Equine ovarian tissue viability after cryopreservation and in vitro culture

Abstract

Ovarian tissue cryopreservation allows the preservation of the female fertility potential for an undetermined period. The objectives of this study were to compare the efficiency of cryoprotective agents (CPAs; dimethyl sulfoxide, DMSO; ethylene glycol, EG; and propylene glycol, PROH) using slow-freezing and vitrification methods, and evaluate the viability of cryopreserved equine ovarian tissue after 7 days of culture. Fresh and cryopreserved ovarian fragments were evaluated for preantral follicle morphology, stromal cell density, EGFR, Ki-67, Bax, and Bcl-2 protein expression, and DNA fragmentation. Vitrification with EG had the highest rate of morphologically normal preantral follicles, while DMSO had the lowest (76.1 ± 6.1% and 40.9 ± 14.8%, respectively; P < 0.05). In slow-freezing, despite that DMSO had the highest percentage of morphologically normal follicles (77.7 ± 5.8%), no difference among the CPAs was observed. Fluorescence intensity of EGFR and Ki-67 was greater when vitrification with EG was used. Regardless of the cryopreservation treatment, DMSO had the highest (P < 0.05) Bax/Bcl-2 ratio; however, DNA fragmentation was similar (P > 0.05) among treatments after thawing. After in vitro culture, the percentage of normal follicles was similar (P > 0.05) between slow-freezing and vitrification methods; however, vitrification had greater (P < 0.05) stromal cell density than slow-freezing. In summary, equine ovarian tissue was successfully cryopreserved, increasing the viability of the cells in the ovarian tissue after thawing when using DMSO and EG for slow-freezing and vitrification methods, respectively. Therefore, these results are relevant for fertility preservation programs.

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

“…Similar results for ovarian tissue cryopreservation using EG 3 M were recently reported for a species closely related to asinine: equine, in which 76.1 ± 6.1% morphologically normal PAFs were obtained after vitrification. However, horse PAFs appear to be more sensitive to DMSO, as its presence dramatically reduces the percentage of normal follicles after vitrification (40.9 ± 14.8%) (Gastal et al., ), unlike that observed for donkeys (81.7 ± 37.5%). Such differences regarding the sensitivity of CPAs could be attributed to the fact that membrane properties such as water loss from the tissues and, consequently, the parameters of membrane permeability of ovarian cells from these two species differ (Kardak, Leibo, & Devireddy, ).…”

Effect of different penetrating and non‐penetrating cryoprotectants and media temperature on the cryosurvival of vitrified in vitro produced porcine blastocysts

“…In addition, DMSO was found to cause an influx of intracellular calcium in mouse oocytes, possibly by interfering with intracellular organelles (Larman, Sheehan, & Gardner, ). Like EG, PROH has a low molecular weight (76.1 g/mol) and is highly permeable (Pedro et al., ), and is commonly used as a cryoprotectant in slow freeze protocols for oocytes, embryos and tissues (Gastal et al., ; Liu et al., ; Zhang, Liu, Xing, Zhou, & Cao, ). However, PROH has been shown to elicit more toxic effects than EG and DMSO when used alone (Somfai et al., ; Szurek & Eroglu, ; Taniguchi et al., ).…”

“…is highly permeable (Pedro et al, 2005), and is commonly used as a cryoprotectant in slow freeze protocols for oocytes, embryos and tissues (Gastal et al, 2017;Liu et al, 2009;Zhang, Liu, Xing, Zhou, & Cao, 2011). However, PROH has been shown to elicit more toxic effects than EG and DMSO when used alone (Somfai et al, 2013;Szurek & Eroglu, 2011;Taniguchi et al, 2011).…”

Cryopreservation of Preantral Follicles

“…It is also important to maintain genetic material from autochthonous breeds, pets, or even production animals. In the latter case, genetically superior animals that show better patterns of production (bovines [24], equines [25], sheep [26], and pigs [27]) are being increasingly studied with a view to enhancing livestock species [28]. This has led to researchers working on the development of cryopreservation protocols specific to different species.…”

“…These permeating CPAs are frequently used for ovarian tissue cryopreservation. So far, DMSO has proved more [45], goats [46], mares [25], and sheep [47] while EG is usually used in association with DMSO [48,49].…”