Overall,
G. amansii treatments did not appear to be detrimental to ruminal fermentation as assessed by
in vitro fermentation parameters. In fact,
G. amansii supplementation has the potential to assist in ruminant feeding for improved gas production and fermentation performance. For example, supplementation with
G. amansii treatments produced pH values remaining in the proper range of 6.37 to 7.35, which is a suitable pH range for cellulose digestion (6.0 to 6.8), protein synthesis (6.3 to 7.4), proteolytic activity (6.5 to 7.0) and VFA productivity (6.0 to 6.6) as suggested by McCullough [
16], as well as for ruminal microbial activity, which is not negatively affected within a pH range of 5.8 to 7.2 [
17]. In addition, % DM disappearance after
G. amansii supplementation was significantly reduced for the whole experimental period; however, total gas production was significantly increased at 12 and 72 h incubation as compared to CON, which may indicate a potential improvement to feed utilization efficiency [
18]. However, when dietary fiber was included in
G. amansii treatments, an increase of total gas production was observed without any reduction of % DM disappearance, which is in agreement with other algae studies involving dietary fiber [
19].
In recent years, extensive studies investigating the potential use of terrestrial plants for nutritional manipulation of enteric methane production have been conducted. Interestingly, one study focusing on algae supplementation reported reduced methane production [
20]; however, the current study provides evidence that
G. amansii supplementation can significantly increase
in vitro methane and carbon dioxide emission. In particular, increased methane production may have partially been due to an alteration in microbial diversity with an increase in the protozoan population (ciliate-associated methanogens) [
21], and a major member of the fibrolytic microorganism population,
R. flavefaciens [
22], resulting from
G. amansii treatments as compared with CON. However, methanogenic archaea,
R. albus, and
F. succinogenes (two other major members of the fibrolytic microorganism population) populations were significantly reduced. Ciliate-associated methanogens may generate up to 37% of methane produced in the rumen [
23]. Therefore, although an increase in ciliate-associated methanogens may help to explain the increase in methane production, a reduction in methanogenic archaea would counter it. With regards to the fibrolytic microorganism population,
R. flavefaciens normally produces succinic acid as a major fermentation product together with acetic and formic acids, H
2, and CO
2. Additionally,
R. albus is a very promising bacterium to produce H
2 from energy forage, with the potential of utilizing the cellulosic and hemicellulosic biomass [
24]. In contrast,
F. succinogenes is a non-H
2-producing species. The increase in the
R. flavefaciens population might be the culprit behind the increase in methane and CO
2 production. A previous study by Chaucheyras-Durand et al [
25] showed that methane production was clearly reduced when the dominant fibrolytic species was a non-H
2-producing species, such as
F. succinogenes, without significantly impairing fiber degradation and fermentations in the rumen. This was not the case in our study. As such, H
2 is of critical concern to the microbial ecosystem in ruminants. H
2 produced during enteric fermentation is the precursor of methane emission from ruminants, and the regulation of H
2, rather than methane, is the key to controlling ruminant methane emission. In addition, 80% of total enteric methane production is generated from carbon dioxide and hydrogen as a substrate [
26], which supports the positive correlation observed with methane and carbon dioxide production in our study. Increased methane emission is indicative of increased methanogen activity. The methanogens utilize mainly hydrogen and carbon dioxide, secondary fermentation products produced by rumen fermentation [
27], as well as acetate, as a substrate for methanogenesis [
28]. By removing hydrogen as the precursor of ruminal methane emission, methanogens allow the microorganisms involved in fermentation to function optimally and support the complete oxidation of substrates [
29]. Overall,
G. amansii supplementation resulted in increased methane production, which can be partially explained by increased methanogen activity (increased ciliate-associated methanogens) resulting from an increase in carbon dioxide production by the
R. flavefaciens populations, acetate concentration and A/P ratio; however, all these parameters are still within optimal fermentation conditions [
30]. In addition to having an effect on methane and carbon dioxide,
G. amansii supplementation also resulted in a significantly higher concentration of total VFA, acetate, propionate, butyrate, and A/P ratio being produced as compared to CON, demonstrating that fermentation was significantly affected.
G. amansii supplementation resulted in a significantly increased amount of microbial growth at 12, 48 and 72 h, as compared to CON, which is in agreement with Ha et al [
18], who suggested that rumen microorganisms need an adaptation period for changing environmental conditions of up to 6 h before their numbers increase, until nutrient depletion and waste products generated from microbial growth in the medium begin to inhibit their growth. Moreover, VFAs are released as the major end products of rumen microbial fermentation instead of glucose. Propionate is the most abundant of the glucogenic acids and the predominant substrate for gluconeogenesis in ruminants [
31]. Interestingly, both propionate and glucose concentration were significantly increased throughout the whole experimental period after
G. amansii supplementation, demonstrating a positive correlation with one another. Overall,
G. amansii supplementation resulted in a higher microbial growth rate, manifesting itself in the form of observed higher total gas and VFA production as compared with CON.
The objective of this study was to investigate and determine whether dietary supplementation of G. amansii could be useful for improving ruminal fermentation, as assessed by in vitro fermentation parameters. Overall, the results of our study indicate that G. amansii supplementation is potentially useful (i.e. may improve ruminant growth performance via increased total gas and VFA production), but does come with some undesirable effects. For example, G. amansii supplementation appears to increase methane production (increased methanogenic activity by ciliate-associated methanogens using increased H2 and CO2 being produced by an increased R. flavefaciens population), which is in disagreement with previous observations on Rhodophyta supplementation under in vitro fermentation conditions. More research is required to demonstrate and elucidate what G. amansii supplementation can do to improve growth performance and its effect on methane production in ruminants.