The Photoperiod-Driven Cyclical Secretion of Pineal Melatonin Regulates Seasonal Reproduction in Geese (Anser cygnoides).

The photoperiod is the predominant environmental factor that governs seasonal reproduction in animals; however, the underlying molecular regulatory mechanism has yet to be fully elucidated. Herein, Yangzhou geese (Anser cygnoides) were selected at the spring equinox (SE), summer solstice (SS), autumn equinox (AE), and winter solstice (WS), and the regulation of seasonal reproduction via the light-driven cyclical secretion of pineal melatonin was investigated. We show that there were seasonal variations in the laying rate and GSI, while the ovarian area decreased 1.5-fold from the SS to the AE. Moreover, not only did the weight and volume of the pineal gland increase with a shortened photoperiod, but the secretory activity was also enhanced. Notably, tissue distribution further revealed seasonal oscillations in melatonin receptors (Mtnrs) in the pineal gland and the hypothalamus-pituitary-gonadal (HPG) axis. The immunohistochemical staining indicated higher Mtnr levels due to the shortened photoperiod. Furthermore, the upregulation of aralkylamine N-acetyltransferase (Aanat) was observed from the SS to the AE, concurrently resulting in a downregulation of the gonadotrophin-releasing hormone (GnRH) and gonadotropins (GtHs). This trend was also evident in the secretion of hormones. These data indicate that melatonin secretion during specific seasons is indicative of alterations in the photoperiod, thereby allowing for insight into the neuroendocrine regulation of reproduction via an intrinsic molecular depiction of external photoperiodic variations.

Phosphoproteome Reveals Extracellular Regulated Protein Kinase Phosphorylation Mediated by Mitogen-Activated Protein Kinase Kinase-Regulating Granulosa Cell Apoptosis in Broody Geese.

Geese have strong brooding abilities, which severely affect their egg-laying performance. Phosphorylation is widely involved in regulating reproductive activities, but its role in goose brooding behavior is unclear. In this study, we investigated differences in the phosphoprotein composition of ovarian tissue between laying and brooding geese. Brooding geese exhibited ovarian and follicular atrophy, as well as significant oxidative stress and granulosa cell apoptosis. We identified 578 highly phosphorylated proteins and 281 lowly phosphorylated proteins, and a KEGG pathway analysis showed that these differentially phosphorylated proteins were mainly involved in cell apoptosis, adhesion junctions, and other signaling pathways related to goose brooding behavior. The extracellular regulated protein kinase (ERK)-B-Cell Lymphoma 2(BCL2) signaling pathway was identified as playing an important role in regulating cell apoptosis. The phosphorylation levels of ERK proteins were significantly lower in brooding geese than in laying geese, and the expression of mitogen-activated protein kinase kinase (MEK) was downregulated. Overexpression of MEK led to a significant increase in ERK phosphorylation and BCL2 transcription in H2O2-induced granulosa cells (p < 0.05), partially rescuing cell death. Conversely, granulosa cells receiving MEK siRNA exhibited the opposite trend. In conclusion, geese experience significant oxidative stress and granulosa cell apoptosis during brooding, with downregulated MEK expression, decreased phosphorylation of ERK protein, and inhibited expression of BCL2.

The First Crested Duck Genome Reveals Clues to Genetic Compensation and Crest Cushion Formation.

The Chinese crested (CC) duck is a unique indigenous waterfowl breed, which has a crest cushion that affects its survival rate. Therefore, the CC duck is an ideal model to investigate the genetic compensation response to maintain genetic stability. In the present study, we first generated a chromosome-level genome of CC ducks. Comparative genomics revealed that genes related to tissue repair, immune function, and tumors were under strong positive selection, indicating that these adaptive changes might enhance cancer resistance and immune response to maintain the genetic stability of CC ducks. We also assembled a Chinese spot-billed (Csp-b) duck genome, and detected the structural variations (SVs) in the genome assemblies of three ducks (i.e., CC duck, Csp-b duck, and Peking duck). Functional analysis revealed that several SVs were related to the immune system of CC ducks, further strongly suggesting that genetic compensation in the anti-tumor and immune systems supports the survival of CC ducks. Moreover, we confirmed that the CC duck originated from the mallard ducks. Finally, we revealed the physiological and genetic basis of crest traits and identified a causative mutation in TAS2R40 that leads to crest formation. Overall, the findings of this study provide new insights into the role of genetic compensation in adaptive evolution.

Chinese Domestic Ducks Evolved from Mallard Duck (Anas platyrhynchos) and Spot-Billed Duck (A. zonorhyncha).

China has a rich genetic resource in its 34 domestic duck breeds. In order to detect the genetic diversity and explore the origin of these indigenous duck populations, the mitochondrial DNA (mtDNA) control region was sequenced and analyzed for 208 individual ducks, including 22 domestic breeds, wild Mallards ducks, Eastern spot-billed ducks, White Muscovy ducks, and Black Muscovy ducks. The haplotype diversity (Hd) was 0.653 and the average nucleotide diversity (Pi) was 0.005, indicating moderate genetic diversity. Sixty haplotypes were detected, and the maximum-likelihood (ML) phylogenetic tree and median-joining (MJ) network were generated from the sequence analyses. In this study, haplotypes from the Mallard duck (Anas platyrhynchos) were detected in most of the Chinese domestic duck breeds. In addition, the Eastern spot-billed duck (A. zonorhyncha) H8 haplotype was detected in two duck breeds. Only two haplotypes were found in Muscovy ducks, suggesting low genetic diversity within this population. The sequence and haplotype analyses revealed that both A. platyrhynchos and A. zonorhyncha contributed to the evolution of domestic ducks in China.

Comparison of carcass traits and nutritional profile in two different broiler-type duck lines.

Cherry Valley ducks (CVDs) and White Kaiya ducks (WKDs) are judged to be fast- and slow-growing lines, respectively. To investigate the carcass traits and nutritional profile at their marketable ages, 12 birds (38 days for CVDs, n = 6; 56 days for WKDs, n = 6) were randomly selected and slaughtered. Indicators such as breast muscle weight, shear force, and proximate composition were comprehensively detected. Although the carcass and breast muscle weight in WKDs were significantly lower, remarkably higher intramuscular fat and tenderness and less moisture content were observed in WKDs. Besides, WKDs contained higher contents of Cu, Zn, and Ca, whereas CVDs contained higher leucine (Leu) and histidine (His) compositions (P < 0.01). Moreover, higher monounsaturated fatty acids (MUFAs) and polyunsaturated fatty acids (PUFAs) along with lower saturated fatty acids (SFAs) were detected in WKDs (P < 0.01). Taken together, despite light carcass and breast muscle weight, WKDs had advantages in nutritional compositions except for amino acid constituents, including intramuscular fat, MUFAs, and PUFAs, as well as Cu, Zn, and Ca. These data would not only provide genetic resources for breeding new duck lines but also offer a useful reference for making decisions on high-nutrient meat consumption.

Effects of T-2 toxin on growth performance, feather quality, tibia development and blood parameters in Yangzhou goslings.

T-2 toxin is a dangerous natural pollutant and widely exists in animal feed, often causing toxic damage to poultry, such as slow growth and development, immunosuppression, and death. Although geese are considered the most sensitive poultry to T-2 toxin, the exact damage caused by T-2 toxin to geese is elusive. In the present study, a total of forty two 1-day-old healthy Yangzhou male goslings were randomly allotted seven diets contaminated with 0, 0.2, 0.4, 0.6, 0.8, 1.0, or 2.0 mg/kg T-2 toxin for 21 d, and the effects of T-2 toxin exposure on growth performance, feather quality, tibia development, and blood parameters were investigated. The results showed that T-2 toxin exposure significantly inhibited feed intake, body weight gain, shank length growth, and organ development (e.g., ileum, cecum, liver, spleen, bursa, and tibia) in a dose-dependent manner. In addition, the more serious feathering abnormalities and feather damage were observed in goslings exposed to a high dose of T-2 toxin (0.8, 1.0, and 2.0 mg/kg), which were mainly sparsely covered with short, dry, rough, curly, and gloss-free feathers on the back. We also found that hypertrophic chondrocytes of the tibial growth plate exhibited abnormal morphology and nuclear consolidation or loss, accompanied by necrosis and excessive apoptosis under 2.0 mg/kg T-2 toxin exposure. Moreover, 2.0 mg/kg T-2 toxin exposure triggered erythropenia, thrombocytosis, alanine aminotransferase, and aspartate aminotransferase activity, as well as high blood urea nitrogen, uric acid, and lactic dehydrogenase levels. Collectively, these data indicate that T-2 toxin had an adverse effect on the growth performance, feather quality, and tibia development, and caused liver and kidney damage and abnormal blood parameters in Yangzhou goslings, providing crucial information toward the prevention and control of T-2 toxin contamination in poultry feed.

Comparative Characterization of Key Volatile Compounds in Slow- and Fast-Growing Duck Raw Meat Based on Widely Targeted Metabolomics.

The volatile aroma compounds in raw duck meat strongly affect consumers' purchase decisions and they vary among breeds with different growth rates. In this study, slow-growing (SG) Liancheng White and fast-growing (FG) Cherry Valley ducks were selected, and their volatile compounds were characterized using electric nose and gas chromatography-mass spectrometry. Furthermore, a widely targeted metabolomics approach was used to investigate the metabolites associated with volatile compounds. The results showed that hexanal, nonanal, octanal, heptanal, and 2-pentylfuran were abundantly present in duck meat, regardless of the breed. The higher nonanal and octanal rates contributed to the fatty and fruity aroma in SG meat than FG meat, while FG meat had a mushroom note resulting from higher octenol. Furthermore, widely targeted metabolomics showed a lower carnitine content in SG meat, which might promote lipid deposition to produce more octanal and nonanal. Higher sugar and amino acid contents led to a meaty aroma, whereas more trimethylamine N-oxide may generate a fishy note in SG meat. Taken together, this study characterized the raw duck meat aroma and provided the basic mechanism of the formation of the key volatile compound.

Effect of different free-range systems on the growth performance, carcass traits, and meat quality of Yangzhou geese.

This study was conducted to investigate the effects of different free-range systems on the growth performance, carcass traits, and meat quality of geese. Grass pasture zones in the study area were selected, and 28 d-old male Yangzhou geese with similar body weights (1.57 ± 0.12 kg) were randomly allocated to one of three conditions: (A) free-range conditions in the apron area during 9:00 a.m.-4:00 p.m. (10-20 m from shed with grass pasture); (B) free-range conditions in the outer range from 9:00 a.m. to 4:00 p.m. (beyond 50 m from shed with grass pasture); and (C) barn system. Free range-reared geese had higher weight gain after 42 days of age than barn-reared geese, regardless of the range area. A lower feed conversion ratio was found in outer range-reared and apron area-reared geese from 28 to 63 days of age. In addition, the highest percentages of leg and breast muscle weights were observed in outer range-reared and apron area-reared geese, respectively. Finally, outer-range rearing resulted in a lower pH and lower moisture content. Therefore, these data suggest that the outer range system benefits growth performance and feed conversion ratio of geese and results in a higher percentage of leg muscle weight, lower pH, and lower moisture content.

Probing the Conductive and Tribological Behaviors of Solid Additives in Multiply Alkylated Cyclopentanes for Sliding Electrical Contact.

Sliding electrical contacts need to be lubricated by conductive lubricants to perform low energy dissipation, high reliability, and long service life. This work studied the thermal stability, anti-corrosion capacity, and conductive, and tribological behaviors of several solid additives in multiply alkylated cyclopentanes (MACs), including carbon nanotubes (CNTs), multilayer graphene (MG), and silver microparticles. The results showed that all the additives possessed favorable thermal stability and corrosion resistance; in particular, CNTs and MG exhibited lower and more stable electrical contact resistance (ECR) and better lubricity abilities than Ag microparticles. Moreover, based on the characterization of the worn surfaces and the film thickness calculation, the favorable conductive and tribological properties of CNTs and MG were related to the high conductivity and specific structure of the additives and the good chemical inertness of MACs.

Effects of Chicken Serum Metabolite Treatment on the Blood Glucose Control and Inflammatory Response in Streptozotocin-Induced Type 2 Diabetes Mellitus Rats.

Chickens can live healthy without adverse effects despite high blood glucose levels. However, the blood biomolecules responsible for maintaining chronic hyperglycemia are unknown. Here, the effects of chicken serum metabolite treatment on blood glucose control and inflammatory response in streptozotocin (STZ)-induced Type 2 Diabetes Mellitus (T2DM) rats were investigated. First, chicken serum treatment reduced the advanced glycation end-products (AGEs) and blood glucose levels in STZ-induced T2DM rats. Second, insulin/glucose-induced acute hypoglycemic/hyperglycemic chickens and the blood biomolecules were screened via nontargeted ultra-performance liquid chromatography with mass spectroscopy (UPLC-MS), identifying 366 key metabolites, including DL-arginine and taurine, as potential markers for chronic hyperglycemia in chickens. Finally, DL-arginine functions for blood glucose control and inflammatory response were evaluated. We found that DL-arginine reduced the levels of blood glucose and AGEs in STZ-induced T2DM rats. In addition, DL-arginine treatment upregulated the glucose transporter type 4 (GLUT4) expression in the muscles and downregulated the advanced glycation end products receptor-1 (AGER1) expression in the liver and nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) expression in the pancreas and thymus tissues. Overall, these results demonstrate that serum metabolite of DL-arginine could maintain blood glucose homeostasis and suppress the inflammatory response in chickens. Therefore, DL-arginine may be a novel target for developing therapeutic agents to regulate hyperglycemia.

Comparative phosphoproteomic provides insights into meat quality differences between slow- and fast-growing broilers.

The selection of broilers for augmented growth rate and breast yield has been accompanied by deterioration in meat quality. To characterise the meat quality differences between slow- (SG) and fast-growing broilers (FG), Xueshan and Ross 308 chickens were employed to determine the mechanisms causing these differences. SG meat was found to display more redness and yellowness, higher shear force, pH24h, and protein content, with lower intramuscular fat (IMF) content than FG meat. Further, based on comparative phosphoproteomic analysis (SG/FG), upregulated phosphorylated myofibrillar proteins resulted in larger fibres, which contributed to lower pressing loss and tenderness. The phosphoproteins of glycolytic enzymes, phosphorylase kinases, and calcium-related proteins were significantly downregulated, which reduced the acidity of the meat. SLC7A5 at Ser21, MRC2 at Ser1359 and CRAT at Ser341, AUP1 at Ser377 positively affected protein and IMF deposition, respectively. Together, these phosphoproteins elicit vital information for the genetic improvement of chicken meat quality.

Effect of marketable age on proximate composition and nutritional profile of breast meat from Cherry Valley broiler ducks.

Marketable age is an important determinant of meat quality. Cherry Valley duck (SM3 medium) is the most efficient Pekin-type duck and is the most widely farmed breed globally. However, whether marketable age determines the meat quality of Cherry Valley ducks is not well documented. The objective of this study was to investigate the effect of marketable age on the proximate composition and nutritional profile of breast meat from Cherry Valley broiler ducks. Ducks at 28, 38, 42, and 45 days old were selected and slaughtered, and their proximate composition, cholesterol and essential mineral compositions, and amino acids and fatty acid profile of breast meat lipid were determined. The results showed higher protein content and lower intramuscular fat content were observed in the 38-day-old ducks than in the 28-day-old birds (P < 0.05). Additionally, 38-day-old ducks contained higher Fe and Mg contents (P < 0.05), whereas 28-day-old birds had higher Zn and Ca contents (P < 0.05). The essential amino acid content in 38-day-old was about 95.29 g/kg, higher than that in 28-day-old birds (P < 0.05). The contents of C20:5 n-3, omega-6 polyunsaturated fatty acids, and polyunsaturated fatty acids were the highest in 38-day-old birds (P < 0.05), whereas the content of C20:4 n-6, DHA (C22:6 n-3), and saturated fatty acids in 28-day-old birds was the lowest (P < 0.05). Finally, a comprehensive evaluation model of multiple traits was developed by applying principal component analysis, and the meat nutrition of 38-day-old ducks was identified as the optimal. Taken together, the meat of 38-day-old ducks had an advantage in proximate composition, minerals content, essential amino acids, and fatty acids, and 38 d might be recommended as an appropriate marketable age to provide duck meat of high nutrition value.

Dual Transcriptomic Analyses Unveil Host-Pathogen Interactions Between Salmonella enterica Serovar Enteritidis and Laying Ducks (Anas platyrhynchos).

Salmonella enteritidis (SE) is a pathogen that can readily infect ovarian tissues and colonize the granulosa cell layer such that it can be transmitted via eggs from infected poultry to humans in whom it can cause food poisoning. Ducks are an important egg-laying species that are susceptible to SE infection, yet the host-pathogen interactions between SE and ducks have not been thoroughly studied to date. Herein, we performed dual RNA-sequencing analyses of these two organisms in a time-resolved infection model of duck granulosa cells (dGCs) by SE. In total, 10,510 genes were significantly differentially expressed in host dGCs, and 265 genes were differentially expressed in SE over the course of infection. These differentially expressed genes (DEGs) of dGCs were enriched in the cytokine-cytokine receptor interaction pathway via KEGG analyses, and the DEGs in SE were enriched in the two-component system, bacterial secretion system, and metabolism of pathogen factors pathways as determined. A subsequent weighted gene co-expression network analysis revealed that the cytokine-cytokine receptor interaction pathway is mostly enriched at 6 h post-infection (hpi). Moreover, a number of pathogenic factors identified in the pathogen-host interaction database (PHI-base) are upregulated in SE, including genes encoding the pathogenicity island/component, type III secretion, and regulators of systemic infection. Furthermore, an intracellular network associated with the regulation of SE infection in ducks was constructed, and 16 cytokine response-related dGCs DEGs (including IL15, CD40, and CCR7) and 17 pathogenesis-related factors (including sseL, ompR, and fliC) were identified, respectively. Overall, these results not only offer new insights into the mechanisms underlying host-pathogen interactions between SE and ducks, but they may also aid in the selection of potential targets for antimicrobial drug development.

Identification of Differentially Expressed Non-coding RNA Networks With Potential Immunoregulatory Roles During Salmonella Enteritidis Infection in Ducks.

Salmonella enterica serovar Enteritidis (S. Enteritidis) is a pathogen that can colonize the preovulatory follicles of poultry, thereby causing both reduced egg production and an elevated risk of foodborne salmonellosis in humans. Although a few studies have revealed S. Enteritidis preferentially invades the granulosa cell layer within these follicles, it can readily persist and proliferate through mechanisms that are not well-understood. In this study, we characterized competing endogenous RNA (ceRNA) regulatory networks within duck granulosa cells following time-course of S. Enteritidis challenge. The 8108 long non-coding RNAs (lncRNAs), 1545 circular RNAs (circRNAs), 542 microRNAs (miRNAs), and 4137 mRNAs (fold change ≥2; P < 0.01) were differentially expressed during S. Enteritidis challenge. Also, eight mRNAs, eight lncRNAs and five circRNAs were selected and the consistent expression trend was found between qRT-PCR detection and RNA-seq. Moreover, the target genes of these differentially expressed ncRNAs (including lncRNAs, circRNAs and miRNAs) were predicted, and significantly enriched in the innate immune response and steroidogenesis pathways. Then, the colocalization and coexpression analyses were conducted to investigate relationships between ncRNAs and mRNAs. The 16 differentially expressed miRNAs targeting 60 differentially expressed mRNAs were identified in granulosa cells at 3 and 6 h post-infection (hpi) and enriched in the MAPK, GnRH, cytokine-cytokine receptor interaction, Toll-like receptor, endocytosis, and oxidative phosphorylation signaling pathways. Additionally, underlying lncRNA-miRNA-mRNA and circRNA-miRNA-mRNA ceRNA networks were then constructed to further understand their interaction during S. Enteritidis infection. Lnc_012227 and novel_circ_0004892 were identified as ceRNAs, which could compete with miR-let-7g-5p and thereby indirectly modulating map3k8 expression to control S. Enteritidis infection. Together, our data thus identified promising candidate ncRNAs responsible for regulating S. Enteritidis infection in the preovulatory follicles of ducks, offering new insights regarding the ovarian transmission of this pathogen.

Quantitative phosphoproteomic analysis unveil the effect of marketable ages on meat quality in geese.

The quality of poultry goose meat is closely related to its marketable ages, with meat quality varying with increasing marketable age. Geese of two marketable ages (70-day and 120-day) were selected to understand the mechanisms behind this effect. Darker and redder meat; chewier and higher water-holding capacity (WHC) as well as greater protein and intramuscular fat (IMF) content were observed in the breast muscle (BM) of 120-day-old geese as compared to 70-day-old geese. Quantitative phosphoproteomics revealed up-regulated phosphorylated myofibrillar proteins and glycolytic enzymes in 120BM contributed to chewier meat with higher WHC. Redder meat might be attributed to phosphorylated mitochondrial proteins interacting with glycolytic enzymes in energy metabolism. Additionally, phosphorylation of PLIN1 and PERM1 might positively affect IMF deposition. Taken together, these data provided a phosphoproteomics perspective for the effect of marketable ages on meat quality and a theoretical strategy for improving meat quality in geese of younger marketable age.

The specificity protein 3 (SP3) gene in ducks (Anas platyrhynchos): cloning, characterization and expression during viral infection.

Specificity Protein 3 (SP3) is a newly identified regulator of tumor growth and invasiveness in humans. In this study, we identified and characterized the function of duck SP3 (duSP3). The full-length cDNA sequence of the duSP3 gene was cloned via rapid amplification of cDNA ends. It contained 2468 nucleotides, including a 111 base pair (bp) 5'-untranslated region (UTR), 215 bp 3'-UTR, and 2142 bp open reading frame (ORF), which encoded a 713 amino acid (AA) strongly conserved with Avian SP3. Tissue specificity analysis demonstrated that duSP3 was constitutively expressed in the eight tissues tested: liver, spleen, lung, heart, kidney, thymus, breast, and leg; and low expression levels were observed in all tissues, except the spleen and thymus. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis revealed that duSP3 expression rapidly increased in vitro after stimulation with both the hepatitis virus (DHV-1) and polyriboinosinic polyribocytidylic acid (poly(I:C)). However, the expression under these treatments varied in kidney and liver tissues; in the liver, duSP3 increased significantly at 36 h after the DHV-1 treatment and peaked at 72 h after poly(I:C) stimulation. These results suggested that SP3 may play a positive role in immune responses against viral infections in ducks.

Genome-wide identification for genes involved in sodium dodecyl sulfate toxicity in Saccharomyces cerevisiae.

BACKGROUND: Sodium dodecyl sulfate (SDS) is one of the most widely used anionic alkyl sulfate surfactants. Toxicological information on SDS is accumulating, however, mechanisms of SDS toxicity regulation remain poorly understood. In this study, the relationship between the SDS-sensitive mutants and their intracellular ROS levels has been investigated. RESULTS: Through a genome-scale screen, we have identified 108 yeast single-gene deletion mutants that are sensitive to 0.03% SDS. These genes were predominantly related to the cellular processes of metabolism, cell cycle and DNA processing, cellular transport, transport facilities and transport routes, transcription and the protein with binding function or cofactor requirement (structural or catalytic). Measurement of the intracellular ROS (reactive oxygen species) levels of these SDS-sensitive mutants showed that about 79% of SDS-sensitive mutants accumulated significantly higher intracellular ROS levels than the wild-type cells under SDS stress. Moreover, SDS could generate oxidative damage and up-regulate several antioxidant defenses genes, and some of the SDS-sensitive genes were involved in this process. CONCLUSION: This study provides insight on yeast genes involved in SDS tolerance and the elevated intracellular ROS caused by SDS stress, which is a potential way to understand the detoxification mechanisms of SDS by yeast cells.

Intron polymorphisms of MAGI-1 and ACSF2 and effects on their expression in different goose breeds.

The goose is one of the most important waterfowl, having lowing laying rate. Previous studies have shown the SNPs in the introns of MAGI-1 (Record-106975) and ACSF2 (Record-106582) significantly associated with egg production in geese. However, the mechanism of those SNPs influencing egg production remains unclear. In this study, the three goose breeds (Yangzhou geese, Zhedong white geese, and Carlos geese) with obviously different egg production were selected, and the allele frequency distribution and functions of those SNPs were investigated. The results suggested that the allele frequency distribution of ACSF2 was significantly different among the three goose breeds (χc2 = 92.377, Pc = 2.29 × 10-22), with the C allele appearing at frequencies of 0.29 in the Yangzhou geese and 0.94 in the Carlos geese. In contrast, the allele frequencies of MAGI-1 were not significantly different among the different goose breeds. Quantitative Reverse Transcription PCR (qRT-PCR) showed that the expression of MAGI-1 with the AG genotype individuals was significantly higher than those of the AA and GG genotype. For ACSF2, the CC genotype had significantly higher expression than both the AC genotype and the AA genotype. The luciferase reporter analysis revealed that the site-directed mutation ACSF2 (A>C) significantly drove the expression activity. Further analysis suggested that the mutation altered the binding site of the transcription factor BARHL2. Binding of BARHL2 to the ACSF2 intron was confirmed by electrophoretic mobility shift assay (EMSA) analysis. Thus, our findings revealed the A>C mutation of ACSF2 (Record-106582) could promote the expression by regulating the binding of BARHL2, resulting in differences in egg performance, which provided molecular insights into the effect of the polymorphism in ACSF2 on egg performance in geese.

Roles of miRNA-1 and miRNA-133 in the proliferation and differentiation of myoblasts in duck skeletal muscle.

MicroRNA (miRNA)-1 and miRNA-133 are derived from the same bicistronic pairs with roles in skeletal muscle development. Many investigations have focused on the role of miRNA-1 and miRNA-133 in the regulation of skeletal muscle development in mammals and fish. However, the mechanisms of miRNA-1 and miRNA-133 underlying the differences in skeletal muscle development between different breeds are not well known. Our study found that the weights of body and breast at 42 days of age were greater in Cherry Valley ducks than in Putian ducks and the areas of breast muscle fibers increased with age; the areas of muscle fibers of Cherry Valley ducks were always greater than those of Putian ducks. Besides, quantitative reverse-transcriptase polymerase chain reaction analysis revealed that relatively high levels of miRNA-1 and miRNA-133 were detected in heart, breast, and leg muscles compared with the liver, spleen, lung, kidney, and the expression levels of miRNA-1 and miRNA-133 remained stable in the embryo stage, and in the growth period, the fluctuation in miRNA expression levels in Putian ducks was considerably higher than that in Cherry Valley ducks, especially from 7 to 28 days. However, in the late growth period, the expression of miRNA-1 and miRNA-133 of Cherry Valley duck was higher than that of Putian duck, which may indicate that miRNA-1 and miRNA-133 play a more important role during the growth period. To determine the function of miRNA-1 and miRNA-133 in skeletal muscle development, we found that the overexpression of miRNA-1, but not miRNA-133, promoted fusion of adjacent myoblasts. By contrast, a repressor of miRNA-1 promoted, whereas a miRNA-133 inhibitor inhibited, myoblast proliferation. Accordingly, the expression levels of myocyte enhancer factor 2D (MEF2D) and myogenic differentiation ( MYOD) were significantly increased by an miRNA-1 mimic and the miRNA-133 inhibitor. In addition, we found that the expression levels of miRNA-1 significantly affected the expression of histone deacetylase 4 ( HDAC4), and miRNA-133 affected serum response factor ( SRF) and transforming growth factor ß receptor 1 ( TGFBR1) levels. However, dual-luciferase reporter assays revealed that only miRNA-1 directly inhibited pGL- HDAC4 luciferase reporter activity, whereas miRNA-133 did not affect pGL- SRF or pGL- TGFBR1 fluorescence activity. Taken together, these results suggest that miRNA-1 targets HDAC4 to promote the differentiation of duck myoblasts and miRNA-133 may affect SRF and TGFBR1 expression to promote proliferation, which indicates that miRNA-1 and miRNA-133 play different important roles in skeletal muscle development.

FTH1 expression is affected by promoter polymorphism and not DNA methylation in response to DHV-1 challenge in duck.

Ferritin heavy polypeptide 1 (FTH1) plays a pivotal role in response to viral infections. FTH1 expression is modulated by various pathogens, but the regulatory mechanisms are unknown. We firstly construct duck hepatitis virus 1 (DHV-1) infection model, including morbid ducklings, non-morbid ducklings and control ducklings. Then the mRNA expression of duck FTH1 (duFTH1) was measured mRNA expression of duck FTH1 (duFTH1) in the liver and spleen after duck hepatitis virus 1 (DHV-1) infection using quantitative polymerase chain reaction (qPCR) and found that duFTH1 mRNA was down-regulated significantly in morbid ducklings (liver, P < 0.01; spleen, P < 0.05) compared with the control ducklings. We also found that duFTH1 expression was significantly higher in the spleen (P < 0.01) and liver (P < 0.05) of non-morbid ducklings than in morbid ducklings. Moreover, DNA methylation of the duFTH1 promoter was examined by bisulfite sequencing (BSP) and we found that the duFTH1 promoter was hypomethylated, the relative methylation was only 5.9% and 2.0% in the morbid ducklings and non-morbid ducklings, respectively. The promoter contained a -55 C/T mutation in 75% of non-morbid ducklings, and this polymorphism affected promoter activity. Further analysis suggested that this mutation altered the binding site of the transcription factor NRF1. Binding of NRF1 to the FTH1 promoter was confirmed by electrophoretic mobility shift assay (EMSA) analysis. Thus, our findings revealed the NRF1 was a negative regulator, and lossed of binding of NRF1 to duFTH1 promoter due to -55C/T mutation enhances duFTH1 expression in non-morbid ducks, which provided molecular insights into the effect of duFTH1 expression via promoter polymorphisms, but not DNA methylation, in response to DHV-1 challenge.