Developmental Changes of Duckling Liver and Isolation of Primary Hepatocytes.

The liver is the main site of fat synthesis and plays an important role in the study of fat deposition in poultry. In this study, we investigated the developmental changes of duckling livers and isolated primary duck hepatocytes. Firstly, we observed morphological changes in duckling livers from the embryonic period to the first week after hatching. Liver weight increased with age. Hematoxylin-eosin and Oil Red O staining analyses showed that hepatic lipids increased gradually during the embryonic period and declined post-hatching. Liver samples were collected from 21-day-old duck embryos for hepatocyte isolation. The hepatocytes showed limited self-renewal and proliferative ability and were maintained in culture for up to 7 days. Typical parenchymal morphology, with a characteristic polygonal shape, appeared after two days of culture. Periodic acid-Schiff (PAS) staining analysis confirmed the characteristics of duck embryo hepatocytes. PCR analysis showed that these cells from duck embryos expressed the liver cell markers ALB and CD36. Immunohistochemical staining and immunofluorescence analysis also confirmed ALB and CK18 expression. Our findings provide a novel insight regarding in vitro cell culture and the characteristics of hepatocytes from avian species, which could enable further studies concerning specific research on duck lipid metabolism.

Effects of linseed oil supplementation duration on fatty acid profile and fatty acid metabolism-related genes in the muscles of Chinese crested white ducks.

Meat rich in polyunsaturated fatty acids is considered beneficial to health. Supplementing the diet with linseed oil promotes the deposition of polyunsaturated fatty acids (PUFAs) in poultry, a conclusion that has been confirmed multiple times in chicken meat. However, fewer studies have focused on the effects of dietary fatty acids on duck meat. Therefore, this study aims to evaluate the effects of the feeding time of a linseed oil diet on duck meat performance and gene expression, including meat quality performance, plasma biochemical indicators, fatty acid profile, and gene expression. For this study, we selected 168 Chinese crested ducks at 28 days old and divided them into three groups, with 56 birds in each group. The linseed oil content in the different treatment groups was as follows: the control group (0% flaxseed oil), the 14d group (2% linseed oil), and the 28d group (2% linseed oil). Ducks in the two experimental groups were fed a linseed oil diet for 28 and 14 days at 28 and 42 days of age, respectively. The results showed that linseed oil had no negative effect on duck performance (slaughter rate, breast muscle weight, and leg muscle weight) or meat quality performance (pH, meat color, drip loss, and shear force) (P > 0.05). The addition of linseed oil in the diet increased plasma total cholesterol and high-density lipoprotein cholesterol levels (P < 0.05), while decreasing triglyceride content (P < 0.05). Furthermore, the supplementation of linseed oil for four weeks affected the composition of muscle fatty acids. Specifically, levels of α-linolenic acid, eicosapentaenoic acid, and docosahexaenoic acid were increased (P < 0.05), while eicosatetraenoic acid content was negatively correlated with flaxseed oil intake (P < 0.05). qRT-PCR analysis further revealed that the expression of FATP1, FABP5, and ELOVL5 genes in the breast muscle, as well as FABP3 and FADS2 genes in the thigh muscle, increased after four weeks of linseed oil supplementation (P < 0.05). However, after two weeks of feeding, CPT1A gene expression inhibited fatty acid deposition, suggesting an increase in fatty acid oxidation (P < 0.05). Overall, the four-week feeding time may be a key factor in promoting the deposition of n-3 PUFAs in duck meat. However, the limitation of this study is that it remains unknown whether longer supplementation time will continue to affect the deposition of n-3 PUFAs. Further experiments are needed to explain how prolonged feeding of linseed oil will affect the meat quality traits and fatty acid profile of duck meat.

Association analysis of polymorphisms of candidate genes for laying traits in Yangzhou geese.

Egg production is an important economic trait in the Chinese goose industry. Due to the low heritability of annual egg production traits in geese, large-scale individual selection based on annual egg production measurements cannot be carried out. Therefore, new selection methods must be applied for large-scale early selections. To screen for effective molecular markers for early Yangzhou geese selection, the genotypes and gene frequencies of mutated loci of five candidate genes related to egg production, MAGI-1, ACSF2, ASTN2, KIAA1462, and ARHGAP21, were detected and analyzed by PCR-direct sequencing.Furthermore, correlation analysis was performed with annual egg mass and body weight at the point of lay and egg weight, and the results were as follows:Magi-1 (Record-106975)was A > G, ACSF2 (Record-106582)was A > C, ASTN2 (Record-111407)was A > T, KIAA1462 (Record-134172)was A > T, and the base of ARHGAP21 (Record-112359) was G > T. At all the five loci above, the Yangzhou geese population followed the Hardy-Weinberg equilibrium (P > 0.05). The results of the association analysis between different genotypes and production performance showed no significant differences in annual egg production, body weight at the point of lay, and egg weight, among different genotypes (P > 0.05) at the mutation loci of MAGI-1 and ASTN2. At the ACSF2 and KIAA1462, the annual egg production of AC was significantly higher than that of AA and CC (P < 0.05), the annual egg production of TT was significantly higher than that of AA (P < 0.05), and there were no significant differences in body weight at the point of lay and egg weight, among the three genotypes (P > 0.05). At ARHGAP21, the body weight at the lay point of the TT genotype was the highest, which was significantly higher than that of GG (P < 0.05); however, there was no significant difference with the heterozygous GT genotype for this trait (P > 0.05). Therefore, Genotype AC at ACSF2 and genotype TT at KIAA1462 could be used as favorable genotypes for egg production, and genotype TT at ARHGAP21 could be used as a favorable genotype for weight in Yangzhou geese.

Metabolomics and Proteomics Characterizing Hepatic Reactions to Dietary Linseed Oil in Duck.

The imbalance in polyunsaturated fatty acid (PUFA) composition in human food is ubiquitous and closely related to obesity and cardiovascular diseases. The development of n-3 PUFA-enriched poultry products is of great significance for optimizing fatty acid composition. This study aimed to improve our understanding of the effects of dietary linseed oil on hepatic metabolism using untargeted metabolomics and 4D label-free proteome analysis. A total of 91 metabolites and 63 proteins showed differences in abundance in duck livers between the high linseed oil and control groups. Pathway analysis revealed that the biosynthesis of unsaturated fatty acids, linoleic acid, glycerophospholipid, and pyrimidine metabolisms were significantly enriched in ducks fed with linseed oil. Meanwhile, dietary linseed oil changed liver fatty acid composition, which was reflected in the increase in the abundance of downstream metabolites, such as α-linolenic acid (ALA; 18:3n-3) as a substrate, including n-3 PUFA and its related glycerophospholipids, and a decrease in downstream n-6 PUFA synthesis using linoleic acid (LA; 18:2n-6) as a substrate. Moreover, the anabolism of PUFA in duck livers showed substrate-dependent effects, and the expression of related proteins in the process of fatty acid anabolism, such as FADS2, LPIN2, and PLA2G4A, were significantly regulated by linseed oil. Collectively, our work highlights the ALA substrate dependence during n-3 PUFA synthesis in duck livers. The present study expands our knowledge of the process products of PUFA metabolism and provides some potential biomarkers for liver health.

Dietary linseed oil affects the polyunsaturated fatty acid and transcriptome profiles in the livers and breast muscles of ducks.

Linseed oil, an important source of dietary α-linolenic acid, is used to provide meat enriched in n-3 PUFA. We investigated the effects of dietary linseed oil (0, 0.5, 1, and 2%) on growth performance, meat quality, tissue fatty acid (FA), and transcriptome profiles in ducks. The result showed that dietary linseed oil had no effect on growth performance. Increasing dietary linseed oil enrichment raised n-3 PUFA and linoleic acid (LA) levels in both the liver and breast muscle, but decreased dihomo-gamma-linolenic acid (DGLA) and arachidonic acid (ARA) levels in the liver. The liver n-3 PUFA content was negatively correlated with duck body weight. Transcriptome analysis showed that dietary linseed oil caused hepatic changes in genes (SCD, FADS1, FADS2, and ACOT6) related to the biosynthesis of unsaturated fatty acids. Besides, dietary linseed oil also affected the expression of genes related to PUFAs and downstream metabolites (such as linoleic acid, steroid hormone, progesterone, etc.) metabolic pathways in both liver and breast muscle. Key genes involved in PUFA synthesis and transport pathways were examined by RT-qPCR, and the results verified that hepatic expression levels of FADS1 and FADS2 decreased, and those of FABP4 and FABP5 increased when 2% linseed oil was added. CD36 expression level increased in breast muscle when 2% linseed oil was added. Thus, 2% dietary linseed oil supplementation produces n-3 PUFA-enriched duck products by regulating the PUFA metabolic pathways, which could be advantageous for health-conscious consumers.

MicroRNA analysis reveals the role of miR-214 in duck adipocyte differentiation.

OBJECTIVE: Fat deposition in poultry is an important factor in production performance and meat quality research. miRNAs also play important roles in regulating adipocyte differentiation process. This study was to investigate the expression patterns of miRNAs in duck adipocytes after differentiation and explore the role of miR-214 in regulating carnitine palmitoyltransferases 2 (CPT2) gene expression during duck adipocyte differentiation. METHODS: Successful systems for the isolation, culture, and induction of duck primary fat cells was developed in the experiment. Using Illumina next-generation sequencing, the miRNAs libraries of duck adipocytes were established. miRanda was used to predict differentially expressed (DE) miRNAs and their target genes. The expression patterns of miR-214 and CPT2 during the differentiation were verified by quantitative real-time polymerase chain reaction and western blot. Luciferase reporter assays were used to explore the specific regions of CPT2 targeted by miR-214. We used a miR-214 over-expression strategy in vitro to further investigate its effect on differentiation process and CPT2 gene transcription. RESULTS: There were 481 miRNAs identified in duck adipocytes, included 57 DE miRNA candidates. And the 1,046 targets genes of DE miRNAs were mainly involved in p53 signaling, FoxO signaling, and fatty acid metabolism pathways. miR-214 and CPT2 showed contrasting expression patterns before and after differentiation, and they were selected for further research. The expression of miR-214 was decreased during the first 3 days of duck adipocytes differentiation, and then increased, while the expression of CPT2 increased both in the transcriptional and protein level. The luciferase assay suggested that miR-214 targets the 3'untranslated region of CPT2. Overexpression of miR-214 not only promoted the formation of lipid droplets but also decreased the protein abundance of CPT2. CONCLUSION: Current study reports the expression profile of miRNAs in duck adipocytes differentiated for 4 days. And miR-214 has been proved to have the regulator potential for fat deposition in duck.

Identification, biogenesis and function prediction of novel circRNA during the chicken ALV-J infection.

Circular RNA (circRNA) is a new non-coding RNA with a highly conserved and stable covalently closed loop structure, and it plays an important role in a variety of biological processes and the occurrence of diseases. Based on the sequencing results, circRNA_3079 had the most significant difference between the infected group and normal group, up to about 8 times. It has attracted our attention and was selected for further verification and analysis. Though the characteristics analysis of circRNA_3079 in chicken, we found circRNA_3079 is a stable, circular transcript, which mainly exists in the cytoplasm. And it is widely expressed in various tissues of chickens, and highly expressed in lung, spleen, lymph and bursa of fabricius. Bioinformatics analysis results showed that circRNA_3079 and the predicted target genes are enriched in many pathways related to immunity or tumors, such as p53 signaling pathway, Jak-STAT signaling pathway and NOD-like receptor signaling pathway, which revealed that circRNA_3079 may indirectly regulate the ALV-J infection process through the regulation of target genes.HIGHLIGHTSCircRNA_3079 is an abundant and stable circular RNA expressed in different tissues and cells in chicken.The circularization of circRNA_3079 does not depend on the reverse complementary repetitive sequence of the flanking sequence.CircRNA_3079 may indirectly regulate the ALV-J infection process.

Effect of stocking density on performance, meat quality and cecal bacterial communities of yellow feather broilers.

Totally, 315 42-day-old male Xueshan chickens were allocated into 3 caging densities, 14, 21 and 28 birds/m2. Each treatment was represented by 5 replicates. The body weight (BW), slaughter performance, meat quality, behavioral assessment, and the cecal microorganisms were detected at the market age. The results showed that the BW of broilers in the low- and medium-density groups was significantly higher (p < 0.05) than that of the high-density group from the age of 10 weeks. Only the feather quality of the broilers in the low-density group improved significantly (p < 0.05) compared with those of the other two groups. And, the abdominal fat percentage and the fat content of thigh muscle of broilers in the low- and medium-density groups were higher (p < 0.05) than those in the high-density group. No significant difference (p > 0.05) was noted in the other traits. The abundance of some microbial like Akkermansiaceae, Lactobacillaceae and Faecalibacterium may be correlated with the BW and fat content of broilers. The findings of this study suggest that increasing the stocking density decreased the final BW, fat content and the feather quality, whereas no evidence was found that stocking density caused changes in other parameters.

Characterization of ovarian morphology and reproductive hormones in Zhedong white geese (Anser cygnoides domesticus) during the reproductive cycle.

Zhedong white goose (Anser cygnoides domesticus) is a native Chinese breed with strong broodiness and low egg production, which is related to the physiology of reproduction. However, thus far, the physiology of goose reproduction has not been well elucidated. In the present study, the ovarian morphology and reproductive hormones of Zhedong white geese were investigated during the reproductive cycle (the laying and brooding periods). The results showed that the surface of the ovary was atrophied and follicular atresia appeared to some extent in the brooding period compared with the laying period. The concentrations of follicle-stimulating hormone, progesterone and luteinizing hormone were significantly higher than those in the brooding period (p < 0.05). In contrast, the concentrations of prolactin (PRL) and anti-Müllerian hormone (AMH) in the laying period were significantly lower than those in the brooding period (p < 0.05). In addition, the mRNA expression levels of PRL, AMH, dopamine-ß-hydroxylase (DßH) and cytochrome P450 side-chain cleavage enzyme (P450scc) were detected in the hypothalamus, pituitary and ovaries by using real-time polymerase chain reaction. The results showed that AMH mRNA was expressed specifically in ovary tissue. The expression levels of DßH and PRL in the brooding period was significantly higher than those in the laying period in the three tissues, especially in the early and middle stages of the brooding period. Moreover, AMH mRNA expression in the ovaries presented the same trend. In addition, P450scc mRNA was highly expressed in both the ovary and pituitary in the laying period. These results revealed the remarkable features of ovarian morphology and characterized the hormonal pattern and expression profile during the reproductive cycle, all of which contribute to understanding the differences in reproductive physiology between the laying and brooding periods in Zhedong white geese.

Circular RNA expression profiling reveals that circ-PLXNA1 functions in duck adipocyte differentiation.

Adipocytes are derived from pluripotent mesenchymal stem cells through adipogenesis. Pre-adipocyte differentiation in poultry greatly influences fat deposition and meat quality. Circular RNAs (circRNAs) have an important function in cancer and some differentiation processes. Herein, high-throughput transcriptome sequencing was used to detect circRNAs present in cherry valley duck pre-adipocyte and adipocyte differentiation over 3 days. We identified 9,311 circRNAs and 141 differentially expressed circRNAs. Sequencing results were verified through qRT-PCR using seven randomly selected circRNAs, and competing endogenous RNA (ceRNA) networks were exhibited by ten important circRNAs in duck adipocyte differentiation. circRNA plexin A1 (circ-PLXNA1) was detected in duck adipocytes and mainly expressed in adipose, leg muscle and liver. Inhibition of circ-PLXNA1 limited the differentiation of duck adipocyte. There were four corresponding miRNAs for circ-PLXNA1 and 313 target genes for those miRNAs. CeRNA"circ-PLXNA1/miR-214/CTNNB1 axis" was focused and verified by a dual-luciferase reporter experiment. After co-transfection of cells with si-circ-PLXNA1 and miR-214 mimics, the expression level of CTNNB1 was down-regulated, triglyceride content and the adipogenic capacity of preadipocytes decreased. While there were no significant change after si-CTNNB1 transfection. All these results provide further insight into the circRNAs, especially for circ-PLXNA1 in duck adipocyte differentiation.

Marginal diversity analysis of conservation of Chinese domestic duck breeds.

The present study aimed to systematically evaluate the genetic diversity of Chinese domestic duck breeds and ensure the most effective allocation and usage of conservation funds. We first performed an analysis of DNA genetic distance in 21 duck breeds by measuring short tandem repeats. Then, we calculated the extinction probability, contribution rate, and marginal diversity for each breed. The results showed that the extinction rate of the Zhongshan duck, Guangxi duck, and Ji'an duck were the highest at 0.67, 0.59, and 0.59, respectively, and that of the Linwu duck, Jinding duck, and Gaoyou duck were the lowest at 0.15, 0.18, and 0.19, respectively. The current diversity of populations was 7.72 and the expected diversity in five hundred years is 5.14 ± 1.15. The marginal diversity of the Chinese Muscovy duck was the largest (-2.20), accounting for 42.61% of the expected diversity, followed by the Guangxi duck (-0.49, 9.44%), whereas the Jinding duck was the smallest (-0.12; 2.32%). The protection potency of the Chinese Muscovy duck was the largest (0.61), followed by Guangxi duck (0.29), whereas the Jinding duck was the smallest (0.02). This study provides a reference for determining the conservation priority of Chinese domestic duck breeds or genetic resources.

Expression patterns of novel circular RNAs in chicken cells after avian leukosis virus subgroup J infection.

Avian leukosis virus subgroup J (ALV-J) is an oncogenic retrovirus that causes severe economic losses to the poultry industry worldwide. Circular RNAs (circRNAs) are a class of non-coding RNAs that has been described in various biological systems and pathogenic processes. However, the immune mechanisms in response to circRNAs remain unknown. In this study, high-throughput transcriptome sequencing was used to detect circRNAs present in chicken macrophage (HD11) and chick embryo fibroblast (CEF) cells infected with ALV-J. We identified 7684 circRNAs from diverse genomic locations in CEF and HD11 after ALV-J infection, these RNAs showed complex expression patterns that differed based on the cells type and infection time. In total, 302 differentially expressed (DE) circRNAs and 164 DE circRNAs were identified in CEF and HD11 after ALV-J infection, respectively. CircRNA7419-associated with KDM4C- and circRNA6679 and circRNA6680-associated with TNFAIP6- were involved in the immune response upon ALV-J infection in CEF. Host genes were analyzed through further bioinformatics analysis. The result confirmed that a large number of DE circRNAs corresponded to several immune-associated or tumor-associated terms and pathways, such as Mucin type O-Glycan biosynthesis, MAPK signaling pathway, B cell receptor signaling, and Wnt signaling pathway in CEF, as well as Jak-STAT signaling pathway, apoptosis, and MAPK signaling pathway in HD11. CircRNAs related to the B cell receptor signaling pathway in CEF, and the Jak-STAT signaling pathway in HD11, were selected for circRNA-miRNA interaction network analyses. Our study indicates that circRNAs expression was altered by ALV-J infection in both CEF and HD11, and may play a key role in the progression of ALV-J infection.

Selectived and Reshaped Early Dominant Microbial Community in the Cecum With Similar Proportions and Better Homogenization and Species Diversity Due to Organic Acids as AGP Alternatives Mediate Their Effects on Broilers Growth.

Understanding the differences in microbial communities shaped by different food selective forces, especially during early post-hatch period, is critical to gain insight into how to select, evaluate, and improve antibiotic growth promoters (AGPs) alternatives in food animals. As a model system, commercial diet-administered OAs (DOAs) and water-administered OAs (WOAs) were used separately or in combination as Virginiamycin alternatives for broiler feeding during two growth phases: 1-21 days and 22-42 days. Among these three OA-treated groups, the DOA group was most similar to the AGP group in the composition and the proportion of these dominant bacterial communities at the level of phylum, family, and genus in cecal chyme of broilers. Sub-therapeutic Virginiamycin decreased the richness, homogenization, and species diversity of gut microbiota, especially in the early growth stage from days 1 to 21. Among these three OA supplementation schemes, it was clear that DOA supplementation was more likely to increase or maintain the richness, homogenization, species diversity, and predicted gene functions of cecal microbiota in treated broilers than either no supplementation or AGP supplementation during two experimental stages. The interference of DOA treatment with early colonization of probiotics and pathogens in broiler cecum was the most similar to AGP treatment, and OAs did not cause the occurrence of Virginiamycin-resistant strains of Enterococcus at the end of this trial. In terms of the predicted gene functions of the microbiota, AGP and DOA treatments provided a similar selective force for microbial metabolism functions in the cecum of broiler chickens, especially in the early growth stage. Noticeably, the relative abundance of some microbiome that was modified by Virginiamycin or DOA supplementation was significantly correlated with body weight gain and KEGG pathway analysis-annotated gene functions such as replication and repair, translation, nucleotide metabolism, and so on. With the comprehensive analysis of these results and practical application, shortened DOA supplementation, after optimization of the amount of addition, would be a suitable alternative to sub-therapeutic Virginiamycin. It was suggested that the programed intestinal microecology under such early selection forces and the effective addition time may be the key elements to focus on the designed alternate strategies of AGPs in food animals.

MicroRNA-122 targets genes related to goose fatty liver.

MicroRNA-122 (miR-122), a completely conserved, liver-specific miRNA in vertebrates, is essential for the maintenance of liver homeostasis. This 22-nucleotide-length RNA regulates diverse functions such as cholesterol, glucose, and lipid metabolism as well as iron homeostasis and infection of hepatitis C virus (HCV). Landes goose, which has a good, fatty liver, has important significance for us in studying miR-122 function in goose fatty liver. In the current study, we identified miR-122 in goose liver and its expression pattern and target genes. We found that miR-122 was highly expressed in goose liver and its expression was down-regulated after overfeeding; some genes related to lipid metabolism, including prolyl 4-hydroxylase subunit alpha 1 (P4HA1); aldolase, fructose-bisphosphate B (ALDOB); and pyruvate kinase, muscle (PKM2), were predicted and validated as target genes of goose miR-122. After overexpression or inhibition of miR-122 in primary goose hepatocytes, the expression of ALDOB and PKM2 was changed, but not that of P4HA1, indicating miR-122 regulates ALDOB and PKM2 expression at the mRNA level. These findings suggest miR-122 play important roles in goose fatty liver by targeting some of the genes related to lipid metabolism.

Expression of mitochondria-related genes is elevated in overfeeding-induced goose fatty liver.

Mitochondrion, the power house of the cell, is an important organelle involving in energy homeostasis. Change in mitochondrial mass and function may lead to metabolic disorders. Previous studies indicate that mitochondrial mass loss and dysfunction are associated with non-alcoholic fatty liver disease (NAFLD) in human and mouse. However, it is unclear whether mitochondrial genes are involved in the development of goose fatty liver. To address this, we determined the response of goose mitochondrial genes to overfeeding and other fatty liver-related factors (e.g., hyperinsulinemia, hyperglycemia, and hyperlipidemia). We first employed RNA-seq technology to determine the differentially expressed genes in the livers from normally-fed vs. overfed geese, followed by bioinformatics analysis and quantitative PCR validation. Data indicated that a majority of mitochondrial genes in the liver were induced by overfeeding. To understand how these genes are regulated in the context of fatty liver, we treated goose primary hepatocytes with high levels of glucose, fatty acids and insulin. The results indicated that these factors had an influence on the expression of some mitochondria related genes. Together, these findings suggest that the induction of mitochondrial gene expression by overfeeding is required for the development of goose fatty liver, and this induction is partially attributable to hyperglycemia, hyperlipidemia and hyperinsulinemia.

cDNA cloning and the response to overfeeding in the expression of stearoyl-CoA desaturase 1 gene in Landes goose.

Stearoyl-CoA desaturase 1 (SCD1) is a rate limiting enzyme in the biosynthesis of monounsaturated fatty acids. It has been cloned from several species: Rattus norvegicus, Mus musculus, Homo Sapiens and Gallus gallus, but not from Anser anser. This study was conducted to isolate the SCD1 cDNA sequence and investigate the effect of overfeeding on SCD1 gene tissue expression in Landes goose. The complete cDNA is 3294 bp in length, with an ORF of 1.083 bp encoding a predicted polypeptide of 360 amino acids and 5'/3'-UTR of 74 and 2137 bp, respectively. Quantitative real time PCR (qPCR) was used to examine SCD1 expression in heart, liver, spleen, lung, kidney, gizzard, glandular stomach, intestine, crureus, pectoral muscle, hypothalamus and adipose tissue (abdominal fat) in both the overfed and control group. SCD1 mRNA was highly expressed in goose fatty liver, and the expression levels of SCD1 in liver and fat of overfeeding group were more than double that of the control group. During the overfeeding period, SCD1 expression in liver and adipose tissue reached the highest level after 70 days, but declined at 79 days. In the control group, after fasting 24h, the expression level of SCD1 gene in tissues declined sharply. However, SCD1 gene expression in hypothalamus was unaffected. The results of this study provide a theoretical basis to study the relationship between SCD1 gene expression and the formation of fatty liver of Landes goose in response to overfeeding.