Mice Embryos Cryopreservation: Vitrification or Ultra-Rapid Freezing?
Acta Scientiae Veterinariae
Mice Embryos Cryopreservation: Vitrification or Ultra-Rapid Freezing?
Autor Correspondente: Jamir Machado Jr | [email protected]
Palavras-chave: glass micropipettes, ops, freezing, murine, morulae
Resumos Cadastrados
Resumo Inglês:
Background: The importance of mice as animal model for research has promoted the surge of many strains with important
characteristics which need to be preserved. Embryo cryopreservation appears as the most suitable technique. However, until
now there is not an effective methodology for this specie. This study aimed to evaluate three methods for mice embryos
cryopreservation.
Materials, Methods & Results: Morulae (n = 565) were obtained from 42 F1 females (Balb C males X C57 Black females) 6 to
8 weeks old, super-ovulated with 10 IU of eCG, and 10 IU hCG 46 h later. Recovered embryos at morulae stage were randomly
allocated in the experimental groups: Fresh controls (CG), vitrification in glass micropipettes (GMV), vitrification in 0.25 mL
straws (SV) and ultra-rapid freezing (UR). For vitrification, embryos were exposed for 60 s to 10% ethylene glycol (EG) + 10%
propylene glycol (PROP), followed by exposure for 25 s to 20% EG + 20% PROP, while were loaded according to the
experimental group (GMV or SV) and immersed in liquid nitrogen (LN2). Re-warming was performed with decreasing sucrose
(SUC) solutions (0.3 M and 0.15 M), for 5 min each. In UR group, embryos were exposed for 5 min in 3.0 M glycerol + 0.5 M
SUC in D-PBS + 20% EMS, loaded in straws and maintained for 1 min 2 cm above the LN2, and then plunged in it. Thawing
was performed in a water-bath at 37ºC, for 20 s, followed by exposure for 5 min in 0.5 M SUC solution. In vitro culture was
performed in D-PBS + 20% EMS for 72 h, at 39ºC and 5% CO2. The first evaluation at 24 h of culture considered blastocyst
stage as viability criteria. The second evaluation was performed at 72 h of culture and considered hatching rate as viability
criteria. Data were analyzed by χ2 test (P <0.05). The GMV group presented the highest blastocyst rate among all treatments
(95.6%), as well as the highest hatching rate (94.9%), similar to the CG (97.8% and 93.4% respectively). The UR group
presented 91.9% of blastocysts, which was higher than SV, but lower than CG. The SV group had lower blastocyst rate (78.5%)
than other treatments. Conversely, the hatching rate presented (76.0%) was higher than the UR (61.3%). Additionally, 126
morulae vitrified in GMV were transferred to 7 pseudo-pregnant recipient females (18/recipient), with two of the recipients
(28.5%) delivered six and nine pups, respectively.
Discussion: The most commonly employed methodologies for mouse embryos cryopreservation are the ultra-rapid freezing
and vitrification. In previous studies we obtained high blastocyst rates (83.6 to 92.9%) with the ultra-rapid freezing of mice
morulae. The embryo stage of development influences viability of cryopreserved embryos, with the highest viability at the
morula stage. In this study, the blastocyst rate obtained after the ultra-rapid freezing (91.9%) characterized the high repeatability
of the technique, as well as highlighted the morula stage as suitable for cryopreservation. The GMV group showed the highest
development rate among the treatments (95.6%), which did not differ from control group (97.8%). Since the GMV and SV
groups used the same cryoprotectant solution, we can infer that divergences in viability between the two groups were
determined by the loading container type, as well as the volume of solution used during cryopreservation. The glass micropipettes
provide higher cooling rates than plastic straws, even though it is possible that the lower cooling rate provided by the straws,
associated to a major volume of extender determined the lower viability of such treatments. It is possible that the SV had
occasioned injuries that might have prevented a significant percentage of morulae from developing to the blastocyst stage.
Conversely, once they had reached this stage, the viability was higher (96.8%). Whereas for the UR treatment, apparently the
injuries did not prevent their development to the blastocyst stage, being the negative effect thus been manifested later, by
reducing hatching rates (61.3%). Data allow us to conclude that vitrification in glass micropipettes is the most suitable method
for cryopreservation of mice morulae, providing viability rates similar to those obtained with fresh embryos, and health live
puppies, after embryo transfer.
