Experimental animals and pastures

All procedures involving the animals were approved by the Swiss Cantonal Committee for Animal Care and Use (approval no. ZG 58/12). Suckling calves were chosen as experimental animals, as suckler beef production is a common use of extensive pastures in Europe. Twenty-four calves (13 steers castrated shortly after birth and 11 heifers) from F1 Angus × Holstein (n = 19) or Limousin × Holstein (n = 5) dams in their 2nd to 6th lactation, all mated with the same Angus sire (Eldorado, CH 120·0728·8751·3), were involved in the present study. The calves weighed 185 ± 22 kg and were 18 ± 2·5 weeks old at the beginning of the experiment. Twelve calves, with their dams, were each allocated to two groups as balanced as possible for the calves’ sex, live weight (LW) and age, and the dams’ genotype and lactation number.

Before the experiment, calves and their dams had been grazing for 1 month on flat pastures of the winter station at 1000 m above sea level (a.s.l.). The experiment started on 20 June 2013 with the transport of animals to the ETH alpine research station Alp Weissenstein, canton of Grisons. From arrival on the alpine research station onwards, the animals were either kept on steep pastures with 35–40% inclination (S-calves with dams; area situated between 1920 and 2040 m a.s.l.) or on flat pastures with 0% inclination (F-calves with dams; 2030 m a.s.l.). The experimental period lasted for approximately 11 weeks, equivalent to the typical duration of the grazing season on high altitude seasonal alpine pastures (Swiss Federal Office of Agriculture 2014). The LW was measured weekly on each calf over the whole period. The two groups were subjected to a rotational pasture management (fenced paddocks of 1–2 ha) in order to avoid substantial changes in forage quality over time. Apart from the intention to generate a large difference in inclination, the sites selected as experimental pastures were anticipated to offer swards with a similar forage quality in order to avoid confounding of physical activity with forage quality. During the experiment, forage samples were collected every week or, alternatively, just before the animals entered a new paddock. The available biomass 20 mm above ground level in three to four strips with an area of 80 × 1200 mm² was selected to be representative of the paddock. It was harvested using a grass shear (ComfortCut Gardena, Husqvarna AB, Stockholm, Sweden). Samples were oven-dried for 48 h at 60 °C, ground to 1 mm and pooled per pasture and sampling date. The pooled forage samples were analysed for contents of dry matter (DM) and ash using a TGA-701 (Leco Corporation, St. Joseph, Michigan, USA) and nitrogen (N) using a C/N analyser Typ FP-2000 (Leco Instrumente GmbH, Kirchheim, Germany). Additionally, neutral detergent fibre (NDF), acid detergent fibre (ADF) and acid detergent lignin (ADL) were analysed according to Van Soest et al. (Reference Van Soest, Robertson and Lewis1991) using the Fibertec System M (Tecator 1020 Hot Extraction, FOSS GmbH, Hamburg, Germany) excluding residual ash content. For NDF analysis, α-amylase was used.

Locomotive and feeding behaviour measurements

Calves were equipped with pedometers (IceTag, IceRobotics Ltd, Edinburgh, Scotland, UK) and rumination sensors (MSR data logger, Seuzach, Switzerland) to assess locomotive activity and feeding behaviour, respectively. Four calves per group carried a pedometer for 3 weeks, and the procedure was carried out three times, using different calves each time. This schedule meant that each calf carried a pedometer for 3 weeks. In total, pedometer data on the locomotive activity of 22 calves for an average of 15 days per calf could be used to calculate times lying down and the number of steps per 24 h. From that, the relationship between the number of total steps per unit of time the calves were standing or walking within the day was computed. With the equipment used, standing and walking could not be satisfactorily separated. Six rumination sensors were available, which were carried alternatively by one experimental group of calves or the other over 5 days. With this setting, each calf carried the rumination sensor at least twice. Finally, a total of 32 datasets of, on average, 4·8 days of recording from 20 different calves could be used for data analysis. From the rumination sensor data, the daily ruminating and eating times (the latter including both grazing and suckling) as well as the number of ruminated boluses were calculated using the software Viewer 2 provided by MSR data logger.

Slaughter procedure, measurements on the carcasses and sampling techniques

In order to allow sufficient time for data recording and sampling at a commercial abattoir, the calves were slaughtered in two sub-groups on 2 and 5 September 2013. For that, each experimental group was split into two sub-groups of six calves, balanced for sex, LW and age at slaughter. The transport from the alpine research station to the abattoir lasted 3 h. The calves were slaughtered within 1 h after arrival. Stunning was performed by percussion using a captive bolt. Blood was taken at exsanguination, preserved in blood sampling tubes containing heparin (Vacuette, Greiner Bio-One GmbH, Kremsmünter, Austria). The haemoglobin concentration was measured within 6 h after slaughter (Hemocue 201+ device, Hemocue AB, Ångelholm, Sweden). Each ventricle of the heart was cut open to let the residual blood drip out, and then the hearts were weighed. Hot carcasses were weighed within 30 min after slaughter. The ratio of the hot carcass weight to the LW measured prior to slaughter was defined as dressing percentage. For each calf, carcass merit was evaluated by an experienced staff member according to the CH-TAX classification applied in Switzerland, which resembles the EUROP classification. In brief, a 7-point scale was applied for carcass conformation (1 = lowest muscularity, 7 = highest muscularity) and a five-point scale for carcass fat cover (1 = too lean, 3 = optimal, 5 = too fat; Proviande 2015).

Samples for determining fibre types and post mortem (p.m.) proteolysis were taken from the left side of the carcass from the longissimus thoracis (LT) muscle between the 8th and the 9th rib and from the biceps femoris muscle (BF) within 40 mm of its cranial extremity. The samples subjected to fibre typing were obtained 1 h p.m. and those to be used for proteolysis analysis were taken 1, 3, 24 h and 7 days p.m. The latter was obtained from a sample harvested from the chilled carcasses at 24 h and stored for a further 6 days in a vacuumed bag at 4 °C. All samples were preserved at –80 °C until analysis. For accuracy of the sampling time, the samples taken at 1 and 3 h p.m. were first frozen in liquid N. The pH was recorded at 1, 3 and 24 h p.m. at a depth of about 20 mm inside the muscle, at the same point where the samples were taken. Samples for meat quality analysis were collected 24 h p.m. and included the LT from the 8th to the 12th rib and the BF from between 40 and 120 mm away from the cranial extremity of the muscle. The cross-sectional area of the LT was assessed with the ImageJ software (http://imagej.net) using a scaled photograph of the transversal cut of the muscle, which was still attached to the rib and spine to avoid deformation, at the level of the 13th rib. This was not possible in the BF as this type of assessment is only reliable when the muscle is supported by a bone to avoid deformation.

A 10 mm-thick slice from both BF and LT of each calf was homogenized in a blender (Moulinette type 643, Moulinex, Ecully, France) and frozen at –20 °C until chemical analysis was performed. The rest of the meat was vacuum packaged and aged for 21 days at 4 °C, which is a common period of ageing in Switzerland (F.J. Steiner, Managing Director of Pro Beef GmbH, personal communication, 2014). After this period, the meat was subjected to determination of physicochemical meat quality. Two 20 mm-thick slices were prepared from the cranial side of the LT after 21 days’ ageing and preserved frozen at –20 °C for later sensory analysis.

Muscle fibre typing and assessment of post mortem proteolysis

Transverse serial sections (10 µm thick) of the LT and BF were prepared using a Cryotome (Shandon Inc., Pittsburgh, USA). Muscle fibres were differentiated as slow-twitch (type I), fast-twitch (type IIX/B) and intermediate (type IIA) by their myosin ATPase activity based on the method described by Solomon & Dunn (Reference Solomon and Dunn1988). Each section was first incubated at pH 9·4 and then at pH 4·6. Three stained sections (containing at least 100 muscle fibres) per muscle and calf were analysed. Fibres were differentiated by type, counted and their area was measured with the software AnalySIS 5·0 (Olympus Europa GmbH, Hamburg, Germany).

The degradation of metavinculin, vinculin, desmin and integrin beta-1D, and the proportion of intact (80 kDa) μ-calpain were used as indicators of p.m. muscle proteolysis. The relative quantity of myofibrillar proteins was determined by chemiluminescence as previously described by Bee et al. (Reference Bee, Biolley, Guex, Herzog, Lonergan and Huff-Lonergan2006) and Berard et al. (Reference Berard, Kreuzer and Bee2008). The proportion of intact μ-calpain was determined by Western blot (procedure described in Bee et al. Reference Bee, Biolley, Guex, Herzog, Lonergan and Huff-Lonergan2006) using a specific μ-calpain antibody (9A4H8D3, Thermo Fisher Scientific, Rockford, Illinois, USA). Metavinculin and vinculin degradation were assessed by the decrease in intensity of the 150 and 117 kDa bands, respectively. For desmin, integrin beta-1D, metavinculin and vinculin, the degradation ratio was calculated as the intensity of each corresponding immunoreactive band over the intensity of the respective bands in a reference sample loaded on each gel and values were then normalized over the 1 h sample intensity. The results for μ-calpain were expressed as a proportion of unautolysed 80 kDa of the total peak area of the calpain bands (80, 78 and 76 kDa). For all other traits, values were related to those obtained 1 h p.m.

Meat analysis

Samples of BF and LT were subjected to the same analytical programme. Meat DM, ash and protein (6·25 × N) contents were analysed as described for forage analysis. Intramuscular fat content was determined by petroleum ether extraction using a Soxhlet extractor (B-811, Büchi, Flawil, Switzerland). Colour was measured on 10 mm-thick slices of fresh meat which had been allowed to bloom for 1 h in the dark at 4 °C. A Chroma Meter (model 300-CR, Minolta, Dietikon, Switzerland) was positioned at three locations on the meat slice to assess the CIE L* a* b* (lightness, redness and yellowness, respectively) colour space with D65 as light source. In addition, chroma and hue were calculated (Warris Reference Warris2010). For later statistical analysis, colour trait measurements were pooled to one dataset per muscle and calf. Drip loss was assessed according to the method of Honikel (Reference Honikel1998) where a 15 mm-thick meat slice was positioned to hang in a net in a sealed plastic bag at 4 °C for 48 h (in duplicate for LT, because there was more material available from this muscle). Cooking loss was measured on a 24 mm-thick meat slice in a vacuumed plastic bag cooked in a water bath for 45 min at 72 °C. Thereafter, the meat was cooled to room temperature for 2 h and weighed. Drip loss and cooking loss were calculated as the weight loss during hanging or cooking, respectively. Shear force was measured in the cooked and cooled meat as well. A Warner–Bratzler shear blade mounted on a texture analyser (TAXT2, Stable Micro System, Surrey, UK) was used for that purpose. Cross head speed was set to 2 mm/s. At least four meat cores of 12·7 mm diameter were obtained perpendicular to the meat slice by cutting with a drilling machine on which a sharp round knife was mounted. The results obtained with individual cores were pooled to one shear force value per muscle and calf. Due to technical problems, shear force values with the LT meat cooked in the water bath are not available. For that reason, shear force in this muscle was determined on LT meat cooked on a grill plate, which was also used for the sensory analysis (see below). In the BF, collagen properties were also determined. The collagen content was calculated from the hydroxyproline content multiplied by eight, according to method 318·1 of the Swiss Food Manual (Swiss Federal Office of Health and Food Commission 1999). Hydroxyproline content was determined by spectrophotometry as described by Loetscher et al. (Reference Loetscher, Albiker, Stephan, Kreuzer and Messikommer2015) and according to the method 317·1 of the Swiss Food Manual (Swiss Federal Office of Health and Food Commission 1999).

Eight experienced panellists performed the sensory analysis. They were first trained during two sessions for the criteria to be assessed during the analysis. These were flavour, juiciness, tenderness and overall liking graded from 0 (minimum grade) to 10 (highest grade). Three sessions with eight samples per session were carried out to analyse the 24 samples of the LT. Each session consisted of two services of four grilled samples each (two samples from S-calves and two samples from F-calves). Samples were prepared from frozen 20 mm-thick slices thawed overnight at 4 °C. The slices were cooked on a preheated (195 °C) aluminium grill plate (Beer Grill AG, Zurich, Switzerland) to an internal temperature of 69 °C as measured with a temperature probe inserted into the core of the meat slice. Just after grilling, each slice was cut into cubes of about 10 × 10 × 20 mm³. Two to three warm cubes per sample were served immediately on pre-warmed plates to each panellist.

Statistical analysis

Proximate forage analyses were compared by analysis of variance with the GLM model of SAS (version 9.3, SAS Institute, Cary, North Carolina, USA). Further statistical analyses were performed by analysis of variance with the MIXED model of SAS. Data were analysed with group as fixed effect, slaughter date as random effect and individual calves as experimental units. As for meat pH and muscle fibre proteolysis, the effect of time of sampling p.m. was not of primary interest for the present experiment, so time p.m. was not included in the model and each time point was analysed independently. Data from pedometers and rumination sensors were analysed considering group, date of measurement and their interaction as fixed effects. Date of measurement was treated as a repeated factor (animals nested within groups). For sensory analysis data, panellist was added as a random effect. Significance was considered at P < 0·05.