Cellular and Molecular Events that Occur in the Oocyte during Prolonged Ovarian Storage in Sheep

SIMPLE SUMMARY: Establishing efficient in vitro embryo production (IVP) protocols in sheep usually requires prolonged transportation of post-mortem ovaries since adult animals are often slaughtered in abattoirs far from laboratories. In this study, different analyses were carried out to investigate important cellular and molecular aspects of hypoxic injury on excised ovaries over time in order to understand the factors jeopardizing the development of competent oocytes during prolonged transport times. We observed that, when ovaries were stored for more than 7 h, the quality and developmental potential of oocytes and cumulus cells were greatly reduced. Moreover, the use of medium TCM199 over saline solution also had deleterious effects. Beyond transport time, strategies aimed at reducing these damages may improve oocyte quality and developmental competence. ABSTRACT: For the past two decades, there has been a growing interest in the application of in vitro embryo production (IVP) in small ruminants such as sheep. To improve efficiency, a large number abattoir-derived ovaries must be used, and long distances from the laboratory are usually inevitable when adult animals are used. In that scenario, prolonged sheep ovary transportation may negatively affect oocyte developmental competence. Here, we evaluated the effect of ovary storage time (3, 5, 7, 9, 11 and 13 h) and the medium in which they were transported (TCM199 and saline solution) on oocyte quality. Thus, live/dead status, early apoptosis, DNA fragmentation, reduced glutathione (GSH) and reactive oxygen species (ROS) content, caspase-3 activity, mitochondrial membrane potential and distribution, and relative abundance of mRNA transcript levels were assessed in oocytes. After in vitro maturation (IVM), cumulus cell viability and quality, meiotic and fertilization competence, embryo rates and blastocyst quality were also evaluated. The results revealed that, after 7 h of storage, oocyte quality and developmental potential were significantly impaired since higher rates of dead oocytes and DNA fragmentation and lower rates of viable, matured and fertilized oocytes were observed. The percentage of cleavage, blastocyst rates and cumulus cell parameters (viability, active mitochondria and GSH/ROS ratio) were also decreased. Moreover, the preservation of ovaries in medium TCM199 had a detrimental effect on cumulus cells and oocyte competence. In conclusion, ovary transport times up to 5 h in saline solution are the most adequate storage conditions to maintain oocyte quality as well as developmental capacity in sheep. A strategy to rescue the poor developmental potential of stored oocytes will be necessary for successful production of high-quality embryos when longer ovarian preservation times are necessary.