Sara Pegolo

Genetic and environmental relationships of detailed milk fatty acids profile determined by gas chromatography in Brown Swiss cows

The aim of this study was to characterize the profile of 47 fatty acids, including conjugated linoleic acid (CLA), 13 fatty acid groups, and 5 Δ(9)-desaturation indices in milk samples from Brown Swiss cows. The genetic variation was assessed and the statistical relevance of the genetic background for each trait was evaluated using the Bayes factor test. The additive genetic, herd-date, and residual relationships were also estimated among all single fatty acids and groups of fatty acids. Individual milk samples were collected from 1,158 Italian Brown Swiss cows and a detailed analysis of fat percentages and milk fatty acid compositions was performed by gas chromatography. Bayesian animal models were used for (co)variance components estimation. Exploitable genetic variation was observed for most of the de novo synthesized fatty acids and saturated fatty acids, except for C4:0 and C6:0, whereas long-chain fatty acids and unsaturated fatty acids (including CLA) were mainly influenced by herd-date effects. Herd-date effect explained large portions of the total phenotypic variance for C18:2 cis-9,cis-12 (0.668), C18:3 cis-9,cis-12,cis-15 (0.631), and the biohydrogenation and elongation products of these fatty acids. The desaturation ratios showed higher heritability estimates than the individual fatty acids, except for CLA desaturation index (0.098). Among the medium-chain fatty acids, C12:0 had greater heritability than C14:0 (0.243 vs. 0.097, respectively). Both C14:0 and C16:0 showed negative additive genetic correlations with the main monounsaturated and polyunsaturated fatty acids of milk fat, suggesting that their synthesis in the mammary gland may be influenced by the presence of unsaturated fatty acids. No correlation was observed between C4:0 and the other short-chain fatty acids (except for C6:0), confirming the independence of C4:0 from de novo mammary fatty acid synthesis. Among the genetic correlations dealing with potentially beneficial fatty acids, C18:0 was positively correlated with vaccenic and rumenic acids and negatively with linoleic acid. Finally, fatty acids C6:0 through C14:0 showed relevant correlations due to unknown environmental effects, suggesting the potential existence of genetic variances in micro-environmental sensitivity. This study allowed us to acquire new knowledge about the genetic and the environmental relationships among fatty acids. Likewise, the existence of genetic variation for most of de novo synthetized fatty acids and saturated fatty acids was also observed. Overall, these results provide useful information to combine feeding with genetic selection strategies for obtaining a desirable milk fatty acids profile, depending on the origin of fatty acids in milk.

Real time RT-PCR analysis of inflammatory mediator expression in recurrent airway obstruction-affected horses

The goal of the present study was to investigate mRNA expression levels of several cytokines and inflammatory mediators in broncho-alveolar lavage (BAL) fluid and respiratory epithelium in recurrent airway obstruction (RAO)-affected horses. RAO, also called heaves, is a common, performance-limiting, equine respiratory disease with clinical signs and pathophysiological similarities to human asthma, and characterized by bronchospasm, neutrophilic infiltration and increased mucus in the airways. Six RAO-affected horses were examined twice within 15 days and seven clinically healthy horses were examined for comparison. Quantitative real-time RT-PCR was used to assess mRNA expression of the inflammatory mediators IL-1β, IL-6, IL-8, IL-13, IL-17, TNFα, INFγ, TGFβ1, NFκ-β and TRL4 in bronchial biopsies and in BAL fluid. Gene expression levels were then compared with clinical signs, endoscopic examination, complete blood cell count, cytology of BAL fluid, histological examination of bronchial tissue and bacteriological and mycological examinations. Expression of IL1β, IL8, TLR4, TNFα, TGFβ1 and NFkβ transcripts was significantly up-regulated in RAO-affected compared to healthy horses. A similar trend, albeit not significant, was showed for IL17 and INFγ. A highly significant correlation was observed among IL-1β, IL8, TGFβ1, NFkβ, TRL4, and INFγ expression patterns as well as between expression levels of these genes and clinical parameters. In the present study, the comparison between clinically healthy and RAO-affected horses gave new insights on the cytokine expression in equine health and disease status. The identification of cytokines implicated in the pathogenesis of RAO may contribute to the diagnosis and treatment of this disease.

High performance liquid chromatography determination of cytochrome P450 1A and 2C activities in bovine liver microsomes

This study reports fluorescence high performance liquid chromatography (HPLC) and UV-Vis HPLC methods for the determination of 7-ethoxyresorufin O-deethylase (EROD) and tolbutamide methylhydroxylase (TMH) activities, respectively, using bovine liver microsomes. The detection limits were 0.022 and 5.5 pmol on the column, respectively; intra-day and inter-day precisions (expressed as relative standard deviation) were <10%. Both methods showed enough sensitivity to allow for an accurate determination of enzyme kinetic parameters according to Michaelis-Menten plots and the results were: K(m)=0.23+/-0.051 microM, V(max)=0.488+/-0.035 nmol/min/mg protein for EROD activity, and K(m)=1010+/-155.7 microM, V(max)=0.089+/-0.006 nmol/min/mg protein for TMH activity. An Eadie-Hofstee plot analysis showed that in bovine liver microsomes, EROD and TMH activities followed a monophasic kinetic pattern. alpha-Naphthoflavone, a cytochrome P450 1A1/2 (CYP1A1/2) inhibitor, and sulfaphenazole, a cytochrome P450 2C9 (CYP2C9) inhibitor, decreased EROD and TMH activities, respectively. The sensitivity of the methods allowed the use of microsomes with low enzyme activity, such as those from veal calf liver. Thus, EROD and TMH activities may be adopted as markers for the evaluation of CYP1A and CYP2C9-like activities in liver microsomes from veal and beef cattle.

Transcriptomic markers meet the real world: finding diagnostic signatures of corticosteroid treatment in commercial beef samples

BackgroundThe use of growth-promoters in beef cattle, despite the EU ban, remains a frequent practice. The use of transcriptomic markers has already proposed to identify indirect evidence of anabolic hormone treatment. So far, such approach has been tested in experimentally treated animals. Here, for the first time commercial samples were analyzed.ResultsQuantitative determination of Dexamethasone (DEX) residues in the urine collected at the slaughterhouse was performed by Liquid Chromatography-Mass Spectrometry (LC-MS). DNA-microarray technology was used to obtain transcriptomic profiles of skeletal muscle in commercial samples and negative controls. LC-MS confirmed the presence of low level of DEX residues in the urine of the commercial samples suspect for histological classification. Principal Component Analysis (PCA) on microarray data identified two clusters of samples. One cluster included negative controls and a subset of commercial samples, while a second cluster included part of the specimens collected at the slaughterhouse together with positives for corticosteroid treatment based on thymus histology and LC-MS. Functional analysis of the differentially expressed genes (3961) between the two groups provided further evidence that animals clustering with positive samples might have been treated with corticosteroids. These suspect samples could be reliably classified with a specific classification tool (Prediction Analysis of Microarray) using just two genes.ConclusionsDespite broad variation observed in gene expression profiles, the present study showed that DNA-microarrays can be used to find transcriptomic signatures of putative anabolic treatments and that gene expression markers could represent a useful screening tool.

Transcriptomic profiling as a screening tool to detect trenbolone treatment in beef cattle.

The effects of steroid hormone implants containing trenbolone alone (Finaplix-H), combined with 17β-oestradiol (17β-E; Revalor-H), or with 17β-E and dexamethasone (Revalor-H plus dexamethasone per os) on the bovine muscle transcriptome were examined by DNA-microarray. Overall, large sets of genes were shown to be modulated by the different growth promoters (GPs) and the regulated pathways and biological processes were mostly shared among the treatment groups. Using the Prediction Analysis of Microarray program, GP-treated animals were accurately identified by a small number of predictive genes. A meta-analysis approach was also carried out for the Revalor group to potentially increase the robustness of class prediction analysis. After data pre-processing, a high level of accuracy (90%) was obtained in the classification of samples, using 105 predictive gene markers. Transcriptomics could thus help in the identification of indirect biomarkers for anabolic treatment in beef cattle to be applied for the screening of muscle samples collected after slaughtering.

Pathway-based genome-wide association analysis of milk coagulation properties, curd firmness, cheese yield, and curd nutrient recovery in dairy cattle

It is becoming common to complement genome-wide association studies (GWAS) with gene-set enrichment analysis to deepen the understanding of the biological pathways affecting quantitative traits. Our objective was to conduct a gene ontology and pathway-based analysis to identify possible biological mechanisms involved in the regulation of bovine milk technological traits: coagulation properties, curd firmness modeling, individual cheese yield (CY), and milk nutrient recovery into the curd (REC) or whey loss traits. Results from 2 previous GWAS studies using 1,011 cows genotyped for 50k single nucleotide polymorphisms were used. Overall, the phenotypes analyzed consisted of 3 traditional milk coagulation property measures [RCT: rennet coagulation time defined as the time (min) from addition of enzyme to the beginning of coagulation; k20: the interval (min) from RCT to the time at which a curd firmness of 20 mm is attained; a30: a measure of the extent of curd firmness (mm) 30 min after coagulant addition], 6 curd firmness modeling traits [RCTeq: RCT estimated through the CF equation (min); CFP: potential asymptotic curd firmness (mm); kCF: curd-firming rate constant (% × min-1); kSR: syneresis rate constant (% × min-1); CFmax: maximum curd firmness (mm); and tmax: time to CFmax (min)], 3 individual CY-related traits expressing the weight of fresh curd (%CYCURD), curd solids (%CYSOLIDS), and curd moisture (%CYWATER) as a percentage of weight of milk processed and 4 milk nutrient and energy recoveries in the curd (RECFAT, RECPROTEIN, RECSOLIDS, and RECENERGY calculated as the % ratio between the nutrient in curd and the corresponding nutrient in processed milk), milk pH, and protein percentage. Each trait was analyzed separately. In total, 13,269 annotated genes were used in the analysis. The Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway databases were queried for enrichment analyses. Overall, 21 Gene Ontology and 17 Kyoto Encyclopedia of Genes and Genomes categories were significantly associated (false discovery rate at 5%) with 7 traits (RCT, RCTeq, kCF, %CYSOLIDS, RECFAT, RECSOLIDS, and RECENERGY), with some being in common between traits. The significantly enriched categories included calcium signaling pathway, salivary secretion, metabolic pathways, carbohydrate digestion and absorption, the tight junction and the phosphatidylinositol pathways, as well as pathways related to the bovine mammary gland health status, and contained a total of 150 genes spanning all chromosomes but 9, 20, and 27. This study provided new insights into the regulation of bovine milk coagulation and cheese ability that were not captured by the GWAS.

Effects of candidate gene polymorphisms on the detailed fatty acids profile determined by gas chromatography in bovine milk

Association analyses between candidate genes and bovine milk fatty acids can improve our understanding of genetic variation in milk fatty acid profiles and reveal potential opportunities to tailor milk fat composition through selection strategies. In this work, we investigated the association of 51 single nucleotide polymorphisms (SNP) selected from 37 candidate genes using a functional and positional approach, with 47 fatty acids, 9 fatty acid groups, and 5 Δ(9)-desaturation indices in milk samples from Brown Swiss cows. Individual milk samples were collected from 1,158 Italian Brown Swiss cows, and gas chromatography was used to obtain detailed milk fatty acid compositions. A GoldenGate assay system (Illumina, San Diego, CA) was used to perform genotype 96 selected SNP located in 54 genes across 22 chromosomes. In total, 51 polymorphic SNP in 37 candidate genes were retained for the association analysis. A Bayesian linear animal model was used to estimate the contribution of each SNP. A total of 129 tests indicated relevant additive effects between a given SNP and a single fatty acid trait; 38 SNP belonging to 30 genes were relevant for a total of 57 fatty acid traits. Most of the studied fatty acid traits (~81%) were relevantly associated with multiple SNP. Relevantly associated SNP were mainly found in genes related to fat metabolism, linked to or contained in previously identified quantitative trait loci for fat yield or content, or associated with genes previously identified in association analyses with milk fatty acid profiles in other cow breeds. The most representative candidate genes were LEP, PRL, STAT5A, CCL3, ACACA, GHR, ADRB2, LPIN1, STAT1, FABP4, and CSN2. In particular, relevant associations with SNP located on bovine chromosome 19 (BTA19) were found. Two candidate genes on BTA19 (CCL3 and ACACA) were relevantly associated with de novo short- and medium-chain fatty acids, likely explaining the high heritability values found for these fatty acids (with the exception of C6:0). Two additional genes on BTA19 (CCL2 and GH1) showed associations with saturated and branched-chain fatty acids. Our findings provide basic information on genes and SNP affecting the milk fatty acid composition of dairy cows. These results may support the possibility of using genetic selection to modify milk fatty acid profiles to promote beneficial health-related effects.

Primary hepatocytes as an useful bioassay to characterize metabolism and bioactivity of illicit steroids in cattle.

Cattle hepatocytes have already been used in veterinary in vitro toxicology, but their usefulness as a multi-parametric screening bioassay has never been investigated so far. In this study, cattle hepatocytes were incubated with illicit steroids/prohormones (boldenone, BOLD; its precursor boldione, ADD; dehydroepiandrosterone, DHEA; an association of ADD:BOLD), to characterize their transcriptional effects on drug metabolizing enzymes (DMEs) and related nuclear receptors (NRs), on cytochrome P450 3A (CYP3A) apoprotein and catalytic activity as well as to determine ADD and BOLD metabolite profiling. DHEA-exposed cells showed an up-regulation (higher than 2.5-fold changes) of three out of six NRs, CYP2B22 and CYP2C87; likewise, ADD:BOLD increased CYP4A11 mRNA levels. In contrast, a reduction of CYP1A1 and CYP2E1 mRNAs (lower than 2.5(-1)-fold changes) was noticed in ADD- and DHEA-incubated cells. No effect was noticed on CYP3A gene and protein expression, though an inhibition of 6β-, 2β- and 16β-hydroxylation of testosterone (higher than 60% of control cells) was observed in ADD- and BOLD-exposed cells. Finally, 17α-BOLD was the main metabolite extracted from hepatocyte media incubated with ADD and BOLD, but several mono-hydroxylated BOLD and ADD derivatives were detected, too. Collectively, cattle hepatocytes can represent a complementary screening bioassay, useful to characterize growth promoters metabolite profiling and their effects upon DMEs expression, regulation and function.

Factors affecting variations in the detailed fatty acid profile of Mediterranean buffalo milk determined by 2-dimensional gas chromatography

Buffalo milk is the worlds second most widely produced milk, and increasing attention is being paid to its composition, particularly the fatty acid profile. The objectives of the present study were (1) to characterize the fatty acid composition of Mediterranean buffalo milk, and (2) to investigate potential sources of variation in the buffalo milk fatty acid profile. We determined the profile of 69 fatty acid traits in 272 individual samples of Mediterranean buffalo milk using gas chromatography. In total, 51 individual fatty acids were identified: 24 saturated fatty acids, 13 monounsaturated fatty acids, and 14 polyunsaturated fatty acids. The major individual fatty acids in buffalo milk were in the order 16:0, 18:1 cis-9, 14:0, and 18:0. Saturated fatty acids were the predominant fraction in buffalo milk fat (70.49%); monounsaturated and polyunsaturated fatty acids were at 25.95 and 3.54%, respectively. Adopting a classification based on carbon-chain length, we found that medium-chain fatty acids (11-16 carbons) represented the greater part (53.7%) of the fatty acid fraction of buffalo milk, whereas long-chain fatty acids (17-24 carbons) and short-chain fatty acids (4-10 carbons) accounted for 32.73 and 9.72%, respectively. The n-3 and n-6 fatty acids were 0.46 and 1.77%, respectively. The main conjugated linoleic acid, rumenic acid, represented 0.45% of total milk fatty acids. Herd/test date and stage of lactation were confirmed as important sources of variation in the fatty acid profile of buffalo milk. The percentages of short-chain and medium-chain fatty acids in buffalo milk increased in early lactation (+0.6 and +3.5%, respectively), whereas long-chain fatty acids decreased (-4.2%). The only exception to this pattern was butyric acid, which linearly decreased from the beginning of lactation, confirmation that its synthesis is independent of malonyl-CoA. These results seem to suggest that in early lactation the mobilization of energy reserves may have less influence on the fatty acid profile of buffalo milk than that of cow milk, probably due to a shorter and less severe period of negative energy balance. Parity affected the profiles of a few traits and had the most significant effects on branched-chain fatty acids. This work provided a detailed overview of the fatty acid profile in buffalo milk including also those fatty acids present in small concentrations, which may have beneficial effects for human health. Our results contributed also to increase the knowledge about the effects of some of the major factors affecting buffalo production traits and fatty acid concentrations in milk, and consequently its technological and nutritional properties.

Toxicogenomic markers for corticosteroid treatment in beef cattle: integrated analysis of transcriptomic data.

In the present work, an integrated analysis was performed on DNA-microarray data of bovine muscle samples belonging to controls, animals treated with various growth promoters (GPs) and unknown commercial samples. The aim was identify a robust gene expression signature of corticosteroid treatment for the classification of commercial samples, despite the effects of biological variation and other confounding factors. DNA-Microarray data from 5 different batches of bovine skeletal muscle samples were analyzed (146 samples). After preprocessing, expression data from animals treated with corticosteroids and controls from the different batches (89 samples) were used to train a Support Vector Machines (SVMs) classifier. The optimal number of gene probes chosen by our classification framework was 73. The SVMs with linear kernel built on these 73 biomarker genes was predicted to perform on novel samples with a high classification accuracy (Matthews correlation coefficient equal to 0.77) and an average percentage of false positive and false negative equal to 5% and 6%, respectively. Concluding, a relatively small set of genes was able to discriminate between controls and corticosteroid-treated animals, despite different breeds, animal ages, and combination of GPs. The results are extremely promising, suggesting that integrated analysis provides robust transcriptomic signatures for GP abuse.