| Mammalian oocytes cryopreservation is one of the most important components in the field of life science. In recent years, although people have carried out extensive research in this regard, the effect of oocyte cryopreservation is still unsatisfactory, especially for immature oocytes. There are so many factors that will influence the effect of vitrification, and the cooling rate has been considered to be the most important factor. Now, the most common cryogen used for oocytes vitrification is liquid nitrogen(﹣196℃). However, the temperature of the liquid helium is ﹣269 ℃, and the research about using liquid helium as a cryogen has rarely been reported. So, the present object was proposed on the basis of liquid nitrogen OPS vitrification to improve the results of bovine immature oocytes cryopreservation by using liquid helium, as a new cryogen to accelerate the cooling rate. Moreover, the aim of this study was to establish and optimize the method for liquid helium vitrification.1. The establishment of liquid nitrogen(LN2) OPS vitrification method for bovine immature oocytes.Experiment 1: To explore the pulling method for open pulled straw(OPS), we used water bath, common alcohol lamp and homemade small alcohol lamp as the heat source, respectively. Results: with water bath and common alcohol lamp, we didn′t get OPS which meets the requirements. With homemade small alcohol lamp, we successfully pulled out this open pulled straw(inner diameter 0.8 mm and wall thickness 0.07 mm).Experiment 2: The comparison of straw and OPS vitrification. Bovine immature COCs were selected and assigned to the following experimental groups randomly: control, straw and OPS group. The COCs in control group were submitted to IVM, IVF and IVC immediately and the other two groups were vitrified by straw an OPS method respectively. The COCs of vitrified-thawed were submitted to IVM, IVF and IVC in sequence. Results: Compared with the normal morphology, maturation, cleavage and blastocyst rates of control group(100%, 86.1%, 69.7% and 38.2%), the development competence of both vitrification methods were significantly reduced(P<0.05). However, the normal morphology rates of group straw and group OPS were 65.2% and 77.8% respectively, the maturation rates were 31.0% and 41.6% respectively, the cleavage rates were 19.9% and 29.8% respectively, the blastocyst rates were 0 and 7.0% respectively. The effect of group OPS was significantly better than group straw(P<0.05).The conclusion: The homemade small alcohol lamp is more suitable for drawing OPS; The effect of OPS method is better than straw method for bovine immature oocytes vitrification.2. The exploration of liquid heliun(LHe) OPS vitrification method for bovine immature oocytes.Bovine immature COCs were selected and divided into three experimental groups randomly: control, LN2 and LHe group. The COCs in control group were submitted to IVM, IVF and IVC immediately and the other two groups were vitrified by LN2 and LHe OPS vitrification respectively. The COCs of vitrified-thawed were submitted to IVM, IVF and IVC in sequence. Results: Compared with the normal morphology, maturation, cleavage and blastocyst rates of control group(100%, 87.0%, 69.5% and 42.0%), the development competence of both vitrification methods were significantly reduced(P<0.05). However, the normal morphology rates of LHe group(84.7%) was significantly higher than LN2 group(78.5%; P<0.05). After IVM, the maturation rates of LHe group and LN2 group were 43.9% and 39.2% respectively, the difference was significant(P<0.05). After IVF and IVC, the cleavage and blastocyst rates of LN2 group(30.0%, 4.1%) were significantly lower than LHe group(35.4%, 6.4%; P<0.05). The conclusion: The results of LHe OPS vitrification is better than LN2 OPS vitrification and the liquid helium as a cyrogen to vitrify bovine immature oocytes is feasible.3. The optimization experiment for LHe OPS vitrificationExperiment 1: Selection for the optimal vitrification solution(VS2). Bovine immature COCs were selected and divided into 6 experimental groups randomly: control, EDS30, EDS35, EDS40, EDS45 and EDS50 group. The COCs in control group were submitted to IVM, IVF and IVC immediately and the other five groups were vitrified by LHe OPS method with 5 different VS2 s. The COCs of vitrifiedthawed were submitted to IVM, IVF and IVC in sequence. Results: Compared with the normal morphology, maturation, cleavage and blastocyst rates of control group(100%, 84.5%, 69.9% and 41.1%), the development competence of all vitrified groups were significantly reduced(P<0.05). However, among 5 vitrified groups, the developmental parameters(90.3%, 51.9%, 41.8% and 10.0%) of vitrified-thawed oocytes in EDS35 group were significantly better than the other 4 groups respectively(P<0.05). The resultst in EDS50 grouop was the worst, lower than the other 4 groups significantly(P<0.05). The difference among EDS30( 83.0%, 42.0%, 33.8% and 6.5%), EDS40(84.9%, 43.9%, 34.9% and 6.4%) and EDS45(82.6%, 42.4%, 33.2% and 5.1%) groups were not significant(P>0.05).Experiment 2: The comparison of different operating temperatures in LHe OPS vitrification. Bovine immature oocytes were divided into 3 groups randomly: control, 25 ℃and 39 ℃group. The COCs in control group were submitted to IVM, IVF and IVC immediately. The only difference between this two LHe vitrified groups was the operating temperature, other operations were all the same. This oocytes were vitrified by LHe OPS method with two different operating temperature and the oocytes of vitrified-thawed were submitted to IVM, IVF and IVC in sequence. Results: Compared with the normal morphology, maturation, cleavage and blastocyst rates of control group(100%, 84.6%, 71.5% and 40.6%; P<0.05), all developmental parameters in this two vitrified groups were significantly decreased(P < 0.05). The normal morphology rates of 39 ℃(89.2%) and 25℃ group(85.8%) didn′t exist significant difference(P>0.05). However, the maturation, cleavage and blastocyst rates in 39 ℃group( 51.4%, 41.2% and 10.0%) were all significantly higher than 25 group(℃ 45.7%, 34.1% and 6.3%; P<0.05).Experiment 3: Selection for the optimal pre-treatment time. Bovine immature oocytes were divided into 4 groups randomly: control, 1min, 3min and 5min pretreatment group. The COCs in control group were submitted to IVM, IVF and IVC immediately. The only difference among this three LHe vitrified groups was the pretreatment time in VS1, other operations were the same. This oocytes were vitrified by LHe OPS method with three different pre-treatment time and the oocytes of vitrifiedthawed and control group were submitted to IVM, IVF and IVC in sequence. Results: Commpared with the normal morphology, maturation, cleavage and blastocyst rates of control group(84.0%, 70.6% and 40.1%), all developmental parameters in this three vitrified groups were significantly decreased(P<0.05). The normal morphology rates of 1min, 3min and 5min group were 87.7%, 87.2% and 86.4% respectively, the difference among this three vitrified groups was not significant(P > 0.05). The maturation, cleavage and blastocyst rates between 1min(49.6%, 39.6% and 10.4%) and 3min pre-treatment group(47.4%, 37.0% and 8.8%) didn′t exist significant difference(P>0.05). However, all this developmental parameters in 1min and 3min groups were significantly(P<0.05) higher than 5 min groups(41.0%, 32.0% and 6.1%; P<0.05).The conclusion: For LHe OPS vitrification, the optimal VS2 is EDS35, the optimal operating temperature is 39℃, and the optimal pre-treatment time is 1 min. |
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