In vitro production of Bali cattle embryos still needs in-depth investigations to produce embryos suitable for transfer. The current study aimed to examine the level of cell apoptosis in Bali cattle embryos produced in vitro and at three stage of oocyte maturation, fertilization, and embryo culture. A total of 107 pairs of ovaries derived from slaughterhouses of Indonesia were collected. The used oocytes were grades A and B (Grade A had compact cumulus oocyte complex (COC) cells surrounded by five or more layers of cumulus cells, and grade B had a non-compact COC and a dark cytoplasm with complements from the complete radiata corona but surrounded by no more than five layers of cumulus cells). Fertilization of oocytes was done using the semen of a Bali bull. Bali cattle semen was frozen in straw semen for 5 minutes at 1500 rpm twice, then the supernatant and spermatozoa were separated and equilibrated for 30 minutes. Fertilization lasted for 5-6 hours in the incubator. Then, oocyte culture was carried out using CR1aa media and evaluated at 48 hours post-insemination (hpi). The result of the current study showed that the development of Bali cattle embryos produced in vitro after 48 hours of culture included 2 cells (31.91%), 4 cells (32.97%), 8 cells (24.46%), and 16 cells (10.63%). The percentage of embryos containing at least one nucleus exhibiting Terminal dUTP nick-end labeling (TUNEL) characteristics of apoptosis entailed 28.33% (2 cells), 41.93% (4 cells), 43.48% (8 cells), and 50% (16 cells). The division ability of embryos aged 48 hpi consisted of 2, 4, 8, and 16 cells. In conclusion, apoptosis in Bali cattle began to be detected in the two-cells stage. The sooner a cell undergoes apoptosis, the lower the level of the cell’s ability to develop further.

Antioxidants are molecular compounds that can give their electron structure to free radical molecules without disturbing them and can break the chain of free radical compounds. Antioxidants that can be used include enzymatic and non-enzymatic antioxidants. Supplementation of antioxidants into maturation mediums or cultures with the right concentration can efficiently improves oocyte maturation, cell division, and embryo quality in bovine. Enzymatic and non-enzymatic antioxidant supplementation of the maturation medium increase the number of oocytes that reach metaphase II (MII). Furthermore, the supplementation of both antioxidants in maturation and culture mediums are also able to increase cell division and embryo that reaches blastocyst. Non-enzymatic antioxidant supplementation is more effective than enzymatic antioxidants in improving the maturation and division of cells in the production of bovine embryos in vitro. In conclusion, non-enzymatic antioxidant supplementation is more effective in supporting embryonic development in vitro.