| Astaxanthin has been widely used as an antioxidant in many fields.Astaxanthin has many forms and functions,not only to eliminate free radicals and can prevent the chemical reactions caused by it.At present,astaxanthin antioxidant effects of experimental research,mostly concentrated in food,health products,drugs and as a feed additive research.However,relatively few studies have been reported on the development of embryos in vitro.Therefore,23-25 g of the ICR female mouse were used to study the effects of different concentrations of astaxanthin on the increase of cleavage rate and blastocyst rate.The relative expression of TGF-? and Bcl-2 genes were detected by real-time PCR.The results of the trial are as follows:(1)Different concentrations of astaxanthin were added to the culture medium in vitro,after96 h of in vitro culture,When the concentration of astaxanthin was 5 ?mol/L and 10 ?mol/L,the cleavage rate was increased.The cleavage rate of 10?mol/L group was significant higher than that of 0 ?mol/L group and 5 ?mol/L group(P<0.05);however,the cleavage rate in the 50 ?mol/L group was significant lower than that in the 0 ?mol/L group(P<0.05),significantly lower than that in the 5 ?mol/L group and 10 ?mol/L group(P<0.01);the results showed that the addition of 10?mol/L of astaxanthin could significantly increase the cleavage rate in the development medium,but the addition of 50 ?mol/L astaxanthin significantly reduced the cleavage rate.The blastocyst rate of astaxanthin 10 ?mol/L group was significant higher than that of 0 ?mol/L group and 25?mol/L(P<0.05),which was significantly higher than that of 50 ?mol/L group(P<0.01);50?mol/L group was significantly lower than the 0 ?mol/L,5 ?mol/L group and 25 ?mol/L group,which indicated that the addition of 10 ?mol/L of astaxanthin could significantly increase the blastocyst development rate of embryos,but when the concentration was 50 ?mol/L,Significantly reduce embryonic blastocyst development.(2)There was no significant difference in the 0 ?mol/L test group and 5 ?mol/L test group in the developmental stages of 2-cell,4-cell,8-cell,morula and blastocyst(P>0.05);compared with the 10 ?mol/L test group,the formation rate of 2-cell,4-cell,8-cell,morula and blastocyst was significantly lower(P<0.05);compared with the 25 ?mol/L test group,it was found that there was no significant difference in the developmental stage(P>0.05);between 0 ?mol/L and 50 ?mol/L in the test group,2-cells,4-cells and 8-cells was significantly different(P<0.05),but in the morula and blastocyst stages were significantly different(P<0.01).Add astaxanthin in each test group comparison we found that between 5 ?mol/L and 10 ?mol/L test group no significant difference(P>0.05),but with 50 ?mol/L test group in each stage were significantly different(P<0.01);the 5 ?mol/L test group compared with 25 ?mol/L test group at different stages were no significant difference(P>0.05),the 10 ?mol/L test group was significantly different in the 2-cell,4-cell and morula stage compared with the 25 ?mol/L test group(P<0.01),but there was significant difference in blastocyst stage(P<0.05);there was no significant difference between the25 ?mol/L test group and the 50 ?mol/L test group in 2-cell and the 4-cell stage(P>0.05),but there was significant difference between the 8-cell,the morula and the blastocyst stage(P<0.05).The results showed that different concentrations of astaxanthin could affect the development of the two stages.The concentration of 10 ?mol/L could significantly increase the formation of 2-cell,morula,blastocyst and improve the development of in vitro fertilized embryos.(3)The blastocysts cell number was the cell number of blastocyst after 96 h in vitro fertilization,and the cell number of blastocyst increased with the concentration of 0-10 ?mol/L,compared with the 0 ?mol/L group,the cell number of blastocyst in the 10 ?mol/L group was statistically different(P<0.05).When the concentration of astaxanthin is further increased,the cell number of blastocyst in the 25 ?mol/L group and the 50 ?mol/L group were lower than that in the10 ?mol/L group,but higher than the 0 ?mol/L group,indicating that with the increase of astaxanthin concentration,which could present a certain toxic effect and reduce the cell number of embryos.The results showed that the blastocysts cell number increased significantly when the concentration of astaxanthin was 10 ?mol/L.(4)The total RNA of blastocysts in 5 groups was extracted and the relative expression of TGF-? and Bcl-2 mRNA were detected by ?-actin as internal reference gene.The relative expression of TGF-? in 0 ?mol/L group was respectively compare with the relative expression of TGF-? in 10 ?mol/L group and 50 ?mol/L group,and the relative expression of TGF-? in 10?mol/L group was significantly higher than that in 0 ?mol/L group and 50 ?mol/L group(P<0.01),there was no significant difference in the relative expression of 0 ?mol/L group and 50 ?mol/L group.The relative expression of Bcl-2 in 0 ?mol/L group was set to 1.The relative expression of Bcl-2 in 10 ?mol/L group and 50 ?mol/L group were compared respectively,the relative expression of Bcl-2 in 50 ?mol/L group was significantly higher than that in 0 ?mol/L group and10 ?mol/L group(P<0.01),there was no significant difference in the relative expression of 0?mol/L group and 10 ?mol/L group.In this study,the addition of 10 ?mol/L astaxanthin to the developmental culture medium could promote the development of embryos,which could significantly increase the cleavage rate?the blastocyst rate of the fertilized eggs in mouse and the cell number of blastocysts.The addition of 10 ?mol/L astaxanthin significantly increased the expression level of TGF-? gene in vitro,and the expression level of Bcl-2 gene was increased by 50 ?mol/L. |
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