In vitro evaluation of ruminal digestibility and fermentation characteristics of local feedstuff-based beef cattle ration [version 2; peer review: 1 approved with reservations]

Background: Consumption of local feedstuff Neptunia plena L. Benth and Leersia hexandra Swartz as a ration by the animal subject is expected to promote cost efficiency and production, as well as provide essential nutrition needs. Therefore, this study aimed to evaluate dry matter digestibility (DMD), organic matter digestibility (OMD), ammonia (NH 3 ) production, and volatile fatty acid (VFA) in beef cattle. Methods: Feed and rumen inoculum samples were prepared and analyzed for their proximate contents. There were five treatment groups based on the diet received by beef cattle, namely: T 1 ( Leersia hexandra Swartz 100 %); T 2 ( Neptunia plena L. Benth 100%); T 3 ( Leersia hexandra Swartz 15% + ( Neptunia plena L. Benth 15% + 70 % Other Feedstuffs); T 4 ( Leersia hexandra Swartz 20% + ( Neptunia plena L. Benth 20% + 60% Other Feedstuffs); T 5 ( Leersia hexandra Swartz 25% + ( Neptunia plena L. Benth 25% + 50% Other Feedstuffs). In vitro approaches were used to determine the DMD, OMD, NH 3 production, and VFA in beef cattle. The data were analyzed using ANOVA at a significance level of 95%, and a Duncan Multiple Range Test. Results: The results showed that the highest DMD (P<0.05) was derived from T 5 (56.47%), followed by T 4 (56.45%) and T 3 (55.90%). T 5 =62.40% significantly (P<0.05) generated the highest OMD followed by T 4 =61.95% and T 3 =60.82%. This treatment had the highest NH 3 value, namely 5.02 mM, compared with T 3 =4.55 mM, T 4 =4.50 mM, T 2 =4.22 mM, and T 1 =3.99 mM. Furthermore, T 5 had the highest VFA (P<0.05) compared with T 4 , T 3 , T 2 , and T 1 with the value of 150.5, 133.0, 130.5, 130.0, and 123.5 mM, respectively. Conclusions: The local feedstuff-based ration contributed to beef cattle production.


Introduction
Livestock, particularly ruminants, is an integral part of the agricultural sector and represents a significant impact on the national economy (Beigh et al. 2017). Ruminants are produced at a more competitive rate than poultry to enhance business sustainability (Silva et al. 2019). They form nutritious food material (meat) from plant fiber (Krizsan et al. 2012). Global food demand for animal protein has been rising significantly, hence some efforts are needed to ensure adequate supply. One of these efforts is to increase livestock productivity through more efficient use of available resources, which are 98% natural (Andriarimalala et al. 2019). Feed is the main constraint faced by breeders in Indonesia to boost beef cattle productivity. Feed deficiency becomes a dominant threat during the dry season (Al-Arif et al. 2017), specifically for forages (Al-Masri 2010). Wild grass and agriculture biomass are consumed as an alternative during the dry season. However, these feedstuffs contain high fiber and low nutrients such as protein, energy, mineral, and vitamin that affect the ruminal microbe fermentation process (Andriarimalala et al. 2019). The maintenance and production needs of beef cattle cannot be fulfilled from a single feed source such as forages (Al-Arif et al. 2017), therefore a balanced or quality ratio is needed (Ramaiyulis et al. 2018).
The beef cattle population in the East Kalimantan Province has reached 119,675 heads (Indonesian statistics 2020). This needs to be increased through some efforts which include enhancement of the feed sector. The optimum productivity is achieved with adequate feed supply, both in terms of quality and quantity (Daru and Mayulu 2020). Local feedstuffs are accessible for breeders due to being available in abundance (Hasan et al. 2020), hence their exploitation is expected to increase feed production sustainability. The local feedstuff sources in East Kalimantan Province, such as Supan-Supan Leguminosae (Neptunia plena L. Benth) and Kolomento grass (Leersia hexandra Swartz) are essential factors in creating a balanced ration for beef cattle (Mayulu et al. 2019).
Neptunia plena L. Benth is a semi-aquatic legume from the Fabaceae family, with compound leaves and a stem that forms a fibrous sponge and taproots to support growth on the water surface, known as floating (Mayulu et al. , 2021. Also, Leersiahexandra Swartz is annual in nature, easily grown (Liu et al. 2011) in inundated wetlands, known as swamps (Lin et al. 2018), tolerant to heavy metal chromium (Cr) (Zhang et al. 2007), and can be cultivated artificially (Ning et al. 2018). This plant possesses the potential for copper phytoextraction on contaminated soil (Lin et al. 2019) and is harvested several times during the growing period. It has dry matter production up to nine tons/ha within 60 days and is used as feed ingredients for the beef cattle ration (Liu et al. 2011).
Knowledge of the potential nutrition contained in local feedstuff ration is expected to increase breeders' willingness to adopt their respective sources. Neptunia plena L. Benth and Leersia hexandra Swartz tend to be developed into a sustainable feedstuff ration for beef cattle due to being abundant throughout the year, specifically during feed scarcity. It is important to measure ruminant digestibility and fermentation level with the feedstuffs, as well as compose these to formulate a perfect ration. Various feedstuffs need to be evaluated in ration formulation (Hasan et al. 2020;Peiretti 2020) because the chemical content presents quality-related information (Forejtová et al. 2005;Al-Arif et al. 2017). Determination of feed nutrient quality requires a fast and accurate method such as chemical and biological analysis (Baran et al. 2017). An in vitro method is a digestibility and fermentation rate test ) that provides animals' biological attributes in a simpler way (Fondevila and Espés 2008). This can be used in daily feeding evaluation which is performed to achieve feed optimization and usage efficiency as well as to minimize nutrient excretion into the environment (Dijkstra et al. 2005). Ideally, the ruminant feed is evaluated in vivo to obtain more accurate results, particularly for nutrient quality, but the method is not practical and cost-effective. Therefore, alternative evaluations need to be performed in laboratory conditions using in vitro methods (Dijkstra et al. 2005;Daru and Mayulu 2020).
Advantages of evaluating ruminal feed digestibility using in vitro methods include testing several feed samples simultaneously to ensure cheaper cost and less time consumption (Dijkstra et al. 2005;Mayulu et al. 2019;Zewdie 2019;Daru and Mayulu 2020). Hence, this research aimed to evaluate the beef cattle ration biologically on a laboratory scale through quantitative assessment or in vitro method.

REVISED Amendments from Version 1
Improvements to this article are in accordance with constructive comments from reviewers.
1. Improvements to the abstract, providing the required details. 2. Adding detailed information on research methods. 3. Added information on "NH 3 " in several places in the discussion. 4. Improvement of the conclusion according to the reviewer's comments.
Any further responses from the reviewers can be found at the end of the article

Methods
This research was carried out in the Laboratory of Feed Nutrient Science, Faculty of Animal Husbandry and Agriculture, Diponegoro University, Semarang. Some of the materials used were feed ration which consisted of Neptunia plena L. Benth and Leersia hexandra Swartz, as well as rice bran, palm cake, and calliandra. The in vitro analysis used beef cattle rumen fluid derived from the Boestaman Semarang animal slaughterhouse, pepsin-HCl solution as the proteindegrading enzyme, McDougall solution (artificial saliva), saturated sodium carbonate (Na 2 CO 3 ), 15% sulfuric acid (H 2 SO 4 ), and 0.5N NaOH, boric acid solution, 0.5% HCl, 1% phenolphthalein indicator, 0.0055N sulfuric acid, vaseline, methyl red and Bromocresol Green, Whatman filter paper 41, Aquadest, CO 2 , and ice for stopping the fermentation process.

Preparation of feed sample
Feedstuff sample materials were prepared through physical treatment consisting of cutting, drying, and milling process, until they were mashed (Fondevila and Espés 2008). These were tested through proximate analysis (Acland 1985), to determine their nutritional content. Local feed resources such as Neptunia plena L. Benth and Leersia hexandra Swartz (used whole stems and leaves), and other rations, namely rice bran, maize, palm oil cake, and calliandra, were obtained from wild grasslands, agricultural by-products, and plantations in Samarinda, East Kalimantan Province.

Preparation of rumen inoculum sample
The rumen fluid was obtained from the Boestaman Semarang Slaughterhouse from an Ongole Peranakan beef cattle with a slaughter weight of 296.4 kg. Cattle are kept conventionally and given forage-based feed with a frequency of twice a day. The rumen fluid was collected in the morning after slaughter. The rumen liquid obtained was then filtered and put into a thermos that had previously been filled with warm water at a temperature of 39°C. This was closed to maintain an anaerobic atmosphere and brought to the laboratory for research observation.

Proximate analysis
The Association of Official Agricultural Chemists (AOAC) procedure (Acland 1985) was applied to determine the observed feedstuffs' nutritional content, namely dry matter (DM), crude fiber (CF), crude protein (CP), ether extract (EE), ash, and nitrogen-free extract (NFE) (Evan et al. 2020). The proximate analysis results were presented in prior research , Table 1).

Experimental design
In this research, a completely randomized design with five treatments was used. The main consideration in ration formulation used was 11%-12% crude protein balance, with ration energy calculated based on the total digestible nutrient (TDN) AE60%. The ration CP balance was in the range of 10% minimum and 14% maximum, and the energy needs TDN was AE60%. The treatments consisted of T 1 (Leersia hexandra Swartz 100%); T 2 (Neptunia plena L. Benth. 100%); T 3 (Leersia hexandra Swartz 15% + (Neptunia plena L. Benth 15% + 70 % Other Feedstuffs); T4 (Leersia hexandra Swartz 20% + (Neptunia plena L. Benth 20% + 60% Other Feedstuffs); T5 (Leersia hexandra Swartz 25% + (Neptunia plena L. Benth 25% + 50% Other Feedstuffs)   Table 2). Tilley and Terry's (1963) in vitro analysis is an alternative method to specifically evaluate ruminants' feed nutrient usage amount to determine the DMD, OMD, NH 3 production, and VFA in a laboratory setting (Gosselink et al. 2004;  The fermenter tube which has been filled with the sample is then filled with CO 2 gas and closed (for anaerobic conditions). The fermenter tube is then put into a rack that has been provided in a waterbath with a temperature of 39°C to be incubated for three hours and shaken every 30 minutes. the fermentation process will be stopped after three hours by moving the fermenter tube from the water bath into a container containing ice cubes, then centrifuged for 15 minutes to separate the residue and supernatant. The supernatant liquid as much as 1 ml was then put into a Conway dish (sterilized and the lips of the cup and the lid were smeared with Vaseline) on the left side of the screen and on the right side of the bulkhead dripped with saturated sodium carbonate (Na 2 CO 3 ) and the middle of the cup was dripped with methyl red and indicator green bromcresol. The filled cup is then closed tightly and shaken (forming a figure eight) slowly until the supernatant and sodium carbonate are homogeneous and allowed to stand for 24 hours at room temperature with the aim that the resulting NH 3 can be bound with boric acid, after 24 hours the titration is carried out with H 2 SO 4 0.0055 N until the color changes from green to pink. Measurement of VFA using Steam Distillation technique. VFA measurements were carried out by taking and inserting 5 ml of supernatant and 1 ml of 15% H 2 SO 4 using a pipette into a distillation tube and inserting it into a 1000 ml Erlenmeyer flask with 800 ml of distilled aquadest, then preparing a 100 ml Erlenmeyer flask to which NaOH solution was added 5 ml of 0.5 N (useful as a catcher for hot steam from the distillation) and tightly closed and heated with Bunsen. The hot steam will push the VFA through the condensed cooling tube and accommodated in a 100 ml Erlenmeyer flask containing 15 ml of 0.5 N NaOH solution until the volume reaches 100 ml, then the Bunsen is turned off. The captured steam is then added with 2 drops of 1% phenolphthalein indicator and titrated with 0.5% HCL solution until the color changes from red to clear (colorless).

Calculation and statistical analysis
Parameters of DMD, OMD, NH 3 fermentation level, and VFA fermentation level were calculated by using the following equations (Hristov et al. 2019;Daru and Mayulu 2020).
DMD equation: OM residue = weight after oven-tanur-filter paper Blank = weight after oven-CP-filter paper NH 3 production equation: Remarks: N=H 2 SO 4 solution normality VFA production equation: Remarks: a = Titrant volume of the blank (mL) b = Titrant volume of the sample (mL) The in vitro method-derived results were analyzed using ANOVA at a significance level of 95%, followed by Duncan Multiple Range Test (DMRT) which applied the Costas program approach.

Results and discussion
Dry matter and organic matter digestibility Beef cattle convert low-quality feed (high fiber) into products containing high nutritional value and quality, such as meat (Deutschmann et al. 2017;Mayulu et al. 2020;Daru and Mayulu 2020). This ability is promoted by a complex digestive system, particularly the stomach which consists of four compartments, namely the rumen, reticulum, omasum, and abomasum (Mayulu et al. 2021). The rumen, sometimes called reticulum-rumen, accommodates about 80% of the total digested amount and contains microbes that digest fibers effectively. Therefore, it enables ruminants to survive with poor nutritional quality and conditions (Mohamed and Chaudhry 2008). Feed deficiency elevates ruminal microbes' degradation rate and increases the metabolic capacity to use energy, both of which lead to an OMD increase (Al-Masri 2010).
Digestibility is defined as the number of nutritional feedstuffs absorbed or used by livestock to satisfy their needs such as production, growth, reproduction, and other functions (Abbasi et al. 2018). It is also an important indicator in measuring the nutritional quality of feed ( Table 3 shows the in vitro DMD and OMD of beef cattle rations formulated from local feedstuffs. ANOVA results showed that T 5 = 56.47% was the highest DMD mean, followed by T 4 = 56.45%, T 3 = 55.90%, T 2 = 42.94%, and T 1 = 41.30%. According to DMRT results, T 5 produced the highest DMD but was not significantly different from T 4 and T 3 . T 5 treatment resulted in a significantly higher DMD (p < 0.05) than T 1 and T 2 . Local feedstuff usage in the ration with percentages of 15, 20, and 25 produced T 5 = 56.47%, T 4 = 56.45%, and T 3 = 55.90%. These values were higher compared with single feedstuff T 1 (100% Leersiahexandra Swartz) and T 2 (100% Neptunia plena L Benth) which had a DMD of 42.94% and 41.30% respectively, as presented in Table 3. Based on Table 1, the low digestibility of single feedstuff in T 1 and T 2 is due to high crude fiber content i.e., 49.23% and 54.76% respectively. This is in line with the results of Mayulu et al. (2021) who stated that high CF contained in the feedstuffs causes low digestibility.
Crude fiber is part of the nutritional components of feedstuffs which is difficult to digest but is needed in the digestive tract for promoting peristalsis, specifically to support ruminal performance ( Organic matter (OM) acts as the energy source for building substances to promote the body's metabolic processes (Mayulu and Sutrisno 2010). OMD is defined as a proportion of OM digested by the digestive tract, which is used to measure available energy, and estimate protein synthesis by ruminal microbes (Al-Arif et al. 2017). This is closely related to DMD since the part of DM consists of OM which contains CF, CP, EE, and NFE ).
Based on the ANOVA results, in vitro OMD means of beef cattle ration based on local feedstuffs from the highest to smallest value were T 5 = 62.40%, T 4 = 61.95%, T 3 = 60.82%, T 1 = 52.89%, and T 2 = 49.31%. The DMRT results showed that the highest OMD was derived from T 5 , but it wasn't significantly different from T 3 and T 4 . T 5 treatment had a significantly higher OMD (P < 0.05) compared to T 1 and T 2 . Organic matter digestibility derived from T 1, T 3, T 4, and T 5 had a higher value than the report by Al-Arif et al. (2017) who obtained an in vitro OMD of 24.98% from a single feed forage and 49.70% from the ration. The low T 2 OMD value of 49.31% was probably due to ruminal microbes' activity or feedstuff nutritional content and extremely small particle, causing a lower rate of feed leaving the rumen and smaller chances of proper degradation ).

Production of NH 3 and VFA
In addition to the digestibility value, feed nutritional content was calculated from the fermentation variable, i.e. NH 3 and VFA concentration. Protein is an essential nutrient that determines the economic success of the beef cattle industry (Chathurika et al. 2019). The beef cattle rumen degrades low biological protein and low-quality fiber into a microbial protein with high biological value (Liu et al. 2019;Chathurika et al. 2019). Ammonia serves as a primary nitrogen source for most ruminal microbes (Imsya et al. 2013), which is responsible for carrying out higher microbial protein synthesis (Supapong et al. 2019;Mayulu et al. 2021). The measurement of this element is employed to estimate protein degradation and usage by ruminal microbes; hence OMD has a strong correlation with microbial protein synthesis (Imsya et al. 2013). NH 3 production reflects the amount of feedstuff protein degraded, and the rate at which this process occurs is an important characteristic for determining protein value (Liu et al. 2019). Ammonia nitrogen is an essential nutrient in promoting microbial growth. High NH 3 production is needed to reach maximum fermentation level and increases feed digestibility (Al-Arif et al. 2017). NH 3 concentration in the rumen is a balance between the produced and absorbed amount, known to be optimal for microbial needs once ranging from 3.57-7.14 mM (Mayulu et al. 2019).
The in vitro NH 3 means of beef cattle ration based on local feedstuffs obtained from ANOVA were T 5 = 5.02 mM, T 3 = 4.55 mM, T 4 = 4.50 mM, T 2 = 4.22 mM, and T 1 = 3.99 mM. The DMRT result showed that the highest NH 3 was produced from T 5 . A high value of NH 3 concentration is probably due to the ration's carbohydrate structure and remnant retention duration inside the rumen (Mayulu et al. 2019). The result of T 5 was significantly higher (P < 0.05) compared with T 3 , T 4 , T 2, and T 1 . The highest NH 3 production, i.e. 5.02 mM, was derived from T 5 which contained 11.68% CP and 59.39% TDN. A higher ammonia value was obtained compared to the report by Al-Arif et al. (2017) who produced an in vitro NH 3 concentration of 3.95 mM with single forage feedstuff and 2.88 mM with the ration. This result was in the optimum range between 3.57-7.14 mM, hence it was expected to promote ruminal microbial biosynthesis. Higher NH 3 concentration reflects more protein decomposition during in vitro fermentation, and this is associated with higher CP content (Wang et al. 2021). The different NH 3 derived in this research tended to be initiated by the amount of feedstuff crude fiber, as well as protein solubility and degradation rate. Low NH 3 production causes slow growing rate of ruminal microbes which leads to decreasing population and inhibited carbohydrate degradation Sarnataro and Spanghero 2020).
VFA is the end product of carbohydrate metabolism by ruminal microbes (Supapong et al. 2019) and acts as an energy source (80%) . VFA is developed through hydrolysis of polysaccharide carbohydrates which are converted into monosaccharides, specifically glucose. These are then converted into acetate (C 2 ), propionate (C 3 ), butyrate (C 4 ), isobutyrate, valerate, isovalerate, methane (CH 4 ), and CO 2 (Abbasi et al. 2018;Kongphitee et al. 2018). OM in a ration that is easily degraded by ruminal microbes is indicated by a high VFA concentration (Mayulu et al. 2019). VFA concentration depends on nutrient digestibility (particularly that of carbohydrates), VFA absorption rate, the ruminal microbial community activity, and degradation rate (Tilahun et al. 2022).
The in vitro VFA means of beef cattle ration based on local feedstuffs obtained from ANOVA were T 5 = 150.5 mM, T 4 = 133.0 mM, T 3 = 130.5 mM, T 2 = 130.0 mM, and T 1 = 123.5 mM. The DMRT results showed that T 5 had a significantly higher value i.e. 150.5 mM (P < 0.05) compared with T 4 , T 3 , T 2 , and T 1 . A high VFA concentration indicates an increased ruminal microbes' activity because more OM is being fermented inside the rumen (Hasan et al. 2020). The result of T 4 was not significantly different once compared to T 3 and T 2 values. The obtained VFA concentration was normal, ranging from 70-150 mM (Tilahun et al. 2022) and 80-160 mM (Mayulu et al. 2019(Mayulu et al. , 2021, with a tendency to promote optimum microbial growth. This is in line with the report by Mayulu et al. (2019Mayulu et al. ( , 2021 and Tilahun et al. (2022) who stated that VFA concentration promotes ruminal microbe biosynthesis. Increasing VFA concentration within the optimum range reflects an effective fermentation process, but an extremely high value causes a balance disorder inside the rumen (Mayulu et al. 2019). VFA concentration is influenced by the ration's carbohydrate content (Supapong et al. 2019), inoculum collecting duration, incubation time, particle size, and inoculum preparation (Patra and Yu 2013), and fiber digestibility.

Conclusions
The results of the study and through the approach of analysis of variance, evaluation of the digestibility value (DMD and OMD) and fermentation level (NH 3 and VFA) of beef cattle consuming local feedstuff-based ration in vitro, it can be concluded that the use of local feed ingredients in quantity is able to contribute to beef cattle production and further research needs to be done both from the author and other researchers, especially by expanding the variables and the stage of direct testing on cattle (in vivo and in sacco).

Yosra Ahmed Soltan
Animal and Fish Production Department, Faculty of Agriculture, Alexandria University, Alexandria, Egypt In the abstract, please add the word "ruminal" before the word "dry matter digestibility...." You don't need to put this sentence in the abstract "The data were analyzed using ANOVA at a significance level of 95%, and a Duncan Multiple Range Test", please remove it.
Remove the word "derived", and write this sentence again.
"This treatment had the highest NH 3...", this sentence has to be rewritten. You cannot start the sentence with this treatment, put its name directly and make the necessary changes.
In the conclusion of the abstract, please remove "contributed" and replace it with "can be used to ensure the sustainable production of beef cattle".
For the "Preparation of feed sample", how were the samples dried?
For the "Proximate analysis", please replace the words "nutritional contents" with "chemical composition". The same observation is in the title of table 1. Please write the statistical analysis in a separate part, and include the experimental unit, number of repetitions/treatment, and the model used to analyze these data. Please insert the full name of all abbreviations when first mentioned (e.g., in the methods of the abstract, NH3-N, VFA...).
In the abstract, what are these five treatments?
Keywords, 'Functional feeds' in place of 'Functional foods'.
In the introduction you must mention the experimental plants that you used, and why you selected them? Their advantages as alternative feed resources for cattle.
For the methods part, you should insert the number of animals that you used their rumen from the slaughterhouse, mention their breed, weight, feed, and life stage.
Which part of Leersia hexandra did you use? Leaves or grains? How did you collected these plants?
The treatments are not clear. Table 1, was the chemical composition DM based? Table 2, what are T1, T2...?
How did you measure NH3-N and VFA?
May you insert the fiber content and NFC for your treatments in table 2? These parameters can be used to explain the obtained results, as NFC is composed mainly of starch, in addition to simple sugars and soluble fiber. Thus, each fraction can be fermented differently while providing various energy sources.
For ruminal microbial growth, and consequently the OMD (Please see Soltan et al., 2021 1 ).
The conclusion needs to be rewritten again, why did you insert the data again? Just refer to their meaning and promote advice from this study.
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