Stem cells from domestic animals are important for deriving therapeutic applications, generating models for human diseases, and developing alternative methods for conservation and preservation of endangered species. Cat embryonic stem like-cells (cESC) have been derived from in vivo and in vitro-produced blastocysts (Gómez et al. 2010 Theriogenology 74, 498). Although cESC colonies can be cultured in an undifferentiated state for several passages, they gradually lose their capacity to maintain pluripotency. Therefore, to maintain pluripotency of cat ESC during in vitro culture, it is necessary to develop a better understanding of the mechanisms involved in self-renewal and differentiation, as well as to enhance in vitro culture conditions. In mouse ESC, the Wnt/β-catenin signalling pathway has been identified as an essential pathway for maintenance of pluripotency and avoidance of differentiation (Kirby et al. 2012). Nonetheless, activation of the Wnt signalling and its role in human ESC remains controversial. Wnt activation is mediated by the cytoplasmic protein – Disheveled – that inactivates a multi-protein complex, including glycogen synthase kinase 3 (GSK3β) and inhibits β-catenin degradation. In the present study, we evaluated the role of Wnt/β-catenin signalling in self-renewal and maintenance of an undifferentiated state of cat ESC. Cat ESC were cultured on mitotically inactivated cat embryonic fibroblasts (CEF) in modified-ESC medium (DMEM-F12, 200 mM l-glutamine + 0.14% β-mercaptoethanol, 1.25% nonessential amino acids, 15% knockout replacement serum, 5% fetal bovine serum, 1000 U mL–1 leukemia inhibitory factor (LIF), and 10 ng mL–1 basic fibroblast growth factor, bFGF) and supplemented with GSK3β inhibitor -SB216763 (10 μM v. 20 μM v. 0 μM). The concentrations of β-catenin and GSK3β in cat ESC colonies were measured by ELISA, and the effect of GSK3β on cat ESC was measured by their cell size, morphology, expression of pluripotent markers at the mRNA and protein level (POU5F1, NANOG, SOX-2), and their ability to differentiate into ectoderm cell lineage. Our results indicated that GSK3β inhibitor inactivates GSK3β, leading to an increase in total β-catenin in cat ESC. Moreover, colonies cultured in the presence of GSK3β inhibitor showed flattened shape and irregular borders (compared with the dome shape and marked borders in nontreated colonies), and both the concentration and the passage significantly reduced the colony cell size, the expression of POU5F1 and SOX-2 at the mRNA and protein level, and lowered their ability to differentiate into neurogenic-like cells compared with that of colonies cultured without the GSK3β inhibitor. Even though we demonstrated that the Wnt/β-catenin signalling pathway influenced the expression of POU5F1 and SOX-2 of cat ESC, it is not clear why the accumulation of β-catenin did not enhance self-renewal. Further studies are required to evaluate the influence of GSK3β inhibitor and other small molecules on self-renewal of cat ESC cultured without the presence of a feeder cell layer.

In many mammalian species, surface markers have been used to obtain enriched populations of spermatogonial stem cells (SSCs) for assisted reproduction and other applications; however, little is known about the expression patterns of feline SSCs. In this study, we assessed expression of the SSC surface markers commonly used in other species, KIT, ITGA6, CD9, GFRalpha1, ADGRA3, and THY1, in addition to the less frequently used pluripotent markers TRA-1-60, TRA-1-81, SSEA-1, and SSEA-4 in SSCs of both prepubertal and adult domestic cats (Felis catus). To further characterize cat SSCs, we sorted cells using SSC-specific markers and evaluated the expression of the pluripotent transcription factors NANOG, POU5F1, and SOX2 and the proto-oncogene MYC within these populations. We concluded that SSC surface markers used in other mammalian species were not specific for identifying cat SSCs. However, the pluripotent markers we evaluated were more specific to cat spermatogonia, and the presence of SSEA-1 and SSEA-4 in fewer and primarily individual cells suggests that these two markers may be used for enrichment of cat SSCs. The expression of pluripotent transcription factors at mRNA level by single-stained cells positive for SSEA-4 and by dual-stained cells positive for both GFRalpha1 and SSEA-4 reflects the undifferentiated stage of cat SSCs. The absence of transcription factors in double-stained cells positive for only one marker implies the loss of the stem cell-like identity with the loss of either GFRalpha1 or SSEA-4. Further investigation is warranted to elucidate the biological characteristics of these spermatogonial subpopulations.

Vitrification remains a promising technique in the preservation of valuable genetic material; however, in the cat, success has varied. Live kittens have been produced from embryos vitrified at early cleavage stages, but phenotypic abnormalities in some kittens suggest possible epigenetic effects of the vitrification process. It has been reported that cryopreservation alters epigenetic events in somatic donor cells, which indirectly influences physical status of cloned offspring. However, extending post-warming in vitro culture of donor cells corrects these epigenetic modifications, resulting in normal embryos/clones. Accordingly, in the present study, vitrification was performed at the pronuclear stage to lengthen pretransfer culture time, and vitrified cat zygotes were assessed by analysing (1) histone acetylation/methylation, (2) global DNA methylation, (3) pluripotent gene expression, (4) in vitro development, and () in vivo viability. In vivo matured/IVF oocytes were vitrified in 15% dimethyl sulfoxide, 15% ethylene glycol, and 0.5 M sucrose at 16 h post-insemination (PI). After warming in 1.0 M sucrose at 38°C, embryos were fixed at 18 h or 40 h PI, and the nuclear intensity of either acetyl/dimethyl-H3K9 or 5-methylcytosine was determined by immunofluorescence. Results showed that at 18 h PI, mean H3K9ac intensity of vitrified embryos (11.8; n = 6) was higher than that of corresponding nonvitrified (fresh) controls (4.5; n = 6) and the fresh (3.2; n = 11) and vitrified (0.6; n = 7) 40-h groups (2-way ANOVA; P < 0.05). H3K9me2 in the fresh (36.9) and vitrified (32.5) 18-h embryos was similar but increased relative to both fresh (10.7) and vitrified (9.2) 40-h groups (P < 0.05). Mean DNA methylation (5MeC) in the fresh (31.6; n = 1) and vitrified (24.7; n = 3) 18-h groups was similar to that of the fresh 40-h group (19.8; n = 4) but higher than that of the vitrified 40-h group (15.0; n = 5; P < 0.05). To assess expression of POU5F1 and Nanog, qRT-PCR was performed on Day 8 blastocysts. Relative to controls (n = 9), mean POU5F1 and Nanog levels in vitrified blastocysts (n = 24) were 1.38- and 1.98-fold higher, respectively (one-way ANOVA; P > 0.05). In terms of in vitro development, Day 2 cleavage of vitrified zygotes (59%; n = 508) was similar to that of controls (66%; n = 340), but Day 8 blastocyst formation was reduced (9 v. 31%; t-test; P < 0.05). In vivo viability was assessed by oviducal transfer of 41 Day 1 embryos into 2 recipients. One pregnancy was established (50%), with 3 live kittens weighing 70, 79, and 131 g delivered without assistance on Day 65 of gestation. The 2 smaller kittens died within a few hours of birth, with the smallest exhibiting an umbilical hernia and organ exteriorization. The third kitten developed into a normal, healthy adult. In summary, mean H3K9me2, 5MeC, and POU5F1/Nanog expression of vitrified zygotes was similar to corresponding controls. H3K9ac increased at 18h PI as a result of vitrification, but was reduced after culture to 40 h PI. Although vitrified zygotes cleaved in vitro at rates similar to controls, blastocyst development was reduced. In vivo viability was demonstrated; however, postnatal survival of kittens produced was low.

In the first successful transfer of cat embryos (Theriogenology 11, 51–62), the uterus was accessed by midventral laparotomy. That surgical approach was the most widely used method for transferring cat embryos for more than two decades. Then, 10 to 15 years ago pregnancies were reported after early cleavage stage embryos were transferred to the oviduct of recipients using a laparoscopic technique. Even though laparoscopic oviducal embryo transfer has produced higher survival/pregnancy rates than were obtained previously there are valid reasons for establishing a minimally invasive, technically simple method for depositing morulae and blastocysts into the uterus of recipients. Thus, the purpose of the present project was to develop a technique for laparoscopic uterine embryo transfer in the cat. Recipients (n = 4) were gonadotropin-treated females (Theriogenology 81, 126–37) from which prevoulatory oocytes (n = 27–42/retrieval) had been recovered 7 or 8 days previously. The procedure for accessing the reproductive tract has been described (Theriogenology 71, 864–71). Briefly, after abdominal insufflation via a Veress needle, two 5-mm ports were inserted in the midline – one ~2.5 cm anterior to the umbilicus and the other between the two most posterior teats. An endoscope/camera and a Babcock forceps were placed in the anterior and posterior ports, respectively. After the left uterine horn was stabilised with the Veress needle, the Babcock forceps were gently applied at ~2–3 cm from the anterior tip. In the first two attempts, a 16 g × 5 cm thin-walled stainless steel (s.s.) trocar/cannula was inserted transabdominally such that it aligned with the anterior portion of the left uterine horn when elevated with the forceps. Then, either a 14-cm, 3.5 Fr tom cat catheter with a s.s. sharp-tipped stylette or a 20/22 g × 6 cm indwelling catheter was passed through the s.s. cannula and inserted into the uterine horn. In each case, the length of the s.s. cannula restricted depth of insertion of the catheter into the horn. Polyethylene tubing (PE10) containing the embryos was threaded through the catheter and embryos were expelled with a 1-mL threaded-plunger syringe. The failure to establish pregnancies after transfer of five or six Day 7 or Day 8 IVF-derived “fresh” embryos into the first two recipients was attributed to technical difficulties. So, for the third and fourth procedures, we shortened the s.s. trocar/cannula to 2.5 cm and, for insertion into the horn, a 20/22 g × 6 cm indwelling catheter was used. With the third procedure, in which cryopreserved d 8 IVF blastocysts were transferred into a Day 7 recipient, the failure was possibility due sub-optimal in vitro development of embryos after thawing on d 7. For the fourth transfer, 6 “fresh” Day 8 IVF blastocysts – 2 expanding and 4 in the early stages of emerging from the zona pellucida – were auto-transferred into a 3-year-old recipient. A singleton pregnancy was diagnosed by ultrasonography on Day 28 and a live, healthy male kitten (119 g) was born on Day 67. In summary, we demonstrated the feasibility of transferring in vitro-derived cat embryos into the uterus of recipients by the minimally invasive technique of laparoscopy.

Transplantation of mesenchymal stem cells (MSCs) isolated from bone marrow or adipose tissue is emerging as a promising tool for cell replacement therapy and regenerative medicine in domestic and endangered animal species. Defining the differentiation capability of adipose-derived mesenchymal stromal/stem cells (AMSCs) collected from different depot sites of adipose tissue will be essential for developing strategies for cell replacement therapy. In the present study, we compared the biological characteristics of domestic cat AMSCs isolated from visceral fat of the abdominal cavity (AB) with AMSCs from subcutaneous (SQ) tissue, and the functional capability of domestic and black-footed cat (Felis nigripes) AMSCs to differentiate into other cell types. Our results showed that both domestic and black-footed cat adipose-derived stromal vascular fractions contained AMSCs. Both domestic cat AB- and SQ-AMSCs showed important clonogenic ability and the minimal MSC immunophenotype as defined by the International Society for Cellular Therapy in humans. However, domestic cat AB-AMSCs had higher percentages of cells positive for MSCs-associated cluster of differentiation (CD) markers CD90(+) and CD105(+) (92% and 80%, respectively) than those of SQ-AMSCs (77% and 58%, respectively). Although these results may suggest that AB-AMSCs may be more multipotent than SQ-AMSCs, both types of cells showed similar expression of pluripotent genes Oct-4 and Klf4, except for higher expression of Nanog than in AB-AMSCs, and equivalent in vitro multilineage differentiation. Under appropriate stimuli, the black-footed cat and both domestic cat AB- and SQ-AMSCs differentiated not only toward mesoderm cell lineages but also toward ectoderm cell lineage, such as neuron cell-like cells. Black-footed cat AMSCs had more capability to differentiate toward chondrocytes. These results suggest that the defined AMSC population (regardless of site of collection) could potentially be employed as a therapeutic agent for both domestic and endangered diseased or injured felids.

Different feeder cells (FC) influence the isolation, proliferation, and self-renewal of cat embryonic stem cells (cat ESC; Gómez et al. 2010 Theriogenology 74, 498–515) possibly by secretion of growth factors that affect intracellular signalling pathways involved in self-renewal. Supplementation of the culture medium with fibroblast growth factor (FGF) stimulates the secretion of Activin A in mouse and human FC, which enhances undifferentiation in human ESC (Eiselleova et al. 2008 Int. J. Dev. Biol. 52, 353-363). Moreover, the Activin/Nodal pathway plays an important role in maintaining pluripotency of hESC through mechanism(s) in which FGF acts as a competence factor (Vallier et al. 2005 J. Cell Sci. 118, 4495–4509). Little is known about secretion of growth factors by cat FC and whether cat ESC use the activin/nodal pathway for their self-renewal. Our previous work has indicated that culturing cat ESC with bFGF enhances the stem cell replication and self-renewal (Gómez et al. 2010 Theriogenology 74, 498–515). Here we evaluated the effect of bFGF supplementation in the culture medium on the abilities of cat embryonic fibroblast (CEF) and mouse embryonic fibroblast (MEF) FC to: (1) secrete Activin A and (2) support undifferentiated growth of cat ESC. For experiment 1, mitomycin-C-treated CEF (n = 2) and MEF (n = 2) were, respectively, cultured with ESC medium supplemented with (1) LIF (1000 IU), (2) bFGF (10 ng mL–1), (3) LIF + bFGF, or (4) no factors. The medium for each condition was collected at 24 h after culture and Activin A protein concentration was detected with a feline Activin A-ELISA kit. Results showed that supplementation of ESC medium with bFGF with or without LIF significantly increased the secretion of Activin A in MEF (5256 and 7048 ng mL–1, respectively; P < 0.001), but not in CEF (150 and 131 ng mL–1, respectively). Moreover, differences in Activin A secretion were observed between both MEF cell lines (10 269 v. 2034 ng mL–1; P < 0.001). For experiment 2, cat ESC were cultured in CEF or MEF in the ESC medium supplemented with bFGF (10 ng mL–1), LIF (1000 UI), and an inhibitor of glycogen synthase kinase-3 β (GSK3-b), SB 216763 (2.1 µM mL–1). Results showed differences in morphology of cat ESC cultured in CEF or MEF, where colonies cultured in CEF had clearly defined borders and a tightly domed shape, with a high nucleus to cytoplasm ratio and prominent nucleoli. In comparison, ESC cultured in MEF had poorly defined borders and a flattened shape. In addition, the mean cell size of colonies at passage 8 (P8) cultured on CEF was larger (612 ± 0.9 µm) than that of those cultured on MEF (360 ± 0.5 µm; P < 0.001). Colonies cultured on MEF differentiated into fibroblast-like cells and other noncharacterised cell types after P8. These results clearly indicated that CEF do not secrete Activin A. The negative effect of Activin A on the morphology of cat ESC cultured on MEF may suggest a synergism between GSK3b inhibitor and Activin A that may induce differentiation, possibly into mesoendodermal cells (Teo et al. 2014 Stem Cell Rep. 3, 5–14). Studies that evaluate the effects of supplementing ESC medium with a lower concentration of Activin A may help to elucidate the importance of the Activin/Nodal pathway in cat ESC.

Chronic kidney disease is a major cause of mortality in cats (Boyd et al. 2008J. Vet. Intern. Med. 22, 1111–1117). Similarly, the black-footed cat (Felis nigripes; BFC) frequently suffers from kidney failure caused by amyloidosis (Terio et al. 2008 Vet. Pathol. 45, 393–400). Adipose tissue-derived mesenchymal stem cells (AMSC) are a valuable cell source in regenerative medicine for treating certain diseases, including those suffered by endangered species. In the domestic cat (DSH), AMSC have been isolated from subcutaneous (SQ; Quimby et al. 2011 J. Feline Med. Surg. 13, 418–426) and epididymal adipose tissue (Zhang et al. 2014 Stem Cell Rev. Rep. 10, 600–611). Whether AMSC isolated from visceral fat of the abdominal cavity (AB) have similar developmental potential has not been studied. In this study, we (1) compared the biological characteristics of DSH-AMSC isolated from AB and SQ adipose tissue, and (2) evaluated the functional capability of DSH and BFC-AMSC to differentiate into other cell types. The AB and SQ adipose tissues were harvested via laparoscopy or from an incision in the ventral abdomen, respectively. Tissues were digested with collagenase II (1 mg mL–1) at 37°C for 20 to 40 min with shaking at 150 rpm for 20 to 40 min. Cells from the stromal vascular fraction were cultured in DMEM-F12 medium with 12% fetal bovine serum under 5% CO2 in air at 38°C. Results showed that AB biopsies were smaller (1.2 ± 0.2 g) than that of SQ biopsies (3.6 ± 0.7 g). The mean number of nucleated cells per gram from AB biopsies (0.6 to 22 × 106) was similar to that of SQ biopsies (0.4 to 24 ×106). The cell-doubling numbers (days) per passage (P1 to P5) in both cell types remained constant (0.9 to 2.6), but SQ-AMSC at P5 required more cell doublings (4.5 ± 2.1) to reach 50% confluence. The AB-AMSC showed more colony-forming units (CFU; 7.0%) after 8 to 10 days of seeding at 8000 per cm2 than did SQ-AMSC (1.5% CFU). The SQ-AMSC did not form colonies at cell densities below 4000 per cm2. However, AB-AMSC colony formation only substantially decreased when the cell densities were below 1000 per cm2 (0.1%). Flow cytometry analysis revealed higher percentages of CD90+ (92%), CD105+ (80%), and CD146+ (17%) cells in AB-AMSC than in SQ-AMSC (77, 57, and 9%, respectively). Both AB and SQ-AMSC showed negative expressions of CD14–, CD45–, CD73–, and HLA-DR–. Gene expression analysis revealed that pluripotent genes Nanog, KLF4, Oct-4, and proto-oncogene C-Myc were expressed by both cell types, while Sox2 was not expressed in either type of AMSC. Under appropriate stimuli, DSH- and BFC-AMSC demonstrated differentiation potential towards adipogenic, osteogenic, chondrogenic, and neurogenic lineages. The AMSC from both species were less responsive towards osteogenesis than adipogenesis, and BFC cells had more capability to differentiate towards chondrocytes. These results suggest that the defined AMSC population (regardless of site of collection) could potentially be employed as a therapeutic agent for diseased or injured felids, both domestic and endangered.

As precursors to germline stem cells and gametes, there are many potential applications for primordial germ cells (PGC). Primordial germ cell-like cells have been generated from mouse embryonic stem cells and induced pluripotent stem cells, which subsequently were used to produce functional spermatozoa, oocytes, and healthy offspring (Hayashi et al. 2012 Science 338(6109), 971–975). Applying this approach to generate sperm and oocytes of endangered species is an appealing prospect. Detection of molecular markers associated with PGC is essential to optimizing the process of PGC induction. In the current study, in vitro-derived domestic cat embryos were assessed at various developmental stages to characterise the expression of markers related to the specification process of cat PGC. In vivo-matured, IVF oocytes were cultured until Days 7, 9, and 12 post-insemination. Then, embryos were assessed by RT-qPCR to determine relative transcript abundance of the pluripotency markers NANOG, POU5F1, and SOX2; the epiblast marker DNMT3B; the primitive endoderm marker GATA4; the PGC marker PRDM14; and the germ cell marker VASA; RPS19 was used as the internal reference gene. To validate the qPCR results, fibroblasts served as the negative control cells, whereas spermatogonial stem cells (SSC) served as the positive control cells for GATA4, PRDM14, and VASA. Total mRNA were isolated using the Cells-to-cDNA™ II Kit (Ambion/Thermo Fisher Scientific, Waltham, MA, USA) from either pools of 2 to 6 embryos or ~25 000 fibroblasts/SSC. A minimum of 2 biological replicates for each sample type was analysed, with transcript abundance detected in 2 technical replicates by SYBR Green chemistry. Student’s t-tests were performed on the ΔCts for statistical analysis. PRDM14, specific to the germ cell lineage, was detected as early as Day 7, suggesting the presence of PGC precursor cells. Compared with their levels at Day 7, PRDM14 expression was 0.34-fold lower in SSC (P < 0.05), whereas expression of VASA and GATA4 were 1964-fold and 144-fold higher, respectively (P < 0.05). This seems to emphasise the relative importance of PRDM14 in pre-germ cell stages. In general, all genes analysed were up-regulated from Day 7 to Day 9. This up-regulation was statistically significant for SOX2 and GATA4 (P < 0.05). Relative to that at Day 9, all transcripts were relatively less abundant at Day 12 (P < 0.05 for NANOG, POU5F1, SOX2, DNMT3B, and PRDM14). The data suggest that PGC specification takes place near Day 9, with peak specification activity concluding by Day 12. Although much needs be explored about PGC specification in the cat before applying induction and in vitro germ cell production techniques, these findings represent the first step towards a new potential strategy for preserving endangered and threatened felids.

In 2003, the first wild felid was produced by interspecies somatic cell nuclear transfer. Since then other wild felid clone offspring have been produced by using the same technique with minor modifications. This chapter describes detailed protocols used in our laboratory for (1) the isolation, culture, and preparation of fibroblast cells as donor nucleus, and (2) embryo reconstruction with domestic cat enucleated oocytes to produce cloned embryos that develop to the blastocyst stage in vitro and, after transfer into synchronized recipients, establish successful pregnancies.

The ability to cryopreserve oocytes is an effective method to retain valuable genetic material of mammals, including that of endangered animals. Embryos of domestic cats are amenable to cryopreservation, whereas their oocytes are much less cryo-tolerant. The capability of oocytes to survive cryopreservation is affected by several factors, one of which has been hypothesized to be the high concentration of intracellular lipids. To test this hypothesis, in this study we polarized lipids of cat oocytes and tested their cooling and freezing sensitivity. We found that the sensitivity of oocytes to cooling and cryopreservation does appear to be related to their high intracellular lipid content, as indicated by higher cryosurvival and development into blastocysts when intracellular lipids of in vitro matured oocytes were polarized before vitrification. However, polarization of all intracellular lipids was detrimental to development of embryos. Cell numbers in blastocysts derived from fully polarized/vitrified oocytes were significantly lower than those of partially polarized/vitrified or non-vitrified/fresh oocytes. Although embryos derived from fully polarized/vitrified oocytes developed to the blastocyst stage at higher rates than those of partially polarized/vitrified or non-centrifuged/vitrified oocytes, their in vivo developmental competence was compromised. When embryos derived from fully polarized/vitrified oocytes were transferred, although two recipients became pregnant, all implanted embryos were reabsorbed. In contrast, when embryos derived from oocytes that were only partially lipid polarized before vitrification and then were transferred, one recipient did become pregnant and produced a live healthy kitten. The present results suggest that other approaches to altering intra-cellular lipid levels in cat oocytes should be evaluated to improve their functional survival after cryopreservation.

The domestic cat is a mammalian species of particular importance due to their evolutionary history, role as companion animals and as a research model. Stem-cell-based therapies have been developed to improve health and well-being in domestic cats, as an alternative approach for the conservation of genetically valuable animals and preservation of endangered animals, and as an efficient method to produce genetically engineered cats for modeling human diseases. Successful application of stem-cell-based therapies in cats is dependent on development of robust methods to efficiently isolate stem cells and a better understanding of the mechanisms that control stem cell fate to differentiate these cells into specific phenotypes. This chapter describes recent progress on the isolation, culture, and characterization of pluripotent and multipotent stem cells and preliminary trials in regenerative medicine in domestic cats and prospects for applications to conservation and veterinary care of endangered felids.

Fishing cats (Prionailurus viverrinus) are small (6–15 kg) spotted cats from dispersed areas of Southeast Asia found mostly in wetland habitats. They are classified by the International Union for Conservation of Nature (IUCN) as endangered, with a decreasing population, due to habitat loss and degradation. Few studies have been done on applying assisted breeding techniques to the species, although the birth of a live kitten after IVF/embryo transfer (ET) has been reported (2006 Theriogenology 66, 1518–1524). Here, we describe the birth of a live fishing cat kitten using the technique of laparoscopic intratubal AI. A ten-year-old female who had served previously as an oocyte donor (5×) following gonadotropin treatment was administered a total of 5 IU of porcine FSH (Sioux Biochemical, Sioux City, Iowa) over 4 days (1×/day) followed by 10 IU of porcine LH on the fifth day. At approximately 28 h after LH treatment, the ovaries/oviduct were accessed by a laparoscopic technique comparable to that used for oviducal embryo transfer (ibid.). To deposit semen into the left oviduct, a 16-guage thin-wall trocar/needle was inserted into the abdominal cavity on the right side, approximately 1 cm lateral to the midline and approximately 2 to 3 cm below the umbilicus. A 14-cm open-end tom cat catheter was inserted into the 16-guage cannula (blunt) and the catheter tip was positioned underneath the fimbria overlaying the ovary. Then, a 50-mm length of 30-guage polytetrafluoroethylene (PTFE) thin-wall tubing containing approximately 30 μL of freshly collected semen was threaded through the catheter and the sample was expelled with positive pressure from a threaded-plunger 1-mL syringe. The left ovary contained 7 to 8 preovulatory (3–4 mm) follicles, 4 of which were manually ruptured immediately after deposition of semen with a 22-guage needle inserted through the 16-guage cannula. Then, with the 16-guage trocar/cannula in the same position (on the right side), the tip was redirected towards the right ovary and approximately 30 μL of semen was deposited underneath the fimbria as described above. The right ovary presented with 5 to 6 preovulatory (3–4 mm) follicles, 2 of which were punctured with the 22-guage needle after insemination. No ovulations were present on either ovary. The semen used for insemination was a fresh sample collected by electroejaculation from a 9-year-old male. The raw sperm concentration was 220 million mL–1, with 70% motility. The number of motile sperm deposited per oviduct was estimated to be approximately 4.6 million. The female was anesthetized 51 days later and radiography was done to determine her pregnancy status. A single fetus was present, so she was moved from an outdoor pen into a large indoor holding pen to allow for video-monitoring during the remainder of gestation. On Day 70, early signs of labour were observed and an elective Caesarean section was done approximately 4 h later. A live, healthy male kitten weighing 204 g was delivered. One year later, gonadotropin treatment/AI were repeated on the same pair. At approximately 30 h post-LH treatment, preovulatory follicles were present, but fewer than the previous treatment (5–6 total). Two fresh ovulation sites were seen on the left ovary. Pregnancy was not established. A reason for the failure was not apparent, unless it was age related.

Captive breeding efforts in felids, including assisted reproduction techniques, have had varied success depending on species. Spermatogonial stem cells (SSC), comprising a small percentage of germ cells in the testis, are progenitor cells with the ability to both self-renew and differentiate into spermatozoa throughout the life of the male. Manipulation of SSC for transplantation (SSCT) may allow the propagation of genetically important males, as demonstrated by the production of ocelot sperm following transplantation of ocelot mixed germ cells to domestic cat testes (Silva et al. 2012 J. Androl. 33, 264–276). Using specific cell surface markers, SSC have been isolated from mixed germ cells in several other species for SSCT, culture, and studying germ cell biology; however, expression may differ with species. Using the domestic cat as a model for exotic felids, we recently began evaluating the expression of surface markers in feline SSC. Previously, we determined that pluripotent markers SSEA-1, SSEA-4, TRA-1–60, and TRA-1–81 were more specific to cat spermatogonia than SSC surface markers GFRα1 and GPR125 used in other species, with SSEA-1 and SSEA-4 expressed in the fewest cells (Powell et al. 2011 Reprod. Fertil. Dev. 24, 221–222; Powell et al. 2012 Reprod. Fertil. Dev. 25, 290–291). Our current goal was to 1) confirm the presence of SSC within SSEA-1+ and SSEA-4+ cell populations by the ability to colonize following SSCT; 2) compare the effectiveness of transplanting SSC purified by flow cytometry versus mixed germ cells; and 3) show that depletion of endogenous germ cells before SSCT, usually performed by irradiation or chemotherapy in other studies, is not necessary when using sexually immature recipients. Mixed germ cells from 8 to 12 adult testes were pooled, stained for SSEA-1 or SSEA-4, and sorted by flow cytometry. SSEA-1+, SSEA-4+, or mixed germ cells were then labelled with the membrane dye PKH26 (Sigma MINI26) and injected into the testes of six 5-month-old and six 6-month-old cats at the site of the external rete testis after carefully microdissecting the head of the epididymis away from the testis. Injections contained an average of 230 000 sorted or 10 × 106 mixed germ cells suspended in 80 μL of DMEM/F12 + 3 μL of Trypan Blue (T8154, Sigma, St. Louis, MO, USA). Testes were harvested 10 to 12 weeks post-SSCT and bisected, half snap-frozen for later cryosectioning and the other half enzymatically digested to loosen seminiferous tubules for immediate evaluation. Fluorescence was detected in the testes of both 6-month-old males that received injections of mixed germ cells, one 6-month-old male injected with SSEA-4+ cells, and two 5-month-old males, one injected with SSEA-4+ cells and one with SSEA-1+ cells. Results indicate that SSC are found in both SSEA-1+ and SSEA-4+ cell populations, but that purification of SSC is not necessary for successful SSCT. Additionally, SSC colonization in cats is possible without depletion of endogenous cells in sexually immature recipients.

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. (C) 2014 Elsevier Inc. All rights reserved.

We evaluated the cortisol response of adult female eland (n=8) that were handled in hydraulic chute daily or 3×/week. Females were divided into two groups and each group (n=4) successively received two estrous cycle synchronization treatments: (1) two injections of prostaglandin (PG-PG) F2α at 11 day intervals and (2) oral administration of altrenogest for 7 days and an injection of PGF2α on day 7 (Alt-PG). Blood samples were collected 3×/week during the synchronization (Synch) and expected luteal phase (Nonintensive) periods, and daily during the expected time of induced (Intensive 1) or natural (Intensive 2) estrus. Overall, mean cortisol levels were highest during Intensive 1, followed by Intensive 2, Synch and Nonintensive periods. Individual eland were the most significant source of variation for cortisol level. The frequency of handling and the synchronization treatment significantly affected cortisol levels in 3/8 and 4/8 females, respectively. In conclusion, in response to increased frequency of handling, eland cortisol levels rose transiently and returned to baseline within few days after more intensive handling. Thus, the eland females were tolerant to and recovered from the effects of repeated daily handling.

The cryopreservation of ovarian tissue is linked to a wide range of possible applications, from oocyte harvesting to allo- and xenotransplantation. These procedures have significant potential for the preservation of valuable genetic material and endangered-species conservation. The objectives of the present study were to (1) compare viability of preantral follicles obtained from fresh v. vitrified feline ovarian cortex, (2) evaluate the effect of apoptotic inhibitors (ROCK inhibitor v. glutathione) on viability of follicles from vitrified samples, and (3) determine the optimal inhibitor concentration for follicle viability. In Experiment 1, 5 × 5 × 1 mm cortical tissue samples were obtained from excised cat ovaries and assigned to either the fresh control or vitrification group. Fresh samples were processed through a 230-micron-pore dissection strainer to collect preantral follicles. Follicles were then stained in Trypan blue to determine membrane integrity and survival rates. Vitrification samples were first equilibrated in 7.5% dimethyl sulfoxide and 7.5% ethylene glycol at ~22°C and then in vitrification solution consisting of 20% dimethyl sulfoxide, 20% ethylene glycol, and 0.5 M sucrose. They were then vitrified on a thin, perforated, metal strip (Cryotissue, Kitazato Biopharma, Fujinomiya, Japan). Samples were later warmed in 1.0 M sucrose at 38°C. Follicles were then collected and assessed for survival. In Experiment 2, follicles were collected from samples vitrified/warmed in cryo-media supplemented with either 3 × 104 nM ROCK inhibitor or 6 nM glutathione. Follicles from samples vitrified/warmed without inhibitor treatment were used as controls. In Experiment 3, tissue samples were vitrified/warmed in cryo-media supplemented with 0, 2, 6, or 10 nM glutathione before follicle viability was determined. Data were evaluated by chi square analysis. In Experiment 1, 637 and 340 follicles were collected from fresh and vitrified samples, respectively. Overall, survival was higher in freshly collected follicles when compared to those from the vitrified group (67 v. 18%, respectively; P < 0.05). Evaluation of apoptotic inhibitors was determined through collection of 314, 354, and 506 follicles from inhibitor-free, ROCK inhibitor, and glutathione-treated media, respectively. Follicles from samples vitrified in inhibitor-free media and in ROCK inhibitor survived at a lower rate than those from glutathione-treated samples (10 and 13% v. 18%, respectively; P < 0.05). In Experiment 3, a total of 539, 641, 625, and 632 follicles were collected from samples treated in 0, 2, 6, and 10 nM glutathione, respectively. There were no statistical differences in follicle survival among the 0, 2, and 6 nM groups. However, follicles treated in 10 nM glutathione survived at a higher rate than those vitrified/warmed in the absence of glutathione (20 v. 14%; P < 0.05). In summary, viability of preantral follicles from ovarian cortical tissue was significantly reduced by vitrification. Despite this, tolerance of such follicles to cryopreservation was improved by vitrifying and warming in cryo-media containing 10 nM glutathione. Partially funded by the LSU/ACRES Collaborative Project.

Spermatogonial stem cells (SSC), progenitor cells capable of both self-renewal and producing daughter cells that will differentiate into sperm, can be manipulated for transplantation to propagate genetically important males. This application was demonstrated in felids by the successful xeno-transplantation of ocelot mixed germ cells into the testes of domestic cats, which resulted in the production of ocelot sperm (Silva et al. 2012 J. Androl. 33, 264–276). Spermatogonial stem cells are in low numbers in the testis, but have been identified and isolated in different mammalian species using SSC surface markers; however, their expression varies among species. Until recently, little was known about the expression of SSC surface markers in feline species. We previously demonstrated that many mixed germ cells collected from adult cat testes express the germ cell markers GFRα1, GPR125, and C-Kit, and a smaller population of cells expresses the pluripotent SSC-specific markers SSEA-1 and SSEA-4 (Powell et al. 2011 Reprod. Fertil. Dev. 24, 221–222). In the present study, our goal was to identify germ cell and SSC-specific markers in SSC from cat testes. Immunohistochemical (IHC) localization of germ cell markers GFRα1, GPR125, and C-Kit and pluripotent SSC-specific markers SSEA-1, SSEA-4, TRA-1-60, TRA-1-81, and Oct-4 was detected in testis tissue from both sexually mature and prepubertal males. Testes were fixed with modified Davidson’s fixative for 24 h before processing, embedding, and sectioning. The EXPOSE Mouse and Rabbit Specific HRP/DAB detection IHC kit (Abcam®, Cambridge, MA, USA) was used for antibody detection. Staining for SSEA-1, SSEA-4, TRA-1-60, TRA-1-81, and Oct-4 markers was expressed specifically at the basement membrane of the seminiferous tubules in both adult and prepubertal testes. The GFRα1 and GPR125 markers were detected at the basement membrane of the seminiferous tubules and across the seminiferous tubule section. However, C-Kit was not detected in any cell. Using flow cytometry from a pool of cells from seven adult testes, we detected 45% GFRα1, 50% GPR125, 59% C-Kit, 18% TRA-1-60, 16% TRA-1-81 positive cells, and a very small portion of SSEA-1 (7%) and SSEA-4 (3%) positive cells. Dual staining of germ cells pooled from 3 testes revealed 3 distinct cell populations that were positive for GFRα1 only (23%), positive for both GFRα1 and SSEA-4 (6%), and positive for SSEA-4 only (1%). Our IHC staining of cat testes indicated that cells along the basement membrane of seminiferous tubules were positive for SSC-specific markers, and flow cytometry analysis revealed that there were different cell populations expressing both germ cell and SSC-specific markers. Flow cytometry results show overlapping germ cell populations expressing SSEA-4 and GFRα1, and IHC results reveal that SSEA-4 positive cells are spermatogonia, whereas GFRα1 positive cells include other stages of germ cells, indicating that the small population of cells positive only for SSEA-4 is undifferentiated cat SSC.