ORIGINAL PAPER Storage of gastrointestinal nematode infective larvae for species preservation and experimental infections C. Chylinski & J. Cortet & G. Sallé & P. Jacquiet & J. Cabaret Received: 11 September 2014 /Accepted: 14 November 2014 /Published online: 4 December 2014 # Springer-Verlag Berlin Heidelberg 2014 Abstract Techniques to preserve the infective third-stage larvae (L3) of gastrointestinal nematodes are of considerable interest to preserve rare species and to maintain a stable source for routine experimental infections. This study compares the relative pros and cons of the two most common techniques, cryopreservation and refrigeration by comparing how they influence consequent infection outcome parameters in terms of life-history traits and fitness as a function of time using the gastrointestinal nematode of sheep Haemonchus contortus as a study species. Establishment capacity was found to be significantly reduced in cryopreserved stocks of L3 compared to refrigerated stocks, but this was followed by significant increases in their fecundity. Refrigeration did not affect L3 stocks consequent fitness by 16 months (the maximum exam- ined) although they did incur a significant reduction in estab- lishment, followed once again by an augmentation in fecun- dity. The study highlights potential areas for bias in comparing studies using L3 larvae maintained for different periods of time under different techniques. Keywords Cryopreservation . Fitness . Haemonchus contortus . Life-history traits . Refrigeration Introduction Techniques to preserve the infective third-stage larvae (L3) of gastrointestinal nematodes (GIN) are of great experimental interest, both to preserve rare species selected for particular aptitudes, i.e. anthelmintic resistance, morphology etc. and as a source for routine experimental infections. Effective preser- vation techniques reduce labour and financial costs associated with maintaining differing isolates in constant cycle through their obligate hosts and further minimize the potential for genetic drift or unexpected selection that may result from repeated passage (Chehresa et al. 1997; Gasnier et al. 1992). Two techniques are commonly used in the preservation of GIN L3 stages. The first is cryopreservation whereby the L3 are ex-sheathed and maintained in vials of physiological se- rum in liquid nitrogen (VanWyk et al. 1977). Studies using the sheep GIN Haemonchus contortus have found that not only are the worms recoverable after 10 years of storage (Rew and Campbell 1983), they also remain infective to sheep (Campbell et al. 1973). The second technique is to maintain stocks of L3 refrigerated at 4 °C (MAFF 1986). This tech- nique takes advantage of GIN L3 physiology. The L3 possess an external protective sheath to provide greater resistance against environmental variables; however, it also prevents the L3 from feeding (O’Connor et al. 2006). The L3 therefore exist on a limited energy budget. While elevated temperatures can cause a rapid decline in their energy reserves by increasing their metabolic rate (Vlassoff et al. 2001), maintaining L3 stocks at cooler temperatures can lower their metabolism to sustain their energy reserves. The refrigeration technique was first outlined in the Ministry of Agriculture, Fisheries and Food (MAFF) manuals (1977, 1986); however, the sources on which this information was based are not documented. While there is some information regarding the efficacy of the cryopreserved techniques (Van Wyk et al. 1977, 2000; Rew and Cambell 1983; Van Wyk and Gerber 2000), no studies have compared this technique against refrigeration. Cryopreservation has a clear duration advantage over re- frigeration. Studies by Van Wyk et al. (2000) found that H. contortus, among other nematode species, could survive C. Chylinski : J. Cortet : G. Sallé : J. Cabaret (*) INRA, UMR 1282, ISP 213, 37380 Nouzilly and F. Rabelais University, 37000 Tours, France e-mail: jacques.cabaret@tours.inra.fr P. Jacquiet Ecole Nationale Vétérinaire de Toulouse, 23 Chemin des Capelles, 31300 Toulouse, France Parasitol Res (2015) 114:715–720 DOI 10.1007/s00436-014-4238-5