The experiment was conducted at the research farm, Khon Kaen University, Khon Kaen Province, Thailand (16.46° N 102.82° E; altitude 169 m above sea level) from January 2021 to June 2021. All animal treatment and related procedures were performed according to the Khon Kaen University Animal Ethics Committee (Record No. IACC-KKU-49/62).
2.1. Animals, Experimental Design, and Diet
A total of thirty-six fattening beef steers, eighteen Charolais crossbred (75% Charolais × 25% native Thai; age of 24.6 ± 2 months initial body weight of 548 ± 9.90 kg) and eighteen Holstein Friesian crossbred (93.19% Holstein Friesian × 6.81% native Thai; average age of 22 ± 0.1 months initial body weight of 496 ± 17.0 kg) steers were used for the experiment. Animal was purchased at the backgrounded stage from local farmers and treated for intestinal and external parasites (1 mL/50 kg body weight; Ivermectin F, Bangkok, Thailand) and vitamins A, D3, and E (10 mL/head; Phenix, Bangkok, Thailand; vitamin A propionate, 300,000 IU/mL; vitamin D3 cholecalciferol, 100,000 IU/mL, vitamin E acetate, 50 mg/mL) before the start of the experiment. Each animal was housed in a pen (2.5 m × 4.5 m) with free feed and drinking water access. The pens were cleaned every morning throughout the experiment. Animals were fed ad libitum twice daily at 08.00 and 15.00 h (50:50 proportion w/w).
The individual animal was considered an experimental unit and was assigned to one of the six blocks (replicates) according to initial body weight using a 2 × 3 factorial arrangement in a randomized complete block design. Factor A was two cattle breeds (Charolais and Holstein Friesian crossbred), and factor B was three dietary metabolizable energy densities (low, medium, and high;
Table 1). The feeding trial was divided into two phases (the early fattening phase lasted 59 days, and the late fattening lasted 104 days before slaughtering) after an adaptation period of 15 days. For each feeding phase, the dietary energy density was calculated to contain metabolizable energy content based on the energy content of feedstuffs according to WTSR [
13] for the early fattening stage (low: 10.5, medium: 10.8, and high: 11.2 MJ ME/kg DM) and late fattening stage (low: 10.5, medium: 11.1, and high: 11.8 MJ ME/kg DM).
According to WTSR [
13], the total mixed ration silage was formulated (
Table 1) and produced to meet the nutrient requirements of fattening beef cattle. The total mixed ration silage was prepared in a horizontal mixer. Approximately 2000 kg of each batch of the dietary treatment mixture was mixed and packed 35 kg per plastic bag silos and, after preparation, was stored outdoors at ambient temperatures of 25 °C to 39 °C for at least seven days [
4,
5].
2.2. Sampling Collection and Laboratory Analysis
Dry matter (DM) content was determined by fan-forced oven of the samples at 105 °C to a constant weight. Subsamples were dried in a fan-forced air oven at 65 °C for 24 h, milled, and passed through a 1 mm screen and chemical analysis. Ash and ether extract (EE) was determined using the AOAC method [
14] (methods 942.05 and 920.39). Dry feed samples weighing approximately 0.1 g were encapsulated in tin foil and analyzed for nitrogen (N) using a nitrogen analyzer (LECO FP828, MI, USA). Crude protein (CP) was calculated as 6.25 × N according to the methods of Etheridge et al. [
15]. Fiber contents, including those of neutral detergent fiber (NDF) and acid detergent fiber (ADF), were analyzed using an Ankom200 Fibre Analyzer (Ankom Technology, Macedon, NY, USA), NDF treated with α-amylase and sodium sulfite according to the method of Mertens [
16]. Non-fiber carbohydrate content was estimated using the 100 − (CP + NDF + EE + Ash) [
17].
During the experiment, each morning, we collected feed-offered samples every week and pooled them for chemical analysis. The daily nutrient and energy intake were calculated as the difference between the amount of feed offered and refused.
The body weight of all cattle was recorded at the start and end of the experiment before the morning feeding. The average daily gain was calculated by the initial and final body divided by the total day of each feeding phase.
Blood and rumen fluid samples were collected from each animal three hours after the morning feeding on day 54 (for the early fattening phase) and day 103 (for the late fattening phase) after starting the experiment. Approximately 10 mL of blood was collected from the jugular vein, packed on ice, and transported to the laboratory (Accreditation No. 4138/57; Khon Kaen TLC Lab Center Co., Ltd., Khon Kaen, Thailand). Plasma metabolites (urea nitrogen, glucose, triglyceride, cholesterol, total protein, and albumin concentrations) were determined using colorimetric method test kits (Roche Diagnostics, Indianapolis, IN, USA) and an automated analyzer (COBAS INTEGRA 400 plus analyzer, Roche Diagnostics, Indianapolis, IN, USA). Two hundred milliliters of rumen fluid were taken from each animal using an esophageal–rumen stomach tube technique [
5]. The ruminal fluid pH was immediately measured with a glass electrode pH meter (Eutech pH 700, Eutech Instruments Pte Ltd., Ayer Rajah Crescent, Singapore). Ruminal fluids were separated from the feed particles through four layers of gauze, and then 100 mL of rumen fluid was put into 150 mL plastic containers with 10 mL of 6 N HCL. These were collected and stored in ice buckets before being transported to the laboratory and stored at −20 °C. The concentration of NH3-N was analyzed using a using Kjeldahl nitrogen analyzer. The lactic acid and volatile fatty acid concentrations were analyzed using gas chromatography (GC2014, Shimadzu, Kyoto, Japan).
Animals were slaughtered at the end of the experiment. They were transported by truck for approximately 8 h (506 km) to a good manufacturing practice (GMP) standard commercial slaughterhouse (Prakob Beef Products Co., Ltd., Ratchaburi, Thailand). They were slaughtered after being fasted overnight but allowed free access to water. The animals were weighed, stunned, bled, skinned, eviscerated, and washed. The dressing percentages of hot carcasses were calculated. After dressing, the carcasses were chilled, aging at 4 °C to 7 d. The chilled carcass percentage was calculated similarly to the hot carcass dressing percentage.
After 7-day chilling, the rib-eye area between the 12th and 13th rib surfaces of the longissimus dorsi muscle was measured using a transfer and graph paper. In contrast, the back fat thickness was measured at 3/4 of the longissimus dorsi muscle length at the 12th rib. At the same time, approximately 1 kg of longissimus dorsi muscle samples were taken from the right side of each cattle between the 12th and 13th ribs, transported to the laboratory at 4 °C, and stored at −20 °C for further physicochemical characteristics and chemical analysis. To analyze physicochemical characteristics, the longissimus dorsi muscle samples were cut in half at a thickness of 2.5 cm steaks and then exposed to air for one hour (blooming) at room temperature. To measure surface meat color after muscle oxygenation, meat pH was using a pH meter (Consort C933 Multi-parameter analyzer, Consort bvba, Belgium), and the meat color surface was measured at three locations that were randomly selected to calculate the average values using a color reader (CR-10, Konica Minolta, Tokyo, Japan). Lightness (L*), redness (a*), and yellowness (b*) were recorded.
To determine the drip loss, 50 g meat samples were hung on a nylon cord in a plastic bag at 4 °C for 24 h. Three 2.5 cm thick steak subsamples were sealed in heat-resistant plastic bags to be boiled in a water bath (WNE 29, Memmert, Schwabach, Germany) at 80 °C until an internal temperature of 75 °C was reached. Following that, the samples, after cooking, were sliced into three subsamples parallel to the fiber orientation (width × length × height, 1 × 3 × 1 cm
3 rectangle) before measuring Warner–Bratzler shear force with a texture analyzer (model TA. HDplusC, Stable Micro Systems, Goldalming, UK). The chemical composition of longissimus dorsi samples was determined following the standard methods of AOAC [
14].
2.3. Statistical Analysis
All data were subjected to analysis of covariance using initial body weight as the covariate to adjust for initial weight differences among animals by the generalized linear model procedure of SAS 9.0 [
18]. Nutrient intake, ruminal fermentation, plasma blood metabolites, carcass characteristics, meat quality, and chemical data were analyzed according to a 2 × 3 factorial arrangement in a randomized complete block design according to the following model:
where Y
ijk is the dependent variable, μ is the overall mean,
cov is the covariate (initial body weight),
Blki is the effect of the block (i = 1 to 6), α
j is the effect of cattle breeds (j = 1 to 2), β
k is the effect of dietary energy density (k = 1 to 3), αβ
jk is the interaction effect (breeds × energy density), and ε
ijk is an error. Within the dietary treatments, polynomial contrasts (linear and quadratic) were performed to examine the effects of the energy density. Significance was declared at
p ≤ 0.05.