Breed differences in animal feed conversion and economic trait performance
Throughout the feed intake measurement period, summary statistics shows animals on test had an average DMI of 1.11 kg/d (SD = 0.18), ADG of 0.27 kg/d (SD = 0.1), FCR of 4.04 kg of DMI/ Kg of ADG (SD = 0.1), start weight of 29.60 kg (SD = 3.7), final live weight of 46.00 kg (SD = 2.9), carcass weight of 20.20 kg (SD = 1.6), and a KO% of 44.1% (SD = 2.3). Average daily gain (P = 0.005), FCR (P = 0.035), CW (P < 0.04) and start weight (P < 0.036) were all significantly affected by breed. Summary statistics, along with comparisons amongst breeds for animal performance, feed intake and feed efficiency are displayed in Table 1. In summary, the Cheviot breed had the lowest FCR and the highest ADG, carcass weight, and start weight among all breeds, with differences in ADG and FCR being significant when compared to the Connemara breed and differences in carcass and start weight being significant when compared to the Lanark breed. In addition, the Cheviot breed had the fastest maturing lambs with 80% of lambs reaching maturity within the first 42 days (data not shown) and a mean LW of 47.1 kg (Table 1).
Overall microbial community structure
After data processing, filtering, and removal of chimeras and lowly sequenced samples a total of 5,411,353 reads remained, with an average of 91.3% of reads surviving. The average number of reads per sample was 64,420, which mapped to 2547 ASVs. After removal of taxa unassigned at the phylum level 2434 ASV’s remained. Analysis of the ASVs across all samples revealed that bacteria and archaea represented 95.4 and 4.6% of the microbial population, respectively. A total of 19 bacterial taxa were classified at the phylum level, with Firmicutes being the most abundant (45.8%), followed by Bacteroidetes (33.4%) and Proteobacteria (8.0%). There were 192 taxa classified at the genus level, with Prevotella_1 (13.1%) and Prevotella_7 (13.1%) being the most dominant followed by Succinivibrio (6.3%). Methanobrevibacter was shown to be the most abundant archaeal genus. (78.1%). In this study, no non-methanogenic archaeal taxa were identified.
Breed effects on bacterial and archaeal populations in the solid ruminal fraction
In the solid ruminal fraction, a total of 1706 bacterial ASVs agglomerated to 227 genera, 89 families, 51 orders, 27 classes and 16 phyla. Firmicutes (48.2%) Bacteroidetes (30.1%), Fibrobacterota (6.1%) were the three most abundant bacteria phyla (Fig. 1). Prevotella_7 (9.7%), Prevotella_1 (9.2%), unclassified Lachnospiraceae (7.6%), Fibrobacter (6.1%) and Ruminococcus_1 (5.6%) were the 5 most dominant bacteria genera (Fig. 2). A total of 27 archaeal ASVs were identified and agglomerated to 4 genera (Methanobrevibacter, Methanosphera, Methanimicrococcus and Candidatus methanomethylophilus), three families, three orders three classes and one phylum. Methanobrevibacter was the most dominant archaeal genus (72.9%).
Stack barchart representing the mean relative abundance of the 5 most dominant phyla across breeds (i.e. Cheviot, Connemara, Lanark, Perth) for solid liquid and epithelial ruminal fractions. Solid (Cheviot n = 8, Connemara n = 5, Lanark n = 9, Perth n = 7), liquid (Cheviot n = 9, Connemara n = 5, Lanark n = 9, Perth n = 5), epithelial (Cheviot n = 9, Connemara n = 3, Lanark n = 6, Perth n = 8).
Stack barchart representing the mean relative abundance of the 10 most dominant genera across breeds (i.e. Cheviot, Connemara, Lanark, Perth) for solid liquid and epithelial ruminal fractions. Solid (Cheviot n = 8, Connemara n = 5, Lanark n = 9, Perth n = 7), liquid (Cheviot n = 9, Connemara n = 5, Lanark n = 9, Perth n = 5), epithelial (Cheviot n = 9, Connemara n = 3, Lanark n = 6, Perth n = 8).
Alpha diversity analysis revealed that breed had an effect on solid associated bacterial and archaeal community richness and bacteria community PD (ANOVA, P < 0.05) (Table 2). Such differences were observed between the Cheviot and Lanark breeds, with the Cheviot exhibiting the least and the Lanark exhibiting the most rumen microbial diversity among the breeds. Based on weighted and unweighted UniFrac distances, beta diversity analysis showed no differences in overall community composition across the breeds (PERMANOVA, P > 0.05) for either bacterial or archaeal communities (Table 3).
The abundance of Sharpea at the genus level, Sharpea azabuensis at the species level and an unclassified ASV (ASV37) belonging to the family Lachnospiraceae were affected by breed (LRT, P.adj < 0.05) (Table 4). Sharpea (Wald, P.adj < 0.001; Log2FC = 4.37) and Sharpea azabuensis (Wald, P.adj < 0.001; Log2FC = 4.58) were higher in Perth compared to Cheviot. ASV37 (family Lachnospiraceae) was more abundant in Cheviot (Wald, P.adj < 0.01; Log2FC = 3.22) and Perth (Wald, P.adj < 0.001; Log2FC = 3.4) compared to Lanark (Table 4). Pairwise analysis between each of the breeds revealed a further 2 bacterial ASVs as differentially abundant. ASV48 classified to the genus Prevotella_9 was higher in Lanark compared to Perth (Wald, P.adj < 0.0001; Log2FC = 7.88) and Cheviot (Wald, P.adj < 0.01; Log2FC = 9.11), and ASV329 classified to the genus Pyramidobacter was higher in Lanark compared to Cheviot (Wald, P.adj < 0.05; Log2FC = 5.52). At the genus level P-2534-18B5_gut group (ASV17), belonging to phylum Bacteroidetes, was higher in the Perth (Wald, P.adj < 0.05; Log2FC = 5.78) and Lanark (Wald, P.adj < 0.05; Log2FC = 6.82) breeds compared to Cheviot, and Candidatus Saccharimonas was higher in the Lanark compared to the Cheviot (Wald, P.adj < 0.05; Log2FC = 5.66). Similarly, at the family level P-2534-18B5_gut group (ASV17) was higher in the Perth (Wald, P.adj < 0.01; Log2FC = 5.88) and Lanark (Wald, P.adj < 0.01; Log2FC = 6.80) breeds compared to Cheviot, and Saccharimonadaceae (ASV317) was higher in the Lanark (Wald, P.adj < 0.01; Log2FC = 5.48) and Connemara (Wald, P.adj < 0.05; Log2FC = 6.89) breeds compared to the Cheviot. At the order level Coriobacteriales was higher in the Lanark compared to the Perth (Wald, P.adj < 0.05; Log2FC = 1.17) (Table 5). One archaea ASV belonging to the genus Candidatus Methanomethylophilus (ASV337) was higher in the Perth (Wald, P.adj < 0.01; Log2FC = 3.12) and Lanark (Wald, P.adj < 0.05; Log2FC = 3.21) compared to Cheviot (Table 5).
Breed effects on bacterial and archaeal populations in the liquid ruminal fraction
For the liquid ruminal fraction, a total of 1790 bacteria ASVs agglomerated to 236 genera, 95 families, 57 orders, 29 classes and 17 phyla. Firmicutes (43.1%), Bacteroidetes (37.1%), and Proteobacteria (8.9%) were the most dominant phyla (Fig. 1). Prevotella 7 (12.2%), Prevotella 1 (11.7%), unclassified Lachnospiraceae (6.2%), Succinivibrio (5.8%) and Succiniclasticum (4.4%) were the five most dominant genera (Fig. 2). Twenty six archaea ASVs were available for analysis, which agglomerated to four genera (Methanobrevibacter, Methanosphera, unclassified Methanomethylophilaceae and Candidatus Methanomethylophilus), two families, two orders two classes and one phylum. Methanobrevibacter was the most dominant genus (78.9%).
Although there was no effect of breed on alpha diversity indices for bacteria communities (ANOVA, P > 0.05), breed did have an impact on the richness of archaeal communities. (ANOVA, P < 0.05) (Table 1). The Lanark breed had the highest level of archaeal community richness, whereas the Cheviot breed had the lowest level (Table 5). Based on weighted and unweighted UniFrac distances, the analysis of beta diversity showed no differences in overall community composition among breeds (PERMANOVA, P > 0.05) for either bacterial or archaeal communities (Table 3).
The likelihood ratio test detected no breed effect (LRT, P.adj > 0.05) on the abundance of bacterial or archaeal taxa across all taxonomic ranks. Pairwise analysis between each of the breeds revealed five taxa at the ASV level as differentially abundant. Two ASVs, ASV23 (Wald, P.adj < 0.01; Log2FC = 5.94) and ASV43 (Wald, P.adj < 0.05; Log2FC = 2.51) classified to the family Muribaculaceae were higher in Cheviot compared to Perth, ASV44 classified to the genus Acetitomaculum was higher in Cheviot (Wald, P.adj < 0.05; Log2FC = 10.26) compared to Connemara, ASV55 classified as Sharpea azabuensis was higher in Perth (Wald, P.adj < 0.01; Log2FC = 4.81) compared to Cheviot, and ASV20 classified to Lachnospiraceae NK3A20 group was higher in the Lanark (Wald, P.adj < 0.05; Log2FC = 3.06) compared to Connemara. At the genus level Sharpea (Wald, P.adj < 0.001; Log2FC = 5.05) was higher in the Perth compared to the Cheviot, ASV223 classified to order Rhodospirillales (Wald, P.adj < 0.01; Log2FC = 7.03) was higher in the Cheviot compared to the Perth, and ASV461 classified to Clostridiales_vadinBB60 group was higher in the Lanark (Wald, P.adj < 0.05; Log2FC = 5.86) compared to the Connemara. At the family level Muribaculaceae (Wald, P.adj < 0.05; Log2FC = 2.23) was higher in Cheviot compared to Perth, ASV223 classified to order Rhodospirillales was higher in the Cheviot compared to the Connemara (Wald, P.adj < 0.05; Log2FC = 7.11) and Perth (Wald, P.adj < 0.01; Log2FC = 6.81), ASV461 classified to Clostridiales_vadinBB60 group was higher in the Cheviot (Wald, P.adj < 0.05; Log2FC = 5.82) and Lanark (Wald, P.adj < 0.01; Log2FC = 6.09) compared to the Connemara, and ASV17 classified to P-2534-18B5_gut group was higher in the Perth (Wald, P.adj < 0.05; Log2FC = 4.75) and Lanark (Wald, P.adj < 0.05; Log2FC = 5.87) compared to the Cheviot. At the order level Rhodospirillales was higher in the Cheviot compared to the Connemara (Wald, P.adj < 0.05; Log2FC = 7.29) and Perth (Wald, P.adj < 0.01; Log2FC = 6.93), and Betaproteobacteriales was higher in the Cheviot compared to the Connemara (Wald, P.adj < 0.05; Log2FC = 3.03). At the class level the abundance of Alphaproteobacteria was higher in the Cheviot compared to the Perth (Wald, P.adj < 0.01; Log2FC = 7.11) and Connemara (Wald, P.adj < 0.05; Log2FC = 7.52). Finally at the phylum level the abundance of Proteobacteria was higher in the Perth compared to the Connemara (Wald, P.adj < 0.05; Log2FC = 2.62) (Table 5).
Breed effects on bacterial and archaeal populations in the epithelial ruminal fraction
In the epithelial ruminal fraction, a total of 1891 bacteria ASVs agglomerated to 231 genera, 89 families, 52 orders, 29 classes and 17 phyla. Firmicutes (46.3%), Bacteroidetes (33.4%), and Proteobacteria (10.1%) were the most dominant phyla (Fig. 1). Prevotella 1 (10.0%), Prevotella 7 (8.9%), Succinvibrio (5.9%), unclassified Lachnospiraceae (5.7%), and Ruminococcus 2 (5.0%) were the five most dominant genera (Fig. 2). Twenty eight archaeal ASVs were available for analysis, which agglomerated to five genera (Methanobrevibacter, Methanosphera, Methanimicrococcus, unclassified Methanomethylophilaceae and Candidatus Methanomethylophilus), three families, three orders, three classes and one phylum. Methanobrevibacter was the most dominant genus (82.0%).
Alpha diversity analysis revealed that while breed had no effect on epithelial associated archaeal community indices, it had a significant effect on bacteria community richness and inverse Simpson diversity (ANOVA, P < 0.05) (Table 2). Beta diversity analysis based on weighted and unweighted UniFrac distances, found no differences in community composition among the breeds (PERMANOVA, P > 0.05), for either bacterial or archaeal communities (Table 3).
The abundance of Family XIII at the family level and an unclassified ASV (ASV379) belonging to Family XIII at the ASV level were affected by breed (LRT, P.adj < 0.01) (Table 4). Family XIII was higher in Lanark compared to Cheviot (Wald, P.adj < 0.05; Log2FC = 1.41) and Perth (Wald, P.adj < 0.05; Log2FC = 1.13), and ASV379, belonging to Family XIII, was higher in the Lanark breed (Wald, P.adj < 0.05; Log2FC = 2.88) when compared to Perth breed (Table 5). Pairwise analysis revealed a further seven bacterial ASVs as differentially abundant. ASV37 classified to the Lachnospiraceae family was higher in the Perth (Wald, P.adj < 0.05; Log2FC = 2.72) compared to the Lanark, ASV123 classified to the genus Prevotella_1 was higher in the Connemara (Wald, P.adj < 0.01; Log2FC = 7.85) compared to the Lanark, ASV633 classified to the genus Ruminococcacese UCG–010 was higher in the Perth (Wald, P.adj < 0.05; Log2FC = 4.0) compared to the Lanark, ASV24 classified to the genus Succiniclasticum was lower in the Connemara compared to Cheviot (Wald, P.adj < 0.0001; Log2FC = 24.14), Lanark (Wald, P.adj < 0.0001; Log2FC = 23.67) and Perth (Wald, P.adj < 0.0001; Log2FC = 25.98) breeds, ASV74 classified to the genus Syntrophococcus was higher in the Lanark breed compared to Connemara (Wald, P.adj < 0.05; Log2FC = 5.42), ASV33 classified to the genus Ruminococcus_1 was higher in Perth (Wald, P.adj < 0.05; Log2FC = 4.81) and Connemara (Wald, P.adj < 0.01; Log2FC = 7.80) compared to Cheviot, and ASV118 also classified to the genus Ruminococcus_1 was higher in Perth (Wald, P.adj < 0.05; Log2FC = 5.52) compared to Lanark. At the genus level, Sharpea was higher in the Perth compared to the Cheviot (Wald, P.adj < 0.05; Log2FC = 3.13), ASV361 classified to Family XIII AD3011 group was higher in the Lanark (Wald, P.adj < 0.01; Log2FC = 2.28) when compared to the Cheviot, while ASV69 classified to Ruminococcaceae UCG-014 (Wald, P.adj < 0.05; Log2FC = 2.77) and ASV406 classified to Family XIII UCG-001 (Wald, P.adj < 0.05; Log2FC = 3.10) were both higher in the Cheviot when compared to the Lanark. At the family level Atopobiaceae was higher in the Lanark (Wald, P.adj < 0.05; Log2FC = 1.96) compared to Perth, and Synergistaceae was higher in the Lanark compared to the Cheviot (Wald, P.adj < 0.05; Log2FC = 2.19) (Table 5).
Effect of ruminal fraction on bacterial and archaeal populations across breeds
Bacterial and archaeal populations across ruminal fractions were investigated for Cheviot, Lanark and Perth breeds, and only included animals where all three ruminal fractions were available. Firmicutes was the most abundant phylum in the Cheviot (mean, solid = 51%, liquid = 41%, epithelial = 45%), Lanark (mean, solid = 44%, liquid = 39%, epithelial = 43%) and Perth (mean, solid = 49%, liquid = 46%, epithelial = 49%) breeds (Fig. 3). In the epithelial fraction Prevotella_7 was the most abundant genus in the Cheviot (10.6%) and Lanark (8.3%) breeds, while Prevotella_1 (10.0%) was the most dominant genus for the Perth breed. In the liquid fraction Prevotella_1 was the most dominant in the Cheviot (12.1%) breed, while Prevotella_7 was most dominant in Lanark (11.1%) and Perth (13.1%) breeds. In the solid ruminal fraction unclassified Lachnospiracheae, Prevotella_1 and Prevotella_7 were most abundant in the Cheviot (9.9%), Lanark (10.5%) and Perth (11.1%) breeds, respectively (Fig. 4).
Stack barchart representing the mean relative abundance of the 5 most dominant phyla across ruminal fractions (i.e. solid, liquid and epithelial) for Cheviot, Lanark and Perth breeds. Cheviot (solid n = 7, liquid n = 5, epithelial n = 5), Lanark (solid n = 7, liquid n = 5, epithelial n = 5), Perth (solid n = 7, liquid n = 5, epithelial n = 5).
Stack barchart representing the mean relative abundance of the 10 most dominant bacterial genera across ruminal fractions (i.e. solid, liquid and epithelial) for Cheviot, Lanark and Perth breeds. Cheviot (solid n = 7, liquid n = 5, epithelial n = 5), Lanark (solid n = 7, liquid n = 5, epithelial n = 5), Perth (solid n = 7, liquid n = 5, epithelial n = 5).
For the Cheviot breed, bacterial community alpha diversity measures were not affected by ruminal fraction (ANOVA, P > 0.1). For the Lanark breed, bacterial community Shannon diversity was affected by ruminal fraction (ANOVA, P < 0.05). For the Perth breed, bacterial community richness (observed ASV) and phylogenetic diversity (PD) were affected by ruminal fraction (ANOVA, P < 0.05), with the rumen epithelial fraction exhibiting greater diversity than solid and liquid ruminal fractions (Table 6). For all three breeds, archaeal community alpha diversity measures were not affected by ruminal fraction (ANOVA, P > 0.1). Beta diversity analysis showed that bacterial and archaeal community composition were also unaffected by ruminal fraction for all breeds analysed (PERMANOVA, P > 0.1) (Table 7).
Overall, ruminal fraction influenced 36 taxonomic groups across all ranks, representing 19 distinct ASVs, in the three breeds studied (LRT, P < 0.05). Ruminal fraction influenced the abundance of 18 taxa (11 distinct ASVs) in the Lanark breed, the most of any of breeds studied. ASV141, classified to the phylum Epsilonbacteraeota and the genus Campylobacter, was affected by ruminal fraction (LRT, P < 0.05) at all taxonomic ranks (i.e. phylum to ASV) and found to be significantly more abundant in the epithelial fraction when compared to the solid fraction. The abundance of ASV449, classified to the genus Desulfobulbus, was affected by ruminal fraction at the order, family and genus taxonomic ranks (LRT, P.adj < 0.05), found to be significantly more abundan in the epithelial ruminal fraction. At the genus level the abundance of Butyrivibrio 2, Fretibacterium, Howardella, and an unclassified ASV (ASV219) belonging to family Neisseriaceae were all affected by ruminal fraction (LRT, P < 0.05) and significantly higher in the epithelial fraction. Conversely, the abundance of Shutterella and two unclassified ASVs belonging to families Family XII UCG-001 and Eggerthellaceae were highest in the solid ruminal fraction (Wald, P.adj < 0.05). At the ASV level, the abundance of two unclassified ASVs; ASV210 and ASV239, belonging to genus Mogibacterium and family Family XIII were affected by ruminal fraction (LRT, P < 0.05) and highest in the epithelial ruminal fraction (Wald, P < 0.05). In the Cheviot breed, the abundance of 15 taxa (8 unique ASVs) were affected by ruminal fraction. ASV141 (Campylobacter) from taxonomic ranks phylum to genus and ASV449 (Desulfobulbus) from order to genus were differentially abundant and significantly more abundant in the epithelial ruminal fraction (Wald, P < 0.05). At the family level, Neisseriaceae and an unclassified ASV, ASV198, belonging to the order Coriobacteriales, were affected by ruminal fraction, with the epithelial and solid ruminal fractions, respectively, containing a higher proportion of these bacteria. At the genus level, the abundance of Mogibacterium, Butyrivibro 2 and two unclassified ASVs, ASV142 (F_Erysipelotrichaceae_UCG-004) and ASV263 (F_Burkholderiaceae), were affected by ruminal fraction (LRT, P < 0.05), with highest abundances observed in the epithelial ruminal fraction. In the Perth breed, the abundance of the bacterial phylum Tenericutes and an unclassified archaeal genus, ASV475, belonging to the family Methanomethylophilaceae were impacted by ruminal fraction (LRT, P0.05) (Table 8) Taken together, the majority of differences in microbial abundance were observed between the solid and epithelial ruminal fractions, as shown in Table 9, which summarises all the results of pairwise analysis between fractions.
Bacterial and archaeal genera associated with FCR and ADG
Spearman’s correlation analysis was performed between the relative abundance of genera and animal production traits; FCR and ADG to find potential drivers of feed efficiency in the solid, liquid and epithelial fractions. After adjusting for repeated hypotheses testing, no genera were determined to be statistically significant. Therefore, putative drivers of FCR and ADG were considered to have a (P < 0.05). In the solid fraction, four bacterial genera showed significant negative correlations with FCR: Succinivibrionaceae (ρ = − 4.1), Lachnospira (ρ = − 3.9), Syntrophococcus (ρ = − 3.8) and an unclassified genus (ASV9) belonging to the order Gastranaerophilales (ρ = − 4.1). Ruminococcaceae UCG-013 (ρ = − 4.1) positively associated with ADG, while Lachnospiraceae NK3A20 group (ρ = − 3.8) was negatively correlated with ADG. In the liquid ruminal fraction the genus Acetitomaculum (ρ = − 3.8) an unclassified ASV belonging to the order Gastranaerophilales (ρ = − 4.4) and the archaeal genus Candidatus Methanomethylophilus (ρ = − 3.8) negatively correlated with FCR. Prevotella 9 (ρ = 3.8), Roseburia (ρ = 4.9), and 5 unclassified genera belonging to the families Ruminococcaceae-UCG-013 (ρ = 4.5), –UCG-002 (ρ = 4.4), –UCG-014 (ρ = 4.1), –UCG-010 (ρ = 4.1), and Lachnospiraceae (ρ = 3.9), and an unclassified genus belonging to order Mollicutes (ρ = 4.9) positively associated with ADG. In the epithelial fraction we observed no significant associations with FCR. Prevotella 9 (ρ = 4.8), 4 unclassified genera belonging to the families Ruminococcaceae-UCG-013 (ρ = 4.9), –UCG-009 (ρ = 4.7), –UCG-014 (ρ = 4.1) and Lachnospiraceae (ρ = 4.5), an unclassified genus belonging to order Mollicutes_RF39 (ρ = 4.6) and the archaeal genus Methanosphera (ρ = 4.6) positively associated with ADG. Mogibacterium (ρ = − 4.1) and 2 unclassified genera belonging to the families Prevotellaceae (ρ = − 4.3) and Christensenellaceae (ρ = 4.3) negatively associated with ADG (Table 10).
Source: Ecology - nature.com
