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.     

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     

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.     

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.     

Serum amyloid A (SAA) mRNA expression in chicken and quails in response to bacterial stress

Monitoring of acute phase proteins such as serum amyloid A at gene expression level may provide quick information about immune status of the host and its susceptibility towards common infections. Present study was carried out to evaluate and compare the mRNA expression of SAA gene in Rhode Island Red chicken (RIR) and Japanese quails using real time PCR analysis in response to inactivated Salmonella gallinarum culture. The results showed that expression of SAA gene was approximately 17–33 folds higher in case of birds administered with bacterial culture when compared to un-inoculated controls and expression was higher and quicker in case of quails than RIR chicken. The SAA genes from chicken and quail were cloned and upon sequence analysis it was observed that deduced amino acid sequence of SAA from chicken and quails were having approximately seven percent variation which might have significance in function of this protein in these species.     

Susceptibility difference between methicillin‐susceptible and methicillin‐resistant Staphylococcus aureus to a bovine myeloid antimicrobial peptide (BMAP‐28)

A bovine myeloid antimicrobial peptide antimicrobial peptide (BMAP‐28) is a member of the cathelicidin family and acts as a component of innate immunity. There are few reports of susceptibility difference of methicillin‐resistant Staphylococcus aureus (MRSA) and susceptible strains (MSSA) against BMAP‐28. This study aims to clarify how a few amino acid substitutions of BMAP‐28 are related to its antimicrobial activity using four analog peptides of BMAP‐28. We also compared cellular fatty acid components of MSSA and MRSA using gas chromatography. We found that a few amino acid substitutions of BMAP‐28 do not change antimicrobial activity. It was also revealed that the percentage of cis‐11‐eicosenoic acid in total detected fatty acids of MRSA was significantly higher than that of MSSA. In addition, the percentage of palmitic acid in total detected fatty acids of MRSA tended to be lower than that of MSSA. Our results will provide new information to deal with the question of differences in bacterial susceptibility against BMAP‐28.     

Developmental stages of the blue‐breasted quail (Coturnix chinensis)

Chickens and Japanese quail (Coturnix japonica) have traditionally been the primary avian models in developmental biology research. Recently, the blue‐breasted quail (Coturnix chinesis), the smallest species in the order Galliformes, has been proposed as an excellent candidate model in avian developmental studies owing to its precocious and prolific properties. However, data on the embryonic development of blue‐breasted quail are scarce. Here, we developed a normal developmental series for the blue‐breasted quail based on developmental features. The blue‐breasted quail embryos take 17 days to reach the hatching period at 37.7°C. We documented specific periods of incubation in which significant development occurred, and created a 39‐stage developmental series. The developmental series for the blue‐breasted quail was almost identical to that for chickens and Japanese quail in the earlier stages of development (stages 1–16). Our staging series is especially useful at later stages of development (stages 34–39) of blue‐breasted quail embryos as a major criterion of staging in this phase of development was the weight of embryos and the length of third toes.     

Intracerebroventricular administration of chicken glucagon‐like peptide‐2 potently suppresses food intake in chicks

Glucagon‐related peptides, such as glucagon‐like peptide (GLP)‐1, GLP‐2 and oxyntomodulin (OXM), are processed from an identical precursor proglucagon. In mammals, all of these peptides are suggested to be involved in the central regulation of food intake. We previously showed that intracerebroventricular administration of chicken OXM and GLP‐1 significantly suppressed food intake in chicks. Here, we show that central administration of chicken GLP‐2 potently suppresses food intake in chicks. Male 8‐day‐old chicks (Gallus gallus domesticus) were used in all experiments. Intracerebroventricular administration of chicken GLP‐2 significantly suppressed food intake in chicks. Plasma glucose concentration was significantly decreased by chicken GLP‐2, whereas plasma nonesterified fatty acid concentration was significantly increased. Intracerebroventricular administration of chicken GLP‐2 did not affect plasma corticosterone concentration. In addition, the anorexigenic effect of GLP‐2 was not reversed by the corticotropin‐releasing factor (CRF) receptor antagonist α‐helical CRF, suggesting that CRF is not a downstream mediator of the anorexigenic pathway of GLP‐2 in chicks. Intracerebroventricular administration of an equimolar amount of GLP‐1 and GLP‐2, but not OXM, significantly suppressed food intake in both broiler and layer chicks. All our findings suggest that GLP‐2 functions as a potent anorexigenic peptide in the brain, as well as GLP‐1, in chicks.     

Effect of dietary supplementation of calcium butyrate on growth performance,carcass traits,intestinal health and pro‐inflammatory cytokines in Japanese quails

The objective of the present study was to evaluate the potential effect of dietary calcium butyrate on growth performance, carcass traits and gut health in Japanese quails. In total, 320 one‐day‐old Japanese quails were randomly assigned to 4 equal treatments, with 8 replicates of 10 Japanese quails, for 4 weeks. The Japanese quails in control treatment were fed control diet whereas in the other treatments the Japanese quails were fed diet supplemented with calcium butyrate at 0.3, 0.5 and 0.7 g/kg diet. Data concerning performance measurements were recorded weekly. In addition, eight Japanese quails (one/replicate) from each treatment were selected randomly for serum collection to measure pro‐ and anti‐inflammatory cytokines. Pooled faecal samples from each replicate of each treatment were also collected at three time points (0, 2 and 4 weeks) for count E. coli and C. perfringens. The results showed that after 7 days of the experimental period, Japanese quails fed calcium butyrate supplemented diet at 0.7 g/kg showed a greater (p < .05) body weight and a favourable (p < .05) feed conversion ratio than the other treatments. Moreover, serum superoxide dismutase and catalase activities were increased (p < .05) in Japanese quails fed calcium butyrate supplemented diet at 0.7 g/kg. Calcium butyrate supplementation at 0.7 g/kg was associated with reduction (p < .05) in TNF‐α, IL‐6 and IL1‐β, while IL‐10 was increased (p < .05). In addition, after 2 weeks of calcium butyrate supplementation, a reduction (p < .05) in E. coli and C. perfringens counts was observed in excreta of Japanese quails fed 0.5 and 0.7 g calcium butyrate/kg diets. It is concluded that calcium butyrate supplementation improves body weight gain, reduces E. coli and C. perfringens counts and has anti‐inflammatory/anti‐oxidant effect in Japanese quails.     

Skeletal development in blue‐breasted quail embryos

The blue‐breasted quail (Coturnix chinensis), the smallest species of quail with short generation interval and excellent reproductive performance, is a potential avian research model. A normal series of skeletal development of avian embryos could be served as a reference standard in the fields of developmental biology and teratological testing as well as in the investigation of mutation with skeletal abnormalities and in the study of the molecular mechanisms of skeletal development through genome manipulation. Furthermore, ossification sequence shows a species‐specific pattern and has potential utility in phylogeny. However, data on the skeletal development of blue‐breasted quail embryos are scarce. Here, we established a series of normal stages for the skeletal development of blue‐breasted quail embryos. Cartilage and ossified bones of blue‐breasted quail embryos were stained blue and red with Alcian blue 8GX and Alizarin red S, respectively. The time and order of chondrification and calcification of their skeletons were documented every 24 hr from 3 to 17 days of incubation, and a 15‐stage series of skeletal development was created. Moreover, a comparative study with the Japanese quail (Coturnix japonica) demonstrated that ossification sequence differed significantly between these two species.     

Effect of low‐ or high‐CP diet with cold‐pressed oil supplementation on growth,immunity and antioxidant indices of growing quail

This study was to investigate the effect of dietary protein levels and supplementation with two cold‐pressed oils on the performance, immunity and antioxidant indices of growing Japanese quails. A 3 × 4 factorial experiment, using 3 dietary crude protein (CP) levels (22%, 24% and 26%) and 4 supplemental herbal oils levels: control without additives, 250 mg marjoram (Mar), 250 mg thyme (Thy) and 125 mg Mar + 125 mg Thy per kg diet. A total of 720 unsexed, 7‐day‐old quail chicks were randomly distributed into twelve treatment groups (4 replicates per group of 15 chicks in each). The live body weight (LBW) on 42 days and body weight gain (BWG) from 7 to 42 days were significantly improved (p < 0.01 and 0.05) in chicks fed 24% or 26% CP compared with those of the 22% CP group. The groups fed diets supplemented with Mar or in combination with Thy oil exhibited significantly better LBW, BWG and feed conversion ratio (FCR) than the control at all periods except BWG from 21 to 42 days and FCR from 7 to 21 days were not significant. Feed intake was gradually decreased with increasing dietary CP level or adding herbal oils during the periods 21–42 and 7–42 days of age. Plasma triglycerides, cholesterol, total lipids and malondialdehyde were decreased by cold‐pressed oils supplementation, but the activity of reduced glutathione and superoxide dismutase was increased (p < 0.01) compared with the control. In conclusion, quails fed a diet containing 24% or 26% CP or diets supplemented with Mar or Thy oils or both exhibited improvement in the performance, lipid profile, immunity and antioxidant capacity without any detrimental impacts on the other studied parameters.     

Antimicrobial activity of a bovine myeloid antimicrobial peptide (BMAP‐28) against methicillin‐susceptible and methicillin‐resistant Staphylococcus aureus

A bovine myeloid antimicrobial peptide (BMAP‐28) is a member of the cathelicidin family which is included in the innate immune system of mammals. Recently, there have been many studies about antimicrobial peptides. This study aims to clarify whether BMAP‐28 has bactericidal activity against methicillin‐resistant Staphylococcus aureus (MRSA) and compares its activity against methicillin‐susceptible S. aureus (MSSA) and MRSA. We found that the peptide was effective in killing MRSA (minimal inhibitory concentration (MIC) range; 5–20 µg/mL). It was also revealed that MSSA (MIC range; 1.25–20 µg/mL) had two levels of susceptibility to BMAP‐28. We also examined the effect of BMAP‐28 on bacterial shape to visually show its activity. After exposure to the peptide, both MSSA and MRSA cells showed the morphological changes on their surfaces. Our results indicate that BMAP‐28 is a promising candidate for medicine against drug‐resistant bacteria.     

Effects of dietary oleuropein supplementation on growth performance,serum lipid concentrations and lipid oxidation of Japanese quails

An in vivo experiment was conducted to compare the effects of dietary supplementation with oleuropein and/or α‐tocopheryl acetate on growth performance, serum lipid concentrations and lipid oxidation of Japanese quail meat during refrigerated storage. Performance and slaughtering parameters were not affected by dietary treatments. The diets supplemented with oleuropein at the levels of 150 or 200 mg/kg were more effective in delaying lipid oxidation in breast and thigh meats compared with the control diet. The dietary inclusion of neither vitamin E nor oleuropein at different levels did not significantly affect the fatty acid compositions of the breast meat compared with the control diet. The diets supplemented with oleuropein at the levels of 150 or 200 mg/kg had significantly the highest polyunsaturated fatty acid and omega‐3 fatty acid contents in thigh meat compared with the vitamin E diet. The ratio of omega‐6 fatty acids to omega‐3 FAs in thigh meat of quails fed diet supplemented with vitamin E at the level of 200 mg/kg was equivalent to those of quails fed the diets supplemented with oleuropein at the levels of 100 and 150 mg/kg . The results showed that the dietary oleuropein supplementation at 150 mg/kg level may be used in quail diets enriched with the polyunsaturated fatty acids of vitamin E as a natural antioxidant.     

Detection of the EGFP sequence in breast muscle of 3‐year‐old chicken after transfection using sonoporation

In our continuing effort to generate transgenic chickens, sonoporation was chosen to insert an exogenous gene into the chicken genome. An EGFP expression vector (pCAG‐EGFPac) and microbubbles were injected into the central disc of stage‐X blastoderm or the germinal crescent of stage‐4 embryos, followed by ultrasonic vibration. Nineteen chicks out of 108 treated embryos hatched, six females and six males out of these 19 chicks grew to sexual maturity and two females and three males lived for 3 years. Genomic DNA from 17 out of 35 gonads from embryos and chicks that died before sexual maturity was EGFP‐positive by PCR. No EGFP sequence was detected in the genomic DNA of 322 embryos from six sexually mature females and the semen from four sexually mature males by PCR. When genomic DNA was obtained from various tissues of five 3‐year‐old chickens, the EGFP sequence was amplified from the genomic DNA of the breast muscle of a female (No. 85). The above sequence was subjected to DNA sequencing and verified to be the EGFP sequence. These results showed that sonoporation is an effective tool for the transduction of exogenous genes into chicken embryos for the generation of transgenic chickens.     

Expression of chicken LEAP-2 in the reproductive organs and embryos and in response to <Emphasis Type="Italic">Salmonella enterica</Emphasis> infection

In recent years host antimicrobial peptides and proteins have been recognised as key mediators of the innate immune response in many vertebrate species, providing the first line of defense against potential pathogens. In chickens a number of cationic antimicrobial peptides have been recently identified. However, although these peptides have been studied extensively in the avian gastrointestinal tract, little is known about their function in the chicken reproductive organs and embryos. Chicken Liver Expressed Antimicrobial Peptide-2 (cLEAP-2) has been previously reported to function in protecting birds against microbial attack. The aim of this study was to investigate the expression of cLEAP-2 gene in the chicken reproductive organs, as well as in chicken embryos during embryonic development, and to determine whether cLEAP-2 expression in the chicken reproductive organs was constitutive or induced as a response to Salmonella enteritidis infection. RNA was extracted from ovary, oviduct, testis and epididymis of sexually mature healthy and Salmonella infected birds, as well as from chicken embryos until day ten of embryonic development. Expression analysis data revealed that cLEAP-2 was expressed in the chicken ovary, testis and epididymis as well as in embryos during early embryonic development. Quantitative real-time PCR analysis revealed that cLEAP-2 expression was constitutive in the chicken epididymis, but was significantly up regulated in the chicken gonads, following Salmonella infection. In addition, expression of cLEAP-2 during chicken embryogenesis appeared to be developmentally regulated. These data provide evidence to suggest a key role of cLEAP-2 in the protection of the chicken reproductive organs and the developing embryos from Salmonella colonization.     

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.     

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.     

Relationship between abdominal fat content and avian uncoupling protein gene expression in skeletal muscle of Japanese quail Coturnix japonica

1. The genetic architecture of the avian uncoupling protein (avUCP) was investigated and the relationship between avUCP gene expression and the amount of abdominal fat of Japanese quail was determined by quantitative real-time PCR.

2. The Japanese quail avUCP gene consists of six exons and five introns. Sequences of nucleotides and amino acids were 94·6% and 86·0% identical to those of the chicken avUCP gene, and phylogenetic analysis showed that the Japanese quail avUCP gene consists of the same clusters as the chicken and turkey avUCP.

3. Expression of the avUCP gene was significantly higher in the Pectoralis major (1·28?±?0·24) than in the Biceps femoris (0·63?±?0·14).

4. A positive correlation coefficient between the avUCP gene expression in the Pectoralis major and Biceps femoris was observed (r?=?0·79, P?=?0·02), whereas a negative correlation coefficient was observed between the abdominal fat percentage (AFP) and gene expression in both the Pectoralis major (r?=??0·82, P?=?0·01) and Biceps femoris (r?=??0·61, P?=?0·11).

5. The avUCP gene was associated with the accumulation of abdominal fat in Japanese quail and it was concluded that modulation of avUCP gene expression could be utilised to control abdominal fat accumulation in poultry.