Symposium

Presenter: Usman Abubakar
Advisor: Zhengguo Xiao

Neonates face significantly higher morbidity and mortality rates from infectious diseases than adults, accounting for over 30% of newborn deaths. In calves, approximately 66% of early morbidity and all first-year mortality occur within the first month of life and are primarily due to gastrointestinal infections. These infections are driven by enteric parasites like gastrointestinal nematodes and bacteria like Escherichia coli; as well as respiratory pathogens such as bovine respiratory syncytial virus (BRSV). Despite this high susceptibility, the mechanisms underlying neonatal immune dysfunction remain incompletely understood. Protection against infection is primarily mediated by the adaptive immune system, in which B cells produce antibodies and T cells coordinate immune responses. CD8+ T cells target intracellular pathogens such as viruses by killing infected cells. In contrast, CD4+ T cells orchestrate immune responses against both intracellular and extracellular pathogens, including viruses, bacteria, and parasites. Upon activation, CD4+ T cells differentiate into specialized functional subsets that direct immune responses by producing cytokines that enhance B cell antibody production and promote pathogen clearance. Among these, Th1 responses are typically associated with immunity to intracellular pathogens, while Th2 responses support antibody-mediated immunity and the expulsion of extracellular pathogens. Given that effective protection against extracellular pathogens relies heavily on CD4+ T cell-mediated immunity, particularly Th2 responses, understanding how these pathways are regulated in cattle is critical. Previously, our lab demonstrated that adult bovine CD4+ T cells exhibit limited IL-4 protein expression even under canonical Th2-polarizing conditions and instead predominantly display an IFNγ-biased or mixed Th0 phenotype, challenging the classical paradigm of T helper 2 (Th2) differentiation defined by interleukin-4 (IL-4) production. These findings highlight fundamental differences between bovine and murine/human Th2 biology and raise critical questions regarding the development and functional identity of Th2 responses in cattle. However, whether these unconventional Th2 characteristics observed in adult cattle are already established in early life or are further constrained by neonatal immune immaturity remains unknown. Therefore, we aim to determine whether developmental constraints shape neonatal CD4+ T cell differentiation and whether the atypical Th2 phenotype observed in adult cattle is established early in life or emerges during immune maturation. To investigate Th2 differentiation in neonatal calves, we will isolate naïve CD4+ T cells (CD4⁺CD25⁻) and culture them under the three-signal model of T cell activation with established Th2-polarizing conditions. Differentiated cells will be assessed using intracellular cytokine staining and transcriptional profiling to define their functional phenotype. These findings will provide critical insights into neonatal immune programming and inform strategies to enhance protective immunity and improve vaccine efficacy in early life.

Advisor: Sunoh Che
Co-authors: Sarai Celestine, Randolph Mitchell

This study evaluated the effects of guanidinoacetic acid (GAA) supplementation on the prevalence of woody breast (WB) and spaghetti meat (SM) and gene expression in the pectoralis major muscle of broilers. We hypothesized that GAA, a precursor of creatine, may influence muscle energy metabolism and susceptibility to myopathies. A controlled experiment was conducted using Ross 708 broilers (42 birds × 36 pens, total n = 1512), assigned to two dietary treatments: a control group (18 pens) and a GAA-treated group at 0.08% (18 pens) under commercial-like conditions. Body weight and feed intake were recorded at four production stages, while myopathy scores were assessed at 6 and 7 weeks through visual and tactile evaluation. Pectoralis major muscle samples from control and GAA-treated birds were collected for transcriptomic analysis, including principal component analysis (PCA), to assess gene expression differences between treatments. Results indicated that GAA supplementation significantly increased body weight and feed intake at 7 weeks (p = 0.03 and p = 0.01, respectively), while FCR and myopathy prevalence were not significantly affected (SM: p = 0.089; WB moderate: p = 0.901; WB severe: p = 0.105). PCA and PERMANOVA showed no overall differences in gene expression between treatment groups. However, differential gene expression analysis revealed that, at week 6, GAA supplementation increased the expression of genes associated with cell proliferation and tissue repair (SKA1, SPARC, EPX). By week 7, the upregulation of HPGD and RhoV suggested reduced inflammatory signaling and cytoskeletal remodeling. These complementary findings suggest that GAA may modulate muscle energy and tissue remodeling, potentially affecting myopathy development, even in the absence of measurable prevalence changes. This study highlights the complex molecular effects of nutritional interventions and identifies gene expression changes that may help develop future mitigation strategies to reduce broiler breast myopathies.

Advisor: Tom Porter
Co-authors: Paige Meisner, Ricardo Sierra Arroyo, Kuan Ling Liu, Diane Hildenberger, Isaac Harford, Kristen Diehl

The objective of this study was to quantify testicular morphology in roosters with high and low sperm mobility (HM and LM, respectively) and to determine if high oleic soybean meal could alter sperm dynamics. Broiler breeder roosters were randomly allocated to the standard broiler breeder commercial diet or high oleic soybean diet (n=28) experimental groups at 21 weeks of age. Semen was collected twice a week and assessed for sperm concentration and mobility. Testes were collected from HM and LM roosters at 35 weeks of age from each diet (n=5). The right testis was weighed, fixed in 10% formalin, and sectioned for histological analysis of the seminiferous tubules (ST) (ST area, ST diameter, lumen width, and epithelial thickness).  Semen metrics, testis weight/diameter, and ST parameters were analyzed by a one-way (diet), two-way (diet; mobility), and three-way ANOVA (diet; mobility; ST location), respectively. The birds fed a high oleic soybean diet had higher sperm mobility (0.57 ± 0.017 AU) than the birds fed a standard soybean diet (0.52 ±0.01 AU). The weight of the left testes was less in LM birds (13.17 ± 0.9 g) than HM birds (18.64 ±  1.39 g). The ST area was greater in HM birds (0.081 ± 0.004 mm²) than LM birds (0.069 ± 0.001 mm²). The ST epithelium thickness was greater in HM birds (95.68 ± 1.28mm) than LM birds (90.77 ± 1.33 mm) and it was also greater in the internal region (96.55 ± 1.17 mm) than the external region (89.90 ± 1.34 mm). The ST diameter was greater in the external region (322.75 ± 4.40 mm) than the internal region (305.48 ±  4.07 mm). The lumen width was greater in birds fed a high oleic diet (120.05 ± 2.83 mm) than the birds fed a standard soybean diet (110.59 ± 2.52 mm) and it was also greater in the external region (127.03 ± 2.92 mm) than the internal region (102.84 ± 2.15 mm). High oleic soybean meal improved sperm mobility and increased ST lumen width. The low mobility phenotype was associated with substantially lower left testes weight, smaller ST area, and smaller ST epithelial thickness. ST location influenced ST diameter, lumen width, and epithelial thickness. Future directions include analysis of differences in genetic or hormonal regulation of testicular growth and function. 

Advisor: Byung-Eun Kim
Co-authors: Yasmin A. Mejia-Guevara, Yining Liu, Ashton Trey Belew, Evan Ying, Najib-El Sayed

Iron (Fe) is essential for adipose tissue development and thermogenic function, yet the mechanisms linking Fe availability to adipogenesis and browning remain poorly understood. Adipose tissue consists of white, brown, and beige depots, with beige adipocytes representing a key therapeutic target for alleviating metabolic disorders. Fe uptake is facilitated by transferrin receptor 1 (TfR1), and while Fe is known to support mitochondrial biogenesis and browning of inguinal white adipose tissue (iWAT), its precise molecular role in thermogenic signaling and postnatal adipose development has remained unresolved. To investigate the role of Fe in iWAT browning, we employed dietary Fe-deficient and adipose-specific TfR1 knockout (TfR1^adi/adi) mice. RNA sequencing of Fe-deficient iWAT following β3-adrenergic stimulation (CL316,243) revealed an Fe-dependent thermogenic gene subset enriched for cAMP response elements (CRE) rather than canonical iron response elements. Further, Fe deficiency impaired intracellular cAMP generation in response to both CL316,243 and direct adenylyl cyclase stimulation, blunting PKA-CREB-driven thermogenic programs. Mechanistically, Fe deficiency induced expression of Adcy3-at, which suppresses full-length ADCY3 function, thereby reducing cAMP production and thermogenic capacity. In parallel, we assessed TfR1's contribution to postnatal adipose development across key developmental stages (P3–P30). TfR1^adi/adi mice exhibited progressive iWAT atrophy coinciding with loss of TfR1-knockout adipocytes, while TfR1 expression peaked in iWAT at P13 which aligns with the reported postnatal beiging window between P10 and P20. BAT maintained consistently high TfR1 expression from P6 to P14, revealing depot-specific dependencies on TfR1. Importantly, Fe supplementation at P3 and P10 rescued both iWAT development and CL316,243 responsiveness, directly linking Fe availability to postnatal adipose remodeling and thermogenic competence. Together, these findings identify Fe and TfR1 as critical regulators of postnatal adipogenesis and thermogenic signaling, uncovering a previously unrecognized molecular link between Fe status, cAMP production, and the browning capacity of iWAT.

Advisor: Byung-Eun Kim
Co-authors: Yuan Yee Lee, Yining Liu, Irene Ki

Copper (Cu) is an essential micronutrient required for enzymatic activity, ATP synthesis, and tissue development. Mutations in Cu transporters disrupt Cu homeostasis, causing Menkes or Wilson’s disease, underscoring the need for regulated Cu transport. The high-affinity Cu transporter CTR1 mediates dietary Cu uptake in intestinal epithelial cells (IECs), yet how its subcellular localization is dynamically regulated across developmental stages, metabolic demands, and systemic Cu status to maintain whole-body Cu homeostasis remains unclear. In the fetal stage, hepatic Cu levels and ATP7A, an ATP-dependent Cu exporter, peak and decline postnatally, suggesting liver-driven Cu supply before dietary intake begins. However, the mechanisms of fetal hepatic Cu storage, postnatal mobilization, and intestinal CTR1 response remain unclear. We hypothesize that CTR1 localizes basolaterally in fetal IECs to import maternal Cu and shifts apically after birth for dietary uptake. To test this, we performed age-dependent immunofluorescence (IF) of CTR1 in mouse intestine and, since constitutive intestinal CTR1 knockout (KO) is postnatally lethal, we generated tamoxifen-inducible, intestine-specific CTR1 KO mice (Villin-CreERT2) to study intestinal CTR1 function in adults. CTR1 deletion elevated CCS protein in the intestine, indicating local Cu deficiency; however, 10 days of CTR1 loss did not induce Cu deficiency in liver, heart, or brain, suggesting compensatory mechanisms or existing Cu stores use. IF across developmental stages revealed CTR1 in a largely sub-apical vesicular pattern at postnatal days 0 and 10, transitioning to dynamic intracellular trafficking in adults. To explore ATP7A’s role in developmental Cu regulation, we characterized its hepatic expression across the perinatal period. ATP7A displayed broad punctate staining at embryonic day 18.5, primarily in albumin-positive hepatocytes, which declined by postnatal day 5 and became restricted near hepatic veins by P14, while Kupffer cells and sinusoidal endothelial cells showed no dramatic changes. These findings suggest coordinated, stage-dependent regulation of intestinal CTR1 trafficking and hepatic ATP7A expression during the perinatal-to-adult transition, advancing our understanding of systemic Cu homeostasis and Cu-related disorder.

Advisor: Tom Porter
Co-authors: A. Bond, R. Sierra Arroyo, B. Kamkrathok, P. Sinpru, and K. Diehl

Pre-incubation egg storage is a standard practice in commercial poultry production, yet extended storage durations are associated with declining hatchability and increased early embryonic mortality during the first three days of incubation. Previously, targeted multiple reaction monitoring-based lipidomic and metabolomic profiling identified storage- and mortality-associated yolk biomarkers. The objective of this study was to evaluate the transcriptional and metabolic responses associated with egg storage in broiler breeder embryos. Fertilized Cobb broiler breeder eggs were stored for either 2 or 10 days at 12 C and then incubated at 37.5 C. Yolk and embryo samples were collected on day 3 of incubation (n=8/storage duration). RNA expression data was analyzed using one-way ANOVA with storage duration as the main effect. Reverse transcription-quantitative PCR (RT-qPCR) was performed to evaluate mRNA levels of 15 genes associated with fatty acid metabolism, oxidative stress, cellular transport and signaling, and cellular differentiation between storage duration groups. Seven genes were significantly (p<0.05) up-regulated in the 10-day storage condition, implicating shifts in mitochondrial fatty acid oxidation (carnitine palmitoyltransferase 1A), oxidative stress (glutathione peroxidase 7), cellular transport (beta tubulin and clathrin heavy chain), and early developmental signaling (inhibitor of DNA binding 2 and polo-like kinase 1). These results indicate that some but not all of the genes analyzed for mitochondrial fatty acid metabolism, oxidative stress, cellular transport and signaling, and cellular differentiation were affected by egg storage. It remains to be determined whether any of the genes whose expression was affected by storage results in embryonic mortality. Future integration of metabolomic datasets may help provide biologically informed strategies for improved hatchability in commercial poultry production.

Advisor: Tom Porter
Co-authors: Parama Bhattacharjee and Nishanth Sunny

Provision of feed after hatching induces a metabolic switch in chicks, where there is a shift from hepatic lipolysis during embryonic development to de novo lipogenesis after hatch. Delayed feeding (DF) delays the metabolic switch and alters gene expression in the hypothalamus—a neuroendocrine organ responsible for appetite and control of feed intake. Our lab has shown that hypothalamic mRNA expression of neuropeptide y (NPY) increases after 48 hours (hr) DF, which is a neuropeptide driving feed intake. Furthermore, our group previously reported that DF increased thyrotropin-releasing hormone receptor mRNA expression in the pituitary gland. While the effects of DF have been studied on the hypothalamus, the effects of specific dietary macronutrients have not been studied. The objectives of this study were to determine the effects of DF and specific dietary macronutrients on hypothalamic mRNA expression. Ross 708 broiler chicks were separated into 7 treatment groups (n=8/group): day of hatch (DOH), fully fed (FF), fasted (DF), and fed semi-purified (SP)-complete, SP-dextrin, SP-casein, and SP-corn oil. FF chicks were fed ad libitum, while the SP diets were orally gavaged every 8 hr for 48 hr and consisted of 85% of its respective macronutrient on an isocaloric basis. Gene expression was measured using reverse transcription-quantitative-PCR and normalized to phosphoglycerate kinase 1 before being statistically analyzed by ANOVA (SAS Version 9.4). The following hypothalamic genes that regulate metabolism and feed intake were analyzed: NPY, deiodinase 2 (DIO2), and thyrotropin-releasing hormone (TRH). Genes downstream from hypothalamic TRH expressed in the liver—deiodinases 1 and 3—were measured and normalized to β-actin. No significant changes in hypothalamic TRH mRNA were found in DF chicks compared to FF chicks. There was a significant increase (P<0.05, relative to FF) in hypothalamic NPY and DIO2 mRNA. DIO1 and DIO3 showed decreased and increased mRNA expression in the liver, respectively. We conclude that fasting newly hatched chicks alters the thyrotropic axis and the regulation of feed intake by the hypothalamus. However, none of the SP-diets given by oral gavage mimicked the FF treatment group. Ongoing research is evaluating the effects of SP diets provided in a crumble form ad libitum.

Advisor: Heewon Seo
Co-author: Joe Cain

The ovine placenta utilizes specialized structures called placentomes that act as the primary sites of maternal-fetal exchange during pregnancy. Each placentome is formed by the extension of tortuous cotyledonary villi into the caruncular endometrium (CE). The mechanisms that regulate this invasion and limit it specifically to the CE, remain poorly understood. We hypothesize that the cellular and extracellular matrix (ECM) components of the endometrium selectively permit invasion into the CE during placentomal development. In the present study, transcriptomic and histological analyses were performed to identify possible mediators of this regulation. Total RNA was extracted from paired caruncular and intercaruncular endometrial (IE) samples collected from ewes on Days 32-34 of gestation. Bulk RNA-seq analysis identified 1407 differentially expressed genes (DEGs; Padj< 0.05, log2FC≥ 1). Of these DEGs, 713 were upregulated in the CE and 694 were upregulated in the IE. Functional enrichment analysis using Gene Ontology indicated that these DEGs were predominantly associated with cell–cell signaling and ECM organization. KEGG pathway analysis further revealed enrichment of pathways related to ECM–receptor interaction, integrin signaling, and focal adhesions, in the CE. These data indicate dynamic crosstalk between cells of the CE and cotyledon, and interactions with the ECM during cotyledonary invasion. Uterine sections from ewes on Days 18-34 of gestation were collected and stained. PAS staining revealed that the basement membrane of the syncytial epithelium remains intact in the IE but is breached at the leading front of invading cotyledonary villi. Additionally, a distinct band of PAS stain was detected at the basal stroma of the CE where it appears to delineate the maximum possible extent of cotyledonary invasion. Masson's trichrome staining detected differences in ECM composition and density in the sub-epithelial stroma between the CE and IE. This variation suggests that differential collagen depositions may establish a permissive microenvironment for invasion, similar to the ECM-dependent regulation of invasion observed in human implantation. Collectively, these results indicate a crucial role for region-specific cell-cell and cell-ECM interactions in facilitating the development of placentomes.

Advisor: Nishanth E. Sunny

Elevated plasma branched chain amino acids (BCAAs; Valine, Leucine, Isoleucine) and changes in their degradation networks interact with lipid and mitochondrial metabolism in insulin resistant muscle, yet the mechanism remains unknown. We hypothesize that increased BCAA degradation induces lipid oxidation and tricarboxylic acid (TCA) cycle metabolism via AMP-activated protein kinase (AMPK). Mouse C2C12 myoblasts were given increasing concentrations (0, 50, 100mM) of 3,6-dichloro-benzo[b]thiophene-2-carboxylic acid (BT2) for 3 days to induce BCAA degradation. Myoblasts were seeded (500,000 cells/well) in low-glucose (5 mM) DMEM containing 250µM 2:1 ¹³C₁₈ Oleate:¹³C₁₆Palmitate and 2mM of 5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR; AMPK activator) for 5 hrs before collection in 100µL cold methanol for gas chromatography–mass spectrometry. Intracellular BCAAs (µg/500,000 myoblasts ± SEM; Isoleucine; 0µM BT2; 0.61±0.06 vs. 100 µM BT2; 0.44±0.05 p = 0.06) and their corresponding ketoacids (ng/500,000 myoblasts ± SEM; α-keto-β-methylvalerate (KMV); 0µM BT2; 1.57±0.16 vs. 100 µM BT2; 1.20±0.08 p = 0.07) trended lower with increased BT2, indicating higher rates of BCAA degradation. Lower levels of intracellular branched chain keto-acids following BT2 priming were significantly correlated with higher M+2 Citrate enrichment (APE; atom percent excess) arising from ¹³C fatty acids (e.g., M+2 Citrate vs. KMV; r (21) = -0.67, p ≤ 0.001), suggesting induced lipid oxidation with higher BCAA degradation. This induction resulted in higher enrichments (APE) of M+1, M+2, and M+3 isotopomers of multiple TCA cycle intermediates, showing a general induction of mitochondrial TCA cycle metabolism (α-ketoglutarate; M+1; 0µM BT2; 0.81±0.18 vs. 100µM BT2; 1.50±0.25 ; M+2; 0µM BT2; 5.76±0.58 vs. 100µM BT2; 8.36±0.87 ; M+3; 0µM BT2; 1.58±0.38 vs. 100µM BT2; 4.26±0.63, p ≤ 0.05). Interestingly, AMPK activation in myoblasts via AICAR enhanced BT2-mediated induction of lipid oxidation and TCA cycle metabolism (Citrate M+2 (APE); 100µM BT2 + No AICAR; 30.81±1.19 vs. 100 µM BT2 + 2mM AICAR; 37.75±1.29 p ≤ 0.001). Priming myoblasts to induce BCAA degradation results in higher mitochondrial lipid oxidation via AMPK. Modulation of BCAA degradation could be a strategy to enhance mitochondrial lipid oxidation and TCA cycle function in myoblasts. 

Advisor: Debabrata Biswas
Co-authors: Anna Phan, Susan Lin

Lactiplantibacillus plantarum (LP) is a well-characterized probiotic with demonstrated antimicrobial and immunomodulatory properties relevant to poultry health. Berry pomace extract (BPE), a polyphenolic plant-derived byproduct, exhibits intrinsic antimicrobial activity and selectively promotes growth of beneficial microorganisms, consistent with prebiotic functionality. The combination of LP and BPE as a synbiotic formulation represents a promising antibiotic alternative strategy against Avian Pathogenic Escherichia coli (APEC), a major extraintestinal poultry pathogen with recognized zoonotic implications. This study evaluated the in vitro antimicrobial efficacy of LP and BPE against APEC and their potential to alter interaction of the pathogen with host macrophages. Inhibitory and bactericidal concentrations of BPE against APEC were determined. LP was grown in BPE supplemented media to obtain  cell-free culture supernatants (CFCS^BPE). APEC growth kinetics, survival, stress-response, and virulence-associated gene expression were assessed following treatment with CFCS^BPE or BPE independently. BPE demonstrated an MIC of 1 mg GAE/mL against APEC and enhanced LP growth at 0.5 mg GAE/mL, supporting its prebiotic potential. CFCS^BPE reduced APEC counts by >3 log CFU/mL within 24h and achieved complete elimination by 48h (p<0.001). These effects were accompanied by significant downregulation of virulence-associated (iss, fyuA), outer membrane (ompC), quorum sensing (luxS), stress-response (rpoS), and cell wall biosynthesis (murD) genes (p<0.05). Further, bacterial membrane integrity and structural alterations were visualized via confocal and scanning electron microscopy. The HD-11 cells were employed to assess the interaction of host macrophage in the presence of LP or BPE. LP pre-treatment significantly enhanced macrophage bactericidal activity (p<0.05) without altering phagocytic uptake efficiency. LP and BPE differentially modulated the expression of pro-inflammatory (IL-1β, IL-6, IL-8) and anti-inflammatory (IL-10) cytokines. Collectively, these findings demonstrate the combined antimicrobial and host immunomodulatory capacity of LP and BPE, supporting their development as antibiotic-free intervention strategies for control of APEC in poultry production. 

Advisor: Iqbal Hamza
Co-authors: Vijaya Pandey, Jean-Martin Harder, Joshua MacDermott, David Bodine, Ute Hellmich, James Wohlschlegel, Iqbal Hamza

Heme, an iron-containing biomolecule, serves as the prosthetic group for proteins and enzymes involved in many essential biological pathways across all living organisms. Due to its hydrophobicity and cytotoxicity, heme transport and trafficking must be tightly regulated. Using Caenorhabditis elegans, we identified the first metazoan heme importer/transporter, HRG-1/SLC48A1, which mediates heme uptake in the worm intestine. In mammals, HRG1 is required for heme-iron recycling in macrophages to sustain new red blood cell production. Given the hydrophobicity and cytotoxicity of free heme, HRG1 likely transports heme by forming ternary complexes with other proteins. Here, we identify the in vivo HRG1 interactome using cross-species proteomic analyses combined with functional screening. By integrating data from transgenic mice, C. elegans, and mammalian cells, we reveal a comprehensive list of HRG1 interactors. By assessing their impact on HRG1 expression, localization, and function in C. elegans, Saccharomyces cerevisiae, and mammalian cell lines we identify ATP-binding cassette transporter ABCB6 as a prominent HRG1 interactor. Using biochemical, cellular, and complementary structural analyses, we demonstrated that HRG1 and ABCB6 forms a stable complex on lysosomal membranes. Our findings provided a functional framework for heme trafficking mechanisms through HRG1-ABCB6 protein complexes.

Advisor: Heewon Seo

Pig conceptuses elongate rapidly through extensive cellular proliferation, migration, and morphological change. Tricarboxylic acid (TCA) cycle flux provides biosynthetic precursors for synthesis of lipids, nucleotides, and amino acids. However, during the peri-implantation period of pregnancy, the low-oxygen intrauterine environment slows electron transport chain (ETC) activity, resulting in the accumulation of NADH and the depletion of NAD⁺, stalling upstream oxidative TCA cycle reactions and reducing the generation of biosynthetic intermediates. There is biological precedence for the use of non-canonical metabolic strategies such as reverse TCA cycle reactions to maintain TCA cycle flux and redox balance in low-oxygen conditions, but these pathways have yet to be examined in the porcine conceptus trophectoderm. In the present study, conceptuses from Days 11 to 17 of pregnancy were collected and subjected to LC-MS/MS. After Day 11, concentrations of pyruvate, citrate, and succinate decreased, while concentrations of lactate and fumarate increased, indicating a metabolic shift in response to low oxygen availability. When porcine trophectoderm cells (pTr2) were cultured with low oxygen (5% O₂), or with antimycin to inhibit ETC function, an increase in the NADH/NAD⁺ ratio was detected, suggesting that low-oxygen levels within the intrauterine environment generate a reductive force capable of reversing TCA cycle flux. Isocitrate dehydrogenase 1 (IDH1) catalyzes the interconversion of isocitrate into αKG in either the forward or reverse direction of the TCA cycle. ATP citrate synthase (ACLY) irreversibly catalyzes the reverse conversion of citrate into oxaloacetate and acetyl-CoA. Immunoreactive IDH1 and ACLY were detected in conceptus trophectoderm during the peri-implantation period, which suggests that pig conceptuses can reverse TCA cycle flux. Consistent with these findings, ex vivo cultures of conceptuses from Day 15 of pregnancy demonstrated that antimycin-mediated inhibition of the ETC causes accumulation of succinate, which is the end-product of the reverse TCA cycle in mammals. Collectively, these results indicate that pig conceptuses likely utilize non-canonical TCA cycle activity to fulfill biosynthetic demands as they elongate in challenging redox conditions.