Parental betaine supplementation promotes gosling growth with epigenetic modulation of IGF gene family in the liver.

Betaine is widely used as a feed additive in the chicken industry to promote laying performance and growth performance, yet it is unknown whether betaine can be used in geese to improve the laying performance of goose breeders and the growth traits of offspring goslings. In this study, laying goose breeders at 39 wk of age were fed basal (Control, CON) or betaine-supplemented diets at low (2.5 g/kg, LBT) or high (5 g/kg, HBT) levels for 7 wk, and the breeder eggs laid in the last week were collected for incubation. Offspring goslings were examined at 35 and 63 d of age. The laying rate tended to be increased (P = 0.065), and the feed efficiency of the breeders was improved by betaine supplementation, while the average daily gain of the offspring goslings was significantly increased (P < 0.05). Concentrations of insulin-like growth factor 2 (IGF-2) in serum and liver were significantly increased in the HBT group (P < 0.05), with age-dependent alterations of serum T3 levels. Concurrently, hepatic mRNA expression of the IGF gene family was significantly increased in goslings derived from betaine-treated breeders (P < 0.05). A higher ratio of proliferating cell nuclear antigen (PCNA)-immunopositive nuclei was found in the liver sections of the HBT group, which was confirmed by significantly upregulated hepatic expression of PCNA mRNA and protein (P < 0.05). Moreover, hepatic expression of thyroxine deiodinase type 1 (Dio1) and thyroid hormone receptor ß (TRß) was also significantly upregulated in goslings of the HBT group (P < 0.05). These changes were associated with significantly higher levels of global DNA 5-mC methylation, together with increased expression of methyl transfer genes (P < 0.05), including betaine-homocysteine methyltransferase (BHMT), glycine N-methyltransferase (GNMT), and DNA (cytosine-5-)-methyltransferase 1 (DNMT1). The promoter regions of IGF-2 genes, as well as the predicted TRß binding site on the IGF-2 gene, were significantly hypomethylated (P < 0.05). These results indicate that gosling growth can be improved by dietary betaine supplementation in goose breeders via epigenetic modulation of the IGF gene family, especially IGF-2, in the liver.

The goose industry plays important roles in economics, cultures, and ecosystems, yet the low laying and growth rates of many indigenous breeds hinders the development of the goose farming. Betaine, an important methyl donor, is commonly used as a feed additive in livestock and poultry to enhance animal growth. Dietary supplementation of betaine in laying hens or gestational sows has been reported to promote the growth of their offspring. Here, we sought to investigate whether and how dietary betaine supplementation affects the growth and development of offspring goslings. In this study, goose breeders, both male and female, were fed a basal diet supplemented respectively with 0, 2.5, or 5 g/kg betaine for 7 wk. Goslings hatched from the breeder eggs of different groups were raised under the same standard condition for assessing the growth performance. Parental betaine increases the growth rate of offspring goslings with decreased DNA methylation on the IGF-2 gene promoter and increased expression of the IGF-2 gene in the liver. These results provide scientific evidence for the inter-generational effect of betaine on gosling growth.

Effects of essential oil mixtures on expression of genes involved in lipogenesis and fatty acid oxidation in geese (Anser anser).

The use of essential oils has recently increased in the poultry sector. The aim of this study was to investigate the effects of essential oil mixture (juniper, mint, oregano and rosemary oil) on fatty acid oxidation and lipogenic gene expression in geese. Research groups were formed as C (control; no additives), EK1 (0.4 ml/l essential oil mixture supplemented) and EK2 (0.8 ml/l essential oil mixture supplemented). Relative expression levels of genes included in lipogenesis (ACCα, ChREBP, FASN, LXRα and SREBP-1) expression levels of genes included in fatty acid oxidation (ACOX1, CPT1, CPT1A, PPARα and PPARγ) were measured using RT-qPCR. Group EK1 upregulates the mRNA expression levels of genes involved in lipogenesis such as ACCα, ChREBP and SREBP-1, while it downregulates the mRNA expression in levels of all genes involved in fatty acid oxidation. Group EK2 increases the mRNA expression levels of genes involved in lipogenesis such as ACCα, FASN and SREBP-1, while it decreased mRNA expression at the levels of all genes involved in fatty acid oxidation, as in the other group. In the study, adding an essential oil mixture to drinking water is predicted to increase fatty liver because it upregulates genes related to fat synthesis (lipogenesis) and downregulates genes related to fat degradation (fatty acid oxidation).

Reference gene selection for expression studies in the reproductive axis tissues of Magang geese at different reproductive stages under light treatment.

In quantitative PCR research, appropriate reference genes are key to determining accurate mRNA expression levels. In order to screen the reference genes suitable for detecting gene expression in tissues of the reproductive axis, a total of 420 (males and females = 1:5) 3-year-old Magang geese were selected and subjected to light treatment. The hypothalamus, pituitary and testicular tissues were subsequently collected at different stages. Ten genes including HPRT1, GAPDH, ACTB, LDHA, SDHA, B2M, TUBB4, TFRC, RPS2 and RPL4 were selected as candidate reference genes. The expression of these genes in goose reproductive axis tissues was detected by real-time fluorescent quantitative PCR. The ΔCT, geNorm, NormFinder and BestKeeper algorithms were applied to sort gene expression according to stability. The results showed that ACTB and TUBB4 were the most suitable reference genes for the hypothalamic tissue of Magang goose in the three breeding stages; HPRT1 and RPL4 for pituitary tissue; and HPRT1 and LDHA for testicular tissue. For all three reproductive axis tissues, ACTB was the most suitable reference gene, whereas the least stable reference gene was GAPDH. Altogether, these results can provide references for tissue expression studies in geese under light treatment.

De Novo Assembly and Comparative Transcriptome Profiling of Anser anser and Anser cygnoides Geese Species' Embryonic Skin Feather Follicles.

Geese feather production and the quality of downy feathers are additional economically important traits in the geese industry. However, little information is available about the molecular mechanisms fundamental to feather formation and the quality of feathers in geese. This study conducted de novo transcriptome sequencing analysis of two related geese species using the Illumina 4000 platform to determine the genes involved in embryonic skin feather follicle development. A total of 165,564,278 for Anser anser and 144,595,262 for Anser cygnoides clean reads were generated, which were further assembled into 77,134 unigenes with an average length of 906 base pairs in Anser anser and 66,041 unigenes with an average length of 922 base pairs in Anser cygnoides. To recognize the potential regulatory roles of differentially expressed genes (DEGs) during geese embryonic skin feather follicle development, the obtained unigenes were annotated to Gene Ontology (GO), Eukaryotic Orthologous Groups (KOG), and Kyoto Encyclopedia of Genes and Genomes (KEGG) for functional analysis. In both species, GO and KOG had shown similar distribution patterns during functional annotation except for KEGG, which showed significant variation in signaling enrichment. Anser asnser was significantly enriched in the calcium signaling pathway, whereas Anser cygnoides was significantly enriched with glycerolipid metabolism. Further analysis indicated that 14,227 gene families were conserved between the species, among which a total of 20,715 specific gene families were identified. Comparative RNA-Seq data analysis may reveal inclusive knowledge to assist in the identification of genetic regulators at a molecular level to improve feather quality production in geese and other poultry species.

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.

Transcriptome analysis of hepatic gene expression and DNA methylation in methionine- and betaine-supplemented geese (Anser cygnoides domesticus).

Dietary methionine (Met) restriction produces a coordinated series of transcriptional responses in the liver that limits growth performance and amino acid metabolism. Methyl donor supplementation with betaine (Bet) may protect against this disturbance and affect the molecular basis of gene regulation. However, a lack of genetic information remains an obstacle to understand the mechanisms underlying the relationship between Met and Bet supplementation and its effects on genetic mechanisms. The goal of this study was to identify the effects of dietary supplementation of Met and Bet on growth performance, transcriptomic gene expression, and epigenetic mechanisms in geese on a Met-deficient diet. One hundred and fifty 21-day-old healthy male Yangzhou geese of similar body weight were randomly distributed into 3 groups with 5 replicates per treatment and 10 geese per replicate: Met-deficient diet (Control), Control+1.2 g/kg of Met (Met), and Control+0.6 g/kg of Bet (Bet). All geese had free access to the diet and water throughout rearing. Our results indicated that supplementation of 1.2 g/kg of Met in Met-deficient feed increased growth performance and plasma homocysteine (HCY) levels, indicating increased transsulfuration flux in the liver. Supplementation of 0.6 g/kg Bet had no apparent sparing effect on Met needs for growth performance in growing geese. The expression of many genes critical for Met metabolism is increased in Met supplementation group. In the Bet-supplemented group, genes involved in energy production and conversion were up-regulated. Dietary supplementation with Bet and Met also altered DNA methylation. We observed changes in the methylation of the LOC106032502 promoter and corresponding changes in mRNA expression. In conclusion, Met and Bet supplementation in geese affects the transcriptional regulatory network and alters the hepatic DNA methylation of LOC106032502.

Molecular cloning and expression analysis of neuregulin 1 (Nrg1) in the hypothalamus of Huoyan goose during different stages of the egg-laying cycle.

Neuregulin 1 (Nrg1) is one of the most active members of the epidermal growth factor (EGF)-like family, which bind to the ErbB tyrosine kinase receptor and play many roles in modulation of synaptic activity, synaptogenesis, GABAergic neurotransmission, neurotransmitter receptor expression and the hormonal control of neuroendocrine reproductive development. In this study, we cloned and characterized the cDNA of goose Nrg1 originating from hypothalamus tissues of Huoyan goose using RACE method, investigated the mRNA expression profiles during different stages of the egg-laying cycle by real-time PCR. Multiple alignments and phylogenetic analyses of the deduced amino acid sequence were conducted using bioinformatics tools. We also determined the profiles of blood serum progesterone, estradiol, FSH and LH content during different egg-laying stages using radioimmunoassay. The cDNA of Nrg1 is consisted of 2061bp open reading frame encoding 686 amino acids. The deduced amino acid sequence of goose Nrg1 contains one EGF domain from amino acid residues 224 to 265 and shows a closer genetic relationship to the avian species than to other mammal species. The expression level of Nrg1 mRNA increased from the pre-laying period to the peak-laying period, reached its peak in the peak-laying period, and then decreased in the ceased period. The concentrations of FSH and estradiol in blood serum have the similar changing trend. These results might suggest a potential correlation between Nrg1/ErbB signaling network with the reproductive neuroendocrine of Huoyan goose.

Transcriptome profiling of the hypothalamus during prelaying and laying periods in Sichuan white geese (Anser cygnoides).

The Sichuan White goose is a Chinese breed well known for the quality of its meat. However, reproductive performance in this goose is not ideal, and little information is available regarding the abundance of transcripts. To better understand the molecular mechanism(s) underpinning prelaying and laying periods in the Sichuan white goose, high-throughput RNA sequencing was performed to analyze the transcriptome in the hypothalamus. After sequencing and annotation, 26,921 unigenes were obtained, with 48 transcripts up-regulated in the prelaying period and 180 transcripts up-regulated during the laying period. These transcripts were primarily related to diseases, cancers, signaling molecules and interactions, the nervous system and the immune system. Eight transcripts were selected for further analyses with quantitative reverse-transcription polymerase chain reaction (qRT-PCR). The qRT-PCR results were mostly consistent with those from the high-throughput RNA sequencing. Among these transcripts, serine/threonine-protein kinase (AMPK), heat shock protein 70 (HSP70) and NADH dehydrogenase 1 (ND1) were differentially expressed during the prelaying and laying periods. The results of this study provide a useful resource for future studies examining the hypothalamus in geese.

Comprehensive analysis of Sichuan white geese (Anser cygnoides) transcriptome.

High-throughput RNA sequencing was performed for comprehensively analyzing the transcriptome of geese. A total of 28,803,759 bp of raw sequence data was generated by 454 GS Flx+. After removal of adaptor sequences, 28,730,361 bp remained and 117,279 reads were obtained, with an average length of 244 bases. Simultaneously, complementary DNA samples from two different reproductive stages of goose ovarian, hypothalamus and pituitary tissue were sequenced separately using Illumina MiSeq platform. A total of 12 688 673 148 bp of raw sequence data were generated by Illumina MiSeq. After removal of adaptor sequences, 8 198 126 562 bp remained and 60 382 786 clean reads were obtained, with an average length of 135 bases. Assembly of all the reads from both 454 Flx+ and Illumina platforms formed 56,839 contigs. The sequence size ranges from 38 to 28,206 bp in size, with an average size of 2584 bp and an N50 of 4624. The assembly produced a substantial number of large contigs: 35,545 (62.5%) were longer than 1 kb, of which 8850 (15.6%) were longer than 5 kb. The sequencing depth was 85 X on average. We performed comprehensive function annotations on unigenes including protein sequence similarity, gene ontology (GO) term classification, and Kyoto Encylcopedia of Genes and Genomes (KEGG) pathway enrichment. GO analysis showed that approximately 63% of the contigs had annotation information, among the 35,953 annotated isotigs in Nr database, 24,783 (68.9%) sequences were assigned with one or more GO terms. There were 14,634 (40.7%) isotigs for biological processes, 10,557(29.3%) isotigs for cellular component, 22,607 (62.9%) isotigs for molecular function. The result of KEGG pathway mapping 8926 sequences had the pathway annotation, and took part in 477 pathways. Additionally, 10,685 simple sequence repeat (SSR) markers were identified from the assembled sequences. The most frequent repeat motifs were trinucleotides, which accounted for 53.03% of all SSRs, followed by dinucleotides (39.9%), tetranucleotides (5.08%), pentanucleotides (1.68%) and hexanucleotides (0.32%). Transcriptome sequencing on mixture issue of the geese yielded substantial transcriptional sequences and potentially useful SSR markers which provide an important data source for geese research.

Differential expression profiling of hypothalamus genes in laying period and ceased period Huoyan geese.

The hypothalamus plays a central role in controlling poultry reproductive activity. To increase our understanding of genes involved in egg laying of Huoyan geese, gene profiles in the hypothalamus of laying period and ceased period Huoyan geese were investigated using suppression subtractive hybridization (SSH) method. A total of 95 differentially expressed sequence tags (ESTs), including 46 up-regulated and 49 down-regulated sequences showed homology to known genes of the non-redundant NCBI databases. Bioinformatic analysis demonstrated that these genes were mainly involved in anatomical structure development, signal transduction, cellular nitrogen compound metabolic process, biosynthetic process, cellular protein modification process, cell differentiation, transport, cell adhesion, and reproduction. Ten ESTs were selected for further analyses by quantitative real-time RT-PCR (qRT-PCR). Whose most part of results were consistent with the SSH results. Of note, AdipoR2, Nrg1, and NCAM1, which related with secretion of GnRH and other hormones, were identified to be differentially expressed between laying period and ceased period. These findings provided a new source for mining genes related to higher laying performance of Huoyan geese, which facilitate our understanding of the reproductive biology of the goose.

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.

Effects of dietary tryptophan on protein metabolism and related gene expression in Yangzhou goslings under different feeding regimens.

This study was conducted to investigate the effects of feeding regimens and dietary Trp levels on protein metabolism and regulation of the related gene expression in Yangzhou goslings. A 2 × 3 factorial completely randomized experiment was applied, and the treatments were designed as 2 feeding regimens (ad libitum vs. restricted feeding), and each contained 3 levels of Trp (low-Trp group, 0.14%; medium-Trp group, 0.22%; high-Trp group, 0.30%). The results show that ADG and feed conversion ratio (FCR) were significantly affected by feeding regimens (P < 0.05); dietary Trp levels influenced ADG and ADFI in the starter and overall period (P < 0.05), and interactions between Trp levels and feeding regimens on ADG, ADFI, and FCR were observed in different growing periods (P < 0.05). Serum total protein, triglycerides, and total cholesterol levels in the ad libitum group were higher than those in the restricted feeding group (P < 0.05), and the concentration of serum total protein, glucose, and insulin-like growth factor-I were higher in the medium-Trp and high-Trp groups (P < 0.05); however, serum uric acid, triglyceride, and cortisol levels were reduced in the high-Trp group (P < 0.05). Feeding regimen and dietary Trp levels affected serum glucose (P < 0.05) interactively. In the ad libitum group, tryptophanyl tRNA synthetase (TTS) mRNA expressed at a higher level in the high-Trp treatment, whereas expression of poultry target of rapamycin (pTOR) and p70 ribosomal protein S6 kinase1 (S6K1) mRNA was upregulated in the low-Trp treatment (P < 0.05). Expression and phosphorylation levels of pTOR were upregulated in thigh tissue with increased dietary Trp, but cathepsin B and 20S protease mRNA expression decreased (P < 0.05). It was concluded that the protein deposition in gosling thigh tissue was affected by dietary Trp through positive regulation of the TTS mRNA and pTOR protein expression and phosphorylation levels for protein synthesis, as well as the suppression of protein degradation-related gene expression.

The phytoestrogen daidzein may affect reproductive performance of Zhedong White geese by regulating gene mRNA levels in the HPG axis.

1. The effect of daidzein, a naturally occurring phytoestrogen, on the reproductive performance of 120 female Zhedong White geese was determined. The geese were divided into 4 groups which were fed on diets containing 0 (Control), 10 (Da1), 20 (Da2) and 30 (Da3) mg daidzein per kg diet. Egg production and weight, fertility and hatchability rates, concentrations of estradiol (E2), triiodothyronine (T3), progesterone (P4), thyroxine (T4) and growth hormone (GH) in serum, and mRNA levels of gonadotropin releasing hormone (GnRH), ß-follicle stimulating hormone (FSHß), follicle stimulating hormone receptor (FSHR), oestrogen receptor1 (ESR1), oestrogen receptor2 (ESR2), prolactin (PRL), prolactin receptor (PRLR), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the hypothalamus-pituitary-gonadal axis (HPGA) were measured. 2. Daidzein increased egg weight and fertility but had no detectable effect on egg production and hatchability. 3. Daidzein affected serum P4 and GH concentrations and T4 rhythm, up-regulated GnRH mRNA and PRLR mRNA levels in the hypothalamus, down-regulated PRLR mRNA in the hypothalamus, PRL mRNA in the pituitary, and ESR2 mRNA levels in the ovary, respectively. The mRNA rhythms of PRLR in the hypothalamus, PRL, PRLR and FSHß in the pituitary, FSHR, ESR1 and ESR2 in the ovary were significantly changed in the Da2 group. 4. It is suggested that an appropriate dose of daidzein might improve reproductive performance by affecting serum hormone concentrations and rhythms and regulating gene mRNA levels in the HPGA of female Zhedong White geese.