Practical application of laparoscopic oviductal artificial insemination for the propagation of domestic cats and wild felids.

AI was first reported in cats almost 50 years ago but, unlike AI in other domesticated animals (e.g. dogs, cattle, horses), has not been widely used for routine propagation by veterinarians or breeders. Anatomical and physiological challenges with cats have hindered the efficiency of AI using standardised transcervical approaches applied to other species. Development of laparoscopic oviductal AI (LO-AI) has helped overcome some of these barriers and, during the past 7 years, produced high pregnancy percentages (>70%) in domestic cats using both fresh collected and frozen-thawed semen and resulted in the birth of full-term offspring in three cat hereditary disease models and six wild cat species (ocelot, Pallas's cat, fishing cat, sand cat, tiger, clouded leopard). The standard approach involves exogenous gonadotrophin treatment (typically equine chorionic gonadotrophin followed by porcine LH) to induce ovarian follicular growth and ovulation, with laparoscopic visualisation of the oviductal ostium for direct intraluminal insemination with low numbers of spermatozoa. Similar ovarian synchronisation and insemination approaches have been used with wild felids, but frequently must be refined on a species-by-species basis. From a practical perspective, LO-AI in domestic cats now has adequate efficiency for applied use as a reproductive service in veterinary practices that possess basic laparoscopy expertise.

Characterization of the behavior and reproductive endocrinology of giant river otters (Pteronura brasiliensis) in managed care.

Propagation of giant river otters (GRO) in zoos is inconsistent: some pairs never reproduce while others are prolific in producing young but can be hindered by low cub survival. Developing effective breeding programs requires understanding normal reproductive parameters and behavior. Fecal samples were collected for 6-16 months from five breeding pairs, two individual females, and one female pair at seven zoos, and analyzed for fecal progesterone, estrogen, testosterone, and glucocorticoid (FGM) metabolites via enzyme immunoassay. Enclosure characteristics and management routines were recorded at six facilities where behavior was assessed over 1 week. Median fecal progestogens during pregnancy and pseudopregnancy were ∼2.5-3.8× greater than basal concentrations. Gestation lasted 66.5 ± 3.5 days (62-70 days); pseudopregnancies lasted 58 ± 11.6 days (41-69 days). Elevated progestogens indicate ovulation but cannot distinguish pregnancy from pseudopregnancy. Periodically sustained, elevated progestogens observed in two females housed without a male indicated spontaneous ovulation. Elevations in fecal estrogens were not associated with estrus, and seasonality in male testosterone was not observed. Wavering scream and contact call vocalizations among reproductively successful males and females, respectively, suggested the importance of social communication. Most facilities housing successful pairs had larger enclosures with more water than land area, vegetation, and limited public exposure. Baseline FGM were negatively correlated with enclosure size and percentage of water area (p < 0.05), and lower baseline FGM were associated with reproductive success (p < 0.05). These results suggest that housing GRO in spacious enclosures with open water and some insulation from disturbance might promote appropriate behavior, lower FGM, and reproduction.

Assessment of the reproductive physiology of the potto (Perodicticus potto) through fecal hormone metabolite analyses and trans-abdominal ultrasonography.

Potto (Perodicticus potto) reproductive biology has been minimally studied. Noninvasive endocrinology and ultrasonography are proven tools for reproductive assessment in other primates. In this study, we used fecal hormone metabolite analysis to monitor one adult male potto and four females at different life stages. Validated testosterone (T), estrone conjugate (EC), and progesterone (P4) enzyme immunoassays (EIA) were used to assess male testicular function and female ovarian and placental activity. The male excreted mean T concentrations of 4.72 (±1.66) µg/g feces, that did not differ (P > 0.05) over time or when paired with alternate females. Baseline concentrations of EC (range: 47.93-78.81 ng/g feces) and P4 (range: 2.29-12.46 µg/g feces) differed among adult females. Follicular phases averaged 9.1 days (±3.43, n = 30 phases), whereas luteal phases averaged 19.89 days (±9.49, n = 19 phases). Gestation length (n = 2 pregnancies) was 170 days. Gestational EC and P4 concentrations were positively correlated (pregnancy A, r (132) = 0.71; pregnancy B, r (145) = 0.76) and returned to non-pregnant luteal phase levels 3-7 days post parturition. Extreme differences between pregnant and non-pregnant EC and P4 concentrations may allow for one-sample pregnancy diagnosis. Trans-abdominal ultrasonography was validated for pregnancy diagnosis with the fetus observed between 100 and 110 days post breeding. To our knowledge, this is the first use of fecal endocrinology and ultrasonography to monitor reproductive function and pregnancy in this species, and the only study in any lorisid to measure progestagens in correlation with reproductive events.

A chromosome-scale fishing cat reference genome for the evaluation of potential germline risk variants.

The fishing cat, Prionailurus viverrinus, faces a population decline, increasing the importance of maintaining healthy zoo populations. Unfortunately, zoo-managed individuals currently face a high prevalence of transitional cell carcinoma (TCC), a form of bladder cancer. To investigate the genetics of inherited diseases among captive fishing cats, we present a chromosome-scale assembly, generate the pedigree of the zoo-managed population, reaffirm the close genetic relationship with the Asian leopard cat (Prionailurus bengalensis), and identify 7.4 million single nucleotide variants (SNVs) and 23,432 structural variants (SVs) from whole genome sequencing (WGS) data of healthy and TCC cats. Only BRCA2 was found to have a high recurrent number of missense mutations in fishing cats diagnosed with TCC when compared to inherited human cancer risk variants. These new fishing cat genomic resources will aid conservation efforts to improve their genetic fitness and enhance the comparative study of feline genomes.

Laparoscopic oviductal artificial insemination improves pregnancy success in exogenous gonadotropin-treated domestic cats as a model for endangered felids.

Artificial insemination (AI) in cats traditionally uses equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG) to induce follicular development and ovulation, with subsequent bilateral laparoscopic intrauterine insemination. However, long-acting hCG generates undesirable secondary ovulations in cats. Uterine AI also requires relatively high numbers of spermatozoa for fertilization (~8 × 10(6) sperm), and unfortunately, sperm recovery from felids is frequently poor. Using short-acting porcine luteinizing hormone (pLH) instead of hCG, and using the oviduct as the site of sperm deposition, could improve fertilization success while requiring fewer spermatozoa. Our objectives were to compare pregnancy and fertilization success between 1) uterine and oviductal inseminations and 2) eCG/hCG and eCG/pLH regimens in domestic cats. Sixteen females received either eCG (100 IU)/hCG (75 IU) or eCG (100 IU)/pLH (1000 IU). All females ovulated and were inseminated in one uterine horn and the contralateral oviduct using fresh semen (1 × 10(6) motile sperm/site) from a different male for each site. Pregnant females (11/16; 69%) were spayed approximately 20 days post-AI, and fetal paternity was genetically determined. The number of corpora lutea (CL) at AI was similar between hormone regimens, but hCG increased the number of CL at 20 days post-AI. Numbers of pregnancies and normal fetuses were similar between regimens. Implantation abnormalities were observed in the hCG group only. Finally, oviductal AI produced more fetuses than uterine AI. In summary, laparoscopic oviductal AI with low sperm numbers in eCG/hCG- or eCG/pLH-treated females resulted in high pregnancy and fertilization percentages in domestic cats. Our subsequent successes with oviductal AI in eCG/pLH-treated nondomestic felids to produce healthy offspring supports cross-species applicability.

Assessment of adrenocortical and gonadal hormones in male spider monkeys (Ateles geoffroyi) following capture, restraint and anesthesia.

The spider monkey (SM) (Ateles geoffroyi) a New World primate species native to Mexican forests, has become endangered in the wild due to environmental perturbations. Little is known about adrenal function and its relationship to reproduction in this species. Our objectives were to assess serum glucocorticoid (GC), mineralocorticoid (MC) and testosterone concentrations in captive SM and evaluate adrenal and testicular responses to potentially stressful animal handling procedures. Seven adult males, housed in a single mixed gender group in an off-exhibit enclosure at the University Park were captured for anesthesia every 2 months over a 1-year period. Blood samples were collected from each male at three time points: (1) ∼5-10 min after ketamine injection in the outdoor enclosure; (2) ∼2 hr later following animal transport to the laboratory and immediately after tiletamine-zolazepam injection; and (3) ∼20-30 min following the second anesthetic injection. Serum samples were frozen and later analyzed for cortisol, corticosterone, aldosterone and testosterone via radioimmunoassay. Cortisol was the primary GC detected in SM serum with much higher mean concentrations than for corticosterone. Capture, restraint and anesthesia resulted in significant increases in both cortisol and corticosterone concentrations. Whereas aldosterone concentrations were unchanged by animal handling procedures, testosterone concentrations significantly declined under anesthesia over time. In summary, these results provide data for the main adrenocortical hormones in male SM and characterize their acute adrenal responses to potentially stressful handling and anesthesia procedures. Our findings also suggest an interaction between acute increases in corticosteroids and decreased concentrations of serum testosterone.

The challenge of assisted reproduction for conservation of wild felids - A reality check.

Threats to the Earth's biodiversity are increasing exponentially, driven by human population growth and resource consumption. As many as one million wildlife species may disappear within the next few decades due to this human-induced extinction event. This represents our current reality and has profound implications for wildlife conservation. Within this context, application of assisted reproductive technology (ART) to conservation management is unlikely to mitigate broad-scale species loss, but for select species, such as wild cats, ART may determine if populations survive or disappear. In North American and European zoos, 20 of the world's 38 wild felid species are managed within structured breeding programs, but most are not sustainable with natural breeding alone. Zoo-based breeding programs are facing tenuous futures due to triage-based responses to this growing sustainability crisis. Theoretically, ART could benefit conservation management, but only by recognizing and addressing its present challenges. The application of ART to wildlife has been rarely successful, with only 62 mammal species (including 15 cat species) ever propagated by AI, and just 35 of these species (6 cats) reproduced following frozen semen AI. Even this most basic form of ART has a minimal impact on wildlife sustainability. The drivers of this deficit include lack of species-specific reproductive knowledge and limited access to animals for study, but also is exacerbated by a science-conservation disconnect that attempts to apply advanced reproductive technologies to species in which basic ART remains unproven. For a few felid species, these scientific challenges have been overcome and AI with frozen semen is becoming feasible as a practical management tool; for other felids, further research is needed. Non-scientific issues also impair our ability to use ART to implement global management plans. Political dysfunction, regulatory barriers and societal indifference create inertia that interferes with achieving meaningful progress in applying ART to wildlife. Collectively, these challenges may seem insurmountable but human resiliency is essential if we are to resolve these issues in a systematic manner. It will require expanding collaborative efforts substantially and intensifying efforts to conserve wildlife species that are literally running out of time. Our goal is to create a new reality that includes a sustainable future for wild felids and other imperiled wildlife species.

Durable contraception in the female domestic cat using viral-vectored delivery of a feline anti-Müllerian hormone transgene.

Eighty percent of the estimated 600 million domestic cats in the world are free-roaming. These cats typically experience suboptimal welfare and inflict high levels of predation on wildlife. Additionally, euthanasia of healthy animals in overpopulated shelters raises ethical considerations. While surgical sterilization is the mainstay of pet population control, there is a need for efficient, safe, and cost-effective permanent contraception alternatives. Herein, we report evidence that a single intramuscular treatment with an adeno-associated viral vector delivering an anti-Müllerian hormone transgene produces long-term contraception in the domestic cat. Treated females are followed for over two years, during which transgene expression, anti-transgene antibodies, and reproductive hormones are monitored. Mating behavior and reproductive success are measured during two mating studies. Here we show that ectopic expression of anti-Müllerian hormone does not impair sex steroids nor estrous cycling, but prevents breeding-induced ovulation, resulting in safe and durable contraception in the female domestic cat.

Cross-species efficacy of a chemically-defined, soy lecithin-based cryomedium for semen banking in imperiled wild felids.

Felid semen has historically been frozen using an egg yolk-based cryopreservation medium. However, the use of egg introduces several potential concerns, such as variability in composition, microbial contamination, and regulatory issues. In the present study, our aim was to compare a chemically-defined, soy-based medium (SOY) to a commercial egg yolk-based medium (TEY) for the cryopreservation of sperm in four imperiled small cat species. Semen was collected from adult male cats (n = 6 black-footed cats; n = 6 sand cats; n = 4 fishing cats; and n = 7 Pallas' cats) via electroejaculation, split into two aliquots, and cryopreserved in SOY or TEY. Frozen-thawed samples were evaluated for sperm motility and rate of progressive motility (up to 24 h post-thaw) and acrosome status (0 and 6 h). No difference in post-thaw traits were observed between treatments in all four species. Heterologous IVF using oocytes collected laparoscopically from domestic cats demonstrated no difference among freezing treatments in percentage of mature oocytes that cleaved or the mean number of blastomeres at 48 h post-insemination. More spermatozoa frozen with SOY were bound to the zona pellucida in the sand cat (P = 0.018), but no treatment effect was observed in the other three species. These findings collectively demonstrate that SOY may be a preferable alternative to TEY for sperm cryopreservation in these four wild felid species.

Analysis of cat oocyte activation methods for the generation of feline disease models by nuclear transfer.

BACKGROUND: Somatic cell nuclear transfer in cats offers a useful tool for the generation of valuable research models. However, low birth rates after nuclear transfer hamper exploitation of the full potential of the technology. Poor embryo development after activation of the reconstructed oocytes seems to be responsible, at least in part, for the low efficiency. The objective of this study was to characterize the response of cat oocytes to various stimuli in order to fine-tune existing and possibly develop new activation methods for the generation of cat disease models by somatic cell nuclear transfer. METHODS: First, changes in the intracellular free calcium concentration [Ca2+]i in the oocytes induced by a number of artificial stimuli were characterized. The stimuli included electroporation, ethanol, ionomycin, thimerosal, strontium-chloride and sodium (Na+)-free medium. The potential of the most promising treatments (with or without subsequent incubation in the presence of cycloheximide and cytochalasin B) to stimulate oocyte activation and support development of the resultant parthenogenetic embryos was then evaluated. Finally, the most effective methods were selected to activate oocytes reconstructed during nuclear transfer with fibroblasts from mucopolysaccharidosis I- and alpha-mannosidosis-affected cats. RESULTS: All treatments were able to elicit a [Ca2+]i elevation in the ooplasm with various characteristics. Pronuclear formation and development up to the blastocyst stage was most efficiently triggered by electroporation (60.5 +/- 2.9 and 11.5 +/- 1.7%) and the combined thimerosal/DTT treatment (67.7 +/- 1.8 and 10.6 +/- 1.9%); incubation of the stimulated oocytes with cycloheximide and cytochalasin B had a positive effect on embryo development. When these two methods were used to activate oocytes reconstructed during nuclear transfer, up to 84.9% of the reconstructed oocytes cleaved. When the 2 to 4-cell embryos (a total of 220) were transferred into 19 recipient females, 4 animals became pregnant. All of the fetuses developed from oocytes activated by electroporation followed by cycloheximide and cytochalasin B incubation; no fetal development was detected as a result of thimerosal/DTT activation. Although heartbeats were detected in two of the cloned fetuses, no term development occurred. CONCLUSION: Electroporation proved to be the most effective method for the activation of cat oocytes reconstructed by nuclear transfer. The combined thimerosal/DTT treatment followed by cycloheximide and cytochalasin B incubation triggered development effectively to the blastocyst stage; whether it is a viable option to stimulate term development of cloned cat embryos needs further investigations.

Characterization of basal seminal traits and reproductive endocrine profiles in North American river otters and Asian small-clawed otters.

In this study, fecal samples were collected from 24 North American river (NARO) and 17 Asian small-clawed otters (ASCO) for 6-36 months and semen collected seasonally from NARO males (n=4/season) via electroejaculation. Our main objectives were to: (1) characterize endocrine parameters by longitudinal monitoring of fecal hormone metabolites and (2) investigate semen collection and basal seminal traits in NARO. NARO demonstrated a distinct seasonality in the spring, with females having a monoestrual estrogen elevation lasting 15.33+/-1.98 (mean+/-SEM) days and males peaking in testosterone production for 25.50+/-7.51 days. Pregnancy was characterized by 7-9 months of basal fecal progesterone, presumably corresponding to embryonic diapause, followed by a rapid increase over the final 68-73 days to term. Pseudopregnancy exhibited a similar late winter progesterone peak of 68-72 days, which could not be differentiated from pregnancy. Geographic latitude possibly influenced the timing of increased testosterone in males and increased progesterone in pregnant/pseudopregnant females. In ASCO, monitoring of fecal estrogens did not allow consistent detection of peak values associated with behavioral estrus. Both pregnancy and pseudopregnancy were characterized by a moderate rise in fecal progesterone for 14-16 days postovulation followed by a marked increase. Total gestation length was 67-77 days compared with 62-84 days for pseudopregnancy. In NARO, optimal sperm recovery and quality occurred only in the spring, corresponding with seasonal increases in testicular volume and fecal testosterone. These findings represent the first comprehensive information on normative endocrine and seminal traits in freshwater otter species.

Refinement of a commercial bench-top relaxin assay for pregnancy diagnosis using urine from domestic and nondomestic felids.

Relaxin, a 6-kDa polypeptide hormone, is excreted in the urine during pregnancy in several mammalian species. A recent study showed that detection of urinary relaxin using a bench-top serum assay (Witness relaxin kit, Synbiotics Corp., San Diego, California 92127, USA) can be diagnostic for pregnancy in domestic cats (Felis silvestris catus), but it is unknown whether the bench-top kit is applicable with urine across felid species. Our objectives were to 1) examine modifications in urine processing to improve kit reliability in pregnant cats, 2) evaluate the impact of concentrating urine via filtration on relaxin detection, 3) assess the effect of sample freezing on relaxin concentrations, and 4) begin quantifying urinary relaxin levels in nondomestic felids. Urine and serum were collected from domestic cats and nondomestic cat species (Pallas' cat, Otocolobus manul; sand cat, Felis margarita; cheetah, Acinonyx jubatus; and lion, Panthera leo) at several times after breeding. Urine samples, subjected to various processing methods, were tested using the bench-top kit, and relaxin levels were later quantified via radioimmunoassay. For domestic cat urine samples, filtration and addition of protein/phosphate buffer improved the consistency of the relaxin kit for early pregnancy diagnosis. Urine freezing caused a slight (approximately 13%) but significant decrease in relaxin concentrations, but frozen-thawed samples still tested positive with the bench-top kit. In nondomestic felids, urinary relaxin immunoreactivity during pregnancy was similar to or higher than that of pregnant domestic cats, suggesting that relaxin is a reliable cross-species marker of pregnancy. Urinary relaxin was detectable using the bench-top kit in pregnant Pallas' cats, but urine samples from other species tested negative, regardless of processing methods. Findings suggest that measurement of urinary relaxin is a promising approach for noninvasive pregnancy diagnosis in exotic felids, but further assessment of urinary relaxin profiles among cat species and modification of the bench-top relaxin kit are warranted to improve cross-species utility.

Comparison of different sperm cryopreservation procedures on post-thaw quality and heterologous in vitro fertilisation success in the ocelot (Leopardus pardalis).

Cryopreservation of spermatozoa from free-living ocelots (Leopardus pardalis) could benefit their conservation by facilitating gene flow between in situ and ex situ populations without requiring removal of additional cats from the wild. The objective of this study was to investigate three different methods of ocelot sperm cryopreservation to identify the most appropriate technique for use in a field environment. Male ocelots (n = 10), housed in North American zoos, were anaesthetised with tiletamine-zolazepam (7 mg kg(-1) bodyweight; i.m.) and subjected to a regimented electroejaculation procedure. Recovered semen was evaluated for sperm concentration, motility and morphology and processed for cryopreservation by three methods: (1) pelleting on dry ice, (2) freezing in straws over liquid nitrogen vapour; and (3) freezing in straws in a dry shipper. Frozen samples were thawed and assessed for post-thaw acrosome status, viability, motility over time and ability to fertilize viable domestic cat oocytes. Although several post-thaw sperm parameters varied (P < 0.05) among freezing methods, frozen-thawed ocelot spermatozoa from all treatments showed a similar (P > 0.05) capacity to bind, penetrate and fertilize viable domestic cat oocytes. These findings suggest that spermatozoa collected from male ocelots under field conditions may be frozen in straws either using liquid nitrogen alone or in a charged dry shipper to retain adequate functional competence after thawing for use with assisted reproductive procedures.

Seminal and endocrine characteristics of male Pallas' Cats (Otocolobus manul) maintained under artificial lighting with simulated natural photoperiods.

Pallas' cats (Otocolobus manul) have a pronounced reproductive seasonality controlled by photoperiod. Previous studies of reproduction in captive Pallas' cats exposed to natural light showed a breeding season of December-April. This study evaluated the impact of artificial lighting timed to simulate natural photoperiods on male reproductive seasonality of four Pallas' cats housed indoors. Semen evaluation, blood collection, and body weight measurements were conducted every 1-2 months from November 2000-June 2001. Fecal samples were collected from each male twice weekly to assess testosterone and corticoid concentrations. Mean values for reproductive traits (sperm attributes, testicular volume) were highest from February-April, the defined breeding season. Fecal testosterone concentrations were highest from mid-January to mid-March. Male Pallas' cats managed indoors under simulated photoperiods experienced a delayed onset of the breeding season by 1-2 months and a decreased length of the breeding season. Over the course of the study, fecal corticoid concentrations did not seem to differ among seasons. Although mating attempts during this study were unsuccessful, subsequent pairings of male and female Pallas' cats in the same research colony during the 2002 and 2003 breeding seasons produced viable offspring. These results suggest that male Pallas' cats, housed indoors under simulated photoperiods, exhibit distinct reproductive cyclic patterns, characterized by a delayed and truncated breeding season. Adrenocortical activity varied among individuals, but did not adversely affect reproductive parameters. Housing Pallas' cats indoors under simulated photoperiods may represent a viable strategy for maintaining breeding success while limiting disease exposure. Zoo Biol 0:1-13, 2007. (c) 2007 Wiley-Liss, Inc.

Interaction of extender composition and freezing method for effective semen cryopreservation in the North American river otter (Lontra canadensis).

Semen cryopreservation and storage in genome resource banks (GRBs), in combination with artificial insemination (AI), could be invaluable for genetic management and conservation of endangered otter species. For any applied conservation benefit, effective methods for otter sperm processing and cryopreservation first must be established. In this study, our objective was to develop an effective semen cryopreservation method for the North American river otter, evaluating the effect of extender composition (i.e., glycerol concentration, Equex STM paste supplementation) and freezing protocol (timing of glycerol addition, pre-freeze cooling rate, freezing/packaging method) on post-thaw sperm motility, longevity and acrosome status. Semen was collected from 14 otters housed at 9 zoos, and following cryopreservation in an egg-yolk based extender, thawed to assess sperm motility and acrosome status immediately post-thaw and during 6 h of in vitro culture. Results indicated that extender containing 4% glycerol was preferable (p < 0.05) to 8% glycerol but the temperature/timing of extender addition containing 4% glycerol did not affect (p > 0.05) post-thaw sperm parameters. Treatments with extender containing Equex and frozen by pelleting on dry ice showed greater (p < 0.05) motility and percentage of intact acrosomes compared to treatments frozen in extender without Equex, regardless of pre-freeze cooling rate. In the absence of Equex, pelleting provided superior post-thaw sperm motility (p < 0.01) and higher (p < 0.001) percentage of sperm with intact acrosomes compared to samples frozen in straws over liquid nitrogen vapor. Results of this study indicate that cryopreservation of otter sperm using an egg-yolk -TEST based extender containing 4% glycerol and 1% Equex, with the pellet freezing method, provided superior post-thaw sperm motility, longevity and acrosomal integrity compared to other combinations. Neither alterations in timing of glycerolated extender addition nor pre-freeze cooling rate had a discernable effect on post-thaw otter sperm parameters. These findings represent the first assessment of semen cryopreservation in any otter species and may be of value as a model for development of semen cryopreservation strategies in other endangered otter species.

Assessment of basic seminal characteristics, sperm cryopreservation and heterologous in vitro fertilisation in the fishing cat (Prionailurus viverrinus).

Conservation of the fishing cat, a threatened south-east Asian felid, could benefit from effective ex situ genetic management and breeding programmes, including the use of assisted reproduction. The aims of the present study were to: (1) characterise basal seminal traits of fishing cats in Thailand zoos; and (2) investigate the effect of cryopreservation on sperm motility, acrosomal integrity and in vitro function. Seminal traits were evaluated in electroejaculates collected from eight males. Spermatozoa were diluted in n-tris(hydroxymethyl)-methyl-2-aminoethanesulfonic acid Tris (TEST)-yolk buffer (TYB) without glycerol, then diluted further with TYB with glycerol (4% final concentration) at either 25 degrees C or after slow cooling to 5 degrees C and frozen in straws over liquid nitrogen vapour. After thawing, sperm function was assessed by insemination of viable domestic cat oocytes. Fishing cat ejaculates averaged (+/- s.e.m.) 43.6 +/- 14.2 x 10(6) motile spermatozoa with 33.5 +/- 6.8% normal sperm morphology. Semen processing had a negligible effect (P > 0.05) on sperm motility and acrosomal integrity, but values were reduced (P < 0.05) after thawing. All thawed samples fertilised domestic cat oocytes, with 62.1% (36/58) of mature oocytes cleaving. Glycerol addition at 5 degrees C resulted in higher (P < 0.05) post-thaw motility and intact acrosomes than glycerol addition at 25 degrees C. In conclusion, good-quality ejaculates can be obtained from Thai fishing cats and their spermatozoa exhibit adequate function after cryopreservation for in vitro fertilisation procedures.

Application of assisted reproduction for population management in felids: the potential and reality for conservation of small cats.

Assisted reproductive technology (ART), using the primary applied tools of AI, ET, and sperm and embryo cryopreservation, has been promoted over the past decades for its potential to conserve endangered wildlife, including felids. However, if the goal is efficient, consistent production of viable offspring for population management, then the 'potential' of ART has yet to become 'reality' for any non-domestic cat species. For the five small-sized felids (i.e., Brazilian ocelot, fishing cat, Pallas' cat, Arabian sand cat, black-footed cat) managed by Species Survival Plans (SSPs) in North American zoos, achieving this potential may be an absolute necessity if genetically viable captive populations are to be maintained into the next century. Modeling programs suggest that current SSP populations are not sustainable without periodic introduction of new founders and improved demographic parameters, including longer generation intervals and larger population sizes. ART provides the means to address each of these management challenges. In each small cat SSP species, fecal hormone metabolite assays and seminal analysis have proven useful for characterizing basal reproductive parameters, a necessary prerequisite to developing ART. Of the five SSP species, ART has been used to produce living offspring only in the ocelot, including after AI with frozen-thawed spermatozoa and following transfer of frozen-thawed IVF embryos. The true efficacy of these techniques, however, is still unknown. To improve the applicability of ART for population management, priorities for immediate research include further investigation of ovarian stimulation protocols, sperm and embryo cryopreservation methods, embryo culture systems, and fetal and neonatal viability following ART.

Conservation science in a terrorist age: the impact of airport security screening on the viability and DNA integrity of frozen felid spermatozoa.

In response to growing terrorism concerns, the Transportation Security Administration now requires that all checked baggage at U.S. airports be scanned through a cabinet x-ray system, which may increase risk of radiation damage to transported biologic samples and other sensitive genetic material. The objective of this study was to investigate the effect of these new airport security regulations on the viability and DNA integrity of frozen felid spermatozoa. Semen was collected from two domestic cats (Felis silvestris catus) and one fishing cat (Prionailurus viverrinus), cryopreserved in plastic freezing straws, and transferred into liquid nitrogen dry shippers for security screening. Treatment groups included frozen samples from each male scanned once or three times using a Transportation Security Administration-operated cabinet x-ray system, in addition to non-scanned samples (i.e., negative control) and samples previously scanned three times and exposed to five additional high-intensity x-ray bursts (i.e., positive control). Dosimeters placed in empty dry shippers were used to quantify radiation exposure. Following treatment, straws were thawed and spermatozoa analyzed for post-thaw motility (percentage motile and rate of progressive movement), acrosome status, and DNA integrity using single-cell gel electrophoresis (i.e., the comet assay). Dosimeter measurements determined that each airport screening procedure produced approximately 16 mrem of radiation exposure. Our results indicated that all levels of radiation exposure adversely affected (P < 0.05) post-thaw sperm motility, but the percentage of acrosome-intact spermatozoa did not differ (P > 0.05) among treatment groups. Results also showed that the amount of double-stranded DNA damage was greater (P < 0.05) in sperm samples from both cat species scanned three times compared to samples scanned once or negative controls. Findings suggest that new airport security measures may cause radiation-induced damage to frozen spermatozoa and other valuable biologic samples transported on passenger aircraft and that alternative modes of sample transportation should be used whenever possible.

Assessment of viral presence in semen and reproductive function of frozen-thawed spermatozoa from Pallas' cats (Otocolobus manul) infected with feline herpesvirus.

Although herpesviruses are known to contaminate the semen of several mammalian species, the occurrence of feline herpesvirus type 1 (FHV-1) in semen of infected cats has not been reported. Our objectives in this study were to investigate the presence of FHV-1 DNA in seminal fluid and frozen-thawed spermatozoa from FHV-1 infected Pallas' cats (Otocolobus manul) and assess the functionality of their frozen-thawed spermatozoa in vitro. Over a 3-yr period, semen (n = 33 ejaculates) was collected periodically via electroejaculation from four Pallas' cats chronically infected with FHV-1. Spermic ejaculates were frozen by pelleting on dry ice and stored in liquid nitrogen. After thawing, sperm motility and acrosome status were assessed over time during in vitro culture. For vitro fertilization (IVF), viable domestic cat (Felis silvestris catus) oocytes were inseminated with frozen-thawed Pallas' cat spermatozoa and evaluated for embryo cleavage. For FHV-1 polymerase chain reaction (PCR) analysis, DNA was extracted from seminal fluid, frozen-thawed spermatozoa, inseminated oocytes, heterologous IVF embryos, and conjunctival biopsies and analyzed for presence of a 322-base pair region of the FHV-1 thymidine kinase gene. Immediately post-thaw, sperm motility and percentage of intact acrosomes were decreased (P < 0.05) compared to fresh samples, and declined further (P < 0.05) during culture. However, all frozen-thawed IVF samples were capable of fertilizing domestic cat oocytes (overall, 46.1 +/- 6.0% cleavage). PCR analysis did not identify FHV-1 DNA in any reproductive sample despite the repeated detection of FHV-1 DNA in conjunctival biopsies. These results suggest that semen collected from Pallas' cats infected with FHV-1 does not contain cell-associated or non-cell-associated virus and that frozen-thawed spermatozoa exhibit adequate function for potential genetic rescue with minimal risk of FHV-1 transmission.

Exploring the ecologic basis for extreme susceptibility of Pallas' cats (Otocolobus manul) to fatal toxoplasmosis.

Recent efforts by North American zoos to establish a genetically viable captive population of Pallas' cats (Otocolobus manul) have been compromised by high newborn mortality (approximately 60%), primarily because of toxoplasmosis. The basis for this extreme susceptibility to toxoplasmosis is unknown. In the present study, the general health status of wild Pallas' cats in Mongolia was evaluated, including assessment of basal hematologic parameters and fecal corticoid metabolite concentrations. The prevalence of exposure to Toxoplasma gondii in Mongolian Pallas' cats, local domestic cats, and prey species also was determined based on serology and/or polymerase chain reaction analysis. Biologic samples (blood, feces, and/or brain tissue) were obtained from 15 wild Pallas' cats, 15 domestic cats, and 45 prey animals (rodents and pikas) captured in Mongolia during the summers of 2000 and 2001. Comparative data were obtained from nine captive Pallas' cats maintained in North American zoos. Based on physical examinations, complete blood counts, and blood chemistry analyses, only minor differences were observed in the general health status of wild and captive Pallas' cats. Fecal cortisol metabolite concentrations did not differ (P > 0.05) between populations, indicating that Pallas' cats in captivity and in the wild have similar basal adrenocortical activity. A pronounced difference (P < 0.01) in seroprevalence to T. gondii was observed between populations. Whereas all captive Pallas' cats exhibited elevated immunoglobulin titers (IgG > 2,048) to T. gondii, only two of 15 (13%) wild Pallas' cats were seropositive, with both cats having lower IgG titers (< 1,024). Furthermore, no evidence of exposure to this parasite was found in any of the Mongolian domestic cats or prey species. These findings suggest that wild Pallas' cats have minimal opportunity for exposure to T. gondii in their natural habitat and, typically, do not become infected with this parasite until being brought into captivity. Accordingly, maintenance of a viable captive population may require implementing effective strategies to prevent exposure of immunologically naive Pallas' cats to T. gondii and to reduce parasite transmission between seropositive females and their highly susceptible offspring.