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Simple Summary: There is a growing demand for the development of optimal protocols for ovarian tissue cryopreservation in different animal models to enable the maintenance of cell morphology. Therefore, it is crucial to understand the effects of conservation on ovarian cell proliferation and apoptosis to improve follicular development after in vitro culture and/or ovarian tissue transplantation. This study compared the effects of three concentrations (1, 1.5, and 3 M) of dimethyl sulfoxide (DMSO) during vitrification to identify optimal conditions for preserving bovine preantral follicles and to contribute to the improvement of the technique and its future application in assisted reproduction procedures. Following vitrification, cryopreserved ovarian fragments exhibited similar percentages of intact follicles, with only the 3 M DMSO concentration differing from the control. Upon analyzing the free radical production, the 3 M concentration exhibited higher oxidative stress levels than lower DMSO concentrations. Exposure to 3 M DMSO for 30 min led to a higher rate of follicular degeneration. After in vitro cultivation of the vitrified fragments, the 1 M DMSO concentration showed higher percentages of morphologically intact follicles than the other concentrations. Accordingly, bovine preantral follicles could be cryopreserved in situ with greater efficiency in 1 M DMSO. Ovarian tissue cryopreservation has been widely investigated for preserving female fertility. In the present study, we aimed to compare the effects of three concentrations (1, 1.5, and 3 M) of dimethylsulfoxide (DMSO) on the vitrification of ovarian tissue. The ovarian cortex was divided into control and vitrified groups: (i) 1 M-DMSO, (ii) 1.5 M-DMSO, and (iii) 3 M-DMSO. Follicles from all fragments were analyzed for DMSO-induced deleterious effects, morphological and morphometric aspects, and concentration of reactive oxygen species. Additionally, the fragments were cultured to assess the integrity and return of follicular development post-vitrification. All DMSO concentrations resulted in a higher percentage of degenerated preantral follicles than before the cryopreservation process. After vitrification, the cryopreserved ovarian fragments showed similar percentages of intact follicles; however, the 3 M DMSO concentration differed from the control. Analyzing free radical production, we found that the 3 M DMSO concentration had higher levels of oxidative stress than the lower DMSO. After in vitro cultivation of the vitrified/warmed fragments, the 1 M DMSO concentration exhibited higher percentages of morphologically intact follicles than the other concentrations. Therefore, we suggest that bovine preantral follicles can be cryopreserved in situ with greater efficiency in 1 M DMSO. [ABSTRACT FROM AUTHOR]