Meat Qualitative Traits of Wild Boar (Sus Scrofa) and Fallow Deer (Dama Dama) Hunted in Central Italy

Dipartimento di Scienze e Tecnologie per l’Agricoltura, le Foreste,

la Natura e l’Energia (DAFNE)

Doctorate (XXVII CYCLE) SSD AGR19 “Scienze e tecnologie per la gestione forestale e ambientale”

Meat qualitative traits of wild boar (Sus scrofa) and fallow deer (Dama dama) hunted in Central Italy

Tutor: Candidate: Dott.ssa Sebastiana Failla

Doctorare coordinator: Prof. Bartolomeo Schirone

CONTENTS

1. ABSTRACT 5

2. RIASSUNTO 7

3. GENERAL INTRODUCTION 11

3.1. The increase of wild animals populations in Italy 13

3.2. Problems consequent to the increase of wild animal populations 15

3.3. Wild animals harvesting and meat availability in Italy 15

3.4. The quality of meat from hunting harvested wild animals 21

1. FIRST STUDY - Some traits of fallow deer and wild boar meat as affected by hunting withdrawal - 46

2. SECOND STUDY - Effects of the hunting method on meat quality from fallow deer and wild boar and preliminary studies for predicting lipid oxidation using visible reflectance spectra. 51

3. THIRD STUDY - Hunting area affects chemical and physical characteristics and fatty acid composition of wild boar (Sus scrofa) meat 58

4. FOURTH STUDY - Organoleptic and nutritional characteristics of wild boar meat as affected by cooking treatment 66

5. GENERAL CONCLUSIONS 88

6. AKNOWLEDGEMENTS 91

1. ABSTRACT

Wild boar (Sus scrofa), roe deer (Capreolus capreolus) and red deer (Cervus elaphus) populations have increased in the last few decades throughout Europe and this trend will probably continue in the coming decade. The increased abundance of ungulates was followed by increased hunting bags and consequent availability of meat for both home consumption and market. The quality of wild ungulate meat varies considerably and depends on many factors including diet, gender, age, fatness of the animal, hunting season, hunting system and conservation processes. Meat from hunted wild ungulates undergoes several steps from the field to the consumer, implying a possible decline in meat quality and uncertainty in the final quality. Furthermore its physicochemical properties, undergoes significant changes during cooking, that affects the final quality of meat products, especially water loss and fatty acid content, and modify the nutritional value of cooked products if compared to raw samples. During the 2011 to 2015 term we performed four studies, three of which published. Objectives of these studies were: to evaluate the influence of hunting technique on main meat quality traits in wild boar (Sus scrofa) and fallow deer (Dama dama); to test the feasibility of visible reflectance spectra to predict the lipid oxidation of meat during the conservation; to investigate how different geographical hunting areas and cooking processing influence chemical composition, quality traits, fatty acid composition and lipid quality indexes of wild boar meat.

First study: The goal of this paper was the evaluation of influence of hunting technique on main meat quality traits in wild ungulates. Twenty-five wild boars and fourteen fallow deer were investigated. They were hunted in two different ways: dog-drive hunting and still hunting, that implies a stressful escape or not. Still hunted animals provided meat with higher water loss: + 1.7 % and + 2.6 % thawing and cooking loss, respectively. Furthermore, meat from dog-drive hunted animals was paler and yellower than the other (L* 38.62 vs 31.74, P<0.05; b 12.65 vs 9.65, P<0.05). Outcomes confirmed stress as source of abnormalities in meat quality. These anomalies might result discordant as a function of the species taken into account.

Second study: This study examined the influence of hunting methods on the colour and lipid oxidation state of meat from wild boar (Sus scrofa) and fallow deer (Dama dama). In addition, the feasibility of using visible reflectance spectra (360 to 740 nm) to predict the lipid oxidation of meat was evaluated. A total of 25 wild boars and 14 fallow deer were hunted with two different methods, dog-drive hunting (DH) and harvest culling (HC), that implies different animal stresses before shooting. Lipid oxidation increased in the meat from both species, which have been frozen for 3 months. The increase of malondialdehyde, substance reactive to thiobarbituric acid (TBARS), was higher in the DH group than the HC group (P<0.05). Our results confirmed the importance of hunting stress on meat quality and lipid oxidation. The multivariate regression analysis showed a high correlation (R2=0.968) between the predicted and measured TBARS values, suggesting that visible spectroscopy should prove useful for predicting meat oxidation.

Third study: The aim of the third study was to investigate the effect of different geographical hunting areas on chemical composition, fatty acid composition and lipid quality indexes of wild boar meat. The study was conducted on 48 boars hunted in three different hunting areas. The geographical hunting area influenced the cooking loss percentage (P<0.05), dry matter and protein content (P<0.05). The main fatty acids in longissimus thoracis muscle of wild boar were oleic (18:1 cis-9), linoleic (18:2 n-6), palmitic (16:0) and stearic (18:0) acids. Palmitic and stearic fatty acids were comprised 20.46% and 14.7% of the total fatty acids, respectively, in all experimental groups. The polyunsaturated linolenic (C18:3 n-3; P<0.001) content as well as the long-chain n-3 polyunsaturated fatty acid content were affected by the hunting area. The geographical hunting area did not affect the pH value at 72 h, the thawing loss or the lipid quality indexes, such as the atherogenic and thrombogenic indexes.

Fourth study: The aim of this study was to evaluate the influence of grill treatment on physical quality, colour, fatty acids composition an lipid oxidation of longissimus thoracis from ten wild boars, hunted in some areas of the Province of Rome. Cooking induced a significant loss of moisture and, consequently, the cooked meat showed significantly higher protein, ash and fat contents than raw samples. The protein and myoglobin degradation, consequent to heat treatment, changed the colour of meat, showing higher lightness (58.59 vs 38.94, P<0.001) and yellowness value (14.71 vs 11.67, P<0.001) and lower redness value (3.90 vs 6.94, P<0.01) than raw samples. Cooked meat showed a lower content of saturated (30.47 vs 32.13 %, P<0.01) and a higher level of polyunsaturated fatty acids if compared to raw meat (27.02 vs 23.03 %, P<0.05). This is probably due to the prominent presence of saturated fatty in neutral lipids, that are more prone to migration during cooking. From the nutritional point of view, the cooking treatment improves wild boar meat fatty acid composition, but increases the amount of lipid oxidation.

Key words: wild boar, fallow deer, Sus scrofa, Dama dama, meat, fatty acid, lipid oxidation, meat physical quality

2. RIASSUNTO

Caratteristiche qualitative della carne di cinghiale (Sus scrofa) e daino (Dama dama) abbattuti in Italia Centrale Le popolazioni di cinghiale (Sus scrofa), capriolo (Capreolus capreolus) e cervo (Cervus elaphus) sono aumentate negli ultimi decenni in tutta Europa e questa tendenza probabilmente continuerà nel prossimo decennio. Pertanto, la caccia agli ungulati aumenterà la disponibilità di carne di selvaggina sia per il consumo domestico, che per la commercializzazione. La qualità della carne di ungulati selvatici varia notevolmente e dipende da molti fattori tra cui la dieta, il sesso, l'età, lo stato di ingrassamento dell'animale, la stagione di caccia, il sistema di caccia e i processi di conservazione. La carne di ungulati selvatici abbattuti attraverso la caccia subisce diversi passaggi dal campo al consumatore, ciò implica un possibile declino della sua qualità e l'incertezza del risultato finale. Inoltre, le sue proprietà fisico-chimiche subiscono variazioni significative durante la cottura che ne possono pregiudicare la qualità, in particolare dovute alla perdita di acqua e alla variazione del contenuto di acidi grassi, modificando il valore nutrizionale del prodotto cotto rispetto alla carne cruda. Durante il periodo 2011-2015 sono stati condotti quattro studi i cui risultati sono riportati in quattro pubblicazioni che costituiscono la presente tesi. Obiettivi di questi studi sono stati: valutare l'influenza della tecnica di caccia sulle principali caratteristiche qualitative della carne di cinghiale (Sus scrofa) e daino (Dama dama), verificare l’utilizzo degli spettri di riflettenza nel visibile per prevedere l'ossidazione dei lipidi della carne durante la conservazione e infine studiare come le diverse aree geografiche di caccia e la cottura influenzino la composizione chimica, acidica e gli indici di qualità dei lipidi della carne di cinghiale.

Primo studio: Obiettivo di questo lavoro è stato la valutazione dell’effetto della tecnica di caccia sulle principali caratteristiche qualitative della carne di ungulati selvatici. Per la prova sono stati utilizzati venticinque cinghiali e quattordici daini. Essi sono stati cacciati in modo diverso: la caccia in braccata con il cane e la caccia da appostamento fisso, che implicano una fuga stressante dei soggetti. Gli animali abbattuti con la braccata hanno mostrato carne con perdite di acqua più alta: + 1,7% e + 2,6% rispettivamente per la perdita di scongelamento e cottura. In ogni caso, gli animali cacciati in braccata hanno mostrato una carne più pallida e più gialla rispetto agli altri (L* 38,62 vs 31,74, P<0,05; b* 12,65 vs 9,65, P<0,05). I risultati confermano lo stress come fonte di anomalie della qualità della carne. Queste anomalie potrebbero risultare discordanti in funzione della specie presa in considerazione.

Secondo studio: Questo studio ha esaminato l'influenza dei metodi di caccia sul colore e sullo stato di ossidazione dei lipidi della carne di cinghiale (Sus scrofa) e daino (Dama dama). Inoltre, si è valutata la possibilità di utilizzare gli spettri di riflettanza nel visibile (360-740 nm) per prevedere l'ossidazione dei lipidi della carne. Un totale di 25 cinghiali e 14 daini sono stati cacciati con due metodi diversi, braccata con cani (DH) e caccia da appostamento fisso (HC), che implicano diverse sollecitazioni degli animali prima di scattare. L’ossidazione lipidica è aumentata nella carne congelata per 3 mesi per entrambe le specie. L'aumento dei valori di malondialdeide, sostanza reattiva all’acido tiobarbiturico (TBARS), è stato più accentuato nel gruppo DH rispetto al gruppo HC (P <0,05). I nostri risultati hanno confermato l'importanza della caccia come fonte di stress, le conseguenze sulla qualità della carne e sulla ossidazione lipidica. L'analisi della regressione multivariata ha mostrato una correlazione elevata (R2 = 0,968) tra le sostanze stimate con la riflettanza ed i valori di TBARS, il che suggerisce che la spettrometria nel visibile potrebbe rivelarsi utile per misurare l’ossidazione della carne.

Terzo studio: Lo scopo del terzo lavoro era quello di studiare l'effetto delle diverse aree geografiche di caccia su composizione chimica, composizione in acidi grassi e indici di qualità dei lipidi della carne di cinghiale. Lo studio è stato condotto su 48 cinghiali abbattuti in tre diverse zone di caccia. La zona di caccia ha influenzato la percentuale di perdita di cottura (P <0,05), di sostanza secca e di proteine (P <0,05). I principali acidi grassi del muscolo longissimus thoracis di cinghiale sono stati: oleico (18:1 cis-9), linoleico (18:2 n-6), palmitico (16:0) e stearico (18:0). Gli acidi grassi palmitico e stearico hanno costituito una percentuale che va da 20,46 a 14,7% sugli acidi grassi totali, rispettivamente in tutti i gruppi studiati. L'α-linolenico (C18:3 n-3; P < 0,001), un acido grasso polinsaturo a lunga catena della serie n-3, è stato influenzato dalla zona di caccia. La zona di caccia non ha influenzato il valore di pH a 72 h, la perdita da scongelamento e gli indici di qualità dei lipidi compreso quello aterogenico e trombogenico.

Quarto studio: Lo scopo di questo studio era di valutare l'influenza del trattamento di cottura alla griglia su qualità fisica, colore, composizione in acidi grassi e ossidazione dei lipidi del longissimus thoracis di dieci cinghiali, abbattuti in alcune zone della provincia di Roma. La cottura ha causato una significativa perdita di liquidi; di conseguenza, la carne cotta ha mostrato una maggiore percentuale di proteine, ceneri e grassi rispetto a quella cruda. La degradazione delle proteine miofibrillari e della mioglobina dovuta al calore ha modificato il colore della carne mostrando un valore maggiore di luminosità (58,59 vs 38,94; P<0,001), un più alto indice del giallo (14,71 vs 11,67; P<0,001) e un minor valore dell’indice del rosso (3,90 vs 6,94; P<0,01) rispetto alla carne cruda. La carne cotta ha presentato un minor contenuto di acidi grassi saturi (30,47 vs 32,13%; P<0,01) e un livello più elevato di acidi grassi polinsaturi rispetto alla carne cruda (27,02 vs 23,03%; P<0,05). Tale osservazione può essere spiegata dal fatto che i grassi saturi sono in gran parte rappresentati nei lipidi neutri e sono più inclini ad essere persi durante la cottura. Dal punto di vista nutrizionale, la cottura migliora la composizione in acidi grassi della carne di cinghiale selvatico, ma aumenta l’entità della ossidazione dei lipidi.

Parole chiave: cinghiale, daino, Sus scrofa, Dama dama, carne, acidi grassi, ossidazione lipidica, qualità fisica della carne.

3. GENERAL INTRODUCTION

In this doctoral thesis I present the four studies conducted during the 2011- 2014 tern in the frame of the PhD in “Scienze e tecnologie per la gestione forestale e ambientale” The thesis is focused on the study of the qualitative characteristics of wild ungulate meat. Growing understanding of the relationship between diet, specific food ingredients and health is leading to new insights into the effect of food components on physiological function and health. This awareness has moved consumers to become more health-conscious, driving a trend towards healthy and nutritious foods with additional health promoting functions. (Henchion et al., 2014) The importance of meat as a source of high biological value proteins (providing all essential amino acids as lysine, threonine, methionine, phenylalanine, tryptophan, leucine, isoleucineand valine) and micronutrients (e.g. vitamins A, B6, B12, D, E, iron, zinc, selenium) is well recognised, however, over the past 10–15 years, these positive attributes have often been overshadowed. Moreover, meat is often considered a problem for human health mainly when food habits imply overconsumption of animal origin feeds (Biesalski, 2005). A new approach of the consumer implies the ethical characteristics of foods, mainly as regards the low environmental impact and animal welfare (de Backer and Hudders, 2015). The meat of wild ungulates rich in substances with nutraceutical functions and extensively produced could represent a niche product that meet consumer requests, but it is necessary to overcome the negative value given to the hunting as activity capable to produce stress for the animal. So, to learn more about its nutritional and hygienic characteristics, and to assure the consumer that the animal has undergone the least possible stress is needed in order to widen the use of wild ungulates meat. The studies performed in this thesis are consequently oriented to this goal.

Since some of these studies have been published in scientific journals and congress proceedings, these parts are attached as reprints of published articles. Unpublished study, still in preparation, is included as manuscript. The published and in progress articles are:

First study Amici, A., Meo Zilio, D., Ficco, A., Primi, R., Serrani, F., Failla, S., Conto,` M. (2011). Some traits of fallow deer and wild boar meat as affected by hunting withdrawal: First results. in Proceeding of 57th I.Co.M.S.T., 148-151.

Second study Cifuni, G.F., Amici A., Contò, M., Viola, V., Failla S. (2014). Effects of the hunting method on meat quality from fallow deer and wild boar and preliminary studies for predicting lipid oxidation using visible reflectance spectra. The European Journal of Wildlife Research, 60, 519–526. doi 10.1007/s10344-014- 0814-3

Third study Amici, A., Cifuni, G. F., Contò, M ., Esposito, L., Failla, S. (2015). Hunting area affects chemical and physical characteristics and fatty acid composition of wild boar (Sus scrofa) meat. Rendiconti Lincei. Scienze Fisiche e Naturali. doi 10.1007/s12210-015-0412-7

Fourth study (in progress) Failla, S., Cifuni, G. F., Contò, M., Amici A., Organoleptic and nutritional characteristics of wild boar meat affected by cooking treatment. (presented as poster in the 21st ASPA congress, Milano June 2015 “ Failla S., Cifuni G. F., Contò M., Viola P., Amici A. Effect of cooking on fatty acid composition, lipid oxidation and cholesterol content of hunted wild boar (Sus scrofa) meat. Proceeding 21st ASPA P012”)

3.1. The increase of wild animals populations in Italy

Unlike what happened in the period between the two world wars in Europe in the last century there is a massive expansion that has continued to this day (Boitani et al., 1995a; Danilkin, 2001) The spread of wild boar was due to the massive restocking and reproductive potential of these animals (Nahlik and Sandor, 2003). In Italy the expansion process of ungulates (mainly roe deer) began in the early 70s. This process started in the north-east regions and rapidly expanded to north- western regions, and then to the northern Apennines. Nowdays roe deer is spreading in the centre and south of the peninsula, where large areas are still available for further expansion (Pedrotti et al., 2001; Carnevali et al., 2009). Even populations of ungulates such as red deer and mouflon are expanding, although with less intensity, also following releases programs (Pedrotti et al., 2001; Carnevali et al., 2009). In Italy the wild boar showed a more complex trend of expansion that started in 60s. It was affected both by natural dispersion processes, as for instance from France, and by release for hunting purposes.

Wild boar survived the 2nd world war in some area of central and southern Italy, but was also translocated from central Europe for meat (husbandry) and/or restocking. The wild boar (Sus scrofa L. 1758) is the wild ungulate more consistent and widely distributed (over 600,000 units) and the growth of populations is expected for the next decade (Ramanzin et al., 2010). Currently, the distribution range of the species cover approximately 64% of the Italian territory (190,000 km2) (Carnevali et al., 2009) and is likely to further expansion (Ramanzin et al., 2010). Also in the rest of In Europe the wild boar (Sus scrofa) is increasingly popular and continues a growth in numbers and distribution area over the past three decades in all of Western Europe (Saez-Royuela and Telleria, 1986; Toigo et al., 2008; Monaco et al., 2003; Scillitani et al., 2010). This growth include not only wild boar but also the other ungulates. The increasing trend of wild boar population are the result of a number of causes. Reforestation processes, for instance, affected the marginal areas as a result of the abandonment of traditional activities (Delibes-Mateos et al., 2009). In addition to the increase of areas representing the optimum suitable habitat, many other factors encouraged the population explosion of wild species; a) the changes in forest management practices (Bobek et al., 1984), b) territorial planning (Milner et al., 2006 ), c) climate change (Mysterud et al., 2001; Root et al., 2003), d) spatial and temporal limits to hunting withdrawal (Servanty et al., 2009), e) the absence of active management within the protected areas (sensu L. 394/91) and, f) illegal release of animals from Central and Eastern Europe (Saez-Rojuela and Telleriia, 1986; Apollonio et al., 1988) which, together with the intercross with the domestic pig (Herrero and Fernández De Luco, 2002), g) induced an increase in the reproductive potential of the species than the native Italian wild boar (Sus scrofa Majori) (Perco, 1987; Massei and Toso, 1993). One factor, often overlooked, that affects the abundance of the populations the legal status of the area (private game, free hunting, protection etc.) and the presence of protected areas or not managed areas, which play a role in the formation of so-called refuge areas (Amici et al., 2012b).

3.2. Problems consequent to the increase of wild animal populations

The increased abundance of some ungulates in many areas has become true overabundance causing conflicts with human activities and biodiversity conservation (Côté et al., 2004). At the present time, wild boar, due to its widespread presence, abundance, its ecological plasticity and opportunistic use of different foods, is believed to be the species most responsible for the economic damage to the agro-livestock-forest sector. Above the adequate values of density, to be evaluated according to habitat and land use, wild boar should be also considered a threat to the conservation of biological diversity (Giménez-Anaya et al., 2008), and an economic cost, often unsustainable for the public administration. In addition, wild boar often negatively affect conservation strategies of protected areas destined to protection and conservation of threatened species. No less important are traffic accidents (Primi et al., 2009), involving human health, and expenditure for damage claims and collision prevention systems.

3.3. Wild animals harvesting and meat availability in Italy

The strong increase in the number of wild ungulates was followed by a strong increase of harvesting (Pedrotti et al., 2001; Carnevali et al., 2009), that consequently induced the increased availability of meat (Ramanzin et al., 2010) which, however, is largely destined to home consumption (Danieli et al., 2012; Ramanzin et al., 2010). A complete figure of wild ungulates in Europe is not available, although some attempts to estimate wild animals as source of food for humans are mentionable (NWFP COST action 1203 - http://www.nwfps.eu/). In Italy, the only affordable data are provided by ISPRA in the so-called BDU (banca dati ungulati) which is edited every few years (Carnevali et al., 2009; Pedrotti et al., 2001). Nonetheless, these data are not updated and are often largely underestimated for some species as for instance for wild boar. Data are expressed on the basis of four macro-regions (Figure 1) as proposed by ISPRA (Carnevali et al., 2009). Wild boar harvest increased by 60% in the last decade as reported in Ramanzin et al. (2010). Although it may be expected that harvesting rate should increase in the future, predictions are uncertain because data are still not provided to the central institutions (ISPRA). For the Eastern Alps, for instance, uncertainty arise due to the legal status of the species. The change of wild boar from pest animal to hunting animal will probably induce a rapid increase of population and harvesting. Large part of roe deer are harvested in the Northern Apennines, 30 % in the Eastern Alps, and less than 10% in the Western Alps. Culling rate was unchanged in the last 10 years in the Eastern Alps (probably the carrying capacity of the area), but increased in Western Alps (+100%) and Northern Apennines (+300%), where the distribution area expanded and increased abundance of the populations (Ramanzin et al., 2010). In Southern Apennines (Adriani et al., 2008) culling is still absent or scares. Roe deer is absent in the Islands. In the future culling rates will remain probably unchanged in the Eastern Alps, but will increase mainly in Southern Apennines, where suitable areas area available (Boitani et al., 2002; Carnevali et al., 2009). Red deer is mainly harvested in the Eastern (70% of total harvesting) and Western Alps (23% of total harvesting). In Northern Apennines it is scarce (7% of total harvesting) and mainly in Tuscany and Emilia Romagna (Carnevali et al., 2009). In the Islands, red deer is present only in Sardinia, with a protected sub- species (Cervus elaphus corsicanus). In most of the Eastern and Western Alps distribution and abundance are still far from carrying capacity (Ramanzin and Sommavilla, 2004), and in Southern Apennines vast areas are suitable for colonisation (Boitani et al., 2002). This process has been artificially facilitated by recent reintroduction projects (Toso, 1999; Carnevali et al., 2009), and now red deer is present across all the Apennines in more or less isolated small populations that will merge into a continuous range, although the winter habitat availability might represent a limiting factor (Amici et al., 2007). In the past decade, culling rates have increased by 240%, and we believe that in the near future red deer culling might surge to two times the present rates. Fallow deer is mainly harvested in Tuscany (Northern Apennines). In Southern Apennines and Islands, data on population abundance and harvesting refer only to small areas and are not relevant nowadays (Amici, pers. com.).

Figure 1 Subdivision of Italy in macro-regions (according to Carnevali et al., 2009)

Eastern Alps: Trentino-Alto Adige, Veneto, Friuli Venezia Giulia; Western Alps: Piemonte, Val d’Aosta, Lombardia; Northern Apennines: Liguria, Emilia Romagna, Toscana, Marche, Umbria; Southern Apennines and Islands: Abruzzo, Molise, Lazio, Campania, Puglia, Basilicata, Calabria, Sicilia, Sardegna; (from Ramanzin et al., 2010, partially modified)

Table 1 Wild ungulates harvesting in Italy expressed as number of heads (ISPRA, 2015 - unpublished data from BDU). The value “0” indicate that the species is non-present or not harvested in the Region. Fallow deer Roe deer Mouflon Chamois Wild boar Red deer Aastern Alps 104 17026 728 8999 3865 6800 Friuli Venezia-Giulia 0 3728 121 422 2733 675 Trentino Alto Adige 0 11315 137 7309 93 4662 Veneto 104 1983470 1268 1039 1463 Northern 3264 48564981 29 121397 1056 Emilia Romagna 961 22442 32 0 11965 500 Liguria 261 15480 29 22655 0 Marche 0 15580 0 6604 0 Toscana 2042 22049949 0 69353 556 Umbria 0 9670 0 10820 0 Southern 349 2790 0 4946 0 Abruzzo 0 0000 0 Basilicata 0 0000 0 Calabria 0 0 0 0 4164 0 Campania 0 0000 0 Lazio 349 2790 0 782 0 Molise 0 0000 0 Puglia 0 0000 0 Sardegna 0 0000 0 Sicilia 0 0000 0 Western Alps 110 5859 204 3861 23386 2176 Lombardia 0 67489 1244 5912 1153 Piemonte 110 2572115 1912 16979 801 Val d'Aosta 0 2613 0 705 495 222 Overall number 3827 71728 1913 12889 153594 10032 *See figure 1

The amount of wild animals’ meat available for Italian consumers is still not known and is probably impossible to estimate. A raw approximation is possible if we consider the above reported data (ungulates) and some data from import (ISTAT- http://agri.istat.it/). This figure is incomplete for two main reasons; a) data collection from local authorities is incomplete (mainly for wild boar) and numbers are dramatically underestimated (Riga, 2015 pers. com.), b) poaching still represent an important way of wildlife harvesting in many regions.

The data on carcass availability includes both home consumption and market availability and was calculated on the basis of a conversion factor calculated on the average body weight of animals harvested for each species according to Ramanzin et al. (2010).

Table 2 Calculated amount of ungulates carcasses* available from hunting in Italy (tons.). The value “0” indicate that the species is non-present or not harvested in the Region. Area*/Region Fallow deer Roe deer Mouflon Chamois Wild boar Red deer Eastern Alps 3.2 212.8 12.4 130.5 131.4 408.0 Friuli Venezia-Giulia 0 46.6 2.1 6.1 92.9 40.5 Trentino Alto Adige 0 141.4 2.3 106.0 3.2 279.7 Veneto 3.2 24.8 8.0 18.4 35.3 87.8 Northern Apennines 99.6 607.1 16.7 0.4 4127.5 63.4 Emilia Romagna 29.3 280.5 0.5 0 406.8 30 Liguria 8.0 19.4 0 0.4 770.3 0 Marche 0 19.5 0 0 224.5 0 Toscana 62.3 275.6 16.1 0 2358.0 33.4 Umbria 0 12.1 0 0 367.9 0 Southern Apennines 10.6 3.5 0 0 168.2 0 Abruzzo 0 0 0 0 0 0 Basilicata 0 0 0 0 0 0 Calabria 0 0 0 0 141.6 0 Campania 0 0 0 0 0 0 Lazio 10.6 3.5 0 0 26.6 0 Molise 0 0 0 0 0 0 Puglia 0 0 0 0 0 0 Sardegna 0 0 0 0 0 0 Sicilia 0 0 0 0 0 0 Western Alps 3.4 73.2 3.5 56.0 795.1 130.6 Lombardia 0 8.4 1.5 18.0 201.0 69.2 Piemonte 3.4 32.2 2.0 27.7 577.3 48.1 Val d'Aosta 0 32.7 0 10.2 16.8 13.3 Overall amount 116.7 896.6 32.5 186.9 5222.2 601.9 * assuming an average carcass weight of 12.5 kg for roe deer, 60.0 kg for red deer; 30.5

In more details, the carcass weight (without blood, skin, head, distal portions of hind- and fore-legs, and offal), for each species, according to field available data and literature was calculated. The dressing percentage was calculated as 83% of

field dressed weight (after bleeding and offal removal) for cervids and bovids, and as 65% of live weight for wild boar (Müller et al., 2000; Tuckwell, 2003; Skewes et al., 2008). Field dressed weights for cervids and bovids were calculated according to Ramanzin et al. (2010). These data have been obtained from hunting statistics of the Alpine and Northern Apennine areas (Andreoli et al., 2004; Ramanzin and Sommavilla, 2004; Soffiantini et al., 2006), while for wild boar hunting statistics of Northern and Southern Apennines were used. The species roe deer contributes with about 900 tons (12.7% of total) to the present availability of ungulate meats (Table 2). For red deer the present contribution of the species to wild ungulate meat availability is about 600 tons; 8.5% of total). For fallow deer and chamois the amount of carcasses represent the 1.7 and 2.6 % respectively of the overall amount (Table 2). An increase for these species is not probably due to the limited extension of distribution area. A little contribution to meat availability is provided by mouflon (0.5% of total amount) probably because of the spot distribution and scarce abundance with the exception of Sardinia where the species is protected. The largest part of meat availability is provided by wild boar throughout the macro-regions (74%). This specie will probably contribute with increasing amounts in the next decade both due to the expansion of the species and to the accuracy of data collection. The estimates of meat availability given in table 2, as also suggested by Ramanzin et al. (2010), indicate that per capita yearly consumption of meat from harvested ungulates in Italy is very low. This amount vary between 0.1-0.3 kg/person and depends on the macro region considered). This amount strongly increases if we consider the largest amount of game meat is consumed by hunters (http://agri.istat.it/) and their families. For hunters/familiar the average per capita yearly consumption is between 1.0- 4.0 kg according to the macro region considered. These data are confirmed by Danieli et al. (2012) in Viterbo Province, Italy but are lower than those registered in Spain by Morales et al. (2011).

The last survey on ungulate farming in Italy (Carnevali et al., 2009) reported 1200 breeding farms with a stock of approximately 2800 wild boars, 3300 fallow deer, 1000 red deer, 200 roe deer and 650 mouflon. Previous data (Salghetti, 1991) estimated the size of farmed stocks at approximately 14,000 wild boar, 10,000 fallow deer, 2,000 mouflon and 1600 red deer, mainly concentrated in the Umbria and Toscana regions. According to FEDFA (2007) these data reflect the negative trend in the last decades. As a general conclusion it should be underlined that the import-export balance should be considered. Data on wild animals meat and carcasses available from ISTAT (http://agri.istat.it/) are variable in the last years. A first survey performed by a scientific commission of ASPA (Associacione per le Scienze e le Produzioni Animali) suggests that the balance could be negative (Amici, pers. comm.).

3.4. The meat quality of hunting harvested wild animals

To lead the consumers towards the consumption of wild ungulates meat is necessary ensure high qualitative standards not only for chemical composition and sensory characteristics, but also to guarantee the hygiene of the products. The meat of wild animals does not always correspond to the organoleptic characteristics to which the consumer is used, but he is aware to these differences on colour, tenderness and flovour (Hoffman and Wiklund, 2006; Hutchison et al., 2010). In fact, over time the man has adapted specific culinary techniques for these particular type of meat, using marinate rich in spices that have a good antioxidant activity and tenderizing power (Dhanda et al., 2003; Żochowska-Kujawska et al., 2012). Wild ungulate meat is darker than that from domestic species (Volpelli et al., 2003; Marsico et al., 2007), but the consumers consider the dark colour a typical feature of game meat. The particular colour is probably due to the high presence in iron, but also to different proportion on white (type IIB fibres) and red (type I

fibres) fibers (Żochowska et al., 2005 and 2012; Sales and Kotrba, 2013). The last one are very abundant in the longissimus muscle of wild boars and deer. It is commonly accepted that physical exercise, due also to continuous alertness to predators, increases oxidative and decreases glycolytic metabolism, obtaining higher percentage of red fibres (Oshima et al., 2009). However, wild boar muscle is characterized by a higher percentage of subtile fibres compared to deer meat, that showing less coarser structure was higher susceptible to tenderization (Żochowska- Kujawska et al., 2012). Game meat in general is believed to be tougher than meat from domestic species. Literature on farmed ungulates emphasises the importance of pre-slaughter stress on meat quality (Smith and Dobson, 1990; Bornett-Gauci et al., 2006) and of some techniques to improve tenderness after shot down using, marinate , electrostimolation, massage or manipulation of rigor temperature (Wiklund et al., 2001; Żochowska-Kujawska et al., 2007, Bekhit et al., 2007) obtaining acceptable level of tenderness early post-mortem with less damaging effect on the colour stability. It is known by the chemical point of view that carcasses and meat of wild ruminants are usually much thinner than those of domestic animals, with content of intramuscular fat (IMF) lower than 1% (Drew, 1985; Casoli et al., 1986; Duranti e Casoli et al., 2005; Poli et al., 1993; Zomborszky et al., 1996; Summer et al., 1997; Secchiari et al., 2001; Volpelli et al. 2002, 2003, Wiklund et al., 2003), consisting predominantly of structural lipid components (polar lipid fraction) that have high proportions of polyunsaturated fatty acids (PUFA). One of the features that gives substantial changes to the nutritional value of meat is the intramuscular fat content to which the percentage variation of proximate composition and the ratio of fatty acids depend. Indeed, fatter meat has a higher content of saturated (SFA) fatty acids and a greater amount of total cholesterol (Quaresma et al., 2012). The protein content on wild ungulates not significantly affected by the region, age or gender (Dunnemberg et al., 2013), it range from 21 to 24% for deer muscles,

and varies from 21 to 23% in wild boar muscles and showes significantly higher value in male animals older than 1 year (Volpelli et al., 2003;Dunnemberg et al., 2013; Sales and Kotrba, 2013; Dahlan and Norfarizan-Hanoon, 2007). The water content is not significantly different in deer and wild boar muscles and ranges from 73% to 76% (Volpelli, 2003; Purchas et al., 2010, Triumf et al., 2012; Sales and Kotrba, 2013). Furthermore, low IMF results in desirable polyunsaturated/saturatedand n–6/n- 3 fatty acid ratio (Hoffman, 2006; Onyango et al., 1998, Cordain et al., 2002), and the high content of 18:3 n-3 (3.3% of total fatty acids) as found in venison from Quaresma et al. (2012) and it is definitely linked to the low intramuscular fat, while usually the content reported in longissimus muscle of wild boar is lower than 1g/100g of total fatty acids (Polak et al., 2008, Purchas et al., 2010) with the exception of the data reported by Dannemberg et al. (2013) for both genders. The best evidence in the change of the physiological natural cycle of the animal and food linked with their habitat causes differences concerning chemical and physical characteristics of meat (Stevenson et al., 1992; Wiklund et al., 2010, Amici et al., 2015). Therefore, the chemical characteristics of meat of wild ungulates depend primarily on the animal’s age and gender, plus sampling place. As well as regards the grazing animals, administration of concentrated food induces the increase of SFA and decrease of PUFA (Manley and Forss, 1979; Poli et al., 1993; Secchiari et al., 2001; Rule et al., 2002; Volpelli et al., 2002, 2003). A wide panoramic on several factors that determine the variability of meat quality in wild ungulates hunted in Germany was reported by Dannemberg et al. (2013). The total SFA proportion in the longissimus muscle of venison ranges from about 32 to 50%, while for wild boar it ranches from 32 to 40% of the total fatty acids and it is affected by the region, gender and age as reviewed in table 3 by different authors. Higher SFA proportions were detected in the muscle of older male and female animals compared to the younger ones. The highest proportions of

single SFA are observed for 14:0 (myristic acid), 16:0 (palmitic acid) and 18:0 (stearic acid) (Dannemberg et al., 2013). Total monoinsatured fatty acid content (MUFA) in wild boar muscle varies widely from about 37 to 51 %. This high quantity of MUFA compared to reed meat depend principally by oleic acid content it could be caused by the high consumption of acorns and chestnuts, which are rich in high oleic acid contents (Quaresma et al., 2011). The sum of the PUFA proportion in venison meat differs significantly between the region and age groups and varies between 23 and 57%. Linolenic acid (LNA, 18:3n-3) varies between 3.4 and 4.6%, while the total PUFA content in longissimus muscle of wild boar ranges from about 18 to 29%. The most abundant long-chain PUFA in the muscle samples is 20:4n−6 followed by 22:5 n-3 and 20:5 n-3. The resulting n-6/n-3 PUFA ratio in the longissimus muscle of wild boar is ≤7 and approximately two times higher compared to the ratio in roe deer muscle that is at most about 4. Concerning the fatty acids n-3 PUFA with high biological importance (18:3 n-3, 20:5 n-3, 22:5 n-3 and 22:6 n-3) the total amount is lower 2% of total fatty acids for wild boar and can reach also 14% in venison meat (considering the authors reported in table 3). CLA isomers are the major intermediates formed during rumen biohydrogenation of C18 PUFA, it exerts positive effects to prevent cancer, cardiovascular disease, diabetes, and improve immune system and bone health (Jahreis et al., 2000)

However, the 18:2cis‐9, trans‐11 content is particularly scarce in wild animals. Vaccenic acid (VA, 18:1trans‐11), an important precursor in the biosynthesis of conjugated linoleic acids, occurres at a low proportion, between 0.8 and 2.1%, of the total fatty acids (Benjamin and Spener 2009) and consistent with values frequently found in intensively fed beef (Raes et al., 2004). The low content of 18:2cis‐9,trans‐11 is probably a consequence of the low IMF content (Jerónimo et al., 2011).

Table 3 Fatty acid composition in different wild ungulate’s meat

Species SFA MUFA PUFA P/S n6/n3 n3*

Wild boar male Skewes et al., 2009 39.64 50.75 9.62 0.25 6.85 3.0 Quaresma et al., 2011 34.7 38.9 25.4 0.60 17 1.4 Dannenberg et al., 2013 32.6 42.2 25.1 0.77 9.3 2.7 Razmaite et al., 2012 38.9 43.65 17.39 0.45 9.01 1.91 Wild boar female Quaresma et al., 2011 34.2 42.6 22.5 0.52 15.5 1.4 Dannenberg et al., 2013 35.2 41.8 22.9 0.65 9.2 4.1 Razmaite et al., 2012 36.3 44.26 18.91 0.53 10.33 1.93 Wild boar young Quaresma et al., 2011 33.3 42.2 23.8 0.55 12.6 1.7 Dannenberg et al., 2013 33.2 37.5 29.2 0.88 9.9 2.9 Deer female Purchas et al., 20 44.75 29.9 25.35 0.57 1.58 9.67 Triunf et al., 2012 40.6 29.0 23.4 0.60 3.63 5.08 Polak et al., 2008 37.3 37.2 25.48 0.70 4.02 4.58 Dannenberg et al., 2013 46.8 23.3 29.6 0.63 2.6 8.5 Deer male Purchas et al., 20 40.1 26.5 32.9 0.82 1.21 14.2 Triunf et al., 2012 41.4 28.2 23.3 0.61 3.67 5.39 Wiklund et al., 2001 42.62 30.0 27.38 0.64 2.2 7.7 Volpelli et al., 2003 31.83 11.25 56.92 1.78 3.30 13.35 Polak et al., 2008 42.42 31.72 25.87 0.63 2.60 6.91 Dannenberg et al., 2013 49.8 22.1 27.8 0.56 2.4 8.3 Deer calves Triunf et al., 2012 45.9 33.2 14.8 0.32 4.24 2.83 Polak et al., 2008 35.59 26.99 34.42 1.06 4.53 7.77 Dannenberg et al., 2013 35.3 19.3 45.1 1.27 2.7 13.7 * assuming n3 as Ʃ ALA(13:3-n3)+EPA(20:5-n3)+DHA(22:6n-3)+DPA(22:4n-3)

Cholesterol content is quite similar or higher with respect to domestic animals. In fallow deer meat, Poli et al. (1993) and Secchiari et al. (2001) reported average values of 80-85 mg/100 g and 102 mg/100 g, respectively.

Differences may be found also among wild ruminants species according to feeding habits. Different diets, either natural or administered in stable, in fact affect the amount of cholesterol in ungulates meat from 50 mg/100 g of meat (Quaresma et al., 2012). reaching even to values of 94 mg / 100 g (Polak et al., 2008). Some studies have suggested the possibility that venison meat may be an healthy food commodity reducing the risk of cancer and prevent coronary disease Ulbricht and Sauthgate (1991). World Health Organization (WHO, 1990) recommends that the daily fat intake be reduced to 30% of the total energy intake, and that saturated fats should be limited to 10% of this caloric intake. It is also advised that cholesterol intake should not exceed 300 mg per day. At the same time, the recommended P/S ratio (PUFA/SFA) should be more than 0.4 (Wood et al., 2008). The ratio of n6/n3 PUFAs is considered to be a risk factor in cancerogenesis and coronary heart disease, and it is recommended that this ratio be less than 4.0 (Enser et al., 2001, Simopoulos 2008). The wild ungulate for low fat content is very close to the recommended values. Therefore the meat of wild ungulate could be considered as functional food. that is a “food that produces benefits on one or more target functions in the body, beyond to bring adequate nutritional effects, in a way that is relevant to either improved state of health and well-being and/or reduction of disease risk”. This definition was included in the EC Project FUFOSE Consensus Document (1999).

Wild ungulate meat presents in addition high percentage of essential trace elements for human health such as zinc and iron and a good quantity of some elements considered of high nutritional value with antioxidant effect as taurine, carnosine, anserine and vitamin E (Stevenson-Barry, 1999; Purcha et al, 2010 , Triunf et al., 2012). The iron concentration in deer muscles ranges between 26.3 and 34.4 mg/kg muscle and in wild boar between 17.3 and 21 mg/kg muscle. The average iron

concentrations in the muscle of wild boar and roe deer are approximately 4 times and 2 times higher than the iron levels reported for the muscles of domestic pigs and beef cattle (Dannenberger et al., 2013). The zinc concentration of roe deer and wild boar muscle is on a similar level and varies between 13.6 and 39.3 mg/kg muscle and 18.1 and 31.9 mg/kg muscle, respectively, depending on the region, age and gender (Dannenberger et al., 2013). But the high value of wild ungulate meat can be compromised by poor preservation. Lipid oxidation is a major cause of deterioration of meat quality (Kanner, 1994) in particular for animal rich in PUFA such as wild ungulates (Okabe et al., 2002). It accelerates in the immediate post-slaughter period and during handling, processing, storage and cooking to produce discoloration, off-flavors, texture defects and potentially toxic compounds. The literature on farmed ungulates emphasizes the important effect that pre-slaughter stress has on meat quality and stability (Bradshaw and Bateson, 2000, Pollard et al., 2002; Bornett-Gauci et al., 2006). In some works (Sales and Kotrba, 2013 and Valencak, 2010) it was reported that wild boar is more prone to oxidative spoilage compared to pork. This might be due to higher contents of unsaturated fatty acids and higher content of heme iron in meat from wild game (Hofbauer and Smulders, 2011; Mielnik et al., 2011; Descalzo and Sancho, 2008). The muscle has a natural defense mechanism against oxidative stress due to non-enzymatic water and lipid soluble compounds like vitamin E, vitamin C, carotenoids, ubiquinols, polyphenols and so on. A positive effect of diet with vitamin E content on lipid oxidation is reported also by Sampels et al., 2006 . The antioxidant effect linked to the type of diet is still reduced by the type of cooking, TBARS in lean meat (fallow deer, wild boar, roe deer) were significantly affected by culinary treatment, more in boiled meat than in grilled meat. The hunting methods affected lipid oxidation in the meat of the wild ungulate as confirmed by our results (Cifuni et al., 2014). However, TBARS value were not affected by the embedded fragments due to bullets into muscle (Irschik et al., 2015). Area surrounding the bullet pathway can show extremely high Pb contamination in the muscle (Hunt et al., 2006; Doborowolska and Melosik, 2008).

Furthermore it should be underlined that, in some studies, muscle or organs of some individuals present levels of some heavy metals (cadmium, lead, copper, chromium, selenium) higher than that required for human consumption in accordance to national legislation (Swiergosz et al., 1993; Santiago et al., 1998; Pompe-Gotal e Pompe-Gotal et al., 2002; Falandysz et al., 2005; Lazarus et al. 2005; Mandas, 2005; Valencak et al., 2006; Amici et al., 2008; Bilandžić et al., 2009; Reglero et al., 2009). The EU limits for Cd and Pb in meat and offal are reported by Regulation (EC) No 1881/2006 (European Commission, 2006), lately amended by Regulation (EC) No 629/2008 (European Commission, 2008a). Also the polychlorinated biphenyls and organoclorines that are synthetic organic compounds widely used in agricultural practices, can be accumulate in the fat of wild animal (Bachou et al., 1998; Alleva et al., 2006, Ramanzin et al. 2010; Amici et al., 2011; Amici et al., 2012). Food safety preservation in wild animal is another problem. Infant delayed after wounding, causes microbial infections, that will spread from gut spilling (Gill, 2007). In addition, badly placed shots may cause carcass damage, and any shot in the gut will cause rapid microbial contamination of the carcass (Gill, 2007; Atassanova et al., 2008). Safety requirements of game meats have been addressed in Regulations (EC) No. 853/2004 and No. 854/2004 (European Commission, 2004b; 2004c). Muscle contamination by microorganisms from the hide or the gastro-intestinal tract may be influenced by numerous factors and it could continue during carcass storage. However, the subsequent handling, transportation and slaughtering processes are very critical steps, especially in warm months (; MacDougall et al., 1979; Gill, 2007; Atassanova et al., 2008).

3.5. General aims of the study

Objectives of these studies were:  to evaluate the influence of hunting technique on main meat quality traits in wild boar (Sus scrofa) and fallow deer (Dama dama);  to test the feasibility of visible reflectance spectra (360 to 740 nm) to predict the lipid oxidation of meat during the conservation;  to investigate how different geo-graphical hunting areas and cooking processing influence chemical composition, quality traits, fatty acid composition and lipid quality indexes of wild boar meat.

3.6. CITED LITERATURE

Adriani, S., Alicicco, D., Serrani, F., Amici, A., 2008. Distribuzione del Capriolo (Capreolus capreolus) in provincia di Frosinone, primi risultati. In: Prigioni C., Meriggi A., Merli E. (ed). VI Congr. It. Teriologia, Hystrix, It. J. Mamm., (N.S.) SUPPL. 2008: 64

http://gis.dipbsf.uninsubria.it/congressi/index.php/atit/atit2008/rt/metadata/92/0

Alleva, E., Francia, N., Pandolfi, M., De Marinis, A. M., Chiarotti, F., Santucci, D., 2006. Organochlorine and Heavy-Metal Contaminants in Wild Mammals and Birds of Urbino-Pesaro Province, Italy: An Analytic Overview for Potential Bioindicators. Arch. Environ. Contam. Toxicol. 51: 123–134. DOI:10.1007/s00244-005-0218-15.

Amici, A., Fasciolo, V., Serrani, F., Adriani, S., Alicicco, D., Ronchi, B., 2007. A deterministic model to predict red deer (Cervus elaphus L.) winter habitat in Cicolano, Central Apennine – Italy. Proc. 1st International Conference on Genus Cervus, Fiera di Primiero (TN), pp 28-29.

Amici, A., Serrani, F., Sabatini, A., 2008. Determinazione dei metalli pesanti nel cinghiale (Sus scrofa). In B. Floris and G. Moniello (eds.) Master di I livello in Conservazione e Gestione della Fauna, Ed. Madrikè, Genova. 53-63

Amici, A., Meo Zilio, D., Ficco, A., Primi, R., Serrani, F., Failla, S., Contò, M., 2011. Some Traits of Fallow Deer and Wild Boar Meat as Affected by Hunting Withdrawal: First Results. Proc. 57th Int. Congr. Meat Scie Tech., Ghent, Belgium n°148

Amici, A., Danieli, P.P., Russo, C., Primi, R., Ronchi, B., 2012. Concentrations of some toxic and trace elements in wild boar (Sus scrofa) organs and tissues in different areas of the Province of Viterbo (Central Italy). Italian

Journal of Animal Science ,11:e65, 354-362 doiOI: 10.4081/ijas.2012.e65

Amici, A., Serrani, F., Rossi, C. M., Primi, R., 2012b. Increase in crop damage caused by wild boar (Sus scrofa L.): the “refuge effect”. Agron Sustain Dev. 32:683–692 doi 10.1007/s13593-011-0057-6

Amici. A., Danieli, P.P., Primi, R., Ronchi, B., 2013. Heavy metals in free- living wild boars harvested in Viterbo (Central Italy): are hunters at risk? In: proc. ASPA 20th Congress Bologna, June 11-13, 2013, Italian Journal of Animal Science 2013; volume 12 supplement 1, pag. 18, Abstr.

Amici, A., Cifuni, G. F., Contò, M ., Esposito, L., Failla, S., 2015. Hunting area affects chemical and physical characteristics and fatty acid composition of wild boar (Sus scrofa) meat. Rendiconti Lincei. Scienze Fisiche e Naturali. doi 10.1007/s12210-015-0412-7

Andreoli, E., Fabbri, F., Mattiello, S., 2004. General conditions and biometry of chamois (Rupicapra rupicapra) culled in the Italian Alps. Proc. 6th International Wildlife Ranching Symposium, Paris, France, pp 120-121.

Apollonio, M., Randi, E., Toso, S., 1988. The systematics of the wild boar (Sus scrofa L.) in Italy. B. Zool. 3:213-221.

Atanassova, V., Apelt, J., Reich, F., Klein, G., 2008. Microbiological quality of freshly shot game in Germany. Meat Science, 78: 414–419. DOI:10.1016/j.meatsci.2007.07.004

Bachour, G., Failing, K., Georgii, S., Elmadfa, I., Brunn H., 1998. Species and organ dependence of PCB contamination in fish, foxes, roe deer, and humans. Arch. Environ. Contam. Toxicol. 35: 666-673. DOI: 10.1007/s002449900429

Beiglböck, C., Steineck, T., Tataruch, F., Ruf, T., 2001. Environmental cadmium induces histopathological changes in kidneys of roe deer. Environ. Toxicol. Chem. 21: 1811-1816. DOI:10.1002/etc.5620210908

Bekhit, A.E.D., Farouk, M.M., Cassidy, L., Gilbert, K.V., 2007. Effects of rigor temperature and electrical stimulation on venison quality. Meat Science, 75: 564–574. doi:10.1016/j.meatsci.2006.09.005

Benjamin, S., Spener, F., 2009. Conjugated linoleic acids as functional food: An insight into their health beneﬁts. Nutr. Metab. 6: 36 doi:10.1186/1743-7075-6- 36

Biesalski, H.K., 2005. Meat as a component of a healthy diet – are there any risks or benefits if meat is avoided in the diet? Meat Science, 70: 509–524.

Bilandžić, N., Sedak, M., Vratarić, D., Perić, T., Šimić, B., 2009. Lead and cadmium in red deer and wild boar from different hunting grounds in Croatia. Sci. Total Environ. 407: 4243-4247. http://www.ncbi.nlm.nih.gov/pubmed/19411089

Bobek, B., Boyce, MS., Kosobucka, M., 1984. Factors affecting Red Deer (Cervus elaphus) population density in south-eastern Poland. J Appl Ecol 21:881– 890

Boitani, L., Corsi, F., Falcucci, A., Maiorano, L., Marzetti, I., Masi, M., Montemaggiori, A., Ottaviani, D., Reggiani, G., Rondinini, C., 2002. Rete Ecologica Nazionale. Un approccio alla conservazione dei vertebrati italiani. Università di Roma "La Sapienza", Dipartimento di Biologia Animale e dell’Uomo; Ministero dell’Ambiente, Direzione per la Conservazione della Natura; Istituto di Ecologia Applicata. Home page address: http://www.gisbau.uniroma1.it/ren_docs/Boitani%20et%20al%202002a.pdf

Boitani, L., Trapanese, P., Mattei, L., 1995. Demographic patterns of a wild boar (Sus scrofa L.) population in Tuscany, Italy. Ibex Journal of Mountain Ecology 3:197-201.

Bornett-Gauci, H.L., Martin, J.E., Arney, D.R., 2006. The welfare of low volume farm animal during transport and at slaughter: a review of current

knowledge and recommendations for future research. AnimWelf 15:299–308

Bradshaw, E.L., Bateson, P., 2000. Welfare implications of culling red deer (Cervus elaphus). Anim. Welfare 9: 3-24. http://firenzeita.library.ingentaconnect.com/content/ufaw/aw/2000/000000 09/00000001/art00002 Carnevali, L., Pedrotti, L., Riga, F., Toso, S., 2009. Banca Dati Ungulati: Status, distribuzione, consistenza, gestione e prelievo venatorio delle popolazioni di Ungulati in Italia. Rapporto 2001-2005. Biol. Cons. Fauna, 117: 1-168.

Casoli, C., Duranti, E., Rambotti, F., 1986. Rilievi alla macellazione e caratteristiche quanti-qualitative della carne di daino (Dama dama L.). Zoot. Nutr. Anim., 5: 411-421.

Casoli, C., Duranti, E., Cambiotti, F., Avellini, P., 2005. Wild Ungulate Slaughtering and Meat Inspection. Vet. Res, Commun. 29 (2): 89-95.

Cifuni, G.F., Amici A., Contò, M., Viola, V., Failla S. (2014). Effects of the hunting method on meat quality from fallow deer and wild boar and preliminary studies for predicting lipid oxidation using visible reflectance spectra. The European Journal of Wildlife Research, 60, 519–526. doi 10.1007/s10344-014- 0814-3

Cordain, L., Watkins, B.A., Florant, G.L., Kelher, M., Rogers, L., Li, Y., 2002. Fatty acids analysis of wild ruminant tissues: evolutionary implications for reducing diet-related chronic disease. Eur. J. Clin. Nutr. 56: 181-191. DOI: 10.1038/sj/ejcn/1601307

Côté, S.D., Rooney, T.P., Tremblay, J-P., Dussault, C., Waller, D.M., 2004. Ecological impacts of deer overabundance. Ann. Rev. Ecol. Evol. Syst. 35:113–47

Dahlan, I, Norfarizan-Hanoon, N.A., 2008. Chemical composition, palatability and physical characteristics of venison from farmed deer. Animal Science Journal,

79:498–503. doi: 10.1111/j.1740-0929.2008.00555.x

Danieli, P.P., Serrani, F., Primi, R., Ponzetta, M. P., Ronchi, B., Amici, A., 2012. Cadmium, Lead and Chromium in Large Game: A Local-Scale Exposure Assessment for Hunters Consuming Meat and Liver of Wild Boar. Archives of Environmental Contamination and Toxicology, In press. DOI: 10.1007/s00244- 012-9791-2 Danilkin, A.A., 2001. The wild boar: an un -precedented spread or restoration of the species range? Dokl. Biol. Sci. 380:457-460.

Dannenberger, D., Nuernberg, G., Nuernberg, K., Hagemann E., 2013. The effects of gender, age and region on macro- and micronutrient contents and fatty acid profiles in the muscles of roe deer and wild boar in Mecklenburg-Western Pomerania-Germany. Meat Science, 94: 39-46

doi: 10.1016/j.meatsci.2012.12.010

De Backer, C. J.S., Hudders, L., 2015. Meat morals: relationship between meat consumption consumer attitudes towards human and animal welfareand moral behavior. Meat Science, 99, 68-74.

Delibes-Mateos, M., Farfán, M.A., Olivero, J., Márquez, A.L., Vargas, J.M., 2009. Long-term changes in game species over a long period of transformation in the Iberian Mediterranean landscape. – Environ. Manage. 43: 1256–1268

Descalzo, A.M., Sancho, A.M., 2008. A review of natural antioxidants and their effects on oxidative status, odor and quality of fresh beef produced in Argentina Meat Science, 79: 423–436

Dhanda, J.S., Pegg, R.B., Shand, P.J., 2003. Tenderness and chemical composition of elk (Cervus elaphus) meat: effects of muscle type, marinade composition and cooking method. Journal of Food Science. 68: 1882-1888. http://www3.interscience.wiley.com/cgi‐bin/fulltext/118832482/PDFSTART

Dobrowolska, A., Melosik, M., 2008. Bullet-derived lead in tissues of the wild

boar (Sus scrofa) and red deer (Cervus elaphus). Eur. J. Wildl. Res. 54: 231–235. DOI: 10.1007/s10344-007-0134-y

Drew, K.R., 1985. Meat production from farmed deer. Roy. Soc. NZ Bull. 22: 285-290.

Duranti, E., Casoli, C. 1993. La produzione di carne di daino (Dama dama L.) allevato in ambiente confinato. Proc. Parliamo di carni complementari (ovine, caprine, di ungulati, equine). Fossano, Italy, 45-62.

EC Project FUFOSE, Consensus Document (1999). Scientific concepts of functional foods in Europe. The British Journal of Nutrition, 81(4), S1−S27.

Enser, M. 2001.The role of fats in human nutrition B Rossell (Ed.), Oils and fats, Vol. 2. Animal carcass fats, Leatherhead Publishing, Leatherhead, Surrey, UK (2001), pp. 77–122

Falandysz, J., Szymczyk-Kobrzyńska, K., Brzostowski, A., Zalewski, K., Zasadowski, A., 2005. Concentrations of heavy metals in the tissues of red deer (Cervus elaphus) from the region of Warmia and Mazury, Poland. Food Addit. Contam. 22: 141-149.

FEDFA, 2007. Deer farming in Italy. Allevamenti di cervidi in Umbria. Proc. Congress Allevamenti di Selvaggina, Nocera Umbra. Home page address: http://www.fedfa.com/index2.html.

Gill, C.O., 2007. Microbiological conditions of meat from large game animals and birds. Meat Sci. 77: 149-160. DOI:10.1016/j.meatsci.2007.03.007

Giménez-Anaya, A., Herrero, J., Rosell, C., Couto, S., García-Serrano, A., 2008. Food habits of wild boars (Sus scrofa) in a Mediterranean coastal wetland. Wetlands 28: 197–203

Henchion, M., McCarthy, M., Resconi, V.C., Troy, D., 2014. Meat consumption: Trends and quality matters. Meat Science 98 (2014) 561–568

Herrero, J. F., De Luco, D., 2002. Wild boars (Sus scrofa L.) in Uruguay: scavengers or predators? Mammalia 67 (4): 485-491.

Hofbauer, P., Smulders, F.J.M., 2011. The muscle biological background of meat quality including that of game species P. Paulsen, F. Bauer, M. Vodnansky, R. Winkelmayer, F.J.M. Smulders (Eds.), Game meat hygiene in focus, Wageningen Academic Publishers, Wageningen , 273–295.

Hoffman, L. C., Wiklund, E., 2006. Game and venison - Meat for the modern consumer. Meat Sci. 74: 197–208 doi:10.1016/j.meatsci.2006.04.005

Hunt, W.G., Watson, R.T., Oaks, J.L., Parish, C.N., Burnham, K.K., Tucker, R.L. Russell L. Belthoff, J.R., Hart, G., 2009. Lead bullet fragments in venison from Rifle-Killed deer: potential for human dietary exposure. PLoS One. 4(4):e5330. Hutchison, C.L., Mulley, R.C., Wiklund, E., Flesch, J.S., 2010. Consumer evaluation of venison sensory quality: Effects of sex, body condition score and carcase suspension method. Meat Science, 86: 311–316. doi:10.1016/j.meatsci.2010.04.031 Irschik, M.S, Paulsen S.P., Tichy, A., Bauer F., 2015. Release of copper from embedded solid copper bullets into muscle and fat tissues of fallow deer (Dama dama), roe deer (Capreolus capreolus), and wild boar (Sus scrofa) and effect of copper content on oxidative stability of heat-processed meat Meat Science 108: 21-27

Jerónimo, E., Alves, S.P., Alfaia, C.M., Prates, J.A.M., Santos-Silva, Bessa, J., R.J.B. 2011. Biohydrogenation intermediates are differentially deposited between polar and neutral intramuscular lipids of lambs. European Journal of Lipid Science and Technology, 11,924–934.

Jahreis, G., Kraft, J., Tischendorf, F., Schone, F., and von Loeffelholz, C., 2000. Conjugated linoleic acids: Physiological effects in animal and man with special regard to body composition. European Journal of Lipid Science and

Technology, 102, 695–703

Kanner, J., 1994. Oxidative processes in meat and meat products: quality implications. Meat Sci 36,:169–189

Lazarus, M., Orct, T., Blanusa, M., Vickovic, I., Sostarić, B., 2008. Toxic and essential metal concentrations in four tissues of red deer (Cervus elaphus) from Baranja, Croatia. Food Addit. Contam. A. 25:270-283.

Lazarus, M., Vikoiæ, I., Šoštariæ, B., Blanuša, M., 2005. Heavy metal levels in tissues of red deer (Cervus elaphus) from eastern Croatia. Arh. Hig. Rada. Toksiko. 56:233-240.

MacDougall, D.B., Shaw, B.G., Nute, G.R., Rhodes, D.N., 1979. Effect of pre-slaughter handling on the quality and microbiology of venison from farmed young red deer. J. Anim. Sci. Food Agric. 30: 1160-1167. DOI: 10.1002/jsfa.2740301208

Mandas, L., 2005. Problemi sanitari della fauna selvatica nelle oasi di protezione faunistica. Regione Autonoma della Sardegna – Ente Foreste della Sardegna; pp 11.

Manley, T.R., Forss, D.A. 1979. Fatty acids of meat lipids from young red deer (Cervuselaphus). J. Sci. Food. Agric. 30: 927-931.

Marsico, G., Forcelli, M.G., Tarricone, S., Rasulo, A., Pinto, F., Celi, R. 2007. Quality of the meat of wild and raised wild boar. Prog. Nutr. 9: 248–252

Massei, G., Toso, S., 1993. Biologia e gestione del Cinghiale. Documenti Tecnici 5. INFS ed., Bologna, Italy.

Mielnik, M.B., Rzeszutek, A., Triumf, E.C., Egelandsdal B., 2011. Antioxidant and other quality properties of reindeer muscle from two different Norwegian regions Meat Science 89, 526–532 doi:10.1016/j.meatsci.2011.05.021

Milner, J.M., Bonenfant, C., Mysterud, A., Gaillard, J-M., Csányi, S.,

Stenseth, N.C., 2006. Temporal and spatial development of red deer harvesting in Europe: biological and cultural factors. J. Appl. Ecol. 43: 721–734. http://cat.inist.fr/?aModele=afficheN&cpsidt=17909798 ISSN: 0021-8901

Monaco, A., Franzetti, B., Pedrotti, L., Toso, S., 2003. Linee guida per la gestione del cinghiale. Ministero Politiche Agricole e Forestali – I.N.F.S.

Morales, J.S.S., Rojas, R.M., Pérez-Rodríguez, F., Casas, A.A., López, M.A.A. 2011. Risk assessment of the lead intake by consumption of red deer and wild boar meat in Southern Spain. Food Addit Contam 28:1021-1033

Müller, E., Moser, G., Bartenschlager, H., Geldermann, H., 2000. Trait values of growth, carcass and meat quality in Wild Boar, Meishan and Pietrain pigs as well as their crossbred generations. J. Anim. Breed. Genet. 117:189-202.

Mysterud, A., Langvatn, R., Stenseth, N.C., 2004. Patterns of reproductive effort in male ungulates. J. Zool. Lond. 264:209-215. DOI:10.1017/S0952836904005618

Nahlik, A., Sandor, G., 2003. Birth rate and offspring survival in a free- ranging wild boar Sus scrofa population. Wildlife Biol. 9:37- 42.

Okabe, Y., Watanabe, A., Shingu, H., Kushibiki, S., Hodate, K., Ishida, M., Ikeda, S., Takeda, T. 2002. Effects of a-tocopherol level in raw venison on lipid oxidation and volatiles during storage Meat Science, 62, 457–462

Onyango, C.A., Izumimoto, M., Kutima, P.M., 1998. Comparison of some physical and chemical properties of selected game meats. Meat Sci., 49: 117-125. DOI:10.1016/S0309-1740(97)00116-2

Orusa, R., Robetto, S., Ferrari, N., Lo Valvo, T., Tarasco, R., Cerise, S., Abete, C., 2005. Contamination with cadmium and lead in wild boar (Sus scrofa) from areas with different anthropogenic impact. Proc 27th Congress of Game

Biologists. Hannover (Germany), pp 429.

Oshima, I., Iwamoto, H., Nakamura, Y.N., Takayama, K., Ono, Y., Murakami, T., Shiba, N., Tabata, S., and Nishimura, S. 2009. Comparative study of the histochemical properties, collagen content and architecture of the skeletal muscles of wild boar crossbred pigs and commercial hybrid pigs. Meat Science, 81, 382– 390.

Paulsen, P., Winkelmayer, R., 2004. Seasonal variation in the microbial contamination of game carcasses in an Austrian hunting area. Eur. J. Wildl. Res. 50: 157–159. DOI:10.1007/s10344-004-0054-z

Pedrotti, L., Dupré, E., Preatoni, D., Toso, S., 2001. Banca Dati Ungulati: status, distribuzione, consistenza, gestione, prelievo venatorio e potenzialità delle popolazioni di Ungulati in Italia. Biol. Cons. Fauna, 109: 1-132.

Perco, F., 1987. Ungulati. Carlo Lorenzini ed., Udine, Italy.

Polak T., Rajar, A., Gasperlin, L., Zlender, B., (2008). Cholesterol concentration and fatty acid profile of red deer (Cervus elaphus) meat Meat Science, 8, 864–869

Poli, B.M., Giorgetti, A., Campodoni, G., Parisi, G., Franci, O., 1993. Caratteristiche qualitative della carne di daini di diverse età. Proc. Parliamo di carnicomplementari, Fossano, Italy, 191-198.

Pollard, J.C., Littlejohn, R.P., Asher, G.W., Pearse, A.J.T., Stevenson-Barry JM, McGregor SK et al (2002) A comparison of biochemical and meat quality variables in red deer (Cervus elaphus) following either slaughter at pasture or killing at a deer slaughter plant. Meat Science, 60:85–94

Pompe-Gotal, J., Crniæ, A.P., 2002. Cadmium in tissues of roe deer (Capreolus capreolus) in Croatia. Veterinarsky Arhiv. 72: 303-310.

http://www.vef.hr/vetarhiv/papers/72‐6/pompe‐gotal.pdf

Primi, R., Pelorosso, R., Ripa, M.N., Amici, A., 2009. A statistical GIs-based analysis of Wild boar (Sus scrofa) traffic collisions in a Mediterranean area. Ital. J. Anim. Sci. 8, 649-651

Purchas, R.W., Triumf, E.C., Egelandsdal, B. 2010. Quality characteristics and composition of the longissimus muscle in the short-loin from male and female farmed red deer in New Zealand. Meat Science, 8: 505–510

Quaresma, M.A.G., Alves, S.A., Trigo-Rodrigues, I., Pereira-Silva, R., Santos, N., Lemos, J.P.C., Barreto, A.S., Bessa, R.J.B, 2011. Nutritional evaluation of the lipid fraction of feral wild boar (Sus scrofascrofa) meat. Meat Science. 89:457-461 DOI: 10.1016/j.meatsci.2011.05.005

Quaresma, M.A.G., Trigo-Rodrigues, I., Alves, S.P., Martins, S.I.V., Barreto, A.S., Bessa, R.J.B. 2012. Nutritiona l evaluation of the lipid fraction ofIber ian red deer (Cervus elaphus hispanicus) tenderloin. Meat Science, 92, 519-524.

Raes, K., De Smet, S., Demeyer, D. 2004.Effect of dietary fatty acids on incorporation of long chain polyunsaturated fatty acids and conjugated linoleic acid in lamb, beef and pork meat: A review Animal Feed Science and Technology, 11, 199–221.

Ramanzin, M., Amici, A., Casoli, C., Esposito, L., Lupi, P., Marsico, G., Mattiello, S., Olivieri, O., Ponzetta, M. P., Russo, C., TrabalzaMarinucci, M., 2010. Meat from wild ungulates: ensuring quality and hygiene of an increasing resource. Italian J. Anim. Sci. 9: 318-331 doi:10.4081/ijas.2010.e61

Ramanzin, M., Sommavilla, G., 2004. Piano Faunistico-Venatorio Provinciale. Aggiornamento 2003-2008. Amministrazione Provinciale di Belluno. Belluno, 96 pp.

Razmaitė, V, Švirmickas, G.J., Šiukščius, A., 2012. Effect of weight, sex and hunting period on fatty acid composition of intramuscular and subcutaneous fat

from wild boar. Italian J. Anim. Sci. 11:174-179 doi:10.4081/ijas.2012.e32

Reglero, M. M., Taggart, M. A., Monsalve-Gonzalez L., Mateo, R. 2009. Heavy metal exposure in large game from a lead mining area: Effects on oxidative stress and fatty acid composition in liver. Environ. Pollut. 157:1388-1395. DOI:10.1016/j.envpol.2008.11.036

Root, T.L., Price, J.T.,. Hall, K. R, Schneider, S.H., Rosenzweig, C. and Pounds, J.A. 2003. Fingerprints of global warming on wild animals and plants Nature 421, 57-60 doi:10.1038/nature01333

Rule, D.C., Broughton, K.S., Shellito, S.M., Maiorano, G.M., 2002. Comparison of beef fatty acid profiles and cholesterol concentrations of bison, beef, cattle, elk and chicken. J. Anim. Sci. 80: 1202-1211.

Saez-Royuela, C., Telleria, J.L., 1986. The increased population of wild boar (Sus scrofa L.). Europe. Mammal. Rev. 16: 97-101. DOI:10.1111/j.1365- 2907.1986.tb00027.x

Sales, J., Kotrba, R., 2013. Meat from wild boar (Sus scrofa L.): A review. Meat Sci. 94: 187–201 DOI: 10.1016/j.meatsci.2013.01.012

Salghetti, A., 1991. Elementi strutturali ed economici degli allevamenti di ungulati selvatici in Italia. Ann. Fac. Med. Vet. Parma 11: 87-156.

Sampels, S., Pickova, J., Wiklund, E., 2005. Influence of production system, age and sex on carcass parameters and some biochemical meat quality characteristics of reindeer. Rangifer, 25, 85–96

Santiago, D., Motas-Guzman, M., Reja, A., Maria-Mojica, P., Rodero, B., Garcia-Fernandez, A. J., 1998. Lead and cadmium in red deer and wild boar from Sierra Morena Mountains (Andalusia, Spain). Bull. Environ. Contam. Toxicol. 61: 730-737. DOI:10.1007/s001289900822

Scillitani, L., Monaco, A., Toso, S., 2010. Do intensive drive hunts affect wild

boar (Susscrofa) spatial behaviour in Italy? Some evidences and management implications. Eur J Wild Res 56: 307–318 Secchiari, P., Boselli, E., Serra, A., Mele, M., Savioli, S., Buccioni, A., Ferruzzi, G., Paoletti, F., 2001. Intramuscular fat quality of wild fallow deer (Damadama) meat. Prog. Nutr. 3 (4): 25-30.

Servanty, S., Gaillard, J.M., Toıgo, C., Brandt, S., Baubet, E., 2009. Pulsed resources and climate-induced variation in the reproductive traits of wild boar under high hunting pressure. Journal of Animal Ecology 78: 1278–12

Simopoulos, A.P., 2008. The importance of the omega-6/omega-3 fatty acid ratio in cardiovascular disease and other chronic diseases. Exp. Biol. Med. 233: 674-688 doi: 10.3181/0711-MR-311

Skewes, O., Morales, R., Mendoza, N., Smulders, F.J.M, Paulsen, P., 2009. Carcass and meat quality traits of wild boar (Sus scrofa s. L) with 2n=36 karyotype compared to those of phenotypically similar crossbreeds (2n=37 and 2n=38) raised under same farming conditions 2. Fatty acid profile and cholesterol. Meat Science. 83: 195-200 doi: 10.1016/j.meatsci.2009.04.017

Skewes, O., Morales, R., González, F., Lui, J., Hofbauer, P., Paulsen, P., 2008. Carcass and meat quality traits of wild boar (Sus scrofa s. L.) with 2n = 36 karyotype compared to those of phenotypically similar crossbreeds (2n = 37 and 2n = 38) raised under same farming conditions. 1. Carcass quantity and meat dressing. Meat Science. 80: 1200-1204. DOI:10.1016/j.meatsci.2008.05.015

Smith, R.F., Dobson, H., 1990. Effect of pre slaughter experience on behaviour, plasma cortisol and muscle pH in farmed red deer. Vet. Rec. 126,155- 158.

Soffiantini, C. S., Lazzini, C., Sabbioni, A., Zanon, A., Beretti, V., 2006. Curve di crescita della popolazione di caprioli della provincia di Massa Carrara. Ann. Fac. Med. Vet. Parma 26: 319-334.

http://www.unipr.it/arpa/facvet/annali/2006/319_334.pdf

Stevenson, J.M., Seman, D.L., Littlejohn, R.P., 1992. Seasonal variation in venison quality of mature, farmed red deer stags in New Zealand. J. Anim. Sci. 70: 1389–1396. http://jas.fass.org/cgi/content/abstract/70/5/1389

Stevenson-Barry, J.M., Duncan, S.J., Littlejohn, R.P., 1999. Venison vitamin E levels and he relationship between vitamin E, iron and copper levels and display life for venison and beef. Proc. 45th Int. Congr. Meat Sci. Technol., Yokohama, Japan, 458-459.

Summer, A., Sussi, C., Martuzzi, F., Catalano, A.L., 1997. Rilievi alla macellazione, prove di sezionamento e composizione chimica della carne di daino (Dama dama) e di cervo (Cervuselaphus). Ann. Fac. Med. Vet. Parma, 1-7.

Swiergosz, R., Perzanowski, K., Makosz, U., Bitek, I., 1993. The incidence of heavy metals and other toxic elements in big game tissues. Sci, Total Environ. Supplement: 225-231. DOI:10.1016/S0048-9697(05)80022-1

Toıgo, C., Servanty, S., Gaillard, J.M., Brandt, S. & Baubet, E., 2008. Survival patterns in an intensivelyhunted wild boar population: disentangling natural from hunting mortality. Journal of Wildlife Management , 72, 1532–1539.

Toso, S., 1999. Cervo Cervus elaphus Linneaus, 1758. In: Spagnesi M. and Toso S. (ed). Iconografia dei mammiferi d’Italia, Ministero dell’Ambiente – Istituto Nazionale per la Fauna Selvatica, Ozzano nell’Emilia (BO), pp 168-169.

Triumf, E.C., Purchas, R.W., Mielnik, M., Maehre, H.K., Elvevoll, E., Slinde, E., Egelandsdal, B. 2012. Composition and some quality characteristics of the longissimus muscle of reindeer in Norway compared to farmed New Zealand red deer. Meat Science, 90, 122–129

Tuckwell, C., 2003. The Deer Farming Handbook. Rural Industries Research and Development Corporation, Canberra. ISBN 0642585970

Ulbricht, T.L.V., Sauthgate, D.A.T., 1991. Coronary heart disease: Seven dietary factors. Lancet, 338: 985–991

Valencak, T.G., Tataruch, F., Steineck, T., Arnold, W. 2006. How healthy is game meat? Fatty acid composition, pollutants, and threat of zoonoses. Internistische Praxis. 46: 911-920.

Volpelli, L.A., Valusso, R., Morgante, M., Pittia, P., Piasentier, E., 2003. Meat quality in male fallow deer (Damadama): effects of age and supplementary feeding. Meat Science 65: 555-562 Volpelli, L.A., Valusso, R., Morgante, M., Piasentier, E., 2002. Produzione di carne di daino. Notiziario ERSA, 5: 47-50.

WHO World Health Organization, 2003. Diet, nutrition and the prevention of chronic diseases. Report of a Joint WHO/FAO Expert Consultation. WHO Technical Report Series n 916. ed. World Health Organization, Geneva, CH

Wiklund, E., Stevenson-Barry, J.M., Duncan, S.J., Littlejohn, R.P., 2001. Electrical stimulation of red deer (Cervus elaphus) carcasses – effects on rate of pH decline, meat tenderness, colour stability and water holding capacity. Meat Sci. 56: 211-220. DOI:10.1016/S0309-1740(01)00077-8

Wiklund, E., Manley, T.R., Littlejohn, R.P., Stevenson-Barry, J.M., 2003. Fatty acid composition and sensory quality of musculus longissimus and carcass parameters in red deer (Cervus elaphus) grazed on natural pasture or fed a commercial feed mixture. J. Sci. Food Agric. 83: 419-424. DOI:10.1002/jsfa.1384

Wiklund, E., Samples, S., Manley, T.R., Pickova, J., Littlejohn, R.P., 2006. Effects of feeding regimen and chilled storage on water-holding capacity, colour stability, pigment content and oxidation in red deer (Cervus elaphus) meat. J. Sci. Food Agric. 86: 98-106. ISSN: 0022-5142 CODEN JSFAAE

Wiklund, E., Dobbie, P., Stuart, A., Littlejohn R.P. 2010. Seasonal variation in red deer (Cervus elaphus) venison (M. longissimus dorsi) drip loss, calpain

activity, colour and tenderness. Meat Science, 86,720–727

Wood, J.D., Enser, M., Fisher, A.V., Nute, G.R., Sheard, P. R., Richardson, R. I., Hughes, S.I., Whittington, F.M, 2008. Fat deposition, fatty acid composition and meat quality: A review. Meat Science. 78: 343–358 doi: 10.1016/j.meatsci.2007.07.019

Żochowska, J., Lachowicz, K., Gajowiecki, L., Sobczak, M., Kotowicz, M., Zych, A., 2005. Effects of carcass weight and muscle on texture, structure and myofibre characteristics of wild boar meat. Meat Sci. 71: 244–248. DOI:10.1016/j.meatsci.2005.03.019

Zochowska-Kujawska, J., Lachowicz, K., Sobczak, M., Gajowiecki, L.,Kotowicz, M., Zych A., Medrala D. 2007. Effects of massaging on hardness, rheological properties, and structure of four wild boar muscles of different fibre type content and age. Meat Science, 75, 595–602. doi:10.1016/j.meatsci.2006.09.018

Żochowska-Kujawska, J., Lachowicz, K., Sobczak, M., 2012. Effects of fibre type and kefir, wine lemon, and pineapple marinades on texture and sensory properties of wild boar and deer longissimus muscle. Meat Science, 92, 675–680. doi:10.1016/j.meatsci.2012.06.020

Zomborszky, Z., Szentmihalyi, G., Sardi, I., Horn, P., Szabo, C.S., 1996. Nutrient composition of muscles in deer and boar. J. Food Sci. 61 (3): 625-627.

1. FIRST STUDY - SOME TRAITS OF FALLOW DEER AND WILD

BOAR MEAT AS AFFECTED BY HUNTING WITHDRAWAL -

Published in:

Amici, A., Meo Zilio, D., Ficco, A., Primi, R., Serrani, F., Failla, S., Contò, M. (2011). Some traits of fallow deer and wild boar meat as affected by hunting withdrawal: First results. in Proceeding of 57th I.Co.M.S.T. Ghent, Belgium, 148.

2. SECOND STUDY - EFFECTS OF THE HUNTING METHOD ON MEAT QUALITY FROM FALLOW DEER AND WILD BOAR AND PRELIMINARY STUDIES FOR PREDICTING LIPID OXIDATION USING VISIBLE REFLECTANCE SPECTRA.

Published in:

3. THIRD STUDY - HUNTING AREA AFFECTS CHEMICAL AND PHYSICAL CHARACTERISTICS AND FATTY ACID COMPOSITION OF WILD BOAR (SUS SCROFA) MEAT

Published in:

4. FOURTH STUDY - ORGANOLEPTIC AND NUTRITIONAL CHARACTERISTICS OF WILD BOAR MEAT AFFECTED BY COOKING TREATMENT

Manuscript in progress:

Failla, S., Cifuni, G. F., Contò, M., Amici A., Organoleptic and nutritional characteristics of wild boar meat affected by cooking treatment. (presented as poster in the 21st ASPA congress, Milano June 2015 “ Failla S., Cifuni G. F., Contò M., Viola P., Amici A. Effect of cooking on fatty acid composition, lipid oxidation and cholesterol content of hunted wild boar (Sus scrofa) meat. Proceeding 21st ASPA P012”)

Organoleptic and nutritional characteristics of wild boar meat affected by cooking treatment

Failla1*, S., Cifuni1, G. F., Contò1, M., Amici2 A.,

1Consiglio per la Ricerca in Agricoltura, CRA-PCM, Via Salaria 31, 0015 Monterotondo (RM), Italy 2Department of Agriculture, Forest, Nature and Energy-DAFNE, University of Tuscia, Via S. Camillo de Lellis, snc, 01100 Viterbo, Italy * Ph.D Sciences and Technologies for the Forest and Environmental Management University of Tuscia, Viterbo, Italy

Abstract

Meat composition, as well as its physicochemical properties, undergoes significant changes during cooking, affecting the final characteristics of meat products, mainly water loss and fatty acid content, and modifying the nutritional value of cooked products if compared to raw samples. The aim of this study was to evaluate the influence of grill treatment on physical quality, colour, fatty acid composition an lipid oxidation of longissimus thoracis from 10 wild boars, hunted in some areas of the Province of Rome. Results indicate that cooking induced a significant loss of water and, consequently, the cooked meat showed significantly higher protein, ash and fat contents than raw samples. The protein and myoglobin degradation, consequent to heat treatment, changed the colour parameters of meat, showing higher lightness (58.59 vs 38.94, P<0.001) and yellowness value (14.71 vs 11.67, P<0.001) and lower redness value (3.90 vs 6.94, P<0.01) than raw meat. Cooked meat showed a lower content of saturated (30.47 vs 32.13 %, P<0.01) and a higher level of polyunsaturated fatty acids compared to raw meat (27.02 vs 23.03 %, P<0.05). This is probably due to the prominent presence of saturated fatty acids in neutral lipids that are more prone to migration during cooking. From the

nutritional point of view, the cooking treatment improves wild boar meat fatty acid composition, but increase the lipid oxidation.

Key words: wild boar, cooked meat, cooking loss, fatty acid composition

Introduction

The use of wild ungulates as alternative source of meat has now a noticeable spreading in some European areas. Particularly wild boar (Sus scrofa) is the most common species hunted in the Centre of Italy where in the last decade shows an excessive numerical growth due to the scarce number of natural predators and to inadequate wildlife management (Ramanzin et al., 2010). Therefore the wild boar hunting is necessary, in order to decrease its density and impact on small game species and to decrease its economic impact on agriculture (Geisser et al., 2004; Hearn et al., 2014). Wild boar hunting provides a noticeable amount of meat with remarkable nutritional characteristics (Quaresma et al., 2011; Ramanzin et al., 2010), although the variability of meat quality of wild boar in wild conditions is considerable and it depends on many factors (Razmaite et al., 2012; Amici et al., 2011; 2015). In addition, large part of the meat is consumed cooked and this process further modifies its organoleptic characteristics and nutritional value, particularly due to the loss of a large amount of its mass in the form of meat juice and other nutritional compounds. Most of the water in the raw meat is held within the myofibrils and cooking induces structural changes and shrinkage because of denaturation of myofibrillar proteins and collagen (Tornberg, 2005). Moisture migrates from the inside of meat to extracellular void as combination of vapor and water due to pressure and concentration gradients generated by shrinkage. The main organoleptic changes caused by cooking are, time, temperature, chemical composition and position (core or crust regions) dependent. However, the basic effect of the heat treatment is the higher coagulation of meat proteins (Tornberg,

2005) and lipid oxidation (Broncano et al., 2009), that also induces some positive physical and chemical changes that improve taste, flavor, texture, safety and digestibility (van Boekel et al., 2010). A good cooking technique implies a limited shrinkage and water losses and the reaching of the internal temperature of 75°C avoiding the presence of excessive brown crust on meat surface, that cause the production of carcinogenic compounds (Sugimura et al., 2004). Several authors pointed out that the cooking process affects the lipid composition of meat, especially the fatty acid content and their oxidative stability, by changing the nutritional value of cooked products in relation to raw samples. However, there is a great variability in changes concerning individual fatty acid in response to different cooking methods (Badiani et al., 2002; Juárez et al., 2010). Recent studies have shown that fatty acid profile of wild boar meat may be beneficial from nutritional point of view, and in line with health recommendations (Dannenberger et al., 2013; Razmaite et al., 2012; Sales and Kotrba, 2013; Amici et al., 2015), but information concerning the effect of cooking treatment on nutritional characteristics and quality of wild boar meat are scarce, therefore the aim of this article was to study the influence of grilling treatment on the physical, chemical and lipid composition of wild boar hunted in Italy

Material and methods

Animals and sample collection

The animals were culled in the Province of Rome, in a flat zone close to the Tyrrhenian sea. Wild boars were hunted in wild conditions in compliance to Italian rules (Scillitani et al., 2010). Meat samples from 10 wild boar males (about 2 years old, estimated from tooth eruption as reported in Boitani and Mattei 1992) were collected between October and November, when the weather conditions were generally good (not

rainy) and without snow cover. All the animals were ear tagged after shooting and a format was filled with the most relevant hunting data. Each carcass was dissected and a portion of longissimus thoracis muscle, between the 2nd and the 6th rib, from left side was taken and stored for 4 days at + 2 °C, then was trimmed of subcutaneous fat and subdivided in two portions (for raw and grilled meat). For each portion, weight and pH were measured, the last one by probe insertion on samples, using a pHmeter Hanna HI98240 equipped with automatic temperature compensation, then the samples were frozen at -80 °C in vacuum packing.

Sample cooking and analysis

Individual meat samples, after 2 months of freezing, were thawed out at 4°C for a night and after slightly dried to the surface, weighed to obtain the percentage of thaw loss by difference in weight. Half of the samples were grilled at 150°C using an electrical griddle (Delonghi, Model CG660) until an internal temperature of 75 ± 2°C was reached. Temperature was controlled with thermocouple with the probe infixed in the middle of the sample (Delta OHM HD 8802). After the cooking process, the meat was cooled for 3 hours and weighed and percentage of cooking loss was calculated, than the samples were subdivided in two part one for physical analysis (colour and shear force) and one for chemical analysis (proximate composition, fatty acids and lipid oxidation). Colour components in CIEL*a*b* space (lightness L*, redness a* and yellowness b*, CIE, 1986) and visual reflectance spectra between 360-740 nm (by steps of 10 nm), using a MINOLTA CM 2600D spectrophotometer with aperture size 8mm, specular component included and D65 illuminant (D65-10°-d/8 SCI) were determined after 1 h of oxygen exposure for raw meat at 10°C. The Kubelka- Munk absorption and scattering coefficients ratio (K/S) were calculated to determine the percentage of deoxymyoglobin, oxymyoglobin and metmyoglobin by the equations suggested by AMSA (1991).

The shear force (WBS) was performed in four samples for each animal with a 1 x 1cm cross section and 2 cm of length, each sample was cut perpendicular to the longitudinal orientation of the muscle fibers with a Warner–Bratzler shear attachment (1 mm thick) using an INSTRON 5543 texturometer. A 50-kg compression load cell and a crosshead speed of 100 mm/min was used. The maximum load was expressed in Newtons (Chrystall et al., 1994). The proximate composition (dry matter, fat, protein and ash) was determined according to AOAC methods (1995) and expressed as percent of meat. True retention (TR) of this components was calculated to evaluate the true compositional changes during cooking according to Murphy et al., (1975) using the following equation:

The lipids were extracted according to the procedure of Folch et al., (1957). Duplicates of lipid extract (10 ml) that corresponded to 100 mg of lipids were methylated by adding 1 ml of n-hexane and 0.05 ml of 2 N methanolic KOH, according to IUPAC procedure (1992). The transmethylation was achieved in 5 min at room temperature. Gas chromatographic analysis was performed on a GC 6890N (Agilent, Inc., California, USA) instrument. A fused silica capillary column coated with 100% cyanopropyl polysiloxane (Supelco, 2560; 100 m, 0.25 mm (i.d.), 0.25 m film thickness) was used to analyse the methylated fatty acid content. Operating conditions were: a helium ﬂow rate of 1 ml/min, an FID detector at 300°C, a split– splitless injector at 250 °C, an injection volume of 1µl. The temperature programme of the column was: 4 min at 140 °C and a subsequent increase to 220 °C at 4 °C/min and then held at 220 °C for 10 min. The individual fatty acid peaks were identified by comparing the relative retention times of FAME (Fatty Acid Methyl Esters) peaks of the samples with fatty

acid methyl ester standards from Sigma (USA) by spiking samples with standards. The fatty acids were expressed as a percent of the total methylated fatty acids. Lipid oxidation was measured on raw and cooked meat by the thiobarbituric acid-reactive substances (TBARS) method according to Bergamo et al., (1998). The samples were homogenized in water supplemented with butylated hydroxytoluene. Proteins were precipitated with ice-cold 10% trichloroacetic acid and removed by centrifugation. The supernatant was incubated, at 90°C for 30 min, in a 0.28% thiobarbituric acid (TBA) mixture. The TBARS adduct was fractionated by reverse phase HPLC and detected by fluorescence (λEX = 515 nm; λEM = 543 nm). Elution was performed at a 1 mL/min flow rate with a mixture of acetonitrile and sodium phosphate at pH 7 (15:85 v/v). The results were expressed as mg of malondialdehyde (MDA) per kg of meat.

Statistical analysis

Data were analyzed by ANOVA using SAS procedure (SAS, 2002) with heat treatment as factor. Mean values were compared by Duncan’s test, and the P-level for significance was 0.05.

Results and Discussion

Physical characteristics and myoglobin pigments

None of the meat samples showed anomalous pH values (5.61 ±0.05 average s.d.). The percentage of water loss in raw meat (Table 1) was resulting from the losses during thaw of samples, this value is usually higher compared to the water lost for dripping, because the freezing action reduce the protein capacity to held water (Leygonie et al., 2012), however water loss recorded in raw meat was quite low if compared to a previous work of Cifuni et al., (2014).

Cooking loss percentage was similar to values reported by Marsico et al., (2007) and Cifuni et al., (2014).

Table 1. Physical characteristics and myoglobin pigments on raw and cooked longissimus thoracis of wild boar .

Raw meat Grillied meat Root M.S.E Significance Water loss (%) 3.30(1) 30.52 1.643 *** WBS (N) 42.62 41.36 3.708 Ns Lightness 38.94 58.59 2.862 *** Redness (a*) 6.94 3.90 2.092 ** Yellowness(b*) 11.67 14.71 0.873 *** Deoximyoglobin % 26.65 25.37 2.489 Ns Oxymyoglobin % 50.40 38.45 2.841 *** Metmyoglobin % 22.95 36.18 0.684 *** * P<0.05; ** P<0.01; *** P<0.001; ns = not significant. NOTE 1= water loss in raw meat is obtained as thaw loss

The water loss is definitely dependent on characteristics of raw meat in terms of type and size of muscle fibers, presence of marbling, amount of soluble collagen and total moisture, considerable as intrinsic factors but also depends on technological factors as conservation, that produces protein degradation and consequently decrease in ionic strength, and cooking process. The maximum load (WBS) showed no differences between cooked and raw samples, probably because the meat has not undergone a marked shrinkage of myofibrils. Similar values were reported by Sales and Kotrba (2013) for animals of 60 kg live weight, and by Żmijewski, and Korzeniowski (2001) for longissimus muscle stored only 24 hours. Nevertheless, parameters such as cooking loss, water holding capacity, Warner Bratzler shear force are expression of the same phenomenon, and they result

correlated, each one underlining a partial contribution of definition of qualitative characteristics of meat (Hughes et al., 2014). High losses of water in fact could imply less juiciness and therefore loss of quality. The cooked meat was more bright (higher L*, P <0.001) and yellow (higher b*; P<0.001), and less red (lower a*, P<0.01) than raw samples (Table 1). Lightness and yellow index showed the largest increases with cooking (+33.5%,+43.8% respectively) according to Pakula and Stamminger, (2012), that consider most dominant changes in surface of the cooked meat occur in the lightness. The colour parameters in raw meat were similar to those found in a previous evidence by Amici et al., (2015), but showed lower values in L* and a*index if compared with the data of Marchiori and de Felício (2003) and Marsico et al., (2007). No data was found in literature on the colour of wild boar cooked meat. The meat of wild boar has an intensive red colour, if compared with domestic pig meat due to an higher concentration of myoglobin as a result of their intense physical activity (Szmańko et al., 2007). After cooking several changes both in the appearance and physical properties of meat occur due heating process. These changes include discoloration of the meat from deep red to light brown colour, due to denaturation of myoglobin pigments as reported by other authors for other species (Liu et al., 2000; Kondjoyan et al., 2014; Hughes et al., 2014). Cooking led to a reduction of the oximyoglobin (P<0.01; reddish loss) and an increase of metmyoglobin (P<0.001; brownish-red) in meat samples. Myoglobin pigments are greatly reduced with cooking, forming other compounds (Swatland, 1995). Considering the percentage of the three myoglobin components, no evident variation was noted in deoximyoglobin percentage, while substantial loss in oxymyoglobin (P<0.001) and increase of denaturated metmyoglobin percentage (p<0.001) were found as reported in Mancini and Hunt (2005).

Figure 1- Reflectance spectrum on visible (360-740nm wavelengths) on raw and cooked longissimus thoracis of wild boar.

The spectral features in the visible region (Figure 1) showed the same changes due to cooking process, when the colour of wild boar meat changed from red to grey-brown. The reflectance curve on visible for the cooked meat showed high values in all wavelengths compared to raw meat curve. Shrinkage of myofibrils in combination with protein denaturation, which creates a more dense protein structure, increases light scattering (Pakula and Stamminger, 2012). Light scattering is the process in which light is diffused or reflected by collisions with particles of the medium, that in meat samples is represented by myofibril, connective tissue, fat, water, and it is influenced by their structural attributes. The reflectance values went up after 650 nm for sulf-myoglobin accumulation due to cooking (Swatland, 1995), furthermore the myoglobin and oxymyoglobin peaks around 540-580 nm disappeared, demonstrating the denaturation of these pigments with heat. Similar pattern of reflectance is reported by García -Segovia, (2007) for beef meat cooked at 80°C.

This lightness in cooked meat is also closely related to the pH and WHC of raw meat, because this parameters drive structural changes such as protein denaturation and aggregation and myofibrillar shrinkage (Kondjoyan et al., 2014; Hughes et al., 2014). Colour metric distance between raw (r ) and cooked meat (c) as proposes by

* CIEL*a*b* (ΔE ab= ) confirmed the assumption that the increase in lightness and b index is dominant, showing an high * value of ΔE ab =20.1.

Proximate composition and chemical characteristics

Cooking led to a significant loss of moisture (Table 2) and, consequently, the cooked meat showed significantly higher protein (P<0.001), ash (P<0.001) and fat (P<0.01) contents than raw samples. Moreover, the decrease in the moisture content has been described as the most prominent change and subsequent increase of the other traits (protein, ash and fat contents) were related to that (García-Arias et al., 2003). In fact, higher water loss determined higher increases in the rest of components (Juárez et al., 2010). Cooking influences the content of several nutrients in meat in different ways depending on the cut and the cooking process. To determine how much of the respective nutrients were gained or lost, to compare the contents in absolute concentration became important, taking into account the concentration effect caused by water loss during cooking (Gerber et al., 2009). Cooked meat showed higher percentage of fat compared to raw samples, but considering the true retention of nutrients (TR %=90.5) the meat during cooking lost fat as reported in Badiani et al. (2002). Therefore, considering the TR% cooked meat increased the nutritional characteristic, in particular for fat loss and protein retention (TR% = 109.5 for protein ), probably due to low solubilisation of collagen. On the contrary the meat lost the ash (TR%=96.4 ), because its high solubility on free water of meat dripped during cooking. Hence retention of nutrients in the

samples varies with the nutrient type, the nature of food and the heating methods used (Jensen et al., 2014).

Table 2. Proximate composition (g/100g meat) on raw and cooked meat .

Raw meat Grilled meat Root M.S.E Significance TR%(1) Moisture 73.78 61.55 1.214 *** Protein 22.54 33.54 1.340 *** 109.5±8.39 Ash 1.37 1.90 0.116 *** 96.4±6.71 Fat 2.31 3.01 0.528 ** 90.5±15.6 * P<0.05; ** P<0.01; *** P<0.001; ns = not significant. Note 1- TR%=True retention (%) of protein, lipid, ash after cooking of longissimus thoracis muscle (values are expressed as mean±standard error) Murphy et al.,(1975)

Quaresma et al., (2011) and Ramanzin et al., (2010) found in wild boar meat a large variation in fat content values. One reason for this wide variability could be the feral conditions of the wild boar, including diet, age, sex and season. Dannenberger et al., (2013) and Sales and Kotrba (2013) showed in raw meat lower fat percentage between 1.2, 1.9 % in animals older than 1 year if compared to our data.

Effect of cooking on fatty acid composition and lipid oxidation

Similarly to other monogastric animals, fatty acid composition of wild boar meat depends largely on the diet provided (Sales and Kotrba 2013). The fatty acid composition on raw meat (Table 3) were within the range reported for wild boar meat in Quaresma et al., 2011, for domesticated wild boar meat in Skewes et al., (2009) and for pork reared on pasture under organic production in Oksbjerg, et al., (2005).

The major saturated fatty acids (SFA) in wild boar were palmitic (16:0) and stearic (18:0), the principal monoinsatured (MUFA) was oleic (18:1 cis-9) and for polynsatured (PUFA) was linoleic (18:2n-6), all these were 82.43 g/100g of total fatty acids detected in raw meat. The PUFA with high nutritional value as conjugated linoleic acid (CLA, 18:2 cis-9,trans-11 isomer), linolenic fatty acid (18:3n-3), eicosapentaenoic acid EPA (20:5n-3) and docasahexaenoic acid DHA (22:6n-3) were present only in traces (2.0 g/100gof fatty acids%). The presence of CLA as in other monogastric animals depend of diet. In our work the n-6/n-3 ratio in raw meat was 2 times higher than the value recommended by British Department of Health, (1994), because of the low presence of n-3 fatty acids, however our result was similar to that obtained by Marsico et al., (2007); Oksbjerg et al., (2005) and Dannenberger et al., (2013) and lower that the value reported in Quaresma et al., (2011). The high P/S ratio (0.72) was more than 0.45, minimum value recommended by the British Department of Health (1994), the low atherogenic and thrombogenic indexes found in this meat, allow to consider this meat as product with valuable dietary characteristics (Sales and Kotrba 2013). The good composition of fatty acid of longissimus thoracis improved further with grilling. Several authors pointed out that the cooking process can affect the lipid composition of meat, especially the fatty acid content, by changing the nutritional value of cooked products in relation to raw samples (Badiani et al., 2002). Cooked meat (Table 3) showed lower content of saturated fatty acids (P<0.01) and higher level of polyunsaturated fatty acids compared to raw meat (P<0.05), no significantly differences were obtained in monoinsatured fatty acids. Concerning saturated fatty acids, the content of myristic (C14:0) and palmitic (C16:0) decreased in cooked meat (P<0.05, P<0.01, respectively).

Table 3. Fatty acid composition (g/100g FAME) on raw and cooked longissimus thoracis of wild boar

Fatty acids Raw meat Grilled meat Root M.S.E Signific. C14:0 0.93 0.83 0.093 * C16:0 20.13 18.39 0.601 ** C16:1 2.75 2.73 0.583 Ns C18:0 10.38 10.08 0.804 Ns C18:1trans11 0.26 0.24 0.078 Ns C18:1cis9 36.82 33.11 3.474 * C18:1n7 4.40 5.71 0.937 * C18:2n6 15.10 17.46 1.958 * C18:3n3 0.99 0.86 0.229 Ns CLA 0.051 0.048 0.010 Ns C20:0 0.14 0.13 0.020 Ns C20:1 0.45 0.58 0.251 Ns C20:3n6 0.38 0.45 0.137 Ns C20:3n3 0.19 0.14 0.043 * C20:4n6 4.41 5.83 1.278 * C20:5n3EPA 0.21 0.31 0.065 ** C22:4n6 0.031 0.029 0.008 Ns C22:5n3DPA 0.034 0.043 0.022 Ns C22:6n3DHA 0.75 0.90 0.234 Ns SFA 32.13 30.47 1.045 ** MUFA 44.84 42.51 3.318 Ns PUFA 23.03 27.02 3.181 * P/S 0.72 0.89 0.102 ** n-3 2.53 2.59 0.283 Ns n-6 20.41 24.34 3.021 * n6/n3 8.07 9.44 1.137 * AI index 0.35 0.32 0.014 ** TI index 0.78 0.72 0.035 ** TBARS (MDA 1.17 2.73 0.368 *** mg/kg) * P<0.05; ** P<0.01; *** P<0.001; ns = not significant. In table the following fatty acids were not reported because present as traces: C12:0;C10:0; C14:1; C15:0; C15:1; C18:2trans9 cis11; C18:2 trans9 cis12; C18:3n6; C21:0; C20:2n6; C22:0; C22:4n6. P/S=PUFA/SFA, Atherogenic index (AI), and thrombogenic index (TI) were calculated according to Ulbricht and Southgate (1991) as follows: AI=(C12:0+4×C14:0+C16:0)/[ΣMUFA+Σ(n‐6)+(Σ(n‐3)] TI=(C14:0+C16:0+C18:0)/[0.5×ΣMUFA+0.5×Σ(n‐6)+3×Σ(n‐3)+Σ(n‐3)/Σ(n‐6)].

For most polyunsaturated fatty acids, their level did not significantly changed after cooking. However, the level of arachidonic acid (C20:4n-6) and eicosapentaenoic acid (C20:5n-3) increased (P<0.05 and P<0.01 respectively) in cooked meat than raw samples. The proportions of n-6 fatty acids (P<0.05) and the n6/n3 ratio also increased (P<0.05) after cooking. Grilled meat showed higher P/S ratio (P<0.01) and lower atherogenic and thrombogenic index (P<0.001 for both) Variations in the fatty acid composition of cooked samples similar to our results have already been reported by Alfaia et al., (2010), who observed significant differences in the fatty acid profile of beef meat in response to cooking methods. Several mechanisms that occur during cooking, such as water loss and lipid oxidation, diffusion and exchange, can lead to relative changes in some fatty acids (Rodriguez-Estrada et al., 1997). Reducing the intake of SFA and increasing the intake of n-3 PUFA, eicosapentaenoic acid (EPA; C20:5 n-3) and docasahexaenoic acid (DHA; C22:6 n- 3) have been demonstrated to have important roles in reducing the risk of cardiovascular disease (Calder, 2004). The oxidation of lipids is one of the most important changes during food storage and processing. It depends on the polyunsaturated fatty acid content, as well on the balance between anti- and pro-oxidant compounds (Nuernberg et al., 2006). TBARS content is commonly evaluated to determine lipid oxidation which is related to meat quality deterioration and to the formation of potentially toxic compounds as malondialdehyde, that have been shown to be linked to the development of degenerative diseases (Boyd and McGuire, 1991), but also cooking method, rate and final temperature of treatment affects the rate of lipid oxidation. The increase in the TBARS values during grilling (P<0.001) was consistent with the result reported by Conchillo et al., (2003); Hernández et al., (1999). An explanation for the high values of TBARS in our samples could be the fact that meat from wild boars contains higher proportion of polyunsaturated fatty acids than other meats and these compounds have lower melting points, and hence are

more susceptible to oxidation than MUFA and SFA. High unsaturation index in meat may affect its oxidative stability. However, several authors observed that some cooking methods did not produce this phenomena, probably due to positive time- temperature interaction (Alfaia et al., 2010).

Conclusion

The grilling treatment changes the organoleptic composition of wild boar meat and from the point of view of nutritional value, it improves the chemical and nutritional composition, in particular regarding the composition in fatty acids. The grilling treatment reduced the saturated myristic and palmitic fatty acid contents responsible of different cardiovascular disease and increased the levels of eicosapentaenoic acid, that has an important role in its prevention. Unfortunately the cooking did not affected the levels n-6/n-3 ratio, but improved other nutritional fatty acid ratios, showing a positive effect on PUFA/SFA ratio compared to raw meat and improved the atherogenic and thrombogenic indexes. On the contrary, the cooking procedure due to high temperature increased the formation of malondialdehyde, a compound obtained from lipid oxidation.

References

Alfaia, C.M.M., Alves, S.P., Lopes, A.F., Fernandes, M.J.E.,Costa, A.S.H., Castro, M.L.F., Bessa, R.J.B., Prates, J.A.M. (2010). Effect of cooking methods on fatty acids, conjugated isomers of linoleic acid and nutritional quality of beef intramuscular fat. Meat Science, 84, 769-777. doi:10.1016/j.meatsci.2009.11.014 Amici, A., Meo Zilio, D., Ficco, A., Primi, R., Serrani, F., Failla, S., Contò, M. (2011). Some traits of fallow deer and wild boar meat as affected by hunting withdrawal: First results. in Proceeding of 57th I.Co.M.S.T., 148.

Amici, A., Cifuni, G. F., Contò, M ., Esposito, L., Failla, S. (2015). Hunting area affects chemical and physical characteristics and fatty acid composition of wild boar (Sus scrofa) meat. Rendiconti Lincei. Scienze Fisiche e Naturali. doi 10.1007/s12210-015-0412-7 AMSA (1991). Guidelines for meat color evaluation. In: Reciprocal Meats Conference Manhattan, KS, USA pp. 1−17 Association of Official Analytical Chemists (AOAC), (1995). Official methods of analysis 16 th edn. AOAC, Arlington. Badiani, A., Stipa, S., Bitossi, F., Gatta, P. P., Vignola, G., and Chizzolini, R. (2002). Lipid composition, retention and oxidation in fresh and completely trimmed beef muscles as affected by common culinary practices. Meat Science, 60, 169−186. doi:10.1016/S0309-1740(01)00119-X Bergamo, P., Fedele, E., Balestrieri, M., Abrescia, P., Ferrara, L. (1998). Measurement of malondialdehyde levels in food by High-Performance Liquid Chromatography with fluorometric detection. Journal of Agricultural Food Chemistry 46, 2171–2176. doi: 10.1021/jf9709919 Boitani, L., Mattei, L., (1992). Aging wild boar by toot eruption. Pp 419-421 in Proceeding International Conference of Ongulés/Ungulates, Paris-Toulouse, France Boyd, N. F., and McGuire, V. (1991). The possible role of lipid peroxidation in breast cancer risk. Free Radical Biology and Medicine, 10 (3–4), 185–190. British Department of Health (1994). Nutritional aspects of cardiovascular diseases. Report on health and social subjects n°46. London: H.M. Stationery Office. Broncano, J. M. Petrón, M.J. Parra, V., Timón M.L. (2009). Effect of different cooking methods on lipid oxidation and formation of free cholesterol oxidation products (COPs) in Latissimus dorsi muscle of Iberian pigs. Meat Science, 83, 431-437. doi:10.1016/j.meatsci.2009.06.021 Calder, P. C. (2004). N−3 fatty acid and cardiovascular disease evidence explained and mechanisms explored. Clinical Science, 107, 1–11.

Chrystall, B.B, Culioli, J, Demeyer, D, Honikel, KO, Moller, AJ, Purslow, et al. (1994) Recommendation of reference methods for assessment of meat tenderness. In Proceedings of the 40th I.Co.M.S.T , S-V06. CIELab.(1986).Colorimetry: Official recommendations of the international commission on illumination. CIE Publ. No. 15.2. CIE Central Bureau,Vienna. Cifuni, G.F., Amici, A., Contò, M., Viola, V., Failla, S. (2014). Effects of the hunting method on meat quality from fallow deer and wild boar and preliminary studies for predicting lipid oxidation using visible reflectance spectra. The European Journal of Wildlife Research, 60, 519–526. doi 10.1007/s10344-014- 0814-3 Cobos, A., Veiga, A. and Díaz, O. (2008). Chemical and lipid composition of deboned pieces of dry-cured pork forelegs as affected by desalting and boiling: the effects of vacuum packaging. Food Chemistry, 106, 951–956. Conchillo, A., Ansorena, D., Astiasarán, I. (2003). Combined effect of cooking (grilling and roasting) and chilling storage (with and without air) on lipid and cholesterol oxidation in chicken breast. Journal of Food Protection, 66, 840–846. Dannenberger, D., Nuernberg, G., Nuernberg, K., Hagemann, E. (2013). The effects of gender, age and region on macro- and micronutrient contents and fatty acid profiles in the muscles of roe deer and wild boar in Mecklenburg-Western Pomerania (Germany). Meat Science, 94, 39–46. doi:10.1016/j.meatsci.2012.12.010 Duckett, S.K. and Wagner, D.G., (1998). Effect of cooking on the fatty acid composition of beef intramuscular lipid. Journal of Food Composition and Analysis, 11, 357–362. Enser, M., Hallet, K., Hewitt, B., Fursey, G.A.J. Wood, J.D. (1996). Fatty acid content and composition of English beef, lamb and pork at retail. Meat Science, 42, 443–456. doi:10.1016/0309-1740(95)00037-2 Folch, J., Lees, M., Stanley, G.H.S. (1957). A simple method for the isolation and purification of lipids from animal tissue. Journal of Biological Chemistry, 226, 497-509.

Geisser, H., Reyer, H., Krausman, U. (2004). Efficacy of hunting, feeding and fencing to reduce crop damage by wild boars. The Journal of Wildelife Management., 68, 939–946. doi:http://dx.doi.org/10.2193/0022- 541X(2004)068[0939:EOHFAF]2.0.CO;2 Gerber, N.,. Scheeder, M.R.L, Wenk, C. (2009). The influence of cooking and fat trimming on the actual nutrient intake from meat. Meat Science 81 , 148–154. doi:10.1016/j.meatsci.2008.07.012 García-Segovia, P., Andrés-Bello, A., and Martínez-Monzó, J. (2007). Effect of cooking method on mechanical properties, color and structure of beef muscle (M. pectoralis). Journal of Food Engineering, 80, 813–821. doi:10.1016/j.jfoodeng.2006.07.010 Hearn, R., Watkins, C., Balzaretti, R. (2014). The cultural and land use implications of the reappearance of the wild boar in North West Italy: A case study of the Val di Vara. Journal of Rural Studies, 36, 52-63. doi:10.1016/j.jrurstud.2014.06.004 Hernández, P., Navarro, J. L., Toldrá, F. (1999). Lipids of pork meat as affected by various cooking techniques. Food Science and Technology International, 5, 501– 508. Hughes, J.M., Oiseth, S.K., Purslow P.P., Warner, R. D. (2014). A structural approach to understanding the interactions between colour, water-holding capacity and tenderness. Meat Science, 98, 520–532. doi:10.1016/j.meatsci.2014.05.022 IUPAC International Union of Pure and Applied Chemistry, (1992). Standard Methods for the Analysis of Oils, Fats and Derivatives. ed. Blackwell Scientific Publications, Oxford, UK. Jensen, I.J., Dort, J., Eilertsen, K.E., (2014). Proximate composition, antihypertensive and antioxidative properties of the semimembranosus muscle from pork and beef after cooking and in vitro digestion. Meat Science, 96, 916– 921. doi:10.1016/j.meatsci.2013.10.014

Juárez, M, Failla, S., Ficco, A., Pena, F., Avilés, C., Polvillo O. (2010). Buffalo meat composition as affected by different cooking methods. Food and Bioproducts Processing, 88, 145–148. doi:10.1016/j.fbp.2009.05.001 Kondjoyan, A., Kohler, A., Realini, C.E., Portanguen. S., Kowalski, R., Clerjon, S., Gatellier, P., Chevolleau, S., Bonny, J.M., Debrauwer, R. (2014). Towards models for the prediction of beef meat quality during cooking. Meat Science, 97, 323–331. doi:10.1016/j.meatsci.2013.07.032 Leygonie, C., Britz, T. J., Hoffman, L.C., (2012). Impact of freezing and thawing on the quality of meat: Review. Meat Science, 91, 93–98. doi:10.1016/j.meatsci.2012.01.013 Liu, Y., Chen, Y. R., Ozaki, Y. (2000). Characterization of visible spectral intensity variations of wholesome and unwholesome chicken meats with two-dimensional correlation spectroscopy. Applied Spectroscopy, 54, 587–594. Mancini, R. A., Hunt, M.C. (2005). Current research in meat color. Meat Science, 71, 100-121. doi:10.1016/j.meatsci.2005.03.003 Marchiori, A. F., and de Felício, P. E. (2003). Quality of wild boar meat and commercial pork. Scientia Agricola, 60, 1–5. Marsico, G., Forcelli, M.G., Tarricone, S., Rasulo, A., Pinto, F., Celi, R., Cagnetta, P. (2007). Quality of the meat of wild and raised wild boar, Progress in Nutrition, 9, 248-252. Murphy, E.W., Criner, P. E., Gray, B. C. (1975). Comparisons of methods for calculating retentions of nutrients in cooked foods. Journal of Agricultural and Food Chemistry, 23, 1153–1157. doi:10.1021/jf60202a021 Nuernberg, K., Kuechenmeister, U., Nuernberg, G., Hartung, M., Dannenberger, D., Ender, K. (2006). Effect of storage and grilling on fatty acids in muscle of pigs fed plant oils. European Journal of Lipid Science and Technology, 108, 554–560. Oksbjerg, N., Strudsholm, K., Kinnndahl, G., Hermansen, J. E. (2005) Meat quality of fully or partly outdoor reared pigs in organic production. Acta Agric. Scand. Section A, 55, 106–112. doi:10.1080/09064700510009324

Pakula, C., and Stamminger, R. (2012). Measuring changes in internal meat colour, colour lightness and colour opacity as predictors of cooking time. Meat Science, 90, 721–727. doi:10.1016/j.meatsci.2011.11.002 Quaresma, M. A. G., Alves, S. P., Trigo-Rodrigues, I., Pereira-Silva, R., Santos,N., Lemos, J. P. C., Barreto, A. S., and Bessa, R. J. B. (2011). Nutritional evaluation of the lipid fraction of feral wild boar (Sus scrofa scrofa) meat. Meat Science, 89, 457–461. doi:10.1016/j.meatsci.2011.05.005 Ramanzin, M., Amici, A., Casoli, C., Esposito, L., Lupi, P., Marsico, G., Mattiello, S., Olivieri, O., Ponzetta, M.P., Russo, C., Trabalza Marinucci, M., (2010). Meat from wild ungulates: ensuring quality and hygiene of an increasing resource. Italian Journal of Animal Science, 9, 318 –331 doi:10.4081/ijas.2010.e61 Razmaite, V., Švirmickas, G. J., Šiukščius, A., (2012). Effect of weight, sex and hunting period on fatty acid composition of intramuscular and subcutaneous fat from wild boar. Italian Journal of Animal Science, 11, 32-37. doi:10.4081/ijas.2012.e32 Rodriguez-Estrada, M. T., Penazzi, G., Caboni, M. F., Bertacco, G., and Lercker, G. (1997). Effect of different cooking methods on some lipid and protein components of hamburgers. Meat Science, 45, 365–375. Sales, J., Kotrba, R. (2013). Meat from wild boar (Sus scrofa L.): A review. Meat Science, 94, 187-201. doi:10.1016/j.meatsci.2013.01.012 SAS Institute, 2002. SAS and STAT User's Guide, Release 91 SAS Institute Inc, Cary, NC. Scheeder, M.R.L., Casutt, M.M., Roulin, M., Escher, F., Dufey, P.A. and Kreuzer, M. (2001). Fatty acid composition, cooking loss and texture of beef patties from meat of bulls fed different fats. Meat Science, 58, 321–328. doi:10.1016/S0309- 1740(01)00037-7 Scillitani, L, Monaco, A, Toso, S. (2010). Do intensive drive hunts affect wild boar (Sus scrofa) spatial behaviour in Italy? Some evidences and management implications. European Journal Wildlife Research, 56, 307–318. doi:10.1007/s10344-009-0314-z

Skewes, O., Morales, R., Mendoza, N., Smulders, F.J.M., Paulsen, P. (2009). Carcass and meat quality traits of wild boar (Sus scrofa s. L.) with 2n=36 karyotype compared to those of phenotypically similar crossbreeds (2n=37 and 2n=38) raised under the same farming conditions 2: Fatty acid profile and cholesterol. Meat Science, 83, 195-200. doi:10.1016/j.meatsci.2009.04.017 Sugimura, T, Wakabayashi, K, Nakagama, H, Nagao, M. (2004). Heterocyclic amines: Mutagens/carcinogens produced during cooking of meat and fish. CANCER SCIENC, 95, 290–299. doi:10.1111/j.1349-7006.2004.tb03205.x Szmańko, T., Górecka, J., Korzeniowska, M., Malicki, A., Eeremenko, E. (2007). Comparison of chosen quality parameters of meat from wild boar and domestic pigs. Polish Journal of Food and Nutrition Sciences, 57, 523–528. Swatland, H., (1995). On-line evaluation of meat Ed. Thechnomic Basel 185-206. Tornberg, E. (2005). Effects of heat on meat proteins – Implication s on structure and quality of meat products Review Article. Meat Science,70, 493-508. doi:10.1016/j.meatsci.2004.11.021 Ulbricht, T.L.V, Sauthgate, D.A.T. (1991). Coronary heart disease: seven dietary factors. Lancet, 338, 985–991 van Boekel, M., Fogliano, V., Pellegrini, N., Stanton, C., Scholz, G., Lalljie, S., Somoza, V., Knorr, D., Jasti, P.R., Eisenbrand, G. (2010). A review on the beneficial aspects of food processing. Molecular Nutrition and Food Research, 54, 1215–1247. doi:10.1002/mnfr.200900608 Żmijewski, T. , Korzeniowski, W. (2001). Thechnological properties of wild boars meat. Electronic Journal of Polish Agricultural Universities. 4: 2. Available Online: http://www.ejpau.media.pl/volume4/issue2/food/art-02.html

5. GENERAL CONCLUSIONS

The increased abundance of some ungulates in many areas of Central Italy cause conflicts with human activities and biodiversity conservation. In particular, wild boar, due to its widespread presence, abundance, its ecological plasticity, and its opportunistic use of different foods, is believed to be the species most responsible for the economic damage to the agro-livestock-forest sector. Therefore, the hunting removal of these animals has become an ecological and economical and environmental need. Meat obtained from wild animals presents a high variability of organoleptic and chemical parameters due to several factors. These factors are both intrinsic to the animal as sex, age and so on, and extrinsic, related to the season, the hunting area, but also the storage system of carcass before the transport to the slaughterhouse, chilling storage, aging, conservation and cooking. In any case, the consumption of meat of wild ungulates is still closely connected with the maintenance of high standards of quality, both for chemical composition and sensory characteristics, and to guarantee the hygiene of the product. Some sources of variability, able to influence this process, have been considered in our studies. These studies provided important information, but still not exhaustive due to the low number of animals considered. No significant differences emerged from comparison between the age for proximate composition and physical quality, both for wild boar and fallow deer. While for gender, females showed less moisture and more fat if compared to males. The largest percentage of fat also affects the higher meat tenderness in female. The meat chilling for a longer time shows a meat tending to be more tender, in particular, for follow deer meat, in addition, post mortem treatment deeply influenced meat water losses in both species.

Hunting technique shows to have a direct and meaningful influence on colour parameters. In fact, L* and b* values resulted to be higher in wild boars and fallow deer harvested using dog drive hunting technique. Hunted animals in general, and in particular those hunted with dogs, undergo considerable stress. Our study confirmed the important effects that pre-slaughter stress has on meat quality and lipid stability, which results in fluid loss, darkening of the meat and higher hardness. From the multivariate regression between the spectrum of colour and lipid oxidation observed in meat we confirm the interconnections between these two variables. Therefore, the use of the spectrum of reflectance in the visible (in particular as regards the 500-550 spectral region indicative of presence of oxymyoglobin and between 630 and 740 nm, indicative of the presence of metmyoglobin and other pigments oxidized) allows us to discriminate animals subjected to excessive stress whose meat has a strong lipid and myoglobin oxidation.

In conclusion, we believe that hunting without driving dogs reduces the pre- slaughter stress and improves the quality of meat from wild ungulates.

The considerable differences in land use of the three areas considered in our studies justify the organoleptic and nutritional differences of wild boar meat. In particular, the percentage of cultivations adjacent to the hunting areas, in which the wild boar occasionally feed, represents the main source of variability of qualitative characteristics of meat. As expected, characteristics of wild animal meat showed very high variability due to the environmental factors. Nevertheless, colour parameters, cooking loss, dry matter, protein content and fatty acid profile of meat showed some differences in relation to hunting area as a probable effect of feeds availability. In terms of human health, meat from animals hunted in hilly sub- Apennine area in the Province of Viterbo and in National Reserve of Castelporziano areas showed desirable fatty acid compositions high contents of linolenic and lower n- 6/n-3 ratio.

The grilling treatment changes the organoleptic composition of wild boar meat and it improves the chemical and nutritional characteristics, in particular regarding the fatty acids composition. The grilling treatment lowered the saturated myristic and palmitic fatty acid contents responsible of different cardiovascular disease and increased the levels of eicosapentaenoic acid, that has an important role in its prevention. Unfortunately the cooking did not affected the levels n-6/n-3 ratio, but improved other nutritional fatty acid ratios, showing a positive effect on P/S ratio compared to raw meat and improved the aterogenic and thrombogenic index. On the contrary, the cooking procedure due to high temperature increased the formation lipid oxidation. High value of TBARS found in cooked meat could be linked to the fact that the meat of wild boar contains higher percentage of polyunsaturated fatty acids compared to other types of meat and polyunsaturated fatty acids are more prone to oxidation. The results obtained in these studies provide important additional information on the chemical composition, physical traits, organoleptic characteristics and nutritional value of meat from wild ungulates harvested in Central Italy. These results partially balance the lack of knowledge on this topic and the scarce available scientific literature. Some results obtained in these studies encourage the inclusion of wild ungulate meat in human diet due to the nutritional value and organoleptic characteristics.

6. AKNOWLEDGEMENTS

The Author thank:

- The National Reserve of Castelporziano, Roma, Italy; - Osservatorio per lo studio e la gestione delle risorse faunstiche dell’Università degli studi della Tuscia - ISPRA for providing data for the killing of ungulates.

- All my collaborators who have participated to these studies: Michela Contò, Giulia Francesca Cifuni, Maurizio Mormile, David Meo Zilio and Antonella Ficco

A special thanks to my tutor, Professor Andrea Amici, who introduced me to this new line of research for me unknown.