Freezing of pejerrey embryos at -14 AND -20°C with microinjection of a cryoprotective solution.

MACORETTA CL; MIRANDA LA

Manaos

Simposio; 11th International Symposium on Reproductive Physiology of Fish; 2018

IntroductionThe pejerrey (Odontesthesbonariensis) is a multiple seasonal spawner with a great economicimportance and is considering appropriated for aquaculture. For this reason isnecessary to apply biotechnologies to optimize its reproduction in captivity.In this context, the objective of this work was to develop a protocol forfreezing pejerrey embryos.  MethodsPejerrey embryos with non-pigmented optical vesicles were selected fromthe Hydrobiological Station of Chascomús (Buenos Aires, Argentina). Embryoswere exposed to 1ml of cryoprotective solution in cryovials for 10 minutes at0° C (equilibrium temperature) and subjected to rapid (-6.6°C/min) or slowfreezing (from 0°C to -7°C; -2°C/min; plateau of 10 min; -0.5°C/min) until a final temperature of -14 or -20°C.At these temperatures the embryos were stored for 1 hour. Two differentsolutions were used. Solution 1 (S1) was composed of 10% v/v methanol; DMSO 10%v/v, Sucrose 10% w/v, NaCl 5g/l; and Solution 2 (S2), with the same compositionof S1 but without DMSO. Moreover, other assays were done microinjecting embryosin the perivitelline space with 32.2nl of S1 or S2 and using the same freezingprotocols described above. The freezing equipment was Cryologic (Australia).The microinjection´s needles were made from glass capillaries using the PC10-Puller(Narishige, Japan) and the microinjection was carried out using themicroinjector Nanoject II (Drummond, USA). In all cases, the cryovials werethawed at 37°C for 1 minute 30 seconds, and the embryos were washed 3 timeswith 5 ml of incubation water. Finally, they were incubated in a programmableincubator (Ingelab, Argentina) at 22°C and photoperiod 12h L: 12h O untilhatching. All the assays were done by triplicate with 10-12 embryos pertreatment. Embryos survival was evaluated daily as well as hatching rate andlarval survival (with starvation condition). Results and Discussion High rates of embryo survival post-thawing were obtained in all assays(80-100%). Particularly, at the slow freezing until -14°C and -20°C and at therapid freezing until -20°C with microinjection of S2 there were nosignificantly differences between the hatching rates of treatment embryos andthe control embryos (-14°C: 86,67±11,55 %; -20°C: 67,93±8,31 %). Even thoughthere were detected different morphological abnormalities in the larvae fromfrozen embryos, its rate survival were no significantly different with the ratesurvival of the control larvae (6,62±1,21 days).                                        ConclusionThese preliminary results show that it is possible to freeze pejerreyembryos and establish the basis for future trials in this field.