- [Show abstract] [Hide abstract] ABSTRACT: Toll-like receptors (TLR) are trans-membrane sensors recognizing invading microbes. Toll-like receptors play a central role in initiating immune responses against several pathogens. In this study, we investigated the response of TLR and downstream genes to Marek's disease virus (MDV) infection. Forty 1-d-old chicks were randomly divided into 2 groups, with 20 chicks infected with MDV and 20 chicks mock-infected. Four chickens were euthanized respectively from infected and age-matched noninfected groups at 4, 7, 14, 21, and 28 d postinfection (dpi). Bursas, spleens, and thymuses were removed. The differential expression of TLR genes, including TLR3, TLR5, TLR7, TLR15, and TLR21, and downstream genes of TLR7, including MyD88, TRAF3, TRAF6, IFNA, IFNB, and IL6, in lymphoid tissues of MDV-infected and noninfected chickens was determined by real-time PCR. The results showed that the change of TLR genes was different in 3 lymphoid tissues. Expression of TLR7 and MyD88 was upregulated at 14 dpi and downregulated at 28 dpi in MDV-infected compared with noninfected spleens. The TRAF6 and IFNB were upregulated, and TRAF3, IFNA, and IL6 genes showed increasing trends in MDV-infected compared with noninfected spleens at 14 dpi. The expression of TLR3 and TLR15 genes was downregulated in MDV-infected compared with noninfected spleens at 28 dpi. The results indicated that TLR7 and its downstream genes were a response to MDV infection at 14 dpi. However, the function of TLR was impaired when the infection entered the tumor transformation phase. In bursas, TLR3 and TLR15 genes were upregulated at 7 and 4 dpi, respectively. It indicated that TLR3 and TLR15 might be involved in response to MDV infection in bursa at early phases. However, no differential expression of TLR genes was observed between MDV-infected and noninfected thymuses, which indicated that the thymus had little response to MDV infection mediated by TLR.
- [Show abstract] [Hide abstract] ABSTRACT: Damaged eggshells result in losses of eggs. Numerous efforts have been carried out to improve eggshell quality, which may lead to increased eggshell thickness. The conventional way of enhancing eggshell strength with thicker eggshell on average may be replaced by a new strategy to improve eggshell uniformity without increasing eggshell thickness. To achieve this, it is necessary to investigate global variation of eggshell thickness. In this study, we used 100 fresh eggs from 52-wk-old layers of a commercial brown-egg variety. To determine the global variation of eggshell thickness, 42 points for each egg along both longitudinal and latitudinal axes were selected to measure thickness using an eggshell thickness gauge. The eggshell thickness from blunt to sharp end varied significantly (P < 0.05). The area surrounding the blunt end was the thinnest (0.341 ± 0.025 mm), whereas the area surrounding the sharp end was the thickest (0.367 ± 0.023 mm). It was found that the thickness of the sharp end was the closest to the average thickness of the whole eggshell and could be considered as a valid measurement of eggshell thickness. A new parameter, eggshell thickness uniformity, was defined as the reciprocal of the coefficient of variation (1/CV) of eggshell thickness from 42 points of each egg and can be used to evaluate the eggshell quality. Eggshell thickness uniformity was positively correlated with breaking strength (r = 0.341; P < 0.01), suggesting that the parameter may be used as a potential selection criterion in breeding program to improve eggshell quality without increasing eggshell thickness.
- [Show abstract] [Hide abstract] ABSTRACT: Chicken is considered to be an excellent model for genetic studies of phenotypic and genomic evolution, with large effective population size, specialized commercial lines, and strong human-driven selection. High-density chicken SNP chips can help to achieve a better understanding of the selection mechanisms in artificially selected populations. We performed the genome-wide tests for the selection signature in 385 White Leghorn hens and mapped positively selected regions to the genome annotations. Ten QTL related to egg production, egg quality, growth, and disease resistance traits were selected for extended haplotype homozygosity tests to give a brief overview of recent selection signatures in chicken QTL. We also reported 185 candidate genes/CDSs showing top P-values and slower decay of haplotype homozygosities. Some of these genes seemed to have significant effects on important economical traits, and most of them have not been reported in chickens. The current study provides a genome-wide map of linkage disequilibrium extents and distributions and selection footprints in the chicken genome. A panel of genes, including PRL, NCKX1, NRF1, LHX2, and SFRP1 associated with egg production, metabolism traits, and response to illumination were identified. In addition, there were more genes identified that have not yet been reported in chickens, and our results provide new clues for further study.
- [Show abstract] [Hide abstract] ABSTRACT: Marek's disease (MD) is a neoplastic disease in chickens, caused by the Marek's disease virus (MDV). To investigate host genetic resistance to MD, we conducted a genome-wide association study (GWAS) on 67 MDV-infected chickens based on a case and control design, including 57 susceptible chickens in the case group and 10 resistant chickens as controls. After searching 38 655 valid genomic markers, two SNPs were found to be associated with host resistance to MD. One SNP, rs14527240, reaching chromosome-wide significance level (P < 0.01) was located in the SPARC-related modular calcium-binding 1 (SMOC1) gene on GGA5. The other one, GGaluGA156129, reaching genome-wide significance (P < 0.05), was located in the protein tyrosine phosphatase, non-receptor type 3 (PTPN3) gene on GGA2. In addition, expression patterns of these two genes in spleens were detected by qPCR. The expression of SMOC1 was significantly up-regulated (P < 0.05), whereas the expression of PTNP3 did not show significance when the case group was compared with the control group. Up-regulation of SMOC1 in susceptible spleens suggests its important roles in MD tumorigenesis. This is the first study to investigate MD-resistant loci, and it demonstrates the power of GWASs for mapping genes associated with MD resistance.
- [Show abstract] [Hide abstract] ABSTRACT: The Wnt signaling pathway plays a crucial role during embryogenesis in vertebrates. In this study, 124 SNP in 31 Wnt signaling pathway genes were selected to genotype 764 individuals in an F2 resource population by reciprocally crossing Silkie fowls and Cornish broilers, and 102 SNP were polymorphic. Pairwise linkage disequilibrium among the SNP within each gene was calculated. Haplotypes were reconstructed from the SNP in strong linkage disequilibrium. The associations of SNP and haplotypes with carcass traits were analyzed respectively, and the SNP contributions to phenotypic variance were estimated. The present study showed that 58 SNP in 24 genes and 8 haplotype blocks within 7 genes were significantly (P < 0.05) associated with at least one carcass trait. Fourteen SNP (among the 58 SNP) explained >2% phenotypic variance, 12 of which had significantly (P < 0.01) additive or dominant effects. Furthermore, both rs15865526 (Wnt9A) and rs14066777 (MAPK9) as well as their corresponding haplotype blocks were significantly associated with shank circumference and wing weight, respectively. In addition, 5 muscle-weight-related SNP explained >7% phenotypic variance, which was much higher than those of others. It was found that the Wnt signaling pathway was strongly associated with chicken carcass traits, and 7 genes were particularly important, namely RHOA and CHP for breast muscle weight, Wnt3A for breast muscle weight percentage over carcass weight, RAC1 for thigh weight percentage and thigh muscle weight percentage over carcass weight, Wnt11 for thigh weight percentage over carcass weight, Wnt9A for shank length, and MAPK9 for shank circumference. It is evident that Wnt signaling plays a major role in regulating carcass characteristics important for production traits in chickens.
- [Show abstract] [Hide abstract] ABSTRACT: Some members of the low-density lipoprotein receptor (LDLR) family play important roles in the regulation of lipoprotein metabolism and egg quality traits. Low-density lipoprotein receptor-related protein 2 (LRP2) gene belongs to the LDLR super family, and widely expresses in many tissues. This work identified and genotyped 1 single-nucleotide polymorphism (SNP), T14347C, at 3'-UTR of the LRP2 using matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS), and analyzed the effects of the SNP (T14347C) on egg-quality traits in 544 dwarf hens from 44 sire families. Frequencies of this SNP in the studied population did not agree with the Hardy-Weinberg equilibrium (P < 0.0001). Egg weight, albumen weight, albumen height, and albumen ratio of the TT genotype were significantly higher than those of the CC genotype (P < 0.05), whereas eggshell ratio of the TT genotype was significantly lower than that of the CC genotype (P < 0.05). The relative expression level of the LRP2 gene in the magnum was determined by real-time quantitative PCR. The gene expression of genotype CC individuals was significantly higher than that of TT and CT birds (P < 0.05). By combining both genetic effects and expression analyses results, we propose that the LRP2 gene is a good candidate gene, exhibiting a key role in albumen formation processes.
- [Show abstract] [Hide abstract] ABSTRACT: Wang, X.T., Zhao, C.J., Li, J.Y., Xu, G.Y., Lian, L.S., Wu, C.X. and Deng, X.M. 2010. Heme oxygenase-1 is important to the formation of eggshell biliverdin in chicken. J. Appl. Anim. Res., 38: 229–232.We postulated that the pathway of the biliverdin in eggshell was similar to that of the biliverdin in bile and attempted to search some proofs. First, it was found that the mean mRNA expression level of heme oxygenase-1 (HO-1) in the shell glands of the Dongxiang blue-shelled hens (n-6) was about 45.8 times more than that of the Dwarf brown-shelled hens (n-6) with the method of real time quantitative PCR. Second, the solution of hemin (40μ mol/ml), which was the inducer of HO-1 expression, was intraperitoneally injected into the Dongxiang blue-shelled hens (n=6), and the saline water (0.68%) was intraperitoneally injected into the control group (n=6), at 1 ml/kg, once a day, for two consecutive days. After injection with hemin solution, one hen in the treatment group laid a very blue egg, and the content of biliverdin in this shell increased to about 3.1 times as compared with before injection of hemin solution. At the same time, the expression level of HO-1 mRNA in the shell gland of this treated hen laid a very blue egg was about 3.0 times the control hens. These results supported our postulation.
- [Show abstract] [Hide abstract] ABSTRACT: Major histocompatibility complex has previously been shown to influence the resistance of chicken to Marek's disease virus (MDV). However, little is known about expression of other genes in the MHC-I and II pathway after MDV infection. This study aimed at investigating 8 immune-related genes in the MHC core region that affects host responses to MDV. Spleens of infected and age-matched uninfected chickens were removed at 4, 7, 14, 21, and 28 d postinfection for gene expression detection using real-time PCR. Different expression patterns of MHC-I and II pathway genes were observed in the spleen. In the MHC-I pathway, the expression of transporter of antigen protein 1 (TAP1), transporter of antigen protein 2 (TAP2), and transporter of antigen protein-binding protein (TAPBP) genes was significantly increased in the spleen of MDV-infected than that of uninfected chickens. It indicated that host antivirus responses were generated to enhance antigen presentation. However, MHC-II pathway genes showed contrary trends. Classical MHC-II β chain major gene (BLB2) and nonclassical class II genes [DM α chain gene (DMA), DM β chain gene-1 (DMB1), and DM β chain gene-2 (DMB2)] had consistent lower transcripts in spleens of MDV-infected than that of uninfected chickens, which reflected that MDV interfered with multiple components of the MHC-II pathway. Overall, expression of most genes in the MHC core region was altered; moreover, the genes in endogenous and exogenous antigen presentation pathways had different expression patterns in the spleen after MDV infection.
- [Show abstract] [Hide abstract] ABSTRACT: The objective of the present study was to evaluate the role of myofiber characteristics and the thickness of 2 major muscle membranes, perimysium and endomysium, in determining the breast meat tenderness of chickens. Birds from 2 breeds (White Leghorn and a line of broiler) were chosen. Chicks were sexed and wing-banded at hatch and were grown in separate cages in a single house. Sixty broilers and 60 White Leghorns were harvested at 6 wk of age, respectively, whereas another 60 White Leghorns were slaughtered at 18 wk of age. An equal number of males and females was maintained for each group. Body weight, breast muscle weight, pH, drip loss, cooking loss, Warner-Bratzler shear force value (SFV), total energy of shear force, fiber diameter, sarcomere length, myofiber density, and the thickness of endomysium and perimysium of the breast were determined for each bird. At 6 wk of age, histological examination indicated that the size of myofiber and thickness of endomysium and perimysium of broilers were larger than that those of White Leghorns (P < 0.01), whereas the SFV, drip loss, and cooking loss of broilers were smaller (P < 0.01). A comparison between the White Leghorns at 18 wk and the broilers at 6 wk, which were at similar BW but different ages, showed that the breast muscle weight of broilers was larger (P < 0.01) than that of White Leghorns. For breast muscle, the endomysium of broilers at 6 wk was thicker than that of White Leghorns at 18 wk (P < 0.01), whereas the perimysium was thinner (P < 0.01). The SFV, drip loss, and the cooking loss of broilers were smaller than those of White Leghorns at similar BW (P < 0.01). Meat tenderness was negatively correlated with myofiber density (-0.27) and the thickness of endomysium (-0.29) and positively correlated with the thickness of perimysium (0.20). It is suggested that muscle membrane should be considered in evaluating meat tenderness of the chicken.
- [Show abstract] [Hide abstract] ABSTRACT: The tyrosinase (TYR) and melanocortin 1 receptor (MC1R) genes have been accepted as major genes involved in the plumage pigmentation of chickens. The co-segregation of plumage coloration and sequence polymorphism in TYR and MC1R genes were investigated using an intercross between black and white plumage color types of the Dongxiang blue-shelled chicken. Profiles of plumage color changing and genes expression levels of TYR and MC1R were observed from hatch to 112 d of age using quantitative real-time reverse transcription-PCR. Intercrossed offspring were classified by phenotypes of plumage colors. The phenotypes of black and amber chicks with genotypes of E_C_ exhibited a black feather pattern, whereas white, gray, and buff chicks with genotypes of E_cc and eecc belonged to the white feather pattern. Although TYR in cooperation with MC1R determined the coloration feather patterns, the different phenotypes did not correspond completely with the genotypes. During the period studied, plumage phenotype changed dramatically, and the buff and gray down were gradually replaced by whiteness feathers. Real-time reverse transcription-PCR studies showed that 1) expression levels of TYR declined dramatically with age, and expression at hatch was highest (P < 0.01) during the ages studied; 2) expression level of MC1R was higher at 28 d than at younger and older ages; and 3) expression of TYR in chickens carrying E/E and E/e alleles on MC1R loci were higher than those carrying e/e alleles from hatch to 28 d.
- [Show abstract] [Hide abstract] ABSTRACT: Low-density lipoprotein receptor-related protein 8 (LRP8), a member of the low-density lipoprotein receptor gene family with a role in clusterin processing, was investigated as a candidate gene for egg quality-related traits. One SNP from C to T at position 1623 of the open reading frame of LRP8 was identified and genotyped by a high-throughput genotyping method, matrix-assisted laser desorption-ionization time-of-flight mass spectrometry in 747 egg-type dwarf layers from 44 sire families. There were no significant differences among genotypes for any interior egg traits measured, except for yolk color, in which color was deeper for the TT genotype than CC or CT (P < 0.05). For shell traits, strength and thickness were greater for TT than CC (P < 0.05), with CT intermediate and not different from either. Shape index was lower for CT than either TT or CC, which did not differ, whereas for shell color, CT was intermediate to the homozygotes, which differed (CC > TT). The present results indicated that LRP8, as a new member of eggshell matrix protein, may be a candidate gene associated with eggshell traits.
- [Show abstract] [Hide abstract] ABSTRACT: Based on the knowledge of the heme bio-synthetic and metabolic pathway and the structures of biliverdin and protoporphyrin, experiments were carried out to compare the difference between the total quality of eggshell pigments in blue-shelled eggs and brown-shelled eggs from the same population (Dongxiang, China) and to analyze the correlation between the quantity of protoporphyrin and biliverdin in the 2 kinds of eggshells. It was found that there was no significant difference between the total quantity of eggshell pigments in Dongxiang blue-shelled eggs and Dongxiang brown-shelled eggs (P = 0.9006), and a highly significant positive correlation between the quantity of protoporphyrin and biliverdin in blue eggshells (P < 0.01) and a significant positive correlation between the quantity of protoporphyrin and biliverdin in brown eggshells (P < 0.05). These results suggested that eggshell protoporphyrin and eggshell biliverdin probably derived from common precursor material.
- [Show abstract] [Hide abstract] ABSTRACT: Blue-shelled eggs are gaining popularity as the consumption demand diversifies in some countries. This study was carried out to investigate the laying performance and egg quality of the blue-shelled egg layers as well as the effects of different housing systems on egg production and quality traits. One thousand pullets from Dongxiang blue-shelled layers were divided into 2 even groups and kept in different housing systems (outdoor vs. cage). Daily laying performance was recorded from 20 to 60 wk of age. External and internal egg quality traits were examined at 26, 34, 42, and 50 wk. Yolk cholesterol concentration and whole egg cholesterol content were measured at 40 wk of age. Average laying rate from 20 to 60 wk for the cage (54.7%) was significantly higher than that of outdoor layers (39.3%). Among all of the egg quality traits, only eggshell color was affected by housing system. Interaction between housing system and layer age was found in egg weight, eggshell color, eggshell ratio, yolk color, and yolk weight. Meanwhile, cholesterol concentration in yolk was 8.64 +/- 0.40 mg/g in the outdoor eggs, which was significantly lower than that of eggs from the cage birds (10.32 +/- 0.48 mg/g; P < 0.05). Whole egg cholesterol content in the outdoor eggs (125.23 +/- 6.32 mg/egg) was also significantly lower than that of eggs from the caged layers (158.01 +/- 8.62 mg/egg). The results demonstrated that blue-shelled layers have lower productivity in the outdoor system than in the cage system. Blue-shelled layers have lower egg weight, larger yolk proportion, and lower cholesterol content compared with commercial layers. In a proper marketing system, lower productivity could be balanced by a higher price for the better quality of blue-shelled eggs.
- [Show abstract] [Hide abstract] ABSTRACT: The chicken Mx gene has been regarded as a candidate gene for resistance to avian influenza virus (AIV). In this study, three groups of chickens with homozygotes (AA, GG) and heterozygotes (AG) of the resistant (A) and susceptible alleles (G) to AIV of the Mx gene were constructed from a line of dwarf egg-type chickens. These chickens were not examined for their resistant activities to AIV because the differential resistance had only been detected in vitro. The birds of the three groups were vaccinated with inactivated H5N2 AIV vaccine and the level of hemagglutination inhibition (HI) antibody to AIV was detected. The association between disease resistant activity to AIV and antibody response to AIV vaccination in the three groups was analyzed. The chickens with homozygous resistant allele A showed the lowest antibody levels, whereas the heterozygous chickens (AG) presented the highest antibody level after the boosting vaccination, which indicates that the efficiency of artificial selection on the resistant allele of Mx gene will be compromised since the homozygotes of the allele presented the weakest antibody response to the corresponding vaccine.
- [Show abstract] [Hide abstract] ABSTRACT: Mammal microsomal glutathione transferase 1 (MGST1) can conjugate many toxic or carcinogenic substances and depress oxidative stress. In this study, Chicken MGST1 and its variants were cloned for the first time and were composed of 956 or 944 nucleotides. The 12 nt deletion in the exon 2 did not alter the GT-AG rule and the ORFs for the two MGST1 variants were the same, which both comprised 465 nucletides and encoded a peptide with 155 amino acids. It was found that the two different splice variants identified using RT-PCR expressed in all three organs investigated of Dwarf Brown Chicken, namely liver, spleen and shell gland. Moreover, the expression level of MGST1 mRNA in the liver of Dwarf Brown chickens was the highest (p
- [Show abstract] [Hide abstract] ABSTRACT: The Mx protein, which confers resistance to orthomyxovirus, has been detected in several organisms, and one nonsynonymous substitution (S631N) of the chicken Mx protein has been shown to affect resistant activities to the avian influenza virus in vitro. In the current study, the genomic sequence and polymorphism of the chicken Mx gene are reported. The full length of the chicken Mx gene spans about 21 kb, with 13 exons on chromosome 1 of the chicken genome. A total of 237 single nucleotide polymorphisms were found in the chicken Mx gene by comparison among 4 directly sequenced Mx genomic DNA sequences, and the reference sequence was inferred from the chicken genome project. The genomic diversity of the chicken Mx gene showed large variation in different regions, with the highest diversity in the 5′ untranslated region and the lowest in the 3′ untranslated region. The genomic structure and variation of sequences gathered here will allow an extensive analysis of the gene function with the aim of improving the antiviral resistance activities of chickens.
- [Show abstract] [Hide abstract] ABSTRACT: Eggshell colour is an important economic trait for egg-type chickens. Three hundred and thirty eggs of 11 different egg-type chicken varieties producing white, tinted, blue and brown eggs were used in the present study. Eight points on each egg were measured using Minolta Chromameter CR-300. The values of lightness (L*), redness (a*), yellowness (b*) and chroma (C*) of eggshells were obtained as objective and quantitative measurements of eggshell colour. The results showed that eggshell colour in different egg-type chicken varieties, different eggs within varieties and different points on the same egg varied. The variation ranges of L* values were 92.9∼97.9, 58.1∼90.2, 68.7∼89.9 and 48.1∼74.4, and the means of L* values were 95.2, 82.3, 84.0 and 63.0 for white, tinted, blue and brown eggs, respectively. The variation range of a* values of white, tinted, blue and brown eggs were -0.50∼0.70, 2.00∼14.9, -10.10∼9.70 and 10.2∼32.5 respectively. The values of L*, a*, b* and C* among different eggshell colour types were significantly different (P<0.05). High and significant correlations among the eight measurements on each egg were found (R>0.98). Result of principal component analysis (PCA) indicated that one single point at the blunt end of egg made the greatest contribution to the overall pigmentation of eggs and could be employed as a simplified measurement, with the accuracy as high as 0.993. At this point, the range of a* value varied from -0.50 to 0.40, 2.20 to 18.7, -8.00 to -0.50 and 10.2 to 24.5 for white, tinted, blue and brown eggs, respectively, therefore the a* value at this single point could be used to classify the three types of egg-shell colour (white, blue, tinted/brown). However, the tinted and brown eggs cannot be distinguished accurately with a* value.
- [Show abstract] [Hide abstract] ABSTRACT: Thyroid hormone responsive Spot 14 (THRSP) is suggested as a transcription factor involved in the regulation of adipogenic enzymes by 3 thyroid response elements in the promoter region. In the chicken genome, THRSP gene was identified to duplicate into 2 paralogs, THRSPalpha and THRSPbeta. In the current study, cDNA sequences of the duplicated duck THRSP genes were cloned by real-time PCR and rapid amplification of cDNA ends. Duck THRSPalpha and THRSPbeta were predicted to encode peptides with 133 amino acids, which had 74 and 68% sequence identity at cDNA level, 78 and 74% identity at amino acid level to the chicken counterparts, respectively. A high percentage (73.1%) of G and C nucleotides were found in the 3' untranslated region of duck THRSPbeta cDNA. Although a low similarity of peptide composition was shared between ducks and mammals, and a moderate similarity was shared between ducks and chickens, many predicted properties of THRSP, including the pI, subcellular localization and functional domains seemed to be highly conserved. The present study demonstrated that the duck THRSP gene duplicates into the 2 paralogs as in chickens. Phylogenetic analysis indicated that the duplication for THRSP paralogs appeared to have taken place preceding the chicken-duck split, and the diverging rate between THRSP paralogs seemed faster in the chicken genome than that in the duck genome. Expression analysis by real-time quantitative PCR showed that THRSP paralogs in ducks were more actively transcribed in fat tissues (i.e., s.c. fat and abdominal fat) than in liver, and the mRNA concentrations of THRSPbeta were higher than that of THRSPalpha in liver and s.c. fat.
- [Show abstract] [Hide abstract] ABSTRACT: Biliverdin is an important pigment in the eggshell of chickens and other avian species. Determination of the biosynthesis site for biliverdin is essential for understanding the biochemical process and genetic basis of eggshell pigmentation. Either blood or the shell gland could be the biosynthesis site of eggshell biliverdin. A segregation population with full-sib sisters genotyped Oo and oo, which laid blue-shelled eggs and light brown eggs, respectively, was constructed in a native Chinese chicken breed. Ultraviolet spectrophotometry and HPLC were used to determine the biliverdin concentration in eggshells, blood, bile, excreta, and shell gland of both groups of chickens. Biliverdin content was significantly different between egg shells of blue-shelled and brown-shelled chickens (P < 0.01). Blood and bile were tested 3 to 4 h before oviposition, and excreta was tested randomly. Results showed no significant difference in biliverdin concentration in blood, bile, and excreta between the 2 groups. In the shell gland, the biliverdin contents for the blue-shelled and brown-shelled chickens were 8.25 +/- 2.55 and 1.29 +/- 0.12 nmol/g, respectively, which showed a significant difference (P < 0.01). Our results demonstrated that blood is not the biosynthesis site of the shell biliverdin. Biliverdin is most likely synthesized in the shell gland and then deposited onto the eggshell of chickens.
China Agriculture University-EastPeping, Beijing, China