ß-Mercaptoethanol in culture medium improves cryotolerance of in vitro-produced bovine embryos.

The objective of this study was to investigate the effects of adding ß-mercaptoethanol (ßME) to culture medium of bovine in vitro-produced (IVP) embryos prior to or after vitrification on embryo development and cryotolerance. In Experiment I, Day-7 IVP blastocysts were vitrified and, after warming, cultured in medium containing 0, 50 or 100 µM ßME for 72 h. Embryos cultured in 100 µM ßME attained higher hatching rates (66.7%) than those culture in 0 (47.7%) and 50 (52.4%) µM ßME. In Experiment II, IVP embryos were in vitro-cultured (IVC) to the blastocyst stage in 0 (control) or 100 µM ßME, followed by vitrification. After warming, embryos were cultured for 72 h (post-warming culture, PWC) in 0 (control) or 100 µM ßME, in a 2 × 2 factorial design: (i) CTRL-CTRL, control IVC and control PWC; (ii) CTRL-ßME, control IVC and ßME-supplemented PWC; (iii) ßME-CTRL, ßME-supplemented IVC and control PWC; or (iv) ßME-ßME, ßME-supplemented IVC and ßME-supplemented PWC. ßME during IVC reduced embryo development (28.0% vs. 43.8%) but, following vitrification, higher re-expansion rates were seen in ßME-CTRL (84.0%) and ßME-ßME (87.5%) than in CTRL-CTRL (71.0%) and CTRL-ßME (73.1%). Hatching rates were higher in CTRL-ßME (58.1%) and ßME-ßME (63.8%) than in CTRL-CTRL (36.6%) and ßME-CTRL (42.0%). Total cell number in hatched blastocysts was higher in ßME-ßME (181.2 ± 7.4 cells) than CTRL-CTRL (139.0 ± 9.9 cells). Adding ßME to the IVC medium reduced development but increased cryotolerance, whereas adding ßME to the PWC medium improved embryo survival, hatching rates, and total cell numbers.

Characterization of the extracellular matrix in the chorioallantoic membrane of water buffalo (Bubalus bubalis) in early gestation.

Placenta is formed by a parenchyma rich in extracellular matrix (ECM), and this structure guarantees the proper development of the embryo and placental functioning. Recently, studies have focused on the characterization of ECM in the placenta and foetal membranes of different species. This work aimed to analyse the composition of the ECM and to quantify the types of collagens in its composition. For this, 33 chorioallantoic membranes were used at different gestational ages, which were grouped into five groups. Subsequently, haematoxylin-eosin staining, Masson trichrome and picrosirius were performed for histological analysis. Through the technique of polarized light, it was possible to quantify the total collagen present in the membranes and finally the immunohistochemical technique was performed to verify the presence of collagens and glycoproteins. It was possible to verify that the chorioallantoic membranes have, in all the gestational periods of the initial third of gestation, the same histological structures, being the most significant difference the membrane thickening that occurs gradually during the gestation. However, we notice the appearance of binucleate cells only from group II. In addition, it was verified that a gradual increase of collagen occurs until the group IV, yet from the group V begins to occur a decrease of this protein. In addition, collagen I, collagen III, fibronectin and laminin were present in all membranes. With this, we concluded that the buffalo chorioallantoic membrane presents ECM in constant remodelling at the beginning of gestation and can be used as biomaterial in works on regenerative biology.