Effect of Tea Leaves Powder Supplementation on Fermented Oil Palm Fronds on Fermentation Characteristics, Rumen Microbial Profile, and Methane Production In vitro

| Tea leaves are types of plant leaves with the potential to be used as an antiprotozoal to increase livestock production. Therefore, this research aimed to evaluate the effect of tea leaves powder supplementation on fermented oil palm fronds (FOPF) on fermentation characteristics, rumen microbial profile, and methane production in vitro. Randomized block design with four treatments were used in this research. Treatments were tea leaves powder supplementation at 0, 2, 4, and 6% and four groups as replications. The observed variables were fermentation characteristics, rumen microbial profile, and methane production in vitro. The results showed that at 2% supplementation, the substrate had the lowest pH value. Meanwhile, at 4-6%, the supplement decreased NH 3 , total VFA, the proportion of propionate, DMD, OMD, protozoa population, and increased A:P ratio. However, there was no effect on bacteria population and methane production. In conclusion, supplementation of tea leaves powder at 2% on fermented oil palm fronds was did not influence fermentation characteristics, rumen microbial profile, and methane production in vitro . The tea can be used in ration to optimize the utilization of oil palm fronds.


INTRODUCTION
I ndonesia is the biggest producer of palm oil worldwide, and in 2019/2020 the production volume was recorded at 42.50 million metric tons (Shahbandeh, 2020). Directorate General of Estate Crops (2019) reported that in 2018 there were 14.33 million hectares of oil palm planted area, with private companies controlling 55.09%, smallholders 40.62%, and state-owned companies, 4.29%. Moreover, Poh et al. (2020) stated that 10 tonnes of oil palm fronds (OPF) are produced in one hectare of oil palm plant area, therefore, in Indonesia, there should be 140.33 mil-lion tons of OPF. In tropical countries, these fronds are used as ruminant feed in the livestock industry (Ng et al., 2011;Ebrahimi et al., 2015). This statement is supported by Azevêdo et al. (2012) which reported that ruminants could convert renewable agro-industrial by-products to high-quality feed. Nurhaita et al. (2014) reported that oil palm fronds (OPF) are feed from a by-product that has low quality and nutritional value, high fiber content, low palatability and digestibility. Furthermore, the major constraints to the use of OPF as livestock feed are the high crude fiber (CF,
The low nutritional and digestibility value of OPF as a by-product could be improved using fermentation technology. This claim is supported by Wizna et al. (2009) which reported that the fermentation process was able to reduce crude fiber content by 32% and increase crude protein content by 360%. Fermented products have better nutritional quality and digestibility than unfermented products (Astuti et al., 2017;Wajiyah et al., 2015). Meanwhile, to improve the quality and modify rumen microbes, fermented OPF (FOPF) products need to be supplemented with bioactive compounds. A study on meta-analysis in vitro by Klevenhusen et al. (2012) and in vivo by Khiaosa and Zebeli (2013) reported that bioactive compound supplementation is used as a rumen microbial modifying agent, and reducing methane gas production has a positive effect on livestock productivity.
Tea leaves are one of the plant leaves that have the potential to be used as antimethanogenic and antiprotozoal agents (Hu et al., 2005;Ramírez-Restrepo et al., 2016) to increase the growth performance of small ruminants (Mao et al., 2010;Zhou et al., 2011). However, there is not much information on the effect of the supplementation of these leaves on FOPF on fermentation characteristics, rumen microbes profile, and methane production. Therefore, this research aimed to evaluate the effect of tea leaves powder supplementation in FOPF on fermentation characteristics, rumen microbes profile, and methane gas production in vitro.

PreParatioN of tea leaves Powder
Tea leaves were obtained from the plantation area of Kepahiang, Bengkulu Province, and sun-dried for 4-5 days. The dry leaves were then powdered using a hammer mill with 80-100 mesh size. (Nurhaita et al., 2016).

PreParatioN of local microorgaNisms
The formula for preparing the local microorganism (MOL) was 2 kg of goat's rumen fluid, 15 L of coconut water, and 4 kg of molasses. Furthermore, these ingredients were put into a jerry can, closed tightly, and mixed evenly. Then a smaller jerry can was prepared with water and closed tightly. The caps of both jerry cans were perforated and connected using a small tube. Finally, the small jerry can that contained water functioned to accommodate the gas formed during the MOL production process (10 days incubation). fermeNted oil Palm froNds (Nurhaita et al.,

2019).
Oil palm fronds were chopped and sun-dried until the water content was at + 60%. Then 10 ml MOL/kg FOPF, 1% urea, and 5% molasses were added to the chopped OPF. Subsequently, the materials were mixed properly, put in a plastic bag, compacted, tied, and fermented for 7 days. The fermented oil palm fronds were then evaluated for their physical quality and powdered using a hammer mill with 80-100 mesh size.

statistical aNalyses
Randomized block design with four treatments was used in this experiment. Treatments were supplementation of tea leaves powder at 0, 2, 4, and 6% and four groups as replications. Data were analyzed using ANOVA and the differences among the means of the treatments were examined using DMRT (Steel & Torrie, 1980).

NutrieNt Profiles of the fermeNted oil Palm froNds, tea leaves, aNd substrate
The nutrient profiles of the OPF, FOPF, tea leaves powder, and substrate are shown in Table 1

fermeNtatioN characteristics In vItro
Data of fermentation characteristics in vitro are shown in Table 2. The Rumen pH value ranged from 6.73-6.65, with the lowest value being due to the supplementation of 2% tea leaves powder. Furthermore, the supplementation of this powder on FOPF decreased NH 3 concentration (7.01-8.99 mM) and total VFA (67.18-97.26 mM), and their lowest value was in the treatment with 6% supplementation (NH 3 at 7.01 mM; VFA total at 67.18 mM). The proportion of acetate (61.66-66.73%) and butyrate (16.71-19.05%) in the rumen was not affected by the dietary treatments. However, supplementation of the powder at 4% caused the lowest proportion of propionate (15.91%) and the highest A/P ratio (4.24). A similar result was obtained for the DMD (43.92%) and OMD (42.71%), where the lowest values were in the treatment with supplementation at 4%. Finally, the ranges of DMD and OMD were

rumeN microbial Profile aNd methaNe ProductioN In vItro
Data for rumen microbial profile and methane production in vitro were presented in Table 3. It was observed that tea leaves powder supplementation up to 6% on FOPF did not affect the total bacteria (3.46-4.11 x 10 9 ), cellulolytic bacteria (6.87-8.80 x 10 7 ), and methane production (7.57-7.82%). However, the supplementation decreased the protozoa population, with the lowest value being caused by the supplementation at 6% (2.57 x 10 5 ).

NutrieNt Profile of the fermeNted oil Palm froNds, tea leaves Powder, aNd substrate
The MOL which consisted of microbes contributed to the increase in the CP of FOPF because these microscopic organisms mostly consist of protein. This was supported by Crueger and Crueger (1984) which stated that different kinds of microbes affect protein content, including bacteria, as they contain 70 to 78% protein (Crueger and Crueger, 1984). Based on the results of microbiology identification in the Indonesian Institute of Sciences (2019), the MOL in this study consisted of 5 species of Bacillus bacteria, namely Bacillus sp, B. aureus, B. altitudinis, B. cereus, and B. megatereum. Oboh (2006) stated that microbial proliferation or multiplication could increase protein content. Also, microorganisms may increase CP due to their ability to synthesize amino acids ( Jokotagha & Amoo, 2012). A similar result was reported by Yao et al. (2018), which showed that the CP in the fermented by-product from the beverage industry with Candida utilis and Bacillus subtilis supplementation was higher than in the unfermented product.
The cell membrane of MOL bacteria contains inorganic compounds which contributed to an increase in ash content (Ahaotu et al., 2013). Moreover, Jokotagha & Amoo (2012) reported that some factors contributed to an increase in fat during the fermentation process such as an increase in microbial population that consists of lipid component, extracellular enzyme (lipase) from the activities of lipolytic microorganisms, and microbial oil substance in the fermentation medium. Fermentation technology could reduce CF content because the bacteria from MOL could degrade fibers. Obueh et al. (2014) reported that enzymes of hydrolysis and oxidation could convert the recalcitrant compounds from waste into utilizable compounds. Furthermore, it was discovered by Ojewumi et al. (2018) that CF content could be reduced through both aerobic and anaerobic fermentation. This was supported by Adeleke et al. (2017) which showed that the fiber content from fermented cassava peels decreased by 33.77 and 23.46%. Bacteria from MOL also degraded tannin and saponin in OPF. The same result was reported by Ojewumi et al. (2018), that after fermentation with Saccharomyces cerevisiae, tannin and saponin from cassava waste reduced by 44 and 27.78% respectively. Nutrient in tea leaves powder is almost similar to shrub legume of Desmodium velutinum that has CP at 16.00%, ash at 6.59%, and tannin at 7.70% (Heinritz et al., 2012). Moreover, supplementation of this powder at 2-6% on FOPF caused the CP to be at 13.27-13.94% and TDN at 65.64-68.78%, which conforms with the beef requirement for fattening in Indonesia (BSN, 2016).

fermeNtatioN characteristics In vItro
Rumen pH value in this research was normal as Dehority (2005) reported that the normal rumen pH value is between 5.4-7.8. The lowest pH which occurred with supplementation at 2% might be due to the higher total VFA production than in the 4-6% supplementation groups. Roca-Fernandez et al. (2020) reported that 50% orchardgrass + 50% birdsfoot trefoil had the lowest pH and highest total VFA production. The increasing supplementation of tea leaves powder on FOPF, the NH 3 concentration decreased progressively. This is probably due to the increasing tannin content on the substrate as more tea leaves powder was added. Similar research was reported by Mohammadabadi & Chaji (2012), that the addition of 30 g/kg DM oak leaves and fruit, and pistachio hull and leaves that contained tannin in sunflower meal decreased ruminal NH 3 concentration compared to the control.
Tannin from tea leaves helps to the protect dietary protein from rumen microbes degradation. This condition has a beneficial effect with respect to ruminant production because the absorption of amino acids in the small intestine could be increased. Waghorn (2008) reported that animal which grazed forage with high condensed tannin (CT) had a higher flow of metabolizable protein and essential amino acids in their small intestine compared to those that grazed forage with low CT.
There was a decrease in total VFA, DMD, and OMD with increasing levels of supplementation of tea leaves powder, probably due to the increasing formation of tannin with macromolecule complexes, which hinders microbial enzymes (McSweeney et al., 2001), therefore, reducing fermentative activities and depressing intestinal digestion (Makkar, 2003). Alipour & Rouzbehan (2010) stated that the addition of tannins might be due to a reduction of microbes in feed particles. Also, Gemeda & Hassen (2014) reported that tannin from a different species of tropical browse plants decreased total VFA and IVOMD. Jaya-

Advances in Animal and Veterinary Sciences
July 2021 | Volume 9 | Issue 7 | Page 975 negara et al. (2019) stated that with higher levels of the compound on Indigofera and Moringa silages, there was a greater reduction in total VFA concentration, DMD, and OMD rumen in vitro.
The increasing levels of supplementation of tea leaves powder, there was a continuous decrease in the proportion of propionate and an increase in A:P ratio, meanwhile, there was no effect on the proportion of acetate and butyrate. A similar result was obtained by Patra et al. (2010), that the addition of 0.50 mL extracts of clove (plant extract rich in tannins) increased A:P ratio and decreased the production of propionate by direct inhibition of Selenomonas ruminantium population. Also, Cieslak et al. (2016) found that the addition of tannin extract from Sanguisorba officinalis caused a significant decrease in propionate (linear P<0.01). Castro-Montoya et al. (2011) in the research with dietary Quebracho, mimosa, and chestnut tannins demonstrated the opposite effect, that there was a significant linear decrease in the proportion of acetate (P=0.01) and A:P ratio (P<0.001) but an increase of propionate proportion descriptively.

rumeN microbial Profile aNd methaNe ProductioN In vItro
Supplementation of tea leaves powder did not affect total and cellulolytic bacteria population and methane production. However, the protozoa population decreased with increasing levels of supplementation. This condition occurred because the leaves contained saponin which has a defaunation effect that enables the lysis of protozoa cells. The study by Ramírez-Restrepo et al. (2016) is in support of this finding as it showed that tea leaves are one of the plant leaves with the potential to be used as an antiprotozoal agent. Saponin reacts with the cholesterol from the protozoa membrane, leading to an increase in the permeability of the cell walls, thus killing the protozoa (Wallace et al., 2002). Hidayah et al. (2020) also confirmed these findings, that the increasing substitution of native grass with jengkol (A. jiringa) peel powder that contained saponin, decreased the protozoa population. Finally, according to Cieslak et al. (2014), the use of S. officinalis root, which is a source of saponin, could reduce the population of protozoa (Cieslak et al., 2014).
Methane production was estimated using VFA profiles, as this method leads to accurate results with a very low RMSPE and a high coefficient of measurement ( Jayanegara et al., 2015). The results showed that the supplementation of tea leaves powder on FOPF until 6% did not decrease the methane gas production. This is supported by Ramírez-Restrepo et al. (2016) which reported that the addition of tea seed (Camellia sinensis) saponin (TSS) did not affect methane gas emissions. Besides, this condition might be due to the level of tannin and saponin in the leaves, which was not sufficient to decrease the production of the gas.
Based on the research of Krueger et al. (2010), low tannins (hydrolysable tannin) have little or no effect on methane production, and on the proportion of acetate, propionate, or butyrate. However, Hassanat and Benchaar (2013), found a contradicting result that the methane gas production decreased compared to the control with the addition of up to 40% of acacia, chestnut or valonea (consist of tannins ≥ 50 g/kg), or quebracho (consist of tannins ≥ 100 g/ kg). The varying impacts of tannin on methane production might be due to its chemical structure and concentration in the plant from which it was obtained (Ramírez- Restrepo et al., 2016).

CONCLUSION
Supplementation of tea leaves powder at 2% on fermented oil palm fronds was did not influence fermentation characteristics, rumen microbial profile, and methane production in vitro. The tea can be used in ration to optimize the utilization of oil palm fronds.