Abstract:
A diluent for the cryogenic storage of bovine spermatozoa includes a phospholipid, a liposoluble vitamin accompanied by an emulsifier, an antioxidant and a polyol.

Description:
BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     The invention relates to a diluent for cryogenic storage of bovine spermatozoa. More specifically, the invention relates to an aqueous diluent medium which contains no substance of animal origin and which can be stored, ready for use, over prolonged periods. 
     2. Description of the Prior Art 
     Diluents for cryogenic storage of bovine spermatozoa are known in the art. FR-B-2 720 407 describes a diluent which contains nutrients, buffers and mineral salts, as well as a protective product consisting of soya lecithin. 
     The diluent described in the abovementioned patent is intended for diluting bovine semen which is then frozen pending subsequent use. This is an essentially “sanitary” approach. This diluent is relatively satisfactory. 
     It is easy to prepare and is very clear. The absence of products of animal origin ensures the absence of any infective agent. 
     However, the question of the biological efficacy of the product was not addressed. The efficacy in the concentration ranges used previously does not provide the flexibility required in an insemination center. 
     Specifically, it is currently required to optimize the production of semen, i.e. to produce a large number of doses stored in a “French straw” with a minimum concentration of spermatozoa. 
     In vivo tests with a very low concentration of spermatozoa in the straw indicate that the diluent of FR-B-2 720 407 does not perform well. 
     One object of the present invention is to provide a diluent for cryogenic storage of bovine spermatozoa which gives improved in vivo results. 
     Another object of the invention is to provide a diluent which assures satisfactory survival of the spermatozoa at low concentrations. 
     The present invention provides a diluent which satisfies the above objects. 
     SUMMARY OF THE INVENTION 
     The diluent for cryogenic storage of bovine spermatozoa of the present invention includes a phospholipid, a liposoluble vitamin accompanied by an emulsifier, an antioxidant and a polyol. 
     The vitamin is vitamin A, for example. 
     The vitamin is accompanied by an emulsifier, for example sorbitan monooleate, which is sold under the trade name Tween 80. 
     The antioxidant can be an amino acid with anti-oxidant properties, for example taurine or glycine. 
     The antioxidant can instead be an antioxidant peptide, for example reduced glutathione. 
     The diluent of the present invention can instead contain a sterol, in particular cholesterol. 
     When a sterol is present in the diluent, the diluent advantageously contains cyclodextrine which render it soluble in an aqueous medium. 
     The diluent of the present invention can additionally contain salts and carbohydrates. 
     The carbohydrates are glucose, fructose or lactose, for example. 
     The salts are buffers in particular, for example trimethylolmethylamine, which is usually referred to as Tris buffer or simply Tris. 
     The diluent of the invention contains an effective dose of a polyol capable of inhibiting the formation of ice crystals. The polyol is advantageously glycerol. 
     To prepare the diluent according to the invention, the following steps are carried out: 
     a) preparing a dispersion of a phospholipid in the form of particles in a polyol at a sufficient temperature, 
     b) stirring the dispersion from step a) to micronize the phospholipid particles, 
     c) leaving the dispersion obtained in step b) to stand for a period of at least about 12 hours to stabilize the emulsion obtained, i.e. to ensure no separation of phases, yielding a preparation referred to as the preparation from step c), 
     d) preparing an aqueous phase including an amino acid with antioxidant properties, an antioxidant peptide and a vitamin, yielding an aqueous phase referred to as the aqueous phase from step d), 
     e) combining the preparation from step c) and the aqueous phase from step d), yielding a preparation referred to as the preparation from step e), and 
     f) sterilizing the preparation from step e). 
     According to one preferred embodiment of the invention, the sterilization in step f) is carried out by ionizing radiation in an irradiation dose range from about 5 Kgy to about 20 Kgy, preferably from about 15 Kgy to about 20 Kgy. 
     Techniques for obtaining the dispersion from step a) include techniques using mechanical dispersants and emulsifiers which act by pressure. 
     In order to obtain a stable emulsion in step c), it is necessary to leave the dispersion to stand for at least 12 hours. 
     The diluent of the invention is preferably formulated with a small amount of water and is diluted for use with sterile water. 
     The diluent advantageously contains an antibiotic agent at the time of use, but if the antibiotic agent is added to the diluent during its preparation, its efficacy can be lost during the sterilization step. 
     According to one preferred embodiment of the present invention, the antibiotic agent is added extemporaneously at the time of use. 
     In this embodiment, the antibiotic-free diluent is advantageously contained in a receptacle closed by a stopper. For example, the antibiotic agent in powder form is present in the stopper and is released into the diluent by exerting pressure on the stopper. 
     The antibiotic agent is gentamycin in sulfate form, for example. 
     The diluent of the invention typically contains the following phospholipids in the weight proportions indicated below: 
     “Lecithin 100” having the following weight proportions of the following phospholipids: 
     phosphatidylcholine 20-24 wt. % 
     phosphatidylethanolamine 18-22 wt. % 
     phosphatidylinositol 12-15 wt. % 
     “Lecithin 130” having the following weight proportions of the following phospholipids: 
     phosphatidylethanolamine 14-20 wt. % 
     phosphatidylinositol 7-13 wt. % 
     phosphatidylcholine 30-35 wt. % 
     Lecithin 130 is a phosphatidylcholine-enriched lecithin. 
     It should be noted that the diluent of the invention is intended exclusively for cryogenic storage of bovine spermatozoa. It is not suitable for cryogenic storage of spermatozoa of other animal species or human spermatozoa. 
     In one preferred embodiment, a diluent according to the invention includes, per 100 ml of water: 
     
       
         
               
               
               
             
           
               
                   
                   
               
             
             
               
                   
                 Tris 
                 1 to 2 g 
               
               
                   
                 Trisodium citrate dihydrate 
                  5 to 10 g 
               
               
                   
                 Potassium chloride 
                 0.2 to 0.5 g 
               
               
                   
                 Fructose 
                 0.6 to 1.0 g 
               
               
                   
                 Lactose monohydrate 
                 0.18 to 0.30 g 
               
               
                   
                 Glycine 
                 2.0 to 3.0 g 
               
               
                   
                 Anhydrous glucose 
                 0.25 to 0.40 g 
               
               
                   
                 Taurine 
                 0.0030 to 0.0040 g 
               
               
                   
                 Gentamycin sulfate 
                 0.20 g to 0.30 g  
               
               
                   
                 Tylosin tartrate 
                 0.028 to 0.040 g 
               
               
                   
                 Lincospectin 100 
                 0.30 to 0.35 g 
               
               
                   
                 Glycerol 
                 30 to 45 g 
               
               
                   
                 Calcium lactate hydrate 
                 0.03 to 0.05 g 
               
               
                   
                 Lecithin 100 
                 2.0 to 3.0 g 
               
               
                   
                 Lecithin 130 
                 0.80 to 1.10 g 
               
               
                   
                 Citric acid monohydrate 
                 0.5 to 2 g   
               
               
                   
                 Ultra-pure water 
                 qs 
               
               
                   
                   
               
             
          
         
       
     
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     The examples which follow illustrate the invention in a nonlimiting manner. 
    
    
     EXAMPLE 1 
     Following the procedure described above, a diluent according to the invention having the composition below was formulated: 
     
       
         
               
               
               
             
           
               
                   
                   
               
             
             
               
                   
                 Tris 
                 1.8490 g 
               
               
                   
                 Trisodium citrate dihydrate 
                 7.3965 g 
               
               
                   
                 Potassium chloride 
                 0.2955 g 
               
               
                   
                 Fructose 
                 0.8875 g 
               
               
                   
                 Lactose monohydrate 
                 0.2220 g 
               
               
                   
                 Glycine 
                 2.7735 g 
               
               
                   
                 Anhydrous glucose 
                 0.3695 g 
               
               
                   
                 Taurine 
                 0.0037 g 
               
               
                   
                 Gentamycin sulfate 
                 0.2400 g 
               
               
                   
                 Tylosin tartrate 
                 0.0330 g 
               
               
                   
                 Lincospectin 100 
                 0.3830 g 
               
               
                   
                 Glycerol 
                 40.2240 g  
               
               
                   
                 Calcium lactate hydrate 
                 0.0455 g 
               
               
                   
                 Lecithin 100 
                 2.6000 g 
               
               
                   
                 Lecithin 130 
                 1.0400 g 
               
               
                   
                 Citric acid monohydrate 
                 1.0400 g 
               
               
                   
                 Ultra-pure water 
                 0.0650 l 
               
               
                   
                   
               
             
          
         
       
     
     The above formulation produced 100 ml of concentrated diluent which was diluted to 500 ml for use. 
     EXAMPLE 2 
     Comparative in vitro tests: anomalies and test of osmotic strength 
     In this example, the diluent of Example 1 of the invention was compared with the diluent of the prior art described in EP-A-685 556 as regards anomalies and osmotic strength in vitro. 
     Following the procedure described in EP-A-685 556, a diluent according to the prior art having the composition below was formulated: 
     
       
         
               
               
               
             
           
               
                   
                   
               
             
             
               
                   
                 Trimethylolmethylamine 
                 3.4 g to 4.2 g 
               
               
                   
                 Trisodium citrate dihydrate 
                  13.7 g to 16.75 g 
               
               
                   
                 Potassium chloride 
                 0.55 g to 0.67 g 
               
               
                   
                 Fructose 
                 1.65 g to 2.0 g  
               
               
                   
                 Glucose 
                 0.68 g to 0.84 g 
               
               
                   
                 Lactose 
                 0.41 g to 0.50 g 
               
               
                   
                 Calcium lactate 
                 0.09 g to 0.11 g 
               
               
                   
                 Glycine 
                 5.15 g to 6.25 g 
               
               
                   
                 Glycerol 
                 64 ml to 78 ml 
               
               
                   
                 Soya lecithin 
                 6.75 g to 8.25 g 
               
               
                   
                   
               
             
          
         
       
     
     This vehicle was diluted with 750 ml to 900 ml of water prior to use. 
     Table I gives comparative results for the diluent of the prior art (DILUENT A) and the diluent of the invention (DILUENT I) for various bulls: NIZAGO, NOMEL, NORRIS, NOVAK, OCARINA and OKAVANGO. 
     
       
         
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE I 
               
               
                   
               
               
                   
                 % major 
                   
                   
               
               
                 IDENTIFICATION 
                 anomalies 
                 % abnormal 
                 HOST 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 NIZAGO DILUENT A 
                 12.0% 
                 23.5% 
                 51.24% 
               
               
                 NIZAGO DILUENT I 
                   
                   
                 65.05% 
               
               
                 NOMEL DILUENT A 
                 13.5% 
                 30.0% 
                 34.31% 
               
               
                 NOMEL DILUENT I 
                   
                   
                 54.50% 
               
               
                 NORRIS DILUENT A 
                 6.0% 
                 17.5% 
                 50.50% 
               
               
                 NORRIS DILUENT I 
                   
                   
                 53.00% 
               
               
                 NOVAK DILUENT A 
                 20.0% 
                 26.0% 
                 34.80% 
               
               
                 NOVAK DILUENT I 
                   
                   
                 47.71% 
               
               
                 OCARINA DILUENT A 
                 7.5% 
                 21.0% 
                 50.00% 
               
               
                 OCARINA DILUENT I 
                   
                   
                 47.76% 
               
               
                 OKAVANGO DILUENT A 
                 8.5% 
                 18.5% 
                 67.68% 
               
               
                 OKAVANGO DILUENT I 
                   
                   
                 67.65% 
               
               
                 Average results for 
                 11.0% 
                 23.0% 
                 48.09% 
               
               
                 DILUENT A at 
               
               
                 7 × 10 6  spz/dose 
               
               
                 Average results for 
                   
                   
                 55.95% 
               
               
                 DILUENT I at 
               
               
                 7 × 10 6  spz/dose 
               
               
                   
               
               
                 HOST = osmotic strength test  
               
               
                 spz = spermatozoa  
               
             
          
         
       
     
     Table I clearly indicates the superiority of the diluent of the invention over the diluent of the prior art since, for 7×10 6  spermatozoa per dose, a very much higher percentage of osmotic strength in vitro is obtained for the diluent of the invention. 
     EXAMPLE 3 
     Comparative in vitro tests, results after thawing and temperature resistance test 
     In this example, the diluent of Example 1 of the invention was compared with the diluent of the prior art described in EP-A-685 556 used in Example 2, as regards results after thawing and in vitro temperature resistance. 
     Table II gives comparative results for the diluent of the prior art (DILUENT A) and the diluent of the invention (DILUENT I) with various bulls: NIZAGO, KOMEL, NORRIS, NOVAK, OCARINA and OKAVANGO. 
     Table II clearly indicates the superiority of the diluent of the invention over the diluent of the prior art since, for 7×10 6  spermatozoa per dose, a higher percentage of mobiles and higher motility are obtained, except for the percentage of mobiles during the thawing after 1 h 30, which is very much inferior. 
     EXAMPLE 4 
     Comparative in vivo tests: results of in vivo artificial insemination for a nonreturn rate at 25 days 
     In this example, the diluent of Example 1 of the invention was compared with the diluent of the prior art described in EP-A-685 556 used in Example 2, as regards the results of in vivo primary artificial insemination (PAI), fertilization by primary artificial insemination (PAI fert) and percentage of fertilization by primary artificial insemination (% PAI fert), for a nonreturn rate (NRR) at 25 days. 
     Table III gives comparative results for the diluent of the prior art (DILUENT A) and the diluent of the invention (DILUENT I) with various bulls: NIZAGO, NOMEL, NORRIS, NOVAK, OCARINA and OKAVANGO. 
     
       
         
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
             
           
               
                   
                 TABLE II 
               
             
             
               
                   
                   
               
               
                   
                   
                   
                   
                   
                 TRT 1 h to 
               
               
                   
                 Thawing 
                 TRT + 30 min 
                 TRT + 1 h 30 
                 Thawing 
                 1 h 45 
               
             
          
           
               
                   
                 % 
                 Moti- 
                 % 
                 Moti- 
                 % 
                 Moti- 
                 % 
                 Moti- 
                 % 
                 Moti- 
               
               
                 IDENTIFICATION 
                 mobiles 
                 lilty 
                 mobiles 
                 lilty 
                 mobiles 
                 lilty 
                 mobiles 
                 lilty 
                 mobiles 
                 lilty 
               
               
                   
               
             
          
           
               
                 NIZAGO DILUENT A 
                 55% 
                 3 
                 50% 
                 2 
                 30% 
                 1.5 
                 55% 
                 3 
                  0% 
                 0 
               
               
                 NIZAGO DILUENT I 
                 55% 
                 3.5 
                 55% 
                 3 
                 45% 
                 2 
                 55% 
                 3 
                 45% 
                 2 
               
               
                 NOMEL DILUENT A 
                 55% 
                 3.5 
                 55% 
                 3 
                 10% 
                 1 
                 45% 
                 3 
                 35% 
                 2 
               
               
                 NOMEL DILUENT I 
                 45% 
                 3 
                 45% 
                 2 
                 10% 
                 0.5 
                 45% 
                 3 
                 40% 
                 3 
               
               
                 NORRIS DILUENT A 
                 40% 
                 2 
                 40% 
                 1 
                 10% 
                 0.5 
                 50% 
                 2.5 
                  0% 
               
               
                 NORRIS DILUENT I 
                 45% 
                 3 
                 45% 
                 2 
                 45% 
                 3 
                 30% 
                 3 
                  5% 
               
               
                 NOVAK DILUENT A 
                 30% 
                 2 
                 30% 
                 1.5 
                 10% 
                 0.5 
                 45% 
                 2.5 
                  0% 
               
               
                 NOVAK DILUENT I 
                 35% 
                 3 
                 35% 
                 2.5 
                 35% 
                 3 
                 40% 
                 3.5 
                  0% 
               
               
                 OCARINA DILUENT A 
                 55% 
                 3.5 
                 50% 
                 3 
                 50% 
                 3 
                 45% 
                 3 
                  5% 
               
               
                 OCARINA DILUENT I 
                 60% 
                 3.5 
                 55% 
                 3 
                 50% 
                 3.5 
                 55% 
                 4 
                 10% 
               
               
                 OKAVANGO DILUENT A 
                 55% 
                 3.5 
                 45% 
                 2.5 
                 30% 
                 2 
                 55% 
                 3 
                 45% 
                 2.5 
               
               
                 OKAVANGO DILUENT I 
                 55% 
                 3.5 
                 60% 
                 3.5 
                 50% 
                 3 
                 55% 
                 3 
                 45% 
                 3 
               
               
                 Average results for 
                 48% 
                 2.92 
                 45% 
                 2.17 
                 23% 
                 1.42 
                 49% 
                 2.83 
                 14% 
                 1.50 
               
               
                 DILUENT A at 
               
               
                 7 × 10 4  spz/dose 
               
               
                 Average results for 
                 49% 
                 3.25 
                 49% 
                 2.67 
                 39% 
                 2.50 
                 47% 
                 3.25 
                 24% 
                 2.67 
               
               
                 DILUENT I at 
               
               
                 7 × 10 6  spz/dose 
               
               
                   
               
               
                 TRT = temperature resistance test  
               
               
                 spz = spermatozoa  
               
             
          
         
       
     
     
       
         
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
               
             
           
               
                   
                 TABLE III 
               
             
             
               
                   
                   
               
               
                   
                 NRR at 25 days 
               
             
          
           
               
                   
                   
                 PAI 
                 % PAI 
                   
                 AI2 
                 % IA2 
                   
                 AI3 + 
                 % AI3 + 
                 Total 
                 Total 
                 Total % 
               
               
                 IDENTIFICATION 
                 PAI 
                 fert 
                 fert 
                 AI2 
                 fert 
                 fert 
                 IA3+ 
                 fert 
                 fert 
                 AI 
                 AI fert 
                 AI fert 
               
               
                   
               
               
                 NIZAGO DILUENT A 
                 66 
                 44 
                 66.67% 
                 48 
                 35 
                 72.92% 
                 74 
                 55 
                 74.32% 
                 188 
                 134 
                 71.28% 
               
               
                 NIZAGO DILUENT I 
                 68 
                 55 
                 80.88% 
                 33 
                 27 
                 81.82% 
                 89 
                 71 
                 79.78% 
                 190 
                 153 
                 80.53% 
               
               
                 NOMEL DILUENT A 
                 76 
                 60 
                 78.95% 
                 45 
                 37 
                 82.22% 
                 71 
                 59 
                 83.10% 
                 192 
                 156 
                 81.25% 
               
               
                 NOMEL DILUENT I 
                 72 
                 59 
                 81.94% 
                 48 
                 40 
                 83.33% 
                 72 
                 60 
                 83.33% 
                 192 
                 159 
                 82.81% 
               
               
                 NORRIS DILUENT A 
                 74 
                 61 
                 82.43% 
                 42 
                 31 
                 73.81% 
                 75 
                 56 
                 74.67% 
                 191 
                 148 
                 77.49% 
               
               
                 NORRIS DILUENT I 
                 60 
                 51 
                 85.00% 
                 41 
                 33 
                 80.49% 
                 73 
                 54 
                 73.97% 
                 174 
                 138 
                 79.31% 
               
               
                 NOVAK DILUENT A 
                 80 
                 63 
                 78.75% 
                 34 
                 31 
                 91.18% 
                 63 
                 50 
                 79.37% 
                 177 
                 144 
                 81.36% 
               
               
                 NOVAK DILUENT I 
                 68 
                 52 
                 76.47% 
                 50 
                 45 
                 90.00% 
                 77 
                 67 
                 87.01% 
                 195 
                 164 
                 84.10% 
               
               
                 OCARINA DILUENT A 
                 73 
                 60 
                 82.19% 
                 41 
                 38 
                 92.68% 
                 73 
                 60 
                 82.19% 
                 187 
                 158 
                 84.49% 
               
               
                 OCARINA DILUENT I 
                 72 
                 65 
                 90.28% 
                 47 
                 37 
                 78.72% 
                 74 
                 59 
                 79.73% 
                 193 
                 161 
                 83.42% 
               
               
                 OKAVANGO DILUENT A 
                 74 
                 57 
                 77.03% 
                 34 
                 29 
                 85.29% 
                 60 
                 49 
                 81.67% 
                 168 
                 135 
                 80.36% 
               
               
                 OKAVANGO DILUENT I 
                 71 
                 52 
                 73.24% 
                 47 
                 39 
                 82.98% 
                 75 
                 63 
                 84.00% 
                 193 
                 154 
                 79.79% 
               
               
                 Average results for 
                 443  
                 345  
                 77.88% 
                 244  
                 201  
                 82.38% 
                 416  
                 329  
                 79.09% 
                 1103  
                 875 
                 79.33% 
               
               
                 Diluent A at 
               
               
                 7 × 10 6  spz/dose 
               
               
                 Average results for 
                 411  
                 334  
                 81.27% 
                 266  
                 221  
                 83.08% 
                 460  
                 374  
                 81.30% 
                 1137  
                 929 
                 81.71% 
               
               
                 Diluent I at 
               
               
                 7 × 10 6  spz/dose 
               
               
                 Average results for 
                 2028  
                 1630  
                 80.37% 
               
               
                 the test bulls over 
               
               
                 the same period at 
               
               
                 20 × 10 6  spz/dose 
               
               
                 DILUENT A 
               
               
                   
               
               
                 NRR = nonreturn rate  
               
               
                 spz = spermatozoa  
               
             
          
         
       
     
     Table III clearly indicates the superiority of the diluent of the invention over the diluent of the prior art, since a very much higher percentage PAI is obtained in all cases but one (for the bull OKAVANGO). The average results for 7×10 6  spermatozoa per dose reveal a very much higher PAI percentage for the diluent of the invention. 
     Surprisingly, the diluent of the prior art shows very inferior average results, even for 20×10 6  spermatozoa per dose, than the diluent of the invention for only 7×10 6  spermatozoa per dose. 
     EXAMPLE 5 
     Comparative in vivo tests: results of in vivo artificial insemination for a nonreturn rate at 60 days 
     In this Example, the diluent of Example 1 of the invention was compared with the diluent of the prior art described in EP-A-685 556 used in Example 2, as regards the results of in vivo primary artificial insemination (PAI), fertilization by primary artificial insemination (PAI fert) and percentage of fertilization by primary artificial insemination (% PAI fert), for a nonreturn rate (NRR) at 60 days. 
     Table IV gives comparative results for the diluent of the prior art (DILUENT A) and the diluent of the invention (DILUENT I) with various bulls: NIZAGO, NOMEL, NORRIS, NOVAK, OCARINA and OKAVANGO. 
     Surprisingly, the diluent of the invention even has, for only 7×10 6  spermatozoa per dose, average results that are better than those for the diluent of the prior art for 20×10 6  spermatozoa per dose. 
     
       
         
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
               
             
           
               
                   
                 TABLE IV 
               
             
             
               
                   
                   
               
               
                   
                 NRR at 60 days 
               
             
          
           
               
                   
                   
                 PAI 
                 % PAI 
                   
                 AI2 
                 % IA2 
                   
                 AI3 + 
                 % AI3 + 
                 Total 
                 Total 
                 Total % 
               
               
                 IDENTIFICATION 
                 PAI 
                 fert 
                 fert 
                 AI2 
                 fert 
                 fert 
                 IA3+ 
                 fert 
                 fert 
                 AI 
                 AI fert 
                 AI fert 
               
               
                   
               
               
                 NIZAGO DILUENT A 
                 55 
                 24 
                 43.64% 
                 40 
                 15 
                 37.50% 
                 68 
                 39 
                 57.35% 
                 163 
                  78 
                 47.85% 
               
               
                 NIZAGO DILUENT I 
                 68 
                 40 
                 58.82% 
                 33 
                 22 
                 66.67% 
                 87 
                 60 
                 68.97% 
                 188 
                 122 
                 64.89% 
               
               
                 NOMEL DILUENT A 
                 72 
                 41 
                 56.94% 
                 43 
                 27 
                 62.79% 
                 64 
                 41 
                 64.06% 
                 179 
                 109 
                 60.89% 
               
               
                 NOMEL DILUENT I 
                 62 
                 38 
                 61.29% 
                 38 
                 24 
                 63.16% 
                 63 
                 36 
                 57.14% 
                 163 
                  98 
                 60.12% 
               
               
                 NORRIS DILUENT A 
                 69 
                 39 
                 56.52% 
                 41 
                 24 
                 58.54% 
                 72 
                 40 
                 55.56% 
                 182 
                 103 
                 56.59% 
               
               
                 NORRIS DILUENT I 
                 55 
                 35 
                 63.64% 
                 34 
                 19 
                 55.88% 
                 65 
                 38 
                 58.46% 
                 154 
                  92 
                 59.74% 
               
               
                 NOVAK DILUENT A 
                 61 
                 36 
                 59.02% 
                 47 
                 37 
                 78.72% 
                 72 
                 47 
                 65.28% 
                 180 
                 120 
                 66.67% 
               
               
                 NOVAK DILUENT I 
                 74 
                 44 
                 59.46% 
                 30 
                 21 
                 70.00% 
                 57 
                 37 
                 64.91% 
                 161 
                 102 
                 63.35% 
               
               
                 OCARINA DILUENT A 
                 44 
                 27 
                 61.36% 
                 31 
                 24 
                 77.42% 
                 53 
                 35 
                 66.04% 
                 128 
                  86 
                 67.19% 
               
               
                 OCARINA DILUENT I 
                 68 
                 50 
                 73.53% 
                 44 
                 28 
                 63.64% 
                 72 
                 46 
                 63.89% 
                 184 
                 124 
                 67.39% 
               
               
                 OKAVANGO DILUENT A 
                 39 
                 26 
                 66.67% 
                 23 
                 15 
                 65.22% 
                 42 
                 27 
                 64.29% 
                 104 
                  68 
                 65.38% 
               
               
                 OKAVANGO DILUENT I 
                 70 
                 36 
                 51.43% 
                 46 
                 29 
                 63.04% 
                 75 
                 52 
                 69.33% 
                 191 
                 117 
                 61.26% 
               
               
                 Average results for 
                 340  
                 193  
                 56.76% 
                 225  
                 142  
                 63.11% 
                 371  
                 229  
                 61.73% 
                 936 
                 564 
                 60.26% 
               
               
                 Diluent A at 
               
               
                 7 × 10 6  spz/dose 
               
               
                 Average results for 
                 397  
                 243  
                 61.23% 
                 225  
                 143  
                 63.56% 
                 419  
                 269  
                 64.20% 
                 1041  
                 655 
                 62.92% 
               
               
                 Diluent I at 
               
               
                 7 × 10 6  spz/dose 
               
               
                 Average results for 
                 1771  
                 1097  
                 61.94% 
               
               
                 the test bulls over 
               
               
                 the same period at 
               
               
                 20 × 10 6  spz/dose 
               
               
                 DILUENT A 
               
               
                   
               
               
                 NRR = nonreturn rate  
               
               
                 spz = spermatozoa  
               
             
          
         
       
     
     EXAMPLE 6 
     Comparative in vivo tests: results of in vivo artificial insemination for a nonreturn rate at 90 days 
     In this example, the diluent of Example 1 of the invention was compared with the diluent of the prior art described in EP-A-685 556 used in Example 2, as regards the results of in vivo primary artificial insemination (PAI), fertilization by primary artificial insemination (PAI fert) and percentage of fertilization by primary artificial insemination (% PAI fert), for a nonreturn rate (NRR) at 90 days. 
     Table V gives comparative results for the diluent of the prior art (DILUENT A) and the diluent of the invention (DILUENT I) with different bulls: NIZAGO, NOMEL, NORRIS, NOVAK, OCARINA and OKAVANGO. 
     Table V clearly indicates the superiority of the diluent of the invention over the diluent of the prior art, since a higher percentage of PAI is obtained in all cases but one (for the OKAVANGO bull). The average results for 7×10 6  spermatozoa per dose reveal a markedly higher percentage of PAI for the diluent of the invention. 
     Surprisingly, the diluent of the invention even shows, for only 7×10 6  spermatozoa per dose, average results that are higher than those for the diluent of the prior art for 20×10 6  spermatozoa per dose. 
     Although the invention has been described for specific embodiments, it will be understood that it encompasses all the implementation variants in the scope of the claims. 
     
       
         
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
               
             
           
               
                   
                 TABLE V 
               
             
             
               
                   
                   
               
               
                   
                 NRR at 90 days 
               
             
          
           
               
                   
                   
                 PAI 
                 % PAI 
                   
                 AI2 
                 % IA2 
                   
                 AI3 + 
                 % AI3 + 
                 Total 
                 Total 
                 Total % 
               
               
                 IDENTIFICATION 
                 PAI 
                 fert 
                 fert 
                 AI2 
                 fert 
                 fert 
                 IA3+ 
                 fert 
                 fert 
                 AI 
                 AI fert 
                 AI fert 
               
               
                   
               
               
                 NIZAGO DILUENT A 
                 65 
                 24 
                 36.92% 
                 47 
                 19 
                 40.43% 
                 74 
                 35 
                 47.30% 
                 186 
                  78 
                 41.94% 
               
               
                 NIZAGO DILUENT I 
                 68 
                 37 
                 54.41% 
                 33 
                 19 
                 57.58% 
                 89 
                 55 
                 61.80% 
                 190 
                 111 
                 58.42% 
               
               
                 NOMEL DILUENT A 
                 76 
                 36 
                 47.37% 
                 45 
                 24 
                 53.33% 
                 71 
                 42 
                 59.15% 
                 192 
                 102 
                 53.13% 
               
               
                 NOMEL DILUENT I 
                 72 
                 37 
                 51.39% 
                 48 
                 22 
                 45.83% 
                 72 
                 38 
                 52.78% 
                 192 
                  97 
                 50.52% 
               
               
                 NORRIS DILUENT A 
                 74 
                 37 
                 50.00% 
                 42 
                 23 
                 54.76% 
                 75 
                 37 
                 49.33% 
                 191 
                  97 
                 50.79% 
               
               
                 NORRIS DILUENT I 
                 60 
                 31 
                 51.67% 
                 41 
                 21 
                 51.22% 
                 73 
                 42 
                 57.53% 
                 174 
                  94 
                 54.02% 
               
               
                 NOVAK DILUENT A 
                 68 
                 33 
                 48.53% 
                 50 
                 35 
                 70.00% 
                 77 
                 43 
                 55.84% 
                 195 
                 111 
                 56.92% 
               
               
                 NOVAK DILUENT I 
                 78 
                 41 
                 52.56% 
                 32 
                 23 
                 71.88% 
                 62 
                 38 
                 61.29% 
                 172 
                 102 
                 59.30% 
               
               
                 OCARINA DILUENT A 
                 72 
                 42 
                 58.33% 
                 41 
                 25 
                 60.98% 
                 73 
                 49 
                 67.12% 
                 186 
                 116 
                 62.37% 
               
               
                 OCARINA DILUENT I 
                 72 
                 46 
                 63.89% 
                 47 
                 27 
                 57.45% 
                 74 
                 42 
                 56.76% 
                 193 
                 115 
                 59.59% 
               
               
                 OKAVANGO DILUENT A 
                 73 
                 37 
                 50.68% 
                 34 
                 20 
                 58.82% 
                 60 
                 34 
                 56.67% 
                 167 
                  91 
                 54.49% 
               
               
                 OKAVANGO DILUENT I 
                 71 
                 31 
                 43.66% 
                 47 
                 29 
                 61.70% 
                 75 
                 47 
                 62.67% 
                 193 
                 107 
                 55.44% 
               
               
                 Average results for 
                 428  
                 209  
                 48.83% 
                 259  
                 146  
                 56.37% 
                 430  
                 240  
                 55.81% 
                 1117  
                 595 
                 53.27% 
               
               
                 Diluent A at 
               
               
                 7 × 10 6  spz/dose 
               
               
                 Average results for 
                 421  
                 223  
                 52.97% 
                 248  
                 141  
                 56.85% 
                 445  
                 262  
                 58.99% 
                 1114  
                 626 
                 56.19% 
               
               
                 Diluent I at 
               
               
                 7 × 10 6  spz/dose 
               
               
                 Average results for 
                 1833  
                 1009  
                 55.05% 
               
               
                 the test bulls over 
               
               
                 the same period at 
               
               
                 20 × 10 6  spz/dose 
               
               
                 DILUENT A 
               
               
                   
               
               
                 NRR = nonreturn rate  
               
               
                 spz = spermatozoa