## 2 Materials and Methods  
Four different methods [8; 14; 15; 16] were considered in order to find the optimum parameters for TBARS determination. The methods were selected based on the matrix type considered for determination (milk, egg yolk, and meat--all animal-origin products); the reagents used (approximately the same reagents for different types of matrices); and the time-consuming determination method (UV-VIS spectrometry). The method parameters are described in Table 1.  
Four parameters (trichloroacetic acid concentration (TCA), antioxidant type, incubation time, and thiobarbituric acid (TBA) concentration) defined in previous methods (Table 1) were modified in order to establish the best experimental sequence for milk TBARS determination. Each sample used for analytical determination consisted of 5 mL of raw cow milk (unprocessed), obtained from a local producer. Milk samples were stored at \(-\)20 \({}^{\circ}\)C until analysis. Determinations were performed after defrosting of samples overnight in the refrigerator. Ten mL of TCA at different concentrations (7.5%, 10%, and 20%) was used in order to establish suitable conditions for the protein-removal step. In addition, two different antioxidants that can slow down the milk lipid oxidation process were tested (0.1% ethylenediaminetetraacetic acid (EDTA) and 0.1% butylated hydroxytoluene (BHT)) by adding 5 mL of each antioxidant to sample solutions. After centrifugation at 3000 rpm for 5 min, the samples were filtered and then 2.5 mL of filtrate was incubated with 1.5 mL of TBA at different concentrations (0.3%, 0.5%, and 0.8%) for 40, 60, and 90 min at 80 \({}^{\circ}\)C.  
After incubation and cooling of samples, absorbance values were read at different wavelengths, specific for milk degradation products (450 nm--saturated aldehydes, 495 nm--dienals, and 532 nm--MDA), using a Jasco V\(-\)530 spectrophotometer (Japan Servo Co., Ltd., Tokyo, Japan). Following this step, the absorption spectrum for MDA \begin{table}
\begin{tabular}{c c c c c} \hline \hline
**TCA 1** & **Antiv oxidant** & **TBA 2** & **Incubation Conditions** & **References** \\ \hline
7.5\% & 0.1\% EDTA 3  & 0.02 mol/L & 40 min; 100 \({}^{\circ}\)C & [14] \\
7.5\% & 0.8\% BHT 4  & 0.80\% & 60 min; 80 \({}^{\circ}\)C & [8] \\
20\% & 0 & 0.01 mol/L & 15 min; 90 \({}^{\circ}\)C & [15] \\
1 g/mL & 0 & 1.45\% & 60 min; 80 \({}^{\circ}\)C & [16] \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Methods parameters for TBARS determination. at 532 nm was obtained. The concentrations, expressed as \(\upmu\)g MDA/L milk (ppb), were calculated using a specific calibration curve. Tetramethoxypropane 1,1,3,3 (TMP) was used for preparation of the MDA standard solution, by analytical weighting of 0.0178 g TMP (as MDA precursor) diluted with 100 mL of 0.1 M HCL. After hydrolysis TMP solution (80 \({}^{\circ}\)C for 10 min), the MDA stock solution (1080 \(\upmu\)g/mL) was obtained. Preparing of MDA solutions was done by diluting of MDA stock solution to appropriate concentrations for the calibration curve. After the optimum analytical conditions for TBARS determination were established, the method was applied to different milk samples (three samples of raw cow milk and three samples of raw sheep milk), obtained from a local producer. Milk samples were stored at \(-20\)\({}^{\circ}\)C until analysis. Determinations were performed after defrosting of samples overnight in the refrigerator, at 4 \({}^{\circ}\)C and after keeping them for 24 h at 20 \({}^{\circ}\)C.  
Determination of fatty acid profiles of sheep and cow milk samples was performed using gas chromatography with a system consisting of a Perkin Elmer\(-\)Clarus 500 gas chromatograph, with a capillary column and a flame ionization detector (FID). The capillary column was filled with a stationary phase with a high polarity, having 60 \(\times\) 0.25 mm interior diameter and 0.25 \(\upmu\)m thickness.