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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. s454
Acta Scientiae Veterinariae, 2011. 39(Suppl 1): Abstracts - 25th Annual Meeting SBTE-Brazil. August 2011. A237 SUPPORTIVE BIOTECHNOLOGIES: CRYOPRESERVATION AND CRYOBIOLOGY, IMAGE ANALYSIS AND DIAGNOSIS, MOLECULAR BIOLOGY AND “OMICS” CRYOTOLER OLERANCE OF BOVINE IN VITRO-PR -PRODUCED EMBRYOS SUBJECTED TO CONJUGA ONJUGATED LINOLEIC ACID Luciana Simões Rafagnin Marinho 1 , Joana Cláudia Mezzalira 1 , Lain Uriel Ohlweiler 1 , Fabiana Forell 1 , Dimas Estrasulas Oliveira 2 & Alceu Mezzalira 1 1 UDESC, LAGES, SC, BRAZIL. 2 UDESC, CHAPECÓ, SC, BRAZIL. In vitro production (IVP) of bovine embryos is an efficient biotechnology to multiply genetically superior animals, being applied on a commercial scale worldwide. Nevertheless, it is limited by the low embryo survival rates after cryopreservation, which restricts the use of surplus embryos obtained in routines. The low cryotolerance of bovine IVP embryos is attributed to excessive accumulation of lipids in the cytoplasm. The isomer of conjugated linoleic acid trans-10, cis-12 CLA is targeted to reduce the lipid content in different cells, including embryos. The aim of this study was to evaluate the effect of trans-10, cis-12 CLA on bovine IVP embryos cryotolerance subjected to vitrification or conventional freezing. Bovine oocytes were selected and subjected to in vitro maturation and fertilization. For in vitro culture, presumptive zygotes were allocated into two groups: CLA group, with 100 µM of trans-10, cis-12 CLA, and control group, free of CLA. Cleavage (D2) and blastocyst (D7) rates were evaluated. Grades I and II expanded blastocysts (n = 156) were subjected to vitrification (20% propanediol + 20% ethylene glycol) or conventional freezing (1,5 M ethylene glycol). After warming, re-expansion and hatching rates were evaluated using the chisquare test with 5% of significance. There was no difference between cleavage rates of control (81.3%) and CLA groups (77.9%) or blastocyst rates between control (30.4%) and CLA groups (28, 5%). After cryopreservation, there was no difference between hatching rates of vitrified control (67.4%) and vitrified CLA groups (65.8%). Both rates were higher than those of frozen control group (28.6%) and frozen CLA group (13.3%), which did not differ among each other (P > 0.05). It can be concluded that, under the conditions of this study, the addition of trans-10, cis-12 CLA to culture medium does not improve cryotolerance of bovine IVP embryos and that vitrification provides greater embryo viability after warming than conventional freezing. Keywords: vitrification, freezing, cla. A238 SUPPORTIVE BIOTECHNOLOGIES: CRYOPRESERVATION AND CRYOBIOLOGY, IMAGE ANALYSIS AND DIAGNOSIS, MOLECULAR BIOLOGY AND “OMICS” BVD VDV V DETECTION BY A POLYMER YMERASE CHAIN REACTION-B CTION-BASED ASSAY IN BOVINE FOLLICULAR FLUID A ndrea Giannotti Galupp aluppo, Ma theus Nunes Web eber er, Rena enata Fon ont oura Budasz udaszew ewsk ski, A ngela Oliv liveir eira Corb orbellini, Lis San ant os Mar arques ques, José Luis Rigo Rodrigues & Claudio Wageck Canal UFRGS, PORTO ALEGRE, RS, BRAZIL. Because of its broad distribution among populations of cattle and association with cells and fluids from infected animals, bovine viral diarrhea vírus (BVDV) represents a potential problem in applications of assisted reproduction (Gard et al. Theriogenology 68: 434-442, N 2007). The use of ovaries and oviducts harvested in abattoirs from unknown health status animals is already an established practice in the production of in vitro embryos. The aim of this study was detect BVDV by RT-PCR from bovine follicular fluid (FF) obtained from in vitro embryo production procedure. The collects were performed between February and December 2010. Bovine ovaries were collected immediately after slaughter at a commercial abattoir and washed with modified phosphate-buffered saline (PBSm). FF was aspirated from ovarian follicles range 2-8 mm in diameter and a 2 mL sample was stored at -80ºC until the moment of use. A total of 22 FF pools were analyzed, from 1844 animals (range 43-136 animals/pool). For the isolation of total RNA was used TRIzol ® LS Reagent (Invitrogen, USA) according to manufacture recommendation. The cDNA was synthesized using the SuperScript ® Reverse Transcriptase Kit (Invitrogen, USA) according to manufacture recommendation. PCR was performed using the primers 324 and 326 (Vilcek et al. Archives of Virology 136: 309–323, 1994). The PCR products were separated by electrophoresis carried out in 2% agarose gel dyed with Blue green loading dye I (LGC Biotecnologia, Brazil). Visualization of amplification products was performed using UV light. It was obtained 3 positive BVDV FF pools (13.63 %). The presence of BVDV has been determined to be detrimental in culture systems, causing a reduction in rates of maturation, fertilization and/or development. Considering that, the use of FF BVDV PCR-assay is an important tool to assure the embryos quality production in vitro. Keywords: bvdv, folicular fluid, bovine. s455
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