M. M. Franco

Post‐hatching development of in vitro bovine embryos from day 7 to 14 in vivo versus in vitro

This study evaluates the post‐hatching development of in vitro‐produced (IVP) embryos until Day 14. On Day 7, IVP embryos were either transferred to recipient uteruses or placed in a post‐hatching development (PHD) system. As a control group, in vivo‐produced (IVV), Day‐7 embryos were also transferred to recipient uteruses. All groups were collected on Day 14 and were morphologically evaluated. Day‐7 and Day‐14 IVV and IVP embryos were used for quantification of eight genes (PLAC8, CD9, SLC2A1, SLC2A3, KRT8, SOD2, HSP1A1, and IFNT2) by reverse transcriptase qPCR. Day‐14 embryos from the PHD system were smaller (2.92 ± 0.45 mm) and had a lower embryonic disk diameter (0.14 ± 0.00 mm) than those produced by IVV (24.18 ± 3.71; 0.29 ± 0.03 mm, respectively) or IVP (19.06 ± 2.43; 0.28 ± 0.01 mm) culture and transferred to the uterus (P > 0.05). Day‐7 IVP embryos had a higher expression of the HSP1A1, SCL2A1, and SCL2A3 genes than IVV embryos. When these embryos were cultured in the uterus, no differences in gene expression were observed on Day 14. Conversely, Day‐14 IVP embryos cultured in the PHD system showed a higher expression of PLAC8, SOD2, and SLC2A3 genes. It is concluded that Day‐7 IVP embryos are different from IVV embryos in regards to gene expression, although exposure to the uterine environment during the elongation period allowed the IVP embryos to overcome this difference. In contrast, IVP embryos cultured in the PHD system were morphologically and molecularly different, being of poorer quality than those cultured in the uterus. Mol. Reprod. Dev. 80: 936–947, 2013.

Vitrification of bovine oocytes at different meiotic stages using the Cryotop method: Assessment of morphological, molecular and functional patterns ☆

This study aimed to investigate the functional, morphological and molecular patterns of bovine oocytes vitrified at different times during in vitro maturation (IVM). Four groups of oocytes were used: non-vitrified control oocytes (CG), oocytes vitrified at 0 h (V0), oocytes vitrified after 8 h of IVM (V8) and oocytes vitrified after 22 h of IVM (V22). After vitrification, the oocytes were warmed and then returned to the incubator to complete a total of 24h of IVM. To evaluate the effect of vitrification, the nuclear maturation and fertilization rates were assessed by lacmoid staining and ultrastructural electron microscopy. The cleavage and blastocyst rates were evaluated at D2, D7 and D8. The expression levels of CASP3, TP53, HDAC2, SUV39H1 and DNMT1 were investigated by RT-qPCR. The nuclear maturation, oocyte fertilization, cleavage and blastocyst rates were higher (P < 0.05) in the CG group (80%; 81.3%; 88.5%; and 35.8%) than in the V0 (44%; 44.6%; 22.7%; and 2.6%), V8 (50%; 63%; 21.5%; and 2.2%) and V22 (55.5%; 66.9%; 24.1%; and 4.6%) groups. Ultrastructural analysis revealed significant damage within the cytoplasm of all vitrified groups, but more severe degeneration was observed in the V22 group. The gene expression profiles were not affected by vitrification (P > 0.05). In conclusion, cytoplasm degeneration seems to be the most severe form of damage caused by vitrification. The use of the Cryotop method for vitrification severely reduces bovine oocyte viability regardless of whether it is performed at GV, GVBD or MII stage.

Molecular markers for oocyte competence in bovine cumulus cells

This study aimed to quantify the expression of candidate genes in cumulus cells (CCs) from cumulus-oocyte complexes (COCs) with high and low potential for in vitro development up to the blastocyst stage. First, the effects of individual culture and biopsy on embryo development were evaluated. Individuals cultured using the well of the well system were compared with individuals cultured in 20 μL droplets (microdroplets) and those cultured in groups (control). Blastocyst rates were lower for the individual culture systems (P < 0.05; well of the well = 17.9%, n = 95; microdrop = 26.3%, n = 95) than for the control group (45.0%, n = 209). Second, the effects of biopsy on embryo production were compared between the control and microdroplet cultures, and no effects (P > 0.05) were observed for either group. Finally, the expression profiles of glypican 4 (GPC4), IGF4-binding protein, follicle-stimulating hormonereceptor, growth hormone receptor, epidermal growth factor receptor, fibroblast growth factor 11, solute carrier family 2 member 1, solute carrier family 2 member 3,sprouty homolog 1, versican, and keratin protein 8 in CCs obtained by biopsy were quantified by real-time polymerase chain reaction. Cumulus cells were categorized on the basis of the fates of the COCs: expanded blastocyst, cleaved and arrested, and uncleaved. The GPC4 gene was overexpressed (P = 0.007) in CCs from oocytes that formed embryos compared with those that produced cleaved and arrested embryos. We concluded that individual culture reduced blastocyst production; however, biopsy did not affect embryo development. The profile of GPC4 expression can be used as a marker to distinguish COCs with potential for embryo development from those with limited developmental potential.

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

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 (Brandao 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.