Simultaneous determination of selected ionophoric coccidiostats and amino acids in feed premixes using high-performance liquid chromatography-ultraviolet detection method.

The combination of ionophoric coccidiostats and amino acids (AAs) is important in poultry feeding to enhance immunity and improve the growth and feed efficiency of birds suffering from coccidiosis. A simple, rapid, and economical high-performance liquid chromatography-ultraviolet detection (HPLC-UV) method for the simultaneous determination of three ionophoric coccidiostats, namely salinomycin (SAL), maduramicin (MAD), and monensin (MON) in addition to three AAs; L-tryptophan (L-TRP), alpha-ketoleucin (KLEU), and L-valine (L-VAL) in feed premixes was developed and validated. Chromatographic separation was achieved in less than 12 min using a phenyl hexyl column with a mobile phase consisting of acetonitrile/methanol/water (25:20:55, v/v/v) adjusted to pH 3 using phosphoric acid. Isocratic elution was performed at a flow rate of 1 mL/min with UV detection at 210 nm. The method showed good linearity in the ranges 0.50-5.0 mg/mL for MON, 0.20-2.0 mg/mL for MAD and SAL, 10.0-100.0 µg/mL for L-TRP and KLEU, and 50.0-500.0 µg/mL for VAL. The developed method was successfully applied to determine the studied analytes in feed premixes with good recoveries and precision. The good validation criteria of the proposed method allow its utilization in quality control laboratories.

Effects of allicin on growth performance, antioxidant profile, and microbiota compared to monensin of growing goats.

Allicin is a sulfur-containing compound extracted from raw garlic (Allium sativum L.). We compared the effect of allicin addition on growth performance, serum biochemical parameters, and rumen microbiota of goats compared to monensin. Twenty-four Anhui white goats were assigned randomly to one of three dietary treatments: 1) a basal diet (CON); 2) the basal diet with allicin addition at 750 mg per head per day (AC); 3) the basal diet with monensin addition at 30 mg per kg of diet (MS). Animals were fed for 8 weeks. Results showed the average daily gain, and feed efficiency was increased with allicin and monensin addition. Serum levels of IgG, total superoxide dismutase, and glutathione peroxidase were higher in the AC group than those in the CON and MS groups. The microbiota analysis revealed that monensin addition mainly affected genera related to carbohydrate and protein metabolism, and allicin mainly affected genera related to energy metabolism and intestinal health. In conclusion, allicin could improve growth performance and have advantages over monensin in improving the antioxidant capacity and immune function of goats. Allicin may be a potential alternative to monensin.

Staphylococcus aureus mutants resistant to the feed-additive monensin show increased virulence and altered purine metabolism.

Ionophores are antibacterial compounds that affect bacterial growth by changing intracellular concentrations of the essential cations, sodium and potassium. They are extensively used in animal husbandry to increase productivity and reduce infectious diseases, but our understanding of the potential for and effects of resistance development to ionophores is poorly known. Thus, given their widespread global usage, it is important to determine the potential negative consequences of ionophore use on human and animal health. In this study, we demonstrate that exposure to the ionophore monensin can select for resistant mutants in the human and animal pathogen Staphylococcus aureus, with a majority of the resistant mutants showing increased growth rates in vitro and/or in mice. Whole-genome sequencing and proteomic analysis of the resistant mutants show that the resistance phenotype is associated with de-repression of de novo purine synthesis, which could be achieved through mutations in different transcriptional regulators including mutations in the gene purR, the repressor of the purine de novo synthesis pathway. This study shows that mutants with reduced susceptibility to the ionophore monensin can be readily selected and highlights an unexplored link between ionophore resistance, purine metabolism, and fitness in pathogenic bacteria.IMPORTANCEThis study demonstrates a novel link between ionophore resistance, purine metabolism, and virulence/fitness in the key human and animal pathogen Staphylococcus aureus. The results show that mutants with reduced susceptibility to the commonly used ionophore monensin can be readily selected and that the reduced susceptibility observed is associated with an increased expression of the de novo purine synthesis pathway. This study increases our understanding of the impact of the use of animal feed additives on both human and veterinary medicine.

Effects of cashew nut shell extract and monensin on in vitro ruminal fermentation, methane production, and ruminal bacterial community.

The objective of this study was to evaluate the effects of cashew nut shell extract (CNSE) and monensin on ruminal in vitro fermentation, CH4 production, and ruminal bacterial community structure. Treatments were as follows: control (CON, basal diet without additives); 2.5 µM monensin (MON); 0.1 mg CNSE granule/g DM (CNSE100); and 0.2 mg CNSE granule/g DM (CNSE200). Each treatment was incubated with 52 mL of buffered ruminal content and 500 mg of total mixed ration for 24 h using serum vials. The experiment was performed as a complete randomized block design with 3 runs. Run was used as a blocking factor. Each treatment had 5 replicates, in which 2 were used to determine nutrient degradability, and 3 were used to determine pH, NH3-N, volatile fatty acids, lactate, total gas, CH4 production, and bacterial community composition. Treatment responses for all data, excluding bacterial abundance, were analyzed with the GLIMMIX procedure of SAS v9.4. Treatment responses for bacterial community structure were analyzed with a PERMANOVA test run with the R package vegan. Orthogonal contrasts were used to test the effects of (1) additive inclusion (ADD: CON vs. MON, CNSE100, and CNSE200); (2) additive type (MCN: MON vs. CNSE100 and CNSE200); and (3) CNSE dose (DOS: CNSE100 vs. CNSE200). We observed that pH, acetate, and acetate:propionate ratio in the CNSE100 treatment were lower compared with CNSE200, and propionate in the CNSE100 treatment was greater compared with CNSE200. Compared with MON, CNSE treatments tended to decrease total lactate concentration. Total gas production of CON was greater by 2.63% compared with all treatments, and total CH4 production was reduced by 10.64% in both CNSE treatments compared with MON. Also, compared with MON, in vitro dry matter degradabilities in CNSE treatments were lower. No effects were observed for NH3-N or in vitro neutral detergent fiber degradability. Finally, the relative abundances of Prevotella, Treponema, and Schwartzia were lower, whereas the relative abundances of Butyrivibrio and Succinivibrio were greater in all treatments compared with CON. Overall, the inclusion of CNSE decreased CH4 production compared with MON, making CNSE a possible CH4 mitigation additive in dairy cattle diets.

Effects of monensin sodium and live attenuated oocyst vaccine as coccidiosis management programs on productive performance, bone quality and mineral utilisation in broiler chickens.

1. The following study was conducted to evaluate the influence of coccidiosis vaccine-induced metabolic stress on the utilisation of minerals in broilers. The starter, grower and finisher phase diets, including macro- and micro minerals at the recommended levels for the breed standards, were fed to chickens between 1 and 39 d of age.2. A total of 486, one-d-old male broilers were randomly distributed into three coccidiosis management programs (CMP) with six replications each. The CMP comprised: monensin sodium (MON), coccidiosis vaccine (VAC), not treated with MON or VAC (CNT).3. No significant differences between CMP were observed for body weight and weight gain among treatments. When compared to the CNT, the VAC program increased feed intake (P < 0.05) between d 1 to 13 and 14 to 26, while FCR worsened in the latter (P < 0.05) and the former (P = 0.05) periods.4. For birds in the MON and VAC programs, tibia bone length at d 13 and bone diameter at d 39 were both enhanced (P < 0.05). Meat yield characteristics were comparable among the CMP.5. Faeces of VAC birds had a lower (P < 0.05) dry matter and ash content than those in CNT program. CMP had no effect on serum or bone mineral concentrations at any point in time. For minerals, Mg, Na, and K faecal excretion was reduced (P < 0.01) as a result of the VAC program at d 13 with a trend at d 26.6. Compared to the CNT, the VAC program decreased the percentage ratio of drip loss (P = 0.08), water holding capacity (P < 0.01) and cooking loss (P < 0.01) in breast meat.7. Overall, the results showed that current broiler industry practices are capable of meeting the mineral needs of broilers vaccinated against coccidiosis.

Effects of monensin supplementation on rumen fermentation, methane emissions, nitrogen balance, and metabolic responses of dairy cows: A systematic review and dose-response meta-analysis.

To investigate the effects of supplemental monensin administration on the metabolic responses of dairy cows, a systematic review and dose-response meta-analysis were conducted. Initially, 604 studies were identified through comprehensive database searches, including Google Scholar, Scopus, Science Direct, and PubMed, using key words related to dairy cows, monensin, and metabolic outcomes. After a 2-stage screening process, 51 articles with a total of 60 experiments were selected for meta-analysis based on criteria such as study implementation date between 2001 and 2022, presence of a control group that did not receive monensin supplementation, reporting of at least 1 outcome variable, and presentation of means and corresponding errors. The meta-analysis used the 1-stage random-effects method, and sensitivity analyses were performed to assess the robustness of the results. The results showed that the administration of monensin at a dosage of 19 to 26 mg/kg was inversely related to methane emissions and that the administration of monensin at a dosage of 18 to 50 mg/kg resulted in a significant decrease in dry matter intake. Administration of monensin at doses of 13 to 28 and 15 to 24 mg/kg also resulted in a significant decrease in ruminal acetate proportion and an increase in propionate proportion, respectively, with no effects on ruminal butyrate, NH3, or pH levels. We found no effects on blood parameters or nitrogen retention, but a significant negative correlation was observed between monensin supplementation and fecal nitrogen excretion. Based on the analysis of all variables evaluated, the optimal dose range of monensin was estimated to be 19 to 24 mg/kg.

Effect of monensin on milk production efficiency and milk composition in lactating dairy cows fed modern diets.

Since the US Food and Drug Administration's approval of monensin in 2004, significant nutritional advances have been made to increase feed efficiency and milk fat production. Recent evidence suggests monensin's adverse effect on milk fat percentage may be absent when diets are formulated to address known diet-induced milk fat depression risk factors. Thus, study objectives were to evaluate effects of monensin level on dry matter intake (DMI), milk production and composition, and efficiency of high-producing cows fed diets formulated to optimize milk fat. Ninety-six lactating Holstein cows (36 primiparous, 60 multiparous; 106 ± 17 d in milk [DIM]) were balanced by parity, DIM, and milk production and were randomly assigned to 1 of 12 pens with 8 cows per pen. All cows received 11 g/t monensin for 5 wk after which pens received 1 of 4 dietary treatments (n = 3) formulated to provide 0 (CON), 11 (R11), 14.5 (R14.5), or 18 (R18) g/t monensin for 9 wk. The basal diet was 54% forage, 27% NDF, 29% starch, and 2.3% rumen unsaturated fatty acid load. Pen was the experimental unit and data were analyzed using the Fit Model Procedure of JMP. Effects of treatment, time, and treatment × time interaction were included as fixed effects and pen as a random effect. Least squares means were determined and linear and quadratic contrasts were tested. Dry matter intake tended to decrease linearly with increasing monensin dose. Milk yield, fat percentage, and protein percentage and yield were unaffected by treatment while fat yield was quadratically increased. Milk de novo and mixed fatty acid (FA) yields (g/d) increased quadratically with monensin whereas preformed FA linearly decreased during the experimental period. Energy-corrected milk (ECM) was quadratically increased by monensin. Milk urea nitrogen concentrations increased linearly with increasing monensin dose. Monensin linearly increased feed efficiency (ECM/DMI, 3.5% fat-corrected milk/DMI, and solids-corrected milk/DMI). Body weight gain did not differ between treatments. Estimated dietary energy tended to increase linearly with increasing monensin level. These data suggest monensin improves component-corrected milk production efficiency, estimated dietary energy, and does not negatively affect milk fat percentage or FA profile.

Monensin poisoning outbreak in free-ranging and captive birds.

Monensin poisoning is uncommon and has been rarely reported in birds. This work aimed to described clinical-pathological aspects of an outbreak of monensin poisoning in captive and free-ranging birds. Thirty-seven of 600 captive birds fed a diet containing 893.19 mg/kg of monensin died within 10 days (mortality 6.17%). There was no ionophore antibiotics on the feed label supplied to captive birds, which established an error in feed production. Necropsies were performed on twelve animals: Muscovy duck (Cairina moschata) (2/12), greater rhea (Rhea americana) (2/12), black-necked swan (Cygnus melancoryphus) (2/12), garganey (Anas querquedula) (1/12), ostrich (Struthio camelus) (1/12), and common pigeon (Columbus livia) (4/12). These four common pigeons were free-ranging birds and died after eating the same contaminated feed. Birds were mainly found dead, however in animals which clinical signs were observed (Columba livia, Rhea americana, Cairina moschata, Anas querquedula, and Struthio camelus), they included incoordination, inability to stand, and intense prostration, that ranged from 24 to 72 h until death. Grossly, five birds had focally extensive pale firm areas in the myocardium and two had in the skeletal muscles, one being concomitant lesions. Histologically, muscle necrosis and degeneration were observed in striated musculature (skeletal and/or heart) in all birds analyzed. Monensin poisoning outbreaks can affect free-ranging birds that are fed on external feeders, as well as captive birds, due to an error in the feed formulation.

Broad spectrum post-entry inhibitors of coronavirus replication: Cardiotonic steroids and monensin.

A small molecule screen identified several cardiotonic steroids (digitoxin and ouabain) and the ionophore monensin as potent inhibitors of HCoV-229E, HCoV-OC43, and SARS-CoV-2 replication with EC50s in the low nM range. Subsequent tests confirmed antiviral activity in primary cell models including human nasal epithelial cells and lung organoids. Addition of digitoxin, ouabain, or monensin strongly reduced viral gene expression as measured by both viral protein and RNA accumulation. Furthermore, the compounds acted post virus entry. While the antiviral activity of digitoxin was dependent upon activation of the MEK and JNK signaling pathways but not signaling through GPCRs, the antiviral effect of monensin was reversed upon inhibition of several signaling pathways. Together, the data demonstrates the potent anti-coronavirus properties of two classes of FDA approved drugs that function by altering the properties of the infected cell, rendering it unable to support virus replication.

Effects of a novel direct-fed microbial on growth performance, carcass characteristics, nutrient digestibility, and ruminal morphology of beef feedlot steers.

The effects of a novel direct-fed microbial (DFM) on feedlot performance, carcass characteristics, digestibility, ruminal morphology, and volatile fatty acid (VFA) profile of finishing steers were evaluated. Single-source Angus-crossbred yearling steers (n = 144; initial body weight (BW) = 371 ±â€…19 kg) were used in a randomized complete block design. Steers were blocked by initial BW and randomly assigned to treatments (12 pens/treatment; 4 steers/pen). Treatments included (A) CONTROL (no DFM, tylosin, or monensin, (B) MONTY (monensin sodium [330 mg/animal-daily] and tylosin phosphate [90 mg/animal-daily]), and (C) MONPRO (monensin sodium [same as previous] and Lactobacillus salivarius L28 [1 × 106 CFU/animal-daily]). Treatments were included in a steam-flaked corn-based finisher diet offered once daily using a clean-bunk management for ~149 d. The digestibility assessment was performed from days 70 to 74. Ruminal fluid and rumen tissue samples were collected at the slaughter for VFA profile and papillae morphology analyses, respectively. Data were analyzed using the GLIMMIX procedure of SAS with pen serving as the experimental unit, treatment as fixed effect, and BW block as random effect. Steers offered MONPRO had on average 5.3% less (P < 0.01) dry matter intake (9.56 kg/d) compared with either CONTROL (10.16 kg/d) or MONTY (9.96 kg/d). The carcass-adjusted final BW (613 kg; P = 0.23), overall average daily gain (1.64 kg/d; P = 0.23), and gain-efficiency (0.165; P = 0.61) were not affected by treatments. Steers offered CONTROL had greater (P < 0.01) marbling score and tended (P = 0.06) to have less carcasses grading Select and tended (P = 0.10) to have more carcasses grading Upper-Choice, while other carcass characteristics and liver-abscesses were not affected (P ≥ 0.23) by treatments. The digestibility of nutrients (P ≥ 0.13) and the ruminal VFA profile (P ≥ 0.12) were not affected by treatments. Steers offered MONPRO tended (P = 0.09) to have 16% greater average papillae number compared to other treatments. Yearlings offered finishing diets containing L. salivarius L28 plus monensin did not affect growth performance, digestibility, or ruminal VFA, but reduced feed intake. Carcass quality was negatively affected by treatments, while animals consuming L. salivarius L28 and monensin tended to improve ruminal morphology. Current findings in ruminal morphology and feed intake may warrant further assessment of diets containing L. salivarius L28 on beef cattle food safety aspects.

Antimicrobial resistance is a growing concern to public health and medically important antibiotics have been listed in the Veterinary Feed Directive. Nutritional technologies, such as direct-fed microbials, are being increasingly studied for the development of an effective use on beef cattle production systems. The newly isolated strain of Lactobacillus salivarius L28 has demonstrated pathogenic inhibition of Escherichia coli, Salmonella, and Listeria monocytogenes on in vitro assessments. The potential benefits have warranted the exploration of L. salivarius L28 in a feedlot setting. Single-source Angus-crossbred yearling steers were offered steam-flaked corn-based finishing diets containing no feed additive, or either a combination of tylosin plus monensin or L. salivarius L28 plus monensin. Steers offered L. salivarius L28 plus monensin consumed 5.3% less feed compared with other treatments, while other growth performance variables and the digestibility of nutrients were not affected. Carcasses from cattle supplemented with monensin had slightly lower carcass quality grades than those not supplemented with monensin. Lactobacillus salivarius L28 plus monensin tended to improve steers ruminal morphology. Current findings may warrant further food safety assessments when cattle are offered diets containing L. salivarius L28.

Luminescent reporter cells enable the identification of broad-spectrum antivirals against emerging viruses.

The emerging viruses SARS-CoV-2 and arenaviruses cause severe respiratory and hemorrhagic diseases, respectively. The production of infectious particles of both viruses and virus spread in tissues requires cleavage of surface glycoproteins (GPs) by host proprotein convertases (PCs). SARS-CoV-2 and arenaviruses rely on GP cleavage by PCs furin and subtilisin kexin isozyme-1/site-1 protease (SKI-1/S1P), respectively. We report improved luciferase-based reporter cell lines, named luminescent inducible proprotein convertase reporter cells that we employ to monitor PC activity in its authentic subcellular compartment. Using these sensor lines we screened a small compound library in high-throughput manner. We identified 23 FDA-approved small molecules, among them monensin which displayed broad activity against furin and SKI-1/S1P. Monensin inhibited arenaviruses and SARS-CoV-2 in a dose-dependent manner. We observed a strong reduction in infectious particle release upon monensin treatment with little effect on released genome copies. This was reflected by inhibition of SARS-CoV-2 spike processing suggesting the release of immature particles. In a proof of concept experiment using human precision cut lung slices, monensin potently inhibited SARS-CoV-2 infection, evidenced by reduced infectious particle release. We propose that our PC sensor pipeline is a suitable tool to identify broad-spectrum antivirals with therapeutic potential to combat current and future emerging viruses.

Accidental monensin poisoning in goats.

Sodium monensin is the most frequently used ionophore as a growth promoter in ruminant diets. It has numerous benefits; however its toxic effects have also been observed in several animal species. Naturally occurring cases have not yet been reported in goats. This study describes an outbreak of accidental poisoning, characterizing its clinical, laboratory and pathological findings. Thirty-seven of 40 Anglo Nubian goat kids became intoxicated after receiving a diet that was erroneously supplemented with sodium monensin. They ingested an estimated toxic dose between 25 and 39 mg/kg BW. Clinical evolution was monitored (n = 27), followed by serum creatine kinase (CK) and aspartate aminotransferase (AST) activities measurements, and blood gas analysis. Postmortem examinations were performed between 1 and 8 days of evolution (n = 14). Clinical signs began 5 h after ingestion and included reticuloruminal hypomotility, lethargy, anorexia, tachycardia, cardiac arrhythmia, wet cough, pulmonary and tracheal crackles, and serous nasal discharge. The morbidity and lethality rates were 92.5 and 62.1%, respectively. CK and AST activities increased, reaching median values of 10,860 and 1596 U/L, respectively; the hyperchloremic metabolic acidosis was mild. The lesions were characterized by degeneration and necrosis of the cardiac and skeletal muscles, pulmonary congestion and edema, and passive liver congestion. The kids essentially developed cardiomyopathy with left and right congestive heart failures. Unlike in other ruminant species, skeletal muscle functional disability was infrequent. It can be concluded that monensin is toxic to goats and should be used with caution in their diet.

Triepoxide formation by a flavin-dependent monooxygenase in monensin biosynthesis.

Monensin A is a prototypical natural polyether polyketide antibiotic. It acts by binding a metal cation and facilitating its transport across the cell membrane. Biosynthesis of monensin A involves construction of a polyene polyketide backbone, subsequent epoxidation of the alkenes, and, lastly, formation of cyclic ethers via epoxide-opening cyclization. MonCI, a flavin-dependent monooxygenase, is thought to transform all three alkenes in the intermediate polyketide premonensin A into epoxides. Our crystallographic study has revealed that MonCI's exquisite stereocontrol is due to the preorganization of the active site residues which allows only one specific face of the alkene to approach the reactive C(4a)-hydroperoxyflavin moiety. Furthermore, MonCI has an unusually large substrate-binding cavity that can accommodate premonensin A in an extended or folded conformation which allows any of the three alkenes to be placed next to C(4a)-hydroperoxyflavin. MonCI, with its ability to perform multiple epoxidations on the same substrate in a stereospecific manner, demonstrates the extraordinary versatility of the flavin-dependent monooxygenase family of enzymes.

Effects of monensin in supplements for beef cattle in tropical grazing systems during the rainy season.

Two experiments were carried out to evaluate the effect of monensin in supplements for grazing heifers. In experiment I, treatments consisted of protein supplements (low intake - 0.5 kg/animal/day and high intake - 1.0 kg/animal/day) associated or not with monensin. Animal performance, nutrient intake, and digestibility were evaluated. Forty crossbred heifers with an initial body weight (BW) of 213.8 ± 4.5 kg were used. There was no difference in average daily gain between treatments (average of 0.588 kg/animal/day). There was no interaction between monensin and supplements for intake parameters and digestibility. Dry matter (DM) intake was equal between treatments (~ 2% of BW). However, there was a reduction in pasture intake with the high-intake supplement. In experiment II, treatments consisted of two types of supplements (protein or mineral) associated or not with monensin. The variables analyzed were productive performance, ingestive behavior, and thermal comfort, evaluated through the index of thermal stress for cows (ITSC). Forty crossbred heifers with a BW of 159.2 ± 1.3 kg were used. The type of supplement did not influence the performance of the animals. However, monensin promoted greater weight gain in the animals (110 g/animal/day). There was an interaction effect between supplementation and monensin addition on behavioral activities, except for idle time. The inclusion of monensin in the protein supplement reduced the grazing time. The ITSC value influenced the activities of ingestive behavior, and the increase of this index reduced the grazing time in all treatments. Forage quality influences the response to monensin use, and the addition of monensin in supplements for grazing cattle during the rainy season is recommended for forages with high CP content.

Occurrence and persistence of antibiotics administered to cattle in a newly established feedlot.

The practice of using therapeutic and prophylactic veterinary antibiotics in livestock farming is a worldwide phenomenon. Over the last decade, there has been a growing concern of antibiotic residues entering the environment via animal manure. Similar studies have focused on the occurrence and biological effects of antibiotics in land-applied animal feedlots; however, limited research has been conducted on the occurrence and persistence of antibiotics in animal feedlots. Therefore, the objective of this study was to evaluate antibiotic persistence, fate, and transport in surface water runoff and feedlot sediment in feedlot pens with livestock either receiving or not receiving antibiotic treatments through injection and feed. The two antibiotics (tylosin and monensin) added to animal feed were observed to persist in the soil environment for more than 30 days along with injected florfenicol. Monensin (5.6× higher) and tylosin (20× higher) were significantly higher in livestock pens receiving antibiotics compared to livestock pens not receiving the antibiotics. Further, rainfall was observed to significantly impact soil surface concentrations of florfenicol. Other antibiotics administrated by injection were not observed to statistically increase in concentrations in runoff or feedlot sediment. Our findings emphasize antibiotics administered in feedlots have the potential to persist and remain in feedlot sediment and runoff, particularly in instances of regular administration in feed.

Effects of cashew nutshell extract and monensin on microbial fermentation in a dual-flow continuous culture.

The objective of this study was to compare cashew nutshell extract (CNSE) to monensin and evaluate changes in in vitro mixed ruminal microorganism fermentation, nutrient digestibility, and microbial nitrogen outflow. Treatments were randomly assigned to 8 fermenters in a replicated 4 × 4 Latin square design with 4 experimental periods of 10 d (7 d for diet adaptation and 3 d for sample collection). Basal diets contained 43.5:56.5 forage: concentrate ratio and each fermenter was fed 106 g of DM/d divided equally between 2 feeding times. Treatments were control (CON, basal diet without additives), 2.5 µM monensin (MON), 0.1 mg CNSE granule/g DM (CNSE100), and 0.2 mg CNSE granule/g DM (CNSE200). On d 8 to10, samples were collected for pH, lactate, NH3-N, volatile fatty acids (VFA), mixed protozoa counts, organic matter (OM), and neutral detergent fiber (NDF) digestibility. Data were analyzed with the GLIMMIX procedure of SAS. Orthogonal contrasts were used to test the effects of (1) ADD (CON vs. MON, CNSE100, and CNSE200); (2) MCN (MON vs. CNSE100 and CNSE200); and (3) DOSE (CNSE100 vs. CNSE200). We observed that butyrate concentration in all treatments was lower compared with CON and the concentration for MON was lower compared with CNSE treatments. Protozoal population in all treatments was lower compared with CON. No effects were observed for pH, lactate, NH3-N, total VFA, OM, or N utilization. Within the 24-h pool, protozoal generation time, tended to be lower, while NDF digestibility tended to be greater in response to all additives. Furthermore, the microbial N flow, and the efficiency of N use tended to be lower for the monensin treatment compared with CNSE treatments. Overall, our results showed that both monensin and CNSE decreased butyrate synthesis and protozoal populations, while not affecting OM digestibility and tended to increase NDF digestibility; however, such effects are greater with monensin than CNSE nutshell.

Effects of feed additives on rumen function and bacterial and archaeal communities during a starch and fructose challenge.

The objective of this study was to improve understandings of the rumen microbial ecosystem during ruminal acidosis and responses to feed additives to improve prudent use strategies for ruminal acidosis control. Rumen bacterial and archaeal community composition (BCC) and its associations with rumen fermentation measures were examined in Holstein heifers fed feed additives and challenged with starch and fructose. Heifers (n = 40) were randomly allocated to 5 treatment groups: (1) control (no additives); (2) virginiamycin (VM; 200 mg/d); (3) monensin (MT; 200 mg/d) + tylosin (110 mg/d); (4) monensin (MLY; 220 mg/d) + live yeast (5.0 × 1012 cfu/d); (5) sodium bicarbonate (BUF; 200 g/d) + magnesium oxide (30 g/d). Heifers were fed twice daily a 62% forage:38% concentrate total mixed ration at 1.25% of body weight (BW) dry matter (DM)/d for a 20-d adaptation period with their additive(s). Fructose (0.1% of BW/d) was added to the ration for the last 10 d of adaptation. On d 21 heifers were challenged once with a ration consisting of 1.0% of BW DM wheat and 0.2% of BW fructose plus their additive(s). A rumen sample was collected from each heifer via stomach tube weekly (d 0, 7, 14) and 5 times over a 3.6 h period at 5, 65, 115, 165, and 215 min after consumption of the challenge ration (d 21) and analyzed for pH, and ammonia, d- and l-lactate, volatile fatty acids (VFA), and histamine concentrations and total bacteria and archaea. The 16S rRNA gene spanning the V4 region was PCR amplified and sequenced. Alpha and ß diversity and associations of relative abundances of taxa with rumen fermentation measures were evaluated. Rumen BCC shifted among treatment groups in the adaptation period and across the challenge sampling period, indicating the feed additives had different modes of action. The monensin-containing treatment groups, MT and MLY often had similar relative abundances of rumen bacterial phyla and families. The MLY treatment group was characterized in the challenge period by increased relative abundances of the lactate utilizing genera Anaerovibrio and Megasphaera. The MLY treatment group also had increased diversity of ruminal bacteria which may provide resilience to changes in substrates. The control and BUF treatment groups were most similar in BCC. A redundancy analysis showed the MLY treatment group differed from all other treatment groups and concentrations of histamine and valerate in the rumen were associated with the most variation in the microbiota, 5.3% and 4.8%, respectively. It was evident from the taxa common to all treatment groups that cattle have a core microbiota. Functional redundancy of rumen bacteria which was reflected in the greater sensitivity for the rumen BCC than rumen fermentation measures likely provide resilience to changes in substrate. This functional redundancy of microbes in cattle suggests that there is no single optimal ruminal microbial population and no universally superior feed additive(s). In summary, differences in modes of action suggest the potential for more targeted and improved prudent use of feed additives with no single feed additive(s) providing an optimal BCC in all heifers.

Monensin induces secretory granule-mediated cell death in eosinophils.

BACKGROUND: Eosinophils contribute to the pathology of several types of disorders, in particular of allergic nature, and strategies to limit their actions are therefore warranted. OBJECTIVE: We sought to evaluate the possibility of targeting the acidic, lysosome-like eosinophil granules as a potential means of inducing eosinophil cell death. METHODS: To this end, we used monensin, an ionophoric drug that has previously been shown to permeabilize the secretory granules of mast cells, thereby inducing cell death. RESULTS: Our findings reveal that monensin induces cell death in human eosinophils, whereas neutrophils were less affected. Blockade of granule acidification reduced the effect of monensin on the eosinophils, demonstrating that granule acidity is an important factor in the mechanism of cell death. Furthermore, monensin caused an elevation of the granule pH, which was accompanied by a decrease of the cytosolic pH, hence indicating that monensin caused leakage of acidic contents from the granules into the cytosol. In agreement with a granule-targeting mechanism, transmission electron microscopy analysis revealed that monensin caused extensive morphological alterations of the eosinophil granules, as manifested by a marked loss of electron density. Eosinophil cell death in response to monensin was caspase-independent, but dependent on granzyme B, a pro-apoptotic serine protease known to be expressed by eosinophils. CONCLUSIONS: We conclude that monensin causes cell death of human eosinophils through a granule-mediated mechanism dependent on granzyme B.

Restricted maternal nutrition and supplementation of propylene glycol, monensin sodium and rumen-protected choline chloride during late pregnancy does not affect muscle fibre characteristics of offspring.

BACKGROUND: Grazing in arid and semi-arid regions faces pregnant ewes with feed restrictions and hence affects the offspring muscle fibre characteristics. Using feed additives that enhance nutrient availability during foetal muscle development is expected to alter offspring skeletal muscle characteristics. OBJECTIVES: This study evaluated the effect of maternal restricted nutrition and supplementation of propylene glycol, monensin sodium and rumen-protected choline chloride on lamb's muscle fibre characteristics. METHODS: Forty-eight Ghezel ewes were randomly allocated to one of six diets (N = 8) during the last 6 weeks of gestation: ad libitum feed intake (AL); restricted feeding (RF); restricted feeding containing propylene glycol (PG); restricted feeding containing propylene glycol and monensin sodium (MS); restricted feeding containing propylene glycol and rumen-protected choline chloride (RPC); restricted feeding containing propylene glycol, monensin sodium and rumen-protected choline chloride (PMC). The muscle samples were obtained from the semitendinosus muscle of 2-week-old male lambs (n = 5/treatment) via biopsy and were stained and classified as fibre types I, IIA and IIB. RESULTS: Pre-parturient maternal feed restriction and administration of propylene glycol, monensin sodium and rumen-protected choline chloride had no significant effect on fibre-type composition, fibre density of muscle, muscle cross-sectional area and volume density of fibres (p > 0.05). CONCLUSIONS: Either maternal dietary restriction or supplementation of nutrient flux-involved additives during late pregnancy did not alter muscle fibre development and had no short-term effects on muscle properties of the resulting offspring as myogenesis occurs in early and mid-gestation, not late gestation. Therefore, maternal nutrition may not be a problematic issue in sheep production in arid and semi-arid areas.

Repurposing Polyether Ionophores as a New-Class of Anti-SARS-Cov-2 Agents as Adjunct Therapy.

The emergence of SARS-CoV-2 and its variants have posed a significant threat to humankind in tackling the viral spread. Furthermore, currently repurposed drugs and frontline antiviral agents have failed to cure severe ongoing infections effectively. This insufficiency has fuelled research for potent and safe therapeutic agents to treat COVID-19. Nonetheless, various vaccine candidates have displayed a differential efficacy and need for repetitive dosing. The FDA-approved polyether ionophore veterinary antibiotic for treating coccidiosis has been repurposed for treating SARS-CoV-2 infection (as shown by both in vitro and in vivo studies) and other deadly human viruses. Based on selectivity index values, ionophores display therapeutic effects at sub-nanomolar concentrations and exhibit selective killing ability. They act on different viral targets (structural and non-structural proteins), host-cell components leading to SARS-CoV-2 inhibition, and their activity is further enhanced by Zn2+ supplementation. This review summarizes the anti-SARS-CoV-2 potential and molecular viral targets of selective ionophores like monensin, salinomycin, maduramicin, CP-80,219, nanchangmycin, narasin, X-206 and valinomycin. Ionophore combinations with Zn2+ are a new therapeutic strategy that warrants further investigation for possible human benefits.