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Vitrification of immature equine oocytes using synthetic ice blockers: ultrastructural injuries and maturation rates
Journal of Equine Veterinary Science 34 (2014) 184
Contents lists available at ScienceDirect
Journal of Equine Veterinary Science journal homepage: www.j-evs.com
Vitrification of immature equine oocytes using synthetic ice blockers: ultrastructural injuries and maturation rates B.R. Curcio 1, *, M.O. Gastal 2, G.R. Pereira 1, C.D. Corcini 1, F.C. Landim-Alvarenga 3, S.S. Barros 4, C.E.W. Nogueira 1, J.C. Deschamps 1, E.L. Gastal 2 1
Federal University of Pelotas, Pelotas, RS, Brazil Southern Illinois University, Carbondale, IL, USA State University of Sao Paulo, Botucatu, SP, Brazil 4 Federal University of Santa Maria, Santa Maria, RS, Brazil 2 3
Low maturation rates have been obtained after vitrification of immature equine oocytes [Tharasanit et al., Reproduction 2006;132:759-69]. Although oocyte cryopreservation technique is critical for equine assisted reproduction programs, this is still an inefficient process, in part, to an inadequate oocyte competence post-thawing which can be compromised by changes resulting from cold shock [Chen et al., Mol Cell Endocrinol 2003;202:101-7]. Thus, investigations are needed for a better understanding of developmental competence of equine oocytes after vitrification. Low concentration of synthetic ice blockers (SIB) inhibits ice crystal formation [Wowk et al., Cryobiology 2000;40:228-36], and has recently been used for vitrification of equine oocytes [de Leon et al., Theriogenology 2012;77:21-27]. The objectives of this study were to identify ultrastructural morphological injuries and maturation rates in equine oocytes submitted to vitrification solutions (VS) with SIB during different exposure times. In Experiment 1, cumulus-oocyte complexes (COCs) were allocated to treatments: (a) Fresh COCs fixed after selection (Control; n ¼ 10); (b) COCs exposed to VS-1 (1.4 M DMSO + 1.8 M EG + 1% SIB) for 3 min and VS-2 (2.8 M DMSO + 3.6 M EG + 0.6 M Sucrose + 1% SIB) for 1 min (Eq-long; n ¼ 10); and (c) COCs exposed to VS-1 for 1.5 min and VS-2 for 30 s (Eq-short; n ¼ 10). COCs were vitrified using open-
* Presenting author
pulled straws, and after thawing and cultured for 1-2 h, the ultrastructural characteristics were evaluated by TEM. In Experiment 2, compact (Cp) and expanded (Ex) COCs were: (a) immediately matured in vitro (Control; n ¼ 161); (b) vitrified using DMSO, EG, and SIB (Eq-short; n ¼ 149); and (c) vitrified using formamide, DMSO, EG, polyvinylpyrrolidone, and SIB (Eq-short-mod; n ¼ 144). After thawing and IVM for 30 h, oocytes were stained with Hoechst for nuclear status assessment. More (P<0.02) oocytes with normal ultrastructural morphology were seen in fresh control (80%) and Eq-short (60%) groups than in Eqlong (30%) group. Metaphase II (MII) rates were higher (P<0.05) for Ex than Cp COCs (53.2 vs 29.3%) in the control group. Ex oocytes showed higher (P<0.05) MII rates than Cp oocytes in both Eq-short (50% and 27.3%, respectively) and Eq-short-mod (45.7% and 21.6%, respectively) groups. In conclusion, reduction of exposure time to the vitrification solutions better preserved the oocyte ultrastructural features and enhanced MII rates for vitrified Ex COCs. This study sheds light on potential alternatives for vitrification of immature equine oocytes. Acknowledgments Supported by CAPES, Brazil.
Contents lists available at ScienceDirect
Journal of Equine Veterinary Science journal homepage: www.j-evs.com
Vitrification of immature equine oocytes using synthetic ice blockers: ultrastructural injuries and maturation rates B.R. Curcio 1, *, M.O. Gastal 2, G.R. Pereira 1, C.D. Corcini 1, F.C. Landim-Alvarenga 3, S.S. Barros 4, C.E.W. Nogueira 1, J.C. Deschamps 1, E.L. Gastal 2 1
Federal University of Pelotas, Pelotas, RS, Brazil Southern Illinois University, Carbondale, IL, USA State University of Sao Paulo, Botucatu, SP, Brazil 4 Federal University of Santa Maria, Santa Maria, RS, Brazil 2 3
Low maturation rates have been obtained after vitrification of immature equine oocytes [Tharasanit et al., Reproduction 2006;132:759-69]. Although oocyte cryopreservation technique is critical for equine assisted reproduction programs, this is still an inefficient process, in part, to an inadequate oocyte competence post-thawing which can be compromised by changes resulting from cold shock [Chen et al., Mol Cell Endocrinol 2003;202:101-7]. Thus, investigations are needed for a better understanding of developmental competence of equine oocytes after vitrification. Low concentration of synthetic ice blockers (SIB) inhibits ice crystal formation [Wowk et al., Cryobiology 2000;40:228-36], and has recently been used for vitrification of equine oocytes [de Leon et al., Theriogenology 2012;77:21-27]. The objectives of this study were to identify ultrastructural morphological injuries and maturation rates in equine oocytes submitted to vitrification solutions (VS) with SIB during different exposure times. In Experiment 1, cumulus-oocyte complexes (COCs) were allocated to treatments: (a) Fresh COCs fixed after selection (Control; n ¼ 10); (b) COCs exposed to VS-1 (1.4 M DMSO + 1.8 M EG + 1% SIB) for 3 min and VS-2 (2.8 M DMSO + 3.6 M EG + 0.6 M Sucrose + 1% SIB) for 1 min (Eq-long; n ¼ 10); and (c) COCs exposed to VS-1 for 1.5 min and VS-2 for 30 s (Eq-short; n ¼ 10). COCs were vitrified using open-
* Presenting author
pulled straws, and after thawing and cultured for 1-2 h, the ultrastructural characteristics were evaluated by TEM. In Experiment 2, compact (Cp) and expanded (Ex) COCs were: (a) immediately matured in vitro (Control; n ¼ 161); (b) vitrified using DMSO, EG, and SIB (Eq-short; n ¼ 149); and (c) vitrified using formamide, DMSO, EG, polyvinylpyrrolidone, and SIB (Eq-short-mod; n ¼ 144). After thawing and IVM for 30 h, oocytes were stained with Hoechst for nuclear status assessment. More (P<0.02) oocytes with normal ultrastructural morphology were seen in fresh control (80%) and Eq-short (60%) groups than in Eqlong (30%) group. Metaphase II (MII) rates were higher (P<0.05) for Ex than Cp COCs (53.2 vs 29.3%) in the control group. Ex oocytes showed higher (P<0.05) MII rates than Cp oocytes in both Eq-short (50% and 27.3%, respectively) and Eq-short-mod (45.7% and 21.6%, respectively) groups. In conclusion, reduction of exposure time to the vitrification solutions better preserved the oocyte ultrastructural features and enhanced MII rates for vitrified Ex COCs. This study sheds light on potential alternatives for vitrification of immature equine oocytes. Acknowledgments Supported by CAPES, Brazil.