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Aflatoxins are poisonous carcinogens produced by fungi, mainly Aspergillus flavus and Aspergillus parasiticus. Aflatoxins can contaminate a variety of livestock feeds and cause enormous economic losses, estimated at between US $52.1 and US$ 1.68 billion annually for the U.S. corn industry alone. In addition, aflatoxin can be transferred from the diet to the milk of cows as aflatoxin M1 (AFM1), posing a significant human health hazard. In dairy cows, sheep and goats, chronic exposure to dietary aflatoxin can reduce milk production, impair reproduction and liver function, compromise immune function, and increase susceptibility to diseases; hence, strategies to lower aflatoxin contamination of feeds and to prevent or reduce the transfer of the toxin to milk are required for safeguarding animal and human health and improving the safety of dairy products and profitability of the dairy industry. This article provides an overview of the toxicity of aflatoxin to ruminant livestock, its occurrence in livestock feeds, and the effectiveness of different strategies for preventing and mitigating aflatoxin contamination of feeds.

We investigated the impact of a rumen-bypass protein (RBP) supplement on growth performance, plasma and urinary N (UN) concentration, hepatic mitochondrial protein complexes, and hepatic mRNA expression of immune genes of beef steers with negative or positive residual feed intake (RFI) phenotype. Forty crossbred beef steers with an average body weight (BW) of 492 ± 36 kg were subjected to a generalized randomized block design over a 42-day experimental period. This study followed a 2 × 2 factorial arrangement of treatments. The factors evaluated were: 1) RFI classification (low-RFI (-2.12 kg/d) vs. high-RFI (2.02 kg/d), and 2) rumen-bypass protein supplement: RBP supplement (RBP; 227 g/steer/d) vs. control diet (CON; 0 g/d), resulting in four distinct treatments: LRFI-CON (n = 10), LRFI-RBP ( n = 10), HRFI-CON (n = 10), and HRFI-RBP ( n = 10). The RBP supplement (84% crude protein) is a mixture of hydrolyzed feather meal, porcine blood meal, and DL-methionine hydroxy analogue. The beef steers were stratified by BW, randomly assigned to treatments, and housed in four pens (1 treatment/pen) equipped with two GrowSafe feed bunks each to measure individual dry mater intake (DMI). Body weight was measured every 7 d. Liver tissue samples were collected on d 42 from all the beef steers. These samples were used for mRNA expression analysis of 16 immune-related genes and for evaluating the mitochondrial protein complexes I ‐ V. No significant effects due to RBP supplementation or RFI × RBP interactions ( P > 0.05) were observed for average daily gain (ADG) and DMI. However, compared to high-RFI steers, low-RFI steers showed a trend towards reduced DMI (12.9 vs. 13.6 kg/d; P = 0.07) but ADG was similar for the two RFI groups. Regardless of RFI status, supplemental RBP increased blood urea nitrogen (BUN) ( P = 0.01), with a lower BUN concentration in low-RFI steers compared to high-RFI ones. A tendency for interaction ( P = 0.07) between RFI and RBP was detected for the UN concentrations; feeding the dietary RBP increased the UN concentration in high-RFI beef steers (209 vs. 124 mM), whereas the concentration was lower than that of the CON group for low-RFI beef steers (86 vs. 131 mM). Interactions of RBP and RFI were observed ( P ≤ 0.05) for mitochondrial activities of complexes IV, V, and mRNA expressions of some immune genes such as TLR2 , TLR3 , and IL23A . In conclusion, while RBP supplementation did not alter growth performance, its observed effects on hepatic immune gene expression, mitochondrial protein complexes, BUN, and UN depended on the beef steers’ RFI phenotype. Therefore, the RFI status of beef steers should be considered in future studies evaluating the effects of dietary protein supplements.

Bacterial expansin-like proteins have synergistically increased cellulose hydrolysis by cellulolytic enzymes during the initial stages of biofuel production, but they have not been tested on livestock feeds. The objectives of this study were to: isolate and express an expansin-like protein (BsEXLX1), to verify its disruptive activity (expansion) on cotton fibers by immunodetection (Experiment 1), and to determine the effect of dose, pH and temperature for BsEXLX1 and cellulase to synergistically hydrolyze filter paper (FP) and carboxymethyl cellulose (CMC) under laboratory (Experiment 2) and simulated ruminal (Experiment 3) conditions. In addition, we determined the ability of BsEXLX1 to synergistically increase hydrolysis of corn and bermudagrass silages by an exogenous fibrolytic enzyme (EFE) (Experiment 4) and how different doses of BsEXLX1 and EFE affect the gas production (GP), in vitro digestibility and fermentation of a diet for dairy cows (Experiment 5). In Experiment 1, immunofluorescence-based examination of cotton microfiber treated without or with recombinant expansin-like protein expressed from Bacillus subtilis (BsEXLX1) increased the surface area by > 100% compared to the untreated control. In Experiment 2, adding BsEXLX1 (100 μg/g FP) to cellulase (0.0148 FPU) increased release of reducing sugars compared to cellulase alone by more than 40% (P < 0.01) at optimal pH (4.0) and temperature (50°C) after 24 h. In Experiment 3 and 4, adding BsEXLX1 to cellulase or EFE, synergistically increased release of reducing sugars from FP, corn and bermudagrass silages under simulated ruminal conditions (pH 6.0, 39°C). In Experiment 5, increasing the concentration of BsEXLX1 linearly increased (P < 0.01) GP from fermentation of a diet for dairy cows by up to 17.8%. Synergistic effects between BsEXLX1 and EFE increased in vitro NDF digestibility of the diet by 23.3% compared to the control. In vitro digestibility of hemicellulose and butyrate concentration were linearly increased by BsEXLX1 compared to the control. This study demonstrated that BsEXLX1 can improve the efficacy of cellulase and EFE at hydrolyzing pure substrates and dairy cow feeds, respectively.

We investigated whole blood and hepatic mRNA expressions of immune genes and rumen microbiome of crossbred beef steers with divergent residual feed intake phenotype to identify relevant biological processes underpinning feed efficiency in beef cattle. Low-RFI beef steers ( n  = 20; RFI = − 1.83 kg/d) and high-RFI beef steers ( n  = 20; RFI = + 2.12 kg/d) were identified from a group of 108 growing crossbred beef steers (average BW = 282 ± 30.4 kg) fed a high-forage total mixed ration after a 70-d performance testing period. At the end of the 70-d testing period, liver biopsies and blood samples were collected for total RNA extraction and cDNA synthesis. Rumen fluid samples were also collected for analysis of the rumen microbial community. The mRNA expression of 84 genes related to innate and adaptive immunity was analyzed using pathway-focused PCR-based arrays. Differentially expressed genes were determined using P -value ≤ 0.05 and fold change (FC) ≥ 1.5 (in whole blood) or ≥ 2.0 (in the liver). Gene ontology analysis of the differentially expressed genes revealed that pathways related to pattern recognition receptor activity, positive regulation of phagocytosis, positive regulation of vitamin metabolic process, vascular endothelial growth factor production, positive regulation of epithelial tube formation and T-helper cell differentiation were significantly enriched (FDR < 0.05) in low-RFI steers. In the rumen, the relative abundance of PeH15, Arthrobacter, Moryella, Weissella, and Muribaculaceae was enriched in low-RFI steers, while Methanobrevibacter, Bacteroidales_BS11_gut_group, Bacteroides and Clostridium_sensu_stricto_1 were reduced. In conclusion, our study found that low-RFI beef steers exhibit increased mRNA expression of genes related to immune cell functions in whole blood and liver tissues, specifically those involved in pathogen recognition and phagocytosis regulation. Additionally, these low-RFI steers showed differences in the relative abundance of some microbial taxa which may partially account for their improved feed efficiency compared to high-RFI steers.

We evaluated the effects of live yeast on ruminal bacterial diversity and metabolome of beef steer. Eight rumen-cannulated Holstein steers were assigned randomly to one of two treatment sequences in a study with two 25-d experimental periods and a crossover design. The steers were housed in individual pens. The dietary treatments were control (CON) or yeast (YEA; CON plus 15 g/d of live yeast product). Bacterial diversity was examined by sequencing the V3-V4 region of 16S rRNA gene. The metabolome analysis was performed using a liquid chromatograph and a mass spectrometry system (LC–MS). Live yeast supplementation increased the relative abundance of eight cellulolytic bacterial genera as well as Anaerovorax and Lachnospiraceae. Proteiniclasticum, Salmonella, and Lactococcus were not detected in the YEA treatment. Live yeast supplementation increased the concentrations of 4-cyclohexanedione and glucopyranoside and decreased the concentrations of threonic acid, xanthosine, deoxycholic acid, lauroylcarnitine, methoxybenzoic acid, and pentadecylbenzoic acid. The bacteroidales BS11, Christensenellaceae R-7, and Candidatus saccharimonas showed positive correlations with the metabolites involved in amino acid biosynthesis and the metabolism of energy substrates; the functions of these bacteria are not fully understood in relation to the mode of action of yeast. This study confirms the usefulness of LC–MS-based metabolomics in deciphering the mode of action of live yeast in the rumen.

Several studies have evaluated the effects of live yeast supplementation on rumen microbial population; however, its effect on differential microbial genes and their functional potential has not been described. Thus, this study applied shotgun metagenomic sequencing to evaluate the effects of live yeast supplementation on genetic and functional potential of the rumen microbiota in beef cattle. Eight rumen-cannulated Holstein steers were randomly assigned to two treatments in a cross-over design with two 25-day experimental periods and a 10-day wash-out between the two periods. The steers were housed in individual pens and fed 50% concentrate-mix and 50% red clover/orchard hay ad libitum . Treatments were (1) control (CON; basal diet without additive) and (2) yeast (YEA; basal diet plus 15 g/d of live yeast product). Rumen fluid samples were collected at 3, 6, and 9 h after feeding on the last d of each period. Sequencing was done on an Illumina HiSeq 2500 platform. Dietary yeast supplementation increased the relative abundance of carbohydrate-fermenting bacteria (such as Ruminococcus albus , R . champanellensis , R. bromii , and R. obeum ) and lactate-utilizing bacteria (such as Megasphaera elsdenii , Desulfovibrio desulfuricans , and D. vulgaris ). A total of 154 differentially abundant genes (DEGs) were obtained (false discovery rate < 0.01). Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation analysis of the DEGs revealed that 10 pathways, including amino sugar and nucleotide sugar metabolism, oxidative phosphorylation, lipopolysaccharide biosynthesis, pantothenate and coenzyme A biosynthesis, glutathione metabolism, beta-alanine metabolism, polyketide sugar unit biosynthesis, protein export, ribosome, and bacterial secretory system, were enriched in steers fed YEA. Annotation analysis of the DEGs in the carbohydrate-active enzymes (CAZy) database revealed that the abundance of genes coding for enzymes belonging to glycoside hydrolases, glycosyltransferases, and carbohydrate binding modules were enriched in steers fed YEA. These results confirm the effectiveness of a live S. cerevisiae product for improving rumen function in beef steers by increasing the abundance of cellulolytic bacteria, lactic acid-utilizing bacteria, and carbohydrate-active enzymes in the rumen.

A 14-day study was conducted to evaluate the effect of litter type (dirty litter, DL; fresh litter, FL) and Salmonella Enteritidis SE challenge (no challenge, NC; challenge, SE) on the growth performance and cecal microbial composition of neonate chicks. Day-old chicks (n = 240, Ross 708 male) were allocated to a 2 × 2 factorial design consisting of four treatments: chicks raised on dirty litter (CONDL), chicks raised on fresh litter (CONFL); and chicks raised on litter types similar to CONDL and CONFL but inoculated with 7.46 × 108 CFU SE/mL at d 1 (CONDLSE and CONFLSE). The performance indices measured included body weight (BW), body weight gain (BWG), feed intake (FI), mortality, and feed conversion ratio (FCR). Cecal SE concentration was assessed on d 3 and 14, and ceca were collected from chicks on day 14 for DNA extraction. The Illumina Miseq platform was used for microbiome analysis of the V3–V4 region of the 16S rRNA gene. The interaction of litter type and SE influenced FCR and FI. CONDL recorded the poorest FCR (1.832). FI was highest and similar in CONFLSE, CONDL, and CONDLSE (0.655, 0.692, and 0.677, respectively). Cecal SE concentration was significantly reduced in CONDLSE at d 3 and 14. Alpha diversity was higher (p < 0.05) in the DL compared to that in NC. Beta diversity showed a separation (p < 0.05) between the DL and the FL. Comparative tree analysis revealed 21 differential significant genera, with 14 prevalent in the DL and 7 in the FL, specifically, bacteria genera such as Lactobacillus, Clostridia_vadinBB60_group, Lachnospira, Oscillospiraceae UCG_005, and Marvinbryantia, which play significant roles relating to improved growth performance, metabolic homeostasis within the gut, energy metabolism, and short-chain fatty acid (SCFA) utilization. Our results concluded that litter management regimen differentially alters the microbiome of chicks, which accounts for the improved performance and exclusion of pathogens in the study.

We examined the effects of two direct-fed microbials (DFM) containing multiple microbial species and their fermentation products on energy status, nutrient digestibility, and ruminal fermentation, bacterial community, and metabolome of beef steers. Nine ruminally cannulated Holstein steers (mean ± SD body weight: 243 ± 12.4 kg) were assigned to three treatments arranged in a triplicated 3 x 3 Latin square design with three 21-d periods. Dietary treatments were 1) control (CON; basal diet), 2) Commence (PROB; basal diet plus 19 g/d of Commence), and 3) RX3 (SYNB; basal diet plus 28 g/d of RX3). Commence and RX3 are both multispecies DFM products. From day 16 to 20 of each period, feed and fecal samples were collected daily to determine the apparent total tract digestibilities of nutrients using indigestible neutral detergent fiber method. On day 21 of each period, blood samples were collected for analysis of plasma glucose and nonesterified fatty acid. Ruminal contents were collected at approximately 1, 3, 6, 9, 12, and 18 h after feeding on day 21 for analysis of volatile fatty acids (VFA), lactate, ammonia-N concentrations, bacterial community, and metabolome profile. Total tract digestibilities of nutrients did not differ (P > 0.05) among treatments. Compared with CON, steers fed either supplemental PROB or SYNB had greater (P = 0.04) plasma glucose concentrations. Compared with CON, total ruminal VFA, propionate, isovalerate, and valerate concentrations increased (P < 0.05) or tended to increase (P < 0.10) with either supplemental PROB or SYNB, but were not different (P > 0.05) between PROB and SYNB. Compared with CON, PROB reduced (P < 0.05) the relative abundance of Prevotella 1 and Prevotellaceae UCG-001 but increased (P < 0.05) the relative abundance of Rikenellaceae RC9, Succinivibrionaceae UCG-001, Succiniclasticum, and Ruminococcaceae UCG-002. Supplemental SYNB decreased (P < 0.05) the relative abundance of Prevotella 1 and Prevotellaceae UCG-001 but increased (P < 0.05) the relative abundance of Prevotella 7, Succinivibrio, Succiniclasticum, and Ruminococcaceae UCG-014. Compared with CON, metabolome analysis revealed that some amino acids were increased (P < 0.05) in steers fed PROB. This study demonstrated that, compared with CON, supplementation of either PROB or SYNB altered the ruminal bacterial community and metabolome differently; however, their effects on the ruminal VFA profile and energy status of the steers were not different from each other.

Subacute ruminal acidosis (SARA) is a metabolic disease of ruminants characterized by low pH, with significant impacts on rumen microbial activity, and animal productivity and health. Microbial changes during subacute ruminal acidosis have previously been analyzed using quantitative PCR and 16S rRNA sequencing, which do not reveal the actual activity of the rumen microbial population. Here, we report the functional activity of the rumen microbiota during subacute ruminal acidosis. Eight rumen-cannulated Holstein steers were assigned randomly to acidosis-inducing or control diet. Rumen fluid samples were taken at 0, 3, 6, and 9 h relative to feeding from both treatments on the challenge day. A metatranscriptome library was prepared from RNA extracted from the samples and the sequencing of the metatranscriptome library was performed on Illumina HiSeq4000 following a 2 × 150 bp index run. Cellulolytic ruminal bacteria including Fibrobacter succinogenes, Ruminococcus albus, and R. bicirculans were reduced by an induced acidotic challenge. Up to 68 functional genes were differentially expressed between the two treatments. Genes mapped to carbohydrate, amino acid, energy, vitamin and co-factor metabolism pathways, and bacterial biofilm formation pathways were enriched in beef cattle challenged with sub-acute acidosis. This study reveals transcriptionally active taxa and metabolic pathways of rumen microbiota during induced acidotic challenge.

We utilized plasma proteomics profiling to explore metabolic pathways and key proteins associated with divergent residual body weight gain (RADG) phenotype in crossbred (Angus × Hereford) beef steers. A group of 108 crossbred growing beef steers (average BW = 282.87 ± 30 kg; age = 253 ± 28 days) were fed a high-forage total mixed ration for 49 days in five dry lot pens (20–22 beef steers per pen), each equipped with two GrowSafe8000 intake nodes to determine their RADG phenotype. After RADG identification, blood samples were collected from the beef steers with the highest RADG (most efficient; n  = 15; 0.76 kg/d) and lowest RADG (least efficient; n  = 15; −0.65 kg/d). Plasma proteomics analysis was conducted on all plasma samples using a nano LC–MS/MS platform. Proteins with FC ≥ 1.2 and false-discovery rate-adjusted p- values (FDR) ≤ 0.05 were considered significantly differentially abundant. The analysis identified 435 proteins, with 59 differentially abundant proteins (DAPs) between positive and negative-RADG beef steers. Plasma abundance of 38 proteins, such as macrophage stimulating 1 and peptidase D was upregulated (FC ≥ 1.2, FDR ≤ 0.05) in positive-RADG beef steers, while 21 proteins, including fibronectin and ALB protein were greater (FC < 1.2, FDR ≤ 0.05) in negative-RADG beef steers. The results of the Gene Ontology (GO) analysis of all the DAPs showed enrichment of pathways such as metabolic processes, biological regulation, and catalytic activity in positive-RADG beef steers. Results of the EuKaryotic Orthologous Groups (KOG) analysis revealed increased abundance of DAPs involved in energy production and conversion, amino acid transport and metabolism, and lipid transport and metabolism in positive-RADG beef steers. The results of this study revealed key metabolic pathways and proteins associated with divergent RADG phenotype in beef cattle which give more insight into the biological basis of feed efficiency in crossbred beef cattle.