Damage to fetal bovine ovarian tissue caused by cryoprotectant exposure and vitrification is mitigated during tissue culture

In contributing to the conservation of wild rodents, the aim of this study was to evaluate the use of distinct cryoprotectants, separately or in combination, for solid surface vitrification (SSV) of red‐rumped agouti ovarian tissue. Ovarian cortex from nine females was recovered and fragmented. Fresh fragments (control) were used to analyse the pre‐antral follicle (PF) morphology using a histologic procedure, viability using the Trypan blue test, cell proliferation by counting the argyrophilic nucleolar organizing regions (Ag‐NORs technique) and DNA integrity using the TUNEL assay. The remaining fragments were vitrified using SSV method with 3 M or 6 M ethylene glycol (EG) or dimethyl sulfoxide (DMSO), or in combination (3 M EG/3 M DMSO), and further evaluated as reported for the fresh samples. All cryoprotectants were effective at preserving PFs morphology compared to the control group (80.7 ± 5.21%), except 6 M EG and 3 M DMSO that provoked a significant (p < .05) decrease on the values of morphologically normal primary (60.0 ± 19.0%) and primordial (44 ± 4.5%) follicles, respectively. Regarding viability, all cryoprotectants provided values similar to that verified for the control group (79.0%), but a significant decrease (p < .05) was observed with EG/DMSO combination (59%). Using Ag‐NORs technique, the highest (p < .05) cell proliferative capacity was detected when using EG at each tested concentration. The TUNEL proved the preservation of DNA integrity regardless of the cryoprotectant. In summary, we suggest the use of 3 M EG for the solid surface vitrification of red‐rumped agouti ovarian tissue.

Section: Discussionsupporting

confidence: 81%

Section: Discussionsupporting

confidence: 78%

Comparison of different intracellular cryoprotectants on the solid surface vitrification of red‐rumped agouti (Dasyprocta Leporina Lichtenstein, 1823) ovarian tissue

Praxedes

Lima

Silva

et al. 2019

“…(): 39% normal). Furthermore, the current study examined the follicular morphology at multiple time points following tissue warming, which demonstrated that post‐warming ovarian tissue culture is necessary to properly examine the effect of varying vitrification protocols on follicular health (Mouttham et al., ).…”

Section: Discussionmentioning

confidence: 99%

“…Ovarian tissue vitrification, followed by transplantation, has resulted in the live birth of human babies (Suzuki et al., ). Furthermore, vitrification was been shown to preserve primordial follicle integrity and function (Mouttham, Fortune, & Comizzoli, ), making it an adequate method of primordial follicle cryopreservation.…”

Section: Introductionmentioning

confidence: 99%

Presence of sucrose in the vitrification solution and exposure for longer periods of time improve post‐warming follicle integrity in cat ovarian tissues

Mouttham

Comizzoli

2016

ContentsOvarian tissue cryopreservation followed by tissue culture is a promising approach to preserving the fertility of biomedical models and endangered species. The objective of this study was to investigate the impact of exposure time to vitrification solution and presence of sucrose using different exposure temperatures and base media on intra-ovarian follicle integrity. Peripubertal ovarian cortical pieces were obtained by isolating the cortex and dissecting it into 1 × 1 × 0.2 mm 3 pieces. The cortical pieces were then exposed to equilibration solution and then vitrification solutions (VS) in one of the conditions mentioned above, plunged directly into liquid nitrogen and stored for ≥24 hr in liquid nitrogen. After thawing, the cortical pieces were cultured in vitro for 0, 1 or 7 days to determine the follicle integrity (through histological assessment) and the ability of the tissue to recover from cryoinjury. Fresh controls maintained a constant level of normal morphology (>60% of the total follicles) throughout the culture period. Cortical pieces exposed to VS with sucrose for 10 min had the highest percentage of normal follicles (approximately 20% after 7 days of culture) throughout the culture period. Other conditions using different base medium, lower exposure temperatures or different thawing methods did not improve the follicle integrity. This protocol provides a solid foundation on which to optimize ovarian tissue cryopreservation in the domestic cat and to investigate the molecular effects of vitrification.

“…Cryoprotectants agents have physical and chemical functions in the tissue, altering freezing conditions, in particular, with the formation of ice crystals (Asgari, Valojerdi, Ebrahimi, & Fatehi, 2015) without intoxicating cells in the process (Mouttham, Fortune, & Comizzoli, 2015). Choosing the right CPAs is essential, which should involve prior evaluation of the benefits of the chosen substance including the permeability of the membranes, CPA concentration, the perfect match of cryoprotectant effect and toxicity, the possibility of combination with other CPAs, among others.…”

Section: Introductionmentioning

confidence: 99%

Vitrification of ovarian tissue of Brazilian North‐eastern donkeys (Equus asinus) using different cryoprotectants

Lopes

Praxedes

Campos

et al. 2018

The aim of this study was to assess a vitrification protocol for asinine ovarian tissue, to preserve preantral follicles using different cryoprotectant solutions, composed of various concentrations (EG 3 M or 6 M) of dimethyl sulfoxide or ethylene glycol isolate, or as a combination (DMSO 3 M + EG 3 M). Ten pairs of ovaries from Brazilian north-eastern breed jennies were obtained through videolaparoscopy, and cortical fragments were submitted to a solid-surface vitrification (SSV) using each cryoprotectant solution. The ovarian tissue was evaluated for follicular morphology and viability, DNA integrity (TUNEL technique) and the presence of nucleolar organizing regions in granulosa cells (AgNOR technique). After thawing, the percentage of normal preantral follicles was significantly reduced in the vitrified ovarian tissue fragments compared to the fresh control (p < 0.05). When comparing treatments, the use of DMSO 3 M (81.7 ± 37.5%), EG 3 M (83.7 ± 27.4%) and the combination of both DMSO 3 M + EG 3 M (81.8 ± 46.8%) allowed a greater percentage of follicular survival in contrast to DMSO 6 M (69.8 ± 16.5%) and EG 6 M (72.3 ± 18.0%; p < 0.05). When vitrified using the DMSO + EG combination, a higher percentage (62.5 ± 29.1%) of viable follicles (trypan blue) was observed in relation to the other vitrification treatments (p < 0.05). The TUNEL technique identified that all treatments tested showed DNA fragmentation in the follicular cells, except in the case of the DMSO 3 M + EG 3 M treatment. When evaluating the presence of NORs, no significant differences were observed in the amount of NORs between the fresh and vitrified groups using DMSO 3 M + EG 3 M (p > 0.05). We concluded that the combination DMSO 3 M + EG was more efficient for the vitrification of ovarian tissue taken from Equus asinus, allowing adequate preservation of PAFs morphology, viability, DNA integrity and cell proliferative capacity.