Effects of dilution procedure and culture conditions after thawing on survival of frozen bovine blastocysts produced in vitro

Massip, A.; Mermillod, Pascal; Wils, C.; Dessy, F.

doi:10.1530/jrf.0.0970065

Cited by 38 publications

(18 citation statements)

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“…In the second experiment, delipidated, sham and control Day-7 blastocysts were frozen using a conventional slow freezing procedure (19). This protocol is used for in vivo-produced embryos and is not optimal for in vitro ones, but the purpose of this freezing thawing process was to submit the different groups of IVP embryos to the same extreme conditions during the freezing process, and to make the differences between the groups more evident.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Delipidating in vitro-produced bovine zygotes: Effect on further development and consequences for freezability

Díez

Heyman

Bourhis³

et al. 2001

Theriogenology

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To study the effect of partial removal of intracytoplasmatic lipids from bovine zygotes on their in vitro and in vivo survival, presumptive zygotes were delipidated by micromanipulation and cocultured with Veto cells in B2+10% FCS. Blastocyst rates of delipidated (n=960), sham (centrifuged but not delipidated, n=830) and control embryos (n=950) were 42.1, 42.3 and 39.9% respectively (P > 0.05). Day 7 blastocysts derived from delipidated zygotes had a mean of 123.9 + 45.6 nuclei compared to 137.5 + 32.9 for control blastocysts (P > 0.05). The full-term development of delipidated blastocysts after single transfer to recipients was similar to that of control IVF blastocysts (41.2% vs 45.4% respectively). To assess the effect of delipidation on the embryo tolerance to freczing/thawing, delipidated (n=73), control (n=67) and shmn (n=50) Day 7 blastocysts were frozen in 1.36 M glycerol + 0.25 M sucrose in PBS. After thawing, embryos were cocultured for 72 h with Vero cells in B2+10% FCS. Survival rates at 24 h were not significantly different between groups. However, in the delipidated group, the survival rate after 48 h in culture was significantly higher than in the control group (56.2 vs 39.8, P < 0.02), resulting in a higher hatching rate after 3 days in culture (45.2 vs 22.4, P < 0.02). Pregnancy rates for delipidated and control frozen/thawed embryos were respectively 10.5 and 22.2% (P > 0.05). Electron microscopic observations showed much fewer lipid droplets (and smaller) in delipated blastocysts than in controls. Taken together, our data show that delipidation of one cell stage bovine embryos is compatible with their normal development to term and has a beneficial effect on their tolerance to freezing and thawing at the blastocyst stage. This procedure, however, alters the developmental potential of such blastocysts, suggesting that maternally inherited lipid stores interfere with metabolic recovery after thawing.

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Section: Discussionmentioning

confidence: 99%

“…In vitro-produced Day 7 blastocysts were frozen according to the protocol of Massip et al (1993), used for in vivo-produced embryos. Our purpose was to submit the different groups of IVP embryos to the same extreme conditions during the freezing process, and to make the differences among the groups more evident.…”

Section: Freezing Proceduresmentioning

confidence: 99%

Delipidating in vitro-produced bovine zygotes: Effect on further development and consequences for freezability

Díez

Heyman

Bourhis³

et al. 2001

Theriogenology

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“…Massip et al (1993a). After thawing (15 s in a 20°C water bath), the contents of each straw was expelled into a petri dish and mixed by shaking rapidly; after 10 min at room temperature, embryos were washed twice in PBS for 10 min.…”

Section: Introductionmentioning

confidence: 99%

Survival and viability of fresh and frozen-thawed in vitro bovine blastocysts

Massip¹,

Mermillod²,

Langendonckt³

et al. 1995

Reprod. Nutr. Dev.

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“…It is likely that the percentage of hatched embryos can be further improved by modifying postthaw culture conditions. Massip et al (1993) found hatching rates for frozen/thawed, in vitro-produced embryos were 41% when culture was performed in the presence of bovine oviductal epithelial cells, whereas hatching rate using other culture conditions not involving coculture was 0-6%. Nonetheless, one would not expect optimal pregnancy rates to be achieved following direct transfer of embryos frozen in glycerol even with the inclusion of cytochalasin B treatment.…”

Section: Discussionmentioning

confidence: 97%

Effects of Hyaluronic Acid in Culture and Cytochalasin B Treatment Before Freezing on Survival of Cryopreserved Bovine Embryos Produced in Vitro

Franco

Hansen

2006

In Vitro Cell Dev Biol Anim

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One limitation to the widespread use of in vitro-produced embryos in cattle is their poor survival following cryopreservation. Two approaches for enhancing survival of in vitro-produced bovine embryos following cryopreservation were evaluated: culture in the presence of hyaluronic acid and alterations in the cytoskeleton through cytochalasin B treatment. The experiment was a 2 x 2 factorial design to test main effects of hyaluronic acid added to culture at day 5 after insemination (+ or -) and cryopreservation treatment (control or cytochalasin B). Embryos used for cryopreservation were blastocysts and expanded blastocysts harvested on day 7 after insemination. Cytochalasin B increased the percent of embryos that re-expanded (P < 0.0001) and that hatched following thawing (P < 0.05). The hatching percent was 29.6% for embryos treated with cytochalasin B versus 9.1% for control embryos. There was no significant effect of hyaluronic acid on survival although there was a tendency for embryos cultured with hyaluronic acid to have higher percent hatching if not treated with cytochalasin B (12.7% for hyaluronic acid versus 4.5% for control; hyaluronic acid x cytochalasin B interaction; P = 0.09). In conclusion, cytochalasin B treatment before freezing improved cryosurvival of bovine embryos produced in vitro. Such a treatment could be incorporated into methods for cryopreservation of bovine embryos provided post-transfer survival is adequate. In contrast, culture with hyaluronic acid was of minimal benefit- the increased cryosurvival in the absence of cytochalasin B was not sufficient to allow an adequate number of embryos to survive.

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Effects of dilution procedure and culture conditions after thawing on survival of frozen bovine blastocysts produced in vitro

Cited by 38 publications

References 4 publications

Delipidating in vitro-produced bovine zygotes: Effect on further development and consequences for freezability

Delipidating in vitro-produced bovine zygotes: Effect on further development and consequences for freezability

Survival and viability of fresh and frozen-thawed in vitro bovine blastocysts

Effects of Hyaluronic Acid in Culture and Cytochalasin B Treatment Before Freezing on Survival of Cryopreserved Bovine Embryos Produced in Vitro

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