卵母细胞体外成熟时间对绵羊核移植效率的影响(英文)

[Objective] The study aimed to provide references for the time of oocyte maturation in vitro and enucleation in the course of sheep nuclear transfer(NT).[Method] Compared the effects of different maturation time of oocytes on enucleation efficiency and reconstructed embryo development by means of blind enucleation and fluorescence microscopy.[Result] Treatment of IVM(in vitro maturation)19-21 h was significantly higher than IVM 16-18 h treatment in oocyte maturation rate(P<0.05)and was significantly higher than IVM 22-24 h treatment in enucleation rate(P<0.05).Three treatments had no significant difference in cleavage rate and blastocyst rate(P>0.05),but IVM 19-21 h treatment was significantly higher than the other 2 treatments in average cell number of blastocysts(P<0.05).[Conclusion] The appropriate in vitro maturation time of oocytes was 19-21 h for sheep nuclear transfer,which could significantly improve the quality of blastocysts according to the cell number per blastocyst(P<0.05).     

Effects of in Vitro Maturation Time of Oocytes on Sheep Nuclear Transfer Efficiency

[Objective] The study aimed to provide references for the time of oocyte maturation in vitro and enucleation in the course of sheep nuclear transfer(NT).[Method] Compared the effects of different maturation time of oocytes on enucleation efficiency and reconstructed embryo development by means of blind enucleation and fluorescence microscopy.[Result] Treatment of IVM(in vitro maturation)19-21 h was significantly higher than IVM 16-18 h treatment in oocyte maturation rate(P<0.05)and was significantly higher than IVM 22-24 h treatment in enucleation rate(P<0.05).Three treatments had no significant difference in cleavage rate and blastocyst rate(P>0.05),but IVM 19-21 h treatment was significantly higher than the other 2 treatments in average cell number of blastocysts(P<0.05).[Conclusion] The appropriate in vitro maturation time of oocytes was 19-21 h for sheep nuclear transfer,which could significantly improve the quality of blastocysts according to the cell number per blastocyst(P<0.05).     

“胰岛素-转铁蛋白-硒钠”对山羊卵母细胞体外成熟及胚胎发育的影响(英文)

[Objective] The research aimed to enhance culture efficiencies of oocyte and embryo of goat in vitro and to explore serum-free culture system in vitro.[Method] At present,the conventional solutions of oocyte maturation and embryo development in vitro were always added into 1% ITS(Insulin-transferrin-selenium) or using 1% ITS to replace FBS in 2 kinds culture solutions for conducting in vitro cultures of goat oocyte and parthenogenetic embryo.The influences of ITS on their developments were detected.[Result] ITS in maturation liquid of oocytes could not increase oocytes maturation rate but significantly increased blastocyst rate (58.06% vs. 48.19%)of parthenogenetic embryo.If FBS in maturation liquid of oocytes was replaced by ITS, the maturation rate, cleavage rate and blastocyst rate were basically unchanged.Adding ITS into embryo medium could increase blastocyst rate (68.30% vs. 56.82%)of parthenogenetic embryo of goat.If FBS in embryo medium was replaced by ITS,the cleavage rate didn’t change basically,while the blastocyst rate in ITS was obviously lower than that in FBS group(42.33% vs.56.82%).[Conclusion] ITS could promote maturation of oocyte in vitro and early embryonic development, in addition,ITS could replace serum in maturation medium of oocyte as serum-free culture system for conducting relevant researches.     

Studies on Nuclear Transplantation in Mammalian Embryos

A series of experiments were conducted to study the major procedures in nuclear transplanation such as oocyte enucleation and activation,electrofusion and developent of the nuclear transplant embryos in the mouse,rabbits and sheep.The important results are as follows:1.In the mouse,only 35% of the oocytes collected 15-16h after hCG had a notable first polar body(FPb) and those without FPb were enucleated by removing cytoplasm from the PVS-wider side and the enucleation rate was similar to that in the oocytes with FPb,and the enucleation rate of removing 1/3 cytoplasm was remarkably higher than that of removing 1/4 cytoplasm.2.Among the three fusion media tested;mannitol and sucrose solutions produced better results than M2 in electrofusion of mouse 2-cell embryos.Under favorable pulse conditions,the osmotic pressure of fusion medium had no motable effect on electrofusion,but as the conditions became so unfavorable that some embryos began to lyse,the fusion rates in hypertonic mannitol solution were significantly higher than those in isotonic or hypotonic solutions.A wide range of pulse strengths (0.31-2.04kv/cm) and durations(10-1280us) were used and 100% of fusion were obtained in many cases.Optimal pulse durations were plotted for field strengths to obtain high fusion rates(96%-100%)in mouse2-cell embryos.3.With one pulse of 0.45kv/cm,satisfactory results of mouse oocyte activation were obatined only when the duration increased to 160us or longer,The activation rate increased as the oocytes got older.Some of the oocytes ar.rested at metaphaseⅢ after electrical stimulation and their proportion to the number of oocytes not activated increased with egg age.4.10% and 31% of the nuclear transplant embryos developed to morula or blastocyst stage in sheep and rabbits,respectively,with Chinese-made hormones and chemicals.     

Effect of Swim-Up and Percoll Treatment on Sperm Quality and In vitro Embryo Development in Yak

This study was designed to determine the effect of different sperm preparation treatments on yak sperm quality and in vitro embryo development.Frozen-thawed semen samples were treated using swim-up or percoll gradient centrifugation methods.Sperm concentration,progressive motility,recovery of motile sperm,membrane integrity,acrosome and chromatin integrity were scored and compared in recovered samples and controls.In addition,the effects of two sperm separation treatments on embryos capable of cleavage and in vitro development to the blastocyst stage were evaluated.Swim-up separated sperm showed a higher motility,while the concentration of spermatozoa recovered and percent recovery of motile sperm were higher with percoll gradient centrifugation separation.According to the optical and electron microscopies,swim-up produced higher percentage of sperm with intact plasma membrane and acrosome than percoll gradient centrifugation separation.However,there was no difference in the percentage of sperm with intact chromatin between two treatment groups.Cell numbers in the blastocysts of two groups were not different.The blastocyst rate was similar in both groups,whereas cleavage rate was higher when swim-up was used.     

Studies on Electrical Activation of Porcine Oocytes Matured in vitro and Embryo Culture Systems

Conditions for electrical parthenogenetic activation of porcine oocytes matured in vitro and in vitro culture systems of porcine embryo were studied. The best results were achieved under the conditions of electrical field strength and the pulse duration at 130Vmm-1/80 μs, with a blastocyst development rate of (20.12 ± 8.18)% (P ＞ 0.05). No significant difference was found between treatments of multiple pulses and a single pulse ( P ＞ 0.05). Parthenogenetic embryos were cultured with different methods and air conditions for 7 days in vitro, blastocyst development rate of embryos with changed culture media [ (26.44 ± 8.35)% ] or changed media with 10% fetal bovine serum (FBS) [ (17.68 ± 5.39)% ] on the fifth day showing no significant difference from that of embryos without change of culture media [ (25.30 ± 7.55) %, P ＞ 0.05 ], while cell numbers of blastocysts from embryos with changed culture media (15.78 ± 5.46 and 14.55 ± 4.81) were significantly lower than number of blastocysts from embryos without change of culture media (18.01 ± 6.79,P ＜ 0.01 ). Blastocyst development rate and blastocyst cell number of embryos cultured in lower O2 (5 % CO2:7%O2:88%N2) also showed no significant difference from those in high O2 (5% CO2 in air) [ (20.78 ± 8.80) % and 17.00 ± 6.12 vs. (25.30 ± 7.55) % and 18.01 ± 6.79, P ＞ 0.05 ]. It is concluded that change of culture media with the same new one or changing over to media with 10% fetal bovine serum (FBS) on the fifth day and low O2 environment are not necessary for porcine embryos development.     

Expression of HSP72 in Mouse Preimplantation Embryos with Heat Shock

The objective of the paper was to detect HSP72 expression and HSP72 gene sequence in heat shocked mouse preimplantation embryos and the effects of different thermo conditions on hatching rates of embryos. The mouse blastocysts cultured in vitro were heat treated at 40℃ and 38℃ for 1 h, 2 h and 3 h and then recovered at 370C for 3 h, 2 h and 1 h, respectively, to detect their HSP72 gene expression by using RT-PCR after the total R.NA extraction. The hatching rate of the blastocysts for different treated groups was recorded and the expression of liSP72 in the blastocysts was determined by Western blot. The results showed that all the groups of blastocysts, including the control, had the expression of HSP72 gene. The expression of HSP72 protein had the highest level in the embryos stressed at 38℃ for 2 h, and it was significantly higher than that in the control group. The expression of HSP72 in the groups of blastocysts treated at 40℃ was not significantly different from that in the control group. The embryos with induction of mild heat shock at 38℃ for 2 h, then subjected to heat shock at 40℃ for 2 h, had a significant higher （P〈0.05） hatching rate of 54.74% compared to 47.85% in the embryos treated directly at 40℃ for 2 h. The above results indicated that the mouse blastocysts were sensitive to heat shock and a mild heat shock induced HSP72 gene expression. Induction of HSP72 expression with mild heat shock helped embryos to tolerate more severe heat shocks.     

Relationship Between the mRNA Expression Level of TGF-β Receptor Genes in Tissues and Ovulation Rate in Hu Sheep

Eight ewes of Hu sheep which bred multi-lamb were used as the high-fecundity group and the other eight ewes of Hu sheep which bred single lamb were used as the control group to investigate the relationship between the mRNA expression level of TGF-β receptor genes in tissues and ovulation rate in Hu sheep. Cloprostenol sodium was injected to make the synchronization of estrus treatment, then all ewes were slaughtered within 24-36 h after empathema and the ovaries were collected. Furthermore, the number of ovulation points was counted to determine ovulation rate for each sheep. Tissue expression analysis was conducted by RT-PCR for one ewe form the high-fecundity group and the relationship between the mRNA expression of genes encoding TGF-β receptors and ovulation rate was detected by real-time fluorescent quantitative PCR. The results showed that the relative expression level of TGF-flR I gene in the reproductive organ was significantly higher than in the lung and muscle （P 〈 0.01）, while relative expression level of TGF-βR H in reproductive organ was significantly higher than that of other tissues （P 〈 0.01）, indicating that these are highly expressed genes in the ovary. In addition, mRNA expression level of TGF-βR I and TGF-flRH in the ovaries of the high-fecundity group were significantly higher （P〈 0.01） and obviously higher （P= 0.011） than the control group, respectively. The mRNA expression level of TGF-βR I and TGF-βR H had a positive correlation with ovulation rate and the correlation coefficients were 0.562 （P〉 0.05） and 0.711 （P〈 0.05）, respectively. It is suggested that TGF-β receptors have close relationship with highfecundity in Hu sheep.     

Effect of NPK Application on Yield,Nutrients and Water Utilization Under Sudangrass and Ryegrass Rotation Regime

Sudangrass and ryegrass rotation is a new type of cropping system in Jianghan Plain,which develops very fast in recent years.So,it is essential to work out the optimal nutrients and water management measures in this new system.The effect of NPK fertilizer on yield of forage grass,nutrients and water utilization under sudangrass and ryegrass rotation was studied through continuous pot experiments for three years.The results showed that NPK combination could significantly improve the yield of forage grasses.The total yields during the periods of 2005-2006,2006-2007 and 2007-2008 were 1 690.0,2 091.3 and 1 770.7 g/pot,respectively.The yields in PK treatment were 700.7,1 256.9 and 856.3% lower than those of NPK treatment,while the yields in NK treatment were 426.3,384.9 and 792.3% lower than those of NPK treatment,respectively.Similarly,the yields in NP treatment were 15.9,4.4 and 10.6% lower than those of NPK treatment,respectively.The NPK combination was found to improve the nutrients uptake,and the total N uptakes during the above periods were 10.0,14.8 and 10.6 times higher than that of PK treatment,respectively,and 3.7,1.8 and 5.1 times higher than those of NK treatment,respectively,but,were similar to NP treatment.The total P uptakes were 4.6,6.8 and 5.3 times higher than those of PK treatment,and were 2.4,2.3 and 2.9 times higher than those of NK treatment,respectively,but were similar to NP treatment.The total K uptakes were 5.2,8.4 and 4.9 times higher than that of PK treatment,3.9,2.3 and 3.9 times higher than those of NK treatment,and 80.9,57.4 and 200.5% higher than those of NP treatment,respectively.Water productivity for three years time periods were 16.5,16.1 and 16.2 kg m-3,and were 3.8,5.4 and 4.2 times higher than those of PK treatment,2.3,1.3 and 2.6 times higher than those of NK treatment,and were 16.4,8.8 and 22.4% higher than those of NP treatment,respectively.The soil pH was decreased and soil organic matter was increased in all treatments with increase in the time of sudangrass and ryegrass rotation system.The total soil N was increased in NP,NK and NPK treatments,while it was stable in PK treatment.The available P in NP,PK and NPK treatments was increased significantly,while it was stable in NK treatment.The slow releasing K and available K were increased significantly in NK and PK treatments,but decreased in NP and NPK treatments.The treatment with NPK combination was found to improve the yield of forage,nutrient uptake and soil fertility in sudangrass and ryegrass rotation system.     

Effect of GPAG Supplement on Porcine Oocyte Maturation and Its Following Embryonic Development by Parthenogenetic Activated

The aim of this study was to compare the effect of GPAG and commonly used FCS on porcine oocyte maturation and subsequent embryonic development after the fertilization. COCs were aspirated from follicles and cultured for 16, 24, 32, 40 and 48 h in TCM-199 medium either with GPAG or FCS. After 24 h with GPAG, 89.4% of oocytes reached M Ⅰ stage while in the medium supplemented with FCS, only 27.7% of oocytes reached the same stage （P〈0.05）. Prolonged incubation for up to 32 h clearly demonstrated that some of oocytes cultured in GPAG medium were at M Ⅱ stage （35.7%）, few of oocytes from FCS medium were at M Ⅱ stage （7.5%） （P〈0.05）. Both groups of oocytes reached the same stage of maturation within 48 h. After 48 h of culture, the oocytes with extruded polar bodies were inseminated. Fertilized oocytes were cultured in PZM3 medium supplemented with 3 mg.mL of BSA. After 7 days, the development and the quality of embryos were evaluated. The results showed that the maturation of oocytes in the presence of GPAG significantly increased their subsequent developmental ability when compared with FCS supplementation （29.2% ： 18.9% of blastocysts, P〈0.05）. However, differential staining revealed that once blastocysts were formed in either group, they had the same total cell number （39 ： 38） and the ICM/total cell ratio （0.26 ： 0.28）     

卵母细胞体外成熟时间对绵羊核移植效率的影响

[目的]为绵羊克隆试验中卵母细胞的体外成熟及去核时间提供参考。[方法]利用盲吸法结合荧光显微镜检查,对绵羊不同成熟时间卵母细胞去核的效率及其后续重构胚的发育进行对比。[结果]体外成熟培养19~21 h的绵羊卵母细胞在成熟率上显著高于体外成熟培养16~18 h(P(0.05),在去核成功率上显著高于体外成熟培养22~24 h(P(0.05);3个试验组在卵裂率、囊胚率上差异不显著(P(0.05),但是体外成熟培养19~21 h的试验组囊胚平均细胞数要显著高于其他2组(P(0.05)。[结论]体外成熟培养19~21 h的卵母细胞较适于作为受体细胞进行绵羊核移植,可以显著提高囊胚的质量。     

不同浓度Leptin对猪卵母细胞体外成熟和早期孤雌发育的影响

[目的]系统比较了在成熟培养液中添加Leptin对猪卵母细胞体外成熟和孤雌激活胚的发育率和囊胚质量,同时比较了成熟培养液和胚胎培养液添加Leptin对孤雌激活胚发育的影响。[方法]成熟培养液中添加不同浓度Leptin对猪卵母细胞体外成熟和早期孤雌发育能力的影响及囊胚的内细胞团细胞数目,并且研究了成熟培养液和胚胎培养液单独或联合添加Leptin对猪早期孤雌胚胎发育能力的影响及囊胚的内细胞团细胞数目。[结果]成熟培养液中添加不同浓度Leptin(0,10,50,100和200 ng/ml),体外成熟44 h,卵母细胞核成熟率统计分析差异不显著(P>0.05);将核成熟卵母细胞进行化学激活,第2天胚胎卵裂率统计分析差异不显著(P>0.05),但100ng/ml组与其他组第7天的囊胚率及囊胚的总细胞数差异显著(P<0.05);成熟液添加Leptin 100 ng/ml、胚胎培养液添加Leptin 10 ng/ml与其他组第7天的囊胚率及囊胚的内细胞团细胞数差异显著(P<0.05)。[结论]Leptin对卵母细胞成熟和孤雌胚胎发育能力上有相辅相成的作用。     

卵泡液对牛卵母细胞发育潜力的影响

为了确定卵泡液(直径大于10mm)对卵母细胞成熟的影响,共收集1759枚卵母细胞进行试验。在成熟液中分别添加0%,10%,30%和50%的卵泡液(bFF,bovinefollicularfluid),成熟培养24h后,孤雌激活或者体外受精和胚胎培养;对比10?F和10%胎牛血清(FBS),以及观察微滴培养对卵母细胞成熟的影响。结果表明,在孤雌激活组中,bFF浓度对卵裂率的影响差异不显著,但胚胎囊胚发育率高于对照组(P<0.05);在体外受精组中随着bFF浓度增加而卵裂率下降(P<0.01),而且囊胚率也下降(P<0.05)。在10%的bFF组中,孤雌激活囊胚率和体外受精囊胚率分别为47.1%和38.0%,囊胚扩张率显著高于其它组。从试验得出,添加10?F有利于卵母细胞胞质成熟,且不会降低到受精率,而且可以替代FBS;微滴培养不利于卵母细胞成熟。     

亮甲酚蓝筛选对广西巴马小型猪孤雌激活及克隆重构胚早期发育的影响

[目的]探讨亮甲酚蓝(BCB)染色是否可以筛选出发育潜能优秀的卵母细胞.[方法]猪卵丘卵母细胞复合体(COCs)在体外成熟培养前用13μmol/L BCB染色,根据卵母细胞胞质蓝色的有无分为阳性(BCB+,蓝色)和阴性组(BCB-,不着色),以在不含BCB的改良杜氏磷酸盐缓冲液(m-DPBS)中处理90 min为控制对照组,直接培育作空白对照,体外成熟培养后统计各组卵母细胞的核成熟率及孤雌激活胚胎、克隆重构胚胎(供体细胞来自广西巴马小型猪)的早期发育率.[结果]BCB+组卵母细胞的核成熟率(80.8%)显著高于BCB-组(44.2%)和空白对照组(72.4%);BCB+组的孤雌囊胚形成率(32.9%)显著高于BCB-组(10.1%),但与对照组(25.8%)之间无显著差异;BCB+组显微操作核移植胚胎的囊胚发育率(14.1%)显著高于BCB-组(3.4%),但与对照组之间无显著差异;各组手工克隆胚胎发育率之间无显著差异.[结论]BCB染色筛选出的卵母细胞具有较好的核成熟发育潜能,但未能显著改善广西巴马小型猪显微操作及手工克隆胚胎的早期发育.     

尿嘧啶和ATP对牛卵母细胞成熟及激活后孤雌胚胎发育的影响

[目的]探究尿嘧啶(Uracil)和三磷酸腺苷二钠盐(ATP)对牛卵母细胞成熟及激活后孤雌胚胎发育的影响。[方法]在体外成熟培养液中添加不同浓度的尿嘧啶和ATP,研究其对牛卵母细胞体外成熟及激活后孤雌胚胎发育能力的影响。[结果]在成熟培养液中添加50μg/ml尿嘧啶能够显著提高卵母细胞的成熟率、分裂率及孤雌胚胎囊胚率;在成熟培养液中分别添加0、250、500、750μg/mlATP,对卵母细胞成熟率影响不大,但添加500μg/ml ATP能显著提高激活后孤雌胚胎囊胚率。[结论]该研究为牛卵母细胞IVM研究提供了参考资料。     

成熟培养基对猪卵母细胞的影响

评估NCSU23和TCM199两种培养基对猪卵母细胞体外成熟、孤雌激活以及核移植重构胚发育的影响。分别以NCSU23和TCM199为基础培养基,向其中添加相同浓度的半胱氨酸、激素和生长因子,作为猪卵母细胞体外成熟培养液,培养44 h(5%CO2、39℃、饱和湿度),挑选第一极体;成熟后的卵母细胞进行孤雌激活和核移植重构胚操作,144 h后观察囊胚数。结果表明,NCSU23组与TCM199组对卵母细胞体外成熟(68.18%和67.55%)、孤雌囊胚率(38.40%和37.60%)与核移植囊胚率(23.79%和21.38%)无显著影响(P>0.05),但是两者对孤雌囊胚细胞数的影响差异显著(分别为38.4和37.6个细胞)(P<0.05)。NCSU23成熟培养基能够使胞质成熟完全,提高囊胚细胞数,有利于妊娠的发生。可见,以NCSU23为基础培养基的体外成熟液有利于猪卵母细胞体外发育。     

“胰岛素-转铁蛋白-硒钠”对山羊卵母细胞体外成熟及胚胎发育的影响

[目的]为提高山羊卵母细胞及胚胎体外培养的效率,探索无血清体外培养体系。[方法]在目前卵母细胞体外成熟及胚胎培养所用的常规培养液中添加1%ITS(胰岛素-转铁蛋白-硒钠)或以1%ITS分别替代2种培养液中FBS,对山羊卵母细胞及孤雌激活胚胎进行体外培养,检测ITS对其发育率的影响。[结果]添加ITS到卵母细胞成熟液中,对卵母细胞成熟率没有明显提高,但显著提高了其激活后孤雌胚胎的囊胚率(58.06%vs.48.19%);以ITS替代成熟液中FBS,取得了与FBS组相似的成熟率、卵裂率和囊胚率。添加ITS到胚胎培养液中,显著提高了山羊孤雌胚胎的囊胚率(68.30%vs.56.82%);以ITS替代胚胎培养液中FBS,卵裂率与FBS组无显著差异,囊胚率显著低于FBS组(42.33%vs.56.82%)。[结论]ITS对山羊卵母细胞体外成熟及胚胎早期发育均有促进作用;另外,ITS可以替代卵母细胞成熟培养液中的血清,作为无血清培养体系用于相关研究。