Folltropin

All embryo transfer procedures were carried out during the breeding season from November to May by one expert as described elsewhere with modifications [26 (link)]. In brief, donors were super-ovulated with a total of 400 NIU of FSH (Folltropin^®, Vetoquinol, Lure, France) in decreasing doses, 12 h apart over a six-day period (4-4, 2.5-2.5, 1.5-1.5, 1-1, 0.5-0.5, 0.5-0.5 mL), and referred for mating 36 h after the last injection. Embryos were recovered 8.5 days after the mating by non-surgical uterine flushing and non-surgically transferred to recipients’ uterine horn on day 7.5 after induction of ovulation. Embryos were evaluated and categorized with regard to the grade (transferable and nontransferable) and shape (spherical and collapsed) [27 (link),28 (link)]. The pregnancy data set included the recipients of transferable embryos with spherical shapes, and pregnancy was confirmed 20 ± 3 days after transfer by observing embryonic fluids in the presence of a viable CL together with embryonic proper or heart beat in ultrasonographic scanning of the uterus.

In vitro maturation of cat oocytes was performed using a method previously described for the domestic cat¹⁰⁶ . Briefly, 50 µl droplets of Quinn’s Advantage Protein Plus Blastocyst Medium, supplemented with 1 µg ml⁻¹ follicle stimulating hormone (Folltropin, Vetoquinol, USA), and luteinizing hormone (Lutropin V, Vetoquinol, USA), were equilibrated under mineral oil at 5% CO₂ and 38 °C for at least 2 hours before use. Fresh or frozen-thawed cat COCs were transferred in groups of 14–28 oocytes to each droplet and incubated for 24 hours. Following IVM, oocytes were washed in PBS containing EDTA (0.1 mM), EGTA (0.1 mM), Imidazole (50 mM), 4% Triton-X, PVP (3 mg mL⁻¹) and PMSF (24 µM). During this step, oocytes were rapidly pipetted up and down to remove cumulus cells, then fixed overnight in 4% paraformaldehyde in PBS at 4 °C.
Oocyte survival, defined as maintaining normal morphology with spherical shape, intact membranes, clear zone without rupture, and uniform cytoplasm, was evaluated immediately after thawing (0 h) and following in vitro maturation (26 h) via light microscopy^{107 (link)}.