Differentiating between metabolic health statuses in Simmental cows and describing related milk fatty acids and relevant associated factors.

The aim of this observational study was to examine differences in milk fatty acid (FA) concentrations for different metabolic health statuses and for associated factors-specifically to examine with which FA concentrations an increased risk for developing a poor metabolic adaptation syndrome (PMAS) was associated. During weekly visits over 51 wk, blood samples were collected from cows between 5 and 50 days in milk. The farmer collected corresponding milk samples from all voluntary milkings. The analysis was performed on n = 2,432 samples from n = 553 Simmental cows. The observations were assigned to five different cow types (healthy, clever, athletic, hyperketonemic, and PMAS, representing five metabolic health statuses), based on the thresholds of 0.7 mmol/L, 1.2 mmol/L, and 1.4 for the concentrations of ß-hydroxybutyrate and nonesterified fatty acids and for the milk fat-to-protein ratio, respectively. Linear regression models using the predictor variables cow type, parity, week of lactation, and milk yield as fixed effects were developed using a stepwise forward selection to test for significant associations of predictor variables regarding FA concentrations in milk. There was a significant interaction term found between PMAS cows and parity compared to healthy cows for C18:1 (P < 0.001) and for C18:0 (P < 0.01). It revealed higher concentrations for PMAS in primiparous and multiparous cows compared to healthy cows, the slope being steeper for primiparous cows. Further, an interaction term was found between PMAS cows and milk yield compared to healthy cows and milk yield for C16:0 (P < 0.05), revealing a steeper slope for the decrease of C16:0 concentrations with increasing milk yield for PMAS compared to healthy cows. The significant associations and interaction terms between cow type, parity, week of lactation, and milk yield as predictor variables and C16:0, C18:0, and C18:1 concentrations suggest excellent opportunities for cow herd health screening during the early postpartum period.

Many Radiographic and Magnetic Resonance Imaging Assessments for Surgical Decision Making in Pediatric Patellofemoral Instability Patients Demonstrate Poor Interrater Reliability.

PURPOSE: To evaluate the interrater reliability of several common radiologic parameters used for patellofemoral instability and to attempt to improve reliability for measurements demonstrating unacceptable interrater reliability through consensus training. METHODS: Fifty patients with patellar instability between the ages of 10 and 19 years were selected from a prospectively enrolled cohort. For measurements demonstrating unacceptable interrater reliability (intraclass correlation coefficient [ICC]: <0.6), raters discussed consensus methods to improve reliability and re-examined a subset of 20 images from the previous set of images. If reliability was still low after the second round of assessment, the measure was considered unreliable. RESULTS: Of the 50 included subjects, 22 (44%) were male and the mean age at the time of imaging was 14 ± 2 years. With 1 or fewer consensus training sessions, the interrater reliability of the following radiograph indices were found to be reliable: trochlea crossing sign (ICC: 0.625), congruence angle (ICC: 0.768), Caton-Deshamps index (ICC: 0.644), lateral patellofemoral angle (ICC: 0.768), and mechanical axis deviation on hip-to-ankle alignment radiographs (ICC: 0.665-0.777). Reliable magnetic resonance imaging (MRI) indices were trochlear depth (ICC: 0.743), trochlear bump (ICC: 0.861), sulcus angle (ICC: 0.684), patellar tilt (ICC: 0.841), tibial tubercle to trochlear groove distance (ICC: 0.706), effusion (ICC: 0.866), and bone marrow edema (ICC: 0.961). CONCLUSIONS: With 1 or fewer consensus training sessions, the interrater reliability of the following patellofemoral indices were found to be reliable for trochlear morphology: trochlea crossing sign and congruence angle on radiograph and trochlear depth, trochlear bump, and sulcus angle on MRI. Reliable patellar position measurements included: Caton-Deshamps index and lateral patellofemoral angle on radiograph and patellar tilt and tibial tubercle to trochlear groove distance on MRI. Additional global measurements (e.g., mechanical axis deviation on standing radiographs) and MRI assessments demonstrated acceptable reliability. LEVEL OF EVIDENCE: II, prospective diagnostic study.

Particulated Juvenile Articular Cartilage and Matrix-Induced Autologous Chondrocyte Implantation Are Cost-Effective for Patellar Chondral Lesions.

PURPOSE: To compare the cost-effectiveness of nonoperative management, particulated juvenile allograft cartilage (PJAC), and matrix-induced autologous chondrocyte implantation (MACI) in the management of patellar chondral lesions. METHODS: A Markov model was used to evaluate the cost-effectiveness of three strategies for symptomatic patellar chondral lesions: 1) nonoperative management, 2) PJAC, and 3) MACI. Model inputs (transition probabilities, utilities, and costs) were derived from literature review and an institutional cohort of 67 patients treated with PJAC for patellar chondral defects (mean age 26 years, mean lesion size 2.7 cm2). Societal and payer perspectives over a 15-year time horizon were evaluated. The principal outcome measure was the incremental cost-effectiveness ratio (ICER) using a $100,000/quality-adjusted life year (QALY) willingness-to-pay threshold. Sensitivity analyses were performed to assess the robustness of the model and the relative effects of variable estimates on base case conclusions. RESULTS: From a societal perspective, nonoperative management, PJAC, and MACI cost $4,140, $52,683, and $83,073 and were associated with 5.28, 7.22, and 6.92 QALYs gained, respectively. PJAC and MACI were cost-effective relative to nonoperative management (ICERs $25,010/QALY and $48,344/QALY, respectively). PJAC dominated MACI in the base case analysis by being cheaper and more effective, but this was sensitive to the estimated effectiveness of both strategies. PJAC remained cost-effective if PJAC and MACI were considered equally effective. CONCLUSIONS: In the management of symptomatic patellar cartilage defects, PJAC and MACI were both cost-effective compared to nonoperative management. Because of the need for one surgery instead of two, and less costly graft material, PJAC was cheaper than MACI. Consequently, when PJAC and MACI were considered equally effective, PJAC was more cost-effective than MACI. Sensitivity analyses accounting for the lack of robust long-term data for PJAC or MACI demonstrated that the cost-effectiveness of PJAC versus MACI depended heavily on the relative probabilities of yielding similar clinical results. LEVEL OF EVIDENCE: III, economic and decision analysis.

Evaluation and management of patellar instability.

Patellar instability is a common clinical problem that primarily affects the adolescent and young adult population. The demographic and anatomic risk factors that predispose patients to patellar instability are multifactorial and include young age, female sex, trochlear dysplasia, elevated tibial tubercle to trochlear groove distance (TT-TG), patella alta, femoral and tibial malalignment, ligamentous laxity, and lack of neuromuscular control. There have been substantial efforts to predict which patients who sustain a first-time dislocation will go on to incur additional dislocations. This is particularly important because with each dislocation event, there is a significant risk of injury to the patellofemoral joint including both medial patellofemoral ligament (MPFL) stretch or rupture and damage to the cartilage which can range from simple fissures to full-thickness cartilage defects and osteochondral fractures. Prediction models have demonstrated that amongst first time dislocators, young patients with trochlear dysplasia are at the highest risk for redislocation. The current standard of care for treatment of first-time dislocators without a loose body or osteochondral fracture is nonoperative management. However, recently there has been a focus on implementing a risk-stratified approach to the surgical indications for a first-time dislocator as the high-risk population might be better treated with early surgical stabilization to prevent or reduce their risk of recurrent dislocation and its associated morbidity. Likewise, for patients with recurrent dislocations, it remains to be determined whether an isolated MPFL reconstruction is sufficient for high-risk patients with several poor prognostic risk factors or if bony realignment procedures should be implemented concurrently.

Reliability of Radiologic Assessments of Clinically Relevant Growth Remaining in Knee MRI of Children and Adolescents With Patellofemoral Instability: Data From the JUPITER Cohort.

BACKGROUND: Surgical decision making and preoperative planning for children and adolescents with patellofemoral instability rely heavily on a patient's skeletal maturity. To be clinically useful, radiologic assessments of skeletal maturity must demonstrate acceptable interrater reliability and accuracy. PURPOSE: The purpose of this study was to examine the interrater reliability among surgeons of varying experience levels and specialty training backgrounds when evaluating the skeletal maturity of the distal femur and proximal tibia of children and adolescents with patellofemoral instability. STUDY DESIGN: Cohort study (diagnosis); Level of evidence, 3. METHODS: Six fellowship-trained orthopaedic surgeons (3 pediatric orthopaedic, 2 sports medicine, and 1 with both) who perform a high volume of patellofemoral instability surgery examined 20 blinded knee radiographs and magnetic resonance images in random order. They assessed these images for clinically relevant growth (open physis) or clinically insignificant growth (closing/closed physis) remaining in the distal femoral and proximal tibial physes. Fleiss' kappa was calculated for each measurement. After initial ratings, raters discussed consensus methods to improve reliability and assessed the images again to determine if training and new criteria improved interrater reliability. RESULTS: Reliability for initial assessments of distal femoral and proximal tibial physeal patency was poor (kappa range, 0.01-0.58). After consensus building, all assessments demonstrated almost-perfect interrater reliability (kappa, 0.99 for all measurements). CONCLUSION: Surgical decision making and preoperative planning for children and adolescents with patellofemoral instability rely heavily on radiologic assessment of skeletal maturity. This study found that initial interrater reliability of physeal patency and clinical decision making was unacceptably low. However, with the addition of new criteria, a consensus-building process, and training, these variables became highly reliable.

Novel conditional plasmids regulated by chemical switches provide versatile tools for genetic engineering in Escherichia coli.

Engineering bacterial genomes or foreign DNA cloned as bacterial artificial chromosomes (BACs) relies on usage of helper plasmids, which deliver the desired tools transiently into the bacteria to be modified. After the anticipated action is completed the helper plasmids need to be cured. To make this efficient, plasmids are used that are maintained by conditional amplicons or carry a counter-selection marker. Here, we describe new conditional plasmids that can be maintained or cured by using chemical induction or repression. Our method is based on the dependency of plasmids carrying ori6Kγ origin of replication on the presence of protein Π. Ori6Kγ based plasmids are tightly regulated conditional constructs, but they require usually special E. coli strains to operate. To avoid this, we placed the Π protein expression under the control of a co-expressed conditional repressor. Regulating the maintenance of plasmids with administration or removal of chemicals is fully compatible with any other conditional amplicons applied to date. Here, we describe methods for inducing sites specific recombination of BACs as an example. However, the same strategy might be used to construct appropriate helper plasmids for any other transient components of genome editing methodologies such as λred recombinases or CRISPR/Cas components.

[Use of serum haptoglobin concentration as an indicator in animal health monitoring of dairy cows]. / Nutzung der Haptoglobinkonzentration im Blutserum als Indikator im Tiergesundheitsmonitoring bei Milchkühen.

OBJECTIVE: To investigate the association between haptoglobin concentration in the blood and the occurrence of ketosis, selected clinical parameters as well as lameness in dairy cows. MATERIAL AND METHODS: The data was collected on 39 dairy farms in Bavaria over a period of 8 months. In 712 Simmental and Brown Swiss cows, clinical examinations as well as milk and blood samplings were performed between 10 and 30 days after calving. In these blood samples, the concentrations of non-esterified fatty acids (NEFA), ß-hydroxybutyric acid (BHBA) and haptoglobin (Hp) were determined. Analysis of the milk included milk constituents (fat, protein, urea, lactose and acetone), BHBA, NEFA and the somatic cell count (SCC). RESULTS: Significant correlations were found between increased Hp-concentration on the one hand and increased NEFA levels in blood and milk (p < 0.001), increased somatic cell count (p < 0.001), lameness (p < 0.001), as well as reduced lactose content (p < 0.001) and protein content in the milk (p = 0.001) on the other hand. Animals sampled during the warmer summer months showed significantly higher serum Hp-concentrations (p < 0,001). Heifers exhibited significantly higher Hp-values than multiparous individuals (p < 0.001). By dividing the examined cows into 4 clusters, a Hp-threshold value could be determined at 0.18 mg/ml. Combined with a SCC threshold of 40 500 cells/ml milk, the majority of animals with subclinical and clinical abnormalities could be identified. CONCLUSION AND CLINICAL RELEVANCE: Measurement of the Hp-concentration in blood is a pertinent approach in animal health monitoring during the postpartum period. In combination with evaluations of milk amount and contents, deviations from the physiological status may be recognized and affected individuals treated early on. Haptoglobin may be used to assess the health status of the individual animal as well as an indicator of herd health in the context of animal health monitoring.

Why and Where to Move the Tibial Tubercle: Indications and Techniques for Tibial Tubercle Osteotomy.

Patellofemoral disorders including pain and instability are common orthopedic problems, particularly in the adolescent population. Patellofemoral pain is usually anterior, poorly localized, and diffuse. Because of its multifactorial etiology, patellofemoral pain can be clinically challenging to diagnose and manage. With regards to instability, predisposing factors include trochlear dysplasia, patella alta, patellar tilt, and an elevated tibial tuberosity and trochlea groove distance. Initially, nonoperative management is recommended to treat patellofemoral maladies such as overload, maltracking, and acute first-time dislocations. However, tibial tubercle transfer (TTT) is commonly used to address cases of symptomatic malalignment and overload and recurrent patellar instability. The tubercle can be translated in multiplanar directions to correct patellar height, maltracking associated with instability, and to offload chondral defects. A thorough understanding of the anatomy and biomechanics of the patellofemoral joint is essential for optimizing results after TTT. Individualizing the direction and degree of tubercle transfer on the basis of patient parameters is critical to producing successful long-term results after surgery. This article will review the indications for performing a TTT and highlight the various techniques.

Herd health monitoring in dairy farms - discover metabolic diseases. An overview.

The transition period, 3 weeks before and 3 weeks after calving, is inevitably accompanied by a negative energy balance, which sometimes causes metabolic disturbances, such as ketosis. Subclinical ketosis (SCK) is defined as an increase in the ß-hydroxybutyrate (BHB) concentration to ≥ 1.2 mmol/l in the blood. According to a recent study, a value of ≥ 0.7 mmol/l of non-esterified fatty acids (NEFA) in the blood indicates the potential development of the poor metabolic adaption syndrome (PMAS). With a herd prevalence of 21 %, and an incidence of approximately 40 % within the first 2 weeks after calving, SCK is a relevant herd health problem. The milk yield decreases in the first 2 weeks postpartum by 3-5.3 kg/d for each ketotic cow, and the total milk reduction through the whole lactation period of 305 days averages 112 kg (SD 89 kg). Although the cow does not display any clinical signs of ketosis at this stage, the risk of developing associated production diseases like retained placenta, metritis, displaced abomasum, lameness and clinical ketosis increases and the expected performance in terms of milk production will decrease. The herd health status deteriorates and the risk for early culling increases. Another impact factor is the financial aspect, which includes costs for early death, reduced milk production, reproduction losses, and associated production diseases. In the literature, the calculated costs per SCK case vary between $ 78 and $ 289. The gold standard diagnostic test for SCK is the photometric measurement of BHB in blood. This method is accurate, but results are delayed due to the required laboratory analysis. There are also some rapid cow-side tests, i. e. urine or milk strip tests available to identify ketotic cows. The common disadvantage of these methods is that they are not suitable for herd health monitoring because of the need to collect samples from each cow manually and the high rates of false negative results. However, Fourier transform infrared spectroscopy is suitable for herd health monitoring. It is already being used for the analysis of milk composition. This inexpensive, rapid and simple technique has a specificity of 83.8 % and a sensitivity of 82.4 %. Therefore, FTIR is an early and easy method for detecting ketotic cows, that could help reduce financial and performance losses associated with ketosis.

Isolated Medial Patellofemoral Ligament Reconstruction for Patellar Instability Regardless of Tibial Tubercle-Trochlear Groove Distance and Patellar Height: Outcomes at 1 and 2 Years.

BACKGROUND: It is unclear which patients with recurrent patellar instability require a bony procedure in addition to medial patellofemoral ligament (MPFL) reconstruction. PURPOSE: To report 1- and 2-year outcomes of patients after isolated MPFL reconstruction performed for patellar instability regardless of patellar height, tibial tubercle-trochlear groove (TT-TG) distance, or trochlear dysplasia. STUDY DESIGN: Case series; Level of evidence, 4. METHODS: All patients with recurrent patellar instability and without significant unloadable chondral defects (Outerbridge grade IV), cartilage defects (especially inferior/lateral patella), previous failed surgery, or pain >50% as their chief complaint were prospectively enrolled beginning March 2014. All patients underwent primary, unilateral, isolated MPFL reconstruction regardless of concomitant bony pathology for treatment of recurrent patellar instability. Information on recurrent subjective instability, dislocations, ability to return to sport (RTS), and outcome scores was recorded at 1 and 2 years. TT-TG distance, patellar height (with the Caton-Deschamps index), and trochlear depth were measured. RESULTS: Ninety patients (77% female; mean ± SD age, 19.4 ± 5.6 years) underwent MPFL reconstruction between March 2014 and August 2017: 72 (80%) reached 1-year follow-up, and 47 (52.2%) reached 2-year follow-up (mean follow-up, 2.2 years). Mean TT-TG distance was 14.7 ± 5.4 mm (range, -2.2 to 26.8 mm); mean patellar height, 1.2 ± 0.11 mm (range, 0.89-1.45 mm); and mean trochlear depth, 1.8 ± 1.4 mm (range, 0.05-6.85 mm). Ninety-six percent of patients at 1 year and 100% at 2 years had no self-reported patellofemoral instability; 1 patient experienced a redislocation at 3.5 years. RTS rates at 1 and 2 years were 90% and 88%, respectively. Mean time to RTS was 8.8 months. All patients had clinically and statistically significant improvement in mean Knee injury and Osteoarthritis Outcome Score-Quality of Life (32.7 to 72.0, P < .001), mean International Knee Documentation Committee subjective form (51.4 to 82.6, P < .001), and mean Kujala score (62.2 to 89.5, P < .001). No difference existed between 1- and 2-year outcome scores (all P > .05). CONCLUSION: At early follow-up of 1 and 2 years, isolated MPFL reconstruction is an effective treatment for patellar instability and provides significant improvements in outcome scores with a low redislocation/instability rate regardless of bony pathologies, including TT-TG distance, Caton-Deschamps index, and trochlear dysplasia. Future data from this cohort will be used to assess long-term outcomes.

L-Erythrulose production with a multideletion strain of Gluconobacter oxydans.

Many ketoses or organic acids can be produced by membrane-associated oxidation with Gluconobacter oxydans. In this study, the oxidation of meso-erythritol to L-erythrulose was investigated with the strain G. oxydans 621HΔupp BP.8, a multideletion strain lacking the genes for eight membrane-bound dehydrogenases. First batch biotransformations with growing cells showed re-consumption of L-erythrulose by G. oxydans 621HΔupp BP.8 in contrast to resting cells. The batch biotransformation with 2.8 g L-1 resting cells of G. oxydans 621HΔupp BP.8 in a DO-controlled stirred-tank bioreactor resulted in 242 g L-1 L-erythrulose with a product yield of 99% (w/w) and a space-time yield of 10 g L-1 h-1. Reaction engineering studies showed substrate excess inhibition as well as product inhibition of G. oxydans 621HΔupp BP.8 in batch biotransformations. In order to overcome substrate inhibition, a continuous membrane bioreactor with full cell retention was applied for meso-erythritol oxidation with resting cells of G. oxydans 621HΔupp BP.8. At a mean hydraulic residence time of 2 h, a space-time yield of 27 g L-1 h-1 L-erythrulose was achieved without changing the product yield of 99% (w/w) resulting in a cell-specific product yield of up to 4.4 gP gX-1 in the steady state. The product concentration (54 g L-1 L-erythrulose) was reduced in the continuous biotransformation process compared with the batch process to avoid product inhibition.

Defining differences in patient characteristics between spasmodic dysphonia and laryngeal tremor.

OBJECTIVE: To compare presenting characteristics of patients with adductor spasmodic dysphonia (ADSD), ADSD with laryngeal tremor (ADSD + LT), and laryngeal tremor without ADSD (LT). DESIGN: Cross-sectional analysis. METHODS: Patients treated for laryngeal movement disorders (1990-2016) were included. Analysis of variance and chi square tests measured differences in patient characteristics across the three disease groups. Using ADSD as the referent, multivariable logistic regression models were used to determine whether potential risk factors including patient demographics, family history, presence of potential inciting events prior to disease onset, and coprevalent movement disorders were associated with ADSD + LT or LT. RESULTS: In all, 652 patients with ADSD (n = 377), ADSD + LT (n = 98), and LT (n = 177) were included. ADSD patients were significantly younger than those with ADSD + LT and LT (52.5 ± 13.4, 63.9 ± 11.3, and 69.3 ± 10.5 years, respectively; P < 0.001). Coprevalent movement disorders were more common in ADSD + LT (38.7%) and LT (57.1%) groups than in the ADSD group (11.5%; P < 0.001). Compared to ADSD, patients with ADSD + LT and LT were more likely to develop an additional movement disorder during follow-up. In multivariable analyses, increasing age, female gender, and having a movement disorder at presentation were associated with significantly greater odds of having ADSD + LT or LT when compared to ADSD. CONCLUSION: ADSD + LT patients demonstrate intermediate gender composition and age distributions between those with ADSD and LT. These findings suggest that ADSD + LT may be a distinct phenotype in the spectrum of laryngeal movement disorders. LEVEL OF EVIDENCE: 4 Laryngoscope, 129:170-176, 2019.

MPFL in First-Time Dislocators.

PURPOSE OF REVIEW: Historically, the standard of care for patients with an acute patella dislocation has been non-operative with the exception being those with a loose body or osteochondral fracture requiring fixation or removal. RECENT FINDINGS: Recent literature has brought into question this standard of care approach and defined a higher risk subset of first-time dislocators who may benefit from early operative treatment. In addition, these studies suggest that operative treatment not only reduces the risk of recurrence but may improve outcomes overall and specifically in the pediatric population. Though the "high risk" population of first-time dislocators has been more clearly defined, how we treat them remains controversial. We continue to need more evidence-based guidelines to help us manage who we should be fixing and how we should be fixing them. We currently have several multi-center studies in progress, including one specifically looking at the question of medial patellofemoral ligament reconstruction in first-time pediatric and adolescent dislocators.

Comparative analysis based on replication banding reveals the mechanism responsible for the difference in the karyotype constitution of treefrogs Ololygon and Scinax (Arboranae, Hylidae, Scinaxinae).

According to the recent taxonomic and phylogenetic revision of the family Hylidae, species of the former Scinax catharinae (Boulenger, 1888) clade were included in the resurrected genus Ololygon Fitzinger, 1843, while species of the Scinax ruber (Laurenti, 1768) clade were mostly included in the genus Scinax Wagler, 1830, and two were allocated to the newly created genus Julianus Duellman et al., 2016. Although all the species of the former Scinax genus shared a diploid number of 2n = 24 and the same fundamental number of chromosome arms of FN = 48, two karyotypic constitutions were unequivocally recognized, related mainly to the distinct size and morphology of the first two chromosome pairs. Some possible mechanisms for these differences had been suggested, but without any experimental evidence. In this paper, a comparison was carried out based on replication chromosome banding, obtained after DNA incorporation of 5-bromodeoxiuridine in chromosomes of Ololygon and Scinax. The obtained results revealed that the loss of repetitive segments in chromosome pairs 1 and 2 was the mechanism responsible for karyotype difference. The distinct localization of the nucleolus organizer regions in the species of both genera also differentiates the two karyotypic constitutions.

Possible interspecific origin of the B chromosome of Hypsiboas albopunctatus (Spix, 1824) (Anura, Hylidae), revealed by microdissection, chromosome painting, and reverse hybridisation.

The B chromosome in the hylid Hypsiboas albopunctatus (2n = 22 + B) is small, almost entirely composed of C-positive heterochromatin, and does not pair with any chromosome of the A complement. B probe, obtained by microdissection and DOP-PCR amplification, was used to search for homology between the B and regular chromosomes of H. albopunctatus and of the related species H. raniceps (Cope, 1862). Reverse hybridisation was also carried out in the investigation. The B probe exclusively painted the supernumerary, not hybridising any other chromosomes in H. albopunctatus, but all H. raniceps chromosomes showed small labelling signals. This result might be an indication that differences exist between the repetitive sequences of A and B chromosomes of H. albopunctatus, and that the chromosomes of H. raniceps and the heterochromatin of the B chromosome of H. albopunctatus are enriched with the same type of repetitive DNA. In meiotic preparations, the B labelled about 30% of scored spermatids, revealing a non-mendelian inheritance, and the painted B in micronucleus suggests that the supernumerary is eliminated from germ line cells. Although our results could suggest an interespecific origin of the B at first sight, further analysis on its repetitive sequences is still necessary. Nevertheless, the accumulation of repetitive sequences, detected in another species, even though closely related, remains an intriguing question.

Cytogenetic analysis of Phyllomedusa distincta Lutz, 1950 (2n = 2x = 26), P. tetraploidea Pombal and Haddad, 1992 (2n = 4x = 52), and their natural triploid hybrids (2n = 3x = 39) (Anura, Hylidae, Phyllomedusinae).

BACKGROUND: Natural polyploidy has played an important role during the speciation and evolution of vertebrates, including anurans, with more than 55 described cases. The species of the Phyllomedusa burmeisteri group are mostly characterized by having 26 chromosomes, but a karyotype with 52 chromosomes was described in P. tetraploidea. This species was found in sintopy with P. distincta in two localities of São Paulo State (Brazil), where triploid animals also occur, as consequence of natural hybridisation. We analyse the chromosomes of P. distincta, P. tetraploidea, and their triploid hybrids, to enlighten the origin of polyploidy and to obtain some evidence on diploidisation of tetraploid karyotype. RESULTS: Phyllomedusa distincta was 2n = 2x = 26, whereas P. tetraploidea was 2n = 4x = 52, and the hybrid individuals was 2n = 3x = 39. In meiotic phases, bivalents were observed in the diploid males, whereas both bivalents and tetravalents were observed in the tetraploid males. Univalents, bivalents or trivalents; metaphase II cells carrying variable number of chromosomes; and spermatids were detected in the testis preparations of the triploid males, indicating that the triploids were not completely sterile. In natural and experimental conditions, the triploids cross with the parental species, producing abnormal egg clutches and tadpoles with malformations. The embryos and tadpoles exhibited intraindividual karyotype variability and all of the metaphases contained abnormal constitutions. Multiple NORs, detected by Ag-impregnation and FISH with an rDNA probe, were observed on chromosome 1 in the three karyotypic forms; and, additionally, on chromosome 9 in the diploids, mostly on chromosome 8 in the tetraploids, and on both chromosome 8 and 9 in the triploids. Nevertheless, NOR-bearing chromosome 9 was detected in the tetraploids, and chromosome 9 carried active or inactive NORs in the triploids. C-banding, base-specific fluorochrome stainings with CMA3 and DAPI, FISH with a telomeric probe, and BrdU incorporation in DNA showed nearly equivalent patterns in the karyotypes of P. distincta, P. tetraploidea, and the triploid hybrids. CONCLUSIONS: All the used cytogenetic techniques have provided strong evidence that the process of diploidisation, an essential step for stabilising the selective advantages produced by polyploidisation, is under way in distinct quartets of the tetraploid karyotype.

Cytogenetic analyses of eight species in the genus Leptodactylus Fitzinger, 1843 (Amphibia, Anura, Leptodactylidae), including a new diploid number and a karyotype with multiple translocations.

BACKGROUND: The karyotypes of Leptodactylus species usually consist of 22 bi-armed chromosomes, but morphological variations in some chromosomes and even differences in the 2n have been reported. To better understand the mechanisms responsible for these differences, eight species were analysed using classical and molecular cytogenetic techniques, including replication banding with BrdU incorporation. RESULTS: Distinct chromosome numbers were found: 2n = 22 in Leptodactylus chaquensis, L. labyrinthicus, L. pentadactylus, L. petersii, L. podicipinus, and L. rhodomystax; 2n = 20 in Leptodactylus sp. (aff. podicipinus); and 2n = 24 in L. marmoratus. Among the species with 2n = 22, only three had the same basic karyotype. Leptodactylus pentadactylus presented multiple translocations, L. petersii displayed chromosome morphological discrepancy, and L. podicipinus had four pairs of telocentric chromosomes. Replication banding was crucial for characterising this variability and for explaining the reduced 2n in Leptodactylus sp. (aff. podicipinus). Leptodactylus marmoratus had few chromosomes with a similar banding patterns to the 2n = 22 karyotypes. The majority of the species presented a single NOR-bearing pair, which was confirmed using Ag-impregnation and FISH with an rDNA probe. In general, the NOR-bearing chromosomes corresponded to chromosome 8, but NORs were found on chromosome 3 or 4 in some species. Leptodactylus marmoratus had NORs on chromosome pairs 6 and 8. The data from C-banding, fluorochrome staining, and FISH using the telomeric probe helped in characterising the repetitive sequences. Even though hybridisation did occur on the chromosome ends, telomere-like repetitive sequences outside of the telomere region were identified. Metaphase I cells from L. pentadactylus confirmed its complex karyotype constitution because 12 chromosomes appeared as ring-shaped chain in addition to five bivalents. CONCLUSIONS: Species of Leptodactylus exhibited both major and minor karyotypic differences which were identified by classical and molecular cytogenetic techniques. Replication banding, which is a unique procedure that has been used to obtain longitudinal multiple band patterns in amphibian chromosomes, allowed us to outline the general mechanisms responsible for these karyotype differences. The findings also suggested that L. marmoratus, which was formerly included in the genus Adenomera, may have undergone great chromosomal repatterning.