Oregano essential oil composition

Compounds in the oregano oil were identified with a gas chromatographer (GC; Hewlett Packard P-6890, California, USA) attached to a mass spectrophotometer (MS; Hewlett Packard 7953, California, USA), using a capillary column (30 m long, 0.25 mm internal diameter, 0.25 µm film thickness; Hewlett Packard 5ms^®, California, USA). Injection port temperature was 240 °C. Initial temperature was 50 °C for 5 min, and was increased 10 °C per minute until reaching 260 °C. The carrier gas was helium. The MS was run in scan mode (m/z range: 30-550) with electronic ionization (70 eV) and a 1.0 ml/min flow rate.

Propolis flavonoids and phenols contents and antioxidant capacity

Flavonoids content was quantified following the aluminum chloride method, total phenols with the Folin-Ciocalteu method and antioxidant capacity with the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical method²⁰.

Experiment location

The evaluation was done at the Veterinary Medicine and Zootechny Academic Unit poultry farm of the Universidad de Nayarit (Compostela Municipality, Nayarit, Mexico).

Animals and experimental diets

Experimental animals were 480, one-d-old Ross line broilers randomly distributed in four treatments (C= control, P= 100 mg propolis/kg feed, A = 100 mg oregano oil/kg feed, PA= 50 mg propolis plus 50 mg oregano oil/kg feed) with four replicates of 30 chickens each per treatment. Starter and finishing diets were based on corn-soybean paste meal (Table 1) provided ad libitum for 42 d. The oregano oil and/or propolis were added during feed processing.

Productive parameters and blood samples

Productive parameters were measured every seven days, and mortality as it occurred. At d 21 and 42, blood samples were taken from the brachial vein of two birds per replicate and 1.8 mg ethylenediaminetetraacetic acid (EDTA) per ml added. These samples were dyed with Wright dye for blood metabolite measurement (Easy-Vet, Desego).

Meat samples

Meat samples were taken from two birds per replicate. After a 6 h fast, the animals were killed by severing the jugular vein and carotid artery following an established protocol (Norma Oficial Mexicana NOM-033-SAG/ZOO-2014)²¹. The carcass was drained of blood for 2 min, placed in water (60 °C) for 120 sec to allow for manual removal of the feathers, and cooled in ice water (0 °C) for one hour. The breast meat was removed from the carcass, the skin and any visible fat removed, and the meat stored in a vacuum and frozen (-20 °C) for approximately one month.

Meat oxidative stability

Oxidative stability was measured using a 30 g sample of meat and adding 30 ml distilled water with 0.2 mL 7% BHT (2,6-di-tert-butyl-4-methyl-phenol, Sigma-Aldrich, Toluca, Mexico) diluted in 96% methyl alcohol (CH₃CH₂OH). This mixture was liquified for 30 sec in a blender (Oster, M4655-813 / 465-42), filtered through a 0.84 mm plastic mesh and left to stand for 30 min at 25 °C in darkness. A 1 mL subsample was taken from the top layer and 2 ml 0.02 M thiobarbituric acid (Sigma-Aldrich, Toluca, Mexico) combined with 15% trichloroacetic acid (TCA) in distilled water added to it. The solution was stirred for 10 sec, placed in water at 80 °C for 10 min and finally at 0 °C for 10 min. Absorbance was measured at 532 nm (Biotek, Epoch, USA), and the resulting values multiplied by 7.8 to express mg malondialdehyde (MDA) per kilo of meat²².

Statistical analysis

The results were analyzed with a completely random design using the Generalized Linear Method (GLM) of the SAS statistics program. The means were compared with a Tukey test at a P≤0.05 significance level. Mortality results were transformed with an arc sine function and expressed as a percentage. The statistical model was:

Where:

Y_ij is feed intake, weight gain, feed conversion, mortality, leukocytes, chemical variables and MDA per kilogram of meat;

µ is the general mean;

Ƭ _i is the effect of the oregano oil, propolis and oregano oil plus propolis;

Ε _ij is the random error.

Oregano oil

The most abundant compounds in the oregano oil were thymol (43.47 %), carvacrol (29.16 %), eucalyptol (6.96 %), caryophyllene (5.38 %) and tetramethyl (2.96 %). It is the thymol and carvacrol contents of oregano oil that slow bacterial growth. Their concentrations can vary due to place of origin, harvest time and plant maturity, but they remain the most abundant compounds in all phenological stages of the oregano plant. These two compounds can account for as much as 80% of oregano oil^23,24. In the present study they accounted for 72 %, more than twice that reported elsewhere^25,26.

Propolis

Flavonoid content in the evaluated propolis was 5.6 mg quercetin equivalents/g propolis, phenols content was 840 µg caffeic acid equivalents/g propolis, and antioxidant capacity was 138 µg Trolox equivalents/g propolis. Biologically active compound contents can vary widely in propolis depending on source region; for example, flavonoid contents ranging from 8 to 188 mg/g propolis and phenols contents ranging from 42.9 to 329.0 mg/g propolis have been reported from China, India, Macedonia and Iran^10,24,25. Reported levels in propolis from Mexico are 379.2 mg flavonoids, 500.9 mg phenols and 54.4 mg Trolox equivalents per gram of propolis²⁰. Compared to the above values, the propolis analyzed here contained limited amounts of these active compounds.

Productive parameters

Inclusion of the oregano oil and/or propolis in the feed had no effect on feed intake, weight gain, feed conversion and mortality (Table 2). Apparently, the animals exhibited no quantifiable response because the inclusion levels were not high enough. Previous reports of productive performance in broilers in response to addition of oregano oil or propolis in poultry diets are variable. In two studies inclusion levels of 25, 50, 300 and 600 ppm propolis per kilogram of feed had no effect on broilers^27,28. In another study, only addition of 15,000 and 20,000 ppm oregano oil to boiler diets increased productive yields²⁹. In the present study the doses of propolis and/or oregano oil did not provide enough active compounds to stimulate digestive enzyme secretion, and the birds consequently showed no changes in productive parameters. Only high doses or larger amounts of active compounds can stimulate digestive enzyme secretion, allowing the birds to better exploit ingested nutrients and thus increase yield³⁰. A serious challenge when comparing studies of production performance in response to natural compounds is that most do not report the amount of active compounds contained in evaluated propolis or essential oils, precluding any comparisons.

Leukocytes

At three weeks, lymphocyte levels were lowest in the P treatment (P≤0.05), and eosinophils were highest in the AP treatment (P≤0.05). The C and A treatments were similar (Table 3). Lymphocytes support immune response when challenged by invading microorganisms. The present results coincide with previous studies in which lymphocyte levels in poultry decreased in response to addition of propolis to the diet because the active compounds in propolis inhibit development of T lymphocytes^31,32. In another study levels as low as 5 µg propolis per milliliter caused negative effects in vitro, due to the flavonoids content³³.

Eosinophil levels were two-fold higher in the AP treatment than in the P treatment. Eosinophils are cells linked to the development of T lymphocytes, the populations of which decrease as bird age increases³⁴. However, when development of the digestive system improves, eosinophils increase; broilers offered feed with added oregano oil or propolis have exhibited improved intestinal flora and stimulation of cytosines which induced eosinophil proliferation³⁵.

Blood metabolites

In six-week-old birds, triglycerides levels were highest in treatment A (P≤0.05) and lowest in AP, while C and P had intermediate values and did not differ between themselves (Table 4). Blood metabolite levels are indicative of general animal health. Addition of propolis (300 ppm) in broiler diets has been reported to lower cholesterol and triglyceride levels³⁶, although this response is inconsistent³⁷. For example, broilers fed diets containing 6% fat levels and added propolis exhibited low cholesterol and triglyceride levels³⁸, whereas in trials with lower energy levels no hypocholesterolemic or hypolipidemic effect was observed with addition of propolis or oregano oil to the diet.

Chicken breast oxidative stability

Oxidation in the breast meat was highest in the AP treatment (P≤0.05), with no difference between the A, P and C treatments (Table 5). Propolis and oregano oil are known to have antioxidant capacity in vitro and in vivo^16,39, but their effects may vary when mixed with other ingredients in a diet. For example, oregano oil combined with acidified soybean oil has no antioxidant effect³⁹, whereas addition of 200 ppm propolis is reported to decrease MDA content in chicken meat⁴⁰. The latter may be due to accumulation of the bioactive compounds in propolis in cell membranes, which could protect them from oxidation^33,36. Apparently, combining oregano oil with propolis accelerates the oxidative process in meat, but when administered independently they have no effect on MDA levels.