Pascottini Osvaldo Bogado, Catteeuw Maaike, Van Soom Ann, Opsomer Geert
Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium.
Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium.
Theriogenology. 2018 Feb;107:63-69. doi: 10.1016/j.theriogenology.2017.10.040. Epub 2017 Nov 4.
Bovine in vitro embryo production (IVP) following Ovum Pick Up (OPU) is all too often hampered by a large time gap between the harvest of oocytes of the first and last OPU session of the day. Immediately after retrieval, oocyte maturation is initiated, resulting in oocytes maturing at different time points which necessitates laborious scheduling of the IVP process. In this study, the potential of a commercial embryo holding medium (EHM; Syngro, Bioniche Inc.) to hold immature bovine oocytes was validated. We assessed the effect of holding time and temperature on (1) oocytes' maturation; (2) blastocyst development and quality at day 8 post insemination; and (3) blastocyst yield in small groups of oocytes/zygotes simulating OPU settings. Oocytes, harvested from slaughterhouse ovaries, were held for 6 h (either at 4 °C, room temperature [RT; 22-25 °C], or 38.5 °C), for 10 h (at 4 °C or RT), and for 14 h (only at RT) in 1 mL sterile glass osmometer tubes filled with EHM prior to standard maturation (22 h at 38.5 °C) and subsequent IVP. Results were compared with controls in which no prior holding was applied. Differences between the treated and control groups were assessed by generalized mixed-effects models and considered significant at P < 0.05. Generally, oocytes held up to 14 h in EHM at different temperatures remained at the germinal vesicle stage. Holding immature oocytes in EHM for 6 h at 38.5 °C and for 10 h at 4 °C significantly decreased maturation (57.1 ± 4.1% VS 80.9 ± 3.2% and 68.6 ± 3.5% VS 80.7 ± 2.9%; respectively), and development (11.0 ± 1.8% VS 36.2 ± 2.8% and 20.1 ± 3.3% VS 40.6 ± 4.6%) (P < 0.05). However, holding in EHM for both 6 and 10 h at RT, did not affect the maturation rates (83.2 ± 2.9% and 78.9 ± 3.2%) nor day 8 blastocyst rates (35.2 ± 2.7% and 40.2 ± 4.5%). Prolonging holding time to 14 h in RT decreased maturation and day 8 blastocyst yield (71.9 ± 3.5% VS 84.5 ± 2.7% and 25.7 ± 2.5% VS 39.5 ± 2.8%, respectively) (P < 0.05). Holding oocytes in EHM did not significantly affect embryonic quality as assessed by differential apoptotic staining in any of the time points. To simulate OPU-settings, small groups of 10 oocytes were held in EHM for 6 or 10 h at RT. When subsequently matured, fertilized and cultured per 8 zygotes, day 8 blastocyst rate was not affected (19.8 ± 3.5% VS 20.6 ± 3.6% and 18.8 ± 3.6% VS 18.3 ± 3.4%). In conclusion, immature bovine oocytes can be successfully conserved in EHM at RT for up to 10 h without compromising their embryonic developmental competence nor quality.
在通过采卵（OPU）进行牛体外胚胎生产（IVP）时，常常会受到一天中首次和末次OPU采卵之间存在较大时间间隔的影响。采卵后，卵母细胞立即开始成熟，导致卵母细胞在不同时间点成熟，这就需要对IVP过程进行繁琐的安排。在本研究中，验证了一种商业胚胎保存培养基（EHM；Syngro，Bioniche公司）保存未成熟牛卵母细胞的潜力。我们评估了保存时间和温度对以下方面的影响：（1）卵母细胞的成熟；（2）授精后第8天的囊胚发育和质量；（3）在模拟OPU设置的小批量卵母细胞/受精卵中的囊胚产量。从屠宰场卵巢采集的卵母细胞，在标准成熟培养（38.5°C下培养22小时）和随后的IVP之前，在装有EHM的1mL无菌玻璃渗透计管中分别保存6小时（4°C、室温[RT；22 - 25°C]或38.5°C）、10小时（4°C或RT）和14小时（仅在RT）。将结果与未进行预先保存的对照组进行比较。通过广义混合效应模型评估处理组和对照组之间的差异，P < 0.05时认为差异具有统计学意义。一般来说，在不同温度下于EHM中保存长达14小时的卵母细胞仍处于生发泡期。在38.5°C下将未成熟卵母细胞在EHM中保存6小时以及在4°C下保存10小时，显著降低了成熟率（分别为57.1±4.1%对80.9±3.2%和68.6±3.5%对80.7±2.9%）和发育率（11.0±1.8%对36.2±2.8%和20.1±3.3%对40.6±4.6%）（P < 0.05）。然而，在RT下于EHM中保存6小时和10小时，既不影响成熟率（83.2±2.9%和78.9±3.2%），也不影响第8天的囊胚率（35.2±2.7%和40.2±4.5%）。在RT下将保存时间延长至14小时会降低成熟率和第8天的囊胚产量（分别为71.9±3.5%对84.5±2.7%和25.7±2.5%对39.5±2.8%）（P < 0.05）。在任何时间点，通过差异凋亡染色评估，在EHM中保存卵母细胞对胚胎质量没有显著影响。为了模拟OPU设置，将10个卵母细胞的小批量在RT下于EHM中保存6小时或10小时。随后每8个受精卵进行成熟、受精和培养时，第8天的囊胚率不受影响（19.8±3.5%对20.6±3.6%和18.8±3.6%对18.3±3.4%）。总之，未成熟牛卵母细胞可以在RT下于EHM中成功保存长达10小时，而不会损害其胚胎发育能力和质量。
