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 Bovine tuberculosis (bTB) 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 Assam State Zoo, 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 tissue smears from granulomatous lesions confirmed the presence of acid-fast bacilli. Microscopic examination of TB 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 nBLAST analysis following Sanger dideoxynucleotide 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 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 three weeks. The semen was divided into five equal portions and mixed with an extender having different concentrations of canthaxanthin: 0.00 µM (control), 5.00 µM, 15.00 µM, 25.00 µM, 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 multiple factors associated with sperm motility, 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 µM, 25.00 µM, 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 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 Menthol is an organic compound derived from mint oils, known for its cooling and soothing properties, often used in various pharmaceutical, cosmetic, and therapeutic applications. It exerts beneficial effects on the digestive system by relaxing smooth muscles, reducing spasms, and improving gastrointestinal motility. This study aimed to evaluate the effects of menthol on bovine ileal smooth muscle contractions in vitro. Ileal tissue segments were collected from adult cattle at an abattoir and subsequently immersed in 25 mL organ baths containing Tyrode’s solution. The baths were maintained at 37 °C and continuously aerated with a gas mixture of 95% O₂ and 5% CO₂. The tissues were subjected to various contractile agents, including potassium chloride (KCl) at concentrations of 30 and 80 mM, carbachol (CCh) at 1 μM and 4 μM, and barium chloride (BaCl₂) at 30 mM. Menthol was cumulatively applied in incremental concentrations to assess its modulatory effects on contraction amplitude. Results demonstrated that menthol elicited a dose-dependent inhibition of smooth muscle contractions across most stimulatory conditions, with the extent of inhibition varying among different stimuli. The Ca2+ channel blocking activity was further confirmed when pre-treatment of isolated ileums with menthol (23 and 200 μg mL⁻¹) caused a rightward shift in the Ca2+ concentration-response curves (CRCs), similar to verapamil. These findings suggest menthol’s spasmolytic action may be mediated through the modulation and inhibition of calcium channels. Menthol effectively attenuates bovine ileal smooth muscle contractions in vitro, indicating its potential as a natural therapeutic agent for controlling gastrointestinal hyperactivity in cattle.

Abstract Porcine parvoviruses (PPVs) are globally recognized as significant contributors to porcine reproductive failure, primarily characterized by fetal death. It can cause infection in pregnant sows which can result in severe reproductive issues, including stillbirth, mummification, embryonic death, and infertility (SMEDI). An RPA assay targeting the variable region of the outer capsid protein gene of the PPV-7 genome was developed and systematically optimized under different reaction conditions. The assay exhibited optimal amplification performance at a constant temperature of 35 °C for 25 minutes, using 0.72 µM of each forward and reverse primer and 14 mM magnesium acetate (MgOAc). This assay demonstrated high sensitivity, detecting as few as 2050 copies of viral nucleic acids in both the conventional and fluorescent dye-based formats. The assay demonstrated high specificity, showing no cross-reactivity with other common porcine pathogens such as porcine sapelovirus, porcine circovirus, and classical swine fever virus.Of the 167 field samples tested, 23 were found to be positive for PPV-7, yielding a positivity rate of 13.7%. Operating at a low, constant temperature, the assay eliminates the requirement for advanced laboratory equipment, making it ideally suited for pen-side diagnostics in field conditions. In conclusion, this novel assay demonstrates strong potential for field-based detection of PPV-7 circulating within the swine population of Haryana, India, marking the first report of its kind from this region. Further validation using samples from clinically affected herds will strengthen its diagnostic applicability.

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 calculation of the cranial cavity volume is important for brain development, pathologies, and health status. In recent years, advancements in clinical applications have made volume calculation methods from radiological images more consistent. In this context, it was aimed to calculate the intracranial volume of the Koçeri sheep's skulls using 3D models based on computed tomography scan (CT scan) sections and stereology. In the study, 7 adult female and 7 male Koçeri sheep were used. The skulls used were scanned with a 64-detector Multi-Detector Computed Tomography device with a section thickness of 0.625 mm, 80 kV, 200 mA, and 639 mGy specifications. Using the MIMICS 20.1 software, three-dimensional models of the skull region were created and intracranial volumes were measured. The same reference points were measured using the Cavalieri principle. As a result of the study, measurements taken using CT scan showed that the cranial volume in females was 133.09 ± 7.81 cm³ and in males was 148.68 ± 16.25 cm³. The intracranial volume calculated using the Cavalieri principle was found to be 133.87 ± 8.43 cm³ in females and 149.77 ± 16.74 cm³ in males. The data were statistically analyzed in terms of two methods and sexual dimorphism. While no difference was found between the methods, a significant statistical difference was determined between the genders. In this context, our study is believed to make significant contributions to clinical sciences through accurate management of cranial disease diagnosis and processes, and to taxonomy through identification of interspecies differences.

Abstract Testicular ischemia-reperfusion is accompanied by elevated production of reactive oxygen species. It has been reported that reactive oxygen species are highly reactive to cellular carbohydrates, DNA, lipids and proteins, and result in testicular ischemia-reperfusion injury. Gastrodin is the principal active ingredient isolated from the medicinal plant Gastrodia elata Blume and has anti-oxidative stress effect. In this study, we explored the potential protective activity of gastrodin in rat testicular ischemia-reperfusion injury model and underlying mechanism. Male rats were randomized into 3 groups (sham control, testicular ischemia-reperfusion injury, testicular ischemia-reperfusion injury along with gastrodin injection) (n = 20). Testicular ischemia-reperfusion injury group received 2-hour period of left testicular torsion (720°, counterclockwise) and 4-hour or 3-month period of testicular detorsion. At the onset of testicular detorsion, gastrodin-treated rats were given 100.00 mg kg-1 gastrodin by intraperitoneal route. Following testicular detorsion, testicular tissues were collected for enzymatic activity analysis, oxidative stress evaluation and histopathological examination. The ipsilateral testicular xanthine oxidase activity (source of reactive oxygen species production) and malondialdehyde level (a precise biomarker of reactive oxygen species) were significantly increased in testicular ischemia-reperfusion injury group versus sham control group, while testicular spermatogenic function was decreased. Furthermore, gastrodin administration reduced xanthine oxidase activity and malondialdehyde level in ipsilateral testicular tissue, while improving testicular spermatogenic function. Consequently, it is suggested that gastrodin plays a protective role in testicular torsion/detorsion-induced ischemia/reperfusion injury through inhibiting xanthine oxidase activity to decrease reactive oxygen species formation.

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 Polycystic kidney disease (PKD) is a hereditary disorder characterized by progressive renal cyst formation, leading to compromised kidney function and an increased risk of chronic kidney disease (CKD). This prospective, 18-month longitudinal study evaluated diagnostic and prognostic markers in 12 PKD-affected cats, utilizing ultrasound imaging alongside biochemical analysis. The study documented a 14% increase in cyst volume, a 7% enlargement in kidney length, and significant elevations in plasma creatinine, phosphorus, and blood urea nitrogen levels (P < 0.05), underscoring their correlation with disease progression. Hematological analysis revealed progressive anemia associated with advanced CKD stages, further delineating the systemic impact of PKD.
These findings underscore the progressive nature of CKD in cats with PKD, even with diligent care provided by pet owners and veterinarians. Furthermore, the importance of routine sonogeraphic evaluation along with hematological and biochemical assessments is highlighted in our study to enhance early detection and improve outcomes in feline PKD. Future longitudinal studies with expanded cohorts are essential to validate these findings and refine diagnostic criteria for PKD-associated CKD.

Abstract This study is the first to apply the BIENE protocol to assess dairy ewe welfare in intensive farms in Algeria. A total of 1,964 ewes from 50 farms in the Algiers region were evaluated between December 2022 and March 2023 using 13 animal-based indicators related to nutrition, housing, health, and behavior. Moderate tooth loss affected 54.67 ± 3.32% of ewes, and 20.38 ± 2.78% had a low body condition score (BCS < 2). Housing problems included fleece moisture (11.28 ± 2.55%) and over 40% soiling of flanks and hindquarters. Health issues such as mild anemia (23.55%), lameness (26.45%), nasal discharge (35.47 ± 4.43%), and labored breathing (8.79 ± 1.47%) were observed.In terms of behavior, most ewes appeared attentive and active, although 40.55% showed fear and 19.9% aggression. Flight distance increased between tests (from 2.50 ± 0.07 s to 3.80 ± 0.07 s), and animals maintained greater distance from the corridor (2.79 ± 0.08 m) than from the observer (1.85 ± 0.06 m). PCA of qualitative behavior assessment identified three clusters: well-being, discomfort, and vitality.Spearman correlation analysis showed that relaxed ewes had better BCS (r = +0.29) and were less likely to be lame (r = –0.32) or anemic (r = –0.37). Vigorous ewes showed fewer signs of respiratory distress (r = –0.32), while apathetic ones were more prone to poor conditioning (r = +0.31) and injuries (r = +0.30) (p < 0.05 or p < 0.01).The BIENE protocol proved effective in detecting major welfare issues in intensive Algerian sheep farming.