Summer Scholars Faculty Mentor Summaries

Dr. Payal Agarwal: Exploration of different immunotherapy modalities in osteosarcoma.
Osteosarcoma (OS) is a highly aggressive and metastatic bone malignancy that primarily affects children and young adults. The survival rates for patients with OS have not improved significantly over the past 20 years, especially for those with metastatic disease. Cancer Immunotherapy has shown promise in treating various cancers and is a potential avenue for treating osteosarcoma. Immunotherapy reshapes the tumor microenvironment from immunologically ‘cold’ to immunologically ‘hot.’ In this study, we are exploring immunotherapies, individually, such as oncolytic virus armed with anti-PD1 anti-PDL1 sdAb and CAR T cell therapy against B7-H3 and also in combination to ascertain the advantages over using individual therapies. Anti-B7H3 CAR T cell therapy and anti-PD1 sdAbs will enhance the existing anti-tumor immunity. Oncolytic viruses will trigger immunity against tumor cells in primary as well as secondary sites. We have developed a next-generation canine adenovirus (CAV2-AU-M3) that infects and lyse tumor cells and secretes anti-PD1 sdAbs in cell vicinity. Our next step is to evaluate these oncolytic viruses for induction of cell lysis and functionality of anti-PD-1 sdAbs in vitro, in 2D and 3D canine OS cell cultures, and in vivo, in a murine xenograft model using canine OS cells. We will also assay the immunogenic cell death-inducing effects of CAV2-AU-M3 by assaying cytokine, chemokine, and DAMP (damage-associated molecular patterns) profiles in OS cells in response to oncolytic virotherapy. We will also explore and characterize the CAR T cells against B7-H3 surface antigen. CAR T cells will be assessed for their functionality (cell killing and cytokine secretion) in vitro, in 2D and 3D canine OS cell cultures, and in vivo, in a murine xenograft model of canine OS. 

Dr. Lindsey Boone and Dr. Deepti Pillai: Evaluation of dose and timing of administration of tissue plasminogen activator (TPA) for dispersal of synovial biofilm aggregates formed during equine synovial sepsis. Equine synovial sepsis is a prevalent condition that leads to significant morbidity and mortality. Factors affecting outcome include etiology, time between infection and treatment, synovial bioburden, lack of culture results, and antimicrobial resistance amongst others. Antimicrobial resistance in equine synovial sepsis is prevalent, which reinforces the need for new treatment strategies. Biofilm dispersal is a promising therapeutical option to dissolve biofilm and restore antimicrobial activity. There is limited research on the clinical application of tissue plasminogen activator (TPA) for biofilm dispersal in equine synovial sepsis and its reported use in vivo remains anecdotical. Our objectives are to evaluate the effect of dosing as well as timing of TPA administration on dispersal of equine synovial fluid biofilm aggregate and bacterial viability. Synovial fluid will be collected and inoculated with four common equine clinical isolates (Staphylococcus aureus, Streptococcus equi subsp. zooepidemicus, Enterococcus faecalis, and Escherichia Coli). On the first phase of the study, TPA will be added at 6 hours post-inoculation at doses of 0.25 mg/ml, 0.5 mg/ml, 0.75 mg/ml and 1 mg/ml and then challenged with amikacin and bacterial viability and biofilm dispersal will be measured. On the second phase of the study, TPA will be administered at 6, 12, 24 and 48 hours post-inoculation and bacterial viability and dispersal measured. Finally, reformation will be assessed at 24, 48 and 72 hours post-treatment. This work can provide clinically relevant guidelines for dosing and timing of administration of intrasynovial TPA in equine synovial sepsis, as well as investigate biofilm reformation post-dispersal. 

Dr. Serena Ceriotti: Comparison Of Different Laboratory Methods For Determining Cell-free Hemoglobin Concentrations In Equine Plasma. Cell free hemoglobin (CFH) increases in sepsis and is widely used as a prognostic indicator in human patients, including bed-side measurement with the HemoCue® device. Whether CFH can provide similar prognostic value in septic horses remains unknown, but it could have meaningful clinical impact if HemoCue® measurements prove reliable in equine practice. Currently, there is little to no published information on plasma CFH concentrations in healthy or clinically ill adult horses, and the performance of commonly used CFH measurement methods has not been systematically evaluated in this species. This summer project will compare and validate multiple laboratory approaches for quantifying CFH in equine plasma, with the goal of identifying accurate and practical methods for research and clinical use. 

Dr. Miria Criado: Research in Avian Disease: Avian Influenza. Are you passionate about veterinary medicine and interested in how infectious diseases impact poultry and other animals? Our lab provides a unique opportunity to gain hands-on experience in research at the intersection of animal health, disease prevention, and public safety. We study avian diseases with a focus on practical applications that directly inform veterinary practice and animal management. Avian influenza (AIV) is one of the most important viral threats to poultry worldwide. Beyond causing significant economic losses, AIV can spread to livestock and domestic animals, creating wider risks for animal populations and, in some cases, humans. Recent outbreaks have highlighted the urgent need to understand, monitor, and manage these infections, making avian influenza a central model for studying disease susceptibility, transmission, and control strategies in veterinary medicine. In our research, students engage with applied and experimental approaches to explore how chickens respond to infection, identify resistance traits, and develop strategies for disease management and biosecurity. We combine classical virology, molecular techniques, and in-vitro and in-ovo models to investigate avian pathogens in ways that have direct veterinary relevance. By bridging basic research and real-world applications, our work supports improved poultry health, food security, and the prevention of emerging infectious diseases. 

Shollie M. Falkenberg: Evaluation of different adjuvants and duration of immunity conferred by novel influenza vaccine for prevention and control of influenza A H5N1 in cattle. Given the H5N1 spillover into cattle, the previous history of influenza A infections reported in cattle, and the high seroprevalence of influenza D antibodies in cattle, a greater understanding of immune responses in cattle is needed for influenza viruses. Vaccination is one of the most important preventive tools and control measures against infectious diseases. The efficacy of a vaccine depends not only on the antigen, but also on adjuvants that are often used to stimulate the immune system in a more effective way. Adjuvants are defined as constituents added to vaccines to improve immune responses towards an antigen. This project aims to compare cattle immunized with the same influenza antigen but formulated with adjuvants that differ in their mode of action. Through this project you will assist with cattle sampling, sample processing, perform enzyme linked (ELISA) assay, and data entry and analysis. Upon conclusion of this study, we aim to better understand response to immunization and duration of immunity associated with different adjuvants to develop control strategies against HPAI in cattle. This study plays a pivotal role in advancing vaccine development for HPAI in cattle and evaluating effective immunization strategies. 

Dr. James Gillespie: Development of Species-Specific Immunocontraceptives in Wild Pigs.
Free-ranging populations of wild pigs, or feral swine, pose a significant economic problem for several regions across the United States and other developed countries, causing severe damage to agricultural crops, wildlife habitats, equipment, water supplies, and private property. Feral swine populations are estimated to include over 4-5 million pigs reported across 35 states and their numbers are expanding rapidly. The need to develop affordable, effective, species-specific contraceptive methods for control of wild pig populations is immediate and global. A species-specific, bacteriophage-based immunocontraceptive technology is proposed as a method to control wild pig populations. The long-term goal of proposed research is to develop, validate, and ultimately deploy a bacteriophage-based, pig-specific immunocontraceptive program for control of wild pig populations. Our short-term goal is to determine if one or more of the bacteriophage clones already selected for binding specificity to ZP obtained from domestic pigs (Sus scrofa domesticus) will also bind specifically to ZP obtained from wild/hybrid pigs (Sus scrofa subspecies) and not bind to unintended targets like wild deer or domestic pets (cats & dogs). Evidence of such specific binding will establish that peptides displayed on selected bacteriophage clones mimic porcine sperm cell surface peptides involved in sperm-ZP binding and, therefore, can be expected to induce production of anti-sperm antibodies in vaccinated, wild/hybrid pigs. Scholars will learn techniques in applied microbiology/biotechnology and molecular biology including generation of recombinant bacteriophages, quantitative PCR, SDS-PAGE, Western blotting, and ELISA. 

Dr. Kristine Griffett: Development of Novel Non-Opioid Compounds for Chronic Pain. This project focuses on the development of characterization of novel, non-opioid compounds targeting the nuclear receptor REV-ERB for analgesia. The opioid crisis is a substantial concern worldwide, and new efficacious pain therapies are desperately needed to reduce use and abuse of opioids. We have developed small molecule agonists for the REV-ERB nuclear receptors that appear to have analgesic properties in our preliminary testing. We believe that the inhibitory effects on inflammation at the site of nerve damage is one path that REV-ERB elicits its analgesic properties. The goal is to develop novel therapeutics that will be used for veterinary and human medicine, reducing our dependency on opioids, and reducing potential for abuse among patients, clients, and even providers. Our current model of efficacy testing is in mice, using mechanical and thermal measures to evaluate efficacy of compounds. This work will focus on evaluating animal tolerance to the compounds, efficacy testing, and evaluation of molecular and histological changes in sensory tissues. Techniques including QPCR, behavioral analysis, RNA-seq, IF/IHC or others may be utilized for this work. For more information regarding our work, please check out our website (www.griffettlab.wordpress.com). 

Dr. Amanda Gross: Viral and non-viral therapies for neurodegenerative diseases. Our laboratory uses viral (adeno-associated virus, AAV) gene therapies and non-viral lipid nanoparticles (polymersomes) to treat genetic neurodegenerative diseases, called GM1 and GM2 gangliosidosis. These diseases occur when a gene that produces an enzyme used in the lysosome to break down lipids, called gangliosides, is mutated. Without the needed enzymes the gangliosides accumulate throughout the body, most detrimentally in the brain, and cause cell death. We use AAV to deliver the functional gene so cells can start producing the enzymes they are missing. We also use polymersomes to deliver functional enzyme to the cells as a standalone or additive therapy alongside AAV. The Scott-Ritchey Research Center (SRRC), at the AU-CVM, has feline models of both GM1 and GM2 that we use to evaluate our treatments. Throughout the lives of the cats, we take neurological, magnetic resonance imaging (MRI), magnetic resonance spectroscopy (MRS), elastography (an ultrasound based method to determine the elasticity of tissues) measurements as well as collect blood and CSF for analysis of enzyme restoration. Following necropsy, we measure enzyme activity and gene expression in tissues from the animals. During this project, scholars will learn how to interpret MRI, MRS, and elastography data, as well as how to analyze samples for enzyme activity and gene expression. There is also the opportunity to be involved in any veterinary procedures occurring in the SRRC cat and dog colonies when not actively engaged in the lab. The results from these studies will be published in peer-reviewed journals and scholars will have the opportunity to assist in manuscript preparation and publication. 

Dr. Katie Horzmann: Developmental toxicity of environmental contaminants. I work with emerging and legacy environmental toxicants and study the effects of developmental exposure to these toxicants using the zebrafish (Danio rerio) biomedical model. We have multiple ongoing projects in the laboratory on topics ranging from micoplastics to chemicals and zebrafish behavioral models of Autism Spectrum Disorder. Previous summer scholars have worked with metabolites of a chemical drinking water contaminant called trichloroethylene (TCE). TCE is an industrial solvent and degreaser that contaminates over half of all Superfund sites, is a known carcinogen, and is linked to other adverse health outcomes including congenital cardiac defects and neurotoxicity. TCE is rapidly metabolized and the metabolites are thought to contribute to the overall toxicity. One option for student scholars would be to complete a project investigating the developmental toxicity of a TCE metabolite in embryonic and larval zebrafish. In addition to learning zebrafish husbandry and handling skills, scholars would evaluate embryonic mortality and hatching, embryonic behavior, larval behavior, and larval morphology. Other projects could focus on craniofacial developmental changes, behavioral assays, or gene expression alterations. 

Dr. Laura Huber, Steven Kitchens and Alinne Santana-Pereira: Bacteriophage-Based Biological Control of Virulent and Antimicrobial-Resistant Rhodococcus equi in Horse-Breeding Farm Environments. This project proposes isolating bacteriophages from soils of horse-breeding farms endemic for Rhodococcus equi to evaluate their potential as biological control agents against virulent and antimicrobial-resistant strains. Following isolation, phages will be plaque-purified, amplified, and stocked, then comprehensively characterized through virulence and adsorption kinetics, host-range testing across multiple R. equi strains and related bacteria, environmental sensitivity assays (pH, light, chlorine), genomic DNA extraction and sequencing, and transmission electron microscopy imaging. Using in vitro soil experiments under varying antimicrobial pressures, we will assess phage effectiveness, feasibility, and ecological impacts, including effects on the native soil microbiome and resistome. Overall, the study aims to determine whether targeted phage biocontrol can sustainably reduce pathogenic and drug-resistant R. equi in endemic farm soils while minimizing unintended ecological consequences. 

Dr. Jeba Jesudoss Chelladurai: Investigating Hookworm Anthelmintic Resistance in Alabama Dogs Using qPCR: Present and Past Comparisons. Hookworms are intestinal nematodes that cause anemia and diarrhea in dogs and can be difficult to control when they become resistant to common dewormers. This project will use qPCR to investigate whether hookworms in Alabama dogs show signs of resistance, both in current infections and in archived samples from past decades. The student will test known hookworm-positive and negative samples using the qPCR assay and compare results across time periods to look for trends in resistance-associated markers. Our findings will help clarify how widespread resistance may be in this region and inform better deworming strategies for veterinary clinics. The veterinary student scholar will learn to extract DNA from samples, set up and run conventional and quantitative PCRs, and interpret amplification curves and cycle threshold values. They will also learn how to organize data, compare present and past samples, and present the results in a scientific poster. Alternatively, a ruminant/equine parasitology related project can be performed if the student is interested (please discuss with mentor about the alternative project if interested). 

Drs. Aime and Jacob Johnson: Profound sedation is often required of feline subjects in both clinical and research settings to facilitate examination and diagnostic procedures. Dexmedetomidine, an alpha-2 agonist, is an approved sedative for felines but causes vasoconstriction, which can impair diagnostics. ZenAlpha; a combination of medetomidine and vatinoxan, a selective alpha-2 inhibitor; is approved for sedation in canine patients. It provides equivalent sedation without the cardiovascular effects. The goal of this project is to compare the pharmacodynamic effects of dexmedetomidine versus ZenAlpha in feline patients after intramuscular injection. Experience with the following procedures is desired but not required: intramuscular injection, intravenous catheterization, phlebotomy, feline handling and restraint, indirect blood pressure assessment. Scholar will be responsible for literature search, testing, data collection, data analysis, manuscript preparation. 

Dr. Constantinos S. Kyriakis: Evaluation of the reverse zoonotic potential of H3N2 subclade K human influenza virus. Influenza A viruses (IAVs) are among the most important animal and human health pathogens, causing significant disease to both humans and animals, affecting communities around the world, and impacting animal production and the economy. Several animal species, including wild birds, domesticated poultry and mammals are reservoirs of different viruses, which occasionally spill over infecting other animals or humans. Due to the nature of their genome, IAV are characterized by two important genetic traits: antigenic drift and genetic shift. They rapidly mutate and evade immune responses or adapt to new species, while when two virus strain infect the same host, they can exchange gene segments, resulting in the emergence of novel viral strains with the capacity to cause epizootics or pandemics. Human and swine IAVs share a common history, with major outbreaks of respiratory disease recorded simultaneously in both humans and pigs during the 1918-19 Spanish Influenza Pandemic. Pigs are natural hosts of IAV, and exhibit similar disease and immune responses with humans. Currently circulating swine IAVs in North America share gene segments with seasonal human viruses, which is the result of frequent reverse zoonotic events that have occurred over the last decades. The emergence of the H3N2 subclade K virus in humans is an example of a novel drifted viral strain, evading preexisting immune responses, causing disease even in vaccinated individuals. The extensive circulation of this virus in the human population increases the chances of spillover events to swine. In this project, we will use a One Health approach to evaluate the reverse zoonotic potential of H3N2 subclade K strains by (a) conducting viral kinetic studies in primary and immortalized porcine respiratory cells, and (b) by investigating the presence of cross-reactive neutralizing antibodies in a broad panel of swine sera, including field samples, as well as samples from experimentally infected or vaccinated animals. 

Dr. Dana LeVine: Immune-mediated hemolytic anemia (IMHA) is a common cause of severe anemia in dogs. IMHA is an autoimmune disease in which autoantibodies develop that target normal red cells for destruction. Unfortunately, IMHA is associated with a tragically high mortality rate (up to 80%) and mortality is mostly attributed to fatal thromboembolic events. The cause of thromboembolism is poorly understood. When activated, neutrophils can extrude neutrophil extracellular traps (NETs), webs of DNA, nucleosomes, histones, and granular proteases. NETs are designed to trap and kill invading microorganisms. However, NETs can also activate clot formation and induce thrombosis. We believe that NETs are important in the pathogenesis of thrombosis in IMHA and we have shown that markers of NETs are increased in the blood of dogs with IMHA. We have preliminary results suggesting that circulating cell-free DNA (cfDNA), a NET marker, is predictive of death in dogs with IMHA. NETs may be both a key prognostic marker in IMHA and may also provide an exciting new therapeutic target for this devastating disease. NET markers are also elevated in other important canine diseases like sepsis, trauma, and cancer, and NET markers may allow for rapid diagnosis and treatment of septic canine patients. Unfortunately, a major limitation in studying NETosis is the lack of a gold standard assay for measuring NETs. Although there are many biomarkers for NETs such as nucleosomes and cfDNA, these markers are not specific for NETosis and can also increase with necrosis or apoptosis. We propose to develop a flow cytometry assay that will be able to specifically measure circulating NETs in dogs and enable better characterization of the role of NETs in thrombosis in IMHA, and in other canine diseases. Developing a flow cytometric assay for NETs has the potential to revolutionize the study of NETosis in both veterinary and human medicine. Through this project you will learn to isolate canine neutrophils and stimulate NETosis (https://www.jove.com/video/54726/a-simple-fluorescence-assay-for-quantification-canine-neutrophil). You will also have the opportunity to practice venipuncture skills and you will become an expert on IMHA and will become familiar with the principles and techniques of flow cytometry. 

Dr. Maria Naskou: Platelet lysate against biofilm formation. This project will evaluate the ability of platelet lysate derived from canine and equine donors to inhibit bacterial biofilm formation. Biofilms, which are bacterial communities resistant to standard treatments, are a major concern in veterinary medicine, particularly in chronic infections and on medical devices. The study will assess whether platelet lysate alone can reduce biofilm formation and whether it has a synergistic effect when combined with commonly used antibiotics. The student will participate in generating platelet lysate, culturing bacterial strains, and performing biofilm assays to measure inhibition and synergy. The findings could provide valuable insights into alternative strategies to prevent or manage biofilm-associated infections in veterinary patients. 

Dr. Kathryn Reif and Dr. Anastasia Cooper: Characterization of mosquito feeding behaviors during heartworm ingestion and transmission. A major knowledge gap exists regarding the blood-feeding behaviors of hematophagous arthropods (e.g., mosquitoes) because these behaviors occur beneath the surface of the host’s skin and cannot be directly observed. Numerous variables—including pathogen infection status, host immune status, and environmental factors—likely influence vector feeding behavior. Our group is developing and applying a technique called electropenetrography (EPG) to elucidate these behaviors in mosquitoes and other blood-feeding arthropods. EPG is a minimally invasive method that indirectly visualizes and quantifies feeding behaviors occurring within opaque host tissues by measuring changes in electrical signals during an arthropod bite. The present study will investigate how Dirofilaria immitis (heartworm) infection may alter the feeding behaviors of Aedes aegypti mosquitoes. Our Summer Scholar will address this question through a combination of in vitro and in vivo experiments that vary the infection status of the vector and host/bloodmeal. Specifically, the scholar will use EPG to compare A. aegypti feeding behavior on uninfected versus microfilaria-infected blood using an in vitro feeding system. The scholar will then use EPG to assess how mosquito infection status influences feeding behavior when mosquitoes feed on either an artificial system or a live host. This investigation will increase our understanding of how pathogen infection in the vector or host can alter vector feeding behaviors in ways that may facilitate pathogen transmission. The project will provide opportunities to work with mosquito and mouse research models, learn EPG techniques, collaborate closely with a postdoctoral researcher (Dr. Cooper), and potentially co-author a research article for publication in a scientific journal. Duties may include animal monitoring and anesthesia, mosquito rearing and electrode attachment, data collection and scoring, statistical analysis, and manuscript preparation. 

Dr. Maninder Sandey: Immune checkpoint inhibitors (ICIs) have shown unprecedented clinical activity in a wide range of malignancies. However, their efficacy remains limited in many malignancies due to primary or acquired resistance. Targeted therapies that activate tumor necrosis factor receptor superfamily (TNFRSF) members, like OX40 & 4-1BB, are currently explored to augment the clinical efficacy of ICIs. In this study, we have constructed a novel-nanobody (Nb) based Agonist-Redirected Checkpoint (ARC) platform that consolidates immune checkpoint blockade (ICB) and TNFRSF agonism in a single biologic. Our long-term goal is to utilize this Nb-based ARC platform to develop novel immunotherapeutics to treat canine and human cancer patients. In this project, we will: (Aim 1) assess the safety and pharmacokinetic (PK) profile of aPD1-Fc-OX40L; (Aim 2) conduct a phase II/III clinical trial of aPD1-Fc-OX40L in canine patients with oral melanoma (OM); and (Aim 3) assess the functional activities of aPD1-Fc-OX40L over monotherapies. We will perform clinical correlative studies (using flow cytometry and immunohistochemistry (IHC)) on tumor and peripheral blood samples collected before and after administration of aPD1-Fc-OX40L to understand the cellular and molecular mechanisms that determine the antitumor immune response. 

Dr. Melissa Singletary: Nose to Tail: An Evaluation of Health, Welfare and Performance in Working Dogs. The Canine Performance Sciences (CPS) Program conducts research and development to tackle one health and national security initiatives. Our efforts include research focused on health and welfare management of working dogs throughout all life stages from breeding and production to field operations and retirement. CPS summer scholar participants usually perform research that connects benchtop laboratory work to direct application in working dog populations. We aim to provide our summer scholars with a well-rounded and hands-on clinical research experience in production, colony management, behavior and health care of performance/working dogs. Working across the program, Summer Scholars at CPS will also learn about early puppy development, foundational training, and operational application of detection dogs. Based on student interests, there are multiple options for conducting a summer research project examining metrics for working dog health, welfare, and performance in a transdisciplinary approach through medical, behavioral/cognitive, physical fitness and olfactory-based tasks. 

Dr. Scarlett Sumner: Wound healing and antimicrobial properties of canine platelet lysate in a chronic and infected wound model. In addition to hemostasis, platelets contain growth factors, cytokines, and antimicrobial peptides. Platelet products such as platelet rich plasma and platelet lysate have been shown to have wound healing and antimicrobial properties. The summer project will focus on testing canine platelet lysate on a bench top chronic wound model and an infected wound model involving the cell culture of canine keratinocytes. The project is part of a series of projects to evaluate platelet lysate as potential clinical therapeutic. The summer scholar will develop bench top laboratory skills and be involved in literature review, lab meetings, cell culture, data collection, and data analysis. The results of this project will be published in a peer-reviewed journal. There will be an opportunity for student participation in the manuscript preparation and authorship. I work in collaboration with Dr. Naskou’s lab, and the summer student will be exposed to additional ongoing projects on platelet lysate. 

Dr. Jordan Towns: Validation of a diagnostic test using the Advia 2120 Hematology Analyzer for Feline Infectious Peritonitis (FIP). Diagnosis of Feline Infectious Peritonitis (FIP) infection in cats is often a diagnostic challenge. There are a variety of diagnostic tests available for FIP, but many of these have poor diagnostic sensitivity and/or specificity. Frequently, multiple diagnostic modalities are required to reach a diagnosis of FIP. In a recent study, researchers determined that the Sysmex XT 2000-iV hematology analyzer could provide useful diagnostic information about body cavity effusions in cats with FIP. This information can help distinguish cats infected with FIP from cats with body cavity effusions for other reasons. The Advia 120 and 2120 hematology analyzers are capable of providing similar information, but this test has not been evaluated or validated on this instrument. These analyzers are used by many Clinical Pathology labs in the United States, including Auburn University’s lab. This study is a retrospective study that will evaluate case records and analyze laboratory data from cats that were diagnosed with FIP, as well as a control group of cats with body cavity effusions for other pathophysiologic reasons. The student will aid in developing the study design, collecting and evaluating laboratory data from cats, and generating figures for a manuscript. This project will not involve hands-on experience with cats, but will provide experience with the scientific method, including developing and refining study design, interpreting laboratory data, performing statistics on sets of data, and generating figures to illustrate that data. Additionally, based on the findings from this study, the student will be involved in helping develop a potential prospective study testing this new diagnostic tests on future cases that present to Auburn University. 

Dr. Chengming Wang. Non-Invasive Fetal Sexing in Large and Small Ruminants. The ability to determine the sex of a fetus early in gestation is a game-changer for modern livestock management. For producers, this technology facilitates: 1) Strategic Herd Planning: Prioritizing resources for dams carrying high-value offspring (e.g., replacement heifers in dairies or males in meat-focused systems); 2) Economic Optimization: Enhancing marketability and streamlining supply chains by predicting production outputs well in advance; 3) Improved Animal Welfare: Reducing unnecessary interventions and allowing for specialized prenatal care based on fetal requirements. Despite the clear benefits, current methods are often limited by narrow gestational windows or cost barriers. This project seeks to overcome these hurdles by focusing on Cell-Free DNA (cfDNA) Analysis via maternal “liquid biopsies.” The core technical approach involves the deployment of highly sensitive quantitative PCR (qPCR) assays designed to detect minute concentrations of fetal DNA circulating in maternal plasma. To ensure maximum diagnostic accuracy and mitigate the risks of low-titer samples, this project will: 1) Utilize Multi-Target Amplification: Rather than relying on a single genetic marker, we will target several highly conserved, Y-chromosome-specific sequences (such as SRY, ZFY, or repetitive DNA elements). 2) Enhance Sensitivity: Implement optimized qPCR protocols capable of detecting fetal sex as early as the first trimester, providing a non-invasive alternative to traditional ultrasound. 3) Validate Biochemical Proxies: Explore supplementary sex-specific metabolites or hormones in milk and blood to create a multi-modal diagnostic framework.