IntroductionTop

The ban on the use of antibiotics as performance-enhancing additives in livestock farming has forced producers to adapt to the new world demands concerning the production of animal protein. As a result, the poultry industry has sought alternative additives, especially natural products which work analogously to chemical growth promoters (Koiyama et al., 2014). Among these alternative additives are essential oils, secondary plant metabolites that act against external agents such as insects, fungi and pathogenic bacteria (Cho et al., 2014; Giannenas et al., 2014; Dantas et al., 2015).

In this scenario, the antimicrobial properties of Lippia gracilis Schauer essential oil (LGSEO) have been investigated due to their inhibitory effect on pathogenic microorganisms such as Escherichia coli, Salmonella sp. and Staphylococcus aureus (Horošová et al., 2006; Oliveira et al., 2007; Wang et al., 2016), as the essential oil contains high concentrations of terpenoids such as thymol and carvacrol. These compounds have pharmacological potential against gram-positive and gram-negative microorganisms by acting on the cell membrane, dispersing polypeptide chains that will be part of the cell membrane constitution (Lobo et al., 2011; Costa Júnior et al.,2016) and thereby reducing microbial growth (Bona et al., 2012).

Essential oils act on the intestinal microbiota, where they reduce pathogenic bacteria and consequently alter the intestinal morphology and influence nutrient absorption (Brenes & Roura, 2010; Behnamifar et al., 2015). When used as additives in quail diets, essential oils may affect feed intake, since they increase digestive enzymes secretions and intestinal motility (El-Hack et al., 2015), these associated factors may lead to change the intestinal epithelium paremnquima, improving bird’s immunity (Rocha et al., 2020). In addition, their antioxidant activity allows to mitigate thermal stress effects, due to the reduction in body and rectal temperatures of treated animals (Daader et al., 2018).

Recent studies have shown that dietary inclusion of Lippia javanica leaf meal improves performance, carcass characteristics, intestinal health and immune development in quail and chickens reared in free-range systems (Mpofu et al., 2016; Mnisi et al., 2017; Matshogo et al., 2018). However, there are few reports on the use of Lippia gracilis essential oil as a growth-promoting additive in quail diets and its potential as an inhibitor of pathogenic microorganism growth. It is important to emphasize that quails are more susceptible to enteropathogens than chickens, caused mainly by Salmonella sp. and E. coli bacteria (Dipineto et al., 2014), showing the need for studies to assess the prevalence of these bacterial strains in these production systems.

In view of the above considerations, the present study proposes to examine the effect of LGSEO as a growth promoter on the performance and organ development of Japanese quail up to 35 days of age as well as its potential as an antimicrobial agent against Salmonella sp. and E. coli bacteria.

Material and methodsTop

The experimental project was approved by the Ethics Committee on Animal Research of the Federal University of Sergipe (UFS) under the protocol no 09/2015.

Essential oil extraction

The plants used for the extraction of the LGSEO were cultivated at the Rural Campus of the UFS. Leaves of Lippia gracillis Schauer varieties LGRA 107 and 108 were used to obtain the oil. The oil was extracted by the hydrodistillation process using a Clevenger apparatus, following the methodology described by Silva et al. (2019). The oil yield was 3% and contains myrcene (2.49%), p-cymene (11.61%), 1.8-cineole (1.36%), ϒ-terpinene (11.17%), thymol methyl ether (5.07%), thymol (4.08%), carvacrol (45.17%) (Santos et al., 2016).

Antibiogram test

The antibiogram test was performed in accordance with Pereira et al. (2017). Salmonella sp. and E. coli strains belonging to the collection at the Laboratory of Bacteriology at the Department of Microbiology, Federal University of Sergipe, Brazil, were used.

The inocula were seeded on the surface of Müller-Hinton agar; 2.0 mL of saline solution with the inocula (3×10⁸ CFU inoculum per plate) were added and smears were performed using sterile swabs, covering the plate’s surface with inoculum. Ten cultured petri dishes were used, f ive for each inoculum (Salmonella sp. or E. coli). Six wells were drilled in each plate (6 mm) using the “hole plate” technique; the control solution, based on sodium hypochlorite at 0.9% to confirm bacterial growth, was deposited in only one of the wells; and at the other five wells 20 µL of diluted LGSEO for 2.5% of dimethyl sulfoxide (Rocha et al., 2020) were deposited. Finally, 25 repetitions were evaluated for each bacterial strain.

Lastly, the plates were incubated in a forced-air oven at a temperature of 37 ºC for 24 h for the first reading, which was repeated 48 h later to check for alterations in the inhibition halos. Readings were taken with a 150-mm digital caliper (Lee Tools).

Performance trial

A total of 504 female Japanese quail (Coturnix coturnix japonica) at an average initial weight of 6.80 ± 0.10 g was used in the study. The birds were evaluated in a completely randomized design with six treatments, namely, a diet with 500 mg/kg bacitracin methylene disalicylate (BMD) and diets with 0, 100, 200, 300 and 400 mg/kg of LGSEO. Seven replicates were performed, using 12 birds per experimental unit. The animals were housed in suspended cages (50 cm wide × 50 cm long × 40 cm high), equipped with feeders and pressure-type drinkers until the 14th day of age. After this period, the pressure-type drinkers were replaced with nipple drinkers. The floor under the cages was adapted and covered with reused wood shavings bedding to increase the health challenge and simulate a commercial farming environment.

In the rearing period of 2 to 14 days, the recorded maximum and minimum temperatures were 36 and 28ºC, respectively, and air relative humidity was 37%. From 14 to 35 days, the maximum and minimum temperatures were 34 and 24ºC, respectively, and air relative humidity was 46%.

The diets were formulated to meet the nutritional re-quirements of Japanese quail as recommended by the Bra-zilian Tables for Poultry and Swine (2011) in the growth phase (Table 1). LGSEO and the growth promoter (BMD) were added to the diets replacing the inert material kao-lin. The LGSEO was diluted in soybean oil prior to being mixed with the diets. Diets were prepared and stored in white buckets with lids for protection against sunlight. Water and feed were available ad libitum.

The chemical composition of the diets was determined at the Animal Nutrition Laboratory of the Animal Science Department (DZO/UFS) Sergipe, Brazil. Crude protein and minerals were analyzed following AOAC (2012) recommendations.

Table 1. Composition of the experimental diets.

^[1] aSoybean meal with 45% of crude protein. bDicalcium phosphate. c Minimum composition per kilogram: folic acid 150 mg, panto-thenic acid 6000 mg, biotin 40 mg, copper 1400 mg, iron 6000 mg, iodine 915 mg. manganese 17 g, niacin 13 g, selenium 300 mg, vitamin A 5000000 IU, vitamin B12 6500 mg, vitamin B2 2000 mg, vitamin B6 250 mg, vitamin D3 1600000 IU, vitamin E 4000 IU, vitamin K3 1000 mg, zinc 38 g. dKaolin. [2] ME: metabolizable energy. SID: standardized ileal digestibility (Brazilian Tables for Poultry and Swine, 2011). [3] LGSEO: Lippia gracilis Schauer essential oil. [4] BMD: Bacitracin methylene disalicylate.

Performance and organ development

Birds and experimental diets were weighed weekly to determine final body weight (g), weight gain (WG, g/bird), feed intake (FI, g/bird) and feed efficiency (FE, g/g).

At 35 days of age, two birds whose weight was closest to the average of the experimental unit were slaughtered by cervical displacement, to evaluate organ development. The proventriculus, gizzard, heart, liver, pancreas, intes-tines and abdominal fat were weighed on a digital scale (0.01-g precision). The length of the small intestine and ceca were measured using a tape measure. The relative weight of the organs was calculated in relation to the weight of the bird (Gul et al., 2018).

Quantitative microbiology

For the microbiological analysis of Salmonella sp. and E. coli counts, another two birds per experimental unit were slaughtered by cervical displacement. The small intestine’s content was harvested, and one pool was formed per experimental unit. The collected mate-rial was weighed and packed in Falcon tubes containing buffered saline solution (5.61 g NaCl, 1 g KH2PO4, 2 g Na₂PO₄ and 0.11 g KCl in 1000 mL distilled water) and 10% glycerol, which was used as a cryoprotectant agent, and frozen at –4 ºC. Subsequently, the samples were sent to the Laboratory of Bacteriobiology at UFS for analy-ses (Rocha et al., 2020).

Serial dilutions (10^-1, 10^-3, 10^-5 and 10^-7) were prepa-red with buffered saline solution. One 1-mL aliquot of each dilution was used for seeding in a Petri dish con-taining Rappaport Vassiliadis medium for Salmonella sp. growth and EC broth for E. coli growth. After the inoculum was spread with Drigalski’s spatula, the dishes were incubated in an oven at 37 ºC for 24 h and then a bacterial count was performed (CFU/g of feces) (Pereira et al. 2017).

The count was determined by the following formula (Rocha et al., 2020): Number of colony forming units × Mean serial dilution value.

Statistical analysis

The results of the antibiogram test and quantitative microbiology were analyzed descriptively. Performan-ce (WG, FI and FE) and organ-development variables were subjected to analysis of variance by the polyno-mial regression model procedure of SAS statistical sof-tware version 9.0 (2002), at the 5% significance level. Dunnett’s test was applied to compare the treatment con-taining BMD to the other treatments.

Table 2. In vitro inhibitory potential of Lippia gracilis Schauer essential oil (LGSEO) against Escherichia coli and Salmonella sp. bacteria.

Inhibition halo (in mm) against Escherichia coli and Salmonella sp. bac-teria from a solution of DSMO + LGSEO by the “hole plate” technique after 24-48 h of exposure at a temperature of 37 ºC. LGSEO: solution with LGSEO + 2.5% dimethyl sulfoxide. Control: solution with 0.9% sodium hypochlorite.