Animal & Avian Sciences
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Item MEDIATION OF CORTICOSTERONE-INDUCED GROWTH HORMONE GENE EXPRESSION IN CHICKEN EMBRYONIC PITUITARY CELLS: IDENTIFICATION OF TRANS-ACTING FACTORS AND A NOVEL PITUITARY CELL TYPE(2024) Liu, Kuan Ling; Porter, Tom E.; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Growth hormone (GH) is responsible for up to 30% of growth in broiler chickens. Somatotrophs, or GH secreting cells, begin to differentiate around embryonic day (e)14, in conjunction with an increase in the primary plasma glucocorticoid (GC) corticosterone (CORT). CORT treatment of e11 chicken embryonic pituitary (CEP) cells induces premature GH secretion. This GC-induced process involves trans-acting factors because the GH gene lacks a canonical GC response element (GRE). In addition to the binding of ETS1 and the GC receptor (GR) to the GC-responsive region (GCRR; 1045/ 964), we hypothesize that there are other regulatory factors necessary for CORT responsiveness. By modifying the pGL3_-1742/+25 GH-luciferase reporter, we have constructed various other GH-luciferase reporters and analyzed them for promoter activity in response to CORT treatment. We identified a putative distal (d) ETS-Like 1 (ELK1) binding site that is necessary. The proximal (p)PIT1 site and pTATA box were also identified to be critical for CORT induction of the GH gene. Interestingly, cloning multiple copies of the extended GCRR (eGCRR; -1067/-900) further increased promoter activity in an additive manner under both basal and CORT treated conditions. Through single-cell RNA sequencing (scRNAseq), 8 members of the ETS family of transcription factors were identified in > 5% of the somatotroph population. Commercial antibodies were validated, and human (h)ETV1, hELF2, hELK3, and hETV6 antibodies were confirmed to recognize their recombinant chicken ortholog and to identify their corresponding protein in e11 CEP cells. Results from chromatin immunoprecipitation quantitative PCR suggest that multiple ETS members are involved in CORT induction of the GH gene with more evidence pointing towards ELF2 and ELK3. Identifying trans-acting factors for the GH gene inducible by CORT allows for better understanding of endogenous GH regulation in chickens. Further analysis of the scRNAseq data from e11 CEP cells revealed a cluster of cells expressing genes for more than one hormone-producing cell type (“premature nebulous” cluster). Within the premature nebulous cluster, a large population (~30%) was co-expressing proopiomelanocortin (POMC) and growth hormone (GH). We named this novel cell population the cortico-somatotrophs. Through RNA fluorescent in-situ hybridization (RNA-FISH) and dual label immunofluorescence, we verified the existence of the cortico-somatotrophs at both the mRNA and protein level, respectively. Cortico-somatotrophs were also shown to share genes for receptors normally specific to both corticotrophs (CRH-R1) and somatotrophs (GHRHR). Additionally, in response to CORT treatment, the cortico somatotrophs showed an increase in GH as well as a decrease in POMC mRNA levels. The discovery of the cortico-somatotrophs suggests a modification to the current dogma on pituitary cell lineages, where corticotrophs and somatotrophs may have overlapping developmental pathways. In conclusion, our discovery of the cortico somatotrophs has furthered our understanding of CEP development and opened the door for further exploration of the cell lineages during pituitary development.Item Phenotypic and Genetic Analysis of Reasons for Disposal in Dairy Cattle(2024) Iqbal, Victoria Audrey; Ma, Li; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Reasons for disposal are defined as why a cow has left the herd during lactation and are documented as termination codes. Dairy cattle termination codes were collected by Dairy Records Processing Centers and stored in the National Cooperator Database maintained by the Council on Dairy Cattle Breeding for analysis. The list of possible termination codes is as follows: code 0 is cow lactation that ended typically without an abortion, code 1 is locomotion problems, code 2 is female transferred or sold, code 3 is low milk yield, code 4 is reproductive problems, code 5 is unspecified reasons, code 6 is death, code 7 is the presence of mastitis, code 8 is abortion, code 9 is udder problems, code A is an unfavorable phenotype, and lastly code B is undesirable temperament. Understanding termination codes is the key to understanding and improving farm management. Unfortunately, the secondary termination codes are not utilized, despite studies saying one reason is too limited. Heifer termination codes should be more utilized, and studies show that this could improve heifer management. The four processing centers' principal termination codes deviated a little from year to year, but processing center D had the most variation in principal termination codes. There were few records with termination codes 9, A, and B. There was low lameness found for Jersey cattle but more fluctuations for their termination codes 6, 7, and 8. Jersey's main reason for disposal was sold and low milk yield. As for Holstein, the main reasons for disposal were low milk production and death. Recommendations include removing termination code 5 (other reasons) and enforcing a secondary termination code for code 2 (sold). Also, including the percentage of animal records used in traits developed at the CDCB was recommended to encourage farmers to add more records to improve breeding selections. Overall, the top main reasons for disposal were low milk yield, death, and reproduction across breeds from 2011 to 2022. To determine whether health traits correlate to termination codes and how health traits change the probability of survival, a multinomial logistic regression was developed, where twelve health traits, breeds, and other factors were used as an independent variable for the termination code, the dependent variable. The output is a regression coefficient list that conveys the effect of each health trait for each termination code. The results show the apparent impacts of animal breeds on different termination codes, such as dairy crossbreeds negatively affecting termination due to reproductive advantages that follow the literature. Lastly, using termination codes as phenotype, this study focuses on developing a genome-wide association study (GWAS) using the Weighted single-step Genomic Best Linear unbiased prediction (WssGBLUP) model to find significant SNPs related to survival in Holstein cows. In summary, this study provided an understanding of reasons for disposal trends, modeled the reasons for disposal, determined the likelihood of termination post-incidence, and found the heritability and important SNPs of each termination code.Item THE STUDY OF ENERGY METABOLISM IN THE PERIPARTURIENT DAIRY COW: INVESTIGATING THE ROLE OF KETONES AND NAD+(2024) da Silva, Mariana; Rico, Jorge Eduardo; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Dairy cows typically experience increased energy demands during the transition from gestation to lactation, and a myriad of metabolic adaptations are set in place to facilitate this transition. This thesis integrates findings from two experiments exploring energy metabolism during this critical phase. In the first experiment, the effects of hyperketonemia on dairy cow health and productivity were investigated. Eight Holstein cows (19.62 ± 5.44 days postpartum) were intravenously infused with either Na-BHB (KET) or NaCl (CON) for 48 hours. The study aimed to assess the impact of elevated beta-hydroxybutyrate (BHB) levels on blood biomarkers and immune response. Despite no significant differences in dry matter intake or overall milk yield, KET cows exhibited lower milk yield post-immune challenge (LPS bolus) and higher plasma NEFA levels. While plasma glucose concentrations remained unchanged, but BHB concentrations were significantly higher in the KET group (P < 0.001). The second experiment focused on the role of Nicotinamide Adenine Dinucleotide (NAD) coenzymes in energy metabolism during the transition from gestation to lactation. Twenty-six peripartal dairy cows were enrolled in an observational study to examine the NAD metabolome in liver, blood, and milk. Liver biopsies were performed 21 days before and 7 days after parturition, and blood samples were collected weekly. The study hypothesized that hepatic NAD levels deplete while blood and milk NAD pools increase postpartum, with a possible influence of residual feed intake (RFI). Measurements of plasma glucose, free fatty acids, BHB, and insulin, along with comprehensive NAD metabolome profiling using HPLC coupled to mass spectrometry, aimed to elucidate the relationship between NAD metabolism, metabolic biomarkers, and production performance. After calving, animals showed decreased glucose and insulin levels, and increased NEFA and BHB levels, with no significant group differences (P = 0.53). Oxidative stress markers (protein carbonyl, 8-OHdG) and total antioxidant capacity were measured on plasma. The DNA and protein oxidative stress markers remained unchanged relative to parturition, but the Low-RFI group tended to display higher antioxidant capacity (P = 0.08). NAD metabolites increased, and NAD precursor concentrations decreased in the liver. Nicotinamide Mononucleotide was higher in the High-RFI group (P = 0.04) and tended to decrease post-calving (P = 0.06). The liver NAD metabolome remained stable (P = 0.83). Both studies underscore the complexity of energy metabolism during the peripartum period in dairy cows. Collectively, our findings expand our understanding of novel aspects of energy metabolism, with potential implications for health, productivity, and disease resilience in dairy cows. Further research is essential to fully understand these mechanisms and improve management strategies for dairy cows during this critical period.Item COORDINATED TRAFFICKING OF HEME TRANSPORTERS BY CARGO SORTING COMPLEXES IS ESSENTIAL FOR ORGANISMAL HEME HOMEOSTASIS(2025) Dutt, Sohini; Hamza, Iqbal IH; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Heme, an iron-containing organic ring, is a vital cofactor responsible for diverse biologicalfunctions and is the major source of bioavailable iron in the human diet. As a hydrophobic and cytotoxic cofactor, heme must be transported in a highly controlled manner through membranes via specific intra- and inter-cellular pathways. However, the genes and pathways responsible for heme trafficking remain poorly understood. Unlike other metazoans, Caenorhabditis elegans cannot synthesize heme but requires heme for sustenance. Thus, C. elegans is an ideal animal model to identify heme trafficking pathways as it permits organismal heme homeostasis to be directly manipulated by controlling environmental heme. Heme is imported apically into the intestine by HRG-1-related permeases and exported basolaterally by MRP-5/ABCC5 to extra- intestinal tissues. Loss of mrp-5 causes embryonic lethality that can be suppressed by dietary heme supplementation raising the possibility that MRP-5-independent heme export pathways must exist. Here we show, by performing a forward genetic screen in mrp-5 null mutants, that loss of the vesicular cargo sorting Adaptor Protein complexes (AP-3) fully rescues mrp-5 lethality and restores heme homeostasis. Remarkably, intestinal heme accumulation due to mrp-5-deficiency causes a concomitant deficit in the lysosomal heme importer HRG-1 abundance and localization. Loss of both MRP-5 and AP-3 subunits resurrects HRG-1 levels and localization, thus underscoring the crucial role of HRG-1 in dictating mrp-5 mutant phenotypes. In the absence of MRP-5, heme is exported by SLC49A3 homolog, a previously uncharacterized transporter. Live- cell imaging reveals vesicular coalescence that facilitates heme transfer between the importers and exporters at the interface of lysosomal-related organelle. These results define a mechanistic model for metazoan heme trafficking and identifies SLC49A3 as a promising candidate for heme export in mammals.Item OVARIAN STROMAL CELLS IMPROVE SURVIVAL, BUT NOT GROWTH, IN PRE- AND EARLY ANTRAL FELINE FOLLICLES(2024) Marks, Batsheva Naomi; Keefer, Carol; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Ovarian stromal cells act as crucial support and regulators for in vivo folliculogenesis; however, less is known about their effect on in vitro grown follicles. The objective of this study was to investigate the impact of ovarian stromal cell co-culture or conditioned medium (CM) on survival and development of cat pre-, early, and antral follicles in vitro. Ovaries were obtained from cats older than six months (n = 3), then enzymatically digested to release stromal cells. The ovarian stromal cells were allowed to grow to confluency in a T75 flask, before being cryopreserved for long term storage in liquid nitrogen. Cells were thawed one week prior to follicular culture onset, and passaged once before CM collection. CM was subsequently removed 24 - 48 hours after feeding, and stored at -80C until used. Ovarian follicles were mechanically isolated from cats older than six months (n = 23 cats, 155 follicles), encapsulated in 0.5% alginate hydrogel. The isolated follicles were then divided into five treatment groups (control, ovarian stromal cell co-culture, 20% CM, 50% CM, and 100% CM in Endothelial Cell Growth Medium), and classified based on initial diameter as preantral (224.4 + 4.7 m), early antral (394.8 + 7.4 m), or antral (592.2 + 18.8 m). Culture subsequently lasted for 13 days, and survival and growth of the follicles were evaluated on Days 0, 4, 6, 8, 11 and 13. At the end of culture, follicles were assessed via qRT-PCR for expression of CYP19A, FSHR, and GDP9 to further quantify development. Statistical analysis was done in R software. Follicles in 100% CM had higher survival up to Day 11 of culture as compared to other treatment groups (Cox proportional hazards model, p < 0.01). Initial stage also influenced survival, with antral follicle survival significantly lower than that of pre- and early antral follicles (p < 0.0001). However, no differences in growth were detected across the treatment groups, nor across initial size classifications (Kruskal-Wallis test, p > 0.05). Post culture qRT-PCR analysis of the three selected genes showed upregulation of CYP19A in 50% CM follicles compared to the control (ANOVA, p < 0.05). However, there were no differences in CYP19A expression between the control and other treatment groups, or in GDF9 and FSHR expression among culture groups (p > 0.05). In summary, the findings demonstrated that conditioned medium collected from primary culture of ovarian stromal cells improves in vitro survival and modulates CYP19A expression of isolated cat follicles. Further research to identify paracrine factors present in conditioned medium will elucidate the roles of ovarian stromal cells pertaining to follicle survival during in vitro folliculogenesis.Item IDENTIFICATION OF KEY MOLECULES IN PLACODE-DERIVED NEURONS THAT COORDINATE CHICK TRIGEMINAL GANGLIOGENESIS(2024) Hines, Margaret; Taneyhill, Lisa; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The trigeminal nerve is the largest of the cranial nerves, possessing three main branches (ophthalmic, maxillary, and mandibular) and relaying sensations of pain, touch, and temperature from the face and head to the brain. Cell bodies of this nerve are positioned in the trigeminal ganglion, which arises from the coalescence of neural crest cells and placode cells. These progenitor cells give rise to trigeminal sensory neurons, with placode cell differentiation occurring first. While the dual cellular origin of the trigeminal ganglion has been known for decades, the molecular mechanisms controlling trigeminal ganglion development remain obscure. To elucidate molecules involved in this process, we performed RNAsequencing on the forming chick trigeminal ganglion when only placode cells contribute neurons and identified Neurogenin2 (Neurog2), Neuronal Differentiation 1 (NeuroD1), and Elongator acetyltransferase complex subunit 1 (Elp1) for further study. While Neurog2, NeuroD1, and Elp1 have established roles in neurogenesis in other systems, their functions in placode cells during trigeminal gangliogenesis had yet to be investigated. To address this, we used the chick embryo due to experimental advantages afforded by this model for the study of trigeminal placode cells and trigeminal ganglion development. Using morpholino antisense oligonucleotides, we depleted Neurog2, NeuroD1, or Elp1 from trigeminal placode cells and demonstrated each are essential for proper trigeminal ganglion development. Knockdown of Neurog2, NeuroD1, or Elp1 reduced trigeminal ganglion size and led to aberrant innervation of the eye by the ophthalmic branch. While depletion of Neurog2 and NeuroD1 had opposite effects on the width of the ophthalmic branch, Elp1 reduction appeared to have no effect. However, Elp1 knockdown led to less compact trigeminal ganglion nerve branches, decreased axon projections, and general disorganization of neurons and neural crest cells. Taken together with prior findings, our results suggest a novel interrelationship among Neurog2, NeuroD1, and Elp1 during trigeminal gangliogenesis. Our results have potential high significance for providing new insights into the function of Neurog2, NeuroD1, and Elp1 in trigeminal ganglion development and the etiology of human and animal diseases arising from defects in neural crest cells and/or placode cells.Item Characterisation of sRNAs enriched in outer membrane vesicles of pathogenic Flavobacterium psychrophilum causing Bacterial Cold Water Disease in rainbow trout(Wiley, 2024-06-19) Chapagain, Pratima; Ali, Ali; Kidane, Destaalem T.; Farone, Mary; Salem, MohamedFlavobacterium psychrophilum (Fp) causes Bacterial Cold Water Disease in salmonids. During host-pathogen interactions, gram-negative bacteria, such as Fp, release external membrane vesicles (OMVs) harbouring cargos, such as DNA, RNA and virulence factors. This study aimed to characterise the potential role of the OMVs’ small RNAs (sRNAs) in the Fp-rainbow trout host-pathogen interactions. sRNAs carried within OMVs were isolated from Fp. RNA-Seq datasets from whole-cell Fp and their isolated OMVs indicated substantial enrichment of specific sRNAs in the OMVs compared to the parent cell. Many of the OMV-packaged sRNAs were located in the pathogenicity islands of Fp. Conservation of sRNAs in 65 strains with variable degrees of virulence was reported. Dual RNA-Seq of host and pathogen transcriptomes on day 5 post-infection of Fp -resistant and -susceptible rainbow trout genetic lines revealed correlated expression of OMV-packaged sRNAs and their predicted host's immune gene targets. In vitro, treatment of the rainbow trout epithelial cell line RTgill-W1 with OMVs showed signs of cytotoxicity accompanied by dynamic changes in the expression of host genes when profiled 24 h following treatment. The OMV-treated cells, similar to the Fp -resistant fish, showed downregulated expression of the suppressor of cytokine signalling 1 (SOCS1) gene, suggesting induction of phagosomal maturation. Other signs of modulating the host gene expression following OMV-treatment include favouring elements from the phagocytic, endocytic and antigen presentation pathways in addition to HSP70, HSP90 and cochaperone proteins, which provide evidence for a potential role of OMVs in boosting the host immune response. In conclusion, the study identified novel microbial targets and inherent characteristics of OMVs that could open up new avenues of treatment and prevention of fish infections.Item USING TRIBUTYRIN, A BUTYRATE PRODRUG, DURING GESTATION TO ALTER PERFORMANCE IN PIGS(2024) Cooper, Elizabeth Jean; Salem, Mohamed; Taneyhill, Lisa A; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Prenatal and postnatal muscle growth and development occurs in a series of three waves. Every stage of the process requires the coordinated actions of myogenic precursor cells (MPC) or satellite cells (SC). Fetal programming of the muscle resident stem cells through maternal dietary intake is a potential method by which to influence muscle fiber growth throughout life. Butyric acid is a potent histone deacetylase (HDAC) inhibitor and has demonstrated strong effects on improving activation and differentiation of skeletal muscle satellite cells. It has also been implicated in serving as an important regulatory mechanism in intestinal cells. In animal models, this activity has resulted in direct promotion of muscle growth and improved resistance to pathogen induced illness. Whether butyrate supplementation during gestation can influence fetal and then lifelong muscle development has not yet been examined. Tributyrin, a pro-drug form of butyrate was utilized in all studies to address technical difficulties associated with using butyric acid as a feed supplement. In the first experiment, we aimed to investigate whether tributyrin at various concentrations in the sow diet could enhance fetal development and the degree to which inclusion levels might be most ideal (1% or 2% butyric acid). We found that tributyrin had a dose-dependent effect on fetal myogenic precursor cell activity and muscle structural organization. An increased proportion of primary fibers identified in the muscle of 2% treated fetal pigs indicated a greater capacity for an increased number of myofibers. Given these results, we repeated the gestational feeding study with more sows which were then allowed to farrow naturally with just one dietary level of tributyrin used (2%). Piglet tissues were collected between postnatal days 3 and 5, and sow milk samples were collected once on day 5 post farrowing to test the fatty acid composition. We found significant changes in the sow’s transition- milk in response to tributyrin. There was an indication of differences in expression of genes associated with SC differentiation in whole Longissimus dorsi (LD) muscle. However, there was no notable change in the muscle fiber size or numbers by day 3 or 5 post farrowing. In our final study, we performed a collaborative study with a contract research organization (CRO) based in the Midwest. This study followed the same design as our first two studies, but post-farrowing offspring growth was monitored. We saw significant improvements to pre-weaning average daily gains in tributyrin treated piglets. A subsample of gilts were selected once they reached 6 months of age for evaluation of carcass traits. Of those selected gilts, there was a significant reduction in overall fat accumulation. Future research is needed to confirm whether this is a universal response for pigs that had prenatal exposure to tributyrin. These findings help support the idea that prenatal tributyrin can enhance muscle growth and lifelong performance in pigs.Item INVESTIGATION OF DISRUPTED INSULIN SIGNALING IN A SWINE MODEL(2024) Markley, Grace Irene; Stahl, Chad H; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Insulin is an anabolic hormone involved in glucose uptake and synthesis of fats, proteins, and glycogen. Domesticated livestock species such as swine require efficient insulin signaling to meet production demands across the world. Insulin signaling is tightly regulated and acts on metabolic tissues such as the liver, skeletal muscle, and adipose tissue. The most well characterized disruption of insulin signaling is insulin resistance that is often caused by obesity induced inflammation. However, insulin signaling can be disrupted via atypical mechanisms such as immune response to a pathogen and adaptor proteins. We aimed to evaluate the impact of pathogen exposure and intrinsic adaptor proteins on insulin signaling in pigs. The first study focuses on the impact of growth factor receptor bound protein 10 (GRB10) as an inhibitor of insulin signaling. In commercial swine, the presence of GRB10 has been linked to growth, reproduction, feed efficiency and lean muscle growth. While insulin induces glucose uptake in typical tissues, such as the liver and skeletal muscle, insulin also acts on other cell types including mesenchymal stem cells (MSC). MSC are adult multipotent stem cells that can self-renew and differentiate into multiple cell types including adipocytes. The process of adipogenesis requires insulin signaling to synthesize new triglycerides and store them in lipid droplets. While GRB10 has been established as a regulator of insulin signaling, the role of GRB10 in swine MSC has yet to be firmly established. We generated GRB10 knockdown (GRB10-KD) MSC to evaluate the impact of GRB10 on insulin signaling and glucose uptake. We observed reduced glucose utilization under basal conditions and reduced insulin signaling when incubated with insulin over 48 hours. We also noticed a two-fold reduction in proliferation rate among GRB10-KD MSC. When differentiated into adipocytes, we observed an increase in transcript abundance with genes associated with insulin signaling and adipogenesis. GRB10 has the potential to regulate insulin signaling in swine MSC and contribute to overall growth and development. The second chapter focuses on the impact of lipopolysaccharide (LPS), an endotoxin produced by gram-negative bacteria, which can induce a severe, systemic immune response. In pigs, chronic LPS exposure has induced insulin resistance. However, the effects of acute exposure to LPS on insulin signaling and resistance have not been elucidated. We found that acute exposure to LPS in crossbred post-weaning pigs induced changes in insulin signaling and glucose metabolism. There was an LPS induced decrease in insulin two hours after injection which was paired with hyperglycemia. At 24 hours post LPS there was a marked insulin resistance indicated by hyperinsulinemia and hyperglycemia. We also noted that there were liver specific decreases in genes associated with glucose metabolism, insulin signaling and fatty acid metabolism. As well as reduction in protein abundance such as protein kinase B (AKT) and phosphoinositide-3 kinase (PI3K) in the liver after LPS administration. During an acute exposure to endotoxin, insulin signaling, and glucose metabolism is reduced in the liver. These results highlight that insulin signaling is a complex and dynamic process that can be controlled through a variety of mechanisms and swine can serve to model these disruptions.Item DIFFERENTIATION AND REGULATION OF BOVINE TH2 CELLS(2024) Kandel, Anmol; Xiao, Zhengguo Zhengguo; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Memory CD4+ T cells, specifically type-2 (Th2) cells, are pivotal in defending against infections caused by extracellular pathogens, including several economically important parasites. However, whether interleukin-4 (IL4) expression is a signature feature of bovine Th2 cells likewise in mice and humans is unclear. Pasture-raised cattle, routinely exposed to extracellular parasites such as Ostertagia ostertagi (OO), are likely to develop a typical Th2 memory response. Therefore, using cytokine induction assay, we evaluated the circulatory memory bovine T cell profile of these cattle and also analyzed if the expression of presumptuous memory marker, CD45RO, is reliable in identifying memory bovine T cells. Surprisingly, the majority of the memory CD4+ T cells dominantly produced interferon-gamma (IFNγ), with only a small fraction co-expressing IL4, and memory bovine T cell identification did not correlate with CD45RO expression. Results suggested that cattle naturally exposed to extracellular parasites do not develop typically IL4 dominant Th2 response. To further investigate these results, resting CD4+ T cells isolated from healthy cattle blood were cultured under simple in vitro Th2 culture. Analysis of differentiated cells through flow cytometry revealed limited IL4 protein detection, which was in line with the lack of upregulation of IL4 and its master regulator GATA3 transcripts shown by the quantitative polymerase chain reaction (qPCR) assay. To validate whether differentiated cells were actually Th2, unbiased proteomic analysis was conducted. Based on differentially expressed 397 proteins between differentiated cells and naïve phenotype, bovine Th2 differentiation was validated; nonetheless, the process was not found to be associated with IL4 induction. Moreover, despite using published strategies from mice and humans, such as reducing T cell receptor (TCR) stimulation strength and adding exogenous recombinant bovine IL4, the expression of IL4 could not be significantly enhanced. Interestingly, differentiated bovine Th2 cells proliferated in the presence of OO antigens, suggesting that extracellular parasites could influence bovine Th2 differentiation, at least in vitro. To validate the results from pathogen-infected tissues and in vitro culture, a panel of anti-parasitic CD4+ single T cell clones was established from five pasture-raised cattle that were infected with OO. Evaluation of memory responses exhibited by the anti-parasitic CD4+ single T cell clones strongly supported IFNγ dominant memory response, and only 20% of them co-expressed IL4 through a small subset of IFN γ + cells. All the data pointed out that bovine CD4+ T cell differentiation is partially distinct from those in mice and humans, and IL4 expression is not a hallmark feature of the bovine Th2 cells.