Weekly trend in body weight among male and female ZDW geeseAt the hatching stage, female ZDW geese showed higher body weight (BW0) than males (P < 0.05). Starting from 14 days of age, male geese consistently displayed significantly higher body weight than females (P < 0.01). The difference between the body weights of male and female geese increased with age. The male geese were 4.16% and 13.73% heavier than females at BW14 and BW70, respectively.Blood biochemistry indices (BCIs) among ZDW geese at the age of 10 weeksAmong all five BCIs, only the blood glucose (GLU) concentration was significantly higher in males than in females (P < 0.05). Although males also showed higher TG concentrations, the difference was not statistically significant. For the BCIs of CHO, HDL, and LDL, females displayed higher levels than males, although the differences were not statistically significant (P > 0.05).Hormone concentrations among ZDW geese at the age of 10 weeksThe concentrations of three hormones in male and female geese were compared. The concentration of E2 was higher in females than in males, whereas those of T and GH were slightly higher in males. However, no significant differences were observed in hormone concentrations between female and male geese (P > 0.05).Histological investigations of muscle fiber properties between male and female geeseTransverse sections and longitudinal sections of male and female leg muscles were stained by hematoxylin and eosin. Areas of transverse sections for each muscle fiber were marked manually and calculated using the software CaseViewer (V2.4, 3DHISTECH, Hungary).Although some male muscle fibers were smaller and thinner (blue arrows in Fig. 1A,D) than those of females, collectively counting the fibers in different fields under the microscope revealed that the areas of leg muscle fibers from male geese were significantly higher than those from female geese (P < 0.05, Fig. 1A–C). To confirm these findings, longitudinal sections of muscles were also investigated. The widths of leg muscle fibers from these sections were significantly higher in male than in female geese (P < 0.01).Figure 1Histological staining for leg muscles from male and female geese. (A) and (D) were from male geese while (B) and (E) were for female geese. A/B and D/E were transections and longisections, respectively. The black horizontal lines in A/B/D/E were the 50 μm plotting scales. (C) were the areas of muscle fibers from the cross sections (μm2) between male and female geese. (D) were the width for the leg muscle fibers from longisections (μm) between male and female geese.DEGs in tissues between male and female ZDW geese at the age of 10 weeksMale and female geese at age of ten weeks were selected because geese showed large difference at the time (Male: 4371.71 ± 399.58; Female: 3771.14 ± 389.11, Table 1). Further, the age of 10 weeks is the time point at which farmers begin to select geese for breeding in next generation and BW10 reflects individual’s growth performance. Breast muscles, leg muscles, and pituitary tissues were selected from eight samples (male: 4; female: 4) for subsequent library construction and transcriptome sequencing. After filtration of the raw data, each sample was covered by more than 20 million read pairs (20,196,145.04 ± 774,767.23). The mean depth was 5.41 (± 0.21). For the leg, breast, and pituitary tissues, 1101 (507 upregulated; 594 downregulated), 773 (311 upregulated; 462 downregulated), and 517 (281 upregulated; 236 downregulated) DEGs were identified in males compared with females (P < 0.01), respectively. Of these DEGs, 245 were common between leg and breast muscle transcriptomes from male and female samples, including IGFALS and NCAPG, among others (Fig. 2A). However, only 99 out of 245 common DEGs were annotated well, whereas the remaining 146 genes did not show clear gene information. More gene information is listed in Tables S1–S3.Table 1 Body weight (g) of male and female ZDW at different ages.Figure 2Volcano plots displayed differentially expressed genes (DEGs) between different comparative transcriptome analyses: (A–C) were DEGs from leg transcriptomes (male vs female), breast muscle transcriptomes (male vs female), and pituitary transcriptomes (male vs female). (D) was the number of common DEGs (cDEGs) among different groups.Of the 245 DEGs, 69 were also present in the comparative transcriptome analysis for all three tissue types between male and female geese, including MRPS27, MRPPS30, and SLC2A9, among others (Fig. 2B). More genes are listed in Tables S1–S3.Gene ontology terms (GOs) enriched by DEGsDEGs between male and female leg, breast muscles, and pituitary tissues were enriched in 1043, 622, and 1068 GO terms, respectively (Tables S4–S6). GOs enriched by DEGs from leg muscle transcriptomes showed hormonal activity (GO: 0,005,179). A total of 109 common GO terms were detected by both DEGs from leg and breast muscle transcriptomes, covering functions such as vitamin transport (GO: 0,051,180), glucose transmembrane transporter activity (GO: 0,005,355), and lipid transport involved in lipid storage (GO: 0,010,877, Tables S5–S7).Several GO terms enriched by DEGs in the pituitary transcriptomes were related to muscle development, such as structural consistency of muscle (GO: 0,008,307), sarcomere (GO: 0,030,017), myofibril (GO: 0,030,016), and contractile fiber (GO: 0,043,292), among others. GOs related to bone development and hormone metabolism, including bone mineralization (GO: 0,030,282), bone growth (GO: 0,098,868), hormone activity (GO: 0,005,179), and response to steroid hormone (GO: 0,048,545), were also detected (Fig. 3). More detected GO terms are listed in Tables S4–S6.Figure 3Gene Ontology enrichment analysis using DEG between leg muscle (A), breast muscle (B) and pituitary tissues (C). Circular plots display six GO terms. (D) showed common GOs detected in all three different tissues. Other significantly enriched GO terms were listed in Table S4-S6.KEGG pathway enrichment analysesBased on upregulated DEGs in male geese, 11, 7, and 11 pathways were enriched in leg muscle, breast muscle, and pituitary tissues, respectively (Fig. 4). In leg muscles, 11 pathways were significantly enriched in males, including vitamin digestion and absorption, cardiac muscle contraction, and bile secretion. Breast muscles showed significant enrichment in seven tested pathways, including neuroactive ligand-receptor interaction. In pituitary tissues, significant enrichment was observed in 11 signaling pathways, including cardiac muscle contraction, hypertrophic cardiomyopathy, and gastric acid secretion, among others. The neuroactive ligand-receptor interaction pathway was consistently detected in all three tissues of male geese.Figure 4The most significantly enriched KEGG pathways enriched using differentially expressed genes between male and female muscle and pituitary tissues. (A) Leg muscle; (B) Breast muscle; (C) Pituitary.Protein–protein interaction (PPI) analysisThe upregulated DEGs in pituitary samples were used to construct a protein–protein interaction network. Six clear gene hubs were formed, with biological functions related to myosin development, mitochondrial respiratory chain, calcium metabolism, bone morphogenesis, and IGF1-involved growth processes (Fig. 5). The sub-network associated with HOXA5 was also observed in the analysis.Figure 5Protein–protein interaction (PPI) network constructed by the STRING web server using DEGs detected in pituitary samples. Node size indicates the degree of each gene.Validation of DEGs by qPCRFor both leg muscle and pituitary tissues, four genes belong to DEGs were randomly selected for qPCR to verify their expression profiles. In breast muscles, upregulated genes (ANO4, KRT222, and SYT4) and downregulated gene (POU1F1) were detected with same expression patterns found in our transcriptome sequencing experiments. The expression levels of POU1F1 in females was higher than males although it showed no statistical significance (top panel in Fig. 5). The significantly upregulated DEGs (APOB, GHRHR, TMEM163, and IGFN1) in male leg muscles showed similar expression differences between males and females (Fig. 6). In pituitary tissues, four genes of HOXA5, BMP6, TYRP1, and IGF1 were all significantly upregulated in male pituitary tissues (Table S1 and S3). Their expression patterns were consistent with results from our RNA-Seq experiments, confirming the reliability of our transcriptome analyses.Figure 6Relative quantitative values for twelve selected DEGs. Expression profiles in leg muscle (panel up) and pituitary (panel down) tissues were tested, while breast muscle tissue was not selected. POU1F1 showed no significantly difference between male and female geese (P > 0.05) while expression difference between genders for other genes among tissues showed statistical significance (P < 0.05).
