Crude nutrients and fermentation products
Significant differences in the factor habitat were found for all nutrient groups (p < 0.001, Total lipids p = 0.037), but this was caused by a few habitats. For example, a significantly high crude protein content in habitats AG and SF, a significantly high NFC content in the alpine habitats, or a significantly high total fibre content in the forest habitats PF, BF and SF (Fig. 1, Table S3). The situation is similar for the seasonal factor (p ≤ 0.005), except for crude ash (p = 0.082) and hemicellulose (p = 0.226). Regarding the season, the nutrient group matters a lot, but significant differences are often determined by winter (Table S4-S10). The gender only causes a significant difference in the crude ash content (p = 0.002). And the age class causes a significant difference in all of the nutrient groups (p ≤ 0.021), with the exception of crude ash (p = 0.108) and NFC (p = 0.14). In most cases, the adult and juvenile animals do not differ significantly from each other. Significant differences were mainly found between subadults and the two other age classes.
Ammonia (p < 0.001) and lactate (p < 0.001) levels differ significantly between habitats (Fig. 2, Table S3). The significant differences are mainly due to the low ammonia content in habitats SF and AMF and the high content in habitats ABF and AG. The lactate content is significantly lower, especially in habitats BF and AMF and highest in habitat ABF. Gender and age class do not make a significant difference. However, the ammonia content differs significantly between the seasons (p < 0.001), the lactate content does not (p = 0.179) (Table S4-S10).
There is a significant difference regarding the factor habitat for all VFA´s (p < 0.01). This is mainly due to low levels of acetic acid in habitat SF, propionic acid in habitat BF and butyric, isobutyric, valeric and isovaleric acid in forest habitats BF, PF and SF (Fig. 3, Table S3). Significantly high concentrations of acetic acid are found in the habitats ABF and AMF, of propionic acid in the habitats ABF and AG, and of butyric and valeric acid mainly in the alpine habitats AMF and GSF. Isobutyric and isovaleric acid are highest in the habitats ABF, AG and GSF. There is also a significant difference between the age classes (P ≤ 0.03), with the exception of propionic acid (p = 0.432), and the seasons (p < 0.001) (Table S4-S10). The significant differences between the seasons are mainly due to low values in winter for all VFA´s. Gender, in turn, does not cause a significant difference in the content of VFA´s.
Habitat AG (Table S3, S4): The crude nutrients in the rumen content had a low total fibre content. The available total fibre content was mainly characterized by a high amount of hemicellulose (16.5%), whereas cellulose with 17% and lignin with 12% are below the average. The NFC content was medium with 14.3% and the crude protein content was the highest of all habitats (26.8%, closely followed by habitat SF). Significant seasonal fluctuations occurred for lignin and NFC. The lignin content was highest in spring (13.8%) and winter (13.8%) and lowest in autumn (9.9%). Antagonistically, the NFC content was the highest in autumn and the lowest in spring. The protein content was highest in spring with 31% and lowest in winter with 23.7%. Ammonia and lactate concentrations have also their lowest point in winter. Ammonia increased again significantly in spring up to 416 mg/l, while lactate was still low and reached its maximum values in summer and autumn (around 700 mg/ml). Propionic, butyric and valeric acid showed similar patterns throughout the year, with the highest concentration in autumn and the lowest in spring.
Habitat ABF (Table S3, S5): Overall, there was a very low total fibre content in the rumen content (46% NDF), with a peak in summer (contrary to the other habitats) and with low amounts of lignin (12.3%). Crude protein (23%) and NFC (16.3%) content were within the Bavarian average. Significant seasonal variations occurred only for the NFC content with highest amounts in autumn (18.1%), when more tree fruits, such as acorns and beechnuts, were consumed. The average concentration of lactate (572 mg/l) and propionic acid (1.88 mg/ml) were above the average of all other investigated habitats. Ammonia concentration was the highest of all habitats with 430 mg/l. Iso-butyric acid (0.06 mg/ml) and iso-valeric acid (0.12 mg/ml) were also significantly enhanced compared to other habitats.
Habitat BF (Table S3, S6): The total fibre content in the rumen was relatively high with 50.2%, NFC (14.6%) and crude protein (22.7%) were in medium range compared to other habitats. We found significant seasonal differences in fibres (especially hemicellulose), proteins and NFC´s. Crude protein and NFC increased significantly during this season. Lactate (172.6 mg/l), propionic (1.42 mg/ml), butyric (0.86 mg/ml) and isovaleric acid (0.05 mg/ml) occur in very low concentrations.
Habitat PF (Table S3, S7): The rumen content had significantly high total fibre contents (54.5% NDF) and all fibre fractions also had their highest proportions in this habitat. The NFC content is only 13.5% and the crude protein content was significantly low and the lowest of all habitats, with 19.8% on average. The proportion of valeric acid was also very low with only 0.15 mg/ml. Strong seasonal fluctuations occurred especially in the fibre content (cellulose and lignin) and crude protein, as well as in some fermentation products. NDF, cellulose and lignin reached their maximum value in winter and their lowest value in summer. The course of the crude protein content is antagonistic to this. Hemicellulose content was highest in spring.
Habitat SF (Table S3, S8): The total fibre content in the rumen was relatively high with 51% NDF, with comparatively few celluloses and more hemicellulose. The crude protein content was nearly the highest with 26.78%, just behind habitat AL, and the NFC content was the lowest of all habitats with 7.9% (peak in autumn with 9.4%). The course of crude protein and total fibre content were antagonistic with the highest content of total fibre in winter and the lowest in summer. The proportion of cellulose (17.5%) was comparatively low compared to other habitats, and the proportion of hemicellulose (16.8%) was high. The curves of the fibre fractions were similar throughout the year, except for summer. While hemicellulose and lignin reached their lowest value here, the cellulose fraction was high in this season. In addition, the concentration of ammonia was the lowest at 237 mg/l. Significant seasonal differences were only found for total lipids and some fermentation products.
Habitat GSF (Table S3, S9): The total fibre (46% NDF) and crude protein content (23%) was slightly below average (∅ 23.6 & 48%). The NFC content, on the other hand, was high with nearly 18%. Regarding the fibre fractions, the hemicellulose content was the lowest of all habitats (10.5%). In contrast, the lignin (15.1%) and the cellulose content (20.4%) was quite high. The total fibre content (NDF), as well as all fibre fractions, had their peak values in winter. Cellulose had a second peak in summer. The crude protein content was highest in spring. Moreover, the butyric acid (1.36 mg/ml) and the valeric acid content (0.28 mg/ml) was the highest of all habitats. Seasonal variations occurred mainly in protein and total fibre content, as well as in some fatty acids.
Habitat AMF (Table S3, S10): The proportion of total fibres was low (44.3% NDF), NDF and all fibre fractions had the highest content in winter. The crude protein content was medium (23.3%) with a peak in spring and the NFC content was highest in this habitat (18.6%), with highest proportions in summer and autumn. Furthermore, the concentration of butyric acid (1.33 mg/ml) and valeric acid (0.27 mg/ml) was high and the concentration of ammonia (286 mg/l) and lactate (302 mg/l) was relatively low. The typical strong seasonal fluctuations in the alpine region were also reflected in the distribution of nutrients. Except for NFC and hemicellulose, there are significant seasonal differences for all groups
Variation in the composition of the rumen microbiota
The bacterial microbiota in the rumen content of roe deer differed significantly in terms of habitat (p < 0.01), season (p < 0.01) and age class (p < 0.01, adult vs. subadult vs. juvenile). Most significant differences exist between the juvenile and subadult age classes. Gender was not causing a significant difference. The principal coordinate analysis (PCO) (Fig. 4A) showed a clustering of samples from habitats SF, GSF, AMF and BF on the left side of the plot. The smallest distance is between habitat GSF and AMF (77% dissimilarity). The second cluster of similarity is formed among habitat AG, ABF and PF on the right side of the plot. The Shannon diversity analysis (Fig. 4B) showed that habitat GSF has the highest ruminal microbial alpha diversity and habitat AG has the lowest.
On average, Firmicutes is the most frequent phylum in the roe deer rumen content (58.7%), followed by the Bacteroidetes (18.4%), Actinobacteria (16%), Candidatus Saccharibacteria (2.2%) (formerly known as TM7), Spirochaetes (1.9%) and Proteobacteria (1.8%). Synergistetes (0.2%), Tenericutes (0.1%), Chloroflexi (0.1%) and the phylum SR1 (0.03%) were detected with a proportion of less than 1%. A low proportion of 0.56% of the sequences are unclassified bacteria and could not even be determined at phylum level. The proportions of Firmicutes, Bacteroidetes and Synergistetes were similar in all habitats. However, significant differences exist between the habitats within all the remaining phyla (Table S11).
Thirty seven out of 55 genera were assigned to unclassified bacteria at various taxonomic levels. These unclassified genera correspond to 87.3% (82–90%, depending on habitat) of the sequences. This suggests a high number of still unknown genera in the roe deer rumen content. At the family level there are 66.8% unclassified sequences; at the order level 24.9%, and at the class level 4.6%.
Among the classified genera, Prevotella, Olsenella, Paraburkholderia, Streptococcus and Ralstonia were dominant. Overall, the unclassified Clostridiales is the most common group with an average of 35.2%, and its therefore also the leading contributor to the core microbiota. The common representative genera among all samples, named core microbiota according to Henderson et al. [22], consists of 12 genera (Fig. 5), forming 87–90% of the total microbiota (Fig. S2).
A habitat-specific discrimination analysis (LEfSe, Linear discriminant analysis Effect Size) was done to identify genera that contributed significantly to the community structure per habitat (Fig. 6). The single genera are named in the following habitat descriptions (Fig. S3, Table S11 & S12).
Habitat AG: The microbiota at phylum level is composed of significantly less SR1 (< SF, AMF, GSF, p ≤ 0.013), Tenericutes (< SF, GSF, p ≤ 0.049) and Spirochaetes (< ABF, p = 0.019) compared to some of the other habitats. Important genera (LEfSe) were Prevotella, Olsenella and Paraburkholderia. Paraburkholderia (> PF, ABF, GSF, AMF, BF, p ≥ 0.031), Clostridium sensu stricto (> BF, p ≥ 0.011) and uncl. Spirochaetaceae (> BF, p ≥ 0.001) occurred in significantly higher abundances.
Habitat ABF: The microbiota at phylum level is composed of significantly less Actinobacteria (lowest of all habitats, < PF, SF, AMF, BF, p ≤ 0.019) and Candidatus Saccharibacteria (lowest of all, < SF, BF, GSF, AMF, p ≤ 0.043) and significant more Spirochaetes (most of all, > BF, SF, PF, AG, p ≥ 0.019) compared to some of the other habitats. Important genera (LEfSe), were unclassified Bacteroidia, unclassified Spirochaetia and Ralstonia. Ralstonia (> SF, AG, BF, p < 0.001; peak in autumn), uncl. Spirochaetia (> BF, SF, AG, PF, p ≤ 0.008), uncl. Spirochaetaceae (> BF, p < 0.001), Enterococcus (> PF, p = 0.007), uncl. Christenellaceae (> AG, p = 0.006), uncl. Muribaculaceae (> BF, GSF, AMF, p ≥ 0.02) occured in significantly higher abundances. Olsenella was significantly less abundant (< BF, p = 0.014).
Habitat BF: The microbiota at phylum level is composed of significantly more Actinobacteria (most off all habitats, > ABF, p < 0.001) and less Spirochaetes (< AMF, GSF, ABF, p ≤ 0.002) and SR1 (< SF, AMF, GSF, p ≤ 0.01), compared to some of the other habitats. At the genera level, significantly more Olsenella (> GSF, p = 0.013), uncl. Clostridia (> AG, p = 0.001), uncl. Coriobacteriia (> ABF, AG, p = 0.045), uncl. Eggerthellaceae (> AG, ABF, p < 0.001), Synthrophococcus (> GSF, p = 0.001), uncl. Actinomycetales (> ABF, AG, AMF, p ≤ 0.025), uncl. Eggerthellales (> ABF, p = 0.001) and uncl. Rhodobacterales (> AG, SF, PF, p ≤ 0.032) occurred. Prevotella was significantly less abundant (> SF, p = 0.043).
Habitat PF: The microbiota at phylum level is composed of significantly less Proteobacteria (lowest of all habitats, < ABF, p = 0.019), Candidatus Saccharibacteria (< BF, GSF, AMF, p ≤ 0.017), Spirochaetes (< ABF, p = 0.002), SR1 (< SF, AMF, GSF, p ≤ 0.008) and Tenericutes (< SF, GSF, p ≤ 0.01), compared to some of the other habitats. In this habitat, also seasonal effects were already reflected at the phylum level. A significant difference in the abundance of Bacteroidetes between winter vs. autumn (p = 0.032) was identified, with a decrease over the year, from winter to autumn. Firmicutes (p = 0.05), unclassified Bacteria (p = 0.039) and SR1 (p = 0.025) differed significantly between autumn vs. spring. Important genera (LEfSe) were uncl. Ruminococcaceae, uncl. Eggerthellaceae and Streptococcus. Streptococci occurred almost exclusively in summer, decreased abundance in autumn, and reached zero in spring and winter. Uncl. Eggerthellaceae (> AG, ABF, p ≥ 0.003) and Synthrophococcus (> GSF, p < 0.001) were significantly higher abundant.
Habitat SF: The microbiota at phylum level is composed of significantly more Spirochaetes (< ABF, p < 0.001) and less Tenericutes (> PF, AG, p ≤ 0.049) compared to some of the other habitats. One important genus (LEfSe) of significant high abundance was uncl. Prevotellaceae (> PF, BF ≤ 0.021). Further genera, that occurred in higher abundance were Paraburkholderia (> PF, ABF, GSF, AMF, BF, p ≤ 0.002), uncl. Eggerthellaceae (> AG, ABF, p ≤ 0.009), uncl. Eggerthellales (> ABF, p = 0.001) and uncl. Mollicutes (> PF, AG, P ≤ 0.049).
Habitat GSF: The microbiota at phylum level is composed of significantly more SR1 (> PF, BF, AG, ABF, p ≤ 0.002) and Tenericutes (> PF, AG, p ≤ 0.002), compared to some of the other habitats. Significant seasonal variations occurred in Spirochaetes (highest in autumn, p = 0.004) and Synergistetes (highest in summer, p = 0.007). Important genera (LEfSe) were uncl. Clostridia (> AG, GSF, ABF, p ≤ 0.015) and uncl. Spirochaetaceae (> BF, PF, p ≤ 0.01), which occurred in significantly high abundances. Moreover, there occur statistically significant higher abundances in Ralstonia (> SF, AG, BF, p ≤ 0.033), Anaerobutyricum (> AG, PF, ABF, p < 0.001), Butyrivibrio (> BF, PF, AG, ABF, AMF, p ≤ 0.001), Caballeronia (> PF, AG, BF, ABF, SF, p ≤ 0.044), Lactobacillus (> PF, AG, ABF, p < 0.001), Roseburia (> AG, PF, ABF, p < 0.001), Mollicutes (> PF, AG, p ≤ 0.002), uncl. Prevotellaceae (> PF, BF, p ≤ 0.007), uncl. Eggerthellaceae (> AG, ABF, p ≤ 0.028), uncl. Coriobactariia (> ABF, p = 0.001) and uncl. Actinomycetaceae (> BF, p < 0.001). Olsenella occurred in significantly less abundances (< BF, p = 0.013).
Habitat AMF: The microbiota at phylum level is composed of significantly more Chloroflexi (most and significant more of all, p < 0.001) and Candidatus Saccharibacteria (> ABF, PF, < 0.001). Significant seasonal variations were only found for Synergistetes between winter and spring, being higher in the latter. One important genus (LEfSe) was uncl. Coriobacteriia (> ABF, AG, PF, p < 0.001), with the highest abundances found in the samples from summer. Moreover, other genera also presented significant differences, among them were Adlerkreutzia (> ABF, AG, PF, BF, p ≤ 0.04), Caballeronia (> PF, AG, BF, ABF, p < 0.001), Roseburia (> AG, PF, ABF, p < 0.001), uncl. Clostridia (> AG, PF, p ≤ 0.016), uncl. Prevotellaceae (> PF, p = 0.007), uncl. Spirochaetaceae ((> BF, PF, p ≤ 0.002), uncl. Spirochaetia (> BF, p = 0.031), uncl. Aggregatilineales (> all habitats, p < 0.001) and uncl. Negativicutes (> AG, BF, PF, p ≤ 0.011).