Forced Collapse of the Blastocoel Cavity Improves Developmental Potential in Cryopreserved Bovine Blastocysts by Slow‐Rate Freezing and Vitrification

Abstract

This study was conducted to evaluate the effectiveness of forced collapse of the blastocoel before slow-rate freezing and vitrification of bovine blastocysts. Cryopreservation of bovine blastocysts has been proposed as a tool to improve the feasibility of cattle production using the embryo transfer technique. However, the low efficiency of frozen-thawed embryos survival and further development is a crucial problem. In this study, bovine in vitro and in vivo blastocysts were slow-rate frozen and vitrified after forced blastocoele collapse (FBC) of the blastocyst cavity by puncturing the blastocoele with a pulled Pasteur pipet. Differences in the developmental potential of frozen-thawed blastocysts derived from FBC and non-FBC groups were found in both slow-rate freezing and vitrification. Furthermore, we found that the total cell number of blastocysts in FBC groups was increased and the index of apoptosis in FBC groups was decreased. Consistent with these results, real-time RT-PCR analysis data showed that expression of the anti-apoptotic Bcl-XL gene was significantly increased by FBC groups, whereas expression of the pro-apoptotic Bax gene was significantly decreased by FBC groups. Our results also showed that pregnancy outcomes in both slow-rate frozen and vitrified bovine in vivo blastocysts could be improved by reducing the fluid content after FBC of the blastocyst cavity. Therefore, we suggest that FBC of the blastocyst cavity with a pulled Pasteur pipet is an effective pre-treatment technique for both slow-rate freezing and vitrification of bovine blastocysts.

Successful Vitrification of In vivo Embryos Collected from Superovulated Japanese Black Cattle (Wagyu)

“…To improve the vitrification procedure in ruminants too, blastocysts have been punctured to flush away the blastocoele fluid and return the embryo to a morula‐like stage (Min et al. ).…”

“…), to be considered as a possible alternative approach to routine slow freezing technology, has been implemented in this study (Min et al. ). In fact, vitrification does not require expensive slow freezer apparatus, cryopreservation equipment and freezing media, which are usually not affordable for most local farmers.…”

Effect of blastocoel fluid reduction before vitrification on gene expression in mouse blastocysts

“…A large blastocoel may, in fact, cause insufficient permeation of cryoprotectant and dehydration, resulting in the formation of ice crystal during vitrification; reducing the volume of blastocoel fluid by artificial collapse before vitrification overcomes this problem. A number of studies demonstrated improved rates of hatching, implantation, and pregnancy of blastocysts following artificial collapse of the blastocoel (Vanderzwalmen et al, ; Son et al, ; Hiraoka et al, ; Mukaida et al, ; Iwayama et al, ; Min et al, ; Van Landuyt et al, )—including ours (Table )—yet the underlying mechanism for this improvement is still unclear (Son et al, ). Creating a physical hole in the zona pellucida during the artificial collapse process might assist hatching, although changes to the energy allocation of expanded blastocyst may also contribute.…”

“…Substantial improvements in vitrification protocols have increased the survival rates of post‐warmed oocytes and embryos (Stehlik et al, ; Liebermann and Tucker, ; Iwayama et al, ), whereas insufficient advances have been made regarding the hatching rate of vitrified blastocysts compared to their fresh counterparts (Cuello et al, ; Sánchez‐Osorio et al, ; Min et al, ). Despite the optimized viability of the vitrified‐warmed blastocysts, a significant decrease was still observed in the hatching rate (Table ).…”

“…These harmful effects of blastocoel fluid can be reduced by depleting the fluid volume, known as artificial collapse of the blastocoel cavity, using a glass micro‐needle (Vanderzwalmen et al, ; Kim et al, ), 29‐gauge needle (Son et al, ), repeated pipetting through different diameter micropipettes (Hiraoka et al, ), microsuction (Chen et al, ), or laser pulse (Mukaida et al, ; Van Landuyt et al, ). Implementing artificial collapse methods before vitrification appears to increase pregnancy and embryo survival rates by decreasing DNA damage (Desai et al, ; Min et al, ) and improving total cell number as well as decreasing apoptosis index (Min et al, ).…”