Genetic parameters, reciprocal cross differences, and age-related heterosis of egg-laying performance in chickens.

BACKGROUND: Egg-laying performance is economically important in poultry breeding programs. Crossbreeding between indigenous and elite commercial lines to exploit heterosis has been an upward trend in traditional layer breeding for niche markets. The objective of this study was to analyse the genetic background and to estimate the heterosis of longitudinal egg-laying traits in reciprocal crosses between an indigenous Beijing-You and an elite commercial White Leghorn layer line. Egg weights were measured for the first three eggs, monthly from 28 to 76 weeks of age, and at 86 and 100 weeks of age. Egg quality traits were measured at 32, 54, 72, 86, and 100 weeks of age. Egg production traits were measured from the start of lay until 43, 72, and 100 weeks of age. Heritabilities and phenotypic and genetic correlations were estimated. Heterosis was estimated as the percentage difference of performance of a crossbred from that of the parental average. Reciprocal cross differences were estimated as the difference between the reciprocal crossbreds as a percentage of the parental average. RESULTS: Estimates of heritability of egg weights ranged from 0.29 to 0.75. Estimates of genetic correlations between egg weights at different ages ranged from 0.72 to 1.00. Estimates of heritability for cumulative egg numbers until 43, 72, and 100 weeks of age were around 0.15. Estimates of heterosis for egg weight and cumulative egg number increased with age, ranging from 1.0 to 9.0% and from 1.4 to 11.6%, respectively. From 72 to 100 weeks of age, crossbreds produced more eggs per week than the superior parent White Leghorn (3.5 eggs for White Leghorn, 3.8 and 3.9 eggs for crossbreds). Heterosis for eggshell thickness ranged from 2.7 to 6.6% when using Beijing-You as the sire breed. No significant difference between reciprocal crosses was observed for the investigated traits, except for eggshell strength at 54 weeks of age. CONCLUSIONS: The heterosis was substantial for egg weight and cumulative egg number, and increased with age, suggesting that non-additive genetic effects are important in crossbreds between the indigenous and elite breeds. Generally, the crossbreds performed similar to or even outperformed the commercial White Leghorns for egg production persistency.

The mRNA-lncRNA landscape of multiple tissues uncovers key regulators and molecular pathways that underlie heterosis for feed intake and efficiency in laying chickens.

BACKGROUND: Heterosis is routinely exploited to improve animal performance. However, heterosis and its underlying molecular mechanism for feed intake and efficiency have been rarely explored in chickens. Feed efficiency continues to be an important breeding goal trait since feed accounts for 60 to 70% of the total production costs in poultry. Here, we profiled the mRNA-lncRNA landscape of 96 samples of the hypothalamus, liver and duodenum mucosa from White Leghorn (WL), Beijing-You chicken (YY), and their reciprocal crosses (WY and YW) to elucidate the regulatory mechanisms of heterosis. RESULTS: We observed negative heterosis for both feed intake and residual feed intake (RFI) in YW during the laying period from 43 to 46 weeks of age. Analysis of the global expression pattern showed that non-additivity was a major component of the inheritance of gene expression in the three tissues for YW but not for WY. The YW-specific non-additively expressed genes (YWG) and lncRNA (YWL) dominated the total number of non-additively expressed genes and lncRNA in the hypothalamus and duodenum mucosa. Enrichment analysis of YWG showed that mitochondria components and oxidation phosphorylation (OXPHOS) pathways were shared among the three tissues. The OXPHOS pathway was enriched by target genes for YWL with non-additive inheritance of expression in the liver and duodenum mucosa. Weighted gene co-expression network analysis revealed divergent co-expression modules associated with feed intake and RFI in the three tissues from WL, YW, and YY. Among the negatively related modules, the OXPHOS pathway was enriched by hub genes in the three tissues, which supports the critical role of oxidative phosphorylation. Furthermore, protein quantification of ATP5I was highly consistent with ATP5I expression in the liver, which suggests that, in crossbred YW, non-additive gene expression is down-regulated and decreases ATP production through oxidative phosphorylation, resulting in negative heterosis for feed intake and efficiency. CONCLUSIONS: Our results demonstrate that non-additively expressed genes and lncRNA involved in oxidative phosphorylation in the hypothalamus, liver, and duodenum mucosa are key regulators of the negative heterosis for feed intake and RFI in layer chickens. These findings should facilitate the rational choice of suitable parents for producing crossbred chickens.

Identification of miRNA Associated with Trichomonas gallinae Resistance in Pigeon (Columba livia).

Trichomonas gallinae (T. gallinae) has a great influence on the pigeon industry. Pigeons display different resistance abilities to T. gallinae, so the study of the molecular mechanism of resistance is necessary in breeding disease resistant lines. MiRNA plays important roles in the immune response, but there are still no reports of miRNA regulating trichomonosis resistance. We used small RNA sequencing technology to characterize miRNA profiles in different groups. T. gallinae was nasally inoculated in one day old squabs, and according to the infection status, the groups were divided into control (C), susceptible (S) and tolerant (T) groups. We identified 2429 miRNAs in total, including 1162 known miRNAs and 1267 new miRNAs. In a comparison among the C, S and T groups, the target genes of differentially expressed miRNAs were analyzed via GO and KEGG annotation. The results showed that the target genes were enriched in immune-response-related pathways. This indicated that the differentially expressed miRNAs had a critical influence on T. gallinae infection. Novel_miR_741, which could inhibit the expression of PRKCQ, was down-regulated in the T group compared to the C group. It was proven that a decreased novel_miR_741 expression would increase the expression of PRKCQ and increase the immune response. This study brings new insights into understanding the mechanism of trichomonosis resistance.

Genome-wide run of homozygosity analysis reveals candidate genomic regions associated with environmental adaptations of Tibetan native chickens.

BACKGROUND: In Tibet, the two most important breeds are Tibetan chicken and Lhasa white chicken, and the duo exhibit specific adaptations to the high altitude thereby supplying proteins for humans living in the plateau. These breeds are partly included in the conservation plans because they represent important chicken genetic resources. However, the genetic diversity of these chickens is rarely investigated. Based on whole-genome sequencing data of 113 chickens from 4 populations of Tibetan chicken including Shigatse (SH), Nyemo (NM), Dagze (DZ) and Nyingchi (LZ), as well as Lhasa white (LW) chicken breed, we investigated the genetic diversity of these chicken breeds by genetic differentiation, run of homozygosity (ROH), genomic inbreeding and selection signature analyses. RESULTS: Our results revealed high genetic diversity across the five chicken populations. The linkage disequilibrium decay was highest in LZ, while subtle genetic differentiation was found between LZ and other populations (Fst ranging from 0.05 to 0.10). Furthermore, the highest ROH-based inbreeding estimate (FROH) of 0.11 was observed in LZ. In other populations, the FROH ranged from 0.04 to 0.06. In total, 74, 111, 62, 42 and 54 ROH islands containing SNPs ranked top 1% for concurrency were identified in SH, NM, DZ, LZ and LW, respectively. Genes common to the ROH islands in the five populations included BDNF, CCDC34, LGR4, LIN7C, GLS, LOC101747789, MYO1B, STAT1 and STAT4. This suggested their essential roles in adaptation of the chickens. We also identified a common candidate genomic region harboring AMY2A, NTNG1 and VAV3 genes in all populations. These genes had been implicated in digestion, neurite growth and high-altitude adaptation. CONCLUSIONS: High genetic diversity is observed in Tibetan native chickens. Inbreeding is more intense in the Nyingchi population which is also genetically distant from other chicken populations. Candidate genes in ROH islands are likely to be the drivers of adaptation to high altitude exhibited by the five Tibetan native chicken populations. Our findings contribute to the understanding of genetic diversity offer valuable insights for the genetic mechanism of adaptation, and provide veritable tools that can help in the design and implementation of breeding and conservation strategies for Tibetan native chickens.

Seminal Plasma Proteome as an Indicator of Sperm Dysfunction and Low Sperm Motility in Chickens.

Molecular mechanisms underlying sperm motility have not been fully explained, particularly in chickens. The objective was to identify seminal plasma proteins associated with chicken sperm motility by comparing the seminal plasma proteomic profile of roosters with low sperm motility (LSM, n = 4) and high sperm motility (HSM, n = 4). Using a label-free MS-based method, a total of 522 seminal plasma proteins were identified, including 386 (∼74%) previously reported and 136 novel ones. A total of 70 differentially abundant proteins were defined, including 48 more-abundant, 15 less-abundant, and seven proteins unique to the LSM group (specific proteins). Key secretory proteins like less-abundant adhesion G-protein coupled receptor G2 (ADGRG2) and more-abundant serine peptidase inhibitor Kazal-type 2 (SPINK2) in the LSM suggested that the corresponding secretory tissues played a crucial role in maintaining sperm motility. Majority (80%) of the more-abundant and five specific proteins were annotated to the cytoplasmic domain which might be a result of higher plasma membrane damage and acrosome dysfunction in LSM. Additionally, more-abundant mitochondrial proteins were detected in LSM seminal plasma associated with lower spermatozoa mitochondrial membrane potential (ΔΨm) and ATP concentrations. Further studies showed that the spermatozoa might be suffering from oxidative stress, as the amount of spermatozoa reactive oxygen species (ROS) were largely enhanced, seminal malondialdehyde (MDA) concentrations were increased, and the seminal plasma total antioxidant capacity (T-AOC) were decreased. Our study provides an additional catalogue of chicken seminal plasma proteome and supports the idea that seminal plasma could be as an indicator of spermatozoa physiology. More-abundant of acrosome, mitochondria and sperm cytoskeleton proteins in the seminal plasma could be a marker of sperm dysfunction and loss of motility. The degeneration of spermatozoa caused by the reduced seminal T-AOC and enhanced oxidative stress might be potential determinants of low sperm motility. These results could extend our understanding of sperm motility and sperm physiology regulation.

Identification of a novel differentially methylated region adjacent to ATG16L2 in lung cancer cells using methyl-CpG binding domain protein-enriched genome sequencing.

Lung cancer is the most common cancer worldwide. Epigenetic modifications like DNA methylation play fundamental roles in the dynamic process of lung cancer. The objective of this study was to use methyl-CpG binding domain protein-enriched genome sequencing (MBD-Seq) to identify novel and high-confidence DNA methylation in lung tumor. We first compared the whole-genome DNA methylation of three lung cancer cell lines, including A549, H1299, and SK-MES-1, against BEAS-2B, a lung/bronchial normal epithelial cell line. We then used pyrosequencing and OneStep qMethyl kit methods to verify the results in the cell line specimens. MBD-Seq identified 279, 8046, and 22 887 differentially methylated regions (DMRs), respectively, with 120 common DMRs among three comparison groups. Three DMRs were consistent with the MBD-Seq results by both pyrosequencing and OneStep qMethyl validations. Furthermore, OneStep qMethyl kit was also performed for functional validation of these three potential DMRs in sputum DNA from clinical participants. We successfully identified one new DMR adjacent to ATG16L2. The novel DMR might have an important function in lung carcinogenesis. Further validation of the finding in clinical specimens of lung cancer patients and functional analysis of this novel DMR in the development of lung cancer through transcriptional silencing of ATG16L2 are warranted.

Supplementation with embryo chicken egg extract improves exercise performance and exerts anti-fatigue effects via AMPK/mTOR signalling pathway in mice.

BACKGROUND: Embryo chicken egg is a nutritional supplement that has been used to enhance physical fitness and promote wound healing according to traditional Chinese medicine for many years. In this study, we evaluated the effects of embryo chicken egg extract (ECE) on the exercise performance and fatigue in mice and the underlying mechanisms. RESULTS: The results indicated that ECE can prolong the exhaustive swimming time, decrease lactic acid, blood urea nitrogen, creatine kinase, and malondialdehyde levels, and increase superoxide dismutase, glutathione peroxidase, and glycogen levels. Additionally, ECE can also regulate the balance of oxidative stress via the adenosine monophosphate activated protein kinase/mammalian target of rapamycin signalling pathway. CONCLUSION: Taken together, these results showed that ECE can improve exercise performance and reduce physical fatigue in mice, which indicates that ECE can be used as a potential supplement to reduce physical fatigue. © 2020 Society of Chemical Industry.

Genome-wide characterization of copy number variations in the host genome in genetic resistance to Marek's disease using next generation sequencing.

BACKGROUND: Marek's disease (MD) is a highly neoplastic disease primarily affecting chickens, and remains as a chronic infectious disease that threatens the poultry industry. Copy number variation (CNV) has been examined in many species and is recognized as a major source of genetic variation that directly contributes to phenotypic variation such as resistance to infectious diseases. Two highly inbred chicken lines, 63 (MD-resistant) and 72 (MD-susceptible), as well as their F1 generation and six recombinant congenic strains (RCSs) with varied susceptibility to MD, are considered as ideal models to identify the complex mechanisms of genetic and molecular resistance to MD. RESULTS: In the present study, to unravel the potential genetic mechanisms underlying resistance to MD, we performed a genome-wide CNV detection using next generation sequencing on the inbred chicken lines with the assistance of CNVnator. As a result, a total of 1649 CNV regions (CNVRs) were successfully identified after merging all the nine datasets, of which 90 CNVRs were overlapped across all the chicken lines. Within these shared regions, 1360 harbored genes were identified. In addition, 55 and 44 CNVRs with 62 and 57 harbored genes were specifically identified in line 63 and 72, respectively. Bioinformatics analysis showed that the nearby genes were significantly enriched in 36 GO terms and 6 KEGG pathways including JAK/STAT signaling pathway. Ten CNVRs (nine deletions and one duplication) involved in 10 disease-related genes were selected for validation by using quantitative real-time PCR (qPCR), all of which were successfully confirmed. Finally, qPCR was also used to validate two deletion events in line 72 that were definitely normal in line 63. One high-confidence gene, IRF2 was identified as the most promising candidate gene underlying resistance and susceptibility to MD in view of its function and overlaps with data from previous study. CONCLUSIONS: Our findings provide valuable insights for understanding the genetic mechanism of resistance to MD and the identified gene and pathway could be considered as the subject of further functional characterization.

Single SNP- and pathway-based genome-wide association studies for beak deformity in chickens using high-density 600K SNP arrays.

BACKGROUND: Beak deformity, typically expressed as the crossing of upper and lower mandibles, is found in several indigenous chicken breeds, including the Beijing-You chickens studied here. Beak deformity severely impairs the birds' growth and welfare. Although previous studies shed some light on the genetic regulation of this complex trait, the genetic basis of this malformation remains incompletely understood. RESULTS: In this study, single SNP- and pathway-based genome-wide association studies (GWASs) were performed using ROADTRIPS and SNP ratio test (SRT), respectively. A total of 48 birds with deformed beaks (case) and 48 normal birds (control) were genotyped using Affymetrix 600 K HD genotyping arrays. As a result, 95 individuals and 429,539 SNPs were obtained after quality control. The P-value was corrected by a Bonferroni adjustment based on linkage disequilibrium pruning. The single SNP-based association study identified one associated SNP with 5% genome-wide significance and seven suggestively associated SNPs. Four high-confidence genes, LOC421892, TDRD3, RET, and STMN1, were identified as the most promising candidate genes underlying this complex trait in view of their positions, functions, and overlaps with previous studies. The pathway-based association study highlighted the association of six pathways with beak deformity, including the calcium signaling pathway. CONCLUSIONS: Potentially useful candidate genes and pathways for beak deformity were identified, which should be the subject of further functional characterization.

Study on LOC426217 as a candidate gene for beak deformity in chicken.

BACKGROUND: The beak deformity (crossed beaks) was found in some indigenous chickens of China, such as Beijing-You (BJY), Qingyuan Partridge, and Huxu Chickens. Birds with deformed beaks have reduced feed intake and drinking, impeded growth rate, and poor production performance. Beak deformity reduces the economy of poultry industry and affects animal welfare as well. The genetic basis of this malformation remains incompletely understood. LOC426217, also named claw keratin-like, was the most up-regulated gene in the deformed beaks from a previous digital gene expression (DGE) analysis and was selected as an important candidate gene for further analysis. RESULTS: In the present study, quantitative real-time PCR (qRT-PCR) was firstly performed to determine the expression pattern of LOC426217 gene in deformed and normal beaks to verify the DGE results. Tissue-specific expression profile of this gene in 14 tissues was also determined using qRT-PCR. The LOC426217 was amplified from the genomic DNA of 171 deformed and 164 normal beaks, and sequenced to detect the single nucleotide polymorphisms (SNPs). The results showed that LOC426217 was significantly high-expressed in the deformed beaks, which was in good agreement with the DGE results. This gene was specifically high-expressed in beaks than other tissues. Eight SNPs were detected in LOC426217: -62G > T, 24 T > C, 36G > C, 192A > T, 204C > T, 222 T > C, 285G > T, and 363 T > C. Genotype frequency of G-62 T, T24C, G36C, T222C, and T363C loci was significant different between deformed and normal beaks. Haplotype analysis revealed one block with SNPs T24C and G36C, and one block with SNPs A192T, C204T, T222C, and G285T in normal birds, while the block with SNPs G36C and A192T in deformed ones. CONCLUSIONS: It was concluded from these results that the over-expression of LOC426217 in the beak maybe related to the malformation. The polymorphisms of LOC426217 gene were associated with the beak deformity trait where the SNPs of G-62 T, T24C, G36C, T222C, and T363C loci maybe used as markers. The specific haplotype block in deformed birds may be a potential linkage marker for this trait.

Folate supplementation modifies CCAAT/enhancer-binding protein α methylation to mediate differentiation of preadipocytes in chickens.

Folate, an essential vitamin participating in 1-carbon metabolism leading to a methyl donor function, is a key factor inducing epigenetic changes. This study sought to determine if folate influences the methylation level of cytosine-guanine (CpG) islands in the promoters of critical adipogenic genes in chickens, and how this might affect gene expression and differentiation of preadipocytes in vitro. Preadipocytes were treated with 0 to 16 mg/L of folate during the induction of differentiation, and cell proliferation and lipid accumulation were assessed. The folate supplementation resulted in enhanced cell proliferation and decreased content of lipid per adipocyte at d 6 of differentiation. The effects of folate on relative expression of genes critical for adipocyte differentiation and 1-carbon metabolism were measured by quantitative reverse-transcription PCR. Folate caused a dose-dependent decrease in transcript abundance of peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer-binding protein α (C/EBPα) gene expression, and the downstream enzyme fatty acid synthase; in contrast, expression of DNA (cytosine-5)-methyltransferase and methylenetetrahydrofolate reductase was obviously upregulated at d 6 of differentiation (P < 0.05). The DNA methylation was examined with the bisulfite sequencing PCR method. Overall CpG methylation in the C/EBPα gene promoter region was 21.8% lower (P < 0.05) and the gene's expression was 2.7-fold higher in the absence of folate, compared with cells treated with 16 mg/L of folate, whereas methylation of the PPARγ promoter was not affected. Overall, the results show that folate increased the proliferation of adipocytes but reduced per-cell lipid accumulation, thereby influencing differentiation; it increased expression of genes involved in 1-carbon metabolism resulting in greater methylation of the C/EBPα promoter during differentiation and decreased that gene's expression, perhaps accounting for decreased expression of PPARγ.

Effects of Supplementing Drinking Water of Parental Pigeons with Enterococcus faecium and Bacillus subtilis on Antibody Levels and Microbiomes in Squabs.

Enterococcus faecium (E. faecium) and Bacillus subtilis (B. subtilis) are widely used as probiotics to improve performance in animal production, but there have been few reports of their impacts on pigeon milk. In this study, twenty-four pairs of parental pigeons were randomly divided into four groups, with six replicates, and each pair feeding three squabs. The control group drank normal water. The E. faecium group, B. subtilis group, and mixed group drank water supplemented with 3 × 106 CFU/mL E. faecium, 2 × 107 CFU/mL B. subtilis, and a mixture of these two probiotics, respectively. The experiment lasted 19 days. The results demonstrated that the IgA and IgG levels were significantly higher in the milk of Group D pigeons than in the other groups. At the phylum level, Fimicutes, Actinobacteria, and Bacteroidetes were the three main phyla identified. At the genus level, Lactobacillus, Bifidobacterium, Veillonella, and Enterococcus were the four main genera identified. In conclusion, drinking water supplemented with E. faecium and B. subtilis could improve immunoglobulin levels in pigeon milk, and this could increase the ability of squabs to resist disease. E. faecium and B. subtilis could be used as probiotics in the pigeon industry.

Research Note: Genetic parameters estimation of egg quality traits in Rhode Island Red and White Leghorn chickens.

The continuous increasing demand for egg quality and quantity, and the expanding market share have enabled the egg industry to achieve significant benefits through genetic improvement. This study aims to estimate the genetic parameters and explore selectable breeding traits in the purebred Rhode Island Red (RIR) and White Leghorn (WL), which are 2 high-yielding layer breeds, and better understand their underlying genetic basis and accelerate genetic progress. The DMU software was utilized to analyze 12 egg quality traits, including egg length (EL), egg width (EW), egg shape index (ESI), egg weight (EWT), albumen height (AH), yolk color (YC), Haugh unit (HU), yolk weight (YW), albumen weight (AW), albumen-to-egg weight ratio (AWR), yolk-to-albumen ratio (YAR), and yolk-to-egg weight ratio (YWR). In RIR, the heritability of egg quality traits ranged from 0.196 to 0.427, while the repeatability ranged from 0.395 to 0.668. In WL, the heritability of egg quality traits ranged from 0.203 to 0.347, and the repeatability ranged from 0.424 to 0.656. In both RIR and WL, highly strong genetic correlations were observed between AW and EW, as well as between AW and EWT. The genetic correlations for AW and EW were 0.902 in RIR and 0.864 in WL, while the genetic correlations for AW and EWT were 0.981 in RIR and 0.960 in WL. The egg quality traits in both breeds showed moderate heritability, indicating great genetic potential for improvement through selective breeding. This can help breeders meet the increasingly diverse egg preferences of consumers through genetic selection. Additionally, there is a highly strong correlation between egg width/egg weight, and albumen weight in both breeds. In practical production, it is feasible to estimate albumen weight by measuring egg width and egg weight, which can simplify the method for measuring albumen weight. In conclusions, our finding provided valuable insights into the genetic architecture of egg quality traits in RIR and WL chickens. They help our understanding of the potential for genetic improvement of these traits through selective breeding programs.

Research note: differential heterosis of spent laying hens' carcass characteristics and meat quality in reciprocal crosses between White Leghorn and Beijing-You chickens.

Hybridization is used extensively in commercial layer production. However, heterosis for carcass performance and meat quality of spent laying hens remains unclear, especially under the trend of extended laying cycles. In this study, indigenous Beijing-You chickens (Y) and elite White Leghorn layers (W) were selected to generate purebreds (WW and YY) and reciprocal crosses (WY and YW). Data on traits including carcass compositions, meat quality, and main nutrients for breast muscle were collected when chickens were fed to 100 wk of age. Results showed that body weight (BW) and dressed weight for WY and YW with positive heterosis were significantly higher than WW (P < 0.05). YW had the heaviest breast and thigh of 232.28 g and 278.48 g, respectively. The abdominal fat weight for WY and YW were greatly higher than that for WW (P > 0.05). The yields of carcass compositions, including the dressed yield, half eviscerated yield, eviscerated yield, breast yield and thigh yield, did not differ among the four genetic groups (P > 0.05), except for the yield of abdominal fat. The largest heterosis differences appeared in breast weight (12.26% in YW vs. -0.46% in WY) and abdominal fat yield (15.26% in YW vs. 24.55% in WY). Although BW for crossbreds were similar, the specific parts of the carcass between them were different. For meat quality, WY had negative heterosis (P < 0.05) with the lowest lightness and yellowness, whereas YW had the completely opposite trend. Neither pH1h nor pH24h values had differences among purebreds and reciprocal crossbreds (P > 0.05). The drip loss and cooking loss were 4.01%-4.77% and 15.59%-21.31% respectively among the four genetic groups. The main nutrients of breast, including moisture, crude protein, intramuscular fat and unsaturated fatty acid, did not differ for purebreds and crossbreds (P > 0.05), except for saturated fatty acid. In general, the crossbreds even at the later laying period still showed divergent heterosis on carcass performance and meat characteristics. In view of the heterosis, Beijing-You chickens can be used as the sire line in the crossbreeding to improve carcass compositions of spent hens.

Regulation of winter wheat-originated antifreeze glycoprotein on rooster spermatozoa freezability.

The freezability of chicken spermatozoa is low, therefore, effective cryoprotectants is desiderated. Antifreeze proteins (AFPs) are widely found in cold-tolerant species and help them to survive in freezing environments. This study was the first to evaluate the effects of different concentrations of plant-originated antifreeze glycoprotein (AFGP) (0, 0.1, 1, and 5 µg/mL) on post-thawed sperm motion characteristics, morphology, mitochondrial function, antioxidant activity, and fertilizing potential in chickens. Results showed that the total motility of 0.1 to 1 µg/mL AFGP groups were significantly higher than those of the 5 µg/mL AFGP group (P < 0.05). The post-thawed sperm viability of 0.1 µg/mL AFGP group was significantly higher than any of test groups (P < 0.05). Higher abnormal morphology rate of post-thawed sperm was observed in the control group (0 µg/mL AFGP) than in the 0.1, 1, and 5 µg/mL AFGP groups (P < 0.05). The concentrations of malondialdehyde (MDA) decreased gradually with the increase of AFGP concentration. ATP was significantly higher in the 0.1 and 1 µg/mL AFGP groups than those of control and any of test groups (P < 0.05). The 0.1 to 1 µg/mL AFGP groups had increased mitochondrial membrane potential (MMP) level (P > 0.05). The 0.1 µg/mL AFGP group had the highest average fertility (61.36%) compared with control group (57.02%) and any of test groups of chickens at 31 wk of age, and the 1 µg/mL AFGP group had the highest average fertility (37.72%) compared with control group (21.73%) and any of test groups of chickens at 65 wk of age. In conclusion, the results from this study suggest lower concentration of AFGP (0.1-1 µg/mL) showed positive effect for sperm function. This study inspires the continuous evaluation and seeking right way of adopting different kinds of AFPs in rooster semen cryopreservation.

Prevalence Study of Trichomonas gallinae in Domestic Pigeons in Northeastern Beijing and Experimental Model of Trichomoniasis in White King Squabs Measuring In Situ Apoptosis and Immune Factors in Crop and Esophagus.

Trichomonas gallinae (T. gallinae) is a flagellated protozoan and the causative agent of trichomoniasis, or canker, in birds. In the current study, the prevalence of T. gallinae was firstly investigated in five breeds. According to the results of the prevalence study, White King pigeons were selected as the experimental animals. A total of 135 White King squabs at one day of age were randomly divided into two groups and raised in separate isolators. The challenged group (N = 100) was challenged intranasally with 5 × 106 parasites/mL of the T. gallinae strain, and the control group (N = 35) was intranasally administered medium of equivalent volume. At 1, 2, 3 and 5 days post infection (DPIs), the crops and esophagi were collected for RNA extraction and formaldehyde fixation. The results showed that prevalence of T. gallinae in the five breeds ranged from 27.13% (White Carneau) to 43.14% (White King). After the challenge, mild microscopic lesions were observed in both tissues. Apoptosis rates were higher in the challenged group than in the control group at 2 and 5 DPIs in the crop and at 1, 2 and 7 DPIs in the esophagus. For both tissues, relative expression of IL-1ß increased dramatically at the beginning and decreased at 5 DPIs, and TGF-ß increased stably in the challenged group.

Multi-tissue transcriptome profiling linked the association between tissue-specific circRNAs and the heterosis for feed intake and efficiency in chicken.

Heterosis has been widely utilized in chickens. The nonadditive inheritance of genes contributes to this biological phenomenon. However, the role of circRNAs played in the heterosis is poorly determined. In this study, we observed divergent heterosis for residual feed intake (RFI) between 2 crossbreds derived from a reciprocal cross between White Leghorns and Beijing You chickens. Then, circRNA landscape for 120 samples covering the hypothalamus, liver, duodenum mucosa and ovary were profiled to elucidate the regulatory mechanisms of heterosis. We detected that a small proportion of circRNAs (7.83-20.35%) were additively and non-additively expressed, in which non-additivity was a major inheritance of circRNAs in the crossbreds. Tissue-specific expression of circRNAs was prevalent across 4 tissues. Weighted gene co-expression network analysis revealed circRNA-mRNA co-expression modules associated with feed intake and RFI in the hypothalamus and liver, and the co-expressed genes were enriched in oxidative phosphorylation pathway. We further identified 8 nonadditive circRNAs highly correlated with 16 nonadditive genes regulating negative heterosis for RFI in the 2 tissues. Circ-ITSN2 was validated in the liver tissue for its significantly positive correlation with PGPEP1L. Moreover, the bioinformatic analysis indicated that candidate circRNAs might be functioned by binding the microRNAs and interacting with the RNA binding proteins. The integration of multi-tissue transcriptome firstly linked the association between tissue-specific circRNAs and the heterosis for feed intake and efficiency in chicken, which provide novel insights into the molecular mechanism underlying heterosis for feed efficiency. The validated circRNAs can act as potential biomarkers for predicting RFI and its heterosis.

Integrated transcriptomic analysis on chicken ovary reveals CYP21A1 affects follicle granulosa cell development and steroid hormone synthesis.

Egg production is an economically important trait in poultry breeding and production. Follicular development was regulated by several hormones released and genes expressed in the granulosa cells, impacting the egg production and fecundity of hens. However, the molecular functions of these candidate genes that modulate these processes remain largely unknown. In the present study, bioinformatics analyses were performed to identify the candidate genes related to egg production in the ovarian tissue of White Leghorns with high egg production and Beijing You chicken with low egg production during sexual maturity and peak laying periods. The ovarian granulosa cells were used to assess the function of CYP21A1 by transfecting with CYP21A1-specific small interfering RNAs (siRNAs) and overexpression plasmids. We identified 514 differentially expressed genes (|Log2(fold change) | >1, P <0.05) between the 2 chicken breeds in both laying periods. Among these genes, CYP21A1, which is involved in the steroid hormone biosynthesis pathway was consistently upregulated in White Leghorns. Weighted gene co-expression network analysis (WGCNA) further suggested that CYP21A1 was a hub gene, which could positively respond to treatment with follicle stimulation hormone (FSH), affecting egg production. The interference of CYP21A1 significantly inhibited cell proliferation and promoted cell apoptosis. Overexpression of CYP21A1 promotes cell proliferation and inhibits cell apoptosis. Furthermore, the interference with CYP21A1 significantly downregulated the expression of STAR, CYP11A1, HSD3B1, and FSHR and also decreased the synthesis of progesterone (P4) and estradiol (E2) in granulosa cells. Overexpression of CYP21A1 increased the synthesis of P4 and estradiol E2 and the expression of steroid hormone synthesis-related genes in granulosa cells. Our findings provide new evidence for the biological role of CYP21A1 on granulosa cell proliferation, apoptosis, and steroid hormone synthesis, which lays the theoretical basis for improving egg production.

Pigeon novel-miR-741 targets OTUD1 to inhibit proliferation and promote apoptosis of crop fibroblasts.

Trichomonas gallinae (T. gallinae) is a globally distributed protozoan parasite and could cause serious damage to the pigeon industry. MiRNAs have important roles in regulating parasite infection, but its impacts on T. gallinae resistance have rarely been reported. In the present study, we identified a new miRNA (novel-miR-741) and its predicted target OTU deubiquitinase 1 (OTUD1) that might be associated with immunity to T. gallinae in pigeon. Novel-miR-741 and OTUD1 over-expression vectors and interference vectors were constructed. Results from dual luciferase activity assay demonstrated that OTUD1 was a downstream target of novel-miR-741. The Cell Counting Kit-8 and apoptosis assays showed that novel-miR-741 inhibited the proliferation and promoted apoptosis of pigeon crop fibroblasts. Meanwhile, mRNA levels of OTUD1 were significantly reduced in novel-miR-741 mimic-transfected fibroblasts, while mRNA levels of OTUD1 were significantly increased in the novel-miR-741 inhibitor-transfected fibroblasts. The regulatory roles of si-OTUD1 on fibroblasts proliferation, apoptosis, and migration were similar to novel-miR-741 mimic. Our findings demonstrated that novel-miR-741 inhibited the proliferation, and migration of crop fibroblasts, while OTUD1 promoted the proliferation and migration of crop fibroblasts. Therefore, the regulation of OTUD1 by novel-miR-741 was proposed as a potential therapeutic strategy for T. gallinae.

Novel insights into the circadian modulation of lipid metabolism in chicken livers revealed by RNA sequencing and weighted gene co-expression network analysis.

The circadian clock is crucial for maintaining lipid metabolism homeostasis in mammals. Despite the economic importance of fat content in poultry, research on the regulatory effects and molecular mechanisms of the circadian clock on avian hepatic lipid metabolism has been limited. In this study, we observed significant diurnal variations (P<0.05) in triglyceride (TG), free fatty acids (FFA), fatty acid synthase (FAS), and total cholesterol (TC) levels in the chicken embryonic liver under 12-h light/12-h dark incubation conditions, with TG, FFA, and TC concentrations showing significant cosine rhythmic oscillations (P<0.05). However, such rhythmic variations were not observed under complete darkness incubation conditions. Using transcriptome sequencing technology, we identified 157 genes significantly upregulated at night and 313 genes significantly upregulated during the 12-h light/12-h dark cycle. These circadian differential genes are involved in processes and pathways such as lipid catabolic process regulation, meiotic cell cycle, circadian rhythm regulation, positive regulation of the MAPK cascade, and glycerolipid metabolism. Weighted gene co-expression network analysis (WGCNA) revealed 3 modules-green, blue, and red-that significantly correlate with FFA, FAS, and TG, respectively. Genes within these modules were enriched in processes and pathways including the cell cycle, light stimulus response, circadian rhythm regulation, phosphorylation, positive regulation of the MAPK cascade, and lipid biosynthesis. Notably, we identified ten hub genes, including protein kinase C delta (PRKCD), polo like kinase 4 (PLK4), clock circadian regulator (CLOCK), steroid 5 alpha-reductase 3 (SRD5A3), BUB1 mitotic checkpoint serine/threonine kinase (BUB1B), shugoshin 1 (SGO1), NDC80 kinetochore complex component (NDC80), NIMA related kinase 2 (NEK2), minichromosome maintenance complex component 4 (MCM4), polo like kinase 1 (PLK1), potentially link circadian regulation with lipid metabolic homeostasis. These findings demonstrate the regulatory role of the circadian clock in chicken liver lipid metabolism homeostasis and provide a theoretical basis and molecular targets for optimizing the circadian clock to reduce excessive fat deposition in chickens, which is significant for the healthy development of the poultry industry.