The number of challenges facing livestock production systems is increasing at an unprecedented rate. There is a growing demand for animal-derived foods in terms of both quality and quantity, particularly in developing countries. Accordingly, there is a need to ensure food and nutrition security to sustain the growth of the human population (1) while simultaneously counteracting the pressures on available resources and the effects of climate change. More sustainable livestock systems, in particular those relating to ruminants, are needed to enhance animal productivity, health, and welfare and increase food production while reducing the negative impact of farms on the environment and food–feed competition (2). Non-conventional (novel or underexploited) feeds are of particular interest as they can effectively contribute to improving ruminant-derived food production, which is in line with the projected transition toward a circular bio-based economy in the European Union. Direct-fed microbial, microalgae, seaweeds, aquatic plants (e.g., duckweed), oilseeds (e.g., camelina), tropical tree and shrub leaves (e.g., cassava, Leucaena sp., and Flemingia sp.), and fruit and vegetable by-products have all been suggested as potential non-conventional ruminant feed resources given their low arable land and water requirements as well as their potential to reduce enteric methanogenesis and promote healthier ruminant-derived foods and more sustainable production systems (3–6).

Feed evaluation studies are needed to fully assess the potential of non-conventional feeds as ingredients or additives in ruminant diets. Studies evaluating production responses and in vivo digestibility require a large number of animals and amounts of feed, require an extensive investment in labor and time, are expensive (7), and raise ethical issues regarding the use of untested non-conventional feeds and the use of animals for experimental purposes. Consequently, in vitro methodologies (e.g., Tilley and Terry method, Ankom Daisy system, Hohenheim gas test, batch culture technique, and rumen simulation technique) are increasingly being used for evaluating the degradability and fermentability of novel feeds, ingredients, and supplements. Despite providing useful data, these methodologies require ruminal microbial inocula to recreate a ruminal fermentation environment and fail to mimic the complete ruminal digestive process (7).

The need for ruminal fluid inocula adds a source of variation owing to differences within and between animals used as rumen digesta donors, which can affect in vitro fermentation results. To reduce this source of variation and error, the ruminal inocula used for in vitro studies should be representative of the in vivo microbiota composition and activity (7, 8), and true replicates must be employed (9). Dietary composition and nutrient availability are the factors that most contribute to variation in rumen microbiota composition and activity (8, 10). Accordingly, the diets of rumen inoculum donors should be similar to those used in in vitro feed evaluation systems (8, 11). However, this finding is difficult to achieve when assessing non-conventional ingredients or additives as feed resources for ruminants, and most in vitro studies use non-adapted ruminal inocula.

Feeding strategies, such as varying the neutral detergent fiber-to-rapidly fermentable carbohydrate ratio [acidogenicity value concept; (12, 13)] or the physically effective neutral detergent fiber content (14) of diets, can effectively modulate rumen function without negatively affecting the health and welfare of animals. Thus, we hypothesized that ruminal inocula with distinct fermentation profiles could be attained by altering the proportion of basal forage in the diets of the ruminal inoculum donors from corn silage to haylage, two of the most commonly used forages in dairy feeding, and that these changes would affect the fermentation profile of non-conventional feeds after 24 h of in vitro incubation with non-adapted ruminal inocula. In this context, increasing levels (0, 5, 10, and 15%, on a dry matter [DM] basis) of a sustainable, non-conventional feed, a commercially available algal blend composed of two microalgal (Chlorella vulgaris and Nannochloropsis oceanica) and two seaweed (Ulva sp. and Gracilaria gracilis) species, were added to a basal substrate (corn silage: haylage, 1:1 DM basis), and each mixture was separately incubated with individual ruminal inocula derived from each of the four diets containing different proportions of corn silage and haylage. The effects on gas and methane production and the fermentation profile were subsequently evaluated after 24 h of in vitro batch incubation.

In vitro rumen batch culture is a technique widely used for predicting the degradability and nutritive value of feed sources (23, 24). It is also used to screen the potential of non-conventional additives and feeds, such as biochars, microalgae, and seaweeds (6, 25, 26), to modulate ruminal fermentation and, in particular, to reduce methane production. Despite its simplicity and the possibility of testing multiple samples at a single batch under similar conditions and without interference from the metabolic process of the host, several factors may influence the consistency of results (11, 23). Among these factors, the ruminal inoculum plays a major role.

Variations in in vitro gas production and fermentability due to the ruminal inoculum have been attributed to differences in the microbiota population profile and activity, among and within donor animals, between days, in sampling times (before or after feeding), and in the dietary composition and nutrient and energy intake of the host animals (7, 8, 27). In our study, we focused on the impact of the composition of the basal forage of diets on the ruminal inoculum of late-lactation cannulated cows. Four diets were formulated with similar ingredients, varying only in the proportions of two types of basal forage of differing chemical composition and particle size. Proper forage particle size is crucial for normal ruminal function and host health as it promotes a stable digesta mat, which increases the retention time of fiber particles, thereby promoting their fermentation by the rumen microbial community and stimulating reticulorumen motility, rumination, and salivation, neutralizing excessive acid production, and preventing a pH drop below levels required for optimal fermentation (28, 29). Along with other factors, particle size also plays an important role in feed intake and, thus, in dietary nutrient and energy digestibility. In the current study, a decrease in DM intake was observed when cows were fed diets with increasing proportions of haylage in substitution of corn silage, with cows fed the 100HL diet consuming 1.6 kg/day less than animals fed the 100CS diet. These differences may be due to the longer time required for eating and chewing haylage-containing diets, greater rumen fill effects, and longer retention times (30). Indeed, one study noted that, in early-lactating cows, the DM intake was reduced by 2.8 and 3.5 kg/day with 50% replacement of corn silage by long chop and short chop ryegrass silage, respectively (31). In contrast, a different study reported that the replacement of 0, 35, 65, and 100% ryegrass silage with corn silage had no impact on DM intake in cows during late lactation (32). These contrasting results highlight the importance of silage chop length on feed intake and performance (31).

Changing the acidogenicity value and the physically effective neutral detergent fiber contents of diets are complementary dietary formulation strategies for effectively manipulating the ruminal acid load (14, 33), with the carbohydrate type (starch vs. fiber) greatly influencing the ruminal microbial population and the fermentation profile (29, 34, 35). In the present study, these changes were reflected in the alterations in the ruminal fluid fermentation profiles observed when corn silage was replaced with haylage and in the concomitant increase in the neutral detergent fiber-to-starch ratio (1.8 to 10.5). These changes resulted in increases in pH values, the acetate molar proportion, and the acetate-to-propionate plus butyrate ratio, consistent with a higher fiber content (36). The ammonia-N content was 1.9 times higher in the ruminal fluid of cows fed 100HL than in that of cows fed 100CS, reflecting the higher crude protein content of the 100HL diet, further suggesting that corn silage-based diets improve the synchronization of energy and N release [synchronism concept, (37)]. Indeed, fermentable carbohydrates have been shown to reduce ammonia production by enhancing the capture of released ammonia-N or amino acids by ruminal microbiota or by reducing amino acid deamination (38, 39). While the total VFA content reflects the ruminal fermentation efficiency, the dietary treatment showed only a tendency (p = 0.088) to affect ruminal total VFA concentrations in the current study. The similar total VFA concentrations found may have been due to the ruminal fluid being collected before the morning meal when VFA production is lower (40) and the microbiota population is more stable (34).

High biomass productivity, a low carbon and water footprint, and significant nutritive and functional values have influenced the interest in the use of algae as a non-conventional and sustainable animal feed. Several studies have assessed the effects of microalgal and seaweed species as supplements and/or ingredients for ruminant feeding [see reviews (41, 42)]. Recently, the combination of microalgae (43, 44) and of seaweeds (45, 46) was reported to improve ruminant nutrition and milk quality. However, to the best of our knowledge, only one recent study evaluated the effects of a mixture of one microalgal (Euglena gracilis) and one seaweed (Asparagopsis taxiformis) species on in vitro ruminal fermentation parameters (47), and none of the studies have investigated the potential effects of combining two microalgal and two seaweed species. Thus, in this study, we assessed, for the first time, the effects of increasing supplementation levels (0, 5, 10, and 15%, DM basis) of a commercially available algal blend composed of two microalgal and two seaweed species on gas and methane production, pH, ammonia-N contents, and total VFA production and the proportion of individual VFA after 24 h of incubation with ruminal inocula obtained from cows fed forage-based diets with different proportions of two basal forages. A wide range of algal supplementation levels are reported in the literature, with microalgae being included at lower levels (up to 10%, 26) and seaweeds at higher levels (up to 25%, 6, 15, 47). As the commercially available algal blend used in the present study was composed of both microalgae and seaweeds, low (5%), average (10%), and high (15%) supplementation levels were assessed to gain further insights into the potential of this non-conventional and sustainable feed in ruminant nutrition.

In short-term in vitro systems such as batch culture technique, the microbiota of the ruminal inoculum cannot adapt to the substrate provided; thus, the rate, extent, and parameter profile of fermentation greatly depend on the composition and activity of the microbial population in the ruminal inoculum. Consequently, although not always feasible, the current recommendations are that the basal substrate should be similar to that in the diets of the donor animals (8, 11). Thus, in the present study, the basal substrate used was a combination (1:1) of the two base forages (corn silage and haylage) of the diets of the ruminal content donors supplemented with increasing algal blend levels; following incubation, in vitro fermentability was evaluated. The ruminal inoculum source was found to have a stronger impact than algal blend inclusion levels of up to 15% (DM basis) on short-term in vitro fermentation parameters. Inocula from cows fed diets in which corn silage was replaced with haylage effectively reduced total gas and methane production by 10.7 and 17.9%, respectively, while increasing the algal blend supplementation level contributed to reductions of 9.3 and 6.2%, respectively. Methane is the predominant greenhouse gas produced by ruminants. In addition to a high energetic cost for the animal, methane production is associated with a high environmental burden (48); indeed, it has been estimated that to limit the global temperature increase to 1.5°C, it is necessary to decrease enteric methane emissions by 20% by 2030, using 2020 as the baseline (49). In addition to reducing methane production, the algal blend used in this study is a sustainable feed as microalgae and seaweeds are produced autotrophically in local photobioreactors and IMTA systems, respectively, in which energy is derived from solar power, and nutrients are obtained from fish aquaculture wastewaters (50, 51). However, owing to the recalcitrant nature of the cell walls of microalgae and seaweeds, the fermentability of the algal blend may have been impaired, as complex algal cell wall polysaccharides are absent in conventional feeds and the microbial populations of the ruminal inocula were not adapted to this unconventional feed source (6, 26). Meanwhile, the effect of the ruminal inoculum source was greater than expected. Although studies have reported great effects of ruminal inocula source on fermentation rates, the extent of fermentation, and, thus, gas production, is often not affected, or only mildly so (52). In addition, in a review of the factors that affect in vitro gas production, Rymer et al. (27) concluded that above a minimum activity level of the microbiota, the ruminal inoculum source did not affect gas production, as long as a diet containing forage and concentrate, ideally in a 60:40 ratio, was fed to the ruminal fluid donors. Even though such a forage-based diet was provided to the ruminal fluid donor animals in the current study, microbial population activity may have been affected as the ammonia-N concentrations after 24 h of incubation was below the minimum level required for efficient microbial production and cellulolytic activity [50 mg/L; (53)].

In this study, the interaction between ruminal inoculum source and algal blend supplementation level affected total VFA production and the proportions of most individual VFA. Total VFA production was found to decrease with increasing algal supplementation levels with all inocula, with the strongest reduction being observed with inocula from cows fed the 30CS70HL diet. Given that VFA are the primary energy source of the ruminant host (54), feed sources with methane mitigation potential that do not reduce ruminal fermentation and total VFA production are of particular interest (49, 55). Acetate, propionate, and butyrate are the main VFA produced in the rumen, and their ratios mainly reflect the fermentation of non-structural and structural polysaccharides. In the current study, the molar proportions of acetate and propionate were, respectively, higher and lower after 24 h of incubation with the algal blend at the 15% supplementation level compared with those observed with the 0 and 5% inclusion levels, but only when the inoculum was derived from cows fed 100% corn silage as the base forage; no interaction effect was observed for butyrate. Higher acetate and butyrate and lower propionate production have been associated with increased gas production (56) and thus also with enhanced fermentability and microbiota production and activity; however, such an association was not observed in our study. The supplementation of corn silage or haylage substrate with microalgal (26) or macroalgal (6) species resulted in different fermentation profiles, with haylage showing lower fermentability. As previously mentioned, diet is known to greatly affect the composition and activity of the ruminal microbiota. Although changes in the ruminal microbiota can partly explain the results obtained in our study, Jami and Mizrahi (57) reported that differences in ruminal bacterial populations among animals fed the same diet were substantially higher than those promoted by different diets. Moreover, the authors concluded that despite the variation in bacterial taxa among animals, the microbiota was phylogenetically highly similar (82%) and thus did not affect fermentation ability. Hua et al. (58) compared the effects of glucogenic and lipogenic diets on ruminal bacterial community structure, fermentation intermediary metabolites, and methane production following 48 h of incubation with a ruminal inoculum. Although several amylolytic and cellulolytic bacteria were found to be sensitive to dietary differences, most highly abundant bacteria were stable or were only marginally affected, thus highlighting the relevance of the microbiota profile of ruminal inocula in short-term in vitro fermentation studies.

Ruminal inoculum source markedly affected the in vitro fermentability of the algal blend, supporting the importance of the diet of rumen donors in the modulation of the microbial ecosystem and its impact on the in vitro evaluation of non-conventional feeds for ruminants. Indeed, the replacement of corn silage with haylage as the basal forage in the diet of late-lactation dairy cows decreased gas and methane production and modified the ruminal fermentation profile after 24 h of in vitro batch incubation. Gas and methane production was also reduced with algal blend supplementation levels of up to 15% (DM basis). The interaction between the ruminal inoculum source and the algal blend supplementation level exerted an effect only on total VFA production and the profiles of most individual VFA; this observation highlights the need to consider the diet of ruminal inoculum donors and the algal blend inclusion level when evaluating the optimal supplementation strategy for promoting ruminant nutrition and production sustainability without impairing ruminal function. Overall, our results highlight the importance of ruminal inocula when assessing the fermentability of a non-conventional feed. Our findings further highlight the potential of the algal blend as a natural modulator of ruminal fermentation in vitro; however, further in vivo studies are needed to reveal its full potential.

The original contributions presented in the study are included in the article/Supplementary material, further inquiries can be directed to the corresponding author.

All procedures involving animals were approved by the Animal Ethics Committee of the School of Medicine and Biomedical Sciences, University of Porto (ICBAS-UP) and licenced by the Portuguese General Directorate of Food and Veterinary Affairs (permit #0421/000/000/2021). All the procedures were performed by trained scientists (FELASA category C) in accordance with the European Union Directive 2010/63/EU relating to the protection of animals used for scientific purposes.

CM: Formal analysis, Investigation, Writing – original draft, Writing – review & editing. MM: Formal analysis, Investigation, Writing – original draft, Writing – review & editing, Conceptualization, Validation. IV: Investigation, Writing – review & editing, Conceptualization. AC: Formal analysis, Writing – review & editing, Conceptualization. AF: Funding acquisition, Writing – original draft, Writing – review & editing, Conceptualization, Resources, Supervision, Validation.