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

Year: 2025

Type: article

Abstract: Nematode infective larvae (L 3 ) of veterinary importance have been cryopreserved in liquid nitrogen (LN 2 ) 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 L 3 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 LN 2 before storage at −150 °C (LN/-150°C); stored directly at −150°C or stored in LN 2 (LN). In vitro survivability of L 3 (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 % CO 2 prior to dead/alive assessment. An in vivo study was undertaken with L 3 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. • Three cryopreservation strategies were assessed for ovine infective larvae. • -150°C freezing was better than liquid nitrogen-based cryopreservation techniques. • -150°C consistently shown ≥ 90 % in vitro survivability for all isolates. • In vivo testing showed differences in establishment with various storage conditions.

Source: Veterinary Parasitology

DOI: 10.1016/j.vetpar.2025.110422

Authors: A.A. Morrison, L.M. Andrews, M. O’Shea, E. Geddes, D.J. Bartley

Institutions: Moredun Research Institute, Roslin Institute, University of Edinburgh

Language: English

Topic: Helminth infection and control

Subfield: Small Animals

Field: Veterinary

Domain: Health Sciences

SDG: Zero hunger

Open Access status: hybrid

Estimated APC paid: $3,680