Effect of different methods of cryopreservation on the cytoskeletal integrity of dromedary camel (Camelus dromedarius

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• 作者：J.A. Skidmore ; E. Schoevers ; T.A.E. Stout
• 关键词：Camel embryos ; Cryopreservation ; Cytoskeleton
• 刊名：Animal Reproduction Science
• 出版年：2009
• 出版时间：July 2009
• 年：2009
• 卷：113
• 期：1-4
• 页码：196-204
• 全文大小：470 K

文摘

This study examined the effect of different methods of cryopreservation on the cytoskeletal integrity of camel embryos. A total of 32 embryos were recovered on Days 6 and 7 after ovulation and measured before being frozen using either a conventional slow-cooling technique (n = 12: six Day 6 and six Day 7 embryos) or vitrification (n = 12: four Day 6 and eight Day 7). The remaining 8 ‘control’ embryos (four Day 6 and four Day 7) were not cryopreserved but instead incubated in holding medium for 30 min. After thawing, warming or incubation, the embryos were stained with 4,6-diamino-2-phenylindole dihydrochloride (DAPI) to identify dead cells. Subsequently, the embryos were fixed in 4 % paraformaldehyde, permeabilized and labelled with Alexa Fluor 488-Phalloidin to enable assessment of cytoskeleton integrity.

Vitrified&#x2013;warmed embryos contained a significantly higher percentage of dead cells than either conventionally frozen embryos or controls (P < 0.05). Although the proportion of dead cells in conventionally frozen embryos tended to be higher than in controls, the difference was not significant (P &#x2265; 0.07). Whereas embryo size did not affect the number of dead cells in conventionally frozen embryos, vitrified&#x2013;warmed embryos >300 μm in diameter had a significantly higher percentage of dead cells than embryos &#x2264;300 μm (P = 0.01). Cytoskeleton integrity was also affected by both freezing method and embryo diameter. All 8 control embryos had a Grade I cytoskeleton, compared with only 2/24 (8.3 % ) frozen or vitrified embryos. Of the 8 slow-frozen or vitrified embryos with a Grade III cytoskeleton post-thaw, 7 had been vitrified and 6 were larger (Day 7) embryos. These results indicate that while both slow-freezing and vitrification of camel embryos lead to cytoskeleton disruption and cell death, embryo quality is better preserved by slow-freezing.