Cloned animals are expected to be more homogenous than outbred lines and could therefore represent a good model for research purposes where a small inter-individual variation is desired. However, only sparse data are available about the phenotypic variation of cloned pigs [9
]. The present study for the first time reports a metabolomic phenotyping of cloned pigs. Using NMR-based metabolomics we have shown that the metabolite profile of plasma and bile, but not urine, differed for cloned pigs and normal outbred pigs. In fact, the bile and plasma concentrations of multiple metabolites differed suggesting that the cloned pigs had an altered metabolic phenotype compared with the control outbred pigs.
For plasma samples it was identified that lactate levels were higher in cloned pigs than in normal outbred pigs. Alanine and glutamate, which are both linked to the citrate cycle, were also found in higher concentrations in cloned pigs. The reason for an increased plasma lactate remains unknown. In a study on heifers, cloned animals were found to have higher oxidative metabolism than control animals as assessed by isocitrate dehydrogenase, cytochrome-c oxidase and beta-hydroxyacyl-CoA dehydrogenase activities in muscle biopsies [20
]. However, the increased plasma lactate levels observed in this study indicate higher anaerobic metabolism. Consequently, the increased lactate levels in the cloned pigs are probably a consequence of another mechanism. Oocytes and embryos are inevitably exposed to oxidative stress generated by reactive oxygen species during in vitro culture [21
], and this might have impact on stress sensitivity post-natally in the cloned pig. The higher plasma lactate levels might therefore reflect higher stress sensitivity in cloned pigs compared with normal pigs due to the in vitro cloning procedure.
Plasma lipid and lipoprotein signals were higher in the control group as compared to the cloned group, which may be related to differences in the regulation of lipoprotein circulation. Likewise creatine, which is involved in cellular energy production, was elevated in control pigs. Thus, several metabolites indicate that the response of cloned pigs and control pigs to a dietary intervention is not the same, and this could affect the applicability in relation to human nutrition.
Furthermore, differences in bile composition, which is also under genetic control, could affect lipid metabolism [22
]. The relative content of bile acids and phospholipids affects the micelle surface and core composition in the small intestine, and thus probably has an impact on the absorption of dietary lipids [23
]. Increasing amounts of phospholipids have also been shown to reduce the critical micelle concentration of bile salts, thus affecting the number of micelles [24
]. In the present study high amounts of bile acids in the bile were associated with an increased content of lipids in plasma, though a direct causal connection could not be established. Studies with rodents have shown that an increase in plasma bile acids induced by diet, reduce liver VLDL secretion and prevent elevated serum triacylglycerol concentration [26
]. These findings seem opposite to our data and further work is needed to elucidate the physiological role of production and circulation of bile acids, which also appears to be important for weight regulation [27
In order to elucidate the potential of a cloned pig model, it is also important to consider the inter-individual variation. Consequently, for all bio-fluids the variance of individual metabolites was calculated and compared with the variance for control outbred pigs. This analysis did not reveal a significant difference in variation between the two groups. Consequently, based on the present study it cannot be concluded that the inter-individual variation in the metabolic phenotype is smaller for cloned pigs compared with normal outbred pigs. While a standardized genotype does not seem to affect the inter-individual variation, the gut-microbiota is known to affect host phenotype [19
]. In the present study no systematic effect of gut-microbiota was expected since all animals received the same diet, and no difference in the contents of metabolites originating from microbial fermentation (e.g. acetic acid, butyric acid) could be demonstrated in the present study. Fluctuations in the host metabolome could arise, however, as a result of the complex interactions between nutrition, immune function, and gut-microbiota, and therefore the gut-microbiomes of cloned and normal pigs are currently being analyzed and will be the subject of additional papers. Therefore, when a low inter-individual variation between subjects is required, a standardization of the gut-microbiota might be more important than standardization of the genotype [28
]. However, the present study only included a limited number of animals, and further studies with larger numbers of subjects are needed to substantiate these results. In addition the metabolites included in the analysis were chosen among the most intense resonances, which may introduce a bias. Less abundant metabolites, which are not easily detected by NMR-based metabolomics, are currently being analyzed by LC-MS-based metabolomics and will be the subject of a subsequent paper