Effect of Two-step Vitrification on Developmental Competence of in vitro and in vivo Produced Bovine Embryos

Abstract

The aim of this study was to establish the effect of two-step vitrification on survival rate of bovine embryos produced in vitro (method A) and in vivo (method B) from Holstein-Friesian cattle. The embryos suitable for vitrification were frozen by a two-step technique, using increasing concentrations of dimethyl sulphoxide (DMSO) and ethylene glycol (EG). After thawing, the quality grade and developmental stage of embryos was assessed. In vitro developmental competence of embryos of different quality grade obtained by method B (n = 82) was significantly higher (p < 0.001) compared to method A (n = 98). The best results were detected when we vitrified the embryos of the grade 1 quality; namely, the hatched blastocyst stage was reached by 6.9% (2/29) of embryos retrieved by method A and by 36.7% (11/30) of embryos retrieved by method B (p < 0.01). In the case of developmental competence of embryos at different developmental stages we reached significantly better results (p < 0.001) when we vitrified the embryos produced by method B (n = 84) in comparison with method A (n = 67). We noted a higher hatching rate at the stage of expanded blastocyst; namely, the hatched blastocyst stage was reached by 7.4% (2/27) of embryos produced by method A and by 30.8% (8/26) of embryos produced by method B (p < 0.05). In general, the hatched blastocyst stage was reached by 15.1% (50/331) of all thawed embryos retrieved by method A and B.In conclusion, when we applied two-step vitrification on the grade 1 quality embryos at the stage of expanded blastocyst produced in vitro or at the stage of morula produced in vivo we achieved the highest hatching rates.

Effect of different penetrating and non‐penetrating cryoprotectants and media temperature on the cryosurvival of vitrified in vitro produced porcine blastocysts

“…The majority of vitrification protocols describe the use of 37°C media, although warmed media may increase the toxic effects of cryoprotectants on embryos (Kuwayama, Gandhi, Kagawala, & Ramani, ). Several studies have described vitrification using room temperature media (22°C) with great success (Hlavicova, Lopatarova, & Cech, ; Kuwayama et al., ; Maehara et al., ; Uchikura et al., ). Kuwayama et al.…”

“…The majority of vitrification protocols describe the use of 37°C media, although warmed media may increase the toxic effects of cryoprotectants on embryos (Kuwayama, Gandhi, Kagawala, & Ramani, 2014). Several studies have described vitrification using room temperature media (22°C) with great success (Hlavicova, Lopatarova, & Cech, 2010;Kuwayama et al, 2005;Maehara et al, 2012;Uchikura et al, 2013). Kuwayama et al (2005) described a vitrification protocol with an extended equilibration time, to allow more complete penetration of the cryoprotectants, and with the vitrification solutions used at room temperature, to reduce the toxic effects that warming to 37°C may cause.…”