Effect of dietary Lippia citriodora extract on productive performance and meat quality parameters in hares ( Lepus europaeus Pall . )

Three different dietary doses of Lippia citriodora natural extract were tested in order to verify their effect on productive performance and the meat quality of intensively-reared hare (Lepus europaeus Pall.). The 240 day-trial was conducted on 20 male hares, divided into 4 homogeneous groups of 5 animals each. A control group received a basal diet without any supplementation, and the other three experimental groups received a natural extract of Lippia citriodora, titrated in verbascoside, in the following amounts: 1 g, 1.5 g and 2 g of natural extract/kg feed in the low (LNE), medium (MNE), and high natural extract (HNE) groups, respectively. Natural extract enhanced the quality of the meat: there was a significant decrease (P<0.05) in saturated fatty acids and a significant increase (P<0.05) in monoand poly-unsaturated fatty acids. The oxidative stability of the meat improved, thus highlighting a possible link between the decrease (P<0.05) in TBARS values and an increase (P<0.05) in lipid vitamin content. The cholesterol content of the meat decreased markedly (P<0.01) after the dietary verbascoside treatment, thus improving the health benefits of the meat. These results clearly show the important role of Lippia citriodora extract, titrated in verbascoside, in improving the quality of the meat of intensively-reared hares. In addition the present paper underlines how the use of natural antioxidant in the animal feeding system may provide healthier and low-oxidized products to the final consumer.


Introduction
Natural antioxidants in food are able to scavenge the free radicals that lead to the oxidation of cellular lipids (Di Benedetto et al. 2010).These compounds also prevent the lipid oxidation of meat, which is one of the primary causes of the deterioration of muscle tissue (Buckley et al. 1995).This is because they reduce the action of oxygen, slow the development of odours and improve the oxidative stability of the meat.Synthetic antioxidants such as butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA), are widely used for the control of meat lipid peroxidation.However their use insecure (Kahl & Kappus 1993), integration of natural antioxidants in the animal diet is a good strategy to enhance the shelf life of meat (Djenane et al. 2004).The use of antioxidants can increase the oxidative stability of polyunsaturated fatty acids thus influencing meat storage and its susceptibility to oxidation (Meineri et al. 2010).Several studies carried out on laboratory animals have shown that many natural compounds (such as polyphenols, carotenoids, anthocyanidins, vitamin C) are also interesting immunostimulants (Katiyar 2002).In fact, they integrate endogenous antioxidant defences especially under stress, particularly during weaning, pre-acclimatization and restocking young hares into the natural environment.Among plant extract compounds, Lippia citriodora extract (containing verbascoside or acteoside) have shown the highest scavenger capacity in the group of phenylpropanoids glycosides (Wang et al. 1996) and the highest antioxidant activity compared to other phenyl compounds.Previous studies have shown that dietary supplementation of Lippia citriodora extract, positively influenced the plasma oxidative status in Lacaune ewes during the peripartum period (Casamassima et al. 2012) and in intensively reared Lepus corsicanus hare (Palazzo et al. 2011).A dietary Lippia citriodora supplement could thus be used in animal feeding to improve animal welfare, meat quality and also have health benefits for consumers.The use of dietary natural extracts can also be useful when the hares are slaughtered as the extracts are rich in antioxidant substances (flavonoids, tannins, polyphenols, terpenes), which then has a positive impact on the shelf life and on food safety (Sánchez-Escalante et al. 2003).These antioxidant substances act through many various physiological mechanisms.Rosemary, for example, chelates metal ions such as Fe -2 , resulting in a reduction in the formation of the activated oxygen (Fang & Wada 1993).The inclusion of antioxidants in food can have positive effect on human health because they protect important cellular components such as DNA, proteins and lipid membranes by reactive oxygen substances (Elmastas et al. 2007).The literature is poor regarding the effect of Lippia citriodora extract on meat quality (Rossi et al. 2013), and there are no data regarding the effect of this extract on hare meat.Skrivanko et al. (2008) recommend the addition of hare meat in the diet, due to its sensory characteristics, its high protein content, its low fat content and its energetic value which is similar to other meats.In order to verify a possible antioxidant effect on animal products using a feed strategy, a dietary Lippia citriodora supplement was evaluated on productive parameters and various meat quality traits of intensively-reared hare.

Animals, diet and experimental protocol
The 240 day-trial was conducted on 20 male hares (Lepus europaeus Pall.), divided into 4 groups of 5 animals each, homogeneous by age (210 ± 5 days) and body weight (2.90 ± 0.315 kg).A control group received a basal diet without any supplementation, and the other three experimental groups received a natural extract of Lippia citriodora leaves (Verbenaceae), titrated in verbascoside (5 mg/g of natural extract), according to the following amounts: 1 g, 1.5 g and 2 g of natural extract/kg feed in the low natural extract (LNE), medium natural extract (MNE), and high natural extract (HNE) groups, respectively.The natural extract supplement did not contain vitamin E and the composition of the natural extract, according to a certificate of analysis provided by the manufacturer (Lombarda Trading, Casalbuttano ed Uniti, Italy), was: gallic acid, 1.75 ± 0.07; 3.4-dihydroxybenzoic acid, 0.45 ± 0.04; methyl gallate, 1.91 ± 0.09; isoverbascoside, 0.43 ± 0.04 and verbascoside, 4.47 ± 0.08 g/kg.The concentrated pellets were provided by Martini S.p.A (Budrio di Longiano, Italy) and the chemical composition of the feed (AOAC 2000) were (per kg of dry matter): crude protein 154 g; crude fat 33 g; crude fibre 195 g; Neutral Detergent Fibre 385 g; Acid Detergent Fibre 240 g; ashes 85 g, moisture 111 g.The feed concentrate and alfalfa hay were administered daily ad libitum.All the hares were reared in a weaning-fattening cage with feeders and automatic water dispensers until slaughter.At slaughter, the relief of body weight and weight of the carcass were registered, and the killing percentages were determined.In addition the following quality parameters were performed: chemical composition, meat fatty acidic profile, cholesterol content, thiobarbituric acid reactive substances (TBARS) content, vitamin A, and vitamin E concentrations of longissimus lumborum muscle.Non-fasting animals were slaughtered at the age of 450 ± 5 d in an experimental slaughterhouse, and immediately before slaughter the body weight of all the hares was recorded.The hares were stunned electrically and sacrificed by bleeding, following the guidelines established by the European Community (n.86/609/EEC) and approved by the Italian Ministry of Health (Law 116/92), in accordance with Italian laws on the slaughter and killing of animals.The carcasses were prepared as reported by Blasco & Ouhayoun (1993) for rabbits by removing the skin, the distal part of the limbs, genital organs, bladder and gastrointestinal tract.Warm carcasses were weighed and the killing percentage was calculated.After the carcasses had chilled at 4 °C for 48 h, the content of TBARS and chemical composition of meat were determined in a sample of longissimus lumborum, cut between the first and the seventh right-side lumbar vertebra.The determination was made following Meineri et al. (2010), andcomplied with ASPA recommendations (1996).The rest of the longissimus lumborum muscle samples were vacuum packaged and frozen at −20 °C until analysis.The fatty acid composition of intramuscular fat was determined after chloroformmethanol extraction (Folch et al. 1957), and fatty acids were determined as methyl esters (FAME) (Dal Bosco et al. 2004), using a gas chromatograph ThermoQuest TRACE 2000 (SAC tm-5 column 300 cm × 0.25 mm, Supelco, USA).The fatty acid percentages were calculated with Chrom-Card software v. 1.17.Cholesterol content was determined according to Du & Ahn (2002) while vitamin A and E contents were determined according to Oriani et al. (2001).

Statistical analysis of the data
After the normal evaluation of frequency distribution, analysis of variance was performed on all variables using the GLM procedure of the statistical package SPSS v. 18 (SPSS Inc., Chicago, IL, USA).The productive data were processed using one-way ANOVA with the dietary treatment as a main effect.All data are expressed as mean ± standard error.The differences were considered significant at P<0.05.

Results and discussion
The dressing percentage and chemical composition data of hare meat are reported in Table 1.The dietary natural extract supplementation did not affect any of the parameters shown in the table.Table 2 reports various qualitative traits and the oxidative stability of longissimus lumborum muscle.Total saturated fatty acids were affected by natural extract (P<0.05),resulting in lower values in groups LNE, MNE and HNE by 12.3 %, 12.6 % and 15.4 % respectively, compared to the control group.Monounsaturated fatty acids were also influenced by dietary treatment with an increase of 9.5 %, 13.5 % and 11.1 % respectively in the LNE, MNE and HNE groups compared to the control group.Lippia citriodora extract significantly influenced (P<0.05) the polyunsaturated fatty acids values, which increased in the LNE, MNE and HNE groups, by 12.4 %, 9.7 % and 16.1 % respectively, compared to the control group.Dietary supplementation naturally led to a variation in the n-3 and n-6 polyunsaturated fatty acids, by significantly decreasing (P<0.05) the n-6/n-3 ratio, compared to the control group.In fact, the ratio decreased by 33.0 %, 14.3 % and 23.7 %, respectively in the LNE, MNE and HNE groups.The meat TBARS content was lower (P<0.05)by 25.4 %, 27.7 % and 37.5 % in the LNE, MNE and HNE groups compared to the control group.Regarding the meat vitamin concentration, vitamin E increased significantly (P<0.01) by 28.4 %, 21.4 % and 64.1 % in the three experimental groups (LNE, MNE and HNE respectively).Vitamin A on the other hand increased significantly (P<0.05)only between the control group and HNE group (37.5 %).The meat cholesterol content was positively influenced (P<0.01) by the dietary treatment, which in the experimental groups was lower by 23.5 % in the LNE group, by 26.4 % in the MNE group and 31.8 % in the HNE group compared to the control group.
The productive data and chemical composition of hare meat (Table 1) resulting from our test, are in agreement with the literature (Vicenti et al. 2003); also the meat fatty acidic profile and the content of individual fatty acids (Table 2) are in line with Vicenti et al. (2003).Table 2 highlights an improvement in the quality traits of the experimental animal groups.In fact, the natural extract reduced the saturated fatty acids and increased the mono-and poly-unsaturated fatty acids with an improvement in the n-6/n-3 ratio.This is particularly important considering that polyunsaturated fatty acids, especially the n-6 and n-3 series, are not synthesized by the human body (Kulasek & Bartnikowska 1994).Our results are in line with Bernardini et al. (1999) who in growing rabbits fed with 160 g flax seeds per kg of food, reported an increase in polyunsaturated fatty acids with a decrease in the n-6/n-3 ratio.Recently, researchers focused on the use of plant-based substances to improve the cholesterol content and to modify the acidic profile in meat.In fact, Habibian Dehkordi et al. (2010) have shown a decrease in cholesterol and lipoproteins in chicken meat when extracts of garlic are used in feed.The decrease in TBARS content and the increase of vitamin E in treated-animal meat, highlight that dietary natural extract supplement increased the shelf life of the meat.In fact, many dietary spices, including oregano (Origanum vulgare L.), rosemary (Rosmarinus officinalis L.) and sage (Salvia officinalis L.) have a high antioxidant capacity (Wojdyło et al. 2007).Natural antioxidants, in fact, delay or inhibit the oxidation of other substances, because of inhibition chain reactions oxidant propagation.Polyphenols, including verbascoside, have a high antioxidant activity through three mechanisms: as a scavenger of free radicals (Zheng et al. 2009), a chelator of transition- metal (Andjelković et al. 2006), and/or through the ability to detect singlet oxygen (Mukai et al. 2005).Some researchers (Botsoglou et al. 2003) reported that the low MDA formation in meat by feeding animals with oregano essential oils, is probably due to the presence of antioxidant compounds which pass to the blood, and are distributed and stored in muscles and other tissues.The decrease in cholesterol content found in the experimental groups, can be attributed to the effect of antioxidant substances which can stimulate or inhibit the hepatic action of HMG-CoA reductase, an enzyme that controls the synthesis of cholesterol (Kowalska & Bielanski 2009).
In conclusion, the use of Lippia citriodora extract positively affected the quality of the meat, with a significant decrease in saturated fatty acid content and a significant increase in mono-and poly-unsaturated fatty acids in treated meat.The oxidative stability of the meat improved, highlighting a possible link between the decrease in TBARS values and the increase in vitamins content.In addition, the cholesterol content of the meat decreased markedly after the dietary natural extract treatment, thus improving the health benefits of the meat.These results (cholesterol and TBARS content, vitamin E concentration) could suggest that the best dose of natural extract supplementation is the highest (2 g/kg feed), but further investigations need to confirm this conclusion.This experiment clearly shows the important role of natural extract in enhancing the health and welfare of intensively-reared hares and also in improving the quality of the meat.

Table 1
Effect of Lippia citriodora extract on dressing percentage and some meat quality parameters in intensivelyreared hares