Porcine In Vitro Fertilization Research Articles

Oviduct epithelial cell‑derived extracellular vesicles promote the developmental competence of IVF porcine embryos.

Assisted reproductive technology has increased the efficiency of animal reproduction. However, polyspermy is a significant limitation of porcine invitro fertilization (IVF). Therefore, reducing the polyspermy rate and improving monospermic embryos is crucial. Recent studies have reported that oviductal fluid, along with its contents of extracellular vesicles (EVs), enhanced the fertilization process and supported embryo development. Consequently, the present study investigated the effects of porcine oviduct epithelial cells (OEC‑EVs) on sperm‑oocyte interactions during porcine IVF and evaluated invitro embryo developmental competence outcomes. During IVF embryo development, the cleavage rate was significantly higher in the group treated with 50ng/ml OEC‑EVs compared with the control group (67.6±2.5 vs. 57.3±1.9; P<0.05). Furthermore, the OEC‑EV group had significantly more embryos (16.4±1.2 vs. 10.2±0.8; P<0.05), and the polyspermy rate significantly decreased (32.9±2.5 vs. 43.8±3.1; P<0.05) compared with that of the control group. Additionally, the fluorescence intensities of cortical granules (3.56±0.47 vs. 2.15±0.24; P<0.05) and active mitochondria (8.14±0.34 vs. 5.96±0.38; P<0.05) were significantly higher in the OEC‑EV group compared with those in the control group. In conclusion, OEC‑EV adsorption and penetration crosstalk between sperm and oocytes was observed. OEC‑EV treatment was demonstrated to significantly improve the concentration and distribution of cortical granules in oocytes. Furthermore, OEC‑EVs also increased oocyte mitochondrial activity, reduced polyspermy and increased the IVF success rate.

Coordination of zygotic genome activation entry and exit by H3K4me3 and H3K27me3 in porcine early embryos.

Histone modifications are critical epigenetic indicators of chromatin state associated with gene expression. Although the reprogramming patterns of H3K4me3 and H3K27me3 have been elucidated in mouse and human preimplantation embryos, the relationship between these marks and zygotic genome activation (ZGA) remains poorly understood. By ultra-low-input native chromatin immunoprecipitation and sequencing, we profiled global H3K4me3 and H3K27me3 in porcine oocytes and in vitro fertilized (IVF) embryos. We found that promoters of ZGA genes occupied sharp H3K4me3 peaks in oocytes, and these peaks became broader after fertilization, and reshaped into sharp again during ZGA. By simultaneous depletion of H3K4me3 demethylase KDM5B and KDM5C, we determined that broad H3K4me3 domain maintenance impaired ZGA gene expression, suggesting its function to prevent premature ZGA entry. By contrast, broad H3K27me3 domains underwent global removal upon fertilization, followed by a re-establishment for H3K4me3/H3K27me3 bivalency in morulae. We also found that bivalent marks were deposited at promoters of ZGA genes, and inhibiting this deposition was correlated with the activation of ZGA genes. It suggests that promoter bivalency contributes to ZGA exit in porcine embryos. Moreover, we demonstrated that aberrant reprogramming of H3K4me3 and H3K27me3 triggered ZGA dysregulation in somatic cell nuclear transfer (SCNT) embryos, whereas H3K27me3-mediated imprinting did not exist in porcine IVF and SCNT embryos. Our findings highlight two previously unknown epigenetic reprogramming modes coordinated with ZGA in porcine preimplantation embryos. Finally, the similarities observed between porcine and human histone modification dynamics suggest that the porcine embryo may also be a useful model for human embryo research.

RNA-Seq Reveals the Underlying Molecular Mechanism of First Cleavage Time Affecting Porcine Embryo Development.

The selection and evaluation of high-quality embryos are the key factors affecting in vitro embryo development and pregnancy outcome. The timing of first embryonic cleavage has been considered a positive indicator of the in vitro developmental potential of embryos, while the underlying molecular mechanism is still not fully understood. In this study, the embryos generated by parthenogenetic activation (PA) or in vitro fertilization (IVF) were monitored and recorded every 2 h and divided into two groups (early cleavage or late cleavage) based on the cleavage rate and blastocyst formation data. RNA sequencing was used to analyze the gene expression pattern of the embryos. We identified 667 and 71 different expression genes (DEGs) in early cleavage and late cleavage porcine PA and IVF embryos, respectively. Further Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses showed that the DEGs are mainly enriched in pathways concerning the proteasome, DNA repair, cell cycle arrest, autophagy, and apoptosis, suggesting that severe endoplasmic reticulum stress (ERS) and DNA damage may be the key factors that led to the low development potential of late cleavage embryos. This study provides a theoretical basis for the following application and offers important information about the understanding of the timely manner of porcine embryo development.

Supplementation with asiatic acid during in vitro maturation improves porcine oocyte developmental competence by regulating oxidative stress

Asiatic acid is a natural triterpene found in Centella asiatica that acts as an effective free radical scavenger. Our previous research showed that asiatic acid delayed porcine oocyte ageing in vitro and improved preimplantation embryo development competence in vitro; however, the protective effects of asiatic acid against oxidative stress in porcine oocyte maturation are still unclear. Here, we investigated the effects of asiatic acid on porcine oocyte in vitro maturation (IVM) and subsequent embryonic development competence after parthenogenetic activation (PA) and in vitro fertilization (IVF). The results of the present research showed that 10 μM asiatic acid supplementation did not affect the expansion of cumulus cells or polar body extrusion of porcine oocytes, while asiatic acid application significantly increased the subsequent blastocyst formation rate and quality of porcine PA and IVF embryos. Hydrogen peroxide (H2O2) is a reactive oxygen species (ROS) that induces oxidative stress in porcine oocytes. As expected, asiatic acid supplementation not only decreased intracellular ROS levels but also attenuated H2O2-induced intracellular ROS generation. Further analysis revealed that asiatic acid supplementation enhanced intracellular glutathione production, mitochondrial membrane potential, and ATP generation at the end of IVM. In summary, our results reveal that asiatic acid supplementation exerts beneficial effects on porcine oocytes by regulating oxidative stress during the IVM process and could act as a potential antioxidant in porcine oocytes matured in vitro production systems.

N-(2-mercaptopropionyl)-glycine enhances in vitro pig embryo production and reduces oxidative stress

This study evaluated the effects of different concentrations (1, 10, 25, 50, and 100 µM) of the antioxidant N-(2-mercaptopropionyl)-glycine (NMPG), during the culture of in vitro-fertilized porcine oocytes. While the highest concentrations of NMPG (50 and 100 µM) were toxic to the developing embryos during the first two days of culture, 25 µM NMPG achieved cleavage rates that were similar to those achieved by the control but did not sustain blastocyst production by Day 7 of culture. Compared to the control culture medium, the culture medium supplemented with 10 µM NMPG increased (P < 0.05) the rates of blastocyst formation, decreased (P < 0.05) the intracellular levels of reactive oxygen substances, and downregulated (P < 0.05) the expression of the oxidative stress related gene GPX1. In conclusion, these results demonstrated that supplementation of porcine embryo culture medium with 10 µM NMPG can attenuate oxidative stress and increase the yield of in vitro production of blastocysts.

Addition of exogenous proteins detected in oviductal secretions to in vitro culture medium does not improve the efficiency of in vitro fertilization in pigs

This work was designed to study whether HSP70-1A, HSP90α, ezrin or PDI4, proteins previously identified in porcine oviductal secretions, have a role in zona pellucida (ZP) resistance to enzymatic digestion, in vitro fertilization (IVF) and sperm viability. In vitro matured porcine cumulus oocyte complexes were denuded and i) incubated for 1 h in TALP medium supplemented or not with each exogenous oviductal protein and in presence or absence of heparin to assess ZP digestion time by pronase; and ii) inseminated with fresh ejaculated boar spermatozoa in medium supplemented or not with each exogenous oviductal protein to assess their effect on fertilization results. Finally, spermatozoa were incubated in Tyrode’s medium (0, 1 and 20 h) supplemented or not with HSP-701A, HSP-90α or ezrin, to assess simultaneously sperm viability and acrosome status by means of flow cytometry. Although all proteins increased the ZP digestion time, this increase was lower than 1 min, being ezrin the protein with a stronger effect. Presence of heparin in the medium reinforced the ZP hardening effect of ezrin and HSP-701A up to one more min, but not HSP-90α nor PDI4. Sperm penetration, but not IVF efficiency, increased when gametes were cocultured in medium containing PDIA4 whereas sperm penetration and polyspermy rates decreased in presence of ezrin and HSP proteins. This reduction was not the result of a detrimental effect of proteins on sperm viability or acrosome reaction. In conclusion, addition of exogenous proteins detected in oviductal secretions to artificial media does not reproduce the effect of adding such secretions nor improve the final efficiency of the porcine IVF system.

Improved early development potence of in vitro fertilization embryos by treatment with tubacin increasing acetylated tubulin of matured porcine oocytes

To improve the developmental potential of in vitro embryos is a long-term concern field for human assisted reproduction and animal in vitro embryo production practice. In the current study, we examined the effects and mechanism of an HDAC6 inhibitor, tubacin, on the maturation of porcine oocytes and in vitro development of porcine IVF embryos. It has been demonstrated the effect of tubacin on the acetylation level of α-tubulin in porcine oocytes. As a result, the maturation rate of porcine oocytes was significantly improved (P < 0.05), and the following development potent of blastocysts forming rate was also significantly increased (P < 0.05). We found that the increased acetylation of α-tubulin significantly reduced the abnormal rate of microtubule, furthermore, the proportion of mitochondria in the vicinity of in vitro fertilization (IVF) nucleus was significantly enhanced in Metaphase I (MI) and Metaphase II (MII) stages. The expression levels of microtubule assembly genes (TUBA1A, αTAT1 and MAP2) significantly up-regulated in MI and MII stages. Together, these results suggest that treatment of porcine oocytes during maturation with tubacin could promote their IVF embryos developmental competence by altering spindle formation, mitochondrial concentration and genes expression patterns of matured porcine oocytes.

Efficient generation of GGTA1-deficient pigs by electroporation of the CRISPR/Cas9 system into in vitro-fertilized zygotes

BackgroundXenoantigens are a major source of concern with regard to the success of interspecific xenografts. GGTA1 encodes α1,3-galactosyltransferase, which is essential for the biosynthesis of galactosyl-alpha 1,3-galactose, the major xenoantigen causing hyperacute rejection. GGTA1-modified pigs, therefore, are promising donors for pig-to-human xenotransplantation. In this study, we developed a method for the introduction of the CRISPR/Cas9 system into in vitro-fertilized porcine zygotes via electroporation to generate GGTA1-modified pigs.ResultsWe designed five guide RNAs (gRNAs) targeting distinct sites in GGTA1. After the introduction of the Cas9 protein with each gRNA via electroporation, the gene editing efficiency in blastocysts developed from zygotes was evaluated. The gRNA with the highest gene editing efficiency was used to generate GGTA1-edited pigs. Six piglets were delivered from two recipient gilts after the transfer of electroporated zygotes with the Cas9/gRNA complex. Deep sequencing analysis revealed that five out of six piglets carried a biallelic mutation in the targeted region of GGTA1, with no off-target events. Furthermore, staining with isolectin B4 confirmed deficient GGTA1 function in GGTA1 biallelic mutant piglets.ConclusionsWe established GGTA1-modified pigs with high efficiency by introducing a CRISPR/Cas9 system into zygotes via electroporation. Multiple gene modifications, including knock-ins of human genes, in porcine zygotes via electroporation may further improve the application of the technique in pig-to-human xenotransplantation.

Fatty acid metabolism as an indicator for the maternal–to–zygotic transition in porcine IVF embryos revealed by RNA sequencing

A number of fatty acids have been found in porcine oocytes and early embryos. Recent studies have indicated the importance of fatty acids in the development of pre-implantation porcine embryos, whether derived from in vivo or somatic cell nuclear transfer. However, the effects of fatty acids on porcine embryos produced by in vitro fertilization (IVF) remain poorly defined. This study aimed to investigate the patterns of gene expression and functions of fatty acids in pre-implantation IVF porcine embryos at different stages using transcriptome sequencing. We found that, in IVF porcine embryos, genes related to fatty acid metabolism were positively expressed during early embryonic development. Additionally, the expression of genes related to lipid metabolism changed dramatically during the maternal–to–zygotic transition (MZT), and the genes associated with lipid metabolism were correlated with zygotic genome activation in porcine IVF embryos, suggesting that fatty acid metabolism plays an important role in MZT. In summary, fatty acid metabolism may be an indicator of MZT in porcine IVF embryos, which presents new considerations for exploring the regulatory mechanisms of this process.

Periovulatory oviductal fluid decreases sperm protein kinase A activity, tyrosine phosphorylation, and in vitro fertilization in pig.

Molecules from the female reproductive tract modulate capacitation and function of sperm cells in vivo. These molecules vary in a quantitative and qualitative manner throughout the estrous cycle. This work evaluates the effect of using various female reproductive fluids on capacitation and fertilization of pig spermatozoa in vitro. The effects of culturing spermatozoa in different fluids on the levels of sperm protein kinase A (pPKA), tyrosine phosphorylation, acrosome reaction, and in vitro fertilization (IVF) were evaluated. The fluids tested were as follows: oviductal fluid (OF) from five phases of the estrous cycle, namely early and late follicular (OF-EF, OF-LF), early and late luteal (OF-EL, OF-LL) and periovulatory (pOF), follicular fluid from medium-sized follicles, and secretions of cumulus-oocyte complexes (conditioned medium). The pPKAs and tyrosine phosphorylation were decreased by OF-EF, OF-LF, OF-EL, and pOF but not by follicular fluid and conditioned medium. OF-EF, OF-LF, and pOF also decreased the sperm acrosome reaction. Moreover, the effect of pOF on pPKAs and tyrosine phosphorylation was reversible. In in vitro fertilization, OF-EF, OF-LF, OF-EL, and pOF reduced the percentage of penetrated oocytes, the mean number of spermatozoa per penetrated oocyte, and increased monospermy. OF from follicular, early luteal, and periovulatory phases of the estrous cycle modulates the sperm protein phosphorylation as well as the acrosome reaction involved in capacitation and increases monospermic fertilization in in vitro fertilization. Our findings suggest that fluids from the female reproductive tract could be used as additives in porcine IVF systems to modulate sperm-oocyte interaction.

Selection based on morphological features of porcine embryos produced by in vitro fertilization: Timing of early cleavages and the effect of polyspermy.

The aim of this study was to examine whether a morphological approach is efficient for selecting high‐quality porcine embryos produced by in vitro fertilization (IVF) under high polyspermy conditions. Frozen‐thawed Meishan epididymal spermatozoa showing moderate and high polyspermy were subjected to IVF (1 × 105 sperms/ml). Under conditions of moderate polyspermy, 4‐cell embryos selected at 48 hr after IVF (single selection) and 8‐cell embryos selected at 79 hr after IVF from the collected 4‐cell embryos (double selection) showed high developmental competence. Likewise, 4‐ and 8‐cell embryos produced by IVF under high polyspermy conditions also showed high competence for development to blastocysts. However, blastocysts derived from high polyspermy conditions had significantly fewer cells than those produced under moderate polyspermy conditions. Furthermore, the frequency of nuclear and chromosomal abnormalities in 4‐ and 8‐cell embryos produced under conditions of high polyspermy was significantly (p < .05) higher in comparison to moderate polyspermy conditions. These findings suggest that although high polyspermy affects the frequency of nuclear and chromosomal anomalies in porcine IVF embryos, subsequent selection based on morphological features of 4‐ and 8‐cell embryos even under high polyspermy conditions, could be an alternative option for selecting porcine IVF embryos with high development ability.

Porcine IVF embryo development and estrogen receptors are influenced by the concentration of percoll gradients during sperm selection.

This study aimed to evaluate the effect of three different concentrations of discontinuous gradients of percoll (90/45, 80/40, and 70/35) in the outcome of porcine in vitro fertilization (IVF) and its influence on further embryo development and quality. Embryo viability was assessed by the expression of estrogen receptors (E2 R) and cleaved caspase-3 (CC3). The highest percoll concentration (90/45) resulted in the lowest embryo production (24.9%) in comparison with 80/40 (37.5%) and 70/35 (40.0%), with the production being similar between the two lowest concentrations. The hatching rate for 90/45 (26.2%) was lower than for 80/40 (45.5%), and both were similar to the Group 70/35 (32.9%). The hatched embryos from the concentration 90/45 showed the lowest proportion of E2 R expression (3.6%), while the Groups 80/40 (22.6%) and 70/35 (39.3%) had a similar proportion of expression. The live embryos that did not hatch until Day 8 of culture presented a higher CC3 proportion for Group 90/45 (18.3%), in comparison with 80/40 (12.7%) and 70/35 (10.7%), with the latter two being similar. In conclusion, adjustments in percoll concentration used for sperm selection before porcine IVF can improve embryo production and competence for pregnancy recognition and establishment.

Generation of CD163-edited pig via electroporation of the CRISPR/Cas9 system into porcine in vitro-fertilized zygotes

CD163 is a putative fusion receptor for virus of porcine reproductive and respiratory syndrome (PRRS). In this study, we introduced a CRISPR/Cas9 system [guide RNAs (gRNAs) with Cas9 protein] targeting the CD163 gene into in vitro-fertilized porcine zygotes by electroporation to generate CD163-modified pigs. First, we designed four types of gRNAs that targeted distinct sites in exon 7 of the CD163 gene. Cas9 protein with different gRNAs was introduced into in vitro-fertilized zygotes by electroporation. When the electroporated zygotes were allowed to develop to blastocysts in vitro and the genome editing efficiency was evaluated using these blastocysts, three (gRNA1, 2, and 4) of the four gRNAs tested successfully edited the CD163 gene. To generate CD163-knockout pigs, a total of 200 electroporated zygotes using these three gRNAs were transferred into the oviducts of oestrous-synchronized surrogate and the surrogate gave birth to eight piglets. Subsequent sequence analysis revealed that one of the piglets carried no wild-type sequence in CD163 gene. The other seven piglets carried only wild-type sequence. Thus, we successfully generated a CD163-edited pig by electroporation of the CRISPR/Cas9 system into in vitro-fertilized zygotes, although further improvement is required to generate genetically modified pigs with high efficiency.

High pre-freezing sperm dilution improves monospermy without affecting the penetration rate in porcine IVF

The high incidence of polyspermy is still an unresolved problem for the production of in vitro-produced porcine embryos. In this work, we modified the usual sperm processing sequence for in vitro fertilization (IVF), and the spermatozoa from four boars were frozen directly at a low sperm concentration of 20 × 106 sperm/mL (high pre-freezing sperm dilution group; F20), thawed and processed for IVF in three replicates. Spermatozoa from the same boars frozen at a conventional concentration (1000 × 106 sperm/mL) were used as the control group. The post-thaw sperm quality evaluation demonstrated that despite there being no differences in the percentage of motile spermatozoa between groups, the proportion of live spermatozoa with intact acrosomes was significantly higher in the F20 group than in the control. The in vitro penetration rate was also similar between groups; however, the co-incubation of oocytes with F20 sperm increased monospermy, IVF efficiency, cleavage rate and the efficiency of blastocyst formation compared with the results for oocytes co-incubated with control spermatozoa. These results indicate, for the first time, that a high pre-freezing sperm dilution increases monospermy without affecting penetration rates, thereby increasing blastocyst formation.

Function of berberine on porcine in vitro fertilization embryo development and differential expression analysis of microRNAs.

The effect of berberine (Ber) on in vitro fertilization (IVF) embryo development in pigs and the associated differential expression of microRNAs (miRNAs) in the embryo were investigated. NCSU-23 embryonic culture medium was used for a control group, while NCSU-23 embryonic culture medium added with Ber was used for a Ber group. The embryo development rates in these groups were determined, and the zygotes, 4- and 8-cell embryos, and blastocysts were collected for cDNA microarray analysis. The development rates of 2-, 4-, 8-cell embryos and blastocysts were significantly higher in the Ber group than those in the control group (p<0.01). The differentially expressed miRNAs in the 8-cell versus the 4-cell stage in control group as well as in the 8-cell Ber group versus the 8-cell control group overlapped, and it was found that nine miRNAs were commonly upregulated and two of them were downregulated, while there was no overlap among the other groups. The target genes of Ber-regulated miRNAs at the 8-cell stage were mainly associated with the molecular pathway of nucleic acid and protein synthesis. These findings suggest that Ber may regulate the expression of miRNAs at the 8-cell stage, which is beneficial to provide material reserves for the maternal to zygote transition of porcine embryos, thereby increasing the porcine IVF embryo development rate.

GDF8 enhances SOX2 expression and blastocyst total cell number in porcine IVF embryo development

Growth differentiation factor 8 (GDF8) is a member of the transforming growth factor-β family and a physiological regulator. According to recent studies, GDF8 can be detected in follicular fluid and the uterus, suggesting that GDF8 may affect preimplantation embryonic development and act in a paracrine manner to improve the success of late-blastocyst implantation in vivo. We investigated the effect of GDF8 supplementation during in vitro culture (IVC) of porcine embryos derived from in vitro fertilization (IVF) and parthenogenetic activation (PA) on cleavage, blastocyst formation rate, and total cell number and analysed gene transcription levels and cell linage specification in the resulting blastocysts. First, the concentration of GDF8 in porcine oviductal fluid was determined to be 139.8 pg/mL. Then, 0, 0.2, 2, or 20 ng/mL GDF8 was added to embryos throughout the entire IVC period. Our results showed that supplementation with GDF8 during porcine preimplantation embryo IVC enhanced blastocyst formation and total cell number and altered the transcriptional patterns of genes that regulate pluripotency and cavitation. Furthermore, using differential immunostaining, we demonstrated that supplementation with GDF8 enhanced the expression of the genuine inner cell mass (ICM) marker SOX2 and the ICM/trophectoderm ratio, improving IVF blastocyst quality. In conclusion, for the first time, we demonstrated the presence of the in vivo oviductal factor GDF8 in oviductal fluid. Furthermore, we found that GDF8 supplementation at 0.2 ng/mL increased the blastocyst total cell number and ICM/trophectoderm ratio by inducing the transcription of genes involved in developmental competence and the expression of genuine ICM marker SOX2 during porcine IVF embryo development in vitro.

Histone 3 lysine 4, 9, and 27 demethylases expression profile in fertilized and cloned bovine and porcine embryos.

Epigenetic modifications in the C-terminal domain of histones coordinate important events during early development including embryo genome activation (EGA) and cell differentiation. In this study, the mRNA expression profile of the main lysine demethylases (KDMs) acting on the lysine 4 (H3K4), 9 (H3K9), and 27 (H3K27) of the histone H3 was determined at pre-, during and post-EGA stages of bovine and porcine embryos produced by in vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT). In IVF embryos, mRNA abundance of most KDMs revealed a bell-shaped profile with peak expression around the EGA period, i.e. Day 3 for porcine (KDM2B, KDM5B, KDM5C, KDM4B, KDM4C, KDM6A, KDM6B, and KDM7A), and Day 4 for bovine (KDM1A, KDM5A, KDM5B, KDM5C, KDM3A, KDM4A, KDM4C, and KDM7A). The mRNA profile of KDM1A, KDM2B, KDM3A, KDM3B, KDM6A, and KDM6B differed between porcine and bovine IVF embryos. Several differences were also observed between SCNT and IVF, which includes a precocious peak in the mRNA expression of KDM1A, KDM3A, KDM4C, KDM5A, KDM5B, KDM5C, KDM6A, and KDM7A in bovine SCNT embryos; absence of mRNA peak for KDM4B, KDM4C, and KDM6A in porcine SCNT embryos; and early decreasing in KDM5B and KDM5C mRNA in porcine SCNT embryos. Based on the mRNA profile, this study has identified several KDMs that are likely involved in the regulation of the EGA transition, KDMs that may have a species-specific role in bovine and porcine embryos, and KDMs that are improperly expressed during cell reprogramming in SCNT embryos.

Dynamic changes of SETD2, a histone H3K36 methyltransferase, in porcine oocytes, IVF and SCNT embryos.

SETD2 (SET domain containing protein 2) acts as a histone H3 lysine 36 (H3K36)-specific methyltransferase and may play important roles in active gene transcription in human cells. However, its expression and role in porcine oocytes and preimplantation embryos are not well understood. Here, we used immunofluorescence and laser scanning confocal microscopy to examine SETD2 expression in porcine fetal fibroblasts, oocytes, and preimplantation embryos derived from in vitro fertilization (IVF), parthenogenetic activation (PA), and somatic cell nuclear transfer (SCNT). In porcine fetal fibroblasts, SETD2 expression was detected in interphase cells, but not in M (mitotic)-phase cells. The SETD2 signal was observed in non-surrounded nucleolus (NSN)-stage oocytes, but not in surrounded nucleolus (SN)-, metaphase I (MI)-, or metaphase II (MII)-stage oocytes. The SETD2 signal was detectable in sperm, and undetectable immediately after fertilization, detectable at the 2-cell stage, and peaked at the 4-cell stage of IVF embryos in which porcine embryonic genome is activated. Similar to the pattern found in IVF embryos, the SETD2 signal was absent from PA embryos at the 1-cell stage, but it was detected at the 2-cell stage and thereafter maintained to the blastocyst stage. Interestingly, unlike the IVF and PA embryos, the SETD2 signal was detected throughout the development of SCNT embryos, including at the 1-cell stage. These data suggest that SETD2 may be functional for embryonic gene transcription in porcine preimplantation embryos. It is further speculated that the aberrant expression of SETD2 at the 1-cell stage of porcine SCNT embryos may be a factor in the low efficiency of cloning in pig.

Improving porcine in vitro fertilization output by simulating the oviductal environment

Differences between the in vitro and in vivo environment in which fertilization occurs seem to play a key role in the low efficiency of porcine in vitro fertilization (IVF). This work proposes an IVF system based on the in vivo oviductal periovulatory environment. The combined use of an IVF medium at the pH found in the oviduct in the periovulatory stage (pHe 8.0), a mixture of oviductal components (cumulus-oocyte complex secretions, follicular fluid and oviductal periovulatory fluid, OFCM) and a device that interposes a physical barrier between gametes (an inverted screw cap of a Falcon tube, S) was compared with the classical system at pHe 7.4, in a 4-well multidish (W) lacking oviduct biological components. The results showed that the new IVF system reduced polyspermy and increased the final efficiency by more than 48%. This higher efficiency seems to be a direct consequence of a reduced sperm motility and lower capacitating status and it could be related to the action of OFCM components over gametes and to the increase in the sperm intracellular pH (pHi) caused by the higher pHe used. In conclusion, a medium at pH 8.0 supplemented with OFCM reduces polyspermy and improves porcine IVF output.

Factors affecting efficiency of introducing foreign DNA and RNA into parthenogenetic or in vitro-fertilized porcine eggs by cytoplasmic microinjection.

Cytoplasmic microinjection (CI) of foreign gene into in vivo fertilized zygotes has emerged as a useful tool for transgenic pig production. In the current study, we investigated factors affecting transgenic efficiency and developmental potential of parthenogenetic (PA) and in vitro-fertilized (IVF) porcine embryos produced by CI. These factors included adding of RNase inhibitor, DNA or RNA concentration, injection time, and different structures of plasmids. Our results showed that adding of 1-4U/μL of RNase inhibitor did not have negative effect on development potential of CI-PA embryos, and RNase inhibitor injection significantly increased EGFP expressing rate of CI-PA embryos. High injection DNA concentration and long injection interval after PA significantly reduced blastocyst formation. Different molecular structures such as DNA or RNA affected CI-PA embryos development, and RNA had little harmful effect on pig's early embryonic development. EGFP expression rate of CI-IVF embryos was improved following the increase of foreign DNA concentration, but blastocyst formation rate was decreased. Injection time after IVF did not show any significant difference on embryonic development, but longer interval resulted in a significantly lower EGFP expressing rate. Cas9 mRNA and myostatin (GDF-8) sgRNA co-injection indicated that the mutation rate of CI-IVF group was significantly higher than that of CI-PA. The CI-IVF-generated embryos were then transferred to six recipient pigs, but no live piglets were obtained. The following pronuclear formation assessment showed more than 76.1% IVF zygotes were polyspermy. These results demonstrate that CI-PA and CI-IVF were effective methods for production of transgenic pig embryos. However, polyspermic fertilization and poor quality of porcine IVF blastocysts are still the main problem of resulting in pregnancy failure.