1486468 Transplanting ovafractionating sperm L L AUGSPURGER 11 Nov 1974 [23 Nov 1973] 48625/74 Heading C6F [Also in Divisions A1 and C5] A process for transplanting ova, or cellular bodies derived from ova, from a female hoofed mammal donor to a recipient foster female for maturation involves receiving ova from the donor, placing the recovered ova in tissue culture medium and storing the recovered ova for a period of time in such a way as to defer normal development of the ova, determining the time of ovulation in the recipient, removing one or more of the individual ova or cellular bodies derived from the stored ova in the culture medium, and transplanting the removed individual ova or cellular bodies to the recipient at a time such that the transplanted ova or bodies reach the blastocyst stage in the recipient at the time the recipient would normally implant a blastocyst had the recipient been fertilized. Male producing cellular bodies are separated from the female producing bodies before transplantation, whereby the recipient may receive only transplants of like sex. The genetic manipulation of the ova and cellular bodies is described in considerable detail. The cellular bodies may be cultured at a temperature between 30?? and 38??C in a culture medium containing glucose, saline solution, amino acids, vitamins and sodium pyruvate prior to their transplantation. Detailed formulations of suitable culture media are provided. A preferred medium includes lipoic acid, 1-glutamine and vitamin B 12 , and is buffered with HERPES buffer and sodium bicarbonate to pH 7-3-7.4. A gaseous cover consisting of 5 vol. per cent carbon dioxide and 95% air or oxygen should be provided. The ova may be kept under liquid nitrogen in a medium containing sodium pyruvate or sodium oxaloacetate. The spermatozoa are also pre-sexed prior to their use in the fertilizing by fractionation into the X and Y chromosome bearing spermatozoa. This may involve percolating the spermatozoa through cation exchange resins (leaving untrapped the spermatozoa capable of producing female offspring) or anion exchange resins (leaving male producing sperm untrapped). Preferred cation exchange materials are copolymers of carboxylic divinyl benzene, copolymers of methacrylic acid with divinyl benzene, and copolymers of maleic anhydride with styrene and divinyl benzene. Preferred anion exchange resins can be produced by nitration and subsequent reduction of styrene divinyl benzene copolymers. Particularly in the case of cattle sperm, due to the great difference in size the of X and Y spermatozoa. fractionation may be achieved in a counterstreaming centrifuge using speeds between 1000 and 1200 rpm and speeds below 1000 rpm for the recovery of the male and female producing fractions, respectively. The percolation or centrifugation is preferably conducted under a vacuum of 15 to 30 cm. Hg differential from the pressure at which the sperm were obtained. Fertilization of the matured oocytes, or "pseudo-fertilization" by sterile sperm or chemically, may be made in vitro.
