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Revista Brasileira de Zootecnia

versão impressa ISSN 1516-3598versão On-line ISSN 1806-9290

R. Bras. Zootec. vol.48  Viçosa  2019  Epub 12-Set-2019

http://dx.doi.org/10.1590/rbz4820180254 

Non-Ruminants

Blood parameters and hepatic histopathology of broilers fed rations supplemented with essential oils

Vanessa Kelly Ferreira do Rosário Santos1 
http://orcid.org/0000-0002-9994-8058

Wedson Carlos Lima Nogueira1 
http://orcid.org/0000-0001-7378-780X

Renato de Lima Santos2 
http://orcid.org/0000-0002-4830-0470

Neide Judith Faria de Oliveira1 
http://orcid.org/0000-0003-0854-892X

Ernane Ronie Martins1 
http://orcid.org/0000-0001-6139-7206

Izabela Lorena Azevedo3 
http://orcid.org/0000-0002-0707-7567

Tatiane Furtado de Carvalho2 
http://orcid.org/0000-0002-3053-7242

Anna Christina de Almeida1  * 
http://orcid.org/0000-0001-9836-4117

1Universidade Federal de Minas Gerais, Instituto de Ciências Agrárias, Montes Claros, MG, Brasil

2Universidade Federal de Minas Gerais, Escola de Veterinária, Belo Horizonte, MG, Brasil

3Instituto Federal de Educação, Ciência e Tecnologia Baiano, Senhor do Bonfim, BA, Brasil


ABSTRACT

This study aimed to evaluate the hematological profile, hepatic function, and histopathology of mixed-sex broilers fed rations supplemented with microencapsulated essential oils from Cymbopogon flexuosus (lemon grass) and Lippia rotundifolia (chá-de-pedestre). One hundred and fifty Cobb chicks were housed in cages from 1 to 42 days of age in a completely randomized design, with six replicates with five chickens in each of five treatments (150 total chicks): basal diet without antibiotic growth promoter (negative control), diet with enramicina and salinomycin (positive control), diet with lemon grass essential oil, diet with L. rotundifolia essential oil, and diet with a mixture of lemon grass and L. rotundifolia essential oils. Mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH) were significantly lower in untreated broilers (negative control group). Gender was not associated with erythrogram values, but aspartate aminotransferase activity (AST) was higher in females. Gamma-glutamyl transferase (GGT) was higher in chicks in the group that received rations with lemon grass oil. Broilers that received L. rotundifolia oil developed more hepatic lesions, although no effect of sex was observed related to the lesion score. Biliary hyperplasia and fibroplasias were observed in all groups, with higher histopathology scores in broilers that received diets containing L. rotundifolia oil. Mixed-sex broilers fed rations supplemented with lemon grass and L. rotundifolia essential oils have normal complete blood counts and unspecific hepatic lesions and are characterized by lipidosis, hyperplasia of the bile ducts, and fibroplasia.

Keywords: animal production; bird; bird nutrition; essential oils; hematology; liver

Introduction

Antibiotics in small dosages added to the avian diet as growth promoters result in better utilization of nutrients by the birds and higher growth rates and feed conversion efficiency. In addition, antibiotics act on the intestinal health of chickens. However, this practice has contributed to the emergence of resistant strains of microorganisms (Attia et al., 2017a). Thus, some countries have banned the use of antibiotics as additives in animal feed to minimize the transmission and spread of resistant bacteria through the food chain (Attia et al., 2018).

In contrast, the use of organic acids, enzymes, pre- and probiotics, and essential oils may result in higher performance and better intestinal health without the inconvenience of antibiotic resistance (Dhama et al., 2015; Zeng et al., 2015; Cho et al., 2014; Krishan and Narang, 2014). Variable results have been obtained using oils for antimicrobial purposes, such as rosemary (Traesel et al., 2011), thyme (Saleh et al., 2014; Toghyani et al., 2011; Toghyani et al., 2010; Tollba et al., 2010; Attia et al., 2017b), and ginger (Mehr et al., 2014) oils. However, the possible toxic effects and/or changes in metabolism caused by essential oils still generate doubts regarding their safety.

Cymbopogon flexuosus (lemon grass) and Lippia rotundifolia (chá-de-pedestre) essential oils can be used as alternative performance enhancers due to their in vitro antimicrobial action, and they can promote diet stability (Assis et al., 2017; Azevedo et al., 2016; Souza et al., 2015). Cymbopogon flexuosus, also known as grass balm, is native to Asia (May et al., 2008). The major components present in lemon grass oil are myrcene, geraniol, and citral (Simões et al., 2010), with the latter being the most important. Lippia rotundifolia is popularly known in Brazil as chá-de-pedestre. Its main components are β-myrcene, farnesol, limonene, and myrcenal (Leitão et al., 2008). Although it is promising as a phytotherapeutic plant, its pharmacological activity is still uncharacterized (Resende et al., 2015). Souza et al. (2015) demonstrated its antimicrobial activity against S. aureus and E. coli isolated from avian intestines.

Even though previous studies have used medicinal plants as performance enhancers for poultry, the subsequent changes affecting the metabolism of birds have not been fully characterized. Current research aims at evaluating the effect of phylogenics and essential oils on biochemistry and blood components. Available results are promising, indicating no changes in physiological parameters and improvement of the health status of treated birds (Attia et al., 2017b; 2018; Bortoluzzi et al., 2018; Chowdhury et al., 2018; Mokhtari et al., 2018).

Thus, this study aimed to evaluate blood parameters and hepatic histopathology of mixed broilers fed rations supplemented with microencapsulated essential oils.

Material and Methods

The present study was conducted in Montes Claros, MG, Brazil (16°41′00″ S, 43°50′00″ W). All procedures were in accordance with ethical standards and were approved by the Ethics Committee on Animal Use under case number 102/2013. The adopted procedures followed Azevedo et al. (2017).

One hundred and fifty one-day-old mixed sex Cobb 500® chicks were housed in 30 cages (60×35×100 cm) containing drinkers and feeders. The experiment was conducted in a completely randomized 2×5 (sexes × treatments) factorial design with three replicates of 10 animals per treatment (five males and five females). The treatments included the following: negative control, rations without antimicrobials or anticoccidials; positive control, rations supplemented with 10 ppm of enramycinand 42 ppm of salinomycin; C. flexuosus essential oil, rations with 120 mg of essential oil per kg of live weight; L. rotundifolia essential oil, rations with 120 mg of essential oil per kg of live weight; and association, rations with a mixture of two essential oils (60 mg of each essential oil). Dosage was based on previously demonstrated in vitro antimicrobial activity by the oils using the disc-diffusion method, minimum inhibitory concentration, and minimum bactericidal concentration as described by Azevedo et al. (2016) and Souza et al. (2015) for C. flexuosus essential oil and L. rotundifolia essential oil, respectively.

Nutritional planning was divided into three phases: initial, from 1 to 21 days; growth, from 22 to 33 days; and finishing, from 34 to 42 days. Rations were provided ad libitum during all experimental periods in ground form following the nutritional levels recommended by Rostagno et al. (2011) and presented by (Azevedo et al., 2017). The major compound of C. flexuosus essential oils was citral (77.42%), while that of L. rotundifolia essential oils was β-myrcene (15.52%) (Azevedo et al., 2017). Essential oils were converted into microcapsules by the coacervation method with edible polymers. Compounds were identified by gas chromatography-mass spectrometry analysis, which ensured the stability of the rations after microencapsulation as described by Azevedo et al. (2017).

At 43 days of age, two birds of each experimental treatment were selected (one male and one female); selection included birds weighing up to 10% above or below the average weight. After fasting for 8 h, blood samples were collected for complete blood count analysis and serum biochemistry. Blood samples (4 mL) from the cutaneous ulnar vein were obtained according to recommendations of the Pan American Health Organization/World Health Organization (PAHO/WHO) (Manual…, 2010) and transferred to tubes to perform complete blood count and serum biochemistry. Hematological profiles were assessed by flow cytometry. Triglycerides were measured by end point colorimetry, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) by the ultraviolet kinetic technique, gamma-glutamyl transferase (GGT) by the kinetic colorimetric method, and glucose by kinetic methodology.

For histopathological analysis, sections of the liver were collected and fixed by immersion in 10% buffered formalin for 24h, followed by dehydration in increasing concentrations of ethanol, diaphonization in xylol, and embedding in paraffin. Sections of 5 μm were stained with hematoxylin and eosin (H&E), for histopathological analysis under optical microscope coupled to a camera, under magnification of 10X and 40X. Lesions were assessed according to intensity and given a score: 0 = no damage, 1 = mild damage, 2 = moderate damage, and 3 = severe damage (Andrade et al., 2014). Histopathological scores are described in Table 1.

Table 1 Criteria for histopathological scores considering lipidosis, fibroplasia and bile duct hyperplasia 

Score Lipidosis1 Fibroplasia2 Bile ducts hyperplasia3
0.5 HLMD FPMM PDMM
1.0 HLMM FPMD PDBD
2.0 HLMO FPMO PDBO
3.0 FPMI

1HLMD: hepatocytes with micro and macro vacuoles, well delimited, compatible with discreet multifocal lipidosis; HLMM: hepatocytes with micro and macro vacuoles, well delimited, compatible with diffuse minimal lipidosis; HLMO: hepatocytes with micro and macro vacuoles, well delimited, compatible with moderate multifocal lipidosis.

2FPMM: periportal minimum multifocal fibroplasia; FPMD: periportal mild multifocal fibroplasia; FPMO: periportal moderate multifocal fibroplasia; FPMI: predominantly multifocal periportal intense fibroplasia.

3PDMM: minimum multifocal bile ducts proliferation; PDBD: multifocal discreet bile ducts proliferation; PDBO: moderate multifocal bile ducts proliferation.

Data were analyzed by analysis of variance in R Statistical software (R Core Team, 2011) with sex and diet as the main effect and pen as an experimental unit. When there were significant interaction effects (P<0.05), averages were compared with Tukey's test. The statistical model used was:

Yijk=μ+αi+βj+(αβ)ij+εijk,

in which Yijk = set of observations of the dependent variable corresponding to the sex i, adding essential oil j and repetition k; µ = set of observations; αi = effect of the sex; βj = effect of addition of essential oils in the diet; (αβ)ij = effect of interaction between sex i and addition of essential oils in the order j; and εijk = experimental error concerning the observation of the effect of sex i, adding essential oils in the diet j and repetition k.

Histopathological score data were non-parametric; therefore, a Kruskal-Wallis test was used to compare the means of each group using the program GraphPad Prism version 5.0.

Results

During the period in which the broilers were housed, no clinical or behavioral changes were observed, indicating normal behavior. In this study, we defined as standard normality reference the results of the analyses of the positive control group and reference standards values for the species.

Mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH) were higher in the treatments than in the negative control (P<0.05; Table 2). The other erythrogram parameters were similar between treatments (P>0.05) and were not influenced by gender. Mean heterophil (Table 3) values were lower in the treatments compared with the negative control.

Table 2 Erythrogram of mixed-sex broilers fed rations supplemented with essential oils 

Treatment1 Sex Erythrocytes(millions/mm3) Hemoglobin(g%) Hematocrit (%) MCV(fl) MCH(pg) MCHC(%)
M 3.11±0.32 10.72±1.13 28.59±3.22 91.66±3.28 34.66±2.27 37.80±1.95
F 3.15±0.57 10.03±1.26 28.15±4.95 90.00±4.05 32.98±3.60 36.80±3.70
Negative control 3.52±0.82 10.38±0.96 30.80±7.31 87.40b±3.21 30.32b±4.72 34.71±5.30
Positive control 3.05±0.36 11.00±1.54 28.28±4.45 92.32a±5.37 36.03a±2.82 38.85±2.36
C. flexuosus 3.08±0.23 9.90±1.75 28.03±2.58 90.99a±2.34 33.73a±0.93 37.07±0.86
L. rotundifolia 2.95±0.27 10.18±1.07 26.68±2.14 90.50a±1.76 35.02a±0.67 38.71±1.21
Association2 3.06±0.20 10.41±0.66 28.01±1.66 92.95a±3.17 34.00a±1.12 37.18±1.12
Negative control M 3.26±0.60 10.40±0.81 29.13±4.92 89.53±2.36 32.34±3.58 36.09±3.53
F 3.78±1.05 10.36±1.28 32.46±10.06 85.26±2.57 28.31±5.54 33.33±7.22
Positive control M 3.28±0.24 12.23±1.15 31.56±3.68 95.93±4.31 37.22±1.02 38.83±1.70
F 2.82±0.33 9.76±0.15 25.00±1.92 88.71±3.82 34.84±3.82 38.86±3.33
C. flexuosus M 3.04±0.28 10.30±0.86 27.43±2.77 90.11±2.37 33.87±1.35 37.58±0.87
F 3.12±0.22 9.50±2.55 28.73±2.76 91.88±2.39 33.59±0.55 36.56±0.57
L. rotundifolia M 2.92±0.10 9.96±0.92 26.43±1.01 90.51±1.36 35.15±0.90 38.85±1.59
F 2.98±0.42 10.40±1.37 26.93±3.20 90.48±2.43 34.88±0.51 38.56±1.03
Association M 3.08±0.26 10.70±0.81 28.40±1.85 92.21±1.96 34.72±0.69 37.65±0.59
F 3.04±0.19 10.13±0.45 27.63±1.73 93.70±4.43 33.29±1.06 36.71±1.46
Source of variation P-value
Treatment (T) 0.26 0.61 0.56 0.03 0.01 0.14
Sex (S) 0.83 0.13 0.77 0.14 0.08 0.36
T×S 0.49 0.32 0.34 0.06 0.67 0.93

MCV - mean corpuscular volume; MCH - mean corpuscular hemoglobin; MCHC - mean corpuscular hemoglobin concentration.

1Treatment = ration negative control: without additives; positive control ration: with antimicrobial and anticoccidial; control diet essential oil of C. flexuosus; control diet essential oil of L. rotundifolia.

2Association: control diet essential oil of C. flexuosus and L. rotundifolia.

a,b - average followed by the same letter in the column does not differ among themselves by Tukey test (P<0.05).

Table 3 Leukogram of mixed-sex broilers fed rations supplemented with essential oils 

Treatment1 Sex Leukocytes Heterophil (Hf) Hf:Lf Eosinophil Lymphocyte (Lf) Monocyte
----------------------------------------------- mm3 -----------------------------------------------
M 28.89±3.31 7.46±2.74 0.10±0.03 11.26±6.35 72.40±7.07 8.86±4.20
F 27.58±4.23 8.00±5.43 0.12±0.12 12.73±6.39 69.46±10.80 9.80±4.66
Negative control 30.39±4.94 12.16b±7.52 0.20±0.17 11.83±7.33 65.66±12.51 10.33±6.68
Positive control 26.74±4.41 7.50a±2.88 0.10±0.04 12.50±7.44 74.33±10.09 5.66±2.58
C. flexuosus 28.30±3.80 7.16a±1.16 0.09±0.01 10.83±5.03 73.50±3.14 8.50±2.42
L. rotundifolia 28.56±3.31 6.16a±0.98 0.08±0.00 12.33±8.91 69.50±10.67 12.00±4.47
Association2 27.19±2.09 5.66a±2.25 0.08±0.03 12.50±3.78 71.66±6.31 10.16±2.63
Negative control M 27.69±3.51 9.66±2.88 0.13±0.03 9.33±9.23 74.00±4.58 7.00±3.60
F 33.09±5.18 14.66±10.69 0.28±0.23 14.33±5.50 57.33±12.74 13.66±8.08
Positive control M 29.86±3.82 10.00±1.00 0.14±0.02 14.33±10.96 70.00±13.89 5.66±4.04
F 23.62±2.18 5.00±1.00 0.06±0.01 10.66±2.88 78.66±2.30 5.66±0.57
C. flexuosus M 28.59±5.87 6.66±1.15 0.09±0.01 11.66±5.03 72.33±2.88 9.33±3.21
F 28.01±1.17 7.66±1.15 0.10±0.01 10.00±6.00 74.66±3.51 7.66±1.52
L. rotundifolia M 29.75±2.55 6.33±1.15 0.08±0.00 9.00±4.00 73.00±8.88 11.66±6.35
F 27.37±4.08 6.00±1.00 0.09±0.00 15.66±12.22 66.00±13.00 12.33±3.05
Association2 M 28.55±1.98 4.66±3.05 0.06±0.04 12.00±2.64 72.66±6.02 10.66±2.51
F 25.83±1.23 6.66±0.57 0.09±0.01 13.00±5.29 70.66±7.76 9.66±3.21
Source of variation P-value
Treatment (T) 0.43 0.04 0.06 0.99 0.44 0.13
Sex (S) 0.32 0.69 0.44 0.57 0.36 0.54
T×S 0.10 0.25 0.17 0.69 0.15 0.45

1Treatments = ration negative control: without additives; positive control ration: with antimicrobial and anticoccidial; control diet essential oil of C. flexuosus; control diet essential oil of L. rotundifolia.

2Association: control diet essential oil of C. flexuosus and L. rotundifolia.

a,b - average followed by the same letter in the column does not differ among themselves by Tukey test (P<0.05)

Gender influenced the enzyme activity of AST (P<0.05) with increased activity in females (Table 4). Gamma-glutamyl transferase was higher in the group that received rations with lemon grass oil (P<0.05). Triglyceride levels were not influenced by the treatments (P>0.05). There was no difference in the mean total protein, albumin, globulin, serum albumin:globulin ratio, total cholesterol, or glucose levels between treatments and the positive control (P>0.05) (Table 4). However, a significant difference was observed between the sexes regarding albumin (P<0.05), with males exhibiting a greater concentration than females.

Table 4 Liver enzymes activity and serum biochemistry of mixed-sex broilers fed rations supplemented with essential oils 

Treatment1 Sex AST ALT GGT TP Albumin Globulin Triglycerides TC
---------------- IU/L ---------------- --------------- g/L --------------- --------- mg/dL ---------
M 297.40b±96.48 9.40±0.82 22.66±6.22 3.20±0.62 1.68a±0.15 1.51±0.70 19.06±9.76 115.40±18.82
F 400.86a±178.23 10.20±1.47 20.46±4.27 2.82±0.39 1.53b±0.25 1.28±0.37 17.00±12.07 105.26±18.42
Negative control 257.66±9.22 9.00±1.09 21.33b±3.66 2.97±0.31 1.44±0.24 1.53±0.26 14.33±8.52 96.33±12.17
Positive control 335.83±200.98 10.00±1.54 17.50b±2.81 2.79±0.62 1.62±0.15 1.17±0.54 25.00±16.58 127.66±14.12
C. flexuosus 301.00±95.34 9.50±0.83 27.16a±5.56 2.83±0.50 1.64±0.19 1.19±0.43 25.33±5.78 105.33±19.28
L. rotundifolia 431.00±174.94 10.00±0.89 20.66b±5.46 3.26±0.51 1.58±0.23 1.67±0.54 15.83±6.24 110.00±14.99
Association2 420.16±154.18 10.50±1.51 21.16b±5.11 3.18±0.72 1.74±0.21 1.43±0.88 9.66±5.75 112.33±22.83
Negative control M 256.66±12.66 8.66±1.15 22.33±4.50 3.08±0.32 1.60±0.06 1.48±0.26 14.33±12.50 106.33±7.63
F 258.66±7.02 9.33±1.15 20.33±3.21 2.87±0.34 1.29±0.27 1.58±0.30 14.33±5.03 86.33±3.51
Positive control M 263.33±32.33 9.33±0.57 19.00±3.46 2.93±0.87 1.74±0.07 1.19±0.80 27.00±9.16 129.00±21.70
F 408.33±290.12 10.66±2.08 16.00±1.00 2.65±0.37 1.50±0.10 1.14±0.27 23.00±24.33 126.33±4.72
C. flexuosus M 266.00±9.64 9.33±0.57 29.66±7.50 3.22±0.36 1.68±0.10 1.54±0.27 25.33±8.62 114.33±20.55
F 336.00±137.69 9.66±1.15 24.66±1.52 2.44±0.21 1.59±0.28 0.84±0.19 25.33±3.05 96.33±16.25
L. rotundifolia M 284.66±28.53 9.66±0.57 21.33±7.37 3.50±0.57 1.69±0.20 1.81±0.75 15.66±7.37 114.00±10.53
F 577.33±107.05 10.33±1.15 20.00±4.35 3.02±0.40 1.48±0.25 1.53±0.34 16.00±6.55 106.00±20.07
Association2 M 416.33±189.43 10.00±1.00 21.00±5.29 3.26±1.05 1.71±0.30 1.54±1.33 13.00±6.24 113.33±31.72
F 424.00±153.29 11.00±2.00 21.33±6.11 3.10±0.44 1.77±0.14 1.33±0.36 6.33±3.21 111.33±17.15
Source of variation P-value
Treatment (T) 0.14 0.32 0.04 0.51 0.19 0.55 0.06 0.06
Sex (S) 0.04 0.09 0.23 0.07 0.04 0.30 0.59 0.12
T×S 0.32 0.96 0.90 0.86 0.55 0.82 0.96 0.84

AST - aspartate aminotransferase; ALT - alanine aminotransferase; GGT - gamma-glutamyl transferase; TP - total protein; TC - total cholesterol.

1Treatment = negative control ration: without additives; positive control ration: with antimicrobial and anticoccidial; control diet essential oil of C. flexuosus; control diet essential oil of L. rotundifolia.

2Association: control diet essential oil of C. flexuosus and L. rotundifolia.

a,b - average followed by the same letter in column does not differ among themselves by Tukey test (P<0.05).

In relation to the histopathological analysis (Figure 1), lesions were observed in chickens of all treatments, although with different scores. Discreet lipidosis was observed in animals in the negative control. The birds in the other treatments received scores that ranged from 2-3 for lipidosis, hyperplasia of the bile ducts, and fibroplasia. Broilers that received chá-de-pedestre oil in the diet exhibited higher damage scores in comparison with those in other groups (P<0.05). No effect of gender was observed on lesion scores (P>0.05). The occurrence of lesions characterized by hyperplasia of the bile ducts (Figure 2A) and fibroplasia (Figure 2B) was found in birds of all treatments, except the negative control, and broilers that received rations containing chá-de-pedestre oil had the highest scores.

Figure 1 Score of liver lesions of mixed-sex broilers fed rations supplemented with essential oils.1 Treatments: negative control: negative control diet without additives; positive control: positive control diet with antimicrobial and anticoccidial; C. flexuosus: control diet with essential oil of C. flexuosus; L. rotundifolia: control diet with essential oil of L. rotundifolia; association: control diet association between the essential oils of C. flexuosus and L. rotundifolia.Kruskal-Wallis Test; * indicates (P<0.0162). 

Figure 2 Lesions in the liver of mixed-sex broilers fed rations supplemented with essential oil of L. roduntifolia).A - Bile ducts hyperplasia and inflammatory infiltrate; B - Fibroplasia, hyperplasia of the bile ducts and inflammatory infiltrate.Hematoxylin-eosin staining (H&E); Bar = 100 µm. 

Discussion

Productivity parameters of experimental batches in this study were within the range considered normal for this commercial lineage as previously described by Azevedo et al. (2017), because the survival was 100% throughout the experimental period and experimental animals remained healthy. Studies with other phytogenic and essential oils added to the diet are in good agreement with our results. The beneficial effects have been associated with the modulating activity of the metabolic pathways of the cecal microbiota, by stimulating the immune system, and improving intestinal digestibility (Attia et al., 2017b; 2018; Bortoluzzi et al., 2018; Chowdhury et al., 2018; Mokhtari et al., 2018).

The hematological parameters results obtained in this research may also be associated with the health of birds and corroborate those of studies conducted with Thymus vulgaris (Toghyani et al., 2010; Ali, 2014; Saleh et al., 2014) and physiological criteria (Attia et al., 2017b; 2018).

The mean heterophil values (Table 3) and mean total protein, albumin, globulin, serum albumin:globulin ratio values (Table 4) indicated that the birds remained within normal ranges during the experiment. Attia et al. (2017b), Attia et al. (2018), Mokhtari et al. (2018), and Traesel et al. (2011) observed similar results with other phytogenics and essential oils, suggesting an improvement in general health. The possible action of essential oils on the avian immune system is still unknown. However, immunomodulating activity has been described for other essential oils in different experimental models (Brenes and Roura, 2010; Dhama et al., 2015). Essential oils acting as immune stimulants have been demonstrated by the use of thyme oil and ginseng in broiler diets (Toghyani et al., 2010; Toghyani et al., 2011; Saleh et al., 2014; Attia et al., 2017b; 2018). According to these authors, thyme oil and ginseng may stimulate innate immunity, increasing the phagocytic activity of heterophils.

Gender influencing AST levels has not been described in studies using phytogenic or essential oils in birds. High AST levels may be related to hepatocyte membrane damage and lesions in bile ducts. However, generation of AST from other tissues, such as muscles, must be considered (Tennant and Center, 2008; Grunkemeyer, 2010; Attia et al., 2017a). High environment temperatures may have affected these results. Notably, broilers and laying hens can suffer liver damage from unfavorable environmental conditions (Attia et al., 2017a).

Conversely, the highest levels of GGT observed in birds of the diet group with lemon grass essential oil (Table 4) may be associated with liver lesions. Gamma-glutamyl transferase is a primary marker for hepatobiliary diseases and can be indicative of intra-hepatic and extra-hepatic cholelithiasis (Tennant and Center, 2008; Grunkemeyer, 2010). The effect of essential oils on the hepatic function of broilers is variable. Cinnamon, oregano, pepper, thyme (Toghyani et al., 2011; Attia et al., 2017b; 2018), and ginseng (Catalan et al., 2013) oils do not influence hepatic biochemical components, such as serum protein, albumin, triglyceride, and AST. However, considering the conditions of this study, GGT levels (Table 4) may indicate hepatic damage in broilers, which may be associated with hepatic fibroplasia and hyperplasia of the bile ducts observed in groups that received rations with essential oils (Figure 1).

Biochemical profile results from a study on the association between hepatic lesions and alterations of hepatic enzymes such as GGT may indicate a loss in the synthesis capacity of the liver or hepatocellular damage, which reflects the ability of the liver to employ proper metabolism of lipids, proteins, and carbohydrates (Grunkemeyer, 2010). The author also notes that GGT changes are nonspecific and may be observed in association with various liver changes.

The results obtained in the present study are similar to those of studies with clove essential oils (Mehr et al., 2014) and thyme (Toghyani et al., 2010). This pattern may be indicative of a balance between intestinal absorption, synthesis, and secretion in hepatocytes and absorption in adipose tissue (Lumeij, 2008).

Lipidosis, as observed in this study, should not be interpreted as a possible effect of essential oils, since it was also observed in broilers that received diets without these oils. However, in other studies, a difference in the degree of lipidosis has been observed, depending on the origin of oils used in the diets (Tufarelli et al., 2015), even when this difference has been reversible, depending on the extent of lipidosis (Blevins et al., 2010). According to Schmidt et al. (2003), lipidosis is frequent in broilers and may be associated with diet, mycotoxins, or the metabolism of hepatic enzymes; however, in this study, only the broilers in the treatment containing lemon grass oil exhibited higher levels of GGT.

In broilers, hyperplasia and fibroplasia of the bile ducts are classified as nonspecific lesions and are associated with changes in hepatic metabolism that occur systematically after lesion of the liver parenchyma. These lesions are of various origins, and most of the time, they are not observed as clinical manifestations prior to death, making it difficult to determine the exact causes (Hochleithner et al., 2005).

Previous studies on the toxicity of C. flexuosus and L. rotundifolia specifically were not found in the literature. However, plants of the same genus have been shown not to be toxic. Costa et al. (2011) analyzed oral toxicity and genotoxicity in Cymbopogon citratus at a dose of 3,500 mg/kg in mice and found it not to be toxic, and Andrade et al. (2014) tested the effects of Lippia origanoides at a dosage of 120 μL/mL, and, likewise, it did not induce acute oral toxicity or subchronic signs in rats.

Despite being used in veterinary and human medicine, there is little information about the absorption of essential oil compounds after oral administration (Kohlert et al., 2000). Few studies have focused on the toxicity of essential oils; however, it has been confirmed that they have toxic effects on eukaryotic cells with variable effects. Raut and Karuppayil (2014) showed the safety of essential oils at low concentrations. However, studies that assess the toxic concentrations for each oil are still needed.

Conclusions

Mixed-sex broilers fed rations supplemented with C. flexuosus and L. rotundifolia essential oils had normal complete blood counts and unspecific hepatic lesions characterized by lipidosis, hyperplasia of the bile ducts, and fibroplasia.

Acknowledgments

The authors are grateful to the Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) (Finance Code 001). RLS is recipient of a fellowship from CNPq (Brazil).

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Recebido: 26 de Dezembro de 2018; Aceito: 03 de Maio de 2019

*Corresponding author:aca2006@ica.ufmg.br

Conflict of Interest

The authors declare no conflict of interest.

Author Contributions

Conceptualization: V.K.F.R. Santos, W.C.L. Nogueira, R.L. Santos, E.R. Martins, I.L. Azevedo and A.C. Almeida. Data curation: V.K.F.R. Santos, W.C.L. Nogueira, R.L. Santos, E.R. Martins and A.C. Almeida. Formal analysis: V.K.F.R. Santos, W.C.L. Nogueira, R.L. Santos, N.J.F. Oliveira, E.R. Martins, T.F. Carvalho and A.C. Almeida. Funding acquisition: E.R. Martins and A.C. Almeida. Investigation: V.K.F.R. Santos, W.C.L. Nogueira, R.L. Santos, N.J.F. Oliveira, E.R. Martins, I.L. Azevedo, T.F. Carvalho and A.C. Almeida. Methodology: V.K.F.R. Santos, W.C.L. Nogueira, R.L. Santos, N.J.F. Oliveira, E.R. Martins, I.L. Azevedo, T.F. Carvalho and A.C. Almeida. Project administration: E.R. Martins and A.C. Almeida. Resources: V.K.F.R. Santos, W.C.L. Nogueira, R.L. Santos, N.J.F. Oliveira, E.R. Martins, I.L. Azevedo, T.F. Carvalho and A.C. Almeida. Writing-original draft: V.K.F.R. Santos, W.C.L. Nogueira, R.L. Santos, N.J.F. Oliveira, E.R. Martins and A.C. Almeida. Writing-review & editing: W.C.L. Nogueira, R.L. Santos, N.J.F. Oliveira, E.R. Martins and A.C. Almeida.

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