Ultrastructure of Bovine Embryos Frozen and Thawed by a Two-Step Freezing Method

Hyttel, Poul; Lehn-Jensen, H.; Greve, T.

doi:10.1159/000146132

Cited by 13 publications

(9 citation statements)

References 11 publications

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“…Also, a great amount of intercellular junctions were seen. In fair and poor quality embryos, our results agree with those of Mohr and Trounson (1981), Hyttel et al (1986), however, these last authors point to disturbances in the zona pellucida of poor quality embryos, that were not found in our study.…”

Section: Discussionsupporting

confidence: 86%

Comparison of Stereoscopy, Light Microscopy and Ultrastructural Methods for Evaluation of Bovine Embryos

Aguilar

Galina

Merchant

et al. 2002

Reprod Domestic Animals

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The most important point in embryo transfer success is the evaluation of the stage of development and quality of embryos. Therefore, the purpose of this study was to compare the morphological evaluation of embryos using stereoscopy, light microscopy and electron microscopy in order to establish the accuracy of former method compared with more invasive and accurate procedures. For this purpose, 23 Brahman x Swiss cows were used and synchronized with Norgestomet 6 mg plus, 5 mg Estradiol valerate (Syncromate B(R), Rhone Merieux, Mexico, Mexico City) and superovulated with Folltropin-V 240 mg (Vetrepharm, Mexico, Mexico City). Non-surgical embryo collection was performed 7.5 days after insemination. Descriptive statistics analysis was used to assess the data. Seventy-eight embryos were collected and classified by stereoscopic microscopy, finding 51.2% (40) of good quality, 25.6% (20) fair and 24.3% (19) poor. Later, under light microscopy observation, evaluation of the same embryos resulted in 25.6% (20) good, 32.0% (25) fair and 42.3% poor quality. Finally, in the evaluation of embryos under electron microscopy 24.3% (19) were found to be of good quality, 29.3% (23) fair and 46.1% (36) poor. Evaluation of embryos with stereoscopic microscopy was found to be very subjective, as nearly 50% of embryos classified by this method as good quality, showed features of degenerative stages under light and electron microscopy. Embryos with these features are generally frozen and transferred, which could be one of the reasons for having low fertility rate in embryo transfer programmes.

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Section: Discussionsupporting

confidence: 86%

Comparison of Stereoscopy, Light Microscopy and Ultrastructural Methods for Evaluation of Bovine Embryos

Aguilar

Galina

Merchant

et al. 2002

Reprod Domestic Animals

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“…However, no severe cellular damage or re-organization of the structure of the embryo was mentioned, although the re-expansion rate of the vitrified embryos using the ethylene glycol-ficoll-sucrose method in that study was ,50%. After conventional slow-rate freezing, Hyttel et al (1986) described an increased number of secondary lysosomes as well as electron-lucent spaces in the cytoplasmic matrix of in-vivo-produced bovine embryos. Similar findings were not done in this experiment probably due to the fact that the embryos frozen by traditional slow-rate freezing methods are to a higher degree exposed to ice crystal formation and shrinkage due to the osmotic damage.…”

Section: Discussionmentioning

confidence: 99%

“…In surviving blastocysts, these changes are almost totally absent following the first 24 hr of culture. Although some studies dealing with the ultrastructural changes of embryos following cryopreservation have been published (Hyttel et al, 1986;Darvelid et al, 1994;Kuwayama et al, 1994;Wiemer et al, 1995), no investigation about the sequential changes after vitrification explaining the above mentioned phenomena have been performed.…”

Section: Introductionmentioning

confidence: 99%

Morphological changes of in-vitro-produced bovine blastocysts after vitrification, in-straw direct rehydration, and culture

Vajta¹,

Hyttel²,

Callesen³

1997

Mol. Reprod. Dev.

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Morphological signs of injury and regeneration following vitrification and warming of bovine embryos were studied by light and electron microscopy. In‐vitro‐produced Day 7 expanded blastocysts (Day 0 = day of insemination) were vitrified by a two‐step equilibration method using ethylene glycol and dimethyl sulphoxide as cryoprotectants. Thawing was performed by in‐straw direct rehydration, followed by in vitro culture on a granulosa cell monolayer. Embryos were processed for transmission electron microscopy immediately after warming (0 hr) as well as after 4 hr or 24 hr of culture following warming. A control group of unfrozen embryos was also processed. At 0 hr after warming, except for a rapid collapse of the blastocoele, only minor changes were detectable by stereomicroscope. However, at the ultrastructural level, signs of extensive injury were seen, including a general distension or shrinkage of mitochondria, disintegration of cell adhesions between adjacent trophoblastic cells, and complete rupture of some cells. At 4 hr, stereomicroscopic investigation revealed collapsed blastocoele and a darkened granular appearance of the cell mass. At the ultrastructural level, signs of regeneration were also observable: cells with minor injuries were re‐assembled in a central area forming a small blastocoele, cell adhesion structures were re‐established, and damage of mitochondria was less severe. The majority of irreversibly damaged cells or cell debris was accumulated in the perivitelline space. At 24 hr, stereomicroscopic investigation of surviving blastocysts showed no signs of the previous injury. At the ultrastructural level, cellular debris in the perivitelline space and some degenerated cells in the blastocoele were the only signs of previous injuries. In conclusion, ultrastructural investigation revealed unexpectedly extensive damage followed by a rapid regeneration and reorganization of the embryonic structure. Mol. Reprod. Dev. 48:9–17, 1997. © 1997 Wiley‐Liss, Inc.

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“…2-16 cells embryos, we observe an increase of the average size of the lipid droplets independent of the culture condition (Table 1). During cryopreservation, larger droplets lead to a reduction of the organization of the cytoplasm and produce irreversible damage to the embryo [24,26]. Thus we do not expect that 2-16 cells embryos tolerance to cryopreservation will be different between embryos cultured with or without serum, as occurs in blastocysts [50].…”

Section: Lipidsmentioning

confidence: 99%

Does serum cause lipid-droplet accumulation in bovine embryos produced in vitro, during developmental days 1 to 4?

Crocco

Kelmansky

Mariano

2013

J Assist Reprod Genet

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SummaryPurpose Serum supplementation has shown to have beneficial effects on in vitro bovine embryo development. However, it is often assumed that serum supplementation may produce mitochondrial damage and this damage would generate lipid accumulation, a major obstacle for cryopreservation. The aim of the present study is to investigate the previous assumptions in early embryonic stages. Methods We considered in vitro produced bovine embryos from day 1 to 4 of development, which were grown in presence of serum from days 1, 2 or 3 or in absence of it. Electron transmission micrographs allowed us to quantify the area occupied by lipid droplets and by the different mitochondrial types to evaluate serum effect. Using confocal microscopy we analyzed mitochondrial activity and location. Results We found no evidence of lipid droplets accumulation or mitochondrial degeneration or reduction of mitochondrial area in serum supplemented media. Further, our results suggest that events of mitochondrial proliferation are taking place even in serum supplemented media.Conclusions Serum does not produce lipid accumulation or mitochondrial damage in bovine embryos from 2 to 16 cells. When serum was added to embryo culture medium on day 3 of development, there were ultrastructural signs of a beneficial effect for embryo development. The lack of serum until day 3 may also avoid the unnecessary exposure to potentially inhibitory factors present on it.

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Ultrastructure of Bovine Embryos Frozen and Thawed by a Two-Step Freezing Method

Cited by 13 publications

References 11 publications

Comparison of Stereoscopy, Light Microscopy and Ultrastructural Methods for Evaluation of Bovine Embryos

Comparison of Stereoscopy, Light Microscopy and Ultrastructural Methods for Evaluation of Bovine Embryos

Morphological changes of in-vitro-produced bovine blastocysts after vitrification, in-straw direct rehydration, and culture

Does serum cause lipid-droplet accumulation in bovine embryos produced in vitro, during developmental days 1 to 4?

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