The harmful effect of removing the extracellular vitrification medium during embryo cryopreservation using a nylon mesh device in rabbit

Abstract

During the last decades, many techniques have been developed to reduce sample volume and improve cooling and warming rates during embryo vitrification. The vast majority are based on the "minimum drop size" concept, in which the vitrification solution around embryos is reduced by aspiration, leaving a tiny part of volume surrounding embryos.However, novel cryodevices were aimed to remove the entire vitrification solution. This study was designed to compare the "minimum drop size" technique using Cryotop® with the nylon mesh as cryodevice on rabbit morula embryos. The outcomes assessed were the in vitro development rates (experiment 1) and the offspring rates at birth (experiment 2).Embryos were vitrified in a two-step procedure; equilibrium (10% EG + 10% Me2SO) for 2 min and vitrification (20% EG + 20% Me2SO) for 1 minutes. In experiment 1, embryos (n= 323) were warmed and subsequently in vitro cultured for 48 h to assess the embryo developmental capability to reach the hatching-hatched blastocyst stage. In experiment 2, embryos were transferred using the laparoscopic technique (n=369) to assess the offspring rate at birth. In this context, rates of in vitro embryo development were similar between vitrified groups (0.73±0.042% and 0.66±0.047% for Cryotop® and nylon mesh device, respectively), but lower than in the fresh group (0.97±0.016%, p<0.05). In experiment 2, there were no significant differences in survival rates (offspring born/total embryos transferred) among the Cryotop® device group and fresh group (0.41±0.049% and 0.49±0.050%, respectively). But significantly lower value was obtained in the nylon mesh device group (0.18±0.030%). These results indicate that nylon mesh is not suitable as cryodevice for rabbit morula vitrification, remaining those using the "minimum drop size" methodology as the best option.

Metabolomic Analysis Reveals Changes in Preimplantation Embryos Following Fresh or Vitrified Transfer

“…In addition, cryopreservation is known for its harmful impacts on embryonic health, placing embryos at risk from a variety of types of cryoinjury during temperature and phase transitions [ 18 , 27 ]. Such survival impairment is in agreement with previous studies performed in rabbits, where manipulations of either fresh or vitrified embryos resulted in negative developmental effects both under in vitro conditions and in vivo [ 22 , 23 , 26 , 28 , 29 , 30 ]. It has been reported that successful hatching is dependent on a sufficiently high number of embryonic cells, which enables blastocyst expansion and zona shedding [ 31 ].…”

Experimental Evidence Reveals Both Cross-Infection and Cross-Contamination Risk of Embryo Storage in Liquid Nitrogen Biobanks

“…In oocyte vitrification, a decrease in cryosurvival rate due to the use of closed devices has been reported [31]. However, Cryotop and mini-straws had a similar embryonic development in rabbit species [24], but slightly less than the in vitro development, compared with fresh embryos [24,32,33]. Bielanski et al [13] showed that Cryotop-closed with a straw cap system and straws had the same embryo development results after a year of storage, with a lower potential danger of embryo contamination.…”