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Production of ovine chimeras
THERIOGENOLOGY PRODUCTION OF OVINE CHIMERAS J. E. ButlerI, G. B. Anderson', R. H. BonDurant* and R. L. Pashen' Department of Animal Science' and Department of Reproduction* University of California, Davis, CA
95616
U.S.A.
Ovine chimeras were produced by injecting inner cell masses (ICMl isolated by immunosurgery from Rambouillet or Finnish Landrace donor embryos into the blastocoeles of Finnish Landrace or Rambouillet recipient embryos, respectively. Zonae pellucidae were removed from donor blastocysts by incubation in 0.5% pronase and gentle pipetting, followed by culture in drops of Whitten's medium containing 0.5% BSA for three hours to allow recovery from the pronase treatment. During culture, embryos were maintained at 37C in an atmosphere of 5% CO in air. Denuded embryos were cultured for 1 hour in rabbit anti-si eep antiserum at a dilution of 1 to 8 (v/v). The a$tiserum was developed by three, weekly intravenous injections of 4x10 sheep spleen cells. Antiserum was harvested by heart puncture 10 days following the last injection. Embryos were next washed three times and placed in a 1 to 4 (v/v) dilution of guinea pig serum for 1 hour. Lysed trophoblastic cells were removed by gentle pipetting. An isolated KM was drawn into a micropipette with an outer diameter of approximately 50 urn attached to a Leitz micromanipulator. The recipient embryo was held at the polar trophectoderm overlying the ICM by a microholding pipette with an outer diameter of 125 urn. The donor ICM was introduced by puncturing the injection pipette through the zona pellucida and trophectoderm into the blastocoele of the recipient blastocyst. The injection pipette was positioned next to the ICM of the recipient embryo and the donor ICM expelled. Blastocysts were cultured overnight and then examined to assess incorporation of the injected cells. Twenty of the 25 recipient embryos were successfully injected (80%). Donor ICMs were incorporated by 17 embryos (85%) which were transferred to nine recipient ewes. Embryos that did not incorporate the injected cells into a single ICM were not transferred. Whenever possible, two embryos were transferred to each recipient. Because of this, one ewe received an unsuccessfully injected blastocyst in addition to a chimeric embryo. All nine recipients became pregnant and all pregnancies were carried to term. A total of 15 lambs were born for a survival rate of 83% (15/181. Parentage was determined at one month of age by analysis of red cell antigens, hemoglobin and blood transferrins. Five lambs were determined to be blood chimeras while three lambs had blood characteristics derived solely from the injected -1CMs. One lamb, although not a blood chimera, was overtly chimeric with identifiable characteristics of the two breeds. Overall survival of injected ICMs was 60% (g/15), while the rate of chimerism was 40% (6/151. This procedure has an advantage over other published methods in which a high degree of chimerism was produced in that it does not require the use of an intermediate recipient. The ability to readily produce chimeras in livestock species should greatly expand their use in the study of embryonic development in livestock.
JANUARY
1985 VOL. 23 NO. 1
183
95616
U.S.A.
Ovine chimeras were produced by injecting inner cell masses (ICMl isolated by immunosurgery from Rambouillet or Finnish Landrace donor embryos into the blastocoeles of Finnish Landrace or Rambouillet recipient embryos, respectively. Zonae pellucidae were removed from donor blastocysts by incubation in 0.5% pronase and gentle pipetting, followed by culture in drops of Whitten's medium containing 0.5% BSA for three hours to allow recovery from the pronase treatment. During culture, embryos were maintained at 37C in an atmosphere of 5% CO in air. Denuded embryos were cultured for 1 hour in rabbit anti-si eep antiserum at a dilution of 1 to 8 (v/v). The a$tiserum was developed by three, weekly intravenous injections of 4x10 sheep spleen cells. Antiserum was harvested by heart puncture 10 days following the last injection. Embryos were next washed three times and placed in a 1 to 4 (v/v) dilution of guinea pig serum for 1 hour. Lysed trophoblastic cells were removed by gentle pipetting. An isolated KM was drawn into a micropipette with an outer diameter of approximately 50 urn attached to a Leitz micromanipulator. The recipient embryo was held at the polar trophectoderm overlying the ICM by a microholding pipette with an outer diameter of 125 urn. The donor ICM was introduced by puncturing the injection pipette through the zona pellucida and trophectoderm into the blastocoele of the recipient blastocyst. The injection pipette was positioned next to the ICM of the recipient embryo and the donor ICM expelled. Blastocysts were cultured overnight and then examined to assess incorporation of the injected cells. Twenty of the 25 recipient embryos were successfully injected (80%). Donor ICMs were incorporated by 17 embryos (85%) which were transferred to nine recipient ewes. Embryos that did not incorporate the injected cells into a single ICM were not transferred. Whenever possible, two embryos were transferred to each recipient. Because of this, one ewe received an unsuccessfully injected blastocyst in addition to a chimeric embryo. All nine recipients became pregnant and all pregnancies were carried to term. A total of 15 lambs were born for a survival rate of 83% (15/181. Parentage was determined at one month of age by analysis of red cell antigens, hemoglobin and blood transferrins. Five lambs were determined to be blood chimeras while three lambs had blood characteristics derived solely from the injected -1CMs. One lamb, although not a blood chimera, was overtly chimeric with identifiable characteristics of the two breeds. Overall survival of injected ICMs was 60% (g/15), while the rate of chimerism was 40% (6/151. This procedure has an advantage over other published methods in which a high degree of chimerism was produced in that it does not require the use of an intermediate recipient. The ability to readily produce chimeras in livestock species should greatly expand their use in the study of embryonic development in livestock.
JANUARY
1985 VOL. 23 NO. 1
183