Comparison of in vitro and in vivo resurrection success of three ovine gastrointestinal nematode species following different cryopreservation strategies

Abstract

Nematode infective larvae (L₃) of veterinary importance have been cryopreserved in liquid nitrogen (LN₂) to minimize the need for continued passage through live animals. Health and safety concerns, combined with increasing pressures to reduce running and maintenance costs have driven the need to explore alternative preservation techniques. Super-cold (-150°C) freezers have been used for long term storage of cell lines, but no published data were available for livestock gastro-intestinal nematodes (GIN). In vitro and in vivo survivability of L₃ of three GIN species (Teladorsagia circumcincta (MTci2), Trichostrongylus colubriformis (MTco1) and Haemonchus contortus (MHco3 and MHco18)) were assessed following three cryopreservation storage methods. In brief, fifty thousand larvae were exsheathed, and cryopreserved using one of three methods; either snap frozen in LN₂ before storage at −150 °C (LN/-150°C); stored directly at −150°C or stored in LN₂ (LN). In vitro survivability of L₃ (dead versus alive) were assessed at approximately 1, 2, 4, 6, 11 and 23 months post −150°C and LN/-150°C storage. Larvae were defrosted and left in PBS overnight at 39.6°C and 10 % CO₂ prior to dead/alive assessment. An in vivo study was undertaken with L₃ following 4 months of storage. The tubes stored directly in −150°C have consistently shown ≥ 90 % in vitro survivability for all isolates, whereas LN/−150°C showed inter species variability (range: 7–63 % survivability). The in vivo assessment demonstrated a significant difference in establishment with overall group mean establishment ranging from 9 % in the LN/−150°C to 62 % of the fresh larvae, with the −150°C and LN groups establishing 25 % and 10 % respectively.