# Cryopreservation and In Vitro Culture of Isolated Porcine Ovarian Follicles

## TL;DR

This study compares two freezing methods for preserving pig ovarian follicles, finding that one method supports better survival and hormone production.

## Contribution

The study introduces a comparison of open and closed vitrification methods for porcine follicle preservation.

## Key findings

- Open vitrification (OPS) showed higher survival rates than the closed method (CT).
- OPS follicles produced less estradiol on Day 2, but no differences were seen on Day 7.
- The culture system supports hormone production but may need refinement for long-term use.

## Abstract

The long-term storage of ovary-originated follicles can serve as a promising approach to conserve genetic material and fertility of female animals, even after death. However, since a standardized protocol is lacking, there is a need for a well-working process providing a high survival rate of frozen–thawed samples. The objective of this study was to analyze the effect of two different freezing methods (open and closed vitrification) on porcine follicles, assessing the survival rate and hormonal production during a 10-day-long culture. Although refinement is needed, our data indicate that our culture media is suitable to maintain the hormonal production of these samples. Regarding the freezing system, open vitrification is an appropriate method to preserve the preantral follicles and provides superior survival rates to the closed method.

Cryopreservation of preantral follicles (PAFs) is a promising tool for gene conservation and fertility preservation. However, standardized protocols for the cryopreservation and in vitro culture of isolated follicles—particularly in pigs—are still lacking. This study aimed to analyze the survival and developmental potential of porcine PAFs vitrified using two different methods: open pulled straw (OPS) and cryotube (CT). Ovaries of Hungarian Large White sows were collected from a local slaughterhouse and enzymatically digested to isolate preantral follicles. Morphologically normal follicles were assigned to three groups: fresh control, OPS-vitrified, and CT-vitrified. All follicles were cultured for 10 days in FSH-supplemented medium, with growth, survival, and estradiol (E2) production monitored. Survival rate was lower in the CT group (83.3%) than that of the control and OPS (97.4% and 94.4%, respectively). The follicular area was consistently larger in control than in CT and OPS, with no difference between vitrified groups. E2 production varied among treatments: OPS follicles showed lower E2 levels on Day 2, no differences were detected on Day 7, and CT follicles produced less E2 on Day 10. These results indicate that OPS is the more suitable vitrification method for porcine PAFs and that the culture system supports hormone production; however, it may require refinement to provide long-term follicle maintenance.