Microbiological load and preantral follicle preservation using different systems for ovarian tissue vitrification in the red-rumped agouti

Abstract

We evaluated the effect of open and closed systems used for ovarian tissue vitrification on the microbiological load and preservation of preantral follicles (PAFs) in the red-rumped agoutis. The ovaries from eight females were recovered and fragmented, with four cortexes fragments immediately fixed and evaluated (fresh group). The other fragments were processed for the solid-surface vitrification method (SSV) or an ovarian tissue cryosystem (OTC) using fetal calf serum, ethylene glycol, and sucrose as cryoprotectants, stored for two weeks, and rewarmed. Subsequently, fragments were subjected to a 24-h in vitro culture and assessed for microbiological load, PAF morphology, and DNA integrity. There was no fungal contamination; however, the vitrified samples from two individuals showed bacterial contamination of 79 200 colony forming units per milliliter (CFU)/mL for SSV and 3120 CFU/mL for OTC. From those samples, a total of eight different types of bacterial colonies were isolated and identified as coagulase-negative Staphylococci and Gram-positive bacilli. Regarding PAF morphology, both systems provided adequate preservation, with values higher than 70% normal follicles observed before and after culture. The TUNEL assay revealed that both SSV (52.39%) and OTC (41.67%) could preserve DNA integrity after vitrification and after 24 h of culture. In summary, both open and closed systems were equally efficient in preserving agouti ovarian tissues, especially concerning the preantral follicle morphology and DNA integrity; however, the OTC seems to provide a less adequate environment for bacterial proliferation.