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1、胚胎在体外的维持演示文稿第一页，共三十八页。（优选）胚胎在体外的维持第二页，共三十八页。动物繁殖学双语教学nTo date, no chemically defined medium has been formulatedformulated阐明阐明to support normal development of bovine embryos satisfactorily for longer than about 24 hours.第三页，共三十八页。动物繁殖学双语教学 However, several media are commercially available (or can be
2、prepared easily) that adequately maintain embryos for the usual interval between collection and transfer. For longer periods, cryopreservationcryopreservation低温贮藏低温贮藏 is recommended.第四页，共三十八页。动物繁殖学双语教学nSTORAGE CONDITIONSnFor short periods of culture, the nutrient properties of the medium are far les
3、s critical than pH, osmolality, temperature, sterility, and lack of toxicity.第五页，共三十八页。动物繁殖学双语教学 An embryos range of tolerance for these properties is narrow. InadequateInadequate不充分的不充分的control of these aspects of culture accounts for many failed transfers. In addition: embryos are damaged if expos
4、ed to excess light (e.g. microscope lights) for prolonged periods, but normal lighting or daylight in the work area is not harmful if embryos are not exposed for more than half an hour. 第六页，共三十八页。动物繁殖学双语教学 Body temperature of cattle is 39, and for certain purposes this temperalure is appropriate. Ho
5、wever, for routine routine 程序程序commercial embryo transfer we do not recommend storage above 37 because thermometers thermometers 温度计温度计, even those on incubatorsincubators孵卵器孵卵器, are frequently incorrect by 1 or 2. Culturing embryos at 41 is extremely damaging, whereas storing them for up to 12 hour
6、s between 18 and 37is not.第七页，共三十八页。动物繁殖学双语教学nMEDIAnThe simplest medium is sterile saline (9 gm NaCl/litre of sterilized, deionizeddeionized脱离子脱离子or distilled water).第八页，共三十八页。动物繁殖学双语教学nSaline will not support embryonic development, but will keep embryos for a few hours; it is better to use saline a
7、nd transfer embryos quickly than to use a more complex medium without a proper buffering缓冲缓冲(作用作用) system.第九页，共三十八页。动物繁殖学双语教学 Moreover, saline can be steam-sterilized (at least 30 minutes at 121 under a pressure of 104 kilopascals千克力千克力, ) if a 0.22-gm biological filtration system is not available.
8、A serious problem with saline, however, is that embryos will floatfloat漂浮漂浮or stick to plastic or glass unless a macromoleculemacromolecule高分子高分子such as bovine serum albumin (BSA) or serum is added, both of which require membrane filtration for sterilization. BSA and serum are sterile when purchased
9、. 第十页，共三十八页。TABLE 3 Recommended culture conditiongs第十一页，共三十八页。第十二页，共三十八页。动物繁殖学双语教学n For most applications, we recommend a modified Dulbeccos phosphatebuffered saline (PBS). It is easy to use because it does not have to be equilibrated and maintained in an atmosphere of 5 percent CO2 in air. 第十三页，共三十
10、八页。动物繁殖学双语教学nIt can be prepared from stock reagents (Table 4) or purchased in either ready-to-use or concentrated form, which must be diluted with sterile, deionizeddeionized脱离子剂脱离子剂or distilled water.第十四页，共三十八页。动物繁殖学双语教学 A frequent error is failure to dilute concentrated medium; embryos contract ma
11、rkedly when exposed to the high osmolality同渗重摩同渗重摩. Commercially available Dulbeccos PBS does not usually contain antibiotics, sodium pyruvate丙酮酸盐丙酮酸盐(或酯或酯), glucose or macromolecules高分子高分子. 第十五页，共三十八页。动物繁殖学双语教学 The least important ingredient因素因素is the Na pyruvate丙酮酸钠丙酮酸钠; neither Na pyruvate nor or
12、ganisms glucose is needed for most applications. The CaC12 and MgSO4 can also be eliminated排排除除if embryos are to be kept in vitro在生物体外在生物体外only a short time. If these are omitted漏检漏检, osmolality同渗重摩同渗重摩should be adjusted by adding extra NaC1.第十六页，共三十八页。动物繁殖学双语教学 Other media used to culture bovine em
13、bryos include Tissue Culture Medium- 199 (TCM-199), Hams F-10 medium, and Brinsters Mouse Ova Culture Medium-3 (BMOC-3). All of these are commercially available. TCM-199 with Hanks salts does not depend on CO2 for buffering, but TCM-199 with Eafies salts as well as Hams F-10 and BMOC-3 must be maint
14、ained under an atmosphere of 5 percent CO2 in air.第十七页，共三十八页。动物繁殖学双语教学nMACROMOLECULAR SUPPLEMENTSnSupplementation of media with a large protein molecule decreases surface tension tension 张力的张力的, which reduces the tendency of the embryo to float float 漂浮漂浮or adhere to plastic or glass, and helps to i
15、nactivateinactivate阻阻止活动止活动 heavy metals and other toxins.第十八页，共三十八页。动物繁殖学双语教学nHowever, macromolecules derived from serum are also a possible vectorvector带菌者带菌者 for viral infection of embryos. BSA and bovine serum are currently recommended sources of macromolecules. PolyvinylPolyvinyl乙烯聚合物乙烯聚合物 alco
16、hol and polyvinyl pyrrolidonepyrrolidone吡咯啉吡咯啉 are being studied as possible non-biological macromolecules to circumvent the danger of infection.第十九页，共三十八页。动物繁殖学双语教学These macromolecules do not function as well as BSA in reducing surface tension or chelatingchelating螯合螯合 toxins. BSA (Fraction V) should be added to medium just before use at a concentration of 0.05-0.1 percent for flushing and 0.4 percent for culture. 第二十页，共三十八页。动物繁殖学双语教学 The powder should be poured very gentlygently逐渐地逐渐地 on the s
