333 EFFECTS OF BOVINE OOCYTE QUALITY ON KINETICS OF NUCLEAR MATURATION AND EMBRYONIC DEVELOPMENT AFTER IN VITRO FERTILIZATION

Abstract

Many factors act together to prepare the immature oocyte for successful development to a competent embryo after fertilization. Defects in oocyte maturation and further development can possibly be caused by the oocyte quality or an inadequate nuclear maturation or even by a failure of both. In the present study, the effect of COCs quality on meiotic development and further embryo-development after in vitro fertilization was evaluated. A total of 3604 COCs was separated according to their morphological aspect and were classified as A, B, and C categories. Briefly, in class A, oocytes possessed compact layers of cumulus cells, being difficult to evaluate their number having a homogenous ooplasm with uniform color. In class B, oocytes show more or equal to five layers of cumulus cells, easily identifiable under a stereomicroscope and/or granulations in the ooplasm. In class C, some granulation was observed in oocytes with about three layers of cumulus cells. The total number of oocytes was divided into two groups (I and II) in which in the group I, COCs (n = 540) were fixed 0, 6, 12, 18, 24, and 30 h following ovarian aspiration, DNA was stained with aceto-orcein, and the nucleus were observed under a phase contrast microscope. In the Group II, COCs (n = 3064) were fertilized with frozen/thawed bull semen after 24 h of maturation, which was made in M199 medium (Sigma, St, Louis, MO, USA). The development of the embryos was evaluated on the third and seventh day after fertilization. Embryos were co-cultured with monolayers of granulosa cells in 45 μL droplets of B2 medium (CCD Laboratory, Paris, France), supplemented with 10% serum under mineral oil, at 39°C and 5% CO₂ in air. It was observed that, other than the oocytes achieved metaphase II at 24 h was greater for the oocytes classified as A (65.4%), and B (61.0%) greater than C (51.2%), no statistical difference was observed between oocyte quality and capability to maturation. As far as the embryonic development is concerned, the same tendency was observed for the cleavage and for the morulae/blastocyst stage after 7 days after fertilization (P < 0.001). The percentages of cleaved oocytes classified as A, B, and C, were respectively 65.2%, 58.4%, and 48.0%. The development to the morulae/blastocyst stage of the cleaved embryos was A = 38.5%/27.4%, B = 33.6%/25.0%, and C = 30.9%/17.2% (Table 1). The results of our study clearly demonstrate that the morphology of the oocytes plays an important role on the in vitro embryonic developmental competence after fertilization.

Table 1.  Development of oocytes according to COCs quality, evaluated 3 and 7 days after fertilization

The first author is supported by the Regional Foundation for Science and Technology of the Azores Government. This study was supported by the IBBA Institute grant number M2.1.2/I/022/2008 CITA-A is fully acknowledged.