Key miRNAs of chicken seminal plasma extracellular vesicles related with sperm motility regulation.

MicroRNAs (miRNAs) are bio-active elements cargoed by seminal plasma extracellular vesicles extracellular vesicles (SPEVs) which are crucial for sperm function and fertility modulation. This study aimed to isolate, characterize, and identify the miRNA expression profiles in the SPEVs from high (HSM) and low sperm motility (LSM) groups that could serve as fertility biomarkers and explain the underlying mechanisms. The isolated SPEVs were round spherical structures of approximately 50-200 nm in diameter expressing molecular markers. A total of 1006 and 1084 miRNAs were detected in HSM and LSM, respectively, with 34 being differentially expressed. Their targeted genes involved in SNARE interactions in vesicular transport, Metabolic pathways, and Apelin signaling pathway, etc. The joint analysis with mRNAs of sperm and sperm storage tubules cells highlighted the cellular communication mediated by SPEVs miRNAs, where they may rule fertility by affecting sperm maturation and amino acid metabolism. SPEVs as additives could improve fertility of fresh and frozen sperm, while the knockdown of one of the differentially expressed miRNAs, miR-24-3p, diminished this effect, indicating its crucial roles. This study expands our understanding of SPEVs miRNAs mediated sperm maturation and fertility modulation, and may help to develop new therapeutic strategies for infertility and sperm storage.

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

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

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

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

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

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

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

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

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.

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

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

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

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

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.

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.

MicroRNAs with non-additive expression in the ovary of hybrid hens target genes enriched in key reproductive pathways that may influence heterosis for egg laying traits.

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-ß, 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.

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.

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.

Transcriptome Analysis Revealed Potential Mechanisms of Resistance to Trichomoniasis gallinae Infection in Pigeon (Columba livia).

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.