A Novel 165 Kb Duplication Involving the α-Globin Gene Cluster Is Identified by Low-Pass Whole Genome Sequencing in a Chinese Thalassemia Intermedia Patient.

Copy number variations (CNVs) involving the α-globin gene cluster can lead to an imbalance in the proportion of α- and ß-globin chains and consequently cause clinical symptoms of ß-thalassemia. In our case, a 6-year-old boy, clinically diagnosed with ß thalassemia intermedia, was admitted for further genetic diagnosis with his family. Targeted sequencing and third generation sequencing (TGS) were used to detect the possible variants of the thalassemia genes. Low-pass whole genome sequencing (lpWGS) was conducted to specify the exact location of relevant CNVs across the genome, which was then validated by multiplex ligation-dependent probe amplification.The results revealed that the patient had a heterozygous ß0 mutation of Codon17 (A > T) and a full duplication of the α-globin gene cluster, inherited from his mother and father, respectively. Besides, a novel point mutation within the 5' untranslated region of ß-Globin (HBB: c. -175 (G > A) was only detected in the patient. This study suggests that lpWGS seems a powerful alternative to detect large CNVs related to thalassemia with second intention for more information of the breakpoints and a simultaneous genome-scale detection of other pathogenic CNVs.

The effect of chronic intermittent hypobaric hypoxia improving liver damage in metabolic syndrome rats through ferritinophagy.

Studies have confirmed that hepatic iron overload is one of the important factors causing liver damage in the metabolic syndrome (MS). As a special form of autophagy, ferritinophagy is involved in the regulation of iron metabolism. Our previous studies have shown that chronic intermittent hypobaric hypoxia (CIHH) can improve the iron metabolism disorder. The aim of this study was to investigate how CIHH improves liver damage through ferritinophagy in MS rats. Male Sprague-Dawley rats aged 8-10 weeks were randomly divided into four groups: control (CON), CIHH (exposed to hypoxia at a simulated altitude of 5000 m for 28 days, 6 h daily), MS model (induced by a 16-week high-fat diet and 10% fructose water feeding), and MS + CIHH (exposed to CIHH after a 16-week MS inducement) groups. Liver index, liver function, iron content, tissue morphology, oxidative stress, ferritinophagy, ferroptosis, and iron metabolism-related protein expression were measured, and the ferritinophagy flux in the liver was further analyzed. Compared with CON rats, MS rats had an increased liver index, damaged liver tissue and function, increased iron content and iron deposition, disrupted iron metabolism, significantly increased oxidative stress indicators in the liver, significantly upregulated expression of ferroptosis-related proteins, and downregulated expression of nuclear receptor coactivator 4 (NCOA4) and ferritinophagy flux. After CIHH treatment, the degree of liver damage and various abnormal indicators in MS rats were significantly improved. CIHH may improve liver damage by promoting NCOA4-mediated ferritinophagy, reducing iron overload and oxidative stress, and thereby alleviating ferroptosis in MS rats.

Machine learning prognosis model based on patient-reported outcomes for chronic heart failure patients after discharge.

BACKGROUND: Patient-reported outcomes (PROs) can be obtained outside hospitals and are of great significance for evaluation of patients with chronic heart failure (CHF). The aim of this study was to establish a prediction model using PROs for out-of-hospital patients. METHODS: CHF-PRO were collected in 941 patients with CHF from a prospective cohort. Primary endpoints were all-cause mortality, HF hospitalization, and major adverse cardiovascular events (MACEs). To establish prognosis models during the two years follow-up, six machine learning methods were used, including logistic regression, random forest classifier, extreme gradient boosting (XGBoost), light gradient boosting machine, naive bayes, and multilayer perceptron. Models were established in four steps, namely, using general information as predictors, using four domains of CHF-PRO, using both of them and adjusting the parameters. The discrimination and calibration were then estimated. Further analyze were performed for the best model. The top prediction variables were further assessed. The Shapley additive explanations (SHAP) method was used to explain black boxes of the models. Moreover, a self-made web-based risk calculator was established to facilitate the clinical application. RESULTS: CHF-PRO showed strong prediction value and improved the performance of the models. Among the approaches, XGBoost of the parameter adjustment model had the highest prediction performance with an area under the curve of 0.754 (95% CI: 0.737 to 0.761) for death, 0.718 (95% CI: 0.717 to 0.721) for HF rehospitalization and 0.670 (95% CI: 0.595 to 0.710) for MACEs. The four domains of CHF-PRO, especially the physical domain, showed the most significant impact on the prediction of outcomes. CONCLUSION: CHF-PRO showed strong prediction value in the models. The XGBoost models using variables based on CHF-PRO and the patient's general information provide prognostic assessment for patients with CHF. The self-made web-based risk calculator can be conveniently used to predict the prognosis for patients after discharge. CLINICAL TRIAL REGISTRATION: URL: http://www.chictr.org.cn/index.aspx ; Unique identifier: ChiCTR2100043337.

[Research progress on the regulation mechanisms of iron metabolism in anemia of chronic disease].

Anemia of chronic disease (ACD), complicated by various chronic inflammatory diseases, is the second most prevalent type of anemia after iron deficiency anemia in the world. ACD significantly reduces the life quality of patients with chronic diseases, and represents an independent poor prognostic factor in certain chronic diseases. A large body of studies has demonstrated that most of anemia is related to abnormal iron metabolism. In the past decade, hepcidin, as a key factor in regulating iron metabolism, has attracted enormous attention due to its important role in the pathogenesis of ACD. This article reviews the research progress on the role and underlying regulatory mechanisms of hepcidin in ACD. We also discuss the potential of hepcidin as an effective therapeutic target for ACD treatment, in order to provide a new maneuver for improving the quality of ACD patients' life.

Transgenic Wuzhishan minipigs designed to express a dominant-negative porcine growth hormone receptor display small stature and a perturbed insulin/IGF-1 pathway.

Growth hormone (GH) is an anabolic mitogen with widespread influence on cellular growth and differentiation as well as on glucose and lipid metabolism. GH binding to the growth hormone receptor (GHR) on hepatocytes prompts expression of insulin growth factor I (IGF-1) involved in nutritionally induced compensatory hyperplasia of pancreatic ß-cell islets and insulin release. A prolonged hyperactivity of the IGF-1/insulin axis in the face of insulinotropic nutrition, on the other hand, can lead to collapse of the pancreatic islets and glucose intolerance. Individuals with Laron syndrome carry mutations in the GHR gene resulting in severe congenital IGF-1 deficiency and elevated GH serum levels leading to short stature as well as perturbed lipid and glucose metabolism. However, these individuals enjoy a reduced prevalence of acne, cancer and possibly diabetes. Minipigs have become important biomedical models for human conditions due to similarities in organ anatomy, physiology, and metabolism relative to humans. The purpose of this study was to generate transgenic Wuzhishan minipigs by handmade cloning with impaired systemic GHR activity and assess their growth profile and glucose metabolism. Transgenic minipigs featuring overexpression of a dominant-negative porcine GHR (GHR(dm)) presented postnatal growth retardation and proportionate dwarfism. Molecular changes included elevated GH serum levels and mild hyperglycemia. We believe that this model may prove valuable in the study of GH functions in relation to cancer, diabetes and longevity.

[Production of GHR double-allelic knockout Bama pig by TALENs and handmade cloning].

DNA editing techniques for targeted genome modification have witnessed remarkable advances and been widely used in various organisms. However, traditional gene targeting and cloning method has been shown to be low efficient, time-consuming and expensive for generating knockout animals, especially for big animals. Here we report the generation of site-specific genome modified pig with the newly developed artificially engineered sequence-specific endonucleases (transcription activator-like effector nuclease, TALENs) and handmade cloning (HMC) methods. First, we constructed the porcine GHR-knockout vector according to TALENs kit protocol. To obtain the nuclear donor, the fetal fibroblast cell of Bama (BM) pig were transfected with GHR-knockout vector in G418 selection medium. We collected 173 cell for further positive identification which showed that 46.2% (78/173) of the clones were GHR-knockout cell strains. We chose one bi-allelic knockout cell strain as nuclear donor to produce reconstructed embryos by HMC. It was shown that the blastocyst rate was 43.5% at the 6(th) day in vitro, then 654 HMC-blastocysts were transplanted to uterus of six recipient sows. Finally, a total of 10 live offspring were delivered including 7 bi-allelic knockout piglets. Fibroblasts were obtained from ear biopsies for GHR knockout detection. The body weight of the piglets was measured consecutively, and it was found that the GHR(-)(/)(-) pigs were only 50% smaller than that of the controls at the 20(th) week. In conclusion, our results indicate that TALENs and HMC technology can rapidly and efficiently produce knockout animals for agricultural and biomedical research.

Histopathological changes of the dural myeloid cells and lymphatic vessels in a mouse model of sepsis-associated encephalopathy.

As a common diffuse encephalopathy caused by sepsis, sepsis-associated encephalopathy (SAE) is closely associated with increased mortality, severe cognition dysfunction and increased cost of health care in patients of sepsis. Accumulating evidence suggests that the dura mater, the outermost meninges of the central nervous system (CNS), plays an important role in CNS immunity, especially with the discovery of meningeal lymphatic vessels (mLVs), as well as a plentiful array of resident or infiltrating immune cells harbored in the dura. Although these findings have significantly enhanced our understanding of the immune function of dura under both steady-state and pathological condition of CNS, whether and how the immune cells and mLVs within dura response to SAE still remains largely unexplored. Here, we established lipopolysaccharide (LPS) intraperitoneal injection-induced SAE model and examined the dural resident immune cells and mLVs. We analysed the histological change in dura by performing hematoxylin and eosin (H&E) and immunofluorescence staining. Results showed that systemic exposure to LPS induced neutrophils recruitment, exudation and gathering around the dural blood vessels. Moreover, resident macrophage altered its shape as well as location, and downregulated major histocompatibility (MHC) class II expression following LPS injection. We also found that LPS exposure induced dorsal meningeal lymphangiogenesis. Together, these findings collectively demonstrated that LPS-induced SAE can stimulate immune cells and mLVs within dura and provided more information about the immune response of the dura in sepsis.

Identification of echocardiographic subgroups in patients with coronary heart disease combined with heart failure based on latent variable stratification.

BACKGROUND: The prognosis of chronic heart failure is poor, and it remains a challenge to classify patients for better personalized intervention. This study aimed to explore potential subgroups in patients with coronary heart disease and chronic heart failure using comprehensive echocardiographic indices. METHODS: 5126 patients with coronary heart disease with chronic heart failure were included. Latent class analysis was applied to identify the grouping patterns of patients based on echocardiographic indices. Network maps and radar charts of echocardiographic indices were drawn to visualize the distribution of echocardiographic findings. The incidence of adverse outcomes was presented on the Kaplan-Meier curve and compared using the log-rank test. The Cox regression model was used to analyze the relationship between subgroups and mortality. RESULTS: Three groups were identified: eccentric hypertrophy, concentric hypertrophy, and decreased diastolic function. Network plots showed a higher correlation between left atrial diameter, left ventricular mass index, and left ventricle ejection fraction in the eccentric hypertrophy group than in the other groups. The Kaplan-Meier curve showed a significant difference in mortality between the three subgroups (P < 0.001). Multivariate Cox analysis indicated that the eccentric hypertrophy group had the highest risk of death (HR = 1.586, 95% CI: 1.310-1.921, P < 0.001) compared with the other groups. CONCLUSION: Patients with coronary heart disease and chronic heart failure can be classified into three subgroups based on echocardiographic indices. This grouping has been shown to be an independent risk factor for mortality in these patients. Accurate subgrouping based on echocardiographic indices is important for identifying high-risk patients.

Chronic intermittent hypobaric hypoxia improves iron metabolism disorders via the IL-6/JAK2/STAT3 and Epo/STAT5/ERFE signaling pathways in metabolic syndrome rats.

AIM: Our previous study demonstrated that chronic intermittent hypobaric hypoxia (CIHH) improved iron metabolism disorder in obese rats through the downregulation of hepcidin. This study aimed to observe the molecular mechanism of CIHH in improving iron metabolism disorders, especially by Janus kinase/signal transducer and activation of the transcription (JAK/STAT) signaling pathway in metabolic syndrome (MS) rats. METHODS: Six-week-old male Sprague-Dawley rats were randomly divided into four groups: CON, CIHH (subjected to hypobaric hypoxia simulating 5000-m altitude for 28 days, 6 h daily), MS (induced by high fat diet and fructose water), and MS+CIHH. The serum levels of glucose, lipid metabolism, iron metabolism, interleukin-6 (IL-6), erythropoietin (Epo) and hepcidin were measured. The protein expressions of JAK2, STAT3, STAT5, bone morphogenetic protein 6 (BMP6), small mothers against decapentaplegic 1 (SMAD1) and hepcidin were examined. The mRNA expressions of erythroferrone (ERFE) and hepcidin were analyzed. RESULTS: The MS rats displayed obesity, hyperglycemia, hyperlipidemia, iron metabolism disorder, increased IL-6 and hepcidin serum levels, upregulation of JAK2/STAT3 signaling pathway, decreased Epo serum levels, downregulation of STAT5/ERFE signaling pathway in spleen, upregulation of BMP/SMAD signaling pathway in liver, and increased hepcidin mRNA and protein expression compared to CON rats. All the aforementioned abnormalities in MS rats were ameliorated in MS + CIHH rats. CONCLUSIONS: CIHH improved iron metabolism disorders, possibly by inhibiting IL-6/JAK2/STAT3 and activating Epo/STAT5/ERFE signaling pathway, thus downregulating hepcidin in MS rats.

Dynamic Trajectory of a Patient-Reported Outcome and Its Associated Factors for Patients with Chronic Heart Failure: A Growth Mixture Model Approach.

Purpose: This study aimed to identify subgroups of chronic heart failure (CHF) patients with distinct trajectories of quality of life (QOL) and to identify baseline characteristics associated with the trajectories. Patients and methods: Two-year, prospective, cohort study including 315 patients with CHF was conducted from July 2017. Information on QOL assessed by CHF-patient-reported outcomes measure (CHF-PROM) was collected at baseline, 6, 12, 18, and 24 months. Demographic and clinical variables were recorded at baseline. Growth mixture model was used to identify distinct trajectories of CHF-PROM and its physical, psychological, social, and therapeutic domains. Single factor analysis was employed to assess the factors associated with development of CHF-PROM over time. Results: Two classes of overall score of CHF-PROM were identified: poorer (14.0%) and better (86.0%). Poorer class tended to be aged, have low diastolic blood pressure, have concomitant atrial fibrillation, diabetes, chronic obstructive pulmonary disease, cancers, and central nervous system diseases, and used nitrates. Three classes of physical scores were identified: unstable-poorer (5.2%), stable-poorer (29.4%) and better (65.4%). Age, NYHA grade, chronic obstructive pulmonary disease, combined with cancers and central nervous system diseases were related to the grouping. Poorer (8.6%) and better (91.4%) classes of psychological scores were identified. Poorer class tended to be female and had concomitant atrial fibrillation. Degenerate class (34.6%) and meliorate class (65.4%) of therapeutic scores were identified. Degenerate class tended to have concomitant chronic obstructive pulmonary disease and use less angiotensin converting enzyme inhibitors. Conclusion: We identified different classes with distinct trajectories of QOL that may help proper evaluate QOL and further improve its status for patients CHF.

Single-cell transcriptional profiling reveals cellular and molecular divergence in human maternal-fetal interface.

Placenta plays essential role in successful pregnancy, as the most important organ connecting and interplaying between mother and fetus. However, the cellular characteristics and molecular interaction of cell populations within the fetomaternal interface is still poorly understood. Here, we surveyed the single-cell transcriptomic landscape of human full-term placenta and revealed the heterogeneity of cytotrophoblast cell (CTB) and stromal cell (STR) with the fetal/maternal origin consecutively localized from fetal section (FS), middle section (Mid_S) to maternal section (Mat_S) of maternal-fetal interface. Then, we highlighted a subpopulation of CTB, named trophoblast progenitor-like cells (TPLCs) existed in the full-term placenta and mainly distributed in Mid_S, with high expression of a pool of putative cell surface markers. Further, we revealed the putative key transcription factor PRDM6 that might promote the differentiation of endovascular extravillous trophoblast cells (enEVT) by inhibiting cell proliferation, and down-regulation of PRDM6 might lead to an abnormal enEVT differentiation process in PE. Together, our study offers important resources for better understanding of human placenta and stem cell-based therapy, and provides new insights on the study of tissue heterogeneity, the clinical prevention and control of PE as well as the maternal-fetal interface.

High efficiency of BRCA1 knockout using rAAV-mediated gene targeting: developing a pig model for breast cancer.

Germline inactivating mutations of the breast cancer associated gene 1 (BRCA1) predispose to breast cancer and account for most cases of familiar breast and/or ovarian cancer. The pig is an excellent model for medical research as well as testing of new methods and drugs for disease prevention and treatment. We have generated cloned BRCA1 knockout (KO) Yucatan miniature piglets by targeting exon 11 using recombinant adeno-associated virus (rAAV)-mediated gene targeting and somatic cell nuclear transfer by Handmade Cloning (HMC). We found a very high targeting rate of rAAV-mediated BRCA1 KO. Approximately 35% of the selected cells were BRCA1 targeted. One BRCA1 KO cell clone (5D1), identified by PCR and Southern blot, was used as nuclear donor for HMC. Reconstructed embryos were transferred to three recipient sows which gave birth to 8 piglets in total. Genotyping identified seven piglets as BRCA1 heterozygotes (BRCA1(+/∆11)), and one as wild type. The BRCA1 expression was decreased at the mRNA level in BRCA1(+/∆11) fibroblasts. However, all BRCA1(+/∆11) piglets died within 18 days after birth. The causes of perinatal mortality remain unclear. Possible explanations may include a combination of the BRCA1 haploinsufficiency, problems of epigenetic reprogramming, presence of the marker gene, single cell clone effects, and/or the special genetic background of the minipigs.

[Production of porcine blastocysts expressed EGFP by handmade cloning].

Production of transgenic animals via somatic cell nuclear transfer (SCNT) has been widely used worldwide. However, the application of SCNT is impeded by overall high costs and low efficiency. Here, we reported a modification of the existing technology in order to overcome some of the disadvantages associated with SCNT. Firstly, a marker gene, enhanced green fluorescent gene (EGFP), was transfected into pig fetal fibroblast cells, and was subsequently screened by fluorescent expression to ensure donor cells expressing EGFP. Porcine embryos expressing EGFP were then produced by a method called handmade cloning (HMC), a simplified method for micromanipulation. To demonstrate the concept, we collected a total of 378 fresh swine oocytes, from which 266 with the nucleus removed, obtained a total of 127 viable recombinant oocytes after fusion with EGFP-expressing cells. In vitro incubation of the 127 recombinant oocytes for approximately 144 hours resulted in successful generation of 65 viable embryos, with an average success rate of 52.1±8.3%. Compared with the traditional SCNT, the method of HMC is not only easy to operate, but also increases the rate of recombinant embryo significantly. Furthermore, the modified method no longer relies on expensive instrument like micromanipulator, facilitating the industrialization of transgenic animal production.

Single-cell transcriptome profiling reveals molecular heterogeneity in human umbilical cord tissue and culture-expanded mesenchymal stem cells.

Human umbilical cord-derived mesenchymal stem/stromal cells (UMSCs) demonstrate great therapeutic potential in regenerative medicine. The use of UMSCs for clinical applications requires high quantity and good quality of cells usually by in vitro expansion. However, the heterogeneity and the characteristics of cultured UMSCs and the cognate human umbilical cord tissue at single-cell resolution remain poorly defined. In this study, we created a single-cell transcriptome profile of human umbilical cord tissue and the cognate culture-expanded UMSCs. Based on the inferred characteristics of cell clusters and trajectory analysis, we identified three subgroups in culture-expanded UMSCs and putative novel transcription factors (TFs) in regulating UMSC state transition. Further, putative ligand-receptor interaction analysis demonstrated that cellular interactions most frequently occurred in epithelial-like cells with other cell groups in umbilical cord tissue. Moreover, we dissected the transcriptomic differences of in vitro and in vivo subgroups and inferred the telomere-related molecules and pathways that might be activated in UMSCs for cell expansion in vitro. Our study provides a comprehensive and integrative study of the transcriptomics of human umbilical cord tissue and their cognate-cultured counterparts, which paves the way for a deeper understanding of cellular heterogeneity and offers fundamental biological insight of UMSCs-based cell therapy.

Stress for stress tolerance? A fundamentally new approach in mammalian embryology.

In vitro culture, storage, and manipulation of gametes and embryos require meticulously adjusted conditions to avoid or minimize the harmful effects of uncontrolled stress. However, recent work indicates that a well-defined and properly applied stress may induce general adaptation and increase tolerance to various in vitro procedures. The aim of this review is to summarize reports on the effects of stress on gametes and embryos of several species. Treatment with sublethal doses of high hydrostatic pressure (HHP), or osmotic, heat, or oxidative stress resulted in increased morphological survival, fertilizing ability, or developmental potential after various in vitro or in vivo procedures. HHP treatment of spermatozoa, oocytes, embryos, and embryonic stem cells increased fertilizing ability, developmental competence, and differentiation and improved results after cryopreservation, parthenogenetic activation, intracytoplasmic sperm injection, and somatic cell nuclear transfer. Osmotic stress of oocytes resulted in higher developmental rates after cryopreservation, parthenogenetic activation, and somatic cell nuclear transfer. Heat shock was reported to increase developmental competence of parthenogenetically activated oocytes. Although cellular and subcellular mechanisms supposedly contributing to these processes require further research, the new principle, i.e., to improve the stress tolerance by a defined sublethal stress, may outline a completely new strategy in mammalian embryology, as well as cryopreservation of other cells and tissues with remarkable theoretical and practical consequences.

An atlas of the protein-coding genes in the human, pig, and mouse brain.

The brain, with its diverse physiology and intricate cellular organization, is the most complex organ of the mammalian body. To expand our basic understanding of the neurobiology of the brain and its diseases, we performed a comprehensive molecular dissection of 10 major brain regions and multiple subregions using a variety of transcriptomics methods and antibody-based mapping. This analysis was carried out in the human, pig, and mouse brain to allow the identification of regional expression profiles, as well as to study similarities and differences in expression levels between the three species. The resulting data have been made available in an open-access Brain Atlas resource, part of the Human Protein Atlas, to allow exploration and comparison of the expression of individual protein-coding genes in various parts of the mammalian brain.

Hemizygous minipigs produced by random gene insertion and handmade cloning express the Alzheimer's disease-causing dominant mutation APPsw.

In an effort to develop a porcine model of Alzheimer's disease we used handmade cloning to produce seven transgenic Göttingen minipigs. The donor fibroblasts had been stably transfected with a plasmid cassette containing, as transgene, the cDNA of the neuronal variant of the human amyloid precursor protein gene with the Swedish mutation preceded by beta-globin sequences to induce splicing and a human PDGF beta promoter fragment to drive transcription. Transgene insertion had occurred only at the GLIS3 locus where a single complete copy of the transgene was identified in intronic sequences in opposite direction. Similar and robust levels of the transgene transcript were detected in skin biopsies from all piglets and the sequence of full-length transcript was verified. Consistent with PDGF beta promoter function, high levels of transgene expression, including high level of the corresponding protein, was observed in brain tissue and not in heart or liver tissues. A rough estimate predicts that accumulation of the A beta peptide in the brain may develop at the age of 1-2 years.

Elevated NaCl concentration improves cryotolerance and developmental competence of porcine oocytes.

High hydrostatic pressure has been reported to improve the fertilizing or developmental ability of mammalian spermatozoa, oocytes and embryos. This study investigated the effect of another stress, temporarily increased NaCl concentration, on cryotolerance and developmental competence of porcine oocytes. In Experiment 1, survival rates were compared after 1 h exposure to seven elevated NaCl concentrations and 1 h recovery time. In Experiment 2, oocytes were exposed to 593 and 1306 mOsmol NaCl, subsequently recovered, vitrified, then subjected to parthenogenetic activation. Both cleavage and blastocyst rates increased after NaCl treatment compared with untreated controls. In Experiment 3, oocytes were treated with 593 mOsmol NaCl followed by 1 and 2 h recovery, respectively, then used as recipients for somatic cell nuclear transfer (SCNT). Cleavage rates were not different from those in untreated controls, but blastocyst rates increased in both NaCl-treated groups. In conclusion, treatment of porcine oocytes with elevated NaCl concentrations improved their developmental competence after vitrification and parthenogenetic activation or SCNT. Further experiments are required to investigate in-vivo consequences, and the effect on gametes and embryos of different mammalian species.

Osmotic stress induced by sodium chloride, sucrose or trehalose improves cryotolerance and developmental competence of porcine oocytes.

Exposure of porcine oocytes to increased concentrations of NaCl prior to manipulation has been reported not only to increase cryotolerance after vitrification, but also to improve developmental competence after somatic cell nuclear transfer (SCNT). In the present study we compared the effects of NaCl with those of concentrated solutions of two non-permeable osmotic agents, namely sucrose and trehalose, on the cryotolerance and developmental competence of porcine oocytes. In Experiment 1, porcine in vitro-matured cumulus-oocyte complexes (COCs; n = 1200) were exposed to 588 mOsmol NaCl, sucrose or trehalose solutions for 1 h, allowed to recover for a further 1 h, vitrified, warmed and subjected to parthenogenetic activation. Both Day 2 (where Day 0 is the day of activation) cleavage and Day 7 blastocyst rates were significantly increased after NaCl, sucrose and trehalose osmotic treatments compared with untreated controls (cleavage: 46 +/- 5%, 44 +/- 7%, 45 +/- 4% and 26 +/- 6%, respectively; expanded blastocyst rate: 6 +/- 1%, 6 +/- 2%, 7 +/- 2% and 1 +/- 1%, respectively). In Experiment 2, COCs (n = 2000) were treated with 588 mOsmol NaCl, sucrose or trehalose, then used as recipients for SCNT (Day 0). Cleavage rates on Day 1 did not differ between the NaCl-, sucrose-, trehalose-treated and the untreated control groups (92 +/- 3%, 95 +/- 3%, 92 +/- 2% and 94 +/- 2%, respectively), but blastocyst rates on Day 6 were higher in all treated groups compared with control (64 +/- 2%, 69 +/- 5%, 65 +/- 3% and 47 +/- 4%, respectively). Cell numbers of Day 6 blastocysts were higher in the control and NaCl-treated groups compared with the sucrose- and trehalose-treated groups. In conclusion, treatment of porcine oocytes with osmotic stress improved developmental competence after vitrification combined with parthenogenetic activation, as well as after SCNT.

High hydrostatic pressure treatment of porcine oocytes before handmade cloning improves developmental competence and cryosurvival.

An innovative technique, called the high hydrostatic pressure (HHP) treatment, has been recently reported to improve the cryosurvival of gametes or embryos in certain mammalian species. The aim of the present study was to investigate the in vitro and in vivo developmental competence and cryotolerance of embryos produced by handmade cloning (HMC) after pressure treatment of recipient oocytes. In vitro-matured porcine oocytes were treated with a sublethal hydrostatic pressure of 20 MPa (200 times greater than atmospheric pressure) and recovered for either 1 or 2 h (HHP1 and HHP2 groups, respectively) before they were used for HMC. After 7 days of in vitro culture, blastocyst rates and mean cell numbers were determined. Randomly selected blastocysts were vitrified with the Cryotop method based on minimum volume cooling procedure. The blastocyst rate was higher in the HHP2 group than in the control group (68.2 +/- 4.1% vs. 46.4 +/- 4.2%; p < 0.01), while there was no difference between HHP1 and control group (52.1 +/- 1.2% vs. 49.0 +/- 2.7%; p > 0.05). Similar mean cell numbers of produced blastocysts were obtained in HHP2 and control groups (56 +/- 4 vs. 49 +/- 5; p > 0.05). Subsequent blastocyst vitrification with the Cryotop method resulted in significantly higher survival rate after thawing in the HHP2 group than in the control group (61.6 +/- 4.0% vs. 30.2 +/- 30.9%; p < 0.01). Fifty-six and 57 day 5 to day 7 fresh blastocysts in HHP1 group were transferred into two recipient sows on day 5 of the estrous cycle. One recipient was diagnosed pregnant and gave birth to two healthy piglets by naturally delivery on day 122 of gestation. This pilot study proved that the sublethal HHP treatment of porcine oocytes before HMC results in improved in vitro developmental competence and cryotolerance, and supports embryonic and fetal development as well as pregnancy establishment and maintenance up to the birth of healthy piglets.