Culture on IVF bovine embryos and hemiembryos.

CARLOS IRALA; DANIEL F. SALAMONE

XXXV Reunião Anual da Sociedade Brasileira de Tecnologia de Embriões (SBTE)

Congreso; Annual Conference of the International Embryo Technology Society, Savannah, Georgia, 10?13 January 2022; 2022

SBTE

Effects of Zona-Free Culture on IVF bovine embryos and hemiembryos. In recent years, IVF have become one of the most applied biotechnologies in livestock production. Monozygotic twin production has been successful in bovine species, by removal of the Zona Pellucida (ZP) and blastomere disaggregation at two cells stage. Since monozygotic twins are genetically identical, it is theoretically possible to duplicate the number of embryos of superior genetic value from a single embryo. However, ZP-Free embryos have less competence to development than ZP-Intact embryos.The objective of this work is to evaluate the effect of the ZP-Free embryo culture and blastomere separation in blastocyst development rate.Ovaries were collected from slaughterhouse and transferred to laboratory in sterile physiologic solution. COCs were aspirated from follicles of 2-8mm using 18G needles. COCs were searched in Tyrode’s Album Lactate Pyruvate Hepes. (TALP-H) with 1% Penecillin-Streptomycin-Fungizone (Gibco, USA), then maturated in vitro in Tissue Culture Medium 199 (TCM 199, Gibco, USA) 100µl drops under mineral oil, 22 hours, in humidified air at 38,5ºC (Thermo Electron, USA). Matured COCs were co-incubated in contact with thawed bull sperm, at 16x10⁶ spz/ml concentration. Diluted in Sperm Wash Solution and Sperm Dilution Solution in the same proportion, in 100µl drops under mineral oil during 5 hours. After that, Presumptive zygotes were deposited in 100µl of TALP-H solution, and agitated 1 minute in order to remove the cumulous cells and spermatozoids adhered. Presumptive zygotes were then separated in two groups. One group was transferred to 50µl drops of Synthetic Oviductal Fluid (SOF), with 2.5% of FBS, 7 days and 38.5ºC and 5% O₂ under mineral oil. Cleavage was evaluated at 30 hours post IVF. The other group was transferred to 100µl TALPH drops covered in mineral oil to remove the ZP. The ZP was removed by adding 10µl of protease 1 minute. ZP-free embryos were washed in TALP-H and individually cultured in microwells in SOF medium and 38,5ºC and 5% O₂ under mineral oil. 30 hours post IVF, cleavage was evaluated. Half of the ZP-free embryos were selected randomly. Two-cells embryos were washed in TALPH and transferred to 50µl Drops of Dulbecco’s phosphate-buffered saline (DPBS) medium (Gibco, USA), with no Ca₂₊, no Mg₂₊, and 20% of FBS covered in mineral oil. Blastomeres were separated by gently pippeting and individually cultured in microwells under same culture conditions. Blastocyst rate were evaluated at Day 7 of embryo culture. Blastocist Rate from Control IVF Embryos, vs ZP-free embryos and hemiembryos were analyzed by Chi-Square Test (P