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: Electroejaculates from eight snow leopards were used to determine how the motility of spermatozoa was influenced by (i) type of media (Ham's F10, PBS, human tubal fluid or RPMI-1640); (ii) holding temperature (23 degrees C versus 37 degrees C); (iii) washing of spermatozoa and (iv) a sperm metabolic enhancer, pentoxifylline. The duration of sperm motility was assessed by evaluating samples in each treatment every hour for 6 h and a sperm motility index (a value combining percentage sperm motility and rate of forward progression) calculated. Spermatozoa from the Ham's F10, PBS and PBS plus pentoxifylline treatments were also co-incubated with zona-intact, domestic cat eggs that were fixed and evaluated for spermatozoa bound to the zona pellucida, penetrating the outer and inner layers of the zona pellucida and within the perivitelline space. During the 6 h co-incubation, the sperm motility index in PBS with pentoxifylline was greater (P < 0.05) than in PBS alone which, in turn, was greater (P < 0.05) than in the other three test media. Washing the spermatozoa enhanced (P < 0.05) motility in both PBS and PBS plus pentoxifylline relative to unwashed samples, but there was no effect (P > 0.05) of holding temperature. Pentoxifylline supplementation enhanced (P < 0.05) the proportion of cat eggs with bound, but not penetrated, snow leopard spermatozoa in the inner layer of the zona pellucida, and there were no spermatozoa in the perivitelline space.(ABSTRACT TRUNCATED AT 250 WORDS)

Abstract: Noninvasive fecal assays were used to study steroid metabolism and ovarian activity in several felid species. Using the domestic cat (Felis catus) as model, the excretory products of injected [14C]estradiol (E2) and [14C]progesterone (P4) were determined. Within 2 days, 97.0 +/- 0.6% and 96.7 +/- 0.5% of recovered E2 and P4 radioactivity, respectively, was found in feces. E2 was excreted as unconjugated estradiol and estrone (40%) and as a non-enzyme- hydrolyzable conjugate (60%). P4 was excreted primarily as non-enzyme- hydrolyzable, conjugated metabolites (78%) and as unconjugated pregnenolone epimers. A simple method for extracting fecal steroid metabolites optimized extraction efficiencies of the E2 and P4 excretion products (90.1 +/- 0.8% and 87.2 +/- 1.4%, respectively). Analysis of HPLC fractions of extracted fecal samples from the radiolabel-injected domestic cats revealed that E2 immunoreactivity coincided primarily with the unconjugated metabolized [14C]E2 peak, whereas progestogen immunoreactivity coincided with a single conjugated epimer and multiple unconjugated pregnenolone epimers. After HPLC separation, similar immunoreactive E2 and P4 metabolite profiles were observed in the leopard cat (F. bengalensis), cheetah (Acinonyx jubatus), clouded leopard (Neofelis nebulosa), and snow leopard (Panthera uncia). Longitudinal analyses demonstrated that changes in fecal E2 and P4 metabolite concentrations reflected natural or artificially induced ovarian activity. For example, severalfold increases in E2 excretion were associated with overt estrus or exogenous gonadotropin treatment, and elevated fecal P4 metabolite concentrations occurred during pregnant and nonpregnant (pseudopregnant) luteal phases. Although overall concentrations were similar, the duration of elevated fecal P4 metabolites during pseudopregnancy was approximately half that observed during pregnancy. In summary, steroid metabolism mechanisms appear to be conserved among these physically diverse, taxonomically related species. Results indicate that this hormone-monitoring approach will be extremely useful for elucidating the hormonal regulatory mechanism associated with the reproductive cycle, pregnancy, and parturition of intractable and endangered felid species.

Abstract: The potential for rescuing immature oocytes from the ovaries of females of rare felid species which die or undergo medical ovariohysterectomy was evaluated. Ovaries were recovered from 13 species representing 35 individuals in good-to-poor health. Although the majority of females were 10 yr of age or older and in fair-to-poor health, a total of 846 oocytes were recovered of which 608 (71.9%) were classified as fair-to- excellent quality. One hundred of these oocytes were used for initial maturation classification and as parthogenetic controls. Overall, of the 508 fair-to-excellent quality oocytes placed in culture, 164 (32.3%) matured to metaphase II in vitro. For species in which 3 or more individuals yielded oocytes, mean oocyte maturation rates were as follows: 36.2%, tiger; 27.9% leopard; and 8.3%, cheetah. In vitro insemination of oocytes resulted in fertilization (2 polar bodies, 2 pronuclei, or cleavage) rates of 9.1% to 28.6% (leopard) using homologous fresh spermatozoa and 4.0% (lion) to 40.0% (puma) using homologous frozen-thawed spermatozoa. Inseminations using heterologous (domestic cat) spermatozoa also resulted in fertilized oocytes in the tiger, leopard, snow leopard, puma, serval, and Geoffroy's cat (range in fertilization rate, 5.0% for leopard to 46.2% for puma). Cleaved embryos resulted from the insemination of leopard oocytes with homologous sperm (n = 1 embryo) and puma oocytes with domestic cat sperm (n = 3 embryos). These results demonstrate that immature ovarian oocytes from rare felid species can be stimulated to mature in vitro despite an excision-to-culture interval as long as 36 h.(ABSTRACT TRUNCATED AT 250 WORDS)