Prolonging hypothermic storage (4 C) of bovine embryos with fish antifreeze protein

Embryos obtained via superovulation are necessary for mammalian artificial reproduction, and viability is a key determinant of success. Nonfreezing storage at 4 C is possible, but currently used storage solutions can maintain embryo viability for only 24–48 h. Here we found that 10 mg/ml antifreeze protein (AFP) dissolved in culture medium 199 with 20% (v/v) fetal bovine serum and 25 mM HEPES could keep bovine embryos alive for 10 days at 4 C. We used a recombinant AFP isolated from the notched-fin eelpout (Zoarces elongatus Kner). Photomicroscopy indicated that the AFP–embryo interaction was enhanced at 37 C. Embryos pre-warmed with the AFP solution at 37 C for 60 min maintained high viability, whereas those that were not pre-warmed could live no longer than 7 days. Thus, short-term storage of bovine embryos was achieved by a combination of AFP-containing medium and controlled pre-warming.     

Nuclear transfer using a bovine mammary epithelial cell line (BMEC)

The developmental potential of nuclei from a bovine mammary epithelial cell line (BMEC) in nuclear transfer was investigated. For nuclear transfer donors, BMEC cells (passage 15) were cultured for 4–5 days after seeding at cell densities of 1.0 × 10⁵ cells/cm² (high‐density group) or 0.8 × 10⁴ cells/cm² (low‐density group). First, the effective electric stimulation for fusion of enucleated oocytes with BMEC cells was examined. Fusion rates reached maximum with two DC pulses of 30 V/150 µm for 20 µs in the high‐density group and with two DC pulses of 25 V/150 µm for 10 µs in the low‐density group. The fusion rate (37.5%) in the high‐density group was significantly (P < 0.005) lower than in the low‐density group (71.4%). Second, the in vitro developmental potential of nuclear transfer embryos derived from BMEC cells was examined. In the high‐density and low‐density groups, 18.8% and 24.1% of fused oocytes, respectively, developed to blastocyst stage. The results of this study indicate that nuclei from BMEC cells support the development of nuclear transfer embryos to the blastocyst stage and that the efficiency of oocyte–cell fusion is affected by the culture conditions of the donor BEMC cells before nuclear transfer.     

过氧化氢对小鼠骨髓微核率和孕鼠胚胎着床及生长的影响

通过体内试验研究了过氧化氢对小鼠骨髓微核率和胚胎着床及孕鼠生长发育的影响。将妊娠的昆明种(KM)小鼠分成5个处理组,分别为环磷酰胺(40.00 mg/kg)、蒸馏水和过氧化氢(500.00,125.00,31.25 mg/kg)组。于妊娠后6~15 d,每天采用灌胃给药,环磷酰胺组于妊娠后11~13 d腹腔一次性注射。每3 d称重1次,于妊娠第18 d称重后剖腹检查。另取雌雄各半KM小鼠分成5个处理组,分别为环磷酰胺、蒸馏水和过氧化氢(500.00,100.00,10.00 mg/kg)组,均采用灌胃染毒,每天1次,连续4 d,第5 d处死小鼠,取股骨髓液常规制片,计算红细胞微核率。结果表明,吸收胎和死胎数蒸馏水组为5.56%,中剂量过氧化氢组增加到19.53%(P<0.01),低剂量组增加到13.14%(P<0.05)。蒸馏水组小鼠嗜多染红细胞微核率为0%~0.002%,过氧化氢组增加到0.017%~0.032%(P<0.01)。在本试验剂量范围内过氧化氢有明显的胚胎毒性和遗传毒性。     

Recurrent Temporary Paralysis Reported After Human Rabies Post‐Exposure Prophylaxis

Adverse events can occur after rabies post‐exposure prophylaxis (PEP), and linkage to causality is often difficult to determine. We report a case of recurrent temporary paralysis that began immediately after the initiation of rabies PEP in a man exposed to a bat. The recurrent temporary paralysis first occurred in the patient after his initial dose and then again after day 3 of his rabies PEP. The PEP was terminated prior to a serologic response. The patient continued to experience numerous discrete episodes of temporary paralysis for over two years.     

调节胚胎期代谢提高蛋鸡抗热应激能力

应用维生素E、维生素C及普鲁卡因组合的生理调节剂对蛋鸡胚胎期进行调控，试验分为对照组和3个不同剂量组合的试验组，即试验1组（VE＋VC）、试验2组（VE＋普鲁卡因）、试验3组（VE＋VC＋普鲁卡因），研究不同剂量及组合的胚胎调节剂对出壳后母鸡在热应激条件下生产性能、血液指标和卵泡的变化。试验结果显示：试验1组应激前后平均蛋重分别提高7．50％和9．82％（P〈0．01）；热应激期产蛋率提高5．81％（P〈0．05）。试验2组应激前后产蛋率分别提高了7．45％和9．69％（P〈0．01）。试验1、2、3组总产蛋量在应激前后均有提高，尤其试验1组和2组（P〈0．01）。常温期试验1组胰高血糖素和试验3组T4水平分别提高55．52％和10．03％（P〈0．01）；试验1、2、3组热应激期T4水平分别提高28．65％、132．74％和72．79％（P〈0．05）或（P〈0．01）。试验2组热应激期白蛋白水平和常温期葡萄糖水平分别提高27．36％和31．16％（P〈0．05）；试验3组常温期葡萄糖水平提高25．15％（P〈0．05）。试验2组异嗜性粒细胞和H／L的比值下降，单核细胞数上升（P〈0．05）。试验2组卵泡中大白泡和小白泡的数量显著增多（P〈0．05）。以上结果表明，补充适量的Ve、Vc和普鲁卡因能提高蛋鸡抗热应激能力，改善产蛋性能。     

分子修饰前后人参皂苷体外抗马立克氏病毒鸡胚成纤维细胞模型的建立

用雏鸡将马立克氏病毒(MDV)血毒复壮,分离发病鸡淋巴细胞并接种于鸡胚成纤维细胞,观察其病变。获得适应鸡胚成纤维细胞(CEF)的MD强毒,通过电镜观察、琼脂扩散实验进一步鉴定病毒,并建立了MDV感染CEF细胞模型,为研究人参皂苷及其衍生物的体外抗病毒作用及其机制奠定基础。     

非人灵长类动物体外受精与胚胎移植的研究进展

简要介绍了非人灵长类动物体外受精与胚胎移植的内容、方法和步骤,影响其体外受精的主要因素,以及对未来非人灵长类动物的研究方向。     

松南结缕草成熟胚愈伤组织的诱导和再生

松南结缕草成熟种子经灭菌和打破休眠后,接种于含有不同浓度2,4-D的MS和N6培养基上进行愈伤组织诱导。结果表明,结缕草种子的休眠主要在于破除种壳。2,4-D浓度在2~5 mg/L对愈伤组织诱导率无明显影响。基本培养基MS和N6诱导效果相近,初生愈伤组织为白色、半透明,质地松软、水浸状。继代培养基中添加一定浓度的甘露醇、麦芽糖和ABA以及延长继代时间有助于愈伤组织的胚性化。基本培养基MS更适于结缕草初生愈伤组织致密胚性化。胚性愈伤组织在MS添加0.1 mg/L 2,4-D培养基上即可分化出大量具有根和茎叶的再生植株。     

无血清培养基IVD101和G1/G2在牛体细胞核移植中的应用研究

通过考察牛体细胞核移植重构胚在无血清培养基（IVD101、G1／G2）条件下培养的囊胚发育率及其质量,从而评估无血清培养基支持牛体细胞核移植重构胚体外发育的能力。采用IVD101和G1／G2对牛体细胞核移植胚胎进行体外培养,并以CR1aa＋5％FBS作为对照组。无血清培养基IVD101和G1／G2的囊胚发育率与对照组无显著性差异（41．2％&#177;9．1％、42．2％&#177;10．8％,48．0％&#177;9．2％,P〉0．05）。通过囊胚差异染色和冷冻／解冻胚胎存活率分析胚胎质量,发现无血清培养基ICM／Total略低于对照组（31．8％&#177;10．5％、29．5％&#177;11．9％vs ．33．0％&#177;14．8％）,但无显著性差异（P〉0．05）;3个组别的囊胚经程序化冷冻／解冻后无血清培养基的存活率（IVD101、G1／G2）略高于对照组,但差异不显著（84．8％、80．4％＇US．77．3％,P〉0．05）。结果证明无血清培养基（IVD101、G1／G2）可以支持体细胞核移植重构胚的体外发育,且其对程序化冷冻的耐受性与添加血清组（CR1aa＋5％FBS）相似。     

山羊胚胎分割及同卵双生试验

选择山羊晚期桑椹胚、囊胚、孵出囊胚和孵出增大胚泡,用简化分割法二分。将19对裸半胚移植于18只受体羊,结果有12只妊娠,其中两只胚胎消失,两只流产,其余8只足月分娩,共产半胚羔11只。晚期桑椹胚、囊胚、孵出囊胚和孵出增大胚泡各组的半胚发育为羔羊的发育率分别为12.5％(1/8)、20％(2/10)、25％(3/12)和62.5％(5/8)。前三组均未获得同卵双生羔羊。在第四组,将4对裸半胚移植于4只受体,有3只妊娠,足月分娩半胚羔5只,其中两对为同卵双生。本研究证明,对称分割山羊孵出增大胚泡,不仅其半胚在体内仍可继续发育形成正常胎儿,而且不装透明带移植其裸半胚,仍能获得较高的同卵双生率。山羊孵出增大胚泡更适宜用简化分割法分割。