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.

Polymorphism in the leucine-rich repeats of TLR7 in different breeds of chicken and in silico analysis of its effect on TLR7 structure and function.

Chicken toll-like receptor 7 (chTLR7) is a viral sensing pattern recognition receptor and detects ssRNA. The ligand binding site comprises leucine-rich repeats (LRRs) located in the ectodomain of chTLR7. Hence, any polymorphism in the binding site would modify its functional interaction with the ligand, resulting in varied strength of immune response. This study first aimed to compare the single nucleotide polymorphisms (SNPs) associated with the ligand binding site of TLR7 in three indigenous chicken breeds namely Aseel, Kadaknath, Nicobari along with an exotic breed White Leghorn. Four synonymous SNPs (P123P, I171I, N339N and L421L) and four non-synonymous SNPs (I121V, S135T, F356S and S447G) were identified among various breeds. We employed in silico tools to screen the pathogenic nsSNPs and one nsSNP was identified as having potential impact on chTLR7 protein. Moreover, sequence and structure-based methods were used to determine the effect of nsSNPs on protein stability. It revealed I121V, F356S, and S447G as decreasing the stability while S135T increasing the stability of chTLR7. Additionally, docking analysis confirmed that I121V and F356S reduced the binding affinity of ligands (R-848 and polyU) to chTLR7 protein. The results suggest that the nsSNPs found in this study could alter the ligand binding of chTLR7 and modify the immune response between different breeds further contributing to disease susceptibility or resistance. Further, in vitro and in vivo studies are needed to analyze the effect of these SNPs on susceptibility or resistance against various viral diseases in poultry.

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.

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.

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.

Apolipoprotein A1 and Ceruloplasmin, the key crosstalk players between the liver and adipose tissue during early postpartum of buffaloes: An in-Silico transcriptome based network analysis.

Physiological transition from pregnancy to lactation during early postpartum creates a high-energy demand in females towards milk production for the survival of new offspring. Ruminant milk contains high amount of lactose, a disaccharide of the glucose and galactose. The milk yield of the animals is also dependent on the lactose amount. In ruminants, glucose is majorly supplied by the liver, which obtains energy from the adipose tissue during energy demands. Therefore, there should be an intricate crosstalk between these two tissues for efficient maintenance of energy for normal physiological needs and milk production. In the present study, we analyzed the transcriptome data previously obtained from the buffalo liver and adipose tissue on the 15th day and 30th day of lactation by using several bioinformatics tools such as PANTHER, Secretome-P, STRING and CPDB. Our analysis identified a total of 24 signaling molecules as interactive players between the liver and adipose tissue during early postpartum of buffaloes. Particularly, the liver appears to interact with the adipose tissue and itself majorly through an endocrine/autocrine molecule, APOA1. Similarly, the adipose tissue appears to interact with the liver and itself majorly through an endocrine/autocrine molecule, CP (ceruloplasmin). The APOA1 and CP may counteract with each other on lipolysis in buffalo adipose tissue because of their common signaling molecules being shared. In addition, the interaction between the adipose derived ceruloplasmin and the liver derived lactoferrin seems to be important during early postpartum of buffaloes. The importance of this interaction needs to be studied in further studies.