C. E. Pope

Cloning endangered felids using heterospecific donor oocytes and interspecies embryo transfer

Somatic cell nuclear transfer (SCNT) offers the possibility of preserving endangered species. It is one of the few technologies that avoids the loss of genetic variation and provides the prospect of species continuance, rather than extinction. Nonetheless, there has been a debate over the use of SCNT for preserving endangered species because of abnormal nuclear reprogramming, low efficiency and the involvement of extra mitochondrial DNA (mtDNA) of a different species in live offspring produced by interspecies SCNT. Despite these limitations, live endangered cloned animals have been produced. In the present paper, we describe recent research on the production of cloned embryos derived by fusion of wild felid fibroblast cells with heterospecific domestic cat cytoplasts and their viability after transfer into domestic cat recipients. In addition, we discuss epigenetic events that take place in donor cells and felid cloned embryos and mtDNA inheritance in wild felid clones and their offspring.

Isolation, culture and characterisation of somatic cells derived from semen and milk of endangered sheep and eland antelope

Semen and milk are potential sources of somatic cells for genome banks. In the present study, we cultured and characterised cells from: (1) cooled sheep milk; (2) fresh, cooled and frozen-thawed semen from Gulf Coast native (GCN) sheep (Ovis aries); and (3) fresh eland (Taurotragus oryx) semen. Cells attached to the culture surface from fresh (29%), cooled (43%) and slow-frozen (1 degrees C/min; 14%) ram semen, whereas no attachment occurred in the fast-frozen (10 degrees C/min) group. Proliferation occurred in fresh (50%) and cooled (100%) groups, but no cells proliferated after passage 1 (P1). Eland semen yielded cell lines (100%) that were cryopreserved at P1. In samples from GCN and cross-bred milk, cell attachment (83% and 95%, respectively) and proliferation (60% and 37%, respectively) were observed. Immunocytochemical detection of cytokeratin indicated an epithelial origin of semen-derived cells, whereas milk yielded either fibroblasts, epithelial or a mixture of cell types. Deoxyribonucleic acid microsatellite analysis using cattle-derived markers confirmed that eland cells were from the semen donor. Eland epithelial cells were transferred into eland oocytes and 12 (71%), six (35%) and two (12%) embryos cleaved and developed to morulae or blastocyst stages, respectively. In conclusion, we have developed a technique for obtaining somatic cells from semen. We have also demonstrated that semen-derived cells can serve as karyoplast donors for nuclear transfer.

Development of in-vitro-derived bovine embryos in protein-free media: effects of amino acids, glucose, pyruvate, lactate, phosphate and osmotic pressure

In experiment 1, the effects of a group of either 20 (i.e. glutamine + essential + non-essential) or 11 (i.e. hamster embryo culture medium (HECM)-6) amino acids were evaluated in modified potassium simplex optimised medium (mKSOM) or basic medium (BM)-3. In experiment 2, the effects of glucose, pyruvate, lactate, phosphate or all four substrates were evaluated in low- or high-osmotic pressure BM-3 (255 and 275 mOsmol respectively) containing 20 amino acids (BM-3-20aa). In experiment 1, mKSOM containing 20 amino acids (mKSOM-20aa) supported the highest frequency of total, expanded (Days 7, 8 and 9) and hatched blastocysts. In experiment 2, supplement type affected the frequency of development to at least the morula stage (Day 7), expanded (Day 8), hatched (Day 9) or total blastocysts and cell number per blastocyst. Osmotic pressure affected the frequency of expanded blastocysts (Day 7) and blastocyst cell number. Regardless of the osmotic pressure, BM-3-20aa containing glucose (0.2 mM) supported the highest frequency of blastocyst development. The interaction between supplement type and osmotic pressure was not significant; however, treatment mean differences were more marked in high- than in low-osmotic pressure medium. In conclusion, the beneficial effects of amino acids on in vitro embryo development are influenced by the base medium. Moreover, glucose-containing media supported a higher frequency of embryonic development than pyruvate- and/or phosphate-supplemented media, indicating that glucose plays more important roles in non-energy generating pathways.

228 Wnt SIGNALLING PATHWAY ACTIVATION IN CAT EMBRYONIC STEM LIKE-CELLS AND ITS ROLE IN MAINTAINING PLURIPOTENCY

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 (Gomez 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.

156 LAPAROSCOPIC UTERINE EMBRYO TRANSFER IN THE CAT

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.

110 ASSISTED CONCEPTION IN THE FISHING CAT BY LAPAROSCOPIC INTRATUBAL INSEMINATION

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.