In ovo sexing of chicken eggs by fluorescence spectroscopy.

Culling of day-old male chicks in production of laying hen strains involves several millions of animals every year worldwide and is ethically controversial. In an attempt to provide an alternative, optical spectroscopy was investigated to determine nondestructively in ovo the sex of early embryos of the domestic chicken. The extraembryonic blood circulation system was accessed by producing a window in the egg shell and the flowing blood was illuminated with a near-infrared laser. The strong fluorescence and the weak Raman signals were acquired and spectroscopically analyzed between 800 and 1000 nm. The increase of fluorescence intensity between 3.5 and 11.5 days of incubation was found to be in agreement with the erythropoietic stages, thus enabling to identify hemoglobin as fluorescence source. Sex-related differences in the fluorescence spectrum were found at day 3.5, and principal component (PC) analysis showed that the blood of males was characterized by a specific fluorescence band located at ∼910 nm. Supervised classification of the PC scores enabled the determination of the sex of 380 eggs at day 3.5 of incubation with a correct rate up to 93% by combining the information derived from both fluorescence and Raman scattering. Graphical abstract The fluorescence of blood obtained in ovo by illumination of embryonic vessels with a IR laser displays spectral differences that can be employed for sexing of eggs in early stage of incubation, before onset of embryo sensitivity and without hindering its development into a healthy chick.

The Incidence and Treatment Outcomes of Macrorhabdus ornithogaster Infection in Budgerigars ( Melopsittacus undulatus) in a Veterinary Clinic.

Macrorhabdus ornithogaster, avian gastric yeast, is a common cause of gastrointestinal disease in budgerigars ( Melopsittacus undulatus). To better understand the clinical disease in budgerigars presented in a practice population, we reviewed the occurrence, clinical signs, and treatment success of M ornithogaster disease in budgerigars during a 2.5-year period at the Clinic for Birds and Reptiles, University of Leipzig (Leipzig, Germany). The yeast was diagnosed by microscopic examination of fresh fecal samples. Male budgerigars of all ages were most affected. Most clinical signs in birds with confirmed positive results were nonspecific, except for the occurrence of undigested seeds in the feces. Although radiographic appearance of a dilated proventriculus is indicative of a M ornithogaster infection, it is difficult to recognize because of the small size of the budgerigars. Birds with positive results were treated with amphotericin B (100 mg/kg PO q12h) for 4 weeks. Treatment was stressful for the birds because of the handling required and the long treatment duration, and therapeutic results were unsatisfactory. Therefore, the indications for treatment with amphotericin B should be carefully considered in birds with positive M ornithogaster results. An increased occurrence of the infection in association with other pathogens was detected.

In Ovo Sexing of Domestic Chicken Eggs by Raman Spectroscopy.

Male birds of egg-laying hen strains have no commercial value and are culled immediately after hatching, raising concerns for animal welfare. Existing experimental methods for in ovo sexing require taking samples and are applicable after embryos' sexual differentiation. We demonstrate that Raman spectroscopy enables contactless in ovo sex determination of the domestic chicken (Gallus gallus f. dom.) already at day 3.5 of egg incubation. A sexing accuracy of 90% was obtained by analyzing the spectra of blood circulating in the extraembryonic vessels. The measurement is damage-free and barely affects the hatching rate. Sex recognition is achieved before the onset of sensitivity. Therefore, Raman spectroscopy provides an alternative to the culling of 1-day-old male chicks in laying hen production.

A method to identify protein antigens of Dermanyssus gallinae for the protection of birds from poultry mites.

The poultry red mite (PRM) Dermanyssus gallinae causes high economic losses and is among the most important parasites in poultry farming worldwide. Different chemical, physical, and biological strategies try to control the expansion of PRM. However, effective solutions to this problem still have to be found. Here, we present a method for the development of an immunological control strategy, based on the identification of mite protein antigens which elicit antibodies with anti-mite activity in the immunized chicken. Hens were immunized with different PRM protein extracts formulated with two different adjuvants, and IgY-antibodies were isolated from the eggs. A PRM in vitro feeding assay which used chicken blood spiked with these IgY-preparations was used to detect antibodies which caused PRM mortality. In vitro feeding of mites with IgY isolated from hens immunized with PRM extract formulated with one of the adjuvants showed a statistically significant increase in the mortality as compared to control mites. After the separation of total PRM extracts in two-dimensional gels, several protein spots were recognized by such IgY preparations. Ten protein spots were subjected to mass spectrometry (MS/MS) for the identification of the corresponding proteins. Complete protein sequences were deduced from genomic and transcriptomic assemblies derived from high throughput sequencing of total PRM DNA and RNA. The results may contribute to the development of an immunological control strategy of D. gallinae.

Testicular biopsy in psittacine birds (Psittaciformes): comparative evaluation of testicular reproductive status by endoscopic, histologic, and cytologic examination.

Knowledge of the reproductive cycle of male parrots is important for examining the male genital tract and for successful breeding, especially of endangered species. To evaluate different diagnostic methods and criteria concerning the classification of reproductive stages, we examined 20 testicular samples obtained at necropsy in psittacine birds of different species and testicular biopsy samples collected from 9 cockatiels (Nymphicus hollandicus) and 7 rose-ringed parakeets (Psittacula krameri) by endoscopy 4 times over a 12-month period. The testicular reproductive status was assessed histologically and then compared with the macroscopic appearance of the testicles and cytologic results. The histologic examination was nondiagnostic in 19 of 59 testicular biopsy samples. By contrast, the cytologic preparations were diagnostic in 57 of 59 biopsy samples. The results of the cytologic examination coincided with the histologic results in 34 of 38 biopsy samples and 18 of 20 necropsy samples. Macroscopic parameters displayed some differences between reproductive stages but provided an unreliable indication of the reproductive status. These results suggest that microscopic examination of a testicular biopsy sample is a reliable method for evaluating the reproductive status of male parrots and is preferable to the macroscopic evaluation of the testicle. Cytologic examination provides fast preliminary results, even when the histologic preparation is not sufficient for evaluation, but results may be erroneous. Thus, a combination of histologic and cytologic examination is recommended for evaluating testicular reproductive status.

Testicular biopsy in psittacine birds (Psittaciformes): impact of endoscopy and biopsy on health, testicular morphology, and sperm parameters.

Histologic examination of a testicular biopsy sample may be required to evaluate the reproductive status of male psittacine birds. The purpose of this study was to evaluate the viability of testicular sampling from live birds by assessing the impact on the birds' health, testicular integrity, and sperm quality. Testicular biopsy samples were obtained by endoscopy 4 times during 12 months from 9 cockatiels (Nymphicus hollandicus) and 7 rose-ringed parakeets (Psittacula krameri). Only 2 of 16 birds showed testicular cicatrization or divided testicular tissue after a single endoscopy. Further complications, such as damage to the air sacs or bleeding, predominantly occurred in subsequent endoscopies. In both species, endoscopy and testicular biopsy caused only minor or transient effects on sperm production and sperm quality. These results support that a single testicular biopsy is a viable method for evaluating the reproductive status of male psittacine birds.

A complicated, metastatic, humeral air sac cystadenocarcinoma in a timneh African grey parrot (Psittacus erithacus timneh).

A 9-year-old male timneh African grey parrot (Psittacus erithacus timneh) was presented because of inability to fly and suspected trauma. The owner also had observed dyspnea, with tail bobbing and open-beak breathing. On clinical examination, a hard, painful mass was palpable in the left proximal humerus and axillary area. Radiographs revealed a radiodense soft tissue mass of the left humerus with no bony involvement, multifocal opacities in lung and air sacs, and an enlarged spleen. An asymmetric, vascularized cyst was detected in the mass by ultrasound examination. Results of biopsy of the mass revealed multifocal cysts composed of unilayer isoprismatic cells laying in vascularized connective tissue. Because of the severity of clinical signs and the poor clinical condition, the bird was euthanatized. On postmortem examination, the findings were air sac cystadenocarcinoma involving the humeral air sac with metastases in the spleen and kidneys, atherosclerosis, pneumoconiosis, and mycotic granulomatous pneumonia and airsacculitis with isolation of Aspergillus niger.

Investigation of the Formal Pathogenesis of Green Liver Discoloration in Organically Reared Female Bronze Turkeys (Melleagris gallovapo).

Green liver discoloration (GL) in fattening turkeys is suspected to be a multifactorial disease complex with a compromised immune system as the key factor. This study aimed to identify the formal pathogenesis of GL and to investigate possible nutritional influences. A total of 360 Bronze turkey hens out of 10 flocks from 5 fattening farms were necropsied for detection of GL during 2 consecutive trials on 2 examination dates each (70th to 75th and 120th to 127th day of fattening, respectively). At each examination date, hematological and clinical chemistry analyses, as well as determination of vitamin E and selenium concentrations in the liver, were carried out in 6 hens with (if applicable) and 6 hens without GL, representing a total of 130 individuals. Raw nutrient, energy, amino acid, bulk and trace element, and vitamin E and D3 concentrations were analyzed in feed samples for each of the five feeding phases during each trial. The results of the hematological analyses, clinical chemistry analyses, and determination of vitamin E and selenium liver concentrations were statistically evaluated between: (i) individuals with and without GL, and (ii) individuals from flocks with and without turkeys with GL. At both fattening stages, the occurrence of GL was characterized by an inflammatory reaction. A subacute inflammatory reaction was detected in the early fattening stage, indicating a viral cause of the disease. In the late fattening stage, acute inflammation indicated a bacterial cause of the disease. The results of the feed sample analyses of the different flocks were generally quite homogeneous. However, the nutrient and energy content of the feed likely contribute to GL pathogenesis.

An Explorative Study of the Causal Pathogenesis of Green Liver Discoloration in Organically Reared Female Bronze Turkeys (Meleagris gallopavo) Considering the Infectious Risk Factors.

A recent study revealed that organically raised Bronze turkeys showed a high prevalence of green liver discoloration. This alteration is commonly associated with the Turkey Osteomyelitis Complex and potentially caused by opportunistic bacteria. Therefore, 360 organically fattened Bronze turkeys were examined post-mortem throughout two fattening trials with two examinations each to determine possible infectious risk factors and reduce disease prevalence. Clinical and pathoanatomical examinations were performed on every hen. Histopathological, bacteriological, parasitological, and virological examinations were performed on at least six hens without and, if applicable, six hens with green livers on each examination date. Overall, 9.0% of all hens had a green liver without a correlation with bacterial or parasitological findings but multiple health impairments. The discoloration correlated significantly with the detection of immunosuppressive turkey hemorrhagic enteritis virus at the early stage and macro- and histological joint/bone lesions at the late fattening stage, indicating the presence of two different predisposing pathogeneses. Flocks not being vaccinated against hemorrhagic enteritis but having a virus-positive sample showed the highest prevalence of green liver discoloration and developed worse in various parameters. In conclusion, an adequate vaccination schedule and the prevention of field infections may lead to a decreased risk of performance reduction and improved animal health.

Gender determination of fertilized unincubated chicken eggs by infrared spectroscopic imaging.

Each year, billions of day-old layer chicks are produced in the world. Since only female chicks are reared for egg production, the chicks must be sexed and the unwanted male layer chicks are culled. The culling of male chicks is a serious problem, both in terms of animal welfare and waste disposal. The germinal disc in fertilized but unincubated eggs contains already several thousands of blastoderm cells. The cellular DNA in birds is different for male and female chicks. The difference in DNA content between male and female chicks is around 2% and is measurable by Fourier transform infrared (FT-IR) spectroscopy. In this study, small amounts of blastoderm cells from 22 chicken eggs were characterized by attenuated total reflection FT-IR spectroscopic imaging and classified by linear discriminant analysis. Polymerase chain reaction (PCR) was used as a reference method to determine the gender. The spectroscopic results demonstrate that male blastoderm cells exhibit a higher content of DNA than cells from female blastoderm. The spectroscopic-based gender determination led to the same result as the PCR analysis. FT-IR spectroscopic imaging allows the gender determination of unincubated eggs within a few seconds based on the accurate determination of the different DNA contents in blastoderm cells of both sexes.

Evaluating the reproductive status of the male budgerigar (Melopsittacus undulatus).

Limited knowledge about male psittacine reproduction reduces the success of breeding programmes. Within the scope of fecundity assessment, classification of male sexual status is essential for effective conservation of the species. The aim of the present study was to investigate the testes of male budgerigars (Melopsittacus undulatus), as psittaciform model species to verify their reproductive status by morphological and immunocytochemical examination. Using light microscopy, gonadal samples were categorized resulting in three reproductive states (active, intermediate, non-active). Calculation of testes weights plus measurement of tubular and interstitial dimensions displayed significant (p ≤ 0.05) differences between all three reproductive stages. Lipids in the testicular tubules, analysed by Sudan black staining and fluorescence microscopy (DAPI(2) mode) were highly present in non-active status. Immunocytochemistry involved two different hydroxysteroid dehydrogenases (HSD), 3ß-HSD and 17ß-HSD-2, as markers for steroidogenesis, as well as steroid receptors for androgens (AR), oestrogen (ER) and progesterone (PR). Both HSDs and AR declined in non-active gonads compared to active and intermediate stages, with a positive signal in germ and somatic cells of testis and epididymis. ER and PR were detected in testicular and epididymal cells, similarly expressed in all three stages. The proliferation rate of germ cells in the testicular tubules, obtained by Ki67, differed significantly in active (38.67%), intermediate (32.40%) and non-active (6.01%) status. According to this morphological study, we have been able to establish markers for the reproductive staging of psittacine testes. This knowledge will be useful to deepen reproductive biology in budgerigars.

Attempted semen collection using the massage technique in blue-fronted Amazon parrots (Amazona aestiva aestiva).

The purpose of this study was to establish a technique for collecting semen from blue-fronted Amazon parrots (Amazona aestiva aestiva) and to evaluate the samples that were collected. The massage method is the most common technique used to collect semen in birds and has been proven successful in several psittacine species; however, collection attempts in larger parrots have been unsatisfactory. Six blue-fronted Amazon parrot males, 3 paired with hens and 3 unpaired, were used in this study. The semen collection technique was revised to allow collection from individual birds by a single person. Semen collection was attempted from the 6 parrots on 52-56 occasions, which totaled 330 single attempts. Nineteen ejaculates were collected, and each bird produced at least 1 ejaculate that contained spermatozoa. Large ranges of sample volume (1-15.4 microL), sperm quality (motility = 2%-60%; live:dead ratio = 2:198 to 185:15), sperm concentration (0.79-3.3 x 10(6) sperm/mL), and contamination rate (0%-100%) were observed. Measured parameters did not appear to be significantly impacted by birds being paired or kept singly. Because of the relatively short acclimation period, the birds appeared to be sexually inactive for the majority of the study. Further research using sexually active birds will be necessary to determine standard spermatological parameters and verify the success of the methodology used here.

[Evaluation of housing conditions of budgerigars (Melopsittacus undulatus) and cockatiels (Nymphicus hollandicus) in Germany]. / Beurteilung der Haltungsbedingungen von Wellensittichen (Melopsittacus undulatus) und Nymphensittichen (Nymphicus hollandicus) in Deutschland.

OBJECTIVES: In order to gain information concerning housing conditions of budgerigars and cockatiels, the most frequently kept pet birds in Germany, a country-wide online survey among non-commercial bird owners was performed within the realm of the EXOPET study. MATERIAL AND METHODS: The received questionnaires (541 for budgerigars and 221 for cockatiels) were initially evaluated descriptively. Based on the suggestions developed by an expert group in the context of the EXOPET-project, the housing conditions were subsequently assessed with regard to socialization, size of cage/aviary, cage accessories, temperature, UV light and amount of free flight. RESULTS: The most striking deviations from the recommendations occurred in both cockatiels and budgerigars in the parameter free flight (deficiencies in 89.3 % and 85.5 % of the evaluated husbandries). In connection with the resulting lack of exercise, obesity was the most common health impairment observed by budgerigar owners (26.4 %). 82.8 % of the cockatiels and 52.2 % of the budgerigars were also kept in cages or aviaries that were too small. 73.1 % resp. 73.3 % of the budgerigar resp. cockatiel owners stated to provide their birds with the possibility for free flight on a daily basis. In this context, 48.3 % of the budgerigars and 34.0 % of the cockatiels were offered free flight for at least 12 hours per day. Other significant deficiencies were found concerning the cage accessories. Here, deviations from the recommendations were found in 77.8 % of the budgerigar and 69.0 % of the cockatiel husbandries. CONCLUSION: In order to improve the partly alarming deficits in housing conditions of budgerigars and cockatiels, owner competence must be improved. It seems necessary to provide the animal owner with standardized information concerning the specific pet bird species as well as their individual housing requirements.

[Aspects to improve animal welfare in fattening turkeys: structure and occupational material - a literature review]. / Aspekte zur Verbesserung des Tierwohls bei Mastputen: Strukturierung und Beschäftigungsmaterial ­ eine Literaturübersicht.

Conventional turkey husbandries frequently fail to provide any structural elements or occupational material apart from the feeding and drinking systems. However, these may carry numerous advantages for animal welfare (spatial separation to exercise natural behavior, retreat options, stimulation of locomotion). Hence these are increasingly discussed in conventional turkey husbandry, especially against the background of minimizing injurious pecking. Studies reported in the literature concerning the effects of structural elements or occupational materials are not exactly comparable to one another due to variations in study design. However, the described positive effects predominate across the different evaluations. Nonetheless, when using such elements consideration must be given to several variables. For example, the use and positive effects of perching options depend on the type and dimensions of their structure used as well as on sex, age and breed of the animals. Straw bales represent one option, which then may also serve as occupational material. When hay or straw is offered, appropriately sized grit must also be administered in order to prevent obstipation of the gastrointestinal tract. Different occupational materials should be offered alternately and in sufficient quantities. The outdoor-climate and run area offer climatic and optic stimuli and additional opportunities for exercise. However, various basic requirements must also be considered here. In principle, the commitment and knowledge of the breeder are of crucial importance for all of the animal welfare improvements mentioned. Therefore, regular further training for this group of people is recommended.

Sexing of turkey poults by Fourier transform infrared spectroscopy.

Fourier transform infrared (FT-IR) spectroscopy was used to probe the molecular composition of germinal cells and to identify the gender of turkey poults. Germinal cells obtained from a feather pulp were characterized by FT-IR micro spectroscopy. The sample set consisted of growing contour feathers from 23 male and 23 female turkey poults. Significant spectral variations were observed in the range between 1,000 and 1,250 cm(-1). The spectra of male turkey poults exhibit a significantly higher content of RNA than those of female turkeys. Spectral classification was performed by a non-supervised method based on the principal component analysis. An evaluation of the first and third PCs led to a classification of female and male poults with an accuracy of more than 95%.

Ultrasonographic diagnosis of an endocarditis valvularis in a Burmese python (Python molurus bivittatus) with pneumonia.

An 11-yr-old Burmese python (Python molurus bivittatus) was presented with a history of respiratory symptoms. Computed tomography and an endoscopic examination of the left lung were performed and revealed severe pneumonia. Microbiologic examination of a tracheal wash sample and an endoscopy-guided sample from the lung confirmed infection with Salmonella enterica ssp. IV, Enterobacter cloacae, and Klebsiella pneumoniae. Computed tomographic examination demonstrated a hyperattenuated structure within the heart. Echocardiographic examination revealed a hyperechoic mass at the pulmonic valve as well as a dilated truncus pulmonalis. As therapy for pneumonia was ineffective, the snake was euthanized. Postmortem examination confirmed pneumonia and infective endocarditis of the pulmonic valve caused by septicemia with Salmonella enterica ssp. IV. Focal arteriosclerosis of the pulmonary trunk was also diagnosed. The case presented here demonstrates the possible connection between respiratory and cardiovascular diseases in snakes.

Diagnostic Imaging of the Avian Urinary Tract.

Due to the special anatomy and physiology of the avian urinary system, the value of diagnostic imaging techniques differs from the use in mammals. The diagnostic imaging methods regularly used in practice to evaluate the avian kidneys are often limited to traditional radiography and ultrasonography, whereas other imaging modalities (urography, scintigraphy, computed tomography, MRI) are rarely used. Furthermore endoscopy may be performed and taking a renal biopsy may be considered. The article describes common indications for imaging techniques used to diagnose urinary tract disease as well as its anatomic and pathologic demonstration.

Characterization of atherosclerosis by histochemical and immunohistochemical methods in African grey parrots (Psittacus erithacus) and Amazon parrots (Amazona spp.).

The aim of the study was to characterize atherosclerotic changes in African grey parrots (Psittacus erithacus) and Amazon parrots (Amazona spp.) by histochemical and immunohistochemical methods. Samples of the aorta ascendens and trunci brachiocephalici from 62 African grey parrots and 35 Amazon parrots were stained by hematoxylin and eosin and Elastica van Gieson for grading of atherosclerosis in these birds. Four different stages were differentiated. The incidence of atherosclerosis in the examined parrots was 91.9% in African grey parrots and 91.4% in Amazon parrots. To evaluate the pathogenesis in birds, immunohistochemical methods were performed to demonstrate lymphocytes, macrophages, smooth muscle cells, and chondroitin sulfate. According to the missing lymphocytes and macrophages and the absence of invasion and proliferation of smooth muscle cells in each atherosclerotic stage, "response-to-injury hypothesis" seems inapplicable in parrots. Additionally, we found alterations of vitally important organs (heart, lungs) significantly correlated with atherosclerosis of the aorta ascendens.

Cardiovascular physiology and diseases of pet birds.

Avian cardiac disease in pet birds occurs more often than previously assumed. The article focuses on anatomic peculiarities of the avian cardiovascular system and common diseases. Diagnostic possibilities are demonstrated, and therapeutic measures are discussed.

Mycoplasmas isolated from stone curlews (Burhinus oedicnemus) used in falconry in the United Arab Emirates.

The aim of this study was to evaluate the risk of transmission of Mycoplasma spp. from quarry to hunting falcons in the Middle East. Groups of 17 houbara bustards (Chlamydotis undulata) and 29 stone curlews (Burhinus oedicnemus) kept at three different private collections in Dubai were evaluated for the presence of Mycoplasma. Additionally, 10 falcons used for hunting were investigated for comparison. The falcons showed no clinical signs and were examined within the scope of a routine health check. From all birds, conjunctival and choanal swabs were taken and analyzed via polymerase chain reaction and culture. Although mycoplasmas were not recovered from choanal and conjunctival swabs taken from the houbara bustards, Mycoplasma gypis and M. falconis were isolated from the majority (28/29; 97%) of the stone curlews from choanal and conjunctival swabs. Most of the birds had no associated pathologic findings. Mycoplasma falconis was also detected in samples collected from 2 of the 10 falcons, and M. buteonis was isolated from the majority of falcons (6/10 falcons) from choanal (n = 5) and conjunctival (n = 1) swabs. Mycoplasma gypis could also be isolated from tissue samples (liver, oviduct, syrinx) of one dead stone curlew. This study presents the first isolation of mycoplasmas from stone curlews.