Maternal lineage of Nicobari pig (Sus scrofa nicobaricus) correlated with migration of Nicobarese, a native tribal population of Andaman and Nicobar Islands, India.

Nicobari pig is reared by Nicobarese, a native tribal population of Andaman and Nicobar Islands. Nicobari pig has maintained its genetic identity due to geographical isolation. This communication is the first report on maternal inheritance of Nicobari pigs. DNA polymorphism data showed seven haplotypes. D-loop sequence information and mitogenome analysis were able to earmark Nicobari pigs to Asian clade. The domestication process of pigs and its expansion pattern help to understand human migration pattern. Based on this hypothesis, this communication elucidates the probable origin of Nicobarese. Earlier studies indicated that Nicobarese had genetic affinities to races distributed in China, Malaysia and Thailand. Our data on maternal inheritance of Nicobari pig correlates with the data on migration of Nicobarese. Moreover, we could establish a novel connection of Nicobarese with people of Northeastern parts of India, Philippines and Vietnam through phylogenetic signal and geographical provenance of Nicobari pig. We further concluded that migration of Nicobarese happened during Western route of migration (WRM) ∼4000 years before present. Therefore, we propose one wave hypothesis of peopling of Nicobar based on our study and existence of Ausrtroasiatic language, Mon-Khmer in these islands.

Folate receptor-1 is vital for developmental competence of goat embryos.

Folate is essential for DNA synthesis and methylation via one-carbon (C1) metabolism during embryonic development. It is transported into the developing oocytes via folate receptors (FOLR1 and FOLR2) and transporters (RFC1) for utilization during embryo development. However, the role of folate receptors during pre-implantation stages of embryos is not well known. Thus, the present study aimed to investigate the expression of folate transport genes and proteins in mature oocytes and pre-implantation embryos and the effect of FOLR1 knockdown in zygotes on blastocyst outcome. For this, immature goat oocytes were matured in maturation medium followed by in vitro fertilization and culture at standard conditions. A group of zygotes was transfected with esiRNA against FOLR1 and in vitro cultured for blastocyst outcome assessment. The transcripts and proteins for FOLR1, FOLR2 and RFC1 were present in oocytes as well as all the stages of pre-implantation embryos. Immunofluorescence revealed the presence of FOLR1 in the nuclei of embryos but not in the metaphase (matured) oocytes. The knockdown of FOLR1 in embryos was effective and significantly reduced the blastocyst production rate. The present study demonstrates the existence of active folate transport in oocytes and pre-implantation goat embryos. FOLR1 is vital for pre-implantation embryo development and may aid in the progression by functioning as a transcription factor.

A Natural Quinazoline Derivative from Marine Sponge Hyrtios erectus Induces Apoptosis of Breast Cancer Cells via ROS Production and Intrinsic or Extrinsic Apoptosis Pathways.

Here, we report the therapeutic potential of a natural quinazoline derivative (2-chloro-6-phenyl-8H-quinazolino[4,3-b]quinazolin-8-one) isolated from marine sponge Hyrtios erectus against human breast cancer. The cytotoxicity of the compound was investigated on a human breast carcinoma cell line (MCF-7). Antiproliferative activity of the compound was estimated by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. MTT assay showed significant inhibition of MCF-7 cells viability with the IC50 value of 13.04 ± 1.03 µg/mL after 48 h. The compound induced down-regulation of anti-apoptotic Bcl-2 protein and increase in the pro-apoptotic Bax/Bcl-2 ratio in MCF-7 cells. The compound activated the expression of Caspases-9 and stimulated downstream signal transducer Caspase-7. In addition, Caspase-8 showed remarkable up-regulation in MCF-7 cells treated with the compound. Moreover, the compound was found to promote oxidative stress in MCF-7 cells that led to cell death. In conclusion, the compound could induce apoptosis of breast carcinoma cells via a mechanism that involves ROS production and either extrinsic or intrinsic apoptosis pathways. The systemic toxic potential of the compound was evaluated in an in vivo mouse model, and it was found non-toxic to the major organs.

Buffalo Leukemia Inhibitory Factor Induces Differentiation and Dome-Like Secondary Structures in COS-1 Cells.

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.

Functional characterization of Mammary Gland Protein-40, a chitinase-like glycoprotein expressed during mammary gland apoptosis.

MGP-40 is a chitinase-like protein which is over expressed during mammary gland involution. However, its physiological function in the mammary gland is poorly understood. In the present investigation, we have reported the functional significance of buffalo specific MGP-40 in the mammary gland by using an in vitro model of the buffalo mammary epithelial cell (BuMEC) line. MGP-40 was highly up regulated in BuMECs in serum starved condition as well as after treatment with prolactin suggesting its role in the stress response. Subsequently, to study the effect of MGP-40 on BuMECs, the cells were transfected with a mammalian expression construct of pCI neo harboring MGP-40 gene. It was observed that over expression of MGP-40 enhanced proliferation of BuMECs and protected the cells from apoptosis under serum free condition. In contrast, MGP-40 attenuated the mitogenic effect of insulin in BuMECs. Besides, over expression of the MGP-40 reduced dome formation, acinar polarization and casein synthesis in BuMECs in the presence of lactogenic hormones, it also induced Stat3 phosphorylation and epithelial to mesenchymal transition (EMT) -like features. Together, our data suggest that MGP-40 is involved in protection of BuMECs under stress conditions, inhibits cellular differentiation and induces EMT-like features. A schematic diagram depicting possible association of MGP-40 in various molecular pathways has been presented.

Molecular characterization of IFN-T expressed in buffalo embryonic trophoblasts and expression of recombinant BuIFN-T1a2 and BuIFN-T8 isoforms in E. coli.

Interferon tau (IFN-T) acts as a signaling molecule for maternal recognition of pregnancy (MRP) in ruminants. Aim of the present study was to identify various Buffalo Interferon tau (BuIFN-T) transcripts in buffalo trophoblast, phylogenetic comparison of these sequences with known mRNA sequences of buffalo, bovine, caprine and ovine and to express and purify the recombinant BuIFN-T (rBuIFN-T) isoforms. Following RNA extraction from trophectodermal cells, RT-PCR was performed using Ifn-t gene specific primers. 13 distinct cDNA variants encoding eight different BuIFN-T proteins were identified. BuIFN-T1a2 and BuIFN-T8 were expressed in prokaryotic expression system at 37 °C, 25 °C and 16 °C with 1 mM IPTG for 12 h and the recombinant proteins expressed at 16 °C were partially purified by Immobilised Metal Affinity Chromatography (IMAC). BuIFN-T isoforms have greater nucleotide and amino acid homology with caprine (98-100%, 96-100%), ovine (94-97%, 90-95%) and bovine (89.6-90.6%, 82-86%). These novel BuIFN-T isoforms contained pronounced nucleotide and amino acid sequence identity with one another (99.1-99.8%, 98-99%) but moderate sequence identity with previously identified buffalo IFN-T (90-92%, 82-86%). Solubility of expressed recombinant isoforms (rBuIFN-T1a2 and rBuIFN-T8) was highest at 16 °C. In conclusion, 13 distinct Ifn-t gene variants exist in trophectoderm of in vitro developed buffalo blastocysts that encode eight different proteins. rBuIFN-T1a2 and rBuIFN-T8 were successfully expressed in soluble form in Escherichia coli expression system at 16 °C with 1 mM IPTG and the resulting recombinant proteins were partially purified by IMAC.

Expression and purification of buffalo interferon-tau and efficacy of recombinant buffalo interferon-tau for in vitro embryo development.

The aim of our study was to optimize growth and induction parameters, for expression and large scale purification of functionally active buffalo interferon tau, and to study its possible impact on in vitro blastocyst development. The buffalo interferon-tau gene (BuIFN-T1) bearing gene bank accession No. JX481984, with signal sequence, was obtained through polymerase chain reaction (PCR) from bovine early embryos and was cloned into pJET vector. After being verified, the fragments without signal sequence, were inserted into the expression vector pET-22b and the recombinant plasmid was induced to express the recombinant protein in a prokaryotic expression system. The recombinant BuIFN-T was confirmed by SDS-PAGE and Western blot and subjected to three steps of large scale purification using His Affinity chromatography, Anion Exchange chromatography and Gel Filtration chromatography. The purified recombinant BuIFN-T protein was validated by mass spectroscopy analysis. To examine the effect of recombinant BuIFN-T protein on developmental competency of buffalo embryos, purified recombinant BuIFN-T protein was added to in vitro embryo culture medium (at concentration of 0, 1µg/ml, 2µg/ml, 4µg/ml) for 9days. Addition of recombinant BuIFN-T (2µg/ml) significantly improved the rate of blastocyst production, 45.55% against 31.1% control (p<0.01). Here we conclude that the recombinant BuIFN-T was successfully purified to homogeneity from a prokaryotic expression system and it significantly increased the blastocyst production rate in buffalo. These findings suggest a potential impact of IFN-T in promoting embryonic growth and development.

Molecular cloning and production of caprine recombinant Oct4 protein for generation induced pluripotent stem cells.

Oct4, pluripotency marker and transcription factor, expresses in embryonic stem cells. It plays a pivotal role in determination of stem cells fate. Up and down regulation of Oct4 causes differentiation of embryonic stem cells. It is one of the main transcription factors which remained concerned in every study related to induced pluripotent stem cell. Here, we report the production of goat Oct4 protein using plasmid and lentiviral based vectors. Firstly, Oct4 ORF was cloned in pAcGFP1-N1 plasmid vector and positive clones were screened with colony PCR. Oct4 was over-expressed in CHO-K1 cell line and expression was confirmed by observing green florescent protein expression in CHO-K1 cells. Secondly, Oct4 lentiviral expression construct has been prepared using pLenti-gw vector. Oct4 ORF was cloned into pLenti4/V5-DEST vector and viral particles were produced in 293FT cells. Oct4 viral particles were used to infect goat fibroblast cells. Oct4 expression was observed and confirmed in transfected goat fibroblast cells using RT-PCR. Detection of Oct4 protein in western blotting assay affirmed the capacity of over-expression of our Oct4 lentiviral vector. The lentiviral expression construct and recombinant Oct4 protein may be used for reprogramming of somatic cell into induced pluripotent stem cell.

Generation of parthenogenetic goat blastocysts: effects of different activation methods and culture media.

The present study was carried out to investigate the effects of different activation methods and culture media on the in vitro development of parthenogenetic goat blastocysts. Calcium (Ca2+) ionophore, ethanol or a combination of the two, used as activating reagents, and embryo development medium (EDM), modified Charles Rosenkrans (mCR2a) medium and research vitro cleave (RVCL) medium were used to evaluate the developmental competence of goat blastocysts. Quantitative expression of apoptosis, stress and developmental competence-related genes were analysed in different stages of embryos. In RVCL medium, the cleavage rate of Ca2+ ionophore-treated oocytes (79.61 ± 0.86) was significantly (P < 0.05) higher than in ethanol (74.90 ± 1.51) or in the combination of both Ca2+ ionophore and ethanol. In mCR2a or EDM, hatched blastocyst production rate of Ca2+ ionophore-treated oocytes (8.33 ± 1.44) was significantly higher than in ethanol (6.46 ± 0.11) or in the combined treatment (6.70 ± 0.24). In ethanol, the cleavage, blastocyst and hatched blastocyst production rates in RVCL medium (74.90 ± 1.51, 18.30 ± 1.52 and 8.24 ± 0.15, respectively) were significantly higher than in EDM (67.81 ± 3.21, 14.59 ± 0.27 and 5.59 ± 0.42) or mCR2a medium (65.09 ± 1.57, 15.36 ± 0.52 and 6.46 ± 0.11). The expression of BAX, Oct-4 and GlUT1 transcripts increased gradually from 2-cell stage to blastocyst-stage embryos, whereas the transcript levels of Bcl-2 and MnSOD were significantly lower in blastocysts. In addition, different activation methods and culture media had little effect on the pattern of variation and relative abundance of the above genes in different stages of parthenogenetic activated goat embryos. In conclusion, Ca2+ ionophore as the activating agent, and RVCL as the culture medium are better than other tested options for development of parthenogenetic activated goat blastocysts.

Isolation and propagation of neural stem cells in caprine (Capra hircus).

Neural stem cells (NSCs) can self-renew and give rise to neurons, astrocytes and oligodendrocytes; they are found in the nervous system of mammalian organisms, representing a promising resource for both fundamental research and therapeutics. There have been few investigations on NSCs in the livestock species. Therefore, we have successfully isolated and characterised NSCs from the foetal brain of a small domestic animal, the goat (called GNSCs). These cells from the foetal brain showed self-renewal, rapid proliferation with a population doubling time of 88 h, were morphologically homogeneous and maintained normal chromosome throughout the culture period. The cells expressed NSC-specific markers (Sox2, Pax6 and Mushashi), but were negative for CD34 and CD45. They were capable of multi-differentiation into neurons, astrocytes, oligodendrocytes, as well as adipocytes and osteocytes. The availability of such cells may hold great interest for basic and applied neuroscience.

Molecular cloning, sequence characterization and recombinant expression of Nanog gene in goat fibroblast cells using lentiviral based expression system.

Nanog is a homeodomain containing protein which plays important roles in regulation of signaling pathways for maintenance and induction of pluripotency in stem cells. Because of its unique expression in stem cells it is also regarded as pluripotency marker. In this study goat Nanog (gNanog) gene has been amplified, cloned and characterized at sequence level with successful over-expression in CHO-K1 cell line using a lentiviral based system. gNanog ORF is 903 bp long which codes for Nanog protein of size 300 amino acids (aas). Complete nucleotide sequence shows some evolutionary mutation in goat in comparision to other species. Protein sequence of goat is highly similar to other species. Overall, gNanog nucleotide sequence and predicted protein sequence showed high similarity and minimum divergence with cattle (96 % identity/4 % divergence) and buffalo (94/5 %) while low similarity and high divergence with pig (84/15 %), human (81/23 %) and mouse (69/40 %) indicating evolutionary closeness of gNanog to cattle and buffalo. gNanog lentiviral expression construct was prepared for over-expression of Nanog gene in adult goat fibroblast cells. Lentiviral expression construct of Nanog enabled continuous protein expression for induction and maintenance of pluripotency. Western blotting revealed the expression of Nanog gene at protein level which supported that the lentiviral expression system is highly promising for Nanog protein expression in differentiated goat cell.

Resveratrol treatment during goat oocytes maturation enhances developmental competence of parthenogenetic and hand-made cloned blastocysts by modulating intracellular glutathione level and embryonic gene expression.

PURPOSE: The aim of the present study was to determine whether supplementation of resveratrol, a stilbenoid antioxidant with therapeutic significance, influences goat (Capra hircus) oocyte maturation and subsequent embryonic development and expression of apoptosis and early embryonic development-related genes. METHODS: Five different concentrations of resveratrol (0.1, 0.25, 0.5, 2.0 and 5.0 µM) were used in in vitro maturation (IVM) medium. Cell tracker blue and 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) fluorescent stains were used to assay intracellular glutathione and reactive oxygen species levels in mature oocytes. Parthenogenetic activation and hand-made cloning were performed to check the developmental potential following resveratrol treatment. We used quantitative real-time PCR to analyze embryonic gene expression. RESULT: Compared to control, no significant improvement was observed in nuclear maturation in resveratrol-treated groups and at 5.0 µM concentration maturation rate decreased significantly (P < 0.05). But resveratrol treatment at the concentrations of 0.25, 0.5 µM significantly reduced intracellular ROS, and increased GSH concentrations. Oocytes treated with 0.25, 0.5 µM resveratrol when subsequently used for PA and HMC, higher extent of blastocyst yields were observed. Expression analysis of proapoptotic (Bax) gene in mature oocytes, cumulus cells, and HMC-derived blastocysts revealed lesser transcript abundances in various resveratrol-treated groups., however no change in the same was observed for antiapoptotic gene (Bcl2). Differential expression of genes associated with developmental competence and nuclear reprogramming was also observed in HMC-derived blastocysts. CONCLUSION: Our results show that resveratrol treatment at optimum concentrations (0.25 and 0.5 µM) during IVM produced beneficial microenvironment within oocytes by increasing the intracellular GSH, decreasing ROS level and this in turn, stimulated embryonic development and regulated gene expression.

Isolation and characterization of neural stem cells from buffalo.

Neural stem cells (NSCs) are primordial, uncommitted cells postulated to give rise to the array of more specialized cells of the central nervous system (CNS). NSCs can self-renew and give rise to neurons, astrocytes and oligodendrocytes. NSCs are found in the CNS of mammalian organisms, and represent a promising resource for both fundamental research and CNS repair. Animal models of CNS damage have highlighted the potential benefit of NSC-based approaches. Present study described that buffalo neural stem cells (Bu-NSCs) were isolated and expanded rapidly from buffalo fetal brain in adherent culture. They were capable of multidifferentiation into neurons, astrocytes, and oligodendrocytes. Bu-NSCs were morphologically homogeneous and possessed high proliferation ability. The population doubled every 128.16 h. Normal buffalo karyotype was unchanged throughout the in vitro culture period. Together, we have isolated and cultured Bu-NSC from fetal brain that showed self-renewal, rapid proliferation and ability to differentiate into cells of nervous system. The availability of such cells may hold great interest for basic and applied neuroscience.

Optimizing crop quality and yield: Assessing the impact of integrated potassium management on Chinese cabbage (Brassica rapa L. subsp. chinensis).

Potassium, a pivotal macronutrient essential for growth, development, and crop yield, serves as a critical determinant of soil productivity. Its depletion disrupts the equilibrium of soil nutrients, prompting an investigation into integrated potassium management strategies to address this challenge. A field experiment was conducted during the winter season of 2020 using a randomized complete block design, with eight treatments, each replicated three times in Chinese cabbage (Brassica rapa L. subsp. chinensis). These treatments comprised standard (100 %) and reduced (75 % and 50 %) rates of the recommended dose of potassium (RDK) via muriate of potash (MOP). Variations in the inclusion and exclusion of plant growth-promoting rhizobacteria (PGPR), farmyard manure (FYM) as 25 % of the potassium recommendation, and foliar spray of nano potash were systematically implemented. Findings unequivocally demonstrated that the treatmentT8, involving 100 % RDK +25 % K through FYM + PGPR + nano K fertilizer spray at 25 and 40 DAS, yielded significant improvements in both green fodder (64.0 t ha-1) and dry fodder (7.87 t ha-1).Moreover, T8 exhibited the highest values for total ash (8.75 %), total ash yield (68.9 ± 2.88 kg ha-1), ether extract (2.85 %), ether extract yield (22.4 ± 0.88 kg ha-1), crude protein (9.71 %), and total crude protein yield (76.4 ± 3.21 kg ha-1). Conversely, a marked reduction was observed in various fiber components and carbohydrate fractions upon application of the T8 treatment. The lowest values of yield, crude protein content, total ash ether extract were recorded in treatment T1 (control) applied with no potassium. This investigation underscores the inadequacy of the recommended potassium dose in achieving optimal productivity, necessitating a re-evaluation of potassium fertilization levels. The integrated approach involving FYM, PGPR, and nano potash, coupled with the recommended potassium dose through MOP, emerges as a promising avenue for augmenting both yield and quality parameters in Chinese cabbage.

Allogenic adipose derived mesenchymal stem cells are effective than antibiotics in treating endometritis.

Endometritis is a uterine inflammatory disease that causes reduced livestock fertility, milk production and lifespan leading to significant economic losses to the dairy industry. Mesenchymal stem cells (MSC) may act as an alternative for inefficacy of antibiotics and rising antibiotic resistance in endometritis. The present study aimed to cure the chronic endometritic buffaloes using allogenic adipose-derived MSCs (AD-MSC). AD-MSCs were isolated from buffalo adipose tissue and characterized by multilineage differentiation as well as MSC-specific markers. The in vivo safety and efficacy were assessed after infusion of AD-MSCs. In safety trial, cells were administered in healthy buffaloes via different routes (IV and IC) followed by examination of clinical and hematological parameters. In efficacy study, AD-MSCs treatments (IV and IC) and antibiotic therapy (ABT) in endometritic buffaloes were comparatively evaluated. AD-MSCs did not induced any immunological reaction in treated buffaloes. PMN count, CRP levels and VDS were significantly (p ≤ 0.05) reduced after AD-MSCs infusions in IV and IC groups and no significant difference was observed in antibiotic group. The IV group was marked with 50% absolute risk reduction in endometritis and 50% live calf births after artificial insemination in comparison with ABT group. Anti-inflammatory cytokines (IL4 and IL10) and anti-microbial peptides (PI3, CATHL4, LCN2 and CST3) expressions were significantly (p ≤ 0.05) upregulated in IV group. The calf delivery rate after the treatments in IV group was higher (50%, 3 calves) than the other groups (IC: 33.3%, 2 calves; ABT: 16.6%, 1 calf). In conclusion, the administration of AD-MSCs through IV route was found to be safe and efficacious for alleviating chronic endometritis in dairy buffaloes.

Peeping into Mitochondrial Diversity of Andaman Goats: Unveils Possibility of Maritime Transport with Diversified Geographic Signaling.

Andaman and Nicobar Islands, a part of South-East Asia, is enriched with the presence of native breeds of livestock (cattle, pig, goat) and poultry. There are two native goat breeds, viz., Andaman local goat and Teressa goat in Andaman and Nicobar Islands. However, to date, the origin and genetic makeup of these two breeds have not been detailed. Therefore, the present study describes the genetic makeup of Andaman goats through analysis of mitochondrial D-loop sequence for sequence polymorphism, phylogeographical signaling and population expansion events. The genetic diversity of the Teressa goat was less compared to the Andaman local goat due to its sole presence on Teressa Island. Out of 38 well-defined haplotypes of Andaman goats, the majority of haplotypes belonged to haplogroup A followed by haplogroup B and haplogroup D. The result of mismatch distribution and neutrality tests indicated no population expansion event of haplogroup A and B. Finally, based on poor geographical signaling, we hypothesize that Andaman goats have been imported to these Islands either through multidirectional diffusion or unidirectional diffusion. We justify our hypothesis of multidirectional diffusion on the basis of observation of the haplotype and nucleotide diversity of Andaman goats. Simultaneously, the probability of unidirectional diffusion of goats in these islands from the Indian subcontinent in different spells of domestication events through maritime routes cannot be ignored.

Microarray analysis and PCR validation of genes associated with facultative parthenogenesis in Meleagris gallopavo (Turkey).

A cDNA microarray containing 43,661 differentially expressed genes was carried out on the blastoderm of fertilized and facultative parthenogenic turkey embryos at different hours of development. The total number of up-regulated (UR) and down-regulated (DR) genes at 0, 12, and 24 h of development were 725 and 1436, 942 and 599, and 589 and 1044, respectively. Common genes between 0 and 12 h, 12 and 24 h, and 0 and 24 h were 55, 67, and 110, respectively. The proportion of genes showing above 50-fold UR and DR at 0, 12, and 24 h of development were 2.0% and 1.5%, 0.5% and 1.2%, and 0.2% and 1.1%, respectively. Eight UR genes were validated (APOA1, THRAP3, ARL14EP, PSAP, MOG, MYBPC2, MTIF3 and EDG4) and relative expression of six of them was significantly higher (P ≤ 0.05) in parthenogenic embryos, while two genes showed non-significant (P ≥ 0.05) variation. The expression of BCL11A, PRP4B, TCP1, and TPI1 genes was significantly (P ≤ 0.05) DR in parthenotes in the micro-array study, while the TCP1 gene was up-regulated, and there was no variation in TPI1 gene expression in the PCR validation study. In conclusion, our findings demonstrate differential expression of a large number of genes in parthenotes at different stages of embryo development compared to fertilized embryos. Up-regulation of APOA1, MYBPC2, TCP1, and THRAP3 genes, suggest their crucial role in spontaneous facultative parthenogenic development in turkey birds.

Allogenic umbilical cord blood-mesenchymal stem cells are more effective than antibiotics in alleviating subclinical mastitis in dairy cows.

Subclinical mastitis is an inflammatory disease that affects the milk production, fertility, and lifespan of animals, leading to significant losses to dairy industry. Antibiotics therapies are resulting in suboptimal benefits in treating subclinical mastitis due to prevalent antibiotic resistance in dairy herds. In a quest to develop alternative therapy, umbilical cord-derived mesenchymal stem cells (UCB-MSCs) and its extracellular vesicles (UCB-MSC-EVs) are used, in the present study, to validate its safety and efficacy as potential therapy for treatment of subclinical mastitis in dairy cows with respect to conventional antibiotic therapy (ABT). We isolated, in vitro cultured, and characterized the UCB-MSCs as well as UCB-MSC-EVs. The repeated infusions of low dose MSCs and EVs were delivered in healthy animals for safety analysis, followed by the same administrations in infected animals for therapeutic efficacy analysis. UCB-MSCs and UCB-MSC-EVs were found to be safe at 2 doses with 7-day gap of 5 × 107 cells/injection and EV equivalent to 500 µg protein in DPBS, respectively. Efficacy trials demonstrated significantly decreased somatic cell count to safe levels in milk samples of UCB-MSCs and UCB-MSC-EVs treated groups compared to antibiotic group. The leucocytes expression of anti-inflammatory cytokines, anti-microbial peptides, and angiogenic genes were significantly upregulated in UCB-MSCs and UCB-MSC-EVs treated groups as compared to antibiotic therapy group. Antibiotic therapy and UCB-MSC-EV groups failed to significantly decrease the gene expression of pro-inflammatory cytokine. In conclusion, the administration of UCB-MSCs and UCB-MSC-EVs through intravenous and local routes was found to be safe and efficacious for alleviating subclinical mastitis in dairy cows.

Folate supplementation during oocyte maturation positively impacts the folate-methionine metabolism in pre-implantation embryos.

Folic acid is vital for DNA synthesis and methylations through one-carbon (C1) metabolism. Thus, it is essential for cell division during embryonic development. Although the oocytes contain endogenous pool of folates for development, the present study investigated the effect of external folic acid supplementation on oocyte maturation, blastocyst development and the expression of folate transporters as well as folate metabolism enzymes in oocytes and pre-implantation embryos of goat. Immature goat oocytes, matured in maturation medium comprising different folic acid concentrations (0, 10, 50, 100 and 150 µM), were in vitro fertilized and cultured. Cumulus expansion markers (PTX3 and PTGS2) in cumulus cells were highly upregulated after 50 µM folic acid supplementation indicating higher degree of maturation. Supplementation of 50 µM folic acid during oocyte maturation resulted in significantly higher blastocyst production rate, reduction in intracellular ROS levels as well as upregulation of the transcripts for folate transporters and key folate-methionine cycle enzymes in comparison to control. The present study demonstrates the existence of active folate-methionine cycle in oocytes and pre-implantation goat embryos. Supplementation of 50 µM folic acid in maturation medium improves oocyte maturation, the blastocyst production rate, reduces ROS production as well as upregulate the expression of FOLR1 and folate metabolism enzyme, MTR.

Production of biologically active recombinant buffalo leukemia inhibitory factor (BuLIF) in Escherichia Coli.

BACKGROUND: Leukemia inhibitory factor (LIF) is a multifunctional cytokine which plays multiple roles in different biological processes such as implantation, bone remodeling, and hematopoiesis. The buESCs are difficult to culture due to lack of proper understanding of the culture conditions. LIF is one of the important factors which maintain the pluripotency in embryonic stem cells and commercial LIF from murine and human origin is used in the establishment of buffalo embryonic stem cells (buESCs). The LIF from a foreign origin is not able to maintain pluripotency and proliferation in buESCs for a long term which is contributed by difference in the binding sites on LIF; therefore, culture medium supplemented with buffalo-specific LIF may enhance the efficiency of buESCs by improving the environment of culture conditions. The high cost of LIF is another major drawback which restricts buESCs research, thus limits the scope of buffalo stem cell use. Various methods have been developed to produce human and murine LIF in prokaryotic system. However, Buffalo leukemia inhibitory factor (BuLIF) has not been yet produced in prokaryotic system. Here, we describe a simple strategy for the expression and purification of biologically active BuLIF in Escherichia coli (E. coli). RESULTS: The BuLIF cDNA from buffalo (Bubalus bubalis) was cloned into pET22b(+) and expressed in E. coli Lemo-21(DE3). The expression of BuLIF was directed into periplasmic space of E. coli which resulted in the formation of soluble recombinant protein. One step immobilized metal affinity chromatography (IMAC chromatography) was performed for purification of BuLIF with ≥ 95% of homogeneity. The recombinant protein was confirmed by western blot and identified by mass spectroscopy. The biological activity of recombinant BuLIF was determined on murine myeloid leukemic cells (M1 cells) by MTT proliferation assay. The addition of BuLIF increased the reduction of MTT by stimulated M1 cells in a dose-dependent manner. The BuLIF induced the formation of macrophage like structures from M1 cells where they engulfed fluorescent latex beads. The recombinant BuLIF successfully maintained pluripotency in buffalo embryonic stem cells (buESCs) and were positive for stem cells markers such as Oct-4, Sox-2, Nanog, and alkaline phosphatase activity. CONCLUSIONS: The present study demonstrated a simple method for the production of bioactive BuLIF in E. coli through single step purification. BuLIF effectively maintained buffalo embryonic stem cells pluripotency. Thus, this purified BuLIF can be used in stem cell study, biomedical, and agricultural research.