The low efficiency of somatic cell nuclear transfer (SCNT) greatly limits its application. Compared with the fertilized embryo, cloned embryos display abnormal epigenetic modification and other inferior developmental properties. In this study, small RNAs were isolated, and miR-34c and miR-125b were quantified by real-time PCR; results showed that these micro-RNAs were highly expressed in sperm. The test sample was divided into three groups: one was the fertilized group, one was the SCNT control group (NT-C group), and the third group consisted of SCNT embryos injected with sperm-borne small RNA (NT-T group). The level of tri-methylation of lysine 9 on histone H3 (H3K9me3) at the 8-cell stage was determined by immunofluorescence staining, and the cleavage ratio, blastocyst ratio, apoptotic cell index of the blastocyst and total cell number of blastocysts in each group were analyzed. Results showed that the H3K9me3 level was significantly higher in the NT-C group than in the fertilized group and the NT-T group. The apoptosis index of blastocysts in the NT-C group was significantly higher than that in the fertilized group and the NT-T group. The total cell number of SCNT embryos was significantly lower than that of fertilized embryos, and injecting sperm-borne small RNAs could significantly increase the total cell number of SCNT blastocysts. Our study not only demonstrates that sperm-borne small RNAs have an important role in embryo development, but also provides a new strategy for improving the efficiency of SCNT in rabbit.

Hyaluronidase is widely used in animal and human assisted reproductive technologies (ARTs) to remove cumulus cells around oocytes. However, adverse effects of hyaluronidase treatment, such as increased rates of degeneration and parthenogenesis, have been found after treatment of human and mouse oocytes. Currently, the mechanism(s) of the detrimental effects are unclear. The present study was initiated to identify the mechanism of adverse responses to hyaluronidase treatment in bovine oocytes and early embryos. Cumulus cells were removed from cumulus–oocyte complexes (COCs) with or without hyaluronidase and the oocytes were subjected to intracytoplasmic sperm injection (ICSI) or in vitro fertilization (IVF). Significantly lower rates of blastocyst formation were obtained in the hyaluronidase treatment group after ICSI (22.4%) and IVF (21.2%) compared with the non-hyaluronidase control groups: 36.1% after ICSI and 30.4% after IVF. Next, we examined the effect of hyaluronidase on parthenogenetic development rates and on the cytoplasmic levels of free calcium ions (Ca2+), reactive oxygen species (ROS) and reduced glutathione (GSH). No differences in parthenogenesis rates were found between treated and untreated groups. Ca2+ levels in oocytes from the hyaluronidase treatment group indicated using mean fluorescence intensity were significantly higher (68.8 ± 5.3) compared with in the control group (45.0 ± 2.5). No differences were found in the levels of ROS or GSH between the treated and untreated groups. We conclude that hyaluronidase might trigger an increase in Ca2+ levels in oocytes, resulting in a decreased potential for normal embryonic development.

Embryos that are produced in vitro frequently present epigenetic modifications. However, maternal supplementation with folic acid (FA) may improve oocyte maturation and embryo development, preventing epigenetic errors in the offspring. We sought to evaluate the influence of FA supplementation during in vitro maturation of grade I (GI) and grade III (GIII) bovine oocytes on embryo production rate and the expression of IGF2 and KCNQ1OT1 genes. The oocytes were matured in vitro with different concentrations of FA (0, 10, 30 and 100 μM), followed by in vitro fertilization and embryo culture. On the seventh day (D7) of culture, embryo production was evaluated and gene expression was measured using real-time qPCR. Supplementation with 10 μM of FA did not affect embryo production for GI and GIII oocytes. Moderate supplementation (30 μM) seemed to be a positive influence, increasing embryo production for GIII (P = 0.012), while the highest dose (100 μM) reduced embryo production (P = 0.010) for GI, and IGF2 expression was not detected. In GIII, only embryos whose oocyte maturation was not supplemented with FA demonstrated detected IGF2 expression. The lowest concentration of FA (10 μM) reduced KCNQ1OT1 expression (P = 0.05) on embryos from GIII oocytes. Different FA concentrations induced different effects on bovine embryo production and gene expression that was related to oocyte quality. Despite the epigenetic effects of FA, supplementation seems to be a promising factor to improve bovine embryo production if used carefully, as concentration is an important factor, especially in oocytes with impaired quality.

The present study was designed to evaluate the effect of the combination of oviduct fluid flush (OFF) and oviduct epithelial cells (OEC) in modulating the incidence of polyspermy in pigs. Therefore, for in vitro fertilization (IVF), oocyte and sperm were co-cultured in Tris-buffered medium (TBM) either supplemented with 10% OFF (OFFD group), or in the presence of a bovine OEC monolayer (OEC group), or the oocytes were exposed to OFF for 30 min before IVF (OFFB group), or in the presence of an OEC monolayer (OFFB + OEC group). Regardless of sperm concentration used (0.5, 1.5, and 4.5 × 105 cells/ml), supplementation of IVF medium with 10% OFF led to an increased (P < 0.05) monospermy rate, without alteration (P > 0.05) of the penetration rate in comparison with the control and OEC groups. When the IVF medium was supplemented with heparin, an overall increase (P < 0.05) of the final output of the IVF system in terms of zygotes with two pronuclei (2PN) was observed in the OFFD group, compared with the control and OEC groups, at a sperm concentration of 4.5 × 105 cells/ml. At this concentration, OFFB improved the monospermy rate but decreased the penetration rate, resulting in low efficiency of monospermic zygotes production. Despite this, no major effect was observed in the developmental competence of the presumed zygotes up to the blastocyst stage. The combination of OFFB with OEC improved the penetration rate, while maintaining the high monospermic rate induced by OFFB. In conclusion, the combination of treatment of oocytes by diluted OFF 30 min before IVF, followed by IVF in the presence of OEC, improved monospermic zygote production without reducing the penetration rate, when the IVF medium was supplemented with heparin.

The worldwide production of in vitro-produced embryos in livestock species continues to grow. The current gold standard for selecting quality oocytes and embryos is morphologic assessment, yet this method is subjective and varies based on experience. There is a need for a non-invasive, objective method of selecting viable oocytes and embryos. The aim of this study was to determine if ooplasm area, diameter including zona pellucida (ZP), and ZP thickness of artificially activated oocytes and in vitro fertilized (IVF) zygotes are indicative of development success in vitro and correlated with embryo quality, as assessed by total blastomere number. Diameter affected the probability of development to the blastocyst stage in activated oocytes on day 7 (P < 0.01) and day 8 (P < 0.001), and had a tendency to affect IVF zygotes on day 8 (P = 0.08). Zona pellucida thickness affected the probability of development on day 7 (P < 0.01) and day 8 (P < 0.001) in activated oocytes, and day 8 for IVF zygotes (P < 0.05). An interaction between ZP thickness and diameter was observed on days 7 and 8 (P < 0.05) in IVF zygotes. Area did not significantly affect the probability of development, but was positively correlated with blastomere number on day 8 for IVF zygotes (P = 0.01, conditional R2 = 0.09). Physical parameters of bovine zygotes have the potential for use as a non-invasive, objective selection method. Upon further development, methods used in this study could be integrated into embryo production systems to improve IVF success.

Recurrent implantation failure (RIF) is considered to be one of the major limiting factors of assisted reproductive technology (ART) programme success. The current study focused on the investigation of matrix metalloproteinases (MMPs), tissue inhibitors of MMPs (TIMPs), cytokines and cell adhesion molecules in peripheral blood (PB) and follicular fluid (FF) obtained from 44 women aged between 25 and 39 years old and undergoing intracytoplasmic sperm injection (ICSI). These women were divided into two groups: 22 RIF women with embryo implantation failures after the transfer of at least four fresh or frozen–thawed good quality embryos in a minimum of three ICSI cycles, and 22 ICSI success women (controls) who achieved a clinical pregnancy at their first ICSI attempt. The PB and FF samples were obtained from each patient on the day of oocyte retrieval. MMP-1, -2, -3, -7, -9, TIMP-1, -2, vascular endothelial growth factor (VEGF), leukaemia inhibitory factor (LIF), vascular cell adhesion molecule 1 (VCAM1) and intercellular adhesion molecules 1 (ICAM1) were analyzed using enzyme-linked immunosorbent assay of PB and FF. Our results showed significant decreases in PB MMP-7 and PB VEGF in the RIF group compared with controls [281.11 (33–614) pg/ml vs 119.92 (27–441) pg/ml; P-value = 0.030] and [82.54 (25.94–210.20) pg/ml vs 30.93 (13.62–193.33) pg/ml; P-value = 0.022; respectively]. Receiver operating characteristic (ROC) curve analysis showed informative area under the curve values for PB MMP-7, as well as for PB VEGF, making them able to be proposed as biomarkers of the RIF. Therefore, circulating MMP-7 and VEGF seem to play an interesting role in embryo implantation in in vitro fertilization (IVF)/ICSI cycles and could be proposed as circulating biomarkers of the RIF. These results could be helpful for clinicians and patients to choose the best rescue strategy and treatment to minimize implantation failure in women undergoing IVF/ICSI procedures after the first attempt.

Triploidization plays an important role in aquaculture and surrogate technologies. In this study, we induced triploidy in the matrinxã fish (Brycon amazonicus) using a heat-shock technique. Embryos at 2 min post fertilization (mpf) were heat shocked at 38°C, 40°C, or 42°C for 2 min. Untreated, intact embryos were used as a control. Survival rates during early development were monitored and ploidy status was confirmed using flow cytometry and nuclear diameter analysis of erythrocytes. The hatching rate reduced with heat-shock treatment, and heat-shock treatments at 42°C resulted in no hatching events. Optimal results were obtained at 40°C with 95% of larvae exhibiting triploidy. Therefore, we report that heat-shock treatments of embryos (2 mpf) at 40°C for 2 min is an effective way to induce triploid individuals in B. amazonicus.

The objective of this study was to investigate the effect of oocyte and sperm cryopreservation on donated eggs submitted to intracytoplasmic sperm injection (ICSI) cycles. Medical charts of 122 oocyte recipients undergoing 152 oocyte recipient ICSI cycles, from 2017 to 2018, in a private university-affiliated in vitro fertilization (IVF) centre, were reviewed in this historical cohort study. Cycles were divided into four groups according to the gamete status: the FO/FS Group, recipients in which fresh oocytes were injected with fresh sperm (n = 19); the FO/CrS Group, recipients in which fresh oocytes were injected with cryopreserved sperm (n = 14); the CrO/FS Group, recipients in which cryopreserved oocytes were injected with fresh sperm (n = 85); and the CrO/CrS Group, recipients in which cryopreserved oocytes were injected with cryopreserved sperm (n = 34). Generalized mixed models fit by restricted maximum likelihood, followed by Bonferroni post hoc test for the comparison of means amongst groups were used to investigate the effect of cryopreservation on recipient ICSI outcomes. The results were expressed as mean differences with 95% confidence intervals and P-values. The main outcome measure was the implantation rate. Normal day 3 cleavage speed, blastocyst development and implantation rates were significantly lower in the CrO/CrS Group compared with the FO/FS Group. In conclusion, embryo developmental competence and implantation potential were reduced when vitrified oocytes were injected with frozen sperm in an egg-sharing donation programme.

Oocyte cryopreservation has become an important component of assisted reproductive technology with increasing implication in female fertility preservation and animal reproduction. However, the possible adverse effects of oocyte cryopreservation on epigenetic status of the resulting embryos is still an open question. This study evaluated the effects of MII-oocyte vitrification on gene transcripts linked to epigenetic reprogramming in association with the developmental competence and epigenetic status of the resulting embryos at 2-cell and blastocyst stages in dromedary camel. The cleavage rate of vitrified oocytes following intracytoplasmic sperm injection was significantly increased compared with the control (98.2 ± 2 vs. 72.7 ± 4.1%, respectively), possibly due to the higher susceptibility of vitrified oocytes to spontaneous activation. Nonetheless, the competence of cleaved embryos derived from vitrified oocytes for development to the blastocyst and hatched blastocyst was significantly reduced compared with the control (7.7 ± 1.2 and 11.1 ± 11.1 compared with 28.1 ± 2.6 and 52.4 ± 9.9%, respectively). The relative transcript abundances of epigenetic reprogramming genes DNMT1, DNMT3B, HDAC1, and SUV39H1 were all significantly reduced in vitrified oocytes relative to the control. Evaluation of the epigenetic marks showed significant reductions in the levels of DNA methylation (6.1 ± 0.3 vs. 9.9 ± 0.5, respectively) and H3K9 acetylation (7.8 ± 0.2 vs. 10.7 ± 0.3, respectively) in 2-cell embryos in the vitrification group relative to the control. Development to the blastocyst stage partially adjusted the effects that oocyte vitrification had on the epigenetic status of embryos (DNA methylation: 4.9 ± 0.4 vs. 6.2 ± 0.6; H3K9 acetylation: 5.8 ± 0.3 vs. 8 ± 0.9, respectively). To conclude, oocyte vitrification may interfere with the critical stages of epigenetic reprogramming during preimplantation embryo development.

This study examined the effects of zinc chloride (ZnCl2) and sodium selenite (Na2SeO3) supplementation in maturation medium on in vitro maturation (IVM) rate, oxidative biomarkers and gene expression in buffalo oocytes. Ovaries from a slaughterhouse were aspirated and good quality cumulus–oocyte complexes (COCs) with at least four layers of compact cumulus cells and evenly granulated dark ooplasm were selected. COCs were randomly allocated during IVM (22 h) to one of four treatment groups: (1) control maturation medium (basic medium), or basic medium supplemented with (2) ZnCl2 (1.5 µg/ml), (3) Na2SeO3 (5 µg/l), or (4) ZnCl2 + Na2SeO3 (1.5 µg/ml + 5 µg/l, respectively). Oocytes were denuded after 22 h of IVM in the first four replicates. Specimens were fixed and stained to evaluate the stage of nuclear maturation. The spent medium was collected for biochemical assays of total antioxidant capacity (TAC), malondialdehyde (MDA) and hydrogen peroxide concentrations. A second four replicates were used for COCs for RNA extraction. The expression levels of antioxidant (SOD1, GPX4, CAT and PRDX1), antiapoptotic (BCL2 and BCL-XL) and proapoptotic (BAX and BID) genes were measured. Supplementation with ZnCl2 and Na2SeO3 during IVM increased the ratio of oocytes reaching metaphase II at 22 h, increased TAC and decreased MDA and H2O2 concentrations in the maturation medium (P < 0.05). Moreover, beneficial effects were associated with complementary changes in expression patterns of antioxidative, antiapoptotic and proapoptotic genes, suggesting lower oxidative stress and apoptosis. Supplementation medium with zinc chloride and sodium selenite improves the maturation rate, reduces oxidative stress and increases expression levels of antioxidative and antiapoptotic genes.

Sperm morphometric and morphologic data have been shown to represent useful tools for monitoring fertility, improving assisted reproduction techniques and conservation of genetic material as well as detecting inbreeding of endangered primates. We provide here for the first time sperm morphologic and morphometric data from Cercopithecus neglectus, Cercopithecus cephus, Papio papio and critically endangered Cercopithecus roloway, as well as comparative data from other Cercopithecinae species, i.e. Allochrocebus lhoesti, Mandrillus sphinx and Papio anubis. Following collection from the epididymis, spermatozoa were measured for each species for the following parameters: head length, head width, head perimeter, head area, midpiece length and total flagellum length, and the head volume, ellipticity, elongation, roughness and regularity were then calculated. Our data are consistent with both the general morphology and the morphometric proportions of Cercopithecinae sperm. Some specificities were observed, with C. cephus displaying a narrow head (width = 2.76 ± 0.26 µM) and C. roloway displaying a short midpiece (6.65 ± 0.61 µM). This data set represents an important contribution, especially for Cercopithecus roloway, one of the most endangered monkeys in the world, and further data on additional specimens coupled to data on mating systems and reproductive ecology should allow a better understanding of the mechanisms underlying these morphological differences across primate species.

Medically assisted reproductive (MAR) treatments using donated oocytes are commonly applied in several countries to treat women who cannot conceive with their own gametes. Historically, in Italy, gamete donation has been prohibited but, in 2014, the law changed and gamete donation became allowed for couples undergoing MAR treatments. Consequently, in the last decade, there has been an increase in application of the oocyte donation programme. This study reports an egg-donation programme’s clinical efficacy, based on importing donated vitrified oocytes from cryo-banks located in a foreign country. For this, we conducted a retrospective analysis of data from a single reproductive unit located in Italy (Donna Salus Women’s Health and Fertility, Bozen). The study group consisted of 681 vitrified oocytes, which were warmed and culture to be replaced in 100 recipients. The survival rate after warming was 79.1% (n = 539/681), whereas the fertilization and blastulation rates were 90.2% (n = 486/539) and 47.9% (n = 233/486), respectively. Positive pregnancy test, clinical pregnancy rates, and live-birth rates per embryo transfer were 37.8%, 31.1% and 28.4%, respectively. The multiple pregnancy rate was 0.7%. This study is one of the first to report on the efficacy of a donor oocyte programme in Italy using imported vitrified oocytes. The above data may reassure women who are undertaking donation programmes using vitrified oocytes imported from commercial egg banks.