Exopolysaccharide ID1 Improves Post-Warming Outcomes after Vitrification of In Vitro-Produced Bovine Embryos

Abstract

This study aimed to assess the cryoprotectant role of exopolysaccharide (EPS) ID1, produced by Antarctic Pseudomonas sp., in the vitrification of in vitro-produced (IVP) bovine embryos. IVP day 7 (D7) and day 8 (D8) expanded blastocysts derived from cow or calf oocytes were vitrified without supplementation (EPS0) or supplemented with 10 µg/mL (EPS10) or 100 µg/mL (EPS100) EPS ID1. The effect of EPS ID1 was assessed in post-warming re-expansion and hatching rates, differential cell count, apoptosis rate, and gene expression. EPS100 re-expansion rates were significantly higher than those observed for the EPS0 and EPS10 treatments, regardless of culture length or oocyte source. EPS100 hatching rate was similar to the one of the fresh blastocysts except for those D7 blastocysts derived from calf oocytes. No differences were observed among EPS ID1 treatments when the inner cell mass, trophectoderm, and total cell number were assessed. Although apoptosis rates were higher (p ≤ 0.05) in vitrified groups compared to fresh embryos, EPS100 blastocysts had a lower number (p ≤ 0.05) of apoptotic nuclei than the EPS0 or EPS10 groups. No differences in the expression of BCL2, AQP3, CX43, and SOD1 genes between treatments were observed. Vitrification without EPS ID1 supplementation produced blastocysts with significantly higher BAX gene expression, whereas treatment with 100 µg/mL EPS ID1 returned BAX levels to those observed in non-vitrified blastocysts. Our results suggest that 100 µg/mL EPS ID1 added to the vitrification media is beneficial for embryo cryopreservation because it results in higher re-expansion and hatching ability and it positively modulates apoptosis.

Cryoprotectant role of exopolysaccharide ID1 in the vitrification/in‐straw warming of in vitro‐produced bovine embryos

“…Day 7‐expanded blastocysts were randomly allocated into three groups: (1) Cryotop, blastocysts were vitrified/warmed following the short equilibration protocol of the Cryotop method (Rizos et al, 2001; Walton et al, 2017); (2) VitTrans, blastocysts were vitrified/warmed following the short equilibration VitTrans protocol described in Martinez‐Rodero et al (Martinez‐Rodero et al, 2021) (Figure 1); (3) VitTrans‐EPS ID1, blastocysts were vitrified/warmed by the VitTans protocol but vitrification media were supplemented with 100 μg/mL EPS ID1 as already described in Ordóñez‐León et al (2022). Non‐vitrified blastocysts served as the fresh control.…”

“…Day 7-expanded blastocysts were randomly allocated into three groups: (1) Cryotop, blastocysts were vitrified/warmed following the short equilibration protocol of the Cryotop method (Rizos et al, 2001;Walton et al, 2017) were supplemented with 100 μg/mL EPS ID1 as already described in Ordóñez-León et al (2022). Non-vitrified blastocysts served as the fresh control.…”