In Vitro Goat Fermentation of PUFA-Diet Supplemented with Yeast and Curcuma Xanthorrhiza Roxb

This in vitro experiment was conducted to evaluate the ruminal performances of polyunsaturated fatty acid (PUFA)-diet (containing PUFA with 80% concentrate and 20% King grass) supplemented with yeast and C. xanthorrhiza Roxb. Experimental design was completely randomized block design of 4 x 4 with ruminal liquor derived from 4 slaughtered goats and 4 treatments (PD0-no supplement, PDY- 0.5% yeast, PDC-2% curcuma, and PDM- 0.5% yeast + 2% curcuma). Variables measured were pH, N-NH3, total and partial VFA (volatile fatty acid), protozoa population, and CH4 (methane). Results showed that the lowest (P<0.05) organic (59.63%) and dry matter (58.00%) digestibilities were found in PDM. In in vitro, this diet was also showing quantitatively low in N-NH3 (8.73 mM) and protozoa population (7.90±4.09 103 cfu/mL). On the other hand, it showed numerically high in VFA production (45.27 mM) and pH (6.74), yet low in CH4 (13.43% v/v). Based on these data, PDM was considered the most potential diet to improve nutrient metabolism in rumen of goat, in vitro.


INTRODUCTION
Ruminal fermentation performance is affected mostly by diet being ingested. Diet containing high concentrate with PUFA may increase production, improve milk fat and milk fatty acid in ruminants through microbial hydrogenation of PUFA in the rumen. However, to make such improvements, it needs any bioadditives, such as yeast and curcuma to be supplemented into diet that would determine the kinetics of rumen fermentation.
Yeast has been known as a rumen enhancer that improves digestion and nutrient metabolism. In vitro fermentation of diet added with Diamond-V XP yeast (XPY) showed higher dry matter digestibility (DMD), organic matter digestibility (OMD), and total VFA than those of diet with A-Max yeast with the same dose (57 g/d) (Miller et al., 2002). Feeding XPY ruminally in 56 g/d (to the concentration of 6 10 11 cfu/steer/d) also tended to increase OMD and acid detergent fiber (ADF) (Lehloenya et al., 2008). Supplementation of Saccharomyces cereviseae culture (1.16 10 4 cfu/g) of 0.35g/L produced higher concentration of acetate (C 2 ) than that of higher dose of 0.73g/L (Lynch & Martin, 2002). Results of meta-analysis on the influence of S. cerevisiae supplementation were variable; however, in average, it increased rumen pH and VFA but did not affect acetate/ propionate ratio (Desnoyers et al., 2009). In transition dairy cows, feeding 50 g/d of S. erevisiae fermentation product (Diamond V Original XP) can improve feed intake and milk production than that of no supplement as reported by Zaworski et al. (2014). Besides yeast as a supplement, some herbals containing bio actives such as tannin and curcumin could affect ruminal dynamics.
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 could decrease protozoal population, methane (CH 4 ) gas production, NH 3 , and VFA; on the other hand, it increased bacterial population in the rumen of dairy cow (Khiaosa-ard et al., 2009). Plants containing both hydrolyzable and condensed tannins were reported to be more effective in suppressing methanogenesis than those containing either one (Bhatta et al., 2009). Tannin (13.84%) extracted from Pakar leaves (Ficus infectoria) was added into diet fed for goat and revealed some higher levels of N-NH 3 , acetate, and total fungi with lower levels of total VFA and propionate (Singh et al., 2011).
Curcumin found in C. xanthorrhiza Roxb was reported to have a potential as an antibacterial for certain strain (Wiryawan et al., 2005). It was also reported that in early identification, curcumin and xanthorrhizol showed very significant activities as antibacterial and effectively slowed down the growth of Staphylococcus aureus, Salmonella paratyphi, Trichophyton gypseum, and Mycobacterium tuberculosis (Benson, 2012). Curcuma xanthorrhiza Roxb supplemented for 1.5% in PUFA-concentrate was apparently interacted with the sources of the PUFA (corn oil or roasted ground corn) in decreasing dry matter and organic matter digestions as well as total digestible nutrient (TDN) in dairy cow (Sulistyowati et al., 2010). Therefore, tannin, curcumin, and yeast that were supplemented into PUFA-diet were expected to work synergistically in the rumen system. Based on these data, a study on PUFA-diet supplemented with yeast and curcuma was conducted to observe in vitro rumen fermentation performance of goats.

Preparation of Yeast, Curcuma Powder, and PUFA-Diet
Yeast, curcuma powder, and PUFA-diet were prepared according to protocol reported in the previ-ous result (Sulistyowati et al., 2013). The PUFA-diet was reformulated as it was in the PUFA-concentrate and then was combined with soybean by-product from local tofu industry. Ground corn was half roasted; while soy bean meal was completely roasted in 80 o C for about 20 min. These ingredients together with corn oil were designated as PUFA sources in the diets. Basic PUFA-diet (PD) composed of rice bran (18.42%), ground corn (15.79%), soybean meal (7.89%), cassava meal (7.89%), soybean byproduct (27.49%), corn oil (2.11%), minerals (0.53%), and King grass (19.88%).
Yeast and or Curcuma powder were then added to the diet as a treatment. The treatments were: PD0 : PUFA-diet without supplementation PDY : PUFA-diet with 0.5% yeast supplementation PDC : PUFA-diet with 2% curcuma powder supplementation PDM : PUFA-diet with 0.5% yeast and 2% curcuma powder supplementations

Chemical Analyses
Nutrient contents were assessed based on proximate analysis (AOAC, 1990); ADF (acid detergent fiber) was analyzed according to Van Soest (1990). Curcumin was analyzed by using HPLC separation performed on a C 18 column by using three solvents i.e., methanol, 2% AcOH, and acetonitrile, with detection at 425 nm (Jayaprakasha et al., 2002). Tannin was analyzed by modification of Folin-Ciocalteu method (Harborne, 1987). Fatty acid . Fatty acid Fatty acid content was detected as fatty acid methyl esters (FAME) by using gas chromatography (GC) (Shimadzu 2010 series), after extraction of diet fat samples according to the method conducted by Palmquist & Jenkins (2003). Individual fatty acid was quantified by using specification of column (SP TM -2560, 100 m x 0.25 mm ID, 0.2 µm film), gas carrier (helium, 20 cm/s), oven temperature (140 o C in 5 min to 240 o C at 4 o C/min), detector (FID, 260 o C), and inject (1 µL, 2600C, split 100:1) and each was identified according to a mixed FAME standard (Supelco 37 component, Supelco Inc).
In vitro procedure was based on Tilley & Terry (1963). Fermentor tubes were loaded with 0.5 g of samples and added with 40 mL of Mc. Dougall solution. Tubes were put into shaker bath in 39 o C and added with 10 mL rumen liquor of slaughtered goats. Tubes were soaked and flowed with CO 2 for 30 s and the pH was checked (6.5-6.9) by using pH meter. The tubes were then probed with ventilated rubber caps and fermented for 48 h. When fermentation was stopped at 4 h, the caps were taken off from the tubes, and 2-3 drops of HgCl 2 was added to stop microbial growth (at this point, analyses were conducted for protozoal population and CH 4 production). Fermenter tubes were then centrifuged at 4.000 rpm for 10 min. Substrates were separated as solid in the bottom and supernatant on the top. The supernatant was used for VFA, and N-NH 3 analyses, while the solid was then added with 50 mL pepsin-HCl 0.2%, centrifuged in 4.000 rpm for 15 min. This substrate was then reincubated for 48 h without probes; and then strained through Whatman paper no 41 (the weight was recorded) by using vacuum pump. The solid left in the paper was transferred into baker glass, put in oven in 105 o C for 24 h. The glass, paper, and residue were taken out from the oven, and put in exicator before weighing to get dry matter content. Afterward, these samples were fired in electrical oven for 6 h in 450-600 o C. Then the samples were weighed 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 sample. The digestibility was calculated as if there was a hydrolytic post ruminal fermentation after two timed 24 h (Tilley & Terry, 1963).
Counting of protozoal population was done by adding 0.5 mL methyl formalin saline (MFS) into the reaction tubes then mixing it with rumen liquor and homogenized. Sample was dropped onto hematocytometer and covered it with glass securely. Protozoa population was evaluated under microscope by using 40x objective lense with 10x ocular lense.

Experimental Design and Data Analysis
Experimental design being applied was completely randomized block design with 4 ruminal liquors derived from four slaughtered goats and 4 treatments (PD0, PDY, PDC, PDM), in 3 replications. Data were tabulated and analysis of variance (Anova) was conducted, any significant differences were then tested by using Duncan Multiple Range Test (DMRT) according to Lentner & Bishop (1986).

Chemical Composition of Diets
Average content of protein in these diets was 12.96±0.23% (Table 1). This level was fulfilled the protein requirement of 12% for 30 kg dairy goat with 1 kg milk production (NRC, 1981). Other result showed that protein content in diet with extruded soybean in dairy goat was much higher i.e., 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, and higher fat than other diets. This result strongly approved that these two additives could improve nutrient content.
Diet with high concentrate in this experiment (80%) could decrease ether extract of the diet that eventually would decrease milk fat. However, since this research used fat sources (roasted ground corn, roasted soy bean meal, and corn oil), the ether extract of these diets were relatively high (4.0%-4.43%), except in control diet (3.75%). This result was in accordance with others reports that diet without extruded soybean contained very low fat (1.38%) while the one with that ingredient showed a high fat content (5.19%) (Schmidely et al., 2005). Diet with corn oil was reported to have higher fat i.e., 5.62% (Bouattour et al., 2008). It seemed that bioactives, curcumin and tannin did not affect the fat content of the diets; however, these supplements did affect fatty acid content in dairy goat milk (Sulistyowati et al., 2013).
It has been known that curcumin plays 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 catabolized. It was reported that curcuminoid as much as 0.0133 mg/mL could slow down peroxidation of linoleic acid (Tonnesen & Karlsen, 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 result might be due to the level of curcumin that was not high enough to catabolize the increasing substrate resulted from yeast fermentation in the diet, when both supplements were combined.
Short chain fatty acids (C10 and C11) were not detected in control, yeast, and curcuma diets; while very little level was shown 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). On the other hand, these fatty acids were reported high in diet with supplementation of forage tannin, tannin extract,   , 2009). Ruminant diet with high linoleic acid will be transformed into rumenic acid (cis-9 trans-11 18:2) through ruminal biohydrogenation (Jenkins et al., 2008) that along the process will produce abundant amount of intermediates, such as vaccenic and stearic acids (Palmquist et al., 2005). Biohydrogenation involving rumen microbes depends upon the type of diet. Lipid supplementation stimulated bacteria from the family of Lachnospiraceae (Belenguer et al., 2010).

Fermentation Characteristics
In vitro fermentation characteristics (Table 2) 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. Both digestibilities of diet without additives (PD0) were significantly higher than those of PDM; while the yeast (PDY) or curcuma (PDC) diet was 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 decreased with the increasing yeast level from 0.37 to 0.73g/l in rumen liquor of young male cattle fed alfalfa hay (Lynch & Martin, 2002). Supplementation of S. cereviseae 1026 significantly decreased N-NH 3 , yet increased protozoa population (Al-Ibrahim et al., 2010). Lower ruminal N-NH 3 with yeast supplementation were also found in other reports (Marden et al., 2008;Moallem et al., 2009).
The A/P ratios in these diets (3.54-3.88) were higher than that of with the same high concentrates (2.08-2.31) in Rusitec fermentors as reported by Martinez et al. (2010). It was known that the type of diet will determine the production of propionate and acetate. Carbohydrate produces higher propionate, while fiber will produce higher acetate. The A/P ratio increased with the longer fermentation, suggesting that the more structural carbohydrate was degraded, therefore, the higher acetate and lower propionate production.
Concentration of N-NH 3 did not significantly decrease from 11.26 mM in PD0 to 8.73 mM in PDM.
This level of NH 3 was much higher than that of NH 3 (8.5-9.6 mM) with pH of around 6.7, in goat fed with extruded soybean with or without NaHCO 3 (Schmidely et al., 2005). The N-NH 3 concentration in dairy cow fed diet containing tannin extract was detected to reach the lowest level (5.1 mM) as compared to that of control diet (17.2 mM) with pH around 7.05-7.09 (Khiaosa-Ard et al., 2009). Decreased N-NH 3 was also detected in in vitro system with essential oils (limonene, guaiacol, thymol, and eugenol), suggesting that deamination of amino acid was disrupted (Castillejos et al., 2006).

Protozoa Population
Protozoa population (Table 3) was not significantly low in PDM (7.90 10 3 cfu/mL), compared to control diet (PD0), that was (32. 10 3 cfu/mL). These results suggested that yeast or curcuma or their combination worked well in protecting the PUFA-diet by suppressing the growth of protozoa that eventually, otherwise will increase biohydrogenation of PUFA sources. The PUFA protection of feed source means to elevate ruminal bypass of α-linolenic acid without being modified by microbial biohydrogenation (Khiaosa-Ard et al., 2009). In vitro bacteria and protozoa counts were significantly higher in goats than that of in sheep. However, fungi population was higher in sheep and fungi existence in rumen system was crucial in digesting dry matter as described by Li & Hou (2007). Supplementation Note: Means in the same row with different superscripts differ significantly (P<0.05). PD0: PUFA-diet with no additives; PDY: PUFA-diet with 0.5% yeast; PDC: PUFA-diet with 2% curcuma; PDM: PUFA-diet with a mixture of 0.5% yeast and 2% curcuma. of Hibiscus tiliaceus leaves (saponin source) for 5% significantly decreased rumen protozoa population (4.50 10 3 cfu/mL), compared to control (16.25 10 3 cfu/mL) as reported by Istiqomah et al. (2011). Protozoa population in this research was affected by tannin. Addition of tannin extracted from Acacia mearnsii was effectively decreased protozoa population to a very low level (1.91 10 3 cfu/mL) and the protozoa population could be higher when the additive was not in the form of extract (Khiaosa-Ard et al., 2009). Meanwhile, protozoa population in this research was lower than that found in diet added with saponin generated from Yucca schidigera (6.17 10 5 cfu/mL) and Quillaja saponaria (6.17 10 5 cfu/mL) in rumen ferementation of dairy cow (Holtshausen et al., 2009). Total protozoa (Entodinium spp, Isotrica spp., Dasytrica spp., Epidinium spp., Ophryoscolex spp., and Diplodinium spp.) detected in rumen fermentation of dairy cow supplemented with yeast XP 56g/d was much higher (8.03 10 5 cfu/mL) (Hristov et al., 2010). This result indicated that the type of plants or additives, the method of administration, and the right dose will determine the effectiveness of the bioactives during the rumen fermentation.
Gas production (CH 4 ) in a mix diet (PDM) and curcuma diet (PDC) was higher (around 13%-14%, respectively) as compared to PDY and PD0 diets (about 12%). This implied that supplementation of tannin and curcumin was not effective in reducing methane (CH 4 ) production in this study. On the other hand, Khiaosa-Ard et al. (2009) reported that tannin extract was able to decrease gas production (CH 4 , CO 2 , and H 2 ) to the lowest level (0.454 L/d). Decreased gas production was also found in diet containing silage and concentrate added with Peltiphyllum peltatum rich in phenol and tannin (Jayanegara et al., 2010). The use of rhubarb (milled rhizomes of Rheum spp.) in the diets of ruminants was also reported effectively to improve ruminal fermentation by reducing methane emission that eventually potentially benefits animal production and environment (Gonzales et al., 2010).
Production of methane gas was reported higher (10.3 g/d) with XP yeast supplementation diet than that in control (9.7 g/d) in dairy cow (Hristov et al., 2010). Methane productions with supplementations (curcuma and mix) were not significantly lower than that in control (12.90% v/v). This result was contradicted to the results that CH 4 emission (g/d) of lactating dairy cows fed dietary supplements (fat, fatty acid, and Ca) decreased 10%. However, it remained unchanged when it was correlated to milk production (Zijderveld et al., 2011).
Emissions of daily CH 4 was also affected by the level of crude fiber in the diet; the increasing corn silage (0% to 27%), the higher the production of CH 4 (487 to 540 g/d) in lactating dairy cow (Benchaar et al., 2014). Methane gas was produced through a mechanism starting from anaerobic degradation; triglyceride hydrolysis, saturation of unsaturated fatty acid (C18:2C18:1C18:0C1 6:0C14:0), and successive β-oxidation of saturated fatty acid (C10-C18). All these reactions produced acetate that eventually reacted with H 2 to produce CH 4 (Sage et al., 2008).

CONCLUSION
In spite of having the lowest organic and dry matter digestibilities as well as N-NH 3 production, as was supported by low protozoa population and high VFA production in the goat rumen fluid, the PUFA-diet with a mixture of yeast (0.5%) and curcuma (2%) additives was considered to be the most potential diet.

ACKNOWLEDGEMENT
This research was part of a series of research granted by Hibah STRANAS-DIKTI-IPB with contract No: SP2H/PL/DITLITABMAS/ V/2013. Authors would like to appreciate for this opportunity. We also thank to Ir. Sigit Mujiharjo MSAE for checking with statistical analysis; and, co-workers for laboratory analyses.  Note: PD0: PUFA-diet with no additives; PDY: PUFA-diet with 0.5% yeast; PDC: PUFA-diet with 2% curcuma; PDM: PUFA-diet with a mixture of 0.5% yeast and 2% curcuma.