In our study, a thorough description of the protein and metabolite components of the animals´ saliva was achieved prior to incubation with rumen fluid. The immunological profiling of fresh saliva from the five goats and one sheep revealed relatively low concentrations of IgA compared to the 5.95 mg/ml recently reported in bovine saliva (Fouhse et al., 2017). Even though some previous works where ELISA was not used for quantification (Porter & Noakes, 1970) had difficulties at detecting IgA even after a 20 fold concentration using dialysis, others such as Mach & Pahud (1971) and Lascelles & McDowell (1974) reported much higher IgA saliva concentrations (560 µg/ml, 157 µg/ml; respectively) than what we detected in our study (37.6 µg/ml). As expected, the average concentration of IgA, which is the major immunoglobulin in ruminants’ saliva (Lascelles & McDowell, 1974), was ~4 fold higher than that of IgG. Interestingly, even though this IgA:IgG ratio was maintained in the sheep’s saliva, both concentrations were notably lower in comparison with the other salivas.
The total identified proteins across the salivas used in our study (195) was much greater than the 33 and 13 proteins annotated in sheep and goat saliva following a two-dimensional gel electrophoresis (2D PAGE) approach with two different spectrometry methods (Lamy et al., 2009, 2011; respectively). Despite the number of annotated proteins was much higher in our study, we hypothesize that this difference could partially be caused by the utilization of a protein database such as TrEMBL which, unlike SwissProt, contains computationally annotated protein features instead of manually reviewed annotated proteins. A comprehensive study of the bovine salivary proteome where similar nontargeted MS-MS approaches were used (Ang et al., 2011) identified an average of 179 proteins across different sample preparation methods, which is similar to our figure and slightly closer to the hundreds of proteins identified in human saliva studies (Loo et al., 2010). Like in our study, variability based on different methodologies used and/or animal specificity in previous works played a significant role in this high rate of detected proteins. Such a wide array of salivary proteins are involved in numerous physiological functions across the animal kingdom (Mandel, 1987). Despite the inter- and intraspecies variability with regards to salivary protein components in ruminants, these proteins seem to be involved in similar physiological functions (Ang et al., 2011). Indeed, the functional profile of the salivary proteins detected in cows (Ang et al., 2011) was pretty consistent with that found in goat and sheep proteins identified in our study, most of which are involved in nutrient-binding, transport, enzymatic activity and, to a less extent, immune response.
A previous in vitro study revealed that pre-incubation of specific diets (such as tannins-rich forages) with either sheep or goat saliva had a positive effect on diet degradation when incubated with rumen fluid (Ammar et al., 2013). On the other hand, other works have reported that the diet provided to ruminants and their saliva composition (including its protein fraction) have only minor effects on the rumen microbial activity (Ammar et al., 2011) and vice-versa (Salem et al., 2013). The lack of substantial effects found in these studies could be caused by the relatively short time of incubation (48 h) but also due to a missing exploration of the salivary proteome and metabolome, which we addressed in our study. In this context, the use of different diets or the inoculation with unique microbial strains have been suggested to induce a number of immunological mechanisms in the GIT (Yáñez-Ruiz et al., 2015). This has been reported to be of particular importance with regards to immunological proteins (mainly Ig), given that their concentration varies significantly depending on their rate of secretion through saliva (Subharat et al., 2016), which greatly depends on the presence of specific microorganisms in the rumen (Sharpe et al., 1977).
The metabolomic profile of the ruminants’ saliva has not been thoroughly explored to date. In general, research on the saliva metabolome has been focused on the identification and characterization of salivary biomarkers that could be used as indicators for the detection of a number of diseases (Yoshizawa et al., 2013). Other studies have attempted to better assess the metabolome composition throughout the gut, and the cross-effects that might take place between this and the host microbiota (Gardner et al., 2019; Nicholson et al., 2012). In our study, substantial amounts of polyethylene glycol derivatives were detected, which could come from the use of commercial sponges for collection. Overall, individual specificity on the saliva metabolome observed across our samples could most likely be driven by the unique microbiota present in each animal (Gardner et al., 2019), that altogether could be shaped by salivary proteins with immunological function (Palma-Hidalgo et al., 2021a). Moreover, the substantial differences between GOAT and SHEEP saliva observed in the proteome and metabolome indicated a species-specificity in the abundance of salivary compounds which could partially explain the rumen microbial differences observed between these two species in previous works (Henderson et al., 2015; Langda et al., 2020).
Our semi-continuous incubation system reached a peak of microbial activity in the first hours of incubation and then remained stable in terms of pH and gas production from 36 hours and thereafter. Through the last days of incubation, gas production was very low in AUT bottles compared with the rest, indicating that untreated saliva from goats or sheep contain bioactive components that enhance fermentative activity. At this stage of incubation, the saliva donor species was the most influential factor in vitro fermentation as the SHEEP saliva promoted the highest levels of fermentative activity (+4% gas production) as well as the greatest bacterial and protozoal concentrations (+2% and +7%, respectively). The high butyrate molar proportion and acetate: propionate ratio in SHEEP samples also suggest that a greater fibrolytic activity could have taken place by the more abundant rumen protozoa present in this treatment (Belanche et al., 2019; Eugène et al., 2004). These differences in in vitro rumen fermentation when incubating with saliva of the two small ruminants species were also reported by Ammar et al. (2013) when using tannins-rich substrates, which again suggests that the unique salivary composition of each species or even individuals may modulate microbial activity differently.
Incubation with AUT saliva led to the most divergent rumen microbial community in terms of overlapping ASVs with other treatments and general microbial composition. At phyla level, the relative abundance of the two main bacteria phyla across all treatments was 53% for Bacteroidetes and 30% for Firmicutes, a ratio (1.76) which is almost half (3.25) of what has been previously described in the rumen of goats (Palma-Hidalgo et al., 2021b). We hypothesize that the salivary proteins promoted the growth of Firmicutes bacteria, which have been demonstrated to be more abundant in the proximal GIT or the oral cavity (Fouhse et al., 2017; Yeoman et al., 2018). The salivary components of GOAT and SHEEP salivas also increased the proliferation of saliva-abundant Actinobacteria (Fouhse et al., 2017) which includes numerous species known for their ability to degrade complex compounds like fiber (Barka et al., 2016).
The three microbial taxa that contributed the most to make the AUT prokaryotic composition differ from the rest (particularly that from SHEEP), were Proteobacteria phylum and Prevotellaceae and Rikenellaceae families. With the exception of the AUT-abundant Succinivibrionaceae family, which has been recently correlated with animal growth and VFA production (Palma-Hidalgo et al., 2021b), Proteobacteria are commonly categorized as early rumen colonizers (Jami et al., 2013) and have been often associated with a suboptimal rumen microbial development. The greater abundance of this phylum in AUT treatment may suggest a deficient regulation by the lack of salivary bioactive components with immunological function, which were most likely denatured by autoclaving (Palma-Hidalgo et al., 2021a). This explanation would also be in line with the lower abundances in AUT samples of Prevotella 1 and Prevotellaceae (-22.3%), which is a cornerstone bacterial genus in the rumen and ruminant’s oral cavity (Rey et al., 2014; Tapio et al., 2016) and plays a pivotal role in the rumen metabolism (Precup & Vodnar, 2019). Given the harmless commensal nature of most Prevotella species in the rumen, it might be possible that its growth could be (directly or indirectly) stimulated when incubating with untreated salivas by modulation of salivary protein components, namely immunoglobulins, as it has been demonstrated with other commensal bacteria in mice (Donaldson et al., 2018; Peterson et al., 2007). Indeed, IgA and IgG and its different isoforms have been shown to modulate bacterial populations throughout the GIT (Tsuruta et al., 2012) to maintain mucosal homeostasis (Mantis et al., 2011). However, IgA tagged bovine oral or rumen microbiota have been reported to include significant lower abundance of Prevotellaceae compared to regular rumen microbiota (Fouhse et al., 2017). The high variability in the Ig concentrations in our study, and particularly the low concentrations (-34% IgA) in the sheep saliva coupled with the high abundances of Prevotellaceae in the SHEEP treatment, suggest that other immunological mechanisms driven by different proteins or molecules (e.g. cytokines, defensins, cathelicidins, miRNA; Yáñez-Ruiz et al., 2015) could also be involved in the stimulation or inhibition of the rumen microbes and their fermentative activity (Palma-Hidalgo et al., 2021a). The specificity of these modulatory mechanisms, which seems to vary moderately across species and individuals, may be partially responsible of the resilience and individual host specificity of the ruminal microbiota reported through complete rumen exchange experiments (Weimer, 2015). In line with this, our results suggest that the bioactive components of saliva, have a positive effect on the proliferation of crucial goat rumen bacteria as well as on the microbiota capable of degrading fibrous feeds. However, these positive effects on rumen microbial composition and activity are not as clear when goat rumen fluid is incubated with the specific salivary components of the same animal (OWN), indicating that the influx of new exogenous salivary elements could have synergistic effects on the rumen microbiome and fermentation. The different effects on the rumen microbial composition and activity seemed to be more notable across different species, which supports the fact that the specificity of the goat’s or sheep’s saliva composition (Lamy et al., 2009) observed in our study leads to the development of distinct microbial communities under similar dietary conditions (Langda et al., 2020).
Previous research on the rumen metabolome (Artegoitia et al., 2017; de Almeida et al., 2018) also resulted in the detection of thousands of distinct ‘raw’ metabolic features. The fact that, after quality filtering, the vast majority of them could not be reliably annotated using different search engines and libraries speaks for the great complexity of the rumen metabolome and how it could be a reservoir of novel compounds. The amount of annotated metabolites (19) compared with the 67 identified by de Almeida et al. (2018) made it difficult to discern clear effects of the incubation with different salivas on the rumen metabolomics profile. Despite this, the variability in the level of detection of the different metabolites indicate how its presence and abundance are most likely driven by salivary components per se or by the distinct microbial community (Gardner et al., 2019) modulated by saliva from different donors. The distinct salivary components of different species seem to have a strongest influence on the rumen content as they shape the rumen metabolome differently depending on whether they are constituents of goat’s or sheep’s saliva. Our data indicate that saliva components may partly responsible for the host species-specific rumen microbiota and related metabolites, potentially due to the co-evolution of the microbiome and host (Koskella & Bergelson, 2020).
The characterization of sheep and goats saliva showed distinct metabolomic and proteomic profiles across individuals and animal species, though the general functions (enzymatic, transport, immune response) remain consistent. Inactivating these compounds (i.e. autoclaving) exhibit an important change in the function of saliva in shaping rumen fermentation and microbial community. This finding together with the differences observed between species suggest that the cross-talk mechanisms between salivary components and rumen microbiota can be specific for individuals and/or species and that may contribute to the host selection of the commensal microbiota and its function.