**1. Introduction**

32 A Bird's-Eye View of Veterinary Medicine

Santiago-Moreno, J. n., Toledano-DÌaz, A., Pulido-Pastor, A., GÛmez-Brunet, A., & LÛpez-

Schenk, J. L., & DeGrofft, D. L. (2003). Insemination of cow elk with sexed frozen semen.

Seager, S. W. J. (1969). Successful pregnancies utilizing frozen dog semen. *AI Digest, 17*, 6-16. Senger, P. L. (2003). *Pathways to Pregnancy and Parturition* (Second ed.): Current

Sieme, H., Sch‰fer, T., Stout, T. A. E., Klug, E., & Waberski, D. (2003). The effects of different insemination regimes on fertility in mares. *Theriogenology, 60*(6), 1153-1164. Singleton, W. L. (2001). State of the art in artificial insemination of pigs in the United States.

Soede, N. M. (1993). Boar stimuli around insemination affect reproductive processes in pigs:

Thomassen, R., Farstad, W., Krogenaes, A., Fougner, J. A., & Andersen Berg, K. (2001). Artificial insemination with frozen semen in dogs: a retrospective study. *Journal of* 

Thomassen, R., Sanson, G., Krogenes, A., Fougner, J. A., Berg, K. A., & Farstad, W. (2006). Artificial insemination with frozen semen in dogs: A retrospective study of 10 years

Vazquez, J. M., Martinez, E. A., Parrilla, I., Roca, J., Gil, M. A., & Vazquez, J. L. (2003). Birth of piglets after deep intrauterine insemination with flow cytometrically sorted boar

Vazquez, J. M., Roca, J., Gil, M. A., Cuello, C., Parrilla, I., Vazquez, J. L., et al. (2008). New developments in low-dose insemination technology. *Theriogenology, 70*(8), 1216-1224. Vidament, M. (2005). French field results (1985-2005) on factors affecting fertility of frozen

Vidament, M., Dupere, A. M., Julienne, P., Evain, A., Noue, P., & Palmer, E. (1997). Equine frozen semen: Freezability and fertility field results. *Theriogenology, 48*(6), 907-917. Wolf, D. P. (2009). Artificial insemination and the assisted reproductive technologies in non-

Wongtawan, T., Saravia, F., Wallgren, M., Caballero, I., & RodrÌguez-MartÌnez, H. (2006). Fertility after deep intra-uterine artificial insemination of concentrated low-volume

Yamashiro, H., Han, Y.-J., Sugawara, A., Tomioka, I., Hoshino, Y., & Sato, E. (2007). Freezability of rat epididymal sperm induced by raffinose in modified Krebs-Ringer

bicarbonate (mKRB) based extender solution. *Cryobiology, 55*(3), 285-294. Youngquist, R. S., & Threlfall, W. R. (2007). *Current Therapy in Large Animal Theriogenology*

using a non-surgical approach. *Theriogenology, 66*(6-7), 1645-1650.

stallion semen. *Animal Reproduction Science, 89*(1-4), 115-136.

A review. *Animal Reproduction Science, 32*(1-2), 107-125.

*Reproduction and Fertility Supplement, 57*, 341-346.

spermatozoa. *Theriogenology, 59*(7), 1605-1614.

human primates. *Theriogenology, 71*, 123-129.

(2nd ed.): Saunders Elsevier.

boar semen doses. *Theriogenology, 65*(4), 773-787.

1226.

*Theriogenology, 66*(2), 283-291.

Conceptions,Inc.

[Abstract]. *Theriogenology, 59*, 514.

*Theriogenology, 56*(8), 1305-1310.

Spanish ibex (Capra pyrenaica) epididymal spermatozoa. *Theriogenology, 66*(5), 1219-

Sebasti·n, A. (2006). Birth of live Spanish ibex (Capra pyrenaica hispanica) derived from artificial insemination with epididymal spermatozoa retrieved after death.

> Hormones are chemicals produced by animals to co-ordinate their physiological activities. They act as messengers, produced in and released from one kind of tissue to gradually stimulate or inhibit some process in a different tissue over a long period.

> Steroid hormones fulfill an important role at different stages of mammalian development comprising prenatal development, growth, reproduction and sexual and social behavior.

> The importance of individual hormones varies between sexes and age and a disruption of the endocrine equilibrium may result in multiple biological effects.

> One hormone can have multiple actions, e.g. the male hormone testosterone controls many processes from the development of the foetus, to libido in the adult. Alternatively, one function may be controlled by multiple hormones, e.g. the menstrual cycle involves oestradiol, progesterone, follicle-stimulating hormone and luteinising hormone.

> Hormones produced by the bodies of humans and animals are called endogenous or natural hormones. Compounds chemically synthesised to mimic the effect of natural hormones are called synthetic or xenobiotic hormones.

> Hormones are vital in normal development, maturation and physiological functioning of many vital organs and processes in the body. However, like any other chemicals of natural or synthetic origin, hormones may be toxic to living organisms under certain circumstances. The toxicity may be due to an excess of its normal ('physiological') action. This may be the result of excessive exposure to the substance, for example following absorption of a large dose, or because the physicochemical nature of the substance gives it greater or more prolonged activity of the same type, or because the hormonal action occurs at an abnormal time during development or adult life, or is an action on an organism of the inappropriate sex. Hormones, like other chemicals, may also exert direct toxic actions not related to their endocrine ('physiological') effects.

> Due to the obvious ability to improve weight gain and feed efficiency in meat producing animals, natural hormones and/or the synthetic surrogates have been used in agricultural practice for several decades (table 1).

Steroid Hormones in Food Producing Animals: Regulatory Situation in Europe 35

Oestradiol is synthesized and secreted in early stages of embryogenesis and has an active role in the normal development of the female sex accessories during the lifetime of females. It has been used to induce parturition (birth) especially in sheep, a species in which an associated oestradiol-induced increase in mothering ability has also been recorded

In non-pregnant animals, oestradiol has been used clinically to increase uterine contractions and cervical softening for the expulsion of unwanted uterine contents in the absence of a corpus luteum (i.e. to remove a dead fetus or infected material especially in cattle) (Elmore,

Oestradiol has been used in the past in turkeys and other poultry to castrate young birds. Implants would be placed subcutaneously at 5-6 weeks of age, or in slightly older birds, but certainly 4 weeks before killing. Alternatively, preparations were available as feed-additives. This approach is not now used in Europe although it was used in slower growing Spanish

Fetal mummifications and macerations occur, as do endometritis and pyometra but

In fish, administration of oestradiol at first-fry feeding (approximately 40-70 days of age depending on species) will induce ovarian development and female characteristics in salmonids, flat-fish and eels (Shepherd & Bromage, 1988). Sex-control in this way depresses or inhibits maturation to ensure that metabolism is channelled into body growth (i.e., more

Another use of very low doses of oestradiol is as a growth promoter via appetite stimulating and increased food-conversion properties. Occasionally in the past, this approach has been taken to advance the onset of puberty and thus alleviate potential gynaecological problems in slower maturing species. However, as use of hormonal growth promoters is prohibited

Testosterone and its more active metabolite, 5-dihydrotestosterone (DHT), are the main sex hormones secreted by males. Testosterone is responsible for the early development, and the appearance and maintenance of male secondary sex accessory organs (prostate, secretory glands, penis size, etc.) during adulthood. Testosterone secretion is also affected by the

Testosterone is metabolized and as a result, metabolites of different activity are generated.

The actions of both testosterone and DHT are mediated through their high affinity and high specificity binding and activation of an intracellular protein, the androgen receptor (AR). This AR protein is a member of the steroid hormone superfamily. The ligand-activated androgen receptor mediates its effects on cell growth and differentiation through the

complex interaction among all endocrine glands, especially with those in the brain.

Some of these metabolites play a more active role in certain organs than in others.

1 See the following paragraph: Background of the European Union legislation

1992; Pepper &, Dobson, 1987; Sheldon & Noakes, 1998).

breeds. There are very few reproductive problems in rabbits.

treatment with oestradiol has not been reported (Flecknell, 2000).

saleable flesh). However, this approach is no longer commercially adopted.

(Poindron, 2005).

within the European Union1.

**1.1.2 Testosterone** 


Table 1. Hormonally-active substance used in animal production (by http://www.fao.org/DOCREP/004/X6533E/X6533E01.htm, modified)

Implanting hormonal growth promoters is currently widespread in the beef cattle industry of many non-EU countries for the better performance in growth and improvement of feed efficiency. These hormonal implants may enhance growth during suckling, growing and finishing stages of production (Mader, 1997; Platter *et al.,* 2003).

Growth hormones are implanted under the skin (usually behind the ear) of the animal in the form of depot capsules, where they release a specific dose of hormones over a fixed period of time.

The five hormone types most widely used in meat production include three natural hormones, oestradiol 17-, testosterone, and progesterone, and two synthetic substances, trenbolone and zeranol.

Oestradiol 17- has oestrogenic action (i.e. responsible for female characteristics); testosterone has androgenic action (i.e. responsible for male characteristics); and progesterone has gestagenic action (i.e. responsible for maintaining pregnancy). The other two hormones, as aforesaid, mimic the biological activity of the natural hormones: trenbolone mimics the action of testosterone, and zeranol mimics oestradiol 17-

#### **1.1 Hormonally active substances**

#### **1.1.1 Oestradiol 17-**

Oestradiol 17- is the most active of the female sex hormones synthesized and secreted mainly by the ovary, the adrenals and the testis.

Oestradiol is synthesized and secreted in early stages of embryogenesis and has an active role in the normal development of the female sex accessories during the lifetime of females.

It has been used to induce parturition (birth) especially in sheep, a species in which an associated oestradiol-induced increase in mothering ability has also been recorded (Poindron, 2005).

In non-pregnant animals, oestradiol has been used clinically to increase uterine contractions and cervical softening for the expulsion of unwanted uterine contents in the absence of a corpus luteum (i.e. to remove a dead fetus or infected material especially in cattle) (Elmore, 1992; Pepper &, Dobson, 1987; Sheldon & Noakes, 1998).

Oestradiol has been used in the past in turkeys and other poultry to castrate young birds. Implants would be placed subcutaneously at 5-6 weeks of age, or in slightly older birds, but certainly 4 weeks before killing. Alternatively, preparations were available as feed-additives. This approach is not now used in Europe although it was used in slower growing Spanish breeds. There are very few reproductive problems in rabbits.

Fetal mummifications and macerations occur, as do endometritis and pyometra but treatment with oestradiol has not been reported (Flecknell, 2000).

In fish, administration of oestradiol at first-fry feeding (approximately 40-70 days of age depending on species) will induce ovarian development and female characteristics in salmonids, flat-fish and eels (Shepherd & Bromage, 1988). Sex-control in this way depresses or inhibits maturation to ensure that metabolism is channelled into body growth (i.e., more saleable flesh). However, this approach is no longer commercially adopted.

Another use of very low doses of oestradiol is as a growth promoter via appetite stimulating and increased food-conversion properties. Occasionally in the past, this approach has been taken to advance the onset of puberty and thus alleviate potential gynaecological problems in slower maturing species. However, as use of hormonal growth promoters is prohibited within the European Union1.

#### **1.1.2 Testosterone**

34 A Bird's-Eye View of Veterinary Medicine

Feed additive Implant Oil solution Implant Implant

Steers, heifers

Steers, sheep

Steers, sheep, calves, poultry

Bulls, steers, calves, sheep

Heifers, calves

Steers Veal calves

Calves Swine Steers Steers

Steers

Substances Form Main use - Animals

**TBA** Implant Heifers, culled cows

Implant Feed additive Implant Implant Implant Implant

Implant

Implanting hormonal growth promoters is currently widespread in the beef cattle industry of many non-EU countries for the better performance in growth and improvement of feed efficiency. These hormonal implants may enhance growth during suckling, growing and

Growth hormones are implanted under the skin (usually behind the ear) of the animal in the form of depot capsules, where they release a specific dose of hormones over a fixed period

The five hormone types most widely used in meat production include three natural hormones, oestradiol 17-, testosterone, and progesterone, and two synthetic substances,

Oestradiol 17- has oestrogenic action (i.e. responsible for female characteristics); testosterone has androgenic action (i.e. responsible for male characteristics); and progesterone has gestagenic action (i.e. responsible for maintaining pregnancy). The other two hormones, as aforesaid, mimic the biological activity of the natural hormones:

Oestradiol 17- is the most active of the female sex hormones synthesized and secreted

trenbolone mimics the action of testosterone, and zeranol mimics oestradiol 17-

**Melengestrol acetate** Heifers

Table 1. Hormonally-active substance used in animal production (by http://www.fao.org/DOCREP/004/X6533E/X6533E01.htm, modified)

finishing stages of production (Mader, 1997; Platter *et al.,* 2003).

**Oestrogens alone:**

**Gestagens alone:**

**Androgens alone:**

**Combined preparations: DES and Testosterone DES and Methyl-testosterone** 

**Hexoestrol and TBA Zeranol and TBA Oestradiol-17 and TBA Oestradiol-17 benzoate and testosterone propionate Oestradiol-17 benzoate and** 

**progesterone** 

of time.

trenbolone and zeranol.

**1.1.1 Oestradiol 17-**

**1.1 Hormonally active substances** 

mainly by the ovary, the adrenals and the testis.

**DES DES DES Hexoestrol Zeranol** 

> Testosterone and its more active metabolite, 5-dihydrotestosterone (DHT), are the main sex hormones secreted by males. Testosterone is responsible for the early development, and the appearance and maintenance of male secondary sex accessory organs (prostate, secretory glands, penis size, etc.) during adulthood. Testosterone secretion is also affected by the complex interaction among all endocrine glands, especially with those in the brain.

> Testosterone is metabolized and as a result, metabolites of different activity are generated. Some of these metabolites play a more active role in certain organs than in others.

> The actions of both testosterone and DHT are mediated through their high affinity and high specificity binding and activation of an intracellular protein, the androgen receptor (AR). This AR protein is a member of the steroid hormone superfamily. The ligand-activated androgen receptor mediates its effects on cell growth and differentiation through the

<sup>1</sup> See the following paragraph: Background of the European Union legislation

Steroid Hormones in Food Producing Animals: Regulatory Situation in Europe 37

its anabolic action via interaction with androgen and glucocorticoid receptors (Danhaive and Rousseau, 1986, 1988). Experiments with cattle tissues have shown that 17β-trenbolone binds to the androgen receptor with similar affinity as dihydrotestosterone. It also binds to the progesterone receptor with an affinity that exceeds that of progesterone. The other metabolites of TBA, including 17α-trenbolone (17α-hydroxy-estra-4,9,11-trien-3-one) and TBO (estra-4,9,11-triene-3,17-dione) show a significantly lower binding affinity to both types

Reports regarding the (mis)use of TBA as an anabolic agent in sports people describe several adverse effects, including liver cell injury with an increase in liver-specific enzymes in serum, cholestatic jaundice, peliosis hepatitis and various neoplastic lesions. Moreover, decreased endogenous testosterone production and spermatogenesis, oligospermia and testicular atrophy may be associated with the repeated use of TBA as anabolic (Bahrke and

Zeranol is derived from the naturally occurring mycoestrogen zearalenone, and is a potent oestrogen receptor agonist *in vivo* and *in vitro* (Leffers *et al.,* 2001; Le Guevel and Pakdel, 2001; Takemura *et al.,* 2007; Yuri *et al.,* 2006). Its actions resemble those of oestradiol. (Leffers

Zeranol stimulates the proliferation of ER-dependent cell proliferation in MCF-7 human breast cancer cells (which are widely used in the assessment of estrogenic activity) and in

It is used alone or in combination with TBA as a hormonal growth promoter in various

The use of hormonal growth promoters in food-producing animals has been a sensitive

Prior to 1981, the EC had no universal policy on the use of growth promoting hormones in

The use of hormones had been banned in Italy since 1961, in Denmark since 1963, and in Germany since 1977. Belgium and Greece had never permitted the use of hormones for fattening purposes. However, Spain, the United Kingdom, France and Netherlands

The move to impose a Europe wide ban was spurred by the worrying discovery in 1977 of breast enlargement in girls and boys attending a school in Milan (Italy) (Scaglioni *et al*., 1978). Although oestrogen contamination was not detected when samples of school meals were tested, an uncontrolled supply of poultry and beef was hypothesized as being the

2 See http://www.fao.org/DOCREP/004/X6533E/X6533E03.htm#refeecstr Accessed August 19, 2011.

transfected cells (Leffers *et al.*, 2001; Le Guevel and Pakdel, 2001; Liu and Ling, 2004).

**2. Background of the European Union legislation** 

issue of debate in the EU and elsewhere for several decades.

cause of this outbreak (Fara *et al*., 1979).

permitted the use of most hormones for speeding growth in beef cattle2.

of receptors (Bauer *et al.,* 2000).

Yesalis, 2004; Maravelias *et al.,* 2005).

**1.1.5 Zeranol** 

*et al.,* 2001).

products.

meat animals.

activation and/or suppression of specific gene transcription in target organs. Androgen receptors are detected in tissues of females, as well as males. The presence of this receptor in organs such as the ovary indicates significant activity of androgens in both sexes. Furthermore, the androgen receptor is thought to be involved in ovarian tumorigenesis, as it has been detected in 67 percent of ovarian tumors. Although current information indicates the presence of only a single androgen receptor, it is known that different subsets of genes may be activated by either testosterone or DHT (Chang *et al*., 1995).

In animals, testosterone or testosterone propionate, alone or in combination with other hormonally active substances, is used primarily to improve the rate of weight gain and feed efficiency. This effect is most likely a consequence of the anabolic action of androgens.
