139 Exogenous activation of bovine oocytes after piezo-intracytoplasmic sperm injection: is it really necessary?

In piezo-intracytoplasmic sperm injection (piezo-ICSI), a blunt injection pipette is used to advance into oocytes by piezo-electric movements, making the injection procedure less traumatic to the oocytes (Kimura et al. 1995). However, the requirement of bovine oocyte activation remains controversial, since contradictory results have been published. In the first part of this study, we sought to assess the effect of the piezo pulses in the removal of bovine sperm plasma and acrosome membranes. For this, two different protocols of electrical pulses were evaluated: (i) mild electrical pulses at intensity 2 and speed 2 (I2V2), and (ii) stronger pulses at intensity 4 and speed 7 (I4V7). A control without pulses was also included. Plasma membrane and acrosomal integrity were analysed with PI/Hoechst and PNA-FITC/Hoechst, respectively, by confocal fluorescence microscopy. Data were evaluated by contingency analysis. In the second part of this study, we evaluated the effect of exogenous oocyte activation after piezo-ICSI. Cumulus-oocyte complexes were aspirated from abattoir ovaries and matured in TCM-199 maturation medium for 20–24 h at 38.5°C and 5% CO₂. Injected oocytes were randomly distributed into three groups: (i) piezo-injected oocytes activated with 5 μM ionomycin for 5 min followed by 1 μg/mL anisomycin for 5 h (piezo-ICSI + A), (ii) piezo-ICSI without activation, and (iii) sham injection. Embryos were cultured in 50-μL drops of KSOM medium at 38.5°C and 5% CO₂, 5% O₂, and 90% N₂. Cleavage was recorded at 72 h and blastocyst rate at 192 h. Data were transformed to arcsine, analysed by ANOVA, and means were compared using Tukey’s test with Statgraphics Plus 2 Software. Sperm subjected to both piezoelectric treatments showed significantly higher percentages of membrane damage than the control (P < 0.05). I4V7 treatment generated 100% plasma membrane damage and ∼60% acrosomal membrane damage, which was also significantly greater than I2V2 treatment (20% and 9%, respectively; P < 0.05). Therefore, I4V7 treatment was later used for the generation of piezo-ICSI embryos. Embryo development results with a total of 376 injected oocytes (99, 145, and 132 for sham, piezo-ICSI and piezo-ICSI + A, respectively) showed significant differences in cleavage (P < 0.05) in both piezo-ICSI treatments (87% and 69%, respectively), while sham injection showed the lowest cleavage rate (21%). However, although the blastocyst rate at 192 h was higher with piezo-ICSI + A (26%), no difference was observed with piezo-ICSI treatment without activation (18%). In conclusion, I4V7 treatment is suitable to damage the sperm plasma and acrosome membranes, facilitating the exchange of factors necessary for fertilisation after ICSI. The results of ICSI show that although activation of piezo-injected oocytes accelerated the cleavage rate, it was not required for blastocyst formation, suggesting that activation after piezo-ICSI is not required. Furthermore, the possibility of generating parthenotes after activation of piezo-injected oocytes strongly suggest avoiding this exogenous activation step.

This research was supported by FONDECYT 1201166 ANID and slaughterhouse Frigorifico Temuco, Chile.