Production of F sub(1) Offspring with Vitrified Sperm from a Live-Bearing Fish, the Green Swordtail Xiphophorus hellerii

This study reports the first production of offspring with vitrified sperm from a live-bearing fish Xiphophorus hellerii. The overall goal of this study was to develop streamlined protocols for integration into a standardized approach for vitrification of aquatic species germplasm. The objectives were to (1) estimate acute toxicity of cryoprotectants, (2) evaluate vitrification solutions, (3) compare different thawing methods, (4) evaluate membrane integrity of post-thaw sperm vitrified in different cryoprotectants, and (5) evaluate the fertility of vitrified sperm. Nine cryoprotectants and two commercial vitrification additives were tested for acute toxicity and glass forming ability, alone and in combination. Two vitrification solutions, 40% glycerol (Gly) and 20% Gly + 20% ethylene glycol (EG) in 500mOsmol/kg Hanks' balanced salt solution (HBSS), were selected for vitrification of 10 mu L sperm samples using inoculating loops plunged into liquid nitrogen. Samples were thawed at 24 degree C (one loop in 5 mu L of HBSS or three loops in 500 mu L of HBSS). Samples thawed in 500 mu L were concentrated by centrifugation (1000 g for 5min at 4 degree C) into 5 mu L for artificial insemination. Offspring were produced from virgin females inseminated with sperm vitrified with 20% Gly + 20% EG and concentrated by centrifugation.