1. Introduction
In recent decades, research on feedlot ruminants has primarily focused on finding technologies that improve feed efficiency and growth rates during the fattening process [
1,
2]. The final phase of finishing is characterized by lower growth efficiency in lambs, mainly because their gain consists predominantly of fat rather than muscle [
3]. Thus, one of the main objectives during finishing is to augment the proportion or weight of muscles while decreasing the fat content in the carcass [
1,
2]. An alternative approach to achieve this goal is through the use of growth promoters, such as gluconeogenic precursors or β-adrenergic agonists (β-AA).
It has been observed that the gluconeogenic precursor calcium propionate (CaPr) alters energy metabolism in two ways when supplemented in ruminant diets: firstly, by altering rumen fermentation through improvements in ruminal dry matter (DM) digestibility, thereby increasing the proportion of ruminal propionate, and decreasing methane production [
4,
5]. Another way is by improving insulin’s action on glucose metabolism [
6], thereby promoting an increase in energy status through enhanced glucose synthesis via gluconeogenesis in the liver [
7]. In this context, improvements in growth performance, feed efficiency, and muscle growth have been reported in finishing lambs supplemented with a daily dose of 10 g of CaPr/d [
8]. Carrillo-Muro et al. [
9], studying finishing diets for lambs, determined that a dose of 10 g of CaPr/lamb/d for 42 d led to the following increases: 13% dry matter intake (DMI), 28% average daily gain (ADG), 17% ADG:DMI ratio, 7% final body weight (FBW) and 4% empty body weight (EBW); in addition, cooling loss percent (CL%) was reduced by 13%, without impacting meat quality variables. Furthermore, the duration of CaPr supplementation selectively impacts the benefits in terms of growth and carcass traits. Carrillo-Muro et al. [
10] assessed varying inclusion durations of 10 g of CaPr/lamb/d, noting the following durations and their corresponding maximal increments: 15 d for DMI (1%), 25 d for FBW (5%) and ADG (27%), 28 d for ADG:DMI ratio (25%), 24 d for hot carcass weight (9%, HCW), and 20 d for dressing percentage (4%, D%). However, the extended inclusion duration (42 d) led to an increase in fat thickness (30%, FT) and a reduction in the proportion of loin (22%). Conversely, it increased the weight of leg (10%) and rack (14%). This suggests that the surplus energy provided by CaPr is stored as fat, resulting in a decrease in the proportion of certain muscles.
Zilpaterol hydrochloride (ZH) is a β-AA. When administered at a rate of 4 to 8 mg/kg diet during the final 20–40 d of fattening, it enhances growth performance, dietary energy utilization efficiency, and carcass traits in lambs [
11]. The advantages of using ZH during the finishing phase of fattening have primarily been attributed to alterations in the composition of tissue gain [
2,
12]. Receptors in muscle and fat are activated by ZH, leading to augmented lipolysis, reduced lipogenesis, and increased protein accumulation, either individually or in combination [
13]. However, these alterations in muscle tissue could impact the meat quality of lambs supplemented with ZH [
14]. In support of this, a reduction of 8% in cook loss percentage (VFA%) has been noted in lambs supplemented with ZH [
15]. Likewise, lambs that were administered 0.15 mg ZH/kg body weight (BW) experienced a 10% increase in Warner−Bratzler shear force [
16].
Given the mechanism of action of CaPr and ZH, their combination could be complementary. Based on this, we hypothesized that supplemental CaPr in lambs receiving ZH during the final phase of fattening can enhance the response to ZH supplementation in terms of productive performance and carcass characteristics. Furthermore, the magnitude of these effects may be linked to the duration of CaPr. Therefore, the objective of the present study was to investigate the impact of varying inclusion durations of CaPr (0, 14, 28, or 42 d before slaughter) at a daily dose of 10 g in lambs finished with supplemental ZH (received ZH 28 d plus 3 d ZH withdrawal prior to slaughter) on productive performance, carcass characteristics, and meat quality.
2. Materials and Methods
The experiment took place at the Small Ruminant Experimental Center and Meat Science and Technology Laboratory, both located within the Unidad Académica de Medicina Veterinaria y Zootecnia at the Universidad Autónoma de Zacatecas (UAMVZ-UAZ), in the state of Zacatecas, Mexico (north-central Mexico). Throughout the experiment (April to May 2023), the ambient air temperature averaged 26.2 °C, with a minimum of 9.4 °C and a maximum of 32 °C.
All experimental procedures involving the lambs were conducted in accordance with the guidelines of the approved Official Mexicans Standards: NOM-051-ZOO-1995; NOM-062-ZOO-1999; NOM-024-ZOO-1995; NOM-033-SAG/ZOO-2014 and NOM-EM-015-ZOO-2002. In addition, the experiment reported herein was approved by the Bioethics and Animal Welfare Committee of UAMVZ-UAZ, with protocol number 2023/04.
2.1. Animal Housing, Basal Diet, Management, and Feed Sampling
Forty-five male non-castrated crossbred Dorper lambs, with an average initial body weight (IBW) of 40.17 ± 0.35 kg and aged 6 months, were used. The lambs’ IBWs were recorded, and they were accommodated in 45 individual pens (1.5 × 1.5 m). Three weeks prior to the trial beginning, all lambs underwent health management, which included the following: (1) identification with a uniquely numbered ear tag; (2) intramuscularly vaccination against
Clostridium spp. and
Pasteurella spp. (Exgon 10, Chinoin Veterinary, Aguascalientes, Mexico); and (3) treatment for endoparasites (Closantel 5%, Chinoin Veterinary, Aguascalientes, Mexico) and ectoparasites (Doramectin 1%, Dectomax, Zoetis, Ciudad de Mexico, Mexico). The lambs had an adaptation of 21 d to both the facilities and the basal diet. The composition of ingredients and the chemical characteristics of the basal diet are presented in
Table 1. The diet was formulated to meet or exceed the finishing lambs’ recommendations for nutrients [
17]. Throughout the study, lambs had unrestricted access to both the basal diet and fresh water. Fresh feed was provided twice daily at 800 and 1600 h in a 40:60 ratio, respectively. Feed offered to each lamb was adjusted to minimize waste (5% of the previous day’s intake, as-fed). Feed bunks were visually assessed between 0740 and 0750 h each morning, and any remaining feed was collected and weighed for determination of dry matter intake (DMI). Adjustments in daily feed delivery were made at the afternoon feeding. Prior to the morning feeding, the lambs were individually weighed at the beginning of the experiment (IBW), at intermediate points (14, 28 d) and at the end of the experiment (42 d). Daily samples of the basal diet were collected and analyzed in triplicate for the following: (1) DM%, dried for 24 h at 100 °C in a forced air-drying oven; (2) crude protein (CP) (FP-528 LECO nitrogen analyzer) [
18]; (3) neutral detergent fiber (NDF) (fiber Ankom analyzer); and (4) Ether extract (EE) (extractor of Ankom
xt15).
2.2. Experimental Design and Treatments
A completely randomized design with five treatments was employed to investigate the effects of CaPr administration during four treatment periods (0, 14, 28, or 42 d before slaughter) in lambs that were finished with ZH (for a fixed period of 28 d before slaughter). We randomly assigned the following treatments to the experimental units using a Microsoft
® Excel
® template: (1) no additives (CTL), (2) 0 days on CaPr plus 28 d on ZH, (3) 14 days on CaPr plus 28 d on ZH, (4) 28 days on CaPr plus 28 d on ZH, and (5) 42 days on CaPr plus 28 d on ZH. At the end of the fattening period, all lambs received a withdrawal period of 3 d before slaughter (
Figure 1). The source of CaPr was Nuprocal
® (Nutryplus, Queretaro, Mexico), originating from the same batch, comprising 20% calcium and 69% acid propionic. The source of ZH was the same batch as the patented trademark Zilmax
® (MSD, Salud Animal Mexico, Estado de Mexico, Mexico). Individual CaPr (10 g/lamb/d) and ZH doses were weighed (precision balance, Pioneer-PX523, Ohaus Corp., Parsippany, NJ, USA); daily ZH dosage for feedlot lambs was 0.150 mg/kg average BW (47.8 kg), which essentially equates to 7.2 mg/lamb/d. The treatments were consistently administered by the same individual, and to prevent any influence on the lambs, the bags containing the doses were only identified with consecutive numbers (1 to 5), as seen in
Figure 1. To ensure the treated group’s total intake, the doses were mixed with 100 g of the basal diet, offered in the morning. After consumption, the remaining portion of the diet was administered.
2.3. Productive Performance Calculus
Based on the data collected during the feeding trial, the following parameters were calculated: (1) ADG = [(FBW − IBW)/number of d on feed]; (2) Average DMI = (Feed offered—Feed refused), which was weighed and recorded daily; and (3) ADG:DMI ratio = (ADG/DMI).
2.4. Slaughter Procedure and Visceral Organ Mass Determination
At the conclusion of the 42 d trial period, the lambs underwent a fasting period (18 h) while having access to water. Pre-slaughter weights were recorded for subsequent calculations. During the slaughter process, non-carcass components, including skin, heart, lungs, liver, spleen, kidney, perirenal fat, and full and digesta-free gastrointestinal tract, were removed and weighed. EBW = (Pre-slaughter BW—Total non-carcass components weight). Visceral organ mass was expressed as g/kg of EBW.
2.5. Carcass Characteristics
The hot carcass weight (HCW) was determined prior to chilling (24 h at 4 °C). After the cooling process, the cold carcass weight (CCW) was measured, including the kidneys and internal fat. Carcass D% was calculated as = ([CCW/EBW] × 100), and the percentage of cooling loss (CL%) = ([HCW-CCW]/HCW) × 100. After a 24 h chilling period, the carcass dimensions were measured using a flexible tape measure, including carcass length, leg length, and chest circumference. Longissimus muscle area (LMA) and FT between the 12th and 13th ribs were assessed on days 0, 14, 28, and 42, employing an Aloka Prosound 2 instrument with a 3.5 MHz linear transducer.
2.6. Whole Cuts and Tissue Composition
The left sides of the carcasses were sectioned in accordance with the guidelines of the Institutional Meat Purchase Specifications (IMPS) and North American Meat Processors Association, to extract the (1) forequarter, which was further subdivided into the neck, shoulder IMPS206, shoulder IMPS207, rack IMPS204, breast IMPS209, ribs IMPS209A; and (2) hindquarter, comprising the loin IMPS231, leg IMPS233, and flank IMPS232 [
19]. The weight of each cut was then recorded and expressed either in g/kg of EBW or as a percentage of the CCW. The carcasses were halved, and the left side was dissected. The tissue composition of the shoulder was determined via a physical dissection process to calculate the percentage of muscle, fat, and bone [
20].
2.7. Meat Quality
The 9th to 10th rib section of the longissimus muscle (LM, approximately 500 g) from the right-half carcass was used for subsequent meat quality analysis (frozen at −20 °C).
Color measurements were taken from the surface of the LM exposed by the 12th/13th-rib cut, using a Minolta CR-400 spectrophotometer (Konica Minolta Sensing, Inc., Osaka, Japan). The configuration included an 8 mm aperture size, observer 10, D65 illuminant, and a blooming time of 1.5 s. The color coordinates, including luminosity (Hunter L* Value), redness (Hunter a* value), and yellowness (Hunter b* value), were measured after 24 h postmortem. The pH of the right LM at the 2nd lumbar vertebra (LM) was determined using a portable digital pH meter (Hanna Instruments, Model HI–9025).
The water-holding capacity percent (WHC%) was assessed following the methodology described by Grau and Hamm, as suggested by Tsai and Ockerman [
21]. In summary, 300 mg of LM was enclosed with filter papers (Whatman #1), positioned between glass plates (15 × 15 cm), and subjected to a consistent pressure of 10 kg for 20 min. LM steaks (2 cm thick) acquired from between the 12th rib and L2 vertebrae were vacuum-sealed in plastic bags and frozen at −20 °C. After being stored for 14 d, the steaks were allowed to temper for 24 h at 4 °C, then gently blotted dry and weighed. One steak was sliced into dimensions of 15 mm × 15 mm × 30 mm, then suspended at 4 °C for 24 and 48 h, to calculate the percentage of purge loss (PRL%). Another steak was vacuum-sealed in a polyethylene bag and heated to 80 °C until the internal temperature reached 70 °C for determination of CKL%. The sealed plastic bag samples were placed individually and submerged in a water bath at 75 °C until they attained an internal temperature of 70 °C. Following the cooking process, the samples were cooled under running tap water, extracted from the packaging, gently blotted, and weighed. The WHC%, PRL%, and CKL% were expressed as percentages of weight loss compared to the initial weight. This was calculated as [(initial weight − final weight)/initial weight] × 100.
Steaks (2.54 cm thick) were thawed for 24 h at 4 °C. They were subsequently broiled on an electric grill, specifically the George Foreman Electronics (Model GR2120B), until they reached an internal temperature of 70 °C [
22]. The internal temperature was monitored using a Kitchen thermometer (Model TP700). After the cooking process, the steaks were allowed to cool at 22 °C for 4 h. In due course, six cores, each measuring 1.27 cm, were extracted parallel to the muscle fiber using a mechanical coring device. These cores were then subjected to shearing using a Texture Analyzer (G-R Manufacturing, New York, NY, USA) equipped with a Warner−Bratzler knife, and peak shear force achieved was recorded. The crosshead speed was set to 200 cm min
–1. The shear force measurements were then averaged and expressed in kg/cm
2.
2.8. Statistical Analyses
The statistical analysis was conducted using SAS OnDemand free software. Normality assumptions were validated through the UNIVARIATE procedure. The data were analyzed using a completely randomized design with the GLM procedure. ADG, DMI, and ADG:DMI ratio were analyzed using the MIXED procedure for repeated measurements. Lambs and carcasses were considered as the experimental units for productive performance and meat characteristics. The CCW was introduced as a covariate for the analysis of carcass characteristics. When significant effects were detected, mean comparisons were carried out employing the Tukey method with the LSMEANS instruction.
The duration of CaPr supplementation was categorized into linear and quadratic orthogonal polynomials, involving four equally spaced levels, using the LSMEANS and ESTIMATE statements. In instances where quadratic polynomials exhibited significance, quadratic equations were computed utilizing the REG procedure. Significance was established when the p-value was ≤0.05, and a trend was considered if the p-value was >0.05 and ≤0.10.