Duroc pigs are currently used in breeding programs as paternal lines, while crosses between Landrace and Large White pigs are used as maternal lines. The different aims of selection for paternal and maternal lines might have significantly shaped feed efficiency and gut microbiome composition. For this reason, to investigate the differences between paternal and maternal lines and between the two maternal lines with the minimum number of orthogonal contrasts, we performed the comparison between Duroc and the average of Landrace and Large White [DR vs (LR and LW)/2] and Landrace versus Large White (LR vs LW). In this study, the results section was divided into four parts. In the first part, we reported differences between breeds in terms of feed efficiency and production traits. In the second part, gut microbiome differences between breeds at each time point were showed. In the third part, the effects of ASV and interaction between ASV and breed on feed efficiency were reported separately at the three time points. Lastly, the associations among gut microbiome and feed efficiency were presented.
Feed efficiency and production traits in the Duroc, Landrace, and Large White breeds
Tables 1 and 2 summarizes descriptive statistics and breed difference estimates for average daily feed intake (ADFI), average daily gain (ADG), feed efficiency, and production traits. Average daily feed intake, ADG, back fat, and loin depth were all lower in Duroc (DR) pigs than the average of Landrace (LR) and Large White (LW) animals. Landrace and LW pigs were similar in terms of ADG, RF1, RF2, and IMF, however LW pigs had significantly higher ADFI, FCR, and backfat.
Table 1
Descriptive statistics for growth, feed efficiency, and production traits.
Item | Duroc | | Landrace | | Large White |
Mean | SD | | Mean | SD | | Mean | SD |
Animals, n | 190 | | 221 | | 204 |
Efficiency: | | | | | | | | |
ADFI, g/d | 2,154.1 | 326.1 | | 2,203.2 | 360.3 | | 2,384.5 | 354.2 |
ADG, g/d | 615.2 | 68.2 | | 628.3 | 75.4 | | 650.9 | 82.1 |
RF1, g | 0.00 | 341.2 | | 0.00 | 287.0 | | 0.00 | 287.1 |
RF2, g | 0.00 | 307.8 | | 0.00 | 279.5 | | 0.00 | 263.8 |
FCR | 3.53 | 0.54 | | 3.51 | 0.58 | | 3.67 | 0.45 |
Production traits: | | | | | | | | |
Back fat, mm | 10.12 | 2.63 | | 11.69 | 2.95 | | 14.04 | 3.20 |
Loin depth, mm | 49.08 | 6.63 | | 48.52 | 6.73 | | 47.07 | 6.59 |
IMF, % | 2.09 | 0.89 | | 1.84 | 0.75 | | 1.93 | 0.79 |
ADFI = average daily feed intake; ADG = average daily gain; RF1 = residuals calculated regressing ADFI on ADG; RF2 = residuals calculated regressing ADFI on ADG and body weight; FCR = average feed conversion ratio calculated as the ratio between ADFI and ADG; IMF = intramuscular fat. |
Table 2
Effect of breed on growth, feed efficiency, and production traits.
| Duroc (DR) | | Landrace (LR) | | Large White (LW) | | Contrasts (P-value) | RMSE |
LSM | SE | LSM | SE | LSM | SE | DR vs (LR + LW)/2 | LR vs LW |
Efficiency: | | | | | | | | | | | | |
ADFI, g/d | 2,185.1 | 51.27 | | 2,213.9 | 51.9 | | 2,396.8 | 51.7 | | 0.061 | 0.015 | 267.4 |
ADG, g/d | 618.4 | 10.9 | | 627.1 | 11.1 | | 649.91 | 11.0 | | 0.142 | 0.149 | 56.8 |
RF1, g | -4.61 | 51.38 | | -4.90 | 52.30 | | 21.17 | 52.50 | | 0.842 | 0.727 | 230.3 |
RF2, g | -1.00 | 45.45 | | -9.59 | 46.14 | | 14.84 | 46.28 | | 0.949 | 0.710 | 219.6 |
FCR | 3.56 | 0.08 | | 3.54 | 0.08 | | 3.70 | 0.08 | | 0.530 | 0.154 | 0.38 |
Production traits: | | | | | | | | | | | | |
Back fat, mm | 10.33 | 0.34 | | 11.43 | 0.34 | | 13.93 | 0.29 | | < 0.001 | < 0.001 | 2.36 |
Loin depth, mm | 49.60 | 0.54 | | 47.97 | 0.54 | | 46.37 | 0.50 | | < 0.001 | 0.033 | 4.44 |
IMF, % | 2.04 | 0.07 | | 1.80 | 0.07 | | 1.96 | 0.06 | | 0.065 | 0.086 | 0.68 |
LSM = least squares mean; SE = standard error; RMSE = root mean square error; ADFI = average daily feed intake; ADG = average daily gain; RF1 = residuals calculated regressing ADFI on ADG; RF2 = residuals calculated regressing ADFI on ADG and body weight; FCR = average feed conversion ratio calculated as the ratio between ADFI and ADG; IMF = intramuscular fat. |
[b] |
Least squares means for ADFI, ADG, feed conversion ratio (FCR), residual feed intake 1 (RF1), and residual feed intake 2 (RF2) in each of the three breeds across three time points (T1, T2, and T3) are reported in Fig. 1. Significant (P < 0.05) differences were observed. Across time points, ADG for the three breeds ranged from 0.48 to 0.77 kg/d. At T2 ADG was significantly higher in LW (0.68 ± 0.058 kg/d) than DR (0.64 ± 0.057 kg/d) and LR (0.65 ± 0.055 kg/d), while no statistically significant differences were observed at T1 and T3. Average daily feed intake ranged from 1,503 to 2,795 g/d across time points. Average daily feed intake was significantly higher in LW at T2 and T3 than DR and LR, while no significant differences were observed at T1. The FCR of the three breeds on average ranged from 3.21 to 3.80 across time points. Feed conversion ratio was significantly lower in LR at T1 and T2 compared to DR and LW. With regard to the residual feed intake, RF1 ranged from − 205 to 95, while RF2 ranged from − 226 to 136 across time points. These significant differences between breeds were confirmed by LDA (Fig. 2). The first component explained 93.4% and the second 6.6% of the total variance. The first component differentiated DR and LR from LW based on FCR (loading 10.4) and IMF (loading − 0.18), while the second component discriminated DR and LR based on FCR (loading 8.4) and backfat (loading − 0.25).
Gut microbiome composition in the Duroc, Landrace, and Large White breeds
Figure 3 illustrates the relative abundance of microbial ASV when aggregated at the family level for the three breeds and the three time points surveyed in this study. Over the three time points, about 80% of ASV were classified into just 7 families: Lactobacillaceae, Clostridiaceae, Streptococcaceae, Prevotellaceae, Ruminococcaceae, Eubacteriaceae, and Lachnospiraceae.
Alpha diversity of pig gut microbiome was measured using the Shannon, Simpson, and Inverse Simpson indices (Fig. 4). On average, the alpha diversity across time points ranged from 4.05 to 4.43, from 0.93 to 0.95, and from 18.7 to 27.2 for the Shannon, Simpson, and Inverse Simpson indices, respectively. Comparing the breeds across time points, Shannon index values were significantly (P < 0.05) higher in LR than DR and LW at T1, T2, and T3. Duroc pigs had lower Shannon index values than LR and LW at T1 and T2, while no statistically differences were observed at T3. Simpson index values were significantly lower in DR than LR and LW at T1 and T3, while no statistically significant differences were observed at T2. The Inverse Simpson index was significantly lower in DR than LR and LW at T1. Landrace had a higher Inverse Simpson index than LW at T2, while no statistically significant differences were observed at T3. Results from this analysis revealed some significant influence of breeds on alpha diversity in our data.
Clustering analyses was focused on identifying cluster (enterotypes) among the fecal samples of pigs collected at each time point. Each of these enterotypes may be driven by specific genera that contribute to microbial compositions. The clusters of samples and genera that significantly separate the enterotypes according to breed and time points are shown in Fig. 5 and Supplementary Figure S1. The optimal number of clusters based on Calinski-Harabasz index maximization was 2, with the exception of T1 and T2 for DR and LW samples where the optimal number of clusters was major than 2. In order to identify specific bacterial genera that were characteristic to the three breeds within each time points, we performed an LDA analysis coupled with LDA Effect Size LEfSe. Figure 5 shows the genera that were differentially represented among the three breeds and time points. Anaerostipes and Turicibacter genera had very high LDA scores across all breeds and enterotypes. At T1 the three enterotypes in DR pigs were distinguished by Dorea, Faecalibacterium, and Anaerovibrio, while the enterotypes for LR and LW pigs were distinguished by Anaerostipes and Turicibacter. At T2 the four enterotypes in DR pigs were distinguished by a total of 12 genera. Of these, Turicibacter, Faecalibacterium, Anaerostipes, and Dorea were most abundant in enterotypes A, B, C, and D, respectively. At the same time point the two enterotypes for LR were significantly distinguished for either Turicibacter or Anaerostipes, while the three enterotypes for LW were significantly distinguished by Turicibacter, Anaerostipes, and Clostridium. Enterotypes A and B for DR and LR at T3 were significantly distinguished by Turicibacter and Anaerostipes, while the two enterotypes for LW were distinguished by 11 genera. Of these, Anaerostipes and Sporobacterium were significantly more abundant. This analysis revealed a different grouping of the samples across time points for DR and LW, while the LR samples were mainly clustered into Anaerostipes and Turicibacter enterotypes. These differences among enterotypes thus reflect different combinations of genera with a probable effect on feed efficiency.
ASV differentially represented in the Duroc, Landrace, and Large White breeds
Analyses of ASV representation performed at each time point revealed the gut microbiomes of DR, LR, and LW pigs to be distinct (Supplementary Table S1). When ASV abundances in DR pigs were compared to the average of those in LR and LW pigs at T1, T2, and T3, a total of 441, 401, and 324 ASV were found to be significantly different in terms of their representation (FDR, 5%). Of these, 261 ASV classified as Firmicutes (177), Bacteroidetes (55), Proteobacteria (12), Spirochaetes (11), Actinobacteria (3), Chlamydiae (1), Fusobacteria (1), and Tenericutes (1) were shared between T1 and T2; 267 ASV classified as Firmicutes (180), Bacteroidetes (59), Proteobacteria (11), Spirochaetes (10), Actinobacteria (5), Fusobacteria (1), and Tenericutes (1) were shared between T2 and T3; and 191 ASV classified as phylum Firmicutes (128), Bacteroidetes (41), Proteobacteria (9), Spirochaetes (8), Actinobacteria (3), Fusobacteria (1), and Tenericutes (1) were shared between T1 and T3.
The relative abundances of 184, 153, and 123 ASV were significantly different between LR versus LW pigs at T1, T2, and T3, respectively. Of these, 56 consistently separated LR from LW pigs at T1 and T2. Specifically: Firmicutes (24), Bacteroidetes (16), Proteobacteria (9), Spirochaetes (3), Actinobacteria (3), and Fusobacteria (1). There were 38 ASV that were systematically different between LR and LW at T2 and T3. These belonged to different phyla such as Firmicutes (24), Bacteroidetes (5), Proteobacteria (5), Actinobacteria (3), and Spirochaetes (1). Thirteen ASV classified as Firmicutes (5), Bacteroidetes (4), Proteobacteria (3), and Actinobacteria (1) were shared between T1 and T3.
The significant ASV in the orthogonal contrasts of DR versus the average of LR and LW, and LR versus LW at all three time points are presented as volcano plots (Fig. 6a-c). A comparison of the Duroc sire line with the combined LR and LW maternal lines at T1 revealed a higher abundance of 6 Firmicutes. The most significant of these were classified as Ruminococcus and Clostridium. In a comparison of the two maternal lines with one another at T1, LR had higher representation than LW of Bacteroidetes and Spirochaetes. Within these phyla the most significant genera were Prevotella, Bacteroides, and Treponema. Landrace also had a lower proportion of several ASV than LW at T1, many of which were classified as Clostridium, Camplylobacter, Blautia, Eubacterium, Lactobacillus, and Roseburia (Fig. 6a).
Significant differences were also observed at T2 when comparing ASV abundances in DR pigs with the average of LR and LW. Duroc pigs had higher proportion of 8 Firmicutes, 1 Actinobacteria, and 1 Bacteroidetes than the two maternal lines. The most abundant genera within these phyla were Catenibacterium, Prevotella and Collinsella. On the other hand, DR compared to the average of LR and LW at T2 had a lower proportion of 8 Firmicutes, 4 Bacteroidetes, and 2 Spirochaetes. Within these phyla, the most significant genera were classified as Clostridium, Prevotella, and Treponema. Remarkable differences in ASV abundances were also observed when comparing LR to LW at T2. Specifically, LR had a higher proportion of 6 Bacteroidetes, 6 Firmicutes, 2 Proteobacteria, and 1 Spirochaetes. Within these phyla, the predominant genera were Anaerococcus, Barnesiella, Clostridium, and Prevotella. Interestingly, the gut microbiome of LR at T2 also had a lower proportion ASV belong to Clostridium and Eubacterium than LW (Fig. 6b).
The orthogonal contrast between paternal and maternal lines at T3 revealed a higher abundance of 13 Firmicutes, 2 Bacteroidetes, and 1 Actinobacteria. Within the Firmicutes phylum, as observed in T1 and T2, the most significant genera were Catenibacterium and Clostridium. The paternal line had a lower abundance of 5 Firmicutes and 1 Spirochaetes. Of these, one of the most significant genera was Turicibacter as observed in T1 and T2. We also discovered differences between the two maternal lines at T3. LR compared to LW had a particularly higher abundance of Bacteroides, Campylobacter, Coprococcus, Enterococcus, and Fusobacterium, as well as a lower abundance of Lactobacillus, Ruminococcus, and Desulfovibrio (Fig. 6c). Of these, the genus Bacteroides was the most significant discriminant of LR and LW across the three time points.
Association between pig phenotypes and gut microbiota
We identified several ASV whose relative abundances correlated significantly with feed efficiency and production traits (Supplementary Table S2). A total of 16, 33, and 93 ASV were significantly associated with feed efficiency and production traits at T1, T2, and T3, respectively. They belonged mainly to 4 phyla: Firmicutes, Bacteroidetes, Proteobacteria, and Spirochaetes. At the genus level, 14 were classified as Ruminococcus, 12 as Clostridium, 10 as Eubacterium, 6 as Lactobacillus, 5 as Bacteroides, and 4 as Prevotella, Twelve ASV were classified as belonging to 7 other minor genera, and 79 were unassigned. We identified 20, 1, 20, 26, 61, 6, and 8 taxa that were significantly associated with ADFI, ADG, RF1, RF2, FCR, backfat, and loin depth at the three different time points.
The interaction effects between ASV and breed were significant for 2 and 48 ASV at T1 and T3 (Supplementary Table S3), respectively, while no significant interaction effects were observed at T2. The significant ASV belonged to the Firmicutes and Bacteroidetes. Eight of these belonged to the Faecalibacterium genus, 4 to the Eubacterium, 4 to the Bacteroides, 4 to the Oscillibacter, 4 to the Ruminococcus, 2 to the Anaerovibrio, and 24 were unassigned. Of the ASV identified, 22, 22, and 4 were significantly associated with ADFI, RF1, and FCR, respectively, at T1 and T3.
We discovered within breed and across time points that the genus Oscillibacter was negatively correlated (rs ⁓ -0.30) with feed efficiency (RF1, RF2, and FCR), backfat, and loin depth in DR at T1. The genera Blautia, Dorea, Eubacterium, Faecalibacterium, Lactobacillus, and Ruminococcus, on the other hand, were positively correlated with feed efficiency (rs ⁓ 0.15) and production traits (rs ⁓ 0.22) in Duroc pigs at T2 and T3. Similarly, a negative correlation was obtained between the genus Sarcina and growth (rs = -0.38) as well as IMF (rs = -0.23) at T3 (Fig. 7a-c).
The genus Oscillibacter was negatively correlated (rs ⁓ -0.17) with ADFI and feed efficiency in LR at T1 and T2. This maternal line also had a positive correlation (rs ⁓ 0.17) at T2 and T3 between ASV classified as genera Clostridium and feed efficiency.
In addition, we found a positive correlation (rs ⁓ 0.20) between four genera (Corynebacterium, Lactobacillus, Finegoldia, and Psychrobacter) and both ADFI and feed efficiency in LW pigs at T2, while the genus Desulfovibrio was negatively correlated (rs ⁓ -0.33) with ADFI, feed efficiency, and backfat at T1 and T3.
We found within time points and across breeds that the genus Oscillibacter was negatively correlated with ADFI and feed efficiency for DR, LR, and LW pigs at T1. The genera Anaerovibrio, Clostridium, Faecalibacterium, Eubacterium, and Ruminococcus showed negative correlations with feed efficiency and backfat in both LR and LW at T1. ASV classified as Dorea, Eubacterium, and Lactobacillus were positively correlated with feed efficiency in DR and LW at T2. Additionally, the genus Blautia was positively correlated with feed efficiency in DR and LR at T2. ASVs classified as Dorea and Lactobacillus were positively correlated with ADFI and feed efficiency in DR and LW at T3.