Doctor of Philosophy (PhD)
Forestry, Wildlife, and Fisheries
Sperm from zebrafish, Danio rerio, and Nile tilapia, Oreochromis niloticus, were microinjected directly into egg cytoplasm to evaluate the potential for developing a novel method of fertilization. In zebrafish, the sperm of two lines (wild-type and gold, long-fin) were injected with or without activation into activated and non-activated eggs. No significant difference (P = 0.997) in fertilization by intracytoplasmic sperm injection (ICSI) was observed between the two lines or when the sperm were activated or not (P = 0.057). There was significance difference in fertilization between activated and non-activated eggs (P = 0.010). The highest fertilization rate was achieved by injection of activated sperm into non-activated eggs (35%). From a total of 188 zebrafish eggs injected, 31 (16%) were fertilized, 10 (5%) developed as abnormal larvae and 3 (2%) developed normally and hatched. Damage of maternal chromosomes by the injection procedure could have caused the developmental abnormalities observed after ICSI. This was investigated by fluorescence microscopy using a DNA-specific stain (Hoechst 33324). Fixed and stained animal poles of zebrafish 30 sec after artificial insemination revealed that female chromosomes were located ~40 mm from the sperm injection site (micropyle). Staining of the animal pole after sperm injection showed no disruption of the formation of the second polar body or its extrusion. Evaluation of two sperm injection sites in zebrafish showed no difference in fertilization rate (P = 0.8264) or reduction of abnormal development. Nile tilapia eggs placed in Hanks' balanced salt solution retained their viability for at least 3 hours after collection. Of a total of 160 Nile tilapia eggs injected with fresh sperm, 16 (10%) were fertilized, 10 (6%) developed abnormally to neurula and 5 (3%) developed normally and hatched, two of which reached adulthood. From 45 eggs injected with cryopreserved sperm, 9 (20%) were fertilized but none developed beyond blastula stage. Injections of sperm fixed in methanol did not yield fertilization. These results demonstrate for the first time that injection of single sperm cells into the cytoplasm of a fish egg allows fertilization and subsequent development of normal larvae to hatching and beyond.
Document Availability at the Time of Submission
Release the entire work immediately for access worldwide.
Poleo, German Antonio, "Intracytoplasmic sperm injection (ICSI) in fishes" (2002). LSU Doctoral Dissertations. 3615.