Analysis of a candidate gene for behavioural trait variability in horses: catechol-O-methyltransferase (COMT)

Catechol-O-methyltransferase (COMT) gene is a candidate for the variability of several traits including anxiety, stress response and pain sensibility. The aim of this work was to study COMT gene regions in horse samples (Equus caballus) to check for polymorphisms. DNA samples from 12 horses of different breeds were amplified with two primer pairs and then sequenced on both strands. We analysed about 1000 bp of the gene including exon 4, 5, and 6 regions, the intron 4 and part of the 3\u2019UTR. One single nucleotide polymorphism located in the 3\u2019-UTR of COMT gene (c.*111G>A) was identified and a new PCR-RFLP protocol was set up. Preliminary results indicated that the most frequent allele was the c.*111G. The mutation for its localisation in the 3\u2019-UTR region might have an effect on the mRNA stability and on the levels of enzymes

Olanzapine counteracts stress-induced anxiety-like behavior in rats

Atypical antipsychotics, such as olanzapine, have been reported to display anxiolytic properties as shown in several preclinical and clinical studies. Furthermore, several experimental evidences have shown that olanzapine reduces fear and anxiety in activated anxiety like-behavior test such as Geller-test, ultrasonic vocalization test and stress-induced EtOH consumption. Here, we hypothesized that the anxiolytic action of olanzapine might be due to via an indirect activation of the gamma-amino butyric acid (GABA)-ergic system through 3 alfa-hydroxy-5 alfa-pregnan-20-0ne [allopregnanolone (ALLO)], a potent neuroactive steroid that positively modulates the GABAA/benzodiazepine receptors complex. To address this question, we used a preclinical animal test to screen for novel anxiolytic compounds \u2013 the elevated plus maze (EPM) - in basal condition and after 45 min-restrain stress after acute or repeated (21 days) administration of olanzapine (0.5 mg/Kg i.p.). In this condition, we therefore study the effect of the 5-alpha-reductase inhibitor finasteride (FIN) (50 mg/kg) after co-administration with olanzapine. FIN is an inhibitor of steroidogenic enzymes which acts by inhibiting type II 5-alpha reductase, the enzyme that converts into 5-alpha reduced metabolites like the GABAA positive neuroactive steroid ALLO. Results showed an anxiolytic effect of the acute, but not of the chronic, treatment with olanzapine only in stressed rats. This anxiolytic effect was counteracted by the coadministration with FIN. These evidences suggest that the anxiolytic effects of olanzapine might be due to possible action of olanzapine on steroid function via activation of GABA system

Study of porcine adiponectin (ADIPOQ) gene and association of a missense mutation with EBVs for production and carcass traits in Italian Duroc heavy pigs

The adiponectin (adiponectin, C1Q and collagen domain containing) encoded by ADIPOQ is adipokine that modulates several biological processes such as lipogenesis, gluconeogenesis, fatty acid oxidation, glucose uptake and insulin sensitivity. An objective of this study was to identify SNPs in the ADIPOQ gene both in cosmopolitan and local pig breeds reared in Italy and in Meishan samples. Resequencing of ADIPOQ gene regions in 16 pigs of 12 breeds revealed only two SNPs (EU489740:g.1611G>A synonymous and the EU489740:g.1735G>A missense mutations) already reported. The g.1735G>A polymorphism was genotyped by PCR-RFLP in 510 pigs from 11 breeds reared in Italy (Italian Large White, Italian Duroc, Italian Landrace, Belgian Landrace, Hampshire, Pietrain, Calabrese, Casertana, Cinta Senese, Mora Romagnola and Nero Siciliano) and from Chinese Meishan breed. The Italian local breeds were homozygous for the g.1735G allele while the Meishan samples resulted homozygous for the g.1735A allele. The polymorphism segregates in the cosmopolitan breeds, except for Hampshire, and the rarer g.1735A allele showed the highest frequency in Italian Duroc (0.12). Association analysis of g.1735G>A polymorphism with estimated breeding values (EBVs) for average daily gain (ADG), feed:gain ratio (FGR), weight of neck and loin (lean cuts, LC), weight of ham (H) and backfat thickness (BFT) were performed in Italian Duroc heavy pigs. The GA vs GG pigs showed higher least square means for ADG (P= 0.003) and LC (P= 0.036) EBVs and lower value for FGR EBV (P= 0.033)

Functional genomic approaches to study three porcine lipogenic genes

Lipogenesis is a complex process mainly influenced by genes coding for enzymes involved in de novo fatty acids synthesis. Changes in mRNA abundance of these genes have been associated to obesity related traits but the genetic regulation of fat deposition is not completely known. The variability at DNA and mRNA level of FASN (Fatty Acid Sinthase), ACL (ATP Citrate Lyase) and ACACA (Acetyl CoA Carboxylase) genes, involved in key steps of fatty acids metabolism, was investigated in porcine skeletal muscle and fat tissues. We genotyped Italian Duroc (IDU) and Italian Large White (ILW) pigs both for the SNP T265C of FASN gene (Munoz et al 2003) and the SNP T2956C of ACL gene that we newly identified by in silico analisys and sequencing. We localized ACL gene on SSC12 (RH panel, LOD: 11.49 with SW943 marker) where FASN and ACACA genes map and where QTLs for fat related traits were detected. The mRNA level of the three genes was determined in skeletal muscle and fat tissues samples of ILW and IDU pigs. Differential expression between breeds (FASN P=0.057; ACL P=0.021; ACACA P=0.041) only in skeletal muscle tissue was observed and IDU pigs showed the higher level. Studies are in progress to investigate on relationship between DNA variation and gene expression. The transcription of FASN, ACL and ACACA genes was found to be regulated in both tissues and breeds suggesting that these genes can represent promising functional and positional candidates for fat deposition in meat and carcass

A comparative serum metabonomic analysis between Italian Duroc and Italian Large White pigs

Extensive metabolic profiles of body fluids, obtained by nuclear magnetic resonance (NMR) spectroscopic or mass spectroscopic data, can provide information to deduce the effects of many different factors on biological processes. In particular, genetic factors can determine differences in physiological processes that are captured by metabolomics or metabonomics analyses. In this study we compared metabonomic profiles obtained from serum of two pig breeds (Italian Large White and Italian Duroc) with the aim to disentangle breed specific biological aspects. Twelve castrated males (6 Italian Large White and 6 Italian Duroc) were raised at the test station of the National Pig Breeders Association (ANAS) in the same conditions till they reached a live weight of about 160 kg. After a period of starvation, early in the morning pigs were transported all together at a commercial slaughterhouse, electrically stunned and jugulated within 10 minutes. Collected blood sample were processed after about 2 hours and serum was stored at -80\ub0C till use. High Resolution Magic Angle Spinning-NMR serum spectra were recorded with a Bruker AVANCE spectrometer. Each sample was analysed in triplicate. Preliminary Principal Component Analysis of the obtained NMR spectra indicated that metabonomic analysis could be able to distinguish breed specific metabolic profiles. These results might represent useful information to disclose genetically determined regulatory pathways contributing to understand breed differences in biological processes indirectly linked with production traits