Abstract
Graphical abstract
Lay summary
The decreasing rate of successful pregnancies, both naturally and through assisted conception, has led to innovations in the way eggs, sperm, and embryos are stored. Despite these advances, the use of assisted reproductive techniques to preserve endangered or rare species remains unexplored. Since the location where samples are collected and facilities are often far apart, we aim to address part of this challenge by comparing different methods to store and handle ovarian tissue before freezing. This may pave the way for further research in preserving endangered species, despite the challenges posed by the distance between sample collection sites and suitable facilities.
Understanding reproductive processes involved in ovarian folliculogenesis and applying this knowledge to different species is essential for biodiversity conservation. Low-temperature tissue storage research has allowed scientists to create tissue banking for scientific research and medical applications (Whaley et al. 2021). Ovarian tissue and follicle preservation rely heavily on the methods of collection, transport, and storage (Duncan et al. 2016, Barberino et al. 2019). Therefore, investigating the ideal medium, time, and transport temperature is needed to successfully maintain viable tissues (Duncan et al. 2016, Barberino et al. 2019). Developing such techniques is especially important in cases where samples are collected in areas where facilities are scarce or far away, which is the reality for most wildlife sample collections. Therefore, our project goal was to investigate the ideal transport and storage conditions that can support the collection of bovine preantral follicles for future gamete rescue.
To investigate the effects of storage time and temperature, bovine whole ovaries were (i) immediately used for follicle isolation (FR_RT0h), (ii) immediately vitrified (VT_ RT0h), (iii) vitrified after 24 h incubation at 4°C (VT_4C24h), (iv) vitrified after 24 h incubation at room temperature (VT_RT24h), or (v) vitrified after 48 h at 4°C (VT_4C48h). Whole ovaries were stored in TCM199 supplemented with 20% fetal bovine serum and 22 mM HEPES, after collection of two pieces (10 × 10 × 1 mm, cut using a 3D-printed slicer; Fig. 1A) each for fresh and immediately vitrified groups. Following incubation, ovarian slices were precisely prepared and subjected to vitrification. The slices were subsequently thawed using same storage solution supplemented with sucrose concentrations of 1 mol/L and 0.5 mol/L, respectively, with each thawing phase lasting 5 min. Follicles were mechanically isolated using a combination of homogenization and filtration. Their viability was assessed using Calcein AM, propidium iodide, and Hoechst stains (see Fig. 1A).
Detailed overview of the experimental plan, follicle viability, and height-to-width ratio. A. Overview of experimental plan with different analysis groups. B. Boxplot showing dead, live, and partially live follicles following 0 h (VT_RT0h, FR_RT0h), 24 h (VT_RT24h, VT_4C24h), and 48 h (VT_4C48h) incubation periods. C. Density plot analysis depicting the circularity (height/width) in function of follicle viability. The figure was created using Biorender.com.
Citation: Reproduction and Fertility 5, 1; 10.1530/RAF-23-0071
The analysis of 1485 follicles (13 animals) demonstrated that follicles isolated from fresh controls had similar viability to follicles isolated from vitrified tissues (76.6% and 62.3% for FR_RT0h (n = 8) and VT_RT0h (n = 8), respectively; P = 0.169; Fig. 1B). However, vitrified ovaries had a tendency to a higher number of partially alive follicles (P = 0.06). Interestingly, incubation time and temperature had no significant effect on isolated preantral follicle viability (60.3%, 64.1%, 58.4% for live follicles of VT_RT24h (n = 5), VT_4C24h (n = 6), and VT_4C48h (n = 5), respectively; P > 0.05; Fig. 1B).
Additionally, it is common knowledge for researchers in the field, that the follicle circularity is correlated with its viability. To establish it as an objective tool to help select follicles without the need to perform staining, we evaluated the height-to-width ratio (H/W) of live, partially live, and dead follicles. Differently from dead and partially live, live follicles tend to keep a rounder shape (P = 0.049 and 0.03, for live vs dead and live vs partially, respectively; Fig. 1C), demonstrating that the H/W ratio can be used for viable follicle selection without the need for using potentially harmful staining methods.
Although previous publications have suggested that tissue incubation parameters can influence gamete and follicle quality in different species (reviewed by Barberino et al. 2019) here, only 6.16%, 6.62%, 7.75%, 6.29%, 12.69% (for FR_RT0h, VT_RT0h, VT_RT24h, VT_4C24h, and VT_4C48h, respectively; P > 0.05; Fig. 1B) of follicles were dead, demonstrating that all conditions analyzed can be used for storage and transport of bovine ovarian tissues previous to cryopreservation for future preantral follicle rescue. Our results agree with other studies where no effects of vitrification on preantral follicle viability were observed (Kagawa et al. 2009, Mogheiseh et al. 2017).
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the study reported.
Funding
This study was supported by the Alexander von Humboldt Foundation in the framework of the Sofja Kovalevskaja Award endowed by the German Federal Ministry of Education and Research.
Author contribution statement
Conceptualization: MAMMF; Methodology: YF; Investigation: YF; Supervision: MAMMF; Writing – original draft: YF and MAMMF; Writing – review and editing: YF and MAMMF.
References
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