Preservation of bovine preantral follicle viability and ultra-structure after cooling and freezing of ovarian tissue

Abstract

Bovine preantral follicles within ovarian fragments were exposed and cryopreserved in absence or presence of 1.5 M glycerol (GLY), ethylene glycol (EG), propanediol (PROH) or dimethyl sulfoxide (DMSO), undergoing a previous cooling at 20 °C for 1 h (protocol 1) or at 4 °C for 24 h (protocol 2) in 0.9% saline solution. At the end of each treatment, preantral follicles were classified as non-viable/viable when they were stained/not stained with trypan blue, respectively. To confirm viability staining, ultra-structure of the follicles was evaluated by transmission electronic microscopy (TEM). Data were compared by Chi-square test (P < 0.05). The storage of the ovaries at 20 °C for 1 h (78%) and 4 °C for 24 h (80%) did not reduce significantly the percentage of viable preantral follicles when compared to the control (75%). Similar results were obtained when ovarian fragments, respectively, for protocols 1 and 2, were exposed to MEM (78 and 77%), 1.5 M EG (78 and 71%), as well as frozen in 1.5 M EG (74 and 77%). Percentages of viable follicles in control were similar to those observed after exposure (75%) and freezing (76%) in presence of 1.5 M DMSO only when protocol 1 was used. The increase of the concentration from 1.5 to 3.0 M, for all cryoprotectants, reduced significantly the percentage of viable preantral follicles after freezing. Ultra-structural analysis has confirmed trypan blue results, showing that not only basement membrane, but also organelles, were intact in viable preantral follicles. In conclusion, ovarian tissue cooling at 4 °C for 24 h before cryopreservation (protocol 2) does not affect the viability of bovine preantral follicles when 1.5 M EG is present in the cryopreservation medium.

Ultrastructural features of agouti (Dasyprocta aguti) preantral follicles cryopreserved using dimethyl sulfoxide, ethylene glycol and propanediol

“…In that regard, the latter was closely related to the organization of ovarian tissue and associated with species-specific characteristics [32], not allowing for extrapolation of protocols for species that are phylogenetically distant.…”

Vitamin E-analog Trolox prevents endoplasmic reticulum stress in frozen-thawed ovarian tissue of capuchin monkey (Sapajus apella)

“…Similarly, previous studies have shown the value of antioxidants in protecting ovarian tissue from ischemia following transplantation (Kim et al 2004) or their usefulness in freezing medium to improve follicular preservation (Melo et al 2011;Sanfilippo et al 2013). Vacuolization has been reported after cryopreservation of ovarian tissue from sheep (Santos et al 2006b;Oskam et al 2010), bovine (Celestino et al 2008) and human (Marsella et al 2008), with indications of damage in the ER (Silva et al 2000). In agreement with this evidence, Oskam et al (2010) found oocyte vacuolization together with an extreme decrease in or even total absence of smooth and rough ER in cryopreserved sheep ovarian tissue.…”

Assessment of follicular development in cryopreserved primate ovarian tissue by xenografting: prepubertal tissues are less sensitive to the choice of cryoprotectant

“…This is in accordance with the results of other groups, who found DMSO to allow for better preservation of primary follicles than PrOH in human tissue (Gandolfi et al 2006), although no effects were found in the population of primordial follicles in that study. Other studies suggest that even though EG is known for its low toxicity when applied as a cryoprotectant of bovine and caprine ovarian tissue (Santos et al 2006b, Celestino et al 2008, DMSO preserves the ultrastructure of ovine early-staged follicles better (Santos et al 2006a).…”