In vitro fertilization with hormonally induced sperm and eggs from sharp-ribbed newts Pleurodeles waltl

Abstract

To address the urgent need for the perpetuation of amphibian genetic variation, particularly in Conservation Breeding programs, we developed reproduction technologies for the hormonal induction and sampling of viable sperm and eggs from the sharp-ribbed newt Pleurodeles waltl, followed by successful in vitro fertilization.We also tested the capacity for the short-term refrigerated storage of spermatozoa at +4°C. We used two gonadotropic hormones, Human Chorionic Gonadotrophin (hCG) and Luteinising Hormone Releasing Hormone analogue (LHRHa). The osmolality of P. waltl urinal sperm varied from 23 mOsmol/kg with 6 × 103 spermatozoa/ml to 58 mOsmol/kg with 22 × 103 spermatozoa/ml with a mean osmolality of 34 ± 4 mOsmol/kg. The volumes of urinal sperm collected at 3, 24, and 48 h after injection of chorionic gonadotropin were higher than the volumes collected within the same time intervals after injection of surfagon. However, concentrations of spermatozoa in urinal sperm collected over the first 48 hrs after injection of surfagon (~15.0 × 106/ml) were higher than after injection of chorionic gonadotropin (~0.4 × 106/ml). Therefore the total amount of spermatozoa collected with the use of surfagon was higher than the total amount collected with the use of chorionic gonadotropin. Spermatozoa maintained motility up to 120 h, with 18% still motile after 72 h. We achieved 76% fertilization of eggs and survival of these to hatch of 67%.