Cleavage Development Research Articles

Application of thin slice technology of loose cuttings sample in early stage of experimental geology

Abstract Thin section of rock is a highly technical work in the laboratory, and its application in logging site and rock identification is helpful to solve geological problems quickly in the process of exploration drilling. The cuttings samples cover a variety of lithologies, including sandstone and volcanic rocks with significant diagenetic differences. Due to the special properties of the cuttings, such as loose, fear of water, moisture absorption, brittleness, large hardness differences, inconsistent particle size, and not easy to penetrate glue, the production process has been challenged. In the temperature and humid environment, the cuttings are very susceptible to delixing, especially in the cuttings with cleavage development, more cracks and poor diagenesis. After the sticking sheet is affected by vibration during rough grinding, it is easy to produce warping edge and warping Angle, which leads to the destruction of the original structure of the mineral. The previous production process cycle is long, the operation is relatively not convenient, especially in the grinding into the international standard 0.03mm thick pieces there are certain difficulties. Therefore, it is very important to improve and optimize the rock wafer technology. This will help improve the efficiency of the production of cuttings and reduce the likelihood of damage to mineral structures, which in turn can be used more effectively in exploration drilling sites and rock identification, providing faster and more accurate support for solving geological problems. The results of this study have important significance for improving the efficiency and convenience of the production process and improving the application prospect of the rock sheet technology.

Bisphenol-A disturbs hormonal levels and testis mitochondrial activity, reducing male fertility.

How does bisphenol-A (BPA) influence male fertility, and which mechanisms are activated following BPA exposure? BPA exposure causes hormonal disruption and alters mitochondrial dynamics and activity, ultimately leading to decreased male fertility. As public health concerns following BPA exposure are rising globally, there is a need to understand the exact mechanisms of BPA on various diseases. BPA exposure causes hormonal imbalances and affects male fertility by binding the estrogen receptors (ERs), but the mechanism of how it mediates the hormonal dysregulation is yet to be studied. This study consisted of a comparative study using mice that were separated into a control group and a group exposed to the lowest observed adverse effect level (LOAEL) (n = 20 mice/group) after a week of acclimatization to the environment. For this study, the LOAEL established by the US Environmental Protection Agency of 50 mg/kg body weight (BW)/day of BPA was used. The control mice were given corn oil orally. Based on the daily variations in BW, both groups were gavaged every day from 6 to 11 weeks (6-week exposure). Before sampling, mice were stabilized for a week. Then, the testes and spermatozoa of each mouse were collected to investigate the effects of BPA on male fertility. IVF was carried out using the cumulus-oocyte complexes from female hybrid B6D2F1/CrljOri mice (n = 3) between the ages of eight and twelve weeks. Signaling pathways, apoptosis, and mitochondrial activity/dynamics-related proteins were evaluated by western blotting. ELISA was performed to determine the levels of sex hormones (FSH, LH, and testosterone) in serum. Hematoxylin and eosin staining was used to determine the effects of BPA on histological morphology and stage VII/VIII testicular seminiferous epithelium. Blastocyst formation and cleavage development rate were evaluated using IVF. BPA acted by binding to ERs and G protein-coupled receptors and activating the protein kinase A and mitogen-activated protein kinase signaling pathways, leading to aberrant hormone levels and effects on the respiratory chain complex, ATP synthase and protein-related apoptotic pathways in testis mitochondria (P < 0.05). Subsequently, embryo cleavage and blastocyst formation were reduced after the use of affected sperm, and abnormal morphology of seminiferous tubules and stage VII and VIII seminiferous epithelial cells (P < 0.05) was observed. It is noteworthy that histopathological lesions were detected in the testes at the LOAEL dose, even though the mice remained generally healthy and did not exhibit significant changes in BW following BPA exposure. These observations suggest that testicular toxicity is more than a secondary outcome of compromised overall health in the mice due to systemic effects. Not applicable. Since the protein expression levels in the testes were validated, in vitro studies in each testicular cell type (Leydig cells, Sertoli cells, and spermatogonial stem cells) would be required to shed further light on the exact mechanism resulting from BPA exposure. Furthermore, the BPA doses employed in this study significantly exceed the typical human exposure levels in real-life scenarios. Consequently, it is imperative to conduct experiments focusing on the effects of BPA concentrations more in line with daily human exposures to comprehensively assess their impact on testicular toxicity and mitochondrial activity. These findings demonstrate that BPA exposure impacts male fertility by disrupting mitochondrial dynamics and activities in the testes and provides a solid foundation for subsequent investigations into the effects on male reproductive function and fertility following BPA exposure, and the underlying mechanisms responsible for these effects. In addition, these findings suggest that the LOAEL concentration of BPA demonstrates exceptional toxicity, especially when considering its specific impact on the testes and its adverse consequences for male fertility by impairing mitochondrial activity. Therefore, it is plausible to suggest that BPA elicits distinct toxicological responses and mechanistic endpoints based on the particular concentration levels for each target organ. This work was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2018R1A6A1A03025159). No competing interests are declared.

Detrimental effects of radiofrequency electromagnetic waves emitted by mobile phones on morphokinetics, oxidative stress, and apoptosis in mouse preimplantation embryos

Due to the increasing use of smart mobile phones, the impact of radiofrequency electromagnetic radiation (RF-EMR) on reproductive health has become a serious concern. This study investigated the effect of mobile phone RF-EMR with frequency 900–1800 MHZ on the mouse embryo morphokinetics and genotoxic effect in laboratory conditions. After ovarian stimulation in mice, the MII oocytes were collected and underwent by in vitro fertilization (IVF) method. The generated zygotes were divided into control and exposed groups. Then, the zygotes with 30 min of exposure to mobile phone RF-EMR, and the control zygotes without exposure, were incubated in the time-lapse for 5 days. The intracellular reactive oxygen species (ROS) level, morphokinetic, embryo viability rate, and Gene expression were evaluated. Exposure of zygotes to RF-EMR by inducing ROS caused a significant decrease in blastocyst viability (87.85 ± 2.86 versus 94.23 ± 2.44), delay in cleavage development (t3-t12) and also increased the time (in hours) to reach the blastocyst stage (97.44 ± 5.21 versus 92.56 ± 6.7) compared to the control group. A significant increase observed in mRNA levels of Hsp70 in exposed animals; while Sod gene expression showed a significant down-regulation in this group compared to the controls, respectively. However, there was no significant change in the transcript level of proapoptotic and antiapoptotic genes in embryos of the exposed group compared to the controls. RF-EMR emitted by mobile phone with a frequency of 900–1800 MHZ, through inducing the production of ROS and oxidative stress, could negatively affect the growth and development as well as the transcript levels of oxidative stress associated genes in the preimplantation embryos of mice.

O-071 Paternal body mass index affects early embryo development but does not impact clinical outcomes: a timelapse study of 7,656 embryos in oocyte donation cycles

Abstract Study question Does paternal body mass index (BMI) affect preimplantation development and clinical outcomes in oocyte donation cycles? Summary answer High paternal BMI was associated with delays in early development and poorer embryo quality on day 3, but this did not adversely affect pregnancy outcomes. What is known already Maternal obesity is known to negatively affect preimplantation development and clinical outcomes. While oocyte competence plays a major role in early embryogenesis, male obesity has been linked to poorer semen parameters and epigenetic alterations in sperm. Such characteristics may inherently compromise preimplantation development and ultimately impair reproductive success. Only a few reports have assessed the effects of male BMI on the progression of cell divisions and embryo morphokinetic patterns. However, current findings remain contradictory, while the effects of male obesity on fertility continue to be understudied. Study design, size, duration This retrospective study included 7,656 embryos from 1,342 oocyte donation ICSI cycles, performed between April 2018 and December 2021 across two fertility centres. Sperm originated from the partner (n = 1087) or donor (n = 255) and was either fresh (n = 460) or frozen (n = 835). Only the first fresh (n = 1,140) or frozen (n = 202) embryo transfer (ET) was considered. Our database included 1,089 single and 238 double ETs performed at either the cleavage (n = 424) or blastocyst stage (n = 903) of development. Participants/materials, setting, methods Males were stratified according to BMI, based on WHO guidelines, defined as: normal weight (BMI&amp;lt;24.9, n = 677); pre-obese, (BMI=25.0–29.9, n = 533); and obese (BMI&amp;gt;30, n = 132). Patients with severe male factor and preimplantation genetic testing cycles were excluded. Univariate analyses and logistic regression were used to evaluate associations between BMI, embryo morphokinetics and clinical outcomes. Analyses were adjusted for various confounders pertaining to patient demographics, sperm and cycle parameters. A p-value of &amp;lt; 0.05 was considered significant. Main results and the role of chance Overall, obese men displayed poorer sperm motility and underwent more day 2-4 embryo transfers and freeze-all cycles, compared to pre-obese and normal weight men (p &amp;lt; 0,05). Fertilization rates were not affected by male BMI (77.0% ± 18.9 normal weight; 77.4% ± 18.1 pre-obese; 75.3% ± 19.9 obese; p = 0.62), also in our adjusted analysis. Several morphokinetic parameters were delayed in embryos from pre-obese and obese patients, including pronuclei fading (tPNf, p = 0.0064) and division timings from the 2 to 5-cell stage (t2-t3-t4-t5, p &amp;lt; 0,05). Successively, the number of good quality cleavage embryos decreased significantly with higher BMI (normal weight, 62%; pre-obese, 59%; obese, 50.8%; p = 0.02). In contrast, developmental events past the 5-cell stage, including time to 8-cells (t8), blastulation (tSB) and expanded blastocyst (tB) were comparable amongst the groups (p &amp;gt; 0.05). Similarly, we observed no effect of male BMI on blastocyst quality (normal weight, 70.2%; pre-obese, 73.4%; obese, 75.5%; p = 0.06). Despite early developmental delays, we observed no significant effects on biochemical pregnancy (normal weight, 64.7%; pre-obese 64.1%; obese, 58.7%; p = 0.45), ongoing pregnancy (normal weight, 60.0%; pre-obese, 58.8%; obese, 52.9%; p = 0.35) and live birth rate (normal weight, 41.9%; pre-obese, 42.0%; obese, 34.9%; p = 0.27). Our adjusted analysis corroborated these findings. Limitations, reasons for caution The main limitation of this study is its retrospective design, which may not account for all confounders. Moreover, lifestyle factors, such as smoking or alcohol consumption were not considered. As such, results cannot be generalized to all patient populations. Wider implications of the findings Obese males show lower motile sperm counts and delayed cleavage development. While these changes impaired day 3 embryo quality, they did not affect blastocyst development and quality. That translates into comparable clinical outcomes, suggesting that specific counselling is not required in obese patients. Trial registration number not applicable

Deformation conditions and 40Ar/39Ar dating of thrusting recorded by clay minerals: An example of the Lakora thrust (west-central Pyrenees)

Newly-formed clay minerals in fault zones can provide crucial information about conditions, mechanisms, and timing of deformation at low-grade metamorphism. The present study focuses on the major Lakora thrust in the west-central Pyrenees, that exposes an assemblage of Paleozoic-Upper Cretaceous rocks of the North Pyrenean Zone in the hanging wall and Campanian-Maastrichtian turbidites of the Axial Zone cover in the footwall. Detailed structural, petrological, mineralogical, and chemical investigations were performed on fault rock samples from the Lakora thrust footwall to unravel the deformation conditions related to thrusting. In addition, an attempt to date the thrust activity was made through direct 40Ar/39Ar step-heating on different fractions of muscovite/illite. The structural and petrographic observations suggest a brittle-ductile deformation in two stages along the fault zone: a first stage of bedding-parallel extension and bedding-perpendicular shortening that is marked by boudinage associated with a bedding-parallel cleavage (S0–S1) and a second stage of oblique shearing as marked by reverse faults, folds and oblique cleavage (S2). Pressure-solution and dissolution-recrystallization in the presence of fluids are the main deformation mechanisms that enhanced the neoformation of chlorite and illite associated with cleavage development during the two stages of deformation. The crystallinity, polytypism, and chemistry of illite show that the synkinematic clay minerals with the S2-stage formed under upper anchizone conditions (∼250–300 °C). Such thermal conditions are higher than those expected from the inferred maximal burial (∼6 km), thus arguing for a possible circulation of hot fluids along the thrust footwall. 40Ar/39Ar step-heating results confirm that the newly-formed illite is more abundant in the finer fractions of the most deformed sample, with a total gas age of c. 61.7 ± 0.5 Ma in the finest <0.2 μm fraction. This age is older than the early-mid Eocene age expected from structural constraints for the emplacement of the Lakora thrust sheet due to the mixing of newly-formed illites with small amounts of inherited micas and/or contamination by excess argon carried by fluids.

U-Pb dating reveals multiple Paleoproterozoic orogenic events (Hamersley orogenic cycle) along the southern Pilbara margin (Australia) spanning the onset of atmospheric oxygenation

Abstract The early Paleoproterozoic was a time of unprecedented change in Earth's climate and surface environment. The key to resolving some of the controversies surrounding the timing and causes of these changes lies with supracrustal sequences, such as the 2.45–2.22 Ga Turee Creek Group in the southern Pilbara craton, northwestern Australia. The group preserves a predominantly siliciclastic sequence; however, its precise age, tectonic setting, and postdepositional history are disputed. Although it is interpreted to have been deposited in a foreland basin setting shortly after 2.45 Ga, the oldest well-recognized deformational event, marked by northward folding and thrusting, is the 2.20–2.15 Ga Ophthalmia orogeny. Evidence for a pre-Ophthalmia fabric-forming tectonic event north of the Archean Sylvania Inlier, southeast Pilbara craton, which is marked by tight to isoclinal folding, has been largely overlooked. In this area, we report in situ U-Pb geochronology of authigenic monazite and xenotime in shale with a well-developed tectonic cleavage from the ca. 2.63 Ga Jeerinah Formation. Monazite porphyroblasts, which are locally wrapped by strain fringes aligned in a tectonic cleavage, yielded weighted mean 207Pb/206Pb ages at 2370 ± 11 Ma and 2312 ± 8 Ma, whereas xenotime, which overprints a crenulation cleavage, gave a weighted mean 207Pb/206Pb age of 2291 ± 11 Ma, constraining fabric development to between 2.31 Ga and 2.29 Ga. Our results confirm the existence of a pre-Ophthalmia deformational event in the southeastern Pilbara craton, herein referred to as the Sylvania orogeny, which is part of an ~300 m.y. interval (2.45–2.15 Ga) of northward-directed compression (“Hamersley orogenic cycle”). This orogenic cycle is marked by east-west and northwest-southeast folding, cleavage development, veining, hydrothermal gold mineralization, and isotopic resetting across the southern Pilbara craton. Our results indicate that the syn–Great Oxidation Event Turee Creek Group was deposited in one or more foreland basins after 2.45 Ga. Our results provide a new tectonostratigraphic and geodynamic framework for understanding the timing and origin of geochemical records in a key succession deposited during an interval of global environmental change.

Tracing wedge-internal deformation by means of strontium isotope systematics of vein carbonates

AbstractRadiogenic strontium isotopes (87Sr/86Sr) of vein carbonates play a central role in the tectonometamorphic study of fold-and-thrust belts and accretionary wedges and have been used to document fluid sources and fluxes, for example, along major fault zones. In addition, the 87Sr/86Sr ratios of vein carbonates can trace the diagenetic to metamorphic evolution of pore fluids in accreted sediments. Here we present 87Sr/86Sr ratios of vein carbonates from the Infrahelvetic flysch units of the central European Alps (Glarus Alps, Switzerland), which were accreted to the North Alpine fold-and-thrust belt during the early stages of continental collision. We show that the vein carbonates trace the Sr isotopic evolution of pore fluids from an initial seawater-like signature towards the Sr isotopic composition of the host rock with increasing metamorphic grade. This relationship reflects the progressive equilibration of the pore fluid with the host rock and allows us to constrain the diagenetic to low-grade metamorphic conditions of deformation events, including bedding-parallel shearing, imbricate thrusting, folding, cleavage development, tectonic mélange formation and extension. The strontium isotope systematics of vein carbonates provides new insights into the prograde to early retrograde tectonic evolution of the Alpine fold-and-thrust belt and helps to understand the relative timing of deformation events.

P–253 Description of a rare spontaneous monozygotic blastocyst splitting into two discrete euploid blastocysts in vitro detected with time-lapse imaging and preimplantation genetic testing (PGT)

Abstract Study question Can spontaneous and complete blastocyst splitting into two, in vitro, be investigated using time-lapse imaging and biopsy of each trophectoderm, for inference of ploidy? Summary answer Time-lapse imaging combined with PGT-A gives insights into the incidence, dynamics and timing of rare blastocyst splitting and the ploidy status of each resulting blastocyst. What is known already It is well known that multiple births occur more often with Assisted Reproductive Technologies (ART) than spontaneous conception, even after single embryo transfer. The mechanism of Monozygotic Twinning (MZT) during ART is still unclear but cryopreservation, extended culture, PGT, maternal age and assisted hatching are reported risk factors. MZT is a rare phenomenon, with an incidence of 0.4% in natural conception compared with up to 4.9% in ART. The timing of embryo splitting dictates the type of twinning, in terms of chorionicity and amnionicty, and this is officially determined using ultrasound scanning. Study design, size, duration This is a case study describing the detection of the complete splitting of an IVF blastocyst at 140 hours post insemination (hpi), using time-lapse imaging. The 40-year-old patient previously experienced biochemical pregnancy and several miscarriages; an ectopic molar pregnancy and a probable cornual ectopic. The 39-year-old male partner was normozoospermic. Participants/materials, setting, methods Facilitative laser breaching was performed, according to standard operating procedure, of the morula at 96hpi of embryo development, prior to PGT. Images were collected every 10 minutes and developmental events and embryos morphology annotated using the EmbryoScope+™ time lapse incubator and software. Main results and the role of chance Over 50,000 hatching blastocysts have been time-lapse imaged, scrutinised and annotated within this group of fertility clinics. This is the first time that such a rare blastocyst splitting event has been recorded and studied. Following observation of two pronuclei following IVF and typical cleavage development to blastocyst, with facilitative zona breaching on, at 106.7hpi, the full blastocyst’s trophectoderm (TE) began to herniate and hatch. By 114.3hpi a second internal blastocoel cavity formed appearing to divide the inner cell mass (ICM) within the zona pellucida (ZP). This resulting blastocyst proceeded to hatch as its discrete ICM migrated out of the ZP, along with its TE. TE cells from the original blastocyst then began to hatch at 117.5hpi at the same breached site in the ZP with its ICM visibly evacuating the ZP. By 140hpi the blastocyst had split into two discrete blastocysts while hatching from the ZP. Both resulting blastocysts had clear and separate ICMs and TEs present. Biopsy of approximately 5 cells was performed for each TE, and the blastocysts were vitrified individually. Next Generation Sequencing (NGS) reported both blastocysts to be euploid. Limitations, reasons for caution This case may have been detectable without time-lapse imaging, as the splitting was completed prior to biopsy. More expert scrutiny of the images may result in earlier signs of twinning in progress being detected. Wider implications of the findings: The nature of this detectable in vitro blastocyst splitting, indicates these embryos (if they implanted) to be monozygotic, dichoriol-diamniotic ‘identical’ twins. However – as single embryo transfer is the preferred treatment plan; they may be born years apart. These observations could shed light on the debated models of monozygotic twinning. Trial registration number Not applicable

Late Paleozoic Gondwanide deformation in the Central Andes: Insights from RSCM thermometry and thermal modeling, southern Bolivia

A widespread unconformity between Ordovician metasedimentary rocks and nonmetamorphosed Mesozoic sedimentary rocks across the Eastern Cordillera of Bolivia provides evidence for significant pre-Late Cretaceous deformation and erosion. In this paper, we refine the middle to late Paleozoic tectonic history of the central Andean segment of Gondwana's western margin. We combine Raman spectroscopy of carbonaceous material (RSCM) thermometry with semi-quantitative deformation temperature estimates from quartz recrystallization microstructures and published illite crystallinity values from Cambrian-Devonian sedimentary rocks that span the Eastern Cordillera of southern Bolivia at ~21°S. Estimates of peak temperature and deformation temperature range primarily between ~220 and ~ 400 °C and show a general increase with depth that defines a metamorphic field gradient between ~37 and ~ 47 °C/km. Temperatures obtained from Ordovician rocks directly below the unconformity require erosion of a > ~5 km overburden prior to the Late Cretaceous, which was likely accomplished by a combination of distributed cleavage development and Paleozoic folding and thrust faulting revealed from cross-section reconstructions. The peak temperature data are incorporated into thermal models along with published illite KAr, zircon (UTh)/He, and zircon fission-track cooling ages that refine the timing of Paleozoic orogenesis in the central Andes. We interpret shortening-related flexural subsidence driven by cratonward growth of a pre-Andean orogenic wedge along the western margin of Gondwana, with peak temperature conditions attained during eastward advance of a foreland basin between ~420 and ~ 318 Ma during the Devonian-Carboniferous Gondwanide Orogeny. Erosional exhumation between ~352 and ~ 294 Ma was the result of continued growth and eastward expansion of the associated Transpampean tectonic highland across the Eastern Cordillera. The growth of this pre-Andean contractional orogen coincided with global plate reorganization and the reestablishment of subduction and arc magmatism along the western margin of Gondwana, as indicated by a significant influx of Carboniferous arc-related detritus into the foreland basin system.

94 Myostatin gene editing by CRISPR/Cas9 technology of Brangus fetal fibroblasts to produce edited embryos by cloning

Some European cattle breeds, such as Charolais and Maine Anjou, have natural mutations in the myostatin gene (MSTN) that inhibit its expression and result in an increase in muscle mass and protein content. An innovative hallmark would be the invitro introduction of this genotype in South America cattle breeds to improve their commercial value. To achieve this, we aimed to disrupt MSTN gene expression in bovine fetal fibroblasts (BFFs) using CRISPR/Cas9 technology and to generate MSTN-edited embryos by somatic cell nuclear transfer (SCNT). BFFs were isolated from a cloned fetus of a Brangus bull with a prized genetic background and nucleofected with the Cas9 ribonucleoprotein-gRNA complex previously assessed to target exon 2 of the bovine MSTN gene. To evaluate MSTN editing, genomic DNA from the wild-type (WT) and nucleofected BFFs were isolated, exon 2 of MSTN was amplified by PCR, and the PCR product was Sanger sequenced. In all cases, the sequencing results were analysed using the indel Synthego software tool. According to indel analysis, MSTN gene editing efficiency of the BFFs was 58.83%±3.2 (n=6). The resulting edit consisted of insertion of thymine in exon 2 of the MSTN gene that shifted the gene reading frame, introducing a premature stop codon and generating a truncated MSTN protein. The nucleofected BFFs were then used to generate embryos by SCNT, and WT BFFs were included as controls. Embryo cleavage and blastocyst development rates were evaluated at Day 2 and 7, respectively (Chi-squared test, P&amp;lt;0.05). Although lower cleavage rates were obtained in the MSTN-edited BFFs group [65.3% (n=273/418) vs. 87.6% (n=169/193)], no differences were observed at the blastocyst stage [19.1% (n=80/418) vs. 25.4% (n=49/193)]. To confirm the efficiency of MSTN editing in the cloned embryos, 10 blastocysts generated with the MSTN-edited BFFs were individually analysed for MSTN exon 2 sequence as described before. The results showed that 30% of the blastocysts (3/10) presented a homozygous biallelic edition, which consisted of a thymine base insertion, as expected. In summary, the strategy we used allowed production of MSTN null cloned Brangus embryos avoiding putative undesired integration of exogenous DNA into the bovine genome, such as plasmid sequence, regardless of off-target occurrences. We conclude that CRISPR/Cas9 is an efficient technique to disrupts MSTN gene expression in bovine embryos; this work represents a step toward improving the production efficiency of South American cattle breeds.

99 Increased expression of microRNAs in sperm of Nelore Bulls with high invitro fertility

The spermatozoon is no longer known only as a cell that delivers the male genetic material to the oocyte, because it also provides molecules such as microRNAs (miRNAs) that play a significant role in fertilization and embryonic development. MiRNAs are small noncoding RNAs capable of modulating mRNA translation, thus affecting important biological processes. Sperm miRNAs may influence embryo development and therefore, might be related to invitro production of embryos (IVP), considering that individual bulls have different fertility rates when used for IVF. The aim of this work was to identify miRNAs expressed in semen of bulls with high and low IVP rates. The composition of groups was based on a retrospective database from a reproductive biotechnology company between the years of 2016 and 2018, generating around 7000 IVP manipulations of 430 Nelore bulls. We only considered IVP manipulations that used a minimum of 30 oocytes and conventional semen selection by Percoll gradient. A total of 87 Nelore bulls fit these criteria. We then ranked bulls based on cleavage rate (number of cleaved structures/number of oocytes), blastocyst rate (number of blastocysts/number of oocytes) and embryo development rate (number of blastocysts/number of cleaved structures). Considering these three rates, we allocated bulls to two groups. The top eight were considered to have high IVP fertility (HF) and the bottom eight were grouped together as low IVP fertility (LF). We performed the T TEST procedure (SAS 9.3 software; SAS Institute Inc.) to compare the groups for cleavage (P&amp;lt;0.0001), blastocyst (P=0.0006), and embryo development (P=0.0001) rates. For miRNA analysis, sperm were separated using Percoll gradient and subjected to RNA extraction and cDNA synthesis. First, quantitative real-time PCR was used to evaluate the abundance of 380 bovine-specific miRNAs in a pool of samples for each group, using QuantStudio 6Flex. Then, 48 miRNAs presenting at least a 3-fold change of normalized cycle threshold values between groups were selected: 23 highly detected in HF, 1 highly detected in LF, 18 exclusively detected in HF, and 2 exclusively detected in LF. Last, we evaluated the abundance of these selected miRNAs in each experimental unit. We identified four miRNAs highly abundant in sperm from HF bulls, bta-miR-10a, bta-miR-383, bta-miR-93, and bta-miR-449b. Our results suggest that these miRNAs could play important roles in bovine embryo development. This work was supported by FAPESP (grant# 2018/03871-6).

Structural Evolution of a 1.6 Ga Orogeny Related to the Final Assembly of the Supercontinent Nuna: Coupling of Episodic and Progressive Deformation

AbstractThe poly‐deformed Georgetown Inlier (GTI) in NE Australia has recently been suggested to record a 1.60 Ga orogenic event related to final Nuna assembly. However, the structural evolution of the inlier has remained poorly constrained at the regional scale, and major tectono‐thermal events occurred at circa 1.55 Ga. The GTI is the type region for conceptualization of crenulation cleavage development and where the foliation intersection axes (FIAs) approach has been applied. We reevaluated both concepts by combining a multiscale petrostructural analysis with recent petrological and geochronological data. Three main deformation events (D1–D3) and associated composite fabrics (S1–S3) are identified in the GTI. The original NE orientation of 1.60 Ga D1 compressional structures is preserved in the low‐grade western domain, and the associated composite S1 fabric is retained as microstructural relicts within circa 1.55 Ga D2 low‐strain domains to the east. Extensional D2 structures, characterized by a pervasive, high‐grade, composite S2 foliation throughout the central and eastern domains, are interpreted as the footwall of a regional N‐S trending, W dipping crustal‐scale detachment zone. Syn‐D2 S‐type granites formed at 1.55 Ga as the detachment evolved. D1 stage was associated with Nuna assembly, whereas D2 represents postcollisional extension. Progressive foliation development occurred twice in the GTI, at 1.60 (D1) and 1.55 Ga (D2), but the previous FIA analysis only records the 1.60 Ga event and cannot be easily reconciled with the regional structural analysis. This study highlights that a multiscale and multidisciplinary approach is required to unravel the structural history of orogenic belts.

PREDICTIVE VALUE OF FOLLICULAR FLUID GHRELIN IN WOMEN WITH POLYCYSTIC OVARIAN DISEASE WHO UNDERGO INTRACYTOPLASMIC SPERM INJECTION

Ghrelin’s role in reproduction was first suggested by its wide expression in many human reproductive tissues including its immunohistochemical expression in the human ovary. Ghrelin expression is identified in the seminiferous tubuli of the interstitial Leyding and Sertoli cells in male testicles, whereas it is identified in the ovarian hilus interstitial cells and mature corpus lutea in females.It is postulated that ghrelin plays a local regulatory role in the male and female gonads. In females, Ghrelin acts centrally by suppressing hypothalamic GnRH release and GnRH-induced gonadotropin secretion by the pituitary; and in gonads it acts by increasing luteolytic factors such as PGF2 alfa, and ghrelin inhibiting estradiol and progesterone biosynthesis in granulose-lutein cells as well as granulosa cell (GC) proliferation.This potential regulatory effect on steroidogenesis and ovarian cellular function led to the hypothesis that ghrelin may mediate the relationship between metabolism and oocyte quality in addition to possible role in the regulation of embryo development in as the cleavage development before the six-cell stage of a preimplantation human embryo depends solely on maternally inherited material. Ghrelin levels can, therefore, be an indicator of an underlying pathology as in humans in states of chronic malnutrition such as anorexia nervosa, serum ghrelin levels may be increased, while in other conditions it could be associated with decreased ghrelin levels such as obesity and polycystic ovarian syndrome (PCOS).

Development of the limpet Testudinalia testudinalis (O.F. Müller, 1776) (Patellogastropoda, Mollusca) from the White Sea, with special reference to the radula

ABSTRACT Development of the limpet Testudinalia testudinalis collected from the White Sea was studied using light and scanning electron microscopy. Cleavage and early development of T. testudinalis were consistent with the general pattern of patellogastropod development, differing in only minor details, such as temperature-dependent developmental rates. A pre-torsional veliger radula was detected by calcofluor white staining of unpolymerized chitin. The veliger radula consisted of nine transverse rows with three teeth per row, which transformed into the radula of newly settled juveniles with five teeth per row (one central and two pairs of lateral teeth). The juvenile radula, with two pairs of lateral and one pair of hook-like marginal teeth, resembled the adult radula in terms of teeth form and co-localization. Radula formation differs between the larva and adult in that each tooth is formed by several odontoblasts and microvillar secretion in the former, whereas it is formed by numerous cells and apocrine secretion in the latter.

The early Palaeogene transition from thin-skinned to thick-skinned shortening in the Potosí uplift, Sierra Madre Oriental, northeastern Mexico

ABSTRACT The central Sierra Madre Oriental in Mexico developed primarily in the Late Cretaceous to early Palaeogene as a thin-skinned fold belt above a décollement hosted in Jurassic evaporite. In some regions shortening transitioned to sub-décollement structural levels, resulting in thick-skinned uplifts such as the Potosí uplift in Nuevo León. Thick-skinned deformation in the Potosí uplift involved folding of sub-décollement strata into an NNW-trending anticlinorium, cleavage development, thrust and conjugate strike-slip faulting, and extension fractures associated with barite mineralization. These structures consistently record subhorizontal shortening, directed ~52–65° in the southern uplift, ~69–72° in the northern uplift. Palaeocene to mid-Eocene zircon (U-Th)/He cooling dates record the timing of exhumation associated with thick-skinned uplift and suggest a continuation of shortening rather than a separate tectonic event. Zircon (U-Th)/He dates across the southern Potosí uplift and the Aramberri uplift, ~50 km to the south, range from ~66–53 Ma, whereas dates in the northern part of the Potosí uplift range from ~49–44 Ma. We attribute the transition to thick-skinned shortening to the mechanical strengthening of a planar décollement as rheologically weak evaporite was evacuated beneath synclinal keels of detachment folds. Along-strike differences in timing of exhumation and shortening directions may relate to differences in mechanical stratigraphy. Thicker intervals of evaporite in the northern uplift allowed thin-skinned shortening to continue while the southern uplift transitioned to thick-skinned shortening as the evaporite décollement was exhausted. As a result, stress-strain trajectories in the northern uplift refracted clockwise during continued deformation. Our findings provide new insight into the structural evolution of the Potosí uplift and may provide a framework for studying other thick-skinned uplifts in the orogen, and more generally orogenic belts that record a transition in deformation styles during progressive shortening.

Tracking progressive deformation of an orogenic wedge through two successive internal thrusts: Insights from structure, deformation profile, strain, and vorticity of the Main Central thrust (MCT) and the Pelling-Munsiari thrust (PT), Sikkim Himalayan fold thrust belt

We compare and contrast the structural evolution of two successive internal thrusts, the Main Central thrust (MCT) and the Pelling-Munsiari thrust (PT), from the crystalline core of the Sikkim Himalaya in the context of progressive deformation involving a footwall Lesser Himalayan duplex; the PT is its roof thrust. The mylonitic foliations in both the shear zones are overprinted by successive cleavages. The youngest cleavage from the PT zone continues within the hangingwall MCT sheet that records one additional cleavage possibly indicating time-transgressive cleavage development. The cleavages become steeper while their intensity decreases structurally higher up within individual thrust sheets. The Rs-values and angular shear strain decrease from the mylonite zones to structurally higher up within the sheets. Both the shear zones are Type II with decelerating strain paths. Rs-θ′ relationships and microstructures indicate thrust-parallel stretch is greater than thrust-perpendicular component. Both the shear zones record strain partitioning with lesser competent mylonite domains recording higher Rxz, and a greater proportion of simple-shear than the protomylonite domains. The shear zones recorded the growth of the duplex during progressive deformation that contributed to pure-shear dominated general-shear, higher flattening strain and greater translation on its roof thrust, the PT, than the overlying MCT.

The vein-hosted copper deposits of the Allihies mining area, SW Ireland; a new structural and chronological evaluation

This paper presents new data for historical vein-hosted copper sulfide deposits in the Late Paleozoic Munster and South Munster basins of SW Ireland. Detailed mapping, 3D modelling, fluid inclusion microthermometry and geochronology from the Allihies area of the Beara Peninsula have led to a new interpretation of the timing and development of ore mineralization. Macro- and microstructural studies reveal that the ore-bearing, mainly east–west-striking quartz veins are directly related to early extensional, basinal normal faults. Molybdenite Re–Os dating of the main-stage Cu lode yields ages from 367.3 ± 5.5 to 366.4 ± 1.9 Ma. This early vein system experienced subsequent late Carboniferous Variscan deformation, including cleavage development, sinistral SW–NE strike-slip faulting, cataclastic deformation and recrystallization. The new timing of Cu mineralization in SW Ireland has major implications for its relationship to the base metal deposits of the Irish Midlands. Supplementary material: A video file showing a 3D model of the major lodes and the underground workings at Mountain Mine, and Excel spreadsheets showing fluid inclusion measurements and sample locations of the Allihies Copper Mining District are available at https://doi.org/10.6084/m9.figshare.c.4875015

85 Regulation of mammalian target of rapamycin signaling post-fertilisation is essential for efficient development of bovine pre-implantation embryos

The phosphatidylinositol-3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling pathway is considered a key regulator of various cellular responses related to growth, proliferation, survival, apoptosis, and autophagy. The expression of mTOR in cumulus cells and all oocyte stages highlights a role in embryonic development, but its function in the context of embryo development is not completely clarified. In the current study, we investigated the effect of mTOR activation on the total cleavage and blastocyst development rates in bovine. Oocytes collected from bovine ovaries were invitro matured (around 50 oocytes per group) and fertilised before incubation with the mTOR activator MHY1485 (1 µM) for 72h at 38.5°C and 5% CO2. The total cleavage rate at Day 4 post-fertilisation and the blastocyst development rate at Day 8 post-fertilisation were recorded. The expression levels of the mTOR and PI3K in Day 8 blastocysts were investigated using immunofluorescence and imaged using confocal laser-scanning microscope, whereas the fluorescence intensity was analysed using ImageJ software. The difference between groups was analysed using Student's t-test (GraphPad Prism). Results of microscopic investigations showed higher rates of Day 4 cleavage (83.7±1.3 vs. 72±0.8% for control) and Day 8 blastocyst development (40.4±1.7 vs. 33±1.8% for control) upon addition of MHY1485 (P&amp;lt;0.05). The immunofluorescence showed higher mTOR and PI3K expression levels in MHY1485-treated group than the untreated control (P&amp;lt;0.05). The total number of cells per blastocyst, using 4′,6-diamidino-2-phenylindole (DAPI) staining of the nuclei, was higher in the mTOR-activated group (194.9±4.8 vs. 171.3±3.5 for control). In conclusion, our data reflect the essential role of the mTOR signaling for bovine pre-implantation embryonic development.

191 Effect of the time of oocyte collection through slicing method on meiosis resumption and invitro embryo production

Oocytes from juvenile goats are collected by slicing the ovary surface because the high percentage of small antral follicles limits follicular aspiration. The time of oocyte collection can impair oocyte developmental competence due to spontaneous resumption of meiosis. The aim of this study was to assess whether the time of slicing period affects oocyte meiosis and embryo development after invitro fertilization. Ovaries from juvenile goats (1-2 months old) were recovered at a local slaughterhouse. Cumulus-oocyte complexes (COCs) were collected by slicing, selected, and kept in the slicing medium at 38.5°C in humidified air with 5% CO2 until analysis or culture. The slicing medium was HEPES-buffered (25mM) TCM-199 with 2.2mgmL−1 NaHCO3 and 50mgmL−1 gentamicin. Two slicing periods were tested: T1 (1 h) and T4 (4 h). After this time, a group of oocytes were stained with 1% orcein in 45% acetic acid solution for assessing meiotic arrest and observed as the rate of germinal vesicle (GV; 61-67 oocytes/group from 5 replicates). The remaining COCs were cultured in our conventional IVM medium (TCM-199 with FSH, LH, oestradiol, sodium pyruvate, glutamine, cysteamine, epidermal growth factor, and fetal bovine serum) at 38.5°C with 5% CO2. After 24h, a sample of oocytes were stained for assessing nuclear maturation (28-29 oocytes/group, 3 replicates), and the rest were invitro fertilized with 4×106 spermmL−1 in BO-IVF medium (IVF Bioscience) for 20h and embryo cultured in BO-IVC medium for 7 days (70-81 oocytes/group, 3 replicates). Blastocysts were stained with Hoechst 33258 for determining the number of cells. Data were analysed with two-way ANOVA with RStudio version 1.2.1335. The time of slicing was set as a fixed factor and the replicate as random variable. Data presented as percentage did not follow a normal distribution and were square root arcsine transformed before analysis. At the end of slicing periods T1 and T4, oocytes at GV were 100% and 84.7±5.0%, respectively (P&amp;lt;0.05). After 24h of IVM, the oocytes at MII were 77.0±7.1% and 88.6±7.3%, respectively, without statistical differences. However, oocytes from T1 produced a higher rate of cleaved oocytes (84.6±0.9%) and expanded blastocysts (11.03±5.2%) than T4 (49.8±7.9%, 0%, respectively; P&amp;lt;0.05). The total blastocyst rate for T1 and T4 was 25.4±5.8% and 9.4±4.9%, respectively (P=0.068). No differences were observed in blastocyst cell number (75.9±4.0 and 67.5±10.9, respectively). In conclusion, oocytes resume meiosis before IVM during a long slicing period, even though the slicing medium is not supplemented with hormones or growth factors. The longer slicing period does not affect nuclear maturation but impairs oocyte competence, observed as lower cleavage and blastocyst development. Further experiments are needed to determine whether the use of meiotic inhibitors in the slicing medium can prevent the negative effect of the long slicing period. This study was funded by the Spanish Ministry of Science, Innovation and Universities (AGL2017-85837-R).

197 The use of photostimulation to enhance oocyte cytoplasmic maturation

Phototherapy uses monochromatic light from low-power lasers and light-emitting diodes (LEDs) to modulate biological processes. It has been proposed that the red-to-near infrared optical region (~600-1000nm) enhances cellular metabolic activity by activation of the mitochondrial respiratory chain. However, photostimulation induces the generation of oxide free radicals and could create oxidative stress in exposed cells. The main objective was to use photostimulation to affect the cumulus-oocyte complex metabolic state, aiming to enhance cytoplasmic maturation rates and subsequent embryonic development. A secondary objective was to determine the toxicity of the proposed photostimulation protocol. Abattoir-derived ovaries were used. All media was from IVF Biosciences (Falmouth, Cornwall, UK). Follicles 2 to 6mm in diameter were aspirated. Oocytes with compact cumulus and homogeneous cytoplasm were selected, and 50 oocytes/well were placed in invitro maturation medium (0h) and incubated at 38.5°C in 5% CO2 in air with high humidity in the presence (treatment=exposure for 2min to super-bright LED 1 and 2h after the beginning of maturation; LED wavelength of 660-665 nm; NTE30041; NTE Electronics Inc.) or absence (Control) of light. After maturation (22h), oocytes were split into two wells (25 oocytes/well) and subjected to IVF with semen from two different bulls for 18 to 20h. Cumulus cells were separated by vortexing, zygotes were placed in invitro culture medium, and incubated at 38.5°C in 5% CO2 in air with high humidity. Culture medium was renewed every 48h. Cleavage, morula, and blastocyst rates were recorded as a percentage of the number of oocytes subjected to IVF per treatment. The experiment was replicated 4 times. Statistical analysis was conducted using the Mixed procedure (SAS 9.4, SAS Institute Inc.) with repeated-measures and autoregressive covariance. The model's random effect was well within treatment. Fixed effects were bull, stage of development, and treatment. There was no difference (P=0.8) between treatments for any stage of development measured (cleavage: 76.4±2.7 vs. 74.8±4.1; morula: 36.1±4.8 vs. 35.9±5.8; blastocyst: 20.8±3.2 vs. 20.6±4.4 for control and treatment respectively; mean±s.e.). Sire affected development: bull 1 had a greater percentage (P&amp;lt;0.05) cleavage (82.9±0.02 vs. 68.3±0.02), morula (42.6±0.05 vs. 29.4±0.04), and blastocyst (27.8±0.04 vs. 13.6±0.01) development than bull 2. There was no treatment×bull interaction (P=0.9). In conclusion, there were no stimulatory or toxic effects of this preliminary photostimulation protocol. Further research is needed to develop an optimal protocol that shows a metabolic effect and, potentially, an enhancement of invitro cytoplasmic maturation rates and subsequent embryonic development.