Application of intracytoplasmic sperm injection (ICSI) for fertilization and development in birds

• We developed ICSI-assisted fertilization and gene transfer in quail.
• The oocyte retrieved from the oviduct and a sperm was injected into the oocyte.
• After 24-h culture, the fertilization and development was assessed by microscopic observation.
• The fertility rate was about 15% but was increased over 2 folds by PLCZ.
• In the gene transfer experiment, GFP expression was observed in the blastoderm of ICSI-treated quail egg.

Intracytoplasmic sperm injection (ICSI) technology in birds has been hampered due to opacity of oocyte. We developed ICSI-assisted fertilization and gene transfer in quail. This paper reviews recent advances of our ICSI experiments. The oocyte retrieved from the oviduct and a quail sperm was injected into the oocyte under a stereomicroscope. The oocyte was cultured for 24 h at 41 °C under 5% CO2 in air. The fertilization and development was assessed by microscopic observation. The fertility rate ranged 12–18% and development varied from stage II to V in trials. To improve the fertility rate, phospholipase C (PLC) zeta was injected with a sperm. It was increased to 37–50%. Furthermore, injection of inositol trisphosphate increased to over 85%. Quail oocyte can be fertilized with chicken sperm and so can testicular elongated spermatid. To extend embryonic development, chicken eggshell was used as a surrogate culture at 37 °C after the 24 h incubation at 41 °C under 5% CO2 in air. It survived up to 2 days thereafter. Finally, gene transfer was attempted in quail egg. The sperm membrane was disrupted with Triton X-100 (TX-100) and was injected with PLCzeta cRNA and enhanced green fluorescent protein (EGFP) gene in oocyte. The GFP expression was evaluated at 24 h incubation at 41 °C under 5% CO2 in air in the embryos. While the expression was not detected in the control oocytes, the experimental treatment induced blastoderm development (44%) of the oocytes and 86% of blastoderm showed fluorescent emission. In addition, PCR analysis detected EGFP fragments in 50% of GFP-expressing blastoderm. Our ICSI method may be the first step toward the production of transgenic birds.

Possible molecular pathway in the quail egg in relation to oocyte activation.(PLC zeta is relaeased from sperm).Figure optionsDownload as PowerPoint slide