Abstract: An extraction and assay procedure to measure fecal estradiol-17-beta and progestin concentrations in several cat species was developed and validated for use for noninvasive monitoring of ovarian function. Fecal samples were collected over a range of 3-20 months from female tigers (three), lions (three), snow leopards (three), cheetahs (two), caracals (two), and domestic cats (five). Samples were extracted with 90% methanol, lipids removed with petroleum ether, and the estradiol and progestins in the methanol measured by radioimmunoassay (RIA). High Performance Liquid Chromatography (HPLC) fractionation and subsequent RIA of the fractions indicated that the estradiol-17-beta antiserum cross-reacted primarily with estradiol-17-beta in the feces of lions and tigers and was assumed to be specific for estradiol-17-beta in the feces of other species as well. However, there were several immunoreactive compounds, presumably progesterone metabolites, excreted in the feces which varied both quantitatively and qualitatively among species. The behavior of tigers, lions, cheetahs, and caracals was visually monitored during the collection period and frequency of sexual behaviors was positively correlated with increases in fecal estradiol in all species observed. The mean fecal estradiol-17-beta peaks were as follows: tigers, 128.0 +- 13.1; lions, 186.0 +- 14.8; snow leopards, 136.7 +- 15.9; cheetahs, 140.9 +- 9.0; caracals, 24.5 +- 4.0; and domestic cats 158.9 +- 19.3 ng/gm. Fecal progestin concentrations rose significantly (P lt 0,001) only after breeding or during pregnancy and were as follows: tigers, 5.6 +- 0.6; lions, 1.9 +- 0.1; cheetahs, 8.4 +- 1.1; and caracals, 2.4 +- 0.4 mu-g/gm. Fecal progestins were elevated for one-half to two-thirds of the gestation length during presumed pseudopregnancy but remained elevated throughout successful pregnancies. These results suggest that ovarian function can be monitored noninvasively in the family Felidae by the measurement of fecal estradiol-17-beta and progestin concentrations.

Abstract: The endangered snow leopard Panthera uncia occurs in rugged, high-altitude regions of Central Asia. However, information on the status of this felid is limited in many areas. We conducted a pilot study to optimize molecular markers for the analysis of snow leopard scat samples and to examine the feasibility of using noninvasive genetic methods for monitoring this felid. We designed snow leopard-specific primers for seven microsatellite loci that amplified shorter segments and avoided flanking sequences shared with repetitive elements. By redesigning primers we maximized genotyping success and minimized genotyping errors. In addition, we tested a Y chromosome-marker for sex identification and designed a panel of mitochondrial DNA primers for examining genetic diversity of snow leopards using scat samples. We collected scats believed to be from snow leopards in three separate geographic regions including north-western India, central China and southern Mongolia. We observed snow leopard scats in all three sites despite only brief 2-day surveys in each area. There was a high rate of species misidentification in the field with up to 54% of snow leopard scats misidentified as red fox. The high rate of field misidentification suggests sign surveys incorporating scat likely overestimate snow leopard abundance. The highest ratio of snow leopard scats was observed in Ladakh (India) and South Gobi (Mongolia), where four and five snow leopards were detected, respectively. Our findings describe a species-specific molecular panel for analysis of snow leopard scats, and highlight the efficacy of noninvasive genetic surveys for monitoring snow leopards. These methods enable large-scale noninvasive studies that will provide information critical for conservation of snow leopards.

Abstract: The purpose of this study was to determine the molecular basis for a relative deficiency in the cat of cytosolic arylamine N- acetyltransferase (NAT), an enzyme family that is important in the metabolism of xenobiotics and that normally consists of at least two related enzymes, NAT1 and NAT2. N-acetyltransferase in feline liver showed high affinity (mean Km = 2.1 microM) for p-aminobenzoic acid, an NAT1 selective substrate in humans and rabbits, but showed a very poor affinity (mean Km > 10 mM) for sulfamethazine, an NAT2 selective substrate in humans and rabbits. Immunoreactive N-acetyltransferase was detected in feline liver, bladder and colon using an NAT1-specific antipeptide antibody, but was not detected in any tissues using an NAT2- specific antibody. Southern blot analysis of genomic DNA demonstrated a single band in domestic cats using each of six restriction digests; single bands were also found on Southern blot analysis of six wild felids. The deduced amino acid sequence of the central portion of feline N-acetyltransferase, obtained by polymerase chain reaction amplification in both domestic cats and seven wild felids (lion, tiger, lynx, snow leopard, bobcat, Asian leopard cat and cheetah), contained three residues, Phe125, Arg127, and Tyr129, which determine NAT1-like substrate specificity in humans. These results support the conclusion that cytosolic arylamine N-acetylation activity is low in the cat because of the presence of a single N-acetyltransferase that has substrate specificity, immunogenicity and sequence characteristics similar to human NAT1, and that the unusual presence of only a single N- acetyltransferase gene appears to be a family wide trait shared by other felids.

Abstract: Most conventional spermatology research involves common mammalian species including livestock, laboratory animals and humans. Yet, there are more than 4500 mammalian species inhabiting the planet for which little is known about basic reproductive biology, including sperm characteristics and function. This information is important, not just as adjunct knowledge, but because the majority of these species are threatened with extinction, largely due to human-induced pressures. The field of conservation is changing rapidly, and global cooperation is emerging among a variety of wildlife enthusiasts, ranging from management authorities of nature reserves to curators of rare zoological collections. Conservation progress depends on systematic, multidisciplinary research first to answer basic questions, with new data then applied to endangered species management plans. The reproductive physiologist is a crucial component of this scheme. Reproduction is the essence of species survival, and enormous effort needs to be directed at these 'untraditional' research species, subspecies and populations. Spermatology research combined with simultaneous efforts in endocrinology, embryology and cryopreservation (among others) can lead to the successful application of assisted reproduction. Examples from this laboratory include an array of wild felid species and a rare cervid and mustelid. Obstacles to success are formidable, including unique species-specificities, diminished genetic diversity and a general lack of resources. Nonetheless, the field offers tremendous opportunities for generating unique knowledge of comparative interest and with conservation utility.