Days 6~7 buffalo SCNT and the IVF blastocysts derived from abattoir ovaries and OPU were cryopreserved by slow freezing and vitrification in different cryoprotectant mediums.The 40%EG,25%EG+25%DMSO and 20%EG+20%DMSO+0.5 mol/L sucrose were adopted for vitrification,while 10% glycerol and 0.05 M trehalose dehydrate+1.8%EG+0.4%BSA were used for slow freezing.The results showed that(1): the efficiency of different vitrification medium was different from each other,20%EG+ 20%DMSO+0.5 mol/L sucrose was the best.As for slow freezing,the efficiency of the two kinds of medium was not different from each other.(2): 76.5% of the slow frozen blastocysts survived in 0~24 h after thawing,which was higher than that of vitrified blastocysts(48.9%).On the contrary,more vitrified blastocysts(42.6%) survived in 24~48 h than the slow frozen blastocysts(23.5%).In conclusion,the efficiency of vittification is higher than slow freezing,and the medium of 20%EG+20%DMSO+0.5 mol/L sucrose is best,while the slow frozen blastocysts survived earlier than the vitrified ones after thawing.

The hand-made cloning(HMC) technique describes a simplified nuclear transfer process without the need for micromanipulators.This study was conducted to investigate the effect of different enucleation,fusion methods and donor cell types on the efficiency of HMC in buffalo.The results indicated that chemically assisted enucleation with cytochalasin b(CB) showed similar enucleation efficiency to Hoechst 33342 staining enucleation;the fusion rate of one-step method was significantly higher than two-steps method(P0.01),while the developmental abilities of the couplets reconstructed by the two methods were no significant different from each other(P0.05).The fusion rate of HMC couplets derived from ear fibroblasts was significantly lower than that of couplets from cumulus cells(P0.01),while the cleavage and blastocyst rates of the embryos from the two cell types were not different from each other(P0.05).The study showed that CB assisted enucleation,one-step fusion,cumulus cells can be adopted for HMC in buffalo to improve the efficiency of cloning.

There are many factors that affect transfer efficiency of cloned embryos developed from somatic cells.Such factors as expression of estrus,embryo types,embryo stage and season were investigated in this paper.The results showed that the estrus method had no significant effect on pregnancy rates following cloned embryo transfer.As far as embryo type,there was no difference on conception rate between fresh embryo and frozen embryo.The conception rate was 9.79% and 14.5%,respectively.The highest rate of pregnancy was observed in either fresh or frozen-thawed embryos at the 6 day stage.Different stages(5,6,7 and 8-10 d)of cloned embryos had no significant difference(fresh embryos P=0.260 7,frozen-thawed embryos P=0.065 2)in terms of pregnancy rates after transfer.The pregnancy rate after cloned embryo transfer was the best in autumn,which reached 16.06%,and had a significant difference compared to those in other seasons(P0.05).The following was in spring,but it had no difference between in summer and winter(P0.05).The above results indicated that the method of the somatic cell cloned embryos in embryo transfer is feasible and relatively low efficiency.

To evaluate the influence of epidermal growth factor(EGF) on buffalo early embryo development and apoptosis in vitro,oocytes from buffalo ovaries at a slaughterhouse were matured and fertilized,and presumptive zygotes were cultured in the medium supplemented with EGF(0,25,50 or 100 ng/mL),then examined cleavage and blastocyst rate,respectively.After staining with Annexin V-Fluos Apoptosis Derection Kit(Annexin-V-Fluos Staining kit),apoptosis and necrosis of blastocyst under fluorescent microscope was examined.The result showed that the hatched rate in addition of 50 ng/mL EGF was significantly higher than that in controls(P0.05),and apoptosis rate and necrosis rate were significantly lower than those in controls(P0.05).While the cleavage rate,blastocyst rate,D7 blastocyst rate and the hatched rate in 100 ng/mL EGF in culture medium were lower than those in other groups,but the apoptosis rate and necrosis rate were higher.In conclusions,incubation of buffalo embryo in the presence of EGF improved the rate of hatched blastocyst and reduced the apoptosis of in vitro produced blastocysts.

本文主要探讨供体细胞类型及含羞草素对水牛体细胞克隆的影响。结果如下:与成纤维细胞相比,卵丘/颗粒细胞可以获得较高的融合率和分裂率,但两者的囊胚率差异不显著;克隆水牛的成纤维细胞能用于核移植,而且对核移植结果没有影响;性别对重构胚的早期发育影响不大;含羞草素处理可以替代血清饥饿处理。

为探讨水牛桑椹胚去致密化和不同分割方法对水牛切割胚发育效果的影响,将体外受精获得的水牛桑椹胚(第5d)和囊胚(第6～7d)分割后进行体外培养,观察其发育情况。结果显示:(1)水牛桑椹胚经去致密化处理后,其分割成功率(67.69%vs.51.71%)和囊胚发育率(58.30%vs.41.13%)均显著高于未处理组(P<0.01),但囊胚细胞数差异不显著(P>0.05);(2)以直接分割法和单臂分割法分别分割桑椹胚和囊胚,其分割成功率、囊胚发育率和囊胚细胞数三者均无显著差异(P>0.05)。可见,水牛桑椹胚去致密化有利于切割胚的成活和发育,而直接分割法和单臂分割法均可用于水牛桑椹胚和囊胚的分割。

The objective of this study was to explore the effects of buffalo follicular fluid(BuFF) and bovine follicular fluid(BoFF) on the IVF of buffalo oocytes obtained from Ovum pick-up(OPU) and abattoir,in order to develop IVF system and improve embryo production efficiency in buffalo.According to the replacement amount of fetal bovine serum(FBS) by FF in IVM medium,oocytes were distributed randomly into 4 groups.group Ⅰ: 0% FF+ 10% FBS,group Ⅱ: 5% FF +5% FBS,group Ⅲ: 10% FF+ 0% FBS and group Ⅳ: 15% FF+ 0% FBS.As for addition of BuFF,the results showed that BuFF did not improve the cleavage rate of OPU and abattoir-derived oocytes after IVF(P0.05).However,addition of 5% and 10% BuFF have significantly improved the develop efficiency of IVF embryos,the blastocyst formation rates of groups Ⅱ and Ⅲ were significantly higher than those of groups Ⅰand Ⅳ(P0.01),and there was no difference between groups Ⅱ and Ⅲ(P0.05).The blastocyst formation rate of group Ⅳ was lower than that of group Ⅰ,but the difference between two groups were not significant(P0.05).As for the addition of BoFF,the cleavage rate and blastocyst formation rate of OPU-derived oocytes in group Ⅲ were significantly higher than those of groups Ⅰand Ⅳ(P0.01),and there was no significant difference between groupsⅠand Ⅱ(P0.05).BoFF did not improve the cleavage rate of abattoir-derived oocytes after IVF(P0.05).However,the blastocyst formation rate of groups Ⅱand Ⅲ were significantly higher than those of groups Ⅰ(P0.05) and Ⅳ(P0.01),and blastocyst formation rate of group Ⅳ was significantly lower than that of group Ⅰ(P0.05),while the blastocyst formation rate was significantly different between groups Ⅱand Ⅲ(P0.05).In conclusion,addition of 5% to 10% BuFF or BoFF in IVM medium in place of FBS can significantly improve the efficiency of IVF embryo production in buffalo,and BuFF shows a better effect.

为探讨采卵季节对水牛活体采卵及其体外受精效果的影响,本研究选自2006-2008年78批次的626头牛活体采卵收集3143枚卵母细胞数据和同一操作者的51批次共培养1366枚卵母细胞的体外受精生产胚胎数据,并根据水牛繁殖特点和发情规律,每年分为3个阶段,即春季(1～4月),夏季(5～7月)和秋冬季(8～12)3个季节进行统计分析。结果发现,水牛活体采卵和体外受精效果受季节影响较大。在秋冬季进行活采时,平均头次获卵母细胞总数和可用卵母细胞数分别为(5.88±2.84)枚和(3.56±1.51)枚,平均头次卵母细胞总数明显高于夏季的(3.72±1.58)枚(P<0.01),但与春季的(4.80±1.77)枚差异不显著(P>0.05),而头均可用卵母细胞数则高于春季的(2.77±1.19)枚(P<0.05)和夏季的(2.11±1.11)枚(P<0.01);季节之间卵母细胞可用率没有差异(P>0.05);虽然每个季节活体采卵体外受精卵分裂率没有差异(P>0,05),但在秋冬季采卵时,其囊胚率(27.09%)则明显高于春季(20.95%)和夏季(20.45%),P<0.05,而春季和夏季之间没有差异(P>0.05)。研究表明:一年四季都可以进行水牛活体采卵体外生产胚胎,但从采卵效果和胚胎生产效率上看,在秋冬季进行活体采卵效果最好。

本文探讨了成纤维细胞的不同处理对水牛亚种间核移植的影响。试验设置3个组:对照组、接触性抑制组及血清饥饿组。结果:对照组在融合率、分裂率上均显著低于血清饥饿组及接触性抑制组(63.0%vs 73.0%、77.0%,43.8%vs 49.9%、50.0%)(P<0.05)。但血清饥饿组和接触性抑制组之间的融合率、分裂率差别不显著(73.0%vs 77.0%,49.9%vs 50.0%)(P>0.05)。这3个处理组之间的囊胚率差异不显著(7.0%,7.6%,9.5%)(P>0.05)。以上表明:对成纤维细胞进行处理可以提高水牛亚种间核移植的效率。

Effects of amino acids on the in vitro development of buffalo embryos derived from parthenogenetic activation(PA) were investigated,Addition of 1.5% MEM nonessential amino acid solution(NEAA) to SOF medium resulted in a significant increase in the cleavage rate of PA(79.8% vs 67.7%,P<0.05).However,supplementation with 3.0% BME essential amino acid solution(EAA) decreased the cleavage rate of IVF embryos(42.2% vs 52.2%,P<0.05),although the hatched rate of PA blastocysts was increased by supplementation with 1.0% EAA(77.3% vs 43.3%,P<0.05).

The objective of the current study was to explore the efficiency of embryo production by sexed sperms and oocytes derived from OPU and abattoir ovaries.Twenty cycling river-type buffaloes(12 Murrah buffaloes,8 Nili-ravi buffaloes) were in the OPU seesions.Retrieval of oocytes from live buffaloes was consecutively carried out once every four days within six weeks and oocytes graded A or B was recovered.Ovaries used for aspiration were derived from buffaloes in abattoir.Oocytes were aspirated from follicles by a syringe equipped with 18G needle and oocytes graded A or B was recovered.Oocytes derived from OPU and abattoir ovaries maturated under the same conditions.Embryo development was evaluated following in vitro fertilized with sexed buffalo sperms.The results revealed that the cleavage and blastocyst development of oocytes derived from OPU and abattoir ovary was not statistically different.And IVF of OPU oocytes with sexed or unsexed sperm was also similar in the cleavage rate and blastocyst development rate,52.80% versus 56.26%(P>0.05) and 20.93% versus 21.81%(P>0.05),respectively.Our results indicated the feasibility of embryo production in vitro using sexed sperm in buffalo species.

水牛分布在世界129个国家和地区,尤其对南亚和东南亚地区的农村经济做出了很大的贡献.水牛的生理特点是初情期迟,初产年龄长,产后久不发情,产犊间隔长,受胎率低.为了解决此问题,利用生物技术如人工授精、超数排卵和胚胎移植技术来提高水牛的繁殖效率.自Drost等(1983)首次报道水牛胚胎移植成功至目前为止已超过20多年,对水牛的超数排卵、胚胎回收研究虽有进展但进展缓慢.超排效果差和胚胎回收率低使沼泽型水牛和河流型水牛卵巢上原始卵泡数分别比奶牛低20%和30%,而且水牛卵泡闭锁率比奶牛高.近年来水牛的胚胎回收率已有很大提高,可用胚胎达2.5～3.0枚,但大多数在1.0～2.0枚范围内.

The appropriate electric fusion parameters for nuclear transfer(NT)of porcine somatic cells were investigated using in vitro matured porcine oosytes as recipients and granulose cells as donors in this study.When the pulse duration was fixed at 40 μs,0.77 kV/cm of field intensity resulted in significantly more oocytes fused(59.3%)in comparison with 1.54 kV/cm(38.6%,P0.05),and more NT embryos cleaved(77.8%)and developed to the morula and blastocyst stage(33.3%)in comparison with 2.31 kV/cm(52.2% and 13.6% respectively,P0.05).When the field intensity was fixed at 0.74 kV/cm,20 μs of pulse duration resulted in more oocytes fused(68.0%)in comparison with 30 μs(42.7%)and 40 μs(35.5%,P0.05),and more oocytes cleaved(89.3%)compared with 40 μs(60.5%,P0.05).When the field intensity was fixed at 0.74 kV/cm and pulse duration was fixed at 20 μs,one pulse resulted in more oocytes fused(72.8%)in comparison with two pulses(56.0%,P0.05).Applying alternating current(1.0 V)before direct pulsedid not affect the fusion rate(70.0% vs 76.0%),cleavage rate(77.3% vs 75.0%)embryo development(17.3% vs 18.4%,P0.05).In conclusion,the appropriate electric parameter for of porcine somatic NT is proved to be 0.77 kV/cm of field intensity,20 μs of pulse duration and 1 direct pulse,which will benefit the porcine somatic NT in future.

Buffalo oocytes matured in vitro for 22 h were enucleated by blind-sucking, point-hitting and Spindle-View System, respectively. The enucleation rate of oocytes by the Spindle-View System (95%) was significantly(P0. 01) higher than that achieved by blind-sucking(65.1%) and the pointhitting(81. 8%). However, there was no significant difference in fusion ration, cleavage rate and blasto cyst yield of the reconstructed embryos among the three enucleation methods(P0.05). Results indicated that the point-hitting can be employed for enucleation of oocytes in consideration of the costs of the Spindle-View System.

Zygotes and early embryos derived from nuclear transfer or gene targeting must be cultured in vitro to the morula and blastocyst stage at which can be transferred into recipients by unsurgery.In this paper,the influence of the ingredients of culture medium on the embryo developing in vitro were reviewed,such as serum bull serum albumin(BSA) or polyvinyl alcohol,growth factors,cytokines and extra-cellular matrix molecules,ethylenediamine tetraacetic acid(EDTA),NaN_3 and 2,4-dinitrophenol.