Improving porcine in vitro blastocyst development using fetal bovine serum, amino acids, and insulin-transferrin-selenium.

In vitro production of porcine-hatched blastocysts is important for various applications. However, the mechanobiology of blastocoel expansion and hatching remains poorly understood. Our study aimed to efficiently produce hatched blastocysts and investigate the hatching mechanics of late-stage porcine embryos using time-lapse assessment. In this study, fetal bovine serum (FBS), amino acids (AA), and insulin-transferrin-selenium (ITS) were added individually or in combination to culture day 5 parthenogenetic morulae and early blastocysts to investigate their developmental capacity. We found that FBS-AA-ITS supplementation significantly promoted blastocoel expansion (4.4-fold), accelerated early hatching (at day 6 vs. day 7.5) and improved hatching rates (39.59 ± 4.34% vs. 0 ± 0% on day 7, and 80.02 ± 2.93% vs. 2.78 ± 1.60% on day 8) compared with non-supplemented controls. It also increased the number of trophectoderm (2.5-fold) and inner cell mass (2.7-fold). Furthermore, FBS-AA-ITS elevated BCL2 expression and reduced BAX expression, potentially inhibiting apoptosis. In in vitro fertilized embryos, the combination also significantly improved the hatching rate (8.3-fold) and cell number (2.7-fold). Analysis of the effects of FBS on hatching dynamics revealed that FBS enhanced hatching by increasing the number of blastomeres and blastocoel diameter. Blastocysts supplemented with FBS had significantly smaller hatching sizes (320.09 ± 5.01 µm vs. 343.95 ± 5.67 µm) and faster rate of hatching progress (5.6-fold) compared with non-FBS groups. In conclusion, FBS-AA-ITS played a pivotal role in supporting the development of late-stage porcine embryos and significantly accelerated their progression through the embryonic developmental phase.