Subsequent Embryo Development Research Articles

Recent advances in the study of the structure and function of the epididymis

Testicular sperm maturation is critical for establishing male fertility. Spermatozoa undergo remodeling of sperm proteins and changes in lipid and ribonucleic acid composition during transport in the epididymal ducts, which play an important role in sperm maturation. The anatomy, epithelial cell types, physiological functions, and epigenetic inheritance of the epididymis are explored, and recent findings in epididymal research are analyzed. Suggesting possible directions for future research on the epididymis. Using the keywords “epididymis”, “sperm”, and “sperm maturation”, a search of the epididymis was performed through databases and official websites of journals related to reproduction. The epididymis was searched in databases and on the official websites of journals related to reproduction. This review introduces the characteristics of the epididymis, as well as the biological functions of cell types such as principal cells, clear cells, and basal cells, providing a detailed description of the overall physiological functions of the epididymis. It highlights current research hotspots in the field of epididymis, including single-cell analysis, epigenetics, and extracellular vesicles, aiming to offer a comprehensive understanding for beginners. The review emphasizes the importance of the epididymis, its impact on sperm maturation and subsequent embryo development, and how it advances research on epididymal diseases while providing new directions for the study and treatment strategies of infertility.

The effects of photobiomodulation on the improvement of sperm parameters: A review study.

The prevalence of male infertility has become a significant clinical concern worldwide, with a noticeable upward trend in recent times. The rates of fertilization and subsequent development of embryos are dependent on many parameters associated with the quality and viability of sperm. Photobiomodulation (PBM) is a promising approach with a great potential for translational applications in the treatment of spermatozoa exhibiting low quality and motility. In this study, a comprehensive analysis of the existing literature, specifically examining the mechanisms of action of PBM has been presented. Our objective was to enhance knowledge in the field of laser light therapy in order to promote the usage of irradiation in clinical settings in a more effective way. Within the realm of reproductive science, the utilization of PBM has been employed to enhance the metabolic processes, motility, and viability of spermatozoa. This is attributed to its advantageous effects on mitochondria, resulting in the activation of the mitochondrial respiratory chain and subsequent synthesis of ATP. This therapeutic approach can be highly advantageous in circumventing the reliance on chemical substances within the culture medium for spermatozoa while also facilitating the viability and motility of spermatozoa, particularly in circumstances involving thawing or samples with significant immotility.

Cat presumptive zygotes assessment in relation to their development

Abstract The evaluation of oocytes and zygotes, based on their size, shape and morphology, is a valuable tool for predicting subsequent embryo development. While this assessment is non-invasive and made possible with time-lapse monitoring systems, not all the assessment criteria used for zygotes with pale cytoplasm can be used for domestic cat zygotes, which have dark cytoplasm. In this study, feline presumptive zygotes were evaluated for shape, size, and morphology. Measurements were also made of the diameter of the entire zygote, its cytoplasm, and the zona pellucida. Differences in the dataset were assessed using the generalized linear model (GLM) procedure. While there was no relationship between a combination of the tested parameters with the potential for cleavage, blastocyst development, and hatching, the parameters of the shape and size of the entire oocyte, and of the zona pellucida, were related to the development potential. The results presented in this study indicate that the assessment procedure for human zygotes has to be adjusted to be used in the cat model, however the relationship between measurements of the diameter of presumptive feline zygotes and the thickness of zona pellucida with their developmental potential deserves further investigation to optimize assessment of cat presumptive zygotes.

Fibronectin 1 supports oocyte in vitro maturation in pigs

Oocyte in vitro maturation (IVM) based on the follicular fluid (FF) environment can exploit untapped resources, however, what FF factors regulate oocyte maturation remains unclear. This work demonstrated that serum and FF significantly promoted oocyte polar body extrusion (PBE) and subsequent embryo development, and FF was especially effective. Fibronectin 1 (FN1) was predicted as one potential candidate to regulate oocyte maturation by proteomics. FN1 transcription obviously decreased, and the protein expression significantly increased and migrated to plasma membrane or even outside during oocyte IVM. Treatment with 10 ng/mL FN1 significantly improved oocyte PBE rate. FN1 significantly upregulated the percentage of regular spindle morphology, downregulated the γ-H2AX level, decreased the levels of ROS and apoptosis, and increased GSH and mitochondrion contents by ameliorating the expression of corresponding genes. Moreover, FN1 significantly increased the p-PI3K level to enhance the activation of PI3K signaling pathway. In conclusion, this study discovers and confirms that FN1 is one factor in FF that significantly enhances oocyte maturation, and the underlying mechanism is that FN1 ameliorates oocyte nuclear and cytoplasmic maturation by promoting the activation of PI3K signaling pathway.

Transcriptomic responses of cumulus granulosa cells to SARS-CoV-2 infection during controlled ovarian stimulation.

Cumulus granulosa cells (CGCs) play a crucial role in follicular development, but so far, no research has explored the impact of SARS-CoV-2 infection on ovarian function from the perspective of CGCs. In the present study, we compared the cycle outcomes between infected and uninfected female patients undergoing controlled ovarian stimulation, performed bulk RNA-sequencing of collected CGCs, and used bioinformatic methods to explore transcriptomic changes. The results showed that women with SARS-CoV-2 infection during stimulation had significantly lower number of oocytes retrieved and follicle-oocyte index, while subsequent fertilization and embryo development were similar. CGCs were not directly infected by SARS-CoV-2, but exhibited dramatic differences in gene expression (156 up-regulated and 65 down-regulated). Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses demonstrated a high enrichment in antiviral, immune and inflammatory responses with necroptosis. In addition, the pathways related to telomere organization and double strand break repair were significantly affected by infection in gene set enrichment analysis. Further weighted gene co-expression network analysis identified a key module associated with ovarian response traits, which was mainly enriched as a decrease of leukocyte chemotaxis and migration in CGCs. For the first time, our study describes how SARS-CoV-2 infection indirectly affects CGCs at the transcriptional level, which may impair oocyte-CGC crosstalk and consequently lead to poor ovarian response during fertility treatment.

Embryo development is impaired by sperm mitochondrial-derived ROS

BackgroundBasal energetic metabolism in sperm, particularly oxidative phosphorylation, is known to condition not only their oocyte fertilising ability, but also the subsequent embryo development. While the molecular pathways underlying these events still need to be elucidated, reactive oxygen species (ROS) could have a relevant role. We, therefore, aimed to describe the mechanisms through which mitochondrial activity can influence the first stages of embryo development.ResultsWe first show that embryo development is tightly influenced by both intracellular ROS and mitochondrial activity. In addition, we depict that the inhibition of mitochondrial activity dramatically decreases intracellular ROS levels. Finally, we also demonstrate that the inhibition of mitochondrial respiration positively influences sperm DNA integrity, most likely because of the depletion of intracellular ROS formation.ConclusionCollectively, the data presented in this work reveals that impairment of early embryo development may result from the accumulation of sperm DNA damage caused by mitochondrial-derived ROS.

Pivotal role for long noncoding RNAs in zygotic genome activation in mice.

Vertebrate life begins with fertilization, and then the zygote genome is activated after transient silencing, a process termed zygotic genome activation (ZGA). Despite its fundamental role in totipotency and the initiation of life, the precise mechanism underlying ZGA initiation remains unclear. The existence of minor ZGA implies the possible critical role of noncoding RNAs in the initiation of ZGA. Here, we delineate the expression profile of long noncoding RNAs (lncRNAs) in early mouse embryonic development and elucidate their critical role in minor ZGA. Compared with protein-coding genes (PCGs), lncRNAs exhibit a stronger correlation with minor ZGA. Distinct H3K9me3 profiles can be observed between lncRNA genes and PCGs, and the enrichment of H3K9me3 before ZGA might explain the suspended expression of major ZGA-related PCGs despite possessing PolII pre-configuration. Furthermore, we identified the presence of PolII-enriched MuERV-L around the transcriptional start site of minor ZGA-related lncRNAs, and these repeats are responsible for the activation of minor ZGA-related lncRNAs and subsequent embryo development. Our study suggests that MuERV-L mediates minor ZGA lncRNA activation as a critical driver between epigenetic reprogramming triggered by fertilization and the embryo developmental program, thus providing clues for understanding the regulatory mechanism of totipotency and establishing bona fide totipotent stem cells.

Chrysoeriol Improves the Early Development Potential of Porcine Oocytes by Maintaining Lipid Homeostasis and Improving Mitochondrial Function.

Our previous study established that chrysoeriol (CHE) can reduce reactive oxygen species (ROS) accumulation, apoptosis, and autophagy in vitro culture (IVC) of porcine embryos. However, the role of CHE in oocyte maturation and lipid homeostasis is unclear. Herein, we aimed to elucidate the effect of CHE on porcine oocyte competence in vitro maturation (IVM) and subsequent embryo development. The study chooses parthenogenetic activated porcine oocytes as the research model. The study revealed that the cumulus expansion index and related gene expressions are significantly elevated after supplementing 1 μM CHE. Although there were no significant differences in nuclear maturation and cleavage rates, the blastocyst formation rate and total cell numbers were significantly increased in the 1 μM CHE group. In addition, CHE improved the expression of genes related to oocyte and embryo development. ROS was significantly downregulated in all CHE treatment groups, and intracellular GSH (glutathione) was significantly upregulated in 0.01, 0.1, and 1 μM CHE groups. The immunofluorescence results indicated that mitochondrial membrane potential (MMP) and lipid droplet (LD), fatty acid (FA), ATP, and functional mitochondria contents significantly increased with 1 μM CHE compared to the control. Furthermore, CHE increased the expression of genes related to lipid metabolism, mitochondrial biogenesis, and β-oxidation.

A molecular mechanism for embryonic resetting of winter memory and restoration of winter annual growth habit in wheat.

The staple food crop winter bread wheat (Triticum aestivum) acquires competence to flower in late spring after experiencing prolonged cold in temperate winter seasons, through the physiological process of vernalization. Prolonged cold exposure results in transcriptional repression of the floral repressor VERNALIZATION 2 (TaVRN2) and activates the expression of the potent floral promoter VERNALIZATION 1 (TaVRN1). Cold-induced TaVRN1 activation and TaVRN2 repression are maintained in post-cold vegetative growth and development, leading to an epigenetic 'memory of winter cold', enabling spring flowering. When and how the cold memory is reset in wheat is essentially unknown. Here we report that the cold-induced TaVRN1 activation is inherited by early embryos, but reset in subsequent embryo development, whereas TaVRN2 remains silenced through seed development, but is reactivated rapidly by light during seed germination. We further found that a chromatin reader mediates embryonic resetting of TaVRN1 and that chromatin modifications play an important role in the regulation of TaVRN1 expression and thus the floral transition, in response to developmental state and environmental cues. The findings define a two-step molecular mechanism for re-establishing vernalization requirement in common wheat, ensuring that each generation must experience winter cold to acquire competence to flower in spring.

Synergistic impact of α-linolenic acid and α-tocopherol on in vitro maturation and culture of buffalo oocytes.

The objective of the current study was to investigate the synergistic impact of α-Tocopherol and α-Linolenic acid (100 µM) on IVM and IVC of Nili Ravi buffalo oocytes. Oocytes were obtained from the ovaries of slaughtered buffaloes within two hours after slaughter and brought to laboratory. Buffalo cumulus oocyte complexes were placed randomly in the five experimental groups included; GROUP 1: Maturation media (MM) + 100 µM ALA (control), GROUP 2: MM + 100 µM ALA + 50μM α-Tocopherol, GROUP 3: MM + 100 µM ALA + 100μM α-Tocopherol, GROUP 4: MM + 100 µM ALA + 200 μM α-Tocopherol and GROUP 5: MM + 100 µM ALA + 300 μM α-Tocopherol under an atmosphere of 5% CO2 in air at 38.5 °C for 22-24 h. Cumulus expansion and nuclear maturation status was determined (Experiment 1). In experiment 2, oocytes were matured as in experiment 1. The matured oocytes were then fertilized in Tyrode's Albumin Lactate Pyruvate (TALP) medium for about 20 h and cultured in synthetic oviductal fluid (SOF) medium to determine effect of α-Linolenic acid (100 µM) and α-Tocopherol in IVM medium on IVC of presumptive zygotes. To study the effect of α-Linolenic acid (100 µM) in IVM media and increasing concentration of α-tocopherol in the culture media on early embryo development (Experiment 3), the presumptive zygotes were randomly distributed into the five experimental groups with increasing concentration of α-tocopherol in culture media. Higher percentage of MII stage oocytes in experiment 1(65.2±2.0), embryos at morula stage in experiment 2 (30.4±1.5) and experiment 3 (22.2±2.0) were obtained. However, overall results for cumulus cell expansion, maturation of oocyte to MII stage and subsequent embryo development among treatments remain statistically similar (P > 0.05). Supplementation of α-tocopherol in maturation media having α-Linolenic acid and/or in embryo culture media did not further enhance in vitro maturation of oocyte or embryo production.

In vitro development of preimplantation cattle embryos obtained from oocytes irradiated with low doses of ionizing radiation

Radiation-induced processes in germ reproductive cells, as well as the effect of low doses of radiation on the reproductive potential of farm animals remain poorly studied. Assessing the radiation effects of low doses on gametes and early embryos will reveal biological features that are important for assisted reproductive technologies in animal husbandry. In addition, the study of the processes of maturation and fertilization of irradiated gametes and the subsequent development of early preimplantation embryos will make it possible to update measures to protect the health and reproductive potential of farm animals in conditions of technogenic radiation risks. In this study, we focused on analyzing the effects of irradiation on the ovaries of cattle and examining the consequences of this exposure on embryo development under in vitro fertilization conditions. We used X-ray radiation of different dose rates with an absorbed dose of 100 mGy. The research results showed a decrease in the frequency of blastocyst formation in all experimental groups of embryos obtained from oocytes of irradiated ovaries. The data obtained can serve as a scientific basis for the development of measures to preserve the reproductive potential of farm animals under technogenic radiation risks, as well as for further search of ways to improve assisted reproductive technologies in animal husbandry.

Piperine improves the quality of porcine oocytes by reducing oxidative stress

Oxidative stress caused by light and high temperature arises during in vitro maturation (IVM), resulting in low-quality embryos compared with those obtained in vivo. To overcome this problem, we investigated the influence of piperine (PIP) treatment during maturation of porcine oocytes on subsequent embryo development in vitro. Porcine oocytes were cultured in IVM medium supplemented with 0, 50, 100, 200, or 400 μM PIP. After parthenogenetic activation, the blastocyst (BL) formation was significantly higher and the apoptosis rate was significantly lower using 200 μM PIP-treated oocytes (200 PIP). In the 200 PIP group, the level of reactive oxygen species at the metaphase II stage was decreased, accompanied by an increased level of glutathione and increased expression of antioxidant processes (Nrf2, CAT, HO-1, SOD1, and SOD2). Consistently, chromosome misalignment and aberrant spindle organization were alleviated and phosphorylated p44/42 mitogen-activated protein kinase activity was increased in the 200 PIP group. Expression of development-related (CDX2, NANOG, POU5F1, and SOX2), anti-apoptotic (BCL2L1 and BIRC5), and pro-apoptotic (BAK, FAS, and CASP3) processes was altered in the 200 PIP group. Ultimately, embryo development was improved in the 200 PIP group following somatic cell nuclear transfer. These findings suggest that PIP improves the quality of porcine oocytes by reducing oxidative stress, which inevitably arises via IVM. In-depth mechanistic studies of porcine oocytes will improve the efficiencies of assisted reproductive technologies.

Automation in vitrification and thawing of mouse oocytes and embryos.

Vitrification is a common technique for cryopreserving oocytes or embryos. However, manual vitrification is tedious and labor-intensive, and can be subject to variations caused by human factors. To address these challenges, we developed an automated vitrification-thawing system (AVTS) based on a cryo-handle. Our study firstly assessed the efficiency of cryoprotectant exchange through comparing the osmolalities of fresh and collected solutions during automated vitrification and thawing, and evaluated the cooling and warming rates of the cryo-handle. We also compared mouse oocyte survival, fertilization and embryo development after thawing and ICSI, and the development of re-frozen cleavage embryos between manual operation and automated system. The results showed that the osmolalities of collected samples were within normal range and comparable to fresh solutions. Furthermore, the automated system could obtain the reliable cooling and warming rates. Particularly, there were no significant differences in oocyte survival rates, fertilization rates, and subsequent embryo development and its quality between two procedures. Our findings suggest that AVTS has no impact on osmolalities of vitrification and thawing solutions, ensuring the proper exchange of cryoprotectants. The cryo-handle also shows the ability to achieve reliable cooling and warming rates, which benefits for the cryopreservation and thawing process. Moreover, the results from mouse oocytes and embryos indicate that automated system has effectively maintained the survival and fertilization of frozen oocytes and supported subsequent embryo development. Therefore, the automated vitrification and thawing system will inevitably represent a superior alternative to manual operation.

Effects of mycobacterium cell wall fraction on embryo development following in vitro embryo production and pregnancy rates following embryo transfer in virgin dairy heifers

An experiment was conducted to determine whether administration of mycobacterium cell wall fraction (MCWF; Amplimune, NovaVive) could enhance embryo developmental competence following in vitro embryo production (IVP) and pregnancy establishment after embryo transfer (ET). Nulliparous, Holstein heifers (n = 40; age 8–15 months) were submitted to two rounds of ovum pick-up (OPU) and IVP in a crossover design. Thirty-six h after follicle wave synchronization, treatments (saline or MCWF, 5 mL, im) were administered in conjunction with a single dose of follicle stimulating hormone (175 IU) and OPU was performed 48–52 h later. Recovered cumulus-oocyte complexes were used for IVP to assess embryo development. For ET, nulliparous, Holstein heifers (n = 225; age 12–18 months) were used as recipients. At 12–24 h after detection of spontaneous estrus, recipients were randomly treated with either saline or MCWF (5 mL, im). The effect of MCWF on pregnancy per ET (P/ET) was assessed in a 2 × 2 factorial design with recipients treated with or without MCWF receiving a fresh IVP embryo from a donor treated with or without MCWF at day 7 or 8 after detected estrus. Blood samples were collected from a subset of donors (n = 8) and recipients (n = 26 to 33 per treatment) prior to treatment and at 6 and 24 h post-treatment to determine serum concentration of interleukin (IL)-1β, IL-6, tumor necrosis factor-α, and interferon-γ. Blood samples were also collected from a group of recipients (n = 31 to 39 per treatment) to assess serum concentration of progesterone at days 4, 7, and 16 post-treatment. Pregnancy status was determined at days 40 and 100 of gestation. Donor treatment with MCWF tended (P < 0.07) to increase the proportion of oocytes that developed into transferable embryos, but there was no effect of MCWF on other parameters of embryo development. The P/ET at days 40 and 100 of gestation and pregnancy loss were not affected by donor treatment or recipient treatment with MCWF and there was no interaction. Serum concentration of proinflammatory cytokines among donors and recipients and serum concentration of progesterone among recipients were not increased by treatment with MCWF. Results of the present study indicate that treatment of donors with MCWF has minimal impact on subsequent embryo development following IVP. Moreover, regardless of whether donors or recipients were treated with MCWF, there was no effect on P/ET following transfer of IVP embryos.

Ellagic acid treatment during in vitro maturation of porcine oocytes improves development competence after parthenogenetic activation and somatic cell nuclear transfer

Ellagic acid (EA) is a natural polyphenol and a free radical scavenger with antioxidant properties. This study investigated the protective effects of EA during in vitro maturation (IVM) of porcine oocytes. To determine the optimal concentration, IVM medium was supplemented with various concentrations of EA. Treatment with 10 μM EA (10 EA) resulted in the highest cleavage rate, blastocyst formation rate, and total cell number per blastocyst and the lowest percentage of apoptotic cell in parthenogenetic blastocysts. In the 10 EA group, abnormal spindle and chromosome misalignment were rescued and the ratio of phosphorylated p44/42 to total p44/42 was increased. Furthermore, the reactive oxygen species and glutathione levels were significantly decreased and increased, respectively, and antioxidant genes (Nrf2, HO-1, CAT, and SOD1) were significantly upregulated in the 10 EA group. mRNA expression of developmental-related (CDX2, POU5F1, and SOX2) and anti-apoptotic (BCL2L1) genes was significantly upregulated in the 10 EA group, while mRNA expression of pro-apoptotic genes (BAK, FAS, and CASP3) was significantly downregulated. Ultimately, following somatic cell nuclear transfer, the blastocyst formation rate was significantly increased and the percentage of apoptotic cell in blastocysts was significantly decreased in the 10 EA group. In conclusion, addition of 10 EA to IVM medium improved oocyte maturation and the subsequent embryo development capacity through antioxidant mechanisms. These findings suggest that EA can enhance the efficiencies of assisted reproductive technologies.

PSIII-4 Effect of Photobiomodulation Treatment on Atp Concentrations in Bovine Oocytes During Maturation

Abstract Oocyte cytoplasmic maturation is critical in determining the success of subsequent embryo development. It has been proposed that light in the red-to-near infrared optical region (~600–1000 nm) enhances cellular metabolic activity through activation of the mitochondrial respiratory chain, increasing intracellular ATP concentrations, and has the potential to modify the oocyte metabolome. Previous research indicated a greater cleavage rate and similar blastocyst rates for bovine oocytes treated with photobiomodulation at hours 16 and 20 from the start of maturation compared with control. For the present study, two separate experiments were conducted to determine ATP concentration in oocytes during maturation after photobiomodulation treatment. A standardized in vitro maturation (IVM) protocol and commercial media (IVF Bioscience) were used. Briefly, bovine ovaries were obtained at a local slaughterhouse, cumulus oocyte complexes (COCs) were collected by aspiration of small (2 to 6 mm) follicles. For experiment 1, compact COCs were cultured in in vitro maturation media (n = 30/well) and treated with no light (control); light (660-665nm) for 10 minutes at hour 16 from start of maturation (L-16) and light for 10 minutes at hour 20 from the start of maturation (L-20). At hour 24 of in vitro maturation, COCs were vortexed to remove cumulus, stained with BioTracker ATP-Red Live Cell Dye (Sigma-Aldrich SCT045), imaged with Cytation-1 (Agilent Technologies Inc.) in 6 replicates. For experiment 2, COCs were cultured in in vitro maturation media (n = 40/well) and treated with no light (control) and light (660-665nm) for 10 minutes at hour 16 from start of maturation (L-16). At hours 18, 20, 22 and 24 of in vitro maturation, COCs were vortexed to remove cumulus, stained with BioTracker ATP-Red Live Cell Dye, and imaged with Cytation-1 in 4 replicates. For experiment 1, one-way ANOVA and Student’s T-Test were performed (JMP software 16.0); in experiment 2, each well was considered as an experimental unit in a model of repeated measures, main effects of time and treatment, blocked by replicate (Mixed procedure, SAS Institute Inc.). In experiment 1, there was a significant (P &amp;lt; 0.05) effect of treatment; ATP concentrations were greater in L-16 than control, and L-20 was intermediate. ATP concentrations were 19,579.3a ± 596.5, 21,652.0b ± 602.7 and 19,710.9ab ± 570.6 relative fluorescence units (RFU) for control, L-16, and L-20 respectively. In experiment 2, there was a significant (P &amp;lt; 0.05) effect of treatment and no significant effect of time. Overall ATP concentrations were 25,668 ± 894.6 and 28,332 ± 894.6 RFU for control and L-16 respectively. In conclusion, photobiomodulation treatment with LED 660-665nm for 10 minutes at hour 16 from start of oocyte maturation increases intracellular ATP concentrations from hour 18 to 24 of maturation.

398 Subclinical Lipopolysaccharide Exposure During Oocyte Maturation Disrupts Early Bovine Embryonic Development

Abstract Reduced conception rates and infertility are associated with bacterial infections in cattle. Chronic and acute gram-negative bacteria release of lipopolysaccharide (LPS) endotoxin elicits robust immune responses capable of disrupting folliculogenesis, steroidogenesis, oocyte competence, and embryo development. However, the effects of subclinical or non-detectable infections on oocyte competence and subsequent embryo development remain to be fully elucidated. Therefore, the objective of this study was to investigate varying concentrations of LPS on oocyte and embryo competence. Bovine oocytes were collected from slaughterhouse-derived ovaries and incubated with vehicle control or increasing doses of LPS (0, 0.01, 0.1, and 1 µg/mL) for 21 h during in vitro maturation. Oocytes (n = 252 in 3 replicates) were fixed and stained for evaluation of nuclear maturation. A subset of oocytes (n = 153 in 3 replicates) was fertilized and cultured in a time-lapse incubator where individual well images were captured every 5 min for 8 d for video compilation and subsequent analysis. A set of embryos from LPS-matured oocytes (n = 1,126 in 5 replicates) were cultured for 8 d to evaluate blastocyst cell counts (n = 163 in 5 replicates). This evaluation included total, trophectoderm, and inner cell mass cell numbers. Results demonstrate no significant difference among 0.01, 0.1, or 1 µg/mL LPS treatment groups and the percent of oocytes at metaphase II (MII), meiosis I (MI), or germinal vesicle (GV) when compared with vehicle-treated controls after 21 h maturation (P = 0.44, 0.26, and 0.90, respectively). Likewise, no differences were detected among 0.01, 0.1, or 1 µg/mL LPS treatment groups and presumptive zygotes that did not cleave after fertilization (P = 0.84), those that cleaved but arrested at the 2-cell stage (P = 0.50), 4-cell (P = 0.76), prior to morula (P = 76), or morula stage (P = 0.60). However, the percentage of blastocysts derived from oocytes matured in 0.01 µg/mL LPS tended to decrease compared with vehicle-treated controls (11.38 ± 8.74 and 25.45 ± 2.73%, respectively, P = 0.09). Similarly, the proportion of oocytes that developed to the blastocyst stage was greater in vehicle-treated controls (25.45%) compared with embryos derived from oocytes matured in 0.1 (5.92 ± 3.62% P = 0.03) and 1 µg/mL (6.55 ± 3.62%, P = 0.03) LPS. Lipopolysaccharide treatment did not affect total blastocyst cell count (P = 0.73), as indicated by the number of cells of the trophectoderm (P = 0.45), and inner cell mass (P = 0.30). Therefore, these data suggest that although nuclear maturation was not affected by low LPS concentrations, LPS-matured oocytes that subsequently underwent in vitro fertilization, experienced decreased competence to develop to the blastocyst stage. Therefore, non-detectable or subclinical bacterial infections have the capability to decrease embryonic development in cattle.

Low-dose lipopolysaccharide exposure during oocyte maturation disrupts early bovine embryonic development

Gram-negative bacteria release of lipopolysaccharide (LPS) endotoxin elicits robust immune responses capable of disrupting normal ovarian function contributing to female infertility. However, effects of subclinical or non-detectable infections on oocyte competence and subsequent embryo development remain to be fully elucidated. The aim of this study was to investigate the effects of exposing bovine oocytes to low LPS doses on oocyte and embryo competence. Bovine oocytes were collected from slaughterhouse-derived ovaries and matured with vehicle-control or increasing doses of LPS (0.01, 0.1, and 1 μg/mL) for 21 h. Oocytes (n = 252) were evaluated for nuclear maturation. A set of embryos from LPS-matured oocytes (n = 300) were cultured for 8 d to evaluate day 3 cleavage rates and day 8 blastocyst rates along with blastocyst cell counts. A subset of oocytes (n = 153) was fertilized and cultured for time-lapse image capture and analysis of embryo development. Results demonstrate no significant treatment differences among treatment groups in percent of oocytes at germinal vesicle (GV; P = 0.90), germinal vesicle breakdown (GVBD; P = 0.13), meiosis I (MI; P = 0.26), or metaphase II (MII; P = 0.44). Likewise, treatment differences were not observed in cleavage rates (P = 0.97), or blastocyst rates (P = 0.88) evaluated via traditional microscopy. Treatment with LPS did not affect total blastocyst cell count (P = 0.68), as indicated by trophectoderm (P = 0.83), and inner cell mass (P = 0.21) cell counts. Time-lapse embryo evaluation demonstrated no differences among control or LPS matured oocytes in number of zygotes that did not cleave after fertilization (P = 0.84), or those that cleaved but arrested at the 2-cell stage (P = 0.50), 4-cell (P = 0.76), prior to morula (P = 0.76). However, embryos derived from oocytes challenged with 0.1 μg/mL LPS tended to have reduced development to the morula stage compared with vehicle-treated controls (P = 0.06). Additionally, the percentage of blastocysts derived from oocytes matured in 0.01 μg/mL LPS tended to decrease compared to vehicle-treated controls (11.38 and 25.45 %, respectively; P = 0.09). Similarly, the proportion of oocytes that developed to the blastocyst stage was greater in vehicle-treated controls (25.45 %) compared with embryos derived from oocytes matured in 0.1 and 1 μg/mL (5.92 and 6.55 %, respectively; P = 0.03) LPS. These data suggest LPS-matured oocytes that subsequently underwent in vitro fertilization, experienced decreased competence to develop to the blastocyst stage.

The development of new biomarkers of spermatozoa quality in cattle.

There is a current need for new biomarkers of spermatozoa quality, that consistently and correctly identify spermatozoa that will successfully contribute to subsequent embryo development. This could improve the standardization of semen analysis, decrease early embryo mortality, and use these biomarkers as a selection tool before servicing females. This study utilized imaging techniques to identify potential biomarkers of sperm quality, using sires previously classified as high (n = 4) or low (n = 4) performing at producing blastocysts in vitro. Spermatozoa were assessed before and following a gradient purification protocol, to understand how populations of cells are impacted by such protocols and may differ between in vivo and in vitro use. Pre-gradient samples from low-performing sires had an increased incidence of DNA damage, although post-gradient samples from high-performing sires were found to have an increased incidence of DNA damage. When evaluating morphology via fluorescent microscopy, the most prevalent defects in pre-gradient samples from high-performing sires were tail defects, which are successfully removed during purification processing. The most prevalent defects in pre-gradient samples from low-performing sires were aggresome defects located in the head, which would be brought into an oocyte upon fertilization and may impair embryo development. Image-based flow cytometry (IBFC) was employed to quantify defect prevalence to evaluate a greater sample size decreasing the variability that exists in manual assessments. Using IBFC, aggresome defects were again identified in the heads of spermatozoa from low-performing sires. Post-gradient samples from low-performing sires had a significantly greater (p < 0.05) incidence of aggresome defects than post-gradient samples from high-performing sires. Additionally, IBFC was used to evaluate spermatozoa viability following gradient purification. Distinct populations of sperm cells were identified. High-performing sires had more spermatozoa in the population deemed most viable than low-performing sires. This study demonstrated that spermatozoa defects vary in populations before and following gradient purification, indicating that it may be beneficial to separately evaluate semen for in vivo and in vitro use. Furthermore, a prevalent defect in low-performing sires that could explain a discrepancy between successful fertilization and embryo development was identified. Therefore, elucidating a malfunction regulated by sire, that could potentially affect early embryo development.

β-nicotinamide mononucleotide rescues the quality of aged oocyte and improves subsequent embryo development in pigs.

Oocyte senescence alters the shape and function, thereby weakening the fertilization potential. Nicotinamide mononucleotide (NMN) reverses age-related dysfunctions in various organs. Studies had shown long-term administration of NMN reduced the physiological decline associated in aged mice and reversed the aging of the ovaries. However, the protective effect of NMN on aged porcine oocytes is still unclear. In this study, we investigated the effects of NMN on aging porcine oocytes and subsequent embryonic development. We established a model of senescence of porcine oocytes after ovulation by extending the culture time in vitro. NMN supplementation significantly reduced reactive oxygen species (ROS) levels in senescence oocytes and increased the mRNA levels of antioxidant genes SOD1 and Cat. The mitochondrial membrane potential of aged oocytes treated with NMN was increased compared with that of untreated oocytes. In addition, the mRNA level of apoptosis-related gene Bax was significantly decreased in senescence oocytes treated with NMN, while the mRNA level of anti-apoptosis-related gene BCL-2 was significantly increased. Furthermore, NMN supplementation enhanced the subsequent development ability of senescent oocytes during in vitro aging. Compared with untreated senescent oocytes, the blastocyst formation rate and pluripotent genes of senescent oocytes treated with NMN were significantly increased. Taken together, these results suggest that NMN is beneficial for delaying the aging process in porcine oocytes.