Permeability of mouse oocytes and embryos at various developmental stages to five cryoprotectants

To assess the permeability of mouse oocytes and embryos, matured oocytes and embryos at various stages of development were placed in five cryoprotectant solutions at 25 C for 25 min. From the cross-sectional areas of the oocytes/embryos, the relative change in volume was analyzed. In oocytes, shrinkage was least extensive and recovery was quickest in the propylene glycol solution, showing that propylene glycol permeates the oocytes most rapidly. Dimethyl sulfoxide, acetamide, and ethylene glycol permeated the oocytes slightly more slowly than propylene glycol. The oocytes in glycerol shrunk extensively and then expanded marginally, indicating slow permeation. The volume changes of 1-cell and 2-cell embryos were similar to those of oocytes, showing little change in permeability. In 8-cell embryos, the volume recovered much faster than in the earlier stages especially in glycerol and acetamide. In morulae, the volume recovery was much faster in glycerol and in ethylene glycol; in ethylene glycol, the extent of shrinkage was small and the recovery was fast, indicating an extremely rapid permeation. Although the permeability of oocytes/embryos generally increased as embryo development proceeded, the degree of increase varied greatly among the cryoprotectants. Interestingly, the volume change in propylene glycol was virtually unaffected by the stage of development. Such information will be valuable for determining a suitable protocol for the cryopreservation of oocytes/embryos at different stages of development.     

Equilibrium vitrification of mouse embryos at various developmental stages using low concentrations of cryoprotectants

We previously developed a new vitrification method (equilibrium vitrification) by which two-cell mouse embryos can be vitrified in liquid nitrogen in a highly dehydrated/concentrated state using low concentrations of cryoprotectants. In the present study, we examined whether this method is effective for mouse embryos at multiple developmental stages. Four-cell embryos, eight-cell embryos, morulae, and blastocysts were vitrified with EDFS10/10a, 10% (v/v) ethylene glycol and 10% (v/v) DMSO in FSa solution. The FSa solution was PB1 medium containing 30% (w/v) Ficoll PM-70 plus 0.5 M sucrose. The state of dehydration/concentration was assessed by examining the survival of vitrified embryos after storage at –80°C. When four-cell embryos and eight-cell embryos were vitrified with EDFS10/10a in liquid nitrogen and then stored at –80°C, the survival rate was high, even after 28 days, with relatively high developmental ability. On the other hand, the survival of morulae and blastocysts vitrified in liquid nitrogen and stored at –80°C for four days was low. Therefore, morulae and blastocysts cannot be vitrified in a highly dehydrated/concentrated state using the same method as with two-cell embryos. However, when blastocysts were shrunken artificially before vitrification, survival was high after storage at –80°C for four days with high developmental ability. In conclusion, the equilibrium vitrification method using low concentrations of cryoprotectants, which is effective for two-cell mouse embryos, is also useful for embryos at multiple stages. This method enables the convenient transportation of vitrified embryos using dry ice.     

Survival and developmental competence of bovine embryos at different developmental stages and separated blastomeres after vitrification in different solutions

Generating techniques to enhance the success of blastomere separation is important for bovine economy, because it increases the number of transferable embryos. This study aimed to identify the optimum cryoprotectants for the vitrification of bovine embryos and the separation of blastomeres at different stages. In experiment 1, expanded blastocysts were vitrified in two different vitrification solutions, either (1) ethylene glycol (EG) + propylene glycol (PG) or (2) EG. The survival rate of blastocysts in the EG + PG was higher than that of the EG. In experiment 2, intact two‐cell and eight‐cell stage embryos were vitrified in the same solutions used in experiment 1. The EG + PG produced more dead embryos than the EG (P < 0.05). In the EG, the rate of blastocyst formation was similar for the vitrified two‐ and eight‐cell embryos and the non‐vitrified ywo‐cell embryos. In experiment 3, separated blastomeres of two‐ and eight‐cell embryos were vitrified in EG. There was no difference in the rate of blastocyst formation and total number of cells between the two vitrified groups. In summary, at the blastocyst stage, EG + PG was superior, based on both survival rates and cell numbers; however, at the 2–8 cell stage, the use of EG alone was better than the other groups.     

Wistar大鼠超数排卵及采集不同时期胚胎的初步研究

为了研究获取Wistar大鼠不同发育阶段胚胎所需超排试剂的最佳剂量以及不同发育期胚胎的采集方法,试验采用10 IU/只、20 IU/只和30 IU/只3种剂量的孕马血清促性腺激素-人绒毛膜促性腺激素(PMSG-HCG)对8～9周龄的Wistar大鼠进行超排处理。结果表明:采集2-细胞期胚胎时,分别获得胚胎(12.1±4.6)枚、(23.1±10.2)枚和(24.9±8.2)枚,其中激素剂量为30 IU/只时采集2-细胞期胚胎超排效果最好;在采集桑葚胚期胚胎时,分别可获得胚胎(10.3±5.0)枚、(23.7±17.2)枚和(17.2±5.9)枚,其中激素剂量为20 IU/只的超排效果最好。     

Transfer of pig embryos to different uterine sites.

Embryo transfer in pigs normally involves surgery. In connection with the development of nonsurgical or endoscopic transfer techniques, it is important to know whether the uterine site to which embryos are transferred has an effect on the success rate. In the present investigation, prepubertal donor gilts were treated with 1,500 IU of PMSG and, 72 h later, with 500 IU of hCG. Gilts were artificially inseminated 24 and 36 h after hCG injection. Embryos at the expanded blastocyst stage were collected from donor gilts. Recipient gilts were treated synchronous with the donors, using 1,000 IU of PMSG followed, 72 h later, with 500 IU of hCG. After a maximum of 3 h in vitro, embryos (n = 15 to 20, mean = 17.3) were transferred surgically to the middle of the uterine horn, to the caudal quarter of the uterine horn, or to the uterine body. Recipients were slaughtered between 28 and 34 d after transfer. The pregnancy rate of the recipients was low when the embryos were deposited in the uterine body (12%), compared with the middle (88%) or the caudal quarter of the uterine horn (81%) (P < .01). The corresponding average number of viable fetuses per pregnant recipient was 8.2 in the uterine body, 5.6 in the middle, and 4.5 in the caudal quarter. Average survival rate of embryos after transfer to the middle of the uterine horn was 41% vs 29 and 3% after transfer to the caudal quarter or the uterine body, respectively (P < .01). Hence, the uterine body seems to be an unsuitable site for embryo transfer in pigs. These results may explain the unsatisfactory results achieved with nonsurgical embryo transfer in the past.     

Open-pulled straw (OPS) vitrification of in vitro fertilised mouse embryos at various stages

The present study was designed to investigate the cryotolerance of in vitro fertilised (IVF) mouse embryos at various preimplantation developmental stages. IVF mouse embryos were vitrified by the open-pulled straw (OPS) method. After warming, embryos were morphologically evaluated and assessed by their development to blastocysts, hatched blastocysts or term. The results showed that a high proportion (93.3-100.0%) of vitrified embryos at all developmental stages were morphologically normal after recovery. The developmental rate of vitrified 1-cell embryos to blastocyst (40.0%) or hatched blastocyst (32.7%) or term (9.3%) was significantly lower than that from other stages (P < 0.05). Vitrified embryos from 2-cell to early blastocyst stage showed similar blastocyst (71.8-89.5%) and hatched blastocyst rates (61.1-69.6%) and could develop to term without a significant loss of survival compared with those of fresh embryos (P > 0.05). Vitrified 2-cell embryos showed the highest survival rate in vivo (50.6%, 88/174), compared with that from other stages (9.3-30.5%, P < 0.05). The data demonstrate that the OPS method is suitable for the cryopreservation of IVF mouse embryos from 2-cell stage to early blastocyst stage without a significant loss of survival. Embryos at the 2-cell stage had the best tolerance for cryopreservation in the present study.     

Ultrastructural characteristics of canine transmissible venereal tumor at various stages of growth and regression.

Ultrastructural study of canine transmissible tumors in developing, mature, and regressing stages from 6 dogs revealed the presence of healthy and degenerating tumor cells in all neoplasms. The total number of neoplastic cells seemed to decrease, and the number of degenerating neoplastic cells seemed to increase in mature tumors. Macrophages, lymphocytes, and plasma cells infiltrated mature and regressing tumors. Alteratons in degenerating tumor cells consisted mainly of cytoplasmic changes in early stages and of both nuclear and cytoplasmic changes in cells in which degeneration was more advanced. Amounts of endoplasmic reticulum and ribosomes were decreased. There were swelling and vacuolation of mitochondria. Nuclear chromatin was clumped along the nuclear envelope, and the perinuclear space was widened. Degenerating cells often contained membrane-bound granules and clusters. Lamellar complexes were observed in tumor cells from 2 dogs. Virus particles were not seen.     

Immunoglobulin G and E responses in various stages of canine leishmaniosis

Pathogenesis in visceral leishmaniosis is associated with depressed cellular immunity and a significant rise of antileishmanial antibodies. We assessed the relative levels of immunoglobulin E anti-Leishmania infantum, together with those of IgG, IgG1 and IgG2, using the enzyme-linked immunosorbent assay (ELISA) test, in non-infected and infected dogs with or without symptoms, and their association with symptoms to differentiate the stages of the infection. The expression of all immunoglobulins (IgG, IgG1, IgG2 and IgE) was higher in symptomatic dogs than in all other categories. IgG and IgG2 expression was higher in the infected asymptomatic group than in the non-infected group, whereas IgG1 and IgE expression was only higher in symptomatic animals. This correlation between the expression of IgG1 and IgE and the pathology of leishmaniosis points to their potential role as markers of the active disease.     

不同发育时期旋毛虫脂肪酸组成的研究

为探讨旋毛虫生长发育过程中脂肪酸组分的变化及外界环境对其影响,本研究采用贝尔曼氏装置分别收集旋毛虫肌幼虫和成虫,同时,将少量骨骼肌和肠黏膜进行酯化处理,通过气相色谱质谱联用技术进行分析。结果显示:肌幼虫和成虫的脂肪酸组分涵盖了C12～C22的22种,主要为16、18和20碳脂肪酸,但旋毛虫肌幼虫与成虫的各脂肪酸相对含量有明显差异(p0.05)。分别对旋毛虫肌幼虫和成虫及其各自寄生部位骨骼肌和肠黏膜中的脂肪酸成份进行比较分析,结果显示:各脂肪酸组成相似,但在相对含量上却有显著差异(p0.05)。表明不同发育时期旋毛虫的脂肪酸组成在种类和相对含量上均存在明显差异,这可能与其所处的不同宿主环境和不同发育时期虫体的特殊生理结构有关。     

Uptake of metronidazole in Artemia at different developmental life stages

The purpose of this study was to examine the capacity of live brine shrimp Artemia spp. to accumulate metronidazole at different developmental life stages. Metronidazole is used in fish as an antiparasitic medication. An effective drug delivery method is to enrich the Artemia with metronidazole and offer them as live feed to the infected fish, usually ornamental species and other small fishes. Artemia cysts were hatched and then soaked in a metronidazole solution (0.05%) at instars 1-3 of larval development. Our findings indicated that Artemia were able to accumulate metronidazole at levels considered therapeutic to other animals and humans (25-100 mg/kg). However, the levels varied depending on the stage of larval development. Artemia accumulated the highest levels of metronidazole (137-143 mg/kg) when they started filter feeding (instar 2), whereas newly hatched Artemia (instar 1) contained the lowest level (85 mg/kg). Based on this study and a review of the literature, a new protocol recommended for enriching Artemia with metronidazole consists of soaking the Artemia in a 0.05% metronidazole solution for 3 h at room temperature. Because metronidazole is relatively insoluble in water, it must first be dissolved in warm water with continuous stirring.     

不同发育时期澳洲坚果果皮抗氧化物质成分分析

以云南省永德县大雪山乡澳洲坚果种植基地广泛种植的优良品种O.C.为研究材料,利用超高效液相色谱-串联质谱（UPLC-MS/MS）的技术方法,对该品种花后30天、60天和90天的果皮进行代谢组学分析,探究不同发育时期澳洲坚果果皮酚酸类物质组分及含量的变化,为果皮中酚酸类抗氧化物质的开发和利用提供参考依据。结果表明：不同发育时期果皮中的酚酸类物质组分及含量有较大差异,在检测到的114个代谢物中,绿原酸 (3-O-咖啡酰奎宁酸)、1-O-阿魏酰-D-葡萄糖和1-O-对香豆酰-β-D-葡萄糖等物质的含量在花后30天最高,表明发育前期需要大量能量物质作为生长代谢原料;熊果苷、6''-咖啡酰熊果苷、红景天苷、龙胆酸、阿魏酸、3,4-二没食子酰莽草酸、没食子苯乙酮、原儿茶酸、水杨酸以及豆腐果苷等多种物质在花后60天及90天含量较高;6''-对香豆酰熊果苷和3,4-二甲氧基肉桂酸为花后90天标志性物质。结论： 本文分析测试了澳洲坚果果实不同发育期果皮中酚酸类物质的种类及含量变化,发现果皮中含有美白、抗菌等活性成分,具有一定的药用价值,为今后澳洲坚果早期落果及采后果皮加工利用、保健品及药物开发等奠定了很好的基础。     

黄花棘豆不同生长时期苦马豆素含量的分析

采用气相色谱法对黄花棘豆(Oxytropis ochrocephala)不同生育期的根、茎、叶、花、果实及地上部分全草中主要有毒成分苦马豆素的含量进行检测,分析苦马豆素的变化规律,调查并计算单位面积黄花棘豆地上全草中苦马豆素的产量。结果表明,黄花棘豆果实中苦马豆素含量最高,达到107.787 mg·kg-1;地上部分全草中苦马豆素含量在结果期最高,达到48.19 mg·kg-1。从4个生长时期黄花棘豆不同部位苦马豆素含量平均值来看,果实＞花＞叶＞茎＞根;从地上部分全草中苦马豆素含量来看,结实期＞盛花期＞枯萎期＞孕蕾期,样地中黄花棘豆地上部分全草苦马豆素产量也符合此规律。     

输卵管和颗粒细胞单层对牛体外受精胚胎发育的影响

以屠宰场牛卵巢为试验材料,研究输卵管细胞单层(OCM)和颗粒细胞单层(GCM)对牛卵母细胞体外成熟(IVM)、体外受精(IVF)和体外培养(IVC)后胚胎发育能力的影响。(1)从卵泡抽取卵丘卵母细胞复合体(COCs),并根据卵母细胞外面卵丘细胞的层数将其分为3类:1级(≥4层);2级(2～3层);3级(0～1层)。作分别在IVM和IVC培养液中添加GCM(1×106个/mL)与不添加的对比试验。结果显示:添加GCM对1级卵母细胞的卵裂率、6～8细胞发育率和囊胚率无明显影响(P>0.05);但添加GCM的2级、3级卵母细胞,受精后的卵裂率、6～8细胞发育率和囊胚率分别高于未添加组(P<0.05)。(2)所有卵母细胞(包括COCs和裸卵)被随机分为3个组,在其IVM和IVC培养液中分别添加OCM、GCM或不添加体细胞(对照组)。结果显示:OCM和GCM组的卵裂率、6～8细胞发育率和囊胚率均高于对照组(P<0.05),而两试验组之间差异不显著。     

不同生长期金荞麦营养成分含量及消化率测定研究

为系统地测定分析不同生长期金荞麦的常规营养成分、氨基酸、微量元素含量及其回肠末端消化率, 本研究用3头安装“T”型瘘管, 并做回-直肠吻合切除盲肠的巴克夏-驯养野猪-高坡黑猪(巴-野-高)三元杂交生长猪作为试验动物, 采用3×3拉丁方试验设计开展消化试验。结果表明, 分枝期、孕蕾期和初花期金荞麦OM、CP、EE、Ca、P、CF含量分别为88.59%、22.72%、2.34%、1.05%、0.39%、13.51%, 89.10%、20.57%、1.69%、1.25%、0.42%、15.50%和89.63%、17.54%、1.37%、1.29%、0.46%、19.75%;EAA、NEAA和TAA含量分别为9285、7982、6244 mg/100 g, 14334、10810、9320 mg/100 g和23619、18792、15564 mg/100 g。随着生长期的后延, 营养成分含量显著降低, 在体和离体消化率也随着生长期的后延而显著降低。分枝期与孕蕾期间营养成分含量差异显著(P<0.05), 分枝期与初花期间营养成分含量差异极显著(P<0.01), 孕蕾期与初花期间营养成分含量差异显著(P<0.05)。从分枝期到孕蕾期, 金荞麦CP、EE、EAA及TAA下降较慢, CF含量升高较慢, 营养成分消化率下降也较慢;从孕蕾期到初花期, 金荞麦CP、EE、EAA及TAA下降较快, CF含量升高也较快, 营养成分消化率下降也较快。以分枝期、孕蕾期和初花期体外法测定常规营养成分及微量元素消化率值为自变量, 以对应的动物饲养试验回肠末端表观消化率值为因变量建立的回归方程拟合度好,可用于推算对应的回肠末端表观消化率。本实验证明, 金荞麦营养丰富、消化率高, 必需氨基酸比例高、适合动物消化吸收。孕蕾期为金荞麦适宜收割期, 在适宜收割期内, 收割时间点应选择孕蕾后期。     

Development of uterine chick embryos after storage at 5 degrees C for 15 hours

Eggs obtained from the uterus of hens by hormonal administration 14 to 10 h before expected oviposition were kept at 5 degrees C for 15 h. Forty-two of these uterine eggs were incubated as whole eggs; in the case of a further 70 eggs the blastoderms were isolated and incubated whilst immersed in liquid egg albumen. Thirty-two of the eggs incubated whole (76%) developed to the primitive streak stage, but only 6 of the incubated blastoderms (9%) reached this stage of development.