92 Using physical parameters of bovine zygotes to predict in vitro development success

Abstract

The objective of this study was to determine if physical parameters of bovine zygotes are correlated with in vitro development success. We examined the relationship between a zygote's outer diameter (OD), cell area, and zona pellucida (ZP) thickness on blastocyst development and blastomere number. Bovine cumulus-oocyte complexes, from abattoir-derived ovaries, were matured in tissue culture medium 199-based maturation medium for 21-23 h. In vitro fertilisation was performed with frozen thawed semen containing a pool of 4 Holstein bulls. After incubation with sperm for 16-18 h, presumptive zygotes (n = 875) were denuded and placed in 5 µL droplets of synthetic oviductal fluid-bovine embryo 1 medium (SOF-BE1) under oil to be individually photographed using an inverted scope with a digital camera. Zygotes were then group-cultured in 50 µL of synthetic oviductal fluid-BE1 droplets in a polyester micromesh to identify individuals. Development to the blastocyst stage was recorded on Day 7 and Day 8 of culture, and Day 8 embryos were fixed and stained with Hoechst 33342 (n = 87). ImageJ was used to measure the OD, area, and ZP from images. Data were analysed with a logistic regression using the lme4 package in R with Day 7 or Day 8 blastocyst development as the response; OD, area, and ZP as predictors; and study replicate (n = 11) and culture droplet as random effects. Residual estimates of area and ZP calculated from OD were used to account for collinearity. Average OD was 151.2 µm, ranging 130.4-171.6 µm. Average ZP was 11.8 µm, ranging 7.2-17.5 µm. Area averaged 9856.7 µm², ranging from 6421.9 to 13 814.8 µm². There was a tendency for an effect of OD on probability of development on Day 8 (P = 0.08) but not Day 7. There was an effect of ZP on the probability of development on Day 8 (P = 0.04) but not Day 7. Blastocyst rates on Day 8 for zygotes with ZP <11.8 µm were 24.3%, whereas zygotes with ZP >11.8 µm were 21.7%. There was an interaction (P < 0.05) between OD and ZP thickness on Day 7 and Day 8. Estimated probability of development of zygotes with ZP <10.7 µm and OD <146.9 µm averaged 28.5%, whereas those with ZP >12.7 µm and OD >155.8 µm averaged 43.3%. The observed blastocyst rates for these two groups were 26.2% and 25%, respectively. Zygotes with OD between 146.9-155.8 µm and ZP between 10.7-12.7 µm had an estimated development probability of 22.2%. The obtained Day 8 blastocyst rate for this group was 22.5%. Area did not affect probability of development, but was positively correlated with total blastomere number at Day 8 (P = 0.01; marginal R² = 0.09). The observed interaction between ZP thickness and OD may be indicative of an optimum ooplasm area before maturation or fertilisation that may enhance development. Area assessment after fertilisation is not correlated with development success. In addition, these data in comparison with our previous data generated in parthenogenetic embryos indicate that artificial activation of oocytes is not an ideal model in place of IVF for studying development. We continue to demonstrate that physical parameters of zygotes may have potential as a noninvasive, objective selection tool of embryos.