title: Effects of Hoechst 33342 staining and ultraviolet irradiation on the developmental competence of in vitro-matured porcine oocytes.
abstract: Hoechst 33342 (H342) in combination with ultraviolet (UV) irradiation is frequently used to assist the enucleation of porcine oocytes in somatic cell nuclear transfer programs. This work evaluated the effects of H342 (5 μg/mL for 12 min) staining and/or exposure to UV irradiation on fertilisability and developmental capacity of porcine oocytes matured in vitro. In Experiment 1, a total of 1388 mature oocytes were distributed in the following groups: Group 1: oocytes without treatment (Control), Group 2: oocytes stained with H342, Group 3: oocytes stained with H342 and UV irradiated for 30 sec, and Group 4: oocytes UV irradiated for 30 sec. Oocytes from each group were exposed to thawed spermatozoa and cultured for 18 h to assess fertilization parameters or for 7 d to evaluate embryo development. Sperm penetration (P < 0.001) and monospermy (P < 0.04) were lower in oocytes exposed to H342/UV (80.7 ± 4.5% and 30.7 ± 5.4%, respectively) than in oocytes from the control group (94.9 ± 4.3 and 50.0 ± 4.9, respectively). The oocytes exposed to H342/UV showed lower (P < 0.001) cleavage (49.8 ± 2.9%) and blastocyst (7.7 ± 2.9%) rates than oocytes from the other groups (range: 73.8 ± 2.9% to 77.7 ± 2.9% and 22.3 ± 2.9% to 30.9 ± 3.0%, respectively). Experiment 2 was designed to evaluate the effect of shorter UV irradiation (5 sec). A total of 1835 mature oocytes were separated into the same groups as those of Experiment 1. The fertilization parameters and the cleavage rates were not influenced by the different treatments. However, the oocytes exposed to H342 and UV irradiation for 5 sec showed a lower (P < 0.02) rate of blastocyst formation (15.2 ± 4.5%) than the oocytes from other groups (range: 26.1 ± 4.5% to 30.7 ± 4.5%). In conclusion, our results demonstrate that the combination of H342 staining with UV irradiation has a clear deleterious effect on the developmental ability of oocytes, with the effects being more intense with increased exposure to UV irradiation.
authors.affiliation: Department of Animal Medicine and Surgery, Faculty of Veterinary Medicine, University of Murcia, Murcia, Spain.
authors.forename: C
authors.initials: C
authors.lastname: Maside
authors.forename: M A
authors.initials: MA
authors.lastname: Gil
authors.forename: C
authors.initials: C
authors.lastname: Cuello
authors.forename: J
authors.initials: J
authors.lastname: Sanchez-Osorio
authors.forename: I
authors.initials: I
authors.lastname: Parrilla
authors.forename: X
authors.initials: X
authors.lastname: Lucas
authors.forename: J N
authors.initials: JN
authors.lastname: Caamaño
authors.forename: J M
authors.initials: JM
authors.lastname: Vazquez
authors.forename: J
authors.initials: J
authors.lastname: Roca
authors.forename: E A
authors.initials: EA
authors.lastname: Martinez
mesh_terms: D000818:Animals; D001562:Benzimidazoles / Q000633:toxicity*; D018929:Cell Culture Techniques / Q000662:veterinary*; D005260:Female; D005307:Fertilization in Vitro / Q000662:veterinary; D053652:Nuclear Transfer Techniques / Q000662:veterinary; D009865:Oocytes / Q000187:drug effects / Q000254:growth & development / Q000528:radiation effects*; D011829:Radiation Dosage; D013084:Sperm-Ovum Interactions / Q000187:drug effects / Q000528:radiation effects; D013552:Swine / Q000196:embryology*; D014466:Ultraviolet Rays
chemical_list: D001562:Benzimidazoles; C017807:bisbenzimide ethoxide trihydrochloride