Mechanisms of steroidal implants to improve beef cattle growth: a review

ABSTRACT

For more than 60 y, beef cattle producers have safely used various types of growth-enhancing technology (GET) such as steroidal implants with anabolic activity and orally active beta-adrenergic agonists to increase skeletal muscle growth rate, improve carcass leanness, increase average daily gain (ADG), and alter dry matter intake (DMI) compared to non-treated cattle. Generally, the use of a GET increases ADG and only moderately affects DMI relative to non-treated cattle; subsequently, this enhances the rate of live weight gain relative to the amount of feed needed to achieve that gain, this is referred to as feed efficiency (G:F). When a producer chooses to utilize a GET, improvements in treated cattle over non-treated cattle are typically in the range of 8% to 28% for ADG and 5% to 20% for G:F. This review of the literature is intended to provide up to date insight into the mechanisms of how steroidal implants with anabolic activity enhance cattle growth and how these technologies have evolved since their introduction to U.S. beef producers nearly 60 y ago.

KEYWORDS:

• Beef
• growth
• steroidal implants

Application of steroidal implants in U.S. beef production

Rationale for use

Fast and economical gains are the primary objective of animal feeding operations. Average daily gain (ADG) is a measure of productivity, and the amount of gain accrued per unit of feed, termed gain to feed (G:F), is a measure of production efficiency. Thus, a primary focus of the field of animal nutrition is faster rates of gain, delivered in the most efficient manner possible, via health management and exploiting growth physiology. For more than 60 y, beef cattle producers in the U.S. have also safely used various types of growth-enhancing technologies (GET) such as steroidal implants with anabolic activity and more recently orally-active β-adrenergic agonists that improve production efficiencies by improving carcass leanness, increasing ADG, and altering dry matter intake (DMI). When a producer chooses to utilize a GET, improvements in treated cattle over non-treated cattle are typically in the range of 8% to 28% for ADG and 5% to 20% for G:F, with the wide ranges occurring due to technology utilized and stage of production when implemented (Johnson and Beckett 2014).

Implants

More than 30 commercially-available implants are marketed in the U.S. for beef cattle production. These implants have been classified into low-, medium-, and high-potency implants (Johnson and Beckett 2014) or coated and non-coated implants. The active ingredients contained in steroidal implants belong to three major categories of endogenously produced hormones: androgens (i.e. male hormones), estrogens (i.e. female hormones), and progestins (i.e. pregnancy hormones). These implant products are administered subcutaneously on the posterior side of the animals’ ear. Once a non-coated implant is correctly administered, the anabolic compound is slowly released into blood circulation from the excipient for 60–120 d (Mader 1998), also referred to as implant payout. Payout of anabolics from the excipient can be altered via various excipient compounds used in the implant formulation (i.e. cholesterol or lactose), the amount of pressure applied to the implants during formation of the implant pellets, as well as various polymers that delay or slow the release of anabolic compunds into circulation from the excipient (Lee et al. 2000; Cady et al. 2002). In the last decade, implant technologies have improved and new coated-implant products have been made commercially available that extend the payout period in excess of 200 d post-implantation (FOIA 2007, 2014, 2017a, 2017b).

Postnatal skeletal muscle hypertrophy

Skeletal muscle is an economically relevant tissue in livestock production. Skeletal muscle in mammals is multinucleated. The cellular unit of skeletal muscle is the individual muscle fibre and muscle fibre number is fixed at birth. Postnatal skeletal muscle growth is by way of increases in size of the existing muscle fibre via hypertrophy, as hyperplastic growth (i.e. increase in cell number) occurs primarily in utero (Johnston et al. 1975). Hypertrophic growth of skeletal muscle in mammals is supported by the addition of new nuclei to the multinucleated muscle fibre (Moss and Leblond 1971). The additional nuclei arise from a pool of previously identified mesenchymal stem cells, these cells were first identified via electron microscopy in frog skeletal muscle between the basal lamina and sarcolemma of the muscle fibre (Mauro 1961). The location between the basal lamina and sarcolemma of the muscle fibre is more appropriately termed the muscle satellite cell niche. The mesenchymal cells that reside in this location are maintained in a quiescent state. When these cells are activated from quiescence, they divide and fuse into the muscle fibre in order to support postnatal skeletal muscle growth. The use of steroidal implants with anabolic activity alter satellite cell populations and maintain the DNA unit throughout growth, while the application of β-AA’s increase skeletal muscle hypertrophy without influencing muscle nuclei number (Hosford et al. 2015). Failure to increase nuclei content of the muscle cell ultimately results in un-sustained skeletal muscle hypertrophy in β-AA fed animals and is partially why β-AA’s are traditionally fed only at the end of the feeding period.

Steroidal hormones with anabolic activity

Overview of steroidal implants used in U.S. beef production

Implant formulations containing steroidal hormones with anabolic activity have been used safely in U.S. beef production since 1956. Commercially available implant formulations for use in U.S. beef cattle production contain naturally occurring estradiol-17β (E₂), the synthetically modified prodrug of E₂, estradiol benzoate (EB), or an estrogen-like compound (i.e. zeranol). These compounds are utilized either alone or in combination with progesterone, testosterone propionate, or trenbolone acetate in differing amounts and ratios of active compounds in the final implant pellets.

The least potent anabolic implant formulations are generally found in a preparation containing a low dose of estrogen alone (i.e. Ralgro; Merck Animal Health, Madison NJ) or in combination with a progestin (i.e. Component E-C, Elanco, Indianapolis, IN or Synovex-C, Zoetis, Parsippany, NJ) for use in both suckling steers and heifer calves, greater than 40 d of age and weighing less than 182-kg at the time of implantation. Greater potency implants are available for cattle weaned at the time of implantation and weighing greater than 182-kg at the time of implantation. These moderate potency implants are generally found in a preparation containing a greater dose of estrogen alone (i.e. Compudose or Encore, both Elanco) or in combination with a progesterone (i.e. Component E-S, Elanco or Synovex-S, Zoetis) for steers, or for use in terminal bound heifers with testosterone propionate (i.e. Component E-H, Elanco or Synovex-H, Zoetis). Another class of implant that fits into the middle potency category is an implant that contains 40 mg TBA and 8 mg of E₂ (i.e. Compoenent TE-G, Elanco or Revalor-G, Merck Animal Health). This combination implant is approved for stocker steers and heifers not fed in confinement. The most potent class of implants available are those reserved for the confined feeding of cattle. The most potent class of implants contain TBA alone (i.e. Component T-H, Elanco, Component T-S, Elanco, or Finiplix-H, Merck Animal Health) or in combination with E₂ or EB (Component TE-IS, Elanco or Revalor-IS, Merck Animal Health; Component TE-IH, Elanco or Revalor-IH, Merck Animal Health; Synovex-Choice, Zoetis; Component TE-S, Elanco or Revalor-S, Merck Animal Health; Component TE-H, Elanco or Revalor-H, Merck Animal Health; Component TE-200, Elanco or Revalor-200, Merck Animal Health; Synovex-Plus, Zoetis).

A new generation of combination TBA + E₂ implants have been made commercially available in the last decade, that are designed to delay (approximately 70 d) or sustain the release of anabolic constituents (approximately 200 d) post-implantation. This new class of combination implants contain TBA and E₂ (Revalor-XS approved for use in steers fed in confinement; Revalor-XH approved for use in heifers fed in confinement; Revalor-XR approved for both steers and heifers fed in confinement, all from Merck Animal Health) or TBA and EB (i.e. Synovex ONE Grass and Synovex ONE Feedlot, approved for both steers and heifers, not fed in confinement or fed in confinement, respectively, both from Zoetis). In these new implant formulations all or some of the implant pellets are coated with a polymer barrier intended to delay entirely for a certain period of time post-implantation or slow the release of anabolic constituents over time (Lee et al. 2000; Cady et al. 2002). In either case, this allows for anabolic stimulation later in the feeding period. This new class of combination implants has been shown to consistently improve gain and gain efficiency for up to 200 d post-implantation relative to non-implanted controls (Cleale et al. 2012). In particular, the use of a Revalor-XS has been shown to provide similar gains associated with an initial Revalor-IS, re-implanted with a Revalor-S, approximately 70 d from the initial implant when on feed for greater than 200 d (Parr et al. 2011b; Nichols et al. 2014). Thus, coated implants can eliminate the need to re-implant cattle when on feed in excess of 200 d. Allowing for producers to use valuable resources (i.e. time and facility use) elsewhere, and also limit disruption to animal performance.

Steroid hormone receptors

Steroid hormone receptors are located in every mammalian tissue depot. Steroid hormones promote a plethora of biochemical actions that can be measured via distinctly different kinetic mechanisms (Filardo and Thomas 2012). Typically, these signaling events are delineated as delayed or genomic and rapid or non-genomic events. In either case, steroid hormones bind to receptors throughout the body and elicit transcriptional responses that are able to be measured as a product of hormonal signaling (Filardo and Thomas 2012).

Classical hormone receptors

The classical effect of hormones on target tissue is initiated following ligand binding to a steroid hormone receptor located in the cytosol of the cell with a high affinity. These receptors are members of the nuclear hormone receptor family that include the glucocorticoid and mineralocorticoid receptors to name a few. These nuclear receptors have many similarities in their overall structure (Yen 2015). Upon ligand binding, the ligand–receptor complex is then translocated to the nucleus of the cell where transcription is initiated. Thus, classical hormone receptors are a class of transcription factors (Yen 2015). These transcription factors are important in a multitude of biological events related to tissue growth, reproduction, and basic animal metabolism (Yen 2015). As a transcription factor, classical hormone receptors have the ability to directly bind to DNA (i.e. hormone response elements) located on various genes throughout the body. In skeletal muscle, one example of this would be the androgen response element located on the IGF-I gene in skeletal muscle (Wu et al. 2007). Another example would be associated with the estrogen response element on the growth hormone-releasing hormone (GHRH) gene in the hypothalamus (Lalmansingh and Uht 2008). Increased GHRH from the hypothalamus can in turn increase acidophilic cell size in the anterior pituitary. This in turn increases growth hormone (GH) output from somatotrophs, thus increasing GH in circulation (Preston 1975; Trenkle 1976). There are two types of nuclear estrogen receptors, ER-α and ER-β (Brandenberger et al. 1997; Couse et al. 1997; Oesterreich et al. 2000; Fu et al. 2014). The ER-α isoform is of the greatest abundance in skeletal muscle (Pfaffl et al. 2001; Fu et al. 2014) in Bos species. Finally, androgens bind to the androgen receptor in a variety of tissues and elicit classical ligand activated transcriptional responses (Davey and Grossmann 2016). Androgen receptors are located throughout most mammalian tissue depots, including skeletal muscle (Davey and Grossmann 2016). In fact, the binding affinity of steroid sex hormones to receptors in skeletal muscle is very similar to the binding affinity in gonadal tissue (Revankar et al. 2005). However, there are far fewer classical hormone receptors in skeletal muscle compared to gonadal tissue (Fu et al. 2014). Understanding the distribution of classical hormone receptors and their potential to bind steroid hormones throughout the body could provide insight to new technologies that might be implemented to improve growth biology and production efficiencies of meat animals.

Mode of action: genomic

The traditional responses of hormones on target tissues are activated following ligand binding to a hormone receptor located in the cytosol of the cell with a high affinity. Upon ligand binding, the ligand–receptor complex is translocated to the nucleus of the cell where transcriptional activity is initiated. These transcription factors are important in a multitude of biological events related to economically important tissue growth (i.e. adipose tissue and skeletal muscle in meat animals) and anabolism of reproduction tissue (Yen 2015) in breeding animals. As a transcription factor, classical hormone receptors have the ability to directly bind to DNA at various loci across the genome throughout tissues with specific responsibilities. In skeletal muscle, one example is the androgen response element located on the promoter region of the IGF-I gene in skeletal muscle (Wu et al. 2007) or with the estrogen response element on the growth hormone-releasing hormone (GHRH) gene in the hypothalamus (Lalmansingh and Uht 2008).

Membrane bound hormone receptors

Membrane bound steroid hormone receptors are nearly all members of the G protein-coupled receptor (GPR) super family (Filardo and Thomas 2012). The role of these transmembrane receptors in vivo is to rapidly activate signal transduction. These receptors are targets for many pharmacological compounds used in oncological and reproductive medicine (Prossnitz and Maggiolini 2009). The GPR’s produce intracellular signals that activate cellular responses via secondary messenger systems following ligand binding to the extracellular receptor domain complex (Prossnitz and Maggiolini 2009; Thomas 2012). The G protein-coupled receptor 30 [now known as G protein-coupled estrogen receptor 1 (GPER1)] is a member of the GPR super family (Revankar et al. 2005; Kamanga-Sollo et al. 2008; Prossnitz and Maggiolini 2009; Filardo and Thomas 2012). The expression of GPER1 has also been identified in the endoplasmic reticulum of skeletal muscle (Revankar et al. 2005; Prossnitz and Maggiolini 2009). The localization in the endoplasmic reticulum is likely due to differing binding affinity for E₂ (Prossnitz and Maggiolini 2009) compared to the ER-α receptor. In fact, it has been demonstrated that GPER1 (Kd ∼6 nM) has nearly 10X lesser affinity for E₂ in circulation than the ER-α (Kd ∼0.5 nM) receptor (Revankar et al. 2005). There is evidence that there are also progestin and androgen membrane receptors in reproductive tissue and skeletal muscle (Filardo and Thomas 2012; Thomas 2012). It has been demonstrated that there is a decrease in IGF-I mRNA when a GPR antagonist is included in the growth media with TBA in bovine satellite cell (BSC) cultures obtained from non-implanted steers (Thornton et al. 2015; Thornton et al. 2016). This indicates a unique mechanism in which steroidal sex hormones with anabolic activity can elicit cellular responses and influence skeletal muscle growth. This mechanism of action differs from their traditional role of ligand activated transcription factors that bind to hormone response elements of various genes in different tissue depots. In the simplest of terms the differences between genomic and non-genomic effects are related to their regulation of transcriptional processes (Simoncini et al. 2004). While genomic actions of steroid hormone regulate transcriptional processes via nuclear translocation and subsequent binding to specific response elements, non-genomic actions of steroid hormones alter transcriptional processes by way of steroid-induced alterations of cytoplasmic and cell membrane bound regulatory proteins (Simoncini et al. 2004).

Mode of action: non-genomic

The non-genomic mechanism of steroid hormone signaling is brought about by various GPR’s (Thomas 2012). Many orphan GPR’s have been proposed to elicit biological responses following stimulation by steroid hormones (Prossnitz and Maggiolini 2009; Thornton et al. 2015; Thornton et al. 2016). The G protein-coupled receptors span the plasma membrane of the cell 7 times, and function by way of a secondary messenger systems, namely cyclic adenosine monophosphate (cAMP) or inositol trisphosphate (IP3) and diacylglycerol (DAG). These secondary messengers are capable of altering physiological responses in the target tissue, and occur in a matter of seconds compared to traditional nuclear hormone responses, that are much slower to develop. Once the ligand binds to the extracellular receptor domain, the initiation of a cascade of events occurs rapidly. Initially, ligand binding causes a conformational change to the intracellular receptor domains. This causes the G protein–complex (α, β, and ϒ subunits) to associate with the intracellular receptor domains. The ensuing step is that the α-subunit (i.e. the active catalytic subunit) of the G protein complex binds to GTP, and stimulates the production of adenylate cyclase that can in turn produce cAMP from ATP. Ultimately, this results in elevated protein kinase A (PKA) activity. The influence of PKA altering the phosphorylation of a variety of proteins following GPR activation is a hallmark sign of GPR signaling. A protection mechanism for chronic ligand stimulation in all GPRs is an associated receptor kinase (Ribas et al. 2007). Much like the protection mechanism associated with β-AR. Receptor desensitization of the β-AR, and down-regulation occurs with β-AA feeding (Hosford et al. 2015) or when β-AA are applied for extended periods of time (Sibley et al. 1987). This protection mechanism of the GPR associated kinase and differing affinities for steroid hormones potentially mediate differing cellular responses in a variety of tissues (Ribas et al. 2007), and is likely associated with alterations in affinity of the steroid hormone for the various types of receptors that bind steroid hormones throughout the body (Revankar et al. 2005).

Biological alterations post-administration

In short, the application of steroidal hormones with anabolic activity in beef cattle results in an increase in mature body weight of cattle. Mature body weight is defined as the point in time that the carcass would contain 50% muscle, 35% fat, and 15% bone (Butterfield 1976; Butterfield 1988). Increased mature body weight is associated with greater lean tissue deposition potential. Additionally, the use of steroidal implants delay chemical maturity (i.e. delay fattening). Implants increase chemically mature body weight (Preston 1975). Chemically mature body weight is the weight at which cattle achieve at least 28% empty body fat (Preston 1975; Guiroy et al. 2001). As a generalization, implants increase chemically mature body weight by about 40–50 kg (Preston et al. 1990; Johnson et al. 1996a; Parr et al. 2011a; Smith et al. 2018).

It was first demonstrated that administration of a combination TBA + E₂ implant resulted in increased longissimus muscle (LM) area and increased protein accumulation in the empty body compared to non-implanted steers (Johnson et al. 1996a). Sera harvested from implanted steers has been shown to increase the proliferation of BSC in vitro (Johnson et al. 1996b). Additionally, BSCs isolated from implanted steers more rapidly fuse into, and form myotubes, upon introduction to differentiation media (Johnson et al. 1998b).

Additionally, E₂ has been shown to influence GHRH production in the hypothalamus, as well as increase the size of acidophilic cells in the anterior pituitary (Preston 1975; Trenkle 1976). Somatotrophs, a group of cells from the acidophilic lineage, are responsible for the production of growth hormone (GH) that is secreted from the anterior pituitary where it then enters circulation and elicits growth responses in various target tissues throughout the body, namely hepatic tissue. Alterations in production of GH at the anterior pituitary level subsequently increase hepatic output of IGF-I. Steroid hormones have also been shown to increase GH binding affinity to GH receptors in the liver (Trenkle 1976). That can in turn increase hepatic output of IGF-I.

The IGF-I molecule is very similar to the anabolic hormone insulin, that is critical for both carbohydrate and amino acid metabolism. Since, IGF-I can bind to the insulin receptor (with much lesser affinity), it is carried throughout circulation via IGF binding proteins (IGFBP’s) in order to prevent deleterious effects if IGF-I was to bind to the classical insulin receptor, namely hypoglycemia (Clemmons 1993). Steroid hormones such as TBA and E₂ have been shown to increase the amounts of IGFBP-3 in circulation (Johnson et al. 1996b). The primary IGFBP isoform observed during postnatal periods is IGFBP-3 (Clemmons 1993). This allows for improved delivery of IGF-I produced at the liver to target tissues throughout the body of the animal (Clemmons 1993). Studies have demonstrated that hepatic knockout of IGF-I production does not completely retard skeletal muscle growth (Sjogren et al. 1999; Yakar et al. 1999). Thus, IGF-I produced via the hepatic route is likely only critical in regards to long bone growth in terminal bound animals. There is also evidence that the use of steroidal implants with anabolic activity can increase the local production of IGF-I in skeletal muscle (Johnson et al. 1998a; Pampusch et al. 2008; Parr et al. 2014). One mechanism is via binding to the androgen response element on the promoter region of the IGF-I gene (Wu et al. 2007). There is no estrogen response element located on the IGF-I gene (Umayahara et al. 1994). However, there is evidence that E₂ can stimulate local IGF-I production (Kamanga-Sollo et al. 2008) via GPER1. Direct binding of androgens and estrogens to skeletal muscle subsequently increases the amount of local IGF-I as evidenced by increased gene expression of IGF-I in longissimus muscle following implantation with TBA and E₂ (Johnson et al. 1998a; Pampusch et al. 2008; Parr et al. 2014). Local IGF-I is critical for the recruitment of satellite cells needed in order to support postnatal skeletal muscle hypertrophy. Locally produced IGF-I in skeletal muscle is able to progress bovine satellite cells through proliferation and differentiation into skeletal muscle fibres. This results in increased protein synthesis and decreased protein degradation (Vernon and Buttery 1978; Lobley et al. 1985; Hunter and Magner 1990). Moreover, it allows for maintenance of the DNA to protein ratio throughout skeletal muscle hypertrophy. Plus, TBA administration has been shown to decrease circulating levels of cortisol in steers, while E₂ does not. Cortisol is a catabolic hormone that causes increased protein degradation, and the use of TBA lowers urinary levels on N-methyl histidine (Hayden et al. 1992), a marker of protein degradation.

Steroid hormones can indirectly increase muscle growth via the influence on cortisol or by increasing circulating levels of IGF-I to support long bone growth, or directly to influence muscle satellite cell populations. This results in the net effect of greater protein synthesis and reduced protein degradation (Vernon and Buttery 1978; Lobley et al. 1985; Hunter and Magner 1990), resulting in increased muscle tissue anabolism due to increased mature body size and more active satellite cell populations.

Application in confinement based U.S. fed cattle operations

APHIS (2013) reported that more than 90% of all cattle entering the feedlot were administered an anabolic implant once, and approximately 80% of steers and 99% of heifers weighing less than 318-kg received 2 or more anabolic implants. Samuelson et al. (2016) indicated that the average time on feed for feedlot cattle in the U.S. is 201 d. The duration of effective anabolic stimulation in non-coated implants is no more than 140 d (Smith et al. 2018). Most commercially available implants have a rapid payout period and anabolic payout is un-orchestrated. Thus, it is common to apply a lower-potency initial implant and then re-implant with a higher-potency implant approximately 70 d later, or apply a single coated implant at feedlot introduction.

Combination trenbolone acetate and estradiol-17β implants in U.S. beef production

Live animal performance

Implants increase ADG, G:F, and decrease USDA marbling scores and YG compared to non-implanted cattle fed for slaughter at equal days on feed. Implants increase frame size and delay fattening. This shift in frame size, requires implanted cattle be fed to greater final shrunk body weight (FSBW) in order to reach similar empty body fat (EBF) percentage as compared to non-implanted cattle. Classical improvements in implanted cattle over non-implanted cattle have been summarized previously and are typically in the range of 8% to 28% for ADG and 5% to 20% for G:F depending on implant potency and stage of production (Johnson and Beckett 2014). Depending upon initial body weight (BW) at feedlot introduction the use of two or more TBA + E₂ implants is a common practice in feedlot production (APHIS 2013). Implants stimulate DMI and steers receiving more than one implant have greater daily intakes over steers receiving a single implant during the feedlot production phase (Parr et al. 2011a; Parr et al. 2011b). Typically, implants improve live basis gain and carcass-adjusted gain efficiency (Preston et al. 1990; Guiroy et al. 2002; Reinhardt 2007; Parr et al. 2011a; Parr et al. 2011b) when harvested at similar chemical EBF percentage.

In the last 11 y, coated and long acting TBA + E₂ implants have been made commercially available to producers. This new technology allows for cattle to be implanted a single time 200 d prior to harvest, effectively eliminating the need to re-implant cattle with first generation, non-coated TBA + E₂ implants. The benefit of not removing cattle from their pen to re-implant allows greater flexibility to feedlot managers by reducing the risk of intake disruption and injury to cattle during processing.

It is well documented that the use of implants, regardless of coating, total dose, or timing of administration increases final live BW measures relative to non-implanted (NI) steers (Reinhardt 2007; Parr et al. 2011a; Parr et al. 2011b; McLaughlin et al. 2013). The altered payout characteristics associated with coated implants have been shown to improve final live BW measures over administration of a single TBA + E₂ implant (Parr et al. 2011a; Parr et al. 2011b). When using a Revalor-XR implant (delayed release coated implant; 200 mg TBA and 20 mg E₂) BW measures and ADG are similar for coated implants versus NI during the initial 70 d period (FOIA 2017b). McLaughlin et al. (2013) reported that steers receiving a long-acting implant (sustained release coated implant: 200 mg TBA + 28 mg estradiol benzoate) had lower ADG and G:F when compared to steers given a conventional implant (non-coated implant: 200 mg TBA + 28 mg estradiol benzoate) during the initial 75 d post-implantation. While steers administered a Revalor-XS (initial and delayed release implant) were intermediate and did not differ from either implant group (McLaughlin et al. 2013). According to the FOIA (2017b), 70 d post-implantation steers receiving a Revalor-XR implant do not differ from negative controls for ADG or G:F. However, from 71 to 200 d post-implantation cattle implanted with Revalor-XR have improved ADG and G:F over negative controls (FOIA 2017b). McLaughlin et al. (2013) indicated that steers receiving a coated implant have a marked improvement for interim period ADG from d 75 to 140 over steers administered a non-coated implant.

Carcass characteristics

The use of a steroidal implant increases the percentage of muscle and decrease the percentage of adipose tissue in the carcass at a constant live weight compared to cattle that have not received a steroidal implant. The use of implants improves hot carcass weight (HCW) and longissimus muscle (LM) area over non-implanted steers harvested at equal BF accumulation (Preston et al. 1990; Johnson et al. 1996a; Guiroy et al. 2002; Reinhardt 2007; Parr et al. 2011a; Parr et al. 2011b). When harvested at equal days on feed, implanted cattle have lower USDA marbling scores and larger LM area than non-implanted cattle (Johnson et al. 1996a; Parr et al. 2011a, 2011b). (Smith et al. 2017) indicated that non-coated and coated implants differentially alter adipogenic gene expression in LM biopsies of steers. (Smith et al. 2017) demonstrated that steers administered a non-coated implant containing 120 mg TBA + 24 mg E₂ had decreased expression of PPARγ, GPR 41, and GPR 43, important genes involved in adipogenesis, in LM biopsies relative to negative controls or steers implanted with a Revalor-XS (coated implant). Bryant et al. (2010) indicated that steers implanted with a non-coated implant containing 80 mg TBA + 16 mg E₂ and re-implanted with a non-coated implant containing 120 mg TBA + 24 mg E₂ on d 56 had decreased marbling scores relative to controls at equal empty body fat percentage and this is likely a function of dilution of intramuscular fat via increased LM area (Parr et al. 2011a; Smith et al. 2017). Preston et al. (1990) indicated that implanted steers need to be fed to 39.5-kg greater FSBW over non-implanted cattle to minimize reductions in USDA marbling scores. (Guiroy et al. 2002) noted that steers administered a non-coated implant containing 80 mg TBA + 16 mg E₂ and re-implanted with a non-coated implant containing 120 mg TBA + 24 mg E₂ required a 42-kg increase in final shrunk BW to reach similar body composition of non-implanted steers. When the biology of growth is understood, and cattle are harvested at acceptable increases in final BW and EBF percentage, implant effects on carcass quality grade are minimized. Yield grade is negatively impacted by BF depth. At a constant body weight, implants decrease yield grade as evidenced by both decreased external BF accumulation and increased LM area. Depending on initial and re-implant timing, shifts in the distribution of USDA quality and yield grades occur, as anabolic exposure throughout various stages of growth alters the development of economically relevant tissue depots such as marbling, LM area and BF (Parr et al. 2011b).

Hormonal and sera metabolite responses

Implantation with a combination of TBA + E₂ increases circulating E₂ concentrations over NI controls (Johnson et al. 1996a; Bryant et al. 2010; Blackwell et al. 2014; Parr et al. 2014). Johnson et al. (1996a) first reported that E₂ values in sera were greatest at d 21 post-implanting in steers given a combination implant that contained 120 mg TBA and 24 mg E₂ and that values declined after that, however, E₂ in sera remained elevated relative to negative controls throughout the entire study. Others have demonstrated using coated and non-coated implants that E₂ in circulation remains greater than NI controls in excess of 100 d (Bryant et al. 2010; Parr et al. 2014). Relevance of sera levels of steroid hormones and how these influence steroidal implant activity have not been clearly established. Depending upon the type of implant used (i.e. coated or non-coated) steroid hormone levels in circulation have been shown to decline rapidly after approximately 3–4 weeks post-implantation in non-coated implants and ADG response is generally the greatest during the initial 28 d period following implantation with a non-coated implant. A better understanding of what these minimum steroid hormone thresholds in circulation are to elicit maximal growth responses deserve further investigation in the future.

Trenbolone acetate, the primary parent androgen molecule used in beef cattle implant formulations is a synthetic molecule. Once released from the excipient, it is rapidly de-acylated into 17β-TbOH, the active anabolic metabolite. The use of TBA implants rapidly increases circulating concentrations of sera 17β-TbOH relative to animals that have not been administered TBA (Johnson et al. 1996a; Blackwell et al. 2014; Parr et al. 2014). Typically increases in sera 17-β TbOH occur in concert with improvements in gain, gain efficiency, and circulating concentrations of sera IGF-I. This provides evidence that 17β-TbOH increases IGF-I output from hepatic tissue. This increased IGF-I output is likely associated with alterations in hypothalamic GHRH output and increased GH production from the anterior pituitary, as well as, improvements in GH binding to GH receptors located in hepatic tissue, much like what has been demonstrated in regards to E₂ (Trenkle 1976).

Implantation with TBA + E₂ increases circulating concentrations of sera IGF-I relative to negative control animals (Johnson et al. 1996b; Bryant et al. 2010; Parr et al. 2014). Bryant et al. (2010) reported increased sera IGF-I values in circulation by 42 d in heifers implanted with TBA and E₂, while steers implanted with TBA and E₂ increased sera IGF-I in circulation by d 21 and 27, respectively (Johnson et al. 1996b; Parr et al. 2014). Reinhardt et al. (2013) reported no differences in circulating concentrations of plasma IGF-I in steers administered an initial and delayed release implant containing [200 mg TBA + 40 mg E₂ (total): 80 mg TBA + 16 mg E₂ (non-coated) and 120 mg TBA + 24 mg E₂ (coated), Revalor-XS, Merck Animal Health] relative to NI steers at 28 d post-implantation. The influence of TBA and E₂ on circulating IGF-I would be directly related to alterations in GHRH at the hypothalamus and GH output from the anterior pituitary. These cellular manipulations that increase GHRH from the hypothalamus and increase GH output from the anterior pituitary, subsequently increase IGF-I production from hepatic tissue.

Elevated NEFA values are a measure of adipose tissue catabolism and implanting with TBA + E₂ has minimal impact on circulating concentrations of NEFA. Parr et al. (2014) detected no differences in circulating concentrations of NEFA in steers given no implant, non-coated implant, or a coated implant. Others have also reported similar results, when implanting heifers with TBA (Heitzman and Chan 1974) or steers with hexoestrol, TBA, or in combination together (Galbraith and Watson 1978). In contrast, Bryant et al. (2010) reported increased sera concentrations of NEFA in heifers implanted with TBA alone or in combination with E₂. Sera NEFA values are an excellent indicator of adipose tissue catabolism. Implants cause a delay in fattening, when DMI is not limiting and energy intake is adequate, sera NEFA might be of little diagnostic value as an indicator of adipose tissue catabolism.

Serum urea-N (SUN) is an indicator of urinary N excretion. When cattle are consuming similar DMI and rumen degradable protein is constant across diets, depression in SUN is generally a well-accepted measure of lean tissue anabolism. It has been demonstrated previously (Heitzman and Chan 1974; Heitzman et al. 1977; Bryant et al. 2010; Parr et al. 2014) that SUN concentrations are decreased by the use of anabolic implants. This would be expected because steroid hormones with anabolic activity increase N retention compared to non-treated cattle. Combination TBA + E₂ steroid implants increase protein accumulation within 40 d of administration relative to animals that have not been administered a steroidal implant (Johnson et al. 1996a). This is due to increased protein synthesis and decreased protein degradation, resulting in increased N retention with the net effect being increased skeletal muscle anabolism (Lobley et al. 1985). This phenomenon is well associated with the application of steroidal implants with anabolic activity and this action would be expected to increase N retention in the body (Lobley et al. 1985). Therefore, depressions in SUN following implant administration have been well documented in the literature (Heitzman and Chan 1974; Heitzman et al. 1977; Bryant et al. 2010; Parr et al. 2014).

Safety of steroidal implants with anabolic activity in beef production

Determination of safety

Any new implant formulation marketed in the U.S. is required to go through a thorough, multi-step scientific review by the FDA in order to ensure animal well-being and safety to the human food supply chain. The use of these compounds must then continually be proven safe for human consumption via random testing for residues in edible tissue and to the environment by way of many independently conducted post-approval environmental impact studies. The United States Department of Agriculture-Food Safety Inspection Services (USDA/FSIS) monitors levels of various residues in tissues such as muscle and liver using maximum residue levels set forth by the Joint Expert Committee on Food Additives of the Food and Agriculture Organization of the United Nations and World Health Organization (JECFA). The primary goal of JECFA is to provide insight into understanding the risk of residues in meat from animals administered veterinary pharmaceuticals. In regards to animal growth promotors, there are differences for natural and synthetic compounds. The JECFA has established maximum residue levels (MRL) for xenobiotic compounds (i.e. trenbolone acetate and zearanol), and has set for average allowable daily intakes (ADI) for natural compounds, such as estradiol-17β and testosterone propionate. Ensuring food safety to the public is based upon calculations from the no-hormonal effect level. Next, a safety factor of 100X is assigned and the resulting value is termed ADI in µg/kg BW. These steps all occur prior to use in commercial practice in order to ensure minimal risk for environmental exposure to humans. The reason there is no MRL for natural compounds is because implanted animals rarely have differing residue levels from non-implanted intact animals following harvest.

Human environmental exposure

Of importance to this review of literature is trenbolone acetate, a synthetic steroid that is not naturally occurring in animals (Blackwell et al. 2014). Anabolic constituents have a rapid half-life in circulation. Once TBA is released from the excipient and enters the bloodstream it is readily de-acylated to 17β-TbOH (Blackwell et al. 2014). This 17β-TbOH metabolite is the active ingredient of the implant pellets and has 10 to 50X greater anabolic activity in mammalian tissue as compared to the naturally produced androgen, testosterone. Plus, it is considerably less androgenic, meaning it has far lesser capability to induce unwanted secondary sexual effects, namely masculinization and un-wanted male behaviour. Additionally, 17β-TbOH, is not aromatized to estrogen, like natural testosterone. Maximum, 17β-TbOH concentrations in sera of steers can occur within approximately 2 d of administering a TBA + E₂ implant (Johnson et al. 1996a; Blackwell et al. 2014; Parr et al. 2014). Once released from the implant, 17β-TbOH has been shown to have a relatively short half-life ranging from 0.5 to 2 h (Pottier et al. 1975). The liver is the organ that is responsible for metabolizing the active TbOH metabolite, 17β-TbOH, into other metabolites in preparation for excretion from the body. Two other metabolites, in addition to 17β-TbOH, that are detectable in feces, sera, and urine following TBA administration are 17α-TbOH and trendione, neither of which are active anabolic metabolites of TbOH (Blackwell et al. 2014). The majority of TbOH excreted from the body in cattle is in the form of 17α-TbOH (Blackwell et al. 2014). The majority of TbOH remaining in edible tissue of cattle is the active anabolic compound, 17β-TbOH (Heitzman and Harwood 1977; Rico 1983). The major routes of hormonal metabolite excretion are fecal and urinary in mammals. The risk for environmental exposure herein lies towards consumption of the active metabolite of TbOH, 17β-TbOH in edible tissue, however, the risk for consumption of doses that would be hormonally active are minimal. The FDA (FOIA 1987) first set the tolerance level for muscle and liver tissue in cattle administered TBA at 50 ppb of 17β-TbOH. These levels were lowered by JECFA in 1988 to 2 ppb in muscle and 10 ppb in liver tissue (Jeong et al. 2010). The MRL’s set forth by JECFA are established at much higher levels than those obtained from independent academic and industrial research, following normal application. Many studies have indicated that the true level of 17β-TbOH residues in meat is much lower (Pottier et al. 1975; Heitzman and Harwood 1977; Rico 1983). Many labs have demonstrated, that cattle administered a single dose of 200–300 mg TBA possess muscle and fat residue levels of 0.05–0.6 ppb from 60 to 90 d after implantation with a TBA implant product (Pottier et al. 1975; Heitzman and Harwood 1977; Rico 1983). The USDA/FSIS monitors levels of various residues in tissues such as muscle and liver using the MRL’s set forth by JECFA. These results are published in the USDA/FSIS National Residue Program Red Book. Access to the USDA/FSIS red book indicates that there has not been any residue violations for 17β-TbOH detected in meat tissue for over 11 y. Another safety factor for ingesting steroid hormones from meat obtained from implanted cattle is that only 0.1–10% of the anabolic compound is absorbed from the human gastrointestinal tract into the bloodstream (Doyle 2000). The judicious use of anabolic compounds and adherence to labelled administration protocols ensure safety to the human food supply chain. The use of approved anabolic growth promoters as intended proves little risk for residues in meat tissue, subsequently, negating potential for human environmental exposure.

Conclusions to review

Steroidal hormones with anabolic activity bind to classical nuclear and membrane bound hormone receptors with different binding affinities in skeletal muscle. The application of steroidal hormones with anabolic activity in beef cattle increases the mature body weight of cattle. Increased mature body weight following the administration of a steroidal implant is directly associated with greater lean tissue deposition. Steroidal sex hormones with anabolic activity increase GH output from the anterior pituitary that in turn increases hepatic IGF-I output into blood circulation (Preston 1975; Trenkle 1976; Johnson et al. 1996b; Bryant et al. 2010; Parr et al. 2014). Steroidal hormones with anabolic activity also increase local IGF-I in LM biopsies of steers (Johnson et al. 1998a; Parr et al. 2014). Local IGF-I in skeletal muscle is important for the proliferation and differentiation of BSC’s needed to support the DNA to protein unit throughout growth. The net effect following implantation with anabolic steroid hormones is greater protein synthesis and reduced protein degradation, resulting in increased muscle tissue anabolism due to increased mature body weight and more active satellite cell populations compared to animals that have not been administered a steroidal implant. These compounds are critical tools required for efficient beef production in North America. Continuous regulatory oversight has proven that the judicious use of anabolic compounds occurs in U.S. animal feeding operations. This ensures safety to the human food supply chain. The use of approved anabolic growth promoters as intended in a normal production environment provides little risk for residues in meat tissue, subsequently, negating potential for human environmental exposure. No other technology is available to producers that match the improvements in animal performance and gain efficiency achieved via implants at an equal chemical fat percentage (Reinhardt 2007).

Disclosure statement

No potential conflict of interest was reported by the author(s).