Abstract
BackgroundTo 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 in vitro produced embryos (IVEP) were vitrified at either early (EBs: n = 74) or fully expanded blastocyst stage (FEBs: n = 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 (n = 102) were maintained in in vitro 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.ResultsBlastocyst re-expansion (2 h) after warming was higher (P < 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 (P < 0.001) in both methods (MS and TS) than vitrified EBs (MS: 56.25%; TS: 42.42%) and was higher (P < 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.ConclusionsA high survival rate of IVP embryos can be achieved by the new “E.Vit” device with hatching rates in vitro comparable with control fresh embryos. This method has the potential for use in direct embryo transfer in field conditions.
Highlights
Over the past decades, the worldwide increase in small ruminant breeding has been supported by the development and improvement of assisted reproductive technologies (ART) [1, 2]
Embryo recovery The embryo recovery rates from the “E.Vit” system are reported in Table 1 comparing the different embryonic stages (EB vs. fully expanded blastocyst (FEB)) and the different methods of cryoprotectant exposure (TS vs. MS)
Survival of embryos after 24 h of post warming in vitro culture Survival rates were significantly (P < 0.001) higher after vitrification of fully expanded blastocysts (FEB) compared with the early blastocyst (EB) in the TS method [FEB: N = 67/80 (83.75%) vs EB: N = 14/33 (42.42%), respectively] and in the MS method [FEB: N = 81/86 (94.19%) vs EB: N = 18/32 (56.25%), respectively)
Summary
The worldwide increase in small ruminant breeding has been supported by the development and improvement of assisted reproductive technologies (ART) [1, 2]. Recent improvements of embryo production and cryopreservation technologies have the potential to allow wider propagation of valuable genetics in small ruminant populations and establishment of flocks. Ledda et al Journal of Animal Science and Biotechnology (2019) 10:90 without risk of disease transmission These technologies could make a substantial contribution to the preservation of endangered species or breeds. To date, documented use and success rates for different cryopreservation techniques and devices in small ruminants are relatively scarce compared with cattle [6,7,8]. Ovine in vitro produced embryos (IVEP) were vitrified at either early (EBs: n = 74) or fully expanded blastocyst stage (FEBs: n = 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. The number of apoptotic cells was determined by TUNEL assay
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
