Experimental Animal Testing

Eighteen Pelibuey male lambs aged 2.5 months and weighing 18 ± 1 kg were acquired from Rodeo Ranch in Jalapa municipality, state of Tabasco, in south-eastern Mexico. Animals were dewormed with closantel 10% (Digavet, Belgium), 1 mL oral albendazole per 10 kg of body weight (Pfizer, USA) and treated with an intramuscular injection of vitamins A, D and E (2 mL 10 kg⁻¹ of bodyweight, Pfizer, USA) following the technical specifications of the products and per accordance with the UK Animals (Scientific Procedures) Act 1986, EU Directive 2010/63/EU for animal experiments, the Guide for the Care and Use of Laboratory Animals (NIH Publications No. 8023, revised 1978) and the Mexican Official Standards (^{SAGARPA 1995}, SAGARPA 2014).

Lambs were fed with a standard diet (SDiet) -12 MJ per day with 16% protein- formulated with milled corn, whole wheat, soy paste, grass, coconut paste, molasses, and a supplement of minerals and vitamins (^{AFRC 1993}). Water was provided ad libitum through 5 L dispensers. After being weighed, animals were randomly divided into three groups of six lambs each, according to the BA they received: SDiet or negative control, ZH group = SDiet+ZH (Zilmax, Merck Sharp & Dohme Corp, USA), and RH group = SDiet+RH (Lapi-Racto. REG. SAGARPA Q-20183-158, Lapisa). Dosage of growth promoters was given in accordance to the NOM-EM-015-Z00-2002 (^{SAGARPA 2002}) and the technical specifications of the product (C115 g ZH, and 125 g RH). The experiment lasted 40 days: growth promoters were supplied daily for 37 days plus the withdrawal period of 72 h. All feeding was with- drawn 12 h before slaughter. The response variables were final live weight gain (FW), average daily weight gain (ADG), feed conversion ratio (FCR), and feed consumption for 40 days. In addition, urine samples were taken from each animal (days 0, 10, 20, 30, and 40) and placed in sterile assay tubes, frozen immediately by using liquid nitrogen and stored at -20 °C until analysis.

All lambs were slaughtered following the Mexican Official Standard on animal welfare, NOM- 033-ZOO-1995 (^{SAGARPA 2014}). For each animal, 350 g of different cuts from the hindquarter were stored at - 20 ^oC until analysis.

Extraction of BA from muscle samples

To extract the BA, 100 g of muscle was blended with 40 mL of acetonitrile and 20 mL of isopropanol during 30 s. NaCl (1.2 g) was added to the blend, mixed on vortex for 2 min, supplemented with 4 g of Na2SO4 and 0.5 g of MgSO4 and mixed again for 2 min (Water, 2012). Lastly, the samples were centrifuged (Hermle Model Z323K, Germany) at 1960 g for 10 min. The supernatant was dried using a rotary evaporator (Büchi R-215, Labortechnik, Switzerland). The pellet was dissolved in 1 mL of 0.1% formic acid, sonicated for 2 min, passed through cartridges (Oasis MCX 3 cc Vac Cartridge, 60 mg Sorbent per cartridge, 30 µm, Waters, USA) and filtered (Membrane Filter, 0.45 µm and 0.22 µm pore size, Merck Millipore, Ireland) (Water 2012).

Determination of BA in urine and meat samples

Identification and quantification of growth promoters (BA) were determined through HPLC using a five-point calibration curve of the standards in the following concentrations for each group: ZH (0.47, 0.23, 0.11, 0.05, 0.02 g mL⁻¹ diluted in 50:50 methanol: water), and RH (0.66, 0.33, 0.16, 0.08, 0.04 g mL⁻¹ diluted in 50:50 methanol: water) (^{Sumano et al. 2002}). After extraction, samples were analyzed according to the ^{USDA (2012)}. Urine samples were injected directly after filtration (0.45 µm). The standards were filtered (0.45µm) and injected into the HPLC (HPLC 1200 (Agilent Technologies, Germany), at 4◦C, in a C-18 column (ZORBAX Eclipse column, XDB 80Å C18, 4.6 x 150 mm, 5 µm, Agilent, USA), with a flow of 20 µL min⁻¹ at 210 nm to 280 nm wavelengths, using as mobile phase (A) water and 1% formic acid, and as a gradient (B) acetonitrile and 1% formic acid (^{Waters 2012}).

Light microscopy

Micrographs of the meat samples from each treatment group were taken through optical microscopy. Samples were cut in 5 mm sections and placed in 35 mL of fixative FAA (30 mL of 40% formaldehyde, 150 mL of ethanol, 15 mL of acetic acid, and 105 mL of distilled water) for 72 h. Afterwards, they were washed and dehydrated with water: ethanol (3:1, 1:1, 1:3) and absolute ethanol, for 40 min. Samples were placed in xylol: ethanol (3:1,1:1,1:3) until absolute xylol was reached, during 20 min; then embedded in paraffin: xylol, set in a microscope slide, and stained with haematoxylin-eosin. Longitudinal and transverse sections were observed under LEYCA RAM optical microscope at 60X, 150X, and 300X (^{Arzate-Vázquez et al. 2011}).

Scanning Electron Microscopy (SEM)

Using a scalpel blade no. 10, muscle tissue cuts were made to obtain longitudinal and transversal sections (1.0 x 1.0 x 0.5 cm). These were fixed in 4% formaldehyde and 25% glutaraldehyde, later washed, and dehydrated with water: ethanol (25:100). Finally, the sample sections were dried until critical point in an SPI-Module Sputter (SPI-supplies, West Chester, PA), carbon coating equipment at 34-35 ^oC and 1200 psi, and covered with gold for 40 s (^{Estrada-Solis et al. 2016}). Micrographs were taken at 5 kV through scanning electron microscope (JSM-7800F, JEOL, Japan).

Digital Image Analysis (DIA)

Ten micrographs were analyzed with ImageJ software (1.52q, National Institutes of Health, USA) to determine muscle thickness. The scale was set to 2.55 pixels/µm based on SEM imaging (5 kV, 200X magnification) previously obtained, and the images threshold was automatically adjusted by the software (approximately 100). Average muscle fiber thickness was determined from a minimum of 20 muscle fibers per micrograph using ImageJ software measuring tool.

Statistical analysis

The results of biometric parameters and muscle fiber thickness were analyzed with an ANOVA test using Sigma Plot 12.5 software (Systat Software, USA) and reported as average ± standard error. The contrast of means was performed through the Tukey Test, assuming a significance level of 0.05.

In vivo assessment of β-agonists in lambs

Table 1 shows a greater DWG in the animals fed with BA (93 and 49% for ZH and RH, respectively), compared with the control group, during the 37 days of BA treatment. DWG pairwise comparisons between all three groups were statistically significant (p ≤ 0.05). FCR was lower in ZH in contrast to RH and the control group (p ≤ 0.05). FE was higher in the ZH group (26.00%) than in the RH group (20.63%), (p ≤ 0.05).

Kinetics and urinary excretion of β-agonists

Firstly, to detect BA on muscle samples, the UV spectrum of each standard was obtained using a UV detector of 190-380 nm; maximum wavelengths for standard samples were: ZH = 210 nm, 8.9 min retention time and RH = 280 nm, 12.49 min retention time.

Secondly, the concentration of BA excreted in urine was evaluated. Higher urinary excretion of BA occurred in the first 10 days of treatment (Table 2). At day 40, the excreted amount of BA was minimal (ZH = 0.14 mg mL¹ and RH = 0.0039 mg mL⁻¹), indicating that a pre-slaughter withdrawal period of 72 h allows proper elimination of ZH and RH.

Determination of β-agonist in meat samples

Analysis of meat samples through HPLC reported the absence of ZH and RH residues, probably due to their urinary excretion and the withdrawal period (72 h) before slaughter.

Optical microscopy of lamb samples

Optical microscopy allowed to characterize different cellular structures of lamb samples - to assess the effect of BA on their lipid content. Optical micrographs of the transverse sections of the lamb samples reflect the intensity of lipolysis in the BA treatment groups, compared to the control group (Figure 1).

Figure 1 Kinetics of BA present in the urine of lambs treated with two growth promoters 

SEM of lamb samples

Figure 2 displays morphological aspects of lamb meat samples in the treated groups. Elongated muscle fibers surrounded by dense connective tissue (epimysium) were identified in groups ZH and RH (1). While the control group, showed contracted fibers with a corrugated appearance (2). this effect may indicate stress on the lambs during slaughter. Abundant connective tissue was detected in ZH treatment samples (3).

Figure 2 Scanning electron longitudinal-section micrographs of lamb samples in Control group, Zilpaterol Hydrochloride (ZH) group, and Ractopamine Hydrochloride (RH) group, 200X magnification (bar scale corresponds to 100 µm). (1) muscle fibers surrounded by their dense connective tissue; (2) contracted fibers with corrugated appearance; (3) connective tissue 

Muscle thickness

200 muscle fibers were measured with DIA software using the longitudinal-section micrographs obtained by SEM. Results show that muscle fiber thickness may be proportional to drug bioavailability. This seems to be correlated with the observations under optical microscopy (Figure 1) in which RH treat-ment might increase fat content in lamb samples. A deeper study is currently in progress to assess differences in fatty acids content of the samples in the BA-treated groups.

In vivo assessment of β-agonists in lambs

Positive effects in DWG were observed from day 13 of BA supplementation due to their capacity to stimulate muscle hypertrophy (^{Mersmann 1998}) through inhibiting proteolysis and lipogenesis. stimulating protein synthesis, and redirecting energetic substrates towards muscle accretion (^{Mills 2002}, ^{Chu et al. 2017}, ^{Claffey et al. 2018}). In the present study, DWG was higher than reported by ^{López-Carlos et al. (2010}) who fed sheep a diet of 28% alfalfa and 47.65% yellow corn, which supplied 22 and 9% of raw protein into the diet, respectively. Differences in weight gain can be caused by multiple factors, for instance, the quality and amount of dietary protein, duration of treatment, sex, and hygiene (^{Parr et al. 2016}, ^{Tang et al. 2016}, ^{Rivera-Villegas et al. 2019}). DWG reflects lean muscle mass gain, because BA act on the calpain-calpastatin complex, diminishing protein degradation (^{Dávila-Ramirez et al. 2015}, ^{González-Ronquillo and Angeles-Hernández 2017}, ^{Smith et al. 2019}). These results are consistent with ^{Resendiz et al. (2013)}, which declared that a high FE is an indicator of the efficiency and profitability of productive systems.

Kinetics of β-agonists excretion in urine

According to ^{Waters (2012)} and ^{Morales-Trejo et al. (2013)}, selective analyses can reliably identify growth promoters in biological matrices of ruminants such as liver, meat, and urine. BA absorption depends on the administration pathway; drug levels in plasma and blood are higher after oral dosing than intramuscular administration. After reaching effective concentrations in muscle, BA are excreted into the urine (^{Meza-Márquez et al. 2012}). Similarly to present findings, ^{Van Hoof et al. (2005)} reported urinary excretion of RH and ZH in calves; the maximum elimination rate (12.6 µg mL⁻¹) of both compounds occurred after the first 5 days of treatment. For this reason, urine is a reliable biological matrix to detect and quantify the elimination rate of BA (Van Hoof et al. 2005).

In all species, BA are mostly excreted through urine (50-58%), but a smaller portion are eliminated through feces (5-30%), and milk (0.9-3%) as non-metabolized products (^{Ma et al. 2018}). ^{Sumano et al. (2002)} indicated that BA are bio transformed and rapidly inactivated by Catechol-O-Methyltransferase (COMT) enzyme, which methylates the hydroxyl groups on the aromatic ring. RH is metabolized by hepatic glucuronidation; hence, it is not a substrate for COMT.

Determination of β-agonists in meat samples

Lamb samples at day 40 were BA-free, after the withdrawal period recommended in the technical specifications for using ZH and RH. ^{Mondragón et al. (2010}), ^{López-Carlos et al. (2011)} and ^{Avendaño-Reyes et al. (2018)}, agree that BA are fully eliminated from meat samples after 72 h.

Optical microscopy of lamb samples

^{Giannetti et al. (2016)} indicated that the decrease of lipids or lipolysis in sheep -produced by lipases that hydrolyze triacylglycerols to glycerol and free fatty acids-facilitates glycolysis, oxidation, and the tricarboxylic acid cycle, which can derive in the synthesis of proteins. ^{Vahedi et al. (2014)} manifested that the addition of BA to the diet of ruminants increases muscle mass and decreases adipose tissue (depending on the cut area).

SEM of lamb samples

Meat has a complex structural organization, and its components impact its quality (^{Lu et al. 2000}). Moreover, meat morphological structure indicates the post-mortem treatment to which the animal was subjected, this may modify quality attributes, such as tenderness (^{Li et al. 1999}). ^{Romero-Peñuela et al. (2011)} stated that stress can only be measured by biomarkers or cortisol levels. The present study’s findings revealed that in ZH groups the sarcoplasm was more compressed (F), compared with RH and control, which showed open basal cisterns that function as calcium storage. After the death of the animal, calcium is released to activate endogenous enzymes such as calpains and catechins that improve meat tenderness (^{Rubio-Lozano et al. 2011}).

Muscle fiber thickness

The greater muscle fiber thickness was observed in the ZH group, which may be due to the bioavailability of the treatments. RH bioavailability ranges between 45-50% in ruminants, and 88% in pigs; while ZH bioavailability is 80% in ruminants (^{Sumano et al. 2002}, ^{Estrada-Solis et al. 2016}). Results suggested that muscle fiber thickness is correlated with the yield in weight gain. as reported by ^{Chulayo and Muchenje (2016)}, who described that the use of growth promoters in ruminants improved protein synthesis, hence, greater muscle thickness. As observed in Table 3, the percentage of increase in fiber thickness was higher after ZH treatment (69.2%) than after RH intervention (15.6%) compared to control. The reason beneath this significant difference is directly related to the increase that RH produced in FW respect to the control, this is, RH increased 14.7% the weight of the sheep, but such increase seems to be explained not only by the increase in muscle fiber thickness because the end percent increase of this parameter was the lowest after RH treatment.

Since the same diet was provided to all lambs, differences in muscle fiber thickness and weight gain depended only on the type of BA that was supplied; like the results of ^{Giannetti et al. (2016)} and ^{Rivera-Villegas et al. (2019)} on using ZH in cattle to promote weight gain. Furthermore, RH treatment increased muscle mass and fat simultaneously, probably due to the rapid metabolism of this BA (^{Sumano et al. 2002}).