Veterinary Research Forum

Abstract There is an increasing interest in exploring plant extracts as potential new treatment options for these ailments. Ziziphus vulgaris L. and Camellia sinensis (L.) Kuntze are medicinal plants widely used in different traditional medical practices, especially in Iranian Traditional Medicine, for addressing a range of health issues. Although their biological properties are well-established, their effectiveness against Giardia duodenalis cysts is still uncertain. This research aimed to assess the impact of Z. vulgaris and C. sinensis extracts on G. duodenalis cysts in vitro and to compare their effectiveness with metronidazole. Cysts were extracted from stool samples and concentrated using a 0.85 M sucrose solution. Extracts of Z. vulgaris and C. sinensis were prepared at concentrations of 25, 50, and 100 mg mL-1. The impacts of these extracts at different concentrations were evaluated at 10, 15, 30, 60, and 180 min, and the results were compared to control groups. The collected data were documented and statistically analyzed. The findings revealed that Z. vulgaris extract at a concentration of 100 mg mL-1and C. sinensis extract at a concentration of 50 mg mL-1 showed similar effectiveness to metronidazole in eliminating Giardia cysts. In addition, the cytotoxic effects of Z. vulgaris and C. sinensis extracts, in comparison to metronidazole, indicated a rise in fatality rates with prolonged exposure times and higher extract concentrations (p < 0.05). Therefore, it can be concluded that extracts of Z. vulgaris and C. sinensis are as effective as metronidazole for killing Giardia cysts in vitro.

Abstract This study evaluated the protective effects of intraperitoneal tannic acid (TA) against ischemia-reperfusion (I/R) injury in a rat model of testicular torsion. Eighteen adult male Wistar rats were randomized into three groups (n=6 each): sham (surgery without ischemia), I/R (3 hr ischemia + 3 hr reperfusion), and I/R+TA (TA 50 mg kg-1; 100 µL i.p. 30 min before reperfusion). Testicular tissues were sampled immediately after reperfusion for biochemical assays to measure malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione peroxidase (GPx) levels. Epididymides were analyzed 60 days later for sperm count, motility, viability, morphology, and DNA integrity. I/R significantly elevated MDA and sperm DNA damage while reducing SOD, GPx, sperm parameters, testicular weight and spermatogenesis. TA administration reversed these changes and restored the parameters to levels close to those of the sham group. Overall, intraperitoneal TA mitigated I/R-induced oxidative stress and preserved reproductive function, indicating its potential therapeutic value in testicular torsion.

Abstract Testicular ischemia-reperfusion (I/R) injury during testicular torsion is strongly influenced by oxidative stress caused by excessive accumulation of uncaptured reactive oxygen species (ROS). Kisspeptin-10, a biologically active fragment of the kisspeptin peptide family, has demonstrated significant antioxidant and anti-apoptotic properties. Recent studies indicate that kisspeptin-10 can mitigate oxidative stress by reducing reactive oxygen species levels and enhancing the activity of endogenous antioxidant enzymes such as superoxide dismutase (SOD) and catalase. This study examined the effects of kisspeptin-10 on I/R injury in testicular torsion/detorsion (T/D) of male rats. Twenty male rats were divided into four groups: the control group and three treatment groups (720° T/D, 720° T/D+ 0.50 µg kg-1 kisspeptin-10, 720° T/D+ 1.00 µg kg-1 kisspeptin-10). After inducing 720° clockwise testicular torsion for 2 hr, various factors such as sperm parameters, number, total motility, viability, DNA damage and hypoosmotic test were evaluated. The results showed that 720° T/D can increase sperm DNA damage. In addition, it also had negative effects on overall motility and other properties such as viability and plasma membrane functionality. The results also showed that administration of kisspeptin-10 to T/D rats can reduce DNA damage. These effects could also increase overall motility, viability and plasma membrane functionality compared to the T/D group. Based on our results, kisspeptin-10 provides significant protection against acute T/D injury to the testis when administered after spermatic cord torsion in rat.

Abstract Candida albicans is a commensal opportunistic yeast colonizing the skin and mucosal surfaces of humans and animals, which, under specific predisposing conditions, can proliferate excessively and lead to clinical manifestations of candidiasis. The widespread prophylactic and therapeutic use of antifungal agents has led to the emergence of drug-resistant strains, necessitating the exploration of novel therapeutic alternatives. Considering the clinical and conformational significance of equine skin health, the present study aimed to evaluate the therapeutic efficacy of ethanol extract of propolis in an experimental model of cutaneous candidiasis in horses. In 2022, two clinically healthy, six-year-old female horses (approximately 400 kg) were selected for the study. Immunosuppression was induced using dexamethasone and four intradermal inoculation sites were created on the shaved thoracic and flank regions of both lateral aspects using C. albicans (CPTC: 5027) suspension (5 × 10⁶ CFU mL-1). The experimental sites on the right thorax were treated with ethanol extract of propolis while those on the left thorax received topical nystatin. Lesions on the right flank were treated with glycerin as a vehicle control, and lesions on the left flank were left untreated. Cutaneous candidiasis was successfully induced within five days post-inoculation. Clinical resolution was observed following five days of treatment with ethanol extract of propolis, whereas, nystatin required eight days to achieve complete lesion resolution. The findings of this study suggested that topical ethanol extract of propolis demonstrated superior efficacy in accelerating the resolution of C. albicans-induced cutaneous lesions in horses compared to nystatin.

Abstract Corneal ulcers, particularly from alkaline chemical injuries, are a leading ocular complication in animals, accounting for about 20% of emergency referrals. These burns damage the conjunctiva and cornea, causing deep ulcers, potential perforation, delayed healing, infections, and blindness. Prompt treatment is crucial to accelerate healing and minimize complications like permanent opacity. This study compared the efficacy of blood serums (autologous, homologous, heterologous) against standard treatment (N-acetylcysteine and ciprofloxacin eye drops) and a control group (Normal saline) in treating induced corneal ulcers in two groups consisting of 10 healthy mixed-bred female dogs. Ulcers were created using 3M sodium hydroxide-soaked filter paper on both eyes. Evaluations at 1, 7, 14, and 21 days assessed quantitative parameters (corneal/stromal thickness, ulcer size) and qualitative symptoms (photophobia, blepharospasm, discharge, congestion, edema, neovascularization, opacity) via ultrasonography and clinical exams, respectively. Results showed significant time- and treatment-dependent improvements. Heterologous serum notably reduced thickness and ulcer size from day 7 compared to others. By days 14-21, all treatments (serums and drugs) lowered stromal thickness versus control. Qualitative parameters improved significantly with all treatments from day 7. These findings support the use of blood-derived serums, especially heterologous serum, to accelerate healing and improve clinical outcomes in canine corneal ulcers.

Abstract This study aimed to examine the effects of varying levels of L-carnitine and omega-3 and omega-9 fatty acids on performance, blood parameters, and testicular histomorphometry in fattening male Moghani lambs. Thirty-five five-month-old male Moghani lambs (27 ± 600 kg) were individually housed and randomly assigned to five dietary treatments. Diets included a control and four diets containing 3% calcium salts of omega-3 or omega-9 fatty acids with 100 or 200 mg/kg DMI of L-carnitine. Lambs were fed for 75 days to evaluate growth performance, nutrient digestibility, blood metabolites, carcass traits, and testicular histomorphometry.The results indicated significant effects on daily weight gain during the second month, average weight gain in the second month, and feed conversion ratio in the third month (P<0.05). However, glucose, total cholesterol, LDL cholesterol, triglycerides, blood urea nitrogen, and HDL cholesterol levels on day 30, as well as triglyceride levels on day 60, were not significantly influenced by dietary treatments. Blood urea nitrogen and HDL cholesterol levels, along with total protein, were significantly affected on day 60, with dietary treatments having a marked influence (P<0.05). Liver enzyme activities, including aspartate aminotransferase (AST) and alanine aminotransferase (ALT), were significantly affected during the first 30 days of the study (P<0.05), while alkaline phosphatase (ALP) activity remained unaffected over the 30 and 60-day periods. Testicular histomorphometry showed significant effects of dietary treatments on spermatogenesis index, seminiferous tubule diameter, and Leydig cell count (P<0.05).

Abstract The present study was conducted to investigate the effect of L-carnitine (LC) supplementation in diets containing different levels of fish oil (FO) on performance, egg quality parameters and egg yolk fatty acids (FAs) profile in aged laying hens. In this study, 432 laying hens (Hy-line-W36, 65 weeks of age) were used allocated in a 3 × 3 factorial design with 6 replications and 8 birds per replication. The experimental treatments included diets containing 3 levels of FO (0, 1.5 and 3% of the diet) and 3 levels of LC (0, 300 and 600 mg/kg of the diet). The results showed that hens treated with 3% FO and 300 and 600 mg LC had the highest egg production rate, egg weight and egg mass, which was significantly higher than the treatments without FO and LC. With increasing usage level of FO to 3% of diet, egg yolk pH decreased significantly (p <0.05), however, yolk pH increased when diet supplemented with 600 mg LC. The percentage of polyunsaturated FAs (PUFA), the ratio of PUFA/saturated FAs (SFA) and the percentage of omega-3 FAs increased significantly with increasing FO usage level in the diet (p <0.05). Therefore, the use of LC in a diet containing FO not only increased egg production performance and egg internal quality (Haugh unit and yolk color) of aged laying hens, but also increased the production and deposition of omega-3 FAs in egg yolk.

Abstract The current finding reports the development of iELISA with recombinant AMA-1 (rAMA-1) to identify an infection of Babesia in naturally infected cattle. The 48 kDa protein-encoding recombinant AMA-1 gene was cloned into the pET-28a (+) expression vector and expressed in E. coli. The resulting congregate protein was refined under native settings. Towards the evaluation of the diagnostic potential of AMA-1 as a sero-diagnostic reagent, a panel of sera samples from Babesia infected cattle; uninfected sera as well as Babesia positive samples with other species including B. bigemina, B. divergens, B. major, B. occultans were utilized. Additionally, the efficacy of rAMA-1-based serological assays was compared with commercially available kits using 200 samples taken from cattle suspected of babesiosis. The results demonstrated that the iELISA using rAMA-1 exhibited a diagnostic sensitivity of 88.89%, when compared to commercially available ELISA kit as the reference test. The specificity of this assay was 76.66%. These findings suggest that the iELISA employing rAMA-1 can be utilized on large-scale epidemiological surveys and clinical detection of Babesia infection in cattle.

Abstract Genetic variations within a partial region of the growth hormone (GH) gene and their associations with growth traits were investigated in European sea bass (Dicentrarchus labrax) using DNA sequencing. Five haplotypes and ten novel single nucleotide polymorphisms (SNPs) were identified in the GH gene of European sea bass. In silico analysis revealed two amino acid substitutions between the reference and our partial protein sequence, specifically S24L and S42T. Both variations were located within the α1 helix, which also contains H34, a zinc-binding residue. The genotypes at the g.1611T>C locus of the GH gene were found to be significantly associated with total weight, fillet weight, and head length. Additionally, the association between GH g.1557A>T genotypes and both preanal and abdominal lengths was statistically significant (P < 0.05). Similarly, the genotypes of g. 1857 C>T loci having the synonymous mutation in phenylalanine amino acid were significantly associated with standard length (P < 0.05). HAP4 reported the highest weight and length traits than the other haplotypes (P<0.05). It is suggested that HAP4 could be used as a potential genetic marker for selective breeding programs of European sea bass under Mediterranean conditions.

Abstract Non-typhoidal Salmonella (NTS) is a predominant etiological agent of foodborne infections globally. Salmonella serovars are present throughout the chicken's digestive tract, particularly in the ceca. This study, conducted from October 2024 to April 2025, employed cultural methods to isolate and identify Salmonella from samples collected at the broiler chicken wet market in Sulaymaniyah province. The research additionally examined the prevalence of Salmonella enterica serovar Typhimurium in these samples by polymerase chain reaction (PCR). 210 samples were obtained from the cecum, cutting boards, knives, body swabs, workers' hands, and water. Salmonella was isolated from the samples and identified by culturing and molecular techniques. Consequently, 103 individuals (49%) tested positive for Salmonella via culture. The PCR results revealed that the contamination rate for Salmonella enterica was 74 (71.85%); 47 (63.51%) were S. Typhimurium, and 33 (70.22%) of S. Typhimurium possessed the hilA gene. The highest prevalence was observed in chopping boards, with 91.67% for S. enterica, whereas the maximum rate for S. Typhimurium was recorded in water, at 100%. The phylogenetic tree indicates that the Iraqi isolate with accession number PV250092 belongs to the principal group of isolates, exhibiting a bootstrap support value of 100%, signifying a robust genetic association with them. To conclude, this research demonstrated a notable prevalence of Salmonella in the broiler chicken processing environment, particularly a high incidence of S. Typhimurium isolates. The placement of the Iraqi isolate within the global isolate clade suggests a recent common ancestor with other isolates, indicating global transmission pathways.

Abstract This study investigated the effects of zinc oxide nanoparticles (ZnO-NPs) on the growth, blood parameters, skin mucus, and serum biochemistry of juvenile common carp (Cyprinus carpio). A total of 300 fish with an average weight of 12 ± 0.33 g were randomly distributed in 12 fiberglass tanks with a capacity of 300 L and fed with diets supplemented with ZnO-NPs at concentrations of 0 (control), 10, 50, and 100 mg kg-1 for 60 days. The results indicated that 50 mg kg-1 ZnO-NPs supplementation significantly improved fish weight, length, specific growth rate, and feed conversion ratio (p<0.05). The highest survival rate was observed in the treatment with 50 mg kg-1 ZnO-NPs (p>0.05). This treatment also led to the lowest red blood cell counts but the highest hemoglobin levels, mean corpuscular hemoglobin concentration, white blood cell counts, and lymphocyte percentages (p>0.05). In contrast, the highest neutrophil counts were observed at 100 mg kg-1 ZnO-NPs (p<0.05). The highest serum biochemical including total immunoglobulin, lysozyme, and zinc levels were observed in the treatment with 50 mg kg-1 ZnO-NPs (p<0.05). While, in the skin mucus, total immunoglobulin, lysozyme, and alkaline phosphatase significantly increased at groups fed with 10 mg kg-1 ZnO-NPs (p<0.05). Overall, the findings suggest that dietary inclusion of 50 mg kg−1 ZnO-NPs can effectively promote growth and enhance immune responses of juvenile C. carpio, indicating their potential application in aquaculture practices. However, caution is advised at higher concentrations due to potential adverse effects on fish health.

Abstract This study aimed to identify and characterize nitrifying bacteria and their associated bacteriophages from swiftlet house environments as a preliminary step toward developing biological control strategies for nitrate contamination in edible bird nests—a food safety concern due to excessive nitrite and nitrate accumulation. A total of 80 environmental samples were collected between October and November 2024 from four swiftlet houses in Central Kalimantan, Indonesia, including 20 samples each from feces, pond water, soil, and edible bird nests (Aerodramus fuciphagus). Phenotypic characterization (culture, Gram staining, biochemical tests) and genotypic identification (PCR and 16S rRNA gene sequencing) were performed. Bacteriophages were screened using spot tests and plaque assays. The study resulted in the isolation and identification of 15 Nitrobacter isolates. Molecular analysis confirmed all isolates belonged to the Nitrobacter genus. One isolate, Nitrobacter AP6, was selected as a representative host for phage screening due to its consistent growth and plaque clarity. Spot tests revealed four bacteriophages capable of lysing Nitrobacter AP6. These findings demonstrate the feasibility of isolating nitrifying bacteria and phages from swiftlet environments and provide a foundation for future biocontrol development, though further in vitro and in situ validation is required to assess effectiveness and safety.

Abstract Bone tissue engineering offers a potential alternative to conventional grafting by combining biocompatible scaffolds with biological stimulants. Polycaprolactone (PCL) is a biodegradable polyester with good mechanical strength and biocompatibility. Hydroxyapatite (HA) is a calcium phosphate mineral that is a major component of bone and has excellent bioactivity and biocompatibility. This research investigated the histopathological effects of PCL-HA nanocomposite scaffolds along with their combination with platelet-rich fibrin (PRF), on the regeneration of bone in rabbit calvarial bone defects. Four circular full-thickness bone defects of 5.00 mm in diameter were created on the calvarial bone of 15 male New Zealand white rabbits. Three defects were filled with PRF, PCL-HA, and PCL-HA/PRF, and one defect was served as a control with no filler. Histopathological evaluations were conducted at 4-, 8-, and 12-weeks post-implantation. Data were evaluated using the Kruskal-Wallis and Mann-Whitney U tests. Significant differences were observed between the treatment and control groups regarding bone regeneration throughout all 12^th weeks studied. In the 4^th weeks, no significant differences in bone regeneration were noted among the treatment groups. In the 8^th weeks, most new bone formation was observed in the PCL-HA/PRF group. Both the PCL-HA and PCL-HA/PRF groups significantly improved bone regeneration compared to the control and PRF groups, with the PCL-HA/PRF group demonstrating the greatest bone formation, and vascularization and the lowest inflammation by the 12^th week. Thus, PCL-HA/PRF could be considered as a suitable alternative to bone grafts and could be increasingly utilized in orthopedic surgery and bone tissue engineering.

Abstract Bovine tuberculosis is a chronic bacterial disease primarily caused by Mycobacterium bovis, a member of the Mycobacterium tuberculosis complex (MTBC), with significant zoonotic implications. This study aimed to detect MTBC in wildlife species, specifically nilgai (Boselaphus tragocamelus) and sambar deer (Rusa unicolor), using gross pathology, histopathology, acid-fast staining, and molecular confirmation. Necropsied tissue samples were collected during post-mortem examination of a nilgai and a sambar deer from the Assam State Zoo, Guwahati, India. Macroscopically, multiple granulomatous tubercles of varying sizes were observed in the lungs and liver, with creamy white caseous material marked upon sectioning. Ziehl-Neelsen staining of the tissue smears from granulomatous lesions confirmed the presence of acid-fast bacilli. Microscopic examination of tuberculosis granulomas revealed a central necrotic mass surrounded by inflammatory cell infiltration, including Langerhans-type giant cells. Molecular confirmation of MTBC infection was achieved by amplifying hsp65 and IS1081 in tissue samples, further validated by Basic Local Alignment Search Tool for nucleotide analysis following Sanger dideoxy sequencing. In conclusion, this study confirmed the presence of tuberculosis in these wildlife species through an integrated approach combining pathology, microbiology, and molecular diagnostics, highlighting the need to understand pathogen entry into the herd and prevent potential spillover.

Abstract Streptococcus agalactiae is a Gram-positive bacterial pathogen accountable for disease outbreaks in tilapia, resulting in substantial economic losses in aquaculture. In this study, a 10-round whole-cell Systemic Evolution of Ligands by Exponential Enrichment procedure was conducted to isolate and characterize single-stranded DNA aptamers with specific binding affinity to S. agalactiae. The enriched single-stranded DNA libraries were sequenced by high-throughput sequencing, yielding a total of 180,987 reads, of which 126,933 included unique aptamer sequences. The most frequent sequences were selected for further analysis. Among them, SA1 had the highest frequency, with 231 reads, whereas SA2 showed a lower number of 16 reads. These findings underscored the validity of combining whole-cell Systemic Evolution of Ligands by Exponential Enrichment, high-throughput sequencing and aptamer-structure analysis to explore the interaction between aptamers and bacterial cell surfaces. The selected aptamers not only showed promise for the detection of S. agalactiae but also served as molecular probes for identifying surface-exposed proteins, studying host-pathogen interactions and guiding targeted drug delivery in aquaculture-related applications.

Abstract Selenium (Se) and gold (Au) nanoparticles (NPs) possess unique properties such as high absorption ability, low toxicity and minimal environmental persistence. These properties, combined with demonstrated anti-parasitic effects against certain parasites, suggest their potential as candidate for house fly control. This study aimed to evaluate the effectiveness of Se and AuNPs in controlling house flies. We investigated the insecticidal effect of Se and AuNPson Musca domestica using a dipping method. Several concentrations (20.00, 60.00, 100, 400, and 1,000 ppm) were prepared using deionized water. Fifteen larvae per concentration were tested, with each test repeated three times. The larvae were dipped in the NP solutions for 30, 60 and 90 sec in 10.00 mL of the tested concentrations. Distilled water was used for the control group. Larval mortality was recorded 24 hr post-treatment. No mortality was observed in house fly larvae 24 hr after exposure to different concentrations of Se and AuNPs, regardless of the incubation time. Thus, Se and AuNPs showed no insecticidal effect on house fly larvae. In conclusion, Se and AuNPs cannot be used to control house flies. Due to the issues associated with chemical control, it is crucial to search for alternative pesticides.

Abstract Physalopteridae nematodes pose a threat to a broad spectrum of animals including amphibians, reptiles, birds and mammals. The current study was the first molecular and phylogenetic characterization of Physaloptera clausa parasitizing long-eared hedgehogs (Hemiechinus auritus) in Iran. A male road-killed H. auritus was collected from Shahriar City, Tehran province in May 2022 and it was subjected to necropsy. After isolating parasites, they underwent morphological analysis using a light microscope and an identification key. For molecular analysis, the genomic DNA was isolated using the FavorPrepTM Tissue Genomic DNA Extraction Mini Kit. The PCR products were sequenced, the sequence data were analyzed and multiple alignments were conducted using the Clustal Omega. For phylogenetic analysis, these nucleotide sequences were aligned in MEGA 11 based on the lowest bayesian information criterion score. A cluster of parasites was found on the mucosa of the hedgehog stomach. All the nematodes were identified as P. clausa. The sequence obtained in this study has been submitted to GenBank^® with the accession number OR088573. The phylogeny analysis revealed that the genus Physaloptera formed a major clade where P. clausa was clustered with various Physaloptera species, closely related to Turgida, Physalopteroides and Skrjabinoptera genera. Our study specified the necessity for additional taxonomic and phylogenetic research on Physaloptera species and related genera to fully understand the evolutionary dynamics and ecological significance of these parasites.

Abstract The excessive production of reactive oxygen species during the freezing and thawing process triggers lipid peroxidation in the sperm membrane, resulting in oxidative harm and a decline in semen quality. The aim of this study was to examine the impact of different concentrations of canthaxanthin on the quality of goat spermatozoa throughout the process of cryopreservation. Semen was collected from each goat using an artificial vagina for a continuous duration of 3 weeks. The semen was divided into five equal portions and mixed with an extender having different concentrations of canthaxanthin: 0.00 (control), 5.00, 15.00, 25.00, and 35.00 µM. The computer-assisted semen analyzer was used to determine the sperm motility and kinetic parameters. The findings indicated that the inclusion of canthaxanthin at a concentration of 5.00 µM resulted in a minimal enhancement in sperm kinematic factors such as overall motility, progressive motility, fast progressive motility, curve-line velocity, distance curve line, amplitude of lateral head displacement, beat-cross frequency, wobble, linearity, and straightness. Nevertheless, these enhancements did not demonstrate statistical significance compared to the control, 15.00, 25.00, and 35.00 µM. The introduction of 5.00 µM of canthaxanthin resulted in a significant rise in straight-line velocity, average path velocity and head activity compared to the control group. Hence, the addition of 5.00 µM of canthaxanthin could be employed to improve the cryopreservation of goat spermatozoa.

Abstract To mitigate metabolic disorders and skeletal abnormalities associated with rapid growth in broilers, and to reduce reliance on antibiotics, strategies involving feed restriction and non-antibiotic dietary additives are increasingly explored. This study investigated the effects of various feeding methods and additives on weight gain, feed intake, feed conversion ratio, carcass characteristics, intestinal morphology and ileal digestibility in broiler chickens. The research involved 480 one-day-old Ross 308 male broiler chickens. A 2 × 5 factorial experiment, arranged in a completely randomized design, incorporating two feeding methods (without and white quantitative restriction) and four types of additives including thyme essential oil (300 g per ton), oregano essential oil (300 g per ton), equal weight ratio of thyme and oregano essential oil (300 g per ton) and probiotic (250 g per ton) along with control diets (without additives). Dietary supplementation with oregano essential oil significantly increased weight gain compared to the other additive groups. While the feeding method, additives and their interactions significantly influenced feed intake in chickens and the feed conversion ratio was primarily affected by the type of additive. Supplementation with any of the tested additives led to reduced total feed intake and improved final feed conversion ratio compared to the control group. Histological analysis revealed that chickens receiving oregano exhibited the highest villus height to crypt depth ratio. In conclusion, this research suggested that quantitative feed restriction, particularly when combined with specific dietary additives such as oregano essential oil could significantly improve broiler growth performance, feed efficiency and intestinal morphology, offering potential alternatives to conventional practices.