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Effects of resazurin on fertilization of bovine oocytes and preimplantation embryo development in vitro
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Theriogenology EFFECTS OF RESAZURIN ON FERTILIZATION OF BOVINE OOCYTES AND PREIMPLANTATION EMBRYO DEVELOPMENT IN VITRO S. Wang, R. G. Holyoak, G. Liu, R.C. Evans and T. D. Bunch Animal Science Department, Utah State University, Logan, Utah 84322-9400, USA
Resazurin (7-hydroxy-3H-phenoxazin-3-one lo-oxide) is a redox dye that when exposed to metabolically active spermatozoa is reduced to resorufin, as manifested by visual color changes from blue (resazurin) to pink (resort&). Resazurin is used for assessing potential fertility of spermatozoa and recently has been used to investigate viability or ftmctional status of eukaryotic cells. Because of its high sensitivity and reproducibility, the resazurin reduction assay has the potential to be developed into a method for noninvasively assessing fertilizing capacity of spermatozoa and embryo viability. This study examined the fertilizing capacity of resazurin-exposed spermatozoa and investigated the effects of resazurin on the development of preimplantation bovine embryos using in vitro maturation (IVM), fertilization (IVF) and embryo culture (IVC) methodologies. Bovine oocytes were aspirated from ovaries collected from a local abattoir and matured in vitro as described by Hawk and Wall (1994, Theriogenology 4 1,157 1- 1583). IVM cultured oocytes were fertilized in vitro as described by Parrish et al (1988, Bio. Reprod. 38, 1171-1180) with modifications according to the experimental treatments. Cryopreserved bovine semen was thawed and the live sperm separated by a Percoll gradient. Capacitation occurred in fert-TALP containing 10 &ml heparin. The resulting zygotes were cultured in a modified CR-2 medium (Wang et al., 1997, Theriogenology 47,304) with modifications according to the experimental treatments. Cleavage rates were established at 48 h after exposure of oocytes to spermatozoa and post-cleavage embryo development was determined at d 6, 8, and 10 of culture. Two experiments in a randomized complete block design were used. Percentage data were angularly transformed and analyzed by the use of a general linear model (GLM) ANOVA. In Experiment 1, Percoll gradient separated live spermatozoa were exposed to 17.6 ,ug resazurin/ml medium (R70 17, Sigma Chemical Company, St. Louis, MO, USA) for 0 (TRTl), 15 (TRT2), 30 (TRT3) and 60 min (TRT4), respectively, in spermTALP. The resazurin-exposed spermatozoa were then used for IVF of in vitro matured oocytes (n=ll94). The cleavage rates were 80.5%, 84.1%, 85.4% and 85.3%; the percentages of morulae at d 6 of culture were 42.3,44.3,38.0 and 39.6; the percentages ofblastocysts at d 8 were 18.1,20.0, 15.5 and 19.7; and the percentages of expanded and hatched blastocyst at d 10 were 12.8,12.7,10.2 and 12.9, for TRTl, TRT2, TRT3 and TRT4, respectively. There was no significant (P>O.O5) difference with respect to cleavage rates and embryo development between treatments. In Experiment 2, cleaved IVMiIVF-derived embryos (n=63 1) were placed in cultme medium containing 0.0 (TRTl), 8.8 (TRTZ), 17.5 (TRT3) and 26.3 (TRT4) Hg resazmin/ml for 6 hand then transferred to normal medium for the rest of the culture period. The percentages of morulae at d 6 of culture were 35.5,42.3,31.8 and 32.8; the percentages ofblastocysts at d 8 were 13.3,14.8,12.1 and 11.2; and the percentages of expanded and hatched blastocysts at d 10 were 7.4,8.8,7.6 and 7.3 for TRTl, TRT2, TRT3, TRT4, respectively. There was no significant (P>O.O5) difference in post-cleavage embryo development between treatments. In conclusion, resazurin had no adverse effects on fertilizing capacity of spermatozoa or on subsequent embryo development under the conditions of this study.
Theriogenology EFFECTS OF RESAZURIN ON FERTILIZATION OF BOVINE OOCYTES AND PREIMPLANTATION EMBRYO DEVELOPMENT IN VITRO S. Wang, R. G. Holyoak, G. Liu, R.C. Evans and T. D. Bunch Animal Science Department, Utah State University, Logan, Utah 84322-9400, USA
Resazurin (7-hydroxy-3H-phenoxazin-3-one lo-oxide) is a redox dye that when exposed to metabolically active spermatozoa is reduced to resorufin, as manifested by visual color changes from blue (resazurin) to pink (resort&). Resazurin is used for assessing potential fertility of spermatozoa and recently has been used to investigate viability or ftmctional status of eukaryotic cells. Because of its high sensitivity and reproducibility, the resazurin reduction assay has the potential to be developed into a method for noninvasively assessing fertilizing capacity of spermatozoa and embryo viability. This study examined the fertilizing capacity of resazurin-exposed spermatozoa and investigated the effects of resazurin on the development of preimplantation bovine embryos using in vitro maturation (IVM), fertilization (IVF) and embryo culture (IVC) methodologies. Bovine oocytes were aspirated from ovaries collected from a local abattoir and matured in vitro as described by Hawk and Wall (1994, Theriogenology 4 1,157 1- 1583). IVM cultured oocytes were fertilized in vitro as described by Parrish et al (1988, Bio. Reprod. 38, 1171-1180) with modifications according to the experimental treatments. Cryopreserved bovine semen was thawed and the live sperm separated by a Percoll gradient. Capacitation occurred in fert-TALP containing 10 &ml heparin. The resulting zygotes were cultured in a modified CR-2 medium (Wang et al., 1997, Theriogenology 47,304) with modifications according to the experimental treatments. Cleavage rates were established at 48 h after exposure of oocytes to spermatozoa and post-cleavage embryo development was determined at d 6, 8, and 10 of culture. Two experiments in a randomized complete block design were used. Percentage data were angularly transformed and analyzed by the use of a general linear model (GLM) ANOVA. In Experiment 1, Percoll gradient separated live spermatozoa were exposed to 17.6 ,ug resazurin/ml medium (R70 17, Sigma Chemical Company, St. Louis, MO, USA) for 0 (TRTl), 15 (TRT2), 30 (TRT3) and 60 min (TRT4), respectively, in spermTALP. The resazurin-exposed spermatozoa were then used for IVF of in vitro matured oocytes (n=ll94). The cleavage rates were 80.5%, 84.1%, 85.4% and 85.3%; the percentages of morulae at d 6 of culture were 42.3,44.3,38.0 and 39.6; the percentages ofblastocysts at d 8 were 18.1,20.0, 15.5 and 19.7; and the percentages of expanded and hatched blastocyst at d 10 were 12.8,12.7,10.2 and 12.9, for TRTl, TRT2, TRT3 and TRT4, respectively. There was no significant (P>O.O5) difference with respect to cleavage rates and embryo development between treatments. In Experiment 2, cleaved IVMiIVF-derived embryos (n=63 1) were placed in cultme medium containing 0.0 (TRTl), 8.8 (TRTZ), 17.5 (TRT3) and 26.3 (TRT4) Hg resazmin/ml for 6 hand then transferred to normal medium for the rest of the culture period. The percentages of morulae at d 6 of culture were 35.5,42.3,31.8 and 32.8; the percentages ofblastocysts at d 8 were 13.3,14.8,12.1 and 11.2; and the percentages of expanded and hatched blastocysts at d 10 were 7.4,8.8,7.6 and 7.3 for TRTl, TRT2, TRT3, TRT4, respectively. There was no significant (P>O.O5) difference in post-cleavage embryo development between treatments. In conclusion, resazurin had no adverse effects on fertilizing capacity of spermatozoa or on subsequent embryo development under the conditions of this study.