A new pellet technique for cryopreserving ram and bull spermatozoa using the cold surface of cattle fat

A technique for freezing ram and bull spermatozoa in pellet form, using the cold surface of cattle fat was compared to other freezing procedures. Three freezing methods were compared to cryopreserve ram spermatozoa: 0.25 ml straws, pellets frozen on the cold surface of paraffin wax and pellets frozen on the cold surface of cattle fat. In addition, two cryoprotectants, glycerol or sucrose, in an egg yolk-Tris diluent were compared. Ram spermatozoa frozen as pellets on cattle fat exhibited higher percentages of motile cells after thawing (54%) than spermatozoa frozen in straws (49%) or as pellets on paraffin wax (42%, S.E.M.=1; P<0.05). However, the percentages of acrosome intact cells were similar for spermatozoa frozen as pellets (49%) and spermatozoa frozen in straws (48%; P>0.05), but higher than for spermatozoa frozen as pellets on paraffin wax (39%, S.E.M.=1; P>0.05). Ram spermatozoa exhibited higher percentages of motile cells after thawing when the cryoprotectant was sucrose (51%) compared to glycerol (46%; P<0.05). Similarly, acrosomal integrity was greater with sucrose (49%) than with glycerol (42%; P<0.05). Bull spermatozoa exhibited higher percentages of motile cells after thawing, when cells were frozen in straws (47%) than in the pellet form, regardless of the surface on which the pellets were frozen (31-37%, S.E.M.=3; P<0.05). However, bull spermatozoa exhibited higher percentages of motile cells when frozen as pellets on the surface of cattle fat (66%) or dry ice (61%), than when frozen on paraffin wax (53%, S.E.M.=4; P<0.05). In conclusion, although bull spermatozoa survive cryopreservation more effectively in straws, ram spermatozoa can be cryopreserved as pellets on the cold surface of cattle fat using sucrose as the cryoprotectant. This technique is simple, requires little equipment, is less expensive than using straws and may prove useful for cryopreserving ram and possibly bull spermatozoa in developing countries.