Studies on the cryopreservation of immature and in vitro matured bovine - oocytes

The developmental potential of mammalian oocytes cryopreserved with procedures similar to those used for embryos has been limited, inasmuch as oocytes differ from embryos in advanced stages of development, both physiologically and morphologically. The objective of this work was to elucidate the precise nature of freeze-thaw damage with the expectation that identification of specific targets will enable devising optimal procedures for cryopreservation of bovine oocytes to prevent specific damage and minimize the loss of developmental capacity.In the first series of experiments, bovine oocytes were vitrified (V-oocytes) or frozen slowly (S-oocytes) at the germinal vesicle (GV) stage or after maturation in vitro (IVM). Survival was assessed morphologically and also by in vitro fertilization (IVF) and culture (IVC). Morphological integrity and developmental capacity were greater in S-oocytes than in V-oocytes (P $<$ 0.05). Transfer of four embryos (2 morulae and 2 blastocysts) derived from post-IVM S-oocytes into a recipient heifer resulted in the birth of twin calves.In the second series of experiments, oocytes (GV and IVM) were exposed to a cryoprotectant solution (DAP213: 2M DMSO, 1M acetamide, 3M propanediol) for 1.5 or 5 min and viability assessed by IVM-IVF-IVC. Oocytes were also examined by transmission electron microscopy (TEM) before (control) or after exposure to the cryoprotectant. DAP213 induced profound premature cortical granule (CG) release and vesiculation. These changes were less pronounced in oocytes exposed to DAP213 only after IVM. The results suggest that: (1) the extrusion of CG is one of the important cytological events affected by the treatment of oocytes with DAP213; (2) GV oocytes are more sensitive to the cryoprotectant than IVM oocytes.In the third series of experiments, GV and IVM oocytes were vitrified with DAP213. On rewarming, DAP213 was removed by a one- or three-step dilution procedure and survival assessed by development after IVM-IVF-IVC. Morphology was assessed by TEM study immediately following DAP213 removal. Both assessments indicated that: (1) IVM oocytes are more tolerant to vitrification than are GV oocytes; (2) the three-step dilution is less damaging than the one-step procedure; (3) changes in the zona pellucida (loss of plasticity) of IVM oocytes following vitrification may result from the premature release of cortical granules