| In modern society,with the improvement of people’s living standards and the improvement of the recognition and value of pets,the demand for cloned pets is also increasing.In many developed countries,dog and cat cloning has been commercialized,showing a huge market prospect.However,although somatic cell cloning technology has been relatively mature in cattle,sheep,pigs and other livestock,there is still little research on pets such as dogs and cats.Due to the differences between species,the cloning system of dogs and cats needs targeted improvement in many technical links.The low efficiency of oocyte maturation in vitro is one of the key factors affecting the efficiency of cat cloning.This study focused on the two urgent problems to be solved in cat cloning technology: there are more lipid droplets in the cytoplasm of cat oocytes,which are prone to oxidative stress and it is difficult to determine the optimal maturation time in vitro.It focused on the optimal culture time of cat oocytes and the effects of cysteine and cysteamine on the in vitro maturation of domestic cat oocytes.1.To explore the optimal culture time of domestic cat oocytes,the time gradients designed in this experiment were 24 h,26 h,28 h,32 h and 36 h.Firstly,we observe the exclusion rate of the first polar body of oocytes in different culture groups,and record different karyotypes through fluorescence staining,so as to determine the best maturation time in oocytes,and then conduct parthenogenetic activation to further verify the subsequent development of oocytes.The results are as follows:(1)When the in vitro maturation culture time was 36 h,the proportion of oocytes developed to MⅡ stage was the highest,reaching 67.92%;when the culture time was 24 h,the proportion was only 35.20%.When the culture time was 28 h,32h and 36 h,the proportion of oocytes maturation was 63.99%,65.50% and 67.92% respectively,with no significant difference(p > 0.05).The degeneration rates of 28 h and 32 h groups were 6.70% and 8.50%respectively,with no significant difference(p > 0.05).The degeneration rate of 36 h group was11.32%,which was significantly different from other groups(P < 0.05).(2)The activation rate of oocytes and the rate of morula were the highest at 28 h,which were 70.87% and 48.54% respectively,but there was no significant difference with 32 h group;the blastocyst rate of 28 h group was the highest,reaching 33.98%,which was significantly different from that of 32 h group(p < 0.05);The activation rate,mulberry rate and blastocyst rate of 36 h group were the lowest,and there were significant differences with other groups(p< 0.05).2.The effects of antioxidants cysteine and cysteamine on the levels of reactive oxygen species(ROS),glutathione(GSH),early apoptosis,mitochondrial activity,spindle morphology,chromosome arrangement and maturation efficiency of in vitro mature cat oocytes were studied.The results are as follows:(1)In the cysteine group,when 0.5 m M cysteine was added,the proportion of oocytes developed to MⅡ stage was the highest,reaching 46.29%;and in the cysteamine group,when100μM cysteamine was added,the oocyte maturation rate was the highest,reaching 63.99%.(2)When we add 0.5 m M cysteine and 100 μM cysteamine,the fluorescence intensity of ROS and GSH in domestic cat oocytes decreased significantly(p < 0.05).(3)In the control group,32.00% oocytes showed apoptosis,using 0.5 m M cysteine and100 μM cysteamine treatment,14.29% and 10.61% oocytes showed apoptosis,and the proportion of apoptosis rate decreased significantly(p < 0.05).(4)In the control group,the fluorescence intensity of mitochondria in domestic cat oocytes was weak and scattered;after adding 0.5 m M cysteine and 100 μM cysteamine,the mitochondrial fluorescence intensity of oocytes was significantly enhanced(p < 0.05),and the distribution of mitochondria was more uniform.Add 0.5 m M cysteine and 100 μM cysteamine,the proportion of oocytes with spindle disorder and chromosome misalignment in mcysteamine group was 14.67% and 13.24%,respectively,which was significantly lower than that in the control group,24.29%(p < 0.05).(5)The parthenogenetic activation rate of oocytes added with 0.5 m M and 1 m M cysteine was higher,and there was no significant difference between the two groups,67.62% and 70.54%respectively;the morula rate and blastocyst rate of oocytes in the group supplemented with0.5 m M cysteine were the highest,40.00% and 27.62% respectively,and there was a significant difference with other groups(p < 0.05).After adding 100 μM and 200 μM cysteamine,the parthenogenetic activation rate of oocytes in the two groups was higher,70.87%and 73.39% respectively,and there was no significant difference between the two groups;add100 μM cysteamine,the morula rate and blastocyst rate of oocytes were the highest,48.54%and 33.98% respectively,and there was significant difference with other groups(p < 0.05).According to the above experiments,the optimal culture conditions of domestic cat oocytes in vitro are as follows: add 100 μM cysteamine and cultured for 28 h.The results were as follows: the cleavage rate of nuclear transferred embryos was 81.82%,the blastocyst rate was 39.68%,and the hatching rate was 28.00%.In conclusion,this study shows that the optimal maturation culture time of cat oocyte nucleus is 28-32 h,and the best culture time to ensure the synchronous maturation of oocyte nucleus and cytoplasm is 28 h.The addition of 0.5 m M cysteine and 100 μM cysteamine can significantly reduce the oxidative stress response in cat oocyte maturation in vitro. |
PDF Full Text Request