C. Dumas

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.

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.