In vitro and in vivodevelopment of mice morulae after storage in non-frozen conditions

Abstract

BackgroundInterchange of genetically modified (GM) mice between laboratories using embryos provides several advantages. Not only is transport stress avoided, but also the health status of the recipient colony is not compromised. Embryos do not need to be shipped in frozen stage, which requires expensive packaging in addition to a certain degree of expertise in order to freeze and thaw them correctly. The aim of this study was to examine different storage conditions and their effect on embryo viability in order to establish the feasibility of practical, non-frozen conditions for embryo shipment.MethodsMouse morulae developed in vivo (collected from donors 2.5d post coitum) or in vitro (zygotes cultured until morulae stage) were stored, combining two different media (KSOMeq or KSOM-H) and temperatures (4 degrees C, 15 degrees C and 37 degrees C) throughout 24 or 48 hours. After storage in vitro viability was assessed determining percentage of development to blastocyst and total cell number. In vivo viability was determined based on the number of implantations and living fetuses after embryo transfer of stored embryos. The storage effect at the molecular level was assessed by studying a gene pool involved in early development by quantitative RT-PCR.ResultsIn vivo-produced morulae stored for 24 hours did not show differences in development up to the blastocyst stage, regardless of the storage type. Even though a decrease in the total cell number in vivo was observed, embryo development after embryo transfer was not affected. All 24 hour storage conditions tested provided a similar number of implantations and fetuses at day 14 of pregnancy. Morulae obtained from in vitro embryo culture collected at the 1-cell stage showed a decreased ability to develop to blastocyst after 24 hours of storage at 15degrees C both in KSOMeq and KSOM-H. Concomitantly, a significant decrease of embryo implantation rates after transfer to recipients was also found. In order to further characterize the effect of non-frozen storage combining a molecular approach with the ordinary in vitro culture evaluation, embryos collected at the morula stage were submitted to the same storage conditions described throughout 48 hours. In vitro culture of those embryos showed a significant decrease in their developmental rate to blastocyst in both KSOMeq and KSOM-H at 15degrees C, which also affected the total number of cells. Gene transcription studies confirmed significant alterations in retrotransposons (Erv4 and Iap) after 48 h of storage at 15degrees C.ConclusionsOur results show that both KSOMeq and KSOM-H can be equally used, and that several temperature conditions allow good survival rates in vitro and in vivo. Some of these storage conditions can substitute freezing in order to maintain embryo viability for 24–48 hours, providing a reliable and less demanding technical alternative for embryo interchanges.

Prevention of hatching of porcine morulae and blastocysts by liquid storage at 20 °C

“…This is not surprising since in our previous work, we observed a certain development delay in porcine morulae preserved for up to 72 h at 25 °C or 37 °C, with the delay being most marked at 25 °C 4,6 . Our data suggest that the metabolism of pig embryos is severely reduced at temperatures below 20 °C, an effect that could be related to the misregulation of genes that play important roles in embryo development and cell proliferation and alterations in other molecular events, as previously reported for mice embryos stored at refrigeration temperatures 13,15 . Although the developmental arrest observed in the stored embryos was temporal, their final developmental stage was equally delayed including a lower total cell number compared to controls.…”

“…Although the developmental arrest observed in the stored embryos was temporal, their final developmental stage was equally delayed including a lower total cell number compared to controls. However, this situation may not be relevant for two main reasons; firstly, we previously reported that developmentally delayed stored pig embryos retained the capacity to develop in vivo 4,6 ; and secondly, although the relevance of a cell number limit is not yet fully understood, embryos with significantly lower cell numbers do not exhibit a compromised implantation capacity, at least in rodents 15 .…”

“…Studies performed in rodents, ruminants and equine have indicated that low, supra-zero temperatures (around 4 °C) induce a temporary arrest of embryonic development 9,12,13,15,41 . Interestingly, our results show that porcine embryos (morulae and blastocysts) did not grow during the 72 h of storage tested, at either 17 °C or 20 °C.…”

“…Cooling to refrigeration temperatures (around 4–5 °C) not only inhibits embryo metabolism but also maintains embryo viability in several species, including bovine 7–9 , ovine 10 , rabbit 11,12 , and rodent 13–15 species and humans 16 . However, such cooled storage shows that embryonic survival rates differ among species, embryo stages and laboratory procedures.…”

Simple transport and cryopreservation of cold-stored mouse embryos

“…The cold-transport system of two-cell embryos is a useful technique to transport genetically engineered mice without the shipment of live animals. There are several studies that have improved the storage medium of two-cell mouse embryos [ 1 , 3 , 4 , 7 , 8 , 11 , 18 , 19 ]. Previously, we reported that NAC improved the developmental rate of two-cell embryos after cold storage and prolonged the storage period up to 96 h [ 6 ].…”