To effectively transfer vitrification technology to the field, the procedures used for the warming and transfer of cryopreserved bovine embryos should be kept as simple as possible. The VitTrans device was designed to facilitate the vitrification/warming technique by including an easy one-step in-straw dilution method followed by direct embryo transfer to the uterus . While we have reported post-warming survival rates of around 60% for D7 expanded blastocysts vitrified using VitTrans, here we modified the two-step vitrification protocol to improve post-warming outcomes. The objective of this study was to investigate the effects of different equilibration times on several post-warming outcome measures in bovine D7 and D8 expanded blastocysts vitrified using the VitTrans procedure. Our results indicate that a short equilibration time (3 min) during vitrification improves post-warming survival and the hatching ability of both D7 and D8 expanded blastocysts, whereas lengthening the equilibration time to 12 min does not seem to offer any further benefits. In addition, the hatching rates of D7-blastocysts vitrified by the SE protocol were similar to those recorded for fresh non-vitrified embryos. Several studies have compared equilibration times used in the vitrification of in vitro produced blastocysts of different species [25–29]. In cattle, Do et al.  found similar re-expansion (24 h post-warming) and hatching rates (48 h post-warming) when bovine expanded blastocysts were vitrified after a short (3 min) or long equilibration (8 min) time possibly explained by differences in temperature and equilibration times. Thus, while the short equilibration time tested by Do et al.  was similar to ours (3 min at 37 °C), their long equilibration protocol consisted of 8 min at room temperature, which may have resulted in reduced cytotoxicity and osmotic stress  and thus minimized differences between the use of their short or long protocol. Consistently, in a study carried out in the dromedary camel, loading of CPAs at 37 °C for a short exposure time (3 min) led to an outcome comparable to that of original processing at room temperature with a longer exposure time (15 min) . When working at room temperature in humans and mice, different equilibration times did not affect post-warming embryo survival [25, 28]. However, lengthening the exposure time to the equilibration solution from 4 to 8 min was found to improve the DNA integrity index after the vitrification of murine blastocysts [25, 28]. Prior to the vitrification of human blastocysts, 9–10 min of exposure to the equilibration solution improved the outcomes clinical pregnancy, embryo implantation and live birth rates compared to shorter exposure times .
Different vitrification outcomes have been recently reported after vitrification of expanded blastocysts using various one-step warming devices and short equilibration times. As we have also observed, one-step in-straw warming/dilution of expanded blastocysts vitrified in fiberplugs returned similar  or higher survival rates  for D7 than D8 blastocysts. However, either lower  or higher  hatching rates were observed at 24 h post-warming when D7 or D8 expanded blastocysts were vitrified in fiberplugs compared to our results. Further, one-step warming of bovine D7 expanded blastocysts vitrified on hand-pulled glass micropipettes as the cryodevice led to higher hatching rates assessed at 72 h post-warming rather than 24 h post-warming [12, 19].
When 24 h post-warming outcomes were compared after the vitrification of blastocysts produced after different times of in vitro culture, our results are consistent with those of others. Thus, significantly higher survival, re-expansion and hatching rates have been described after the vitrification of D7 compared to D8 IVP bovine blastocysts [20, 21, 30, 31] such that cryotolerance diminishes as the length of embryo culture increases. In the present study, although the hatching ability of D7 blastocysts vitrified/warmed within the SE protocol was comparable that of fresh non-vitrified D7 blastocysts, Day 8 vitrified/warmed blastocyst gave rise to under half of the hatching yield observed in the fresh control group. Early developing embryos are better at surviving than later embryos. This has been highlighted in prior work in which vitrified/warmed earlier cryopreserved IVP bovine blastocysts returned higher survival, hatching and pregnancy rates [20, 21].
The correct distribution of cells in the ICM and outer TE layer of the blastocyst is crucial for embryo development. However, while it is accepted that a minimal number of embryonic cells is needed to establish pregnancy , optimal ICM and TE cell numbers and distributions in the blastocyst remain unclear. Thus, higher ICM cell counts may lead to increased pregnancy rates  and an excessive number of cells allocated to the TE may lead to pregnancy abnormalities [34, 35]. Here, the TUNEL assay combined with differential staining for ICM and TE cells revealed significantly lower TCN and TE-cell numbers and a higher apoptosis rate in vitrified/warmed D7 re-expanded blastocysts compared to fresh ones while no differences emerged in ICM cell numbers, suggesting that the main site of cryopreservation-related membrane damage was the trophectoderm. Similar  or reduced total cell counts have been reported after bovine embryo vitrification [37, 38], mainly due to a low cell count in the TE. This effect is consistent with a greater accumulation of lipids in the TE than ICM , as cytoplasmic lipid contents appear strongly related to survival of cryopreservation . In contrast, Gomez et al.  described that vitrification seemed to exert a detrimental effect on the ICM, while TE cells survived cryopreservation in numbers comparable to those counted in embryos before vitrification. However, we detected no differences in TCN, or in ICM and TE cell numbers between D7 blastocysts vitrified after SE and fresh blastocysts, while D7 blastocysts vitrified after LE showed significantly lower TCN and numbers of TE cells. This suggests that D7 expanded blastocysts vitrified using our SE protocol suffered less cryodamage or were able to recover from any damage at 24 h post-warming, showing similar hatching rates and embryo quality as those of fresh ones. However, among the D8 embryos subjected to vitrification/warming, TCN, and ICM and TE cell numbers were significantly lower in hatching blastocysts when compared to fresh blastocysts, regardless of the equilibration time. The timing of blastocyst formation is a good marker of embryo quality determining that early-cavitating embryos are of better quality than later cavitating embryos in terms of total cell numbers, inner cell mass and trophectoderm cell distributions, and cryosurvival [30, 34]. While we still lack reliable blastocyst stage morphological predictors of competence after embryo transfer, it is accepted by many research groups and commercial companies that best pregnancy rates are achieved after the transfer of day 7 expanded bovine blastocysts whether fresh or cryopreserved (reviewed by [41, 42]).
Apoptosis has been frequently used as a marker for embryo quality as high rates of apoptotic cells have been linked to the reduced developmental competence of both in vivo or in vitro produced embryos [43–45]. Vitrification requires adequate dehydration and a high viscosity across all blastomeres and blastocele which is difficult given the characteristics of the blastocyst (multicellularity, presence of blastocele with high water content). This determines that vitrification leads to a post-warming increase in apoptosis . Our results revealed that both equilibration solution exposure times induced apoptosis in surviving blastocysts by the time of their re-expansion and hatching. However, while the apoptosis rate for D7 expanded blastocysts vitrified via the VitTrans LE protocol was similar to that reported previously by Morató and Mogas , the apoptotic cell rate was significantly higher for the LE than SE protocol or control embryos. This finding suggests that the high toxicity effect of CPAs produced at high temperature can be avoided to some extent by reducing the time of exposure to the cryoprotectant . Moreover, D8 embryos induced higher percentages of apoptotic cells that D7 embryos, in agreement with results observed when expanded blastocysts were vitrified/warned using a one-step direct transfer procedure .
When genes related to apoptosis were analysed, a significantly higher abundance of BCL2L1 transcripts was observed in both expanded and hatched blastocysts derived from the SE protocol when compared to fresh embryos or those vitrified after the long equilibration period, while there were no differences in BAX gene expression among treatments. Yang and Rajamahendran  related a higher expression level of Bcl-2 to better quality embryos less prone to apoptosis. However, the levels of BCL2L1 gene expression observed in our study were inconsistent with apoptosis levels assessed by TUNEL in fresh or vitrified D7 blastocysts, suggesting that apoptosis detected by TUNEL is independent of the expression of BCL2L1 or BAX genes, as observed previously . Similarly, mRNA levels of SOD1 were upregulated after SE treatment, indicating that a shorter exposure time may reduce oxidative stress by improving the activity of antioxidant enzymes and improving the quality of vitrified/warmed embryos . In addition, a trend although not significant (P = 0.07) was observed towards greater CX43 and AQP3 gene expression in blastocysts subjected to SE compared to LE. In effect, high expression of CX43, a gene related to cell compaction and adhesion , has been linked to better quality and more cryotolerant embryos . AQP3 plays an important role in the transport of cryoprotectants and fluids during the cryopreservation of bovine embryos . The presence of mRNA encoding this protein has been also related to better embryo cryotolerance . While not always significant, the differences in gene expression observed in surviving blastocysts derived from D7 blastocysts vitrified after the SE treatment could be indicative of better embryo quality. In effect, these blastocysts showed an improved hatching ability together with higher TCN, and TE cell numbers and a lower apoptosis rate.