A Simple Method for Selection of Cumulus-Oocyte Complexes from Bovine Ovaries by Sedimentation with Percoll

Abstract

Abstract. Selection of bovine cumulus-oocyte complexes (COCs) for in vitro embryo production (IVP) is generally based on the morphological characteristics of the cumulus cells surrounding the oocytes and the ooplasm under microscopic observation. The purpose of this study was to examine a simple method for selection of COCs by sedimentation with Percoll solutions. COCs were aspirated from ovaries derived from a local slaughterhouse, and the COCs were classified by the morphology of their cumulus cell layers, as follows: Class A, compact and thick; Class B, compact but thin; Class C, partially denuded and thin; and Class D, denuded. Percoll solutions were prepared by diluting Percoll to 7.5, 10, 12.5, 15, 17.5, 20, 22.5, 25, 27.5 and 30% solutions, respectively. COCs were placed on the surface of the Percoll solution for 3 min, and the precipitated COCs were transferred to stepwise high density solution. The percentage of Percoll solution just before buoyancy was considered to the specific sedimentary value of the COC and oocyte. The mean sedimentary value of Class A COCs was higher than those of the other classes (P<0.01). The mean sedimentary values of denuded oocytes from Classes A and B were higher than those from Classes C and D (P<0.01). Our results show that sedimentation of COCs and denuded oocytes was generally related to the morphological quality of the COCs, although the sedimentary values ranged widely for one class of COCs and oocytes. The Percoll method can be used for simple selection of COCs. Key words: Bovine, Cumulus-oocyte complex, Percoll, Sedimentation (J. Reprod. Dev. 53: [971][972][973][974][975][976] 2007) n vitro maturation, fertilization and culture technologies for bovine oocytes are beneficial for increasing the number of embryos transferable to recipient cows. Various factors, including oocyte quality, culture media, protein source, growth factors and oxygen tension for the in vitro embryo production system (IVP), affect preimplantation embryo development in vitro [1][2][3]. Oocyte quality is an important factor affecting the success of an IVP system. Selection of oocytes for in vitro maturation is generally based on the quality of the cytoplasm and the characteristics of the cumulus cell investment around the oocyte [4,5]. These morphological criteria are routinely used to select the cumulus-oocyte complexes (COCs) with the most competent oocytes for embryo development. However, subjective evaluation of oocyte quality by morphological criteria under microscopic observation is thought to differ among evaluators. Recently, other means of characterizing bovine oocytes have been examined, such as evaluating the corona radiata density of cumulus-corona-oocyte complexes [6], distribution of cortical granules in oocytes [7] and expression of the LH receptor in

Simple separation of good quality bovine oocytes using a microfluidic device

“…In contrast, Yotsushima et al . reported that the speed of sedimentation of cumulus-oocyte complexes (COCs) and denuded oocytes in hypertonic solution correlated with the morphological quality of COCs 10 . This report indicated that the sedimentation method could be a useful method to objectively select good quality oocytes.…”