In vitro embryo production in the Iberian lynx (Lynx pardinus) using oocytes recovered post-mortem and cryopreserved spermatozoa

The Iberian lynx ( Lynx pardinus ), once considered the most endangered felid in the world, has become a remarkable example of conservation success thanks to intensive ex situ and in situ management efforts. However, the long-term viability of the species remains under threat. The implementation of assisted reproductive technologies (ARTs) would contribute to ensure the species viability, but the use of ARTs is limited in this species and there are still important gaps in the knowledge of its physiology. Here, we report the first in vitro fertilization (IVF) and early embryo development in the Iberian lynx using oocytes retrieved post-mortem and cryopreserved spermatozoa. Oocytes from six females were subjected to in vitro maturation using two sets of conditions. In the first series, oocytes incubated in vitro for 24 h, exhibited a very low proportion (0–37 %) reaching metaphase II (MII). Attempted IVF with cryopreserved spermatozoa resulted in no fertilization. In a second series, oocytes were incubated in vitro for 28 h; grade I-II oocytes exhibited 40 % maturation rate (MII), whereas grade III oocytes showed 28 % maturation rate. Matured oocytes from one female were co-incubated with cryopreserved sperm for 18 h resulting in cleavage rates of 39.3 % (grade I–II) and 7.1 % (grade III) and the opportunity to vitrify one blastocyst and six morulae. These results demonstrate the feasibility of using ARTs in this species, validating domestic cat protocols for Iberian lynx gametes, and providing a foundation for further refinement of IVF protocols, embryo cryopreservation, and ultimately, embryo transfer strategies for use in conservation programs. • Iberian lynx oocytes collected post-mortem were successfully matured in vitro . • Cryopreserved Iberian lynx sperm were able to fertilize IVM homologous oocytes generating embryos. • Assisted reproduction could contribute to genetic rescue in the Iberian lynx.