The Silage Quality of Pennisetum purpureum Cultivar Gamma Umami Mixed with Calliandra calothyrsus and Lactiplantibacillus plantarum

This study aimed to determine the effect of Calliandra calothyrsus supplementation and inoculation of Lactiplantibacillus plantarum (L. plantarum) on Penisetum purpureum cv. Gamma Umami grass fermentative and chemical quality and nutrient degradability. The study used a completely randomized design with 3 × 3 factorial patterns. The first factor was Calliandra supplementation levels at 10%, 20%, and 30%; the second was L. plantarum inoculation levels at 0%, 2%, and 4%. The variables measured included chemical fermentation profiles, chemical composition, and rumen fermentation and degradability parameters. The result showed that a higher level of C. calothyrsus supplementation concomitant increased silage pH and NH 3 -N concentration (p<0.05), while L. plantarum inoculation significantly decreased the silage pH and ammonia concentration (p<0.05). The silage contents of dry matter, organic matter, crude protein, and ether extract in silage significantly (p<0.05) increased. Inoculation of L. plantarum decreased (p<0.05) crude fiber, ether extract, and total tannin content of silage. The total volatile fatty acids, acetate, propionate concentrations, and rumen microbial protein synthesis were significantly increased with Calliandra supplementation (p<0.05). L. plantarum inoculation treatment only increased the proportion of acetate (p<0.05) and tended to increase the volatile fatty acids of rumen fluid, the proportion of acetate, propionate, and butyrate. The rumen ammonia concentration decreased with Calliandra supplementation and L. plantarum inoculation. It is concluded that 30% Calliandra supplementation and 2% L. plantarum inoculation and their combination were the treatments that produced the best chemical fermentation, rumen fermentation, and degradability parameters.


INTRODUCTION
Pennisetum purpureum cv.Gamma Umami (P.purpureum cv.Gamma Umami) is used as superior forage because of its high level of productivity.This grass was developed at the Faculty of Animal Science Universitas Gadjah Mada and has received a variety list mark (889/pvhp/2020) for protecting agricultural plant varieties from the Indonesian Ministry of Agriculture and can support feed adequacy.P. purpureum cv.Gamma Umami grass is available throughout the year because of its high tolerance to drought or lack of water sources and even in abundance during the rainy season; however, the nutrient content, especially protein, is relatively low (Sanjaya et al., 2022).To address this problem, especially to meet the adequate protein needs et al., 2022).It is important to ensure that the forage used for silage has enough NSC.When the amount of NSC is low, the lactic acid bacteria may not have enough substrate to produce adequate lactic acid.If necessary, additional carbohydrates can be added to the silage to increase the NSC content.
Preserving forage with the addition of legumes can indeed be challenging.Xue et al. (2020) suggest that using legumes can increase silage protein content but can also cause undesirable fermentation, proteolysis, and poorly utilized nitrogen by ruminants.According to Ni et al. (2018), legumes in silage contribute to a high buffering capacity and make it more difficult to suppress pH during ensilage compared to grass species under natural fermentation conditions; legumes have a lower sugar content than grass species.The buffering capacity in legumes is 500-550 mE/kg of DM (McDonald et al., 1991).The legume addition may prevent the pH from decreasing to the level expected for the silage process to take place properly, which is between pH 3.8 to 4.2.As other researchers reported, legume silage with a DM content of approximately <35%-55% results in a pH value of approximately 4.3-5.0(Kung et al., 2018).
The silage quality is determined not only by anaerobic conditions but also by the degree of acidity.A high degree of acidity (low pH) is determined by the number of lactic acid bacteria (LAB) colonization and the consequent production of lactic acid.Elevated lactic acid production will reduce silage acidity (pH) and help inhibit the growth and development of spoilage microbes and pathogens, which are detrimental because they use nutrients in silage (Soundharrajan et al., 2021).An essential factor in making silage is the moisture content of the raw material.Silage with high moisture content (>80%) has a slimy texture and is soft and moldy, whereas silage with low moisture content (<30%) has a dry texture and is overgrown with fungus.Forage with high water content can impair fermentation (Guo et al., 2013).
Lactic acid bacteria strain Lactiplantibacillus plantarum (L.plantarum) is a lactic acid bacteria (LAB) strain that usually grows when forage plants are fermented (epiphyte bacteria).The applicant rate of L. plantarum in silage can vary depending on the specific purpose of the fermentation.However, a standard guideline is that well-preserved silage requires no less than 10 5 cfu/g FM of LAB, which includes L. plantarum (Dong et al., 2022).Another Study revealed that LAB inoculant doses ranged from 0 cfu/g, 1 x 10 6 cfu/g, 1 x 10 7 cfu/g, and 1 x 10 8 cfu/g on three tropical forages (cassava foliage, P. purpureum, and B. papyrifera) with the result that the addition of L. plantarum improved the fermentation quality and bacterial community of silage, which reduced part of mycotoxin content and abundance of harmful bacteria (Liu et al., 2022).
Therefore, it is necessary to study the effect of Calliandra mixed with P. purpureum cv.Gamma Umami grass as one of the new varieties in this grass group.To our knowledge, a high proportion of legumes can increase the nutritional value of silage but can also reduce its quality; therefore, adding inoculants to enrich the fermentation results is necessary.There is no informa-tion regarding the preservation strategy of P. purpureum cv.Gamma Umami from the tropics was applied with Calliandra and L. plantarum inoculation.Therefore, this study was conducted to observe whether P. purpureum cv.Gamma Umami with Calliandra supplementation and L. plantarum inoculation in mixed silage on fermentation characteristics of silage, nutritional quality, digestibility, and rumen fermentation profile.

Ethical Clearance
The research ethics committee of the Faculty of Veterinary Medicine Universitas Gadjah Mada approved this study, with Ethical Approval number 027/ EC-FKH/Eks./2022.

Experimental Design
The study used a completely randomized design with a 3 x 3 factorial pattern.The first factor was Calliandra supplementation, and the second was L. plantarum inoculation.The following treatments were used: 10%, 20%, or 30% Calliandra supplementation on fresh matter (FM) basis of P. purpureum cv.Gamma Umami, and 0%, 2%, or 4% L. plantarum inoculation.Inoculum was prepared with L. plantarum concentrations of 0 cfu/g FM forage for 0% inoculation treatment, 2 x 10 5 cfu/g for 2% inoculation treatment, and 4 x 10 5 cfu/g FM forage for 4% inoculation treatment.Each treatment was replicated five times and fermented for 21 days in a sterilized room with daily temperatures of 24 °C -27 o C and about 55%-65% relative humidity.Room sterilization was carried out by emptying the room of equipment not used during the fermentation process, and the entire surface of the room was sprayed with 90% alcohol.The silo used was stored in a jar with a 2 kg capacity.

Forage Harvested and Inoculation Preparation
The grass (P.purpureum cv.Gamma Umami) used in this study was cut from the forage field, which was 60 days old, at the Faculty of Animal Science Universitas Gadjah Mada.Calliandra was obtained from the Garden of the Regional Technical Implementation Unit of Agriculture Services of the Special Region of Yogyakarta.The Inoculants of L. plantarum were a commercial product from the National Research and Innovation Agency (BRIN, Indonesia), containing L. plantarum concentration of 10 10 cfu/L.
The grass was cut to a size of 2-5 cm.Wilting was done by aerating for ±4 days until the moisture content was 65% and the dry matter (DM) reached 35%.Wilting was done by placing grass standing in an open warehouse.Calliandra was harvested in the edible parts two days before use.The ingredient composition of the silage was grass with 35% dry matter (DM), 90% organic matter (OM), 11.2% crude protein (CP), 30.3% crude fiber (CF), 3.03% ether extract (EE), while Calliandra contained 60% DM, 93.55% OM, 23.78% CP, 17% CF, 4.8% EE, and 6.20% total tannins (TT) after two days withering.

Chemical Composition, Fermentation Quality, and In Vitro Analysis Procedures
The DM, OM, CF, CP, and EE silage contents were determined using AOAC (2005), and total tannin content followed the procedure Wiryawan et al. (1999).The pH value was measured with a pH meter (Hanna brand) (Nahm, 1992).Analysis of the ammonia nitrogen (NH 3 -N) concentration followed the procedure of Chaney & Marbach (1962).In vitro digestibility was analyzed using the methods developed by Tilley & Terry (1963).
A total of 0.5 g of Calliandra calothyrsus leaves was taken for total tannin analysis.The sample was finely crushed, and 20 mL of 70% acetone solution containing 0.1 ascorbic acid was added.Then, centrifugation was performed for 10 minutes at 7000 rpm at 4 o C. The supernatant sediment was re-extracted with 70% acetone solution twice and centrifuged to retrieve the supernatant.The supernatant obtained was rotovaporized to evaporate the acetone.The resulting rotavapor was extracted with diethyl ether 3 times in a ratio of 1:1 using a column separator to separate chlorophyll from the water fraction.Finally, the water fraction was frozen by freeze drying to obtain tannins, which were then calculated in concentration.
In vitro tubes were filled with samples consisting of rumen fluid and McDougall solution with 1:4 ratios.
McDougall solution was prepared for 1 liter; the ingredients and method of preparation were as follows: weighed 9.8 g of NaHCO 3 , 10 g of Na 2 HPO 4 12 H 2 O, 0.57 g of KCl, 0.47 g of NaCl, 0.12 g of MgSO 4 7H 2 O.The ingredients that have been weighed are then dissolved with 500 mL of distilled water in a beaker glass (1000 mL capacity) (solution A).Dissolution was carried out at 39 o C and using a magnetic stirrer to speed up the process.Furthermore, about 5.3 g of CaCl 2 was put into a graduated glass and dissolved with 100 mL of distilled water (solution B).Subsequently, 1 mL of Solution B was added to solution A and stirred until homogeneous (solution C).Finally, distilled water was added to Solution 3 until the volume became 1000 mL (McDougall, 1948).Carbon dioxide gas (CO 2 ) was provided simultaneously to enable the anaerobic condition in tubes that will be incubated.Incubation was conducted in the water bath at 39-40 o C for 48 hours.
Rumen fermentation parameters, including NH 3 -N concentration in rumen fluid, were measured following the technique of Chaney & Marbach (1962).In vitro rumen fluid of as much as 1 mL was mixed with distilled water to a volume of 5 mL, then centrifuged at 3000 rpm for 15 minutes, and then 2 mL was taken to be mixed with 1 mL of 10% sodium tungstate and 1 mL of 1N H 2 SO 4 .The mixed solution was then centrifuged at 10,000 rpm for 10 minutes.The sample liquid added 2.5 mL of LC and 2.5 mL of LD solution in another tube.The final step is incubation at 40 o C for 30 minutes using a water bath.After the blue color is formed, the reading is done on a spectrophotometer with a wavelength of 630 nm.
The concentration of volatile fatty acids (VFA) was measured following the method of Filípek & Dvořák (2009).The VFA was determined by adding 25% metaphosphoric acid at a ratio of 4 : 1 (v/v).This mixture was centrifuged at 3000 g for 10 minutes, and 0.5 mL of 20 mM/L methyl n-valeric acid was added as an internal standard.Concentrations of VFA were determined via a gas chromatograph (model GC-8A; Shimadzu, Kyoto, Japan) equipped with a flame ionization detector and CP-FFAP CB column (Agilent Technologies, Inc. Santa Clara, CA, USA) (25 m x 0.32 mm inner diameter, 0.30 µm film thickness).
The Lowry method determined Rumen microbial protein synthesis (MPS) (Plummer, 1971).A total of 1 mL of in vitro rumen fluid from each treatment was placed in a tube, and 5 mL of lowry B, the solution, remained for 10 minutes.Subsequently, lowry A solution was added as a volume of 0.5 mL and allowed to settle for 30 minutes.Then, the absorbance was recorded at a wavelength of 750 nm using a UV-VIS Spectrophotometer T60U.

Data Analysis
The data obtained from this study were analyzed using the general linear model (GLM) procedure of IBM SPSS version 20 at a significance level of 5%.The statistical model used was the analysis of variance (ANOVA): Where yijk represents the observed value of each individual, µ denotes the overall mean, αi represents the effect of the Calliandra supplementation factor, βj represents the effect of the L. plantarum inoculation factor, αβ(ij) signifies the interaction between these two factors, and ɛij denotes the residual error (variation among replicates for each treatment).

Silage Chemical Composition
As shown in Table 1, Calliandra supplementation and its interactions significantly (p<0.05)affect the DM content of P. purpureum cv.Gamma Umami silage.However, inoculation of L. plantarum did not affect it.Calliandra supplementation at 10% produced the lowest DM content of 23.65%, significantly less than that produced with the 20% and 30% supplementation treatments of 24.65% and 25.10% DM, respectively (p<0.05).
The DM and OM contents did not differ significantly (p>0.05) because of the influence of the L. plantarum inoculation.However, different results were obtained with 30% Calliandra supplementation, which produced the highest DM content (25.10% vs. 24.65%;23.65%) and OM content (88.77% vs. 88.28%;87.31%, p<0.05) compared with other group treatments.Each treatment factor and their combined effect significantly influenced the CP, CF, and EE contents (p<0.05;Table 1).
The CP content was influenced by the level of supplementation and the dose of inoculation, and both showed a linear trend of increasing CP content with increasing levels of supplementation and increasing doses of L. plantarum inoculation (p<0.05).Treatment with 30% Calliandra supplementation produced the highest CP content (17.13%) compared to the other treatments.The optimal combination effect between the two treatment factors that produced the highest CP of 17.71% (p<0.05) was obtained with 30% Calliandra supplementation and 2% inoculation dose.
A positive response was also shown in the CF content, where increasing the supplementation and inoculation dose and their interactions reduced the CF content of grass silage (p<0.05).Similarly, the treatment factors significantly affected the EE contents and were between 2.09% and 2.71% (p<0.05).

Silage Fermentative Quality
The pH value of the P. purpureum cv.Gamma Umami silage significantly (p<0.05)increased with Calliandra addition (Table 2).The 10% Calliandra supplementation resulted in a pH of 3.72, substantially different from that of the 20% and 30% supplementation treatments at a pH of 3.80 and 3.95 (p<0.05),respectively.Furthermore, a significant (p<0.05)difference in pH value occurred due to the influence of L. plantarum inoculation dose (p<0.05).The pH value concomitantly decreased with the increase in the inoculation dose.Treatment with 4% L. plantarum inoculation significantly reduced the pH value of the silage to the lowest value obtained (pH 3.65).
A significant effect was shown by the interaction of the two treatment factors (p<0.05).The test results showed that the lowest pH value was obtained with 10% Calliandra supplementation and 4% L. plantarum inoculation dose (pH 3.72) (Table 2).However, the pH value was not significantly different from that found with the interaction of 20% or 30% Calliandra supplementation treatment at a dose of 4% L. plantarum inoculation.
The concentration of NH 3 -N silage was significantly (p<0.05)increased with the increasing levels of Calliandra supplementation (p<0.05).Calliandra supplementation at 10% level resulted in significantly (p<0.05)lower NH 3 -N concentration (5.26 mg/100 g) compared to Calliandra supplementation at 20% (5.50 mg/100 g) and 30% supplementation (5.88 mg/100 g).For all doses of L. plantarum inoculation, the P. purpureum cv.Gamma Umami grass silage showed significant concentrations  of NH 3 -N silage (p<0.05).Among the inoculation doses, the lowest concentration of NH 3 -N was with 2% inoculation, which produced 5.12 mg/100 g.The negative interaction of both treatment factors showed a significant effect.

Digestibility and Rumen Fermentation Characteristics
Table 3 shows that Calliandra supplementation and doses of L. plantarum inoculation and their interaction did not affect the rumen pH produced with P. purpureum cv.Gamma Umami grass silage.The resulting rumen pH ranged from 7.18 to 7.42 (p<0.05).However, a positive effect was obtained when the supplementation and inoculation dose increased, increasing the total VFA and the concentrations of acetic acid, propionate acid, and acetate: propionate ratio in the rumen fluid (p<0.05).A significant effect was only present with the Calliandra supplementation factor for butyric acid.
The current result shows that the digestibility of dry matter (DMD) and organic matter (OMD) increased concomitantly with the increase of Calliandra supplementation and L. plantarum dose (p<0.05).Likewise, the interaction effect significantly affected the DMD and OMD of silage (p<0.05).The interaction effect showed that the best digestibility was obtained at the treatment dose of 2% L. plantarum inoculation at all levels of Calliandra supplementation with average DMD of 53.04%, 54.00%, and 48.92%, and OMD values of 50.86%, 53,63%, and 47.48% respectively (p<0.05).Partially, the DMD and OMD values with Calliandra supplementation at 10% were 51.13% and 49.21%, while 20% were 51.30% and 49.62%, while 30% were 49.16% and 47.41% (p<0.05).
The total VFA concentration obtained due to Calliandra supplementation ranged from 49.69 mmol/L to 55.25 mmol/L, with 20% supplementing treatment producing the highest total VFA concentration (p<0.05).In comparison, the effect of inoculation dose treatment ranged from 51.24 mmol/L to 53.71 mmol/L of rumen fluid, with the highest VFA concentration obtained at the 4% inoculation dose (p<0.05).There was a significant interaction between two factors on total VFA concentration (p<0.05).The highest total VFA concentration was obtained in combination with 20% Calliandra supplemented and 2% inoculation L. plantarum (56.57mmol/L) compared to all combination treatments (p<0.05).
The results also showed that the partial concentrations of acetic acid experienced a linear increasing trend as the level of supplementation and inoculation dose increased (p<0.05).The acetic acid obtained in our study ranged from 55.39% to 57.56% of molar (p<0.05).The analogous pattern was also demonstrated by the L. plantarum inoculation treatment, which provided the highest acetic acid concentration of 56.93% molar at a 4% dose of inoculation than 2%, which produced acetic acid of 55.39% of molar (p<0.05).However, neither treatment had any interaction effect on acetic acid concentration (Table 3).
Table 3 showed that increasing Calliandra supplementation and dose L. plantarum inoculation increased propionic acid concentrations (p<0.05).The highest propionic acid concentration was obtained in the 30% Calliandra supplementation, which dramatically decreased in the 20% and 10% supplementation treatments (35.03% of molar vs. 32.51% and 31.62% of molar, respectively, p<0.05).Regarding L. plantarum inoculation treatment, inoculated doses of 2% and 4% showed no difference, but both showed significant differences with 0% inoculation treatment (33.49% and 33.32% of molar vs. 32.35% of molar, respectively, p<0.05).The interaction of both treatment factors produced the highest propionic acid concentration obtained when combined with Calliandra supplementation of 30% and 2% inoculation dose, providing propionic acid of 36.03% of molar (p<0.05).Except for 30% Calliandra supplementation and 4% L. plantarum inoculation dose, that treatment showed a significant difference with propionic acid concentration in the other treatments (p<0.05).
Application of different levels of Calliandra supplementation and doses of L. plantarum inoculation in P. purpureum cv.Gamma Umami grass silage resulted in significant differences in acetate: propionate ratio (A: P ratio) (p<0.05).The highest A: P ratio was found in the 10% supplementation treatment, followed by 20% supplementation, and the lowest in the 30% supplementation treatment (1.76, 1.71, and 1.64, respectively, p<0.05).On the other hand, silage without L. plantarum inoculation significantly produced the highest A: P ratio compared to the other treatments (1.74, 1.70, and 1.67, respectively, p<0.05).An interaction effect was found in the A: P ratio with Calliandra application level at all inoculation doses of L. plantarum, where the highest value was found in the combination of 10% supplementation treatment at all inoculation doses (p<0.05;Table 3).
Our current study showed that treatment and their interaction significantly affected the NH 3 -N of rumen fluid (p<0.05).The NH 3 -N of rumen fluid ranged from 12.86 mmol/L to 25.51 mmol/L.Based on the partial effect, the increase of Calliandra supplementation linearly decreased the NH 3 -N concentration of silage (20.35 mmol/L to 17.64 mmol/L; p<0.05).A similar phenomenon also occurred due to increasing the inoculation dose of L. plantarum (24.00 mmol/L to 13.13 mmol/L; p<0.05).On the other hand, the lowest concentration of NH 3 -N was found in the interaction of all levels of Calliandra supplementation and the inoculation dose of L. plantarum 2%-4% (p<0.05), while for the interaction effect without the inoculation, treatment resulted in significantly higher NH 3 -N concentrations.
Analysis of variance showed that Calliandra supplementation significantly influenced rumen microbial protein synthesis (MPS) (p<0.05).In contrast, the inoculation dose of L. plantarum and its interaction had no effect on rumen microbial protein synthesis within the range of MPS production 7.89% DM to 9.85% DM (Table 3).Partially, the highest MPS production was obtained in the 30% Calliandra supplementation treatment, followed by the 20% supplementation treatment, and the lowest MPS production was obtained in the 10% supplementation treatment (9.98% DM vs. 8.78% DM and 8.90% DM, respectively, p<0.05).

Silage Chemical Composition
Increasing the levels of Calliandra supplementation concomitant increased the DM and OM of the P. Purpureum cv.Gamma Umami grass silage (p<0.05,Table 1).Mixing several feed ingredients provides advantages, such as increasing the nutrient content; in this case, the CP content of silage linearly increases (see Table 1).Increasing the CP content of the silage increases the OM content and ultimately improves the content of the DM silage.Wang et al. (2017) reported an increased DM content with a mixture of corn stover and different portions of legume herbage after 45 days of ensiling.
The OM content showed a linear relationship with the DM content.Calliandra supplementation at 30% produced the highest OM content of 88.87% (p<0.05).The increase in OM silage content because of Calliandra supplementation was caused by the contribution of OM from Calliandra itself.Unfortunately, the OM content was not affected by the inoculation dose of L. plantarum inoculation and their interaction (p>0.05).Using L. plantarum in Bengal grass silage (Panicum maximum) with an incubation period of 15 days also produced an OM content that did not differ from that in silage that was not inoculated (Adesoji et al., 2010).Furthermore, using L. buchneri inoculum combined with L. plantarum increased the aerobic stability of silage and inhibited yeast activity, reduced pH, NH 3 -N concentration, and loss during fermentation.However, it showed no significant difference in DM, OM, and neutral detergent fiber (NDF) contents (Filya, 2003).
Calliandra supplementation in P. purpureum cv.Gamma Umami grass silage significantly affected the CP content (p<0.05).The CP content of the silage increased with increasing levels of Calliandra supplementation and significantly differed between supplementation levels.These results agree with a previous study, which reported that 37% Calliandra addition in cactus silage produced silage with the highest N content of 40 g/kg DM, significantly different from other treatments (Gusha et al., 2015).The increase in Calliandra supplementation caused an increase in their CP content; Calliandra has a high CP content of 23.51%, and the higher supplementation contributes to the increase in CP content.These occurred as an associative effect of nutrients from feed ingredients that have been mixed.McDonald et al. (2011) explained that the nitrogen (N) content was higher in mixed silage than in silage prepared from cereals.
L. plantarum inoculation did not affect silage CP content (p>0.05).The resulting CP content was between 13.22% to 17.71% in all treatments.Hu et al. (2009) reported a lack of effect of L. plantarum inoculation on the CP content of corn silage.L. plantarum inoculation in silage preparation was primarily aimed at helping achieve a rapid decrease in pH and optimizing the silage process rather than increasing the nutritional content of the silage.The difference in CP content can be attributed to the low pH and NH 3 -N concentration (Table 1) in the whole silage treatment that indirectly produced the low proteolytic activity.
Calliandra supplementation in the P. purpureum cv.Gamma Umami grass silage significantly affected the CF content (p<0.05).Calliandra supplementation at 30% significantly decreased CF content (25.07%DM) compared with that at 20% and 10% levels (25.72% and 26.29% DM, respectively; p<0.05).The low content of CF at 30% supplementation was caused by the increase in the portion of Calliandra to basal material that reduced the proportion of grass used and thus decreased the CF content it contributed.Another researcher also reported the same trend (Wang et al., 2017;Sutaryono et al., 2023).They showed a decrease in the CF content of corn stover silage by 10% to 30%, in line with the increase in legume administration.
The low content of CF in the treatment with 2% L. plantarum may be due to hemicellulose hydrolysis during the silage process, where almost half of the hemicellulose can be degraded during the silage process (Putra et al., 2017;Li et al., 2019).Inoculation of L. plantarum in corn silage of different maturity stages showed decreased NDF content (Haghparvar et al., 2012).The decrease in CF content may be caused by degradation by plant or bacterial enzymes and hydrolysis by organic acids during the fermentation process (Widiyastuti et al., 2014;Li et al., 2019).
The current result showed that the Calliandra calothyrsus supplementation and L. plantarum inoculation and their combination significantly affected the EE content of the grass silage (p<0.05).The content of EE in all treatments was between 2.09% to 2.71%.The EE content in these treatments remained within the normal EE acceptance range in the rumen, as more than 5% fat in the feed will interfere with rumen biological processes.A high EE feed content will interfere with the activity of rumen microbes, especially cellulose-digesting bacteria.In addition, the high content of EE in the feed will also interfere with the VFA absorption process.Raes et al. (2004) explained that the availability of fat in feed is not only a source of energy but is also needed to support several biological functions of the rumen to ensure healthy performance.
The significantly low EE content in the silage that received the 4% dose of L. plantarum inoculation was due to the EE in the silage used by L. plantarum during the ensiling process.McDonald et al. (1991) reported that the reduced content of unsaturated fatty acids in silage was due to the biohydrogenation process during ensilage.Furthermore, the use of L. plantarum as an inoculant was shown a decrease in the concentration of C18:3n-3 in grass silage (Boufaïed et al., 2003).However, in another study, a reduction in EE content in barley silage that received LAB inoculation did not show any difference with the treatment without the addition of LAB inoculation (3.63% vs 4.08% DM, respectively) (Kim et al., 2015).
The total tannin (TT) content of Calliandra used in the study was 6.20% after two days of withering.Total tannin concentration increased directly to the increase in Calliandra supplementation level (p<0.05).It was understood that the increased addition of Calliandra, besides contributing to the increase in silage protein content, also contributed to the increase in silage tannin content.This agrees with Gao et al. (2022), who noted that when legumes are ensiled, the tannins they contain become part of the silage.Contrary to the Calliandra supplementation, L. plantarum inoculation reduced the TT content of P. purpureum cv.Gamma Umami grass silage (p<0.05).The decrease in TT content of silage in this study is presumably due to the ability of L. plantarum to reduce tannin by producing the tannase enzyme.In their study, Tefa et al. (2019) found that L. plantarum could reduce the tannin content of Canna edulis Kerr from 2.53 mg/mL to 0.84 mg/mL under optimum conditions.
The interaction effect of Calliandra supplementation and L. plantarum inoculation influenced the TT content of P. purpureum cv.Gamma Umami grass silage (p<0.05).The interaction effect in this case at least provides benefits among others: Calliandra plays a role in increasing the CP content of silage, and L. plantarum in improving the overall fermentation quality of silage, but also contributed by reducing the amount of TT input from Calliandra through its tannase enzyme activity.However, the influential effect of L. plantarum on reducing tannins and other anti-nutritional factors may depend on various factors, including the specific strain of L. plantarum used, the composition of the diet, and the physiological condition of the ruminant (Li et al., 2022;Wang et al., 2022).Therefore, further research may be required to optimize the use of L. plantarum in reducing tannins in mix or sole legume silage for specific applications.

Silage Fermentative Quality
The silage pH value determines the success rate and reflects the fermentation dynamics during the ensiling process (Zhang et al., 2017;Mu et al., 2020).At a critical pH, lactic acid is the dominant acidic product that inhibits the growth of other bacteria.Successful ensiling of silage containing legumes is quite challenging to achieve.Legumes contain high amounts of protein, which act as a buffering capacity, and are low in water-soluble carbohydrates (WSC) (Dewhurst et al., 2003;Liu et al., 2012).Low pH value obtained in this study with both the inoculated and uninoculated treatments was thought to be due to the suitability of the L. plantarum or LAB epiphytic inoculant source with the silage material or forage.These enabled the adaptation process and inoculant colonization of the silage material to proceed relatively quickly based on the overall pH value of the silage produced in all treatments (Table 2).
Well-preserved silage should have a pH value of at least 4.5 (McDonald et al., 1991).Combining both factors proved to decrease the concentration NH 3 -N with a pH value of 3.87-3.88 in all treatments, thus meeting the criteria of well-preserved silage.These results agree with previously reported values that the inoculation of Lactobacillus species affected the pH of silage ranging from 4.15 to 5.53 (Paradhipta et al., 2019;Li et al., 2020;Wu et al., 2022).Low pH values indicated that lactic acid production sufficiently suppressed the pH and inhibited microbial activity, thereby preserving the organic matter.Enterobacteria, Clostridium, Listeria, and other harmful organisms can continue to grow, perform further fermentation, and produce various metabolic products if the lactic acid formation is insufficient to lower the pH to a level that inhibits them (Ávila & Carvalho, 2020).The pH value obtained in our study was lower than that found in a combination of Guinea grass and Stylo-legume silage (pH 4.45) (Bureenok et al., 2016).
The silage NH 3 -N concentration was similar in all treatments, ranging from 4.60 to 6.17 mg/100 g (Table 2).This condition shows that the level of Calliandra supplementation increased the concentration of NH 3 -N silage (p<0.05), with the lowest concentration obtained with a 10% level of Calliandra supplementation (5.26 mg/100 g).The NH 3 -N concentration increases concomitantly with the increase in the pH value of the P. purpureum cv.Gamma Umami grass silage.These conditions illustrate that the protein content in Calliandra can act as a pH buffer to inhibit the rapid decrease in pH at the beginning of the fermentation phase.The higher the pH of the resulting silage, the higher the proteolytic activity.Muhandiram et al. (2023) stated that a rapid decrease in pH can help inhibit proteolysis, thereby maintaining the protein content of the silage.Low pH values at the beginning of the ensiling process help prevent protein breakdown into NH 3 -N.Our result was slightly higher than found in the mixed silage of high moisture amaranth and rice straw treated with L. plantarum NH 3 -N of (3.07 mg/100 g) (Kim et al., 2017b), which might be due to different silage base materials used in each of these studies.However, the concentration of NH 3 -N obtained in this study met the critical attention of NH 3 -N in the silage (<10 g/100 g of total N) (Lima et al., 2010).
Alternatively, the increase in the NH 3 -N concentration may be due to the amount dissolved N obtained from supplementation with Calliandra, which caused an increase in the NH 3 -N concentration in silage, which indicates the proportion of protein degraded during the ensiling process.The concentration of NH 3 -N is closely related to the number of products and the type of fermentation during the silage process.Thus, the concentration of NH 3 -N is associated with the level of protein degradation both by plant enzymes and microbial enzyme activity, which acts as a secondary fermenter by Enterobacteria, Clostridia (butyric acid producer), and yeasts (ethanol producer) (Kim et al., 2021).However, the efficiency of rumen microbial N-synthesis can be increased by supplementing the silage with N-protein rather than non-protein nitrogen (NPN); thus, reduced proteolysis during ensilage is essential (Muhandiram et al., 2023).

Rumen Fermentation Profile
The effect of treatments on the pH of in vitro rumen fluid is shown in Table 3. Calliandra supplementation, L. plantarum inoculation, and the interaction between both factors did not affect rumen pH.Adding Calliandra silage at 30%, 40%, and 60% to the basal feed of rice straw in bulls dairy cows also did not affect the pH compared with the control treatment (Giang et al., 2016).In this study, the rumen pH value at all levels of Calliandra addition ranged from 7.24 to 7.3; however, the in vitro rumen pH value obtained was relatively higher than the ideal pH of rumen fluid of 5.5 to 6.9 (McDonald et al., 2011).The increased range of rumen pH values was thought to be caused by the type of feed used that contained low energy or nonstructural carbohydrates, which then affected the low production of VFA.
Table 3 demonstrated that the DMD of P. purpureum cv.Gamma umami grass silage increased linearly with increasing levels of Calliandra supplementation (p<0.05).The same increasing trend was observed for the OMD.The increased digestibility of silage in our results is thought to be due to the increased availability of CP in the silage.This condition intensifies rumen microbial activity to degrade silage during digestibility testing.Similar results reported by Sutaryono et al. (2023) showed an increase in the digestibility of corn stover silage concomitant with an increase in the Leucaena portion.They further explained that legumes in the feed provide a nitrogen source for rumen microbes.For rumen microbes, the availability of nitrogen, carbon, and ATP sources can encourage intensive cell multiplication (Dilaga et al., 2022).Another study by Xue et al. (2020) under in vitro assessment showed that there was an increase in DMD and OMD of mixed silage of Orchardgrass and Alfalfa, which was caused by an increase of protein content and a decrease in the fiber fraction as the proportion of alfalfa was added.
Our current study also showed that the inoculation dose of L. plantarum significantly increased the OMD and OMD of P. purpureum cv.Gamma umami grass silage (p<0.05).Several researchers have also confirmed the increase in silage digestibility due to L. plantarum inoculation.For example, Cao et al. (2011) showed that using L. plantarum inoculant in vegetable waste silage increased DMD and decreased CH 4 gas production under in vitro conditions.Furthermore, Paradhipta et al. (2019) showed increased digestibility of high moisture sorghum-sudangrass silage due to inoculation of Lactobacillus plantarum R48-27 and Lactobacillus buchineri R4-26 at ratio 1:1.Inoculation of L. plantarum in silage at least provides benefits as a potential probiotic and its interaction with rumen microbes in improving rumen performance.Lactic acid bacteria (LAB) inoculants can improve livestock performance by changing rumen fermentation patterns (Mohammed et al., 2012;Li et al., 2023).
Due to interaction effects, the high DMD and OMD values are caused by the suitability between the availability of energy and protein to meet the needs of rumen microbes for these two substrates.Thus supporting the rumen fermentation process and indirectly increasing the activity of silage degradation.These conditions lead to faster rumen microbial proliferation, improving overall rumen performance.Qu et al. (2013) showed that higher CP content and lower fiber content in legumes than in grasses may affect digestion.
The production of VFA in the rumen is related to the carbohydrate fermentation process and is directly related to livestock productivity because most of the VFA in the rumen originates in the fermented carbohydrate feed (Kariyani et al., 2021).The digestibility rate can be used to predict feed quality.Our results indicated that an increase in the level of Calliandra supplementation from 10% to 20% increased the VFA concentration.However, the 30% level of supplementation decreased the VFA concentration (Table 3).This decrease at 30% supplementation was caused by the lower carbohydrate levels indirectly influenced by the tannins contained in Calliandra (Table 2), although the 30% treatment had a higher level of CP, which could contribute to the increase in VFA concentration when deaminated in the rumen (France & Dijkstra, 2005;Weimer, 2022).Jayanegara et al. (2009) indicate that the presence of tannin at 0.5 mg/ mL in hay rations significantly decreased the total rate of gas production, organic matter digestibility (OMD), total VFA, and iso-VFA of rumen fluid under in vitro assessment.
Overall, the total VFA obtained in this study was relatively low compared with the average concentration in the rumen of 70 mM-130 mM (McDonald et al., 2011).The total VFA concentration of the results of this study is consistent with Noviandi et al. (2014) showed the total VFA concentration in rumen fluid from a mixed feed Alfafa, Lotus corniculatus, and Astragalus cicer L. and tall fescue grass (Festuca arundinaceae) with a combined ratio of 25%, 50%, and 75% inclusion of legumes produced total VFA concentrations between 47.8 to 56.1 mM under in vitro continuous culture conditions.
The proportion of acetic acid at both 20% and 30% Calliandra supplementation levels was similar but significantly higher than that at 10% of supplemented (p<0.05).The concentration of acetic acid obtained in this study was slightly lower than the concentration of acetic acid (30.7 mM-31.9mM) in a previous study that used a mixed feed of legumes (Alfafa, Lotus corniculatus, and Astragalus cicer L.) and tall fescue grass (Festuca arundinaceae) with a combined ratio of added legumes (to 25%), which was digested using the in vitro continuous culture method (Noviandi et al., 2014).The difference in the amount of acetic acid between both studies was caused by differences in the quality of the forage used, where the silage feed in this study originated from a tropical area.
Increasing dose inoculation of L. plantarum showed a linear increase in the rumen acetic acid concentration (p<0.05).The increase in acetic acid concentration in this study might be attributed to the activity of L. plantarum, which produces organic acids, during the ensilage process and the possibility of producing enzymes from the fiber degradation group.In their study, Kim et al. (2017a) reported that Lactobacillus plantarum R48-27, a silage inoculant, can release cellulose, xylinase, chitinase, and esterase enzymes.In addition, feeding with LAB inoculants, such as L. plantarum species, can change the rumen fermentation pattern through their interaction with rumen microbes, increasing the amount of digested OM and ruminant performance (Hristov et al., 2013).
Supplementation of 20% Calliandra and 2% inoculation of L. plantarum showed significant interaction in producing the highest acetic acid concentration (p<0.05).This interaction shows a positive supporting condition, where the addition of calliandra directly affects the increase in the proportion of degradable fiber, and on the other hand, with the inoculation of L. plantarum, which works to produce organic acid compounds such as lactic acid and the possibility of producing enzymes to reducing structural carbohydrates (Li et al., 2022), by degrading the complex bonds of lignocellulose which in turn facilitates rumen microbes to degrade them.According to Weimer (2022), the degradation of structural carbohydrates such as cellulose and hemicellulose in the rumen by anaerobic microbes produces volatile fatty acids, including acetate.However, Ungerfeld (2020) emphasizes that other factors, such as feed fermentation rate and methanogen growth, can also influence the concentration of acetate and other volatile fatty acids in the rumen.
The proportion of propionic acid was significantly affected by the interaction of both treatment factors (p<0.05).Our study showed that increasing the level of calliandra supplementation led to an increase in propionic acid concentration from rumen fermentation, with 30% calliandra supplementation with 2% and 4% dose of inoculation resulting in the highest propionic acid concentration among the treatments.Calliandra is a protein source feed ingredient from the legume group.Fermentation of legumes as feed ingredients in the rumen is more directed toward producing propionic acid (Gonzalez-Garcia et al., 2017;Ammar & Philippidis, 2021), which explains the low or absent variation in the proportion of butyric acid in all treatments.Chen et al. (2020) noted that stimulating ruminant propionate fermentation can reduce methane emissions.Unlike acetate and butyrate, propionate is a gluconeogenic VFA and thus can increase energy availability for production.
In this study, the A: P ratio of in vitro rumen fluid increased in the 10% Calliandra supplementation treatment at all L. plantarum inoculation doses but conversely decreased when the supplementation level was applied at all inoculation doses.This condition may be due to the effect of the concentration of acetic acid and propionate, which both increased in this study so that it does not provide a gap in the value of the increase in the ratio of A: P ratio of the silage.Sun et al. (2022) indicate that the balance of volatile fatty acids, including the ratio of acetate, butyrate, and propionate, can vary depending on the type of carbohydrate being fermented.For example, starch fermentation generally promotes propionate formation, which is associated with less methane production.This balance is essential as it can affect the energy supply to the ruminant.For example, propionate is a significant source of glucose in ruminant, which is crucial for meeting their energy needs.
Silage prepared with high protein content was identified with high NH 3 -N concentration consequences feed protein degraded during the ensilage process.Our current result showed that the NH 3 -N concentration is affected by Calliandra supplementation and a dose of inoculation, and their interaction was demonstrated in the normal range of NH 3 -N concentration of 12.86 to 25.51 mmol/L of rumen fluid.Following McDonald et al. (2011), the ideal concentration of NH₃-N in rumen fluid leading to support microbial growth ranged from 10.21 to 35.76 mmol/L.Overall, our current result showed that the NH 3 -N concentration was affected by Calliandra supplementation and a dose of inoculation, and their interaction was demonstrated in the normal range of NH 3 -N concentration of 12.86 to 25.51 mmol/L of rumen fluid.The ideal concentration of NH 3 -N in rumen fluid leading to support microbial growth ranged from 10.21 to 35.76 mmol/L (McDonald et al., 2011).
In our study, the declined two conditions probably caused NH₃-N concentration: 1) Rumen microbes efficiently incorporate ammonia to meet their N requirements for cell synthesis in line with an increased level of Calliandra supplementation, 2) limiting protein degradation activity of proteolytic bacteria caused by tannin content; as in this present study, there was an increase in total tannin content by increasing the level of Calliandra supplementation.Other researchers have also reported decreased NH 3 -N concentration in line with the legume increase (Min et al., 2002;Copani et al., 2014;Mudhita et al., 2016).Tannin compounds have two pathways in influencing rumen NH 3 -N concentration, as follows: 1) reducing feed protein degradation through the formation of tannin-protein-complex formation or reducing the solubility of proteins (Min et al., 2002), and 2) inhibiting the activity of proteolytic bacteria or proteolytic enzymes or inhibiting the activity of both (Patra, 2012).Nevertheless, the specific effects may depend on various factors, including the type and concentration of tannins, diet composition, and physiological condition of the ruminant (Ningrat et al., 2016;Sun et al., 2022).
A similar trend was also shown by dose inoculation treatment.The difference (p<0.05) in NH 3 -N concentration of rumen fluid between silage treated with L. plantarum inoculation and without inoculation is thought to be closely related to the increased VFA concentration of both L. plantarum inoculation treatments so that it can describe the availability of energy which is then incorporated with NH 3 -N through the biochemical process of rumen microbial cells.However, different results were reported by Hapsari et al. (2016) showed that inoculation of L. plantarum 1A-2 singly in elephant grass silage produced rumen NH 3 -N concentrations that were not different from the treatment without inoculation or the combination treatment of L. plantarum 1A-2 plus formic acid.
The best interaction was shown by the treatment of 30% Calliandra supplementation with a 2% L. plantarum inoculation dose, which resulted in NH 3 -N concentration of 15.71 mmol/L.Although the interaction of the 20% and 30% Calliandra supplemented treatment with a 4% inoculation dose resulted in lower NH 3 -N concentrations of 13.16 mmol/L and 13.38 mmol/L, respectively, the efficiency of using L. plantarum inoculation needs to be considered.Likewise, the interaction between the 30% Calliandra supplementation treatment with uninoculated of L. plantarum was not chosen as the best treatment interaction due to considerations in terms of silage feed conservation in this study, as explained earlier that the NH 3 -N concentration in the 30% Calliandra supplementation, and without L. plantarum inoculation treatment resulted in higher NH 3 -N silage.
Calliandra supplementation significantly affected microbial protein synthesis (MPS) (p<0.05).Microbial protein synthesis increased and showed significant differences between groups as the level of Calliandra supplementation increased.In this study, increased MPS due to Calliandra supplementation occurred due to synchronization between energy and protein in the rumen fluid.This synchronization of energy and protein availability is indicated by the increase in VFA concentration and the decrease in rumen NH 3 -N concentration (Table 3), indicating an increase in microbial biomass that performs degradation activities of the silage.Noviandi et al. (2014) reported an increase in the level of legume use in the treatment feed resulted in increased microbial protein synthesis, namely obtained microbial N of 7.64% DM at a legume-grass ratio of 25:75 and obtained 9.00% DM at a legume-grass ratio of 75:25.
On the other hand, the increase in rumen MPS is also thought to be caused by the activity of tannin compounds contained by Calliandra, which, in this case, causes a decrease in the protozoal population in the rumen fluid.Patra (2012) stressed that tannins also affect the efficiency of rumen MPS.The decrease in protozoa was followed by a decrease in microbes from the archaea group that act as CH 4 producers.This study indicates this condition by the low A: P ratio value produced (Table 3).The previous research by Tan et al. (2011) showed reduced CH 4 and CO 2 production concomitantly increased condensed tannins from Leucaena leucocephala.

CONCLUSION
The present study concluded that the supplementation levels of Calliandra at 20% to 30% effectively increased the chemical composition of P. purpureum cv.Gamma Umami grass silage.Similarly, the dose of 2% L. plantarum inoculation positively contributed to the quality of the resulting silage.Overall, a synergistic interaction occurred between Calliandra and L. plantarum in increasing the chemical composition, silage fermentation quality, and improving digestibility and rumen fermentation parameters with the optimal combination obtained at 20% Calliandra supplementation with a dose of L. plantarum of 4%.Further, an in vivo study should be performed to investigate the direct effect of the overall performance of these feeds on cattle production.

Table 3 .
Degradability and rumen fermentation characteristics of Pennisetum purpureum cv.Gamma Umami silage with different levels of Calliandra calothyrsus and different doses of Lactiplantibacillus plantarum inoculants