Insulin-like growth factor binding protein-2 (IGFBP-2) regulates a broad spectrum of biological activities involved in growth, development, and differentiation. This study aimed at comparing polymorphisms in intron2 of the IGFBP-2 gene among four chicken breeds and at analyzing the associations between its genotypes and body weight in Jinghai Yellow chicken by using PCR-SSCP technique. For primer P2, three genotypes (AA, AB and BB) were observed in the four chicken breeds. Gene sequencing revealed one insertion/deletion (the inserted/deleted TC after position 552bp) in the intron 2 of IGFBP-2 gene. For primer P5, three genotypes were identified in Jinghai Yellow chickens, and named CC, CD and DD. Gene sequencing revealed two SNPs (C1107G, C1130T) and one inserted/deleted GCCAGGT after 1115bp in the intron 2 of IGFBP-2 gene. The results of the linear model analysis showed that Jinghai Yellow chickens with AA genotype had significantly heavier body weight, at hatch and 12 weeks of age, than those of the AB genotype (p<0.05). The A allele had a positive effect on body weight. We speculate that mutations in intron 2 could be used as genetic markers for body weight in Jinghai Yellow chicken. This study provides valuable information for the protection of genetic resources and for breeding of Jinghai Yellow chicken.

The aim of this study was to investigate the effects of Myostatin (MSTN) and MyoGenin (MyoG) on goose skeletal muscle growth. In this study, MSTN and MyoG gene expression in breast and leg muscle of Zi and Rhine goose were detected by Real-time Polymerase Chain Reaction (PCR), and the correlations between genes expression levels and carcass traits were investigated. The results showed that the breast muscle weight and breast muscle percentage of Rhine goose were significantly higher than Zi goose (p<0.01). MSTN mRNA and MyoG mRNA expression in breast muscle of Zi goose were significantly higher than that of Rhine goose and the level of MSTN in leg muscle of Rhine was significantly higher than that of Zi goose (p<0.01). There was a significant difference between MSTN mRNA expression in breast muscle and in leg muscle of Zi goose (p<0.01). MSTN mRNA expression in leg muscle was significantly higher than that of breast muscle of Rhine goose (p<0.05). There was a significant difference between MyoG mRNA expression in breast muscle and in leg muscle of Zi goose and Rhine goose (p<0.01). There was a negative correlation between MSTN mRNA expression in breast muscle and body weight, breast muscle weight and breast muscle percentage.

Egg production in chickens is a quantitative trait. The aim of this study was to investigate the effect of promoter methylation of the Zona pellucida 2 (ZP2) gene on egg production. Real-time fluorescence quantification showed that the expression of the ZP2 gene in the ovaries of 300-day-old Jinghai yellow chickens in the high-laying group was significantly higher than that in the low-laying group (p < 0.01). A series of deletion fragments of the ZP2 gene promoter in Jinghai yellow chickens had different promoter activities in DF-1 cells, and the core region of the ZP2 gene promoter was found to be between −1552 and −1348. Four CpG islands in the promoter region of the ZP2 gene were detected by software prediction. The overall degree of methylation of the ZP2-1 amplified fragment was negatively correlated with mRNA expression to some extent (R = −0.197); the overall degree of methylation of the ZP2-2 amplified fragment was also negatively correlated with mRNA expression to some extent (R = −0.264), in which the methylation of methylcytosine (mC)-9, mC-20, and mC-21 sites was significantly negatively correlated with mRNA expression (p < 0.05). In addition, the mC-20 and mC-21 sites are located on the Sp1 transcription factor binding site, and it is speculated that these two sites may be the main sites for regulating transcription. In summary, the methylation sites mC-20 and mC-21 of the ZP2 gene may inhibit the binding of Sp1 and DNA, affect the transcription of the ZP2 gene, and then affect the number of eggs produced by the Jinghai yellow chickens.

The clutch is defined as consecutive days of oviposition. Clutch length is an index that reflects ovulation persistence, and is highly correlated with egg production in birds. To identify the genetic markers associated with clutch length in geese, two consecutive experiments were conducted. In the first experiment, 200 Zi geese were selected, all 230 days old, were selected from the same batch and raised individually in the same environment. Data of egg-laying and clutch traits were recorded. After the laying period, three geese with the longest clutch lengths were selected to form the length clutch group (LCG) and three geese with the shortest clutch lengths were formed the short clutch group (SCG). In the second experiment, the ovaries of six geese were collected for transcriptomic and metabolomic analyses. The results showed that large clutch length (LCL) and average clutch length (ACL) were positively correlated with egg number (EN) (P < 0.01; r = 0.63 and 0.60, respectively). Large clutch number (LCN) was significantly correlated with the peak egg number (PEN) (r = 0.58, P < 0.01) and EN (r = 0.60, P < 0.01). EN, LCN, LCL, and ACL showed significant differences (P < 0.01) between the two clutch length groups. Transcriptomic analysis identified 424 differentially expressed genes (DEGs). Functional enrichment analysis revealed that these DEGs were mainly involved in neuroactive ligand-receptor interactions, ovarian steroidogenesis, and calcium signaling pathways. Further, AVPR1A, FGF14, and LHCGR were predicted as the key genes regulating LCL. Metabolomic analysis identified 349 differential metabolites (DMs) in both the positive and negative ion modes. Pyruvate, isocitric acid, D/L‑serine, 3-phospho-d-glycerate, succinate, glycine, and glutamic acid were identified as the key metabolites mainly enriched in the signaling pathways of the TCA cycle. Integration of transcriptomic and metabolomic data revealed critical gene-metabolite pairs, including ACSL4–phosphoenolpyruvate, implicated in LCL regulation. In summary, this study provides new insights into the genes and molecular markers affecting LCL in Zi geese.

The proliferation and apoptosis of ovarian granulosa cells (GC) is critical for follicular development and ovulation, especially for egg-laying performance in female birds. In geese, follicular atrophy is accompanied by follicular GC apoptosis during brooding stages. MicroRNAs are involved in follicular development, atrophy, ovulation, and degeneration. Our previous high-throughput sequencing study of ovaries from laying and brooding geese revealed that miR-192-2 may be involved in follicle growth and development, as well as follicle cell proliferation and apoptosis in geese. To further investigate the effect of miR-192-2 on GC in geese, we screened the target gene of miR-192-2 (IGFBP2) and constructed a miR-192-2 overexpression and interference vector, synthesized a small interfering RNA for IGFBP2. The results showed that after miR-192-2 overexpression, the mRNA and protein expression of proliferation-related genes (PCNA, CDK2, CCND1, and CCND2) were significantly decreased, the mRNA and protein expression of apoptosis-related genes (Caspase3, Caspase8, and Caspase9) were significantly increased, and the mRNA and protein expression of anti-apoptosis gene Bcl-2 were significantly decreased. While the mRNA and protein expressions of PCNA, CDK2, CCND1, and CCND2 were significantly decreased after the downregulation of IGFBP2 expression, the mRNA and protein expressions of Caspase3, Caspase8, and Caspase9 were significantly increased, and the mRNA and protein expression of Bcl-2 was significantly decreased. In conclusion, miR-192-2 inhibited proliferation and promoted apoptosis of follicular GC by targeting IGFBP2 in Zhedong White geese. Apoptosis of GC leads to follicular atresia, which in turn leads to brooding behavior in female geese. Therefore, it is possible to explore inhibiting the expression of miR-192-2 in production to alter the brooding behavior of Zhedong White Geese, thereby improving egg production and economic returns for producers.

The Zi goose is an excellent local goose breed in China, characterized by strong tolerance to roughage, high cold resistance, and high egg production. Anthocyanins are natural water-soluble pigments with numerous biological functions such as growth promotion, antioxidant activity, and immune regulation. However, there are few reports on whether anthocyanins have an improving effect on Zi goose semen. This study aimed to explore the effects of anthocyanins on semen quality, semen antioxidant capacity, and sperm apoptosis of Zi geese, so as to provide references for the large-scale breeding and industrial utilization of Zi geese. Sixty 12-month-old Zi geese were selected for the experiment, and their semen was collected by the massage method. Semen diluent containing different concentrations of anthocyanins was added to the mixed semen, which was then stored at 37 °C for detections. The results showed that the sperm survival rate of Zi geese was the highest when 30 mg/L of anthocyanins was added to the diluent. Compared with the control group, the anthocyanin group showed significantly higher sperm survival rate, sperm motility, plasma membrane integrity rate, acrosome integrity rate, mitochondrial activity, and DNA integrity (p < 0.05), while the sperm mortality rate was significantly decreased (p < 0.05). There were no significant differences in semen pH value, sperm density, and sperm abnormality rate (p > 0.05). The contents of superoxide dismutase and glutathione peroxidase were significantly increased (p < 0.05), while the levels of reactive oxygen species and malondialdehyde were significantly decreased (p < 0.05) in the anthocyanin group. Anthocyanins significantly increased the mRNA and protein expression of Bcl-2 (p < 0.05), and significantly decreased the mRNA and protein expression of Bax, Caspase-3, and P53 (p < 0.05). In conclusion, adding 30 mg/L anthocyanins to semen diluent can improve semen quality in Zi geese.