Electroporation Delivery of Cas9 sgRNA Ribonucleoprotein-Mediated Genome Editing in Sheep IVF Zygotes.

The utilization of electroporation for delivering CRISPR/Cas9 system components has enabled efficient gene editing in mammalian zygotes, facilitating the development of genome-edited animals. In this study, our research focused on targeting the ACTG1 and MSTN genes in sheep, revealing a threshold phenomenon in electroporation with a voltage tolerance in sheep in vitro fertilization (IVF) zygotes. Various poring voltages near 40 V and pulse durations were examined for electroporating sheep zygotes. The study concluded that stronger electric fields required shorter pulse durations to achieve the optimal conditions for high gene mutation rates and reasonable blastocyst development. This investigation also assessed the quality of Cas9/sgRNA ribonucleoprotein complexes (Cas9 RNPs) and their influence on genome editing efficiency in sheep early embryos. It was highlighted that pre-complexation of Cas9 proteins with single-guide RNA (sgRNA) before electroporation was essential for achieving a high mutation rate. The use of suitable electroporation parameters for sheep IVF zygotes led to significantly high mutation rates and heterozygote ratios. By delivering Cas9 RNPs and single-stranded oligodeoxynucleotides (ssODNs) to zygotes through electroporation, targeting the MSTN (Myostatin) gene, a knock-in efficiency of 26% was achieved. The successful generation of MSTN-modified lambs was demonstrated by delivering Cas9 RNPs into IVF zygotes via electroporation.

Cottonseed meal protein hydrolysate influences growth performance, carcass characteristics, serum biochemical indices, and intestinal morphology in yellow-feather broilers.

This study investigated the effects of cottonseed meal protein hydrolysate (CPH) on the growth performance, carcass characteristics, serum biochemical indices, intestinal morphology, and enzyme activities of yellow-feather broilers. We randomly divided 240 chicks into four groups, each with six replicates: a basal diet with 0% (CON), 1% (LCPH), 3% (MCPH), or 5% (HCPH) CPH. The trail spanned 63 days and included three phases: Days 1-21, 22-42, and 43-63. Increased average daily gain (ADG) and decreased ratio of feed to gain (F/G) with LCPH were observed in 21-day-old broilers (P < 0.05). MCPH led to higher ADG and average daily feed intake (ADFI) in 42-day-old broilers (P < 0.05). Additionally, CPH supplementation resulted in increased dressing percentage, percentage of half-eviscerated yield, percentage of eviscerated yield, breast muscle rate, and leg muscle rate were observed (P < 0.05) with diet. The serum levels of total protein (TP), high-density lipoprotein cholesterol (HDL-C), calcium (Ca), and phosphorus (P) were enhanced, and blood urea nitrogen (BUN) and triglyceride (TG) levels decreased with diet and CPH (P < 0.05). CPH increased the length of the jejunum and ileum and the weight of the duodenum, jejunum, and ileum in 21-day-old broilers (P < 0.05). Alterations in the duodenal villus structure in broilers occurred on Days 21 and 42, and the CPH groups performed better; however, a similar change occurred in the jejunum on Days 42 and 63 (P < 0.05). MCPH and HCPH enhanced trypsin activity in the duodenum of 21-day-old and 63-day-old broilers (p < 0.05). Chymotrypsin activity increased (P > 0.05) in the duodenum of 63-day-old broilers fed MCPH. Lipase activity increased (P < 0.05) in the jejuna of 21-day-old broilers treated with HCPH. CPH increased trypsin activity in the ilea of 21-day-old broilers (P < 0.05). These results showed that CPH influenced the growth performance, carcass characteristics, serum biochemical indices, and intestinal morphology of yellow-feather broilers, which are related to growth stage. The recommended CPH level in broilers is 1% before 21 days of age and 3% after 21 days of age.

Effects of dietary supplementation with multispecies probiotics on intestinal epithelial development and growth performance of neonatal calves challenged with Escherichia coli K99.

BACKGROUND: Probiotics exhibit antibiotic properties and are capable of treating certain bacterial infections, including diarrhea. Therefore, the aim of this study is to investigate the effects of dietary supplementation with multispecies probiotic (MSP) on diarrhea, average daily gain (ADG) and intestinal development of neonatal calves challenged with Escherichia coli K99. RESULTS: Thirty-six neonatal Holstein calves were randomly assigned to three treatment groups. After E. coli K99 challenge, calves in the control (C) and MSP treatment groups had significantly higher ADG and feed efficiency, and significantly lower fecal scores than those of calves in the diarrhea (D) group. The mean time of diarrhea resolution was 4.5 and 3.1 days for calves in the D and MSP treatment groups, respectively. Furthermore, the structures of the various segments (duodenum, jejunum and ileum) of the small intestine of the calves, activities of several small intestinal enzymes, and expression of several energy metabolism-related genes in the small intestine segments were significantly affected by MSP treatments. CONCLUSION: Dietary supplementation of MSP had a positive effect in treating calf diarrhea; it improved ADG and feed efficiency and promoted development of the small intestine. © 2022 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Effects of Clostridium butyricum on growth performance, metabonomics and intestinal microbial differences of weaned piglets.

BACKGROUND: Weaning stress of piglets causes a huge economic loss to the pig industry. Balance and stability of the intestinal microenvironment is an effective way to reduce the occurance of stress during the weaning process. Clostridium butyricum, as a new microecological preparation, is resistant to high temperature, acid, bile salts and some antibiotics. The aim of present study is to investigate the effects of C. butyricum on the intestinal microbiota and their metabolites in weaned piglets. RESULTS: There was no statistical significance in the growth performance and the incidence of diarrhoea among the weaned piglets treated with C. butyricum during 0-21 days experimental period. Analysis of 16S rRNA gene sequencing results showed that the operational taxonomic units (OTUs), abundance-based coverage estimator (ACE) and Chao index of the CB group were found to be significantly increased compared with the NC group (P < 0.05). Bacteroidetes, Firmicutes and Tenericutes were the predominant bacterial phyla in the weaned piglets. A marked increase in the relative abundance of Megasphaera, Ruminococcaceae_NK4A214_group and Prevotellaceae_UCG-003, along with a decreased relative abundance of Ruminococcaceae_UCG-005 was observed in the CB group, when compared with the NC group (P < 0.05). With the addition of C. butyricum, a total of twenty-two significantly altered metabolites were obtained in the feces of piglets. The integrated pathway analysis by MetaboAnalyst indicated that arginine and proline metabolism; valine, leucine and isoleucine biosynthesis; and phenylalanine metabolism were the main three altered pathways, based on the topology. Furthermore, Spearman's analysis revealed some altered gut microbiota genus such as Oscillospira, Ruminococcaceae_NK4A214_group, Megasphaera, Ruminococcaceae_UCG-005, Prevotella_2, Ruminococcaceae_UCG-002, Rikenellaceae_RC9_gut_group and Prevotellaceae_UCG-003 were associated with the alterations in the fecal metabolites (P < 0.05), indicating that C. butyricum presented a potential protective impact through gut microbiota. The intestinal metabolites changed by C. butyricum mainly involved the variation of citrulline, dicarboxylic acids, branched-chain amino acid and tryptophan metabolic pathways. CONCLUSIONS: Overall, this study strengthens the idea that the dietary C. butyricum treatment can significantly alter the intestinal microbiota and metabolite profiles of the weaned piglets, and C. butyricum can offer potential benefits for the gut health.

DNA methylation of CpG sites in the chicken KLF7 promoter and Exon 2 in association with mRNA expression in abdominal adipose tissue and blood metabolic indicators.

BACKGROUND: Our previous study found that chicken KLF7 was an important regulator in formation of adipose tissue. In the present study, we analyzed the association for DNA methylation in chicken KLF7 with its transcripts of abdominal adipose tissue and blood metabolic indicators. RESULTS: The KLF7 transcripts of the adipose tissue of Chinese yellow broilers were associated with age (F = 6.67, P = 0.0035). In addition, the KLF7 transcripts were negatively correlated with blood glucose levels (r = - 0.61841, P = 0.0140). The DNA methylation levels of 26 CpG loci in the chicken KLF7 promoter and Exon 2 were studied by Sequenom MassArray. A total of 22 valid datasets were obtained. None of them was significantly different in relation to age (P > 0.05). However, the DNA methylation levels in the promoter were lower than those in Exon 2 (T = 40.74, P < 0.01). Correlation analysis showed that the DNA methylation levels of PCpG6 and E2CpG9 were significantly correlated with KLF7 transcripts and blood high-density lipoprotein levels, respectively, and many CpG loci were correlated with each other (P < 0.05). The methylation data were subjected to principal component analysis and factor analysis. The six principal components (z1-z6) were extracted and named Factors 1-6, respectively. Factor analysis showed that Factor 1 had a higher load on the loci in the promoter, and Factors 2-6 loaded highly on quite different loci in Exon 2. Correlation analysis showed that only z1 was significantly correlated to KLF7 transcripts (P < 0.05). In addition, an established regression equation between z1 and KLF7 transcripts was built, and the contribution of z1 to the variation on KLF7 transcripts was 34.29%. CONCLUSIONS: In conclusion, the KLF7 transcripts of chicken abdominal adipose tissue might be inhibited by DNA methylation in the promoter, and it might be related to the DNA methylation level of PCpG6.

Cottonseed meal fermented by Candida tropical reduces the fat deposition in white-feather broilers through cecum bacteria-host metabolic cross-talk.

In the present study, effects of cottonseed meal fermented by Candida tropicalis (FCSM) on fat deposition, cecum microbiota, and metabolites and their interactions were studied in broilers. A total of 180 1-day-old broilers were randomly assigned into two groups with six replicates of 15 birds in each. The birds were offered two diets consisted one control, i.e., supplemented with 0% FCSM (CON) and an experimental, with 6% FCSM (FCSM). Illumina MiSeq sequencing and liquid chromatography-mass spectrometry were used to investigate the profile changes of the cecum microbes and metabolites and the interactions among fat deposition, microbes, and metabolites. Results showed that at the age of 21 days, both the abdominal fat and subcutaneous fat thickness of the experimental birds decreased significantly (P < 0.05) in response to the dietary FCSM supplementation. The predominant microbial flora in cecum consisted Bacteroidetes (53.55%), Firmicutes (33.75%), and Proteobacteria (8.61%). FCSM diet increased the relative abundance of Bacteroides but decreased obese microbial including Faecalibacterium, Lachnospiraceae, Ruminococcaceae, and Anaerofilum. Cecum metabolomics analysis revealed that lipids, organic acids, vitamins, and peptides were significantly altered by adding FCSM in diet. Correlation analysis showed that abdominal fat and subcutaneous fat thickness related negatively with Bacteroides while the same related positively with Faecalibacterium, Lachnospiraceae, and Ruminococcaceae. Moreover, abdominal fat and subcutaneous fat thickness were related negatively with nicotinic acid, sebacic acid, thymidine, and succinic acid. These findings indicated that FCSM reduced the fat deposition by regulating cecum microbiota and metabolites in broilers. The results are contributory to the development of probiotics and the improvement in the production of broilers.

Effect of a functional recombinant cytochrome P450 enzyme of Helicoverpa armigera on gossypol metabolism co-expressed with NADPH-cytochrome P450 reductase in Pichia pastoris.

Gossypol is a polyphonic toxic compound that is present in cotton plants. The P450 cytochromes CYP6AE14 and CYP9A12 of Helicoverpa armigera are highly induced by gossypol and have been reported to be possibly involved in gossypol degradation. To determine whether the candidate H. armigera CYP6AE14 and CYP9A12 enzymes could metabolize gossypol in vitro, functional recombinant H. armigera CYP6AE14 and CPR (CYP9A12 and CPR) enzymes were successfully expressed in Pichia pastoris (P. pastoris). UPLC-QTOF/MS demonstrated the following results: (1) Free gossypol was spontaneously degraded to the gossypol metabolites G1 (m/z 265) and G2 (m/z 293) without the addition of any enzyme. (2) Free gossypol was observed following the addition of the endogenous or recombinant H. armigera P450 cytochrome CYP6AE14/CYP9A12 enzyme: in the first pathway, free gossypol was dehydroxylated and decarboxylated to G3 (m/z 453), and in the second pathway, the aldehyde group of gossypol and its metabolite were covalently bound with the amine products to form G4 (m/z 437) and G5 (m/z 783). (3) In addition to the gossypol binding pathways, the recombinant H. armigera CPR and CYP9A12 enzymes was found that could further decarboxylate the gossypol intermediate demethylated reduction of gossypolonic acid (m/z 294) and demethylated gossic acid (m/z 265) to G0 (m/z 209) and G0' (m/z 249) respectively.

Branched Chain Amino Acids: Beyond Nutrition Metabolism.

Branched chain amino acids (BCAAs), including leucine (Leu), isoleucine (Ile), and valine (Val), play critical roles in the regulation of energy homeostasis, nutrition metabolism, gut health, immunity and disease in humans and animals. As the most abundant of essential amino acids (EAAs), BCAAs are not only the substrates for synthesis of nitrogenous compounds, they also serve as signaling molecules regulating metabolism of glucose, lipid, and protein synthesis, intestinal health, and immunity via special signaling network, especially phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) signal pathway. Current evidence supports BCAAs and their derivatives as the potential biomarkers of diseases such as insulin resistance (IR), type 2 diabetes mellitus (T2DM), cancer, and cardiovascular diseases (CVDs). These diseases are closely associated with catabolism and balance of BCAAs. Hence, optimizing dietary BCAA levels should have a positive effect on the parameters associated with health and diseases. This review focuses on recent findings of BCAAs in metabolic pathways and regulation, and underlying the relationship of BCAAs to related disease processes.

Clostridium butyricum Prevents Diarrhea Incidence in Weaned Piglets Induced by Escherichia coli K88 through Rectal Bacteria-Host Metabolic Cross-Talk.

This study aimed to evaluate the effects of Clostridium butyricum (C. butyricum) on the prevention of the diarrhea rates and growth performances of weaned piglets induced by Escherichia coli K88 (E. coli K88). Twenty-four weaned piglets (6.92 ± 0.11 kg) were randomly assigned to one of three treatment groups for a period of 21 days. Each group consisted of eight pigs, with each pig being housed in an individual pen. Group I received the control diet along with normal saline, Group II received the control diet along with E. coli K88, and Group III received the control diet supplemented with 5 × 108 CFU/kg of C. butyricum and E. coli K88. We examined alterations in rectal microbiota and metabolites, analyzed the incidence of diarrhea, and investigated the interactions between microbiota and metabolites through the application of Illumina MiSeq sequencing and liquid chromatography-mass spectrometry. The results showed that, from days 14 to 21, the diarrhea incidence in Group III decreased significantly by 83.29% compared to Group II (p < 0.05). Over the entire experimental duration, the average daily feed intake of Group III decreased significantly by 11.13% compared to Group I (p < 0.05), while the diarrhea incidence in Group III decreased by 71.46% compared to Group II (p < 0.05). The predominant microbial flora in the rectum consisted of Firmicutes (57.32%), Bacteroidetes (41.03%), and Proteobacteria (0.66%). Administering E. coli K88 orally can elevate the relative abundance of Megasphaera (p < 0.05). Conversely, the supplementation of C. butyricum in the diet reduced the relative abundance of Megasphaera (p < 0.05), while increasing the relative abundance of unclassified_f_Lachnospiraceae (p < 0.05). Rectal metabolomics analysis revealed that supplementing C. butyricum in the feed significantly altered the amino acids and fatty acids of the piglets infected with E. coli K88 (p < 0.05). The correlation analysis showed that the occurrence of diarrhea was inversely related to adipic acid (p < 0.05) and positively associated with (5-hydroxyindol-3-YL) acetic acid and L-aspartic acid (p < 0.05). Prevotella_1 exhibited a negative correlation with octadecanoic acid (p < 0.05). Prevotellaceae_UCG-005 showed a negative correlation with (5-hydroxyindol-3-YL) acetic acid (p < 0.05). The findings from this research study aid in probiotic development and the enhancement of healthy growth in weaned piglets.

Combined transcriptomics and cellular analyses reveal the molecular mechanism by which Candida tropicalis ZD-3 adapts to and degrades gossypol.

Microbial degradation techniques are often considered an environmentally friendly and cost-effective strategy for reducing gossypol toxicity. However, the mechanism by which Candida tropicalis degrades gossypol remains unclear. In the current study, we aimed to establish the mechanisms of biodegradation and adaptation mechanisms by C. tropicalis ZD-3. The toxicological evaluation results revealed that ZD-3 adapts to gossypol primarily by activating the antioxidant defense system to alleviate the oxidative stress response induced by gossypol. Transcriptomic analyses further suggested that ZD-3 protects against gossypol toxicity via cell wall remodeling. The intracellular enzyme CTRG_04744 gene was significantly up-regulated under gossypol stress, and then expressed in Pichia pastoris. The purified AKR_Z1 degraded 92 % of gossypol within 48 h. In addition, the aldehyde group of gossypol was effectively eliminated to achieve the desired detoxification. Collectively, these results provide theoretical guidance for the continued development of bio-efficient strategies capable of degrading gossypol.

Capsaicin Modulates Hepatic and Intestinal Inflammation and Oxidative Stress by Regulating the Colon Microbiota.

We aimed to investigate the role of capsaicin (CAP) in modulating lipopolysaccharide (LPS)-induced hepatic and intestinal inflammation, oxidative stress, and its colonic microflora in mice. Thirty healthy male Kunming mice with similar body weights were randomly assigned to three groups: the control group (CON), the LPS group, and the CAP group, with ten mice in each group. The CON and the LPS groups received a daily dose of normal saline, respectively, while the CAP group received an equivalent dose of CAP. On the 28th day of the experiment, the LPS and the CAP groups were intraperitoneally injected with LPS, while the CON group was injected with an equal volume of normal saline. The results lead to the following conclusions. Compared to the LPS group, CAP improved the loss of hepatic lobular structure and significantly increased the duodenal villus length and ratio of villus length to crypt depth. CAP increased hepatic and colon interleukin-10 (IL-10) and decreased IL-6, IL-1ß, and tumor necrosis factor (TNF-α) levels. CAP also increased hepatic catalase (CAT), glutathione peroxidase (GSH-Px), and superoxide dismutase (SOD) expression, and decreased malondialdehyde (MDA) levels. CAP significantly increased the relative abundances of Mucispirillum, Helicobacter, Prevotellaceae-UCG-001, Colidextribacter, unclassified-f-Oscillospiraceae, and Odoribacter, some of which were closely related to hepatic and colonic immune and oxidative markers. CAP also decreased the overall content of short-chain fatty acids, except for propionic acid. Overall, CAP can regulate the colon microbiota and exert anti-inflammatory and antioxidant effects. Whether CAP exerts its anti-inflammatory and antioxidant effects by modulating the colonic microflora, mainly Mucispirillum spp. and Helicobacter spp., requires further investigation.

Candida tropicalis ZD-3 prevents excessive fat deposition by regulating ileal microbiota and bile acids enterohepatic circulation in broilers.

Introduction: Evidence suggests that the dietary intake of Candida tropicalisZD-3 (ZD-3) has various health benefits, but the treatment mechanisms and effects remain unclear. The aim of this study investigates the effect of ZD-3 on reducing fat deposition in broilers and the underlying mechanism. Methods: 180 one-day-old, yellow-feathered broilers were randomly divided into three groups: control (CON) group fed a basal diet, an active Candida tropicalis ZD-3 (ZD) group supplemented with ZD, and a heat-inactivated Candida tropicalis ZD-3 (HZD) group supplemented with HZD. The experiment lasted for 28 d. Results: The ZD and HZD treatments significantly reduced the abdominal fat index (p < 0.05), decreased TG levels in serum and liver (p < 0.05), altered the ileal microbial composition by reducing the Firmicutes to Bacteroidetes (F/B) ratio. Additionally, the ZD and HZD treatments reduced liver cholesterol by decreasing ileal FXR-FGF19 signaling and increasing liver FXR-SHP signaling (p < 0.05). The ZD and HZD treatments also changed liver PC and TG classes lipid composition, regulating liver lipid metabolism by promoting TG degradation and modulating the signal transduction of the cell membrane. Discussion: Overall, ZD-3 was effective in improving lipid metabolism in broilers by regulating the ileal microbial composition and BAs enterohepatic circulation. This study provides a theoretical basis for the development and application of ZD-3 for the regulation of lipid metabolism in broilers.

Dietary succinate reduces fat deposition through gut microbiota and lipid metabolism in broilers.

Succinate has been shown to be a potentially beneficial nutritional supplement with a diverse range of physiological functions. However, it remains unknown whether succinate supplementation regulates lipid metabolism in chickens. The aim of this study was to explore how succinate affects fat deposition and the underlying mechanism involved in broilers and to determine the most appropriate level of succinate supplementation in the diet. A total of 640 one-day-old male yellow-feathered broilers were randomly divided into 4 groups with 8 replicates and 20 broilers per replicate. A basal diet was provided to the control group (CON). The experimental broilers were fed diets containing 0.2% (L), 0.4% (M), or 0.6% (H) succinate and the study was lasted for 21 d. The linear (l) and quadratic (q) effects of succinate addition were determined. The results indicated that supplementation with 0.4% succinate reduced ADFI, serum triglycerides (l, q; P < 0.05), glucose (q; P < 0.05), and increased high-density lipidprotein cholesterol (l, q; P < 0.05) concentrations in broilers. Moreover, 0.4% succinate affects lipid metabolism by decreasing the abdominal fat percentage and adipocyte surface area, the expression of genes that promote liposynthesis in the abdominal fat and liver, as well as increasing the expression of genes that promote lipolysis in the abdominal fat and liver. In addition, increased cecal propionic acid content (q, P < 0.05) was found in the M group compared to the CON group. The 16S rRNA sequence analysis showed that group M altered cecum microbial composition by increasing the abundance of genera such as Blautia and Sellimonas (P < 0.05). LC-MS metabolomic analysis revealed that the differential metabolites between the M and CON groups were enriched in amino acid-related pathways. In conclusion, the optimum level of succinate added to broiler diets in the present study was 0.4%. Succinate can potentially reduce fat accumulation in broilers by modulating the composition of the gut flora and amino acid metabolism related to lipid metabolism.

Effect of the supplementation of exogenous complex non-starch polysaccharidases on the growth performance, rumen fermentation and microflora of fattening sheep.

The objective of this study was to evaluate the effects of exogenous non-starch polysaccharidases (a mixture of cellulase, xylanase, ß-glucanase and mannanase) on the growth performance and nutrient digestibility, rumen fermentation, and rumen microflora of sheep. The animal trial was conducted using 36 5-month-old female fattening hybrid sheep (Duolang♂ × Hu♀) who were randomly assigned into four groups comprising nine sheep per treatment: CON, T1, T2, and T3, with 0, 0.1, 0.3, and 0.5% NSPases/kg DM of TMR, respectively. This complex enzyme product was screened for optimal ratios based on previous in vitro tests and responded positively to the in vitro fermentation of the TMR. When treated with NSPases, there was a non-linear effect of average daily gain and feed conversion rate, with the greatest improvement observed in the T2 group. There were no significant differences (p > 0.05) in nutrient intake or apparent digestibility among the NSPase-supplemented groups. In addition, T2 group had a significantly higher acetate to propionate ratio and pH (p < 0.05) than the other groups, and NH3-N and microbial protein concentrations showed a quadratic curve. The results revealed that both immunoglobulins and serum hormones increased linearly with addition (p < 0.05). As the T2 group showed the best growth performance, the CON and T2 groups were subjected to rumen metagenomic analysis. The results showed higher abundance of bacteria and lower abundance of Viruses in the rumen microbiota of the T2 group compared to the CON group. In addition, Uroviricota and Proteobacteria abundance was significantly lower in the T2 group than in the CON group at the phylum level (p < 0.05). These results suggest that the supplementation of high-concentrate rations with NSPases enhance immunity, reduces virus abundance in the rumen, improves rumen health, and promotes rumen fermentation. Our findings provide novel insights for improving growth performance and alleviating inflammatory responses arising from high concentrate feeding patterns in ruminants. However, the biological mechanisms cannot be elucidated by exploring the composition of rumen microbe alone, and further studies are required.

Differential Microbial Composition and Interkingdom Interactions in the Intestinal Microbiota of Holstein and German Simmental × Holstein Cross F1 Calves: A Comprehensive Analysis of Bacterial and Fungal Diversity.

Calf intestines are colonized by rich and complex microbial communities, playing a crucial role in animal physiology, metabolism, nutrition, and immune function. In this study, we provide insight into the composition of fecal microbial bacteria and fungi, respectively, as well as the cross-kingdom interactions. We investigated the intestinal microbiota of different breeds of calves by characterizing the bacterial and fungal communities in the rectal feces of Holstein calves and German Simmental × Holstein cross F1 generation (GXH) using 16S rRNA and ITS amplicon sequencing techniques. PICRUSt2 (version 2.2.0) were used to determine microbial diversity and function and explore the reasons why Holstein calves are more susceptible to diarrhea. The results revealed no significant difference in the diversity of fecal microbiota among the groups (p > 0.05). We identified Firmicutes, Bacteroidetes, and Proteobacteria as the dominant bacterial phyla in the fecal bacterial communities of the two breeds of calves. Ascomycota and Basidiomycota play important roles in the fungal community but differ in relative abundance. Bacteroides was the dominant genus at the group level for calf fecal microbiota in both breeds. The relative abundance of Prevotella, Escherichia-Shigella, Peptostreptococcus, and Butyricicoccus was higher in Holstein calves, and the relative abundance of Faecalibacterium, Megamonas, Butyricicoccus, and Alloprevotella was lower than GXH group. Aspergillus and Cladosporium were the dominating genera of fecal fungi in both groups of calves. LEfSe analysis revealed 33 different bacteria and 23 different fungi between the two groups, with more differential strains found in GXH. In addition, the feces fungi-bacteria interkingdom interactions varied among breeds. Thus, the composition and structure of bacterial and fungal communities in calf feces varied by breed, indicating a potential association between breed and microbial communities. We also found differences in the network between bacterial-fungal kingdoms. We explain the reasons for Holstein calves being more prone to diarrhea. This indicated that breed makes differences in calf diarrhea rates by influencing gut microbial composition and interactions.

Effects of two strains of Lactobacillus isolated from the feces of calves after fecal microbiota transplantation on growth performance, immune capacity, and intestinal barrier function of weaned calves.

Introduction: Weaning stress seriously affects the welfare of calves and causes huge economic losses to the cattle breeding industry. Probiotics play an important role in improving animal growth performance, enhancing immune function, and improving gut microbiota. The newly isolated strains of Lactobacillus reuteri L81 and Lactobacillus johnsonii L29 have shown potential as probiotics. Here, we studied the probiotic properties of these two strains on weaned calves. Methods: Forty calves were randomly assigned to four groups before weaning, with 10 calves in each group, control group (Ctrl group), L. reuteri L81 supplementation group (2 g per day per calf), L. johnsonii L29 supplementation group (2 g per day per calf), L. reuteri L81 and L. johnsonii L29 composite group (2 g per day per calf), and the effects of Lactobacillus reuteri L81 and Lactobacillus johnsonii L29 supplementation on growth performance, immune status, antioxidant capacity, and intestinal barrier function of weaned calves were evaluated. Results: The results showed that probiotics supplementation increased the average daily weight gain of calves after weaning, reduced weaning diarrhea index (p < 0.05), and increased serum IgA, IgM, and IgG levels (p < 0.05). L. reuteri L81 supplementation significantly decreased IL-6, increased IL-10 and superoxide dismutase (SOD) levels at 21 d after weaning (p < 0.05). Moreover, probiotics supplementation significantly decreased serum endotoxin (ET), diamine oxidase (DAO), and D-lactic acid (D-LA) levels at different time points (p < 0.05). In addition, supplementation with L. reuteri L81 significantly reduced the crypt depth and increased the ratio of villus height to crypt depth (p < 0.05) in the ileum, increased gene expression of tight junction protein ZO-1, Claudin-1 and Occludin in jejunum and ileum mucosa, reduced the gene expression of INF- γ in ileum mucosa and IL-8 in jejunum mucosa, and increased the abundance of beneficial bacteria, including Bifidobacterium, Lactobacillus, Oscillospira, etc. Discussion: verall, these results showed that the two strains isolated from cattle feces after low concentration fecal microbiota transplantation improved the growth performance, immune performance, antioxidant capacity, and intestinal barrier function of weaned calves, indicating their potential as supplements to alleviate weaning diarrhea in calves.

Impacts of sodium butyrate on intestinal mucosal barrier and intestinal microbial community in a weaned piglet model.

Objective: Butyrate is thought to enhance intestinal mucosal homeostasis, but the detailed mechanism remains unclear. Therefore, further investigation on the mechanism of butyrate regulation of intestinal mucosal homeostasis was performed. Materials and methods: This study used weaned piglets with similar intestinal metabolic function to humans as a research model. The dietary supplemented 0.2% sodium butyrate group (0.2% S) and negative control group (CON) were established to detect the effects of butyrate on growth performance, intestinal tissue morphology, mucosal barrier function, and intestinal microbial community structure in weaned piglets. Results: There was an increase in average daily gain (ADG) during three different experimental periods and a reduction in average daily feed intake (ADFI) and feed-to-gain ratio (F:G) during days 1-35 and days 15-35 in 0.2% S compared with CON (P > 0.05). Furthermore, villus height in the ileum and duodenum was increased, and crypt depths in the colon and jejunum were reduced in both groups (P < 0.05). Moreover, the ratio of villus height and crypt depth (V/C) in 0.2% S both in the ileum and jejunum was significantly increased (P < 0.05) compared with CON. The relative mRNA expression of PKC, MUC1, CLDN1, and ITGB1 was upregulated in the ileum of 0.2% S compared with CON (P < 0.05). The digesta samples of 0.2% S, both in the ileum (P < 0.05) and colon, contained greater intestinal bacterial abundance and diversity of probiotics, including Lactobacillus, Streptococcus, Megasphaera, and Blautia, which promoted amino acid metabolism and energy production and conversion in the colon and the synthesis of carbon-containing biomolecules in the ileum. Conclusion: In summary, dietary supplementation with 0.2% sodium butyrate was shown to have a tendency to improve the growth performance of weaned piglets and enhance intestinal mucosal barrier function via altering the gut microbiota.

Biodegradation of Free Gossypol by Helicoverpa armigera Carboxylesterase Expressed in Pichia pastoris.

Gossypol is a polyphenolic toxic secondary metabolite derived from cotton. Free gossypol in cotton meal is remarkably harmful to animals. Furthermore, microbial degradation of gossypol produces metabolites that reduce feed quality. We adopted an enzymatic method to degrade free gossypol safely and effectively. We cloned the gene cce001a encoding carboxylesterase (CarE) into pPICZαA and transformed it into Pichia pastoris GS115. The target protein was successfully obtained, and CarE CCE001a could effectively degrade free gossypol with a degradation rate of 89%. When esterase was added, the exposed toxic groups of gossypol reacted with different amino acids and amines to form bound gossypol, generating substances with (M + H) m/z ratios of 560.15, 600.25, and 713.46. The molecular formula was C27H28O13, C34H36N2O6, and C47H59N3O3. The observed instability of the hydroxyl groups caused the substitution and shedding of the group, forming a substance with m/z of 488.26 and molecular formula C31H36O5. These properties render the CarE CCE001a a valid candidate for the detoxification of cotton meal. Furthermore, the findings help elucidate the degradation process of gossypol in vitro.

Comparison of Bacterial and Fungal Community Structure and Potential Function Analysis of Yak Feces before and after Weaning.

Weaning is one of the most stressful periods in yak growth. However, the impact of weaning on microbial diversity, structure, and potential function of yak feces is not clear. In this study, 12 Xinjiang yaks aged 3, 4, 5, and 6 months old were selected to collect fresh feces before and after weaning. Through 16S rRNA and ITS high-throughput sequencing, the dynamic distribution and potential function of yak fecal, bacterial, and fungal communities in each month were revealed. The study found that the richness of fungi had a significant impact on weaning. At the phylum level, Firmicutes, Bacteroidetes, Ascomycota, and Basidiomycota, and at the genus level, 5-7N15, Oscillospira, Roseburia, Dorea, Preussia, Neoascochyta, Naganishia, and Sporormiella were enriched in yak feces of different months old. The abundance and proportion of bacteria Firmicutes, Bacteroidetes, 5-7N15, and fungi Mucoromyceta changed significantly before and after weaning. With the increase of months, Verrucomicrobia and Akkermansia have shown a downward trend. Through the prediction and analysis of fecal microbial function, it was found that at the level of primary pathways, weaning has a significant impact on cellular processes, environmental information processing, genetic information processing, metabolism, and organismal systems. At the level of secondary metabolic pathways, weaning has a significant impact on cell motility, signal transduction, folding, sorting and degradation, translation, amino acid metabolism, glycan biosynthesis and metabolism, metabolism of terpenoids and polyketides, and xenobiotics biodegradation and metabolism. In addition, by analyzing the differences in functional pathways and microbial composition between sample groups of different months, it was found that the differences in functional pathways were related to the abundance differences of some microorganisms. In general, the changes in the composition and structure of yak fecal microflora may reflect the adaptability of the intestinal microbiota.

Fermented grape seed meal promotes broiler growth and reduces abdominal fat deposition through intestinal microorganisms.

The fermentation of grape seed meal, a non-conventional feed resource, improves its conventional nutritional composition, promotes the growth and development of livestock and fat metabolism by influencing the structure and diversity of intestinal bacteria. In this study, the nutritional components of Fermented grape seed meal (FGSM) and their effects on the growth performance, carcass quality, serum biochemistry, and intestinal bacteria of yellow feather broilers were investigated. A total of 240 male 14-day-old yellow-feathered broilers were randomly selected and divided into four groups, with three replicates of 20 chickens each. Animals were fed diets containing 0% (Group I), 2% (Group II), 4% (Group III), or 6% (Group IV) FGSM until they were 56 days old. The results showed that Acid soluble protein (ASP) and Crude protein (CP) contents increased, Acid detergent fiber (ADF) and Neutral detergent fiber (NDF) contents decreased, and free amino acid content increased in the FGSM group. The non-targeted metabolome identified 29 differential metabolites in FGSM, including organic acids, polyunsaturated fatty acids, and monosaccharides. During the entire trial period, Average daily gain (ADG) increased and Feed conversion ratio (FCR) decreased in response to dietary FGSM supplementation (p < 0.05). TP content in the serum increased and BUN content decreased in groups III and IV (p < 0.05). Simultaneously, the serum TG content in group III and the abdominal fat rate in group IV were significantly reduced (p < 0.05). The results of gut microbiota analysis showed that FGSM could significantly increase the Shannon and Simpson indices of broilers (35 days). Reducing the relative abundance of Bacteroidetes significantly altered cecal microbiota composition by increasing the relative abundance of Firmicutes (p < 0.05). By day 56, butyric acid content increased in the cecal samples from Group III (p < 0.05). In addition, Spearman's correlation analysis revealed a strong correlation between broiler growth performance, abdominal fat percentage, SCFAs, and gut microbes. In summary, the addition of appropriate levels of FGSM to rations improved broiler growth performance and reduced fat deposition by regulating gut microbes through differential metabolites and affecting the microbiota structure and SCFA content of the gut.