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.

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.

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.

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.

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.

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.

Effect of a Multispecies Probiotic Mixture on the Growth and Incidence of Diarrhea, Immune Function, and Fecal Microbiota of Pre-weaning Dairy Calves.

The effects of different doses of a multispecies probiotic (MSP) mixture on growth performance, the incidence of diarrhea rate and immune function, and fecal microbial diversity and structure were evaluated in pre-weaning Holstein dairy calves at WK2, WK4, WK6, and WK8. Forty Chinese Holstein female newborn calves were randomly assigned to four treatments with 10 calves in each group, C (control group), T1 (0.5 g MSP/calf/day, T2 (1 g MSP/calf/day), and T3 (2 g MSP/calf/day) groups. The experimental period was 56 days. Feed intake and health scoring were recorded every day until the end of the experiment. Fecal contents and blood samples were sampled at WK2, WK4, WK6, and WK8. Growth performance, incidence of diarrhea, and total serum concentrations (IgA, IgG, and IgM) were analyzed. Bacterial 16S rRNA and fungal ITS genes were high-throughput sequenced for fecal microbiota. The relationships among the populations of the principal fecal microbiota at WK2 and the growth performance or serum immunoglobulin concentrations were analyzed using Pearson's rank correlation coefficients. The MSP supplementation reduced the incidence of diarrhea in the first 4 weeks of life, and serum IgA, IgG, and IgM concentrations increased between WK2 and WK8 in the T3 group. There was an increase in growth performance and reduction in the incidence of diarrhea until WK4 after birth in T3 group, compared with the control, T1, and T2 groups. The results of fecal microbiota analysis showed that Firmicutes and Bacteroides were the predominant phyla, with Blautia, Ruminococcaceae_UCG-005, norank_f__Muribaculaceae, Bacteroides, Subdoligranulum, and Bifidobacterium being the dominant genera in calf feces. Aspergillus, Thermomyces, and Saccharomyces were the predominant fungal phyla. Compared with the control, in T1 and T2 groups, the MSP supplementation reduced the relative abundance of Bacteroidetes and increased the relative abundance of Bifidobacterium, Lactobacillus, Collinsella, and Saccharomyces at WK2 in group T3. Thus, the fecal microbial composition and diversity was significantly affected by the MSP mixture during the first 2 weeks of the calves' life. MSP mixtures reduced the incidence of diarrhea in pre-weaning calves (during the first 4 weeks of life). There was a significant improvement in growth performance, reduction in calf diarrhea, balance in the fecal microbiota, and an overall improvement in serum immunity, compared with the control group. We, therefore, recommend adding 2 g/day of multispecies probiotic mixture supplementation in diets of dairy calves during their first 4 weeks of life before weaning.

Amino acids metabolism by rumen microorganisms: Nutrition and ecology strategies to reduce nitrogen emissions from the inside to the outside.

For the ruminant animal industry, the emission of nitrogenous substances, such as nitrous oxide (N2O) and ammonia (NH3), not only challenges environmental sustainability but also restricts its development. The metabolism of proteins and amino acids by rumen microorganisms is a key factor affecting nitrogen (N) excretion in ruminant animals. Rumen microorganisms that affect N excretion mainly include three types: proteolytic and peptidolytic bacteria (PPB), ureolytic bacteria (UB), and hyper-ammonia-producing bacteria (HAB). Microbes residing in the rumen, however, are influenced by several complex factors, such as diet, which results in fluctuations in the rumen metabolism of proteins and amino acids and ultimately affects N emission. Combining feed nutrition strategies (including ingredient adjustment and feed additives) and ecological mitigation strategies of N2O and NH3 in industrial practice can reduce the emission of nitrogenous pollutants from the ruminant breeding industry. In this review, the characteristics of the rumen microbial community related to N metabolism in ruminants were used as the metabolic basis. Furthermore, an effective strategy to increase N utilisation efficiency in combination with nutrition and ecology was reviewed to provide an inside-out approach to reduce N emissions from ruminants.

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.

Effects of Multispecies Probiotic on Intestinal Microbiota and Mucosal Barrier Function of Neonatal Calves Infected With E. coli K99.

Altered gut microbiota are implicated in inflammatory neonatal calf diarrhea caused by E. coli K99. Beneficial probiotics are used to modulate gut microbiota. However, factors that mediate host-microbe interactions remain unclear. We evaluated the effects of a combination of multispecies probiotics (MSP) on growth, intestinal epithelial development, intestinal immune function and microbiota of neonatal calves infected with E. coli K99. Twelve newborn calves were randomly assigned as follows: C (control, without MSP); D (E. coli O78:K99 + gentamycin); and P (E. coli O78:K99 + supplemental MSP). All groups were studied for 21 d. MSP supplementation significantly (i) changed fungal Chao1 and Shannon indices of the intestine compared with group D; (ii) reduced the relative abundance of Bacteroides and Actinobacteria, while increasing Bifidobacteria, Ascomycetes, and Saccharomyces, compared with groups C and D; (iii) improved duodenal and jejunal mucosal SIgA and total Short Chain Fatty Acids (SCFA) concentrations compared with group D; (iv) increased relative ZO-1 and occludin mRNA expression in jejunal mucosa compared with group D; and (v) enhanced intestinal energy metabolism and defense mechanisms of calves by reducing HSP90 expression in E. coli K99, thereby alleviating the inflammatory response and promoting recovery of mucosal function. Our research may provide direct theoretical support for future applications of MSP in ruminant production.

Dietary Montmorillonite Improves the Intestinal Mucosal Barrier and Optimizes the Intestinal Microbial Community of Weaned Piglets.

The study investigated the impact of dietary montmorillonite on the growth performance, intestinal mucosal barrier, and microbial community in weaned piglets with control group (CON) and dietary supplementation of 0.2% montmorillonite (0.2% M). Compared with the CON group, 0.2% M feed in the diet increased the average daily gain (ADG) on days 15-35 and day 1-35 and the average daily feed intake on days 1-35 (ADFI) (0.05 < P < 0.1). Besides, higher villus height of the duodenum and jejunum and lower crypt depth of duodenum and colon were revealed in the 0.2% M group than in the CON group (P < 0.05). Moreover, the V/C (ratio of the villus height and crypt depth) in the 0.2% M group was increased compared to that in the CON group both from the duodenum and ileum (P < 0.05). The relative mRNA expression of mucin-1, ITGB1 (ß1-integrins), and PKC (protein kinase C) of ileum in the 0.2% M group were upregulated (P < 0.05) compared to that in the CON group. The digesta sample of ileum from piglets in the 0.2% M group contained greater (P < 0.05) intestinal bacterial diversity and abundances of probiotics, such as Streptococcus, Eubacterium_rectale_group, and Lactobacillus, which could promote the synthesis of carbon-containing biomolecules. Overall, dietary supplementation of 0.2% M was shown to have a tendency to improve the growth performance of weaned piglets and may enhance their intestinal mucosal barrier function via altering the gut microbiota.

Antiviral effects of Bovine antimicrobial peptide against TGEV in vivo and in vitro.

BACKGROUND: In suckling piglets, transmissible gastroenteritis virus (TGEV) causes lethal diarrhea accompanied by high infection and mortality rates, leading to considerable economic losses. This study explored methods of preventing or inhibiting their production. Bovine antimicrobial peptide-13 (APB-13) has antibacterial, antiviral, and immune functions. OBJECTIVES: This study analyzed the efficacy of APB-13 against TGEV through in vivo and in vitro experiments. METHODS: The effects of APB-13 toxicity and virus inhibition rate on swine testicular (ST) cells were detected using 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT). The impact of APB-13 on virus replication was examined through the 50% tissue culture infective dose (TCID50). The mRNA and protein levels were investigated by real-time quantitative polymerase chain reaction and western blot (WB). Tissue sections were used to detect intestinal morphological development. RESULTS: The safe and effective concentration range of APB-13 on ST cells ranged from 0 to 62.5 µg/mL, and the highest viral inhibitory rate of APB-13 was 74.1%. The log10TCID50 of 62.5 µg/mL APB-13 was 3.63 lower than that of the virus control. The mRNA and protein expression at 62.5 µg/mL APB-13 was significantly lower than that of the virus control at 24 hpi. Piglets in the APB-13 group showed significantly lower viral shedding than that in the virus control group, and the pathological tissue sections of the jejunum morphology revealed significant differences between the groups. CONCLUSIONS: APB-13 exhibited good antiviral effects on TGEV in vivo and in vitro.

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.

Impacts of Dietary Protein from Fermented Cottonseed Meal on Lipid Metabolism and Metabolomic Profiling in the Serum of Broilers.

Dietary protein from fermented cottonseed meal (FCSM), widely used in poultry diets in China, had regulating effects on lipid metabolism. To understand the effects of FCSM on lipid metabolism in broilers, we analyzed the biochemical indexes, enzyme activity, hormone level and metabolites in serum responses to FCSM intake. One hundred and eighty 21-d-old Chinese yellow feathered broilers (536.07±4.43 g) were randomly divided into 3 groups with 6 replicates and 3 diets with 6 % supplementation of unfermented CSM (control group), FCSM by C. Tropicalis (Ct CSM) or C. tropicalis plus S. Cerevisae (Ct-Sc CSM). Result showed that: (1) FCSM intake decreased significantly the content of triglyceride (TAG), total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) (P<0.05) in serum; (2) FCSM intake could significantly increase enzyme activity of acetyl CoA carboxylase (ACC), lipoprotein lipase (LPL), fatty acid synthase (FAS) and hormone sensitive lipase (HSL) (P<0.05); (3) Ct-Sc CSM intake increased significantly the levels of adiponectin (ADP) (P<0.05); (4) FCSM intake caused significant metabolic changes involving glycolysis, TCA cycle, synthesis of fatty acid and glycogen, and metabolism of glycerolipid, vitamins B group and amino acids. Our results strongly suggested that FCSM intake could significantly affect lipid metabolism via multiple pathways. These findings provided new essential information about the effect of FCSM on broilers and demonstrated the great potential of nutrimetabolomics, through which the research complex nutrients are included in animal diet.

Regulation of Probiotics on Metabolism of Dietary Protein in Intestine.

Proteins are indispensable components of living organisms, which are derived mainly from diet through metabolism. Dietary proteins are degraded by endogenous digestive enzymes to di- or tripeptides and free amino acids (AAs) in the small intestine lumen and then absorbed into blood and lymph through intestinal epithelial cells via diverse transporters. Microorganisms are involved not only in the proteins' catabolism, but also the AAs, especially essential AAs, anabolism. Probiotics regulate these processes by providing exogenous proteases and AAs and peptide transporters, and reducing hazardous substances in the food and feed. But the core mechanism is modulating of the composition of intestinal microorganisms through their colonization and exclusion of pathogens. The other effects of probiotics are associated with normal intestinal morphology, which implies that the enterocytes secrete more enzymes to decompose dietary proteins and absorb more nutrients.

Effect of Fermented Cottonseed Meal on the Lipid-Related Indices and Serum Metabolic Profiles in Broiler Chickens.

This study aimed to investigate the changes of lipid-related gene and serum metabolites in broiler chickens fed with fermented cottonseed meal (FCSM) diet, through quantitative real-time PCR and metabolomics analysis. Totally, 180 1-day-old Cobb broilers were randomly assigned to two groups with six replicates of 15 birds in each. The two diets consisted of a control diet supplemented with 0% FCSM (CON group) and an experimental diet with 6% FCSM (fermented by Candida tropicalis) replacing the soybean meal (FCSM group). The results showed that both abdominal fat content and subcutaneous fat thickness significantly reduced (p < 0.05) in response to dietary FCSM supplementation at the age of 21 d. Serum concentrations of glucose, triglyceride, and low-density lipoprotein cholesterol decreased (p < 0.05) in FCSM fed broilers compared with CON fed broilers, while the levels of epinephrine and growth hormone in serum, liver and abdominal fat tissue were higher (p < 0.05) in FCSM than in CON fed broilers. The activity of hormone-sensitive esterase and lipoprotein lipase (LPL) in the liver and abdominal fat were higher (p < 0.05) in FCSM than CON group. Additionally, compared with the CON group (p < 0.05), the expression of peroxisome proliferator-activated receptor alpha and LPL genes were upregulated in the livers of FCSM group broilers. Gene expressions of hormone-sensitive lipase and LPL in the abdominal fat tissue were also upregulated (p < 0.05) with the broilers fed with FCSM diets. A total of 20 significantly different metabolites were obtained in the serum of different dietary FCSM supplemented fed broilers. The mainly altered pathways were clustered into organic acid metabolism, fatty acid metabolism, and amino acid metabolism. These results not only provide a better understanding of broilers' lipid metabolism with FCSM but also can be helpful in further improvement of the broilers' healthy production and utilization of FCSM.

Optimization of the process parameters for reduction of gossypol levels in cottonseed meal by functional recombinant NADPH-cytochrome P450 reductase and cytochrome P450 CYP9A12 of Helicoverpa armigera.

Gossypol is a toxic polyphenolic product that is derived from cotton plants. The toxicity of gossypol has limited the utilization of cottonseed meal (CSM) in the feed industry. The gene, Helicoverpa armigera CYP9A12, is a gossypol-inducible cytochrome P450 gene. The objective of our study was to obtain the functional recombinant H. armigera CYP9A12 enzyme in Pichia pastoris and to verify whether this candidate enzyme could decrease gossypol in vitro. Free and total gossypol contents were detected in the enzyme solution and in CSM. The H. armigera CYP9A12 enzyme degraded free concentration of gossypol. After optimization of the single-test and response surface method, free gossypol content could be decreased to 40.91 mg/kg in CSM by the H. armigera CYP9A12 enzyme when the initial temperature was 35 °C, the enzymatic hydrolysis time lasted 2.5 h, the enzyme addition was 2.5 mL, and the substrate moisture was 39%.

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.