Angiogenesis and follicular development in ovarian tissue of cattle following vitrification and post-warming culture on chicken chorioallantoic membrane

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Abstract

The purpose of this study was to assess the viability and growth of follicles in ovarian tissues of cattle vitrified using two non-permeating cryoprotectants (sucrose and trehalose) and two cryodevices (cryovial and cryotop). Cortical slices (1−2 mm3) from cattle ovaries (n = 5) were assigned to one of the 14 treatment groups. Cortical slices were vitrified in a TCM199 medium supplemented with ethylene glycol, DMSO, calf serum and either 0.5 M sucrose or trehalose, in cryovials or on cryotops. After warming, cortical slices were either fixed immediately for histology or grafted on a chorioallantoic membrane (CAM) of 10-day old chick embryos. Angiogenesis in ovarian tissues was determined. Viable and atretic preantral (primordial, primary and secondary) follicle densities were examined histologically. There was angiogenesis (chicken) in cortical slices grafted on the CAM by day 5 of culture, however, there was no difference for blood vessel densities when there was use of non-permeating cryoprotectants or cryodevices. Total, viable and atretic follicle densities did not differ (P > 0.05) with use of non-permeating cryoprotectants or cryodevices. The proportion of viable follicles was greater (P < 0.001) in fresh-control than CAM culture-control or vitrification groups. The inclusion of sucrose in the vitrification solution resulted in a larger number of atretic follicles than in the fresh-control group (P < 0.05). In summary, sucrose and trehalose, and cryotop and cryovial were equally suitable for vitrification of ovarian tissues of cattle. Vitrification of ovarian tissues of cattle with subsequent use of CAM culture adversely affected follicular development.

Introduction

Long-term storage of ovarian tissues is a promising approach for genetic conservation of females from endangered species and valuable animals. Ovarian cortical tissues contain a large population of primordial and preantral follicles. Unlike fully-developed preovulatory oocytes (120 μm diameter; the largest mammalian cell), oocytes contained in primordial and preantral follicles are more suitable for cryopreservation due to the small size (30 μm diameter), absence of zona pellucida and metabolic quiescence (Newton et al., 1996). Furthermore, oocytes from these follicles have the capacity to repair sub-lethal damage, resulting from cryopreservation procedures, because of the long growing phase during development subsequent to cryopreservation (Shaw et al., 2000). The development of a reliable cryopreservation method for the ovarian cortex is important for implementation of this important conservation strategy. There has been less attention on cryopreservation of ovarian tissue of cattle as compared to semen and embryos.

Vitrification is a simple, quick and inexpensive method of cryopreservation compared with slow freezing. Vitrification, is accomplished when cells are cooled at an ultra-rapid rate as a result of transformation of the viscous portion of cells to the glass-like state avoiding the harmful intracellular ice crystal formation which occurs during slow freezing. During vitrification, there is use of concentrated membrane-permeating cryoprotectants (DMSO, ethylene glycol etc.) which bind with free intracellular water and interact with cytoplasmic organelles, however, these are considered toxic (Fahy et al., 1990). The addition of non-permeating cryoprotectants, such as sucrose or trehalose (disaccharides), into the vitrification solution also dehydrate tissues/cells by increasing the extracellular osmolality (Posillico et al., 2010). The inclusion of sugar in the vitrification solution reduces the concentration and thus associated toxicity of permeating cryoprotectants (Liebermann et al., 2002). Trehalose binds more tightly with water molecules than other sugars (Crowe et al., 1996; Sano et al., 1999). At temperatures that exist during cryoprotection, the aggregation of membrane proteins is prevented and transformation of membranes from a liquid to gel state is delayed when there is inclusion of trehalose which stabilizes the cell plasma membrane (Kandror et al., 2002; Crowe et al., 2003). It was hypothesized that trehalose is a superior non-permeating cryoprotectant as compared with sucrose to maintain normal morphology of follicles in the vitrified ovarian tissue of cattle.

With vitrification, tissues/cells undergo ultra-rapid cooling and warming rates in rapidly transitioning through the critical temperature zone (between 15 and −5 °C) and as a result there is a decrease in damage to tissues/cells integrity as a result of chilling and potential osmotic damage (Massip, 2003). The packaging approach used for cryo-protection of cells/tissue is an important factor in vitrification because of the effects on cooling rate (Huang et al., 2008). Two distinct packaging systems are used for vitrification of tissues: 1) the open system (e.g., cryotop) which when used there is an ultra-rapid cooling rate, however, with tissues/cells there potentially are microorganisms present in liquid nitrogen which may result in transmission of diseases (Cutting et al., 2009), and 2) closed system (e.g., the cryovial) that allows for vitrification without the risk of microbial transmission occurring, however, there is a lesser cooling rate due to a physical barrier between liquid nitrogen and the tissues/cells sample (Amorim et al., 2011). In the present study, it was hypothesized that there was a more rapid cooling rate in a cryotop that results in this being a more effective method for vitrification with the result being a greater post-warming viability of ovarian tissues of cattle than with the use of a cryovial.

The overall goal with the present study was to develop a vitrification protocol for ovarian tissues of cattle using the chick chorioallantoic membrane (CAM) for short term culture. The specific objectives of this study were to compare the effects of non-permeating cryoprotectants (sucrose compared with trehalose) and cryodevices (cryovial compared with cryotop) on the angiogenesis and development of preantral follicles in cortical ovarian tissues (slices) of cattle after vitrification and a 5-day CAM culture.

Section snippets

Treatment groups and ovarian tissue collection

Ovaries were collected from beef cows (> 5 years old) at a slaughterhouse 130 km from the Cryobiology Laboratory, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, SK, Canada. Ovaries were placed between ice packs at 4 °C in a styrofoam box and transported to the laboratory within 6 h of collection. In the laboratory, after removing the surrounding connective tissue, ovaries were washed twice with cold (4 °C) 1X Dulbecco’s phosphate buffer saline (DPBS). Ovaries (n

Results

During vitrification, cooling rates of ovarian tissue slices were 7481 ± 206 °C/min and 664 ± 26 °C/min with the cryotop and cryovial, respectively (Fig. 1; P < 0.05).

Discussion

This is a first report on the comparison of avian-origin angiogenesis and follicular development in fresh and vitrified-warmed ovarian tissues of cattle after 5-day of CAM culture. Furthermore, the effectiveness of using the vitrification system was evaluated by using two different non-permeating cryoprotectants (sucrose and trehalose) and two different cryodevices (cryovial and cryotop). In the present study, control and vitrified ovarian tissues were infiltrated with chicken blood vessels and

Declarations of Competing Interest

None

Acknowledgments

This research was funded by the Canadian Animal Genetic Resource Program, Agriculture and Agri-Food Canada (Grant # AGR-14227). Authors are grateful to Dr. Reuben Mapletoft, Distinguished Professor Emeritus, Western College of Veterinary Medicine, University of Saskatchewan for editing this manuscript.

References (33)

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