Abstract
Simple SummaryEstablishing 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.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.
Highlights
Assisted reproduction technologies (ARTs) in small ruminants, such as sheep, have great potential for genetic improvement and dissemination programs, since they allow for a rapid and sustainable increase in animals of great genetic merit
Though the main lines of investigation in small ruminants have focused on germplasm banks and artificial insemination [2], over the past few decades, slight advances have been made toward the use of in vitro embryo production (IVP) [1,2,3]
A significantly higher level (p < 0.05) of reactive oxygen species (ROS) was recorded in immature sheep oocytes recovered from ovaries stored for 3 h (42.35 ± 3.69) compared to oocytes obtained from ovaries stored for 13 h
Summary
Assisted reproduction technologies (ARTs) in small ruminants, such as sheep, have great potential for genetic improvement and dissemination programs, since they allow for a rapid and sustainable increase in animals of great genetic merit. Though the main lines of investigation in small ruminants have focused on germplasm banks and artificial insemination [2], over the past few decades, slight advances have been made toward the use of in vitro embryo production (IVP) [1,2,3]. Improving the efficiency of IVP protocols in these species entails the use of ovaries of dead animals because a large number of samples should be collected. Unlike their in vivo counterparts, oocytes retrieved from dead animals exhibit reduced developmental potential [4]. Considering that the quality of oocytes determines the developmental potential of embryos after fertilization [5], the preservation of oocyte integrity from the moment the animal dies until the ovaries are processed is of critical importance
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