142 ZONA PELLUCIDA TAGGING WITH BARCODES ALLOWS THE TRACEABILITY OF BOVINE EMBRYOS CULTURED IN GROUP

Abstract

The low number of oocytes collected from unstimulated donors by ovum pick-up means that embryos produced from each female have to be cultured in very small groups. Because embryo quality and development rates are reduced in individual and small group culture, several methods to culture embryos individually but sharing the same medium have been designed. However, these systems prevent embryo movements, interfering with paracrine factors transmission and gradient changes. Here, we present an alternative in vitro culture method to allow the co-culture of embryos from different origins, without movement restriction and preserving their pedigree, by labelling the zygotes with polysilicon barcodes attached to the outer surface of the zona pellucida (ZP). Barcodes (10 × 6 × 1 µm) with 8 rectangular bits of binary codification (256 possible combinations), which can be read under a standard inverted microscope, were fabricated using silicon microtechnologies. To provide the barcodes with a ZP-binding capacity, they were biofunctionalized by self-assembled monolayers with the wheat germ agglutinin (WGA) lectin, which recognizes specific saccharides highly abundant in the ZP of most mammalian species. As a proof of concept, the culture method was tested on bovine zygotes produced from slaughterhouse-derived cow oocytes matured and fertilized in vitro. Using a mouth-controlled pipette, presumptive zygotes were individually rolled over WGA-biofunctionalized barcodes (8 barcodes/embryo) previously placed at the bottom of a drop of manipulation media. Four different barcodes, each one with a different codification, were used to encode 25 embryos (6–7 embryos/barcode codification), which were then cultured together in the same drop of medium. Day 7 (D7) and Day 8 (D8) blastocyst, and barcode retention rates were assessed. In addition, D7 expanded blastocysts were vitrified by the cryotop method and post-warming survival was determined as re-expansion rate at 24 h in culture. Finally, the quality of D8 blastocysts was assessed by differential staining and counting of inner cell mass (ICM) and trophectoderm (TE) cells. In all the experiments, a control group without barcodes was cultured and vitrified-warmed. Data were analyzed by chi-square and Mann–Whitney tests. The presence of barcodes attached to the ZP did not affect in vitro embryo development (D8 blastocysts: 29.7% control n = 309, 36.2% encoded n = 315), post-warming survival (86.4% control n = 66, 80.5% encoded n = 82), or blastocyst quality (IMC/TE: 22.1 ± 1.4/64.5 ± 5.7 control n = 18, 22.2 ± 1.7/64.1 ± 6.1 encoded n = 23). The labelling system was effective until D8 of culture, as all the embryos maintained barcodes attached (4 ± 1.8 barcodes/embryo) and could be identified, even after undergoing vitrification and warming. In conclusion, identification of co-cultured embryos by biofunctionalized barcodes attached to the ZP is feasible and will allow to culture embryos from different donors in the same drop, keeping the benefits of collective culture.

Support was provided by Spanish MEC (TEC2011-29140-C03; RZ2010-00015-0-00; AGL2010-19069), Generalitat Catalunya (2009 SGR 282 and 621), and PIF-UAB Fellowship.