Single-nucleus transcriptional and chromatin accessible profiles reveal critical cell types and molecular architecture underlying chicken sex determination.

INTRODUCTION: Understanding the sex determination mechanisms in birds has great significance for the biological sciences and production in the poultry industry. Sex determination in chickens is a complex process that involves fate decisions of supporting cells such as granulosa or Sertoli cells. However, a systematic understanding of the genetic regulation and cell commitment process underlying sex determination in chickens is still lacking. OBJECTIVES: We aimed to dissect the molecular characteristics associated with sex determination in the gonads of chicken embryos. METHODS: Single-nucleus RNA-seq (snRNA-seq) and ATAC-seq (snATAC-seq) analysis were conducted on the gonads of female and male chickens at embryonic day 3.5 (E3.5), E4.5, and E5.5. RESULTS: Here, we provided a time-course transcriptional and chromatin accessible profiling of gonads during chicken sex determination at single-cell resolution. We uncovered differences in cell composition and developmental trajectories between female and male gonads and found that the divergence of transcription and accessibility in gonadal cells first emerged at E5.5. Furthermore, we revealed key cell-type-specific transcription factors (TFs) and regulatory networks that drive lineage commitment. Sex determination signaling pathways, dominated by BMP signaling, are preferentially activated in males during gonadal development. Further pseudotime analysis of the supporting cells indicated that granulosa cells were regulated mainly by the TEAD gene family and that Sertoli cells were driven by the DMRT1 regulons. Cross-species analysis suggested high conservation of both cell types and cell-lineage-specific TFs across the six vertebrates. CONCLUSIONS: Overall, our study will contribute to accelerating the development of sex manipulation technology in the poultry industry and the application of chickens as a unique model for studying cell fate decisions.

Comprehensive Atlas of Alternative Splicing Reveals NSRP1 Promoting Adipogenesis through CCDC18.

Alternative splicing (AS) plays a crucial role in regulating gene expression, function, and diversity. However, limited reports exist on the identification and comparison of AS in Eastern and Western pigs. Here, we analyzed 243 transcriptome data from eight tissues, integrating information on transcription factors (TFs), selection signals, splicing factors (SFs), and quantitative trait loci (QTL) to comprehensively study alternative splicing events (ASEs) in pigs. Five ASE types were identified, with Mutually Exclusive Exon (MXE) and Skipped Exon (SE) ASEs being the most prevalent. A significant portion of genes with ASEs (ASGs) showed conservation across all eight tissues (63.21-76.13% per tissue). Differentially alternative splicing genes (DASGs) and differentially expressed genes (DEGs) exhibited tissue specificity, with blood and adipose tissues having more DASGs. Functional enrichment analysis revealed coDASG_DEGs in adipose were enriched in pathways associated with adipose deposition and immune inflammation, while coDASG_DEGs in blood were enriched in pathways related to immune inflammation and metabolism. Adipose deposition in Eastern pigs might be linked to the down-regulation of immune-inflammation-related pathways and reduced insulin resistance. The TFs, selection signals, and SFs appeared to regulate ASEs. Notably, ARID4A (TF), NSRP1 (SF), ANKRD12, IFT74, KIAA2026, CCDC18, NEXN, PPIG, and ROCK1 genes in adipose tissue showed potential regulatory effects on adipose-deposition traits. NSRP1 could promote adipogenesis by regulating alternative splicing and expression of CCDC18. Conducting an in-depth investigation into AS, this study has successfully identified key marker genes essential for pig genetic breeding and the enhancement of meat quality, which will play important roles in promoting the diversity of pork quality and meeting market demand.

Uncovering Evolutionary Adaptations in Common Warthogs through Genomic Analyses.

In the Suidae family, warthogs show significant survival adaptability and trait specificity. This study offers a comparative genomic analysis between the warthog and other Suidae species, including the Luchuan pig, Duroc pig, and Red River hog. By integrating the four genomes with sequences from the other four species, we identified 8868 single-copy orthologous genes. Based on 8868 orthologous protein sequences, phylogenetic assessments highlighted divergence timelines and unique evolutionary branches within suid species. Warthogs exist on different evolutionary branches compared to DRCs and LCs, with a divergence time preceding that of DRC and LC. Contraction and expansion analyses of warthog gene families have been conducted to elucidate the mechanisms of their evolutionary adaptations. Using GO, KEGG, and MGI databases, warthogs showed a preference for expansion in sensory genes and contraction in metabolic genes, underscoring phenotypic diversity and adaptive evolution direction. Associating genes with the QTLdb-pigSS11 database revealed links between gene families and immunity traits. The overlap of olfactory genes in immune-related QTL regions highlighted their importance in evolutionary adaptations. This work highlights the unique evolutionary strategies and adaptive mechanisms of warthogs, guiding future research into the distinct adaptability and disease resistance in pigs, particularly focusing on traits such as resistance to African Swine Fever Virus.

Research on Chinese consumers' shell egg consumption preferences and the egg quality of functional eggs.

The purpose of this study is to investigate the characteristics of egg consumption in China and the production of functional eggs, and finally enrich the types of shell egg products. Trial 1 explored the influence of egg quality on Chinese consumers' willingness to purchase eggs through a questionnaire, which investigated 1,317 consumers' preferences for egg appearance, factors influencing egg purchase, and purchase of functional eggs. The results showed that about 65% of respondents ate more than 4 eggs per wk, pink eggs were the most popular in China, about 65% of consumers preferred eggs with an egg weight of 48 to 58 grams. For functional eggs, 75.32% of consumers have never heard of them. Preferences for eggshell color and yolk color varied by geographic region, with darker colors preferred in Northeast China. Based on the survey results of functional eggs consumption in Trial 1, the dwarf layers of China Agricultural University were used in Trial 2 to produce functional eggs. The eggs are small and pink in color, which is in line with the preferences of Chinese consumers. Three hundred dwarf layers were divided into 4 groups, using the linseed oil added, marigold extract added, and yeast selenium added diets to produce normal, n-3 fatty acid-enriched, lutein-enriched and selenium-enriched eggs by feeding for 28 d, determined the eggs' nutrient content and egg quality. The results showed that the n-3 fatty acid, lutein and selenium contents of the eggs of dwarf layers were significantly increased by changing the diets and did not affect the egg weight, eggshell strength, Haugh units or the proportion of egg parts. The results of this study are helpful to understand the trend of egg consumption preferences in China, and on this basis to produce functional eggs that meet the consumers' expectations.

Young SINEs in pig genomes impact gene regulation, genetic diversity, and complex traits.

Transposable elements (TEs) are a major source of genetic polymorphisms and play a role in chromatin architecture, gene regulatory networks, and genomic evolution. However, their functional role in pigs and contributions to complex traits are largely unknown. We created a catalog of TEs (n = 3,087,929) in pigs and found that young SINEs were predominantly silenced by histone modifications, DNA methylation, and decreased accessibility. However, some transcripts from active young SINEs showed high tissue-specificity, as confirmed by analyzing 3570 RNA-seq samples. We also detected 211,067 dimorphic SINEs in 374 individuals, including 340 population-specific ones associated with local adaptation. Mapping these dimorphic SINEs to genome-wide associations of 97 complex traits in pigs, we found 54 candidate genes (e.g., ANK2 and VRTN) that might be mediated by TEs. Our findings highlight the important roles of young SINEs and provide a supplement for genotype-to-phenotype associations and modern breeding in pigs.

FMO3 deficiency of duck leads to decreased lipid deposition and increased antibacterial activity.

BACKGROUND: Most duck eggs possess a fishy odor, indicating that ducks generally exhibit impaired trimethylamine (TMA) metabolism. TMA accumulation is responsible for this unpleasant odor, and TMA metabolism plays an essential role in trimethylaminuria (TMAU), also known as fish odor syndrome. In this study, we focused on the unusual TMA metabolism mechanism in ducks, and further explored the unclear reasons leading to the debilitating TMA metabolism. METHODS: To achieve this, transcriptome, proteome, and metagenome analyses were first integrated based on the constructed duck populations with high and low TMA metabolism abilities. Additionally, further experiments were conducted to validate the hypothesis regarding the limited flavin-containing monooxygenase 3 (FMO3) metabolism ability of ducks. RESULTS: The study demonstrated that liver FMO3 and cecal microbes, including Akkermansia and Mucispirillum, participated in TMA metabolism in ducks. The limited oxidation ability of FMO3 explains the weakening of TMA metabolism in ducks. Nevertheless, it decreases lipid deposition and increases antibacterial activity, contributing to its survival and reproduction during the evolutionary adaptation process. CONCLUSIONS: This study demonstrated the function of FMO3 and intestinal microbes in regulating TMA metabolism and illustrated the biological significance of FMO3 impairment in ducks.

Long-lasting effects of lipopolysaccharide on the reproduction and splenic transcriptome of hens and their offspring.

Lipopolysaccharide (LPS) is ubiquitous in the environment and is released after the death of gram-negative bacteria, which may be related to inflammation and immunosuppression. However, its impact on the reproduction of animals and their offspring, especially the underlying mechanism need further elucidation. Here, we used laying hens as a model organism to investigate the effects of maternal exposure to LPS (LPS maternal stimulation) on animal and their offspring's immunity and reproductive performance, as well as the regulatory role of the transcriptome. We found that the LPS maternal stimulation could reduce the egg-laying rate of hens and their offspring, especially during the early and late laying stages. The transcriptome study of the spleen in F0, F1 and F2 generations showed that the maternal stimulation of the LPS affects the patterns of gene expression in laying hens, and this change has a long-lasting effect. Further analysis of DEGs and their enrichment pathways found that the LPS maternal stimulation mainly affects the reproduction and immunity of laying hens and their offspring. The DEGs such as AVD, HPS5, CATHL2, S100A12, EXFABP, RSFR, LY86, PKD4, XCL1, FOS, TREM2 and MST1 may play an essential role in the regulation of the immunity and egg-laying rate of hens. Furthermore, the MMR1L3, C3, F13A1, LY86 and GDPD2 genes with heritable effects are highly correlated with the egg-laying rate, may have an important reference value for further research. Our study reveals the profound implications of LPS exposure on immunity and reproduction of offspring, elaborating the impact of immune alteration on the egg-laying rate, emphasizing the regulatory role of intergenerational transmission of the transcriptome, implying that the environment parents being exposed to has an important impact on offspring.

Choline diet improves serum lipid parameters and alters egg composition in breeder ducks.

BACKGROUND: Choline is an important nutrient, playing key roles in numerous metabolic pathways relevant to animal health. OBJECTIVES: The objective of this study was to evaluate the effect of dietary choline on the lipid parameters, cardiovascular health (CVH), and levels of egg trimethylamine (TMA) and cholesterol in breeder ducks during the late laying period. METHODS: A total of 60 Jingjiang ducks were randomly separated into six replicates of 10 ducks each. After peak production until 65 weeks of age, the birds were fed a control basal diet. The same ducks served as the control group until 65 weeks of age, when the same ducks served as the choline-supplemented group, after 15 days of dietary choline supplementation at 2955 mg/kg choline above and over the basal diet initially provided. The 15 days of choline supplementation included an initial 5-day acclimatisation period. RESULTS: Dietary choline supplementation increased serum TMA (p < 0.01), high-density lipoprotein cholesterol, very low-density lipoprotein, and triglyceride levels in older breeder ducks. However, it did not change the levels of trimethylamine N-oxide but decreased the atherosclerosis index compared with those of the control group (p < 0.01). Moreover, it increased (p < 0.01) the egg yolk TMA levels but did not change the concentrations of cholesterol in egg yolk. CONCLUSIONS: Dietary choline supplementation had a beneficial effect on lipid parameters and CVH in older breeder ducks, although it increased the serum and egg yolk TMA levels.

Antibacterial Properties of TMA against Escherichia coli and Effect of Temperature and Storage Duration on TMA Content, Lysozyme Activity and Content in Eggs.

Studies on trimethylamine (TMA) in egg yolk have focused on how it impacts the flavor of eggs, but there has been little focus on its other functions. We designed an in vitro antibacterial test of TMA according to TMA concentrations that covered the TMA contents typically found in egg yolk. The change in TMA content in yolk was analyzed at different storage temperatures and for different storage durations. The known antibacterial components of eggs, including the cuticle quality of the eggshell and the lysozyme activity and content in egg white, were also assessed. The total bacterial count (TBC) of different parts of eggs were detected. The results showed that the inhibitory effect of TMA on Escherichia coli (E. coli) growth increased with increasing TMA concentration, and the yolk TMA content significantly increased with storage duration (p < 0.05). The cuticle quality and lysozyme content and activity significantly decreased with storage time and increasing temperature, accompanied by a significant increase in the TBC on the eggshell surface and in the egg white (p < 0.05). This work reveals a new role for trace TMA in yolks because it reduces the risk of bacterial colonization, especially when the antibacterial function of eggs is gradually weakened during storage.

Cuticle deposition duration in the uterus is correlated with eggshell cuticle quality in White Leghorn laying hens.

The cuticle formed in the uterus is the outermost layer as the first defense line of eggshell against microbial invasions in most avian species, and analyzing its genetic regulation and influencing factors are of great importance to egg biosecurity in poultry production worldwide. The current study compared the uterine transcriptome and proteome of laying hens producing eggs with good and poor cuticle quality (GC and PC, the top and tail of the cuticle quality distribution), and identified several genes involved with eggshell cuticle quality (ESCQ). Overall, transcriptomic analysis identified 53 differentially expressed genes (DEGs) between PC versus GC group hens, among which 25 were up-regulated and 28 were down-regulated. No differences were found in the uterine proteome. Several DEGs, including PTGDS, PLCG2, ADM and PRLR related to uterine functions and reproductive hormones, were validated by qPCR analysis. Egg quality measurements between GC and PC hens showed GC hens had longer laying interval between two consecutive ovipositions (25.64 ± 1.23 vs 24.94 ± 1.12 h) and thicker eggshell thickness (352.01 ± 23.04 vs 316.20 ± 30.58 µm) (P < 0.05). Apart from eggshell traits, other egg quality traits didn't differ. The result demonstrated eggshell and cuticle deposition duration in the uterus is one of the major factors affecting ESCQ in laying hens. PTGDS, PLCG2, ADM and PRLR genes were discovered and might play crucial roles in cuticle deposition by regulating the uterine muscular activities and secretion function. The findings in the present study provide new insights into the genetic regulation of cuticle deposition in laying hens and establish a foundation for further investigations.

Comparative study of eggshell antibacterial effectivity in precocial and altricial birds using Escherichia coli.

In this study, we compared the antibacterial effectivity of the eggs of six precocial and four altricial bird species using Escherichia coli, based on their eggshell traits. The ultrastructure of eggshell was observed using a scanning electron microscope (SEM). According to SEM results, eggs from precocial birds (chicken, turkey, quail, duck, ostrich, and goose) had cuticle on the eggshells, while eggs from altricial birds (pigeon, budgerigar, munia, and canary) did not. The environment/selection pressure may induce the divergent evolution process in eggs of precocial and altricial birds. The E. coli experiment results showed that chicken, turkey, quail, duck, and goose eggs, with a high cuticle opacity, exhibited a much lower E. coli penetration rate. In contrast, the eggs with poor (ostrich) or without (pigeon, budgerigar, munia, and canary) cuticle exhibited a higher penetration rate. It is suggested that cuticle is a main barrier against bacterial penetration in precocial birds' eggs. Turkey and quail eggs showed the lowest E. coli contamination rate (3.33% and 2.22%, respectively), probably because of the tightly connected nanosphere structure on their cuticle. As for altricial birds' eggs, the eggs of budgerigar, munia, and canary with small pore diameter (0.57 to 1.22 µm) had a lower E. coli penetration rate than pigeon eggs (45.56%, 66.67%, 50%, and 97.78%, respectively, P < 0.05), indicating that pore diameter played a significant role in defending against bacterial trans-shell invasion. We found that eggshell thickness and pore area decreased with egg size. The cuticle quality had no relationship with egg size, but was closely related to the bird species. The E. coli penetration rate of altricial birds' eggs was significantly higher than that of precocial birds' eggs, mainly because the pores are exposed on the eggshell surface and cuticle protection is absent. This study provides detailed information on the eggshell cuticle, which gives insight into the cuticle evolution process that occurred in precocial and altricial bird species. Moreover, the results of E. coli penetration may help understanding the antibacterial behavior in birds.

Analysis of fishy taint in duck eggs reveals the causative constituent of the fishy odor and factors affecting the perception ability of this odor.

Fresh duck eggs normally have an unpleasant fishy odor when compared with eggs from other poultry. However, the cause of this unpleasant smell remains unknown. Besides, we have limited knowledge regarding the perception ability of this odor. Therefore, we aimed to identify the causative substances responsible for the fishy odor in duck eggs, and further evaluate the factors affecting the perception ability of this odor. We detected and quantified the volatile substances in the yolks of Jingjiang duck and Beijing duck eggs using headspace gas chromatography, and evaluated the fishy odor rating scores. We performed association studies on the relationship between the relative content of volatile substances and fishy odor rating scores. The fishy odor of egg yolks from different species/breeds was also characterized. Furthermore, we assessed the species/breeds of poultry eggs and the effects of gender and region of the evaluators on the fishy odor rating score by using an ordinal logistic regression model. Results showed that trimethylamine (TMA) is responsible for the fishy odor of duck eggs, which was confirmed with subsequent verification (P < 0.001). The fishy odor of duck eggs was stronger than that of chicken eggs; the fishy odor of Jingjiang duck eggs was the strongest. Results also indicated that the fishy odor perception ability was affected by the gender and region of the evaluators (P < 0.001). Overall, these data clarified that TMA imparted the fishy odor to duck eggs; factors such as gender and region may affect the fishy odor perception ability.

Impact of cuticle quality and eggshell thickness on egg antibacterial efficiency.

The cuticle is the first barrier against bacterial trans-shell penetration. To evaluate the cuticle quality among different colored eggshells, an efficient and reliable method is proposed in this paper on the basis of the opacity theory. It is shown that the Spearman's rank correlation coefficients between the proposed opacity method and the reported reflectance and ΔE*ab methods are all more than 0.90 in measuring the cuticle deposition for the same breed eggs, indicating that the result measured by the opacity method is credible. In addition, the validity of opacity method is also verified by the Escherichia coli penetration experiments performed on the eggs of Dwarf Layer (tinted-shelled), Hy-Line Brown (brown-shelled), and White Leghorn (white-shelled) birds. Results show that the Dwarf Layer eggs, with the thickest cuticle deposition, have the lowest bacterial penetration ratio than the eggs of the other 2 investigated breeds. We found that eggshell thickness is another important factor influencing bacterial penetration apart from the cuticle. Therefore, enhancing cuticle deposition and eggshell thickness would significantly improve the eggs' antibacterial efficiency. It is found that the antibacterial efficiency will reach as high as 98% in such a case where the cuticle opacity is over 27.5% and the eggshell is thicker than 340 µm. No significant difference was found in pore density between the penetrated and non-penetrated eggs. Moreover, according to the data for 3 regions of the penetrated eggs, we found that the sharp end shows the highest proportion of bacterial contamination compared with the blunt end and equator. The cuticle quality at equator shows a strong positive correlation between the blunt and sharp end (r2 = 0.8 and 0.7, respectively, P < 0.05). The cuticle at the equator can be selected as an indicator for cuticle breeding to improve the cuticle quality at the blunt and sharp end simultaneously. These results are of significance for the improvement of egg quality and safety in modern commercial breeding.

Fishy Odor and TMA Content Levels in Duck Egg Yolks.

The differences between the trimethylamine (TMA) content levels in duck and chicken egg yolks under normal dietary conditions were compared. Moreover, the association between the polymorphisms of the duck FMO3 gene and TMA content levels in duck egg yolks was analyzed. Then, to detect the mutations associated with the fish-flavor trait, duck populations were selected for a high-choline diet experiment, which was followed by full-length sequencing of the FMO3 exons. The results showed that the TMA content levels in duck eggs (3.60 µg/g) were significantly higher than those in chicken eggs (2.35 µg/g) under normal dietary conditions (P < 0.01). With regard to the high-choline diet, the average TMA content levels in duck egg yolks (9.21 µg/g; P < 0.01) increased significantly. Furthermore, 5 SNPs reported in Ensembl database were detected in duck FMO3 exons. However, no mutation loci were found to be significantly associated with the TMA content levels in duck egg yolks. Besides, duck liver FMO3 mRNA expression levels were not associated with the TMA content levels. The results indicated that excessive TMA deposition in duck eggs is one of main factors causing the fishy odor in duck eggs, and the addition of choline in the ducks' diets was responsible for inducing an increase in the TMA content levels in duck eggs. PRACTICAL APPLICATION: Our study can help to diminish the fishy taste in duck eggs by reducing the amount of supplemented choline. Furthermore, this study laid a solid foundation for revealing the genetic factors involved in the fishy odor in duck eggs.

Salmonella Contamination in Layer Farms in China: Detection and Genetic Analysis.

Salmonella is the most common cause of foodborne illnesses worldwide. Poultry eggs are a major contamination source of Salmonella. The prevalence of Salmonella has been effectively reduced since a series of measures were taken to reduce contamination in egg-laying houses. In the present study, 1,512 environmental samples obtained from layer farms of different production scales were screened in a voluntary Salmonella survey study. Contaminations were detected using a PCR method. Genetic relationships among Salmonella samples were specified using molecular typing by enterobacterial repetitive intergenic consensus (ERIC)-PCR. The survey results showed that two layer farms, located in the Shandong and Hebei provinces, were contaminated with Salmonella. Thirty-one samples from these two farms, including feed, drinking nipples, egg collection belt, air inlets and outlets, air, overshoes, and eggshells, were identified as Salmonella-positive. It was observed that certain samples within the henhouses as well as in the egg collecting areas showed relatively high genetic similarities. The survey conclusively revealed minor Salmonella contamination in northern China. Moreover, various areas within the layer farms were identified as part of the propagation chain of Salmonella. Furthermore, evidence of cross-contamination of Salmonella was found in the laying houses and egg collection areas, even between these two regions. Therefore, it is necessary to establish routine Salmonella detection and subsequent environmental control measures in order to decrease the prevalence of Salmonella.

The Distribution Characteristics and Applications for Maternal Cells on Chicken Egg Vitelline Membrane.

The major components of vitelline membrane (VM) are ovomucin, VM outer (VMO) I and VMO II. At present, the distribution pattern of maternal cells on the VM has not been described in detail. In this study, the existence and distribution characteristics of maternal cells on VM were observed. There were more than 3.2 × 105 somatic cells on VM, which were uneven distributed. The calcein AM/PI staining of the maternal cells on the VM showed that the cells' viability changed with the freshness of the eggs, and that the maternal cells gradually underwent apoptosis and became degraded. The results of morphology of different tissues indicated that the most of maternal cells on the VM were granulosa cells. Moreover, the karyotype of the cultured granulosa cells, which is the main source of cells on VM, were identified as the normal diploid karyotype of chicken. Furthermore, the VM DNA extracted from chickens and quails, which represent the eggs of different size, was adequate for further genetic analysis. The VM DNA was easily accessible and relatively constant, without cross-contamination. Therefore, the VM DNA could potentially be applied for the molecular traceability between eggs and chickens, and be beneficial in avian ecology research studies.