Comparative Analysis of Nuclear Transfer Embryo Derived Mouse Embryonic Stem Cells. Part I: Cellular characterization

Embryonic stem cells derived from nuclear transfer embryos (ntESCs) are particularly valuable for regenerative medicine, as they are a patient-specific and histocompatible cell source for the treatment of varying diseases. However, currently, little is known about their cellular and molecular profile. In the present study, in a mouse model different donor cell-derived ntESCs from various genetic backgrounds were compared with reference ESCs and analyzed comprehensively at the cellular level. A number of pluripotency marker genes were compared by flow cytometry and immunocytochemistry analysis. Significant differences at the protein level were observed for POU5F1, SOX2, FGF4, NANOG, and SSEA-1. However, such differences had no effect on in vitro cell differentiation and cell fate: derivatives of the three germ layers were detected in all ntESC lines. The neural and cardiac in vitro differentiation revealed minor differences between the cell lines, both at the mRNA and protein level. Karyotype analyses and cell growth studies did not reveal any significant variations. Despite some differences observed, the present study revealed that ntESC lines had similar differentiation competences compared to other ESCs. The results indicate that the observed differences may be related to the genotype rather than to the nuclear transfer technology

Expression profiles of the pluripotency marker gene POU5F1 and validation of reference genes in rabbit oocytes and preimplantation stage embryos

<p>Abstract</p> <p>Background</p> <p>The surge in the number of gene expression studies and tendencies to increase the quality of analysis have necessitated the identification of stable reference genes. Although rabbits are classical experimental model animals, stable reference genes have not been identified for normalization. The aims of this study were to compare the expression profiles of the widely used reference genes in rabbit oocytes and preimplantation stage embryos, and to select and validate stable ones to use as reference.</p> <p>Results</p> <p>Quantitative real time PCR method was used to evaluate 13 commonly used references (<it>Actb, Gapdh, Hprt1, H2afz, Ubc, Ppia, Eef1e1, Polr2a, Tbp, G6pdx, B2m, Pgk1</it>, and <it>Ywhaz</it>) and <it>POU5F1 (Oct4) </it>genes. Expressions of these genes were examined in multiple individual embryos of seven different preimplantation developmental stages and embryo types (<it>in vivo </it>and <it>in vitro</it>). Initial analysis identified three genes (<it>Ubc, Tbp</it>, and <it>B2m) </it>close to the detection limit with irregular expression between the different stages. As variability impedes the selection of stable genes, these were excluded from further analysis. The expression levels of the remaining ten genes, varied according to developmental stage and embryo types. These genes were ranked using the geNorm software and finally the three most stable references (<it>H2afz, Hprt1</it>, and <it>Ywhaz</it>) were selected. Normalization factor was calculated (from the geometric averages of the three selected genes) and used to normalize the expressions of <it>POU5F1 </it>gene. The results showed the expected expression patterns of the POU5F1 during development.</p> <p>Conclusion</p> <p>Compared to the earlier studies with similar objectives, the comparison of large number of genes, the use of multiple individual embryos as compared to pools, and simultaneous analyses of <it>in vitro </it>and <it>in vivo </it>derived embryo samples were unique approaches in our study. Based on quantification, pattern and geNorm analyses, we found the three genes (<it>H2afz, Hprt1</it>, and <it>Ywhaz</it>) to be the most stable across developmental stages and embryo types, and the geometric averages of these genes can be used for appropriate normalization.</p

Quantitative evaluation and selection of reference genes in mouse oocytes and embryos cultured in vivo and in vitro

BACKGROUND: Real-time PCR is an efficient tool to measure transcripts and provide valuable quantitative information on gene expression of preimplantation stage embryos. Finding valid reference genes for normalization is essential to interpret the real-time PCR results accurately, and understand the biological dynamics during early development. The use of reference genes also known as housekeeping genes is the most widely applied approach. However, the different genes are not systematically compared, and as a result there is no uniformity between studies in selecting the reference gene. The goals of this study were to compare a wide selection of the most commonly used housekeeping genes in mouse oocytes and preimplantation stage embryos produced under different culture conditions, and select the best stable genes for normalization of gene expression data. RESULTS: Quantitative real time PCR method was used to evaluate 12 commonly used housekeeping genes (Actb, Gapdh, H2afz, Hprt, Ppia, Ubc, Eef1e1, Tubb4, Hist2h2aa1, Tbp, Bmp7, Polr2a) in multiple individual embryos representing six different developmental stages. The results were analysed, and stable genes were selected using the geNorm software. The expression pattern was almost similar despite differences in the culture system; however, the transcript levels were affected by culture conditions. The genes have showed various stabilities, and have been ranked accordingly. CONCLUSION: Compared to earlier studies with similar objectives, we used a unique approach in analysing larger number of genes, comparing embryo samples derived in vivo or in vitro, analysing the expression in the early and late maternal to zygote transition periods separately, and using multiple individual embryos. Based on detailed quantification, pattern analyses and using the geNorm application, we found Ppia, H2afz and Hprt1 genes to be the most stable across the different stages and culture conditions, while Actb, the classical housekeeping gene, showed the least stability. We recommend the use of the geometric averages of those three genes for normalization in mouse preimplantation-stage gene expression studies

Impact of Technology Adoption through Dissemination Innovation Platforms (IP) on Yield, Food Security and Poverty: Evidence from Major Wheat Producing Regions of Ethiopia

Despite the high wheat production potential of Ethiopia, domestic production has been unable to match domestic demand, forcing the country to be a net wheat importer. Several development activities have aimed at increasing improved technological adoption to boost wheat yield. These have used linear and top-down approaches to disseminate different technologies. Recently, a new approach known as a technology dissemination innovation platform (IP) has been tried in four of the major wheat producing regions by the Support to Agricultural Research for Development of Strategic Crops (SARD-SC) wheat project. Despite this, there has no empirical investigation of this approach. This paper details the result of investigating the impact of wheat technology adoption through a technology dissemination IP approach. It uses two period survey data collected from 506 sample households in 2012 and 2016. Household Food Insecurity Access Scale (HFIAS) measures were used to investigate the impact of the intervention on food security and a simple poverty scorecard approach to assess the impact on poverty. Both propensity score matching and difference-in-difference econometric models were utilized to investigate the impact on wheat yields. The results reveal that the intervention brought about a significant and positive impact on food security, poverty and wheat yield. Adapting an innovation platform approach would, therefore, have a positive impact on yield increment, food security and poverty in implementing projects that have national importance. In addition, it is clear the current widely used, linear, top-down extension approach should be replaced by an approach that follows a technology innovation platform and which would provide a positive impact in these areas

Amino acids in the uterine luminal fluid reflects the temporal changes in transporter expression in the endometrium and conceptus during early pregnancy in cattle

In cattle, conceptus-maternal interactions are critical for the establishment and maintenance of pregnancy. A major component of this early interaction involves the transport of nutrients and secretion of key molecules by uterine epithelial cells to help support conceptus development during the peri-implantation period of pregnancy. Objectives were to: 1) analyze temporal changes in the amino acid (AA) content of uterine luminal fluid (ULF) during the bovine estrous cycle; 2) understand conceptus-induced alterations in AA content; 3) determine expression of AA transporters in the endometrium and conceptus; and 4) determine how these transporters are modulated by (Progesterone) P4. Concentrations of aspartic acid, arginine, glutamine, histidine, lysine, isoleucine, leucine, phenylalanine and tyrosine decreased on Day 16 of the estrous cycle but increased on Day 19 in pregnant heifers (P < 0.05). Glutamic acid only increased in pregnant heifers on Day 19 (P,0.001). Asparagine concentrations were greater in ULF of cyclic compared to pregnant heifers on Day 7 (P < 0.05) while valine concentrations were higher in pregnant heifers on Day 16 (P < 0.05). Temporal changes in expression of the cationic AA transporters SLC7A1 SLC7A4 and SLC7A6 occurred in the endometrium during the estrous cycle/early pregnancy coordinate with changes in conceptus expression of SLC7A4, SLC7A2 and SLC7A1 (P < 0.05). Only one acidic AA transporter (SLC1A5) increased in the endometrium while conceptus expression of SLC1A4 increased (P < 0.05). The neutral AA transporters SLC38A2 and SLC7A5 increased in the endometrium in a temporal manner while conceptus expression of SLC38A7, SLC43A2, SLC38A11 and SLC7A8 also increased (P < 0.05). P4 modified the expression of SLC1A1, -1A4, -1A5, -38A2 , -38A4, -38A7, -43A2, -6A14, -7A1, -7A5 and -7A7 in the endometrium. Results demonstrate that temporal changes in AA in the ULF reflect changes in transporter expression in the endometrium and conceptus during early pregnancy in cattle, some of which are modified by P4. © 2014 Forde et al

Genetic merit for fertility traits in Holstein cows: VI. Oocyte developmental competence and embryo development

peer-reviewedThe hypothesis of this study was that cows with good genetic merit for fertility traits (Fert+) would produce oocytes and embryos of greater quality than cows with poor genetic merit for fertility traits (Fert−) and that mRNA expression of candidate genes would reflect the observed differences in quality. The aim of the study, therefore, was to determine the effect of genetic merit for fertility traits on morphological classification and mRNA abundance of key genes in immature oocytes and cumulus cells following ovum pick-up and in embryos following superovulation, artificial insemination (AI), and uterine flushing. In experiment 1, 17 Fert+ and 11 Fert− cows, ranging from 54 to 84 d in milk, were submitted to ovum pick-up on 4 occasions during a 2-wk period. Recovered cumulus–oocyte complexes (COC) were morphologically graded. Oocytes and cumulus cells were separated, and mRNA abundance of genes associated with oocyte developmental competence was measured. There was no effect of genotype on the distribution of COC grades or on the mRNA abundance of the candidate genes in grade 1 COC. In experiment 2, 20 Fert+ and 19 Fert− cows, ranging from 71 to 189 d in milk, were submitted to superovulation and AI. The uteri of cows that responded to the superovulation protocol (17 Fert+ and 16 Fert− cows) were nonsurgically flushed 7 d postovulation. Recovered embryos were morphologically graded, and mRNA abundance of genes associated with embryo development was measured in grade 1 blastocysts. The response to the superovulation protocol was assessed by counting the number of codominant follicles on the day of AI, which was similar for both genotypes (22.0 ± 9.7 and 19.8 ± 8.2 for Fert+ and Fert− cows, respectively). There was no effect of genotype on the proportion of transferable embryos recovered or on the mRNA abundance of the candidate genes tested in the grade 1 blastocysts. Of the total embryos classified as blastocysts, however, the Fert+ cows tended to have a greater proportion of grade 1 blastocysts compared with Fert− cows (90% vs. 64%, respectively). In conclusion, genetic merit for fertility traits had a no effect on mRNA abundance of the candidate genes that were examined in immature oocytes and cumulus cells and in embryos recovered after superovulation. The observed differences in morphological blastocyst quality following superovulation would suggest that the superior reproductive performance of Fert+ cows could arise during the later stages of embryo development from d 7 until maternal recognition of pregnancy

Fisheries in Lake Tinishu Abaya (Ethiopia) could be managed using dietary nature of Nile tilapia (Oreochromis niloticus L. 1757)

Lake Tinisu Abaya is home to some fish species. The lake’s native fish species include Barbus and Tilapia zilli. Tilapia fish (Oreochromis niloticus L.) were stocked in Lake Tinishu Aabaya in 1997. This study aimed to investigate the feeding behaviors of O. niloticus in Lake Tinishu Abaya to develop an appropriate fisheries management approach in the lake. 428 O. niloticus fish samples (2.5 to 30.9 cm and 1.1 to 475 g) were collected to calculate the diet composition. Out of the total of 428 gut contents examined, 55 (12.85%) were found to be empty and 373 (87.15%) to be containing one or more food items. The diet behavior of the stocked fish in the study lake revealed that phytoplankton (39.5% by volume) and macrophytes (25.81% by volume) were the most noteworthy food items followed by detritus (14.39%) and zooplankton (12.95%). With increasing fish size, the importance of phytoplankton, macrophytes, and detritus increased while the contribution of zooplankton, insects, and other foods with an animal origin decreased. Seasonal variation in the diet composition of fish was evident (t-test; p &lt; 0.05). Macrophytes, zooplankton, and detritus were the dominating food items during the wet season, while phytoplankton predominated during the dry season. This study demonstrated that O. niloticus had phytoplanktivores that primarily consumed phytoplankton and that the seasons and fish size had a significant impact on their feeding preferences. The diet of O. niloticus in Lake Tinishu Abaya comprised foods with both plant and animal origins. It concludes that the dietary habit of O. niloticus in the lake is, generally, the omnivorous type

Experiential Learning in Soil Science: Evaluating Soil Quality in South Wollo, Ethiopia

This study abroad program provided an opportunity to enhance the international experience and research skills of University of Nebraska-Lincoln (UNL) students within a multidisciplinary team’s research-education goal on improving food security in Ethiopia. The experiential study component of the project included preparatory sessions during the Fall 2015 Semester at the UNL followed by a four- to five-week fieldwork experience in Ethiopia. Teams from the UNL and an Ethiopian university participated in survey data collection and field soil evaluations from farms in the Gerado area of South Wollo, Ethiopia. Having students interact with farmers increased student self-confidence and enhanced their leadership skills. Soil fertility evaluation provided the opportunity for students to apply soil health concepts in a practical setting, thus giving them a fuller understanding of these commonly challenging concepts. Throughout the study experience in Ethiopia, two UNL students wrote reflective journal entries each day and participated in daily wrap-ups. Students also gave permission to staff at Wollo University and to fellow students and faculty at the UNL. In addition they completed a final paper integrating the journal material and the experiential research work in Ethiopia. Students experienced cultural immersion while participating in this ongoing research project

Current fishery status in Ethiopian reservoirs: challenges and management

Ethiopia is branded as the Water Tower of Africa because of its abundant inland water bodies that may be used sustainably to boost the economy. This review article’s goal is to examine the current status and future potential of Ethiopia’s fisheries, with a special focus on reservoirs. There are 200 known fish species (194 native, 40 endemic, and 11 exotic) in in Ethiopia’s inland water. Ethiopia’s fish production has increased by a factor of more than five in the past three decades (3,500 tons in 1983 to 15,681 tons in 2000 and 18,058 tons in 2010). However, fish produced in reservoirs is too low (8,059 t/year). The current fish harvests from reservoirs are significantly below the predicted potential yield, accounting for only 35% of the calculated fish production capacity. In this review, 14 potential reserves were revealed. A total of 48 fish species have been reported in these, of which 26 species (or around 54%) are present in Alwero reservoir. 19 of the 48 species that have been officially recognized belong to the Cyprinidae family and made up nearly 40% of the fish population in Ethiopian reservoirs. There has been, however, a decline in the productivity of Ethiopia’s reservoir fish. Hence, actively start fishing in the numerous reservoirs that have been neglected yet, link the potential of fisheries and the benefits of the fish farming supply chain, let’s begin building several other reservoirs similar to the Grand Ethiopian Renaissance Dam and beginning mechanized-based fish harvesting

Assessment of barriers to the implementation of community-based data verification and immunization data discrepancies between health facilities and the community in Tach Gayint district, Northwest Ethiopia.

AbstractIntroduction: While community-based data verification (CBDV) is critical for effective implementation of immunization programs, limited evidence exists detailing its implementation at the local levels thereby threatening data quality which is used to guide decision making.Aim: To explore the barriers to proper implementation of CBDV and determine the level of immunization data discrepancy between the health facilities and community levels in Tach Gayint district of Northwest Ethiopia.Methods: A Mixed methods approach was used. Interviews with twenty-six key informants’ (health experts) in immunization data, and an additional a sample of 324 infants were recruited. All health centers in the district (6) and 2 health posts from each health center (12 in total) were selected using Simple Random Sampling. Key informant interviewees were purposely included from all health facilities. For quantitative data, samples of infants were proportionally allocated for each health facility as per their DPT/Pentavalent-1 vaccine report. Thematic analysis of the qualitative data and descriptive quantitative analysis were performed using statistical software open-code v-4.02 and STATA v14.1 respectively.Results: Only few health facilities implemented CBDV and consider it to be their routine task. Also, barriers to effective implementation of CBDV such as lack of prioritization, poor capacity among health staff, and conflicting job roles were identified. The highest immunization data discrepancy among community and health facilities was observed for the measles-one vaccine (35.4%), and the minimum was for DPT/Pentavalent-1 (25.6%).Conclusion: This study revealed a poor level of CBDV implementation and barriers to its effective implementation which include lack of prioritizing CBDV, limited capacity among health staff in performing CBDV, and conflicting job roles among health staff. There was a high level of immunization data discrepancy for measles-1 and DTP/Pentalent-3 vaccines. Based on our finding, we make the following recommendations: building skills among health workers to perform CBDV, enhancing availability and use of standard CBDV tools, ensuring monitoring, and control mechanism, and setting clear definition of roles regarding CBDV, as well as closing the gap in level of immunization data discrepancy could help foster effective implementation of CBDV. [Ethiop. J. Health Dev. 2021; 35(SI-3):09-15]Key words: Immunization, CBDV, Data discrepancy, Data qualit