Animal Science

Immune response and blood biochemistry in broilers fed tithonia forage meal at the finishing stage

0000-0001-9880-0310Lourdes Lucila Savón-Valdés^{1  *}  , 0000-0003-0740-9346Bárbara Rodríguez¹  , 0000-0003-1769-3416Ysnagmy Vázquez¹  , 0000-0002-9516-7182Idania Scull¹  , 0000-0002-2641-1815Magaly Herrera-Villafranca¹  , 0000-0002-1690-1140Tomás E. Ruiz¹ 

¹Instituto de Ciencia Animal, Apartado Postal 24, San José de las Lajas, Mayabeque, Cuba

ABSTRACT

A total of 40 male broilers EB₃₄, 42 days old, were used to evaluate the effect of Tithonia diversifolia forage meal intake, plant material 10, on indicators of immune response and blood biochemistry. A completely random design was used. The animals were distributed in five treatments with eight repetitions: corn/soybean concentrate control, 5, 10, 15 and 20 % substitution of the concentrate by tithonia forage meal. For the analysis of the immune response, the lymphoid organs (spleen, Bursa of Fabricius and thymus) were extracted and the relative weights (g/g LW x 100 g of the organs) were determined. Blood was extracted from the jugular vein and serum indicators of blood biochemistry, cholesterol, glucose, triglycerides, total protein, albumin, alanine amino transferase enzymes, and aspartate amine transferase and uric acid were analyzed. From the biological point of view (P=0.0593) lower relative weight of the spleen was found for the chickens that received 20 % tithonia forage meal (0.13, 0.12, 0.12, 0.13 and 0.07 g/g LW x100) for 0, 5, 10, 15 and 20 % substitution of corn/soybean by tithonia forage meal plant material 10, respectively. The intake levels of tithonia forage meal in broilers did not influence on the indicators of blood biochemistry, with the exception of uric acid, which increased with 15 % and 20 % substitution. These results suggest the possibility of using Tithonia diversifolia forage meal plant material 10, in a corn/soybean diet up to levels of 10 % for broilers in the finishing stage, without causing damage to the animals health.

Key words: Tithonia diversifolia; forage meal; health; birds

Currently, according to the ^{FAO (2018)}, the use of tree and shrub plants to replace traditional sources, such as soybean and corn, in the diet of monogastric species, including poultry, constitutes a very attractive food alternative because it reduces the rations cost due to import substitution, and promotes a more diverse and sustainable feeding system. Among these plants, Tithonia diversifolia plant material 10 highlighted, which is characterized by its availability and excellent nutritional value (^{Scull et al. 2019}).

In the countries of the Latin American and Caribbean region, such as Mexico, Colombia and Cuba, researches to establish the level of tithonia intake, as leave meal or foliage in broilers were carried out (^{Gutiérrez-Castro and Hurtado 2019}). Tithonia was also used as forage meal (leaves + young stems) in broilers (^{Rodríguez et al. 2020}) or in laying hens and their replacements (^{Rodríguez et al. 2018}, ^{Fuentes-Martínez et al. 2019} and ^{Vázquez et al. 2021}). Most of the studies refer to nutritional aspects, such as productive performance and carcass yield. However, very few deal with what is related to the physiological response of the digestive system and its accessory organs in each of these categories, and the analysis of the blood biochemical profile is rarely considered (Gutiérrez-Castro and Corredor Mateus 2019), and much less the immune response, as health indicators in birds.

The objective of this research was to determine the effect of the intake of different levels of forage meal (leaves + young stems) of Tithonia diversifolia, plant material 10, on indicators of the immune response and blood biochemistry of broilers in the finishing stage.

MATERIALS AND METHODS

Preparation of tithonia forage meal. The plant material was harvested in red ferrallitic soil (^{Hernández et al. 2019}), in Unidad Experimental de Pastos y Forrajes Miguel Sistach Naya from Instituto de Ciencia Animal (ICA), Cuba Republic. The leaves and young stems of the plant were used, with cutting ages between 60 and 70 d, at a height of 15 cm (^{Ruiz et al. 2017}). The fresh forage was milled to a particle size between 5 and 8 mm, before being exposed to the sun for 72 h. In the drying period, the material was spread on the plate at a bed height that did not exceed 30 cm, and it was turned several times a day with a rake to achieve uniformity. Subsequently, it was passed through a hammer mill until a particle size of 1 mm was obtained. The material was packed in 50 kg jute bags and kept indoors and ventilated until use.

Location of the experiments. The experiment was carried out in the poultry experimental unit from Instituto de Ciencia Animal, located in San José de las Lajas municipality, Mayabeque province, Cuba, at 22º 53 North latitude and 82º 02 West latitude, at 80 m a. s. l. (^{Herrera 1976}).

Animals and diets. A total of forty male chickens of the Cuban hybrid EB₃₄, 42 days old, were used, distributed in five treatments, with 8 repetitions each. These were: control (corn/soybean) and treatments with 5, 10, 15 and 20 % substitution of corn/soybean by T. diversifolia plant material 10 forage meal, respectively. The chickens were from a productive performance experiment with 875 animals, seven repetitions, of 25 birds each, which were given the same treatments. They had free access to water during the research time.

The diets were formulated isoprotein and isoenergetic, according to the ^{Rostagno et al. (2017)} recommendations for broilers. In the bromatological composition of tithonia forage meal of plant material 10, a contribution of 24.6 % crude protein, 20.3 % ash, 2.38 % Ca, 36.07 % NDF, 32.48 % ADF, 25.87 % cellulose was found (^{Scull et al. 2019}), which characterizes it as an alternative protein source high in fiber. The experimental diets are shown in table 1.

Table 1 Composition of the experimental diets for broilers in the finishing stage 

Raw matters	Tithonia forage meal levels %
	0	5	10	15	20
Corn meal	61.43	55.95	50.88	45.81	41.00
Soybean meal	32.45	31.10	30	28.5	27.23
Tithonia forage	0	5	10	15	20
Vegetable oil	2.20	4.15	5.60	7.35	8.65
Monocalcium phosphate	1.43	1.45	1.41	1.40	1.38
Calcium carbonate	1.60	1.30	1.02	0.75	0.50
Common salt	0.30	0.30	0.30	0.30	0.30
Methionine	0.16	0.22	0.24	0.28	0.31
Lysine	0	0.10	0.12	0.18	0.21
Choline	0.13	0.13	0.13	0.13	0.13
Premixture^1	0.30	0.30	0.30	0.30	0.30
Calculated contributions, %
CP	19.22	19.27	19.37	19.34	19.38
ME, MJkg	3003	3018	3009	3018	3001
Ca	0.95	0.95	0.95	0.95	0.95
Available phosphorus	0.42	0.42	0.42	0.42	0.42
CF	2.80	3.33	3.88	4.42	4.97
Lysine	1.01	1.03	1.01	1.01	1.00

¹Each kg contains vitamins A 13,500 IU, D3 3,375 IU, E 34 mg, B2 6 mg, pantothenic acid 16 mg, nicotinic acid 56 mg, Cu 2,000 mg, folic acid 1.13 mg, vitamin B12 34 μg, Mn 72 mg, Zn 48 mg

Experimental procedure. Broilers were slaughtered at 42 d of age. A total of 8 birds/treatment were selected, and always based on the average weight of the group of birds in a range of 10 %.

Immunological indicators. The birds were sacrificed two hours and thirty minutes after food ingestion by the jugular vein exsanguination method, described by ^{Sánchez (1990)}. Subsequently, the abdominal cavity was opened and the spleen, thymus and Bursa of Fabricius were removed. The morphometric data of these organs, expressed as relative weight (g of the organ/g of live weight x 100), were evaluated as indicators of the immune response. The live weight (LW) was established at slaughter (^{Savón et al. 2020}).

Indicators of blood biochemistry and health. The blood sample was taken directly from the jugular vein, slightly tilting the collection tube so that it rolls along the walls. This prevents hemolysis of the serum that will be used later. The indicators of blood biochemistry, cholesterol, glucose, triglycerides, total proteins, albumin, and the enzymes alanine amino transferase, aspartate amino transferase and uric acid were analyzed. All determinations were performed on blood serum using a Cobas integra 400PLUS automatic analyzer (Roche Diagnostic System).

Statistical analysis. A completely random design was used, with four treatments and eight repetitions. For the analysis of the results, the computerized statistical package INFOSTAT (^{Di Rienzo et al. 2012}) was applied. The mean values were compared using ^{Duncan (1955)} test in the necessary cases.

RESULTS AND DISCUSSION

There were no differences between treatments in the relative weights of the Bursa of Fabricius and the thymus, with the inclusion of tithonia forage meal in broilers (table 2).

Table 2 Relative weight of lymphoid organs of finishing broilers receiving tithonia forage meal, g/gLWx100 

Indicators	Tithonia forage meal,%					SE (±) Sign.
	0	5	10	15	20
Bursa of Fabricius	0.26	0.28	0.29	0.3	0.36	0.03 P=0.2655
Spleen	0.13	0.12	0.12	0.13	0.07	0.01 P=0.0593
Thymus	0.51	0.60	0.45	0.53	0.46	0.07 P=0.5067

The relative weight of the Bursa of Fabricius showed values between 0.26-0.36, so the animals were immunocompetent. According to ^{Savón et al. (2020)}, this condition is reached when the ratio between the absolute weight of the organ with respect to live weight is between (0.20-0.40). For Contreras and Fernández (1999), cited by ^{Savón et al. (2020)}, the immune system of birds show an evolution, and the Bursa and the thymus in the first stage of life are the central organs of immunity. In the first, B lymphocytes are produced, responsible for humoral immunity (antibody production by B lymphocytes), and in the second, cell-mediated immunity (cytokine and cell production by lymphocytes). In birds, the peripheral tissues of the immune system are the spleen, cecal tonsils, Harder's glands, and bone marrow, which are populate during bird growth by B and T lymphocytes. As these animals approach maturity, the bursa and thymus regress and the bird's immunocompetence becomes dependent on the peripheral immune system. The spleen plays a very important role, facing the challenges linked to the immune response throughout the bird’s life (^{Tizzard 2018}).

The relative weight of the spleen did not statistically differ between treatments (table 2), but from the biological point of view, the chickens that received 20 % substitution in the diet had lower relative weight (P=0.0593). Among the causes of not having found statistical differences, it can be consider the sample size or errors that were not controlled, inherent to the sampling, either in the weighing of organ or in not completely eliminating the adhered fat, among others. The lower relative weight of the spleen could show an immunosuppressive effect, conditioned by the presence of secondary metabolites in T. diversifolia forage meal, plant material 10. Although ^{Scull et al. (2019)} expressed the presence of low levels of functional groups, such as triterpenes, ^{González et al. (2014)} found the presence of others, such as coumarins and saponins, which can have toxic effects. The inclusion of tithonia forage meal at 20 % could increase the toxic effect and negatively influence on the organ weight. This result should be the subject of further research, with studies at the histological level.

Blood biochemistry indicators. The blood biochemical indicators are shown in table 3. The serum indicators of protein metabolism: total proteins, albumin and albumin/globulin ratio did not differ between treatments, except for uric acid. Regarding total proteins, 30-50 g/L (^{Miranda et al. 2007}) and 35.6 ± 0.34 g/L (^{Nunes et al. 2018}) are reported as normal values, while ^{Café et al. (2012)} showed figures of 26.5 g/L, which are close to those of this research. The differences may be due to the influence of various factors, such as diets, breed and environmental conditions in which the experiments were carried out.

Table 3 Indicators of the blood biochemistry of broilers that received tithonia forage meal in the finishing stage 

Variables	Tithonia forage meal, %					SE (±) Signif.
	0	5	10	15	20
A/G	0.80	0.79	0.88	0.86	0.84	0.07 P=0.8574
TP, g/L	25.26	28.30	25.36	26.19	28.61	1.76 P=0.5122
ALB, g/L	10.78	11.96	11.08	12.45	12.39	0.56 P=0.1421
ALAT, U/L	2.63	2.13	2.00	3.00	2.38	0.42 P=0.4596
ASAT, U/L	224.38	258.88	243.88	251.13	235.50	12.36 P=0.3360
CHOl, mmol/L	3.42	3.53	3.45	4.03	3.80	0.18 P=0.0936
GLU, mmol/L	12.30	12.68	12.43	13.40	12.87	0.75 P=0.8551
TG, mmol/L	1.39	1.50	1.56	1.69	1.68	0.17 P=0.7126
UA, µmol/L	278.50^b	305.38^b	275.00^b	396.63^a	330.50^ab	29.91 P=0.0424

¹A/G: albumin/globulin ratio, TP: total protein, ALB: albumin, TG: triglycerides, UA: uric acid, Chol: cholesterol, GLU: glucos, ASAT: alanine aspartic transferase, ALAT= alanine amino transferase

^a,b Means in the same row with different letters differ at P < 0.05 (^{Duncan 1955})

Albumin values were within the known range for this indicator (10.8-16 g/L), according to ^{Gálvez et al. (2009)}. This suggests that tithonia forage meal levels did not alter liver protein synthesis. Regarding globulins, considering that they are calculated as the difference between total proteins and albumin, they showed figures of 14.48, 16.34, 14.28, 13.76 and 16.32 g/L for the control treatments, 5, 10, 15 and 20 % tithonia forage meal, respectively. These values were very low compared to those indicated by these authors for this indicator (20-29 g/L) and those obtained by Gutiérrez-Castro and Corredor-Mateus (2019) (17-20 g/L) with tithonia meal (does not specify if it is leaf or forage). ^{Café et al. (2012)} obtained values of only 10.8 g/L.

The albumin/globulin ratio is considered quite favorable and indicative that the birds were fine from a nutritional point of view, since figures close to unity were obtained, which is the optimum ratio.

It could be showed that serum uric acid depends on the quality as well as the quantity of protein supplied in the diet. The highest figure of this metabolite was shown with the level of 15 % of tithonia forage meal, which differed (P<0.0424) in 118.13 µ moles/L from the control, although it was similar to the 20 % inclusion of this food that, in turn, did not differ from that one. In relation to this, it is necessary to point out that the standard error was high (29.91), and this could contribute that there was no significant difference between both treatments.

The determination of uric acid is carried out to evaluate the renal function of animals, since this is the main product of protein catabolism in birds, and constitutes approximately 60 to 80 % of the total nitrogen excreted in the urine, so serum concentrations higher than 308.57 µ moles /L could suggest some alteration of renal function (^{Rodríguez 2012}). Looking at table 3, only the 10 % substitution level of tithonia forage meal is below that figure.

The alanine amino transferase and aspartate amino transferase enzymes showed normal values for all treatments, so the intake of levels of up to 20 % of tithonia forage meal did not cause health disorders in the animals. These enzymes refer the state of liver function. From these results it can be inferred that, since there were no significant differences between treatments, the birds did not have liver problems that could be attributed to the supplied diets.

The indicators of energy metabolism, glucose, cholesterol and triglycerides, did not differ between treatments. The first two were in the normal range for the species: glucose (11-21 mmol/L) and cholesterol (2.58-5.17 mmol/L), according to ^{Gálvez et al. (2009)} and ^{Holguin et al. (2009)}, respectively. For triglycerides, the figures were higher than 0.45-1.35 µ mol/L, reported by ^{Nunes et al. (2018)}. It should be highlighted that the liver is the main organ of lipid metabolism in birds. It is the main place of lipogenesis and cholesterol is synthesized in it, in addition to its main product are triglycerides (^{Osorio and Flores 2011} and ^{Osorio and Flores 2018}). It is known that triglycerides can change their figures according to the diet.

CONCLUSIONS

The results showed that in corn/soybean diets, intake levels up to 10 % of Tithonia diversifolia forage meal in broilers in the finishing stage do not harm the health status evaluated by the immune response or the biochemical sanguine profile. The inclusion of 10 % Tithonia diversifolia forage meal, plant material 10, for broilers in the finishing stage is recommended in corn/soybean diets.

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Vázquez, Y., Rodríguez, B., Savón, L. & Ruiz, T. E. 2021 “Efecto de la harina de forraje de Tithonia diversifolia en indicadores productivos de reemplazo de ponedoras White Leghorn L-33”. Livestock Research for Rural Development, 33, Article #110ISSN: 0121-3784. Available: http://www.lrrd.org/lrrd33/9/33110ysnag.html.

Received: October 13, 2021; Accepted: June 28, 2022

^*Email: [e-mail]

Conflict of interest: The authors declare that there are no conflicts of interests among them

Author´s contribution: Lourdes L. Savón-Valdés: Conceptualization, Investigation, Data curation, Writing - original draft, Bárbara Rodríguez: Investigation, Ysgnamy Vázquez: Investigation, Idania Scull: Investigation, Magaly Herrera: Formal analysis, Tomás Ruiz: Funding acquisition