Conservation & Research Center, Smithsonian National Zoological Park, Front Royal, VA, USA
Assisted reproductive technologies including artificial insemination (AI), in vitro fertilization (IVF) and embryo transfer have the potential to be important tools in the management of many endangered cats. Developing successful assisted reproductive techniques requires knowledge of the female reproductive cycle and precise control of ovarian activity. Control of ovulation is one key step in coordinating procedures to optimize reproductive success.
Numerous exogenous hormones are available to induce ovarian follicular growth and ovulation in mammals, with several adapted to felids. The most common hormones used to initiate folliculogenesis are porcine follicle stimulating hormone (pFSH) and equine chorionic gonadotropin (eCG). Folliculogenesis has been successfully stimulated with a 3- or 4-day course of daily pFSH injections in the domestic cat,1 caracal,2 jaguarandi,1 tiger,3 cheetah,4 leopard, lion,4 and puma4. Equine chorionic gonadotropin (eCG), which has a longer half-life, is given as a single injection and has been used in the domestic cat,5 leopard cat,6 cheetah,7 clouded leopard,7 tiger,8 puma,9 ocelot,10 and snow leopard11. Once follicles are mature, ovulation can be induced with multiple injections of luteinizing hormone (LH)3,12 or a single injection of human chorionic gonadotropin (hCG).6,13 Less commonly, gonadotropin-releasing hormone (GnRH) or GnRH agonists also can be used to induce ovulation.14,15
Felids exhibit a highly variable response to exogenous gonadotropins used to stimulate follicular growth and ovulation. This has made development of assisted reproduction difficult. Over the past 15 years, research has identified species differences in ovulation strategies and dose-responses to gonadotropic hormones that have contributed to the variability in response to ovarian stimulation. Cats are generally considered induced or reflex ovulators and would be expected to ovulate only following cervico-vaginal stimulation during intromission. However, several species have proven to be susceptible to occasional spontaneous ovulation in the absence of mating stimuli. Species prone to spontaneous ovulation episodes include the domestic cat,16 lion,17 clouded leopard,18 margay,19 and fishing cat20. Felids are the only known taxon to exhibit such variability in ovulation mechanisms across individuals. The felids also show great diversity in sensitivity to exogenous gonadotropin administration. For example, the 9-kg ocelot requires more than twice as much eCG and hCG to induce ovulation then does the 35-kg cheetah.7,10 In short, there is no relatedness between species body mass and the dose of eCG/hCG required to artificially induce ovulation.
The variability between species of felids in ovarian control mechanisms is reflected in the success of assisted reproductive technologies. In some species, such as the cheetah where ovulation induction after eCG/hCG is fairly consistent, more than 50% of females become pregnant after AI. In contrast, those species with the most variable responses to exogenous gonadotropins experience AI success rates of less than 10%.7,12
This variation occurs not only among species, but among individuals within a species. Species prone to spontaneous ovulation produce intermittent corpora lutea (CL) that blunt ovarian responses to eCG/hCG therapy that, in turn, lead to reduced AI/IVF success.21,22 A variable female response also occurs because the exogenous gonadotropins can perturb the endocrine environment, which disrupts oocyte maturation, fertilization, embryo development and implantation. Perturbations in ovarian stimulation resulting in decreased fertility in felids include ovarian hyper-stimulation episodes that cause elevated or protracted estrogen concentrations, premature or hyper-progesterone secretion and luteal insufficiency/premature luteolysis.3,23-26
Recent studies have investigated the use of ovarian inhibition prior to ovulation induction to create a more predictable response. The ability to suppress cyclic activity before gonadotropic therapy has improved assisted reproduction success in other species.27-30 A suppressed ovary is more likely to give a uniform response to exogenous gonadotropins. To this purpose, the progestin levonorgestrel (Norplant) recently was compared to the GnRH antagonist, antide, as a pretreatment to gonadotropin stimulation in the domestic cat. Pretreatment with levonorgestrel, but not antide, prior to gonadotropin stimulation more than doubled the embryo yield after IVF31,32 and resulted in a uniform excellent response for AI33 compared to gonadotropin stimulation alone.
Although pretreatment with levonorgestrel implants before gonadotropin stimulation has improved ovarian response to eCG/hCG therapy in the domestic cat, results in other species have been species specific. Preliminary trials have been conducted in the fishing cat, cheetah and clouded leopard. Results in the fishing cat and cheetah are promising. Two fishing cats given Norplant followed by eCG and hCG stimulation exhibited fresh ovulation sites with minimal numbers of unovulated follicles, a perfect response for AI (Bauer, unpublished data). Similarly, three of three cheetahs pretreated with Norplant ovulated after gonadotropin stimulation, and only one had significant numbers of unovulated follicles (Howard, unpublished data). Unlike in the domestic cat, fishing cat and cheetah, however, pretreatment with Norplant in the clouded leopard appeared to interfere with ovarian response to gonadotropin stimulation. Only three of eight (37.5%) clouded leopards given Norplant before eCG/hCG ovulated, and all females had multiple unovulated ovarian follicles (Pelican, unpublished data). Further research will be required to elucidate the species differences in ovarian response to similar gonadotropin protocols.
Exogenous gonadotropins also may be useful in treating pathologic conditions, including persistent estrus or secondary anestrus associated with ovarian follicular cysts. In any case of abnormal estrous cycling, a thorough history and physical examination should rule out nonreproductive causes of dysfunction including neoplasia, nutrition or stress-related alterations in reproductive activity. In cases of persistent estrus, a thorough ultrasound examination should be undertaken to identify the source of elevated estrogens (follicular cysts versus neoplasia) and to evaluate the condition of the uterus. Fecal hormone analysis also can be used to verify anestrus or hyper-estrogenism.
Follicular cysts have been documented in the domestic cat,34,35 but the prevalence of this condition in nondomestic felid species is unknown. Even in the domestic cat, the etiology of this condition is unclear, and the recommended treatment is ovariectomy. Clearly, this is not a desirable option in most rare wild felid species. In other species, a variety of hormonal treatments have been used with variable success. Ovulation induction hormones including GnRH, LH and hCG can be administered to artificially rupture the anovulatory follicles.36,37 However, the dysfunctional follicles are often insensitive to ovulatory stimuli.38,39 Recent research indicates that follicular cysts in cattle develop following an inadequate luteal phase with low levels of progesterone.40 Exposure to high progesterone concentrations successfully re-sets the hypothalamo-pituitary-ovarian axis and returns the animal to normal estrous cycling.40 In cats, ovulation induction and progesterone treatment must be undertaken with caution because prolonged estrogen exposure followed by elevations in progesterone due to treatment or ovulation could result in cystic endometrial hyperplasia and pyometra. Again, more research is required to determine the most successful treatments for ovarian follicular cysts in felids.
In conclusion, ovulation induction is important for timing assisted reproductive procedures appropriately and for treating anovulatory conditions secondary to ovarian follicular cysts. Significant progress has been made toward developing a consistent ovulation protocol for felids. Species differences are significant, and it is relatively easy to cause ovarian hyperstimulation and altered endocrine profiles that, in turn, can compromise reproduction. Fundamental studies directed at each species, however, generally have been effective at elucidating efficient ovulation induction protocols. Furthermore, recent investigations into using progestogen pretreatment to improve ovarian response to exogenous gonadotropins have yielded promising results.
The authors sincerely thank all of the domestic cat, fishing cat, cheetah and clouded leopard keepers and staff who contributed time and effort to this research. We also thank the Fishing Cat, Cheetah and Clouded Leopard Species Survival Plan Management Committees for their cooperation.
1. Pope, C.E., C.A. Johnson, M.A. McRae, G.L. Keller and B.L. Dresser, 1998. Development of embryos produced by intracytoplasmic sperm injection of cat oocytes. Anim. Reprod. Sci. 53: 221–236.
2. Pope, C.E., M.C. Gomez, A.M. Davis, R.F. Harris, S.K. Mikota, E.H. Boyd and B.L. Dresser, 2001. Oocyte retrieval, in vitro fertilization and embryo transfer in the caracal (Caracal caracal). Theriogenology 55: 397.
3. Crichton, E.G., E. Bedows, A.K. Miller-Lindholm, D.M. Baldwin, D.L. Armstrong, L.H. Graham, J.J. Ford, J.O. Gjorret, P. Hyttel, C.E. Pope, G. Vajta and N.M. Loskutoff, 2003. Efficacy of porcine gonadotropins for repeated stimulation of ovarian activity for oocyte retrieval and in vitro embryo production and cryopreservation in Siberian tigers (Panthera tigris altaica). Biol. Reprod. 68: 105–113.
4. Phillips, L.G., L.G. Simmons, M. Bush, J.G. Howard and D.E. Wildt, 1982. Gonadotropin regimen for inducing ovarian activity in captive wild felids. JAVMA 181: 1246–1250.
5. Howard, J.G., M.A. Barone, A.M. Donoghue and D.E. Wildt, 1992. The effect of preovulatory anaesthesia on ovulation in laparoscopically inseminated domestic cats. J. Reprod. Fertil. 96: 175–186.
6. Howard, J.G., 1999. Assisted reproductive techniques in nondomestic carnivores. In: Zoo and Wild Animal Medicine: Current Therapy IV. Fowler, M.E., Miller, R.E., eds. Philadelphia: W. B. Saunders Co., 449–457.
7. Howard, J.G., T.L. Roth, A.P. Byers, W.F. Swanson and D.E. Wildt, 1997. Sensitivity to exogenous gonadotropins for ovulation induction and laparoscopic artificial insemination in the cheetah and clouded leopard. Biol. Reprod. 56: 1059–1068.
8. Donoghue, A.M., L.A. Johnston, D.L. Armstrong, L.G. Simmons and D.E. Wildt, 1993. Birth of a Siberian tiger cub (Panthera tigris altaica) following laparoscopic intrauterine insemination. J. Zoo Wildl. Med. 24: 185–189.
9. Barone, M.A., D.E. Wildt, A.P. Byers, M.E. Roelke, C.M. Glass and J.G. Howard, 1994. Gonadotrophin dose and timing of anesthesia for laparoscopic artificial insemination in the puma (Felis concolor). J. Reprod. Fertil. 101: 103–108.
10. Swanson, W.F., J.G. Howard, T.L. Roth, J.L. Brown, T. Alvarado, M. Burton, D. Starnes and D.E. Wildt, 1996. Responsiveness of ovaries to exogenous gonadotrophins and laparoscopic artificial insemination with frozen-thawed spermatozoa in ocelots (Felis pardalis). J. Reprod. Fertil. 106: 87–94.
11. Roth, T.L., D.L. Armstrong, M.T. Barrie and D.E. Wildt, 1997. Seasonal effects on ovarian responsiveness to exogenous gonadotrophins and successful artificial insemination in the snow leopard (Uncia uncia). Reprod. Fertil. Devel. 9: 285–295.
12. Pope, C.E., 2000. Embryo technology in conservation efforts for endangered felids. Theriogenology 53: 163–74.
13. Donoghue, A.M., L.A. Johnston, L. Munson, J.L. Brown and D.E. Wildt, 1992. Influence of gonadotropin treatment interval on follicular maturation, in vitro fertilization, circulating steroid concentrations, and subsequent luteal function in the domestic cat. Biol. Reprod. 46: 972–980.
14. Wildt, D.E., P.K. Chakraborty, D. Meltzer and M. Bush, 1984. Pituitary and gonadal response to LH releasing hormone administration in the female and male cheetah. J. Endocrinol. 101: 51–56.
15. Goodrowe, K.L. and D.E. Wildt, 1987. Ovarian response to human chorionic gonadotropin or gonadotropin releasing hormone in cats in natural or induced estrus. Theriogenology 27: 811–817.
16. Gudermuth, D.F., L. Newton, P. Daels and P. Concannon, 1997. Incidence of spontaneous ovulation in young, group-housed cats based on serum and faecal concentrations of progesterone. J. Reprod. Fertil. Suppl. 51: 177–184.
17. Schramm, R.D., M.B. Briggs and J.J. Reeves, 1994. Spontaneous and induced ovulation in the lion (Panthera leo). Zoo Biol.: 301–307.
18. Brown, J.L., D.E. Wildt, L.H. Graham, A.P. Byers, L. Collins, S. Barrett and J.G. Howard, 1995. Natural versus chorionic gonadotropin-induced ovarian responses in the clouded leopard (Neofelis nebulosa) assessed by fecal steroid analysis. Biol. Reprod. 53: 93–102.
19. Moreira, N., E.L. Monteiro-Filho, W. Moraes, W.F. Swanson, L.H. Graham, O.L. Pasquali, M.L. Gomes, R.N. Morais, D.E. Wildt and J.L. Brown, 2001. Reproductive steroid hormones and ovarian activity in felids of the Leopardus genus. Zoo Biol. 20: 103–116.
20. Moreland, R., J. Brown, D.E. Wildt and J.G. Howard, 2002. Basic reproductive biology of the fishing cat (Prionailurus viverrinus). Biol. Reprod. Suppl. in press.
21. Swanson, W.F., T.L. Roth, J.L. Brown and D.E. Wildt, 1995. Relationship of circulating steroid hormones, luteal luteinizing hormone receptor and progesterone concentration, and embryonic mortality during early embryogenesis in the domestic cat. Biol. Reprod. 53: 1022–1029.
22. Brown, J.L. and D.E. Wildt, 1997. Assessing reproductive status in wild felids by non-invasive faecal steroid monitoring. Int. Zoo Yb. 35: 173–191.
23. Goodrowe, K.L., J.G. Howard and D.E. Wildt, 1988. Comparison of embryo recovery, embryo quality, oestradioll 7β and progesterone profiles in domestic cats (Felis catus) at natural or induced oestrus. J. Reprod. Fertil. 82: 553–561.
24. Graham, L.H., W.F. Swanson and J.L. Brown, 2000. Chorionic gonadotropin administration in domestic cats causes an abnormal endocrine environment that disrupts oviductal embryo transport. Theriogenology 54: 1117–1131.
25. Roth, T.L., B.A. Wolfe, J.A. Long, J.G. Howard and D.E. Wildt, 1997. Effects of equine chorionic gonadotropin, human chorionic gonadotropin, and laparoscopic artificial insemination on embryo, endocrine, and luteal characteristics in the domestic cat. Biol. Reprod. 57: 165–171.
26. Swanson, W.F., B.A. Wolfe, J.L. Brown, T. Martin-Jimenez, J.E. Riviere, T.L. Roth and D.E. Wildt, 1997. Pharmacokinetics and ovarian-stimulatory effects of equine and human chorionic gonadotropins administered singly and in combination in the domestic cat. Biol. Reprod. 57: 295–302.
27. Hattab, S.A., A.K. Kadoom, R. Palme and E. Bamberg, 2000. Effect of Crestar on estrus synchronization and the relationship between fecal and plasma concentrations of progestagens in buffalo cows. Theriogenology 54: 1007–1017.
28. Morrow, C.J., B.A. Wolfe, T.L. Roth, D.E. Wildt, M. Bush, E.S. Blumer, M.W. Atkinson and S.L. Monfort, 2000. Comparing ovulation synchronization protocols for artificial insemination in the scimitar-homed oryx (Oryx dammah). Anim. Reprod. Sci. 59: 71–86.
29. Albano, C., J. Smitz, H. Toumaye, H. Riethmuller-Winzin, A. Van Steirteghem and P. Devroey, 1999. Luteal phase and clinical outcome after human menopausal gonadotrophin/gondotrophin releasing hormone antagonist treatment for ovarian stimulation in in-vitro fertilization/intracytoplasmic sperm injection cycles. Hum. Reprod. 14: 1426–1430.
30. Thompson, K.V. and S.L. Monfort, 1999. Synchronization of oestrous cycles in sable antelope. Anim. Reprod. Sci. 57: 185–197.
31. Pelican, K.M., R.E. Spindler, D.E. Wildt, M.A. Ottinger and J.G. Howard, 2001. Short term ovarian suppression with levonorgestrel before gonadotropin stimulation enhances IVF embryo production in the domestic cat. Biol. Reprod. Suppl. 64: 52.
32. Pelican, K.M., D.E. Wildt, M.A. Ottinger and J.G. Howard, 2002. Short term ovarian suppression with levonorgestrel before gonadotropin stimulation improves ovarian response for IVF in the domestic cat. Theriogenology Supplement 57: 679.
33. Pelican, K.M., 2002. The Progestin Levonorgestrel is Superior to the GnRH Antagonist Antide for Ovarian Suppression Prior to Gonadotropin Stimulation in the Domestic Cat. Department of Animal and Avian Sciences. College Park, Maryland: University of Maryland, 280.
34. Bloom, F., 1954. Pathology in the Dog and Cat. Evanston, IL: American Veterinary Publishers.
35. van der Kolk, F.R., 1985. [A case of cystic ovarian follicles in the cat]. Tijdschr Diergeneeskd 110: 98.
36. Kawate, N., H. Yamada, T. Suga, T. Inaba and J. Mori, 1997. Induction of luteinizing hormone surge by pulsatile administration of gonadotropin-releasing hormone analogue in cows with follicular cysts. J. Vet. Med. Sci. 59: 463–466.
37. Davidson, A.P. and E.C. Feldman, 1995. Ovarian and estrous cycle abnormalities in the bitch. In: Textbook of Veterinary Internal Medicine. Ettinger, S.J., Feldman, E.C., eds. Philadelphia: W.B. Saunders Company, 1607–1613.
38. Brown, J.L., T.B. Hildebrandt, W. Theison and D.L. Neiffer, 1999. Endocrine and ultrasound evaluation of a noncycling African elephant : Identification of an ovarian follicular cyst. Zoo Biol. 18: 223–232.
39. Jou, P., B.C. Buckrell, R.M. Liptrap, A.J. Summerlee and W.H. Johnson, 1999. Evaluation of the effect of GnRH on follicular ovarian cysts in dairy cows using trans-rectal ultrasonography. Theriogenology 52: 923–937.
40. Gumen, A., R. Sartori, F.M. Costa and M.C. Wiltbank, 2002. A GnRH/LH surge without subsequent progesterone exposure can induce development of follicular cysts. J. Dairy Sci. 85: 43–50.