Polyspermy Rates Research Articles

Single-cell multi-omics analysis of in vitro post-ovulatory aged oocytes revealed aging-dependent protein degradation

Once ovulated, the oocyte has to be fertilized in a short time window, or it will undergo post-ovulation aging (POA), whose underlying mechanisms are still not elucidated. Here, we optimized single-cell proteomics methods and performed single-cell transcriptomic, proteomic and phosphoproteomic analysis of fresh, POA, and melatonin-treated POA oocytes. POA oocytes showed down-regulation of most differentially expressed proteins, with little correlation with mRNA expression, and the protein changes can be rescued by melatonin treatment. MG132 treatment rescued the decreased fertilization and polyspermy rates, and up-regulated fragmentation and parthenogenesis rates of POA oocytes. MG132-treated oocytes displayed health status at proteome, phosphoproteome and fertilization ability similar to fresh oocytes, suggesting that protein stabilization might be the underlying mechanism for melatonin to rescue POA. The important roles of proteasome-mediated protein degradation during oocyte POA revealed by single-cell multi-omics analyses offer new perspectives for increasing oocyte quality during POA, and improving assisted reproduction technologies.

How to beat the bull: Lycopene as a tool to improve in vitro fertilization efficiency in bulls with high polyspermy

High levels of reactive oxygen species (ROS) derived from in vitro conditions compromise oocyte quality and subsequent polyspermy prevention by the zona and membrane block. Antioxidant supplementation, like lycopene, during in vitro maturation (IVM) mitigates ROS effects, yet, its efficacy in blocking polyspermy remains uncertain. This study aims to evaluate the effect of lycopene supplementation during IVM on oocyte maturation, fertilization, and developmental parameters. To this end, bovine oocytes were supplemented with 0.2 µM lycopene and fertilized with semen from three bulls. The three bulls showed different fertilization potential in vitro, with bull 1 showing the highest penetration and polyspermy rates and the lowest in vitro fertilization (IVF) efficiency. Interestingly, in bull 1, the treatment with lycopene improved IVF efficiency (p = 0.043) and reduced the polyspermy rate (p = 0.028). However, none of these effects were observed in bulls 2 and 3. Bulls with higher penetration rates exhibited better blastocyst rates although those rates did not seem to be associated with polyspermy or IVF efficiency. Oocyte mitochondrial distribution and activity and cortical granule migration and distribution were not influenced by lycopene. In conclusion, we demonstrated that lycopene addition during oocyte maturation had a positive impact on IVF efficiency by reducing polyspermy rates in a bull-dependent manner. The reduction in polyspermy rates was not caused by changes in cortical granule migration or oocyte mitochondrial distribution. Lycopene must therefore induce other changes in the oocyte that lower the in vitro penetration rates of specific bulls prone to polyspermy.

The mammalian sperm factor phospholipase C zeta is critical for early embryo division and pregnancy in humans and mice.

The mammalian sperm factor phospholipase C zeta is critical for early embryo division and pregnancy in humans and mice.

Morphokinetic Profiling Suggests That Rapid First Cleavage Division Accurately Predicts the Chances of Blastulation in Pig In Vitro Produced Embryos.

The study of pig preimplantation embryo development has several potential uses: from agriculture to the production of medically relevant genetically modified organisms and from rare breed conservation to acting as a physiologically relevant model for progressing human and other (e.g., endangered) species' in vitro fertilisation technology. Despite this, barriers to the widespread adoption of pig embryo in vitro production include lipid-laden cells that are hard to visualise, slow adoption of contemporary technologies such as the use of time-lapse incubators or artificial intelligence, poor blastulation and high polyspermy rates. Here, we employ a commercially available time-lapse incubator to provide a comprehensive overview of the morphokinetics of pig preimplantation development for the first time. We tested the hypotheses that (a) there are differences in developmental timings between blastulating and non-blastulating embryos and (b) embryo developmental morphokinetic features can be used to predict the likelihood of blastulation. The abattoir-derived oocytes fertilised by commercial extended semen produced presumptive zygotes were split into two groups: cavitating/blastulating 144 h post gamete co-incubation and those that were not. The blastulating group reached the 2-cell and morula stages significantly earlier, and the time taken to reach the 2-cell stage was identified to be a predictive marker for blastocyst formation. Reverse cleavage was also associated with poor blastulation. These data demonstrate the potential of morphokinetic analysis in automating and upscaling pig in vitro production through effective embryo selection.

Fertilization, pregnancy, and neonatal outcomes after IVF, rescue ICSI, and ICSI in unexplained infertility: A retrospective study

AbstractConventional intracytoplasmic sperm injection (ICSI) is not recommended for nonmale infertile patients to avoid fertilization failure (FF) in view of controversies regarding safety issues. Among the strategies that may help to promote the use of in vitro fertilization (IVF) for women with a high risk of FF, rescue ICSI (R‐ICSI) has attracted attention. This study aimed to evaluate the efficacy of short‐term insemination combined with early R‐ICSI in the treatment of unexplained infertility. A total of 952 controlled ovarian stimulation (COS) cycles for unexplained infertility were divided into short‐term IVF (short‐term insemination without R‐ICSI, n = 500), R‐ICSI (short‐term insemination with R‐ICSI, n = 141), and ICSI (conventional ICSI, n = 311) groups. Patients underwent consecutive transfer cycles until live birth, or until all embryos from the first COS cycle were used. Laboratory data and clinical outcomes from short‐term IVF, R‐ICSI, and ICSI groups were compared. Short‐term IVF and ICSI groups were superior to R‐ICSI group in polyspermy rate, available embryo rate, and top‐quality embryos rate. Short‐term IVF, R‐ICSI, and ICSI groups underwent 705 (500 fresh and 205 frozen‐thawed cycles), 190 (141 fresh and 49 frozen‐thawed cycles), and 445 (311 fresh and 134 frozen‐thawed cycles) transfer cycles, resulting in 294, 76, and 190 live birth cycles, respectively. Comparison of pregnancy outcomes among these three groups demonstrated similar clinical pregnancy rates and live birth rates in fresh and frozen‐thawed embryo transfer cycles. There was no significant difference in the cumulative live birth rate among these three groups. R‐ICSI group showed similar neonatal outcomes compared with short‐term IVF and ICSI groups, including the rates of low birth weight, fetal macrosomia, small for gestational age and large for gestational age. Short‐term insemination combined with early R‐ICSI achieved satisfactory pregnancy and neonatal outcomes, albeit with a high polyspermy rate, which was an effective alternative to avoid excessive use of ICSI in unexplained infertility.

Early cumulus cell removal increases cumulative live birth rate while having no negative effect on the malformation rate in in vitro fertilization: a propensity score-matched cohort study.

The aim of this study was to investigate the efficacy and safety of early cumulus cell removal (ECCR) during human in vitro fertilization (IVF). A retrospective analysis was performed between January 2011 and December 2019. The study enrolled 1131 couples who underwent IVF treatment with ECCR. After propensity score matching at a 1:1 ratio, 1131 couples who underwent overnight coincubation of gametes were selected. The main outcome measure was the cumulative live birth rate. Secondary outcome measures included the cumulative pregnancy rate, polyspermy rate, available embryo rate, miscarriage rate, malformation rate, time to live birth, and oocyte-to-baby rate. There were no significant differences found between the two groups in the polyspermy rate, available embryo rate, miscarriage rate, time to live birth, oocyte-to-baby rate, and neonatal congenital anomalies rate. The results of the study showed that ECCR was associated with a significantly higher cumulative live birth rate and cumulative pregnancy rate, along with a significantly lower fertilization rate. ECCR tended to confer increased cumulative live birth rate and had no negative effect on the neonatal malformation rate.

Correlation between the number of oocytes and the increase of polyspermy rate in IVF cycles

Aims: To investigate the association between the number of oocytes and the polyspermy rate following in vitro fertilization (IVF) treatment. Materials and methods: 376 IVF cycles with gonadotropin-releasing hormone (GnRH) antagonist protocol in the reproductive center of our hospital were retrospectively included in the analysis, which were divided into five groups according to the number of oocytes retrieved. Group A (78 cases):1–5 oocytes, group B (118 cases): 6–10 oocytes, group C (94 cases): 11–15 oocytes, group D (55 cases): 16–20 oocytes, group E (31 cases): ≥21 oocytes. According to polyspermy rate, 376 IVF cycles were then divided into two groups. Normal level polyspermy group (170 cases): polyspermy rate<6%, and high level polyspermy group (206 cases): polyspermy rate ≥ 6%. The variables with p < .10 in univariate analysis were incorporated into the multiple logistic regression model to control the confounding, and the effect of the number of oocytes on the increase of polyspermy rate was analyzed. Results: Multiple logistic regression analysis showed that after adjustment for confounding factor, the increase risk of polyspermy rate in group B, C, D and E was 1.763, 3.804, 2.021 and 3.208 times of that in group A respectively (OR = 1.763, p = .085; OR = 3.804, p = .001; OR = 2.021, p = .158; OR = 3.208, p = .068, respectively). Conclusion: This result demonstrated that when the oocyte number is 15 or less, the more the oocyte number, the greater the increase risk of polyspermy rate. While, there appears to be little increase risk of polyspermy rate when the oocyte number is more than 15.

Oviduct epithelial cell‑derived extracellular vesicles promote the developmental competence of IVF porcine embryos.

Assisted reproductive technology has increased the efficiency of animal reproduction. However, polyspermy is a significant limitation of porcine invitro fertilization (IVF). Therefore, reducing the polyspermy rate and improving monospermic embryos is crucial. Recent studies have reported that oviductal fluid, along with its contents of extracellular vesicles (EVs), enhanced the fertilization process and supported embryo development. Consequently, the present study investigated the effects of porcine oviduct epithelial cells (OEC‑EVs) on sperm‑oocyte interactions during porcine IVF and evaluated invitro embryo developmental competence outcomes. During IVF embryo development, the cleavage rate was significantly higher in the group treated with 50ng/ml OEC‑EVs compared with the control group (67.6±2.5 vs. 57.3±1.9; P<0.05). Furthermore, the OEC‑EV group had significantly more embryos (16.4±1.2 vs. 10.2±0.8; P<0.05), and the polyspermy rate significantly decreased (32.9±2.5 vs. 43.8±3.1; P<0.05) compared with that of the control group. Additionally, the fluorescence intensities of cortical granules (3.56±0.47 vs. 2.15±0.24; P<0.05) and active mitochondria (8.14±0.34 vs. 5.96±0.38; P<0.05) were significantly higher in the OEC‑EV group compared with those in the control group. In conclusion, OEC‑EV adsorption and penetration crosstalk between sperm and oocytes was observed. OEC‑EV treatment was demonstrated to significantly improve the concentration and distribution of cortical granules in oocytes. Furthermore, OEC‑EVs also increased oocyte mitochondrial activity, reduced polyspermy and increased the IVF success rate.

Early Cumulus Cell Removal Increases Cumulative Live Birth Rate while Having No Negative Effect on the Malformation Rate in In vitro Fertilization: A Propensity Score-Matched Cohort Study

Objective: The aim of this study was to investigate the efficacy and safety of early cumulus cell removal (ECCR) during human in vitro fertilization (IVF). Methods: A retrospective analysis was performed between January 2011 and December 2019. The study enrolled 1,131 couples who underwent IVF treatment with ECCR. After propensity score matching at a 1:1 ratio, 1,131 couples who underwent overnight coincubation of gametes were selected. The main outcome measure was the cumulative live birth rate. Secondary outcome measures included the cumulative pregnancy rate, polyspermy rate, available embryo rate, miscarriage rate, malformation rate, time to live birth, and oocyte-to-baby rate. Results: There were no significant differences found between the two groups in the polyspermy rate, available embryo rate, miscarriage rate, time to live birth, oocyte-to-baby rate, and neonatal congenital anomalies rate. The results of the study showed that ECCR was associated with a significantly higher cumulative live birth rate and cumulative pregnancy rate, along with a significantly lower fertilization rate. Conclusion: ECCR tended to confer increased cumulative live birth rate and had no negative effect on the neonatal malformation rate.

Brief and long co-incubation of sperm and oocytes for in vitro fertilization: a meta-analysis of randomized controlled trials

BackgroundThere is still no consensus on the optimal time of oocyte–sperm co-incubation during in vitro fertilization and embryo transfer (IVF-ET). The aim of this meta-analysis was to compare the effects of brief (1-6 h) and long (16-24 h) gametes co-incubation time on IVF outcomes.MethodsThe study protocol was registered online through PROSPERO (CRD42022337503) and PRISMA guidelines were followed in the present study. The following databases were searched from inception to May 2022 for randomized controlled trials (RCTs): PubMed, Embase, Cochrane library, Web of Science, using search terms related to IVF, gametes, time of co-incubation and reproductive outcome measure. Studies comparing outcomes of brief co-incubation to that of long co-incubation during IVF, and reporting primary outcome (live birth rate), secondary outcomes (clinical pregnancy rate; ongoing pregnancy rate; miscarriage rate; normal fertilization rate; polyspermy rate; top-quality embryo rate; implantation rate) were searched. A total of 11 studies were included in the meta-analysis. Combined odds ratio (OR) and 95% confidence interval (CI) were calculated for the data. Statistical heterogeneity analysis between studies was assessed by Cochran Q and I2 statistic with a significant threshold of P < 0.05. Methodologic quality assessment of RCTs was made for potential risk of bias with Cochrane Risk of Bias Tool.ResultsCompared to long-term co-incubation, brief co-incubation had an advantage in increasing implantation rate (OR: 1.97, 95% CI: 1.52–2.57), ongoing pregnancy rate (OR: 2.18, 95% CI: 1.44–3.29) and top-quality embryo rate (OR: 1.17, 95% CI: 1.02–1.35). However, brief co-incubation of gametes had no advantages in the live-birth rate (OR: 1.09, 95% CI: 0.72–1.65), miscarriage rate (OR: 1.32, 95% CI: 0.55–3.18), clinical pregnancy rate (OR: 1.36, 95% CI: 0.99–1.87) and polyspermy rate (OR: 0.80, 95% CI: 0.48–1.33) than long-term co-incubation. Additionally, the brief co-incubation was associated with lower normal fertilization rate (OR: 0.89, 95% CI: 0.80–0.99), compared with long co-incubation.ConclusionsBrief co-incubation of gametes had the advantages in increasing implantation rate, ongoing pregnancy rate and top-quality embryo rate than long-term co-incubation. However, the live-birth rate displayed no difference between the two in vitro fertilization methods. Gametes co-incubation time should be individualized according to each patient’s IVF history, infertility causes and the semen parameters.

The Effect of Acidic and Alkaline Seawater on the F-Actin-Dependent Ca2+ Signals Following Insemination of Immature Starfish Oocytes and Mature Eggs.

In starfish, the addition of the hormone 1-methyladenine (1-MA) to immature oocytes (germinal vesicle, GV-stage) arrested at the prophase of the first meiotic division induces meiosis resumption (maturation), which makes the mature eggs able to respond to the sperm with a normal fertilization response. The optimal fertilizability achieved during the maturation process results from the exquisite structural reorganization of the actin cytoskeleton in the cortex and cytoplasm induced by the maturing hormone. In this report, we have investigated the influence of acidic and alkaline seawater on the structure of the cortical F-actin network of immature oocytes of the starfish (Astropecten aranciacus) and its dynamic changes upon insemination. The results have shown that the altered seawater pH strongly affected the sperm-induced Ca2+ response and the polyspermy rate. When immature starfish oocytes were stimulated with 1-MA in acidic or alkaline seawater, the maturation process displayed a strong dependency on pH in terms of the dynamic structural changes of the cortical F-actin. The resulting alteration of the actin cytoskeleton, in turn, affected the pattern of Ca2+ signals at fertilization and sperm penetration.

Coculture with porcine luteal cells during in vitro porcine oocyte maturation affects lipid content, cortical reaction and zona pellucida ultrastructure.

In pigs, in vitro fertilisation (IVF) is associated with high polyspermy rates, and for this reason, in vitro embryo production (IVP) is still an inefficient biotechnology. Coculture with somatic cells is an alternative to improve suboptimal in vitro maturation (IVM) conditions. This study was conducted to test a coculture system of porcine luteal cells (PLC) and cumulus-oocyte complexes (COC) to improve oocyte metabolism. COC were matured in vitro with PLC. Oocyte lipid content, mitochondrial activity, zona pellucida (ZP) digestibility and pore size, cortical reaction and in vitro embryo development were assessed. Coculture reduced cytoplasmic lipid content in the oocyte cytoplasm without increasing mitochondrial activity. Although ZP digestibility and ZP pore number were not different between culture systems, ZP pores were smaller in the coculture. Coculture impacted the distribution of cortical granules as they were found immediately under the oolemma, and more of them had released their content in the ZP. Coculture with porcine luteal cells during IVM increased monospermic penetration and embryo development after IVF. The coculture of COC with PLC affects the metabolism of the oocyte and benefits monospermic penetration and embryo development. The coculture system with PLC could be an alternative for the conventional maturation medium in pigs.

Role of the bovine PRAMEY protein in sperm function during in vitro fertilization (IVF).

Preferentially expressed antigen in melanoma (PRAME) is a cancer/testis antigen (CTA) that is predominantly expressed in normal male gonad tissues and a variety of tumors. PRAME proteins are present in the acrosome and sperm tail, but their role in sperm function is unknown. The objective of this study was to examine the function of the bovine Y-linked PRAME (PRAMEY) during spermatozoal capacitation, the acrosome reaction (AR), and fertilization. Freshly ejaculated spermatozoa were induced to capacitate and undergo AR in vitro. Western blotting results revealed a decrease in the PRAMEY protein in capacitated spermatozoa, and the release of the PRAMEY protein from the acrosome during the AR, suggesting its involvement in sperm capacitation and AR. IVF was performed using in vitro matured bovine oocytes and cauda epididymal spermatozoa either treated with PRAMEY antibody, rabbit IgG, or DPBS. Sperm-egg binding and early embryos were examined at 6 and 45h post IVF, respectively. The number of spermatozoa that bound per oocyte was nearly two-fold greater in the PRAMEY antibody treatment group (34.4) when compared to both the rabbit IgG (17.6) and DPBS (18.1) controls (P < 0.01). Polyspermy rate in the antibody-treated group (18.9%) was three-fold greater than the rabbit IgG control (6.0%) (P < 0.01). The results indicate that PRAMEY may play a role in anti-polyspermy defense. This study thus provides the initial evidence for the involvement of the PRAME protein family in sperm function and fertilization.

Hypothalamic-Pituitary-Adrenal Hormones Impair Pig Fertilization and Preimplantation Embryo Development via Inducing Oviductal Epithelial Apoptosis: An In Vitro Study.

Previous studies show that stressful events after ovulation in sows significantly impaired the embryo cleavage with a significant elevation of blood cortisol. However, the effects of corticotropin-releasing hormone (CRH), adrenocorticotropic hormone (ACTH) and cortisol on fertilization and embryo development remain to be specified, and whether they damage pig embryos directly or indirectly is unclear. This study demonstrated that embryo development was unaffected when pig parthenotes were cultured with different concentrations of CRH/ACTH/cortisol. However, embryo development was significantly impaired when the embryos were cocultured with pig oviductal epithelial cells (OECs) in the presence of CRH/cortisol or cultured in medium that was conditioned with CRH/cortisol-pretreated OECs (CRH/cortisol-CM). Fertilization in CRH/cortisol-CM significantly increased the rates of polyspermy. CRH and cortisol induced apoptosis of OECs through FAS and TNFα signaling. The apoptotic OECs produced less growth factors but more FASL and TNFα, which induced apoptosis in embryos. Pig embryos were not sensitive to CRH because they expressed no CRH receptor but the CRH-binding protein, and they were tolerant to cortisol because they expressed more 11-beta hydroxysteroid dehydrogenase 2 (HSD11B2) than HSD11B1. When used at a stress-induced physiological concentration, while culture with either CRH or cortisol alone showed no effect, culture with both significantly increased apoptosis in OECs. In conclusion, CRH and cortisol impair pig fertilization and preimplantation embryo development indirectly by inducing OEC apoptosis via the activation of the FAS and TNFα systems. ACTH did not show any detrimental effect on pig embryos, nor OECs.

Optimizing swine in vitro embryo production with growth factor and antioxidant supplementation during oocyte maturation

Porcine in vitro fertilization often results in low embryo development rates compared to other livestock species, which is often associated with either a low fertilization rate or high incidence of polyspermy. Since the quality of oocyte maturation is known to play a significant role in oocyte competence, we investigated the impact of supplementing in vitro maturation (IVM) medium containing porcine follicular fluid (pFF) with the growth factors FGF2, LIF and IGF1 (FLI), along with different combinations of cysteine, melatonin and ITS, on cumulus cell expansion, oocyte meiotic maturation, fertilization outcome, embryo development and blastocyst cell numbers. Maturation medium containing pFF yielded the greatest cumulus expansion. Compared to pFF and FLI individually, using pFF and FLI together resulted in the best embryo development rates over total oocyte placed in IVF (12.5% vs. 15.0% vs. 26.6%, respectively). Supplementation of IVM medium containing pFF and FLI with either cysteine, melatonin or insulin-transferrin-selenium, revealed that cysteine was essential to improve embryo development, while melatonin and ITS had a limited impact on improving blastocyst rates. Finally, we observed that pig oocytes matured in medium supplemented with pFF, FLI, cysteine and melatonin had a high proportion of monospermic zygotes (68.2%) and low proportion of polyspermic zygotes (15.9%) following IVF and yielded superior cleavage (78.2%) and blastocyst (32.0%) rates.

Bulk vitrification of in vitro produced bovine zygotes without reducing developmental competence to the blastocyst stage

Cryopreservation of mammalian zygotes can be advantageous since it enables their flexile use in time and space for alternative purposes such as genome editing. Here we report a simple, quick and inexpensive vitrification protocol for in vitro produced bovine zygotes which enables their bulk preservation. Slaughterhouse-derived oocytes were subjected to in vitro maturation and fertilization (IVF). Ten h after IVF, cumulus-enclosed zygotes were equilibrated in 2% (v/v) ethylene glycol + 2% (v/v) propylene glycol for 13–15 min then vitrified in groups of 52–100 in 2 μL microdrops of 17.5% (v/v) ethylene glycol + 17.5% (v/v) propylene glycol supplemented with 0.3 M sucrose and 50 mg/mL polyvinylpyrrolidone. The presence of cumulus cells is important for the success of the process. Therefore, we applied a modified IVF protocol using a short (30 min) co-incubation interval which allowed zygote culture with attached cumulus cells until vitrification and even reduced polyspermy rates without affecting the total fertilization rate. Vitrified zygotes were similar to their non-vitrified counterparts in terms of survival, post-warming development to the blastocyst stage and blastocyst quality measured by cell numbers and cryo-survival. In conclusion, our vitrification protocol integrated with the modified IVF system enabled the quick cryopreservation of bovine zygotes in large groups without reducing their developmental competence to the blastocyst stage.

Effects of intracytoplasmic sperm injection timing and fertilization methods on the development of bovine spindle transferred embryos

Cytoplasmic replacement by spindle transfer (ST) technique can be applied to improve the developmental competence of poor qualitied or aged oocytes. In cattle, ST technology has not been well established for producing embryos and the calves successfully using intracytoplasmic sperm injection (ICSI) or in vitro fertilization (IVF). The objective of this study was to develop a novel procedure for producing bovine ST embryos, which could be fundamental to applying ST technology in other mammals. In the present study, the efficacies of performing ICSI before (ICSI-ST) or after (ST-ICSI) and IVF on the development of ST bovine embryos were investigated. Results indicated that the blastocyst rate of ST embryos produced by ICSI-ST (24.7%) was higher (P < 0.05) than that produced by ST-ICSI (5.9%). On the other hand, ST-IVF had the highest fertilization rate (97.3%), polyspermy rate (24.7%), and lowest blastocyst rate (22.7%) when compared to denuded oocytes (DO), zona cut oocytes (ZC), and cumulus-oocyte complexes (COCs)-IVF groups. Finally, the in vitro development rates of ICSI-ST (24.5%) and ST-IVF (25.2%) were not significantly different (P > 0.05). However, the pregnancy rate (46.7%) and birth rate (33.3%) of ST-IVF were higher (P < 0.05) than those of ICSI-ST (6.3% and 0%, respectively). The percentage of mitochondrial DNA (mtDNA) heteroplasmy derived from donor karyoplasts of the 5 claves was between 2% and 18%. Taken together, performing ICSI prior to ST can improve the embryonic development of ST bovine embryos. Moreover, using IVF, instead of ICSI, for ST oocyte fertilization dramatically increased the pregnancy rate and birth rate of ST calves, in which mtDNA heteroplasmy derived from donor karyoplasts exists.

Paternal effect does not affect in vitro embryo morphokinetics but modulates molecular profile

The use of different sires influences in vitro embryo production (IVP) outcome. Paternal effects are observed from the first cleavages until after embryonic genome activation (EGA). Little is known about the mechanisms that promote in vitro fertility differences, even less about the consequences on embryo development. Therefore, this study aimed to evaluate the paternal effect at fertilization, embryo developmental kinetics, gene expression and quality from high and low in vitro fertility bulls. A retrospective analysis for bull selection was performed using the In vitro Brazil company database from 2012 to 2015. The dataset was edited employing cleavage and blastocyst rates ranking a total of 140 bulls. Subsequently, the dataset was restricted by embryo development rate (blastocyst/cleaved rate) and ten bulls were selected as high (HF; n = 5) and low (LF; n = 5) in vitro fertility groups. IVP embryos derived from high and low fertility bulls were classified according to their stage of development (2 cells, 3–4 cells, 6 cells, 8–16 cells), at 24, 36, 48, 60, 72 hpi, respectively, to evaluate embryo kinetics. Pronuclei formation (24 hpi), cleavage rate (Day 3), development rate, and blastocyst morphology (Grade I and II – Day 7) were also assessed, as well as the abundance of 96 transcripts at 8–16 cell stage and blastocysts. There was no difference in early embryo kinetics (P > 0.05), and cleavage rate (HF = 86.7%; LF = 84.9%; P = 0.25). Nevertheless, the fertilization rate was higher on HF (72%) than LF (62%) and the polyspermy rate was lower on HF compared to LF (HF:16.2% LF:29.2%). As expected, blastocyst rate (HF = 29.4%; LF = 16.0%; P < 0.0001) and development rate (HF = 33.9% LF = 18.9%; P < 0.0001) were higher in HF than LF. At the 8–16 cell stage, 22 transcripts were differentially represented (P ≤ 0.05) between the two groups. Only PGK1 and TFAM levels were higher in HF while transcripts related to stress (6/22, ∼27%), cell proliferation (6/22, ∼27%), lipid metabolism genes (5/22, ∼23%), and other cellular functions (5/22, ∼23%) were higher on LF embryos. Blastocysts had 9 differentially represented transcripts (P ≤ 0.05); being only ACSL3 and ELOV1 higher in the HF group. Lipid metabolism genes (3/9, 33%) and other cellular functions (6/9, 67%) were higher in the LF group. In conclusion, the timing of the first cleavages is not affected by in vitro bull fertility. However, low in vitro fertility bulls presented higher polyspermy rates and produced 8–16 cells embryos with higher levels of transcripts related to apoptosis and cell damage pathways compared to high in vitro fertility ones. Evidence such as polyspermy and increase in apoptotic and oxidative stress genes at the EGA stage suggest that embryo development is impaired in the LF group leading to the reduction of blastocyst rate.

Elevated toxicity of resuspended mine tailings over time

Resuspension of disposed mine tailings is an important secondary source of heavy metal pollution in affected regions. UK copper mine tailings were continuously resuspended in seawater over 14 days to understand potential metal dissolution and associated ecotoxicological impacts. Aqueous concentrations of arsenic increased 859%, nickel 85%, manganese and cadmium over 40%, and vanadium and zinc over 20%. Green sea urchin (Psammechinus miliaris) gametes were exposed to 18-h (18hr) and 14-day (14d) resuspension elutriates. Fertilisation success dropped from 72% (18hr) to 21.06% (14d). Toxicity Index (TI) values (reduced fertilisation membrane quality and fertilisation success) increased from 1.6 (18hr) to 2.8 (14d). Embryo polyspermy frequencies increased from 17.58% (18hr) to 21.13% (14d). Higher TI values and polyspermy rates indicate impacts may go beyond standard bioassay endpoints. Current predictions of mine tailing impacts may be underestimated. This is important for ecosystem resilience when considering other anthropogenic stressors such as climate change. CapsuleDisturbance of metal contaminated sediments over prolonged periods results in ever increasing dissolution concentrations, and elevated toxicity of the water medium to biological receptors.

Sperm migration, selection, survival, and fertilizing ability in the mammalian oviduct†.

In vitro fertilization (IVF) gives rise to embryos in a number of mammalian species and is currently widely used for assisted reproduction in humans and for genetic purposes in cattle. However, the rate of polyspermy is generally higher in vitro than in vivo and IVF remains ineffective in some domestic species like pigs and horses, highlighting the importance of the female reproductive tract for gamete quality and fertilization. In this review, the way the female environment modulates sperm selective migration, survival, and acquisition of fertilizing ability in the oviduct is being considered under six aspects: (1) the utero-tubal junction that selects a sperm sub-population entering the oviduct; (2) the presence of sperm binding sites on luminal epithelial cells in the oviduct, which prolong sperm viability and plays a role in limiting polyspermic fertilization; (3) the contractions of the oviduct, which promote sperm migration toward the site of fertilization in the ampulla; (4) the regions of the oviduct, which play different roles in regulating sperm physiology and interactions with oviduct epithelial cells; (5) the time of ovulation, and (6) the steroid hormonal environment which regulates sperm release from the luminal epithelial cells and facilitates capacitation in a finely orchestrated manner.