72 Reconstructing bovine embryos following individual blastomere reduction

Blastomere complementation is a very useful technique to produce multiple blastocysts from a single embryo with high genetic merit. However, systematic evaluation of embryo complementation efficacy from continued individually blastomere reduction remains unexplored in cattle. Here, we aimed to evaluate the capacity of individual blastomeres from different developmental stages of bovine preimplantation embryos to form blastocysts via cell mass reduction. Bovine IVF embryos were produced using IVF Bioscience media under 38°C and 5% O₂ and incubated with protease to remove zona pellucida. Individual blastomeres were isolated and cultured in a well system for up to 7 days. The sequential cell reduction from each stage of preimplantation was performed, and the developmental potential to blastocyst was evaluated. The markers CDX2 (trophectoderm) and SOX2 (inner cell mass) were analyzed by immunofluorescence to determine the total cell numbers for each lineage. Raw images were processed in Fiji, and the total number of cells was calculated based on DAPI spots. In the first round of cell reduction, we found blastomeres from embryos at 2-, 4-, and 8-cell stages were able to develop into blastocysts (first reduction of 2-cell: n = 15, blastomere/blastocyst: 30/19, blastocyst rate = 63.3%; first reduction of 4-cell: n = 15, blastomere/blastocyst: 56/29, blastocyst rate = 51.8%; first reduction of 8-cell: n = 11, blastomere/blastocyst: 88/28, blastocyst rate = 31.8%). During the sequential blastomere reduction, at the 2-cell stage, we observed that the blastocyst rate was 77.8% from the second reduction of reconstructed 2-cell (n = 9, blastomere/blastocyst = 18/14), 25% from the third reduction of reconstructed 2-cell (n = 9, blastomere/blastocyst = 9/36), and 19.2% from the fourth reduction of reconstructed 2-cell (n = 9, blastomere/blastocyst = 52/10). At the 4-cell stage, we observed that the blastocyst rate was 41.7% from second reduction of reconstructed 4-cell (n = 6, blastomere/blastocyst = 10/24) and 20% from third reduction of reconstructed 4-cell (n = 6, blastomere/blastocyst = 8/40). At 8-cell stage, we were only able to progress one more blastomere reduction, and the blastocyst development rate was 45% (second reduction of reconstructed 8-cell: n = 3, blastomere/blastocyst = 11/24). Additionally, there was a gradual reduction of total cell number of reconstructed blastocysts associated with cleavage and multiple cell reduction across 2-, 4-, and 8-cell embryos, with a decrease of CDX2-positive and an increase of SOX2-positive cell numbers. We also evaluated the totipotency to pluripotency transitions across development stages, and we found that the expression of a totipotent marker (ZSCAN4) was present in 2-, 4-, and 8-cell IVF embryos but gradually lost signal with development progression. In contrast, the pluripotent gene SOX2 first emerged from IVF morulae and blastocysts, while it was present throughout reconstructed 2-, 4-, and 8-cell embryos. In summary, we demonstrated that the bovine blastocyst can be reconstructed from individual blastomeres until 8-cell embryos. There was a reduced complementation efficiency and developmental competence to reach the blastocyst stage across 2-, 4-, and 8-cell embryos and from multiple cell reductions.