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.

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.

Transcriptome analysis of ovarian tissues highlights genes controlling energy homeostasis and oxidative stress as potential drivers of heterosis for egg number and clutch size in crossbred laying hens.

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.

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.

Extracellular vesicle-coupled miRNA profiles of chicken seminal plasma and their potential interaction with recipient cells.

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.

Research Note: Genetic parameters for egg production and clutch-related traits in indigenous Beijing-You chickens.

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.

Supplementation of exogenous bile acids improve antitrichomonal activity and enhance intestinal health in pigeon (Columba livia).

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.

The In Vitro Evaluation of Rooster Semen Pellets Frozen with Dimethylacetamide.

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.

Analyses of circRNAs profiles of the lactating and nonlactating crops in pigeon (Columba livia).

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.

miR-193-5p negatively regulates PIK3CD to promote crop fibrocyte proliferation in pigeon (Columba livia).

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.

Effects of feed systems on growth performance, carcass characteristics, organ index, and serum biochemical parameters of pigeon.

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.

Research Note: Heterosis for egg production and oviposition pattern in reciprocal crossbreeds of indigenous and elite laying chickens.

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.

Poultry genetic heritage cryopreservation and reconstruction: advancement and future challenges.

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.

Effect of Dimethylacetamide Concentration on Motility, Quality, Antioxidant Biomarkers, Anti-Freeze Gene Expression, and Fertilizing Ability of Frozen/Thawed Rooster Sperm.

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.

Genetic Basis of Sexual Maturation Heterosis: Insights From Ovary lncRNA and mRNA Repertoire in Chicken.

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.

Effect of glycerol concentration, glycerol removal method, and straw type on the quality and fertility of frozen chicken semen.

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.

Analysis of DNA Methylation Profiles in Mandibular Condyle of Chicks With Crossed Beaks Using Whole-Genome Bisulfite Sequencing.

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.

Monochromatic green light stimulation during incubation shortened the hatching time via pineal function in White Leghorn eggs.

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.

Phenotype characterization of crossed beaks in Beijing-You chickens based on morphological observation.

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 < 0.05) and caused the mandible deviated to the same direction. Meanwhile, the short-side MR prevented the occlusion, leading the jugal arch deformity, which in turn resulted in a bent maxillary horizontally. Similarly, chicks with side beaks also had asymmetry in MR length and the deformities of the jugal arch after dissection. In summary, asymmetric growth of bilateral MR induced crossed beaks and side beaks; the mandibular condyle could be an ideal sample for the related molecular mechanism studies underlying this trait.

Effects of monochromatic green light stimulation during embryogenesis on hatching and posthatch performance of four strains of layer breeder.

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 < 0.01). In addition, hatch window and peak hatching period were not extended by the green light stimulation (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 < 0.05) and the difference disappeared from week 14. The results demonstrate that 12L:12D monochromatic green light stimulation during embryogenesis shortens the hatching time with no negative effects on hatching and posthatch performance. These effects were consistent among the 4 layer strains.