Evidence of Damage in Cryopreserved and Fresh Bovine Embryos Using the Tunel Technique

Abstract

The objective of the present study was to evaluate the quality of bovine embryos cryopreserved in different years in Chiapas, Mexico. The embryos were obtained from a government institution (FIMEGEN) dedicated to promoting embryo transfer among dual-purpose cattle farmers. Forty-three embryos frozen in 1988, 1989, 2000 and 2002 were analysed with the Tunel technique to detect programmed cell death (apoptosis). Eleven fresh embryos were used as controls. Analysis of variance was used in embryos stored in the different years with averages tested using Tukey's test. Student's t-test was employed to compare fresh and frozen cells. Embryos with shorter storage time presented a lower number (p < 0.001) of Tunel-positive cells compared with embryos stored for longer time. On the contrary, when comparing the number of apoptotic cells between frozen and fresh embryos a higher number of positive cells (p < 0.05) were found in the former. The present results suggest that the cryopreservation per se caused damage that compromises the viability of the embryo. Another explanation for the lower pregnancy rate found in the tropics could be irreversible damage caused by poor storage technique in these large operations.

Artificial Collapse of Blastocysts Before Vitrification: Mechanical vs. Laser Technique and Effect on Survival, Cell Number, and Cell Death in Early and Expanded Blastocysts

“…This method has been used by various investigators to assess cell death in frozen embryos subjected to varying culture conditions and growth factor additives. 18,[28][29][30] Use of DNA damage as an early marker for cell death was a very valuable tool in elucidating differences between treatment groups even as early as 3 h after warming. Minimal damage was seen with either method of collapse (3-5%) in contrast to the non-collapsed control blastocysts (13%).…”

Development of vitrified-warmed mouse embryos co-cultured with polarized or non-polarized uterine epithelial cells using sequential culture media

“…Columns within a variate with different letters are significantly different (ANOVA, P<0.05) intact blastocysts, some cells spontaneously undergo apoptosis during embryonic development [50]. Recently, the number of apoptotic cells in the frozen embryos was shown to increase after thawing compared with that of fresh embryos [11]. Therefore, the damage in the cell organelles and genetic material of embryos resulting from the exposure to cryoprotectants may trigger the apoptotic cascade leading to a decrease in the developmental competence of embryos.…”

“…As shown in previous studies, cryopreservation increases the number of apoptotic cells compared with that of fresh embryos [11]. It has been well known that the embryos can be severely altered or damaged by being cooled below physiological temperature, and DNA fragmentation is associated with cooling or cryosensivity [12].…”

Can Ethanol Enhance the Cleavage and Development of Blocked Two-cell Embryos?

“…Keeping embryos in low temperatures causes DNA fragmentation and cell death [22], [23]. Last studies have indicated that ethanol can activate oocyte and causes parthenogenesis.…”