Temperament Type Specific Metabolite Profiles of the Prefrontal Cortex and Serum in Cattle

In the past decade the number of studies investigating temperament in farm animals has increased greatly because temperament has been shown not only to affect handling but also reproduction, health and economically important production traits. However, molecular pathways underlying temperament and molecular pathways linking temperament to production traits, health and reproduction have yet to be studied in full detail. Here we report the results of metabolite profiling of the prefrontal cortex and serum of cattle with distinct temperament types that were performed to further explore their molecular divergence in the response to the slaughter procedure and to identify new targets for further research of cattle temperament. By performing an untargeted comprehensive metabolite profiling, 627 and 1097 metabolite features comprising 235 and 328 metabolites could be detected in the prefrontal cortex and serum, respectively. In total, 54 prefrontal cortex and 51 serum metabolite features were indicated to have a high relevance in the classification of temperament types by a sparse partial least square discriminant analysis. A clear discrimination between fearful/neophobic-alert, interested-stressed, subdued/uninterested-calm and outgoing/neophilic-alert temperament types could be observed based on the abundance of the identified relevant prefrontal cortex and serum metabolites. Metabolites with high relevance in the classification of temperament types revealed that the main differences between temperament types in the response to the slaughter procedure were related to the abundance of glycerophospholipids, fatty acyls and sterol lipids. Differences in the abundance of metabolites related to C21 steroid metabolism and oxidative stress indicated that the differences in the metabolite profiles of the four extreme temperament types could be the result of a temperament type specific regulation of molecular pathways that are known to be involved in the stress and fear response

Comparing Two Intestinal Porcine Epithelial Cell Lines (IPECs): Morphological Differentiation, Function and Metabolism

The pig shows genetical and physiological resemblance to human, which predestines it as an experimental animal model especially for mucosal physiology. Therefore, the intestinal epithelial cell lines 1 and J2 (IPEC-1, IPEC-J2) - spontaneously immortalised cell lines from the porcine intestine - are important tools for studying intestinal function. A microarray (GeneChip Porcine Genome Array) was performed to compare the genome wide gene expression of IPECs. Different significantly up-regulated pathways were identified, like “lysosome”, “pathways in cancer”, “regulation of actin cytoskeleton” and “oxidative phosphorylation” in IPEC-J2 in comparison to IPEC-1. On the other hand, “spliceosome”, “ribosome”, “RNA-degradation” and “tight junction” are significantly down-regulated pathways in IPEC-J2 in comparison to IPEC-1. Examined pathways were followed up by functional analyses. ATP-, oxygen, glucose and lactate-measurement provide evidence for up-regulation of oxidative phosphorylation in IPEC-J2. These cells seem to be more active in their metabolism than IPEC-1 cells due to a significant higher ATP-content as well as a higher O2- and glucose-consumption. The down-regulated pathway “ribosome” was followed up by measurement of RNA- and protein content. In summary, IPEC-J2 is a morphologically and functionally more differentiated cell line in comparison to IPEC-1. In addition, IPEC-J2 cells are a preferential tool for in vitro studies with the focus on metabolism

Annotation and classification of serum metabolites with high relevance in the classification of temperament types.

<p>Annotation and classification of serum metabolites with high relevance in the classification of temperament types.</p

Annotation and classification of prefrontal cortex metabolites with high relevance in the classification of temperament types.

<p>Annotation and classification of prefrontal cortex metabolites with high relevance in the classification of temperament types.</p

Significant differences between temperament types in the abundance of selected excitatory and inhibitory neurotransmitters in the prefrontal cortex.

<p>Significant differences (<i>p</i> < 0.05) between temperament types are indicated by arrows.</p><p>Significant differences between temperament types in the abundance of selected excitatory and inhibitory neurotransmitters in the prefrontal cortex.</p

Principal component score plot for prefrontal cortex and serum metabolites.

<p>Principal component score plot of the first two principal components for (A) the complete prefrontal cortex data and (B) the complete serum data. Symbols indicate the different temperament types, rhombs the fearful/neophobic-alert, circles the interested-stressed, triangles the outgoing/neophilic-alert, squares the subdued/uninterested-calm and crosses the indistinct temperament type.</p

Hierarchical clustering of metabolites with high relevance.

<p>Two way hierarchical clustering of (A) prefrontal cortex and (B) serum metabolite features with a high relevance in the classification of temperament types. Symbols indicate the different temperament types, rhombs the fearful/neophobic-alert, circles the interested-stressed, triangles the outgoing/neophilic-alert, squares the subdued/uninterested-calm and crosses the indistinct temperament type.</p

Metabolites with high relevance in the classification of temperament types.

<p>Metabolite names, measuring technique and their VIP-Scores, number of occurrences in the outer cross-validation runs and nominal <i>p</i>-values for the Kruskal-Wallis-Test are shown for serum (S) and prefrontal cortex (PC) metabolites with high relevance in the classification of temperament types.</p><p>Metabolites with high relevance in the classification of temperament types.</p

Polarisation of IPEC-1 and IPEC-J2, confocal microscopy and transmission electron microscopy.

<p>(A) Ten days old IPEC-1 and IPEC-J2 cells were fixed and stained for ZO-1 (<i>Zonula occludens</i>, green) and beta-catenin (<i>Zonula adherens</i>, red). Both cells lines showed a polarised structure with ZO-1 immunoreactivity at the apical pole and the underlying beta-catenin expression. (B) Important genes of a polarised brush border were analysed using microarray and qPCR. Here, significant differences were found between the cell lines. VIL1 (villin-1), VIL2 (villin-2 = ezrin), TLR4 (toll like receptor 4), MUC4 (mucin 4) and ESPN (espin) were significant up-regulated in IPEC-J2 in comparison to IPEC-1 in microarray as well as in qPCR. Villin1 and villin2 (= ezrin) were followed up by Western blot analyses. No villin-1 was detected in IPEC-1 but a strong expression in IPEC-J2 over time. Villin-2 was expressed in both cell lines. (C) Cells were cultured for 10 or 31 days and analysed using transmission electronmicroscopy. Both cell lines showed well-developed tight junctions at day 10 and 31 (red arrows). Desmosomes (blue arrows) and interdigitation (yellow arrow) were also observed. No differences were detected concerning microvilli length between both cell lines and at both time points. In contrast, the number of microvilli differed between the cell lines (IPEC-J2>IPEC-1).</p

Anchorage independent growth.

<p>Both cell lines were seeded in “Soft agar” (S, 0.33%) on “Feeder agar” (F, 0.5%) with (w) or without (wo) the application of EGF and ITS. Caco-2 cells were used as positive control. No anchorage independent growth was detected in IPEC-1 and IPEC-J2. On the other hand, Caco-2 showed an anchorage independent growth which did not depend on the additives EGF and ITS (bar = 20 μm). The results represent at least three independent experiments (n = 3).</p