A G R I C U LT U R A L A N D F O O D S C I E N C E M. Białek et al. (2020) 29: 405–419 405 Diets enriched in fish and rapeseed oils, carnosic acid, and different chemical forms of selenium affect fatty acid profile in the periintestinal fat and indices of nutritional properties of selected tissues of lambs Małgorzata Białek, Marian Czauderna and Kamil Zaworski The Kielanowski Institute of Animal Physiology and Nutrition, Polish Academy of Sciences, 05-110 Jabłonna, Poland e-mail: m.czauderna@ifzz.pl The aim of our study was to investigate the impact of carnosic acid (CA), selenate (VISe) or selenized yeast (YSe) on concentrations of fatty acids (FA), tocopherols, cholesterol and malondialdehyde in the periintestinal fat (PIF) and muscles of lambs. Male lambs were fed the control diet containing rapeseed (RO) and fish (FO) oils, the CA diet con- taining RO, FO and CA, the YSe-CA diet with RO, FO, CA and YSe, and the VISe-CA diet with RO, FO, CA and VISe. The experimental diets with CA, irrespective of the presence of YSe or VISe, decreased sums of saturated FA (SFA) and the thrombogenic SFA in the PIF compared to the control. The experimental diets increased the Δ9-desaturation capacity in the PIF compared to the control. The experimental diets with YSe or VISe reduced sums of long-chain polyunsaturated FA in the PIF compared to the control and CA diets. The PIF and muscles of lambs fed the VISe-CA diet were characterised by the highest hypocholesterolemic/hypercholesterolemic-FA ratio, and lower modified atherogenic index compared to the control. Key words: ovine periintestinal fat, muscles, tocopherols, cholesterol, oxidative stress, modified atherogenic index Introduction The oxidation of polyunsaturated fatty acids (PUFA), cholesterol and proteins in tissues is one of the most preva- lent modifications caused by reactive oxygen and nitrogen species (ROS and RNS) in animal organisms (Saheem et al. 2017). Numerous studies have indicated that ruminants’ tissues are susceptible to oxidation of lipid unsat- urated fatty acids (UFA), sulphur-amino acids in proteins or cholesterol in cell membranes (Krajewska-Bienias et al. 2017, Morán et al. 2017, Przybylski et al. 2017). Dietary antioxidants (like seleno-compounds, polyphenols, ca- rotenoids, lycopene or tocopherols) may prevent or delay some types of oxidative damage in tissues of animals and humans (Choe and Min 2009, Rozbicka-Wieczorek et al. 2014, El-Ramady et al. 2015, Gargiulo et al. 2017). In fact, studies have shown that selenium (Se) exerts its antioxidative properties through Se-proteins/enzymes of which there may be more than 35 in mammals (Edens and Sefton 2016, Collins 2017). Recent studies have shown that Se is not only an essential part of antioxidants but also a regulator of gene expression (Juszczuk-Kubiak et al. 2016, Zhao et al. 2016). For instance, Se supplementation promotes higher levels of the gene expression of the lipoprotein lipase and apolipoprotein E, particularly in skeletal muscle and possibly in fatty acid utilisation and triacylglyceride metabolism; as a consequence, dietary Se alters lipid metabolism and protein synthesis in the tissues of mammals. Moreover, Se-enzymes suppress pro-inflammatory cell metabolisms by reducing oxidative degradation in intracellular fluid; therefore, Se-compounds have been found to improve immunity in mammals (Raman 2000, El-Ramady et al. 2015). Endogenous Se is present in the tissues and fluids of animals and humans mostly as Se-cysteine (Se-Cys), which is the crucial functional site of ~35 Se-proteins/enzymes (like thioredoxin reductases, isozymes of the glutathione-peroxidase family or iodothyronine deiodinases) or Se-methionine (Se- Met), which can be bound non-specifically to Se-Met-proteins (Collins 2017). It is worth stressing that organic chemical forms of Se (like selenized yeast) are more efficiently incorporated in rumen microbiota and tissues of ruminants compared to selenate (VISe) or selenite (IVSe) (Navarro-Alarcon and Cabrera-Vique 2008, Čobanová et al. 2017, Czauderna et al. 2018). Compared to VISe, IVSe efficiently reacts with dietary components and metabolites of ruminal microbiota (especially those with thiol groups and R-S-S-R’, etc. (Czauderna and Samochocka 1981), as well as IVSe is directly reduced to elemental Se (oSe) by ruminal microorganisms. Unfortunately, oSe is unreactive in the anaerobic ruminal environment (Romero-Pérez et al. 2010). Similarly, Se absorption in mammals’ tissues is significantly higher from dietary VISe than from dietary IVSe (Van Dael et al. 2001). But most importantly, concern has been raised about the potential pro-oxidative properties of IVSe and its lower stability in comparison with VISe when added to diets (Van Dael et al. 2001). Manuscript received August 2020 A G R I C U LT U R A L A N D F O O D S C I E N C E M. Białek et al. (2020) 29: 405–419 406 Recent studies have indicated that inorganic and organic Se-compounds affect microbial populations and mi- croorganism activity (Ošt’ádalová 2012, Kišidayová et al. 2014, Čobanová et al. 2017), as well as fatty acid (FA) metabolism (like enzymatic isomerisation and biohydrogenation of UFA) in the rumen of ruminants or in in vitro incubated ruminal fluids (Czauderna et al. 2012a, 2012b, 2014, 2015, 2018, Miltko et al. 2016, Rozbicka-Wieczorek et al. 2016a, 2016b, 2016c, Białek et al. 2020). Our previous studies have indicated that dietary carnosic acid (CA), selenized yeast (YSe) or VISe affect the concentrations of FA, total cholesterol (TCh), tocopherols and malondialde- hyde (MDA, the marker of PUFA per-oxidation) in the liver, brain, muscles, blood and subcutaneous fat of lambs (Czauderna et al. 2009b, 2011, 2012a, 2012b, Rozbicka-Wieczorek et al. 2016b, 2016c). Moreover, recent studies have shown that CA (a catecholic diterpene) is used as an antioxidant and preservative in foods of animal origin (Rozbicka-Wieczorek et al. 2016b, 2016c, Morán et al. 2012a, 2012b, 2013, 2017, Ortuño et al. 2017). In fact, CA protects cellular biomolecules (like proteins, lipids, RNA and DNA) against chemical stressors, such as reactive ox- ygen species (ROS), paraquat (an agrochemical) or 6-hydroxydopamine. Moreover, an earlier study has reported the inhibition of pro-inflammatory mediator secretion by lipopolysaccharide-stimulated macrophages using high doses of CA (Hadad and Levy 2012). Recent studies have also shown that dietary CA can modify a rumen micro- organism’s profile, resulting in changes of microbiota metabolism, and the yield of biohydrogenation (BH) of UFA in the rumen (Jordán et al. 2013, Miltko et al. 2016, Rozbicka-Wieczorek et al. 2016c). For instance, CA, being rich in Rosmarinus officinalis L. leaves, decreased the ruminal abundance of protozoa, archaea, Prevotella spp., Rumi- nococcus albus and Clostridium aminophilum, whereas increased (though only numerically) the abundance of Ru- minococcus flavefaciens (Cobellis et al. 2016). Interestingly, CA has the most powerful antioxidant potency among diterpenes (like carnosol, rosmanol or iso-rosmanol) in rosemary (Masuda et al. 2001). CA (a natural antioxidant) has a typical o-diphenol structure, so it is easily oxidised to carnosol, the product of the oxidative biotransforma- tion of CA (Masuda et al. 2001, Johnson 2011, Ortuño et al. 2017). Considering the above, special attention should be paid to CA as a dietary supplement. We intend to examine the effect of one natural diterpene (not a mixture of diterpenes) with or without Se-compounds on FA and tocopherols assimilation in the periintestinal fat (PIF) of lambs. The PIF belongs to visceral fat that is stored within the abdominal cavity around a number of very impor- tant internal organs (such as the intestines, the pancreas and the liver). Storing higher amounts of visceral fat (like the PIF), however, is associated with increased risks of a number of health problems, much more so than subcu- taneous fat. Considering all of the above, we hypothesise that the inclusion of CA (a lipophilic antioxidant) with/ without Se (the essential part of Se-antioxidants) in a diet with rapeseed oil (RO) and fish oil (FO) may increase the contents of tocopherols and UFA, and especially the highly unsaturated long chain PUFA (LPUFA), in the PIF and in other selected tissues of lambs. Moreover, we hypothesise that CA and Se (as YSe or VISe) simultaneously added to the lambs’ diet enriched in FO would reduce contents of MDA, as well as increase the values of the hy- pocholesterolemic/hypercholesterolemic FA ratio in the PIF and other tissues. Thus, we expected that CA and YSe or VISe added to a diet with RO and especially FO (rich in n-3LPUFA) would improve animal health and welfare. Therefore, the main aim of our study is focused on animal nutrition (especially FA and tocopherols assimilation by lambs). Considering the above, we plan to investigate the effect of CA and the different chemical forms of Se (as YSe and VISe) added to a basal diet with RO and FO (rich in n-3LPUFA) on the contents of selected FA (especially n-3LPUFA), tocopherols, TCh and MDA, as well as on values of the hypocholesterolemic/hypercholesterolemic FA ratio, and the atherogenic and thrombogenic indices in the PIF of lambs. Materials and methods Animals, housing, experimental design, diets, management and sampling Lamb welfare guidelines and all handling procedures accepted by the 3rd Local Commission of Animal Experiment Ethics at the University of Life Sciences (Warsaw, Poland) were strictly followed throughout the preliminary period and for the duration of the experiments. Twenty-four male Corriedale lambs with an average initial body weight (BW) of 30.4±2.5 kg were individually penned as described in our previous publication (Czauderna et al. 2017). All experiments on the lambs and tissue collections were carried out at The Kielanowski Institute of Animal Physiology and Nutrition (Jabłonna, Poland). Briefly, during a 3-week preliminary period, the lambs were fed the basal diet (BD) (the standard concentrate–hay diet with min- eral premix and vitamins; Table 1) (NRC 2007) enriched in 2% RO and 1% odourless FO (Table 2). The content of Se in the basal diet was 0.16 mg of Se in 1 kg of BD. After the preliminary period, the lambs were divided into 4 groups of 6 animals; a 35-day experiment was conducted, during which animals were fed the basal diet supplemented with 2% RO and 1% FO (the control diet) or with 2% RO, 1% FO and antioxidant(s) (i.e. 0.1% CA and/or 0.35 mg of Se as YSe or VISe in 1 kg of BD) (Table 3). About 83% of the total Se-amount of dietary YSe is found in the chemical form of Se-Met, whereas 5% of Se is in the form of Se-Cys incorporated into the proteins of Saccharomyces cerevisiae. A G R I C U LT U R A L A N D F O O D S C I E N C E M. Białek et al. (2020) 29: 405–419 407 1 1 kg of the mineral and vitamin mixture comprised: 285 g Ca, 16 g phosphorus, 56 g Na, 42 mg Co as carbonate, 10 mg iodine as iodate, 1 g Fe as sulphate, 6 mg Se as selenite, 0.5 g Cu as sulphate, 5.8 g Mn as sulphate, 7.5 g Zn as sulphate; vitamins: A (500000 IU kg-1), D3 (125000 IU kg-1), and E as α-tocopherol (25000 IU kg-1). 20 g of the mineral and vitamin mixture was added to 1 kg of the basal diet (BD); 2 The gross energy (MJ per kg of dry matter [DM]): barley meal: 16.3, soybean meal: 17.8, wheat starch: 16.7; 3 the gross energy: 17.1 MJ per kg of DM Table 2. The concentrations (mg kg-1) of the main fatty acids in the components of the lambs’ diet: concentrate, meadow hay, rapeseed oil (RO) and odourless fish oil (FO)1 Fatty acids Concentrate Meadow hay RO FO C8:0 - 83 - - C12:0 - 142 - 82 C14:0 104 239 56 12345 C15:0 - - - 477 c9C14:1 - 131 - 215 C16:0 3189 4034 13091 56947 c7C16:1 - - - 318 c9C16:1 - 184 33 420 ∑C16:2 - - - 15586 C17:0 - - - 493 c9C17:1 - - - 193 C18:0 1425 459 5490 9452 c6C18:1 - - 6 188 c7C18:1 - - - 842 c9C18:1 774 1266 385859 290592 c12C18:1 - 72 786 15834 c14C18:1 - - - 159 C18:2n-6 (LA) 29163 13100 282394 114512 C18:3n-3 (αLNA) 1014 4178 83474 20968 c7c9c12c15C18:4 - - - 473 C20:0 - 58 430 - c11C20:1 - 74 - 24206 c11c14C20:2 - - - 2270 c8c11c14C20:3 - - - 258 C20:4n-6 (AA) - - - 304 c8c11c14c17C20:4 - - - 607 C20:5n-3 (EPA) - - - 6792 C22:0 - 101 153 139 Table 1. Chemical composition (%) of the concentrate-hay diet (the basal diet) with vitamins and mineral mixture1 fed to lambs Item Meadow hay 3 Concentrate 2 Barley meal Soybean meal Wheat starch Dry matter 88.4 87.6 89.7 87.3 Crude protein 9.50 9.94 41.81 0.90 Crude fibre 27.29 2.87 4.34 – Crude fat 3.40 2.50 2.25 0.09 Ash 4.85 1.84 6.16 0.12 Neutral detergent fiber 59.17 18.02 18.81 – Acid detergent fiber 32.08 4.61 6.44 – Acid detergent lignin 4.47 1.14 1.49 – c11C22:1 - - - 1704 c13C22:1 - - - 11036 A G R I C U LT U R A L A N D F O O D S C I E N C E M. Białek et al. (2020) 29: 405–419 408 All details of the composition (including fatty acid compositions) of the control and experimental diets and rape- seed oil were presented in recent publications by the authors (Czauderna et al. 2017, 2018). The Se status of lambs fed the control and experimental diets has been presented in our latest papers (Czauderna et al. 2017, 2018). The control and experimental diets were offered in two equal meals, as described previously (Czauderna et al. 2017); drinking water for animals was available ad libitum. The initial BW of lambs and body weight gain (BWG) are summarised in Table 3. After the 35-day experiment (i.e. at 0700–0800 h), all lambs were deprived of con- sciousness by intramuscular injections of xylazine (0.2–0.4 mg kg-1 of BW) and then slaughtered (Czauderna et al. 2017). The lambs were slaughtered in accordance with the European Union Council Regulations (EC) No 1099/2009 dated 24.09.2009 for the protection of animals at the time of slaughter in small experimental slaughterhouses. Next, tissues of PIF (78±9 g), subcutaneous fat (SCF) (92±11 g), musculus biceps femoris (MBF) (174±14 g) and musculus longissimus dorsi (MLD) (653±77 g) were immediately removed from each lamb. All samples were ho- mogenised straight away using a tissue homogeniser (IKA®T18 basic, Ultra-Turrax®, Germany). The contents of MDA were determined in freshly homogenised PIF samples (Czauderna et al. 2011). All homogenised tissue samples were transferred into tightly sealed vessels and stored at −32 °C for further chromatographic analyses. All samples were analysed individually. The contents of FA, TCh, MDA and tocopherols in tissue samples were expressed per g of fresh matter. Chemicals and analytical methods Commercial rapeseed oil and odourless fish oil (rich in highly unsaturated LPUFA) were purchased from Company AGSOL (Pacanów, Poland), CA was purchased from Hunan Geneham Biomedical Technology Ltd (Changsha Road, Changsha, Hunan, China), while selenized yeast (Se-Saccharomyces cerevisiae) was donated by Sel-Plex (Alltech Inc., Nicholasville, KY, USA). The vitamin and mineral premix (ID number: aPL 1405002p) was purchased from POL- FAMIX OK (Grodzisk Mazowiecki, Poland). Methanol (≥ 99.9%), HPLC-acetonitrile (≥ 99.9%) and n-hexane (≥ 99%) were purchased from Lab-Scan (Dublin, Ireland). A conjugated linoleic acid (CLA) isomer mixture, a nonadecanoic acid (as the internal standard) and other c13c16C22:2 - - - 95 c7c10c13c16C22:4 - - - 144 DPA - - - 1560 DHA - - - 26570 C24:0 - 69 - - c15C24:1 - 71 61 397 1 The iodine value of FO: 50–65 g/100 g FO; the acid value of FO: 20 mg KOH g-1 FO. The energy content of FO and RO was 36.8 and 37.0 MJ kg−1 oil, respectively. BWG = body weight gain; BD = basal diet; RO = rapeseed oil; FO = fish oil; YSe = selenized yeast; VISe = selenate; BW initial = the initial body weight of lambs after the preliminary period; a, b Different letters within a column indicate significant differences at p < 0.05; 1 For the 3-week of preliminary period lambs were fed the diet with 2% RO and 1% FO. 2 The average body weight gain (BWG, kg) of lambs fed the control or experimental diets for 35 days of the experimental period: BWG = (BW 35days – BW initial ), where BW 35days - the body weight of lambs after 35 days of experiment. The average daily diet intake was 1.08 kg per lamb. 3 The concentration of selenium in the control and CA diets was 0.16 mg of Se in 1 kg of diets. 4 The concentration of selenium in the YSe-CA and VISe-CA diets was 0.51 mg of Se in 1 kg of diets. Table 3. The experimental scheme, the chemical composition of the control and experimental diets, the initial body weight (BW initial ) and the body weight gain (BWG, kg) of lambs Group 1 Additives added to the basal diet BWinitial kg BWG 2 kg Control group 3 2% RO and 1% FO (The control diet) 30.6±2.4 7.2±0.3ab CA group 3 2% RO, 1% FO and 0.1% CA (The CA diet) 30.6±2.6 6.6±0.3a YSe-CA group 4 2% RO, 1% FO, 0.1% CA and 0.35 mg of Se as YSe in 1 kg of BD (The YSe-CA diet) 30.3±2.7 6.6±0.3a VISe-CA group 4 2% RO, 1% FO, 0.1% CA and 0.35 mg of Se as VISe in 1 kg of BD (The VISe-CA diet) 30.3±3.0 8.2±0.4b A G R I C U LT U R A L A N D F O O D S C I E N C E M. Białek et al. (2020) 29: 405–419 409 37 fatty acid standard mixture (FAME), α-tocopherol, α-tocopheryl acetate, cholesterol, sorbic acid, 2,6-di‑tert‑bu- tyl‑pcresol, 25% aqueous 1,5-pentanedialdehyde solution, 2,4-dinitrophenylhydrazine (containing ~30% water), 1,1,3,3-tetramethoxypropane (99%), trichloroacetic acid and 25% BF 3 in methanol were obtained from Sigma- Aldrich (St Louis, MO, USA). KOH, NaOH, Na 2 SO 4 , chloroform and dichloromethane were purchased from Avantor Performance Materials (Gliwice, Poland). All other chemicals were of analytical grade. Preparation of fatty acid methyl esters (FAME) in ovine tissues The homogenised samples of the PIF (~10 mg), SCF (~10 mg) and muscles (40–50 mg) were saponified using a KOH solution according to methods described by Czauderna et al. (2007); nonadecanoic acid (as the internal standard) was added to each saponified biological sample. Then, mild base- and acid-catalysed methylations were intro- duced for the preparation of FAME in processed biological samples (Czauderna et al. 2007). Fatty acids (as FAME) in assayed biological samples were then determined using capillary-gas chromatography with mass spectrome- try according to the methods presented by Rozbicka-Wieczorek et al. (2014). All analyses were performed on a Shimadzu GC-MS-QP2010 Plus EI equipped with a BPX70 fused silica column (120 m [length] × 0.25 mm [i.d.] × 0.25 μm [film thickness]) and a quadruple mass selective detector (Model 5973 N). FAME identification was vali- dated based on the electron impact ionisation spectra of FAME and compared to authentic FAME standards and the NIST 2007 reference mass spectra library (National Institute of Standard and Technology, Gaithersburg, MD, USA) (Rozbicka-Wieczorek et al. 2014). Determination of tocopherols, TCh and MDA in ovine tissues TCh, α-tocopherol (αT), δ-tocopherol (δΤ), γ-tocopherol (γΤ) and α-tocopheryl acetate (αTAC) were quantified in the homogenised samples of the PIF, SCF and muscles (MLD and MBF) using a liquid chromatographic system (SHI- MADZU, Tokyo, Japan) according to methods described by Czauderna et al. (2009a). The liquid chromatographic instrument used consisted of an ultra-fast liquid chromatography system, incorporating two pumps, an autosam- pler, a CBM-20A communications bus module, a column oven, a Kinetex C18-column (2.6 μm; Hydro-RP, 150 mm × 2.1 mm; Phenomenex, Torrance, CA, USA) in conjunction with a guard column, a degasser and a photodiode array detector (Białek and Czauderna 2019). The concentration of MDA in the PIF samples was determined after saponification followed by derivatisation ac- cording to methods described by Czauderna et al. (2011). The chromatographic separations of derivatised MDA from endogenic species of the processed PIF samples were conducted using an ultra-fast liquid chromatography system and a photodiode array detector (Czauderna et al. 2011). Atherogenic and thrombogenic indices were calculated according to the equations given by Morán et al. (2013). The hypocholesterolemic/hypercholesterolemic fatty acid (h/H-Ch) ratio was calculated using the equation given by Fernández et al. (2007). Statistical analysis All statistical analyses of the effects of dietary additives were carried out using the Statistica 12.5 PL software package (StatSoft Inc., Tulsa, OK, USA). Differences were considered significant at p < 0.05. The obtained results are shown as means and SEM (standard error of mean). The influence of dietary modifications on the contents of analytes in all biological samples for variables with normal distribution was tested with one-way ANOVA and the post-hoc Honestly Significant Difference (HSD) Tukey test. For variables without normal distribution, the re- sults were tested with Kruskal-Wallis, which is a non-parametric equivalent of one-way ANOVA, with a post-hoc multiple comparison test. Results Concentrations of selected saturated fatty acids (SFA) in the PIF of lambs Experimental data reflecting the contents of SFA in the PIF of lambs fed the experimental diets are summarised in Table 4. We found that the diets enriched in CA, irrespective of the presence of YSe or VISe, resulted in a de- crease in the contents of C8:0, C15:0, C17:0, C18:0, C20:0, C22:0 and the sum of thrombogenic SFA (T-SFA) in the PIF compared to the control diet. Moreover, the CA and VISe-CA diets reduced the concentration of C16:0 in the PIF as compared to the control diet. All experimental diets reduced the content sum of SFA (ΣSFA) in the PIF in A G R I C U LT U R A L A N D F O O D S C I E N C E M. Białek et al. (2020) 29: 405–419 410 comparison with the control diet. Moreover, the experimental diet with CA, irrespective of the presence of YSe or VISe, decreased the ratios of ΣSFA/ΣUFA and ΣSFA/ΣFA in the PIF compared to the control diet. Conversely, all ex- perimental diets increased the contents of C21:0 and C23:0 in the PIF compared to the control diet. The diet with VISe caused the highest increase in the content of C21:0 in the PIF compared to the control and other experimen- tal diets. All experimental diets had no effect on the contents of C11:0, C12:0, C13:0, C14:0, the atherogenic SFA (A-SFA) and the sum of medium-chain SFA (Σ medium SFA) in the PIF. The content ratio of A-SFA to the sum of FA (A-SFA/ΣFA) in the PIF of lambs fed the VISe-CA diet was lower than in the PIF of lambs fed the control, CA and YSe-CA diets. The CA and YSe-CA diets elevated the values of the A-SFA/ ΣFA ratio in the PIF compared to the control diet. To the contrary, all experimental diets, especially the YSe-CA diet, reduced the values of the T-SFA/ΣFA ratio in the PIF in comparison with the control diet. Concentrations of unsaturated fatty acids in the PIF of lambs The influence of the experimental diets enriched in CA with or without Se (as YSe or VISe) on the contents of mono- unsaturated fatty acids (MUFA) and PUFA are presented in Tables 5 and 6. The results of our experiments demon- strated that VISe added to the diet with CA reduced the contents of c7C16:1, c9C16:1, c6C18:1 and c11C20:1 in comparison with the control and YSe-CA diets (Table 5). We found that the experimental diet supplemented only with CA resulted in a decrease in the contents of c9C14:1, c7C16:1, c9C16:1, c6C18:1 and c11C20:1 in the PIF com- pared to the control diet. Conversely, no noticeable differences in the content of t11C18:1 (TVA) and c9C18:1, the sum of all assayed MUFA (ΣMUFA) and the ratio of ΣMUFA/ΣFA were found between the control and all experi- mental groups of lambs. SEM = standard error of the mean; UFA = unsaturated fatty acids; PUFA = polyunsaturated fatty acids. a, b Different letters within a row indicate significant differences at p < 0.05. 1 The sum: C12:0, C14:0 and C16:0; 2 The sum: C14:0, C16:0 and C18:0; 3 The sum: C8:0, C10:0, C12:0 and C14:0; 4 The sum: C8:0, C10:0, C11:0, C12:0, C13:0, C14:0, C15:0, C16:0, C17:0, C18:0, C20:0, C21:0, C22:0 and C23:0 Table 4. The concentrations (mg g-1 PIF) of selected individual saturated fatty acids (SFA), atherogenic-SFA1 (A-SFA), thrombogenic- SFA2 (T-SFA), the sums of medium-chain SFA (Σ medium SFA)3, all assayed SFA (ΣSFA)4 and the ratios of ∑SFA to the sum of PUFA (ΣSFA/∑PUFA), UFA (∑SFA/ΣUFA) and all assayed FA (∑SFA/∑FA) in the periintestinal fat (PIF) of lambs Item Additive – CA YSe + CA VISe + CA SEM p-value Group Control CA YSe-CA VISe-CA C8:0 0.071b 0.041a 0.039a 0.040a 0.002 0.03 C10:0 2.22b 1.88a 1.89a 1.93ab 0.07 0.03 C11:0 0.017 0.021 0.015 0.019 0.005 0.31 C12:0 3.16 2.97 3.38 3.23 0.6 0.09 C13:0 0.119 0.106 0.099 0.124 0.009 0.27 C14:0 31. 93 30.79 36.86 30.12 1.18 0.17 C15:0 4.71c 3.35a 4.09b 4.18b 0.08 0.03 C16:0 172c 152ab 164bc 146a 3 0.03 C17:0 10.6b 8.8a 9.3a 9.0a 0.2 0.02 C18:0 226c 185a 186a 197a 5 0.03 C20:0 0.346b 0.240a 0.257a 0.267a 0.014 0.02 C21:0 0.001a 0.073b 0.178c 0.245d 0.014 0.00 C22:0 0.270b 0.199a 0.204a 0.211a 0.012 0.00 C23:0 0.0007a 0.0092b 0.0081b 0.0084b 0.0003 0.01 A-SFA 207 186 205 180 4 0.28 A-SFA/ΣFA 0.263b 0.271c 0.273c 0.254a 0.002 0.02 T-SFA 431 b 368a 387a 373a 9 0.02 T-SFA/ΣFA 0.545c 0.535b 0.517a 0.529b 0.002 0.02 Σ medium SFA 37.3 35.6 42.1 35.3 0.5 0.31 ΣSFA 451b 386a 407a 393a 8 0.02 ΣSFA/ΣUFA 1.311d 1.279c 1.185a 1.244b 0.012 0.03 ΣSFA/ΣFA 0.571c 0.561b 0.542a 0.556ab 0.002 0.04 A G R I C U LT U R A L A N D F O O D S C I E N C E M. Białek et al. (2020) 29: 405–419 411 Indices of Δ9-desaturation of C18:0 (C18:1∆9-index) and Δ9-desaturation of C16:0 and C18:0 (Σ∆9-index) were higher in the PIF of lambs fed the experimental diets than in the PIF of the control lambs. Moreover, the diet supplement- ed with YSe most efficiently elevated the C18:1∆9-index and Σ∆9-index in the PIF. Similarly, all experimental diets, particularly those supplemented with YSe or VISe, increased the values of the PUFA elongase index (Elongindex) in the PIF compared to the control diet. To the contrary, all experimental diets, especially the VISe-CA diet, reduced the index values of Δ9-desaturation of TVA (CLA∆9-index) in the PIF in comparison with the control diet. The values of Δ4-desaturation (∆4-index) of c7c10c13c16c19C22:5 (DPA) were smaller in the PIF of lambs fed the experimental diets, including extra Se (as YSe or VISe), than in the PIF of lambs fed the control and CA diets. As can be seen from the results in Table 6, all experimental diets affected the contents of PUFA in the PIF. Indeed, the contents of c9t11CLA, c11c14C20:2 and c5c8c11c14c17C20:5 (EPA) were smaller in the PIF of lambs fed the ex- perimental diets than the control diet. Moreover, the experimental diets supplemented with YSe or VISe considerably reduced the contents of c11c14C20:2, c5c8c11c14C20:4, c5c8c11c14c17C20:5 (EPA) and c4c7c10c13c16c19C22:5 (DHA) in the PIF, in comparison with the control and CA diets. In contrast, the diets with YSe or VISe caused a strong increase in the concentration sum of ct/tcCLA (∑ct/tcCLA) in the PIFcompared with the control and CA diets. The experimental diets, regardless of the presence of YSe or VISe, had no effect on the contents of c9c12C18:2 (LA), c6c9c12C18:3 (γLNA), c6c9c12c15C18:4, c8c11c14c17C20:4, c7c10c13c16C22:4, c7c10c13c16c19C22:5 (DPA), the sums of n-6PUFA (Σn-6PUFA) and PUFA (ΣPUFA), and the values of the ratio of Σn-6PUFA to Σn-3PUFA (n-6/n-3) in the PIF. It was found that the values of the content ratio of ΣPUFA to ΣFA (ΣPUFA/ΣFA) were higher in the PIF of lambs fed the YSe-CA or VISe-CA diets than the control and CA diets. Alternatively, the diets with YSe or VISe reduced the content sums of n-6LPUFA (Σn-6LPUFA), n-3LPUFA (Σn-3LPUFA) and LPUFA (ΣLPUFA) in the PIF compared to the control and CA diets. As a consequence, the experimental diets with YSe or VISe reduced the ratio values of Σn-3LPUFA/ΣFA and ΣLPUFA/ΣFA in the PIF compared to the control and CA diets. Table 5. The concentrations (mg g-1 PIF) of selected individual monounsaturated fatty acids (MUFA), the concentration sum of all assayed MUFA (ΣMUFA)1 and index values of ∆9-desaturases (C18:1∆9-index2, Σ∆9-index3 and CLA∆9-index4) in the periintestinal fat (PIF) of lambs Item Additive – CA YSe + CA VISe + CA SEM p-value Group Control CA YSe-CA VISe-CA c7C14:1 0.11 0.10 0.09 0.07 0.02 0.21 c9C14:1 2.15c 1.57a 1.83b 1.64ab 0.11 0.02 isoC15:0 0.09 0.08 0.08 0.06 0.01 0.19 c7C16:1 5.52b 4.75a 6.70c 4.63a 0.180 0.03 c9C16:1 7.45b 6.66a 7.58b 6.01a 0.20 0.04 c9C17:1 0.34 0.19 0.27 0.29 0.07 0.31 t9C18:1 0.53 0.52 0.58 0.47 0.08 0.26 t11C18:1 (TVA) 3.67 3.10 3.91 3.65 0.17 0.18 c6C18:1 25.5b 16.3a 16.4a 12.8a 1.2 0.01 c7C18:1 5.1 5.3 3.4 4.6 0.2 0.18 c9C18:1 222 201 233 213 6 0.32 c11C18:1 0.91 0.79 0.66 0.82 0.6 0.41 c12C18:1 13.2 12.0 12.0 14.4 0.5 0.27 c11C20:1 2.27c 1.53b 1.50b 1.36a 0.13 0.02 c11C22:1 0.002 0.003 0.001 0.002 0.0003 0.48 c13C22:1 0.009c 0.008c 0.003b 0.001a 0.001 0.01 c15C24:1 0.0009 0.0007 0.0006 0.0008 0.0002 0.39 ΣMUFA 291 254 290 266 7 0.33 C18:1∆9-index 0.497a 0.521b 0.556c 0.519b 0.004 0.02 Σ∆9-index 0.365a 0.380b 0.407c 0.390b 0.002 0.02 CLA∆9-index (CLA/TVA) 0.398d 0.310c 0.298b 0.269a 0.004 0.00 ΣMUFA/ΣFA 0.366 0.369 0.385 0.374 0.003 0.37 SEM = standard error of the mean; CLA = conjugated linoleic acid isomers. a, b Different letters within a row indicate significant differences at p < 0.05. 1 The sum: c7C14:1, c9C14:1, isoC15:1, c7C16:1, c9C16:1, c9C17:1, t9C18:1, t11C18:1, c6C18:1, c7C18:1, c9C18:1, c11C18:1, c12C18:1, c11C20:1, c11C22:1, c13C22:1 and c15C24:1. 2 C18:1∆9-index = c9C18:1/(c9C18:1 + C18:0). 3 Σ∆9-index = (c9C16:1 + c9C18:1)/(c9C16:1 + C16:0 + c9C18:1 + C18:0). 4 CLA∆9-index = c9t11CLA/(c9t11CLA + TVA) (see Table 4). A G R I C U LT U R A L A N D F O O D S C I E N C E M. Białek et al. (2020) 29: 405–419 412 Concentrations of TCh, tocopherols and MDA, and values of atherogenic, thrombogenic and hypocholesterolemic indices in selected tissues of lambs The experimental results reflecting the concentrations of TCh, δ-tocopherol (δT), γ-tocopherol (γT), α-tocopherol (αT), α-tocopheryl acetate (αTAc) and MDA are summarised in Table 7. The diets enriched in CA with or without VISe reduced the content of TCh in the PIF compared to the control and YSe-CA diets. In sheep fed the diet enriched with CA and Se (as YSe or VISe), the contents of δT and γT in the PIF decreased in comparison with the control and CA diets. In contrast, the contents of αT and αTAc were higher in the PIF of lambs fed the diets enriched with YSe or VISe than in the PIF of the control lambs. The contents of αT, and the sums of αT and αTAc (∑(αT+αTac)) and all assayed tocopherols (ΣTs) were higher in the PIF of lambs fed the YSe-CA diet than the control and VISe-CA diets. Table 6. The concentrations (mg g-1) of c9t11CLA, other ct/ctCLA isomers (Σct/tcCLA)1, the sum of CLA isomers (ΣCLA)2, selected individual PUFA, the sums of n-6PUFA (∑n-6PUFA)3, n-3PUFA (∑n-3PUFA)4, n-6LPUFA (∑n-6LPUFA)5, n-3LPUFA (∑n-3LPUFA)6, LPUFA (∑LPUFA)7, all PUFA (∑PUFA)8, the ratios of ∑n-6PUFA to ∑n-3PUFA (n-6/n-3), ∑PUFA to ∑SFA (∑PUFA/∑SFA), ∑PUFA to ΣFA9 (∑PUFA/∑FA) and ∑n-3LPUFA to ∑FA (∑n-3LPUFA/∑FA)10, and elongation and ∆4-desaturation indices in the periintestinal fat (PIF) of lambs Item Additive – CA YSe + CA VISe + CA SEM p-value Group Control CA YSe-CA VISe-CA c9t11CLA 2.55c 1.43a 1.72b 1.48a 0.09 0.02 Σct/tcCLA 0.019a 0.021a 0.527b 0.824c 0.20 0.00 ΣttCLA 0.011ab 0.009a 0.013b 0.016c 0.001 0.03 ΣCLA 2.58c 1.46a 2.26b 2.32bc 0.10 0.04 c9c12C18:2 (LA) 50.0 44.7 50.6 47.1 1.0 0.37 c6c9c9C18:3 (γLNA) 0.043 0.027 0.033 0.040 0.009 0.51 c9c12c15C18:3 (αLNA) 3.40c 3.04b 3.21bc 2.69a 0.09 0.02 c6c9c12c15C18:4 0.015 0.011 0.012 0.009 0.003 0.18 c11c14C20:2 0.200c 0.110b 0.003a 0.002a 0.019 0.00 c8c11c14c17C20:4 0.017 0.009 0.012 0.013 0.003 0.21 c5c8c11c14C20:4 (AA) 0.339b 0.357b 0.219a 0.240a 0.017 0.03 c5c8c11c14c17C20:5 (EPA) 0.130c 0.109b 0.021a 0.012a 0.011 0.02 c7c10c13c16C22:4 0.010 0.013 0.015 0.017 0.004 0.32 c7c10c13c16c19C22:5 (DPA) 0.212 0.193 0.211 0.217 0.010 0.47 c4c7c10c13c16c19C22:5 (DHA) 0.035c 0.030c 0.001a 0.006b 0.003 0.02 Σn-6PUFA 50.6 45.1 50.9 47.3 1.1 0.13 Σn-3PUFA 3.65c 3.26b 3.42b 2.91a 0.09 0.04 Σn-6PUFA/Σn-3PUFA (n-6/n-3) 14.4a 14.5a 15.2b 17.0c 0.3 0.03 Σn-6LPUFA 0.339b 0.357b 0.219a 0.240a 0.010 0.02 Σn-3LPUFA 0.376c 0.332b 0.231a 0235a 0.012 0.02 Σn-3LPUFA/ΣFA 0.476c 0.483d 0.308a 0.332b 0.001 0.03 ΣLPUFA 0.715b 0.689b 0.450a 0.475a 0.032 0.04 ΣPUFA 56.8 49.9 56.5 52.5 1.1 0.47 ΣLPUFA/ΣFA 0.905c 1.003d 0.600a 0.671b 0.009 0.03 ∑PUFA/∑FA 0.0721a 0.0726a 0.0754c 0.0737b 0.0002 0.03 ∆4-index 11 0.099c 0.110d 0.003a 0.019b 0.010 0.01 EPA-Elongindex 12 0.620a 0.638b 0.909c 0.948d 0.003 0.02 SEM = standard error of the mean. a, b Different letters within a row indicate significant differences at p < 0.05. 1 The sum of ct/tcCLA isomers: cis-transCLA: 11-13, 12-14; trans-cisCLA: 7-9, 8-10, 9-11, 10-12, 11-13 and 12-14. 2 The sum: c9t11CLA, ct/tcCLA isomers, ttCLA isomers (trans-trans: 7-7, 8-10, 9-11, 10-12, 11-13 and 12-14) and ccCLA isomers (cis-cis: 8-10, 9-11, 10-12 and c11-12). 3 The sum: LA, c6c9c12C18:3 c11c14C20:2, AA and c7c10c13c16C22:4. 4 The sum: αLNA, c6c9c12c15C18:4 and Σn-3LPUFA. 5 The sum: c11c14C20:2, AA and c7c10c13c16C22:4. 6 The sum: c8c11c14c17C20:4, EPA, DPA and DHA. 7 The sum: Σn-6LPUFA and Σn-3LPUFA. 8 The sum: ΣCLA, Σn-3PUFA and Σn-6PUFA. 9 The sum of all fatty acids (ΣFA). 10 The concentration ratio of ∑n-3LPUFA (µg/g PIF) to ∑FA (mg/g PIF). 11 ∆4-index = c4c7c10c13c16c19C22:6/ (c4c7c10c13c16c19C22:6 + c7c10c13c16c19C22:5). 12 EPA-Elongindex = c7c10c13c16c19C22:5/(c7c10c13c16c19C22:5 + c5c8c11c14c17C20:5). A G R I C U LT U R A L A N D F O O D S C I E N C E M. Białek et al. (2020) 29: 405–419 413 The experimental diets, especially the CA diet, reduced the content of MDA and the PUFA peroxidation index (MDA index ) in the PIF compared to the control diet. Moreover, the content of MDA was smaller in the PIF of lambs fed the diet with VISe than in the PIF of animals fed the diet with YSe. Interestingly, we found that the lowest level of MDA, and the value of MDA index was in the PIF of lambs fed the CA diet. Investigation of the impact of the contents of tocopherols, A-SFA, n-6PUFA and n-3PUFA in the PIF on the atherogenic index showed that all experimental diets, especially the diet with YSe, reduced the value of our modified atherogen- ic index ( index ASFA+Toc) in the PIF, subcutaneous fat (SCF), MLD and MBF in comparison with the control diet (Table 8). SEM = standard error of the mean; MDA = malondialdehyde; a, b Different letters within a row indicate significant differences at p < 0.05. 1 Concentrations of MDA were determined immediately after the homogenization of PIF samples. 2 The sum: αΤ and αΤAc; 3 The sum: δΤ, γΤ, αΤ and αΤAc; 4 The concentration ratio of MDA (ng g-1 PIF) to ΣPUFA (mg g-1 PIF); MDA index = MDA/ΣPUFA. Table 7. The concentrations (µg g-1 PIF) of total cholesterol (TCh), tocopherols and MDA (ng g-1 PIF)1 and values of the PUFA peroxidation index (MDA index ) in the periintestinal fat (PIF) of lambs fed the control and experimental diets Item Additive – CA YSe + CA VISe + CA SEM p-value Group Control CA YSe-CA VISe-CA TCh 72.3c 42.7a 71.8c 53.9b 0.2 0.04 δ-tocopherol (δΤ) 1.48b 1.38b 1.17a 1.17a 0.01 0.02 γ-tocopherol (γΤ) 1.17b 1.17b 0.97a 0.95a 0.01 0.02 α-tocopherol (αΤ) 2.38a 2.45ab 3.20c 2.51b 0.02 0.03 α-tocopheryl acetate (αΤAc) 3.47a 4.50c 4.04b 3.81b 0.02 0.02 Σ(αΤ+αΤAc) 2 5.85a 6.95c 7.24c 6.32b 0.02 0.01 ΣTs 3 8.52a 9.48b 9.38b 8.42a 0.05 0.04 MDA, 5.08d 1.74a 4.50c 3.17b 0.04 0.02 MDA index 4 0.106c 0.036a 0.080b 0.077b 0.002 0.03 Table 8. The ∑PUFA/∑SFA ratio (∑PUFA/∑SFA), thrombogenic ( index TSFA)1 and atherogenic ( index ASFA)2 indices in the PIF of lambs. The hypo-cholesterolemic/hypercholesterolemic fatty acid (h/H-Ch) ratio3 and the modified atherogenic index ( index ASFA+Toc)4 in the PIF, SCF, musculus longissimus dorsi (MLD) and musculus biceps femoris (MBF) of lambs fed the control and experimental diets Item Additive – CA YSe + CA VISe + CA SEM p-value Group Control CA YSe-CA VISe-CA the periintestinal fat (PIF) ∑PUFA/∑SFA 0.127a 0.130a 0.140c 0.134b 0.001 0.02 h/H-Ch ratio 1.355a 1.361a 1.429b 1.494c 0.017 0.03 index TSFA 2.124c 2.068b 1.933a 2.068b 0.017 0.03 index ASFA 0.888b 0.923c 0.918c 0.859a 0.014 0.03 index ASFA+Toc 0.104c 0.092b 0.087a 0.094b 0.001 0.02 the subcutaneous fat (SCF) index ASFA+Toc 0.071c 0.066b 0.062ab 0.059a 0.001 0.03 h/H-Ch ratio 1.791b 1.796b 1.733a 1.831c 0.014 0.02 musculus longissimus dorsi (MLD) index ASFA+Toc 0.249c 0.096ab 0.085a 0.102b 0.002 0.01 h/H-Ch ratio 1.653a 1.734b 1.665a 1.841c 0.012 0.03 musculus biceps femoris (MBF) index ASFA 0.662b 0.716c 5 0.719c 5 0.617a 5 0.011 0.03 index ASFA+Toc 0.144d 0.123a 0.139c 0.130b 0.002 0.04 h/H-Ch ratio 1.719b 1.638a 1.645a 1.776c 0.024 0.04 SEM = standard error of the mean. a, b Different letters within a row indicate significant differences at p < 0.05. 1 The thrombogenic index = (C14:0 + C16:0 + C18:0) / [(0.5 × ΣMUFA + 0.5 × Σn-6PUFA + 3 × Σn-3PUFA) / Σn-6PUFA)] (Morán et al. 2013); 2 The atherogenic index = (C12:0 + 4 × C14:0 + C16:0) / (ΣMUFA + Σn-6PUFA + Σn-3PUFA) (Morán et al. 2013); 3 h/H-Ch = (c9C18:1 + c11C18:1 + c9c12C18:2 + c9c12c15C18:3 + c6c9c12C18:3 + c8c11c14C20:3 + c11c14c17C20:3 + c5c8c11c14C20:4 + c8c11c14c17C20:4 + c5c8c11c14c17C20:5 + c7c10c13c16C22:4 + c7c10c13c16c19C22:5 + c4c7c10c13c16c19 C22:6) / (C14:0+C16:0) (Fernández et al. 2007); 4 The modified atherogenic index ( index ASFA+Toc) = index ASFA /(1.49 x C αΤ + 1.36 x C αΤAc + 0.15 x C γΤ + 0.05 x C δΤ ), where: index ASFA - the atherogenic index ; C αΤ − the concentration of α-tocopherol; C αΤAc - the concentration of α-tocopheryl acetate; C γΤ − the concentration of γ-tocopherol; C δΤ - the concentration of δ-tocopherol. Tocopherol concentrations - µg g-1. 5 Results were published in the previous paper (Rozbicka-Wieczorek et al. 2016b). A G R I C U LT U R A L A N D F O O D S C I E N C E M. Białek et al. (2020) 29: 405–419 414 Conversely, compared to the control diet, only the experimental diet with VISe decreased the atherogenic ( index ASFA) index (Morán et al. 2013) in the PIF (Table 8), MLD (Jaworska et al. 2016) and MBF (Table 8; Rozbicka-Wieczorek et al. 2016b), whereas in the SCF, index ASFA was higher in lambs fed the CA and YSe-CA diets than in the control and VISe-CA diets (Krajewska-Bienias et al. 2017). As can be seen from the results in Table 8, all experimental diets fed to lambs decreased index TSFA in the PIF in comparison with the control diet. It has been found that the experimental diets enriched in YSe or VISe increased the ΣPUFA/ΣSFA ratio in the PIF, compared to the control and CA diets. The YSe-CA diet most effectively increased the value of the ΣPUFA/ΣSFA ratio in the PIF. As can be seen from the current results, the VISe-CA diet fed to lambs resulted in an increase in the h/H-Ch ratio in the PIF, SCF, MLD and MBF compared to the control and other experimental diets (Table 8). The addition of YSe to the experimental diet increased h/H-Ch only in the PIF, in comparison with the control and CA diets. Discussion Our studies show that short-term dietary supplementation of CA and Se as VISe or YSe (rich in Se-Met) can be used to modify the concentration of FA, TCh and tocopherols in the PIF, SCF and muscles of lambs without adverse- ly influencing performance or causing physiological disorders in the liver, blood and brain (Eun et al. 2013, Roz- bicka-Wieczorek et al. 2016b, 2016c, Czauderna et al. 2017, 2018, Krajewska-Bienias et al. 2017, Przybylski et al. 2017, Białek et al. 2018). Se-compounds are metabolised to intermediates and then utilised for the biosynthesis of Se-Cys- or Se-Met-containing proteins/enzymes in the tissues of mammals (Collins 2017). The most import- ant physiological functions of half of these Se-proteins/enzymes are to maintain the appropriate metabolism of c5c8c11c14C20:4, as well as low levels of free radicals or pre-oxides within cells, thus decreasing oxidative stress and peroxidative damage of UFA, DNA, cholesterol or sulphur-amino acids in proteins in the tissues of mammals (Choe and Min 2009, Čobanová et al. 2017, Czauderna et al. 2011, Saheem et al. 2017). In this context, special attention should be paid to the role of CA from Rosmarinus officinalis. CA is an abietane diterpenoid that is abi- eta-8,11,13-triene substituted by hydroxy groups at positions 11 and 12, and a carboxy group at position 20. In- terestingly, CA exhibits neuroprotective, antiangiogenic, antineoplastic, antioxidant, antimicrobial and anti-HIV activity (Ibarra et al. 2011, Morán et al. 2012b, Jordán et al. 2013, Sasaki et al. 2013, Morán et al. 2017). What is more, CA added to diets has the ability to modify the microbial population in the rumen and, hence, the bio- synthesis yields of volatile compounds, and UFA isomerisation and biohydrogenation in the rumen (Morán et al. 2012a, 2013, Miltko et al. 2016). Our current study and other studies have shown that CA added to diets with or without Se (as YSe or VISe) affected fatty acids profiles, the contents of cholesterol, its oxidation products and the accumulation of carbonyl moieties on proteins produced by oxidative stress in animal tissues (Ibarra et al. 2011, Morán et al. 2012a, Jordán et al. 2013, Rozbicka-Wieczorek et al. 2016a, 2016c). In accordance with the above, all experimental diets reduced the content of MDA, as well as the values of the PUFA peroxidation index in the PIF and SCF (Krajewska-Bienias et al. 2017), compared to the control diet. Impact of the experimental diets on the concentrations of fatty acids in the PIF Our results, summarised in Tables 4–6, showed that all experimental diets reduced the contents of even-long-chain SFA, c11C20:1, c9t11CLA, c11t14C20:2 and EPA in the PIF compared to the control, whereas the YSe-CA and VISe-CA diets decreased the levels of c11t14C20:2, c5c8c11c14C20:4, EPA and DHA, as well as the value of the ∆4-desat- urase index in the PIF in comparison with the control and CA diets. Based on the above results, it can be argued that YSe or VISe added to the diet decreased the capacity of Δ6-, Δ5- and especially Δ4-desaturase required for the biosynthesis of highly unsaturated LPUFA as EPA, DPA and DHA (bio-synthesised from αLNA), and c5c8c11c14C20:4 and c7c10c13c16C22:4 (biosynthesised from LA). This is consistent with the results of a previous study (Krajews- ka-Bienias et al. 2017), in which the YSe-CA and VISe-CA diets also reduced the contents of n-6LPUFA and n-3LPU- FA in the SCF in comparison with the control diet. Moreover, in an earlier study conducted on lambs, it was also found that VISe added to a diet with linseed oil reduced the content of PUFA in the SCF and perirenal fat compared to a diet with linseed oil alone (Czauderna et al. 2012a). As can be seen from the results summarised in Table 5, the experimental diets, especially the YSe-CA diet, fed to lambs resulted in an increase in the capacity of Δ9 desaturation in the PIF and SCF (Krajewska-Bienias et al. 2017) compared to the control diet. Considering the above, we suggested that dietary CA and particularly VISe add- ed to the diet increased Δ9-desaturase capacity via stimulation of stearoyl-CoA desaturase mRNA expression in the PIF. Furthermore, the regulation of stearoyl-CoA desaturase activity is sensitive to diet composition, dietary supplements (like phenolic compounds, minerals or peroxisomal proliferators), as well as hormonal imbalances, A G R I C U LT U R A L A N D F O O D S C I E N C E M. Białek et al. (2020) 29: 405–419 415 developmental processes or temperature changes (Ntambi 1999). In addition, our results reinforce the finding that Δ9-desaturation capacity depends upon the chemical form of a substrate (Rozbicka-Wieczorek et al. 2014). In fact, the experimental diets, particularly those with YSe or VISe, decreased the index of Δ9 desaturation of t11C18:1 (CLA∆9-index) in the PIF compared to the control diet (Table 5); thus, the content of c9t11CLA (the product of Δ9- desaturase) was lower in the PIF (Table 6) and the SCF of lambs fed the experimental diets in comparison with the control diet (Krajewska-Bienias et al. 2017). What is more, dietary YSe or VISe increased the capacity of enzymatic elongation of PUFA in the PIF; in fact, the PUFA elongase index was higher in the PIF of lambs fed the experimental diets with YSe or VISe than the control and CA diets (Table 5). As a consequence, the content of c11c14C20:2 (the substrate for PUFA elongase) was con- siderably lower in the PIF, SCF (Krajewska-Bienias et al. 2017) and MBF (Table 8) of lambs fed the diets enriched in YSe or VISe than the control and/or CA diets. In accordance with the above, the experimental diets, especially the YSe-CA and VISe-CA diets, considerably increased the contents of C21:0 and C23:0 (long-chain SFA). Considering the above, we suggested that CA added to the diet increased the capacity for the enzymatic elongation of odd-chain SFA (the substrates for elongase: C15:0, C17:0, C19:0 and C21:0) in the PIF compared to the control diet. Indeed, these enzymes are regulated/affected by dietary supplements, developmental processes, hormones and chron- ic diseases. As a consequence, changes in elongase activities impact fatty acid profiles in mammal tissues (Jump 2009). Moreover, YSe and especially VISe added to the experimental diet enhanced the capacity of odd-chain fatty acid elongases compared to the diet with only CA. Hypocholesterolemic/hypercholesterolemic FA ratio, and atherogenic and thrombogenic indices of lambs’ tissues Our current and previous studies have suggested that the experimental diets containing CA with or without YSe stimulated atherogenic properties in the PIF and SCF (Krajewska-Bienias et al. 2017). In fact, these experimental diets increased the concentration of A-SFA in the SCF, as well as the values of index ASFA in the PIF and SCF. Further- more, atherogenesis, a multi-factor process, is stimulated by A-SFA (especially C14:0), and reduced by dietary n-3PUFA, as well as tocopherols (antioxidants). Recent studies have documented that diets enriched in tocopherols and plant oils rich in n-3PUFA have an antiatherogenic effect (i.e. reduced aortic cholesterol content, intimal lipid infiltration and discrete alterations to the middle layer of the arterial wall) (Saini et al. 2012, Haliga et al. 2015). In fact, “the oxidation theory” of antioxidant systems and atherosclerosis argues that oxidation of low density lipo- proteins (LDL) significantly contributes to atherogenesis. Dietary antioxidants (i.e. tocopherols or tocotrienols) and coantioxidants (ascorbate and ubiquinol-10) prevent the oxidation of LDL, cholesterol and UFA in lipids, thus, de- laying the atherogenesis process in animals and humans (Salvayre et al. 2016). Considering the above, we argued that the atherogenic index ( index ASFA) should be calculated based on the contents of pro-atherogenic saturated fatty acids (i.e. A-SFA), MUFA, n-6PUFA and n-3PUFA, as well as the contents of anti-atherogenic tocopherols in tissues. Thus, it seems reasonable to assume that the modified atherogenic index ( index ASFA+Toc) should be calculated in the following manner: index ASFA+Toc = index ASFA / (1.49 × C αΤ + 1.36 × C αΤAc + 0.15 × C γΤ + 0.05 × C δΤ ) where: C αΤ , C αΤAc , C γΤ and C δΤ – the concentrations of α-tocopherol, α-tocopheryl acetate, γ-tocopherol and δ-to- copherol respectively; 1.49, 1.36, 0.15 and 0.05 are coefficients of the biological activity of tocopherols (Leth and Søndergaard 1977). In fact, all experimental diets, especially the YSe-CA diet, reduced the values of the modified atherogenic index ( index ASFA+Toc) in the PIF, SCF and MLD compared to the control diet. Moreover, our current stud- ies (Table 7) and previous investigations of cholesterol biosynthesis in the liver (Rozbicka-Wieczorek et al. 2016c) documented that VISe and/or CA added to the diet revealed anti-cholesterolemic activity. Indeed, supplementation with VISe or especially with selenite reduced the activity of HMG-CoA reductase (3-hy- droxy-3-methyl-glutaryl-coenzyme A reductase) (Nassir et al. 1997). This enzyme is the rate-controlling enzyme of the mevalonate pathway, the metabolic pathway that synthesizes cholesterol as well as other isoprenoids. Interestingly, HMG-CoA reductase possesses cysteine residues and is subject to regulation by thiol-disulfide ex- change (Nassir et al. 1997). As a consequence, supplementation with CA (the effective antioxidant), VISe or par- ticularly with selenite (Czauderna and Samochocka 1981) affects thiol-disulfide exchange equilibria and disul- fide bond stability of HMG-CoA reductase. Moreover, the effect of dietary CA on cholesterol reduction may be attributed to a decrease in the micellar solubilisation of cholesterol in the digestive tract, to a stimulation in bile flow, bile cholesterol and bile acid content and to a subsequent increase in the faecal excretion of steroids A G R I C U LT U R A L A N D F O O D S C I E N C E M. Białek et al. (2020) 29: 405–419 416 (Ibarra et al. 2011, Afonso et al. 2013). Our current study and previous investigations suggest that dietary CA pro- tects against hypercholesterolemia-induced oxidative stress, increasing the activities of antioxidant enzymes and decreasing the concentrations of reactive substances (Afonso et al. 2013, Jordán et al. 2013). Therefore, we argued that CA with or without Se (as YSe and VISe) reduced the concentrations of radicals (like ROS and RNS) in ovine tis- sues. As a consequence, the contents of MDA and the values of MDA index were lower in the PIF (Table 7) and SCF (Krajewska-Bienias et al. 2017) of lambs fed the experimental diets than the control diet. Moreover, we suggested that the experimental diets enriched in antioxidants reduced the oxidative degradation of tocopherols and stimu- lated the regeneration of degraded tocopherols in ovine tissues (Choe and Min 2009, Morán et al. 2012a, 2012b, 2017, Jordán et al. 2013, Haliga et al. 2015, Čobanová et al. 2017). In our opinion, compared with the atherogenic index ( index ASFA), the modified atherogenic index ( index ASFA+Toc) provides more detailed insights into the mechanisms of atherogenesis and, thus, possesses better predictive properties. Indeed, the effect of experimental diets enriched in antioxidant(s) on the values of index ASFA+Toc is more consistent than on the values of index ASFA in all assayed tissues. The values of our modified atherogenic index ( index ASFA+Toc) confirmed that the experimental diets including antiox- idants (i.e. CA, VISe and especially YSe) reduced the risk of atherogenesis (Saini et al. 2012, Salvayre et al. 2016). In this and earlier studies, we have confirmed that the CA and YSe-CA diets reduced the thrombogenic proper- ties in the PIF, SCF (Krajewska-Bienias et al. 2017), the rumen-surrounding fat (Białek and Czauderna 2019) and MLD (Jaworska et al. 2016) compared to the control. In fact, the CA and YSe-CA diets reduced index TSFA in these tis- sues more efficiently than the control diet. Similarly, compared with the control diet, the VISe-CA diet decreased index TSFA or T-SFA in the PIF, SCF and MLD, as its values were lower than in the control tissues. Our current and our recent studies have also showed that the experimental diet with VISe increased the ∑PUFA/∑SFA ratio in the PIF, MLD and MBF, as well as the h/H-Ch ratio in the PIF, SCF, MLD and MBF of lambs compared to the control. Con- sidering the above, we argued that the experimental diets, especially the YSe-CA diet, improved the health and welfare of lambs (which probably applies to other ruminants), as well as the nutritional value of lambs’ meat (i.e. MLD and MBF) and the SCF for humans. Indeed, the high ∑PUFA/∑SFA and h/H-Ch ratios and the low values of atherogenic and thrombogenic indices in MLD and MBF (Jaworska et al. 2016, Rozbicka-Wieczorek et al. 2016b) documented their suitability for healthier diets, since these diets are successful in slowing down the atheroscle- rosis and thrombogenesis processes. As a consequence, the meat of lambs fed the YSe-CA diet in particular may decrease the threat of atrial fibrillation, coronary thrombosis and the risk of cardiovascular disorders in humans (Fernández et al. 2007, Haliga et al. 2015, Salvayre et al. 2016). Conclusion The current study is important for the understanding of the influence of dietary CA, YSe and VISe on the contents of FA, cholesterol and tocopherols in the adipose tissues and muscles of lambs. Alongside earlier studies performed on ruminal microbiota, the present research confirmed that dietary YSe and VISe, as well as CA, have the ability to modify the metabolism of FA in ruminal microorganisms, adipose tissues and muscles of lambs. Our data indi- cated that dietary CA combines antioxidant and hypocholesterolemic activities. The modified atherogenic index strongly suggests that the experimental diets containing CA, irrespective of the presence of YSe or VISe, decreased the atherogenic capacity of the PIF, SCF, MLD and MBF compared to the control diet. In our opinion, the modified atherogenic index ( index ASFA+Toc) better assesses the atherogenic capacity of tissues, as it takes into consideration the contents of pro-atherogenic SFA, as well as the contents of anti-atherogenic tocopherols in the analysed tis- sues. Therefore, we argued that the modified atherogenic index is a better assessment tool for the atherogenic properties of tissues than the commonly used atherogenic index ( index ASFA). Our studies have shown that the PIF, MLD and MBF of lambs fed the VISe-CA diet were characterised by the highest ΣPUFA/ΣSFA and hypocholester- olemic/hypercholesterolemic FA ratios, as well as lower atherogenic and thrombogenic indices and contents of TCh and MDA compared to the control and other experimental diets. Therefore, we argued that the experimental diet with CA and VISe in particular, improved lambs’ health and welfare, as well as the dietary properties of ani- mal products for human consumption. Further studies are necessary to determine if diets containing CA and Se (as YSe or VISe) along with FO and RO in- duce changes in the content of FA, tocopherols, cholesterol and MDA in the perirenal fat of lambs. Moreover, new studies are necessary to determine if dietary supplementations with Se (as YSe or VISe) and carnosol (the oxida- tion product of CA) affect the bioaccumulations of FA (especially n-3LPUFA and conjugated PUFA), tocopherols, tocotrienols and peroxidation products of cholesterol and steroid hormones in adipose tissues, muscles and, in particular, in the liver, brain and heart of lambs. The current studies also provides useful knowledge for nutrition- ists carrying out further studies aimed at improving the health and welfare of farm animals, as well as the pro- healthy properties of animal products for human consumption. A G R I C U LT U R A L A N D F O O D S C I E N C E M. Białek et al. (2020) 29: 405–419 417 Acknowledgments This study was in part supported by the National Science Centre (Grant No. 2013/09/B/NZ9/00291) and by the statutory funds from The Kielanowski Institute of Animal Physiology and Nutrition, PAS (Poland). 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Journal of Nutrition 146: 1625–1633. https://doi.org/10.3945/jn.116.229955 Diets enriched in fish and rapeseed oils, carnosic acid, anddifferent chemical forms of selenium affect fatty acid profilein the periintestinal fat and indices of nutritional properties ofselected tissues of lambs Introduction Materials and methods Animals, housing, experimental design, diets, management and sampling Chemicals and analytical methods Preparation of fatty acid methyl esters (FAME) in ovine tissues Determination of tocopherols, TCh and MDA in ovine tissues Statistical analysis Results Concentrations of selected saturated fatty acids (SFA) in the PIF of lambs Concentrations of unsaturated fatty acids in the PIF of lambs Concentrations of TCh, tocopherols and MDA, and values of atherogenic,thrombogenic and hypocholesterolemic indices in selected tissues of lambs Discussion Impact of the experimental diets on the concentrations of fatty acids in the PIF Hypocholesterolemic/hypercholesterolemic FA ratio, and atherogenic andthrombogenic indices of lambs’ tissues Conclusion Acknowledgments References