Preliminary findings of age and male sexual characteristics andand potential effect to semen characteristics and cryopreservation of the critically endangered Bornean orangutan in Malaysia.

Bornean orangutan is a critically endangered non-human primate; however, the threat of extinction is not merely from poaching and habitat loss. Orangutan survival is also threatened by the genetic loss and genetic bottleneck due to the low effective population, prompting the dire need for an immediate genetic preservation program through systematic biobanking and assisted reproductive technology (ART). This study aims to provide integral data to the semen characteristics, extension, and cryopreservation of the Bornean orangutan and the potential relationship to male traits. Five captive orangutans from Sepilok Orangutan Rehabilitation Centre (SORC) with a mean body weight of 52.81 ± 7.00 kg were used for this study. Semen collection was performed using electroejaculation (EE) under complete general anesthesia. Semen was subjected to macroscopic and microscopic evaluation while testicular measurement was obtained using digital calipers. The semen characteristics of the orangutans are volume (778 ± 250.21 µl), pH (7.80 ± 0.25), concentration (32.38 ± 17.40 × 106 sperm/ml), total motility (61.00 ± 12.88%), adjusted motility index (48.76 ± 11.32%), live spermatozoa (77.75 ± 6.94%) and normal spermatozoa (11.48 ± 11.34%). Analysis of variance statistical analysis test was used to compare the significant difference between means, at (p < 0.05). Spermatozoa concentration was the only significant different parameter between individuals. Testes biometry parameters are statistically significant between the flanged and unflanged individuals. Live spermatozoa are different in adult and sub-adult individual while teratospermia was found to be consistently high in all individuals. Chilled and post-thaw quality after cryopreservation suggests promising survivability of spermatozoa. Semen collection with EE yields a consistent and acceptable quality of spermatozoa for cryopreservation, biobanking purposes, and potential application of ART.

A Preliminary Study on Semen Collection, Its Evaluation, and Testicular and Sperm Morphometries in The Wild Proboscis Monkey (Nasalis Larvatus).

INTRODUCTION: The proboscis monkey (Nasalis larvatus) is an endangered species with a declining population. This article describes the first successful attempt at sperm collection and evaluation, and the testicular and sperm morphometries of the wild proboscis monkey in Sabah, Malaysia. MATERIAL AND METHODS: Eight semen collection procedures using electro-ejaculation and digital manipulation were conducted in three wild adult male proboscis monkeys. A total of 21 ejaculates were collected. The testicular biometry was measured with the aid of ultrasonography. Sample evaluation included semen volume and pH and sperm concentration, viability, and abnormality. The sperm morphometry was undertaken using phase contrast microscopy. RESULTS: The mean (±SD) total testicular volume of these animals was 5.77 cm3 (±1.58). Semen collection by electro-ejaculation resulted in an 84% success rate, while digital manipulation did not result in any ejaculation. Each animal showed different semen characteristics, where the volume was 5-540 µL, pH 8-9, and sperm concentration 0.041-83.00 ×106/mL. The percentage of abnormal sperm was high at 76.8% (±89.60), largely due to midpiece abnormality. Normal sperm had a spherical head and long tail with a head : midpiece : tail length ratio of 1 : 2: 8. CONCLUSION: The social status of these animals may contribute to the generally low quality of the semen. The techniques and data from this study are useful for future conservation and application of assisted reproductive technology in this species.

The ART of bringing extinction to a freeze - History and future of species conservation, exemplified by rhinos.

The ongoing mass extinction of animal species at an unprecedented rate is largely caused by human activities. Progressive habitat destruction and fragmentation is resulting in accelerated loss of biodiversity on a global scale. Over decades, captive breeding programs of non-domestic species were characterized by efforts to optimize species-specific husbandry, to increase studbook-based animal exchange, and to improve enclosure designs. To counter the ongoing dramatic loss of biodiversity, new approaches are warranted. Recently, new ideas, particularly the application of assisted reproduction technologies (ART), have been incorporated into classical zoo breeding programs. These technologies include semen and oocyte collection, artificial insemination, and in-vitro embryo generation. More futuristic ideas of advanced ART (aART) implement recent advances in biotechnology and stem-cell related approaches such as cloning, inner cell mass transfer (ICM), and the stem-cell-associated techniques (SCAT) for the generation of gametes and ultimately embryos of highly endangered species, such as the northern white rhinoceros (Ceratotherium simum cottoni) of which only two female individuals are left. Both, ART and aART greatly depend on and benefit from the rapidly evolving cryopreservation techniques and biobanking not only of genetic, but also of viable cellular materials suitable for the generation of induced pluripotent stem cells (iPSC). The availability of cryopreserved materials bridges gaps in time and space, thereby optimizing the available genetic variability and enhancing the chance to restore viable populations.

Repeated Evaluations of Testes and Semen Characteristics in Two Binturongs (Arctictis binturong).

The binturong is a medium size carnivore belonging to the Viverrid family that lives in dense forests of South-East Asia. In addition to the protection of this vulnerable species in its natural habitat (in situ), conservation breeding efforts (ex situ) aim at maintaining a good genetic diversity while increasing the number of individuals to reinforce wild populations. Both approaches require a solid understanding of binturong's basic biology. However, there is still a lack of precise information about reproduction. The objective of this brief research report was to analyze testicular sizes and semen characteristics at different times of the year to better understand the reproductive physiology and inform future conservation efforts. A secondary objective was to describe sperm cryotolerance for the first time in that species. Examinations of testes and semen collections were conducted on two adult males. While testicular measurements were relatively constant across multiple examinations, semen characteristics (volume, viability, sperm concentrations, sperm motility) varied between samples. However, incidence of sperm morphological abnormalities was consistently high. Sperm cryotolerance appeared to be poor but further studies are warranted. The present dataset will be useful for future research on binturong reproduction and for the development of assisted reproductive techniques and biobanking of germplasms in that species.

The Comparison of Semen Collection in Electroejaculation, Rectal Massage and Combination of Both Methods in the Critically Endangered Malayan Pangolin, Manis javanica.

This article describes the semen characteristics from different collection methods between captive and confiscated Malayan pangolins, Manis javanica. Semen was collected from 15 pangolins; two captive and 13 confiscated individuals at the mean weight of 9.36 ± 1.94 kg. The three semen collection methods employed were electroejaculation, rectal massage and a combination of both techniques. The semen characteristics (mean ± standard deviation) of the Malayan pangolin are volume (73.75 ± 144.57 µL), pH (7.63 ± 0.53), spermatozoa concentration (997.19 ± 728.98 × 106 /mL), total motility (59.60% ± 30.00%), progressive motility (48.95% ± 30.93%), mass motility (3.50 ± 1.50) and live spermatozoa (80.25% ± 13.45%). There was no significant difference in semen characteristics between the three collection methods. The percentages of live spermatozoa were significantly different, suggesting better samples from captive compared to confiscated animals. However, there was no significant difference in spermatozoa kinetics between the captive and confiscated samples, suggesting the potential of utilizing confiscated individuals for gamete recovery to conserve the genetic pool of pangolins. All three methods of semen collection were successfully performed in pangolins and should be considered; however, electroejaculation remains the most consistent method of obtaining semen from the species.

Two variants of uterine leiomyoma in Malaysia's last Sumatran rhinoceros (Dicerorhinus sumatrensis).

Following its capture in March 2014, an adult female Sumatran rhinoceros frequently showed profuse vaginal bleeding. An ultrasonography suggested the presence of multiple reproductive lesions, including two uterine masses which were suspected to be leiomyomas. Soon after, an open pyometra was confirmed. Later in November 2019, the patient died and necropsy confirmed the presence of two uterine masses; one was located at the cervico-uterine junction and another in the uterine body, with pyometra, and cystic endometrial hyerplasia. Based on histological, special stains, and immunohistochemical examination, it was shown that one of the masses was composed of large, ovoid and polyhedral neoplastic mesenchymal cells with eosinophilic cytoplasm and a few binucleated cells surrounded by collagen fibres. It was tested positive for SMA and vimentin, while negative for desmin, cytokeratin AE1/AE3, EMA, CD34, and S100. The other mass was composed of mesenchymal cells undergoing myxoid degeneration as evidenced by the presence of glycosaminoglycan-rich matrix. It was tested positive for SMA, vimentin, partially positive for desmin, and negative for the other markers. With the aid of human medical nomenclature, these masses were diagnosed as epithelioid leiomyoma and myxoid leiomyoma, respectively. This report provides a clinical presentation, and histologic descriptions of the two variants of leiomyomas that have not been reported in veterinary medicine.

First Evaluations and Cryopreservation of Semen Samples from Sunda Clouded Leopards (Neofelis diardi).

A better understanding of semen characteristics and resilience to freezing temperatures is necessary before developing assisted reproductive techniques and systematic biobanking for the Sunda clouded leopard. The objective of this study was to evaluate for the first time the semen and sperm quality (in fresh and frozen samples) of two captive Sunda clouded leopards in Malaysia. A total of 17 examinations of the reproductive tract (using ultrasonography) and electro-ejaculations were performed on the two leopards over a 2-year period. Samples obtained from Leopard 1 (8 years old) varied in terms of volume (402 ± 92 µL), pH (7.9 ± 0.9), sperm motility (54.5 ± 24.2%), sperm concentration (122.4 ± 84.7 × 106 sperm/mL), normal morphology (23.9 ± 12.3%), and viability (55.2 ± 18.2%). Midpiece defects represented the most common structural abnormality followed by abnormal tail and head defects. Samples from Leopard 2 (11 year old with abnormal testicular tissue) were of lesser quality. Two frozen semen samples from Leopard 1 were thawed and examined for acrosome integrity. Post-thawed samples contained <10% of motile spermatozoa but almost 50% of abnormal acrosomes. The present results emphasized the high incidence of structurally-abnormal spermatozoa, similar to the mainland clouded leopard. Post-thaw evaluations showed that the few surviving spermatozoa could potentially be used for in vitro fertilization or sperm injection. However, more individuals must be studied to validate those first findings that are exciting but still preliminary.

Identification of hybrids of painted and milky storks using FTA card-collected blood, molecular markers, and morphologies.

Suspicious hybrids of painted storks and milky storks were found in a Malaysian zoo. Blood of these birds was sampled on FTA cards for DNA fingerprinting. Of 44 optimized primers, 6 produced diagnostic markers that could identify hybrids. The markers were based on simple, direct PCR-generated multilocus banding patterns that provided two sets of genetic data, one for each of the two stork species and another for the hybrids. It also revealed that large DNA fragments (3,000 bp) could be amplified from blood collected on FTA cards. When the results of each individual bird's DNA fingerprint were compared with plumage characters, the hybrids were found to express a range of intermediate phenotypic traits of the pure breeds with no dominant plumage characteristic from either parental species.

Blood meal analysis of tabanid fly after it biting the rare Sumatran rhinoceros.

OBJECTIVE: To demonstrate a noninvasive large mammalian genetic sampling method using blood meal obtained from a tabanid fly. METHODS: Blood meal was recovered from the abdomen of an engorged tabanid fly (Haematopota sp.) which was captured immediately after biting a Sumatran rhino in captivity. The blood was applied on to a Whatman FTA(®) blood card. Subsequent laboratory work was conducted to extract, amplify and sequence the DNA from the sample. Validation was done by sampling the hair follicles and blood samples from the rhinoceros and subjecting it to the same laboratory process. RESULTS: BLAST search and constructed phylogenetic trees confirmed the blood meal samples were indeed from the rhino. CONCLUSIONS: This method could be used in the field application to noninvasively collect genetic samples. Collection of tabanids and other haematophagous arthropods (e.g. mosquitoes and ticks) and other blood-sucking parasites (e.g. leeches and worms) could also provide information on vector-borne diseases.