Influence of Embryonic Size and Manipulation on Pregnancy Rates of Mares After Transfer of Cryopreserved Equine Embryos

Many years of poor results of equine embryo cryopreservation has produced a lack of confidence in this technique. Embryo cryopreservation has been successfully used for more than 20 years in other species like bovine and human. The large size of the embryos and the presence of a capsule impermeable to cryoprotectants have been the two main reasons for the failure. In the last few years, a mayor breakthrough for this technique was obtained when large equine embryos could be successfully cryopreserved after breaching the capsule and collapsing the blastocoel cavity. In the present study, we compared the pregnancy rates obtained by vitrification or cryopreservation by slow freezing of embryos smaller than 300 μm. No difference was found between vitrification and slow freezing of embryos <180 μm (pregnancy rate on day 16: 34/61, 55.7%; 6/8, 75%) but produced very low results for embryos between 180 and 300 μm in diameter (0/11, 0%; 1/7, 14.3%). Embryos larger than 300 μm were collapsed before cryopreservation, and two different types of carriers, hemi-straw or Stripper-Tip, were used for vitrification. High pregnancy rates were obtained when the hemi-straw was used as a carrier (7/10, 70% vs. 0/5, 0%), demonstrating that a minimum vitrification volume was essential to preserve the embryo viability. These findings establish that, due to the large range in diameter, equine embryos need to be cryopreserved using different protocols depending on their size.     

Influence of Ovulation Status,Seasonality and Embryo Transfer Method on Development of Cloned Porcine Embryos

To improve pig cloning efficiency, the present study evaluated the effect of ovulation status, seasonality and embryo transfer (ET) method on in vivo development of cloned porcine embryos. Cloned embryos were transferred to surrogate mothers on the same day of somatic cell nuclear transfer. In pre‐ovulation stage (PO), pregnancy rate (PR) and delivery rate (DR) were 36.3% and 9.4%, respectively. In post‐ovulation stage, 22.7% PR and 2.1% DR were recorded (both PR and DR are significantly higher in PO). When ET was performed during winter (December–February), spring (March–May), summer (June–August) and autumn (September–November), the PRs were 13.4%, 37.3%, 24.6% and 51.0%, while DRs were 0%, 12.7%, 4.3% and 7.8%, respectively. The highest PRs were recorded in autumn groups. However, DRs were significantly lower in autumn (7.8%) group compared with spring (12.7%) group. The PR was the lowest and no piglets were born in winter group, which might be because of the effect of low temperature during ET. To overcome the low PR in winter group, 0.25 ml straws were used for ET to minimize exposure time of embryos to ambient temperature. The straw ET group showed significantly higher PR in the winter group (23. 9%) compared with the conventional catheter‐loading group (7.7%). We suggest that using PO recipient and ET in spring is the best condition for pig cloning. In addition, alternative method to reduce cold shock during ET in winter is necessary.     

Radiographic Estimation of Pregnancy in Goats

Contents: The onset and progression of goat fetal skeletal radio opacity were studied to identify the stage of pregnancy and the number of fetuses present. Lateral radiographs using 75 KVP, 200 Ma, 0.25 s and 100 FFD was found to be good values for detection of pregnancy in goats. Lengths taken for skull, femur and vertebral column of the fetus were used as a parameter for determination of the stage of pregnancy. Counting the number of fetal skulls were used for estimating the number of fetuses present. Accurate radiological diagnosis of pregnancy in goat was possible after 10 weeks of gestation .
Inhalt: Radiographische Bestimmung der Trächtigkeit bei der Ziege
Der Beginn und die Weiterentwicktung fötater Skelettbildung wurde radiologisch bestimmt, um das Stadium der Trächtigkeit und die Zahl der Föten zu ermitteln. Laterale Röntgenbilder wurden mit 75 KVP, 200 Ma., 0,25 Sek. und 100 FFD ermittelt, wobei sich diese Einstellung als günstig für die Trächtigkeitserkennung bei Ziegen erwies. Die Ermittlung der Schädellänge, des Femurs und des Rückenmarks beim Fötus wurden als Parameter für die Bestimmung des Trächtigkeitsstadiums benutzt. Die Zahl der Fötalschädel ergab die Zahl der Föten. Genaue radiologische Diagnose der Trächtigkeit bei Ziegen war ab etwa 10 Wochen der Trächtigkeit möglich .     

细胞松弛素B对猪孤雌胚胎和体外克隆胚胎发育能力的影响

为探讨细胞松弛素B（cytochalasin B,CB）对猪孤雌胚胎和克隆胚胎发育能力的影响,本研究通过在猪体外胚胎培养基中添加不同浓度CB以及不同孵育时间的处理,筛选出CB对猪早期胚胎发育的最适浓度和最佳孵育时间,同时通过Hoechst33342染色检测猪体外囊胚孵化期的细胞数差异,进一步研究CB对孤雌胚胎和克隆胚胎发育的影响。结果显示,培养基中添加CB浓度为7.5 μg/mL时孤雌胚胎和克隆胚胎的卵裂率分别为85.00%和90.23%,囊胚率为35.68%和42.58%,均显著高于其他各组（P < 0.05）;采用7.5 μg/mL CB处理电激活后的孤雌胚胎和克隆胚胎,孤雌胚胎孵育4 h组的卵裂率（83.80%）和囊胚率最高（35.39%）,与其他各组差异显著（P < 0.05）,而克隆胚胎孵育6 h组的卵裂率（83.98%）和囊胚率最高（55.62%）,与其他各组差异显著（P < 0.05）。此外,Hoechst33342染色结果显示,未添加CB处理的孤雌胚胎在囊胚孵化期的细胞平均数为28个,CB处理组的孤雌胚胎和克隆胚胎细胞平均数分别为36和52个,处理组和未处理组细胞数差异显著（P < 0.05）。结果表明,猪体外孤雌胚胎用7.5 μg/mL CB 处理4 h可获得较高的卵裂率和囊胚率;体外克隆胚胎用7.5 μg/mL CB 处理6 h卵裂率及囊胚率最高,且囊胚期内细胞团细胞总数最多。CB处理有利于体外胚胎早期发育,提高克隆胚胎移植受孕率。     

B超妊娠诊断技术在转基因山羊生产中的应用

用配有7．5MHz探头的超声扫描仪，以胎盘子叶作为主要的判断依据。对310只转基因胚胎移植的受体山羊进行早期妊娠诊断，对检出的怀孕母羊进行了相应的处理。结果表明；胚胎移植后60～65天试验母羊妊娠诊断阴性(B超判断未怀孕)的准确率为98．1％，妊娠诊断阳性准确率为100％。     

Development In Vitro and Mitochondrial Fate of Interspecies Cloned Embryos

Although the technique of interspecies somatic cell nuclear transfer can be used to increase the population size of endangered mammals, the mitochondrial heteroplasmy in cloned embryos and animals makes this idea doubtful. In present study, goat–sheep cloned embryos were constructed by fusing goat foetal fibroblasts (GFFs) into sheep oocytes and then cultured in vitro to investigate the capability of sheep oocyte dedifferentiating GFF nucleus. Moreover, at each stage of 1‐ (immediately after fused), 2‐, 4‐, 8‐, 16‐cell, morula and blastocyst, the copy number of mtDNA from GFF and sheep oocyte was examined using real‐time PCR. The results showed that: 7.4% of the fused cloned embryos can develop to the blastocyst stage; in the process of one cell to the morula stage, the copy number of two kinds of mtDNA was stable relatively; however, in the process of morula to the blastocyst stage, the decreasing in the copy number of GFF‐derived mtDNA, while the increasing in sheep oocyte‐derived, resulted in their ratio of decreasing sharply from 2.0 ± 1.0% to 0.012 ± 0.004%. This study demonstrates that: (i) the goat–sheep cloned embryos have the ability to develop to blastocyst in vitro; (ii) from the morula stage to the blastocyst stage of goat–sheep cloned embryos, goat derived mitochondria can be gradually replaced with those from sheep oocyte.     

猪体细胞克隆胚胎体外发育过程中的凋亡规律

本试验旨在研究比较猪体细胞克隆胚胎体外发育与凋亡变化规律,探讨其体外发育潜能与细胞凋亡间的关系。将体外成熟培养获得的猪MⅡ期卵母细胞随机分为2组,分别进行体细胞核移植(SNCT)与体外受精(IVF)处理,采用彗星试验(Comet assay)和Real-time RT-PCR技术对体外发育过程中各时期胚胎的细胞凋亡事件及抗凋亡基因Bcl-2 mRNA的表达水平进行研究。结果表明,两组在卵裂率上无显著差异(77.6%vs 80.3%,P0.05),在囊胚发育率上,SCNT组胚胎明显低于IVF组,但无统计学差异(18.9%vs 24.5%,P0.05);随着胚胎发育,SCNT与IVF组胚胎的凋亡率均呈现不断增加的趋势,Bcl-2 mRNA表达量则逐渐下降;到16-细胞以后,SCNT组胚胎的凋亡率均显著高于IVF组,同时Bcl-2 mRNA表达量均显著低于IVF组(P0.05)。本试验结果表明,猪克隆胚胎生产效率低下可能与胚胎发育过程中的细胞凋亡有关。     

硒对妊娠早期山羊子宫局部细胞因子分泌的调节

为了探索硒对妊娠早期子宫局部细胞因子IL-2、IL-4、TGF-β1及TGF-β2的调节作用,将妊娠山羊分为试验Ⅰ、Ⅱ、Ⅲ组和对照组。Ⅰ、Ⅱ、Ⅲ组山羊分别以0．3、0．5和0．7mg／kg剂量的硒灌服亚硒酸钠溶液,对照组灌服1mL／kg的蒸馏水,每周1次,连续3周。用ELISA试剂盒检测子宫局部细胞因子IL-2、IL-4、TGF-β1及TGF-β2的动态变化以观察硒对其分泌的调节作用。结果表明：（1）妊娠早期（15～19d）山羊两侧子宫角中IL-2、IL-4、TGF-β1及TGF-β2水平无显著差异。妊娠早期山羊两侧子宫角的子宫液IL-2水平在15～18d呈上升趋势,19d时趋于下降;IL-4水平一直呈上升趋势;TGF-β1水平较高,呈不规律变化;TGF-β2呈现先升高、后趋于平稳的趋势。（2）灌服亚硒酸钠溶液,可以显著降低妊娠16～18d子宫液IL-2的水平（P〈0．05或P〈0．01）;同时可显著升高IL-4的水平（P〈0．05或P〈0．01）,且呈一定的剂量依赖关系。（3）0．5和0．7mg／kg剂量组显著升高妊娠16～18d子宫液TGF-β1水平（P〈0．05或P〈0．01）;同时对妊娠16～17d子宫液TGF-β2水平也有显著的升高作用（P〈0．05或P〈0．01）,但对18d时的TGF-β2影响不显著。试验表明,硒对妊娠早期山羊子宫局部IL-2、IL-4、TGF-β1及TGF-β2的分泌具有一定的调节作用。     

Factors Affecting Recipients' Pregnancy,Pregnancy Loss,and Foaling Rates in a Commercial Equine Embryo Transfer Program

During 11 breeding seasons, 351 7- to 10-day-old horse embryos were nonsurgically transferred into recipients that ovulated between 3 and 10 days earlier. Pregnancy rates at 14 and 40 days and foaling rates were 77.8% (273/351), 69.2% (243/351), and 64.4% (226/351), respectively. Pregnancy loss between 14 and 40 days was 11% and between 40 days and delivery was 7%. The transfer of quality grade 3 to 4 embryos resulted in a significantly lower pregnancy rate at 14 days compared with the transfer of grade 1 to 2 embryos (46.2% vs. 79%; P < .05). Eight-day-old embryos resulted in significantly lower pregnancy losses than day 9 or 10 embryos, as occurred for embryos between 400 and 1200 μm compared with embryos <400 μm. Embryos recovered from mares >20 years resulted in a significantly higher pregnancy loss rate than those recovered from younger mares. The same happened for embryos coming from mares affected by reproductive pathologies compared with healthy mares performing sport activity. None of the evaluated parameters influenced recipients' foaling rate significantly.     

用猪用妊娠诊断剂对山羊进行早期妊娠诊断试验

选择配种后 2 0± 2d的黄淮母山羊 16 4只 ,随机分为试验组 90只和对照组 74只 ,探讨“猪用妊娠诊断剂”对山羊早期妊娠诊断效果。结果表明 ,试验组肌注妊娠诊断剂 ,检出空怀准确率为 92 % (P <0 0 1) ,检出妊娠准确率为 30 % ,而分娩率和双胎率分别低于对照组 5 2 %和33 4 % (P >0 0 5 )。综合分析认为雌激素类妊娠诊断剂不适合于山羊早期妊娠诊断。     

An Inter‐Subspecies Cloned Buffalo (Bubalus bubalis) Obtained by Transferring of Cryopreserved Embryos via Somatic Cell Nuclear Transfer

The aim of this study was to explore the feasibility of cryopreservation of inter‐subspecies cloned embryos in buffalo. In our experiment, river buffalo ear fibroblast nucleus was fused into swamp buffalo oocyte cytoplasm. The blastocyst formation rate for nuclear transfer of freshly thawed cells was not different from those of growing cells, confluent or serum‐starved cells. A total of 122 cloned blastocysts derived from cryopreserved fibroblasts were cryopreserved and thawed, 37 were survived, the cryosurvival rate was 30.3%. The survived blastocysts were transferred into 15 recipient buffalos. Five of the recipients established pregnancy, but four of them aborted on day 53, 59, 145 and 179 of gestation respectively. One cross‐bred buffalo (Murrah × Swamp buffalo (2n = 49) received three embryos delivered a 40.5 kg female calf by natural delivery on day 320 of gestation. Up to now (13‐month old), the cloned calf has been growing well with no abnormity observed. These results demonstrated that cryopreservation of inter‐subspecies cloned embryos is feasible to produce buffalo offspring.     

体细胞核移植生产梅山猪

本研究以中国优良地方品种梅山猪为材料,采用胰酶消化法获得猪胎儿成纤维细胞,通过使用Y染色体SRY基因引物SRY-1、SRY-2进行PCR扩增,结果发现,雄性胎儿样本能扩增出250 bp的Y染色体特异性基因片段,而雌性胎儿样本未扩增出此条带;G1、G2、G3、G4和G5代的融合效率差异不显著(P>0.05),但G5代作为供体细胞核移植的重组胚胎卵裂率显著高于G1、G2、G3和G4代(P<0.05);使用G5代的胎儿成纤维细胞作为供核细胞,通过手术移植的方法,将重构胚胎移植到二元后备母猪体内,结果成功地获得了体细胞克隆梅山仔猪,并且仔猪全部为雄性胎儿,说明该性别鉴定方法不但操作简单,而且准确度高;经微卫星DNA 多态性鉴定,确定克隆猪来自供核细胞,与代孕母猪无亲缘关系。本研究将为中国优质猪品种改良、保种、性别控制及建立人类疾病模型等研究提供有效可行的方法,为批量生产克隆优秀种猪提供了坚实的理论基础。     

牛胚胎玻璃化超快速冷冻一步法移植试验

应用 30 %乙二醇 0 .3mol/ L 蔗糖 - m- PBS液 (VS1)、30 %乙二醇 0 .3m ol/ L 蔗糖 5 %葡聚糖 (T- 5 0 0 ) - m-PBS液 (VS2 )、30 %乙二醇 0 .3mol/ L蔗糖 10 %葡聚糖 (T- 5 0 0 ) - m- PBS液 (VS3)玻璃化超快速冷冻奶牛胚胎 ,一步法移植。结果显示 ,VS1、VS2、VS3组玻璃化超快速冷冻奶牛胚胎解冻后的形态正常率分别为 10 0 % (2 0 / 2 0 )、95 %(19/ 2 0 )和 5 5 .6 % (5 / 9) ;培养存活率分别为 70 % (14 / 2 0 )、75 % (15 / 2 0 )和 2 2 .2 % (2 / 9) ;囊胚孵化率分别为 0、2 0 % (4/2 0 )和 0。VS1、VS2组玻璃化冷冻奶牛胚胎解冻后的形态正常率极显著地高于 VS3组 (P<0 .0 1) ;VS1、VS2组玻璃化冷冻奶牛胚胎解冻后的培养存活率分别显著 (P<0 .0 5 )和极显著 (P<0 .0 1)地高于 VS3组。 VS1组冷冻的胚胎经一步法移植了 5头 (1枚 /头 ) ,未获得妊娠 (0 / 5 ) ;VS2组冷冻的胚胎用一步法移植了 10头 (1枚 /头 ) ,结果获得 2头妊娠(2 / 10 ) ,并产下 2头正常犊牛。     

Pregnancy Rates to Fixed Embryo Transfer of Vitrified IVP Bos indicus,Bos taurus or Bos indicus × Bos taurus Embryos

The pregnancy rates obtained after the transfer of cryopreserved in vitro‐produced (IVP) embryos are usually low and/or inconsistent. The objective of this study was to evaluate the pregnancy rates of Holstein, Gyr and Holstein × Gyr cattle after the transfer of vitrified IVP embryos produced with X‐sorted sperm. Seventy‐two Gyr and 703 Holstein females were subjected to ovum pickup (OPU) sessions, followed by in vitro embryo production using semen from sires of the same breeds. Embryos (1636 Holstein, 241 Gyr and 1515 Holstein × Gyr) were exposed to forskolin for 48 h prior to vitrification. The pregnancy rate achieved with Gyr dam and sire was 46.1%, which was similar (p = 0.11) to that of Holstein dam and Gyr sire (40.3%). Crossing Gyr dams with Holstein sires resulted in a pregnancy rate of 38.9% and did not differ (p = 0.58) from the pregnancy rate obtained with the cross between Holstein dams and Gyr sires. The rate obtained with Holstein dam and sire was 32.5%. The average pregnancy rate was 36.6%, and no difference was found in the proportion of female foetuses (88.8%, in average) among breeds (p > 0.05). In conclusion, transfer of cryopreserved X‐sorted embryos represents an interesting choice for dairy cattle. Despite the small differences between pregnancy rates, we highlight the efficiency of this strategy for all of the racial groups studied.     

供体细胞对猪体细胞克隆胚胎早期发育的影响

以中国农业大学实验用小型猪香猪胎儿成纤维细胞、成年耳成纤维细胞和颗粒细胞3种细胞系为供体细胞进行核移植。比较了血清饥饿法和接触抑制法处理胎儿成纤维细胞诱导进入G0／G1期的效率，发现二者差异不显著（P〉0．05），血清饥饿2d和4d差异不明显，同样接触抑制2d和4d差异也不显著（P〉0．05）。系统研究了影响克隆胚胎发育的供体因素：血清饥饿与否、细胞形态、细胞类型及个体差异等，结果表明：血清饥饿处理对克隆胚的早期发育没有明显的促进作用；圆形光滑细胞有利于细胞融合，对早期发育无显著影响（P〉0．05）；不同个体、不同类型的供体细胞对克隆胚囊胚发育率有一定的影响。     

体细胞克隆猪的研究与展望

用体细胞核移植的方法生产的克隆猪诞生至今已近5年，虽然同胚胎细胞核移植相比，体细胞核移植技术难度大，成功率低，但近年来用体细胞核移植技术克隆猪有了更深入的研究，在体细胞核移植基本机理和关键技术上取得了一定的进展。综述了用核移植的方法生产体细胞克隆猪的技术关键和难点及应用情况，并对提高猪体细胞核移植效率的策略进行了分析。     

Cellular Composition and Viability of Cloned Bovine Embryos Using Exogene-Transfected Somatic Cells

The present study compared the efficiency of transgenic (TG) cloned embryo production by somatic cell nuclear transfer (SCNT) with fetal-derived fibroblast cells (FFCs) which were transfected with pEGFP-N1 to in vitro-fertilized (IVF), parthenogenetic and SCNT counterparts by evaluating the rates of cleavage and blastocyst formation, apoptosis rate at different developmental stages, cell number, ploidy and gene expression in blastocysts. In SCNT and TG embryos, the rates of cleavage and blastocyst formation were significantly lower (p < 0.05) than those of IVF controls, but it did not differ between SCNT and TG embryos. In IVF control, 86.7% embryos displayed diploid chromosomal complements and the rates were significantly (p < 0.05) higher than those of SCNT and TG embryos. Most TG embryos (79%) with FFCs expressed the gene by both PCR and under fluorescence microscopy. The expression of apoptosis by TUNEL was first detected at six to eight cell stages in all embryos of IVF, SCNT and TG groups, but the expression rate at each developmental stages was significantly higher (p < 0.05) in SCNT and TG embryos than in IVF counterparts. The expression rate in inner cell mass (ICM) of TG embryos was significantly higher (p < 0.05) than in SCNT and IVF embryos. These results indicate that the high occurrence of apoptosis observed in SCNT and TG embryos compared with IVF counterparts might influence the developmental competence. Moreover, the SCNT embryos derived using non-transfected donor cells exhibited a lower apoptosis expression in ICM cells than in TG embryos derived using pEGP-N1-transfected donor cells suggesting a possible role of negative gene effect in TG embryos.     

Effect of Meloxicam on Pregnancy Rate of Recipient Heifers Following Transfer of In Vitro Produced Embryos

The main objective of this study was to determine if administration of meloxicam, a cyclooxygenase (COX) two inhibitor, to heifers in which embryo transfer (ET) is more difficult and requires a greater manipulation of the tract, would be beneficial. Nulliparous recipient heifers were divided in two groups: CON (n = 102), in which animals received 10 ml of saline IM (the same volume of meloxicam) and MEL (n = 105) animals that were treated with meloxicam. According to the degree in passing the catheter, recipients from both groups were classified as Grade I, easy (< 60 s), and Grade II (more than 80 s), difficult. Immediately after embryo transfer, MEL recipients received an injection of 200 mg of meloxicam (10 ml).There was no difference in the pregnancy rates on Day 35 considering animals which presented Grade I cervix independently whether the treatment was performed or not (p = 0.22). There was a statistical difference in the pregnancy rates (p < 0.01) between both groups (49.0% and 66.7% for CON and MEL, respectively) when cervical grade was not considered, on Day 35. Considering the animals that presented Grade II cervix, the pregnancy rate was higher for MEL (21.15% and 78.84%, respectively) in both examinations (p < 0.01).The authors concluded that meloxicam had a positive influence on general pregnancy rate of treated heifers in comparison to non‐treated heifers. It was also observed that pregnancy rate was not influenced by meloxicam administration in Grade I heifers. Treatment increased the pregnancy rate of Grade II heifers.     

布尔山羊胚胎移植技术的研究与应(简报)

1999年10～11月份,分3批使用阴道栓+FSH超数排卵供体布尔山羊13只.在放入阴道栓的第8～10 d,连续3d递减量肌肉注射FSH(澳大利亚)320 mg.9只供体羊发情、配种、采胚(有效率69.23%).平均采胚数18.11±5.18枚,其中可用胚平均数15.44±6.31枚(可用胚率85.28%).将139枚7日龄可用胚移植受体关中奶山羊89只,妊娠50只,妊娠率56.18%.其中鲜胚移植妊娠率61.11%(44/72),冻胚移植妊娠率41.67%(5/12),二分割胚移妊娠率20%(1/5).50只妊娠受体羊共产羔68只,每只供体羊平均获羔羊7.56只.供体羊采胚后,平均39.9 d发情,配种,全部妊娠产羔,产羔率200%.胚胎移植羔羊性别、初生重、发病率和布尔山羊自繁羔羊无显著差异,P＞O.05.本次布尔山羊移植产生明显的经济效益,技术成熟,可推广应用,逐步产业化.