Characterization of the cathelicidin cluster in the Japanese quail (Coturnix japonica)

The Japanese quail has several advantages as a low‐fat meat bird with high immunity against diseases. Cathelicidins (CATHs) are antimicrobial peptides that play an important role in innate immunity. The aim of this study was to characterize the CATH cluster in the Japanese quail (Coturnix japonica). The Japanese quail CATH (CjCATH) cluster, contains four CATH genes, as in the chicken. The coding sequences of CjCATHs exhibited >85.3% identity to chicken CATHs. The predicted amino acid sequences of the four CjCATH genes contained the cathelin‐like domain characteristic of CATH proteins. Polymorphisms were detected in the open reading frames (ORFs) of all CjCATH sequences. Two amino acid substitutions were observed in the antimicrobial region of the mature peptide of CjCATH2, and predicted to influence peptide function. CjCATH1 is expressed in lung, heart, bone marrow and bursa of Fabricius (BF). CjCATH2 is expressed in bone marrow. CjCATH3 is expressed in lung, heart, bone marrow, BF, tongue and duodenum. CjCATHB1 is expressed in bone marrow and BF. This study is the first to characterize CATH genes in the Japanese quail, and identifies novel antimicrobial peptide sequences belonging to the cathelicidin family, which may play a role in immunity in this species.     

Immunohistochemical and ultrastructural characterization of the ovarian surface epithelium of Japanese quail (Coturnix japonica)

Contrary to humans, most ovarian tumors in other species do not arise from the ovarian surface epithelium but are of follicular‐, stromal‐ or germ‐cell origin. One of the few species where ovarian cancer arises from the ovarian surface epithelium (OSE) is chicken (Gallus domesticus). Little is known about the morphology of the OSE in other avian species. In our study we analyzed the OSE morphology of Japanese quail (Coturnix japonica) using ultrastructural and histochemical techniques. Carbohydrate residues have been studied by using a panel of fluorescein isothiocyanate labeled lectins. Japanese quails are commonly used animal models in biomedical research as their housing is comparatively inexpensive and they show a short generation interval. Our ultrastructural and histochemical results demonstrate that the quail ovarian surface epithelium shows characteristic features which resemble the epithelia of both chicken and human. Additionally, the ovarian surface epithelium of the Japanese quail contains cytokeratin as well as vimentin intermediate filaments in their cytoplasm. Therewith and among other parts the quail OSE shows many characteristic features also seen in those of humans, which may qualify quail's ovary as a potential animal model for human ovarian carcinomas.     

Analysis of 46-kDa protein in the perivitelline membrane of Japanese quail (Coturnix japonica)

The extracellular matrix surrounding the oocyte before ovulation is called the perivitelline membrane (PL) in avian species. The PL is constructed with two major glycoproteins, ZPC and ZP1, which are synthesized in the ovarian granulosa cells and the liver, respectively. Although the properties of the major components in the PL have been examined, knowledge about the nature of its minor constituents is lacking. In this study we focused on PL protein, which migrates at 46‐kDa in the gel of SDS‐PAGE. N‐terminal sequence analysis demonstrated that the 46‐kDa protein is the C‐terminal fragment of ZP1. Analysis of lysylendopeptidase digests or cyanogens bromide‐degraded fragments of ZP1 confirmed this postulate. Western blot analysis using antiserum against 46‐kDa protein indicated the absence of 46‐kDa protein in the serum. Moreover, small immunoreactive bands, thought to be cleaved fragments of ZP1, were detected in the PL lysate by western blot analysis using antiserum against the N‐terminal peptide of ZP1. These results indicated that the N‐terminal proteolytic processing of ZP1 might take place after the arrival of ZP1 at the ovary, and the resulting product, 46‐kDa protein, is incorporated into the PL.     

Effects of probiotic Bacillus cereus toyoi on abdominal fat accumulation in the Japanese quail (Coturnix japonica)

Effects of a commercial probiotic, Bacillus cereus toyoi on abdominal fat accumulation in the Japanese quail fed either a commercial control diet (crude protein (CP), 23.5%; metabolizable energy (ME), 11.7MJ/kg) or a high‐energy diet (CP, 23.0%; ME, 13.8MJ/kg) were investigated. Four‐week‐old male birds (n = 108) were divided into four diet groups: control diet, control with probiotic, high‐energy diet and high‐energy diet with probiotic. At 8 weeks and 12 weeks of age (4 and 8 weeks of probiotic supplementation period, respectively), abdominal fat, muscles and the liver were weighed. The weight of total fat and protein in the muscles and liver were also determined. A rectal temperature was recorded weekly. Bodyweight and feed intake in both diets were not affected by probiotic supplementation. At 8 weeks of age, birds fed the control diet with probiotic had significantly less abdominal fat than those fed without the probiotic, and a similar tendency was seen for both diet groups at 12 weeks of age. The decrease in abdominal fat coincided with an increase in rectal temperature. These results suggest that greater metabolizable energy was consumed through elevation of heat production by supplementation of B. cereus toyoi, with hardly any energy stored as body fat. Probiotic supplementation appears to induce greater muscle weight, higher protein and lower lipid content in muscles by 8 weeks of age, and lower lipid content in muscles and greater liver weight by 12 weeks of age, in the Japanese quail.     

Characterization and expression of non‐polymorphic liver expressed antimicrobial peptide 2: LEAP‐2 in the Japanese quail,Coturnix japonica

Liver‐expressed antimicrobial peptide 2 (LEAP‐2) is a cationic peptide that plays an important role in innate immunity for host defense. The aim of this study was to characterize the LEAP‐2 gene in the Japanese quail (Coturnix japonica). Japanese quail LEAP‐2 (CjLEAP‐2) was identified from the Japanese quail draft genome database by a local BLAST analysis using chicken LEAP‐2 (GgLEAP‐2). The exon‐intron structure of CjLEAP‐2, analyzed from three quails, is composed of three exons, as is the chicken LEAP‐2 homolog (GgLEAP‐2). An analysis of the coding sequence revealed that CjLEAP‐2 is 231 bp long, like GgLEAP‐2, and 93% identical to GgLEAP‐2 at the nucleic acid level. The predicted amino acid sequence of CjLEAP‐2 contained the liver‐expressed antimicrobial peptide 2‐precursor domain and four cysteine residues characteristic of the LEAP‐2 protein. The amino acid sequence of the mature peptide of CjLEAP‐2 was 100% identical to that of GgLEAP‐2. We confirmed that CjLEAP‐2 was transcribed in at least seven tissues, including the digestive system. Additionally, the mature peptide region of CjLEAP‐2 exhibited no polymorphisms in 99 quails from six strains. Taken together, these findings indicate that CjLEAP‐2 is non‐polymorphic and therefore, it likely plays an important role in the innate immunity of quail as it does in chicken.     

Basic characterization of avian β‐defensin genes in the Japanese quail,Coturnix japonica

In this study, we identified a cluster of 14 avian β‐defensins (AvBD; approximately 66 kbp) in the Japanese quail, Coturnix japonica. Except for AvBD12 (CjAvBD12) and ‐13, the CjAvBDs coding sequences exhibited greater than 78.0% similarity to the respective orthologous chicken AvBD genes (GgAvBD). The putative amino acid sequence encoded by each CjAvBD contained six cysteine residues and the GXC (X1‐2) motif considered essential for the β‐defensin family. Each CjAvBDs also formed a sub‐group with the respective orthologous genes of various bird species in a phylogenetic tree analysis. Synteny between the CjAvBD cluster and GgAvBD cluster was confirmed. The CjAvBD cluster was mapped on the long‐arm end of chromosome 3 by linkage analysis based on single nucleotide polymorphisms (SNPs) of CjAvBD1 and CjAvBD12 (approximately 46kbp), as well as GgAvBD cluster. We also confirmed that CjAvBD1, ‐4, ‐5, ‐9, and ‐10 are transcribed in 20 tissues, including immune and digestive tissues. However, our experimental data indicated that the CjAvBD cluster lacks the AvBD3 and ‐7 loci, whereas the CjAvBD101α, ‐101β, and ‐101θ loci arose from gene duplication of the AvBD6 orthologous locus in the CjAvBD cluster after differentiation between Coturnix ‐ Gallus.     

Polymerase chain reaction-restriction fragment length polymorphism method for identifying carriers of hemophilia A in Japanese brown cattle

Hemophilia A is a severe congenital bleeding disorder characterized by subcutaneous hematoma and hemorrhage into muscles resulting from a deficiency of blood coagulation factor VIII. The authors have recently reported two cases of hemophilia A in Japanese Brown cattle and identified a nucleotide substitution in the factor VIII gene, resulting in an amino acid substitution of Leu to His, as a possible cause of the deficiency. In the present study, a simple and effective polymerase chain reaction (PCR)‐based diagnostic method was developed to identify carriers of this disorder, using a mismatch primer in combination with restriction enzyme digestion. The PCR reaction amplified a 118 bp fragment, which was not digested by the BspT104I restriction enzyme in affected animals but was digested into two fragments in normal animals. Both digested and undigested fragments were observed in carrier animals. This method was applied to identify the carriers of hemophilia A in a population of Japanese Brown cattle. By screening 155 DNA samples from Japanese Brown cattle, except for the dam of the two probands, no carriers were identified. It was therefore concluded that the probands represent isolated cases of hemophilia A, and that the frequency of the mutant allele in the Japanese Brown cattle population is very low.     

Primary analysis of DNA polymorphisms in the TRIM region (MHC subregion) of the Japanese quail,Coturnix japonica

Based on sequences of two cosmid clones from Japanese quail (Coturnix japonica, Coja), we confirmed that the syntenic cluster, GNB2L1～BTN1～BTN2, is located in the quail TRIM subregion of the quail major histocompatibility complex (MHC Coja) region. These cosmids also included four CjBG loci and one CjLEC locus; therefore, the quail TRIM subregion was thought to be adjacent to the BG/LEC subregion. We then identified three polymorphic markers – CjHEP21, CjTRIM39.2 and CjBTN2 – in the TRIM subregion that may be useful for the functional analysis of the MHC‐Coja region. We examined MHC‐Coja sequences from 321 individual quails sampled from 11 inbred strains, and we found eight alleles for each of the three genes – CjHEP21, CjTRIM39.2 and CjBTN2. These polymorphisms represent the first avian DNA markers in the TRIM subregion. Additionally, we discovered a quail‐specific VNTR (variable number of long tandem repeats, 133–137 bp) in intron 7 of CjBTN2. We identified 25 haplotypes in the sample of 321 quail; these haplotypes comprised combinations of all 24 alleles of the three polymorphic genes. We suggest that there are two recombination hotspots, one between each pair of adjacent loci. All strains, except AMRP, contained multiple haplotypes; the AMRP strain contained a single, apparently fixed haplotype.     

Hypocholesterolemic effect of karaya saponin in Japanese laying quails (Coturnix coturnix japonica)

The effect of karaya saponin supplementation on the serum and egg yolk cholesterol and fatty acid composition in egg yolk were investigated in Japanese quails. A total of 80 Japanese quails aged 5 weeks were equally divided into four groups of 20. Four levels (0, 25, 50 and 75 mg/kg feed) of karaya saponin were included in the basal diet and experiment was lasted for 6 weeks. The cholesterol fractions in the egg yolk and serum were measured by enzymatic assay, and the fatty acid composition in egg yolk was determined by gas chromatography. The results revealed that the supplementation of 75 mg/kg karaya saponin significantly reduced (p < 0.05) cholesterol and triglycerides concentration in serum and egg yolk. High-density lipoprotein-cholesterol was increased, low-density lipoprotein-cholesterol and the atherogenic index were decreased (p < 0.05) by the dietary supplementations. Hepatic cholesterol was reduced (p < 0.05) by the 25 mg/kg karaya saponin. A higher degree of yolk colour was improved (p < 0.05) when 75 mg/kg saponin was supplemented in the diet. The concentration of polyunsaturated fatty acids (PUFA) in egg yolk was increased (p < 0.05) in a dose-dependent manner in quails fed the supplemented diet than the control diet. The ratio of PUFA to saturated fatty acids in egg yolk was improved (p < 0.05) by 75 mg/kg feed karaya saponin-supplemented diet. Therefore, the dietary supplementation of 75 mg/kg karaya saponin may be a feasible means of producing quail eggs with lower cholesterol and higher PUFA content for health conscious consumers.     

Intestinal mucosa develops in a sex-dependent manner in Japanese quail (Coturnix japonica) fed Saccharomyces cerevisiae

ABSTRACT

1. The aim of study was to investigate whether the impact of the yeast Saccharomyces cerevisiae on the histological structure of the intestine, innervation of the small intestine wall, and basal biochemical serum parameters in Japanese quail was sex dependent.

2. One-day-old healthy male and female Japanese quail were fed either a basal diet containing no yeast (control group) or the basal diet plus 1.5% (15 g/kg of diet) of yeast (S. cerevisiae inactivated by drying). Samples from the duodenum and jejunum were taken from each bird at the age of 42 days. Blood samples were collected at this age and the concentrations of glucose, total protein, creatinine, uric acid, lipid profile (total cholesterol, low density lipoproteins (LDL), high density lipoproteins (HDL) and triacylglycerols (TG)), alanine aminotransferase (ALAT), aspartate aminotransferase (AspAT), lactate dehydrogenase (LDH), amylase (AMY), calcium, phosphorus and iron were determined.

3. Female quail fed diets supplemented with yeast had significantly lower total cholesterol and amylase activity than the control females. The concentration of HDL was higher in the male quail than in the females, irrespective of the treatment. An opposite effect was observed in LDL. The diet treatments influenced the activity of AspAT, which was significantly less in the male quail fed diets with 1.5% yeast.

4. Supplementation with S. cerevisiae increased the myenteron, submucosa and mucosa thickness, villus length and thickness and size of absorptive surface, while the number of villi and enterocytes were decreased in the duodenum in males. Female quail showed an increased absorptive surface in the jejunum. The Meissner (submucosal) plexuses were influenced by the feeding and sex to a greater extent than the Auerbach plexus (in the muscularis propria).

5. The results demonstrated that S. cerevisiae (1.5%) in the diet caused significant positive effects in Japanese quail, exerting an effect on the morphology of the small intestine in a sex-dependent manner.     

Influence of animal age on body concentrations of minerals in Japanese quail (Coturnix japonica)

Mathematical modelling of the relationships between mineral inputs and outputs would enable the prediction of mineral requirements of poultry under a wide range of conditions. To establish the feasibility of possible modelling of mineral requirements, the current study aimed to describe the individual mineral concentrations of whole bodies of quail over the life cycle from hatching to 70 days of age. Quail were reared indoors without any restrictions that could limit growth. Sampling of birds (n = 6–18) was carried out at 0, 3, 7, 14, 21, 35, 49 and 70 days after hatching. Freeze‐dried samples of whole bodies (digestive contents removed) were analysed for ash, and macrominerals (calcium, magnesium, phosphorus, potassium, sodium) and microminerals (copper, iron, manganese, nickel, selenium, zinc). Ash concentration followed a curvilinear trend, with a maximum of 101.7 g/kg dry matter at 32.77 days. Individual mineral concentrations, expressed as a proportion of ash, were fluctuating over time, with the most prominent changes at 3 days and again at either 14 or 21 days. Dissimilar patterns in individual mineral concentrations resulted that ratios between minerals followed inconsistent patterns over time. Although mineral contents in absolute quantities can be described through modelling over the entire life cycle of the bird, it can be concluded that variable concentrations of individual minerals could complicate further model development.     

CXCL14-like immunoreactivity in somatostatin-producing cells of the Japanese quail (Coturnix japonica) pancreas

This study examines chemokine CXCL14-like peptide distribution in the Japanese quail (Coturnix japonica) pancreas using a specific anti-human CXCL14 antibody. CXCL14-immunoreactive cells were observed in the pancreatic islet peripheral region. The staining was abolished after pre-absorbing the antibody with recombinant human CXCL14. CXCL14-immunoreactive cells were immuno-positive for somatostatin, but not glucagon and insulin. CXCL14 secreted from somatostatin-producing cells might participate in insulin secretion modulation together with somatostatin. In addition, CXCL14 might participate in glucose homeostasis in co-operation with somatostatin and growth hormone.     

A Growth Performance and Nonlinear Growth Curve Functions of Large- and Normal-Sized Japanese Quail (Coturnix japonica)

This study aimed to evaluate the differences between the growth patterns of large- and normal-sized Japanese quail strains and their F₁ progeny, by fitting their growth parameter values to five nonlinear regression growth models (Weibull, Logistic, Gompertz, Richards, and Brody). The Richards model presented the best fit for both sexes of the large-sized quail strain, whereas the Gompertz model presented the best fit for both sexes of the normal-sized quail strain, based on goodness-of-fit criteria (higher adjusted R² and lower Akaike and Bayesian information criteria). Both sexes of F₁ birds derived from the cross between normal-sized females and large-sized males were best fitted by the Richards model. In contrast, growth parameters of the F₁ birds derived from the cross between large-sized females and normal-sized males were best fitted to the Gompertz model. The data could be fitted nearly as well to the Weibull and Logistic models as to the Richards and Gompertz models. The Brody model presented the poorest fit for the growth parameter values. The results indicated that the Richards and Gompertz models could best describe the growth characteristics of both large- and normal-sized quails. Moreover, the observed growth pattern of the F₁ birds was likely inherited from the male parental strain. To the best of our knowledge, this is the first study comparing the growth curves of the reciprocal F₁ generations with their parental strains in quails.     

Histological and histochemical studies on the oviduct microcirculation of the laying Japanese quail (Coturnix japonica)

The health of oviduct circulation is very important for normal egg production. The present work describes the histology and histochemistry of vessels of different types on the oviduct of quail. Arteria, venae and lymphatics of the oviduct were described clearly. In addition, special vascular elements were observed presenting circular, longitudinal and oblique smooth muscle fibres on the blood vessel wall. Glomus cells were observed at typical glomus vessels, atypical glomus vessels and on the wall of some of arteria and arteriolae. Also, spirally oriented blood vessels were observed. Dark brown pigment was detected in the wall of arteria, venae, lymphatics and the special types of blood vessels.     

The effects of transportation stress on Japanese quail (Coturnix Coturnix japonica) fed corn‐based diet in comparison with wheat‐based diet supplemented with xylanase and phytase

Harvesting, handling and transporting quails to the slaughterhouses, other farms and laboratories might covertly reduce their welfare. The aim of this study was to evaluate the effects of two major sources of energy in poultry nutrition on reducing transportation stress in Japanese quail (Coturnix Coturnix japonica). Male quails (n = 60) were divided into two groups. The first group was fed corn‐based diet, and the second was fed wheat‐based diet supplemented with xylanase and phytase. At the end of the experiment (day 35), quails were subjected to 80 km of transportation. Immediately on arrival and after 24 h, heterophil counts, lymphocyte counts and H:L ratios were measured. On arrival, H counts were lower, L counts were higher, and H:L ratios were lower for corn‐fed group. After 24 h, wheat‐fed group showed lower increment of H counts, greater increment of L counts and also decrement of H:L ratios rather than corn‐fed group which showed increment of H:L ratios. However, these ratios were still lower in corn‐fed group. Results indicate that corn‐based diets can help Japanese quail to better resist transportation stress, although it seems that feeding wheat‐based diets supplemented with xylanase and phytase could have positive effects for coping better with stress after journeys.     

Effect of a single polymorphism in the Japanese quail NK‐lysin gene on antimicrobial activity

NK‐lysins are cationic peptides that play important roles in host protection, and are an important constituent of innate immunity. We identified nine single‐nucleotide polymorphisms (SNPs) in the NK‐lysin open reading frame (ORF) from 32 Japanese quails in six strains: A, B, ND, K, P, and Y. The G to A substitution at nucleotide position 272 in the ORF resulted in a Gly (G) to Asp (D) amino acid substitution (Cj31G and Cj31D alleles). The Cj31D allele was detected in P (frequency 0.76) and Y (frequency 0.03) strains. We compared the antimicrobial activities of four synthetic peptides from the helix 2‐loop‐helix 3 region of avian NK‐lysins against Escherichia coli: Cj31G and Cj31D from quail and Gg29N and Gg29D from chicken. The antimicrobial activities of the four peptides decreased in the following order: Gg29N > Cj31G > Gg29D > Cj31D (P < 0.05). Although there were no differences in the predicted secondary structure of the Cj31G and Cj31D, the net charge of the Cj31G was higher than that of Cj31D. These data indicated that the antimicrobial activity of CjNKL is influenced by net charge, similar to that which has been observed in chicken. © 2015 Japanese Society of Animal Science