Nutrient composition, ruminal fermentation characteristics, methane production and in vitro digestibility of fermented cactus feed for ruminants

Rezki Amalyadi¹, Khairil Anwar¹, Aminurrahman¹ and Imam Munandar²

¹Department of Animal Science, Faculty of Animal Science, University of Mataram, Mataram City, 83115, West Nusa Tenggara, Indonesia
rezkiamalyadi@staff.unram.ac.id
²Department of Animal Science, Faculty of Life Science and Technology, Sumbawa University of Technology, Sumbawa City, 84371, West Nusa Tenggara, Indonesia

Abstract

This study evaluated the nutrient composition, protein solubility, ruminal fermentation characteristics, methane production and in vitro digestibility of fermented cactus (Opuntia ficus-indica) feed as an alternative feed resource for ruminants under tropical dryland conditions. A completely randomized design was used with four fermentation durations (0, 7, 14 and 21 days). Chemical analyses included dry matter, organic matter, crude protein, crude fiber, neutral detergent fiber, acid detergent fiber and soluble protein fractions. Ruminal fermentation characteristics were evaluated through ammonia nitrogen (NH₃-N), individual volatile fatty acids (acetate, propionate and butyrate), total VFA concentration and methane production during in vitro incubation. Fermentation significantly improved (p < 0.05) nutritional quality and digestibility of cactus feed. Crude protein and soluble protein contents increased progressively with fermentation duration, while crude fiber, NDF and ADF decreased. In vitro dry matter digestibility and organic matter digestibility were enhanced in fermented treatments compared with the control. Fermentation also increased NH₃-N concentration and altered VFA profiles by increasing propionate production and reducing the acetate-to-propionate ratio. Methane production tended to decrease with increasing fermentation duration, indicating improved fermentation efficiency. The results suggest that fermentation enhances nutrient availability and ruminal utilization of cactus feed while potentially reducing enteric methane emissions. Fermented cactus therefore represents a promising climate-resilient feed resource for sustainable ruminant production in drought-prone environments.

Keywords: cactus bioconversion, fermented cladodes, rumen degradability, dryland feed innovation, tropical alternative forage

Introduction

The increasing frequency of drought, land degradation and seasonal forage scarcity has become a major challenge for sustainable ruminant production systems, particularly in tropical and semi-arid regions. Conventional forage resources are increasingly unable to meet the nutritional requirements of livestock throughout the year, resulting in reduced animal productivity and increased feeding costs. Consequently, the exploration of climate-resilient alternative feed resources has become an important research priority in livestock nutrition science. Among the promising drought-tolerant feed resources, cactus species, particularly those belonging to the genera Opuntia and Nopalea, have received considerable attention due to their high biomass production, exceptional water-use efficiency and adaptability to marginal environments (Dubeux Jr et al 2021).

Cactus cladodes are widely recognized as an alternative energy-rich feed source for ruminants in arid and semi-arid ecosystems. Their crassulacean acid metabolism (CAM) photosynthetic pathway allows efficient water conservation and sustained biomass production under prolonged drought conditions. In addition, cactus contains high concentrations of soluble carbohydrates and non-fibrous carbohydrates, which contribute positively to rumen fermentation and animal energy supply (Pastorelli et al 2022). However, despite these advantages, fresh cactus generally contains low dry matter, low crude protein and limited structural fiber, which may restrict its direct utilization as a sole feed ingredient for ruminants (Dubeux Jr et al 2021).

Previous studies demonstrated that the nutritional composition and digestibility of cactus are strongly influenced by species, cultivar, maturity stage, agronomic management and post-harvest processing techniques. Pessoa et al (2022) reported substantial variation in crude protein, lignin, pectin and in vitro dry matter digestibility among several Opuntia and Nopalea cultivars. Similarly, D. V. Pessoa et al (2020) observed that mature cladodes generally exhibited higher neutral detergent fiber concentrations but variable digestibility responses depending on cactus genotype. These findings indicate that improving the feeding value of cactus requires appropriate processing technologies capable of enhancing nutrient availability and fermentation quality.

Fermentation technology has emerged as an effective biological approach to improve the nutritional quality, palatability, preservation and digestibility of unconventional feed resources. Microbial fermentation can reduce anti-nutritional compounds, increase microbial protein synthesis, improve fiber degradation and stabilize high-moisture feed materials through organic acid production. In cactus-based feed systems, fermentation is particularly relevant because the high moisture and soluble carbohydrate contents of cactus provide favorable substrates for microbial growth during ensiling or controlled fermentation processes (Gannuscio et al 2024; Vastolo et al 2020). Furthermore, fermented cactus feed may improve ruminal fermentation efficiency and nutrient utilization in ruminants compared with fresh cactus material.

The evaluation of in vitro digestibility represents an important laboratory-based approach for assessing the feeding potential of alternative feed resources before in vivo application. In vitro techniques provide reliable estimates of nutrient degradation, ruminal fermentation characteristics and feed utilization efficiency under controlled conditions. Several studies have reported that cactus-based diets exhibit relatively high organic matter digestibility because of their elevated soluble carbohydrate fractions and pectin content (Cerrillo & Juarez, 2004; Inácio et al 2019). Nevertheless, information regarding the nutrient composition and in vitro digestibility of fermented cactus feed remains limited, especially under tropical livestock production systems where feed scarcity and climate stress are increasingly severe.

In recent years, the utilization of cactus as a sustainable feed resource has become increasingly relevant within the framework of climate-smart livestock production. Opuntia-based feeding systems have been associated with improved resilience of smallholder farming systems, reduced dependence on conventional forage crops and enhanced feed availability during prolonged dry seasons (Pastorelli et al 2022). Moreover, the integration of locally available cactus resources into ruminant feeding strategies may contribute to sustainable livestock intensification and rural food security in dryland ecosystems.

Despite the growing interest in cactus utilization for ruminant feeding, studies focusing specifically on the effects of fermentation on nutrient composition and in vitro nutrient digestibility are still relatively scarce. Comprehensive evaluation of fermented cactus feed is therefore necessary to determine its nutritional potential and practical applicability in ruminant production systems. Therefore, this study aimed to evaluate the nutrient composition and in vitro digestibility of fermented cactus feed as an alternative feed resource for ruminants.

Materials and methods

Study area and experimental design

This study was conducted at the Integrated Laboratory, Sumbawa University of Technology, located in a tropical dryland region of Indonesia. The experiment evaluated the nutritional composition and in vitro digestibility of fermented cactus-based feed intended for ruminant feeding systems. A completely randomized design (CRD) was applied with four fermentation treatments and four replications for each treatment.

The experimental treatments consisted of different fermentation periods of cactus feed materials, namely:

P0 = non-fermented cactus feed (control),

P1 = fermented cactus feed for 7 days,

P2 = fermented cactus feed for 14 days and

P3 = fermented cactus feed for 21 days.

The selection of fermentation duration was based on previous studies indicating that microbial fermentation can significantly alter nutrient fractions and improve feed digestibility through structural carbohydrate degradation and enhanced microbial activity (Dubeux Jr et al 2021; Vastolo et al 2020).

Preparation of cactus feed materials

Fresh cladodes of cactus (Opuntia ficus-indica) were collected from local dryland farming areas. The cactus cladodes were cleaned to remove dirt and spines, chopped into approximately 2–3 cm pieces and air-dried for 24 h to reduce excessive moisture content. The chopped cactus material was subsequently mixed with rice bran and molasses at proportions of 15% and 5% (dry matter basis), respectively, to improve fermentation substrate quality and carbohydrate availability for microbial growth. A commercial microbial inoculant containing lactic acid bacteria (Lactobacillus plantarum) was added at a concentration of 1 × 10^6 CFU g^-1 fresh material. The mixed feed materials were thoroughly homogenized and packed into airtight polyethylene silos for anaerobic fermentation according to the designated treatment periods.

Fermentation technology was selected because previous studies demonstrated that biological processing improves the preservation quality, nutrient availability and feeding value of cactus-based feed resources through organic acid production and fiber degradation (Pastorelli et al 2022; Vastolo et al 2020).

Chemical composition analysis

At the end of each fermentation period, representative feed samples were collected and oven-dried at 60°C for 48 h until a constant weight was obtained. The dried samples were subsequently ground using a Wiley mill fitted with a 1-mm screen to ensure homogeneity prior to laboratory analyses. Chemical composition was determined according to the procedures of the Association of Official Analytical Chemists (AOAC., 2019). The analyzed parameters included dry matter (DM), organic matter (OM), crude protein (CP), ether extract (EE), crude fiber (CF) and ash content. Fiber fractions were evaluated using the Van Soest method, including neutral detergent fiber (NDF) and acid detergent fiber (ADF). Hemicellulose concentration was calculated as the difference between NDF and ADF values. To further assess nitrogen availability, soluble protein concentration was determined using the buffer-soluble protein procedure described by Rekowski et al (2021). Briefly, feed samples were incubated in a borate-phosphate buffer solution to extract the soluble nitrogen fraction, which was subsequently quantified using the Kjeldahl method. Protein solubility was expressed as a percentage of total crude protein and was used as an indicator of the proportion of nitrogen readily available for ruminal microbial utilization.

In vitro digestibility and methane production analysis

In vitro digestibility was evaluated using the two-stage Tilley and Terry technique as modified by Van Soest. Rumen fluid was collected before the morning feeding from two fistulated Bali cattle receiving a standard forage-concentrate diet. Immediately after collection, the rumen fluid was filtered through four layers of cheesecloth and continuously flushed with CO₂ to maintain anaerobic conditions throughout the preparation process. Approximately 0.5 g of each feed sample was transferred into fermentation tubes containing 40 mL of McDougall’s buffer solution and 10 mL of freshly collected rumen fluid. The tubes were incubated anaerobically at 39°C for 48 h in a shaking water bath to simulate ruminal fermentation. During the incubation period, total gas production was monitored using calibrated gas-tight syringes. Methane concentration in the fermentation gas was subsequently determined by gas chromatography equipped with a flame ionization detector and methane production was expressed as mL CH₄ g⁻¹ dry matter incubated.

Following the ruminal incubation phase, fermentation residues were treated with pepsin-HCl solution and incubated for an additional 48 h to simulate post-ruminal digestion. The remaining undigested residues were then filtered, oven-dried and weighed to determine in vitro dry matter digestibility (IVDMD) and in vitro organic matter digestibility (IVOMD). The in vitro digestibility technique is widely recognized as a reliable method for estimating ruminal degradation characteristics and evaluating the feeding value of conventional and unconventional feed resources under controlled laboratory conditions (Inácio et al 2019). In the present study, fermented cactus feed was expected to exhibit improved digestibility and fermentation efficiency as a result of enhanced microbial degradation of structural carbohydrates during the fermentation process.

Fermentation characteristics

Fermentation characteristics included pH, ammonia nitrogen (NH₃-N), methane production and volatile fatty acid profiles. Individual VFAs including acetate, propionate and butyrate were quantified using gas chromatography. The acetate-to-propionate ratio was subsequently calculated as an indicator of ruminal fermentation efficiency.

Statistical analysis

All experimental data were analyzed using one-way analysis of variance (ANOVA) under a completely randomized design using IBM SPSS Statistics version 26. When significant differences among treatments were detected ( p < 0.05), Duncan’s Multiple Range Test was applied to compare treatment means.

The statistical model used was: Y_ij= µ + τ _i + Ɛ _ij

Where:
Y_i=observed variable,

µ =overall mean,

τ _i =treatment effect and Ɛ _ij =experimental error.

Results and discussion

Nutrient composition of fermented cactus feed

Fermentation (Table 1) significantly influenced the chemical composition of cactus-based feed (p< 0.05), particularly dry matter (DM), crude protein (CP), neutral detergent fiber (NDF) and acid detergent fiber (ADF) contents. The improvement in nutrient quality observed after fermentation indicated that microbial activity contributed to structural carbohydrate degradation and enhanced nutrient availability.

Soluble protein increased significantly with fermentation duration, indicating enhanced proteolytic activity and partial hydrolysis of complex protein structures into more readily available nitrogen fractions. The increase from 24.15% in the control treatment to 39.76% after 21 days of fermentation suggests improved nitrogen availability for ruminal microorganisms and may partly explain the higher NH₃-N concentration and digestibility observed in fermented treatments. Similar responses have been reported in cactus silages and other biologically fermented feed resources where microbial activity increased soluble nitrogen fractions and improved nutrient utilization.

The increase in crude protein content following fermentation was likely associated with microbial biomass synthesis and nitrogen enrichment during the fermentation process. Similar findings were reported by da Silva Brito et al (2020), who observed increased crude protein and improved fermentation quality in cactus pear silage supplemented with Gliricidia sepium. The reduction in NDF and ADF contents further indicated partial hydrolysis of structural carbohydrates by fermentative microorganisms, improving feed degradability and nutrient accessibility.

The decline in crude fiber fractions after 14 and 21 days of fermentation suggested active cellulolytic and hemicellulolytic microbial activity. This finding is consistent with previous observations that fermentation can modify lignocellulosic structures and improve the nutritive value of unconventional feed resources (Todaro et al 2020; Vastolo et al 2020).

Moreover, the relatively high organic matter content and reduced fiber fractions indicated that fermented cactus feed could serve as an energy-rich feed resource for ruminants under tropical dryland conditions. Cactus contains high levels of soluble carbohydrates and pectin, which contribute positively to ruminal fermentation efficiency and microbial growth (Siqueira et al 2021).

In vitro digestibility of fermented cactus feed

Table 2 presents the effects of fermentation duration on in vitro digestibility and ruminal fermentation characteristics of cactus feed. Fermentation significantly improved (p < 0.05) in vitro dry matter digestibility (IVDMD), in vitro organic matter digestibility (IVOMD), ammonia nitrogen (NH₃-N), volatile fatty acid production and several indicators of ruminal fermentation efficiency.

The higher digestibility values observed in fermented treatments may be attributed to the breakdown of complex fiber structures into simpler carbohydrates that are more readily utilized by rumen microorganisms. Ensiling and microbial fermentation are known to improve nutrient accessibility through partial degradation of cellulose and hemicellulose components (da Silva Brito et al 2020). Consequently, IVDMD increased from 61.28% in the control treatment to 73.17% after 21 days of fermentation, while IVOMD increased from 58.94% to 70.62%, indicating improved nutrient availability and substrate utilization.

The increase in NH₃-N concentration with fermentation duration indicated enhanced protein degradation and greater nitrogen availability for microbial growth. According to Liang et al (2020), adequate ammonia nitrogen concentration is essential for optimal microbial protein synthesis and ruminal fermentation efficiency. The increase in NH₃-N observed in fermented treatments therefore suggests improved synchronization between energy and nitrogen availability within the rumen ecosystem.

Total volatile fatty acid production increased progressively with fermentation duration, reflecting enhanced fermentability of the cactus substrate. VFAs constitute the primary source of metabolic energy for ruminants and are closely associated with carbohydrate degradation in the rumen. The elevated total VFA concentration observed in fermented treatments confirms improved ruminal fermentation characteristics and greater utilization of fermentable substrates.

Beyond the increase in total VFA concentration, fermentation also modified the profile of individual VFAs. Acetate concentration increased slightly, whereas propionate exhibited a more pronounced increase as fermentation duration progressed. As a consequence, the acetate-to-propionate ratio declined significantly from 3.09 in the control treatment to 2.18 after 21 days of fermentation. This reduction suggests a shift toward a more energetically efficient ruminal fermentation pattern. Propionate serves as the principal gluconeogenic precursor in ruminants and is frequently associated with enhanced feed efficiency because its formation acts as an alternative hydrogen sink, thereby reducing hydrogen availability for methanogenic microorganisms.

Similarly, butyrate concentration increased with fermentation duration, indicating greater microbial fermentation activity. Butyrate plays an important role in maintaining ruminal epithelial health and contributes to energy metabolism within the digestive tract. The simultaneous increase in propionate and butyrate concentrations therefore suggests that fermentation improved both the intensity and efficiency of ruminal fermentation.

Methane production decreased significantly as fermentation duration increased. Methane output declined from 24.63 mL g⁻¹ DM in the control treatment to 17.95 mL g⁻¹ DM after 21 days of fermentation. The reduction in methane production may be associated with the greater formation of propionate, which competes with methanogenesis for metabolic hydrogen within the rumen ecosystem. Lower methane output indicates improved fermentation efficiency and reduced energy losses during microbial degradation of feed substrates. These findings support the potential role of fermented cactus feed as a climate-smart feeding strategy capable of improving nutrient utilization while mitigating enteric methane emissions from ruminants. The relationship between methane production and the acetate-to-propionate ratio is presented in Figure 1.

Figure 1. Relationship between methane production (mL/g DM) and acetate:propionate ratio

The strong positive association observed between methane production and the acetate-to-propionate ratio indicates that treatments characterized by lower acetate-to-propionate ratios consistently produced less methane. This finding suggests that fermentation promoted metabolic pathways favoring propionate synthesis rather than methanogenesis. Similar responses have been reported in cactus-based feeding systems where readily fermentable carbohydrates and pectin stimulated propionate production and reduced methane emissions (Siqueira et al 2021). Reduced methane formation represents an important advantage because methane production constitutes a loss of dietary energy and contributes to greenhouse gas emissions from ruminant livestock.

Despite the increase in fermentation activity, ruminal pH remained within the normal physiological range for fiber digestion. The slight decline in pH from 6.92 to 6.57 indicates greater fermentation intensity while maintaining stable ruminal conditions. Similar responses were documented in sheep fed spineless cactus silage, where ensiling improved nutrient digestibility and microbial protein synthesis without negatively affecting rumen stability (Pereira et al 2021). Overall, these findings demonstrate that fermentation enhanced nutrient digestibility, improved fermentation efficiency, modified VFA profiles toward greater propionate production and reduced methane generation, thereby increasing the nutritional and environmental value of cactus feed for ruminant production systems.

Relationship between fermentation and nutrient availability

The positive relationship between fermentation duration and digestibility parameters suggested that biological processing effectively enhanced the feeding value of cactus-based feed. Fermentation not only improved preservation quality but also increased substrate accessibility for rumen microorganisms through biochemical modification of plant cell walls.

The increase in digestibility following fermentation may also be associated with improved microbial colonization of feed particles during rumen incubation. Fermented feed substrates generally exhibit softer tissue structure and reduced lignification, facilitating microbial attachment and enzymatic degradation (Ahmadi et al 2019).

Additionally, fermentation may contribute to improved palatability and feed conservation under tropical environments where high moisture feed materials are susceptible to rapid spoilage. Previous studies demonstrated that cactus silage exhibits favorable aerobic stability and reduced nutrient losses when properly fermented (da Silva Brito et al 2020; Horibe, 2021).

The current findings therefore support the potential application of fermented cactus feed as a sustainable alternative feed resource for ruminants in dryland and climate-vulnerable regions. The ability of cactus to maintain biomass production under drought stress, combined with improved digestibility after fermentation, provides important opportunities for resilient livestock feeding systems. Recent studies have emphasized the strategic role of cactus-based feeding systems in enhancing feed security and reducing dependence on conventional forages in semi-arid livestock production systems (Siqueira et al 2021).

Conclusion

Fermentation improved not only nutrient composition and digestibility but also protein solubility and ruminal fermentation efficiency. The increase in soluble protein fractions, enhanced propionate production, reduced acetate-to-propionate ratio and lower methane production indicate that fermented cactus feed can improve nutrient utilization while contributing to environmentally sustainable livestock production. Fermentation for 21 days produced the most favorable responses and demonstrates the potential of cactus as a climate-resilient feed resource for ruminants in tropical dryland systems.

Acknowledgments

The authors thank the local smallholder farmers for their cooperation, the technical staff at the Sumbawa University of Technology and Animal Nutrition Laboratory. Constructive suggestions from anonymous reviewers are greatly appreciated.

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