Colonic Microbiota Improves Fiber Digestion Ability and Enhances Absorption of Short-Chain Fatty Acids in Local Pigs of Hainan.

Compared to commercial breeds, Chinese local pig breeds have a greater ability to digest dietary fiber, which may be due to differences in intestinal microbiota. In this study, we fed Ding'an and DLY pigs high and low levels of dietary fiber, respectively, to investigate factors contributing to high dietary fiber adaption in Ding'an pigs. Twelve Ding'an pigs and DLY pigs were randomly divided into a 2 (diet) × 2 (breed) factorial experiment (n = 3). Compared with commercial pigs, Ding'an pigs have a stronger ability to digest dietary fiber. Prevotella was more prevalent in Ding'an pigs than in DLY pigs, which may be an important reason for the stronger ability of fiber degradation in Ding'an pigs. When the effects of feed and breed factors are considered, differences in abundance of 31 species and 14 species, respectively, may result in a greater ability of fiber degradation in Ding'an pigs. Among them, Prevotella. sp. CAG:520 may be a newly discovered bacterium related to fiber degradation, which positively correlated with many fiber-degrading bacteria (r > 0.7). We also found that the concentration of plant metabolites with anti-inflammatory and antioxidant effects was higher in the colonic chyme of Ding'an pigs after increasing the fiber content, which resulted in the downregulated expression of inflammatory factors in colonic mucosa. Spearman's correlation coefficient revealed a strong correlation between microbiota and the apparent digestibility of dietary fiber (r > 0.7). The mRNA expressions of SLC16A1, PYY, and GCG were significantly increased in the colonic mucosa of Ding'an pigs fed on high-fiber diets, which indicates that Ding'an pigs have an enhanced absorption of SCFAs. Our results suggested that an appropriate increase in dietary fiber content can reduce the inflammatory response and improve feed efficiency in Ding'an pigs, and differences in the intestinal microbial composition may be an important reason for the difference in the fiber degradation capacity between the two breeds of pigs.

Study on the Characteristics of Coarse Feeding Tolerance of Ding'an Pigs: Phenotypic and Candidate Genes Identification.

Ding'an (DA) pig, a prominent local breed in Hainan Province, exhibits notable advantages in coarse feeding tolerance and high-quality meat. To explore the potential genetic mechanism of coarse feeding tolerance in DA pigs, 60-day-old full sibling pairs of DA and DLY (Duroc-Landrace-Yorkshire) pigs were subjected to fed normal (5%) and high (10%) crude fiber diets for 56 days, respectively. The findings showed that increasing the crude fiber level had no impact on the apparent digestibility of crude fiber, intramuscular fat, and marbling scores in DA pigs, whereas these factors were significantly reduced in DLY pigs (p < 0.05). Through differential expression analysis and Weighted Gene Co-expression Network Analysis (WGCNA) of the colonic mucosal transcriptome data, 65 and 482 candidate genes with coarse feeding tolerance in DA pigs were identified, respectively. Joint analysis screened four key candidate genes, including LDHB, MLC1, LSG1, and ESM1, potentially serving as key regulated genes for coarse feeding tolerance. Functional analysis revealed that the most significant pathway enriched in differential genes associated with coarse feeding tolerance in Ding'an pigs was the signaling receptor binding. The results hold substantial significance for advancing our understanding of the genetic mechanisms governing coarse feeding tolerance in Ding'an pigs.

Effects of dietary cecropin on growth performance, diarrhea rate and intestinal health of nursery Hainan pigs.

Antimicrobial peptides could inhibit the growth of harmful bacteria and promote the growth performance in weaned piglets. Here, we investigated the effects of dietary supplementation with cecropin antimicrobial peptides (CAP) on growth performance, diarrhea rate, intestinal health in nursery Hainan piglets. For this, 120 healthy nursery Hainan male piglets (13.29 ± 0.29 kg, 44 days old) were randomly divided into 5 groups-a control (CON) group (fed a basal diet), an antibiotic control (AC) group (fed a basal diet supplemented with 250 mg/kg colistin sulfate); and 3 experimental groups (provided the basal diet supplemented with 250, 500, or 1,000 mg/kg CAP). Pre-feeding lasted 7 days and the official period lasted 40 days. The results showed that compared with the CON group, dietary supplementation of 500 mg/kg CAP had significantly increased the average daily gain (ADG, p < 0.05), while the feed conversion ratio (FCR) and diarrhea rate were markedly reduced (p < 0.05), serum total protein (TP), albumin, IgA, IgM, and globulin concentrations were significantly increased (p < 0.05), where serum aspartate aminotransferase (AST) level was significantly reduced (p < 0.05), and it also increased the villus height and the villus height-to-crypt depth ratio in the jejunum, reduced the serum D-lactic acid concentrations and diamine oxidase activity, and increased the expression level of ZO-1 and occludin in the jejunum and ileum (p < 0.05), the relative abundance of Firmicutes, Lactobacillus, and Limoslactobacillus in the colon were increased (p < 0.05), whereas that of Streptococcus and Escherichia-Shigella were reduced (p < 0.05). These results indicated that dietary supplementation with 500 mg/kg CAP could improve the growth performance, reduce the diarrhea rate, improve the serum immunity, intestinal health of nursery pigs.

Protective effect of Enterococcus faecium against ethanol-induced gastric injury via extracellular vesicles.

Recently, Enterococcus has been shown to have gastric protective functions, and the mechanisms by which Enterococcus modulates gastric function are still being investigated. Herein, we investigated how Enterococcus faecium (Efm) and E. faecium-derived extracellular vesicles (EVs) (EfmEVs) exert protective effect against ethanol-induced gastric injury by investigating the effect of EfmEVs on gastric mucosal ulcer scoring, histological lesion, mucosal glycoprotein production, acidity, anti-oxidative function, and inflammatory responses in rat. Pretreatment with Efm showed significant reduction of ethanol-induced gastric injury, as evidenced by the lowering of ulcer index, histological lesion, gastric pH, and inflammatory responses and the enhancement of mucosal glycoprotein production and anti-oxidative function. Further functional studies on three bioactive components [inactivated Efm, EfmEVs (EVs), and EV-free supernatants] of the bacterial culture showed that EVs are mostly responsible for the gastroprotective effect. Moreover, EV secretion is beneficial for the gastroprotective effect of Efm. Hence, EVs mediated the protective effect of Efm against ethanol-induced gastric injury by lowering inflammatory responses and enhancing anti-oxidative function and may be a potent anti-inflammatory and anti-oxidative strategy to alleviate hyperinflammatory gastrointestinal tract conditions.IMPORTANCEThis study indicated that Enterococcus faecium provided a protective effect against rat gastric injury, which involved improvement of the mucosal glycoprotein production, anti-oxidative function, and inflammatory responses. Furthermore, we confirmed that three bioactive components (inactivated Efm, extracellular vesicles, and EV-free supernatants) of E. faecium culture also contributed to the gastroprotective effect. Importantly, E. faecium-derived EVs showed an effective impact for the gastroprotective effect.

The Genetic Selection of HSPD1 and HSPE1 Reduce Inflammation of Liver and Spleen While Restraining the Growth and Development of Skeletal Muscle in Wuzhishan Pigs.

Wuzhishan (WZS) pigs, which are minipigs native to Hainan Province in China, are characterized by strong resistance to extreme hot temperatures and humidity. The relationship between their immune response and growth still needs to be clarified. In this study, we used whole genome sequencing (WGS) to detect variations within 37 WZS pigs, 32 Large White (LW) pigs, and 22 Xiangxi black (XXB) pigs, and ~2.49 GB of SNPs were obtained. These data were combined with those of two other pig breeds, and it was found that most of the genes detected (354) were located within the distinct genetic regions between WZS pigs and LW pigs. The network that was constructed using these genes represented a center including 12 hub genes, five of which had structural variations (SVs) within their regulatory regions. Furthermore, RNA-seq and RT-qPCR data for 12 genes were primarily consistent in liver, spleen, and LDM tissues. Notably, the expression of HSPs (HSPD1 and HSPE1) was higher while that of most genes involved in the JAK3-STAT pathway were lower in liver tissue of WZS pigs, compared with LW pigs. This likely not only reduced inflammation-related immune response but also impaired their growth. Our findings demonstrated the role of HSPs in the connection between inflammation and growth rate, while also providing the fundamental genetic selection of the adaptability of WZS pigs.

Diverse WGBS profiles of longissimus dorsi muscle in Hainan black goats and hybrid goats.

BACKGROUND: Goat products have played a crucial role in meeting the dietary demands of people since the Neolithic era, giving rise to a multitude of goat breeds globally with varying characteristics and meat qualities. The primary objective of this study is to pinpoint the pivotal genes and their functions responsible for regulating muscle fiber growth in the longissimus dorsi muscle (LDM) through DNA methylation modifications in Hainan black goats and hybrid goats. METHODS: Whole-genome bisulfite sequencing (WGBS) was employed to scrutinize the impact of methylation on LDM growth. This was accomplished by comparing methylation differences, gene expression, and their associations with growth-related traits. RESULTS: In this study, we identified a total of 3,269 genes from differentially methylated regions (DMR), and detected 189 differentially expressed genes (DEGs) through RNA-seq analysis. Hypo DMR genes were primarily enriched in KEGG terms associated with muscle development, such as MAPK and PI3K-Akt signaling pathways. We selected 11 hub genes from the network that intersected the gene sets within DMR and DEGs, and nine genes exhibited significant correlation with one or more of the three LDM growth traits, namely area, height, and weight of loin eye muscle. Particularly, PRKG1 demonstrated a negative correlation with all three traits. The top five most crucial genes played vital roles in muscle fiber growth: FOXO3 safeguarded the myofiber's immune environment, FOXO6 was involved in myotube development and differentiation, and PRKG1 facilitated vasodilatation to release more glucose. This, in turn, accelerated the transfer of glucose from blood vessels to myofibers, regulated by ADCY5 and AKT2, ultimately ensuring glycogen storage and energy provision in muscle fibers. CONCLUSION: This study delved into the diverse methylation modifications affecting critical genes, which collectively contribute to the maintenance of glycogen storage around myofibers, ultimately supporting muscle fiber growth.

Dietary Supplementation with Putrescine Improves Growth Performance and Meat Quality of Wenchang Chickens.

This study was to investigate the effects of dietary supplementation with putrescine on the growth performance and meat quality of chickens. A total of 480 eighty-day-old female Wenchang chickens were randomly assigned into four groups, with 8 replications per group and 15 animals per replicate. The chickens in the control group (Con) were fed a basal diet; the 3 experimental groups were fed a basal diet with 0.01%, 0.03%, and 0.05% putrescine, respectively. The experiment lasted for 40 days. The results showed that dietary supplementation with 0.05% putrescine increased (p < 0.05) the final body weight and average daily weight gain, and decreased the ratio of feed intake to the body weight gain of Wenchang chickens. Dietary supplementation with putrescine decreased the concentrations of putrescine, spermidine, and spermine in serum (p < 0.05). The contents of methionine, phenylalanine, lysine, aspartic acid, tyrosine, total essential amino acids, and total amino acids in breast muscle were higher (p < 0.05) in 0.03% and 0.05% groups than those in Con group. However, the contents of undecanoic acid, lauric acid, tridecanoic acid, myristic acid, pentadecanoic acid, arachidic acid, docosanoic acid, tricosanic acid, lignoceric acid, erucic acid, cis-11,14,17-eicosatrienoate, linoleic acid, and total n-6 monounsaturated fatty acids in breast muscle were lower (p < 0.05) in 0.03% and 0.05% groups than those in Con group. In addition, putrescine supplementation decreased (p < 0.05) the ratio of n-6/n-3 polyunsaturated fatty acids in breast meat. Overall, dietary supplementation with 0.05% putrescine enhanced the growth performance and meat quality of Wenchang chickens.

Novel Haplotype in the HHEX Gene Promoter Associated with Body Length in Pigs.

The screening of important candidate genes and the identification of genetic markers are important for molecular selection in the pig industry. The hematopoietically expressed homeobox (HHEX) gene plays an important role in embryonic development and organogenesis; however, the genetic variation and expression pattern of the porcine HHEX gene remains to be clarified. In this study, semiquantitative RT-PCR and immunohistochemistry results showed the specific expression of the HHEX gene in porcine cartilage tissues. A novel haplotype consisting of two SNPs rs80901185 (T > C) and rs80934526 (A > G) was detected in the promoter region of the HHEX gene. The expression of the HHEX gene was significantly higher in Yorkshire pigs (TA haplotype) than in Wuzhishan pigs (CG haplotype), and a population analysis showed that this haplotype was significantly associated with body length. An analysis subsequently revealed that the -586 to -1 bp region of the HHEX gene promoter showed the highest activity. Furthermore, we found that the activity of the TA haplotype was significantly higher than that of the CG haplotype by changing the potential binding of transcription factors YY1 and HDAC2. In summary, we conclude that the porcine HHEX gene may contribute to the breeding of pigs for body length traits.

Interactive effects of high temperature and crude protein levels on growth performance, nitrogen excretion, and fecal characteristics of broilers.

In the present study, we aimed to explore the interactive effects of high temperature (HT) and dietary crude protein (CP) levels on nitrogen (N) excretion, fecal characteristics, and growth performance of broilers. A total of 288 broilers (Arbor Acres) were divided into six groups with eight replicates (six broilers per replicate). Two temperatures (ambient temperature: AT, 23 °C; HT: 28 ~ 32 ~ 28 °C) and three diets (CP: 14.90%, 18.18%, or 21.19%, with equal amounts of essential amino acids) were examined in a 2 × 3 factorial design. The experiment arrangement was from 4 to 6 weeks of age. The results showed that HT led to a significant decrease in the N excretion (P < 0.0001), average daily feed intake (P < 0.0001), and weight gain of broilers (P < 0.0001), while it markedly increased the fecal pH (P = 0.015), fecal moisture (P = 0.0014), uric acid (UA) contents (P = 0.0018), and feed/gain ratio (P < 0.0001). A low CP diet significantly decreased the N excretion (P < 0.001), fecal pH (P = 0.016), fecal moisture (P < 0.0001), and UA contents (P < 0.0001), while it markedly increased the feed/gain ratio (P < 0.001). In conclusion, HT had a negative impact on the fecal characteristics and growth performance of broilers but showed positive effects on N excretion. Moreover, decreased CP levels had a positive effect on the N excretion and fecal characteristics in broilers.

Plant miR167e-5p promotes 3T3-L1 adipocyte adipogenesis by targeting ß-catenin.

Adipogenesis is important in the development of fat deposition. Evidence showed that plant microRNAs (miRNAs) could be absorbed by the digestive tract and exert regulatory effects on animals' physiological processes. However, the regulation of plant miRNA on host lipogenesis remains unknown. This study explored the potential function of plant miRNA, miR167e-5p, in adipogenesis in vitro. The presentation of plant miR167e-5p improved lipid accumulation in 3T3-L1 cells. Bioinformatics prediction and luciferase reporter assay indicated that miR167e-5p targeted ß-catenin. MiR167e-5p could not only negatively affect the expression of ß-catenin but also showed a positive effect on several fat synthesis-related genes, peroxisome proliferator-activated receptor gamma (Pparγ), CCAAT/enhancer-binding protein α (Cebpα), fatty acid-binding protein 4 (Ap2), lipolysis genes, adipose triglyceride lipase (Atgl), and hormone-sensitive lipase (Hsl) messenger RNA levels. Meanwhile, lipid accumulation and the expression of the ß-catenin and other five fat synthesis-related genes were recovered to their original pattern by adding the miR167e-5p inhibitor in 3T3-L1 cells. The immunoblot confirmed the same expression pattern in protein levels in ß-catenin, PPAR-γ, FAS, and HSL. This research demonstrates that plant miR167e-5p can potentially affect adipogenesis through the regulation of ß-catenin, suggesting that plant miRNAs could be a new class of bioactive ingredients in adipogenesis.

Effects of dietary supplementation with dandelion tannins or soybean isoflavones on growth performance, antioxidant function, intestinal morphology, and microbiota composition in Wenchang chickens.

Many benefits have been found in supplementing tannins or soybean isoflavones to poultry, including increased body weight gain, antioxidant activity, and better intestinal morphology. However, few studies tested the influence of dandelion tannins or soybean isoflavones supplementation on Wenchang chickens. This study investigates the effects of dietary supplementation with dandelion tannins or soybean isoflavones on the growth performance, antioxidant function, and intestinal health of female Wenchang chickens. A total of 300 chickens were randomly divided into five groups, with six replicates per group and 10 broilers per replicate. The chickens in the control group (Con) were fed a basal diet; the four experimental groups were fed a basal diet with different supplements: 300 mg/kg of dandelion tannin (DT1), 500 mg/kg of dandelion tannin (DT2), 300 mg/kg of soybean isoflavone (SI1), or 500 mg/kg of soybean isoflavone (SI2). The experiment lasted 40 days. The results showed that the final body weight (BW) and average daily gain (ADG) were higher in the DT2 and SI1 groups than in the Con group (P < 0.05). In addition, dietary supplementation with dandelion tannin or soybean isoflavone increased the level of serum albumin (P <0.05); the concentrations of serum aspartate aminotransferase and glucose were significantly higher in the SI1 group (P < 0.05) than in the Con group and the concentration of triglycerides in the DT1 group (P < 0.05). The serum catalase (CAT) level was higher in the DT1 and SI1 groups than in the Con group (P < 0.05). The ileum pH value was lower in the DT2 or SI1 group than in the Con group (P < 0.05). The jejunum villus height and mucosal muscularis thickness were increased in the DT2 and SI1 groups (P < 0.05), whereas the jejunum crypt depth was decreased in the DT1 or DT2 group compared to the Con group (P < 0.05). In addition, the messenger RNA (mRNA) expression level of zonula occludens 1 (ZO-1) in the duodenum of the SI1 group and those of occludin, ZO-1, and claudin-1 in the ileum of the DT2 and SI1 groups were upregulated (P < 0.05) compared to the Con group. Moreover, the DT2 and SI1 groups exhibited reduced intestinal microbiota diversity relative to the Con group, as evidenced by decreased Simpson and Shannon indexes. Compared to the Con group, the relative abundance of Proteobacteria was lower and that of Barnesiella was higher in the DT2 group (P < 0.05). Overall, dietary supplementation with 500 mg/kg of dandelion tannin or 300 mg/kg of soybean isoflavone improved the growth performance, serum biochemical indexes, antioxidant function, and intestinal morphology and modulated the cecal microbiota composition of Wenchang chickens.

MiR-125b-2 knockout increases high-fat diet-induced fat accumulation and insulin resistance.

Obese individuals are more susceptible to comorbidities than individuals of healthy weight, including cardiovascular disease and metabolic disorders. MicroRNAs are a class of small and noncoding RNAs that are implicated in the regulation of chronic human diseases. We previously reported that miR-125b plays a critical role in adipogenesis in vitro. However, the involvement of miR-125b-2 in fat metabolism in vivo remains unknown. In the present study, miR-125b-2 knockout mice were generated using CRISPR/CAS9 technology, resulting in mice with a 7 bp deletion in the seed sequence of miR-125b-2. MiR-125b-2 knockout increased the weight of liver tissue, epididymal white fat and inguinal white fat. MiR-125b-2 knockout also increased adipocyte volume in HFD-induced obese mice, while there were no significant differences in body weight and feed intake versus mice fed a normal diet. Additionally, qRT-PCR and western blot analysis revealed that the expression of the miR-125b-2 target gene SCD-1 and fat synthesis-associated genes, such as PPARγ and C/EBPα, were significantly up-regulated in miR-125b-2KO mice (P < 0.05). Moreover, miR-125b-2KO altered HFD-induced changes in glucose tolerance and insulin resistance. In conclusion, we show that miR-125b-2 is a novel potential target for regulating fat accumulation, and also a candidate target to develop novel treatment strategies for obesity and diabetes.

Oral Administration of Bovine and Porcine Milk Exosome Alter miRNAs Profiles in Piglet Serum.

Breast milk is the most important nutrient source for newborn mammals. Studies have reported that milk contains microRNAs (miRNAs), which are potential regulatory components. Currently, existing functional and nutritional two competing hypotheses in milk field though little date have been provided for nutritional hypothesis. In this study, we used the qRT-PCR method to evaluated whether milk miRNAs can be absorbed by newborn piglets by feeding them porcine or bovine milk. The result showed that miRNA levels (miR-2284×, 2291, 7134, 1343, 500, 223) were significantly different between bovine and porcine milk. Four miRNAs (miR-2284×, 2291, 7134, 1343) were significantly different in piglet serum after feeding porcine or bovine milk. After separated milk exosomes by ultracentrifugation, the results showed the selected milk miRNAs (miR-2284×, 2291, 7134, 1343) were present in both exosomes and supernatants, and the miRNAs showed the coincidental expression in IPEC-J2 cells. All our founding suggested that the milk miRNAs can be absorbed both in vivo and in vitro, which will building the foundation for understanding whether these sort of miRNAs exert physiological functions after being absorbed and provided additional evidence for the nutritional hypotheses.

Genotype identification and phylogenetic analysis of Enterocytozoon bieneusi in farmed black goats (Capra hircus) from China's Hainan Province.

Enterocytozoon bieneusi is an important pathogen commonly found in humans and animals. Farmed animals with close contact to humans are important hosts of E. bieneusi. The role of goats in the transmission of E. bieneusi, however, remains unclear. In this study, 341 fresh fecal samples of black goats were collected from five locations in Hainan Province, China. Enterocytozoon bieneusi was identified and genotyped by sequences of the internal transcribed spacer (ITS) region. Phylogenetic analysis was performed by constructing a neighbor-joining tree of the ITS gene sequences. The average prevalence of E. bieneusi in black goats was 24.0% (82/341) with rates ranging from 6.3% (4/63) to 37.2% (32/86) across the locations (χ2 = 17.252, p < 0.01). Eight genotypes of E. bieneusi were identified, including six known genotypes: CHG5 (n = 47); CHG3 (n = 23); CHG2 (n = 4); CM21 (n = 3); D (n = 2); and AHG1 (n = 1), and two novel genotypes termed HNG-I (n = 1) and HNG-II (n = 1). In the phylogenetic tree, genotype D was clustered into Group 1 and the other identified genotypes were included in Group 2. This represents the first report identifying E. bieneusi in black goats from Hainan Province, with a high prevalence and wide occurrence demonstrated. The two new genotypes identified provide additional insights into the genotypic variations in E. bieneusi. Due to the small percentage of zoonotic genotypes in these animals, there is minimal risk of zoonotic transmission of E. bieneusi.

TITLE: Identification du génotype et analyse phylogénétique d'Enterocytozoon bieneusi chez des chèvres noires (Capra hircus) de la province de Hainan, en Chine. ABSTRACT: Enterocytozoon bieneusi est un agent pathogène important que l'on trouve couramment chez l'homme et les animaux. Les animaux d'élevage, en contact étroit avec l'homme, sont des hôtes importants d'E. bieneusi. Le rôle des chèvres dans la transmission d'E. bieneusi reste toutefois incertain. Dans cette étude, 341 échantillons de fèces fraîches de chèvres noires ont été prélevés dans cinq sites de la province de Hainan, en Chine. Enterocytozoon bieneusi a été identifié et génotypé par des séquences de la région de l'espaceur interne transcrit (ITS). L'analyse phylogénétique a été réalisée en construisant un arbre de jonction voisine des séquences du gène ITS. La prévalence moyenne d'E. bieneusi chez les chèvres noires était de 24,0 % (82/341), avec des taux allant de 6,3 % (4/63) à 37,2 % (32/86) dans tous les sites (χ2 = 17,252, p < 0,01). Huit génotypes d'E. bieneusi ont été identifiés, dont six génotypes connus: CHG5 (n = 47) ; CHG3 (n = 23) ; CHG2 (n = 4) ; CM21 (n = 3) ; D (n = 2) ; AHG1 (n = 1) et deux nouveaux génotypes appelés HNG-I (n = 1) et HNG-II (n = 1). Dans l'arbre phylogénétique, le génotype D appartenait au groupe 1 et les autres génotypes identifiés étaient inclus dans le groupe 2. Il s'agit du premier rapport identifiant E. bieneusi chez des chèvres noires de la province de Hainan, avec une prévalence élevée et une occurrence étendue. Les deux nouveaux génotypes identifiés fournissent des informations supplémentaires sur les variations génotypiques chez E. bieneusi. En raison du faible pourcentage de génotypes zoonotiques chez ces animaux, le risque de transmission zoonotique d'E. bieneusi est minime.

Revelation of mRNAs and proteins in porcine milk exosomes by transcriptomic and proteomic analysis.

BACKGROUND: Milk is a complex liquid that provides nutrition to newborns. Recent reports demonstrated that milk is enriched in maternal-derived exosomes that are involved in fetal physiological and pathological conditions by transmission of exosomal mRNAs, miRNAs and proteins. Until now, there is no such research relevant to exosomal mRNAs and proteins in porcine milk, therefore, we have attempted to investigate porcine milk exosomal mRNAs and proteins using RNA-sequencing and proteomic analysis. RESULTS: A total of 16,304 (13,895 known and 2,409 novel mRNAs) mRNAs and 639 (571 known, 66 candidate and 2 putative proteins) proteins were identified. GO and KEGG annotation indicated that most proteins were located in the cytoplasm and participated in many immunity and disease-related pathways, and some mRNAs were closely related to metabolisms, degradation and signaling pathways. Interestingly, 19 categories of proteins were tissue-specific and detected in placenta, liver, milk, plasma and mammary. COG analysis divided the identified mRNAs and proteins into 6 and 23 categories, respectively, 18 mRNAs and 10 proteins appeared to be involved in cell cycle control, cell division and chromosome partitioning. Additionally, 14 selected mRNAs were identified by qPCR, meanwhile, 10 proteins related to immunity and cell proliferation were detected by Western blot. CONCLUSIONS: These results provide the first insight into porcine milk exosomal mRNA and proteins, and will facilitate further research into the physiological significance of milk exosomes for infants.

In low protein diets, microRNA-19b regulates urea synthesis by targeting SIRT5.

Ammonia detoxification, which takes place via the hepatic urea cycle, is essential for nitrogen homeostasis and physiological well-being. It has been reported that a reduction in dietary protein reduces urea nitrogen. MicroRNAs (miRNAs) are major regulatory non-coding RNAs that have significant effects on several metabolic pathways; however, little is known on whether miRNAs regulate hepatic urea synthesis. The objective of this study was to assess the miRNA expression profile in a low protein diet and identify miRNAs involved in the regulation of the hepatic urea cycle using a porcine model. Weaned 28-days old piglets were fed a corn-soybean normal protein diet (NP) or a corn-soybean low protein diet (LP) for 30 d. Hepatic and blood samples were collected, and the miRNA expression profile was assessed by sequencing and qRT-PCR. Furthermore, we evaluated the possible role of miR-19b in urea synthesis regulation. There were 25 differentially expressed miRNAs between the NP and LP groups. Six of these miRNAs were predicted to be involved in urea cycle metabolism. MiR-19b negatively regulated urea synthesis by targeting SIRT5, which is a positive regulator of CPS1, the rate limiting enzyme in the urea cycle. Our study presented a novel explanation of ureagenesis regulation by miRNAs.

Porcine milk-derived exosomes promote proliferation of intestinal epithelial cells.

Milk-derived exosomes were identified as a novel mechanism of mother-to-child transmission of regulatory molecules, but their functions in intestinal tissues of neonates are not well-studied. Here, we characterized potential roles of porcine milk-derived exosomes in the intestinal tract. In vitro, treatment with milk-derived exosomes (27 ± 3 ng and 55 ± 5 ng total RNA) significantly promoted IPEC-J2 cell proliferation by MTT, CCK8, EdU fluorescence and EdU flow cytometry assays. The qRT-PCR and Western blot analyses indicated milk-derived exosomes (0.27 ± 0.03 µg total RNA) significantly promoted expression of CDX2, IGF-1R and PCNA, and inhibited p53 gene expression involved in intestinal proliferation. Additionally, six detected miRNAs were significantly increased in IPEC-J2 cell, while FAS and SERPINE were significantly down-regulated relative to that in control. In vivo, treated groups (0.125 µg and 0.25 µg total RNA) significantly raised mice' villus height, crypt depth and ratio of villus length to crypt depth of intestinal tissues, significantly increased CDX2, PCNA and IGF-1R' expression and significantly inhibited p53' expression. Our study demonstrated that milk-derived exosomes can facilitate intestinal cell proliferation and intestinal tract development, thus giving a new insight for milk nutrition and newborn development and health.

Characterization of the Methylation Status of Pax7 and Myogenic Regulator Factors in Cell Myogenic Differentiation.

Epigenetic processes in the development of skeletal muscle have been appreciated for over a decade. DNA methylation is a major epigenetic modification important for regulating gene expression and suppressing spurious transcription. Up to now, the importance of epigenetic marks in the regulation of Pax7 and myogenic regulatory factors (MRFs) expression is far less explored. In the present study, semi-quantitative the real-time polymerase chain reaction (RT-PCR) analyses showed MyoD and Myf5 were expressed in activated and quiescent C2C12 cells. MyoG was expressed in a later stage of myogenesis. Pax7 was weakly expressed in differentiated C2C12 cells. To further understand the regulation of expression of these genes, the DNA methylation status of Pax7, MyoD, and Myf5 was determined by bisulfite sequencing PCR. During the C2C12 myoblasts fusion process, the changes of promoter and exon 1 methylation of Pax7, MyoD, and Myf5 genes were observed. In addition, an inverse relationship of low methylation and high expression was found. These results suggest that DNA methylation may be an important mechanism regulating Pax7 and MRFs transcription in cell myogenic differentiation.

Two new species of Eunice Cuvier, 1817 (Polychaeta, Eunicidae) from the coral reefs of Hainan Island with a key to 16 species of Eunice from China seas.

A taxonomic study of Eunice species based on material deposited in the Marine Biological Museum of the Chinese Academy of Sciences (MBMCAS) including recently collected specimens from coastal regions of Hainan Island, yielded two new species: Eunice hainanensis n. sp. and E. carrerai n. sp. Both species were collected from dead coral rocks in the reefs of the coastal region of Hainan Island, northern South China Sea. Eunice hainanensis has translucent bidentate subacicular hooks and branchiae present over an extensive region of the body. Within the Eunice group possessing these characters, the new species highly resembles E. schizobranchia Claparède, 1870 in having a numerous chaetigers and a very late start of branchiae (348-570 chaetigers with branchiae from chaetigers 69-72 vs. 731 chaetigers with branchiae from chaetiger 67). However, the two species differ by the presence of the maxillary plate VI (MxVI) in the new species (vs. absent in the latter). Besides, E. hainanensis is much smaller (1.7-1.9 mm vs. 5 mm in maximal width). Eunice carrerai belongs to the Eunice group that has dark bidentate subacicular hooks and branchiae present over an extensive region of the body. It can be distinguished from similar congeners that have the branchiae starting from chaetigers 3-4 and prostomial appendages with moniliform articulations by a combination of characters such as the presence of MxVI, notopodial articulations limited to anterior chaetigers, peristomial cirri articulated and extending to anterior edge of first peristomial ring. A key to 16 species of Eunice identified from China seas in the material examined with notes on their distribution is provided. The major characters of these species are briefly summarized.

Distribution and linkage disequilibrium analysis of polymorphisms of MC4R, LEP, H-FABP genes in the different populations of pigs, associated with economic traits in DIV2 line.

PCR-RFLP was used to analyze the polymorphisms of MC4R, LEP, H-FABP genes in a swine breed composite (DIV2) and 4 swine breeds (Yorkshire, Landrace, Meishan, Bamei). The association study of these polymorphisms with several economic traits was carried out on a DIV2 population. The results obtained showed that MC4R/TaqI genotype had an effect for average backfat thickness (P < 0.05) and lean meat percentage (P < 0.05). At locus LEP/HinfI animals of AA genotype had lower test daily gain than that of BB (P < 0.01) or AB genotype (P < 0.05). At the H-FABP/HaeIII locus lean meat percentage of the individuals with genotype DD were higher than that with genotype dd (P < 0.05). Linkage disequilibrium analysis among MC4R, LEP and H-FABP revealed that these genes were independent. This represented two or more genes that could be combined together within one genotype in order to facilitate breeding for objective traits. In addition, a method allowing simultaneous detection of fragments of MC4R and LEP gene was developed.