Conjugated Linoleic Acid (CLA) are a group of positional and geometric isomers of Linoleic Acid characterized by a carbon chain containing 18 carbon atoms and two double bonds, not in the classic position (cis), but conjugated from the carbons atoms 9, 10 or 11. Double bonds have different position in the carbon chain ([7,9], [8,10], [9,11], [10,12], [11,13] and [12,14]) and four different geometric distribution (cis/trans, trans/cis, cis/cis and trans/trans). In total 24 possible isomers are identify, but the two most present and often most identified are: C18:2cis9,trans11 (60-85% of the isomers identify in meat and >90% in milk) and C18:2trans10,cis12 isomers. Observing meat samples other isomers are commonly identified, as example, C18:2trans7,cis9 and C18:2trans11,cis13. In 1987, CLA have been discovered thanks to the research group of Dr. Pariza that studying some carcinogenic components in grilled meat, identify these molecules with anticancer activity, called Conjugated Linoleic Acid. After the discovery, they still receive many attention because of their biological activities and implication on human health. The biological effect are due to the separate actions of the main isomers (C18:2cis9,trans11 and C18:2trans10,cis12) and sometimes by the synergistic action of both. Also, minor isomers have biological effect, as examples, C18:2trans9,trans11 that inhibits platelet aggregation and has anti-proliferative effect and C18:2cis9,cis11 that showed anticancer effect. In animal model, CLA not only reduce initiation, promotion and progression steps of cancer development, but also reduce metastasis. Nevertheless, was show that CLA isomers are important because of they have effect on animal performance and the principal effects are: prevent chemically-induced tumors, protect against the catabolic effects of immune stimulation, improve feed efficiency, reduce excess body weight gain, reduce body fat, increase lean body mass and lower blood lipids. In human diet, the main sources of CLA are representing by ruminants products, in particular milk, dairy product and beef. The higher concentration of CLA in these products is linked to the presence of rumen that through microbial biohydrogenation (Butyrivibrio fibrisolvens) can transform Linoleic Acid to the major isomer C18:2cis9,trans11 (Rumenic Acid). Conjugated Linoleic Acid (CLA) can be analyze with different methods of lipid extraction and derivatization, but always after transformation in methyl ester derivatives of fatty acids (FAMEs) that is carried out in a simple reaction. This simple reaction becomes more complicated because of conjugated fatty acids are involved and the presence of conjugated double bond makes them unsuitable for the most common techniques employed for fatty acids analysis. The presence of double bonds may increase isomerization and epimerization of these bonds, increasing CLA with trans/trans configuration and reducing cis/trans or trans/cis configuration. For this reason is very important find a suitable method for lipid extraction (and the resulting determination of crude fat) and subsequently for the transformation in methyl ester derivatives of fatty acids (FAMEs). For this reason at the first part of my thesis was given a methodological approach in order to understand the differences between methods and which is the most suitable (Chapter 2 and Chapter 7). All the data used, belonging to a trial carried out at “Lucio Toniolo”, the Experimental Farm of the University of Padova in Legnaro (Padova, Italy). Animals used are a crossbreed between Belgian Blue bulls and Brown Swiss dairy cows fed with one of 3 experimental diets. Diets were composed by unifeed and differ depending on the rumen protected CLA supplementation (rpCLA), overall, three supplementation are available 0, 8 and 80 g/d/animal. The trial ended in March 2011 when animals were slaughtered outside the faculty in a slaughterhouse located in Pergine province of Trento (Trentino Alto Adige Region). Samples were collected and in particular three tissues were subjected to analysis for fatty acids profile and CLA content: muscle Longissimus Thoracis, Subcutaneous Fat and Liver. In the main Chapter (Chapter 2) tissues (Longissimus Thoracis, Subcutaneous Fat and Liver) were analyzed for determine fatty acids profile comparing three different methods of extraction (Folch (1957), Accelerated Solvent Extraction (ASE) and Jenkins (2010)) using as chromatography technique two dimensional GC (GC×GC). The purpose of this trial was identify among the different methods of extraction, one method that is able to identify CLA isomers without causing isomerization. Folch (1957) is one of the older and most used methods. It was born for analyze samples rich in phospholipids (as lipids of brain). It works at room temperature using a mixture of solvents composed by chloroform/methanol (2:1, v/v). Accelerated Solvent Extraction (ASE) was used with the purpose to compare a room temperature method with a method that works at high temperature and pressure (120°C and 20MPa) with the same mixture of solvents (chloroform/methanol, 2:1, v/v). This method, which have recently been introduced; reduce the use of solvent and saving time at work, giving results that were similar or better if compared with the conventional Folch (1957) extraction. On the contrary, it is expected to increase the isomerization of double bonds and the isomers with trans/trans configuration. The last method, Jenkins (2010), is a direct method, which reduces the length of the total procedure, saving time at work, reducing the sample amount, reducing the use of solvents, giving analysis less expensive and easier. The main characteristic is that lipid extraction step is avoided and fatty acids are extracted and trans-esterified in the same time. The statistical analysis was performed in four steps: resolution power and assessment of the number of undetected FA, study the main sources of variation, using Levene’s test explored the variances homogeneity for the main sources of variations and relationships between methods. The incidence of undetectable values on the total number of expected observation, which depend on the sensitivity of the method used, for liver, fat and muscle, ranged 0.04 to 0.08, 0.05 to 0.06, and 0.05 to 0.12, respectively, with incidences greater for the Jenkins method compared to the other two for liver and muscle samples but not for subcutaneous fat. In liver the highest incidence of null values with the Jenkins method was mainly observed for short chain FA (C8:0 and C10:0), whereas in the case of muscle the highest incidence of undetectable values was mainly observed for the C24:0, for two C20:1t unknown isomers, and for Ω3 (C20:3n3, C20:4n3, C22:5n6). Many sources of variation result high significant (P<0.001). Diet was significant in particular for the FA that are the main components of the supplementation (C18:0, C18:1cis9, C18:2cis9,trans11 and C18:2trans10,cis12). The results of the Levene’s test evidenced, except for diet, as the variances among levels within tissue, method or method×tissue were not homoscedastic for the large majority of the FA. The second part of the methodological approach is reported in Chapter 7 (Minor Chapter) and considers the effect of the chromatography technique on CLA isomers content. Despite the high resolution power of GC×GC this technique allows to identify all the FA and the CLA isomers recognized by internal standard. However, it is not able to identify the other isomers and for this reason is necessary to find a new method that allows a clear and complete identification of CLA. Silver Ion High Performance Liquid Chromotography (Ag+HPLC) is currently the most effective way to separate and quantitate individual isomers of CLA in beef. This part was performed with the contribution of the Leibniz Institute for Farm Animal Biology (Dummerstorf, Germany) and in particular the Muscle and Biology Growth Unit. Tissue analyze in this trial were Longissimus Thoracis, Subcutaneous Fat and Liver. Lipids were extracted using Folch (1957) and methylated using and acid-base catalysis. Data were analyzed considering as main sources of variation: diet, tissue and repetition. Tissue resulted always significant (P<0.001) with a tissue depending distribution of the isomers and a higher concentration in subcutaneous fat. Diet was significant for the main isomers (C18:2cis9,trans11 and C18:2trans10,cis12) because of constituents rpCLA supplementation.
In the second part of my thesis are considered the effects that can modify FA profile, such as, breed, gender, diet, type of birth, age and tissue. Animals used belonging to four native alpine sheep breeds: Alpagota, Brogna, Foza and Lamon. All these breeds represent an important genetic resources and the purpose is created a program of conservation for increase the number of animals in these populations. For this reason Veneto Agricoltura created the experimental farm “Villiago” (Belluno province, Veneto Region, North Italy). Animals used for this research belongs to two flocks undergoing an in situ conservation program between “Lucio Toniolo” Experimental Farm of the University of Padova and the Experimental Farm of Veneto Agricoltura located in Villiago. In total 115 animals were used and reared in the period between December 2010 and July 2012. Animals are slaughter at different age and they are considered belonging to three different categories: 31 suckling lambs, 36 lambs, 24 heavy lambs and 24 ewes. The different trial were characterized by different diets: pasture (PAS), penned in the open barn and fed with hay, concentrate and supplemented with rumen protected Conjugated Linoleic Acid (rpCLA) product (CLA+) and penned in an open barn and fed with hay and concentrate (CLA-). In trials with “lambs” and “heavy lambs” animals were supplement with 8.0 g/d/animal of rpCLA. In “suckling lambs” and “ewes” animals were supplement with 4.0 g/d/animal of rpCLA and 12 g/d/animal of rpCLA, respectively. From the results, is possible observe that diet is highly significant (P<0.001) and in particular pasture. Fatty acids are statistically influenced by pasture and mainly fatty acids with odd chain and branched fatty acids (iso and anteiso) that tend to be lower in diet at pasture. The reason is that the diet can influence ruminal Ph and microorganisms that consequently change reactions and final products. Pasture is also important because it can influence the amount of long chain fatty acids (LC-PUFA), increasing Ω3 (respectively, PAS=2.70, CLA-=1.46 and CLA+=1.54), reducing Ω6 (respectively, PAS=3.76, CLA-=4.41 and CLA+=4.75) and Ω6/Ω3 ratio. CLA isomers content is higher in pasture than in diet with concentrate supplement with rpCLA (respectively, PAS=0.80, CLA-=0.56 and CLA+=0.71). In the other two trials characterized by CLA+ and CLA- diet was considered an important and significant effect. rpCLA supplementation reported significant effect (P<0.001 and P<0.01) for FA constituents of supplementation, such as C18:0 and C18:2trans10,cis12. In suckling lambs results were different from other trials, in particular for CLA isomers, such as, C18:2cis11,trans13 (P<0.01), C18:2cis11,cis13 (P<0.001) and C18:2cis9,cis11 (P<0.001). The reason is the different distribution is ewe’s milk that was used in suckling lambs diet and could have influence their fatty acids profile. Breed was not a significant effect, such as, gender and age at slaughter. Observing data about orthogonal contrast FA and CLA isomers distribution is tissue specific and there are many differences between lean tissues (muscle) and fat tissues. Liver has particular characteristics, in fact, its content of PUFA and particular Ω3 and CLA is higher than in the others tissues. Differences of this tissue are linked to its specific lipid composition and metabolic process which lead to the production and absorption of fatty acids.
In Chapter 5 and Chapter 6 were evaluate effects that CLA supplementation can have on animal performance because of many research have been conduct in vitro, but other research are needed.
In Chapter 5 was examined the effect of rpCLA supplementation in lactating ewes on their milk composition and Milk Coagulation Properties (MCPs) of sheep’s milk. Animal used in this trial are the same of the trial describe above and in particular of the trial call “suckling lambs” and “ewes”.
This study allowed to know that rpCLA supplementation in sheep can change the composition and cheese-making properties of milk, as example, delaying gelation, slowing curd firming and accelerated syneresis and future studies are necessary to know the effect of CLA on cheese yield/ quality. rpCLA supplementation affect milk composition, reducing protein content, solid non-fat content, casein index and increasing SCS. rpCLA supplementation had negative effects on parameters of coagulation and curd firming.
In Chapter 6 was evaluate the effect of breed and sex on growth rate, slaughter traits and meat quality traits of lambs of Alpagota, Brogna and Foza breeds. Animals used in this trial are the same of the trial describe above and in particular of the trial call “lambs”. These results are part of a bigger study which comprises also growth rate, slaughter traits and meat quality of the trial call “heavy lambs”, “suckling lambs” and “ewes”. Observing the results of this trial is possible obtain lamb carcasses and meat with valuable characteristics that can be exploited through typical products and food preparation in local markets and gastronomy, according to tradition. The valorisation of these productions can be an important tool for the in situ conservation of these breeds. As example, Slow Food organization has recognized “Agnello Alpagoto” (lambs of Alpagota breeds) as a Slow Food Presidium.Dal 1987 anno in cui il Dott. Pariza e il suo gruppo di ricerca scoprirono i Coniugati dell’Acido Linoleico (CLA) molti sono stati gli studi che hanno cercato di definire le principali caratteristiche di queste molecole. Caratterizzati da un alto valore biologico gli furono attribuiti molti effetti benefici sulla salute umana, come l’effetto anticancerogeno, la riduzione del rischio di malattie cardiovascolari e la riduzione del rischio di sviluppo dell’aterosclerosi. In seguito gli fu attribuita anche importanza nel miglioramento delle performance animali, come l’aumento delle capacità di accrescimento, dell’efficienza alimentare e una riduzione della deposizione di grasso con conseguente aumento della massa magra. I Coniugati dell’Acido Linoleico (CLA) sono un gruppo d’isomeri geometrici e posizionali dell’Acido Linoleico caratterizzati da una catena di 18 atomi di carbonio contenente due doppi legami non in posizione classica (cis), ma coniugati dal carbonio 9, 10 o 11. I doppi legami possono presentare diversa disposizione spaziale dando origine a quattro diverse configurazioni: cis/trans, trans/cis, cis/cis e trans/trans. Secondo i carboni ai quali sono legati, possono avere diverse posizioni: ([7,9], [8,10], [9,11], [10,12], [11,13] e [12,14]) con un totale d’isomeri identificati pari a 24. I due più presenti e più identificati sono il C18:2cis9,trans11 (60-85% degli isomeri presenti nella carne e >90% nel latte) e il C18:2trans10,cis12. Altri isomeri molto presenti nella carne sono anche il C18:2trans7,cis9 e il C18:2trans11,cis13. Grazie alla capacità del rumine di produrre acidi grassi e in particolare CLA, si possono trovare soprattutto nei prodotti di origine animale (latte, prodotti lattiero caseari e carne). La presenza del doppio legame rende i CLA delle molecole complesse da identificare, perché può essere facilmente soggetto a fenomeni d’isomerizzazione o epimerizzazione che possono portare a un aumento delle forme di tipo trans/trans con conseguente riduzione delle forme cis/trans o trans/cis. Diversi studi hanno cercato di definire quale sia il metodo più adatto per l’estrazione del grasso (determinazione estratto etereo, EE) e per la successiva trasformazione in composti volatili, ovvero, esteri metilici degli acidi grassi (FAME). Per questo motivo parte della mia tesi è stata improntata su un approccio metodologico allo scopo di capire tra i tanti metodi quale fosse il più idoneo e che differenze i diversi metodi potessero avere. I dati contenuti nei contributi legati all’approccio metodologico sono stati raccolti da vitelloni maschi nati da un incrocio tra vacche da latte di razza Bruna e tori di razza Bianca Blu del Belgio. Questi animali sono stati allevati presso l’azienda sperimentale dell’Università degli Studi di Padova Lucio Toniolo. Durante tutta prova sono stati allevati in azienda e alimentati con una dieta a base di unifeed caratterizzata da tre diverse integrazioni di CLA rumino protetti (rpCLA): 0, 8 e 80 g di CLA al giorno per ogni animale. La prova è terminata con la macellazione avvenuta in un macello esterno all’Università situato a Pergine (Provincia di Trento, Trentino Alto Adige). I tessuti utilizzati per le analisi sono stati prelevati in macello ed in laboratorio di Qualità carne durante lo svolgimento delle analisi di qualità. I tessuti prelevati e studiati sono stati tre: muscolo Longissimus Thoracis, grasso sottocutaneo e fegato. Nel primo contributo (Chapter 2) i tessuti (Longissimus Thoracis, grasso sottocutaneo e fegato) sono stati analizzati allo scopo di determinare il profilo acidico, confrontando tre diversi metodi di estrazione del grasso (Folch (1957), Acellerated Solvent Extraction (ASE) e Jenkins (2010)) e utilizzando come tecnica cromatografica la Gas Cromatografia a due dimensioni (GCxGC). La scelta di eseguire un confronto metodologico è legata al fatto che secondo il metodo d’analisi utilizzato i risultati sono diversi. Di conseguenza, uno degli obiettivi era trovare un metodo che fosse in grado di salvaguardare i CLA senza provocare isomerizzazioni. A questo è dovuta la scelta del metodo Folch (1957), uno dei più antichi e più utilizzati in matrici di diversa natura. Nato per essere usato in campioni con grasso molto ricco di fosfolipidi (come il grasso presente nel cervello), lavora a temperatura ambiente utilizzando una miscela di solventi composta da cloroformio:metanolo (2:1, v/v). Il metodo Acellerated Solvent Extraction (ASE) è stato utilizzato allo scopo di confrontare una metodica che lavora con alte temperature e pressioni (120°C e 20 MPa) con una miscela di solventi identica a quella usata nel metodo Folch (1957) (cloroformio:metanolo (2:1, v/v)). Grazie alle caratteristiche positive il metodo ASE, negli ultimi anni si è molto diffuso. Esso è caratterizzato da una maggiore velocità d’estrazione, un ridotto utilizzo di solventi e una minore laboriosità da parte degli operatori. Si pensa che le condizioni (temperatura e pressione) a cui lo strumento lavora possano provocare isomerizzazioni incrementando gli isomeri con configurazione trans/trans. L’ultimo è un metodo diretto che in seguito ad una fase preparativa (di liofilizzazione del campione fatta con lo scopo di rimuovere l’acqua) permette di ottenere gli esteri metilici degli acidi grassi (FAME) da poter analizzare in GCxGC. Questo metodo richiede un’esterificazione di tipo acido-basico. Il lavoro è stato suddiviso in quattro passaggi in modo da: identificare il potere di risoluzione e l’incidenza di picchi non identificati, lo studio delle fonti di variazione, test di Levene per determinare l’omoscedasticità o eteroscedasticità delle varianze e infine degli studi per valutare la correlazione tra metodi. L’incidenza di valori non identificati è legata al numero di picchi osservati, che dipendono dalla sensibilità del metodo e dal tessuto analizzato: fegato, grasso e muscolo (da 0.04 a 0.08, da 0.05 a 0.06 e da 0.05 a 0.12, rispettivamente). La maggiore incidenza di valori nulli è stata osservata nel fegato e in particolare negli acidi grassi a corta/media catena (C8:0 e C10:0) e nel muscolo negli acidi grassi C24:0, C20:1t1t unknown isomers e nei PUFAn3 (C20:3n3, C20:4n3, C22:5n6) sempre con il metodo Jenkins (2010). Tra tutte le fonti di variazione analizzate lo scopo, era considerare le due principali che sono state molto significative (P<0.001) e sono rappresentate dal tessuto, dal metodo e dalla loro interazione. L’effetto della dieta è stato significativo per gli acidi grassi facenti parte dell’integrazione (C18:0, C18:1cis9 e C18:2trans10,cis12). Dai test di Levene è emerso che le varianze sono eteroscedastiche, tranne la dieta che è risultata, omoscedastica.
Il passo successivo è stato considerare una tecnica cromatografica alternativa al GCxGC che fosse più specifica per lo studio degli isomeri dei CLA (Chapter 7) Nonostante, il GCxGC abbia un maggiore potere di risoluzione non è in grado di identificare tutti gli isomeri dei CLA, ma solo i due principali (C18:2cis9,trans11 e C18:2trans10,cis12). Per questo motivo sono presenti altre tecniche cromatografiche che permettono un’identificazione più precisa come la cromatografia liquida su colonna d’argento (Ag+HPLC). Il lavoro è stato svolto in collaborazione con il Leibniz Insitute for Farm Animal Biology (Dummerstorf, Germany) e in particolare l’unità di Muscle and Biology Growth. L’obiettivo in questo caso era identificare il maggior numero d’isomeri dei CLA presenti nel campione. I tessuti analizzati erano: muscolo (Longissimus Thoracis), grasso sottocutaneo e fegato. Il grasso (estratto etereo, EE) è stato ottenuto con il metodo Folch (1957) cui è seguita poi un’esterificazione acido-basica. Le analisi statistiche sono state eseguite considerando gli effetti di dieta, tessuto e ripetizione sulla distribuzione degli isomeri. La prima cosa che differenzia questa metodica dal GCxGC è il numero di picchi identificati che son
