Abstract

The domestic cat is a valuable model for the generation of assisted reproductive techniques that might be used in the conservation of endangered wild felids. However, the in vitro embryo production systems in the domestic cat stillhave a low efficiency. The oocyte competence is an important factor that determine the successful in the in vitroembryo production systems. In humans, the mild follicular stimulation with gonadotropins (or priming) prior to thein vitro maturation (IVM), has been used to increase the oocyte maturation and blastocyst formation rates. The objective of this research was to evaluate the mild follicular stimulation with eCG in the in vitro fertilization system(IVF) in the domestic cat. For this purpose, nine domestic cat were treated with a subcutaneous dose of 200 IU ofeCG and were subjected to ovariohysterectomy 4 days later for ovaries recovery and cumulus-oocyte complexes(COCs) collection. Additionally, others two cats were synchronized for embryo transfer procedure with 200 IU ofeCG and an intramuscular dose of 100 IU of hCG 4 days later. Each cat correspond to an individual biological replicate, for this reason, the COCs recovered from each cat were matured, fertilized and cultured separately. For IVM, only grade I and II COCs were selected and matured in TCM-199 Earle’s salts medium supplemented with 4mg/mL BSA, 0.1 IU FSH-LH (Pluset), 0.36 mM sodium pyruvate, 2 mM glutamine, 2,2 mM calcium lactate, 1μg/mL 17-β estradiol, 20 μg/mL EGF and 50 μg/mL gentamycin for 28-30 hours in a 5% CO2, 5% O2 and 90% N2 humidified atmosphere to 38.5ºC. The IVF was realized using epididymal cat sperm which was refrigerated to 4ºCfor 24 hours, 1.5 – 2.5 x 106 spermatozoa /mL were incubated with 20-30 COCs in TALP medium supplemented with 6 mg/mL BSA, 0.36 mM sodium pyruvate, 1 mM glutamine, 2.2 mM calcium lactate, 1% MEM non essentialamino acids (NEAA), 0.01 mg/mL heparin sodium salt and 50 μg/mL de gentamycin for 18 hours in a 5% CO2humidified atmosphere to 38.5ºC. The presumed zygotes were cultured in SOF medium in a 5% CO2, 5% O2 and 90% N2 humidified atmosphere to 38.5ºC during 7-8 days. The cleavage, morula, blastocyst and hatching blastocyst rates were estimated. Once finished the culture, the blastocysts and hatching blastocysts were fixed and stained with Hoechst for total cell counting. Additionally, a total of 23 blastocysts were transferred into the uterine horn of thetwo previously synchronized cats (15 and 8 blastocysts per cat, respectively). The descriptive statistic was realized using the statistical software Infostat. Regarding to in vitro embryo production, the results of this research demonstrated that the domestic cat oocytes recovered after eCG priming are capable to develop in vitro after IVF until the blastocyst stage. Cleavage rate was 155/239 (64.9%), morula rate 115/155 (74.2%), total blastocyst rate 51/155 (32.9%) and hatching blastocyst rate 15/155 (9.7%). Furthermore, the embryo staining revealed the total cell number (mean ± standard deviation) of the blastocysts (182.8 ± 76.9) and hatching blastocysts (420.2 ± 106.1)generated. Finally, one gestational vesicle was detected at the 25 days of gestation in the cat that received 15 blastocysts and a healthy female kitten born after 64 days of gestation. No implantation was detected in the cat that received 8 blastocysts. In conclusion, the mild follicular stimulation might be a useful alternative for the in vitro and in vivo embryo development in the domestic cat and could be applicable in wild felid species.