Veterinary Research Forum

Abstract Dicrocoeliasis is a globally significant condition impacting both economic and public health. The lack of effective vaccines and the emergence of drug-resistant flukes have prompted research into alternative treatments. Metallic nanoparticles have recently been studied for their potential as anthelmintic agents. This research examines the anthelmintic activity of copper oxide (CuO-NPs) and zinc oxide nanoparticles (ZnO-NPs) against Dicrocoelium dendriticum in vitro. Using adult motility inhibition tests and oxidative stress biomarkers, including glutathione peroxidase (GSH), glutathione S-transferase (GST), superoxide dismutase (SOD), and malondialdehyde (MDA), this study evaluated the effects of CuO-NPs and ZnO-NPs. Flukes were treated with various concentrations of nanoparticles (1, 4, 8, 12, and 16 ppm) for 24 hr. CuO-NPs and ZnO-NPs demonstrated concentration- and time-dependent anthelmintic activity. Higher concentrations (12 and 16 ppm of CuO-NPs; 16 ppm of ZnO-NPs) significantly inhibited worm motility compared to controls. The nanoparticles induced oxidative stress in the flukes, with decreased SOD, GST, and GSH activity and increased MDA levels. Based on these findings, CuO-NPs and ZnO-NPs exhibit potential as therapeutic agents for controlling and treating D. dendriticum. However, further studies are necessary to assess their safety and efficacy in vivo for managing parasitic infections.

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 Thymoquinone (TQ), the bioactive compound found in black seed, possesses beneficial properties. In the present study, the effects of oral administration (PO) of TQ were investigated on acute corneal and orofacial pain in rats. To clarify the central mechanism of action, muscarinic cholinergic, cannabinergic 1 (CB1) and 5-hydroxytryptamine 3 (5-HT3) receptor antagonists were delivered into the 4th ventricle of the brain after PO administration of TQ. Acute corneal and orofacial pain were induced by dropping of hypertonic saline (50.00 µL, 5.00 M) on the corneal surface and subcutaneous (SC) injection of capsaicin (1.50 µg, 20.00 µL) in the vibrissa pad, respectively. The eye wiping number and face rubbing duration were recorded as corneal and orofacial pain behavioral responses. Locomotor activity was measured using an open-field test. The TQ (5.00 mg kg-1) had no effects, while it reduced pain responses at 10.00, 20.00 and 40.00 mg kg-1. Intracerebroventricular (ICV) injections of naloxone (an antagonist opioid receptors), AM251 (an antagonist CB1 receptors), atropine (an antagonist of muscarinic cholinergic receptors) and ondansetron (an antagonist of 5-HT3 receptors) at a similar dose of 10.00 µg kg-1 inhibited corneal and orofacial pain suppression caused by 40.00 mg kg-1 TQ. The tested drugs did not affect locomotor activity. It is concluded that TQ caused analgesia in the acute corneal and orofacial pain. Central opioid, cholinergic muscarinic, CB1 and 5-HT3 receptors might be involved in the antinociceptive effects of TQ.

Abstract Cadmium (Cd) is a highly toxic environmental pollutant known to cause severe damage to the male reproductive system. This study aimed to investigate the protective effects of bee bread (BB), a natural product with antioxidant, anti-apoptotic, and anti-inflammatory properties, against Cd-induced testicular toxicity in male Wistar Albino rats. A total of 32 rats were divided into four groups: Control, BB (0.5 g/kg of BB), Cd (5 mg/kg of Cd), and Cd + BB (0.5 g/kg of pBB + 5 mg/kg of Cd) groups. Administrations via oral gavage were performed for 4 weeks. Semen analysis revealed significant reductions in sperm motility and density, along with increases in abnormal and dead sperm ratios in the Cd and Cd+BB groups compared to controls. Histopathological examination showed severe degeneration and desquamation of germ cells, tubular atrophy, and a decrease in spermatozoa in the Cd-treated groups. Polymerase chain reaction analysis indicated upregulation of apoptotic markers (Caspase-3, -8, and -9) and oxidative stress enzymes [catalase (CAT), superoxide dismutase (SOD1)] in the Cd and Cd+BB groups, while AR levels were reduced, signifying disrupted testicular function. BB administration partially mitigated Cd-induced damage, as evidenced by less severe histopathological changes and moderated gene expression alterations. However, the protective effects of BB were not sufficient to completely counteract the toxic impact of Cd. The study concludes that while BB has some potential in reducing Cd-induced testicular toxicity, its protective efficacy is limited, warranting further research to explore its therapeutic potential in combination with other protective agents.

Abstract Effective reproductive management in cattle, such as cows and buffaloes, requires early and accurate pregnancy detection. Early identification of pregnancy enables farmers to promptly identify non-pregnant animals for treatment and/or rebreeding, thereby reducing the calving interval. This study aimed to standardize the expression of the CCL8 and CXCL10 genes as markers for early pregnancy detection in Murrah buffaloes. Blood samples were collected on day 16 post-artificial insemination (A.I.) for gene expression analysis and on days 0, 7, 14, and 21 post-A.I. for progesterone concentration measurement. Buffaloes were categorized as pregnant (n = 6) or non-pregnant (n = 6) based on the resumption of estrus. Gene expression levels in peripheral blood leukocytes (PBLs) were analyzed using qRT-PCR with SYBR green dye. Amplicons of CCL8, CXCL10, and GAPDH genes measured 108, 117, and 158 bp, respectively. Results showed that CCL8 mRNA expression in pregnant buffaloes was 5.13 and 12.21 times higher compared to non-pregnant buffaloes, while CXCL10 mRNA expression was 4.19 and 22.17 times higher. These findings indicate significantly elevated CCL8 and CXCL10 mRNA expression levels in PBLs of pregnant buffaloes on day 16. Progesterone levels in the pregnant group increased significantly (p < 0.05) from day 0 to day 21, while no significant differences were observed between groups on days 0, 7, and 14. Pregnancy was further confirmed via per-rectal examination on day 45 post-A.I. Therefore, CCL8 and CXCL10 gene expression profiling on day 16 can serve as reliable early pregnancy markers in Murrah buffaloes.

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 Aim: The aim of this study, conducted between 2020 and 2023, was todetect Coxiella burnetii, Chlamydia abortus, and Brucella species in abomasal contents of aborted foetuses of ruminants from different parts of the Central Anatolia region in Türkiye using PCR.
Methods and Results: The abomasal contents of a total of 97 aborted fetuses from cattle, sheep, and goats with a history of abortion, collected between the years 2020 and 2023, were tested in this study. As a result of PCR analysis of 97 abomasal contents, 4 (4.1%;95%CI:1.33–10.82) of them were C.abortus, including 3 sheep and 1 goat. Two (2.1%; 95% CI: 0.36–7.96) of them were C. burnetii, including 1 sheep and 1 cattle. A total of 60 (61.9%;95% CI:51.4–71.37) samples from 47 cattle, 9 sheep, and 4 goats were determined by Brucella genus-specific PCR. The multiplex PCR was used for identification of different species of Brucella spp. Following multiplex PCR analysis of the positive Brucella spp. samples, 39 (65%;95%CI:51.52–76.55) samples were identified as B. abortus, including 2 sheep, 1goat, and 36cattle. Additionally, 19 (31.7%;95%CI:20.6–45.09) isolates were identified as B.melitensis, including 5 sheep, 2 goats, and 12 cattle. In two sheep samples, both B.melitensis and C.abortus were identified from the same animals.
Conclusions: In conclusion, Brucella spp. was found to be the most prevalent pathogen causing abortions, with C.abortus contributing substantially. Effective control strategies under the One Health approach are essential to prevent the uncontrolled spread and interspecies transmission of these zoonotic agents in the region and country.

Abstract Abstract
Biofilm formation is a key virulence factor in Staphylococcus aureus, contributing to bacterial persistence, antimicrobial resistance, and chronic infections. This study aimed to investigate the presence of biofilm-associated genes (fib, fnbA, fnbB, clfA, and clfB) in S. aureus isolates from dogs in Ilam, Iran. From December 2022 to September 2023, 250 swab samples were collected from nasal, oral, and rectal sites of dogs, yielding 81 S. aureus isolates confirmed by polymerase chain reaction (PCR) amplification of the nuc gene. The prevalence of biofilm-associated genes varied, with clfA, clfB, and fnbA detected in 98.8% of isolates, fib in 63.0%, and fnbB in 16.0%. Notably, fnbA, clfA, and clfB were present in all rectal isolates, while fnbB was absent in this group. The findings highlight the widespread presence of biofilm-related genes in S. aureus from dogs, suggesting their potential role in colonization and zoonotic transmission. The high prevalence of adhesion-associated genes underscores the need for monitoring biofilm-forming S. aureus in companion animals to mitigate antimicrobial resistance and public health risks.

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 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 postmortem prevalence of EGGD and other gastric lesions in Colombian equids, specifically horses, donkeys, and mules. The study utilized a sample of 300 equine stomachs, with 100 specimens from each group, collected immediately after slaughter. The postmortem examination included a thorough inspection of the ventral fundus, the adjacent area of the margo plicatus, and the 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 68.3% on average, with specific rates of 45% in horses, 77% in donkeys, and 83% in mules, with lesions predominantly affecting the fundus and pyloric antrum regions. Inflammatory processes were observed to be similar between donkeys and mules, while horses exhibited distinct inflammatory patterns in the glandular mucosa. Additional gastric findings, such as gastritis (85.3%), gastric impaction (10%), and parasitic infections (13.3%), were also documented during the study. The findings suggest that EGGD is prevalent across all three groups of equids. The inflammatory conditions and their distribution are similar in horses and donkeys, although differences in the location, frequency, and severity of mucosal lesions were observed among the three species.

Abstract The effects of substituting whole egg yolk (WEY) in a Tris-citrate-based extender with two derived fractions—a buffer-soluble fraction (BSF) and an ammonium sulfate insoluble yolk fraction (ASIF)—on the freezability of bull sperm were investigated. The BCF and ASIF levels of egg yolk in the respective diluents were consistent with their extracted values from an equivalent volume (20%) of egg yolk in the control diluent. The extenders were then enriched with 0.00%, 5.00%, and 10.00% (v/v) of pasteurized skim milk. Semen samples were collected weekly over five consecutive weeks from six adult bulls, and the standard ejaculates were pooled. The pooled semen was subsequently divided into seven experimental extenders and frozen in 0.5 ml French straws. Various sperm quality parameters, including kinematics, acrosome integrity, capacitation, and DNA fragmentation, were evaluated post-thawing. Results indicated that sperm kinematics and the percentage of acrosome-reacted sperm in experimental extenders were not significantly different from those in the control group. The milk-free ASIF extender exhibited higher percentages of capacitated but lower percentages of spermatozoa with intact DNA compared to the WEY extender sperm (p < 0.05). The addition of 10.00% milk into the BSF diluent resulted in a significant increase in the proportion of sperm with intact DNA (p < 0.05) and a notable decrease in the percentage of sperm with partially fragmented DNA compared to the control (p < 0.05). In conclusion, a BSF extender enriched with 10.00% cow's skimmed milk is recommended as a substitute for WEY in the cryopreservation of bull semen.

Abstract Inflammatory bowel disease (IBD) encompasses a group of chronic inflammatory conditions that primarily impact the gastrointestinal system. While ulcerative colitis and Crohn's disease are the principal manifestations in humans, animals frequently exhibit lymphocytic-plasmacytic enteritis/colitis and eosinophilic enteritis/colitis. Growing evidence suggests a complex interplay among genetic predisposition, gut microbiota imbalance and abnormal immune responses to intestinal microbes in susceptible individuals. This intricate involvement results in remarkably similar clinical presentations across species. Patients often experience symptoms such as diarrhea, vomiting, weight loss and anemia. Extraintestinal manifestations including uveitis, skin rash and arthritis may also occur. Endoscopy and biopsy typically serve as the gold standard for confirming the diagnosis and differentiating it from other gastrointestinal disorders in humans and animals. The treatment approach generally focuses on managing disease activity through immunosuppressive medications such as glucocorticoids, administered at appropriate dosages. However, the precise cause of IBD remains a topic of ongoing research. With the emergence of additional treatment options like herbal compounds and fecal microbiota transplantation, which have demonstrated effectiveness in restoring gut health in IBD patients, there is optimism for novel therapeutic strategies. Ultimately, conclusion is that chronic gastrointestinal conditions like IBD are complex in both human and veterinary medicine. These diseases share numerous common pathophysiological features, yet, diagnostic and treatment challenges continue to exist.

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 examined the effects of alpha-ketoglutaric acid (AKG; 2.00, 4.00, and 8.00 mM L^-1) and folic acid (FA; 50.00 nM L^-1) on bull sperm cells under ammonia stress induced by 175 µM L^-1 ammonium chloride. Sperm parameters including kinematic motility, survival rate, membrane integrity, DNA integrity, superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT), total antioxidant capacity (TAC) activities, and apoptosis were assessed in the groups. Sperm motility indices, survival rate, plasma membrane integrity, SOD, CAT, TAC, and GPx enzymes activity, DNA damage, and apoptosis in the treated groups were significantly differed from those in the control groups. The AKG concentrations of 4.00 and 8.00 mM L^-1 co-supplemented with 50.00 nM L^-1 FA mitigated the negative effects of ammonia on sperm cells. This study indicated that supplementation with AKG and FA at the desired concentrations counteracted the adverse effects of ammonia toxicity that preceded clinical signs. Further studies are needed to evaluate the fertility of these sperms, either in vitro or in vivo.