Cryopreservation induces higher oxidative stress levels in Bos indicus embryos compared with Bos taurus.

Freezing and thawing of Bos indicus embryos affect their quality for embryo transfer. The objective of this study was to compare the levels of reactive oxygen species between Bos indicus and Bos taurus embryos produced in vivo, before and after conventional freezing, as well as to analyze damage caused by apoptosis and lipid peroxidation. Bos indicus has higher levels of reactive oxygen species than Bos taurus embryos, both fresh (14.32 ± 1.41 auf vs 8.07 ± 1.15 auf (arbitrary units of fluorescence), P < 0.05) and after freezing (20.91 ± 1.21 auf vs 14.39 ± 0.58 auf, P < 0.05). The number of apoptotic nuclei is also significantly higher in Bos indicus embryos than Bos taurus (8.28 ± 0.80 vs 1 ± 0.57, P < 0.05) but highlighting a notable increase after the freeze-thaw process in both subspecies (Bos indicus from 8.28 ± 0.80 to 10.71 ± 0.42, P < 0.05; Bos taurus from 1 ± 0.57 to 5.5 ± 1.15, P < 0.05). Finally, although lipid peroxidation is lower in Bos indicus embryos before freezing in comparison with Bos taurus (2.46 ± 0.14 vs 4.20 ± 0.51), the effect after the freeze-thaw process showed an increase of 4.34 in Bos indicus than Bos taurus embryos (51.45 ± 5.52 auf vs 11.85 ± 2.88 auf, P < 0.05). In conclusion, in comparison with Bos taurus, Bos indicus embryos undergo greater oxidative stress causing increases in the cryopreservation process, promoting major cell damage and lowering embryonic viability.

Lipid droplets in clusters negatively affect Bos indicus embryos during cryopreservation.

Embryo transfer using cryopreserved B. indicus embryos results in low pregnancy rates. The low viability of B. indicus embryos is likely a result of their low freezability relative to embryos from other species, notably B. taurus. Freezability is probably related to the quantity of lipid droplets in the embryonic cells. However, the mechanism underlying this association is unknown. Using multiphoton and transmission electron microscopy, the proportion, volume, morphology and composition of lipid droplets in fresh and cryopreserved B. indicus and B. taurus embryos were studied. B. indicus embryos have more droplets than B. taurus and a greater percentage of lipid droplets in clusters (34% vs. 24%; p < 0.05). Also, B. indicus individual droplets are of greater volume than those of B. taurus (47 µm3 vs. 34 µm3 ; p < 0.05). After freezing, the percentage of clusters decreased in B. indicus (11% vs. 5%; p < 0.05), while in B. taurus they increased (2% vs. 7%; p < 0.05). Freezing influenced the lipid droplet morphology, increasing the number of cracked droplets in B. indicus embryos (33% fresh vs. 62% frozen; p < 0.05), but not in B. taurus embryos (35% fresh vs. 34% frozen; ns). Regarding composition, saturated lipid droplets prevail in B. indicus embryos, contrary to B. taurus embryos. We observed differences in lipid droplet organization, proportion, volume, morphology and composition between B. indicus and B. taurus embryos which are altered in opposite directions following freezing.