Effect of weaning age on piglet weight and occurrence of diarrhea
Forty-eight Large White piglets (23 females and 25 males) were divided into four groups of 12 animals that were weaned at different ages: 14 days (W14), 21 days (W21), 28 days (W28), and 42 days (W42). These groups are hereafter referred to as the weaning groups. Animals presenting diarrhea were unevenly distributed across groups, with a strong reduction in the proportion of affected animals in the groups W28 and W42: 3/10 (30%) in the W14 group, 5/12 (41%) in the W21 group, 1/12 (8%) in the W28 group, and 0/11 (0%) in the W42 group. A Chi-square test confirmed that these differences were significant (p < 0.05).
To characterize piglet growth, we monitored the weight of pigs in each weaning group from birth (day 0) to 62 days of age (weight was measured at 5, 12, 20, 27, 33, 48, 55, and 62 days of age). Using ANOVAs, we found that the weaning groups differed in weight across time and that patterns of differences varied (Additional file 5: Table S1). In general, after weaning, the mean weight for the W14 group was consistently lower than the mean weights for the other groups (Fig. 1). In addition, piglets in the groups W14 (at day 20), W21 (at day 27), and W28 (at day 33) lost weight immediately after weaning. Indeed, three animals from the W14 group were euthanized because they were lethargic and failed to grow (decision made in accordance with the project’s established ethical guidelines). On day 62, the mean weights for the groups W21, W28, and W42 were statistically similar to each other, and they all differed from the weight for the W14 group (p < 0.05).
Fecal microbiota sequencing, OTU identification and annotation
The piglets’ fecal microbiota were analyzed by sequencing the bacterial 16S rRNA gene using an Illumina MiSeq Sequencer. Samples with fewer than 10,000 reads following quality control procedures were removed from the analysis, resulting in sample sizes of 3–12 piglets per sampling point (see the Methods section). After performing quality control, a mean of 63,716 reads were available for each sample. Sequences from the whole sample set were successfully clustered into 1121 operational taxonomic units (OTUs), and only 0.26% of the OTUs could not be assigned to a given phylum. Overall, 539 of the 1121 OTUs (48%) were assigned to a genus. The phyla Firmicutes (700/1121) and Bacteroidetes (340/1121) represented 62 and 30% of the OTUs, respectively. Within the phylum Firmicutes, 95% (665/700) of the OTUs were assigned to the order Clostridiales, 40% (265/665) to the family Ruminococcaceae, and 23% (153/665) to the family Lachnospiraceae. Within the phylum Bacteroidetes, 53% (179/340) were assigned to the genus Prevotella. Other phyla were also represented, but they were less common (e.g., Proteobacteria: 5%, Spirochaetes: 0.45%, Fusobacteria: 0.45%, Actinobacteria: 0.35%, Deferribacteres: 0.27%, and Tenericutes: 0.01%; Fig. 2a). At the phylum (Fig. 2a) and genus (Fig. 2b) levels, the overall abundance of diverse OTUs varied based on weaning age and among sampling points within weaning groups (see the following sections). When we examined the 75% most prevalent taxa in each group at the three sampling points, we found that, out of the 1121 OTUs observed overall, 760 OTUs were present in the W14 group, 807 OTUs were present in the W21 group, 882 OTUs were present in the W28 group, and 933 OTUs were present in the W42 group. This result illustrates that OTU richness increased with age at weaning.
Effect of weaning age on fecal microbiota diversity and composition before and after weaning
Alpha diversity, beta diversity, and richness were calculated using the rarefied OTU counts for each group and then compared among weaning groups and sampling points (Fig. 3). ANOVAs and Tukey’s honest significant difference (HSD) tests were used to assess any resulting differences (Additional file 6: Table S2). Overall, there were significant differences (p < 0.05) in alpha diversity and richness among sampling points within all the weaning groups except W42. In the W42 group, only beta diversity differed significantly among sampling points. The results for alpha diversity and richness reflect the diversification that takes place in the gut microbiota during and after weaning. The results for beta diversity fit with the idea that microbiota heterogeneity declines as animals grow older. The Tukey’s HSD tests highlighted that the significant differences mainly originated from differences in diversity and richness between the pre- and post-weaning sampling points. Moreover, we observed that beta diversity declined between 7 days post weaning and 60 days of age, except in the W14 group (Fig. 3b).
Non-metric multidimensional scaling (NMDS) analyses were carried out using Bray-Curtis dissimilarity values quantifying overall differences in gut microbiota composition between samples collected before weaning, 7 days after weaning, and at 60 days of age for piglets in each weaning group (Fig. 4). For the groups W14, W21, and W28, there were clear differences between the results for the three sampling points. For the group W42, in contrast, the centroid for the pre-weaning data was distinct from the centroids for the data from 7 days post weaning and 60 days of age, which overlapped.
We used the metagenomeSeq package in R to identify differentially abundant (DA) OTUs within the full dataset (1121 OTUs) for each weaning group; we specifically compared the pre-weaning data and the data obtained 7 days after weaning. In the W14 group, there were 224 DA OTUs (Additional file 7: Table S3). In the W21 group, this number increased to 484 (Additional file 8: Table S4). In W28 and W42, there were 395 DA OTUs (Additional file 9: Table S5) and 461 OTUs (Additional file 10: Table S6), respectively. There was some degree of overlap among the DA OTUs (Additional file 1: Figure S1), although there were unique OTUs in all the weaning groups (W14: 44, W21: 106, W28: 71, and W42: 107). Overall, Bacteroides, Ruminococcus, Oscillospira, and Clostridium were more abundant before weaning and Succinivibrio, Prevotella, and Campylobacter were more abundant 7 days after weaning. Interestingly, Faecalibacterium prausnitzii was found to be highly abundant after weaning in all the weaning groups.
Effect of weaning age on F. prausnitzii abundance before and after weaning
In the full dataset, three OTUs were annotated as F. prausnitzii (OTU IDs 851,865, 350,121, and 525,215). Since at least one of these OTUs was DA in most comparisons, we decided to explore the overall abundance of F. prausnitzii by summing the abundances of the three OTUs for each sample. We had previously normalized these data by log scaling the cumulative sum scaling (CSS) values obtained in metagenomeSeq. For each weaning group, there was a clear increase in F. prausnitzii abundance over time, and the highest abundances were observed in the W42 group (Fig. 5). In the groups W14 and W21, there was a marked increase in abundance between weaning and 60 days of age; in the groups W28 and W42, abundance tended to be more stable 7 days post weaning. At weaning, F. prausnitzii was most abundant in the W42 group, equivalently abundant at lower levels in the W21 and W28 groups, and least abundant in the W14 group. There were significant differences among the four weaning groups (ANOVA: p < 0.05), and F. prausnitzii was more abundant before weaning in piglets weaned at a later age (Additional file 11: Table S7). Indeed, piglets weaned at 14 days of age had the lowest abundance of F. prausnitzii before weaning, a pattern that persisted until 60 days of age. Post-hoc analysis found differences in the abundance of F. prausnitzii between the groups W14 and W42 before weaning and between various combinations of the weaning groups at 7 days post weaning and 60 days of age (Additional file 12: Table S8).
Effect of weaning age on fecal microbiota diversity and composition before weaning
Before weaning, alpha diversity was significantly higher in the W42 group than in the other three groups (Tukey’s HSD: p < 0.05) (Additional file 13: Table S9; W42 versus W14, W42 versus W21, and W42 versus W28). The same pattern was seen for richness, with an additional significant difference between the groups W14 and W28 (Additional file 13: Table S9). Beta diversity was only significantly different between the W42 group and the groups W14 and W21 (Additional file 13: Table S9). In the NMDS analysis, there were significant associations with litter and weaning group (p < 0.05) (Additional file 2: Figure S2A).
Furthermore, before weaning, there were 165 DA OTUs for the four weaning groups combined (Additional file 14: Table S10). These OTUs belonged to the phyla Firmicutes, Bacteroidetes, and Proteobacteria and the genera Bacteroides, Ruminococcus, and Prevotella. There was some overlap among groups: 44 of the DA OTUs were shared (Additional file 3: Figure S3).
Among the weaning groups, there was differential abundance of the phyla Tenericutes, Spirochaetes, Deferribacteres, and Fusobacteria (Additional file 15: Table S11) and the genera Paludibacter, Comamonas, Helicobacter, Peptostreptococcus, Streptococcus, Treponema, Catenibacterium, and Dorea (Additional file 16: Table S12).
Effect of weaning age on fecal microbiota diversity and composition at seven days post weaning
Seven days after weaning, there was no difference in alpha diversity and richness among the four weaning groups (Additional file 13: Table S9). Beta diversity was significantly higher in the W14 group than in the other three groups, and the W42 group had the lowest beta diversity. The NMDS analysis found no differences among the groups (Additional file 2: Figure S2B). There were a total of 165 DA OTUs (Additional file 17: Table S13) that mainly belonged to the phyla Firmicutes, Bacteroidetes, and Proteobacteria and the genera Prevotella, Ruminococcus, Bacteroides, and Oscillospira. One of the F. prausnitzii OTUs was more abundant in the groups W28 and W42. The weaning groups shared 25 OTUs (Additional file 4: Figure S4), which were more heterogeneous than the OTUs shared by the groups prior to weaning; they belonged to the orders Clostridiales and Bacteroidales. In the analyses at the phylum and genus levels, only the genera Actinobacillus, Peptostreptococcus, and Klebsiella were differently abundant among the weaning groups (Additional file 18: Table S14).
Effect of weaning age on fecal microbiota diversity and composition at 60 days of age
When the piglets were 60 days old, alpha diversity was significantly different between the groups W21 and W42 (p < 0.05); richness and beta diversity did not vary based on weaning age (Additional file 13: Table S9). Similarly, the NMDS analysis found no differences among weaning groups (Additional file 2: Figure S2C). There were 54 DA OTUs (Additional file 19: Table S15) that belonged to phyla Firmicutes, Bacteroidetes, and Proteobacteria and, for the most part, the genera Prevotella, Ruminococcus, and Bacteroides.