title: Blastocyst development after intracytoplasmic sperm injection of equine oocytes vitrified at the germinal-vesicle stage.
abstract: We evaluated the meiotic and developmental competence of GV-stage equine oocytes vitrified under different conditions. In a preliminary study, using dimethyl sulfoxide (D), ethylene glycol (EG) and sucrose (S) as cryoprotectants, the maturation rate was higher for cumulus-oocyte complexes (COCs) held overnight before vitrification (37%) than for those vitrified immediately (14%; P < 0.05). Thereafter, all COCs were held overnight before vitrification. In Experiment 1 we compared 1 min (1m) and 4 min (4m) exposure to vitrification and warming solutions; oocytes that subsequently matured were fertilized by ICSI. The maturation rate was similar between timing groups (29-36%), but was significantly lower than that for controls (73%). The 1m treatment yielded one blastocyst (11%), vs. 19% in controls. In Experiment 2, propylene glycol (PG) and trehalose (T) were also used. We compared two base solutions: M199 with 10% FBS (M199+), and 100% FBS; three cryoprotectant combinations: D-EG-S; PG-EG-S; and PG-EG-T; and two timings in vitrification solution: ∼30 s (30s) and 1 min (1m). The most effective treatment (FBS/PG-EG-T/30s) yielded 42% maturation, 80% cleavage and 1 blastocyst (10%), vs. 49%, 93% and 29%, respectively for controls (P > 0.1). In Experiment 3, we evaluated the toxicity of the M199/D-EG-S/1m and FBS/PG-EG-T/30s treatments, without actual vitrification. These treatments did not affect maturation but both significantly reduced blastocyst development (0% and 0%, vs. 21% for controls). This represents the second report of blastocyst development after vitrification of GV-stage equine oocytes, and presents the highest developmental competence yet achieved; however, more work is needed to increase the efficiency of this system.
authors.affiliation: College of Veterinary Medicine & Biomedical Sciences, Texas A&M University, College Station, TX 77843-4466, United States.
authors.forename: Heloísa Siqueira
authors.initials: HS
authors.lastname: Canesin
authors.affiliation: College of Veterinary Medicine & Biomedical Sciences, Texas A&M University, College Station, TX 77843-4466, United States.
authors.forename: Joao Gatto
authors.initials: JG
authors.lastname: Brom-de-Luna
authors.affiliation: College of Veterinary Medicine & Biomedical Sciences, Texas A&M University, College Station, TX 77843-4466, United States.
authors.forename: Young-Ho
authors.initials: YH
authors.lastname: Choi
authors.affiliation: College of Veterinary Medicine & Biomedical Sciences, Texas A&M University, College Station, TX 77843-4466, United States.
authors.forename: Isabel
authors.initials: I
authors.lastname: Ortiz
authors.affiliation: Department of Clinical Sciences, Faculty of Veterinary Medicine, University of Montreal, Saint-Hyacinthe, QC, J2S 2M2, Canada.
authors.forename: Mouhamadou
authors.initials: M
authors.lastname: Diaw
authors.affiliation: College of Veterinary Medicine & Biomedical Sciences, Texas A&M University, College Station, TX 77843-4466, United States. Electronic address: khinrichs@cvm.tamu.edu.
authors.forename: Katrin
authors.initials: K
authors.lastname: Hinrichs
mesh_terms: D000818:Animals; D001755:Blastocyst / Q000187:drug effects; D015925:Cryopreservation / Q000379:methods* / Q000662:veterinary; D003451:Cryoprotective Agents / Q000494:pharmacology; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D047108:Embryonic Development / Q000187:drug effects; D019855:Ethylene Glycol / Q000494:pharmacology; D005260:Female; D006736:Horses; D009865:Oocytes; D019946:Propylene Glycol / Q000494:pharmacology; D020554:Sperm Injections, Intracytoplasmic / Q000379:methods* / Q000662:veterinary; D013395:Sucrose / Q000494:pharmacology; D014199:Trehalose / Q000494:pharmacology; D058989:Vitrification
chemical_list: D003451:Cryoprotective Agents; D013395:Sucrose; D019946:Propylene Glycol; D014199:Trehalose; D019855:Ethylene Glycol; D004121:Dimethyl Sulfoxide
keywords: Blastocyst; COCs; Equine; ICSI; Maturation; Oocyte; Vitrification