Moringaceae family (Kumar et al., 2013); it has been used mainly for human consumption, since it has acquired great importance as a nutritional supplement and medicinal plant; this brings as a consequence the increase in its cost; therefore, to be used in animal feed, it should be promoted that more trees are planted in the productions where it is required (Etalem et al., 2014). This plant is important as a forage crop due to its nutritional characteristics and its high yield in fresh biomass production (Padilla et al., 2014). The leaves of the genus M. oleifera are distinguished by their high content of macronutrients such as protein and energy; and micronutrients such as vitamins and minerals. However, it is worth mentioning that it also has phenols, anti-nutritional factors such as tannins, saponins, phytates and oxalates (Teteh et al., 2013).

One of the factors that it is important to take into account in animals is stress, which can affect the modulation of the immune response, among other things; which causes changes in the circulating immune cells in blood. The most common causes of stress in animals are due to feeding and environmental conditions. It has been proven that M. oleifera is a tree rich in vitamins, minerals and antioxidants, which promotes health and improves the immune response. A study was conducted to evaluate the effect of the M. oleifera leaf as an immunomodulator in mice, and it was shown to have significant potential as an immunomodulatory agent (Al-Majali et al., 2017).

It has been shown that the pro-inflammatory mediators produced during the inflammatory response aggravate the pathological development of some chronic diseases and showed that M. oleifera is a potent inhibitor of inflammation (Channarong et al., 2011)

Also, it is important to take into account the development of the intestine, which is reflected in the integrity of its morphology; it has a direct relationship with the growth and development of the animal. A study was conducted in which the effects of dust from the leaves of M. oleifera as a dietary supplement were evaluated to evaluate intestinal morphometry in rats without obtaining significant differences (Zvinorova et al., 2015). The intestinal mucosa development shows a relationship with the presence or absence of exogenous agents which is reflected in the change in height and density of the villi and in depth of the crypts, among others (Gomide, 2004).

The importance of this work lies in looking for a viable alternative available from a food source, which has an effect on the immune system and can be incorporated into the diets of poultry production. It is expected that the inclusion of the M. oleifera leaf in broiler feed will have a positive effect on its immunological constants.

The objective of this work was to evaluate the effects of the inclusion of M. oleifera leaf on the immunological constants of broiler chickens.

MATERIAL AND METHODS

They were used 180 Cobb line chicks, one day old females were distributed in 3 treatments of 6 repetitions each and 10 replicates per repetition: 1. Control, 2. M10 (10 % inclusion of M. oleifera), 3. M20 (20 % inclusion of M. oleifera). They lived in pens of 1 m2 per 10 birds (Cobb 500, 2013, Cobb 500, 2015). The diets were formulated at 95 % of that stipulated by the NRC (1994) (table 1).

Table 1 Inclusion levels of ingredients in initiation and completion diets. 

Ingredient Control M10 M20
I F I F I F
Oil 6.00 6.00 6.00 6.00 6.00 6.00
Sorghum 48.18 59.28 46.91 58.20 45.31 58.23
Soybean paste 35.92 26.34 32.80 24.50 30.50 22.00
Leaf of M. oleifera - - 4.50 3.00 8.50 5.50
Carbonate of Ca 1.21 1.10 1.00 0.97 0.79 0.88
Dicalcium phosphate 0.68 0.28 0.80 0.34 0.90 0.39
Premix 8.00 7.00 8.00 7.00 8.00 7.00

I = Initiation stage diet, F = Completion stage diet.

For the microscopic morphometric evaluation, samples were taken from the duodenum and jejunum of the birds destined for humane euthanasia, at 21 and 42 days of age (SEMARNAT, 1995); the samples were preserved in 10 % formaldehyde. The paraffin inclusion technique was performed; subsequently, histological sections 4 microns thick and hematoxylin eosin staining were performed. The plates were read with micrometric objective microscopes, then the height of the villi and the depth of the intestinal crypts were measured; then the Motic Images Plus ® program was used (Rodríguez, 2012).

The experimental design was used completely at random; to then perform ANDEVA. When there was a statistical difference between the treatments, the Tukey test was performed to compare means with a confidence level of 95 %. The data was analyzed in the statistical package Minitab (2014) ®. 3 ml of blood was extracted from 3 birds per pen, at 21 and 42 days of age, and the samples were placed in collection tubes; these were allowed to stand until complete coagulation and then centrifuged at 3500 rpm for 10 min. Sera were separated and transferred to 1.5 ml microvials; then, two aliquots of each were made.

The quantification of IgY was carried out by means of a Sandwich ELISA method, the standards available in the kit were used; samples were added to the polystyrene microtiter wells (included in the kit) containing Anti-IgY antibodies adsorbed to the bottom, and allowed to bind for 30 min. Subsequently, four washes were made to remove proteins that did not bind; Anti-IgY antibody conjugated to horseradish peroxidase (HRP) was added at the dilution recommended by the kit, and incubated at 37 °C for the time indicated in the kit. Again, 4 washes were carried out and subsequently chromogenic substrate 3, 3', 5, 5' - tetramethylbenzidine (TMB) was added and incubated for 10 min in the dark. Finally, a solution was added to stop the reaction, and the absorbance was read; a filter for 450 nm was used in an ELISA reader. The amount of antibodies contained in the sample is directly proportional to the absorbance at 450 nm (MyBioSource®).

To perform the statistical analysis, a linear trend of coincidence with known data points was used; using the method of least squares to determine the values in ng/ml. Once these values were obtained, ANOVA was performed; when there was a statistical difference between the treatments, the Tukey test was used with a level of significance of 95 %.

RESULTS AND DISCUSSION

With respect to the height of villi in the duodenum at 21 days, the treatment that had the highest values was M20, with a value of 1620 μm, which was higher than the M10 group and Control at 156 and 366 μm, respectively. On the other hand, when comparing the Control group, it was lower by 210 μm with respect to the M10 group. The height of the villi of the jejunum showed a similar behavior, since the M20 group presented a value of 1537 μm, which was higher in 165 and 271 μm to the M10 and Control groups, respectively. The differences were statistically significant and are presented in Table 2. For the measurement of the crypt depth of the duodenum at 21 days, an opposite behavior was presented, since the M20 group obtained a value of 213 μm; which is smaller than Control and M10 by 38 and 44 μm, respectively; these values are statistically significant. In Jejunum there was no statistically significant difference between treatments; these results are shown in Table 2. At 42 days of age, in the height of villi of the duodenum, the Control group obtained a value of 1485 μm, which was higher by 170 and 160 μm to the groups M10 and M20, respectively; these differences are statistically significant. However, the height of the villi of the jejunum was significantly higher in the M10 treatment, with a value of 1579 μm compared to the M20 and Control treatments, which obtained 155 and 164 μm less, respectively; this difference is statistically significant (table 2).

However, for the depth measurement of duodenum crypts at 42 days of age, the same pattern was not present, since no statistically significant difference was found between the treatments; On the contrary, in the jejunum there was a statistically significant difference between the Control treatment and M20 with values of 190 and 167 μm, respectively (Table 2).

Table 2 Height of villi and depth of intestinal crypts at 21 and 42 days 

Control M10 M20 P-Value-P EE
Height of villi at 21 days (μm)
Duodenum 1263 c 1473 b 1629 a <0.001 56.27
Jejunum 1266 b 1372 b 1537 a <0.001 68.90
Depth of crypts at 21 days (μm)
Duodenum 251 a 257 a 213 b <0.001 15.30
Jejunum 262 a 227 a 258 a 0.051 18.33
Vellosity height at 42 days (μm)
Duodenum 1485 b 1655 a 1645 a <0.001 52.13
Jejunum 1415 b 1579 a 1424 b 0.002 61.98
Depth of crypts at 42 days (μm)
Duodenum 193 a 177 a 181 a 0.098 9.52
Jejunum 190 a 176 ab 167 b 0.015 10.05

a, b Different literals per row indicate a statistically significant difference (P <0.05). EE = Standard error of the deviation

This tendency of behavior that increases the height of villi and decreases depth of crypts suggests a greater absorption of nutrients and leads to a better digestion (Lipismita et al., 2015). Zvinorova et al. (2015) measured intestinal villi height and depth of crypts in rats, diets with 14 and 20 % of M. oleifera were used and did not obtain significant differences between villus length, depth of crypts, nor in the proportion of both

When analyzing the results of the amount of IgY on day 21, the control treatment obtained a value of 92 ng/ml, lower by 85 and 121 ng/ml to groups M10 and M20; these values are statistically significant; however, between the M10 and M20 treatment there was no significant statistical difference. At 42 days, the Control group had a higher value of 52 ng/ml with respect to the M10 group this difference is statistically significant. However, there was no significant statistical difference between the Control and M20 treatments, as between the M10 and M20 treatments (Table 4). There are no works in which the amount of immunoglobulins in birds has been determined when using M. oleifera.

Table 3 Different inclusion levels of the M. oleifera leaf on the amount of IgY expressed in ng / mL at 21 and 42 days of age 

Traetments 21 days 42 days
Control 92 a 160 a
M10 177 b 108 b
M20 213 b 140 ab
Valor P <0.001 0.048
EE 29.13 30.10

a, b Different literals per row indicate a statistically significant difference (P <0.05). EE = Standard error of the deviation

CONCLUSION

Intestinal villi development was better in animals fed M. oleifera, since longer villi and shallower crypts were obtained. M. oleifera had a positive effect as an immunostimulant at 21 days, since it raises the levels of IgY in the blood.

ACKNOWLEDGEMENT

Thanks to National Council of Science and Technology (CONACYT). University of Guadalajara.

BIBLIOGRAPHY

AOAC. 2000. Official Methods of Analysis. Association of Official Analytical Chemists. Inc. Washington, D.C. E.U.A. ISBN 10: 0935584676 -ISBN 13: 9780935584677 [ Links ]

AL-MAJALI IS, Al-Oran SA, Hassuneh MR, Al-Qaralleh HN, Abu Rayyan W, Al-Thunibat OY, Mallah E, Abu-Rayyan A, Salem S. 2017. Immunomodulatory effect of Moringa peregrina leaves, ex vivo and in vivo study. Central European Journal of Immunology. 42:3. Pp. 231-238. ISSN: 1426-3912. DOI: 10.5114/CEJI.2017.70964. [ Links ]

CHANNARONG M, Pimajai C, Phawachaya P, Saovaros S, Siriporn T. 2011. M. oleifera Pod inhibits inflammatory mediator production by Lipopolysaccharide-Stimulated RAW 264.7 Murine Macrophage Cell Lines. 35:2 Pp. 445 - 455. ISSN: 0360-3997, EISSN: 1573-2576. DOI: 10.1007/s10753-011-9334-4. [ Links ]

GOMIDE JM, Vinícius SE, Macari M, Boleli IC. 2004. Use of scanning electron microscopy for the evaluation of intestinal epithelium integrity. Revista Brasileira de Zootecnia. 33: 6. ISSN: 1806-9290. [ Links ]

KUMAR DD, Dora J, Kumar A, Kumar GR. 2013. A Multipurpose Tree - Moringa oleifera. International Journal of Pharmaceutical and Chemical Sciences. 2: 415 - 422. ISSN: 2277-5005 [ Links ]

LIPISMITA S, Vishwa BC, Guttula S, Ramesh S, Ashok KP. 2015. Prebiotic potential of Jerusalem artichoke (Helianthus tuberosus L.) in Wistar rats: effects of levels of supplementation on hindgut fermentation, intestinal morphology, blood metabolites and immune reponse. J Sci Food Agric. 95: 1689-1696. DOI: 10.1002/jsfa.6873. [ Links ]

MARTÍN C, Martín G, García A, Fernández T, Hernández E, Puls J. 2013. Potenciales aplicaciones de Moringa oleifera. Una revisión crítica. Pastos y Forrajes. 36 (2): 137 - 149. ISSN: 0864-0394. [ Links ]

MINITAB STATISTICAL SOFTWARE, 2014. State College, PA. [ Links ]

MyBioSource ®, Chicken IgY ELISA Kit, Immunoperoxidase Assay for Determination of IgY in Chicken Samples, MBS564008. [ Links ]

NATIONAL RESEARCH COUNCIL, 1994. Nutrient Requirements of Poultry: Ninth Revised Edition, 1994. Washington, DC: The National Academy Press. ISBN: 978-0-309-04892-7. [ Links ]

PADILLA C, Fraga N, Scull I, Tuero R, Sarduy L. 2014. Efecto de la altura de corte en indicadores de la producción de forraje de Moringa oleifera vc. Plain. Revista Cubana de Ciencia Agrícola. 48 (4): 405 - 409. ISSN: 0034-7485 [ Links ]

RODRÍGUEZ JC. 2012. Respuesta morfométrica intestinal de pollos alimentados con diferentes niveles de morera (Morus alba). Revista Citecsa. 3 (4). ISSN: 2027-6745. [ Links ]

SAGARPA (Secretaría de Agricultura, Ganadería, Desarrollo Rural, Pesca y Alimentación). 2017. Comunicado de prensa, La Paz, Baja California Sur, 13 de octubre de 2017. Disponible en la Web: http://www.sagarpa.gob.mx/Delegaciones/bajacaliforniasur/boletines/2017/octubre/Documents/2017BS336.pdfLinks ]

SEMARNAT (Secretaría de Agricultura, Ganadería y Desarrollo Rural). 1996. Sacrificio humanitario de los animales domésticos y silvestres. NOM-033-ZOO-1995, México: Diario Oficial de la Federación, 17 p. Disponible: http://www.dof.gob.mx/nota_detalle.php?codigo=5376424&fecha=18/12/2014Links ]

TALHA E. 2013. The use of Moringa oleifera in poultry diets. Turkish Journal of Veterinary and Animal Sciences. 37: 492 - 496. DOI: 10.3906/vet-1211-40 [ Links ]

TETEH A, Lawson E, Tona K, Decuypere E, Gbeassor M. 2013. Moringa oleifera Leave: Hydro-Alcoholic Extract and Effects on Growth Performance of Broilers. International Journal of Poultry Science. 12 (7): 401 - 405. ISSN: 1682-8356. [ Links ]

YAMÉOGO CW, Bengaly MD, Savadogo A, Nikiema PA, Traore SA. 2011. Determination of Chemical Composition and Nutritional Values of Moringa oleifera Leaves. Pakistan Journal of Nutrition. 10 (3): 264 - 26. ISSN: 1680-5194. [ Links ]

ZVINOROVA PI, Lekhanya L, Erlwanger K, Chivandi E. 2013. Dietary effects of Moringa oleifera leaf powder on growth, gastrointestinal morphometry and blood and liver metabolites in Sprague Dawley rats. Journal of Animal Physiology and Animal Nutrition. 99. 21-28. DOI: 10.1111/jpn.12182 [ Links ]

Received: March 01, 2018; Accepted: June 26, 2018

*Responsible and correspondence author: Sánchez-Chiprés David Román. Department of Animal Production, University Center of Biological and Agricultural Sciences; Camino Ingeniero Ramón Padilla Sánchez # 2100, Predio Las Agujas, Nextipac, Zapopan, Jalisco, Mexico, C.P. 45200. Email: chipresw99@hotmail.com

Creative Commons License This is an open-access article distributed under the terms of the Creative Commons Attribution License