IN VITRO GOAT FERMENTATION OF PUFA- DIET

LIST OF APPENDIX 1 Breed and lactation state of each dairy goat used in the research 64 2 Body measurements of dairy goat used in the research 65 3 Goat and treatment arrangement during in vivo experiment 66 4 Environment condition around housing at Cordero Farm in Ciapus, Bogor 67 5 Anova of PUFA-concentrate moisture 70 6 DMRT of PUFA-concentrate moisture 70 7 Anova of PUFA-concentrate dry matter 70 8 DRMT of PUFA-concentrate dry matter 70 9 Anova of PUFA-concentrate ash 71 10 Anova of PUFA-concentrate organic matter 71 11 DMRT-week of PUFA-concentrate organic matter 71 12 DMRT-treatment of PUFA-concentrate organic matter 71 13 ANOVA of PUFA-concentrate ether extract 72 14 ANOVA of PUFA-concentrate crude protein 72 15 ANOVA of PUFA-concentrate crude fiber 72 16 DMRT of PUFA-concentrate crude fiber 72 17 ANOVA of PUFA-concentrate NFE 73 18 DMRT-week of PUFA-concentrate NFE 73 19 DMRT-treatment of PUFA-concentrate NFE 73 20 ANOVA of PUFA-concentrate ADF 73 21 ANOVA of PUFA-concentrate ADF 74 22 DMRT-week of PUFA-concentrate ADF 74 23 DMRT-treatment of PUFA-concentrate ADF 74 24 ANOVA of PUFA-concentrate saccharomyces 74 25 DMRT of PUFA-concentrate saccharomyces 75 26 ANOVA of PUFA-concentrate bascillus 75 27 DMRT of PUFA-concentrate bascillus 75 28 ANOVA of dry matter consumption 75 29 ANOVA of organic matter consumption 75 30 ANOVA of crude protein consumption 76 31 NOVA of crude fiber consumption 76 32 ANOVA of ether extract consumption 76 33 DMRT of ether extract consumption 76 34 ANOVA of NFE consumption 76 35 ANOVA of gross energy consumption 77 36 ANOVA of NDF consumption 77 37 DMRT of NDF consumption 77 38 ANOVA of ADF consumption 77 39 ANOVA of Ca consumption 77 40 DMRT of Ca consumption 78 41 ANOVA of P consumption 78 42 DMRT of P consumption 78 43 ANOVA of dry matter digestibility 78 44 DMRT of dry matter digestibility 78 45 ANOVA of organic matter digestibility 79 46 DMRT of organic matter digestibility 79 47 ANOVA of crude protein digestibility 79 48 DMRT of crude protein digestibility 79 49 ANOVA of crude fiber digestibility 79 50 DMRT of crude fiber digestibility 80 51 ANOVA of ether extract digestibility 80 52 DMRT of ether extract digestibility 80 53 ANOVA of NFE digestibility 80 54 DMRT of NFE digestibility 80 55 ANOVA of gross energy digestibility 81 56 DMRT of gross energy digestibility 81 57 ANOVA of NDF digestibility 81 58 DMRT of NDF digestibility 81 59 ANOVA of ADF digestibility 81 60 DMRT of ADF digestibility 82 61 ANOVA of Ca digestibility 82 62 DMRT of Ca digestibility 82 63 ANOVA of P digestibility 82 64 DMRT of P digestibility 82 65 ANOVA of TDN 83 66 DMRT of TDN 83 67 ANOVA of milk production 83 68 ANOVA of caproic acid C6:0 in milk 83 69 DMRT of caproic acid C6:0 in milk 83 70 ANOVA of undecanoic acid C11:0 in milk 83 71 DMRT of undecanoic acid C11:0 in milk 84 72 ANOVA of lauric acid C12:0 in milk 84 73 DMRT of lauric acid C12:0in milk 84 74 ANOVA of tridecanoid acid C13:0 in milk 84 75 DMRT of tridecanoic acid C13:0 in milk 84 76 Milk production of goats fed PUFA- diets along the research 85 77 Milk production of goats in block 1 fed PUFA-diets along the research 85 78 Milk production of goats in block 2 fed PUFA-diets along the research 85 79 Milk production of goats in block 3 fed PUFA-diets along the research 85 80 Milk production of goats in block 4 fed PUFA-diets along the research 85

1. INTRODUCTION Background

Milk production in Indonesia is increasing yearly, however, it has not fulfilled milk demand. In 2011, milk production was 926 10 3 tons, while the total demand of milk and milk product was 3,903 10 3 tons DGLAH 2011. The lack of this milk demand would be imported for about 76.27 a year; with an international milk price of 400ton, it would cost around 1,190,800,000yr. On the other hand, with 17,483 10 3 population of goat in 2011, supposed 0.5 out of them was dairy goat and 30 of them was in lactation with 0.5 kgd milk production, in one lactation period would be producing milk about 1,967 tons. There might be a potential alternative of milk production as much as 0.05yr to supply national milk demand. This assumption might be too low compared to data released by FAO that was about 1.56 goat milk contributed by Indonesia to the world goat milk Thiruvenkadan 2012. This huge gap of milk consumption and milk production should be narrowed. At the same time, the quality of the milk needs to be improved as well. For these purposes there has to be nutrition manipulation that could be applied to the dairy ruminants, such as dairy goat. In terms of fatty acid short, medium- chain, saturated branched, mono- polyunsaturated, cis- trans conjugated contents in animal product, including milk, have been receiving a lot of attention concerning of human health Chilliard et al. 2003. Therefore, it needs efforts in improving rumen kinetics relating to nutrients of the feed provided, especially in improving the polyunsaturated fat, PUFA. This could be achieved by applying diet rich in PUFA such as roasted corn grain, roasted soybean meal, and corn oil supplemented with temulawak Curcuma xanthorrhiza Roxb and yeast. Curcuma xanthorrhiza Roxb, within its root, contains some bioactives such as curcuminoids 3 of dry matter consisting of curcumin C, demethoxycurcumin, and bisdemethoxycurcumin Rukayadi et al. 2008; and xanthorrizol 33.2 of rhizome oil reported by Sirat et al. 2008. Curcumin has molecular weight of C 21 H 20 O 6 that would decrease due to the changes of poliphenol weight during radiation process of curcuma powder and curcuma simplicia. This condition resulted in degradation of covalent bond that changed to free phenol becoming highly active in antioxidant compared to that of in curcumin tablet and fresh curcuma Nurlidar and Chosdu 2008. Bioactive of curcumin also functions as antibiofilm Rukayadi et al. 2008, antimicrobial, anti- inflammation, anticancer apoptosis, antiangiogenesis, detoxification, neuro protection, and antiaging Hwang 2008; elevates bile production, lessens tissue inflammation and plasma LDL low density lipoprotein in rabbit Wientarsih and Meulen 2008. It was also reported that in early identification, curcumin and xanthorrhizol worked very strong as antibacterial and effectively slow down the growth of Staphylococcus aureus, Salmonella paratyphi, Trichophyton gypseum, and Mycobacterium tuberculosis Benson 2012. Curcumin interacts with adiposity pancreas cells, heart cells, macrophage, and muscle in suppressing proinflammatory transcription factors Aggarwal 2010. As anti inflammation and anti pathogen, curcumin is expected to decrease inflammation in mammary gland causing mastitis. The indicators of this are decreasing SCC somatic cell count, Staph. aureus, and E. coli contents in milk. In organic less use of antibiotic synthetic dairy farm, it was found decreasing CNS coagulase negative staphylococci and environment Streptococcus sp in milk sample Suriyasataporn 2010. Normal SCC level of dairy cow milk was less than 1 x 10 5 cfuml; while the infected cow was 1 x 10 6 cfuml BytyQi et al. 2010. It has been reported also that SCC consisting of 75 leucocytes: neutrophils, macrophages, lymphocytes, erythrocytes, monocytes; and 25 epithelial cells in infected dairy cows were facing potential loss of milk production for up to1200 kglactation with SCC of 2.2 - 4.5 x 10 6 cellsml Sharma et al. 2011. There was correlation between high level of SCC and mastitis associated with decreasing lactose, α-lactalbumin, and milk fat Harmon 1994. Combination of polyherbal in low level 125 mgkg body weight produced milk optimally in dairy goat Mirzaei and Prasad 2011. Curcuma, including C. xanthorrhiza Roxb, also contains bioactive steroid that served as lactagogum, conserving the continuity of cell differentiation of epithel cells in normal tissue, mucose secretion, alveolus proliferation, and ductul growth in mammary gland. Hormones relating to this process such as prolactin is the most secreted hormon right before partus Larson 1985. Concentration of prolactin in dairy goat blood decreased from 18.55 ngml in 15 days of lactation to 5.88 ngml in 150 days of lactation Singh and Ludri 2002. Therefore, curcuma besides maintaining lactation, also containing other secondary compound, such as tannin and saponin that function in microbial rumen activity. Ruminal kinetics are expressed by microbial rumen activity in digesting fiber that its dynamics were affected by protozoa functioning as predator for bacteria Gutierez 2007. Saponin triterpenoid and steroid saponin generated from any materials were identified detrimental toward protozoa antiprotozoa and as defaunating agent in rumen with the effect of detergent foaming on the surface of cell membrane Francis et al. 2002. Saponin generated from lerak Sapindus rarak extract of 0.8 mgml rumen liquor decreased protozoa population and affected composition and total bacteria population after 24 hours in vitro fermentation. Population of F. succinogenes bacteria was dropped with increasing consentrate; whereas P. ruminicola increased markedly with increasing Sapindus . Decreasing protozoa was associated with decreasing activity of methanogen; modifying H 2 to be propionate supported by bacteria with certain amount of population and appropriate type as well as abundant amount of substrate Sugiarti et al. 2011. Beside saponin, tannin is an effective poliphenol in decreasing methan CH4 gas production that was produced about 10 out of energy , in vitro or in vivo . The efficacy of tannin depends on type of carrier materials, easily hydrolyzed easily accumulated, toxic or condensed bound, safe. Tannin from chestnut was the most reasonable in reducing CH 4 gas per digested organic matter with the lowest C2C3 ratio Jayanegara et al. 2008. Methane gas is affected during fermentation process of fat source of diet and metabolized it through lipid hydrolysis process with end results of triglyceride, free fatty acid, glycerol, then continued in β- oxidation to yield acetate and H 2 , as the precursors of methane gas production Sage et al. 2008. Lipid supplementation produced fatty acid, including CLA conjugated linoleic acid based on its source; high in cooking oil and fresh grass, but, low in unprotected grain oil Chilliard et al. 2003. Free fatty acid and CLA in the rumen will be biohydrogenized hydrolyzed by bacteria with addition of 2 ion H + to be saturated; in conversely, it should be bypassed then biosynthesized as unsaturated fat in milk. Eventually, it is transported into mammary gland as in precursor, changed from linolenic acid C18:3 cis 9, cis 12, cis 15 in several steps to become stearate C18:0, then ultimately converted into oleate in mammary gland. This process is optimazed by SCD steoroyl- CoA desaturase enzyme. Fatty acid content of milk of goat fed sunflower and soy bean oil was lowered in saturated fat and atherogenicity index 1.21- 1.71, but increased in CLA compared to control. This suggested that unsaturated fatty acid in blood vessel was going up that made this index down as expected Chilliard et al. 2003. Yeast, mostly mentioned as Saccharomyces cereviseae in any forms, dried or liquid, is widely used as rumen enhancer or fermenting feed supplement with variable effects on dry matter and organic matter digestibility, ruminal microbes, and ruminal fermentation activity pH rumen, VFA, lactate, milk production, and fatty acid as described by Desnoyers et al. 2009. Lynch and Martin 2002 reported that yeast, both in culture and cells showed no different activities; however with high dose 0.73 gl produced lower CH4 and C2C3 in in vitro fermentation of diet containing Bermuda grass. Viability of yeast cell in dry form was decreasing from 9.88 x 10 10 cfug to 5.43 x 10 10 cfug after 3 months of storage in 40 C Sullivan and Bradford 2011. Supplementation of RumiSacc, commercial yeast, was not significantly different from control in affecting BUN blood urea nitrogen, cholesterol, triglyceride, and glucose of dairy cow Yalcin et al . 2011. Synergy of temulawak curcuma, yeast, and PUFA- concentrate has been gradually conducted in lactating ruminants. Milk production of lactating Bali cow could increase 6.6- 9.3 times higher with 5 pasta of fermented cassava and temulawak C. xanthorrhiza Roxb liquid, reported by Sulistyowati 1999; milk yield has doubled with Tabut abbreviation for fermented cassava and curcuma block 300g in lactating Bali cows Sulistyowati et al. 2001; in FH dairy cow with Tabut block, its milk production increased with increasing amount 0- 450g of the block following this model: Y: 6.66 + 0.05 x, r: 0.90 Sulistyowati and Erwanto 2009. Microbial population of Tabut block decreased with increasing curcuma 15-25 during storage of 3- 12 weeks Sulistyowati et al. 2008 b , while 15 curcuma level was optimal for milk production Sulistyowati et al. 2008 a in FH dairy cows; 2yeast supplementation in FH dairy cows produced the highest milk yield Sulistyowati et al. 2010 b ; concentrate containing 10.5C. xanthorrhiza Roxb was optimal for milk production in FH dairy cows Sulistyowati et al. 2010 a ; PUFA- concentrate containing 4.5 roasted ground corn produced the highest milk production with lower milk fat with increased PUFA and ratio of n6n3 was about 2.14 in milk of dairy cow Sulistyowati et al. 2010 c with high TDN Sulistyowati et al. 2010 d ; curcuma concentrate in lactating dairy cow Sulistyowati et al. 2011. Meanwhile, milk production of dairy goat supplemented with 3gd of yeast was optimal Sulistyowati and Mega 2002; whereas, dairy goat fed pasta of fermented cassava and curcuma liquid could increase milk production for 0.1 kgd with significant reduction in milk fat to 2.09 Sulistyowati 2009. However, along with these researches, there has not been studied on ruminal activity, blood metabolites, milk fatty acid quality and milk production, especially in dairy goat, moreover during late lactation. Therefore, three experiments had been conducted nutrient quality of stored PUFA-concentrate, in vitro ruminal fermentation, and in vivo application of PUFA-diet supplemented with yeast and C. xanthorrhiza Roxb in late lactating dairy goat. Problems Based on above description, it has been analyzed several problems that need to be improved: 1. Milk production of dairy goat was very low compared to its actual capacity. 2. Milk quality, especially milk fatty acid was high in saturated fat. 3. Ruminal fermentation needs to be manipulated with nutrient originated from PUFA –diet supplemented with curcuma and yeast. Research Purposes Based on the identified problems, three experiments were designed to meet those three purposes: 1. To evaluate nutritional quality of PUFA- concentrate containing roasted ground corn, roasted soybean meal, corn oil supplemented with curcuma and yeast stored in 2, 4, and 6 weeks. 2. To analyze performance of PUFA- diet supplemented with curcuma and yeast in in vitro ruminal fermentation.

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

women in lactation, curcuma and yeast on nutrient digestibility, blood metabolites, mammary health, production and fatty acid quality of milk of late lactating dairy goat. Research Outcomes Out of each experiment, there would be a result that could be applied, those are: 1. One PUFA – concentrate formula that is durable in nutritional quality during the 2- 6 weeks of storage. 2. One PUFA- diet formula that is optimal in in vitro ruminal fermentation that is optimal metabolically. 3. One formula of PUFA- diet that is optimal for mammary health, production and fatty acid quality of milk of late lactating dairy goat. Research Hypothesis 1. Curcumin and tannin within curcuma are able to protect nutrients of PUFA- concentrate stored for up to 6 weeks. 2. Curcumin, tannin, yeast Saccharomyces cereviseae, and PUFA sources roasted ground corn, roasted soybean meal, and corn oil in PUFA- diet were able to reduce unwanted microbes, such as protozoa in such a way that would improve metabolic rumen in biohydrogenation process. 3. Curcumin, tannin, yeast Saccharomyces cereviseae, and PUFA sources roasted ground corn, roasted soybean meal, and corn oil in PUFA- diet were able to improve metabolic rumen in biohydrogenation process that eventually will improve mammary health, production and fatty acid quality of milk of late lactating dairy goat. 2.THE NUTRITIVE PERFORMANCES OF PUFA- CONCENTRATE SUPPLEMENTED WITH YEAST AND Curcuma xanthorrhiza Roxb STORED IN 2-6 WEEKS ABSTRACT The objective of this experiment was to evaluate the nutritive performances of PUFA- concentrate supplemented with yeast and Curcuma xanthorrhiza Roxb stored in 2-6 weeks. There were four PUFA- concentrates, namely, no supplement PC0, 0.5yeast PCY, 2curcuma powder PCC, and 0.5yeast with 2curcuma powder PCM. Yeast containing 3.6 x 10 7 cfug and curcuma powder containing 0.8 curcumin were self made. These concentrates were evaluated for nutrition and fatty acid contents during 2 and 6 weeks of storage. Results showed that moisture and Saccharomyces cereviseae 20.68 x 10 6 cfug increased significantly p0.05 in 6 weeks of storage; whereas, dry matter DM and organic matterOM, crude fiber CF, nitrogen free extract NFE decreased significantly p0.05. The total PUFA P, PS, Monounsaturated fatty acid MUFA, and long chain fatty acid LCFA contents were found higher in PUFA- concentrate with 2curcuma powder. Whereas, the PUFA- concentrate with 0.5yeast and 2curcuma powder was higher in unsaturated U fat and the ratio of US. Combining all nutrient performances during the storage of 2- 6 weeks, the PUFA- concentrate with 0.5yeast and 2 curcuma powder was considered nutritionally healthy. Key words: curcuma, nutrient, PUFA-concentrate, yeast, storage INTRODUCTION Concentrate is the main nutrient source for livestock, including dairy goats. Its nutritional values are importantly taken into account in order to fulfill the requirement for improved production and good quality of milk produced. Several feed supplements such as yeast have been applied extensively. It was reported as optimal level in a rate of 3gdairy goat Sulistyowati and Mega 2002, 20gdairy cow Sulistyowati et al. 2010 b , 56gdairy cow Hristov et al. 2010, and 50g Rumisacc dairy cow Yalçın 2011. Yeast contain active viable cells, especially Saccharomyces cereviseae that would be beneficial for nutrient digestion through fermentation within the concentrate all the way to rumen system. Herbs are considered as local plants medicine and as feed supplement. Temulawak Javanese- Indonesian or Curcuma xanthorrhiza Roxb as other Curcuma sp, containing curcuminoids curcumin, demethoxycurcumin, and bisdemethoxycurcumin known for its biological activities, such as antioxidant, anti-inflamantory, antimicrobial Jayaprakasha et al. 2002. Supplementation of this curcuma powder of 15gkg concentrate increased milk production of dairy cow Sulistyowati et al. 2010 a and 60g curcuma diluent300g block decreased total bacteria and fungi Sulistyowati et al. 2008 b . Polyunsaturated fatty acid PUFA supplementation in concentrate is incompletely bio hydrogenised later in the bio system of ruminants which eventually will reduce fat, short and medium chain fatty acid into long chain fatty acid in milk Whitlock et al. 2002 and a b c d Figure 2.2 Steps in preparing C. xanthorrhiza Roxb powder: a shredding; b drying; c oven; d grinding Schmidely et al. 2005. Concentrate supplemented with roasted ground corn, yeast, and curcuma powder showed optimal levels in PUFA, ratio of PUFAsaturated PS, and n6n3 designated for dairy cows Sulistyowati et al. 2010 b . Dairy goats and their products are getting popular in terms of preference and nutrition for human health. Therefore, a part of this research was designed to evaluate concentrate containing roasted corn grain, roasted soy bean meal, and corn oil as PUFA sources supplemented with yeast and Curcuma xanthorrhiza Roxb, stored for 2, 4, and 6 weeks MATERIALS AND METHODS Feed Supplements Preparation Yeast supplement was prepared by modification on a procedure of Pusbangtepa 1981. The ingredients were 500g of rice flour, 50g of cassava tuber,10.5of sugar, 10g of garlic, 20.5of Alpinia galanga Sw, 10g of lemon juice, 10g of local Bengkulu, Indonesia yeast, and 500g of water; mixed, mounted in 10g each, then sun dried. This yeast contained 3.6 10 7 cfug. These are ingredients and steps in preparing the yeast; shown in Figure 2.1. a b c d Figure 2.1. Ingredients and steps in preparing the yeast: a yeast ingredients; b mixing; c dough; d mouting-drying Curcuma supplement was made of C. xanthorrhiza Roxb tuber which was sliced thinly about 1 mm thick, sun dried for about 4 hours, then oven dried in 60° C for 48 hours. It was grounded and refined as powder, which was then about 26.6 ww out of fresh curcuma. Bio actives detected in this curcuma were 0.8 curcumin and 1.58 tannin . In this treatment, the curcuma powder was added as much as 2 or 20gkg of PUFA- concentrate, mixed thoroughly. Steps in preparing the curcuma powder were presented in Figure 2.2.