Ledda Sergio, Kelly Jen M, Nieddu Stefano, Bebbere Daniela, Ariu Federica, Bogliolo Luisa, Natan Dity, Arav Amir
1Department of Veterinary Medicine, University of Sassari, Sassari, Italy.
2South Australian Research and Development Institute, Turretfield Research Centre, 129 Holland Road, Rosedale, SA 5350 Australia.
J Anim Sci Biotechnol. 2019 Nov 1;10:90. doi: 10.1186/s40104-019-0390-1. eCollection 2019.
To advance the use of embryo vitrification in veterinary practice, we developed a system in which embryo vitrification, warming and dilution can be performed within a straw. Ovine produced embryos (IVEP) were vitrified at either early (EBs: = 74) or fully expanded blastocyst stage (FEBs: = 195), using a new device named "E.Vit", composed by a 0.25-mL straw with a 50-μm pore polycarbonate grid at one end. Embryos at each stage (EBs and FEBs) were vitrified by either Two-step (TS) or Multi-step (MS; 6 different concentrations of vitrification solutions) protocol. Non-vitrified embryos ( = 102) were maintained in culture as a control. Warming consisted of placing the straws directly into 1.5 mL tubes containing a TCM-199 solution with three decreasing concentrations of sucrose. Blastocyst re-expansion, embryo survival and hatching rate were evaluated at 2, 24 and 48 h post warming. The number of apoptotic cells was determined by TUNEL assay.
Blastocyst re-expansion (2 h) after warming was higher ( < 0.05) in FEBs group, vitrified with the MS and TS methods (77.90% and 71.25%, respectively) compared with the EBs group (MS: 59.38% and TS: 48.50%, respectively). Survival rates of vitrified FEBs after 24 h IVC were higher ( < 0.001) in both methods (MS and TS) than vitrified EBs (MS: 56.25%; TS: 42.42%) and was higher ( < 0.05) in the MS method (94.19%) compared with those in TS (83.75%). After 48 h of culture the hatching rate for FEBs vitrified in MS system (91.86%) was similar to control (91.89%), but higher than FEB TS (77.5%) and EBs vitrified in MS (37.5%) and TS (33.33%). Number of apoptotic cells were higher in EBs, irrespective of the system used, compared to FEBs. The number of apoptotic cells in FEBs vitrified with MS was comparable to the control.
A high survival rate of IVP embryos can be achieved by the new "E.Vit" device with hatching rates comparable with control fresh embryos. This method has the potential for use in direct embryo transfer in field conditions.
为了推动胚胎玻璃化技术在兽医实践中的应用，我们开发了一种系统，可在同一根细管内完成胚胎玻璃化、复温及稀释操作。使用一种名为“E.Vit”的新装置，将绵羊体外生产的胚胎（IVEP）在早期囊胚阶段（EBs：n = 74）或完全扩张囊胚阶段（FEBs：n = 195）进行玻璃化处理，该装置由一端带有50μm孔径聚碳酸酯网格的0.25 mL细管组成。每个阶段（EBs和FEBs）的胚胎通过两步法（TS）或多步法（MS；6种不同浓度的玻璃化溶液）方案进行玻璃化处理。未玻璃化的胚胎（n = 102）作为对照进行培养。复温是将细管直接放入含有三种蔗糖浓度递减的TCM - 199溶液的1.5 mL试管中。在复温后2、24和48小时评估囊胚再扩张、胚胎存活率和孵化率。通过TUNEL法测定凋亡细胞数量。
与EBs组（MS：59.38%，TS：48.50%）相比，采用MS和TS方法玻璃化处理的FEBs组在复温后2小时的囊胚再扩张率更高（P < 0.05）（分别为77.90%和71.25%）。在两种方法（MS和TS）中，玻璃化FEBs在体外培养24小时后的存活率均高于玻璃化EBs（MS：56.25%；TS：42.42%），且MS方法（94.19%）高于TS方法（83.75%）（P < 0.05）。培养48小时后，MS系统中玻璃化FEBs的孵化率（91.86%）与对照组（91.89%）相似，但高于FEBs的TS组（77.5%）以及MS和TS方法玻璃化处理的EBs组（分别为37.5%和33.33%）。无论采用何种系统，EBs中的凋亡细胞数量均高于FEBs。采用MS方法玻璃化处理的FEBs中的凋亡细胞数量与对照组相当。
新型“E.Vit”装置可实现较高的体外生产胚胎存活率，其孵化率与对照新鲜胚胎相当。该方法具有在野外条件下直接进行胚胎移植的应用潜力。
