2011 (SBTE) 25th Annual Meeting Proceedings - International ...

Acta Scientiae Veterinariae, 2011. 39(Suppl 1): Abstracts - 25th Annual Meeting SBTE-Brazil. August 2011.
A235 SUPPORTIVE BIOTECHNOLOGIES: CRYOPRESERVATION AND CRYOBIOLOGY, IMAGE ANALYSIS AND DIAGNOSIS, MOLECULAR BIOLOGY AND “OMICS”
CRYOPRESER
OPRESERVATION OF SOMATIC CELLS IN 0.5 ML OR 0.25 ML STRAWS S AND PROPYLENE GLYCOL OR ETHYLENE GLYCOL
Monica Urio 1 , Claudia Regina Appio Duarte 1 , Renata Cristina Fleith 1 , Maria Luiza Munhoz 1 , Fabiano Carminatti Zago 1 , Fabiana Forell 1 Alceu Mezzalira 1 ,
Marcelo Bertolini 2 & Ubirajara Maciel da Costa 1
1
CAV - UDESC, LAGES, SC, BRAZIL. 2 UNIFOR, FORTALEZA, CE, BRAZIL.
The standard protocol for the cryopreservation of somatic cells makes use of cryovials as classic containers for the storage of
cells, and dimethyl sulfoxide (DMSO) as the cryoprotectant agent of choice. However, cryovials take significant space into LN2 tanks, and
DMSO can be highly toxic and induce differentiation in cells in culture. Thus, the aim of this study was to establish a protocol for
cryopreservation of somatic cells in straws comparing the efficiency of three cryoprotectant agents: DMSO, Ethylene Glycol (EG) and
Propylene Glycol (PG). Primary cell cultures of bovine fibroblasts were maintained in DMEM + 10% FCS in an incubator at 5% CO2 in air
and saturated humidity. Such cells were used for nine experimental groups varying the cryoprotectant agent (DMSO, EG, PG) and the
container (0.25 and 0.5 mL straws and cryovials). Cells were frozen in a solution containing 10% of one of the cryoprotectants in culture
medium. For this, cells were loaded into the container and exposed to the cryoprotectant at 4° C for 15 min before freezing. Straws were kept
in LN2 vapor for 5 min and then immersed in LN2, whereas cryovials were cooled at -1°C/min down to -80°C, using Mr. Frosty ® (Nalgene),
and then transferred to LN2. The experiment was separated in two steps for the survival study, evaluated by the Trypan blue staining. In the
first step, the survival rate was evaluated immediately after thawing (seven replications). In the second step, cell survival was evaluated after
24 h of culture (only for the groups usning straws, in nine replications). Results were evaluated by the ÷2, for P < 0.05. The rate of cell survival
immediately after thawing showed no significant differences between the cryoprotectants and containers, with survival rates for DMSO, EG
and PG being 83%, 86% and 87% in 0.5 mL straws ; 81%, 83% and 87% in 0,25 mL straws; and 87%, 90% and 91% in cryovials, respectively.
Survival rates after 24 h of culture also showed no significant difference between cryoprotectants (DMSO, EG or PG), with survival rates
being 91%, 90% and 91% for 0.5 mL straws, and 91%, 91% and 93% for 0.25 mL straws, respectively. In summary, the freezing of somatic
cells in straws was proven practical and feasible, facilitating storage of larger batches of cells in LN2 tanks, also providing the same cell viability
after freezingas cryovials. Moreover, EG and PG can be readily used as cryoprotectant agents for somatic cell freezing with the same cell
survival efficiency as for DMSO.
Keywords: somatic cell, cryoprotectants, straws.
A236 SUPPORTIVE BIOTECHNOLOGIES: CRYOPRESERVATION AND CRYOBIOLOGY, IMAGE ANALYSIS AND DIAGNOSIS, MOLECULAR BIOLOGY AND “OMICS”
OVARIAN
TISSUE CRYOPRESER
OPRESERVATION FROM BITCHES:
MORPHOLOGIC OGIC EVAL
ALUATION OF PRE-ANTRALS ALS FOLLICLES
José Luiz Jivago de Paula Rôlo 1 , Michelle Silva Araújo 2 , Fernanda Paulini 1 & Carolina Madeira Lucci 1
1
PPG BIOLOGIA ANIMAL, IB, UNB, BRASILIA, DF, BRAZIL. 2 FACULDADE DE AGRONOMIA E MEDICINA VETERINÁRIA, UNB, BRASILIA, DF, BRAZIL.
The ovarian tissue can be used as an storable source of oocytes, especially enclosed in pre-antral follicles (PAF), and its
cryopreservation is well established in some species, with few reports in dogs (ISHIJIMA et al., 2006, J. Reprod. Dev. 52, 293-299). The slow
freezing (SF) is the most used technique, but the vitrification (V) is an alternative that offers advantages in convenience. There are no studies
comparing the two methods in bitch ovarian tissue. The aim of this study was to compare the effects of SF and V techniques in the bitch
ovarian tissue in the morphology of PAF. Ten ovaries from five healthy bitches were collected after elective ovariohysterectomy and
transported to the lab in buffered saline solution at 37°C. The ovaries were cut in fragments of 1x1x3 cm and randomly assigned to the
procedures of SF, V or immediate fixation (control, C). For SF, the tissue samples were equilibrated in a programmable freezer (Biocom) for
20 min at 10°C in a solution of 1.5M DMSO, 0.4% sucrose, 10% FBS in MEM. Then submitted to the following freezing curve: -1°C/min
until -7°C, when seeding was performed, and -0.3°C/min until -30°C. At the end, the samples were stored in liquid nitrogen. The V method
was based on ISHIJIMA et al. (2006), but the vitrification was done in a solid surface. After 7 days the samples were placed at 37°C and
equilibrated in 0.4% sucrose, 10% FBS and MEM, and washed 3 times with decreasing sucrose concentration. Thawed fragments and the C
were fixed in Carnoy and processed for light microscopy with HE staining for the morphological evaluation of PAF. The percentages of normal
follicles (NF) in the different treatments were analyzed by ANOVA and Tukey´s test. The total percentage of normal PAF was 93.66±6.81%
for C, 86.16±11.05% for SF and 68.14±12.75% for V. For primordial follicles the percentage of NF was 96.69±4.72% for C, 89.51±10.39%
SF and 75.32±9.23% in V. For primary and secondary follicles the values were, respectively, 94.80±6.91% and 87.62±17.12% for C,
86.8±12.15% and 76.35±26.34% for SF and 61.53±14.78 and 52.25±22.13% for V. The percentage of NF in V was significantly lower (P <
0.05) than in the other two treatments, except for the secondary follicles, where no significant difference between SF and V was observed.
There was no significant difference between SF and C for any follicular class. In conclusion, SF is the best cryopreservation method for PAF
in bitches ovarian tissue, and the V protocol needs improvement for a better preservation of the morphological characteristics of the follicles.
Keywords: slow freezing, vitrification, oocyte.
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