The effectiveness of the stereomicroscopic evaluation of embryo quality in vitrified–warmed porcine blastocysts: An ultrastructural and cell death study

The objective of this study was to analyze the validity of the stereomicroscopic evaluation of vitrified–warmed (V–W) porcine blastocysts. Unhatched blastocysts were obtained from Large-white gilts (n = 10). Blastocysts (n = 156) were vitrified using the Open Pulled Straw technology. After warming, V–W blastocysts were cultured for 24 h (V24). Then, their developmental progression was morphologically assessed by stereomicroscopy and classified as: V24 viable re-expanded blastocysts; V24 viable hatched blastocysts or V24 degenerated. Blastocysts which re-expanded or hatched after warming were considered viable. Some fresh blastocysts were not vitrified and were evaluated after 24 h in culture (F24). By stereomicroscopic analysis all the fresh blastocysts were considered viable. Some F24, V24 re-expanded viable, V24 hatched viable and V24 degenerated blastocysts were processed for transmission electron microscopy (n = 13, 19, 9 and 9, respectively) or assessed by TUNEL for cell-death evaluation (n = 16, 21, 11 and 21, respectively). All V24 hatched blastocysts showed similar ultrastructure to fresh blastocysts. However, some V24 re-expanded blastocysts considered viable (6/19) revealed ultrastructural alterations. Degenerated V24 blastocysts showed ultrastructural disintegration. Hatched V24 blastocysts did not differ (p > 0.05) from F24 hatched blastocysts with regard to the ratio of dead cells (2.8 ± 0.5% versus 1.9 ± 0.3%, respectively). However, V24 expanded blastocysts had higher (p < 0.01) cell death levels (4.3 ± 3.4%) than those observed in the F24 expanded blastocysts (1.1 ± 0.3%). The degenerated blastocysts showed the highest cell-death index (19.4 ± 6.3%). In summary, V–W blastocyst hatching during in vitro culture appears to coincide with good ultrastructure and low cell-death index, suggesting that the hatching rate assessed by stereomicroscopy is more appropriate than embryo re-expansion for an evaluation of V–W blastocyst quality.