Introduction and Overview

To date, there is no single approach for rapid induction of estrus that has been tested sufficiently to permit recommendation for clinical use. Many protocols depend on use of hormone preparations or drugs not readily available in some countries. Potentially useful success rates in limited numbers of bitches have been reported for protocols involving administration of eCG (i.e., PMSG) alone, of eCG followed by HCG to induce ovulation at the induced estrus, of FSH and estrogen in a prescribed sequence, of estrogen, LH and FSH in a prescribed sequence, of estrogen alone, of GnRH, or of a GnRH agonist (Wright, 1982; Shille et al., 1989; Concannon, 1992; Vanderlip et al, 1987; Concannon et al., 1997). Human menopausal gonadotrophin treatment has also been used successfully (Wanke et al., 1997). Also, likely to be useful clinically are non-rapid methods that do not involve direct stimulation of the ovaries. These include the use of prostaglandins to shorten the luteal phase and cause a resulting shortening of metestrus (diestrus) and of anestrus (Romagnoli et al., 1996), and the use a dopamine agonist to terminate anestrus and cause a premature proestrus (Verstegen et al., 1994). However, new GnRH-agonist therapies for acute induction of proestrus may become clinically useful in the near future. The natural transition from anestrus to proestrus involves increased pulsatile secretion of GnRH and LH for several days, a resulting elevation in mean LH concentrations with or without a further increase in already-elevated concentrations of FSH (Concannon, 1993). Endogenous regulation of this transition is not well understood, but can be affected by exposure to estrus pheromones and possibly other external stimuli. It apparently requires prolonged withdrawal of progesterone, based on the > 2 month obligate anestrus seen following the end of the 2 month luteal phase or progestin contraception. It likely also involves prolactin and or changes in dopaminaergic activity in the hypothalamus, based on the fore-shortening of anestrus by prolactin-lowering doses of dopamine agonists. The role of LH is probably greater than that of FSH in initiating the transition, based on induction of estrus and/or proestrus by administration of highly purified LH alone, but not by FSH alone (Verstegen et al, 1997).

Rapid Estrus Induction with Gonadotropins

PMSG (Pregnant mares serum gonadotropin). PMSG at the usual dose of 20 IU/kg/day, given for 10 days during anestrus and followed by an "ovulatory dose" of HCG (500-1000 IU), rapidly results in an estrus frequently characterized by low fertility, hyper-estrogenism, cystic ovaries, anovulation, inadequate luteal phase and/or pregnancy resorption. The same PMSG doses given for 5 days and immediately followed by HCG resulted in high fertility in some studies (Arnold et al, 1989) but not in another (Concannon, unpublished observations). Where successful, the HCG injection after 5 days of PMSG appears to stimulate further follicle development (rather than ovulation) with ovulation occurring spontaneously about one week later. Factors complicating use of this protocol include potential differences among preparations of PMSG and biopotency estimates, and availability in the different countries. Other doses, frequencies or durations have not been well studied. Alternate day PMSG administration did not induce proestrus (vanHaaften et al., 1989).

HMG (Human menopausal gonadotropin). HMG is marketed for human use in North America, but is available as a veterinary drug in some countries. In one study, HMG administered to anestrous dogs induced proestrus in 9 of 10 dogs and 4 became pregnant (Wanke et al., 1997). The protocol involved nine daily injections of 75 IU of HMG using a preparation (Pergovet *RTM) that was determined to have 1 IU of LH and 1 IU of FSH in each IU of HMG. The 40% pregnancy rate suggests HMG may be as effective as PMSG in dogs. Efficacy my be related to the relatively high LH activity compared to eCG.

Purified Pituitary Gonadotrophins: FSH or LH. Unlike the situation in livestock species, administration of FSH alone to dogs has apparently not resulted in fertile estrus. In contrast, purified porcine LH at doses of 0.1 IU/kg, t.i.d., for 7 days resulted in proestrus (100%), estrus (70%) and pregnancy (35%) in anestrous dogs (Verstegen et al., 1997). Those results have been interpreted to suggest that it is LH that is the endocrine factor which is deficient in anestrus and the more important of the gonadotrophins in the process of transition from anestrus to proestrus. Use of available preparations of HCG for such application has not been reported and commercial pituitary LH (PLH) is no longer readily available commercially in most countries.

Estrogen and FSH. Modifications of a protocol apparently used by greyhound breeders and reported to be successful include administration of diethylstilbestrol (DES) tablets (5 mg/day, PO) daily until 3 days of obvious proestrus signs, and administration of FSH (10 mg, IM) on days 5, 9 and 11 of the induced proestrus (Bouchard et al., 1991). In 13 dogs this resulted in estrus, ovulations and pregnancies in 9, 6 and 4 dogs, respectively, with ovulation occurring about 8 days after end of DES treatment. Earlier study using PLH (5 mg) in place of the first FSH injections yielded 100% success, but failed when HCG was substituted for the no-longer available PLH (Shille et al., 1989). More recently, the use of DES administration alone (7.4±1.1 days) was reported to be successful in each of 5 dogs when initiated at 95-129 days after parturition or prostaglandin-induced luteolysis. Ovulations occurred about 14 days after the end of DES injections (Bouchard et al., 1993). One possibility is that exogenous estrogen is acting in these cases like clomiphene in humans-i.e., by negative feedback suppression of gonadotrophin secretion, with a burst of LH and FSH secretion occurring when the estrogen is discontinued. However, in beagle bitches not previously pregnant and not pre-treated with PGF, the use of DES alone at the same doses did not result in fertile estrus (Concannon, 1996, Unpublished observations).

Rapid Induction of Estrus With GnRH and GnRH Agonists

GnRH (Gonadotrophin releasing hormone). GnRH given as intravenous pulses of 20-450 ug/kg every 90 minutes for 9-14 days had success rates of 63-88% in inducing fertile proestrus and ovulation by the end of treatment when initiated in anestrus (Vanderlip et al, 1987; Concannon et al., 1997). While useful for research, it is probably not clinically practical due to the expensive of a battery-operated, portable infusion pump connected to a jugular cannula. The treatment induces pulsatile LH release similar to that which normally occurs at the end of anestrus. However, a non-physiological, continuous elevation in LH induced by constant or infrequent administration of GnRH-superagonist can also induce proestrus.

GnRH-Agonists and Superagonists. Super-agonist analogs that are 100-200 as potent as GnRH (nafarelin, lutrelin, deslorelin) cause prolonged release of LH after single injection, and when infused constantly cause a surge of LH release followed by progressively reduced elevations in LH, and eventually suppression of LH by down-regulation of GnRH receptors. Injection of a nafarelin-like agonist every 8 h (at 1 ug/kg for 11 days and 0.5 ug/kg for 3 days) induced fertile proestrus in 80% of anestrus bitches within 9-11 days (Cain et al, 1990). Infusion of lutrelin via S.C. osmotic mini-pump for 8-14 days was successful in inducing fertile proestrus in anestrus bitches and in persistent anestrus bitches (Concannon, 1993; Concannon, unpublished data).

More recently injections of long-acting preparations of GnRH-agonists have been used to induce estrus in anestrus bitches. One protocol, reported by Cinone et al. (1996), involved subcutaneous injection of a mixture of the GnRH agonist buserelin mixed with a liquid silastic polymer. The slow release agonist from the injection site resulted in 27 of 29 anestrus or prepubertal bitches exhibiting proestrus and becoming pregnant, with estrus occurring within 10-15 days of injection. In a study by Inaba et al. (1998), a micro-capsule formulation of the GnRH agonist leuprolide acetate was used to induce proestrus and estrus. A subsequent injection of an aqueous preparation of a related GnRH agonist was then used to induce ovulation at the onset of the behavioral estrus. In 6 early anestrus, 6 late-anestrus bitches, and 6 prepubertal bitches the pregnancy rates were 50%, 100% and 83%. Another GnRH agonist, deslorelin, in the form of a long acting implant (Ovuplant) has been used to induce potentially fertile proestrus in anestrus bitches (Kutzler, 2002). Removal of the implant after 10 days appears to improve the pregnancy rate following breeding during estrus of the induced cycle. One difficulty in using 'continuous' administration of a GnRH agonist is that doses that induce proestrus may also result in down-regulation of LH release sufficient to compromise the occurrence of a spontaneous preovulatory LH surge (Concannon, 1993). Therefore, methods for withdrawal of treatment "on demand" or for actively inducing ovulation following proestrus-induction may be required

Shortening of Cycles with Prostaglandin Induced Luteolysis

Prostaglandin F2a (PGF). PGF-induced luteolysis alone, during the early or mid-luteal phases, reduces interestrus intervals, but not in a predictable time frame. PGF (50-200 ug/kg, bid, SC or IM, for 4-9 days, and to effect) has been used to prevent or terminate pregnancy, and to shorten luteal phases. One study reported 208 ± 39 day interestrus intervals in normal cycles, and intervals of only 134 ± 40 days for cycles in which PGF was used to induce luteolysis around day 20 after onset of estrus (Romagnoli et al., 1996).

Termination of Anestrus with Dopamine Agonists

Prolactin-lowering doses of dopamine agonists administered beginning at days 90-135 of the cycle can result in premature proestrus and fertile estrus with pro-estrus occurring by 17-50 days of treatment. Bromocriptine (Parlodel, Sandoz) is marketed as a human drug for treatment of hyper-prolactinemia and is not approved for veterinary use. Cabergoline (Galastop, Vetem) is marketed in some European countries as a veterinary drug for treatment of pseudopregnancy in dogs. Bromocriptine administered P.O. and b.i.d., at a low dose of 20 ug/kg induced proestrus in 47 ± 2 days (Okkens et al, 1985) while higher doses of 50 ug/kg induced proestrus in 17-28 days (Concannon, 1993). Similar results were also obtained with 100 ug/kg bromocriptine administered orally, once or twice daily (Concannon, unpublished data). Bromocriptine often causes emesis in early stages of treatment, can cause anorexia, and is not approved for veterinary use. Cabergoline, marketed in Europe for use in pseudopregnant dogs, has also been reported to shorten the interval to proestrus when administered to bitches in anestrus, but with fewer and less severe side effects. Cabergoline, at doses of 5 ug/kg/day, P.O., induced normal, fertile proestrus in 15-25 days, 10-20 days, or 4-10 days when administered to bitches in early, middle or late anestrus, respectively (Verstegen et al, 1994). The reason for the more rapid response in late anestrus is not known. Similar treatment has also been reported to be effective in bitches with persistent anestrus (Jochle et al.,1989). Thus, results to date show that a physiological but premature proestrus can be induced by the administration a dopamine agonist in anestrous bitches. This would appear to be clinically useful in some situations.

In summary, use of gonadotropins to induce estrus can to result in low fertility and abnormal luteal function. The use of GnRH agonists, either injected several times per day or as a depot injection or implant appears to have considerable promise and merits further study. The use of dopamine agonists to prematurely terminate anestrus typically results in a physiological estrus and spontaneous ovulation, but the duration of treatment required can vary greatly depending on the stage of the cycle.

References

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