Figure 1.
Immunohistochemical staining of ovine myosin heavy chains and laminin in serial sections of semimembranosus muscle.
Representative myofibers are indicated: s, type 1 (slow oxidative); a, type 2A (fast twitch oxidative-glycolytic); c, type 2C (type 1–type 2A intermediate); ax, type 2AX (type 2A–type 2X intermediate). Non-labelled myofibers represent type 2X (fast twitch glycolytic). The top, middle and bottom panels were stained with myosin heavy chains (MHC) antibodies S5 8H2, WB-MHCs and MY32, respectively. Note the larger type 2X myofibres in the semimembranosus muscle of Callipyge (NmatCpat genotype) (right column) compared with normal (NmatNpat genotype) (left column) lambs, and the absence in the sections shown here and the overall very low prevalence of type 2A and type 2AX myofibres in Callipyge compared to normal semimembranosus muscle (see Table 1).
Table 1.
Myofibre characteristics of muscle affected (semimembranosus and semitendinosus) and not affected (supraspinatus) by the Callipyge mutation, determined using myosin heavy chain (MHC) immunohistochemistry in normal (NmatNpat) and Callipyge (NmatCpat) lambs at 11 weeks of age.
Figure 2.
Orthogonal partial least squares – discriminant analysis (OPLS–DA) of lamb plasma metabolic profiles.
Panel A: Scores plot of the comparison of plasma samples at 8 weeks (red dots) and 12 weeks (black dots) of age. Samples are separated by their age in dimension t[1], while the first orthogonal dimension to[1] contains orthogonal intra-group variation unrelated to age. Panel C: Scores plot of the comparison of plasma samples from NmatNpat (triangles) and NmatCpat (open triangles) lambs at 12 weeks of age. Samples are separated by genotype in dimension t[1], while the first orthogonal dimension to[1] contains intra-group variation unrelated to genotype. Panels B and D: corresponding 1D back-scaled loadings plots (0.5–5.1 ppm) for panels A and C, respectively, with the identity of several metabolites annotated. Weights of variables are shown by the colour scale. 3HB, 3-hydroxybutyrate; NAC, N-acetyl glycoproteins; TMAO, trimethylamine N-oxide; U3/U4, unknown metabolites 3/4.
Table 2.
Changes in relative concentrations of key metabolites in lamb plasma during postnatal developmenta.
Figure 3.
The metabolic pathways are represented as circles according to their scores from enrichment (vertical axis) and topology analyses (pathway impact, horizontal axis) using MetaboAnalyst 2.0 [56]. Darker circle colors indicate more significant changes of metabolites in the corresponding pathway. The size of the circle corresponds to the pathway impact score and is correlated with the centrality of the involved metabolites. The metabolic pathways involved in age differences are shown in panels A and B, and the pathways perturbed due to the Callipyge mutation are summarized in panels C and D. Panels A and C were generated using the Homo sapiens library, while the Bos taurus library was selected for production of panels B and D, respectively. Pathways were annotated by numbering when the P values calculated from the enrichment analysis were <0.05. The annotated pathways include: 1, Aminoacyl-tRNA biosynthesis; 2, Arginine and proline metabolism; 3, Biotin metabolism; 4, Cyanoamino acid metabolism; 5, D-Glutamine and D-glutamate metabolism; 6, Galactose metabolism; 7, Glycine, serine and threonine metabolism; 8, Lysine degradation; 9, Methane metabolism; 10, Nitrogen metabolism; 11, Synthesis and degradation of ketone bodies. The color of each metabolic pathway is related to the P value obtained from enrichment analysis and its size represents the fold enrichment score i.e. −ln(P).
Figure 4.
Metabolic pathways involved in postnatal lamb development.
Metabolites indentified in Table 2 are summarized according to their occurrence in metabolic pathways as annotated by KEGG metabolic pathways. ↓, metabolites decreasing with age (blue); ↑, metabolites increasing with age (red). TMAO, trimethylamine N-oxide; HCHO, formaldehyde; CH4, methane. The reactions contained in the boxed section are likely to be derived from rumen microbial metabolism, except for reactions denoted by **, which occur in mammalian tissues [63], [84], and reactions denoted by *, which can occur both in mammalian tissues or the gut microbiome [85]. This representation of the data recognises that plasma metabolites report the combined metabolic activities of all major tissues as well as the rumen microbiome.
Table 3.
Comparison of relative concentrations of metabolites in plasma from Callipyge (NmatCpat) and normal (NmatNpat) lambs at 12 weeks of agea.
Figure 5.
Metabolic pathways affected by the Callipyge mutation in lambs at 12 weeks of age.
Metabolites indentified in Table 3 are summarized according their occurrence in metabolic pathways as annotated by KEGG metabolic pathways. ↓, metabolites decreased in plasma from NmatCpat lambs (blue); ↑, metabolites increased in plasma from NmatCpat lambs (red).