This review consist of a complete recompilation of advances made in in vitro embryo production (IVP) and cellular therapies in the domestic cat and wild felids species. Actually, the domestic cat is considered a valuable model for the development of assisted reproductive techniques and cellular regenerative therapies that might be used in the conservation of endangered felids. The in vitro embryo production technologies such as in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI) and somatic cell nuclear transfer (SCNT) have been applied in different species of small wild felid and big felids, resulting in live birth in some cases. However, more studies are needed to improve the efficiency of these techniques and maximize their use in the reproduction and conservation of wild felids. On the other hand, the mesenchymal stem cells (MSCs) therapies have been increasingly used in the veterinary medicine. Recent studies had reported the use of MSCs in the treatment of some chronic diseases in the domestic cat. For these reasons, the MSCs therapies are projected as a promising alternative for the treatments of chronic diseases that might affect wild felids that live in zoos, which could be caused by their captive environment conditions.
- wild felids
- domestic cat
- in vitro fertilization
- somatic cell nuclear transfer
- mesenchymal stem cells
Actually, the domestic cat is considered a valuable model for the development of assisted reproductive technologies (ARTs) and cellular regenerative therapies that might be used in the conservation of endangered felids [1, 2]. At present, according to the Red List of Endangered Species of the IUCN, 17 species of wild felid are classified as endangered and 8 as near threatened . This has led to the implementation of programs based in ARTs for the genetic preservation of endangered felids in zoological institutions from the USA and countries of Latin America like Mexico and Brazil [4, 5]. The
Regarding cellular therapies, cellular resources that might be potentially used in cell therapy, have been obtained in different species. The use of methodologies for cellular reprogramming has allowed the production of pluripotent and multipotent cells. However, it has been preferred that those methods that do not implicate a dependence on the expression of exogenous genes or changes at the cellular level may affect the viability of the cell for the use in therapy.
The use of stem cell for regenerative therapy in cats has been stimulated by the possibility to treat severe diseases such as chronic renal failure that is the main cause of death in felines, asthma and chronic feline gingivostomatitis. The domestic cats also represent a good model for the study of the efficacy of stem cell therapies for human diseases. Furthermore, it has been postulated that the use of a less differentiated cell type, as the mesenchymal stem cells, like nucleus donor, can improve the efficiency of the SCNT technique, which could be applicable in the conservation of endangered felid species.
In vitroembryo production systems in the domestic cat and wild felids
The studies related to the use of ARTs in felids have increased considerably during the last years . In 1990s, several studies reported the live birth of different big felid as the cheetah (
In vitrofertilization (IVF)
The first IVF report in felids was made in the domestic cat in 1970, in which cat oocytes were co-cultured
In vitrofertilization in the domestic cat
A series of studies have been made to improve the efficiency of the IVF. However, the domestic cat embryos generated by IVF have a reduced developmental capacity compared to the
|Cleavage rate (%)||Morula rate (%)||Blastocyst rate (%)||Pregnancy rate (%)||Live birth rate (%)|
|62.9–88.2||40–47||33.2–71.3||31–83.3||31–83.3||[2, 8, 22, 26, 29, 34]|
|50.2–71.5||52.2–62.7||30.0–61.1||40||20||[2, 27, 28, 105]|
|eCG + IVM||64.9||74.2||32.9||66.6||33.3||[Unpublished data]|
One of the biggest problem of the IVP systems in the domestic cat, is the low competence of the
Our research group evaluated the individual effect of both gonadotrophins (pFSH and eCG) in the morphological quality and gene expression pattern of cumulus-oocyte complexes (COCs). Regarding the pFSH treatment, our study demonstrated that pFSH enhanced the morphological quality of COCs and increased the expression of the gonadotrophin receptor
Actually, our research group has used the mild ovarian stimulation with eCG for the
This protocol was used in the production of embryos by IVF (Table 1). Additionally, we evaluated the
In general, the transfer of felid embryos has been successfully made at earlier stages, at the 1–2 cell stage  and morula or early blastocyst stages [16, 32]. Kanda et al. evaluated the transfer of cat embryos at the morula stage, early blastocyst stage after 4–6 days of IVC and blastocyst stage after 7 days of IVC. All the recipients that received embryos at the morula stage (4/4, 100%) and three recipients that received early blastocyst (3/5, 60%) became pregnant. However, no pregnancies were established after the transfer of 7-days blastocysts (0/3) . It has been postulated that this might be due to the zona pellucida integrity of 7-days cat blastocysts, most of the blastocysts begin to hatch the zona pellucida after day 6 and this might affect negative
In vitrofertilization in small wild felids
The first live birth report was made in the Indian desert cat (
|Fertilization rate (%)||Cleavage rate (%)||Morula rate (%)||Blastocyst rate (%)||Pregnancy rate (%)||Live birth rate (%)|
|Indian desert cat||67.0||—||—||—||25.0||25.0|||
|African wildcat||74.0||—||—||—||66.6||33.3||[2, 35]|
In vitrofertilization in big felids
Actually, only a few studies have reported the embryo production by IVF in big felid. The first successful report of IVF was made in tiger (
IVF have been performed in the Puma as well; the semen samples collected from male specimens and used for the IVF showed high degree of pleomorphism (82–99%). Despite that, an overall fertilization rate of 43.5% was obtained . Subsequently, the IVF in the Cheetah was described; after IVF an overall fertilization rate was only 26.6%. However, the fertilization rate varied from 0 to 73.3% among males . Previous reports have described a high proportion of abnormal spermatozoa/ejaculate in the Cheetah (31–97%) [45, 46]. A decade later, it was described the IVF in the lion (
In resume, the IVF has been used more in small wild felids than in big felids, this is mainly due to the easier manipulation of small wild felids during the laparoscopic oocyte retrieval and semen collection procedure. Furthermore, in the case of some small felids species, the generated embryos can be transferred to a domestic cat recipient producing live offspring. Additionally, the high pleomorphism incidence in the spermatozoa of some big felid species, reduces the efficient of IVF in those species.
2.2. Intracytoplasmic sperm injection (ICSI)
Actually, the fertilization techniques assisted by micromanipulation are used widely in the human fertility clinics, allowing the
2.2.1. Intracytoplasmic sperm injection in the domestic cat
The first ICSI-derived domestic cat embryos were produced using
2.2.2. Intracytoplasmic sperm injection in small wild felids and big felids
No many studies related to the ICSI in wild felids have been published. The first report was made in the jaguarundi (
|Cleavage rate (%)||Morula rate (%)||Blastocyst rate (%)||Pregnancy rate (%)||Live birth rate (%)|
|Domestic cat||57.0–81.6||50.8–81.0||6.6–42.9||33.3–50.0||22.2–50.0||[8, 9, 50]|
It has been described that the generation of lion embryos by ICSI using sperm collected by percutaneous epididymal sperm aspiration (PESA) from two vasectomized male lions. After ICSI, all the cleaved embryos reached the morula stage at day 5 of culture . Subsequently, it was described that the lion oocytes are able to mature
More research is needed to evaluate the ICSI in big felids. The ICSI allows the
2.3. Somatic cell nuclear transfer (SCNT)
During the SCNT, the oocyte cytoplasmic factors are capable to reprogram the expression pattern of a somatic cell into a pluripotent embryonic state, allowing it to initiate the early development . The fact that a differentiated cell must be reprogramed to a pluripotent stage made the SCNT a complex technique with a reduced efficiency. Different factors may affect the efficiency of the SCNT, some of these are: the quality of the donor nucleus or the recipient oocyte and the synchronization between the donor nucleus and the oocyte. These factors can lead to an inadequate reprogramming of the donor genome and to a reduced embryo development . Despite this low efficiency, the SCNT is the only technique that may generate genetically identical individuals, which bring the possibility to rescue the genetic material of endangered species.
2.3.1. Somatic cell nuclear transfer in the domestic cat
The first domestic cat produced by SCNT was made using cumulus cells as nucleus donors for metaphase II enucleated oocytes. A live kitten was born 66 days after the embryo transfer . Subsequently, it was described the use of cat fetal fibroblasts and adult fibroblasts as nucleus donors for SCNT. No statistical differences were observed in the fusion, cleavage and blastocyst rates when fetal or adult fibroblasts were used. Furthermore, live birth was obtained using both type of cells .
In addition, the advances of the SCNT in felids have led the production of transgenic animals. A domestic cat that expresses the red fluorescent protein was generated using a retroviral vector for the incorporation of the transgene in the somatic cells used as nucleus donor . A year later, it was reported the birth of a transgenic cat that express the green fluorescent protein, which was generated using a lentiviral vector to modify the donor cells . The objective of these studies was to potentiate the use of the domestic cat as a biomedical model for the study of analogues diseases in humans. This proves that these techniques could be implemented to generate genetically identical animals that have integrated coding genes for specific human diseases.
2.3.2. Somatic cell nuclear transfer in small wild felids
The scares availability of gametes is one of the biggest problem for the
In the leopard cat (
2.3.3. Somatic cell nuclear transfer in big felids
There are scarce reports regarding to the iSCNT in big felids. The first report was made in the tiger, in which pig oocytes were used as recipient cytoplast. However, a blastocyst rate of only 0.7% was obtained . More recently, leopard, tiger and lion cloned embryos were generated by iSCNT, using rabbit oocytes as cytoplast recipients. This study described that the rabbit oocytes were capable to reprogram big felid somatic cells. However, the blastocyst rate after
On the other hand, several methods have been implemented to improve the low efficiency of the SCNT such as embryo aggregation. This consists of the
|Clevage rate (%)||Morula rate (%)||Blastocyst rate (%)||Prenancy rate (%)||Live birth rate (%)|
|Domestic cat||57.6–98.2||4.0–49.5||2.0–47.7||20.0||20.0||[13, 14, 54, 63]|
|African wildcat||79.0–89.0||35.0–51.0||17.0–33.0||40.0–50.0||25.0||[13, 14]|
In resume, the iSCNT have a reduced efficiency and it seems that these problems are more evident when the species used as nucleus donor are more distant phylogenetically from the domestic cat. This might be related to an incomplete donor nucleus reprogramming. Regarding the big felids, it has been proved that pig and rabbit oocytes are able to reprogram big felid donor cells, but they are not as efficient as domestic cat oocytes. The embryo aggregation has been postulated as a method that improves the developmental capacity of big cat cloned embryos. However, the developmental capacity of these embryos is reduced compared to domestic cat cloned embryos. More studies are needed to improve the reprograming events that allow the development of iSCNT-derived embryos in big felids.
2.4. Gene expression analysis as indicator of developmental capacity in felid embryos
During the early development, the expression of specific genes allows the embryo progress from one stage to the next . For this reason, the relative quantification of mRNA from crucial genes during the early development is considered an adequate indicator of the embryo quality . The pluripotency markers
It has been described that the gonadotrophin treatments in the domestic cat improve the gene expression pattern in the COCs and in the produced embryos. The ovarian stimulation of domestic cat with pFSH enhances the embryo developmental capacity and increases expression of
Furthermore, in the domestic cat, it has been described that the
3. Cellular regenerative therapies in felids
The challenge in feline cell therapy is to offer treatment options to different diseases and to use stem cells in endangered animals is another challenge. This latter challenge is even more complicated because of the low availability of tissue to isolate stem cells in these species. However, biotechnology can bring us closer to the possibility of improving techniques for obtaining stem cells for therapy and tissue regeneration that may also contribute to the conservation of these individuals. This review tries to cover the main aspects of stem cells in domestic cats and its future application in wild cats, as well as their obtaining and characterization.
3.1. Induced pluripotent feline stem cells
The induced pluripotent stem cells (iPSCs) are generated by the induced expression of pluripotency transcription factors in cells that normally do not express. In this way, stem cells from somatic and differentiated cells can be obtained. Takahashi and Yamaka were the first to develop and implement this technique in 2006, who induced somatic cells mouse in iPSC by transfection of the cells with the transcription factors “OSKM” corresponding to transcription factor binding octamer 3/4 (Oct3/4), high protein group mobility-related gene Sry2 (Sox2), Kruppel factor 4 (Klf4) and c-Myc oncoprotein .
To date, there are reports of iPSC generated from endangered feline species such as snow leopard, tiger, jaguar and African serval [70, 71]. Protocols for induction to pluripotency in wild cats conclude that Nanog is a key factor in reprogramming . For this reason, reprogramming cocktails included the four Yamanaka factors plus Nanog and use culture medium supplemented with LIF and SFB. When Nanog was removed from the cocktail, the reprogramming efficiency was greatly reduced and the colonies reached only up to the seventh pass (P7) . On the other hand, the inclusion of Nanog made that the iPSCs colonies could be expanded
Despite all these difficulties, the therapeutic and conservation potential that can be reached with cellular reprogramming in domestic and wild felids is promising.
3.2. Mesenchymal stem cells (MSC)
Mesenchymal stem cells (MSC), also called by some authors, as mesenchymal stromal cells are unspecialized cells that have the ability to self-renew by cell division and differentiate into specialized cells . The MSCs are involved in the tissue regeneration by two different mechanisms. They may contribute directly to tissue repairing by differentiation into specific cell phenotypes such as, ligaments, tendons or alternatively fibroblasts, but not necessarily exclusive. These adult stem cells have the ability to produce extracellular matrix and bioactive proteins such as growth factors, anti-apoptotic and chemotactic agents that have an important effect on the dynamic cellular, production of anabolic effects, stimulate neovascularization and additional recruitment of stem cells the site of injury. Stem cells recruited at these sites can differentiate and/or produce biologically active peptides . MSCs have a fibroblast-like morphology, must be adherent to plastic, express surface molecules like CD105, CD73 and CD90 and have a negative expression of CD45, CD34, CD14 o CD11b, CD79α o CD19 and HLA-RD. In addition, the MSCs are capable to differentiate
3.2.1. Tissues source of mesenchymal stem cells (MSCs) in cats
In animals and humans, different tissues represent a potential resource for obtaining MSCs. The bone marrow was the first tissue explored; one of the best sources of production and so far the most widely used in cell therapy. However, other sources such as adipose tissue, have been investigated and successfully used. In the field of veterinary medicine, depending on the target species, the tissue from which these cells are obtained is crucial and can be influenced by the weight, size and the handling of the patient. However, the method for obtaining these tissues does not always correlate with a good source of MSCs. Therefore, the investigation of other resources for obtaining MSCs in different species is important, considering that the results between species cannot be extrapolated in all cases.
220.127.116.11. Bone marrow
MSCs isolated from bone marrow (BM-MSCs) has been described in many animal species such as mice, rabbits, horses, pigs, cattle, dogs and cat [78–83]. Maciel and colleagues conducted a study of the morphology of BM-MSC in felines and they noted the predominance of two types of cells, spindled and elongated . It has been reported that feline BM-MSCs have the potential to differentiate toward neuronal lineage . Feline BM-MSC after the fourth passage becomes more homogeneous and express surface markers like CD44, CD105 and CD29.
18.104.22.168. Peripheral blood
Peripheral blood is another tissue of which has been documented obtaining of MSCs. Compared to bone marrow, it is a safer and less painful resource with less subsequent complications. Feline peripheral blood (fPBMSCs) has been isolated from venous blood of cats and expanded in culture . The morphology and capacity for differentiation into mesoderm lineages of these MSCs is similar to others obtained from different feline tissues. However, these MSCs require a special culture medium containing 5% autologous plasma (AP) and 10% fetal bovine serum (FBS), showing a similar growth curve that others reports because its proliferation is limited to the seventh passage . These cells express CD44 and CD90 surface markers and are negative to major histocompatibility class II and CD4.
22.214.171.124. Fetal membranes
The interest of MSCs derived from fetal and extra-embryonic tissues has increased because of its usefulness in the field of regenerative medicine [75, 92–94]. In felines, these MSCs are obtained from tissues discarded after birth or from procedures such as cesarean operation and ovariohysterectomy. These cells have been isolated and successfully expanded. It was reported that these cells have an adequate level of homogeneity at the passage 3, as it was found in canines . Unlike other tissues, the cell population of the feline AMSC increases considerably after the passage 7, with higher values than those reported in horses and dogs, the cell number tends to increase along with number passages . Additionally, the viability of these cells after cryopreservation was similar to the viability of fresh cells, which is ideal for cell banks and future applications in cell therapy . AMSCs feline express CD73 and CD90 surface markers but did not express hematopoietic specific markers CD34, CD45 and CD79 .
126.96.36.199. Adipose tissue
Adipose tissue is abundant and more accessible than other tissues and is the most commonly used in cellular veterinary therapies . Cellular therapies for animals using cells derived from adipose tissue (AMSC) have been used to treat osteoarthritis, injuries of ligaments and tendons in canine and equine, feline gingivostomatitis with good results and other pathologies that are being studied as chronic renal failure, asthma and chronic enteropathy in domestic cats [91, 96–98]. Feline AMSCs have been isolated and characterized from black-footed cat, an endangered feline . Both cat and black-footed cat AMSCs showed potential adipogenic, osteogenic and chondrogenic differentiation, but the surface marker expression of black-footed cat AMSCs is unknown and it could be difficult to evaluate because of the specificity of the antibodies. However, these results obtained from endangered felines can be used in ARTs for their conservation such as SCNT, or potentially be used in cell therapy of individuals of the same species.
188.8.131.52. Potential sources of MSCs
Brain, muscle, synovial fluid, tendon, placenta and dental pulp are others potential sources for obtaining MSCs in several species [99–101]. Some of these tissues could eventually be used to isolate mesenchymal stem cells in felines to perform extensive characterization procedures, which could defined if these cells have any potential to be employed in cell therapy. Considering that stem cells can be isolated for other purposes such as species conservation, spermatogonial stem cells (SSCs) can be isolated from testicular tissue. In domestic cats, these cells were successfully isolated and cultured
3.3. Doses and routes of administration for MSC treatment in domestic cats
To date, mesenchymal stem cells are the only ones that have been evaluated in treatments of certain pathologies in domestic cats. The establishment of the treatment doses of MSCs in cats has been a main point to obtain the expected therapeutic effect and to reduce the adverse effects that may occur. After the adequate characterization of MSCs depending on their place of origin, the challenge is to evaluate the dose response and the route of administration. It has been reported that the intravenous route is the most chosen in the treatments with MSCs. Allogenic and autologus administration of MSCs in cats have not shown any side effects [96, 98, 104]. More studies are still required to determine alternative administration routes that may increase the treatment efficacy. Furthermore, the doses used are still a topic of discussion, but some doses are proposed for certain treatments (Table 6). The effects of reported treatments suggest that they can be seen since the third dose of MSCs administration .
|domestic cat||3 doses of 2 × 106 cells/kg||Intravenous||Chronic renal failure||10–15 years||Allogenic|||
|Domestic cat||2 doses of 2 × 106 cells/kg||Intravenous||Enteropathy||7–15 years||Allogenic|||
|Domestic cat||1 doses of 5 × 106 cells/kg||Intravenous||Gingivostomatitis||1–14 years||Autologus|||
Great advances have been made in the