Metabolomic phenotyping of a cloned pig model

Abstract

Background: Pigs are widely used as models for human physiological changes in intervention studies, because of the close resemblance between human and porcine physiology and the high degree of experimental control when using an animal model. Cloned animals have, in principle, identical genotypes and possibly also phenotypes and this offer an extra level of experimental control which could possibly make them a desirable tool for intervention studies. Therefore, in the present study, we address how phenotype and phenotypic variation is affected by cloning, through comparison of cloned pigs and normal outbred pigs.

Results: The metabolic phenotype of cloned pigs (n = 5) was for the first time elucidated by nuclear magnetic resonance (NMR)-based metabolomic analysis of multiple bio-fluids including plasma, bile and urine. The metabolic phenotype of the cloned pigs was compared with normal outbred pigs (n = 6) by multivariate data analysis, which revealed differences in the metabolic phenotypes. Plasma lactate was higher for cloned vs control pigs, while multiple metabolites were altered in the bile. However a lower inter-individual variability for cloned pigs compared with control pigs could not be established.

Conclusions: From the present study we conclude that cloned and normal outbred pigs are phenotypically different. However, it cannot be concluded that the use of cloned animals will reduce the inter-individual variation in intervention studies, though this is based on a limited number of animals.

Figure 1

NMR spectra and assignments. Representative ¹H NMR spectra of bile, plasma and urine obtained from a cloned pig. All spectra were acquired on a 600 MHz spectrometer. Less abundant amino acids (Glu, Gln, Lys, and Arg) with complex spectra could not be unambiguously assigned in the spectral region 1.6 - 2.6 ppm in urine. Assignments: 1, bile acids, cholesterol; 2, lipids (-CH₂-); 3, bile acids cholesterol, lipids; 4, bisallyllic protons; 5, conjugated taurine; 6, choline or phosphatidylcholine (PC) (-N-(CH₃)₃); 7, glycine/taurine conjugated cholate (CA), glycine/taurine conjugated chenodeoxy cholate (CDCA); 8, Conjugated deoxycholate (H-3β), conjugated taurine (H-25); 9, PC (CH₂-N); 10, Conjugated glycine (H-25); 11, conjugated cholate and deoxycholate, PC-glycerol (3-CH₂); 12, PC-glycerol (1-CH); 13, PC (-O-CH2-); 14, CH = CH, cholesterol (6-CH), PC-glycerol (2-CH); 15; Conj bile acids (-NH-); 16, LDL/VLDL; 17, leucine; 18, valine; 19, isoleucine; 20, lactate; 21, alanine; 22, adipate; 23, acetate; 24, N-acetyl glycoproteins; 25, O-acetyl glycoproteins; 26, glutamine/glutamate; 27, pyruvate; 28, glutamate; 29, creatine; 30, choline; 31, trimethylamine-N-oxide (TMAO); 32, glucose; 33, creatinine; 34, tyrosine; 35, phenylalanine; 36, isovaleraldehyde; 37, α-hydroxyisobutyrate; 38, unknown; 39, citrate; 40, dimethylamine; 41, trimethyl amine; 42, taurine; 43, glycine; 44, phenylacetylglycine; 45, hippurate; 46, unknown; 47, alantoin; 48, urea; 49, unknown; 50, guanine. For further information about identified metabolites, please refer to Additional file 1

Figure 2

Urine PCA. PCA score plots of urine samples from cloned pigs (closed symbols) and control pigs (open symbols). n = 11. Cumulated explained variance, R²X = 0.57, Cumulated cross validated explained variance, Q²(cum) = -0.17 for the three component model made from normalized data.

Figure 3

Bile PCA. PCA score plot of bile samples from cloned pigs (closed symbols) and control pigs (open symbols) (a) and PC1 loading plot (b). n = 11. R²X = 0.57, Q²(cum) = 0.36 for the one component model. For visualization principal component 2 is shown in the score plot. Major discriminatory resonances are marked with numbers that correspond to the identifications in figure 1.

Figure 4

Plasma PCA. PCA score plot of plasma samples from cloned pigs (closed symbols) and control pigs (open symbols) (a) and PC2 loading plot (b). n = 112. R²X = 0.97, Q²(cum) = 0.74 for the 10 component model. Major discriminatory resonances are marked with numbers that correspond to the identifications in figure 1. Asterisk denotes presumed threonine, which could not be resolved in the 1-D NMR spectrum.

Figure 5

Plasma O-PLS. O-PLS score plot of all plasma samples from cloned pigs (closed symbols) and control pigs (open symbols) (a) and S-plot for plasma metabolites (b). R²X = 0.72, R²Y = 0.83, Q²(cum) = 0.65. Variables with high discriminatory ability are identified in the S-plot and metabolites related to these variables are enlarged and coded with colours according to the legend. For clarity other variables are shown as dots.