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Characterization of freshly retrieved preantral follicles using a low-invasive, mechanical isolation method extended to different ruminant species

Published online by Cambridge University Press:  17 July 2014

A. Langbeen ,
E.P.A. Jorssen ,
E. Fransen ,
A.P.A. Rodriguez ,
M. Chong García ,
J.L.M.R. Leroy  and
P.E.J. Bols
A. Langbeen*
Affiliation:
University of Antwerp, Faculty of Pharmaceutical, Biomedical and Veterinary Sciences, Laboratory of Veterinary Physiology and Biochemistry, Universiteitsplein 1, Gebouw U, B-2610 Wilrijk, Belgium.
E.P.A. Jorssen
Affiliation:
University of Antwerp, Faculty of Pharmaceutical, Biomedical and Veterinary Sciences, Laboratory of Veterinary Physiology and Biochemistry, Universiteitsplein 1, Gebouw U, B-2610 Wilrijk, Belgium.
E. Fransen
Affiliation:
StatUa Center for Statistics, University of Antwerp, Prinsstraat 13, B-2000 Antwerp, Belgium.
A.P.A. Rodriguez
Affiliation:
Research Center for Biotechnology Transfer and Innovation, Laboratory of Manipulation of Oocytes and Preantral Follicles, Veterinary Faculty, State University of Ceará, Av. Paranjana 1700, Campus do Itaperi, Fortaleza, 60740–903, CE, Brazil.
M. Chong García
Affiliation:
University of Antwerp, Faculty of Pharmaceutical, Biomedical and Veterinary Sciences, Laboratory of Veterinary Physiology and Biochemistry, Universiteitsplein 1, Gebouw U, B-2610 Wilrijk, Belgium. CIMAGT Centro de Investigaciones para el Mejoramiento Animal de la Ganaderia Tropical, Calle 101, No. 6401 entre 64 y 100, Loma de Tierra, Cotorro, Ciudad de la Habana, Cuba.
J.L.M.R. Leroy
Affiliation:
University of Antwerp, Faculty of Pharmaceutical, Biomedical and Veterinary Sciences, Laboratory of Veterinary Physiology and Biochemistry, Universiteitsplein 1, Gebouw U, B-2610 Wilrijk, Belgium.
P.E.J. Bols
Affiliation:
University of Antwerp, Faculty of Pharmaceutical, Biomedical and Veterinary Sciences, Laboratory of Veterinary Physiology and Biochemistry, Universiteitsplein 1, Gebouw U, B-2610 Wilrijk, Belgium.
*
All correspondence to: A. Langbeen. University of Antwerp, Faculty of Pharmaceutical, Biomedical and Veterinary Sciences, Laboratory of Veterinary Physiology and Biochemistry, Universiteitsplein 1, Gebouw U, B-2610 Wilrijk, Belgium. Tel: +32 3 265 23 98. Fax: +32 3 265 24 33. e-mail: an.langbeen@uantwerpen.be
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Summary

Due to the increased interest in preantral follicular physiology, non-invasive retrieval and morphological classification are crucial. Therefore, this study aimed: (1) to standardize a minimally invasive isolation protocol, applicable to three ruminant species; (2) to morphologically classify preantral follicles upon retrieval; and (3) to describe morphological features of freshly retrieved follicles compared with follicle characteristics using invasive methods. Bovine, caprine and ovine ovarian cortex strips were retrieved from slaughterhouse ovaries and dispersed. This suspension was filtered, centrifuged, re-suspended and transferred to a Petri dish, to which 0.025 mg/ml neutral red (NR) was added to assess the viability of the isolated follicles. Between 59 and 191 follicles per follicle class and per species were collected and classified by light microscopy, based on follicular cell morphology. Subsequently, follicle diameters were measured. The proposed isolation protocol was applicable to all three species and showed a significant, expected increase in diameter with developmental stage. With an average diameter of 37 ± 5 μm for primordial follicles, 47 ± 6.3 μm for primary follicles and 67.1 ± 13.1 μm for secondary follicles, no significant difference in diameter among the three species was observed. Bovine, caprine and ovine follicles (63, 59 and 50% respectively) were graded as viable upon retrieval. Using the same morphological characteristics as determined by invasive techniques [e.g. haematoxylin–eosin (HE) sections], cumulus cell morphology and follicle diameter could be used routinely to classify freshly retrieved follicles. Finally, we applied a mechanical, minimally invasive, follicle isolation protocol and extended it to three ruminant species, yielding viable preantral follicles without compromising further in vitro processing and allowing routine follicle characterization upon retrieval.

Keywords

Follicle classificationLow-invasive follicle isolationMorphological characterizationPreantral folliclesRuminant reproduction
Type
Research Article
Information
Zygote , Volume 23 , Issue 5 , October 2015 , pp. 683 - 694
Copyright
Copyright © Cambridge University Press 2014 

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