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This study aimed to understand the molecular characteristics of buffalo leukemia inhibitory factor (BuLIF) and the generation of a stably transfected COS-1_BuLIF cell line for its functional characterization. Cumulus cells, isolated from oocytes, were separated, and total cDNA was prepared. The BuLIF gene was ligated into the cloning vector pJET1.2/blunt and expression vector pAcGFP-N1 which was transfected into COS-1 cells and confirmed by qRT-PCR and Western blot. BuLIF was immunoprecipitated and evaluated through a MTT assay. qRT-PCR of STAT3 was performed. The multiple sequence alignment of BuLIF showed high similarity with sheep (98.77%) and cattle (96.62%) compared with other species. The BuLIF gene has an open reading frame of 609 nucleotides coding for 202 amino acids. BuLIF was integrated into the genome of COS-1 cells and resulted in the formation of dome-like secondary structures which are indicative of its functional role mediated through STAT3 proteins. In conclusion, this cell line is suitable for understanding LIF-mediated biological functions.

© 2015 Brahma et al. Nucleotide binding and oligomerization domain (NOD)-like receptors (NLRs) are innate immune receptors that recognize bacterial cell wall components and initiate host immune response. Structure and function of NLRs have been well studied in human and mice, but little information exists on genetic composition and role of these receptors in innate immune system of water buffalo-a species known for its exceptional disease resistance. Here, a comparative study on the functional domains of NOD1 and NOD2 was performed across different species. The NOD mediated in-vitro cellular responses were studied in buffalo peripheral blood mononuclear cells, resident macrophages, mammary epithelial, and fibroblast cells. Buffalo NOD1 (buNOD1) and buNOD2 showed conserved domain architectures as found in other mammals. The domains of buNOD1 and buNOD2 showed analogy in secondary and tertiary conformations. Constitutive expressions of NODs were ubiquitous in different tissues. Following treatment with NOD agonists, peripheral lymphocytes showed an IFN-γ response along-with production of pro-inflammatory cytokines. Alveolar macrophages and mammary epithelial cells showed NOD mediated in-vitro immune response through NF-κB dependent pathway. Fibroblasts showed pro-inflammatory cytokine response following agonist treatment. Our study demonstrates that both immune and nonimmune cells could generate NOD-mediated responses to pathogens though the type and magnitude of response depend on the cell types. The structural basis of ligand recognition by buffalo NODs and knowledge of immune response by different cell types could be useful for development of non-infective innate immune modulators and next generation anti-inflammatory compounds.

Turkey hens show spontaneous parthenogenesis (embryo development without any male contribution) which is influenced by genetic and environmental factors. Chromosome pattern and differential expression of genes associated with parthenogenetic development in turkey eggs were investigated in the present study. The metaphase spread obtained from parthenogenetic embryos was classified as haploid, diploid, polyploidy or aneuploidy based on the proportion of ‘n’ number of chromosomes. With the advancement of embryonic age, per cent of haploid cell (38.73 to 20.44) or other ploidy decreased while those of diploid cell increased (21.10 to 42.06) and the transition of ploidy continued till 48 h of embryo dveleopment. Early developmental stages presented higher ratios of W chromosomes in comparison to Z chromosomes while ZW combination was absent. Freshly laid parthenogenic eggs had higher Sox2 gene expression, but 24hrs old embryo had higher Sox3, GATA-4 or PouV genes expression. Expression of male specific genes (DMRT and AMH) was higher in 12 h or older parthenogenetic embryos, but the female specific genes, ASW and P450 were expressed more in freshly laid parthenogenetic eggs. It is concluded that transition of ploidy from haploid to diploid or poly-ploidy continued till 48 h or beyond parthenogenetic development. Significantly higher expression of Sox3 or GATA-4 gene in parthenogenetic embryos could potentially be used as marker gene for indication of parthenogenesis in turkey.

With an aim to isolate, culture and characterize goat embryonic stem cell-like cells derived from in vitro fertilized goat blastocysts, slaughterhouse derived goat oocytes were in vitro matured in maturation medium in 5% CO2 air at 38.5 degrees C. Matured oocytes were fertilized in vitro with fresh capacitated spermatozoa. Total 636 (36.5%) cleaved embryos were obtained which were further co-cultured with goat oviductal epithelial cells (GOEC) for 7-10 days. GOEC culture system was better for formation of morula (150; 44.3%) and hatched blastocyst (13; 3.8%) than embryo development medium culture system, [morula (69; 23.1%) and hatched blastocyst (5; 1.6%)]. Out of total blastocysts (48) the primary colonies were formed in 23.3% (7/30) blastocysts, and 66.6% (12/18) of hatched blastocysts. The cells of the inner cell mass (ICM) derived primary colonies were small, aggregated and tightly packed in nature forming embryoid bodies on further subculture. The colonies were stained to see the expression of alkaline phosphatase and positive result was obtained. Goat embryonic stem cell like outgrowths were also characterized for Oct-4 expression and positive result was found. It could be concluded that ICM cells were isolated from in vitro fertilized goat blastocysts and cultured for embryonic stem cell-like cells and expression of alkaline phosphatase and Oct-4 in these cells were positive.