Developmental potential of early bovine zygotes submitted to centrifugation and microinjection following in vitro maturation of oocytes

Gagné, Marc; Pothier, F.; Sirard, Marc‐André

doi:10.1016/0093-691x(90)90533-y

Cited by 11 publications

(2 citation statements)

References 23 publications

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“…It is possible that zygotes forming pronuclei early after fertilization are inherently more competent to develop. Microinjection of in vitro fertilized bovine embryos has generally been carried out from 18-24 h after insemination (Gagne et al, 1990;Hill et al, 1992). The present study was designed to investigate the effect of injection at various times after insemination on embryo development and DNA detection rates.…”

Section: Introductionmentioning

confidence: 99%

Influence of time of gene microinjection on development and DNA detection frequency in bovine embryos

Krisher

Gibbons

Canseco

et al. 1994

Transgenic Research

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The effect of DNA microinjection at various times after in vitro insemination on DNA detection and survival rates of bovine embryos was investigated. Oocytes were inseminated 24 h after maturation with frozen/thawed semen prepared with a Percoll separation procedure. At 11, 15 and 19 h after insemination, embryos were centrifuged to visualize pronuclei and microinjected with a murine whey acidic protein-human protein C genomic DNA construct. After culture for 7 days on Buffalo Rat Liver cells, embryos were assessed for stage of development and assayed for the presence of the transgene by polymerase chain reaction. Of zygotes in the 11 h after insemination treatment, 16% (25/152) of non-injected and 7% (11/161) of injected embryos developed to the morula or blastocyst stage. Comparable development of non-injected and injected embryos treated at 15 h after insemination was 15% (23/158) and 4% (6/159) and treated at 19 h after insemination was 14% (23/162) and 1% (1/165), respectively. Development of injected embryos was greater (p < 0.05) when injection was performed at 11 h after insemination compared to 19 h after insemination. Development of non-injected embryos was greater (p < 0.01) than that of injected embryos. There was no difference in transgene detection frequency in embryos of all developmental states between treatments (53% at 11; 50% at 15; 48% at 19 h after insemination). Injected embryos testing positive for the presence of the transgene exhibited increased development over negative embryos (p < 0.01). Greater development efficiencies can be obtained in microinjected bovine embryos when injection is performed early in pronuclear formation.

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Section: Introductionmentioning

confidence: 99%

Influence of time of gene microinjection on development and DNA detection frequency in bovine embryos

Krisher

Gibbons

Canseco

et al. 1994

Transgenic Research

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“…A total of 105 1-cell, 49 2-cell, and 43 4-cell embryos were microinjected with whey acidic protein-human protein C; 50 1-cell, 8 2-cell, and 19 4-cell embryos remained uninjected. Five (3%) 1-cell embryos were destroyed by DNA microinjection.…”

Section: Resultsmentioning

confidence: 99%

Effects of Time of Deoxyribonucleic Acid Microinjection on Gene Detection and In Vitro Development of Bovine Embryos

Canseco

Russell

Johnson

et al. 1994

Journal of Dairy Science

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In vivo fertilized embryos were surgically collected from superovulated dairy cows to evaluate microinjection on embryo development and utilized the polymerase chain reaction technique for selection of transgenic embryos. Seventy-two percent of the embryos with visible pronuclei or nuclei were microinjected with DNA, and the remaining 28% served as uninjected controls. All embryos were cocultured with bovine oviductal epithelial cells. Mean final development scores of embryos within the same initial cell stage at collection were unaffected by microinjection. After 144 h of culture, 45% of the microinjected embryos developed to the morula or blastocyst stage. The transgene was detected in 50, 10, and 9% of demimorulae from embryos microinjected at the 1-, 2-, and 4-cell stages. Frequency of transgene detection was higher in morulae from 1-cell embryos than in morulae from 2-and 4-cell embryos. Use of in vitro coculture, embryo bisection, and polymerase chain reaction technique facilitated selection of bovine embryos that carried the transgene. (Key words: deoxyribonucleic acid microinjection, bovine embryo culture) Abbreviation key: OEC = oviductal epithelial cells, PCR = polymerase chain reaction.

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Electroporation of bovine spermatozoa to carry foreign DNA in oocytes

Gagné¹,

Pothier

Sirard³

1991

Molecular Reproduction Devel

101

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In the present study, electroporation was used to test the ability of spermatozoa to carry foreign DNA into the bovine oocytes. Frozen-thawed bovine spermatozoa (10(7)/ml) were electroporated using six different combinations of voltage (500, 1,000, or 1,500 V) and capacitance (1 or 25 microFarads) in the presence of 1 mg/ml of plasmid pRGH527. The portions of plasmids retained by sperm cells after three washings (stable for ten washings) were 4.3, 5.5, 5.1, 6.0, 6.8, and 5.8% for 1 microFarad, 500, 1,000, and 1,500 V and 25 microFarads, 500, 1,000, and 1,500 V, respectively. Nonelectroporated cells have retained only 1% of plasmids. In the same experiment, electroporated spermatozoa were acrosome reacted by treatment with ionophore A23187 to evaluate the fraction of marked plasmids joined at the acrosomal membrane. The results show that 3.5, 5.0, 4.4, 5.0, 6.3, and 4.4% remain tied to the ionophore-treated sperm. Only 0.7% of plasmid was retained after removal of the acrosome of nonelectroporated cells. Acrosome reaction was not significantly induced by the electrical field (EF) (P less than 0.005). EF decrease motility significantly for greater than 100 V in 0.3 M mannitol (M) and mannitol-TALP (MT) (1/1) media and greater than or equal to 500 V (P less than 0.05) in TALP medium. The retained plasmid rate was compared between TALP medium M and MT media and resulted in a percentage of 1.0, 2.5, 6.5 at 1 microFarads, 100 V, and 0.9, 3.8, and 3.8 at 25 microFarads, 100 V in TALP, MT, and M medium, respectively. Sperm cells electroporated at 1 microFarad, 500 or 1,000 V, 25 microFarad, 500 V or 1,000 in TALP medium hold plasmids in proportion of 5.2, 5.4, 7.4, and 6.0%. Electroporation above 100 V in M and MT killed the cells. In a part of this experiment, spermatozoa electroporated in the presence of radiolabeled plasmids have been treated with DNase I and results revealed that 35, 28, 54, 58, and 3% of marked DNA remains in sperm cells following digestion after electroporation in TALP (1,000 V, 1 and 25 microFarads), M medium (100 V, 1 and 25 microFarads), and control, respectively. Using in vitro matured bovine oocytes, the electroporation conditions were correlated with the fertilization rate (85% for control and 55% for electroporated spermatozoa). Autoradiography of embryos following fertilization indicated the presence of plasmids in the cytoplasm and in the zona pellucida.(ABSTRACT TRUNCATED AT 400 WORDS)

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Developmental potential of early bovine zygotes submitted to centrifugation and microinjection following in vitro maturation of oocytes

Cited by 11 publications

References 23 publications

Influence of time of gene microinjection on development and DNA detection frequency in bovine embryos

Influence of time of gene microinjection on development and DNA detection frequency in bovine embryos

Effects of Time of Deoxyribonucleic Acid Microinjection on Gene Detection and In Vitro Development of Bovine Embryos

Electroporation of bovine spermatozoa to carry foreign DNA in oocytes

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