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.

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.

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.

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.

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

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

Analysis of Long Non-Coding RNAs and mRNAs Associated with Lactation in the Crop of Pigeons (Columba livia).

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.

Identification of microRNA-Associated-ceRNA Networks Regulating Crop Milk Production in Pigeon (Columba livia).

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.

Characterization of gonadotropin releasing hormone receptor gene in Sokoto and Kalahari Red goats.

The objective of this study was to characterize GnRHR gene in Sokoto (n = 70) and Kalahari Red (n = 70) goats. Three SNPs, (g.-29T > G, g.48 G > A and g.209 T > G), were detected in Sokoto Red (SR) and one (g.48 G > A) in Kalahari Red (KR) goats. All the mutations occurred within the 5'UTR and Exon one of the gene and the g.209 T > G was non-synonymous and, therefore, resulted in an amino acid change from methionine to arginine at Position 70 of the GnRHR polypeptide. The homozygous mutant genotypes at the three SNP loci were not detected in both breeds but minor allele frequencies were ≥ 0.1 for the three SNP loci in SR goats. Frequency of the T allele, however, was 0.93 at the only SNP locus detected in KR goats. There was a strong linkage disequilibrium (LD; r2>0.98) among the detected mutations in SR goats resulting in two haplotypes (T-G-T and G-A-G) with a frequency of 86% and 13%, respectively. There was no significant association between genotypes at the polymorphic loci and litter size (P > 0.05) in the two breeds. The non-synonymous mutation (g.209T>G) appears to have changed the nucleotide binding region and area spanning exposed/buried regions on the predicted secondary structure of the two variants of the receptor. This change led to variation in the tertiary structure between the two variants of the receptor and can influence the function of the transmembrane receptor. Comparison of the GnRH receptor domains for goats, sheep, cattle and swine confirmed that the seven transmembrane domains of the receptor are conserved in all the farm animals considered.