Dietary supplementation of coated sodium butyrate improves growth performance of laying ducks by regulating intestinal health and immunological performance.

Introduction: This study was conducted to assess the effects of dietary supplementation of coated sodium butyrate (CSB) on the growth performance, serum antioxidant, immune performance, and intestinal microbiota of laying ducks. Methods: A total of 120 48-week-old laying ducks were randomly divided into 2 treatment groups: the control group (group C fed a basal diet) and the CSB-treated group (group CSB fed the basal diet + 250 g/t of CSB). Each treatment consisted of 6 replicates, with 10 ducks per replicate, and the trial was conducted for 60 days. Results: Compared with the group C, the group CSB showed a significant increase in the laying rate (p<0.05) of the 53-56 week-old ducks. Additionally, the serum total antioxidant capacity, superoxide dismutase activity and immunoglobulin G level were significantly higher (p<0.05), while the serum malondialdehyde content and tumor necrosis factor (TNF)-a level were significantly lower (p<0.05) in the serum of the group CSB compared to the group C. Moreover, the expression of IL-1b and TNF-a in the spleen of the group CSB was significantly lower (p<0.05) compared to that of the group C. In addition, compared with the group C, the expression of Occludin in the ileum and the villus height in the jejunum were significantly higher in the group CSB (p<0.05). Furthermore, Chao1, Shannon, and Pielou-e indices were higher in the group CSB compared to the group C (p<0.05). The abundance of Bacteroidetes in the group CSB was lower than that in the group C (p<0.05), while the abundances of Firmicutes and Actinobacteria were higher in the group CSB compared to the group C (p<0.05). Conclusions: Our results suggest that the dietary supplementation of CSB can alleviate egg-laying stress in laying ducks by enhancing immunity and maintaining the intestinal health of the ducks.

Effects of Tributyrin Supplementation on Liver Fat Deposition, Lipid Levels and Lipid Metabolism-Related Gene Expression in Broiler Chickens.

The objective of this study was to investigate the effects of tributyrin supplementation on liver fat metabolism in broiler chickens. Two hundred and forty broilers were randomly allocated into two experimental groups (6 replicates per treatment; 20 chickens in each replicate): the control group (CN), which received a basal diet, and the tributyrin group (TB), which received a basal diet supplemented with 1 g/kg of tributyrin. The experimental period lasted 37 days. The results showed that in the liver, broilers supplemented with tributyrin had higher content of high-density lipoprotein cholesterol (HDL-C) (p < 0.05). Liver hepatic lipase (HL), lipoprotein lipase (LPL) and total lipid (TL) activity were significantly lower than in the TB group than that in the NC group. Meanwhile, the diet supplemented with tributyrin had more lipid droplets than the NC group, whereas the TB and NC groups showed no histological abnormalities in the liver. Furthermore, the mRNA expression levels of peroxisome proliferators-activated receptor α (PPARα), proliferators-activated receptor γ (PPARγ), fatty acid synthase (FAS), LPL and adipose triglyceride lipase (ATGL) in the liver were significantly upregulated in the TB group (p < 0.05), while those of the long-chain acyl-CoA-synthetase 1 (ACSL1) mRNA between the TB group and the NC group were not different (p > 0.05). These findings indicated that the diet supplemented with tributyrin could increase fat deposition appropriately by promoting fat synthesis without causing liver tissue damage, which demonstrated that tributyrin can be considered a valid feed additive for broiler chickens.

Methodological Considerations in N-of-1 Trials of Traditional Chinese Medicine.

More and more scholars choose N-of-1 trials for TCM clinical research. However, the quality of the experimental designs was uneven. Accumulating more than eight years of experience in exploring the N-of-1 trials of TCM, the authors and their team searched the related literature in main Chinese and English databases, referenced to relevant Chinese and international guidelines. The design, implementation, and data analysis of N-of-1 trials of TCM are still in in-depth exploration and practice. "Carryover effect" may affect the design and quality of the trials. Individualized treatment should be guided by the classic theories of TCM. It is expected to formulate reasonable observation periods and pairs and closely integrate individual and group statistical analysis.

A Series of N-of-1 Trials for Traditional Chinese Medicine Using a Bayesian Method: Study Rationale and Protocol.

Background. Our previous studies showed that N-of-1 trials could reflect the individualized characteristics of traditional Chinese medicine (TCM) syndrome differentiation with good feasibility, but the sensitivity was low. Therefore, this study will use hierarchical Bayesian statistical method to improve the sensitivity and applicability of N-of-1 trials of TCM. Methods/Design. This is a randomized, double-blind, placebo-controlled, three-pair crossover trial for a single subject, including 4-8 weeks of run-in period and 24 weeks of formal trial. In this study, we will recruit a total of 30 participants who are in the stable stage of bronchiectasis. The trial will be divided into three pairs (cycles), and one cycle contains two observation periods. The medications will be taken for three weeks and stopped for one week in the last week of each observation period. The order of syndrome differentiation decoction and placebo will be randomly determined. Patient self-reported symptom score (on a 7-point Likert scale) is the primary outcome. Discussion. Some confounding variables (such as TCM syndrome type and potential carryover effect of TCM) will be introduced into hierarchical Bayesian statistical method to improve the sensitivity and applicability of N-of-1 trials of TCM, and the use of prior available information (e.g., "borrowing from strength" of previous trial results) within the analysis may improve the sensitivity of the results of a series of N-of-1 trials, from both the individual and population level to study the efficacy of TCM syndrome differentiation. It is the exploration of improving the objective evaluation method of the clinical efficacy of TCM and may provide reference value for clinical trials of TCM in other chronic diseases. This trial is registered with ClinicalTrials.gov (ID: NCT04601792).

Effects of Compound Probiotics on Cecal Microbiome and Metabolome of Shaoxing Duck.

This experiment was conducted to investigate the effects of compound probiotics on intestinal microflora and metabolome of Shaoxing ducks. A total of 640 1-day-old Shaoxing ducks were randomly divided into two treatments with eight replicates and forty ducks for each replicate. The ducks were fed basal diet (Ctrl) and basal diet supplemented with 0.15% compound probiotics (MixP). The experiment lasted for 85 days. The results showed that the abundance of Bacteroidetes and Bacteroides in MixP was higher than that in Ctrl (P < 0.05). However, the abundance of Firmicutes and Oscillospira and Desulfovibrio in MixP was lower than that in Ctrl (P < 0.05). Concentrations of 71 metabolites differed significantly (P < 0.05) between the MixP and the Ctrl groups; for example, Pyridoxal (Vitamin B6), L-Arginine, and Betaine aldehyde were up-regulated (P < 0.05), and 7-oxocholesterol, 3-hydroxy-L-kynureni-ne, and N-acetyl-d-glucosamine were down-regulated (P < 0.05). KEGG was enriched in 15 metabolic pathways. The pathways of Vitamin B6 metabolism, Vascular smooth muscle contraction, Vitamin digestion and absorption, and Protein digestion and absorption were influenced by compound probiotics supplementation. Thus, supplementation of compound probiotics improved cecal heath through shifts in the cecal microbiome and metabolome.

Molecular cloning, expression and mimicking antiviral activity analysis of retinoic acid-inducible gene-I in duck (Anas platyrhynchos).

Intracellular double-stranded RNA (dsRNA) is a chief sign of replication for many viruses. Pattern recognition receptors(PRRs) of the innate immune system detected the dsRNA and initiate the antiviral responses. Retinoic acid-inducible gene I (RIG-I), a member of PRRs, plays an essential regulatory role in dsRNA-induced signalling. In this study, the full-length complementary DNA (cDNA) of duck RIG-I (duRIG-I) was cloned using the reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of the cDNA ends (RACE). The cDNA of duRIG-I contained 97-bp 5'UTR, 141-bp 3'-UTR and 2802 bp complete open-reading frame (ORF) encoding 933 amino acids. Multiple sequence alignments showed that duRIG-I shared high similarity with RIG-I from other vertebrates. Quantitative real-time PCR (qRT-PCR) analysis revealed that duRIG-I mRNA was expressed in all tested tissues, with high levels in the liver, heart, spleen, kidney and thymus, while lower in the duodenum. duRIG-I could be induced by treatment with poly(I:C). Further, overexpression of duRIG-I significantly activated the transcription of poly(I:C)-induced IFN-b, IRF7, TRIF, Mx, STAT1 and STAT2 mRNA, and duRIG-I knockdown showed the opposite results. Overall, our results suggested that duRIG-I could be an important receptor for mimicking antiviral state in duck, which warrant further studies to show the possible mechanism.

Digital gene-expression profiling analysis of the fatty liver of Landes geese fed different supplemental oils.

The goose liver is an ideal model for deciphering lipogenesis molecular mechanisms. This study was designed to investigate the effect of different lipid sources on hepatic lipogenesis in overfed geese. Sixty Landes geese were fed ad libitum with no fat (control) or overfed diets containing 2% goose fat (GF) or rapeseed oil (RO) for 20 days. We measured fatty acid composition of the liver at day 20 of overfeeding. We performed a transcriptomic comparison of fatty liver between GF and RO-fed geese to gain insights into the molecular and cellular events mediating lipogenesis activity. The results showed that there was no substantial effect on fatty liver performance between GF- and RO-fed geese. Significant differences in fatty acid composition were detected between GF- and RO-fed geese. Total ω-6 PUFAs increased and saturated fatty acid decreased (P < 0.05) with RO supplementation when compared with GF, but ω-3 PUFAs did not differ between the two diets. Concentrations of C16:1, C18:1, C18:2, C20:2, and C22:1 were higher (P < 0.05) in the fatty liver of RO-fed geese compared to those in the GF group. Analysis of transcriptome sequencing showed that there were 124 up-regulated and 129 down-regulated differentially expressed genes in the fatty liver of RO and GF-fed geese. Many of these genes code for proteins involved in the lipid metabolic process, including bile secretion, adipocytokine signalling pathway, biosynthesis of unsaturated fatty acids, linoleic acid metabolism, fatty acid elongation and fatty acid biosynthesis, and fat digestion and absorption. Moreover, genes involved in lipid-related pathways such as peroxisome, steroid biosynthesis, steroid hormone biosynthesis, retinol metabolism, and apoptosis were altered, suggesting that the fatty liver of goose fed different oils undertakes both an oxidation function and hormone-related metabolic function. In conclusions, these data suggest that RO supplementation reduces liver lipid oxidation and improves lipogenesis. These findings provide new insights into the molecular mechanisms involved in fatty liver formation and provide valuable resources for analysing mechanisms underlying the effects of oils from different sources on the goose fatty liver.

Original Research: Effect of various dietary fats on fatty acid profile in duck liver: Efficient conversion of short-chain to long-chain omega-3 fatty acids.

Omega-3 fatty acids, especially long-chain omega-3 fatty acids, have been associated with potential health benefits for chronic disease prevention. Our previous studies found that dietary omega-3 fatty acids could accumulate in the meat and eggs in a duck model. This study was to reveal the effects of various dietary fats on fatty acid profile and conversion of omega-3 fatty acids in duck liver. Female Shan Partridge Ducks were randomly assigned to five dietary treatments, each consisting of 6 replicates of 30 birds. The experimental diets substituted the basal diet by 2% of flaxseed oil, rapeseed oil, beef tallow, or fish oil, respectively. In addition, a dose response study was further conducted for flaxseed and fish oil diets at 0.5%, 1%, and 2%, respectively. At the end of the five-week treatment, fatty acids were extracted from the liver samples and analyzed by GC-FID. As expected, the total omega-3 fatty acids and the ratio of total omega-3/omega-6 significantly increased in both flaxseed and fish oil groups when compared with the control diet. No significant change of total saturated fatty acids or omega-3 fatty acids was found in both rapeseed and beef tallow groups. The dose response study further indicated that 59-81% of the short-chain omega-3 ALA in flaxseed oil-fed group was efficiently converted to long-chain DHA in the duck liver, whereas 1% of dietary flaxseed oil could produce an equivalent level of DHA as 0.5% of dietary fish oil. The more omega-3 fatty acids, the less omega-6 fatty acids in the duck liver. Taken together, this study showed the fatty acid profiling in the duck liver after various dietary fat consumption, provided insight into a dose response change of omega-3 fatty acids, indicated an efficient conversion of short- to long-chain omega-3 fatty acid, and suggested alternative long-chain omega-3 fatty acid-enriched duck products for human health benefits.

Effects of dietary fats on egg quality and lipid parameters in serum and yolks of Shan Partridge Duck.

The effects of different dietary fats with variable levels of polyunsaturated fatty acids (PUFAs) on egg quality of Shan Partridge Duck, serum, and yolk lipid parameters were examined in this study. A flock of 585 optimal produced ducks were selected and diets enriched with 0.5%, 1%, or 2% fish oil (F)/flaxseed oil (FL)/rapeseed oil (R)/tallow (T) plus basal diet were supplied through a 28-d period. Supplemental fat source and fat level had no effects on egg qualities. Proportions of yolk total cholesterol (TC), saturated fatty acids (SFAs), monounsaturated fatty acids (MUFAs) were reduced (P < 0.001), while polyunsaturated fatty acids (PUFAs), ω-6 polyunsaturated fatty acids (n-6 PUFAs), ω-3 polyunsaturated fatty acids (n-3 PUFAs), Docosahexaenoic Acid (DHA), and Eicosapentaenoic Acid (EPA) were increased by fish oil, flaxseed oil, or rapeseed oil. Effects of supplementation increasing DHA and EPA were detected in F, FL, and R. Compared with C, fish oil significantly increased low-density lipoprotein cholesterol (LDL-C) in serum, flaxseed oil significantly reduced TC and increased very low-density lipoprotein cholesterol (VLDL-C), rapeseed oil significantly reduced TC and LDL-C in serum and increased VLDL-C, tallow significantly increased LDL-C. It is concluded that unsaturated fatty acids rich diets (fish oil, flaxseed oil, and rapeseed oil) might increase yolk PUFAs, reduce yolk cholesterol, and change serum lipid parameters without evident effect on egg qualities.

Identification of differentially expressed known and novel miRNAs in broodiness of goose.

MicroRNAs (miRNAs) are endogenous small noncoding RNAs plays a critical role in posttranscriptional regulation of gene expression. Broodiness is observed in most avian species and influences egg production. Several genes are known to play an important role in regulating the progress of reproduction. The goose is one of the most important waterfowls. However, the involvement of miRNAs in the broodiness behavior of Anser cygnoides (Swan Goose) is unknown. High-throughput sequencing and bioinformatics analysis were used to identify the miRNAs involved in egg-laying and brooding behavior of geese in our study. The results showed 38 up-regulated and 14 down-regulated known miRNAs/miRNA*s with reads>1,000 in at least one group and a fold change of >2.0, compared with those of the egg-laying group (P<0.001). We also identified 114 and 94 novel miRNAs in the broody and egg-laying groups, respectively. Of these, 4 novel miRNAs were differentially expressed between the two groups. The study showed the expression of small RNAs in goose reproduction and identified known and novel miRNAs regulated in broodiness. The results reveal that these differentially expressed miRNAs may be involved in broodiness of A. cygnoides.

Antioxidant effect of a phytoestrogen equol on cultured muscle cells of embryonic broilers.

Previous studies have shown that the in ovo injection of equol can markedly improve the water-holding capacity of muscles of broilers chickens at 7 wk of age through promotion of the antioxidant status. We aimed to investigate directly the antioxidant effects of equol on muscle cells in broilers. Muscle cells were separated from leg muscle of embryos on the 11th day of incubation and treated with equol and H(2)O(2), either alone or together. Cells were pretreated with medium containing 1, 10, or 100 µM equol for 1 h prior to the addition of 1 mM H(2)O(2) for a further 1 h. Photomicrographs of cells were obtained. Cell viability, malondialdehyde (MDA) content, and L-lactate dehydrogenase (LDH) activity in the cell supernatant, as well as intracellular total superoxide dismutase (T-SOD) and glutathione peroxidase (GSH-Px) activities were determined. Treatment with 1 mM H(2)O(2) caused serious damage to cells, indicated by comets with no clear head region but a very apparent tail of DNA fragments. Pretreatment with low (1 µM) but not high concentrations of equol (10 µM) inhibited cell damage, while 100 µM equol caused more serious damage than H(2)O(2) alone. Pretreatment with 1 µM equol had no effect on cell viability, while pretreatment with 10 and 100 µM equol significantly decreased cell viability in a dose-dependent manner. Compared with H(2)O(2) alone, pretreatment with low-dosage equol markedly decreased LDH activity and MDA production in the supernatant, significantly increased intracellular T-SOD activity (P < 0.05) and tended to increase intracellular GSH-Px activity (0.05 < P < 0.1). Pretreatment with high-dosage equol (10 and 100 µM) significantly enhanced LDH activity, but had no effect on MDA content, T-SOD or GSH-Px activity induced by H(2)O(2,) except for an obvious increase in GSH-Px activity caused by 10 µM equol. These results indicate that equol at low dosage can prevent skeletal muscle cell damage induced by H(2)O(2), while pretreatment with high-dosage equol shows a synergistic effect with H(2)O(2) in inducing cell damage.

Effect of dietary fatty acids on serum parameters, fatty acid compositions, and liver histology in Shaoxing laying ducks.

The effects of different fatty acid (FA) contents in diet on serum parameters, FA compositions of eggs and meat, and liver morphological changes were studied in Shaoxing laying ducks. A total of 264 ducks at 17 weeks were fed a control diet or a diet containing 30 g/kg fish oil (FO), 25 g/kg sunflower oil (SO), or 30 g/kg palm oil with 20 g/kg beef tallow (PBO). Malondialdehyde (MDA) content in the liver and the serum of ducks fed the PBO diet was significantly (P<0.05) higher than that of ducks fed the other diets. Triglyceride (TG) and total cholesterol (TC) levels were significantly lower (P<0.05) in ducks fed the FO diet. Serum TC also was lower in ducks fed the SO diet. Superoxide dismutase (SOD) activity was also affected by diets. The contents of polyunsaturated FAs (PUFAs) in eggs and meat were significantly higher (P<0.001) in ducks fed the FO and SO diets than in ducks fed the control diet. The level of C22:6 (n-3) FA in ducks fed the FO diet was significantly higher than that in ducks fed the other diets. However, the conversion efficiency of the longer-chain C20:5 (n-3) FA was higher than that of C22:6 (n-3). Ducks fed the PBO diet exhibited lipid droplet accumulation in the liver. These results demonstrate that a diet enriched with different FAs has strong effects on serum lipid levels and the deposition of PUFAs into tissue lipids.

[Expression characteristics of prolactin gene in Eastern Zhejiang white geese].

This study was conducted to clone the prolactin gene (PRL) in Eastern Zhejiang White Geese and to investigate the PRL gene expression characteristics during egg-laying, out-of-lay and incubating periods by real time PCR. Comparisons were made respectively of concentration of prolactin mRNA in the hypothalamus, pituitary gland and ovary of the adult female geese at different reproductive periods. The result indicated that there were significant differences (P<0.05) in PRL mRNA expression between different reproductive periods of the geese. The lowest level of PRL expression was found in out-of-lay geese, higher in the egg-laying geese, and the highest in incubating geese. Furthermore, the analysis of PRL expression in different tissues indicated that the highest levels of PRL was expressed in the pituitary gland, followed in hypothalamus, and the least in ovary of the geese. There were significant difference (P<0.01) expression of PRL between the pituitary gland/hypothalamus and ovary of the geese, whereas no any difference was observed between the pituitary gland and hypothalamus (P>0.05). In summary, the PRL mRNA expression had variance in different reproductive periods of the geese.

Dietary daidzein influences laying performance of ducks (Anas platyrhynchos) and early post-hatch growth of their hatchlings by modulating gene expression.

Our previous studies demonstrated that dietary supplementation of daidzein improves egg production in duck breeders during late periods of the laying cycle. The present study was aimed to clarify whether the growth of ducklings hatched from eggs laid by daidzein-treated hens would be affected, and to elucidate the mechanisms underlying potential trans-generational effects, by determining changes of hormone levels and mRNA expression of relevant genes. Daidzein was added to the basal diet of 415-day-old duck breeders at the level of 5 mg/kg. During 9 weeks of daidzein treatment, laying rate increased by 7.70%, average egg mass tended to increase, whereas yolk/albumen ratio decreased significantly. These changes were accompanied by significantly elevated plasma T4 and E2 levels, enhanced gonadotropin releasing hormone (GnRH) mRNA, but diminished estrogen receptor (ER)-beta mRNA expression in hypothalamus of daidzein-treated hens. Ducklings hatched from daidzein-treated eggs were significantly smaller at hatching, but they caught up with their control counterparts by 4 weeks of age. Serum levels of T4, pituitary GH, hepatic GH receptor (GHR) and type-1 IGF receptor (IGF-1R) mRNA expression were all suppressed markedly in the daidzein-treated group at hatching, but this suppression proved to be temporary, as at 4 weeks of age, expression levels of all investigated genes were restored. However, it is noteworthy that at 4 weeks of age an obvious down-regulation of hypothalamic GnRH mRNA expression was detected in ducklings maternally exposed to daidzein. Our results provide evidence that maternal exposure to daidzein affects post-hatch growth in the duck with accompanying changes in the secretion of metabolic hormones and expression of growth-related genes. Although the negative effect of maternal daidzein on embryonic growth could be eliminated 4 weeks after hatching, the long-term effect of maternal daidzein on reproductive function is not to be ignored and awaits further investigation.