Vitrification of Bovine Oocytes: Effect of the Meiotic Stage on Nuclear and Cytoplasmic Maturation.

Abstract

Cryopreservation of the female gamete is an important tool for the assisted reproductive technologies. However, because of their large size and marked sensitivity to cooling, cryopreservation of oocytes is very difficult. Although vitrification has been indicated as the election method no suitable results have yet been reported. The aim of this study was to evaluate nuclear and cytoplasmic maturation of oocytes vitrified at different moments of in vitro maturation using cryotop method. Bovine oocytes obtained from slaughterhouse ovaries, were select and allocated to four groups: non vitrified, used as control (CG), vitrified immediately after selection (V0), vitrified at 8 hours (V8) and vitrified at 22 hours (V22) of in vitro maturation. After warming, oocytes were placed in the incubator to complete 24 h of maturation. At the end of maturation period oocytes were denuded, fixed and stained with lacmoid or were in vitro fertilized and cultured. Oocytes of all treatments that showed metaphase II plate were classified as mature, those with any chromosomal aberrations as abnormal, and those with diffusely stained cytoplasm or undefined chromatin, as degenerate. Cleavage rate was evaluated on D2 after in vitro insemination (pi) and blastocyst rates on D7 and D8 pi. Data were compared using Chi-square analysis (P<0.05). Vitrified treatments, V0 (n = 51), V8 (n = 50) and V22 (n = 39) presented lower (P < 0.05) percentage of oocytes reaching metaphase II compared to CG (n = 36), with maturation rates of 47%, 50%, 56% and 83%, respectively. All cryopreserved groups showed a higher (P<0.05) percentage of abnormal (V0: 33%; V8: 30% and V22: 23%) and degenerated chromatin (V0:19%; V8:18% and V22:17%) than CG (11%; 5%). Cleavage and blastocyst rate on D7 and D8 were similar (P<0.05) among vitrified groups V0 (19%, 2.6% and 2.6%; n = 112), V8 (18%, 2.6%, and 2.6%; n = 115) and V22 (21%, 3.6% and 3.6%; n = 109). However, CG (n = 119) showed a higher (P<0.05) cleavage (93%) and a higher (P < 0.05) percentage of oocytes that developed to blastocyst on D7 (34%) and D8 (37%). The results suggest that, stage of meiosis in which oocytes are vitrified did not minimize the deleterious effect of cryopreservation on nuclear and cytoplasmic maturation of bovine oocytes. However, even with low rates, they were capable of been fertilized and develop to blastocyst stage. Research supported by CNPq and Embrapa-Cenargen. (poster)