Excretal Near Infrared Reflectance Spectrometry to monitor the nutrient content of diets of grazing young ostriches (Struthio camelus)

Feeding systems in which young ostriches feed on pasture but have access to concentrates provide better welfare than confined systems but are sustainable only if nutrition is carefully controlled. This study was conducted to evaluate the potential of "excretal NIRS", a methodology that associates excretal spectral information in the near infrared (NIR) region with dietary attributes, in predicting dietary quality and nutrient intake in grazing ostrich chicks. Sixty sets of excretal and dietary information from chicks fed only concentrate or also grazing lucerne, barley, sulla or natural pastures, were used. The coefficient of determination (R2) and the standard error of cross validation (SECV) served to evaluate calibration quality. The prediction of dietary concentrate content ranging 420 to 1000 g/kg of DMI, was highly linear (R2 = 0.96), with SECV of 63 g/kg. Similar R2 values were noted for the dietary contents of crude protein (CP), acid detergent fibre (ADF) and ash; that for the prediction of neutral detergent fibre (NDF) was lower (0.87). Ash, CP, NDF and ADF were predicted with SECV values of 14.8, 5.0, 8.9 and 10.7 g/kg DM diet, respectively. The calibration for apparent total organic matter digestibility was of poor quality. Good (R2 = 0.95) and acceptable (R2 = 0.86) calibrations were obtained for the daily intakes of pasture and concentrate, respectively, with SECVs of 75 and 131 g/d. Predictions of ash (R2 = 0.85, SECV = 11 g/d) and ADF (R2 = 0.80, SECV = 19 g/d) intakes had mediocre accuracy, and calibrations for CP and NDF intakes were even poorer. These results suggest that excretal NIRS may be useful to predict dietary intake and composition for grazing ostriches when applied to a known nutritional environment attended with calibration standards. Keywords: Ratites, faecal NIRS, nutrition; pasture, herbivorySouth African Journal of Animal Science Vol. 36 (4) 2006: pp. 248-25

Validation of Faecal NIRS for Monitoring the Diet of Confined and Grazing Goats

Goats are used for brush control and ecological management of Mediterranean grazing lands. Farmers are willing to cooperate with communities but they need an easy method to evaluate the daily intake of nutrients. A calibration of the chemical attributes of goats\u27 diets was set-up, based on faecal near infrared (NIR) spectra (Landau et al., 2004; Table 1). The accuracy of this methodology was estimated by using the standard error of cross-validation (SECV), which represents the variability in the difference between predicted and reference values when the equation is applied sequentially to subsets of data from the calibration data set. This procedure is justified in situations with calibration samples that are randomly selected from a natural population, but may give over-optimistic results, in particular if data are replicated. The standard error of prediction (SEP) represents the variability in the difference between predicted and reference values when the equation is applied to an external (i.e., not used in any step of the calibration) validation data set. (Naes et al., 2002). The aim of the present study was to test the robustness of predicting dietary CP, in vitro dry matter digestibility (IVDMD), and NDF percentages in goats\u27 diets, using faecal samples totally external to calibrations

Variations of X Chromosome Inactivation Occur in Early Passages of Female Human Embryonic Stem Cells

X chromosome inactivation (XCI) is a dosage compensation mechanism essential for embryonic development and cell physiology. Human embryonic stem cells (hESCs) derived from inner cell mass (ICM) of blastocyst stage embryos have been used as a model system to understand XCI initiation and maintenance. Previous studies of undifferentiated female hESCs at intermediate passages have shown three possible states of XCI; 1) cells in a pre-XCI state, 2) cells that already exhibit XCI, or 3) cells that never undergo XCI even upon differentiation. In this study, XCI status was assayed in ten female hESC lines between passage 5 and 15 to determine whether XCI variations occur in early passages of hESCs. Our results show that three different states of XCI already exist in the early passages of hESC. In addition, we observe one cell line with skewed XCI and preferential expression of X-linked genes from the paternal allele, while another cell line exhibits random XCI. Skewed XCI in undifferentiated hESCs may be due to clonal selection in culture instead of non-random XCI in ICM cells. We also found that XIST promoter methylation is correlated with silencing of XIST transcripts in early passages of hESCs, even in the pre-XCI state. In conclusion, XCI variations already take place in early passages of hESCs, which may be a consequence of in vitro culture selection during the derivation process. Nevertheless, we cannot rule out the possibility that XCI variations in hESCs may reflect heterogeneous XCI states in ICM cells that stochastically give rise to hESCs

A Novel SALL4/OCT4 Transcriptional Feedback Network for Pluripotency of Embryonic Stem Cells

Background: SALL4 is a member of the SALL gene family that encodes a group of putative developmental transcription factors. Murine Sall4 plays a critical role in maintaining embryonic stem cell (ES cell) pluripotency and self-renewal. We have shown that Sall4 activates Oct4 and is a master regulator in murine ES cells. Other SALL gene members, especially Sall1 and Sall3 are expressed in both murine and human ES cells, and deletions of these two genes in mice lead to perinatal death due to developmental defects. To date, little is known about the molecular mechanisms controlling the regulation of expressions of SALL4 or other SALL gene family members. Methodology/Principal Findings: This report describes a novel SALL4/OCT4 regulator feedback loop in ES cells in balancing the proper expression dosage of SALL4 and OCT4 for the maintenance of ESC stem cell properties. While we have observed that a positive feedback relationship is present between SALL4 and OCT4, the strong self-repression of SALL4 seems to be the “break” for this loop. In addition, we have shown that SALL4 can repress the promoters of other SALL family members, such as SALL1 and SALL3, which competes with the activation of these two genes by OCT4. Conclusions/Significance: Our findings, when taken together, indicate that SALL4 is a master regulator that controls its own expression and the expression of OCT4. SALL4 and OCT4 work antagonistically to balance the expressions of other SALL gene family members. This novel SALL4/OCT4 transcription regulation feedback loop should provide more insight into the mechanism of governing the “stemness” of ES cells

Expression profiling in transgenic FVB/N embryonic stem cells overexpressing STAT3

BACKGROUND: The transcription factor STAT3 is a downstream target of the LIF signalling cascade. LIF signalling or activation is sufficient to maintain embryonic stem (ES) cells in an undifferentiated and pluripotent state. To further investigate the importance of STAT3 in the establishment of ES cells we have in a first step derived stable pluripotent embryonic stem cells from transgenic FVB mice expressing a conditional tamoxifen dependent STAT3-MER fusion protein. In a second step, STAT3-MER overexpressing cells were used to identify STAT3 pathway-related genes by expression profiling in order to identify new key-players involved in maintenance of pluripotency in ES cells. RESULTS: Transgenic STAT3-MER blastocysts yielded pluripotent germline-competent ES cells at a high frequency in the absence of LIF when established in tamoxifen-containing medium. Expression profiling of tamoxifen-induced transgenic FVB ES cell lines revealed a set of 26 genes that were markedly up- or down-regulated when compared with wild type cells. The expression of four of the up-regulated genes (Hexokinase II, Lefty2, Pramel7, PP1rs15B) was shown to be restricted to the inner cell mass (ICM) of the blastocysts. These differentially expressed genes represent potential candidates for the maintenance of pluripotency of ES cells. We finally overexpressed two candidate genes, Pem/Rhox5 and Pramel7, in ES cells and demonstrated that their overexpression is sufficient for the maintenance of expression of ES cell markers as well as of the typical morphology of pluripotent ES cells in absence of LIF. CONCLUSION: Overexpression of STAT3-MER in the inner cell mass of blastocyst facilitates the establishment of ES cells and induces the upregulation of potential candidate genes involved in the maintenance of pluripotency. Two of them, Pem/Rhox5 and Pramel7, when overexpressed in ES cells are able to maintain the embryonic stem cells in a pluripotent state in a LIF independent manner as STAT3 or Nanog

Kinesin Light Chain 1 Suppression Impairs Human Embryonic Stem Cell Neural Differentiation and Amyloid Precursor Protein Metabolism

The etiology of sporadic Alzheimer disease (AD) is largely unknown, although evidence implicates the pathological hallmark molecules amyloid beta (Aβ) and phosphorylated Tau. Work in animal models suggests that altered axonal transport caused by Kinesin-1 dysfunction perturbs levels of both Aβ and phosphorylated Tau in neural tissues, but the relevance of Kinesin-1 dependent functions to the human disease is unknown. To begin to address this issue, we generated human embryonic stem cells (hESC) expressing reduced levels of the kinesin light chain 1 (KLC1) Kinesin-1 subunit to use as a source of human neural cultures. Despite reduction of KLC1, undifferentiated hESC exhibited apparently normal colony morphology and pluripotency marker expression. Differentiated neural cultures derived from KLC1-suppressed hESC contained neural rosettes but further differentiation revealed obvious morphological changes along with reduced levels of microtubule-associated neural proteins, including Tau and less secreted Aβ, supporting the previously established connection between KLC1, Tau and Aβ. Intriguingly, KLC1-suppressed neural precursors (NPs), isolated using a cell surface marker signature known to identify cells that give rise to neurons and glia, unlike control cells, failed to proliferate. We suggest that KLC1 is required for normal human neural differentiation, ensuring proper metabolism of AD-associated molecules APP and Tau and for proliferation of NPs. Because impaired APP metabolism is linked to AD, this human cell culture model system will not only be a useful tool for understanding the role of KLC1 in regulating the production, transport and turnover of APP and Tau in neurons, but also in defining the essential function(s) of KLC1 in NPs and their progeny. This knowledge should have important implications for human neurodevelopmental and neurodegenerative diseases

Data mining old digestibility trials for nutritional monitoring in confined goats with aids of fecal near infra-red spectrometry

There is currently no economically viable method to monitor the nutrition of individual goats reared in confinement. Fecal nearinfrared reflectance spectroscopy (\u201cfecal NIRS\u201d) enables to predict dietary attributes, based on the analysis of the reflectance of feces in the Near Infrared. Fecal NIRS calibration datasets comprise fecal NIR spectra paired with their associated dietary information. Dedicated trials to generate such datasets are costly, but such datasets are also the outcome of digestibility trials conducted by the hundreds throughout the world. We asked whether data-mining past digestibility trials in which fecal samples were retained could be used to construct fecal NIRS calibrations. This depends on the stability of fecal NIR spectra over years. Using fecal samples that were NIR-scanned in 2003, kept in dry storage at room temperature for 3 years and then scanned again in 2006, we found that the spectra changed, in particular in NIR regions related with CP and digestibility, but the five principal component scores, explaining almost all the spectral variability, were not modified. Fecal NIRS calibration equations based on 375 fecal samples collected in three countries from dairy goats between 1978 and 2001 were less precise and accurate than those based on a subset of 134 samples collected after 1988, which predicted the dietary percentages of hay, silage, corn stover, and beet pulp with high R2 cal > 0.97 and accuracy values \u2013 estimated by the standard error of cross-validation (SECV) \u2013 of 4.1%, 3.2%, 6.0%, and 2.2% of DM, respectively. The R2 cal and SECV for dietary percentage of concentrate was 0.89 and 6.4%, respectively. All dietary chemical percentages were predicted with R2 cal values above 0.93. The SECV values for dietary percentages of CP, NDF, ADF, and ADL were 0.9%, 2.9%, 1.7%, and 0.60% of DM. External validations of NDF and ADF were satisfactory. The digestibility of OM and NDF featured R2 cal values of 0.88 and 0.94, with SECV values of 2.7% and 4.1%. The intakes ofDMand CP were predicted with R2 cal > 0.91 and SECV values of 264 and 45 g d 121, respectively. These results suggest that monitoring the feeding efficiency and nutritional traceability of individual goats by fecal NIRS is potentially feasible. Recent fecal samples should be used, but moderately aged samples (<18 years) may be generally used and older samples can contribute to some calibrations. The challenge for the goat industry would be to organize cooperatively the collection and calibration of fecal NIRS data-sets and exploiting them cooperatively

Defined culture conditions of human embryonic stem cells

Human embryonic stem cells (hESCs) are pluripotent cells that have the potential to differentiate into any tissue in the human body; therefore, they are a valuable resource for regenerative medicine, drug screening, and developmental studies. However, the clinical application of hESCs is hampered by the difficulties of eliminating animal products in the culture medium and/or the complexity of conditions required to support hESC growth. We have developed a simple medium [termed hESC Cocktail (HESCO)] containing basic fibroblast growth factor, Wnt3a, April (a proliferation-inducing ligand)/BAFF (B cell-activating factor belonging to TNF), albumin, cholesterol, insulin, and transferrin, which is sufficient for hESC self-renewal and proliferation. Cells grown in HESCO were maintained in an undifferentiated state as determined by using six different stem cell markers, and their genomic integrity was confirmed by karyotyping. Cells cultured in HESCO readily form embryoid bodies in tissue culture and teratomas in mice. In both cases, the cells differentiated into each of the three cell lineages, ectoderm, endoderm, and mesoderm, indicating that they maintained their pluripotency. The use of a minimal medium sufficient for hESC growth is expected to greatly facilitate clinical application and developmental studies of hESCs