Department of Animal Science, Division of Applied Life Science (BK21), Jinju, GyeongNam Province, Republic of Korea.
Department of Animal Science and Biotechnology, Sangji Youngseo College, Wonju, Republic of Korea.
Theriogenology. 2014 Feb;81(3):467-73. doi: 10.1016/j.theriogenology.2013.10.024. Epub 2013 Nov 5.
In this study, we investigated whether vitrification of an embryo by attachment to the inner surface of a plastic straw, which requires a small volume of vitrification solution, improves the survival of thawed embryos. In vitro-produced Korean native cattle blastocysts were randomly assigned into four groups: (1) blastocysts attached to the inner surface of a plastic straw (aV); (2) blastocysts loaded into the column of a plastic straw (cV); (3) blastocysts directly dropped into liquid nitrogen (dV); and (4) nonvitrified blastocysts (control). The postthaw recovery rate did not significantly differ among the aV, dV, and cV groups (98.3% vs. 81.5% vs. 91.4%). The postthaw survival rate was greater in the control, aV, and dV groups than in the cV group (100%, 87.7%, and 81.8% vs. 26.4%, P < 0.05), but did not significantly differ among the control, aV, and dV groups. The total number of cells per blastocyst did not significantly differ among the groups (134.4 ± 38.9 in control vs. 114 ± 48.1 in aV, 105.6 ± 33.9 in dV, and 102 ± 35.1 in cV group). However, the number of apoptotic cells per blastocyst was higher in the dV and cV groups than in the control group (10.9 ± 9.6 and 14.5 ± 9.5 vs. 0.4 ± 1.4; P < 0.05), but did not significantly differ between the control and aV groups (0.4 ± 1.4 vs. 6.6 ± 9.5). In addition, the blastocoel of each blastocyst was left intact or was mechanically punctured to reduce its volume, and the blastocysts were then vitrified using the aV method. At 12 hours after thawing, the re-expansion rate did not significantly differ among the control, punctured aV, and nonpunctured aV groups (93.3% vs. 85.2% vs. 82.8%). However, at 24 hours after thawing, the hatching rate was greater in the control and punctured aV groups than in the nonpunctured aV group (75% and 62.9% vs. 37.1%; P < 0.05). The total number of cells per blastocyst was greater in the control group than in the nonpunctured aV group (143 ± 37.2 vs. 94.5 ± 18.6; P < 0.05), but did not significantly differ between the control and punctured aV groups (143 ± 37.2 vs. 119.4 ± 19.7). The number of apoptotic cells per blastocyst was greater in the nonpunctured aV group than in the control and punctured aV groups (11.3 ± 6.1 vs. 5.9 ± 5.8 and 6.3 ± 4.4; P < 0.05). Taken together, the data show that the aV method improved the survival and quality of vitrified-thawed blastocysts. Furthermore, puncture of the blastocoel increased the hatching rate and reduced the number of apoptotic cells in vitrified-thawed blastocysts generated using the aV method.
在这项研究中，我们研究了胚胎通过附着在塑料吸管内表面的玻璃化是否可以改善解冻胚胎的存活率，因为这种方法需要的玻璃化溶液体积较小。我们将韩国本土牛的体外生产的囊胚随机分为四组：（1）附着在塑料吸管内表面的囊胚（aV）；（2）装入塑料吸管柱的囊胚（cV）；（3）直接放入液氮中的囊胚（dV）；和（4）未玻璃化的囊胚（对照）。aV、dV 和 cV 组的解冻后恢复率没有显著差异（98.3% vs. 81.5% vs. 91.4%）。与 cV 组相比，对照组、aV 组和 dV 组的解冻后存活率更高（100%、87.7%和 81.8% vs. 26.4%，P<0.05），但对照组、aV 组和 dV 组之间没有显著差异。每个囊胚的细胞总数在各组之间没有显著差异（对照组 134.4±38.9 个，aV 组 114±48.1 个，dV 组 105.6±33.9 个，cV 组 102±35.1 个）。然而，dV 和 cV 组每个囊胚的凋亡细胞数量高于对照组（10.9±9.6 和 14.5±9.5 比 0.4±1.4；P<0.05），但对照组和 aV 组之间没有显著差异（0.4±1.4 比 6.6±9.5）。此外，通过机械穿刺使每个囊胚的囊胚腔保持完整或减小其体积，然后使用 aV 方法对囊胚进行玻璃化处理。解冻后 12 小时，对照组、穿刺 aV 组和未穿刺 aV 组的再扩张率没有显著差异（93.3% vs. 85.2% vs. 82.8%）。然而，解冻后 24 小时，对照组和穿刺 aV 组的孵化率高于未穿刺 aV 组（75%和 62.9% vs. 37.1%；P<0.05）。对照组的每个囊胚的细胞总数多于未穿刺 aV 组（143±37.2 个，94.5±18.6 个；P<0.05），但对照组和穿刺 aV 组之间没有显著差异（143±37.2 个，119.4±19.7 个）。未穿刺 aV 组每个囊胚的凋亡细胞数量多于对照组和穿刺 aV 组（11.3±6.1 比 5.9±5.8 和 6.3±4.4；P<0.05）。综上所述，数据表明 aV 方法提高了玻璃化冷冻和解冻囊胚的存活率和质量。此外，对囊胚腔的穿刺增加了使用 aV 方法产生的玻璃化冷冻和解冻囊胚的孵化率，并减少了凋亡细胞的数量。
