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7 May 2026

11 Pages

Potential of Alaria esculenta Silages as Ruminant Feed: Ruminal Fermentation, Methane Production, and Nutritive Value

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1
Departamento de Producción Agraria, Escuela Técnica Superior de Ingeniería Agronómica, Agroalimentaria y de Biosistemas, Universidad Politécnica de Madrid, 28040 Madrid, Spain
2
Departamento de Producción Animal, Facultad de Veterinaria, Universidad Complutense de Madrid, 28040 Madrid, Spain
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Departamento de Producción Sostenible de Rumiantes, Estación Experimental del Zaidín (CSIC), 18100 Granada, Spain
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Faculty of Biosciences and Aquaculture, Nord University, 8026 Bodø, Norway
Phycology2026, 6(2), 48;https://doi.org/10.3390/phycology6020048
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Abstract

Brown seaweed Alaria esculenta has potential as a sustainable feed for ruminants, but it deteriorates quickly after harvesting. This study evaluated silage as a preservation method and analyzed its effects on nutritive value and anti-methanogenic properties. Four treatments were tested: no additive, formic acid, lactic acid bacteria (LAB), and pre-wilting at 30% dry matter plus LAB (PLAB). Chemical composition and in vitro fermentation using sheep rumen fluid were analyzed. Ensiling reduced fiber components and polyphenols but did not affect gross energy, and increased gas production and total volatile fatty acids, indicating improved fermentability, especially in PLAB treatment. However, overall nutritive value remained lower than that of alfalfa hay and concentrate. Methane production increased after ensiling due to higher fermentation levels. Despite this, the methane-to-VFA ratio in all the seaweed samples was about one-third of that of conventional feeds, indicating anti-methanogenic properties. No differences in this ratio were found between fresh and ensiled seaweed, indicating that these anti-methanogenic properties were preserved. Overall, ensiling is a viable method to stabilize A. esculenta, improving its fermentability and maintaining its capacity to reduce methane emissions, although its nutritional value remains relatively low compared to traditional ruminant feeds.

1. Introduction

Seaweeds have been used in some coastal areas to feed sheep throughout history [1]. Interest in seaweeds as ruminant feed has increased in recent years because they are more sustainable than land-based crops, as their cultivation does not require freshwater, fertilizers, or pesticides [2] and it can reduce CO2 levels [3]. From a nutritional perspective, some seaweeds are rich in nutrients such as protein, complex carbohydrates, polyunsaturated fatty acids, minerals and biocompounds which may enhance immunity and antioxidant status in cattle, sheep and goats, and improve meat and milk quality [4]. Additionally, some biocompounds in some seaweeds, such as polyphenols or bromoform, may possess anti-methanogenic properties, as demonstrated in both in vitro and in vivo experiments [5,6]. Indeed, emissions of CH4 from livestock ruminants account for up to 16% of global greenhouse gas emissions and 30–32% of anthropogenic CH4 emissions [7]. Consequently, feeding seaweed to ruminant animals is being evaluated as a strategy to mitigate CH4 emissions. Among these, brown seaweed Alaria esculenta has gained particular attention for its high biomass yield (150–200 t fresh biomass per ha and year) [8], and relatively high content of polyphenols and other biocompounds with potential anti-methanogenic properties [5,8]. However, its potential as ruminant feed has not been fully exploited because they deteriorate due to their high water content, which limits their use in livestock feeding [8]. Silage, which is based on anaerobic acid fermentations, has been proposed as a viable method to preserve seaweeds and prevent their rapid post-harvest spoilage [8,9]. Although seaweeds are challenging to ensile because of its high buffering capacity and low initial number of epiphytic lactic acid bacteria [9,10], this process can be improved with additives such as formic acid or lactic acid bacteria. A previous study [8] analyzed the fermentation products of A. esculenta silages, but information on the potential effects of ensiling A. esculenta on its anti-methanogenic properties, chemical composition and nutritive value is lacking. This information is essential for formulating balanced diets and efficiently incorporating seaweeds while maximizing their environmental benefits. Therefore, the objective of this study was to evaluate the effects of different silage treatments on chemical composition and in vitro CH4 production and ruminal fermentation of A. esculenta silages.

2. Materials and Methods

2.1. Alaria Esculenta Samples and Silages Preparation

Fresh A. esculenta biomass was obtained at a commercial floating aquaculture facility located in Lofoten in June 2018 (Norway, 68.293, 15.131). After harvesting, the seaweed was maintained in 600 L water tanks with running seawater set to 7 ± 1 °C and constant aeration. Biomass was maintained at 150 μmol photons m−2 s−1, measured at the water surface of the tanks. The silages were prepared within 48 h after harvesting. The biomass was washed three times (10 s each) in 3 sequential water baths with decreasing salinity (100% seawater, 70% seawater, and freshwater, 10 s at each step); excess of water was drained, and the biomass was hand chopped (about 1 × 3 cm). During chopping, two representative samples of fresh A. esculenta, composed of biomass from different individuals, were taken and immediately frozen (−40 °C) until further analysis. Four different A. esculenta silages were prepared: ensiled without additives (CON), with formic acid (FA; 4 g formic acid/kg seaweed), with lactic acid bacteria (LAB; 2.5 × 109 colony forming unit (CFU) Lactobacillus plantarum and 2.5 × 109 CFU Lactobacillus fermentum per kg seaweed), and with LAB treatment after pre-wilting and oven-drying (37 °C with forced-air ventilation) of seaweed to 30% dry matter (DM) content; PLAB). The silages were prepared in vacuum-sealed bags (6 bags per treatment) each containing 2 kg of seaweed from different individuals. A detailed description of silage preparation is given by Yen et al. [8]. After 90 days of storage, bags were opened and the content of 3 random bags from each treatment were pooled to obtain 2 samples per silage treatment that were frozen (−40 °C). Therefore, the sample size for this study was n = 2. All samples (fresh seaweed and silages) were freeze-dried and ground to 1 mm before chemical composition analysis and in vitro incubations. In addition, samples of the feeds (alfalfa hay and concentrate) fed to donor sheep were included in the study as reference feeds for comparative purposes, and their chemical composition is given in Table 1.
Table 1. Chemical composition (g/kg dry matter (DM) unless otherwise stated) of alfalfa hay and concentrate fed to sheep used as donors of ruminal fluid for in vitro incubations and used as reference feeds.

2.2. Donor Animals and In Vitro Trials

The care and handling of the sheep used as donors of ruminal fluid for the in vitro incubations were carried out by trained personnel according to the European guidelines for the protection of experimental animals.
Ruminal fluid from four rumen-fistulated Lacaune sheep (64.2 ± 1.95 kg body weight) was independently used as the source of ruminal microorganisms to produce four replicates. Sheep were fed a diet based on alfalfa hay and a commercial concentrate based on cereals and soybean meal at a 90:10 ratio. Animals were individually housed in pens with free access to drinking water, and the diet was supplied twice a day at 45 g DM/kg0.75 to prevent feed selection. Prior to the morning feeding, approximately 300 g of ruminal content was collected from each sheep and filtered through four layers of cheesecloth to obtain four different ruminal fluids. Each individual fluid was independently mixed with a pre-heated (39 °C) buffer solution (without trypticase, Goering and Van Soest [11]) in a 1:4 ratio under continuous CO2 flushing to obtain four replicates in the in vitro incubations.
Two in vitro trials were conducted. The first trial lasted for 120 h and aimed to determine the gas production kinetics and to evaluate degradation rates of the fresh seaweed, the silages and the alfalfa hay. Briefly, 500 mg of DM of each sample was weighed in 120 mL glass bottles that were filled with 50 mL of the ruminal fluid–buffer mixture of each of the 4 sheep, making a total of 4 vials per sample. Bottles were sealed with rubber stoppers, incubated at 39 °C, and the gas produced was measured at 2, 4, 6, 9, 12, 24, 30, 48, 54, 72, 96 and 120 h using a pressure transducer (Delta Ohm DTP704-2BGI, Herter Instruments SL, Barcelona, Spain) and a calibrated plastic syringe. After each measurement, the gas was released from the bottles. Two bottles without substrate (blanks) were included per inoculum to correct for endogenous gas production. In addition, all samples were also incubated for 120 h in an Ankom Daisy II incubator to determine the DM potential degradability (PDMD). Briefly, 300 mg of each sample were weighed in duplicate into polyester bags (Ankom Corp #57 bags, 30 µm pore size; Ankom Technology Corp., Fairport, NY, USA) and incubated in the mixture of the pooled ruminal fluid of the four sheep and the buffer solution described previously under continuous rotation at 39 °C. At the end of the incubation, bags were washed with cold water, dried at 60 °C and weighted to determine the loss of DM.
The second in vitro trial was carried out following the same methodology used to determine the gas production kinetics, but only lasted for 24 h [12]. After 24 h, gas production was measured and a gas sample (10 mL) was collected in vacuum tubes (Terumo Europe NV, Leuven, Belgium) for CH4 analysis. Finally, 2 mL of bottles’ content were mixed with 2 mL of 0.5 M HCl and frozen (−20 °C) for volatile fatty acid (VFA) and NH3-N analysis.

2.3. Chemical Analyses

The DM content (ID 934.01), ash content (ID 942.05), ether extract (EE; ID 920.39) and total starch (ID 996.11) of the fresh A. esculenta, its silages, and the alfalfa hay used as reference were determined according to the AOAC procedures [13]. The total nitrogen (N) content was analyzed using a Leco FP258 analyzer (Leco Corporation, St. Joseph, MI, USA) according to the Dumas method. The sequential fiber method described by Van Soest et al. [14] was utilized to determine the neutral detergent fiber (NDF; with heat-stable amylase), and acid detergent fiber (ADF) content and results were expressed ash-free. Lignin content was determined as described by Robertson and Van Soest [15]. The content in total extractable polyphenols (TEP) and total soluble sugars were analyzed through colorimetry following the procedures described by Julkunen-Tiito [16] and Yemm and Willis [17], respectively. The gross energy was measured with an adiabatic calorimeter (6400 Automatic Isoperibol Calorimeter, Parr Instruments, Moline, IL, USA). Concentrations of VFA were determined by gas chromatography using a Shimadzu GC 2010 (Shimadzu Europe GmbH, Duisburg, Germany) gas chromatograph equipped with an autosampler and a TR-FFAP column (30 m × 0.53 mm × 1 μm; Supelco, Madrid, Spain). Determination of CH4 concentration was determined by gas chromatography using a Shimadzu GC 14B gas chromatograph (Shimadzu Europe GmbH, Duisburg, Germany), heated at 200 °C and equipped with a flame ionization detector and a column packed with Carboxen 1000 (Supelco, Madrid, Spain). Helium was used as carrier gas and peaks were identified by comparison with a standard of known methane concentration. The concentration of NH3-N was determined following the colorimetric method described by Weatherburn [18] and using a spectrophotometer (Genesys 10 uV Scanning, Thermo Scientific, Madison, WI, USA).

2.4. Calculations and Statistical Analyses

Gas production data were fitted to the exponential model: Gas = PGP × (1 − e(−c×(t−lag))). In this model, PGP is the potential gas production, c is the gas production rate, lag is the delay before gas production starts, and t is the time of gas measurement, using the NLIN procedure of SAS 9.3 [19]. The average gas production rate (AGPR) was calculated as AGPR = PGP × c/[2 × (ln2 + c × lag)]. Finally, the dry matter effective degradability (DMED) was calculated as DMED = [(PDMD × c)/(c + kp)] e(−c×lag), assuming a particulate passage rate (Kp) of 4.2% per h adequate for low-producing animals [20]. These equations have been validated and used in previous studies [6,9].
The experiment was conducted as a completely randomized design in which the treatment (fresh seaweed and four silage treatments) was the only factor analyzed. Chemical composition data were analyzed using the PROC GLM of SAS 9.3 [19], considering the treatment as the main effect. Gas production kinetics and fermentation parameters data were analyzed using the PROC MIXED of SAS 9.3 [19], where silage treatment (fresh seaweed and four silage treatments) was considered a fixed effect, and the ruminal inoculum was considered a random effect. When significant differences were detected (p ≤ 0.05), means were compared by Tukey’s multiple comparison test.

3. Results and Discussion

As previously reported by Yen et al. [8], the silages tested in the present study had pH values below 4.90 (4.84, 3.69, 4.62 and 4.42 for Control, FA, LAB and PLAB, respectively) and no butyrate content was detected. Both results indicate that no clostridial fermentation occurred and, therefore, reflect good-quality silages [9,21]. Elevated ammonia concentrations might negatively influence pH and palatability, but NH3 levels were low or not detected (0.126, 0.004, 0.041 g/kg DM and not detected for Control, FA, LAB and PLAB, respectively) [21].

3.1. Chemical Composition

As expected, DM in PLAB silage was significantly higher (p < 0.05; Table 2) than that in both fresh A. esculenta and the rest of the silages due to pre-wilting performed before ensiling. In agreement with previous reports [3,6], fresh A. esculenta had high ash content (215 g/kg DM), which was unaffected by ensiling (p = 0.237). Similarly, ensiling did not affect N content (p = 0.109), suggesting no N losses during silage’s storage. These results agree with other studies in which Ulva rigida [22], Saccharina latissima [9,22,23] and Porphyra umbilicalis [9] were ensiled. Significant differences (p ≤ 0.021) were observed among the silages in NDF, ADF, soluble sugars, and starch, while lignin content showed no differences (p = 0.226). Both CON and FA silages had lower (p < 0.05) content than fresh A. esculenta (237 and 209 g/kg DM in fresh A. esculenta vs. 199 and 177 g/kg DM in CON and FA as average, respectively). In contrast, LAB and PLAB silages had similar values to fresh A. esculenta, indicating that these fiber fractions remain unchanged during ensiling. Decrease in NDF and ADF fractions after ensiling of other seaweeds such as S. latissima, P. umbilicalis, Gracilaria vermiculophylla and U. rigida have been noticed [10,22], but other studies reported no changes in fiber fractions [9] or even observed increases in NDF content by ensiling S. latissima [22]. Seaweeds contain non-conventional carbohydrates, such as laminarin and mannitol [8,24], which can be as high as 15% of the dry weight in A. esculenta [25]. These non-conventional carbohydrates can be fermented by lactic acid bacteria during ensiling [26] and might be quantified as NDF and ADF fractions, helping to explain the observed decreases in NDF and ADF fractions over the ensiling process. Nonetheless, it should be noted that analyzing carbohydrates and interpreting fiber fractions in seaweeds is challenging [10,23].
Table 2. Chemical composition (g/kg dry matter (DM) unless otherwise stated) of fresh Alaria esculenta and different A. esculenta silages (CON, FA, LAB and PLAB).
Although there were significant differences (p ≤ 0.005) among the silages in soluble sugars and total starch content, no clear effect of ensiling on these fractions was observed and differences were minor (the largest difference between treatments was 8.7 g/kg DM) and probably of no practical significance. Differences in the EE content were also observed (p = 0.001), but it should be noted that fat levels in seaweeds are minimal and often considered negligible [27]. Reported TEP content in this study for the fresh seaweed was lower than that reported by Roleda et al. [28] for A. esculenta biomass sampled in either summer or autumn (28–56 mg PGE/g DM) in the vicinity of Bodø (Norway), but similar to that reported by de la Moneda et al. [3] for A. esculenta biomass sampled in autumn (28.1 tannic acid/g DM) in Bodø (Norway). In this study, samples were also collected in summer, but gallic acid was used as standard reference while Roleda et al. [28] used phloroglucinol, and de la Moneda et al. [3] used gallic acid, which could also help explain the disparities. Previous studies have reported large seasonal variability in the TEP content of A. esculenta (22.3 and 33.2 mg PGE/g DM [5], and 8.91 and 50.3 tannic acid/g DM [6] for biomass sampled in spring and autumn, respectively). In any case, concentration of total polyphenols in A. esculenta is larger than that observed in other brown species, such as S. latissima or Palmaria palmata, but lower than that reported in Ascophyllum nodosum or Fucus sp. [5,28]. Although some of the anti-methanogenic compounds present in seaweeds are unknown, it has been suggested that TEP content could be a good indicator of the anti-methanogenic activity [5]. Compared with fresh A. esculenta, all the silages had lower (p < 0.05) TEP content, particularly the PLAB silage that showed the lowest value. Similarly, Marcos et al. [23] observed that TEP content decreased after ensiling S. latissima. Lactobacillus sp. and other microorganisms can degrade polyphenols [29], which could help explain these results. Gross energy remained unaffected (p = 0.338) despite the observed changes in the chemical composition after ensiling. Reported effects of ensiling other brown seaweeds on chemical composition vary across studies [10,23], likely due to the high variability in seaweed chemical composition and characteristics, which are highly influenced by factors such as location, season, light exposure, and cultivation method [6].
The N, NDF, ADF and EE content in A. esculenta and its silages were lower than that observed in the alfalfa hay, but ash and TEP content was higher. Compared with the concentrate, fresh seaweed and its silages had lower N and EE content, but greater ash, NDF, ADF content. The gross energy content of the fresh seaweed and the silages was lower than in both reference feeds, which can be related to their high ash and low EE content.

3.2. In Vitro Trials

The PGP values are indicative of the potential of feed for ruminal fermentation, as the amount of gas is directly related to the amount of organic matter fermented [30]. As shown in Table 3, the PGP value for the fresh seaweed was similar to that reported by Molina-Alcaide et al. [6] for A. esculenta harvested in autumn, but lower than those reported by de la Moneda et al. [3] when A. esculenta was harvested in either spring or autumn. Compared to the fresh seaweed, ensiling increased (p < 0.05) the PGP, with the PLAB silage showing the highest value (Table 3), indicating that ensiling increased the ruminal degradability of A. esculenta. In contrast, the rate of gas production (c) and lag values were unaffected (p ≥ 0.061) by ensiling. Both FA and PLAB silages had higher (p < 0.05) AGPR values for the fresh seaweed and CON silage. Ensiling might have facilitated ruminal degradation by weakening chemical bonds within the cell wall. The lower NDF, ADF and TEP concentrations in the silages compared with the fresh seaweed could also help to explain these results, as fiber is less fermentable than other carbohydrates [31] and polyphenols can inhibit ruminal fermentation [32]. Pandey et al. [5] suggested that TEP content is a major factor determining seaweed degradability. However, in our study, TEP concentrations in the fresh seaweed and silages were not correlated with their DMED (PROC CORR of SAS 9.3 [19], r = −0.39, p = 0.270, n = 10). Nevertheless, both PGP and AGPR in all the silages were less than half that of alfalfa hay and much lower than that of concentrate, which indicates low potential fermentability. The low gas production values in the silages may be partly related to their high ash and lignin content, to microbial inhibition [32], or to the type of carbohydrates of A. esculenta that can be difficult to degrade and ferment [27].
Table 3. Parameters of gas production kinetics and ruminal degradability of fresh Alaria esculenta, different A. esculenta silages (CON, FA, LAB and PLAB) and feeds (alfalfa hay and concentrate) used as reference.
The PDMD of all the silages was greater (p < 0.05) than that of fresh A. esculenta, which agrees well with the greater PGP values observed in all the silages. Moreover, the DMED of the silages was on average 21.0% larger than that of the fresh seaweed. Other authors have also observed an increase in the ruminal degradability of different seaweeds after ensiling [9]. The DMED of the silages was similar to that of the alfalfa hay (383 and 379 g/kg DM, respectively), but lower than that of the concentrate (0.546 g/kg).
In accordance with that observed for gas production, ensiling increased (p < 0.05) total VFA production by 22.3% (as average), indicating an increase in ruminal fermentability, especially in the PLAB silage that had the highest (p < 0.05) VFA production (Table 4). However, total VFA production of A. esculenta and its silages was only 0.47 and 0.57 (average value for all the silages), respectively, of that observed for alfalfa hay, and 0.37 and 0.46 of total VFA production of the concentrate. These results indicate low nutritional values of both A. esculenta and its silages for ruminants. Nonetheless, the nutritive value of seaweeds may be highly variable [3,6]. Other studies have reported that A. esculenta could replace up to 20% DM of high-quality forages without affecting total VFA production [5].
Table 4. Fermentation parameters of fresh Alaria esculenta, different A. esculenta silages (CON, FA, LAB and PLAB) and feeds (alfalfa hay and concentrate) used as reference.
Significant differences (p < 0.001; Table 4) were observed for molar proportions of all individual VFA analyzed, although differences were small and of little practical importance. The PLAB silage had the lowest (p < 0.05) propionate and the highest (p < 0.05) butyrate and minor VFA (calculated as the sum of isobutyrate, isovalerate, and valerate) proportions. With the exception of FA silage, ensiling significantly increased the proportions of minor VFA and it was greatest for PLAB (p < 0.001). Some minor VFA (isovalerate and isobutyrate) are produced by the microbial deamination of some amino acids [33] and these results might, therefore, indicate greater protein degradation. In agreement with this hypothesis, NH3-N concentrations increased after ensiling and were greatest (p < 0.05) in the PLAB silage, which also showed the greatest concentrations of minor VFA.
The acetate/propionate ratio in the seaweed samples (fresh and ensiled) ranged from 2.46 for CON silage to 2.70 for PLAB silage, being lower than those found in alfalfa hay (3.29) and concentrate (2.84), indicating a distinct fermentative profile. The VFA profile differed from that of reference feeds, although it was more similar to the concentrate than to alfalfa hay. Pandey et al. [5] suggested that TEP may modulate ruminal fermentation. In addition, the different carbohydrates present in seaweeds compared to those in the reference feeds might also have influenced ruminal microorganisms.
Production of CH4 was greater (p < 0.05; Table 4) in all the silages than in the fresh A. esculenta, but this increase might have been due to increased fermentation. In fact, no differences (p = 0.338) among fresh A. esculenta and all the silages were observed in the CH4/VFA ratio, indicating that the increased CH4 production in the silages was due to their higher fermentability. The amount of CH4 in the seaweed samples was about one third of that in the alfalfa hay and concentrate, but the differences could be due to the lower fermentation of the seaweed samples. Nonetheless, the CH4/VFA ratio of fresh A. esculenta was 0.65 and 0.75 of those of the alfalfa hay and the concentrate, respectively, indicating anti-methanogenic activity in the seaweed. The CH4/VFA ratio reflects the relationship between methane production (energetic loss) and energy yield (VFA is the main energy source for the host ruminant) and, thus, indicates energy efficiency and highlights potential anti-methanogenic properties. The similar CH4/VFA ratios observed in both fresh A. esculenta and all the silages indicate that the anti-methanogenic activity was maintained over ensiling. Marcos et al. [23] reported similar CH4/VFA ratios when evaluating Saccharina latissima silages, although the TEP content was significantly lower. This suggests that not only the content but also the type of polyphenolic compounds may be important when evaluating the anti-methanogenic activity of brown seaweeds. Pandey et al. [5] did not observe differences in the in vitro 48 h CH4 production after replacing 20% DM of maize silage with A. esculenta harvested in spring, but the CH4/VFA ratio was reduced by up to 13%. The reduction in the CH4/VFA ratio reported by Pandey et al. [5] is notable, particularly, given the low NDF content of the maize silage used (402 g/kg DM) and the 48 h incubation period, during which fibrous substrates with higher CH4/VFA ratios may be extensively fermented. The anti-methanogenic activity of A. esculenta previously reported is consistent with our results, even though our incubation period was shorter (24 h).

4. Conclusions

Ensiling Alaria esculenta resulted in small changes in chemical composition but improved in vitro ruminal fermentation and ruminal degradability. However, both gas and total VFA productions of A. esculenta silages were low, suggesting a low nutritive value, although this would largely depend on the characteristics of the fresh seaweed. The pre-wilting of seaweed to 30% DM content before addition of lactic acid bacteria would be the recommended treatment for ensiling A. esculenta. Anti-methanogenic properties of A. esculenta were not negatively affected by ensiling. Confirmation of the anti-methanogenic properties of A. esculenta under in vivo conditions is still required.

Author Contributions

T.d.E.: methodology, formal analysis, investigation, writing—original draft; C.N.M.: methodology, formal analysis, investigation, supervision, writing—original draft; E.M.-A.: conceptualization, methodology; M.N.-G.: conceptualization, resources, project administration, funding acquisition; Y.Y.: Investigation; J.E.F.-Y.: investigation; M.D.C.: conceptualization, methodology, supervision, writing—original draft; All authors contributed to Writing—review and editing. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Research Council of Norway ES 631289, Ensiled Cultivated Macroalgae as a Sustainable Ruminant Feedstuff-EnMac.

Institutional Review Board Statement

All experimental procedures were performed in compliance with relevant laws and institutional guidelines and have been approved by the Institutional Animal Care and Use Committee of the Comunidad Autónoma de Madrid of Spain (Approval number PROEX 035/17, 4 April 2017).

Data Availability Statement

Data are contained within the article.

Conflicts of Interest

The authors declare that they have no competing financial, non-financial, or personal interests that could have influenced this study.

Abbreviations

The following abbreviations are used in this manuscript:
ADFacid detergent fiber
AGPRaverage gas production rate
cgas production rate
CFUcolony forming unit
CONensiled without additives
DMdry matter
DMEDdry matter effective degradability
EEether extract
FAensiled with 4 g of formic acid per kg seaweed
LABensiled with Lactobacillus fermentum (2.5 × 109 CFU/kg seaweed) and Lactobacillus plantarum (2.5 × 109 CFU/kg seaweed)
lagtime before fermentation starts
Nnitrogen
NDFneutral detergent fiber
PDMDdry matter potential degradability
PLABseaweed pre-wilted to 30% dry matter content and ensiled as LAB
PGPpotential gas production; c: rate of gas production
SEMstandard error of the mean
TEPtotal extractable polyphenols
VFAvolatile fatty acids

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