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Fertilization by sperm injection in cattle
FERTILIEATION BY SPERM INJECTION IN CATTLE Goto, A. Kinoshita, Y. Takuma and K. Ogawa Department of Animal Science Faculty of Agriculture Kagoshima University Kagoshima 890 Japan
K.
In vitro capacitated bovine spermatozoa were killed by freezing-thawing and then microinjected into the ooplasm of in vitro matured bovine oocytes in order to determine whether oocytes fertilized by sperm injection could undergo normal development to blastocyst stage. Ovaries were collected from cows at a local slaughter house and brought Follicular to the laboratory in physiological saline at 32-38'C. oocytes (oocyte-cumulus complexes) were matured as described previously Matured oocyte-cumulus complexes were (J. Reprod. Fert. 83, 753). treated with hyaluronidase (1 mg/ml) and the cumulus cells surrounding oocytes were partially removed by pipetting in order to help the injection procedure. When a small portion of zona pellucida became cumulus-free the pipetting was stopped. Care was taken not to remove the oocytes. Whole, in vitro excess cells from capacitated, frozen-thawed spermatozoa were injected into the ooplasm, and the injected oocytes were cocultured with cumulus cells (J. Reprod. Fert. 83, 753) for an additional 8 days. The cumulus cells surrounding the embryos were removed by pipetting after 72 h of injection but the cumulus cell layer attached to the bottom of the culture dish was not cocultured with this layer. removed and the embryos were If sham-injected oocytes were activated by a 10 min incubation in 50 PM calcium ionophore (A23187), the oocytes cleaved parthenogenetically (2.4%, l/41) but did not develop beyond 6-cell. If sperm injected oocytes were activated by a 10 min incubation in 50 fl A23187, development to 2-4 cell, 6-12 cell, morula and blastocyst was 18.5% (17/92), 15.2% (14/92), 9.9% (g/92) and 10.5% (4/38), respectively. Whether any of these embryos would have developed to term if transferred into a cow uterus remains to be examined. However, the appearance of the cleavage stages, morulae and blastocysts in vitro was normal. Furthermore the time required to reach 2-4 cell, 6-12 cell, morula and blastocyst stage after injection in vitro was similar to that of in vitro fertilized bovine oocytes which has resulted in the birth of a live calf after transfer (J. Reprod. Fert. 83, 753). (Supported by Grant from Inamori Foundation to K.G.)
JANUARY
1990 VOL. 33 NO. 1
K.
In vitro capacitated bovine spermatozoa were killed by freezing-thawing and then microinjected into the ooplasm of in vitro matured bovine oocytes in order to determine whether oocytes fertilized by sperm injection could undergo normal development to blastocyst stage. Ovaries were collected from cows at a local slaughter house and brought Follicular to the laboratory in physiological saline at 32-38'C. oocytes (oocyte-cumulus complexes) were matured as described previously Matured oocyte-cumulus complexes were (J. Reprod. Fert. 83, 753). treated with hyaluronidase (1 mg/ml) and the cumulus cells surrounding oocytes were partially removed by pipetting in order to help the injection procedure. When a small portion of zona pellucida became cumulus-free the pipetting was stopped. Care was taken not to remove the oocytes. Whole, in vitro excess cells from capacitated, frozen-thawed spermatozoa were injected into the ooplasm, and the injected oocytes were cocultured with cumulus cells (J. Reprod. Fert. 83, 753) for an additional 8 days. The cumulus cells surrounding the embryos were removed by pipetting after 72 h of injection but the cumulus cell layer attached to the bottom of the culture dish was not cocultured with this layer. removed and the embryos were If sham-injected oocytes were activated by a 10 min incubation in 50 PM calcium ionophore (A23187), the oocytes cleaved parthenogenetically (2.4%, l/41) but did not develop beyond 6-cell. If sperm injected oocytes were activated by a 10 min incubation in 50 fl A23187, development to 2-4 cell, 6-12 cell, morula and blastocyst was 18.5% (17/92), 15.2% (14/92), 9.9% (g/92) and 10.5% (4/38), respectively. Whether any of these embryos would have developed to term if transferred into a cow uterus remains to be examined. However, the appearance of the cleavage stages, morulae and blastocysts in vitro was normal. Furthermore the time required to reach 2-4 cell, 6-12 cell, morula and blastocyst stage after injection in vitro was similar to that of in vitro fertilized bovine oocytes which has resulted in the birth of a live calf after transfer (J. Reprod. Fert. 83, 753). (Supported by Grant from Inamori Foundation to K.G.)
JANUARY
1990 VOL. 33 NO. 1