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RESEARCH ARTICLE

In vitro growth and development of isolated secondary follicles from vitrified caprine ovarian cortex

Érica S. S. Leal A , Luis A. Vieira A , Naíza A. R. Sá A , Gerlane M. Silva A , Franciele O. Lunardi A , Anna C. A. Ferreira A , Cláudio C. Campello A , Benner G. Alves A , Francielli W. S. Cibin B , Johan Smitz C , José R. Figueiredo A and Ana P. R. Rodrigues A D
+ Author Affiliations
- Author Affiliations

A Faculty of Veterinary Medicine, LAMOFOPA, PPGCV, State University of Ceará, Av. Dr Silas Munguba, 1700 – Campus of Itaperi, Fortaleza – CE – CEP 60741-903, Brazil.

B University Federal of Pampa, Uruguaiana-Rio Grande do Sul, Av. General Osório, 900 – São Jorge Bagé, RS – CE – 96400-100, Brazil.

C Follicle Biology Laboratory, Center for Reproductive Medicine, UZ Brussel, Laarbeeklaan 101, B-1090 Brussels, Belgium.

D Corresponding author. Email: aprrodriguespapers@gmail.com

Reproduction, Fertility and Development 30(2) 359-370 https://doi.org/10.1071/RD16487
Submitted: 14 May 2016  Accepted: 17 June 2017   Published: 3 August 2017

Abstract

The aim of this study was to evaluate the viability, antrum formation and in vitro development of isolated secondary follicles from vitrified caprine ovarian cortex in a medium previously established for fresh isolated secondary follicles, in the absence (α-minimum essential medium (α-MEM+) alone) or presence of FSH and vascular endothelial growth factor (VEGF; α-MEM++FSH+VEGF). Ovarian fragments were distributed among five treatments (T1 to T5): fresh follicles were fixed immediately (T1), follicles from fresh tissue were cultured in vitro in α-MEM+ (T2) or α-MEM++FSH+VEGF (T3) and follicles from vitrified tissue were cultured in vitro in α-MEM+ (T4) or α-MEM++FSH+VEGF (T5). After 6 days of culture, treated follicles (T2, T3, T4 and T5) were evaluated for morphology, viability and follicular development (growth, antrum formation and proliferation of granulosa cells by Ki67 and argyrophilic nucleolar organiser region (AgNOR) staining). The levels of reactive oxygen species (ROS) in the culture media were also assessed. Overall, morphology of vitrified follicles was altered (P < 0.05) compared with the fresh follicles. Follicular viability, antrum formation and ROS were similar between treatments (P > 0.05). The average overall and daily follicular growth was highest (P < 0.05) in T3. Granulosa cells in all treatments (T1, T2, T3, T4 and T5) stained positive for Ki67. However, fresh follicles from T3 had significantly higher AgNOR staining (P < 0.05) compared with follicles of T1, T2, T4 and T5. In conclusion, secondary follicles can be isolated from vitrified and warmed ovarian cortex and survive and form an antrum when growing in an in vitro culture for 6 days.

Additional keywords: folliculogenesis, hormone, preantral follicle, vitrification.


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