Introduction

As more effective drugs become available and fewer new breakthrough drugs emerge, clinical investigation objectives change. Clinical investigations often seek non-inferiority or equivalency effects of new drugs, rather than superiority, compared with existing effective standard drugs in active controlled trials, and the known efficacy of the standard treatment is then transformed to the new treatment. Since the interest is primarily one-sided, such trials are named “non-inferiority” trials to show that the new treatment is not inferior to the standard treatment by more than a small, predefined margin.¹

Demand for bovine meat has steadily increased in recent decades,² and pharmacological intervention with zilpaterol hydrochloride (ZH) has been used to increase production. This drug has been approved by health authorities in the USA,³ Mexico,⁴ South Africa⁵ and Canada.⁶ Feed supplementation with ZH by Zilmax®, the pioneer brand (Merck, Sharp & Dohme Corp. MSD, México) improves feed conversion rates and carcass yields at the expense of muscle mass,⁷ while reducing overall fat deposition.⁸ Increased protein synthesis in muscle fibres is due to altered transcriptional activity in the myosin heavy-chain isoforms.⁹ Nutrients that usually form fat are shifted to increase muscle synthesis.¹⁰ ZH is rapidly and completely absorbed within 12 h after oral administration with food. Elimination occurs through the urine (86 %) in a biphasic manner: first with a half-life of 15.3 ± 1.8 h, then a 98 % clearing of the drug in 48 h.¹¹ The second phase is slower and allows detecting zilpaterol after a withdrawal time of 8 d; however, a withdrawal time of 2 to 4 d has been set as the standard for meat production.¹²

Recently, generic commercial ZH brands have become available in Mexico, but no information exists on their comparative efficacy regarding fattening efficiency and meat quality features, and no published information is available on ZH use in humid, tropical conditions, which are common in Mexican meat production. Moreover, most studies have been conducted on Bos taurus livestock, while cattle utilized for meat production in Mexico are mainly of the Bos indicus species.¹³ Thus, this study performed a non-inferiority assay to compare the pharmacodynamic responses of the reference ZH preparation (Zilmax® MSD) and a generic ZH brand (Zipamix® PiSA Agropecuaria S.A. de C.V., Mexico) on meat production and basic meat quality parameters, under a specific setting: Bos indicus cattle in humid tropical conditions.

Materials and methods

Procedures and bull management followed official Mexican regulations for animal care.^15,16 The trial was approved by the Care Committee for Animal Use of Universidad Nacional Autónoma de México, based on the VICH GL9 guidelines for medical veterinary products.¹⁷ The trial was performed on bulls from a feedlot production located in the Tamuín region of San Luis Potosí, Mexico (22° 05›30.6» N, 98° 37’21.2” W). In this tropical region, temperatures are consistently high (ranging from 19.3 °C to 48.5 °C) with a mean of 25.5 °C throughout the year. Annual rainfall ranges from 800 to 1,500 mm and two weather seasons stand out: the drought season (late November through late May) and the rainy season (June to early November).¹⁸ This study was conducted during the rainy season, from August 10th, 2016 to September 14th, 2016. Humidity during this time ranged from 29 % to 96 %, and the temperature ranged from 33 °C to 49 °C. This region is classified as tropical savannah climate. Warm all year, with dry season (Aw), according to the climatic classification of Köppen¹⁹ with an average precipitation ranging from 1000 mm to 1400 mm/year.

Results and discussion

This study aimed to determine whether the pharmacodynamic effects of a generic ZH statistically differed from the reference ZH formulation, using a non-inferiority assay²⁷ based on weight gain, secondary productive parameter variables, and the sensorial characteristics of the meat. Table 2 shows the Wald values for each model, their degrees of freedom (d.f.) and P-values. The observed results between ZHg and ZHr (Table 3) are statistically indistinguishable with a 95 % CI.

An average weight gain of 7.76 kg was observed in the treated bulls compared with the control bulls (Table 3). The CCW performance showed a mean increase of 7.37 kg in the ZH-treated bulls, compared with the untreated animals (Cg). In contrast, feed conversion (G:F) did not significantly differ between treated and untreated bulls (note the 95 % Wald confidence intervals (CI), Table 3). These values confirm that ZH promotes growth. The cold carcass mean weight and carcass yield in the treatment groups, ZHg and ZHr, were statistically indistinguishable (95 % Wald CI). Given the large sample size, the test powers were between 0.66 and 0.99. Figure 1 shows the means and 95 % confidence intervals of the average daily gain, dressing and conversion rate (G:F; kg/kg). Note that for conversion rate, an overlap clearly exists between the latter groups, unlike for the other two variables in which the control groups did not overlap with the treated groups. ZHr-treated animals varied greatly in conversion rate. This was corrected in the GzLM model. Maximum likelihood was used with GzLM model to lower the adjusted mean variation (Figure 1).

Figure 1 Means and 95 % confidence intervals for dressing percentage, average daily gain and conversion rate (kg/kg) by treatment as follows: ZHg = zilpaterol hydrochloride from Zipamix® (Pisa Agropecuaria México, Guadalajara, Mexico); ZHr = zilpaterol hydrochloride from Zilmax (MERCK, SHARP & DOHME CORP. MSD Salud Animal México, Mexico City, Mexico). 

Proximal chemical analysis data for the Longissimus dorsi muscle are presented in Table 4. The findings indicate that supplementing either commercial ZH brand does not modify the moisture, fat or ash content in the muscle (P = 0.32; Wald c² test = 2.5; d.f.= 2), compared with the ZH-free animal meat (1-β = 0.63). The ZHr protein percentage was higher than that of the ZHg and control groups (24.16 and 23.52 %, respectively), and the test powers were between 0.6 and 0.82.

Zilpaterol supplementation (ZHg and ZHr) did not modify the consumers’ scores for odour, taste and softness of the Longissimus dorsi muscle (P = 0.50; Wald χ² test = 0.8; d.f. = 2). Table 5 summarizes these data. For these attributes, more than 65 % of consumers assigned scores ranging from 5 to 7 (i.e., I liked it lightly, and I really liked it) as observed in Figure 2. Meat juiciness from animals treated with either ZHg or ZHr presented lower scores (≤ 5) compared with the untreated animal meat (P = 0.046; χ²= 2; N = 219).

Figure 2 Sensorial hedonic scale percentages for aroma, flavour, tenderness and juiciness per treatment as follows: ZHg = zilpaterol hydrochloride from Zipamix® (Pisa Agropecuaria México, Guadalajara, Mexico); ZHr = zilpaterol hydrochloride from Zilmax (MSD). 

For most variables assessed, differences between the groups treated with ZH and the control group were highly significant (P < 0.01). This demonstrates the efficacy of both ZH commercial brands. However, subtracting the mean values of the generic or the reference product from the untreated group reveals a higher difference favouring the generic commercial brand. This is illustrated in Table 3, where the difference between the final weight of the animals treated with the reference product and the control group is 6.13 kg. This value is 9.38 kg for the generic group; however, the difference between these groups was not statistically significant, indicating that the effectiveness of the ZHg treatment is not inferior to that of the reference product, ZHr. In other words, in all variables assessed between the ZHg and ZHr groups, the results confirmed the non-inferiority hypothesis. CCW and CCY mean values for the bulls treated with the generic ZH product did not differ from their corresponding values in the ZHr group; however, in this trial, the weight gain increments were 11.7 % in the ZHg group and 8.3 % in the ZHr group. Nevertheless, non-inferiority criteria require a minimum difference of at least 20 %.²⁷

Predictably, both treatments were statistically superior to the control. The magnitude of improvement in final body weight (22 %), lies near the maximum value reported in the literature (25 %), using the reference brand ZH. ^4,31-39 For example, weight gain coincides greatly with other studies conducted on Bos taurus bulls.^34,40 Elam et al. treated Bos taurus bulls with 8.33 mg ZH/kg feed as dry matter for 30 days and obtained final weight increments of 9.3 kg compared with untreated bulls.⁴² These values are similar to those obtained in this trial for Bos indicus (i.e., final weight increments of 7.75 kg at a dose of 6 mg of generic ZH/kg feed). The mean final weight reported by Avendaño et al. using a generic ZH, was higher than the one achieved here²¹; however, the weight gain reported by these authors was similar to the corresponding value obtained during this trial (e.g., 77.80 vs. 53.16, respectively).

Differences in final weight (e.g., 528.83 kg vs 518.4 kg, respectively) could be due to differences in age, diet and climate. Most trials using the reference ZH brand were conducted in temperate/cold areas, which have predominant Bos taurus bulls^33,42-44. After a thorough literature review, only two trials could be compared with the one described here. One occurred in Baja California, Mexico, which is a geographical area characterized by high environmental temperatures (annual mean of 25.3 °C with a range of 5 °C to 45 °C)¹⁸ and a BWh region (i.e., dry climate, with the driest season during winter and an average annual temperature higher than 18 °C), ²¹ corresponding to a desert-like scenario. When the study was conducted, the temperature oscillated approximately 20.3 °C with 57.6 % humidity,¹⁹ and zebu-type cattle were tested as well as 75/25 % Bos indicus/Bos taurus bulls. In that study, the total weight gain achieved with the generic brand was only 8.3 %, while in the present trial the same value reached 21.5 %. The second study was conducted in Yucatán, Mexico.²⁰ This region has similar weather conditions to those in our trial (i.e., a humid climate ranging from 30.92 % to 69.08 % and high temperatures year-round, where the annual mean is 28 °C with a range of 16 °C to 40 °C).¹⁶ The region has been classified as Aw (i.e., humid tropical climate with a dry season during winter and at least one month with a monthly precipitation < 60 mm).¹⁹ The zebu bulls included in the research of Castellanos et al. 2006 were younger and lighter than those in our study, and no generic ZH brand was tested²⁰; only the reference ZH from Zilmax® and an untreated control group were studied. These methodological differences hinder direct comparisons with the present trial; however, Castellanos et al. reported a final weight gain of only 1.05 % of the ZH-treated group compared with the control group (e.g., 48.6 kg vs 51 kg, respectively).²⁰ Comparisons among studies are difficult as many factors could have influenced the results, such as diet digestibility, surface area of the provided shade, and weather conditions. Nevertheless, one issue appears to be clear in these three studies: the presence of particularly unfavourable climate conditions for optimal weight gain.⁴⁵ Furthermore, competitive production variables were noticeably improved. For example, the mean temperature during this trial was 29.3 °C at 8:00 hours and 35.0 °C at 1:00 p.m., with relative humidity of 83.8 % and 53.2 %, respectively. Although the study was conducted during the rainy season, only three days experienced heavy rain. The bulls were supplemented with ZH only for 30 days, but this supplementation has been used up to 40 days.³⁴ It appears that production variables are similar in either dosing scheme.^{7,20,21,32,34} The accepted meat withdrawal period in countries that have authorized ZH use for 30 to 40 days is 3 to 4 days.^46,47 Within these dosing ranges, ZH supplementation appears to be economically profitable.^19-48

In cattle, β-adrenergic agonist drugs are reported to modify the adipose tissue metabolism and decrease fat by increasing lipolysis and decreasing lipogenesis. In muscle tissue, these drugs increase protein by reducing its degradation and increasing protein synthesis.⁴⁹ The obtained values from the proximal chemical analysis agree with the moisture, protein, fat and ash ranges for the Longissimus dorsi muscle of Mexican cattle,^50,51 although the results found during this trial showed slightly higher protein and fat. In contrast, these data differ from values reported in the literature outside Mexico. Shook et al. 2002, evaluated the meat of British and British × Continental bulls treated with 8.3 mg ZH/kg feed as dry matter for 20 d.⁵⁰ These authors found that the protein percent was higher in the meat from ZH-treated bulls, compared with untreated animals (23.41 vs 22.87 %), but fat deposition remained statistically indistinguishable. In contrast, Rathmann et al. 2009, found that ZH-treated animal meat had less fat and more muscle moisture and protein content compared with untreated animals.⁹ Holmer et al. 2009, mentioned that moisture was unaffected, but the amount of fat was lower in the measured muscles (Triceps brachii, Gluteus medius and Longissimus lumborum) from animals treated for 30 d with ZH.⁴³ Hilton et al. 2009 observed a decrease in the fat percentage of the Longissimus lumborum muscle, but the protein and humidity percentages remained unaltered in ZH-supplemented bulls.⁵² Differences in the fat amount, protein content and humidity among the studies outside Mexico and the data gathered in this trial may be partly explained by the genetic contributions of Bos taurus and Bos indicus from each group of bulls, as Bos indicus have lower fat deposition rates than Bos taurus.⁵⁴

Although consumers detected differences in the Longissimus dorsi muscle juiciness for both ZH treatments, the overall pleasing rating was unaffected (P = 0.046; Wald χ² test = 6.2; d.f = 1). This agrees with the results of Garmyn³⁶, who indicated that ZH does not modify Longissimus dorsi muscle taste from Holstein bulls compared with control samples. Despite sustained juiciness and general softness scores being affected, it is likely that these latter changes were related to the lesser amount of intramuscular fat in the ZH-treated animal meat.⁵⁴ This effect was not demonstrated here; however, the reduced juiciness and general softness across all studies has been linked to muscle fibre hypertrophy.^55,56

Finally, generic drug preparations fulfil a debatable but sometimes clear social benefit by reducing costs of reference-drug preparations, which occurred in this study. Additionally, ZH use dissuades clenbuterol use as an illegal agent to improve carcass yield, thus preventing human clenbuterol toxicity outbreaks.⁵⁶ The lack of pharmacodynamic similarity is often seen as an argument for discrediting generic preparations; however, in this assay, carcass yield and meat quality from both ZH-tested preparations were indistinguishable.

Conclusions

Few countries have approved ZH use for the final phase of fattening beef cattle, and until now, non-inferiority data for generic preparations were lacking. One exception is an essay that estimated carcass quality when using a generic ZH brand (Grofactor® Virbac, Mexico)²¹; however, as environmental conditions are key factors for the well-being and fattening efficiency of bulls, that study failed to disclose details on the cattle housing such as shade surface or how the feed was served. Consequently, this is the first carefully structured trial to compare productive variables from a non-inferiority perspective, conducted under humid tropical conditions in cattle with a marked Bos indicus genotype.

Regarding meat quality, few differences (not statistically significant) were observed between reference and generic ZH preparations, and both improved meat production compared with the untreated animals. This evidence confirms the non-inferiority nature of the generic ZH preparation.