Phytobiotic Effect of Anacardium occidentale L. Leaves Powder on Performance, Carcass Traits, and Intestinal Characteristics in Broilers

30/July/2020 Approved: 22/November/2020 ABSTRACT In order to evaluate the phytobiotic effect of cashew (Anacardium occidentale L.) leaves powder (CLP) on growth performance, carcass traits, lymphoid organ weights and cecal lactic-acid bacteria in broilers, a total of 720 unsexed broilers of the Ross 308® genetic line were distributed according to a completely randomized design with three experimental treatments, five repetitions and 48 chickens per repetitions. The experimental treatments consisted of a basal diet (DB; T0) and DB + supplementation with 0.50 (T1) and 0.75% (T2) of CLP. At 32 days, the body weight, feed intake and feed conversion ratio were not modified ( p >0.05) by the experimental groups; however, T1 increased ( p <0.05) the viability compared to T0. Additionally, T2 improved ( p <0.05) the carcass and leg yields, although without significant changes ( p >0.05) for the other edible portions. Moreover, this natural product (A. occidentale) increased ( p <0.05) the ash content and the range of the red tone and decreased ( p <0.05) the L* in the breast, although moisture, fat, pH, and b* were not affected by the experimental diets ( p >0.05). Similarly, T1 favored ( p <0.05) the growth of green bacterial colonies with a white halo, and both treatments with CLP the total cecal acid-lactic bacteria. No significant changes were observed ( p >0.05) in the relative weight of the immune organs (thymus, bursa of Fabricius, and spleen). Dietary supplementation with cashew leaves powder enhanced the viability of the batch (0.5%), some edible portions (0.75%), the breast pigmentation, and the cecal beneficial bacteria


INTRODUCTION
The World Health Organization-WHO (2018) as a consequence of the use of antibiotics and their impact on the environment has determined that the frequency of resistance among bacterial pathogens is increasing worldwide and is considered as one of the biggest threats to health. Although, the inappropriate use of antibiotics in human medicine is considered the main driver of this phenomenon, the excessive use of antibiotics in livestock farming contributes greatly to increasing this threat (ECDC, 2015). As a result, the improper use of antibiotics in livestock activity is currently severely criticized by international health authorities and the media (WHO, 2018), therefore has generated interest in different areas to improve management practices in different productive sectors. Thus, the researchers' current premise is to obtain natural alternatives to decrease the indiscriminate use of antibiotics and improve performance in birds (Aroche et al., 2018).
Consequently, in this post-antibiotic stage, phytonutrients, phytobiotics, or additives have gained increasing interest due to their important biological activities, including anti-inflammatory, antioxidant, antimicrobial, and immunomodulatory functions (Windisch et al., 2007).

Phytobiotic Effect of Anacardium Occidentale L. Leaves Powder on Performance, Carcass Traits, and Intestinal Characteristics in Broilers
Phytobiotic products are rich in beneficial secondary metabolites such as tannins, coumarins, flavonoids, flavones, anthocyanins, and reducing sugars that in small concentrations on poultry diets have a significant impact on the animal response (Ogbuewu et al., 2020). Compared to synthetic antibiotics, natural products (such as phytobiotics) used in animal diets generally have mild and no residual toxicity and microbial resistance, thus, they are ideal candidates for the use in antibiotics-free poultry productions (Diaz-Sanchez et al., 2015).
In this sense, cashew (Anacardium occidentale L.) is a native plant of the northwest of Brazil and has been widely studied due to its pharmacological properties (Farias dos Santos et al., 2018). Specifically, the leaves of this plant (cashew) have a high concentration of important polyphenolic compounds and other beneficial secondary metabolites with antimicrobial, antioxidant, anti-inflammatory, and antidiarrheal activity (Sunderam et al., 2019). Previous results demonstrated that the dietary use of cashew leaves powder, up to 1.5%, increased body weight and pullet uniformity, and improved productivity and egg quality of laying hens (Martínez et al., 2012 a,b). The use of this natural product in broilers could benefit the biological activity and the final product (breast) of this poultry production. Thus, the aim of the study was to determine the phytobiotic effect of cashew (Anacardium occidentale L.) powder on growth performance, carcass traits, relative weight of lymphoid organs and cecal acid-lactic bacteria in broilers.

Study site and experimental design
The study was carried out during the months of June-July of the year 2019, in the Poultry Research and Teaching Center of the Pan-American Agricultural School (Zamorano University), San Antonio de Oriente, Honduras, located at an altitude of 800 meters above sea level with an annual average temperature of 24 °C.

Preparation of the phytobiotic from the cashew leaves
The cashew leaves were collected randomly from 10 trees of approximately 10 years of age in the periurban areas of the municipality of Palestina -Patuca in the department of Olancho, Honduras. For the collection of the leaves, the diversity in size and structure were considered, as well as the optimal classification of the leaves, identified in the Agricultural Science and Production Department, Pan-American Agricultural School, Honduras. The leaves were deposited and stored in a fiber bag to avoid the increase in humidity due to transpiration during transportation to the preparation area (Aroche et al., 2018).
Distilled water was used to wash the leaves and remove impurities. Subsequently, the layer of leaves (1-2 cm) was placed on a dry wide surface, protected with paper, and left to dry naturally inside for 5 days according to the methodology of Martínez et al. (2012 c). Once dry, the leaves were transported to the Food Analysis laboratory, Pan-American Agricultural School, Honduras and grounded with a blade mill to a grain size of 1 mm (Martínez et al., 2012 b). The dry matter (DM), crude protein (CP) and crude fiber were determined in triplicate (AOAC, 2006). The concentrations of calcium (Ca), sodium (Na), manganese (Mn), zinc (Zn), copper (Co) and phosphorous (P) were determined in triplicate by inductively coupled plasma-optical emission spectrometry (ICP-OES, Perkin Elmer Optima 3200RL) (Table 1). Furthermore, in previous works we determined that the leaves of this plant have a significant concentration of flavonoids (mainly cyanidin 3-O-xylosyl-rutinoside), tannins, beta-sitosterol, coumarins, reducing sugars and anthocyanidins (Martínez et al., 2012 c,d).

Experimental design, animals, and treatments
A total of 720 unsexed broilers of the Ross 308® genetic line were distributed according to a completely

Experimental conditions
Each replication consisted of a pen with a deep wood chip bed and 11 birds/m 2 . Feed and water were offered ad libitum in hopper feeders and nipple waterers, respectively. The temperature and ventilation inside the ship were controlled by gas brooders, curtain management and fans. The barn was disinfected according to environmental quality standards of Poultry Research and Education Center Protocol, 24 hours before the chicks entered the experimental area, this was disinfected with quaternary ammonium (5%). No medications or therapeutic veterinary care were used throughout the experimental stage. The birds were vaccinated against Newcastle, Gumboro diseases, and infectious bronchitis on the first day.

Growth performance
In each feeding phase (starter, grower, and finisher) the indicators of the productive performance of the broilers were determined. Viability was determined by the total count of live animals from the population size for each repetition at the start of the experiment. The initial and final body weight (BW) of each stage was performed for each animal with a Mettler Toledo® IND226 industrial scale (China) with an accuracy of ± 1.00 g. The feed intake (FI) was determined daily through eRBCA-2020-1362 4 Martínez Y, Tobar LA, Lagos HM, Parrado CA, Urquía AM, Valdivié M

Phytobiotic Effect of Anacardium Occidentale L. Leaves Powder on Performance, Carcass Traits, and Intestinal Characteristics in Broilers
the offer and reject method. The feed conversion ratio (FRC) was calculated by the relationship among the amount of feed eaten and the gain of 1 g of body weight.

Carcass traits and lymphoid organ weights
At 32 days of age, 10 male broilers per treatment were sacrificed by the bleeding method through the jugular vein, the birds were fasted for six hours. Initially, the broilers were weighed before slaughter on the Truweigh Blaze digital scale BL-100-01-BK (China) with an accuracy of ± 0.1 g. Afterwards, the carcass, the edible viscera (liver, heart, and gizzard), breast, liver, abdominal fat, bursa of Fabricius, thymus and spleen were weighed.
The analysis of the chemical composition of the breast was performed five times in the Food Analysis Laboratory of the Zamorano University. The skinless breasts were thawed and ground to a paste with a homogenizing blender, except for the colorimetry analysis, which used a small portion of the entire muscle. Afterwards, 1 g of ground meat was weighed and 10 ml of distilled water were added, the sample was homogenized for two minutes with a vortex and with a Bantex model 300 A digital potentiometer, calibrated with buffer solutions (pH 4 to 7), and then the meat pH was determined.
In addition, the color test was determined with a sphere spectrophotometer Minolta CR-400/410 chromameter (Konica Minolta Sensing Inc., Osaka Japan). For the latter, samples were placed inside the equipment and each muscle was sampled twice. Chromatic coordinates L* (luminosity), a* (red index) and b* (yellow index) were evaluated. Additionally, in the samples the content of moisture, fat, ash, and protein were prescribed, according to the methodology described by AOAC (2006).

pH and cecal lactic acid bacteria
At the time of slaughter, five left cecum and five right cecum were taken for each treatment. In the left cecum, pH was determined using an Oakton ® model pH 700 (USA) digital potentiometer, calibrated using buffer solutions (pH 1.68, 4.01, 7.00, 10.01, and 12.45).
Besides, the left cecum of five birds/treatment was taken, and the mucosa was scraped with a scalpel for microbiological culture. Each sample's cecal content was placed in a sterile tube; weight was recorded and diluted with Butterfield's phosphate-buffered dilution water to a 1:9 ratio (w:v). Diluted cecal contents were homogenized, and serial dilutions (1/10) were made from it until dilution 10^5. Aliquots of 0.1 ml of each dilution were spread plated on the surface of MRS agar (Neogen Acumedia, Mich.) supplemented with methylene blue (0.016 g/1000 ml) at 37 °C with a pH of 5.6 for 48 hours in anaerobiosis (Gas Pak system, BBL, Cockeysville, USA). Counts of lactic acid bacteria were reported as Log CFU/g by colonies' morphology on MRS + MB agar. Gram stain and catalase activity were tested on each type of colonies reported (Molina et al., 2019). The microbiological tests were performed in the Food Microbiology Laboratory of the Zamorano University.

Statistical analysis
Data was processed by simple classification analysis of variance (ANOVA) in a completely randomized design. Prior to this, the normality of the data was verified by the Kolmogorov Smirnov test and for the uniformity of variance, the Bartlett test was used. In some cases, the Duncan's test was used to determine the differences between means. Viability was determined by comparison of proportions. All analyzes were carried out according to the SPSS statistical software version 21.0. Table 3 shows the growth performance of broiler when CLP is used as a dietary supplement. At 10 days of age, T2 increased (p<0.05) body weight and feed intake compared to T0, although no changes were found for the viability and feed conversion ratio among treatments (p>0.05). In the grower period (11-24 days), T1 showed the highest percentages for viability in relation to T0 (p<0.05), the other indicators did not change (p>0.05) because of the natural product (A. occidentale), the same happened in the other productive stages (25-32 days and 0-32 days). Likewise, the experimental groups with CLP increased (p<0.05) the viability in the finisher stage (25-32 days), and throughout the production stage (0-32 days) T1 showed the best result for this productive indicator (p<0.05).

RESULTS AND DISCUSSION
The objective of the experimental work was to determine whether the use of this natural product (cashew) with high in dry matter and protein (Table  1) in broilers had the same positive effects previously found in laying hens and pullets (Martínez et al., 2012 a;Martínez et al., 2013), considering that the broiler has a more susceptible digestive system due to rapid growth. effect on viability, this could be attributed to the fact that CLP has a high content of essential minerals (Table  1), as well as beneficial secondary metabolites such as coumarins, flavonoids, triterpenes, anthocyanins, phenols, and saponins that in small concentrations have antimicrobial, anti-inflammatory, and antioxidant effects, which benefit the animal response (Martínez et al., 2012 c).
In this sense, Pawar et al. (2000); da Silveira-Vasconcelos et al. (2015) and Awakan et al. (2018) reported that the leaves of this medicinal plant are rich in the organic compound oleamide (9-octadecenamide), and become responsible for its anti-inflammatory activity in animal models. Other studies reported similar results in viability when they used CLP as part of a mixed medicinal powder in broilers (Aroche et al., 2018), which clearly shows that this natural product (up to 0.75%) does not cause symptoms related to anti-nutritional factors, because an excess of secondary metabolites (mainly tannins) inhibits the absorption of nutrients, which can cause metabolic disorders and negative effects for the production of broilers (Savón et al., 2007). Conversely, Huang et al. (2017) informed that small concentrations of tannins on broiler diets could be used as an alternative to in-feed antibiotics due to its anti-inflammatory and antimicrobial effect. Thus, phytobiotic products represent a promising non-antibiotic tool that improves the intestinal health and general health of animals, leading to better viability (Rossi et al., 2020).
During the first 10 days of age, T2 promoted the body weight compared to basal diet, and both supplementations with CLP promoted the feed intake with respect to T0 (Table 3), however, these production indicators in the following stages remained statistically unchanged (p>0.05). It is known that the first days of broilers are the most critical because they have an immature digestive, enzymatic and immune system, being the justification for the use of subtherapeutic antibiotics from the early stages in broilers. Apparently, due to the phytochemical and bactericidal characteristics, CLP exerted a natural growth-promoting effect on these animals. According to Aroche et al. (2018) this natural product (cashew) has antibacterial effects against strains of E. coli and S. aureus, therefore the supplementation of this phytobiotic can reduce these bacteria in the gastrointestinal tract, which could contribute to better intestinal health of the bird. Table 4 shows the effect of dietary supplementation with CLP on the carcass traits of broilers at 32 days. T2 increased (p<0.05) carcass and leg yields compared to T0 and T1. However, the breast, abdominal fat, liver, heart, and gizzard yields did not show statistical differences between treatments (p>0.05).

Phytobiotic Effect of Anacardium Occidentale L. Leaves Powder on Performance, Carcass Traits, and Intestinal Characteristics in Broilers
The fact that T2 increases carcass and leg yields showed that this natural product has beneficial chemical characteristics that can increase higher muscle synthesis compared to broilers-control group (Table 4). According to Farag & El-Rayes (2016), the changes that occur in performance due to the effect of natural products in broiler diets could in turn influence the carcass traits. Likewise, botanicals as dietary supplements can increase the proliferation of some groups of beneficial intestinal microorganisms, which affect the microbial competitive exclusion with a direct impact on the performance and carcass quality (Petracci et al., 2015). Furthermore, the positive effects of the phytogenics on broiler carcass yields have been related to digestibility and absorption of nutrients, mainly amino acids such as lysine, methionine, and threonine (Bess et al., 2012;Azad et al., 2013;Tumbagahon, 2019). The presence of flavonoids, anthocyanidins, and oleamide in the CLP could increase the nutrients digestibility, organic functioning, and antioxidant activity (Martínez et al., 2012c), as well as stimulate the immune system and the secretion of anti-inflammatory cytokines (Kong et al., 2007). Authors as El-Ghousein & Al-Beitawi (2009), Safamehr et al. (2012), and Mashayekhi et al. (2018) informed an increase of the carcass yield when they used leaves of Thumus valgaris, Urtica dioca and Eucalyptus globulus, as dietary supplemental of broiler diets. However, Al-Beitawi & El-Ghousein (2008) and Vase-Seyyed et al. (2018) with the dietary use of Nigella sativa, Rhus coriaria, Heracleum persicum and Mentha peperita found no changes in the carcass characteristics of broilers, respectively. The aforementioned, determines that, the intakes of leaves powder through the diet in the different feeding phases could generate better use of the nutrients and, therefore, improve the carcass yield; although it will depend on the chemical nature of the secondary metabolites and their concentration in the diet.
Another important fact is that the liver and heart did not increase their relative weight due to the dietary use of CLP (Table 4), similar results were found in a previous study when we used this natural product (A. occidentale) up to 1.5% in pullet diets (Martínez et al., 2013). Meanwhile, Woyengo et al. (2011) reported that diets rich in glucosinolate increased the liver relative weight; however, Molina et al. (2019) found a gradual reduction in the relative weight of this organ in broilers to a higher dietary supplementation with Ganoderma lucidum fungus rich in polysaccharides. Furthermore, Cañete et al. (2017) had related the changes in the heart relative weight with the diarrheal syndrome of birds due to the increased activity of the circulatory system caused by hemoconcentration and hypovolemia, something that did not occur in our experiment. It seems that the secondary metabolites cashew leaves powder had no apparent detrimental effect on these organs because according to Savón et al. (2007) excess phytochemical compounds change the relative weight and functionality of the liver and heart. Table 5 shows the effect of dietary supplementation up to 0.75% with cashew leaves powder on the chemical composition and colorimetry of the breast. This natural product (cashew) increased (p<0.05) the ash content and a* and decreased the L* in the breast. However, the moisture, protein, pH, and b* were not statistically different due to the effect of the treatments (p>0.05).
Previous studies in laying hens found that dietary supplementation with CLP (0.5%) improved the external and internal egg quality (Martínez et al., 2012 b), these results show that this phytobiotic can also modify the carcass traits, especially the minerals concentration and red pigments in the breast (Table  5). In this sense, Tumbagahon (2019), found that a natural product derived from cashew, as a phytobiotic additive in broiler diets, improved digestibility, nutrient Means within the same row with different superscript differ significantly (p<0.05). , which have justified their use as a restorative of these minerals in diarrheal syndrome (Cañete et al., 2017). The ash content is directly related to the amount of minerals present in CLP (Table 1), therefore, the treatments with cashew leaves powder (T1 and T2) could have increased the assimilation and translocation of these minerals to the breast, since T1 and T2 increased ash by 0.10 and 0.21% compared to T0, respectively (Table 5). According to Martínez et al. (2012 c) cashew leaves powder has anthocyanidins, these secondary metabolites are water-soluble pigments responsible for the red, purple, and blue color in the leaves, flowers, and fruits, which have been attributed anti-inflammatory and antioxidant properties (Khoo et al., 2017). Our previous results reported that the use of this phytobiotic (A. occidentale) in the diets of laying hens increased the pigmentation of the egg yolk (Martínez et al., 2012 b).
In accordance, this experiment demonstrated that the dietary use of CLP in broilers also increases the range of red pigments in the breast. According to , the pigmentation of the skin of broilers is achieved through diets that contain pigments that are soluble in lipids or water, which can be transferred to the tissue of animals. Furthermore, the results found have a concordant relationship between a* and L* (Table 5). Grashorn (2016), found that the luminosity (L *) decreases as the red pigments increase (a*; Table   5). The results show that CLP has the potential as a phytobiotic additive in broiler diets to improve some characteristics of chicken skin, being a purchasing decision factor in some countries (Kennedy et al., 2005).
Dietary supplementation with cashew leaves powder did not change (p>0.05) the relative weight of the lymphoid organs (Bursa de Fabricius, thymus and spleen) in broilers (Table 6).
Dietary supplementation with CLP was not sufficient to change the relative weight of the lymphoid organs in broilers (p>0.05), especially bursa of Fabricius and thymus, which are the primary lymphoid organs that involuted from the 60 days of age (Taha-Abdelaziz et al., 2018). These organs participate in the immune response against pathogens through the maturation and differentiation of B and T lymphocytes (Lillehoj et al., 2011). Perhaps, these results may be due to the acceptable conditions of tenure, biosecurity and animal welfare of the experiment, since the increased activity and growth of these hematopoietic organs has been related to stressors and the presence of pathogenic bacteria (Lillehoj et al., 2011;Kim & Lillehoj, 2018). Other results with 0.5% of cashew leaves powder on pullet diets indicated an increase in the relative weight of the bursa of Fabricius and thymus, however, a higher supplementation provoked a significant reduction in the productive response and in the weight relative of these immune organs related to antinutritional factors (Martínez et al., 2013). Means within the same row with different superscript differ significantly (p<0.05).
T0: basal diet (BD); BD+0.50% of cashew leaves powder (T1); BD+0.75% of cashew leaves powder (T2).  Table 7 shows the effect of dietary supplementation with cashew leaves powder on the count of lactic acid bacteria (LABs) and cecal pH. T1 promoted (p<0.05) the growth of green bacillus with white halo and both treatments with CLP increased (p<0.05) the total cecal lactic acid bacteria. However, no significant differences were observed among treatments (p>0.05) for the irregular white bacilli, irregular white coconuts and cecal pH in broilers.
The intestinal flora plays a fundamental role in the absorption of nutrients, which improves health and protects the broiler chicken from the appearance of Means within the same row with different superscript differ significantly (p<0.05).
1 The count was expressed as Log 10 , 2 irregular white colonies; 3 green colonies with white halo; 4 white colonies.
enterobacteriaceae (Prado-Rebolledo et al., 2017). Some LABs can regulate the intestinal microflora, given their antagonistic action against specific groups of microorganisms, as well as improve the function of the immune system and intestinal integrity, which is positively correlated with nutritional demand (Xi et al., 2019;Betancur et al., 2020). Clearly, it is observed that the dietary supplementation with CLP improved the growth of the cecal LABs and gut health in broilers, which is related to the higher percentage of viability with T1, as well as some changes in the carcass traits in the treatments where this natural product was used (Table 3, 4 and 5). According to Rafeeq et al. (2016) and Giannenas et al. (2018) the use of phytobiotic additives (from Cuminum cyminum L., Foeniculum vulgare Mill. and Achillea wilhemsii C. Koch; Magnifera indica L., Withania somnifera L., Phyllanthus emblica L. and Ocimum sanctum L., respectively), increased the cecal lactic acid bacteria (LABs) count, which improved intestinal permeability and nutrient digestibility. Furthermore, Diaz-Sanchez et al. (2015) reported that, although the action mechanisms of phytobiotics have not been fully elucidated, some authors suggest that these phytochemical compounds stimulate the immune system, specifically by activating lymphocytes, macrophages, and NK cells, in addition to protecting the intestine from the colonization of pathogenic bacteria and promoting bacterial competitive exclusion (Hong et al., 2019). Thus, the use of this natural product could be used in environmentally friendly productions (antibiotic-free), which would contribute to the new demands of consumers.
Although the growth of LABs was higher with CLP, this was not enough to reduce the cecal pH, similar results were found by Martínez et al. (2012a) and Martínez et al. (2013) when they used 0.5% of CLP on pullet diets. However, Ripon et al. (2019) indicated a proportional decrease in intestinal pH to higher supplementation of a phytobiotic extract in broiler diets. Also, according to Ding et al. (2011) the phytobiotic products can lower intestinal pH through the growth of cecal LAB in non-ruminants, with higher emphasis on pigs than in broilers. A higher dietary concentration of cashew leaves powder appears to be necessary to modify the cecal pH in broilers. It is important to note that metabolic disorders, microbial dysbiosis, and high concentration of antinutritional compounds cause an increase in intestinal pH, something that did not occur in this experiment (Latorre et al., 2015). The findings of the present research increase understanding of the positive effects of botanicals products as alternatives to subtherapeutic antibiotics and their beneficial role in the gut microbiota, viability, and carcass traits of broilers.

CONCLUSIONS
Dietary supplementation with cashew (Anacardium occidentale L.) leaves powder (T2) increased body weight and feed intake up to 10 days of age, after that only viability improved with T1, without modifying the other productive indicators and the relative weight of the immune organs. Also, the carcass and leg yields increased with T2 with relation to T0, and this natural product enhanced the ash content, L* and a* in the