Slow-controlled freezing versus speed-cooling for cryopreservation of whole guinea pig ovaries

Xu, Zhihui; Wang, Xiaojiao; Wu, Yonggen; Meng, Yuanyuan; Wu, Fangfang; Zhou, Na; Chen, Wenwei; Ye, Bilu; Liu, Jun; Zhou, Ying

doi:10.1016/j.theriogenology.2011.08.017

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Volume 77, Issue 3 , Pages 483-491, February 2012

Slow-controlled freezing versus speed-cooling for cryopreservation of whole guinea pig ovaries

Zhihui Xu
- Centre for Reproductive Medicine, the First Affiliated Hospital of Wenzhou Medical College, Wenzhou, Zhejiang, 325000, China
Xiaojiao Wang
- Centre for Reproductive Medicine, the First Affiliated Hospital of Wenzhou Medical College, Wenzhou, Zhejiang, 325000, China
Yonggen Wu
- Centre for Reproductive Medicine, the First Affiliated Hospital of Wenzhou Medical College, Wenzhou, Zhejiang, 325000, China
Yuanyuan Meng
- Centre for Reproductive Medicine, the First Affiliated Hospital of Wenzhou Medical College, Wenzhou, Zhejiang, 325000, China
Fangfang Wu
- Centre for Reproductive Medicine, the First Affiliated Hospital of Wenzhou Medical College, Wenzhou, Zhejiang, 325000, China
Na Zhou
- Centre for Reproductive Medicine, the First Affiliated Hospital of Wenzhou Medical College, Wenzhou, Zhejiang, 325000, China
Wenwei Chen
- Centre for Transplantation, the First Affiliated Hospital of Wenzhou Medical College, Wenzhou, Zhejiang, 325000, China
Bilu Ye
- Centre for Reproductive Medicine, the First Affiliated Hospital of Wenzhou Medical College, Wenzhou, Zhejiang, 325000, China
Jun Liu
- Department of Histology and Embryology, Nantong Medical College, Nantong University, Nantong, Jiangsu, 226220, China
- Corresponding authors. Tel.: 86-577-88069649; fax: 86-577-88069393 (Y. Zhou); fax: 86-513-81160706 (J. Liu)
Ying Zhou
- Centre for Reproductive Medicine, the First Affiliated Hospital of Wenzhou Medical College, Wenzhou, Zhejiang, 325000, China
- Department of Histology and Embryology, Wenzhou Medical College, Wenzhou, Zhejiang, 325000, China
- Corresponding authors. Tel.: 86-577-88069649; fax: 86-577-88069393 (Y. Zhou); fax: 86-513-81160706 (J. Liu)

Received 17 April 2011; received in revised form 5 August 2011; accepted 13 August 2011. published online 30 September 2011.

Abstract

The objective of this study was to evaluate the feasibility of whole-ovary perfusion, and to compare the effects of speed-cooling and slow-controlled freezing of whole guinea pig ovaries. Slow-freezing and speed-cooling procedures were performed after perfusion of guinea pig ovaries with cryoprotectants. Ink perfused via the vascular pedicles was present in the microvessels around various follicles at various stages of development in the cortical and medullar regions, thereby confirming that perfusion was effective. Vascular damage was essentially confined to the cannulated artery. Based on histological examination, there were (mean ± SEM) 93.1 ± 4.2, 79.0 ± 2.0, and 54.7 ± 8.5% healthy follicles in the fresh, slow-freezing and speed-cooling groups, respectively (each group differed from the other two, P < 0.05). Trypan blue staining of isolated follicles confirmed that cellular damage was greater following speed-cooling than slow-freezing (58.6 vs 29.2%, P < 0.05). Based on a TUNEL assay, speed-cooling caused more apoptotic granulosa and theca cells in antral follicles than slow-freezing. In conclusion, the present study provided evidence that guinea pig whole ovaries could be perfused with cryoprotectant and cryopreserved in vitro. Furthermore, the slow-freezing protocol resulted in less cellular damage in thawed tissues than speed-cooling.

Keywords: Ovary , Perfusion , Blood vessel , Cryopreservation , Guinea pig