<div>Background: The mutations in POU1F1 gene result in deficiency of GH, PRL and TSH-&acirc; expression and thus have an indirect effect on development and reproduction. The present study was conducted to determine and compare the polymorphism of POU1F1 gene in Mandya and NARI-Suwarna Sheep.&nbsp;&nbsp; 
 
Methods:&nbsp;Blood samples were collected from 50 each of Mandya and NARI-Suwarna sheep and genomic DNA was isolated following Miller&rsquo;s high salt method. PCR-RFLP analysis was performed for exon 3 and exon 4 regions of POU1F1 gene, whereas, PCR-SSCP analysis was performed for exon 6 region of POU1F1 gene.&nbsp; 
 
Result: The PCR products of sizes 365 bp, 508 bp and 501 bp for exon 3, exon 4 and exon 6 regions of POU1F1 gene, respectively were successfully amplified. PCR-RFLP analysis of exon 3 and exon 4 regions of POU1F1 gene with AluI and EcoRI restriction enzymes, respectively revealed monomorphism in both Mandya and NARI-Suwarna sheep. The PCR-SSCP analysis of exon 6 region of POU1F1 gene revealed two patterns, P1 and P2 with frequencies of 98 and 2 per cent, respectively in Mandya sheep and 90 and 10 per cent, respectively in NARI-Suwarna sheep. The alignment of P1 and P2 pattern sequences revealed two SNPs (109 G&gt;C and 112 T&gt;A) in Mandya sheep, whereas 3 SNPs (218 T&gt;G, 225 G&gt;A and 264 T&gt;A) were revealed in NARI-Suwarna sheep. Polymorphism was successfully established at exon 6 region of POU1F1 gene.&nbsp;</div>

Polymorphism Study of POU1F1 Gene in Mandya and NARI-Suwarna Sheep

<div>Background: POU1F1 is known as a major candidate gene coding for pituitary-specific transcription factor-1 regulating the production of anterior pituitary hormones like GH, TSH, LH, FSH and prolactin in small ruminants. However, little is known about the genetic variability of caprine POU1F1 gene in Indian breeds. Hence, the present study aimed at the identification of single nucleotide polymorphisms in the exon 3 region of POU1F1 and their association with body weight and milk production in Malabari goats.&nbsp; 
&nbsp;</div>

<div style="text-align:justify">Methods: The present study was conducted during 2016-2018 on 100 Malabari goats at the University Goat and Sheep Farm, Mannuthy. Genomic DNA was extracted using standard phenol-chloroform method. The POU1F1 exon 3 locus was PCR amplified using appropriate primers. The 234 bp PCR product was subjected to single-strand conformation polymorphism (SSCP) analysis on 12% PAGE gel and silver-stained. The different SSCP patterns were sequenced and homologous sequences identified using Blast algorithms. Allelic-genotypic frequencies and the observed-expected heterozygosity at the locus were estimated. Data on body weights at birth, three, six, nine months of age and parity-wise lactation milk yield were used to find the association of POU1F1 exon 3 variants with body weights and milk yield using one-way ANOVA in General Linear Models for fixed effects.&nbsp;</div>

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<div style="text-align:justify">Result: PCR-SSCP revealed GG, GA genotypes with frequencies of 0.44 and 0.56 and G and A alleles with frequencies of 0.72 and 0.28. The observed and expected heterozygosity for the locus were 0.5657 and 0.4077 respectively. Sequencing of the PCR products revealed a synonymous single nucleotide polymorphism (g. 42C&gt;T). GG homozygotes had higher body weight while GA heterozygotes had higher milk yield (p&lt;0.05). It was concluded that the polymorphic exon 3 locus significantly contributed to variation in pre-pubertal body weight and milk yield and hence may be employed in the marker-assisted selection programmes for Malabari goats.&nbsp;</div>

<div>Genetic Variability in Exon 3 Region of POU1F1 Gene and its Effect on Body Weight and Milk Yield in Malabari Goats</div>

Background: In vitro fertilization (IVF) the technique is useful for understanding early mammalian development and maximizing and speeding up genetic improvement in livestock. Genes that function as genetic markers and are involved in the pre-implantation development of embryos may be used to evaluate the quality of an embryo and the expression of these genes is correlated with the timing of embryonic genomic activation.&nbsp; 
 
Methods: Maturing the oocytes of bovine, then fertilizing these matured oocytes and finally culturing the fertilized oocytes to develop into embryos. Determined the gene expression of different certain genes HSP70, OCT4, GLUT1, BAX and DNMT1 in these different cell stages of embryos that were produced.&nbsp; 
 
Result: The mRNA expression of these genes was estimated after collecting the embryos listed above that were produced by the in vitro technique. The results showed statistically significant differences in the relative abundance between some of the genes mentioned above in each embryo stage. The timing of the zygotic genome activation process (ZGA) can result in differences in gene expression levels between the early embryonic genes in each developmental stage of in vitro-produced bovine embryos.

Comparison of Gene Expression for Preimplantation Genes in Bovine Embryos Fertilized in vitro

<div>Background: Pasteurella multocida is the causative agent of many economically important diseases in a wide range of hosts. The mechanisms by which these bacteria can invade the mucosa, evade innate immunity and cause systemic disease are slowly being elucidated. Many key virulence factors are yet to be identified, including those required for initial attachment and invasion of host cells and for persistence in a relatively nutrient poor and hostile environment. This has led to intensive research to understand host adaptation mechanisms and virulence factors in order to develop effective vaccines.&nbsp; 
 
Methods: The present study was carried out to know the distribution of virulence genes viz., haemoglobin binding proteins (hgbA and hgbB), outer membrane protein (ompH), fimbrial antigen (ptfA), filamentous haemagglutinin (pfhA) and transferrin binding protein (tbpA) by PCR in P. multocida CapA isolates from apparently healthy or carrier animals and CapB isolates from field Haemorrhagic septicemia (HS) cases to monitor the epidemiological associations of virulence genes in Cap A and Cap B isolates. 
 
Result: The study revealed that all the six virulence associated genes were present in Cap B isolates. None of the Cap A isolates harboured tbpA and pfhA genes. These two genes were closely related to serotype B causing Haemorrhagic septicemia and were epidemiologically associated with disease status.</div>

<div>Virulence Gene Pattern of Pasteurella multocida Isolates of Buffalo in Association to Capsule Biosynthesis Genes</div>

<div>Present investigation was undertaken to detect and characterize the PPR virus from different clinical tissue samples of 14 sheep and 17 goats with respiratory disease from Maharashtra, India. All animals were tested by Sandwich ELISA, of which 70.96% were found positive carrying high PPR virus inwhich 12 were sheep and 10 were goats respectively. For confirmation of PPR, molecular detection was performed with RT-PCR using F gene and N gene specific primers. Intestine samples accounted for highest percent positivity (75%) followed by blood (66.66%) and lymph node (62.5%) for presence of virus. Unusually higher positivity was observed in heart, liver and Kidney (60%, each) than normal predilection sites such as lungs (57.84%) and spleen (50%). While nasal swabs and blood were individually processed with F gene and N gene specific PCR, the triturated organs were pooled for processing into &lsquo;Sample A&rsquo; comprised of heart, kidney, liver and intestine combined together and &lsquo;Sample B&rsquo; comprising of lung, spleen and lymph nodes combined for the molecular detection of PPR yielding the products each of 372bp and 463bp sizes, respectively. Out of total 40 samples tested, 09/12 each from both sample A and B, while 02/10 nasal swabs resulted positive, respectively and all 06 blood samples remained negative. (F as well as N gene PCR methods were found best suitable for detection of PPR virus from tissue samples of small ruminants).&nbsp;</div>

<div>F Gene and N Gene based Reverse Transcription PCR for Molecular Characterization of Peste des Petits Ruminants Virus</div>

<div>Background: Salmonella Typhimurium (S.Typhimurium) adapts to the broad fluctuations of oxygen concentrations encountered in the host. The transition from aerobic to microaerobic/anaerobic condition encountered in the intestine is mainly regulated by fumarate and nitrate reductase (fnr) regulatory gene and aerobic respiratory control A (arcA) gene. Aim is to appraise the role of fnr gene under anaerobic conditions. 
 
Methods: In this study, we deleted fnr gene from S.Typhimurium using lambda red-recombinase mediated gene knockout protocol. Further carried out in vitro characterization and analyzed the differential protein expression in wild type (WT) and isogenic &Delta;fnr null mutant (&Delta;fnr) using SDS-PAGE and MALDI-TOF mass spectrometry under anaerobic conditions. 
 
Result: In growth competition, WT strain outcompeted the &Delta;fnr and biofilm-forming ability of &Delta;fnr was significantly reduced compared to WT strain. Swimming motility was reduced in &Delta;fnr strain. Besides, differential protein expression revealed the global changes in the expression of many proteins in fnr strain. One differentially expressed protein was identified as TolA, an inner membrane envelope protein. It points out that fnr may regulate the genes responsible for motility and biofilm formation. FNR protein positively regulates TolA, which is important for bacterial virulence, maintenance of membrane integrity, LPS production and replication of bacteria.</div>

<div>Regulatory Role of fnr Gene in Growth and tolA Gene Expression in Salmonella Typhimurium</div>

Background: MicroRNA-449b (miR-449b) is a ~22-base pair long nucleotide, that post-transcriptionally affects processes like proliferation, differentiation and apoptosis by directly targeting the HDAC and DNMTs family genes that regulate DNA methylation. 
 
Methods: This study aimed to investigate the effect of miR-449b mimic, inhibitor and scrambled sequence on buffalo fibroblast cells containing the human insulin (hINS) gene. A transgenic buffalo cell line containing human insulin gene (hINS) was treated with 40 nM miR-449b mimic and inhibitor by transfection method (lipofectamine-3000). 
 
Result: miR-449b mimic treated cells showed a significant decrease (P&lt;0.05) in the expression of epigenetic-related genes and found a significant reduction (P&lt;0.05) in the expression of HDAC1, DNMT3A and DNMT3B genes. In contrast, DNMT1 showed no significant change (P&lt;0.05) in their expression level as compared to control whereas miR-449b inhibitor-treated cells found significantly increased (P&lt;0.05) expression levels of HDAC1, DNMT 3A, DNMT 3B and DNMT1 genes as compared to control. In apoptosis-related genes, miR-449b mimic significantly increased (P&lt;0.05) the expression of BCL-XL and MCL-1, while miR-449b inhibitor significantly increased (P&lt;0.05) the expression of MCL-1 gene whereas there was no significant change (P&lt;0.05) in the expression of BCL-XL. These findings indicate that treatment of miR-449b mimic on transgenic cells reduces the DNA methylation and apoptosis levels. This reduced methylation level helps in the nuclear reprogramming of transgenic cells. Thus, these cells can be a better choice as donor somatic cells for efficient handmade transgenic cloned embryo production.

Microrna-449b Reduces Methylation and Apoptosis-related Gene Expression in Fibroblast Cells Containing Human Insulin Gene

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<div>The objective of the study was to identify suitable reference genes that can be used for quantitative real-time PCR (qPCR) analysis in mung bean (Vigna radiata). Therefore, 10 potential reference genes were selected and the results showed that ubiquitin-conjugating enzyme was suitable as reference under drought and pathogen infection stress; elongation factor 1-&aacute; was the most stable gene under waterlogging; and actin performed the best under saline stress. These selected reference genes were further confirmed by analysis of the expression profiles of catalase and peroxidase under waterlogging. Our results will contribute to the improvement of the accuracy of gene expression evaluation in mung bean.</div>

<div>Reference Genes for Quantitative Real-Time PCR Analysis of Gene Expression in Mung Bean under Abiotic Stress and Cercospora canescens Infection</div>

<div>Background: E. coli is a Gram negative bacterium commonly found in the intestine of animals and humans. Most E. coli are harmless but some can cause infection. Antimicrobial resistance is a public health problem, hence this study was carried out to estimate the antimicrobial resistance pattern and virulotyping of&nbsp;E. coli isolates. 
 
Methods: During the period 2018-19, a total of 100 faecal samples from goats (n=50) and sheep (n=50) were processed for isolation and identification of E.coli. All the E. coli isolates were tested for resistance against 10 different antibiotics. The prevalence of various antibiotic resistance genes viz bla TEM, tetA and tetB and virulence genes Stx1, Stx2 and hlyA in all E. coli isolates was determined by PCR. 
 
Result: Out of 100 samples, 40 isolates of E.coli (24 from goats and 16 from sheep) were isolated, identified biochemically and by PCR. Majority of the E.coli isolates were found resistant to penicillin (100%) followed by ampicillin (93%), tetracycline, (68.8%), gentamicin (37.4%), ceftriazone (18.8%), amikacin (16.7%), chloramphenicol (4.2%), co- trimoxazole (4.2%) and ofloxacin (4.2%). Out of all the isolates, 12 isolates were positive for blaTEM, 10 for tetB, 19 for tetA, five for Stx1 and hlyA and none carried Stx2 gene.</div>

Molecular Detection of Antimicrobial Resistance Genes and Virulence Genes in E. coli Isolated from Sheep and Goat Faecal Samples

<div>Background: Osmanabadi is the premium meat type goat breed of Maharashtra, known for its adaptability and reproductive efficiency. Identification of superior germplasm and incorporating them into breeding programme is the need of hour for augmenting productivity of Osmanabadi as well as other indigenous goat breeds. Hence, the present research was aimed to study Pituitary Specific Transcription Factor I (POU1F1) gene polymorphism and its associations with body weight of Osmanabadi goat.&nbsp; 
 
Methods: Genomic DNA was extracted from 217 blood samples of randomly selected Osmanabadi goats. The 450 bp fragment of POU1F1 gene encompassing exon-6 and its flanking region (3&rsquo;UTR) was PCR amplified.&nbsp;The PCR amplicon was subjected to RFLP using AluI and PstI restriction enzymes to identify polymorphism at nucleotide position 174 (T&gt;C) in exon 6 and at nucleotide position 110 (T&gt;C) in the 3&rsquo;UTR of POU1F1 gene, respectively. The association of the observed allelic variants / genotypes with body weight of the animals was performed using One-way ANOVA.&nbsp; 
 
Result: The AluI PCR-RFLP of 450 bp amplicon of POU1F1 gene in Osmanabadi goats revealed polymorphism with three different genotypes viz., &lsquo;CC&rsquo;, &lsquo;TT&rsquo; and &lsquo;TC&rsquo;. The observed frequencies for TT, TC and CC genotypes were 0.604, 0.355 and 0.041 respectively. The frequencies of T and C alleles were found to be 0.781 and 0.219, respectively. However, PstI PCR-RFLP revealed monomorphic 3&rsquo;UTR of POU1F1 gene with single TT genotype. We observed significant differences (P&lt;0.01) between various genotypes at AluI exon-6 locus of POU1F1 gene and body weights at birth, 3 and 6 months of Osmanabadi goat. Highest body weight was recorded for TT genotype followed by TC and CC genotypes in all three age groups. Our findings indicate that the AluI PCR-RFLP locus of the exon-6 of POU1F1 gene can be used as a potent molecular marker for selection of superior stock of Osmanabadi goats.&nbsp;&nbsp;</div>