OUTs cluster and abundance
16S rRNA gene (V3 -V4 regions) amplicon was used to determine the fecal microbiota composition of sheep and goats at different ages. The number of 16 s rRNA raw reads was varied among the animals. The total number of 16 s rRNA raw reads was 115,181. After quality-filtering with Geneious Prime, a total of 5,938 16S rRNA sequence reads were retained with read numbers per animal/fecal sample ranging from 106 to 2,420 (Table 1). The average length of the quality-checked and filtered sequences was 250 bp. All reads that passed quality filtering were clustered into OTUs at 95% identity. A total of 166 OTUs were generated. The number of OTUs per sample was ranged from 27 to 54. The number of OTUs reflects the richness of the sample. Ech 4 revealed the highest number of OTUs. Generally, sheep fecal samples were highly OTUs abundant than goats fecal samples even at the same age (Fig. 1).
Fecal microbiota composition and abundance
To describe the fecal microbiota composition of sheep and goats, a taxon-dependent analysis was carried out using the Sequence Classifier tool, (Table 2).
At the phyla level, a total of four bacterial phyla were identified in both sheep and goats regardless of age. Firmicutes and Proteobacteria were defined as the core phyla. At all ages, Firmicutes was the most abundant phylum (from 95.37–37.87%), followed by Proteobacteria (from 62.03–4.54%). The low-abundant phyla were Actinobacteria and Bacteroidetes, (Fig. 2). At 6-month-old, the phylum Firmicutes dominated all bacterial communities (93.01% in sheep, 37.87% in goat); while the phylum Proteobacteria was dramatically higher in goat (62.03%) than in sheep (6.88%). Noteworthy, the phylum Actinobacteria shared the same abundance percentage in both sheep and goat (0.09). The phylum Bacteroidetes was absent in both sheep and goat. At 12-month-old, the abundance of the phylum Firmicutes in goat (95.37%) was noticeably more than sheep (72.76%). On the other hand, the phylum Proteobacteria was more abundant in sheep (26.83%) than goat (4.54%). Furthermore, the phylum Actinobacteria was less abundant in goat (0.10%) than sheep (0.35%). %). Although the phylum Bacteroidetes was absent in goat, the phylum Bacteroidetes had the least abundance in sheep (0.06%).
At the class level, a total of seven bacterial classes were identified in sheep and goats. Clostridia, Bacilli, and Gammaproteobacteria were defined as the core classes. Clostridia and Bacilli were more abundant, followed by Gammaproteobacteria. On the other hand, Coriobacteriia, Tissierellia, Erysipelotrichia, and Negativicutes were less abundant. At 6-month-old, most dominant classes were Clostridia (4.71% in sheep, 0.41% in goat), Bacilli (3.77% in sheep, 0.28% in goat) and Gammaproteobacteria (0.94% in sheep, 0.08% in goat). It is important to highlight that Clostridia and Bacilli were more abundant in sheep than goats; while, Tissierellia, Erysipelotrichia, and Negativicutes were only found in goat at an abundance rate of 0.17%, 0.04%, and 0.04%, respectively. Furthermore, the class Coriobacteriia was absent in both sheep and goat. At 12-month-old, the class composition was similar to the animals of the 6-month-old. The dominant classes were Clostridia (0.88% in sheep, 0.86% in goat), Bacilli (0.54% in sheep, 0.51% in goat) and Gammaproteobacteria (0.10% in sheep, 0.22% in goat). Although the classes Coriobacteriia and Negativicutes were absent in goat, the class Coriobacteriia had a low abundance in sheep (0.05%).
At the order level, there were nine bacterial classes identified in both sheep and goats. Regardless of which age were, Clostridiales (4.71–0.41%), Lactobacillales (2.83–0.05%) and Bacillales (0.94% to 0.12) were defined as the core orders. At 6-month-old, Clostridiales (4.71% in sheep, 0.41% in goat), Lactobacillales (2.83% in sheep, 0.17% in goat) and Bacillales (0.94% in sheep, 0.12% in goat) were the most dominant orders. Notably, they were more abundant in sheep than goats. The classes Tissierellales (0.17%), Enterobacterales (0.08%), Bifidobacteriales (0.08%) and Erysipelotrichales (0.04%) only found in goat. Whereas, the classes Coriobacteriales and Bacteroidales were absent in both sheep and goat. At 12-month-old, the results showed that most abundant orders were Enterobacterales (0.05% in sheep, 0.22% in goat), Lactobacillales (0.22% in sheep 0.05, 0.29% in goat), Clostridiales (0.88% in sheep, 0.79% in goat), Bacillales (0.25% in sheep, 0.29% in goat), Tissierellales (0.05% in sheep, 0.07% in goat),Erysipelotrichales (0.05% in sheep, 0.07% in goat) and Bifidobacteriales (0.05%in sheep, 0.07% in goat). An interesting contrast, the order Coriobacteriales only found in sheep but the order Bacteroidales only found in goat.
At the family level, twenty families were identified in both sheep and goats. Despite of the age difference, the families, Enterococcaceae (1.88–0.05%), Lachnospiraceae (0.94–0.14%), Peptostreptococcaceae (0.94%to 0.08%) and Bacillaceae (0.94–0.08%) were defined as the core orders. At 6-month-old, the families Enterococcaceae (1.88% in sheep, 0.08% in goat), Lachnospiraceae (0.94% in sheep, 0.17% in goat), Bacillaceae (0.94% in sheep, 0.08% in goat) and Peptostreptococcaceae (0.94% in sheep, 0.08% in goat) were core families in both species. Although the family Clostridiaceae had the highest abundance percentage (3.77%), the family Clostridiaceae only found in sheep. Some families not found in both sheep and goats including Aerococcaceae, Ruminococcaceae, Carnobacteriaceae, Oscillospiraceae, Rikenellaceae, Bacteroidaceae, Atopobiaceae and Gottschalkiaceae. At 12-month-old, the dominant families in both sheep and goats were Enterococcaceae (0.05% in sheep, 0.14% in goat), Lachnospiraceae (0.25% in sheep, 0.14% in goat), Bacillaceae (0.19% in sheep, 0.14% in goat), Peptostreptococcaceae (0.25% in sheep, 0.29%in goat), Streptococcaceae (0.10% in sheep, 0.07%in goat), Enterobacteriaceae (0.10% in sheep, 0.29% in goat), Bifidobacteriaceae (0.05% in sheep, 0.07% in goat), Erysipelotrichaceae (0.05% in sheep, 0.07% in goat),Oscillospiraceae (0.10% in sheep, 0.07% in goat) and Bacteroidaceae (0.05% in sheep, 0.07% in goat). In contract, the family Sporomusaceae was found neither in sheep nor in goats.
At the genus level, a total of 36 genera was identified. Of these, 10 were identified in both sheep and goats regardless to age including Escherichia (40.12–5.49%), Clostridium (19.38–0.22%), Enterococcus (5.03–1.73%), Lysinibacillus (76.95–7.25%), Streptococcus (2.83–1.88%), Anaerocolumna (1.63–0.94%), Anaerotignum (35.95–4.93%), Muricomes (0.99–0.21%), Tissierella (0.91–0.39%) and Bifidobacterium (0.51–0.21%), (Fig. 3). At 6-month-old, the core genera were Escherichia, Enterococcus, Lysinibacillus, Streptococcus, and Anaerocolumna. On the other hand, the genera Vagococcus, Shigella, Paraclostridium, Paeniclostridium, Bacillus, Lactonifactor, Mageeibacillus, Kroppenstedtia, Alistipes, Faecalicatena, Intestinimonas, Atopobium, Gracilibacter, Bacteroides, Butyricicoccus, Mogibacterium, Abiotrophia, Papillibacter, Coprococcus, and Coprococcus were not found either in sheep or goats. Notably, Lysinibacillus in sheep (76.42%), as well as Anaerotignum in goats (35.95%), were significantly more abundant. Figure 4 demonstrates sheep and goats fecal microbiota composition at the genus level at 6-month-old. At 12-month-old, the dominant genera that identified in both sheep and goats were Escherichia, Clostridium, Enterococcus, Lysinibacillus, Streptococcus, Romboutsia, Paeniclostridium, Bacteroides and Bifidobacterium. Differently, the genera Falcatimonas, Sporobacter and Desnuesiella were not found either in sheep or goats. It is worth to mention that Escherichia (21.99%) and Clostridium (19.38%) remarkably more frequent but only in sheep. Figure 5 demonstrates Sheep and goats fecal microbiota composition at the genus level at 12-month-old.
Microbial community richness and diversity
Alpha diversity is applied for analyzing the complexity of species diversity for a sample and/or group of samples. Three indices were used; the Shannon index, the observed species, and the Chao1 index. The indices indicated that the highest level for the complexity of species diversity was detected in sheep fecal samples at 12 months age, while the lowest level of complexity was observed in the goat fecal samples at the same age. The group α-diversity analysis result designated sheep feces as more complex regarding its species diversity (Table 3), (Fig. 6).