Ultra-Structural Alterations inIn VitroProduced Four-Cell Bovine Embryos Following Controlled Slow Freezing or Vitrification

Abstract

Cryopreservation is the process of freezing and preserving cells and tissues at low temperatures. Controlled slow freezing and vitrification have successfully been used for cryopreservation of mammalian embryos. We investigated the effect of these two cryopreservation methods on in vitro produced four-cell stage bovine embryos which were classified according to their quality and separated into three groups. The first group was maintained as untreated controls (n = 350). Embryos of the second (n = 385) and the third (n = 385) groups were cryopreserved either by controlled slow freezing or by vitrification. Embryos in groups 2 and 3 were thawed after 1 day. Hundred embryos were randomly selected from the control group, and 100 morphologically intact embryos from the second and third group were thawed after 1 day and cultured to observe the development up to the blastocyst stage. The blastocyst development rate was 22% in the control group, 1% in the slow-freezing group and 3% in the vitrification group. Remaining embryos of all three groups were examined by light microscopy, transmission electron microscopy and immunofluorescence confocal microscopy with subsequent histological staining procedures. Cryopreservation caused degenerative changes at the ultra-structural level. Compared with vitrification, slow freezing caused an increased mitochondrial degeneration, cytoplasmic vacuolization, disruption of the nuclear and plasma membrane integrity, organelle disintegration, cytoskeletal damage, a reduced thickness of the zona pellucida and a formation of fractures in the zona pellucida. Further studies are required to understand and decrease the harmful effects of cryopreservation.

Assisted hatching of vitrified-warmed blastocysts prior to embryo transfer does not improve pregnancy outcomes

“…In addition, with current methods of vitrification, thawed blastocysts may not benefit from AH as much as they did with past freeze-thaw techniques. Indeed, there is evidence in bovine embryos that the ZP displays different ultra-structural alterations after slow freeze compared to vitrification [ 35 ].…”

Comparison between in vitro embryo production using Y-sorted sperm and timed artificial insemination with non-sorted sperm to produce crossbred calves

“…In the present study, the pregnancy rates when FTET was performed with fresh embryos or vitrified embryos were similar (P = 0.79, Table 2). Results of several studies indicate that there are negative effects of cryopreservation on embryo development after warming (Cavusoglu et al, 2016), but the effects of cryopreservation may vary with animal species, breed, origin (in vitro or in vivo), lipid composition of the embryo, size and stage of development, quality of the embryo/oocyte, cell permeability, toxicity and osmotic properties of cryoprotectants and cooling and warming rates (Almiñana and Cuello, 2015). In contrast, results of other studies indicate that there can be acceptable pregnancy rates after FTET of embryos derived with use of IVEP and fertilization with sex-sorted semen, both fresh and vitrified (Xu et al, 2006;Pontes et al, 2011;Trigal et al, 2012) or only vitrified/warmed (Marinho et al, 2015).…”

Comet assay on thawed embryos: An optimized technique to evaluate DNA damage in mouse embryos

“…Nevertheless, we can assume that embryo loss was not as high as in the present study. Our hypothesis is that the important embryo loss we observed with Protocol 1 could be related to the alteration of ZP due to the freezing-thawing protocol: dehydratation due to cryprotectant could induce hardening and thinning of the ZP (Cavusoglu et al, 2016;Trounson and Mohr, 1983). These alterations could modify the interaction between the ZP and the agarose, and increase the embryo loss during the steps of coverslip removal and/ or lysis.…”