In a study published in the journal Archives Animal Breeding, Zhixiang Wang of Guangxi University and colleagues systematically tested how ESR1 controls the workhorse cells of milk production, the buffalo mammary epithelial cells. Using two complementary genetic manipulation techniques in cultured cells, small interfering RNA to silence the gene and a lentiviral vector to overexpress it, the team showed that ESR1 sits at a regulatory crossroads governing milk fat synthesis, milk protein production, cell proliferation, and cell survival. When ESR1 activity was dialed down, the cells grew more slowly and produced less fat and protein machinery; when it was dialed up, the opposite occurred, with the effects on gene expression mirroring each other almost perfectly in the two experimental directions.

The experimental material came from lactating Murrah buffaloes at the Buffalo Research Institute of the Chinese Academy of Agricultural Sciences in Nanning, Guangxi. The researchers collected mammary gland tissue from mid-lactation animals aged five to six years, then isolated mammary epithelial cells using a tissue explant culture method. Fibroblast contamination was removed through repeated rounds of differential adhesion purification, and the resulting cell populations were verified as genuine mammary epithelial cells by confirming that they expressed characteristic marker genes such as CSN2, CSN3, and LALBA at levels far higher than in buffalo fibroblast controls. The purified cells displayed the expected oval epithelial morphology and a typical growth curve, moving from a lag phase into rapid logarithmic growth before reaching confluence.

With a validated cell system in hand, the team designed siRNA molecules targeting the buffalo ESR1 messenger RNA and delivered them into the cells, confirming successful uptake through green fluorescence signals and verifying silencing efficiency by quantitative real-time PCR. Proliferation was then measured in two independent ways: the CCK-8 assay, which tracks metabolic activity as a proxy for cell number over 24, 48, and 72 hours, and the EdU assay, which directly labels cells in the process of copying their DNA. Both approaches converged on the same conclusion. Suppressing ESR1 significantly reduced proliferative activity, while the absorbance values in the interference group dropped markedly at 24 and 48 hours compared with controls.

The gene expression data revealed how deeply ESR1’s influence runs through the milk synthesis machinery. Silencing ESR1 significantly downregulated a suite of genes responsible for lipid uptake, transport, and storage, including PPARG, LPL, PLIN2, VLDLR, ADFP, and CD36, although the fatty acid synthesis gene ACACA remained unchanged. Genes tied to milk protein synthesis also fell, among them the casein genes CSN1S1 and CSN1S2 and key components of the mTOR signaling pathway such as RPS6, RHEB, EIF4E, and AKT1. Proliferation-associated genes, including STAT5A, MTOR, ELF5, CCND1, EGF, and IGF1, were likewise suppressed. Meanwhile, the pro-apoptotic genes BAX and CASP9 rose while the anti-apoptotic gene BCL2 fell, indicating that losing ESR1 pushes the cells toward self-destruction.

Overexpression told the mirror-image story. When the researchers infected the cells with a lentiviral vector carrying the buffalo ESR1 gene, ESR1 transcript levels rose dramatically, and the EdU and CCK-8 assays showed significantly enhanced proliferation at 48 and 72 hours. The lipid transport genes PPARG, LPL, PLIN2, VLDLR, ADFP, and CD36 all climbed, as did the casein genes and the mTOR pathway components. The proliferation genes STAT5A, MTOR, ELF5, CCND1, EGF, and IGF1 were upregulated, BCL2 increased, and the pro-apoptotic genes BAX and CASP9 declined. The complete symmetry between the knockdown and overexpression results strengthens the case that ESR1 is a genuine driver of these cellular programs rather than a passive bystander.

The fat-related findings fit neatly with earlier work in other species. Milk fat synthesis depends on three linked stages: uptake of fatty acids from circulating lipoproteins, their synthesis and modification, and storage in lipid droplets before secretion. LPL breaks down triglycerides in chylomicrons and very-low-density lipoproteins into free fatty acids for cellular uptake, CD36 ferries long-chain fatty acids across the membrane, and VLDLR delivers fatty acids and cholesterol through endocytosis. The authors note that ESR1 can recruit the co-activator PPARGC1B, which boosts the transcriptional activity of PPARG, a master regulator of lipid metabolism that in turn upregulates CD36 and LPL. The synchronized expression patterns observed in the buffalo cells suggest that ESR1 amplifies the lipid metabolic network largely through this PPARG-centered cascade, while its positive effects on the lipid droplet proteins PLIN2 and ADFP may help stabilize the droplets that milk fat secretion requires.

The protein synthesis results point to a two-pronged mechanism. In dairy cow cells, previous research showed that ESR1 indirectly upregulates CSN1S1 and CSN1S2 through an ERα–NFκB1–mTOR cascade, and the buffalo data align closely with that model. The PI3K-AKT-mTOR pathway is a central hub for milk protein synthesis, and ESR1’s positive regulation of AKT1, RHEB, RPS6, and EIF4E suggests it feeds energy and building blocks into the protein translation machinery through this route. Intriguingly, ESR1 also negatively regulated PDK1, an enzyme that shunts pyruvate away from the mitochondria into glycolysis. The authors propose that when high ESR1 expression lowers PDK1 levels, the pyruvate dehydrogenase complex regains activity, allowing pyruvate to enter the tricarboxylic acid cycle. That metabolic shift would enhance oxidative phosphorylation, supplying the ATP and amino acid precursors that the energy-hungry mTOR-driven protein synthesis program demands.

The study’s authors are careful to frame the work as a foundation rather than a finished tool. Profiling ESR1 expression patterns and identifying functional genetic variants could yield candidate molecular markers for marker-assisted selection and genomic selection programs, allowing breeders to screen for buffalo individuals with inherently high milk yield and improved composition. But they also flag real risks: artificially excessive activation of mammary cell proliferation and milk synthesis could impose metabolic stress and negative energy balance on lactating animals, and because ESR1 is a pleiotropic receptor with essential roles in systemic reproductive regulation, overactivation might disturb endocrine homeostasis, impair fertility, or raise susceptibility to mammary gland disorders. In vivo studies to define the optimal range of ESR1 activity are the clear next step. For now, the study delivers something buffalo genetics has long lacked: a mechanistic, experimentally validated picture of how a single estrogen-responsive gene orchestrates the cellular engines of one of the world’s most cherished milks.

Subject of Research: Regulation of lactation traits by the estrogen receptor alpha gene ESR1 in buffalo mammary epithelial cells

Article Title: The role of ESR1 in regulating lactation traits in buffalo mammary epithelial cells

Article References: Wang, Z., Chen, X., Qin, C., Jiang, J., Li, L., Liu, Q., Teng, C., Xiong, J., Wang, L., Amin, A., & Li, Z. (2026). The role of ESR1 in regulating lactation traits in buffalo mammary epithelial cells. Archives Animal Breeding, 69(2), 347-361. https://doi.org/10.5194/aab-69-347-2026

Image Credits: AI Generated

DOI: 10.5194/aab-69-347-2026

Keywords: ESR1, estrogen receptor alpha, buffalo, mammary epithelial cells, lactation, milk fat synthesis, milk protein synthesis, mTOR signaling, siRNA, gene overexpression, cell proliferation, animal breeding

The team’s methodology combined classical bacteriology with molecular confirmation, a dual approach that proved essential for accuracy. Of the 400 nasal swabs, 49 samples yielded colonies consistent with P. multocida on culture and biochemical screening, suggesting a presumptive prevalence of 12.25 percent. However, when the researchers applied polymerase chain reaction assays targeting species-specific genes, only 29 of those isolates were molecularly confirmed, yielding a PCR-confirmed prevalence of 7.25 percent. The discrepancy between the two methods underscores a persistent challenge in field microbiology: phenotypic identification can overestimate true prevalence because closely related commensal bacteria may mimic the colony morphology and biochemical profile of P. multocida, while PCR specificity comes at the cost of occasional sensitivity losses in samples with low bacterial DNA loads or inhibitory compounds.

Perhaps the most consequential molecular finding was capsular typing. Every PCR-positive isolate belonged to capsular type B, the serogroup associated with serotype B:2, one of the two strains—alongside E:2—responsible for devastating hemorrhagic septicemia outbreaks across Asia. Hemorrhagic septicemia is among the most lethal bacterial diseases of cattle and buffaloes in the region, producing fever, edema, respiratory distress, and rapid death. Because recovered and subclinically infected animals can harbor the organism in the lymphatic tissues of the upper respiratory tract and intermittently shed it in nasal secretions, the identification of type B organisms in nasal carriers carries significant epidemiological weight. Carrier animals act as a reservoir from which the bacterium can spread to susceptible herdmates and even to other indigenous species through aerosols, secretions, and direct contact.

The geographic distribution of positive samples varied considerably across the study sites, hinting at the influence of local husbandry and hygiene practices. Zakiganj recorded the highest detection rates, with 19.64 percent culture positivity and 14.29 percent molecular confirmation, followed closely by Fenchuganj. At the other extreme, no PCR-positive samples were recovered from Companiganj despite a small number of culture-positive swabs there, and Golapganj showed the lowest confirmed prevalence at 1.89 percent. The researchers did not find a statistically significant association between study area and infection status, but the spatial heterogeneity they observed aligns with the idea that management intensity, biosecurity, and animal movement patterns shape local reservoirs of infection in resource-limited farming systems.

Animal-level risk factors told a clearer story. Buffaloes aged between one and three years carried significantly higher odds of infection than calves under one year of age, with an odds ratio of 3.94. The researchers attribute this pattern to the confluence of immunological and management stressors that young adult animals face: weaning, increased movement and mixing with other animals, the onset of draught work or fattening regimens, and dietary and environmental changes that can suppress immune defenses. Meanwhile, calves may retain protection from maternally derived antibodies acquired through colostrum in areas where dams have been vaccinated or exposed, and older adults in endemic regions may have developed acquired immunity that reduces detectable carriage. Sex showed no significant effect, with males at 8.79 percent and females at 5.96 percent prevalence, suggesting that biological sex is far less determinative than age, stress, and management context.

The single strongest predictor of nasal carriage was clinical status. Buffaloes presenting with respiratory symptoms were 8.88 times more likely to test positive for P. multocida than apparently healthy animals, with a prevalence of 18.48 percent among symptomatic animals compared with just 2.49 percent among healthy ones. This association, highly significant statistically, reinforces the pathophysiological role of P. multocida as both a primary and an opportunistic respiratory pathogen. Under conditions of stress—poor nutrition, transport, adverse weather, or concurrent infections—the bacterium proliferates in the upper respiratory tract and can descend into the lower airways, initiating pneumonia or the systemic catastrophe of hemorrhagic septicemia. Once clinical signs appear, treatment is often ineffective, which is why the carrier state identified in this study represents such a critical intervention point.

The antimicrobial susceptibility results are where the study delivers its most alarming message. Among the 29 confirmed isolates, resistance to ampicillin reached 96.55 percent, oxytetracycline 93.10 percent, and penicillin-G 89.65 percent—three drugs that constitute the backbone of empirical treatment in many Bangladeshi livestock systems. Resistance to streptomycin, sulfamethoxazole-trimethoprim, gentamicin, and chloramphenicol also exceeded 50 percent. Only cefixime, a third-generation cephalosporin to which 93.10 percent of isolates remained susceptible, and azithromycin, with 68.96 percent susceptibility, retained meaningful activity. The researchers link the resistance patterns to decades of indiscriminate antibiotic use in a country where veterinary oversight is limited, informal animal health providers are widespread, and farmers frequently administer drugs without susceptibility testing, imposing relentless selective pressure on bacterial populations.

Correlation analysis of the resistance profiles revealed patterns that point to shared underlying mechanisms. Penicillin-G and ampicillin resistance were strongly and positively correlated, consistent with class-wide β-lactam resistance driven by β-lactamase production or altered penicillin-binding proteins. Gentamicin resistance correlated strongly with oxytetracycline resistance, and ampicillin resistance likewise tracked with tetracycline resistance, a pattern the authors interpret as evidence of co-selection: tetracycline and aminoglycoside resistance determinants are commonly carried together on mobile genetic elements such as plasmids and transposons, so selection pressure from one drug class can maintain resistance to another. The lone notable negative correlation, between cefixime and gentamicin, fits the observation that cephalosporins, rarely used in veterinary medicine, have escaped the co-resistance cascade that has eroded the utility of older drugs.

The study’s limitations are acknowledged by its authors. Molecular characterization was confined to species confirmation and capsular typing; more granular tools such as virulence gene profiling, multi-locus sequence typing, and whole-genome sequencing were not applied, and resistance was assessed phenotypically rather than by identifying the specific genes or mobile elements involved. Even so, the practical implications are immediate. The findings argue for routine susceptibility testing before treatment, targeted vaccination programs, improved biosecurity and husbandry to reduce stress in high-risk young adult animals, and strengthened national antimicrobial stewardship. Because cefixime and azithromycin remain effective but are considered critically important antimicrocrobials for human medicine, the authors caution that their use in animals must be judicious to preserve that efficacy.

For Bangladesh, where buffaloes supply milk, meat, and draught power to rural communities operating with limited veterinary infrastructure, the study provides the first systematic molecular characterization of nasal P. multocida carriage in the species. It demonstrates that the reservoir for hemorrhagic septicemia extends beyond visibly sick animals into the herds themselves, and that the bacteria circulating in that reservoir have accumulated resistance to the drugs farmers reach for first. Integrating molecular diagnostics with antimicrobial resistance monitoring, the researchers conclude, will be essential to controlling pasteurellosis and preventing resistant strains from spreading further through buffalo production systems—and, by extension, through the food animal populations on which millions of livelihoods depend.

Subject of Research: Prevalence and antimicrobial resistance of Pasteurella multocida carried in the nasal cavity of buffaloes in Bangladesh

Article Title: Prevalence, Molecular Identification, and Antimicrobial Resistance Traits of Pasteurella multocida Isolated From Nasal Swabs of Buffaloes in Bangladesh

Article References: Ghosh, P. K., Rahman, A., Roy, M., Sakib, M. A. N., Tuha, M. M.-E.-A., Ali, M., Chowdhury, M. S. R., Uddin, M. B., Rahman, M. M., & Hossain, M. M. (2026). Prevalence, Molecular Identification, and Antimicrobial Resistance Traits of Pasteurella multocida Isolated From Nasal Swabs of Buffaloes in Bangladesh. MicrobiologyOpen, 15(5), Article e70432. https://doi.org/10.1002/mbo3.70432

Image Credits: AI Generated

DOI: 10.1002/mbo3.70432

Keywords: Pasteurella multocida, buffalo, Bangladesh, hemorrhagic septicemia, antimicrobial resistance, nasal swabs, capsular type B, PCR, respiratory disease, livestock, multidrug resistance, veterinary microbiology

The research team, led by scientists at Guangxi University in collaboration with the Guangxi Buffalo Research Institute and international partners, performed an integrated transcriptomic analysis on milk-derived RNA from Murrah buffaloes at mid-lactation. The animals were divided into two clearly separated groups based on lactation performance. High-yield buffaloes produced an average of roughly 3,804 kilograms of milk per lactation, while low-yield animals averaged about 1,928 kilograms, a difference of nearly a factor of two. By sequencing the complete RNA content of milk from both groups, the researchers set out to identify the transcriptional features that accompany, and possibly underpin, this dramatic divergence in productivity.

The choice of milk as the sampling medium is one of the study’s most practical innovations. Mammary epithelial cells and other milk-associated cells are shed continuously into milk, meaning a simple sample of the fluid carries a molecular record of the secretory activity inside the udder. Unlike a mammary gland biopsy, milk collection is non-invasive, repeatable, and stress-free for the animal, which matters both for animal welfare and for the feasibility of scaling such measurements to breeding programs. The study’s authors note that no animals were euthanised, anaesthetised, or subjected to invasive procedures, and that the work was approved by the Animal Experiment Ethics Review Committee of Guangxi University.

Sequencing the milk RNA revealed a remarkably complex transcriptomic landscape. In total, the team identified 17,735 messenger RNAs, the protein-coding workhorses of the cell, alongside 2,641 long non-coding RNAs, 6,668 circular RNAs, and 578 microRNAs. This catalogue of four distinct RNA classes is what makes the study integrated rather than a conventional single-layer gene expression analysis. Long non-coding RNAs and circular RNAs can act as molecular sponges that bind microRNAs, thereby modulating how much protein gets produced from messenger RNA transcripts, a regulatory architecture known as the competing endogenous RNA, or ceRNA, network. Capturing all of these layers simultaneously from the same samples allowed the researchers to begin mapping how they interact.

The comparative analysis between high-yield and low-yield animals produced a coherent biological story. Buffaloes with higher milk output showed coordinated upregulation of genes involved in oxidative phosphorylation, the mitochondrial process that converts nutrients into cellular energy in the form of ATP. Genes tied to glucose metabolism, cellular metabolism in general, and protein synthesis were also elevated in the high-yield group. Taken together, these patterns point to an enhanced bioenergetic and biosynthetic state in the milk-associated cells of productive animals. In essence, the secretory cells of high-yielding buffaloes appear to run a hotter metabolic engine, burning more glucose through their mitochondria and building more protein, which is exactly what one would expect from tissue tasked with secreting roughly twice the volume of milk.

Equally telling was what was turned down. Transcripts associated with signal transduction, transporter activity, and apoptosis-related processes showed reduced expression in the high-yield group. Lower expression of apoptosis-related genes suggests that the milk cells of productive animals may resist programmed cell death, potentially sustaining secretory activity for longer. Reduced signalling and transporter transcripts hint at a shift in cellular priorities: rather than communicating with their environment or shuttling molecules across membranes, the cells of high-yield animals appear to channel resources into synthesis and secretion. The researchers are careful to frame these as associations rather than proven causes, describing the work as an exploratory framework, but the consistency of the metabolic signature across functional categories lends it weight.

By integrating the messenger RNA data with the non-coding RNA layers, the team constructed putative ceRNA networks that connect microRNAs, their messenger RNA targets, and the long non-coding and circular RNAs that sequester them. Within these networks, two candidate genes emerged as potentially associated with milk yield-related cellular pathways: SNX25, which encodes sorting nexin 25, a protein involved in intracellular trafficking, and DHPR, which encodes dihydropyridine reductase, an enzyme in the metabolic recycling pathway. These genes now stand as priority targets for future functional validation, the experimental step needed to determine whether they merely correlate with high yield or actively contribute to it.

The implications for buffalo breeding are potentially significant. Traditional genetic selection for milk yield in buffalo has been slowed by long generation intervals, limited pedigree and genomic records in many producing countries, and the sheer difficulty of measuring lactation traits early in an animal’s life. A transcriptomic marker panel derived from milk, measurable without harming the animal and potentially early in a lactation, could complement genomic selection by providing a functional readout of the actual secretory machinery. If the metabolic and ceRNA signatures identified here hold up in larger cohorts and across breeds, they could help identify high-potential animals sooner and guide the development of genetic improvement strategies tailored to buffalo biology rather than borrowed wholesale from dairy cattle.

The study also contributes a substantial public resource to the emerging field of livestock transcriptomics. Cataloguing nearly 18,000 messenger RNAs and thousands of non-coding transcripts from buffalo milk provides a reference for other researchers studying mammary biology, lactation physiology, and even milk quality traits, since many of the same metabolic pathways that drive yield also influence fat and protein composition. The raw sequencing data have been deposited in the Sequence Read Archive, allowing independent verification and reuse. The work was supported by the Guangxi Key Research and Development Program and the National Natural Science Foundation of China, reflecting the strategic importance of buffalo dairy production in southern China and across South Asia.

As with any exploratory transcriptomic study, caveats remain. The findings describe molecular features associated with divergent yield phenotypes at a single lactation stage in one breed, and the ceRNA networks are computational predictions that require experimental confirmation in mammary cells. The authors themselves position the work as a framework and a source of candidate targets rather than a definitive mechanism. Even so, the study marks a step toward a future in which a routine milk sample, already collected on every dairy farm every day, doubles as a molecular diagnostic window into the animal’s productive capacity. For an industry built on the biology of secretion, reading the transcriptome in the milk itself may prove to be one of the most elegant shortcuts breeding science has found in decades.

Subject of Research: Transcriptomic analysis of milk-derived RNA linked to divergent milk yield in buffalo

Article Title: Integrated transcriptomic analysis of milk-derived RNA associated with divergent milk yield phenotypes in buffalo

Article References: Integrated transcriptomic analysis of milk-derived RNA associated with divergent milk yield phenotypes in buffalo. (n.d.). https://doi.org/10.1186/s12864-026-13363-w

Image Credits: AI Generated

DOI: 10.1186/s12864-026-13363-w

Keywords: buffalo, milk yield, transcriptomics, RNA sequencing, non-coding RNAs, ceRNA network, oxidative phosphorylation, lactation, molecular breeding, gene expression, Murrah buffalo, BMC Genomics