BackgroundNecrotic enteritis (NE) in broiler chickens leads to significant economic losses in poultry production. This study examined the inhibitory effects of usnic acid and tannic acid on coccidia, sporozoite, and Clostridium perfringens and assessed their influence on growth performance and intestinal health in NE-challenged broilers through in vitro and in vivo experiments.MethodsThe in vitro experiment included 5 treatment groups: the negative control (NC), 2 μmol/L diclazuril (DZ), 30 μmol/L usnic acid (UA), 90 μmol/L tannic acid (TA), and 15 μmol/L usnic acid + 45 μmol/L tannic acid (UTA) groups. The in vivo experiment involved 320 broilers divided into four groups: PC (NE-challenged), SA (500 mg/kg salinomycin premix + NE-challenged), UA (300 mg/kg usnic acid + NE-challenged), and UTA (300 mg/kg usnic acid + 500 mg/kg tannic acid + NE-challenged) groups.ResultsIn the in vitro study, the UA, TA, and UTA treatments significantly increased apoptosis in coccidian oocysts and sporozoites, lowered the mitochondrial membrane potential (P < 0.05), and disrupted the oocyst structure compared with those in the NC group. UA and TA had inhibitory effects on C. perfringens, with the strongest inhibition observed in the UTA group. The in vivo results demonstrated that the SA group presented significantly improved growth performance on d 13, 21, and 28 (P < 0.05), whereas the UA and UTA groups presented improvements on d 13 and 21 (P < 0.05). The SA, UA, and UTA treatments reduced the intestinal lesion scores by d 28 and the fecal coccidian oocyst counts from d 19 to 21 (P < 0.05). Compared with the PC group, the UA and UTA groups presented lower intestinal sIgA levels and CD8+ cell percentages (P < 0.05), with a trend toward a reduced CD3+ cell percentage (P = 0.069). The SA, UA, and UTA treatments significantly reduced the serum diamine oxidase activity, crypt depth, and platelet-derived growth factor levels in the intestinal mucosa while increasing the villus height to crypt depth ratio and number of goblet cells (P < 0.05). The UTA treatment also significantly increased the acetate and butyrate concentrations in the cecum (P < 0.05). With respect to the gut microbiota, significant changes in β diversity in the ileum and cecum were observed in the SA, UA, and UTA groups, indicating that the microbial community compositions differed among the groups. Romboutsia dominated the SA group, Bacillales dominated the UA group, and Lactobacillales and Lachnospirales dominated the UTA group in the ileal microbiota. In the cecal microbiota, Lactobacillus, Butyricicoccus, and Blautia abundances were significantly elevated in the UTA group (P < 0.05).ConclusionUsnic acid and tannic acid induce apoptosis in coccidia and sporozoites by lowering the mitochondrial membrane potential. Both usnic acid alone and in combination with tannic acid alleviate NE-induced adverse effects in broilers by modulating intestinal immunity, altering the microbial composition, and improving intestinal barrier function. Compared with usnic acid alone, the combination of usnic acid and tannic acid had superior effects, providing a promising basis for the development of effective feed additive combinations.

BackgroundThis study was carried out to investigate the individual and combined contamination of aflatoxin B1 (AFB1), deoxynivalenol (DON), and zearalenone (ZEN) in feeds in China between 2021 and 2024. A total of 23,003 feed samples, including 17,489 feedstuff samples and 5,514 complete feed samples, were collected from different provinces of China for mycotoxin analysis.ResultsThe analyzed mycotoxins displayed considerably high contamination in the feed samples, with the individual contamination of AFB1, DON, and ZEN were 20.0%–100%, 33.3%–100%, and 85.0%–100%, respectively. The average concentrations of AFB1, DON, and ZEN were 1.2–728.7 μg/kg, 106–8,634.8 μg/kg, and 18.1–3,341.6 μg/kg, respectively. Notably, the rates over China’s safety standards for AFB1, DON, and ZEN in raw ingredients were 9.7%, 2.7%, and 15.7%, respectively. Meanwhile, 3.5%, 1.1%, and 8.7% of analyzed complete feeds exceeded China’s safety standards for AFB1, DON, and ZEN, respectively. Moreover, the co-contamination rates of AFB1, DON, and ZEN in more than 70% of raw ingredients and 87.5% of complete feed products were 60.0%–100% and 61.5%–100%, respectively.ConclusionThis study reveals that the feeds in China have commonly been contaminated with AFB1, DON, and ZEN alone and their combination during the past four years. These findings highlight the significance of monitoring mycotoxin contaminant levels in domestic animal feed and the importance of carrying out feed administration and remediation strategies for mycotoxin control.

BackgroundCoccidiosis, caused by Eimeria parasites, is a major enteric disease in poultry, significantly impacting animal health, production performance, and welfare. This disease imposes a substantial economic burden, costing the global poultry industry up to $13 billion annually. However, effective mitigation strategies for coccidiosis remain elusive. While different chicken breeds exhibit varying resistance to coccidiosis, no commensal bacteria have been directly linked to this resistance.MethodsTo assess relative resistance of different breeds to coccidiosis, 10-day-old Fayoumi M5.1, Leghorn Ghs6, and Cobb chickens were challenged with 50,000 sporulated Eimeria maxima oocysts or mock-infected. Body weight changes, small intestinal lesions, and fecal oocyst shedding were evaluated on d 17. Ileal and cecal digesta were collected from individual animals on d 17 and subjected to microbiome analysis using 16S rRNA gene sequencing. ResultsFayoumi M5.1 chickens showed the lowest growth retardation, intestinal lesion score, fecal oocyst shedding, and pathobiont proliferation compared to Ghs6 and Cobb chickens. The intestinal microbiota of M5.1 chickens also differed markedly from the other two breeds under both healthy and coccidiosis conditions. Notably, group A Lactobacillus and Ligilactobacillus salivarius were the least prevalent in both the ileum and cecum of healthy M5.1 chickens, but became highly enriched and comparable to Ghs6 and Cobb chickens in response to coccidiosis. Conversely, Weissella, Staphylococcus gallinarum, and Enterococcus durans/hirae were more abundant in the ileum of healthy M5.1 chickens than in the other two breeds. Despite being reduced by Eimeria, these bacteria retained higher abundance in M5.1 chickens compared to the other breeds.ConclusionsFayoumi M5.1 chickens exhibit greater resistance to coccidiosis than Leghorn Ghs6 layers and Cobb broilers. Several commensal bacteria, including group A Lactobacillus, L. salivarius, Weissella, S. gallinarum, and E. durans/hirae, are differentially enriched in Fayoumi M5.1 chickens with strong correlation with coccidiosis resistance. These bacteria hold potential as probiotics for coccidiosis mitigation.

BackgroundInsect meals have been identified as innovative and sustainable feedstuffs that could be used in ruminant nutrition. However, current research on the effects that their processing may have on rumen digestibility and fatty acid (FA) biohydrogenation is scant. This trial aims to investigate the effects (i) of drying temperature of full-fat Hermetia illucens (HI) and Tenebrio molitor (TM) meals, and (ii) of residual ether extract (EE) content of defatted HI and TM meals, on their fermentation characteristics and FA of rumen digesta after 24-h in vitro rumen incubation.MethodsThe tested full-fat meals included four HI and four TM meals obtained applying drying temperatures ranging from 30 °C to 70 °C, while the tested defatted meals consisted of five HI and two TM meals containing a residual EE content ranging from 4.7 to 19.7 g EE/100 g dry matter (DM). The applied statistical models (GLM ANOVA) tested the effects of insect species, drying temperature (full-fat meals) or EE content (defatted meals), and their interaction.ResultsDrying temperature had minor effects on in vitro ruminal digestibility and FA profile of rumen digesta. Irrespective of insect species, increasing the drying temperature led to a reduction of in vitro degradation of proteins from insect meals, as outlined by the significant decrease in ammonia production (−0.009 mmol/g DM and −0.126 g/100 g total N for each additional 1 °C). Irrespective of insect species, defatting increased total gas, volatile fatty acids (VFA) and CH4 productions, and the proportions of total saturated and branched-chain FA in rumen digesta (+0.038 mmol/g DM, +0.063 mmol/g DM, +12.9 µmol/g DM, +0.18 g/100 g FA, and +0.19 g/100 g FA for each reduced 1 g EE/100 g DM, respectively), and reduced the proportion of total PUFA (−0.12 g/100 g FA).ConclusionsThe applied drying temperatures of full-fat insect meals are too low to exert impactful effects on rumen digestibility and FA biohydrogenation. Fat lowered fermentation activity, probably because of an inhibitory effect on rumen microbiota. The increased ruminal digestibility of defatted insect meals suggests that they can be more suitable to be used in ruminant nutrition than full-fat ones.

The intestinal tract, a complex organ responsible for nutrient absorption and digestion, relies heavily on a balanced gut microbiome to maintain its integrity. Disruptions to this delicate microbial ecosystem can lead to intestinal inflammation, a hallmark of inflammatory bowel disease (IBD). While the role of the gut microbiome in IBD is increasingly recognized, the underlying mechanisms, particularly those involving endoplasmic reticulum (ER) stress, autophagy, and cell death, remain incompletely understood. ER stress, a cellular response to various stressors, can trigger inflammation and cell death. Autophagy, a cellular degradation process, can either alleviate or exacerbate ER stress-induced inflammation, depending on the specific context. The gut microbiome can influence both ER stress and autophagy pathways, further complicating the interplay between these processes. This review delves into the intricate relationship between ER stress, autophagy, and the gut microbiome in the context of intestinal inflammation. By exploring the molecular mechanisms underlying these interactions, we aim to provide a comprehensive theoretical framework for developing novel therapeutic strategies for IBD. A deeper understanding of the ER stress-autophagy axis, the gut microbial-ER stress axis, and the gut microbial-autophagy axis may pave the way for targeted interventions to restore intestinal health and mitigate the impact of IBD.Graphical

BackgroundLactate is a classical byproduct of glucose metabolism, and the main lactate production pathway depends on glycolysis. Lactate stabilized HIF1α by inhibiting PHD activity, leading to hypoxic stress response and exacerbating glycolysis in multiple tissues. However, the redox induction mechanism of lactate in mammary gland has not been understood yet. Herein, we describe a lactate-responsive HIF1α/circadian control mechanism in oxidative stress in the mammary glands of dairy cows.ResultsThe in vivo study showed that dairy cows with high lactate concentrations are associated with reduced milk yield and more ROS accumulation in mammary gland. Western blot results in MAC-T cells showed positive correlation between lactate concentrations, expression of HIF1α and oxidative stress indicators, but not circadian core components. To test how lactate-mediated HIF1α dysfunction leads to cell protection process, we investigated altered expression of circadian core related genes following HIF1α stabilization. We found that stabilized HIF1α by lactate inhibited stimulated expression of circadian core components due to the similarity of HRE and E-box transcription elements. Furthermore, we found that lactate treatment strengthened the binding of HIF1α with BMAL1, HMOX1 and FOXO3 in MAC-T cells. Moreover, HIF1α knockdown altered expression of circadian rhythm related genes and reduced oxidative stress state.ConclusionIn summary, our study highlights the central role of competitive transcriptional element occupancy in lactate-mediated oxidative stress of mammary gland, which is caused by HIF1α stabilization and circadian rhythm dysfunction. Our findings introduce a novel nutritional strategy with potential applications in dairy farming for optimizing milk production and maintaining mammary gland health.

BackgroundEarly embryo development plays a pivotal role in determining pregnancy outcomes, postnatal development, and lifelong health. Therefore, the strategic selection of functional nutrients to enhance embryo development is of paramount importance. In this study, we established a stable porcine trophectoderm cell line expressing dual fluorescent reporter genes driven by the CDX2 and TEAD4 gene promoter segments using lentiviral transfection.ResultsThree amino acid metabolites—kynurenic acid, taurine, and tryptamine—met the minimum z-score criteria of 2.0 for both luciferase and Renilla luciferase activities and were initially identified as potential metabolites for embryo development, with their beneficial effects validated by qPCR. Given that the identified metabolites are closely related to methionine, arginine, and tryptophan, we selected these three amino acids, using lysine as a standard, and employed response surface methodology combined with our high-throughput screening cell model to efficiently screen and optimize amino acid combination conducive to early embryo development. The optimized candidate amino acid system included lysine (1.87 mmol/L), methionine (0.82 mmol/L), tryptophan (0.23 mmol/L), and arginine (3 mmol/L), with the ratio of 1:0.43:0.12:1.60. In vitro experiments confirmed that this amino acid system enhances the expression of key genes involved in early embryonic development and improves in vitro embryo adhesion. Transcriptomic analysis of blastocysts suggested that candidate amino acid system enhances early embryo development by regulating early embryonic cell cycle and differentiation, as well as improving nutrient absorption. Furthermore, based on response surface methodology, 400 sows were used to verify this amino acid system, substituting arginine with the more cost-effective N-carbamoyl glutamate (NCG), a precursor of arginine. The optimal dietary amino acid requirement was predicted to be 0.71% lysine, 0.32% methionine, 0.22% tryptophan, and 0.10% NCG for sows during early gestation. The optimized amino acid system ratio of the feed, derived from the peripheral release of essential amino acids, was found to be 1:0.45:0.13, which is largely consistent with the results obtained from the cell model optimization. Subsequently, we furtherly verified that this optimal dietary amino acid system significantly increased total litter size, live litter size and litter weight in sows.ConclusionsIn summary, we successfully established a dual-fluorescent high-throughput screening cell model for the efficient identification of potential nutrients that would promote embryo development and implantation. This innovative approach overcomes the limitations of traditional amino acid nutrition studies in sows, providing a more effective model for enhancing reproductive outcomes.

BackgroundIn this study, the effects of L-leucine (Leu) on rumen fermentation parameters, rumen epithelium development, amino acid composition, rumen bacterial communities and rumen metabolites in beef cattle were investigated. Twenty-four fattening Angus females of similar initial weight (575.5 ± 22.1 kg) were randomly assigned to 2 treatments with 4 replicate pens (3 cattle per pen). They were fed either a basal diet or a basal diet supplemented with 6.0 g L-Leu/100 kg BW/d for 120 d.Results(1) Leu increased the ruminal concentrations of total volatile fatty acid (VFA) (P = 0.017), propionate (P = 0.023), isovalerate (P = 0.001), and branched-chain volatile fatty acid (BCVFA) (P = 0.01) at 4 h post-feeding. It also tended to increase acetate (P = 0.083) and decrease the ammonia-N (NH3-N) concentration (P = 0.055), but it did not affect ruminal pH (P > 0.1). Leu also increased microbial crude protein (MCP) (P = 0.026) at 4 h post-feeding, but decreased MCP at 8 h post-feeding (P = 0.010). (2) Supplementation with L-Leu increased the ruminal concentrations of phenylalanine (P = 0.011), lysine (P = 0.034), and tyrosine (P = 0.033), while decreasing the cystine concentration (P = 0.010). (3) Leu increased the thickness of the stratum spinosum and basal (P < 0.05), while decreasing the thickness of the stratum granulosum (P < 0.05). (4) Leu upregulated the relative mRNA abundance of genes involved in tight junction proteins (P < 0.05) and VFA absorption and metabolism (P < 0.01) in the rumen epithelium. This upregulation was positively correlated with the concentrations ruminal isovalerate and BCVFA (P < 0.01). (5) L-Leu did not affect the diversity and richness of ruminal microbes (P > 0.05), but differential bacterial biomarkers (LEfSe, LDA > 2) were either positively or negatively correlated with ruminal MCP, NH3-N, and BCVFA concentrations (P < 0.001). Additionally, differential bacterial metabolites (OPLS-DA, VIP > 1.5) were primarily enriched in the amino acid metabolism pathway and the cofactors and vitamins metabolism pathway (P < 0.05).ConclusionsDietary supplementation with L-Leu altered rumen fermentation parameters and patterns, improved rumen epithelial morphology, and enhanced the expression of genes related to VFA absorption and metabolism in the rumen epithelium of beef cattle.

BackgroundCoumarins are toxic phytochemicals found in a variety of plants and are known to limit microbial degradation and interfere with nutrient cycling. While the degradation of coumarins by fungi has been studied in an environmental context, little is known about their degradation in the gastrointestinal system of herbivores after ingestion.ResultsIn this study, we investigated in vitro fermentation by microbial enrichment, transcriptome sequencing, and high-resolution mass spectrometry to evaluate the ability of rumen anaerobic fungi to degrade coumarins. The results showed that despite the low abundance of anaerobic fungi in the rumen microbiota, they were able to effectively degrade coumarins. Specifically, Pecoramyces ruminantium F1 could tolerate coumarin concentrations up to 3 mmol/L and degrade it efficiently via metabolic pathways involving alpha/beta hydrolases and NAD(P)H oxidoreductases within the late growth phase. The fungus metabolized coumarin to less toxic compounds, including o-coumaric acid and melilotic acid, highlighting the detoxification potential of anaerobic fungi.ConclusionsThis study is the first to demonstrate the ability of rumen anaerobic fungi to degrade coumarin, providing new insights into the use of anaerobic fungi in sustainable agricultural practices and environmental detoxification strategies.

BackgroundHeat stress (HS) poses a significant threat to male goat reproduction. Sertoli cells (SCs) provide both structural and nutritional support necessary for germ cells. HS induces physiological and biochemical changes in SCs. Nevertheless, the molecular mechanisms involved are still not fully understood. Melatonin is a classic antioxidant that can alleviate HS-induced male reproductive damage. However, the underlying molecular mechanisms by which melatonin mitigates damage to goat testicular SCs remain unclear and require further investigation.ResultsIn this study, an in vivo heat stress model was established in goats. The results showed that HS exposure led to testicular injury, abnormal spermatogenesis and apoptosis of SCs. To elucidate the mechanism of HS-induced SC apoptosis, primary SCs were isolated and cultured from goat testes, then exposed to HS. HS exposure increased the production of reactive oxygen species (ROS), decreased adenosine triphosphate (ATP) synthesis, and reduced mitochondrial membrane potential in SCs. Additionally, HS increased the expression of mitochondrial fission proteins 1 (FIS1) and dynamin-related protein 1 (DRP1) while decreasing the expression of mitochondrial fusion proteins Mitofusin 1 (MFN1), Mitofusin 2 (MFN2), and optic atrophy 1 (OPA1). This resulted in excessive mitochondrial fission and mitochondria-dependent apoptosis. Mdivi-1 (DRP1 inhibitor) reduces mitochondria-dependent apoptosis by inhibiting excessive mitochondrial fission. Mitochondrial fission is closely related to mitophagy. HS activated upstream mitophagy but inhibited autophagic flux, disrupting mitophagy and exacerbating mitochondria-dependent apoptosis. Finally, the classical antioxidant melatonin was shown to reduce mitochondria-dependent apoptosis in SCs exposed to HS by decreasing ROS levels, restoring mitochondrial homeostasis, and normalizing mitophagy.ConclusionsIn summary, these findings indicated that the mechanism of HS-induced mitochondria-dependent apoptosis in SCs is mediated by hyperactivation of the ROS-DRP1-mitochondrial fission axis and inhibition of mitochondrial autophagy. Melatonin inhibited HS-induced mitochondria-dependent apoptosis in SCs by restoring mitochondrial homeostasis. This study enhances the understanding of the mechanisms through which heat stress triggers apoptosis and provides a vision for the development of drugs against HS by targeting mitochondria in goats.Graphical