Collection of gametes from live axolotl, Ambystoma mexicanum, and standardization of in vitro fertilization

• Published in: Theriogenology 2011-01, Vol.75 (2), p.354-361
• Main Authors: Mansour, N., Lahnsteiner, F., Patzner, R.A.
• Format: Article
• Language: English
• Subjects: abdominal massage semen collection technique; Ambystoma; Ambystoma mexicanum; Ambystoma mexicanum - physiology; Amphibians; Animals; Axolotl; Cell Separation; embryogenesis; Female; Fertilization; Fertilization in Vitro - methods; Fertilization in Vitro - standards; Fertilization in Vitro - veterinary; germ cells; Germ Cells - cytology; Handling (Psychology); human chorionic gonadotropin; in vitro fertilization; Male; Oocyte Retrieval - methods; Oocyte Retrieval - standards; Oocyte Retrieval - veterinary; osmolality; ovulation; Quality Control; Reference Standards; salamanders and newts; Semen; simulation models; spawning; Sperm Retrieval - standards; Sperm Retrieval - veterinary; Urodele
• ISSN: 0093-691X; 1879-3231
• DOI: 10.1016/j.theriogenology.2010.09.006

This study established the first protocol for collection of gametes from live axolotl, Ambystoma mexicanum, by gentle abdominal massage and in vitro fertilization. To stimulate spermiation and ovulation, human chorionic gonadotrophin (hCG) and Ovopel pellets, which are commercially used to stimulate spawning in fish, were tested. The hCG was more effective than Ovopel pellets and yielded a higher semen volume in the injected males and a shorter response time in the females. Collected semen by this method was already motile and fertile. Fertile eggs could be collected in 3–4 successive collection times after the female has started the typical spawning behaviour. The fertilization condition that yielded the highest hatching rate was mixing semen with eggs before the addition of a fertilization saline solution (20 mmol/l NaCl, 1 mmol/l KCl, 1 mmol/l Mg 2SO 4, 1 mmol Ca 2Cl, 3 mmol NaHCO 3, 10 mmol/l Tris, pH 8.5 – Osmolality = 65 mosmol/kg). When the pH of the fertilization solution was increased to ≥ 10, the hatching rate was significantly increased. The use of fertilization solutions with osmolalities of ≥ 150 and ≥ 182 were accompanied with a significant decrease in hatching rates and the appearance of deformed larvae, respectively. In conclusion, a reliable protocol for gamete collection from live axolotl is established as a laboratory model of in vitro fertilization for urodele amphibians. This protocol may be transferable to endangered urodeles.