Holding of bovine blastocysts at suprazero temperatures using small molecules

Abstract

Although assisted reproductive technology (ART) currently exists, the only embryo preservation technology that is available is cryopreservation. In the present study, small molecules were used to hold embryos at room temperature. The basic medium for embryo holding for a short period of time at 4 °C, 10 °C and 20 °C consisted of 1% BSA non-cryopreservation medium (BNC) instead of fetal bovine serum. To maintain survival and prevent damage during embryo incubation, three candidate small molecules were selected-CHIR99021, Y-27632 and Thiazovivin-and their concentrations were optimized. The viability and hatching rate of embryos incubated at 10 °C were greater for Y-27632-BNC and CHIR99021+Y-27632-BNC compared to BNC. However, the rate was lower for Thiazovivin-BNC compared to BNC. Although there were no surviving embryos after incubation at 20 °C, the viability and hatching rate of embryos significantly increased in Y-27632-BNC and CHIR99021+Y-27632-BNC compared to BNC. The pregnancy rate of embryos incubated at 20 °C was also greater in the CHIR99021+Y-27632-BNC group compared to that in the frozen group. The mechanism by which small molecules enhance survival of embryos during incubation was investigated, and expression of heat shock protein 70 was observed to increase. The findings of this work may be useful in improving ART in the agricultural field.

Figure 1

The effects of three small molecules on bovine embryo incubation at 4 °C for 96 h. (a) After incubation at 4 °C for 96 h, the morphology of embryos in three different small molecule groups were comparable to those in the BNC group, appearing dark and shrunken. (b) After in vitro culture for 48 h (recovering period), expanded and hatched embryos were shown in all experimental groups without any morphological differences. Abbreviations are the same as in Table 2. Scale bar = 100 μm.

Figure 2

The effects of small molecules on bovine embryo incubation at 20 °C for 72 h. (a) After incubation at 20 °C for 72 h, the morphology of the embryos incubated in three different small molecule groups was comparable to those incubated in BNC, appearing dark and shrunken. (b) After in vitro culture for 48 h (recovering period), expanded and hatched embryos were shown in Y−, C+Y− and C+T-BNC media with no morphological differences. However, there were no living embryos after incubation in BNC. Abbreviations are the same as in Table 2. Scale bar = 100 μm.

Figure 3

The expression of heat shock protein 70 (HSP70) after incubation with small molecules at 4, 10 and 20 °C. HSP70 expression (green) was observed in embryos incubated in C+Y-BNC at 4, 10 and 20 °C (a,a′, c,c′, e,e′, f and f′), but not BNC (b,b′, d, and d′). The arrows point to elevated expression of HSP70 in trophoblasts and the arrow heads point to elevated HSP70 expression in the inner cell mass. The nucleus was stained with DAPI (blue). The negative control was a stained embryo without primary anti-body (g). Abbreviations are the same as in Table 2. Scale bar = 200 μm.