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

Abstract

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.

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

“…Moreover, it is pointed that samples exposed to some CPAs combinations can remain at high concentrations (DMSO: 9.8 mg/g, EG: 9.8 mg/g) in the tissues, even after heating (Nakamura et al, ), which could impair the viability of some tissues as evident for the agouti ovary. Even if positive results derived from intracellular CPAs combination have been reported for the preservation of ovarian tissue derived from several species, such as donkeys (Lopes et al, ) and cats (Brito et al, ), this is not a consensus. Efficient results have also been highlighted for the use of an individual intracell CPA, such as the EG associated with only the sucrose present in the vitrification solution (Lima et al, ), as described for agoutis in the present study.…”

Resurrecting biodiversity: advanced assisted reproductive technologies and biobanking

“…An alternative approach to freezing oocytes is the cryopreservation of immature-oocyte-containing ovarian tissue, which has been conducted in a number of species ( Table 3 ) ( Martinez 2017 ). Since the first mouse was born in 1996 following in vitro growth of primordial follicles ( Eppig & O’Brien 1996 ), there have been many published studies on this technique in a number of species including white-tailed deer ( Odocoileus virginianu ) ( Gastal et al 2018 ), domestic cat ( Felis catus ) ( Mouttham & Comizzoli 2016 ), collared peccary ( Pecari tajacu ) ( Lima et al 2019 ), yellow-toothed cavies ( Galea musteloides ) ( Praxedes et al 2017 ), brown trout ( Salmo trutta ) ( Lujić et al 2017 ), donkey ( Equus asinus ) ( Lopes et al 2018 ) and domestic cattle ( Figueiredo et al 1993 , 1994 a , b , Hulshof et al 1995 , Vasconcelos et al 2013 ). Following death, euthanasia or neutering, a portion of the ovary can be cryopreserved; the immature follicle-containing cortex is dissected and cut into small strips under sterile conditions before slow freezing ( Benesova & Trefil 2016 , Hinkle et al 2021 ).…”

Biobanking and use of gonadal tissues - a promising strategy for conserving wildlife from the Caatinga biome

“…For Spix’s yellow-tooth cavy ( Table 3 ), a pioneering study on the species using SSV demonstrated that 3.0 M DMSO was efficient in preserving follicular morphology and DNA integrity ( Praxedes et al, 2017 ). As for northeastern donkeys, the best results regarding ovarian tissue cryopreservation were achieved when using the combination of EG and DMSO in the SSV ( Lopes et al, 2018 ).…”

“…Preservation of ovarian and testicular tissues have already been attempted in those species. Additionally, domestic species such as the donkeys ( Lopes et al, 2018 ) and domestic cat ( Amelkina et al, 2022 ) have been proposed as models for wild species that are phylogenetically close.…”