Loss of aggregation capacity of bovine in vitro produced embryos and blastocyst derived trophoblast from day 6 of development

YAURI FELIPE M; ALBERIO V; SALAMONE DF

Nueva York

Congreso; 46th IETS Annual Conference; 2020

IETS

Embryo aggregation consists of placing more than one zona free (ZF) embryo in contact during development to obtain a unique structure. It has been reported in different species that aggregated cloned embryos show certain benefits compared to non-aggregated embryos. One way to obtain these benefits in IVF embryos, would be to generate a transient chimera by the introduction of trophoblastic cells. Bovine trophoblastic cells can be obtained by embryo bisection of blastocysts, cutting asymmetrically to use trophoblasts (Tr) for aggregation and leaving aside the portion that contains the ICM. Taking all this into account, the objectives of this work are to study the aggregation of Tr at different days of development and to determine the appropriate time of aggregation. To this aim, COCs collected from slaughterhouse ovaries were matured in TCM 199 containing 10% fetal bovine serum, 10 µg/mL FSH, 0.3 mM sodium pyruvate, 100 mM cysteamine, and 2% antibiotic-antimycotic for 24 hours, at 6.5% CO2 in humidified air and 38.5 °C. IVF was performed with 16 x 106 spermatozoa per ml for 5 hours. Afterwards, presumptive zygotes were cultured in Synthetic Oviductal Fluid (SOF) for 7 days at 38.5 °C and 5% O2. In experiment 1, embryo bisection of day 7 blastocysts was performed manually under stereoscopic observation with a microblade to obtain Tr. Then, these were aggregated with the bisected part containing the ICM (n=22) or with ZF embryos of days 4 (n=23), 5(n=25), 6 (n=22) and blastocysts (n=25) and placed in microwells in a 100 ul SOF drop covered by mineral oil (Gambini et al., 2012, Bio Rep 1;15). In experiment 2, ZF synchronous whole embryos were aggregated at different developmental days: day 3 (n= 18), 4 (n= 18), 5 (n= 47), 6 (n= 48) and 7 (n= 45) in microwells. In both experiments, aggregation was assessed at day 8. As results of Experiment 1, no aggregation was observed between the trophoblasts and the embryos or the bisected ICM. Experiment 2 showed embryo aggregation on days 3 (55%), 4 (27%) and 5 (61%), while on days 6 and 7 no aggregation was observed. According to these results, we can conclude that, in our culture conditions, trophoblasts obtained by blastocysts bisection have no capacity of aggregation. In addition, day 6 and 7 whole ZF embryos also do not aggregate. As a general conclusion, there is a period from day 0 to day 5 of the in vitro development of bovine embryos in which aggregation is possible. Aggregation of blastocyst-derived trophoblasts to cloned or high-value IVF embryos aiming their quality improvement is not an effective strategy.