Cryotop vitrification of buffalo (Bubalus bubalis) in vitro matured oocytes: effects of cryoprotectant concentrations and warming procedures.
The aim of the work was to evaluate the in vitro developmental competence of in vitro-matured buffalo oocytes after Cryotop vitrification (CTV) and in vitro fertilization (IVF). To optimize parameters, two cryoprotectant (CP) concentrations and two warming-dilution procedures were applied. Oocytes were vitrified in 16.5% ethylene glycol (EG), 16.5% dimethylsulphoxide (DMSO) and 0.5 M sucrose in Groups A and C, and in higher CP concentrations (20% EG, 20% DMSO and 0.5 M sucrose) in Groups B and D. Warming was performed in 1.25 M sucrose for 1 min, then in 0.62, 0.42 and 0.31 M sucrose, 30 s each (Groups A and B), or in 0.25 M sucrose for 1 min and in 0.15 M sucrose for 5 min (Groups C and D). After warming, the oocytes were fertilized and cultured in vitro. Survival rate post-warming was lower in Group D (83.6%) than in Groups A and B (92.4 and 92.8%, respectively), while intermediate values were found in Group C (85.7%). Survival rates at 24 h decreased in Groups C and D (52.0% and 50%, respectively) and remained high in Groups A and B (84.0% and 85.6%, respectively), thus indicating that the dilution of CP after warming is critical for buffalo oocyte cryopreservation. Similar differences were also observed in cleavage rates (42.7%, 55.3%, 28.4% and 36.3% for Groups A, B, C and D, respectively) whereas no differences in blastocyst rates were found among groups (6.4%, 7.8%, 5.9% and 6.9% for Groups A, B, C and D, respectively). Blastocyst production after IVF of vitrified oocytes proves the feasibility of CTV in buffalo species.
Attanasio
L
L
DISCIZIA, Faculty of Veterinary Medicine, Federico II University, Naples, Italy.
Boccia
L
L
Vajta
G
G
Kuwayama
M
M
Campanile
G
G
Zicarelli
L
L
Neglia
G
G
Gasparrini
B
B
D000818:Animals; D002020:Buffaloes / Q000502:physiology*; D018929:Cell Culture Techniques; D003451:Cryoprotective Agents / Q000008:administration & dosage* / Q000494:pharmacology*; D003470:Culture Media / Q000737:chemistry; D005260:Female; D005307:Fertilization in Vitro; D006358:Hot Temperature; D009865:Oocytes / Q000187:drug effects* / Q000502:physiology; D058989:Vitrification
D003451:Cryoprotective Agents; D003470:Culture Media