Although IVF has been performed routinely for many years to help couples with fertility problems and in relation to modern breeding of farm animals, pregnancy rates after transfer to a recipient have not improved during the last decade. Early prediction of the viability of in-vitro developed embryos before the transfer to a recipient still remains challenging. Presently, the predominant non-invasive technique for selecting viable embryos is based on morphology, where parameters such as rates of cleavage and blastocyst formation as well as developmental kinetics are evaluated mostly subjectively. The simple morphological approach is, however, inadequate for the prediction of embryo quality, and several studies have focused on developing new non-invasive methods using molecular approaches based particularly on proteomics, metabolomics and most recently small non-coding RNA, including microRNA. This review outlines the potential of several non-invasive in-vitro methods based on analysis of spent embryo culture medium.

The pig has been proposed as a relevant model for human obesity-induced inflammation, and cloning may improve the applicability of this model. We tested the assumptions that cloning would reduce interindividual variation in gene expression of innate immune factors and that their expression would remain unaffected by the cloning process. We investigated the expression of 40 innate immune factors by high-throughput quantitative real-time PCR in samples from liver, abdominal subcutaneous adipose tissue (SAT), visceral adipose tissue (VAT), and neck SAT in cloned pigs compared to normal outbred pigs.The variation in gene expression was found to be similar for the two groups, and the expression of a small number of genes was significantly affected by cloning. In the VAT and abdominal SAT, six out of seven significantly differentially expressed genes were downregulated in the clones. In contrast, most differently expressed genes in both liver and neck SAT were upregulated (seven out of eight). Remarkably, acute phase proteins (APPs) dominated the upregulated genes in the liver, whereas APP expression was either unchanged or downregulated in abdominal SAT and VAT. The general conclusion from this work is that cloning leads to subtle changes in specific subsets of innate immune genes. Such changes, even if minor, may have phenotypic effects over time, e.g., in models of long-term inflammation related to obesity.

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The transcription factor, tonicity-responsive enhancer binding protein (TonEBP), is involved in the adaptive response against hypertonicity. TonEBP regulates the expression of genes that catalyze the accumulation of osmolytes, and its transcriptional activity is increased by hypertonicity. The goal of the present investigation was to investigate whether cell shrinkage or high intracellular ionic concentration induced the activation of TonEBP. We designed a model system for isotonically shrinking cells over a prolonged period of time. Cells swelled in hypotonic medium and performed a regulatory volume decrease. Upon return to the original isotonic medium, cells shrank initially, followed by a regulatory volume increase. To maintain cell shrinkage, the RVI process was inhibited as follows: ethyl-isopropyl-amiloride inhibited the Na(+)/H(+) antiport, bumetanide inhibited the Na(+)-K(+)-2Cl(-) cotransporter, and gadolinium inhibited shrinkage-activated Na(+) channels. Cells remained shrunken for at least 4 h (isotonically shrunken cells). The activity of TonEBP was investigated with a Luciferase assay after isotonic shrinkage and after shrinkage in a high-NaCl hypertonic medium. We found that TonEBP was strongly activated after 4 and 16 h in cells in high-NaCl hypertonic medium, but not after 4 or 16 h in isotonically shrunken cells. Cells treated with high-NaCl hypertonic medium for 4 h had significantly higher intracellular concentrations of both K(+) and Na(+) than isotonically shrunken cells. This strongly suggested that an increase in intracellular ionic concentration and not cell shrinkage is involved in TonEBP activation.

The usefulness of Göttingen minipigs as models for obesity and obesity-related pathologies is well established. The low-grade inflammation associated with obesity involves a range of innate immune factors; however, to our knowledge, the impact of obesity on innate immune factor expression has not been studied in Göttingen minipigs. Therefore, we studied the expression of innate immune genes in liver and adipose tissues as well as serum concentrations of cytokines and acute phase proteins in obese vs. lean Göttingen minipigs. In the liver, of 35 investigated genes, the expression of nine was significantly different in obese pigs (three up-regulated, six down-regulated). Of 33 genes in adipose tissues, obesity was associated with changed expression of 12 genes in the visceral adipose tissue (VAT) (three up-regulated), 11 in the abdominal retroperitoneal adipose tissue (RPAT) (seven of these up-regulated) and eight in the subcutaneous adipose tissue (SAT) from the neck (five of which were up-regulated). Obesity-associated expression changes were observed for three genes in all adipose tissues, namely chemokine (C-C motif) ligand 3-like 1 (up-regulated), CD200 molecule (down-regulated) and interleukin 1 receptor antagonist (up-regulated) with interleukin 1 receptor antagonist being the most highly regulated gene in both VAT and RPAT. Looking at patterns of expression across the three types of adipose tissues, obesity was associated with an increased number of acute phase proteins differentially expressed between adipose tissues and a decreased tissue-specific expression of cytokines and chemokines. In contrast to obese humans, no changes in serum concentrations of haptoglobin, C-reactive protein, serum amyloid A, tumor necrosis factor-α and interleukin 6 were found in obese Göttingen minipigs.