BOVINE EMBRYO IN VITRO CULTURE

Abstract: A culture method and system has been realized for in vitro development of bovine embryos from early stage 1- to 2-cell embryos to morula or blastocyst stage. The embryos are placed in a culture system of a buffered balanced salt solution with a carbohydrate energy source medium and bovine oviductal epithelial cells in suspension. Embryo development is enhanced by the addition of bovine or ovine oviductal fluid as well as non-essential amino acids to the system. The method and system can be used to culture genetically manipulated embryos used in transgenic procedures and nuclear transfer.

Description: BOVINE EMBRYO IN VITRO CULTURE Baci:cround of the Invention The in vitro culture of embryos of domestic animals has been studied and is of interest particularly with new techniques of genetic manipulation of early stage embryos. In vitro culture involves removing an egg from the animal' reproductive tract. Fertllization can occur before cr aft±: removal. Later stage embryos are also cultured In vItro which have been removed from the animal at a time after fertilization. The removed embryos are then placed in a culture medium for further development. A number of domestic and laboratory animal embryos have been used for in vitro culture experiments. Wright and Bondioli, "Aspects of In Vitro Fertilization and Embryo Culture in Domestic Animals," J. Anim. Sci., vol. 53, No. 3, pp.702-729 (1981). The embryos have been removed at very early stages, 1- to 2-cell, and later uterine stages, around 32-cell, and cultured in vitro. Generally a high proportion of the later stage embryos from domestic and laboratory animals will develop in vitro to a blastocyst stage and many till hatch from the zona pellucid. In bovine in vitro culture a "blocking" phenomenon has been observed at the 8- to 16-cell stage when culturing from 1-cell embryos. ante, "Culture Media and Culture of Early Embryos, Theriosenology, vol. 27, No. 1, pp. :.9-57 (1987); Eyestone and First, "A Study of the 8- to 16-Cell Developmental Block in Bovine Embryos Cultured In itro," Therioaenoloay, vol. 25, No. 1, p. 152 (1986). The bovine embryos generally fail to develop past the 8- to 16-cell stage to the blastocyst stage when cultured in tissue culture media wit various supplements. In one study 2- and 8-cell stage embryos were cultured in a various media. The 8-cell embryos were successfully cultured to blastocyst stage in vitro in selected medium, however none of the 2-cell embryos underwent more than one complete cell division in vitro. right et awl., "Successful Culture In Vitro cf Bovine Embryos to the Blastocyst Stage, Biol. ReDrod., vol. 14, pp.157-162 (1976). In another study, three of twenty-two i- to 2-cell stage bovine embryos cultured in medium developed into blastocysts, but this work has not been reportedly reproduced. Wight et al., "Blastocyst Expansion and Hatching of Bovine Ova Culture In vitro," J. Anim. Sci., vol. 43, p.170 (1976). The conventional method of culturing bovine embryos which hare been removed at the 1- te 2-cell stage is to transfer the embryo to a sheep oviduct for in vivo culture which overcomes the blocking observed in vitro. The embryos are later flushed from the sheep oviduct and transferred to recipient cow. The conventional method of bovine embryo culture involves the additional steps of culturing for a period of time in sheep, removal from sheep and re-introduction into the recipient cow. During the course 0 the transfers, there can be damage to or loss of the embryos. Another alternative for culturing bovine embryos is 2 co-culture system which includes the use ot a layer of cells cultured from embrvonic or adult tissue with a culture medium. The co-culture technique has promoted successful development for early stage embryos. However, the success rate for development to blastocyst or morula is higher when culturing embryos older than 1- to 2-cell. Camous et al, "Cleavage Beyond the Block Stage and Survival After Transfer of Early Bovine Embryos Cultured With Trophoblastic Vesicles", J. Repro. Fert., vol. 72, pp. 479-485 (1984) . In some cases, co-culture systems have been used only with bovine embryos at the 4-cell and older stages. Eyestone et al, "Co-Culture of Early Bovine Embryos With Oviductal Epithelium", Therioaenolocy, vol. 27, No. 1, p. 228 (1987). Summary of the Invention The present technique and components realize a system which cultures in vitro a substantial percentage of early stage bovine embryos to morula and blastocyst stage. The system uses a culture medium of buffered balanced salt solution with a carbohydrate energy source and bovine oviductal epithelial cells in a suspension. The early stage, 1- to 2-cell embryos are collected and placed in a drop of the medium with about 5% to 15% volume of cells in the fluid medium. The culture system drop is covered under a film such as silicone or paraffin oil. The suspension system is incubated at about 38 CC. in a 5 CO and 95% air humidified atmosphere. The cells develop to blastocyst stage in about seven days. At this point the embryos can be transferred to a recipient cow by non-surgical means. The culture medium can be supplemented with amino acids, growth factors and vitamins. The addition of non-essential amino acids increases the development rate of the embryos. The culture system may be supplemented with oviductal fluid from bovine or ovine. The oviductal fluid is mixed with the culture medium. A ':1 ratio of culture medium and oviductal fluid with the bovine oviductal epithelial cells system produces a substantial percentage of early stage embryos developing into blastocysts. The system of this invention can be used for bovine embryos and eggs that are subject to genetic research. In the nuclear transplantation studies, the genetic material from a selected cell is transferred with an egg that has been subjected to an enucleation procedure. The egg with the new genetic material is fused and cultured. Using the system of this invention, the culture process can be in vitro and the embryos can be observed during the critical early stages. For the nuclear transfer procedures, a system with demonstrated success for culturing 1-cell throuch blastocyst is 2 necessity. After the embryos are cultured, they may be transferred to recipient cows as desired. Similarly for research involving gene transfer, an in vitro system which is successful with early stage embryos is needed. In gene transfer work, foreign DNA is inserted into the nuclear material of preferably l-cell embryos. A culturing method is then used to grow the embryo to determine whether a transgenic embryo develops with the foreign gene incorporated and expressed. An in vitro technicue would allow the investigator to observe which embryos were developing and make further determination of the progress of culture. Description of the Preferred Methods and EmbodIment The following is a description of the preferred method and alternatives for culturing in vitro bovine embryos. The procedures have been used to culture early stage embryos from 1- and 2-cell until development to the morula or blastocyst stage. The techniques were utilized with early stage embryos used for transgenic research, the introduction of foreign DNA with the bovine embryo, and nuclear transfer research wherein nuclear donor material was transferred to an enucleated egg. The method was also used to develop bovine embryos which were not subject to any genetic manipulatIon. The method is successful for in vitro culture of manip- ulated and non-manipulated bovine embryos from the 1-cell stage to past the 8- to 16-cell stage. The preferred method and culture system utilizes a buffered balanced salt solution with a carbohydrate energy source medium. The balanced salt solution includes inorganic salts maintained at a physiological pH by a bicarbonate -CO2 buffer system. The carbohydrate energy source can include glucose, lacate, pyruvate or acetate. A medium of choice was Brinsters Mouse ova culture medium modified by the addition of sodium acetate and supplemented with bovine serum albumin. The medium was also supplemented with essential amino acids, non-essential amino acids and a combination of essential and non-essential amino acids. The components of the medium with non-essential amino acids included are listed in Table 1. Table 1 PREFERRED CULTURE MEDIUM FOR IN VITRO CULTURE OF BOVINE EMBRYOS Grams/Liter Unless Component Otherwise Noted NaCl 5.546 KC1 .356 CaCl2H2O .250 KH2PO4 .162 MgSO4.7H2O .294 NaHC03 2.106 Glucose 1.000 Na Lactate 3.6 ml of 60% syrup Na Pyruvate .056 EDTA .034 Na Acetate .050 Phenol Red 1 ml of .05t solution L-Alanine* 8.9 mg/l =-Asparagine H20* 15.0 mg/1 L-Aspartic Acid* 13.30 mg/l L-Glutamic Acid* 14.70 mg/l Glycine* 7.5 mg/1 L-Proline* 11.50 mg/l L-Serine* 10.50 mg/1 bovine serum albumin 6.0 mg/l penicillin 10,000 units/l strep-tomycin 10,000 mcg/1 *Non-essential amino acids. The basic culture medium was the components listed in Table 1 less the non-essential amino acids. The preferred method utilizes bovine oviductal epithelial cells in the culture system which are recovered from oviductal flushings when early stage embryos are recovered. The cells chosen are predominantly ciliated epithelial cells. The oviductal ciliated epithelial cells are washed ounce in phosphate buffered saline by allowing the clumps of cells to settle in a tube. The cells were then watched twice in culture media by pipetting. The cells were generally used the same d of collection. The cells have been fro=en and stored in liquid nitrogen and used successfully after storage. The culture system can be comprised of a mixture of the buffered balanced salt with a carbohydrate energy source medium and the cells. A 25 ul drop of medium will have about 5E to 15% by volume cf cells or about 2 to 4 s of cells added to the drops in a suspension-type system In the preferred method oviductal fluid collected from a cow or sheep is part of the culture system along wIth the medium and cells. In some cases a superovulated cow which had occulted oviducts produced a large amount fluid which had accumulated. ; superovulated animal was listed surgically on one or both ends of the oviduct and fluid was recovered. The bovine oviductal fluid (BOF) was recovered in a sterile manner. Any cellular debris was removed by centrifugation. The BOF can be frozen until utilized. Sheep oviductal fluid has been used with some success. In the preferred method and preferred culture sstem a 1:1 mixture of medium described in Table 1 and BOF is used. The amount -of BOF can be decreased since medium alone can be used A culture drop of 25 > 1 of the mixture with 2 ul of loosely packed cells added make up a drop of the culture system. The embryos were placed in a drop of the culture system. Preferably 1 to 10 embryos are contained in one drop. The embryos were washed once in a drop of the culture mixture before being placed in the culture drop. The culture system drop is covered with a film of paraffin, silicone oil or other inert covering. The culture system with embryos is kept at about 38"C under a 5% CO2 and 95% air humidified system. Larger volume culture drops of 1 ml or higher can be used. With a larger culture drop the oil covering can be omitted if desired. The oil covering is used to prevent evaporation of medium from a small volume. The culture is maintained for seven days during which time the normally developing embryos will be at the morula cr blastocyst stage. The embryos can be transferred non surgically to recipient cows for full development. The development to blastocysts, approximately 60 to 100 cells, exceeds the previously reported "blocking" in bovines at the 8- to 16-cell stage. This procedure has been used to culture embryos that have not been the subject of any manIpulation to determine the successful development from l-cell to blastocyst stage. Separate experiments were run with combinations of medium (as shown in Table 1 with and without the non-essential amino acids listed therein), BOF and cells in the culture system. In these experiments, it is not possible to determine at a 1-cell stage whether fertilization has been successful so that failure of development due to lack of fertilization can occur. The following Table 2 shows the results of the in vItro method and various culture systems. The development was termnated after seven days of culture to record the cellular count. Table 2 CULTURE SYSTEM RESULTS Medium Medium, Medium, Medium, Onlv Cells BOF BOF, Cells Number of Embryos 35 35 35 35 Percentage Developing After 7 Days 3% 43% 17% 608 The results show the development to the morula or blastocyst stage was greatest in the mixture of medium, BOF, and cells with 605 of the embryos developing. A system o medium and the cells showed a 43E development to morula cr blastocyst stage. The culture medium was varied to include combinations of essential and non-essential amino acids, vitamins and other additives as described below. A culture medium with the basic components of Table 1 (components of Table 1 less the non-essential amino acids) plus all of the essential and non-essential amino acids listed in Table 3 below was prepared. Table 3 COMBINED ESSEttTIAL AND NON-ESSENTIAL AMINO ACIDS Amino Acid m/1 of medium L-alanine g.o L-arginine HC1 211.0 L-asparaglne-H20 15.01 L-aspartic acid 13.0 L-cysteine 25.0 L-glutamic acid 14.7 L-glutamine 146.0 Glycine 7.51 L-histidine HC1-H20 23.0 L-isoleucine 2.6 L-leucine 13.0 L-lysine HC1 29.0 L-methionine 4.e8 L-phenyl alanine 5.0 L-proline 11.5 L-serine 10.5 L-threonine 3.57 L-tryptophan .6 L- rosine 1.81 L-valine 3.5 The basic culture medium was also supplemented with vitamins and other additives as shown In Table 4. Table 4 MEDIUM SUPPLEMENTS VITAMINS i OTHER ADDITIVES mo/1 Hypoxanthine 4.o Lipoic acid .2 Thymidine .7 Biotin .024 D-Ca: Pantothenate .715 Choline chloride .698 Folic acid 1.320 Inositol .541 Niacinamide .615 Pyridoxine-HCl .206 Riboflavin .376 Thiamine 1.0 Vitamin B12 1.36 Hypoxanthine, lipoic acid and thymidine are not generally considered vitamins but were included in this mixture referred to as a vitamin mixture. Hypoxanthine, a nucleotide precursor; thymidine, a nucleotide and lipcic acid, a coenzyme were also added. Systems using various culture media were tested with oviductal cells in suspension but without BOF. The media used were: (1) basic medium without any amino acids, (2) basic medium with vitamins (Table 4) , (3) basic medium with all amino acids in Table 3, and (4) basic medium with vitamins (Table 4) and all amino acids in Table 3. The results are reported in Table 5 showing the number of embryos developing to the morula or blastocyst stage. Table 5 (1) (2) (3) (4) No Amino Medium + Acids Medium + Medium + Vitamins t Medium Vitamins Amino Acids Amino Acids Number of Embryos 31 35 3 32 Number Developing 12 13 24 20 Percentage Developing 39% 37% 735 63% The results indicated that the development was improve when the medium is supplemented with amino acids. Additional culture systems were prepared using media supplemented with various amino acid mixtures and cells. The basic medium has the components listed in Table 1 less the non-essential amino acids. The various media tested were the basic medium of Table 1 plus essential and non-essential amino acids of Table 3. A medium was prepared with the basic medium of Table 1 and the essential amino acids used in MEM medium as shown in Table 6 below. Table 6 MEM ESSENTIAL AMINO ACIDS Amino Acids mg/l L-arginine HC1 126.40 L-cystine 24.0 L-histidir.e HC1-H20 42.00 L-isoleucine 2 52.50 L-leucine 52.40 L-lysir.e HC1 72.50 L-mezhionine 15.10 L-phenylalanine 33.00 L-threonine 47.60 L-tryptophan 10.20 L-tyrcsine 36.00 L-valine 46.80 The culture medium cf Table 1 includes the non-essential amino acids which are used in MEM tissue culture medium. Another medium was prepared combining the MEM essential and non-essential amino acids of Tables 1 and 6 with the other basic medium components of Table 1. The culturing method as described herein was followed using the various media and oviductal cells in the suspension system. Table 7 records the results of culture in vitro to morula or blastocyst stage for bovine embryos. Table 7 CULTURE RESULTS AMINO ACID SUPPLEMENTS Basic Basic Medium Medium Basic + MEM + All Medium + Essential ME.w Amino Amino Acids Medium Amino Acids Acics Table 3 Table i Table 6 Tables l;ó Number of Embryo 26 27 27 25 Number Developing 10 1t9 9 11 Percentage Developing 39E 70% 33% 44 The medium of Table 1 with the non-essential amino acids of MEM medium exhibited the best results. The in vitro development technique has been used for bovine embryos that have been subject to transgenic proce- dures. In these cases the pronucleus of a presumably fertilized embryo was injected with various foreign DNAs to determine if the foreign gene could be incorporated and expressed. The injection of foreign DNA can be lethal to the embryo. Also, not all embryos are fertilized. The injected embryos were cultured according to the preferred method in a system including medium of Table 1 in a 1:1 ratio with BOF and about 2 ul of cells in a suspension. About 25% of the embryos developed to morula or blastocysts. Upon transfer to recipient cows by non-surgical methods, about 30E of these embryos resulted in pregnancies.

Claims: WHAT IS CLAIMED IS: 1. A method to culture bovine embryos in vitro comprising steps of a) collecting early stage embryos; b) placing the early stage embryos in a suspension culture system of buffered balanced salt solution with a carbohydrate energy source medium and boine oviductal epithelial cells; c) holding the suspension system at about 38"C and about 5 CO2 and 95% air humidified atmosphere; and d) culturing the embryos until morula or blastocyst stage. 2. A method to culture bovine embryos in vitro of claim 1 wherein 1n step (a) the examining of the early stage embryos and selecting 1- to 2-cell embryos for culturing. 3. A method to culture bovine embryos of claim 1 including the step of transferring the cultured embryo to a recipient cow. 4. A method to culture bovine embryos of claim 1 including the step of covering the suspension system under a ilm after step (b). 5. A method to culture bovine embryos in vitro comprising the steps of a) collecting early stage embryos; b) placing the embryos in a mixture of bovine or ovine oviductal fluid, buffered balanced salt solution with a carbohydrate enter source medium and bovine oviductal epithelial cells in a suspension system; c) holding the suspension system at about 3E C and about 5 CO2 and 95% air humidified atmosphere; and d) culturing the embryos until morula or blastocyst stage. C. A method to culture bovine embryos in vitro of Claim 5 including the step of transferring the cultured embryo to a recipient cow. 7. A method to culture bovine embryos of Claim 5 including the step of covering the suspension system under a film after step (b) 8. A method to culture bovine embryos in vitro comprising the steps of a) collecting early stage embryos; b) placing the embryos in a mixture of bovine or ovine oviductal fluid, buffered balanced salt solution with a carbohydrate energy source medium supplemented with amino acids and bovine oviductal epithelial cells in a suspension system; c) holding the suspension sstem at about 38"C and about 5% C02 and 95 air humidified atmosphere; and culturing the embryos until morula or blastocyst stage. 9. An in vitro culture system for bovine embryos comprisIng a mixture of a buffered balanced salt solution with a carbohydrate energy source and bovine oviductal epithelial cells in a suspension system. 10. An in vitro culture system of bovine embryos of claim 9 whereIn said buffered balanced salt solution with carbohydrate energy source is suppleiiiented with amino acids. 11. An in vitro culture system for bovine embryos comprising a mixture of a buffered balanced salt solution with a carbohydrate energy source with oviductal fluid selected from the group of bovine or ovine and oviductal epithelial cells in 2 suspension system. 12. An in vitro culture system for bovine embryos of claim 11 wherein the buffered balanced salt solution with a carbohydrate energy source medium and oviductal fluid is in a 1:1 mixture. 13. An in vitro culture system for bovine embryos of claim 11 wherein the oviductal epithelial cells are about 5% to 15 volume of the fluid medina. 14. An in vitro culture system for bovine embryos of claim 11 wherein the buffered balanced salt solution with a carbohydrate energy source is supplemented with amino acids. 15. An in vitro culture system for bovine embryos of claim 11 wherein said buffered balanced salt solution with carbohydrate energy source is supplemented with amino acids.

Inventors: BONDIOLI KENNETH R, BONDIOLI, KENNETH, R.
Applicants: GRANADA GENETICS INC, GRANADA GENETICS, INC.
Application year: 1988
Main classification: C12N5
IPC codes: A01K67, C12N5