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The aim of this study was to assess the presence and distribution of apoptosis in porcine cumulus‐oocyte complexes (COCs) and its relations with COC morphology and developmental competence. The COCs were obtained from slaughterhouse ovaries, classified into A1 (top category), A2, B1, B2, C, and D based on their morphology. A1, A2, and B1 were matured and fertilized in vitro, and blastocyst rate was compared among them. Before and after in vitro maturation (IVM), annexin‐V staining, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays were performed to assess early and late apoptosis, respectively. There was a significant increase in both annexin‐V (+) oocytes and TUNEL (+) cumulus cells as morphology further deteriorated. There were no statistical differences regarding annexin‐V (+) oocytes within immature and post‐IVM COCs, but TUNEL (+) oocytes were only observed in post‐IVM COCs. Early and late apoptosis was detected in cumulus cells of all categories of immature and post‐IVM COCs. However, the difference was only significant for annexin‐V (+). There were no significant differences in embryo development. Therefore, apoptosis increases as the morphological features of the immature COCs decrease. In conclusion, the selection of COCs from Categories A1, A2, and B1 may be used as a selection criterion for in vitro development.

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Coculture with somatic cells is an alternative to improve suboptimal invitro culture conditions. In pigs, IVF is related to poor male pronuclear formation and high rates of polyspermy. The aim of this study was to assess the effect of a coculture system with porcine luteal cells (PLCs) on the IVM of porcine cumulus–oocyte complexes (COCs). Abattoir-derived ovaries were used to obtain PLCs and COCs. COCs were matured invitro in TCM-199 with or without the addition of human menopausal gonadotrophin (hMG; C+hMG and C-hMG respectively), in coculture with PLCs from passage 1 (PLC-1) and in PLC-1 conditioned medium (CM). In the coculture system, nuclear maturation rates were significantly higher than in the C-hMG and CM groups, but similar to rates in the C+hMG group. In cumulus cells, PLC-1 coculture decreased viability, early apoptosis and necrosis, and increased late apoptosis compared with C+hMG. PLC-1 coculture also decreased reactive oxygen species levels in cumulus cells. After IVF, monospermic penetration and IVF efficiency increased in the PLC-1 group compared with the C+hMG group. After invitro culture, higher blastocysts rates were observed in the PLC-1 group. This is the first report of a coculture system of COCs with PLCs. Our model could be an alternative for the conventional maturation medium plus gonadotrophins because of its lower rates of polyspermic penetration and higher blastocysts rates, key issues in porcine invitro embryo production.

Abattoir ovaries, which are the main source of oocytes for reproductive biotechnologies, arrive at the laboratory under ischaemic conditions. Reoxygenation generates reactive oxygen species (ROS) in ischaemic tissues, which could affect oocyte quality. The aim of this study was to evaluate the effect of supplementation of media with dimethylthiourea (DMTU) during the collection and washing of cumulus–oocyte complexes (COC) on ROS levels, COC apoptosis and oocyte nuclear and cytoplasmic maturation. Thus, the collection (TCM-199) and washing (TCM-199 with 10% porcine follicular fluid, sodium pyruvate and antibiotics) media were supplemented with 1 and 10mM DMTU. In the control group, the media were not supplemented with DMTU. Intracellular ROS levels decreased significantly in the DMTU-treated groups (P<0.05). Although no effects on rate of nuclear maturation were observed, DMTU significantly increased sperm penetration rates without increasing polyspermy (P<0.05). The addition of 10mM DMTU to the collection and washing media enhanced IVF efficiency. DMTU did not modify the early or late apoptosis of oocytes. Both concentrations of DMTU significantly increased viability and decreased the apoptosis of cumulus cells (P<0.05). These results suggest that the addition of 1 or 10mM of DMTU to the media during the collection and washing of porcine COCs is useful for decreasing cumulus apoptosis mediated by ROS and for optimising the IVF of porcine oocytes.

The aim of this study was to evaluate the effect of the antioxidant dimethylthiourea (DMTU) as a supplement during the collection and washing of cumulus–oocyte complexes (COC) on the apoptosis in cumulus cells (CC) and the competition for oocyte development in vitro. The COC were obtained by follicular aspiration. The aspiration and washing medium was TCM-199 with 10% porcine follicular fluid, 0.3 mM sodium pyruvate, and penicillin/streptomycin. Treated groups were supplemented with 2 or 20 mM DMTU. After 44 h of in vitro maturation (IVM), the percentage of nuclear maturation was determined by staining of the denuded oocytes with Hoechst 33442 (n = 357). After 18 h of in vitro fertilization (IVF) with 15°C refrigerated spermatozoa from boars of proven fertility, the suspected zygotes were stained with Hoechst 33342 to evaluate the percentages of sperm penetration (SP), monospermic penetration (MP), male pronucleus formation (MPN), and IVF efficiency (monospermic oocytes/total oocytes) (n = 260). In post-IVM CC, flow cytometry was performed after staining with annexin V-propidium iodide to evaluate apoptosis and viability (n = 90,000). Data were analysed with Flowing V 2.5.1 (http://flowingsoftware.btk.fi/). A comparison of proportion among categories was used (Fisher’s exact test), and significant differences were determined at P < 0.05. No effects were observed in the percentages of nuclear maturation. Treatments with DMTU significantly increased the SP (Control: 35%; 2 mM: 72.9%; 20 mM: 67.8%) without modifying the MP. Although in both treatments, the amount of MPN was always greater than in the control, the percentages of MPN formation did not show significant differences. The 20 mM treatment significantly increased the efficiency of IVF (control: 22.2%; 2 mM: 31.9%; 20 mM: 45.7%). In the CC of the treated groups, a significant increase in viability (control: 7.4%; 2 mM: 12.2%; 20 mM: 14.7%) and a decrease in early apoptosis (control: 60.4%; 2 mM: 55.5%; 20 mM: 57.1%) were observed. Late apoptosis decreased in the 20 mM treatment (27.3%) compared with the other experimental groups (control: 31.3%; 2 mM: 31.2%). The addition of DMTU during COC collection and washing reduce the ROS-mediated apoptosis in CC and optimize the efficiency of IVF. Besides, CC quality reflects the effect of DMTU on oocytes, so viability and apoptosis analysis on CC constitutes an interesting noninvasive method to assess oocyte competence.

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