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Cryopreservation of isolated bovine blastomeres
THERIOGENOLOGY CRYOPRESERVATION OF ISOLATED BOVINE BLASTOMERES A. Lucas-Hahn and H. Niemann lnstitut fur Tierzucht und Tierverhalten (FAL), Mariensee, 3057 Neustadt 1, F.R.G.
Cryopreservation of isolated blastomeres from preimplantation embryos would significantly facilitate cloning, the use of DNA-probes for sex determination and diagnosis of genetic disorders. The objective of this study was to investigate if isolated bovine blastomeres could survive freezing and thawing. Holstein Friesian cows were superovulated and 8-cell embryos were recovered at slaughter by flushing the oviducts 3 days after insemination (= day 0). The zona pellucida of morphologically intact embryos was mechanically removed. After a short incubation in Ca+ ’ and Mg+ + -free PBS-medium individual blastomeres were separated by gentle pipetting with a 40 - 50 pm fire-polished glass pipette. After isolation, single blastomeres were either transferred into an empty zona pellucida or left denuded. Subsequently, all blastomeres were equilibrated in a two-step procedure with 1.5 M 1,2-propanediol (PROH) for 15 min and 1.5 M PROH plus 0.2 M sucrose for 5 min in PBS supplemented with 20% newborn calf serum (NBCS). Individual blastomeres were then sucked into 0.25 ml straws and cooling was started at -7°C in an ethanol freezer. Seeding was induced manually at -7°C and the samples were cooled at 0.8C/min to -30°C. The samples were held at -30°C for 30 min and were then plunged into liquid nitrogen. The blastomeres were thawed in air at room temperature and the cryoprotectants were removed by placing the blastomeres into a 0.75 M 1.2-propanediol + 0.7 M sucrose solution for 5 to IO min, then transferred to a 0.7 M sucrose solution for 5 min, followed by several washes in PBS supplemented with 20% NBCS. The blastomeres were evaluated morphologically under a stereomicroscope and by vital fluorescence staining (Fluorescein-diacetyl = FDA). Blastomeres were considered to have survived when the cytoplasm still displayed a fine granulated structure and membranes were intact as demonstrated by FDA-fluorescence.. The results are shown in the table.
Group
With zona Denuded
No. blastomeres frozen
120 77
Recovery rate after thawing n
(%)
94 60
(78.3) (77.9)
Morphologically intact blastomeres/ recovered blastomeres n (%) 73a 24b
(77.7) (40.0)
Chi square a:b p< 0.01
The agreement between morphological and fluorescence evaluation was 90%. The results indicate, that the presence of the zona pellucida is required for post-thaw survival of bovine blastomeres.
JANUARY 1991 VOL. 35 NO. 1
235
Cryopreservation of isolated blastomeres from preimplantation embryos would significantly facilitate cloning, the use of DNA-probes for sex determination and diagnosis of genetic disorders. The objective of this study was to investigate if isolated bovine blastomeres could survive freezing and thawing. Holstein Friesian cows were superovulated and 8-cell embryos were recovered at slaughter by flushing the oviducts 3 days after insemination (= day 0). The zona pellucida of morphologically intact embryos was mechanically removed. After a short incubation in Ca+ ’ and Mg+ + -free PBS-medium individual blastomeres were separated by gentle pipetting with a 40 - 50 pm fire-polished glass pipette. After isolation, single blastomeres were either transferred into an empty zona pellucida or left denuded. Subsequently, all blastomeres were equilibrated in a two-step procedure with 1.5 M 1,2-propanediol (PROH) for 15 min and 1.5 M PROH plus 0.2 M sucrose for 5 min in PBS supplemented with 20% newborn calf serum (NBCS). Individual blastomeres were then sucked into 0.25 ml straws and cooling was started at -7°C in an ethanol freezer. Seeding was induced manually at -7°C and the samples were cooled at 0.8C/min to -30°C. The samples were held at -30°C for 30 min and were then plunged into liquid nitrogen. The blastomeres were thawed in air at room temperature and the cryoprotectants were removed by placing the blastomeres into a 0.75 M 1.2-propanediol + 0.7 M sucrose solution for 5 to IO min, then transferred to a 0.7 M sucrose solution for 5 min, followed by several washes in PBS supplemented with 20% NBCS. The blastomeres were evaluated morphologically under a stereomicroscope and by vital fluorescence staining (Fluorescein-diacetyl = FDA). Blastomeres were considered to have survived when the cytoplasm still displayed a fine granulated structure and membranes were intact as demonstrated by FDA-fluorescence.. The results are shown in the table.
Group
With zona Denuded
No. blastomeres frozen
120 77
Recovery rate after thawing n
(%)
94 60
(78.3) (77.9)
Morphologically intact blastomeres/ recovered blastomeres n (%) 73a 24b
(77.7) (40.0)
Chi square a:b p< 0.01
The agreement between morphological and fluorescence evaluation was 90%. The results indicate, that the presence of the zona pellucida is required for post-thaw survival of bovine blastomeres.
JANUARY 1991 VOL. 35 NO. 1
235