Unidade de Biotecnologia e Recursos Geneticos

Quinta do Anjo, Portugal

Unidade de Biotecnologia e Recursos Geneticos

Quinta do Anjo, Portugal
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Romao R.,Unidade de Biotecnologia e Recursos Geneticos | Romao R.,University of Évora | Marques C.C.,Unidade de Biotecnologia e Recursos Geneticos | Baptista M.C.,Unidade de Biotecnologia e Recursos Geneticos | And 10 more authors.
Theriogenology | Year: 2015

The low survival of sheep invitro-produced (IVP) embryos after cryopreservation is a key limiting step to the widespread of this technology. In the present work, different approaches for enhancing cryosurvival of these embryos were compared: embryo delipidation by centrifugation in the absence or presence of cytochalasin D, a cytoskeleton stabilizer or by embryo culture in the presence of different doses of the trans-10 cis-12-conjugated linoleic acid isomer (CLA). Three experiments were conducted. In experiment 1, IVP blastocysts before vitrification were randomly distributed into four groups: control; centrifuged (cent), cytochalasin D (cyto-D), centrifuged+cytochalasin D (cent+cyto-D). In experiment 2, different doses of CLA (25, 50, and 100μM) were supplemented during embryo culture before vitrification of blastocysts. A control group ran simultaneously. A third experiment was performed to compare both approaches from the previous ones but without the groups with the worst results (groups: control, cyto-D, cent+cyto-D, CLA25, CLA50). In all experiments, embryos integrity and reexpansion were assessed after warming and after 3hours of culture. In experiment 1, the postwarming integrity rate was the lowest (P<0.05) in embryos from the cent group (cent: 50.6±10.3% vs. control: 74.6±9.2%, cyto-D: 92.3±9.7%, and cent+cyto-D: 90.5±11.2%), whereas the best (P<0.05) reexpansion scores were obtained in cent+cyto-D embryos (cent+cyto-D: 2.6±0.28 vs. control: 1.8±0.08, cent: 1.9±0.2, and cyto-D: 1.8±0.31). In experiments 2 and 3, higher (P<0.05) cleavage rates were observed in CLA25 (50.9±6.2% and 49.2±5.6%, respectively) and CLA50 (48.9±6.2% and 47.6±5.6%, respectively) than those in the control (41.8±6.1% and 40.4±5.4%, respectively) group. In experiment 2, CLA100 presented the lowest (P<0.002) Day-6 and -7 embryo production rate and quality. After warming, superior (P<0.02) expansion scores were achieved in CLA25 (3.1±0.29) and CLA50 (3.8±0.17) than in the control (1.9±0.10) group. Similar results were attained in experiment 3. However, although cent+cyto-D embryos showed higher (P=0.008) postwarming expansion scores than the control (2.8±0.29 vs. 1.9±0.07) group, this score was lower (P=0.0009) than that in CLA50 embryos (3.8±0.17). In conclusion, our results showed that different protocols of lipid reduction can be successfully applied to improve the cryotolerance of IVP sheep embryos. © 2015 Elsevier Inc.


PubMed | Unidade de Biotecnologia e Recursos Geneticos, University of Lisbon and University of Évora
Type: Journal Article | Journal: Theriogenology | Year: 2015

The low survival of sheep in vitro-produced (IVP) embryos after cryopreservation is a key limiting step to the widespread of this technology. In the present work, different approaches for enhancing cryosurvival of these embryos were compared: embryo delipidation by centrifugation in the absence or presence of cytochalasin D, a cytoskeleton stabilizer or by embryo culture in the presence of different doses of the trans-10 cis-12-conjugated linoleic acid isomer (CLA). Three experiments were conducted. In experiment 1, IVP blastocysts before vitrification were randomly distributed into four groups: control; centrifuged (cent), cytochalasin D (cyto-D), centrifuged + cytochalasin D (cent + cyto-D). In experiment 2, different doses of CLA (25, 50, and 100 M) were supplemented during embryo culture before vitrification of blastocysts. A control group ran simultaneously. A third experiment was performed to compare both approaches from the previous ones but without the groups with the worst results (groups: control, cyto-D, cent + cyto-D, CLA25, CLA50). In all experiments, embryos integrity and reexpansion were assessed after warming and after 3 hours of culture. In experiment 1, the postwarming integrity rate was the lowest (P < 0.05) in embryos from the cent group (cent: 50.6 10.3% vs.74.6 9.2%, cyto-D: 92.3 9.7%, and cent + cyto-D: 90.5 11.2%), whereas the best (P < 0.05) reexpansion scores were obtained in cent + cyto-D embryos (cent + cyto-D: 2.6 0.28 vs.1.8 0.08, cent: 1.9 0.2, and cyto-D: 1.8 0.31). In experiments 2 and 3, higher (P < 0.05) cleavage rates were observed in CLA25 (50.9 6.2% and 49.2 5.6%, respectively) and CLA50 (48.9 6.2% and 47.6 5.6%, respectively) than those in the control (41.8 6.1% and 40.4 5.4%, respectively) group. In experiment 2, CLA100 presented the lowest (P < 0.002) Day-6 and -7 embryo production rate and quality. After warming, superior (P < 0.02) expansion scores were achieved in CLA25 (3.1 0.29) and CLA50 (3.8 0.17) than in the control (1.9 0.10) group. Similar results were attained in experiment 3. However, although cent + cyto-D embryos showed higher (P = 0.008) postwarming expansion scores than the control (2.8 0.29 vs. 1.9 0.07) group, this score was lower (P = 0.0009) than that in CLA50 embryos (3.8 0.17). In conclusion, our results showed that different protocols of lipid reduction can be successfully applied to improve the cryotolerance of IVP sheep embryos.

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