Co-contamination and interactions of multiple mycotoxins and heavy metals in rice, maize, soybeans, and wheat flour marketed in Shanghai City.

Mycotoxins and heavy metals extensively contaminate grains and grain products, posing severe health risks. This work implements validated ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) and inductively coupled plasma mass spectrometry (ICP-MS) methods to quantify the concentration of 12 mycotoxins and five heavy metals in rice, maize, soybeans, and wheat flour samples marketed in Shanghai. The mixed contamination characteristics were analyzed using correlation cluster analysis and co-contamination index, and the probabilities of all cross combinations of contaminations were analyzed using a self-designed JAVA language program. The results showed that grains and grain products were frequently contaminated with both mycotoxins and heavy metals, mostly with deoxynivalenol (DON), 3-acetyl-deoxynivalenol (3-ADON), 15-acetyl-deoxynivalenol (15-ADON), ochratoxin A (OTA), aflatoxins, fumonisin B1 (FB1), fumonisin B2 (FB2), fumonisin B3 (FB3), arsenic (As), chromium (Cr) and cadmium (Cd). All the samples (100 %) were contaminated with two or more contaminants, and 77.3 % of the samples were co-contaminated with more than four contaminants. In cereals and cereal products, the following combinations were closely associated: (FB3 +3-ADON), (FB1 +As), (FB1 +FB2), (DON+FB1), (DON+Cd), (As+Cd), (DON+Cd+As), (FB1 +FB2 +As), and (DON+3-ADON+15-ADON). The results indicated that mycotoxins and heavy metals frequently co-occurred in Shanghai grains and grain products, and they provided primary data for safety assessments, early warnings, and regulatory measures on these contaminants to protect public health.

Growth performance, lipid metabolism, and systemic immunity of weaned piglets were altered by buckwheat protein through the modulation of gut microbiota.

Tartary buckwheat protein (BWP) is well known for the wide-spectrum antibacterial activity and the lipid metabolism- regulating property; therefore, BWP can be applied as feed additives to improve the animal's nutritional supply. With the aim to investigate the bioactive actions of the BWP, growth performance, lipid metabolism and systemic immunity of the weaned piglets were measured, and the alterations of pig gut microbiota were also analyzed. According to the results, the growth performances of the weaned piglets which were calculated as the average daily gain (ADG) and the average daily feed intake (ADFI) were significantly increased when compared to the control group. Simultaneously, the serum levels of the total cholesterol (TC), triglycerides (TG), and low-density lipoprotein cholesterol (LDL-C) were decreased, while the levels of high-density lipoprotein cholesterol (HDL-C) were increased in the BWP group. Moreover, the relative abundances of Lactobacillus, Prevotella_9, Subdoligranulum, Blautia, and other potential probiotics in the gut microbiota of weaned piglets were obviously increased in the BWP group. However, the relative abundances of Escherichia-Shigella, Campylobacter, Rikenellaceae_RC9_gut_group and other opportunistic pathogens were obviously decreased in the BWP group. In all, BWP was proved to be able to significantly improve the growth performance, lipid metabolism, and systemic immunity of the weaned piglets, and the specific mechanism might relate to the alterations of the gut microbiota. Therefore, BWP could be explored as a prospective antibiotic alternative for pig feed additives.

Fecal microbial composition associated with testosterone in the development of Meishan male pigs.

Introduction: The gut microbiota closely relates to host health, whereas the relationship between gut microbiota and testosterone during the development of Meishan male pigs remains unclear. This study investigated the fecal microbiota composition and testosterone level during development in Meishan male pigs. Methods: Fresh fecal samples of 20 healthy Meishan male pigs were individually collected at 10 and 22 weeks (wk) of age for testosterone content detection and bacteria pyrosequencing analysis. Anaerobic culture experiment of fecal bacteria in vitro was performed for bacteria pyrosequencing analysis. Results: The fecal testosterone content increased significantly from 10 weeks (wk) to 22 wk of age (P < 0.05). Meanwhile, the boars at 22 wk had a lower abundance of phylum Bacteroidetes and Proteobacteria, and genus Alloprevotella, Prevotella_1, Prevotellaceae_NK3B31_group, and Streptococcus in the fecal microbiota composition (P < 0.05). but higher proportions of the phylum Actinobacteria, Firmicutes, Kiritimatiellaeota, and Tenericutes, and genus Clostridium_sensu_stricto_1, Muribaculaceae and Terrisporobacter than that at 10 wk (P < 0.05), and the Firmicutes to Bacteroidetes ratio was higher at 22 wk than 10 wk (P < 0.05). Moreover, the fecal testosterone level significantly correlated with the relative abundance of the phylum Actinobacteria, Firmicutes, and Tencuteseri, and genus Alloprevotella, Clostridium_sensu_stricto_1, Muribaculaceae, Prevotella_1 and Streptococcus. Furthermore, the in vitro experiments indicated that the abundance of the phylum Proteobacteria and genus Escherichia-Shigella reduced with the increase of supplemental testosterone level. In contrast, the proportion of Firmicutes phylum increased with additional testosterone levels. Discussion: Testosterone could modulate the microflora structure. Meanwhile, the bacteria could degrade the testosterone in a dose testosterone-dependent manner. These results provide us with new insights into the relationship between the gut microbiome and testosterone and the contributions of the gut microbiome in physiological regulation in response to gonad development.

Effects of Chinese herbal feed additives on the sperm quality and reproductive capacity in breeding boars.

Currently, Chinese herbal feed additives (CHFA) are commonly utilized in domestic pig farms. However, their impact on the sperm quality and reproductive capacity of imported breeding boars has yet to be thoroughly explored. In this study, the effect of CHFA on the sperm quality and reproductive capacity of the imported Duroc boars was investigated. Sixteen boars were randomly divided into control group and experimental (CHFA treated) group and fed normal or CHFA-levels containing diets, respectively. The sperm quality and reproductive hormone levels were periodically tested, and the reproductive capacity with breeding sows were evaluated. The results showed that the CHFA treated group boars significantly improved sperm volume, sperm concentration, and motility and reduced the sperm abnormalities. Furthermore, the serum levels of reproductive hormone such as follicle-stimulating hormone (FSH), luteinizing hormone (LH), and testosterone (T) in the CHFA treated group were significantly higher than those in the control group. Although there was no significant difference in the initial birth weight of piglets between the two groups, the CHFA treated group had a significantly higher average number of piglets born, the average number of piglets born alive, the number of piglets weaned at 28 days, and the weaning weight compared to the control group. These findings suggest that CHFA can significantly improve the sperm quality of breeding boars and enhance their reproductive hormone levels as well as the reproductive capacity, providing direct evidence for the further application of CHFA in the management of breeding boars in China.

Melatonin mediates via melatonin receptor 1 in a temperature-dependent manner regulating ATP metabolism and antioxidative enzyme activity of boar spermatozoa in vitro.

Researches indicated that melatonin (MLT) could ameliorate oxidative damage and increase sperm motility for conservation sperms, whereas the underlying mechanism remains unknown. And elucidating this, present study was designed to investigate the energy metabolism of sperm throughout preservation at 17 °C and activating with 37 °C. Herein, semen of ten Landrace boars (16∼18 months of age) were diluted and treated with gradient concentrations of MLT (0 nM, 1 nM, 10 nM, 1 µM, 1 mM and 5 mM), and kept away from light in the incubator of 17 °C for 5 days (preservation), and the sperm motility was evaluated after 37 °C incubation for 10 min (activating). The results showed that 1.0 µM MLT treated-sperms had higher motility than other treatments from the 2nd onward. On the 3rd day 17 °C storage, 1.0 µM MLT significantly decreased the ATP content and the mRNA abundance of mitochondrial DNA encoding genes, including NADH dehydrogenase subunits (NDs) 1-4 and 4L, cytochrome b (CYTB), cytochrome c oxidases (COXs) 1 and 3, ATP synthase 6, but increased the hexokinase activity (HK) and the malondialdehyde (MDA) content compared to that of the control. Whereas after 10 min 37 °C incubation, 1.0 µM MLT increased the ATP content, and the mRNA expressions of ND2 and 4L, CYTB, COX1, ATP6, and the activities of mitochondrial respiratory chain complex I and superoxide dismutase (SOD), but depressed the pyruvate kinase (PK) activity in contrast to that at 17 °C. Furthermore, 1.0 µM MLT treated sperms had a higher MLT receptor 1 (MT1) protein level at 17 °C storage than that after 10 min 37 °C incubation, no changes of the MT2 expression were observed at different temperature. This study suggests that MLT might mediate via MT1 in a temperature-dependent manner regulating sperm ATP generation and antioxidative enzyme activity in vitro.

Comparative Transcriptome Analysis Provides Insight into Spatio-Temporal Expression Characteristics and Genetic Regulatory Network in Postnatal Developing Subcutaneous and Visceral Fat of Bama Pig.

The depot differences between Subcutaneous Fat (SAF) and Visceral Fat (VAF) are critical for human well-being and disease processes in regard to energy metabolism and endocrine function. Miniature pigs (Sus scrofa) are ideal biomedical models for human energy metabolism and obesity due to the similarity of their lipid metabolism with that of humans. However, the regulation of differences in fat deposition and development remains unclear. In this study, the development of SAF and VAF was characterized and compared in Bama pig during postnatal development (infancy, puberty and adulthood), using RNA sequencing techniques (RNA-Seq). The transcriptome of SAF and VAF was profiled and isolated from 1-, 3- and 6 months-old pigs and identified 23,636 expressed genes, of which 1,165 genes were differentially expressed between the depots and/or developmental stages. Upregulated genes in SAF showed significant function and pathway enrichment in the central nervous system development, lipid metabolism, oxidation-reduction process and cell adhesion, whereas genes involved in the immune system, actin cytoskeleton organization, male gonad development and the hippo signaling pathway were preferentially expressed in VAF. Miner analysis of short time-series expression demonstrated that differentiation in gene expression patterns between the two depots corresponded to their distinct responses in sexual development, hormone signaling pathways, lipid metabolism and the hippo signaling pathway. Transcriptome analysis of SAF and VAF suggested that the depot differences in adipose tissue are not only related to lipid metabolism and endocrine function, but are closely associated with sexual development and organ size regulation.

Genome-Wide Characterization and Comparative Analyses of Simple Sequence Repeats among Four Miniature Pig Breeds.

Simple sequence repeats (SSRs) are commonly used as molecular markers in research on genetic diversity and discrimination among taxa or breeds because polymorphisms in these regions contribute to gene function and phenotypically important traits. In this study, we investigated genome-wide characteristics, repeat units, and polymorphisms of SSRs using sequencing data from SSR-enriched libraries created from Wuzhishan (WZS), Bama (BM), inbred Luchuan (LC) and Zangxiang (ZX) miniature pig breeds. The numbers and types of SSRs, distributions of repeat units and polymorphic SSRs varied among the four breeds. Compared to the Duroc pig reference genome, 2518 polymorphic SSRs were unique and common to all four breeds and functional annotation revealed that they may affect the coding and regulatory regions of genes. Several examples, such as FGF23, MYF6, IGF1R, and LEPROT, are associated with growth and development in pigs. Three of the polymorphic SSRs were selected to confirm the polymorphism and the corresponding alleles through fluorescence polymerase chain reaction (PCR) and capillary electrophoresis. Together, this study provides useful insights into the discovery, characteristics and distribution of SSRs in four pig breeds. The polymorphic SSRs, especially those common and unique to all four pig breeds, might affect associated genes and play important roles in growth and development.

Pyrodextrin enhances intestinal function through changing the intestinal microbiota composition and metabolism in early weaned piglets.

Pyrodextrin (PD) is prepared from starch by heat treatment and is resistant to amylase. We hypothesized that PD might have prebiotic potential affecting the microbiota composition, because it contains a non-digestible portion that may behave as dietary fiber. This study investigated the effects of PD supplementation on growth performance, gut morphology, short-chain fatty acids (SCFAs), and the bacterial community in weaned piglets receiving dietary supplementation of 0.5% PD. The piglets in the PD (treated) groups showed greater antioxidant capacity and feed efficiency (P < 0.05), as well as improved intestinal morphology in comparison with the piglets in the weaned (control) group. Gut microbiota profiles were assessed through 16S rRNA sequencing on the ileum contents and feces of early weaned piglets. Several genus-level enrichments and depletions were observed in response to PD treatment. Of note, PD supplementation decreased the relative abundance of pathogenic organisms, including Defluviicoccus and Gardnerella, while markedly increasing that of commensal bacteria (genera Psychrobacter and Prevotella), which have important roles in nutrient absorption and immune response regulation. The most notable effect in the PD treatment groups was increased production of SCFAs in the feces of PD-treated weaned piglets. Correlation analysis revealed that the improvement in SCFAs was positively correlated with the increase in SCFA-producing bacteria. Overall, this study provides a more comprehensive understanding of the effects of PD supplementation on the fecal microbial community and the modulation of SCFA production in early weaned piglets, thus indicating that PD can be used to alleviate weaning stress in piglets.

Crude fiber modulates the fecal microbiome and steroid hormones in pregnant Meishan sows.

The beneficial effects of dietary fiber on the reproductive performance and welfare of sows have been discussed broadly, but few researches examined the causal changes and the association of gut microbiota and the steroid hormones, the main regulators of reproductive function. To shed light on this, thirty-six Meishan sows were allocated into 2.5% crude fiber (CF) group and 7.5% CF group respectively for an entire farrowing interval. On the 90th day of gestation, the saliva and fresh stool of sows were individually collected in the morning (06:00-07:00) for steroid hormones, short-chain fatty acids (SCFAs) and microbiome analysis. In addition, the parameter of pregnant behavioral and farrowing performance was recorded and evaluated. We observed that, as compared with the 2.5% CF treatment, 7.5% CF significantly increased the litter size (p = 0.01), reduced the stereotypic behaviors including sham chewing, rolling tongue and licking ground (p = 0.02, 0.04, 0.01) at later gestation stage, but increased lying time (p = 0.00). In coincide with this, 7.5% CF diet increased the salivary progesterone (p = 0.00), fecal estradiol and progesterone (p = 0.01, 0.02) level, fecal water and SCFAs content (p = 0.02, 0.03), decreased the salivary and fecal cortisol (p = 0.01, 0.00) level. Further, 7.5% CF diet increased the fecal microbiota richness (ACE, p = 0.04; Chao, p = 0.07) and diversity (Shannon, p = 0.01; Simpson, p = 0.04), the proportion of genus Ruminococcus, Butyrivibrio, Lactobacillus and Fibrobacter (p = 0.02, 0.05, 0.04, 0.00), whereas reduced the proportion of genus Clostridium, Streptococcus, Bacteroides and Escherichia-Shigella (p = 0.00, 0.00, 0.04, 0.04). These results indicate that, fibrous diet can regulate the steroid hormones secretion and modulate the gut with more cellulose-degrading and probiotic bacterium, but less opportunistic pathogens, and this may contribute to the improvement of reproductive performance and welfare in sows.

Butyric acid regulates progesterone and estradiol secretion via cAMP signaling pathway in porcine granulosa cells.

Butyric acid (BA), one of the short chain fatty acids (SCFAs), has positive actions on the metabolism, inflammation, etc. However, whether it influences the reproductive physiology and if so the detail mechanism involved has not yet been determined. In this study, the porcine granulosa cells (PGCs) were treated with gradient concentrations of BA. After 24h culture, 0.05mM BA significantly stimulated the progesterone (P4) secretion (P<0.05), 5mM and 10mM BA significantly inhibited the P4 secretion (P<0.05). Simultaneously, BA up-regulated the estradiol (E2) secretion in a dose dependent manner, 5mM and 10mM BA significantly promoted the E2 level (P<0.05). In addition, 10mM BA significantly promoted the G-protein-coupled receptor 41/43 mRNA (P<0.05). Interestingly, 5mM BA treatment significantly down-regulated cyclic adenosine monophosphate (cAMP) content (P<0.05), steroidogenic acute regulatory (StAR), steroidogenic factor 1 (SF1), P450scc in the mRNA and/or protein level (P<0.05), and these actions were reversed by cAMP activator forskolin (FK). Moreover, the co-treatment of 5mM BA and bupivacaine (BPC, the cAMP inhibitor) significantly accumulated the inhibition action of BPC on cAMP, the secretion of P4, and the abundance of StAR mRNA (P<0.05), inhibited the up-regulation of 5mM BA on the E2 secretion (P<0.05). Further, the Global Proteome and KEGG pathway analysis found that 5mM BA significantly up-regulated the I3LM80 proteins (P<0.05), which is involved in the steroid biosynthesis signaling pathway. 5mM BA significantly decreased the F2Z5G3 protein level (P<0.05), and the cAMP signaling pathway. In conclusion, present findings for the first time demonstrated that BA could regulate the P4 and E2 hormone synthesis in PGCs via the cAMP signaling pathway.