Abstract
The current protocols of in vitro fertilization and culture in sheep rely on paradigms established more than 25 years ago, where Metaphase II oocytes are co-incubated with capacitated spermatozoa overnight. While this approach maximizes the number of fertilized oocytes, on the other side it exposes them to high concentration of reactive oxygen species (ROS) generated by active and degenerating spermatozoa, and positively correlates with polyspermy. Here we set up to precisely define the time frame during which spermatozoa effectively penetrates and fertilizes the oocyte, in order to drastically reduce spermatozoa-oocyte interaction. To do that, in vitro matured sheep oocytes co-incubated with spermatozoa in IVF medium were sampled every 30 min (start of incubation time 0) to verify the presence of a fertilizing spermatozoon. Having defined the fertilization time frame (4 h, data from 105 oocytes), we next compared the standard IVF procedures overnight (about 16 h spermatozoa/oocyte exposure, group o/nIVF) with a short one (4 h, group shIVF). A lower polyspermic fertilization (> 2PN) was detected in shIVF (6.5%) compared to o/nIVF (17.8%), P < 0.05. The o/nIVF group resulted in a significantly lower 2-cell stage embryos, than shIVF [34.6% (81/234) vs 50.6% (122/241) respectively, P < 0.001]. Likewise, the development to blastocyst stage confirmed a better quality [29% (70/241) vs 23.5% (55/234), shIVF vs o/nIVF respectively] and an increased Total Cell Number (TCN) in shIVF embryos, compared with o/n ones. The data on ROS have confirmed that its generation is IVF time-dependent, with high levels in the o/nIVF group. Overall, the data suggest that a shorter oocyte-spermatozoa incubation results in an improved embryo production and a better embryo quality, very likely as a consequence of a shorter exposure to the free oxygen radicals and the ensuing oxidative stress imposed by overnight culture.
Highlights
The current protocols of in vitro fertilization and culture in sheep rely on paradigms established more than 25 years ago, where Metaphase II oocytes are co-incubated with capacitated spermatozoa overnight
Having confirmed and quantified the high levels of reactive oxygen species (ROS) produced in In Vitro Fertilization (IVF) medium and finely tracked the early fertilization event, we have observed that restricting gamete co-incubation to only 4 h resulted in a significant amelioration in embryo production outcomes, both quantitatively and qualitatively
Matured oocytes were co-incubated with capacitated spermatozoa in IVF medium, and aliquots of oocytes were monitored for spermatozoa-oocyte interaction (Fig. 1), previous enzymatic/mechanical cumulus cells removal
Summary
The current protocols of in vitro fertilization and culture in sheep rely on paradigms established more than 25 years ago, where Metaphase II oocytes are co-incubated with capacitated spermatozoa overnight While this approach maximizes the number of fertilized oocytes, on the other side it exposes them to high concentration of reactive oxygen species (ROS) generated by active and degenerating spermatozoa, and positively correlates with polyspermy. Having defined the fertilization time frame (4 h, data from 105 oocytes), we compared the standard IVF procedures overnight (about 16 h spermatozoa/oocyte exposure, group o/nIVF) with a short one (4 h, group shIVF). Having confirmed and quantified the high levels of ROS produced in IVF medium and finely tracked the early fertilization event, we have observed that restricting gamete co-incubation to only 4 h resulted in a significant amelioration in embryo production outcomes, both quantitatively and qualitatively
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