Supplementing Psophocarpus scandens leaves and yeast-fermented rice in ammoniated rice straw as basal substrate reduces methane production and enhance volatile fatty acid in in vitro incubation

L T B Phuong, L A Tuyet, N T M Nhan, B Vongkhamchanh¹, S Inthapanya² and T R Preston³

Nong Lam University, HCMC, Vietnam
phuong.lethuybinh@hcmuaf.edu.vn
¹ Faculty of Agriculture and Forestry, Champasack University, Champasak, Lao PDR
² Faculty of Agriculture and Forest Resources, Souphanouvong University, Luang Prabang, Lao PDR
³ Centro para la Investigación en Sistemas Sostenibles de Producción Agropecuaria CIPAV, Carrera 25 No 6-62 Cali, Colombia

Abstract

The study was conducted to determine methane production and improved efficiency of in vitro fermentation by supplementing with Psophocarpus scandens (PS) leaves as protein and propionic acid derived from yeast-fermented rice (YFR) in ammoniated rice straw (ARS) as basal diet. The substrates were arranged and mixed in four treatments and three replications including (i) ammoniated rice straw as control treatment (ARS), (ii) ARS plus 30% PS leaves (ARS-PS), (iii) ARS plus 4% YFR (ARS-YFR), and (iv) ARS plus combination of 30% PS leaves and 4% YFR (ARS-PS-YFR). The result shows that supplementation with PS leaves or with both PS leaves and YFR results in lower methane per unit soluble DM compared to ARS alone and ARS plus YFR, while the corresponding total gas volume tends to be higher. However, compared to the ARS replacement level in the treatment, the addition of PS leaves was the main factor in reducing methane emission (8.7%), while the contribution of YFR to the reduction in methane was unclear. Total VFA was highest in the ARS-PS-YFR treatment, followed by ARS-PS and lowest in the ARS alone and ARS-YFR treatments. Among molar percentages, the addition of PS leaves, YFR, or their combination in the ARS diet had a trend in reducing proportion of acetic acid compare to ARS alone with borderline significance ( p = 0.077), while propionic acid was followed in more enhancing order by ARS-YFR, ARS-PS, ARS-PS-YFR compared to ARS alone. Butyric acid was not affected by treatments. The ratio of acetic acid to propionic acid was positively correlated with methane production, reflecting the accumulation of propionic acid associated with reduced methane production.

Keywords: propionic acid, methane emission, enteric fermentation, bypass protein

Introduction

Rumen fermentation is the primary source of methane emissions from enteric fermentation, a major contributor to greenhouse gases. Therefore, developing feeding systems that enhance feed conversion efficiency can reduce methane emissions from both enteric fermentation and manure (feces and urine), thereby improving environmental sustainability and animal productivity. Studies have shown that feed materials capable of bypassing post-ruminal digestion can reduce methane production (Phonethep et al, 2016; Do et al, 2013), and highly digestibility and animal productivity are largely influenced by the bypass characteristics of dietary proteins (Preston and Leng, 1987). Straw is a major source of roughage for ruminants, and its dry form can be stored to meet feed demands during the dry season or periods of feed scarcity. Treatment of raw rice straw with urea or anhydrous ammonia prior to its inclusion in ruminant diets enhances fiber digestibility, improves the nutritional composition of the straw (Trach et al, 2001), and reduces methane production. However, ammoniated rice straw alone is rarely sufficient to support optimal production (e.g., growth or milk yield), as its energy density and rumen-available protein content remain limited even after treatment. Psophocarpus scandens leaves have a high protein content of up to 27.7% of dry matter (DM) (Kambashi et al, 2014). Nguyen Thi Hong Nhan et al (2014) showed that supplementing Psophocarpus scandens and Tithonia diversifolia leaves as protein sources in para grass as basal diet improved feed intake, digestibility and daily weight gain of goats compared to supplementing only Tithonia diversifolia leaves. Psophocarpus scandens (PS) leaves may have good bypass properties in rumen fermentation, with this property PS leaves may also bring the expectation of reducing methane and increasing digestibility coefficient in ruminants when they use ammoniated rice straw as basal diet.

Besides the potential protein source for animal growth, the energy provided from propionic acid metabolism in rumen fermentation of ruminants is important because it directly affects growth and productivity of animals. Phuong et al (2023) showed that the increase in propionic acid was evident from 0 to 1.5% of fermented rice straw and continued to increase slightly from 1.5 to 6% of supplementation.

The hypothesis supporting this research was that rumen fermentation would improve by supplementing them with “rumen-escape protein” and propionic acid derived from yeast-fermented broken rice via the pathway: hydrolysed yeast cell wall, beta-glucan - lactic acid – propionic acid. Both supplements would be expected to contribute to the formation of propionic acid as a means of reducing emissions of rumen methane and to improvements in productivity in terms of meat and milk.

Material and method

Materials preparation and treatments

Psophocarpus scandens leaves used in the experiment were grown at the Center for Research and Technology Transfer (CRTT). Rice straw was cut into 5–6 cm pieces, and 500 g of the chopped material was treated with 3% urea (on a dry matter basis) and 500 mL of water to achieve a moisture content of approximately 40–45%. The ammoniation process was carried out for 21 days. Yeast-fermented rice was prepared following the procedure described by Phuong et al (2023). Briefly, 1 kg of polished white rice was soaked in 1.5 L of tap water for 5 hours, then milled and inoculated with Saccharomyces cerevisiae at 0.5% of DM (1 × 10¹⁰ CFU/g). The mixture was packed into 2 kg plastic bags, sealed, and incubated for 7 days to allow fermentation.

The in vitro incubation was used an ammoniated rice straw (ARS) as control treatment. Experimental treatments were supplemented by Psophocarpus scandens (PS) leave as protein source and/or yeast fermented rice (YFR) as source of energy for propionic acid. The sample of substrates as dry matter were mixed and arranged following treatments and three replications:

ARS: Ammoniated rice straw as control treatment

ARS-PS: Ammoniated rice straw and 30% PS leaves

ARS-YFR: Ammoniated rice straw and 4% YFR

ARS-PS-YFR: Ammoniated rice straw plus 30% PS leaves and 4% YFR

The crude protein of treatments was adjusted to around 12.82 % on a DM basis. The composition of treatments is presented in Table 1.

In vitro incubation

The thermos flask intended for rumen fluid collection was pre-warmed with hot water (40°C) prior to arrival at the slaughterhouse. Rumen fluid from goats was collected immediately after slaughter at the abattoir. The rumen contents were pooled and filtered through two layers of cheesecloth to remove excess feed particles; hot water previously used for pre-warmed flask was then utilized to keep the filtrate container warm during filtration. Filtrated fluid was then transferred into the pre-warmed flask and then rapidly transported to the laboratory to mix with the substrates. 12 g of substrate as DM was mixed with 0.24 liters of filtered rumen fluid, followed by 0.96 liters of buffer solution (Table 2). This mixture was contained in incubated bottles, which were flushed with carbon dioxide gas CO₂ and incubated in a water bath at 39^oC for 24 hours. The fermentation reaction in the incubated system was stopped by placing ice in a water bath for 10-15 minutes.

Analytical methods and data collection

The chemical composition of all experimental feed ingredients was analyzed for Dry Matter (DM) and Crude Protein (CP) following the AOAC (2001) method. Crude fiber, ADF, NDF and ADL were determined following by methods AOCS Ba-6a-05:2017 (VFA), GE029.2021(Ref.ANKOMTechnology method 12:2015) (VF), GE030.2021(Ref.ANKOM Technology method 13:2015) (VF), GE234.2021(Ref.ANKOMTechnology method 8:2013) (VF) respectively. The soluble crude protein of PS leaves and YFR was determined by immersing 3g DM of sample in 100ml dissolved NaCl solution (58g NaCl filled with distilled water up to 1000 ml). This mixture was then stirred for 2h, allowed to settle for 30 min, decent the water and collect the bottom insoluble matter to dry until getting constant weight. The soluble crude protein fraction was calculated as the difference between the total crude protein and the insoluble fraction.

Total gas volume in vitro fermentation was measured by the displacement of water level from the receiving bottle suspended in water. The percentage of methane (CH₄) in gas after 24 hours of fermentation was measured using a Crowcon analyzer (Crowcon Instruments Ltd, UK).

The pH measurements were taken immediately after opening the fermentation vessel, measured directly in the well-mixed fermentation broth using a portable pH meter before filtration procedure. The residue DM after fermentation of each in vitro bottle was separated by filtering through cloth and non-absorbent cotton wool. The solid residue was then dried to a constant weight and DM truly solubility was calculated by subtracting the remaining DM from the total initial grams incubated (12 g DM). While filtrated liquid samples were acidified by 1M H₂SO₄ (10:1, vol:vol), centrifuged at 10,000 × g for 15 min. The supernatant was collected and stored at −20 °C for total VFA, molar proportion of acetic acid, propionic acid, and butyric acid as described by Thanh et al (2022).

The data were analyzed with the general linear model (GLM) option in the ANOVA program of the Minitab software (Minitab 18). Sources of variation were treatments and error.

Results

Ammoniated rice straw does improve fiber degradability and supplies non-protein nitrogen (NPN) compared to pure rice straw, but energy and protein needs were still restricted for rumen microbes. Therefore, supplementing Psophocarpus scandens leaves as bypass protein source (with CP up to 27.2 % in DM but low solubility 15.1%) and yeast fermented rice could improve protein and energy demand for fermented rumen.

The effects of PS leaves and/or YFR supplementation on total gas volume and methane production were shown in Table 1. Total gas volume after 24 hours of fermentation did not differ significantly among treatments, but a trend was observed that the combined addition of PS leaves and YFR increased gas volume more than no supplementation or individual supplementation. The presence of PS leaves or combination of PS and YFR in the treatment improved DM solubility percentage rather than YFR and ARS alone. A similar trend was observed with individual PS or a combination of PS and YFR in the ARS diet, showing an effective reduction in methane per unit DM solubilized, while this impact was not unclear by adding 4% YFR.

Although ARS was replaced by YFR at 4%, by PS at 25% and by PS-YFR at 29.1% in the ARS-YFR, ARS-PS, ARS-PS-YFR treatments, the percentage reduction in methane production based on methane per unit DM solubilized of these diets was still higher than the replacement level (Table 2). In which, the difference between methane reduction and % replacement was highest in ARS-PS-YFR (10.2%), followed by ARS-PS (8.7%), but this difference was not significant in ARS-YFR.

Total volatile fatty acid concentrations in Table 3 differed among treatments after 24 hours of fermentation, with the combination of PS leaves and YFR in the ARS diet being highest, followed by ARS-PS, and lowest in the ARS alone and ARS-YFR treatments. Among molar percentages, supplementation of PS, YFR, or their combination in the ARS diet tended to reduce acetic acid content more than that of ARS alone with borderline significance (p = 0.077), while propionic acid was followed in a more enhancing order by ARS-YFR, ARS-PS, ARS-PS-YFR compared to ARS alone. Butyric acid was not affected by treatments. The ratio of acetic acid to propionic acid was positively correlated with methane production (Figure 1), reflecting that propionic acid accumulation is associated with reduced methane production.

Figure 1. Correlation between methane volume per DM solubility (ml/g) and ratio of acetic acid and propionic acid

Discussion

The results appeared to be inversely proportional in the gas volume and methane (ml/g) among treatments. Supplementation with PS leaves or with both PS leaves and YFR resulted in lower methane per unit soluble DM compared to ammoniated rice straw (ARS) alone and ARS plus YFR, while total gas volume tended to be higher. This indicates improved fermentation efficiency with supplementary PS or PS plus YFR to the ARS diet. However, different diets contribute variably to methane emissions per unit of digested dry matter. Therefore, the replacement of ARS in the treatment with YFR and/or PS leaves was evaluated based on the reduction in methane emissions per unit of soluble dry matter, thereby assessing the relative contribution of PS leaves and YFR to lowering methane emissions compared with ARS alone. PS supplementation to the ARS diet appeared to be the main factor in reducing methane emissions (8.7%), while the contribution of YFR to this reduction was unclear. Yeast fermented rice straw (YFR) was expected to release glucose polymers such as beta-glucan, which in turn serve as a substrate for propionic acid production (Phuong et al, 2023), but perhaps YFR was supplemented at low levels (4% of DM basis) in a rice straw-poor diet, so its effects are unclear.

Total VFA was affected by the treatments, in which supplementation with PS leaves or with the PS-YFR combination resulted in higher total VFA compared to ARS alone. VFA accumulation also slightly reduced the pH in ARS-PS and ARS-PS-YFR. This indicates improved fermentation efficiency with the addition of PS or PS-YFR. Individual VFA affects methane production. Jiang et al. (2024) examined methane production at different propionate concentrations (15, 30, and 60 mM) and showed that higher propionate concentrations reduced methane production, and propionate degradation led to acetate accumulation. In the present study, increasing molar percentage of propionic acid along with a decreasing trend of acetic acid explained the lower methane production found in the diets containing PS and the PS-YFR combination. However, the effect of YFR was not evident as propionic acid, acetic acid, ratio of Ac:Pr and pH value (Table 4) showed no difference between PS and PS-YFR or between with YFR and without YFR in ARS diet.

Psophocarpus scandens leaves are considered a source of protein in basal diets using ammoniated rice straw. Mechanistically, soluble protein is rapidly converted to ammonia via hydrogen, thereby reducing the hydrogen sink available for methane formation during in vitro fermentation (Preston et al 2013). However, the low soluble protein content of Psophocarpus scandens leaves (15.1%) contributes to only an 8.7% reduction in methane emissions compared to ammoniated rice straw alone. In terms of in vivo conditions, Psophocarpus scandens leaves represent a potential feed component for sustainable ruminant diets, as they can improve nitrogen utilization efficiency due to their high proportion of rumen-undegradable protein (Thu et al 2025). This characteristic makes them appropriate for inclusion in diets based on low-nutrient by-products such as rice straw.

Conclusion

Supplementation with Psophocarpus scandenin ammoniated rice straw as basal diet reduced 8.7% methane production, lower ratio of acetic acid and propionic acid while higher total VFA concentration compared to using ammoniated rice straw alone.

Supplementation of YFR at 4% in the diet had no clear effect on reducing methane production and volatile fatty acid production compared to ARS alone.

Acknowledgments

The authors acknowledge support for this research from research funding of Nong Lam University, Ho Chi Minh City, Vietnam and equipment from Center for Research and Technology Transfer are acknowledged for providing the facilities to carry out this research.

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