| This paper studied the effects of laminin on fertilization in vitro and embryonic formation of dairy cattle. And the way to increase embryonic quality and quantity also was studied for building a perfect IVP system. Mainly contented the maturation in vitro, in vitro fertilization, the effect of integrinβ1 during sperm-oocyte fusion, in vitro culture and improving the way value the quantity of blastocysts. The results were as follows: 1. Effects of extracellular and discrete Cumulus Cells on in vitro maturation of Bovine Oocytes.The aim of the present study was to determine the effects of cumulus cell on in vitro maturation rate of cumulus-oocytes-complexs(COCS) and artificial denuded oocytes(DOs), and its pathway and strength was also investigated. The results showed that: Co-culture with discrete cumulus cells significantly improved the in vitro maturation rate of denudes oocytes(57.98±14.27 vs 35.53±14.00, P<0.05), but had no effects on COCS(76.66±5.77 vs 66.76±9.46, P>0.05); Co-culture with extracellular cumulus cell did not significantly improve the in vitro maturation rate of denudes oocytes(35.83±18.32 vs 35.53±14.00, P>0.05). The analysis results showed that: Extracellular cumulus cell promoted the in vitro maturation of oocytes by the pathway of gap-junction, however discrete cumulus cells worked by the pathway of paracrine; Three or more layers cumulus improved the mean maturation ability of their surrounded oocytes by 85.34%, Added discrete cumulus cells separately improved the mean maturation ability of COCs and DOs by 16.18% and 60.61%. 2.The effects of different IVF mediums and with or without cumulus cells on cleavage rate and embryonic formation in vitro matured.Three different IVF mediums, IVF-100, BO and TALP, were tested in the present study, and the oocytes with or without cumulus cells were also investigated in cleavage rate and embryonic formation. The result showed that the cleavage rate in medium IVF-100 was higher that in medium BO(0.7055±0.0745 VS 0.5794±0.0956,P<0.05). But no significant different was found between medium TALP and others. Though the cleavage rate of NOs was lower than that of COCs, no significant different was found. Additional, NOs, fertilized in vitro, could reach the blastocyst stage, but the rate significantly lower than COCs. In concluding, medium BO and TALP still need to be improved and the capacity of embryonic development formed from NOs is extremely lower than that formed from COCs. 3. Improving bovine embryonic development by supplementing exogenous laminin to in vitro culture medium.The present study examined the effect of natural mouse laminin on the development of in vitro fertilized bovine embryos. The result showed that: in Experiment 1, the expression of laminin, in oocytes and early embryo from 0 to 8 day, was firstly detected at the 8-cell stage by immumofluorescence method and displayed increase tendency from 16-cell stage and abundant amount at morula and blastocyst stage. Addtionally, the distribution of laminin also could be detected in trophoderm cells in expended blastocyst. In Experiment 2, the presumptive zygotes were divided and transferred to CR1 aa medium supplemented the different concentration(0μg/ml, 5μg/ml, 10μg/ml and 20μg/ml) of laminin, adding 10μg/ml laminin group displayed significantly higher cleavage rate than control group(80% vs 70%, P<0.05) and 20μg/ml group(67%, P<0.01) and no difference was found between 5μg/ml group and other groups. Blastocyst rate in 10μg/ml laminin group was significantly higher than 0(48% vs 37%,P<0.05), 5 and 20μg/ml groups(34% and 30%, P<0.01). The blastocyst in 5μg/ml and 10μg/ml groups showed significantly higher cell numbers than control(97.00±8.24, 98.71±13.59 vs 87.86±6.22, P<0.05). But no difference was found between 20μg/ml group and other groups.These results suggested that the expression of laminin plays an important role in embryo development. Thus, supplementing 10μg/ml exogenous laminin to in vitro culture medium could improve efficiently bovine in vitro embryonic development. 4. The Exploration of simple Double- staining in Bovine Blastocyst.The objective of this study was to build a reliable way for the blastocyst double staining in bovine,depended on others’ methods which has been reported before. In our experment the Method 1、Method 3 and Method 4 all got a big success in distinguishing between the Inner cell mass(ICM) and trophoblast cell(TE) of the bovine blastocyst. The different didn’t display between the Method 3 and the Hoechst33342 staining(84.33±9.98 VS 83.63±9.74,P>0.05) in the sum of blastula cells,and so as to the Method 4(93.00±10.50 VS 83.63±9.74,P>0.05).The ratio of the ICM to the sum of blastula cells were 0.3382 and 0.3356 in Method 3 and Method 4,and all approximated to 0.33. The result of our study indicated that the Method 3 and Method 4 can be used in the double staining of bovinea as a reliable way. 5. The effect of integrinβ1 on the fusion of sperm-oocyte.Laminin and anti-β1 were used in this experiment to study the effect of integrinβ1 on the fusion of sperm-oocyte and the probable reason that laminin disappear during fertilization was also analyzed. The result showed that when the integrinβ1 of oocytes were blocked up by laminin, the fertilization(10μg/ml:67.38±5.41, 20μg/ml:71.51±4.96 VS 86.04±3.73, P<0.01) and cleavage rate(10μg/ml:41.43±8.33 VS 59.30±7.85, P<0.01) were significantly lower. But no influence for anti-β1. When eliminated the influence of heparin receptor by adding laminin to IVF medium, the effect on fertilization was a dose dependent(10μg/ml: 80.36±1.92 VS 86.04±3.73, P>0.05; 20μg/ml:64.95±7.61 VS 86.04±3.73, P<0.01) but no different in cleavage. While significantly lower rates were found when adding anti-β1 to it. The results also found that when the integrinβ1 of sperm were blocked up by laminin or anti-β1 or both sperm and oocyte blocked by either, the fertilization and cleavage rate were significantly lower(P<0.01) than the control group. These results suggested that sperm-oocyte fusion can be extremely disturbed by blocking up the integrinβ1 of sperm, not oocytes. And the reason for laminin’s disappear during IVF may for inhibiting fertilization by blocking up the integrinβ1 of sperm or heparin. |
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