Hatched Blastocyst Stage Research Articles

Nuclear transfer leads to aberrant cell cycle regulation and translation, as determined by gene expression, and selection of mitochondrial DNA in porcine blastocysts

Abstract Nuclear transfer technologies, such as metaphase II spindle transfer (MII-ST), are being developed to overcome poor oocyte quality and prevent children from acquiring severe mitochondrial DNA (mtDNA) disease. However, it is unclear whether these technologies perturb gene expression; and mtDNA carryover can be reduced to minimal levels. Using our pig model, we isolated individual karyoplasts with limited mtDNA carryover (306 ± 49 copies) from mature oocytes. Firstly, we reintroduced the spindle into its original cytoplast (autologous), fertilised, and cultured the resultant embryos to the hatching blastocyst stage of development. Following RNA-seq, 64 genes were differentially expressed at FDR < 0.05; and 1327 genes at P < 0.05. Pathways associated with cell cycle and epigenetic regulation and genes associated with nuclear-mitochondrial interactions were upregulated whilst protein translation pathways were downregulated. We then transferred karyoplasts to third party cytoplasts (heterologous) and undertook mtDNA next generation sequencing of 2-cell to hatching blastocyst stage preimplantation embryos. Nine of 20 embryos possessed two mitochondrial genomes. Our outcomes show that autologous nuclear transfer alone significantly changes hatching blastocyst gene expression; very low levels of mtDNA carryover can be preferentially replicated during early development; and nuclear and mitochondrial synchrony has not been reestablished.

Using extended growth of cattle embryos in culture to gain insights into bovine developmental events on embryonic days 8 to 10

We have previously described an extended embryo culture system, based on uterine composition, growth factors and the cell culture additive B27, for growing cattle embryos in vitro beyond embryonic day 7. Here, extended in vitro embryos are compared to embryos developed in the uterus and are used to establish a developmental staging framework useful for understanding developmental events occurring until Day 10. Immunofluorescence or mRNA expression of the ICM/epiblast markers OCT4, SOX2 and NANOG, hypoblast markers GATA6, SOX17 and GATA4 and trophoblast genes CDX2, GATA3, ASCL2 and IFNT revealed the presence of four stages during this period that can be molecularly distinguished. These are expanded blastocyst, hatched blastocyst, hypoblast layering and early hypoblast migration. Interestingly NANOG and SOX17 show reciprocal expression at the expanded blastocyst stage, well before SOX2 and GATA6 expression refines to a similar so-called “salt and pepper” mutually exclusive expression in the ICM at the hatched blastocyst stage. GATA4 expression is only seen from stages when the hypoblast starts migrating around the blastocyst cavity. Intriguingly, trophoblast still expresses GATA6 and OCT4 in all cells during the expanded blastocyst phase, while SOX2 and SOX17 are seen in only some trophoblast cells. By the hypoblast-epiblast layering stage no trophoblast expression remains except for that of OCT4 protein, which starts waning in trophoblast once the hypoblast begins migrating. Lastly, it is shown that cultured embryos exhibit increased expression of the stress marker TP53 in the epiblast and hypoblast at late stages in comparison to embryos produced in the uterine environment.

Estrogen signaling encourages blastocyst development and implantation potential.

Estrogen is well-known for preparing uterine receptivity. However, its roles in regulating embryo development and implantation are unclear. Our objective was to characterize estrogen receptor 1 (ESR1) in human and mouse embryos and determine the effect of estradiol (E2) supplementation on pre- and peri-implantation blastocyst development. Mouse embryos, 8-cell through hatched blastocyst stages, and human embryonic days 5-7 blastocysts were stained for ESR1 and imaged using confocal microscopy. We then treated 8-cell mouse embryos with 8nM E2 during in vitro culture (IVC) and examined embryo morphokinetics, blastocyst development, and cell allocation into the inner cell mass (ICM) and trophectoderm (TE). Finally, we disrupted ESR1, using ICI 182,780, and evaluated peri-implantation development. ESR1 exhibits nuclear localization in early blastocysts followed by aggregation, predominantly in the TE of hatching and hatched blastocysts, in human and mouse embryos. During IVC, most E2 was absorbed by the mineral oil, and no effect on embryo development was found. When IVC was performed without an oil overlay, embryos treated with E2 exhibited increased blastocyst development and ICM:TE ratio. Additionally, embryos treated with ICI 182,780 had significantly decreased trophoblast outgrowth during extended embryo culture. Similar ESR1 localization in mouse and human blastocysts suggests a conserved role in blastocyst development. These mechanisms may be underappreciated due to the use of mineral oil during conventional IVC. This work provides important context for how estrogenic toxicants may impact reproductive health and offers an avenue to further optimize human-assisted reproductive technology (ART) to treat infertility.

Multiple ovulation and embryo transfer in sheep: Effects of embryo developmental stage and quality on viability in vivo under farm conditions.

Multiple ovulation and embryo transfer (MOET) technologies are integral to genetic improvement programs in the sheep industries. Despite the protocols being well established, previous findings regarding the effects of embryo properties on MOET success remain contradictory. The objective of this study was to determine the effects of embryo developmental stage and quality on embryo viability following transfer to recipient ewes. Data including details of 377 embryos collected from 45 Merino donor ewes were obtained from MOET trials conducted on three separate farms on day 6 after laparoscopic artificial insemination (AI). A total of 270 embryos were classified as being of transferrable grade (grade 1: n = 233; grade 2: n = 37). One or two transferrable grade embryos were transferred to each of 256 synchronised recipient ewes and pregnancy diagnosis was performed on day 36 after embryo transfer. Embryos at the hatched blastocyst stage tended to have greater viability in vivo compared to embryos at the late morula stage (59.0 ± 10.6% vs. 36.2 ± 9.7%; P = 0.083). The viability of grade 1 embryos was greater than that of grade 2 embryos (53.6 ± 7.8% vs. 35.9 ± 10.2%; P < 0.05). The results suggest that the success of the MOET trials was influenced by the transfer of embryos at the late morula stage, almost half of which were classified as grade 2 embryos. These findings highlight the importance of following strict embryo quality grading criteria to inform the most economical management of recipient ewes and maximize pregnancy outcomes.

A successful vitrification technique for goat morulae conservation

The aim of this study was to evaluate the effect of different vitrification and warming processes on the in vitro embryo survival of caprine morulae, considering the day of recovery. A total of 136 morulae of Criolla-Neuquina goats recovered on Days 7 or 8 after sponge removal, were exposed to three different vitrification processes; V1 (n = 48): glycerol (G) + ethylene glycol (EG); V2 (n = 44): EG + 0.5 M sucrose and V3 (n = 44): G + EG + 0.5 M sucrose. The morulae of each vitrification process were randomly assigned to three warming processes; W1 (n = 45): 0.5 M sucrose at 25 °C; W2 (n = 44): 0.5 M sucrose at 39 °C; and W3 (n = 47): solution containing half the concentration of the cryoprotectants + 0.5 M sucrose at 25 °C. After, embryos were cultured in 100 μL TCM 199 drops under mineral oil, at 39 °C and a 6.5% CO2 atmosphere for 72 h according to the different treatments. There were no viable embryos in V1 and V2 in none of their three respective warmings. Only V3 showed an embryo survival rate to hatched blastocyst stage of 59.1%. When considering embryo survival according to the warming processes, the survival rate was higher in V3W2 (76.9%) and V3W3 (66.7%) groups compared to the V3W1 group (37.5%; P < 0.05). The embryo survival of V3 for Day 8 after sponge removal (81.3%) was higher compared to Day 7 (46.4%; P < 0.05). In conclusion, a successful embryo survival is obtained by using a combination of cryoprotectants (G + EG) with addition of sucrose in the vitrification process for conservation of caprine morulae in embryo transfer programs. The survival rates in vitro of vitrified-warmed morulae in goats were influenced by their recovery day. Further studies should be conducted to determine if these results are reproducible in vivo embryo transfer on field situations.

Effects of oxygen tension and humidity on the preimplantation development of mouse embryos produced by in vitro fertilization: analysis using a non-humidifying incubator with time-lapse cinematography.

To examine the effects of oxygen tension and humidity on early embryonic development, the preimplantation development of mouse embryos produced by in vitro fertilization was assessed by time-lapse cinematography to evaluate morphokinetic development with higher precision. Zygotes were produced from spermatozoa and oocytes from ICR mice and cultured in KSOM under low or high oxygen tension in a non-humidified incubator with time-lapse cinematography (CCM-iBIS). The developmental rates of embryos to the 4-cell and blastocyst stages under lower oxygen tension in CCM-iBIS were significantly higher than those under higher oxygen tension in CCM-iBIS. Ninety-six hours after insemination, a large number of embryos cultured under low oxygen tension developed to the hatching blastocyst stage. Embryonic development was more synchronized under lower oxygen tension. Non-humidified cultures did not affect embryonic development. On average, mouse embryos cultured at lower oxygen tension reached 2-cell at 18 h, 3-cell at 39 h, 4-cell at 40 h, initiation of compaction at 58 h, morula at 69 h, and blastocyst at 82 h after insemination. In conclusion, lower oxygen tension better supports preimplantation development of mouse embryos fertilized in vitro, and non-humidified culture conditions do not influence the embryonic development in vitro.

Transcriptome dynamics in early in vivo developing and in vitro produced porcine embryos

BackgroundThe transcriptional changes around the time of embryonic genome activation in pre-implantation embryos indicate that this process is highly dynamic. In vitro produced porcine blastocysts are known to be less competent than in vivo developed blastocysts. To understand the conditions that compromise developmental competence of in vitro embryos, it is crucial to evaluate the transcriptional profile of porcine embryos during pre-implantation stages. In this study, we investigated the transcriptome dynamics in in vivo developed and in vitro produced 4-cell embryos, morulae and hatched blastocysts.ResultsIn vivo developed and in vitro produced embryos displayed largely similar transcriptome profiles during development. Enriched canonical pathways from the 4-cell to the morula transition that were shared between in vivo developed and in vitro produced embryos included oxidative phosphorylation and EIF2 signaling. The shared canonical pathways from the morula to the hatched blastocyst transition were 14–3-3-mediated signaling, xenobiotic metabolism general signaling pathway, and NRF2-mediated oxidative stress response. The in vivo developed and in vitro produced hatched blastocysts further were compared to identify molecular signaling pathways indicative of lower developmental competence of in vitro produced hatched blastocysts. A higher metabolic rate and expression of the arginine transporter SLC7A1 were found in in vitro produced hatched blastocysts.ConclusionsOur findings suggest that embryos with compromised developmental potential are arrested at an early stage of development, while embryos developing to the hatched blastocyst stage display largely similar transcriptome profiles, irrespective of the embryo source. The hatched blastocysts derived from the in vitro fertilization-pipeline showed an enrichment in molecular signaling pathways associated with lower developmental competence, compared to the in vivo developed embryos.

Balanced Notch-Wnt signaling interplay is required for mouse embryo and fetal development

This study investigated the role of Notch and Wnt cell signaling interplay in the mouse early embryo, and its effects on fetal development. Developmental kinetics was evaluated in embryos in vitro cultured from the 8-16-cell to the hatched blastocyst stage in the presence of signaling inhibitors of Notch (DAPT) and/or Wnt (DKK1). An embryo subset was evaluated for differential cell count and gene transcription of Notch (receptors Notch1-4, ligands Dll1, Dll4, Jagged1-2, effectors Hes1-2) and Wnt (Wnt3a, Lrp6, Gsk3β, C-myc, Tcf4, β-catenin) components, E-cadherin and pluripotency and differentiation markers (Sox2, Oct4, Klf4, Cdx2), whereas a second subset was evaluated for implantation ability and development to term following transfer into recipients. Notch and Wnt blockades had significant opposing effects on developmental kinetics - Notch blockade retarded while Wnt blockade fastened development. This evidences that Notch and Wnt regulate the pace of embryo kinetics by respectively speeding and braking development. Blockades significantly changed the transcription profile of Sox2, Oct4, Klf4 and Cdx2, and Notch and double blockades significantly changed embryonic cell numbers and cell ratio. The double blockade induced more severe phenotypes than those expected from the cumulative effects of single blockades. Implantation ability was unaffected, but Notch and double blockades significantly decreased fetal development to term. Compared to control embryos, Notch blockade and Wnt blockade embryos originated, respectively, significantly lighter and heavier fetuses. In conclusion, Notch and Wnt signaling interplay in the regulation of the pace of early embryo kinetics, and their actions at this stage have significant carry-over effects on later fetal development to term.

Laser assisted hatching of human embryos at fertilization check, on day 1 of culture, allows for continuous uninterrupted time-lapse incubation of 5+ days, while producing hatching blastocysts as expected

Objective Human embryos must fully hatch from the Zona Pellucida (ZP) for implantation. Furthermore, studies show that implantation rates increase when the embryo has low energy expenditure. Extended culture of embryos to the blastocyst stage does not allow the natural process of hatching to occur, thus necessitating assisted hatching (AH) to breach the ZP for optimal embryo development. This AH is generally performed on day 3 of culture, however this necessitates interrupting any continuous culture of embryos during incubation from day 1 to 7, especially during Time-Lapse. We set out to determine if human blastocysts possess the ability to reach the hatching stage during 5+ days of continuous culture after laser AH is performed two days earlier than standard; at the Day 1 fertilization check. Design A retrospective data analysis at a single large private fertility center to analyze blastocyst laser hatching performed on Day 1 when embryos undergo 5+ days of continuous culture in a Time-Lapse incubator. Materials and methods Oocytes were retrieved and inseminated using standard clinical practice protocols. 16-18 hours post insemination, the day 1 fertilization check was performed. Embryos with abnormal fertilization (3PN and MPN) were donated for research and underwent AH using a Hamilton Thorne Lykos Laser at a Pulse setting of 290µs per our protocol. The laser hatched embryos were placed into a Time-Lapse Embryoscope in order to monitor their ability to form blastocysts and to undergo hatching. Results A total of 41 abnormally fertilized embryos were cultured for 5+ days, with only 12 embryos undergoing blastulation, which is an expected rate for abnormal embryo development. All 12 embryos that formed blastocysts, hatched successfully (100%). Initial hatching was observed as early as 95 hours post insemination (3.96 days of culture) and the latest observed at 140 hours (5.83 days) with a mean time to start hatching of 112.31 hours (4.68 days). Conclusions This study demonstrates that laser AH performed on day 1 at the fertilization check can produce hatching blastocysts at the expected rate. This allows practices to place embryos into Time-Lapse incubation with completely uninterrupted continuous culture for 5+ days, until the desired hatching blastocyst stage has been achieved. Disclosure None Funding None

Establishment of day 7 blastocyst freezing criteria using blastocyst diameter for single vitrified-warmed blastocyst transfer from live birth outcomes: a single-center, large cohort, retrospectively matched study.

To establish blastocyst freezing criteria for day 7 blastocyst (day 7 BL) for single vitrified-warmed blastocyst transfer (SVBT) by examining the diameter of blastocysts. Patients who underwent day 7 BL transfer cycles (1143 cycles, mean age: 38.5 ± 3.5) and randomly selected patients after 1:1 matching who underwent day 6 BL transfer cycles and day 2-single-embryo transfer (SET) cycles were used for analysis. Comparison of the miscarriage (per clinical pregnancy) and live birth rates were made among day 2-SET, day 7 BL, and day 6 BL. These blastocyst groups were stratified into six groups based on blastocyst diameter, namely, 180 μm, 190 μm, 200 μm, 210 μm, over 220 μm, and hatched, for making the freezing criteria. For each diameter, 180 μm, 190 μm, 200 μm, 210 μm, over 220 μm, and hatched, the live birth rates of day 7 BL after SVBT were 9.0%, 11.9%, 11.5%, 15.6%, 20.0%, and 19.9%, respectively. Compared with the 14.6% live birth rate of the day 2-SET group, the live birth rate of 220 μm day 7 BL was significantly higher (P < 0.05) and was around the same in other diameter groups. Our study demonstrates that sufficient live birth rates can be obtained after SVBT even from blastocysts on day 7 when blastocysts were vitrified at expanded blastocyst stage of over 180 μm of diameter or at hatched blastocyst stage and were transferred at the optimal time. This is the first study to establish a day 7 blastocyst freezing criteria using blastocyst diameter, which is an objective assessment way.

Derivation of oocyte-like cells from putative embryonic stem cells and parthenogenetically activated into blastocysts in goat

Germ cells are responsible for the propagation of live animals from generation to generation, but to surprise, a steep increase in infertile problems among livestock poses great threat for economic development of human race. An alternative and robust approach is essential to combat these ailments. Here, we demonstrate that goat putative embryonic stem cells (ESCs) were successfully in vitro differentiated into primordial germ cells and oocyte-like cells using bone morphogenetic protein-4 (BMP-4) and trans-retinoic acid (RA). Oocyte-like cells having distinct zonapellucida recruited adjacent somatic cells in differentiating culture to form cumulus-oocyte complexes (COCs). The putative COCs were found to express the zonapellucida specific (ZP1 and ZP2) and oocyte-specific markers. Primordial germ cell-specific markers VASA, DAZL, STELLA, and PUM1 were detected at protein and mRNA level. In addition to that, the surface architecture of these putative COCs was thoroughly visualized by the scanning electron microscope. The putative COCs were further parthenogenetically activated to develop into healthy morula, blastocysts and hatched blastocyst stage like embryos. Our findings may contribute to the fundamental understanding of mammalian germ cell biology and may provide clinical insights regarding infertility ailments.

Efficiency of Dimethyl Sulphoxide and Ethylene Glycol on Subsequent Development of Vitrified Awassi Sheep Embryos

The use of cryoprotectants in vitrification would reduce the critical damages to the embryos, thus increase the survival rates. This research was conducted in the laboratory of reproductive biotechnology at the faculty of Agriculture of Aleppo University. The study aimed to evaluate the viability and survivability of early Syrian Awassi embryos under the influence of dimethyl sulphoxide (DMSO) and ethylene glycol (EG) following vitrification. Embryos were vitrified in three solutions of cryoprotectants (A: DMSO (3 ml), B: EG (3 ml), and C which was composed of a combination of DMSO (1.5 ml) and EG (1.5 ml)). After thawing, embryos that had been vitrified in C solution achieved the highest rates of cleavage (P&amp;lt; 0.01) comparing with A and B solutions for 2-16 cell stage (50.00% Vs 30.77% and 36.36%), morula (9.00% Vs 44.44% and 40.00%) and blastocyst stage embryos (92.86% Vs 58.33% and 50.00%) respectively. Down to the hatching blastocyst stage, 2-16 cell stage vitrified embryos in C solution achieved an encouraging rate comparing with A and B solutions (39.20% Vs23.08% and 22.73% respectively). The rates of arrested embryos decreased significantly (P&amp;lt; 0.05) after thawing across the three solutions especially the morula and blastocyst stage (0.00 and 3.70% respectively) (C solution). No significant differences were observed in the three types of embryos across all stages and solutions despite the large range among these rates. Given the apparent benefit of the participatory effect of cytoprotectants, it is advised to use a mixture of DMSO and EG (1:1) in vitrification of ovine embryos.

Experimental Evidence Reveals Both Cross-Infection and Cross-Contamination Risk of Embryo Storage in Liquid Nitrogen Biobanks.

Simple SummaryThis study was conducted to demonstrate the potential hazards of cross-infection and cross-contamination of embryos during storage in liquid nitrogen biobanks. For the harmless and successful cryopreservation of embryos, the vitrification method must be chosen meticulously to guarantee not only a high post-thaw survival of embryos, but also to reduce the risk of disease transmission when those embryos are in storage for long periods.In recent decades, gamete and embryo cryopreservation have become routine procedures in livestock and human assisted reproduction. However, the safe storage of germplasm and the prevention of disease transmission continue to be potential hazards of disease transmission through embryo transfer. This study aimed to demonstrate the potential risk of cross-infection of embryos from contaminated liquid nitrogen, and cross-contamination of sterile liquid nitrogen from infected embryos in naked and closed devices. Additionally, we examined the effects of antibiotic-free media on culture development of infected embryos. The study was a laboratory-based analysis using rabbit as a model. Two experiments were performed to evaluate both cross-infection (liquid nitrogen to embryos) and cross-contamination (embryos to liquid nitrogen) of artificially inoculated Salmonella Typhimurium, Staphylococcus aureus, Enterobacter aerogenes, and Aspergillus brasiliensis. Rapid cooling through vitrification was conducted on rabbit embryos, stored for a year, thawed, and cultured. In vivo produced late morulae–early blastocyst stages (72 h) embryos were used (n = 480). Embryos were cultured for 1 h in solutions with and without pathogens. Then, the embryos were vitrified and stored in naked and closed devices for one year in two liquid nitrogen biobanks (one pathogen-free and the other artificially contaminated). Embryos were warmed and cultured for a further 48 h, assessing the development and the presence of microorganism (chromogenic media, scanning electron microscopy). Embryos stored in naked devices in artificially contaminated liquid nitrogen became infected (12.5%), while none of the embryos stored in closed devices were infected. Meanwhile, storage of artificially infected embryos incurred liquid nitrogen biobank contamination (100%). Observations by scanning electron microscopy revealed that all the microorganisms were caught in the surface of embryos after the vitrification-thawed procedure. Nevertheless, embryos cultured in antibiotics and antimycotic medium developed to the hatched blastocyst stage, while artificially infected embryos cultured in antibiotic-free medium failed to develop. In conclusion, our findings support that both cross-contamination and cross-infection during embryo storage in liquid nitrogen biobanks are plausible. So, to ensure biosafety for the cryogenic storage, closed systems that avoid direct contact with liquid nitrogen must be used. Moreover, it seems essential to provide best practice guidelines for the cryogenic preservation and storage of gametes and embryos, to define appropriate quality and risk management procedures.

Selected Amino Acids Promote Mouse Pre-implantation Embryo Development in a Growth Factor-Like Manner.

Groups of amino acids, and some selected amino acids, added to media used for culture of pre-implantation embryos have previously been shown to improve development in various ways including survival to the blastocyst stage, increased blastocyst cell number and improved hatching. In this study, we cultured 1-cell mouse embryos for 5 days to the hatching blastocyst stage in isosmotic medium (270 mOsm/kg) at high density (10 embryos/10 μL), where autocrine/paracrine support of development occurs, and low density (1 embryo/100 μL), where autocrine/paracrine support is minimized and development is compromised. When 400 μM L-Pro or 1 mM L-Gln was added to embryos at low density, the percentage of embryos reaching the blastocyst stage and the percentage hatching increased compared to low-density culture without these amino acids, and were now similar to those for embryos cultured at high density without amino acids. When L-Pro or L-Gln was added to embryos at high density, the percentage of embryos reaching the blastocyst stage didn’t change but hatching improved. Neither embryo culture density nor the presence of these amino acids had any effect on blastocyst cell number. D-Pro and the osmolytes Gly and Betaine did not improve embryo development in low- or high-density culture indicating the mechanism was stereospecific and not osmotic, respectively. L-Pro- and L-Gln-mediated improvement in development is observed from the 5-cell stage and persists to the blastocyst stage. Molar excess of Gly, Betaine or L-Leu over L-Pro eliminated improvement in development and hatching consistent with them acting as competitive inhibitors of transporter-mediated uptake across the plasma membrane. The L-Pro effect is dependent on mTORC1 signaling (rapamycin sensitive) while that for L-Gln is not. The addition of L-Pro leads to significant nuclear translocation of p-AktS473 at the 2- and 4-cell stages and of p-ERK1/2T202/Y204 nuclear translocation at the 2-, 4-, and 8-cell stages. L-Pro improvement in embryo development involves mechanisms analogous to those seen with Pro-mediated differentiation of mouse ES cells, which is also stereoselective, dependent on transporter uptake, and activates Akt, ERK, and mTORC1 signaling pathways.

Dietary Selenium Supplementation Ameliorates Female Reproductive Efficiency in Aging Mice.

Female reproductive (ovarian) aging is distinctively characterized by a markedly reduced reproductive function due to a remarkable decline in quality and quantity of follicles and oocytes. Selenium (Se) has been implicated in playing many important biological roles in male fertility and reproduction; however, its potential roles in female reproduction, particularly in aging subjects, remain poorly elucidated. Therefore, in the current study we used a murine model of female reproductive aging and elucidated how different Se-levels might affect the reproductive efficiency in aging females. Our results showed that at the end of an 8-week dietary trial, whole-blood Se concentration and blood total antioxidant capacity (TAOC) were significantly reduced in Se-deficient (0.08 mg Se/kg; Se-D) mice, whereas both of these biomarkers were significantly higher in inorganic (0.33 mg/kg; ISe-S) and organic (0.33 mg/kg; OSe-S) Se-supplemented groups. Similarly, compared to the Se-D group, Se supplementation significantly ameliorated the maintenance of follicles and reduced the rate of apoptosis in ovaries. Meanwhile, the rate of in vitro-produced embryos resulting from germinal vesicle (GV) oocytes was also significantly improved in Se-supplemented (ISe-S and OSe-S) groups compared to the Se-D mice, in which none of the embryos developed to the hatched blastocyst stage. RT-qPCR results revealed that mRNA expression of Gpx1, Gpx3, Gpx4, Selenof, p21, and Bcl-2 genes in ovaries of aging mice was differentially modulated by dietary Se levels. A considerably higher mRNA expression of Gpx1, Gpx3, Gpx4, and Selenof was observed in Se-supplemented groups compared to the Se-D group. Similarly, mRNA expression of Bcl-2 and p21 was significantly lower in Se-supplemented groups. Immunohistochemical assay also revealed a significantly higher expression of GPX4 in Se-supplemented mice. Our results reasonably indicate that Se deficiency (or marginal levels) can negatively impact the fertility and reproduction in females, particularly those of an advancing age, and that the Se supplementation (inorganic and organic) can substantiate ovarian function and overall reproductive efficiency in aging females.

9 Pregnancy loss in Holstein lactating recipient cows diagnosed pregnant by pregnancy-associated glycoprotein test in blood

The objective of this study was to evaluate the pregnancy loss (PL) between Day 30 (P30) and Day 80 (P80) of pregnancy in lactating Holstein recipients that received an invivo- (flush) or invitro-produced (IVF) embryo. The recipient cows were located at Maddox Dairy in Riverdale, CA, USA, a Holstein herd that milks 3500 cows with a 305-day mature-equivalent milk production of 12 800 kg. First-lactation cows were enrolled in a Presynch-Ovsynch oestrus-synchronization program and scheduled to be artificially bred for the first time at 80 days after calving or to receive an embryo 7 or 8 days after the expected heat (recipients). The data from 590 pregnancies (1045 embryo transfers) from embryos transferred between January 2018 and March 2019 was analysed. Only grade 1 embryos (from morula to hatched blastocyst stage) produced invivo or invitro from Holstein donor heifers, lactating or dry cows, were transferred fresh (invivo or invitro) or frozen-thawed (invivo), and pregnancy rates are shown in Table 1. The ruminant trophoblast produces pregnancy-associated glycoproteins (PAG) that can be detected by enzyme-linked immunoassay (ELISA) in the blood of pregnant cattle as early as 28 days after insemination. Various dairy herds in the USA have been using this test to supplement or replace the use of transrectal ultrasonography for early pregnancy diagnosis. Blood was sampled on P30 after expected heat day (23 days after embryo transfer) from the recipient cows and sent to IDEXX for the PAG Bovine Pregnancy Test, which was reconfirmed on P80 of pregnancy by transrectal ultrasonography. Pregnancy loss was considered to have occurred when a cow was pregnant on P30 but not pregnant on P80. The variable PL was analysed by binary logistic regression in the MINITAB program, and the model included effects of donor status (heifer vs. milk vs. dry) and embryo type. The total PL was 12.2% (72/590) and the details are shown in Table 1. No effect of donor status (P=0.80) was detected. However, there was effect of embryo type (P=0.004). The IVF embryos had a PL of 18.0% compared with 9.5% for the invivo-produced embryos. Further research should be performed to study heifer embryos PL, because currently more embryos are being produced from very young donor cows and sires due to intensive use of genomic testing. In conclusion, there is a higher PL in lactating dairy recipients receiving IVF fresh embryos compared with fresh or frozen invivo-produced embryos. Table 1.Embryo transfer (ET) pregnancy rates (PR, P30) in first-lactation Holstein cows (top) and pregnancy loss (PL) from Day 30 (P30) to 80 (P80) in first-lactation recipient Holstein cows (bottom) Item Heifer donor Lactating donor Dry donor Total ET P30 PR% ET P30 PR% ET P30 PR% ET P30 PR% Invivo - fresh 6 3 50.0 43 25 58.1 329 206 62.6 378 234 61.9 Invivo - frozen 75 38 50.7 221 129 58.4 296 167 56.4 IVF - fresh 123 61 49.6 91 42 46.2 157 86 54.8 371 189 50.9 Total 129 64 49.6 209 105 50.2 707 421 59.5 1045 590 56.5 P30 P80 PL% P30 P80 PL% P30 P80 PL% P30 P80 PL% Invivo - fresh 3 3 0.0 25 24 4.0 206 186 9.7 234 213 9.0 Invivo - frozen 38 35 7.9 129 115 10.9 167 150 10.2 IVF - fresh 61 48 21.3 42 35 16.7 86 72 16.3 189 155 18.0 Total 64 51 20.3 105 94 10.5 421 373 11.4 590 518 12.2

PREGNANCY RATE OF RECIPIENT COWS AFTER TRANSFER OF IN VITRO-PRODUCED NELLORE EMBRYOS

ABSTRACT The objective of this study was to evaluate the pregnancy rate (PR) of cows of six farms in Bolivia and in the states of Acre and Rondônia in Brazil after the transfer of 4,321 in vitro Nellore embryos produced by a private company, in 2015 to 2016. The effects of the farm location, year of embryo transfer (ET), season of ET, number of previous ET per recipient, and embryo development stage on the PR of the recipient cow (chi square p&lt;0.05) were evaluated. The PR of the six evaluated farms were similar, with an average of 45.15%. The farm location, year of ET (2015 or 2016), and season of ET (rainy or dry season) did not affect the PR (p&gt;0.05). The PR found after embryo transfer for recipient cows that had already been used once (45.58%) or twice (43.40%) for this purpose were higher than that of recipient cows used three to eight times (29.01%) (p&lt;0.05). ET at expanded or hatched blastocyst stage resulted in higher PR (47.35%) when compared to the ET at morula or initial blastocyst (41.06%) and at blastocyst stage (43%) (p&lt;0.05), which did not differ from each other (p&gt;0.05). Under the conditions of this study, the transfer of in vitro-produced Nellore embryos at advanced development stages (expanded or hatched blastocyst) results in higher PR in cows that had been used once or twice for this purpose.

Does culture of embryos in an ultra-low (2%) oxygen environment yield better blastocyst development than 6% oxygen using time-lapse morphokinetics?

Objective To evaluate if culture of mouse embryos in an ultra-low oxygen environment could enhance blastocyst development in terms of time-lapse morphokinetics. Design Prospective study Materials and methods A total of 214 commercially obtained frozen mouse embryos (B6D2F1 & B6C3F1 hybrid) were thawed and cultured in One-Step medium with 10%SPS using an EmbryoScope time lapse incubator at 37°C, 5.5% CO2, and either (1) 6.0% O2 (n=106) or (2) 2.0% O2 (n=108) Embryo images were recorded every 10 minutes for 6 days of culture. Time lapse videos were annotated for the following time points: 2cell (t2), 3cell (t3), 4cell (t4), 5cell (t5), 6cell (t6), 7cell (t7), 8cell (t8), start of compaction (tSC), morula (tsM), start of blastulation (tSB), blastocyst (tB), expanded blastocyst (tEB) and hatching blastocyst (tHB). The 2-cell stage was considered as time zero since the exact time of insemination was unknown. Time points were statistically compared between the two groups. Blastocyst development rates for both culture environments were also compared. Results There were no statistically significant differences between the two groups in any of the time points measured up to the 8-cell stage. However, after the 8-cell stage, the 2% O2 group showed significantly slower embryo development for each time point up to the hatching blastocyst stage. There was no difference in the blastocyst development rate between the 6% O2 and 2% O2 environments (99.1% vs 95.4%). Conclusions Our data did not show any benefit of culturing embryos in a 2% oxygen environment. Although there was no difference in the blastocyst development rate between culturing in 2% and 6% oxygen, the 2% oxygen group took significantly longer to reach the blastocyst stage than the 6% oxygen group. Disclosures Nothing to disclose Funding None

Prostaglandin F2\u03b1 (PGF2\u03b1) production possibility and its receptors expression in the early- and late-cleaved preimplantation bovine embryos

BackgroundProstaglandin F2α (PGF2α) is an important component for the physiology of female reproductive processes. In the literature, the data pertaining to the synthesis and action of PGF2α in early embryonic bovine development are limited. In our study, we used the bovine in vitro culture model based on the time of first cleavage to determine the mRNA expression and immunolocalization of PGF2α synthase and its receptor in bovine embryos from the 2-cell stage to the hatched blastocyst stage. We also evaluated PGF2α production at 2, 5 and 7 days of in vitro culture.ResultsWe found a significantly higher proportion of blastocysts obtained from the early-cleaved embryos than from the late-cleaved embryos (37.7% vs. 26.1% respectively, P < 0.05). The PGFS mRNA expression was significantly higher in the late-cleaved group than in the early-cleaved group at the 2-, 4- and 16-cell stages (P < 0.05). For PTGFR, we observed that within the late-cleaved group, the mRNA abundance was significantly higher in embryos at the 2- and 16-cell stages than in embryos at the 4- and 8-cell stages (P < 0.05). We observed that PTGFR mRNA expression was significantly higher in the 2- and 16-cell embryos in the late-cleaved group than that in the early-cleaved group embryos (P < 0.05). Among the blastocysts, the PGFS and PTGFR expression levels showed a trend towards higher mRNA expression in the late-cleaved group than in the early-cleaved group. Analysis of PGF2α production showed that within the early-cleaved group, the content of PGF2α in the in vitro culture medium was significantly higher on day 7 than it was on day 2 (P < 0.05).ConclusionsThe mRNA expression levels of PGF2α synthase and its receptor depend on the developmental stage and the embryo quality. Analyses of PGFS and PTGFR expression in bovine blastocysts and of PGF2α embryo production suggest that prostaglandin F2α can act in an autocrine and paracrine manner in bovine in vitro-produced preimplantation embryos. Moreover, the tendency of PTGFR and PGFS mRNA expression to be upregulated in embryos with low developmental potential can indicate a compensation mechanism related to high PGFS and PTGFR mRNA expression levels in low-quality embryos.

Expression of enzymes involved in the synthesis of prostaglandin E2 in early- and late-cleaved bovine embryos at different stages of preimplantation development

Prostaglandin (PG) E2 plays a role in numerous aspects of mammalian reproduction, such as oviductal transport of gametes, hatching from the zona pellucida in blastocysts and early embryonic development. Despite the evident role of PGE2 in the regulation of female reproductive processes, in the literature, there is very little information concerning the expression of PGE2 synthesizing enzymes and the exact amount of PGE2 produced by bovine embryos in vitro. In the present study, we aimed to determine the mRNA levels and immunolocalization of the enzymes responsible for PGE2 synthesis (PTGS2, mPGES1, mPGES2 and cPGES) in embryos at the 2-cell, 4-cell, 8-cell, 16-cell, morula, early blastocyst, blastocyst, expanded blastocyst and hatched blastocyst stages, using a well-defined bovine model of oocyte developmental competence based on the time of first cleavage. PTGS2, mPGES2 and cPGES transcripts and proteins were detected in all stages of embryos, whereas the mPGES1 transcript and protein were not detected in embryos from the 2- to 16-cell stage. The results showed different transcription profiles of the enzymes involved in PGE2 synthesis in early- and late-cleaved embryos during the early stages of their in vitro preimplantation development. We also found that all the analysed stages of bovine preimplantation embryos released PGE2, with the highest concentration on Day 7 of culture in both the early- and late-cleaved groups. The present study is the first to demonstrate PGE2 synthesis and production by bovine early- and late-cleaved embryos at different stages of preimplantation development. Bovine embryos can produce PGE2, which may exert paracrine regulation during development. The transcription levels of PGE2 synthases were affected by the embryonic stage of development and quality. Our results indicate that the different transcription profiles of PTGS2, mPGES1, mPGES2 and cPGES, as well as PGE2 concentration, in early-versus late-cleaved embryos are dependent on the quality of the oocytes from which the embryos were obtained, which could reveal the association of PGE2 production during bovine preimplantation development with more advanced stages of embryo development.