Felis Nigripes Research Articles

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.

Nuclear transfer in cats and its application

Nuclear transfer (NT) technology is typically used for generating identical individuals, but it is also a powerful resource for understanding the cellular and molecular aspects of nuclear reprogramming. Most recently, the procedure has been used in humans for producing patient-specific embryonic stem cells. The successful application of NT in cats was demonstrated by the birth of domestic and non-domestic cloned kittens at a similar level of efficiency to that reported for other mammalian species. In cats, it has been demonstrated that either in vivo or in vitro matured oocytes can be used as donor cytoplasts. The length of in vitro oocyte maturation affects in vitro development of reconstructed embryos, and oocytes matured in vitro for shorter periods of time are the preferred source of donor cytoplasts. For NT, cat somatic cells can be synchronized into the G0/G1 phase of the cell cycle by using different methods of cell synchronization without affecting the frequency of in vitro development of cloned embryos. Also, embryo development to the blastocyst stage in vitro is not influenced by cell type, but the effect of cell type on the percentage of normal offspring produced requires evaluation. Inter-species NT has potential application for preserving endangered felids, as live offspring of male and female African wildcats (AWC, Felis silvestris lybica) have been born and pregnancies have been produced after transferring black-footed cat ( Felis nigripes) cloned embryos into domestic cat ( Felis silvestris catus) recipients. Also, successful in vitro embryo development to the blastocyst stage has been achieved after inter-generic NT of somatic cells of non-domestic felids into domestic cat oocytes, but no viable progeny have been obtained. Thus, while cat cytoplasm induces early nuclear remodeling of cell nuclei from a different genus, the high incidence of early embryo developmental arrest may be caused by abnormal nuclear reprogramming. Fetal resorption and abortions were frequently observed at various stages of pregnancy after transfer of AWC cloned embryos into domestic cat recipients. Abnormalities, such as abdominal organ exteriorization and respiratory failure and septicemia were the main causes of death in neonatal cloned kittens. Nonetheless, several live domestic and AWC cloned kittens have been born that are seemingly normal and healthy. It is important to continue evaluating these animals throughout their lives and to examine their capability for natural reproduction.

77 LOSS OF SAND CAT CLONED EMBRYOS DURING EARLY PREGNANCY MAY BE ASSOCIATED WITH ABERRANT Oct-4 EXPRESSION

Genetic incompatibilities between the donor nucleus and the recipient cytoplast of inter-species cloned embryos may be associated with a low frequency of development to the blastocyst stage and/or in vivo developmental competence. We have established pregnancies after the transfer of inter-species Black-footed cat (BFC; Felis nigripes) cloned embryos and live offspring after the transfer of inter-species African Wildcat (AWC; Felis silvestris lybica) cloned embryos into domestic cat recipients (G�mez et al. 2006 Theriogenology 66, 72; 2004 Cloning Stem Cells 6, 217). However, many of the pregnancies were re-absorbing before Day 30 of gestation. According to Arat et al. (2003 Mol. Reprod. Dev. 66, 334), inter-generic mouse–bovine cloned embryos do not express the POU transcription factor Octamer-4 (Oct-4), and this abnormal gene expression may compromise pluripotency and contribute to the failure of cloned embryos to undergo post-implantation development (Boiani et al. 2002 Genes Dev. 16, 1209). The purpose of the present study was to evaluate (1) the in vivo developmental competence of nuclear transfer embryos derived by fusion of Sand cat (SC; Felis margarita) fibroblasts with domestic cat (DSH; Felis catus) cytoplasts after transfer into domestic cat recipients, and (2) protein distribution of Oct-4 in derived cloned blastocysts. Fibroblast cell lines were established from skin biopsies of 2 adult SC (both males). Cloned embryos were produced as described by G�mez et al. (2004 Cloning Stem Cells 6, 217). Reconstructed SC (n = 1113) Day 1 NT embryos were transferred by laparoscopy into the oviducts of 29 gonadotropin-treated DSH recipients on Day 1 after induced ovulation. Pregnancy was assessed by ultrasonography on Day 22. Seven (24%) cats receiving somatic cell nuclear transfer (SCNT) embryos became pregnant. Two SC cloned embryos developed to term; one kitten died in utero on Day 61 of gestation and the second kitten died 2 h after being delivered by caesarean section on Day 62 of gestation. The clonal status of the SC kittens was assessed by multiplex PCR amplification of 20 microsatellite markers. Immunocytochemical localization of Oct-4 was determined on Day 8 SCNT (n = 16) and DSH/IVF (n = 10) blastocysts. Embryos were incubated in 1 : 100 anti-human Oct-4 polyclonal antibody for 1 h, followed by incubation in 1 : 128 FITC-labeled anti-goat IgG for 1 h. Embryos were then counter-stained with propidium iodide, and examined with epifluorescence microscopy. Most cloned blastocysts had a detectable signal of Oct-4 (69%), but some exhibited aberrant expression in the trophectoderm (TPD; 44%). In contrast, the signal in most DSH/IVF blastocysts was restricted to the inner cell mass (ICM; 80%). Oct-4 expression was not detected in 2-cell DSH/IVF embryos. These observations suggest that aberrant Oct-4 expression may contribute to the embryonic loss observed in the present study.

36 IMPROVING THE APPLICATION OF NUCLEAR TRANSFER FOR PRODUCING NON-DOMESTIC FELIDS

One of the most remarkable aspects of somatic cell nuclear transfer (NT) is the possibility of avoiding extinction when there are few remaining animals of a specific felid population. Previously, we produced live male African Wildcat (AWC; Felis lybica) cloned kittens using inter-species nuclear transfer (Gomez et al. 2004 Cloning and Stem Cells 6, 217–228). The production of females is a primary objective of most breeding programs. Therefore, the purpose of the present study was to determine (1) if we could produce live female AWC cloned kittens at a proportion similar to that previously demonstrated with males, and (2) if our inter-species NT technique used to produce AWC is applicable to in vitro production of another non-domestic felid species. Specifically, we evaluated the in vivo developmental competence of NT embryos derived by fusion of Black footed cat (BFC, Felis nigripes) and AWC fibroblasts with domestic cat (DSH, Felis catus) cytoplasts, after transfer into domestic cat recipients. Fibroblast cell lines were established from skin biopsies of BFC (6-year-old), and AWC (12-year-old) adult females. After at least three passages, cells were serum-starved for 5 days and injected into the perivitelline space of enucleated domestic cat oocytes. Fusion of cell-cytoplast couplets was induced by applying a 3-s AC pre-pulse of 20 V, 1 MHz, followed by two 30-μs DC pulses of 240 V/mm. Fused couplets were activated 2 to 3 h after fusion by exposure to two 60 μsec DC pulses of 120 V/mm, followed by 4 h incubation with 10 μg/mL cycloheximide and 5 μg/mL cytochalasin B. Reconstructed BFC (n = 16) and AWC (n = 536) NT Day 1 embryos were transferred by laparoscopy into the oviducts of 1 and 12 gonadotrophin-treated DSH recipients, respectively, on Day 1 after induced ovulation. Pregnancy was assessed by ultrasonography on Day 22. One cat (100%) receiving BFC NT embryos and 5 (41.6%) cats receiving AWC NT embryos became pregnant. Twenty-three AWC cloned embryos implanted and 11 kittens were born. Three BFC NT embryos implanted and the pregnancy is currently ongoing. AWC cloned kittens were phenotypically and genetically identical to their somatic cell donor. Their clonal identity was assessed by multiplex PCR amplification of 20 microsatellite markers, including seven markers that are known to be on the X chromosome. In summary, these results indicate that female AWC cloned kittens can be produced and BFC pregnancy can be established in domestic cat recipients. The embryo implantation rate and viability of AWC female cloned embryos was higher than that observed after the transfer of AWC male cloned embryos. The difference may be due to improvements in the NT procedure, rather than to differences in the sex of the cell lines. Table 1. Implantation rate and fetal survival to term of AWC and BFC NT embryos in pregnant domestic cat recipients

Bartonella henselaein African Lion, South Africa

To the Editor: Four members of the bacterial genus Bartonella, Bartonella henselae, B. clarridgeiae, B. koehlerae, and B. bovis, have been isolated from felids, mostly domestic cats (1,2). Of these four species, B. henselae and B. clarridgeiae are recognized human pathogens, which cause many illnesses, including endocarditis, prolonged fever, various ocular infections and, most commonly, cat scratch disease (1). In 1994, domestic cats (Felis domesticus) were found to be a reservoir for B. henselae; subsequent surveys have shown that a large proportion of the domestic cat population worldwide has been exposed to, or infected with, bartonellae (1). The epidemiologic features of Bartonella infection in other felid species has been explored; a high prevalence of seropositivity has been found in free-ranging and captive wild cats from California and Florida (3), as well as panthers from Florida (4). B. henselae has been isolated from a captive cheetah in Zimbabwe (5). During 2002, blood samples were collected from 65 African lions that inhabited three ranches in the Free State Province of South Africa. These ranches breed and rear lions specifically for game. Although the lions are contained within vast (several km2) enclosures, they are free to move about and interact with one another. The lions have minimal contact with humans or other animals, except carcasses of horses and donkeys that are provided as food. The lions do not receive any other food, food supplements, growth enhancers, or antiparasite prophylaxis. All three ranches are deep in the veld, at least 20 km from any settlements. Blood samples were drawn from the lions as part of an ongoing health surveillance program conducted by the African Large Predator Research Unit, University of Bloemfontein. Whole blood samples were drawn aseptically from each lion into EDTA tubes, stored at 4°C before being returned to the laboratory, and then frozen at –70°C in the laboratory. Subsequently, blood samples were thawed, and an aliquot was plated onto 10% sheep blood–enriched agar and incubated at 37°C in a 5% CO2 atmosphere for a maximum of 45 days. One culture yielded putative bartonellae (small, smooth, white-gray colonies) after 14 days' incubation. A crude DNA extract was prepared from this isolate and used as a template in previously described polymerase chain reaction–based assays to detect and identify Bartonella species which targeted fragments of the 16S rRNA encoding gene and 16S/23S intergenic spacer region (6). Amplification products of the expected size were obtained from the DNA extract. The nucleotide base sequence of each product showed that each shared 100% similarity with sequences of other B. henselae isolates held in GenBank. The 16S rRNA gene sequence was identical to that of type II variants. Antisera from 62 of the 65 samples were tested for the presence of anti-Bartonella immunoglobulin G antibodies using an enzyme-linked immunosorbent assay previously evaluated to detect antibodies in domestic cats (7). Eighteen of the samples had matrix scores above the upper limit of the normal range of values observed in uninfected cats, thus indicating past exposure to Bartonella species. No serum from the B. henselae culture-positive animal was available for testing. Our findings confirm that lions are susceptible to infection by B. henselae, but their role as reservoirs for this species remain unclear. The observed prevalence of infection (1.5%) and exposure rate (29%) in our study are lower than those typically observed in domestic cats, particularly in warmer regions of the world. Nonetheless, our serologic data do suggest that a substantial proportion of the lions are exposed to bartonellae. Although limited, our assessment of the lion B. henselae isolate suggests that it is within the genetic spectrum of strains associated with domestic cats, and lions may serve as an extension to this reservoir. The extent of contact between domestic cats, or their ectoparasites, and the farmed lions we studied is likely to be minimal, given the remoteness of the enclosures (the infected lion lived on a cat-free ranch). However, the lions may have contact with other wild-living felids such as the African wild cat (Felis silvestris lybica), small spotted cat (Felis nigripes), and the caracal (Caracal caracal) which are endemic to the region.

Home range size and social organisation of black-footed cats

Summary A total of 17 black-footed cats (7 males, 10 females) were studied by telemetry and direct observation on a game farm near Kimberley, South Africa. Total annual ranges of five adult resident males averaged 20.7km2(100% Minimum Convex Polygon (MCP100)) or 16.1 km2(95% MCP=MCP95) incorporating all fixes taken, and were significantly larger than the annual ranges of seven adult females with 10 km2(MCP100) or 8.6 km2(MCP95). Male seasonal ranges were significantly larger than those of all females. Adult resident males overlapped with up to four female's ranges and encompassed on average 66.7% of each females' range during winter 1997 or 60.2% during summer 1998, respectively. Intra–sexual overlap in the 1998 summer non–mating season was slight for adult males (12.9% for MCP100, n=3) but was considerable for females (40.4% for MCP100, n=5). The range of one female lay entirely within the home range of another, but despite this overlap all black–footed cats hunted solitarily. Consecutive calculated seasonal range centres of black–footed cats shifted by 835±414 m and MCP95ranges were overlapped by the next season's range by 68±11%, and were thus relatively stable. The population density for the 60 km2study area in summer 1998 was 0.17 adult cats/km2.

The influence of olfactory enrichment on the behaviour of captive black-footed cats, Felis nigripes

The value of olfactory enrichment for captive-housed animals is now well recognised. Large cats have been shown to benefit from the introduction of odours to their captive environment, but to date the effect of odour introduction on the behaviour of small cats remains unknown. This study investigated the behaviour of six zoo-housed black-footed cats, Felis nigripes, in response to four odours (no odour [control], nutmeg, catnip and body odour of prey) introduced individually on cloths into the animals’ pens over a period of 5 days. It was hypothesised that the cats’ behaviour would differ significantly between the control and experimental odours and that interest in the experimental odours would wane over time. All of the experimental odours influenced the cats’ behaviour, resulting in an increase in the amount of time that the animals spent in active behaviours, i.e. moving (average increase of 8.3%), grooming (average increase of 5.9%), exploring the cloth (average increase of 10.9%) and exploring the pen (average increase of 9.2%). The experimental odours also resulted in a decrease in the amount of time that the cats spent in sedentary behaviours, i.e. standing (average decrease of 2.8%), sitting (average decrease of 5.2%) and resting (average decrease of 25.9%). Nutmeg exerted less of an effect on the cats’ behaviour than catnip or odour of prey. The cats’ response to all of the experimental odours waned over the course of the 5-day observation period, suggesting that the animals habituated to the stimuli. The results highlight the potential for odour to be employed as a method of environmental enrichment for small captive-housed felids, if presented in an appropriate manner.

The role of scent marking in a free‐ranging, female black‐footed cat (Felis nigripes)

AbstractA radio‐collared, female black‐footed cat (Felis nigripes) was followed for a total period of 575 hours, over seven observation periods spanning 18 months, in the Kimberley region of the Northern Cape Province, South Africa. Data on reproduction and scent marking were collected by direct observation. The predominant form of scent marking was urine‐spraying, the frequency of which fluctuated during the year and seemed to be related to changes in the reproductive cycle. Marking frequency increased dramatically approximately one and a half months before mating, and conception of the first litter of the breeding season occurred at a low frequency during pregnancy, and was entirely absent when the cat was rearing young. This strongly indicates a primary function in the advertisement of female reproductive condition, although an additional role in the maintenance of social spacing is suggested. Scent marks were not restricted to the borders of the animal's range, but corresponded to those areas used most intensely during the year.

The role of scent marking in a free-ranging, female black-footed cat (Felis nigripes)

A radio-collared, female black-footed cat (Felis nigripes) was followed for a total period of 575 hours, over seven observation periods spanning 18 months, in the Kimberley region of the Northern Cape Province, South Africa. Data on reproduction and scent marking were collected by direct observation. The predominant form of scent marking was urine-spraying, the frequency of which fluctuated during the year and seemed to be related to changes in the reproductive cycle. Marking frequency increased dramatically approximately one and a half months before mating, and conception of the first litter of the breeding season occurred at a low frequency during pregnancy, and was entirely absent when the cat was rearing young. This strongly indicates a primary function in the advertisement of female reproductive condition, although an additional role in the maintenance of social spacing is suggested. Scent marks were not restricted to the borders of the animal's range, but corresponded to those areas used most intensely during the year.

In situ and ex situ observations and management of Black‐footed cats Felis nigripes

The international studbook for the Black‐footed cat Felis nigripes shows a steady increase in the captive population between 1974, when four animals were recorded, and 1990, when the total captive population was 108, although numbers have decreased since then. High mortality and an unequal sex ratio pose serious husbandry and management problems. Since 1993 research into the behaviour and ecology of this small nocturnal cat has been carried out in a 110 km2 area 10 km south‐east of Kimberley, South Africa. Radio‐collars fitted to nine cats made it possible to obtain data on size of home range and distribution. This paper describes the history, management and husbandry of Black‐footed cats in captivity. Information is also given on hunting technique, diet, interaction with non‐prey species, communication and reproduction, obtained from four animals observed in the wild for a total of 800 hours,

Resolution of recent radiations within three evolutionary lineages of felidae using mitochondrial restriction fragment length polymorphism variation

Patterns of mitochondrial restriction fragment length polymorphism (RFLP) variation were used to resolve more recent relationships among the species of the Felidae ocelot lineage, domestic cat lineage, and pantherine lineage. Twenty-five of 28 restriction enzymes revealed site variation in at least 1 of 21 cat species. The ocelot lineage was resolved into three separate sistertaxa groups: Geoffroy's cat (Oncifelis geoffroyi) and kodkod (O. guigna), ocelot (Leopardus pardalis) and margay (L. wiedii), and pampas cat (Lynchailurus colocolo) and most of the tigrina samples (Leopardus tigrina). Within the domestic cat lineage, domestic cat (Felis catus), European wild cat (F. silvestris), and African wild cat (F. libyca) formed a monophyletic trichotomy, which was joined with sand cat (F. margarita) to a common ancestor. Jungle cat (F. chaus) and black-footed cat (F. nigripes) mtDNAs diverged earlier than those of the other domestic cat lineage species and are less closely related. Within the pantherine lineage, phylogenetic analysis identified two distinct groups, uniting lion (P. leo) with leopard (P. pardus) and tiger (P. tigris) with snow leopard (P. uncia).

Comparison of co-culture and conditioned medium on expansion and hatching of in vitro-derived bovine blastocysts

A brief overview of the progress made during the past approximately 40 years on the development of methods for in vitro production of cat embryos and intra- and interspecies embryo transfer is described. The presentation is focused primarily on research done over the past 30 years at the Cincinnati Zoo (1980–1995) and at the Audubon Nature Institute, New Orleans (1996–present) beginning with original studies on determining optimal doses of porcine FSH for ovarian stimulation and uterine embryo recovery, cryopreservation, and transfer. A key early finding was the ability of cats to respond to multiple gonadotropin (porcine FSH) treatments by repeated stimulation of follicular development. With a ≥6-month interval between FSH treatments, over the past 15 years (1998–2013), we have done 1603 laparoscopic oocyte retrievals on 337 cats and recovered >38,000 mature oocytes (mean = 24.1 per laparoscopic oocyte retrieval). The limited information available on in vivo blastocyst development in the cat during the latter portion of the preimplantation period (approximately Days 8 to 12 after coitum or approximately Days 7 to 11 after ovulation) was assembled for the purpose of comparing and contrasting it with the growth, expansion, and zona functioning of in vitro-derived blastocysts. Also, results of transferring morulae and/or blastocysts into synchronous recipients are described to emphasize evidence that appears to allude to an essential role for an intact zona pellucida in successful implantation and subsequent development in the cat. Until 2003, our in vitro-derived embryos were transferred into the uterine horns of recipients to determine the feasibility of producing offspring from such primary methods as IVF, intracytoplasmic sperm injection, SCNT, and embryo cryopreservation. With the exception of SCNT embryos, pregnancy rates were satisfactory, but embryo survival rates were not. Subsequently, after finding that SCNT embryo survival rate could be improved using laparoscopic transfer of early cleavage stage embryos into the oviduct, we applied the technique to embryos derived using IVF with sex-sorted sperm, oocyte vitrification, and embryo cryopreservation. Overall, a pregnancy rate of 67% (14/21) has resulted. Most recently, with the oviductal embryo transfer technique, two litters of Black-Footed cat kittens have been born from intra- and interspecies transfer of cryopreserved embryos.