Dietary 5-hydroxytryptophan improves sheep growth performance by enhancing ruminal functions, antioxidant capacity, and tryptophan metabolism: in vitro and in vivo studies.

Background: Hydroxytryptophan (5-HTP) can regulate the synthesis of 5-Hydroxytryptamine (5-HT) and melatonin (MT). In a previous metabolome analysis, we found that 5-HTP is an effective ingredient in yeast culture for regulating rumen fermentation. However, research on the effect of this microbial product (5-HTP) as a functional feed additive in sheep production is still not well explained. Therefore, this study examined the effects of 5-HTP on sheep rumen function and growth performance using in vitro and in vivo models. Methods: A two-factor in vitro experiment involving different 5-HTP doses and fermentation times was conducted. Then, in the in vivo experiment, 10 sheep were divided into a control group which was fed a basal diet, and a 5-HTP group supplemented with 8 mg/kg 5-HTP for 60 days. Results: The results showed that 5-HTP supplementation had a significant effect on in vitro DMD, pH, NH3-N, acetic acid, propionic acid, and TVFA concentrations. 5-HTP altered rumen bacteria composition and diversity indices including Chao1, Shannon, and Simpson. Moreover, the in vivo study on sheep confirmed that supplementing with 8 mg/kg of 5-HTP improved rumen fermentation efficiency and microbial composition. This led to enhanced sheep growth performance and increased involvement in the tryptophan metabolic pathway, suggesting potential benefits. Conclusion: Dietary 5-HTP (8 mg/kg DM) improves sheep growth performance by enhancing ruminal functions, antioxidant capacity, and tryptophan metabolism. This study can provide a foundation for the development of 5-HTP as a functional feed additive in ruminants' production.

Metabolomic analysis of rumen-protected branched-chain amino acids in primiparous dairy cows.

Introduction: Peripartal cows are susceptible to a negative energy balance due to inadequate nutrient intake and high energy requirements for lactation. Improving the energy metabolism of perinatal dairy cows is crucial in increasing production in dairy cows. Methods: In this study, we investigated the impact of rumen-protected branched-chain amino acid (RPBCAA) on the production performance, energy and lipid metabolism, oxidative stress, and immune function of primiparous dairy cows using metabolomics through a single-factor experiment. Twenty healthy primiparous Holstein cows were selected based on body condition scores and expected calving date, and were randomly divided into RPBCAA (n = 10) and control (n = 10) groups. The control group received a basal diet from calving until 21 d in milk, and the RPBCAA group received the basal diet and 44.6 g/d RPLeu, 25.14 g/d RPIle, and 25.43 g/d RPVal. Results: In comparison to the control group, the supplementation of RPBCAA had no significant effect on milk yield and milk composition of the dairy cows. Supplementation with RPBCAA significantly increased the concentrations of insulin, insulin growth factor 1, glucagon, and growth hormones, which are indicators of energy metabolism in postpartum cows. The very low density lipoprotein, fatty acid synthase, acetyl coenzyme A carboxylase, and hormone-sensitive lipase contents of the RPBCAA group were significantly greater than that of the control group; these metrics are related to lipid metabolism. In addition, RPBCAA supplementation significantly increased serum glutathione peroxidase and immunoglobulin G concentrations and decreased malondialdehyde concentrations. Liquid chromatography-mass spectrometry (LC-MS) analysis revealed 414 serum and 430 milk metabolic features. Supplementation with RPBCAA primarily increased concentrations of amino acid and lipid metabolism pathways and upregulated the abundance of serotonin, glutamine, and phosphatidylcholines. Discussion: In summary, adding RPBCAA to the daily ration can influence endocrine function and improve energy metabolism, regulate amino acid and lipid metabolism, mitigate oxidative stress and maintain immune function on primiparous cows in early lactation.

Mechanistic insights into inositol-mediated rumen function promotion and metabolic alteration using in vitro and in vivo models.

Inositol is a bioactive factor that is widely found in nature; however, there are few studies on its use in ruminant nutrition. This study investigated the effects of different inositol doses and fermentation times on rumen fermentation and microbial diversity, as well as the levels of rumen and blood metabolites in sheep. Rumen fermentation parameters, microbial diversity, and metabolites after different inositol doses were determined in vitro. According to the in vitro results, six small-tailed Han sheep fitted with permanent rumen fistulas were used in a 3 × 3 Latin square feeding experiment where inositol was injected into the rumen twice a day and rumen fluid and blood samples were collected. The in vitro results showed that inositol could increase in vitro dry matter digestibility, in vitro crude protein digestibility, NH3-N, acetic acid, propionic acid, and rumen microbial diversity and affect rumen metabolic pathways (p < 0.05). The feeding experiment results showed that inositol increased the blood concentration of high-density lipoprotein and IgG, IgM, and IL-4 levels. The rumen microbial composition was significantly affected (p < 0.05). Differential metabolites in the rumen were mainly involved in ABC transporters, biotin metabolism, and phenylalanine metabolism, whereas those in the blood were mainly involved in arginine biosynthesis and glutathione and tyrosine metabolism. In conclusion, inositol improves rumen function, affects rumen microorganisms and rumen and blood metabolites and may reduce inflammation, improving animal health.

Unlocking the potential of N-acetylcysteine: Improving hepatopancreas inflammation, antioxidant capacity and health in common carp (Cyprinus carpio) via the MAPK/NF-κB/Nrf2 signalling pathway.

N-acetylcysteine (NAC) positively contributes to enhancing animal health, regulating inflammation and reducing stress by participating in the synthesis of cysteine, glutathione, and taurine in the body. The present study aims to investigate the effects of dietary different levels of NAC on the morphology, function and physiological state of hepatopancreas in juvenile common carp (Cyprinus carpio). 450 common carps were randomly divided into 5 groups: N1 (basal diet), N2 (1.5 g/kg NAC diet), N3 (3.0 g/kg NAC diet), N4 (4.5 g/kg NAC diet) and N5 (6.0 g/kg NAC diet), and fed for 8 weeks. The results indicated that dietary 3.0-6.0 g/kg NAC reduced hepatopancreas lipid vacuoles and nuclear translocation, and inhibited apoptosis in common carp. Simultaneously, the activities of hepatopancreas alanine aminotransferase and aspartate aminotransferase progressively increased with rising dietary NAC levels. Dietary NAC enhanced the non-specific immune function of common carp, and exerted anti-inflammatory effects by inhibiting the MAPK/NF-κB signaling pathway. Additionally, dietary 3.0-6.0 g/kg NAC significantly improved the antioxidant capacity of common carp, which was associated with enhanced glutathione metabolism, clearance of ROS and the activation of Nrf2 signaling pathway. In summary, NAC has the potential to alleviate inflammation, mitigate oxidative stress and inhibit apoptosis via the MAPK/NF-κB/Nrf2 signaling pathway, thereby improving hepatopancreas function and health of common carp. The current findings provide a theoretical basis for promoting the application of NAC in aquaculture and ecological cultivation of aquatic animals.

Impact of deoxynivalenol on rumen function, production, and health of dairy cows: Insights from metabolomics and microbiota analysis.

Deoxynivalenol contamination in feed and food, pervasive from growth, storage, and processing, poses a significant risk to dairy cows, particularly when exposed to a high-starch diet; however, whether a high-starch diet exacerbates these negative effects remains unclear. Therefore, we investigated the combined impact of deoxynivalenol and dietary starch on the production performance, rumen function, and health of dairy cows using metabolomics and 16 S rRNA sequencing. Our findings suggested that both high- and low-starch diets contaminated with deoxynivalenol significantly reduced the concentration of propionate, isobutyrate, valerate, total volatile fatty acids (TVFA), and microbial crude protein (MCP) concentrations, accompanied by a noteworthy increase in NH3-N concentration in vitro and in vivo (P < 0.05). Deoxynivalenol altered the abundance of microbial communities in vivo, notably affecting Oscillospiraceae, Lachnospiraceae, Desulfovibrionaceae, and Selenomonadaceae. Additionally, it significantly downregulated lecithin, arachidonic acid, valine, leucine, isoleucine, arginine, and proline metabolism (P < 0.05). Furthermore, deoxynivalenol triggered oxidative stress, inflammation, and dysregulation in immune system linkage, ultimately compromising the overall health of dairy cows. Collectively, both high- and low-starch diets contaminated with deoxynivalenol could have detrimental effects on rumen function, posing a potential threat to production performance and the overall health of cows. Notably, the negative effects of deoxynivalenol are more pronounced with a high-starch diet than a low-starch diet.

Effects of antimicrobial peptide and tributyrin on fecal microflora and blood indices of female calves.

This study evaluated the effects of antimicrobial peptide (AMP) and tributyrin (TB) on dairy calves in terms of growth performance, immunity, oxidative stress, and intestinal microflora. A total of 40 female calves were divided into four treatment groups (n = 10): basal diet +0.015% essential oil, basal diet +0.03% AMP, basal diet +0.15% TB, and basal diet +0.03% AMP + 0.15% TB. AMP and TB supplementation increased the average daily gain (ADG) and weaning weight, while reducing diarrhea occurrence. Additionally, AMP and TB supplementation reduced the levels of reactive oxygen species (ROS) and malonaldehyde (MDA), while increasing superoxide dismutase (SOD) levels and serum immunoglobulin M (IgM) levels. However, the combined use of AMP and TB did not significantly affect the average daily feed intake, ADG, weaning weight, or diarrhea incidence but decreased ROS levels, while increasing SOD levels as well as MDA and IgM levels. Moreover, AMP and TG supplementation increased the relative abundance of several beneficial fiber- and mucin-degrading bacteria in the gut, in contrast to combined AMP and TB supplementation. The 16S rRNA results showed that AMP supplementation significantly increased the relative abundance of Rikenellaceae_RC9_gut_group, Ruminococcaceae_UCG-014 and [Eubacterium]_coprostanoligenes group (p < .01), and significantly decreased the relative abundance of Ruminococcaceae_UCG-005 and Christensenellaceae_R-7_group (p < .01). The TB supplementation significantly increased the abundances of Rikenellaceae_RC9_gut_group and Ruminococcaceae_UCG-005 (p < .01), and significantly decreased the relative abundances of Ruminococcaceae_UCG-014, [Eubacterium]_coprostanoligenes group and Christensenellaceae_R-7_group (p < .01). The combined use of AMP and TB significantly increased the relative abundance of Rikenellaceae_RC9_gut_group and Bacteroides (p < .01), and significantly decreased the relative abundance of Ruminococcaceae_UCG-014, [Eubacterium]_coprostanoligenes group and Christensenellaceae_R-7_group (p < .01). In summary, diets supplemented with either AMP or TB improved the intestinal microflora, growth performance, and health of weaned calves, but combined use was detrimental to calf performance.

Gastrointestinal digestion and absorption of soybean ß-conglycinin in an early weaned piglet model: An initial step to the induction of soybean allergy.

To date, it still remains unknown how ß-conglycinin, a major soybean allergen, crosses intestinal epithelial barrier to reach immune cells. The purpose of this study was to elucidate the pathway and molecular mechanism of ß-conglycinin absorption and transport across intestinal mucosal epithelium using a ß-conglycinin allergic piglet model. Ten-day old piglets were orally sensitized with diets containing 2% and 4% ß-conglycinin. The digestion, absorption and transport of ß-conglycinin in gastrointestinal tract was investigated. The results showed that ß-conglycinin had a certain resistance to gastrointestinal digestion, and the digestion-resistant subunits and fragments were absorbed into the intestinal mucosa and then induced an anaphylaxis in early weaned piglets. The absorption occurred in the form of IgE-allergen immune complex through transcellular pathway with CD23 as the receptor. These results provided important clues for using the pathway and molecule as inhibitor target to prevent and alleviate soybean ß-conglycinin allergy in infants.

Effects of ultra-high-temperature processes on metabolite changes in milk.

Processing can affect milk properties and alter the composition of milk metabolites, which has corresponding effects on milk flavor and quality. It is quite important to study the safe quality control of milk processing. Therefore, the purpose of this study was to identify metabolites at different steps of ultra-high-temperature-sterilized (UHT) milk processing using gas chromatography-mass spectrometry (GC-MS). These steps included raw milk, pasteurized milk (80°C for 15 s), semi-finished milk (after pasteurizing, it was homogenized at 75°C with pressure of 250 bar), UHT milk (at 140°C for 10 s), and finished milk (homogenized UHT milk). A total of 66 metabolites were identified across all samples, including 30 metabolites in the chloroform layers of the milk samples and 41 metabolites in the water layers; 5 metabolites were found in both layers. The metabolites were primarily fatty acids, amino acids, sugars, and organic acids. For example, pasteurized and ultra-high-temperature-sterilized kinds of milk had lactose contents similar to those of raw milk, with increases in saturated fatty acids such as hexadecanoic acid and octadecanoic acid. Additionally, these findings indicated that these methods of processing can affect the contents of some components of milk. Therefore, from the perspective of milk's nutritional value and consumer health, the excessive heating of dairy products should be avoided and the milk heat treatment process should be standardized from the source.

LC/MS analysis of mushrooms provided new insights into dietary management of diabetes mellitus in rats.

Mushrooms possess antihyperglycemic effect on diabetic individuals due to their nonfibrous and fibrous bioactive compounds. This study aimed to reveal the effect of different types of mushrooms on plasma glucose level and gut microbiota composition in diabetic individuals. The effects of five different mushroom species (Ganoderma lucidum, GLM; Pleurotus ostreatus, POM; Pleurotus citrinopileatus, PCM; Lentinus edodes, LEM; or Hypsizigus marmoreus, HMM) on alloxan-induced diabetic rats were investigated in this study. The results indicated that LEM and HMM treatments showed lower plasma glucose levels. For the microbiota composition, ACE, Chao1, Shannon, and Simpson were significantly affected by PCM and LEM treatments (p < .05), while ACE, Shannon, and Simpson indexes were affected by HMM treatment (p < .01). Simpson index was affected in positive control (C+) and POM groups. All these four indices were lower in GLM treatment (p < .05). Dietary supplementation of mushrooms reduced plasma glucose level directly through mushrooms' bioactive compounds (agmatine, sphingosine, pyridoxine, linolenic, and alanine) and indirectly through stachyose (oligosaccharide) and gut microbiota modulation. In conclusion, LEM and HMM can be used as food additives to improve plasma glucose level and gut microbiome composition in diabetic individuals.

The apoptotic effects of soybean agglutinin were induced through three different signal pathways by down-regulating cytoskeleton proteins in IPEC-J2 cells.

Soybean agglutinin (SBA) is a main anti-nutritional factor in soybean. SBA exhibits its anti-nutritional functions by binding to intestinal epithelial cells. Keratin8 (KRT8), Keratin18 (KRT18) and Actin (ACTA) are the representative SBA-specific binding proteins. Such cytoskeletal proteins act a crucial role in different cell activities. However, limited reports reveal what the signal transduction pathway of apoptosis caused by SBA when binding to KRT8, KRT18 and ACTA. We aimed to evaluate the effects of SBA on cell apoptosis and the expression of the cytoskeletal protein (KRT8, KRT18 and ACTA), reveal the roles of these cytoskeletal proteins or their combinations on SBA-induced cell apoptosis in IPEC-J2 cell line, evaluate the influences of SBA on the mitochondria, endoplasmic reticulum stress and death receptor-mediated apoptosis signal pathway and to show the roles of KRT8, KRT18 and ACTA in different apoptosis signal pathways induced by SBA. The results showed that SBA induced the IPEC-J2 cell apoptosis and decreased the mRNA expression of KRT8, KRT18 and ACTA (p < 0.05). The degree of effect of three cytoskeleton proteins on cell apoptosis was ACTA > KRT8 > KRT18. The roles of these three cytoskeletal proteins on IPEC-J2 apoptotic rates had a certain accumulation effect. SBA up-regulated mitochondrial fission variant protein (FIS1) and fusion protein (Mfn2) promoted CytC and AIF in mitochondria to enter the cytoplasm, activated caspase-9 and caspase-3, damaged or declined mitochondrial function and reduced ATP synthesis (p < 0.05). Also, SBA up-regulated the expression of GRP78, XBP-1, eIF2α, p-eIF2α and CHOP (p < 0.05), down-regulated the expression level of ASK1 protein (p < 0.05). SBA led to the recruitment of FADD to the cytoplasmic membrane and increased the expression of FasL, resulting in caspase-8 processing. SBA up-regulated the expression level of Bax protein and decreased cytosolic Bcl-2 and Bid (p < 0.05). In addition, there was a significant negative correlation between the gene expression of cytoskeleton proteins and apoptosis, as well as the expression of key proteins of apoptosis-related signal transduction pathways. In conclusion, SBA induced the activation of the mitochondria, endoplasmic reticulum stress and the death receptor-mediated apoptosis signal pathway and the crosstalk between them. The effect of SBA on these three pathways was mainly exhibited via down-regulation of the mRNA expression of the three cytoskeletal expressions. This study elucidates the molecular mechanism and signaling pathway of SBA that lead to apoptosis from the perspective of cell biology and molecular biology and provides a new perspective on the toxicity mechanism of other food-derived anti-nutrients, medical gastrointestinal health and related cancer treatment.

Dietary protein to starch metabolizable energy ratios alter growth performance and gastrointestinal microbiota of calves.

The diet structure is very important for the growth and development of calves. This study aimed to investigate the effects of dietary protein-to-starch metabolizable energy ratios (DPSRs) on growth performance, blood index, and gastrointestinal microbiota of calves. Forty-eight Holstein bull calves were fed six dietary DPSRs including A20-35 (20% CP and 35% starch), B20-30, C20-25, D22-35, E22-30, and F22-25 at d 4 to d 60, and then changed to another six dietary DPSRs at d 61 to d 180 (A18-30, B18-27, C18-24, D20-30, E20-27, and F20-24). Twelve calves (d 60) from groups A20-35, C20-25, D22-35, and F22-25 (n = 3) and another twelve calves (d 180) from groups A18-30, C18-24, D20-30, and F20-24 (n = 3) were euthanized. The growth performance parameters were measured. Blood, ruminal fluid, and cecum digesta were collected for further analysis. Results showed heart girth gain of B18-27 was significantly higher than A18-30, C18-24, and heart girth gain (d 180) was significantly affected by protein × starch (DPSRs; p < 0.05). Blood urea nitrogen (BUN; d 60) in C20-25 was significantly higher than A20-35 and B20-30 (p < 0.05). The BUN (d 180) in D20-30 was significantly higher than A18-30 (p < 0.05). The BUN was significantly affected by protein × starch (p < 0.05) on d 60. The albumin (ALB) levels in C20-25 and C18-24 were significantly higher than that in A20-35 on d 60 and A18-30 on d 180, respectively (p < 0.05). The ALB level in D22-35 on d 60 and E20-27 on d 180 was significantly higher than that in other groups (p < 0.05). The ALB level was significantly affected by protein and starch, respectively, on d 60 (p < 0.05). In the rumen, the genera Roseburia (C20-25) and Dialister (D22-35), Prevotellaceae UCG-001 (C18-24), Erysipelotrichaceae UCG-002, and Anaerovorax (F20-24) were found in significant higher relative abundances than those in other groups (p < 0.05). In the cecum, the genera Bacteroides and Eisenbergiella (F22-25), Ruminiclostridium_1 and Candidatus Stoquefichus (A18-30), Erysipelotrichaceae UCG-004 and Tyzzerella 4 (D20-30), and Prevotellaceae UCG-003 and Klebsiella (F20-24) were found in significant higher abundances than those in other groups (p < 0.05). Collectively, these results indicated that the heart girth, BUN, ALB, and gastrointestinal microbiota responded distinctly to differing DPSRs.

Fructo-oligosaccharides Ameliorate Intestinal Mechanical Barrier Injury in Piglets Induced by Soybean Antigen in vitro and in vivo.

BACKGROUND: Fructose oligosaccharides (FOS) have been shown to reduce soybean antigeninduced hypersensitivity in piglets, but their effects on intestinal epithelial barrier function have not been characterized. Therefore, this study aimed to determine the effects of FOS on intestinal barrier injury induced by soybean antigen in piglets in vitro and in vivo. METHODS: We studied the protective effects of FOS against mechanical barrier dysfunction induced using ß-conglycinin or glycinin in porcine intestinal epithelial cells (IPEC-J2), and measured the serum concentrations of diamine oxidase (DAO), D-lactic acid, and endotoxin, and the expression of tight junction (TJ) proteins, in piglets. RESULTS: We found that FOS concentration dependently increases cell activity, trans-epithelial electrical resistance, and TJ protein expression (P < 0.05) and reduces alkaline phosphatase (AP) activity (P < 0.05) in vitro. In addition, the serum DAO, D-lactic acid, and endotoxin concentrations were reduced by FOS administration in piglets (P < 0.05). Both in vitro and in vivo, the expression levels of TJ proteins (zona occludens 1 and occludin) were increased significantly by FOS (P < 0.05). CONCLUSION: Therefore, FOS protect against intestinal injury induced by soybean antigen in piglets, which may provide a basis for the prevention of allergy.

Effects of diets with different amino acid release characteristics on the gut microbiota and barrier function of weaned pigs.

BACKGROUND: The absorption and utilization of proteins by animals is affected by the amino acid (AA) release characteristics of their diets. In the present study, we aimed to determine the effects of diets with various amino acid release characteristics on the intestinal barrier function and diversity of gut microbiota of weaned pigs. RESULTS: Forty-eight pigs (7.45 ± 0.58 kg) were fed with diets having different amino acid release characteristics during a period of 28 days. We used a 2 × 3 full-factor (two protein levels and three protein sources with differing amino acid release characteristics) experimental design, with normal (standard terminal ileal digestibility of 17.5%) or low (standard terminal ileal digestibility of 14.9%) protein levels as the first factor. Casein (CAS), corn gluten meal (CGM) and a MIX diet were used as protein sources. Due to the more balanced release of amino acids, the diamine oxidase (DAO) concentrations in the CAS and MIX groups were significantly lower than those in the CGM group (P < 0.05); Reducing the dietary protein content from 17.5% to 14.9% had no significant effects on the levels of serum DAO or D-lactic acid. By contrast, it increased the microbial diversity (chao1 and ACE values) and the number of Lactobacillus in the jejunum (P < 0.05). The CAS-containing diet and the MIX diet resulted in significantly higher microbial diversity (Simpson and Shannon) than the CGM-containing diet in the jejunum. CONCLUSION: The balanced release of amino acids in CAS and MIX diets maintained intestinal barrier function and increased gut microbiota diversity. These findings could potentially provide a scientific reference for the rational preparation of piglet feed.

The physiological dissimilarities of Holstein dairy cows with different milk yields.

BACKGROUND: Even if breed, parity, dietary and environmental management are same, dairy cows still have notable differences in milk yield that may be underpinned by physiologic differences. OBJECTIVES: This study aimed to investigate the physiological dissimilarities of dairy cows with different milk yields. METHODS: Thirty cows were sorted into high milk-yielding cows (group H: 58.93±2.31 kg/day), moderate milk-yielding cows (group M: 44.99±0.54 kg/day), and low milk-yielding cows (group L: 24.99±6.83 kg/day) according to milk yield. Blood was collected and serum parameters were assessed. Rumen fluid was collected for the evaluation of rumen fermentation parameters (RFPs) and bacterial community composition (BCC). RESULTS: Serum prolactin, growth hormone, glutathione peroxidase, immunoglobulin A and non-esterified fatty acid had a significantly positive correlation with milk yield (p < 0.05), whereas serum glucagon and total antioxidant capacity had a significantly negative correlation with milk yield (p < 0.05). The concentration of valeric acid and the ratio of acetic acid to propionic acid in the rumen fluid in group H was significantly lower than that in group L (p < 0.05). The concentration of acetic acid and butyric acid in group H was significantly lower than that in groups M and L (p < 0.05). The relative abundances of Ruminococcaceae_NK4A214_group, Prevotella_1, Rikenellaceae_RC9_gut_group, Christensenellaceae_R-7_group, Muribaculaceae, and Ruminococcus_2 were negatively correlated with milk yield, whereas the relative abundance of Succinivibrionaceae_UCG-001, Lachnospiraceae_NK3A20_group, Shuttleworthia and Dialister were positively correlated with milk yield (p < 0.05). CONCLUSIONS: This study indicates that dairy cows with different milk yields have clear divergence in serum indicators, RFPs, BCC and rumen microbial metabolism.

Effects of yeast culture and oxalic acid supplementation on in vitro nutrient disappearance, rumen fermentation, and bacterial community composition.

Hemicellulose is an important polysaccharide in ruminant nutrition, but it has not been studied as thoroughly as cellulose. Further research is needed to explore supplements that can improve its digestibility and ruminal buffering effects. Our previous research demonstrated the efficacy of oxalic acid (OA) as an essential nutrient in yeast culture (YC) for improving rumen fermentation performance. Consequently, we conducted in vitro rumen digestion experiments to examine the effects of YC and OA on rumen fermentation and bacterial composition. Two diets containing different levels of hemicellulose were formulated: diet 1 with 10.3% and diet 2 with 17% hemicellulose. Three levels of YC (0.00, 0.625, and 1.25 g/kg) and three doses of OA (0.0, 0.4, and 0.8 g/kg, DM) were added into each diet with a 3 × 3 factorial design. A comprehensive assessment was conducted on a total of 18 experimental treatments at fermentation periods of 0, 6, 12, 24, and 48 h. In the first experiment (diet 1), the supplementation of YC, OA, and their interaction significantly increased in vitro DM disappearance (IVDMD) and NDF disappearance (IVNDFD; p < 0.001). In the second experiment (diet 2), the supplementation of OA and the interaction between YC and OA (p < 0.001) increased IVDMD and IVCPD, but had no significant effects on IVNDFD. The interactions of YC and OA significantly increased ammonia nitrogen (p < 0.001). The production of acetic acid, propionic acid, and total volatile fatty acids (TVFA), and pH levels were significantly higher in treatments supplemented with YC and OA (p < 0.001). YC and OA in both diets significantly altered the rumen bacterial community leading to increased Shannon and Simpson diversity indices (p < 0.001). In both diets, OA supplementation significantly increased the relative abundance of the phylum Bacteroidetes and Prevotella genus. The result also showed a positive correlation between the Prevotella and Selenomonas genera with IVDMD, IVNDFD, propionic acid, and TVFA production, suggesting that these dominant bacteria enhanced nutrient disappearance in the rumen. In conclusion, adding YC and OA resulted in modifications to the bacterial community's composition and diversity, and improved nutrient disappearance. These changes indicate improved rumen fermentation efficiency, which is promising for future in vivo studies.

Proteomics Insights into the Gene Network of cis9, trans11-Conjugated Linoleic Acid Biosynthesis in Bovine Mammary Gland Epithelial Cells.

The objective of the study was to elucidate the stearoyl-coenzyme A desaturase (SCD1)-dependent gene network of c9, t11-CLA biosynthesis in MAC-T cells from an energy metabolism perspective. The cells were divided into the CAY group (firstly incubated with CAY10566, a chemical inhibitor of SCD1, then incubated with trans-11-octadecenoic acid, (TVA)), the TVA group (only TVA), and the control group (without CAY, TVA). The c9, t11-CLA, and TVA contents were determined by gas chromatography. The mRNA levels of SCD1 and candidate genes were analyzed via real-time PCR. Tandem mass tag (TMT)-based quantitative proteomics, bioinformatic analysis, parallel reaction monitoring (PRM), and small RNA interference were used to explore genes involved in the SCD1-dependent c9, t11-CLA biosynthesis. The results showed that the SCD1 deficiency led by CAY10566 blocked the biosynthesis of c9, t11-CLA. In total, 60 SCD1-related proteins mainly involved in energy metabolism pathways were primarily screened by TMT-based quantitative proteomics analysis. Moreover, 17 proteins were validated using PRM analysis. Then, 11 genes were verified to have negative relationships with SCD1 after the small RNA interference analysis. Based on the above results, we concluded that genes involved in energy metabolism pathways have an impact on the SCD1-dependent molecular mechanism of c9, t11-CLA biosynthesis.

Relationship between dietary amino acid release kinetics and nitrogen deposition in growing pigs.

Although the protein content of swine diets is formulated based on the ileal digestibility of protein and amino acids (AA) under current nutrition requirements, the nitrogen utilization efficiency of swine varies based on protein source, which may be related to AA release kinetics. In this experiment, a 2 × 2 factorial arrangement with casein (CAS)-enriched or corn gluten meal (CGM)-enriched protein sources at different digestible crude protein levels (normal [N], 13%; and low [L], 11%) were applied to 24 crossbred (Duroc × Landrace × Yorkshire) growing pigs (average body weight = 43.3 ± 3.5 kg) in 4 treatments (N.CAS, L.CAS, N.CGM, L.CGM, respectively) to investigate the effects of AA release kinetics on nitrogen deposition in growing pigs. Standardized ileal digestible AA in all diets were balanced by adding individual AA to meet the nutrient requirements. The AA release kinetics were detected in vitro by measuring the hydrolysis of various protein diets under pepsin and trypsin conditions. The results demonstrated that the time of AA release peak in the CGM diet was 12 h later than that in the CAS diet. The synchronization indices of dietary AA release in N.CAS, N.CGM, L.CAS, and L.CGM were 23.73%, 29.37%, 23.40%, and 26.07%, respectively. The N.CGM had the poorest AA release synchronism while the N.CAS had the greatest among the 4 diets. However, within the pigs, L.CAS and N.CGM showed the highest (81.08%) and lowest (73.54%) nitrogen biological values, respectively, despite the standard ileal digestible AA levels being equal for all diets. These results indicate that the release kinetics of dietary AA had great effect on nitrogen deposition. To optimize nitrogen deposition, AA release kinetics and composition should be taken into consideration when formulating diets for growing pigs.

Protective Mechanism of Leucine and Isoleucine against H2O2-Induced Oxidative Damage in Bovine Mammary Epithelial Cells.

Leucine and isoleucine possess antioxidative and anti-inflammatory properties. However, their underlying protective mechanisms against oxidative damage remain unknown. Therefore, in this study, the protective mechanism of leucine and isoleucine against H2O2-induced oxidative damage in a bovine mammary epithelial cell lines (MAC-T cells) were investigated. Briefly, MAC-T cells exposed or free to H2O2 were incubated with different combinations of leucine and isoleucine. The cellular relative proliferation rate and viability, oxidative stress indicators, and inflammatory factors were determined by specific commercial kits. The genes related to barrier functions was measured by real-time quantitative PCR. The protein expression differences were explored by 4D label-free quantitative proteomic analyses and validated by parallel reaction monitoring. The results revealed that leucine and isoleucine increased cell proliferation, total antioxidant status (TAS), and the relative mRNA expression of occludin, as well as decreased malondialdehyde (MDA), total oxidant status (TOS)/TAS, IL-6, IL-1ß, and TOS. When leucine and isoleucine were combined, MDA, TOS/TAS, and the relative mRNA expression levels of claudin-1, occludin, and zonula occludens-1 increased when compared to leucine or isoleucine alone. Proteomics analyses revealed that leucine significantly upregulated the propanoate metabolism; valine, leucine, and isoleucine degradation; and thermogenesis pathways, whereas isoleucine significantly upregulated the peroxisome and propanoate metabolism pathways. In conclusion, leucine protected MAC-T cells from H2O2-induced oxidative stress by generating more ATP to supplement energy demands, and isoleucine improved the deficit in peroxisome transport and promoted acetyl-CoA production. The findings of this study enhance our understanding of the protective mechanisms of leucine and isoleucine against oxidative damage.

Target-Binding Accelerated Response for Sensitive Detection of Basal H2O2 in Tumor Cells and Tissues via a Dual-Functional Fluorescence Probe.

Aberrant production of H2O2 is involved in cancer. The levels of H2O2 are significantly higher in tumor cells than in normal cells. It is important to develop fluorescent probes to image basal H2O2 selectively in tumor cells. So far, a cancer cell-targeting probe to image basal H2O2 has not been reported. Thus, we developed a fluorescent probe, BBHP, which contains benzil as a H2O2-recognition site and biotin as a target binding motif for the selective and sufficient detection of H2O2 in tumor cells. BBHP enables a selective fluorescence turn-on response to H2O2. The binding of the probe with biotin receptors can greatly accelerate the fluorescence response to H2O2. As a result, BBHP can sufficiently image basal H2O2 in biotin receptor-positive cancer cells and tumor tissues. Finally, BBHP was successfully applied to discriminate between cancerous and normal tissues.

Effects of protein content and the inclusion of protein sources with different amino acid release dynamics on the nitrogen utilization of weaned piglets.

OBJECTIVE: We aimed to investigate the effect of the differing amino acid (AA) release dynamics of two protein sources on the growth performance, nitrogen deposition, plasma biochemical parameters, and muscle synthesis and degradation of piglets when included in their diets at normal and low concentrations. METHODS: Forty-eight piglets (Duroc×Landrace×Large White) with initial body weight of 7.45±0.58 kg were assigned to six groups and fed one of 6 diets. The 6 dietary treatments were arranged by 3×2 factorial with 3 protein sources and 2 dietary protein levels. They are NCAS (a normal protein content with casein), NBlend (a normal protein content with blend of casein and corn gluten meal), NCGM (a normal protein content with corn gluten meal), LCAS (a low protein content with casein), LBlend (a low protein content with blend of casein and corn gluten meal), LCGM (a low protein content with corn gluten meal). The release dynamics of AA in these diets were determined by in vitro digestion. The digestibility, utilization and biological value of nitrogen in piglets were determined by micro Kjeldahl method. Plasma insulin was measured by enzyme-linked immunosorbent assay kits. The protein expression of mediators of muscle synthesis and degradation was determined by western blotting. RESULTS: Although the consumption of a low-protein diet supplemented with crystalline AA was associated with greater nitrogen digestion and utilization (p<0.05), the final body weight, growth performance, nitrogen deposition, and phosphorylation of ribosomal protein S6 kinase 1 and eIF4E binding protein 1 in the muscle of pigs in the low-protein diet-fed groups were lower than those of the normal-protein diet-fed groups (p<0.05) because of the absence of non-essential AA. Because of the more balanced release of AA, the casein (CAS) and Blend-fed groups showed superior growth performance, final body weight and nitrogen deposition, and lower expression of muscle ring finger 1 and muscle atrophy F-box than the CGM-fed groups (p<0.05). CONCLUSION: We conclude that the balanced release of AA from CAS containing diets and mixed diets could reduce muscle degradation, favor nitrogen retention, % intake and improve growth performance in pigs consuming either a normal- or low-protein diet.