Keywords: fatty liver, force feeding, stimulation, geese

The present experiment aimed at comparing fatty livers obtained from overfeeding or from an alternative method. Indeed, it has previously been shown that, under specific handling conditions and without overfeeding , it was possible to obtain in Greylag geese (Anser anser) a spontaneous liver steatosis. The livers were compared for chemical and lipid composition, quality indicators, sensory traits and acceptability by consumers. Two hundred and thirty male geese were reared, but for practical reasons only ninety five livers were analyzed in this experiment. From hatching, geese were subdivided in three groups, and each group followed different rearing treatments. Eighty--five birds followed a standard overfeeding method, one hundred and twenty five birds followed an alternative one, based on the simulation of premigratory period, and a third group of twenty birds was used as control.

After treatment all animals were slaughtered, respectively at 17 (overfed), 31 (stimulated) an 19 weeks (control) of age. During the treatment there was an increase in body and liver weight, for both treatments groups as compared to control. The average values of liver weight were strongly higher in overfed birds (1102 g) than in the stimulated (445 g). Lipid analysis shows that overfed birds' livers have higher lipid content (56%) than stimulated livers (53%), whereas free glucose and glycogen are higher in stimulated birds' livers. Neutral lipid profile, obtained by Gas Chromatography (GC), showed some differences between the two groups, mainly in term of diglycerides and cholesterol esters. Sensory analysis indicated that fatty livers obtained under standard overfeeding conditions were globally more appreciated than those obtained by stimulation. Results for an alternative production of fatty liver are encouraging, however the variability in the individual response remains very high. Further research is needed to better understand the origin of this variability.

Ringraziamenti

Ringrazio il Prof. Paolo Catellani e la Prof. Antonella Dalle Zotte, per avermi dato la possibilità di vivere questa eccezionale esperienza francese, per i consigli e per tutto il loro appoggio.

Un grazie anche alla mia famiglia, Francesco ed i miei amici, che mi hanno sempre sostenuta anche a più di mille chilometri di distanza.

RIASSUNTO

La tradizione del force feeding per la produzione del foie gras è una pratica molto antica, risalente probabilmente ai tempi degli Egizi (Guemene et al., 2004). Al giorno d'oggi, il foie gras costituisce un prodotto di largo consumo durante le occasioni di festa, soprattutto in Francia, che ne è anche il primo produttore a livello mondiale. La pratica del force feeding è tuttavia molto contestata da gruppi animalisti, che vedono in questa tradizione una forma di tortura per gli animali. Il presente progetto è parte di un piano sperimentale promosso dal centro di ricerca francese INRA (Toulouse, Francia), volto a dimostrare che è possibile indurre, in oche maschio della specie Greylag ( Anser anser), il naturale sviluppo di iperfagia e della conseguente statosi epatica, sfruttando una stimolazione alimentare e luminosa che simuli il periodo pre migratorio. Il core di questo lavoro di tesi è rappresentato dal confronto, sotto il profilo chimico e sensoriale, di foie gras ottenuti mediante metodiche standard di overfeeding con foie gras ottenuti attraverso una stimolazione luminosa e alimentare. I risultati ottenuti mostrano chiaramente che l'aumento di peso, non soltanto a livello corporeo, ma soprattutto a livello epatico, è nettamente a favore del metodo standard di allevamento; tuttavia i contenuti in lipidi a livello epatico, mostrano valori simili per i due trattamenti. Per entrambi i gruppi sperimentali, inoltre, la maggioranza de lipidi epatici accumulati appartengono alla classe dei trigliceridi, segno di una steatosi epatica in atto (Robbins and Cotran, 2010). Il profilo in lipidi neutri, ottenuto mediante Gas Cromatografia (GS) evidenzia delle differenze per i due gruppi di allevamento, in termini di digliceridi e esteri del colesterolo. Altri parametri considerati, quali ad esempio, glucosio libero e glicogeno, mostrano concentrazioni epatiche più elevate a seguito del trattamento di stimolazione. Sotto il profilo sensoriale, sono state condotte due analisi, un test sensoriale ed uno di accettabilità. In entrambi i casi è emerso che il foie gras ottenuto mediante metodica standard risulta essere molto più apprezzato.

In conclusione possiamo affermare che i risultati di questa pratica alternativa di stimolazione, volta a produrre foie gras senza alimentazione forzata, sono soddisfacenti; resta tuttavia una grande variabilità individuale nella risposta al trattamento di stimolazione. Saranno necessari ulteriori studi per capire quale sia la causa di tale variabilità.

INTRODUCTION 1.1 The product

The fatty liver, internationally known and called foie gras, is a food product obtained by the overfeeding (gavage) of geese and ducks; according to French law it is defined as "the liver of a duck or a goose that has been specially fattened by gavage" . According to the French Regulation Nf 1538/91 of the commission dated the 5th of June 1991 (JO N°L 143, 7th of June, P. 11; JO N°L233, 22nd of August 1991, p. 31) foie gras is defined by the minimal weights of the livers after overfeeding 400g for the geese and 300g for the ducks. The physiological basis for the fatty liver production is the natural capacity of migrating birds to over consume food in order to store energy before long travels (Bairlein, 1987). Moreover they have the capacity to ingest a high quantity of food and store it, either whole or pre--digested, in the crop, which is an enlarged portion of the oesophagus, while awaiting digestion in the stomach. The storage of fat in the liver, as a consequence of high energy intake, produces steatosis of liver cell. Under natural conditions ducks and geese can double their weight in the autumn, storing fat throughout much of their body, especially on the liver, in preparation for winter migration. In poultry production, to obtain the foie gras, farmed geese and ducks are overfed to obtain the same thing that you can find in the wild. The production of foie gras is highly developed in France, which is the first consumer and producer with 19000 tons in 2010, that represent more than 70% of the world production (Comité Interprofessionel du Foie Gras, 2011). From 2006 the foie gras is part of the cultural and gastronomic patrimony of France (Law n°2006--11 of 5 January 2006 of the orientation agricole ajoutée à l'article L654--27--1 au Code rural français). In France the fatty liver is consumed primarily on special events and in many others country it represents a gourmand product. There are European and French regulations to define the foie gras production and, from the 1st January 1994, the French legislation regulates also all the appellation of food based on foie gras produced in France.

According to the Act of 9 August, 1993 (Decret n°93--999 du 9 Aout 1993 relatif aux preparation à base de foie gras), that defines the different types of product prepared with foie gras and another text, published on 2008 that complete the first one by describing methods for the analysis for different "preparations", we can found different type of this product, described as follow:

• "Foie gras entier", (whole fat liver), prepared with one or two lobes of fat liver with the addition of stuffing;

• "Foie gras", parts of liver agglomerated, with the addition of stuffing;

• "Bloc de foie gras", fat liver processed by mechanical devices and the chucks of liver are not visible;

• "Parfait de foie", includes at least 75% of fat liver processed mechanical devices;

• "Medallion de foie" and "pâté de foie", product with at least 50% of fat liver, that is in chunks or mechanically prepared;

• "Gelatin de foie", product with 50% of fat liver mixed with stuffing;

• "Mousse de foie", product with 50% of fat liver mixed with stuffing and presented as a "mousse";

• "Produits au foie gras", products with foie gras which contains at least 20% of fat liver.

History and production

The practice of overfeeding is a very ancient tradition, originating from Egypt 2500 years BC; the ancient Egyptians, in fact, took advantage from the natural ability of migrating birds to over consume food to store energy before the migration, in order to produce energy food for human consumption (Odum, 1960). Greeks and Romans adopted this method of production very fast and the fatty liver became quickly a much appreciated product; in the middle age the overfeeding of birds got very popular and expanded in a lot of European countries like Poland, Hungary and France, most likely spread by the Jewish population (Guy et al., 2010). Nowadays the practice of overfeeding has disappeared in Egypt and the initial goal has changed; the main objective of overfeeding is now to produce foie gras (fatty liver) of high quality to satisfy the demand of this gourmand product. The fatty liver is traditionally produced by overfeeding ducks and geese; this modality of production is the central point of a high number of controversies for the animal welfare. Recent studies show that the harmful consequences for the birds are limited (Guéméne et Guy, 2004;Guéméne et al., 2006) (2004), have enacted explicit bans against overfeeding; however there are still few nations where foie gras is still produced and consumed, which are for example France, Spain, Hungary and Bulgaria.

Hepatic metabolism

In avian species, as for the humans, the liver is the main site of metabolic process and his metabolism is the source of many substances essential to their health and survival.

The liver has an important role, not only for the metabolic processing of chemical and pharmaceutical products, but also for the elaboration of substances that are provided by the diet. The liver is in fact the organ responsible for the processing of fats and other lipids that are provided by the diet; it can convert the excess of protein and carbohydrates into triglycerides, which are the primary form in which animals store fats. If the body needs energy the liver can break triglycerides down into free fatty acids that are sent to the bloodstream and that can be used by the other tissues as an energy source.

The liver is also the site of another important process, called glycogenesis, that allows the metabolism of glucose and the conversion into glycogen, which is stored as a compact energy reserve; when the body needs energy the liver can convert the glycogen back into glucose (glycogenolysis). If the glucose is not available the liver can synthesize glucose from other substances by a process called gluconeogenesis. The liver is also responsible of the exportation of lipids as lipoprotein to the peripheral tissues.

Using of alimentary lipids

The principal forms of lipids stored in the liver or in the peripheral tissues (adipose tissue, muscles) are the triglycerides; to be absorbed and utilized, the dietary triglycerides must be hydrolyzed into mono/diglycerides, fatty acids and glycerol by the intervention of gastric and pancreatic lipases. Then the monoglycerides and the free fatty acid (FFA) will be associated with cholesterol and biliary salts to realize the micelles, that will be absorbed passively by the enterocytes or by the intervention of FABP (Fatty Acid Binding Protein) (Besnard et al., 1997). At the level of the intestinal mucosa a part of the long--chain fatty acids re--esterifies into triglycerides, which will be secreted by the enterocyte as portomicron, that are lipoproteins rich in triglycerides corresponding to the mammals chylomicrons ( Griffin et al., 1982); the portomicron will reach the liver by the portal vein. The short--chain fatty acids are instead directly secreted in the hepatic portal system and associated with serum albumin to reach the liver (Hocquette et Bauchart, 1999).

Figure 1. Mechanisms of hepatic steatosis in palmipeds [START_REF] Hermier | La stéatose hépatique des palmipèdes gavés : bases métaboliques et sensibilité génétique[END_REF]

Glucidic metabolism

The principal constituent of the birds diet, either for overfeeding or not, is corn that is very rich in starch (approximately 60%). The dietary starch is hydrolysed into dextrins and maltose thanks to the action of pancreatic amylases; to reach the liver, dextrins and maltose are then hydrolyzed into glucose by isomaltase and maltase.

If the body does not need energy, the glucose that arrived at the liver can be stored as glycogen (glicogenogenesis); if there is a request of energy the glucose can be transformed into ATP by the glycolyse and the Krebs's cycle. In that case through the succession of enzymatic reaction of glycolysis, glucose is converted to Acetyl--CoA. The Acetyl--CoA can participate to the formation of ATP (Krebs' cycle) and also be used as substrate for de novo lipogenesis for the synthesis of fatty acid and lipids.

De novo lipogenesis

The regulation of de novo lipogenesis is related to the body demand during feeding and fasting periods. For birds, the capacity of de novo synthesis of lipids is very limited, either in peripheral tissue, and in the adipose tissue [START_REF] Leveille | Lipid biosynthesis in chick. A consideration of site of synthesis, influence of diet and possible regulatory mechanisms[END_REF]; the most important site for the lipogenesis is the liver (Evans et al., 1972;[START_REF] Leveille | Lipid biosynthesis in chick. A consideration of site of synthesis, influence of diet and possible regulatory mechanisms[END_REF]Saadoun et Leclerq, 1983et 1987).

The lipogenesis is a hormonal process and it depends on the insuline/ glucagon ratio; as the insulin can stimulate it (by the activation of acetyl--CoA Carboxylase, ACC), the glucagon inhibits it Goodridge, 1987).

For the synthetis of fatty acid the Acetyl--CoA is converted into malonyl--CoA by the action of a carboxylase (ACC) in presence of ATP; then the fatty acid synthase catalyses the reaction for the formation of the palmitic acid [START_REF] Mayes | Biosynthèse des acides gras[END_REF].

Starting from the palmitic acid, or from the other essential fatty acids, it is possible to syntheyse all of the other long--chain fatty acids by the action of elongase and desaturase of the Endoplasmathic Reticulum (ER).

Depending on the desaturation site there are four enzymes (∆9--, ∆6--, ∆5--and ∆4-desaturases) that play different roles at different steps in the process of long-chain fatty acid synthesis. However, there are some essential fatty acids, like linoleic and α--linoleic acid, which have to be introduced by diet because it is not possible to synthetize them; starting from these fatty acids it is then possible, by the action of desaturase and elongase, to synthetize other fatty acids, such as arachidonic acid, essential for the phospholipids membranes.

At the same time also the saturated, the mono-- and the poly--unsaturated long-chain fatty acids can be esterified as triglycerides, phospholipids and cholesterol esters.

The triglycerides constitute the major part of storage lipids for all the animals; in particular, for overfeed geese and ducks that have developed hepatic steatosis, more than 95% of hepatic lipids are triglycerides synthetized in the hepatocyte from dietary starch (Théron, 2011).

For the cell's structure, the most important phospholipids are phosphatidylcholin (PC) and phosphatidylethanolamin (PE); these two phospholipids have the same synthesis' pathway and, in the liver, the conversion of PE into PC is mediated by a methyl transferase that operates a progressive methylation on the nitrogen of ethanolamine residual. Another important constituent of cells' membranes is the cholesterol; it is very important, not only for the membranes but also for the harmonic equilibrium. In fact, it is the precursor of the D3 vitamin, steroid hormones and biliar salts. The origins of cholesterol are different, as it can be introduced with the diet or be synthesized by the organism in the cytoplasm of intestine's and liver's cells.

Lipids exportation

The dietary lipids and those originating from de novo lipogenesis exported from the liver to the peripheral tissues by the action of lipoproteins. There are different types of lipoproteins according to their different modality of action and to their collocation in the organism.

The lipoproteins involved in the carriage process of long--chain fatty acid are the portomicrons, secreted by the enterocytes; their action is to transport the fatty acids to the peripheral tissues through the portal system. For the transport of fatty acid from the liver to the peripheral tissues the lipoproteins involved are the VLDL (Very Low Density Lipoproteins), which are rich in triglycerides and poor on cholesterol; the fatty acids that are transported by that pathway can be stored or utilized for energy production. In particular the triglycerides that are transported by these lipoproteins have to be hydrolyzed by the action of lipoprotein lipase (LPL) to pass the cellular membranes. It is the post prandial activity of the LPL that allows to incorporate the triglycerides.

For the cholesterol the specific lipoproteins are the IDL (Intermediate Density Lipoproteins), the LDL (Low Density Lipoproteins) and the HDL (High Density Lipoproteins); the first one derives from the degradation of VLDL, the second and the third one are secreted by the liver, with the difference that HDL can be recuperated by the liver.

Hepatic steatosis

In humans, fatty liver is a pathological condition that occurs when there is an excessive and abnormal accumulation of triglycerides and other lipids in the cells of the liver. There are lot of factors than can be involved in the development of this condition, like metabolic syndromes, the excessive consumption of alcohol or a surgical intervention. [START_REF] Torres | Diagnosis and therapy of nonalcoholic steatohepatitis[END_REF].

In birds, however, hepatic steatosis is not a pathological condition since they can naturally develop a fatty liver in the premigratory period, to build up energy reserves for the long travel of migration.

The hepatic steatosis reflects the accumulation of lipids in the liver. For the wild animals this accumulation is a physiological adaptation and it is very important for them to survive in particular conditions, like migration or hibernation; however, for the domestic animals, like for the humans, the hepatic steatosis is a pathological condition. The palmipeds represent an exception in that sense and for them a controlled induction of hepatic statosis by overfeeding is at the origin of the production of foie gras (fatty liver).

In fact, according to the definition of Labie and Tournout (cited by [START_REF] Hermier | Plasma lipoproteins and liver lipids in two breeds of geese with different susceptibility to hepatic steatosis: changes induced by development and force--feeding[END_REF], one can say that the fatty liver of palmipeds is the expression of a reversible hepatic steatosis that has a natural origin.

Mechanism of hepatic steatosis in palmipeds

The diet of palmipeds during the overfeeding period is strongly based on a high proportion of glucides and a lesser extent of lipids; in the liver there is a high stimulation of de novo lipogenesis and a consequent disequilibrium between the synthesis and the exportation of lipids by the VLDL. It is known that during the overfeeding period there is an increase in the production of VLDL and HDL, but this is not enough to inhibit the development of hepatic steatosis ( Fournier et al., 1997).

There are different hypotheses to explain the reason of the development of this condition during the overfeeding period; in fact it can be caused by a defective action of LPL that induces a consequent return of lipids to the liver, or by a defective formation of VLDL, caused by disequilibrium in the utilization of phspholipids that induces cellular hypertrophy [START_REF] Hermier | Plasma lipoproteins and liver lipids in two breeds of geese with different susceptibility to hepatic steatosis: changes induced by development and force--feeding[END_REF] Moreover, it is important to say that during overfeeding the animals are never fasting, so the insulin stimulation is always active and that can inhibit the triglycerides' hydrolysis and stimulate the storage at hepatic level [START_REF] Hermier | La stéatose hépatique des palmipèdes gavés : bases métaboliques et sensibilité génétique[END_REF].

Modifications associated with hepatic steatosis in palmipeds

The hepatic steatosis in palmipeds due to overfeeding is a natural and reversible condition; in fact the overfeeding does not cause structural or metabolic lesions even if is possible to observe some changes (Blum et al., 1990;Bénard et Labie, 1998).

As shown by Baudonnet (1993), the practice of overfeeding induces an important increase on liver weight (factor of 2,5) during the first period of overfeeding and this increase reflects an accumulation of lipids, in particular triglycerides. Also in the fatty acid profile, it is possible to notice some changes; in fact there is an important increase in oleic, palmitic and palmitoleic acid during the first three days of overfeeding and, on the other hand, there is a decrease of essential fatty acids (linoleic and linolenic acids) and stearic acid. This kinetic reflects the activity of the enzymes involved in the process of fatty acids synthesis (Théron, 2011).

Natural induction of hepatic steatosis in geese

In 2008, INRA decided to support researches on the alternative methods of overfeeding for the production of fattened livers. A research project was elaborated by INRA scientists in order to investigate the possibility to induce a spontaneous fattening of the liver.

The initial aim was also to demonstrate the natural ability of domestic waterfowls, at least geese, to spontaneously develop liver steatosis in breeding conditions. To study that, two experiments have been carried out in 2010 and 2011.

The researches started considering that geese were closer to their migratory ancestor than the ducks are. Indeed, for the production of duck fatty liver, a hybrid (Mule duck) between a Muscovy male duck (Cairina moschata) and a common female (Anas Plathyrynchos) is mostly used. It was supposed that geese would be more prone to develop spontaneous hyperphagia and subsequent liver steatosis under specific handling condition than ducks. Indeed, in birds travelling for long distances, the pre--migratory increase in body weight can reach +100% of the lean mass and the major store of energy for migration is fat (Bairlein, 1987). However, there are different opinions about it. As an example, another school of thinking states that birds that are used for foie gras production are different from the wild migratory birds (Guy et al., 2012). In addition, it is generally admitted that the spontaneous pre--migratory fattening is mostly localized in subcutaneous tissue and few energy is stored in the liver (Pond, 1978).

Previous experiments and results

Starting from the hypothesis discussed above, in 2009 the experiments began; the trial was carried out from July 1 st 2009 to February 2 nd 2010, at the INRA unit of waterfowl (Benquet, France).

The type of geese used for the experiment was the Greyag Landaise (Anser anser) because these birds are known to have a good capacity to develop liver steatosis (Guy et al., 1995); that ability is related to a high lipogenic capacity of the liver that, for the birds, is the principal site of de novo lipogenesis [START_REF] Leveille | Lipid biosynthesis in chick. A consideration of site of synthesis, influence of diet and possible regulatory mechanisms[END_REF].

Considering the environmental factors involved in the pre--migratory period, the most important factor controlling migration itself and the pre--migratory fattening process is the photoperiod (review by Bairlein and Gwinner, 1994); in particular it is know that the shortening of day length occurring in wild life before winter migration accelerates the process of fat deposition in the migratory birds.

In order to verify this hypothesis geese were moved from the park to a building where it was possible to control the photoperiod and where the temperature could be recorded. Another factor that was manipulated was the diet and the feeding system; in fact, for the experiment, the feeding system consisted on alternate periods of ad libitum feeding and feeding restriction. The food used was fresh corn.

Animals were slaughtered at different times; in particular for the first experiment the slaughter occurred at the age of 19, 23, 25, 27, 29 or 31 wks, for the second one geese were slaughtered at 19, 27, 30, 32 or 36 wks of age.

The overall design for the breeding and stimulation of the birds is shown in Figure 3. In the graphics below it is possible to see the development of liver weight and the percentage of dry matter in function of the weeks of treatment.

For the first experiment, carried out during winter 2009--2010 (Figure 4a), it is possible to notice that the liver weight increased from week 19 (before the start of experimental treatment) to week 32; at week 27 and 30 the liver weights show respectively, a three-- fold and a four-- fold increase compared to week 19.

At week 36, at the end of the first experimental treatment, the liver weight decreased. For the second experiment (Figure 4b), where it was decided to stop the experimental treatment at 31 weeks, we can notice the same increasing of liver weight showed in the previous experiment, with the difference that in the second case, the increase is slower. It is interesting notice that, at the and of both experiments, the livers exhibited a weight over 400g, that is the legal limit in France for the denomination foie gras given to goose liver fattened after overfeeding in commercial conditions.

As shown in the Figures 4a and4b the pattern of changes in dry matter content closely followed the changes in the liver weight; the content of dry matter, in fact, rose from 30% (w 19; lean livers) to about 60% at the end of treatment ( 36 and 31 wks). This increase was mostly due to the rise of lipid content (Guy et al., 2012).

According to the results of these experiments, we can say that after 12 weeks of breeding under the conditions described above, the livers of geese developed spontaneous fattening and their weight had a five--fold increase (from 100 to 500g); under standard conditions of overfeeding in Landaise geese, liver weight usually reach values above 800g with lipid contents of 50--55% (Salichon et al., 1994;Leprette et al., 1997). These values for lipids content are very close to those recorded in the two presents studies for spontaneously fatty liver (Guy et al., 2012).

What's my project?

These two experiments clearly demonstrated that geese could spontaneously fatten their livers up to some levels which are very close to those encountered in livers from overfed geese. Though the average liver weight remain lower (400--500g vs 1000g and above after force feeding), lipid level rise in the same magnitude to rich about 55 % of the liver weight (results not shown directly but visible in the dry matter charts shown above, a and b). Several questions were raised after these two experiments: do the spontaneous fattened livers show similar chemical composition than "conventional" (namely, from overfed geese) livers, are the lipids the same though their level is similar? How do these two types of livers compare on sensory point of view? A third experiment was planned with the aim of comparing the two types of livers and this work constituted the core of my training period.

AIM OF THE PROJECT

This master's thesis work was done during my six month Erasmus exchange mobility at the Department of "Animal tissue, Nutrition, Digestion, Ecosystem and Metabolism" (UMR 1289 INRA/INPT) at the National Institute of Agronomy--ENSAT--(Toulouse, France). This project was part of an experimental plan that aimed at demonstrating that the Greylag geese (Anser anser) commonly used for the production of fatty liver, are able to develop spontaneous hyperphagia and subsequent liver steatosis, under specific handling conditions and without overfeeding. Combining a short photoperiod and a stimulation of a high energy food intake, it was demonstrated that spontaneous fattening could be initiated, and the livers produced present similar lipid contents (> 50 %) than those obtained by standard overfeeding, though they were lighter.

The main purpose of this study was to evaluate chemical and biochemical composition of livers obtained by geese "stimulation" and to compare these livers with those obtained by the standard overfeeding method. The study concerned the analysis of dry matter and lipid component, the glucose, lactate and glycogen liver concentration, the lipid classes and Triglycerides profiles of liver by Gas Chromatography (GC) and High Performance Liquid Chromatography (HPLC), respectively. In addition, sensory and acceptability tests were carried out in order to compare, from a sensory point of view, livers obtained by the standard overfeeding or the alternative method.

MATERIAL AND METHODS

Experimental plan and animals

Two hundred and thirty Graylag Landaise male geese (Anser anser) one day years old were subdivided in three experimental groups (Table 1). One group corresponded to birds that were reared according to the standard overfeeding method (Overfed group), the second one was reared with the stimulation method (Stimulated group), and the last one corresponded to the group of birds slaughtered just before corn feeding, and were considered as control group before the experimental stimulation (Control group).

The slaughtering age ( The trial was carried out from July 2011 to February 2012 at the INRA experimental unit on waterfowl (Benquete, France).

Rearing program and diet

The trial was carried out from July 2011 to February 2012 at the INRA experimental unit on waterfowl (Benquete, France). Birds of stimulation program and controls were fed a commercial diet from birth to nineteen weeks of age (2800 Kcal/kg ME and 180 g/kg CP from birth to six weeks , and 2800 Kcal/kg ME and 160 g/kg CP from seven to nineteen weeks: Maïsadour, Benquet, France) to meet the NRC requirements (NRC, 1994). From week 20 onward, stimulated birds were fed a fresh harvest corn stored in a flexible silo (preservation in oxygen free conditions). The characteristics of this corn are given in appendix.

The choice of this type of corn was based on its high palatability for geese (Guy, unpublished observations) and its high starch content, which favour fattening. Birds of overfed group were fed the same commercial diet used for the other two groups, from birth to thirteen weeks of age. From week 14 onward, birds were submitted to overfeeding. All the details for each rearing condition are explained in the sections below.

Rearing program of the stimulated group

Feeding program

The feeding program was based on a period of feed amount restriction (from week 8 to week 20) just before the stimulation, which aims to ensure a normal growth while preventing excessive fattening (McLandress and Raveling, 1981) and to induce hyperphagia when stopped. The details of the alimentary schedule are reported in Table 2. The starter food D15 corresponds to 2800 Kcal/kg et 18% MPB and the grower food D25 corresponds to 2800 Kcal/kg and 16% of MPB.

Housing and management conditions of the stimulated group

From birth to fifteen weeks of age the 125 animals of the alternative production were submitted to natural lighting; during this period the duration of daylight fell from 14h11' (beginning of July) to 11h13' (15 th September). At fifteen weeks of age, the free access to the outdoor area was suppressed and birds were then kept under artificial light following a cycle of 10h/day (from 7:00 am to 17:00 pm). At the beginning of week 21 to week 23, the light duration was gradually decreased by 30 minutes every two days, until reaching 7h/day of light. This short photoperiod was maintained up to the end of the experiment (week 31). During the experimental period, no additional heating was provided in the pens, and the temperature was registered daily at 7:30 am. The individual live weight was registered at fourteen and nineteen weeks of age.

Rearing program of for the overfed group

Feeding program The eighty five birds of overfed group followed a standard overfeeding program; until the week 7 birds had free access to food, onward 8 weeks of age they were subjected to feed restriction (hourly restriction) until the end of treatment. The details of the alimentary schedule are reported in Table 3. The starter (D15)and the grower (D25)diets are the same of stimulated and controls birds.

D15 D15 D25 D25 D25 D25 D25
ad libitum ad libitum rationing (from 8am to 5pm) rationing (from 8am to 2pm) rationing (from 8am to 12am) rationing (from 8am to 10am) rationing (from 8am to 12am)

Housing and management conditions of the overfed group

For the animals of overfed group there are not particularly conditions of breeding, in terms of day length and temperature; they are submitted to naturally day length and the building is heating only during the first 4 weeks. For the overfeeding period geese are transferred to a specific building to be overfed, from week 14 until slaughtering. Birds were weighted at 14 weeks of age.

Slaughtering, sampling and measurement

Birds of control group were slaughtered at 19 weeks of age. Overfed and stimulated birds were slaughtered at 17 and 31 weeks of age, respectively. The birds were crated in transport cages (4 birds/cage) and transferred to the experimental slaughterhouse. The animals were weighted and the data obtained were considered as slaughter weight. They were then suspended individually from a shackle with their head downwards and electrically stunned using a head-only scissors tongs (90 V, 50 Hz AC current for 5sec ). All birds were slaughtered by the same slaughtering procedure, that is by ventral cut of neck blood vessels, within 5 second after the end of stun. After bleeding and plucking the carcasses were weighted before the evisceration, (carcass weight).

The liver colour was measured immediately after slaughter, using the trichromatic CIE Lab coordinates system (L*a*b*, CIE, 1976); for each liver three measurements have been done at three different sites along the ventral face of the big lobe (top, middle and bottom) with a CR 300 Minolta chromameter. From the central part of the big lobe a sample of about 50 g was taken, and divided in two parts, frozen in liquid nitrogen, vacuum packed and stored at --80 °C for further chemical analysis. The day after slaughter, all the carcasses were anatomically dissected and the weight of breast muscle (Pectoralis major), breast skin + subcutaneous fat, leg (thigh + drumstick) meat and bones, and leg skin + subcutaneous fat was registered.

Selection and preparation of samples for chemical and biochemical analyses

For practical reasons, the chemical and biochemical analyses were not carried out on all the two hundred and thirty livers. A group of 95 livers were selected as described below.

Selection of liver samples

Livers were assigned to four weight classes, according to their weight measured at slaughter (Table 4). The number of livers in the different weight--classes strongly differed between the two experimental groups (overfed and stimulated). From overfed group 0, 4, 32 and 9 livers were selected, respectively; from the stimulated group 15, 16, 11 and 0 livers were selected, respectively; whereas eight out of the twenty livers of the class < 600 g were selected from the control group (Table 4).

Raw fatty liver preparation

The ninety-- five 50 g raw fatty liver samples were taken from the freezer (--80°C) and ground in liquid nitrogen with a ball--grinder. The aim of this procedure was to obtain a fine, homogeneous and representative powder for each sample that was stored in specific and numerated plastic cups at -80°C.

Chemical analysis

Determination of dry matter

The dry matter (DM) of fatty liver was measured by drying homogenate in an oven for 24h at an average temperature of 103 °C to eliminate residual humidity (J.O.C.E., 1971).

From each homogenate liver, two samples of about 1,5 g were used; the powder was put into glass cups, which contained about 4,5 g of sand, in order to increase the exchange surface. The precise weight of cup containing the sand was taken before putting the sample and represents the tare weight. Then the cups were put in a drying oven at about 103 °C for 24h.

The day after the cups were cooled in a desiccator for about 10--15 minutes to prevent the reacquisition of moisture, and then weighted on a precision balance (Sartorius Analytic, Germany).

The DM rate was determined by the formula:

DM= ((dry weight -tare) / test sample) * 100, expressed as %

Lipids extraction

The total lipid extraction was performed according to Folch method (Folch et al., 1957); to evaluate the lipids content of fatty liver the method exploits a mix of Chloroform : Methanol (2:1, v:v) to separate the lipid and hydrophilic extracts.

A different quantity of samples was taken as a function of the weight of liver using a precision balance(Sartorius Analytic, Germany). For all the overfed livers were weighted about 0.5 g of sample powder, for the stimulated livers 0.5 g (liver >800 g), 0.7 g (liver 600--800 g) and 1 g (liver <600g); for all the livers of control group was weighted 1 g of sample powder.

The sample was put in a polypropylene tube of 50 ml and then 30 ml of Folch mix were added; after about a 30 seconds homogenization with Ultra--Turrax T25 (JK--IKA Labortechnik, German) were added 10 ml of Folch mix, to reach a final volume of 40 ml. The tubes were put overnight at 4°C to obtain a complete extraction and to limit the phenomena of oxidation and evaporation.

The following day the homogenate was filtered using quantitative filter papers (Fischer Bioblock, FR) in tubes placed in refrigerated bath (0--4 °C) and containing 8 ml of NaCl solution (0.73%) to allow the separation; after decantation (about 3 hours) the lipid phase was drawn and put into Wheaton tube of 10 ml (2 tubes for any sample). The quantification of lipids was done gravimetrically after total evaporation of the 10 mL of chloroform contained in the tubes in a Speed -Vac (RC10--10, Jouan SA, FR). The total lipid content was calculated by the formula:

% total lipids= (2 x V x R

Extraction

For the extraction about 2 g of sample were ground in the Ultra--Turrax in 10 ml of perchloric acid 0.5 M and then, two rinsings of the rod were done with 2.5 ml of solution. The homogenate was centrifuged for 20 minutes at 4 °C (600 g) and then, the tubes were placed at a temperature of --20 °C for 10 minutes, to allow the solidification of the "fat cake" formed during the centrifugation. After removing the "fat cake", the samples were resuspended and 4 aliquots of 0.5 ml were sampled for the dosage of glycogen (obtained by the dosage of total glucose). The homogenate was centrifuged another time for 15 minutes (4 °C, 2500 g) and then six 1 ml aliquots were sampled for the dosage of lactate. All the aliquots were stored at a temperature of --20 °C until analysis.

Glycogen hydrolysis

The hydrolysis was realized using the method of Dalrymple and Hamm (1973). A volume of 50 µl of KOH 30% was added in each tube containing 0.5 ml aliquot to allow the neutralization. After agitation by vortex, 1ml of amyloglucosidase solution (2mg/ml) in acetate buffer 0.2M (pH 4.8) was added; the tubes were then placed in a bath for incubation (3hours) at 38 °C and they were regularly agitated by inversion. After incubation, 100 µl of percloric acid solution (3M)

were added to stop the enzymatic reactions, and the tubes were placed for 10 minutes in melting ice. The tubes were then centrifuged for 15 minutes (4000 t/m) and the supernatant was filtered using glasswool; the tubes were then stocked at --20 °C until the dosage analysis.

Dosage of glucose

The dosage of glucose was realized according to the method of Bergmeyer (1974), adding 2 ml of NAD buffer to 100 µl of test sample. The NAD buffer preparation was realized as follow: starting from tiethanolamin hydrochloric buffer 0.1 M (pH = 7.6), EDTA (2 g/l) and MgCl 2 (2 g/l) were added and the pH was adjusted at 7.6 (adding NaOH); after also NAD (40 mg/100 ml of buffer) and ATP (170 mg/ 100 ml of buffer) were added. The enzymes were added to buffer at the end: 100 UI/ml of buffer for both the glucose--6 phosphate dehydrogenase (G6PDH) and the Hexokinase. The absorbance were measured by a spectrophotometer (Lambda20, Perkin Elmer, USA) at a wavelength of 340 nm, corresponding to the absorbance of NADH, H+ formed by the action of G6PDH on the glucose--6 phosphate. To validate the data, the results were compared with a calibration range in order to obtain the concentration of glucose in the samples.

Dosage of lactic acid

The dosage of lactate was realized according to the method of Bergmeyer (1974); a volume of 100 µl of sample testing was added to 2 ml of Tris Base buffer 0.2 M, Hydrazine 0.2 M and NAD 2 mg/ml (pH = 9). The lactate dehydrogense was added at a concentration of 10 U/essay. The absorbance were measured using a spectrophotometer (Lambda20, Perkin Elmer, USA) at a wavelength of 340 nm, corresponding to the absorbance of NADH, H+ formed by the action of lactate dehydrogenase on the L--lactate. To validate the data, the results were compared with a calibration range in order to obtain the concentration of lactate in the samples.

Analysis of lipid fraction of fatty liver

Neutral lipid profile

Neutral lipids (or non polar lipids) are mainly represented by mono--, di--and triglycerides, free fatty acids, cholesterol and cholesterol esters.

To realize the neutral lipid profile, the separation of lipids was carried out by Gas Chromatography (GC) according to the method of Myher and Kuksis (1984). The analysis were carried out using a chromatograph Hewlett Packard 5890 Series II, associated with an automatic injector (6890, Agilent Technologies, USA).

For both the groups stimulated and overfed, starting from the lipid extract obtained by Folch et al. method (1957), a certain quantity of extract was drawn in relation to the quantity (g) of total lipids observed in the sample: for all the sample showing a quantity of total lipids greater than 0.08 g/10 ml of chloroform were drawn 20 µL of extract; between 0.07 and 0.08 g/10ml of chloroform 30µl; for a quantity lower than 0.07 g/10ml of chloroform, were drawn 40µl.

For the control group, before preparing the sample to inject, we separated neutral and polar lipids, using a specific silica membrane (SepPak®) and different flows of chloroform and methanol: after imbibitions of the cartridge with 5ml of chloroform, we deposed 200 µL of sample on the cartridge and then we washed it with a flow of chloroform passed corresponding to 5 ml (flow 0.7 ml/min); to separate the polar lipids we washed the same cartridge with a flow of methanol, corresponding to 6 ml (flow 0.7 ml/min). The solvent of neutral lipid fraction (chloroform) was removed by evaporation under vacuum (SpeedVac) and lipids were resuspended in a know volume of chloroform (4 ml). To do the analysis, were always drawn 50 µl of this extract. The polar lipid fraction was stored at --20 °C for future analysis.

The extract was then put into a vial, provided with a reducer tube of 150 µl, and 20 µl of internal standard were added (Tri--Caprin, Tri C10, 50.5 mg/100 ml heptane); after the evaporation of chloroform by gaseous nitrogen (about 3minutes), 100 µl of Tri--Sil BSA in Pyridin (Thermo Scientific, 49012) were added.

The vial was closed with a Teflon cap (in order to prevent the evaporation), agitated by vortex and put in a desiccator, since the sylilated derivates are much unstable in humid environment. A volume of 1 µl of sample was injected on-- column (injection temperature: 103 °C). The column utilized was DBS--15mx0.32mmx0.1µm (123--5011 Agilent Technologies, USA); and the oven program is presented in Table 5. The lipid composition was quantified by using calibration range:

Free fatty acids: C16:0, C16:1, C18:0, C18:1, C18:2

Monoglycerides : MonoC16:0, MonoC18:1 Diglycerides: DiC16:0, DiC18:1

Cholesterol

Cholesterol esters: CholC16:0, CholC18:1

Triglycerides: TriC16:0, 1,3DiC16:0--2,C18:1, 1,3DiC18:1--2C16:0, TriC18:1

For data analysis (software: GC Chemstation, Agilent Technologies, USA) was done the summation within the lipid classes, in order to compare them to have the neutral lipid profile of the samples.

Triglycerides profile

The triglycerides separation was carried out by High Performance Liquid Chromatography (HPLC), according to [START_REF] Héron | Method development for a quantitative analysis performed without any standard using an evaporative light scattering detector[END_REF] method. The HPLC chain (Agilent Technologies, USA) was associated with an Evaporative Light--Scattering Detector (ELS--D 2100, Polymer Laboratories) in which the solvent stream containing the solute material is nebulized and carried by a gas flow through an evaporation chamber. The solvent is volatilized; leaving a mist of solute particles that scatters light to a photosensitive device. The signal is amplified and a voltage output provides the concentration of solute particles passing through the light; the response, elaborated by the specific software, can be linear, sigmoidal or logarithmical; in our case we had a logarithmic signal, made by the summation of refracted and non--refracted light.

With the HPLC is possible to separate the Triglycerides (TG) in function of the fatty acids which esterifies the glycerol. In this analysis the identification of different TG, were carried out basing on a calibration range of the following TG: Signal integration was carried out using a specific software (GC Chemstation, Agilent Technologies, USA) and to analyze the data, the integrate signal of sample was compared with a specific calibration range.

In the present study, for this analysis was recorded a problem with the dilutions to realize the calibration range; it was not possible to validate the results, so they will not be shown and discussed.

Sensory analysis and acceptability test

In order to have a complete perspective of the alternative fatty livers production by stimulation, a sensory analysis (following the norm NF ISO 13299) and an acceptability test (following the norms XPV09--501 and XPV09--500) were realized under COFRAC accreditation (accreditation number 1--1414, www.cofrac.fr). In these studies the organoleptic profiles of two stimulated references are defined comparing it to one overfed reference profile.

The objective of the study was (i) to compare the sensory profile and acceptability of livers obtained from force feeding and from the stimulation, and (ii) to compare fatty livers issued from spontaneous fattening but showing different weights. Therefore, for the analysis INRA UMR TANDEM provided three classes of livers: classes A and B of stimulated livers and class C of overfed livers, that have been judged by a jury of 11 qualified subjects. The groups corresponded to the following livers :

A: stimulated livers weighting between 600 and 800 g, and corresponding to the weight class that it is aspired to reach after standardization of the alternative production system.

B: stimulated livers weighting between 800 and 1000 g, a weight class corresponding to the most represented weight class of livers obtained after overfeeding and allowing thus the comparison of production system at the same weight.

C: liver from overfed geese within the most represented weight class 800 -1000 g.

Sensory analysis

For the analysis INRA UMR TANDEM provided 3 classes of livers: class A and B stimulated livers and class C overfed livers that have been judge by a jury of 11 qualified subjects. The fatty livers were ridded of their melting fat, were sliced in order to obtain a certain number of slides that had the same thickness; the slices were then stored at a temperature of 4 °C until the analysis.

The samples were presented one by one, following a different order for the different evaluators; the jury tasted the samples one after another and for any characteristic analyzed, gave a score between 0 and 10, following the questionnaire shown in the appendix (appendix c, page 69). The evaluators had not the possibility to revert to a characteristic already evaluated. The results were analyzed by a T-- Student Test, with a threshold of significance fixed at 5%.

Acceptability test

For the analysis INRA UMR TANDEM provided 3 classes of livers: class A and B stimulated livers and class C "overfed" livers that have been judged by a jury of 69 subjects, habitual consumers of fatty liver products (43 women and 26 men).

The object of the study was to evaluate the acceptability level of an alternative product comparing it to a product obtained by standard method. The judges were chosen in order to cover all ranges of employment and all age groups, between 18 and 70 (and more) years old. The samples were prepared and served following the same modality of the sensory analysis; the jury gave a score between 0 (very bad) and 10 (excellent) and they had not the possibility to revert to a characteristic already evaluated and they had to eat at least ½ of the sample (questionnaire appendix d, page 71). The results were analyzed by a T-- Student Test, with a threshold of significance fixed at 5% and with a confidence interval (CI) of 5%.

Statistical analysis

All data, except those from the sensory and acceptability tests, are shown as mean ± standard error of the mean. All data, except those from the sensory and acceptability tests were analyzed using the GLM (General Linear Model) procedure of SAS (Statistical Analysis System). Variance analysis was carried out to evaluate the significance of the effect of production system (alternative vs overfeeding) or the effect of the experimental group (groups A, B or C, as described in the section concerning sensory analysis). When appropriated (significance of the variance analysis model, effects with more than 2 levels), means were compared using multiple-range Duncan test from the SAS system. Levels of significance were reported as: ***, p < 0,001; **, p < 0,01; *, p < 0,05; NS, p > 0,10.

RESULTS AND DISCUSSION

The results are here subdivided in four sections: in the first section the zootechnical parameters are presented, in a second section the liver composition and the biochemical data are shown, in a third section the lipids profile are presented and then, the last section shows the data of sensory and acceptability tests.

A first statistical analysis compared the data of the three experimental groups (overfed, stimulated and controls); in a second analysis were compared the data of the three groups chosen in order to properly represent liver weights between 600 and 1000g, as previously described in the material and methods section.

Zootechnical results

a) Overfeeding and stimulation performance

The results of variance analysis for the two treatments are showed in Table 7. Overfed birds exhibited higher slaughter weight than stimulated ones (9287 vs 8242 g; p< 0.001; Table 7) due to a greater weight gain during the treatment, even though the difference was not statistically significant. Considering the liver weight the overfed birds showed a two--fold higher weight than the stimulated birds. No mortality was observed throughout the duration of the experiment. The presented results confirmed that, under these experimental conditions, it is possible to initiate hyperphagia after feed restriction (Guy et al., 2012), and as a consequence body and liver fattening can be recorded. The increase is more important for the standard overfeeding method (+39%), but comparing the stimulated birds with those of the control group, it is clearly demonstrated that it is possible to obtain a weight gain both for the body (+29%) and the liver with the alternative method.

Considering the liver weight it emerged that the stimulation method is able to enhance liver fattening. Anyway, the increase in liver weight was larger for overfed geese. It is interesting to notice that livers of birds reared with the alternative method and slaughtered at week 31 exhibited an average liver weight over 400 g, this weight being the legal limit in France for the denomination "foie gras" given to goose liver fattened after force feeding in commercial conditions. However, all overfed livers weight more than 700 g, while more than one third of stimulated livers were lighter than 300 g.

Comparing the data of the three groups of liver weight range (Table 8) no significant differences among the slaughter weight of birds of the three groups (A, B, C) were observed. This analysis showed a statistically significant difference between the liver weight of the groups A and C, but not between the groups B and C. The three groups were indeed constructed on the basis of their liver weight, to allow a better comparison between the overfeeding and stimulation treatments. Group A corresponds probably to the liver weight performance that could be reached with the alternative method after further works for its optimization. Group C corresponds to the average liver weight performance in overfed geese nowadays. That is the reason why comparison was carried out between group C and group B, which correspond to liver obtained from stimulation, but with similar weights. The weight gain during the experimental period showed similar values for the two liver weight classes A and B, and also comparing these classes with C there were no statistically significant difference. Moreover, for both slaughter weight and weight gain, overfed birds showed more homogenous values as shown by the difference in standard error of the mean (Table 8). This aspect gains importance when large amount of products has to be produced and standards have to be met. It is interesting to notice that classes B and C, corresponding to livers of the same range of weight, showed similar values of slaughter weight and weight gain; in the class A livers, where the weight range was different and the livers were lighter, the slaughter weight presented no significant differences compared to the other two groups; this similarity in slaughter weight is probably due to a peripheral fat accumulation (i.e. abdominal fat) rather than liver fat accumulation during the stimulation treatment.

b) Body composition and liver quality

Results for body composition analysis (Table 9) showed some differences between the birds of the two treatment groups and the controls. These parameters, together with those of overfeeding and stimulation performance, allowed to better understand how different treatments change the body compositions of geese. Data about the weight of breast muscle suggested that this muscle is less developed in overfed than control birds; it is interesting to notice that, despite increasing in total body weight, this lean mass is the only parameter that is lower in overfed birds than controls. This aspect cannot be seen in the stimulated birds, where the body weight increasing is also related to breast muscle mass increasing. For the other weight indicators there are direct relationships between weight increasing and muscle/skin development. In other words, for overfed birds body weight gain was more related to an increase in liver weight (i.e. as the other compartments' indicators do not differ between stimulated and overfed birds, slaughter weight was significantly higher for this last group). Tight skin was the only exception, as it showed a lower value for the stimulated group. At the end of experimental period overfed geese showed average values of 5.8% abdominal fat and, for the stimulated birds, this parameter reaches 7% at week 31 (slaughter age). These values are similar to those found by [START_REF] Hermier | La stéatose hépatique des palmipèdes gavés : bases métaboliques et sensibilité génétique[END_REF] in previous studies on overfed geese. As for other parameters, despite the fact that slaughter weight was lower for stimulated birds, these animals showed an abdominal fat accumulation that was similar (or slightly higher, ns) to those overfed. It is possible to state that the capacity of liver to export lipids towards peripheral tissues (abdominal or sub--cutaneous) was similar between the two groups. The difference in liver fattening could be due to the fact that, with overfeeding, the daily food supply was higher. Therefore, liver lipogenesis was stronger, and subsequently, lipid accumulation was higher.

The liver cooking yield parameter exhibited average values of 88.3 and 82.0% for overfed and stimulated birds, respectively. Livers from stimulated birds, despite the fact that they were lighter, showed more fat loss during cooking. The reason for this fat loss is not known but it was already observed that reduction of overfeeding period, usually lead to an increasing in liver cooking yield (Théron, 2011). In accordance with these observations, stimulated livers obtained after 12 weeks stimulation compared to 17 days of overfeeding showed higher fat loss values. Data about livers colour are expressed as L*a*b coordinates. The lightness (L*) was higher for livers of overfed birds, while yellowness (b*) and redness (a*) were significantly higher in stimulated birds' livers. That fact that stimulated livers are more yellow could be due to the fact that they accumulated more yellow pigments contained in corn since they ate only corn for 12 weeks, as compared to 17 days of overfeeding. For the redness parameter, it results more difficult to explain, but it may be related to higher blood retention was in stimulated livers as observed at the time of evisceration (Fernandez, data not published).

Comparing the groups A, B and C depending on liver weight classes (Table 10) there are still differences between the groups A--B (stimulated) and the group C (overfed) in term of abdominal fat. These results seem to confirm what previously discussed about the different fattening "pattern" for overfed and stimulated birds. Despite a lower slaughter weight, stimulated birds were characterized by higher incidence of abdominal fat.

Lightness parameter (L*) differed significantly between overfed and stimulated birds. Redness parameter (a*) showed similar values between the group C and A, but these values were significantly higher than value for group B. For the yellowness (b*) were recorded higher values for the stimulated livers.

For the other parameters there are not significant differences between the groups.

Liver composition: chemical and biochemical results a) Lipids and dry matter content

The lipid content (Figure 5) closely followed the liver weight gain during fattening; the lipid content of livers of the control group, that were lean livers obtained by birds slaughtered at 19 weeks of age, was of 5% only, whereas for the other two groups, overfed and stimulated, the lipid values reached on average 56% and 53%, respectively. Under standard overfeeding conditions in Landaise geese liver weight usually reaches values above 800 g, with a lipid content of 50--55% (Salichon et al., 1994;Leprette et al., 1997). In our study, birds reared by alternative method exhibit an average liver weight of 445 g, with values ranging from 69 g up to a maximum of 1130 g (data not shown). Even if there is an high weight variability among livers in stimulated birds and the average weight of these livers was lower (445 g vs 800 g), it is likely that the average liver lipid content value in stimulated birds (approx. 53%) was very close to that recorded in the study above reported on standard overfed livers production. The dry matter content (Figure 5) increased during the overfeeding and stimulating period. Birds of control group exhibited, at 19 weeks of age, a dry matter content of approx. 29%, compared to the 67 and 63% found on overfed and stimulated birds' livers, respectively. This increase in dry matter content of liver was mostly due to the rise in lipid content. A demonstration of this link is given in Figure 6. There is indeed a very strong linear relationship between dry matter and lipid contents in the livers from stimulated (R 2 =0.94) and overfed (R 2 =0.82) birds. In the analysis of the three liver weight classes A, B, C no significant differences in lipid content were found, nor between livers of different weight (C vs A), neither between livers of the same weight range (C vs B) (Figure 7). The average lipid content for all the weight classes was close to the values of 50--55% found for livers of birds reared with standard overfeeding method (Salichon et al., 1994;Leprette et al., 1997). The dry matter content reached average values of 64%, 65% and 66% for the groups A, B and C, respectively; also for this parameter there were not statistical differences between the three groups and the dry matter content increase was essentially related to the rise of lipid content. This link is shown in Figure 8, where groups A and C exhibited strong linear relationship between dry matter and lipid content (R 2 = 0.97 and R 2 =0.93, respectively); for the group B this relationship was less strong, probably due to a single outlier liver or to problems during the analysis. For the two treatments a lactate concentration of approx. 15 µmol/ g whole liver was registered, whereas for the control group the concentration reached on average 26 µmol/g whole liver (Figure 9). Guy et al. (2012) reported that in Landaise male geese reared in same conditions, during the last three weeks of stimulation treatment (from week 29 to week 31) there is an increase in the concentration of lactate. Our data showed with strong statistically significant difference that the concentration of lactate registered for the control groups (birds slaughtered at 19 weeks of age), when expressed over grams of fat free liver, was lower than that registered for overfed and stimulated birds, slaughtered after fattening period. Concentration of lactate in the two treated groups (overfed and stimulated) was similar thus it can be regarded as independent from the fattening method. According to Guy et al. (2012) this lactate content increase observed at the end of treatment could be related to a better uptake of circulating lactate involved in the process of liver gluconeogenesis and lipogenesis through its oxidation to pyruvate. The fact that lactate content in whole liver was higher in the control birds, could depend on the fact that livers from fattened geese contains a large amount of fat and that lactate cumulates mainly in the lean part of the liver. At the same time the concentrations of free glucose (Figure 10) and glycogen (Figure 11) in the fat free livers of stimulated birds were higher than that of overfed and control birds. Considering the concentration of free glucose (Figure 10), it is possible to notice that it increased during the stimulating treatment, from week 19 (control group) to the end of the treatment. This variation in the concentration could be explained by different activity of glycogenolysis, or a higher gluconeogenesis, as suggested by the potential higher lactate uptake suggested by results in Figure 9. 2012) reported glycogen levels of approximately 90 µmol/g of whole liver weight and 175 µmol/g of fat free liver. This concentrations are higher than those registered in this study (Figure 11) where the content of glycogen reached on average 48 µmol/g of whole liver and 102 µmol/g of fat free liver. In both experiments the food was available until the animal were collected for slaughter but probably the time between collection and slaughter was different in the two studies. Indeed, some authors have shown that glycogen levels decrease very quickly in geese livers as the time elapsing from the last meal increases (Leprette et al., 1997, Salichon et al, 1994). The comparison of the results for the lactate content of livers of the three groups A, B and C (Figure 12) did not show any statistically significant difference related to liver weight classes or treatment. At the same time the concentration of free glucose (Figure 13) reached higher values in the stimulated geese' groups A and B ( 21 and 20 µmol /g of whole liver, respectively) than in the overfed group (13 µmol /g of whole liver). The The glycogen concentration was not affected by the liver weight classes (Figure 14) within stimulated birds, whereas its concentration remained significantly higher in stimulated compared to overfed livers , considering either whole or fat free livers. In more detail, the concentration of glycogen in livers ranked in classes A and B values that were more than two--fold higher than the values registered in overfed livers of class C. It could be hypothesized that this difference is due to a shorter time elapsing between the last meal and the slaughter; however, it seems that the same interval was respected for the slaughter of overfed and stimulated birds. On the other hand, it has been shown that premigratory hyperphagia leads to an increase in liver glycogen (that is the primary fuel for takeoff) which can be related to the values we recorded (see review of Barlein and Gwinner, 1994).

Lipid profile

The lipid profile obtained by gas chromatography (GC) shows the different contribution of neutral lipid classes in the total composition of liver lipids. Some statistically significant differences were evidenced between stimulated and overfed geese in the neutral lipid classes (Table 11). Diglycerides were significantly more abundant in overfed liver than in stimulated ones 1.73 vs 1.37% total neutral lipids; p<0.001), the reverse was observed for the cholesterol esters (1.38 vs 2.20%total neutral lipids, respectively; p<0.001) (Table 11).

In control birds, the relative content of Free fatty acids (FFA) was significantly higher than in overfed and stimulated livers (13.8 vs 0.42 and 0.31% total neutral lipids, respectively; p<0.001). The same pattern was observed for monoglycerides and cholesterol. Control birds also show higher contents of diglycerides and cholesterol esters.

In fattened birds' livers, the most representative neutral lipid class was the triglycerides, with percentages around 96% of the total neutral lipids, whereas in control birds triglycerides represent approximately 55 % of total lipids. These levels reflect the status of hepatic steatosis, that is characterized by a high accumulation of triglycerides in the liver. In the second analysis (Table 12), where liver weight classes A, B and C were compared, the effect of treatments appeared to be less significant. A significant difference was found only for the two lipid classes of diglycerides and cholesterol esters. Indeed, cholesterol esters were relatively more abundant in groups A and B than in group C, whereas the reverse was observed for Diglycerides. These differences were apparently not related to the total lipid content of livers, since data about this parameter were not significantly different (Figure 7) among the three experimental groups; neither it was related to the different liver weights, since the two classes C and B (that were represented by livers of the same weight range), were significantly different in terms of percentage of cholesterol esters and diglycerides classes. The difference between overfed and stimulated groups in terms of free fatty acids could be explained by the fact that the level of hepatic free fatty acids and the activity of LPL change in function of the digestive post prandial condition of animals (Bouiller Oudot et al., 2004). An higher amount of diglycerides an free fatty acids could be related to ah higher rate of lipigenesis. The class of triglycerides were still the most representative for both treatments and for all the three groups A, B and C, with no differences between groups. No monoglycerides were detected in the three groups and there were no significant differences for the Cholesterol level in the three groups analyzed. The statistical analysis of the scores given to livers of classes A and B for the sensory properties, showed that the texture of fatty livers of class B was more heterogeneous, the taste was more persistent and the product was less smooth than that of class A fatty livers. This difference could not be explained on the basis of different chemical or biochemical compounds composition, since these analysis did not highlight differences in compositions for these livers.

The comparison between the groups C and B scores, showed some significant differences between the two classes. Firstly, considering the sensory properties, the livers' colour was darker in class B and, this result confirms data obtained by trichromatic CIE Lab coordinates (L*a*b*) discussed in the "Overfeeding and stimulation performance section" (page 37). The yellow colour of the fat component was more pronounced in B class livers; moreover, the livers of this class presented a more intense global smell. Class C livers were more prone to melting, less bitter and presented more heterogeneous texture. It is possible to suppose that these changes on sensory parameters reflect the different composition of the two kind of livers obtained by two different modality of animal rearing.

Lastly, the T--Student test for the scores of groups C and A showed the same significant differences found when comparing B and C classes livers; moreover, for the sensory profile there were also other significant differences in terms of global and lean liver flavour ( A more intense than C) and the fat amount of slices was higher in class A livers. The fat resulted, in fact more yellow and that could have been a consequence of a different fat accumulation, in term of lipid classes, that may corresponded to different flavour perceptions, or to differences in the accumulation of corn pigments (carotenoids in particular).

CONCLUSIONS

The aim of this project was to compare two types of fatty livers, one obtained by "standard overfeeding" and the other one obtained by "stimulation" rearing method, that exploit Landaise geese natural capacity of fattening under proper stimulation of photoperiod and feeding.

Data presented in this study clearly show that under specific management conditions of feeding and photoperiod , the geese are able to fatten and initiate spontaneous liver steatosis. Tough some livers may reach weight similar to those obtained by standard overfeeding (over 800 g), the variability in the response remain very high and the average levels of body fattening and liver steatosis reached under these experimental conditions, are still far below the standards of overfed method. These differences in term of chemical and biochemical composition have also effects on sensory qualities, in fact overfed livers results to be globally more appreciated, on a sensory point of view, than fatty livers obtained by stimulation. This work represents another headway for the development of an "alternative " production of fatty livers. Further studies will be necessary to understand which is the source of the individual variability in the capacity to develop spontaneous liver steatosis and to evaluate the precise moment and conditions in which the light stimulation must be used.

If the individual variability was found to be related to genetic factors, it will be maybe possible to select geese with high aptitude for spontaneous steatosis.

Finally natural stimulation could be efficient enough to replace standard overfeeding practice, that nowadays represents the object of strong criticisms on an animal welfare concern.

• Bairlein, F. 1987. Nutritional requirements for maintenance of body weight and fat deposition in the long--distance migratory garden warbler Sylvia borin (Boddaert). Comparative Biochemistry and Physiology A, 86: 337--347.

• Bairlein, F. and E. Gwinner.1994. Nutritional mechanisms and temporal control of migratory energy accumulation in birds. Annual Review of Nutrition, 14: 187--215.

• Baudonnet, C. 1993. facteurs de variation de la composition biochimique et de la qualité technologique des foies gras de canards. Thèse de Doctorat, Institut National Polytecnique de Toulouse, 182p. • Evans, A. J. 1972.In vitro lipogenesis in the liver and adipose tissue of the female Aylesbury duck at different stages. British Poultry Science. 13 : 595--602.

• Folch J., Lees M. and Sloane Stanley G.H., 1957. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226: 497--509.

• Fournier, E., Peresson, R., Guy, G., Hermier, D. 1997. Relationship between storage and secretion of hepatic lipids in two breeds of geese with different susceptibility to liver steatosis. Poultry Science. 76 : 599--607.

• French 'Code rural', Article L654--27--1 5 (consultato il 3 agosto 2012)

• Goodridge, A.G. 1987. Dietary regulation of gene expression: enzymes involved in carbohydrates and lipid metabolism. Annual Review of nutrition. 7 : 157--185.

• Griffin, H., Grant, G., Perry, M. 1982. Hydrolysis of plasma triacylglycerolrich lipoproteins for immature and laying hens ( Gallus domesticus) by lipoprotein lipase in vitro. The Biochemical Journal. 206 : 647--654.

• Guéméne, D., Guy, G. 2004. The past, present and future of force--feeding and "foie gras" production. World' Poultry Science Journal, Vol. 60.

• Guéméne, D., Guy, G., Noirault, J., Destombes, N., Faure, J. M. 2006.

Rearing conditions during force--feeding period in male mule ducks and their impact upon stress and welfare. Anim Res 55.

• Guy, G., D. Rousselot--Pailley and D. Gourichon. 1995. Comparaison des performances de l'oie, du canard mulard et du canard de Barbarie soumis au gavage. Annales de zootechnie, 44 : 297--305.

• Guy, G., Fortun--Lamothe, L., Bérnard, G., Fernandez, X. 2012. A natural induction of liver steatosis in Greylag Landaise geese. Accepted for publication in Journal of Animal Science.

• Guy, G., Lamothe, L., Fernandez, X.

  Je remercie chaleureusement Mr. Xavier Fernandez, Directeur des Recherches à l'INRA, qui m'a accueillie dans son laboratoire, pour son soutien et sa confiance. Aussi je remercie toute l'équipe TANDEM :Hervé Rémignon et Caroline Molette-pour toute leurs aide pour ma future permanence en France, Michel --pour sa patiente, ses enseignements et les the' glacées, Nathalie, Corinne et Stéphane-pour tout leur sourires, leur aide et amitié, Hélène--pour sa gentillesse, amitié et ses conseilles scientifiques et moins scientifiques, Cécile pour son aide précieuse avec Excel et pour l'initiation au fresbee. Un grand merci au jeunes du labo: Sahar, Yoannah, Nadia, Carole, Anaïs, Florian, Christine, Julien et Mickël. Merci pour votre amitié, vos sourires et votre patience avec moi et mon française!

Figure 2 .

 2 Figure 2. Mechanisms of hepatic steatosis in ducks (after Saez, 2009)

Figure 3 .

 3 Figure 3. Alternative overfeeding method. Scheme of the experiments carried out in 2012 and 2011

Figure 4 .

 4 Figure 4. Time related changes of liver weight and dry matter content. Winter 2009--2010 (a) and winter 2010--2011 (b)

  x 100) / (3 x Vlip x TS) With: V= solvent volume (40 ml, Folch mix) R= Residual lipids (in the Wheaton tube, after evaporation) Vlip= 10ml (volume in the Wheaton tube) TS= Test Sample 3.6 Dosage of lactic acid and glycogen

  di--oleyl 2,3, glycerol (POO), Palmityl 1,3, oleyl 2, glycerol (POP), Tripalmitine (PPP),

Figure 5 .

 5 Figure 5. Comparison of lipids and dry matter content (%) among overfed, stimulated and control geese

Figure 6 .

 6 Figure 6. Relationship between lipid content and dry matter in livers of stimulated and overfed geese

Figure 7 .

 7 Figure 7. Comparison of lipids and dry matter content (%) between different liver weight classes and treatment

Figure 8 .

 8 Figure 8. Relationship between lipid content and dry matter in geese livers of different weight classes and treatment

Figure 9 .

 9 Figure 9. Lactate content of the livers (µmol/g) for overfed, stimulated and control geese

Figure 10 .

 10 Figure10. Free glucose content of the livers (µmol/g) for overfed, stimulated and control geese

Figure 11 .

 11 Figure 11. Glycogen content of the livers (µmol/g) for overfed, stimulated and control geese

Figure 12 .

 12 Figure 12. Lactate content of the livers (µmol/g) for different liver weight classes and treatment

  found in the comparison among treatment groups overall (Figure10).

Figure 13 .

 13 Figure 13. Free glucose content of the livers (µmol/g) for different liver weight classes and treatment

Figure 14 .

 14 Figure 14. Glycogen content of the livers (µmol/g) for different liver weight classes and treatment

  but the discussion is still open. The production of fatty liver has developed in many European countries, like France, Hungary, Spain and Bulgaria, but also in the Unites States and China. France is the leading producer and consumer of foie gras in the world. In France, by tradition, the production areas of foie gras are the Alsace and the south--west of the Country, including the regions of Aquitaine and Midi--Pyrénées. For the European Union to be recognized as foie gras, the fatty liver has to be produced according to traditional farming method in the south west of France. Commercial policies based on a geographical indication of provenance (Indication Géographique Protégée,IGP) have recently emerged in the south west of France.

1.1.2 Europe and overfeeding

Nowadays the production of foie gras in Europe is object of discussion for the animal welfare; the Council of Europe has issued regulations that ban overfeeding in all countries when it is not already practiced, and requires researchs on alternative methods which do not include overfeeding (Official Journal of the European Communities, L221/23, COUNCIL DIRECTIVE 98/58/EC, 20 July 1998 concerning the protection of animals kept for farming purposes). Some Europeans countries, like

Denmark (1991), Germany (1993)

and Italy

  Table 1) was different in the three group; the overfed birds were slaughtered at 17 weeks of age , the stimulated birds at the age of 31 weeks, because it was demonstrated by Guy et al. (2012) that this is the age corresponding to the the optimum stage for a heaviest liver, without mortality. Birds of control groups were slaughtered at the age of 19 weeks, to have a "reference group" before the corn feeding.

	Table 1. Characteristics of the experimental groups
	experimental groups	Number (n)	Slaughtering age (weeks)
	Overfed	85	17
	Stimulated	125	31
	Controls	20	19

Table 2

 2

	. Feeding program of stimulated birds
		Feeding program
	Age (weeks)	Food type	Amount
	0 to 4	D15	ad libitum
	5	D15	300 g
	6	D15	350 g
	7 to 8	D25	350 g
	9 to 10	D25	300 g
	11 to 13	D25	280 g
	14 to 20	D25	250 g
	After 20	fresh corn	ad libitum

Table 3 .

 3 Feeding program of overfed birds

	Age (weeks)	Food type	Amount
	6
	6 and 7
	8 (for2 days)
	8 (for2 days)
	8 (for2 days)
	9 to 13
	14 to 17

Table 4 .

 4 Weight classes of livers for the three treatments groups.

	The number of

Table 5

 5

	. Oven program for Gas Chromatography analysis
			Parameters
	Level	Rate (°C/min)	Final temp. (°C)	Final time (min)
	0		100	3.00
	1	30.0	170	1.00
	2	20.0	250	0.00
	3	8.0	345	11.4

Table 7 .

 7 Fattening performance of the experimental groups (m ± SEM) Groups

	Parameters	Overfed	Stimulated	Controls	Significance
		(n=45)	(n=42)	(n=8)
	Slaughter weight (g) 9287 ± 69 a 8242 ± 98 b 6199 ± 118 c	***
	Weight gain (g)	2603 ± 32	1866 ± 82	--	ns
	Liver weight (g)	1102 ± 23 a	445 ± 24 b	101 ± 3 c	***
	Different letters indicate significant differences among the treatments for the
	parameters considered;
	ns: not significant *: p<0.05 **:p<0.01 ***:p<0.001

Table 8

 8

	. Fattening performance of the experimental groups at different liver weight
	classes (m ± SEM)
			Groups
	Parameters	Overfed C	Stimulated A	Stimulated B	Significance
		(800--1000 g)	(600--800 g)	(800--1000 g)
		(n=18)	(n=28)	(n=9)
	Slaughter weight (g)	9191 ± 120	8683 ± 193	8923 ± 459	ns
	Weight gain (g)	2406 ± 68	2255 ± 169	2244 ± 427	ns
	Liver weight (g)	914 ± 10 a	685 ± 10 b	907 ± 18 a	***
	Different letters indicate significant differences among the weight classes for the
	parameters considered;
	ns: not significant *: p<0.05 **:p<0.01 ***:p<0.001

Table 9 .

 9 Body composition analysis of overfed, stimulated and control geese Groups

	Parameters	Overfed	Stimulated	Controls	Significance
		(n=45)	(n=42)	(n=8)
	Abdominal fat (g)	530 ± 9 a	579 ± 14 a	222 ± 10 b	***
	Carcass weight (g)	5850 ± 50 a	5802 ± 60 a	4986 ± 103 b	***
	Breast muscle (g)	274 ± 2 c	327 ± 5 a	308 ± 6 b	***
	Breast skin (g)	224 ± 3 a	220 ± 4 a	104 ± 4 b	***
	Thigh muscle & bones (g)	420 ± 4 a	408 ± 3 a	367 ± 8 b	***
	Thigh skin (g)	328 ± 59 a	277 ± 6 b	149 ± 5 c	***
	Liver Cooking yield (%)	88.3 ± 1.3 a	82.0 ± 2.1 b	--	*
	Liver colour
	L*	69.2 ± 0.2 a	54.8 ± 0.7 b	--	***
	a*	8.0 ± 0.1 b	9.5 ± 0.2 a	--	***
	b*	27.6 ± 0.3 b	29.8 ± 0.7 a	--	**
	Different letters indicate significant differences among the tratments for the parameters
	considered;
	ns: not significant *: p<0.05 **:p<0.01 ***:p<0.001

Table 10 .

 10 Body composition analysis of the experimental groups at different liver weight classes.

	Groups

Different letters indicate significant differences among weight classes for the parameters considered; ns: not significant *: p<0.05 **:p<0.01 ***:p<0.001

Table 11 .

 11 Composition of neutral lipid classes in livers of overfed, stimulated and control geese Groups

	Parameters	Overfed	Stimulated	Controls	Significance
		(n=45)	(n=42)	(n=8)
	Free Fatty acids	0.42 ± 0.03 b	0.31 ± 0.01 b 13.8 ± 1.0 a	***
	Monoglycerides 0.004 ± 0.004 b	0 b	0.75 ± 0.10 a	***
	Cholesterol	0.45 ± .008 b	0.54 ± 0.01 b 21.8 ± 1.9 a	***
	Diglycerides	1.73 ± 0.06 b	1.37 ± 0.04 c 3.27 ± 0.24 a	***
	Cholesterol esters 1.38 ± 0.04 c	2.2 ± 0.08 b 5.95 ± 0.43 a	***
	Triglycerides	96.0 ± 0.08 a	95.6 ± 0.1 a	54.5 ± 3.2 b	***
	Different letters indicate significant differences between the treatment for the
	parameters considered;
	ns: not significant *: p<0.05 **:p<0.01 ***:p<0.001

Table 12 .

 12 Composition of neutral lipid classes in livers of different weight classes and treatmentGroups

	Parameters	Overfed C	Stimulated A	Stimulated B	Significance
		(800--1000g)	(600--800g)	(800--1000g)
		(n=18)	(n=28)	(n=9)
	Triglycerides	95.8 ± 0.2	95.5 ± 0.2	95.8 ± 0.2	ns
	Diglycerides	1.93 ± 0.18 a	1.34 ± 0.04 b	1.15 ± 0.07 b	***
	Monoglycerides	0	0	0	ns
	Free Fatty acids	0.44 ± 0.05 a	0.33 ± 0.02 b	0.28 ± 0.03 b	*
	Cholesterol	0.49 ± 0.03	0.544 ± 0.022	0.50 ± 0.02	ns
	Cholesterol esters 1.36 ± 0.10 a	2.32 ± 0.13 b	2.23 ± 0.22 b	***
	Different letters indicate significant differences among treatments for the parameters
	considered;
	ns: not significant *: p<0.05 **:p<0.01 ***:p<0.001

Table 13 .

 13 Results of sensory tests for different sensory classes. Scores are expressed as mean ± SD. Significance refers to couple comparison between groups (see appendix for more detailed results) Groups

	Parameters	Overfed C	Stimulated A	Stimulated B	Significance
		(800--1000g)	(600--800g)	(800--1000g)
		(n=18)	(n=28)	(n=9)
	Appearance
	Colour intensity 3.45 ± 1.13	5.45 ± 1.57	6.82 ± 1.76	***
	Colour homogeneity	4.27 ± 2.05	5.27 ± 1.68	5.09 ± 1.81	ns
	Fat amount	1.55 ± 1.57	3.73 ± 1.90	2.55 ± 1.37	*
	Fat yellow colour	2.91 ± 2.12	5.00 ± 2.10	4.64 ± 2.25	**
	Smell
	Global smell	3.82 ± 1.89	5.18 ± 1.83	5.45 ± 2.02	*
	Alcohol smell	0	0.18 ± 0.60	0	ns
	Texture
	Mellowness	5.91 ± 1.64	4.91 ± 1.92	4.18 ± 2.48	ns
	Melting	6.45 ± 1.51	5.64 ± 1.75	4.36 ± 2.62	S10
	Smoothness	5.82 ± 1.83	5.27 ± 1.27	3.73 ± 2.41	*
	Homogeneity	7.00 ± 1.41	6.36 ± 1.50	4.55 ± 1.81	**
	Taste
	Salty	3.55 ± 1.21	4.09 ± 1.30	4.18 ± 1.54	ns
	Bitter	1.27 ± 2.20	3.09 ± 2.43	4.36 ± 3.01	*
	Flavour
	Global flavour	5.64 ± 2.11	6.55 ± 1.13	6.45 ± 1.75	S10
	Persistence	6.09 ± 1.92	6.55 ± 1.44	7.18 ± 1.54	*
	Lean liver	3.27 ± 2.53	4.45 ± 2.50	3.91 ± 2.59	*
	Goose	5.09 ± 2.47	5.36 ± 1.63	4.64 ± 2.62	ns
	Pepper	2.18 ± 1.54	2.55 ± 1.37	2.55 ± 1.97	ns
	Alcohol	0	0	0.09 ± 0.30	ns
	Strange	0	0.09 ± 0.30	0	ns

ns: not significant S10: p<0.1 *: p<0.05 **:p<0.01 ***:p<0.001

•

  Bénard, G., Labie, C. 1998. Evolution histologique du foie des palmipèdes au curs du gavage. In: 3émes Journées de la Recherche sur les Palmipèdes à Foie Gras. Bordeaux, France. 31--35. • Bergmeyer, H. U. 1974. in Methods of enzymatic analysis (ed. GH Bourne), pp. 1127, 1196, 1239, 1464. Academic Press, New York. • Besnard, P., Niot, I., Poirier, H., Bernard, A., Caseeli, C. 1997. Aspects cellulaires et moléculaires de l'absorption intestinale des acides gras. Oléagineux, Corps gras et Lipides. 4 : 185--191. • Blum, J.C., Labie, C., Raynaud, P. 1990. Influence du poids et de la composition chimique du foie gras d'oie sur la fonte mesurée après stérilisation à 104°C. Science des Aliments. 10 : 543--554. • Bouiller--Oudot, M., Leprettre, S., Dubois, J. P., Babilé, R. 2004. Evolution de la composition hépatique lors du refroidissement post mortem de foies gras d'oies dans la carcasse. 6 èmes Journeés de la recherché sur les Palmipèdes à Foie Gras. Arcachon, France. 203--206. • Comité Interprofessionnel des Palmipèdes à Foie Gras (CIFOG).2011. Assemblée générale du 24 juin 2011. Rapport économique de l'année 2010. 75p • Dalrymple, R. H., Hamm, R. 1973. A method for the extraction of glycogen and metabolites from a single muscle sample. Journal of food technology.

8: 439--444.

  2010. L'engraissement spontané des Palmipèdes comme alternative au gavage: socle des connaissances scientifiques actuelles. b) Level of significance for couple comparisons between groups A, B and C for sensory test

			Comparisons
	Parameters	A vs B	A vs C	B vs C
	Appearance
	Colour intensity	0.1465	0.0027 **	0.0006 ***
	Colour homogeneity	0.8068	0.2192	0.4509
	Fat amount	0.1153	0.0119 *	0.1530
	Fat yellow colour	0.3705	0.0106*	0.0113 *
	Smell
	Global smell	0.7433	0.0162 *	0.0225 *
	Alcohol smell	0.3409	0.3409	--
	Texture
	Mellowness	0.3774	0.2124	0.1092
	Melting	0.1856	0.1581	0.0481 *
	Smoothness	0.0605	0.2578	0.0579
	Homogeneity	0.0285 *	0.0455 *	0.0047 **
	Taste
	Salty	0.8100	0.0251 *	0.1717
	Bitter	0.4016	0.0479 *	0.0240 *
	Flavour
	Global flavour	0.7961	0.0429 *	0.1901
	Persistence	0.0261 *	0.2425	0.0142 *
	Lean liver	0.1669	0.0046 **	0.1901
	Goose	0.4355	0.4933	0.6809
	Pepper	>0.9999	0.1039	0.5423
	Alcohol	0.3409	--	0.3409
	Strange	0.3409	0.3409	--

Stearyl 1, di--oleyl 2, 3, glycerol (SOO), Palmityl 1, oleyl 2, stearyl 3, glycerol (POS), Di--palmityl 1,2, stearyl 3, glycerol (PPS), Palmityl 1, di--stearyl 2,3, glycerol (PSS)/ palmityl 2, di--stearyl 1,3, glycerol (SPS).

For the groups stimulated and overfed, starting from the lipid extract obtain by Folch et al. method (1957), the sample to inject was prepared operating different dilutions in function of the quantity (mg/ml chloroform) of total lipids observed in the sample. For the control group, before preparing the sample to inject, we separated neutral lipids and phospholipids using a specific cartridge (SepPak®), as for the CG analysis. The samples were manually injected in the circuit through an injection valve of 20 µl; the flow was set to 1 ml/min and the temperature of column was maintained at 15 °C. The injection was realized into a column Phenomenex™ 2.6µ C18 100Å, 150x4.6mm (Kinetex); a good separation of different lipid classes is assured by using a binary gradient of migration acetonitril -dichlorometan (Table 6). 31.00 20 80 In the detector of light diffusion, the temperatures of nebulization and evaporation were respectively 60 °C and 80 °C, the gas flow and the gain were set at 1.0 and 2.1.

Sensory and acceptability test results

In this section the results of sensory and acceptability tests are presented as average values of the scores (from 0 to 10) given by the panel, as previously described in the material and method section.

a) Sensory test

With this test three types of fatty livers were compared: two obtained by the stimulation method (A and B, that represent two different weight classes) and one obtained by standard overfeeding (C).

b) Acceptability test

With this test the three types of fatty livers A, B and C were compared, as performed with the sensory analysis.

The results are presented in Figure 15. The livers of A and B classes showed no significant differences for any of the analyzed parameter, with a threshold level of 5%. Allowing a threshold level of 10% the class B livers can be regarded as more appreciated than class A livers.

For the others two comparisons (B vs C and A vs C) all parameters considered differed at 5% threshold level. For more detailed statistical data see the appendix.

Foie gras produced by overfeeding method was globally more appreciated than that obtained by alternative method. From a commercial point of view this result assumes a great importance, particularly if the product is intended to compete with an already existing one. On the basis of these last results, alternative fattening cannot totally substitute overfeeding in foie gras production. • Myher, J. J., Kuksis, A. 1984. Determination of plasma total lipid profiles by capillary gas--liquid chromatography. Journal of Biochemical and Biophisical Methods. 10 : 13--23.

• National Research Council. 1994. Nutrient requirement of poultry, 9 th rev. ed. Natl. Acad. Press, Washington, DC.

• Odum, E.P. 1960. Premigratory hyperphagia in birds. American Journal of Clinical Nutrition, 8: 621--629. • Salichon Y., 1991. Elevage et gavage des palmipèdes pour la production du foie gras. Armand Colin Editeur, Paris.

• Salichon, M. R., Guy, G., Russelot, D., and Blum JC. 1994. Composition des 3 types de foie gras: oie, canard mulard, and canard de Barbarie. Ann. Zootech. 43 : 213--220.