Experimental Design and Diet: In this study 40 Akkaraman male yearling sheep, 4-months-old, were used following approval of local ethics committee (University of Firat, 18.04.2007 date and 2007/15 decision). All animals used in the study were vaccinated (against foot and mouth disease, enterotoxaemia) and treated against internal and external parasites prior to the experiment. Yearling sheep were allotted in 4 equal groups (n = 10 in each group) according to the diet regimen, and their initial body weights were homogeneous between groups. The control group was fed with a diet of wheat straw whereas the 3 other groups received alfalfa in the fresh form (group AF), silage (group AS) or in a dried form (group AD). Rations were constituted by wheat straw or alfalfa, and their concentrates were designed to be isocaloric and isonitrogenous (Table
1). The experiment was carried out in individual cages using the facilities at the Veterinary Control and Research Institute in Elazig. The yearling sheep were adjusted to experimental feed for 10 days and following 98 days of sampling period. Feedstuffs and water were offered ad libitum throughout the study. The animals were fed twice a day, at 8.00 am and 6.00 pm.
At the end of the study, six animals in each group were slaughtered. Following the slaughtering, about 100 g of muscle samples from the M. semimembranosus (MSM), M. gluteobiceps (MG), M. longissimus dorsi (MLD), M. deltoideus (MD) muscles and 100 g of tail fat were taken from each animals, and stored -20ºC until analysis.
Chemical Analysis: Crude protein was analyzed according to AOAC10.
Analysis of Cholesterol and Vitamin (A, E) amount with HPLC Device: Cholesterol level was measured by using the method described by Katsanidis and Addis11. One section of lipid extraction phase which was divided into two sections was put into tubes with caps, and 5% KOH solution was added (KOH solution was prepared in 100% ethanol). After mixing thoroughly, it was kept at 85ºC for 15 minutes. Tubes were cooled at room temperature, 5 mL pure water was added and fluid was vortexed. After phase separation, upper hexane phase was taken and its solvent was evaporated. Then it was solved with nitrogen flow in acetonitryl/methanol mixture (50% + 50%, v/v) taken to autosampler vials, and prepared for analysis.
For the mobile phase, acetonitryl/methanol (60% + 40%, v/v) mixture was used. Mobile phase flow speed was 1 ml/min. A UV detector was used for the analysis at 202 nm wave length. Supelcosil LC 18 (15 x 4.6 cm, 5 μm; Sigma, USA) column was used.
Chromatographic analysis was performed using an analytical scale (15 cm× 0.45 cm I.D.) Supelcosil LC 18 DB column with a particle size 5 µm (Sigma, USA). HPLC conditions were as follows: mobile phase 75:25 (v/v/): acetonitrile: methanol; a flow rate of 1 ml/min; column temperature 30 ºC. The detection was performed in UV dedector (Shimadzu, SPD,10AVP), 326 nm for retinol, and 215 nm for α-tocopherol and cholesterol.
Statistical Analysis: Data were subjected to analysis of variance. Significant differences were further subjected to Duncan’s multiple range test of SPSS 11.5 program for Windows12. Results were considered as significant when p values were less than 0.001.
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