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 \u3b49-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

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 \u3b49-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

Integration of GWAS, pathway and network analyses reveals novel mechanistic insights into the synthesis of milk proteins in dairy cows

The quantities and proportions of protein fractions have notable effects on the nutritional and technological value of milk. Although much is known about the effects of genetic variants on milk proteins, the complex relationships among the set of genes and pathways regulating the different protein fractions synthesis and secretion into milk in dairy cows are still not completely understood. We conducted genome-wide association studies (GWAS) for milk nitrogen fractions in a cohort of 1,011 Brown Swiss cows, which uncovered 170 significant single nucleotide polymorphism (SNPs), mostly located on BTA6 and BTA11. Gene-set analysis and the network-based Associated Weight Matrix approach revealed that the milk proteins associated genes were involved in several biological functions, particularly ion and cation transmembrane transporter activity and neuronal and hormone signalling, according to the structure and function of casein micelles. Deeper analysis of the transcription factors and their predicted target genes within the network revealed that GFI1B, ZNF407 and NR5A1 might act as master regulators of milk protein synthesis and secretion. The information acquired provides novel insight into the regulatory mechanisms controlling milk protein synthesis and secretion in bovine mammary gland and may be useful in breeding programmes aimed at improving milk nutritional and/or technological properties.info:eu-repo/semantics/publishedVersio

ProYoungStock: un progetto per promuovere il legame naturale tra vacca e vitello

This web article informs ruminant farmers and ruminant enthusiasts about the ProYoungStock project, with a brief insight into the main objectives and activities

Using high density EEG to assess TMS treatment in patients with schizophrenia.

To access publisher's full text version of this article, please click on the hyperlink in Additional Links field or click on the hyperlink at the top of the page marked DownloadWe present preliminary results from the ongoing study entitled "Icelandic AVH-TMS" which aim is to study the effectiveness of repetitive transcranial magnetic stimulation (rTMS) treatment for patients with schizophrenia and with persistent auditory verbal hallucinations (AVH) using symptoms and psychometric scales and high-density EEG system (256 channels). The aim of the present work was to describe cortical topography of the auditory evoked responses like P50 and N100-P300 complex in healthy participants and patients with schizophrenia and to define a robust methodology of signal quantification using dense-array EEG. Preliminary data is shown for three healthy participants and three patients in baseline conditions and for two patients we show the results recorded before and after 10 days rTMS treatment. Our results show differences in sensory gating (P50 suppresion) and a stronger N100-P300 response to rare audio stimulus after the treatment. Moreover we show the value of assessing brain electrical activity from high-density EEG (256 channels) analyzing the results in different regions of interest. However, it is premature and hazardous to assume that rTMS treatment effectiveness in patients with AVH can be assessed using P50 suppression ratio. Keywords: P300; P50; Transcranial magnetic stimulation; high density EEG; schizophrenia

Enzimi farmaco-metabolizzanti epatici in specie di interesse veterinario

In veterinary medicine, the characterisation of the P450 isoenzymes is still incomplete and moreover, the substrates used were selected on the basis of the available knowledge from experiments in human or laboratory species without any kinetic or inhibition study (Fink-Gremmels, 2008). Defining the contribution of a P450 isoform in the metabolism of a specific drug is of great interest not only for veterinary pharmacology and toxicology but also for human health, for the potential presence of toxic residues in food animal products. During the PhD course, projects dealing with the evaluation of cytochrome P450 activities in veterinary species have been developed. The aim was to increase knowledge about drug metabolizing enzymes in veterinary species and about the modulation of their activity. The modulation may be due to factors as breed but also exposure to environmental contaminants or somministration of illicit drugs used to enhance growth and consequently increase profit (Johnson, 2007). Data collected from these in vitro studies can be actually used to evaluate the effects of drug-drug interactions and for the plan of experimental projects aiming to identify in vivo markers of exposure or treatment. I) Determination of CYP1A and CYP2C activities in bovine liver. In this study, accuracy, precision, LOD and LOQ of High Performance Liquid Chromatography (HPLC) methods previously developed in the lab for the evaluation of ethoxyresorufin-O-deethylase (EROD) e tolbutamide methyl-hydroxylase (TMOH) activities, have been determined. The aim was to calculate the enzyme kinetic parameters: respectively 0.23 ± 0.051 and 1010 ± 155.7 μM for Km and 0.488 ± 0.035 and 0.089 ± 0.006 nmol/min/mg protein for Vmax. The methods were proved to be so sensitive to determine also very low levels of enzyme activity as those of veal calves fed with milk replacers and low iron content diet. Finally, to verify the presence of intra-specific differences in enzyme activity, EROD and TMOH were determined with liver microsomes from Charolais (CH), Blonde D’Aquitaine (BD) and Piedmontese (PM). The results of EROD evidenced higher activity in CH vs PM (p<0.05). So, for the first time, the presence of breed differences in this enzyme activity was established, confirming also what reported by other authors for CYP3A-dependent activities (Dacasto et al., 2005). These differences may influence bioavailability and clinical efficacy of xenobiotics (Sallovitz et al., 2002) and may be of particular interest in veterinary species for the potential presence of toxic residues in food animal products. II) Testosterone hydroxylation in bovine liver: enzyme kinetic and inhibition study. This study evaluated, by HPLC analysis, the kinetic of testosterone (TST) hydroxylase activities in vitro, with liver microsomes from cattle. Kinetic parameters for 6b–, 16b– and 2b –TST hydroxylase activities (OHT) were 93.4±13.8, 36.4±6.1 and 110.8±15.2 ±M respectively for Km and 0.558±0.03, 0.280±0.013 and 0.338±0.017 nmol/min/mg protein for Vmax.. Moreover, chemical inhibition studies with a CYP3A inhibitor (ketoconazole) and CYP2B inhibitors (orphenadrine and 9-ethynylphenanthrene) were performed to establish the involvement of CYP3A and potentially of CYP2B in these enzyme reactions. To further confirm the results obtained, an anti-peptide antibody against bovine CYP3A4 was also used. The antibody specificity with bovine liver microsomes was evaluated and then immunoinhibition studies were conducted, evidencing > 90% inhibition for 16b– and 2b –OHT activities and > 80% for 6b–OHT. These results seemed to suggest the predominant involvement of CYP3A in the production of the three main TST-hydroxylated metabolites in bovine liver while CYP2B seemed to be not involved. III) Effects of illicit treatments on CYP3A in bovine liver and in primary cultures of bovine hepatocytes. These experiments intended to prove the applicability of the HPLC method and TST substrate for evaluating CYP3A activity, aiming to actually verify illicit treatments effect and collect more data respect to previous studies in veal calf and beef cattle. In the first experiment, 6b-, 16b- and 2b-OHT were determined in bovine treated with dexamethasone (D) and dexamethasone+estradiol (DE). The results obtained evidenced a significant induction for 6b- and 16b-OHT in D and DE vs control (K, p<0.05) and for 2b–OHT in D and DE vs K (p<0.05 and p<0.01, respectively), confirming what reported for human (McCune et al., 2000). These results were confirmed by Immunoblotting which evidenced increasing in CYP3A protein expression in D and D+E vs K (p<0.01 and p<0.05, respectively). In the second experiment, 6b-, 16b- and 2b-OHT were determined in bovine hepatocytes after 24 h incubation with boldenone (B), androsta-1,4-diene-3,17-dione (A) and B + A. The results showed an inhibitory trend in hepatocytes incubated with growth promoters vs controls (K). In particular, 6b and 16b OHT were inhibited in A and B vs K while 2b-OHT in A vs K (p<0.05). At the protein level instead, no significant difference was found in CYP3A expression (Immunoblotting). The inhibitory effect observed may be due to the high doses used; inhibitory/toxic effects of high doses of inducing agents were evidenced for instance by Kostrubsky et al. (1999) who used human hepatocytes to study induction of cytochrome P450, stressing the importance of large dose-response studies as well as the need to assess toxicity in these investigations. The results obtained in these experiments confirmed the modulation of expression and regulation of cytochrome P450 enzymes by estrogens, corticosteroids and anabolic steroids. There is the need however, to explain deeply for bovine the mechanism through that these compounds act at pre- and post-transcriptional level. IV) Determination of CYP1A activity in the liver of Zosterisessor ophiocephalus as biomarker of pollution in the Venice Lagoon. Recently, it was proved that biomarkers (BM) are very useful to evidence early biological changes in environmental pollution. One of the most common BM used in fishes is the induction of the P450 system and in particolar ethoxyresorufin O-deethylase activity (EROD) is a marker of fish exposure to potentially toxic compounds, i.e. poli-chlorinated biphenils and polycyclic aromatic hydrocarbons (Burgeot et al., 1994). The aim of this study was to evaluate by HPLC EROD activity in Zosterisessor ophiocephalus, a benthic fish that may represent an useful bioindicator of environmental stress in the Venice Lagoon. Fishes were collected in spring and autumn in three different areas of the Venice Lagoon: Porto Marghera (PM), Valle di Brenta (VB) and Porto Canale (PC); the samples were then classified in females, males and sneakers (small males, < 1 years-old). Factorial analysis of EROD activity results evidenced that site, fish category and season were significant (p12), intermediate in the area of Valle di Brenta (EF=5-12) and low around Porto Canale (EF=1-5; Guerzoni et al., 2004). However, site effect depended on the fish category and in particular differences were found between males and females, due more probably to the analyzed samples than to real differences in induction. From these results, EROD activity may be considered an useful biomarker even if, considering the multifactorial response of individuals, it is important to use several significant biomarkers to accurately evaluate the effects of environmental pollution in the aquatic ecosystem. V) Luminescence high-throughput methods for determining CYP3A and CYP2C activities in horse liver. In the last part of the thesis, results of kinetic and inhibition studies to evaluate CYP2C and CYP3A activities in horse liver through Glo assays were reported. The aim was to evaluate the performances of this technique for drug metabolism or drug-drug interaction studies. Enzyme kinetic parameters for CYP3A and CYP2C activities were respectively 14.5 ± 7.3 and 175.9 ± 16.2 μM for Km and 0.022 ± 0.0004 and 0.025 ± 0.0009 nmol/min/mg protein for Vmax. Ketoconazole (CYP3A inhibitor) evidenced IC50 values of 0.035 μM, confirming good specificity of the luciferin (L)-derivative substrate used for CYP3A (L-IPA). Sulfaphenazole (CYP2C inhibitor) evidenced IC50 values of 47.2 μM, suggesting the need to conduct further studies to verify substrate specificity (L-H), for instance using other selective inhibitors for the CYP2C subfamily as ibuprofen or diclofenac. Luminescence methods allowed to analyse many samples in short time, to monitor more than one isoform simultaneously in the same plate and showed good sensitivity confirmed by LOD values: 0.2 and 0.11 nM for L-H and L-IPA, respectively. So, these results may represent a good starting point to apply this technique in drug metabolism and drug-drug interaction studies in veterinary speciesNel corso dei tre anni di dottorato sono stati sviluppati progetti relativi alla valutazione dell’attività epatica di isoforme di citocromo P450 in specie di interesse veterinario. In medicina veterinaria, la caratterizzazione del sistema P450 è ancora incompleta; inoltre, i substrati utilizzati sono stati selezionati sulla base delle conoscenze disponibili a partire da esperimenti condotti nell’uomo o nelle specie di laboratorio senza studi di cinetica enzimatica o di inibizione (Fink-Gremmels, 2008). Definire il contributo di un’isoforma di citocromo P450 nel metabolismo di un farmaco specifico è importante non solo per la farmacologia e la tossicologia veterinarie ma anche per la salute dell’uomo, per la possibile presenza di residui nei prodotti di origine animale. L’obiettivo della presente tesi di dottorato è stato dunque quello di aumentare le conoscenze sugli enzimi che metabolizzano i farmaci in specie di interesse veterinario e sulla modulazione della loro attività. Tale modulazione può essere dovuta a fattori come la razza ma anche all’esposizione ad inquinanti ambientali oppure alla somministrazione di sostanze illecite utilizzate allo scopo di determinare incremento ponderale, riduzione degli indici di conversione alimentare e quindi aumento complessivo del reddito (Johnson, 2007). Le informazioni ottenute con tali studi in vitro dunque possono essere utili per valutare l’effetto di interazioni tra farmaci e per il disegno sperimentale di progetti volti all’identificazione di marker in vivo di esposizione o trattamento. I) Valutazione dell’attività di CYP1A e CYP2C nel fegato di bovino. Nel presente lavoro, sono stati verificati accuratezza, precisione, LOD e LOQ di metodi in High Performance Liquid Chromatography (HPLC) precedentemente messi a punto nel laboratorio per la valutazione dell’attività di etossiresorufina-O-deetilasi (EROD) e tolbutamide metil-idrossilasi (TMOH) in microsomi epatici di bovino allo scopo di determinare i parametri di cinetica enzimatica: rispettivamente 0,23 ± 0,051 e 1010 ± 155,7 μM per la Km e 0,488 ± 0,035 e 0,089 ± 0,006 nmol/min/mg proteina per laVmax. La sensibilità dei metodi messi a punto ha permesso di valutare anche livelli di attività enzimatica molto bassi come quelli dei vitelli a carne bianca che vengono alimentati con una dieta a base di sostitutivi del latte e basso contenuto di ferro. Infine, per verificare l’esistenza di differenze intra-specifiche nell’attività enzimatica, le attività di EROD e TMOH sono state determinate utilizzando microsomi epatici di bovini di razza Charolais (CH), Blonde D’Aquitaine (BD) e Piemontese (PM). I risultati ottenuti per l’attività di EROD hanno evidenziato una maggiore attività nei bovini di razza CH vs PM (p<0,05). E’ stata dunque dimostrata, per la prima volta, l’esistenza di differenze intra-specifiche dovute alla razza relative a questa attività enzimatica, confermando quanto evidenziato da altri autori in relazione ad attività CYP3A-dipendenti (Dacasto et al., 2003). Tali differenze potrebbero influenzare la biodisponibilità e l’efficacia clinica di xenobiotici (Sallovitz et al., 2002) e potrebbero essere di particolare interesse nelle specie di interesse veterinario per l’ipotetica presenza di residui potenzialmente tossici negli alimenti di origine animale. Per quanto riguarda invece l’attività di TMOH non sono state evidenziate differenze statisticamente significative. II) Valutazione dell’attività di idrossilazione del testosterone nel fegato di bovino: studio di cinetica enzimatica e di inibizione. In questo studio è stata valutata, tramite analisi in HPLC, la cinetica delle reazioni di idrossilazione del testosterone (TST) in vitro, con microsomi epatici di bovino. I parametri di cinetica enzimatica per le attività di 6b–, 16b– e 2b –TST idrossilasi (OHT) sono risultati 93,4±13,8, 36,4±6,1 e 110,8±15,2 ±M rispettivamente, per la Km e 0,558±0,03, 0,280±0,013 e 0,338±0,017 nmol/min/mg proteina per la Vmax.. Inoltre sono stati effettuati degli studi di inibizione chimica con inibitori di CYP3A (chetoconazolo) e di CYP2B (orfenadrina e 9-etinilfenantrene) per definire il coinvolgimento di CYP3A e potenzialmente di CYP2B in queste reazioni enzimatiche. I risultati ottenuti sono stati ulteriormente confermati con l’impiego di un anticorpo anti-peptide contro CYP3A4 di bovino. Dopo aver valutato la sua specificità con microsomi epatici di bovino, sono stati effettuati degli studi di immunoinibizione, che hanno evidenziato un’inibizione > 90% per le attività di 16b– e 2b –OHT e > 80% per 6b–OHT. Nel complesso, questi risultati sembrerebbero suggerire il coinvolgimento predominante di CYP3A nella produzione dei tre principali metaboliti del TST nel fegato di bovino mentre CYP2B non sembrerebbe essere coinvolto. III) Effetti di trattamenti illeciti su CYP3A nel fegato del bovino da carne e in colture primarie di epatociti di bovino. L’obiettivo principale di entrambe le prove sperimentali descritte è stato quello di dimostrare l’applicabilità del metodo HPLC e del substrato TST per la valutazione dell’attività enzimatica CYP3A-dipendente, allo scopo di verificare l’effetto del trattamento e di acquisire ulteriori evidenze sperimentali rispetto ai risultati di studi precedentemente coinvolti dal nostro gruppo di ricerca sul vitello a carne bianca e sul vitellone. In un primo esperimento, sono state determinate le attività di 6b-, 16b- e 2b-OHT in microsomi epatici di bovini trattati con desametazone (D) e desametazone + estradiolo (DE). I risultati ottenuti hanno evidenziato un’induzione statisticamente significativa nelle attività di 6b- e 16b-OHT in D e DE vs controlli (K, p<0,05) e nell’attività di 2b–OHT in D e DE vs K (p<0,05 e p<0,01, rispettivamente), confermando quanto riportato in letteratura per l’uomo (McCune et al., 2000). Tali risultati sono stati convalidati ulteriormente dall’analisi in Immunoblotting che ha evidenziato un aumento dell’espressione di CYP3A in D e D+E vs K (p<0,01 e p<0,05, rispettivamente). In un secondo esperimento, sono state determinate le attività di 6b-, 16b- e 2b-OHT in epatociti di bovino dopo 24 h di incubazione con boldenone (B), androsta-1,4-diene-3,17-dione (A) e B + A. I risultati ottenuti hanno evidenziato un trend inibitorio negli epatociti incubati con promotori di crescita rispetto a K. In particolare, le attività di 6b e 16b OHT sono state inibite in A e B vs K mentre l’attività di 2b-OHT in A vs K (p<0,05). A livello di proteina invece, non è stata riscontrata alcuna differenza statisticamente significativa nell’espressione di CYP3A (Immunoblotting). L’effetto inibitorio osservato potrebbe essere dovuto alle dosi utilizzate; un caso di effetto inibitorio/tossico di alte dosi di sostanze inducenti è riportato da Kostrubsky et al. (1999) che hanno utilizzato epatociti di uomo per studiare l’induzione del sistema citocromo P450 evidenziando la necessità di effettuare degli studi dose-risposta in un ampio range di concentrazioni delle sostanze utilizzate e di valutarne la tossicità. Nel complesso, i risultati ottenuti in questi due esperimenti hanno confermato la modulazione da parte di estrogeni, corticosteroidi ed anabolizzanti dell’espressione e la regolazione degli enzimi citocromo P450-dipendenti. Chiaramente, rimane comunque la necessità di spiegare più approfonditamente per quanto riguarda il bovino i meccanismi tramite i quali queste sostanze agiscono sia a livello pre-trascrizionale che post-trascrizionale. IV) Valutazione dell’attività di CYP1A nel fegato di Zosterisessor ophiocephalus come biomarcatore di inquinamento nella laguna di Venezia. Recentemente, è stato dimostrato che i biomarcatori (BM) sono molto utili per rilevare dei cambiamenti biologici precoci nell’inquinamento ambientale. Uno dei BM più comunemente usati nei pesci è l’induzione dell’attività di etossiresorufina-O-deetilasi (EROD) che è un indicatore dell’esposizione dell’organismo a composti potenzialmente tossici, come ad esempio i policloro-bifenili e gli idrocarburi aromatici policiclici (Burgeot et al., 1994). Di conseguenza, lo scopo del presente lavoro è stato quello di valutare l’attività di EROD tramite HPLC in Zosterisessor ophiocephalus, una specie bentonica che date le sue caratteristiche, può rappresentare un utile indicatore biologico dei livelli di stress ambientale presente nella Laguna di Venezia. I pesci sono stati raccolti in primavera ed in autunno in tre aree della Laguna di Venezia: Porto Marghera (PM), Valle di Brenta (VB) e Porto Canale (PC); i campioni sono stati poi suddivisi in femmine, maschi e sneakers (maschi giovani). L’analisi fattoriale ha evidenziato la significatività dei fattori sito, categoria e stagione (p12), intermedia nei pressi di Valle di Brenta (EF=5-12) e ridotta nel territorio contiguo al sito di Porto Canale (EF=1-5; Guerzoni et al., 2004). L’effetto del sito dipendeva comunque dalla categoria di animali considerata ed in particolare sono state evidenziate delle differenze tra maschi e femmine, dovute probabilmente comunque al campione analizzato più che a differenze reali in termini di induzione. Dai risultati ottenuti, l’attività di EROD è da considerarsi un buon biomarcatore anche se, considerando la natura multifattoriale della risposta dell’individuo, è necessario l’utilizzo congiunto di più biomarcatori significativi per effettuare una valutazione accurata degli effetti dell’inquinamento nell’ecosistema acquatico. V) Messa a punto di metodi high-throughput in luminescenza per la valutazione dell’attività di CYP3A e di CYP2C nel fegato di cavallo. Nell’ultimo capitolo della tesi, sono riportati i risultati relativi agli studi di cinetica enzimatica e di inibizione chimica volti alla valutazione delle attività di CYP2C e CYP3A nel fegato di cavallo mediante l’utilizzo di saggi Glo. Lo scopo è stato quello di valutare le performance di questa tecnologia allo scopo di applicarla in studi di metabolismo e di interazioni tra farmaci. I parametri di cinetica enzimatica per le attività di CYP3A e di CYP2C sono risultati rispettivamente 14,5 ± 7,3 e 175,9 ± 16,24 μM per la Km e 0,022 ± 0,004 e 0,025 ± 0,0009 nmol/min/mg proteina per la Vmax. Chetoconazolo (inibitore di CYP3A) ha evidenziato un valore di IC50 pari a 0,035 μM, confermando quindi una buona specificità del substrato luciferina (L)-derivato utilizzato per CYP3A (L-IPA). Sulfafenazolo (inibitore di CYP2C) ha evidenziato un valore di IC50 pari a 47,2 μM, suggerendo dunque la necessità di effettuare ulteriori studi volti a verificare la specificità di substrato (L-H), ad esempio utilizzando altri inibitori selettivi per tale sottofamiglia come l’ibuprofene o il diclofenac. I metodi in luminescenza messi a punto permettono di analizzare un numero elevato di campioni in tempi brevi, di monitorare più di un’isoforma contemporaneamente nella stessa piastra e sono caratterizzati da una buona sensibilità, confermata dai valori di LOD ottenuti: 0,2 e 0,11 nM per L-H e L-IPA, rispettivamente. I risultati ottenuti dunque possono costituire un ottimo punto di partenza per poter applicare la tecnologia di tali saggi in studi relativi alle specie di interesse veterinario con vantaggi di costi e tempi di realizzazione delle analis

Phenotypic and genetic variation of ultraviolet-visible-infrared spectral wavelengths of bovine meat

Spectroscopic predictions can be used for the genetic improvement of meat quality traits in cattle. No information is however available on the genetics of meat absorbance spectra. This research investigated the phenotypic variation and the heritability of meat absorbance spectra at individual wavelengths in the ultraviolet-visible and near-infrared region (UV-Vis-NIR) obtained with portable spectrometers. Five spectra per instrument were taken on the ribeye surface of 1185 Piemontese young bulls from 93 farms (13,182 Herd-Book pedigree relatives). Linear animal model analyses of 1481 single-wavelengths from UV-Vis-NIRS and 125 from Micro-NIRS were carried out separately. In the overlapping regions, the proportions of phenotypic variance explained by batch/date of slaughter (14 +/- 6% and 17 +/- 7%,), rearing farm (6 +/- 2% and 5 +/- 3%), and the residual variances (72 +/- 10% and 72 +/- 5%) were similar for the UV-Vis-NIRS and Micro-NIRS, but additive genetics (7 +/- 2% and 4 +/- 2%) and heritability (8.3 +/- 2.3% vs 5.1 +/- 0.6%) were greater with the Micro-NIRS. Heritability was much greater for the visible fraction (25.2 +/- 11.4%), especially the violet, blue and green colors, than for the NIR fraction (5.0 +/- 8.0%). These results allow a better understanding of the possibility of using the absorbance of visible and infrared wavelengths correlated with meat quality traits for the genetic improvement in beef cattle

379 ASAS-EAAP Talk: Precision Phenotyping using Infrared Spectroscopy to Improve the Quality of Animal Products

There is an ever-growing interest in research oriented towards the improvement of quality of animal products. In this context, one major operational bottleneck is the possibility to collect quality indicators over the meat and dairy chains and for selective breeding purposes. The use of near-infrared (NIR) and the Fourier-transformed infrared (FTIR) spectroscopy techniques have been proven to be powerful precision phenotyping tools for high-throughput meat and milk quality assessment. Such technologies allow scoring large number of animals and/or derived-products for novel (predicted) phenotypes and indicator traits to set-up potential new payment systems and boost the genetic improvement. One important step in the use of NIR and FTIR tools is the definition of the \u201cgold standard\u201d as the infrared-based predictions could act only as indicators traits. Indeed, the definition of a robust calibration set, the assessment of repeatability and reproducibility of the reference (i.e., gold standard) as well as the detection of random and systematic errors are crucial steps. Once the reference phenotype has been defined, different statistical methodologies could be applied to infrared spectra data. For instance, the partial least squares regression (PLS) is a multivariate regression method commonly used to build up prediction models using NIR and FTIR spectra data. However, the implementation of advanced statistical approaches, such as Bayesian approaches and machine learning methods, might allow us to achieve more robust and accurate predictions. In this talk, we will describe and discuss some of the challenges and potentials of NIR and FTIR tools for large-scale precision phenotyping. Some examples include the use of NIR and Visible-NIR (Vis-NIR) for assessing meat quality parameters (also using portable instruments able to collect spectra directly from the muscle surface at the slaughterhouse) and the use of FTIR for predicting several traits related to fine milk composition and technological traits in dairy cattle