83 Validation of propidium iodide dye for live-dead staining of bovine blastocysts: Preliminary results

Abstract

A widely used criterion routine laboratories utilise for the selection and classification of oocytes and embryos is morphological appearance. In order to make an objective validation of produced embryos, it is necessary to have a method that can help to distinguish viable cells. One simple and easy-to-perform method is the live-dead staining protocol. Usually, ethidium homodimer (EthD) is used to stain the dead cells, in conjunction with Hoechst 33342 for total cell numbers (Stinshoff et al. 2011). However, EthD is a harmful, toxic, and expensive staining dye. Therefore, the aim of this study was to evaluate propidium iodide (PI) as a viable alternative to EthD. To assess the suitability of PI, groups of 20 cumulus-oocyte complexes were collected from abattoir-derived bovine ovaries and matured in tissue culture medium containing bovine serum albumin and equine chorionic gonadotropin/human chorionic gonadotropin for 24 h with 5% CO₂. The cumulus-oocyte complexes were then fertilised in Fert-TALP (Tyrode's albumin lactate pyruvate) for 19 h with 5% CO₂ and finally cultured (n = 968) in SOFaa (synthetic oviductal fluid with amino acids) with 5% CO₂ and 5% O₂ with all steps being performed at 39°C. The development rate on Day 8 was on average 25.4%. Expanded Day 8 blastocysts were then utilised to validate the two different staining protocols. Expanded blastocysts were collected from culture drops and washed in PBS containing polyvinyl alcohol. The blastocysts were then stained with the selective dye and finally counterstained to determine live and dead cells. Four alternative parameters were evaluated: (A) PI 1:5 for 15 min, (B) PI 1:10 for 15 min, (C) PI 1:5 for 5 min, and (D) EthD 1:10 for 15 min (control). Counterstaining with Hoechst 33342 was performed for 3 min in all groups tested. A minimum number of 10 replicate stainings ((A) n = 13, (B) n = 13, (C) n = 12, (D) n = 10) was carried out. The evaluation was performed under a fluorescence microscope (Nikon Eclipse Ci) with the NIS-Elements imaging software (Nikon). The statistical analysis was assessed using R. All data were analysed for normal distribution using a Shapiro-Wilk test. Evaluation of the data was made through the application of t-tests. The total cell numbers observed were in the known range for expanded blastocysts: (A) 106.3 ± 15.3, (B) 109.5 ± 18.8, (C) 119.3 ± 15.0, and (D) 113.1 ± 21.8. The number of dead cells did not differ significantly from the control: (A) 13.3 ± 7.7, (B) 8.5 ± 5.0, (C) 11.8 ± 8.7, and (D) 9.3 ± 2.3. The study indicates that PI could be used as an adequate alternative for EthD. Furthermore, it is possible that the concentration or the incubation time could be reduced to generate similar results to EthD. Further replicates are currently being examined in order to corroborate these results.

We gratefully acknowledge our colleague M. Vopel.