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.

The effects of antibiotic-free supplementation on the ruminal pH variability and methane emissions of beef cattle under the challenge of subacute ruminal acidosis (SARA).

Subacute ruminal acidosis (SARA) in feedlot cattle during the feed transition to grain-based diets is a significant constraint to animal health and productivity. This experiment assessed an antibiotic-free supplement (ProTect®) effects on ruminal pH variability and methane (CH4) emissions of cattle during the challenge of SARA. Ten 18-month-old Angus steers (472 ± 4.8 kg) were randomly allocated into monensin (n = 5) and ProTect® groups (n = 5) and progressively introduced to grain diets incorporating monensin or ProTect® for 36 days of the experiment [starter (7 days; 45% grain), T1 (7 days; 56% grain), T2 (7 days; 67% grain), finisher (15 days; 78% grain)]. The pH variability on the finisher period was reduced by the ProTect® supplement (6.6% vs. 5.2%; P < 0.01), with CH4 emissions being significantly higher relative to the monensin group [88.2 g/day (9.3 g CH4/kg DMI) vs. 133.7 g/day (14.1 g CH4/kg DMI); P < 0.01]. There was no difference between treatments in the time spent on the ruminal pH < 5.6 or < 5.8 (P > 0.05). The model evaluation for the ruminal pH variation indicated that the mean absolute error (MAE) proportion for both groups was good within the same range [4.05% (monensin) vs. 4.25% (ProTect®)] with identical root mean square prediction error (RMSPE) (0.34). It is concluded that the ProTect® supplement is an effective alternative to monensin for preventing SARA in feedlot cattle by managing ruminal pH variation during the transition to high-grain diets. Both monensin and ProTect® supplemented cattle exhibited lower CH4 yield compared to cattle fed forages and low-concentrate diets.

Monensin supplementation downregulated the expression signature of genes involved in cholesterol synthesis in the ruminal epithelium and adipose tissue of lambs.

To understand the metabolic mechanisms regulating lipid metabolism by monensin, Afshari male lambs (n = 16) with 41.0 ± 2.4 kg body weight (BW, mean ± SD) at approximately 180 days of age were randomly assigned equally to two dietary treatments. After a 21-day pre-adaptation period, all animals in two groups continued to receive the basal diet, but one group received no monensin supplementation (control) while the other group received 30 mg/day of monensin per animal. Individual BW was recorded weekly to determine the average daily body weight gain (ADG). At the end of the 56-day experimental period, lambs were weighed and slaughtered. Monensin supplementation did not affect BW, ADG, and rumen fermentation characteristics. However, monensin significantly downregulated the sterol regulatory element-binding protein (SREBP)-2 gene expression in all sample tissues (p < 0.05). Also, monensin downregulated expressions of SREBP-1c and peroxisome proliferator-activated receptor (PPAR)-γ in back fat tissues. Monensin increased the expression of 3-hydroxy-3-methylglutaryl-CoA synthase (HMGCS)-2, but it decreased the mRNA abundance of HMGCS-1 in the rumen epithelial tissues (p < 0.05). Our data suggest that monensin downregulates cholesterol synthesis via inhibition of HMGCS-1 and impairment of the SREBP pathway, probably due to a crosstalk among different tissues to control energy metabolism.

Prosopis juliflora piperidine alkaloid extract levels in diet for sheep change energy and nitrogen metabolism and affect enteric methane yield.

BACKGROUND: Ionophore antibiotics improve the efficiency of energy metabolism, which has driven their use as a feed additive in ruminants for decades. Currently, they have not been approved in many countries, generating a challenge for the immediate search for plant extracts with a similar mode of action on rumen metabolism. This study evaluated the effects of enriched Prosopis juliflora (mesquite) piperidine alkaloid extract (MPA) levels as an alternative phytoadditive to sodium monensin (MON) in sheep. RESULTS: The MPA diet did not differ from MON with regard to nutrient intake. A quadratic effect (P < 0.05) was observed for organic matter and neutral detergent fibre digestibility, with respective maximum point at 25.40 and minimum point at 0.95 mg kg-1 MPA. The MPA levels linearly decreased (P < 0.05) faecal nitrogen loss. MPA did not differ from MON with regard to nutrient digestibility, and MPA levels increased (P < 0.05) the proportion of digestible energy and metabolizability from dietary gross energy. The MPA levels linearly decreased (P < 0.05) enteric CH4 production, the yield showing lower (P < 0.05) energy loss as CH4 than MON. CONCLUSION: The results show that MPA levels of 17.3 and 27.8 mg kg-1 are enteric CH4 inhibitors and enhance energy and protein utilization, indicating a promising alternative to MON for ruminants. © 2022 Society of Chemical Industry.

Supplementation of Ascophyllum nodosum meal and monensin: Effects on diversity and relative abundance of ruminal bacterial taxa and the metabolism of iodine and arsenic in lactating dairy cows.

Previous research has shown that the brown seaweed Ascophyllum nodosum (ASCO) has antimicrobial and antioxidant properties and also increases milk I concentration. We aimed to investigate the effects of supplementing ASCO meal or monensin (MON) on ruminal fermentation, diversity and relative abundance of ruminal bacterial taxa, metabolism of I and As, and blood concentrations of thyroid hormones, antioxidant enzymes, and cortisol in lactating dairy cows. Five multiparous ruminally cannulated Jersey cows averaging (mean ± standard deviation) 102 ± 15 d in milk and 450 ± 33 kg of body weight at the beginning of the study were used in a Latin square design with 28-d periods (21 d for diet adaptation and 7 d for data and sample collection). Cows were fed ad libitum a basal diet containing (dry matter basis) 65% forage as haylage and corn silage and 35% concentrate and were randomly assigned to 1 of the following 5 dietary treatments: 0, 57, 113, or 170 g/d of ASCO meal, or 300 mg/d of MON. Supplements were placed directly into the rumen once daily after the morning feeding. Diets had no effect on ruminal pH and NH3-N concentration, which averaged 6.02 and 6.86 mg/dL, respectively. Total volatile fatty acid concentration decreased linearly in cows fed incremental amounts of ASCO meal. Supplementation with ASCO meal did not change the ruminal molar proportions of volatile fatty acids apart from butyrate, which responded quadratically with the lowest values observed at 56 and 113 g/d of ASCO supplementation. Compared with the control diet or diets containing ASCO meal, cows fed MON showed greater molar proportion of propionate. Diets did not affect the α diversity indices Shannon, Simpson, and Fisher for ruminal bacteria. However, feeding incremental levels of ASCO meal linearly decreased the relative abundance of Tenericutes in ruminal fluid. Monensin increased the relative abundance of the CAG:352 bacterial genus in ruminal fluid compared with the control diet. Linear increases in response to ASCO meal supplementation were observed for the concentrations and output of I in serum, milk, urine, and feces. Fecal excretion of As increased linearly in cows fed varying amounts of ASCO meal, but ASCO did not affect the concentration and secretion of As in milk. The plasma activities of the antioxidant enzymes and the serum concentrations of thyroid hormones did not change. In contrast, circulating cortisol decreased linearly in diets containing ASCO meal. The apparent total-tract digestibilities of dry matter, organic matter, and crude protein increased linearly with ASCO meal, but those of neutral and acid detergent fiber were not affected. In summary, feeding incremental amounts of ASCO meal decreased serum cortisol concentration, and increased I concentrations and output in serum, milk, feces, and urine.

Effects of lysophospholipids on short-term production, nitrogen utilization, and rumen fermentation and bacterial population in lactating dairy cows.

An experiment was conducted to examine effects of supplemental lysophospholipids (LPL) in dairy cows. Eight ruminally cannulated lactating Holstein cows were used in a replicated 4 × 4 Latin square design. Dietary treatments were (1) a dairy ration [CON; 55% forage and 45% concentrate on a dry matter (DM) basis], (2) a positive control diet supplemented with monensin (MON; 16 mg/kg in dietary DM; Elanco Animal Health, Greenfield, IN], (3) a control diet supplemented with low LPL (0.05% of dietary DM; Lipidol Ultra, Easy Bio Inc., Seoul, South Korea), and (4) a control diet supplemented with high LPL (0.075% of dietary DM). Experimental periods were 21 d with 14-d diet adaptation and 7-d sample collection. Daily intake and milk yield were measured and rumen contents were collected for fermentation characteristics and bacterial population. Spot urine and fecal samples (8 samples/cow per period) were collected to determine nutrient digestibility and dietary N utilization. All data were analyzed using the MIXED procedure of SAS (SAS Institute Inc., Cary, NC; group and cow within group were random effects and treatments, time, and their interaction were fixed effects). Preplanned contrasts were made to determine effect of MON versus CON, effect of LPL versus MON, and linear effect of increasing LPL. In the current study, responses to MON generally agreed with effects of monensin observed in the literature (increased milk yield and feed efficiency but decreased milk fat content). Supplementation of LPL to the diet did not alter DM intake but linearly increased milk yield, resulting in increases in feed efficiency (milk yield/DM intake) and milk protein and fat yields. However, total-tract digestibility of DM and organic matter tended to be lower (60.9 vs. 62.2% and 61.8 vs. 63.1%, respectively) for LPL compared with CON. Linear increases in milk N secretion and decreases in urinary N excretion were observed with increasing LPL in the diet. A slight decrease in acetate proportion in the rumen for LPL was found. Relative to MON, very few bacteria in the rumen were affected with increasing LPL. In conclusion, LPL is a potential feed additive that can increase milk yield and components and dietary N utilization. However, more studies with large numbers of animals are needed to confirm the effect of LPL on production. Similar positive effects on production were observed between LPL and MON, but individual mechanisms were likely different according to ruminal fermentation characteristics. Further studies are needed to explore the mode of action of LPL in dairy cows.

High content screening identifies monensin as an EMT-selective cytotoxic compound.

Epithelial-to-mesenchymal transition (EMT) is implicated in cancer metastasis and drug resistance. Specifically targeting cancer cells in an EMT-like state may have therapeutic value. In this study, we developed a cell imaging-based high-content screening protocol to identify EMT-selective cytotoxic compounds. Among the 2,640 compounds tested, salinomycin and monensin, both monovalent cation ionophores, displayed a potent and selective cytotoxic effect against EMT-like cells. The mechanism of action of monensin was further evaluated. Monensin (10 nM) induced apoptosis, cell cycle arrest, and an increase in reactive oxygen species (ROS) production in TEM 4-18 cells. In addition, monensin rapidly induced swelling of Golgi apparatus and perturbed mitochondrial function. These are previously known effects of monensin, albeit occurring at much higher concentrations in the micromolar range. The cytotoxic effect of monensin was not blocked by inhibitors of ferroptosis. To explore the generality of our findings, we evaluated the toxicity of monensin in 24 human cancer cell lines and classified them as resistant or sensitive based on IC50 cutoff of 100 nM. Gene Set Enrichment Analysis identified EMT as the top enriched gene set in the sensitive group. Importantly, increased monensin sensitivity in EMT-like cells is associated with elevated uptake of 3H-monensin compared to resistant cells.

Effects of monensin on growth performance of beef heifers consuming warm-season perennial grass and supplemented with sugarcane molasses.

The objective of this study was to evaluate the effects of monensin on performance of beef heifers fed warm-season forages. Brangus heifers (n = 24) were stratified by BW and BCS, and randomly assigned into 1 of 12 bahiagrass pastures (1.2 ha and 2 heifers/pasture). Heifers were offered 14 kg of sugarcane molasses and 3.5 kg of cottonseed meal weekly from day 0 to 84. Treatments were randomly assigned to pastures (6 pastures/treatment) and consisted of heifers supplemented with or without 200 mg/day of monensin. On d 85, heifers were allocated to individual drylot pens, provided free choice access to bermudagrass hay, and received their respective treatment for 10 d of adaptation and 11 d of data collection. Monensin did not impact (P ≥ 0.13) heifer BW, BCS, overall ADG, bahiagrass IVDOM, CP, herbage mass, and allowance. Supplement disappearance after 10 and 34 h of supplementation was greater for control vs. monensin heifers (P = 0.04) and tended to be greater for control vs. monensin heifers 24 h post-supplementation (P = 0.07). Plasma concentrations of glucose, IGF-1, and BUN (P ≥ 0.24) did not differ between treatments. From d 85 to 106, forage and total DM intake, in vivo DM digestibility, and heifer growth performance did not differ between treatments (P ≥ 0.12). Therefore, adding monensin to sugarcane molasses-based supplements decreased supplement consumption rate, but did not impact growth and blood parameters of heifers grazing warm-season grasses with limited nutritive value.

Effect of the ionophore monensin and tannin extracts supplemented to grass silage on populations of ruminal cellulolytics and methanogens in vitro.

This study examined whether the methane-decreasing effect of monensin (∼21%) and different hydrolysable tannins (24%-65%) during in vitro fermentation of grass silage was accompanied by changes in abundances of cellulolytics and methanogens. Samples of liquid (LAM) and solid (SAM) associated microbes were obtained from two rumen simulation technique experiments in which grass silage was either tested in combination with monensin (0, 2 or 4 mg d-1) or with different tannin extracts from chestnut, valonea, sumac and grape seed (0 or 1.5 g d-1). Total prokaryotes were quantified by 4',6-diamidino-2-phenylindol (DAPI) staining of paraformaldehyde-ethanol-fixed cells and relative abundances of ruminal cellulolytic and methanogenic species were assessed by real time quantitative PCR. Results revealed no change in absolute numbers of prokaryotic cells with monensin treatment, neither in LAM nor in SAM. By contrast, supplementation of chestnut and grape seed tannins decreased total prokaryotic counts compared to control. However, relative abundances of total methanogens did not differ between tannin treatments. Thus, the decreased methane production by 65% and 24% observed for chestnut and grape seed tannins, respectively, may have been caused by a lower total number of methanogens, but methane production seemed to be also dependent on changes in the microbial community composition. While the relative abundance of F. succinogenes decreased with monensin addition, chestnut and valonea tannins inhibited R. albus. Moreover, a decline in relative abundances of Methanobrevibacter sp., especially M. ruminantium, and Methanosphaera stadtmanae was shown with supplementation of monensin or chestnut tannins. Proportions of Methanomicrobium mobile were decreased by monensin in LAM while chestnut and valonea had an increasing effect on this methanogenic species. Our results demonstrate a different impact of monensin and tannins on ruminal cellulolytics and gave indication that methane decrease by monensin and chestnut tannins was associated with decreased abundances of M. ruminantium and M. stadtmanae.

Effect of monensin withdrawal on rumen fermentation, methanogenesis and microbial populations in cattle.

This study was designed to obtain information on the residual influence of dietary monensin on ruminant fermentation, methanogenesis and bacterial population. Three ruminally cannulated crossbreed heifers (14 months old, 363 ± 11 kg) were fed Italian ryegrass straw and concentrate supplemented with monensin for 21 days before sampling. Rumen fluid samples were collected for analysis of short chain fatty acid (SCFA) profiles, monensin concentration, methanogens and rumen bacterial density. Post-feeding rumen fluid was also collected to determine in vitro gas production. Monensin was eliminated from the rumen fluid within 3 days. The composition of SCFA varied after elimination of monensin, while total production of SCFA was 1.78 times higher than on the first day. Methane production increased 7 days after monensin administration ceased, whereas hydrogen production decreased. The methanogens and rumen bacterial copy numbers were unaffected by the withdrawal of monensin.

Urea-N recycling in lactating dairy cows fed diets with 2 different levels of dietary crude protein and starch with or without monensin.

Rumensin (monensin; Elanco Animal Health, Greenfield, IN) has been shown to reduce ammonia production and microbial populations in vitro; thus, it would be assumed to reduce ruminal ammonia production and subsequent urea production and consequently affect urea recycling. The objective of this experiment was to determine the effects of 2 levels of dietary crude protein (CP) and 2 levels of starch, with and without Rumensin on urea-N recycling in lactating dairy cattle. Twelve lactating Holstein dairy cows (107 ± 21 d in milk, 647 kg ± 37 kg of body weight) were fed diets characterized as having high (16.7%) or low (15.3%) CP with or without Rumensin, while dietary starch levels (23 vs. 29%) were varied between 2 feeding periods with at least 7d of adaptation between measurements. Cows assigned to high or low protein and to Rumensin or no Rumensin remained on those treatments to avoid carryover effects. The diets consisted of approximately 40% corn silage, 20% alfalfa hay, and 40% concentrate mix specific to the treatment diets, with 0.5 kg of wheat straw added to the high starch diets to enhance effective fiber intake. The diets were formulated using Cornell Net Carbohydrate and Protein System (version 6.1), and the low-protein diets were formulated to be deficient for rumen ammonia to create conditions that should enhance the demand for urea recycling. The high-protein diets were formulated to be positive for both rumen ammonia and metabolizable protein. Rumen fluid, urine, feces, and milk samples were collected before and after a 72-h continuous jugular infusion of (15)N(15)N-urea. Total urine and feces were collected during the urea infusions for N balance measurements. Milk yield and dry matter intake were improved in cows fed the higher level of dietary CP and by Rumensin. Ruminal ammonia and milk and plasma urea nitrogen concentrations corresponded to dietary CP concentration. As has been shown in vitro, Rumensin reduced rumen ammonia concentration by approximately 23% but did not affect urea entry rate or gastrointestinal entry rate. Urea entry rate averaged approximately 57% of total N intake for cattle with and without Rumensin, and gastrointestinal rate was similar at 43 and 42% of N intake for cattle fed and not fed Rumensin, respectively. The cattle fed the high-protein diet had a 25% increase in urea entry rate and no effect of starch level was observed for any recycling parameters. Contrary to our hypothesis, Rumensin did not alter urea production and recycling.

Performance, histomorphology, and toll-like receptor, chemokine, and cytokine profile locally and systemically in broiler chickens fed diets supplemented with yeast-derived macromolecules.

The turnover of intestinal epithelial cells is a dynamic process that includes adequate cell proliferation and maturation in the presence of microbiota and migration and seeding of immune cells in early gut development in chickens. We studied the effect of yeast-derived macromolecules (YDM) on performance, gut health, and immune system gene expression in the intestine of broiler chickens. One thousand eighty 1-d-old birds, with 60 birds per pen and 6 pens per treatment, were randomly assigned to 3 treatment diets; a diet containing monensin (control), control diet supplemented with bacitracin methylene disalycylate (BMD), and BMD diet supplemented with YDM. Feed intake, BW, mortality, ileum histomorphology, and gene expression of Toll-like receptors (TLR2b, TLR4, and TLR21), cytokines [interferon (IFN)-γ, IFN-ß, IL-12p35, IL-1ß, IL-6, IL-10, IL-8, IL-2, IL-4, and transforming growth factor (TGF)-ß4], and cluster of differentiation (CD)40 in the ileum, cecal tonsil, bursa of Fabricius, and spleen were assessed. No significant overall difference in performance in terms of feed intake, BW gain, and G:F was observed among treatments (P > 0.05). The YDM diet resulted in significantly higher villi height and villi height:crypt depth ratio compared with BMD and control diets (P < 0.05). A significantly lower mortality was observed in the YDM treatment compared with both control and BMD treatments. Compared with the control, gene expression analysis in YDM treatment showed no major change in response in the ileum, whereas higher CD40, IFN-ß, IL-ß, IL-6, TGF-ß4, IL-2, and IL-4 in the cecal tonsil; TLR2b, TLR4, TLR21, and TGF-ß4 in the bursa of Fabricius; and TLR4, IL-12p35, IFN-γ, TGF-ß4, and IL-4 in the spleen was observed (P < 0.05). In conclusion, supplementation of YDM supports pro- and anti-inflammatory cytokine production via T helper type 1 and 2 (Th1 and Th2) cell-associated pathways both locally and systemically with a stronger additive effect in the cecal tonsil in the presence of BMD in the diet of chickens.

Effects of Saccharomyces cerevisiae and monensin on digestion, ruminal parameters, and balance of nitrogenous compounds of beef cattle fed diets with different starch concentrations.

This study was carried out aiming to evaluate the effects of yeast or monensin supplementation on dry matter intake, nutrients digestibility, ruminal volatile fatty acids profile, ruminal pH and ammonia concentration, microbial protein synthesis, and the balance of nitrogen compounds of cattle fed high concentrate diet (80 % dry matter (DM) basis) with two different levels of starch. Eight crossbred beef steers fitted with rumen cannula were assigned to two simultaneous 4 × 4 Latin squares arranged in a 4 × 2 factorial design. Two different starch levels (23 and 38 % of DM) were assigned to each Latin square, independently. Within each Latin square, four treatments were randomly assigned to the experimental animals (control; monensin; 1-g yeast [1 g/100 kg body weight (BW)/day] treatment; and 2.5-g yeast [2.5 g/100 kg BW/day] treatment). Feed additives did not influence ruminal pH (P > 0.05). Total ruminal volatile fatty acids (VFA) concentration was greater (P<0.05) in the diet with the lowest starch level. Similarly, monensin and 1-g yeast treatments resulted in greater (P<0.05) VFA concentration in the rumen. Monensin inclusion in the diet with the highest starch level led to a decrease (P<0.05) in lactate concentration in the rumen. However, acetate levels were increased (P<0.05) by the inclusion of 1 g of yeast in the diet with lowest starch level. Ruminal concentrations of propionate and butyrate, and ammonia-N were not influenced (P>0.05) by none of the additives evaluated. However, propionate concentration was greater (P<0.05) in the low-starch diets. Low-starch diets resulted in lower ruminal ammonia-N concentration and greater neutral detergent fiber digestibility (P<0.05). The excretion of urinary nitrogenous compounds, purine derivatives, synthesis of microbial protein, microbial efficiency, and balance of nitrogenous compounds were not affected by treatments evaluated (P>0.05). Monensin or yeast inclusion in high concentrate beef cattle diets in tropical regions as in Brazil is not justified by do not alter nutrient digestibility, nitrogen balance, and main ruminal parameters.

Eimeria acervulina infection in chicks fed excess copper in the presence or absence of excess dietary methionine.

Five experiments were conducted with young, male, crossbred chicks to investigate the effects of experimental Eimeria acervulina infection on the efficacy of supplemental Cu and (or) methionine when added to a corn-soybean meal diet. Duodenal coccidiosis (due to E. acervulina) and supplemental Cu (500 and 750 mg/kg) depressed weight gain and efficiency of feed utilization. Supplemental Cu increased concentrations of Cu in liver and gallbladder; experimental coccidiosis resulted in a two- to fourfold increase in Cu deposition in these tissues compared with the respective controls. Excess supplemental methionine (.5%) had little effect on Cu toxicity in either healthy or infected chicks. Monensin, an ionophorous coccidiostat, alleviated the coccidiosis-induced Cu deposition.