*2.4. Carcass Classification*

At the end of the experiment, the bulls were transported by truck to a local commercial abattoir. At the end of the slaughter line, warm carcass weight (CW) was recorded, and carcass classification, i.e., evaluation of conformation and fatness, was performed by an accredited classification body using the European Union beef carcass classification system (EUROP). For the statistical analysis, scores of the conformation (E, U, R, O, and P) and fatness (1–5) scale were transformed into numerical classification units on a 15-point scale. Age at slaughter (AS) was calculated as the difference between the date of birth and the date of slaughter. The dressing percentage (DP) was calculated as the ratio of CW to its live weight at slaughter (LWS). A measuring tape was used to record the carcass length and chest depth as described by Campion, Keane, Kenny and Berry [36].

#### *2.5. Measurements of Meat Quality Traits*

A day after slaughter, samples of *Longissimus dorsi* (LD) were taken from the left carcass at the level of the last rib to measure meat quality traits. The muscle was cut into two 5 cm-wide large sections. One part was used for analysis of fresh meat, whereas the other was weighed, vacuum-packed, and subjected to vacuum aging for 14 days at 4 ◦C (matured sample). Fresh samples were used to measure pH value; color parameters (L\*, a\*, and b\*) chemical composition by NIR; marbling; water-holding capacity (WHC); and tenderness.

The pH value was measured at the center of the LD muscles at 24 and 48 h postmortem using an MP120 Mettler Toledo pH meter fitted with a combined glass electrode InLab427 (Mettler-Toledo, GmbH; 8603 Schwarzenbach, Switzerland). The samples were evaluated in two replicates at two different sites [37].

To evaluate meat color, a Minolta L\*a\*b\* colorimeter was used. Analysis was performed on a freshly cut surface of LD exposed for 60 min to bloom [38]. Measurements were taken in triplicate using a Minolta Chroma Meter CR-300 (Minolta Co., Ltd., Osaka, Japan). Color stability measurements were performed 24 h after slaughter.

Determination of moisture, intramuscular fat (IMF), and protein content (chemical composition) were determined by near-infrared spectroscopy (NIR Systems 6500 Monochromator; Foss NIR System, Silver Spring, MD, USA), as described previously by Prevolnik et al. [39].

Water-holding capacity (WHC) was determined by three different methods: drip loss, cooking loss, and thawing loss. Drip loss was determined using the EZ-DripLoss method as described by Christensen [40]. Two cylindrical pieces (2.5 cm) were cut from the central area of the LD. The samples were weighed and sealed in plastic sealable cups (Sarstedt AG & Co., Nümbrecht, Deutschland meat extract collector), and weight loss was recorded after 24 and 168 h of storage [41].

Samples for determining cooking loss were first weighed and then cooked in a thermostatic water bath (ONE 7-45), until the interior of the sample reached 72 ◦C. After cooking, the samples were dried with paper towels and reweighed to obtain data on water loss during cooking.

To determine thawing loss, samples were weighed, vacuum packed, and frozen at −20 ◦C. Samples were then thawed overnight at 4 ◦C, gently dried with a paper towel, and reweighed [42].

After cooking, the samples were left to cool and objective determination of tenderness was performed by determining the shear force (N) with a TA Plus texture analyzer (Ametek Lloyd Instruments Ltd., Fareham, UK). The same procedure was repeated for the aged samples.

To assess marbling, cross-sections of LD were visually compared using a reference standard scale ranging from 1 (devoid of marbling) to 10 (abundantly marbled). Two operators independently evaluated each sample, and an average of the two scores was obtained [43].

#### *2.6. Fecal Clostridia Analysis*

The fecal samples were collected (using individual plastic sleeves) from each animal on the day before the start of the trial and on the day of slaughter. The first fecal samples were obtained on a scale by catching feces of individual animals into a clean plastic bucket. At the end of the fattening trial, colon fecal samples were collected from the slaughterhouse. The fecal samples were stored at 4 ◦C until freezing (–72 ◦C). Samples were then sent by express mail in controlled conditions to the laboratory for further analysis (Miprolab GmbH, Göttingen, Germany).

Two methods were applied for the analysis of *Clostridia*. With the "Most Probable Number" (MPN), the total number of sulfite-reducing *Clostridia* was determined (pathogenic and non-pathogenic species) to ge<sup>t</sup> an overall count of all viable *Clostridia*. Selective *Clostridia* enrichment and counting analyses allowed for the detection, identification, and semi-quantitative counting of the relevant pathogenic *Clostridia* species.

#### 2.6.1. Total Number of *Clostridia*

The total number of viable sulfite-reducing *Clostridia* was determined using the MPN adhering to the principles of EN 26461-1 in a miniaturized design. Briefly, 50 g of each sample was added to 100 mL of differential reinforced *Clostridia*l medium (DRCM, Heipha, Germany) and homogenized using two cycles of 30 s in a paddle blender (Stomacher 400, Seward, UK). From each homogenized sample, 1 mL was pipetted into 9 mL of DRCM and thoroughly mixed. Further dilutions were performed in 96-well plates with a transfer volume of 25 μL

and a total volume of 250 μL per well. Each sample was tested in five replicates. The plates were covered and incubated at 37 ◦C for 14 days. The incubation atmosphere consisted of 85% N2, 10% CO2, and 5% H2 generated with an Anoxomat (Mart, The Netherlands). All black wells were counted as positive for the growth of sulfite-reducing *Clostridia*, and the total number was calculated using the MPN table taking into account the initial dilution. *Clostridia* counts are given as bacteria per gram of sample.

#### 2.6.2. Selective *Clostridia* Enrichment and Counting Analysis

To selectively identify and count relevant pathogenic bacteria (*C. perfringens* and *Clostridium sporogenes*), samples were spread on egg yolk lactose agar (Heipha, Germany). Aliquots of the homogenized fecal samples were heated at 80 ◦C for 10 min to activate the spores and to reduce the interfering accompanying flora. Plates were incubated for 48 h at 37 ◦C in an anaerobic atmosphere as described above. The incubation time was extended to 72 h in the case of insufficient growth. Bacterial colonies were identified by colony properties, including lipase and lecithinase activity. Colonies of the same type, appearance, and macromorphology were randomly selected for confirmation by Remel RapID ANA (Thermo Fisher Scientific, Lenexa, KS, USA) and Gram strain. *C. perfringens* was additionally tested by PCR. Colonies of the various species were counted semi-quantitatively on the plates, and the results were grouped into four classes (Number 0 = no growth; Number 1 = 1–10 colonies; number 2 = 11–50 colonies; number 3 = >50 colonies) between treatments. A quantitative counting of the colonies was not performed, because plate cultures were obtained from pre-enriched cultures and were only able to provide a semiquantitative assessment of the distribution of the individual bacterial species. However, the pre-enrichment process still reflected the initial concentration ranges. The range for the semi-quantitative data was calculated as follows: 0, no growth; range 1, 1–10 colonies; range 2, 11–50 colonies; range 3, >50 colonies. All four ranges reflect the initial concentrations in the samples.
