45 † Corresponding author © 2014 Conscientia Beam. All Rights Reserved. SELENO-CYSTINE AFFECTS THE FATTY ACID PROFILE IN IN VITRO INCUBATED OVINE RUMINAL FLUID CONTAINING -LINOLENIC ACID Marian Czauderna1† --- Rozbicka Wieczorek A.J.2 --- Więsyk E.3 1, 2, 3The Kielanowski Institute of Animal Physiology and Nutrition, Polish Academy of Sciences, Poland ABSTRACT The influence of seleno-cystine (CySe2) added to ovine ruminal fluids containing -linolenic acid (LNA) on the profile of fatty acids (FA) was investigated. Fluids were incubated in vitro at 39°C under CO 2 either alone (RF) or with LNA (1.67 mg/ml) or with a combination of LNA with either a low (1.34 μg/ml) or high (3.33 μg/ml) level of Se as CySe2. Fluids were removed after 0, 6, 12, 18, 24 hrs of incubation and then analyzed to determine FA levels. LNA added to the fluids without/with CySe2 decreased the C180 concentration for incubation at all times from 6 hrs compared with the RF or the fluid containing CySe2. LNA added to the fluids without/with CySe2 decreased the biohydrogenation yield to C18:0. CySe2 added to the fluids decreased the C18:0 concentration and the index of the biohydrogenation to C18:0 compared with the RF. The higher concentration of CySe2 in the fluids with LNA reduced the accumulation of trans11C18:1 for incubation at all times from 18 hrs compared with the fluids with LNA, irrespective of the presence of the lower concentration of CySe2. The lowest concentration of trans11C18:1 in the fluids with LNA and the higher concentration of CySe2 correlated with the lowest yield of the isomerization of LNA into cis9trans11cis15C18:3 and the lowest yield of the initial biohydrogenation of cis9trans11cis15C18:3 to trans11cis15C18:2 in the fluids containing LNA and the higher concentration of CySe2. CySe2 added to the fluids with LNA decreased the ratio of polyunsaturated FA to saturated FA for incubation at all times from 12 hrs compared with the fluids containing LNA. CySe2 in the fluids without/with LNA reduced the FA sum in the fluids. Keywords: Selenium, -Linolenic acid, Ovine ruminal fluids, Fatty acids, Biohydrogenation, Isomerization. Contribution/ Originality Our original study documents that CySe2 added to the ovine ruminal fluids, irrespective of the presence of αLNA, affects concentrations of fatty acids, the capacity of the bacterial isomerases and the biohydrogenation yield of unsaturated fatty acids in in vitro incubated ruminal fluids compared with the control fluid. Animal Review 2014 Vol. 1, No. 3, pp. 45-56 ISSN(e): 2409-6490 ISSN(p): 2412-3382 © 2014 Conscientia Beam. All Rights Reserved. Animal Review, 2014, 1(3): 45-56 46 © 2014 Conscientia Beam. All Rights Reserved. 1. INTRODUCTION A large proportion of selenium (Se) in feedstuffs is present in organic compounds, as seleno- cysteine (CySe) and especially as seleno-methionine (Se-Met), and forms a part of the amino acid structure of protein molecules in the feed [1, 2]. Rumen microorganisms can incorporate CySe and Se-Met into their own proteins, but can also reduce an excess of dietary Se-compounds into inorganic forms (like selenide or Seo) that are largely unavailable to the ruminant [3, 4]. Ruminal microbial Se concentrations were enriched relative to the concentration of Se in rations. Indeed, the concentration of Se was significantly higher than that of the ration, whether considered relative to diet dry matter (average of 46-fold), nitrogen (average of 11.3-fold) or sulfur abundance (average of 26-fold) [5]. Moreover, the enrichment of Se in microbial cells (2-78-fold) was greater than the enrichment of either nitrogen (average of 4-fold) or sulfur (average of 1.6- fold). Ruminal microorganisms reduce much of dietary inorganic Se (as selenite or selenate) to unabsorbable elemental Se or inorganic selenide forms. Bacteria are also able to synthesize Se- Met and CySe, and then these Se-amino acids (Se-AA(s)) are incorporated into microbial proteins. Thus, dietary Se finds its way into a form metabolizable by ruminants, and so the way to improve the healthiness of ruminant meat and milk by increasing the concentration of CySe and Se-Met [6, 7]. Recent investigations demonstrated that n-3 polyunsaturated fatty acids (PUFA) like - linolenic acid (LNA) and long-chain PUFA (LPUFA) possessed also several potential health benefits, including cancer prevention, decreased atherosclerosis, improved immune response and altered fatty acids (FA) and protein metabolism [8-11]. Numerous in vitro and in vivo studies documented that ruminant dietary C18-PUFA like linoleic acid (LA) or LNA are direct incorporated into the ruminal bacteria, isomerized to other geometrical and positional isomers, metabolized into conjugated linoleic acid (CLA) isomers [8, 10, 12-14], as well as biohydrogenated to trans11C18:1 (t11C18:1) and finally to C18:0. Indeed, C18-PUFA, especially LPUFA, have toxic effects on cellulolytic bacteria and protozoa, act against ruminal lactate producers thereby favouring propionate producers [15-17]. Interestingly, unsaturated fatty acids (like LNA, LA or cis9C18:1) inhibited bacterial enoyl-acyl carrier protein reductase, an essential component of bacterial fatty acid synthesis [18]. Unsaturated fatty acids (UFA) are more toxic than saturated fatty acids (SFA) and can inhibit fermentation in the rumen more intensively [19]. UFA are especially toxic to G+ bacteria, whereas G- bacteria are less sensitive to fatty acids at the same concentration [20]. Biohydrogenation is a microbial pathway in ruminal contents designed to reduce unsaturation of lipids found within plant matter, and is likely an evolutionary adaptation to protect the microbial population from antimicrobial effects of unsaturated fatty acids (UFA) [8, 13]. Current studies indicated that t11C18:1, the intermediate biohydrogenation product of αLNA and LA formed in a rumen, may also be of benefit in the prevention of cancer due to endogenous conversion of t11C18:1 to cis9,trans11CLA (c9t11CLA) in mammalian tissues [21-24]. In our recent studies it was found that the concentration of UFA in a body of animals, as well as in membrane of microorganisms cells were positively correlated with the Se level in a diet [25-28]. Indeed, Se is an integral component of antioxidant enzymes (e.g. glutathione Animal Review, 2014, 1(3): 45-56 47 © 2014 Conscientia Beam. All Rights Reserved. peroxidases) that can decrease the risk of PUFA peroxidation in particular [29, 30]. Moreover, our recent studies revealed that of selenate or selenite changed concentrations of FAs and CLA isomers in particularly in incubated ovine ruminal fluid muscle [23, 24]. Considering the above, we hypothesized that Se-cystine (CySe2) added to the ovine ruminal fluids affected bacterial isomerization of -linolenic acid (LNA) and reduced the biohydrogenation of UFA in in vitro incubated ruminal fluid. Therefore, the major objective of the current study was to examine the hypothetically effect of CySe2 on the concentration of UFA, especially LNA, their geometric and positional isomers, in in vitro incubated ovine ruminal fluids containing LNA. 2. MATERIALS AND METHODS 2.1. Animals and Diets Eight ruminally fistulated adult sheep received a mixed diet comprising grass hay, barley, molasses, soybean meal and minerals and vitamins, at 500, 299.5, 100, 91 and 9.5 g/kg dry matter respectively, fed in equal meals of 500 g at 8.00 and 16.00 h [23]. Ruminal digesta samples were taken before feeding in the morning from each sheep. The ruminal fluid was kept at 39C and strained through linen cloth before use. 2.2. Reagents CySe2, LNA and fatty acid methyl ester standards were purchased from Sigma (Poole, Dorset, UK). Other reagents were of analytical grade and were from POCh (Gliwice, Poland). Water used for the preparation of mobile phases and chemical reagents was prepared using an ElixTM water purification system (Millipore). 2.3. Incubations with Ruminal Fluid in Vitro Strained ruminal fluids (SRF) were incubated either alone or with a combination of LNA and two concentrations of CySe2 to determine the interactions in the metabolism of LNA. All in vitro experiments were performed on four different days using samples withdrawn from eight different sheep. In general, 1 ml of strained ruminal fluid was added under CO2 to Pyrex tubes (120 x 11 mm) containing 0.2 ml of water solution containing either alone (the positive control groups) or with a combination of LNA, a low (l) or high (h) level of CySe2 (Table 1). The tubes with the ovine ruminal fluids were incubated at 39°C. The tubes were removed after 0, 6, 12, 18 or 24 hrs of in vitro incubation, heated to inactivate ruminal microbial enzymes for 9–10 min in a block heater at 100°C and stored at –20°C before being submitted for the quantification of FA. The FA were extracted, methylated and analyzed as described below. 2.4. Fatty Acid Extraction and Preparation of Fatty Acid Methyl Esters (FAME) The method of alkaline saponification and extraction was as described previously [24]. Derivatization of the extracted free fatty acids to FAME was carried out using a procedure that Animal Review, 2014, 1(3): 45-56 48 © 2014 Conscientia Beam. All Rights Reserved. contained mild, base- and acid-catalyzed methylation steps that minimized positional and geometrical isomerization of UFA [24]. FAME were then analysed using gas chromatography (GC) according to Czauderna, et al. [24]. The analyses of all FAME in ruminal fluid samples were performed on a SHIMADZU GC-MS QP2010 Plus EI equipped with a BPX70 fused silica capillary column (120 m x 0.25 mm i.d. x 0.25 µm film thickness; SHIM-POL), a quadrupole mass selective detector (Model 5973N) and an injection port. FAME identification was validated based on the electron impact ionization spectra of FAME and compared with authentic FAME standards and the NIST 2007 reference mass spectra library. 2.5. Statistical Analyses Statistical analyses were performed using the Statistica software package (StatSoft, Version 10, 2010). Statistical analyses of the effects of LNA and CySe2 on the concentrations of selected FA in in vitro incubated ruminal fluids were conducted using the non-parametric Mann-Whitney U test. The results are presented as the means of the individually analyzed ruminal fluid samples. Mean values in the columns with different superscripts are significantly different at a,bP < 0.05 and A,BP < 0.01. 3. RESULTS 3.1. The Influence of CySe2 on the Accumulation of Selected Saturated Fatty Acids (SFA) in in Vitro Incubated Ruminal Fluids with  LNA Although factors altering the microbial population and ruminal fermentation are undoubtedly keys to controlling the yield of the biohydrogenation and synthesis of positional or geometric isomers of fatty acids very few studies have directly associated production of fatty acid isomers and their products of the biohydrogenation in ruminal fluids enriched in Se-compounds [12-14, 23, 24, 31, 32]. Therefore, in vitro studies were conducted to determine the effect of CySe2 on the concentrations of selected fatty acids in ovine ruminal fluids containing extra LNA. The results summarised in Table 2 documented that the addition of LNA to the incubated ruminal fluids, irrespective of the presence of CySe2, decreased the concentration of C180 from 6 to 24 hrs of in vitro incubation compared with the control ruminal fluid (RF) or the fluid containing only CySe2, regardless of its concentration. On the other hand, CySe2 added to the ruminal fluids revealed minute influence on the accumulation of C18:0 compared with the RF; indeed, CySe2, in dose dependent manner, slightly reduced the concentration of C18:0 from 12 to 24 hrs compared with the RF. Of interest is the observation that the addition of LNA to the fluids with CySe2, irrespective of its concentration, slightly stimulated the accumulation of C14:0 from 6 to 24 hrs compared with the RF and other ruminal fluids with LNA or CySe2. Moreover, LNA added to the ruminal fluid, irrespective of the presence of CySe2, stimulated the accumulation of C20:0 and C22:0 especially from 18 to 24 hrs of incubation compared with the RF and the fluids with CySe2. On the other hand, all additives in the incubated fluids revealed negligible influence on the concentrations of C15:0 and C16:0 as well as the concentration sum of all assayed SFA (SFA) compared with the RF. Animal Review, 2014, 1(3): 45-56 49 © 2014 Conscientia Beam. All Rights Reserved. 3.2. The Influence of CySe2 on the Accumulation of Selected Mono- and Poly-Unsaturated Fatty Acids in in Vitro Incubated Ruminal Fluids with LNA As can be seen from the results summarized in Table 3, all additives in the incubated ruminal fluids revealed negligible influence on the concentrations of c9C14:1 and c9C16:1 compared with the RF. Similarly, the addition of CySe2 to the fluids enriched in LNA revealed negligible influence on the concentrations of c9C18:1 and t11C18:1 (TVA) as well as the concentration sums of cMUFA and tC18:1 compared with the fluids containing only LNA. The concentrations of c9C18:1, t11C18:1 and cMUFA were higher from 6 to 24 hrs of incubation in the fluids with CySe2 and LNA than in the fluids containing only CySe2. As can be seen from results summarized in Table 4, CySe2 or/and LNA added to the ruminal fluids affected the concentrations of PUFA in in vitro incubated ruminal fluids. The concentration of t11c15C18:2 (tcC18:2) increased throughout the incubation in the ruminal fluids enriched in LNA, whereas decreased in the RF and the fluids containing only CySe2. The addition of CySe2 to the fluids with LNA reduced the concentrations of tcC18:2 and c9t11c15C18:3 (ctcC18:3) from 18 to 24 hrs of incubation compared with the fluids containing only LNA. The concentrations of ctcC18:3, t9t11c15C18:3 (ttcC18:3) and LNA were quantitatively detected in the fluids containing LNA; the addition of CySe2 to the fluids with LNA revealed negligible influence on the concentrations of ttcC18:3 and LNA as well as on the concentration sum of PUFA (PUFA) compared with the fluids with LNA. The addition of CySe2 to the ruminal fluids with LNA slightly decreased the concentration ratio of PUFA to SFA (PUFA/SFA) from 12 to 24 hrs of incubation compared with the fluids with only LNA. Moreover, CySe2 added to the ruminal fluids without or with LNA, decreased the concentration sum of all assayed fatty acids (FA) in incubated fluids compared with the RF and the fluids with only LNA, respectively. As observed from the obtained results (Table 4), LNA or/and CySe2 added to the ruminal fluids changes the indexes of the initial, intermediate and final biohydrogenation in incubated fluids compared with the RF. 4. DISCUSSION Wina, et al. [33] documented that the contents of the major ruminal microorganisms, including protozoa, were found to be kept at higher levels than after 24 hrs in in vitro incubation. Considering the above, the in vitro incubation periods in the current investigation were set to 6, 12, 18 and 24 hrs to determine the influence of CySe2 and/or LNA on the concentration of FA in incubated ovine ruminal fluids. Interestingly, the concentration of protozoa in incubated fluids was highest at 12 hrs of in vitro incubation, whereas only slightly lower (15%) after 24 hrs of incubation compared with the initial concentration of protozoa [33]. Our studies and other investigations have shown that pH of ruminal fluids containing LNA slightly decreases after 24 hrs of in vitro incubation of ovine fluids; the pH value is reduced by approx. 0.5 [34, 35]. Ruminal microorganisms may use the biohydrogenation as a method of defence against toxicity of UFA, especially PUFA (like LNA or LA). Therefore, the first step of the biohydrogenation consists of the enzymatic bacterial isomerisation, which turns cis bonds into Animal Review, 2014, 1(3): 45-56 50 © 2014 Conscientia Beam. All Rights Reserved. trans bonds. This isomerisation step is rapid compared with the biohydrogenation step [8]. As can be seen from data summarized in Table 2, LNA added to the ruminal fluids, irrespective of the presence of CySe2, decreased the yield of the final biohydrogenation (fBH) to C18:0. Considering the above, we argue that LNA decreased the activity of group B bacteria in a rumen [36]. Indeed, group A included the ruminal bacteria that hydrogenate PUFA (e.g. LNA or LA) into t11C18:1 where their effect was thought to end; on the other hand, group B ruminal bacteria were thought to be capable of converting the same PUFA as those of group A bacteria, but they are more capable of biohydrogenating a wider range of fatty acids, ending with C18:0 flow from the rumen [36]. As consequence, the addition of LNA to the fluids, regardless of the presence of CySe2, revealed negligible influence on the concentration of C14:0, C15:0 and C16:0 in incubated fluids. Indeed, these saturated fatty acids are not the products of the fBH [8]. The current results documented that added CySe2, in dose depended manner, reduced the formation of C22:0 in the fluids containing LNA compared with the fluids with only LNA from 18 to 24 hrs of the incubation; so, our results suggested that added CySe2 decreased the capacity of the elongation of saturated fatty acids in the incubated ruminal fluids enriched in LNA. Our investigations revealed that CySe2 added to the fluids without or with LNA reduced the synthesis of fatty acids or/and increased their oxidation. Therefore, the concentration of FA was lower in the incubated fluids containing CySe2 without or with LNA than in the RF or the ruminal fluids with LNA. Our current studies documented that the higher concentration of CySe2 in the ruminal fluids enriched in LNA reduced the accumulation of t11C18:1 from 18 to 24 hrs of incubation compared with the fluids containing LNA, irrespective of the presence of the lower concentration of CySe2 (Table 3); t11C18:0 is the product of isomerization and/or the intermediate biohydrogenation of unsaturated fatty acids (C18:2), like t11c15C18:2 [25]. Indeed, the higher concentration of CySe2 in the ruminal fluids containing LNA most efficiently reduced the index of the initial biohydrogenation of c9t11c15C18:3 and t9t11c15C18:3 to t11c15C18:2 from 18 to 24 hrs of incubation (Table 4). Moreover, the lowest concentration of t11C18:1 in the fluids with LNA and the higher concentration of CySe2 well correlated with the lowest yield of the bacterial isomerization of LNA into c9t11c15C18:3 (ctcC18:3) and the lowest yield of the initial biohydrogenation of ctcC18:3 to t11c15C18:2 (tcC18:2) in the incubated fluids containing LNA and the higher concentration of CySe2 (Table 4). Therefore, the concentration of ctcC18:3 and tcC18:2 in the fluids enriched in LNA and the higher concentration of CySe2 was lower than in the fluids with LNA, irrespective of the presence of the lower concentration of CySe2, from 12 to 24 hrs of the incubation. Considering the above results, we argued that CySe2 added to the incubated fluids with LNA reduced the yield of the bacterial isomerization of LNA (Table 3) as well as the initial biohydrogenation of ctcC18:3 in dose dependent manner from 18 to 24 hrs of the incubation (Table 4). Moreover, we suggest that LNA added to the incubated fluids, irrespective of the presence of CySe2, reduced the capacity of the final biohydrogenation to C18:0 compared with the RF (Tables 2 and 4). Moreover, CySe2 added to the ruminal fluids decreased the concentration of C18:0 as well as the index of the final biohydrogenation to C18:0 compared with Animal Review, 2014, 1(3): 45-56 51 © 2014 Conscientia Beam. All Rights Reserved. the RF. Current observations are also consistent with our recent in vitro studies that have reported that selenate or selenite added to the ruminal fluids containing LNA reduced the capacity of the bacterial isomerization of C18-PUFA and lowered the yield of the final biohydrogenation to C18:0 compared with the fluids with only LNA [24]. Interestingly, LNA or/and CySe2 added to the ruminal fluids revealed an inconsistent effect on the index values of the intermediate biohydrogenation (Table 4) involved in the formation of t11C18:1 (TVA). We suggest that this inconsistent effect may be due to the fact that t11C18:1 is the substrate which is consumed during the final biohydrogenation. Generally, the index values of the intermediate biohydrogenation to t11C18:1 were higher in the fluids containing LNA and the higher or lower concentration of CySe2 compared with the ruminal fluids with only LNA. On the other hand, our results suggest that the higher concentration of CySe2 in the fluids with LNA usually stimulated the yield of elongation of c9c12C18:2 to c13c16C22:2 (i.e. the unsaturated fatty acids) compared with the incubated fluids containing only LNA. Surprisingly, CySe2 added to the ruminal fluids with LNA decreased the concentration ratio of PUFA to SFA (PUFA/SFA) for incubation at all times from 12 hrs compared with the fluid containing LNA (Table 4). 5. 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New York: HNB Publishing, 2005. Animal Review, 2014, 1(3): 45-56 54 © 2014 Conscientia Beam. All Rights Reserved. Table-1. The scheme of in vitro experiments on ovine ruminal fluids1 Group Additive Additive concentration Number (n) of fluid samples individually analysed in every time2 RF3 Water - n=6 L LNA 1.67 mg/ml n=6 Sel CySe2 1.34 g/ml n=5 Seh CySe2 3.33 g/ml n=5 L-Sel CySe2 LNA 1.33 g/ml 1.67 mg/ml n=6 L-Seh CySe2 LNA 3.33 g/ml 1.67 mg/ml n=6 1 1 ml of ovine ruminal fluids was added to 0.2 ml of water solution containing either solely -linolenic acid (LNA), seleno-cystine (CySe2) at different concentrations (l - low; h - high), or their mutual combinations 2 n – ovine ruminal fluid samples collected from n sheep at different times and individually analyzed. All results in Tables 2-4 are mean values from n fluid samples individually analysed in every time 3 RF = the negative control group (in vitro incubated 1ml of the ovine ruminal fluid with 0.2 ml of water (the control ruminal fluid) Table-2. Effects1 of two levels (low, l; high, h) of Se-cystine (CySe2) on metabolism of LNA and the concentration of selected saturated fatty acids (SFA) and the concentration sum of SFA (SFA) (g/ml) in in vitro incubated ruminal fluids Group and in vitro incubation time, hrs C 1 4 :0 C 1 5 :0 C 1 6 :0 C 1 8 :0 C 2 0 :0 C 2 2 :0  S F A RF2 0 3.8 L 0 3.4a Sel 0 3.5a Seh 0 3.7a L-Sel 0 3.7a L-Seh 0 3.7a 23.5 24.0a 24.7a 24.2a 24.6a 24.7a 66 72a 66a 69a 74a 73a 93 99a 97a 94a 100a 100a -3 - - - - - 4.3 0.7Bb 4.5a 4.6a 0.8b - 191 199ab 195ab 195ab 203ab 202ab RF 6 4.4 L 6 5.1ab Sel 6 4.5a Seh 6 4.6a L-Sel 6 5.4b L-Seh 6 5.3ab 25.9 25.6a 24.4a 24.6a 25.8a 25.2a 79 99ab 76a 77a 103b 98ab 105 101a 106a 105a 104a 100a 0.9 7.8B - - - - 4.9 1.4b 4.7a 4.7a - 2.7b 220 239abc 216abc 216abc 238bc 231abc RF 12 5.4 L 12 5.5b Sel 12 5.3ab Seh 12 5.3ab L-Sel 12 5.9b L-Seh 12 6.4b 25.6 25.3a 24.7a 25.0a 26.6a 25.3a 94 102ab 90ab 91ab 106b 104b 127 101a 121b 119ab 104ab 102a 1.1 6.3B 1.0Aa - 10.9B 4.8Bb 3.2 2.8b 4.2a 4.4a 4.4a 2.6b 256 243bc 246bc 244bc 258bcd 245bcd RF 18 5.7 L 18 5.9b Sel 18 6.1b Seh 18 5.9b L-Sel 18 6.3b L-Seh 18 6.4b 25.1 25.8a 25.6a 24.7a 25.2a 25.6a 95 105b 98ab 96ab 107b 111b 132 100a 130b 124ab 104ab 102a 1.0 4.4B 1.0Aa 1.0Aa 8.4B 8.9B 4.5 9.0c 4.4a 4.5a 6.3ac 6.1ac 263 250cd 265cd 257cd 257cd 260cd RF 24 5.8 L 24 6.4b Sel 24 6.2b Seh 24 6.0b L-Sel 24 6.5b L-Seh 24 7.0b 26.9 26.3a 25.4a 24.6a 24.4a 26.5a 102 106b 100ab 99ab 106b 111b 142 101a 135b 131ab 101a 106ab 2.0 6.8B 1.9Aa 1.8A 3.7ABb 3.9ABb 3.8 12.8Ac 4.1b 3.4ab 10.3c 5.9a 283 260cd 273cd 266cd 252bcd 260cd 1 means in columns with the different letters are significantly different at (a,b)P < 0.05 or at (A,B)P < 0.01 2 RF – in vitro incubated ruminal fluids without the additives (the control ruminal fluid) 3 below the quantification limit (LOQ); LOQ was defined as 10 times the average noise level (LOQ was determined according to Lin and McKeon [37]) Animal Review, 2014, 1(3): 45-56 55 © 2014 Conscientia Beam. All Rights Reserved. Table-3. Effects1 of two levels (low, l; high, h) of Se-cystine (CySe2) on metabolism of LNA and the concentration of selected monounsaturated fatty acids (MUFA) and the concentration sums of cMUFA (cMUFA)2 and tC18:1 (tC18:1)3 (g/ml) in in vitro incubated ruminal fluids Group and in vitro incubation time, hrs c9 C 1 4 :1 c 9 C 1 6 :1 c9 C 1 8 :1 t 9 C 1 8 :1 t1 1 C 2 1 8 : 1  cM U F A  tC 1 8 :1 RF 0 4.5 L 0 4.4a Sel 0 4.4a Seh 0 4.6ab L-Sel 0 4.5a L-Seh 0 4.5ab 4.5 1.4b 4.4ad 4.6ad 2.5ab 2.6ab 7.7 -4 7.6a 8.2a 2.8Ab 2.5Ab 3.8 2.9a 6.0b 3.9ab - - 1.9 - - 2.2a - - 16.7 5.8Bb 16.5a 17.4a 9.8b 9.6b 5.8 2.9Ab 6.0a 6.1a - - RF 6 5.5 L 6 6.9ab Sel 6 5.3ab Seh 6 5.5ab L-Sel 6 7.1b L-Seh 6 6.9ab 5.5 6.1cd 5.2cd 5.2cd 6.4cd 6.0cd 8.4 16.1Bc 7.3a 7.8a 13.9Bc 14.7Bc 5.3 - 7.6b 8.2bc - 3.1a 2.3 7.3b - - 9.3Ab 3.2a 19.4 29.1A 17.8a 18.5a 27.4Ac 27.6Ac 7.6 7.3a 7.6a 8.2a 9.3a 6.3a RF 12 6.0 L 12 7.2b Sel 12 5.8ab Seh 12 6.0ab L-Sel 12 7.5b L-Seh 12 7.2b 6.4 4.8ad 6.0cd 6.1cd 6.8cd 6.9cd 6.3 16.8Bc 5.9a 6.8a 17.3Bc 16.9Bc 6.6 - 6.2b 7.2b - - 4.0 9.2b 4.2a 4.0a 9.9b 9.4a 18.7 28.8Cc 17.7a 18.9a 31.7Cc 31.0Cc 10.5 9.2a 10.4a 11.2a 9.9a 9.4a RF 18 6.2 L 18 7.5b Sel 18 6.5ab Seh 18 6.3ab L-Sel 18 7.6b L-Seh 18 7.5b 7.7 7.7cd 6.6cd 6.3cd 7.0cd 7.1cd 2.5 17.2Bc 3.1Ab 3.9Ab 17.3Bc 14.4Bc 5.9 2.1a 6.2b 6.8b - 2.6a 3.9 15.1Bc 4.3a 5.3a 15.5Bc 14.1Bc 16.5 32.4Cc 16.2a 16.5a 31.9Cc 29.0Cc 9.8 17.2Ba 110.4a 12.0a 15.5Ba 16.7Ba RF 24 6.7 L 24 8.0b Sel 24 6.4ab Seh 24 6.4ab L-Sel 24 7.8b L-Seh 24 8.0b 9.2 7.0cd 6.6cd 6.4cd 7.5cd 7.3cd 1.0 17.0Bc 1.9Ab 2.6Ab 16.3Bc 11.9Bc 6.2 4.6ab 3.7a 3.1a 3.0a 15.1d 1.1 19.8Bc 5.7ab 6.2b 19.8Bc 14.9Bc 16.9 32.0Cc 14.9a 15.4a 31.6Cc 27.1Ac 7.3 24.4Bd 9.3a 9.2a 22.8Bd 30.0Ccd 1 means in columns with the different letters are significantly different at (a,b)P < 0.05 or at (A,B)P < 0.01; all abbreviations as in Table 2 2 cMUFA = c9C14:1 + c9C16:1 + c9C18:1 3 tC18:1 = t9C18:1 + t11C18:1 (TVA) 4 below the quantification limit (LOQ) Animal Review, 2014, 1(3): 45-56 56 © 2014 Conscientia Beam. All Rights Reserved. Table-4. Effects1 of two levels (low, l; high, h) of Se-cystine (CySe2) on metabolism of LNA and the concentrations of t11c15C18:2 (tcC18:2), c9t11c15C18:3 (ctcC18:3), t9t11c15C18:3 (ttcC18:3), c13c16C22:2 (C22:2), the concentration sums of PUFA (PUFA) and all assayed fatty acids (FA), the concentration ratio of PUFA to SFA (PUFA/SFA) and the indexes of the initial, intermediate and final biohydrogenation in in vitro incubated ruminal fluids 1 means in columns with the different letter are significantly different at (a,b)P < 0.05 or at (A,B)P < 0.01; all abbreviations as in Table 2 2 below the quantification limit (LOQ) 3 the index of the initial biohydrogenation of c9t11c15C18:3 (ctcC18:3) and t9t11c15C18:3 (ttcC18:3) to t11c15C18:2 (tcC18:2) (indextcC18:2 = tcC18:2/(tcC18:2 + ctcC18:3 + ttcC18:3) 4 the index of the intermediate biohydrogenation of t11c15C18:2 to t11C18:1 (TVA) (indexTVA= t11C18:1/(t11C18:1+t11c15C18:2) 5 the index of the final biohydrogenation of t11C18:1 to C18:0 (indexC18:0 = C18:0/(C18:0+t11C18:1) Views and opinions expressed in this article are the views and opinions of the author(s), Animal Review shall not be responsible or answerable for any loss, damage or liability etc. caused in relation to/arising out of the use of the content.