Natural Versus Chorionic Gonadotropin-Induced Ovarian Responses in the Clouded Leopard (Neofelis Nebulosa) Assessed by Fecal Steroid Analysis1

Abstract

The clouded leopard (Neofelis nebulosa) is an endangered species difficult to propagate in captivity because of sexual incompatibility between paired individuals. Fecal estradiol (E2) and progesterone (P4) metabolites were quantified in 14 females to noninvasively monitor seasonal reproductive patterns and compare ovarian responses to natural mating vs. exogenous gonadotropins. Increased E2 excretion was associated with behavioral estrus or eCG treatment, whereas elevated P4 metabolites were observed during natural and hCG-induced pregnant and nonpregnant luteal phases. On the basis of fecal E2 profiles, duration of the estrous cycle was 24 +/- 2 days, with estrus lasting 6 +/- 1 days. Mean gestation length was 89 +/- 2 days, whereas duration of the nonpregnant luteal phase was 47 +/- 2 days. Females experienced a seasonal anestrus during the late summer and early fall. One female demonstrated a lactational anestrus after the birth of three cubs. On nine occasions, clouded leopards ovulated spontaneously (based on elevated fecal P4 metabolite concentrations) in the absence of mating. Patterns of eCG-stimulated E2 excretion were similar to those associated with natural estrus and were unaffected by eCG dosage (25, 50, or 100 IU). In contrast, post-hCG P4 metabolite profiles varied considerably, with responses including anovulation, attenuated luteal P4 metabolite production, and hyperstimulated luteal function. In some females, compromised luteal function after ovulation induction appeared to be due to the presence of mature CL from previous spontaneous (without copulation) ovulations at the time of gonadotropin treatment. Duration of post-hCG P4 metabolite excretion was reduced approximately 40% in these individuals compared to females with no evidence of active luteal activity. In sum, these are the first data describing the ovarian cycle of this endangered species. The information is important because it is based on the longitudinal assessment of multiple females using a completely atraumatic approach, thereby eliminating the potential confounding impact of stress. Data indicate that spontaneous ovulations and the presence of active luteal tissue on the ovary can profoundly affect ovarian responses to exogenous gonadotropin therapy. Therefore, fecal steroid monitoring can serve as a useful adjunct to developing assisted reproduction techniques, especially the hormonal induction of ovulation for planned artificial insemination.

Reproductive biotechnologies for endangered mammalian species

“…Fewer than 100 mammalian species have been studied among monitoring play an important role in optimizing the success of breeding programs [61]. Fecal steroid metabolite analysis have been used to estimate the pregnancy rate of free ranging herds [63], or to assess the reproductive status of males and females for various species such as the sika deer, wild black rhinoceros (Diceros bicornis minor) and clouded leopard (Neofelis nebulosa) [8,22,75]. The ovarian cycle may also be characterized by steroid and peptide analysis in the urine [55].…”

Approaches and efficacy of artificial insemination in felids and mustelids

“…Interestingly, the cheetah [23], tiger (Panthera tigris) [17,24], tigrina (Leopardus tigrinus) [25] and ocelot (Leopardus pardalis) [25] appear to be exclusively induced ovulators, similar to the puma (Puma concolor) [26], Siberian tiger (Panthera tigris altaica) [27] and snow leopard (Uncia uncia) [28] assessed in earlier studies using blood analyses. In contrast, spontaneous ovulation during natural estrous cycles has been reported in the clouded leopard [29], lion (Panthera leo) [30], leopard (Panthera pardus) [31]; margay (Leopardus wiedii) [25], Pallas' cat [20] and fishing cat (Prionailurus viverrinus) [32,33].…”

“…Therefore, we have speculated that AI efficiency in cats could be enhanced (especially in those species that tend to spontaneously ovulate) by artificially creating a quiescent ovary prior to gonadotropin administration. This may be especially important for the clouded leopard, where up to 43% of females spontaneously release ova in the absence of mating (based on fecal steroid data) [29]. In the presence of mature, functional CL (Fig.…”

“…6), even the ideal eCG/hCG dose fails to elicit normal follicle development and ovulation (Fig. 7) [29,34].…”

Wildlife conservation and reproductive cloning

“…Where intensive efforts have been made to develop such protocols, these are often far more complex projects than originally envisaged. For example, approximately a decade ago a focused research programme was initiated that aimed at developing AI techniques for snow leopard and clouded leopard (Barone et al 1994, Brown et al 1995, Swanson et al 1996. Breeding leopards by the use of in vitro fertilisation and embryo transfer (IVF-ET), or simply by AI, is of interest because behavioural incompatibilities often preclude the use of natural breeding.…”