Abstract
Vitrification of bovine cumulus-oocyte complexes (COCs) is not as successful as bovine embryos, due to oocyte's complex structure and chilling sensitivity. Synchrotron X-ray diffraction (SXRD), a powerful method to study crystal structure and phase changes, was used to detect the glass or ice formation in water, tissue culture medium (TCM)-199, vitrification solution 2 (VS2), and vitrified bovine COCs and morulae. Data revealed Debye's rings and peaks associated with the hexagonal ice crystals at 3.897, 3.635, 3.427, 2.610, 2.241, 1.912 and 1.878 Å in both water and TCM-199, whereas VS2 showed amorphous (glassy) appearance, at 102K (−171°C). An additional peak of sodium phosphate monobasic hydrate (NaH2PO4.H2O) crystals was observed at 2.064 Å in TCM-199 only. All ice and NaH2PO4.H2O peaks were detected in the non-vitrified (control) and vitrified COCs, except two ice peaks (3.145 and 2.655 Å) were absent in the vitrified COCs. The intensities of majority of ice peaks did not differ between the non-vitrified and vitrified COCs. The non-vitrified bovine morulae in TCM-199 demonstrated all ice- and NaH2PO4.H2O-associated Debye's rings and peaks, found in TCM-199 alone. There was no Debye's ring present in the vitrified morulae. In conclusion, SXRD is a powerful method to confirm the vitrifiability of a solution and to detect the glass or ice formation in vitrified cells and tissues. The vitrified bovine COCs exhibited the hexagonal ice crystals instead of glass formation whereas the bovine morulae underwent a typical vitrification.
Highlights
Cryopreservation of mammalian oocytes and embryos is important for conservation of female genetics in domestic animals and endangered species [1, 2], and for assisted reproduction in humans [3]
Cryoloop was used for the Synchrotron X-ray diffraction (SXRD) analysis of water, tissue culture medium (TCM)-199 and vitrification solution 2 (VS2), whereas cryotop was used for the vitrified bovine cumulus-oocyte complexes (COCs) and morulae
This study provided an evidence of the ice crystal formation in water and TCM199, and the glass formation in a commonly used VS2 for bovine COCs and embryos
Summary
Cryopreservation of mammalian oocytes and embryos is important for conservation of female genetics in domestic animals and endangered species [1, 2], and for assisted reproduction in humans [3]. Mammalian oocytes and early embryos are commonly cryopreserved by conventional slow freezing or vitrification method. Vitrification has become a popular method of cryopreservation for mammalian oocytes and embryos as it avoids chilling injury and damage due to the intracellular ice formation [15, 16]. It is fairly cheap, simple, quick and superior to slow freezing [1, 17]. Vitrification causes the lysis of cumulus cells and oocyte, and the misplacement of cortical granules in bovine germinal vesicle (GV) stage COCs [22]. It causes the disorganization of metaphase plate, condensation of chromosomes and clustering of cortical granules in metaphase II (MII) stage oocytes [23, 24]
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