Veterinary Research Forum

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 the 16th day post-artificial insemination for gene expression analysis and on days zero, seven, 14, and 21 post-artificial insemination 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 were analyzed using Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR) with SYBR green dye. Amplicons of CCL8, CXCL10, and GAPDH genes were 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 indicated significantly elevated CCL8 and CXCL10 mRNA expression levels in peripheral blood leukocytes of pregnant buffaloes on the 16th day. Progesterone levels in the pregnant group were increased significantly from day zero to day 21, while no significant differences were observed between groups on days zero, seven and 14. Pregnancy was further confirmed via per-rectal examination on the 45th day post-artificial insemination Therefore, CCL8 and CXCL10 gene expression profiling on the 16th day could serve as reliable early pregnancy markers in Murrah buffaloes.

Abstract This study aimed to obtain a narrow-spectrum antimicrobial peptide. A peptide XMK-8 was designed based on the amino acid sequence of goose MyHC1 protein from positions 1919 to 1936 (some parameters do not meet the requirements of antimicrobial peptides through bioinformatics analysis) using bioinformatics tools and amino acid substitution method. The minimum inhibitory concentration was determined using liquid double dilution method, the hemolysis rate was determined using dilution method, and the effects of temperature, acid-base, enzyme, and salt ions on its antimicrobial activity were evaluated using liquid double dilution method. The results showed that the designed peptide was a cationic hydrophilic peptide with high amphiphilicity and low hemolytic activity on mouse red blood cells. It had no antimicrobial activity against Escherichia coli, Salmonella, Staphylococcus aureus, and Aeromonas hydrophila. The minimum inhibitory concentration against Pasteurella multocida was 250 μg mL^-1, and the minimum inhibitory concentration against Haemophilus parasuis was 1.00 mg mL^-1. The antimicrobial activity of the narrow-spectrum antimicrobial peptide XMK-8 can still be detected after treatment with temperature (0.00 - 100 ˚C), salt ions (sodium ions and potassium ions; 50.00 - 200 mmol L^-1), pH (4.00 - 10.00), and protease K (20.00 - 100 μg mL^-1). Antimicrobial peptide XMK-8 was expected to become a new alternative to antibiotics and would have good application prospects in the prevention and treatment of P. multocida and H. parasuis infections.

Abstract Hyperuricemia, caused by impaired uric acid excretion, poses significant health risks. Urate oxidase (UOX) from Aspergillus flavus offers therapeutic potential by converting uric acid into soluble allantoin; however, its instability limits clinical applications. This study investigated the effects of osmolytes, including proline and glycine, on the kinetics and thermostability of recombinant A. flavus UOX. Following the expression of UOX coding sequence in Escherichia coli BL21, it was purified using Ni2+-NTA agarose affinity chromatography and confirmed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The enzyme maintained its activity up to 35.00 ˚ C and lost its activity at higher temperatures as it lost 70.00 % of its activity after 60 min at 40.00 ˚ C, and the enzyme with proline and glycine additives maintained 73.00% and 30.00% of the activity, respectively. The inactivation rate constant of enzyme (k_in) was decreased in the presence of proline, indicating that the enzyme was more stable with proline, but glycine had no effect on kin. Half-life of enzyme was raised to 86 min in the presence of proline and the Michaelis constant (K_m) was decreased significantly by both osmolytes, as well. These results demonstrated that proline stabilized UOX by mitigating thermal denaturation, likely through preferential hydration and hydrophobic interactions, while glycine enhanced substrate binding. The stabilizing capacity of proline highlighted its utility for inclusion in biopharmaceutical formulations, offering a solution to the persistent challenge of UOX instability in therapeutic contexts. These findings yielded practical strategies for enhancing both structural integrity and catalytic performance of enzymes in pharmaceutical development.

Abstract Cathelicidin-1, an antimicrobial peptide, has garnered attention for its potential role as a biomarker in detecting anestrus in cows, providing insights into bovine reproductive health. This study aimed to analyse cathelicidin-1 within the urinary proteome and evaluate its effectiveness as a diagnostic tool for anestrus in cows. The study employed tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis and matrix-assisted laser desorption ionization-time of flight mass spectrometry techniques to identify and characterize cathelicidin-1 in the context of anestrus in cows. The analysis confirmed the presence and distinct profile of cathelicidin-1, highlighting its significance in reflecting the physiological and pathophysiological states associated with anestrus. Cathelicidin-1 as a promising diagnostic biomarker for anestrus in cows could revolutionize bovine reproductive management by offering more precise and advanced detection methods compared to the traditional, time-consuming, and sometimes inaccurate approaches, like behavioral observation or hormonal assays. If cathelicidin-1 can be detected non-invasively, it would not only enhance early detection and timely intervention but also reduce the need for invasive procedures, thereby improving animal welfare.

Abstract Aflatoxins are toxic chemicals produced by Aspergillus fungi. Reports exist on the relationship of aflatoxin exposure via contaminated food and feed to hepatotoxicity and liver cancer. Aflatoxin B1 (AFB1) and Aflatoxin G1 (AFG1) are two dangerous types of aflatoxins for human health. Bovine α-lactalbumin (ALA) is the second major whey protein in milk which bear diverse biological functions. In this study, the interaction of AFB1 and AFG1 with the ALA protein was studied using fluorescence spectroscopy, molecular docking and molecular dynamic (MD) simulation. The spectroscopy experiments showed that the interaction with AFB1 and AFG1significantly quenched the intrinsic fluorescence emission of ALA via a static quenching mechanism. The free energy of binding and binding constant (Ka) obtained from the intrinsic fluorescence results were –5.32 kcal per mol and 0.80 × 10⁴ L mol^-1 for AFB1 and -5.64 kcal per mol and 1.35 × 10⁴ l mol^-1 for AFG1, respectively. Molecular docking studies were conducted before and after the MD simulation to estimate the binding sites, Ka s and binding mode. Results from the molecular docking showed that AFB1 and AFG1 bound to ALA via hydrophobic interaction and hydrogen bond. After MD simulation, the precision of the Ka obtained from the docking results was improved and it was more similar to the experimental results of fluorescence spectroscopy. Other simulation results were aligned well with the molecular docking and fluorescence spectroscopy results. Accordingly, AFB1and AFG1 could form complex with ALA, however, AFG1 showed higher affinity for binding to ALA and more compact complex structure.

Abstract In this study, the effects of Urtica dioica hydro-alcoholic extract were investigated on the blood glucose and lipid profiles of female ovariectomized and non-ovariectomized rats. In total, 32 adult female rats were divided into four groups (eight each) including control and ovariectomy groups as well as non-ovariectomy and ovariectomy groups treated with 200 mg kg^-1 of Urtica dioica extract orally in the last five weeks of the study starting from the week 56^th. The duration of the study was 60 weeks. Glucose, serum lipid profiles and pancreatic pathological alterations were determined in these groups at the end of experiment. Serum glucose, triglyceride (TG), very-low-density lipoprotein (VLDL), and TG/high-density lipoprotein (HDL) ratio indicated a significant increase in the healthy female rats under treatment with Urtica dioica extract compared to others. The TG, cholesterol, HDL, low-density lipoprotein (LDL) and VLDL showed a significant increase in menopaused rats compared to others. The interaction of consuming Urtica dioica extract and ovariectomy caused significant decreases in glucose, TG, VLDL, HDL/LDL ratio and TG/HDL ratio. Consumption of Urtica dioica extract by non-menopaused rats damaged the beta cells in Langerhans islets. Results of the present study revealed that the consumption of Urtica dioica extract is not beneficial and has diabetogenic effects in female non-ovariectomized rats compared to ovariectomized ones.

Abstract Myocardial infarction is commonly considered as a leading cause of cardiovascular disease taking the lives of seven million people annually. Liver dysfunction is associated with cardiac diseases. The profile of abnormal liver functions in heart failure is not clearly defined. This study was designed to investigate the protective effects of betaine on liver injury after myocardial infarction induced by isoprenaline in rats. Forty-eight male rats were divided into four groups: the control group received normal diet and the experimental groups received 50, 150, and 250 mg kg^-1 body weight of betaine daily through gastric gavages for 60 days. All of experimental and control groups experienced myocardial infarction, induced by subcutaneous injection of 100 mg kg^-1 isoprenaline in two consecutive doses )8:00 AM to 8:00 PM). Liver enzymes including aspartate transaminase (AST) and alanine transaminase (ALT) were significantly reduced in the groups treated with betaine, compared with the control group. The total antioxidant capacity in the experimental groups, treated with betaine, showed a significant increase, compared with the control group. In the control group, severe lesions were created in the liver tissue, while degenerative changes of liver tissue significantly reduced in groups treated with different doses of betaine, showing the repair of liver tissue. Betaine decreased apoptosis in the experimental groups in comparison with the control group. Betaine showed a protective effect against biochemical and histological changes in liver tissue caused by the induction of myocardial infarction via isoprenaline injection.

Abstract Bulk tank somatic cell count (BTSCC) is a gold standard test for identification of milk quality, but its results are influenced by several interventional factors. Recently, application of acute phase proteins and especially milk amyloid A (MMA) has been considered as accurate parameters for milk quality study. The current research was done to evaluate the accuracy of MMA, BTSCC, fat, protein and lactose for identification of milk quality. Ninety bulk tank milk samples were collected from 30 randomly selected dairy herds and classified into two groups of samples with BTSSC > 200000 cells per mL and those with BTSSC < 200000 cells per mL. Protein, fat, lactose and MAA contents of samples were analyzed. Average amount of the MAA in healthy and mastitic milk samples were 5.15 and 504.35 ng mL^-1, respectively. Statistically significant difference was seen for MAA and total protein contents between two groups. Clinical accuracy of MAA-, total protein-, fat- and lactose–based methods was 0.937, 0.757, 0.665 and 0.547, respectively. The MAA method at concentration of 20.78 ng mL^-1 had the highest sensitivity (97.30%) and specificity (46.70%). Evaluation of MAA is recommended as a rapid and accurate method for determination of the unfavorable changes in milk quality and early subclinical mastitis diagnosis.

Abstract Blood selenium and trace minerals play an important role in animal’s health and production. The aims of this study were to determine selenium effect on blood glutathione peroxidase (GPX) activity, trace minerals and weight gain in lambs. Twelve female Makuei breed were studied for 63 days in groups of control, nanoselenium (NanoSe) and sodium selenite (NaSe). Mean concentrations of GPX, Cu and Fe in selenium supplemented groups were higher than in control group but the differences were not significant. Mean GPX and selenium was significant among the bleeding times, for Cu and Zn significant occasionally while not for weight gain. The percentages of weight gain in groups were 34.20, 38.90 and 36.30, respectively, which was not different. The individual comparison of parameters among groups showed differences for GPX, selenium and Cu. Correlations were observed between weight & Fe, weight & GPX, weight & selenium, Zn & Fe and GPX & selenium in NanoSe group. Weight gain showed negative correlations with Fe and positive correlation with GPX. In conclusion, selenium compounds increased GPX activity and selenium in which it was predominant in NanoSe than in NaSe group. Selenium compounds showed no effects on Cu, Zn and Fe but caused weight gain to increase. NanoSe revealed correlations between weight gain, GPX, Fe and selenium and was preferable to NaSe. Thus, the effect of NanoSe on reducing the oxidative stress and increased weight gain was acceptable and probably an option to NaSe administration in lambs.

Abstract To assess the effects of pre-analytical handling (storage time and temperature) on selected hematological parameters, whole blood samples were collected in EDTA coated tubes from each of 30 clinically normal male adult beagle dogs. Each sample was separated in 2 aliquots, of which one was stored in ambient temperature (25 ˚C) and the other one was refrigerated (2 to 4 ˚C). Complete blood counts were performed in 1, 2.5, 5, 12, 24, 36 and 60 hr post-sampling for each aliquot of every sample using a flow cytometer. Packed cell volume values remained stable in the samples kept in room temperature (RT), whereas a significant increase was noted in the refrigerated ones 24 hr post-sampling. Statistically significant increases in red blood cell counts were noted after 24hr in the samples stored in 2 to 4 ˚C and after 12 hr in those kept in RT. No significant changes were observed in haemoglobin concentration. A significant decrease was evident only 60 hr post-sampling for the white blood cells kept in RT, but not for those kept in 2 to 4 ˚C. Platelet counts significantly decreased after 24 hr in the refrigerated aliquots and after 5 hr in those kept in RT. The results of this study indicate that storage of blood samples for up to 24 hr in 2 to 4 ˚C is associated with the least artifactual changes.

Abstract Hydrogen sulfide (H₂S) has been shown to protect the gastric mucosa through several protective mechanisms but till now its effect on mRNA expression of sodium bicarbonate cotransporter 1 (NBC1), trefoil factor1 (TFF1) and trefoil factor2 (TFF2) was not investigated. This study was aimed to evaluate the effect of H₂S on mRNA expression of NBC1, TFF1 and TFF2 in rat gastric mucosa in response to gastric distention. Thirty two rats were randomly assigned into four equal groups. They were control (C), distention (D), propargylglycine (PAG)-, and NaHS-treated groups. To evaluate the effect of exogenous and endogenous H₂S on gene expression of NBC1, TFF1 and TFF2, two groups of rats were received H₂S donor, intra-peritoneal NaHS (80 µg Kg^-1), and PAG (50 mg kg^-1), accompanied to stimulate the gastric acid secretion, respectively. Under general anesthesia and laparotomy, a catheter was inserted into the stomach through duodenum for instillation of isotonic saline for gastric distention. Ninety min after beginning the experiment, animals were sacrificed and the gastric mucosa was collected to determine total acid content of gastric effluents and to quantify the mRNA expression of studied genes by quantitative real-time polymerase chain reaction (qRT-PCR). Results showed that A) gastric distention increased the level of mRNA expressions of NBC1, TFF1 and TFF2; B) these levels in NaHS-treated rats were significantly higher than those in Distention group; and C) PAG decreased the expression levels of NBC1 and TFF1. The Findings showed H₂S upregulated gene expression of NBC1, TFF1 and TFF2 in gastric mucosa.

Abstract This study was conducted to evaluate the effect of soy milk on serum 17- β estradiol level and number of neurons in cerebral cortex and hippocampus as well as determination of the ratio of neurons in cortical and hippocampal regions in neonatal ovariectomized rats. Thirty female rats (one day old) were divided into six groups of five. At day 7, ovariectomy surgery was performed in four groups and two other groups were assumed as sham and control groups. Three groups of ovareictomaized rats were fed with soy milk at the doses of 0.75, 1.50 and 3.00 mL kg^-1 per day since they were 14. At day 60, the blood samples were collected to measure the17- β estradiol concentration, and then the brain of rats were prepared for histological studies. The serum 17- β estradiol level significantly increased in ovariectomized rats fed with soy milk compared to ovariectomized rats with no soy milk supplementation. In addition, the results showed that soy milk significantly increased the number of neurons in CA1, CA2 and dentate gyrus regions of hippocampus and granular layer of cerebral cortex in ovariectomized rats, whereas there was no significant change in number of neurons in CA3 zone of hippocampus and molecular, pyramidal and multiform layers of cerebral cortex in ovariectomized rats fed with soy milk. The ratio of cerebral cortex neurons to hippocampal neurons had no significant changes among the experimental groups.

Abstract Mycophenolate mofetil (MMF) is a selective inhibitor of Inosine-5′-monophosphate dehydrogenase. Gastrointestinal (GI) disturbances in immature ones are reported for MMF-induced compilations, which in the case of occurrence dose reduction is required. Thus, in the present study, the fructooligosaccharide raftilose^® (RFT) was co-administrated with MMF to estimate the protective effect of RFT against MMF-induced GI complications. Thirty six immature male Wistar rats were divided into six groups including: Control (normal saline), RFT-treated (100 mg kg^-1), MMF-treated (20 mg kg^-1), MMF + LRFT (50 mg kg^-1), MMF + MRFT (100 mg kg^-1) and MMF + HRFT (200 mg kg^-1) groups. The hematocrit (Hct), lymphocyte/total WBC, feces water content and pH were analyzed. Moreover, the hepatic functional tests, kidney-related biomarkers, lipid and protein profiles, total antioxidant capacity (TAC), malondialdehyde (MDA) and nitric oxide (NO) contents were assessed. Co-administration of RFT stabilized the MMF-reduced body weight. The MMF significantly diminished Hct and lymph/total WBC (p < 0.05). Only MRFT enhanced the lymphocyte/total WBC. Increased water content, no changes in feces pH, increased serum ALT and AST, no alteration in urea and mild enhancement in creatinine were demonstrated in MMF-received animals. However, RFT at low dose ameliorated the feces parameters and reduced ALT. No significant changes were demonstrated for serum lipid and protein profiles in MMF- and RFT + MMF-treated groups. The RFT enhanced the serum TAC, reduced MDA and NO contents. In conclusion, our data suggested that RFT could be considered as an effective agent to subsidize the MMF-induced clinical, hematological and biochemical disorders.