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The expression and function of bone morphogenetic protein 4 (BMP4) gene in bovine cumulus cells (CCs) was investigated to reveal the mechanisms by which it regulated cell apoptosis and proliferation. The mRNA and protein expression of BMP4 were detected using quantitative PCR (qPCR) and immunofluorescence staining in CCs. The effective siRNAs against BMP4 gene were screened using qPCR and western blotting. The mRNA expression levels of apoptosis-related genes and proliferation-related genes were estimated by qPCR after knocking-down the BMP4 gene in bovine CCs. Cell apoptosis, proliferation and cell cycle were measured with Annexin V-FITC, CCK-8 and propidium iodide staining by flow cytometry. Results showed that the BMP4 gene was expressed and its protein was in the cytoplasm and nuclei of bovine CCs. The BMP4 knockdown increased the cell apoptosis rate and upregulated the mRNA levels of apoptosis genes CASPASE-3 and BAX with downregulation of the anti-apoptosis gene BCL-2 (P 0.05). The proliferation rate declined and the mRNA expression levels of proliferation-related genes PCNA, CDC42 and CCND2 were downregulated in the bovine CCs with BMP4 low expression (P 0.05). The BMP4 knockdown significantly increased the percentage of G0/G1 phase cells while decreased that of S phase cells. Therefore, the expression of BMP4 and its biological functions on the cell proliferation, apoptosis and cell cycle of bovine CCs were first studied. BMP4 knockdown induced cell apoptosis, cell cycle arrest and inhibited proliferation of bovine CCs.

This article presents a time-domain macromodeling algorithm for buried transmission lines subject to coupling of an incident field and terminated with linear and nonlinear loads. Different from overhead lines, the determination of the transient ground admittance is of great importance due to the complexity of the analytical expressions and the necessity of assuring a good approximation over a wide frequency range. The proposed macromodel adopts the analog behavioral modeling (ABM) library of Spice solvers to model frequency-dependent transmission line parameters directly, and calculate convolution in time domain, making it simple and convenient to treat time-domain field-to-line coupling equations without the need of time and spatial discretization and curve fitting used in other time-domain numerical algorithms. Besides, the model complexity is not limited by the line length. Hence, the proposed algorithm can significantly simplify the modeling process and improve simulation efficiency, especially for long multiconductor transmission lines. Furthermore, an effective and simplified expression for self and mutual ground impedance and admittance of underground lines is proposed in this article. With the proposed expression, the time-consuming numerical integration can be avoided, leading to a saving of 98% of the computational time. Finally, several validation examples of field coupling to buried power cables terminated with linear and nonlinear devices are investigated. The comparison of calculated waveshapes and CPU times shows the accuracy and efficiency of the proposed method.

Melatonin (MT) increases oocyte maturation by reducing reactive oxygen species level and enhancing oocyte antioxidant capacity. However, the mechanisms via which MT works are still poorly understood. In the present study, the effects of MT on the maturation rate and development ability of bovine oocytes were investigated. Then, the transcriptome of oocytes treated by MT was sequenced. Finally, the expression of gap junction protein alpha 4 (GJA4) protein and cAMP level were detected in bovine oocytes, and isoprenaline (enhancer of gap junctional intercellular communication (GJIC)) and heptanol (inhibitor of GJIC) were used to investigate the effect of MT on GJIC activity in bovine oocytes. Our results showed that MT significantly improved the maturation, developmental ability and mRNA expression of GJA4 of bovine oocytes. Meanwhile, MT significantly increased GJA4 protein level and cAMP level in bovine oocytes. In contrast to heptanol, both isoproterenol and MT significantly increased GJIC activity, nuclear maturation and the development ability of bovine oocytes. However, MT significantly restored the nuclear maturation and developmental ability of oocytes treated by heptanol. In conclusion, our results showed that MT improves the maturation and developmental ability of bovine oocytes by enhancing GJIC activity via up-regulating GJA4 protein expression in IVM progress.

The traveling-wave Marx generator was first proposed by Carl Baum to analyze the switch-closure propagation process in fast pulsed-power systems. To improve the performance of the transistorized sub-nanosecond pulse generator, the traveling-wave Marx circuit with inter-stage transmission lines is studied. Simulations indicate that the delay time and the impedance of inter-stage transmission lines [microstrip lines on printed circuit board (PCB)] are main factors affecting the pulse waveform. Based on it, the series connection of modularized Marx circuits is proposed, which has the advantages of adjustable amplitude and bendable circuit layout. A compact high-amplitude pulse generator is developed with the amplitude of 8.2 kV, the rise time of 150 ps, and the PCB length of 35 cm. For higher pulse repetition rate, the heat dissipation design of PCB is necessary; however, due to the variations in the microstrip line structure, the commonly used metallic heat sink may cause a significant decrease in pulse amplitude. Thus, a novel heat dissipation design is proposed by optimizing the layout of metallic sinks and introducing heat-conducting ceramic sinks. A high-repetition-rate pulse generator is developed with the maximum pulse repetition rate of 600 kHz, the amplitude of 1.1 kV, and the rise time of 160 ps.

Objective Epigallocatechin-3-gallate (EGCG) is a major ingredient of catechin polyphenols and is considered one of the most promising bioactive compounds in green tea because of its strong antioxidant properties. However, the protective role of EGCG in bovine oocyte in vitro maturation (IVM) has not been investigated. Therefore, we aimed to study the effects of EGCG on IVM of bovine oocytes. Methods Bovine oocytes were treated with different concentrations of EGCG (0, 25, 50, 100, and 200 μM), and the nuclear and cytoplasmic maturation, cumulus cell expansion, intracellular reactive oxygen species (ROS) levels, total antioxidant capacity, the early apoptosis and the developmental competence of in vitro fertilized embryos were measured. The mRNA abundances of antioxidant genes (nuclear factor erythriod-2 related factor 2 [NRF2], superoxide dismutase 1 [SOD1], catalase [CAT], and glutathione peroxidase 4 [GPX4]) in matured bovine oocytes were also quantified. Results Nuclear maturation which is characterized by first polar body extrusion, and cytoplasmic maturation characterized by peripheral and cortical distribution of cortical granules and homogeneous mitochondrial distribution were significantly improved in the 50 μM EGCG-treated group compared with the control group. Adding 50 μM EGCG to the maturation medium significantly increased the cumulus cell expansion index and upregulated the mRNA levels of cumulus cell expansion-related genes (hyaluronan synthase 2, tumor necrosis factor alpha induced protein 6, pentraxin 3, and prostaglandin 2). Both the intracellular ROS level and the early apoptotic rate of matured oocytes were significantly decreased in the 50 μM EGCG group, and the total antioxidant ability was markedly enhanced. Additionally, both the cleavage and blastocyst rates were significantly higher in the 50 μM EGCG-treated oocytes after in vitro fertilization than in the control oocytes. The mRNA abundance of NRF2, SOD1, CAT, and GPX4 were significantly increased in the 50 μM EGCG-treated oocytes. Conclusion In conclusion, 50 μM EGCG can improve the bovine oocyte maturation, and the protective role of EGCG may be correlated with its antioxidative property.

Many experiments show that vitrification significantly reduces the fertilization capacity of mammalian oocytes, restricting the application of vitrified oocytes. It has been proven that the JUNO protein plays a vital role in mammalian oocytes fertilization. However, little information is available about the effects of vitrification on the JUNO protein and the procedure to protect it in bovine oocytes. Here, the present study was designed to investigate the effect of vitrification on the JUNO protein level in bovine oocytes. In this study, MII oocytes were treated with cholesterol-loaded methyl-β-cyclodextrin (CLC; 0, 10, 15, 20 mM) for 45 min before vitrification and methyl-β-cyclodextrin (MβCD; 0, 2.25, 4.25, 6.25 mM) for 45 min after thawing (38-39°C). Then, the expression level and function of JUNO protein, cholesterol level in the membrane, the externalization of phosphatidylserine, sperm binding capacity and the developmental ability of vitrified bovine oocytes were examined. Our results showed that vitrification significantly decreased the JUNO protein level, cholesterol level, sperm binding capacity, development ability, and increased the promoter methylation level of the JUNO gene and apoptosis level of bovine oocytes. Furthermore, 15 mM CLC + 4.25 mM MβCD treatment significantly improved the cholesterol level and increased sperm binding and development ability of vitrified bovine oocytes. In conclusion, the combination treatment of cholesterol-loaded methyl-β-cyclodextrin and methyl-β-cyclodextrin significantly improves the fertilization capacity of vitrified bovine oocytes by protecting fertilization protein JUNO.

This study aims to investigate the expression and function of absent, small, or homeotic 1-like (ASH1L) methyltransferase in bovine cumulus cells in order to reveal by which mechanisms ASH1L regulates epigenetic modification and apoptosis in cumulus cells. The location of ASH1L and the methylation pattern of H3K36 were detected using immunofluorescence staining in cumulus cells. Quantitative PCR (qPCR) and western blotting were used to screen for effective siRNA targeting the ASH1L gene. Also, the mRNA expression levels of apoptosis-related genes and polycomb inhibitory complex genes were estimated by qPCR after knocking down the ASH1L gene in bovine cumulus cells. Cell proliferation and apoptosis were measured with the CCK-8 method and Annexin V-FITC by flow cytometry, respectively. The results of immunofluorescence analysis showed that ASH1L is located in the nucleus of bovine cumulus cells and is distributed in a dotted pattern. ASH1L knockdown in cumulus cells induced a decrease in the levels of H3K36me1/2/3 methylation (P 0.05). Additionally, ASH1L knockdown inhibited cell proliferation, increased the apoptosis rate, and upregulated the expression of apoptosis genes CASPASE-3, BAX and BAX/BCL-2 ratio (P 0.05). Meanwhile, the mRNA expression levels of EZH2 and SUZ12, two subunits of PRC2 protein, were increased in cells with ASH1L knockdown (P 0.05). Therefore, the expression of ASH1L methyltransferase and its function in on the apoptosis of bovine cumulus cells were first studied. The mechanism by which ASH1L regulates the histone methylation and apoptosis in cumulus cells was also revealed.

We measured differential expression profiles of genes and long non-coding RNA (lncRNA) using RNA sequencing in bovine embryos with or without glutathione (GSH) treatment. Bovine embryos fertilized in vitro were treated with GSH to blastocyst. Embryos at the 8-16-cell and morula stages were collected, with embryos without GSH treatment as the control. RNA was isolated, amplified, and sequenced. Differentially expressed genes (DEGs) and lncRNAs (DElncRNAs) were identified and bioinformatic analyses carried out. Transcript levels were confirmed using quantitative RT-PCR. A total of 4100 DEGs were identified, of which 3952 were in GSH-treated morulae and 884 in untreated morulae. More gene ontology (GO) terms were associated with GSH treatment than with control conditions. KEGG analysis showed that glutathione metabolism, citrate cycle, and metabolic pathways involving glycine, serine, and threonine were observed only in GSH-treated embryos. Among 4273 DElncRNAs identified, 59 were potentially important in GSH-treated embryo development, including 14 involved in glutathione metabolism. The 59 DElncRNAs co-expressed with protein-coding mRNAs involved similar GO terms and pathways as the DEGs. This appears to be the first comprehensive profiling of DEGs and DElncRNAs in bovine embryos fertilized in vitro with or without GSH, and the first systematic screen of potential lncRNAs in bovine embryos.

The fertilization capacity of sex-sorted sperms is seriously decreased, which inhibits its wide application. However, little information is still available about the effect of vitamin C (VC) and lycopene (Lyc) on the fertilization capacity of sex-sorted bull sperm. In this study, the washing medium and fertilization medium of sex-sorted sperm from three bull individuals were supplemented with different concentrations of VC (0, 1 × 10-3 , 1 × 10-4 , 1 × 10-5 , 1 × 10-6 M) or Lyc (0, 1 × 10-4 , 1 × 10-5 , 1 × 10-6 , 1 × 10-7 ). After washing twice and incubation for 1.5 hr, the malondialdehyde (MDA) level, phosphatidylserine (PS) translocation, membrane potential (Δψm) and IVF (in vitro fertilization) ability of sex-sorted sperm were investigated. For the sex-sorted sperm of bulls A, B and C, 1 × 10-3 M VC or 1 × 10-4 M Lyc treatment significantly decreased their MDA levels and PS translocation and increased their Δψm levels and cleavage rates after IVF. When blastocysts were concerned, 1 × 10-4 M Lyc significantly improved the blastocyst rates and their IFN-tau expression of bulls A and C. In conclusion, supplementation of 1 × 10-3 M VC or 1 × 10-4 M Lyc in washing and fertilization medium contributed greatly to improving the fertilization capacity of sex-sorted bull sperm during IVF procedure.

The aim of this study was to test the effects of five different concentrations (0, 10−3, 10−4, 10−5, and 10−6 M) of resveratrol (Res) supplementation in bull sperm washing and fertilisation medium on levels of reactive oxygen species (ROS), phosphatidylserine (PS) externalisation, mitochondrial membrane potential (Δψm), ATP and malondialdehyde (MDA), acrosomal integrity, blastocyst rate, and blastocyst quality after in vitro fertilisation (IVF). The results for sex-sorted sperm from three bulls showed: (1) ROS and MDA levels in 10−3 M and 10−4 M Res groups were significantly lower than those of controls (P −3 M and 10−4 M Res groups were significantly higher than those of controls (P −4 M Res group were significantly higher than those of controls (P −4 M Res supplementation in washing and fertilisation medium of sex-sorted bull sperm significantly decreased ROS, PS externalisation, and MDA, and protected mitochondrial function and acrosomal integrity, thereby increasing blastocyst percentage and quality following IVF.

Neonatal respiratory distress is a major mortality factor in cloned animals, but the pathogenesis of this disease is rarely investigated. In this study, four neonatal cloned cattle, born after full-term gestation, exhibited symptoms of neonatal respiratory distress syndrome (NRDS), which included symptoms of hyaline membrane disease as well as disordered surfactant homeostasis in their collapsed lungs. No differences in DNA methylation or histone modifications correlated with the suppressed SPB and SPC transcription observed in the cloned cattle group (p > 0.05), whereas TTF-1 occupancy at SPB and SPC promoter regions in cloned cattle was significantly reduced to 24% and 20% that of normal lungs, respectively (SPB, p

Neonatal respiratory distress is a major mortality factor in cloned animals; however, the pathogenesis of this disease has rarely been investigated. Previous studies have shown that miRNAs regulate critical genes related to lung development, cell differentiation, surfactant synthesis, secretion and lung disease. This study aimed to examine differentially expressed miRNAs in collapsed lungs of cloned bovine neonates and normal lungs in order to identify key pathways and functions that might be related to the pathogenesis of neonatal respiratory distress. In this study, miRNA transcriptomes of collapsed lungs of neonatal cloned bovines and normal lungs were analysed by next-generation sequencing and the results were validated using quantitative real-time PCR (qRT-PCR). A total of 177 differentially expressed miRNAs were identified in the two groups (fold change > 2, RPM ≥ 5), some of which were associated with type II cell differentiation, for example, mmu-miR-29a-5p_L-2R+1, hsa-miR-200c-5p_L-1R+1 and mmu-miR-18a-3p_R+1. The differentially expressed miRNAs were predicted to 6,031 target genes. By Gene Ontology (GO) and Kyoto Encyclopeida of Genes and Genomes (KEGG) DATA base, 133 significant GO terms (p < .05) and 13 significant KEGG pathways (p < .05) were obtained. Many of them were associated with lung development and surfactant homoeostasis, such as lipid biosynthetic processes, protein transport, endocytosis, lysosome, endosome, Golgi apparatus and membrane. Our results of miRNAs express profiles may partially explain the respiratory distress and lung collapse in neonatal bovine clones and could provide novel insights into roles of miRNAs in regulation of lung collapse and neonatal respiratory distress in cloned farm animals.

The present study investigated the effects of melatonin on bovine oocyte maturation and subsequent embryonic development in vitro. Results showed that the nuclear and cytoplasmic maturation, characterized by first polar body extrusion, normal distribution of cortical granules and mitochondria, as well as increased mitochondrial membrane potential, were significantly improved in 10-9 mol/L melatonin-treated oocytes. Melatonin supplementation reduced intracellular reactive oxygen species level and enhanced glutathione production. Meanwhile, the presence of melatonin (10-9 mol/L) during oocyte maturation resulted in a decreased early apoptotic rate in oocytes. After in vitro fertilization, oocytes receiving melatonin supplementation exhibited a significantly higher blastocyst formation rate and yielded a markedly lower number of apoptotic cells. Mechanistic explorations showed that addition of 10-9 mol/L melatonin to in vitro maturation media significantly attenuated the transcript level of caspase-3, while the expressions of BCL-2, XIAP, CAT and HSP70 were significantly reinforced in the resultant embryos. Taken together, melatonin ameliorates bovine oocyte maturation potential, and the beneficial effects can affect subsequent embryonic development. The protective role of melatonin may be due to its anti-apoptotic and anti-oxidative activities.

Contents Vitrification has been shown to decrease the developmental capacity of mammalian oocytes, and this is closely associated with the abnormal mRNA expressions of vitrified oocytes. However, the effect of vitrification on transcriptional machinery of oocytes examined by RNA sequencing (RNA-seq) has yet to be defined. In the present study, the mRNA transcriptomes of fresh and vitrified bovine oocytes were analysed by Smart-seq2 with the differently expressed genes determined by DEseq2 (an adjusted p-value of .05 and a minimum fold change of 2). The differentially expressed mRNAs were then searched against the Gene Ontology (GO) and Genomes (KEGG) database. Finally, the mRNA expressions of 10 candidate genes were validated using quantitative real-time PCR (qRT-PCR). Approximately 12,000 genes were detected in each sample of fresh or vitrified oocytes. Of these, the expression levels of 102 genes differed significantly in vitrified groups: 12 genes mainly involved in cell cycle, fertilization and glucose metabolism were upregulated, and 90 genes mainly involved in mitochondria, ribosomal protein, cytoskeleton, transmembrane protein, cell cycle and calcium ions were downregulated. GO analysis showed that these genes were mainly enriched in terms of membrane-bounded organelles, macromolecular complex, and intracellular part. The mRNA expression levels of 10 candidate genes selected randomly were in agreement with the results of the RNA-seq. In conclusion, our results showed that vitrification affected the mRNA transcriptome of bovine oocytes by downregulating genes, which contributed to the decreased developmental capacity of vitrified oocytes. Our findings will be useful in determining approaches to improve the efficiency of vitrified oocytes.

SUMMARY We aimed to investigate the effect of melatonin on bovine frozen-thawed semen and its impact on fertilization outcome. Plasma membrane integrity, mitochondrial activity, acrosome integrity, and levels of intracellular reactive oxygen species (ROS) were measured in spermatozoa treated with different concentrations of melatonin. Melatonin-treated spermatozoa were then used for in vitro fertilization, followed by analysis of subsequent embryo development and the expression of apoptosis- and antioxidant-related genes. The results revealed that 10−5 and 10−3 M melatonin led to higher plasma membrane integrity, mitochondrial activity, and acrosome integrity, and significantly decreased intracellular ROS levels (P

The rabbit is considered an important model animal from which to obtain embryonic stem cells because of the utility of this animal in physiology and reproductive research. Here, we derived rabbit ES-like (rES-like) cells from blastocysts of superovulated Japanese white rabbits using culture medium containing 10-7 M melatonin, 10 ng/mL basic fibroblast growth factor, and 1,000 IU/mL human leukemia inhibitory factor. This concentration of melatonin had the most significant positive effects on the proliferation inner cell mass-derived cells (improving rates from 19.97% to 34.57%) and the longevity of passaging rES-like cells. Melatonin also enhanced the expression of pluripotent genes-including alkaline phosphatase, Pou5f1, Sox2, Klf4, c-Myc, Nanog, Line28a, and surface marker proteins-in fifth-passage rES-like cells. In vitro, these rES-like cells could spontaneously differentiate into some somatic cells, such as beating cardiomyocytes; formed embryoid bodies; expressed markers of the three germ layers after differentiation; and formed teratomas after injection into non-obese diabetic-severe combined immune deficient (NOD-SCID) mice. Thus, melatonin helped coax ES-like cells from rabbit blastocysts, which raises intriguing questions about the relationship between pluripotency and proliferation in rabbit embryonic stem cells. Mol. Reprod. Dev. 83: 1003-1014, 2016 © 2016 Wiley Periodicals, Inc.

To explore the mechanisms leading to the poor quality of IVF blastocysts, the single-cell whole-genome methylation sequencing technique was used in this study to analyse the methylation patterns of bovine blastocysts derived from invivo, fresh (IVF) or vitrified (V_IVF) oocytes. Genome methylation levels of blastocysts in the IVF and V_IVF groups were significantly lower than those of the invivo group (P

Vitrification of oocytes has been shown to be closely associated with increased levels of reactive oxygen species (ROS) and apoptotic events. However, little information is available the effect of melatonin on the ROS levels and apoptotic events in vitrified oocytes. Therefore, we studied the effect of melatonin on ROS and apoptotic events in vitrified bovine oocytes by supplementing vitrification solution or in vitro maturation (IVM) and vitrification solution with 10(-9) m melatonin. We analyzed the ROS, mitochondrial Ca(2+) (mCa(2+) ) and membrane potential (ΔΨm), externalization of phosphatidylserine (PS), caspase-3 activation, DNA fragmentation, mRNA expression levels of Bax and Bcl2 l1, and developmental potential of vitrified bovine oocytes. Vitrified bovine oocytes exhibited increased levels of ROS, mCa(2+) , Bax mRNA, and caspase-3 protein and higher rates of PS externalization and DNA fragmentation, and decreased ΔΨm and Bcl2 l1 mRNA expression level. However, melatonin supplementation in vitrification solution or IVM and vitrification solution significantly decreased the levels of ROS, mCa(2+) , Bax mRNA expression, and caspase-3 protein, and PS externalization and DNA fragmentation rates, and increased the ΔΨm and Bcl2 l1 mRNA expression level in vitrified oocytes, resulting in an increased developmental ability of vitrified bovine oocytes after parthenogenetic activation. The developmental ability of vitrified oocytes with melatonin supplementation in IVM and vitrification solution was similar to that of fresh ones. This study showed that supplementing the IVM and vitrification medium or vitrification medium with 10(-9) m melatonin significantly decreased the ROS level and inhibited apoptotic events of vitrified bovine oocytes, consequently increasing their developmental potential.

In artificial insemination, semen quality is considered to be closely related to the resultant pregnancy rate and the ratio of embryo development into fetus. The aim of this study was to evaluate the quality of sex-sorted versus unsorted semen used for in vitro fertilization (IVF). Semen was collected from seven bulls (A)–(G); each sample was divided in two, sex-sorted or not. Following IVF, the fertilization rate, embryonic cleavage rate, blastocyst formation rate, total cells number (TCN) of blastocysts, inner cell mass (ICM)/TCN ratio and apoptotic cell ratio (ACR) were determined. The results showed that except for bull C, the unsorted semen yielded a significantly higher IVF rate than sex-sorted semen (p 0.05). Also, there was no significant difference in the expression of IGF1 gene in bovine embryos produced with sorted or unsorted semen (p>0.05). However, the GDF9 gene expression in sexed embryos from bull D was significantly higher than in the others (p

The objective of this study was to establish an efficient system of producing early monozygotic twin bovine embryos in vitro using the blastomere separation and coculture technique. In this study, early eight-cell embryos were chosen to optimize the separation method, and multi-coculture tactics were applied to improve the efficiency of this production system. Bovine embryo blastomeres (groups of at least 30 at the eight-cell stage) were separated into eight segments (to regard an eight-cell embryo as a tangerine, a blastomere as one segment) and one, two and four segments (blastomeres) were cultured respectively in microwells on the bottom of the four-well dish (Nunc, Denmark) with 400 μL of culture medium under paraffin oil. Four different types of coculture tactics (cocultured with nothing, intact embryos, bovine cumulus cells (bCCs), intact embryos & bCCs) were applied to the group of four segments (blastomeres). Finally, diameter and inner cell mass (ICM):trophectoderm (TE) cell ratio was measured as a criterion to assess the quality of the twin embryos which were derived from bovine separated blastomeres. Our results showed that rate of blastocyst formation of the four segments group was significantly greater than one or two group (P0.05). Thus, these results suggest that combined with intact embryos & bCCs coculture system, culturing four isolated segments (blastomeres) per microwell is an efficient system of producing early monozygotic twin bovine embryos. Furthermore, our results also indicate that the quality of blastocysts derived from separated blastomere may be similar to those derived from intact eight-cell embryos.

To determine the beneficial effects and mechanisms of exogenous glutathione (GSH) during IVC on the embryonic development, bovine IVF zygotes were cultured in medium containing different concentrations of GSH, and the rate of cleavage and blastocyst development, total cell number of blastocysts, the inner cell mass:total cell number ratio, and intracellular GSH and reactive oxygen species concentrations were investigated. Gene expressions associated with embryonic development and GSH metabolism were measured using quantitative real-time polymerase chain reaction. At the concentrations of 1, 3, and 5 mM, GSH significantly increased the blastocyst rate and embryo quality. The highest blastocyst rate (51%) and the best embryo quality appeared in the 3-mM GSH treatment. Intracellular content of GSH of embryos at the two- to four-cell stage significantly increased, and the reactive oxygen species level decreased accordingly with 3-mM GSH treatment, but no significant differences were found in the four- to eight-cell stage and blastocysts. Gene expression analysis of the embryo regulator genes (OCT4, SOX2, and KLF4), GSH synthesis genes (GCLM, GCLC, and GSS), and GSH utilization genes (GSTP, GSTM, and GPX) showed that the GSH had no significant impact on these genes. In conclusion, exogenous GSH during IVC improved developmental potential and quality of bovine IVF embryos, which was probably caused by the ability of GSH to maintain the redox balance.

Vitrification reduces the fertilisation capacity and developmental ability of mammalian oocytes; this effect is closely associated with an abnormal increase of cytoplasmic free calcium ions ([Ca2+]i). However, little information about the mechanism by which vitrification increases [Ca2+]i levels or a procedure to regulate [Ca2+]i levels in these oocytes is available. Vitrified bovine oocytes were used to analyse the effect of vitrification on [Ca2+]i, endoplasmic reticulum Ca2+ (ER Ca2+), and mitochondrial Ca2+ (mCa2+) levels. Our results showed that vitrification, especially with dimethyl sulfoxide (DMSO), can induce ER Ca2+ release into the cytoplasm, consequently increasing the [Ca2+]i and mCa2+ levels. Supplementing the cells with 10 μM 1,2-bis (o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA-AM or BAPTA) significantly decreased the [Ca2+]i level and maintained the normal distribution of cortical granules in the vitrified bovine oocytes, increasing their fertilisation ability and cleavage rate after in vitro fertilisation (IVF). Treating vitrified bovine oocytes with 1 μM ruthenium red (RR) significantly inhibited the Ca2+ flux from the cytoplasm into mitochondria; maintained normal mCa2+ levels, mitochondrial membrane potential, and ATP content; and inhibited apoptosis. Treating vitrified oocytes with a combination of BAPTA and RR significantly improved embryo development and quality after IVF.

Compound air pollution is becoming one of three major problems of urban ecological security in China which is experiencing a rapid growth. This paper examined how to establish the "10 Portions Force" model so as to build up the promoting mechanism of government environmental regulation behaviors’ ability, which based on the time and spatial effects of China's haze pollution.

Melatonin is a well-characterized antioxidant that has been successfully used to protect oocytes from reactive oxygen species during in vitro maturation (IVM), resulting in improved fertilization capacity and development ability. However, the mechanism via which melatonin improves oocyte fertilization capacity and development ability remains to be determined. Here, we studied the effects of melatonin on cytoplasmic maturation of bovine oocytes. In the present study, bovine oocytes were cultured in IVM medium supplemented with 0, 10–7, 10–9, and 10−11 mol/L melatonin, and the cytoplasmic maturation parameters of MII oocytes after IVM were investigated, including redistribution of organelles (mitochondria, cortical granules [CGs], and endoplasmic reticulum [ER]), intracellular glutathione (GSH) and ATP levels, expression of endogenous antioxidant genes (Cat, Sod1, and GPx), and fertilization-related events (IP3R1 distribution and expression of CD9 and Juno). Our results showed that melatonin significantly improved the cytoplasmic maturation of bovine oocytes by improving the normal distribution of organelles, increasing intracellular GSH and ATP levels, enhancing antioxidant gene expression levels, and modulating fertilization-related events, all of which resulted in increased fertilization capacity and developmental ability. Meanwhile, melatonin also increased the mRNA and protein expression levels of the Tet1 gene and decreased the Dnmt1 gene mRNA and protein levels in bovine oocytes, indicating that melatonin regulates the expression of the detected genes via demethylation. These findings shed insights into the potential mechanisms by which melatonin improves oocyte quality during IVM.

The paper made unary linear regression and structure deviation degree analyses with the data of Shanxi province in China from 1992 to 2010. The results show that when the coal output increases by 1% , the employment will increase by 1.202%. And the structure deviation degree in the coal industry fell from-0.11 to-0.56 between 1992 and 2001, then it aggrandized to - 0.15 in 2010. Which presented a note of discord surfaced during industrial structure and employment structure adjusting in Shanxi province. This suggests that there are the disguised unemployment within the coal industry. So this paper emphasizes that the government should implement the coal property right reform policy, and simultaneously develop effective measures to guide the workers to flow from the coal industry to the other secondary industries or the third industry.

Respiratory distress is a major cause of mortality in cloned neonatal animals, but its pathogenesis remains poorly understood. Here, we used necropsy and histology procedures to evaluate the lungs of cloned neonatal bovines dying of respiratory distress, finding incomplete lung dilation, alveolar collapse, and thickened alveolar walls. Comparison of the transcriptomes between collapsed lungs of cloned bovines and their normal counterparts revealed 1373 differentially expressed genes in collapsed lungs (p 1.5 or

// Shan-Jiang Zhao 1, * , Yun-Wei Pang 1, * , Xue-Ming Zhao 1 , Wei-Hua Du 1 , Hai-Sheng Hao 1 , Hua-Bin Zhu 1 1 Embryo Biotechnology and Reproduction Laboratory, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, China * These authors contributed equally to this work Correspondence to: Hua-Bin Zhu, email: zhuhuabin@caas.cn Keywords: lipopolysaccharide, oocyte, cytoskeleton, oxidative stress, epigenetic modifications Received: September 24, 2016 Accepted: December 07, 2016 Published: December 16, 2016 ABSTRACT Lipopolysaccharide disturbs the secretion of gonadotropin, endometrial function and implantation efficiency. However, there is little information regarding the effects of lipopolysaccharide on cyclic ovary activity, especially oocyte maturation. Therefore, we aimed to investigate the effects of lipopolysaccharide on the maturation potential of bovine oocytes. We found that lipopolysaccharide exposure significantly decreased the first polar body extrusion rate and delayed the cell cycle progression. The abnormal spindle rate was significantly increased in lipopolysaccharide treatment group, accompanied by disrupted localization and level of phosphorylated mitogen-activated protein kinase (p-MAPK). Moreover, lipopolysaccharide treatment significantly increased intracellular reactive oxygen species (ROS) levels and the early apoptotic rate in oocytes. The pro-apoptotic caspase-3 and Bax mRNA levels and caspase-3 protein level were significantly increased, whereas the anti-apoptotic Bcl-2 and XIAP transcript abundance were significantly decreased in lipopolysaccharide exposure group. Furthermore, the dimethyl-histone H3 lysine 4 (H3K4me2) level was significantly increased, while the DNA methylation (5-mC) and dimethyl-histone H3 lysine 9 (H3K9me2) levels were markedly decreased in oocytes treated with lipopolysaccharide. In conclusion, lipopolysaccharide exposure inhibits the maturation potential of bovine oocytes by affecting cell cycle, cytoskeletal dynamics, oxidative stress, and epigenetic modifications.

The paper made a regression analysis with the panel data of 29 provinces and cities in China from 1996 to 2010 based on Cobb-Douglas production function introducing the tax variable. The results show that the growth of per unit resource tax may extend the economic growth1.0325, 1.0243, and 1.0661 times in eastern, middle and western areas respectively. Meanwhile, when the ratio of the resource tax among the general fiscal income increase by 1%, the output elasticity of capital may increase by10.21%, 15.59% and 14% in tune, the output elasticity of labor may decrease by 22.02%, 17.47% and 13.95%. This suggests that from the perspective of perfecting the resource tax to study environmental tax levy can better rationalize resource price mechanism, and environmental tax is a very important economic method to carry out the stratagem of regional sustainable development, but there is unbalanced effect on different areas.

Paraquat (PQ), a broad-spectrum agricultural pesticide, causes cellular toxicity by increasing oxidative stress levels in various biological systems, including the reproductive system. PQ exposure causes embryotoxicity and reduces the developmental abilities of embryos. However, there is little information regarding the toxic effects of PQ on oocyte maturation. In this study, we studied the toxic effects of PQ exposure and the effects of melatonin on PQ-induced damage in bovine oocytes. PQ exposure disrupted nuclear and cytoplasmic maturation, which was manifested as decreased cumulus cell expansion, reduced first polar body extrusion, and abnormal distribution patterns of cortical granules and mitochondria. In addition, PQ treatment severely disrupted the ability of the resulted in vitro-produced embryos to develop to the blastocyst stage. Moreover, PQ exposure significantly increased the intracellular reactive oxygen species (ROS) level and early apoptotic rate, and decreased the glutathione (GSH) level, antioxidative CAT and GPx4 mRNA, and apoptotic-related Bcl-2/Bax mRNA ratio. These results indicated that PQ causes reproductive toxicity in bovine oocytes. Melatonin application resulted in significant protection against the toxic effects of PQ in PQ-exposed oocytes. The mechanisms underlying the role of melatonin included the inhibition of PQ-induced p38 mitogen-activated protein kinase (MAPK) activation, and restoration of abnormal trimethyl-histone H3 lysine 4 (H3K4me3) and trimethyl-histone H3 lysine 9 (H3K9me3) levels. These results reveal that melatonin serves as a powerful agent against experimental PQ-induced toxicity during bovine oocyte maturation and could form a basis for further studies to develop therapeutic strategies against PQ poisoning.

Objective To investigate the effect of vitrification on Dnmt1o, Hat1, and Hdac1 promoter CpG island methylation patterns and messenger RNA (mRNA) expression levels in mouse metaphase II (MII) oocytes. Design In vitro study. Setting Academic institution. Animal(s) Kunming white mice. Intervention(s) After vitrification, surviving mouse MII oocytes subjected to methylation and expression analysis with fresh oocytes used as a control. Main Outcome Measure(s) Expression levels of mRNA as measured by real-time reverse-transcriptase polymerase chain reaction of methylation patterns of the CpG islands in the Dnmt1o , Hat1 , and Hdac1 promoters analyzed by bisulfite mutagenesis and sequencing. Result(s) The methylation patterns of the promoter CpG islands in Dnmt1o , Hat1 , and Hdac1 were not statistically significantly when comparing vitrified oocytes and fresh oocytes. The expression levels of Hat1 and Hdac1 mRNA were not statistically significantly different in comparing in vitrified oocytes and fresh oocytes. The expression of Dnmt1o mRNA was statistically significantly lower in vitrified oocytes compared with fresh oocytes. Conclusion(s) Vitrification did not statistically significantly alter the methylation patterns of the promoter CpG islands in Dnmt1o , Hat1 , or Hdac1 , but did statistically significantly decrease the expression of Dnmt1o mRNA in mouse MII oocytes.

SummaryWe investigated the effect mouse cumulus cells (mCCs) on the in vitro maturation (IVM) and developmental potential of bovine denuded germinal vesicle oocytes (DOs). Cumulus–oocyte complexes (COCs), DOs and DOs cocultured with either mCCs (DOs + mCCs) or bovine cumulus cells (bCCs; DOs + bCCs) were subjected to IVM. The meiosis II (MII) rates of DOs, glutathione (GSH) contents, zona pellucida (ZP) hardening and parthenogenetic blastocyst rates of MII oocytes were determined. The relative expression levels of bone morphogenetic protein 15 (BMP-15) and growth differentiation factor 9 (GDF-9) in MII oocytes were measured using quantitative real-time polymerase chain reaction (PCR). mCCs significantly increased the MII rate of DOs from 53.5 ± 3.58% to 69.67 ± 4.72% (p < 0.05) but had no effect on the GSH content (2.17 ± 0.31 pmol/oocyte with mCCs, 2.14 ± 0.53 pmol/oocyte without mCCs). For the DOs + mCCs group, the BMP-15 and GDF-9 expression levels were significantly higher and the ZP dissolution time was significantly lower (162.49 ± 12.51 s) than that of the DOs group (213.95 ± 18.87 s; p < 0.05). The blastocyst rate of the DOs + mCCs group (32.56 ± 4.94%) was similar to that of the DOs group (31.75 ± 3.65%) but was significantly lower than that of the COCs group (43.52 ± 5.37%; p < 0.05). In conclusion, mCCs increased the MII rate of DOs and expression of certain genes in MII oocytes, and decreased the ZP hardening of MII oocytes, but could not improve their GSH content or developmental potential.

Take for example the BRIC economies: Brazil, Russia, India and China. We investigated the time series data on the relationship between carbon dioxide emission and economic growth in these fast-growing developing countries by both comparative statics and comparative dynamics. The results show that there is the monotonic relationship between total carbon dioxide emissions, carbon dioxide emissions per capita and per capita GDP in any one of the BRIC countries. And there is decreasing relationship between the carbon dioxide emissions per unit GDP and per capita GDP.

We aimed to investigate the effect of melatonin on bovine frozen-thawed semen and its impact on fertilization outcome. Plasma membrane integrity, mitochondrial activity, acrosome integrity, and levels of intracellular reactive oxygen species (ROS) were measured in spermatozoa treated with different concentrations of melatonin. Melatonin-treated spermatozoa were then used for in vitro fertilization, followed by analysis of subsequent embryo development and the expression of apoptosis- and antioxidant-related genes. The results revealed that 10

Contents Research on bovine embryonic stem cells (bESCs) has been hampered because bESCs are cultured in conditions that are based on information obtained from culturing mouse and human inner cell mass (ICM) cells. The aim of this study was to compare gene expression in ICM and trophectoderm (TE) cell lineages of bovine embryos and to discuss the findings relative to information available for mice and humans. We separated a high-purity (>90%) ICM and TE from bovine blastocysts by magnetic-activated cell sorting and analysed their transcriptomes by single cell RNA-seq. Differentially expressed genes (DEGs) were assessed using Gene Ontology (GO) and Kyoto Encyclopaedia of Genes and Genomes (KEGG) databases. Finally, qRT-PCR was performed to validate the RNA-seq results. From 207 DEGs identified (adjusted p ≤ .05; fold change ≥2), 159 and 48 had greater expression in the ICM and TE cells respectively. We validated 27 genes using qRT-PCR and found their expression patterns were mostly similar to those of RNA-seq, including 12 novel ICM-dominant (HNF4A, CCL24, FGFR4, IFITM3, PTCHD2, GJB5, FN1, KLK7, PRDM14, GRP, FGF19 and GCM1) and two novel TE-dominant (SLC10A1 and WNT4) genes. Bioinformatics analysis showed that these DEGs are involved in many important pathways, such as MAPK and cancer cell pathways, and these pathways have been shown to play essential roles in mouse and human ESCs in the self-renewal and pluripotent maintenance. As a conclusion, there were sufficient differences to allow us to conclude that the control of pluripotency in bovine ICM cells is species-specific.

To our knowledge, no single study has systemically compared cryopreservation efficiencies of bovine blastocysts derived from in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI) and somatic cell nuclear transfer (SCNT) by controlled freezing and vitrification. This experiment, therefore, was designed to compare the cryopreservation of these blastocysts with controlled freezing and OPS vitrification. Adenosine-5′-triphosphate (ATP) content and reactive oxygen species (ROS) level in blastocysts were also analyzed. Firstly, for each type of blastocyst (IVF, ICSI or SCNT), significant differences were observed between the survival rates of the controlled freezing ((81.56±2.33), (68.18±4.72) or (47.89±5.83)%) and OPS vitrification groups ((92.24±4.54), (82.40±3.76) or (78.71±5.91)%; P

Preimplantation embryos are sensitive to oxidative stress-induced damage that can be caused by reactive oxygen species (ROS) originating from normal embryonic metabolism and/or the external surroundings. Paraquat (PQ), a commonly used pesticide and potent ROS generator, can induce embryotoxicity. The present study aimed to investigate the effects of melatonin on PQ-induced damage during embryonic development in bovine preimplantation embryos. PQ treatment significantly reduced the ability of bovine embryos to develop to the blastocyst stage, and the addition of melatonin markedly reversed the developmental failure caused by PQ (20.9% versus 14.3%). Apoptotic assay showed that melatonin pretreatment did not change the total cell number in blastocysts, but the incidence of apoptotic nuclei and the release of cytochrome c were significantly decreased. Using real-time quantitative polymerase chain reaction analysis, we found that melatonin pre-incubation significantly altered the expression levels of genes associated with redox signaling, particularly by attenuating the transcript level of Txnip and reinforcing the expression of Trx. Furthermore, melatonin pretreatment significantly reduced the expression of the pro-apoptotic caspase-3 and Bax, while the expression of the anti-apoptotic Bcl-2 and XIAP was unaffected. Western blot analysis showed that melatonin protected bovine embryos from PQ-induced damage in a p38-dependent manner, but extracellular signal-regulated kinase (ERK) and c-JUN N-terminal kinase (JNK) did not appear to be involved. Together, these results identify an underlying mechanism by which melatonin enhances the developmental potential of bovine preimplantation embryos under oxidative stress conditions.

The developmental competence of embryos cloned from somatic cells depends on the cellular event and molecu-lar process, such as separation of chromosomes and reorganization of spindle after nuclear transfer. Centrosome, the main microtubule organizing centers in a cell, is crucial for reorganization of spindle and normal separation of chromosomes during mitosis. Aberrant of centrosomes will lead to aneuploidy of blastomere and developmental failure of embryo. This paper expounded the situation of animal somatic cell nuclear transfer (SCNT) and biological functions of centrosome and analyzed the inheritance mechanism of centrosome during gametogenesis and fertilization. Additionally, the study condition of centrosome and its associated proteins in SCNT embryos were introduced, which provided a new clue to study the de-velopmental abnormality of cloned embryos and animals.

Vitrification had a significantly negative impact on the mitochondrial function of bovine oocytes. However, 40 μg/mL cyclosporine pretreatment before vitrification contributed greatly to maintaining mitochondrial membrane potential and adenosine triphosphate content, decreasing reactive oxygen species level, and thereby increasing the developmental ability of vitrified oocytes.

SummaryThis study was designed to determine the effect of melatonin on thein vitromaturation (IVM) and developmental potential of bovine oocytes denuded of the cumulus oophorus (DOs). DOs were cultured alone (DOs) or with 10−9M melatonin (DOs + MT), cumulus–oocyte complexes (COCs) were cultured without melatonin as the control. After IVM, meiosis II (MII) rates of DOs, and reactive oxygen species (ROS) levels, apoptotic rates and parthenogenetic blastocyst rates of MII oocytes were determined. The relative expression of ATP synthase F0 Subunit 6 and 8 (ATP6andATP8), bone morphogenetic protein 15 (BMP-15) and growth differentiation factor 9 (GDF-9) mRNA in MII oocytes and IFN-tau (IFN-τ),Na+/K+-ATPase, catenin-beta like 1 (CTNNBL1) andAQP3mRNA in parthenogenetic blastocysts were quantified using real-time polymerase chain reaction (PCR). The results showed that: (1) melatonin significantly increased the MII rate of DOs (65.67 ± 3.59 % vs. 82.29 ± 3.92%;P <0.05), decreased the ROS level (4.83 ± 0.42 counts per second (c.p.s) vs. 3.78 ± 0.29 c.p.s;P <0.05) and apoptotic rate (36.99 ± 3.62 % vs. 21.88 ± 2.08 %;P <0.05) and moderated the reduction of relative mRNA levels ofATP6,ATP8,BMP-15andGDF-9caused by oocyte denudation; (2) melatonin significantly increased the developmental rate (24.17 ± 3.54 % vs. 35.26 ± 4.87%;P <0.05), and expression levels ofIFN-τ,Na+/K+-ATPase,CTNNBL1andAQP3mRNA of blastocyst. These results indicated that melatonin significantly improved the IVM quality of DOs, leading to an increased parthenogenetic blastocyst formation rate and quality.

This study utilized three staining assays (Annexin V, mitochondrial membrane potential (JC-1) and TUNEL) for flow cytometric analysis of apoptosis in sex-sorted sperm from four different bulls (A, B, C and D). Correlations between sperm quality and IVF efficiency were then assessed to determine which assay provided the best prediction of IVF efficiency. The results of the Annexin V assays, as well as measures of viable sperm, early apoptosis, necrotic sperm and mitochondrial membrane potential (∆ψm) showed that the sex-sorted sperm collected from bull A significantly differed from those of the other three bulls (p < 0.05). In addition, the levels of DNA fragmentation in sex-sorted sperm from bull A were significantly lower than those from bulls B and C (p < 0.05). The percentage of cells reaching the cleavage and blastocyst stages in sex-sorted sperm from bull A were significantly greater than those from the other bulls (p < 0.05). A significant positive correlation was observed between viable sperm and the percentage of cells at the cleavage or blastocyst stages (p < 0.05). In contrast, a negative correlation was found between early apoptotic sperm and the percentage of cells at the cleavage or blastocyst stages (p < 0.05). In conclusion, these results indicated that the Annexin V assay was the most reliable technique for the prediction of the IVF success of sex-sorted bovine sperm.

The process of differentiated somatic cells to a pluripotent state or to another unrelated cell type is referred to as cellular reprogramming. This can be achieved through somatic cell nuclear transfer, cell fusion, specific transcription-factor transduction, and exposure to cell extracts. Cellular reprogramming using cell extracts has gained increasing attention in recent years and has been applied to acquire the target cell types, recognize the functional factors, explore the reprogramming mechanism. In this review, we described different methods of reprogramming somatic cells, which mainly focused on the advances in cellular reprogramming using cell extracts.

The present study was designed to determine the effects of vitrification on promoter methylation and the expression levels of pluripotency and differentiation genes in mouse blastocysts. Promoter region CpG methylation patterns and the expression levels of octamer-binding transcription factor (Oct4), Nanog homeobox (Nanog), caudal-type homeobox 2 (Cdx2), and heart and neural crest derivatives-expressed transcript 1 (Hand1) were analyzed in fresh and vitrified mouse blastocysts. Methylation was measured by bisulphate mutagenesis and sequencing; gene expression was determined by real-time reverse transcription-PCR. The results showed that vitrification significantly reduced the methylation levels of the Oct4 (85% vs. 62.5%), Nanog (77.5% vs. 55%), and Cdx2 promoters (4.6% vs. 1.4%; P 0.05) in mouse blastocysts, which correlated with increased expression of Oct4 and Nanog in vitrified blastocysts. Hand1 promoter methylation was not significantly different in the fresh (17.9%) versus vitrification group (21.4%; P 0.05). The expression levels of Cdx2 and Hand1 were not significantly different in fresh and vitrified blastocysts. In conclusion, vitrification significantly decreased Oct4, Nanog, and Cdx2 promoter methylation in mouse blastocysts, which correlated with increased expression of Oct4 and Nanog.

The epidermal growth factor receptor (EGFR), a transmembrane protein receptor, is a member of ErbB family with signal-transducing tyrosine kinase activity. After combined with the ligand, EGFR homologous or heterologous dimers are formed to induce intracellular signal transduction, activate downstream signal transduction pathways, and then produce a series of biological effects. RAF/MEK/RAS/ERK pathway is relevant to cell proliferation, differentiation and apoptosis; while PDK1/AKT /PI3K pathway is involved in cell migration and adhesion. EGFR can promote the maturity of pulmonary type II epithelial cells and the synthesis and secretion of pulmonary surfactant. EGFR shows the effect on mammal lungs in a time-space and dose-dependent manner. The down-regulated expression of it will lead to immature lung development, while the over-expression can promote the cell proliferation, invasion and metastasis of the lung cancer cells. This paper reviewed advances in the study for EGFR and its signal pathway, as well as the relationship among EGFR, atelectasis and lung cancer.

Objective To determine the effect of vitrification and 5-aza-2′-deoxycytidine (5-aza-dC) on the methylation levels of the putative imprinted control region (ICR) of H19 and H19 expression in bovine two-cell embryos and their derived blastocysts. Design Experimental animal study. Setting Academic institution. Animal(s) Abattoir-derived bovine ovaries. Intervention(s) Vitrified two-cell embryos were cultured in vitro to blastocysts with 0.01 μM 5-aza-dC (5-aza-dC group) or without 5-aza-dC (vitrification group). Fresh embryos and their derived blastocysts were used as control. Main Outcome Measure(s) Putative ICR methylation of H19 was measured by bisulfate mutagenesis and sequencing, blastocyst development rate; total cell number were determined; and H19 expression was measured by real-time reverse transcriptase-polymerase chain reaction (PCR). Result(s) Vitrification significantly increased putative ICR methylation of H19 in two-cell embryos and their derived blastocysts; 5-aza-dC significantly reduced putative ICR methylation of H19 in vitrified two-cell embryos and their derived blastocysts. The H19 expression level was significantly higher in blastocysts from the 5-aza-dC group than the vitrification group. The blastocyst development rate and total cell number in the 5-aza-dC and vitrification groups were similar. Conclusion(s) Putative ICR methylation levels of H19 significantly increased in vitrified two-cell embryos and their derived blastocysts; 5-aza-dC significantly reduced putative ICR methylation of H19 and increased H19 expression in blastocysts derived from vitrified two-cell embryos.