The present study was conducted to study the effect of cumulus-oocyte complex (COC) morphology on subsequent in vitro embryo development and to assess the proteome of their corresponding cumulus cells (CC). Cow ovaries were obtained at an abattoir and COC aspirated from 3–8 mm follicles. The COC were defined as type I (TI): homogeneous ooplasma and ≥4 layers of compact CC; type II (TII): granular ooplasma and ≥4 layers of slight expanded CC. Fifty COC had ~500,000 CC. Cumulus cells were frozen in ammonium bicarbonate and immediately lyophilized for proteome analysis. Other selected COC were matured in vitro in TCM-199-supplemented media for 24 h. After maturation, CC were collected (T24) and processed as described above. The remaining COC were fertilized with sperm from a fertile bull and zygotes, cultured in vitro until Day 7. Ten blastocysts per group were stained (Hoechst 33342) and blastomeres, counted for assessment of embryo quality. The CC proteins were obtained from the following groups: immature type I (TIT0) and type II (TIIT0), and in vitro matured type I (TIT24) and type II (TIIT24). For protein extraction, we used sonication (30 min, 4°C), freezing and unfreezing in liquid nitrogen, and maceration. The CC proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and identified by ESI-MS/MS. Differences in cleavage, embryo rates, and blastomere numbers were analysed by t-test. Protein expression difference was set at 2.5-fold (P < 0.05). In silico protein interactions were investigated using STRING v. 10.0. There were no differences in cleavage (88 ± 4 v. 89 ± 8%) and embryo rates (36 ± 7 v. 33 ± 8%) between COC of TI (n = 220) and TII (n = 161), respectively. Blastomeres were also similar in TI (101) and TII (104) groups. Major proteins expressed in all CC were α-enolase, β-actin, oestradiol 17-β-dehydrogenase 1, glutathione S-transferase, glyceraldehyde 3-phosphate dehydrogenase, heat shock protein β-1, histone H2B type 1-N, histone H4, mitochondrial malate dehydrogenase 2, protein disulfide isomarase A6, triosephosphate isomerase, tubulin α-1C chain, and vimentin. Glyceraldehyde 3-phosphate dehydrogenase appeared to be more expressed in TIT0, whereas tubulin α-1C chain and vimentin had greater expression in TIIT24. As evidenced by in silico analysis, most CC proteins interact among themselves, participating in complex networks involving intracellular signalling and other events. In conclusion, there are no difference in embryo development when using compact and early-expanded COC, indicating that both types can be selected for IVP. Protein profile of cumulus cell may serve as a marker for in vitro embryo competence.

The morphological selection of cumulus-oocyte complexes (COCs) is an important step for in vitro embryo production. It has been suggested that COC showing signs of atresia have the ability to generate embryos. The objective of this work was to evaluate the effect of COC morphology from Bos indicus animals with signs of early atresia versus no signs of atresia on in vitro embryo production. COC were classified in: Type I (TI): homogeneous ooplasm with ≥ 4 layers of compact cumulus cells (CC) and Type II (TII): granular ooplasm and ≥ 4 layers of CC slightly expanded. The COC were matured in vitro for 24 hours in TCM199 medium and subsequently fertilized in vitro for 18 h. The suspected zygotes were cultured in vitro for seven days in modified SOFaa medium. Embryonic quality was determined by blastomeric count following staining with Hoechst 33342. Student test was used to determine statistical differences for cleavage, blastocyst rate and blastomeric counts between types of COC. The cleavage rate for TI (n = 220) and TII (n = 161) was 88 ± 4 % and 89 ± 8 % respectively (p> 0.05); embryo development rate was 36 ± 7 % and 33 ± 8 % (p>0.05) respectively. The blastomeric count for both groups was 101 and 104 cells for TI and TII respectively (n = 10), (p>0.05). These results demonstrate that there is no difference in the quantity and quality of embryos produced in vitro using COC type I or type II, suggesting that both types could be used for bovine in vitro embryo production in Bos indicus cows. Keywords: atresia, competence, cumulus-oocyte complex.Resumen La selección morfológica de los complejos cúmulo-oocito (COC) es crucial para la producción de embriones in vitro. Se ha sugerido que COC que muestran signos de atresia poseen capacidad de generar embriones. El objetivo de este trabajo fue evaluar el efecto de la morfología de los COC provenientes de animales Bos indicus con signos de atresia temprana y sin signos de atresia sobre la producción de embriones in vitro. Se clasificaron COC obtenidos de ovarios de faenado en dos grupos: Tipo I (TI): ooplasma homogéneo con ≥ 4 capas de células del cumulo (CC) compactas y Tipo II (TII): ooplasma