152 POST-HATCHING EMBRYO DEVELOPMENT IN VITRO UNTIL DAY 15 IN AGAR GEL DILUTED IN PBS OR IN MILLIQ WATER

Abstract

Post-hatching (PH) in vitro embryo culture is a procedure that allows the establishment of more accurate tools for evaluating embryo developmental potential. An important step in the PH system is the tunnels preparation, since they will hold the developing embryo in advanced stages during culture. However, if the diluent used to dissolve agarose for tunnels preparation can influence the diffusion of nutrients from culture medium to the gel compromising embryo development, is not known. The aim of this study was to compare developmental kinetics and gene expression of Day 15 embryos cultured in the PHD system using PBS and MilliQ water to dilute the agar gel used for tunnels construction. Bovine oocytes obtained from slaughterhouse ovaries were matured, fertilized, and cultured in vitro for 8 days in SOFaaci with 5% fetal bovine serum (FBS) at 39°C in 5% CO₂ in air. On Day 9, PHD (Brandão et al. 2004 Biol. Reprod. 71, 2048-2055) medium was added in culture droplets and on Day 11 embryos were measured. Only morphologically normal with ≥0.5 mm of diameter Day 11 blastocysts were placed individually in the tunnels. The tunnels were produced using agar gel 2.4% diluted either in PBS or MilliQ water, supplemented with 10% FBS. Morphology and length of embryos were evaluated on Day 11, Day 12.5, Day 14, and Day 15. Elongated embryos in Day 15 were used for extraction of total RNA using Trizol Reagent to verify gene expression by qPCR. A total of 4 pools containing 3 Day 15 embryos in each pool were used to evaluate the expression of genes involved in glucose metabolism [glucose-6-phosphate dehydrogenase (G6PDH), solute carrier family 2 member 1 (SLC2A1), solute carrier family 2 member 3 (SLC2A3), phosphoglycerate kinase 1 (PGK1)], placenta development [placenta-specific 8 (PLAC8), keratin proteins 8 (KRT8)], heat stress [heat shock transcription factors 1(HSF1)], and maternal recognition of pregnancy [interferon tau (IFNT)]. Embryo growth and gene expression data were examined by the t-test for parametric or Mann-Whitney for non-parametric (P < 0.05). From a total of 2,933 oocytes used, 1,216 (41%) reached the blastocyst stage on Day 8 and of those 72% hatched on Day 9 (n = 873). On Day 11, 39.5% (345/873) of the hatched blastocysts had diameter ≥0.5 mm and 286 were loaded into the MilliQ water (154) and PBS tunnels (132). No difference was observed in the proportion of elongated embryos between PBS (54%, n = 71) and MilliQ water treatment (42%, n = 65) by chi-square analysis. Final size at Day 15 as well as increase in length of the embryos between Days 11 and 15 were similar in PBS (2.37 ± 0.13 mm; 1.68 ± 0.13 mm) and MilliQ water tunnels (3.03 ± 0.23 mm; 2.33 ± 0.22 mm). Regarding gene expression analysis, only SLC2A1 had a tendency (P = 0.08) to be higher expressed in embryos cultured in PBS tunnels. The results suggested that water can be use to construct agar gel tunnels for the PHD system without interfering in embryo developmental kinetics until Day 15 of culture.

Embrapa, CNPq, CAPES.