Detection of lymphoproliferative disease virus in Iowa Wild Turkeys (Meleagris gallopavo): Comparison of two sections of the proviral genome.

An accurate diagnostic test is an essential aspect of successfully monitoring and managing wildlife diseases. Lymphoproliferative Disease Virus (LPDV) is an avian retrovirus that was first identified in domestic turkeys in Europe and was first reported in a Wild Turkey (Meleagris gallopavo) in the United States in 2009. It has since been found to be widely distributed throughout North America. The majority of studies have utilized bone marrow and PCR primers targeting a 413-nucleotide sequence of the gag gene of the provirus to detect infection. While prior studies have evaluated the viability of other tissues for LPDV detection (whole blood, spleen, liver, cloacal swabs) none to date have studied differences in detection rates when utilizing different genomic regions of the provirus. This study examined the effectiveness of another section of the provirus, a 335-nucleotide sequence starting in the U3 region of the LTR (Long Terminal Repeat) and extending into the Matrix of the gag region (henceforth LTR), for detecting LPDV. Bone marrow samples from hunter-harvested Wild Turkeys (n = 925) were tested for LPDV with the gag gene and a subset (n = 417) including both those testing positive and those where LPDV was not detected was re-tested with LTR. The positive percent agreement (PPA) was 97.1% (68 of 70 gag positive samples tested positive with LTR) while the negative percent agreement (NPA) was only 68.0% (236 of 347 gag negative samples tested negative with LTR). Cohen's Kappa (κ = 0.402, Z = 10.26, p<0.0001) and the McNemar test (OR = 55.5, p<0.0001) indicated weak agreement between the two gene regions. We found that in Iowa Wild Turkeys use of the LTR region identified LPDV in many samples in which we failed to detect LPDV using the gag region and that LTR may be more appropriate for LPDV surveillance and monitoring. However, neither region of the provirus resulted in perfect detection and additional work is necessary to determine if LTR is more reliable in other geographic regions where LPDV occurs.

PCR identification and prevalence of Eimeria species in commercial turkey flocks of the Midwestern United States.

The present study used a PCR approach to characterize prevalence of coccidial species in fecal samples obtained from 40 individual Midwestern turkey flocks to characterize distribution of species in commercial flocks. Each sample was screened for 6 prominent Eimeria species using species-specific primers and was supplemented with a primary nested-PCR approach for amplification of mitochondrial cytochrome c oxidase subunit gene I where initial sample DNA concentrations were low. All samples were positive for at least one species of Eimeria, while most presented 2 (20/40) or 3 (14/40) species in total. Prevalence across farms was primarily dominated by E. meleagrimitis (97.50%), E. adenoeides (95%), and E. gallopavonis (40%). Of the samples positive for E. adenoeides and E. meleagrimitis, almost half (17/40) contained additional species. Data presented here offer insight into Eimeria species currently challenging the Midwestern US turkey industry and potential need to evaluate flocks for species prior to implementing vaccination programs.

Listeriosis with viral coinfections in 8 gray foxes, 8 wild turkeys, and 2 young cervids in the southeastern United States.

Listeria monocytogenes is a bacterium that can cause disease in many species, including humans, livestock, and wildlife. Increased interactions via shared habitats may promote pathogen transmission among these groups. Our objectives were to evaluate the Southeastern Cooperative Wildlife Disease Study diagnostic data to characterize and compare L. monocytogenes-induced lesions and comorbidities in gray foxes and wild turkeys, and to describe cases of listeriosis in 2 cervids. From 1991-2020, 8 gray foxes, 8 wild turkeys, a neonatal elk, and a white-tailed deer fawn from several eastern states in the United States were diagnosed with listeriosis. All 8 foxes had hepatitis and/or hepatic necrosis with intralesional gram-positive bacilli, and concurrent canine distemper virus (CDV) infection; 2 of the foxes had been vaccinated recently for CDV. L. monocytogenes was cultured from the liver (6 of 8) or lung (2 of 8) of foxes. Lesions in wild turkeys included hepatocellular necrosis (3 of 8), heterophilic hepatitis (1 of 8), heterophilic granulomas (1 of 8), intrasinusoidal gram-positive bacilli without hepatic lesions (1 of 8), granulomatous dermatitis (1 of 8), and/or granulomatous myocarditis (2 of 8). Lymphoproliferative disease viral DNA was detected in 5 of 6 turkeys tested; reticuloendotheliosis viral DNA was detected in 2 of 3 turkeys tested. Both cervids had systemic listeriosis, with L. monocytogenes isolated from liver. Immunohistochemistry for Listeria spp. on select cases revealed immunolabeling in affected organs. Listeriosis was thus established as a cause of morbidity and mortality in 3 wildlife species, which often suffered from concurrent infections and likely immunosuppression.

Transcriptome and Proteome Analysis Revealed Key Pathways Regulating Final Stage of Oocyte Maturation of the Turkey (Meleagris gallopavo).

In birds, the zona pellucida (ZP) matrix that surrounds the ovulated oocyte-called the inner perivitelline layer-is involved in sperm-zona interaction and successful fertilization. To identify the important genes and proteins connected with the final step of egg development, next-generation sequencing and two-dimensional electrophoresis, combined with mass spectrometry, were used for the analysis of mature oocytes at the F1 developmental stage. A total of 8161 genes and 228 proteins were annotated. Six subfamilies of genes, with codes ZP, ZP1-4, ZPD, and ZPAX, were identified, with the dominant expression of ZPD. The main expression site for ZP1 was the liver; however, granulosa cells may also participate in local ZP1 secretion. A ubiquitination system was identified in mature oocytes, where ZP1 was found to be the main ubiquitinated protein. Analysis of transcripts classified in estrogen receptor (ESR) signaling indicated the presence of ESR1 and ESR2, as well as a set of estrogen-dependent genes involved in both genomic and nongenomic mechanisms for the regulation of gene expression by estrogen. Oxidative phosphorylation was found to be a possible source of adenosine triphosphate, and the nuclear factor erythroid 2-related factor 2 signaling pathway could be involved in the response against oxidative stress. Oocyte-granulosa cell communication by tight, adherens, and gap junctions seems to be essential for the final step of oocyte maturation.

Characterization and biological role of cysteine-rich venom protein belonging to CRISPs from turkey seminal plasma†.

Turkey semen contains cysteine-rich secretory proteins (CRISPs) that belong to the dominant seminal plasma proteins. We aimed to isolate and characterize CRISP from turkey seminal plasma and evaluate its possible involvement in yellow semen syndrome (YSS). YSS, which is well characterized, causes reduced fertility and hatchability. The protein was purified using hydrophobic interaction, gel filtration, and reverse phase chromatography. It then was subjected to identification by mass spectrometry, analysis of physicochemical properties, and specific antibody production. The biological function of the isolated protein was tested and included its effects on sperm motility and migration and sperm-egg interactions. Sperm motility was measured with the CASA system using Hobson Sperm Tracker. The reproductive tract of turkey toms was analyzed for gene expression; immunohistochemistry was used for protein localization in the male reproductive tract, spermatozoa, and inner perivitelline layer. The isolated protein was identified as cysteine-rich venom protein-like isoform X2 (CRVP X2; XP_010706464.1) and contained feature motifs of CRISP family proteins. Turkey CRVP X2 was present in both spermatozoa and seminal plasma. The extensive secretion of CRVP X2 by the epithelial cells of the epididymis and ductus deferens suggests its involvement in post-testicular sperm maturation. The internally localized CRVP X2 in the proximal part of the sperm tail might be responsible for stimulation of sperm motility. CRVP X2 on the sperm head might be involved in several events prior to fusion and may also participate in gamete fusion itself. Although the mechanisms by which CRVP X2 mediates fertilization are still unknown, the involvement of complementary sites cannot be excluded. The disturbance of CRVP X2 expression can serve as an etiologic factor of YSS in the turkey. This study expands the understanding of the detailed mechanism of fertilization in birds by clarifying the specific role of CRVP X2.

Discovering lethal alleles across the turkey genome using a transmission ratio distortion approach.

Deviation from Mendelian inheritance expectations (transmission ratio distortion, TRD) has been observed in several species, including the mouse and humans. In this study, TRD was characterized in the turkey genome using both allelic (specific- and unspecific-parent TRD) and genotypic (additive- and dominance-TRD) parameterizations within a Bayesian framework. In this study, we evaluated TRD for 23 243 genotyped Turkeys across 56 393 autosomal SNPs. The analyses included 500 sires, 2013 dams and 11 047 offspring (trios). Three different haplotype sliding windows of 4, 10 and 20 SNPs were used across the autosomal chromosomes. Based on the genotypic parameterizations, 14 haplotypes showed additive and dominance TRD effects highlighting regions with a recessive TRD pattern. In contrast, the allelic model uncovered 12 haplotype alleles with the allelic TRD pattern which showed an underrepresentation of heterozygous offspring in addition to the absence of homozygous animals. For regions with the allelic pattern, only one particular region showed a parent-specific TRD where the penetrance was high via the dam, but low via the sire. The gene set analysis uncovered several gene ontology functional terms, Reactome pathways and several Medical Subject Headings that showed significant enrichment of genes associated with TRD. Many of these gene ontology functional terms (e.g. mitotic spindle assembly checkpoint, DRM complex and Aneuploidy), Reactome pathways (e.g. Mismatch repair) and Medical Subject Headings (e.g. Adenosine monophosphate) are known to be related to fertility, embryo development and lethality. The results of this study revealed potential novel candidate lethal haplotypes, functional terms and pathways that may enhance breeding programs in Turkeys through reducing mortality and improving reproduction rate.

Transcriptome analysis of the hypothalamus and pituitary of turkey hens with low and high egg production.

BACKGROUND: High egg producing hens (HEPH) show increased hypothalamic and pituitary gene expression related to hypothalamo-pituitary-gonadal (HPG) axis stimulation as well as increased in vitro responsiveness to gonadotropin releasing hormone (GnRH) stimulation in the pituitary when compared to low egg producing hens (LEPH). Transcriptome analysis was performed on hypothalamus and pituitary samples from LEPH and HEPH to identify novel regulators of HPG axis function. RESULTS: In the hypothalamus and pituitary, 4644 differentially expressed genes (DEGs) were identified between LEPH and HEPH, with 2021 genes up-regulated in LEPH and 2623 genes up-regulated in HEPH. In LEPH, up-regulated genes showed enrichment of the hypothalamo-pituitary-thyroid (HPT) axis. Beta-estradiol was identified as an upstream regulator regardless of tissue. When LEPH and HEPH samples were compared, beta-estradiol was activated in HEPH in 3 of the 4 comparisons, which correlated to the number of beta-estradiol target genes up-regulated in HEPH. In in vitro pituitary cell cultures from LEPH and HEPH, thyroid hormone pretreatment negatively impacted gonadotropin subunit mRNA levels in cells from both LEPH and HEPH, with the effect being more prominent in HEPH cells. Additionally, the effect of estradiol pretreatment on gonadotropin subunit mRNA levels in HEPH cells was negative, whereas estradiol pretreatment increased gonadotropin subunit mRNA levels in LEPH cells. CONCLUSIONS: Up-regulation of the HPT axis in LEPH and upstream beta-estradiol activation in HEPH may play a role in regulating HPG axis function, and ultimately ovulation rates. Thyroid hormone and estradiol pretreatment impacted gonadotropin mRNA levels following GnRH stimulation, with the inhibitory effects of thyroid hormone more detrimental in HEPH and estradiol stimulatory effects more prominent in LEPH. Responsiveness to thyroid hormone and estradiol may be due to desensitization to thyroid hormone and estradiol in LEPH and HEPH, respectively, due to up-regulation of the HPT axis in LEPH and of the HPG axis in HEPH. Further studies will be necessary to identify possible target gene desensitization mechanisms and elicit the regulatory role of the HPT axis and beta-estradiol on ovulation rates in turkey hens.

Differential expression of cell-cell junction proteins in the testis, epididymis, and ductus deferens of domestic turkeys (Meleagris gallopavo) with white and yellow semen.

Tight, adherens, and gap junctions are involved in the regulation of reproductive tissue function in male mammals. In birds, including domestic turkeys, intercellular interactions performed by junctional networks have not yet been studied. Furthermore, the cellular and molecular basis of yellow semen syndrome (YSS) in the turkey population remains poorly understood. Thus, the aim of the present study was 2-fold: first, to provide new information on the localization and expression of cell-cell junction proteins in the testis, epididymis, and ductus deferens of domestic turkeys and second, to compare expression of junctional protein genes between 2 turkey population, one that produces white normal semen (WNS) and the other that produces yellow abnormal semen. Expression of occludin, zonula occludens-1 (ZO-1), connexin 43 (Cx43), N- and E-cadherin, and ß-catenin genes were investigated using 3 complementary techniques: quantitative real-time PCR, western blot, and immunohistochemistry. Compared to WNS testis, epididymis, and ductus deferens, YSS tissues exhibited downregulation of occludin and ß-catenin mRNA (P < 0.05) and protein (P < 0.05 and P < 0.01, respectively) and upregulation of N- and E-cadherin mRNA (P < 0.001, P < 0.05, P < 0.01, respectively) and protein (P < 0.01, P < 0.05, and P < 0.05, respectively). In contrast, ZO-1 and Cx43 mRNA and protein were upregulated in YSS testis (P < 0.05 and P < 0.001, respectively) but not in epididymis and ductus deferens; both mRNAs and proteins were downregulated (P < 0.05) compared to the respective WNS epididymis and ductus deferens. Altered staining intensity of immunoreactive proteins in YSS vs. WNS reproductive tissue sections confirmed the gene expression results. The present study is the first to demonstrate altered levels of junctional protein gene expression in reproductive tissues of male YSS turkeys. These findings may suggest that subtle changes in junctional protein expression affect the microenvironment in which spermatozoa develop and mature and thus may have an impact on the appearance of yellow semen in domestic turkeys.

Differential Transcriptome Responses to Aflatoxin B1 in the Cecal Tonsil of Susceptible and Resistant Turkeys.

The nearly-ubiquitous food and feed-borne mycotoxin aflatoxin B1 (AFB1) is carcinogenic and mutagenic, posing a food safety threat to humans and animals. One of the most susceptible animal species known and thus a good model for characterizing toxicological pathways, is the domesticated turkey (DT), a condition likely due, at least in part, to deficient hepatic AFB1-detoxifying alpha-class glutathione S-transferases (GSTAs). Conversely, wild turkeys (Eastern wild, EW) are relatively resistant to the hepatotoxic, hepatocarcinogenic and immunosuppressive effects of AFB1 owing to functional gene expression and presence of functional hepatic GSTAs. This study was designed to compare the responses in gene expression in the gastrointestinal tract between DT (susceptible phenotype) and EW (resistant phenotype) following dietary AFB1 challenge (320 ppb for 14 days); specifically in cecal tonsil which functions in both nutrient absorption and gut immunity. RNAseq and gene expression analysis revealed significant differential gene expression in AFB1-treated animals compared to control-fed domestic and wild birds and in within-treatment comparisons between bird types. Significantly upregulated expression of the primary hepatic AFB1-activating P450 (CYP1A5) as well as transcriptional changes in tight junction proteins were observed in AFB1-treated birds. Numerous pro-inflammatory cytokines, TGF-ß and EGF were significantly down regulated by AFB1 treatment in DT birds and pathway analysis suggested suppression of enteroendocrine cells. Conversely, AFB1 treatment modified significantly fewer unique genes in EW birds; among these were genes involved in lipid synthesis and metabolism and immune response. This is the first investigation of the effects of AFB1 on the turkey gastro-intestinal tract. Results suggest that in addition to the hepatic transcriptome, animal resistance to this mycotoxin occurs in organ systems outside the liver, specifically as a refractory gastrointestinal tract.

Genetic basis of leg health and its relationship with body weight in purebred turkey lines.

The aims of this study were to estimate the genetic parameters for leg and foot health and mobility in purebred turkey lines and their genetic correlations with BW. Traits were gait score (GS) as an overall measure of leg health, footpad dermatitis (FPD), and 2 skeletal leg health traits, namely, valgus and varus deformities (VVD) and tibial dyschondroplasia (TD). Data from 4 different lines, comprising 3 yr of phenotypic records and 4 yr of pedigree information per line, were used. The sex average BW for the lines at 18 wk ranged from 19.1 kg (line A) to 12.4 kg (line D). The prevalence of VVD ranged from 5.2 to 14.6% and for TD from 4.1 to 23.2%. The average score for FPD on a scale of 0 to 100 ranged from 48.5 to 61.1. Gait Score was scored on a scale of 1 to 5, standardized to a mean of 3 and SD of 1. Heritabilities were estimated at 0.08 to 0.13 for GS, 0.01 to 0.07 for VVD, 0.06 to 0.12 for TD, and 0.10 to 0.15 for FPD (all SE ≤ 0.02). Estimates of the genetic correlations between VVD and TD ranged from 0.03 to 0.21 (all SE ≤ 0.08), and estimates of these with GS ranged from 0.07 to 0.87 (all SE ≤ 0.09). The genetic correlations of FPD with GS ranged from 0.00 to 0.34 (all SE ≤ 0.04), and with the skeletal leg health traits from -0.06 to 0.33 (all SE ≤ 0.06). Body weight showed estimated genetic correlations ranging from 0.28 to 0.51 (all SE ≤ 0.06) with GS, -0.06 to 0.50 (all SE ≤ 0.13) with VVD/TD and 0.05 to 0.34 (all SE ≤ 0.05) with FPD. The results suggest that selection for improved leg health can be incorporated effectively in a commercial turkey breeding program using balanced breeding goals, in which production traits and leg health traits are considered simultaneously.

Proteomic analysis of white and yellow seminal plasma in turkeys (Meleagris gallopavo).

Yellow semen syndrome (YSS) is endemic within domestic turkey populations. Yellow semen is of lower quality and, when used for insemination, results in reduced fertility and hatchability. Little is known about the etiology of YSS. The aim of this study was to compare the proteome of white and yellow seminal plasma of turkeys using 1) 2-dimensional difference gel electrophoresis (2D-DIGE) to quantify seminal plasma proteins and 2) matrix-assisted laser desorption/ionization mass spectrometry to identify the proteins that are differentially abundant in white and yellow seminal plasma. A total of 49 protein spots (30 upregulated and 19 downregulated) were differentially expressed in yellow seminal plasma compared with white seminal plasma. Transthyretin and serum albumin-like showed a 3-fold increase in seminal plasma from males with YSS, and the latter was validated using Western blot analysis. A 3-fold increase was observed for hemopexin-like and immunoglobulin light chain V-J-C region. Pantetheinase-like showed a 1.3-fold increase. Ovotransferrin, hepatocyte growth factor activator, cysteine-rich secretory protein 3-like, and ferritin heavy chain-like showed a significant decrease (at least a 1.3-fold decrease) in yellow semen. Further studies are necessary to evaluate the precise function of the above-mentioned proteins in YSS and to establish quality markers of turkey semen to predict the reproductive potential of individual turkeys.

The effect of toe trimming on production characteristics of heavy turkey toms.

Trimming the 3 anterior toes on both feet at day of hatch to remove the claws, reduce bird scratching, and improve carcass grades is a common practice in the turkey industry. Changes in the method of trimming and the growth potential of turkeys since the majority of research on this topic was completed motivated this study with the objective of establishing the effects of microwave toe treatment on production characteristics of tom turkeys. Turkey toms (306 in total) were either toe trimmed at the hatchery using a microwave claw processor (T) or were sham treated only (NT). Poults were randomly assigned to 1 of 9 replicate pens for each treatment. Average BW, feed consumption, and feed efficiency were determined from BW and feed intake measured by pen on d 0, 7, 21, 42, 56, 70, 91, 126, and 140. On d 140, toms were sent to a commercial processing facility where 5 carcasses from each pen were examined for scratching and other externally visible damage. Average BW was higher for NT toms on d 91, 126, and 140, with final weights of 21.70 and 21.15 kg for NT and T birds, respectively. The T birds had lower feed consumption than their NT counterparts during the first and last week of production, but feed efficiency was unaffected. Carcass scratching (T, 13.33% of carcasses scratched vs. NT, 15.56%) and other carcass damages were not affected by treatment. Although overall mortality was not affected by treatment, the incidence of mortality due to skeletal causes, especially rotated tibia, was increased in T toms. Negative effects on performance and no effect on carcass quality suggest that toe trimming may not be required or recommended for heavy tom turkeys.

A DNA prime-protein boost vaccination strategy targeting turkey coronavirus spike protein fragment containing neutralizing epitope against infectious challenge.

The present study was undertaken to determine immune response and protection efficacy of a spike (S) protein fragment containing neutralizing epitopes (4F/4R) of turkey coronavirus (TCoV) by priming with DNA vaccine and boosting with the recombinant protein from the corresponding DNA vaccine gene segment. Turkeys were vaccinated by priming with either one dose (G1-750DP) or two doses (G3-750DDP) of 750µg DNA vaccine expressing 4F/4R S fragment and boosting with one dose of 200µg 4F/4R S fragment. One dose of 100µg DNA vaccine mixed with polyethyleneimine (PEI) and sodium hyaluronate (HA) followed by one dose of 750µg DNA vaccine and one dose of 200µg 4F/4R S fragment were given to the turkeys in group G2-100DPH. After infectious challenge by TCoV, clinical signs and TCoV detected by immunofluorescence antibody (IFA) assay were observed in less number of turkeys in vaccination groups than that in challenge control groups. TCoV viral RNA loads measured by quantitative real-time reverse transcription-PCR were lower in vaccinated turkeys than those in challenge control turkeys. The turkeys in G3-750DDP produced the highest level of TCoV S protein-specific antibody and virus neutralization (VN) titer. Comparing to the turkeys in G1-750DP, significantly less TCoV were detected by IFA in the turkeys in G2-100DPH receiving an extra dose of 100µg DNA mixed with PEI and HA. The results indicated that DNA-prime protein-boost DNA vaccination regimen targeting TCoV S fragment encompassing neutralizing epitopes induced humoral immune response and partially protected turkeys against infectious challenge by TCoV.

Effects of 17ß-estradiol on turkey myogenic satellite cell proliferation, differentiation, and expression of glypican-1, MyoD and myogenin.

The hypothesis of this study was that 17ß-estradiol (estradiol) stimulates turkey skeletal muscle growth by influencing myogenic satellite cell proliferation, differentiation, and the gene expression of selected proteins important in regulating growth and development. Increasing levels of estradiol were administered in basal medium containing additional nutrients. Female-derived pectoralis major (PM) satellite cell proliferation was stimulated by estradiol at a level of 10(-9)M following 4days of treatment. Male PM and biceps femoris (BF) satellite cell proliferation was increased at 10(-12)M estradiol. Turkey embryonic myoblast proliferation, however, decreased with 10(-9)M and 10(-5)M estradiol following 3days under these conditions. Estradiol had no effect on the differentiation of any of the 4 groups of cells. Likewise, glypican-1 expression was unaffected by estradiol treatment. MyoD expression decreased in male PM but not BF cells. MyoD expression in female PM cells and embryonic myoblasts were also unaffected by estradiol administration. Estradiol decreased myogenin expression in male satellite cells, but had no effect on female cells. There was a slight decrease in myogenin expression in embryonic myoblasts. The results demonstrate a direct effect of estradiol on avian satellite cell proliferation independent of glypican-1, and decreased expression of MyoD and myogenin in some myogenic cells, coinciding with increased cellular proliferation.

Molecular cloning and tissue-specific expression of Toll-like receptor 5 gene from turkeys.

Toll-like receptors (TLRs), a family of transmembrane and cytosolic proteins, detect microbial patterns, initiating innate immune responses in various organisms. Although they are abundant, genetic characterization and functional differences of TLRs in economically important avian species such as chickens and turkeys have not been investigated in detail. In this study, the putative TLR5 coding region from turkey genome was sequenced, and its homology to other vertebrate species was analyzed. Secondary structure analysis revealed protein motifs typical of the chicken TLR5 protein structure, with 97% amino acid identity between them. mRNA expression profiling in adult turkeys revealed abundant TLR5 expression in a broad range of tissues. Stimulation with the TLR5 ligand flagellin resulted in the production of the inflammatory mediators interleukin (IL)-1beta, IL-6, and nitric oxide in peripheral blood mononuclear cells. To our knowledge, this is the first complete turkey TLR5 coding DNA sequence reported in sequence databases.

Dopamine and mesotocin neurotransmission during the transition from incubation to brooding in the turkey.

We investigated the neuroendocrine changes involved in the transition from incubating eggs to brooding of the young in turkeys. Numbers of mesotocin (MT; the avian analog of mammalian oxytocin) immunoreactive (ir) neurons were higher in the nucleus paraventricularis magnocellularis (PVN) and nucleus supraopticus, pars ventralis (SOv) of late stage incubating hens compared to the layers. When incubating and laying hens were presented with poults, all incubating hens displayed brooding behavior. c-fos mRNA expression was found in several brain areas in brooding hens. The majority of c-fos mRNA expression by MT-ir neurons was observed in the PVN and SOv while the majority of c-fos mRNA expression in dopaminergic (DAergic) neurons was observed in the ventral part of the nucleus preopticus medialis (POM). Following intracerebroventricular injection of DA or oxytocin (OT) receptor antagonists, hens incubating eggs were introduced to poults. Over 80% of those injected with vehicle or the D1 DA receptor antagonist brooded poults, while over 80% of those receiving the D2 DA receptor antagonist or the OT receptor antagonist failed to brood the poults. The D2 DA/OT antagonist groups also displayed less c-fos mRNA in the dorsal part of POM and the medial part of the bed nucleus of the stria terminalis (BSTM) areas than did the D1 DA/vehicle groups. These data indicate that numerous brain areas are activated when incubating hens initially transition to poult brooding behavior. They also indicate that DAergic, through its D2 receptor, and MTergic systems may play a role in regulating brooding behaviors in birds.

Isolation, characterization and cDNA sequencing of acrosin from turkey spermatozoa.

Acrosin (EC 3.4.21.10) is serine proteinase localized in the sperm acrosome and considered to play an essential role in fertilization. In contrast to mammalian, there are only limited data concerning avian acrosin, mostly focused on the characterization of mature enzyme. In the present study, acrosin was isolated from turkey spermatozoa using gel filtration in the presence of 4 M urea at acidic pH. N-terminal Edman sequencing allowed the identification of the first 26 N-terminal amino acids: VVGGTEALHG SWPWIVSIQNPRFAGT. This sequence was used to construct primers and obtain a cDNA sequence from the testis. The amino acid sequence deduced from the cDNA shows that turkey acrosin is initially synthesized as prepro-protein with 19-residue signal peptide. This signal sequence is followed by a 327-residue sequence corresponding to the acrosin zymogen. Turkey proacrosin does not contain a proline-rich segment at the C-terminal portion. Mature turkey acrosin is a two-chain molecule consisting of light and heavy chains and was found to be glycoprotein. The proacrosin/acrosin system exists in turkey spermatozoa and this system can be activated similarly to that of mammals. The high value of association constant strongly suggests that acrosin activity in turkeys can be controlled by a seminal plasma Kazal inhibitor under physiological conditions.

A mitogenomic perspective on the ancient, rapid radiation in the Galliformes with an emphasis on the Phasianidae.

BACKGROUND: The Galliformes is a well-known and widely distributed Order in Aves. The phylogenetic relationships of galliform birds, especially the turkeys, grouse, chickens, quails, and pheasants, have been studied intensively, likely because of their close association with humans. Despite extensive studies, convergent morphological evolution and rapid radiation have resulted in conflicting hypotheses of phylogenetic relationships. Many internal nodes have remained ambiguous. RESULTS: We analyzed the complete mitochondrial (mt) genomes from 34 galliform species, including 14 new mt genomes and 20 published mt genomes, and obtained a single, robust tree. Most of the internal branches were relatively short and the terminal branches long suggesting an ancient, rapid radiation. The Megapodiidae formed the sister group to all other galliforms, followed in sequence by the Cracidae, Odontophoridae and Numididae. The remaining clade included the Phasianidae, Tetraonidae and Meleagrididae. The genus Arborophila was the sister group of the remaining taxa followed by Polyplectron. This was followed by two major clades: ((((Gallus, Bambusicola) Francolinus) (Coturnix, Alectoris)) Pavo) and (((((((Chrysolophus, Phasianus) Lophura) Syrmaticus) Perdix) Pucrasia) (Meleagris, Bonasa)) ((Lophophorus, Tetraophasis) Tragopan))). CONCLUSIONS: The traditional hypothesis of monophyletic lineages of pheasants, partridges, peafowls and tragopans was not supported in this study. Mitogenomic analyses recovered robust phylogenetic relationships and suggested that the Galliformes formed a model group for the study of morphological and behavioral evolution.

Structure, genetic mapping, and function of the cytochrome P450 3A37 gene in the turkey (Meleagris gallopavo).

Cytochromes P450 (P450 for protein; CYP for gene) are a superfamily of membrane-bound hemoproteins that oxidize a large number of endogenous and exogenous compounds. Through oxidation reactions, these enzymes are often responsible for the toxic and carcinogenic effects of natural food-borne toxicants, such as the mycotoxin aflatoxin B1 (AFB1). Previous studies in our laboratory have shown that the extreme sensitivity of turkeys to AFB1 is in part explained by efficient hepatic P450-mediated epoxidation to the toxic and reactive metabolite the exo-AFB1-8,9-epoxide (AFBO). Using 3'-5'-rapid amplification of cDNA ends (RACE), we amplified CYP3A37 from turkey liver RNA, the E. coli-expressed protein which efficiently epoxidates AFB(1). Turkey CYP3A37 has an ORF of 1512 bp, and the protein is predicted to be 504 amino acids with 97% homology to chicken CYP3A37. The turkey gene is organized into 13 exons and 12 introns. A single nucleotide polymorphism in the 11th intron was used to assign CYP3A37 to turkey linkage group 10 (corresponding to chicken chromosome 14, GGA14). Because of the important role of P450s in the extreme sensitivity of turkeys to the toxic effects of AFB(1), this study will contribute to the identifying allelic variants of this important gene in poultry.

Cloning of PRL and VIP cDNAs of the Java sparrow (Padda oryzivora).

Complementary DNA (cDNA) of prolactin (PRL) and vasoactive intestinal polypeptide (VIP) of the Java sparrow were cloned and sequenced. The proximal region of the PRL promoter was also identified. Java sparrow PRL was found to have 88.3, 88.3, and 89.1% sequence identity at the cDNA level to PRL of chicken, turkey, and duck, respectively. The predicted amino acid sequence had an overall similarity with a comparable region of chicken (91.4%), turkey (88.9%) and duck (92.0%) PRL. Based on the cDNA sequence and genomic structure of the chicken PRL gene, the proximal promoter was characterized. Sequence analysis of the proximal region of Java sparrow PRL promoter revealed a high degree of similarity to that of chicken, turkey and duck PRL promoters. Moreover, cDNA of prepro-VIP was also cloned and sequenced. Java sparrow prepro-VIP shows high similarity to chicken and turkey prepro-VIP. However, the region upstream of the 5' untranslated region of Java sparrow prepro-VIP did not show similarity to that of chicken. These results suggest that the mechanisms, which regulate expression of the VIP gene, may be different between precocial and altricial birds, but expression of the PRL gene may be widely conserved in avian species.