Improved Training and Semen Collection Outcomes Using the Closed Box Chair for Macaques.

Collaborative semen collection in monkeys is a valuable tool in research, animal collection management, and conservation efforts. To obtain samples, monkeys are often restrained in open restraint chairs (ORC) with the "pole and collar" technique. While commonly used, this restraint is not tolerated by all individuals; some become anxious or aggressive towards the poles and people. In an effort to refine this procedure and improve welfare of the monkeys, we examined the use of a "closed box chair" (CBC), a clear, plexiglass box in which the monkey is trained to sit for sperm collection. The CBC does not require pole and collar, and although legs are secured, the arms and neck are not restrained. The use of CBCs has increased in recent years; however, there are few studies demonstrating its effects on scientific outcomes. We used positive reinforcement techniques to train 34 adult male rhesus macaques (Macaca mulatta) to provide semen samples using either the ORC or the CBC. While all CBC monkeys (n = 14) were reliably trained for this procedure, only 75% of ORC (n = 20) males completed the training (p = 0.04). It took significantly less time to train animals in the CBC than the ORC (201.0 vs. 412.4 min; p <0.001). In a controlled subset, males restrained with ORC (n = 7) produced a significantly lower ejaculatory volume than those collected by CBC (n = 10) (297.6 µL vs. 522.1 µL respectively; p = 0.04) and had a lower concentration of sperm (186.0 × 106/mL vs. 367.5 × 106/mL respectively; p = 0.017), although there were no differences with respect to sperm motility (p = 0.15). Our data suggest the closed box chair technique reduces stress on the animals while enhancing semen quality, supporting the use of the CBC as an important refinement.

Cryopreservation and Preparation of Thawed Spermatozoa from Rhesus Macaques (Macaca mulatta) for In Vitro Fertilization.

Advances in assisted reproductive technologies in rhesus macaques have allowed the development of valuable models of human disease, particularly when combined with recent techniques for gene editing. While the ability to perform in vitro fertilization (IVF) in rhesus macaques is well established, this procedure has not yet been optimized. Specifically, damage to the sperm caused by cryopreservation (cryodamage) may lead to unsuccessful artificial insemination and low fertilization and blastocyst formation rates in vitro. To address this, we systematically assessed 2 cryopreservation methods and 4 recovery methods in the following 3 interdependent experiments: 1) comparing sperm survival after vitrification or slow-freezing; 2) comparing simple wash (SW), density gradient centrifugation (DGC), swim-up (SU), and glass wool filtration (GWF) for removal of cryoprotectants and isolation of motile sperm after thawing; and 3) evaluating the efficacy for IVF of the 2 best methods of isolating thawed sperm. We found that after vitrification, only 1.2 ± 0.3% of thawed sperm were motile, whereas after slow-freezing, 42 ± 5% of thawed sperm were motile. SW was significantly better than all other isolation methods for the recovery of total sperm and for the recovery of sperm with an intact plasma membrane. The isolation methods had no significant differences in the recovery of motile sperm or sperm with progressive motility. However, IVF of ova with sperm recovered by DGC resulted in 5% more embryos and 25% more blastocysts than did IVF with sperm recovered by SW. Although additional studies are required to optimize sperm cryopreservation in rhesus macaques, our study showed that slow-freezing, coupled with DGC, provided the highest efficacy in providing functional sperm for in vitro use.

Thrombectomy for Stroke in the Public Health Care System of Brazil.

BACKGROUND: Randomized trials involving patients with stroke have established that outcomes are improved with the use of thrombectomy for large-vessel occlusion. These trials were performed in high-resource countries and have had limited effects on medical practice in low- and middle-income countries. METHODS: We studied the safety and efficacy of thrombectomy in the public health system of Brazil. In 12 public hospitals, patients with a proximal intracranial occlusion in the anterior circulation that could be treated within 8 hours after the onset of stroke symptoms were randomly assigned in a 1:1 ratio to receive standard care plus mechanical thrombectomy (thrombectomy group) or standard care alone (control group). The primary outcome was the score on the modified Rankin scale (range, 0 [no symptoms] to 6 [death]) at 90 days. RESULTS: A total of 300 patients were enrolled, including 79 who had undergone thrombectomy during an open-label roll-in period. Approximately 70% in the two groups received intravenous alteplase. The trial was stopped early because of efficacy when 221 of a planned 690 patients had undergone randomization (111 to the thrombectomy group and 110 to the control group). The common odds ratio for a better distribution of scores on the modified Rankin scale at 90 days was 2.28 (95% confidence interval [CI], 1.41 to 3.69; P = 0.001), favoring thrombectomy. The percentage of patients with a score on the modified Rankin scale of 0 to 2, signifying an absence of or minor neurologic deficit, was 35.1% in the thrombectomy group and 20.0% in the control group (difference, 15.1 percentage points; 95% CI, 2.6 to 27.6). Asymptomatic intracranial hemorrhage occurred in 51.4% of the patients in the thrombectomy group and 24.5% of those in the control group; symptomatic intracranial hemorrhage occurred in 4.5% of the patients in each group. CONCLUSIONS: In this randomized trial conducted in the public health care system of Brazil, endovascular treatment within 8 hours after the onset of stroke symptoms in conjunction with standard care resulted in better functional outcomes at 90 days than standard care alone. (Funded by the Brazilian Ministry of Health; RESILIENT ClinicalTrials.gov number, NCT02216643.).

Pathogen-specific epitopes as epidemiological tools for defining the magnitude of Mycobacterium leprae transmission in areas endemic for leprosy.

During recent years, comparative genomic analysis has allowed the identification of Mycobacterium leprae-specific genes with potential application for the diagnosis of leprosy. In a previous study, 58 synthetic peptides derived from these sequences were tested for their ability to induce production of IFN-γ in PBMC from endemic controls (EC) with unknown exposure to M. leprae, household contacts of leprosy patients and patients, indicating the potential of these synthetic peptides for the diagnosis of sub- or preclinical forms of leprosy. In the present study, the patterns of IFN-γ release of the individuals exposed or non-exposed to M. leprae were compared using an Artificial Neural Network algorithm, and the most promising M. leprae peptides for the identification of exposed people were selected. This subset of M. leprae-specific peptides allowed the differentiation of groups of individuals from sites hyperendemic for leprosy versus those from areas with lower level detection rates. A progressive reduction in the IFN-γ levels in response to the peptides was seen when contacts of multibacillary (MB) patients were compared to other less exposed groups, suggesting a down modulation of IFN-γ production with an increase in bacillary load or exposure to M. leprae. The data generated indicate that an IFN-γ assay based on these peptides applied individually or as a pool can be used as a new tool for predicting the magnitude of M. leprae transmission in a given population.

Reference intervals of plasma calcium, phosphorus, and magnesium for African grey parrots (Psittacus erithacus) and Hispaniolan parrots (Amazona ventralis).

Calcium (Ca), phosphorus (P), and magnesium (Mg) are important elements for body homeostasis in several diseases associated with imbalances in the plasma concentration of these ions. This is the first published report of reference intervals for Mg in association with Ca and P levels for psittacine species. One milliliter of blood was collected from 26 Hispaniolan parrots (Amazona ventralis) and 24 African grey parrots (Psittacus erithacus). The plasma concentrations of Ca, P, and Mg were determined for each sample. Statistical analyses were performed including all data (analysis 1) and after exclusion of the subjects with Ca > or = 14.00 mg/dl (3.5 mmol) (analysis 2). The data from analysis 1 have a narrower interval than that observed in analysis 2. Following the normality test (Shapiro-Wilk, alpha = 0.05), the univariate and mean procedures were run. For the reference intervals, the lower and upper values were used, after elimination of the outliers calculated by Blom scores from the ranked variables. The analysis 1 references for the Hispaniolans were Ca = 8.80-10.40 mg/dl (2.20-2.60 mmol/L), P = 1.80-4.40 mg/dl (0.58-1.42 mmol/L), Mg = 1.80-3.10 mg/dl (0.74-1.27 mmol/L), and Ca:P ratio = 2.62-5.39; for the African greys analysis 1 references were Ca = 8.20-20.20 mg/dl (2.05-5.05 mmol/L), P = 2.50-5.90 mg/dl (0.81-1.91 mmol/L), Mg = 2.10-3.40 mg/dl (0.82-1.4 mmol/L), and Ca:P ratio = 1.81-3.77. The analysis 2 references for the Hispaniolans were Ca = 8.80-10.30 mg/dl (2.20-2.58 mmol/L), P = 1.80-3.80 mg/dl (0.58-1.23 mmol/L), Mg = 1.90-3.00 mg/dl (0.82-1.07 mmol/L), Ca:P ratio = 2.62-5.39; for the African greys analysis 2 references were Ca = 1.07 mmol/L), Ca:P ratio = 1.67-3.50. The results of this study are important for evaluating Mg concentrations in relation to the Ca and P parameters in psittacines. This information will be particularly helpful for veterinarians evaluating the hypocalcemic syndrome in African grey parrots and other disease processes associated with Ca, P, and Mg physiologic imbalances.

Carbonyl cyanide m-chlorophenylhydrazone induced calcium signaling and activation of plasma membrane H(+)-ATPase in the yeast Saccharomyces cerevisiae.

The plasma membrane H(+)-ATPase from Saccharomyces cerevisiae is an enzyme that plays a very important role in the yeast physiology. The addition of protonophores, such as 2,4-dinitrophenol (DNP) and carbonyl cyanide m-chlorophenylhydrazone (CCCP), also triggers a clear in vivo activation of this enzyme. Here, we demonstrate that CCCP-induced activation of the plasma membrane H(+)-ATPase shares some similarities with the sugar-induced activation of the enzyme. Phospholipase C and protein kinase C activities are essential for this activation process while Gpa2p, a G protein involved in the glucose-induced activation of the ATPase, is not required. CCCP also induces a phospholipase C-dependent increase in intracellular calcium. Moreover, we show that the availability of extracellular calcium is required for CCCP stimulation of H(+)-ATPase, suggesting a possible connection between calcium signaling and activation of ATPase.