Intracytoplasmic injection of frozen-thawed epididymal spermatozoa in a nonhuman primate model,the cynomolgus monkey (Macaca fascicularis)

Intracytoplasmic sperm injection (ICSI) with frozen-thawed epididymal spermatozoa was performed in the cynomolgus monkey (Macacafascicularis) to produce embryos in vitro. Eleven sexually mature females were hyperstimulated with an GnRH agonist (1.8 mg active triptorelin per 2 kg body weight), followed (2 weeks later) by rFSH (37.5 IU per 2 kg daily) for 12 days, and finally 1000 IU of hCG. Epididymal spermatozoa were collected from a single adult male monkey. The first stimulation cycle resulted in 90 oocytes; 70% of which were metaphase II (MII). Sixty-four percent of these MII oocytes were fertilized. Comparing ovarian response of five monkeys that underwent a second stimulation cycle there was an increase in oocyte quantity (13.2 versus 9.2 oocytes per monkey) but the percentage of MII oocytes remained the same at 58%. Fertilization and cleavage rates were also reduced but there was an increase in the number of embryos available for transfer. Overall, four monkeys became pregnant resulting in the birth of two healthy infants and two abortions. These findings show that ovarian stimulation by GnRH-rFSH did not compromise the developmental competence of the oocytes; effective combination of cryopreservation of epididymal spermatozoa and ICSI is possible in nonhuman primate reproduction, and thus has potential application in the conservation of highly endangered nonhuman primate species, and the cynomolgus monkey is a reliable biomedical research model to study the potential risks and benefits associated with assisted reproductive techniques prior to approval for clinical trials on humans.     

Extraosseous osteosarcoma in a nonhuman primate (Macaca mulatta)

A case of a very rare malignant bone-forming sarcoma, which was located in the soft tissue but had no anatomic attachment to bone, is presented. This tumor originated in the subcuticular tissue over the right scapula and was histologically composed of sheets of anaplastic spindle cells that produced osteoid. The tumor recurred twice before the animal was killed. The recurrent masses produced less osteoid and were generally less differentiated histologically than the initial tumor.     

Androgen receptors in the fetus of a primate (Macaca fascicularis) : preliminary results

Skin fibroblasts raised from Macaca fascicularis skin samples were tested for specific androgen receptors. By the 100th day of gestation male and female skin fibroblasts androgen receptors were detected. Higher levels of androgen receptors were found in sex skin fibroblasts. These preliminary results suggest that this primate could be used as a good model for the study of androgen receptors ontogenesis and regulation.     

Alkaptonuria in a cynomolgus monkey (Macaca fascicularis)

An eight year old wild caught Cynomolgus monkey was diagnosed as having alkaptonuria, a condition characterized by the passage of a normal-colored urine which darkens upon standing. The underlying cause is the congenital lack of homogentisic acid oxidase with subsequent passage of homogentisic acid in the urine. No other clinical manifestations, such as deposition of pigment in the skin or mucous membranes or development of an ochronitic arthritis, were observed in this animal.     

Measuring pad-pad pinch strength in a non-human primate: Macaca fascicularis.

The primary purpose of this study was to establish a methodology for determining and perhaps predicting (via regression analysis of anthropometric measures) Macaca fascicularis isometric pinch strength for a specific task. The larger purpose of this work was to properly scale a pinching task for the monkeys in order to study dose-response relationships in a non-human primate model for carpal tunnel syndrome. Three female and one male macaque (n = 4) of varying size and age were trained to perform a left-handed pad–pad pinch. The task required 60° of wrist flexion at a static pinching distance of 3 cm between the thumb and fingers. Subjects were trained for a period of 20-weeks. After that time, an analysis of performance gradients found that they had each reached a plateau in their force output. Pinch strength for the four animals ranged from 29.4 to 59.8 N. Regression analysis revealed that body mass (kg) and wrist circumference (cm) were both predictive of pinch strength, exhibiting adjusted R² values of 0.93 (p = 0.024) and 0.96 (p = 0.015), respectively. Thus, the results suggest that maximal pinch strength could be acceptably estimated in future subjects using either the wrist circumference or the body mass measures, as both were strong predictors of pad–pad pinch strength.     

Ependymal cyst in a cynomolgus macaque (Macaca fascicularis)

Background A focal hypointense intracranial lesion was detected by magnetic resonance imaging in the right caudal occipital lobe of the cerebrum in an asymptomatic cynomolgus macaque (Macaca fascicularis). Results Following euthanasia, gross evaluation of the lesion revealed a 1 cm diameter, wedge‐shaped intracranial cavitation without apparent communication with the ventricles. Histologically, the lesion was lined by ciliated cuboidal to low columnar epithelium that showed immunopositivity for cytokeratin, S‐100, and GFAP. Conclusion Based upon the gross, histologic, and immunohistochemical findings, this lesion was classified as an ependymal cyst. In the human patients, ependymal cysts are benign, non‐infectious, non‐inflammatory lesions of the central nervous system that are believed to originate from aberrant migration of ependymal cells during development. To our knowledge, this is the first report of an ependymal cyst in a non‐rodent veterinary species and the first report of a benign intracranial cyst of any classification in a non‐human primate.     

Ameloblastic odontoma in a cynomolgus monkey (Macaca fascicularis)

An ameloblastic odontoma occurred in an adult cynomolgus monkey (Macaca fascicularis). The neoplasm involved the upper left maxilla as a disfiguring, fleshy growth. The tissue and cellular changes were consistent with an ameloblastic odontoma which is rare in man, nonhuman primates (NHP), and other vertebrate animals. The monkey was one of 50 adult, single-caged housed cynomolgus monkeys. No additional clinical signs of disease were present. Hematology and serum chemistries were within normal limits. There have been three reports in the literature of ameloblastic odontomas in NHP; however, this is the first reported case of an ameloblastic odontoma in a cynomolgus monkey.     

Spontaneous endometriosis in a cynomolgus monkey (Macaca fascicularis)

An adult female Macaca fascicularis developed anorexia and minimal fecal production. Abdominal palpation was unrewarding. Rectal palpation revealed a rectal stricture and a large soft tissue mass in the area of the uterus. Clinical pathology tests were within normal limits. Exploratory surgery and histopathology confirmed a diagnosis of endometriosis.     

Experimental Infection of Cynomolgus Macaques (Macaca fascicularis) with Aerosolized Monkeypox Virus

Monkeypox virus (MPXV) infection in humans results in clinical symptoms very similar to ordinary smallpox. Aerosol is a route of secondary transmission for monkeypox, and a primary route of smallpox transmission in humans. Therefore, an animal model for aerosol exposure to MPXV is needed to test medical countermeasures. To characterize the pathogenesis in cynomolgus macaques (Macaca fascicularis), groups of macaques were exposed to four different doses of aerosolized MPXV. Blood was collected the day before, and every other day after exposure and assessed for complete blood count (CBC), clinical chemistry analysis, and quantitative PCR. Macaques showed mild anorexia, depression, and fever on day 6 post-exposure. Lymphadenopathy, which differentiates monkeypox from smallpox, was observed in exposed macaques around day 6 post-exposure. CBC and clinical chemistries showed abnormalities similar to human monkeypox cases. Whole blood and throat swab viral loads peaked around day 10, and in survivors, gradually decreased until day 28 post-exposure. Survival was not dose dependent. As such, doses of 4×10⁴ PFU, 1×10⁵ PFU, or 1×10⁶ PFU resulted in lethality for 70% of the animals, whereas a dose of 4×10⁵ PFU resulted in 85% lethality. Overall, cynomolgus macaques exposed to aerosolized MPXV develop a clinical disease that resembles that of human monkeypox. These findings provide a strong foundation for the use of aerosolized MPXV exposure of cynomolgus macaques as an animal model to test medical countermeasures against orthopoxviruses.     

Fatal pneumococcal meningitis in a colony-born monkey (Macaca fascicularis)

This report describes the clinical course and pathological findings in a four-year-old male cynomolgus monkey (Macaca fascicularis) which succumbed to fulminant pneumococcal meningitis. The extremely rapid course of this infection emphasizes the importance of early recognition of the clinical manifestations of meningitis in the correct diagnosis and rapid treatment of the condition.     

Experimental infection of Cynomolgus Macaques (Macaca fascicularis) with human varicella-zoster virus

Varicella-zoster virus (VZV) is a member of the alphaherpesvirus family and the causative agent of chickenpox and shingles. To determine the utility of cynomolgus macaques (Macaca fascicularis) as a nonhuman primate model to evaluate VZV-based simian immunodeficiency virus/human immunodeficiency virus (SIV/HIV) vaccines, we experimentally inoculated 10 animals with the parental Oka (Oka-P) strain of VZV derived from MeWo or Telo-RF cells. VZV DNA could be detected in the lungs as late as 4 days postinfection, with replicating virus detected by shell vial culture assay in one case. Infection did not result in any overt clinical symptoms but was characterized by humoral and cell-mediated immunity in a time frame and at a magnitude similar to those observed following VZV vaccination in humans. The cell line source of VZV inoculum influenced both the magnitude and polyfunctionality of cell-mediated immunity. Animals mounted a vigorous anamnestic antibody response following a second inoculation 12 weeks later. Inoculations resulted in transient increases in CD4(+) T-cell activation and proliferation, as well as a sustained increase in CD4(+) T cells coexpressing CCR5 and α4β7 integrin. In contrast to previous failed attempts to successfully utilize attenuated VZV-Oka as an SIV vaccine vector in rhesus macaques due to suboptimal infectivity and cellular immunogenicity, the ability to infect cynomolgus macaques with Oka-P VZV should provide a valuable tool for evaluating VZV-vectored SIV/HIV vaccines.     

Mitogenomic phylogeny of the common long-tailed macaque (Macaca fascicularis fascicularis)

Background

Long-tailed macaques (Macaca fascicularis) are an important model species in biomedical research and reliable knowledge about their evolutionary history is essential for biomedical inferences. Ten subspecies have been recognized, of which most are restricted to small islands of Southeast Asia. In contrast, the common long-tailed macaque (M. f. fascicularis) is distributed over large parts of the Southeast Asian mainland and the Sundaland region. To shed more light on the phylogeny of M. f. fascicularis, we sequenced complete mitochondrial (mtDNA) genomes of 40 individuals from all over the taxon’s range, either by classical PCR-amplification and Sanger sequencing or by DNA-capture and high-throughput sequencing.

Results

Both laboratory approaches yielded complete mtDNA genomes from M. f. fascicularis with high accuracy and/or coverage. According to our phylogenetic reconstructions, M. f. fascicularis initially diverged into two clades 1.70 million years ago (Ma), with one including haplotypes from mainland Southeast Asia, the Malay Peninsula and North Sumatra (Clade A) and the other, haplotypes from the islands of Bangka, Java, Borneo, Timor, and the Philippines (Clade B). The three geographical populations of Clade A appear as paraphyletic groups, while local populations of Clade B form monophyletic clades with the exception of a Philippine individual which is nested within the Borneo clade. Further, in Clade B the branching pattern among main clades/lineages remains largely unresolved, most likely due to their relatively rapid diversification 0.93-0.84 Ma.

Conclusions

Both laboratory methods have proven to be powerful to generate complete mtDNA genome data with similarly high accuracy, with the DNA-capture and high-throughput sequencing approach as the most promising and only practical option to obtain such data from highly degraded DNA, in time and with relatively low costs. The application of complete mtDNA genomes yields new insights into the evolutionary history of M. f. fascicularis by providing a more robust phylogeny and more reliable divergence age estimations than earlier studies.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1437-0) contains supplementary material, which is available to authorized users.     

Neuronal ceroid-lipofuscin storage in a cynomolgus monkey (Macaca fascicularis)

The light and electron microscopical characteristics of non-age-related neuronal ceroid-lipofuscinosis in a young cynomolgus monkey (Macaca fascicularis) are described. The pigment in the neuronal cytoplasm was PAS-positive, sudanophilic, weakly acid-fast, and positive with Schmorl's and aldehydefuchsin stains for lipofuscin. Ultra-structurally, it appeared as membrane-bound, electron-dense aggregates within the cytoplasm of neurones of both brain and spinal cord.     

Polyostotic fibrous dysplasia in a cynomolgus Macaque (Macaca fascicularis)

A 2.3-y-old female cynomolgus macaque (Macaca fascicularis) presented with a broken right tibia and fibula. Radiographs showed multiple cyst-like defects in all long bones. We suspected that both fractures were pathologic because they occurred through these defects. Ultrasonography, MRI, and dual X-ray absorptiometry revealed that the defects were filled with soft tissue. Grossly, the bones were abnormal in shape, and a gelatinous material filled the defects and the surrounding marrow cavity. Histologically, the gelatinous material was composed of fibrin and cartilage; few normal bone cells were seen. Genetic testing revealed extra material on the short arm of chromosome 8 in all tissues examined, but no copy number alterations of likely clinical significance were observed, and no abnormalities were found that were unique to the lesions. In light of the clinical signs and radiographic and pathologic findings, polyostotic fibrous dysplasia was diagnosed. This report represents the first documented case of fibrous dysplasia in a cynomolgus macaque.