**3.3. GnRH agonists**

The GnRH agonists are intended to suppress the pulsatility of GnRH and indirectly that of LH and FSH [23]; consequently, these compounds depress the follicular activity in the ovaries and reduce to baseline the secretion of sexual steroids. Shortly, they reset the bitch into a prepubertal stage or in anestrus. By suppressing the secretion of progesterone, these substances will minimize the risk for uterine or mammary diseases found in the progestins contraception. Furthermore, they are beneficial in controlling unwelcome sexual behavior associated with the female season and animal aggressiveness. However, these products are usually expensive, which limits their wider utilization and reduces their competitiveness compared to progestins.

The available commercial products include azagly-nafarelin implant (Gonazon®), deslorelin acetate implants (Suprelorin® or Ovuplant®), and leuprolide acetate implant (Lupron Depot®), although the application of buserelin (50 µg, single administration) may also accomplish the GnRH downregulation. However, the length of the induced anestrus is less precise and the individual variation in the response is higher.

GnRH agonists are presented today as a subcutaneous implant, allowing a prolonged controlled release of the drug into the system. Implants are injected subcutaneously at the interscapular or the postumbilical area [24]. This sustained exposition to the GnRH agonist would override the endogenous secretion of this hormone. The agonist acts at the level of the GnRH receptors [23], inducing the receptor downregulation, internalization and signal uncoupling [4], resulting in the termination of the signaling cascade triggered GnRH in cells. However, GnRH agonists act in a dual phase mechanism. Before acting as described before, the first effect of implants is to stimulate the pituitary axis ("flare-up" effect), triggering an initial release of FSH and LH [25], thus shortly originating a new season in treated bitches. The "flare-up" effect is usually observed within 1 month after the insertion of the implant [26]. This is, to date, the most significant drawback identified for the use of GnRH agonists in dog contraception: to first induce estrus before preventing it [27].

The flare-up effect is more frequent when treatment starts in late anestrus [28, 29], compared to any other stage of the canine cycle. Whether or not it may be decreased when the female is implanted in diestrus or given exogenous progestogen [30–32] is still controversial [33, 34].

females for breeding after the interruption of treatment and dismissal from the racetrack [22]. Anyway, these drawbacks compromised the use of androgens in current clinical practice [7].

Few studies exist on the use of androgens as estrus suppressor in dogs. The oral administration of androgens is described for methyl testosterone (25 mg/dog, once a week [2]), orandrone (0.5 mg/kg, daily [20]), methyl testosterone associated with ethinylestradiol (7 mg/kg, daily, for 5– 10 days.[20]), and mibolerone, a synthetic weak androgen (Cheque Drops, at a dose of 30–180 mcg/day, starting 30 days before the onset of heat; it should not be administered for more than 2 years [7]). Injectable androgen therapeutic options include the intramuscular administration of testosterone cypionate (1 mg/kg, every 2 weeks [22]), testosterone phenylpropionate (110 mg/dog, weekly or alternatively at 0.5–1 mg/kg, every 7–10 days [2] and [20], respectively) or a composition of four different testosterone esters (Durateston®, at a dose of 2.5–5 mg/kg every

The GnRH agonists are intended to suppress the pulsatility of GnRH and indirectly that of LH and FSH [23]; consequently, these compounds depress the follicular activity in the ovaries and reduce to baseline the secretion of sexual steroids. Shortly, they reset the bitch into a prepubertal stage or in anestrus. By suppressing the secretion of progesterone, these substances will minimize the risk for uterine or mammary diseases found in the progestins contraception. Furthermore, they are beneficial in controlling unwelcome sexual behavior associated with the female season and animal aggressiveness. However, these products are usually expensive, which limits their wider utilization and reduces their competitiveness compared to progestins.

The available commercial products include azagly-nafarelin implant (Gonazon®), deslorelin acetate implants (Suprelorin® or Ovuplant®), and leuprolide acetate implant (Lupron Depot®), although the application of buserelin (50 µg, single administration) may also accomplish the GnRH downregulation. However, the length of the induced anestrus is less precise and the

GnRH agonists are presented today as a subcutaneous implant, allowing a prolonged controlled release of the drug into the system. Implants are injected subcutaneously at the interscapular or the postumbilical area [24]. This sustained exposition to the GnRH agonist would override the endogenous secretion of this hormone. The agonist acts at the level of the GnRH receptors [23], inducing the receptor downregulation, internalization and signal uncoupling [4], resulting in the termination of the signaling cascade triggered GnRH in cells. However, GnRH agonists act in a dual phase mechanism. Before acting as described before, the first effect of implants is to stimulate the pituitary axis ("flare-up" effect), triggering an initial release of FSH and LH [25], thus shortly originating a new season in treated bitches. The "flare-up" effect is usually observed within 1 month after the insertion of the implant [26]. This is, to date, the most significant drawback identified for the use of GnRH agonists in dog

6 months [20]).

**3.3. GnRH agonists**

146 Canine Medicine - Recent Topics and Advanced Research

individual variation in the response is higher.

contraception: to first induce estrus before preventing it [27].

Additional adverse side effects reported following the insertion of GnRH agonist implants include persistent estrus associated with the formation of ovarian cysts due to anovulation [35], galactorrhoea, metropathies, vomiting, cystitis, and allergic reactions [36]. These effects occur with variable incidence suggesting the existence of individual idiosyncratic factors. These may also comprise preexisting problems that remained clinically undiagnosed. Consequently, before injection of the implant, the female should be subjected to a thorough clinical and reproductive examination to exclude any ovarian and uterine pathology [24].

GnRH agonists can be used as short or long-term contraceptives in domestic carnivores [37, 38] and may be applied in prepubertal females to postpone the reproductive activity without apparent side effects to delay puberty [39, 40]. The effect of the implants may be fully reverted and fertility regained after the 12-month period of the implant action [23, 41]. However, in some cases, the implant effect can last up to 27 months [23, 32]. An anticipated removal of the implant would also permit the withdrawn of the effects over the hypothalamic-pituitary axis, and regain cyclicity earlier. Recently, it has stretched that Superlorin® presented no detrimental effects on the bitch fertility whether a short-term or the long-term treatment was implemented [24].

**Figure 3** provides a summary analysis to GnRH agonists as contraceptives in dogs.

**Figure 3.** SWOT analysis to the use of GnRH agonists to control the bitch reproduction.

### **3.4. GnRH antagonists**

The GnRH antagonists limit its action by a competitive block of the hormone receptors achieving the annulation of the effects of circulating GnRH. Consequently, the function of the hypothalamus-pituitary-gonadal axis is impaired. Long-term effects of GnRH antagonists also include the down-regulation of the GnRH receptors [4, 42]. Either way, follicular waves are suppressed and ovulation is compromised [19]. Several generations of peptides with GnRH antagonist activity have been tested in dogs, but their use is still limited all over the world, mainly because they present a rather low efficiency as contraceptives. Also, the first generation compounds showed several important side effects, derived from the need for higher doses of these peptides to reach the desired effect. Peptide GnRH antagonists act only for short-term estrus suppression [19, 43], which make them a poor agent when longer periods of contraception are foreseen. Therefore, its use is mostly restricted to the short-term contraception in show or work dogs [19].

**Figure 4.** SWOT analysis to the use of GnRH antagonists as a contraceptive in dogs.

In dogs, the third generation GnRH antagonist, Acylin®, is the most used antagonist. It should be administered within the first 3 days of proestrus, subcutaneously, at a dose of 100 µg/kg. Suspension of the follicular stage is obtained, and ovulation inhibited; however, the bitch is expected to enter a new cycle within 3 weeks of treatment [4].

Companies are now exploiting some nonpeptide molecules as GnRH antagonists, alike those tested for humans, aiming to obtain the long-term release formulations that may be applied for the long-term contraception. However, to our best knowledge, no information is yet available on these molecules.

**Figure 4** provides a summary analysis to the use of GnRH antagonists as contraceptives in dogs.
