Location of the experiment

All management and care procedures of animals were carried out according to the Mexican Official Norms NOM-051-ZOO-1995 (Humane care of animals during mobilization) and NOM-033-ZOO-1995 (Slaughter of domestic and wild animals). The study was performed in spring at the sheep experimental unit of the Instituto de Ciencias Agrícolas, Universidad Autónoma de Baja California, located in the Valley of Mexicali, Baja California, México (32.8^o N, 114.6^o W).

Animals and their experimental management

Thirty entire male lambs from the Dorper × Pelibuey cross (initial LW= 36.9 ± 6.9 kg and age= 5 mo) were utilized, having been adapted to individual pens and basal diet (Table 1) for 20 days previous to the onset of the experiment. The animals received two injections at the beginning of the adaptation period, an intramuscular one administering 1.0 ml of vitamins (Vigantol ADE Fuerte; Bayer, Mexico City) and a subcutaneous one with 0.5 ml of ivermectine (Sanfer, Mexico City). The individual pens were provided with food and water troughs and shade. The basal diet was formulated for a daily weight gain of 300 g in finishing lambs (2.8 Mcal of metabolizable energy per kg of dry matter [DM] and 16 % crude protein)¹².

Feed samples were collected on a weekly basis, and at the end of the experimental period they were brought together in order to obtain two subsamples, which were analyzed to determine its chemical composition^13,14,15. In general, the diet was offered twice a day (at 0700 and 1800 h) during both the adaptation and the experimental period, guaranteeing a daily rejection rate of at least 10 %, while water was available ad libitum.

Experimental design

A 32-d productive performance test was carried out after the adaptation period. On the first day of the test, all fasting male lambs were weighed individually (after 12 h without feed or water) and then grouped into 10 blocks, each containing three lambs of similar initial LW (blocking factor). Thus, the male lambs in each block were randomly assigned to the treatments, which consisted in offering them a basal diet that included: 1) 0 mg of ZH / kg of LW (control); 2) 0.10 mg of patent ZH / kg of LW (Zilmax^TM, Intervet, Mexico City, Mexico; PZH), and 3) 0.10 mg of generic ZH per kg of LW (Grofactor^TM, Virbac Mexico, Guadalajara, Mexico; GZH). All male lambs were individually weighed every 10 d in order to adjust the amount of ZH in PZH and GZH treatments. The daily dose of the product was mixed with 30 g of ground wheat grains and offered in the morning, before the basal diet. The control group was fed 30 g ground wheat grains at the same time as in the PZH and GZH groups. Through the feedlot test, treatments were offered during the first 30 d, and the last two days were utilized as withdrawal period. The fasting male lambs were then transported to the meat workshop (located at a distance of 200 m from the pens), where they were slaughtered by the disgorging method.

Productive performance

The variables evaluated for productive performance were initial and final LW (kg), average daily gain (DWG= TWG/32; kg/d/animal), total weight gain (TWG= final LW - initial LW; kg/period), daily dry matter intake (DMI= kilograms of fresh food ingested × [% DM/100], kg/animal), and feed efficiency (DWG/DMI). Lambs were fasting when the individual LW on day 1 (initial) and 33 (final) of the feedlot test was recorded, and these data were then used to calculate DWG and TWG.

Body offal and carcass characteristics

The evaluation of offal and carcass characteristics was carried out as described by Avendaño-Reyes et al¹⁰. After slaughter, the bodies were eviscerated, and the weights of each organ, viscera and offal (skin, head, feet, testicles, blood, heart, liver, kidneys, lungs, spleen, full and empty gastrointestinal tract, rumen, intestines, and omental and mesenteric fat, as well as the fat surrounding kidneys, pelvic cavity and heart [KPH]) were recorded. The gastrointestinal content was estimated by difference between the weights of the empty and full gastrointestinal tract, while empty LW was calculated by subtracting the gastrointestinal content weight from the final LW. Thus, the weights of all organs, viscera and offal were expressed as percentages of the empty LW.

The hot carcass was weighed (HCW) and then cooled at 4 ºC during 24 h in order to record cold carcass weight (CCW), conformation, carcass length, thoracic depth, leg length and perimeter, Longissimus dorsi muscle (LM) area, and fat thickness. The carcass conformation was assessed on an 8-point scale where 1 is bad and 8 is excellent¹⁶. A flexible measuring tape was utilized to take the carcass morphometric measurements¹⁷. Both LMA and fat thickness were measured between the 12^th and 13^th rib, performing a perpendicular cut to the loin at that height. The LM area was measured using a dot square grid (64 mm²), while the fat thickness was determined with a caliper. Finally, the carcass yield was estimated by expressing the HCW as a percentage of the empty LW.

Primary cut yields

Carcasses were cut along the middle line and the right half carcass was then divided into the following primary cuts¹⁸: forequarter, hindquarter, neck, shoulder, ribs, loin, plain loin, leg, and breast and flank. The right half-carcass and each primary cut were weighed; the yield of each cut was then calculated by expressing its weight as a percentage of the half-carcass weight.

Meat quality

The meat quality was assessed in the LM. A piercing electrode (HACH model PHW57-SS, Colorado, USA) attached to a pH meter (HACH model H160G, HACH, Colorado, USA) was inserted into the carcass loin, between the 12^th and the 13^th rib, in order to record pH at 45 min and 24 h post mortem. Subsequently, the LM was dissected from the loin primary cut, and color parameters (a* [redness], b* [yellowness], L* [luminosity], C* [color saturation index], and h* [hue angle]) were measured at 24 h post mortem, using a portable colorimeter (X-rite model SP60, Michigan, USA). Finally, the LM was vacuum-packed and refrigerated at 4 ºC during 14 d, after which pH and color parameters were measured again, likewise shear force in matured meat was also measured. So, after the aging period, the LM was unpacked and exposed to blooming during 30 min before quality measurements. The pH was measured in a homogenized mixture of 5 g of meat and 25 ml of distilled water, using a liquid potentiometer (Hanna Instruments Digital model HI-2210, Woonsocker, Rhode Island). Color parameters were measured using the methodology described above in the evaluation at 24 h post mortem. Color measurements were carried out in triplicate, and, finally, the average for each parameter was estimated and recorded. For shear force, two (2.5 cm thick) LM steaks were cooked on an electric grill until they attained an internal temperature of 71 °C; the steaks were then cooled to ambient temperature (~25 ºC), and five prisms with a 1.27 cm diameter were obtained, in which the shear force was measured using a Warner-Bratzler shear machine (Salter 235, Manhattan, KS, USA). The shear force per sample was recorded, averaging the three most homogenous values (variation coefficient <5 %).

Statistical analysis

All data were analyzed using the ANOVA procedure of the SAS statistic software¹⁹, applying the statistical model of a randomized complete block design. The model included to initial LW as blocking factor and the type of ZH as treatment. Means were compared through two orthogonal contrasts, establishing P≤0.05 as differences, and 0.05>P≤1.0 as trend. The first contrast compared the control group against the use of any source of ZH (C1: control vs PZH+GZH), and the second contrast compared between the sources of ZH (C2: PZH vs GZH).

Control versus zilpaterol hydrochloride

Regardless of the source of ZH, the dietary addition of this β₂-adrenergic agonist (β₂-AA) did not affect (P≥0.15) the productive performance of animals (Table 2). In carcass, ZH increased carcass yield (P≤0.01) and LM area (P≤0.05); likewise, it exhibited a tendency (P=0.09) to improve conformation, but it did not affect (P=0.09) HCW, CCW, carcass morphometric measurements, or any variable associated with internal or external fat deposition (Table 3). As for the primary cut yields, ZH increased (P≤0.03) yields of forequarter, leg and plain loin, but reduced (P≤0.01) yields of hindquarter and shoulder, without affecting (P≥0.27) any of the other primary cuts (Table 4). In body offal (weights expressed as a percentage of the empty LW), the dietary addition of ZH reduced (P≤0.05) the weights of skin, liver, kidneys, spleen, and rumen, and exhibited a tendency (P=0.06) to decrease the feet weight (Table 5). The weight of the rest of body offal did not vary (P≥0.12) with the ZH inclusion.

In the case of meat quality, ZH did not affect (P≥0.23) the post-mortem pH at 45 min, 24 h, or 14 d, but decreased (P≤0.01) a* and C* values, and tended (P=0.07) to reduce b* values at 24 h (Table 6). After 14 d post mortem, dietary inclusion of ZH reduced (P<0.01) a* values and increased (P=0.05) h* values, while it had no effects (P=0.95) on shear force. Values of L* were not affected (P≥0.65) by ZH in any of the evaluated time periods.

Patent versus generic zilpaterol hydrochloride

Productive performance (Table 2), carcass characteristics (except for carcass yield; Table 3), primary cut yields (Table 4), weight of the offal expressed as a percentage of the empty LW (Table 5), and meat quality (Table 6) exhibited no variation (P≥0.23) between PZH and GZH. The dietary addition of PZH showed a tend (P = 0.07) to increase carcass yield compared to GZH.

Control versus zilpaterol hydrochloride

The addition of ZH to the finishing diet did not improve productive performance or carcass weight in hair male lambs. These results were not expected, as most studies including this β₂-AA report a greater DWG, feed efficiency, HCW, and CCW, with little consistent effects on DMI^{4,5,8,18,20-22}. One might think that having administered a dose below the one recommended by the company (0.10 vs 0.15 mg/kg of LW) was the cause of this lack of effects, as most of those studies that report improvement added the product according to the indications on the label. Nevertheless, a previous study evidenced that 0.10 mg/kg of LW is an optimal dose for finishing male lambs in feedlot when this type of generic ZH is used¹⁰. Notably, those studies in which ZH improved productive performance and carcass weight, the control group exhibited lower DWG (168-280 g/d) than that determined in the present study. So, results in terms of productive performance and carcass weight may be due to the fact that the lambs expressed their maximum genetic potential for growth without the need to use ZH and, in consequence, this promoter had a limited margin to exert its positive effects on DWG and carcass weight. The literature indicates that hair lambs have a DWG of approximately 250 g/d (23), i.e., 50 g less than that observed in the control group of this study which are also hair sheep. According to this finding, Mersmann²⁴ mentions that the effectiveness of β₂-AA for improving body mass gain may be limited by the animal genetic potential.

While ZH did not affect LW gain or carcass weight in male lambs; this product improved carcass yield, LM area and yields of certain primary cuts (i.e., leg and plain loin). This suggests that the β₂-AA promoted muscular hypertrophy and, therefore, increased muscular mass deposition, although to a limited extent. The increase in carcass yield may be a consequence of the greater muscle mass deposition in body regions as plain loin and legs. These findings agree with results of other studies^5,10; still in these, the effect was more pronounced which further supports the idea that external factors were more important than ZH itself in inducing the maximum expression of genetic growth potential by the sheep, limiting the mechanism of action of the β₂-AA²⁴.

The dietary inclusion of ZH did not affect the body internal or external fat deposition, but reduced the weights of some offal (skin, liver, kidneys, spleen, rumen, and feet). This suggests that the muscular hypertrophy observed in loin and legs of the male lambs was caused by an β₂-AA-promoted redistribution of nutrients from offal tissue toward the formation of skeletal muscle as previously reported with the addition of generic ZH^8,10,22.

On the other hand, feeding male lamb with ZH has been mainly associated with an increase in meat pH, which causes a reduction in color values and an increase in meat toughness^6,7,22. However, there is evidence that ZH decreases meat color even with a normal pH²⁵. In the present study, ZH did not affect post mortem pH (45 min, 24 h, or 14 d); this could partially explain because shear force and luminosity of the meat did not change with the daily inclusion of this β₂-AA. Given results for pH, the meat from ZH-treatment unexpectedly discolored without losing luminosity at 24 h post mortem; this effect persisted to a lesser extent after 14 d of maturation. Although the pH at 24 h and 14 d post mortem exhibited no significant change, the pH values in ZH meat (>5.9) remained above the normal range (≤5.8)²⁶. This may have caused an increase in the mitochondrial respiration of oxidative fibers and a greater competition for oxygen with oxymyoglobin, increasing desoxymyoglobin and metmyoglobin concentrations-pigments which favor meat discoloration²⁷.

Patent versus generic zilpaterol hydrochloride

Male lambs fed PZH and GZH exhibited no differences in their productive performance, carcass characteristics, primary cut yields, or meat quality, suggesting that both molecules are similarly effective as growth promoters at a daily dose of 0.10 mg per kg of LW in this type of animals. Although, this twofold finding must be taken with caution, since, as stated above, the dietary addition of patent and generic ZH in the sheep feeding promoted muscular hypertrophy, though not at a sufficient level to result in higher DWG, feed efficiency, or carcass weight. Nevertheless, the fact that generic ZH at the dose used promotes a degree of muscular hypertrophy comparable to that observed with patent ZH in finishing male lambs is rescued. This may be due to the fact that both products are similar in bioequivalence, chirality, and isomeric determination⁹. Despite this, they differ as they are attached to the vehicle (ground cob); while the patent ZH molecule is attached around the granules of the vehicle, the generic ZH molecule remains unattached to this¹⁰. This could reduce the bioavailability and, consequently, the effectiveness of the product. Results of this study suggest that the manner in which the molecule is attached in the vehicle, it is not a factor that decreases or enhances the functioning of ZH on the growth of hair male lambs finished in feedlot.

As in the present study, other authors⁸ found no difference in productive performance, carcass characteristics, wholesale cut yields, or noncarcass component weights between lambs fed patent and generic ZH. On the other hand, in bulls, the source of ZH did not affect growth, carcass characteristics or meat quality⁹.

It should be noted that neither of the sources of ZH promoted lipolysis in order to exert its hypertrophic effects on skeletal muscle²⁵. This had already been documented using generic ZH^8,10,22; however, patent ZH had regularly caused a lipolytic effect in fattening sheep^4,20,28. This suggests that the mechanism of lipolytic action associated to patent ZH depends on the dose, as doses of 0.15 mg per kg of LW were used in previous studies, while the present study used 33.3 % less. Nevertheless, further research is required to confirm this finding.