Supplementation of Heit-Chrose Into Dairy Cow Feed Improves in Vitro Rumen Fermentation

The objective of the study was to evaluate the effect of Heit-Chrose (HC) supplementation using in vitro method on ruminal fermentation of dairy cattle. HC is a feed supplement containing allicin, saponin and organic minerals ( Se , Cr and Zn). This research was conducted using completely randomized design, with 6 treatments and 4 replications. The treatments were : 1). C = dairy cattle feed (CP 15.38%, CF 23.38%, TDN 61.26%); 2). HC-0 = C+ organic minerals (0.3 ppm Se + 0.15 ppm Cr + 40 ppm Zinc-lysinat) + 0 ppm of HC; 3). HC-15= C + 15 ppm HC; 4).HC-30 =C+ 30 ppm of HC; 5). HC-45 =C+ 45 ppm of HC; 6). HC-60 =C + 60 ppm of HC. Data obtained were analyzed using analysis of variance of SPSS program. HC supplementation increased the DMD, OMD, VFA, but reduced total gas, methane and protozoa count. HC supplementation greater than 30 ppm did not further improve ruminal fermentation. Supplementation at 30 ppm of HC to dairy cow feed was the appropriate level to improve the efficiency of rumen fermentation.


Introduction
The aims of manipulation rumen ecosystem areto improve the crude fiberdigestibility in feed, reduce methane production and nitrogen excretion by ruminants. This is done to improve performance and as the most important goal of animal nutritionists (Patra et al., 2006). Extracts of plants with high concentrations of metabolites are the best way to achieve one or more of these goals (Teferedegne, 2000;Wanapat et al., 2008;Prayitno et al., 2013;Bunglavan et al., 2014). Garlic has the complete content such as allicin (C6H10S2O), diallyl sulfide (C6H10S), diallyl disulfide (C6H10S2), allyl mercaptan (C3H6S) and other components (Lawson, 1996), 20.13% saponin (Prayitno et al., 2013). These components can manipulate rumen fermentation such as reducing the proportion of acetate and propionate and increasing butyrate, as inhibitors of methanogenesis and lowering the ratio of CH4: VFA (Busquet et al., 2005;Prayitno et al., 2014). Kim et al. (2009) reported that garlic peel had a 7-fold polyphenol content than that of garlic bulbs, among others, allicin, which plays role in the decline of methanogens. Feed fermentation in the rumen will result in a decreased availability of mineral (Prayitno and Widiyastuti, 2010). Micro minerals such as Selenium, Chromium, and Zinc can improve the efficiency of rumen fermentation (Prayitno et al., 2013;Jayanegara et al., 2013 ). Selenium supplementation of 0.3 ppm + 1.5 ppm of Chromium + 40 ppm of Zinc in dairy cattle feed improve feed efficiency although methanogens that are formed are still in the level of 25.4 mM (Prayitno and Widiyastuti, 2010). The purpose of this study was to assess the effect of supplementation with Heit Chrose in dairy cattle feed on the efficiency of in vitro rumen fermentation.

Materials and Method
The experiment was conducted using a completely randomized design (CRD), with 6 treatments and 4 replications. Feed consisting of 60% forage (Elephant grass 95% and 5% Gliricidia leaves) and concentrate ( coconut meal, soybean meal, pollard, cassava, minerals, CGF (Corn Gluten Feed), and CGM (Corn Gluten Meal). Feed formulation showed in Table 1.
Measuring methane gas (Candraatmadja, 1981) : (1). The apparatus were arranged to form connector system T; (2) gas was taken using syringe; (3) gas in syringe was injected to the tube that was serially connected and contained 5 M NaOH; (3) then the escape gas was collected in the second volume-measuring syringe to contain CH4 gas.

Results and Discussions
The results showed that there was a highly significant effect (P<0.01) of the supplementation treatments on the DMD and the OMD. The results of the study are shown in Table 1. The value of dry matter and organic matter can be used as an indicator of the ease of feeding the digestibility either degradation by rumen microbes or by digestive enzymes and pascarumen digestion. The higher the DMD and the OMD, and the higher the chances of nutrients that can be used both for livestock production and other activities. This study showed that supplementation with HC was able to improve rumen microbial activity.
Compared to previous studies, the value of dry matter and organic matter of this study was low (Prayitno and Hidayat, 2013 ) .

Total VFA
The results showed that there was a highly significant effect (P<0.01) of the treatment. The mean concentration of Volatile Fatty Acids (VFA) in total treatment feed ranged between 132-209 mM. The value exceeded adequacy of VFA as states by Kellems and Church (2010) that the sufficient range of VFA concentration for rumen microbial growth is 80-160 mM. Supplementation of HC in the substrate significantly affected the concentration of total VFA through increased DMD and DMO so there was more dietary available nutrients to be converted into C2 (acetate), C3 (propionate), and C4 (butyrate) through the process of carbohydrate metabolism. Kellems and Church (2010) stated that the VFA is the most important source of metabolic energy for ruminants and the source of the carbon chain for microbial synthesis because VFA is able to supply 55-60 % of the energy needed by cattle.
This study showed HC supplementation increased the production of VFA, or manipulated the fermentation process to increase efficiency. VFA production increase occurred with supplementation of 30 ppm of HC. VFA production is reflected the process of carbohydrates fermentation and related to the productivity of livestock because most of the VFA in the rumen derived from the fermentation of feed carbohydrate (Hungate, 1966). Ration HC-30 had the highest total VFA, this treatment was thought to contain nonstructural carbohydrate (starch) more than the other treatments to contribute to the fermentation of feed, resulting in higher VFA production. Jouany (1994 ) stated that the nonstructural carbohydrates contribute VFA production that is higher than structural carbohydrates.
The results showed that the treatment affected (P<0.05) the concentration of ammonia. It showed that supplementation of HC in the dairy affected microbial activity in the rumen metabolism of proteins to produce ammonia.
The average concentrations of ammonia ranged from 13-16 mM. The values exceeded the adequacy of ammonia concentration in the rumen. One of the causes high concentrations of NH3 was no absorption of ammonia in an in vitro systems resulting in the accumulation of ammonia in the syringe. There was residual ammonia which was not used in the synthesis of microbial protein together with the carbon skeleton derived from the fermentation of carbohydrates, and also could be from donations of protein lysis from microbes. The results in Table 2 show that the average values of ammonia on the successive treatments were 14.72±1.652 mM (HC-0), 14.20±1.000mM (HC-15), 13.02±1.579mM (HC-30), 16.88±1.439mM (HC-45), 15.58±1.895mM (HC-60). Kurniawati (2009) observed the concentration of NH3 generated from all treatments with the addition of Lerak extracts (Sapindus rarak) ranged between 15.85 to 16.31 mM and the values were still optimal for rumen microbial growth. The NH3 concentration of less than 3.57 mM may inhibit rumen microbial growth (McDonald et al., 1995).

NDF digestibility
The results showed a decline in NDF digestibility significantly (P<0.01) in Table 1. NDF digestibility in this study ranged between 29.43 -36.58 %. NDF digestibility was highest in the treatment of HC-15 amounted to 36.58% and the lowest for the treatment NDF digestibility HC-60 amounted to 29.43%. The high digestibility of NDF in the treatment of R2 assumed to be was due to the ability of the rumen microbes to degrade the fiber fraction. This study showed that supplementations of dairy cows feed with HC to the extent of 30 ppm were able to increase the efficiency of rumen fermentation. This condition occured presumably because of the increased rumen microbial activity and decreased production of CH4. Heit-Chrose with active ingredient of allicin and saponins and Se, Cr, and Zn minerals allegedly was able to improve rumen fermentation. The results of this study showed that increasing levels Chrose Heit will lower crude fiber digestibility. The decline in crude fiber alleged suppression of the activity of cellulolytic rumen bacteria.

Total gas
The results showed that supplementation of Heit-Chrose affected rumen fermentation of liquid phase when the feed of dairy cows substrate contained adequate organic minerals. Gas production varied and (tended to be lower) than in control (HC-0 and HC-15) (Table2). The highest gas production occurred in HC-60 treatment. The total gas was a result of fermented feed, especially organic materials into VFA performed by rumen microbes. The amount of gas produced showed high or low digestibility of the feed. High production of gas indicated inefficiency usage of feed, thereby, increased the gas production. The little amount of gas produced in this study showed that fermented organic materials were used for microbial protein synthesis (Kellems and Church (2010). Gas resulted from rumen fermentation consisted of CO2, N2, H2 and CH4.

Methane
Result showed that Heit-Chrose supplementation in dairy cow feed affected the concentration of methane gas, by decreasing 20-25% methane gas. This study shows that supplementation Heit-Chrose on feed dairy cows affected the concentration of methane gas. Supplementation Heit-Chrose was able to decrease methane gas between 20 and 50%. The most significant decrease was reached at 30 ppm Heit-Chrose at certain percentage, while 60 ppm Heit-Chrose did not affect the concentration of methane gas. The result also indicated that the active substance in Heit-Chrose was effective to limit methanogen performance, thereby slowing down methane production. It was reported that allicin extracted from garlic could function more effectively than that from plant (Busquet et al., 2007). In ruminants, the H2 is normally removed by the reduction of CO2 to form methane. Silivong et al. (2013) stated that the longer incubation time would increase methane gas concentration at 24-hour incubation. Methane gas concentration decreased as the nitrate was increased from 52.0 ml/g to 15.6 ml/g.

Protozoa population
Protozoa population in these treatments had varied values and tended to be lower than the control, except for R2. This indicated that the saponins in HC (20.13%) could break down the cell membrane of protozoa (Prayitno et al., 2013). On the other hand, the HC could inhibit the formation of methane, which was of allicin origin that inhibited the enzyme, HMG-CoA reductase (Busquet et al., 2005). As a result, the availability of H2 in the rumen was not used for the synthesis of methane, but for the synthesis of propionate (Prayitno et al., 2013).
The results of analysis of variance showed that the treatment affected highly significantly (P <0.01) on the protozoa population. The highest average value on protozoa population was reached by the control feed (C) is 2.05 ± 0.50 (10 6 cells/ml rumen fluid) and the lowest average was achieved by control feed + 15 ppm of HC (HC-15),0.94 ± 0.423 (10 6 cells/ml rumen fluid). Protozoa population declined due to reduced population of methanogens that are symbiotic with protozoa due to saponins and allicin role of HC. Kongmun et al. (2011) adds, the saponin contained in the flour of garlic oil can reduce methane bacteria, therefore, the protozoa population decreases. Research result informed that increasing Heit-Chrose supplement was not linear with the decreasing protozoa population. Relation between protozoa population and total gas is presented in Figure 1. Previous research on garlic powder supplement to beef cattle feed which based on ammoniated paddy straw reported the decreasing protozoa population up to 48% (Prayitno and Hidayat, 2013).

Total bacteria
The results showed that the treatment affected highly significantly (P<0.01) on total rumen bacteria. Increased total rumen bacteria presumably because of the feed treatment that  was capable of providing nutrients for the growth of the microbes. In addition, it could be affected by the presence of declined protozoa population due to inhibition of methanogenic activity that wasin symbiotic with protozoa (Newbold et al., 1995). The average values of total bacteria ranged from 10.32 to 10.52 (log 10/0.05 gr DM). The mean of total bacteria from all treatments was shown in Table 1. Based on Table 1, treatment HC-60 with a level of 60 ppm of HC had a value of highest total bacteria, thus the active ingredient in allicin and organosulfur of garlic efficiently inhibited the performance of the enzyme of HMG -CoA in suppressing the methanogenic population, so that feed was used more efficiently and total rumen bacteria increased. A research by Kongmun et al. (2011) showed administration of 50 grams of garlic powder on buffaloes were able to increase the total cellulolytic bacteria by 42.2% in the 4-hour observation. The increase of fiber degrading bacteria population will result in an increase in rumen fermentation products. The increasing population of microbes in the rumen will increase the amount of microbial protein.

Conclusions
Heit-Chrose supplementation in feed of dairy cows by in vitro is able to increase the efficiency of rumen fermentation, as indicated by the increasing feed digestibility, VFA and decreasing total gas production, methane gas and protozoa populations.