Cerium oxide nanoparticles (CeO₂ NPs) are able to store and release oxygen, conferring them scavenger activity against oxidative stress. However, their effects in reproductive systems are not yet well understood. The aim of the study was to investigate the effects of exposure of refrigerated ram semen to CeO₂ NPs for 96 h on the main structural and kinematic parameters of spermatozoa. The ejaculates of 5 Sarda rams were collected, pooled and diluted in a soybean lecithin extender. Samples were exposed to increasing doses of CeO₂ NPs (0, 44 and 220 μg/mL) and stored at 4 °C for 96 h. Analyses of kinematic parameters (computer assisted sperm analysis, CASA), integrity of membranes (PI/PSA staining), ROS production (H₂DCFDA staining) and DNA damage (sperm chromatin structure assay with acridine orange, SCSA) were performed every 24 h (0, 24, 48, 72 and 96 h of incubation). The experiment was carried out in 6 replicates. Data were analysed by repeated measures ANOVA with Bonferroni’s as post hoc test. When the assumption of normality was not met (ROS), non-parametric Kruskal-Wallis rank test was carried out. Exposure of ram spermatozoa to increasing doses of CeO₂ NPs had a beneficial effect on the main motility parameters from 48 h of incubation onward. Velocity of sperm cells was enhanced in the groups exposed to CeO₂ NPs compared to the control. Incubation with NPs had beneficial effects on the integrity of plasma membranes of spermatozoa, with higher percentage of damaged cells in the control group compared to the exposed ones. Production of ROS was not affected by exposure to NPs and its levels rose at 96 h of incubation. The integrity of DNA remained stable throughout the 96 h of storage regardless of co-incubation with NPs. We reported beneficial effects of CeO₂ NPs on kinematic and morphologic parameters of ram semen, such as motility and membrane integrity following 96 h of exposure. Furthermore, we also proved no genotoxic effects of CeO₂ NPs. These effects could not be related to an antioxidant activity of CeO₂ NPs, since ROS levels in exposed cells were similar to those of unexposed ones.

To date, large‐scale use of multiple ovulation and embryo transfer (MOET) programmes in ovine species is limited due to unpredictable results and high costs of hormonal stimulation and treatment. Therefore, even if considered reliable, they are not fully applicable in large‐scale systems. More recently, the new prospects offered by in vitro embryo production (IVEP) through collection of oocytes post‐mortem or by repeated ovum pick‐up from live females suggested an alternative to MOET programmes and may be more extensively used, moving from the exclusive research in the laboratory to field application. The possibility to perform oocytes recovery from juvenile lambs to obtain embryos (JIVET) offers the great advantage to significantly reduce the generation interval, speeding the rate of genetic improvement. Although in the past decades several studies implemented novel protocols to enhance embryo production in sheep, the conditions of every single stage of IVEP can significantly affect embryo yield and successful transfer into the recipients. Moreover, the recent progresses on embryo production and freezing technologies might allow wider propagation of valuable genes in small ruminants populations and may be used for constitution of flocks without risks of disease. In addition, they can give a substantial contribution in preserving endangered breeds. The new era of gene editing might offer innovative perspectives in sheep breeding, but the application of such novel techniques implies involvement of specialized operators and is limited by relatively high costs for embryo manipulation and molecular biology analysis.

The sub-cortical maternal complex (SCMC), located in the subcortex of mouse oocytes and preimplantation embryos, is composed of at least four proteins encoded by maternal effect genes: OOEP, NLRP5/MATER, TLE6 and KHDC3/FILIA. The SCMC assembles during oocyte growth and was seen to be essential for murine zygote progression beyond the first embryonic cell divisions; although roles in chromatin reprogramming and embryonic genome activation were hypothesized, the full range of functions of the complex in preimplantation development remains largely unknown. Here we report the expression of the SCMC genes in ovine oocytes and pre-implantation embryos, describing for the first time its expression in a large mammalian species. We report sheep-specific patterns of expression and a relationship with the oocyte developmental potential in terms of delayed degradation of maternal SCMC transcripts in pre-implantation embryos derived from developmentally incompetent oocytes. In addition, by determining OOEP full length cDNA by Rapid Amplification of cDNA Ends (RACE) we identified two different transcript variants (OOEP1 and OOEP2), both expressed in oocytes and early embryos, but with different somatic tissue distributions. In silico translation showed that 140 aminoacid peptide OOEP1 shares an identity with orthologous proteins ranging from 95% with the bovine to 45% with mouse. Conversely, OOEP2 contains a premature termination codon, thus representing an alternative noncoding transcript and supporting the existence of aberrant splicing during ovine oogenesis. These findings confirm the existence of the SCMC in sheep and its key role for the oocyte developmental potential, deepening our understanding on the molecular differences underlying cytoplasmic vs nuclear maturation of the oocytes. Describing differences and overlaps in transcriptome composition between model organisms advance our comprehension of the diversity/uniformity between mammalian species during early embryonic development and provide information on genes that play important regulatory roles in fertility in nonmurine models, including the human.

The present study investigated whether supplementation with different doses of cerium dioxide nanoparticles (CeO2 NPs) during in vitro maturation (IVM) of prepubertal ovine oocytes influenced their embryonic development in vitro. Cumulus–oocyte complexes derived from the ovaries of slaughtered prepubertal sheep underwent IVM with CeO2NPs (0, 44, 88 or 220 µg mL–1). Matured oocytes were fertilised in vitro and zygotes were cultured for 7 days. The results demonstrated that CeO2NPs were internalised in the cumulus cells and not in the oocyte. The treatment with CeO2NPs did not affect nuclear maturation or intracellular levels of reactive oxygen species of the oocytes. The percentage of oocytes with regular chromatin configuration and cytoskeleton structures when treated with 44 µg mL–1 CeO2NPs was similar to oocytes matured in the absence of CeO2NPs and significantly higher than those treated with 88 or 220 µg mL–1 CeO2NPs. The relative quantification of transcripts in the cumulus cells of oocytes matured with 44 µg mL–1 CeO2NPs showed a statistically lower mRNA abundance of BCL2-associated X protein (BAX), B-cell CLL/lymphoma 2 (BCL2) and superoxide dismutase 1 (SOD1) compared with the 0 µg mL–1 CeO2 NPs group. A concentration of 44 µg mL–1 CeO2NPs significantly increased the blastocyst yield and their total, inner cell mass and trophectoderm cell numbers, compared with the 0 and 220 µg mL–1 groups. A low concentration of CeO2NPs in the maturation medium enhanced in vitro embryo production of prepubertal ovine oocytes.