Establishing a 3D-cultured system based on alginate-hydrogel embedding benefits the in vitro maturation of porcine Oocytes.

The in vitro maturation (IVM) quality of oocytes is directly related to the subsequent developmental potential of embryos and a fundamental of in vitro embryo production. However, conventional IVM methods fail to maintain the gap-junction intercellular communication (GJIC) between cumulus-oocyte complexes (COCs), which leads to insufficient oocyte maturation. Herein, we investigated the effects of three different three-dimensional (3D) culture methods on oocyte development in vitro, optimized of the alginate-hydrogel embedding method, and assessed the effects of the alginate-hydrogel embedding method on subsequent embryonic developmental potential of oocytes after IVM and parthenogenetic activation (PA). The results showed that Matrigel embedding and alginate-hydrogel embedding benefited the embryonic developmental potential of oocytes after IVM and PA. With the further optimization of alginate-hydrogel embedding, including crosslinking and decrosslinking of parameters, we established a 3D culture system that can significantly increase oocyte maturation and the blastocyst rate of embryos after PA (27.2 ± 1.5 vs 36.7 ± 2.8, P < 0.05). This 3D culture system produced oocytes with markedly increased mitochondrial intensity and membrane potential, which reduced the abnormalities of spindle formation and cortical granule distribution. The alginate-hydrogel embedding system can also remarkably enhance the GJIC between COCs. In summary, based on alginate-hydrogel embedding, we established a 3D culture system that can improve the IVM quality of porcine oocytes, possibly by enhancing GJIC.

Effects of intracellular Ca2+ on developmental potential and ultrastructure of cryopreserved-warmed oocyte in mouse.

Maintaining appropriate intracellular calcium of oocytes is necessary to prevent ultrastructure and organelle damage caused by freezing and cryoprotectants. The present study aimed to investigate whether cryoprotectant-induced changes in the calcium concentrations of oocytes can be regulated to reduce damage to developmental potential and ultrastructure. A total of 33 mice and 1381 oocytes were used to explore the effects of intracellular calcium on the development and ultrastructures of oocytes subjected to 2-aminoethoxydiphenyl borate (2-APB) inhibition or thapsigargin (TG) stimulation. Results suggested that high levels intracellular calcium interfered with TG compromised oocyte survival (84.4 % vs. 93.4 %, p < 0.01) and blastocyst formation in fresh and cryopreservation oocytes (78.1 % vs. 86.4 %, and 60.5 % vs. 72.5 %, p < 0.05) compared with that of 2-APB pretreated oocytes in which Ca2+ was stabilized even though no differences in fertilization and cleavage was detected (p > 0.05). Examination by transmission electron microscopy indicated that the microvilli decreased and shortened, cortical granules considerably decreased in the cortex area, mitochondrial vesicles and vacuoles increased, and the proportion of vacuole mitochondria increased after oocytes were exposed to cryoprotectants. The cryopreservation-warming process deteriorated the negative effects on organelles of survival oocytes. By contrast, a low level of intracellular calcium mediated with 2-APB was supposed to contribute to the protection of organelles. These findings suggested oocyte injuries induced by cryoprotectants and low temperatures can be alleviated. More studies are necessary to confirm the relationship among Ca2+ concentration of the cytoplasm, ultrastructural injuries, and disrupted developmental potential in oocytes subjected to cryopreservation and warming.

Transcriptomic analysis of Amaranthus retroflex resistant to PPO-inhibitory herbicides.

Amaranthus retroflexus L. is one of the malignant weeds which can cause a reduction in the soybean yield. We found a population of A. retroflexus (R-Q) resistant to fomesafen through the initial screening of whole-plant dose response bioassay in the research. The resistance index of the population (R-Q) was 183 times of the sensitive population (S-N). The resistant and sensitive populations were used as experimental materials in the paper. Strand-specific RNA-Seq analyses of R‒Q and S‒N populations obtained from herbicide-treated and mock-treated leaf samples after treatment were conducted to generate a full-length transcriptome database. We analyzed differentially expressed genes (DEGs) among the R-Q and S‒N A. retroflexus populations treated with recommended dose and mock-treated on the 1st (24 h) and 3rd (72 h) days to identify genes involved in fomesafen resistance. All 82,287 unigenes were annotated by Blastx search with E-value < 0.00001 from 7 databases. A total of 94,815 DEGs among the three group comparisons were identified. Two nuclear genes encoding PPO (PPX1 and PPX2) and five unigenes belonging to the AP2-EREBP, GRAS, NAC, bHLH and bZIP families exhibited different expression patterns between individuals of S‒N and R-Q populations. The A. retroflexus transcriptome and specific transcription factor families which can respond to fomesafen in resistant and susceptible genotypes were reported in this paper. The PPX1 and PPX2 genes of the target enzyme were identified. The study establishes the foundation for future research and provides opportunities to manage resistant weeds better.

Vitamin C down-regulates the H3K9me3-dependent heterochromatin in buffalo fibroblasts via PI3K/PDK1/SGK1/KDM4A signal axis.

The success of reprogramming is dependent on the reprogramming factors enriched in the cytoplasm of recipient oocytes and the potential of donor nucleus to be reprogrammed. Histone 3 lysine 9 trimethylation (H3K9me3) was identified as a major epigenetic barrier impeding complete reprogramming. Treating donor cell with vitamin C (Vc) can enhance the developmental potential of cloned embryos, but the underlying mechanisms still need to be elucidated. In this study, we found that 20µg/mL Vc could promote proliferation and inhibit apoptosis of BFFs, as well as down-regulate the H3K9me3-dependent heterochromatin and increase chromatin accessibility. Inhibited the expression of KDM4A resulted in increasing apoptosis rate and the H3K9me3-dependent heterochromatin, which can be restored by Vc. Moreover, Vc up-regulated the expression of KDM4A through PI3K/PDK1/SGK1 pathway. Inhibiting any factor in the signal axis of this PI3K pathway not only suppressed the activity of KDM4A but also substantially increased the level of H3K9me3 modification and the expression of the HP1α protein. Finally, Vc can rescue those negative effects induced by the blocking the PI3K/PDK1/SGK1 pathway. Collectively, Vc can down-regulate the H3K9me3-dependent heterochromatin in BFFs via PI3K/PDK1/SGK1/KDM4A signal axis, suggesting that Vc can turn the chromatin status of donor cells to be reprogrammed more easily.

Individual variation in buffalo somatic cell cloning efficiency is related to glycolytic metabolism.

Mammalian individuals differ in their somatic cell cloning efficiency, but the mechanisms leading to this variation is poorly understood. Here we found that high cloning efficiency buffalo fetal fibroblasts (BFFs) displayed robust energy metabolism, looser chromatin structure, high H3K9 acetylation and low heterochromatin protein 1α (HP1α) expression. High cloning efficiency BFFs had more H3K9ac regions near to the upstream of glycolysis genes by ChIP-seq, and involved more openness loci related to glycolysis genes through ATAC-seq. The expression of these glycolysis genes was also found to be higher in high cloning efficiency BFFs by qRT-PCR. Two key enzymes of glycolysis, PDKs and LDH, were confirmed to be associated with histone acetylation and chromatin openness of BFFs. Treatment of low cloning efficiency BFFs with PS48 (activator of PDK1) resulted in an increase in the intracellular lactate production and H3K9 acetylation, decrease in histone deacetylase activity and HP1α expression, less condensed chromatin structure and more cloning embryos developing to blastocysts. These results indicate that the cloning efficiency of buffalo somatic cells is associated with their glycolytic metabolism and chromatin structure, and can be improved by increasing glycolytic metabolism.

Inhibition of the liquefaction of alkali-induced egg white gel by sodium ascorbate.

Effects of sodium ascorbate (1%, 2%, 3%) on the liquefaction of alkali-induced egg white gel (EWG) were investigated. Results showed hardness and water holding capacity (WHC) gradually decreased at 1%. However, hardness and WHC declined and then rose at 2% and 3%. Microstructural changes further confirmed the effects of sodium ascorbate on hardness and WHC. Electrophoresis showed sodium ascorbate caused the cross-linking between proteins, which was more resistant to degradation. Fourier transform infrared spectroscopy (FTIR) and surface hydrophobicity indicated sodium ascorbate significantly changed protein structure, especially at 2% and 3% resulted in protein reaggregation, increasing ß-sheet, and decreasing surface hydrophobicity in the later stage. In general, sodium ascorbate didn't inhibit the liquefaction of alkali-induced EWG at 1%, but did effectively at 2% and 3%. Therefore, high concentrations of sodium ascorbate possess the potential to inhibit the "alkali injury liquefaction" of preserved egg whites without heavy metals.

Histone Demethylase KDM4D Could Improve the Developmental Competence of Buffalo (Bubalus Bubalis) Somatic Cell Nuclear Transfer (SCNT) Embryos.

Somatic cell nuclear transfer (SCNT) holds vast potential in agriculture. However, its applications are still limited by its low efficiency. Histone 3 lysine 9 trimethylation (H3K9me3) was identified as an epigenetic barrier for this. Histone demethylase KDM4D could regulate the level of H3K9me3. However, its effects on buffalo SCNT embryos are still unclear. Thus, we performed this study to explore the effects and underlying mechanism of KDM4D on buffalo SCNT embryos. The results revealed that compared with the IVF embryos, the expression level of KDM4D in SCNT embryos was significantly lower at 8- and 16-cell stage, while the level of H3K9me3 in SCNT embryos was significantly higher at 2-cell, 8-cell, and blastocyst stage. Microinjection of KDM4D mRNA could promote the developmental ability of buffalo SCNT embryos. Furthermore, the expression level of ZGA-related genes such as ZSCAN5B, SNAI1, eIF-3a, and TRC at the 8-cell stage was significantly increased. Meanwhile, the pluripotency-related genes like POU5F1, SOX2, and NANOG were also significantly promoted at the blastocyst stage. The results were reversed after KDM4D was inhibited. Altogether, these results revealed that KDM4D could correct the H3K9me3 level, increase the expression level of ZGA and pluripotency-related genes, and finally, promote the developmental competence of buffalo SCNT embryos.

Activation of Wnt/ß-Catenin Signaling Pathway Enhances the Derivation of Buffalo (Bubalus bubalis) Embryonic Stem Cell-Like Cells.

Wnt/ß-Catenin signaling pathway plays an important role in maintaining self-renewal and pluripotency of human and mouse embryonic stem cells (ESCs). Activation of Wnt/ß-Catenin signaling pathway by glycogen synthase kinase-3 (GSK3) inhibitor, the Wnt signaling agonist, could maintain the pluripotency of human and mouse ESCs in the presence of serum. However, the role of signaling pathway in the derivation of buffalo ESCs remains unclear. In this study, we used GSK3 inhibitors (6-bromoindirubin-3'-oxime [BIO] and CHIR99021) and investigated the effect of Wnt/ß-Catenin activation on colony formation, proliferation, self-renewal, and pluripotency of Chinese swamp buffalo (buffalo) embryonic stem cell-like cells (ES-like cells), which were isolated from blastocysts. The results showed that buffalo ES-like cells displayed typical morphological characteristics of pluripotent stem cells: positive for alkaline phosphatase staining, expression of pluripotent markers, including OCT4, SOX2, SSEA-1, SSEA-4, LIN28, CH1, NANOG, and the proliferative markers, PCNA and C-MYC. Furthermore, activation of Wnt/ß-Catenin signaling pathway by GSK3 inhibitors could promote colony formation and proliferation of buffalo ES-like cells and maintain their undifferentiated state, and upregulate the expression levels of pluripotent-related genes and proliferation-related genes. These results indicated that Wnt/ß-Catenin signaling pathway plays an important role in the derivation and pluripotency of buffalo ES-like cells.

Prevalence of Percutaneous Injuries and Associated Factors Among a Sample of Midwives in Hunan Province, China.

Background: Percutaneous injuries and blood-borne-related infections pose occupational hazards to healthcare professionals. However, the prevalence and associated factors for these hazards among midwives in Hunan Province, China are poorly documented. Methods: A cross-sectional study was conducted among a sample of 1,282 eligible midwives in the cities of Yongzhou, Chenzhou, Hengyang, and Changsha in Hunan Province, China, from January 2017 to July 2017. The association of selected independent variables with percutaneous injuries was investigated using binary logistic regression. Results: 992 participants responded (77.3%), and within the previous 12 months, 15.7% experienced percutaneous injuries. In multivariate analysis, hospital size, age, length of employment as a midwife, weekly working hours, and three aspects of Hospital Safety Climate Scale were associated with percutaneous injuries. The risk of percutaneous injuries among the midwives working in hospitals with ≤399 beds was higher than that among those working in hospitals with ≥400 beds by nearly 3 times. Furthermore, the percutaneous injury prevalence of midwives decreased as age increased. Moreover, the probability of percutaneous injuries among the midwives with weekly working hours of >40 was 4.35 times higher compared with that among midwives with weekly working hours of ≤40. Conclusion/Application to practice: The prevalence of percutaneous injuries among midwives in the study hospitals was substantial. Our results further proved that risk mitigation strategies tailored to midwives are needed to reduce this risk. These strategies include ensuring a positive organizational climate, providing highly safe devices, and reducing the workload.

Comparison of Long Non-Coding RNA Expression Profiles of Cattle and Buffalo Differing in Muscle Characteristics.

Buffalo meat consist good qualitative characteristics as it contains "thined tender" which is favorable for cardavascular system. However, the regulatory mechanisms of long non-coding RNA (lncRNA), differences in meat quality are not well known. The chemical-physical parameters revealed the muscle quality of buffalo that can be equivalent of cattle, but there are significant differences in shearing force and muscle fiber structure. Then, we examined lncRNA expression profiles of buffalo and cattle skeletal muscle that provide first insights into their potential roles in buffalo myogenesis. Here, we profiled the expression of lncRNA in cattle and buffalo skeletal muscle tissues, and 16,236 lncRNA candidates were detected with 865 up-regulated lncRNAs and 1,296 down-regulated lncRNAs when comparing buffalo to cattle muscle tissue. We constructed coexpression and ceRNA networks, and found lncRNA MSTRG.48330.7, MSTRG.30030.4, and MSTRG.203788.46 could be as competitive endogenous RNA (ceRNA) containing potential binding sites for miR-1/206 and miR-133a. Tissue expression analysis showed that MSTRG.48330.7, MSTRG.30030.4, and MSTRG.203788.46 were highly and specifically expressed in muscle tissue. Present study may be used as a reference tool for starting point investigations into the roles played by several of those lncRNAs during buffalo myogenesis.

Fatty acid biosynthesis and transcriptional regulation of Stearoyl-CoA Desaturase 1 (SCD1) in buffalo milk.

BACKGROUND: Buffalo milk is considered as a highly nutritious food owing to its higher contents of fatty acids (FA) and rich nutrient profile. Higher fat contents of buffalo milk make it suitable for processing to develop various healthy and nutritious products. Moreover, buffalo milk contains more unsaturated FAs (UFA) such as oleic and linolenic acid, which are important from the human health point of view owing to their desirable physiological effects. However, inadequate information is available about the chemical composition and mechanism of FA synthesis in buffalo milk. In this study, we hypothesized that expression of SCD1 gene could alter the biosynthesis of FA in epithelial cells of mammary gland and subsequently affect the FA contents in buffalo milk. We investigated the transcriptional and biological role of Stearoyl-CoA Desaturase 1 (SCD1) in the buffalo mammary epithelial cells (BMECs) during FA and triacylglycerol (TAG) synthesis. RESULTS: Results revealed that unsaturated fatty acid contents were much higher in concentration in buffalo milk as compared to Holstein cow. Significant increase in the expression level of FAS, ACACA, SREBP1, PPARG, GPAT, and AGPAT genes was observed in response to altered expression of SCD1 in buffalo milk. Moreover, change in SCD1 gene in BMECs also mediated the expression of genes related to FA biosynthesis subsequently leading to alter the FA composition. Overexpression of SCD1 significantly increased the expression of genes associated with FA and TAG synthesis leading to enhance FA and unsaturated FA contents in BMECs. However, down-regulation of SCD1 exhibited opposite consequences. CONCLUSION: Our study provides mechanistic insights on transcriptional regulation of SCD1 to alter FA and TAG synthesis through directly or indirectly mediating biosynthesis and metabolic pathways in BMECs. We provide preliminary findings regarding engineering of FA contents in buffalo milk through SCD1 signaling.

The Psychological Impact and Associated Factors of COVID-19 on the General Public in Hunan, China.

PURPOSE: This study aimed to assess the psychological impact of the COVID-19 pandemic among the general public in Hunan Province, China, which could help develop psychological interventions and mental health programs. PARTICIPANTS AND METHODS: This online cross-sectional study recruited 571 participants through snowball sampling between February 2 and February 5, 2020. Data were collected through a general information questionnaire, the Public Emergency Psychological State Questionnaire, the Simple Coping Style Questionnaire, and the Public Disease Awareness on COVID-19 Scale. RESULTS: The total mean score of the public emergency psychological state of the sample was 0.27 (0.31) points, with only 5.78% of participants (n = 33) developing psychological distress. Avoidant coping style and disease awareness were weakly positively correlated (rs = 0.257, p < 0.01) and weakly negatively correlated (rs = -0.124, p <0.01) with psychological responses, respectively. There were significant psychological differences among the following variables: occupation, symptoms of fever or fatigue, discernment of the authenticity of COVID-19 information, and level of concern regarding COVID-19 (p < 0.05). CONCLUSION: The COVID-19 pandemic appears to have had a minor psychological impact on the general population in Hunan Province. However, psychological health promotion in the general public is still required, especially for employees (such as company employees, migrant workers, and businessmen), individuals with COVID-19-like symptoms, limited discernment competence and unconcerned attitudes. IMPLICATIONS: The initiatives for improving psychological health among the general public could focus on delivering COVID-19 knowledge and alleviating avoidant coping styles. Our findings could provide important insight for the development of psychological support strategies in China, as well as in other places affected by the epidemic.

Understanding divergent domestication traits from the whole-genome sequencing of swamp- and river-buffalo populations.

Domesticated buffaloes have been integral to rice-paddy agro-ecosystems for millennia, yet relatively little is known about the buffalo genomics. Here, we sequenced and assembled reference genomes for both swamp and river buffaloes and we re-sequenced 230 individuals (132 swamp buffaloes and 98 river buffaloes) sampled from across Asia and Europe. Beyond the many actionable insights that our study revealed about the domestication, basic physiology and breeding of buffalo, we made the striking discovery that the divergent domestication traits between swamp and river buffaloes can be explained with recent selections of genes on social behavior, digestion metabolism, strengths and milk production.

Brain-derived neurotrophic factor (BDNF) is expressed in buffalo (Bubalus bubalis) ovarian follicles and promotes oocyte maturation and early embryonic development.

Brain-derived neurotrophic factor (BDNF) has been discovered and characterized for several decades, yet its expression pattern in non-neuronal tissues like ovary and potential mechanism during oocyte maturation are still poorly understood. Thus the present study was devised to determine the expression pattern and mechanism of BDNF during buffalo oocyte maturation. The results revealed that BDNF was presented at different stages of buffalo ovarian follicles as well as during oocyte maturation and early embryo development. BDNF's receptor p75 was detected in granulosa cells, cumulus cells, oocytes, and early embryos, while another receptor neurotrophic tyrosine kinase receptor, type2 (NTRK2) was only identified in granulosa cells and cumulus cells. To determine the effect of BDNF on oocyte maturation and early embryo development, different concentrations (0, 1, 10, 100 ng/mL) of BDNF were added into the in vitro maturation media, respectively. It was divulged that 10 ng/mL BDNF promoted the in vitro maturation rate of buffalo oocytes and the blastocysts rate of embryos cultured in vitro (P < 0.05). Then through using NTRK2 inhibitor K-252a, we found BDNF and its receptor NTRK2 in cumulus cells played an essential role during oocyte maturation. Moreover, to further investigate the underlying mechanism by which BDNF enhances oocyte maturation, RT-qPCR was performed. 10 ng/mL BDNF treatment could decrease the expression level of apoptosis-related genes CCASP9, FAS, up-regulate the expression level of receptor gene NTRK2, cell proliferation-related genes CCNB1, PCNA, gap junction-related genes GJA4, GJA1 as well as cumulus cells expansion-related genes HAS2, PTX3 and TNFAIP6 (P < 0.05). Altogether, our results showed for the first time that BDNF was expressed throughout buffalo ovarian follicle development, oocyte maturation and early embryogenesis. Furthermore, BDNF treatment could improve the efficiency of buffalo oocyte maturation through regulating genes expression in cumulus cells and then promote early embryo development.

Roles of Polyethylenimine Ethoxylated in Efficiently Tuning the Thermoelectric Performance of Poly(3,4-ethylenedioxythiophene)-Rich Nanocrystal Films.

The regulation of oxidation levels is of great importance as an efficient way to optimize the thermoelectric (TE) performance of conducting polymers. Many efforts have been devoted to the acquisition of a high TE performance for poly(3,4-ethylenedioxythiophene):poly(styrene sulfonate) (PEDOT:PSS) by oxidation/reduction post treatment to achieve an effective compromise. However, a strong oxidant/reductant is usually employed to tune the TE performance of PEDOT:PSS with high electrical conductivity (σ) and Seebeck coefficient ( S), and it also presents a number of operational challenges depending on a fast reaction rate. Herein, nontoxic polyethylenimine ethoxylated (PEIE) served as a reductant to successfully realize an enhanced S for PEDOT:PSS, besides playing a significant anion-blocking role in enabling the efficient modulation of the oxidation level by sulfuric acid (H2SO4) with a longer operating time. Eventually, a good PEDOT-rich nanocrystal is achieved by a sequential dipping process in PEIE/ethylene glycol and H2SO4 solutions. The large TE power factor of 133 µW m-1 K-2 can be ascribed to the good formation of PEDOT-rich nanocrystals and an effective compromise between σ and S of PEDOT:PSS films. A mechanism was elucidated for the efficient regulation of σ and S enabling high performance of organic TE materials.

Optimization of parthenogenetic activation of rabbit oocytes and development of rabbit embryo by somatic cell nuclear transfer.

The present study explored a suitable parthenogenetic activation (PA) procedure for rabbit oocytes and investigated the developmental potential of somatic cell nuclear transfer (SCNT) embryos using rabbit foetal fibroblasts (RFFs). The electrical activation had the optimal rate of blastocyst (14.06%) when oocytes were activated by three direct current (DC) pulses (40 V/mm, 20 µs each) followed by 6-dimethylaminopurine (6-DMAP) and cycloheximide (CHX) treatment; the blastocyst rate of ionomycin (ION) + 6-DMAP + CHX (12.07%) activation was higher than that of ION + 6-DMAP (8.6%) activation or ION + CHX (1.24%) activation; there was no significant difference in blastocyst rate between ION + 6-DMAP + CHX and DC + 6-DMAP + CHX groups. The blastocyst rate of ION + 6-DMAP + CHX-activated oocytes in the basic rabbit culture medium (M-199) + 10% foetal bovine serum (FBS; 14.28%) was higher than that in buffalo conditioned medium (5.75%) or G1/G2 medium (0), and the blastocyst rate was increased when M-199 + 10% FBS was supplemented with amino acids. Refreshing culture medium every day or every other day significantly increased the blastocyst rate. Treatment of donor cells with 0.5% FBS for 3-5 days increased blastocyst rate of SCNT embryos (33.33%) than no serum starvation (22.47%) or 0.5% FBS treatment for 6-9 days (23.61%); the blastocyst rate of SCNT embryos derived from nontransgenic RFFs was higher than that derived from transgenic RFFs by electroporation. The blastocyst development ability of SCNT embryos derived from RFFs by electroporation (32.22%) was higher than that of liposome (19.11%) or calcium phosphate (20.00%) transfection, and only the embryos from electroporation group have the EGFP expression (24.44%). In conclusion, this study for the first time systematically optimized the conditions for yield of rabbit embryo by SCNT.

Cytoplasmic volume of recipient oocytes affects the nucleus reprogramming and the developmental competence of HMC buffalo (Bubalus bubalis) embryos.

The present study was undertaken to examine the effects of cytoplasmic volume on nucleus reprogramming and developmental competence of buffalo handmade cloning (HMC) embryos. We found that both HMC embryos derived from ~150% cytoplasm or ~225% cytoplasm resulted in a higher blastocyst rate and total cell number of blastocyst in comparison with those from ~75% cytoplasm (25.4 ± 2.0, 27.9 ± 1.6% vs. 17.9 ± 3.1%; 150 ± 10, 169 ± 12 vs. 85 ± 6, P<0.05). Meanwhile, the proportions of nuclear envelope breakdown (NEBD) and premature chromosome condensation (PCC) were also increased in the embryos derived from ~150 or ~225% enucleated cytoplasm compared to those from ~75% cytoplasm. Moreover, HMC embryos derived from ~225% cytoplasm showed a decrease of global DNA methylation from the 2-cell to the 4-cell stage in comparison with those of ~75% cytoplasm (P<0.05). Furthermore, the expression of embryonic genome activation (EGA) relative genes (eIF1A and U2AF) in HMC embryos derived from ~225% cytoplasm at the 8-cell stages was also found to be enhanced compared with that of the ~75% cytoplasm. Two of seven recipients were confirmed to be pregnant following transfer of blastocysts derived from ~225% cytoplasm, and one healthy cloned calf was delivered at the end of the gestation period, whereas no recipients were pregnant after the transfer of blastocysts derived from ~75% cytoplasm. These results indicate that the cytoplasmic volume of recipient oocytes affects donor nucleus reprogramming, and then further accounted for the developmental ability of the reconstructed embryos.

Efficient Generation of Transgenic Buffalos (Bubalus bubalis) by Nuclear Transfer of Fetal Fibroblasts Expressing Enhanced Green Fluorescent Protein.

The possibility of producing transgenic cloned buffalos by nuclear transfer of fetal fibroblasts expressing enhanced green fluorescent protein (EGFP) was explored in this study. When buffalo fetal fibroblasts (BFFs) isolated from a male buffalo fetus were transfected with pEGFP-N1 (EGFP is driven by CMV and Neo is driven by SV-40) by means of electroporation, Lipofectamine-LTX and X-tremeGENE, the transfection efficiency of electroporation (35.5%) was higher than Lipofectamine-LTX (11.7%) and X-tremeGENE (25.4%, P < 0.05). When BFFs were transfected by means of electroporation, more embryos from BFFs transfected with pEGFP-IRES-Neo (EGFP and Neo are driven by promoter of human elongation factor) cleaved and developed to blastocysts (21.6%) compared to BFFs transfected with pEGFP-N1 (16.4%, P < 0.05). A total of 72 blastocysts were transferred into 36 recipients and six recipients became pregnant. In the end of gestation, the pregnant recipients delivered six healthy calves and one stillborn calf. These calves were confirmed to be derived from the transgenic cells by Southern blot and microsatellite analysis. These results indicate that electroporation is more efficient than lipofection in transfecting exogenous DNA into BFFs and transgenic buffalos can be produced effectively by nuclear transfer of BFFs transfected with pEGFP-IRES-Neo.

Low warfarin resistance frequency in Norway rats in two cities in China after 30 years of usage of anticoagulant rodenticides.

BACKGROUND: Anticoagulant rodenticides have been widely used in rodent control in China for >30 years and resistant Norway rats have been reported. Mutations in the vitamin K epoxide reductase complex, subunit 1 (Vkorc1) gene can cause anticoagulant resistance in rodents. In this study, we analyzed the Vkorc1 polymorphisms of 681 Norway rats collected in Zhanjiang and Harbin Cities in China from 2008 to 2015 and evaluated the warfarin resistance frequency. RESULTS: Analysis revealed four mutations, including three not previously reported. Two new synonymous mutations, His68His and Leu105Leu, are not associated with warfarin resistance. One new nonsynonymous mutation, Ala140Thr, was found in Zhanjiang rat samples collected in 3 years with low frequencies (3.3-4.0%) and is probably associated with warfarin resistance. Laboratory resistance tests suggested low warfarin resistance frequencies in rats from Zhanjiang (4.9-17.1%) and Harbin (0-2.5%) Cities. CONCLUSIONS: Both genetic analysis and laboratory resistance tests suggested low warfarin resistance frequencies in rats from Zhanjiang and Harbin Cities. The alternate usage of first-generation anticoagulant rodenticides (FGARs) and second-generation anticoagulant rodenticides (SGARs) might represent an effective strategy against the development of warfarin resistance in Norway rats in China. © 2018 Society of Chemical Industry.

Basic fibroblast growth factor is critical to reprogramming buffalo (Bubalus bubalis) primordial germ cells into embryonic germ stem cell-like cells.

Primordial germ cells (PGCs) are destined to form gametes in vivo, and they can be reprogrammed into pluripotent embryonic germ (EG) cells in vitro. Buffalo PGC have been reported to be reprogrammed into EG-like cells, but the identities of the major signaling pathways and culture media involved in this derivation remain unclear. Here, the effects of basic fibroblast growth factor (bFGF) and downstream signaling pathways on the reprogramming of buffalo PGCs into EG-like cells were investigated. Results showed bFGF to be critical to buffalo PGCs to dedifferentiate into EG-like cells (20 ng/mL is optimal) with many characteristics of pluripotent stem cells, including alkaline phosphatase (AP) activity, expression of pluripotency marker genes such as OCT4, NANOG, SOX2, SSEA-1, CDH1, and TRA-1-81, and the capacity to differentiate into all three embryonic germ layers. After chemically inhibiting pathways or components downstream of bFGF, data showed that inhibition of the PI3K/AKT pathway led to significantly lower EG cell derivation, while inhibition of P53 activity resulted in an efficiency of EG cell derivation comparable to that in the presence of bFGF. These results suggest that the role of bFGF in PGC-derived EG-like cell generation is mainly due to the activation of the PI3K/AKT/P53 pathway, in particular, the inhibition of P53 function.