Applications of omics and nanotechnology to improve pig embryo production in vitro.

The use of omics data to understand the requirements for in vitro culture systems and also to improve the developmental competence of SCNT-produced embryos has been a valuable tool. Along with this, the use of an innovative nanotechnology-based approach for tagging and site-specific delivery of molecules improving their bioavailability, stability, and cellular uptake brings forth news alternatives for ART. Based on the fact that genetically-modified pigs are becoming increasingly utilized for agricultural and biomedical purposes, there is a great potential to integrate nanotechnology approaches in the in vitro pig embryo production system. The availability of biodegradable and lipid-based nanocarriers that do not impair embryo development has promising applications and, subsequently, will open new horizons in the pig reproductive biology field.

Embryo production in vitro is an important tool to understand the mechanisms underlying early embryo development and consequently increase reproductive efficiency in livestock species ( Dyck et al., 2014 ; Gupta et al., 2009 ). Unfortunately, despite advancements, contact with a suboptimal culture environment produces less developmentally-competent embryos than their in vivo- derived counterparts ( Whitworth et al., 2004 ; Whitworth et al., 2016 ). Chemical and physical factors, such as oxygen levels, temperature, pH, osmolarity, metabolite concentrations, and light, can adversely impact gametes and embryos during culture ( Lane & Gardner, 2005 ; Wale & Gardner, 2016 ). Additionally, the inadequacies of media formulations have negative effects on embryonic development ( Absalón-Medina, Butler, & Gilbert, 2014 ; Heras et al., 2016 ). Preimplantation embryos exhibit remarkable metabolic plasticity, rendering it difficult to define the optimal conditions for development ( Absalón-Medina et al., 2014 ; Krisher, Heuberger, et al., 2015 ). In regard to pigs, there are no standardized protocols for embryo production in vitro , and the success rates of different assisted reproductive technologies (ART) protocols are variable ( Romar, Cánovas, Matás, Gadea, & Coy, 2019 ). Pig embryos produced in vitro have a higher frequency of polyspermy and a reduced ability to support development during the initial cleavage divisions ( Grupen, 2014 ; Rath, Niemann, & Torres, 1995 ) than many other mammalian species. Improvements in the porcine embryo culture system are warranted as genetically modified pigs are becoming increasingly utilized for agricultural and biomedical purposes ( Lai et al., 2002 ; Rogers et al., 2008 ; Schook, Collares, Hu, & Liang, 2015 ; Whitworth et al., 2016 ). To increase the efficiency of pig embryo in vitro systems, it is essential to thoroughly understand the metabolic substrates and genetic pathways that are involved in the development of high quality embryos. The combination of transcriptomic, epigenetic, and metabolomic signatures as well as morphokinetics analyses are thought to be acceptable embryo quality indicators and improve embryo selection ( Krisher, Schoolcraft, & Katz-Jaffe, 2015 ; Romar et al., 2019 ). Previous studies by using expressed sequence tags (ESTs) ( Whitworth et al., 2004 ), DNA microarrays ( Whitworth et al., 2005 ) and innovative next-generation sequencing technology from Illumina platforms ( Bauer et al., 2010 ; Spate et al., 2014 ) have provided valuable information regarding which transcripts are differentially expressed between in vivo -derived and in vitro -cultured oocytes and embryos. Datasets from genome-wide DNA methylation and transcription analyses in single porcine blastocysts ( in vivo and in vitro ) have brought new alternatives to decrease the harmful effects induced by ART ( Canovas et al., 2017 ). Moreover, the advent of metabolomic approaches has created a link from genomic and transcriptomic data to phenotypic characteristics, providing detailed information about embryo metabolism ( Krisher, Heuberger, et al., 2015 ; Tachibana, 2014 ). Besides the importance of controlling culture external conditions, supplementation in vitro to correct specific molecular deficiencies has been reported to increase the efficiency of assisted reproduction techniques ( Chen et al., 2018 ; Heras et al., 2016 ; Moussa, Shu, Zhang, & Zeng, 2015 ; Redel et al., 2016 ; Yuan et al., 2017 ). Recent studies have explored the potential of specific growth factors and amino acids to optimize maturation and embryo culture media ( Chen et al., 2018 ; Krisher & Prather, 2012 ; Redel et al., 2016 ; Redel, Tessanne, Spate, Murphy, & Prather, 2015 ; Yuan et al., 2017 ). However, due to the different chemical and biological composition of supplements, strategies to enable more efficient use of these additives by preventing premature degradation, increasing their bioavailability and facilitating their delivery are desired. Recently, a new approach, based on the use of nanomaterials, has been shown to be a valuable tool for intra-gamete/embryo delivery of target molecules ( Komninou et al., 2016 ; Lucas et al., 2015 ; Remião et al., 2016 ). Nanoparticulated systems have attracted strong interest by different areas of research due to many factors that include stability and encapsulation loading capacity ( Pohlmann et al., 2013 ). These systems are able to pass through biological barriers and target selected pathways ( Barkalina, Jones, Kashir, et al., 2014 ). The growing number of nanomaterials exhibiting biocompatibility with gametes and embryos have created diverse applications for the reproductive field ( Barkalina, Jones, & Coward, 2016 ; Kim et al., 2018 ; Remião et al., 2018 ). Besides the use as nanocarriers for intra-gamete/embryo delivery of supplements, other significant applications of nanotechnology to the swine industry and biomedical science could be the delivery of exogenous DNA to sperm cells ( Barkalina et al., 2016 ; Campos et al., 2011 ) and the delivery of Cas9 or sgRNA plasmids for gene editing ( Wang et al., 2018 ). Herein, we provide an overview about the current applications of omics technologies to further understand preimplantation porcine embryonic development and consequently produce more developmentally-competent embryos in vitro . In addition, we discuss the potential benefits and concerns of nano-sized systems for use in reproductive biology.

Nanotechnology is a prominent field that has improved the existing approaches used in many different areas of research ( Barkalina, Jones, Wood, & Coward, 2015 ). In a biological perspective, nano-objects, which are objects with one or more external dimensions in the nanoscale (1–100 nm) ( ISO, 2015 ) permits establishment of nano-sized platforms that are similar in size to protein structures and other subcellular components ( Lim, Moon, & Lee, 2009 ). These platforms enable long distance transport of molecular cargo and precise delivery in a specific tissue or cell population via innate uptake mechanisms ( Barkalina, Jones, & Coward, 2014 ). Nano-objects have been generated from many materials, including inorganic compounds such as carbon, gold, silver and silica or polymers, liposomes, and dendrimers, which can mimic biological constructs. In addition, different shapes (spherical, discoid, cylindrical, and star-shaped) and sizes can directly influence synthesized nano-objects behavior ( Feugang, 2017 ; Jacobs & Vemuri, 2017 ; Wolfbeis, 2015 ). To date, a variety of nano-sized platforms, such as liposomes, micelles, polymeric nanoparticles (NPs), and carbon nanotubes, have been used for medical purposes, especially for treatment and diagnosis of neurological diseases and cancer (Huang et al., 2017; Sengupta, 2017 ; Wang, Yu, Kong, & Sun, 2017 ). Frequently, nanoparticles, which are nano-objects with all three external dimensions in the nanoscale range and of similar length ( ISO, 2015 ), are based on a central core surrounded by a protective shell and an organic coating layer (i.e., polymers and peptides) that allows surface functionalization with biomolecules for the desired application ( Feugang, 2017 ). A large proportion of central nervous system diseases remain untreatable by using traditional strategies because the blood-brain barrier (BBB) acts as a physical and transport barrier that restricts drugs from entering into the brain ( Pardridge, 2002 ). The use of nanoparticles facilitated penetration through the BBB and enhanced selective binding to amyloid fibrils ( Ruff, Hüwel, Kogan, Simon, & Galla, 2017 ). Regarding cancer therapy and diagnosis, substantial progress has been made toward the use of the nanotechnology. Targeted delivery avoids problems associated with conventional anticancer therapies, such as drug-resistant phenotypic growth, poor water solubility, and severe systemic toxicity ( Ashley et al., 2011 ; Gwinn & Vallyathan, 2006 ). Recently, Wang et al., 2018 demonstrated the great potential of PEGylated nanoparticles (named P-HNPs) for the delivery of Cas9 and sgRNA expression plasmids to mammalian cells and tumor tissues. The P-HNPs system successfully mediated multiplex gene knock-out, gene knock-in, and gene activation in vitro , and gene disruption and tumor growth suppression in vivo , providing advantages over the polycationic transfection agents that are traditionally employed. In the emerging field of nanotechnology, nanoparticle platforms have also demonstrated great potential for detection, diagnosis, and treatment of human infertility. Most reports have explored the use of nanomaterials for the detection and therapy of reproductive cancers and non-cancer conditions, such as uterine leiomyoma ( Shalaby et al., 2016 ) and endometriosis ( Liu et al., 2017 ; Zhao et al., 2016 ). However, a growing number of studies have applied nanotechnology in reproductive biology to target and sort specific populations of gametes and to optimize gene transfer and editing methodologies ( Barkalina, Charalambous, Jones, & Coward, 2014 ; Remião et al., 2018 ). In addition, nanoparticulated systems have shown great potential as carriers to deliver molecules into gametes and embryos to improve their developmental competence in vitro ( Barkalina, Charalambous, et al., 2014 ; Barkalina et al., 2015 ; Komninou et al., 2016 ; Lucas et al., 2015 ; Remião et al., 2016 ) The encouraging data have shown a direct influence of nanomaterials on the efficacy of many existing and pioneering methodologies. Successful development of efficient non-toxic and biodegradable nano-sized platforms have been propelling the application of nanotechnology into other important areas, such as in the reproductive biology field. Having a variety of nano-sized platforms with diverse chemical and biological characteristics, combined with loading and binding capacity, creates unprecedented opportunities to manipulate and determine mechanisms related to early stages of embryo development. The search for advancements in the reproductive sciences has emerged as notable area for nanotechnology studies across a variety of animal species. Nanoparticles have been applied in tagging and identification of embryos, intra-gamete and embryo delivery, sperm enrichment and sorting, sperm-mediated gene transfer, and more recently, in a three-dimensional culture system of secondary follicles. Applications of nanotechnology in studies involving pig oocytes and embryos has not yet been explored. However, results from studies involving other mammalian species, such as mice and bovine, have revealed new alternatives to improve pig embryo production in vitro . Nanoparticles have been tested as a suitable alternative to tag and identify embryos during ART and as a strategy to improve the delivery of different components during in vitro maturation and embryo culture. Fynewever et al., (2007) showed that polystyrene-based nanoparticles were able to tag mouse preimplantation embryos during culture. Moreover, it was observed that external embryo exposure to poly(acrylonitrile)-based nanoparticles demonstrated better embryo development when compared to injected nanoparticles. During bovine IVM, Lucas et al., (2015) and Remião et al., (2016) observed beneficial effects through the use of a nanoencapsulation strategy as a drug-delivery system. IVM medium supplementation with tretinoin (TTN)-loaded lipid-core nanocapsules (TTN-LNC) decreased ROS production and increased the percentage of embryos that developed to the blastocyst stage when compared to non-encapsulated TTN. In addition, nanoencapsulation allowed for the use of lower concentrations of TTN, minimizing the risk of cytotoxic effects related with its use ( Lucas et al., 2015 ). Similarly, melatonin-loaded lipid-core nanocapsules (Mel-LNC) added during bovine IVM and IVC reduced ROS levels and apoptosis in embryos, increased the cleavage and blastocyst rates, up-regulated antioxidant genes, and down-regulated pro-apoptotic genes when compared to non-encapsulated melatonin (Mel) ( Komninou et al., 2016 ; Remião et al., 2016 ). LNCs synthesized by using the Rhodamine B fluorophore-PCL conjugated to label the nanocarrier (Mel-LNC-RB) achieved successful delivery into bovine oocytes and remained inside the cells until the blastocyst stage ( Remião et al., 2016 ). Recently, a three-dimensional (3D) culture system, based on magnetic levitation with precise assembly of nanoparticles promoted the in vitro development of bovine secondary follicles. In this study, magnetic nanoparticles coated with gold, iron oxide and poly-L-lysine were used to facilitate follicular attachment and levitation. It was observed that the 3D culture system enhanced development of secondary follicles when compared to a 2D control treatment (follicles individually culture in 24-well plate). Also, after in vitro maturation, the 3D system improved oocyte viability and meiotic resumption when compared to the control group ( Antonino et al., 2019 ). Data obtained from the porcine model regarding the use of nanostructures in boar sperm have formed the basis for ethical approval of similar experiments in human sperm. Although mice have been the model of choice for most ART studies, there has been a tendency to use bull and boar sperm to evaluate nanomaterial delivery efficiency and effects. Bull and boar sperm, as human sperm, do not need to be surgically retrieved and present similarities regarding morphology and maturation profiles ( Barkalina et al., 2016 ). Characterization of sperm and identification of potential targets, such as proteins related to acrosome integrity ( Cross & Watson, 1994 ) and genes linked to freezability ( Vilagran et al., 2015 ), make the use of nanoparticles interesting for applications in studies involving sperm enrichment and sorting ( Feugang, 2017 ). Mesoporous silica, quantum dots, iron oxide, zinc oxide, gold, and silver nanostructures have been shown to interact with cattle, human, mouse, and pig spermatozoa ( Barkalina, Jones, Kashir, et al., 2014 ; Feugang et al., 2012 ; Feugang, Youngblood, Greene, Willard, & Ryan, 2015 ; Moretti et al., 2013 ; Taylor et al., 2015 ; Yoisungnern et al., 2015 ). In the pig, Barkalina et al., 2016 observed that mesoporous silica nanoparticles (MSNPs) functionalized with polyethileneimine (PEI) and aminopropyltriethoxysilane were able to stably bind one in five boar sperm with no negative effects on the main parameters of sperm function (total and progressive motility, motion parameters, viability, acrosome morphology, or DNA fragmentation index). Moreover, MSNPs loaded with proteins or nucleic acids were able to bind to the boar sperm and to the zona pellucida of mouse oocytes, demonstrating their potential for intracellular delivery. To improve MSNP binding, they targeted MSNP with a specific cell-penetrating peptide, C105Y, resulting in a significantly faster achievement of stable binding rates and not affecting sperm function parameters. Furthermore, Kim et al., (2010) demonstrated that exogenous DNA associated with magnetic iron nanoparticles (MNPs) could be transfected into boar sperm in the presence of an external magnetic field (‘magnitofection’) at a higher efficiency compared to protocols that used naked DNA or other common transfection methods. Recently, by using the same strategy, transgenic mice harboring the enhanced green fluorescent protein (eGFP) gene were generated after Fe 3 O 4 MNPs were used to deliver an exogenous plasmid into mouse sperm under a magnetic field. A higher transfection efficiency was observed when compared to conventional liposome methods ( Wang, Zhao et al., 2017 ). In bovine sperm, Campos et al. (2011) observed an increase in transgene transmission to embryos using semen transfected with exogenous DNA associated with a commercial nanotransfectant or halloysite clay nanotubes. Approximately 40% of embryos contained the transgene and did not exhibit negative effects during development. Due to the fact that there is an increased demand for genetically modified pigs for agricultural and biomedical purposes, the application of nanomaterials during sperm-mediated gene transfer protocols could become a simple and low-cost alternative to porcine micromanipulation procedures. In brief, these methods would avoid the use of agents that disrupt the plasma membrane and would increase the sperm transfection efficacy. As another application, nanoparticles can target spermatozoa during standard sperm purification approaches and improve insemination outcomes. Specifically, in the pig, magnetic iron oxide nanoparticles (Fe 3 O 4 ) coated with annexin V or lectins were able to bind and remove apoptotic and acrosome reacted spermatozoa from semen through a technique termed “nanopurification”. The proportion of motile spermatozoa in the nanopurified groups were significantly higher than the control group (non-purified), and no difference in viability characteristics were observed. After insemination by using control or nanopurified semen, there was no difference in the number of piglets born, suggesting that nanopurification does not impair the fertilizing ability ( Durfey et al., 2019 ; Feugang, Liao, et al., 2015 ). In cattle, nanopurification protocols have increased pregnancy rates after artificial insemination (AI) and IVF ( Odhiambo et al., 2014 ). Additionally, nanoparticles have been tested as non-photobleaching alternative labels for flow cytometry-based sex-sorting procedures which has limitations, such as reduction in sperm fertility. Gold nanoparticles (AuNPs), which are widely used in biomedical applications, conjugated with locked nucleic acids (LNAs) and cell-penetrating peptides could be a suitable tool for sperm sex sorting ( Barchanski et al., 2011 ; Gamrad et al., 2017 ). Functionalization of AuNPs with bull Y-chromosome specific triplex target sequences were successfully hybridized with genomic DNA and the condensed sperm nuclear matrix ( Gamrad et al., 2017 ). Future improvements regarding the use of nanoparticles and molecular probes may represent a promising strategy for sex-sorting of pig sperm. These studies indicate a powerful potential use of nano-based technology in the reproductive biology field and more specifically, for the pig embryo in vitro production system. However, nanotoxicology tests to provide the safety profile of nanomaterials are warranted. Exposure to LNCs during bovine IVM showed that they did not alter embryo development, oocyte maturation, or cumulus cell expansion when different concentrations of LNCs were added in maturation medium ( Lucas et al., 2017 ). Additionally, LNCs decreased ROS production and apoptosis rates, demonstrating its promising application as a nanocarrier ( Lucas et al., 2017 ). In bovine sperm cells, LNC and chitosan-coated lipid-core nanocapsules (LNC-CS) below 40% (v/v) did not impact sperm viability; however, sperm membrane integrity was compromised at and above 40% (v/v) ( Silva et al., 2017 ). In a recent publication, Kim et al., (2018) demonstrated in mice that poly (lactic-co-glycolic acid) (PLGA) nanoparticles coated with PEI and conjugated to tetramethylrhodamine isothiocyanate (TRITC), named TRITC-labeled nano-tracer (TnT), were successfully delivered into spermatozoa, germinal vesicle (GV)-stage oocytes, and 2-cell stage embryos. TnT did not impair the number of cells in the inner cell mass (ICM), apoptosis rate, or blastocyst development. Furthermore, negative health effects as well as morphological and chromosomal abnormalities in the pups exposed to TnT were not observed. The resulting offspring by breeding a male mouse with a female exposed to these nanoparticles during early embryogenesis did not have any negative effects on sex ratio, development, nor phenotype, suggesting a lack of transgenerational toxicity. The successful development of embryos in vitro involves many crucial steps which are susceptible to external perturbations and, consequently, suitable to predict adverse effects. Although nanotechnology applications in the reproductive field are in their infancy stages, they have demonstrated continual advances and promising benefits. The exploration of nanotechnology along with all of these findings underlying porcine embryonic development exhibits favorable features that can bring new alternatives for ART ( Figure 2 ). Nanoparticles can tag embryos and be used to delivery important molecules to improve pig embryo in vitro production and cloning efficiency. Also, nanocarriers associated with exogenous DNA to mediate sperm delivery and to carry genome editing tools, such as the CRISPR/Cas9 system, could facilitate the generation of genetically modified animals. Recently, the use of nanoparticles to model 3D culture systems also creates a new alternative to reproduce an environment more suitable for embryo production in vitro .

In pigs, despite a plethora of research to optimize the in vitro environment, the ability to produce animals through an in vitro system is still limited. The selection of adequate external conditions and appropriate media for oocyte maturation, fertilization and embryo culture are crucial for the success of this process. In general, porcine gametes have been cultivated in a humidified incubator under 5% CO 2 , 20% O 2 and 75 % N 2 at 38.5–39˚C ( Romar et al., 2019 ). After fertilization, the embryos are cultured at 38.5˚C in a humidified atmosphere of 5% CO 2 in air overnight, and then the next day (day 1), they are moved to a humidified atmosphere of low oxygen (5% O 2 , 5% CO 2 , and 90% N 2 ) until day 6 ( Chen et al., 2018 ). Recently, García-Martínez et al., 2018 demonstrated that the use of the same O 2 conditions as those found in the pig reproductive tract (7% O 2 during IVF and embryo culture) were beneficial for embryo production in vitro . For in vitro maturation, the most common medium is Tissue Culture Medium (TCM)-199 followed by other media, such as Waymouth MB 752/1, North Carolina State University (NCSU-37), or modified Tyrode’s solution (mTLP-PVA) ( Motta, Chaves, & Bhat, 2018 ). The basal media used for porcine IVF are the modified Tris-buffered medium (mTBM), TCM-199, Tyrode’s albumin lactate pyruvate (TALP), and the porcine gamete medium (PGM) ( Romar, Funahashi, & Coy, 2016 ). In regards to embryo culture, the most common media used are based on Porcine Zygote Medium (PZM) and NCSU-23; however, embryos cultured with other media, such as Beltsville Embryo Culture Medium (BECM) and Whitten’s Medium (WM) have been able to develop to the blastocyst stage ( Fowler, Mandawala, Griffin, Walling, & Harvey, 2018 ; Motta et al., 2018 ). Several reports have demonstrated that culture with PZM-3 resulted in increased IVF and SCNT-derived blastocyst development compared to NCSU-23 ( Im et al., 2004 ; Nánássy, Lee, Jávor, & Macháty, 2008 ; Wang et al., 2009; Yoshioka, Tanaka, Anas, Suzuki, & Iwamura, 2002 ). Once media are selected, they are always supplemented with different additives to improve the efficiency of these techniques. The use of omics approaches to compare in vivo -derived and in vitro -cultured embryos to search for specific additives that markedly improve pig embryo production in vitro have been the focus of many studies ( Figure 1 ). Omics technologies have revolutionized the biological sciences, by providing the possibility to monitor the dynamics of the living system. This approach is able to provide information from DNA (genome), RNAs (transcriptome), proteins (proteome), and metabolites (metabolome) to assemble a knowledge base with the goal of answering critical biological questions ( Meyer, Zanger, & Schwab, 2013 ). In this section, we discuss the use of different omics platforms to design culture conditions that recapitulate in vivo profiles based on supplementation with different molecules or natural components ( Table 1 ). Also, we highlight the mechanisms that regulate preimplantation embryos metabolism and the factors involved in epigenetic reprogramming of SCNT-derived embryos. The porcine transcriptome has been extensively investigated to identify changes in gene expression between in vivo -derived (IVV) and in vitro -cultured (IVC) embryos. Initial studies utilizing ESTs and DNA microarrays showed alterations in the abundance of transcripts mainly associated with cellular metabolism and transcriptional regulation ( Miles et al., 2008 ; Whitworth et al., 2005 ; Whitworth et al., 2004 ). Interestingly, high abundance of message for ionotropic N-methyl-D-aspartate 3A ( GRIN3A ), a component of the N-methyl-D-aspartic acid (NMDA) receptor, was observed in IVC and IVV embryos when compared to germinal vesicle oocytes, suggesting that NMDA could be beneficial to embryos during preimplantation development ( Whitworth et al., 2005 ). Supplementation with this molecule and/or homocysteine to restore DNA methylation levels enhanced development demonstrating its potential as a bovine serum albumin (BSA) replacement and, consequently, for the formulation of a chemically-defined culture medium ( Spate, Redel, Brown, Murphy, & Prather, 2012 ). Additionally, results from profiling experiments based in pig reproductive tissues revealed that an extracellular matrix protein, known as osteopontin (SPP1), seemed to have an important function in cell-cell interactions ( Green, Kim, Whitworth, Agca, & Prather, 2006 ). SPP1 was added during in vitro fertilization (IVF) and was shown to induce modifications known as “zona hardening” in the zona pellucida (ZP) of porcine oocytes after fertilization. This process reduced sperm penetration and resulted in a decrease in polyspermy during IVF ( Hao et al., 2006 ). Several studies have been performed to determine gene expression patterns using next generation sequencing (NGS) ( Bauer et al., 2010 ; Machuca & Martinez, 2016 ; Mertes et al., 2015 ; Spate et al., 2014 ). This technology has allowed for advances in the field of molecular biology through the possibility of conducting research previously considered economically and logistically infeasible ( Marguerat, Wilhelm, & Bähler, 2008 ). Transcriptome analysis by deep sequencing was performed on pig oocytes derived from in vitro or in vivo maturation. It was observed that the Wingless-type MMTV integration site (WNT) signaling components were overrepresented in oocytes matured in vitro . To inhibit the WNT pathway, the WNT signaling antagonist, Dickkopf-related protein 1 (DKK1), was added to the in vitro maturation medium, thereby improving oocyte maturation rates and enhancing total cell number in blastocysts ( Spate et al., 2014 ). Also, results from RT-PCR analysis showed that activation of the mitogen-activated protein kinase (MAPK) pathway in cumulus cells seems to have an important role in cumulus expansion and meiotic maturation of porcine COCs. Based on this, Yuan et al., (2017) supplemented the maturation medium with three cytokines (FGF2, LIF, and IGF1), named “FLI”, and observed a 4-fold increase in the efficiency of piglet production. Supplementation of the IVM medium with FLI enhanced nuclear maturation and led a 2-fold increase in blastocyst production after IVF. Deep sequencing analysis of IVV or IVC porcine blastocysts identified 1,170 differentially expressed transcripts between these two conditions with 588 of these transcripts exhibiting at least a 2-fold difference ( Bauer et al., 2010 ). Changes in abundance of transcripts involved in amino acid transport and metabolism were frequently observed, indicating that IVC modulates metabolic pathways as a consequence of culture medium components. Genes related to folate metabolism were downregulated in IVC embryos implying possible involvement during development ( Bauer et al., 2010 ). Supplementing culture medium with folate increases trophectoderm and total cell number of blastocysts ( Redel, Spate, Brown, & Prather, 2011 ). In contrast, transcriptional profiling revealed that message abundance of the glycine transporter ( SLC6A9 ) and the arginine transporter ( SLC7A1 ) were up-regulated by at least 25 and 63-fold, respectively, in IVC embryos compared to their IVV counterparts ( Bauer et al., 2010 ; Redel et al., 2016 , 2015 ). Addition of arginine to the culture medium caused a decrease in SLC7A1 to levels comparable to the IVV embryo and improved the percentage of embryos that developed to the blastocyst stage as well as their total cell number ( Redel et al., 2015 ). Similarly, glycine supplementation reduced SLC6A9 abundance, increased total cell number, and decreased the apoptotic index. However, in contrast to the arginine-treated embryos, embryos cultured with supplemental glycine did not establish pregnancies after 11 embryo transfers ( Redel et al., 2016 ). Similar to the arginine story, supplementation of glutamine to the porcine embryo culture medium improved development to the blastocyst stage, reduced transcript levels of a glutamate transporter, SLC1A1 , to a level more similar to in vivo -derived embryos, promoted leucine consumption from the medium, and resulted in birth of live piglets ( Chen et al., 2018 ). Furthermore, the subunit of the granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor (CSFR) was more abundant in IVC embryos when compared to IVV embryos ( Bauer et al., 2010 ). The addition of GM-CSF during culture in vitro increased blastocyst rates and total cell number of IVF and SCNT-derived embryos. Moreover, when SCNT-derived porcine embryos cultured with GM-CSF were transferred to surrogates, they were able to establish pregnancies and resulted in the birth of healthy piglets ( Lee et al., 2013 ). Recently, in a study conducted by Canovas et al., 2017 , porcine genome-wide analyses by RNA-Seq and Bisulfite-Seq were performed to compare gene expression and DNA methylation changes between single in vivo -derived blastocysts collected by flushing the uteri of animals and single in vitro -cultured blastocysts without (C-IVF) or in the presence of natural reproductive fluids (Natur-IVF). As a result of this supplementation, a higher mean number of cells in Natur-IVF and in vivo -derived blastocysts were observed compared to C-IVF embryos. However, there were no differences in blastocyst rates between C- IVF or Natur-IVF groups. In relation to the gene expression profiles, Natur-IVF blastocysts exhibited a pattern more similar to the in vivo -derived group than C-IVF embryos. The C-IVF group had down-regulation of genes related to epigenetic reprogramming, such as DNMT1 and DNMT3B , and higher methylation percentages when compared to the other groups, confirming previous observations that ART-derived embryos have higher levels of methylation than those that are in vivo -derived. C-IVF embryos demonstrated different methylation patterns in three candidate imprinted regions ( ZAC1 , PEG10, and NNAT ) when compared to in vivo -derived blastocysts, and in two regions ( PEG10 and NNAT ) when compared to Natur-IVF. PEG10 has been shown to be related to embryonic development in mice, and NNAT may play a role in brain development and nervous system formation. ZAC1 and IGF2R defects in methylation have been observed in patients with imprinted disorders whether or not ART was used. The reduced methylation in IGF2R and its up-regulation in the C-IVF group could indicate a possibility of LOS-likely syndromes. Genes related to embryonic development, cell cycle regulation and DNA repair were altered in the two in vitro -derived groups, but differences in the methylation levels were not observed. In this study, they suggested that Natur-IVF treatment would generate an offspring healthier than the C-IVF group. The beneficial effects of using natural fluids was also observed by Batista et al., 2015 . In this study, they noted that the use of oviductal fluid (OF) before and during pig IVF increased the efficiency of monospermic zygote production. Oviductal glycoprotein 1 (OVGP1), which was identified as one of the main factors in oviductal secretions, has been shown to be involved in the zona pellucida hardening process, resulting in a reduction of polyspermic fertilization in pigs ( Algarra et al., 2016 ; Coy et al., 2008 ). However, the use of natural fluids as a supplement creates difficulty in the development of standardized protocols because the composition is undefined. The use of chemically defined media, which has become the goal of many embryo laboratories, can improve the biosafety by preventing virus transmission as well as the reliability and reproducibility of results ( Bavister, 1995 ). Additional work based on metabolomics technologies have been performed to better understand metabolic pathways and preferred substrates by embryos ( Krisher, Heuberger, et al., 2015 ; Krisher, Schoolcraft, et al., 2015 ). The use of gas or liquid chromatography (GC and LC, respectively) and matrix-assisted laser desorption ionization (MALDI) platforms coupled with mass spectrometry (MS) were able, in a semitarget approach, to identify some metabolites present in the culture medium ( Krisher, Heuberger, et al., 2015 ). It was shown that carbohydrate sources, such as glucose, lactate, pyruvate, and citrate, normally supplied to human, murine, bovine, and pig in vitro -produced blastocysts were not significantly consumed by the embryos. Also, it was observed that pig embryos depended more on glutamine metabolism than human, murine and bovine embryos ( Krisher, Heuberger, et al., 2015 ). As previously described, uptake and production of amino acids have an important role in preimplantation development ( Bauer et al., 2010 ; Brison et al., 2004 ; Chen et al., 2018 ; Hemmings, Leese, & Picton, 2012 ; Prather, Redel, Whitworth, & Zhao, 2014 ; Redel et al., 2016 , 2015 ). Metabolomics profiling data can indicate potential biomarkers of embryo quality to improve implantation and pregnancy rates after culturing embryos in vitro ( Krisher, Schoolcraft, et al., 2015 ; Wale & Gardner, 2012 ). Addition of different energy sources to porcine embryo culture media has been the subject of extensive investigation. In vitro -produced embryos from several species have demonstrated impaired development in response to high glucose concentrations (>5 mM) ( Conaghan, Handyside, Winston, & Leese, 1993 ; Seshagiri & Bavister, 1991 ; Takahashi & First, 1992 ); however, porcine embryos do not exhibit the same sensitivity ( Hagen, Prather, Sims, & First, 1991 ; Petters & Wells, 1993 ; Swain et al., 2002 ). Glucose consumption by in vivo -derived porcine embryos increased after compaction, but this was not observed in in vitro -produced porcine embryos ( Swain et al., 2002 ). Contrarily, Sturmey and Leese (2003) detected an increase in glucose, lactate and oxygen consumption in in vitro -produced porcine blastocysts. Furthermore, transcriptional profiling of porcine blastocysts cultured in low (5%) versus high, or atmospheric, (20%) oxygen revealed that abundance of SLC2A2 , a high-capacity glucose transporter, was increased which may explain this metabolic shift ( Redel et al., 2012 ). Culture with pyruvate and lactate as the primary energy sources has been shown to increase blastocyst rates and total cell numbers, but a bias towards development of male embryos has also been observed ( Kim et al., 2004 ; Torner, Bussalleu, Briz, Yeste, & Bonet, 2013 , 2014 ). Currently, two formulations of porcine embryo culture media are predominantly utilized: NCSU-23 and porcine zygote medium (PZM) variants ( Canovas et al., 2017 ; Chen et al., 2018 ; Petters and Wells, 1993 ; Redel et al., 2015 ; Spate, Brown, Redel, Whitworth, & Prather, 2015 ; Suzuki, Yoshioka, Sakatani, & Takahashi, 2007 ; Yoshioka et al., 2002 ). NCSU-23 contains glucose (5.5 mM) and glutamine (1 mM), but certain variants utilize pyruvate (0.5 mM) and lactate (5 mM) for the first 48 h which is replaced by glucose (5.5 mM) for the remaining duration of culture. All PZM variants do not contain glucose but do contain pyruvate (0.2 mM), lactate (2 mM), and glutamine (1 to 3.75 mM). Regardless, addition of energy sources to the culture medium is essential to support proliferation which is distinguished by adaptive mechanisms utilized by the embryo. Proliferation during cleavage stages is characterized by extensive nucleic acid synthesis without increases in cellular biomass; therefore, the blastomeres become progressively smaller with each division. To support rapid proliferation, preimplantation embryos exhibit aspects of the Warburg Effect (WE). This phenomenon was first observed by Otto Warburg (1956) whereby cancer cells direct pyruvate away from the tricarboxylic acid (TCA) cycle and towards lactate production even in the presence of oxygen. Moreover, glycolytic intermediates are shuttled towards the Pentose Phosphate Pathway (PPP) to increase concentrations of ribose 5-phosphate and NADPH for nucleic acid synthesis and redox regulation, respectively. In porcine embryos, the greatest PPP activity has been observed at the 4-cell stage ( Flood & Weibold, 1988 ). Importantly, abundance of transcripts involved in the WE phenotype were shown to be increased in porcine blastocysts when cultured in low oxygen compared to high oxygen, such as hexokinase 2 ( HK2 ), pyruvate kinase M2 ( PKM2 ) and pyruvate dehydrogenase kinase 1 ( PDK1 ) ( Redel et al., 2012 ). HK2 and PKM2 drive the buildup of glycolytic intermediates that can enter the PPP ( Christofk, Vander Heiden, Wu, Asara, & Cantley, 2008 ; Wang et al., 2014 ; Wolf et al., 2011 ), and PDK1 prevents conversion of pyruvate to acetyl-CoA, thus increasing lactate production and inhibiting flux through the TCA cycle ( Liu & Yin, 2017 ). This buildup and entrance of glycolytic intermediates into the PPP may be especially important to produce ribose-5-phosphate (R5P) for synthesis of nucleic acids and to increase levels of NADPH, which can act as a reducing agent for thioredoxin (TRX) and glutathione (GSH) to protect against reactive oxygen species (ROS) ( Krisher, Heuberger, et al., 2015 ). Decreased flux through glycolysis further reduces ATP production from this pathway. Alternatively, fatty acid oxidation (FAO) from endogenous lipid stores or glutamine catabolism to α-ketoglutarate may support activities of the TCA cycle and oxidative phosphorylation for ATP production in porcine embryos. However, triglyceride levels have been shown to be consistent from the zygote to the hatched blastocyst; while ATP production increased at the early blastocyst stage ( Sturmey & Leese, 2003 ). Moreover, delipated porcine embryos retain the ability to form blastocysts and piglets after transfer ( Nagashima et al., 2007 , 1994 ), and expression of solute carrier family 2 member 1 ( SLC2A1 ), a high-affinity glucose transporter, was increased ( Wang et al., 2015 ). Several types of cancer cells that exhibit the WE also demonstrate “glutamine addiction” whereby glutamine is readily consumed from the microenvironment to support ATP production and proliferation ( Eagle, 1955 ; Wise & Thompson, 2010 ). Petters et al (1990) demonstrated that porcine zygotes cultured with glutamine as the sole carbon source do not arrest at the 4-cell stage and can develop to the blastocyst stage. Additionally, glutamine supplementation (3.75 mM) to porcine embryo culture medium increased mitochondrial membrane potential in blastocysts, suggesting that glutamine supports oxidative metabolism in the embryos ( Chen et al., 2018 ). Because embryos exhibit characteristics of the WE, methods of inducing these characteristics in donor cells used for somatic cell nuclear transfer (SCNT) may improve reprogramming efficiency to an embryonic state and increase clone litter sizes. Fibroblast cells derived from porcine fetuses were pharmacologically treated with different compounds to induce the WE. PS48 (5-(4-Chloro-phenyl)-3-phenyl-pent-2-enoic acid) was a candidate compound to activate phosphoinositide-dependent protein kinase 1 for enhanced glycolysis. Previously, PS48 has been shown to increase phosphorylation of v-akt murine thymoma viral oncogene homolog (AKT) in porcine blastocysts and act as a replacement for bovine serum albumin ( Spate et al., 2015 ). Another candidate was CPI-613 (6, 8-bis(benzylthio)octanoic acid) to inhibit pyruvate dehydrogenase and α-ketoglutarate dehydrogenase, resulting in decreased mitochondrial metabolism. Treatment with the compounds did not promote proliferation of the donor cells but increased secretion of pyruvate into the medium which is a characteristic of the reverse WE ( Mordhorst, Murphy, Ross, et al., 2018 ; Pavlides et al., 2009 ). Furthermore, donor cells exposed to hypoxic culture conditions (1.25% O 2 ) demonstrated increased abundance of transcripts involved in glycolysis and smaller mitochondria compared to control cells cultured at 5% O 2 , indicating that this method may be effective for inducing the WE ( Mordhorst, Murphy, Schauflinger, et al., 2018 ). When hypoxia-treated donor cells were used for SCNT, reconstructed embryos had increased development to the blastocyst stage, total cell numbers in the blastocysts, and survival probability after transfer compared to control donor cells ( Mordhorst et al., 2019 ). Metabolic studies in other rapidly proliferating cells, such as cancer cells, have provided insight into the metabolism of preimplantation embryos. For example, investigating hallmarks of the WE in porcine preimplantation embryos and applying methods of inducing a WE-like phenotype have improved development in vitro . With this knowledge, techniques to promote the WE in donor cells for SCNT are gaining interest to increase cloning efficiency as well. Development of cloned pigs by SCNT with specific genetic modifications has become a powerful tool for biomedical research and livestock production. Since establishment of embryonic stem cell (ESC) lines remains to be a challenge in pigs, SCNT is a commonly used method to produce cloned genetically modified pigs ( Hou et al., 2016 ). However, due to inadequate nuclear reprogramming of donor cells, the cloning efficiency is still low, approximately 1–5% ( Mao et al., 2015 ). Defects in DNA methylation and histone modifications have often been associated with reduced developmental potential in cloned embryos ( Luo, Ju, Muneri, & Rui, 2015 ). Transcriptional profiling to identify the mechanism underlying this abnormal epigenetic reprogramming has been performed in an attempt to improve the developmental competence of SCNT embryos ( Bonk et al., 2008 ; Whitworth et al., 2015 ; Whitworth, Zhao, Spate, Li, & Prather, 2011 ). Searching for expression patterns more closely related to a normal in vivo -derived embryo and the identification of aberrantly reprogrammed transcripts could allow for targeted correction of gene expression during the cloning process ( Whitworth et al., 2011 ). Several studies evaluated the potential of histone deacetylase inhibitors (HDACi), such as 6-(1,3-dioxo-1H, 3H-benzo[de]isoquinolin-2-yl)-hexanoic acid hydroxyamide (Scriptaid) ( Whitworth et al., 2011 ; Zhang et al., 2017 ; Zhao et al., 2010 ; Zhao et al., 2009), suberoylanilide hydroxamic acid (SAHA), 4-iodo-SAHA (ISAHA) ( Whitworth et al., 2015 ), (2E)-5-[3-[(phenylsufonyl) aminol phenyl]-pent-2-en-4- ynohydroxamic acid (Oxamflatin) ( Mao et al., 2015 ; Park et al., 2012 ) and trichostatin A (TSA) ( Huan et al., 2014 ; Zhao et al., 2010 ) to improve pig cloning efficiency. It is thought that hyperacetylation will enhance transcription and reprogramming by opening the chromatin structure and allowing RNA polymerase II to access transcription start sites more easily ( Thuan et al., 2009 ). Scriptaid treatment for 14 h postactivation for reconstructed pig SCNT embryos increased development to the blastocyst stage and was able to correct a few aberrantly expressed transcripts to the same level as in vivo -derived embryos ( Whitworth et al., 2011 ). Activated SCNT mini-pig embryos treated with Scriptaid for 15 h, besides developmental improvements, had a decrease in apoptosis levels and an upregulation of development-related genes when compared to untreated embryos ( Zhang et al., 2017 ). Zhao et al., 2009 demonstrated that Scriptaid treatment for 14–16 h postfusion and activation increased cloning efficiency in inbred National Institutes of Health miniature pigs from 0% to 1.3% when fetal fibroblasts were used. After 10 embryo transfers for the Scriptaid-treated group and 8 embryo transfers for the untreated group, 14 and 0 live piglets were obtained, respectively, representing an increase in embryo viability after Scriptaid treatment. Scriptaid also improved cloning efficiency when adult ear fibroblasts were used, and the liveborn rate increased from 0% to 3.7% ( Zhao et al., 2010 ). Treatment of reconstructed embryos with SAHA or ISAHA for 14–16 h immediately after activation resulted in the birth of healthy piglets after SCNT. Scriptaid, SAHA or ISAHA upregulated lysosomal transcripts and induced a few variations in expression of histone and pluripotency-related genes compared to IVV, IVF, and untreated SCNT embryos ( Whitworth et al., 2015 ). Also in the pig, treatment with Oxamflatin for 16 h postactivation enhanced blastocyst formation of SCNT embryos and produced more live piglets than the Scriptaid group which was treated under the same conditions. Also, methylation profiles of POU5F1 regulatory elements and centromeric repeat elements were reduced in the Oxamflatin-treated group when compared to the untreated control group ( Mao et al., 2015 ). Oxamflatin treatment for 6 hours postfusion and activation increased blastocyst rates and total cell number in the blastocysts when compared to the untreated group. Moreover, when two different cell lines were used for SCNT, the cloning rates increased to 0.9% to 3.2%, respectively, which was represented by the total number of piglets born ( Park et al., 2012 ). TSA treatment improved cloning efficiency, genomic methylation reprogramming and normalized gene expression profiles similar to IVF counterparts ( Huan et al., 2014 ). However, due to the high percentage of abnormalities observed in TSA-treated cloned pigs, further studies are needed to fully elucidate its mechanism ( Zhao et al., 2010 ). In cattle, in an attempt to mimic the natural conception process, mitochondrial DNA (mtDNA) was depleted from donor cells, and the effects of TSA during embryo culture were evaluated. TSA treatment improved blastocyst formation of SCNT embryos originating from mtDNA-depleted donor cells to rates observed for embryos derived from nondepleted cells ( Srirattana & St John, 2017 ). However, supplementation with mtDNA in depleted donor cells enhanced reprogramming of SCNT embryos when compared to the co-treatment with TSA or TSA alone ( Srirattana & John, 2018 ). Other novel HDACi, such as LAQ824 (Dacinostat, (E)-N-hydroxy-3-[4-[[2-hydroxyethyl-[2-(1H-indol-3-yl) ethyl] amino]-methyl]phenyl]prop-2-enamide) and quisinostat, have demonstrated promising results. Treatment with LAQ824 during the culture of SCNT pig embryos improved blastocyst rates, enhanced the levels of histone acetylation markers, and reduced DNA methylation levels at different developmental stages. Moreover, LAQ824 improved blastocyst quality by decreasing expression of pro-apoptotic genes ( BAX , CASP3, and BAK ) and increasing total cell number and the expression of anti-apoptotic and development-related genes ( POU5F1 , NANOG , SOX2 , GLUT1, and BCL2 ) ( Jin, Lee, Taweechaipaisankul, Kim & Lee 2017 ). Similarly, quisinostat-treated pig SCNT embryos showed an enhancement in their in vitro developmental competence which has been suggested to be related to an increased in acetylation of histone H3 at lysine 9 (H3K9) and to the regulation of genes involved in pluripotency and apoptosis. After three embryo transfers, one sow became pregnant in both treated and untreated groups, generating 6 and 1 fetuses, respectively ( Jin, Guo, et al., 2017 ). Previously reports have also documented an abnormal histone methylation profile during porcine SCNT preimplantation development. Recently, MM-102, a H3K4 histone methyltransferase inhibitor, was added to the culture system after SCNT and was able to reduce the abnormal H3K4me3 levels in NT-embryos at the 4-cell and blastocyst stages as well as up-regulate genes important for pluripotency establishment when compared to IVF counterparts. In addition, MM-102 increased blastocyst formation rates and total cell numbers, contributing to the production of cloned porcine embryos ( Zhang et al., 2018 ). Tao et al., 2017 observed that the selective inhibitor EPZ00477 (EPZ) of a histone H3K79 methyltransferase, DOT1L, restored H3K79me2 levels to those observed in IVF embryos, increased the blastocyst rate of porcine SCNT embryos, and elevated expression of genes associate with pluripotency. Similarly, co-treatment with TSA and BIX-01294, an inhibitor of G9a histone methyltransferase that reduces H3K9me2 levels in somatic cells, corrected abnormal H3K9me2 and 5mC (DNA methylation) levels in 4-cell stage and trophectoderm lineage of SCNT blastocysts and increased the expression of genes related to lineage differentiation when compared to in vivo and in vitro -fertilized embryos ( Cao et al., 2017 ). Taken together, the evaluation of nuclear reprogramming, developmental competence, and acetylation/methylation status can successfully allow for the identification of specific factors that affect the competence of SCNT-derived embryos. The application of these treatments has shown remarkable improvements in cloned embryo development in vitro ; however, to select the best epigenetic reprogramming agent and optimize its effects, more experiments need to be performed.