This study developed a protocol for freezing pejerrey embryos using microinjection of cryoprotective solutions. High hatching rates (80-100%) were obtained when embryos with optic vesicles and no pigment were frozen for 1 hour at -14°C or -20°C after microinjection of solutions containing methanol, DMSO, and sucrose. While some morphological abnormalities occurred in larvae from frozen embryos, larval survival rates were at least 40% for most conditions tested. The results establish a basis for further optimization of cryopreservation methods in pejerrey embryos.

1. FREEZING OF PEJERREY EMBRYOS AT –14 AND –20°C
WITH MICROINJECTION OF A CRYOPROTECTIVE SOLUTION
MACORETTA, C.1
, MIRANDA, L.1
1
Laboratorio de Ictiofisiología y Acuicultura, IIB-INTECH, CONICET-UNSAM
(Chascomús, Buenos Aires, Argentina)
The pejerrey (Odontesthes bonariensis) is a multiple seasonal spawner with a great
economic importance and is considering appropiated for aquaculture. For this
reason is necessary to apply biotechnologies to optimize its reproduction in captivity.
In this context, the objective of this work was to develop a protocol for freezing
pejerrey embryos.
1- INTRODUCTION 3- RESULTS
2- METHODS
Lic. Christian Macoretta, PhD Student
(University of Buenos Aires); clmacoretta@intech.gov.ar
 Microinjector Nanoject II (Drummond, USA)
 Glass capillaries
 PC-10 Puller (Narishige, Japan)
 Cryoprotective solutions (CS: S1 & S2)
S1: Methanol 10% v/v; DMSO 10% v/v; Sucrose 10% w/v; NaCl 5g/l
S2: Methanol 10% v/v; Sucrose 10% w/v; NaCl 5g/l
 Phenol Red 0.5% KCl 100mM (1μl dye/10μl Cryoprotective solution)
 Agarose matrix
 Cryovials 2ml
 Freezing equipment (Cryologic, Australia)
 Programable incubator (Ingelab, Argentina)
 It was possible to freeze pejerrey embryos for 1 hour at two different
temperatures (-14 and –20°C) obtaining high hatching rates.
 This work showed that microinjection is a viable method for the introduction of
cryoprotective solutions into the perivitelline space of pejerrey embryos with optic
vesicles without pigment.
 These preliminary results establish the basis for future trials in this field. It
would be convenient to try other cryoprotective solutions, freezing curves and also
early embryonic stages.
Selection of embryos with optic vesicles without pigment (n=10-12, triplicate)
Without microinjection
Microinjection of 32nl of
S1 or S2
Without any treatment
Exposition to S1 or S2 (1ml)
Freezing at -14°C Freezing at –20°C
Incubation: 22°C 12h L: 12h O
Washings: 3 x 5ml Incubation wáter (Tap wáter + NaCl 5g/l)
High rates of embryo survival post-thawing were obtained in the major part of the
assays (80-100%). The data was expressed as means (±SD) and one-way
ANOVA and Dunn´s multiple comparisons test were used to analyze them
(*: p<0.05; **: p<0.01).
A– Freezing at –14°C
Particularly, at the fast freezing with the S1 and at the slow freezing with both
solutions there were no significant differences between the hatching rates of
treatment embryos and the control embryos (S1 Fast:80%: S1 slow: 86.67±11.55%;
S2 Slow: 86.67±15.28%; Control: 93.33±8.16%).
However, there were detected different
morphological abnormalities in the larvae
from frozen embryos. Even though this
situation, the rate of swimming larvae was
at least of 40% in three of the four
conditions tested (S2 Fast: 57.14±24.74%;
S1 Slow: 38.33±25.05%; S2 Slow:
43.49±12.28%) and there was not found
any significant difference between the
larvae survival curves .
Fast freezing Slow freezing
Evaluation of embryos survival, hatching rate and larval survival (with starvation condition)
Thawing: 37°C 1min 30sec
In the case of the freezing at –20°C
without microinjection, hatching rates
above 60% were obtained in three of
the four conditions tested. Moreover
these hatching rates did not show
significant difference with the control
(S2 Fast: 60±10%;
S1 Slow: 66.67±25.17%;
S2 Slow: 63.33±15.28%;
Control: 93.33±5.16%).
On the other hand, in the case of the
freezing at –20°C with microinjection
the hatching rates were lower than in
the trial without microinjection.
However, in one of the four conditions
tested there was not significant
difference between its hatching rate
and the hatching rate of the control
(S2 Fast: 67.93±8.31%; Control:
92.59±7.73%).
B– Freezing at –20°C
4- CONCLUSIONS