104 COMPARATIVE ANALYSIS OF THE YOLK SAC OF BOVINE EMBRYOS AT 30 DAYS OF PREGNANCY DERIVED FROM ARTIFICIAL INSEMINATION OR IN VITRO FERTILIZATION

Abstract

The yolk sac has importance related to hematopoiesis, because it carries blood cells, and is part of the circulatory system in early embryos. The yolk sac is related to proper embryonic development because the organ is responsible for transmitting metabolic, nutrition, protection, and training of key organs, such as the intestine, in early embryos before formation of the placenta. This study attempted to compare the yolk sac of bovine embryos derived from fixed-time artificial insemination (TAI) and in vitro fertilization (IVF), relating them to their stage of development and growth at 30 days of gestation. Morphological, histological and immunological diseases were also compared. Yolk sacs from TAI or IVF bovine embryos at 30 days of gestation were collected (n = 2/group). The yolk sacs were immersed in fixative solution of 10% formaldehyde and embedded in Paraplast for light microscopy and immunohistochemical techniques. Sample cuts of 5 μm thickness were stained with hematoxylin and eosin (HE) and exposed to primary (anti-K_i-67; 1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and secondary (LSAB + Peroxidase Kit – K0690, DakoCytomation, Glostrup, Denmark) antibodies. To reveal immunoreactivity, diaminobenzidine (DAB, Dako) was used and the background was counter-stained with Harris hematoxylin. On Day 30 of gestation, the yolk sac of embryos from either TAI or IVF were quite developed, yellowish coloured, and presented signs of early regression. Moreover, they showed a central compact area that was clearly visible next to the embryo. The free ends were elongated and accompanied by umbilical vessels towards the ends of the gestational sac. There were no signs of fusion between the chorion and allantois, but the juxtaposition was evident with the allantois. The yolk sac is composed of three layers: the endoderm, mesothelium (simple mesothelial), and an intermediate layer (vascular mesenchymal). The layer corresponding to the mesenchyme presented thin and elongated cells with characteristics of synthesising cells. The K_I-67 is a marker of cell proliferation, present in all active forms of the cell cycle and absent during quiescence. This expression reflects proliferative activity; however, in all samples subjected to immunohistochemical reaction, there was no nuclear staining of the antibody. We conclude that on Day 30 of gestation, the yolk sac shows no differences in morphological and microscopic characteristics between TAI and IVF groups. It also appears that the process of regression had not occurred as indicated by staining patterns of the K_i-67 antibody.