Animal-derived products are currently used to cryopreserve sperm cells. However, these products represent potential risks of contamination by pathogens. Optidyl® (IMV Technologies, L’Aigle, France), containing egg yolk, is a reference product in Europe used routinely in bovine insemination centers. Commercial media such as soy lecithin or liposome-based media have been used to replace extenders containing products derived from animals. However, their protective effect could be called into question because of their non-synthetic or unstable properties. Despite these innovative extenders on the market, it might be necessary for sanitary reasons to cryopreserve bull semen in a stable and synthetic extender. CRYO3 (Stem Alpha, Saint-Genis l’Argentière, France), a serum-free and protein-free medium used for cryopreserving somatic and stem human cells, is a potential medium to cryopreserve reproductive cells. Recently, CRYO3 improved cryopreservation of in vitro-produced bovine embryos compared with fetal calf serum and BSA-based media. Thus, it could be interesting to test this medium on sperm cells. The objective of this study was to compare 2 in vitro freezing media on bull semen: a commercial egg yolk-based medium and a synthetic medium containing 20% CRYO3. Sperm from 5 bulls were collected in Auriva station (Brindas, France). A sample of each ejaculate was used to assess fresh semen quality. The remaining sperm of each bull was split and diluted in 2 media: Optidyl® and a CRYO3-based medium. Semen was equilibrated at least 4 h at 4°C before being packaged in 0.25-mL French straws, and then frozen into a programmable freezer and plunged into liquid nitrogen. Osmolarity and pH were respectively 1462 mOsm/kg and 6.9 for Optidyl® and 1286 mOsm/kg and 6.8 for CRYO3 medium. Viability (with SYBR-14 and propidium iodide) and high mitochondrial membrane potential (hMMP) (with JC-1) were assessed using flow cytometry. A hypo-osmotic swelling test was performed to assess functional membrane integrity (FMI). The motility parameters were evaluated by computer-assisted sperm analysis. Statistical analysis was performed using Wilcoxon test with the R software. Fresh sperm showed 52% viability, 64% hMMP, 74% FMI, and 62 and 76% progressive and total motility, respectively. For all parameters measured, no significant difference was observed between extenders and between fresh and frozen–thawed sperm (P > 0.05). However, Optidyl® showed clearly better survival than CRYO3 (45% v. 16% for viability, 58% v. 20% for hMMP, 67% v. 25% for FMI, 51% v. 14% for progressive motility and 72% v. 32% for total motility, respectively). These results show that it is possible to freeze bovine semen in synthetic extender though the low survival rate after freezing-thawing. Indeed, it is known that motility and flow cytometry parameters are not necessarily good indicators of fertility. Artificial inseminations will be done to verify the fertility of the sperm cryopreserved in CRYO3. Repetitions with more bulls from different breeds will be performed to complete this preliminary work.