Enhancing ruminant feed through in vitro assessment of sago hampas fermentation as an onggok substitute

Heru Ponco Wardono^1,2, Andriyani Astuti¹, Nono Ngadiyono¹ and Ali Agus¹

¹ Faculty of Animal Science, Universitas Gadjah Mada, Jl. Fauna 3, Bulaksumur, Yogyakarta-55281, Indonesia
² Research Center for Animal Husbandry, National Research and Innovation Agency, Cibinong Science Center, Bogor, Indonesia
aliagus@ugm.ac.id

Abstract

Sago hampas, a by-product of starch extraction, is promised as a ruminant feed despite inherent nutritional limitations. This study assesses the effectiveness of fermented sago hampas (FSH) as a substitute for onggok in ruminant feed. Proximate analysis post-3-day fermentation with SBP^® (saus burger pakan) revealed that FSH increased crude protein (CP) content by 1.22% and decreased crude fiber (CF) content by 4.46%, aligning with prior research on enhanced protein and fiber degradation in fermented sago hampas. Formulating five complete feed rations with varying levels of onggok substitution (0%, 25%, 50%, 75% and 100%), allowed for in vitro analyses encompass nutrient digestibility and rumen fermentation profiles. Results indicated that FSH did not significantly alter enzyme activity, demonstrating comparable efficiency to onggok as a source of carbohydrates and protein. FSH substitution did not affect the digestibility of nutrients such as dry matter, organic matter, CP and CF, indicating its potential as an alternative feed source. The investigation extended to the impact on rumen fermentation indicators, including pH, acetate, propionate, butyrate, total volatile fatty acids (VFA), ammonia (NH₃), microbial proteins, enzyme proteins, methane (CH₄) and carbon dioxide (CO₂). The study found no significant differences across these parameters, suggesting that FSH does not compromise rumen function and microbial ecology. In conclusion, this research advocates for the use of FSH as a viable substitute for onggok in ruminant feed, showcasing its potential to enhance nutritional content without compromising in vitro digestibility and rumen fermentation.

Keywords: cassava waste, complete feed, in vitro, sago waste, silage

Introduction

Sago (Metroxylon sago) serves as a crucial source of starch in eastern Indonesia, particularly in Papua and Maluku (Ginting and Pase 2018; Singhal et al 2008; Tiro et al 2018). The extraction process of sago plant stems yields sago starch and a by-product locally known as "sago hampas" (Abd-Aziz 2002; Amin et al 2019; Awg-Adeni et al 2010; Muhsafaat et al 2015; Singhal et al 2008; Vikineswary et al 1994; Zulkarnain et al 2016). Sago hampas, with approximately 60% starch (Lai et al 2013; Vikineswary et al 1994; Zulkarnain et al 2016) and 20% cellulose (Hasanah et al 2020; Zulkarnain et al 2016), holds potential as an alternative feed source for ruminants. However, its low protein content (2-4%) (Muhsafaat et al 2015; Nafiu et al 2018; Setiawan et al 2022; Tiro et al 2018), high fiber content (15-40%) (Abd-Aziz 2002; Nafiu et al 2018; Sumardiono et al 2018; Wardono et al 2021) and 80% moisture content (Lai et al 2013) present challenges in terms of palatability, consumption and digestibility for ruminants (Susanti et al 2022; Vikineswary et al 1994; Wardono et al 2021).

To enhance the nutritional qualities of sago hampas as animal feed, various treatment methods are under consideration, with fermentation being one of them (Ginting and Pase, 2018; Setiawan et al 2022; Vikineswary et al 1994). Fermentation, the conversion of organic matter into simpler compounds by microorganisms (fungi, bacteria, or yeast), especially under anaerobic conditions (Nkhata et al 2018; Nout 2014; Sadh et al 2018; Şanlier et al 2019; Sun et al 2022; Susanti et al 2022), has demonstrated the potential to enhance the nutritional value of sago hampas. This enhancement includes a reduction in crude fiber content and an increase in crude protein content (Ginting and Pase 2018; Nafiu et al 2018; Sumardiono et al 2018; Sumiana et al 2020; Wizna et al 2008), improved digestibility (Li et al 2022; Sadh et al 2018; Sumiana et al 2020) and the production of metabolites such as organic acids and enzymes (Li et al 2022; Sadh et al 2018; Susanti et al 2022). Prior research has explored the effects of fermentation on sago hampas using various single-culture microorganisms, including Aspergillus niger (Daniel et al 2023; Muhsafaat et al 2015), Saccharomyces cerevisiae (Subashini et al 2011), Bacillus amyloliquefaciens(Wizna et al 2008) and Trichoderma sp(Said et al 2022; Sumardiono et al 2018) . However, information regarding the impact of fermented sago hampas (FSH) on rumen fermentation characteristics in vitro remains limited.

Rumen fermentation is a complex process involving the interaction of rumen microorganisms and feed substrates to produce volatile fatty acids (VFA), ammonia (NH₃), microbial proteins, methane (CH₄) and carbon dioxide (CO₂) (Hamid et al 2021; Hasanah et al 2020; Li et al 2022; Samadi et al 2020). These products have important effects on ruminant nutrition and metabolism, as well as their environmental impact (Castillo-González et al 2014; Hamid et al 2021; Kim et al 2018; Moss et al 2000). The fermentation characteristics of the rumen can be influenced by the type and quality of feed ingredients, as well as their interaction in the ration (Hao et al 2021; Holter and Young, 1992; Kim et al 2018; Muhsafaat et al 2015). Therefore, evaluating the effect of FSH on rumen fermentation in vitro is crucial before its application in feeding practices.

This study aims to assess the impact of FSH as a substitute for onggok (Manihot esculenta) in complete feed on nutrient digestibility and rumen fermentation characteristics in vitro. Onggok, a locally named fiber waste from the cassava flour industry, serves as a prevalent energy source for ruminant feed, particularly in Indonesia (Antari et al 2014; Bakrie et al 2018; Marsetyo et al 2021). Onggok shares a composition and nutrient content similar to sago hampas (Antari et al 2014; Bakrie et al 2018; Cowley et al 2020; Zain et al 2023) and its availability continues to decline (Zakaria et al 2020). FSH is anticipated to be an alternative ingredient to replace onggok in ruminant feed.

Materials and methods

Ethical approval

The Faculty of Veterinary Medicine, Universitas Gadjah Mada, Yogyakarta, Indonesia approved this experimental work (Approval Number: 00072/EC-FKH/Eks./2021).

Preparation of sago hampas and fermentation

Sago hampas, sourced from the Kalimantan sago processing industry, underwent sun drying until their moisture content reached approximately 14%. Fermentation employed the solid-state fermentation method with the local probiotic, Saus Burger Pakan (SBP^®), produced by CV. Agromix Lestari – Yogyakarta, Indonesia. Sterilized sago hampas (400g) was mixed with microbial inoculum (activated in a urea-molasses-aquadest solution three hours before use) at a dose of 0.05% (0.05 mL/100g), achieving a mixed moisture content of 40%. This mixture was anaerobically compressed in a mini-silo (1 kg sterile packaging plastic), tightly sealed and fermented for three days. Post-fermentation, the fermented sago hampas (FSH) was oven-dried at 55°C (Memmert GmbH + Co. KG, Germany) for 72 hours until it reached the moisture content of about 10%. The FSH was ground into FSH powder using a Wiley mill (Thomas Scientific, USA) and then filtered by a 1 mm sieve and preserved in a plastic bag for later use. The proximate composition of FSH including dry matter (DM), organic matter (OM), crude protein (CP) and crude fiber (CF), was assessed following the AOAC method.

Formulation and preparation of complete feed

Five formulations of complete feed were developed by substituting onggok (Manihot esculenta) with FSH at different levels: 0%, 25%, 50%, 75% and 100%. Onggok served as the primary energy source in the feed, while FSH was utilized as an alternative energy source. Other concentrate ingredients included cassava chips, ground corn cobs, coffee husks, bran pollard, copra meal, palm kernel meal and premix (a blend of minerals, urea and salt). These ingredients were thoroughly mixed in a feed mixer to achieve homogeneity, followed by drying at 60°C for 24 hours and storage in plastic bags until further use. The nutrient content of the feed ingredients in the treatment was analyzed and shown in Table1 and Table 2 displayed the formulation and composition of complete feed.

In vitro

Digestibility analysis and rumen fermentation

The complete feed underwent in vitro testing with five treatments:

(R1) Concentrate (- onggok) + 100% onggok + 0% FSH + forage

(R2) Concentrate (- onggok) + 75% onggok + 25% FSH + forage

(R3) Concentrate (- onggok) + 50% onggok + 50% FSH + forage

(R4) Concentrate (- onggok) + 25% onggok + 75% FSH + forage

(R5) Concentrate (- onggok) + 0% onggok + 100% FSH + forage

The rumen fluid was taken from Bali cattle weighing about 300 kg fed a diet comprising 3% of their body weight, consisting of Mott elephant grass and concentrate (70:30 DM basis), twice daily (at 08:00 and 15:00) for a week before sampling. Rumen fluid was filtered with four layers of fine cloth and blended with 474 ml H₂O, 237 ml buffer solution, 237 ml macro-mineral solution, 0.12 ml micro-mineral solution, 1.22 ml resazurin and 49.5 ml reducing solution in a 2 L Erlenmeyer flask before the morning feed. The mixture was constantly flushed with CO₂ under anaerobic conditions in the preparation process before moving it to a syringe jar. The rumen fluid and its medium were blended at a 1:2 (v/v) ratio. About 300 mg of each test feed was put into a glass syringe with 30 ml of fermentation media. In-vitro fermentation was performed using gas production (Menke and Steingass 1988) and the two-step method of Tilley and Terry for 96 hours (Tilley and Terry 1963).

The fermentation products were filtered after 48 hours of incubation and the residue was evaluated for nutrient digestibility. The pH of the rumen fluid was measured using a pH meter that was calibrated with pH 4 and pH 7 buffers at the end of fermentation. Ammonia concentration (Chaney and Marbach 1962), microbial protein (Plummer 1987) and VFA (Filípek and Dvořák 2009) were determined by centrifuging the rumen fluid (3000 g/10 minutes). The supernatant was centrifuged again (10,000 g/10 minutes) to distinguish microbial cells and the supernatant with enzymes. The methods of (Bergmeyer et al 1974) were followed to estimate amylase and CMC-ase activities and the protease activity was measured using (Halliwell 1961).

Digestibility in the post-rumen phase was assessed following the 48-hour incubation, incorporating three mL of 20% HCL and one mL of 5% pepsin. This was succeeded by an additional 48-hour incubation period. The filtrate from the syringe was separated and the residual content underwent analysis for DM, OM and CP to ascertain the digestibility of DM, OM and CP.

Data analysis

Data underwent analysis employing Analysis of Variance (ANOVA) with a Randomized Complete Block Design (RCBD) of 5x3 through RStudio software version 4.2.0 (R Core Team, 2022). The experimental treatment involved five levels of substituting ASF for onggok in the complete feed ration and the blocks were characterized by three distinct rumen fluid sampling times. Variations among treatments were assessed using Duncan's Multiple Range Test at a 5% significance level.

Results and discussion

Proximate composition of FSH and complete feed ration

The composition and nutritional value of the closest constituents in the complete feed treatment, along with the results of sago hampas fermentation using SBP^® inoculants, are shown in Table 1. Fermentation resulted in a 1.22% increase in CP content, rising from 1.14% to 2.36% and a 4.46% decrease in CF content, dropping from 23.40% to 18.94%. This aligns with prior research demonstrating enhanced protein and fiber degradation in fermented sago hampas (Ginting and Pase 2018; Nafiu et al 2018; Sumardiono et al 2018; Sumiana et al 2020; Wizna et al 2008). The primary objective of fermentation is to boost enzyme production (Ghoshal et al 2012; Pandey et al 1999; Sadh et al 2018; Sun et al 2022). The elevated protein content results from the synthesis of microorganism biomass and enzymes, while the reduction in fiber content is attributed to the hydrolysis of cellulose and hemicellulose by microorganism enzymes (Daniel et al 2023; Hua et al 2022; Nafiu et al 2018; Sumiana et al 2020).

Comparing CF content, FSH exceeds onggok by 3.19%, indicating onggok's potential as a superior fiber source to FSH. While CF is known to decrease digestibility in feed ingredients, it is essential for sustaining the normal physiological function of the gastrointestinal tract (Antari et al 2014; Hasanah et al 2020; Maktabi et al 2016; Mayulu et al 2013; Mirzaei-Aghsaghali and Maheri-Sis 2011; Nuswantara et al 2023; Santoso et al 2017; Wang et al 2016). The DM and OM content of FSH are marginally lower compared to unfermented sago hampas, attributed to the loss of some soluble carbohydrates during fermentation (Nkhata et al 2018; Susanti et al 2022; Vikineswary et al 1994). The substitution level does not influence the DM and OM rations, indicating similarities in water content, nutrient composition and interaction between onggok and FSH components. The ration meets the complete feed requirements for beef cattle, with CP ranging from 9-14% and CF less than 25% (Hasan et al 2020; Lee et al 2021; Mayulu et al 2013; Nuswantara et al 2023; Parish and Rhinehart 2018; Tuturoong et al 2020).

Table 1 illustrates a gradual increase in CP content with FSH substitution for onggok in complete feed rations. This indicates that FSH has a higher CP content than onggok. However, different results are observed in CF materials among treatment rations, showing no significant difference. The 3.19% difference in CF content between FSH and onggok may not be sufficient to impact the CF content of treatment rations. Additionally, the variation in CF among other ration ingredients may influence the overall CF content of the treatment ration, consistent with the notion that the nutritional content of complete feed depends on the constituent feed ingredients (Greenwood 2021; Hasan et al 2020; Owen, 1984; Reddy et al 2002; Tuturoong et al 2020).

In vitro analysis of fermentation and digestibility In the rumen

Tables 2, 3 and 4 provide an intricate overview of the rumen fermentation and in vitro digestibility features of the complete feed ration. The substitution of onggok with FSH did not impact rumen fluid enzyme activity in vitro, as evidenced by the absence of significant differences between treatments (p>0.05). Enzyme activity, specifically CMCase, amylase and proteases, maintained a consistent mean of 4.98 U/g, 24.92 U/g and 37.25 U/g, respectively, falling within the normal range observed in previous studies (Hu et al 2012; Karasov and Douglas 2013; Rey et al 2012).

These findings suggest that FSH exhibits enzyme activity comparable to onggok in serving as a source of carbohydrates and protein in the ration. This similarity is attributed to the comparable chemical composition between FSH and onggok, particularly in terms of starch, CF and CP content. Table 1 outlines the CF and FSH protein content at 18.94% and 2.36%, respectively, while onggok shows 15.75% and 2.02%. Previous research notes the starch content of onggok ranging from 30-77% (Bussolo de Souza et al 2018; Ferreira-Leitao et al 2010; Kosugi et al 2009; Musita 2018; Pandey et al 2000), crude protein from 0.3-2.0% (Bussolo de Souza et al 2018; Fiorda et al 2013; Musita 2018; Pandey et al 2000) and crude fiber as much as 14-50% (Musita 2018; Pandey et al 2000), while FSH contains starch from 30-73% (Awg-Adeni et al 2013; Lai et al 2013; Muhsafaat et al 2015), crude protein from 3.22-6.79% and crude fiber from approximately 9.44-38.57% (Ginting and Pase 2018; Nafiu et al 2018; Rianza et al 2019; Wardono et al 2021). (Hao et al 2021) suggests that differences in rumen microbial enzyme activity can result from factors such as feed substrates, genetics, livestock type, microbial quantity, rumen pH and temperature. Thus, FSH and onggok in this study likely offer similar substrate availability and degradation rates, resulting in comparable enzyme activity.

The substitution of onggok with ASF across all rations (R1 to R5) demonstrated no impact on the in vitro digestibility of DM, OM, CP and CF at both rumen and total stages (p>0.05). The average digestibility at the rumen stage was 54.65% for DM, 58.26% for OM, 39.11% for CP and 44.76% for CF. Total digestibility in vitro averaged 62.10% for DM, 62.66% for OM, 48.74% for CP and 53.60% for CF. While high digestibility generally indicates better feed quality and energy content (Reddy et al 2002; Santoso et al 2017; Tuturoong et al 2020; Van Dung et al 2013), it may not always reflect high energy content (De La Torre et al 2019; Hamid et al 2021; Kaewpila et al 2018; Sampaio et al 2012), influenced by specific components such as easily digestible carbohydrates (Gómez et al 2016; Shabat et al 2016; Nozière et al 2010) and fat (Allen 2014; Ghani et al 2017; Rajneesh et al 2020). This implies that FSH, as a substitute for onggok, maintains comparable digestibility for ruminants without altering nutrient availability.

The substitution of onggok with ASF also did not influence pH, acetate, propionate, butyrate, total VFA, NH₃, microbial proteins, enzyme proteins, CH ₄ and CO₂of rumen fluid buffer mixture after incubation (p>0.05). Rumen pH in this study maintained an optimal range (6.53-6.69) for fermentation (Hristov et al 2009; Kamra 2005; Liu et al 2017; Marlida et al 2023; Santoso et al 2020; Wang et al 2016). Livestock actively maintains rumen pH close to neutral to facilitate the proper development of cellulolytic and fibrolytic bacteria (Castillo-González et al 2014; Hua et al 2022; Kim et al 2018). VFA concentrations, including acetate (31-110 mM), propionate (21-65 mM) and butyrate (13-33 mM), were consistent across rations, aligning with previous findings for complete feed (Hao et al 2021; Kusmartono et al 2022; Marlida et al 2023; Nuswantara et al 2023; Santoso et al 2017, 2020). Discrepancies in results across studies are attributed to factors such as feed type, measurement methods, supplementation levels, livestock age and environmental conditions (Maktabi et al 2016; Wang et al 2016). The acetate:propionate ratio was notably lower at R1, R3, R4 and R5 than at R2, indicating more efficient carbohydrate fermentation in the rumen (Hao et al 2021; Hasanah et al 2020; Kim et al 2018; Maktabi et al 2016; Santoso et al 2020). However, a higher acetate:propionate ratio can also be seen as positive, promoting the growth of cellulolytic bacteria, enzyme activity and the degradability of crude fiber and protein (Wang et al 2016).

The NH₃concentration in this study ranged from 19.29-22.07 mg/dl, surpassing the 3.5 mg/dl threshold deemed sufficient for microbial protein synthesis (Erdman et al 1986; Hamid et al 2021; Kim et al 2018; Kondo et al 2016; Marlida et al 2023; Pengpeng and Tan, 2013; Santoso et al 2020). Microbial protein and enzyme protein concentrations, ranging from 0.54 to 0.65 mg/ml and 0.89 to 0.96 mg/ml, respectively, were consistent across various rations, aligning with findings from previous research (Hasanah et al 2020; Miura et al 2021; Zhao et al 2018). Variations in results among different studies can be attributed to multiple factors, encompassing physical (rumen temperature, pH, osmolarity and interactions), chemical (NH₃, VFA, minerals and toxins), feed-related (type, quality, quantity and processing), biological (bacteriophages, protozoan predation and bacterial lysis) and livestock-related (age, species, physiological status, sex and stress) aspects (Harun and Sali 2019). CH₄and CO₂concentrations were also similar among rations, ranging from 10-11% and from 57% to 61%, respectively. These values fall within the reported range for complete feed, i.e., 10-27% for CH ₄ (Hamid et al 2021; Samadi et al 2020) and 50-60% for CO₂ (Ungerfeld 2020). Variations in results across studies stem from diverse factors, including genetics, feed composition, feed consumption, energy consumption, rumen microbes, rumen pH, age, body weight, growth rate, production rate and environmental conditions (Broucek 2014; Goopy and Hegarty 2004; Laporte-Uribe 2019; Martin et al 2010). The results suggest that FSH and onggok exert similar effects on microbial ecology in the rumen, demonstrating that FSH can be a viable substitute for onggok in ruminant rations without adverse effects on enzyme activity, nutrient digestibility and rumen fermentation parameters in vitro.

Conclusion

The fermentation of sago hampas with SBP^® significantly enhances the protein content while reducing the fiber content, positioning it as a viable alternative feed source for ruminants. Substituting onggok with ASF in the complete feed ration exhibits no adverse effects on rumen fermentation and in vitro digestibility parameters, except for a notable reduction in the acetate:propionate ratio at specific substitution rates. This reduction suggests the more efficient energy utilization by ruminants. Consequently, ASF proves to be a suitable substitute for onggok in ruminant feed, even up to 100%, without compromising feed quality and rumen function. To comprehensively assess the impact of ASF on animal performance, health and product quality in practical feeding conditions, further research is imperative.

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