Sacrifice and slaughter of animals

When reaching the sacrifice age (about two years) the calves were transported to a commercial slaughterhouse 45 km from the farm and were housed for 14 h in a single pen with available water until the moment of their sacrifice. The slaughter of the carcasses was performed according to European regulations (^{Reglamento CE 1099/2009}) for the sacrifice of supply animals. Immediately after the sacrifice, the carcasses were weighed (CHW) with the kidney, tail and kidney fat; the weight of the carcass was calculated cold (CDW) with the formula: CDW=CHWx0.98; and the carcasses were refrigerated for 7 d in a refrigerator chamber at 4 °C (98 % of relative humidity). Afterwards, the Longissimus dorsi muscle was extracted from the left half-carcass (from the 3^rd thoracic vertebrae to the 6^th lumbar vertebrae) and was transported to the Animal Production laboratory at ETSIA in Universidad de Sevilla (Spain). There, the muscle was divided into three equal portions in weight: cranial (T₇), medial (T₁₄) and caudal (T₂₁). The T₁₄ and T₂₁ portions were vacuum-packed in individual bags and continued their maturation under refrigeration at 4 °C (95 % of relative humidity) until 14 and 21 d, respectively. In this way, samples were obtained to evaluate the quality of meat at three maturation times: 7, 14 and 21 d.

Physical-chemical analysis of the meat

The meat’s pH was determined in the portion T7 with a CRISON potentiometer (pH meter 507) adapted to a penetration electrode. In this portion the moisture (ISO-1442-1997), ash (ISO-R-936), protein (UNE 55-020) and fat (ISO-1443-1973) as well as myoglobin (^{Hornsey, 1956}) contents, were determined. To determine the relative content of fatty acids in the meat of this T7 portion, a gas chromatography analysis was carried out with the method proposed by ^{Aldai et al. (2006)} and a gas chromatograph was used (GC, Agilent 6890N, Inc., California, USA) with a flame ionization detector and an automatic injector (HP 7683, Inc., California, USA) associated to a capillary column HP-88 (100 m, 0.25 mm i.d., 0.2 μm film thickness, Agilent Technologies Spain, S. L., Madrid, Spain). The conditions of the chromatography analysis are detailed in ^{Horcada et al. (2013)}.

In order to evaluate the changes in the variables of meat qualities during maturation, in portions T₇, T₁₄ y T₂₁ , the meat color was evaluated through the determination of physical coordinates of color L* a* b* (^{CIE, 1976}). For this purpose, a Minolta CM-700d (Konica Minolta Co., Japan) spectrophotometer with illuminant D65 and standard observer of 10° was used. In each sample, seven determinations were made with different inclination of the apparatus in several points of a 2 cm thick fillet, approximately, after 1 h of exposure to the air.

The changes in water-holding capacity were determined in the three moments of maturation through the method proposed by ^{Grau and Hamm (1957)}. For this purpose, water losses were calculated (percentage with regard to fresh weight of the meat) through pressure.

To study the changes in meat hardness during maturation, 1.5 cm thick steaks were vacuum-packed from the T₇, T₁₄ and T₂₁ portions, and frozen at -21 °C until the time of analysis. For the test, the samples were thawed for 24 h in a refrigerator regulated at 4 °C, the vacuum-packed samples were immersed in hot water bath until 70 °C inside, and they were cooled under running cold water until reaching room temperature. Later, each sample was cut into prisms of 1x2x1 cm in parallel to the muscle fibers, to perform the measurements of strength at cut with a texturometer (TA.XT plus, UK), with a Warner-Bratzler blade of angle 50° and with the following conditions: blade speed of 150 mm min^-1, sampling speed of 4 points s^-1 and load cell of 30 kg. With each sample, five analytic determinations were performed.

Sensory analysis

To understand the opinion of consumers regarding the sensory characteristics and the acceptability of the Pajuna breed meat at three moments of maturation, a preference test was performed with 180 consumers (85 men and 95 women), randomly chosen, untrained, older than 18 years, and used to calf meat consumption. Each consumer was offered a batch of meat samples of about 150 g from portions T₇, T₁₄ and T₂₁, and an evaluation questionnaire. The vacuum-packed samples, frozen and identified, were delivered all at once to each consumer so they would be cooked in their home on a pan in the habitual manner, without additives (except salt and olive oil). The consumers were informed only that they were going to consume boneless calf meat. The opinion of each consumer was recorded in an assessment questionnaire where the tenderness, juiciness, flavor and aroma, and general acceptability were evaluated according to a numerical scale of 8 points (1 = I extremely dislike it; 8 = I extremely like it) (^{Sosa et al., 2008}).

Statistical analysis

The experimental design was completely random and with a factorial arrangement of 2x3: production system factor (Sem and Ex), and maturation time factor (T₇, T₁₄ and T₂₁ d of maturation). The results were analyzed with SPSS Statistics 17.0 (SPSS Inc., Chicago, USA, 2008). For the productive variables, pH, proximal composition, myoglobin content and fat composition, a t-Student test was carried out with independent samples in function of the productive system (Sem and Ex). For the changes in meat quality (color, water-holding capacity and resistance to cutting), during maturation and the consumers’ assessment, a GLM procedure was performed with the production system (Sem and Ex) and the maturation time (7, 14 and 21 d) as fixed effects, and with the following model:

where, Y_ijk = coordinates of color L* a* b*, water-holding capacity, resistance to cutting, sensory analysis of tenderness, juiciness, flavor and aroma, general acceptability; μ=value of the least square means; S_i =fixed effect of the production system (i=1: semi-extensive; i=2: extensive); M_j =fixed effect of the maturation time (j=1: T₇, j=2: T₁₄, j=3: T₂₁); S_ixM_j interaction between the effect of the production system and the maturation time; e_ijk =random residue. Within each variation factor (production system and maturation time), the differences between means were calculated through a Tukey test (p<0.05).

Physical chemistry of the meat

The pH values (Table 2) were in the interval expected for calf meat without prior stress (^{Albertí et al., 1995}). This must be taken into account in the Pajuna breed because it is a rustic breed from which a higher adaptation to the stress situation prior to the sacrifice is expected, compared to the improved breeds. The proximal composition of the Pajuna calf meat is within the intervals described for cattle (^{Schweigert, 1994}).

Table 2 Effect of the production system on the pH values, proximal composition and heminic pigments of the meat (Longissimus dorsi) of Pajuna breed calves in Semi-extensive and Extensive systems. 

EEM: standard error of the mean.

There were no significant differences in the meat water content between the two production systems. From the chemical constituents of meat (water, ash, protein, fat), the water content shows least variation in function of the production systems (^{Forrest et al., 1979}). However, the mineral (ash), protein and fat content was significantly higher in the Sem group calves compared to those from the Ex group. These results agree with ^{Wood et al. (2008)}, who point out that the meat of calves that receive concentrated feed during the finishing phase present higher infiltration fat content, compared to those that consume grass or forages.

Myoglobin is the principal responsible for meat color and includes a heminic group in its structure (^{Warriss et al., 1990}). The values of myoglobin content observed in Pajuna calves (Table 2) are within those expected for cattle, considered as red meat (^{Beriain et al., 2009}). The content of this pigment was significantly higher in the meat from the Ex group calves than those from the Sem group (p=0.002). This could be because the herb ingested by calves in group Ex contributes important amounts of Fe⁺⁺ available for myoglobin synthesis (^{McCaughey and Cliplef, 1996}).

The percentages of saturated and unsaturated fatty acids (FA) in the Pajuna calf meat (Table 3) were similar to those described for native cattle breeds in the Spanish market (Pirenaica breed, ^{Indurain et al., 2006}; Tudanca breed, ^{Humada et al., 2012}); these authors point out that oleic acid (9c 18:1) is main one in the fat composition (35.1-37.8 %). The relative content of saturated and monounsaturated FA was significantly higher in the Sem group than in the Ex group (p=0.002 and p=0.036, respectively). However, the polyunsaturated FA content in meat from group Ex was higher than that in group Sem (p=0.001), which is related to the type of diet of calves in the two groups. Thus, according to ^{Scerra et al. (2007)} and ^{Blanco et al. (2010)}, the diets based on grass and forage increase the polyunsaturated FA content in meat, compared to the finalization diets based on concentrated feed. The polyunsaturated FA content has beneficial effects on human health compared to the consumption of saturated FA (^{Enser et al., 1996}; ^{Nuernberg et al., 2005}), and the polyunsaturated n-3 FA deserve special attention because of their influence on the prevention of coronary disease (^{Muchenje et al., 2009}; ^{Alfaia et al., 2009}). The production system affected significantly (p=0.001) the percentage of n-3 FA (18:3n-3, EPA, DPA and DHA), which was higher in meat from Ex group calves, compared to those from the Sem group. ^{French et al. (2000)} showed that the contribution of herb to bullock diet increased the content of n-3 FAs that is beneficial to human health.

Table 3 Effect of the production system on the relative content of fatty acids (expressed as total percentage of unidentified fatty acids) of the Pajuna breed calves in Semi-extensive and Extensive systems. 

EEM: standard error of the mean.

Changes in the meat quality parameters during maturation

The values observed for trichromatic coordinates in meat of Pajuna breed calves (Figure 1) are within those described by ^{Ruiz de Huidobro et al. (2003)} and ^{Panea et al. (201)1} for cattle sacrificed at the commercial weights of the Spanish market (between 36-41, 12-18 and 3-20 for L*, a* y b*, respectively). The meat of Pajuna breed calves presented values similar to those described for the Spanish native breeds of Pirenaica or Avileña-Negra Ibérica which are protected with the European distinctions of quality Indicación Geográfica Protegida Ternera de Navarra (^{BOE, 2002}) and Indicación Geográfica Protegida Carne de Ávila (^{BOE, 2008}).

Figure 1 Changes in the trichromatic coordinates (L*, a* and b*) of the meat of Pajuna breed calves during 7, 14 and 21 days of maturation. 

The effect of the production system on the meat color was significant in all moments of maturation for L* (p=0.003). Thus, the calf meat from the Sem group was more luminous throughout maturation than the meat from the Ex group. During maturation of the meat, the value of all the chromatic coordinates analyzed increased (Figure 1). According to ^{Echevarne et al. (1990)}, the prolonged maturation periods decreased the consumption of oxygen by tissues, changing the meat color. This observation was more prevalent on days 0 to 14 of maturation, and after 14 d the evolution of the chromatic variables L*, a * and b* was less intense.

The water-holding capacity of Pajuna calves’ meat (Figure 2) is within the values described by ^{Serra et al. (2008)} for native Spanish breeds in the national markets (Bruna de los Pirineos, Avileña-Negra Ibérica or Morucha). As it happens in normal meat maturation processes, the maturation time of Pajuna calf meat influenced (p=0.003) the water-holding capacity. In the two production systems there was a decrease in the amount of water liberated by the meat as the maturation processes developed. These changes were more noticeable in the meat of calves fed with concentrate feed (Sem) than in those with continuous grazing (Ex).

Figure 2 Changes in the water-holding capacity (percentage of water liberated) of the meat of Pajuna breed calves during 7, 14 and 21 days of maturation. 

The hydration of the muscle fibers depends on the final pH of the meat, which in turn depends on animal management, and the electronic environment of the muscle fibers is the primary determinant of water adsorption in muscle fibers. In the Pajuna calves it is also evident and the water-holding capacity of meat increases during the maturation processes, although in a different way depending on the prior management.

The values of resistance to cutting of Pajuna calf meat during maturation (Figure 3) were similar to those described by ^{María et al. (2003)} for calves of the Spanish breeds selected for meat production (4-5 kg cm^-2) and by ^{Campo et al. (2000)} for calves of Spanish native breeds sacrificed with approximately one year of age.

Figure 3 Changes in resistance to cutting of the meat of Pajuna breed calves during 7, 14 and 21 days of maturation. 

At all moments of the maturation process of the meat, there were no significant differences in the resistance to meat cutting of calves from the Sem or Ex groups (p>0.05); thus, the production systems proposed do not affect the meat toughness in Pajuna calves. This assertion is related to the idea that meat toughness depends to a great extent on the activity of animals during their productive process, on the management prior to the sacrifice, and on the final pH values of the meat. Therefore, the absence of significant differences observed in meat pH between the Sem and Ex groups (Table 2) is related to the absence of significant differences in the meat toughness between the two production groups. However, in both production systems, the maturation time of the meat influenced the resistance to cutting (p=0.002), as was described by ^{Maria et al. (2003)} in calf meat maturated for 14 d. During the period of 7 to 14 d of maturation, the resistance to cutting of the meat was reduced in 14.1 %, whereas at 21 d it was reduced in 28.5 %. These results could respond to the processes described by ^{Nishimura et al. (1996)} regarding the gradual disintegration of the muscle structure that takes place during maturation of the meat.

Sensory analysis

As judged by consumers, the effect of the production system on the attributes assessed in sensory terms (tenderness, juiciness, flavor and aroma) and the general acceptability was not significant (p>0.05) (Table 4). However, the effect of maturation on the meat was significant in all the attributes analyzed (p≤0.01) in the two production systems. This result agrees with ^{Monsón et al. (2005)} who observed that the effect of maturation is more important than the effect of the genotype on the positive valuation of the characteristics of the cattle meat by the consumer. The valuation of tenderness is the main attribute that the consumer considers at the moment of consumption. In both production systems the meat that was best evaluated was matured for 21 d at 4 °C. This observation was corroborated by the instrumental determination in the meat matured for 21 d. According to ^{Campo et al. (2000)}, prolonged maturation periods improve the tenderness of the meat due to the structural changes of disintegration of muscle proteins. In both production systems, the highest grades in juiciness were for the meat matured for 14 and 21 d. As it was described, this observation could be related to the reduction of water liberation in the meat subjected to prolonged maturation periods (Figure 2). Regarding meat flavor and aroma, the best assessment mark by consumers was for meat with 14 d of maturation, which is significant for the meat of calves that received concentrated feed (Sem). However, in calves raised on permanent grazing (Ex), there were no significant differences in the assessment of flavor and aroma for the meat between 14 and 21 d of maturation.

Table 4 Scoring by consumer of the Pajuna breed calf meat in function of the production system (S) and maturation (M) of the meat. 

Grades on a scale of 8 points (1: I extremely dislike it; 8: I extremely like it). a, b, c: Different letters in a line indicate differences in maturation within a production system (p≤0.05). EEM: standard error of the mean.

Studies related to sensory appreciation indicate that fat affects the aroma and flavor of the meat in two ways: through oxidation of the fatty acids when carbonyl compounds are originated that contribute to the aroma, and when it acts as a deposit of odoriferous compounds released during heating or during storage of the meat. In the Pajuna breed, this effect on fat transformations at 14 d of maturation of the meat is quite valued by consumers. In the same way as what was observed for the appreciation of juiciness, the highest marks for general acceptability were for the meat matured for 14 d.