**2. Materials and Methods**

#### *2.1. Meat Samples, Animals, and Treatments*

Given the special culinary preferences of Polish consumers for two parts of cervid meat, e.g., the loin and leg [32,33], the research material consisted of longissimus lumborum (LL) and semimembranosus (SM) muscles dissected from 12 carcasses of fallow deer reared on an organic farm and 12 carcasses of fallow deer kept in a conventional farming system. In both groups of animals, there were six does and six bucks with an equal age ratio of ca. 18 and 30 months. The farms were located in the area of the Beskid Niski Mts., Podkarpackie Province. The organic farm followed the requirements of Regulation (EU) 2018/848 of the European Parliament and Council of 30 May 2018 [34] and the Act on organic farming (Journal of Laws 2009, No. 116, item 975) [35]. The organic farming certificate covered the grazing area, animals, fodder, and all other rearing procedures. The staple food for the animals was provided by a natural grazing ground with a density of 0.42 LU (large livestock units)/ha. The 191 plant species identified in the pasture [36] represented the following floristic groups: grasses— 10.99%, legumes—22.51%, sedges—2.09%, rushes—2.62%, dicotyledonous herbs—44.50%, deciduous trees—6.28%, coniferous trees—1.05%, blackberries—1.05%, shrubs—4.71%, shrublets—2.62%, ferns—0.53%, and horsetails—1.05%. In winter, the animals received hay, straw, cereal grains, and carrots ad libitum.

The animals from the conventional farming system were reared at a density of 0.67 LU/ha following the DEFRA [37] and FEDFA [38] recommendations. The pasture comprised 72 plant species [36] from the following floristic groups: grasses—22.22%, legumes—11.11%, sedges—1.39%, herbaceous dicotyledons—43.05%, deciduous trees— 5.56%, coniferous trees—2.78%, blackberries—1.39%, shrubs—6.94%, shrublets—4.17%, and horsetails—1.39%. In winter, the animals received hay, haylage, straw, and fodder beets ad libitum.

The animals were slaughtered by shooting from October to December upon the consent and supervision of veterinary services. The post-slaughter treatments, e.g., evisceration, skinning, and veterinary inspection of carcasses, were carried out after transporting the carcasses in a refrigeration truck to an authorized processing plant. During the dissection of cooled carcasses (cooling for 48 h at a temperature of 4 ◦C), three equal-sized samples of LL and SM were collected and an approx. 500-g pooled sample placed in polyethylene bags was transported in an isothermal container to the laboratory. The meat was stored at −200 C until laboratory analyses.

#### *2.2. Chemical Analysis*

Fat was extracted with the method proposed by Folch [39]. Approximately 5 g of meat were homogenized with 5 mL of methanol. The samples were extracted using an automated Soxhlet extractor (Soxtec Avanti, Tecator). The extracted lipids were converted into fatty acid methyl esters (FAME). Fatty acids were saponified with 0.5 N KOH in methanol at

80 ◦C and then esterified with boron trifluoride/methanol in accordance with the PN-ISO 1444:2000 standard [40]. Separation and quantification of the fatty acid methyl esters was carried out using a gas chromatograph (Varian 450-GC with an FID detector) equipped with a flame ionization detector and fitted with a Select™ Biodiesel for FAME capillary column (30 m × 0.32 mm internal diameter, and 0.52 μm film thickness, Shinwa Inc.) A split/splitless injection system (split ratio of 1:50) and helium as a carrier gas at a flow rate of 1.5 mL/min were used. The injection port and the detector were maintained at 250 and 270 ◦C, respectively. The column oven temperature was programmed at 100 ◦C, and finally held at 240 ◦C for 20 min. The identification of individual FAMEs was based on a standard mixture of 37 Component FAME Mix-CRM47885, St. Louis, MO, USA. The Galaxie ™ Chromatography Data System software was used to convert the results. All samples were analyzed in triplicate. The results were expressed as g/100 g of total identified fatty acids.

The cholesterol content in the muscles was determined with the SOP M.023a method (2011) [41]. The analysis was performed with a gas chromatograph (GC—2010 Shimadzu) equipped with an on-column capillary injector and a flame ionization detector. A capillary column (Zebron ZB-5, L = 30 mm, I.D. = 0.25 mm; df. = 0.5 μm) and a ramped oven temperature were used (increased to 150 ◦C from 100 ◦C at 30 ◦C/min, then increased to 360 ◦C at 15 ◦C/min). The cholesterol content was expressed as mg/100 g of fresh meat.

Using the content of individual fatty acids (FA), the following parameters were calculated: SFA—saturated fatty acids, UFA—unsaturated fatty acids, MUFA—monounsaturated fatty acids, PUFA—polyunsaturated fatty acid, PUFA n-6—polyunsaturated fatty acid n-6, PUFA n-3—polyunsaturated fatty acid n-3, OFA—hypercholesterolemic fatty acids = (C12:0 + C14:0 + C16:0), and SFA/UFA; MUFA/SFA; PUFA/SFA; PUFA n-6/PUFA n-3.

The assessment of the nutritional quality of lipids was based on calculation of the following parameters: DFA—desirable fatty acids = (MUFA + PUFA + C18:0), AI—atherogenic index = [C12:0 + 4×C14:0 + C16:0]/[MUFA + PUFA] [42], TI—thrombogenic index = [C14:0 + C16:0 + C18:0]/[0.5×MUFA + 0.5×n-6 + 3×n-3 + n-3/n-6] [42], h/H—ratio of hypo- and hypercholesterolemic fatty acids = [C18:1c9 + C18:2n-6 + C18:3n-3 + C20:3n-6 + C20:4n-6 + C20:5n-3 + C22:5n3]/[C12:0 + C14:0 + C16:0] [7], NV—nutritional value = [C12:0 + C14:0 + C16:0]/[C18:1c9 + C18:2n-6] [43], DHA + EPA—sum of docosahexaenoic acid and eicosapentaenoic acids, HPI—health-promoting index = UFA/[C12:0 +(4 × C14:0) + C16:0] [44], CI—cholesterol index = 1.01 (g of SFA 100 g−<sup>1</sup> of fresh matter—0.5 × g of PUFA 100 g−<sup>1</sup> of fresh matter) + (0.06 × mg of cholesterol 100 g−<sup>1</sup> of fresh matter) [45], and CSI—cholesterol-saturated fat index = (1.01 × g of SFA 100 g−<sup>1</sup> of fresh matter) + (0.05 × mg of cholesterol 100 g−1 fresh matter) [46]. The activities of Δ9-desaturase C16 = [C16:1/(16:0 + C16:1)] × 100, Δ9-desaturase C18 [C18:1n9c/(18:0 + C18:1n9c)] × 100, and elongase = [C18:0 + C18:1n9c]/[C16:0 + C16:1+ C18:0 + C18:1n9c] × 100 [29] were calculated as well.

The nutritional contribution of 100 g of fallow deer meat in the diet for adults was estimated by comparison of the total fat and fatty acid content to the recommendations of FAO (2010) [47] and EU (2011) [48]. The cholesterol level was compared with to the recommendations of WHO/FAO (2003) [49].
