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ABSTRACT: 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.

ABSTRACT: 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.

ABSTRACT: 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.

ABSTRACT: 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.

ABSTRACT: This study aimed to systematically determined the effect of 28 h ahemeral light cycle on production performance, egg quality, blood parameters, uterine morphological characteristics, and gene expression of hens during the late laying period. At 74 wk, 260 Hy-Line Brown layers were randomly divided into 2 groups of 130 birds each and in duplicates. Both a regular (16L:8D) and an ahemeral light cycle (16L:12D) were provided to the hens. The oviposition pattern in an ahemeral cycle shifted into darkness, with oviposition mostly occurring 3 to 5 h after light out. Production performance was unaffected by light cycle (P > 0.05). Nonetheless, compared to the normal group, the ahemeral group exhibited increased egg weight, eggshell weight, eggshell percentage, yolk percentage, eggshell thickness, and eggshell strength (P < 0.05). There were rhythmic changes in the uterine morphological structure in both cycles, however, the ahemeral group maintained a longer duration and had more uterine folds than the normal group. In the ahemeral cycle, the phases of the CLOCK and PER2 genes were phase-advanced for 3.96 h and 4.54 h compared to the normal cycle. The PHLPP1 gene, which controls clock resetting, exhibited a substantial oscillated rhythm in the ahemeral group (P < 0.05), while the expression of genes presenting biological rhythm, such as CRY2 and FBXL3, was rhythmically oscillated in normal cycle (P < 0.05). The ITPR2 gene, which regulates intracellular Ca2+ transport, displayed a significant oscillated rhythm in ahemeral alone (P < 0.05), while the CA2 gene, which presents biomineralization, rhythmically oscillated in both cycles (P < 0.05). The ahemeral cycle caused 2.5 h phase delays in the CA2 gene compared to the normal cycle. In conclusion, the 28 h ahemeral light cycle preserved the high condition of the uterine folds and changed the uterine rhythms of CLOCK, PER2, ITPR2, and CA2 gene expression to improve ion transport and uterine biomineralization.

ABSTRACT: 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.

ABSTRACT: 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

Abstract 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.

Abstract 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.

Many local chicken breeds are rapidly declining and even facing extinction due to a variety of factors such as indiscriminate crossbreeding, climate fluctuations, epidemic outbreak, and environmental changes. Developing effective preservation strategies is important to address this situation. The special reproductive characteristics and gamete morphology of chickens pose challenges for preserving genetic heritage through the cryopreservation of genetic materials. Currently, gonad and primordial germ-cell cryopreservation and transplantation are the most promising approaches, especially for the genetic information in the W chromosome of female birds. The study of establishing sterile recipients is crucial for increasing the efficiency of the colonization of transplanted donor tissues and cells. Several classical methods, including ovariectomy and testectomy, busulfan, and irradiation, have been employed to deplete endogenous germ cells in recipient males before transplantation. These methods rely on the toxicity of chemical reagents and physical stimulation to kill germ cells. Recent advances in gene-editing technology have introduced sterile hosts via the knocking out of genes relevant to germ cells’ development. This review explores state-of-the-art technologies for preparing infertile avian recipients (mainly chickens) and aims to provide guidance for the conservation of poultry genetic material and breed restoration.

ABSTRACT: Heterosis is the major benefit of crossbreeding and has been exploited in laying hens breeding for a long time. This genetic phenomenon has been linked to various modes of nonadditive gene action. However, the molecular mechanism of heterosis for egg production in laying hens has not been fully elucidated. To fill this research gap, we sequenced mRNAs and lncRNAs of the ovary stroma containing prehierarchical follicles in White Leghorn, Rhode Island Red chickens as well as their reciprocal crossbreds that demonstrated heterosis for egg number and clutch size. We further delineated the modes of mRNAs and lncRNAs expression to identify their potential functions in the observed heterosis. Results showed that dominance was the principal mode of nonadditive expression exhibited by mRNAs and lncRNAs in the prehierarchical follicles of crossbred hens. Specifically, low-parent dominance was the main mode of mRNA expression, while high-parent dominance was the predominant mode of lncRNA expression. Important pathways enriched by genes that showed higher expression in crossbreds compared to either one or both parental lines were cell adhesion molecules, tyrosine and purine metabolism. In contrast, ECM-receptor interaction, focal adhesion, PPAR signaling, and ferroptosis were enriched in genes with lower expression in the crossbred. Protein network interaction identified nonadditively expressed genes including apolipoprotein B (APOB), transferrin, acyl-CoA synthetase medium-chain family member (APOBEC) 3, APOBEC1 complementation factor, and cathepsin S as hub genes. Among these potential hub genes, APOB was the only gene with underdominance expression common to the 2 reciprocal crossbred lines, and has been linked to oxidative stress. LncRNAs with nonadditive expression in the crossbred hens targeted natriuretic peptide receptor 1, epidermal differentiation protein beta, spermatogenesis-associated gene 22, sperm-associated antigen 16, melanocortin 2 receptor, dolichol kinase, glycine amiinotransferase, and prolactin releasing hormone receptor. In conclusion, genes with nonadditive expression in the crossbred may play crucial roles in follicle growth and atresia by improving follicle competence and increasing oxidative stress, respectively. These 2 phenomena could underpin heterosis for egg production in crossbred laying hens.

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-1β increased dramatically at the beginning and decreased at 5 DPIs, and TGF-β increased stably in the challenged group.

Sperm motility, the percentage of sperm with forward progressions, is one of the semen quality traits that is highly related with male fertility potential in farm animals. The proportion of roosters with low sperm motility impedes the reproduction efficiency. This study aimed to determine crucial testicular proteins linked to sperm motility defects as causes of subfertility in roosters. iTRAQ was performed with the testis from five low sperm motility roosters and three high ones. A total of 2345 proteins were identified, of which, 86 were differentially expressed (DEPs), including 5 up-regulated and 88 down-regulated proteins in the low group. These DEPs were mainly enriched in cytoskeleton and cilium cell component, and biological processes related to sperm motility, and protein transport, and cellular protein localization. A cluster of 13 down-regulated proteins such as IFT88, TEKT1, ACTN2, DNAH5, RSPH9, and SPAG6, were associated with axoneme assembly. Further western blot and immunohistochemical analysis confirmed the down-regulated SPAG6 expression in low group, and indicated its expression in other cell types in testis beyond round spermatids, and that its pattern was in accordance with testis development and recession pace. Silencing transcription factor SOX5 down-regulated SPAG6 transcripts and impaired the cell proliferation and migration. In summary, this study highlights that down-regulated sperm flagellar structure associated proteins were the potential cause of low sperm motility. More specifically, the highly conserved SPAG6 protein across species is a positive regulator for testis development, spermatogenesis, and sperm motility regulation in chickens.

Objective The better understanding of laying pattern of birds is crucial for developing breed-specific proper breeding scheme and management. Methods Daily egg production until 50 wk of age of six chicken breeds including one layer (White Leghorn, WL), three dual-purpose (Rhode Island Red, RIR; Columbian Plymouth Rock, CR; and Barred Plymouth Rock, BR), one synthetic dwarf (DY), and one indigenous (Beijing-You Chicken, BYC) were used to characterize their clutch traits and egg production. The age at first egg, egg number, average and maximum clutch length, pause length, and number of clutches and pauses were calculated accordingly. Results The egg number and average clutch length in WL, RIR, CR, and BR were higher than those in DY and BYC (p

ABSTRACT: The presence of EVs in seminal plasma (SPEVs) suggests their involvement on fertility via transmitting information between the original cells and recipient cells. SPEVs-coupled miRNAs have been shown to affect sperm motility, maturation, and capacitation in mammals, but rarely in poultry species. The present study aims to reveal the profile of SPEVs miRNAs and their potential effect on sperm storage and function in poultry. The SPEVs was successfully isolated from 4 different chicken breeds by ultracentrifugation and verified. Deep sequencing of SPEVs small RNA library of each breed identified 1077 miRNAs in total and 563 shared ones. The top 10 abundant miRNAs (such as miR-10-5p, miR-100-5p, and miR-10a-5p etc.) accounted for around 60% of total SPEVs miRNA reads and are highly conserved across species, predisposing their functional significance. Target genes prediction and functional enrichment analysis indicated that the most abundantly expressed miRNAs may regulate pathways like ubiquitin-mediated proteolysis, endocytosis, mitophagy, glycosphingolipid biosynthesis, fatty acid metabolism, and fatty acid elongation. The high abundant SPEVs-coupled miRNAs were found to target 107 and 64 functionally important mRNAs in the potential recipient cells, sperm and sperm storage tubules (SST) cells, respectively. The pathways that enriched by target mRNAs revealed that the SPEVs-coupled miRNA may rule the fertility by affecting the sperm maturation and regulating the female's immune response and lipid metabolism. In summary, this study presents the distinctive repertoire of SPEVs-coupled miRNAs, and extends our understanding about their potential roles in sperm maturation, capacitation, storage, and fertility, and may help to develop new therapeutic strategies for male infertility and sperm storage.

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.

ABSTRACT: Egg products from indigenous chickens have growing market shares as consumers are pursuing differentiation in egg consumption. The genetic improvement in egg production performance of those breeds is crucial for increasing the economic profit. This study aimed to estimate genetic parameters for egg production and clutch-related traits in indigenous Beijing-You chickens for understanding the genetic architecture and exploring proper biological traits for selection. Data on traits including age at first egg (AFE), egg number (EN), average clutch length (ACL), maximum clutch length (MCL), number of clutches (NC) and pauses (NP), and average pause length (APL) were collected from 4 generations of purebred Beijing-You chickens based on the 43-wk and 66-wk of individual egg production record. The heritabilities, genetic and phenotypic correlations were analyzed by the DMU software with the restricted maximum likelihood method in a multivariate animal model. The results showed that the AFE of Beijing-You chickens was 174.45 d of age, and its heritability was as high as 0.62. The heritability was 0.26 for EN43 and 0.18 for EN66. The clutch traits including ACL, MCL, NC, and NP were moderate to high heritable (h2 = 0.15–0.39), but APL was very low heritable (h2 = 0.05). Genetic correlations were high between AFE and EN (rG(AFE, EN43) = −0.79, rG(AFE, EN66) = −0.39), whereas low between AFE and ACL (rG(AFE, ACL43) = −0.08, rG(AFE, ACL66) = 0.01) and MCL (rG(AFE, MCL) = −0.07). EN had higher correlations with ACL (rG(EN43, ACL43) = 0.59, rG(EN66, ACL66) = 0.40) than that with MCL (rG(EN43, MCL43) = 0.56, rG(EN66, MCL66) = 0.32). The heritability for ACL43 (h2 = 0.38) was higher than that for MCL43 (h2 = 0.33). ACL43 had a positive correlation with EN66 (rG(ACL43, EN66) = 0.62). These results indicated that the egg production of whole laying period could be improved by early selection for AFE and ACL at the same time in Beijing-You chickens.

ABSTRACT: The study investigated the effects of supplementation of bile acids in drinking water on antitrichomonal activity, growth performance, immunity and microbial composition of pigeon. A total of 180 pairs of White King parent pigeons were randomly assigned to 5 treatments of 6 replications with 6 pairs of parent pigeons and 12 squabs in each replicate. The control (CON) group drank water without any additions. The metronidazole (MTZ) group drank water with 500 μg/mL metronidazole for 7 d and without any additions in other days. The else groups drank water with 500, 750, and 1,250 μg/mL bile acid (BAL, BAM, BAH) for 28 d. The results showed that Trichomonas gallinae (T. gallinae) in MTZ, BAL, BAM, and BAH groups were lower than that in CON group at 14, 21, and 28 d of parent pigeons (P < 0.05) and at 21 and 28 d of squabs (P < 0.05). Albumin and alanine transaminase in CON group were higher than those in MTZ, BAL, and BAH groups (P < 0.05). The levels of soluble CD8 were higher in MTZ and BAH groups compared with CON group (P < 0.05). The lesions in oral mucosa, thymus, liver, and spleen tissues of CON group could be observed. Abundance-based coverage estimator (ACE) index in BAH group was higher than that in CON and MTZ groups. Simpson index in CON and BAH groups was higher than MTZ group (P < 0.05). Lactobacillus was the highest colonized colonic bacteria in genera that were 77.21, 91.20, and 73.19% in CON, MTZ, and BAH, respectively. In conclusion, drinking water supplemented with 500, 750, and 1,250 μg/mL bile acid could inhibit growth of T. gallinae in both parent pigeons and squabs. Squabs infected with T. gallinae in control group had higher mortality rate and more serious tissue lesions. Squabs in bile acids treated group had more sCD8 in serum and abundant intestinal morphology. Bile acids could be an efficient drinking supplements to inhibit T. gallinae and improve pigeon adaptive immunity and intestinal health.

Abstract Poultry genetics resources, including commercial selected lines, indigenous breeds, and experimental lines, are now being irreversibly lost at an alarming rate due to multiple reasons, which further threats the future livelihood and academic purpose. Collections of germplasm may reduce the risk of catastrophic loss of genetic diversity by guaranteeing that a pool of genetic variability is available to ensure the reintroduction and replenishment of the genetic stocks. The setting up of biobanks for poultry is challenging because the high sensitiveness of spermatozoa to freezing–thawing process, inability to cryopreserve the egg or embryo, coupled with the females being heterogametic sex. The progress in cryobiology and biotechnologies have made possible the extension of the range of germplasm for poultry species available in cryobanks, including semen, primordial germ cells, somatic cells and gonads. In this review, we introduce the state-of-the-art technologies for avian genetic resource conservation and breed reconstruction, and discuss the potential challenges for future study and further extending of these technologies to ongoing and future conservation efforts.

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.

ABSTRACT: Pigeon has the specific biological ability to produce pigeon milk (also known as crop milk) by its crop. Circular RNAs (circRNAs) are important noncoding RNAs acting as the sponges of miRNAs, but the molecular mechanism of circRNAs regulating crop milk production has not been reported in pigeon. We compared expression profiles of crops during lactating and nonlactating crops, and networks of competing endogenous RNAs (ceRNAs) were constructed. The results showed a total of 8,723 circRNAs were identified, and there were 770 differentially expressed circRNAs (DECs) between these two different periods of crops. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis revealed that the host genes of DECs were enriched in GnRH, MAPK, Insulin, Wnt, and AMPK signaling pathways. Furthermore, gga_circ_0000300 interacted with miR-92-2-5p, which targeted genes participating in lactation and milk composition synthesis. Gga_circ_0003018, gga_circ_0003019 and gga_circ_0003020 could bind with let-7c-5p regulating SOCS3 in crop milk production. These findings provide the circRNAs expression profiles and facilitate the analysis of molecular mechanism of crop milk production in pigeon.

ABSTRACT: The crop of pigeon has specific characteristics as producing crop milk in the lactating period. However, the exact mechanisms underlying the regulation of crop lactation remain unclear. miRNAs, the essential regulators of gene expression, are implicated in various physiological and biological activities. In this study, we discovered a new miRNA that regulated phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit delta (PIK3CD) and crop fibrocyte proliferation. Results of the luciferase reporter assay suggested that miR-193-5p suppressed PIK3CD expression by targeting a conserved binding site in the 3′-untranslated region (UTR) of PIK3CD mRNA. MiR-193-5p promoted crop fibrocyte proliferation and migration, whereas PIK3CD inhibited these effects. These findings suggested an important regulatory role of miR-193-5p in crop fibrocyte proliferation, suggesting that miR-193-5p and PIK3CD might be important regulators of crop milk production.

Abstract 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.

ABSTRACT: This study aimed to investigate the effects of feed systems in parent pigeons on the growth performance, carcass characteristics, organ index, and serum biochemical parameters of squabs. A total of 60 pairs of parent pigeons were selected and divided into 2 groups randomly. The parent pigeons were fed with two feed systems that were whole grains plus granulated feed (WGG) and complete-formula granulated feed (CFG) for 21 d. The results showed that CFG diet could increase carcass yield, heart index, content of trypsin, and growth hormone of squabs (P < 0.05), but decrease feed intake, gizzard index, b* value, malondialdehyde concentration, and uric acid concentration significantly (P < 0.05) comparing with WGG diet. There were no significant differences among the 2 groups in feed intake from d 1 to d 21, abdominal fat yield and body weight changes of squabs and parent pigeons (P > 0.05). It can be concluded from these observations that CFG was beneficial to squab which could improve digestive enzyme and antioxidant ability in the serum, so the CFG should be suggested in practice.

Heterosis has been widely utilized in chicken breeding to improve economically important traits. However, few studies focused on revealing the factors contributing to egg production heterosis. In this study, White Leghorn and Beijing-You chickens were used as parental breeds to generate purebreds (WW, YY) and reciprocal crossbreeds (WY, YW) to characterize heterosis for egg production traits including age at first egg (AFE), clutch traits, oviposition pattern, and egg quality traits. Results showed that egg number until 35 wk of age (EN35) was higher in crossbreeds than the average of purebreds (P < 0.05) and exhibited positive heterosis of 4.03% and 2.84%) in WY and YW respectively. Crossbreeds commenced laying earlier than the average of purebreds (P < 0.05) with negative heterosis of −1.24% and −0.92% for WY and YW respectively. Moreover, EN35 had negative correlation with AFE (r = −0.85) and positive correlation with average clutch length (ACL) (r = 0.48) and maximum clutch length (MCL) (r = 0.66). However, negative heterosis for ACL (−19.62%, −16.51%) and MCL (−22.88%, −18.97%) were obtained in WY and YW, respectively. This may be due to the positive heterosis for number of pauses, which was highly correlated with ACL (r = −0.68) and MCL (r = −0.74). The crossbreeding improved the oviposition pattern. Percent egg laying that occurs between 7:00 and 14:00 was 91.50% (WW), 68.28% (YY), 76.87% (WY), and 79.68% (YW) in the experimental populations. On the other hand, oviposition interval (OI) had negative heterosis in crossbreeds and was negatively correlated with EN35 (r = −0.60). Positive heterosis for egg weight of 2.63% and 3.94% and yolk weight of 4.74% and 6.07% were observed in WY and YW, respectively. Taken together, egg production related traits did not contribute equally to EN heterosis. The AFE and OI exhibited significant correlation with EN indicating that they would be important drivers for EN heterosis.

Heterosis has been extensively exploited in chicken breeding to improve laying traits in commercial hybrid stock. However, the molecular mechanisms underlying it remains elusive. This study characterizes the miRNAome in the pre-hierarchical follicles of purebred and hybrid laying hens, and investigate the functions of miRNAs with non-additive expression in the pre-hierarchical follicles as they modulate heterosis for egg number and clutch size. To achieve that aim, White Leghorn and Rhode Island Red chicken lines were reciprocally crossed to generate hybrids. The crossbreds demonstrated heterosis for egg number and clutch size, and pre-hierarchical follicles from 4 birds of each genotype were collected at 53 weeks of age. Mode of miRNA expression was characterized after miRNA sequencing. A total of 50 miRNAs including 30 novel ones, were found to exhibit non-additive expression. Dominance was the predominant mode of expression exhibited by majority of the miRNAs. Functional analysis of target genes of the known miRNAs with non-additive expression revealed Gene Ontology terms related to regulation of transcription, metabolic processes and gene expression. KEGG and REACTOME pathways including hedgehog, cellular senescence, wnt, TGF-β, progesterone-mediated oocyte maturation, oocyte meiosis, GnRH signaling, signal transduction and generic transcription, which can be linked to primordial follicle activation, growth and ovulation, were significantly enriched by target genes of miRNAs with non-additive expression. Majority of the genes enriched in these biological pathways were targeted by gga-miR-19a, gga-miR-19b, gga-miR-375, gga-miR-135a, and gga-miR-7 and 7b, thus, revealing their synergistic roles in enhancing processes that could influence heterosis for egg number and clutch size in hybrid hens.

Sexual maturation is fundamental to the reproduction and production performance, heterosis of which has been widely used in animal crossbreeding. However, the underlying mechanism have long remained elusive, despite its profound biological and agricultural significance. In the current study, the reciprocal crossing between White Leghorns and Beijing You chickens were performed to measure the sexual maturation heterosis, and the ovary lncRNAs and mRNAs of purebreds and crossbreeds were profiled to illustrate molecular mechanism of heterosis. Heterosis larger than 20% was found for pubic space and oviduct length, whereas age at first egg showed negative heterosis in both crossbreeds. We identified 1170 known lncRNAs and 1994 putative lncRNAs in chicken ovary using a stringent pipeline. Gene expression pattern showed that nonadditivity was predominant, and the proportion of nonadditive lncRNAs and genes was similar between two crossbreeds, ranging from 44.24% to 49.15%. A total of 200 lncRNAs and 682 genes were shared by two crossbreeds, respectively. GO and KEGG analysis showed that the common genes were significantly enriched in the cell cycle, animal organ development, gonad development, ECM-receptor interaction, calcium signaling pathway and GnRH signaling pathway. Weighted gene co-expression network analysis (WGCNA) identified that 7 out of 20 co-expressed lncRNA-mRNA modules significantly correlated with oviduct length and pubic space. Interestingly, genes harbored in seven modules were also enriched in the similar biological process and pathways, in which nonadditive lncRNAs, such as MSTRG.17017.1 and MSTRG.6475.20, were strongly associated with nonadditive genes, such as CACNA1C and TGFB1 to affect gonad development and GnRH signaling pathway, respectively. Moreover, the results of real-time quantitative PCR (RT-qPCR) correlated well with the transcriptome data. Integrated with positive heterosis of serum GnRH and melatonin content detected in crossbreeds, we speculated that nonadditive genes involved in the GnRH signaling pathway elevated the gonad development, leading to the sexual maturation heterosis. We characterized a systematic landscape of ovary lncRNAs and mRNAs related to sexual maturation heterosis in chicken. The quantitative exploration of hybrid transcriptome changes lays foundation for genetic improvement of sexual maturation traits and provides insights into endocrine control of sexual maturation.

Sperm cryopreservation is an effective technique for conserving animal genetic diversity and transmitting superior genetic backgrounds, maintained via a non-invasive sampling and collection of huge quantities of sperm. Nevertheless, cryopreservation in avian species is not commercially viable because of the rooster sperm’s susceptibility to damage. This study aims to estimate the impact of dimethylacetamide (DMA) as a cryoprotectant at different levels (3%, 6%, or 9%) on the post-thawed sperm quality, motility, antioxidant-biomarkers, and the expression of anti-freeze related genes. Semen samples were collected twice a week from twelve roosters aged 40 wk, weighing 3400 ± 70 g, and belonging to the Cairo-B2 chicken strain. Fresh semen samples were rapidly appraised, pooled, diluted with two volumes of a basic extender, and divided equally into three groups. The diluted groups were chilled at −20 °C for 7 min, then gently supplemented with 3, 6, or 9% pre-cooled DMA and equilibrated at 5 °C for a further 10 min. Semen pellets were formed by pipetting drops 7 cm above liquid nitrogen (LN2), which were then kept inside cryovials in the LN2. Thawing was performed 2 months later by taking 3–4 pellets of the frozen semen into a glass tube and warming it in a water bath for 8 s at 60 °C. The results showed that 3% DMA increased the proportion of total motile sperm, progressivity, viability, and plasma membrane integrity (%) compared to the 6% and 9% DMA groups. The lipid peroxidation and antioxidant enzyme activity were improved in the 3% group. At the same time, some anti-freeze-related genes’ (including ras homolog family member A (RHOA), heat shock protein 70 (HSP70), and small nuclear ribonucleoprotein polypeptide A (SNRPA1)) expressions were upregulated within the 3% DMA group relative to other groups. In conclusion, the 3% DMA group maintained higher post-thawed sperm quality than the other tested groups.

The long-term semen cryopreservation is increasingly crucial for conservation of endangered livestock and poultry species. Glycerol is the most widely used cryoprotectant for freezing chicken semen. Continuous improvement in details with glycerol may help increase the fertility of post-thawed semen. Two experiments were performed in the present study to investigate the effects of glycerol concentration, removal method, and straw type on the quality of post-thawed sperm. In experiment 1, glycerol concentration (3%, 5%, 7%, 9%, 11%, and 13%) and glycerol removal method (final dilution ratio 1:1, 1:2, 1:4, 1:8, 1:16, and 1:20) combination groups were investigated for post-thawed sperm quality, residual glycerol concentration, and fertility to find the best combinations. Experiment 2 was performed to evaluate the effects of straw type (0.25 and 0.5 mL) and glycerol concentration (3%, 5%, 7%, 9%, 11%, and 13%) on the post-thawed sperm quality. Results showed that post-thawed sperm motility of 6 glycerol concentration groups were different (P < 0.01). Sperm motility of 5%, 7%, 9%, 11% and 13% was higher than that of 3% (P < 0.01). There was no difference among different concentrations of glycerol in VSL, VCL, VAP, ALH, WOB, BCF, LIN, or STR (P > 0.05). As for the glycerol removal method, sperm motility of 1:8 dilution was the highest, followed by 1:1 and 1:2, while the difference among groups was not statistically significant (P = 0.11). Glycerol concentration and removal method had no interaction effect on sperm motion parameters (P > 0.05). The highest fertility (48.70%) was found for the 5% and 1:2 combination. There was no difference for sperm motility between 0.25 and 0.5 mL straws (P > 0.05). Glycerol concentration and straw type had no interaction effect on the sperm motion parameters (P > 0.05). It can be concluded from these observations that the combination of 5% glycerol and 1:2 dilution rendered higher fertility should be suggested in practice, and that both 0.25 and 0.50 mL straws fit the present procedure.

Abstract Background Effect of monochromatic green light illumination on embryo development has been reported in chickens. The avian pineal gland is an important photo-endocrine organ formed by a mediodorsal protrusion during embryonic development. However, the involvement of pineal gland in the light transduction process remains to be elucidated. In the present study, we investigated the influence of monochromatic green light on hatching time and explored the possible mechanism via pineal function. Results A total of 600 eggs of White Leghorn (Shaver strain) were incubated under photoperiods of either 12 h of light and 12 h of darkness using monochromatic green light (12L:12D group) or 24 h of darkness (0L:24D group) for 18 d. Compared to 0L:24D group, the green light stimulation shortened the hatching time without extending the hatch window or impairing hatchability. The liver of embryos incubated in the 12L:12D light condition was heavier than those of the 0L:24D group on d 21 post incubation which may be linked to the observed increase in the serum concentration of insulin-like growth factor 1 (IGF-1); primary secretion of the liver. Histological structure analysis of pineal gland demonstrated that the light stimulation increased follicle area, wall thickness and lumen area on d 10 and d 12 post incubation. Rhythmic function analysis demonstrated that three clock related genes (brain and muscle ARNT-like-1, BMAL1; circadian locomotor output cycles kaput, CLOCK; and cryptochrome-1, CRY1) and a melatonin rate-limiting enzyme related gene (arylalkylamine N-acetyltransferase, AANAT) were rhythmically expressed in the pineal gland of the 12L:12D group, but not in the 0L:24D group. Simultaneously, the light stimulation also increased the concentration of melatonin (MT), which was linked to hepatocyte proliferation and IGF-1 secretion in previous studies. Conclusions The 12L:12D monochromatic green light stimulation during incubation shortened hatching time without impairing hatching performance. Pineal gland’s early histological development and maturation of its rhythmic function were accelerated by the light stimulation. It may be the key organ in the photo-endocrine axis that regulates embryo development, and the potential mechanism could be through enhanced secretion of MT in the 12L:12D group which promotes the secretion of IGF-1.

This study compared floor (FS) with cage systems (CS) for slow-growing meat-type chickens in terms of muscle fiber characteristics, carcass characteristics, and meat quality. Following the design of a factorial arrangement of 2 housing systems × 2 genders, 180 male and 180 female Beijing You chickens of 8 weeks old were allocated to FS and CS. At the end of 17 weeks, five males and five females from each replicate were selected for measurement. No difference was observed in body, carcass, or eviscerated weight (p > 0.05). FS birds showed higher muscle yield and lower abdominal fat composition (p < 0.05). Inosine-5′-monophosphate (IMP) content was not affected by housing system or gender (p > 0.05). On the contrary, intramuscular fat (IMF) content was affected by both in a way that CS birds and females had higher IMF content (p < 0.05). FS birds had a higher percentage of white muscle fibers (p < 0.05). In conclusion, carcass characteristics, meat quality, and muscle fiber type of slowing-growing broilers are influenced: rearing on the floor may improve muscle development and reduce fat deposition without impairing marketing weight and rearing in cages may improve IMF content.

Ex situ preservation is an important method in the preservation of chickens, and cryopreservation of semen is the only method for gamete preservation at present. During the last two decades, many studies have been performed to develop standard chicken semen cryopreservation technology and achieve great progress. Many attempts and methods were investigated to adapt subspecies or different breeds. In this paper, we firstly reviewed the main factors affecting cryopreservation of chicken sperm, including the unique structure and characteristics of the spermatozoa. Secondly, the studies on key points of the chicken sperm cryopreservation technology, including semen dilution, cryoprotectants, equilibration time, packaging types, and freezing and thawing rates were summarized to generate the optimal parameters. Then, the mechanism underlying freezing damage and freezability revealed by recent omics methods relevant to the efficiency of cryopreservation were discussed. This review will provide relevant reference for the future investigation of poultry semen cryopreservation technology.

Light presents an important exogenous factor for poultry. This study examined effects of LED lights with different defined spectrums on growth and reproduction of indigenous Beijing-You chickens. A total of 576 one-day old female chicks were divided into 16 rooms, and each were exposed to four different lights: LED A (21% green light, 30% blue light, 24% yellow light, and 25% red light), B (35%, 35%, 18%, and 12%), C (27%, 30%, 22%, and 21%), or compact fluorescent lamps (CFL, 15%, 28%, 41%, and 16%). Results showed that feed intake and feed conversion ratio were comparable among treatments throughout the 17 week rearing period (p > 0.05). LED C showed similar body weight gain with CFL, but higher than LED A and B. The CFL birds start to lay on 132.25 d, while LED B did not lay until 148.25 d. The age at 50% egg production did not vary among groups (p = 0.12). Total egg number until 43 week of LED B was higher than others (p < 0.05). Therefore, LED lights with defined spectral proportion have different effects on chickens’ growth and reproduction. The LED C promotes the prepubertal growth, and the LED B provides proper sexual maturation age and better egg-laying persistence.

Crossbreeding advantage in hybrids compared with their parents, termed heterosis, has been exhaustively exploited in chicken breeding over the last century. Reports for crossbreeding of elite laying chickens covering rearing and laying period remain infrequent. In this study, resource populations of Rhode Island Red (RIR) and White Leghorn (WL) pure-bred chickens were reciprocally crossed to generate 4 distinct groups that were evaluated for prelaying growth, egg production, and egg quality. Birds monitored for prelaying growth consists of 105 (RIR), 131 (WL), 207 (RIR × WL) and 229 (WL × RIR), and 30 pullets from each group were evaluated. Egg laying records were collected from 102, 89, 147, and 191 hens in the 4 populations, respectively. In addition, expression of 5 candidate genes for egg production in the ovarian follicles was measured by RT-qPCR. Results showed that BW of hatched chicks in the WL line was higher than the other populations. However, the 2 crossbreds grew faster than WL purebred throughout the prelaying period. Low to medium heterosis was observed for BW and body length before the onset of lay. White Leghorn and the hybrids commenced laying earlier than RIR pullets and egg production traits were favorable in the crossbreds compared with purebreds. Heterosis for egg number and clutch size was moderate in WL × RIR but low in RIR × WL hens. Expression of antimullerian hormone gene was high in WL and RIR × WL hybrids, suggesting WL parent-specific enhancing dominant expression. Shell weight was higher in the crossbreds than purebreds at 52 wk of age, but RIR hens laid eggs with higher shell ratio than the other populations (P

Providing green light during incubation has been shown to accelerate the embryo development and shorten the hatching time in broilers. Few studies have concentrated on the exact effects on layer breeders in the aspects of hatching and posthatch performance. In this study, 4 strains of layer breeder eggs, namely White Leghorn, Rhode Island Red, Columbia Rock, and Barred Rock were used to assess the effects of monochromatic green light during embryogenesis on hatching performance, chick quality, and pubertal growth. Each strain of 600 eggs was incubated under photoperiods of either 12 h of light and 12 h of darkness (12L:12D, light group) or 0 h of light and 24 h of darkness (0L:24D, dark group) for 18 D, with 2 replicates for each treatment. The results showed hatch time, time reaching 90% hatch, and average hatch time were significantly shorter among the 4 strains in the light group (P 0.05). There was no significant difference in hatchability of fertile eggs, chick weight/egg weight, or chick quality among the 4-strain eggs between the light group and dark group (P > 0.05). There was no difference (P > 0.05) in posthatch BW between different light treatments of the 3 strains (White Leghorn, Columbia Rock, and Barred Rock), whereas the BW of Rhode Island Red was higher in light group than that of the dark group at 8 to 12 wk of age (P

Semen quality is important for roosters' fertility. The correlations between semen quality traits and fertility has less been analyzed, impeding the selection of effective parameters for roosters selection. This research aimed to investigate the variation in semen quality traits in relation to fertilization capacity between 2 chicken breeds. A total of 156 Rhode Island Red (n = 78) and White Leghorn (n = 78) roosters of 53 wk were selected for semen quality estimation including semen volume, pH, sperm concentration, motility, viability, abnormal sperm percentage, and sperm kinematic parameters. Individual fertility was measured by artificial insemination using each 30 birds from 2 breeds. Significant variations were observed between breeds in semen volume, pH, sperm motility (MOT), viability, and abnormal sperm percentage (P

The prevalence of crossed beaks ranging from 0.2 to 7.4% was documented in at least 12 chicken strains. Previous studies focused largely on candidate molecules, whereas the morphological observation was missing. This study reported a detailed phenotype and prevalence of crossed beaks based on morphological observation in nine thousand nine hundred 1-day-old female Beijing-You chicks. Affected chicks were classified into 2 categories based on the direction of the mandibular deformation: left and right. Each category was selected to sacrifice for the measurement of length, width, and thickness of the bilateral mandibular ramus (MR). The normal chicks were used as controls. Paraffin section was made for the bilateral MR of a crossed beak and a normal control for histology analysis. A total of 97 out of 9,900 chickens showed beak deformity including 71 crossed beaks (0.72%) and 26 side beaks (0.26%) for which the upper and lower beak were both bent in the same direction. There was no difference in the direction of the bend of the lower beak in crossed beaks (P > 0.05). The incidence of crossed beaks increased quickly from 0 to 56 d and no new incidence after 56 d. The angle of the crossed beaks was below 5° in the first week and had grown more severe with age until 56 d. The mandible structure showed that condyle served as a growth center for the MR extension. The short-side MR of crossed beaks was thicker than normal ones (P

The objective of this study was to investigate the effects of replacing antibiotics with a combination of plant essential oils on the growth performances and gastrointestinal health of broilers. A total of 720 1-day-old male AA broilers were randomly divided into 3 treatments: the control treatment (CON), the Aureomycin supplementation treatment (AGP), and the combined plant oils supplementation treatment (POC), with a 42-D period feeding procedure. Growth performances, carcass performances, intestinal sections, and cecal microbiota were investigated. Results indicated that POC supplementation decreased the feed conversion ratio compared with CON and AGP treatments, though not significantly. No significant differences were found for feed intake, BW gain, and culling rate among the 3 treatments (P > 0.05). In addition, no significant differences were seen on carcass performance. For the aspects of intestinal section, POC supplementation did not make significant effects on intestinal wall thickness, villus heights, crypt depths, and the ratio of villus heights/crypt depths compared with CON and AGP treatments. Cecal microbiota results demonstrated that bacterial diversity and some representative probiotic bacteria were significantly increased in numbers (P < 0.05) after POC supplementation. In conclusion, the combination of essential oils promoted intestinal health through improving gut bacterial diversity and probiotic bacteria, as well as improving feed conversion ratio of broilers. These results indicated that the combination of essential oils may benefit the gastrointestinal health and be applied as an antibiotic alternative.

Abstract 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.

ABSTRACT: The objective of this study was to determine the effect of age at photostimulation on sexual maturity and performance of layer breeders. A total of 192 fourteen-wk-old White Leghorn (WL) breeder hens were randomly allocated to 4 treatments of 48 birds each, with 2 replicates per treatment. The birds were photostimulated at 16 (PS16), 18 (PS18), 20 (PS20), and 22 (PS22) wk of age. Four birds per treatment were randomly selected to evaluate sexual organ development at 1 D before photostimulation and 2, 4, and 6 wk after photostimulation. The ovary weight, large yellow follicles number (LYF), oviduct weight, and oviduct length of PS18 increased sharply after photostimulation. Conversely, the increase in PS16 was not observed until 2 wk after photostimulation. There was no difference in age at sexual maturity between treatments (P > 0.05). The PS16 had the longest interval (28 D) from photostimulation to 5% egg production, while PS22 reached 5% egg production 7 D before photostimulation. The PS22 had lower peak production (P = 0.02) and less egg production (P = 0.02) than other treatments. The PS16 had more broken and abnormal eggs (P = 0.01) and lower hatchability (P = 0.04) than other treatments. In conclusion, photostimulation at 16 and 22 wk of age decreases hatchability and egg production, respectively, and photostimulation at 18 wk is appreciated for the WL breeder hens.

Crossbreeding is normally applied to improve the economical traits of chickens. However, feed efficiency and its relationship with egg quality have rarely been explored in crossbreds. Herein, White Leghorn and Beijing You chickens were selected to generate purebreds (WW, YY) and reciprocal crossbreds (YW, WY), which were evaluated in terms of daily feed intake (DFI), feed conversion ratio (FCR), and residual feed intake (RFI) at 43 to 46 and 69 to 72 weeks of age, respectively. We found that WY was more efficient than YW in both laying periods. The correlation analysis showed that RFI was highly correlated with DFI (0.49 to 0.84) but unrelated to egg mass, and FCR was negatively correlated with egg mass (−0.77 to −0.43) in both purebreds and crossbreds. Moreover, RFI was not correlated with egg quality traits within each genetic group, except for the egg yolk ratio (0.27) in WW. FCR was negatively correlated with eggshell weight and thickness (−0.33 to −0.19) in WW and WY. Compared to FCR, selection for RFI could improve feed efficiency without significant changes in egg mass and quality in chickens. These findings provide new insights into the improvement of feed efficiency using rational parental lines in chicken crossbreeding.

Sperm cryopreservation is of great importance for the poultry industry but still needs to be optimized. The high susceptibility of poultry sperm to cryodamage leads to low fertility rates after cryopreservation. Therefore, the present study aimed at evaluating the effect of including a cryoprotectant, dimethylacetamide (DMA), in the chicken semen freezing extenders at a final concentration of 3%, 6%, or 9% on the post-thawed sperm motility, quality, antioxidant biomarkers, anti-freeze gene expression, and fertilizing ability. Results showed that the total motile sperm, progressivity, and viability were quadratically increased (p < 0.05) in the 6% DMA group. The antioxidant enzyme activity and lipid peroxidation were negatively (p < 0.05) affected by the increase in DMA concentration. Furthermore, some anti-freeze-associated genes such as heat shock protein 70 (HSP70) and ras homolog family member A (RHOA) were linearly and quadratically down-regulated (p < 0.05) with the high concentration of DMA. Finally, the fertility and hatchability rates did not indicate statistical differences between DMA groups. It can be concluded that using the low concentration of 3–6% DMA in the freezing semen extender is preferable to obtain acceptable results in the post-thawed sperm quality and fertility.

Trichomoniasis gallinae (T. gallinae) is one of the most pathogenic parasites in pigeon, particularly in squabs. Oral cavity is the main site for the host-parasite interaction. Herein, we used RNA-sequencing technology to characterize lncRNA and mRNA profiles and compared transcriptomic dynamics of squabs, including four susceptible birds (S) from infected group, four tolerant birds (T) without parasites after T. gallinae infection, and three birds from uninfected group (N), to understand molecular mechanisms underlying host resistance to this parasite. We identified 29,809 putative lncRNAs and characterized their genomic features subsequently. Differentially expressed (DE) genes, DE-lncRNAs and cis/trans target genes of DE-lncRNAs were further compared among the three groups. The KEGG analysis indicated that specific intergroup DEGs were involved in carbon metabolism (S vs. T), metabolic pathways (N vs. T) and focal adhesion pathway (N vs. S), respectively. Whereas, the cis/trans genes of DE-lncRNAs were enriched in cytokine-cytokine receptor interaction, toll-like receptor signaling pathway, p53 signaling pathway and insulin signaling pathway, which play crucial roles in immune system of the host animal. This suggests T. gallinae invasion in pigeon mouth may modulate lncRNAs expression and their target genes. Moreover, co-expression analysis identified crucial lncRNA-mRNA interaction networks. Several DE-lncRNAs including MSTRG.82272.3, MSTRG.114849.42, MSTRG.39405.36, MSTRG.3338.5, and MSTRG.105872.2 targeted methylation and immune-related genes, such as JCHAIN, IL18BP, ANGPT1, TMRT10C, SAMD9L, and SOCS3. This implied that DE-lncRNAs exert critical influence on T. gallinae infections. The quantitative exploration of host transcriptome changes induced by T. gallinae infection broaden both transcriptomic and epigenetic insights into T. gallinae resistance and its pathological mechanism.

Crossed beaks have been observed in at least 12 chicken strains around the world, which severely impairs their growth and welfare. To explore the intrinsic factor causing crossed beaks, this study measured the length of bilateral mandibular ramus of affected birds, and investigated the genome-wide DNA methylation profiles of normal and affected sides of mandibular condyle. Results showed that the trait was caused by impaired development of unilateral mandibular ramus, which is extended through calcification of mandibular condyle. The methylation levels in the CG contexts were higher than that of CHG and CHH, with the highest methylation level of gene body region, followed by transcription termination sites and downstream. Subsequently, we identified 1,568 differentially methylated regions and 1,317 differentially methylated genes in CG contexts. Functional annotation analysis of Gene Ontology and Kyoto Encyclopedia of Genes and Genomes showed that these genes were involved in bone mineralization and bone morphogenesis. Furthermore, by combining the WGBS and previous RNA-Seq data, 11 overlapped genes were regulated by both long non-coding RNA and DNA methylation. Among them, FIGNL1 is an important gene in calcification of mandibular condyle. Generally, because the affected genes play key roles in maintaining mandibular calcification, these changes may be pivotal factors of crossed beaks.

The 2 × 4 factorial experiment was designed to determine the effect of strain and photostimulation age on sexual maturity and reproductive performance of rooster breeders. A total of 96 White Leghorn (WL) and 120 Beijing You Chicken (BYC) roosters were randomly allocated to 4 treatments at 14 wk of age. The treatments represent photostimulation at 16, 18, 20, and 22 wk of age, respectively (PS16, PS18, PS20, and PS22), in both strains. Photostimulation was achieved by increasing the day length from 8L:16D to 14L:10D and by increasing lighting intensity from 10 lx to 80 lx. Three birds from each interaction were sacrificed to characterize the comb and testis weights at 4 time points: 1 d before photostimulation and 2, 4, and 6 wk after photostimulation. Semen quality and hatching performance with the semen of the experimental roosters were measured at 30 and 45 wk of age, respectively. Results showed that the testis weight of PS20 and PS22 in WL and BYC was 6.4- and 2.9-fold higher than that of PS18 before photostimulation, while testis weight of PS18 in both strains increased sharply after photostimulation. The diameter of seminiferous tubules increased in the photostimulated roosters as compared with the nonphotostimulated ones, and mature spermatozoa were produced 4 wk after photostimulation and at 20 wk of age for PS16. The WL had lower semen volume and total sperm count than BYC (P 0.05). In addition, semen quality traits were not affected by age at photostimulation (P > 0.05) in both strains. The fertility and hatching performance were not affected by strain or photostimulation age (P > 0.05). In summary, the sexual maturation of rooster breeders can be advanced by photostimulation at an early age, which does not lead to a difference in semen quality or hatching performance at adult stage.

Abstract 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.

Abstract Sperm motility is the most important indicator in evaluating roosters’ fecundity. However, the genetic mechanisms underlying chicken sperm motility is not yet clear. Long non-coding RNA (lncRNA) play epigenetic roles in reproduction. In this study, RNA sequencing was employed to profile the testis transcriptome (lncRNA and mRNA) of six Beijing-you cocks divergent in sperm motility. In total, 2,597 lncRNAs were identified in the chicken testis, including 1,267 lincRNAs, 975 anti-sense lncRNAs, and 355 intronic lncRNAs. They shared similar features with previous studies. Of these lncRNAs, 124 were differentially expressed. Among 17,690 mRNAs detected in this study, 544 were differentially expressed, including a bunch of genes with known functions on sperm motility. GO annotation analysis revealed these genes were involved in ATP binding, cilium assembly, and oxidation-reduction processes. Integrating analysis of lncRNA and mRNA profiles predicted 10 lncRNA-gene pairs, including 8 co-regulated and 2 inversely-regulated pairs. To the best of our knowledge, this is the first genome-wide investigation of the lncRNAs in the chicken testis associated with sperm motility. Our results provided a catalog of chicken testis lncRNAs and genes worthy of further studies to understand their roles in cocks’ reproductive performance regulation.

Pigeon belongs to altrices. Squab cannot forage independently. Nutrition can only be obtained from crop milk secreted by male and female pigeon. miRNA could regulate many biological events. However, the roles of miRNA and ceRNA in regulating crop milk production are still unknown. In this study, we investigated the miRNAs expression profile of female pigeon crop, explored the potential key genes, and found the regulatory mechanisms of crop milk production. A total of 71 miRNAs were identified differentially expressed significantly. Meanwhile, miR-20b-5p, miR-146b-5p, miR-21-5p, and miR-26b-5p were found to be the key miRNAs regulating lactation. Target genes of these miRNAs participated mainly in cell development; protein and lipid synthesis; and ion signaling processes, such as cell-cell adhesion, epithelial cell morphogenesis, calcium signaling pathway, protein digestion, and absorption. In the ceRNA network, miR-193-5p was located in the central position, and miR-193-5p/CREBRF/LOC110355588, miR-460b-5p/GRHL2/MSTRG.132954, and miR-193-5p/PIK3CD/LOC110355588 regulatory axes were believed to affect lactation. Collectively, our findings enriched the miRNA expression profile of pigeon and provided novel insights into the microRNA-associated-ceRNA networks regulating crop milk production in pigeon.

Pigeons have the ability to produce milk and feed their squabs. The genetic mechanisms underlying milk production in the crops of ’lactating’ pigeons are not fully understood. In this study, RNA sequencing was employed to profile the transcriptome of lncRNA and mRNA in lactating and non-‘lactating’ pigeon crops. We identified 7066 known and 17,085 novel lncRNAs. Of these lncRNAs, 6166 were differentially expressed. Among the 15,138 mRNAs detected, 6483 were differentially expressed, including many predominant genes with known functions in the milk production of mammals. A GO annotation analysis revealed that these genes were significantly enriched in 55, 65, and 30 pathways of biological processes, cellular components, and molecular functions, respectively. A KEGG pathway enrichment analysis revealed that 12 pathways (involving 544 genes), including the biosynthesis of amino acids, the propanoate metabolism, the carbon metabolism and the cell cycle, were significantly enriched. The results provide fundamental evidence for the better understanding of lncRNAs’ and differentially expressed genes’ (DEGs) regulatory role in the molecular pathways governing milk production in pigeon crops. To our knowledge, this is the first genome-wide investigation of the lncRNAs in pigeon crop associated with milk production. This study provided valuable resources for differentially expressed lncRNAs and mRNAs, improving our understanding of the molecular mechanism of pigeon milk production.

Marek’s disease (MD) is a T cell lymphoma disease induced by Marek’s disease virus (MDV), a highly oncogenic α herpesvirus primarily affecting chickens. MD is a chronic infectious disease that threatens the poultry industry. However, the mechanisms of genetic resistance for MD are complex and not completely understood. In this study, to identify high-confidence candidate genes of MD genetic resistance, high throughput sequencing (RNA-seq) was used to obtain transcriptomic data of CD4+ T cells isolated from MDV-infected and non-infected groups of two reciprocal crosses of individuals mating by two highly inbred chicken lines (63 MD-resistant and 72 MD-susceptible). After RNA-seq analysis with two biological replicates in each group, we identified 61 and 123 single nucleotide polymorphisms (SNPs) (false discovery rate (FDR) < 0.05) annotated in 39 and 132 genes in intercrosses 63 × 72 and 72 × 63, respectively, which exhibited allele-specific expression (ASE) in response to MDV infection. Similarly, we identified 62 and 79 SNPs annotated in 66 and 96 genes in infected and non-infected groups, respectively. We identified 534 and 1543 differentially expressed genes (DEGs) (FDR < 0.05) related to MDV infection in intercrosses 63 × 72 and 72 × 63, respectively. We also identified 328 and 20 DEGs in infected and non-infected groups, respectively. The qRT-PCR using seven DEGs further verified our results of RNA-seq analysis. The qRT-PCR of 11 important ASE genes was performed for gene functional validation in CD4+ T cells and tumors. Combining the analyses, six genes (MCL1, SLC43A2, PDE3B, ADAM33, BLB1, and DMB2), especially MCL1, were highlighted as the candidate genes with the potential to be involved in MDV infection. Gene-set enrichment analysis revealed that many ASE genes are linked to T cell activation, T cell receptor (TCR), B cell receptor (BCR), ERK/MAPK, and PI3K/AKT-mTOR signaling pathways, which play potentially important roles in MDV infection. Our approach underlines the importance of comprehensive functional studies for gaining valuable biological insight into the genetic factors behind MD and other complex traits, and our findings provide additional insights into the mechanisms of MD and disease resistance breeding in poultry.