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.

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.

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.

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.