Impact of tropical red seaweed (Palisada perforata) on in vitro methane mitigation and fermentation profiles: The role of forage-to-concentrate ratios

Impacto da alga vermelha tropical (Palisada perforata) na mitigação do metano in vitro e nos perfis de fermentação: O papel das proporções de forragem para concentrado

ABSTRACT

Palisada perforata is one of tropical seaweed that is promising as an additive to reduce rumen methane production, but in total mixed rations (TMR) as a basal feed not reported yet. A randomized factorial block design with two factors (3 x 2) and five replications was applied, time to collect rumen fluid from fistulated Bali cattle as a block. The first factor was the TMR type: TMR 1 (80% roughage, 20% concentrate), TMR 2 (70% roughage, 30% concentrate), and TMR 3 (60% roughage, 40% concentrate). The second factor was the P. perforata enrichment level (0.00 and 5.00% organic matter, OM). Interactions between the TMR type and P. perforata enrichment level were not significant for most variables, except microbial protein synthesis. TMR 3 enriched with 5.00% OM P. perforata resulted in the highest microbial protein synthesis. Methane production was lowest (31.99-36.32%; P < 0.01) in TMR 3, which also exhibited a reduced protozoa population, lower acetate and butyrate proportions, and a reduced acetate-to-propionate ratio, while yielding a higher (P < 0.01) total volatile fatty acid, propionate proportion, and NH₃ concentration. Enrichment with P. perforata at 5.00% OM further decreased methane production, reducing the acetate proportion and acetate-to-propionate ratio while increasing the propionate proportion. In conclusion, a TMR consisting of 60% roughage and 40% concentrate combined with P. perforata at 5.00% OM was the most effective treatment, reducing methane emissions while enhancing rumen fermentation. Dietary P. perforata represents a promising strategy to optimize rumen fermentation efficiency and simultaneously mitigate ruminants’ methane emissions.

Index terms:
Feed efficiency; greenhouse gas emissions; macroalgae additive; rumen microbes; ruminant nutrition; volatile fatty acids.

RESUMO

A Palisada perforata é uma alga tropical promissora como aditivo para reduzir a produção de metano no rúmen, rações totais misturadas (RTM) ainda não foi relatado. Um delineamento em blocos fatoriais casualizados, com dois fatores (3 x 2) e cinco repetições, foi aplicado. As interações entre o tipo de RTM e o nível de enriquecimento com P. perforata não foram significativas para a maioria das variáveis, exceto para a síntese de proteína microbiana. A RTM 3 enriquecida com 5,00% de MO de P. perforata resultou na maior síntese de proteína microbiana. A produção de metano foi menor (31,99-36,32%; P < 0,01) na Ração Total Misturada 3 (RTM 3), que também apresentou uma população reduzida de protozoários, menores proporções de acetato e butirato e uma menor relação acetato/propionato, enquanto apresentou maior (P < 0,01) concentração total de ácidos graxos voláteis, proporção de propionato e concentração de NH₃. O enriquecimento com P. perforata a 5,00% de matéria orgânica (MO) reduziu ainda mais a produção de metano, diminuindo a proporção de acetato e a relação acetato/propionato, enquanto aumentou a proporção de propionato. Em conclusão, uma RTM composta por 60% de volumoso e 40% de concentrado, combinada com P. perforata no nível de inclusão de 5,00% de MO, foi o tratamento mais eficaz, reduzindo as emissões de metano e melhorando a fermentação ruminal. A inclusão de P. perforata na dieta representa uma estratégia promissora para otimizar a eficiência da fermentação ruminal e a síntese de proteína microbiana, mitigando simultaneamente as emissões entéricas de metano em ruminantes.

Termos para indexação:
Eficiência alimentar; emissões de gases de efeito estufa; aditivo de macroalgas; microbiota ruminal; nutrição de ruminantes; ácidos graxos voláteis.

Introduction

Methane (CH₄) production in the rumen represents a major challenge to ruminant productivity because it not only contributes significantly to global greenhouse gas emissions (Króliczewska et al., 2023; Samsudin, 2024) but also reflects an energy loss of approximately 3-12% of gross energy intake (Mayberry et al., 2019). During ruminal fermentation, carbohydrates are converted into volatile fatty acids (VFAs), primarily acetate, propionate, and butyrate, which serve as the main energy sources for the host animal (Zhu et al., 2026). However, methanogenic archaea utilize hydrogen (H₂) and carbon dioxide (CO₂) generated during fermentation to produce CH₄ as a metabolic byproduct, which is a common methanogenesis process, thereby diverting energy that could otherwise be directed toward productive metabolic pathways (Shima et al., 2020; Mauerhofer et al., 2021). Excessive methanogenesis is often associated with lower rumen fermentation efficiency (Guyader et al., 2017) when hydrogen is preferentially used for methane synthesis rather than propionate formation, a more energetically favorable pathway for the animal. Furthermore, rumen efficiency is closely linked to microbial protein synthesis (Badhan et al., 2025), which provides high-quality protein to the host via microbial biomass entering the small intestine. Optimizing rumen fermentation to increase propionate production, enhance microbial protein synthesis, and reduce methane emissions is therefore essential for improving feed utilization efficiency, animal performance, and environmental sustainability in ruminant production systems.

Feed additives, such as vitamins, amino acids, fatty acids, minerals, pharmaceutical compounds, fungal products, and steroidal compounds, are primarily used to decrease CH₄ production and increase animal productivity. These reduce the amount of CH₄ produced (g/day) by either preventing methanogenesis or altering the rumen environment (Honan et al., 2022). Nowadays, seaweed has garnered interest from the public and media as a potential way to lower ruminant enteric CH₄ emissions (Ahmed & Nishida, 2024). Seaweed inclusion in ruminant diets effectively mitigates CH₄ production and enhances their growth rate, feed utilization, and animal productivity (Min et al., 2021). Bromoform and other secondary metabolites (e.g., flavonoids and other phenolic compounds) in seaweed contribute to this effect (Ahmed & Nishida, 2024). Allen et al. (2014) and Costa and Leigh (2014) revealed that methyl coenzyme M reductase and coenzyme M transferase are two important methanogenic enzymes whose activity is inhibited or blocked by bromoform.

The use of red seaweed in ruminants can reduce CH₄ production by 60-90%, and brown and green seaweed reduce CH₄ production by 20% and 45%, respectively, in in vitro studies (Bhowmick & Hayes, 2023). The red seaweed group from the genusAsparagopsis, with speciesA. taxiformisandA. armata,stands out for its potent efficacy in inhibiting methanogenesis. Supplementation at 2-5% organic matter (OM) to basal feed (grass or total mixed ration (TMR)) was able to reduce CH₄ production by 80-98% (Roque et al., 2019; Kinley et al., 2020; Min et al., 2021). Recent research conducted by Mihaila et al. (2022) to determine the effectiveness of other red seaweed species in reducing CH₄ production revealed that the addition of Bonnemaisonia hamifera at 2%, 6%, and 10% OM can reduce enteric CH₄ by 17.1%, 95.4%, and 98.8%, respectively, compared to that of the basal feed (perennial ryegrass) control.

Studies on the effectiveness of seaweed species in reducing CH₄ production remain limited to subtropical seaweed species, and Indonesia is one of the largest tropical seaweed producers in the world (38.7%) after China (47.9%) (Food and Agriculture Organization - FAO, 2018). Erniati et al. (2016) detailed that the seaweed diversity in Indonesia is the largest compared to that of other countries. An expedition conducted by Van Bosse in the Siboga Sea in 1899-1900 reported that Indonesian waters were a source of seaweed germplasm, encompassing approximately 555 species from approximately 8000 species of seaweed globally, that grow well in the Indonesian region (Soetjipto et al., 2019). However, utilizing various tropical seaweed species for ruminant feed supplementation in Indonesia is not common, and thus, few studies have been reported thereon.

The initial research by Hidayah et al. (2024) reported that one of the tropical red seaweed species, Palisada perforata (Bory) K.W. Nam, recovered from Gunungkidul, Yogyakarta Coast, Indonesia, shows potential for use as a feed additive to decrease ruminant CH₄ emissions. This seaweed species has the highest degradable material (crude protein (19.33% dry matter/DM), organic matter (83.47% DM), and nitrogen-free extract (54.15% DM)) and secondary metabolites (phenol (0.80 mg/g DM) and flavonoid (4.26 mg/g DM) compared to other species studied by Hidayah et al. (2023; 2024). Another study, also reported by Hidayah et al. (2025), evaluated this seaweed species as a feed additive to Napier grass. It was found that P. perforata (Bory) K.W. Nam supplementation at 5.00% OM resulted in the greatest CH₄ reduction (83.8% and 74.4%; P < 0.01) and the lowest protozoa count (3.83 × 10⁴/mL). The treatment also increased total VFA and decreased the acetate-to-propionate ratio. This promising result needs to be evaluated further on different basal diets. The use of a basal diet high in concentrate (high in nutrients) will optimize bacterial growth and activity in the rumen. Concentrates consist of non-structural carbohydrates, such as starch, which are rapidly fermented in the rumen, increasing VFA and propionate production (Agle et al., 2010). Propionate is an alternative H₂ absorber, thus reducing CH₄ production (McAllister & Newbold, 2008). Conversely, Ungerfeld (2020) stated that a basal diet high in forage will increase CH₄ production because forage consists of structural carbohydrates (cellulose and hemicellulose) that stimulate acetate and butyrate synthesis, thereby increasing the amount of H₂ available for methanogenesis. (Li et al., 2022) stated that fiber-rich diets favor acetate and butyrate production, while starch-rich diets promote propionate synthesis and reduce methane emissions. Therefore, this study aimed to evaluate the effects of supplementing total mixed ration (TMR) with different concentrate-forage ratios as basal diets with P. perforata (Bory) K.W. Nam on rumen CH₄ production, CH₄ reduction, protozoa population, and fermentation characteristics.

Material and Methods

Experimental design

Rumen fluid collection time was used as a blocking factor in a randomized factorial block design with two factors and five replications employed in this study. The first factor was the total mixed ration (TMR) types based on dry matter (DM): TMR 1: 80% roughage (R) and 20% concentrate (C); TMR 2: 70% R and 30% C; and TMR 3: 60% R and 40% C. The second factor was the P. perforata (Bory) K.W. Nam enrichment level: 0 and 5% OM. The use of this dose is based on the best results from previous studies (Hidayah et al., 2025).

Material preparation

The red seaweed, P. perforata (Bory) K.W. Nam, was collected from Sepanjang Beach, Gunungkidul, Yogyakarta, Indonesia, in October 2022. The seaweed was cleaned and rinsed with water three times to reduce the salt content, sorted to remove other seaweed species, and any sand or other dirt was discarded. Eventually, P. perforata (Bory) K.W. Nam was dried using a freeze dryer (Buchi, Lyovapor, L-200) to preserve bioactive compounds and reduced to a fine powder with an 80-100 mesh size using a hammer mill. The roughage used was Pennisetum purpureum cv. Gama Umami, which was collected from a farm in the Animal Science Faculty of Universitas Gadjah Mada (UGM), Yogyakarta, Indonesia, and dried in an oven at 55 °C. The dehydrated samples (roughage and seaweed) were processed into a powder, placed in sealed plastic bags, and kept at −20 °C until analysis.

The TMR formulation included P. purpureum cv. Gama Umami as roughage and concentrate as the energy-protein source. Total mixed ration had the following nutrient content: 10.69% crude protein (CP) and 69.93% total digestible nutrients (TDN) based on DM. There were iso total digestible nutrients and iso crude protein in the TMR nutrient content. The concentrate composition and nutrient profiles of the experimental TMRs with P. perforata (Bory) K.W. Nam enrichment are shown in Tables 1 and 2, respectively. Nutrient content was analyzed using the (AOAC - Association of Official Analytical Chemists, 2005).

Table 1:
Formula and nutrients of the concentrate and roughage on a dry matter basis.

Ingredients (%)	C1	C2	C3	Nutrient (%)	C1	C2	C3	R
Rice brand	11.25	19.25	23.25	OM	82.64	82.32	82.16	84.15
Wheat pollard	40	40	40	Ash	16.36	16.68	16.84	15.85
Soybean meal	18.25	10.25	6.25	CP	21.13	17.76	16.08	11
Coconut cake meal	14.50	14.50	14.50	EE	5.97	6.12	6.20	1.85
Corn gluten feed	10	10	10	CF	7.38	8.24	8.67	34.26
Molasses	5	5	5	NFE	56.92	58.64	59.50	37.04
Premix	1	1	1	TDN*	82.13	80.94	80.34	52.95

• C1: Concentrate 1, C2: Concentrate 2, C3: Concentrate 3, R: Roughage, OM: Organic matter, CP: Crude protein, EE: Ether extract, CF: Crude fiber, NFE: Nitrogen free extract, TDN: Total digestible nutrient, NFE = 100 - (% CP + % CF + % EE + % ash), TDN = 70.6 + 0.259 CP + 1.01 EE - 0.76 CF + 0.091 NFE (Sutardi, 1980).

Table 2:
Nutrient of different total mixed rations with P. perforata (Bory) K.W.Nam supplementation in dry matter basis.

Nutrient / P. perforata (Bory) K.W.Nam	TMR 1		TMR 2		TMR 3
	0% OM	5% OM	0% OM	5% OM	0% OM	5% OM
OM	87.28	87.10	86.68	86.83	87.93	87.88
Ash	12.72	12.90	13.32	13.17	12.07	12.12
CP	12.62	12.94	12.04	12.16	12.59	13.06
EE	2.14	3.02	2.37	2.90	2.60	2.99
CF	27.25	27.38	27.55	28.08	27.13	27.72
NFE	45.27	43.75	44.72	43.69	45.61	44.11
TDN*	59.81	60.53	59.61	59.67	60.39	60.31

• TMR: total mixed ration, C: Concentrate, R: Roughage, TMR 1: 80% R + 20% C, TMR 2: 70% R + 30% C, TMR 3: 60% R + 40% C, OM: Organic matter, CP: Crude protein, EE: Ether extract, CF: Crude fiber, NFE: Nitrogen free extract, TDN: Total digestible nutrient, NFE = 100 - (% CP + % CF + % EE + % ash), TDN = 70.6 + 0.259 CP + 1.01 EE - 0.76 CF + 0.091 NFE (Sutardi, 1980).

In vitro evaluation

Five runs of the in vitro experiment included six treatments performed in duplicate, with two blanks and two standards. The running time was set to a block to reduce variation between runs that may be caused by variations in rumen fluid intake. The rumen fluid was collected before the morning feeding of two rumen-fistulated Bali cattle (bull and cow), and the sample fluid was filtered through cheesecloth (four layers). Fistulated-Bali cattle diets based on Pennisetum purpureum cv. Gama Umami and wheat pollard (ratio 60:40% DM) were fed to fistulated cattle twice a day (08.00 and 16.00 WIB) with water ad libitum. The procedure to collect rumen fluid was reviewed and approved by the Committee of Animal Ethics from the Faculty of Veterinary Medicine, UGM, Yogyakarta, Indonesia (No. 052/EC-FKH/Eks./2022). The rumen fluid sample was combined with a buffer solution at a 1:2 v/v ratio for a total of 30 mL and 200 mg of substrates according to the treatments. All in vitro procedures were performed according to the method detailed by Menke and Steingass (1988).

Samples of methane (CH₄) production were taken from the gas produced (10 mL in a vacuum tube) at 24 and 48 h of incubation. The sample was measured using gas chromatography (GC 14B, Shimadzu Corp., Kyoto, Japan) with an FID detector and Paropak column (50.0 m × 0.20 mm × 0.30 μm) according to the method described by Fievez et al. (2005). Calculation model for methane analysis ((internal standard concentration x standard peak area)/ methane sample peak area)). The fermentation characteristics (pH, volatile fatty acid/VFA, ammonia/NH₃, and rumen microbial protein) and number of protozoa were measured from rumen fluid samples after 72 h of incubation. Every rumen fluid sample, aside from the pH and protozoa samples, was put into a 1.50 mL microtube, centrifuged at 3000 rpm for 15 minutes, and stored in a freezer until further analysis.

Rumen fermentation characteristics were evaluated in vitro following standard procedures. The pH of the rumen fluid was measured using a portable pH meter (Hanna Instruments, USA). VFA was determined by gas chromatography (GC 2010 Plus, Shimadzu Corp., Kyoto, Japan) equipped with an HP-FFAP column (50.0 m × 0.20 mm × 0.30 μm) and an FID detector, and n-propanol for internal standard, according to the method described by Cottyn and Boucque (1968). The NH₃ was measured using a UV-Vis microplate spectrophotometer set at 750 nm, with a standard curve prepared from (NH₄)₂SO₄ (Merck; y = 0.028018x − 0.01108, r² = 0.99) according to the method detailed by Chaney and Marbach (1962). Microbial protein concentration was determined using the same spectrophotometer at 630 nm, with a standard curve prepared from bovine serum albumin (BSA, Sigma; y = 2.5075x + 0.0824, r² = 0.99) following the method outlined by Plummer (1967). Meanwhile, the number of protozoa cells was calculated using the Diaz et al. (1993) method.

Data analysis

The data were analyzed using ANOVA, and the different treatment means were examined with the Duncan multiple range test. This was analyzed using statistical software package IBM SPSS Statistics version 26 with GLM procedure for mathematical equation model.

Results and Discussion

Methane production, reducing methane, and protozoa population

No significant interaction was observed between the TMR types and the level of P. perforata (Bory) K.W. Nam enrichment on methane (CH₄) emission, CH₄ reduction, and protozoa population. The lowest (P < 0.01) CH₄ production occurred in the highest concentrate proportion (total mixed ration/TMR 3), followed by the protozoa population (Table 3). Adding the highest concentrate proportion (TMR 3) contributed to reducing (P < 0.01) CH₄ production by 31.99-36.32% from that of TMR 1 at 24 and 48 h of incubation (Figure 1). Meanwhile, P. perforata (Bory) K.W. Nam supplementation at 5.00% organic matter (OM) produced lower CH₄ than that without P. perforata (Bory) K.W. Nam inclusion. This supplementation reduced (P < 0.01) CH₄ production by 27.94% in TMR 1 and 26.60% in TMR 3 (Figure 2).

Table 3:
Methane production and protozoa population with Palisada perforata (Bory) K.W. Nam enrichment on the total mixed ration.

Type of TMR	Levels of P. perforata (Bory) K.W.Nam	Methane (CH4) production				Protozoa population (Cell x 104/ml)
		24 hours (ml)	48 hours (ml)	24 hours (ml/g DM)	48 hours (mL/g DM)
Interaction of TMR type and Palisada perforata (Bory) K.W.Nam enrichment levels
TMR 1	0.00% OM	5.25	6.35	26.87	32.37	6.12
	5.00% OM	3.99	5.28	20.59	27.26	5.90
TMR 2	0.00% OM	4.39	5.35	22.17	26.99	4.50
	5.00% OM	3.71	4.51	19.10	23.20	4.37
TMR 3	0.00% OM	3.56	4.37	18.80	23.10	3.15
	5.00% OM	2.83	3.60	14.19	18.08	4.64
SEM		0.87	1.02	4.36	5.07	1.48
p-value		0.27	0.79	0.24	0.78	0.07
Type of TMR effect
TMR 1		4.62c	5.82c	23.73c	29.82c	6.01b
TMR 2		4.05b	4.93b	20.63b	25.10b	4.44a
TMR 3		3.19a	3.99a	16.49a	20.59a	3.90a
p-value		0.00	0.00	0.00	0.00	0.00
Levels of Palisada perforata (Bory) K.W.Nam enrichment effect
0.00% OM		4.40b	5.36b	22.61b	27.49b	4.59
5.00% OM		3.51a	4.46a	17.96a	22.85a	4.97
p-value		0.00	0.00	0.00	0.00	0.25

• Different superscripts in the same column show very significant differences (P<0.01); TMR: total mixed ration; DM: dry matter; OM: organic matter; TMR 1: 80% R (Roughage) + 20% C (Concentrate); TMR 2: 70% R + 30% C, TMR 3: 60% R + 40% C.

Figure 1:
Reduced methane production compared to that of from total mixed ration (TMR) 1. TS0: TMR 2 with no Palisada perforata (Bory) K.W. Nam seaweed enrichment, TS1:TMR 3 with no P. perforata (Bory) K.W. Nam seaweed enrichment, TS2: TMR 2 with 5.00% P. perforata (Bory) K.W. Nam seaweed enrichment, TS3: TMR 3 with 5.00% P. perforata (Bory) K.W. Nam seaweed enrichment.

Figure 1:
Reduced methane production compared to that of from total mixed ration (TMR) 1. TS0: TMR 2 with no Palisada perforata (Bory) K.W. Nam seaweed enrichment, TS1:TMR 3 with no P. perforata (Bory) K.W. Nam seaweed enrichment, TS2: TMR 2 with 5.00% P. perforata (Bory) K.W. Nam seaweed enrichment, TS3: TMR 3 with 5.00% P. perforata (Bory) K.W. Nam seaweed enrichment.

The increasing concentrate proportion shifted the rumen fermentation pattern with increasing propionate and decreasing acetate butyrate proportions (Table 4). Concentrates that are comprised of non-structural carbohydrates, such as starch, raise the propionate concentration (Agle et al., 2010). Newbold et al. (2005) stated that the propionate uptake of H₂ elevates metabolic energy and serves as a precursor for glucose and lactose. McAllister and Newbold (2008) also explained that propionate is an alternative H₂ sink to methanogenesis because its production requires reducing equivalents. Thus, the CH₄ production decreased. Meanwhile, the forage consists of structural carbohydrates, such as cellulose and hemicellulose, thereby promoting the synthesis of acetate and butyrate, which increases the amount of H₂ available for methanogenesis (Ungerfeld, 2020).

Table 4:
Total and partial volatile fatty acids in the total mixed ration with Palisada perforata (Bory) K.W. Nam enrichment.

Type of TMR	Levels of P. perforata (Bory) K.W.Nam	VFA total (mM)	Acetate (%)	Propionate (%)	Butyrate (%)	Acetate / Propionate
Interaction of TMR type and Palisada perforata (Bory) K.W.Nam enrichment levels
TMR 1	0.00% OM	25.61	73.41	14.18	12.40	5.20
	5.00% OM	26.28	69.83	16.11	14.07	4.40
TMR 2	0.00% OM	47.35	70.23	21.45	8.32	3.29
	5.00% OM	48.66	65.83	25.84	8.34	2.58
TMR 3	0.00% OM	55.80	66.02	26.11	7.87	2.54
	5.00% OM	56.64	63.51	29.03	7.46	2.19
SEM		13.61	3.93	5.78	2.77	1.17
p-value		0.98	0.63	0.38	0.10	0.46
Type of TMR effect
TMR 1		25.95a	71.62c	15.15a	13.24c	4.80c
TMR 2		48.00b	68.03b	23.65b	8.33b	2.93b
TMR 3		56.22c	64.76a	27.57c	7.66a	2.37a
p-value		0.00	0.00	0.00	0.00	0.00
Levels of Palisada perforata (Bory) K.W.Nam enrichment effect
0.00% OM		42.92	69.89b	20.58a	9.53	3.68b
5.00% OM		43.86	66.37a	23.66b	9.95	3.06a
p-value		0.51	0.00	0.00	0.30	0.00

• Different superscripts in the same column show very significant differences (P<0.01); TMR: total mixed ration; OM: organic matter; VFA: volatile fatty acid; TMR 1: 80% R (Roughage) + 20% C (Concentrate); TMR 2: 70% R + 30% C, TMR 3: 60% R + 40% C.

The high starch content in the concentrate is rapidly fermented in the rumen, resulting in high volatile fatty acid (VFA) (Table 4) and lactate concentrations. Under these conditions, protozoal numbers are reduced and rumen methanogen growth is suppressed, which restricts the flow of hydrogen from protozoa to methanogens (Van Kessel & Russell, 1996; Williams & Coleman, 1988). The decreased protozoa numbers allow the bacteria population to increase (Table 4). Protozoa have a predative effect on rumen bacteria to obtain protein (Newbold et al., 2015). Singh et al. (2014) reported that CH₄ emission (L/day and L/day/kg W^0.75) and methane energy (kcal/day) in crossbred cattle and buffaloes were significantly higher in feed that included a lower concentrate proportion. Meta-analysis results reported by Spanghero et al. (2022), correlation coefficient of R² = 0.604 between reducing methane production and decreased protozoa number, both in absolute terms. The regression study predicts that the complete removal of protozoa results in a reduction of about 33 % of methane yields.

In this study, increasing the TMR concentrate proportion from 20% to 40% reduced CH₄ production by 31.99-36.32%. Van Wyngaard et al. (2018) reported that increasing concentrate levels from 0 to 8 kg/day in grazing dairy cows reduced CH₄ production from 20.4 to 15.9 g of CH₄/kg of milk yield. A similar result was reported by Van Gastelen et al. (2019), who reviewed 24 studies to examine the effects of lowering the forage-to-concentrate ratio. This revealed that CH₄ yield was reduced by 26% in beef cattle, 14% in dairy cattle, and 6% in sheep when the concentrates were increased by an average of 386 g/kg dry matter (DM). For every animal type, the CH₄ intensity was also reduced (31% for beef cattle, 27% for dairy cattle, and 10% for sheep).

Supplementing the TMR with P. perforata (Bory) K.W. Nam at 5.00% OM significantly decreased CH₄ production. This supplementation reduced CH4 production by 27.94% in TMR 1 and by 26.60% in TMR 3 (P < 0.01). This is related to the seaweed’s phenolic compounds and might be due to the presence of halogenated compounds in the species. Hidayah et al. (2024) reported that P. perforata (Bory) K.W. Nam contains phenols, tannins, and flavonoids at 0.80, 0.76, and 4.26 mg/g DM, respectively. Beauchemin et al. (2020) stated that ruminant CH₄ production might be inhibited by phenolic compounds, which can affect ruminal fermentation. Moreover, phenolic compounds have anti-methanogenic qualities that either directly or indirectly target the methanogens and protozoa with which some methanogens are associated.

Tannins can directly inhibit methanogens to reduce CH₄ production and indirectly reduce fiber digestion (Beauchemin et al., 2020). Bhatta et al. (2009) reiterated that hydrolyzable tannins could change rumen fermentation. It can decrease the proportion of butyric and acetic acids while increasing the proportion of propionic acid. Meanwhile, flavonoids, such as quercetin, have strong anti-methanogenic activity, which can prevent the synthesis of the nucleic acid cell wall and the function of the methanogen cytoplasmic membrane (Patra, 2012). Furthermore, quercetin reduced the total methanogen and protozoa numbers (Oskoueian et al., 2013).

Halogen compounds (such as bromoform) are only present in seaweed and not in terrestrial plants (Mihaila et al., 2022), are anti-methane substances that can prevent the MCR (methyl-coenzyme reductase) enzyme from functioning during methanogenesis (Allen et al., 2014). Increased VFA production will result from a decrease in CH4 production, which will increase energy formation (Choi et al., 2022). According to recent studies, seaweed’s metabolite content, particularly the halogen compound, effectively lowers CH4 emissions in ruminants (Min et al., 2021; Abbott et al., 2020; Kinley et al., 2020; Almeida et al., 2021).

Fermentation characteristics

No significant interaction was observed between the TMR types and the P. perforata (Bory) K.W. Nam enrichment level on all fermentation characteristic parameters, except protein microbial synthesis. The highest protein microbial synthesis occurred when TMR 3 was combined with 5.00% OM P. perforata (Bory) K.W. Nam. The TMR 3 with the highest concentrate proportion also had the highest (P < 0.01) total volatile fatty acid (VFA), propionate proportion, and ammonia (NH₃) concentration, followed by the lowest acetate and butyrate proportion, as well as the lowest acetate-to-propionate ratio.

Enrichment with P. perforata (Bory) K.W. Nam at 5.00% OM reduced (P < 0.01) the acetate proportion and acetate-to-propionate ratio and increased the propionate proportion. Combining the highest concentrate proportion (40% in the TMR formula as a basal diet) with 5.00% OM P. perforata (Bory) K.W. Nam supplementation acted synergistically to facilitate the highest microbial protein synthesis. The high proportion of concentrate provided a high proportion of fermentable carbohydrates and available protein for degradation. This was revealed by the highest VFA total and NH₃ concentration (Tables 4 and 5). Bezerra et al. (2025) stated that synchronizing fermentable carbohydrates and available protein for degradation ensures that rumen microbes have simultaneous access to both energy and nitrogen, improving nitrogen capture efficiency and reducing ammonia escape, promoting more efficient utilization of rumen ammonia nitrogen. Meanwhile, seaweed provides mineral sulfur and phosphorus that influence microbial growth. Hidayah et al. (2024) reported that P. perforata (Bory) K.W. Nam contains sulfur and phosphorus at 0.81 and 0.09% DM, respectively, and the combination of these materials promotes microbial protein synthesis optimization.

Table 5:
pH, NH₃, and microbial protein synthesis with Palisada perforata (Bory) K.W.Nam enrichment on some total mixed rations.

Type of TMR	Levels of P. perforata (Bory) K.W.Nam	pH	NH3 (mg/100 mL)	Microbial protein synthesis (mg/100 mL)
Interaction of TMR type and Palisada perforata (Bory) K.W.Nam enrichment levels
TMR 1	0.00% OM	6.93	15.08	122.92ab
	5.00% OM	6.97	14.43	127.39b
TMR 2	0.00% OM	6.99	16.05	129.25b
	5.00% OM	6.97	17.38	116.97a
TMR 3	0.00% OM	6.97	20.02	144.11c
	5.00% OM	6.95	18.99	152.73d
SEM		0.13	2.38	13.96
p-value		0.25	0.13	0.00
Type of TMR effect
TMR 1		6.95	14.76a	125.16
TMR 2		6.98	16.71b	123.11
TMR 3		6.96	19.51c	148.42
p-value		0.41	0.00	0.00
Levels of Palisada perforate (Bory) K.W.Nam enrichment effect
0.00% OM		6.96	17.05	132.10
5.00% OM		6.96	16.93	132.36
p-value		0.97	0.82	0.91

• Different superscripts in the same column show very significant differences (P<0.01); TMR: total mixed ration; OM: organic matter; NH₃: ammonia; TMR 1: 80% R (Roughage) + 20% C (Concentrate); TMR 2: 70% R + 30% C, TMR 3: 60% R + 40% C.

Hildebrand et al. (2012) reported that diets with a higher concentrate proportion generally enhance the efficiency of microbial crude protein synthesis compared to that of diets with a lower concentrate proportion. Similar results were reported byOlafadehan et al. (2016), where the ruminal pH, NH₃-N, microbial protein supply, and total VFA increased linearly (P < 0.05) with decreasing forage proportions in the diet. The basal diet consisted of timothy hay (60%) and corn grain (40%) on a DM basis, and this was supplemented with 0.25 mg/mL of red seaweed (Amphiroa anceps, Asparagopsis taxiformis, Chondracanthus tenellus, Grateloupia elliptica, and Gracilaria parvispora) extracts, which tended to increase the propionate proportion and decrease the acetate-to-propionate ratio (Choi et al., 2022).

The best dose (5.00% organic matter, OM) of Palisada perforata (red tropical seaweed) supplementation in this research was higher than Asparagopsis taxiformis and Asparagopsis armata (red subtropical seaweed, dose at 0.50-2.00% OM) (Kinley et al., 2020; 2022); Ahmed and Nishida (2024); Mihaila et al. (2022) and lower than Bonnemaisonia hamifera (red subtropical seaweed, dose at 6.00-10.00% OM) (Mihaila et al., 2022). This is because these seaweed different kinds and percentages of bioactive content that contribute to decreased methane production. Using this seaweed as a supplementation to in vivo trial is a challenge due to the palatability.

Conclusions

In conclusion, the 60:40 forage-to-concentrate ratio provided the optimal substrate environment for rumen fermentation when supplemented with 5.00% organic matter (OM) of P. perforata. This combination significantly suppressed methane production and protozoa populations while enhancing microbial protein synthesis by more efficiently utilizing ammonia. These results suggest that P. perforata has strong potential as a functional feed additive to improve rumen efficiency and mitigate environmental impact. However, further in vivo studies are required to confirm these effects on animal performance, palatability, and long-term metabolic health.

Acknowledgment

The Indonesian Education Scholarship (BPI), the Center for Higher Education Funding and Assessment (PPAPT) Ministry of Higher Education, Science, and Technology of Republic of Indonesia, and the Indonesian Endowment Funds for Education (LPDP) all contributed funds to this study.

Data Availability Statement

Data available upon request to authors.

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Editor de seção:

Renato Paiva http://orcid.org/0000-0001-5107-0285

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Corresponding author: [e-mail]

Publication Dates

• Publication in this collection
  17 Aug 2026
• Date of issue
  2026

History

• Received
  29 Mar 2026
• Accepted
  15 July 2026