Establishment of a pheasant ( Phasianus colchicus ) spermatogonial stem cell line for the production of interspecies germ line chimeras

Background: Spermatogonial stem cells (SSCs) are important for the production of interspecies germ line chimeras. The interspecies germ cell transfer technique has been suggested as a way to conserve endangered birds. Our objective was to develop a technique for restoring endangered birds by developing interspecies germ line chimeras between pheasant ( Phasianus colchicus ) and chicken ( Gallus gallus ) with SSCs. Results: SSCs were isolated fromthe surgically removed testis of a pheasant. Growth conditions for pheasant SSCs were established by co-culturing STO (SIM mouse embryo-derived thioguanine and ouabain resistant) cells and pheasant SSCs. The colony-forming cells divided and proliferated stably to yield an established SSC line. Pheasant SSCs showed strong reactivity for GDNF family receptor alpha1 (GFR\u3b11) marker. Finally, production of germline chimeras was attempted by transferring pheasant SSCs into recipient embryos. Although final embryo survival was 5.6% (20/354), the initial survival rate was 88% (312/354). To measure the percent transfer of donor SSC to gonads, the pheasant SSCs were labeled with PKH 26 fluorescent dye. We observed 30% donor cells and 9.48% c-kit/CD117-positive cells in the gonads of recipient chickens. Donor SSCs were thus stably engrafted in the recipient gonads. Conclusions: This study showed that SSCs can be used as a tool for the conservation of endangered birds and the production of germline chimeras. Our findings yield insights into howwe may use the pheasant spermatogonial stem cell line for efficient production of interspecies germ line chimeras and ultimately, to the restoration of endangered birds

Awareness, knowledge, and risks of zoonotic diseases among livestock farmers in Punjab

Aim: The present study was conducted to assess the awareness, knowledge, and risks of zoonotic diseases among livestock farmers in Punjab. Materials and Methods: 250 livestock farmers were selected randomly and interviewed with a pretested questionnaire, which contained both open and close ended questions on different aspects of zoonotic diseases, i.e., awareness, knowledge, risks, etc. Knowledge scorecard was developed, and each correct answer was awarded one mark, and each incorrect answer was given zero mark. Respondents were categorized into low (mean − ½ standard deviation [SD]), moderate (mean ± ½ SD), and high knowledge (Mean + ½ SD) category based on the mean and SD. The information about independent variables viz., age, education, and herd size were collected with the help of structured schedule and scales. The data were analyzed by ANOVA, and results were prepared to assess awareness, knowledge, and risks of zoonotic diseases and its relation with independent variables. Results: Majority of the respondents had age up to 40 years (70%), had their qualification from primary to higher secondary level (77.6%), and had their herd size up to 10 animals (79.6%). About 51.2% and 54.0% respondents had the history of abortion and retained placenta, respectively, at their farms. The respondents not only disposed off the infected placenta (35.6%), aborted fetus (39.6%), or feces (56.4%) from a diarrheic animal but also gave intrauterine medication (23.2%) bare-handedly. About 3.6-69.6% respondents consumed uncooked or unpasteurized animal products. About 84.8%, 46.0%, 32.8%, 4.61%, and 92.4% of livestock farmers were aware of zoonotic nature of rabies, brucellosis, tuberculosis, anthrax, and bird flu, respectively. The 55.6%, 67.2%, 52.0%, 64.0%, and 51.2% respondents were aware of the transmission of zoonotic diseases to human being through contaminated milk, meat, air, feed, or through contact with infected animals, respectively. The transmission of rabies through dog bite (98.4%), need of post-exposure vaccination (96.8%), and annual vaccination of dogs (78%) were well-known facts but only 47.2% livestock owners were aware of the occurrence of abortion due to brucellosis and availability of prophylactic vaccine (67.6%) against it as a preventive measure. About 69.2% respondents belonged to low to medium knowledge level categories, whereas 30.8% respondents had high knowledge (p<0.05) regarding different aspects of zoonotic diseases. Age, education, and herd size had no significant effect on the knowledge level and awareness of farmers toward zoonotic diseases. Conclusion: Therefore, from the present study, it may be concluded that there is a need to create awareness and improve knowledge of livestock farmers toward zoonotic diseases for its effective containment in Punjab

Identification and In-silico Annotation of Functional Single Nucleotide Polymorphisms (SNPs) of the Candidate Gene Association with the Canine Transmissible Venereal Tumor Disease

ABSTRACT Canine transmissible venereal tumor (CTVT) is a histiocytic tumor of the dog that mainly affects the external genitalia, commonly found at tropical and subtropical zones. In the present investigation, we undertook this work mainly to perform a computational analysis of snSNP in the BTNL2 to identify the possible mutations and proposed the model structure of the mutant protein. Four deleterious mutations were identified in BTNL2 in 109 and 319 residual positions. Moreover, we constructed the homolgus structure of native and mutant proteins to predicate the stability. I-Mutant was used for routine analysis of protein stability and for the single site mutation analysis. It was found that mutation of L to S at residual position 109 and A to T at 319 residue position has shown maximum negative effect on the protein stability and considered for further analysis. The mutational effect on the protein function was analyzed by project HOPE. It was found that the wild-type residue is very conserved, but a few other residue types have been observed at this position too. Based on conservation scores this mutation is probably damaging to the protein. The present investigation was further used for molecular expect of the CTVT infection which might be useful in diagnosis and prevention of CTVT in canine

Establishment of a pheasant (Phasianus colchicus) spermatogonial stem cell line for the production of interspecies germ line chimeras

Background: Spermatogonial stem cells (SSCs) are important for the production of interspecies germ line chimeras. The interspecies germ cell transfer technique has been suggested as a way to conserve endangered birds. Our objective was to develop a technique for restoring endangered birds by developing interspecies germ line chimeras between pheasant (Phasianus colchicus) and chicken (Gallus gallus) with SSCs. Results: SSCs were isolated from the surgically removed testis of a pheasant. Growth conditions for pheasant SSCs were established by co-culturing STO (SIM mouse embryo-derived thioguanine and ouabain resistant) cells and pheasant SSCs. The colony-forming cells divided and proliferated stably to yield an established SSC line. Pheasant SSCs showed strong reactivity for GDNF family receptor alpha1 (GFRα1) marker. Finally, production of germ line chimeras was attempted by transferring pheasant SSCs into recipient embryos. Although final embryo survival was 5.6% (20/354), the initial survival rate was 88% (312/354). To measure the percent transfer of donor SSC to gonads, the pheasant SSCs were labeled with PKH 26 fluorescent dye. We observed 30% donor cells and 9.48% c-kit/CD117-positive cells in the gonads of recipient chickens. Donor SSCs were thus stably engrafted in the recipient gonads. Conclusions: This study showed that SSCs can be used as a tool for the conservation of endangered birds and the production of germ line chimeras. Our findings yield insights into how we may use the pheasant spermatogonial stem cell line for efficient production of interspecies germ line chimeras and ultimately, to the restoration of endangered birds

Epigenetic Reprogramming of Adult Mammalian Cells into Induced Pluripotent Stem Cells (iPSCs) - An Emerging Paradigm

ABSTRACT The field of stem-cell biology has been catapulted forward by the startling development of reprogramming technology. The ability to restore pluripotency to somatic cells through the ectopic co-expression of reprogramming factors has created powerful new opportunities for modelling human diseases and offers hope for personalized regenerative cell therapies. Worldwide increases in life expectancy have been paralleled by a greater prevalence of chronic and ageassociated disorders, particularly of the cardiovascular, neural and metabolic systems. Patient-specific induced pluripotent stem (iPS) cells are an emerging paradigm that may address this. Reprogrammed somatic cells from patients are already applied in disease modelling, drug testing and drug discovery, thus enabling researchers to undertake studies for treating diseases &apos;in a dish&apos;, which was previously inconceivable. Although there are currently several strategies to deliver reprogramming factors to induce iPSCs. In this study we have focus is on utilize plasmid vector to reprogramm because of convenience, reasonable efficiency and zero genes fingerprints and xeno free production of iPSCs. This virus-free technique reduces the safety concern for iPScell generation and application, and provides a source of cells for the investigation of the mechanisms underlying reprogramming and pluripotency

Differentiation dynamics of mammary epithelial stem cells from Korean holstein dairy cattle under ECM-free conditions

<div><p>The “stem cells” are commonly defined as “cells capable of self-renewal through replication and differentiating into specific lineages”. The mammary gland contains functional stem/progenitor cells. The current study was planned with the objectives to study the differentiation dynamics of Korean Holstein mammary epithelial stem cells (KHMESCs) under the optimum culture conditions. Lineage negative KHMESCs isolated from mammary tissue of lactating cows have shown the typical differentiation dynamics with formation of lobulo–alveolar structures in <i>in vitro</i> culture. This suggests the existence of bipotential mammary epithelial stem cells in the mammary gland. The strong mRNA expression of pluripotency factors indicates stemness, whereas expression of milk protein genes and epithelial cell-specific gene indicate their differentiation capabilities. Further, immunostaining results have shown the differentiation capabilities of KHMESCs into both luminal and basal lineages under the extracellular matrix (ECM, matrigel) free environment. However, under matrigel, the differentiation process was comparatively higher than without matrigel. Immunostaining results also suggested that differentiated cells could secrete milk proteins such as β-casein. To our knowledge, these data represent the first report on the differentiation dynamics and establishment of mammary epithelial stem cells from Korean Holstein with typical stemness properties. It was observed that isolated KHMESCs had normal morphology, growth pattern, differentiation ability, cytogenetic and secretory activity even without ECM. Therefore, it is concluded that established KHMESCs could be used for further studies on Korean Holstein dairy cows related to lactation studies, as non-GMO animal bioreactors and stem cell-based management of bovine mastitis including post-mastitis damage.</p></div

An Approach to Identify SNPs in the Gene Encoding Acetyl-CoA Acetyltransferase-2 (<i>ACAT</i>-<i>2</i>) and Their Proposed Role in Metabolic Processes in Pig

<div><p>The novel liver protein acetyl-CoA acetyltransferase-2 (ACAT2) is involved in the beta-oxidation and lipid metabolism. Its comprehensive relative expression, <i>in silico</i> non-synonymous single nucleotide polymorphism (nsSNP) analysis, as well as its annotation in terms of metabolic process with another protein from the same family, namely, acetyl-CoA acyltransferase-2 (ACAA2) was performed in <i>Sus scrofa</i>. This investigation was conducted to understand the most important nsSNPs of ACAT2 in terms of their effects on metabolic activities and protein conformation. The two most deleterious mutations at residues 122 (I to V) and 281 (R to H) were found in ACAT2. Validation of expression of genes in the laboratory also supported the idea of differential expression of <i>ACAT2</i> and <i>ACAA2</i> conceived through the <i>in silico</i> analysis. Analysis of the relative expression of <i>ACAT2</i> and <i>ACAA2</i> in the liver tissue of Jeju native pig showed that the former expressed significantly higher (P<0.05). Overall, the computational prediction supported by wet laboratory analysis suggests that ACAT2 might contribute more to metabolic processes than ACAA2 in swine. Further associations of SNPs in ACAT2 with production traits might guide efforts to improve growth performance in Jeju native pigs.</p></div

Expression analysis of mRNA and protein levels of <i>ACAA2</i> and <i>ACAT2</i> in swine and bovine species.

<p>a) Expression of mRNA after RT-PCR using 1% agarose gel. b) Relative quantitative expression of <i>ACAA2</i> and <i>ACAT2</i> genes; bars with different superscripts show significantly different expression in the two species (P<0.05). c) Representative blot expressions of ACAA2, ACAT2, and β-actin proteins obtained by western blotting.</p

Additional file 2: of An integrated in silico approach for functional and structural impact of non- synonymous SNPs in the MYH1 gene in Jeju Native Pigs

The ARRIVE Guidelines Checklist Animal Research: Reporting In Vivo Experiments. (PDF 386 kb