Veterinary Research Forum

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 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 accessory gene regulator(agr) operon as a part of the quorum-sensing (QS) system, is responsible for the regulation of numerous virulence factors in Staphylococci. Furthermore, the ability of the opportunistic pathogen, Staphylococcus pseudintermedius to form biofilm is a significant contributor to chronic diseases. Our aim was evaluation of the association between different agr groups and the expression of icaD gene related to biofilm formation among S. pseudintermedius from nasal samples. Fifty nasal swabs were collected from broiler chickens; then phenotypic and molecular tests performed for isolation of S. pseudintermedius from samples and their agr genotype was also investigated. The biofilm production capacity of the isolates was determined using both microtiter plate assay and amplification of icaD gene. S. pseudintermedius was detected in 86.84% of samples. The assessment of agr typing results related that most of the strains (69.70%) were classified in agr group ɪ. The relative frequency of biofilm producing S. pseudintermedius isolates was 75.76% and all of them harbored icaD gene. The expression level of icaD determinant was significantly higher among isolates in agr group ɪ than other agr groups. The results suggest that broiler chickens may act as a reservoir for transmission of S. pseudintermedius to humans which should be considered a potential zoonotic bacterium able to produce different virulence factors like biofilm.

Abstract Ocular ultrasonography is a safe, painless and non-invasive diagnostic tool that provides veterinarians with detailed images of the eye and surrounding structures. This study evaluated 30 clinically healthy Turkmen horses, divided into two age groups: Group 1 under 36 months (3 males, 8 females), and Group 2: 36 months and older (8 males, 11 females).
Significant differences in several ocular parameters were identified across different age groups in Turkmen horses, as determined by the independent samples T-test and Mann-Whitney U test.
Specifically, the mean intercanthal distance, globe diameter, ciliary body volume and area, iris thickness, corpora nigra distance, volume and area, vitreous chamber, and retinal/choroidal/scleral composite size demonstrated statistically significant variations between age categories (p < 0.05). No significant differences were detected between sexes within any of the age groups for above measured parameters.
Of the ocular parameters evaluated, only anterior chamber depth, central lens thickness, and ciliary body thickness demonstrated statistically significant differences based on both sex and age.
The average corneal thickness of the left and right eyes, as measured in sagittal and dorsal planes, was similar in both male horses under 36 months and those 36 months or older, when compared to their respective female counterparts.
No significant differences were observed between measurements of the left and right eyes, nor were there significant discrepancies between sagittal and dorsal measurements.
These findings provide a baseline for understanding normal ocular ultrasonographic features in Turkmen horses and may aid in the diagnosis and management of ocular diseases specific to this

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 to 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. 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 vs. 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 SDG 2 (Zero Hunger) by contributing to reproductive efficiency in livestock, and SDG 12 (Responsible Consumption and Production) through development of cost-effective, sustainable sperm sexing technologies.

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 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 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 Equine glandular gastric disease (EGGD) has limited prevalence studies and scarce understanding of the predisposing factors and pathophysiological mechanisms involved. The objectives of this study were to determine and compare the post-mortem prevalence of EGGD and other gastric lesions in Colombian equids, specifically horses, donkeys, and mules. The study utilized a sample of 300 equids stomachs, with 100 specimens from each group, collected immediately after slaughter. The post-mortem examination included a thorough inspection of the ventral fundus, adjacent area of the margo plicatus, and pyloric antrum of the glandular mucosa, carefully identifying any lesions or abnormalities. The results showed that the overall macroscopic presence of EGGD in the equid population was 68.30% on average, with specific rates of 45.00% in horses, 77.00% in donkeys, and 83.00% in mules, with lesions predominantly affecting the fundus and pyloric antrum regions. Inflammatory processes were observed to be similar between donkeys and horses, while mules exhibited distinct inflammatory patterns in the glandular mucosa. Additional gastric findings, such as gastritis (85.30%), gastric impaction (10.00%), and parasitic infections (13.30%), were also documented during the study. The findings suggested that EGGD was prevalent across all three groups of equids. Although the inflammatory conditions and their distribution were similar in horses and donkeys, differences in the location, frequency, and severity of mucosal lesions were observed among the three species.

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 This study is the first to apply the behaviour, immunity, nutrition and environment (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 (< 2). Housing problems included fleece moisture (11.28 ± 2.55%) and over 40.00% 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 19.90% aggression. Flight time increased from 2.50 ± 0.07 sec to 3.80 ± 0.07 sec, and animals maintained greater distance from the corridor (2.79 ± 0.08 m) than from the observer (1.85 ± 0.06 m). Principal component analysis of qualitative behavior assessment identified three clusters, including well-being, discomfort, and vitality. Spearman correlation analysis showed that relaxed ewes had better body condition score (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).The BIENE protocol proved effective in detecting major welfare issues in intensive Algerian sheep farming.

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.