title: Cryopreservation of in vitro-produced sheep embryos: Effects of different protocols of lipid reduction.
abstract: 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 μM) 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.
  
  
  METHODS
  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.
  
  
  METHODS
  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.
authors: Unidade de Biotecnologia e Recursos Genéticos, INIAV-Santarém, Quinta da Fonte Boa, Vale de Santarém, Portugal; Escola de Ciências e Tecnologia, ICAAM-Instituto de Ciências Agrárias e Ambientais Mediterrânicas, Universidade de Évora, Núcleo da Mitra, Évora, Portugal. Electronic address: rjromao@uevora.pt.
authors: R
authors: R
authors: Romão
authors: Unidade de Biotecnologia e Recursos Genéticos, INIAV-Santarém, Quinta da Fonte Boa, Vale de Santarém, Portugal.
authors: C C
authors: CC
authors: Marques
authors: Unidade de Biotecnologia e Recursos Genéticos, INIAV-Santarém, Quinta da Fonte Boa, Vale de Santarém, Portugal.
authors: M C
authors: MC
authors: Baptista
authors: Unidade de Biotecnologia e Recursos Genéticos, INIAV-Santarém, Quinta da Fonte Boa, Vale de Santarém, Portugal; CIISA, Faculdade de Medicina Veterinária, Universidade de Lisboa Avenida da Universidade Técnica, Lisboa, Portugal.
authors: J P
authors: JP
authors: Barbas
authors: Unidade de Biotecnologia e Recursos Genéticos, INIAV-Santarém, Quinta da Fonte Boa, Vale de Santarém, Portugal.
authors: A E M
authors: AE
authors: Horta
authors: Unidade de Biotecnologia e Recursos Genéticos, INIAV-Santarém, Quinta da Fonte Boa, Vale de Santarém, Portugal; CIISA, Faculdade de Medicina Veterinária, Universidade de Lisboa Avenida da Universidade Técnica, Lisboa, Portugal; Escola Universitária Vasco da Gama, Av. José R. Sousa Fernandes, Campus Universitário - Bloco B, Lordemão, Coimbra, Portugal.
authors: N
authors: N
authors: Carolino
authors: Escola de Ciências e Tecnologia, ICAAM-Instituto de Ciências Agrárias e Ambientais Mediterrânicas, Universidade de Évora, Núcleo da Mitra, Évora, Portugal.
authors: E
authors: E
authors: Bettencourt
authors: Unidade de Biotecnologia e Recursos Genéticos, INIAV-Santarém, Quinta da Fonte Boa, Vale de Santarém, Portugal; CIISA, Faculdade de Medicina Veterinária, Universidade de Lisboa Avenida da Universidade Técnica, Lisboa, Portugal; Escola Universitária Vasco da Gama, Av. José R. Sousa Fernandes, Campus Universitário - Bloco B, Lordemão, Coimbra, Portugal.
authors: R M
authors: RM
authors: Pereira
mesh_terms: D000818:Animals; D015925:Cryopreservation / Q000379:methods / Q000662:veterinary*; D047108:Embryonic Development; D005307:Fertilization in Vitro / Q000379:methods / Q000662:veterinary*; D050356:Lipid Metabolism; D012756:Sheep / Q000196:embryology*; D014461:Ultracentrifugation / Q000662:veterinary
keywords: Conjugated linoleic acid isomer; Cryopreservation; Cytochalasin; Lipid reduction; Sheep embryo; Ultracentrifugation