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12 pages, 617 KiB

Open AccessArticle

MicroRNAomic Analysis of Spent Media from Slow- and Fast-Growing Bovine Embryos Reveal Distinct Differences

by Paul Del Rio, Sierra DiMarco and Pavneesh Madan

Animals 2024, 14(16), 2331; https://doi.org/10.3390/ani14162331 - 13 Aug 2024

Viewed by 695

Abstract

In bovine embryos, the microRNA (miRNA) expression has been profiled at each stage of early development in vitro. The miRNAomic analysis of spent media has the potential to reveal characteristics of embryo health; however, applications are limited without categorizing miRNA profiles by embryo [...] Read more.

In bovine embryos, the microRNA (miRNA) expression has been profiled at each stage of early development in vitro. The miRNAomic analysis of spent media has the potential to reveal characteristics of embryo health; however, applications are limited without categorizing miRNA profiles by embryo quality. Time-lapse imaging has shown the timing of embryo development in vitro may be indicative of their developmental potential. The study aimed to characterize miRNAs in the spent media of bovine embryos with different growth rates during the pre-implantation phase. Bovine cumulus–oocyte complexes were aspirated from ovaries, fertilized, and cultured to blastocyst stage of development. At the 2-cell, 8-cell, and blastocyst stage, each microdrop of 30 presumptive zygotes were classified as slow- or fast-growing based on the percentage of embryos that had reached the desired morphological stage. A comparative analysis was performed on the spent media of slow- and fast-growing embryos using the results of a GeneChip miRNA 4.0 array hybridization. In total, 34 differentially expressed miRNAs were identified between the comparison groups: 14 miRNAs were found in the 2-cell samples, 7 in the 8-cell samples, and 12 in the blastocyst samples. The results demonstrate distinct miRNAs populations can be identified between slow- and fast-growing embryos, highlighting the novel biomarkers of developmental potential at each stage of pre-implantation development. Full article

(This article belongs to the Special Issue Early Embryo Development in Agricultural Animals)

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12 pages, 2267 KiB

Open AccessArticle

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

by Lucy M. Hillyear, Louisa J. Zak, Tom Beckitt, Darren K. Griffin, Simon C. Harvey and Katie E. Harvey

Animals 2024, 14(5), 783; https://doi.org/10.3390/ani14050783 - 2 Mar 2024

Viewed by 1296

Abstract

The study of pig preimplantation embryo development has several potential uses: from agriculture to the production of medically relevant genetically modified organisms and from rare breed conservation to acting as a physiologically relevant model for progressing human and other (e.g., endangered) species’ in [...] Read more.

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

(This article belongs to the Special Issue Early Embryo Development in Agricultural Animals)

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18 pages, 2783 KiB

Open AccessArticle

Identification of SWI/SNF Subcomplex GBAF Presence, Intra-Complex Interactions, and Transcriptional Dynamics during Early Porcine Development

by Sarah Innis, Aktan Alpsoy, Jennifer Crodian, Yu-Chun Tseng, Emily Dykhuizen, Birgit Cabot and Ryan Cabot

Animals 2024, 14(5), 773; https://doi.org/10.3390/ani14050773 - 29 Feb 2024

Viewed by 1334

Abstract

Understanding the complex interplay between genetics and environmental factors is vital for enhancing livestock production efficiency while safeguarding animal health. Despite extensive studies on production-specific genes in livestock, exploring how epigenetic mechanisms and heritable modifications govern animal growth and development remains an under-explored [...] Read more.

Understanding the complex interplay between genetics and environmental factors is vital for enhancing livestock production efficiency while safeguarding animal health. Despite extensive studies on production-specific genes in livestock, exploring how epigenetic mechanisms and heritable modifications govern animal growth and development remains an under-explored frontier with potential implications across all life stages. This study focuses on the GBAF chromatin remodeling complex and evaluates its presence during embryonic and fetal development in swine. Immunocytochemistry and co-immunoprecipitation techniques were employed to investigate the presence and interactions of GBAF subunits BRD9 and GLTSCR1 in porcine oocytes, preimplantation embryos, and cell lines, and transcriptional dynamics of GBAF subunits across these key developmental stages were analyzed using existing RNA-seq datasets. BRD9 and GLTSCR1 were identified across all represented stages, and an interaction between GLTSCR1 and BAF170 was shown in PTr2 and PFF cells. Our findings highlight the ubiquitous presence of GBAF in porcine early development and the potentially novel association between GLTSCR1 and BAF170 in swine. The transcriptional dynamics findings may suggest GBAF-specific contributions during key developmental events. This study contributes to the growing understanding of epigenetic regulators in both swine and mammalian development, emphasizing the implications of GBAF as a modulator of key developmental events. Full article

(This article belongs to the Special Issue Early Embryo Development in Agricultural Animals)

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20 pages, 7139 KiB

Open AccessArticle

Differentially Expressed Candidate miRNAs of Day 16 Bovine Embryos on the Regulation of Pregnancy Establishment in Dairy Cows

by Vanmathy R. Kasimanickam and Ramanathan K. Kasimanickam

Animals 2023, 13(19), 3052; https://doi.org/10.3390/ani13193052 - 28 Sep 2023

Viewed by 1127

Abstract

Recent advances in high-throughput in silico techniques translate experimental data into meaningful biological networks through which the role of individual proteins, interactions, and their biological functions are comprehended. The study objective was to identify differentially expressed (DE) miRNAs between the day 16 competent, [...] Read more.

Recent advances in high-throughput in silico techniques translate experimental data into meaningful biological networks through which the role of individual proteins, interactions, and their biological functions are comprehended. The study objective was to identify differentially expressed (DE) miRNAs between the day 16 competent, elongated embryo from normal cows and the day 16 noncompetent, tubular embryos from repeat breeder cows, assimilate DE-miRNAs to their target genes, and group target genes based on biological function using in silico methods. The 84 prioritized bovine-specific miRNAs were investigated by RT-PCR, and the results showed that 19 were differentially expressed (11 up- and 8 down-regulated) in the competent embryos compared to noncompetent ones (p ≤ 0.05; fold regulation ≥ 2 magnitudes). Top-ranked integrated genes of DE-miRNAs predicted various biological and molecular functions, cellular processes, and signaling pathways. Further, analysis of the categorized groups of genes showed association with signaling pathways, turning on or off key genes and transcription factors regulating the development of embryo, placenta, and various organs. In conclusion, highly DE-miRNAs in day 16 bovine conceptus regulated the embryogenesis and pregnancy establishment. The elucidated miRNA-mRNA interactions in this study were mostly based on predictions from public databases. Therefore, the causal regulations of these interactions and mechanisms require further functional characterization. Full article

(This article belongs to the Special Issue Early Embryo Development in Agricultural Animals)

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15 pages, 3378 KiB

Open AccessArticle

6-Gingerol Improves In Vitro Porcine Embryo Development by Reducing Oxidative Stress

by Wenjie Yu, Yanxia Peng, Xinyue Peng, Ze Li, Chang Liu, Liu Yang, Yan Gao, Shuang Liang, Bao Yuan, Chengzhen Chen, Nam-hyung Kim, Hao Jiang and Jiabao Zhang

Animals 2023, 13(8), 1315; https://doi.org/10.3390/ani13081315 - 11 Apr 2023

Cited by 3 | Viewed by 2048

Abstract

6-Gingerol, the main active ingredient in ginger, exhibits a variety of biological activities, such as antioxidant, anti-inflammatory, and anticancer activities, and can affect cell development. However, the effects of 6-gingerol on mammalian reproductive processes, especially early embryonic development, are unclear. This study explored [...] Read more.

6-Gingerol, the main active ingredient in ginger, exhibits a variety of biological activities, such as antioxidant, anti-inflammatory, and anticancer activities, and can affect cell development. However, the effects of 6-gingerol on mammalian reproductive processes, especially early embryonic development, are unclear. This study explored whether 6-gingerol can be used to improve the quality of in vitro-cultured porcine embryos. The results showed that 5 μM 6-gingerol significantly increased the blastocyst formation rates of porcine early embryos. 6-Gingerol attenuated intracellular reactive oxygen species accumulation and autophagy, increased intracellular glutathione levels, and increased mitochondrial activity. In addition, 6-gingerol upregulated NANOG, SRY-box transcription factor 2, cytochrome c oxidase subunit II, mechanistic target of rapamycin kinase, and RPTOR independent companion of MTOR complex 2 while downregulating Caspase 3, baculoviral IAP repeat containing 5, autophagy related 12, and Beclin 1. Most importantly, 6-gingerol significantly increased the levels of p-extracellular regulated protein kinase 1/2 while reducing the levels of p-c-Jun N-terminal kinase 1/2/3 and p-p38. These results indicate that 6-gingerol can promote the development of porcine early embryos in vitro. Full article

(This article belongs to the Special Issue Early Embryo Development in Agricultural Animals)

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14 pages, 2873 KiB

Open AccessArticle

Maslinic Acid Supplementation during the In Vitro Culture Period Ameliorates Early Embryonic Development of Porcine Embryos by Regulating Oxidative Stress

by Ting-Ting Yang, Jia-Jia Qi, Bo-Xing Sun, He-Xuan Qu, Hua-Kai Wei, Hao Sun, Hao Jiang, Jia-Bao Zhang and Shuang Liang

Animals 2023, 13(6), 1041; https://doi.org/10.3390/ani13061041 - 13 Mar 2023

Cited by 2 | Viewed by 1622

Abstract

As a pentacyclic triterpene, MA exhibits effective free radical scavenging capabilities. The purpose of this study was to explore the effects of MA on porcine early-stage embryonic development, oxidation resistance and mitochondrial function. Our results showed that 1 μM was the optimal concentration [...] Read more.

As a pentacyclic triterpene, MA exhibits effective free radical scavenging capabilities. The purpose of this study was to explore the effects of MA on porcine early-stage embryonic development, oxidation resistance and mitochondrial function. Our results showed that 1 μM was the optimal concentration of MA, which resulted in dramatically increased blastocyst formation rates and improvement of blastocyst quality of in vitro-derived embryos from parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT). Further analysis indicated that MA supplementation not only significantly decreased the abundance of intracellular reactive oxygen species (ROS) and dramatically increased the abundance of intracellular reductive glutathione (GSH) in porcine early-stage embryos, but also clearly attenuated mitochondrial dysfunction and inhibited apoptosis. Moreover, Western blotting showed that MA supplementation upregulated OCT4 (p < 0.01), SOD1 (p < 0.0001) and CAT (p < 0.05) protein expression in porcine early-stage embryos. Collectively, our data reveal that MA supplementation exerts helpful effects on porcine early embryo development competence via regulation of oxidative stress (OS) and amelioration of mitochondrial function and that MA may be useful for increasing the in vitro production (IVP) efficiency of porcine early-stage embryos. Full article

(This article belongs to the Special Issue Early Embryo Development in Agricultural Animals)

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14 pages, 4176 KiB

Open AccessArticle

Effect of miR-143-3p from Extracellular Vesicles of Porcine Uterine Luminal Fluid on Porcine Trophoblast Cells

by Yue Ding, Qun Hu, Jianyu Gan, Xupeng Zang, Ting Gu, Zhenfang Wu, Gengyuan Cai and Linjun Hong

Animals 2022, 12(23), 3402; https://doi.org/10.3390/ani12233402 - 2 Dec 2022

Cited by 3 | Viewed by 1749

Abstract

Extracellular vesicles (EVs) in uterine luminal fluid (ULF) can reportedly affect the proliferation and migration function of porcine trophoblast cells (PTr2 cells) by mediating the maternal–fetal exchange of information. miR-143-3p is considered a crucial miRNA in early pregnancy in mammals; however, little is [...] Read more.

Extracellular vesicles (EVs) in uterine luminal fluid (ULF) can reportedly affect the proliferation and migration function of porcine trophoblast cells (PTr2 cells) by mediating the maternal–fetal exchange of information. miR-143-3p is considered a crucial miRNA in early pregnancy in mammals; however, little is currently known about how it regulates the function of PTr2 cells. This study aimed to investigate the effects of ssc-miR-143-3p in ULF-EVs on the function of PTr2 cells during porcine embryo implantation. The uptake of ULF-EVs by PTr2 cells was confirmed, which significantly increased the expression of ssc-miR-143-3p. Ssc-miR-143-3p was found to facilitate the proliferation and migration of PTr2 cells in the CCK-8, EdU and wound-closure assays, while the opposite findings were observed after the knockdown of ssc-miR-143-3p. Bioinformatics analysis and the luciferase reporter assay showed that glycerol-3 phosphate dehydrogenase 2 (GDP2) was directly targeted by miR-143-3p. Inhibition of miR-143-3p was validated in mice to inhibit embryo implantation. In summary, ssc-miR-143-3p in ULF-EVs affects the proliferation and migration of PTr2 cells by mediating GPD2, thereby affecting embryo implantation. Full article

(This article belongs to the Special Issue Early Embryo Development in Agricultural Animals)

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15 pages, 2418 KiB

Open AccessArticle

Embryo–Uterine Cross-Talk: Exploration of the Immunomodulatory Mechanism in Buffalo

by Lakshmi Devi Huidrom, Shital Nagargoje Dhanaji, Sriti Pandey, Vikash Chandra and Taru Sharma Gutulla

Animals 2022, 12(22), 3138; https://doi.org/10.3390/ani12223138 - 14 Nov 2022

Cited by 3 | Viewed by 2325

Abstract

Understanding the molecular cross-talk between the embryo and uterine endometrium is crucial for the improvement of IVF outcomes. The present work was undertaken to investigate the effect of pre-implantation embryo on the expression profile of immune-related genes in uterine epithelial cells (UECs) and [...] Read more.

Understanding the molecular cross-talk between the embryo and uterine endometrium is crucial for the improvement of IVF outcomes. The present work was undertaken to investigate the effect of pre-implantation embryo on the expression profile of immune-related genes in uterine epithelial cells (UECs) and PBMCs in buffalo. UECs were isolated from slaughterhouse-derived non-gravid uteri, cultured ex vivo and characterized, and buffalo embryos were produced in vitro from slaughterhouse-derived ovaries. Embryos co-cultured with steroid-treated UECs significantly stimulated (p < 0.05) the relative mRNA abundance of PTGS2, ISG15, OAS1, MX2, IFNAR1 and IFNAR2 in UECs while they significantly suppressed the mRNA expression of NFkβIA, NFkβ2, TNFα and IL1B, with no significant change in TGFβ1 and IL10 in the co-culture of embryos with UECs. In vitro treatment of PBMCs with conditioned media (CM) derived from embryos as well as UEC–embryo co-culture upregulated the mRNA abundance of ISG15, TGFβ1, PTGS2OAS1, MX2 and STAT1 while it downregulated IL17 and TNFα expression. The expression of IFNAR1 and IFNAR2 was elevated in PBMCs cultured in embryo-derived CM, but there was no significant change in PBMCs cultured in UEC–embryo co-culture CM. Thus, it can be concluded that the developing embryo and its secretions modulate the expression of immune responses by inducing an anti-inflammatory action in uterine epithelial cells for acceptance of the semi-allogenic embryo in the uterus to sustain pregnancy in buffalo. Full article

(This article belongs to the Special Issue Early Embryo Development in Agricultural Animals)

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Review

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29 pages, 2314 KiB

Open AccessReview

Early Embryonic Development in Agriculturally Important Species

by Fuller W. Bazer and Gregory A. Johnson

Animals 2024, 14(13), 1882; https://doi.org/10.3390/ani14131882 - 26 Jun 2024

Viewed by 1599

Abstract

The fertilization of oocytes ovulated by pigs, sheep, cows, and horses is not considered a limiting factor in successful establishment of pregnancy. Pig, sheep, and cow embryos undergo cleavage to the blastocyst stage, hatch from the zona pellucida, and undergo central-type implantation. Hatched [...] Read more.

The fertilization of oocytes ovulated by pigs, sheep, cows, and horses is not considered a limiting factor in successful establishment of pregnancy. Pig, sheep, and cow embryos undergo cleavage to the blastocyst stage, hatch from the zona pellucida, and undergo central-type implantation. Hatched blastocysts of pigs, sheep, and cows transition from tubular to long filamentous forms to establish surface area for exchange of nutrients and gases with the uterus. The equine blastocyst, surrounded by external membranes, does not elongate but migrates throughout the uterine lumen before attaching to the uterine luminal epithelium (LE) to begin implantation. Pregnancy recognition signaling in pigs requires the trophectoderm to express interleukin 1 beta, estrogens, prostaglandin E2, and interferon gamma. Sheep and cow conceptus trophectoderm expresses interferon tau that induces interferon regulatory factor 2 that inhibits transcription of estrogen and oxytocin receptors by uterine epithelia. This prevents oxytocin-induced luteolytic pulses of prostaglandin F2-alpha from regressing the corpora lutea, as well as ensuring the secretion of progesterone required for maintenance of pregnancy. The pregnancy recognition signal produced by equine blastocysts is not known. Implantation in these species requires interactions between extracellular matrix (ECM) proteins and integrins as the conceptus undergoes apposition and firm attachment to the uterine LE. This review provides details with respect to early embryonic development and the transition from spherical to filamentous conceptuses in pigs, sheep, and cows, as well as pre-implantation development of equine blastocysts and implantation of the conceptuses. Full article

(This article belongs to the Special Issue Early Embryo Development in Agricultural Animals)

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18 pages, 1277 KiB

Open AccessReview

Unraveling the Consequences of Oxygen Imbalance on Early Embryo Development: Exploring Mitigation Strategies

by Thamiris Vieira Marsico, Mara Viana Silva, Roniele Santana Valente, Kelly Annes, Vitor Braga Rissi, Werner Giehl Glanzner and Mateus José Sudano

Animals 2023, 13(13), 2171; https://doi.org/10.3390/ani13132171 - 1 Jul 2023

Cited by 5 | Viewed by 2253

Abstract

Although well-established and adopted by commercial laboratories, the in vitro embryo production system still requires refinements to achieve its highest efficiency. Early embryonic development is a dynamic event, demanding suitable conditions to provide a high number of embryos with quality and competence. The [...] Read more.

Although well-established and adopted by commercial laboratories, the in vitro embryo production system still requires refinements to achieve its highest efficiency. Early embryonic development is a dynamic event, demanding suitable conditions to provide a high number of embryos with quality and competence. The first step to obtaining an optimized in vitro environment is to know the embryonic metabolism and energy request throughout the different stages of development. Oxygen plays a crucial role in several key biological processes necessary to sustain and complete embryonic development. Nonetheless, there is still controversy regarding the optimal in vitro atmospheric concentrations during culture. Herein, we discuss the impact of oxygen tension on the viability of in vitro-produced embryos during early development. The importance of oxygen tension is addressed as its roles regarding essential embryonic traits, including embryo production rates, embryonic cell viability, gene expression profile, epigenetic regulation, and post-cryopreservation survival. Finally, we highlight the damage caused by in vitro unbalanced oxygen tensions and strategies to mitigate the harmful effects. Full article

(This article belongs to the Special Issue Early Embryo Development in Agricultural Animals)

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36 pages, 2301 KiB

Open AccessReview

Pre-Implantation Bovine Embryo Evaluation—From Optics to Omics and Beyond

by R. A. Chanaka Rabel, Paula V. Marchioretto, Elizabeth A. Bangert, Kenneth Wilson, Derek J. Milner and Matthew B. Wheeler

Animals 2023, 13(13), 2102; https://doi.org/10.3390/ani13132102 - 24 Jun 2023

Cited by 7 | Viewed by 4535

Abstract

Approximately 80% of the ~1.5 million bovine embryos transferred in 2021 were in vitro produced. However, only ~27% of the transferred IVP embryos will result in live births. The ~73% pregnancy failures are partly due to transferring poor-quality embryos, a result of erroneous [...] Read more.

Approximately 80% of the ~1.5 million bovine embryos transferred in 2021 were in vitro produced. However, only ~27% of the transferred IVP embryos will result in live births. The ~73% pregnancy failures are partly due to transferring poor-quality embryos, a result of erroneous stereomicroscopy-based morphological evaluation, the current method of choice for pre-transfer embryo evaluation. Numerous microscopic (e.g., differential interference contrast, electron, fluorescent, time-lapse, and artificial-intelligence-based microscopy) and non-microscopic (e.g., genomics, transcriptomics, epigenomics, proteomics, metabolomics, and nuclear magnetic resonance) methodologies have been tested to find an embryo evaluation technique that is superior to morphologic evaluation. Many of these research tools can accurately determine embryo quality/viability; however, most are invasive, expensive, laborious, technically sophisticated, and/or time-consuming, making them futile in the context of in-field embryo evaluation. However accurate they may be, using complex methods, such as RNA sequencing, SNP chips, mass spectrometry, and multiphoton microscopy, at thousands of embryo production/collection facilities is impractical. Therefore, future research is warranted to innovate field-friendly, simple benchtop tests using findings already available, particularly from omics-based research methodologies. Time-lapse monitoring and artificial-intelligence-based automated image analysis also have the potential for accurate embryo evaluation; however, further research is warranted to innovate economically feasible options for in-field applications. Full article

(This article belongs to the Special Issue Early Embryo Development in Agricultural Animals)

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20 pages, 399 KiB

Open AccessReview

Developmental Hurdles That Can Compromise Pregnancy during the First Month of Gestation in Cattle

by Savannah L. Speckhart, Mary A. Oliver and Alan D. Ealy

Animals 2023, 13(11), 1760; https://doi.org/10.3390/ani13111760 - 25 May 2023

Viewed by 2015

Abstract

Several key developmental events are associated with early embryonic pregnancy losses in beef and dairy cows. These developmental problems are observed at a greater frequency in pregnancies generated from in-vitro-produced bovine embryos. This review describes critical problems that arise during oocyte maturation, fertilization, [...] Read more.

Several key developmental events are associated with early embryonic pregnancy losses in beef and dairy cows. These developmental problems are observed at a greater frequency in pregnancies generated from in-vitro-produced bovine embryos. This review describes critical problems that arise during oocyte maturation, fertilization, early embryonic development, compaction and blastulation, embryonic cell lineage specification, elongation, gastrulation, and placentation. Additionally, discussed are potential remediation strategies, but unfortunately, corrective actions are not available for several of the problems being discussed. Further research is needed to produce bovine embryos that have a greater likelihood of surviving to term. Full article

(This article belongs to the Special Issue Early Embryo Development in Agricultural Animals)

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