97 Loss of aggregation capacity of bovine in vitro-produced embryos and blastocyst-derived trophoblasts from Day 6 of development

Abstract

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 with nonaggregated 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 inner cell mass (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, cumulus-oocyte complexes (COCs) collected from slaughterhouse ovaries were matured in tissue culture medium 199 containing 10% fetal bovine serum, 10 µg mL⁻¹ FSH, 0.3 mM sodium pyruvate, 100 mM cysteamine and 2% antibiotic-antimycotic for 24 h, at 6.5% CO₂ in humidified air and 38.5°C. We performed IVF with 16 × 10⁶ spermatozoa per mL for 5 h. Afterwards, presumptive zygotes were cultured in synthetic oviductal fluid (SOF) for 7 days in a humidified atmosphere at 38.5°C, 5% O₂, 5% CO₂, and 90% N₂. In Experiment 1, embryo bisection of Day 7 blastocysts was performed manually under stereoscopic observation with a microblade to obtain Tr. These were aggregated, with the bisected part containing the ICM (n = 22) or with ZF embryos of Days 4 (n = 23), 5(n = 25), or 6 (n = 22) and blastocysts (n = 25), and placed in microwells in a 100-μL SOF drop covered by mineral oil (Gambini et al. 2012 Biol. Reprod. 87, 15; https://doi.org/10.1095/biolreprod.112.098855). In Experiment 2, ZF synchronous whole embryos were aggregated in microwells at different developmental days: Day 3 (n = 18), 4 (n = 18), 5 (n = 47), 6 (n = 48), and 7 (n = 45). In both experiments, aggregation was assessed at Day 8. In Experiment 1, no aggregation was observed between the Tr and the embryos or the bisected ICM. Experiment 2 showed embryo aggregation on Days 3 (55%), 4 (27%), and 5 (61%), whereas on Days 6 and 7 no aggregation was observed. According to these results, we can conclude that, in our culture conditions, Tr obtained by blastocyst bisection have no capacity for aggregation. Day 6 and 7 whole ZF embryos also do not aggregate. As a general conclusion, there is a period from Days 0-5 of the in vitro development of bovine embryos in which aggregation is possible. Aggregation of blastocyst-derived Tr to cloned or high-value IVF embryos, aiming for quality improvement, is not an effective strategy.