Intracytoplasmic Sperm Injection in the Rhinoceros

Assisted reproductive technologies (ART) have become routine remedies for human infertility. One ART, known as intracytoplasmic sperm injection, or ICSI, is especially useful for the production of embryos when sperm numbers are severely limited or when sperm motility is impaired. Because ICSI involves the microinjection of a single sperm into each ovum, it is an excellent means of conserving valuable sperm from endangered species.

When southern white rhinoceros Ujima died at the San Diego Zoo's Wild Animal Park, her ovaries were removed during a routine necropsy. Six morphologically normal immature oocytes were extracted from her ovaries and placed into cell culture for in vitro maturation (IVM) by the Reproductive Physiology Division staff at CRES. Because no protocols have been developed for IVM in the rhinoceros, the culture medium and conditions used for the closely related domestic horse provided the basics, and modifications were made to tailor the procedure to the rhinoceros.

Each day the six oocytes were examined under the microscope for evidence of maturation or degeneration. Four days after the initiation of cell culture the five surviving oocytes were carefully stripped of their granulosa cells, revealing two oocytes with polar bodies. Because conditions for in vitro fertilization (IVF) in the rhinoceros are unknown, and the oocytes had already been in culture for four days, ICSI was performed to ensure rapid entry of the sperm and initiation of the fertilization process. Each mature oocyte was immobilized with gentle suction on a holding pipette while an individual sperm was aspirated into a tiny injection needle. The needle was plunged directly into the oocyte and the sperm was released into the egg's cytoplasm. The oocytes were returned to culture in a standard fertilization medium without rhinoceros follicular fluid.

The day after ICSI the two injected oocytes were examined microsopically. One exhibted two pronucleii, evidence that fertilization had occurred. The maternal chromosomes left in the oocyte cytoplasm after the extrusion of the polar body formed the female pronucleus, indicating that the oocyte was functionally mature and the cytoplasm had been activated by the ICSI procedure. The chromosomes from the injected sperm formed the male pronucleus and indicated that the selected sperm was capable of chromosome decondensation, a prerequisite for zygote formation.

Althought this rhinoceros oocyte did not continue to develop in culture, the success of in vitro maturation and ICSI was a significant step toward in vitro embryo production from postmortem collected oocytes and sperm. Future work will focus on optimizing protocols for harvesting sperm and oocytes, as well as improving in vitro culture conditions for the rhinoceros and many other endangered species.