In Vitro Rumen Fermentation of Ration Supplemented with Protected Vegetable Oils

This experiment was designed to evaluate the effects of protected vegetable oils supplementation on in vitro fermentation characteristics, rumen microbial population, and methane production in cattle. The treatments were arranged in a complete randomized block design involving 2 factors i.e. oil type (sesame, canola, and flaxseed) and protection methods (non protected, calcium soap, and microencapsulation). Variables observed were rumen pH, N-NH3, total and molar proportion of VFA, dry matter and organic matter digestibility, population of protozoa and total bacteria, methane production, and hydrogen balance. Data were tested using Analysis of Variance (ANOVA) and the differences among treatments means were examined by Duncan Multiple Range Test. The oil type did not affect all variables measured. The protection method using microencapsulation significantly increased N-NH3 concentration. There was an interaction between oil type and protection method on total VFA concentration, molar proportion of VFA, and methane production. The supplementation of calcium soap-flaxseed oil significantly increased total VFA production, while the supplementation of microencapsulated flaxseed oil had the highest propionate concentration and H2 utilization, the lowest A:P ratio, and methane production. It is concluded that microencapsulated flaxseed oil was the best treatment to optimize rumen fermentation.


INTRODUCTION
National meat production from beef cattle could not fulfill the demand and there is still deficit about 400 000 ton in 2014. Program of increasing beef cattle population and productivity by 23% in 2014 is the Government target to suppress import of beef cattle. The target should be followed with improving of beef quality. Beef is red meat containing high saturated fatty acids (SFA) associated with the risk of cardiovascular and cancer diseases when consumed in high level. High SFA content in beef is normally occurs due to biohydrogenation process in the rumen which transforms the PUFA to SFA. This rumen biohydrogenation process is a detoxification mechanism to avoid bacteriostatic effects of unsaturated fatty acids which could disrupt membrane integrity and decrease growth of microbes. Butyrivibrio fibrisolvens is a major microbe which plays role in this biohydrogenation process (Maia et al., 2010).
Previous studies showed that supplementation of vegetable oil (high PUFA content) could decrease saturated fatty acid content and increase unsaturated fatty acid content in beef. Some potential vegetable oils to use are sesame, canola, and flaxseed oil (Manso et al., 2005;Aharoni et al., 2005;Beauchemin et al., 2007). Supplementation of 10% flaxseed oil in cattle ration significantly increased PUFA and omega 3 proportion on intramuscular fat (Kim et al., 2009). Duckett & Gillis (2010) reported that supplementation of 4% canola oil in ration significantly increased (P<0.001) oleic acid, linoleic acid, and decreased palmitic acid on biohydrogenation process than corn oil. Supplementation of 4% flaxseed oil in the form of non protected, lipase-treated, or soapstock in ration increased linoleic acid concentration in beef tissue (Quinn et al. 2008). However, this oil needs to be protected prior to supplementation to avoid biohydrogenation process by rumen microbes, to decrease rumen microbial growth and activity, and to improve feed digestibility. Some protection methods that can be applied are calcium soap (Block et al., 2005;Wynn et al., 2006;Huang et al., 2009) and microencapsulation ( Kanakdande et al., 2007;Calvo et al., 2010;Agnihotri et al., 2012). However, optimization for rumen fermentation, oil type and protection method should be determined. The objective of this research was to evaluate the effects of supplementation of three kinds of vegetable oils (sesame, canola, and flaxseed) protected using calcium soap and microencapsulation on in vitro fermentation characteristics, rumen microbial population, methane production, and hydrogen balance.

Preparation of Calcium Soap and Microencapsulation
The sesame oil, canola oil, and flaxseed oil were produced by MH Farm Bogor Company, Golden Bridge, and Green Tosca, respectively. Calcium soap from these three kinds of vegetable oils was made according to the method by Kumar et al. (2006). The main ingredients for calcium soap were NaOH (to hydrolize the fatty acids of vegetable oil) and CaCl 2 . and CaCl 2 . Sodium hydroxide solution (in line with saponification value) was added into hot vegetable oil, heated and stirred on a hotplate until the fat was completely dissolved. Calcium chloride (2.35 g) were dissolved in 4.7 mL of water and then added slowly into the water soluble soap while being stirred to aid the precipitation of calcium soap. Calcium soap was then dried overnight in a oven at 60 o C.
The microencapsulation was done according to the method by Calvo et al. (2010). Sodium caseinate (protein source) from Sigma Aldrich (Singapore) and lactose (carbohydrate source) of commercial grade were used for microencapsulation wall. The process included making emulsion, mixing the wall materials, and mixing it with oil until homogenous. The ratio of oil and wall materials used was 1% (oil): 2% (1% sodium caseinate and 1% lactose). The emulsion prepared was spray dried using a laboratory scale Buchi spray drier (Mini Spray drier B-190) with 100 mesh or 0.149 mm of nozzle diameter. The pressure of compressed air for the flow of the spray was adjusted to 5 bars. The inlet and outlet air temperatures were maintained at 175±5 o C and 55±5 o C, respectively.

In Vitro Fermentation
In vitro fermentation was conducted according to the method of Tilley and Terry (1963). Into each 100 mL fermentation tube, 500 mg substrate, 40 mL McDougall buffer, and 10 mL rumen fluid were added at conducted at 39 o C. The substrate contained 60% king grass and 40% concentrate mixture (cassava by product, wheat pollard, soybean meal, coconut cake meal, molasses, CaCO 3 , premix, urea, and 4% vegetable oil either non-protected, calcium soap, or microencapsulated) with 15%-17% CP and 69%-74% TDN ( Table 1). The rumen fluid for this experiment was collected after 3 h morning feeding from the 3 rumens fistulated Ongole crossbred beef cattle with Ethical Approval from Animal Care and Use Committee (AUAC) 01-2013b IPB. Samples from aliquol were taken after 4 h incubation for pH, VFA, NH 3, protozoa, total bacterial analysis and after 48 h incubation for dry matter and organic matter digestibility analysis.

Statistical Analysis
The experiment was conducted in a factorial randomized block design with 2 factors and 3 replicates.
The first factor was kinds of vegetable oil (sesame, canola, and flaxseed) and the second factor was protection methods (non protected, calcium soap, and microencapsulation). Data were tested using Analysis of Variance (ANOVA) and the differences among treatments' means were examined by Duncan Multiple Range Test (Steel & Torrie, 1995).

Characteristics of Calcium Soap and Microencapsulation Products
The calcium soap products were a little mushy so a carrier was needed to be able to use it, while the micro-encapsulation product was in the form of powder and was very small in size ( Figure 1). The yield of calcium soap product from sesame, canola, and flexseed oils were 95.60%, 96.81%, and 97.30%, respectively, while microencapsulation of sesame, canola, and flexseed oils were 64.17%, 53.74%, and 51.64%, respectively. Similar finding was reported by Calvo et al. (2010) in which microencapsulation yield was reported at 49.49% with the ratio of the coating material from combination of carbohydrates (lactose) and protein (sodium caseinate) of 1:1 and 1:2 for core material and coating material. Microencapsulation yield was lower than calcium soap yield because proportion of oil and coating used in microencapsulation was 1:2 while calcium soap was 9:1.

Fermentation Characteristics and Population of Rumen Microbes
There were no interaction between type of vegetable oils and the protection method on rumen pH, ammonia (NH 3 ) concentration, dry matter digestibility (DMD) and organic matter digestibility (OMD), protozoa and total bacterial population. Kinds of vegetable oil did not affect fermentation characteristic and rumen microbe population. Supplementation of non protected vegetable oil significantly decreased (P<0.05) rumen pH. significantly increased (P<0.05) N-NH 3 concentration.

Supplementation of microencapsulated vegetable oil
Supplementation of vegetable oil without and with protection using calcium soap or microencapsulation methods did not affect dry matter digestibility (DMD) and organic matter digestibility (OMD), protozoa and total bacterial population (Table 2). Decrease in rumen pH value after supplementation of non-protected vegetable oil was presumably due to less rumen microbial activity, especially protozoa. Jenkins (1993) stated that supplementation of fat on feed can disturb membrane and cellular function, activity and expression of microbial hydrolytic enzymes.
Decrease in protozoa activity will reduce its ability to stabilize rumen pH. Protozoa has ability to stabilize rumen pH value and decrease redox potential of rumen digestibility. Similar finding was reported by Bhatt et al. (2011) in which supplementation of coconut oil at 7.5% on feed of Malpura sheep in vivo tend to decrease the rumen pH value (P= 0.108) linearly compared to supplementation of coconut oil at 2.5% and 5% (6.31 to 6.62). Different result was reported by Jalc et al. (2007) in which supplementation of PUFA (oleic, linoleic, and α-linolenic) at 3,5% on diet containing 80% lucerne and 20% barley did not influence rumen pH value (6.73-6.93). The differences in these responses might be due to differences of the level and profile concentration of fatty acid sources used in these researches. Rumen pH value in this research was 6.00-6.33 which was still considered normal. Dehority (2005) reported that normally rumen pH value was 5.4-7.8.
Increasing ammonia (N-NH 3 ) concentration in response to supplementation of microencapsulated vegetable oil was caused by sodium caseinate as a coating material that increased crude protein content of microencapsulation product (27.64%-30.22%). This microencapsulation product would increase protein content of the ration (19.49%-22.64%), so that linearly increasing ammonia production. McDonald et al. (2002) stated that the amount of protein in ration is one of the factors that influence ammonia production. Concentration of ammonia with addition of non protected oil lower than microencapsulation protection. This was presumble because addition at 4% non protected oil has started disturbing rumen microbial activity, especially proteolytic bacteria and protozoa. Hristov et al. (2004) reported that concentration of ammonia is highly correlated with the total number of protozoa and bacterial activity in the rumen. The addition of fat especially MCFA (Medium Chain Fatty Acid) decreased the growth and activity of the protozoa, proteolysis and ammonia concentrations in vitro and inhibit the activity of polysaccharide degradation.
Supplementation of non protected vegetable oil at 4% did not disturb feed digestibility (DMD and OMD) and rumen microbial population (protozoa and bacteria), but it started to show a decrease in the activity. This might be due to the low level of vegetable oil added on concentrate. Purushothaman et al. (2008) reported that addition at 6% calcium salt of palm oil in the concentrate mixture of lactating crossbred cows did not decreased dry maetter and organic metter digest- calcium soap at 15% on ration contained 59.5 parts sorghum straw and 40.5 parts concentrate mixture did not disturb dry metter and organic metter digestibility on Decani sheep (Ramana et al., 2003). Bhatt et al. (2011) reported that protozoa population decreased linearly (P= 0.006) alongside with the increase in coconut oil added (0% : 99.7x10 4 cells/ml), (2.5% : 74.6x10 4 cells/ml), (5% : 57.7x10 4 cells/mL), and (7.5% : 8.6x10 4 cells/ml). In this study, supplementation of non protected vegetable oil at 4% did not significantly decrease protozoa population, but the population was still lower compared to that of vegetable oil protected with calcium soaps or microencapsulation. Sitoresmi et al. (2009) reported that supplementation of oil at 5.0% gave significant effect in decreasing protozoa population. Different response was reported by Adawiyah (2007) in which supplementation of non-protected fish oil at 1.5% was highly significant in decreasing (P<0.01) total bacterial population, but the population did not decrease in response to 3% supplementation of fish oil protected with calcium soaps (1.71 and 3:53 x 109/mL). The supplementation of non protected fish oil at low level significantly decreased total bacterial population. Fish oil contains EPA and DHA which are the most toxic component in disturbing the growth of rumen bacteria. Maia et al. (2007) stated that EPA and DHA were not metabolized by bacteria so that EPA and DHA were more toxic than linoleic (LA) and

ibility. Supplementation of red palm oil in the form
linolenic acid (LNA). The addition sesame oil decreased the lowest protozoa population than others. This was presumably the highest lauric acid content in sesame oil can reduced protozoa. Hristov et al. (2004) reported that lauric acid was the most toxic of MCFA to the protozoa.
Lauric acid increased the sensitivity of microbial cell wall structure so can inhibit of the activity ciliate protozoa and gram-positive archaea (Machmuller, 2006).

Total VFA Concentrations, Molar Proportion of VFA, Methane Production, and Hydrogen Balance
There was interaction (P<0.01) between the kinds of vegetable oils and protection methods on total VFA concentration, molar proportion of VFA, methane production, and H 2 utilization (Table 3, 4, and 5). The supplementation of calcium soap flaxseed oil significantly increased total VFA production, on the other hand the supplementation of microencapsulated flaxseed oil produced the highest propionate concentration and H 2 utilization, the lowest A:P ratio and methane production.
The high total VFA production with supplementation of calcium soaps indicated that flaxseed oil protected with calcium soap method was able to contribute the highest energy source for ruminants. This was presumably because of flaxseed oil is high linolenic acid content (C18 : 3) compared to other oils, so that need high amount of calcium to be bound. High availability of calcium might be stimulate of the growth rumen bacterial population and their activities that will lead to increase feed fermentation. Ruckebusch & Thivend (1980) stated that calcium plays a role in the synthesis and stability of the microbial cell wall structure and able to activate a wide range of microbial enzymes such as α-amylase and is needed by the rumen microbes to digest cellulose. Additionally, high TDN in the ration with calcium soap flaxseed oil supplementation was potential to increase the availability of nutrients for rumen bacterial degradation process. Bhatt et al. (2013) reported that the addition of 4 % rice bran oil in the form of calcium soaps in vivo can increase (P<0.05) total VFA production, body weight gain, body weight, dry matter intake, and lower feed conversion ratio (FCR) compared to the addition of oil in non protected or control (without the addition of oil) .
Supplementation microencapsulated flaxseed oil resulted in the lowest proportion of acetate and the ratio of A : P, and the highest proportion of propionate. This was presumably because microencapsulated flaxseed oil supplementation can stimulate the growth of bacteria propionate producers in the rumen system so that the ruminal propionate production increased. High production of propionate was correlated with low methane production and high used of H 2 . This was presumably because the propionate formation pathway was a ruminal metabolic pathway that used H 2 (Moss et al., 2000). Thus, it would reduce the bond of H 2 and CO 2 that caused CH 4 production to decrease. Besides, the effect of linolenic fatty acid (C18 : 3) from microencapsulated flaxseed oil was of slow release which will decrease the activity of methanogenic archae. Dan Li et al. (2012) reported that the addition of linoleic fatty acid (C18 : 2) and linolenic fatty acid (C18 : 3) was highly significant (P<0.01) in decreasing Methanobacterium formicicum population compared to the addition of oleic fatty acid. Dan Li et al. (2012) stated that anti methanogenic activity of fatty acids can be caused by the toxic effects of fatty acids on methanogens and by methanogenic competition in using H2 in the process of biohydrogenation of unsaturated fatty acids. Zhang et al. (2008) stated that the decrease in methane production increased with the increase of degree of unsaturated fatty acids. Czerkawski et al. (1966) reported that oleic acid with one double chain / mole could decrease methane production by 1.70 moles / mole of fatty acid. Linoleic acid with 1.72 double chain / mole could decrease methane production by 1.79 moles / mole of fatty acid and linolenic acid with 2.4 double chains / mole could decrease the methane production by 2:05 moles / mole fatty acid.

CONCLUSION
Flaxseed oil and microencapsulation method are the best vegetable oil and protection method to optimize rumen fermentation. Supplementation of microencapsulated flaxseed oil produces the highest propionate concentration and H 2 utilization, the lowest A:P ratio and methane production, and did not disturb rumen microbial activity.