Retrieval of Porcine Ovarian Follicles by Different Methods

Moon Hwan Choi,Jeong Mook Lim,Seung Pyo Gong

doi:10.5713/ajas.2008.60728

Abstract

A series of studies has been conducted to establish a base infrastructure for an ovarian follicle culture system in the porcine and this study was designed to develop an effective retrieval protocol of preantral follicles. Five different methods using collagenase type I (A) or IV (B, Cl, C2 and C3), which employed different treatment durations and/or conditions, were employed and sliced ovarian tissue of prepubertal gilts was provided for the retrieval. A significant increase in total number of follicles retrieved was detected when collagenase IV (methods B or C) was used. In total, more ovarian follicles were retrieved by method B undertaking agitation and method C2 without the agitation than method Cl and C3, while the number of preantral follicles collected was the largest in method B. Neither incubation in 5% CO 2 in air atmosphere instead of the agitation nor increased duration of enzymatic treatment up to 120 minutes improved the efficiency of follicle retrieval. There were no differences in the number of follicles retrieved from intact ovaries and from used ovaries for oocyte collection. These results demonstrate the collagenase IV treatment with agitation is effective for retrieving porcine preantral follicles from the ovaries.

Highlights

Studies on porcine reproduction have become important for developing novel biotechnology, as well as for livestock improvement (Denning and Priddle, 2003; Li et al, 2003; Shang et al, 2007; Xu et al, 2007)
The ovary consists of dense interstitial tissue (Smitz and Cortvrindt, 2001) including fibrous matrix, which mechanical isolation cannot be applied without enzymatic digestion (Telfer, 1996)
There seems to be a follicle-specificity in enzymatic treatment and digestion protocol and in this study, collagenase IV was superior to collagenase I for retrieving large number of oocytes without losing morphological normality

Summary

Introduction

Studies on porcine reproduction have become important for developing novel biotechnology, as well as for livestock improvement (Denning and Priddle, 2003; Li et al, 2003; Shang et al, 2007; Xu et al, 2007). Producing transgenic pig and establishing pluripotent porcine embryonic stem (ES) cells were major targets of the studies, and securing large number of oocyte is a prerequisite factor for the development. There has been a limitation to retrieve sufficient number of the oocytes (Smitz and Cortvrindt, 2001). Numerous efforts have been made to overcome such limitation (Eppig et al, 1989; Eppig et al, 1996; Cortvrindt et al, 1998; Lenie et al, 2004) and many of studies have been exerted to optimize oocyte retrieval system from the livestock (Akshey et al, 2005; Yang et al, 2005; Choi et al, 2006). Mature oocytes derived from the follicle culture have a potential to develop blastocysts and further derive ES cells following subculture of the inner cell mass cells of the parthenotes. Approximately 60% of cultured follicles yield developmentally-competent oocytes and more than 20% of

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