Fatty acid and lipid contents differentiation in cuts of rabbit meat

ABSTRACT The study aimed at quantifying fatty acids (FA), especially the health-promoting ones, in cuts of rabbit meat. Loin, containing mostly glycolytic muscles, had less lipids than cuts containing oxidative (hind leg) or intermediate muscles (foreleg and brisket). Brisket had the highest polyunsaturated FA (PUFA), odd- and branched-chain FAs (OBCFA) and palmitoleic acid C16:1 n-7 contents, better n-6/n-3 proportion, advantageous nutritional indexes (AI, TI, H/H) than other cuts. Hind leg contained less atherogenic saturated FAs (LCSFA) than other cuts. Foreleg was rich in two important n-3 LC-PUFAs: C20:5 and C22:5. The results suggest that apart from the preferred cuts (loin and hind leg) used in gastronomy, brisket and foreleg should be included for being rich in nutritionally valuable PUFA, especially from the n-3 family. Daily portion (250 g) of foreleg would cover 16% of the requirement for EPA and DHA, and the same amount of brisket or hind leg—11%.


Introduction
Meat and meat products are a source of essential amino acids (Bohrer, 2017) as well as of macro-and micronutrients, e.g. zinc, iron, magnesium, phosphorus and cobalt and of vitamins (Biesalski, 2005;Dalle Zotte & Szendrö, 2011). On the other hand, meat and meat products are often regarded negatively due to high contents of fat, saturated fatty acids (SFA), cholesterol and sodium, that may lead to cardiovascular diseases, hypertension, obesity and diabetes (Dalle Zotte & Szendrö, 2011). Recent meta-analyses showed that high consumption of red and processed meat, in contrast to white meat (poultry, fish), moderately increase the risk for colorectal colon cancer (Corpet, 2011;Norat, Lukanova, Ferrari, & Riboli, 2002).
Dalle Zotte (2002) defined two groups of factors affecting rabbit carcass and meat quality: those having moderate or high effect on rabbit meat carcass and meat quality from the consumer's point of view. First group includes environmental conditions, especially temperature, season and rearing techniques, important for the productive and slaughtering performance, i.e. the preslaughter conditions. The higheffect factors are of genetic nature; these affect the growth capacity of rabbits and biological factors, such as body mass. This group includes also feeding regimen that not only influences growth performance, but also affects chemical composition and quality of carcasses. Technological factors play an important role for a combination of carcass processing treatments may have a greater effect on meat quality than production factors.
The composition of rabbit meat lipids is very favourable due to high contents of mono-(MUFA) and polyunsaturated (PUFA) fatty acids (FA), as well as of valuable odd-numbered straight-chain and methyl-branched-chain FAs (OBCFA), not present in meat of typical species like pork, beef or poultry (Leiber et al., 2008). The chemical composition of rabbit meat, mainly that related to lipid content and FA profile, exhibit a relatively pronounced diversity and depends on feeding, age, genotype, breeding and/or physical activity of animals, as well as on the muscle type and sex of the animals in question (Peiretti, 2012). Most studies are devoted on feeding regimen or supplementation effects on FA profile of rabbit meat and focused on FA composition of two main muscles: Longissimus dorsi and Biceps femoris (Dal Bosco et al., 2015Forrester-Anderson et al., 2006;Kowalska & Bielanski, 2009;Peiretti, 2012;Rasińska, Czarniecka-Skubina, Rutkowska, Przybylski, & Brzozowski, 2017). Alasnier, Rémignon, and Gandemer (1996) specified intramuscular lipid characteristics related to the metabolic type of muscles in rabbit carcass: two glycolytic (Longissimus lumborum and Psoas major), two oxidative ones (Soleus and Semimembranosus) and an intermediate one (Gastrocnemius). Those muscles are included mainly in two carcass cuts: loin and hind leg (Alasnier & Gandemer, 1998;Alasnier et al., 1996). Loin and hind leg are considered the most representative cuts of the carcass and most valuable because of a substantial protein content and low collagen amount (Daszkiewicz, Gugołek, Janiszewski, Chwastowska-Siwiecka, & Kubiak, 2011;Kowalska, Gugołek, & Bielanski, 2014;Pla, Pascual, & Arino, 2004). However, other parts of rabbit carcass are used in gastronomy and in meat products. Regarding Polish market, only whole carcasses are available for consumers. This prompted us to study the FA profile of rabbit meat derived from other cuts-e.g. foreleg and brisket, with special emphasis on the contents of health-promoting fatty acids.

Sampling of cuts and preparation
Sixty healthy, weaned New Zealand White breed rabbits (30 males and 30 females), 35 ± 2 days old, weighing 960 g ± 88 g, were kept at the University's experimental farm in Obory. The rabbits were fed ad libitum commercial pelleted diet containing 16.5% protein, 15.4% crude fiber and 3.20% lipids. Lipid fraction contained high amount of PUFA (50.5 g/100 g FA) with dominating linoleic acid: 41.35 g/100 g FA, MUFA (25.3 g/100 g FA) and SFA (17.7 g/100 g FA). Animals were kept indoors in wire cages (0.90 m long x 0.65 m wide x 0.40 m high), 5 rabbits of the same sex per cage. After the fattening period, at the age of 92 ± 2 days, 10 male and 10 female rabbits were selected for having their body mass within a narrow range. The animals were slaughtered in an experimental slaughterhouse by subjecting them to electrical stunning and sacrificed by bleeding, following the guidelines established by the European Community (1099/ 2099/EC 2009).
After slaughter, the carcasses were prepared by removing blood, skin, distal portions of legs, distal part of the tail, organs located in the thorax and neck (lungs, oesophagus, trachea, thymus, heart), genital organs, urinary bladder and gastrointestinal tract, liver and kidneys to obtain the reference carcass. Also, organ fat and subcutaneous fat were removed. Then carcasses were chilled at + 4°C in a ventilated room for 24 h.
Carcasses were weighted (mass of cold carcass ranging 1168-1232 g) and divided into cuts (technological joints): hind and forelegs, loin and brisket. Every cut was carefully deboned and fragmented; muscles with intramuscular fat were homogenized in a domestic mincer (Tefal NE210140, Poland); samples for analysis were vacuum packaged, immediately frozen and kept at −18°C until assayed.
Peaks were identified by comparison with Supelco 37 No. 47,885-U (Sigma, Aldrich, USA) and BR2, BR3 (Larodan, Sveden) standards. The FAs were quantified in relation to the internal standard, nonadecanoic FA (C19:0) (Sigma, Aldrich, USA) which was added before transesterification to lipid samples. Then, amounts of individual FAs were multiplied by percent fat content in given meat sample in order to express them per 100 g of meat.

Data analysis
One-way ANOVA followed by Scheffé's post-hoc test was applied to the data using Statistica 10PL software (StatSoft INc., PL, 2010), after having verified data conformity to the model. Male and female data were combined as on the market they are mixed and the consumers have no sexrelated choice. All FAs were processed independently. The level of p ≤ 0.05 was considered significant.

Results and discussion
Rabbit cuts differed significantly (p < 0.05) in water content from 70.5% in hind leg to 46.7% in brisket (Table 1). Similar results were reported by Pla et al. (2004) who studied rabbit carcasses from food chain stores in Spain, and by Szkucik and Libelt (2006). A somewhat higher water content (76%) was reported by Daszkiewicz et al. (2011) for hind leg and loin (longissimus lumborum) in male carcasses of New Zealand White rabbits fed standard pelleted diet like in our study and of similar age at slaughter.
The cuts differed significantly (p < 0.05) in their protein content, loin and hind leg showing highest, and brisket-lowest values (Table 1). Our results are in accordance with those of Pla et al. (2004), who also found the lowest protein content in the thorax and highest in the main loin muscle (Longissimus dorsi).
As stated by many authors, rabbit meat is considered lean, i.e. containing less fat than, e.g. pork, beef or lamb (Cavani et al., 2004;Dal Bosco et al., 2014;Enser, Hallet, Hewitt, Fursey, & Wood, 1996;Pla et al., 2004). Also in this study, the fat content was low in all cuts except brisket (Table 1). Highest content of fat in brisket and much higher than in our study (12.8%) was reported also by others (Kowalska et al., 2014;Pla et al., 2004). That difference Table 1. Mass of cuts and mean contents (± SD) of chemical components in meat of cuts of rabbit carcasses (n = 20).
Tabla 1. Masa de cortes y contenido medio (± DE) de los componentes químicos en la carne de cortes de conejo en canal (n = 20 Identical superscripts mean no significant differences at p < 0.05.
could have been due to differences in housing conditions. Those authors also reported the lowest fat content in loin compared with other cuts (1.20-1.52%). The fat contents in hind leg cuts shown in Table 1 were similar to those reported by Pla et al. (2004) but much higher than in the New Zealand breed (0.69%; Daszkiewicz et al., 2011;Kowalska et al., 2014). Our results are confirmed by those reported by Alasnier et al. (1996) who found that glycolytic muscles, dominating in the loin (Longissimus dorsi), contain less lipids than oxidative muscles, dominating in hind legs (eg. Semimembranosus, Biceps femoris, Gastrocnemius). However, a much higher lipid content was found in brisket and foreleg, containing mainly intermediate muscles; this might be explained by the propensity of those muscles to accumulate fat cells in the extravascular area (Alasnier et al., 1996). Application of the high-resolution gas chromatography enabled determining the contents of 39 FAs (SCSFA, LCSFA, MUFA, PUFA and OBCFA) in fat extracted from the cuts of rabbit carcasses; the contents of most FAs differed significantly between cuts (Tables 2 and 3).
Many papers were devoted to FA contents of rabbit meat, mainly regarding feeding and rearing factors, the results being expressed as lipid fractions (Dal Bosco et al., 2014, 2015Forrester-Anderson et al., 2006;Kowalska & Bielański;Papadomichelakis, Karagiannidou, Anastasopoulos, & Fegeros, 2010;Rasińska et al., 2017). Yet, from the consumer's point of view, and for nutritional reasons, it is important to present FA contents per 100 g of meat (Kouba, Benatmane, Blochet, & Mourot, 2008). Besides, the reports focused on two main cuts: hind leg and loin, and on their main muscles -Longissimus dorsi and Biceps femoris. Thus, bearing in mind the importance of the FAs profile for human health, we presented their contents as per 100 g of meat cuts (Table 4).
In human diet, meat is perceived as the main source of unfavourable LCSFAs. Their total contents per 100 g fat were very much alike, ranging 35.5-37.8 g, the between-cut differences being, however, significant (p < 0.05) (Table 3). Also, Alasnier et al. (1996) found that the total LCSFA content in muscle fat of glycolytic, oxidative and intermediate muscles was alike. Similarly, loin (Longissimus dorsi) and hind leg (Semimembranosus) muscles did not differ significantly in the content of the main SFA-palmitic FA C16:0 (Kouba et al., 2008). Since carcass cuts differed greatly in fat content, the total LCSFA contents per 100 g of meat differed accordingly between cuts (564-2022 mg; Table 4) and the same was true for all its components.
Feeding regimen is considered the main factor affecting LCSFA and C16:0 contents in fat extracted from rabbit meat (loin and hind leg): a decrease of them was achieved by supplementing standard pelleted diet with fish oil, alfalfa and flax sprouts, extruded linseed and pasture (Dal Bosco et al., 2015;Forrester-Anderson et al., 2006;Kouba et al., 2008;Kowalska & Bielanski, 2009). That decrease had, however, no effect on the content of palmitic acid per 100 g of meat as confirmed by Kouba et al. (2008).
Hind leg fat contained significantly (p < 0.001) more stearic acid (C18:0; 6.52 g/100 g FA), the second most abundant SFA in rabbit meat, compared with other cuts (5.65 g/ 100 g FA, on average). As previously stated by Alasnier et al. (1996), the content of C18:0 in hind leg muscle fat was higher than in the loin containing glycolytic muscles (Longissimus lumborum and Psoas major). A higher content of C18:0 in hind leg fat extracted from the Semimembranosus muscle, than in the loin (Longissimus dorsi muscle), was confirmed also by Kouba et al. (2008), who studied meat from rabbits fed linseed diet. However, when expressed per 100 g meat, hind leg was inferior to brisket in that respect ( Table 4).
As expected, the SCSFA contents in cuts were low, whether expressed per 100 g FA (0.90-1.22 g) or per 100 g of meat (15.5-52.9 mg), that is typical for rabbit meat (Tables  3 and 4).
Analysis of FAME by gas chromatography enabled separation of eight MUFAs, the major MUFA being oleic acid (C18:1 9c). The content of MUFA (28 g/100 g FA, on average), as well as oleic FA in extracted fat, did not vary significantly between cuts (Tables 2 and 3) and was lower than reported by Pla (2008) for fat extracted from meat of conventionally (35.6 g/ 100 g FA) and organically reared rabbits (29.4 g/100 g FA).
Our results differ from those of Alasnier et al. (1996), who Table 3. Mean contents (± SD; g/100 g FA) of fatty acid categories and nutritional indexes in fat extracted from cuts of rabbit carcasses (n = 20).
Cuts differed significantly in the content of vaccenic FA: C18:111t, which has been reported to reduce plasma triacylglycerols, cell growth and/or tumor metabolism and improve immune function on cell culture and animal models (Vahmani, Meadus, Duff, Rolland, & Dugan, 2017;Wang, Jacome-Sosa, & Proctor, 2012). Significantly (p < 0.001) highest content of C18:111t was assayed in fat extracted from foreleg (0.11 g/100 g FA), the lowest one in fat of the loin (0.02 g/100 g FA; Table 2), and the same was true when expressing per 100 g of meat (Table 4). Differentiation in vaccenic FA content between cuts (two main muscles: Longissimus lumborum and Biceps femoris) was also found by Papadomichelakis et al. (2010) who assayed higher content than in our experiment 0.33 and 0.49 g/100 g FA, respectively, in extracted fat (because of feeding rabbits with high digestible fibre diet). It should be mentioned that the abovementioned authors also found a higher content of C18:111t in the glycolytic (Longissimus lumborum) muscle than in other muscles.
Fat extracted from rabbit meat is a source of OBCFAs, hind leg contained significantly more OBCFA than other cuts (Table 3). Similar findings were reported by Papadomichelakis et al. (2010). However, when the OBCFA content was expressed per 100 g of meat, hind leg, foreleg and brisket contained as much as 92-138 mg, that was nutritionally significant (Table 4).
Studies on animals showed a favourable effect of branched-chain fatty acids (BCFA) on limiting or even blocking inflammatory processes in the premature intestine by increasing the expression of cytokines and improving intestinal microbiota (Ran-Ressler, Bae, Lawrence, Wang, & Brenna, 2014). A study in rat pups showed that BCFA reduced the incidence of necrotizing enterocolitis, a devastating intestinal disease affecting premature infants (Ran-Ressler et al., 2014). In our study we identified eight BCFAs; hind leg fat had significantly (p < 0.001) higher content of the anticarcinogenic anteiso C15:0 (0.33 g/100 g FA) than fat from other cuts. However, the contents of other iso-branched BCFA in fat extracted from cuts distinguished by apoptotic properties were not significantly different (iso C16:0), or the differences were nutritionally insignificant (iso C14:0, iso C15:0, iso C17:0; see Table 2). In contrast, the contents of main BCFA per 100 g of meat differed greatly between cuts; brisket had significantly (p < 0.05) highest contents of three important BCFA: iso C15:0, anteiso C15:0, iso C17:0, compared with other cuts (Table 4).
The total content of PUFAs in fat extracted from cuts varied significantly (p < 0.001) from 24.45 (in hind leg containing oxidative muscles) to 29.22 g/100 g FA (in brisket containing mostly intermediate muscles; see Table 2). Brisket had also significantly (p < 0.05) highest PUFA content per 100 g of meat (Table 4). Lower content of PUFA in the oxidative than in other muscles was also reported by Table 4. Mean contents (± SD) of fatty acid categories and main fatty acids (mg/100 g of meat), in meat of cuts of rabbit carcasses (n = 20).
The PUFA group was represented by eleven compounds -n-6 and n-3 FAs, the contents of most of them differing significantly (p < 0.001) between cuts ( Table 3). The quantitatively principal PUFA-linoleic acid (C18:2 9c12c) dominated in brisket both in extracted fat (25.20 g/100 g FA) and in meat (1438 mg/100 g meat; Tables 2 and 4). Our results support the thesis that the lipid composition of meat is related to their metabolic type. Fat extracted from oxidative muscles (hind leg) contained significantly (p < 0.001) lower amount of linoleic FA (n-6 family) and higher of LC-PUFA (n-3 family), as compared with glycolytic muscles of the loin (Alasnier et al., 1996).
The α-linolenic acid (C18:3 9c12c15c), and especially the longchained (LC) PUFAs: C20:5 EPA, C22:5 DPA, C22:6 DHA, received most attention in recent years due to their importance for human health and well-being (Chikwanha, Vahmani, Muchenje, Gugan, & Mapiye, 2017). LC-PUFAs are effective in reducing triacylglycerol in blood and in limiting cardiovascular diseases. EPA and DHA reduce inflammation and may play a role in reducing the risk of childhood allergic diseases. Also, EPA and DHA exert a range of biological activities that may influence tumor cell proliferation and viability; for example, DHA can promote tumor cell apoptosis, possibly by inducing oxidative stress (Calder, 2015;Miles & Calder, 2014;Merendino et al., 2013).
Moreover, brisket fat had better n-6/n-3 proportion compared with other cuts and the same applied to the AI, TI and H/H indexes (Table 3). The values of the TI and H/H nutritional indexes in brisket fat were similar to those computed for the main muscle of the loin (Longissimus lumborum) derived from rabbits fed pelleted diet supplemented with fresh alfalfa (Capra et al., 2013;Dal Bosco et al., 2014). Many studies reported higher n-3 family PUFA content in rabbit meat derived from grass-based diet (Dal Bosco et al., 2015Forrester-Anderson et al., 2006). According to EFSA recommendations, an intake of 250 mg of EPA plus DHA per day appears sufficient for primary prevention in healthy subjects (EFSA, 2010). Thus, an example daily portion of foreleg (250 g), containing 39 mg of those two fatty acids, would cover 16% of the requirement, and the same amount of brisket or hind leg-11%. Human body can synthesize EPA and DHA from α-linolenic acid and, as mentioned earlier, rabbit meat from three cuts is a good source of α-linolenic acid (Table 4). Thus, 250 g of rabbit meat may supply 138-386 mg of α-linolenic acid.

Conclusions
The presented results confirm that loin, containing mostly glycolytic muscles, had less lipids than cuts having mostly oxidative (hind leg) or intermediate (foreleg and brisket) muscles. The results suggest that apart from the preferred cuts (loin and hind leg) used in gastronomy, brisket and foreleg ought to be included as being rich in nutritionally valuable FAs: PUFA, especially α-linolenic, OBCFAs and palmitoleic acid C16:1 n-7. It is to be stressed that fat extracted from brisket not only contained significantly more total PUFA, but had advantageous nutritional indexes (AI, TI, H/H) and better n-6/n-3 proportion than other cuts. Daily portion (250 g) of foreleg would cover 16% of the requirement for EPA and DHA, and the same amount of brisket or hind leg-11%. Thus, supplementing minced rabbit meat products with brisket and foreleg meat may markedly improve their FA profile.

Disclosure statement
No potential conflict of interest was reported by the authors.