Preliminary analysis of vitrified in vitro produced embryos.

Embryo cryopreservation by vitrification, as an alternative approach to traditional slow freezing, has been successful in different domestic species such as bovine and ovine. There are no reports available, to our knowledge, on successful cryopreservation of in vitro produced buffalo embryos. Therefore the aim of this study was to assess the viability of buffalo embryos after vitrification. In this preliminary trial 81 selected buffalo embryos, produced by in vitro maturation, fertilization and culture (IVM/IVF/IVC) of COCs recovered from slaughterhouse ovaries, were used. Embryos were classified under a dissecting microscope according to their stage of development: blastocysts (BL), expanded plus hatching blastocysts (XBL + HBL) and hatched blastocysts (HBL) were vitrified at day 6, 7 and 8 of culture (day 0 = IVF day). All vitrification solutions were prepared using a basic saline solution (PBS) supplemented with 0.3 mM sodium pyruvate, 3.3 mM glucose and 10 % FCS (Sigma). Blastocysts were allocated in 200 mcl drops of glycerol 1.4 M for 5 minutes, then into 200 mcl drops of glycerol 1.4 M and ethylene glycole 3.6 M for 5 minutes before being transferred into a 25 mcl column of glycerol 3.4 M and ethylene glycole 4.6 and loaded into the center of 0.25-ml plastic insemination straws. In the straws the embryos in the vitrification solution were separated by 2 air bubbles from 2 90-mcl columns of 0.5 M sucrose solution. The straws were sealed and plunged immediately into LN2. For thawing, the straws were held for 10 seconds in air before transferring them into a water bath at 20 degree for a further 10 seconds. The blastocysts were then allocated into 200 mcl of a 0.25 M sucrose solution for 5 minutes in order to remove the cryoprotectants, rinsed several times in PBS and cultured in SOF medium, in a humidified atmosphere with 5 % CO2, 7 % O2 and 88 % N2 at 38.5 degree for 48 hours. The viability of vitrified embryos was assessed at 24 and 48 hours post-thawing and based on the re-expansion of the blastocoele and the retention of normal gross morphology. The proportional data for the survival rate were analysed (by ANOVA) using the Generalised Linear Models (GLM) procedure within SPSS 8.0 statistical package (1999). Ageing (2 levels = < 7 d and > 7 d) and developmental stages (3 levels= BL, XBL and HBL) were used as factors in the statistical model. The survival rates recorded at 24 h and 48 h were respectively 63 % and 51.9 %, comparable to results reported by other authors for bovine in vitro produced embryos vitrified with different methods. The stage of development did not affect tolerance to vitrification.