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Beijing You Chicken, a valuable local chicken breed from Beijing, China, was once listed as an endangered breed. From the point of view of conservation, the preservation of this breed is an important task for the local researchers. Semen cryopreservation is a popular method to maintain valuable species. However, during cryopreservation, semen is susceptible to oxidative damage. Melatonin is a potent antioxidant and free radical scavenger, so it has been selected to improve the efficiency of sperm cryopreservation. In this study, the chicken semen was treated with different concentrations of melatonin in the cryopreservation solution. The results showed that melatonin at concentrations of 10−3 M and 10−5 M significantly improved sperm progressive motility and total motility, respectively, compared to the control (P < 0.05). Melatonin at 10−3 M also significantly improved the plasma membrane and acrosome integrity of spermatozoa compared to the control. The mechanisms are that melatonin significantly reduces the level of ROS and preserves sperm mitochondrial membrane potential. Most importantly, the melatonin-treated cryopreserved chicken sperm after artificial insemination significantly increased the hatching rate of chicks compared to the control (p < 0.05). The results show that melatonin has a positive effect on the quality of the cryopreserved spermatozoa. These results provide the theoretical and practical basis for using melatonin to improve Beijing You Chicken conservation, and they may also be applicable to poultry as a whole. [ABSTRACT FROM AUTHOR]

Sperm competition and cryptic female choice (CFC) are 2 significant mechanisms of postcopulatory sexual selection that greatly impact fertilization success in various species. Despite extensive research has conducted on sperm competition and the evolution of sperm traits in internal fertilization, our understanding of the female preferences in selecting sperm is still limited. Here, we aimed to investigate the characteristics of CFC in chickens by utilizing artificial insemination with mixed semen to control for variations in male fertilization success caused by female perception of male quality and mating order. Our results revealed that the offspring from multiple-mated females exhibited mixed paternity. Although the males had an equal number of viable sperm, 1 male consistently exhibited a 15% higher success rate on average, regardless of whether the insemination was performed with fresh or diluted semen. This result suggested that this male demonstrates superior performance in sperm competition, and exhibited a potential advantage in fertilization success. While the dominant male generally made a greater genetic contribution to most offspring, the degree of this advantage varied greatly, ranging from 11.11 to 75%. Furthermore, our study provided evidence of female preferences influenced the precedence of sperm from certain males over others. Interestingly, this bias is not consistently observed among all individuals, as offspring derived from some females were predominantly sired by an overall disadvantaged male while others were predominantly by a different disadvantaged male. Overall, these results underscored the complex processes involved in sperm selection and emphasized the importance of females in sexual selection theory. [ABSTRACT FROM AUTHOR]

Background: In poultry, the smooth transition of follicles from the preovulatory-to-postovulatory phase impacts egg production in hens and can benefit the poultry industry. However, the regulatory mechanism underlying follicular ovulation in avians is a complex biological process that remains unclear. Results: Critical biochemical events involved in ovulation in domestic chickens (Gallus gallus) were evaluated by transcriptomics, proteomics, and in vitro assays. Comparative transcriptome analyses of the largest preovulatory follicle (F1) and postovulatory follicle (POF1) in continuous laying (CL) and intermittent laying (IL) chickens indicated the greatest difference between CL_F1 and IL_F1, with 950 differentially expressed genes (DEGs), and the smallest difference between CL_POF1 and IL_POF1, with 14 DEGs. Additionally, data-independent acquisition proteomics revealed 252 differentially abundant proteins between CL_F1 and IL_F1. Perivitelline membrane synthesis, steroid biosynthesis, lysosomes, and oxidative phosphorylation were identified as pivotal pathways contributing to ovulation regulation. In particular, the regulation of zona pellucida sperm-binding protein 3, plasminogen activator, cathepsin A, and lactate dehydrogenase A (LDHA) was shown to be essential for ovulation. Furthermore, the inhibition of LDHA decreased cell viability and promoted apoptosis of ovarian follicles in vitro. Conclusions: This study reveals several important biochemical events involved in the process of ovulation, as well as crucial role of LDHA. These findings improve our understanding of ovulation and its regulatory mechanisms in avian species. [ABSTRACT FROM AUTHOR]

Background: Embryonic diapause (dormancy) is a state of temporary arrest of embryonic development that is triggered by unfavorable conditions and serves as an evolutionary strategy to ensure reproductive survival. Unlike maternally-controlled embryonic diapause in mammals, chicken embryonic diapause is critically dependent on the environmental temperature. However, the molecular control of diapause in avian species remains largely uncharacterized. In this study, we evaluated the dynamic transcriptomic and phosphoproteomic profiles of chicken embryos in pre-diapause, diapause, and reactivated states. Results: Our data demonstrated a characteristic gene expression pattern in effects on cell survival-associated and stress response signaling pathways. Unlike mammalian diapause, mTOR signaling is not responsible for chicken diapause. However, cold stress responsive genes, such as IRF1, were identified as key regulators of diapause. Further in vitro investigation showed that cold stress-induced transcription of IRF1 was dependent on the PKC-NF-κB signaling pathway, providing a mechanism for proliferation arrest during diapause. Consistently, in vivo overexpression of IRF1 in diapause embryos blocked reactivation after restoration of developmental temperatures. Conclusions: We concluded that embryonic diapause in chicken is characterized by proliferation arrest, which is the same with other spices. However, chicken embryonic diapause is strictly correlated with the cold stress signal and mediated by PKC-NF-κB-IRF1 signaling, which distinguish chicken diapause from the mTOR based diapause in mammals. [ABSTRACT FROM AUTHOR]

Summary: Chicken plumage color, the genetic basis of which is often affected by epistasis, has long interested scientists. In the current study, a population of complex epistasis was constructed by crossing dominant White Leghorn chickens with recessive white feather chickens. Through a genome‐wide association study, we identified single nucleotide polymorphisms and genes significantly associated with white and colored plumage in hens at different developmental stages. Interestingly, white plumage in adulthood was associated with the recessive white feather gene (TYR), whereas white feathers at birth stage were associated with the dominant white feather gene (PMEL), indicating age‐related roles for these genes. TYR was shown to exert an epistatic effect on PMEL in adult hens. Additionally, TYR had an epistatic effect on barred plumage, while barred plumage had an epistatic effect on black plumage. TYR had no epistatic effect on the yellow plumage. We confirmed that the barred plumage gene is CDKN2A, as reported in previous studies. Golgb1 and REEP3, which play important roles in the Golgi network and affect the formation of feather pigments, are important candidate genes for yellow plumage. The candidate genes for black plumage are CAMKK1 and IFT22. Further research is warranted to elucidate the molecular mechanisms underlying these traits. [ABSTRACT FROM AUTHOR]

An amendment to this paper has been published and can be accessed via a link at the top of the paper. [ABSTRACT FROM AUTHOR]

Background: Female sperm storage (FSS), the maintenance of sperm inside the female reproductive tract for an extended period of time, is pervasive among organisms with internal fertilization. Because FSS enables asynchronous mating and fertilization, it could be extremely important to reproduction. However, the physiological mechanisms underlying prolonged preservation and maintenance are poorly understood. Here, we used chicken, a typical oviparous animal, to determine the mechanisms ensuring sperm functionality in sperm storage tubules (SSTs). Results: We performed an insemination experiment on over two thousand hens at two periods, and found that the FSS capabilities varied widely among individuals. Except for the differences in the SST density between the two groups with distinct FSS abilities, we quantitatively profiled small-molecule metabolites derived from SST cells, and identified 28 metabolites with differential expression. In particular, high levels of lipids, fatty acids and lipid peroxidation product were observed in hens with low FSS capability. Pathway analysis showed that these differential metabolites were significantly enriched in the biosynthesis of unsaturated fatty acids. Moreover, we detected the total antioxidant capacity and lipid peroxidation level of SSTs, and found that chickens with a lower FSS ability had a significantly higher content of lipid peroxidation end-product, which was 2.4-fold greater than chickens with a higher FSS capability, and no significant difference was found in the total antioxidant capacity between these two groups. Conclusions: Our findings reveal that the long-term storage of sperm and the maintenance of their function in the female reproductive tract require an adequate microenvironment. The superabundance of fatty acids secreted by SST cells had detrimental effects on sperm storage in the female reproductive tract. Lipid peroxidation produces toxic biological substances that may cause irreversible damage to resident spermatozoa, resulting in short-term sperm retention and decreased fertility. Our findings provide new avenues for studying sperm storage and sustaining fertility. [ABSTRACT FROM AUTHOR]

The Huiyang beard chicken is a well-known Chinese local breed known for its elongated feathers gathered from both sides of the face (muffs) and below the beak (beard), as well as short wattles (SW). The muff and beard (Mb) mutation is caused by ectopic upregulation of the homeobox B8 ( HOXB8 ) gene in the mandibular skin; and the chi-square test showed a significant correlation between SW and Mb genotypes. However, the underlying molecular mechanisms that regulate Mb and SW variations remain unclear. In this study, we investigated the transcriptomes of the mandibular skin and wattles of chickens with and without the Mb genotype to elucidate the molecular basis of these traits. Our results show that HOXB8 is expressed at significantly higher levels in both the mandibular skin and wattles of Mb chickens than in those of wild-type chickens, indicating that HOXB8 regulates both the Mb and SW phenotypes. Key genes for keratin synthesis were highly expressed in the mandibular skin of Mb chickens, suggesting that HOXB8 may play a role in feather development. In wattles, changes in the expression of extracellular matrix synthesis genes may contribute to SW traits. DNA-binding motif analyses revealed that differentially expressed genes were likely to be directly regulated by HOXB8 binding, indicating that HOXB8 may directly or indirectly regulate feather follicle development and wattle growth. Our study identified both known and novel targets, including several genes not previously implicated in feather development and mesenchymal formation. These findings provide insights into the molecular mechanisms of skin appendage variation in birds and offer potential applications in breeding poultry breeds with unique phenotypes. [ABSTRACT FROM AUTHOR]

The eggshell blueness is an interesting object for chicken genetic studies and blue-shelled chicken industry, especially after the discovery of the causative mutation of chicken blue eggshell. In the present study, genome wide association study (GWAS) was conducted in Chinese Dongxiang blue-shelled chicken underlying four traits of blue eggshell pigments: quantity of biliverdin (QB), quantity of protoporphyrin (QP), quantity of total pigment (QT), and color density trait (CD). A total of 139 individuals were randomly collected for GWAS. We detected two SNPs in genome-wise significance and 35 in suggestive significance, 24 out of the 37 SNP were located either within intron/exon or near 15 genes in a range of ~1.17 Mb on GGA21. For further confirmation of the identified SNP loci by GWAS, the follow-up replication studies were performed in two populations. A total of 146 individuals of the second generation derived from the former GWAS population, as well as 280 individuals from an alternative independent population were employed for genotyping by MALDI-TOF MS in a genotype-phenotype association study. Eighteen SNPs evenly distributed on the GGA21 significant region were successfully genotyped in the two populations, of which 4 and 6 SNP loci were shown significantly associated with QB, QT and QP in the two repeat populations, respectively. Further, the SNPs were narrowed down to a region of ~ 653.819 Kb on GGA21 that harbors five candidate genes: AJAP1, TNFRSF9, C1ORF174, CAMTA1, and CEP104. Shell gland of chickens laying dark and light blue eggshell was chosen for detection of mRNA expression of the five candidate genes. The results showed differential expression levels of these genes in the two groups. The specific function of these genes has not yet been defined clearly in chickens and further in-depth studies are needed to explore the new functional role in chicken eggshell blueness. [ABSTRACT FROM AUTHOR]

Piao chicken, a Chinese indigenous rumpless chicken breed, lacks pygostyle, caudal vertebra, uropygial gland and tail feathers. The rumplessness in Piao chicken presents an autosomal dominant inheritance pattern. However, the molecular genetic mechanisms underlying the rumplessness in Piao chicken remains unclear. In this study, whole-genome resequencing was performed for 146 individuals from 10 chicken breeds, including 9 tailed chicken breeds and Piao rumpless breed. Tailbone CT scan for Piao chickens and WL chickens, revealed that some Piao chicken tails were normal in number, and for a few Piao chickens tail length and tail bone numbers were between the rumpless and the normal tailed chickens. The results showed that the rumpless phenotype has not been completely fixed in Piao chicken breed. Using selection signature analysis and structural variation detection, we found a 4174 bp deletion located in the upstream region of IRX1 gene on chromosome 2 related to rumpless phenotype. Structural variation genotyping showed that the deletion was present in all 32 rumpless Piao chickens (del/del, wild/del) and absent from all 112 tailed chickens included in the dataset for the other 9 breeds and 2 tailed Piao chickens (wild/wild). In summary, all rumpless Piao chickens tested here carry this deletion mutation, to show a complete linkage association with rumplessness trait. We suggested that the 4174 bp deletion could be causative for rumpless phenotype in Piao chicken since this is the only mutation to show the complete linkage disequilibrium with rumplessness on whole genome level across all of 146 chickens from the 10 breeds. This study could facilitate a better understanding of the genetic characteristics of Piao chicken. • Piao chicken structural variation detection. • In Piao Chicken, a deletion variant was found to be associated with rumpless. • This study could facilitate a better understanding of tail degeneration in chicken. [ABSTRACT FROM AUTHOR]

Under the pressure of natural and artificial selection, domestic animals, including chickens, have evolved unique mechanisms of genetic adaptations such as high-altitude adaptation, hot and arid climate adaptation, and desert adaptation. Here, we investigated the genetic basis of cold tolerance in chicken by integrating whole-genome and transcriptome sequencing technologies. Genome-wide comparative analyses of 118 chickens living in different latitudes showed 46 genes and several pathways that may be involved in cold adaptation. The results of the functional enrichment analysis of differentially expressed genes proved the important role of metabolic pathways and immune-related pathways in cold tolerance in chickens. The subsequent integration of whole genome and transcriptome sequencing technology further identified six genes — dnah5 (dynein axonemal heavy chain 5), ptgs2 (prostaglandin-endoperoxide synthase 2), inhba (inhibin beta A subunit), irx2 (iroquois homeobox 2), ensgalg00000054917 , and ensgalg00000046652 — requiring more detailed studies. In addition, we also discovered different allele frequency distributions of five SNPs (single nucleotide polymorphisms) within ptgs2 and nine SNPs within dnah5 in chickens in different latitudes, suggesting strong selective pressure of these two genes in chickens. We provide a novel insight into the genetic adaptation in chickens to cold environments, and provide a reference for evaluating and developing adaptive chicken breeds in cold environments. • The genetic basis of cold tolerance in indigenous chickens long-living in the cold environment were explored. • Two important genes associated with cold tolerance were identified. • Provide a precious source for evaluating and developing adaptive chicken breeds to face climate change. [ABSTRACT FROM AUTHOR]

Polydactyly is one of the most common hereditary congenital limb malformations in chickens and other vertebrates. The zone of polarizing activity regulatory sequence (ZRS) is critical for the development of polydactyly. The causative mutation of polydactyly in the Silkie chicken has been mapped to the ZRS; however, the causative mutations of other chicken breeds are yet to be established. To understand whether the same mutation decides the polydactyly phenotype in other chicken breeds, we detected the single-nucleotide polymorphism in 26 different chicken breeds, specifically, 24 Chinese indigenous breeds and 2 European breeds. The mutation was found to have fully penetrated chickens with polydactyly in China, indicating that it is causative for polydactyly in Chinese indigenous chickens. In comparison, the mutation showed no association with polydactyly in Houdan chickens, which originate from France, Europe. Based on the different morphology of polydactyly in Chinese and European breeds, we assumed that the trait might be attributable to different genetic foundations. Therefore, we subsequently performed genome-wide association analysis (GWAS) to locate the region associated with polydactyly. As a result, a ~0.39 Mb genomic region on GGA2p was identified. The region contains six candidate genes, with the causative mutation found in Chinese indigenous breeds also being located in this region. Our results demonstrate that polydactyly in chickens from China and Europe is caused by two independent mutation events that are closely located in the chicken genome. [ABSTRACT FROM AUTHOR]

Genomic imprinting is a phenomenon characterized by parent-of-origin-specific gene expression. While widely documented in viviparous mammals and plants, imprinting in oviparous birds remains controversial. Because genomic imprinting is temporal- and tissue-specific, we investigated this phenomenon only in the brain tissues of 1-day-old chickens (Gallus gallus). We used next-generation sequencing technology to compare four transcriptomes pooled from 11 chickens, generated from reciprocally crossed families, to the DNA sequences of their parents. Candidate imprinted genes were then selected from these sequence alignments and subjected to verification experiments that excluded all but one SNP. Subsequent experiments performed with two new sets of reciprocally crossed families resulted in the exclusion of that candidate SNP as well. Attempts to find evidence of genomic imprinting from long non-coding RNAs yielded negative results. We therefore conclude that genomic imprinting is absent in the brains of 1-day-old chickens. However, due to the temporal and tissue specificity of imprinting, our results cannot be extended to all growth stages and tissue types. [ABSTRACT FROM AUTHOR]

The Silky Fowl (SF) is known for its special phenotypes and atypical distribution of melanocytes among internal organs. Although the genes associated with melanocyte migration have been investigated substantially, there is little information on the postnatal distribution of melanocytes in inner organs and the effect of hyperpigmentation on the development of SF. Here, we analyzed melanocyte distribution in 26 tissues or organs on postnatal day 1 and weeks 2, 3, 4, 6, 10, and 23. Except for the liver, pancreas, pituitary gland, and adrenal gland, melanocytes were distributed throughout the body, primarily around blood vessels. Interaction between melanocytes and the tissue cells was observed, and melanin was transported by filopodia delivery through engulfed and internalized membrane-encapsulated melanosomes. SFs less than 10 weeks old have lower indices of spleen, thymus, and bursa of Fabricius than White Leghorns (WLs). The expression levels of interferon-γ and interlukin-4 genes in the spleen, and serum antibody levels against H5N1 and infectious bursal disease virus were lower in SF than in WL. We also found immune organ developmental difference between Black-boned and non-Black- boned chickens from SFs and WLs hybrid F2 population. However, degeneration of the thymus and bursa of Fabricius occurred later in SF than in WL after sexual maturity. Analysis of apoptotic cells and apoptosis-associated Bax and Bcl-2 proteins indicated that apoptosis is involved in degeneration of the thymus and bursa of Fabricius. Therefore, these results suggest that hyperpigmentation in SF may have a close relationship with immune development in SF, which can provide an important animal model to investigate the roles of melanocyte. [ABSTRACT FROM AUTHOR]

Background: Gene expression variation is a key underlying factor influencing phenotypic variation, and can occur via cis- or trans-regulation. To understand the role of cis- and trans-regulatory variation on population divergence in chicken, we developed reciprocal crosses of two chicken breeds, White Leghorn and Cornish Game, which exhibit major differences in body size and reproductive traits, and used them to determine the degree of cis versus trans variation in the brain, liver, and muscle tissue of male and female 1-day-old specimens. Results: We provided an overview of how transcriptomes are regulated in hybrid progenies of two contrasting breeds based on allele specific expression analysis. Compared with cis-regulatory divergence, trans-acting genes were more extensive in the chicken genome. In addition, considerable compensatory cis- and trans-regulatory changes exist in the chicken genome. Most importantly, stronger purifying selection was observed on genes regulated by trans-variations than in genes regulated by the cis elements. Conclusions: We present a pipeline to explore allele-specific expression in hybrid progenies of inbred lines without a specific reference genome. Our research is the first study to describe the regulatory divergence between two contrasting breeds. The results suggest that artificial selection associated with domestication in chicken could have acted more on trans-regulatory divergence than on cis-regulatory divergence. [ABSTRACT FROM AUTHOR]