Effect of culture methods on cumulus and oocyte morphology and meiotic competence of bovine oocytes from early antral follicles

L Kątska-Książkiewicz,H Alm

doi:10.5194/aab-48-562-2005

Abstract

Abstract. The objective of the present study was to establish a culture system that would maintain the three-dimensional structure of bovine early antral follicles (EAF) or isolated cumulus-oocyte-granulosa complexes (COCGs) and increase the resulting portion of COCs with normal morphology for subsequent IVM. The morphological quality and meiotic competence of oocytes originating from early antral bovine ovarian follicles (0.2 to 0.7 mm and 0.4 to 0.7 mm diameter) were evaluated following culture in vitro for 14 and 7 d, respectively, and subsequent in vitro maturation. Growth culture modifications included culture in the well of the well (WOW) system; a microdroplet of collagen gel (2 x 5 μl vs. 2 x 400 μl; standard system) and culture of EAF in hanging drops of medium (inverted system). Significantly higher (P<0.01) proportions of COCs with normal morphology (60.4%) were obtained from COCGs compared to EAF (4.8%) grown in the WOW system. Embedding of COCGs in microdrops of collagen gel significantly increased proportion of COCs with normal morphology (63.2%) compared to those embedded in standard volume gels (35.3%). Recovery rate of COCs with normal morphology from cultured EAF was improved both by using microdrops of gel (44%) and by culture in the inverted system (39.3%) over that found for the standard system (8.5%).

Highlights

The mammalian ovary contains thousands of oocytes which are enclosed in preantral follicles
In most bovine follicle culture systems, intact follicles or cumulus oocyte complexes surrounded by parietal granulosa cells (COCGs) are cultured under a gase phase of 5% CO2 in air (~ 20% oxygen), between two coats of collagen gel placed on the bottom of culture dish or well, which is covered by medium and, in some cases an overlay of oil (TELFER, 1998; YAMAMOTO et al, 1999; ITOH et al, 2002)
We have investigated the use of different systems for growth culture of follicles and cumulus-oocyte-granulosa complexes (COCGs) and compared it with the system used in other experiments (TELFER, 1998; ITOH et al, 2002)

Summary

Introduction

The mammalian ovary contains thousands of oocytes which are enclosed in preantral follicles. Other attempts to culture bovine oocyte-granulosa cell complexes originating from early antral follicles on the flat substratum in medium supplemented with PVP allowed to improved efficiency of development to blastocyst stage to 12% (HIRAO et al, 2004). It has been observed that during long term culture in collagen gels that most granulosa cells migrate out of the follicle or follicle-like structures, and the oocyte becomes denuded of cumulus cells, which could drastically reduced its quality and competence (MIYANO et al, 1998; YAMAMOTO et al, 1999; KĄTSKA-KSIĄŻKIEWICZ, unpublished observations). In this study our goal was to establish a culture system that would be able to maintain the three-dimensional structure of bovine early antral follicles or COCGs for a long period, reduce the migration of cumulus cells and increase the proportion of cumulusoocyte complexes (COCs) with normal morphology, and as a consequence increase meiotic competence

Objectives

Methods

Results

Discussion

Conclusion