Veterinary Research Forum

Abstract Urinary bladder cancer, the tenth most common cancer globally, exhibits significant variability in the reported expression of estrogen receptor alpha (ERα), a key prognostic marker. Accurate and reproducible assessment of ERα is critical for elucidating its role in bladder cancer biology and identifying therapeutic targets. Aim: This study compared manual scoring with automated analysis using ImmunoRatio software for quantifying ERα expression in a chemically induced bladder cancer model in rats, using N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN). Methods: Twenty male Wistar rats were randomly assigned to two groups: a control group (n=8) and a group treated with BBN (BBN; n=12), administered in drinking water at 0.05% for 20 weeks. Animals were sacrificed and urinary bladders were collected for macroscopic and microscopic evaluation. Samples underwent standard histological processing and immunohistochemical analysis to assess ERα expression, quantified both manually and automatically. Results: The immunoexpression of ERα, detected by ImmunoRatio was found to be significantly higher than that obtained through manual counting in both groups (p < 0.0001). ImmunoRatio analysis indicated a higher ERα expression in the control group than in the BBN group (p < 0.001), whereas manual counting showed the opposite tendency, without statistical significance (p > 0.05). Conclusion: Our findings demonstrated that ImmunoRatio resulted in significantly higher ERα immunoexpression values than manual counting in both control and BBN-treated groups, with inconsistent group-wise trends between methods. These findings suggest that, although digital pathology tools offer advantages in standardization and reproducibility, their application requires tissue-specific calibration and validation to ensure accurate and reliable biomarker quantification.

Abstract Foot-and-mouth disease virus (FMDV) continues to pose a major threat to the livestock industry worldwide due to its high transmissibility and substantial economic consequences. For diagnostic and vaccinal purposes, necessitate the exploration of novel immunogenic targets is pivotal. Here, antigenic determinant (an epitope) sequence comprising core of 12 amino acids from VP1 protein of foot-and-mouth disease virus (FMDV) was designed by immuno informatics. To improve the physio-chemical and stability characteristics of the final peptide, additional residues were incorporated until it resulted in the synthesis of a 23-amino acid peptide (VARTLPTSFNYGAIKATRVTDEC, 2.5 Kda and PI 8.17). After chemical synthetizing with 95 % purity, the peptide was conjugated to bovine serum albumin (BSA) via EDC/NHS cross linker (Peptide: EDC: Sulfo-NHS: BSA (carrier protein): 10:40:50:1 in MES buffer (50 Mm)) and formulated with an aluminum hydroxide adjuvant. Twelve male New Zealand White rabbits were randomly assigned into three groups and main group received the peptide-BSA conjugate. Sera were direct to Dot blot, double immunodiffusion (DID) and serum neutralization (SN) tests, and also indirect peptide ELISA. The synthetic peptide elicited strong antibody response by primary positive results in dot blot and DID test (positive in 1:4 serum dilution) as well as it showed cross-reactivity with whole antigen. Although only low SN titers (1:8) were observed. Moreover, the peptide ELISA confirmed specific antibody responses against O serotype (OD of 2.4 at day 55 in 1/10 dilutions). Altogether, rationally designed epitope are promising regimes for the future development of FMDV novel diagnostic tools and vaccine cocktails.

Abstract This study explored the effects of different levels of hempseed meal(HSM) supplementation in laying hen diets, with and without a multienzyme supplement, on egg production, quality, yolk fatty acid composition. A total of 144 White Leghorn hens, aged 42 weeks, were divided into eight groups with six replicates each, and the experimental groups were fed with four HSM levels (0%,5%,10%,15%) with or without 1000mg multienzyme supplement per kg feed for a period of 84 days trial. Results indicated that with or without multienzyme did not significantly affect egg production, weight, mass, feed conversion ratio. Feed intake was consistent across HSM levels, but cubic interaction between multienzymes and HSM was observed(p<0.05). Eggshell parameters were unaffected by HSM or multienzyme supplementation. Yolk color(L* value) showed a cubic response to HSM levels, peaking in 5% treatment. The addition of multienzymes and HSM significantly decreased the a*value(p<0.05), while the b*value increased quadratically with HSM supplementation(p<0.05). Yolk fatty acid composition exhibited linear and cubic effects from HSM and multienzyme HSM interaction(p<0.05), with increased total polyunsaturated fatty acids(ƩPUFA) and decreased total monounsaturated fatty acids(ƩMUFA)(p<0.05). The omega-6/omega-3 ratio was linearly influenced by HSM supplementation(p<0.05). In summary, multienzyme supplementation alone did not significantly alter production or quality parameters, but interacted with HSM to affect feed intake, yolkcolor(a* value), and fatty acid composition. These findings suggest that HSM supplementation may improve the quality of egg yolk, although it does not affect overall egg production, and may therefore be viable option to improve the fatty acid composition of eggs.

Abstract Superficial digital flexor (SDF) tendonitis is a leading cause of lameness in race and polo horses. Traditional therapies do not always prove effective in eliminating tendinopathy, thus culminating in tendon re-injury, persistent lameness, and performance shortfalls in affected horses. This study assessed therapeutic outcomes of conventional therapies versus extracorporeal shockwave therapy (ESWT), applied on a total of 18 race and polo horses affected with chronic tendinitis. The horses were divided into three groups (n=6): Group A (control, sham treated), Group B (blister treated), and Group C (shock wave treated with 500–1500 pulses, every two weeks for a total of 4-6 treatments). Follow-up clinical examinations revealed that warmth, swelling, and pain scores were significantly lower for the shock wave Group C (P<0.01). Shock wave therapy proved superior in providing an early analgesic effect (day 28) versus the late analgesia provided by blister group B. Lameness scores were likewise significantly lower for the shock wave group C (P<0.01), with an early return to full performance (day 84) versus delayed performance (day 98) and persistent lameness in the blister group B. Assessment of the injured, treated site, through color Doppler ultrasound at the maximum injury zone (MIZ), revealed significant Doppler signals suggestive of vascularization and early healing in the ESWT Group C (P<0.01) versus the absence of such signals in the blister and control groups (B and A), respectively. Conclusively, shockwave therapy proves to be superior to blistering, owing to its greater efficacy, humane approach, early regeneration, and lack of reported complications.

Abstract The aim of this study was to evaluate if amino acids (aa) or endogenous lipids may be used as unique oxidative substrates for porcine oocyte in vitro maturation (IVM). Immature porcine cumulus-oocyte complexes were in vitro matured in NCSU-37 (without pyruvate and glucose, negative control), NCSU-37+L-carnitine (lipid catabolism stimulator), NCSU-37+etomoxir (lipid catabolism inhibitor), NCSU-37+aa, NCSU-37+aa+salicylate (aa catabolism inhibitor), or NCSU-37+glucose (positive control). Oocyte nuclear and cytoplasmic maturation were determined by metaphase II rate and cleavage and blastocyst production rates, respectively. The use of endogenous lipids was determined by evaluating oocyte lipid content after IVM using Nile Red stain, while the use of aa was evaluated measuring ammonia production using spectrophotometry. The medium supplemented with L-carnitine showed a higher nuclear maturation rate (35.3%) compared to those without oxidative substrates (8%) or added with etomoxir (0%) and an increase in lipid consumption respect to the other treatments. Supplementation with aa during IVM resulted in a faster meiotic progression (52.4% of metaphase II) and a higher level of residual ammonia compared with the other groups. No significant differences, however, were detected for cytoplasmic maturation rates between oocytes matured in the presence of aa, L-carnitine or glucose. Our results indicate that both endogenous lipids or aa may be used as unique substrates for porcine oocyte metabolism during IVM, sustaining nuclear and cytoplasmic maturation.

Abstract The occipital region plays a critical functional role in the cranio-cervical junction through its involvement in head carriage, articulation with the atlas, muscle attachments, and balance control. Despite extensive research on canine cranial morphology, potential shape differences in the occipital region related to functional use have not been sufficiently investigated. The present study aimed to evaluate three-dimensional shape variation in the occipital region of mesocephalic dog skulls according to functional categories (hunting, guarding, shepherd/herding) using geometric morphometric methods. A total of 64 skull models were analyzed using 11 anatomical landmarks digitized on three-dimensional .stl files. Following Procrustes superimposition, Principal Component Analysis (PCA), Procrustes ANOVA, regression analysis, and Discriminant Function Analysis (DFA) were performed. PC1 and PC2 explained 23.715% and 13.676% of total shape variation, respectively. While Procrustes ANOVA revealed a significant difference in centroid size (p=0.0191), no statistically significant difference in shape was detected (p=0.2069). Regression analysis demonstrated a significant effect of size on occipital shape (p<0.0002), indicating that allometry is the primary determinant of variation. DFA results showed high misclassification rates (41.6–47.7%), further supporting the absence of distinct functional grouping. These findings suggest that the occipital bone and foramen magnum morphology in mesocephalic dogs are not significantly differentiated according to functional use. Instead, variation appears to be predominantly associated with allometric scaling and overall cranial size rather than specific task-related adaptations. The results emphasize the importance of considering multifactorial developmental and evolutionary influences when evaluating occipital morphology in domestic dogs.

Abstract Hypoderma sinense larvae, as tissue-invasive parasites, migrate through host tissues for months before completing their development in the subdermal tissue of the back. This migratory behavior induces lesions in surrounding tissues, compromising meat quality and economic value. To quantify the tissue damage and characterize the associated microbial communities, this study collected second- and third-stage larvae along with diseased tissues from infected yak skin.
A systematic analysis correlated these larval stages with parasite abundance and tissue damage extent. Histopathological examination evaluated structural changes in affected tissues, while bacterial isolates from lesion sites were obtained through culture-based methods. Bacterial species were identified by sequencing the 16S rDNA gene and conducting phylogenetic analysis.
Third-stage larvae caused significantly greater tissue damage in yaks than second-stage larvae. Histopathological analysis using hematoxylin-eosin and Masson staining revealed multi-layered lesions along the migration paths of third-instar larvae, extending from the epidermis to muscular layers. Key pathological features included: (i) infiltration of inflammatory cells, primarily neutrophils and macrophages; (ii) granuloma formation with fibrous encapsulation; and (iii) scar tissue deposition in perilesional areas.
Microbial analysis identified four bacterial genera—Escherichia, Enterococcus, Staphylococcus, and Bacillus—representing at least five distinct species, with a minimum of four being conditional pathogens. These bacteria vary in their involvement in lesion progression and may contribute to risks of secondary infection and disease transmission.
This study characterized the histopathological features and bacterial profiles of lesions associated with Hypoderma sinense third-stage larval infections, providing insights for clinical diagnosis and management of the disease and its associated microbial pathogens.

Abstract In chick embryos, liver histogenesis begins with an outgrowth of the foregut endoderm (hepatic diverticulum). To date, no studies have examined liver development during embryogenesis in turkeys; therefore, this research was undertaken to address this gap.
Fifty fertilized turkey eggs were placed in an incubator under controlled conditions to allow normal embryonic development. Embryos were sampled on days 7, 10, 15, 18, 21, 24, and 27 of incubation, with five eggs collected at each time point. The sections were stained with Hematoxylin and Eosin (H&E) for general structural observation. Histogenesis parameters were quantitatively evaluated. For functional analysis, the activities of liver specific enzymes namely ALT, AST, ALP, LDH and Rhodanese were measured in tissue homogenates using commercial colorimetric kits
By embryonic day 10, the first recognizable hepatic structures had emerged, including hepatocyte cords, sinusoidal endothelial cells, and the central vein. By day 15, the major hepatic cell populations, including Kupffer cells, were clearly distinguishable, and the liver exhibited a more mature tissue architecture.
Biochemical analysis of liver homogenates demonstrated dynamic changes in enzyme activities during late embryogenesis (days 18–27). The activities of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and rhodanese increased progressively throughout this period. In contrast, lactate dehydrogenase (LDH) activity declined consistently, whereas alkaline phosphatase (ALP) activity was lowest on day 18 and reached its maximum on day 24. These temporal changes likely reflect alterations in metabolic demands, energy utilization, and substrate availability accompanying hepatic maturation and the progressive acquisition of physiological functions by the embryonic liver.

Abstract Aquaculture has a fundamental role in supporting global food security. In response to the escalating human requirement for protein, the production of aquatic species is expanding rapidly. Conversely, aquatic animals are frequently subjected to a variety of husbandry stress, including handling and high stocking densities. They trigger physiological stress responses negatively affecting biological processes, such as somatic growth, reproduction, and immune function, ultimately resulting in inferior performance and high mortality rates. Although antibiotics are still extensively employed, their persistent administration is associated with the emergence of bacterial resistance, destabilizes endogenous microbiota, and causes chemical residues accumulation within aquatic environments. Therefore, the industry has shifted towards eco-friendly therapeutic strategies. In this context, plant essential oils have emerged as promising feed additives. The EOs contain a rich array of bioactive compounds, possessing anti-bacterial, anti-oxidant, and immuno-stimulatory activities. The administration of EOs has been evaluated in a wide range of aquatic species to characterize their therapeutic potential and establish optimal inclusion levels. Research findings indicate that plant EOs represent effective substitutes for antibiotics, playing prominent roles in growth promotion, oxidative stress mitigation, and immune modulation. The present context reviews the beneficial roles of essential oils in modern aquaculture.

Abstract Swine colibacillosis, caused by colonization of the piglet small intestine with enterotoxigenic strains of Escherichia coli, imposes a significant economic burden on the swine industry. Due to high mortality rates and the emergence of antimicrobial resistance, it is considered one of the major welfare concerns in the livestock industry. This study elucidated the genetic basis and expression patterns of key antibiotic resistance genes in porcine E. coli isolates with colibacillosis. Intestinal contents from five affected piglets were subjected to high-throughput sequencing, revealing 26 resistance genes across seven antibiotic classes. Four strains (428, 430, 432, 433) were further analyzed for mRNA expression levels of six resistance genes (blaTEM, tetA, tetB, tetM, qnrB, and qnrS) under exposure to ampicillin, tetracycline and ciprofloxacin at three concentrations (0.01, 0.001, 0.0001% w/v). Our findings revealed five distinct expression patterns: (1) dose dependent upregulation/downregulation, (2) inverse response to concentration changes, (3) maximal downregulation at intermediate dose, (4) minimal down-regulation at intermediate dose, and (5) sustained upregulation. Strain 428 uniformly followed pattern one for all resistance genes, in strain 430, tetB followed pattern three while blaTEM matched pattern one, strain 432 exhibited patterns two for all tetracycline- and β-lactam-resistance genes, and pattern four for qnrB; strain 433 showed patterns three for both fluoroquinolone-resistance genes, pattern four for tetB, and pattern five for blaTEM. These findings highlighted strain-specific and dose-dependent gene expression responses in swine colibacillosis. This work underscored the need for tailored antibiotic strategies, targeted interventions and well-validated monitoring frameworks for antibiotic resistance in veterinary.

Abstract Continuous exposure to pesticides may affect ovarian function and folliculogenesis by altered oxidative statute, steroidogenesis, and oocyte quality. Sweet apricot kernels (SAK) have been traditionally used as phytotherapeutic agents known for their broad pharmacological potential, including anti-oxidant, anti-inflammatory, and anti-cancer properties. This study aimed to assess the protective effect of SAK against ovarian toxicity induced by sub-chronic dietary exposure to a pesticide mixture, consisting of trifloxystrobin and thiacloprid, in female rats. Thirty-two female Wistar rats were randomly assigned into four groups of eight. Samples in 0.50 mL were given by oral gavage for 100 consecutive days. The control group received distilled water, SAK group included untreated rats received SAK extract (50.00 mg kg-1), Mix group received trifloxystrobin (0.30 mg kg^-1) + thiacloprid (0.20 mg kg^-1), and SAK + Mix group included Mix treated rats supplemented with SAK extract (50.00 mg kg^-1). Ovarian tissue samples were collected at the end of experiment for biochemical, histopathological, and immunohistochemical analyses. Pesticide mixture induced ovarian toxicity, characterized by a significant decrease in serum estradiol levels, ovarian total anti-oxidant capacity, ovarian follicle counts, Bcl-2 immuno-staining, and ovarian superoxide dismutase and catalase activities. In addition, ovarian malondialdehyde levels, serum interleukin-6 concentrations, and p53 immuno-staining were increased significantly. The co-administration of SAK extract significantly attenuated malondialdehyde, restored total anti-oxidant capacity levels, and modulated inflammation, estrogen levels, apoptosis-related proteins expression, and catalase and superoxide dismutase enzymes activities. Overall, these results suggest that SAK may protect against pesticide-induced ovarian toxicity through antioxidant, anti-inflammatory, and antiapoptotic mechanisms.

Abstract Tumors in cattle are uncommon, partly due to their relatively shorter lifespan, and vaginal neoplasms are rarely documented. Among vaginal and vulvar tumors, fibropapillomas are the most common, while leiomyomas, leiomyosarcomas, fibrosarcomas, and fibroleiomyosarcomas occur far less frequently. We describe the clinical presentation, surgical management, and pathological characterization of a vaginal fibroleiomyosarcoma in a 5-year-old Holstein cow. Clinical examination revealed a firm intravaginal mass attached to the dorsal wall. Surgical excision was performed under epidural anesthesia, followed by appropriate post-operative antibiotic and analgesic therapy. The cow recovered without complication, and no recurrence was observed during six months of follow-up. Macroscopically, the tumor measured 65.00 × 52.00 × 40.00 mm, and it was firm in consistency and gray in color. Histopathologically, it was composed of spindle-shaped neoplastic cells arranged in fascicles and bundles, displaying marked pleomorphism, blunt-ended nuclei, eosinophilic cytoplasm, and numerous mitotic figures. The stromal component showed variable collagen deposition, giving the lesion a biphasic fibromatous and leiomyomatous pattern. Immunohistochemically, neoplastic cells showed strong cytoplasmic positivity for α-smooth muscle actin and vimentin, confirming both smooth muscle and mesenchymal differentiation. Based on these findings, the tumor was diagnosed as a primary vaginal fibroleiomyosarcoma. To the best of our knowledge, this represents the second report of such a tumor in cattle and the first described in a Holstein cow. The case highlights the diagnostic value of combining histopathological and immunohistochemical analyses for accurate identification of rare bovine vaginal tumors.

Abstract Pasteurella multocida is a widespread zoonotic pathogen associated with severe hepatic inflammatory responses. Although traditional medicinal plants like Tribulus terrestris L. (TL) possess anti-inflammatory properties, their protective effects against P. multocida-induced liver injury remain insufficiently studied. This study established a P. multocida-induced liver injury model to evaluate the protective efficacy of TL extract. Thirty-two male Kunming mice were randomly assigned to four groups: control, model, penicillin-streptomycin, and TL extract. Interventions included daily intra-peritoneal injection of P. multocida (50.00 CFU mL^-1) followed by treatment with antibiotics or TL extract (1,000 mg kg^-1 daily) for 7 consecutive days. Therapeutic potential was evaluated using histopathological analyses (H&E and Masson's staining), RT-qPCR, western blot, immunohistochemistry, and immunofluorescence to assess inflammatory cytokines and NFκB pathway activation. TL extract significantly attenuated hepatic fibrosis and inflammatory cell infiltration. Following infection, NFκB pathway activation was indicated by up-regulated NFκB1 and down-regulated inhibitor of nuclear factor kappa B alpha (IκBα) expressions, alongside increased pro-inflammatory cytokines TNF-α and IL-1β. Treatment with antibiotics or TL reversed these changes. TL likely suppressed NFκB activation by down-regulating the tumor necrosis factor receptor superfamily member 1A (TNFRsf1a) receptor. Furthermore, decreased phosphorylation of IκBα and reduced TNF-α and IL-1β after treatment indicated effective inhibition of NFκB signaling and inflammation, effects comparable to antibiotic treatment. In conclusion, our research reveals that TL extract alleviates P. multocida-induced hepatic inflammation by inhibiting NFκB activation, highlighting its potential as a plant-derived therapeutic agent for bacterial liver injury.

Abstract Interstitial cells of Cajal (ICC) play a crucial role in regulating gastrointestinal motility, functioning as pacemakers in the digestive system of equids. This study described and compared the ICC populations in different regions of the stomachs of equids (horses, donkeys, and mules). A total of 30 healthy stomachs from horses, donkeys, and mules were evaluated using immunohistochemistry and high-resolution microscopy to assess various regions of the stomach in these species. The results showed the presence of ICC in all three equid species, with a predominance in the submucosa across the squamous mucosa, glandular mucosa, and pyloric antrum. The density and morphology of ICC varied slightly between species, although no significant differences were found. Among the equids studied, donkeys showed the lowest ICC density, while horses exhibited the highest in the evaluated tissues. Four cellular morphologies were identified, three of which were included in the analysis, as toluidine staining revealed that one corresponded to mast cells. Further studies are needed to elucidate dynamics and possible alterations in response to gastric pathologies.

Abstract Phthalates are endocrine-disrupting chemicals widely used in numerous consumer products, making human and animal exposure a significant public health concern. This study evaluated the protective effects of royal jelly (RJ) against dibutyl phthalate (DBP)-induced ovarian damage. Forty female Wistar albino rats were randomly allocated into eight groups (n = 5 per group): Control (corn oil), DBP (500 mg kg^-1), RJ (200 mg kg^-1), Quercetin (QCN; 50.00 mg kg^-1), DBP + RJ (100, 200 and 300 mg kg^-1) and DBP + QCN (50.00 mg kg^-1) for 28 consecutive days by gavage. QCN was used as a positive antioxidant control. The RJ was dissolved in normal saline, whereas, QCN and DBP were dissolved in corn oil. The levels of testosterone, dehydroepiandrosterone and tumor necrosis factor alpha in the serum were measured. Oxidative and nitrosative stress biomarkers (malondialdehyde, nitric oxide, total thiol molecules) were assessed in the ovarian tissue. The histopathological alterations of ovarian tissue and ovarian index were also evaluated. The RJ significantly reduced DBP-induced elevations in serum testosterone and dehydro-epiandrosterone levels. Compared to the DBP group, RJ treatment significantly restored the ovarian index and reduced serum tumor necrosis factor alpha level. The RJ treatment significantly attenuated DBP-induced oxidative and nitrosative stress in ovarian tissue. The DBP disrupted normal ovarian architecture, characterized by increased atretic and cystic follicles and reduced corpora lutea formation. These alterations were markedly ameliorated by RJ. In conclusion, RJ attenuated DBP-induced ovarian toxicity as evidenced by reduced oxidative/nitrosative stress, decreased serum tumor necrosis factor alpha and lowered serum testosterone as well as dehydroepiandrosterone levels.

Abstract This study aimed to determine whether the sperm acrosome-associated 1 (SPACA1) gene exhibits differential expression between X- and Y-bearing spermatozoa of Bali bulls and assess its potential as an RNA biomarker for sperm sexing. Sexed frozen semen (X or Y fractions) from Bali bulls was obtained from the Singosari National Artificial Insemination Center, Malang, Indonesia. Post-thaw semen quality was evaluated for motility, viability, abnormality, concentration, and plasma membrane integrity. Total RNA was extracted separately from X and Y fractions using easy-BLUE reagent, and SPACA1 expression was quantified by RT-qPCR using GAPDH as an internal control. Relative expression was calculated by the 2^−ΔΔCt method, and group differences were analyzed with the Mann-Whitney U test. The SPACA1 expression was numerically higher in X-bearing than Y-bearing spermatozoa (1.00 versus 0.51), but the difference was not significant. Thus, SPACA1 transcripts do not provide a reliable molecular marker for sperm sex differentiation. Future work should focus on protein-level characterization of SPACA1 and exploration of sex-biased surface proteins for immunological sexing. This research supports Sustainable Development Goal 2: Zero Hunger, by contributing to reproductive efficiency in livestock, and Sustainable Development Goal 12: Responsible Consumption and Production, through development of cost-effective, sustainable sperm sexing technologies.