Method for freezing bovine embryos
BACKGROUND OF THE INVENTION 
     The present invention relates to a method for freezing bovine embryos. More particularly, the present invention relates to a method for freezing bovine embryos obtained by in vivo or in vitro fertilization, or bovine embryos subjected to biopsy for gene diagnosis or chromosome analysis. 
     DESCRIPTION OF THE PRIOR ART 
     In a step-wise method conventionally employed for freezing bovine embryos, frozen embryos are thawed and then a cryoprotectant is removed under a microscope. This method requires not only a fairly long time and much labor after the thawing but also a microscope. Therefore, a more convenient method is desired in working sites. The conception rate in the aforesaid method is 50 to 60%. Furthermore, when this method is applied to embryos freed from zona pellucida by biopsy, the conception rate is decreased to 30 to 40% (Bondioli et al., Theriogenology, 31, 95-104, 1989; Nibart et al., 12th ICAR, Abstract No. 212, 727-729, 1992). 
     There have come to be carried out gene examinations, for example, sexing of embryos by a PCR method which is now expected to be most practical. For investigating the genes of embryos, biopsy of the embryos is indispensable. A large hole is formed in the zona pellucida by the biopsy, so that the zona pellucida is completely or substantially removed. When the embryos thus lose the zona pellucida, their cryo-preservation becomes difficult in practice. 
     In addition, there is a method (a step-wise method in which a dextran is added in an amount of 10%) which gives such a good result that the conception rate of embryos subjected to biopsy for sexing (embryos freed from zona pellucida) is 63.0% (17/27) (Herr &amp; Reed, Theriogenology, 35, 45-54, 1991). But, the present inventor examined this method to find that this method tends to give different results, depending on developmental stage. Moreover, this method is not a direct method and hence cannot be widely generalized. Accordingly, the present invention was made. 
     SUMMARY OF THE INVENTION 
     The present invention is a method for freezing bovine embryos in which a dextran and supplemented calf serum are added to bovine embryos. According to this method, a fairly high conception rate can be attained even in transplantation of embryos subjected to biopsy. Furthermore, this method is a simple method which permits direct transfer into recipients after thawing. Needless to say, a higher conception rate can be attained in the case of embryos not subjected to biopsy. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     FIG. 1 shows a cross-sectional view of a straw enclosing an embryo according to the present invention. The main body of the straw (A), cotton plug (B), solution to be frozen (C), air (D), solution to be frozen and embryo (E), and seal (F) are shown in the FIGURE. 
    
    
     DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     The present invention is further illustrated with the following examples. 
     EXAMPLE 1 
     (1) Solution to be frozen: 
     The composition of a solution to be frozen which was directly transferable into recipients and was prepared by adding supplemented calf serum (or a material obtained by adding the same growth factors and trace elements as described below to bovine fetal serum or the like) and a dextran to a cryoprotectant, was as follows: 
     4% (v/v) ethylene glycol 
     4% (v/v) propelene glycol 
     20% (v/v) supplemented calf serum * 
     1% (v/V) dextran ** 
     base medium: DPBS (Dulbecco&#39;s phosphate buffer solution 
    
    
     (2) Container for freezing: 
     a 0.25-ml plastic straw 
     (3) Method for insertion into the straw: 
     As shown in FIG. 1, the following were inserted so as to be located in the numerical order from the cotton plug end in the straw. 
     1. the solution to be frozen 
     2. air 
     3. the solution to be frozen and embryos 
     4. air 
     5. the solution to be frozen 
     (4) Program freezer: manufactured by Tokyo Rika 
     (5) Ice seeding temperature: -6° C. 
     (6) Cooling rate: 0.5° C./min 
     (7) Temperature at the immersion in liquid nitrogen: -32.5° C. 
     (8) Thawing method: 
     The straw was taken out of liquid nitrogen, held in air (at room temperature) for 5 to 10 seconds, and then thawed in warm water at 35° C. 
     (9) Method for recovering and cultivating embryos in a cultivation test: 
     The whole contents of the straw were introduced in DPBS+0.2M sucrose+10% calf serum and the mixture was allowed to stand for 5 minutes. Thereafter, the embryos were cultured for 48 hours together with bovine granulosa cells by using TCM 199 medium+5% supplemented calf serum. 
     (10) Transplantation method in a transplantation test: 
     The straw was set in a transplanter as it was, followed by transplantation. 
     
                       TABLE 1                                                     
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Freezing and thawing method                                               
(Comparison with a conventional method)                                   
Method     Freezing solution                                              
                            Thawing method                                
______________________________________                                    
Conventional                                                              
           10% Glycerol + 0.4%                                            
                            3 steps (6.7%,                                
method     BSA              3.3%, 0%,                                     
(SW method*)                Glycerol + 0.3M                               
                            sucrose + 0.4%                                
                            BSA)                                          
Method of the                                                             
           4% Ethylene glycol +                                           
                            Direct trans-                                 
invention  4% propylene glycol +                                          
                            plantation                                    
           20% supplemented                                               
           calf serum + 1%                                                
           dextran                                                        
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 *step-wise method as prior art                                           
 
    
     
                       TABLE 2-1                                                   
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Freezing test on bovine embryos obtained by                               
in vitro fertilization (cultivation results)                              
               2 Hours           48 hours                                 
Freezing       after thawing     after thawing                            
and            Number of   Number of                                      
                                   Number of                              
thawing        surviving   transferable                                   
                                   hatched                                
method   n     embryos     embryos embryos                                
______________________________________                                    
SW method                                                                 
         7      7 (100%)   0 (0.0%)                                       
                                   3 (42.9%)                              
Method   12    12 (100%)   2 (16.7%)                                      
                                   6 (50.0%)                              
of the                                                                    
invention                                                                 
______________________________________                                    
 
    
     
                       TABLE 2-2                                                   
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Freezing test on bovine embryos freed                                     
from zona pellucida (cultivation results)                                 
               2 Hours after thawing                                      
Freezing and         Number of Number of                                  
thawing              surviving transferable                               
method     n         embryos   embryos                                    
______________________________________                                    
SW method  20         5 (25.5%)                                           
                               0 (0.0%)                                   
Method of  11        10 (90.9%)                                           
                               5 (45.5%)                                  
the invention                                                             
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                       TABLE 3                                                     
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Freezing test on bovine embryos freed from                                
zona pellucida (transplantation results)                                  
Freezing and         Number of                                            
thawing method n     implanted embryos                                    
______________________________________                                    
SW method      24    2 (8.3%)                                             
Method of the  20    13 (61.9%)                                           
invention                                                                 
______________________________________                                    
 *The implantation of embryos was confirmed by ultrasonic diagnosis 60 day
 after the estrus of recipients (the most usual number of days at which   
 implantation is confirmed).                                              
 
    
     As is clear from the above, as compared with the conventional method, the method of the present invention gave better results in all of the cultivation tests on embryos obtained by in vitro fertilization and embryos freed from zona pellucida and the transplantation test on the embryos freed from zona pellucida (Tables 1 to 3). Particularly in the case of the transplantation results shown in Table 3, there could be obtained a conception rate of 61.9% (13/20) which is equal to the usual conception rate of frozen bovine embryos and is the best result for embryos subjected to biopsy. 
     It can be conjectured that dextrans accelerate the dehydration of cells and that since they increase the viscosity of a solution, they are effective in protecting the cell surface of, in particular, embryos which have lost their zona pellucidas. The above-mentioned method of Herr et al. uses a characteristic solution to be frozen which has a very high viscosity because it contains a dextran in an amount of as much as 10%. Thus, it can be speculated that owing to such a solution to be frozen, different results are obtained depending on developmental stage (bad results are obtained in the case of enlarged blastocysts having an enlarged blastcoel). In addition, the concentration of 10% gives too high a viscosity, so that sterilization by filtration becomes difficult. Even when the concentration was 5%, it was necessary to change the poresize and the filtration area. 
     EXAMPLE 2 
     For investigating the optimum concentration of dextran, a test was carried out according to the methods described in Example 1. As can be seen from the results shown below, a desirable tendency was observed in the growth of the 1% group. 
     
                       TABLE 4-1                                                   
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Freezing test on bovine embryos                                           
obtained by in vitro fertilization                                        
         2 Hours         48 Hours                                         
         after thawing   after thawing                                    
Dextrran       Number of  Number of                                       
                                   Number of                              
concentration  surviving  transferable                                    
                                   hatched                                
(v/v)    n     embryos    embryos  embryos                                
______________________________________                                    
0%       21    18 (85.7%) 3 (14.3%)                                       
                                   4 (19.0%)                              
 1%*     21    17 (81.0%) 3 (14.3%)                                       
                                   9 (42.9%)                              
5%       14     9 (64.2%) 0 (0.0%) 3 (21.4%)                              
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 *This group corresponds to the present invention.                        
 
    
     
                       TABLE 4-2                                                   
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Freezing test on bovine embryos                                           
freed from zona pellucida                                                 
         2 Hours         48 Hours                                         
         after thawing   after thawing                                    
Dextran        Number of  Number of                                       
                                   Number of                              
concentration  surviving  transferable                                    
                                   growing                                
(v/v)    n     embryos    embryos  embryos                                
______________________________________                                    
0%       9     6 (66.7%)  2 (22.2%)                                       
                                   3 (33.3%)                              
 1%*     9     7 (77.7%)  4 (44.4%)                                       
                                   5 (55.6%)                              
5%       9     8 (88.9%)  1 (11.0%)                                       
                                   3 (33.3%)                              
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 *This group corresponds to the present invention.                        
 
    
     EXAMPLE 3 
     There was carried out a test for comparing dextran with sucrose and trehalose which are often used as non-penetrable cryoprotectants. 
     
                       TABLE 5                                                     
______________________________________                                    
Freezing test on bovine embryos                                           
obtained by in vitro fertilization                                        
         2 Hours         48 Hours                                         
         after thawing   after thawing                                    
               Number of   Number of                                      
                                   Number of                              
               surviving   transferable                                   
                                   hatched                                
Additives                                                                 
         n     embryos     embryos embryos                                
______________________________________                                    
--       21    18 (85.7%)  3 (14.3%)                                      
                                   4 (19.0%)                              
Sutrose  20    18 (85.7%)  2 (10.0%)                                      
                                   7 (35.0%)                              
Trehalose                                                                 
         21    17 (81.0%)  2 (9.5%)                                       
                                   5 (23.8%)                              
Dextran  21    17 (81.0%)  3 (14.3%)                                      
                                   9 (42.9%)                              
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     The concentration of each additive was 1% (v/v). The dextran group corresponds to the present invention. 
     EXAMPLE 4 
     There was carried out a test for comparing supplemented calf serum with BSA (bovine serum albumin) and calf serum. 
     
                       TABLE 6                                                     
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Freezing test on bovine embryos                                           
obtained by in vitro fertilization                                        
         2 Hours         48 Hours                                         
         after thawing   after thawing                                    
               Number of  Number of                                       
                                   Number of                              
               surviving  transferable                                    
                                   hatched                                
Additives                                                                 
         n     embryos    embryos  embryos                                
______________________________________                                    
BSA      12     9 (75.0%) 1 (8.3%) 5 (41.7%)                              
Calf serum                                                                
         12    10 (83.3%) 3 (25.0%)                                       
                                   5 (41.7%)                              
Supplemented                                                              
         21    17 (81.0%) 8 (38.1%)                                       
                                   9 (42.9%)                              
Calf serum*                                                               
______________________________________                                    
 *Corresponding to the method of the present invention.                   
 
    
     
                       TABLE 6-2                                                   
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Freezing test on bovine embryos                                           
freed from zone pellucida                                                 
         2 Hours         48 Hours                                         
         after thawing   after thawing                                    
               Number of  Number of                                       
                                   Number of                              
               surviving  transferable                                    
                                   hatched                                
Additives                                                                 
         n     embryos    embryos  embryos                                
______________________________________                                    
BSA      9     9 (100%)   4 (44.4%)                                       
                                   3 (33.3%)                              
Calf serum                                                                
         9     8 (88.9%)  3 (33.3%)                                       
                                   3 (33.3%)                              
Supplemented                                                              
         9     7 (77.8%)  4 (44.4%)                                       
                                   5 (55.6%)                              
Calf serum*                                                               
______________________________________                                    
 *Corresponding to the method of the present invention.                   
 
    
     The terms used in the above tables are explained below. 
     The term &#34;bovine embryos freed from zona pellucida&#34; means embryos (at a stage of morula or blastocyst 7 to 8 days after fertilization) collected from a live cow, from which the zona pellucida has been removed by biopsy or binary splitting. 
     The term &#34;bovine embryos obtained by in vitro fertilization&#34; means embryos at the early stage to a stage of enlarged blastocyst 7 days after in vitro fertilization. 
     The term &#34;surviving embryos 2 hours after thawing&#34; means embryos whose survival was confirmed regardless of their quality. 
     The term &#34;transferable embryos 2 hours after thawing&#34; means embryos among surviving embryos, which were judged to be so good in quality that they were transferable. 
     The term &#34;growing embryos 48 hours after thawing&#34; means embryos showing satisfactory growth by cultivation. 
     The term &#34;hatched embryos 48 hours after thawing&#34; means embryos hatched by (in vitro culture).