| The techniques on cryopreservation of mouse and porcine expanded blastocysts by vitrification were investigated in this study to pursue the optimal protocol for cryopreservation of porcine expanded blastocysts. The experiments were carried out as the following: 1) the effects of cryoprotectants treatment on the survival of mouse expanded blastocysts at room temperature, 2) vitrification of mouse expanded blastocysts, 3) the toxic test of cryoprotectants to porcine IVM oocytes and vitrification of porcine parthenogenetic expanded blastocysts.The survival rates in vitro were 100% after the mouse expanded blastocysts were exposed to mPBS,30%Ficoll and 0.5mol/L sucrose respectively. It indicated that these chemicals were no toxicity to the embryos. At 25 the survival rates in vitro did not differ from that of the control group after the mouse expanded blastocysts were treated in 10% EG or 10% EG and 10% dimethyl sulfoxide (DMSO) for less than 5 min as well as in EFS or EDFS for 0.5 min (p>0.05). Meanwhile, at 37 the survival rate didn't differ from the control group either after the mouse expanded blastocysts were exposed to 10% EG or 10% EG and 10% DMSO for less than 2.0 min as well as in EFS or EDFS for 0.5min (p>0,05). The results implied that the pretreatments of above solutions prior to vitrification were not harmful to the mouse expanded blastocysts. Furthermore, the results demonstrated that the toxicity of vitrification solutions was from EG and DMSO and the toxicity was increased with the concentration rising, equilibration time extending and the pretreatment temperature elevating.The frozen results of the mouse expanded blastocysts by straw were compared to by open pulled straw (OPS). The survival rate was 87.0% in the best group of 2-step method by straw at 25 . It was higher than that of the one-step method by straw (74.4%), but significantly lower than that of the control (100%, p<0.05). The efficiency of vitrification by OPS was better than by straw. At 37 the survival rate of the hatched blastocysts frozen by OPS was 97.8%. It was not significantly different from that of the control (100%, p>0.05). Three methods were used for diluting the cryoprectants from the embryos frozen in EDFS or EFS after thawing. Method A: the embryos were rinsed in 0.5mol/L sucrose. Method B: the embryos were rinsed in 0.5mol/L sucrose for 2 min, then in 0.3mol/L sucrose for 5 min. Method C: the embryos were rinsed in 0.3 mol/L sucrose for 5 min, then 0.15mol/L sucrose for 2 min. The results showed that method C was the best when the embryos were frozen in EDFS. The method C was also effective for diluting the cryoprotectants from the embryo frozen in EDFS30 by straw or OPS. No significant difference existed in three methods when the embryos were frozen in EFS.133 embryos could be obtained by straw method at 25 and 154 embryos could be obtained by OPS method at 37 . And then transfer respectively into 12 and 10 pseudopregnant recipients for 3~4d, six established pregnancies. The rate of developed into live young was respectively 35.5% and 39.1 %, which was similar to that of the control(39.8%,37/93)(p > 0.05).The porcine IVM oocytes were treated respectively in mPBS, 30%Ficpll and 0.5mol/L sucroce for 2 mins at 37 to check the effects of these chemicals on the viability of the oocytes. The rates of cleavage and blastocyst were 5 1.4%, 48.0%, 5 1.8% and 20.6%, 18.2%, 18.8% respectively after the treated oocytes were eletro-activated and cultured in NCSU23. The results were not significantly different from the control (54.5% and 22.8%, p>0.05). The porcine IVM oocytes were first treated in 10%EG or 10%EG+10%DMSO within 2 min, then in EPS or EDFS for 30s. The treatments didn't affect the developmental abilities of the oocytes after eletro-activation. However, there was significant difference between other treated and the control (p<0.01).The OPS method was adopted to cryopreserve the porcine parthenogenetic blastocysts. A total of 149 blastocysts were frozen in liquid nitrogen after equilibrati...
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