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 Mycobacterium avium subspecies paratuberculosis (MAP), the causative agent of Johne’s disease and a potential contributor to Crohn’s disease, presents a significant challenge due to its resistance to conventional antibiotics. This necessitates the development of innovative strategies for prevention and treatment. This study aimed to evaluate the antibacterial activity of pathogen-specific antibodies derived from chicken egg yolks (IgY) and the postbiotics from lactic acid bacteria against MAP. Immunoglobulin Y antibodies were produced by immunizing hens with formalin-killed MAP strain antigens. The IgY was extracted, purified, and the anti-MAP titers were quantified by indirect ELISA. The Minimum Inhibitory Concentration (MIC) of different concentrations of specific anti-MAP IgY and the mixture of postbiotics (from four different probiotic strains Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus acidophilus, and Pediococcus acidilactici) individually and in combination against MAP was determined at various time intervals. Anti-MAP IgY titers in egg yolks increased within two weeks of immunization, reaching peak levels at six weeks. Growth inhibition assays revealed that postbiotics concentration as low as 6.25 mg mL-1 effectively inhibited MAP growth. Anti-MAP IgY demonstrated antibacterial activity with a MIC of 50 mg mL-1, while the combined IgY-posbiotics treatment achieved MAP growth inhibition at a MIC of 3.125 mg mL-1. The findings of the study suggests that combination therapy with specific IgY and postbiotics may be a promising preventive strategy for controlling MAP infections. Further in vivo studies are needed to elucidate the underlying mechanisms and optimize the application of this approach for broader use in veterinary and human medicine.

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 This study provides a comprehensive analysis of 145 urinary bladder stone cases diagnosed in dogs and cats over a five-year period at a private veterinary hospital. The aim was to evaluate demographic patterns, urolith composition, and species-specific trends to enhance understanding and improve management of urolithiasis in companion animals. A retrospective study was conducted on clinical records, including patient demographics (species, breed, age, sex), urolith composition, and stone size. Chemical composition of uroliths was determined using qualitative chemical analysis. Statistical analyses were performed to identify significant associations between patient demographics and urolith composition.
Of the 145 cases, 80 were dogs (55.2%) and 65 were cats (44.8%). In dogs, calcium oxalate stones predominated (65%), followed by struvite stones (30%) and mixed compositions (5%). Conversely, cats exhibited a higher prevalence of struvite stones (55%), with calcium oxalate accounting for 40% and mixed stones for 5%. Larger stones (10–20 mm) were more common in female dogs, while cats generally presented with fewer and smaller stones. No statistically significant temporal trend in urolith composition was observed for either species during the study period. Stone color distribution varied by species, with cream and brown stones being most common in dogs and cats, respectively.
This study establishes baseline epidemiological data on urolithiasis for this region, revealing significant species-specific differences in stone composition. The high prevalence of calcium oxalate in dogs and struvite in cats highlights the need for tailored clinical management and provides a crucial benchmark for future regional and global comparative studies.

Abstract Leishmaniasis, a disease caused by Leishmania species and transmitted via sandflies. Current control strategies against reservoir hosts and vectors are not eco-friendly. Using Harmaline (HA) from Peganum harmala, and meglumine antimoniate (MA) could be a promising therapy. The study aimed to explore the potential treatment outcomes and action mechanisms of HA and MA against L. major stages by investigating their effectiveness through molecular docking, anti-leishmanial effects, safety assessment, and apoptotic profile evaluation. According to the molecular docking results, the PLIP ligand interaction profiler (PLIP) identified that Bcl2 interacts with HA mainly through hydrogen bonds, while Bax uses both hydrogen and hydrophobic interactions, indicating a stronger binding of HA to Bax compared to Bcl2. HA, combined with MA (HA/MA) showed potent anti-leishmanial activity without toxicity. In vitro studies significantly demonstrated that HA inhibited the growth of promastigotes and amastigotes. HA/MA was more effective in inhibiting parasite growth. Based on the study findings, HA and HA/MA mixture can be considered a viable treatment option for cutaneous Leishmaniasis.

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 Sterilization in animals serves multiple purposes, such as behavior control, performance improvement, and population management. Chemical sterilization has emerged as a promising non-surgical alternative to traditional methods. This study aimed to investigate the effects of intra-testicular injection of zinc-doped carbon dots (Zn-CDs) nanoparticles as a chemical sterilant in mature rats. Twenty-five rats were randomly divided into five groups, including a control group without injection, a sham group receiving 0.50 mL distilled water, and three treatment groups administered respectively 0.50, 2.00, and 8.00 mg kg^-1 of Zn-CDs synthesized through a hydrothermal process. Following anesthesia with ketamine and xylazine, and aseptic preparation, intra-testicular injections were administered bilaterally. At 60th day post-injections, blood samples were collected to measure serum testosterone levels using chemiluminescence immunoassay. The rats were then surgically castrated to assess sperm parameters and testicular histopathology. Testicular oxidant/anti-oxidant status was also evaluated. The results revealed a dose-dependent reduction in sperm viability, membrane integrity, and motility, accompanied by increased sperm DNA damage. The highest Zn-CDs dose caused the most significant decrease in sperm concentration, as well as severe testicular tissue damage. In addition, anti-oxidant capacity, seminiferous tubules maturation, testosterone production, and spermatogenesis declined with increasing Zn-CDs concentrations in a dose-dependent manner. These findings indicate that intra-testicular injection of Zn-CDs effectively induces infertility in mature rats and holds potential as a chemical sterilization method. With further studies to evaluate safety and efficacy, this approach could be developed as a practical solution for large-scale in situ castration, offering a non-surgical alternative for over-population control programs.

Abstract Infectious bronchitis virus (IBV) is an important pathogen in the poultry industry causing avian infectious bronchitis that is, an acute but highly contagious disease affecting the upper respiratory tract, kidneys and reproductive tract. The 3rd passage of a polymerase chain reaction confirmed nephropathogenic IBV isolate was used for this study. Heat stability for 5, 10, 15, 20, and 30 min at 56.00 ˚C, pH sensitivity at pH 3.00, 7.00, 9.00, and 11.00 ultraviolet (UV) irradiation for 10, 15, 20, and 30 min, and chloroform sensitivity were studied. The IBV isolate was found to be susceptible to a temperature of 56.00 ˚C for 5 min and above, UV irradiation within 10 min, chloroform treatment and to pH 11.00 while being resistant to pH 3.00 and 9.00. The second part of the study investigated in vitro effectiveness of the disinfection potential of several commercially used disinfectants in Pakistan against the IBV isolate. For this purpose, Virkon S, Bromosept, and Beloran were employed for the virus inactivation test. Following the IBV challenge for contact time of 1, 5, 10, and 30 min, we counted the number of embryos that died after incubation. Results showed that suitable dilution of disinfectant for the recommended contact period could kill the virus. The maximum susceptibility was seen in the case of Virkon S which killed the virus in just 1 min. Thus, IBV could be killed using commercially available Virkon S, Beloran, and Bromosept after being used in recommended concentrations for recommended contact time.

Abstract Hepatic lipidosis (fatty liver) is a rare condition in dogs, primarily caused by improper feeding and care practices. It involves the accumulation of triacylglycerols in hepatocytes due to various toxic factors and dietary carbohydrate deficiencies. This case report describes the clinical, hematological, and pathological findings of an abandoned 8-year-old dog suffering from hepatic lipidosis that was referred to an animal shelter. The dog was unconscious, immobile, depressed, and severely obese. Pronounced abdominal distension and icteric mucous membranes were immediately noticeable during the initial examination. A blood sample was collected, and the case was referred to a veterinary hospital for diagnostic imaging. Unfortunately, before the referral could be completed, the dog suddenly died. Immediately following the death, a postmortem necropsy was performed to determine the cause. Necropsy revealed an extremely enlarged liver with a yellowish discoloration and abnormal kidneys with nodular lesions. Based on these findings, fatty liver was strongly suspected. Complete blood count and biochemical parameters, including triglycerides, cholesterol, aspartate transferase, alanine transaminase, and alkaline phosphatase, were subsequently evaluated. Complete blood count results indicated leukocytosis, thrombocytopenia, and an increase in red cell distribution width. Biochemical analyses revealed hypertriglyceridemia and elevated levels of alanine transaminase, aspartate transferase, and alkaline phosphatase. Histopathological examination confirmed severe fatty degeneration in the liver, alongside marked degenerative and necrotic changes in the kidneys. The findings of the present study contribute to the limited data on canine hepatic lipidosis and underscore the necessity of raising awareness about its clinical presentation, diagnostic markers, and histopathological characteristics.

Abstract This study compared the effects of a dose of human chorionic gonadotropin (hCG) administration at the time of insemination on the reproductive outcome of estrus-synchronized Mongolian ewes subjected to two artificial insemination methods during the breeding season. All females were treated with a polyurethane intravaginal sponge impregnated with 45.00 mg of flurogestone acetate for 12 days, followed by an intramuscular injection of 330 IU of equine chorionic gonadotropin at sponge removal. In Experiment 1, 150 ewes were inseminated using a laparoscopic intrauterine artificial insemination method 48 hr after sponge removal. The ewes were randomly assigned to the hCG group which received 500 IU of hCG at the time of insemination, and the control group which received 1.00 mL of sterile saline solution. In Experiment 2, 85 ewes were inseminated twice at 48 and 60 hr after sponge removal using a cervical artificial insemination method. The ewes were randomly assigned to the hCG group, which received 500IU of hCG at the time of the first insemination and the control group which received 1.00 mL of sterile saline solution. The pregnancy rate in the control group was not significantly different compared to the hCG group in Experiment 1 and the pregnancy rate in the control group was significantly higher compared to the hCG group in Experiment 2. In conclusion, the administration of hCG at the time of insemination could not be recommended in ewes when implementing a fixed time artificial insemination protocol during the breeding season.

Abstract Newcastle disease (ND) is a highly contagious and severe disease that affects birds, including domestic poultry, causing significant economic losses to the poultry industry. The disease is caused by the ND virus (NDV). Despite extensive vaccination efforts against NDV, controlling the disease remains challenging, primarily due to the emergence of new genotypes. Recent attention has focused on calcium phosphate nanoparticles (CaP NPs) as promising adjuvants for vaccines. This study aimed to design and construct CaP NPs containing inactivated NDV genotype VII and evaluate their safety and humoral immunogenicity in chickens. Following virus propagation in specific-pathogen-free eggs and inactivation with formalin, CaP NPs containing inactivated NDV were prepared using in situ and adsorption methods. The NPs were characterized for shape, size, polydispersity index and surface charge. Chickens were immunized subcutaneously with CaP NPs containing inactivated NDV and the humoral immune response against NDV was assessed using HI and enzyme-linked immunosorbent assays. Results showed a significant increase in the mean antibody titer against NDV in chickens treated with both NP structures compared to control groups. No significant difference was observed between the two NP preparation methods. The safety of CaP NPs containing inactivated NDV was confirmed. However, mucosal immunization with these NPs did not yield satisfactory results indicating the need for further research. Overall, this study confirmed the positive role of CaP as an adjuvant in enhancing the humoral immune response against NDV via injection and highlighted the need for continued research on mucosal immunization with the NPs.

Abstract A 4.5-year-old, nulliparous female New Zealand White Rabbit (Oryctolagus cuniculus) was presented for evaluation of a mammary mass located in right axillary mammary gland. The surgically excised mammary mass was submitted for histopathological and immunohistochemical evaluation. Histopathologically, neoplastic cells were arranged in sheets and nests of irregular sizes. The tissue was composed of amorphous eosinophilic material and necrotic cells. The neoplastic cells had round to oval nuclei with prominent nucleoli, scant basophilic cytoplasm and high mitotic activity. Cytokeratin and vimentin expressions of neoplastic cells were observed in immunohistochemical findings. Based on the microscopic characteristics and immunohisto-chemical staining, a diagnosis of a non-invasive comedocarcinoma was established. In this case, cytokeratin positivity confirmed the epithelial origin, whereas the vimentin positivity supported the presence of epithelial-mesenchymal transition and/or increased malignancy.

Abstract The amygdala is the nucleus of the brain that is largely responsible for perceiving danger and plays a role in emotion, behavior, control and learning. A small amygdala has been associated to aggression. Horned ewes are expected to be more aggressive and have a smaller amygdala. Both horned and hornless ewes exhibit intraspecific head-butting behavior and both species are at risk for traumatic brain injury. The aim of this study was to investigate the neuronal density, glial cells and blood-brain barrier (BBB) of the amygdala in horned and hornless ewes. Four horned and six hornless ewe heads (age: 16.00 ± 4.00 months) were obtained from the abattoir. The brains were carefully removed and preserved in 10.00% formalin for 5 days. Bilateral amygdalae were sectioned. The samples were stained with Hematoxylin and Eosin, immunohistochemical (glial fibrillary acidic protein) and Terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling methods, and the histological structures of the amygdala were examined by light microscopy. The Mann-Whitney U test was used to analyze the data. Neuronal density was estimated to be 143,230 ± 12,540 per mm3 in horned and 152,230 ± 18,430 per mm3 in hornless ewes. Horned subjects had reduced numbers of neurons, damaged BBB and localized inflammatory areas. More apoptotic neurons were observed in horned ewes. Further studies are needed to determine whether these differences in neuronal density, glial cells, and BBB are acquired (due to trauma) or congenital. The results of this study might need further similar studies to be conducted in the future.