23 Biopsied in vitro-produced bovine blastocysts survive vitrification better than slow freezing

Abstract

Trophectoderm (TE) biopsying for single nucleotide polymorphisms (SNPs) analysis is being implemented as a tool for the selection of elite bovine embryos. This biopsy method renders ample cells for analysis without compromising the inner cell mass (ICM), and the blastocyst recovers quickly after biopsy. To use the SNP data for embryo selection before the transfer, the blastocysts need to be cryopreserved either with vitrification or slow-freezing. In intact in vitro-produced (IVP) blastocysts, vitrification has already proven optimal regarding embryo survival and pregnancy rates. This study aimed to investigate which cryopreservation approach is superior regarding blastocyst re-expansion rate as well as ICM, TE, and dead cell numbers after biopsying bovine IVP blastocysts. All IVP media and vitrification kits were from IVF Bioscience, and ethylene glycol with sucrose was from EggTech. Oocytes from slaughterhouse ovaries were used for blastocyst IVP. At Day 7, the blastocysts were pooled and randomised into 3 groups: (1) biopsy-control (BC), (2) biopsy-vitrification (BV), (3) biopsy-slow freeze (BSF). Subsequently, 5 to 10 TE cells were biopsied in BO-transfer medium using a 25-µm (inner diameter) biopsy pipette and flicking against the holding pipette. The BC group was incubated at 6% CO₂ and 38.8°C for 5 h. After scoring the re-expansion rate, the blastocysts were fixed with 4% paraformaldehyde/sucrose for further analyses. The BV and BSF groups were subjected to cryopreservation/thawing protocols according to the manufacturers’ instructions. Both groups, recovered under the same culture conditions as BC, were subsequently scored for re-expansion rate and finally fixed. For cell counting, the embryos were stained with Hoechst (DNA) and CDX2 (TE), combined with the TUNEL staining. ImageJ software (National Institutes for Health) was used for cell counting. P < 0.05 was considered statistically significant and was determined using Fisher’s exact test for blastocyst re-expansion rate and Student’s t-test for cell numbers. The re-expansion rate in BV was 81% (61/75), which was significantly lower than in BC (95%; 225/236; P < 0.005). In BSF, the re-expansion rate was 52% (28/54), which was significantly lower than in both BV and BC (both P < 0.005). For cell counts, 18, 18, and 14 embryos, pooled from 3 to 4 independent IVP replicates, were analysed in BC, BV, and BSF, respectively. The average numbers of ICM cells in BV and BSF were 36 ± 5 and 37 ± 11, respectively, and neither was statistically different from BC (34 ± 7; P > 0.05). The average cell counts for TE cells in BV and BSF were 59 ± 6 and 47 ± 10, respectively, and neither was statistically different from BC (56 ± 8; P > 0.05). The average numbers of dead cells in BV and BSF were 8 ± 3 and 9 ± 2, respectively, and neither was statistically different from BC (10 ± 4; P > 0.05). In summary, the biopsied bovine IVP blastocysts recovered better after vitrification, and neither of cryopreservation methods had any effect on the numbers of ICM, TE, or dead cells.

This project was supported by Innovation Fund Denmark and the Danish Milk Levy Foundation.