The effectiveness of dried seaweed Eucheuma cottonii on in vitro rumen fermentation and enteric methane production

A eficácia da alga marinha Eucheuma cottonii na fermentação ruminal in vitro e na produção de metano entérico

Abstract

Seaweed plays a significant role in reducing green-house gasses (GHG) emitted by ruminant animals, in particular methane gas (CH₄) generated during rumen fermentation of the feed. Euchaema cottonii is one of potential cultivated seaweed in tropical country. A preservation procedure is necessary for further application in CH₄ reduction program. The study looked at how three drying methods (sun drying, oven drying, and freeze drying) affected the E.cottonii ability in reducing CH₄ methane as measured by an in vitro technique. The addition of dried E. cottonii up to 4% dry matter (DM) was tested using rice straw diets mixed with concentrate in the ratio of 70%:30% in DM base. Measurements was conducted on gas production, degradability and end product of rumen fermentation during 48-hour incubation using a gas production system and ANKOM Daisy II techniques. Overall, the three dried products of E. cottonii found to considerably lower production of CH₄, increased DM and OM degradability as well as total VFA production (P<0.05). The three treatments have no effect on NH₃ concentration and the bacterial population, but decreased protozoa population (P<0.01). The lowering of CH₄ gas production in the rumen is impacted by all three drying methods in the same way. It is concluded that dried E.cottonii addition up to 4% DM effective in depressing enteric CH₄ production, without causing negative effect on DM and OM degradability as well as did not depress total VFA formation and bacterial activities. The three different drying-methods have similar effect on reducing CH₄ production, with the most affective is freeze-drying method

Keywords:
seaweed; Euchaema cottonii; enteric methane; mitigation; degradability

Resumo

As algas desempenham um papel significativo na redução dos gases de efeito estufa (GEE) emitidos pelos ruminantes, em particular o gás metano (CH₄) gerado durante a fermentação ruminal do alimento. Euchaema cottonii é uma espécie de alga potencialmente cultivada em países tropicais. Um procedimento de conservação é necessário para uma aplicação futura no programa de redução de CH₄. O estudo analisou como três métodos de secagem (secagem solar, secagem em forno e secagem por congelamento) afetaram a capacidade de E.cottonii em reduzir o metano CH₄, medida por uma técnica in vitro. A adição de E. cottonii seco até 4% de matéria seca (MS) foi testada usando dietas de palha de arroz misturadas com concentrado na proporção de 70%:30% na base de MS. Foram realizadas medições da produção de gás, degradabilidade e o produto final da fermentação ruminal durante 48 horas de incubação usando um sistema de produção de gás e técnicas da ANKOM Daisy II. No geral, os três produtos secos de E. cottonii foram encontrados para reduzir consideravelmente a produção de CH₄, aumentar a degradabilidade de MS e MO, bem como a produção total de VFA (P<0.05). Os três tratamentos não tiveram efeito na concentração de NH₃ e na população bacteriana, mas diminuíram a população de protozoários (P<0.01). A redução da projeção de gás CH₄ no rúmen foi afetada por todos os três métodos de secagem da mesma forma. Conclui-se que a adição de E.cottonii até 4% de MS é eficaz na redução da produção de CH₄ entérico, sem causar efeito negativo na degradabilidade de MS e MO, bem como não reduziu a formação total de VFA e atividades bacterianas. Os três diferentes métodos de secagem têm efeito semelhante na redução da produção de CH₄, sendo o método de secagem por congelamento o mais eficaz.

Palavras-chave:
alga marinha; Euchaema cottonii; metano entérico; mitigação; degradabilidade

1. Introduction

The world’s need for meat and milk is predicted to increase by about 58% as well as 73% by 2050 significantly from 2010 levels (Beauchemin et al., 2020). The United Nations (UN) predicts that the worldwide population will grow to 9.8 billion in 2050 and 11.2 billion in 2100 as well as expects to see a corresponding rise in the need for meat and milk products of 1.04 million tones and 465 million tones, respectively (Tseten et al., 2022) because of population growth, the emergence of a middle class, rising incomes, and urbanization. However concerns about climate change are raised by the development of animal husbandry, which raises atmospheric concentrations of greenhouse gases (GHG) (Beauchemin et al., 2020). Nevertheless, a worldwide contribution of between 7 and 18% of all anthropogenic GHG emissions is commonly acknowledged (Tapio et al., 2017). Anthropogenic GHG emissions have increased considerably in recent years owing mostly to waste management, transportation, energy generation, industrial operations, and agriculture. Such gases hold thermal energy, raising global temperatures, and are, hence, the primary contributors to global warming. Moreover, under a situation of very high GHG emissions, temperatures might increase by up to 5.7 °C worldwide at the end of the 21^st century compared to temperatures in the years 1850-1900 (Subedi et al., 2022).

Ruminant animals play a vital role in efficient agricultural systems; however, they also contribute greatly to GHG emissions. Methane (CH₄) is the major GHG released by ruminants, with intestinal fermentation accounting for more than 90% of total emissions (Doyle et al., 2019). Enteric CH₄ from ruminant accounts for approximately 6% of worldwide anthropogenic GHG emissions and 40% of total livestock emissions. Moreover, ruminal CH₄ generation leads to a waste of energy ranging from 2 to 12% of the total amount of calories consumed (Tseten et al., 2022; Tapio et al., 2017). Methane is a GHG that has a 28-fold greater chance of causing global warming than carbon dioxide (CO₂) (Tapio et al., 2017). Methane is a prime candidate for short-term global warming mitigation owing to its shorter half-life in the atmosphere than CO₂, which is 8.6 years. Furthermore, ruminants provide around 25% of the world's enteric CH₄ output, with dairy cattle making up about 24.8% of it or 0.54% of all GHG emissions (Judy et al., 2019).

The rumen, where a community of methanogenic archaea transforms the H₂ and CO₂ formed by a complex ecosystem of bacteria, anaerobic fungi, and ciliate protozoa is thought to be the source of more than 87% of the CH₄ produced by sheep (Tapio et al., 2017). Numerous studies have been undertaken to evaluate the possibility of GHG mitigation measures, including genetics, plant extracts, chemical supplements, and nutritional feed additives, and to inhibit methanogenesis. Several studies have described the benefits of plant secondary compounds, including essential oils, saponin, and tannins, as alternative feed additions to modulate ruminal fermentation, and antimicrobial activity to enhance animal productivity and decrease CH₄ generation (Min et al., 2021).

Numerous methods for decreasing enteric CH₄ emissions have been introduced in recent decades, with an emphasis on aspects like management, genetic factors, and animal nutrition. Among these approaches, the most effective strategy involves enhancing animal productivity. For instance enhancing the proportion of legumes and grains in the diet, along with adding lipid and oil supplements to the feed, leads to a decrease in CH₄ production within the rumen (Vijn et al., 2020; Widiawati and Hikmawan, 2021). In addition, the utilization of additives in animal feed, such as 3-nitrooxypropanol, has proven capable of diminishing emissions in both dairy and beef cattle by as much as 30% (Vijn et al., 2020). This chemical 3-NOP inhibits the enzyme methyl-coenzyme M-reductase (MCR), which catalyzes the last step of rumen archaea methanogenesis (Roque et al., 2021).

Seaweeds are being researched for their capacity to lower CH₄ emissions in the diet of ruminants (Abbott et al., 2020). Seaweeds colonize aquatic environments that are mostly consumed by coastal communities. A total of 10,100 species of seaweed have been identified on a global scale, and they inhabit various marine environments, with certain types of seaweed even thriving in freshwater habitats (Morais et al., 2020). Seaweeds serve as valuable alternative food options for livestock, mainly providing advantageous nutrients. These include chelated microminerals, which are more easily accessible than inorganic forms, prebiotic functions with complex carbohydrates and pigments along health-beneficial polyunsaturated fatty acids that positively impact consumer health (Makkar et al., 2016).

Several kinds of microalgae (seaweed) consisting of brown (Phaeophyceae), green (Chlorophyta) and Red (Rhodophyta) (Cheong et al., 2023) have been shown to reduce CH₄ generation, although with notable variations in outcomes (Vijn et al., 2020). Significant reduction in CH₄ production has been observed in vitro in certain brown seaweed species like Cystoseira trinodis and Dicotya bartayresii, as well as green seaweed species within the Ulva family. This also led to decreased overall gas production. Except for Asparagopsis, the potential of red seaweeds to inhibit CH₄ production has received limited exploration.

Red seaweeds produce a variety of halogenated secondary compounds, some of which are potential candidates for exerting anti-methanogenic effects (Mihaila et al., 2022). Nevertheless, there has been recent research on the ability of red seaweeds, notably Asparagopsis taxiformis, to mitigate CH₄ emissions (Abbott et al., 2020) acquired utilizing the bioactive bromoform present in the red macroalgae (seaweed) known as A. taxiformis, serving as an additive for animal feed (Nilsson and Martin, 2022). Furthermore, a study conducted in Indonesia suggests that certain types of seaweed mainly cultivated along the coast on many islands in Indonesia is E. cottonii, which contains the hydrocolloid carrageenan and bioactive bromoform. This substance has the potential to reduce CH₄ production within the ruminants (Widiawati and Hikmawan, 2021). Since A. taxiformis rarely cultivated in Indonesia, then the purpose of this study was to investigate the impact of E. cottonii to mitigate enteric CH₄ emissions on ruminants tested by in vitro method. Research on E. cottonii is currently restricted, and further investigation is necessary to explore into the potential bioactive compounds within E. cottonii that could effectively reduce CH₄ emissions in ruminants. Moreover, Indonesia is a potential coastal country for developing seaweed as feed and preventing the issue of global warming. Simple processing technology to get seaweed material into feed and prevent the issue of global warming is important. This research is a development of existing research, but can contribute to science and appropriate technology for the development of functional feed.

The moisture level of fresh seaweed varies, with a high concentration of approximately 80%. There are some issues with time, technical storage, and quality stability when using fresh seaweed as an ingredient in the feed industry. Seaweed's shelf life can be extended and its ability to withstand deterioration and fungal growth inhibited by drying it. Seaweed has been dried using a variety of methods in the food sector, such as sun, freeze, and oven drying (Kadam et al., 2015). Heat-sensitive bioactive chemicals and nutrient content may be adversely affected by this technique. The seaweed's advantageous phenolic chemicals may be diminished by these drying methods (Badmus et al., 2019). Although the three drying methods have been used for seaweed in the food sector, there hasn't been much research done on the use of dried seaweed in the feed industry. Consequently, more research is required to determine the optimal method for drying E.cottoni and its effect on the potential to lower intestinal CH₄ emissions from ruminants.

2. Methodology

2.1. Sample collection and preparation

Seaweed E. cottonii samples were obtained from Lontar village in Serang City, Banten, Tangerang, Indonesia, an area renowned for seaweed cultivation. Following the harvest, the seaweeds were meticulously placed into individual polythene bags, each weighing approximately 1 kg (1000 grams). These bags were subsequently stored in a freezer maintained at -4 degrees Celsius within the Genomic Laboratory of BRIN, Indonesia. The E. cottonii seaweed was then subjected to three distinct drying methods: oven drying, sun drying, and freeze drying.

For the oven-drying process, the seaweed samples were subjected to incubation in ovens at 60 °C for a duration of 2-3 days until they achieved complete dryness. This procedure was repeated in multiple cycles to yield a sufficient quantity of material required for subsequent extraction processes. In the case of sun-dried seaweed samples, they were exposed to natural sunlight until they reached the desired level of dryness.

Freeze-drying samples involved weighing 5 plastic boxes, each containing 50 grams, and one polythene bag containing 30 grams. These seaweed portions were stored at -80 °C. The dried seaweeds obtained from sun drying, freeze drying, and oven drying were subsequently ground into a fine powder, filtered through a 1mm sieve, and preserved in the freezer for future use.

2.2. Donor animal

The procedure of study conducted followed the animal ethics approval number 193/KE.02/SK/10/2023 issued by The National Research and Innovation Agency's Commission Ethics for Maintenance and Animal Used for Research.

Rumen inoculum was obtained from two male crossbred Ongole cattle, which were cannulated ruminally. The cattle were kept in separate pens at the research facilities of Research Centre for Animal Husbandry at National Research and Innovation Agency. Fresh natural grass and concentrate made up the cattle's diet. The animals had unrestricted access to drinking water every day. The daily meal was split into two equal portions and served twice a day, at roughly 8:00 in the morning and 2:00 in the afternoon.

According to the Nutrient Requirement in Beef Cattle Nutrition Series-2018, the ration's nutrients was calculated to meet the maintenance needs of male beef cattle, comprising 11.7% crude protein (CP), 63.7% total dry matter (DM), NEm 0.335 Mcal/kg, Calsium (Ca) 0.49%, and Phosphorus (P) 0.24%, on DM basis.

2.3. Preparation of rumen inoculum

Before the morning feeding time, the whole contents of each beef cattle’s rumen were gathered in the early morning. Within 30 minutes of being collected, they were combined, put into a thermos that had been preheated, and then brought to the laboratory. The entire contents of the rumen were blended for around three minutes in the laboratory, and the filtrate, which would be utilized as the inoculum, was then collected by passing it through two layers of cheesecloth. After that, the filtrate was placed in an Erlenmeyer and flushed with CO₂ gas to maintain the inoculum in an anaerobic state while it was maintained at 39 °C in a water bath.

2.4. In vitro studies

Two techniques were used for the in vitro incubation: first, tight gas culture bottles were used in accordance with (Paya et al., 2012; Theodorou et al., 1994) to collect CH₄ and total gas production, as well as to measure the digestibility of dry matter (DM) and organic matter (OM), as well as the generation of NH₃ and VFA and the microbial population. An ANKOM Daisy II incubator was used as the second method to assess the digestibility of neutral detergent fiber (NDF). The basal diet for the in vitro incubation was total mixed ratio (TMR), which was composed of concentrate and rice straw in a 70:30 ratio.

The four treatments for the Theodorou’s in vitro method were as follows: Control (TMR + no seaweed); oven-dried (TMR 0.5 g + oven-drying seaweed powder 0.02 g); sun-dried (TMR 0.5 g + sun-drying seaweed powder 0.02 g); and freeze-dried (TMR 0.5 g + freeze-drying seaweed powder 0.02 g). Every treatment was duplicated five times, and two blank bottles were utilized in place of TMR. Twenty-two treatments, comprising two blankos, were filled with a 50 mL mixture of rumen fluid and McDougall's buffer solutions. The bottles were then incubated in a water-bath at 39 °C for approximately 48 hours (Bekele et al., 2022).

At 24 and 48 hours into the incubation period, the gas was measured. Glass syringes fitted with 21G needles were used to measure the total amount of gas generated by inserting them into the empty portions of bottles. The entire amount of gas produced was recorded using the gas volume collected in the syringes. The entire collected gas was then put into a 10-milliliter vacuum vial bottle using a needle fitted with three-way taps. All vial bottles containing gas were kept in the freezer during the incubation period in order to analyze the concentration of CH₄.

Following a 48-hour incubation period, a liquid sample was taken out of each bottle and placed into a unique centrifuge tube. The tubes were then spun for 10 minutes at 3000 rpm. There were two repetitions of the centrifugation procedure. A volume of roughly 5 milliliters per set of 22 micro-tubes was used to hold the filtrate liquid from each bottle. The populations of bacteria, protozoa, NH₃, and volatile fatty acids (VFAs) generated during the 48-hour incubation period will be examined in further detail for each set. After the centrifuge tubes containing the residues were fully dry, they were stored in an oven set at 60 °C for two to three days in order to assess the digestibility of DM.

The ANKOM Daisy II incubator equipment was utilized as the second in vitro approach to assess the digestibility of NDF. This apparatus functions as a simulated rumen for in vitro research. After rinsing the F57 filter bags in acetone for three to five minutes, they were allowed to air dry (ANKOM, 1920; Tassone et al., 2020). Four feed treatments, fourteen bag replicates for each treatment, and two blankos for each treatment were placed inside 64 filter bags. Control (TMR + no seaweed), oven-dried (TMR 0.5 g + oven-drying seaweed powder 0.02 g), sun-dried (TMR 0.5 g + sun-drying seaweed powder 0.02 g), and freeze-dried (TMR 0.5 g + freeze-drying seaweed powder 0.02 g) were the feed treatments utilized for the ANKOM Daisy II incubator.

One jar held a total of 16 F57 filter bags (14 feed treatment bags and 2 blankos). As a result, four jars were used and put within the incubator device of the ANKOM Daisy II. The 400 mL combination of buffer solution and rumen fluid was put into each jar. The entire procedure took place in a water bath at 39 °C with CO₂ flushing. According to (Tassone et al., 2020), the jars were then set up on each rotation rack and incubated for 48 hours at 39 °C. The jars were pulled from the incubator after 48 hours. The contents of every jar were thrown away. After thoroughly rinsing the bags from each jar under running water from the faucet, they were placed in the freezer to await additional NDF analysis.

2.5. Analysis of parameters

2.5.1. Concentration of CH₄

Gas collected in vacuum vial bottles during the 48-hour incubation period was submitted to another laboratory for gas chromatography (GC) examination of the CH₄ content. The Carboxen TM 1000 (45/60, 2 m × 1/8 in.) column (Supelco, USA) and flame ionization detector (FID) are fitted with the Shimadzu GC-14 B GC (Shimadzu, Japan). The CH₄ concentration in the gas collected during the incubation was examined using GC 14B Shimadzu.

2.5.2. Ammonia concentration (NH₃)

Conway's microdiffusion technique was used to evaluate samples for NH₃ concentration analysis that had been previously stored in micro-tubes from the Theodoro’s in vitro experiment (Park and Lee, 2020).

2.5.3. Concentration of Volatile Fatty Acids (VFA)

Total and partial VFA concentrations were determined using gas chromatography (Chrompack CP-9002, Chrompack, Inc., Raritan, New Jersey, USA) using samples for VFA concentration measurement that had been previously stored in micro-tubes from the Theodoro’s in vitro experiment.

Population of bacteria and protozoa. The bacterial population was recorded using the roll tube method from (Ogimoto and Imai, 1981), and the microbial population, or the number of protozoa, was determined using a hemacytometer. Samples from the Theodoro’s in vitro experiment that had been previously kept in micro-tubes were used for the microbial population investigation.

3. Results

3.1. Gas production and degradability

This study investigated the influence of drying methods (oven-dried, sun-dried, and freeze-dried) on the effectiveness of E. cottonii seaweed as a feed supplement for reducing enteric methane production in in vitro fermentation system. Measurement on the results of gas produced and degradability of DM and OM during the 48 hours of incubation are presented in Table 1.

Table 1
Gas produced and degradability of feed supplemented with either oven-dried, sun-dried, or freeze-dried of seaweed E.cottonii during a 48 hours of incubation time using in vitro method.

Parameters	Treatments
	Control	Oven-dried	Sun-dried	Freeze-dried	P value
Gas production (mL) (24 h)	43.00 ± 2.35c	45.80 ± 1.30cb	47.80 ± 1.92b	51.80 ± 2.59a	0.000
Gas production (mL) (48 h)	27.60 ± 0.55 c	28.20 ± 0.84bc	29.00 ± 0.71ab	29.20 ± 0.45ab	0.005
Cumulative Total Gas production (mL)	70.60 ± 2.41c	74.00 ± 1.87cb	76.80 ± 1.79b	81.00 ± 2.92a	0.000
CH4 gas concentration (%) (24 h)	3.08 ± 0.30a	0.50 ± 0.12c	0.33 ± 0.23c	1.60 ± 0.30b	0.000
CH4 gas concentration (%) (48 h)	2.97 ± 0.58ab	3.62 ± 0.81a	3.42 ± 0.32a	2.23 ± 0.59b	0.050
CH4 gas production (mL) (24 h)	1.33 ± 0.15a	0.23 ± 0.05b	0.16 ± 0.11b	0.83 ± 0.16b	0.010
CH4 gas production (mL) (48 h)	0.82 ± 0.16b	1.02 ± 0.23a	0.99 ± 0.10a	0.65 ± 0.18c	0.000
Cumulative CH4 gas production (mL)	2.14 ± 0.17a	1.25 ± 0.25b	1.15 ± 0.17b	1.48 ± 0.16ab	0.013
CH4 gas per Total gas produced (%)	3.04 ± 0.25a	1.69 ± 0.36b	1.50 ± 0.22b	1.83 ± 0.15b	0.010
DM Degradability (%)	44.53 ± 0.91b	46.59 ± 1.96ab	46.97 ± 0.96a	46.56 ± 1.27b	0.044
OM Degradability (%)	46.39 ± 0.68b	49.32 ± 1.43a	49.63 ± 0.74a	49.39 ± 0.95a	0.000
NDF Degradability (%)	43.00 ± 0.99b	47.77 ± 1.53a	46.05 ± 1.48a	45.21 ± 0.37a	0.000
Total gas produced/1g DM degraded (mL/g)	316.4 ± 13.0b	302.7 ± 14.7c	310.6 ± 9.5b	328.4 ± 9.1a	0.000
Total gas produced/1g OM degraded (mL/g)	363.9 ± 13.7c	342.5 ± 12.8b	352.3 ± 9.8ab	371.1 ± 10.4a	0.000
CH4 gas produced/1g DM degraded (mL/g)	9.61 ± 0.81a	5.13 ± 1.13b	6.65 ± 0.68b	6.00 ± 0.58b	0.000
CH4 gas produced/1g OM degraded (mL/g)	11.05 ± 0.92a	5.80 ± 1.25b	5.27 ± 0.77b	6.78 ± 0.68b	0.000

• Values with a different superscripts indicate a significant (P < 0.01 or P<0.05) differences between treatments.

All seaweed treatments significantly increased total gas production compared to the control group at both 24 and 48 hours of incubation (P<0.05), with freeze-dried seaweed demonstrating the most pronounced effect (P<0.01). The drying method significantly also impacted enteric CH₄ produced during the incubation time. At 24 hours, oven-dried seaweed displayed the lowest CH₄ concentration, while the control and freeze-dried groups had the highest (P<0.01). This pattern shifted at 48 hours, with the control showing the lowest CH₄ concentration and the oven-dried group having an intermediate value (P<0.05).

In terms of cumulative enteric CH₄ production, sun-dried and oven-dried methods of the seaweed treatments resulted significant lower values compared to the control group at both time points (P<0.01), compared to that generated in freeze-dried treatment. The proportion of enteric CH₄ gas relative to total gas production also followed a similar trend, with seaweed supplementation generally reducing the percentage of enteric CH₄ produced (P<0.01).

Regarding DM and OM degradability, sun-dried and freeze-dried seaweed achieved the highest degradability values. Although only OM degradability showed a statistically significant difference (P<0.01). The NDF degradability was also highest with oven-dried seaweed, but only freeze-dried differed significantly from the control (P<0.01).

The efficiency of gas produced per gram of DM or OM degraded was significantly increased by seaweed supplementation, with freeze-dried seaweed showing the greatest improvement (P<0.01). Similarly, enteric CH₄ production per gram of degraded DM or OM was consistently lower in all seaweed treatments compared to the control group (P<0.01). So, the current study emphasized that supplementing ruminants with E. cottonii seaweed may improve total gas production and degradability, while reducing enteric CH₄ emissions. Both the total gas produced and the CH₄ gas profile appear to be influenced by the drying methods, with freeze-dried seaweed showing the most promise in terms of lowering the production of enteric CH₄.

3.2. End-product of rumen fermentation kinetics

Table 2 shows the results of rumen fermentation end-product when basal feed was supplemented with specific powder of seaweed E. cottonii dried using three distinct methods. The total concentration of volatile fatty acids (VFAs) displayed a trend towards an increase with seaweed supplementation. The freeze-dried seaweed showing the highest value, although the difference compared to the control was not statistically significant (P>0.05). However, the proportions of individual VFAs within the total VFA were significantly affected by the drying method.

Table 2
The end product of rumen fermentation kinetics of feed supplemented with either oven-dried, sun-dried, or freeze-dried of seaweed E. cottonii during a 48 hours of incubation time using in vitro method.

Parameters	Treatments
	Control	Oven-dried	Sun-dried	Freeze-dried	P value
Total VFA concentration (mM)	121.23 ± 20.13b	130.26 ± 6.42b	143.04 ± 21.42a	124.47 ± 10.60b	0.085
Acetic Acid (C2) concentration (mM)	102.25 ± 16.18b	110.82 ± 6.10ab	127.10 ± 20.68a	109.89 ± 7.52ab	0.074
Propionic Acid (C3) concentration (mM)	11.27 ± 2.66	11.16 ± 0.92	10.52 ± 1.42	9.67 ± 1.76	0.498
Butyric Acid (C4) concentration (mM)	5.41 ± 1.31a	4.80 ± 0.88a	4.21 ± 0.38a	3.78 ± 0.79b	0.052
Ratio C2/C3	9.21 ± 1.03b	9.97 ± 0.72b	12.07 ± 1.05a	11.56 ± 1.36ab	0.002
Percentage C2 from Total VFA	84.42 ± 2.33b	85.06 ± 1.31b	88.70 ± 1.53a	88.39 ± 1.53a	0.002
Percentage C3 from Total VFA	9.23 ± 0.79a	8.56 ± 0.51ab	7.39 ± 0.54b	7.72 ± 0.80b	0.002
Percentage C4 from Total VFA	4.44 ± 0.57a	3.68 ± 0.67ab	2.98 ± 0.28b	3.01 ± 0.39b	0.001
NH3 concentration (mM)	2.72 ± 0.23	2.40 ± 0.40	2.80 ± 0.24	2.40 ± 0.37	0.134
Bacteria Population (x 109 cell/mL)	5.09 ± 0.64	5.50 ± 0.39	5.59 ± 0.59	5.23 ± 0.67	0.417
Protozoa Population (x 105 cell/mL)	35.45 ± 3.32a	26.80 ± 3.48b	23.35 ± 4.90c	31.60 ± 7.13ab	0.007

• Values with a different superscripts indicate a significant (P < 0.01 or P<0.05) differences between treatments.

The proportion of acetic acid (C₂) and the C₂/C₃ ratio were both highest with freeze-dried seaweed supplementation, indicating a potential shift towards more efficient energy production for rumen microbes (P<0.01). Conversely, the proportion of butyric acid (C₄) decreased significantly with all seaweed treatments compared to the control (P>0.05). While, the propionic acid (C₃) proportion did not differ statistically among treatments.

Seaweed supplementation did not significantly impact ammonia (NH₃) concentration, where the NH₃ concentration was similar for all group. Although the values tend to lower in oven-dried and freeze-dried treatment compared to control group. Similar result also found in total bacterial population. There is no significant effect of seaweed supplementation on the total bacteria population (P>0.05). Interestingly, addition of seaweed significantly affects the protozoa population. The result showed that protozoal population was significantly lower in oven-dried and sun-dried treatments compared to the control group, with the sun-dried seaweed showing the strongest effect (P<0.01). While the freeze-dried treatment has small effect on the population protozoa, which is showed by the same value with control group and also with oven-dried treatment.

4. Discussion

4.1. Gas production and degradability

The study determines the effect of addition red seaweed E. cottonii for mitigate enteric CH₄ production as well as end product of rumen fermentation. The seaweed was dried using three different methods on enteric CH₄ production and end product of rumen fermentation. The novelty of our study is using E. cottonii, a red seaweed which is the most cultivated in Indonesian waters, which at the same time elevate the DM degradability of feed.

All seaweed treatments significantly increased total gas production compared to the control group during the 48 hours of incubation, with the increasing was range from 2.17% to 18.6%. Among the drying methods applied, the freeze-dried seaweed demonstrating the most pronounced effect with the highest increasing for about 18.6% during the 24 hours of incubation, and also about 14.73% during the total of 48 hours incubation time. The study also showed that addition of seaweed E. cotonii significantly impacted enteric CH₄ produced during the incubation time. Addition of three different dried seaweed up to 4% dry matter reduced the percentage of CH₄ per total gas produced during the incubation time for about 39.14% for freeze-drying method up to 50.66% for the sun-drying method. These results are consistent with the study of Hidayah et al. (2024), which showed that the different drying method influenced the concentration of enteric CH₄ gases produced when Palisada perforate was added as additive. Furthermore, it is found that freeze-drying method is better than the method of drying seaweed P. perforata in a shaded place in related to its potential as a feed additive to mitigate enteric CH₄ in ruminant.

The CH₄ production reduction generated from the current study were lower than when dried Asparagopsis taxiformis was added at the level of 5% dry mater, which can reduce from about 74% (Brooke et al., 2020) up to 99% (Roque et al., 2021) of CH₄ produced. The different species of seaweed used as well as different dose of seaweed added were the most reason for the differences. The current study used E. cotonii with the level of 4% dry matter, while in the previous study used species A. taxiformis with the doses of 5% dry matter. There were also various results of CH₄ reduction when different species of seaweed used as feed additive, such as Mastocarpus stellatus (de la Moneda et al., 2019), Halymenia foresii ((Dubois et al., 2013), Bonnemaisonia hamifera ((Mihaila et al., 2022), Chondrus crispus (Machado et al., 2016), and Gracilaria vermiculophylla (Maia et al., 2016).

The production of gases produced during the process of fermentation of feed materials in the rumen describes the amount of DM or OM that is degraded by rumen microbes. In the current study, addition of seaweed increased DM and OM degradability, which also in line with increasing in the total gas produced per unit of DM and OM degraded. Addition of seaweed increased the volume of gas produced when the DM and OM degraded increased, with the highest value when sun-dried and freeze-dried seaweed were included. Similar finding by Quiroz Guerrero et al. (2020) showed that the lowest gas production was followed by the less DM and OM degraded of forages during in vitro test. Consistent results also reported by Hidayah et al. (2024), which indicated that the drying techniques of seaweed P. perforata influences the concentration of enteric CH₄ gas produced.

Our finding showed that addition of E. cottonii decreased enteric CH₄ production is in line with Thalib et al. (2011) and Widiawati and Hikmawan (2021) . The study suggested that seaweed can be used to mitigate CH₄ gas emissions from ruminant animals, by provide seaweed as a feed additive for the animals (Sultana et al., 2023). Some bioactive substances contained in the E. cottonii is mostly play a major role and responsible in prohibiting the production of CH₄ during feed digestion in the rumen. Previous study by Widiawati et al. (2024) in analyzing the bioactive compound of E. cottoni described that extract fresh and sun-dried E. cottonii contain alkaloid, flavonoid and saponin either extracted using water, dichloromethane and hexane. The flavonoid and saponin compounds are substance that can be used as mitigation agent, which in the same time also increase DM degradability (Ku-Vera et al., 2020; Sommai et al., 2021). According to studies by Machado et al. (2014) and Choi et al. (2021), seaweeds are rich in bioactive secondary metabolites, halogen and phlorotannin compounds, which are the best feed additives for lowering enteric CH₄ emissions (Choi et al., 2021; Roque et al., 2019; Winarni et al., 2021). As an antimethanogenic substance, the halogen compound has the ability to inhibit the enzyme methyl-coenzyme reductase (MCR) during the methanogenesis process (Allen et al., 2014; Costa and Leigh, 2014; Kinley et al., 2016; Machado et al., 2016). Another substance that may be used to lessen the rumen's synthesis of CH₄ is phlorotannin from seaweed (Yang et al., 2022).

4.2. End-product of rumen fermentation kinetics

The effect of supplementation of dried E. cottonii seaweed on end product of rumen fermentation was examined using in vitro test. Observing the end-product of rumen fermentation, such as VFA as well as the population of microbial rumen, due to the addition of seaweed is very important. Because it is closely related to the function of the end-products of rumen fermentation for livestock production. Microbial changes in the rumen will also have a major impact on the microbial activities in digesting feed and producing the end product of rumen fermentation.

The current study demonstrated that reduction on CH₄ production when seaweed E. cottonii supplemented was followed by increasing the total VFA concentration, in particular an increasing in the molar of acetate, but not to the molar of propionate. It is understood that inhibition of CH₄ production has correlated with the increasing in the availability of H₂ in the rumen, which will incorporate into VFA formation (Ungerfeld, 2015). Our study showed an increasing in VFA concentration when E. cottonii added at the level 4% of DM, increase the VFA concentration up to 17.99%. Although our results are different with the reports of Machado et al. (2016) and Terry et al. (2023), which showed decreasing in total VFA concentration when CH₄ production reduced. However, the current results are coherent with those finding that addition of A. taxiformis reduce CH₄ production but did not decrease the total VFA production (Roque et al., 2019). Metabolite seconder such as flavonoid contained in E. cottonii could be has a role in shifting the proportion of molar VFA partial, in particular propionate and acetate (Olagaray and Bradford, 2019). In our study, decreasing in CH₄ production followed by an increased in the proportion of acetate rather than production of propionate, as also showed by high C2:C3 ratio.

There is no effect of seaweed E. cottonii supplementation on NH₃ concentration during the in vitro study. The concentration was similar between control and treatment group. This result was different with the study by Choi et al. (2021), which showed decreasing of NH₃ concentration when a brown seaweed species Ascophyllum nodosum was supplemented. This species was identified has an anti-methanogenic effect without affecting the microbial activities in the rumen. The difference between the two study could be due to the secondary compound contained in the A. nodosum, which can inhibit degradation of protein to release NH₃. According to the metabolite seconder analysis, the E.cottonii did not contain tannin (Widiawati et al., 2024). Thus, it has no inhibition effect on protein degradation in the rumen.

Effect of seaweed addition on the bacteria and protozoa population in the current study is similar with the report of Choi et al. (2021), which mentioned that seaweed extracts changed the amount of rumen microbiota and reduced the production of enteric CH₄. Secondary metabolites contained in seaweed, such as saponins, in addition to decreasing the CH₄ gas, have also been shown to decrease the population of protozoa. This suggests that there is direct interaction between the archaea that produce CH₄ gas and the population of protozoa. The results of the study showed that the administration of E. cottonii containing saponins reduced the population of protozoa population by 10.61%; 24.19% and 33.95%, when freeze-dried, oven-dried, and sun-dried of E. cottoni were added to the feed, respectively. These reductions were followed by the reduction of CH₄ produced. It seems that different drying methods did not affect the function of saponin in reduction of protozoa population. The role of saponin in depress the protozoa population has been widely examined, with variation of saponin sources (Ku-Vera et al., 2020; Ramos-Morales et al., 2014).

Based on observed results of various studies compared to the result of current study, there are variation on the decreasing of enteric CH₄ production due to the administration of the seaweed. This is due to the different species of seaweed used in each study and also the location origin of the seaweed. Beside the large CH₄ reduction potential of seaweed as feed supplement for ruminant (Morais et al., 2020), the species and location origin for seaweed grown must be considered in determining the dose level of seaweed used. This is due to the different content of bioactive such as halogen compound as an anti-methanogenic compound containing in each species of seaweed (Putra et al., 2020).

The result of current study emphasized that supplementing ruminants with E. cottonii seaweed may improve total gas production and degradability, while reducing enteric CH₄ emissions. Both the total gas produced and the CH₄ gas profile appear to be influenced by the drying methods, with freeze-dried seaweed showing the most promise in terms of lowering the production of enteric CH₄. This result is in line with Regal et al. (2020) report, which showed that freeze-drying is the best technique for preserve seaweed A. taxiformis. Moreover, a proper handling way and drying technique of seaweed is essential for quality and product safety and quality.

5. Conclusion

In conclusion, the present study demonstrated that dried E.cottonii addition up to 4% DM effective in depressing enteric CH₄ production, without causing negative effect on DM and OM degradability as well as did not depress total VFA formation and bacterial activities. Depression of CH₄ formation has related to the reduction of protozoa population. The three different drying-methods have similar effect on reducing CH4 production, with the most affective is freeze-drying method.

Acknowledgements

The authors express their gratitude to the National Research and Innovation Agency for the funding support through Research funding under Rumah Program Bibit Unggul Year 2023. This study received partial support from the AgResearch New Zealand through Project number 2022-Indonesia Program Phase2_BRIN.

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Publication Dates

• Publication in this collection
  15 Nov 2024
• Date of issue
  2024

History

• Received
  24 May 2024
• Accepted
  12 Sept 2024