280

BULBUL (T.) AND COLLABORATORS

Effect of myrtle (Myrtus communis L.) oil on performance, egg quality, some biochemical values and hatchability in laying quails T. BULBUL1*, D. YESILBAG2, E. ULUTAS3, H. BIRICIK2, S. S. GEZEN2, A. BULBUL3 Department of Animal Nutrition and Nutritional Diseases, Faculty of Veterinary Medicine, Afyon Kocatepe University, 03200, Afyonkarahisar, TURKEY. Department of Animal Nutrition and Nutritional Diseases, Faculty of Veterinary Medicine, 16059, Uludag University, Bursa, TURKEY. 3 Department of Physiology, Faculty of Veterinary Medicine, 03200, Afyon Kocatepe University, Afyonkarahisar, TURKEY. 1 2

*Corresponding author: [email protected]

SUMMARY

RÉSUMÉ

The aim of this study was to evaluate the effect of myrtle (Mytrus communis L.) oil when added to the diet of laying quails on performance, egg quality, some biochemical values and hatchability. A total of 375 quails (250 females and 125 males; Coturnix coturnix japonica), aged eight weeks old, were randomly allocated to five dietary treatments (five replicates of ten females and five males). The birds were fed either a basal diet or the basal diet supplemented with myrtle oil at a dose of 500, 1000, 2000 or 5000 mg/kg feed. The experiment was conducted for eight weeks. At the end of the experiment, no significant differences were found among the groups in terms of initial live weight, feed consumption, egg weight, egg quality (fracture strength, haugh unit, shape index, yolk index, albumen index), serum biochemical values, hatchability, early embryonic death, late embryonic death and submembranous death. After 8 weeks, egg production was decreased (p< 0.01), whereas feed conversion rate (FCR) was increased (p< 0.05) in birds with a diet supplementation with myrtle oil doses of 5000 mg/kg. Eggshell thickness was decreased (p< 0.05) of groups receiving myrtle oil doses of 2000 and 5000 mg/kg. Yolk color index was affected positively by addition of myrtle oil (p< 0.001). Hatch performance was highest of the groups with diet supplemented with mytrle oil doses of 1000 mg/kg, whereas it was lowest of the groups received 5000 mg/kg mytrle oil (p< 0.01). The addition of myrtle oil to the diets caused significantly decrease in serum total cholesterol (p< 0.01), Ca (p< 0.01) and Malondialdehyde (MDA; p< 0.001) while determined significantly increase in blood urea (p< 0.001) and serum β-carotene (p< 0.001) concentrations. Serum creatine kinase-MB (CK-MB) was increased (p< 0.001) a dose of 5000 mg/kg, whereas albumin concentration decreased (p< 0.01). Egg yolk MDA concentration was decreased (p< 0.01) in all groups that received myrtle oil supplementation in their diets on days 15th and 30rd of storage (p< 0.001). In conclusion, mytrle oil supplementation was changed laying performance and biochemical values in laying quails depending on supplemented quantity and duration. It is recommended to supplement diets with 1000 mg/kg mytrle oil as egg production, egg quality, yolk MDA concentrations and hatching parameters were taken into consideration.

Effets de l’huile essentielle de myrte (Myrtus communis L.) sur les performances, la qualité de l’œuf, certaines valeurs biochimiques et l’éclosion chez les cailles de ponte

Keywords: Myrtle, performance, peroxidation, quails.

hatchability,

lipid

Le but de cette étude était d’évaluer l’effet de l’huile essentielle de myrte (Mytrus communis L.) sur les performances, la qualité de l’œuf, certaines valeurs biochimiques et l’éclosion lorsqu’elle est ajoutée à l’alimentation des cailles de ponte. Un total de 375 cailles (250 femelles et 125 males: Coturnix coturnix japonica), âgées de huit semaines, ont été réparties au hasard en cinq groupes homogènes (cinq réplicas de dix femelles et cinq mâles). Les animaux ont reçu une alimentation de base complétée par l’huile essentielle de myrte à des doses de 0, 500, 1000, 2000 et 5000 mg/kg d’aliments. L’expérience a été menée pendant huit semaines. A la fin de l’expérience, aucune différence significative n’a été perçue entre les groupes en termes de poids vif initial, de consommation d’aliments, du poids et la qualité de l’œuf, les valeurs biochimiques du sérum, l’éclosion, la mort précoce ou tardive de l’embryon et la mort sub-membraneuse. Après 8 semaines, la production d’œufs avait diminué (p< 0.01) alors que l’indice de consommation avait augmenté (p< 0.05) chez les cailles ayant reçu une alimentation complétée par la dose de 5000 mg/kg d’huile essentielle de myrte. L’épaisseur des coquilles avait diminué (p< 0.05) chez les groupes recevant des doses d’huile essentielle de myrte de 2000 et 5000 mg/kg. L’indice de la couleur jaune a été affecté positivement par l’ajout de l’huile essentielle de myrte (p< 0.001). La performance de l’éclosion était plus élevée chez les groupes ayant reçu une alimentation complétée avec des doses d’huile essentielle de myrte de 1000 mg/kg alors qu’elle était plus basse chez les groupes ayant reçu 5000 mg/kg (p< 0.01). L’ajout de l’huile essentielle de myrte à l’alimentation a significativement diminué le cholestérol (p< 0.01), Calcium (p< 0.01) et malondialdéhyde (MDA; p< 0.001) sérique et augmenté significativement les concentrations d’azote uréique (p< 0.001) et de β-carotène (p< 0.001). La créatine kinase sérique (CK-MB) a augmenté (p< 0.001) à la dose de 5000 mg/kg alors que la concentration d’albumine a diminué (p< 0.01). La concentration MDA du jaune d’œuf a diminué (p< 0.01) dans tous les groupes qui ont reçu un complément d’huile essentielle de myrte à leur alimentation aux 15ème et 30ème jours de conservation (p< 0.001). En conclusion, la complémentation par de l’huile essentielle de myrte a changé la performance de ponte et les valeurs biochimiques chez les cailles de ponte selon la quantité et la période d’apport. La prise en compte des paramètres de production et qualité des œufs, de concentration en MDA dans le jaune d’œuf et le taux d’éclosion conduisent à recommander de compléter l’alimentation des cailles pondeuse à la teneur de 1000 mg/kg d’huile essentielle de myrte.

Mots-clés : Myrte, performance, éclosion, peroxydation lipidique, cailles.

Introduction Adding aromatic plants or their extracts, separately or as a mixture, to animal diets has antibacterial [13, 44], antifungal [6, 47], anticoccidial [25], antioxidant [12] and antilipidemic

[19] effects. There are also growth promoting effects that are due to the antimicrobial properties and positive influence on the microflora of the digestive tract [32]. Thus, aromatic plants and their extracts are accepted as natural and safe substances for birds. Revue Méd. Vét., 2014, 165, 9-10, 280-288

USE OF MYRTLE OIL IN LAYING QUAILS DIET As an aromatic plant, Myrtus communis L. belongs to the Myrtaceae family. It is widely used in medicine and the pharmaceutical industry, because it contains volatile fatty acids (VFAs) and other compounds. Volatile fatty acids of M. communis L. include myrtenol, myrtenyl acetate, limonene, linalool, α-pinene, 1,8-cineole, β-caryophyllenein, p-cymene, geraniol, nerol, phenylpropanoid and methyleugenol [38]. It has been reported that derivates of some myrtus extracts, such as beta-triketones [30], tannens, myricetin, gallic acid and ellgic acid, have antibacterial effects and the plant has strong antioxidant activity due to its galloyl derivates [43]. Also, it has antifungal [23], hypoglycaemic [46], anticonvulsant [18], anticarcinogenic [15], anti-inflammatory [20] and antimutagenic [28] effects. Studies involving M. communis L. have focused mostly on its volatile components and the composition of phenolic compounds in leaves and fruit. However, no available scientific data were found about the utilisation of the plant’s versatile benefits in the diets of laying quails. The aim of this research was to investigate the effects on performance, egg quality, some biochemical values and hatchability of laying quails that received varying concentrations of myrtle oil added to their diets.

Ingredients

Corn Full fat soybean Sunflower meal Soybean meal Corn gluten Meat and bone meal Vegetable oil CaCO3 Dicalcium phosphate Salt NaHCO3 L-Lysine DL-Methionine Vitamin mineral premixa Chemical composition Dry matter % Crude protein % Crude fat % Crude fiber % Crude ash % Nitrogen free extract % Calcium % Phosphorus % Metabolisable energyb (MJ/kg)

281

Material and Methods ANIMALS, DIETS AND EXPERIMENTAL DESIGN This experimental study was carried out in The Avian Research Farm at the Animal Research Center of Afyon Kocatepe University, Turkey, following the ethical committee approval. A total of 375 quails (250 females and 125 males; Coturnix coturnix Japonica), aged eight weeks old, were randomly allocated to receive one of five dietary treatments. The birds were fed either a basal diet or the basal diet with supplemented with myrtle oil doses of 500, 1000, 2000 or 5000 mg/kg feed. Each treatment consisted of five replicates of ten females and five males. The birds were housed in cages kept inside a windowed poultry house with a light regimen of 16 hours light and 8 hours dark. Feed and water were provided ad libitum. The experiment was conducted for duration of eight weeks. The content of the basal diet is shown in Table I. The basal diet was formulated to meet or slightly exceed the nutrient requirements recommended by the NATIONAL RESEARCH COUNCIL [37], and contained 12.15 MJ/kg Metabolisable Energy (ME) and 21.68% crude protein. The diet was free of antibiotics, coccidiostats and growth promoters. The nutrient composition of the basal diet, including moisture, crude protein, crude fat, crude fiber, crude ash, calcium and phosphorus contents was

% 47.23 25.00 9.32 6.20 0.60 4.00 1.00 4.60 0.40 0.25 0.25 0.10 0.70 0.35 92.16 21.68 8.06 4.29 6.32 51.81 2.55 0.40 12.15

a Added per kg of diet: retinol acetate, 4.1 mg; cholecalciferol, 0.075 mg; DL-α-tocopherylacetate, 11 mg; menadione, 1 mg; thiamin, 2.8 mg; riboflavin sodium phosphate, 5.8 mg; niacin, 44 mg, pyridoxine, 4 mg; cyanocobalamin, 0.026 mg; Ca-D-pantothenate, 8.8 mg; folic acid, 1 mg; choline, 220; biotin, 0.11 mg; Cu, 6 mg; I, 1.1 mg; Fe, 30 mg; Se, 0.1 mg; Mn, 60 mg; Zn, 50 mg b Metabolisable energy content of diets was estimated using the equation devised by Carpenter and Clegg [33]

Table I: Ingredients and chemical composition of the basal diet Revue Méd. Vét., 2014, 165, 9-10, 280-288

282 determined according to the AOAC [4]. Metabolisable energy of each diet was estimated using the following equation devised by Carpenter and Clegg [33]: ME, kcal/ kg = 53 + 38 [(crude protein, %) + (2.25 x crude fat, %) + (1.1 x starch, %) + (sugar, %)]. Group feeding was applied for all treatment groups. Moreover, specific gravity values were considered when calculating the amount of myrtle oil to add to the diets. The specific gravity value of myrtle oil (0.8897 g/mL) was determined by the suppliers (NBT Company, Alanya, Turkey). The essential oils were extracted by hydrodistillation. Hydrodistillation is a simple form of steam distillation. High pressure steam is forced through crushed plant, picking up the oil. The vapor containing the oil is then condensed, producing a liquid containing a mixture of water and the plant oil. The oil is then separated from the water [7]. Then the composition of myrtle oil was determined by GS-MS. Mytrle oil was added to a small portion of the diet and then sprayed on to the total diet. All diets were prepared freshly every week.

LAYING PERFORMANCE Quails were weighed individually at the beginning and end of the study. Eggs were collected daily and egg production was calculated on % production for egg numbers. Egg weight was recorded daily for each replicate. Feed consumption was recorded every two weeks. The feed conversion rate (FCR) was calculated as kilograms of feed per kilogram of eggs.

EGG QUALITY TRAITS Twenty eggs from each group (five eggs from each replicate) were collected on the second, fourth, sixth and eighth weeks of the experiment to determine the quality of the interior and exterior of the eggs at monthly intervals. Egg quality traits were measured within 24 hours of collection. The eggs were examined for weight (g), length (mm), width (mm), egg shell thickness (mm), fracture strength, albumen index (%), yolk index (%), Haugh Unit (HU), egg shape index and yolk color index. Egg weights were measured to the nearest 0.1 g using a digital scale. Egg width, egg length, yolk width, albumen length and albumen width were measured by digital compass to the nearest 0.01 mm. Measurements of the shell thickness of dried shells with the membrane still intact were obtained from two sides in the equatorial region, as well as on the blunt and the pointed edges with a micrometer to the nearest 0.01 mm. Also, yolk and albumen heights were measured by micrometer to the nearest 0.01 mm. The egg yolk visual color score was determined by matching the yolk with one of the 15 bands of the “1961, Roch Improved Yolk Color Fan”. Egg quality traits were calculated using the following formulas [27,54]: Shape index (%) = [egg width (mm)/egg length (mm)] x 100 Yolk index (%) = [yolk height (mm)/yolk width (mm)] x 100 Haugh Unit = 100 log [albumen height (mm) + 7.57 - 1.7 0.37 x egg weight (g)]

BULBUL (T.) AND COLLABORATORS Albumen index (%) = albumen height (mm)/[(average albumen length (mm) + width (mm) /2) x 100]

DETERMINATION OF SERUM BIOCHEMICAL VALUES At the end of experimental period , blood specimens collected from two animals per replicate, from a total of 10 animals, into dry tubes were centrifuged 1500 g for 15 minutes at 4°C. Then, the sera were stored at -18 ˚C for further analysis. The serum concentrations of calcium (Ca; limit of quantitation, 0.50 mmol/L; intra- and interassay coefficients of variation, 0.7% and 2.1%), alkaline phosphatase (ALP; sensitivity of assay, 1 U/L; intra- and interassay coefficients of variation, 0.8% and 2.9%), alanine aminotransferase (ALT; sensitivity of assay, 1 U/L; intra- and interassay coefficients of variation, 0.8% and 1.1%), aspartate aminotransferase (AST; sensitivity of assay, 1 U/L; intra- and interassay coefficients of variation, 0.7% and 2.9%), creatinine (sensitivity of assay, 1.52 µmol/L; intra- and interassay coefficients of variation, 1.1% and 1.6%), urea (sensitivity of assay, 0.357 mmol/L; intra- and interassay coefficients of variation, 1% and 1.6%), creatine kinase - MB (CK-MB; sensitivity of assay, 10 U/L; intra- and interassay coefficients of variation, 1.9% and 3.6%), albumin (sensitivity of assay, 0.2 g/L; intra- and interassay coefficients of variation, 1.5% and 3.6%, total cholesterol (sensitivity of assay, 0.0259 mmol/L; intra- and interassay coefficients of variation, 1.6% and 2.6%), high-density lipoprotein cholesterol (HDL-C; sensitivity of assay, 0.0259 mmol/L; intra- and interassay coefficients of variation, 1.7% and 2.2%) and low-density lipoprotein cholesterol (LDL-C; sensitivity of assay, 0.0259 mmol/L; intra- and interassay coefficients of variation, 1.5% and 2.2%) (Cormay, Poland) concentrations using autoanalyser (Tokyo Boeki Prestige 24i, Japan) [29,34,45]. Serum MDA [17] and β-carotene concentrations were determined using the method of [51] were determined with enzyme-linked immunosorbent assays and a spectrophotometric reader (MWGt Lambda Scan 200, Bio-Tek Instruments, USA). Precision of the assay was assured by use of a quality control sample, which was included on each plate.

ANALYZING EGGS CONCENTRATIONS

MALONDIALDEHYDE

(MDA)

Malondialdehyde was measured as a secondary oxidation product according to the TBA method described by [31] using spectrophotometry with some modifications. At the end of the experimental period, 100 egg yolk specimens (20 egg yolk specimens from each group) were tested. The lipid oxidation value of raw egg yolk specimens stored at +4oC was determined at 1., 7., 15., and 30. days of storage. A modified 2-thiobarbituric acid method was used, and the results were expressed as the amount of 2-thiobarbituric acid reactive substances (mg MDA). This method is based on observing the red color that is created by the oxidation of unsaturated fatty acids with thiobarbituric acid (TBA) after heating MDA. For the analyses, 10 g of the specimen was homogenized with distilled water in a blender and then transferred to a Kjeldahl Revue Méd. Vét., 2014, 165, 9-10, 280-288

USE OF MYRTLE OIL IN LAYING QUAILS DIET

283

flask, where the specimen was distilled to aggregation by adding 2.5 mL of 4 N HCl (Merck, Germany) and 1 mL of Antifoam A. The reactant, 5 mL of TBA (Merck, Germany), was added to 5 mL distillate and incubated in a water bath for up to 30 min. The final solution and a blank were measured in a spectrophotometer at 538 nm. The final value was expressed as mg MDA/kg specimen (yolk).

Results

DETERMINATION OF HATCHABILITY

It was observed that addition of 500 to 2000 mg/kg of M. communis L. oil to the diets had no effect on final LW; however, supplementation of 5000 mg/kg reduced the final LW in both male and female quails (p< 0.05). M. communis L. oil added diets did not altered feed consumption of the groups. Supplementation of 5000 mg/kg M. communis L. oil reduced egg production in whole study except second week (p< 0.001). However, supplementation of 1000 mg/kg M. communis L. oil to the diets increased egg production in entire study. Supplementation of 5000 mg/kg M. communis L. oil to the diets had negative effects on FCR during whole study although there was no difference in egg weight between groups (Table III).

A total of 160 eggs were collected per group (32 eggs from each replicate) at weeks four and eight of the study. The eggs that had suitable characteristics for brooding were placed in a brooder on a group basis. The number of hatched chicks was recorded for three days after the 17 days of brooding. Then, the remaining non-hatched eggs were cracked, and the number of fertile and unfertile eggs and number of embryonic deaths were recorded. The hatchability characteristics were calculated using the following formulas: Fertility rate (%) = (fertile egg count / hatched egg count) x 100 Hatching performance (%) = (number of eggs hatched out / hatched egg count) x 100 Hatchability (%) = (number of eggs hatched out / hatched fertile egg count) x 100 Fertility checks were performed during the early (less than six days) and late (7 to 15 days) periods. Embryonic and submembranous deaths at 16 to 17 days plus those that died after egg perforation were determined by breaking the egg shell of unhatched eggs at the end of the hatching period.

STATISTICAL ANALYSIS The effect of different treatment doses of myrtle oil on laying performance, egg quality traits, serum biochemical values and egg yolk MDA concentration were subjected to ANOVA procedures appropriate for a completely randomized design. All replicates were the experimental unit for all analysis. When differences (p< 0.05) among means were found, means were separated using Tukey test. A Chi square test was used to evaluate hatching properties. The values were given in median and range.

Components α-pinene 1,8-cineole Limonene Linalool α-terpineol/ α-terpinyl acetate Linalyl acetate Geraniol β-caryophylle Table II: Chemical composition (%) of the volatile oil of Myrtus communis L. Revue Méd. Vét., 2014, 165, 9-10, 280-288

The ingredients and chemical composition of the diets are presented in Table I. The volatile oil composition of myrtle oil is summarized in Table II. The main active components of myrtle oil are α-pinene (31.2%), 1,8-cineole (24.2%) and limonene (13.8%).

During eighth week of experiment, it was found that eggshell thickness was 35,23 mm in control group, whereas the groups supplemented with 500, 1000, 2000 and 5000 mg/kg/day myrtle oil had 34.55, 33,78, 31.30 and 32.21 mm thickness, respectively. Furthermore, yolk color index in control and experimental groups (500, 1000, 2000 and 5000 mg/kg/day) were 6.1 and 7.80, 8.40, 7.80, 8.20, respectively. However, diet supplemented with myrtle oil doses of 2000 and 5000 mg/kg were decreased eggshell thickness (p< 0.05). But addition of myrtle oil to laying quail diets increased yolk color index at eight weeks. Other egg quality traits (fracture strength, haugh unit, shape index, yolk index and albumin index) were not affected by myrtle oil addition to the diets. Biochemical results obtained during the study are shown in Table IV and egg yolk MDA concentrations are shown in Table V. As presented, the addition of myrtle oil to the diets decreased serum Ca concentrations (p< 0.01), whereas it increased urea (p< 0.001) and β-carotene (p< 0.001) concentrations in all groups. Supplementation with 5000 mg/kg myrtle oil was increased CK-MB activity (p< 0.001) and decreased albumin concentration (p< 0.05); during all doses (500-5000 mg/kg) of myrtle oil supplementation, % 31.2 24.2 13.8 8.8 4.9 3.9 1.4 1.0

187.3 (180.2-193.4) 172.1 (168.1-176.3) 209.9 (204.7-216.4)a 193.36 (185.5-193.3)a 31.32 (28,34-32,91) 84.57(77.12-89.14)ab 2.72 (2.62-2.80)b 13.68 (13.06-13.78)

188.4 (181.1-195.6) 175.3 (165.9-178.5)

210.4 (203.9-211.0)a 190.2 (183.3-197.8)a 31,44 (27.44-33.12) 83.14 (80.12-86.91)c 2.78 (2.70-2.89)b 13.60 (12.80-13.87)

204.8 (202.1-213.2)ab 186.7 (183.0-192.0)ab 31.93 (27.11-33.78) 84.21 (81.45-85.88)bc 2.81 (2.70-2.84)ab 13.46 (13.10-13.98)

188.3 (182.5-194.5) 168.12 (165.9-174.5)

2000

Dietary myrtle oil supplementation (mg/kg/day) 500 1000 2000 b b 3060 (2164-3412) 3412 (2345-3821) 2711 (2043-3567)b b a 33.40 (24.00-42.11) 47.16 (23.44-61.23) 45.10 (33.21-58.12)a a a 18.8 (16.4-19.7) 18.6 (17.2-20.2) 17.1 (16.0-18.3)ab b b 9.87 (9.30-10.26) 10.11 (8.50-10.16) 8.32 (7.39-10.08)b 2.25 (1.86-3.57)cb 2.44 (1.69-3.22)b 3.76 (2.64-4.91)a b ab 1.79 (1.53-1.91) 2.45 (2.15-4.11) 3.13 (1.62-3.48)a b b 5.55 (4.82-6.53) 6.00 (4.80-7.20) 4.70 (4.10-7.35)b a a 0.313 (0.268-0.387) 0.295 (0.281-0.299) 0.351 (0.261-0.387)a 2.87 (2.64- 3.36)b 2.54 (2.24-2.93)bc 2.56 (2.21-2.96)bc

Table IV: Effects of Myrtus communis L. oil diets on some serum biochemical values

CK-MB: creatine kinase-MB, HDL-C: High-density lipoprotein cholesterol, LDL-C: Low-density lipoprotein cholesterol, MDA: Malondialdehyde. Letters (a, b,c) in the same line indicate significant differences between different letters, NS = not significant

CK-MB (U/L) Urea (mmol/L) Albumin (g/L) Total cholesterol (mmol//L) HDL-C (mmol/L) LDL-C (mmol//L) Ca (mmol/L) β-carotene (µmol/L) MDA (µmol/L)

0 2667 (2123-3212)b 16.30 (12.18-21.13)c 19.3 (16.5-19.8)a 10.62 (9.30-14.97)a 1.39 (0.46-2.07)c 1.86 (1.53-2.09)b 7.67 (6.80-8.52)a 0.245 (0.207-0.262)b 3.56 (3.21-4.95)a

Table III: Effects of Myrtus communis L. oil diets on laying performance

207.5 (198.6-214.3)ab 190.4(185.1-193.3)a 33.04 (29,88-35.78) 89.03 (84.62-93.56)a 2.78 (2.64-2.86)b 13.32 (13.04-13,82)

187.1 (179.8-193.2) 172.5 (163.4-178.1)

Dietary myrtle oil supplementation (mg/kg/day) 500 1000

FCR: Food conversion ratio (feed consumed, (g)/egg production (g). Letters (a, b, c, d) in the same line indicate significant differences between different letters. NS = not significant

Initial live weights, g Female Male Final live weights, g Female Male Feed consumption, g/day Egg production, % FCR, Egg weight, g

0

5000 5413 (4213-6798)a 54.50 (32.45-65.72)a 15.0 (14.0-15.8)b 9.38 (7.44-10.65)b 3.10 (2.53-4.08)b 2.93 (2.56-3.31)a 5.28 (4.94-5.57)b 0.369 (0.294-0.396)a 2.11 (1.88-2.82)c

199.3 (193.8-205.02)b 184.8 (173.1-189)b 30.78 (28.11-33.34) 76.70(70.84-80.56)d 3.02 (2.89-3.10)a 13.48 (13.23-14.12)

188.7 (183.1-196.7) 172.77 (163.4-178.3)

5000

p< 0.001 0.001 0.01 0.01 0.001 0.01 0.01 0.001 0.001

0.05 0.05 NS 0.01 0.05 NS

NS NS

p
0.05)

1st day 7th day 15th day 30th day

0 0.08 (0.05-0.14)a 0.13 (0.09-0.23)a 0.24 (0.14-0.32)a 0.61 (0.30-0.78)a

differences in serum indicators originating from the liver (ALP, ALT, AST) in this study. Enzyme CK has high activity in skeletal muscles and in the myocardium. The isoenzyme CKMB is present in many tissues, mostly in the myocardium. CK is more active in skeletal muscles than in myocardium. An increase in serum CK and CK-MB activities is generally linked to defects in skeletal muscles and myocardium [29,52]. Elevated concentrations of CK-MB and urea indicate the presence of muscle tissue degeneration as well [29]. In the present study, CK-MB (p< 0.001), urea (p< 0.01) activities were raised, whereas albumin concentration decreased (p< 0.05, Table IV) in the groups fed a diet supplemented with 5000 mg/kg myrtle oil showing that myrtle oil may be cause muscle degeneration. Also, decreased LW in these groups was in accordance with biochemical results. In the present study, it was observed that yolk MDA concentrations were decreased on days 15th and 30rd in all myrtle groups (p< 0.001). It was reported that the antioxidant effects of aromatic plants are due to their phenolic compounds [48]. Phenolic compounds act as antioxidants by trapping free radicals, which reduces singlet oxygen formation by making compounds with metallic ions [42]. Also, 1.8-cineole α-pinen and β-pinenin, which are some major components of myrtle oil, are known to have antioxidant activity [53]. Similarly, antioxidant characteristic of myrtle oil was also reported [2]. Due to the presence of phenolic OH groups; these groups act as hydrogen donors to the peroxy radicals produced in the first step in lipid oxidation, thus retarding the formation of hydroxyl peroxide. In the present study, it was observed that the addition of 1000 mg/kg M. communis L. oil to the diet increased the hatch performance statistically at eight weeks (p< 0.01). ALI et al. [3] reported that individually added 1% thyme, 1% rosemary, 1% oregano and 0.5 to 1% curcuma longa to chicken diets increased fertility and hatchability. Oxidative metabolism increased, especially in the final few days before hatching, as a normal result of embryonic growth. It is reported that over increment lipid peroxidation may lead to tissue damage [49], whereas diets with added antioxidants may protect the embryo and therefore increase survival rate [50]. As a conclusion, this study suggested that myrtle oil supplementation, especially at a concentration of 500 and 1000 mg/kg may be considered a potential natural growth promoter. However, more studies are needed to define the

Dietary myrtle oil supplementation (mg/kg/day) 500 1000 2000 0.04 (0.031-0.09)b 0.07 (0.06-0.14)a 0.08 (0.07-0.12)a 0.13 (0.08-0.19)a 0.07 (0.05-0.12)b 0.11 (0.06-0.16)a b c 0.14 (0.09-0.28) 0.10 (0.07-0.16) 0.08 (0.05-0.13)c b b 0.21 (0.16-0.27) 0.21 (0.13-0.28) 0.11 (0.06-0.30)b

5000 0.08 (0.03-0.13)a 0.05 (0.09-0.15)b 0.09 (0.05-0.15)c 0.15 (0.09-0.26)b

p< NS 0.01 0.01 0.001

Letters (a, b) in the same line indicate significant differences between different letters Table V: Effects of Myrtus communis L. oil diets on egg yolk MDA concentration (mg MDA /kg specimen) in raw egg specimens at different storage times (at +4°C) Revue Méd. Vét., 2014, 165, 9-10, 280-288

USE OF MYRTLE OIL IN LAYING QUAILS DIET effect of myrtle oil supplementation on the performance of poultry with regard to environmental conditions, effective dosage, active oil substances, dietary ingredients and nutrient density. Furthermore, this study indicated that the use of myrtle oil as a natural antioxidant may improve poultry egg quality and may extend the shelf life of poultry egg products.

References 1. -  ABD EL MOTAAL A.M., AHMED A.M.H., BAHAKAIM A.S.A., FATHI M.M.: Productive performance and immunocompetence of commercial laying hens given diets supplemented with Eucalyptus. Int. J. Poult. Sci., 2006, 7, 445-449. 2. -  ALAMANNI M.C., COSSU M.: Radical scavenging activity and antioxidant activity of liquors of myrtle (Myrtus communis L.) berries and leaves. Ital. J. Food Sci., 2004, 2, 197-208. 3. -  ALI M.N., HASSAN M.S., ABD EL-GHANY F.A.: Effect of strain, type of natural antioxidant and sulphate ion on productive, physiological and hatching performance of native laying hens. Int. J. Poult. Sci., 2007, 6, 539-554. 4. -  A.O.A.C.: Official methods of analysis. 17th ed. Association of Official Analytical Chemists, 2000, Maryland. USA. 5. -  ATTIA Y.A., EL-DIN A.E.E.T., ZEWEIL H.S., HUSSAIN A.S., QOTA E.M.S., ARFAT M.A.: The effect of supplementation of enzyme on laying and reproductive performance in Japanese quail hens fed Nigella sativa. J. Poult. Sci., 2008, 45, 110-115. 6. -  BANG K.H., LEE D.W., PARK H.M., RHEE Y.H.: Inhibition of fungal cell wall synthesizing enzymes by trans-cinnamaldehyde. Biosci. Biotechnol. Biochem., 2000, 64, 1061–1063. 7. -  BIRICIK H., YESILBAG D., GEZEN S.S., BULBUL T.: Effects of dietary myrtle oil (Myrtus communis L.) supplementation on growth performance, meat oxidative stability, meat quality and erythrocyte parameters in quails. Revue Med. Vet., 2012, 3, 131-138. 8. -  BOLUKBASI S.C., KUDDUSI-ERHAN M., URUSAN U.: The effects of supplementation of bergamot oil (Citrus bergamia) on egg production, egg quality, fatty acid composition of egg yolk in laying hens. J. Poult. Sci., 2010, 47, 163-169. 9. -  BOLUKBASI S.C., URUSAN H., KUDDUSI-ERHAN M., KIZILTUNC, A.: Effect of dietary supplementation with bergamot oil (Citrus bergamia) on performance and serum metabolic profile of hens, egg quality and yolk fatty acid composition during the late laying period. Arch. Geflügelkun., 2010, 74, 172-177. 10. -  BORTOLOTTI G.R., NEGRO J.J., SURAI P.F., PRIETO P.: Carotenoids in eggs and plasma of red-legged partridges: Effects of diet and reproductive output. Physiol. Biochem. Zool., 2003, 76, 367-374. 11. -  BOTSOGLOU N., FLOROU-PANERI P., BOTSOGLOU E., DOTAS V., GIANNENAS I., KOIDIS A., MITRAKOS P.: The effect of feeding rosemary, oregano, saffron and α-tocopheryl acetate on hen performance and oxidative stability of eggs. S. Afr. J. Anim. Sci., 2005, 35, 143-151. Revue Méd. Vét., 2014, 165, 9-10, 280-288

287 12. -  BOTSOGLOU N.A., FLOROU-PANER P., CHRISTAKI E., FLETOURIS D.J., SPAIS A.B.: Effect of dietary oregano essential oil on performance of chickens and on iron-induced lipid oxidation of breast, thigh and abdominal fat tissues. Br. Poult. Sci., 2002, 43, 223-230. 13. -  BOUZOUITA N.A., KOCHOURI F., HAMDI M., CHAABOUNI M.M.: Antimicrobial activity of essential oils from Tunisian aromatic plants. Flavour Fragr. J., 2003, 18, 380-383. 14. -  CABUK M., BOZKURT M., ALCICEK A., CATLI A.U., BASER K.H.C.: Effect of a dietary essential oil mixture on performance of laying hens in the summer season. S. Afr. J. Anim. Sci., 2006, 36, 215-221. 15. -  CASSADY J.M., SUFFNESS M.: Terpenoid antitumor agents. In: Cassady JM, Douros JD (eds.), Anticancer agents based on natural product models. Academic Press., 1980, 201-269. 16. -  DOLDER S., HOFSTETTER W., WETTERWALD A., MUHLBAUER R.C., FELIX R.: Effect of monoterpenes on the formation and activation of osteoclasts in vitro. J. Bone Miner. Res., 2006, 21, 647-655. 17. -  DRAPER H.H., MCGIRR L.G., HADLEY M.: The metabolism of malondialdehyde. Lipids. 1986, 21, 305307. 18. -  ELISHA E.E., MOUSSA S.O., ABED S.K.: Effects of the leaves of Myrtus communis on the CNS I: The central depressant and anticonvulsant actions. J. Biol. Sci., 1988, 19, 545-560. 19. -  FARAH K.: Effect of coriander seeds as diet ingredient on blood parameters of broiler chicks raised under high ambient temperature. Int. J. Poult. Sci., 2011, 10, 82-86. 20. -  FEISST C., FRANKE L., APPENDINO G., WERZ O.: Identification of molecular targets of the oligomeric nonprenylated acylphloroglucinols from Myrtus communis and their implication as anti-inflammatory compounds. J. Pharmacol. Exp. Therapeut., 2005, 315, 389-396. 21. -  FLAMINI G., CIONIL P.L., MORELLI I., MACCIONI S., BALDINI R.: Phytochemical typologies in some populations of Myrtus communis L. on caprione Promontory (East Liguria, Italy). Food Chem., 2004, 85, 599-604. 22. -  FLOROU-PANERI P., NIKOLAKAKIS I., GIANNENAS I., KOIDIS A., BOTSOGLOU E., DOTAS V., MITSOPOULOS I.: Hen performance and egg quality as affected by dietary oregano essential oil and tocopheryl acetate supplementation. Int. J. Poult. Sci., 2005, 7, 449454. 23. -  GARG S.C., DENGRE S.L.: Antifungal activity of the essential oil of Myrtus-communis-var-microphylla. Herba Hungarica., 1988, 27, 123-126. 24. -  GHASEMI R., ZAREI M., TORKI M.: Adding dedicinal herbs including garlic (Allium sativum) and thyme (Thymus vulgaris) to diet of laying hens and evaluating productive performance and egg quality characteristics. Am. J. Anim. Vet. Sci., 2010, 5, 151-154. 25. -  GIANNENAS I., FOROU-PANERI P., PAPAZAHARIADOU M., CHERISTAKI E., BOTSOGLOU N.A., SPAIS A.B.: Effect of dietary supplementation

288 with oregano essential oil on performance of broilers after experimental infection with Eimeria tenel. Br. Poult. Sci., 2003, 57, 99-106. 26. -  GOODWIN T.W.: Nature and distribution of carotenoids. Food Chem., 1980, 5, 3-13. 27. -  HAUGH R.: The Haugh unit for measuring egg quality. U.S. Egg Poult. Magazine., 1937, 43, 552-555. 28. -  HAYDER, N., SKANDRANI, I., KILANI, S., BOUHLEL, I., ABDELWAHED, A., BEN, AMMAR, R., MAHMOUD, A., GHEDIRA, K., CHEKIR-GHEDIRA, L.: Antimutagenic activity of Myrtus communis L., using Salmonella microsome assay. J. S. Afr. Bot., 2008, 74, 121-125. 29. -  HOCHLEITNER M.: Biochemistries. In: B.W. Ritchie, G.S. Harrison, L.R. Harrison (éd.): Avian medicine: Principles and application, Winger publishing, Florida, 1994, 223-246. 30. -  KASHMAN Y., ROTSTEIN A., LIFSHITZ A.: The structure determination of two new acylphloroglucinols from Myrtus communis. Tetrahedron., 1974, 30, 991997. 31. -  KEP J., ACKMAN P.J., LINKE B.H., NASH D.M.: Differential lipid oxidation in various parts of frozen mackerel. Int. J. Food Sci. Tech., 1977, 12, 37-47. 32. -  LEE K.W., EVERTS H., KAPPERT H.J., FREHNER M., LOSA R., BEYNEN A.C.: Effects of dietary essential oil components on growth performance, digestive enzymes and lipid metabolism in female broiler chickens. Br. Poult. Sci., 2003, 44, 450-457. 33. -  LEESON S., SUMMERS J.D.: Nutrition of the chicken. 4th ed. University Books, Guelph, 2001. 34. -  MAHMOUD H. M., HAGGAG A. M. H., EL-GEBALY H.S.: Toxicological studies of malathion on Japanese quail (Coturnix Japonica). Life Sci., 2012, 9, 1725-1732. 35. -  MIMICA-DUKIC N., BUGARIN D., GRBOVIC S., MITIC-CULAFIC D., VUKOVIC-GACIC B., ORCIC D., JOVIN W., COULADIS M.: Essential oil of Myrtus communis L. as a potential antioxidant and antimutagenic agents. Molecules., 2010, 15, 2759-2770. 36. -  MOHEBBIFAR A., TORKI M.: Effects of adding mixed powder of garlic and thyme to diets included graded levels of rice bran on productive performance of laying hens and egg quality characteristics. Adv. Environ. Biol., 2010, 4, 469-476. 37. -  NATIONAL RESEARCH COUNCIL.: Nutrient requirements of poultry. 9th ed. National Academy Press, Washington, 1994. 38. -  OZEK T., DEMIRCI B., BASER K.H.C.: Chemical composition of Turkish myrtle oil. J. Essent. Oil Res., 2000, 12, 541-544. 39. -  QURESHI A.A., MANGELS W.R., DIN Z.Z., ELSON C.E.: Inhibition of hepatic mevalonate biosynthesis by the monoterpene, d-limonene. J. Agr. Food Chem., 1988, 36, 1220-1224. 40. -  RADWAN-NADIA L., HASSAN R.A., QOTA E.M., FAYEK H.M.: Effect of natural antioxidant on oxidative stability of eggs and productive and reproductive performance of laying hens. Int. J. Poult. Sci., 2008, 7, 134-150.

BULBUL (T.) AND COLLABORATORS 41. -  RASOOLI I., MOOSAVI M.L., REZAEE M.B., JAIMAND K.: Susceptibility of microorganisms to Myrtus communis L. essential oil and its chemical composition. J. Agr. Sci. Tech., 2002, 4, 127-133. 42. -  RICE-EVANS C.A., MILLER J., PAGANGA G.: Antioxidant properties of phenolic compounds. Trends Plant Sci., 1997, 2, 152-159. 43. -  ROMANI A., COINU R., CARTA S., PINELLI P.C., GALARDI C., VINCIERI F.F., FRANCONI F.:. Evaluation of antioxidant effect of different extracts of Myrtus communis L. Free Radic. Res., 2004, 38, 97-103. 44. -  SARICA S., CIFTCI A., DEMIR E., KILINÇ K., YILDIRIM Y.: Use of an antibiotic growth prometer and two herbal natural feed additives with and without exogenous enzymes in wheat based broiler diets. S. Afr. J. Anim. Sci., 2005, 35, 61-72. 45. -  SCHOLTZ N., HALLE I., FLACHOWSKY G., Sauerwein H.: Serum chemistry reference values in adult Japanese quail (Coturnix coturnix japonica) including sex-related differences. Poult. Sci., 2009, 88, 1186-1190. 46. -  SEPICI A., GURBUZ I., CEVIK C., YESILADA E.: Hypoglycaemic effects of myrtle oil in normal and alloxan-diabetic rabbits. J. Ethnopharmacol., 2004, 93, 311-318. 47. -  SHIN S., LIM S.: Antifungal effects of herbal essential oils alone and in combination with ketaconazole against Trichophyton spp. J. Appl. Microbiol., 2004, 97, 12891296. 48. -  SKERGET M., KOTNIK P., HADOLIN M., HRAS A.R., SIMONIC M., KNEZ Z.: Phenols, proanthocyanidins, flavones and flavonols in some plant materials and their antioxidant activities. Food Chem., 2005, 89, 191-198. 49. -  SURAI P.F.: Tissue-specific changes in the activities of antioxidant enzymes during the development of the chicken embryo. Br. Poult. Sci., 1999, 40, 397-405. 50. -  SURAI P.F., SPARKS N.H.C.: Comparative evaluation of the effect of two maternal diets on fatty acid, vitamin E and carotenoid in the chick embryo. Br. Poult. Sci., 2001, 42, 252-259. 51. -  SUZIKI J., KATOH N.: A simple and cheap methods for measuring serum vitamin A in cattle using only a spectrophotometer. Jpn. J. Vet. Sci., 1990, 52, 1281-1283. 52. -  VER-ELST K., SPAPEN H.D., NGUYEN D.N., GARBAR C., HUYGHENS L.P., GORUS F.K.: Cardiac troponins I and T are biological markers of left ventricular dysfunction in septic shock. Clin. Chem., 2000, 46, 650-657. 53. -  WANG W., WU N., ZU Y.G., FU Y.J.: Antioxidative activity of Rosmarinus officinalis L. essential oil compared to its main components. Food Chem., 2008, 108, 1019-1022. 54. -  YANNAKOPOULOS A.L., TSERVENI-GOUSI A.S.: Quality characteristics of quail eggs. Br. Poult. Sci., 1986, 27, 171-176. 55. -  YESILBAG D., EREN M., AGEL H.E., KOVANLIKAYA A., BALCI F.: Effects of dietary rosemary, rosemary volatile oil and vitamin E on broiler performance, meat quality and serum SOD activity. Br. Poult. Sci., 2011, 52, 472-482.

Revue Méd. Vét., 2014, 165, 9-10, 280-288