Vitrification of canine ovarian tissues with polyvinylpyrrolidone preserves the survival and developmental capacity of primordial follicles

Mayako Fujihara,Takehito Kaneko,Miho Inoue-Murayama

doi:10.1038/s41598-019-40711-6

Abstract

Ovarian tissue cryopreservation combined with immature follicle development can preserve female fertility in wildlife, regardless of age or reproductive timing. To investigate the effects of different cryopreservation methods and cryoprotectants on follicular survival and developmental capacity, ovarian cortical pieces from 15 dogs were cryopreserved by slow freezing or vitrification with different additional cryoprotectants as follows: dimethyl sulfoxide (DMSO), polyvinylpyrrolidone (PVP), combined DMSO and PVP (each at half the concentration of when used independently), or none (control). Cryopreserved ovarian tissues were evaluated by neutral red staining, histology, and xenotransplantation assays. Among cryopreservation conditions tested, vitrification with combined DMSO and PVP significantly improved the maintenance of follicular morphology compared to that in control. Furthermore, ovarian tissues vitrified using this condition maintained follicle morphology and developmental capacity 9 weeks after grafting, as shown by an increased percentage of primary and secondary follicles and a significant decrease in the transition stage from primordial to primary stage follicles 5 and 9 weeks after grafting. In contrast, slow freezing and control groups lost intact follicles by 5 weeks after grafting. The described cryopreservation techniques, which preserve canine follicle development, will build the foundation of ovarian tissue cryopreservation to preserve female fertility in wild canids.

Highlights

Many animal species are threatened and endangered mainly due to habitat loss and direct human activities
We first investigated the effect of different cryopreservation methods and cryoprotectants on follicle viability and morphological maintenance
Based on many attempts using different cryoprotectants for vitrification, Mouttham and Comizzoli found that a combination of fetal bovine serum (FBS), sucrose, ethylene glycol (EG), and dimethyl sulfoxide (DMSO) was effective to vitrify feline ovarian tissues[27]

Summary

Introduction

Many animal species are threatened and endangered mainly due to habitat loss and direct human activities. Slow freezing is the most commonly employed cryopreservation technique for ovarian tissue cryopreservation[4,7] This process is associated with very little toxicity and osmotic damage due to the use of low concentrations of cryoprotectants. Vitrification is another cryopreservation method that involves the use of large concentrations of multiple cryoprotectants that can prevent the formation of ice crystals[8] This method is a simple and low-cost technique because of the direct plunging/storing in liquid nitrogen and the fact that it does not require specific freezing machines. Needle immersion vitrification is a promising method of ovarian tissue vitrification[9] This technique is attractive for use in conservation biology because of the ease of handling, and because it provides uniform exposure to cryoprotectants and an increased cooling rate by direct cooling in liquid nitrogen[9]. PVP is not widely used for cryopreservation, and its impact on primordial follicles within ovarian tissues is unclear

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