Deciphering the Drug Delivery Potential of Milk Exosome Nanovesicles for Aminobenzylpenicillin Therapeutic Efficacy against Contagious Staphylococcus Aureus in Bovine Mastitis.

The emergence of antimicrobial resistance and failure of antibiotic treatment are challenging tasks for managing bovine mastitis, which is mainly caused by the contagious Staphylococcus aureus (S. aureus).To overcome these difficulties, there is an urgent need for a novel drug system. In the present study, the aim is to develop next-generation therapeutics against S. aureus by harnessing the drug delivery potential of milk nanovesicles called milk exosomes (mENs). In the present work, a drug system is developed by encapsulating aminobenzylpenicillin (AMP) in mENs (mENs-AMP). Electron microscopy and zeta-sizer results indicate that the size of mENs-AMP ranged from 55.79 ± 2.8 to 85.53 ± 7.4 nm. The AMP loading efficiency in mENs is 88.61% with its sustained release. Fluorescence spectroscopy results indicated that mENs are biocompatible with mammary epithelial cells. In vitro studies show that the antibacterial activity and the minimum inhibitory concentrations of mENs-AMP are eleven times greater and four times lower than that of unencapsulated AMP, respectively. The mENs-AMP exhibit significantly higher therapeutic efficacy than AMP at the same dosage and treatment frequency. Validation of this approach is demonstrated in mastitis-affected animals through an observation in the reduction of somatic cell counts and bacterial loads in the milk of treated animals.

Taurine dynamics in serum during the oestrous cycle in buffaloes.

Estrus identification is one of the common issues in buffaloes because of their short estrus duration and silent estrus problem. Hence, specific biomarkers facilitating in identifying the estrus stage would be helpful to buffalo farmers and researchers. In our previous studies, taurine, a non-protein amino acid that helps in the secretion of reproductive hormones such as GnRH, was found to be associated with postpartum anestrus in buffaloes. Therefore, the present study was conducted to explore the level of taurine in serum during different stages of the oestrous cycle in healthy cyclic buffaloes. Blood samples were collected from healthy cyclic buffaloes (n = 4), and taurine was estimated at the estrus (0th day), proestrus (-2nd day), metestrus (3rd day) and diestrus (+10th day) stages using TLC method. The days of the oestrous cycle were determined by ultrasonography and observation of behavioural signs by trained professionals. The results revealed that taurine was consistently present in the serum. However, the highest concentration of taurine was observed at the proestrus (0.20 ± 0.03 mg/mL) stage, which was greater (p < .05) than metestrus (0.10 ± 0.05 mg/mL) and diestrus (0.13 ± 0.03 mg/mL) stages, but comparable with the estrus stage. These results were also validated in the simulated population datasets of population size 6 to 10,000. Further, ROC curve analysis for the large simulated population indicated the efficiency of taurine to distinguish proestrus from metestrus and diestrus stages at a lower cutoff value of <0.1643 mg/mL with 60% sensitivity and specificity. Therefore, the present study concludes that serum taurine concentration could help in detecting proestrus stage of buffalo estrous cycle.

Mucin and salt combination simulate typical fern-like pattern of buffalo saliva smear at estrus.

Estrus detection in buffaloes primarily relies on behavioral and physiological signs. Especially during summer, these signs are less prominent to recognize. Thus, estrus detection is a pronounced challenge within the realm of buffalo husbandry, particularly in the summer. Therefore, a simple and accurate estrus detection method is required for buffalo farmers. The observation of fern-like salivary crystallization patterns is one such simple method to detect estrus in buffaloes, bactrian camels, beagle bitches, and cows. However, the exact mechanism for the formation of typical fern-like is not known. We hypothesized that it might be because of the estrus-specific mucins and salts. To test this hypothesis, we prepared the smears by combining different concentrations of mucin type -2 (MUC2) and -3 (MUC3) with sodium chloride (NaCl). Microscopic examination confirmed that fern-like patterns resulted from a combination of the MUC3 and NaCl produced more realistic fern patterns than that of MUC2 or BSA with salt. To predict possible mucin and salt concentration showing natural fern-like patterns at the estrus stage in buffalo saliva, we constructed a guide tree of artificially generated fern-like patterns using an image analysis online tool. This computation analysis revealed that most of the natural buffalo estrus saliva samples showing typical fern-like patterns clustered in the cluster 2 of the guide tree comprising of 13 clusters. In the cluster 2, MUC3 in combination with the salt concentrations of 100, 150, and 250 mM was commonly found in a close proximity to the natural typical fern-like patterns of saliva smear of buffaloes at estrus. Conclusively, the buffalo saliva at estrus is predicted to have a gel-forming heavily glycosylated protein such as mucin along with at least 100 mM of NaCl. RESEARCH HIGHLIGHTS: Glycoprotein and salts combination replicates fern-like pattern of buffalo saliva at estrus. MUC3 and NaCl salt combination produces more realistic fern-like patterns compared with MUC2 or BSA and salt combination. MUC3 with NaCl at 100, 150, and 250 mM consistently resembled natural estrus saliva fern-like patterns. During estrus, buffalo saliva is expected to contain heavily glycosylated mucin and at least of 100 mM NaCl.

A synonymous single nucleotide polymorphism (g.36417726C > A) in the Lama2 gene influencing fat deposition is associated with post-partum anestrus interval in Murrah buffalo.

Postpartum absence of estrus exhibition known as postpartum anestrus interval (PPAI) for more than 90 days after calving is a concerning issue for dairy buffalo farmers' economy. The PPAI duration is influenced by both management practices and animal genetics. Investigating genetic markers associated with PPAI is crucial for incorporating them into marker-assisted selection programs. Towards this goal, our study focused on exploring potential genetic markers from early postpartum adipose tissue gene networks. We successfully identified 24 Single Nucleotide Polymorphisms (SNPs) within 9 candidate genes. In our initial analysis involving 100 buffaloes, we detected a significant association (P = 0.02267) between a specific synonymous SNP within the Lama2 gene (g.36417726C > A) and PPAI. This finding was subsequently validated (P = 0.02937) in a larger cohort of 415 buffaloes, where the SNP explained 1.36 % of the genetic variance. Intriguingly, buffaloes with the CC genotype of this SNP exhibited a PPAI that was 12.71 ± 3.21 days longer compared to buffaloes with AA and CA genotypes. To gain insight into the functional relevance of this SNP, a computational analysis was performed which indicated that the C allele of the SNP (g.36417726C > A) increased the stability of LAMA2 mRNA compared to the A allele. This computational prediction was corroborated by observing a significant increase (P = 0.01798) in Lama2 gene expression (greater than 8-fold) and higher fat percentage (P < 0.05) in adipose tissue of CC genotypes (48.78 ± 1.87 %) compared to AA genotypes (33.59 ± 4.5 %). Furthermore, we noted a significant (P < 0.05) upregulation of C/ebpß, Pparγ, Fasn, C/ebpα, and Pnpla2 genes, along with the downregulation of Bmp2 and Ptch1 in CC genotypes as opposed to AA genotypes. This observation suggests the involvement of the Pparγ-mediated pathway in both adipogenesis and lipolysis within CC genotypes. In summary, our comprehensive analysis involving association and functional validation underscores the potential of the SNP (g.36417726C > A) within the Lama2 gene as a promising genetic marker against extended PPAI in Murrah buffalo.

Detection and quantification of TIMP1 and miR-141 through RT-LAMP and TT-LAMP in serum samples during estrous cycle in buffalo.

Estrus identification is a common problem in the reproductive management of farm animals. Hence, several studies have been conducted to explore biomarkers for estrus detection. One of our previous studies identified the abundance of RNA biomarkers such as TIMP1 and miR-141 in buffalo saliva during the estrus stage. However, the level of these RNA biomarkers in buffalo serum during estrous cycle is undetected. Therefore, the present study was designed to quantify TIMP1 and miR-141 in serum during buffalo estrous cycle. Blood samples were collected in different stages of estrous cycle from four healthy cyclic buffaloes. The quantification of TIMP1 and miR-141 was performed with direct serum using RT-LAMP and TT-LAMP technologies, respectively. The LAMP amplification was confirmed by agarose gel electrophoresis and the color change was quantified in comparison to a non-template control using ImageJ software. A decreased abundance of TIMP1 at the diestrus stage and a decreasing trend of miR-141 from proestrus to diestrus stages were observed, which was further reinforced by simulated random populations generated with R programming. Specifically, TIMP1 was found significantly (P < 0.0001) abundant at estrus and metestrus stages as compared to the diestrus stage, whereas miR-141 was significantly (P < 0.001) higher during the proestrus stage as compared to the other stages of estrous cycle. The ROC curve analysis showed miR-141 to be a better biomarker than TIMP1 as it distinguished the proestrus stage from diestrus with a sensitivity and specificity of 83 % and 98 %. This study also marked the first use of TT-LAMP technology for rapid miRNA detection in livestock.

Aflatoxin M1 decreases the expression of genes encoding tight junction proteins and influences the intestinal epithelial integrity.

Aflatoxin M1 (AFM1) is a mycotoxin that is commonly found as a milk contaminant, and its presence in milk has been linked to cytotoxicity. The present study aimed to evaluate the acute cytotoxic effects of AFM1 on intestinal Caco-2 cells. Initially, we checked the morphology and viability of Caco-2 cells after treatment with different concentrations of AFM1 (5 ng/L, 50 ng/L, 250 ng/L, 500 ng/L, 1000 ng/L, and 2000 ng/L) for different time intervals (6 h, 12 h, and 24 h). It was found that AFM1 did not show any effect on cell morphology, but 10% decrease in viability above 1000 ng/L after 12 h. Furthermore, DCFDA assay showed increased ROS production after 6 h treatments. qPCR analysis showed an increased expression of epithelial-specific cytoskeleton marker genes, Cytokeratin, Villin, Vimentin, and JAM-1, and a decreased expression of tight junction protein genes, Claudin-1, Occludin, and ZO-1. Similarly, we found an increased expression of Cyp1a1 transcript with an increasing AFM1 concentration and incubation time. This gene expression analysis showed AFM1 can cause disruption of tight junctions between intestinal cells, which was further confirmed by a transwell experiment. In conclusion, consumption of AFM1-contaminated milk does not show any effect on cells morphology and viability but decreases the expression of intestinal barrier transcripts that may lead to the disruption of intestinal barrier function and leaky gut.

Salivary TIMP1 and predicted mir-141, possible transcript biomarkers for estrus in the buffalo (Bubalus bubalis).

Successful reproductive management of buffaloes depends primarily upon timely estrus identification. However, 50% of the estrus events are undetected in buffaloes with the available estrus identification methods, leading to huge financial loss to buffalo farmers. Hence, there is an urgent need to develop an alternative and accurate estrus identification method, particularly on the basis of biomarkers in non-invasive fluids. Thus, the present study aimed to identify RNA based estrus biomarkers in cell free saliva in Bubalus bubalis, so that they can be used for future field applicable RT-LAMP colour reactions. RNA-Seq analysis of cell free salivary RNA showed 49 differentially abundant mRNAs between the estrus and diestrus stages. Among five mature miRNAs predicted from the RNA-Seq data, four were found differentially altered at the estrus stage than the diestrus stage. Validation study by direct salivary transcript analysis (DSTA) on 6 selected mRNAs (PPARGC1a, TIMP1, PEBP4, CSPG5, PRHR and ATOH7) and 5 miRNAs (bta-miR-92b, bta-miR-302d, bta-miR-141, bta-miR-27a and bta-let-7a-5p) showed significantly higher levels of TIMP1 (3.46 fold; P < 0.5) and bta-mir-141 (1.33 fold; P < 0.5) in cell-free saliva at the estrus stage compared to the diestrus stage. Hence, TIMP1 and miR-141 appear to be the possible transcript biomarkers for estrus in the cell free saliva of the buffalo. However, further validation studies are required in a large population of buffaloes to determine their estrus biomarker potential before considering them for RT-LAMP colour reaction.

A single nucleotide polymorphism of the thyrotropin releasing hormone degrading ectoenzyme (TRHDE) gene is associated with post-partum anestrus in Murrah buffalo.

Thyrotropin releasing hormone degrading enzyme (TRHDE) gene is implicated in Thyrotropin releasing hormone (TRH) mediated prolactin secretion. It has been shown that the prolactin secretion alters the Gonadotropin-releasinghormone(GnRH) mediated estrous cycle. Therefore, TRHDE may also regulate postpartum anestrus. Earlier studies reported the role of non-synonymous single nucleotide polymorphism (SNPs) in various pathophysiological conditions by altering the structure and function of the proteins. Hence, in the present study, we identified SNPs in the putative promoter, first exon, middle exon and 3'-UTR containing the last exon of TRHDE gene and determined their association with postpartum anestrus (PPA) in Murrah buffaloes. We found one non synonymous SNP (G > C at 118095875 bp on chromosome 4) in the first exon of TRHDE and performed its association analysis in a population sample of 50 extreme PPA (residual PPAI: 123.06 ± 12.98 days) and 50 normal (residual PPAI: -80.46 ± 3.19 days) buffaloes. The residual PPAI value was the observed PPAI adjusted for the effect of 38 non-genetic factors. The analysis showed a significant (P < 0.004167) association of this SNP with PPA in buffaloes. Molecular dynamics simulations (MDS) also supported that the C allele altering Glutamine to Histidine at the amino acid 148 of TRHDE could enhance the stability and rigidity of TRHDE protein, which may lower its activity, increase TRH and prolactin, and reduce GnRH in PPA buffaloes. The MDS analysis further strengthens the association of the SNP (G > C) in the TRHDE gene with PPA condition in Murrah buffaloes. However, further investigation is needed to prove the MDS observations.

Meprin A1 subunit beta gene polymorphism is associated with the length of post-partum anestrus interval in Murrah buffaloes.

Postpartum anestrus interval (PPAI) is the interval between parturition and the first postpartum estrus exhibition in animals. Appearance of both normal and PPA buffaloes under the same farm conditions indicates the role of possible genetic predisposition to PPA. To identify the genetic and non-genetic factors associated with PPA in buffaloes, we collected data on PPAI and other 38 non-genetic variables from 575 Murrah buffaloes in the field conditions and identified the PPA associated non-genetic factors in our previous study. To explore the genetic factors associated with the unexplained variation in PPAI residuals, the present study identified 41 single nucleotide polymorphisms (SNPs) in 13 candidate genes using Sanger sequencing. Exploration of their association with the PPAI residuals of 50 extreme PPA and 50 normal buffaloes identified the significant (P < 0.01) association of the SNP (g.37219977A>G) in the 3'-UTR region of the Meprin A 1 subunit beta (Mep1b) gene with PPAI, which was further validated (P = 0.058) in a large population sample (n = 417). Bioinformatics analysis of the 3'-UTR region has identified three miRNA, bta-miR-2420, bta-miR-2325b and bta-miR-453 that could regulate Igf-1 in the plasma of animals with different genotypes (GG, AG and AA). The higher Igf-1 levels in the GG genotypes than that of AA and AG genotypes of this SNP (g.37219977A>G) further suggest the association of Mep1b gene with PPA condition in Murrah buffaloes. As a result of this study, we propose that buffaloes with protective alleles at this SNP be selected to improve the herd's reproductive efficiency.

Regulation of granulosa cell functions through NRP-1 mediated internalization of follicular fluid non-exosomal miR-210.

Crosstalk between follicular fluid (FF) and granulosa cells (GCs) plays a vital role in the regulation of folliculogenesis, ensuring regular reproductive cycle in mammals. This crosstalk is primarily mediated by hormones and signaling molecules, such as cytokines and chemokines. Recently, extracellular microRNAs (miRNAs) have gained a lot of attention in cell-to-cell communication. Extracellular miRNA transportation occurs through exosomes, a kind of micro-vesicles produced from almost all cells. However, the mode of non-exosomal miRNA internalization is not much studied. In the present study, we explored the role of neuropilin-1 (NRP-1) as a receptor in internalizing FF non-exosomal miRNAs in GCs. We first confirmed the expression of NRP-1 in GCs during follicular development followed by its role in the internalization of miR-210, a non-exosomal miRNA. This study showed that incubation of GCs with a non-exosomal fraction of FF increased the content of miR-210 in GCs as compared to their control. To illustrate the role of NRP-1 as a receptor, NRP-1 was knockdown using siRNA. Silencing experimental results showed a significant decrease in uptake of miR-210 in NRP-1 knockdown GCs. Furthermore, downstream expression analysis of miR-210 target genes (CYP19A1, PCNA, and EFNA3) also confirmed the NRP-1 mediated miR-210 internalization. Results of the present study clearly demonstrated that FF non-exosomal miR-210 can be internalized through the NRP-1 receptor. Furthermore, differential expression of NRP-1 in GCs suggests its role in follicular development. Overall, these findings suggest that FF non-exosomal miRNA plays an important role in GC functions and female reproduction.

Organochlorine pesticide dieldrin upregulate proximal promoter (PII) driven CYP19A1 gene expression and increases estrogen production in granulosa cells.

Organochlorine pesticides are highly persistent environmental pollutants, generally shown to act through estrogen receptor alpha and alter estrogen biosynthesis. However, the molecular mechanism of regulation of estrogen biosynthesis by these pesticides is not clear. Estrogen is main female fertility hormone regulated by rate-limiting enzyme aromatase. It is encoded by the CYP19A1 gene, which is expressed using specific promoters. In the present study, the attempt has been made to elucidate the effect of dieldrin on the promoter-specific CYP19A1 gene expression and estrogen hormone production in buffalo granulosa cells. The buffalo granulosa cells were cultured and treated with dieldrin in a dose (100,150 and 200 ng/mL) and time (6, 12, and 24 h) dependent manner, followed by analysis of CYP19A1, promoter-specific CYP19A1 transcript expression, and estrogen production. Results showed that dieldrin significantly increased the expression of the CYP19A1 gene after 6 and 12 h while its expression was decreased after 24 h. To understand the upregulation of CYP19A1 gene, promoters' specific CYP19A1 transcript analysis was done. The finding showed that dieldrin significantly increased the proximal promoter specific CYP19A1 transcript while there was no effect on distal promoter specific CYP19A1 transcripts. This specific-promoter activity was quantified by chromatin immunoprecipitation assay (ChIP). Results confirmed the involvement of the proximal promoter in the overexpression of CYP19A1 gene. Furthermore, a significant increase in estradiol-17ß level was also observed. Overall, the present study demonstrated the stimulatory effect of dieldrin on the CYP19A1 gene and will help to understand the toxicological role of dieldrin on the reproductive system.

Establishment of Repertoire of Placentome-Associated MicroRNAs and Their Appearance in Blood Plasma Could Identify Early Establishment of Pregnancy in Buffalo (Bubalus bubalis).

Precise early pregnancy diagnosis in dairy animals is of utmost importance for an efficient dairy production system. Not detecting a dairy animal pregnant sufficiently early after the breeding results to extending the unproductive time of their milk production cycle and causes substantial economic loss for a dairy producer. At present, the most conventional and authentic pregnancy confirmation practice in cows and buffaloes is rectal palpation of the reproductive organs at Days 35-40 after insemination, which sometime leads to considering an animal as false pregnant. Other alternative methods available for early pregnancy diagnosis lack either accuracy or reproducibility or require elaborate instrumentation and laboratory setup not feasible to practice at farmers' doorstep. The present study was aimed at establishment of the microRNA (miRNA) repertoire of the placentome in buffaloes, which could capture the event of the cross talk between a growing embryo and a dam, through fetal cotyledons and maternal caruncles, and thus could hint at the early pregnancy establishment event in ruminants. Total RNA was isolated from buffalo placentome tissues during early stages of pregnancy (at Day < 25 and Days 30-35), and global small RNA analysis was performed by using Illumina single-end read chemistry and Bubalus bubalis genome. A total of 2,199 miRNAs comprising 1,620 conserved and 579 non-conserved miRNAs were identified. Stringent functional miRNA selection criteria could predict 20 miRNAs worth evaluating for their abundance in the plasma of pregnant, non-pregnant, cyclic non-bred, and non-cyclic prepubertal animals. Eight of them (viz., miR-195-5p, miR-708-3p, miR-379-5p, miR-XX1, miR-XX2, miR-130a-3p, miR-200a-3p, and miR-27) displayed typical abundance patterns in the plasma samples of the animals on Day 19 as well as Day 25 post-insemination, thus making them ambiguous candidates for early pregnancy detection. Similarly, higher abundance of miR-200a-3p and miR130a-3p in non-pregnant animals was indicative of their utility for detecting the animals as not pregnant. Most interestingly, miR-XX1 and miR-XX2 were very characteristically abundant only in pregnant animals. In silico target prediction analysis confirmed that these two miRNAs are important regulators of cyclooxygenase-2 (COX-2) and cell adhesion molecule-2 (CADM-2), both of which play a significant role in the implantation process during feto-maternal cross talk. We interpret that circulatory miR-XX1 and miR-XX2 in blood plasma could be the potential biomarkers for early pregnancy detection in buffaloes.

Validation of salivary ferning based estrus identification method in a large population of water buffaloes (Bubalus bubalis) using Foldscope.

Missing an estrus event is an economic problem in buffaloes because of lack of a simple and accurate estrus identification method. Saliva, a non-invasive fluid available every time, showed typical fern-like crystallization patterns at early estrus in buffaloes. However, to implement this salivary ferning based estrus identification method in the field conditions, the present study, for the first time, validated this method in four buffalo population samples (PS) representing four different field scenarios, an organized herd (PS1, 10 buffaloes monitored daily for a year (149 estrus events)), artificial insemination (AI) centers (PS2, 114 buffaloes brought for AI), induced estrus condition (PS3, 44 buffaloes) and farmers' doorsteps (PS4, 275 random buffaloes with unknown reproductive history and no estrous signs). Totally, 582 saliva samples were collected from 443 buffaloes. Salivary smears were observed under a simple microscope and/or a less expensive (< 1USD) paper microscope, Foldscope. On the basis of salivary fern-like patterns, the proportions of estrus identification were significantly different among PS. Specifically, the proportions in the PS1 (0.74, P < 0.0001) and PS4 (0.08, P < 0.05) were significantly higher than their population proportion estimates, 0.5 and zero, respectively. Therefore, this estrus identification method is much useful in the PS1 and PS4, the practical field scenarios requiring an accurate estrus prediction method, compared to the PS2 and PS3. Especially, this method is 91 % confirmatory to detect early estrus in PS4. Therefore, salivary ferning is a useful technique to identify early estrus in buffaloes in the field conditions at farmers' doorsteps.

Urinary Cell-Free miR-99a-5p as a Potential Biomarker for Estrus Detection in Buffalo.

Accurate estrus detection method is the need of the hour to improve reproductive efficiency of buffaloes in dairy industry, as the currently available estrus detection methods/tools lack high sensitivity and specificity. Recently, circulating miRNAs have been shown as non-invasive biomarkers by various studies. Hence, in order to evaluate their potential as estrus biomarkers, the objective of this study was to identify and compare the levels of 10 hormone-responsive miRNAs in the urine collected at proestrus (PE), estrus (E), and diestrus (DE) phases of buffaloes (n = 3) pertaining to a discovery sample. Among 10 urinary miRNAs, the levels of bta-mir-99a-5p (E/PE 0.5-fold, P < 0.05; DE/PE 1.9-fold), bta-miR-125b (E/PE 0.5-fold; DE/PE 0.7-fold), bta-mir-145 (E/PE 1.5-fold; DE/PE 0.7-fold), bta-mir-210 (E/PE 1.2-fold, DE/PE 0.7-fold), mir-21 (E/PE 1.5-fold, DE/PE 2-fold), and bta-mir-191 (E/PE 1.3-fold; DE/PE 0.8-fold) were found to be altered during different phases of buffalo estrous cycle. In contrast, bta-mir-126-3p, bta-let-7f, bta-mir-16b, and bta-mir-378 were undetected in buffalo urine. Furthermore, a validation study in an independent group of 25 buffalo heifers showed the increased levels of urinary bta-mir-99a-5p during the DE (3.92-fold; P < 0.0001) phase as compared to the E phase. Receiver operating characteristic curve analyses also revealed the ability of urinary miR-99a-5p in distinguishing the E from the DE phase (area under the curve of 0.6464; P < 0.08). In silico analysis further showed an enrichment of miR-99a-5p putative targets in various ovarian signaling pathways, including androgen/estrogen/progesterone biosynthesis and apoptosis signaling, implicating the role of miR-99a-5p in ovarian physiology. In conclusion, significantly lower levels of bta-mir-99a-5p at the E phase than the DE phase in buffalo urine indicate its biomarker potential, which needs to be further explored in a large cohort in the future studies.

De novo pathway is an active metabolic pathway of cysteine synthesis in Haemonchus contortus.

Haemonchus contortus, commonly known as Barber's pole worm, is an economically important gastrointestinal nematode of sheep and goats especially in tropical and sub-tropical regions of the world. Cysteine synthesis is a very important metabolic pathway for the parasite, however the functional aspects of cysteine synthesis in parasite are largely unknown. The key question which we have investigated in the study is; whether the parasite uses a de novo pathway of cysteine synthesis, which is unknown in multicellular organisms of the animal kingdom and known to be absent in mammals. Directional cloning of the cysteine synthase (CS) gene was done in pET303 champion vector using restriction sites XbaI and XhoI. The CS gene of the H.contortus was closely related to CS-A protein of Oesophagostomum dentatum and a hypothetical protein of Ancylostoma ceylanicum. Recombinant protein of the H contortus CS (rHC-CS) gene was expressed using pET303 vector in pLysS BL21 strain of E.coli and subsequently purified by Ni-NTA affinity chromatography. Western blot using anti-His tag antibody confirmed the presence of rHC-CS. Biochemical assay, FTIR and enzyme kinetics studies revealed that rHC-CS used O-acetyl serine as substrate to produce cysteine using de novo pathway and CS activity was also confirmed with the homogenate of H.contortus. Upregulation of CS transcripts in the adult and its downregulation in the L3 larval stage suggests that de novo pathway contributes to the cysteine requirement of mature H.contortus. It is concluded that de novo pathway is an active metabolic pathway in H.contortus.

Faecal microbiome analysis reveals Clostridiales and Bacteroidales as signature gut microbes during estrus of buffalo.

The non-pathogenic intestinal microbes that conquer our intestines are not an accidental jumble of organisms, but rather a disparate community of microbes that coexist, and sustain a mutualistic and symbiotic relationship with the host. The gut microbiome has been shown to be influenced by animal physiology and vice versa. However, information is still scanty. The present study aimed to analyse the variation between faecal bacteria of three different stages (proestrus, estrus and postestrus) of the estrous cycle of Murrah buffalos. A phylogenetic study of buffalo faeces derived from three different stages of estrous cycle was conducted in order to compare the bacterial diversity among these three stages. We performed an exploratory microbiome analysis of buffalo faeces using 16S rRNA sequencing during these stages of the buffalo estrous cycle. A total of three bacterial phyla with six different bacterial orders and twenty-three different genera were identified among all the three comparative phases of the estrous cycle. Among them, the Clostridiales were found to be the most abundant, and Bacteroidales were present exclusive during the estrus phase. As faeces is a source of gut microbes and a non-invasive representative of the metabolic steroids and perceptible pheromones, the profiling of gut microbes during estrous cycle would provide clues towards the major microbes contributing to the perceptible pheromones during estrus stage. To the best of our knowledge, this is the first ever report describing the faecal bacterial diversity during estrous cycle of any ruminant species. Although future studies are required to understand the role of Clostridiales and Bacteroidales in faecal pheromone metabolism.

Profiling and integrated analysis of whole-transcriptome changes in uterine caruncles of pregnant and non-pregnant buffaloes.

Improved reproductive performance in buffaloes can be achieved by understanding the basic mechanism governing the embryonic attachment and feto-maternal communication. Considering this, trascriptomic profiling and integrative analysis of long intergenic non-coding RNAs were carried out in the uterine caruncles of pregnant and non-pregnant buffaloes. Transcriptome data of pregnant and non-pregnant uterine caruncles after quality control was used to perform the analysis. Total of 86 novel lincRNAs expressed in uterine caruncular tissues were identified and characterized. Differential expression analysis revealed that 447 mRNAs and 185 mRNAs were up- and down- regulated, respectively. The number of up- and down- regulated lincRNAs were 114 and 13, respectively. Of the identified 86 novel lincRNAs, six novel lincRNAs were up-regulated in the pregnant uterine caruncles. GO terms (biological process) and PANTHER pathways associated with reproduction and embryogenesis were over-represented in differentially expressed genes. Through miRNA interaction analysis, interactions of 16 differentially expressed lincRNAs with mi-RNAs involved in reproduction were identified. This study has provided a catalogue of differentially expressed genes and novel regions previously unknown to play a significant role in buffalo reproduction. The results from the current study extends the buffalo uterine lncRNAs database and provides candidate regulators for future molecular genetic studies on buffalo uterine physiology to improve the embryo implantation and successful completion of pregnancy.

Transcriptome analysis of buffalo granulosa cells in three dimensional culture systems.

Hanging drop (HD) three-dimensional (3D) culture model for buffalo granulosa cells (GC) was reported to mimic the preovulatory stage of ovarian follicles in our previous study. To further verify its reliability, the present study attempted a comparative transcriptome profile of buffalo GC freshly isolated from ovarian follicles (<8 mm diameter) (FC) and their cultures in normal culture dish (ND or 2D), polyHEMA coated dish (PH) and HD culture systems (3D). Out of 223 significantly (-log2 fold change: >3; p < .0005; false discovery rate [FDR]: <0.1) differentially expressed genes (SDEGs) among different culture systems, 137 were found unannotated, and 94, 29, and 66 were exclusively expressed in FC, PH, and HD, respectively. However, on eliminating the fixed points of p values and FDR from the entire raw data, only 11 genes related to long noncoding RNA, 12 genes related to luteinization, and 3 genes related to follicular maturation were exclusively expressed in FC, PH, and HD culture systems, respectively. The quantitative real time-PCR validation and the next generation sequencing data had more than 90% correlation. Bioinformatics analyses of the exclusively expressed SDEG revealed that the freshly aspirated GCs were a true representative of GCs from small follicles (<8 mm diameter), the GC spheroids under PH maintained mitochondrial function, and those cultured in HD system for 6 days simulated the inflammatory milieu required for ovulation. Therefore, the comparative transcriptome profile also reinforced that HD culture system is better in vitro culture method than the other methods analyzed in this study for buffalo GC.

Reverse transcription-loop mediated isothermal amplification (RT-LAMP) assay for detection of AhR receptor responsive xenobiotics.

Dioxins are a group of highly toxic environmental persistent organic pollutants, which are lipophilic in nature. 2, 3, 7, 8- tetrachlorodibenzo-p-dioxin (TCDD) is the most toxic representative of this class. TCDD causes several human health effects like endocrine disruption, carcinogenesis and reproductive toxicity mediated by aryl-hydrocarbon receptor. Current detection methods of dioxins like gas chromatography-mass spectrometry, liquid chromatography-mass spectrometry etc. are costly and time consuming. Therefore, the present study aims to develop a relatively faster and cheaper technique called reverse transcription-loop mediated isothermal amplification (RT-LAMP) assay to detect dioxins. Cultured granulosa cells used as a model system were treated with different doses (5, 10 and 15 pg/mL) of aryl hydrocarbon receptor (AhR)responsive xenobiotic, TCDD, in accordance with maximum residue limit values. Cells were treated for 6, 12 and 24 h, respectively to study the gene expression of TCDD receptor called AhR and AhR responsive genes, CYP1A1 and CYP1B1, in a dose and time dependent manner. All targeted genes expression significantly increased after 6 and 12 h by 1.3-8 folds. For the development of RT-LAMP assay, CYP1A1 gene was used with 6 h TCDD treatment. RT-LAMP assay was standardized with optimal color change at 30 min using 50 ng of cellular RNA. In all the cases, we could distinguish RT-LAMP-positive condition from one sample to another sample due to intensity of color. The method was also validated by spectrometric method. In conclusion, the developed method will be used to screen AhR receptor responsive xenobiotics by observing the color change in RT-LAMP assay like dioxin used in the present study.

Epitope-based in silico peptide design yields peptide-directed antibodies that recognize the buffalo luteinizing hormone.

We present a novel peptide sequence identified through in silico epitope design and the later generation of peptide-directed antibodies recognizing the buffalo luteinizing hormone. Peptides and antibodies, specific to reproductive hormones, are valuable tools for developing point-of-care immunodiagnostic tools. The study predicted an epitope peptide in silico from buffalo luteinizing hormone and the generation of polyclonal antibodies against this peptide sequence. In this quest, we identified a novel epitope peptide sequence (luteinizing hormone peptide, LHP) through bioinformatics tools. The peptide was further synthesized and characterized. The polyclonal antibodies (anti-LHP) were raised against the peptide in the rabbit. Thereafter, we explored a strategy for detecting buffalo luteinizing hormone (LH) using the anti-peptide antibodies developed. The affinity of the peptide, bovine lutropin beta, and crude LH (prepared from buffalo pituitary) towards the raised antibodies was established by dot blot and ELISA. Specific recognition of the luteinizing hormone by the raised polyclonal antibodies highlights the ability of the identified peptide (LHP) and developed polyclonal antibodies (anti-LHP) as suitable diagnostic reagents for sensing the buffalo luteinizing hormone. Through this work, we analyzed and translated the "-omics" information in the LH gene sequence for the development of a novel peptide and antibodies as valuable immuno-reagents.