To evaluate PUFA- diet added with Asifit tablet a commercial tablet for

was then extracted by soxhlet method in 96 ethanol, continued with evaporation using rotavapor. Residue from this process was washed with aquadest then centrifuged; the solid was crystallized by adding MeOH. This crystal with orange- brown color is curcuminoid, in which curcumin was part of it Sutrisno et al . 2008. Tannin was analyzed by extracting 3g of sample in boiling water, then strained and added with 1 FeCl 3. Whenever green, blue, and black colors were detected in Spectrophotometer , this means positive indicator for tannin. Microbial analyses for bacteria population was quantified by weighing 1g of fineground sample, diluted in 10 ml of aquadest. This solution was serially diluted in 6 folds 10 6 , then 0.1 ml was taken appropriately and inoculated onto 10 ml NA Nutrient Agar plate, made in double. Plates were incubated unaerobic at 40 C for 2 days. Fungi analysis was conducted with the same procedure, except it was inoculated onto 10 ml PDA Potato Dextrose Agar. Population of colony forming unit cfu found in each plate divided by sample weight timed dilution frequencies. The mean value from the double plate counts was used for statistical analysis. Fatty acid methyl esters FAME were determined by using gas chromatography GC Shimadzu 2010 series. There are several calculations of total short chain fatty acid C4- C10, medium chain fatty acid C12-C16, long chain fatty acid C16, mono unsaturated, poly unsaturated, saturated, unsaturated, ratio of PUFAsaturated, and unsaturatedsaturated fatty acid. Fatty acid ratio of n-6n-3 was calculated using this formula Schmidely et al. 2005 : n6n3 = linoleic acid + arachidonic acidlinolenic acid While, atherogenicity score was calculated with this formula Ulbrict and Southgate 1991: Atherogenicity index : C12 + 4C14 + C16 total unsaturated fat These all analyses were conducted in week -2 and week- 6 of the storage of PUFA- concentrate. Temperatures were recorded three times at 06 am, 12 noon, and 03 pm daily for 6 weeks, using thermometer. Averages of temperatures during the 2 and 6 weeks were averaged. RESULTS AND DISCUSSION Moisture, Dry and Organic Matter PUFA- concentrate supplemented with yeast and C. xanthorrhiza Roxb stored for 2- 6 weeks are presented in several tables. In Table 2.3 most of the treatments were higher in moisture 16.38, lower in dry matter -2.05, and lower in organic matter -2.81 significantly P0.05 during 6 weeks of storage, compared to those in week 2 and week 4 in three treatments, except PUFA- concentrate supplemented with yeast and curcuma PCM. Table 2.3 Moisture, dry matter, organic matter, and ash in PUFA-concentrate supplemented with yeast and C. xanthorrhiza Roxb stored for 2-6 weeks Nutrient PC0 PCY PCC PCM Average Moisture 2 weeks 8.46 x 8.78 x 8.79 x 8.79 8.70 X 4 weeks 8.24 x 8.48 x 8.41 x 8.16 8.32 X 6 weeks 11.16 y 10.77 y 11.41 y 8.78 10.53 Y Average 9.29 9.35 9.53 8.58 Dry matter 2 weeks 91.54 x 91.22 x 91.22 x 91.21 91.30 XY 4 weeks 91.77 x 91.52 x 91.59 x 91.84 91.68 X 6 weeks 88.84 y 89.23 y 88.60 y 91.22 89.47 Y Average 90.71 90.65 90.47 91.42 Ash 2 weeks 6.88 6.66 6.35 6.63 6.63 4 weeks 6.57 7.31 6.94 7.05 6.97 6 weeks 6.95 6.78 8.07 6.84 7.16 Average 6.80 6.91 7.12 6.84 Organic matter 2 weeks 84.66 x 84.56 x 84.87 x 84.58 84.67 X 4 weeks 85.20 x 84.21 x 84.65 x 84.79 84.71 X 6 weeks 81.89 yab 82.45 yb 80.52 ya 84.38 c 82.31 Y Average 83.91 AB 83.74 A 83.35 A 84.58 B These relationships showed that the longer the concentrates stored, the higher the water produced from microbial activity in organic materials; therefore the moisture was increasing and at the same time the dry and organic matter were decreasing. However, the PUFA- concentrate supplemented with yeast and curcuma PCM seemed to remain stable in moisture, dry matter, ash, and organic matter composition during the storage of 2, 4, and 6 weeks. In spite of these facts, this treatment was showing significantly p0.05 lowest moisture 8.78, highest dry matter 91.22, and the highest organic matter 84.38 in 6 weeks of storage compared to those in week 2 of storage among treatments. Based on these data, the PUFA- concentrate supplemented with yeast and C. xanthorrhiza Roxb PCM with the highest organic matter is considered the most sustained along with this period of storage 6 weeks. This meant that supplementation of yeast and curcuma powder stabilized moisture, dry and organic matters of the PUFA- concentrate. Moisture content of the concentrate would be another important reason for the reduction in colony forming unit later. This is in conjunction with data on the viability of the cells as it is displayed in Table 2.7; that it showed a positive relationship when the lowest moisture happened in the 6 weeks of storage resulted in the highest concentration of Saccharomyces cereviseae as shown in PUFA- concentrate supplemented with yeast and curcuma PCM. Increasing water availability and high temperature will activate yeast cells that eventually will be ceased because of inadequate available nutrients Sullivan and Bradford 2011. Results in this experiment approved that dry matter and organic matter were decreasing with higher moisture content in the PUFA- concentrate with no supplements, yeast, or curcuma only, resulting in lower concentrations of S. cereviseae. Ether Extract, Crude Protein, and Gross Energy Other nutrients, such as crude protein, N-free extract NFE, and gross energy in PUFA- concentrate supplemented with yeast and curcuma were not significantly changed within 2- 6 weeks of storage Table 2.4. Table 2.4 Ether extract, crude protein, crude fiber, N-free extract, and gross energy in PUFA-concentrate supplemented with yeast and C. xanthorrhiza Roxb stored for 2-6 weeks Nutrient PC0 PCY PCC PCM Average Ether extract 2 weeks 8.96 8.41 8.98 8.17 8.63 4 weeks 8.09 8.28 9.05 9.05 8.62 6 weeks 8.10 8.14 8.56 8.33 8.28 Average 8.38 8.28 8.86 8.51 Crude protein 2 weeks 12.26 12.00 11.79 11.60 11.91 4 weeks 11.54 11.65 11.69 11.58 11.62 6 weeks 11.86 11.27 11.76 11.32 11.55 Average 11.89 11.64 11.74 11.50 Crude fiber 2 weeks 13.51 x 13.30 13.14 13.48 13.36 X 4 weeks 12.91 xy 12.83 13.06 12.88 12.92 XY 6 weeks 12.57 y 12.15 12.64 12.12 12.37 Y Average 13.00 12.76 12.95 12.83 NFE 2 weeks 49.94 x 50.85 50.96 x 51.33 50.77 XY 4 weeks 52.66 y 51.45 50.85 x 51.28 51.56 X 6 weeks 49.36 xab 50.89b c 47.57 ya 52.60 c 50.11 Y Average 50.65 A 51.07 AB 49.79 A 51.74 B Gross energy kcalg 2 weeks 4124 4091 3986 4378 4145 4 weeks 3992 4067 3946 4023 4007 6 weeks 3974 3825 3752 3915 3867 Average 4030 3994 3895 4105 Different superscript in a row a,b,c or within column x,y,z, different significantly P 0.05. PC0: PUFA -concentrate with no supplementation; PCY: PUFA -concentrate with 0.5 yeast; PCC: PUFA - concentrate with no yeast and 2 curcuma; PCM:PUFA -concentrate with 0.5 yeast and 2 curcuma. However, ether extract contents in week-2 of PC0 8.96, PCY 8.41, and PCC 8.98 were significantly higher P0.05 than those in week- 6. While, in PCM concentrate, its ether extract was remained relatively the same during 2- 6 weeks storage. However, this concentrate showed the lowest ether extract in the 2 weeks storage. The crude fiber and NFE of 2 and 4 weeks storage were higher significantly p0.05 than that of in 6 weeks. Reductions were quantitatively found in crude protein -8.01 and gross energy -6.65 within 6 weeks of storage of all PUFA- concentrates. These data suggested that the longer the PUFA- concentrates with or without yeast and curcuma supplementation stored, the less nutritional values shown. In conjunction with these facts Table 2.3 that the average moisture content was increasing while the organic and dry matters were decreasing; consequently, other nutritional contents within the organic matter such as ether extract, crude fiber, crude protein, and gross energy were found diminishing as well. This suggested that during storage, fermentation process was happening where nutrients were broken down significantly. These carbon contents in the nutrients were being taken up for microbial growth, such as it happened in coincidence with higher population of Saccharomyces sp 2.35 10 7 cfug and Bacillus sp 11.43 10 7 cfug as it can be seen in the next table . Acid Detergent Fiber, Ca, and P Within 2 weeks of storage, ADF contents of all PUFA- concentrates were not significantly different as can be seen in Table 2.5. Only ADF was affected, in week 2 and 4 were higher P0.05 than that of during 6 weeks of storage. Table 2.5 Neutral detergent fiber, ADF, Ca, and P in PUFA-concentrate supplemented with yeast and C. xanthorrhiza Roxb stored for 2-6 weeks Nutrient PC0 PCY PCC PCM Average ADF 2 weeks 25.16 x 24.73 23.84 25.37 24.78 X 4 weeks 36.38 y 30.47 27.06 29.13 30.76 Y 6 weeks 33.95 xy 25.16 24.06 22.54 26.43 XY Average 31.83 B 26.78 A 24.99 A 25.68 A Ca 2 weeks 0.38 0.36 0.44 0.47 0.41 4 weeks 0.59 0.57 0.70 0.74 0.65 6 weeks 0.52 0.53 0.57 0.60 0.56 Average 0.50 0.49 0.57 0.60 P 2 weeks 0.66 0.62 0.61 0.63 0.63 4 weeks 0.45 0.55 0.60 0.61 0.55 6 weeks 0.64 0.67 0.64 0.75 0.68 Average 0.58 0.61 0.62 0.66 Different superscript in a row a,b,c or within column x,y,z , different significantly P 0.05. PC0: PUFA -concentrate with no supplementation; PCY: PUFA -concentrate with 0.5 yeast; PCC: PUFA -concentrate with no yeast and 2 curcuma; PCM:PUFA - concentrate with 0.5 yeast and 2 curcuma. In this week, PUFA- concentrate without any supplement PC0 showed the highest ADF 33.95. While, the lowest ADF 22.54 carried by PUFA- concentrate with both supplements PCM. In all treatments, the differences between week-2 and week-6 of storages were found in ADF 4.35, Ca 32.84, and P 7.23. In overall, all three treated PUFA- concentrates were lower P0.05 in ADF compared to that of in control diet. Suggesting that these supplements yeast, curcuma, and their combination worked well in reducing fiber. The PCM was considered the most nutritional since it showed significantly P0.05 the lowest ADF with the highest Ca and P. Other study showed that basal diet for dairy cows in which 56g XP yeast incorporated, was having 31 NDF, 20.4 ADF, 0.96 Ca, and 0.39 P Hristov et al. 2010. Compared to our data for, the NDF and P were more than doubled, while Ca was one third of those in XP diet. Tannin and Curcumin Bio actives detected in the C. xanthorrhiza Roxb used in this experiment were 0.8 curcumin and 1.58 tannin. Tannin levels in four treatments of PUFA- concentrate quantitatively were decreasing as much as 32.78 in 6 weeks storage compared to those in 2 weeks storage Table 2. 6. Among the treatments, PUFA- concentrate with no supplements PC0 quantitatively contained the lowest tannin; while PUFA- concentrate with yeast and curcuma supplements PCM had the highest one during 2- 6 weeks of storage. This suggested that curcuma powder contributed some amount of tannin in the concentrate. Tannins in all type of concentrate found in this study were not as high as it was in polyherbal combination 3.69 as reported by Mirzaei and Prasad 2011. Curcumin levels in both treatments, PCC and PCM were found the same in week -2, yet decreasing in week-6; however, it was slightly higher in PCM. These results showed that tannin and curcumin concentrations were found not as much high as than those in their original sources when other feedstuffs were combined together and depended upon its level added as well. Curcuminoids in Table 2. 6 Tannin and curcumin in PUFA-concentrate containing yeast and C. xanthorrhiza, Roxb during 2-6 weeks storage Bioactive PC0 PCY PCC PCM Average Tannin 2 weeks 0.23 0.27 0.29 0.30 0.27 4 weeks 0.20 0.20 0.19 0.19 0.20 6 weeks 0.17 0.18 0.24 0.24 0.21 Average 0.20 0.22 0.24 0.24 Curcumin 2 weeks - - 0.10 0.10 0.10 4 weeks 0.06 0.06 0.06 6 weeks - - 0.07 0.09 0.08 Average 0.08 0.08 PC0: PUFA -concentrate with no supplementation; PCY: PUFA -concentrate with 0.5 yeast; PCC: PUFA -concentrate with no yeast and 2 curcuma; PCM:PUFA -concentrate with 0.5 yeast and 2 curcuma . C. longa contained curcumin 1.06- 5.65, demethoxycurcumin 0.83- 3.36, and bisdemethoxycurcumin 0.42 – 2.16 as observed by Jayaprakasha et al. 2002. Microbial Viability This self made yeast contained 3.6 10 7 cfug. The viable cell was within the range of other commercial yeast products 2.93 10 7 – 1.43 10 13 cfug that had been surveyed by Sullivan and Bradford 2011. They reported that there was a decrease in the viability of the cell approximately 90 per month when the active dry yeast was kept under high temperature of 40 C; however, there was no significant difference of viable cells stored during spring and summer as the variability of the samples were so wide due to different distribution. In previous research, the yeast was prepared in the ambient temperature of around 31 C then added into PUFA- concentrate and kept in another ambient temperature of around 26.94 C with average moisture of 78.83. These places of yeast preparation were certainly lower in the ambient temperature compared to the previous temperature. Results in Table 2.7 showed that Saccharomyces sp was not significantly different within 2 weeks and 6 weeks; however, they were significantly P0.05 higher in 6 weeks compared to those in 2 weeks of storage. Being the highest one was PUFA- concentrate with yeast and curcuma or PCM 23.5 10 6 cfug or 2.35 10 7 cfug in 6 weeks of storage. This amount of viable cell seemed to be well protected by the addition of curcumin in the concentrate, while it was stored under suitable ambient temperature. This kind of concentrate would be beneficial for ruminants. Bacillus sp were found significantly different P0.05 within 2 and 4 toward 6 weeks, while population in week 4 was about the same as in week 2. However, diet with combination of yeast and curcuma PCM in 4 weeks showed the highest P0.05 Bacillus sp. This might happen as there was yeast that could Table 2.7 Microbes population in PUFA-concentrate supplemented with yeast and C. xanthorrhiza Roxb stored for 2-6 weeks Microbe PC0 PCY PCC PCM Average 10 6 cfug Saccharomyces sp 2 weeks 1.57 x 1.20 x 2.43 x 4.00 x 2.30 X 4 weeks 0.07 x 1.03 x 0.43 x 5.87 x 1.85 X 6 weeks 18.83 y 19.77 y 20.63 y 23.50 y 20.68 Y Average 6.82 7.33 7.83 11.12 Bacillus sp 2 weeks 6.93 3.67 0.37 9.20 5.04 4 weeks 5.90 a 2.90 a 1.93 a 28.73 b 9.87 6 weeks 19.47 8.43 7.87 11.43 11.80 Average 10.77 AB 5.00 A 3.39 A 16.46 B Different superscript in a row a,b,c or within column x, y, z, different significantly P 0.05. PC0: PUFA -concentrate with no supplementation; PCY: PUFA -concentrate with 0.5 yeast; PCC: PUFA - concentrate with no yeast and 2 curcuma; PCM: PUFA -concentrate with 0.5 yeast and 2 curcuma. increase the availability of nutrient; together with nutrient available from curcuma then made this diet as a good source of Bacillus sp as well as Saccharomyces sp to grow more than that in separated supplement diets. These S. cereviseae and Bacillus sp in PUFA- concentrate with curcuma powder in this experiment were higher compared to those in Tabut block 300g containing 20 curcuma diluents and 35 yeast-fermented cassava stored in 12 weeks, were 9.5 10 4 cfug and 1.5 10 7 cfug, respectively Sulistyowati et al. 2008 b . Further study showed that Saccharomyces cereviseae supplementation was reported to increase organic matter digestibility generating a higher energy availability for milk yield in buffalos Campanile et al. 2008. Correlation between Moisture Content and Other Nutrient in PUFA- Concentrate The correlations between moisture content and other nutrient in PUFA- concentrate supplemented with yeast and C. xanthorrhiza Roxb PCM were in negative manner. Higher correlations were found in DM, OM, ether extract, ADF, and bacillus; while the lower correlation was found in crude fiber; as shown in Table 2.8. Fatty Acid Profile Fatty acid methyl esters FAME contents in these concentrates Table 2.9 and Table 2.10 were showing specific amount even though they contained the same level and sources of PUFA roasted ground corn, roasted soy bean meal, and corn oil. This might represent the presence of yeast and curcuma or their combination in reaction with fatty acid content in concentrate during the bio hydrogenation reaction. Total fatty acid of PUFA- concentrate with 2 curcuma PCC seemed to be the highest 79.25, while the one without supplement PC0 was the lowest 33.74, in 2 weeks of storage. In the 6 weeks of storage, the same treatment PCC showed the highest total fatty acid 62.64, while the PUFA- concentrate with 0.5 yeast and 2 curcuma PCM had the lowest total fatty acid 46.54. This described that curcumin only, with the amount of 0.07- 0.10 in PUFA- concentrate would be optimal in hydrogenation process such that it would yield the highest total fatty acid. Table 2.8 Correlation between moisture and other nutrient in PUFA- concentrate supplemented with yeast and C. xanthorrhiza Roxb Y2C20 Moisture - nutrient Equation Y Correlation r Organic matter Y= -0.485x + 88.74 0.87 Ether extract Y= -1.266x +19.37 0.99 Crude protein Y= -0.192x + 13.15 0.46 Crude fiber Y= -0.101x + 13.70 0.10 ADF Y = -8.151x + 95.59 0.89 Saccharomyces cereviseae Y = 121.4x- 930.2 0.41 Bacillus sp Y = -292.6x + 2674 0.99 As shown in the highest total fatty acid, polyunsaturated fatty acid PUFA was also found the highest in concentrate with 2 curcuma, both in 2 weeks 41.77 and 6 weeks 24.07 of storage. Obviously, it was decreasing with the longer time of storage. The total PUFA of PUFA-concentrate with yeast was almost twice 29.25 as it was in PUFA-concentrate with no supplements in 2 weeks of storage. This, in part, could be described that yeast in RumiSacc contained PUFA 26.42 that might contribute to the total PUFA of concentrate or diet as reported by Yalçın et al. 2011. Previous study Sulistyowati et al. 2010 c of concentrate containing 0.5yeast, 10.5curcuma powder, with palm oil, corn oil, and roasted ground corn showed that total PUFA was ranging from 77.23 in 1.5 corn oil and 3 roasted ground corn to 79.19 in 4.5 palm oil. These are about 2- 3 times higher than the results in the present experiment. Table 2.9 Fatty acid contents in PUFA-concentrate supplemented with yeast and C. xanthorrhiza Roxb stored for 2 weeks Fatty acid total fat PC0 PCY PCC PCM SED Capric Acid, C10:0 nd nd n.d 0.05 nd Lauric Acid, C12:0 0.016 nd 0.03 0.02 0.01 Myristic Acid, C14:0 0.09 0.11 0.15 0.18 0.04 Palmitic Acid, C16:0 5.22 6.60 0.01 0.02 3.45 Palmitoleic Acid, C16:1 0.05 0.07 9.23 11.17 5.91 Heptadecanoic Acid, C17:0 0.03 0.04 0.11 0.10 0.04 Stearic Acid, C18:0 1.17 1.48 0.05 0.07 0.74 Elaidic Acid, C18:1n9t 0.03 0.00 2.08 2.45 1.31 Oleic Acid, C18:1n9c 11.41 17.43 24.94 24.42 6.42 Linolelaidic Acid, C18:2n9t n.d 0.03 0.03 0.02 0.01 Linoleic Acid, C18:2n6c 14.15 26.85 38.44 28.89 9.99 Linolenic Acid, C18:3n3 1.05 2.33 3.24 2.07 0.90 Arachidic Acid, C20:0 0.15 0.19 0.28 0.31 0.08 Cis-11-Eicosenoic Acid, C20:1 0.08 0.11 0.16 0.15 0.04 Cis-11,14-Eicosedienoic Acid, C20:2 0.07 0.04 0.06 0.10 0.03 Heneicosanoic Acid, C12, C21:0 0.02 nd 0.03 0.02 0.01 Behenic Acid, C22:0 0.11 0.13 0.19 0.21 0.05 Tricosanoic Acid, C23:0 0.02 0.02 0.03 0.05 0.01 Lignoceric Acid, C24:0 0.10 0.13 0.18 0.22 0.05 Total fatty acid 33.74 55.54 79.25 70.52 19.92 Total SCFA C4- C10 nd nd nd 0.05 - Total MCFA C12- C16 5.38 6.78 9.42 11.39 2.69 Total LCFA CC16 28.39 48.78 69.82 59.08 17.65 Total MUFA 11.52 17.54 27.18 27.02 7.66 Total PUFAP 15.27 29.25 41.77 31.08 10.89 Total Saturated fat S 6.93 8.77 10.29 12.42 2.33 Total Unsaturated U 93.07 91.23 89.71 87.58 2.33 Rasio PS 2.20 3.34 4.06 2.50 0.84 Rasio US 13.44 10.40 8.72 7.05 2.73 n6n3 13.48 11.52 11.86 13.96 1.19 Atherogenicity 0.06 0.08 0.11 0.14 0.03 PC0: PUFA -concentrate with no supplementation; PCY: PUFA -concentrate with 0.5 yeast; PCC: PUFA -concentrate with no yeast and 2curcuma; PCM: PUFA -concentrate with 0.5 yeast and 2 curcuma, nd: not detected. The fatty acid profile of PCC was considered the highest among other treatments; C18:2 38.47 of total FA, C18:1 27.02, and lower levels of C18:3 3.24 and C18:0 0.05 in 2 weeks. In 6 weeks, PCC was still showing the highest fatty acid profile; C18:1 21.94, C18:2 21.88, C18:3 and C18:0 were lower. Curcumin in this concentrate containing corn oil, roasted soy bean meal, and roasted corn, seemed to be able to protect unsaturated fat from further hydrogenation. This is more in positive way since there is no fermentation effect from yeast in this treatment, so that it would not cause a certain level of rancidity that might lead to hydrogenation of much more unsaturated fat. Fatty acids were also detected in soy bean oil that the highest contents were found in C18:2 54 and C18:122, as reported by Bouattour et al. 2008. In accordance with the highest total PUFA in 2 and 6 weeks of storage; medium MCFA and long chain fatty acid LCFA as well as monounsaturated fatty acid MUFA were found to be the highest in the same PUFA- concentrate with curcuma PCC. Saturated S fat increased gradually from 6.93 to 12.42 with the more completed supplement. In contrast, the unsaturated U fat decreased in that manner during the 2 weeks storage. During 6 weeks of storage, this trend worked the other way, PUFA- concentrate with yeast and curcuma PCM showed the lowest saturated fat 28.49 with the highest unsaturated fat 71.51. Ratio of PS, ratio US, n6n3, and atherogeneicity of all treatments in 2 weeks compared to those in 6 weeks of storage were quantitatively down or up in the averages of -81.94, -80.37, 15.74, and 221.74, respectively. The PS ratio and n6n3 ratio in the previous experiment with similar treatment showed 3.7 and 1.59 in average, respectively Sulistyowati et al. 2010 c . It means that, only the PS ratio in 2 week that was close to this; whereas, the n6n3 was 8 – 9 times higher in this current research. The ratio of n6n3 in the 2 weeks 12.71 and 6 weeks 14.59 were much higher than those in soy bean oil 4.69- 6.12, reported by Bouattour et al. 2008. These ratios in PUFA- concentrate were far above the recommended range of n6n3 for human wise is between 5:1 and 10:1. However, it is necessary to check this ratio in the milk of goat fed with this concentrate. Table 2.10 Fatty acid contents in PUFA-concentrate supplemented with yeast and C. xanthorrhiza Roxb stored for 6 weeks Fatty acid total fat PC0 PCY PCC PCM SED Caprilic acid, C8:0 0.21 nd nd nd nd Capric Acid, C10:0 0.05 0.03 0.05 0.04 0.01 Undecanoic acid, C11:0 nd 0.02 nd nd nd Lauric Acid, C12:0 0.03 0.03 0.02 0.02 0.01 Myristic Acid, C14:0 0.23 0.27 0.21 0.16 0.05 Pentadecanoic acid, C15:0 0.04 0.04 0.03 0.03 0.01 Palmitic Acid, C16:0 14.95 15.78 12.89 9.72 2.7 Palmitoleic Acid, C16:1 0.1 0.09 0.11 0.08 0.01 Heptadecanoic Acid, C17:0 0.1 0.09 0.11 nd 0.01 Stearic Acid, C18:0 3.2 3.15 2.64 2.05 0.54 Elaidic Acid, C18:1n9t 0.02 0.04 0.06 0.04 0.02 Oleic Acid, C18:1n9c 21.69 21.52 21.88 16.16 2.77 Linolelaidic Acid, C18:2n9t 0.02 0.02 0.03 nd 0.01 Linoleic Acid, C18:2n6c 15.8 14.81 21.85 16.32 3.17 Linolenic Acid, C18:3n3 1.06 0.93 1.5 1.14 0.24 Arachidic Acid, C20:0 0.41 0.4 0.35 0.24 0.08 Cis-11-Eicosenoic Acid, C20:1 0.17 0.16 0.17 0.12 0.02 Cis-11,14-Eicosedienoic Acid, C20:2 0.08 0.12 0.08 0.04 0.03 Cis-5,8,11,14,17-Eicosapentaenoic acid,C20:5n3 0.03 0.03 0.05 nd 0.01 Heneicosanoic Acid, C12, C21:0 0.06 0.04 0.12 0.02 0.04 Behenic Acid, C22:0 0.28 0.26 0.27 0.18 0.05 Tricosanoic Acid, C23:0 nd nd nd nd Nd Lignoceric Acid, C24:0 0.3 0.29 0.25 0.19 0.05 Nervonic acid, C24:1 0.02 0.02 nd nd Total fatty acid 58.85 58.13 62.64 46.54 6.95 Total SCFA C4- C10 0.04 0.04 0.03 0.03 0.01 Total MedCFA C12- C16 18.35 19.11 15.75 11.85 3.28 Total LongCFA CC16 39.94 38.64 46.61 34.45 5.04 Total MUFA 15.85 14.86 21.93 16.32 3.19 Total PUFAP 17.55 16.39 24.07 17.84 3.46 Total Saturated fat S 40.78 41.4 38.32 28.49 5.99 Total Unsaturated U 59.22 58.6 61.68 71.51 5.99 Ratio - PS 0.43 0.4 0.63 0.63 0.12 Ratio- US 1.45 1.42 1.61 2.51 0.52 Ratio n6n3 14.50 15.43 14.10 14.32 0.59 Atherogenicity 0.31 0.33 0.26 0.17 0.07 PC0: PUFA -concentrate with no supplementation; PCY: PUFA -concentrate with 0.5 yeast; PCC: PUFA –conc.with no yeast and 2 curcuma; PCM:PUFA –conc. with 0.5 yeast and 2 curcuma. Nd: not detected. For a comparison, the ratio of PS were 0.88 and 1.69, respectively in saturated fat prilled hydrogenated fat containing diets and unsaturated fat Ca soaps of long chain fatty acid as reported by Harvatine and Allen 2006. These suggested that the type of fat supplemented in concentrate would determine its type of fatty acid. Different PUFA are not secreted freely from each other, therefore, any other vegetable oil as supplement would respond differently on its fatty acid content Chilliard et al. 2006. The atherogeneicity index increased in PUFA- concentrate with the more complete supplement in 2 weeks; on the other hand, it decreased in PUFA- concentrate with the same treatments in 6 weeks of storage. This index supposedly dealing with unhealthy saturated fat that might lead to some kind of blood vessel disease Ulbricht and Southgate, 1991. However, other researchers Mensink et al. 2003; Knopp and Retzlalaff 2004, Chilliard et al. 2006; Bouattour et al. 2008 declared that there is little fact that the medium chain fatty acid C12:0, C14:0, and C16:0 caused in atherogenic effect. This saturated fat is even better compared to high carbohydrate with low fat; and it would be of concern only if fat consumption is excessive. The average index in this experiment was 0.10 and 0.27 during the 2 and 6 weeks of storage, respectively. These could be considered as low levels in the feed that will be healthy for ruminants and eventually into their product that will be beneficial for human health. CONCLUSION Storage of concentrate containing polyunsaturated fatty acid PUFA sources corn oil, roasted ground corn and soybean meal supplemented with yeast, C. xanthorrhiza Roxb or a combination of these supplements up to 6 weeks increased moisture contents therefore decreased dry and organic matters in all concentrates. However, other nutrients, ether extract, crude protein, gross energy, NDF, Ca, P, tannin, and curcumin as well as PUFA contents relatively unaffected during the storage. In terms of treated concentrates, the one with mix supplement showed highest organic matter and nitrogen free extract in 6 weeks of storage and highest acid detergent fiber in 4 weeks. In contrast, this concentrate was also showing the highest Saccharomyces sp in 6 weeks of storage. The higher contents of PUFA, ratio of PUFA to saturated, MUFA, and long chained fatty acid were shown by PUFA- concentrate with 2 curcuma. In terms of overall nutrient composition, supplementation of 0.5 yeast in combination with 2 curcuma in PUFA concentrate designed for dairy goat is considered as a reasonable effort to maintain its nutritive performance during 6 weeks of storage.

3. IN VITRO GOAT FERMENTATION OF PUFA- DIET SUPPLEMENTED WITH YEAST AND C. xanthorrhiza Roxb

ABSTRACT This experiment, in vitro, was conducted to evaluate the ruminal performances of PUFA- diet containing PUFA-concentrate with soybean byproduct of tofu industry and King grass supplemented with yeast and C. xanthorrhiza Roxb.. Experimental design being applied was Randomized Complete Block of 4 ruminal liquor derived from four slaughtered goats and 4 treatments PD0-no supplement, PDY- 0.5 yeast, PDC-2 curcuma, and PDM- 0.5 yeast + 2 curcuma, in 3 replications. Variables being analyzed were pH, N-NH3, total and partial VFA, TPC, and CH4. Results showed that in spite of having the lowest organic and dry matter digestibilities as well as N- NH3; however, supported by low protozoa population and high VFA production in the goat rumen fluid, the PUFA- diet with a mixture of yeast and curcuma additives was considered the most potential diet to improve nutrient metabolism in rumen Key words : curcuma, in vitro fermentation, PUFA- diet, yeast. INTRODUCTION Ruminal fermentation performance was affected mostly by diet being ingested. Diet containing high concentrate with polyunsaturated fatty acid PUFA may increase production, improve milk fat and milk fatty acid in ruminants, including goat. However, to make such improvements, it needs any bioaditives, such as yeast and curcuma to be supplemented into diet that would determine kinetics of rumen fermentation. Yeast has been known as rumen enhancer. In vitro fermentation of diet added with Diamond- V XP yeast showed higher in dry matter digestibility DMD, organic matter digestibility OMD, and total volatile fatty acid VFA than those of diet with A- Max yeast with the same dose 57gd as reported by Miller- Webster et al. 2002; while supplementation of Saccharomyces cereviseae culture of 0.35gl produced higher concentration of acetate C2 than that of higher dose of 0.73gl Lynch and Martin 2002. Tannin derived from Peltiphyllum peltatum was also utilized to manipulate rumen by decreasing protein degradability in the rumen and make it available in small intestine Jayanegara et al. 2010. Tannin extract decreased protozoal population, methane CH4 gas production, NH3, and VFA; on the other hand, it increased bacterial population in the rumen of dairy cow Khiaosa- ard et al. 2008. Tannin 13.84 extracted from Pakar leaves Ficus infectoria was added into diet fed for goat revealed some higher levels in N-NH, acetate, and total fungi; whereas lower levels in total VFA and propionate Singh et al. 2011. Curcumin and tannin found in Curcuma sp, such as C. domestica and C. xanthorrhiza Roxb were reported to decrease populations of fungi and bacteri in Tabut block, containing fermented yeast and other ingredients Sulistyowati et al. 2008 b . Based on these data, a study on in vitro fermentation of PUFA- diet supplemented with yeast and curcuma PUFA- diet had been conducted to observe in vitro goat rumen fermentation performance. MATERIALS AND METHODS Yeast, Curcuma Powder, and PUFA- Diet Preparation Yeast was prepared from the previous first experiment according to Pusbangtepa 1981. Curcuma powder was also provided from experiment I through the processes of slicing, drying, grinding, and powdering. The PUFA- diet was reformulated as it was in the PUFA- concentrate then combined with soybean by-product from local tofu industry. Ground corn was half roasted; while soy bean meal was all roasted in 80 C for about 20 minutes. These ingredients together with corn oil were designated as PUFA sources in the diets. The PUFA- diets were formulated accordingly to their treatments as can be seen in Table 3.1. Nutrient contents were assessed based on proxymate analysis AOAC, 1990; ADF was analysed according to Van Soest 1990. Curcumin was analysed using maseration method Sutrisno et al. 2008, tannin was analysed by modification of Folin- Ciocalteu method Harborne 1987. Calcium was detected using Atomic Absorbance AA7000 Shimadzu Co. Serial no A 306647-00345. Phosphor was analysed in wet ashing method then quantified by using Spectrophotometer UV-200 RS UV VIS LW Scientific. Fatty acid content was detected as fatty acid methyl esters FAME using gas chromatography GC Shimadzu 2010 series. In vitro procedure was based on Tilley and Terry 1963, as followed. Fermentor tubes was loaded with 0.5 g of samples, added with 40ml of Mc. Dougall solution. Tubes were put into shaker bath in 39 o C, added with 10 ml rumen liquor of slaughtered goats. Tubes were soaked and flowed with CO 2 for 30 seconds, checked for pH 6.5 – 6.9 using pH meter; tubes were then probed with ventilated rubber caps, fermented for 48 hours. When fermentation done, the Table 3.1 Composition of ingredients in PUFA-diet supplemented with yeast and C. xanthorrhiza Roxb used in in vitro goat ruminal fermentation Ingredients PD0 PDY PDC PDM Rice bran 18.42 18.42 18.42 18.42 Ground corn 15.79 15.79 15.79 15.79 Soy bean meal 7.89 7.89 7.89 7.89 Cassava meal 7.89 7.89 7.89 7.89 Soy bean- byproduct 27.49 27.49 27.49 27.49 Corn oil 2.11 2.11 2.11 2.11 Minerals 0.53 0.53 0.53 0.53 King grass 19.88 19.88 19.88 19.88 Yeast - 0.5 - 0.5 Curcuma powder - - 2.0 2.0 PD0: PUFA- diet with no additives; PDY: PUFA- diet with 0.5 or 0.5yeast; PDC: PUFA- diet with 2 or 2curcuma; PDM: PUFA- diet with mix of 0.5 or 0.5yeast and 2 or 2curcuma caps were taken of from the tubes, add with 2 -3 drops of HgCl 2 to stop microbial growth at this point, analyses were conducted for protozoa population, total plate count, TPC, and methane gas, CH4. Fermentor tubes were then centrifuged in 4.000 rpm for 10 minutes. Substrates were separated as solid in the bottom and supernatant above. The supernatant was used for volatile fatty acid, VFA, and N- ammonia, N-NH3analyses; while the solid was then added with 50ml solution of pepsin-HCl 0.2, centrifuged in 4.000 rpm for 15 minutes. This substrate was then reincubated for 48 hours without probes; then strained through whatman paper no 41 that has been known for its weight using vacuum pump. The solid left in the paper was transferred into baker glass, ovened in 105 ⁰C for 24 hours. The glass, paper, and residue were taken out from the oven, put them in excicator then weighed them to get its dry matter content. Afterward, these samples were ashed in electrical tanur for 6 hours in 450 – 600 o C. Then weighing them to get their dry and organic matters to calculate the in vitro dry matter digestibility IVDMD and in vitro organic matter digestibility IVOMD. Blanco was prepared from the residue of the fermentation without samples. These digestibilities were calculated as if that of in hidrolytic post ruminal fermentation after two 24 hour period Metode Tilley and Terry 1963. Analysis on N- NH3 was conducted using micro diffusion Conway General Laboratory Procedure 1966. Analysis of VFA in partial containing of acetate C2, propionate C3, isopropionat, butirate C4, isobutirat, valerate, and isovalerate were detected using gas chromatography GC Chromopack 9002 with centrifuge IEC micromac RF type 3593, column capillary WCOT fused silica ID coating FFAP-CB. Methane CH4 was measured using GC Shimadzu 8-APT detector TCD and C- R6A Chromatopack. Analysis of total plate count TPC was conducted by weighing 50 g sample, added with 450 ml butterfield phosphate solution. Out of this solution, take 10 ml sample, added with 90 ml of butterfield phosphate solution as 10 1 dilution, then any dilution 10 1 ,10 2 ,10 3 or more was done by pippetting 1 ml sample into 9 ml of butterfield phosphate solution, soaked using vortex. Putting 1 ml out of this sample onto PCA agar media, incubated in 35 o C for 48 hours, placed the glass in upside down positition. These are Figure 3.1 some of the activities conducted in this in vitro experiment. a b c d e f Figure 3.1 Some activities conducted in the in vitro experiment: a PUFA-diet; b in vitro fermentation; c TPC test; d methane test; e tannin test; f NH3 test Experimental Design and Data Analysis Experimental design being applied was Randomized Complete Block of 4 diets, 4 ruminal liquor derived from four slaughtered goats, 4 treatments PD0, PDY, PDC, PDM , in 3 replications. Data tabulation was performed and analysis of variance Anova was conducted on sets of data; any significant differences were then tested by using Duncan Multiple Range Test DMRT according to Lentner and Bishop 1986. RESULTS AND DISCUSSION Chemical Composition of Diets Average content of protein in these diets was 12.96 ± 0.23 Table 3.2. This level fulfilled the protein requirement for 30 kg dairy goat with 1 kg milk as stated in NRC 1981, that was 12. Other result showed that protein content in diet with extruded soybean in dairy goat was much higher 18.5 ± 0.38 or 42.75 higher Schmidely et al. 2005. In quantity, PDM, diet with a mixture of yeast and curcuma, contained higher organic matter, lower ADF, higher Ca, and higher fat than other diets. This result approved that these two additives could improve nutrient content. Diet with high concentrate in this experiment was about 80 could decrease ether extraxt of the diet that eventually decreased milk fat. However, since in this research used fat sources roasted ground corn, roasted soy bean meal, and corn oil, the ether extraxt of these diets were relatively high 4.0- 4.43, except in control diet 3.75. This result was in accordance with others that diet without extruded soybean contained very low fat 1.38, while the one with that ingredient showed high fat content 5.19 as reported by Schmidely et al. 2005. Diet with corn oil was reported to have higher fat, 5.62 Bouattour et al. Table 3.2 Nutrient contents of PUFA-diet supplemented with yeast and C. xanthorrhiza Roxb fermented in vitro in goat rumen liquor Nutrient PD0 PDY PDC PDM Dry matter 93.14 93.07 94.42 94.46 Ash 8.34 7.70 7.73 6.64 Organic matter 84.54 85.97 86.81 88.27 Protein 12.86 13.11 13.18 12.67 Ether extract 3.75 4.20 4.43 4.0 Crude fiber 27.32 27.69 26.93 27.74 ADF 54.68 50.77 52.78 43.20 Gross energy Mcalkg 3.84 3.86 3.83 3.90 Ca 0.68 0.60 0.84 0.91 P 0.36 0.43 0.26 0.26 Tannin 0.487 0.492 0.491 0.491 Curcumin - - 0.13 0.11 PD0: PUFA- diet with no additives; PDY: PUFA- diet with 0.5yeast; PDC: PUFA- diet with 2 curcuma; PDM: PUFA- diet with mix of 0.5 yeast and 2 curcuma. 2008. It seemed that bioactives, curcumin and tannin did not affect the fat content of the diets. Fatty Acid Content in PUFA- Diets Among other diets Table 3.3, the one with a mixed additives PDM quantitatively had the lowest saturated fatty acid SFA, highest unsaturated fatty acid UFA, highest UFASFA ratio, lowest n6n3 ratio and atherogenicity. Table 3.3 Fatty acid contents in PUFA-diet supplemented with yeast and C. xanthorrhiza Roxb Fatty acid of total fat PD0 PDY PDC PDM Capric Acid, C10:0 0.01 Undecanoic acid, C11:0 0.02 Lauric Acid, C12:0 0.23 0.21 0.17 0.23 Tridecanoic Acid, C13:0 0.02 Myristic Acid, C14:0 0.29 0.18 0.15 0.21 Pentadecanoic acid, C15:0 0.39 0.05 0.04 0.06 Palmitic Acid, C16:0 3.9 10.92 8.94 12.68 Palmitoleic Acid, C16:1 0.4 0.08 0.06 0.09 Heptadecanoic Acid, C17:0 0.38 0.1 0.08 0.12 Stearic Acid, C18:0 9.07 3.05 2.3 3.48 Elaidic Acid, C18:1n9t 0.06 0.04 Oleic Acid, C18:1n9c 2.21 15.54 11.7 17.37 Linolelaidic Acid, C18:2n9t 0.07 0.05 Linoleic Acid, C18:2n6c 0.92 13.42 10.8 14.85 Linolenic Acid, C18:3n3 0.07 2.31 1.94 2.55 Arachidic Acid, C20:0 0.29 0.66 0.47 0.71 Cis-11-Eicosenoic Acid, C20:1 11 0.08 0.12 Cis-11,14-Eicosedienoic Acid, C20:2 0.07 0.09 0.1 Cis-5,8,11,14,17-Eicosapentaenoic acid,C20:5n3 0.02 .04 Heneicosanoic Acid, C12, C21:0 0.02 0.05 0.03 0.06 Behenic Acid, C22:0 0.46 0.59 0.44 0.67 Tricosanoic Acid, C23:0 0.04 0.11 0.08 0.12 Lignoceric Acid, C24:0 0.3 0.39 0.27 0.45 Nervonic acid, C24:1 0.01 Total Fatty acid 19.17 48.02 37.74 54.16 Total SCFA 0.01 Total MCFA 4.83 11.36 9.3 13.18 Total LCFA 13.83 47.37 28.32 40.7 Total MUFA 2.61 26.68 11.88 17.59 Total PUFAP 1.06 15.82 12.83 17.59 Total Saturated fat S 15.39 16.31 12.97 18.82 Total Unsaturated U 84.61 83.69 87.03 81.18 Rasio PS 0.07 0.97 0.99 0.93 Rasio US 5.50 5.13 6.71 4.31 n6n3 13.14 5.83 5.57 5.86 Atherogenicity 1.44 1.06 0.87 1.23 PD0: PUFA- diet with no additives; PDY: PUFA- diet with 0.5yeast; PDC: PUFA- diet with 2 curcuma; PDM: PUFA- diet with mix of 0.5 yeast and 2 curcuma. However, curcuma supplementation in diet PDC numerically decreased monounsaturated fatty acid MUFA and polyunsaturated fatty acid PUFA compared to that of in yeast diet PDY. It has been known that curcumin played a role in disturbing lipid peroxidation, when the process of demethylation finished, consequently its function as antioxidant was over as well. It means that, curcumin will be functioning depends on the availability of the substrate that is going to be catabolysed. It was reported that curcuminoid as much as 0.0133mgml could slow down peroxidation of linoleic acid Tonnesen 1988. The implication of the result in this research was that curcumin in C. xanthorrhiza, Roxb functioned much stronger in fatty acid reduction when it was singly; in contrast, its effect would be weaker when it was combined with yeast. This can be seen in the level of linoleic acid in PDC that was lower 10.8 compared to that of PDM 14.85. Short chain fatty acids C10 and C11 were not detected in control, yeast, and curcuma diets, very little level showed in the mixed PUFA- diet. Long chain fatty acids C18:0 to C18:3n3 were higher in both yeast PDY and yeast + curcuma diet PDM. These fatty acids were reported high in diet with supplementation of forage tannin, extract tannin, or extract saponin, was about 83.720 Khiaosa- Ard et al. 2008. Atherogenicity index being numerically the lowest was in PDC 0.87, that was accordingly due to the ability of curcumin in reducing medium fatty acid MCFA C12, C14, C16 making the unsaturated fatty acid increased. However, when curcuma was combined with yeast PDM, this index would increase, suggesting that there was fermentation process going on as the effect of the yeast. The fermentation effect with yeast PDY was also increased the index higher than that of in PDC. Indexes in this research were much lower the highest was 1.44 in PD0 compared to control diet 2.20 and 20 extruded soybean 3.48 as reported by Schmidely et al. 2005. Implication of the lower index in diet containing PUFA, moreover, supplemented with curcuma, will indicate lower risk of any detrimental effect caused by saturated fat being consumed by animal that eventually came up in its product such as milk that would be consumed as a healthier food for human. Fermentation Characteristics In vitro fermentation characteristics Table 3.4 of diet containing – PUFA concentrate, yeast, curcuma, and a mixture of these additives in goat rumen liquor were mostly not different significantly, except in IVDM and IVOM were signinificantly different p0.05 among treatments. Both digestibilities of diet with no additives PD0 was significantly higher than those of PDM; while the yeast PDY or curcuma PDC diets were remained the same. Other results showed that IVDMD 53.8- 56.9 and total VFA 43.8- 59.5 mM with PMX70SBK live cells yeast which then decreased with increasing yeast level from 0.37 to 0.73gl in rumen liquor of young male cattle fed alfalfa hay Lynch and Martin 2002. The AP ratio in these diets 3.54- 3.88 were about in the same range as that of with alfalfa hay 3.77- 3.81; whereas diet with high carbohydrate gave lower ratio of 1.87- 2.40. This has been known that type of diet will determine its production of propionate and acetate; carbohydrate produces higher propionate, while fiber will produce higher acetate. The AP ratio increased with the longer fermentation, suggesting that the more structural carbohydrate was degraded, thefore, produced higher acetate and lower propionate. Tannin contents in all PUFA-diets were relatively the same 0.487 - 0.491. Tannin and curcumin added in PDM yielded the highest total VFA 45.27 mM and the lowest isobutirate 0.66. A different result showed that tannin 13.84 from Pakar leaves Ficus infectoria fed to goats showed some lower variables, such as in total VFA 4mM and propionate 11.86; but higher in N-NH3 5.29mM, asetat 83.56, and total fungi 28.16 than those found in feed lower in tannin 8.6 as reported by Singh et al. 2011. Concentration of N- NH3 was decreasing from 11.26 mM in PD0 to 8.73 mM in PDM. This level of NH3 was much higher than that of NH3 8.5- 9.6 mM with pH of around 6.7, in goat fed with extruded soybean with or without NaHCO3 Schmidely et al., 2005. The N-NH3 concentration in dairy cow fed diet containing tannin extract was detected the lowest 5.1 mM compared to that of control diet 17.2 mM with pH around 7.05- 7.09 Khiaosa- Ard et al. 2008. Microbial Activity Total plate count TPC of the fermented diet Table 3.5 was affected decreasing by curcumin PDC or PDM compared to basal diet 107.28 10 5 cfuml. Protozoa population found numerically the lowest 7.90 10 3 cfuml was in PDM, while in control diet PD0 was the highest 32. 10 3 cfuml. These results suggested that yeast or curcuma or their combination worked well in protecting the PUFA- diet. Table 3.4 In vitro dry matter and organic matter digestibility IVDMD and IVOMD and fermentation characteristics of PUFA-diet supplemented with yeast and C. xanthorrhiza Roxb in goat rumen liquor Variable PD0 PDY PDC PDM P pH 6.71 ± 0.02 6.73 ± 0.07 6.73 ± 0.05 6.74 ± 0.04 NS IVDMD 62.72 a ± 1.66 60.26 ab ± 1.90 60.77 a ± 1.63 58.00 b ± 1.05 IVOMD 64.85 a ± 2.22 61.74 bc ± 2.07 62.82 ab ± 1.36 59.63 c ± 0.59 NH3 mM 11.26 ± 3.82 10.28 ± 3.82 9.31 ± 3.75 8.73 ± 3.16 NS VFA total mM 42.13 ± 3.45 43.95 ± 11.09 44.82 ± 12.30 45.27 ±12.92 NS Acetate A, mM 67.13 ± 6.39 67.68 ± 4.43 68.82 ± 4.98 68.46 ± 5.73 NS Propionate P, mM 19.78 ± 4.24 19.35 ± 2.60 18.25 ± 3.20 18.64 ± 3.10 NS Butirate B, mM 11.33 ± 2.90 11.21 ± 3.23 11.13 ± 2.85 11.05 ± 3.86 NS Iso Butirate , mM 1.27 ± 0.17 1.11 ± 0.24 1.08 ± 0.18 0.66 ± 0.58 NS Valerate V, mM 1.76 ± 0.66 1.76 ± 0.81 1.80 ± 0.79 1.86 ± 0.85 NS IsoValerate , mM 2.66 ± 0.30 2.48 ± 0.58 2.46 ± 0.48 2.08 ± 0.87 NS A:P 3.54 ± 0.97 3.55 ± 0.55 3.88 ± 0.82 3.76 ± 0.77 NS PD0: PUFA- diet with no additives; PDY: PUFA- diet with 0.5 yeast; PDC: PUFA- diet with 2 curcuma; PDM: PUFA- diet with mix of 0.5 yeast and 2 curcuma. Averages with different superscript, differed significantly P0.05. NS: not significant