99 SURVIVAL RATEAND IN VITRO DEVELOPMENT OF IN VIVO-PRODUCED AND CRYOPRESERVED DOG EMBRYOS

Abstract

Dogs have been used as an experimental model for human genetic diseases and for research applied to endangered Canidae. Moreover, application of assisted reproductive technologies (ART) by dog breeders is increasing. The aim of this study was to evaluate the survival rate and in vitro development of in vivo-produced and cryopreserved dog embryos. Seven cross-bred bitches were submitted to ovariohysterectomy 12 days after first mating or artificial insemination, and embryos were recovered by uterine horn flushing with 30 mL of PBS/horn (Nutricell, Campinas, SP, Brazil). Grade 1 and 2 morulae (MO; n = 7; IETS) and blastocysts (BL; n = 14) were frozen. For freezing, embryos were immersed in glycerol 10% (GLY; Nutricell, Campinas, SP, Brazil) or ethylene glycol 3,0 M (EG; Nutricell, Campinas, SP, Brazil) for 10 min. Straws were placed in the machine at -7.0°C (TK 3000, Uberaba, MG, Brazil), and equilibrated for 2 min. Seeding was performed at -7.0°C, and another equilibrium period of 2 min was performed. A cooling rate of -0.5°C/min until -32.0°C was used. Embryos were stored in N2L until thawing. Prior to in vitro culture, embryos were removed from N2L, kept at room temperature for 10 s, and put in a water bath at 25°C for 20 s. Embryos frozen in GLY were washed for 5 min in each thawing solution for cryoprotectant removal (0.6 M sacarose + glycerol 5%; 0.6 M sacarose + glycerol 2.5% and 0.6 M sacarose; Nutricell, Campinas, SP, Brazil). After that, embryos were washed 10 times in holding solution (Holding Plus, Bioniche, Pullman, WA, USA). Embryos frozen in EG were kept at room temperature for 10 s, put in a water bath at 25°C for 20 s, and were directly washed 10 times in holding solution. Comparison among groups was performed by ANOVA. After thawing, 9/11 (81.8%) embryos frozen in EG had rupture of zona pelucidae and 2/11 (18.2%) were intact, whereas 9/10 (90.0%) embryos frozen in GLY were intact (P < 0.05). All intact embryos (n = 11) were morphologically normal and were transferred to SOF medium (Nutricell, Campinas, SP, Brazil), cultured for 168 h at 38.3°C, and evaluated at 24-h intervals. After the last evaluation, for both cryoprotectants, hatching rate was 0.0%, but all embryos were morphologically normal. The results of this study suggest that dog embryos have a high in vitro survival rate in standard protocols used for mammalian embryo cryopreservation when glycerol 10% is used as cryoprotectant and that a long period of culture (possibly more than 10 days) is required for dog embryo hatching. On the other hand, this long period for in vitro hatching may reflect the delayed hatching and implantation that occurs in vivo in this species.

Financial by FAPES/MCT/CNPq/CT-INFRA n. 19/2006; Processo n. 36600737/2007, ES- Brasil.