Metagenomic analysis for exploring the potential of Lactobacillus yoelii FYL1 to mitigate bacterial diarrhea and changes in the gut microbiota of juvenile yaks.

Diarrhea in calves is a common disease that results in poor nutrient absorption, poor growth and early death which leads to productivity and economic losses. Therefore, it is important to explore the methods to reduce diarrhea in yak's calves. Efficacy of lactic acid bacteria (LAB) for improvement of bacterial diarrhea is well recognized. For this purpose, two different doses (107 CFU, 1011 CFU) of Lactobacillus yoelii FYL1 isolated from yaks were fed to juvenile yaks exposed to E. coli O78. After a trial period of ten days fresh feces and intestinal contents of the experimental yaks were collected and metagenomics sequencing was performed. It was found that feeding a high dose of Lactobacillus yoelii FYL1 decreased abundance of phylum Firmicutes in the E. coli O78 infected group whereas, it was high in animals fed low dose of Lactobacillu yoelii FYL1. Results also revealed that counts of bacteria from the family Oscillospiraceae, genus Synergistes and Megasphaera were higher in control group whereas, order Bifidobacteriales and family Bifidobacteriaceae were higher in infected group. It was observed that bacterial counts for Pseudoruminococcus were significantly (P < 0.05) higher in animals of group that were given high dose of Lactobacillus yoelii FYL1 (HLAB). Compared to infected group multiple beneficial bacterial genera such as Deinococus and Clostridium were found higher in the animals that were given a low dose of Lactobacillus yoelii FYL1 (LLAB). The abundance of pathogenic bacterial genera that included Parascardovia, Bacteroides and Methanobrevibacter was decreased (P < 0.05) in the lower dose treated group. The results of functional analysis revealed that animals of LLAB had a higher metabolism of terpenoids and polyketides compared to animals of infected group. Virus annotation also presented a significant inhibitory effect of LLAB on some viruses (P < 0.05). It was concluded that L. yoelii FYL1 had an improved effect on gut microbiota of young yaks infected with E. coli O78. This experiment contributes to establish the positive effects of LAB supplementation while treating diarrhea.

Baicalin inhibits apoptosis and enhances chondrocyte proliferation in thiram-induced tibial dyschondroplasia in chickens by regulating Bcl-2/Caspase-9 and Sox-9/Collagen-II expressions.

Avian tibial dyschondroplasia (TD) is a skeletal disease affecting fast growing chickens, resulting in non-mineralized avascular cartilage. This metabolic disorder is characterized by lameness and reduced growth performance causing economic losses. The aim of this study was to investigate the protective effects of baicalin against TD caused by thiram exposure. A total of two hundred and forty (n = 240) one day-old broiler chickens were uniformly and randomly allocated into three different groups (n = 80) viz. control, TD, and baicalin groups. All chickens received standard feed, however, to induce TD, the TD and baicalin groups received thiram (tetramethylthiuram disulfide) at a rate of 50 mg/kg feed from days 4-7. The thiram induction in TD and baicalin groups resulted in lameness, high mortality, and enlarged growth-plate, poor production performance, reduction in ALP, GSH-Px, SOD, and T-AOC levels, and increased AST and ALT, and MDA levels. Furthermore, histopathological results showed less vascularization, and mRNA and protein expression levels of Sox-9, Col-II, and Bcl-2 showed significant downward trend, while caspase-9 displayed significant up-regulation in TD-affected chickens. After the TD induction, the baicalin group was orally administered with baicalin at a rate of 200 mg/kg from days 8-18. Baicalin administration increased the vascularization, and chondrocytes with intact nuclei, alleviated lameness, decreased GP size, increased productive capacity, and restored the liver antioxidant enzymes and serum biochemical levels. Furthermore, baicalin significantly up-regulated the gene and protein expressions of Sox-9, Col-II, and Bcl-2, and significantly down-regulated the expression of caspase-9 (p < 0.05). Therefore, the obtained results suggest that baicalin could be a possible choice in thiram toxicity alleviation by regulating apoptosis and chondrocyte proliferation in thiram-induced tibial dyschondroplasia.

Dietary Supplementation with Rutin Alters Meat Quality, Fatty Acid Profile, Antioxidant Capacity, and Expression Levels of Genes Associated with Lipid Metabolism in Breast Muscle of Qingyuan Partridge Chickens.

Consumer demand for tasty and quality meat has been quickly increasing. This study investigated how dietary supplemented rutin affects meat quality, muscle fatty acid profile, and antioxidant capacity in the Chinese indigenous Qingyuan partridge chicken. A cohort of 180 healthy 119-day-old chickens was subjected to a randomized assignment into three groups, identified as the control, R200, and R400 groups, with respective supplementation of 0, 200, and 400 mg/kg of rutin. The results revealed insignificance in growth performance, namely, average daily gain, average daily feed intake, and feed-to-gain ratio, across the various treatment groups (p > 0.05). Nevertheless, dietary rutin supplementation increased (p < 0.05) breast muscle yield and intramuscular fat content in breast muscle and decreased (p < 0.05) drip loss in breast muscle. Rutin supplementation increased (p < 0.05) the content of high-density lipoprotein but decreased (p < 0.05) the contents of glucose, triglyceride, and total cholesterol in serum. Rutin supplementation increased (p < 0.05) the levels of DHA (C22:6n-3), total polyunsaturated fatty acids (PUFAs), n-3 PUFAs, decanoic acid (C10:0), the activity of Δ5 + Δ6 (22:6 (n - 3)/18:3 (n - 3)), and the ratio of PUFA/SFA in breast muscle but decreased (p < 0.05) the level of palmitoleic acid (C16:1n-7), the ratio of n-6/n-3 PUFAs, and the activity of Δ9 (16:1 (n - 7)/16:0). Rutin treatment also reduced (p < 0.05) the contents of malondialdehyde in serum and breast muscle, and increased (p < 0.05) the catalase activity and total antioxidant capacity in serum and breast muscle and the activity of total superoxide dismutase in serum. Additionally, rutin supplementation downregulated the expression of AMPKα and upregulated the expression of PPARG, FADS1, FAS, ELOVL7, NRF2, and CAT in breast muscle (p < 0.05). Convincingly, the results revealed that rutin supplementation improved meat quality, fatty acid profiles, especially n-3 PUFAs, and the antioxidant capacity of Qingyuan partridge chickens.

Puerarin enhance vascular proliferation and halt apoptosis in thiram-induced avian tibial dyschondroplasia by regulating HIF-1α, TIMP-3 and BCL-2 expressions.

Tetramethyl thiuram disulfide (thiram) is a dithiocarbamate pesticide used for crop protection and storage. But, it's widespread utilization is associated with deleterious growth plate cartilage disorder in broilers termed as avian tibial dyschondroplasia (TD). TD results in non-mineralized and less vascularized proximal tibial growth plate cartilage causing lameness and poor growth performance. This study investigated the therapeutic potential of puerarin against thiram toxicity in TD affected chickens. One-day-old broiler chickens (n = 240) were alienated into three equal groups i.e. control, TD and puerarin (n = 80) and were offered standard feed. Additionally, TD and puerarin groups were offered thiram at 50 mg/kg of feed from 4 to 7 days for TD induction followed by puerarin therapy at 120 mg/kg to puerarin group only from 8 to 18 days for TD treatment. Thiram feeding to TD and puerarin group chickens caused lameness, mortality, and increased the aspartate aminotransferase (AST), alanine aminotransferase (ALT), malondialdehyde (MDA) levels and growth plate (GP) size and upregulated HIF-1α expression. Besides, the production parameters, alkaline phosphatase (ALP), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) levels and the expressions of TIMP-3 and BCL-2 were decreased (p < 0.05). Puerarin alleviated lameness, enhanced angiogenesis and growth performance and serum and antioxidant enzymes, decreased apoptosis and recuperated GP width by significantly downregulating HIF-1α and upregulating the TIMP-3 and BCL-2 mRNA and protein expressions in puerarin group chickens (p < 0.05). In conclusion, the toxic effects associated with thiram can be mitigated using puerarin.

Ameliorative effect of naringin against thiram-induced tibial dyschondroplasia in broiler chicken.

Tetramethyl thiuram disulfide (thiram) is widely used in agricultural production as an insecticide and fungicide, which can also lead to tibial dyschondroplasia (TD) in poultry. TD is characterized by leg disorders and growth performance retardation, and no targeted drugs have been found to treat TD until now. Therefore, the objective of the present study was to explore the ameliorative effect of traditional Chinese medicine naringin on thiram-induced TD chickens. A total of 180 one-day-old Arbor Acres (AA) broiler chickens were randomly divided into three equal groups (n = 60): control group (standard diet), thiram-induced group (thiram 50 mg/kg from day 3 to day 7), and naringin-treated group (naringin 30 mg/kg from day 8 to day 18). During the 18-day experiment, the growth performance, tibial bone parameters, antioxidant property of liver, serum biochemical changes and clinical symptoms were recorded to evaluate the protective effect of naringin in thiram-induced TD broiler chickens. Additionally, mRNA expressions and protein levels of Ihh and PTHrP genes were determined via quantitative real-time polymerase chain reaction and western blot. Administration of naringin showed significant results by alleviating lameness, increased growth performance, recuperated growth plate (GP) width, and improved functions and antioxidant enzyme level of liver in broilers affected by TD. Moreover, naringin treatment restored the development of damaged tibia bone via downregulating Ihh and upregulating PTHrP mRNA and protein expressions. In conclusion, our study determines naringin could be used as an effective medicine to treat TD.

Plastrum Testudinis Extract Mitigates Thiram Toxicity in Broilers via Regulating PI3K/AKT Signaling.

Tibial dyschondroplasia (TD) negatively affects broilers all over the world, in which the accretion of the growth plate (GP) develops into tibial proximal metaphysis. Plastrum testudinis extract (PTE) is renowned as a powerful antioxidant, anti-inflammatory, and bone healing agent. The current study was conducted to evaluate the efficacy of PTE for the treatment of thiram-induced TD chickens. Broilers (day old; n = 300) were raised for 3 days with normal feed. On the 4th day, three groups (n = 100 each) were sorted, namely, the control (normal diet), TD, and PTE groups (normal diet+ thiram 50 mg/kg). On the 7th day, thiram was stopped in the TD and PTE group, and the PTE group received a normal diet and PTE (30 mg/kg/day). Plastrum testudinis extract significantly restored (p < 0.05) the liver antioxidant enzymes, inflammatory cytokines, serum biochemicals, GP width, and tibia weight as compared to the TD group. The PTE administration significantly increased (p < 0.05) growth performance, vascularization, AKT (serine/threonine-protein kinase), and PI3K expressions and the number of hepatocytes and chondrocytes with intact nuclei were enhanced. In conclusion, PTE has the potential to heal TD lesions and act as an antioxidant and anti-inflammatory drug in chickens exposed to thiram via the upregulation of AKT and PI3K expressions.

Influence of dietary supplementation with Bacillus velezensis on intestinal microbial diversity of mice.

Yaks are an aboriginal breed of the Qinghai-Tibet plateau (3000 m), which are highly adaptable to cold and hypoxic environments. It is noticed that hypoxia and hypothermia can induce changes in intestinal microbial structure in animals. Increasing evidences suggested that probiotics supplementation can improve the balance of gut microbiota of animals. However, so far, very few studies have emphasized on the probiotics isolated from yaks in the Qinghai-Tibet Plateau. Therefore, a potential probiotic strain Bacillus velezensis was isolated from yaks. In the present study, a total of 18 Kunming mice (15-18 g) were equally distributed into two groups; control and probiotic treated groups (1 × 109 CFU/day). During the experimental period, all the mice from both groups were given standard normal diet ad libitum. At the end of the experiment, mice were euthanized and the intestines (duodenum, jejunum, ileum, and cecum) were removed for high-throughput sequencing. The results demonstrated that Bacillus velezensis supplementation showed beneficial effects on the gut microbiota of mice. Specifically, Bacillus velezensis supplementation increased the population of Lactobacillus and Ruminococcus in the duodenum, and Candidatus Arthromitus in the jejunum. Additionally, Acinetobacter in the duodenum and Helicobacter in the cecum were decreased after feeding Bacillus velezensis. Altogether, these findings suggested that Bacillus velezensis isolated from Tibetan yaks can improve gut microbiota of mice.

Osthole: A Coumarin Derivative Assuage Thiram-Induced Tibial Dyschondroplasia by Regulating BMP-2 and RUNX-2 Expressions in Chickens.

Avian tibial dyschondroplasia affects fast growing broiler chickens accounting for almost 30% of leg ailments in broilers. The present project was designed to assess the efficacy of osthole against avian tibial dyschondroplasia (TD). Two hundred and forty chickens were equally allocated into control, TD and osthole groups (n = 80). The TD and osthole group chickens were challenged with tetramethylthiuram disulfide (thiram) at 50 mg/kg of feed from 4-7 days, followed by osthole administration at 20 mg/kg orally to the osthole group only from 8-18 days. Thiram feeding resulted in lameness, increased mortality, and decreased production parameters, alkaline phosphatase (ALP), superoxide dismutase (SOD), total antioxidant capacity (T-AOC), and glutathione peroxidase (GSH-PX) levels, along with significantly increased aspartate aminotransferase (AST), alanine aminotransferase (ALT), malondialdehyde (MDA) levels, and growth plate size. Moreover, the genes and protein expressions of BMP-2 and RUNX-2 were significantly down-regulated in TD affected chickens (p < 0.05). Osthole administration showed promising results by alleviating lameness; increased ALP, SOD, T-AOC, and GSH-Px levels; and decreased the AST, ALT, and MDA levels significantly. It restored the size of the growth plate and significantly up-regulated the BMP-2 and RUNX-2 expressions (p < 0.05). In conclusion, the oxidative stress and growth plate anomalies could be assuaged using osthole.

Probiotics isolated from yaks improves the growth performance, antioxidant activity, and cytokines related to immunity and inflammation in mice.

BACKGROUND: Yaks living in the high-altitude hypoxic environment of Tibetan plateau (3600 m) have special gut microbes. However, it is still little research on yak probiotics until now. Therefore, the purpose of our study was to evaluate the growth promoting effect, antioxidant capability, immune effect, and anti-inflammatory ability of Bacillus subtilis and Bacillus velezensis isolated from Tibetan yaks in mice model. RESULTS: The results showed that the isolated strains supplementation not only improve the growth performance but also increased the length of villus in the small intestine and intestinal digestive enzyme activity. Importantly, we observed that the T-AOC, SOD, and GSH-PX levels were increased and the MDA content was reduced in probiotic-treated mice, which implied that probiotics supplementation can ameliorate the antioxidative activity of mice. The levels of AST and ALT correlated with the hepatic injury were reduced and the levels of AKP, TP, GLB, ALB, Ca, and P were markedly higher than those in the control group. Additionally, mice treated with probiotics exhibited higher serum IgG, IgM and IgA, which can reflect the immune status to some extent. At the same time, the major pro-inflammatory factor TNF-α, IL-6, and IL-8 were down-regulated and the anti-inflammatory factor IL-10 was up-regulated compared with the control groups. CONCLUSIONS: In conclusion, these results demonstrated that Bacillus subtilis and Bacillus velezensis supplementation can increase overall growth performance and ameliorate the blood parameters related to inflammation and immunity of mice.

Protective effect of Astragaloside IV to inhibit thiram-induced tibial dyschondroplasia.

Tibial dyschondroplasia (TD) is most the common tibiotarsal bone disease in rapidly growing birds throughout the world. There is accumulating evidence that COX-2 abnormal expression in tibia plays an important role in TD progression. So, the regulation of COX-2 is an ever more appealing target for therapeutic intervention in TD. Astragaloside IV has an indispensable role in maintaining COX-2 expression in many diseases. So, we designed this study to use Astragaloside IV (AST-IV) against TD-affected chickens. A total of 180 Arbor Acres chickens were randomly divided in the control group, TD group, and Astr (AST-IV-treated chickens) group. During the experiment, mortality, feed conversion ratio, physiological changes, biochemical criterion, liver antioxidant enzymes, and gene expression of COX-2 were examined in all the chicken groups at various days. The results showed that AST-IV administration restored the growth performance and tibia lesions and decreased the mortality as compared with TD chickens. The biochemical criterion (ALP, AST, and ALT) of serum and liver antioxidant enzymes (SOD, GSH-Px, MDA, and T-AOC) improved after the administration of AST-IV. The COX-2 gene was upregulated significantly (P < 0.05) in TD chickens. Whereas, AST-IV treatment downregulated both gene and protein expression of COX-2 significantly (P < 0.05) in TD-affected chickens. AST-IV recovered tibial dyschondroplasia chickens by increasing the growth performance, ameliorating tibial cartilage damage, and decreasing COX-2 expression. In conclusion, AST-IV can be used to prevent thiram-induced TD in chickens.

Proanthocyanidins Alleviates AflatoxinB1-Induced Oxidative Stress and Apoptosis through Mitochondrial Pathway in the Bursa of Fabricius of Broilers.

Aflatoxin B1 (AFB1) is a serious threat to the poultry industry. Proanthocyanidins (PCs) demonstrates a broad range of biological, pharmacological, therapeutic, and chemoprotective properties. The aim of this study was to investigate the ameliorative effects of PCs against AFB1-induced histopathology, oxidative stress, and apoptosis via the mitochondrial pathway in the bursa of Fabricius (BF) of broilers. One hundred forty-four one-day old Cobb chicks were randomly assigned into four treatment groups of six replicates (6 birds each replicate) for 28 days. Groups were fed on the following four diets; (1) Basal diet without addition of PCs or AFB1 (Control); (2) basal diet supplemented with 1 mg/kg AFB1 from contaminated corn (AFB1); (3) basal diet supplemented with 250 mg/kg PCs (PCs); and (4) basal diet supplemented with 1 mg/kg AFB1 + 250 mg/kg PCs (AFB1+ PCs). The present study results showed that antioxidant enzymes activities of total superoxide dismutase (T-SOD), catalase (CAT), glutathione peroxidase (GSH-Px), and glutathione S-transferase (GST) in AFB1 treated group were (p < 0.05) decreased, whereas malondialdehyde (MDA) contents were significantly increased in comparison with the control group. Furthermore, we found that dietary PCs treatment ameliorated AFB1-induced oxidative stress in the BF through inhibiting the accumulation of MDA content and enhancing the antioxidant enzymes activities (T-SOD, CAT, GSH-Px, and GST). Similarly, PCs markedly enhanced messenger RNA (mRNA) expression of antioxidant genes (SOD, CAT, GPx1, and GST) in comparison with AFB1 group. Moreover, histological results showed that PCs alleviated AFB1-induced apoptotic cells in the BF of broilers. In addition, both mRNA and protein expression results manifested that mitochondrial-apoptosis-associated genes (Bax, caspase-9, caspase-3, and p53 and cytochrome c) showed up-regulation, while (Bcl-2) showed down-regulation in AFB1 fed group. The supplementation of PCs to AFB1 diet significantly reversed the mRNA and protein expression of these apoptosis-associated genes, as compared to the AFB1 group. Our results demonstrated that PCs ameliorated AFB1-induced oxidative stress by modulating the antioxidant defense system and apoptosis in the BF through mitochondrial pathway in broilers.

Ligustrazine recovers thiram-induced tibial dyschondroplasia in chickens: Involvement of new molecules modulating integrin beta 3.

Tetramethyl thiuram disulfide (thiram) is a dithiocarbamate, which is extensively used in agriculture as pesticide and fungicide for treating grains intended for seed purposes and also for storing food grains. One of the most evident and detrimental effect produced by thiram is tibial dyschondroplasia (TD) in many avian species, by feeding diets containing thiram, a growth plate cartilage disease. TD is characterized by the lack of blood vessels and impaired vascular invasion of the hypertrophic chondrocyte resulting in the massive cell death. This study investigated the effects of ligustrazine on the treatment and control of thiram induced-TD. A total of 210 chicks were divided into three equal groups (n = 70): control group (received standard diet), TD group (feed on thiram containing diet from day 3-7), and ligustrazine group (feed on thiram containing diet from day 3-7 and after that ligustrazine @ 30 mg/kg from day 8 to day 18). During the experiment, the lameness, production parameters, tibia bone indicators, pathological index changes and integrin beta 3 (ITGB3) expressions were examined. The results reveal that ligustrazine plays an important role in improving angiogenesis and decreasing chondrocytes damage in TD chicks via a new molecule modulating ITGB3. So, the administration of ligustrazine can be an important way to cope with the losses and costs associated with TD in commercial poultry farming and animal welfare issue due to environmental contamination of thiram.