RCPV (2013) 108 (587-588) 127-136

O efeito da sazonalidade, do músculo e da raça nos teores de colesterol total, β-caroteno e vitamina E em bovinos da raça , , Barrosã e criados de acordo com o sistema de certificação DOP.

The effect of season, muscle and breed on total cholesterol, β-carotene and vitamin E contents in Alentejana, Arouquesa, Barrosã and Mertolenga bovine breeds reared under the PDO certification system

Mário A.G. Quaresma1*, Inês. Trigo-Rodrigues1, José N. Costa1, Rui J.B. Bessa1,2

1Centro de Investigação Interdisciplinar em Sanidade Animal (CIISA), Faculdade de Medicina Veterinária, Universidade de Lisboa 2Unidade de Produção Animal, L-INIA, Instituto Nacional de Recursos Biológicos

Summary: The objective of this study was to assess the ef- amostras de 3 músculos (longissimus thoracis, longissimus lum- fect of season, breed and muscle on total cholesterol and ma- borum e semitendinosus). O teor de colesterol total foi significati- jor lipid-soluble antioxidants (α-tocopherol, γ-tocopherol and vamente influenciado pela raça, estação e músculo, tendo variado β-carotene) contents. This study embraces 4 Portuguese bovine entre 37,1e 52,8 mg/100 g de carne. A carne de vitela Arouque- native breeds slaughtered as beef (Alentejana and Mertolenga) sa foi a raça que apresentou teores de a-tocoferol e b-caroteno and veal (Arouquesa and Barrosã), all raised in accordance with significativamente superiores às outras raças (5.2 e 0.26 μg/g de the certification of Protected Denomination of Origin (PDO). carne, respectivamente), tendo os seus teores de a-tocoferol fica- Sampling was performed in two different seasons (spring and do acima, do limiar necessário para retardar a oxidação lipídica e autumn), each animal was sampled in 3 muscles (longissimus da mioglobina (3,5 μg/g de carne), o que se verificou em ambas thoracis, longissimus lumborum and semitendinosus). The total as estações apesar do efeito da sazonalidade observado na raça. cholesterol content ranged between 37.1-52.8 mg/100 g of meat Nas outras raças, e apesar de não se ter observado o efeito da sa- and showed significant differences between different breeds, dif- zonalidade, o teor de a-tocoferol ficou abaixo do limiar em uma ferent muscles and even in the same breed slaughtered at dif- época (Mertolenga and Barrosã) ou mesmo nas 2 épocas, como ferent seasons. Arouquesa veal was the only breed associated foi o caso da raça Alentejana (com teores médios de 1,9 μg/g de with seasonal significant differences of both α-tocopherol and carne). Das 4 raças em comparação, a vitela Arouquesa foi a única b-carotene, and it was also the breed displaying the highest val- a revelar teores de antioxidantes acima do limiar necessário para ues of α-tocopherol and b-carotene (5.2 and 0.26 μg/g of meat, garantir adequada protecção antioxidante à carne. respectively) which were significantly higher than the contents displayed by all other breeds. Arouquesa veal was the only meat Palavras-chave: a-tocoferol, g-tocoferol, b-caroteno, Alenteja- displaying, in both seasons, an α-tocopherol content above the na, Arouquesa, Barrosã, Mertolenga threshold necessary to delay lipid and myoglobin oxidation (3.5 μg/g of meat). Conversely, and despite the absence of significant seasonal differences, the meat α-tocopherol content of Merto- Introduction lenga and Barrosã was below the threshold in one season, while the α-tocopherol content of Alentejana breed was below the The Protected Denomination of Origin (PDO), qual- threshold in both seasons (averaging 1.9 μg/g of meat).There- fore, Arouquesa veal was the only breed displaying the essential ification seal of EEC Regulation 2081/92 and 2082/92, antioxidant protection to preserve meat nutritional quality. is based on characteristics and qualities arising essen- tially or exclusively from a geographically restricted Keywords: a-tocopherol, g-tocopherol, b-carotene, Alentejana, origin, including natural, animal and human factors. The Arouquesa, Barrosã, Mertolenga PDO certification has afforded Portuguese bovine meats Resumo: O objectivo do estudo foi avaliar o efeito da estação, an increased value and market acceptability (Costa et raça e músculo no teor de colesterol total e principais antioxidan- al., 2011). On the other hand, Portuguese bovine PDO tes lipossolúveis (a-tocoferol, g-tocoferol e b-caroteno). O estu- meats represent an important socio-economic factor of do inclui 4 raças autóctones, abatidas como novilho (Alentejana wealth to rural populations, rewarding meat authenticity e Mertolenga) e vitela (Arouquesa e Barrosã), criadas de acordo and presumable higher meat quality, while contributing com as especificações do sistema de certificação Denominação de Origem Protegida (DOP). A amostragem foi realizada em diferen- to the preservation of Portuguese bovine autochthonous tes estações (primavera e outono) e de cada animal recolheu-se breeds (Rodrigues et al., 1998). Besides unique and differentiated genetic back- *Correspondência: [email protected] ground associated with each of the autochthonous bo- Tel: +(351) 213602042; Fax: +(351) 21365281 vine breeds, their feeding management is also unique

127 Quaresma M et al. RCPV (2013) 108 (587-588) 127-136 and dependent of regional traditions and differences The seasonality effect on the lipid profile of autoch- in edaphoclimatic conditions. The Portuguese climate thonous bovine breeds raised under the PDO certifica- usually classified as Mediterranean, suffers an impor- tion have been the objective of several studies (Alfaia tant Atlantic influence, especially near the western et al., 2007a; Alfaia et al., 2007c; Alfaia et al., 2006b; coast, while the inner south (Alentejo) has a strong Alfaia et al., 2006c; Alfaia et al., 2007d; Costa et al., Continental influence (Rodrigues et al., 1998). Differ- 2006b; Pestana et al., 2012a; Pestana et al., 2012b). ences in edaphoclimatic conditions between the Portu- The objective of this study was to evaluate the ef- guese provinces should influence the quality and avail- fect of breed, season and muscle, on total cholesterol ability of forages throughout different seasons, which content and major lipid-soluble antioxidants (a-toco- could reproduce on meat characteristics. The PDO pherol, g-tocopherol b-carotene) content in meat of beef and veal are supposedly associated with unique four Portuguese PDO-meats. characteristics (Council Regulation CE No. 2081/92 of 14/07), particularly in terms of their lipid profile (Al- faia et al., 2006a; Alfaia et al., 2007a; Alfaia et al., Material and methods 2007b; Costa et al., 2011). Production systems based on grass feeding produce All animals used in this study were purebred bovines beef with a favourable lipid profile (Nuernberg et al., of Alentejana, Arouquesa, Barrosã and Mertolenga 2005; Wood et al., 2008; Wood et al., 2003). However, breeds, fifteen animals of each breed and season were meat from pasture-fed cattle may have an increased de- selected for the study, providing 30 animals per breed mand for endogenous anti-oxidants because of its high and a total of 120 animals and 360 samples (3 different content of polyunsaturated fatty acids, which in turn, may muscles from each animal). affect its color and lipid stability (Yang et al., 2002b). Meat samples were obtained at the commercial The amount of antioxidants in muscle is of particu- slaughter house (Matadouro Regional do Alto Alen- lar interest, since the control mechanisms that with- tejo located in Sousel for the sampling of Alentejana stand oxidation reactions in vivo are less effective after and Mertolenga breeds and Pec-Nordeste located in slaughter and oxidation proceeds in a comparatively Penafiel for the sampling of Arouquesa and Barrosã uncontrolled manner (Kerry et al., 2000). breeds). The sampling performed for the Spring season Meat oxidation is a major cause for quality dete- was throughout June, while sampling for the Autumn rioration (Buckley et al., 1995; Descalzo and Sancho, season was performed in November. Meat samples 2008) affecting meat major attributes as colour, flavour, were taken from the ribeye portion (T1–T3) of the long- odour and texture (Gray et al., 1996). The impairment issimus dorsi (LD) muscle, the sirloin portion (L1-L3) of meat pigments stability during oxidation conduces of the longissimus lumborum (LL) muscle and distal to meat discoloration thereby decreasing consumer ac- region of semitendinosus (ST) muscle, of the left side ceptability, since meat colour is the foremost impor- of the carcasses. Sampling was performed 24 hours tant factor influencing consumer decision at the time of after slaughter, meat samples were transported under purchase (Bekhit et al., 2003; Gray et al., 1996; Smith refrigeration to the laboratory (<5 °C) and kept in re- et al., 1996). Moreover, the oxidation of polyunsatu- frigeration until processing (an overall period of 24 h rated fatty acids and cholesterol may result in nutri- from time of sampling). Meat samples were trimmed tional quality loss and in the production of potentially of connective and adipose tissues before blending in toxic compounds, exhibiting mutagenic, carcinogenic a food processor, vacuum packed and stored frozen at and cytotoxic properties (Esterbauer, 1993; Homma et -70ºC until analysis. al., 2004; Kubow, 1993). One of the compounds determining the oxidative stability of muscle is α-tocopherol, the major vitamin Production systems E homologue in beef and the foremost important lipid- soluble antioxidant (Decker et al., 2000). The oxida- In this study we are comparing four different tive stability of meat is important for maintaining good breeds, two representing the Northwest and two rep- color stability and for slowing rancidity development resenting the southeast regions of Portugal, but they (Descalzo et al., 2005b; Descalzo and Sancho, 2008). represent 4 different practices in terms of animal feed- Another natural important antioxidant is b-Carotene, ing and animal management. a strong antioxidant localised within the hydrophobic Arouquesa and Barrosã veal are both raised in region of biological membranes, contrasting with the Northwest, mountainous region of Portugal, their pro- scavenging activity of α-tocopherol close to the mem- duction system shares some similarities, but also some brane surface (Olson, 1988). important differences. They are both allowed to suckle Cholesterol, a structural molecule present in all ani- their dam’s milk from the day they are born to the day mal tissues has become an important health issue for con- of slaughter. Arouquesa calves are born in the byre and sumers, since it have been directly associated with disease allowed to go to pasture with their dams when they are development (Biesalski, 2005; Chizzolini et al., 1999). three weeks old (with the exception of winter period,

128 Quaresma M et al. RCPV (2013) 108 (587-588) 127-136 when they are kept indoors), while Barrosã veal are a G1316A Agilent thermostatted column compart- born and kept in the byre away from their dams, which ment with cooling, a G1313A Agilent auto-sampler, are left on pasture throughout day time returning to the a G1315B Agilent UV-visible photodiode array detec- byre in the evening. Barrosã veal diet during day time tor, and a G1321A Agilent fluorescence detector. The is fed on the manger with green fodder, hay, cornflour liquid chromatographic system was controlled and the and other seasonal agriculture byproducts (as potatoes, data collected and processed by the HP ChemStation pumpkins and fruit), depending on seasonal availabil- for LC 3D software (Rev. A.09.01, Agilent Technolo- ity. gies Inc., Palo Alto, CA, U.S.A.). Alentejana and Mertolenga beef are both raised The simultaneous analysis of cholesterol, tocophe- on the southeast region of Portugal, in the province rols and β-carotene in meat was performed using a nor- of Alentejo. They are both raised with their dams in mal-phase silica column (Zorbax RX-Sil with the cor- extensive pasture rearing until weaning, at the age of responding 12.5 mm analytical guard column, 4.6 mm 6 months. Afterwards, their diet is dependent of fod- ID x 250 mm, 5 μm particle size, Agilent Technologies der availability, receiving wheat straw and concentrate Inc., Palo Alto, CA, U.S.A.), with fluorescence detec- feeding throughout their growing and finishing peri- tion for tocopherols (excitation wavelength of 295 nm ods. There is quite variability among herds in the pro- and emission wavelength of 325 nm) and UV-visible duction system of both breeds, dependent of animal photodiode array detection for cholesterol (202 nm) density and agricultural practices. and β-carotene (450 nm) in series. The solvent (1% v/v isopropanol in n-hexane) flow rate was 1 ml/min, the run last for 17 min and the temperature of the column Total cholesterol, b-carotene and vitamin oven was adjusted at +20ºC. The injection volumes E extraction, chromatography analysis used varied between 20 and 100 μl in order to get val- and quantification ues inside the linearity range of the standard curves. Cholesterol and b-carotene standards were acquired The saponification procedure and the chromato- from Sigma-Aldrich Ltd. (St. Louis, MO, USA), while graphic procedure used for the quantification proc- α-, β-, γ- and δ-tocopherols standards were obtained ess was previously described in detail (Prates et al., from Calbiochem (Merck Biosciences, Darmstadt, 2006). Briefly, for saponification, 0.75 g of homog- Germany). enised meat was weighted, to which 0.2 g L-ascorbic The contents of total cholesterol, tocopherols and acid and 5.5 ml saponification solution were added. β-carotene in meat were calculated, in duplicate for The saponification solution, freshly prepared each each muscle sample (values accepted for CV <6%), week, contained 11% w/v potassium hydroxide in a based on the external standard technique, from a stand- mixture of 55% v/v absolute ethanol and 45% v/v dis- ard curve of peak area vs. concentration. tilled water. The sample was then vortexed to assure ascorbic acid complete dissolution. After vortexing, the air was eliminated from the reaction, by displace- Statistical analysis ment with nitrogen gas. The saponification was car- ried out in a shaking water bath (200 rpm) at +80 ºC, Data on total cholesterol, α-tocopherol, γ-tocopherol for 15 min. and β-carotene was analyzed in two separate and com- After saponification, samples were cooled in tap plementary ways. Firstly, data was subjected to analysis water for 1 min. Following cooling 1.5 ml of distilled of variance (ANOVA), considering the breed and season water and 3 ml of 25 μg/ml BHT solution in n-hexane as independent variables, using the GLM procedure of were added (final proportions of 4.5 2 mlH O : 3 ml SAS (SAS, 2004). A second statistical analysis was ac- ethanol : 3 ml n-hexane; the meat sample was assumed complished using the MIXED procedure of SAS, ver- to contribute with 0.5 ml of water). The samples were sion 9.1 (SAS, 2004). The model considers the effects vigorously vortexed for 2 min and centrifuged at 1500 of breed (Arouquesa, Barrosã, Alentejana and Merto- g for 5 min, in order to accelerate phases separation. lenga), muscle type (LT, LL and ST) and the interac- An aliquot of the upper layer (n-hexane) was trans- tion between breed and muscle type. Since measure- ferred into a small screw teflon-lined cap tube and a ments on different muscles from the same animal are spatletip of anhydrous sodium sulphate was added. Fi- not independent observations, muscle type was treated nally, the tube was briefly shaken and an aliquot of the as repeated measure on animal within slaughter season n-hexane layer was filtered through a 0.45 μm hydro- group as subject. Least square means were presented phobic membrane into an amber screw-cap vial with and compared using the LSMEANS/PDIFF option teflon septa. when interaction effect was significant (P<0.05). Means The HPLC system used was an Agilent 1100 Se- were reported as least square means with standard er- ries (Agilent Technologies Inc., Palo Alto, CA, U.S.A.) ror of mean (SEM). Correlations between α-tocopherol, composed by a G1311A Agilent quaternary pump, a γ-tocopherol and β-carotene were also determined using G1322A Agilent vacuum solvent delivery degasser, the Pearson correlation method.

129 Quaresma M et al. RCPV (2013) 108 (587-588) 127-136

Results and Discussion

Table 1 – Total cholesterol, vitamin E homologues (a-tocopherol and g-tocopherol) and β-carotene in beef (Alentejana and Merto- lenga) and veal (Arouquesa and Barrosã) from Portuguese bovine native breeds, raised in accordance with their PDO standards and slaughtered in different seasons (Autumn or Spring)

Beef Veal Significance Alentejana Mertolenga Arouquesa Barrosã Autumn Spring Autumn Spring Autumn Spring Autumn Spring SEM Period Breed P*B Cholesterol1 41.4c 37.4d 41.2c 41.5b,c 50.9a 50.8a 52.4a 43.9b 0.605 <0.001 <0.001 <0.001 a-tocopherol2 2.1d 1.7d 3.4c 4.19b,c 5.7a 4.7b 3.63c 3.03c,d 2.10 0.036 <0.001 0.007 g-tocopherol2 0.17a 0.15a 0.14a,b 0.13b 0.12b 0.07c 0.14a,b 0.13b 0.003 <0.001 <0.001 0.010 b-carotene2 0.04 0.07 0.04 0.09 0.23 0.28 0.08 0.09 0.006 <0.001 <0.001 0.21 1mg/100 g of meat; 2µg/g of meat; different superscripts in the same row correspond to statistical significant differences (P<0.05)

Table 2 – Total cholesterol, vitamin E homologues (a-tocopherol and g-tocopherol) and β-carotene in longissimus thoracis (LT), long- issimus lumborum (LL) and semitendinosus (ST) muscles from beef (Alentejana and Mertolenga) and veal (Arouquesa and Barrosã) of Portuguese bovine native breeds, raised in accordance with their PDO standards

Beef Veal Significance Alentejana Mertolenga Arouquesa Barrosã LT LL ST LT LL ST LT LL ST LT LL ST Muscle B*M Cholesterol1 41.95f,g 38.91h 37.11h 42.96e,f 40.56g 40.58g 52.75a 49.41c 50.33b,c 50.85a,b 47.90c,d 45.59d,e <0.001 0.013 a-tocopherol2 2.17e 1.93e 1.66f 4.10c 3.82c 3.59c 6.04a 4.63b,c 4.88b 3.80c 2.98d 3.20d <0.001 <0.001 g-tocopherol2 0.18a 0.16b 0.14c,d 0.14c,d 0.13d,e 0.12e 0.10e,f 0.08f 0.09f 0.15b,c 0.12e,f 0.13d,e <0.001 <0.001 b-carotene2 0.067e,f 0.057e,f 0.035f 0.073c,d 0.065e,f 0.048f 0.30a 0.23b 0.24b 0.10c 0.08c,d 0.07d,e <0.001 0.003 1mg/100 g of meat; 2µg/g of meat; different superscripts in the same row correspond to statistical significant differences (P<0.05)

Total cholesterol content bovine meats in analysis (37.4 mg/100 g of meat). The lowest total cholesterol content found in veal (Bar- Total cholesterol content displayed significant in- rosã breed from spring; 43.9 mg/100 g of meat) and teractions between breed and season (P<0.0001) and the highest total cholesterol found in beef (Mertolenga between breed and muscle (P=0.0135) as depicted in breed from spring; 41.5mg/100 g of meat) showed no Tables 1 and 2. The effect of season on total cholester- significant differences in between (P>0.05). ol content was limited to Barrosã veal and Alentejana The analysis of the muscle effect revealed than in all beef, in both breeds, the total cholesterol content found four breeds in comparison, the total cholesterol content in meat from animals slaughtered in spring was signifi- revealed a similar pattern of differences. The total cho- cantly inferior to the cholesterol content found in meat lesterol content found in LT muscle was significantly su- from animals slaughtered in autumn, such increased perior to the cholesterol concentration found in the other in total cholesterol content reached 11% in Alentejana two muscles (P<0.05), and no significant difference was beef and 19% in Barrosã veal, while no significant sea- observed between LL and ST muscles (P>0.05). sonal differences in the total cholesterol content were Cholesterol, a steroid-based alcohol with a hydro- found in both Arouquesa veal and Mertolenga beef. carbon side-chain, is an essential structural component The highest total cholesterol content occurred in of mammalian cell membranes, required to establish Barrosã veal slaughtered in autumn (52.4 mg/100 g of the proper membrane permeability and fluidity (Ohls- meat) and in Arouquesa veal (averaging 50.9 mg/100 son, 2010). In meat, cholesterol is present in both mus- g of meat, without significant differences in between cle and adipose tissues, because of its structural role seasons). The cholesterol content from Barrosã veal on cell membranes. In muscle cells, cholesterol is pre- slaughtered in spring displayed a significantly lower dominantly found on cell membranes, but in adipose value than Barrosã veal killed in autumn and Arou- cells cholesterol is principally found in lipid droplets quesa veal slaughtered in both seasons, but it was not stored as cholesterol esters and in a minor scale in cell significantly different from Mertolenga beef slaugh- membranes (Dessi and Batetta, 2003; Hoelscher et al., tered in the same season. On the other hand, beef from 1988). Beef with increased lipid content has a superior Mertolenga breed (from both seasons) and Alentejana accumulation of cholesterol, since the cholesterol ac- breed slaughtered in autumn showed similar choles- cumulation in fat fractions is 50% (30 mg/100 g) to terol values, ranging between 41.2 and 41.5 mg/100 g 100% (60 mg/100 g), greater than that of muscle frac- of meat, while beef from Alentejana breed slaughtered tions in beef (Abu-Tarboush and Dawood, 1993; Lan in spring displayed the lowest cholesterol value of all et al., 1993).

130 Quaresma M et al. RCPV (2013) 108 (587-588) 127-136

The total cholesterol content presented here in veal is another possible explanation to justify differences in and beef (ranged from 37.1 to 52.8 mg/100 g of meat), the total cholesterol content obtained from the same which is in accordance with the range of cholesterol muscle of related animals (breed, herd), this justifica- contents found in veal and beef from Portuguese auto- tion goes beyond the muscle metabolic type and is de- chthonous bovine breeds (Alfaia et al., 2006b; Costa et pendent of the intramuscular fat content, as previously al., 2011; Costa et al., 2006a; Costa et al., 2008; Mon- mentioned cholesterol accumulation in fat fractions is teiro et al., 2012; Pestana et al., 2012a) and in the range 50-100% greater than that of muscle fractions in beef of values for different bovine meat cuts (Chizzolini et (Abu-Tarboush and Dawood, 1993; Lan et al., 1993). al., 1999). Seasonal differences on the cholesterol con- Considering the meat cholesterol contents obtained tent have previously been found in autochthonous bo- in this study, a daily intake of 200 g of bovine meat vine breeds reared under the PDO certification system per day represents a daily ingestion of 37.5-52.5 mg of (Alfaia et al., 2006b; Pestana et al., 2012a) and also in cholesterol, which corresponds to 25-35% of the rec- organic beef (Pestana et al., 2012b). In these studies as ommended maximum daily cholesterol intake, which in ours, spring was always the season associated with has been estimated in 300 mg (Krauss et al., 2000). the lower cholesterol contents in the LL muscle, but not in ST muscle, while the total lipid contents showed Vitamin E homologues no seasonality influence. We have no available infor- mation to explain the reason underlying such result. After slaughter the control mechanisms that with- Accordingly to Chizzolini et al. (1999) differences stand oxidation reactions in vivo are progressively less in cholesterol content can be seen among different effective and oxidation proceeds in a comparatively species and muscles, but their magnitude is generally uncontrolled manner (Kerry et al., 2000). Therefore, low. Our results showed that significant differences meat is very susceptible to oxidation, and this is a ma- could be found between different muscles and differ- jor cause for quality deterioration (Descalzo and San- ent breeds and even in the same breed slaughtered at cho, 2008; Lee et al., 2005; Nuernberg et al., 2005) af- different seasons. Differences between muscles are fecting meat sensorial and nutritional qualities. There majorly associated with dissimilarities in the muscle is however another group of antioxidants that exert fibre composition and the relative proportions of dif- their antioxidant function by non-enzymatic reactions ferent fibre type in each muscle (Alasnier et al., 1996; and remains effective after slaughter for a long period. Chizzolini et al., 1999), since glycolytic and oxidative Among these antioxidants we find the ascorbic acid muscle fibres are associated with different cholesterol and the lipophilic antioxidants (a-tocopherol, g-toco- content and each muscle has its proportion of glyco- pherol and b-carotene) (Descalzo and Sancho, 2008; lytic and oxidative fibre types (Chizzoliniet al., 1999). Faustman et al., 1998; Insani et al., 2008; Liu et al., In this study, we have used the longissimus thoracis, 1995; Muramoto et al., 2003), however their content longissimus lumborum and semitendinosus muscles, in meat is variable and dependent of several factors, all considered to be predominantly glycolytic muscles and diet is one of foremost importance (Descalzo et (Kirchofer et al., 2002; Pratiwi et al., 2006). However, al., 2005a; Yang et al., 2002a; Yang et al., 2002b). the longissimus thoracis muscle displayed, in all four Veal and beef from Portuguese autochthonous breeds in comparison, significantly greater cholesterol breeds displayed two major vitamin E homologues, content than the other two muscles. The greater cho- a- and g-tocopherol. a-Tocopherol was the predomi- lesterol content in the longissimus thoracis is prob- nant vitamin E (Table 1) homologue in both veal and ably consequence of one out of two possible effects: beef, independently of the slaughter season, being re- 1) greater percentage of oxidative fibres or 2) greater sponsible for 92.2-96.8% of total vitamin E in beef and lipid content at intramuscular level. Considering that 96.1-98.3% of total vitamin E in veal. The contents of cholesterol is predominantly found in cell membranes, a- and g-tocopherols in meat showed a statistical sig- differences in cell size and number of cells per unit of nificant interaction between breed and season (P<0.05; muscle volume or weight can lead to a divergence in Table 1) and between muscle and breed (P<0.0001; Ta- total membrane surface area and ultimately in the con- ble 2). For both a- and g-tocopherols, the seasonality tent of membrane components, including cholesterol effect was only expressed in Arouquesa veal and not in (Chizzolini et al., 1999). Another theory sustaining the the other breeds. Regarding muscle it was not observed superior total cholesterol content of oxidative muscles a single pattern of similarities/differences among mus- is known as the metabolic hypothesis, which defends cle types between different breeds. that oxidative muscles are richer in phospholipids and The highest a-tocopherol content of all bovine from the finding that the higher the phospholipid con- meats in comparison was found in Arouquesa veal (5.7 tent of the muscle, the higher the cholesterol content. and 4.7 mg/g of meat for calves slaughtered in autumn The relationship between cholesterol and phospholip- and spring, respectively), while the lowest values of ids is explained by the physical properties of choles- a-tocopherol were found in Alentejana beef (2.1 and terol, on the ordering of phospholipid chains, which 1.7 mg/g of meat for steers slaughtered in autumn and contribute to maintain membrane fluidity in a narrow spring). On the other hand, the a-tocopherol content range (Alasnier et al., 1996). On the other hand, there quantified in Barrosã veal and Mertolenga beef showed

131 Quaresma M et al. RCPV (2013) 108 (587-588) 127-136 no significant differences in between, independently of is preferentially distributed throughout body tissues the season (averaging 3.56 mg/g of meat). relatively to other vitamers E. γ-Tocopherol is usually, Concerning the average g-tocopherol content of in quantitative terms, the second most important vita- both seasons, Alentejana beef displayed the highest min E homologue in meat (Ponte et al., 2008; Prates value of all breeds (0.16 mg/g of meat), Arouquesa veal et al., 2006; Quaresma et al., 2011; Quaresma et al., displayed the lowest value of all breeds (0.10 mg/g of 2012a; Quaresma et al., 2012b; Sampels et al., 2004). meat), while Barrosã veal and Mertolenga beef dis- γ-Tocopherol has a relevant antioxidant function, played an intermediary value of g-tocopherol content which can be classified as complementary antioxidant (0.14 mg/g of meat). The influence of season on the function, since it has the ability to protect against oxy- g-tocopherol contents was limited to Arouquesa veal, gen and nitrogen reactive substances (RNS and ROS, as previously observed for a-tocopherol. respectively), while α-tocopherol antioxidant protec- Relatively to differences between muscles, the LT tion is limited to ROS (Jiang et al., 2001). muscle of Arouquesa and Barrosã breeds displayed a In beef, it was established the α-tocopherol content greater a-tocopherol content than LL and ST muscles, necessary to delay meat oxidation (3.5 μg/g of meat) which showed no significant differences in between. (Faustman et al., 1989; Mitsumoto et al., 1991). The On the other hand, Mertolenga beef showed no signifi- α-tocopherol content in Alentejana (averaged 1.9 μg/g cant differences between muscles in the a-tocopherol of meat) was rather below 3.5 μg/g of meat, indepen- content, whereas in Alentejana beef the ST muscle dently of the season. Conversely, the α-tocopherol displayed a lower a-tocopherol content than LT and content in Arouquesa veal was quite above the recom- LL muscles, which showed no significant differences mended concentration (averaged 5.2 μg/g of meat). On in between. In the g-tocopherol, differences between the other hand, despite the absence of significant sea- groups could be divided into three different patterns: sonal differences in Mertolenga beef and Barrosã veal, 1) in Alentejana beef it was observed that LT and ST the observed differences in the α-tocopherol content muscles displayed the highest and lowest values of g- were of relevant value, since the α-tocopherol content tocopherol content, while the LL muscle displayed an reached levels below 3.5 μg/g of meat in one season intermediary value, but significantly different (P<0.05) (spring in Mertolenga beef and autumn in Barrosã from the other two muscles; 2) in Mertolenga beef it veal). Beef or veal with an α-tocopherol content below was observed that LT and ST muscles displayed the 3.5 μg/g of meat do not possess the required antioxi- highest and lowest values of g-tocopherol content, dant protection to sustain lipid oxidation and preserve while the LL muscle displayed an intermediary value meat nutritional quality. and not significantly different from the other two mus- In both seasons, it was observed a strong positive cles (P>0.05); 3) in veal from Arouquesa and Barrosã and highly significant correlation (P<0.001) between breeds, the LT muscle displayed a significantly supe- a-tocopherol and b-carotene (r = 0.70 in spring and rior content of g-tocopherol than LL and ST muscles, r = 0.71 in autumn), and a highly negative correlation which showed no significant differences in between (P<0.001) between b-carotene and g-tocopherol (r = (P>0.05). -0.56 in spring and r = -0.29 in autumn). In spring, it Vitamin E, and particularly the a-tocopherol, is was also observed a highly significant negative corre- the primary lipid-soluble antioxidant in biological sys- lation (P<0.001) between a-tocopherol and g-tocoph- tems, being responsible for scavenging lipid peroxy erol (r = -0.38). The positive correlations between a- radicals (Liu et al., 1995; Lynch et al., 1999; Müller tocopherol and b-carotene suggests that pasture is the et al., 2010). Due to its lipophilic structure, vitamin prime source of both a-tocopherol and b-carotene, in E tends to accumulate in cellular membranes and fat both seasons. In fact, b-carotene is primarily provided deposits, where it exerts its scavenger function (Thakur by pasture, while a-tocopherol could be delivered by and Srivastava, 1996). In this study, it was possible to pasture ingestion or by concentrate feeding supple- detect and quantify two vitamin E homologues, a- and mented with a-tocopherol. The negative correlations g-tocopherol, which are the only two tocochromanols observed between b-carotene and g-tocopherol and be- usually found in bovine meat (Prates et al., 2006; Qua- tween a-tocopherol and g-tocopherol may indicate that resma et al., 2012b), despite several other tocochro- green pasture is not the prime source of g-tocopherol, manols have been found in other meat types (Ponte which is in agreement with the results of Ponte et al. et al., 2008; Quaresma et al., 2011; Quaresma et al., (2008), which showed that g-tocopherol was found 2012a). The superior a-tocopherol contents found in in pasture in a much lower concentration than it was muscle relatively to all other tocochromanols is de- found in cereals. pendent of a-tocopherol transfer protein (a-TTP), a Accordingly to Daley et al. (2010), the concentra- key-role protein in vitamin E metabolism present in tion of a-tocopherol in dried grains is typically below the liver cells, which has a considerable higher affinity 10 μg/g, whereas green pasture contains up to 300 towards a-tocopherol comparatively to other vitam- μg/g dry matter, consequently the a-tocopherol con- ers E (Decker et al., 2000), and a-TTP is responsible centration found in grain-fed beef ranges between 0.75 for vitamin E packing to VLDL proteins, major trans- to 2.92 μg/g of meat whereas pasture-fed beef ranges porter in the blood stream, consequently a-tocopherol from 2.1 to 7.73 μg/g of meat, depending on the type of

132 Quaresma M et al. RCPV (2013) 108 (587-588) 127-136 forage made available to the animals. Grass finishing est b-carotene contents in ST muscle and no signifi- increases a-tocopherol levels three-fold over grain-fed cant differences were observed between the LT and beef and places grass-fed beef well within range of the LL muscles. Considering that b-carotene deposition muscle a-tocopherol levels needed to extend the shelf- is largely dependent on intake of carotenoids and that life of retail beef (Descalzo et al., 2005b; Descalzo and b-carotene is predominantly found in meat from cattle Sancho, 2008; Insani et al., 2008; Realini et al., 2004; fed on green pasture. Thus, the production system of Yang et al., 2002a). Arouquesa veal was associated with increased inges- The lowest a-tocopherol content found in Alente- tion of green pasture than other PDO bovine breeds in jana beef (averaging 1.9 mg/g of meat) is in agreement study. On the other hand, tissue deposition of b-caro- with the a-tocopherol content found in beef obtained tene occurs preferentially in liver, and in less extension from animals raised with a grain-based diet or finished in adipose tissue and even fewer in muscle tissue (Yang on a grain-fed diet without a-tocopherol supplemen- et al., 2002a). Therefore, for animals fed with similar tation (Quaresma et al., 2012b). Veal from Arouquesa amount of green forage, the higher the intramuscular and Barrosã displayed greater a-tocopherol in autumn fat content the higher is b-carotene content. (although such supremacy was not statistical significant b-Carotene is a strong antioxidant localised within in Barrosã), such superiority is probably consequence the hydrophobic region of biological membranes, con- of agricultural practices and land availability for graz- trasting with the scavenging activity of α-tocopherol ing. In the mountainous regions of the Northwest of close to the membrane surface (Olson, 1988), it is, Portugal, where Arouquesa and Barrosã breeds are therefore, it is possible to say that b-carotene and a- raise, agriculture is held in smallholdings, and during tocopherol may exert a complementary action in the spring the land is being used to produce the main crops protection of cell membranes. b-Carotene is the best of the region, potatoes, and rye, while other portions quencher of singlet oxygen, and is also considered a of land are being prepared for the production of maize, provitamin A because it can be converted to active vi- for these reasons, during spring cattle has to graze in tamin A (Olson, 1988). the most mountainous lands, where pasture quality is Pasture-fed steers incorporated significantly higher lower. On the other hand, in autumn there is a greater amounts of b-carotene into muscle tissues than grain- availability of land for cattle to graze and pasture of fed steers (Daley et al., 2010). However, meat from better quality and in greater amount. Differences ob- grass-fed animals has been associated with considera- served between Barrosã and Arouquesa breeds in the bly variability on b-carotene contents (ranging between a-tocopherol content are probably consequence of dif- 0.16 and 0.74 μg/g of meat), which was also observed ferent husbandry practices, since Arouquesa calves are in meat from grain-fed steers (ranging between 0.01 allowed to follow their mother to the pasture (3 weeks and 0.16 μg/g of meat) (Descalzo et al., 2005a; Insani after birth), while Barrosã calves are kept on the byre et al., 2008; Yang et al., 2002a). The variability associ- and fed on the manger with green fodder, hay, corn ated with meat b-carotene contents in animals raised flour and other seasonal agriculture byproducts. This on pasture is dependent on differences on botanical di- suggestion is also supported by differences observed versity, development stage and availability of the pas- between breeds in b-carotene contents. ture throughout different seasons and time on grazing. Differences in the intramuscular fat content could be another factor influencing -carotene contents in beef, b-Carotene b since b-carotene is predominantly stored in fat depots The b-carotene content in meat was significantly (Strachan et al., 1993). influenced by both the breed (P<0.0001) and season The low b-carotene content found in Barrosã veal and (P<0.0001), as shown in Tables 1 and 2. In all four in beef from both the Alentejana and Mertolenga breeds breeds in analysis, spring was the season with the high- could be consequence of the finishing period, in which est b-carotene content (P<0.0001). Arouquesa veal grain or concentrate feeding are the most common op- was the breed with the greatest accumulation of b-car- tions. Recently, we have demonstrated that steers finished otene (0.26 mg/g of meat), the second major b-carotene on concentrate feeding for 2 or 4 month periods was as- content was found in Barrosã veal (0.09/mg/g of meat), sociated with the loss of 57% and 86% of the β-carotene while beef from Mertolenga and Alentejana breeds dis- content, respectively (Quaresma et al., 2012b). played the lowest contents of b-carotene (0.07 and 0.06 In conclusion, it is possible to say that seasonal dif- /mg/g of meat, respectively). ferences in the total cholesterol content were observed Relatively to the accumulation of b-carotene in dif- in some but not all the breeds in study. In all the four ferent muscles, it was possible to observe three differ- breeds in comparison, the LT muscle had higher total ent patterns: 1) Alentejana beef displayed no signifi- cholesterol content than LL and ST muscles, which cant differences in b-carotene contents between mus- showed no significant differences in between. cles; 2) Mertolenga beef and Arouquesa veal presented Arouquesa veal displayed a significantly higher the highest b-carotene contents in LT muscles, while of a-tocopherol and b-carotene content than all other no significant differences were observed between LL three breeds in comparison, which showed no signifi- and ST muscles; 3) Barrosã veal displayed the low- cant differences in between.

133 Quaresma M et al. RCPV (2013) 108 (587-588) 127-136

In all the breeds in comparison, a seasonal effect Alfaia, C.M.M., Quaresma, M.A.G., Castro, M.L.F., Martins, was observed for b-carotene, since higher b-carotene S.I.V., Portugal, A.P.V., Fontes, C.M.G.A., Bessa, R.J.B., content was found in cattle slaughtered in spring than Prates, J.A.M., 2006b. Fatty acid composition, including in autumn. isomeric profile of conjugated linoleic acid, and choles- The absence of seasonal influence on a- and g- terol in Mertolenga-PDO beef. Journal of the Science of tocopherol contents for Barrosã veal and beef from Food and Agriculture 86, 2196-2205. Alfaia, C.M.M., Ribeiro, V.S.S., Lourenco, M.R.A., Qua- Alentejana and Mertolenga breeds and the low -car- b resma, M.A.G., Martins, S.I.V., Portugal, A.P.V., Fontes, otene content quantified in meat from these breeds C.M.G.A., Bessa, R.J.B., Castro, M.L.F., Prates, J.A.M., is probably consequence of the finishing period with 2006c. Fatty acid composition, conjugated linoleic acid feeding materials rather different from green pasture, isomers and cholesterol in beef from crossbred bullocks which do not provide similar amounts of a-tocophe- intensively produced and from Alentejana purebred bullo- rol and b-carotene. Contrastingly, Arouquesa veal cks reared according to Carnalentejana-PDO specifica- was the only breed displaying seasonal significant dif- tions. Meat Science 72, 425-436. ferences in all antioxidants in study, despite that fact Alfaia, C.P.M., Castro, M.L.F., Martins, S.I.V., Portugal, was the only one displaying the required antioxidant A.P.V., Alves, S.P.A., Fontes, C.M.G.A., Bessa, R.J.B., protection in meat. Prates, J.A.M., 2007d. Influence of slaughter season and muscle type on fatty acid composition, conjugated lino- leic acid isomeric distribution and nutritional quality of Acknowledgments intramuscular fat in Arouquesa-PDO veal. Meat Science 76, 787-795. This research was supported by the Grant CII- Bekhit, A.E.D., Geesink, G.H., Ilian, M.A., Morton, J.D., Bi- SA2005/76 and received laboratory assistance (Ana ckerstaffe, R., 2003. The effects of natural antioxidants on Maria Ferreiro) and HPLC support (Dra. Cristina Al- oxidative processes and metmyoglobin reducing activity in beef patties. Food Chemistry 81, 175-187. faia and Prof. Dr. José Prates) are acknowledged. Biesalski, H.K., 2005. Meat as a component of a healthy diet Sampling assistance (ANCRA; CAPOLIB; CAR- - are there any risks or benefits if meat is avoided in the NALENTEJANA and MERTOCAR) are acknowl- diet? Meat Science 70, 509-524. edged. Buckley, D.J., Morrissey, P.A., Gray, J.I., 1995. Influence of dietary vitamin E on the oxidative stability and quality of pig meat. J Anim Sci 73, 3122-3130. Bibliography Chizzolini, R., Zanardi, E., Dorigoni, V., Ghidini, S., 1999. Calorific value and cholesterol content of normal and low- Abu-Tarboush, H.M., Dawood, A.A., 1993. 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