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Transforming growth factor-beta (TGF-β) plays a critical role in regulating trophoblast invasion and proliferation. Growth differentiation factor-8 (GDF-8) is a member of the TGF-β superfamily and is categorized as a myostatin subtype. It is primarily a secreted protein synthesized in skeletal muscle cells. It is expressed in the placenta, reproductive tissues, and cells. In this study, we investigated the role of GDF-8 in the development and hatching rate of bovine embryos. We noted a notable elevation ( p < 0.05) in the development and hatching rates compared to the control embryos. Furthermore, the GDF-8 group showed a significantly improved total cell number ( p < 0.05) and an increase in trophectoderm ratio inner cell mass (trophectoderm: inner cell mass) cells ( p < 0.001) compared to the control group. Additionally, blastocysts treated with GDF-8 exhibited significantly higher mRNA levels of caudal-type homeobox 2 ( CDX2 ) ( p < 0.05). The trophoblast invasion area was significantly larger in the GDF-8 group than in the control group ( p < 0.01). Furthermore, qRT-PCR analysis revealed significantly higher mRNA levels ( p < 0.05) of matrix metalloproteinases 9 ( MMP9 ) and follistatin-like 3( FSTL3 ), both of which are associated with the ALK5-SMAD2/3 signaling pathway, in the GDF-8 group than those in the control group. The mRNA expression levels of genes related to tight junctions (TJ) and adherent junctions were higher in the GDF-8 group than those in the control group ( p < 0.05). After 24 h of thawing, blastocysts were analyzed using 4-kDa FITC-dextran, which revealed a higher TJ integrity in the GDF-8 group ( p < 0.01). Thus, GDF-8 plays a crucial role in bovine embryonic development, in vitro implantation, and cryotolerance., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Kang, Idrees, Perera, Lee, Zhang, Yu, Jin and Kong.)