Embryo technology in conservation efforts for endangered felids

Most of the 36 species of wild cats are classified as threatened, vulnerable or endangered due to poaching and habitat loss. The important role of assisted reproduction techniques (ART) as part of a multifaceted captive breeding program for selected wild cat species is gradually gaining acceptance. This recognition is a result of the progress made during the last decade in which the feasibility of oocyte recovery from gonadotropin-treated females, in vitro fertilization, embryo cryopreservation and embryo transfer (ET) was demonstrated in the domestic cat (Felis catus). Additionally, embryos have been produced in vitro from oocytes matured in vitro after recovery from ex situ ovaries of both domestic and non-domestic cat species and domestic kittens have been born following transfer of these embryos. In vitro fertilization has been successful in at least one-third of wild cat species and kittens were born after transfer of Indian desert cat (Felis sylvestris ornata) embryos into a domestic cat and con-specific transfer of tiger (Panthera tigris) embryos. The domestic cat is not only a valuable model for development of in vitro techniques but may serve as a recipient of embryos from several species of small wild cats.     

家猫及其他猫科动物的生殖工程研究

在脊椎动物中,猫科动物是一类比较特殊的动物类群,共有37种动物,除家猫外,其余均为珍稀或临近灭绝的濒危动物,因而了解家猫的生殖习性和生殖规律,尤其是了解家猫胚胎工程领域,如猫卵母细胞的体外成熟（in vitro maturation IVM),体外受精（in vitro fertilization,IVF),显微受精等,对于利用现代生殖技术研究和探讨非家猫猫科动物的繁殖和保护具有极为重要的借鉴意义。     

Ovarian control for assisted reproduction in the domestic cat and wild felids

Of the 37 felid species, all but the domestic cat are classified as threatened with extinction in all or part of their native range. Additionally, the domestic cat is a valuable model for human biomedical research. Propagating some wild felids as well as domestic cat populations serving as human models is a major challenge primarily due to difficulties in transporting animals between facilities to ensure the pairing of genetically matched individuals, behavioral incompatibility between pairs and low fertility. Artificial insemination (AI) and in vitro fertilization/embryo transfer (IVF/ET) are powerful tools for helping manage rare populations. Developing successful assisted reproductive techniques requires knowledge of the female reproductive cycle and precise control of ovarian activity. Successful ovarian stimulation for AI and IVF/ET has been achieved in at least one-third of all cat species. However, sensitivity to a given gonadotropin treatment appears highly species-specific, and poor responses are common, particularly in felid species that exhibit spontaneous ovulations. Furthermore, current gonadotropin regimens have been demonstrated to perturb female reproductive function often leading to reduced fertility. Overall, ovarian response to exogenous hormonal stimulation has been highly variable, and pregnancy success after AI or IVF/ET remains low (<20%) in most species. Therefore, there is an immediate need to develop improved regimens that would allow more predictable ovarian responses in felids. We contend that recent research involving the use of progestins to control the ovary prior to gonadotropin stimulation shows promise for providing consistent ovarian stimulation in felids.     

Repetitive sequences in Eurasian lynx (Lynx lynx L.) mitochondrial DNA control region

Mitochondrial DNA (mtDNA) control region (CR) of numerous species is known to include up to five different repetitive sequences (RS1-RS5) that are found at various locations, involving motifs of different length and extensive length heteroplasmy. Two repetitive sequences (RS2 and RS3) on opposite sides of mtDNA central conserved region have been described in domestic cat (Felis catus) and some other felid species. However, the presence of repetitive sequence RS3 has not been detected in Eurasian lynx (Lynx lynx) yet. We analyzed mtDNA CR of 35 Eurasian lynx (L. lynx L.) samples to characterize repetitive sequences and to compare them with those found in other felid species. We confirmed the presence of 80 base pairs (bp) repetitive sequence (RS2) at the 5' end of the Eurasian lynx mtDNA CR L strand and for the first time we described RS3 repetitive sequence at its 3' end, consisting of an array of tandem repeats five to ten bp long. We found that felid species share similar RS3 repetitive pattern and fundamental repeat motif TACAC.     

Current progress on assisted reproduction in dogs and cats: in vitro embryo production

The objective of the development of assisted reproduction techniques in dogs and cats is their application to non-domestic canine and feline species, most of which are considered threatened or endangered. Among these techniques, an entirely in vitro system for embryo production is effectively an important tool for conservation of wildlife. In the last decade, progress has been made in embryo production in carnivores. It has been shown that canine oocytes can resume meiosis in vitro and that these oocytes can be fertilized and developed in vitro, although at a much lower rate than most other domestic animal oocytes. The reason lies in the dissimilarities of reproductive physiology of the dog compared to other species and the lack of precise information concerning the oviductal environment, in which oocyte maturation, fertilization and early embryonic development take place. Successful in vitro embryo production in the domestic cat has been attained with oocytes matured in vitro, and kittens were born after transfer of IVM/IVF derived embryos. On the basis of these results the in vitro fertilization of oocytes has also been applied in several non-domestic feline species. The effectiveness of such protocols in the preservation of genetic material of rare species can be improved by developing better techniques for long-term storage of gametes. In dogs and cats sperm cells have been successfully frozen and the cryopreservation of oocytes would greatly increase their availability for a range of reproductive technologies. Cryopreserved cat oocytes can be fertilized successfully and their development in vitro after fertilization is enhanced when mature oocytes are frozen. Thus refined techniques of oocyte maturation and fertilization in vitro coupled with oocyte cryopreservation could allow for an easy establishment of genetic combinations when male and female gametes in the desired combination are not simultaneously available, and the propagation of endangered carnivores would be facilitated.     

The early stages of follicular development: activation of primordial follicles and growth of preantral follicles

Although enormous progress has been made in understanding the events and regulation of the later stages of ovarian follicular development, the early stages of development, to a large extent and particularly in large mammals, remain a mystery. Mechanisms that regulate the initiation of follicular growth (follicle activation) and the ensuing growth and differentiation of preantral follicles are of considerable interest, since their elucidation is a prerequisite to use of the primordial pool to enhance reproductive efficiency in domestic animals, humans, and endangered species. This review is an attempt to summarize the approaches that have been taken to further this goal and the results thus far of these efforts. Preantral follicular development can be divided into three stages: activation of primordial follicles, the primary to secondary follicle transition, and the development of secondary follicles to the periantral stage. The activation of primordial follicles in vitro has been achieved thus far in rodents, cattle, and primates, where it occurs spontaneously without the addition of growth factors or hormones. The ovaries of rodents are small enough to be cultured intact and, in that experimental situation, some follicles activate, while many remain in the resting pool, and the addition of specific factors can increase or decrease the number of follicles that leave the resting pool in vitro. In contrast, follicular activation in cattle and primates has been studied by culturing small pieces of the ovarian cortex, rich in primordial follicles, and the great majority of the primordial follicles activate in that situation, suggesting the importance of inhibitory factors to the normal, gradual exit of follicles from the resting pool. In cultured rodent ovaries, follicles appear to pass easily and spontaneously from the primary to the secondary stage, whereas few of the activated follicles in cultured cortical pieces from cattle or primates progress from the primary to the secondary stage. Understanding the requirements for the primary to secondary transition is critical for growing follicles activated in vitro to the late preantral and antral stages. In contrast, the requirements for the continued growth of larger preantral follicles, which can be isolated for in vitro studies, have been extensively explored in rodents and to a lesser extent in domestic species. A number of hormones and factors have been implicated and will be discussed. Taken together, the results highlight the need for a better understanding of the earliest stages of follicular development in domestic ruminants, particularly follicle activation and the primary to secondary follicle transition.     

Molecular genetics and evolution of melanism in the cat family

Melanistic coat coloration occurs as a common polymorphism in 11 of 37 felid species and reaches high population frequency in some cases but never achieves complete fixation. To investigate the genetic basis, adaptive significance, and evolutionary history of melanistic variants in the Felidae, we mapped, cloned, and sequenced the cat homologs of two putative candidate genes for melanism (ASIP [agouti] and MC1R) and identified three independent deletions associated with dark coloration in three different felid species. Association and transmission analyses revealed that a 2 bp deletion in the ASIP gene specifies black coloration in domestic cats, and two different "in-frame" deletions in the MC1R gene are implicated in melanism in jaguars and jaguarundis. Melanistic individuals from five other felid species did not carry any of these mutations, implying that there are at least four independent genetic origins for melanism in the cat family. The inferred multiple origins and independent historical elevation in population frequency of felid melanistic mutations suggest the occurrence of adaptive evolution of this visible phenotype in a group of related free-ranging species.     

Genetic and phylogenetic divergence of feline immunodeficiency virus in the puma (Puma concolor).

Feline immunodeficiency virus (FIV) is a lentivirus which causes an AIDS-like disease in domestic cats (Felis catus). A number of other felid species, including the puma (Puma concolor), carry a virus closely related to domestic cat FIV. Serological testing revealed the presence of antibodies to FIV in 22% of 434 samples from throughout the geographic range of the puma. FIV-Pco pol gene sequences isolated from pumas revealed extensive sequence diversity, greater than has been documented in the domestic cat. The puma sequences formed two highly divergent groups, analogous to the clades which have been defined for domestic cat and lion (Panthera leo) FIV. The puma clade A was made up of samples from Florida and California, whereas clade B consisted of samples from other parts of North America, Central America, and Brazil. The difference between these two groups was as great as that reported among three lion FIV clades. Within puma clades, sequence variation is large, comparable to between-clade differences seen for domestic cat clades, allowing recognition of 15 phylogenetic lineages (subclades) among puma FIV-Pco. Large sequence divergence among isolates, nearly complete species monophyly, and widespread geographic distribution suggest that FIV-Pco has evolved within the puma species for a long period. The sequence data provided evidence for vertical transmission of FIV-Pco from mothers to their kittens, for coinfection of individuals by two different viral strains, and for cross-species transmission of FIV from a domestic cat to a puma. These factors may all be important for understanding the epidemiology and natural history of FIV in the puma.     

Rescue and maturation in vitro of follicular oocytes collected from nondomestic felid species.

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)     

Research in nondomestic species: experiences in reproductive physiology research for conservation of endangered felids

Tremendous strides have been made in recent years to broaden our understanding of reproductive processes in nondomestic felid species and further our capacity to use this basic knowledge to control and manipulate reproduction of endangered cats. Much of that progress has culminated from detailed scientific studies conducted in nontraditional laboratory settings, frequently at collaborating zoological parks but also under more primitive conditions, including in the field. A mobile laboratory approach is described, which incorporates a diverse array of disciplines and research techniques. This approach has been extremely useful, especially for conducting gamete characterization and function studies as well as reproductive surveys, and for facilitating the development of assisted reproductive technology. With continuing advances in assisted reproduction in rare felids, more procedures are being conducted primarily as service-related activities, targeted to increase effectiveness of species propagation and population management. It can be a challenge for both investigators and institutional animal care and use committees (IACUCs) to differentiate these service-based procedures from traditional research studies (that require IACUC oversight). For research with rare cat species, multi-institutional collaboration frequently is necessary to gain access to scientifically meaningful numbers of study subjects. Similarly, for service-based efforts, the ability to perform reproductive procedures across institutions under nonstandard laboratory conditions is critical to applying reproductive sciences for managing and preserving threatened cat populations. Reproductive sciences can most effectively assist population management programs (e.g., Species Survival Plans) in addressing conservation priorities if these research and service-related procedures can be conducted "on the road" at distant national and international locales. This mobile laboratory approach has applications beyond endangered species research, notably for other scientific fields (e.g., studies of hereditary disease in domestic cat models) in which bringing the laboratory to the subject is of value.     

Assessing the Status of Wild Felids in a Highly-Disturbed Commercial Forest Reserve in Borneo and the Implications for Camera Trap Survey Design

The proliferation of camera-trapping studies has led to a spate of extensions in the known distributions of many wild cat species, not least in Borneo. However, we still do not have a clear picture of the spatial patterns of felid abundance in Southeast Asia, particularly with respect to the large areas of highly-disturbed habitat. An important obstacle to increasing the usefulness of camera trap data is the widespread practice of setting cameras at non-random locations. Non-random deployment interacts with non-random space-use by animals, causing biases in our inferences about relative abundance from detection frequencies alone. This may be a particular problem if surveys do not adequately sample the full range of habitat features present in a study region. Using camera-trapping records and incidental sightings from the Kalabakan Forest Reserve, Sabah, Malaysian Borneo, we aimed to assess the relative abundance of felid species in highly-disturbed forest, as well as investigate felid space-use and the potential for biases resulting from non-random sampling. Although the area has been intensively logged over three decades, it was found to still retain the full complement of Bornean felids, including the bay cat Pardofelis badia, a poorly known Bornean endemic. Camera-trapping using strictly random locations detected four of the five Bornean felid species and revealed inter- and intra-specific differences in space-use. We compare our results with an extensive dataset of >1,200 felid records from previous camera-trapping studies and show that the relative abundance of the bay cat, in particular, may have previously been underestimated due to the use of non-random survey locations. Further surveys for this species using random locations will be crucial in determining its conservation status. We advocate the more wide-spread use of random survey locations in future camera-trapping surveys in order to increase the robustness and generality of inferences that can be made.     

The AB blood group system in wild felids

The blood type of 131 non-domesticated felids belonging to 26 felid species was surveyed in this study. Based upon a tube hemagglutination assay established for domestic cats, 80% of felids had type-A, 18% type-B, and 2% type-AB blood. Felids in the Puma group and African and Asian golden cats had blood type B, whereas all other species were found to have blood type A. Two cheetahs and one bobcat had type-AB blood. Red cell glycolipids analysed by high performance thin layer chromatography revealed a similar ganglioside pattern in wild cats as reported in domestic cats. Independent of the AB blood group system, incompatible blood crossmatch reactions were detected between different felid groups. In conclusion, wild felids display the same AB-erythrocyte antigens as domestic cats, and the same blood typing procedures can be applied for wild and domestic felids.     

Sex determining of cat embryo and some feline species

Sex identification in mammalian preimplantation embryos is a technique that is used currently for development of the embryo transfer industry for zootechnical animals and is, therefore, a resource for biodiversity preservation. The aim of the present study was to establish a rapid and reliable method for the sexing of preimplantation embryos in domestic cats. Here we describe the use of nested PCR identify Y chromosome-linked markers when starting from small amounts of DNA and test the method for the purpose of sexing different species of wild felids. To evaluate the efficiency of the primers, PCR analysis were performed first in blood samples of sex-known domestic cats. Cat embryos were produced both in vitro and in vivo and the blastocysts were biopsied. A Magnetic Resin System was used to capture a consistent amount of DNA from embryo biopsy and wild felid hairs. The results from nested PCR applied on cat blood that corresponded to the phenotypical sex. Nested PCR was also applied to 37 embryo biopsies and the final result was: 21 males and 16 females. Furthermore, beta-actin was amplified in each sample, as a positive control for DNA presence. Subsequently, nested PCR was performed on blood and hair samples from some wild felines and again the genotyping results and phenotype sex corresponded. The data show that this method is a rapid and repeatable option for sex determination in domestic cat embryos and some wild felids and that a small amount of cells is sufficient to obtain a reliable result. This technique, therefore, affords investigators a new approach that they can insert in the safeguard programmes of felida biodiversity.     

Earliest “Domestic” Cats in China Identified as Leopard Cat (Prionailurus bengalensis)

The ancestor of all modern domestic cats is the wildcat, Felis silvestris lybica, with archaeological evidence indicating it was domesticated as early as 10,000 years ago in South-West Asia. A recent study, however, claims that cat domestication also occurred in China some 5,000 years ago and involved the same wildcat ancestor (F. silvestris). The application of geometric morphometric analyses to ancient small felid bones from China dating between 5,500 to 4,900 BP, instead reveal these and other remains to be that of the leopard cat (Prionailurus bengalensis). These data clearly indicate that the origins of a human-cat ‘domestic’ relationship in Neolithic China began independently from South-West Asia and involved a different wild felid species altogether. The leopard cat’s ‘domestic’ status, however, appears to have been short-lived—its apparent subsequent replacement shown by the fact that today all domestic cats in China are genetically related to F. silvestris.     

Glycolytic enzyme activity is essential for domestic cat (Felis catus) and cheetah (Acinonyx jubatus) sperm motility and viability in a sugar-free medium

We have previously reported a lack of glucose uptake in domestic cat and cheetah spermatozoa, despite observing that these cells produce lactate at rates that correlate positively with sperm function. To elucidate the role of glycolysis in felid sperm energy production, we conducted a comparative study in the domestic cat and cheetah, with the hypothesis that sperm motility and viability are maintained in both species in the absence of glycolytic metabolism and are fueled by endogenous substrates. Washed ejaculates were incubated in chemically defined medium in the presence/absence of glucose and pyruvate. A second set of ejaculates was exposed to a chemical inhibitor of either lactate dehydrogenase (sodium oxamate) or glyceraldehyde-3-phosphate dehydrogenase (alpha-chlorohydrin). Sperm function (motility and acrosomal integrity) and lactate production were assessed, and a subset of spermatozoa was assayed for intracellular glycogen. In both the cat and cheetah, sperm function was maintained without exogenous substrates and following lactate dehydrogenase inhibition. Lactate production occurred in the absence of exogenous hexoses, but only if pyruvate was present. Intracellular glycogen was not detected in spermatozoa from either species. Unexpectedly, glycolytic inhibition by alpha-chlorohydrin resulted in an immediate decline in sperm motility, particularly in the domestic cat. Collectively, our findings reveal an essential role of the glycolytic pathway in felid spermatozoa that is unrelated to hexose metabolism or lactate formation. Instead, glycolytic enzyme activity could be required for the metabolism of endogenous lipid-derived glycerol, with fatty acid oxidation providing the primary energy source in felid spermatozoa.     

Use of embryonic and somatic cells for production of transgenic domestic animals

In contrast to the highly developed genetic modification systems available for manipulating the mouse genome, at this time only simple gain of function modifications can be undertaken in domestic species. Clearly, the greatest barrier to gene targeting in domestic species has been the unavailability of cell lines that can be modified in vitro and still be used to generate a living organism. In the mouse, the embryonic stem (ES) cells and embryonic germ (EG) cells have fulfilled that role. While the nuclear transfer procedures have solved this problem in sheep and cattle, in swine ES and EG cells are still needed. In addition, targeting in domestic species is affected by the need to develop targeting constructs containing isogenic DNA regions. As a result, it is necessary to isolate the gene of interest, sequence required regions, and develop isogenic targeting constructs by technologies such as long-range PCR. On the positive side, enrichment protocols developed in the mouse can be applied to domestic species, thus facilitating the identification of correctly modified cell lines. Hence, progress in mammalian cloning, the development of EG cell lines, and advances in gene targeting presently allows the introduction of precise genetic modifications into the domestic animal genome.     

Long term effects of ART: What do animals tell us?

Early stages of mammalian embryonic development are now known to be very sensitive to their microenvironment, with long term effects on fetal, postnatal, and adult health, thus extending to these early stages the concept of Developmental Origin of Health and Disease (DoHaD). In this scientific context, and with 3% of births in developed countries, safety of Assisted Reproductive Techniques procedures becomes a matter of concern. Besides, embryo technologies in domestic mammals, using huge number of embryos, do not seem to evidence heavy impacts on adult phenotypes. This paper first discusses what can or cannot be concluded from farm animal data, then develops long term effects of ART procedures (ovarian stimulation, in vitro fertilization and embryo culture) evidenced in model species (mainly mouse model). Recent literature demonstrates both individual and cumulative effects of each ART procedure on fetal and postnatal phenotypes. In a second part, because they are sources for further perturbations, immediate effects of ART on early embryo phenotypes at the cellular and molecular levels are described in both farm animals and model species. Mechanistic hypotheses supporting these ART induced phenotypic alterations are subsequently considered. Finally, taking into account interspecies differences in the mechanisms likely to be involved, the relevance of results obtained in animal models for human ART are discussed.     

The application of transgenic techniques for the improvement of domestic animal productivity

The application of transgenesis techniques to domestic animals has now been achieved in all the major species of domestic animals. In this review, the progress towards genuine practical applications of the technique is examined. Areas which appear to have made progress during the past several years include the evaluation of animals with elevated growth hormone levels, the introduction of novel metabolic pathways, the expression of transgenes in the mammary glands of pigs and sheep, and the real possibility that functional immunoglobulin genes might be used to confer genetically-inherited disease resistance to commercially-valuable animal breeds.     

Babesia leo n. sp. from lions in the Kruger National Park, South Africa, and its relation to other small piroplasms.

Babesia leo, a small piroplasm isolated from lions in South Africa is described as a distinct species based on a phylogenetic analysis of the 18S rRNA gene. Intraerythrocytic trophozoite and merozoite stages of B. leo are morphologically indistinguishable from other small piroplasms of felids. Previous studies showed that B. leo was biologically and antigenically distinct from B. felis, which is known to infect wild and domestic felids in South Africa. Molecular characterization showed strong support for the phylogenetic seperation of B. leo as a distinct species from B. felis and other felid piroplasms. Phylogenetic analysis also showed that Babesia microti and all of the felid piroplasms from Africa with known 18S rRNA gene sequences available, including B. leo, formed a single, separate clade, sister to the other babesial and theilerial piroplasm parasites.     

Reproductive biology and a genome resource bank of Felidae

The main achievements in applying modern reproductive technologies to the banking of the genetic resources of the Felidae family are reviewed. The classification of felids at the level of species and subspecies is revised in the light of recent molecular data. Special emphasis is made on such mainstream technologies as semen collection and cryopreservation followed by artificial insemination, as well as on in vitro maturation and fertilization of oocytes combined with the culture of in vitro-derived felid embryos.