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Authors requiring further information regarding Elsevier’s archiving and manuscript policies are encouraged to visit: http://www.elsevier.com/copyright Author's personal copy General and Comparative Endocrinology 166 (2010) 409–416 Contents lists available at ScienceDirect General and Comparative Endocrinology journal homepage: www.elsevier.com/locate/ygcen Oral progestin induces rapid, reversible suppression of ovarian activity in the cat R.A. Stewart a,b,1, K.M. Pelican a,2, J.L. Brown a, D.E. Wildt a,*, M.A. Ottinger b, J.G. Howard a a Department of Reproductive Sciences, Center for Species Survival, Smithsonian’s National Zoological Park, Conservation & Research Center, Front Royal, VA 22630, USA b Department of Animal and Avian Sciences, University of Maryland, College Park, MD 20742, USA article info abstract Article history: The influence of oral progestin (altrenogest; ALT) on cat ovarian activity was studied using non-invasive Received 28 July 2009 fecal steroid monitoring. Queens were assigned to various ALT dosages: (1) 0 mg/kg (control; n = 5 cats); Revised 7 December 2009 (2) 0.044 mg/kg (LOW; n = 5); (3) 0.088 mg/kg (MID; n = 6); or (4) 0.352 mg/kg (HIGH; n = 6). Fecal estro- Accepted 28 December 2009 gen and progestagen concentrations were quantified using enzyme immunoassays for 60 days before, Available online 4 January 2010 38 days during and 60 days after ALT treatment. Initiation of follicular activity was suppressed in all cats during progestin treatment, whereas controls continued to cycle normally. Females (n = 6) with elevated Keywords: fecal estrogens at treatment onset completed a normal follicular phase before returning to baseline and Cat remained suppressed until treatment withdrawal. All cats receiving oral progestin re-initiated follicular Progestin Altrenogest activity after treatment, although MID cats experienced the most synchronized return (within 10– Ovarian suppression 16 days). Mean baseline fecal estrogens and progestagens were higher (P < 0.05) after treatment in HIGH, Fecal hormone monitoring but not in LOW or MID cats compared to pre-treatment values. The results demonstrate that: (1) oral pro- Estrous cycle gestin rapidly suppresses initiation of follicular activity in the cat, but does not influence a follicular phase that exists before treatment initiation; and (2) queens return to normal follicular activity after pro- gestin withdrawal. This study provides foundational information for research aimed at using progestin priming to improve ovarian response in felids scheduled for ovulation induction and assisted breeding. Ó 2010 Elsevier Inc. All rights reserved. 1. Introduction attempts have failed in the fishing cat (Bauer et al., 2004) and Pallas’ cat (Brown et al., 2002). Endangered felids have benefited from assisted reproduction Although a clear etiology for pregnancy failure after AI has not strategies designed to maintain or increase genetic diversity in been established, one contributor appears to be inconsistent ovar- small populations managed ex situ (Wildt and Roth, 1997; Wildt ian response after exogenous gonadotropins are used to stimulate et al., 2010). Approaches such as artificial insemination (AI) and the ovary before insemination (Howard and Wildt, 2009). Most fe- in vitro fertilization/embryo transfer can improve reproductive effi- lid ovulation induction protocols involve administering a species- ciency in rare cat species when natural breeding fails (Howard, specific dosage of equine chorionic gonadotropin (eCG) to initiate 1999; Swanson, 2003; Howard and Wildt, 2009). A laparoscopic follicular growth followed by human chorionic gonadotropin intrauterine AI technique has been successful, producing offspring (hCG) to complete meiotic maturation of the oocyte(s) and ovula- in the cheetah (Howard et al., 1992, 1997), clouded leopard tion (Howard, 1999). However, eCG can stimulate ovulation if gi- (Howard et al., 1996, 1997), tiger (Donoghue et al., 1993), puma ven during estrus, and hCG has folliculogenic properties in the (Barone et al., 1994), leopard cat (Wildt et al., 1992), snow leopard cat (Swanson et al., 1997). Thus, when eCG and hCG are adminis- (Roth et al., 1997a), tigrina (Swanson and Brown, 2004) and ocelot tered in tandem at random intervals in the estrous cycle, a hy- (Swanson et al., 1996b). However, pregnancy success is variable per-follicular state can occur (Swanson et al., 1996a), resulting in across species. AI efficiency is 45% in the cheetah (Howard abnormally high and protracted circulating estrogen profiles, re- et al., 1997), but success remains low (<5%) in the clouded leopard duced embryo quality and delayed embryo transport through the (Howard et al., 1997) and tiger (Graham et al., 2006), and multiple oviduct (Roth et al., 1997b; Graham et al., 2000). Felids are also notorious for exhibiting unusually high variation in ovulation ‘type’. Traditionally considered induced ovulators (Wildt et al., 1980), more recent investigations have confirmed spontaneous * Corresponding author. Fax: +1 540 635 6506. ovulation in laboratory-housed domestic cats (Lawler et al., E-mail address: [email protected] (D.E. Wildt). 1993; Gudermuth et al., 1997; Graham et al., 2000; Pelican et al., 1 Present address: Center for the Integrative Study of Animal Behavior, Indiana 2005) and certain non-domestic species, including the fishing cat, University, Bloomington, IN, USA. 2 Present address: Department of Veterinary Population Medicine, College of margay and clouded leopard (Brown et al., 2001; Moreira et al., Veterinary Medicine, University of Minnesota, St. Paul, MN, USA. 2001; Brown, 2006). Spontaneous ovulation further complicates 0016-6480/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.ygcen.2009.12.016 Author's personal copy 410 R.A. Stewart et al. / General and Comparative Endocrinology 166 (2010) 409–416 artificial control of the ovary because corpora lutea presence and in a domestic cat (Brown et al., 1994; Pelican et al., 2005). We parallel elevations in circulating progesterone reduce (or prevent) presumed that suppression for this interval would ensure lysis of the effective use of exogenous gonadotropins (Pelican et al., existing corpora lutea (if present) and promote sufficient ovarian 2006, 2008). quiescence by the end of treatment. Each cat received two different For assisted reproduction to be maximally efficient in felids, a dosages (in Trial 1 versus Trial 2) separated by an 8-month inter- ‘quiescent’ ovary at the time of ovulation induction is required. val. Resumption of normal follicular activity after Trial 1 was con- This environment increases the chance for a consistent and uni- firmed by fecal hormone analyses before beginning Trial 2. Cats form folliculogenic response to ovarian stimulation while greatly were randomly assigned to the two treatments in a balanced order diminishing the risk of an altered endocrine milieu. This concept to avoid ‘carry-over’ effects between the consecutive trials. appears especially valid in felids as illustrated by the cheetah, a Altrenogest oral suspension (Regu-MateÒ; 2.2 mg/ml; Intervet consistent induced ovulator with frequent periods of acyclicity Inc., Millsboro, DE) was stored at room temperature (21 °C) in (Brown et al., 1996). Both these traits no doubt have contributed an opaque container. During each trial, the progestin was mixed to ‘normal’ ovulation numbers post-eCG/hCG and a comparatively into 5 g of wet food (FriskiesÒ; Nestlé Purina PetCare Co.) and high incidence of AI success in the cheetah (Howard et al., 1992, administered daily for 38 consecutive days. In Trial 1, each cat 1997) as well as in another induced ovulator, the ocelot (Swanson was assigned to one of four ALT treatments: (1) 0 mg/kg (control; et al., 1996b). In non-felids that spontaneously ovulate, including wet food only; n = 3 cats); (2) 0.088 mg/kg (n = 3); (3) 0.176 mg/ domestic livestock (Lofstedt, 1988; Wood et al., 1992; Deligiannis kg (n = 3); or (4) 0.352 mg/kg (n = 4). Based on Trial 1 results, the et al., 2005) and wild ungulates (Monfort et al., 1993; Morrow 0.176 mg/kg treatment was removed from the experimental design et al., 2000), progestins are routinely administered prior to assisted and replaced with a new test dosage (0.044 mg/kg) to better refine breeding to temporarily suppress follicular activity and improve the lowest effective dosage. In Trial 2, each cat was assigned to one ovarian response. Although the progestins melengestrol acetate of four treatments: (1) 0 mg/kg (n = 2); (2) 0.044 mg/kg (n = 5); (3) (MGA), megestrol acetate and medroxyprogesterone acetate are 0.088 mg/kg (n = 3); or (4) 0.352 mg/kg (n = 2). well-known contraceptive agents in felids (Romatowski, 1989; Munson, 2006), their ability to temporarily suppress ovarian activ- 2.3. Fecal hormone extraction ity for the purpose of assisted reproduction has not been investi- gated. Our laboratory confirmed the efficacy of the progestin Over the course of the study, fecal samples were collected daily, levonorgestrel (NorplantÒ) delivered as an implant for short-term sealed in plastic bags labeled with the individual’s name/date and suppression of ovarian activity in the cat (Pelican et al., 2005). stored at À20 °C. Fecal aliquots from 60 days before, 38 days dur- However, this approach requires anesthesia to insert and remove ing and 60 days after each treatment period were extracted to the implants and, more importantly, results in variable returns to quantify estrogen and progestagen concentrations using assays ovarian cyclicity after implant removal (ranging from 2 weeks validated for the domestic cat (Brown et al., 1994).