Investigation on eggshell apex abnormality (EAA) syndrome in France: isolation of Mycoplasma synoviae is frequently associated with Mycoplasma pullorum.

BACKGROUND: Mycoplasma synoviae (MS) is known to cause Eggshell Apex Abnormality (EAA) syndrome characterized by an altered shell surface with increased translucency on the apex. However, no large-scale studies have been conducted to obtain prevalence data of EAA and MS isolates associated to this syndrome. This manuscript reports the results of two field studies performed in the French poultry industry (2015-2017): focusing mainly on investigation of presence and prevalence of EAA in different types of laying hen flocks (phase 1), and isolation of MS strains from EAA-infected flocks (phase 2). RESULTS: The first survey included 77 farms of commercial layers in three French egg-production regions, hosting 40 flocks in alternative systems (ALT) and 56 in furnished cages (FC). Seven flocks (4 FC and 3 ALT) presented EAA clinical signs, giving a prevalence of 7.3% in this studied sample. A second independent field study was conducted to identify MS by in vitro cultivation and PCR in samples from 28 flocks with clinical signs of EAA. Different types of biological specimens were collected in EAA-affected flocks and submitted to the laboratory. M. synoviae was detected in 25/28 flocks, from both production systems (5/5 ALT and 20/23 FC). Detection of MS was significantly higher in tracheal swabs (59%) than in cloacal (10.5%), albumen (3.6%) and egg yolk (1.1%) swabs. It is worth to mention that attempts to clone MS from positive samples were often hampered by the presence of another Mycoplasma species, which showed fast growing behaviour in the selective media used in this study (Frey Medium 4 and Frey Medium 4 supplemented with erythromycin). The use of MALDI-TOF mass spectrometry in combination with next-generation sequencing (NGS) results allowed the identification of this fast growing mycoplasma as Mycoplasma pullorum, which was detected in 14 of the 25 (56%) MS-positive flocks. CONCLUSIONS: These results confirmed the presence of the EAA syndrome in MS-positive flocks of layers in France, reared in different regions and in different production systems (ALT and FC). Studies need to be conducted to test whether M. pullorum may influence the expression of clinical signs of EAA in MS-infected layer farms.

Network prediction of the potential molecular mechanisms in birds of prey exposed to organochlorine pesticides / Red de predicción de potenciales mecanismos moleculares en aves de presa expuestas a pesticidas organoclorados

Abstract In the 1960s, organochlorine compounds were responsible for the decline of birds of prey populations such as Haliaee- tus leucocephalus and Falco peregrinus. Pesticides similar to DDT cause bioaccumulation in birds, affecting their eggshell com- position and compromising their development. Using system biology tools, the goal of this study was to better comprehend how organochlorines act on birds. We performed a literature review, using the STITCH 5.0 platform, searching for the terms DDT and TCDD. The sub-networks were amplified in 100 interactions in STRING 10.5 and joined by the Cytoscape 3.4.0 Merge software, using the experimental animal model Gallus gallus. Clusterization, gene ontology, and centrality were the parameters evaluated in the resulting network. The resulting network had 1,417 interactions and 137 nodes. The clusterization indicated four clusters and the gene ontology pointed to biological processes related to cell signaling and morphological development. The centrality analysis indicated ESR1 and HSP90AB1 as hub/bottleneck proteins involved in the estrogen pathway and calcium transport. Therefore, it is possible that HSP90 proteins have increased expression in birds contaminated with organochlorine pesticides, favoring ESRI-organochlorines interaction and disturbing the calcium availability related to the eggshell formation. The presence or absence of heat shock proteins, such as HSP90, influences several aspects of reproduction in many species. Therefore, the relationship between the HSP90 protein expression and thin-shell syndrome was identified for the first time in this in silico study.

Resumen En los años 60, los organoclorados fueron responsables del declive de aves de rapiña como Haliaeetus leucocephalus y Falco peregrinus. Pesticidas como el DDT, causan biomagnificación en las aves, afectando las cáscaras de los huevos y dañando su desarrollo. El objetivo de este trabajo fue, a través de herramientas de biología de sistemas, comprender cómo los organoclorados actúan en el organismo de las aves. A través de una revisión bibliográfica se incluyeron dos compuestos, DDT y TCDD. Estos fue ron prospectos en la plataforma STITCH 5.0. Las subredes encontradas fueron aumentadas en 100 interacciones en la plataforma STRING 10.5 y unidas por la herramienta Merge del programa Cytoscape 3.4.0, usando el modelo experimental Gallus gallus. En la red resultante se analizaron la clusterización, la ontología génica y la centralidad. La red resultante presentó 137 nudos y 1.417 interacciones. El análisis de clusterización indicó 4 clusters, siendo que el análisis y ontología génica apuntó procesos biológicos ligados a la señalización y al desarrollo morfológico. El estudio de centralidad apuntó a ESR1 y HSP90AB1 como los hubs-bottle- neck proteínas que estaban involucradas en la vía de recepción de estrógeno y en el transporte de calcio. De acuerdo con los resultados podemos inferir que las proteínas HSP90 tienen su expresión aumentada, en aves contaminadas con pesticidas organoclo rados, favoreciendo la interacción entre ESRI y DDT / TCDD. Con ello, la interacción ESRI y la hormona estrógeno se compromete perjudicando el transporte de calcio y consecuentemente la formación de la cáscara del huevo en aves expuestas. La expresión de proteínas de choque térmico ha sido asociada a varios aspectos de la reproducción en muchas especies, sin embargo, una asociación entre HSP90 y el síndrome de la cáscara fina del huevo fue identificada por primera vez en este experimento in silico.

Eggshell apex abnormalities caused by two different Mycoplasma synoviae genotypes and evaluation of eggshell anomalies by full-field optical coherence tomography.

BACKGROUND: Mycoplasma synoviae (MS) is an important poultry pathogen worldwide. This bacterium may cause eggshell changes including an altered shell surface, thinning, and increased translucency in different areas, which leads to a greater incidence of eggshell cracks and breaks. In the present study the association between experimental infection of birds with two field strains of MS from different genotypes and the production of abnormal eggs is described. The analysis of those eggshells using a full-field optical coherence tomography (FF OCT) scanner is also reported. RESULTS: Eggshell samples were obtained from three experimental groups of chickens: one control and two infected tracheally with field strains of MS which produced abnormal eggs. In both experimental groups infected with MS a reduction of mean daily egg production by 11% was observed compared to the control group, which started at 21 to 42 dpi. Eggshell apex abnormalities increased to 24.5% of eggs and in some cases, soft-shelled eggs were produced. This study provides the first analysis of shells from anomalous eggs carried out using FF OCT, which allows three-dimensional structural imaging of an investigated sample at micrometre scale. FF OCT showed ultrastructural changes in eggshells and a smaller number of pores on the entire surface of the affected shells. CONCLUSIONS: The eggshell pathology and the concomitant egg production losses that result from infections highlight the economic significance of MS in commercial poultry. There are differences in the strains of MS which may induce eggshell apex abnormalities (EAA) and egg production losses. The use of FF OCT, which is a noninvasive measurement method based on analysis of the light backscattered from the measured object, will confer the ability to control the quality of eggshells in flocks infected with MS.

Two strains of Mycoplasma synoviae from chicken flocks on the same layer farm differ in their ability to produce eggshell apex abnormality.

Mycoplasma synoviae (Ms) is considered to be an economically important poultry pathogen. Although the full economic costs of infection in layer chickens are still under debate, the prevalence of Ms is known to be high in some countries and earlier reports have shown a correlation between infection and Eggshell Apex Abnormality (EAA). This work is a continuation of an earlier study of a clinical case of EAA on a layer hen farm where the presence of two different strains of Ms, based on the sequence of the 5' end of the vlhA gene, was demonstrated. Both strains could be detected in the trachea but only one (designated strain PASC8) appeared able to colonize the oviduct, while the other (designated TRACH) was not found in the oviduct and has not been related to EAA. The PASC8 partial vlhA gene sequence differs from that of the TRACH in having a 39 nucleotide deletion in the proline rich region and three point mutations in the RIII region. Based on this information an experimental infection was performed in SPF chickens using groups infected with either the PASC8 or the TRACH strain and a non-infected control group. Both Ms strains were detected in the trachea of infected birds, but only the PASC8 strain was found in the oviduct. Furthermore, EAA developed only in the group infected with PASC8 strain. Compared to the control group, both strains produced an adverse impact on egg production: a decrease in the numbers laid and in their average weight (P<0.05) This work demonstrates a difference in oviduct tropism between two Ms strains and a possible relationship to the production of EAA in experimental conditions.

Associação de carboidrases e fitase em dietas valorizadas e seus efeitos sobre desempenho e qualidade dos ovos de poedeiras leves / Association of carbohydrases and phytase in enriched diets ranked and its effects on performance and quality of eggs from light laying hens

Foram utilizadas 350 aves da linhagem comercial Bovans White, com o objetivo de avaliar a associação de carboidrases e fitase em dietas valorizadas sobre o desempenho e qualidade dos ovos de poedeiras leves. O delineamento experimental foi inteiramente ao acaso, com 5 tratamentos e 7 repetições. Os tratamentos foram: T1- Controle positivo sem adição de enzimas e sem valorização dos nutrientes; T2- Controle negativo (CN) 1, valorizando em 1,5% e 6% a EMA (kcal/kg), respectivamente para o milho e o farelo de soja, e em 2% a proteína bruta (PB) e os aminoácidos digestíveis limitantes mais a matriz completa para a enzima fitase; T3- CN 2, valorizando em 1,5% e 6% a EMA (kcal/kg), respectivamente para o milho e o farelo de soja, e em 2% a proteína bruta (PB) e os aminoácidos digestíveis limitantes mais a matriz incompleta para a enzima fitase; T4- suplementado com 100g ton-1 de carboidrase e 30g ton-1 de fitase; T5- suplementado com 100g ton-1 de carboidrase e 30g ton-1 de fitase. Os tratamentos controle positivo, T2, T3, T4 e T5, com ou sem suplementação enzimática, garantiram o desempenho no que diz respeito ao consumo de ração, produção, peso e perdas de ovos e qualidade da casca.

A total of 350 commercial Bovans White laying hens were used to evaluate the association of carbohydrases and phytase in enriched diets and its effects on performance and egg quality of laying hens. The experiment used a randomized design with five treatments and seven replicates. The treatments were: 1. Positive control without added enzymes and without nutrient enrichment, 2. Negative control (NC) 1 with 1.5% and 6% AME (kcal/kg) enrichment for corn and soybean meal respectively, 2% crude protein (CP) enrichment, and digestible limiting digestible amino acids plus the full matrix for the phytase enzyme; 3. NC 2 with 1.5% and 6% AME (kcal/kg) enrichment, respectively, for corn and soybean meal and 2% crude protein (CP) enrichment, and digestible limiting amino acids plus the sparse matrix for the phytase enzyme, 4. NC 1 supplemented with 100 g ton-1 carbohydrase and 30g ton-1 phytase, 5. NC 2 supplemented with 100 g ton-1 carbohydrase and 30g ton-1 phytase. According to the results, the positive control treatments, NC1 and NC2, with or without enzyme supplementation, showed guaranteed performance for feed intake, egg yield, weight, egg loss and shell quality.

Chlorinated, brominated and fluorinated organic pollutants in African Penguin eggs: 30 years since the previous assessment.

The African Penguin population has drastically declined over the last 100 years. Changes in food availability due to over-fishing and other oceanographic changes seem to be major causes. However, it has also been 30 years since organic pollutants as a potential factor have been assessed. We analysed penguin eggs collected in 2011 and 2012 from two breeding colonies 640 km apart: Robben Island near Cape Town on the Atlantic Ocean coast, and Bird Island near Port Elizabeth on the Indian Ocean coast of South Africa. We quantified organochlorine pesticides, brominated flame retardants, and perfluorinated compounds (PFCs). Compared to 30 years ago, concentrations of ΣDDT have remained about the same or slightly lower, while ΣPCBs declined almost four-fold. The use of DDT in malaria control is unlikely to have contributed. PFCs were detected in all eggs. Indications (non-significant) of eggshell thinning associated with ΣDDT and ΣPCB was found. It seems therefore that the concentrations of measured organic pollutants the African Penguin eggs are not contributing directly to its current demise, but concerns remain about thinner shells and desiccation. Effects of combinations of compounds and newer compounds cannot be excluded, as well as more subtle effects on reproduction, development, and behaviour.

Genetic parameters of egg defects and egg quality in layer chickens.

Genetic parameters were estimated for egg defects, egg production, and egg quality traits. Eggs from 11,738 purebred brown-egg laying hens were classified as salable or as having one of the following defects: bloody, broken, calcium deposit, dirty, double yolk, misshapen, pee-wee, shell-less, and soft shelled. Egg quality included albumen height, egg weight, yolk weight, and puncture score. Body weight, age at sexual maturity, and egg production were also recorded. Heritability estimates of liability to defects using a threshold animal model were less than 0.1 for bloody and dirty; between 0.1 and 0.2 for pee-wee, broken, misshapen, soft shelled, and shell-less; and above 0.2 for calcium deposit and double yolk. Quality and production traits were more heritable, with estimates ranging from 0.29 (puncture score) to 0.74 (egg weight). High-producing hens had a lower frequency of egg defects. High egg weight and BW were associated with an increased frequency of double yolks, and to a lesser extent, with more shell quality defects. Estimates of genetic correlations among defect traits that were related to shell quality were positive and moderate to strong (0.24-0.73), suggesting that these could be grouped into one category or selection could be based on the trait with the highest heritability or that is easiest to measure. Selection against defective eggs would be more efficient by including egg defect traits in the selection criterion, along with egg production rate of salable eggs and egg quality traits.

Induction of eggshell apex abnormalities in broiler breeder hens.

Recently, the causal relationship between eggshell apex abnormalities (EAA) and Mycoplasma synoviae was described. This eggshell pathology has only been documented in table egg layers both spontaneously and experimentally infected with M. synoviae, suggesting that meat-type layers are less prone to this condition. In this study the susceptibility of specified pathogen free (SPF) broiler breeder hens to produce eggs with EAA after M. synoviae infection was assessed. Five groups of 12 hens each were made: a negative control group, a group inoculated intratracheally (i.t.) with a M. synoviae EAA strain at 19 weeks of age, a group inoculated i.t. with this strain at 19 and 26 weeks of age, a group inoculated with M. synoviae i.t. at 19 weeks of age and infected 5 days earlier with infectious bronchitis virus D1466 (IBV), and a fifth group similar to the former but inoculated i.t. twice with an M. synoviae EAA strain at 19 and 26 weeks of age. Eggs with EAA were only produced after a single i.t. inoculation with the M. synoviae EAA strain if preceded by an infection with IBV. The production of eggs with EAA started 6 weeks after M. synoviae EAA inoculation and the proportion of eggs with EAA during the experiment was 9/449 (2%), which was much lower than that in SPF layer hens (14-22%). The present results suggest that broiler breeder hens are less susceptible to producing eggs with EAA after an infection with a M. synoviae EAA strain preceded by an IBV infection, compared with table egg layers. Similar to SPF egg layers, the mean daily egg production per hen was significantly reduced by the M. synoviae EAA strain and there was a general negative effect on eggshell strength by this strain, suggesting it could also have a detrimental effect on hatching egg quality.

Effect of a live Mycoplasma synoviae vaccine on the production of eggshell apex abnormalities induced by a M. synoviae infection preceded by an infection with infectious bronchitis virus D1466.

An experimental study was conducted to assess the effect of a live Mycoplasma synoviae vaccine (Vaxsafe MS; Bioproperties Pty Ltd, Ringwood, Victoria, Australia) on M. synoviae-induced eggshell apex abnormalities (EAA). Four experimental groups of specified-pathogen-free white laying hens were made. All groups were inoculated with infectious bronchitis virus D1466 at 18 weeks of age. One group did not receive further treatment (non-vaccinated non-challenged (NVNC)). Two groups were vaccinated at 14 weeks of age against M. synoviae, and one of these groups was also challenged with an EAA-inducing M. synoviae strain 5 days after infectious bronchitis virus challenge (vaccinated non-challenged (VNC) and vaccinated challenged group (VC), respectively). The fourth group was not vaccinated but was challenged with M. synoviae (non-vaccinated challenged (NVC)). Eggs with EAA eggs were produced only in the NVC and VC groups. However, the proportion of eggs with EAA and the mean daily production of eggs with EAA per chicken was significantly lower (P<0.05) in the VC group (88/741 (11.9%) and 0.09+/-0.01 eggs per hen) compared with the NVC group (148/646 (22.9%) and 0.14+/-0.01 eggs per hen). The mean daily egg production per chicken was significantly lower in the NVC group (0.48+/-0.03 eggs) compared with that of the NVNC group (0.60+/-0.03 eggs), but not significantly different from other groups. The eggshell strength of eggs with EAA (22.8 N) was significantly lower (P<0.05) than non-affected eggs from the other groups (33.7 to 39.5 N). Furthermore, the eggshell strength of non-affected eggs in the NVC group was significantly lower (P<0.05) compared with that of non-affected eggs from the flock of origin (33.7 versus 41.2 N), but not different from the other groups. It can be concluded from the present study that vaccination with a live M. synoviae vaccine reduces the occurrence of M. synoviae-induced EAA significantly.

Ensaios de penetração de Salmonella Enteritidis em ovos de galinha com diferentes qualidades de casca, submetidos ou não a lavagem industrial e a duas temperaturas de armazenagem / Penetration of Salmonella Enteritidis in chicken eggs, comparing different shell quality, industrial washing and temperature of storage

Estudou-se o comportamento da Salmonella Enteritidis quando em contato com a superfície de ovos de consumo, classificados de acordo com a qualidade da casca ou com tratamentos (lavados ou não), usando fezes de aves como veículo de contaminação, e armazenados a 8ºC e 70 por cento de umidade relativa ou a 30ºC e 90 por cento de umidade relativa. A 8ºC, o microrganismo permaneceu viável na superfície dos ovos, ao longo do tempo, enquanto a 30ºC foi possível identificar a sua inibição e até sua eliminação da superfície. A contaminação do conteúdo de ovos com defeito de casca foi mais frequente quando os ovos foram submetidos a temperaturas inadequadas de armazenagem (30ºC).

Salmonella Enteriditis behavior was studied when present on the surface of consume eggs, classified according to the shell quality or treatments (washed or not). Stools of fowls were used as a vehicle for contamination. The eggs were stored at 8ºC and 70 percent relative humidity or at 30ºC and 90 percent of relative humidity. At 8ºC, the microorganism stayed viable on the surface of the eggs throughout the study, while at 30ºC it was inhibited and eliminated from the egg shell. The contamination of content of the eggs with shell defect was more frequent when they were submitted to inappropriate temperatures of storage (30ºC).

Reproductive and developmental effects of transovarian exposure to o,p'-DDT in Japanese quails.

Avian species have the possible risk of embryonic exposure to persistent, lipophilic environmental contaminants, such as dichlorodiphenyltrichloroethane (DDT), by transfer of chemicals accumulated in mother birds to eggs. To model developmental and reproductive disorders of wild birds living in contaminated areas, we exposed Japanese quails in ovo to o,p'-DDT prior to incubation. A positive estrogenic substance diethylstilbestrol (DES; 1 and 10 ng/g of egg) and o,p'-DDT (1-100 microg/g of egg) were injected into the yolk before incubation. Treatment with o,p'-DDT (10 or 100 microg/g) but not with DES significantly reduced the hatchability of eggs. After sexual maturation, o,p'-DDT affected eggshell formation in female quails but had little influence on laying; high doses of o,p'-DDT significantly reduced eggshell strength, shell weight, and shell thickness, and several females treated with 100 microg o,p'-DDT/g laid eggs lacking shells. Diethylstilbestrol decreased egg production itself but had little effect on the eggshell. Both o,p'-DDT and DES caused dose-dependent shortening of the left oviduct and abnormal development of the right oviduct in females, while testis asymmetry was observed in males treated with a high dose of DES. In the uterus of the oviduct, the mRNAs for calcium-regulating factors osteopontin and calbindin D28K were reduced by both treatments, particularly that with o,p'-DDT. The results indicated that transovarian exposure to o,p'-DDT could bring about population declines in avian species through loss of fecundity caused by depression of hatchability and dysfunction of the reproductive tract.

Induction of eggshell apex abnormalities by Mycoplasma synoviae: field and experimental studies.

A novel eggshell pathology, characterized by an altered shell surface, thinning, increased translucency, and cracks and breaks in the eggshell apex, has become increasingly common in layer flocks of various breeds in The Netherlands. Two field studies found an association between the eggshell apex abnormalities (EAA) and infection with Mycoplasma synoviae. M. synoviae was isolated from the oviduct of birds that produced abnormal eggs, but not from birds in control flocks, although both affected and control birds had agglutinating antibodies against M. synoviae. After a single injection with long-acting oxytetracycline, the production of abnormal eggs ceased, but then reoccurred about 12 days later. A causal relationship between EAA and M. synoviae infection was subsequently demonstrated experimentally. EAA occurred after intratracheal inoculation of birds with M. synoviae, and even more frequently in birds infected with infectious bronchitis virus 5 days before inoculation of M. synoviae. EAA also occurred, although less frequently, in birds inoculated intravenously with M. synoviae and infected with infectious bronchitis virus. EAA did not occur in birds only inoculated intravenously with M. synoviae. M. synoviae was only isolated from the oviducts of birds producing abnormal eggs. The mean daily egg production was reduced in all groups infected with M. synoviae. Examination of the eggshells by scanning electron microscopy revealed that the mammillary layer of the calcified zone was absent and that the inner eggshell membranes were thicker. Isolates of M. synoviae from the oviducts of birds from farms producing abnormal eggs were examined by amplified fragment-length polymorphism analysis and did not appear to be clonal.

Influência do trincamento da casca do ovo sobre sua qualidade comercial / Eggshell cracking influence on its commercial quality

Com o objetivo de verificar a influência do trincamento da casca do ovo sobre sua qualidade comercial, foi realizado umexperimento com 340 ovos tipo extra, íntegros, divididos em dois grupos de 170, sendo que os ovos de um dos grupos tiveramsuas cascas trincadas artificialmente. Todos os ovos foram acondicionados em temperatura ambiente. Consecutivamentenos 10 primeiros dias e, depois, periodicamente, 20 ovos, 10 trincados e 10 íntegros, foram analisados, num total de 17 diasde análises, onde foram avaliados: peso do ovo inteiro; tamanho da câmara de ar; índice da gema; volume drenado da espumada clara; pH da clara e da gema; e unidade Haugh. Os resultados obtidos demonstraram que o trincamento da casca do ovoinfluenciou negativamente em sua qualidade comercial, visto que os ovos trincados demonstraram maiores quedas nosfatores de qualidade que os ovos íntegros. Isto foi melhor verificado no decréscimo de peso, no aumento da câmara de ar, e,no volume drenado da clara, mais intensos nos ovos trincados. Sendo assim, foi possível concluir que o trincamento da cascado ovo tem influência direta na sua qualidade comercial, evidenciando, portanto, a importância da adoção de cuidados durantesua manipulação e transporte

With the objective of verifying the influence of the cracking of the eggshell on its commercial quality, an experiment was accomplishedwith 360 eggs type extra, intact, divided in two groups of 180. In one of the groups, the eggs were submitted to an artificialcracking on the eggshells. Later, all the eggs were conditioned in room temperature. Consecutively in the first 10 days and, then,periodically, 20 eggs, 10 cracked and 10 intact, were used in interior and exterior quality analyses until the 27o day, in a total of 17days of analyses. The following factors were analyzed: weight of the whole egg, depth of air cell, yolk index, drained volume ofalbumen, pH of yolk and albumen, and Haugh unit. The results indicated that the cracking of the eggshell influenced negativelyin its commercial quality, because the decreasing of the cracked eggs quality factors was higher than the intact eggs. This wasspecially observed in relation to the weight of the egg, that decreased strongly along the days; to the depth of the air cell, that hadits size increased substantially; and to the drained volume of albumen, that became more intense. On the basis of this work itcould be concluded that eggshell cracking has a direct influence on its commercial quality and it is important to be sure aboutits careful manipulation ant transport

Organochlorine contaminants and biomarker response in double-crested cormorants nesting in Green Bay and Lake Michigan, Wisconsin, USA.

Double-crested cormorant (Phalacrocorax auritus) eggs at pipping and sibling 10-day-old chicks were collected from two colonies in Green Bay, WI, one colony in Lake Michigan, WI, and reference colonies in South Dakota and Minnesota. Egg contents and chicks were analyzed for organochlorine contaminants including polychlorinated biphenyl (PCB) congeners. Livers of embryos and chicks were assayed for hepatic microsomal ethoxyresorufin-O-dealkylase (EROD) activity. Eggshell thickness and the physical dimensions of embryo brains were measured. Concentrations of organochlorines, including p,p'-DDE (p,p'-dichlorodiphenyldichloroethylene), PCBs, and PCB congeners were generally an order of magnitude higher in eggs and chicks from Wisconsin than from reference locations. Total PCBs averaged 10-13 microg/g wet weight in eggs from three Wisconsin colonies compared to 0.9 microg/g PCBs from reference locations. Double-crested cormorant chicks accumulated on average 33-66 microg PCBs/day and 7-12 microg p,p'-DDE/day in the Wisconsin colonies compared to 0 microg PCBs/day and 1 microg p,p'-DDE/day in the reference colonies. At pipping, EROD activity in the livers of cormorant embryos was significantly higher in the Wisconsin colonies and significantly correlated with PCBs and the toxic equivalents (TEQs) of aryl hydrocarbon-active PCB congeners relative to 2,3,7,8-tetrachlorodibenzo-p-dioxin. However, in 10-day-old chicks EROD activity was not consistently different among colonies and was not correlated with PCBs or TEQs. A significant negative relationship between embryo brain asymmetry and the size of the egg suggested that physical constraint might be an important factor influencing the response of this bioindicator. Thinner eggshells in two colonies located near Door County, Wisconsin, suggested that historic p,p'-DDE residues associated with orchards are still an important source of p,p'-DDE in the local environment.

Effect of beta-aminoproprionitrile on eggshell formation.

1. Eggshells are bioceramic-biopolymer composites made by a cell-mediated deposition of an extracellular matrix which drives the organisation of the inorganic phase. Ultrastructurally, eggshells are composed of shell membranes, mammillary knobs, palisade, and cuticle. Shell membranes are two nets of type X collagen-containing fibrils. On to these membranes, the mammillary knobs, that is, the crystal nucleation sites, are deposited. Type X collagen is highly cross-linked and insoluble. 2. In order to evaluate the role of type X collagen cross-linking on eggshell formation, hens were injected with different doses of beta-aminoproprionitrile, which specifically interferes with cross-link formation. 3. Changes in egg size and shape were observed. Scanning electron micrographs analysis of these eggs demonstrated marked changes in crystal growth and shell membrane structure and arrangement. A dot-blot analysis, using a monoclonal antibody against chicken type X collagen, shows a dose-dependent increase in shell membrane collagen extractability. 4. It is concluded that the formation of beta-aminoproprionitrile-sensitive cross-links among the type X collagen molecules of the shell membranes play an essential role in normal eggshell formation.

Tonic immobility reaction and heterophil to lymphocyte ratio in hens from three Spanish breeds laying pink eggshells.

Tonic immobility reaction and heterophil:lymphocyte ratio were studied in hens laying pink eggs from three brown egg Spanish breeds (Barred Red Vasca, Red Vasca, Red Villafranquina). There was no significant difference among breeds in terms of tonic immobility duration or heterophil:lymphocyte ratio. Tonic immobility duration was significantly shorter in the pink (220 + or - 23 s) than in the normal brown shell group of hens (416 + or - 23 s; P < 0.001). Differences were consistent across the breeds, tonic immobility of hens laying pink eggs being shorter than that of control hens in all three breeds. Birds of the group with pink shells appeared to be less fearful when defined by either the duration of tonic immobility or the number of inductions to achieve it. There was a significant difference in the heterophil:lymphocyte ratio between the two groups of hens (P < 0.001), mean value being significantly higher in the pink shell (0.73 + or - 0.03) than in the control group (0.35 + or - 0.03). The heterophil:lymphocyte ratio from the pink shell hens increased significantly in the three breeds. Hens that laid pink eggs had significant heterophilia and lymphopenia (P < 0.001). Thus, hens laying pink eggs were less fearful and more stressed than control hens, as indicated by the tonic immobility reaction and the heterophil:lymphocyte ratio, respectively. A significant negative correlation was found between both measurements (r = A).28; P < 0.01). A similar significant negative association (P < 0.001) was found between pink shell color and shell strength.