第六章胚胎在体外的维持.ppt

1、动物繁殖学双语教学English-Chinish of Reproductiong in Farm Animal,Trainingmanuai for embryo transfer in cattle,第六章 胚胎在体外的维持,动物繁殖学双语教学,Maintaining维持 embryos in vitroChapter 6 Embryos derive得自nutrients from the fluid in which they are bathed; moreover, of perhaps more importance, embryos depend on the ambient周

2、围环境 fluid to maintain their physiological integrity完整性.,动物繁殖学双语教学,To date, no chemically defined medium has been formulated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

3、easily) that adequately maintain embryos for the usual interval between collection and transfer. For longer periods, cryopreservation低温贮藏 is recommended.,动物繁殖学双语教学,STORAGE CONDITIONS For short periods of culture, the nutrient properties of the medium are far less critical than pH, osmolality, temper

4、ature, sterility, and lack of toxicity.,动物繁殖学双语教学,An embryos range of tolerance for these properties is narrow. Inadequate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 per

5、iods, 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 程序commercial embryo transfer we do not recommend sto

6、rage above 37 because thermometers 温度计, even those on incubators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,MEDIA The simplest medium is sterile saline (9 gm NaCl/litre of sterilize

7、d, deionized脱离子or distilled water).,动物繁殖学双语教学,Saline 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(作用) system.,动物繁殖学双语教学,Moreover, saline can be ste

8、am-sterilized (at least 30 minutes at 121 under a pressure of 104 kilopascals千克力, ) if a 0.22-gm biological filtration system is not available. A serious problem with saline, however, is that embryos will float漂浮or stick to plastic or glass unless a macromolecule高分子such as bovine serum albumin (BSA)

9、 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,For most applications, we recommend a modified Dulbeccos phosphatebuffered saline (PBS). It is easy to use because it does not

10、have to be equilibrated and maintained in an atmosphere of 5 percent CO2 in air.,动物繁殖学双语教学,It can be prepared from stock reagents (Table 4) or purchased in either ready-to-use or concentrated form, which must be diluted with sterile, deionized脱离子剂or distilled water.,动物繁殖学双语教学,A frequent error is fai

11、lure to dilute concentrated medium; embryos contract markedly when exposed to the high osmolality同渗重摩. Commercially available Dulbeccos PBS does not usually contain antibiotics, sodium pyruvate丙酮酸盐(或酯), glucose or macromolecules高分子.,动物繁殖学双语教学,The least important ingredient因素is the Na pyruvate丙酮酸钠; n

12、either Na pyruvate nor organisms glucose is needed for most applications. The CaC12 and MgSO4 can also be eliminated排除if embryos are to be kept in vitro在生物体外only a short time. If these are omitted漏检, osmolality同渗重摩should be adjusted by adding extra NaC1.,动物繁殖学双语教学,Other media used to culture bovine

13、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 mai

14、ntained under an atmosphere of 5 percent CO2 in air.,动物繁殖学双语教学,MACROMOLECULAR SUPPLEMENTS Supplementation of media with a large protein molecule decreases surface tension 张力的, which reduces the tendency of the embryo to float 漂浮or adhere to plastic or glass, and helps to inactivate阻止活动 heavy metals

15、and other toxins.,动物繁殖学双语教学,However, macromolecules derived from serum are also a possible vector带菌者 for viral infection of embryos. BSA and bovine serum are currently recommended sources of macromolecules. Polyvinyl乙烯聚合物 alcohol and polyvinyl pyrrolidone吡咯啉 are being studied as possible non-biologi

16、cal 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 chelating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 fo

17、r culture.,动物繁殖学双语教学,The powder should be poured very gently逐渐地 on the surface of the medium and be allowed to dissolve 溶解 for about 20 minutes without stirring活跃的 or shaking 摇动, (otherwise it tums 小丘into a glutinous blob粘性水滴的 or makes the medium unusably frothy空洞的).,动物繁殖学双语教学,After the BSA has diss

18、olved, 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 wate

19、r.,动物繁殖学双语教学,If serum is used, steer向.前进 or calf blood should be harvested into sterile, sealable containers directly from the vein (to avoid contaminants from skin and hair). After coagulation 凝结物, clots 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,C

20、lots are then filtered out and the serum centrifuged at 2 000 X g for 12-15 minutes. The sediment沉淀物 is discarded可废弃的 and the supernatant浮在表面的 recentrifuged换发. This step is repeated once again. The final supematant is sterilized by passage through a 0.22-g bacteriological filter into sterile contain

21、ers of convenient size.,动物繁殖学双语教学,In order to inactivate, 阻止活动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 conta

22、iners 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 spec

23、ific-pathogen-free herds, additional sterilization of serum by gamma微克irradiation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 sto

24、rage.,动物繁殖学双语教学,CONTAINERS容器 Embryos should be stored in small (5 ml), sterile, transparent透明的, sealable密封性能, inert惰性的container.,动物繁殖学双语教学,Small test tubes, Petri dishes 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 mov

25、ed any distance before loading装填into straws.,动物繁殖学双语教学,If 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 paraffin石蜡oil to prevent evaporation and contamination and to regulate the rate of gas exchange

26、 between the medium and the surrounding atmosphere.,动物繁殖学双语教学,Paraffin oil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,Except while they are being manipu

27、lated, 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,PIPETTES吸移管 Many devices装置, have been used to manipulate embryos. Standard Pasteur pipettes are much too large and the t

28、ips require fire polishing to prevent damage to embryos.,动物繁殖学双语教学,We prefer to make our own pipettes from Pyrex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 bumer本生灯and pulled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