Study On The Freezing And Rewarming Process Of Oocytes Cryopreserved By Vitrification

Oocyte cryopreservation is the technology that preserve oocytes for a long timewithout losing their activity by inhibiting the metabolism of oocytes under lowtemperatures. This technology is quite significant to the human-assisted reproductionand preservation of fine varieties of livestock. However, the survival rate anddevelopmental rate of vitrified oocytes is really low, and it is far away from clinicalapplication.The freezing and rewarming process of oocyte cryopreservation were studied inthe thesis. By focusing on speeding up the cooling rate and reducing recrystalizationduring rewarming, the aim of this thesis is to reduce harm done to oocytes by crystalsand improve the survival rate and developmental potential. Cryotop is considered asone of the best methods to vitrify oocytes up to date, but there is still room forimprovement in both cooling rate during freezing and recrystalization duringrewarming. Researches were done based on the fact mentioned above as follows.（1）The influential factors of cooling rate during freezing were ananlyzed withnumerical simulation method. It is found out that the cooling rate increased graduallywith the decrease of thickness of carrier, drip volume and cold source temperature aswell as with the increase of heat transfer coefficient. When the carrier was60μm thick,the radius of solution was120μm, cold source temperature was66K and heat transfercoefficient was15,000W/(m2K)，the highest cooling rate of Cryotop reached172,000K/min. Then a fast temperature measurement system was established withdigital oscillometer and Type T thermocouple of25μm diameter. The cooling rates ofPVC Cryotop carrier with different thicknesses were measured when the drip was0.5μL. It is discovered that the cooling rate increased with the decrease of carrier’sthickness, and reached the16,000±1,100K/min when the thickness was60μm. Thecooling rate of100μm PVC in slush nitrogen reached17,000±1,200K/min,41.7%higher than in liquid nitrogen. The cooling rate of100μm cooper carrier in liquidnitrogen reached16,000±1,200K/min,33.3%higher than that of100μm PVC.Thereis quite a gap between value of measurement and simulation, resulting from the sizeof drips and different heat transfer coefficients used.(2) Porcine oocytes in MII phase were vitrified with different thicknesses ofCryotop carrier. The survival rate of oocytes with carrier thickness of60μm and80μmwas71.7±3.5％，69.3±6.5％respectively, nearly the same with100μm standardthickness (70.7±4.9％); Porcine oocytes in MII phase were vitrified with standardcryotop carrier in slush nitrogen at-201℃and-207℃respectively, and the survivalrates in the two experiment groups are68.6±8.2%and68.8±2.6%respectively. Nosignificant difference in survival rate was found in comparison with porcine oocytesfrozen in liquid nitrogen (69.2±4.0%). Mouse oocytes in GV phase were vitrified with100μm cooper carrier, The survival rate was only15.6±5.0％, much lower than thecontrol group with standard Cryotop carrier(83.3±3.3%).(3)The recrystallization properties of vitrified CPA in warming process werestudied with cryomicroscope. It is found that with the increase of warming rate,recrystallization in warming process was reduced and the dangerous crystal zone was narrowed. In addition, the crystallization morphology of concentrated CPA wasrelatively safe to oocytes. The recrystallizaiton properties of CPA with differentconcentrations of HA nanoparticles in warming process was studied withcryomicroscope. It was revealed if0.05%HA nanoparticles was added in warmingprocess, the recrystallization of CPA could be reduced and no threat could be posed tooocytes. If no HA nanoparitcles was added, recrystallization occured in rewarmingprocess and it was likely to threaten oocytes. Also, large quantity of crytals formedwhen CPA with0.5%HA nanoparticles was added, posing threaten to oocytes. Thecrystallization quantity of CPA with HA nanoparticles was measured by DifferentialScanning Calorimetry(DSC), the results showed the gross of crystalization of CPAreduced dramatically if0.1%HA nanoparticles were added.（4）Ultrasonic was used to disperse SiO2、HA、Al2O3、TiO2in CPA. Thefactors that influence the dispersibility effect of nanoparticles in CPA were analyzed.The results showed that the dispersibility of TiO2and Al2O3was better and thedispersibility of HA and SiO2was less good. HA and Al2O3had poor stability andshould be used as soon as they are made, while SiO2and TiO2had better stability andcan be stored for a long time; The dispersibility of HA increased with pH. Thedispersibility of TiO2and Al2O3was best when pH was equal to7, the dispersibility ofSiO2has little to do with pH; The dispersibility of HA nanoparticles in CPA was bestwhen concentration of CPA reached40%. While the dispersibility of SiO2and Al2O3was better at high CPA concentration, and the dispersibility of TiO2was best whenconcentration of CPA reached80%.(5)Nanocryoprotectant was employed to vitirify oocytes, the toxicity ofnanoparticiples as well as its effect on survival rate and developmental potential ofprocine oocytes were studied. The results showed that the range of toxicity on oocyteswere different for differnt types of nanoparticles. No toxicity is demonstrated whenconcentration of HA is lower than0.5%, while for SiO2、Al2O3、TiO2`the thresholdwas0.1%. In addition, HA nanoparticles can promote vitrification effect significantly,when0.1%HA was added in CPA, the developmental rate of GV phase porcineoocytes can reach as high as22%.20nm、40mm、60nm HA nanoparticles can allenhance the developmental rate of GV phase porcine oocytes, which is26.7%、28.7%、28.7%respectively. No significant difference was found among groups. CPA with0.01%~0.5%HA nanoparticles can enhance the developmental rate of GV-phaseporcine oocytes, the developmental rate reached30.4%when0.05%HA was added.CPA with0.1%20nm、40nm、60nm HA can improve the survival rate of MII phaseporcine oocytes, the survival rate of three groups are higher than80%, No significantdifference was found among groups.