Mid-pregnancy ewe shearing and the effects on fetus liver and muscle glycoprotein deposits

The reason why shearing ewes in midpregnancy does increase the lamb birth weight is not completely clears. Therefore, we focused on the analyses of the deposition of glycogen in different fetal tissues to investigate this issue. Thirteen pregnant Australian Merino ewes, raised in native pasture, were separated in two groups. One group (n = 7) was shorn (SE) at 70 days of pregnancy, whereas another group (n = 6) remained unshorn (NSE). Cesarean section was conducted in all the ewes at near parturition, when placenta and fetuses sampling were collected. Placenta, liver and muscle samples were fixed and stained with glycoproteinreactive acid-Schiff acid for analysis under light microscopy. The quantification of these glycoproteins was performed with the support of a program that analyzes the measurement of the intensity of staining by field. Five random fields from each sample were used, where statistical analyzes was used as normal test T. Among the analyzed regions, the deposition of glycoprotein between SE and NSE groups was statistically different in the hepatic portal vein (54,499.23 µm2 in SE and 34,830.73 µm2 in NSE) and in the total muscle area of the sample fragment (41,128, 7 µm2 and 31,942.7 µm2 , respectively; P < 0.05). We conclude that shearing ewes at the 70th day of gestation lead to accumulation of glycoproteins in the liver and muscle of fetuses, which may be responsible for the increase in birth weights in that group.(AU)

Mid-pregnancy ewe shearing and the effects on fetus liver and muscle glycoprotein deposits

The reason why shearing ewes in midpregnancy does increase the lamb birth weight is not completely clears. Therefore, we focused on the analyses of the deposition of glycogen in different fetal tissues to investigate this issue. Thirteen pregnant Australian Merino ewes, raised in native pasture, were separated in two groups. One group (n = 7) was shorn (SE) at 70 days of pregnancy, whereas another group (n = 6) remained unshorn (NSE). Cesarean section was conducted in all the ewes at near parturition, when placenta and fetuses sampling were collected. Placenta, liver and muscle samples were fixed and stained with glycoproteinreactive acid-Schiff acid for analysis under light microscopy. The quantification of these glycoproteins was performed with the support of a program that analyzes the measurement of the intensity of staining by field. Five random fields from each sample were used, where statistical analyzes was used as normal test T. Among the analyzed regions, the deposition of glycoprotein between SE and NSE groups was statistically different in the hepatic portal vein (54,499.23 µm2 in SE and 34,830.73 µm2 in NSE) and in the total muscle area of the sample fragment (41,128, 7 µm2 and 31,942.7 µm2 , respectively; P < 0.05). We conclude that shearing ewes at the 70th day of gestation lead to accumulation of glycoproteins in the liver and muscle of fetuses, which may be responsible for the increase in birth weights in that group.

Probability, odds and random chance: the difficult task of modulating the epigenetic profile of cloned embryos

Since the beginning of modern embryology, scientists have wondered about how a small number of totipotent embryonic cells can become an individual with a wide variety of organs and tissues with distinct functions. Also, the idea of generating a cloned animal using a nucleus from a donor cell is not recent. However, it has taken years of research to achieve this goal, especially regarding mechanisms of cell reprogramming required to return a differentiated cell to totipotency. Cloning by somatic cell nuclear transfer (SCNT) has been a valuable tool to understand epigenetic mechanisms related to cellular reprogramming. However, cloning efficiency is still low, with a low percentage of embryos resulting in healthy animals. The high attrition rate is associated with incomplete or abnormal epigenetic reprogramming, such that many cloned embryos have DNA methylation patterns different than controls, resulting in faulty gene expression and subsequent developmental failures. Attempts to improve genome reprogramming by modulation of oocyte quality and/or somatic cell plasticity, thereby increasing cloning efficiency and preventing detrimental effects on development, have proven ineffective. The recent development of DNA editing techniques may facilitate an improved understanding of cellular reprogramming and the role of DNA methylation in development. These novel tools may lead to new means to modulate epigenetic programming and inheritance, and hold great promise to assist in epigenetic remodeling of the donor nucleus. Such strategies are likely to improve the odds for successful cloning.

Probability, odds and random chance: the difficult task of modulating the epigenetic profile of cloned embryos

Since the beginning of modern embryology, scientists have wondered about how a small number of totipotent embryonic cells can become an individual with a wide variety of organs and tissues with distinct functions. Also, the idea of generating a cloned animal using a nucleus from a donor cell is not recent. However, it has taken years of research to achieve this goal, especially regarding mechanisms of cell reprogramming required to return a differentiated cell to totipotency. Cloning by somatic cell nuclear transfer (SCNT) has been a valuable tool to understand epigenetic mechanisms related to cellular reprogramming. However, cloning efficiency is still low, with a low percentage of embryos resulting in healthy animals. The high attrition rate is associated with incomplete or abnormal epigenetic reprogramming, such that many cloned embryos have DNA methylation patterns different than controls, resulting in faulty gene expression and subsequent developmental failures. Attempts to improve genome reprogramming by modulation of oocyte quality and/or somatic cell plasticity, thereby increasing cloning efficiency and preventing detrimental effects on development, have proven ineffective. The recent development of DNA editing techniques may facilitate an improved understanding of cellular reprogramming and the role of DNA methylation in development. These novel tools may lead to new means to modulate epigenetic programming and inheritance, and hold great promise to assist in epigenetic remodeling of the donor nucleus. Such strategies are likely to improve the odds for successful cloning.(AU)

Effects of oocyte source, cell origin, and embryo reconstruction procedures on in vitro and in vivo embryo survival after goat cloning

The birth of cloned goats has been well documented, but the overall goat cloning efficiency by somatic cell nuclear transfer procedures is still low, which may be further intensified in extreme environments. The aim of this study was to produce cloned goats under the conditions of the Brazilian Semi Arid region, in a transgenic program for the expression of human lysozyme in the milk to target childhood diarrhea and malnutrition, comparing the effects of oocyte source, cell type, and embryo reconstruction procedures on in vitro and in vivo embryo survival after cloning by micromanipulation or by handmade cloning. The use of in vitro-matured oocytes resulted in more viable embryos after cloning than in vivo-matured cytoplasts, but no differences in pregnancy rates on day 23 were seen between oocyte sources (77.5 vs. 77.8%, respectively). The presence or absence of the zona pellucida for embryo reconstruction (78.8 vs. 76.0%, respectively) did not affect pregnancy outcome after transfer. However, pregnancy rate on day 23 was higher for embryos chemically activated by a conventional than a modified protocol (88.1 vs. 50.0%), and for embryos reconstructed with mesenchymal stem cells and fetal fibroblasts (100.0 and 93.3%) than with adult fibroblasts (64.7%). Although most pregnancies were lost, the birth of a cloned female was obtained from embryos reconstructed by micromanipulation using non-transgenic control cells and in vitro-matured oocytes with intact zona pellucida, after conventional activation and transfer at the 1-cell stage.

Effects of oocyte source, cell origin, and embryo reconstruction procedures on in vitro and in vivo embryo survival after goat cloning

The birth of cloned goats has been well documented, but the overall goat cloning efficiency by somatic cell nuclear transfer procedures is still low, which may be further intensified in extreme environments. The aim of this study was to produce cloned goats under the conditions of the Brazilian Semi Arid region, in a transgenic program for the expression of human lysozyme in the milk to target childhood diarrhea and malnutrition, comparing the effects of oocyte source, cell type, and embryo reconstruction procedures on in vitro and in vivo embryo survival after cloning by micromanipulation or by handmade cloning. The use of in vitro-matured oocytes resulted in more viable embryos after cloning than in vivo-matured cytoplasts, but no differences in pregnancy rates on day 23 were seen between oocyte sources (77.5 vs. 77.8%, respectively). The presence or absence of the zona pellucida for embryo reconstruction (78.8 vs. 76.0%, respectively) did not affect pregnancy outcome after transfer. However, pregnancy rate on day 23 was higher for embryos chemically activated by a conventional than a modified protocol (88.1 vs. 50.0%), and for embryos reconstructed with mesenchymal stem cells and fetal fibroblasts (100.0 and 93.3%) than with adult fibroblasts (64.7%). Although most pregnancies were lost, the birth of a cloned female was obtained from embryos reconstructed by micromanipulation using non-transgenic control cells and in vitro-matured oocytes with intact zona pellucida, after conventional activation and transfer at the 1-cell stage.(AU)

Comparative expression profiles of genes related to cellular stress and development in oocyte of goats fed with distinct concentrations of lipids

Background: Lipotoxicity is characterized by an excess of saturated fatty acids in the blood stream, in which other nonadipose cells begin to store them, thereby altering the expression of genes related to endoplasmic reticulum stress, whoseeffects have been associated with decreased oocyte quality in several species, decreased mitochondrial activity, and increasedapoptosis. The present study was conducted to investigate the lipotoxicity effect of diets with increasing fat levels on geneexpression in goats oocyte and granulosa cells.Materials, Methods & Results: Thirty does were divided into three groups of 10 animals each, which received forage andconcentrate to provide respectively 2.7% of lipids (LL group), 3.9% lipids (LI group) and 5.1% lipids (LH group) for 28days. Three days before oocyte harvest, follicular wave was synchronized by 1 mL PGF2α intramuscularly, followed bythe insertion of an intravaginal progesterone release device. Viable oocytes and granulosa cells were subjected to qRT-PCRto determine the expression of PLIN2, ATF4, CHOP10, BAX, BCL2, HSP70 genes, were checked, evaluated using thedissociation curve analysis, which obtained the following values: 77.8, 80.2, 83.6, 78.0, 81.0 and 82.0ºC, respectively. Thesteps of qPCR thermal cycle was: denaturation and polymerase activation, annealing and final extension.Throughout theexperimental period, blood samples were taken for cholesterol, triglycerides and total lipids measurements. Total plasmalipids determination was calculated with the following equation: 2 x (cholesterol + triglycerides) × 1.1 with sensitivity ofthe assay for cholesterol was 1.472 mg/dL and for triglycerides, 2.845 mg/dL. In LH group, it was recorded a more pronounced increase in total lipids (+ 60.1%) (P < 0.05), cholesterol (+ 18.8%) and triglycerides (+ 8.5%). These increaseswere three times higher than that in LL and LI groups. All genes were expressed in oocytes...(AU)

Comparative expression profiles of genes related to cellular stress and development in oocyte of goats fed with distinct concentrations of lipids

Background: Lipotoxicity is characterized by an excess of saturated fatty acids in the blood stream, in which other nonadipose cells begin to store them, thereby altering the expression of genes related to endoplasmic reticulum stress, whoseeffects have been associated with decreased oocyte quality in several species, decreased mitochondrial activity, and increasedapoptosis. The present study was conducted to investigate the lipotoxicity effect of diets with increasing fat levels on geneexpression in goat’s oocyte and granulosa cells.Materials, Methods & Results: Thirty does were divided into three groups of 10 animals each, which received forage andconcentrate to provide respectively 2.7% of lipids (LL group), 3.9% lipids (LI group) and 5.1% lipids (LH group) for 28days. Three days before oocyte harvest, follicular wave was synchronized by 1 mL PGF2α intramuscularly, followed bythe insertion of an intravaginal progesterone release device. Viable oocytes and granulosa cells were subjected to qRT-PCRto determine the expression of PLIN2, ATF4, CHOP10, BAX, BCL2, HSP70 genes, were checked, evaluated using thedissociation curve analysis, which obtained the following values: 77.8, 80.2, 83.6, 78.0, 81.0 and 82.0ºC, respectively. Thesteps of qPCR thermal cycle was: denaturation and polymerase activation, annealing and final extension.Throughout theexperimental period, blood samples were taken for cholesterol, triglycerides and total lipids measurements. Total plasmalipids determination was calculated with the following equation: 2 x (cholesterol + triglycerides) × 1.1 with sensitivity ofthe assay for cholesterol was 1.472 mg/dL and for triglycerides, 2.845 mg/dL. In LH group, it was recorded a more pronounced increase in total lipids (+ 60.1%) (P < 0.05), cholesterol (+ 18.8%) and triglycerides (+ 8.5%). These increaseswere three times higher than that in LL and LI groups. All genes were expressed in oocytes...

Production of recombinant therapeutic proteins in genetically engineered animals: the dawn of a new era / Produção de proteínas terapêuticas recombinantes em animais geneticamente modificados: o surgimento de uma nova era

Background: The production of transgenic animals has been envisioned as a viable strategy to improve food quality, animal yield, and for the production of bioproducts that can be used for the benefit of the human and animal population. Transgenic animals have been used to improve production traits, to add value to animal products, to minimize the impact on the environment, to promote disease resistance, and most notably, to produce recombinant proteins in natural fluids, such as milk, that can be collected, purified and used as biomedical products (biopharming). This review aims to discuss past and recent technological advances in animal transgenesis, and the perspective for biopharming in Brazil.Review: Since the production of recombinant human insulin from Escherichia coli in the 1970s, continuous development of new platforms has allowed a significant expansion in the biopharmaceutical market. The animal platform has been shown to be highly competitive by adding value as low cost implementation, production and scale up, as well as high productivity of synthesized proteins. The expression of recombinant proteins in milk represents the most developed system for production of biopharmaceutical drugs in animals, with two approved biopharmaceuticals for human use: Atryn®, a recombinant antithrombin produced in the milk of goats, approved in 2006 by European Medicines Agency (EMA) and in 2009 by US Food and Drug Administration (FDA), and more recently, Ruconest®, a recombinant human C1 esterase inhibitor protein (C1INH) produced in the milk of rabbits, first approved by EMA in 2012, followed by the FDA approval in 2014. Transgenic animals have been produced by many strategies that have gradually evolved over the decades, including the use of embryo microinjection, viral vectors and transposable elements, sperm-mediated gene transfer, and cloning by somatic cell nuclear transfer (SCNT).[...](AU)

Production of recombinant therapeutic proteins in genetically engineered animals: the dawn of a new era / Produção de proteínas terapêuticas recombinantes em animais geneticamente modificados: o surgimento de uma nova era

Background: The production of transgenic animals has been envisioned as a viable strategy to improve food quality, animal yield, and for the production of bioproducts that can be used for the benefit of the human and animal population. Transgenic animals have been used to improve production traits, to add value to animal products, to minimize the impact on the environment, to promote disease resistance, and most notably, to produce recombinant proteins in natural fluids, such as milk, that can be collected, purified and used as biomedical products (biopharming). This review aims to discuss past and recent technological advances in animal transgenesis, and the perspective for biopharming in Brazil.Review: Since the production of recombinant human insulin from Escherichia coli in the 1970s, continuous development of new platforms has allowed a significant expansion in the biopharmaceutical market. The animal platform has been shown to be highly competitive by adding value as low cost implementation, production and scale up, as well as high productivity of synthesized proteins. The expression of recombinant proteins in milk represents the most developed system for production of biopharmaceutical drugs in animals, with two approved biopharmaceuticals for human use: Atryn®, a recombinant antithrombin produced in the milk of goats, approved in 2006 by European Medicines Agency (EMA) and in 2009 by US Food and Drug Administration (FDA), and more recently, Ruconest®, a recombinant human C1 esterase inhibitor protein (C1INH) produced in the milk of rabbits, first approved by EMA in 2012, followed by the FDA approval in 2014. Transgenic animals have been produced by many strategies that have gradually evolved over the decades, including the use of embryo microinjection, viral vectors and transposable elements, sperm-mediated gene transfer, and cloning by somatic cell nuclear transfer (SCNT).[...]

Efficient RNAi-induced protein knockdown in somatic cells using diced or chemically produced small interfering RNAs (siRNA)

Background: RNA interference (RNAi) is a post-transcriptional gene silencing process in which double-stranded RNA (dsRNA) directs the degradation of a specific corresponding target mRNA. The mediators of this process are small dsRNAs of approximately 21 to 23 bp in length, called small interfering RNAs (siRNAs), which can be prepared in vitro and used to direct the degradation of specific mRNAs inside cells. Hence, siRNAs represent a powerful tool to study and control gene and cell function. Rapid progress has been made in the use of siRNA as a means to attenuate the expression of any protein for which the cDNA sequence is known. Individual siRNAs can be chemically synthesized, in vitro-transcribed, or expressed in cells from siRNA expression vectors. However, screening for the most efficient siRNAs for post-transcriptional gene silencing in cells in culture is a laborious and expensive process. In this study, the effectiveness of two siRNA production strategies for the attenuation of abundant proteins for DNA repair were compared in human cells: (a) the in vitro production of siRNA mixtures by the Dicer enzyme (Diced siRNAs); and (b) the chemical synthesis of very specific and unique siRNA sequences (Stealth RNaiTM). Materials, Methods & Results: For in vitro-produced siRNAs, two segments of the human Ku70 (167 bp in exon 5; and 249 bp in exon 13; NM001469) and Xrcc4 (172 bp in exon 2; and 108 bp in exon 6; NM003401) genes were chosen to generate dsRNA for subsequent "Dicing" to create mixtures of siRNAs. The Diced fragments of siRNA for each gene sequence were pooled and stored at -80ºC. Alternatively, chemically synthesized Stealth siRNAs were designed and generated to match two very specific gene sequence regions for each target gene of interest (Ku70 and Xrcc4). HCT116 cells were plated at 30% confluence in 24- or 6-well culture plates. The next day, cells were transfected by lipofection with either Diced or Stealth siRNAs for Ku70 or Xrcc4, in duplicate, at various doses, with blank and sham transfections used as controls. Cells were harvested at 0, 24, 48, 72 and 96 h post-transfection for protein determination. The knockdown of specific targeted gene products was quantified by Western blot using GAPDH as control. Transfection of gene-specific siRNA to either Ku70 or Xrcc4 with both Diced and Stealth siRNAs resulted in a down regulation of the targeted proteins to approximately 10 to 20% of control levels 48 h after transfection, with recovery to pre-treatment levels by 96 h. Discussion: By transfecting cells with Diced or chemically synthesized Stealth siRNAs, Ku70 and Xrcc4, two highly expressed proteins in cells, were effectively attenuated, demonstrating the great potential for the use of both siRNA production strategies as tools to perform loss of function experiments in mammalian cells. In fact, down-regulation of Ku70 and Xrcc4 has been shown to reduce the activity of the non-homologous end joining DNA pathway, a very desirable approach for the use of homologous recombination technology for gene targeting or knockout studies. Stealth RNAiTM was developed to achieve high specificity and greater stability when compared with mixtures of enzymatically-produced (Diced) siRNA fragments. In this study, both siRNA approaches inhibited the expression of Ku70 and Xrcc4 gene products, with no detectable toxic effects to the cells in culture. However, similar knockdown effects using Diced siRNAs were only attained at concentrations 10-fold higher than with Stealth siRNAs. The application of RNAi technology will expand and continue to provide new insights into gene regulation and as potential applications for new therapies, transgenic animal production and basic research.

In vitro development and cell allocation after aggregation of syngeneic wild type and fluorescence-expressing bovine cloned embryos

Background: The in vitro production (IVP) of embryos by in vitro fertilization or cloning procedures has been known to cause epigenetic changes in the conceptus that in turn are associated with abnormalities in pre-and postnatal development. Handmade cloning (HMC) procedures and the culture of zona-free embryos in individual microwells provide excellent tools for studies in developmental biology, since embryo development and cell allocation patterns can be evaluated under a wide range of embryo reconstruction arrangements and in in vitro embryo culture conditions. As disturbances in embryonic cell allocation after in vitro embryo manipulations and unusual in vivo conditions during the first third of pregnancy appear to be associated with large offspring, embryo aggregation procedures may allow a compensation for epigenetic defects between aggregated embryos or even may influence more favorable cell allocation in embryonic lineages, favoring subsequent development. Thus, the aim of this study was to evaluate in vitro embryo developmental potential and the pattern of cell allocation in blastocysts developed after the aggregation of handmade cloned embryos produced using syngeneic wild type and/or transgenic somatic cells. Materials, Methods & Results: In vitro-matured bovine cumulus-oocyte complexes (COC) were manually bisected after cumulus and zona pellucida removal; then, two enucleated hemi-oocytes were paired and fused with either a wild type (WT) or a GFP-expressing (GFP) fetal skin cell at the 11th and 19th passages, respectively. Following chemical activation, reconstructed cloned embryos and zona-free parthenote embryos were in vitro-cultured in microwells, for 7 days, either individually (1 x 100%) or after the aggregation of two structures (2 x 100%) per microwell, as follows: (G1) one WT cloned embryo; (G2) two aggregated WT embryos; (G3) one GFP cloned embryo; (G4) two aggregated GFP embryos; (G5) aggregation of a WT embryo and a GFP embryo; (G6) one parthenote embryo; or (G7) two aggregated parthenote embryos. Fusion (clones), cleavage (Day 2), and blastocyst (Day 7) rates, and embryonic cell allocation were compared by the x² or Fisher tests. Total cell number (TCN) in blastocysts was analyzed by the Student's test (P < 0.05). Fusion and cleavage rates, and cell allocation were similar between groups. On a per WOW basis, development to the blastocyst stage was similar between groups, except for lower rates of development seen in G3. However, when based on number of embryos per group (one or two), blastocyst development was higher in G1 than all other groups, which were similar between one another. Cloned GFP embryos had lower in vitro development to the blastocyst stage than WT embryos, which had more TCN than parthenote or aggregated chimeric WT/GFP embryos. Aggregated GFP embryos had fewer cells than the other embryo groups. Discussion: The in vitro development of GFP cloned embryos was lower than WT embryos, with no effects on cell allocation in resulting blastocysts. Differences in blastocyst rate between groups were likely due to lower GFP-expressing cell viability, as GFP donor cells were at high population cell doublings when used for cloning. On a per embryo basis, embryo aggregation on Day 1 resulted in blastocyst development similar to non-aggregated embryos on Day 7, with no differences in cell proportion between groups. The use of GFP-expressing cells was proven a promising strategy for the study of cell allocation during embryo development, which may assist in the elucidation of mechanisms of abnormalities after in vitro embryo manipulations, leading to the development of improved protocols for the in vitro production (IVP) of bovine embryos.

Fetal membranes stem cells application in pets

Background: Stem cells are precursor cells that have the capacity for self-renewal and could generate cells with characteristics similar to cells and differentiation, generating varied cell lines. Considering the plasticity of cells can be classified into totipotent, pluripotent or multipotent. According to the isolation period, the stem cells can be classified as embryonic, fetal and adult. In the embryo stage are considered totipotent because they can rebuild any tissue in the body and adulthood are considered multipotent, since they have a more limited plasticity. The fetal tissues and the fetus is a potential source for stem cells, since they expand more rapidly compared to the cells after birth. Stem cells of fetal membranes are derived from extraembryonic tissues with high capacity to differentiate into various tissues. The cord blood stem cells have mesenchymal and hematopoietic, and mesenchymal cells have the potential to proliferate and differentiate into multiple cell lineages. The yolk sac in dogs is morphologically composed of three layers: a single layer of endoderm, a simple mesothelium, and intermediate to them, the vascular mesenchyme. Work identified a population of pluripotent cells in the yolk sac can differentiate into hematopoietic cells, however, can be isolated mesenchymal stem cells. In this review we aim to focus new isolations of cells from umbilical cord blood and yolk sac of dogs, reviewing the main literature on this species. The importance of using dogs out of work has intensified in recent years, since many diseases can manifest itself in a similar way to humans. Additionally, the dog is a pet, and interest in the treatment of diseases and improved quality of life of this species has been accentuated in veterinary medicine. Thus, identifying the cellular sources in the dog opens new horizons for preclinical studies and new therapies for veterinary medicine. Review: This study is related to morphological biology multipotent stem cells, focusing its expansion and use in cell therapy in animal models that have different pathologies. A widely studied model for muscular dystrophy is the GRMD (Golden Retriever Muscular Dystrophy), which is homologous to DMD (Duchenne Muscular Dystrophy) that affects humans. It is a recessive genetic disease, X chromosome which affects approximately 1 in every 3500 boys. It is characterized by a progressive muscle degeneration, resulting from the absence or reduction in the production of dystrophin protein present in the sarcoplasmic membrane of muscle fibers. Conclusion: The use of cells derived from fetal tissue are strong candidates for veterinary regenerative medicine, since they have high capacity for cellular differentiation. The use of fetuses and fetal tissues of humans still has limitations, so the dog is a viable alternative for studies of fetal stem cells. Thus, it is extremely important to know the characteristics of morphology and proliferation of cells derived from fetuses and fetal annexes canines, including yolk sac and umbilical cord as well as know the feasibility of clinical application of these cells in preclinical testing in animal models and eventually in human medicine, thus contributing to regenerative medicine.