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.

[Endocrinologic studies of male psittacine birds for the evaluation of their reproductive status]. / Endokrinologische Untersuchung männlicher Papageienvögel zur Beurteilung ihres Reproduktionsstatus.

OBJECTIVE: Evaluating methods of hormone measurement in different specimens of male parrots in order to assess their reproductive status and stress axis. MATERIAL AND METHODS: Cockatiels and rose-ringed parakeets were chosen as psittaciforme representatives and their endocrine profiles were examined. In various pre-experiments, suitable techniques for the determination of testosterone in plasma, saliva and faeces of male parrots were established. Before analysing the samples by enzyme immunoassay, blood and faeces were extracted using diethyl ether, while saliva could be tested without extraction. Based on the excretion of mainly conjugated testosterone metabolites, parrots' faecal samples were also hydrolysed with ß-glucuronidase/arylsulfatase before extraction. In addition, the levels of the stress hormone corticosterone were determined by radioimmunoassay in order to assess possible relationships between stress and the secretion of testosterone. RESULTS: The examined psittacine species displayed different seasonal secretion patterns for both testosterone and corticosterone. Cockatiels had maximum plasma testosterone levels in February, in contrast, rose-ringed parakeets showed highest concentrations in November. As a consequence of cumulative excretion, both species showed much higher faecal than plasma testosterone concentrations. In rose-ringed parakeets, the levels of corticosterone in plasma were exceptionally high compared to the cockatiels. CONCLUSION: According to this study, we have been able to establish suitable methods for testosterone analysis in blood and faeces of cockatiels and rose-ringed parakeets, supporting the assessment of their reproductive status. At present saliva does not appear to be an ideal medium for reliable hormone level measurement, thus further investigations are required concerning this subject. CLINICAL RELEVANCE: By means of process analysis, it will be possible to detect increasing testosterone levels and/or pathological alterations, which could be considered in breeding programmes.

Flow cytometric analysis of virus-specific T lymphocytes: practicability of detection of HCMV-specific T lymphocytes in whole blood in patients after stem cell transplantation.

The detection and quantification of specific T lymphocytes against human cytomegalovirus (HCMV) has proven an important laboratory marker in the monitoring of patients after stem cell transplantation (SCT). In these patients HCMV infections may cause severe disease and death. However, the determination of HCMV-specific T lymphocytes may be limited by lymphopenia occurring after transplantation. We evaluated a commercial test kit for the reliable determination of HCMV-specific T lymphocyte development in lymphopenic patients after stem cell transplantation. Using a whole blood protocol for the flow cytometric detection of antigen-specific CD4(+) T-helper and CD8(+) cytotoxic T lymphocytes this test kit measures intracellular cytokine production after stimulation with HCMV antigen. The measurement of HCMV-specific T lymphocytes was feasible when at least 3,000 CD4(+) or 1,000 CD8(+) T cells could be counted by flow cytometry. Detection of HCMV-specific T lymphocytes was possible, on average, 67 (SD+/-61) days after transplantation for CD4(+) cells and 27 (SD+/-13) days for CD8(+) cells, thus being still within the critical time for HCMV reactivation. In conclusion, the use of modern test kits permits the measurement of HCMV-specific T lymphocytes in stem cell transplant recipients and may be included in the HCMV monitoring system after SCT.

Antibody selection for immunocytochemical characterization of the male reproductive system in Psittaciformes.

The success of breeding programs is limited by the sparse knowledge about endocrine regulation and biochemical reactions in the psittacine male tract. The immunocytochemical analysis of parrots' testicular tissues provides an insight into their reproductive system but is often hampered by the lack of reliable antibodies. In the present study, we tested a large panel of antibodies raised against steroid receptors, steroidogenic enzymes, relaxin peptides including their receptors, and proliferation markers on paraffin sections of testicular tissue from eight psittacine genera representing three continents. All investigated species displayed the tested markers in somatic and germ cells of testis and epididymis, even though cell distribution was partly heterogenous and in species-specific patterns. The 17ß-hydroxysteroid-dehydrogenase-2, 3ß-hydroxysteroid-dehydrogenase, and smooth muscle actin allowed the cross-species differentiation between active and nonactive gonads. The remaining steroidogenic enzymes, steroid receptors, relaxin peptides, and Ki67 proved to be suitable to define reproductive activity depending on the parrot species. Adapting immunocytochemical methods to different psittacines was successful, though various cellular expression patterns do not allow the transfer of results among different parrot species. However, the availability of a reliable repertory of sexual markers is important to examine reproductive biology of psittacine birds.

Novel approach for genotyping varicella-zoster virus strains from Germany.

In this study, we present a novel genotyping scheme to classify German wild-type varicella-zoster virus (VZV) strains and to differentiate them from the Oka vaccine strain (genotype B). This approach is based on analysis of four loci in open reading frames (ORFs) 51 to 58, encompassing a total length of 1,990 bp. The new genotyping scheme produced identical clusters in phylogenetic analyses compared to full-genome sequences from well-characterized VZV strains. Based on genotype A, D, B, and C reference strains, a dichotomous identification key (DIK) was developed and applied for VZV strains obtained from vesicle fluid and liquor samples originating from 42 patients suffering from varicella or zoster between 2003 and 2006. Sequencing of regions in ORFs 51, 52, 53, 56, 57, and 58 identified 18 single-nucleotide polymorphisms (SNPs), including two novel ones, SNP 89727 and SNP 92792 in ORF51 and ORF52, respectively. The DIK as well as phylogenetic analysis by Bayesian inference showed that 14 VZV strains belonged to genotype A, and 28 VZV strains were classified as genotype D. Neither Japanese (vaccine)-like B strains nor recombinant-like C strains were found within the samples from Germany. The novel genotyping scheme and the DIK were demonstrated to be practical and simple and allow the highly efficient replication of phylogenetic patterns in VZV initially derived from full-genome DNA sequence analyses. Therefore, this approach may allow us to draw a more comprehensive picture of wild-type VZV strains circulating in Germany and Central Europe by high-throughput procedures in the future.

NK sensitivity of neuroblastoma cells determined by a highly sensitive coupled luminescent method.

The measurement of natural killer (NK) cells toxicity against tumor or virus-infected cells especially in cases with small blood samples requires highly sensitive methods. Here, a coupled luminescent method (CLM) based on glyceraldehyde-3-phosphate dehydrogenase release from injured target cells was used to evaluate the cytotoxicity of interleukin-2 activated NK cells against neuroblastoma cell lines. In contrast to most other methods, CLM does not require the pretreatment of target cells with labeling substances which could be toxic or radioactive. The effective killing of tumor cells was achieved by low effector/target ratios ranging from 0.5:1 to 4:1. CLM provides highly sensitive, safe, and fast procedure for measurement of NK cell activity with small blood samples such as those obtained from pediatric patients.