Intracytoplasmic sperm injection(ICSI) plays an important role in equine embryo production. Commercially, immature equine cumulus-oocyte complexes(COCs) are collected from mares via oocyte pickup and placed into holding media for transportation to the ICSI facility, with a recommended upper limit of 24h.However, the effect of prolonged in-vitro holding on oocyte viability and oxidative stress remains unknown.This study aimed to evaluate the effects of holding duration on oocyte viability, oxidative stress, and oxidative DNA damage.
COCs were removed from abattoir-derived ovaries 4-8h post-slaughter, washed, and placed in M199(Hanks salts) supplemented with 3% BSA and held for 0h, 12h, 24h, or 36h at room temperature to mimic commercial conditions.At each time point, COCs were denuded, and oocytes were stained with Live/Dead Violet(viability) and CellROX Green (ROS marker), then fixed in 4% paraformaldehyde. Oocytes were permeabilized with 0.25% Triton X-100, blocked using 3% BSA, and incubated with antibodies against 4-hydroxynonenal(4HNE; lipid peroxidation) and 8-hydroxy-2’-deoxyguanosine(8-OHdG). Samples were mounted for fluorescence microscopy, and images were analysed using ImageJ.
Data were analysed using one-way ANOVA to ascertain the percentage of viable oocytes and the mean fluorescence intensity(FI) of oxidative stress and DNA damage markers compared to the initial(0h) time point. Normality of the data was assessed using the Shapiro-Wilk test. The percentage of viable oocytes decreased significantly at 36h (P≤0.05). Mean FI for 4HNE increased significantly by 12h (P≤0.05) and then decreased significantly by 24h (P≤0.05). The mean FI for CellROX was significantly higher after 24h (P≤0.05). No significant difference in mean FI was observed for 8OHdG at any time point. In conclusion, prolong holding of equine oocytes negatively affects viability and oxidative stress, though no increase in DNA damage was observed. These results suggest that the upper limit for holding time ought to be reduced to 12h to reduce lipid peroxidative damage and adverse ICSI outcomes.