动物繁殖学双语教学动物繁殖学双语教学English-Chinish of Reproductiong in Farm Animal Trainingmanuai for embryo transfer in cattle n第六章 胚胎在体外的维持动物繁殖学双语教学nMaintainingMaintaining维持维持 embryos in vitroChapter 6nEmbryos derivederive得自得自nutrients from the fluid in which they are bathed; moreover, of perhaps more importance, embryos depend on the ambientambient周围环周围环境境 fluid to maintain their physiological integrityintegrity完整性完整性.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.动物繁殖学双语教学 However, several media are commercially available (or can be 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.动物繁殖学双语教学nSTORAGE CONDITIONSnFor short periods of culture, the nutrient properties of the medium are far less critical than pH, osmolality, temperature, sterility, and lack of toxicity.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 exposed 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. 动物繁殖学双语教学 Body temperature of cattle is 39, and for certain purposes this temperalure is appropriate. However, 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 hours between 18 and 37is not.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).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 and transfer embryos quickly than to use a more complex medium without a proper buffering缓冲缓冲(作用作用) system.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. 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. TABLE 3 Recommended culture conditiongs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. 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脱离脱离子剂子剂or distilled water.动物繁殖学双语教学 A frequent error is failure to dilute concentrated medium; embryos contract markedly 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(或或酯酯), glucose or macromolecules高分子高分子. 动物繁殖学双语教学 The least important ingredient因因素素is the Na pyruvate丙酮酸钠丙酮酸钠; neither Na pyruvate nor organisms glucose is needed for most applications. The CaC12 and MgSO4 can also be eliminated排除排除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同同渗重摩渗重摩should be adjusted by adding extra NaC1.动物繁殖学双语教学 Other media used to culture bovine embryos 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 maintained under an atmosphere of 5 percent CO2 in air.动物繁殖学双语教学nMACROMOLECULAR SUPPLEMENTSnSupplementation of media with a large protein molecule decreases surface tensiontension 张张力的力的, which reduces the tendency of the embryo to float float 漂浮漂浮or adhere to plastic or glass, and helps to inactivateinactivate阻止活动阻止活动 heavy metals and other toxins.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乙烯乙烯聚合物聚合物 alcohol and polyvinyl pyrrolidonepyrrolidone吡咯啉吡咯啉 are being studied as possible non-biological macromolecules to circumvent the danger of infection.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. 动物繁殖学双语教学 The powder should be poured very gentlygently逐渐地逐渐地 on the surface of the medium and be allowed to dissolve dissolve 溶溶解解 for about 20 minutes without stirringstirring活跃的活跃的 or shaking shaking 摇动摇动, (otherwise it tumstums 小丘小丘into a glutinous glutinous blobblob粘性水滴的粘性水滴的 or makes the medium unusably frothyfrothy空洞的空洞的). 动物繁殖学双语教学 After the BSA has dissolved, the container should be inverted gently five or six times to mix in the BSA completely. The medium should then be sterilized by passage through a 0.22-g bacteriological filter. Purer types of BSA are also acceptable. BSA can also be purchased as an aqueous solution, e.g. 7 percent BSA in water.动物繁殖学双语教学n If serum is used, steersteer向向.前进前进 or calf blood should be harvested into sterile, sealable containers directly from the vein (to avoid contaminants from skin and hair). After coagulationcoagulation 凝凝结物结物, clots 血（凝血（凝）are cut every 2-3 cm with a sterile knife, and the blood stored overnight at 5. 动物繁殖学双语教学Clots are then filtered out and the serum centrifuged at 2 000 X g for 12-15 minutes. The sedimentsediment沉淀物沉淀物 is discardeddiscarded可可废弃的废弃的 and the supernatant浮在表面的浮在表面的 recentrifuged换发换发. This step is repeated once again. The final supematant is sterilized by passage through a 0.22-g bacteriological filter into sterile containers of convenient size. 动物繁殖学双语教学nIn order to inactivate, 阻止阻止活动活动the protein complement, which can be toxic to embryos, serum must be treated in a 56 water bath for exactly 30 minutes after the serum has reached 56.动物繁殖学双语教学 Serum can be frozen and stored for up to eight months provided that the containers are tightly sealed. For quality control, one aliquot from each batch of serum should be incubated overnight in a sterile, sealed container at 37and examined the next day for bacterial contamination. 动物繁殖学双语教学 For some situations, such as international movement of embryos or maintenance of specific-pathogen-free herds, additional sterilization of serum by gamma微微克克irradiation放射放射 is recommended (Manual of the International Embryo Transfer Society, 1987). Heat-inactivated, sterilized serum is added to medium instead of BSA at a concentration of 0.5-1 percent for flushing and 10 percent for storage.动物繁殖学双语教学nCONTAINERS容器容器nEmbryos should be stored in small (5 ml), sterile, transparenttransparent透透明的明的, sealablesealable密封性能密封性能, inertinert惰性的惰性的container.动物繁殖学双语教学nSmall test tubes, Petri Petri dishes dishes 陪替氏培养皿陪替氏培养皿or multi-well plates are convenient for routine use (Figure 13), but small test tubes are recommended if embryos must be moved any distance before loadingloading装填装填into straws.动物繁殖学双语教学nIf the embryos are to be stored in uncovered containers/or more than 20 minutes, however, the medium should be covered with a thin layer of non-toxic paraffinparaffin石蜡石蜡oil to prevent evaporation and contamination and to regulate the rate of gas exchange between the medium and the surrounding atmosphere.动物繁殖学双语教学Paraffin oil石蜡石蜡can also be used with other types of containers and is recommended if sealable containers are not used. FIGURE 13Left: Plastic multi-well plate with 16 x 17-mm wells (lid to left)动物繁殖学双语教学n Except while they are being manipulated, embryos should be stored in a dark, dust-free incubator or cabinet (e.g. a refrigerator or an ice chest with a tightly-fitting lid set at room temperature or 37).动物繁殖学双语教学nPIPETTES吸移管吸移管n Many devices装置装置, have been used to manipulate embryos. Standard Pasteur pipettes are much too large and the tips require fire polishing to prevent damage to embryos. 动物繁殖学双语教学nWe prefer to make our own pipettes from PyrexPyrex耐热玻璃耐热玻璃glass tubing with a 4-mm outside diameter. Glass, in 15-cm lengths, is heated in the centre with a Bunsen Bunsen bumerbumer本生灯本生灯and pulled拉拉to make an outside diameter of less than 1 mm.动物繁殖学双语教学This is then scored with a diamond pencil and broken to make two pipettes. All ends are fire polished. After pipettes have been washed and rinsed 冲洗器冲洗器thoroughly, they are placed in clean glass test tubes with screw转动转动, tops and sterilized (and dried) by dry heat. 动物繁殖学双语教学nFor use, pipettes are connected to a 0.5- or 1-cc syringe洗涤器洗涤器or plastic mouth piece接合接合with rubber tubing (see Figure 32). Pipettes are made in batches of several hundred.动物繁殖学双语教学nThey can be reused if debris碎片碎片does not adhere粘附粘附and they are placed in a soapy water bath immediately after use. In practice, we discard most of them after one use.动物繁殖学双语教学 Al tematives羟基安定羟基安定to making pipettes are to use tomcat catheters, 0.25-cc French straws (which, however, are too large for good washing procedures), disposable可可任意使用的任意使用的 micropipettes, or pipette tips of various sorts. Care must be taken to wash, sterilize, and rinse 冲洗冲洗these devices with sterile medium prior to use (see Chapters 15 and 18).动物繁殖学双语教学