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Vertebrate reproductive science and technology
RESEARCH ARTICLE

84 COMPARISON BETWEEN IN VIVO AND IN VITRO PRODUCED EMBRYOS WITH FORSKOLIN AFTER VITRIFICATION

D. M. Paschoal A , M. J. Sudano A , T. S. Rascado A , L. C. O. Magalhães A , L. F. Crocomo A , J. F. Lima-Neto A , A. Martins Júnior B and F. C. Landim-Alvarenga A
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- Author Affiliations

A School of Veterinary Medicine and Animal Science, São Paulo State University, UNESP, Botucatu, São Paulo, Brazil;

B School of Odontology, São Paulo State University, UNESP, Araçatuba, São Paulo, Brazil

Reproduction, Fertility and Development 23(1) 147-147 https://doi.org/10.1071/RDv23n1Ab84
Published: 7 December 2010

Abstract

The high concentration of lipids on embryos reduces their viability after cryopreservation. The use of drugs to modify their metabolism has been used to produce embryos with greater resistance to cryopreservation. The present experiment aimed to induce cytoplasmic lipolysis in in vitro produced (IVP) bovine embryos using forskolin (Sigma-Aldrich, St. Louis), which raises the levels of intracellular cAMP. Nelore cow cumulus–oocyte complexes (COC) were matured in TCM 199 and fertilized with frozen Nelore bull semen. Presumptive zygotes were cultured in SOFaa supplemented with BSA in the presence of 2.5% FCS. Embryos were kept in a humidified atmosphere with 5% CO2, 5% O2, and 90% N2 at 38.5°C. On Day 6, embryos were divided into 2 groups: 2.5% FCS (2.5% FCS without forskolin) and 2.5% FCS + F (2.5% FCS plus 10 μM forskolin). Embryo cleavage was recorded on Day 3 (IVF: D0), and blastocyst production on Day 7. Embryo viability was estimated by the index of total number of cells per embryos observed after the staining with propidium iodite and Hoechst 33342. In vitro produced embryos were compared with embryos obtained in vivo from Nelore cows. Embryos were vitrified using the protocol developed by Campos-Chillòn et al. (2006). After thawing, the re-expansion rate and cell number were again estimated (after 12 h). For statistical analysis, percentage cleaved and percentage blastocyst, percentage re-expansion, and total number of cells were transformed using the arcsine transformation (√y/100) and analysed using ANOVA followed by Tukey’s test. The level of significance adopted was 5%. No statistical differences were observed between IVP embryos concerning cleavage (2.5% FCS: 87.48 ± 9.52 and 2.5% FCS + F: 85.13 ± 7.57) and blastocyst formation rates (2.5% FCS: 46.8 ± 13.28 and 2.5% FCS + F: 46.31 ± 11.39). Also, no differences were detected in total number of cells per embryos (2.5% FCS: 162.4 ± 43.3; 2.5% FCS + F: 147.6 ± 35.3 and in vivo: 143.5 ± 11.5) when IVP and in vivo produced embryos were compared. After vitrification the re-expansion rate was similar between IVP and in vivo produced embryos (2.5% FCS: 75.07 ± 9.81; 2.5% FCS + F: 81.09 ± 10.90 and in vivo: 86.40 ± 18.62). But the total cell number of IVP embryos was significantly lower than the in vivo produced embryos [2.5% FCS: 42.6 ± 17.2a (P < 0.001); 2.5% FCS + F: 59.9 ± 46.2a (P < 0.05) and in vivo: 124.2 ± 12.9b]. If one side the viability cell between IVP and in vivo produced embryos is the same, after vitrification in vivo produced embryos showed greater resistance after the vitrification. The addition of forskolin did not interfere in embryo production or quality, but this drug was not efficient in increasing embryo tolerance after vitrification.

FAPESP 07/53505-1.