title: Exposure of in vitro-matured porcine oocytes to SYBR-14 and fluorescence impairs their developmental capacity.
abstract: Staining with Hoechst 33342 followed by ultraviolet irradiation is frequently used to aid or confirm the enucleation of recipient oocytes in porcine somatic cell nuclear transfer programs. However, the procedure has a clearly deleterious effect on the developmental ability of oocytes. This study evaluated the effectiveness of a longer-wavelength fluorochrome (SYBR-14) for visualizing maternal chromosomes in in vitro-matured porcine oocytes and the effects of this dye in combination with fluorescence excitation on the subsequent in vitro fertilization and embryo development of the oocytes. In the first experiment, the oocytes were exposed to different concentrations (1, 3, 5 and 7 μg/mL) of SYBR-14 at different incubation times (5, 10 and 30 min) in a 4 × 3 factorial design. The optimal condition for proper metaphase-II plate and first polar body visualization was a 10-min incubation with 5 μg/mL of SYBR-14. In the second experiment, the degeneration rate of the oocytes 18 h after exposure to SYBR-14 (5 μg/mL for 10 min) and fluorescence excitation for 5 or 30s was significantly higher (p<0.002) than that obtained for non-exposed oocytes. The fertilization parameters were not influenced by the treatments. The cleavage and blastocyst rates during culture were lower (p<0.001) for the oocytes exposed to SYBR-14 and fluorescence than for those in the non-exposed group. These results indicate that the exposure of mature oocytes to SYBR-14 and fluorescence for periods as short as 5s increased the rate of oocyte degeneration and limited their subsequent developmental competence.
authors: Department of Animal Medicine and Surgery, Faculty of Veterinary Medicine, University of Murcia, Spain.
authors: C
authors: C
authors: Maside
authors: M A
authors: MA
authors: Gil
authors: C
authors: C
authors: Cuello
authors: J
authors: J
authors: Sanchez-Osorio
authors: I
authors: I
authors: Parrilla
authors: J M
authors: JM
authors: Vazquez
authors: J
authors: J
authors: Roca
authors: E A
authors: EA
authors: Martinez
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D046149:Embryo Culture Techniques / Q000662:veterinary; D047108:Embryonic Development / Q000187:drug effects* / Q000502:physiology; D005260:Female; D005307:Fertilization in Vitro / Q000662:veterinary; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000494:pharmacology; D059471:In Vitro Oocyte Maturation Techniques / Q000379:methods / Q000662:veterinary; D053652:Nuclear Transfer Techniques / Q000662:veterinary; D009865:Oocytes / Q000187:drug effects* / Q000502:physiology; D009866:Oogenesis / Q000187:drug effects / Q000502:physiology; D009930:Organic Chemicals / Q000494:pharmacology; D013552:Swine / Q000502:physiology
chemical_list: D005456:Fluorescent Dyes; D009930:Organic Chemicals; C093207:SYBR-14