Different chromatin and energy/redox responses of mouse morulae and blastocysts to slow freezing and vitrification

Abstract

BackgroundThe ability to cryopreserve mammalian embryos has become an integral part of assisted reproduction, both in human and veterinary medicine. Despite differences in the size and physiological characteristics of embryos from various species, the embryos have been frozen by either of two procedures: slow freezing or vitrification. The aim of our study was to compare the effect of slow freezing and vitrification to the chromatin structure, energy status and reactive oxygen species production of mouse morulae and blastocysts.MethodsMouse morulae and blastocysts were randomly allocated into vitrification, slow freezing and control groups. For slow freezing, Dulbecco phosphate buffered saline based 10% glicerol solution was used. For vitrification, G-MOPS™ based solution supplemented with 16% ethylene glycol, 16% propylene glycol, Ficoll (10 mg/ml) and sucrose (0.65 mol/l) was used. After warming, the chromatin integrity, mitochondrial distribution pattern and energy/oxidative status were compared among groups.ResultsCryopreservation affected chromatin integrity at a greater extent at the morula than the blastocyst stage. Chromatin damage induced by slow freezing was more relevant compared to vitrification. Slow freezing and vitrification similarly affected mitochondrial distribution pattern. Greater damage was observed at the morula stage and it was associated with embryo grade. Cryopreservation altered the quantitative bioenergy/redox parameters at a greater extent in the morulae than in the blastocysts. Effects induced by slow freezing were not related to embryo grade or mitochondrial pattern, as affected embryos were of all grades and with both mitochondrial patterns. However, effects induced by vitrification were related to mitochondrial pattern, as only embryos with homogeneous mitochondrial pattern in small aggregates had reduced energy status.ConclusionsThis study shows for the first time the joint assessment of chromatin damage and mitochondrial energy/redox potential in fresh and frozen mouse embryos at the morula and blastocyst stage, allowing the comparison of the effects of the two most commonly used cryopreservation procedures.

Successful Vitrification of In vivo Embryos Collected from Superovulated Japanese Black Cattle (Wagyu)

“…; Somoskoi et al. ). In this case, the capacity of a blastocyst to withstand to a certain degree cell damage caused by ice crystals is higher than a morula due mainly to its higher cell number.…”

Effect of different penetrating and non‐penetrating cryoprotectants and media temperature on the cryosurvival of vitrified in vitro produced porcine blastocysts

“…Sucrose is most commonly used as a non-penetrating cryoprotectant because of its high glass-forming potential. Ficoll is also regularly used, as the inclusion of this macromolecule has facilitated the successful vitrification of human, bovine, murine, equine and buffalo embryos (Hufana-Duran et al, 2004;Lagares et al, 2009;Makarevich, Kubovicova, & Chrenek, 2014;Somoskoi et al, 2015;Vanderzwalmen et al, 2003). Trehalose reportedly has the greatest ability to lower the mobility of water molecules hydrogen-bonded with saccharides, making it more effective than sucrose at maintaining the structural integrity of membranes during reduced water states (Ishida et al, 1997;Kawai, Sakurai, Inoue, Chujo, & Kobayashi, 1992 Fernandez Taranco, & Bo, 2014;Zhang, Yi, Yan, & Zhou, 2012;Zhou, Al, Sun, & Lonergan, 2010).…”

Cryosurvival and pregnancy rates: One‐step protocol for freezing–thawing Shangri‐la Yak (Bos grunniens) Embryos

“…This protocol requires longer time to induce TA B L E 1 Effects of postwarming on the developmental potential of one-step embryos in different stages the formation of ice crystals. During slow-rate freezing, intracytoplasmic ice crystal formation harms the structures and organelles of embryonic cells, resulting in cell damage or cell death (Kopeika, Thornhill, & Khalaf, 2015;Somoskoi et al, 2015). Therefore, the capacity of a blastocyst to withstand a certain degree of cold damage is higher than a morula due to its higher cell number.…”