Division of Animal Sciences, University of Missouri, Columbia, Missouri, United States of America.
PLoS One. 2013 Jun 7;8(6):e65545. doi: 10.1371/journal.pone.0065545. Print 2013.
Cryopreservation of swine embryos is inefficient. Our goal was to develop a non-invasive method for "relatively" high-throughput cryopreservation of in vivo-produced swine embryos. Since removal of the lipid droplets within early swine embryos improves cryosurvival we wanted to apply a technique of high osmolality treatment followed by centrifugation that was first developed for in vitro-produced swine embryos to in vivo-produced swine embryos. The first aim was to determine how sensitive the in vivo-produced zygote and 2-cell stage embryo was to various high osmolality conditions for a short duration. Culture for 6, 12 or 18 min at 300, 400 or 500 milliosmoles (mOsm) had no detectable affect on the resulting blastocyst stage embryos (number of inner cell mass nuclei, trophectoderm nuclei, total number of nuclei, ratio of the trophectoderm to inner cell mass nuclei or percent blastocyst). However there was an effect of gilt on each of these parameters. For the second aim we focused on 300 mOsm for 6 min, 400 mOsm for 12 min, 500 mOsm for 12 min, and 500 mOsm for 18 min. The embryos were centrifuged for the duration of high osmolality treatment, then cultured to the blastocyst stage and vitrified. After vitrification and thawing the 500 mOsm for 18 min had the highest percent re-expansion with no difference in the total number of nuclei. While requiring a different base culture medium than in vitro-produced embryos, in vivo-derived embryos also survive cryopreservation without damage to their zona pellucida.
猪胚胎的冷冻保存效率不高。我们的目标是开发一种非侵入性方法，用于对体内产生的猪胚胎进行“相对”高通量的冷冻保存。由于早期猪胚胎中脂质滴的去除可以提高冷冻保存的存活率，我们希望将最初为体外产生的猪胚胎开发的高渗透压处理后离心的技术应用于体内产生的猪胚胎。第一个目的是确定体内产生的受精卵和 2 细胞期胚胎对各种高渗透压条件的短期敏感性。在 300、400 或 500 毫渗透压摩尔（mOsm）下培养 6、12 或 18 分钟对随后的囊胚期胚胎（内细胞团核、滋养层核、总核数、滋养层与内细胞团核的比例或囊胚百分比）没有可检测的影响。然而，每个参数都受到母猪的影响。对于第二个目的，我们专注于 300 mOsm 处理 6 分钟、400 mOsm 处理 12 分钟、500 mOsm 处理 12 分钟和 500 mOsm 处理 18 分钟。胚胎在高渗透压处理期间离心，然后培养至囊胚阶段并进行玻璃化。玻璃化和解冻后，500 mOsm 处理 18 分钟的再扩张率最高，核总数没有差异。虽然需要与体外产生的胚胎不同的基础培养基，但体内来源的胚胎也可以在不损害其透明带的情况下冷冻保存而不受损害。
