Protective Effect Of Antifreeze Protein ? On Vitrified Frozen Porcine Oocytes

In order to explore the protective effect of different concentrations of antifreeze protein ?(AFP ?)on the vitrification of porcine oocytes,and to looked for the most suitable concentration of antifreeze protein in the freezing liquid.In this study,porcine oocytes in GV and MII were taken as research objects.And AFP ? were added with different concentrations(250 ng/m L,500ng/m L,750 ng/m L,1000 ng/m L,1200 ng/m L)respectively to the freezing liquid to analyze the development of oocytes,the changes of reactive ROS content and DNA damage of oocytes before and after freezing with a view to providing a theoretical basis for improving the cryopreservation of oocytes of pigs and other low-temperature sensitive species,and further improving the efficiency of oocyte vitrification cryopreservation.Results found:1.In the freezing liquid treatment experiment,the addition of AFP III concentration of 250 ng/m L,500 ng/m L,750 ng/m L could increase the division rate and blastocyst rate of GV stage oocytes,and the split rate of 500 ng/m L group was significantly higher than that of the frozen liquid treatment group(P<0.05),while the split rate and blastocyst rate were significantly lower than that of the other groups when the concentration of AFP ? was 1200 ng/m L(P<0.05).2.In the vitrification freeze-resuscitation experiment,adding 250 ng/m L,500 ng/m L,and 750 ng/m L AFP ? to the freezing solution increased the division rate and blastocyst rate of GV phase oocytes after freezing resuscitation.When the concentration of AFP III was 500 ng/m L,the rate of oocyte division and blastocyst were significantly higher than that of the frozen control group(P<0.05).Based on the results of 1,2,the addition of 500 ng/m L AFP ? had the best protection effect on the vitrified frozen GV stage pig oocytes.3.In the freezing liquid treatment experiment,each concentration group of AFP III could increase the blastocyst rate of MII stage oocytes,and the concentration of AFP ? added was 500 ng/m L,750 ng/m L,1000 ng/m L,1200ng/m L,the blastocyst rate was significantly higher than that of the frozen liquid treatment group(P <0.05),while the blastocyst rate of the 750 ng/m L group was significantly higher than that of other concentration groups(P <0.05).4.In the vitrification freezing-resuscitation experiment,adding different concentrations of AFP III,the oocyte division rate was higher than that of the frozen control group,and when adding AFP III concentration of 750 ng/m L,the oocyte division rate was significantly higher than that of the frozen control Group and other AFP III concentration groups(P <0.05);based on the results of3 and 4,the best protection effect was achieved when the AFP ? concentration in the freezing solution was 750 ng/m L for vitrified frozen M? stage pig oocytes.5.Vitrification could cause oxidative stress in pig oocytes,and the ROS content of the frozen group with AFP ? added to the freezing solution is significantly lower than that of the frozen control group without AFP III added(P <0.05).Therefore,AFP ? can effectively reduce the oxidative stress level of vitrified frozen porcine oocytes and protect the vitrified frozen oocytes.6.Vitrification cause severe DNA damage to porcine oocytes,but the tail DNA(Tail DNA%)and OTM Tail Moment values of the frozen group with AFP III were significantly lower than those of the frozen control group without AFP III(P <0.05),so we know that AFP ? can reduce the DNA damage caused by vitrification to oocytes.In summary,AFP III could improve the development ability of oocytes after vitrification in vitro,reduce the ROS content of oocytes after freezingresuscitation and reduce the DNA damage level of oocytes caused by vitrification.