Background: Sex-related genes play a crucial role in gonadal differentiation into testes or ovaries. However, the genetic control of gonadal differentiation in Muscovy ducks remains unknown. Therefore, the objective of our study was to screen new candidate genes associated with ovarian and testicular development.Results: In this study, 24 males before gonadal differentiation (MB), 24 females before gonadal differentiation (FB), 24 males after gonadal differentiation (MA) and 24 females after gonadal differentiation (FA) were selected from Putian Muscovy ducks, forming 4 groups. RNA-Seq revealed 101.76 Gb of clean reads and 2800 differentially expressed genes (DEGs), including 46 in MB vs FB, 609 in MA vs FA, 1027 in FA vs FB, and 1118 in MA vs MB. A total of 146 signalling pathways were enriched by KEGG analysis, among which 20, 108, 108 and 116 signalling pathways were obtained in MB vs FB, MA vs MB, MA vs FA and FA vs FB, respectively. In further GO and KEGG analyses, a total of 21 candidate genes related to gonad differentiation and development in Muscovy ducks were screened. Among these, 9 genes were involved in the differentiation and development of the testes, and 12 genes were involved in the differentiation and development of the ovaries. In addition, RNA-Seq data revealed 2744 novel genes.Conclusions: RNA-Seq data revealed 21 genes related to gonadal differentiation and development in Muscovy ducks. We further identified 12 genes, namely,WNT5B,HTRA3, RSPO3, BMP3, HNRNPK, NIPBL, CREB3L4, DKK3, UBE2R2, UBPL3KCMF1, ANXA2,andOSR1, involved in the differentiation and development of ovaries. Moreover, 9 genes, namely,TTN, ATP5A1, DMRT1, DMRT3, AMH, MAP3K1, PIK3R1, AGTandADAMTSL1, were related to the differentiation and development of testes. Moreover, after gonadal differentiation,DMRT3, AMH, PIK3R1, ADAMTSL1, AGTandTTNwere specifically highly expressed in males.WNT5B, ANXA2andOSR1were specifically highly expressed in females. These results provide valuable information for studies on the sex control of Muscovy ducks and reveal novel candidate genes for the differentiation and development of testes and ovaries.

1. Prolactin (PRL) as a polypeptide hormone which plays a crucial role in egg production traits. 2. Polymorphisms of the PRL gene were analysed with DNA sequencing and polymerase chain reaction-single-strand conformation polymorphism methods in two Chinese domestic laying duck breeds (Jinding, n = 400, Youxian, n = 400, respectively). 3. The results showed that one polymorphism was detected (A-412G) in intron 1 of the PRL gene, with three genotypes: AA, AG and GG. Association analysis showed that the ducks with the GG genotype had significantly greater egg production and egg weight than those with AG and AA genotype (p < 0.05). Hence, the 412A > G polymorphism of the PRL gene in intron 1 is a potentially valuable genetic marker for laying duck breeding programmes.

The diversity of avian eggshell colour plays important biological roles in ensuring successful reproduction. Eggshell colour is also an important trait in poultry, but the mechanisms underlying it are poorly understood in ducks. This study aimed to provide insights into the mechanism of blue-green eggshell colour generation. Here, white-shelled ducks (HBR) and blue-green-shelled ducks (HQR) were selected from Putian black ducks, and white-shelled ducks (BBR) were selected from Putian white ducks. Transcriptional changes in the shell gland were analysed using RNA-sequencing on the Illumina HiSeq 2500. Twenty-seven individual cDNA libraries were sequenced and generated an average of 7.35 million reads per library; 70.6% were mapped to the duck reference genome, yielding an average of 13,794 genes detected, which accounted for approximately 86.39% of all 15,967 annotated duck genes. A total of 899 differentially expressed genes (DEGs) were detected between the HQR and BBR groups, and 373 DEGs were detected between the HQR and HBR groups. We analysed the DEGs in the HQR-vs-BBR and HQR-vs-HBR comparisons. None of these DEGs were directly involved in the eggshell pigmentation process in HQR-vs-HBR, while UDP-glucuronosyltransferase 2A2 (UGT2A2) and UDP-glucuronosyltransferase 1–1-like (UGT1–1-like), which participate in biliverdin breakdown, were two of the DEGs in HQR-vs-BBR. In the RT-qPCR results, delta-aminolevulinic acid synthase 1 (ALAS1) and EPRS glutamyl-prolyl-tRNA synthetase were significantly upregulated in the HBR group compared with the HQR and BBR groups (P < 0.05). Haem oxygenase (HMOX1) was significantly downregulated in BBR compared with HQR and HBR (P < 0.05). Biliverdin reductase A (BLVRA), GUSB glucuronidase beta, cytochrome c-type haem lyase, protohaem IX farnesyltransferase and UGT2A2 were significantly upregulated in HBR and BBR compared with HQR (P < 0.05). We conducted a comparative transcriptome analysis of the shell glands of Putian white ducks and Putian black ducks. None of the differentially regulated pathways were directly involved in the eggshell pigmentation process in the HQR-vs-HBR comparison, while 2 DEGs related to biliverdin breakdown were found in HQR-vs-BBR. Based on the RT-qPCR results, we can speculate that both HQR and HBR can produce biliverdin, but HBR cannot accumulate it. Compared with HQR, BBR produced less biliverdin and did not accumulate it.

This study aimed to investigate the temporal dynamics of physiological and gut microbial responses in Mule ducks (M-D) during force-feeding (F-F), with the goal of identifying potential regulatory targets to reduce feeding stress. Male M-Ds were subjected to either F-F or ad libitum feeding. We conducted longitudinal analysis at 72, 78, and 84 days of age to assess growth performance, serum biochemical profiles, and intestinal inflammatory markers, while assessing gut microbiota composition through 16S rDNA sequencing. The F-F group exhibited superior growth performance. Initial physiological responses at day 72 included significantly reduced serum corticotropin-releasing hormone (CRH) and jejunal tumor necrosis factor-alpha (TNF−α). Conversely, F-F induced a persistent and profound alteration in the gut microbiome by day 84, characterized by reduced alpha diversity and a significant enrichment of the genus Limosilactobacillus. Correlation analysis identified Limosilactobacillus as a keystone taxon, strongly associated with intestinal metabolites. Our findings demonstrate that M-Ds undergo time-dependent metabolic and immunological adaptations in response to F-F stress, which correlates with distinct alterations in gut microbiota composition, particularly the enrichment of Limosilactobacillus. These findings provide a theoretical basis for developing microbiota-targeted strategies to alleviate F-F stress in foie gras production.