During this study, a total of 50 caecal pools (25 per breed) were collected, processed and sequenced. No clinical signs were observed, and the productive parameters obtained were in accordance with the breed standards. There were no statistical differences between replicates (P-value > 0.05).
16 rRNA profiling of fast and slow-growing breeds
The MiSeq sequencing of the 50 samples produced a total of 14 143 246 sequencing reads with an average of 282 864.9 reads per sample. Quality and chimera filtering produced a total of 12 661 675 filtered reads with an average of 253 233.5 reads per sample and ranging from 109 447 to 356 331 reads.
Assessment of rarefaction curves based on the Chao1 biodiversity index calculated for the six sequence read groups (day-old chicks, mid-period and slaughter day results for fast and slow-growing breeds) indicated that four of the curves tended to reach a plateau. However, samples from groups 1 and 2 (day-old chicks from both breeds) are at the limit of the rarefaction, leaving a rarefaction number of 72 060 reads (Fig. 1). The Chao1 alpha diversity index reveals a notable difference between the caecal microbiota depending on the age of the animals (Table 1). Statistically significant differences (P-value < 0.05) were found between these groups; samples from day-old chicks of both breeds (88.3 and 111.9 for the fast and slow-growing breed, respectively) displayed a lower level of complexity of the microbiota compared to that found at mid-period (384.4 and 373.8), and samples from mid-period animals displayed a lower level of complexity than the samples from the end of the growing period (420.3 and 447.2 for the fast and slow-growing breed, respectively). Finally, there were statistically significant differences in gut microbiota diversity between both breeds at the end of the growing period (P-value < 0.05).
Table 1
Alpha diversity according to time of the growing period in fast (FG) and slow-growing (SG) breeds.
SAMPLING TIME | Fast-growing | Slow-growing |
Arrival day | 88.3a | 111.9a |
Mid-period | 384.4b | 373.8b |
End | 420.3c | 447.2d |
a, b, c, d: Different superscripts means significant differences with a P-value < 0.05. |
Differential gut microbiota composition
Inspection of predicted taxonomic profiles at phylum level for all samples is summarised in Table 2 and represented in Fig. 2. This analysis exhibited that Firmicutes represented the dominant phylum of the caecal community in both breeds at all sampling times in the production cycle (P-value < 0.05). At the onset of the growing period, Proteobacteria was the second prevalent phylum for fast and slow-growing breeds, outnumbering the Bacteroidetes phylum. However, during the rest of the production cycle, Bacteroidetes phylum was more abundant than Proteobacteria in both breeds. The longitudinal study showed that there were no statistically differences between breeds throughout the growing period (P-value > 0.05).
For the fast-growing breed, there were statistically significant differences depending on the time of sampling. Proteobacteria and Bacteroidetes phyla were more abundant at the arrival day (36.4% and 5%, respectively) and at the slaughter day (1.5% and 5.7%, respectively), though the high Firmicutes percentage was observed at mid-period (95.1%).
For the slow-growing breed, Bacteroidetes (5.7% and 9.3% at arrival and slaughter days, respectively) and Firmicutes (95.2% at mid-period) showed the same pattern as in the fast-growing breed. However, statistically significant differences were shown between day-old chicks and mid-period percentage of Proteobacteria (32.8% and 1.2%, respectively), which subsequently remained stable until the end of the cycle (1.7%).
Table 2
Taxonomic profiles at phylum level according to time of growing period in fast (FG) and slow-growing (SG) breeds.
Sampling time | Arrival day | Mid-period | End |
Breed | FG | SG | FG | SG | FG | SG |
Actinobacteria | 0% | 0.2% | 0.3% | 0.3% | 0.5% | 0.4% |
Bacteroidetes | 5.0% | 5.7% | 1.9% | 1.9% | 5.7%b | 9.3%a |
Cyanobacteria | 0% | 0% | 0.5% | 0.4% | 0.7%b | 1.1%a |
Firmicutes | 58.6% | 61.1% | 95.1% | 95.2% | 90.3%a | 85.6%b |
Proteobacteria | 36.4% | 32.8% | 1.3% | 1.2% | 1.5%b | 1.7%a |
Tenericutes | 0% | 0.2% | 0.3%b | 0.4%a | 0.6% | 1.1% |
Unassigned | 0% | 0% | 0.6% | 0.6% | 0.8% | 0.8% |
a, b: Different superscripts between breeds in the different sampling times means significant differences with a P-value < 0.05. |
Furthermore, 46 taxa were identified at genus level (Fig. 3). Whereas 20 appeared to be present in all samples, 3 appeared to only be present in caecum samples of slow-growing breed and 23 only appeared at some times of the growing period. The most predominant genera identified were Oscillospira spp. (7.5%), Ruminococcus spp. (3.6%), Coprococcus spp. (2.9%), Lactobacillus spp. (2.5%) and Bacteroides spp. (2.0%). In the longitudinal study, the only statistical differences were between breeds in Lactobacillus spp. at the end of the growing period (2.9% and 2.8% in fast and slow-growing breed, respectively). In order to further identify differences in microbiota composition between breeds, we focused on 33 genera, which were shown to be present at an average relative abundance of more than 0.5% in at least one sample group [35].
For the fast-growing breed, the results for the genera analysis are shown in Table 3. At the arrival day, predominant bacteria of microbiota were Unclassified members (U. m.) of the Enterobacteriaceae family (36.4%), U. m. of Clostridiaceae family (6.2%), U. m. of the Ruminococcaceae family (5.7%), U. m. of Lachnospiraceae family (4.9%), Clostridium spp. (4.1%), U. m. of Enterococcaceae family (3.7%), Oscillospira spp. (3.5%) and Enterococcus spp. (3.0%).
Table 3
Taxonomic profiles at genus level according to sampling time in fast-growing breed.
Phylum | Family | Genus | Arrival day | Mid-period | End |
Unassigned | 0.0% | 0.6% | 0.8% |
Bacteroidetes | Bacteroidaceae | Bacteroides | 1.5% | 0.5% | 3.1% |
Porphyromonadaceae | Parabacteroides | 1.2% | 0.4% | 0.7% |
Rikenellaceae | - | 2.0% | 1.1% | 1.2% |
Odoribacteraceae | Butyricimonas | 0.3% | 0.0% | 0.7% |
Cyanobacteria | - | - | 0.0% | 0.5% | 0.7% |
Firmicutes | Plaococcaceae | - | 0.0% | 0.5% | 0.4% |
Enterococcaceae | - | 3.7% | 0.0% | 0.0% |
Enterococcus | 3.0% | 0.2% | 0.1% |
Lactobacillaceae | Lactobacillus | 0.9% | 3.9% | 2.8% |
- | - | 0.2% | 0.5% | 0.6% |
- | - | 13.7% | 29.4% | 28.9% |
Christensenellaceae | - | 0.0% | 0.2% | 0.6% |
Clostridiaceae | - | 0.6% | 0.0% | 0.3% |
- | 5.6% | 0.2% | 0.2% |
Clostridium | 4.1% | 0.5% | 0.5% |
Lachnospiraceae | - | 4.9% | 10.4% | 10.2% |
Blauria | 0.7% | 2.0% | 2.1% |
Coprococcus | 1.6% | 4.0% | 3.5% |
Dorea | 0.2% | 1.4% | 1.1% |
Epulopscium | 2.6% | 0.0% | 0.0% |
[Ruminococcus] | 2.5% | 3.3% | 2.9% |
Ruminococcaceae | - | 5.7% | 18.1% | 17.7% |
Anaerotruncus | 0.0% | 0.5% | 0.4% |
Faecalibacterium | 0.9% | 1.5% | 2.0% |
Oscillospira | 3.5% | 9.6% | 8.8% |
Ruminococcus | 2.1% | 5.0% | 4.4% |
Erysipelotrichaceae | - | 0.9% | 0.9% | 0.4% |
Coprobacillus | 0.4% | 0.9% | 0.5% |
cc_115 | 0.0% | 0.9% | 0.6% |
Proteobacteria | Enterobacteriaceae | - | 36.4% | 1.3% | 1.5% |
At mid-period, the predominant genera in caecal samples were U. m. of the Ruminococcaceae family (18.1%), U. m. of Lachnospiraceae family (10.4%), Oscillospira spp. (9.6%), Coprococcus spp. (4.0%), Lactobacillus spp. (3.9%) and [Ruminococcus] spp. (3.3%). Finally, at the end of the growing period, the most prevalent bacteria were U. m. of the Ruminococcaceae family (17.7%), U. m. of Lachnospiraceae family (10.2%), Oscillospira spp. (8.8%), Coprococcus spp. (3.5%) and Bacteroides spp. (3.1%).
For the slow-growing breed, the results for the genera analysis are shown in Table 4. The pattern for day-old chicks was similar to that observed at this sampling time for the fast-growing breed. The most abundant bacteria were U. m. of the Enterobacteriaceae family (32.6%), U. m. of the Ruminococcaceae family (7.5%), U. m. of Lachnospiraceae family (6.5%), Oscillospira spp. (5.8%), U. m. of Clostridiaceae family (4.8%) and U. m. of Enterococcaceae family (3.6%). At mid-period, predominant genera were U. m. of the Ruminococcaceae family (18.4%), U. m. of Lachnospiraceae family (10.3%), Oscillospira spp. (9.6%), Coprococcus spp. (3.8%), Lactobacillus spp. (3.4%) and [Ruminococcus] spp. (3.3%). Lastly, at slaughter day, U. m. of the Ruminococcaceae family (17.0%) were the most abundant bacteria, followed by U. m. of Lachnospiraceae family (8.6%), Oscillospira spp. (7.7%), Coprococcus spp. (3.2%), Bacteroides spp. (4.1%) and Parabacteroides spp. (3.1%).
Table 4
Taxonomic profiles at genus level according to sampling time in slow-growing breed.
Phylum | Family | Genus | Arrival day | Mid-period | End |
Unassigned | 0.0% | 0.6% | 0.8% |
Bacteroidetes | Bacteroidaceae | Bacteroides | 2.6% | 0.4% | 4.1% |
Porphyromonadaceae | Parabacteroides | 1.0% | 0.5% | 1.1% |
Rikenellaceae | - | 2.0% | 1.1% | 3.1% |
Odoribacteraceae | Butyricimonas | 0.0% | 0.0% | 1.1% |
Cyanobacteria | | | 0.0% | 0.4% | 1.1% |
Firmicutes | Plaococcaceae | - | 0.2% | 0.5% | 0.4% |
Enterococcaceae | - | 3.6% | 0.0% | 0.0% |
Enterococcus | 1.0% | 0.2% | 0.4% |
Lactobacillaceae | Lactobacillus | 1.2% | 3.4% | 2.9% |
- | - | 0.4% | 0.6% | 0.3% |
- | - | 14.6% | 29.9% | 30.0% |
Clostridiaceae | - | 4.8% | 0.2% | 0.3% |
Clostridium | 2.7% | 0.4% | 0.4% |
Lachnospiraceae | - | 6.5% | 10.3% | 8.6% |
Blauria | 0.8% | 1.8% | 1.5% |
Coprococcus | 1.6% | 3.8% | 3.2% |
Dorea | 0.8% | 1.3% | 0.7% |
Epulopscium | 2.4% | 0.0% | 0.0% |
Ruminococcus | 2.1% | 3.3% | 2.3% |
Ruminococcaceae | - | 7.5% | 18.4% | 17.0% |
Anaerotruncus | 0.0% | 0.5% | 0.3% |
Faecalibacterium | 1.5% | 1.8% | 1.5% |
Oscillospira | 5.8% | 9.6% | 7.7% |
Ruminococcus | 1.7% | 5.1% | 3.6% |
Erysipelotrichaceae | - | 1.0% | 0.9% | 0.6% |
Coprobacillus | 0.4% | 0.9% | 0.5% |
cc_115 | 0.0% | 0.8% | 0.6% |
Proteobacteria | Enterobacteriaceae | - | 32.6% | 1.2% | 0.9% |
Tenericutes | - | - | 0.2% | 0.4% | 0.7% |
Finally, in order to assess differences in microbiota between breeds, we analysed the beta diversity based on unweighted UniFrac for these groups, after which the UniFrac distance matrix was represented through Principal Coordinate Analysis (PCA) (Additional file 1). Statistically significant differences only appeared between breeds at the end of the growing period (P-value < 0.05). Moreover, the comparisons of beta diversity and genera presence between both breeds in the different sampling times are represented in Fig. 4, and genera data details are summarised in Additional file 2.