Effects of vitrification cryoprotectant treatment and cooling method on the viability and development of buffalo oocytes after intracytoplasmic sperm injection

In vitro matured (IVM) buffalo oocytes at the metaphase of the second meiotic division (MII) were vitrified in 20% Me₂SO: 20% EG (v/v) and 0.5M sucrose (VA), or 35% EG (v/v), 50mg/mL polyvinylpyrrolidone (PVP), and 0.4M trehalose (VB), either on cryotops or as 2μL microdrops. The viability was assessed after warming by fluorescein diacetate (FDA) staining and all surviving oocytes were subjected to ICSI and ethanol activation. All vitrified groups had similar recovery rates but both VA groups had significantly higher survival and pronuclear formation rates than either of the VB groups. Non treated control oocytes and non cryopreserved oocytes exposed to FDA had significantly higher survival, 2nd polar body extrusion, PN and blastocyst formation rates than any of the four vitrified groups (P<0.05). In conclusion The cryotop and microdrop methods are equally effective for buffalo oocyte vitrification, and although vitrification in VA solution yielded higher rates of survival and formation of 2 pronuclei than VB, the rate of blastocyst formation was comparable for both solutions. A detailed analysis of oocytes that extruded the second polar body after ICSI and activation revealed that only a minority (7-20% of the vitrified and 46-48% of the control oocytes) also had two pronuclei, indicating that normal activation is compromised by vitrification.