Sheep Plasma Research Articles

In vitro inhibitor effect and molecular docking of thiamine (vitamin B1), riboflavin (vitamin B2), and reference inhibitor captopril on angiotensin-converting enzyme purified from sheep plasma

Objective Angiotensin-converting enzyme (ACE, EC 3.4.15.1) is a very important factor in the regulation of blood pressure. Also, the inhibition of ACE with natural compounds has been a very important research area in the treatment of high blood pressure. ACE was purified and characterized from sheep plasma. Molecular docking studies and the inhibition effect of thiamine, riboflavin, and captopril on ACE were investigated. Methods Herein, ACE was purified from sheep plasma by affinity chromatography. The effect of thiamine and riboflavin on ACE was researched. Molecular docking studies were performed to understand the molecular interactions between thiamine, riboflavin, and captopril with ACE. Results The purification coefficient was found to be 8636 fold. The binding energy of thiamine, riboflavin, and captopril was found to be -6.7 kcal/mol, -8.1 kcal/mol, and -5.5 kcal/mol, respectively. Thiamine conformed to three conventional hydrogen bonds with ASP:415, HIS:513, and LYS:454. Riboflavin formed four conventional hydrogen bonds with GLN:281, GLU:376, THR:282, and TYR:520. Captopril formed two conventional hydrogen bonds with ARG:124, one conventional hydrogen bond with TYR:62 and ASN:85, and one carbon-hydrogen bond with ASN:66. Molecular docking results showed that thiamine, riboflavin, and captopril interacted with ACE through hydrogen bonding and hydrophobic interactions. Thiamine and riboflavin indicated significant inhibition effects on ACE. The IC50 values of thiamine, riboflavin, and captopril were found as 960.56 µM, 11.02 µM, and 1.60 nM, respectively. Ki values for thiamine, riboflavin, and captopril were determined as 1352.04 µM, 12.30 µM, and 1.06 nM, respectively. Conclusion In this work, it was concluded that thiamine and riboflavin may have preventive and therapeutical impacts against high blood pressure with their ACE inhibitor effect. Thiamine and riboflavin showed a lower inhibitory effect with a higher IC50 than captopril. However, when the inhibitory effect of thiamine and riboflavin vitamins is compared to captopril, it is concluded that they may be natural inhibitors with fewer side effects.

Short communication: Metabolic synthesis of vitamin D2 by the gut microbiome

The origin of vitamin D2 in herbivorous animals was investigated in vivo in sheep and in bovine as well as mouse gastrointestinal tracts. A high concentration of 25-hydroxyvitamin D2 in blood plasma of sheep both in summer and winter appeared to be incompatible with the undetectable level of vitamin D2 in the pasture on which the sheep were grazing. Studies with bovine rumen contents from a cow grazing the same pasture as the sheep, demonstrated an increased concentration of vitamin D2 on anaerobic incubation in a ‘Rusitec’ artificial rumen, which was further enhanced when cellulose powder was added as a fermentation substrate. The colon contents of mice that were fed from weaning on a vitamin D-free diet were found to contain vitamin D2. The results of these comparative studies in 3 animal species indicated that vitamin D2 was being generated by microbial anaerobic metabolism in the gastrointestinal tract.

Comparative lipidome study of maternal plasma, milk, and lamb plasma in sheep

Lipids play a critical role in neonate development and breastmilk is the newborn’s major source of lipids. Milk lipids directly influence the neonate plasma lipid profile. The milk lipidome is dynamic, influenced by maternal factors and related to the maternal plasma lipidome. The close inter-relationship between the maternal plasma, milk and neonate plasma lipidomes is critical to understanding maternal-child health and nutrition. In this exploratory study, lipidomes of blood and breast milk from Suffolk sheep and matched lamb blood (n = 13), were profiled on day 34 post birth by untargeted mass spectrometry. Comparative multivariate analysis of the three matrices identified distinct differences in lipids and class of lipids amongst them. Paired analysis identified 346 differential lipids (DL) and 31 correlated lipids (CL) in maternal plasma and milk, 340 DL and 32 CL in lamb plasma and milk and 295 DL and 16 CL in maternal plasma and lamb plasma. Conversion of phosphatidic acid to phosphatidyl inositol was the most active pathway in lamb plasma compared to maternal plasma. This exploratory study illustrates the partitioning of lipids across maternal plasma, milk and lamb plasma and the dynamic relationship between them, reiterating the need to study these three matrices as one biological system.

Novel antioxidant peptides from sheep plasma protein hydrolysates: Purification, identification and cytoprotective effects against H2O2-induced oxidative stress.

This study sought to purify and identify antioxidant peptides from sheep (Ovis aries) plasma protein hydrolysates and assess their protective impacts on H2O2-induced Caco-2 cells. The purification process involved reversed high-performance liquid chromatography, anion-exchange chromatography, and Sephadex G-25. Three peptides, namely Trp-Glu-Glu-Pro-Ala-Met (WEEPAM), Ser-Leu-His-Phe-Met-Glu (SLHFME), and His-Cys-Thr-Thr-Phe-Met-Ile, with molecular weights of 761.84, 762.87, and 852.03Da, respectively, were identified by liquid chromatography with tandem mass spectrometry. Among the three antioxidant peptides, superoxide radical (O2 -) radical scavenging capacity of WEEPAM and SLHFME was not significantly different from glutathione (GSH) (p>0.05), while their 1,1-diphenyl-2-picrylhydrazyl radical scavenging capacity was greater than GSH (p<0.05). WEEPAM revealed increased antioxidant activity after pepsin and trypsin hydrolysis under an in vitro digestion model. In addition, WEEPAM inhibited oxidative damage in Caco-2 cells by significantly reducing reactive oxygen species accumulation, early apoptosis, malondialdehyde formation, and increasing intracellular superoxide dismutase, glutathione peroxidase, and catalase activities.

Assessment of functional efficacy of sheep plasma protein hydrolysates and their utilization in mutton sausage

The present study examines the bioactive potential of sheep plasma protein hydrolysates (SPPH) produced by in-vitro gastrointestinal digestion as antioxidants, antimicrobials, anti-obesity agents, and inhibitors of lipid oxidation in sausage to address the oxidative stability and shelf-life issues of mutton. The antioxidant and antimicrobial activities, indicate a positive relationship between the degree of hydrolysis and digestion duration. The study finds that SPPH has a potent inhibitory effect on pancreatic lipase and cholesterol esterase. It has higher oil holding capacity than sheep plasma protein, observed at one hour of hydrolysis time. SPPH exhibit an improved behavior in foaming properties along alkaline pH and digestion time while display lower emulsifying activity and stability with hydrolysis advancement. The SPPH act as a natural preservative in developing functional mutton sausage by inhibiting lipid-oxidation. This study showed that the recovery of SPPH can be a cost-effective and sustainable strategy for generating available ingredients for enhanced shelf-life of meat products.

Comparative analysis of hemostasis indices of sheep and lambs obtained taking into account body temperature

This paper shows the results of the study of the effect of temperature on the rate of coagulation processes in the blood plasma of small ruminants. The aim of the work was to compare the results of the study of blood coagulation indicators of sheep and lambs of Romanov and Edilbaev breed, obtained using medical and veterinary coagulometer, taking into account the age of the animal and its body temperature. The study was conducted in the Vologda region. The following parameters of coagulogram were analyzed: thrombin time, prothrombin time, activated partial thromboplastin time, amount and activity of fibrinogen. It was found that at physiologic temperature on veterinary coagulometer in plasma of sheep and lambs there is an increase of prothrombin time by 8,54 s and 15,24 s respectively, at that correlation-regression and one-factor dispersion analysis shows a positive moderate relationship between prothrombin time and plasma incubation temperature (r=0,3). In the plasma of lambs at the study with the setting of physiological temperature on the veterinary coagulometer there is an increase in the level of fibrinogen and its activity by 0.35 g and 4.53 sec, respectively, Correlation-regression and one-factor dispersion analysis showed a reliable high positive relationship between fibrinogen level and plasma incubation temperature (r=0,9) and a reliable high negative relationship between fibrinogen activity and plasma incubation temperature (r= -0,9). In this regard, a veterinary coagulometer with the possibility of setting the necessary temperature for adequate assessment of hemostasiological processes in sheep of different age groups can be recommended.

Extraction and analysis of moxidectin in wombat plasma and faeces

Sarcoptic mange in wombats results from a skin infestation by Sarcoptes mites and if untreated, results in a slow and painful death. Moxidectin is a pesticide used to treat internal and external parasites in cattle, but has shown to effectively treat other animals, including wombats. Two methods were developed to analyse wombat plasma, and methods were also developed to analyse faeces and fur. Moxidectin-D3 was used as an internal standard and behaved almost identically to moxidectin, resulting in recoveries of 95–105 % across the three matrices, even when matrix interferences caused signal suppression as high 20 %, or when moxidectin loss was high. This was presumably due to the high binding efficiency of plasma for MOX and MOX-D3. Moxidectin limits of detection were 0.01 ng/mL in plasma, 0.3 ng/g dry weight equivalent for faeces and 0.5 ng/g for fur. This study also developed a method to isolate plasma macromolecules, allowing the extraction of bound moxidectin for quantitative purposes, with an LoQ of 0.05 ng/mL. This method was subsequently used to determine that moxidectin was 97–99.4 % bound to lipoproteins in wombat plasma and 98–99 % bound in sheep, cow and horse plasma. The method reported for plasma was quick, cheap, and conducive to large sample batches, while providing high sensitivity. While faecal samples required additional cleanup steps to reduce the matrix effect, co-extracted matrix components such as undigested chlorophyll continued to result in ionisation suppression in the MS/MS. The methods reported here were used to monitor moxidectin in wombats treated with a single pour-on treatment, and confirmed that the moxidectin concentration in wombat plasma had decreased by more than 90 % by 28 days after application, while providing protection against sarcoptic mites over the majority of their life cycle. Clearance of moxidectin occurred via faecal elimination over the four week period and while moxidectin accumulated on fur due to application as a pour-on, concentrations declined rapidly by the four week period as fur fell out and was replaced by fresh fur that did not contain moxidectin.

Levels of Synaptic Proteins in Brain and Neurofilament Light Chain in Cerebrospinal Fluid and Plasma of OVT73 Huntington's Disease Sheep Support a Prodromal Disease State.

Synaptic changes occur early in patients with Huntington's disease (HD) and in mouse models of HD. An analysis of synaptic changes in HD transgenic sheep (OVT73) is fitting since they have been shown to have some phenotypes. They also have larger brains, longer lifespan, and greater motor and cognitive capacities more aligned with humans, and can provide abundant biofluids for in vivo monitoring of therapeutic interventions. The objective of this study was to determine if there were differences between 5- and 10-year-old OVT73 and wild-type (WT) sheep in levels of synaptic proteins in brain and in neurofilament light chain (NfL) in cerebrospinal fluid (CSF) and plasma. Mutant huntingtin (mHTT) and other proteins were measured by western blot assay in synaptosomes prepared from caudate, motor, and piriform cortex in 5-year-old and caudate, putamen, motor; and piriform cortex in 10-year-old WT and OVT73 sheep. Levels of NfL, a biomarker for neuronal damage increased in many neurological disorders including HD, were examined in CSF and plasma samples from 10-year-old WT and OVT73 sheep using the Simoa NfL Advantage kit. Western blot analysis showed mHTT protein expression in synaptosomes from OVT73 sheep was 23% of endogenous sheep HTT levels at both ages. Significant changes were detected in brain levels of PDE10A, SCN4B, DARPP32, calmodulin, SNAP25, PSD95, VGLUT 1, VAMP1, and Na+/K+-ATPase, which depended on age and brain region. There was no difference in NfL levels in CSF and plasma in OVT73 sheep compared to age-matched WT sheep. These results show that synaptic changes occur in brain of 5- and 10-year-old OVT73 sheep, but levels of NfL in biofluids are unaffected. Altogether, the data support a prodromal disease state in OVT73 sheep that involves the caudate, putamen and cortex.

Comparative Thrombin Generation in Animal Plasma: Sensitivity to Human Factor XIa and Tissue Factor.

Preclinical evaluation of drugs in animals helps researchers to select potentially informative clinical laboratory markers for human trials. To assess the utility of animal thrombin generation (TG) assay, we studied the sensitivity of animal plasmas to triggers of TG, human Tissue Factor (TF), and Activated Factor XI (FXIa). Pooled human, mouse, rat, guinea pig, rabbit, bovine, sheep, and goat plasmas were used in this study. TF- or FXIa-triggered TG and clotting were measured via fluorescence and optical density, respectively. Thrombin peak height (TPH) and time (TPT), clot time (CT), and fibrin clot density (FCD) were all analyzed. The trigger low and high sensitivity borders (LSB and HSB) for each assay parameter were defined as TF and FXIa concentrations, providing 20 and 80% of the maximal parameter value, unless the baseline (no trigger) value exceeded 20% of the maximal, in which case, LSB was derived from 120% of baseline value. Normal human samples demonstrated lower TPH HSB than most of the animal samples for both TF and FXIa. Animal samples, except mice, demonstrated lower TPT LSB for FXIa versus humans. Most rodent and rabbit samples produced baseline TG in the absence of TG triggers that were consistent with the pre-activation of blood coagulation. FCD was not sensitive to both TF and FXIa in either of the plasmas. Animal plasmas have widely variable sensitivities to human TF and FXIa, which suggests that optimization of trigger concentration is required prior to test use, and this complicates the extrapolation of animal model results to humans.

Metabolomic Analysis of Plasma in Huntington's Disease Transgenic Sheep (Ovis aries) Reveals Progressive Circadian Rhythm Dysregulation.

Metabolic abnormalities have long been predicted in Huntington's disease (HD) but remain poorly characterized. Chronobiological dysregulation has been described in HD and may include abnormalities in circadian-driven metabolism. Here we investigated metabolite profiles in the transgenic sheep model of HD (OVT73) at presymptomatic ages. Our goal was to understand changes to the metabolome as well as potential metabolite rhythm changes associated with HD. We used targeted liquid chromatography mass spectrometry (LC-MS) metabolomics to analyze metabolites in plasma samples taken from female HD transgenic and normal (control) sheep aged 5 and 7 years. Samples were taken hourly across a 27-h period. The resulting dataset was investigated by machine learning and chronobiological analysis. The metabolic profiles of HD and control sheep were separable by machine learning at both ages. We found both absolute and rhythmic differences in metabolites in HD compared to control sheep at 5 years of age. An increase in both the number of disturbed metabolites and the magnitude of change of acrophase (the time at which the rhythms peak) was seen in samples from 7-year-old HD compared to control sheep. There were striking similarities between the dysregulated metabolites identified in HD sheep and human patients (notably of phosphatidylcholines, amino acids, urea, and threonine). This work provides the first integrated analysis of changes in metabolism and circadian rhythmicity of metabolites in a large animal model of presymptomatic HD.

Comparison of SPEED, S-Trap, and In-Solution-Based Sample Preparation Methods for Mass Spectrometry in Kidney Tissue and Plasma

Mass spectrometry is a powerful technique for investigating renal pathologies and identifying biomarkers, and efficient protein extraction from kidney tissue is essential for bottom-up proteomic analyses. Detergent-based strategies aid cell lysis and protein solubilization but are poorly compatible with downstream protein digestion and liquid chromatography-coupled mass spectrometry, requiring additional purification and buffer-exchange steps. This study compares two well-established detergent-based methods for protein extraction (in-solution sodium deoxycholate (SDC); suspension trapping (S-Trap)) with the recently developed sample preparation by easy extraction and digestion (SPEED) method, which uses strong acid for denaturation. We compared the quantitative performance of each method using label-free mass spectrometry in both sheep kidney cortical tissue and plasma. In kidney tissue, SPEED quantified the most unique proteins (SPEED 1250; S-Trap 1202; SDC 1197). In plasma, S-Trap produced the most unique protein quantifications (S-Trap 150; SDC 148; SPEED 137). Protein quantifications were reproducible across biological replicates in both tissue (R2 = 0.85-0.90) and plasma (SPEED R2 = 0.84; SDC R2 = 0.76, S-Trap R2 = 0.65). Our data suggest SPEED as the optimal method for proteomic preparation in kidney tissue and S-Trap or SPEED as the optimal method for plasma, depending on whether a higher number of protein quantifications or greater reproducibility is desired.

In Vitro Detection of Virulence Factors of Candida albicans &amp; non-albicans Candida species Isolated from the Samples in ICU Patients

Aim: To isolate and identify Candida albicans and non-albicans Candida from various clinical specimen of Intensive care Unit and to investigate some virulence factors and perform their Anti- fungal susceptibility. Materials and Methods: Overall 32 isolates (12 Candida albicans and 20 non- albicans Candida spp.) were included in this study. Virulence factors were studied. In isolates, egg yolk agar was used for determining phospholipase activity, while sheep and human plasma used to determine coagulase activity, Sabouraud dextrose agar with sheep blood for hemolysin activity. Biofilm formation was detected by the tube adherence method. Results: In both Candida spp., it was found that hemolytic activity were higher next to phospholipases activity. It was also found that Coagulase activity were detected in Candida species found to be higher with sheep plasma compared to human plasma. Biofilm production was found to be less and mostly were from blood culture isolates. Conclusion: Knowledge of these virulence factors is an important tool to understand pathogenesis of candidiasis and in addition will help to explore new antifungal drug targets for improved therapeutic regimens.

RF22 | PMON10 Exposure to a Real Life Environmental Chemical Mixture Alters the Metabolite Profile in the Plasma of Sheep: An Untargeted Metabolomics Study.

Abstract Humans are exposed to a mixture of endocrine disrupting chemicals (EDCs) in everyday life, believed to contribute to a range of non-communicable diseases. While the adverse health impacts of exposure to individual EDCs have been extensively investigated, studies on effects of real-world exposures characterized by chronic low doses of a mixture of chemicals, are only recently emerging. Biosolids derived from human wastes, exemplify both the array and dosage of chemicals humans are exposed to in real life and represents a novel model to investigate the risk posed by the EDCs. Previous studies in sheep have demonstrated aberrant reproductive and metabolic phenotypic effects in offsprings, as a result of maternal biosolid exposure. We hypothesize that EDC-mediated disruption in the maternal metabolic milieu is a potential mediator of the aberrant phenotypic outcomes in offsprings in the biosolid-exposure model. Recent untargeted metabolomic studies provide evidence of the technique's ability to unravel biomarkers of both exposure and effect by identifying the disrupted endogenous metabolite profile and xenobiotic mixture, respectively. In this study, we sought to identify biosolid exposure-induced maternal metabolic fingerprints that could influence fetal programming. Ewes were grazed on either organic fertilizer or biosolid-treated pastures from 1 month before mating until close to parturition. Plasma from blood samples collected on day 90 of the 147-day gestation period, from ewes carrying singleton pregnancies (Control n=15, Treatment n=15) were subjected to untargeted metabolomics using liquid chromatography-quadrupole time-of-flight mass spectrometry. Metabolites were identified by matching the retention time, mass and isotope profile to authentic standards or metabolome databases using Agilent's MassHunter Find by Molecular Feature workflow (v7.0). Untargeted metabolomics identified 1006 and 1295 annotated features from the negative and positive mode respectively. Principal component analysis and orthogonal signal correction partial least-squares discriminant analysis discriminated the metabolomes of control sheep and sheep grazed in biosolid-treated pasture. The key differential metabolites identified by variable importance in projection score primarily belonged to the super-class organic acids, fatty acids, benzenoids, glycerophospholipids and organoheterocyclic compounds. These differentially expressed metabolites are implicated in metabolic pathways including carnitine synthesis, alanine metabolism, glucose alanine cycle, phosphatidyl choline biosynthesis and glutathione metabolism. Some of the top differential metabolites- oleamide, pyroglutamic acid and 15-HETrE have been implicated in fetal development. Oleamide, an endocannabinoid, has growth inhibitory properties. 15-HETrE modulates arachidonic acid metabolism and upregulate PPARγ expression, which plays a pivotal role in fetal development and metabolic outcomes. These findings indicate that biosolids exposure results in alterations in the maternal metabolome which likely contributes to the aberrant phenotypic outcomes reported in offspring of biosolid-exposed mothers. This is the first study to apply metabolomics to validate the effect of real-life EDC exposure in sheep, a translationally relevant precocial model with a developmental trajectory similar to humans. Presentation: Sunday, June 12, 2022 1:00 p.m. - 1:05 p.m., Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

Central nervous system distribution of buprenorphine in pregnant sheep, fetuses and newborn lambs after continuous transdermal and single subcutaneous extended-release dosing

Buprenorphine is used during pregnancy for the treatment of opioid use disorder. Limited data exist on the central nervous system (CNS) permeation and distribution, and on the fetal exposure to buprenorphine. The aim of our study was to determine the extent of buprenorphine distribution to CNS in the pregnant sheep, and their fetus at steady-state, and their newborn lambs postdelivery, using three different dosing regimens. Twenty-eight pregnant ewes in late gestation received buprenorphine via 7-day transdermal patch releasing buprenorphine 20 µg/h (n=9) or 40 µg/h (n=11), or an extended-release 8 mg/week subcutaneous injection (n=8). Plasma, cerebrospinal fluid, and CNS tissue samples were collected at steady-state from ewes and fetuses, and from lambs 0.33 – 45 hours after delivery. High accumulation of buprenorphine was observed in all CNS tissues. The median CNS/plasma concentration -ratios of buprenorphine in different CNS areas ranged between 13 and 50 in the ewes, and between 26 and 198 in the fetuses. In the ewes the CNS/plasma -ratios were similar after the three dosing regimens, but higher in the fetuses in the 40 µg/h dosing group, medians 65 – 122, than in the 20 µg/h group, medians 26 – 54. The subcutaneous injection (theoretical release rate 47.6 µg/h) produced higher concentrations than observed after 40 µg/h transdermal patch dosing. The median fetal/maternal concentration -ratios in different dosing groups ranged between 0.21 and 0.54 in plasma, and between 0.38 and 1.3 in CNS tissues, respectively, with the highest ratios observed in the spinal cord. Buprenorphine concentrations in the cerebrospinal fluid were 8 – 13 % of the concurrent plasma concentration in the ewes and 28 % in the fetuses. Buprenorphine was quantifiable in the newborn lambs’ plasma and CNS tissues two days postdelivery. Norbuprenorphine was analyzed from all plasma, cerebrospinal fluid, and CNS tissue samples but was nondetectable or below the LLOQ in most. The current study demonstrates that buprenorphine accumulates into CNS tissues at much higher concentrations than in plasma in pregnant sheep, fetuses, and their newborn lambs even 45 hours after delivery.

Development and Validation of a Sensitive HPLC Assay for Determination of Sparfloxacin According to the European Medicines Agency Guideline

Sparfloxacin, a synthetic antibiotic belonging to the third-generation fluoroquinolones, has numerous pharmacokinetic and microbiological advantages which can make it an excellent candidate for the treatment of infections in sheep. The objective of this study was to develop and validate an analytical HPLC method to quantify sparfloxacin in sheep plasma, following the recommendations set out in the Guideline on Bioanalytical Method Validation of the European Union (EMEA/CHMP/EWP/192217/2009). The HPLC mobile phase consisted of acetonitrile and monopotassium phosphate buffer (1.36 g/L) 49:51 (v/v). Genabilic acid was used as internal standard. Mean retention times for sparfloxacin and genabilic acid were 2.6 and 5.8 min, respectively. The method met all specifications of the EMA guideline, being selective and linear in the range of 0.2–10 µg/mL (R2 ≥ 0.99). Within-run precision ranged between 0.00 and 0.88%, with an accuracy of 90.3–118.0% for the lower limit of quantitation (LLOQ). The LLOQ was 0.2 µg/mL, and no interference from the biological matrix was found. The stability of sparfloxacin in the biological matrix was demonstrated under different storage conditions. Therefore, the method can be used to determine sparfloxacin concentrations in sheep plasma in different types of studies.

Effect of daily or intermittent addition of fenugreek (Trigonella foenum graecum L.) seeds to concentrate on intake, digestion, and growth performance of Barbarine lamb

An 82-day experiment was carried out to determine the response of growing lambs to the intermittent administration of fenugreek (FG) seeds (Trigonella foenum graecum L.) into concentrate. Twenty-Four 3-month-old Barbarine lambs (initial body weight 16.6 ± 0.42 kg) were divided into four equal groups (n = 6). All lambs received 600 g alfalfa pellets and 250 g concentrate containing or not 12% ground FG seeds. Control concentrate (CC) was composed (as fed) of barley (200 g), faba bean (40 g) and mineral and vitamin supplement (MVS, 14 g). The experimental concentrate (CE) contained barley (200 g), FG seeds (30 g) and MVS (14 g). The two concentrates have the same crude protein content. The control group (FG-0) received CC while groups FG-D, FG-2D and FG-4D received CE daily, or in alternation with concentrate CC at 2 or 4-day intervals (FG-2D or FG-4D, respectively). The feeding trial was followed by a 5-day in vivo digestibility trial. The frequency of distribution of FG-containing concentrate (CE) had no effect (P > 0.05) on diet intake and diet apparent digestibility of dry matter, organic matter, crude protein and cell wall (NDFom), nitrogen balance and the concentrations of urea, total protein and glucose in sheep plasma. The intermittent supply of FG seeds in concentrate tended to increase (P = 0.087)the average daily gain in FG-2D and FG-4D lambs and the microbial nitrogen synthesis (P = 0.052)with the FG administration. However, it decreased (P < 0.001) ruminal protozoa count by 33%, 44% and 66% in the FG-D, FG-2D and FG-4D lambs, respectively compared to FG-0 group. Similarly, the intermittent administration of FG seeds decreased ruminal ammonia nitrogen (NH3-N) concentrations (P < 0.001) by 28%, 31% and 35% in the FG-D, FG-2D and FG-4D, respectively. The antiprotozoal activity of FG seeds could have been induced by secondary compounds like saponins. This activity was more efficient when the frequency of incorporation of FG seeds into the concentrate is wider. It is concluded that the intermittent administration of 120 g/kg to concentrate (DM basis), had an antiprotozoal effect but failed to improve diet digestibility and growth performance of Barbarine lambs. Therefore, there is a need to emphasize further investigation to identify the active components responsible for the antiprotozoal activity of FG seeds.

Gel &amp; three-dimensional printing properties of sheep plasma protein-surimi induced by transglutaminase

The objective of the present work was to investigate the effects of transglutaminase (TGase) on rheological property, three-dimensional (3D) printing property, textural property, water holding capacity, water distribution and secondary structures of sheep plasma protein-surimi gels. The results from 3D printing experiments showed that compared with a control group, sheep plasma protein-surimi gels added with 0.2% and 0.3% TGase presented good printing properties. During the heating process, high G′ values were achieved in the formation of sheep plasma protein-surimi gels due to the enhancement of connections between proteins by TGase. Compared with the control group, the hardness, springiness, and chewiness of the 3D printed sheep plasma protein -surimi gels reached the maximum with the addition of 0.4% TGase, which increased by 15.28%, 14.09%, 32.46% respectively. Furthermore, the concentration of the added TGase was directly proportional to the breaking force of the surimi gels. Adding 0.4% TGase increased the gel strength to a maximum value of 185.04 N × mm. The water-holding capacity of the 3D printed surimi gel was also significantly increased after the addition of TGase, as evidenced by low-field nuclear magnetic resonance. The addition of TGase restrained more residual water and increased the T 21 relaxation time. The Raman spectroscopy analysis showed that the α-helix percentage increased significantly with the addition of TGase. Taken together, the results suggest that the 3D printability and gel properties of sheep plasma protein-surimi gel can be effectively improved by TGase. • Transglutaminase (TGase) improves the gel properties of surimi containing sheep plasma protein. • TGase impacts the 3D printability of sheep plasma protein-surimi. • 3D printing technology capable of creating a surimi product.

Determination of Menbutone: Development and Validation of a Sensitive HPLC Assay according to the European Medicines Agency Guideline

Menbutone is often used to stimulate the hepato-digestive activity in case of digestive disorders and hepatic insufficiency in different species (cattle, sheep, goats, pigs, horses, dogs) because it has choleretic and cholagogue effects and increases gastric and pancreatic juices. The objective was to develop and validate an HPLC method to quantify menbutone in sheep plasma using sparfloxacin as internal standard. The HPLC mobile phase consisted of acetonitrile:monopotassium phosphate solution. The method was validated according to EMA guideline (EMEA/CHMP/EWP/192217/2009). Mean retention times of menbutone and sparfloxacin were 4.5 and 2.2 min, respectively. The method met all specifications of the EMA guideline, being selective and linear in the range of 0.2–100 µg/mL (R2 ≥ 0.99). The within-run precision range was 0.19–8.21%, with an accuracy of 102.99–119.52% for the lower limit of quantitation (LLOQ). For the other values (LOW, MED, HIGH) the precision range was 0.01–4.77%, with an accuracy of 85.17–109.67%. The LLOQ was 0.2 µg/mL, and no interference from the biological matrix was found. Stability of menbutone in the biological matrix at different storage conditions was also demonstrated. Thus, the method can be used to determine menbutone concentrations in plasma sheep in different types of studies.

Proteinogram of Immunized Sheep with Detoxified Tiyus serrulatus Scorpion Venom

Background: Scorpionism is a worldwide medical issue, especially relevant in the tropical and subtropical countries. Tityus serrulatus is the species responsible for most cases in Brazil. Antivenom administration to victims is the sole specific therapy obtained from donor animals. Most of these donors suffer with symptoms of the poisoning, debilitating their health and reducing their life expectancy. The aim of the present research was to evaluate whether the immunogens prepared from the crude and detoxified venom of T. serrulatus promoted different changes in fractionated sheep plasma proteins, during a scorpion antivenom serum production.Materials, Methods &amp; Results: Twelve sheep, healthy, mean weight of 30 kg, were distributed into 3 groups (n = 4): G1 (control), G2 (crude venom) and G3 (detoxified venom). The adopted immunization protocol (first cycle) had 6 doses, 3 using Freund's adjuvant, with a 21-day interval between each one (day 0, 22 and 43), and 3 doses with no adjuvant (booster) and 0.2 mg of antigen (reinforcement), spaced 3 days between each other (day 50, 53 and 56). Group control (G1) received 6 immunizations with phosphate buffered saline (PBS) associated with Freund's adjuvant (1:1), while the other 2 groups received 0.5 mg of venom (G2) and detoxified venom (G3), respectively, diluted in PBS, associated with the Freund adjuvant. The boosters were 1/3 of the initial dose, diluted only PBS. At baseline (T0) and at 24 and 48 h after immunization, all animals underwent clinical examinations. Blood samples were collected at day 0, 22, 43, 53 and 56 for proteinogram analysis. Total protein, albumin and globulins fractions were measured. Plasma albumin concentration at T0 ranged from 3.41-4.86 g/dL, with a mean value of 4.12 g/dL. There was no statistical difference between the 3 experimental groups. The normal values determined for α-globulin range from 0.14 to 0.54 g/dL, with a mean of 0.31 g/dL (T0). There was a significant increase in the 3rd immunization and its respective interval (24-48 h), with values above normal in all groups: G1 (0.66 g/dL), G2 (0.62 g/dL) and G3 (0.65g/dL). The β-globulin was subdivided into β1 and β2 globulin. At T0, the normal values of β1 ranged from 0.45 to 1.05 g/dL, with a mean of 0.664 g/dL, and no significant change was observed in this classification. On the other hand, there was an abrupt increase in β2 in all groups after the first immunization, compared to the baseline value in T0 (0.37 g/dL mean value). From the third to the 6th immunization, there was an important reduction in β2 fraction when compared with baseline value. The γ-globulins fraction ranged from 0.80 g/dL to 76 g/dL. In the 6th immunization, there was a significant difference between G1 and the groups that received venom (G2 and G3). Therefore, all animals presented an acute inflammatory response, evidenced by the significant reduction of plasma albumin and an increase in α-globulin and β2-globulin. It is important to point out that T. serrulatus detoxified venom did not cause alterations in ovine proteinogram during the first cycle of immunization.Discussion: The fact that both groups (G2 and G3) presented acute inflammatory response, indicates that this alteration is caused by the adjuvant present in the immunization protocol. Tityus serrulatus venom detoxified with glutaraldehyde did not ~cause significant alterations in ovine proteinogram in the early stages, suggesting that itmay be used as an alternative antigen for the production of antivenom, improving clinical conditions of donor animals.Keywords: scorpion, clinical pathology, ruminants, inflammation, antiserum.Título: Proteinograma de ovelhas imunizadas com veneno detoxificado do escorpião Tityus serrulatusDescritores: escorpião, patologia clínica, ruminantes, inflamação, antisoro.

Antithrombotic Activity of Heparinoid G2 and Its Derivatives from the Clam Coelomactra antiquata.

Clam heparinoid G2 (60.25 kDa) and its depolymerized derivatives DG1 (24.48 kDa) and DG2 (6.75 kDa) prepared from Coelomactra antiquata have been documented to have excellent fibrinolytic and anticoagulant activity. In this study, to further explore the antithrombotic activity of G2, DG1 and DG2, azure A, sheep plasma, and clot lytic rate assays were used to determine their anticoagulant and thrombolytic activity in vitro. The results indicated that the anticoagulant titer of G2 was approximately 70% that of heparin and the thrombolytic activity of DG2 was greater than G2, DG1, and heparin activities. Moreover, in a carrageenan-induced venous thrombosis model, oral administration of G2 and DG1 each at 20 mg/kg and 40 mg/kg for 7 days significantly reduced blacktail thrombus formation, increased tissue-type plasminogen activator, fibrin degradation products, and D-dimer levels, decreased von Willebrand factor and thromboxane B2 levels, and restored phylum and genus abundance changes of intestinal bacteria. DG2 had no antithrombotic effect. At 20 mg/kg, G2, DG1, and heparin had comparable antithrombotic activities, and DG1 at 40 mg/kg had more muscular antithrombotic activity than G2. Thus, DG1 could be an antithrombotic oral agent owing to its more robust antithrombotic activity and lower molecular weight.