Generation of induced pluripotent stem cells from Bornean orangutans.

Introduction: Orangutans, classified under the Pongo genus, are an endangered non-human primate (NHP) species. Derivation of induced pluripotent stem cells (iPSCs) represents a promising avenue for conserving the genetic resources of these animals. Earlier studies focused on deriving orangutan iPSCs (o-iPSCs) from Sumatran orangutans (Pongo abelii). To date, no reports specifically target the other Critically Endangered species in the Pongo genus, the Bornean orangutans (Pongo pygmaeus). Methods: Using Sendai virus-mediated Yamanaka factor-based reprogramming of peripheral blood mononuclear cells to generate iPSCs (bo-iPSCs) from a female captive Bornean orangutan. In this study, we evaluate the colony morphology, pluripotent markers, X chromosome activation status, and transcriptomic profile of the bo-iPSCs to demonstrate the pluripotency of iPSCs from Bornean orangutans. Results: The bo-iPSCs were successfully derived from Bornean orangutans, using Sendai virus-mediated Yamanaka factor-based reprogramming of peripheral blood mononuclear cells. When a modified 4i/L/A (m4i/L/A) culture system was applied to activate the WNT signaling pathway in these bo-iPSCs, the derived cells (m-bo-iPSCs) manifested characteristics akin to human naive pluripotent stem cells, including high expression levels of KLF17, DNMT3L, and DPPA3/5, as well as the X chromosome reactivation. Comparative RNA-seq analysis positioned the m-bo-iPSCs between human naive and formative pluripotent states. Furthermore, the m-bo-iPSCs express differentiation capacity into all three germlines, evidenced by controlled in vitro embryoid body formation assay. Discussion: Our work establishes a novel approach to preserve the genetic diversity of endangered Bornean orangutans while offering insights into primate stem cell pluripotency. In the future, derivation of the primordial germ cell-like cells (PGCLCs) from m-bo-iPSCs is needed to demonstrate the further specific application in species preservation and broaden the knowledge of primordial germ cell specification across species.

BACTERIAL IDENTIFICATION AND ANTIBIOTIC SENSITIVITY FROM THE ABSCESSES OF CAPTIVE TORTOISES-CLINICAL ANTIBIOTIC RECOMMENDATIONS.

Bacterial abscesses are commonly seen in tortoises. The morbidity and the resultant mortality are high. Multifactorial problems, antibiotics misapplication. and antibiotic-resistant bacteria make abscess treatment complicated and ineffective. This study identifies the etiological bacterial species and determines the best antibiotics for abscess treatment in captive tortoises. Sterile swab specimens from 40 tortoises with abscesses were analyzed using the Analytical Profile Index (API) system. Sixty-five bacteria species were identified covering facultative anaerobic gram-negative (n = 30, 46.2%), facultative anaerobic gram-positive (n = 19, 29.2%), and aerobic gram-negative bacteria (n = 16, 24.6%). The antibiotic sensitivity of these bacteria to 30 antibiotics was assessed using the Kirby-Bauer disk-diffusion method. Greater than 80% anaerobic gram-negative bacterial species showed sensitivity to amikacin and ceftazidime. Greater than 80% anaerobic gram-positive bacterial species were sensitive to amoxicillin, ampicillin, carbenicillin, and penicillin. In addition, more than 80% aerobic gram-negative bacterial species were sensitive to ceftazidime, colistin sulphate, amikacin, gentamicin, kanamycin, polymyxin B, and tobramycin. This study provides clinicians significant information for initial antibiotic options, which could elevate the abscess therapy success rate and improve the life quality of tortoises.

Novel Low-Voltage Electro-Ejaculation Approach for Sperm Collection from Zoo Captive Lanyu Miniature Pigs (Sus barbatus sumatranus).

Semen collection can be achieved via hand penile massage or rectal stimulation using electro-ejaculation methods. Traditional electro-ejaculation procedure applied relatively high voltage of 3-15 volts with a maximum current of 900 mA. However, these manipulations often result in great stress and discomforts in animals. In this study, we showed low-voltage electro-ejaculation procedure using 2-3 volts with a maximum current of 500 mA can efficiently stimulated ejaculations in zoo captive lanyu miniature pigs with a high success rate of 81.3% (13/16). Besides normal semen properties (semen volume, pH, sperm concentration), we demonstrated that low-voltage electro-ejaculation caused less stress in the animals, and sperm cells obtained via low-voltage electro-ejaculation exhibit low abnormality (10.3%), high viability (84.3%), motility (75.7%), progressive motility (63.7%), and acrosome integrity (88%). However, cryopreservation protocol used in the current study requires further optimization, as sperm mitochondrial function was partially compromised during freezing procedures. Taken together, we demonstrated in this study that a low-voltage electro-ejaculation approach can be used to obtain quality sperm cells from zoo captive lanyu miniature pig with less physical stress during electro-ejaculation procedure.

The effects of type I collagenase on the degelification of chimpanzee (Pan troglodytes) semen plug and sperm quality.

BACKGROUND: Semen from the chimpanzee species becomes a colloidal solid after ejaculation. The formation of this copulatory plug is believed to prevent additional spermatozoa of subsequent mating events from accessing the ova. However, this naturally preserved strategy hampers the processes for sperm preparation. In this study, we investigated whether collagenase can be used to degelify the semen plug and accelerate the semen liquefaction process in zoo captive chimpanzee species (Pan troglodytes). RESULTS: We showed that incubation of chimpanzee ejaculates with 0.1% type I collagenase efficiently and significantly (p < 0.05) releases 2.7-fold more spermatozoa from the coagulated ejaculates, and this degelification process did not alter sperm morphology or viability; nor did it stimulate spontaneous capacitation or an acrosome reaction as assessed by tyrosine phosphorylation and peanut agglutinin stains; moreover, based on computer assisted sperm analysis assay, motility-related parameters remained similar to those of untreated spermatozoa. When collagenase effects were evaluated on cryopreserved sperm samples, we observed post collagenase treatment in which 2.5% glycerol, as a cryoprotectant, preserved sperm acrosome integrity better than 7.8%; however, 7.8% glycerol, as a cryoprotectant, maintained sperm motility better than that of 2.5% glycerol. CONCLUSIONS: Our results demonstrated for the first time that type I collagenase can be used to obtain a significantly higher number of spermatozoa from colloid chimpanzee semen ejaculate without affecting the physiological properties of spermatozoa, and these results are critical for the subsequent gamete development. Our results would benefit sperm preparation processes and cryopreservation efficiency per ejaculate, as more unaffected spermatozoa can be released from the semen plug within a shorter period of time. These results would also benefit the genetic diversity of the chimpanzee species, using sperm cells from less dominant individuals, and for achieving better pregnancy success in primates with significantly higher amounts of sperm for artificial insemination.

A 20-year disease survey of captive formosan serows (Capricornis swinhoej) at the Taipei Zoo (1991-2011).

The Formosan serow (Capricornis swinhoei) is endemic to Taiwan. The wild population has declined dramatically over the past few decades and the species is listed as a "precious and rare species" protected under law in Taiwan. Disease investigations have been rare except for sporadic observations of wild individuals, and no long-term disease survey has been performed on this species. The objective of this study was to identify and report on the most common diseases in captive Formosan serows and determine the potential causes. Medical records of Formosan serows (n = 62) housed at the Taipei Zoo over a 20-yr period (1991-2011) were collected and analyzed for this study. The most common diseases affected the gastrointestinal system and the skin. Parasitic etiologies accounted for greater than 85% of these diseases, and coinfection was common. Coccidia and lice were the most common endo- and ectoparasites, respectively. High mortality was noted in serows less than 1 yr old associated with parasitism. The results from this study could provide vital information on disease prevention and species management, which may greatly help in rehabilitation of captive and wild populations.

An alternative staining method for counting red-eared slider turtle (Trachemys scripta) blood cells using crystal violet in cells diluted with 0.45% sodium chloride.

Various staining methods are available for reptilian species blood cell quantification. However, these methods have shown inaccurate differentiation limitations. The current study evaluates staining effects and blood cell counting results using an alternative method, counting blood cells diluted with 0.45% sodium chloride solution and stained with crystal violet. Blood samples from 8 red-eared slider turtles (Trachemys scripta) were collected. Red and white blood cell counts were performed using different methods: the unstained method, the Unopette method, Liu stain, and crystal violet method using blood cells diluted in various sodium chloride solution osmolarities. The staining properties and blood cell count results were compared. The crystal violet method using blood cells diluted in 0.45% sodium chloride solution delivered the best staining and counting results among all of the tested methods, with the lowest average coefficient of variance. The proposed method can easily be performed, serving as a feasible method for blood cell counting in chelonians.

Comparison of xylazine-ketamine and dexmedetomidine-ketamine anesthesia in captive Formosan serows (Capricornis swinhoei).

The Formosan serow (Capricornis swinhoei) is a rare endemic species found in Taiwan. Few studies, including studies on anesthetic protocols, have been conducted on this species. This study evaluates the anesthetic effectiveness of intramuscular xylazine-ketamine and dexmedetomidine-ketamine on captive Formosan serows. Fifty-seven anesthetic events were performed on 22 adults using a combination of xylazine (2.6 ± 0.5 mg/kg) and ketamine (3.7 ± 1.0 mg/kg). Forty-eight anesthetic events were performed on 29 adults using a combination of dexmedetomidine (33.9 ± 4.3 µg/kg) and ketamine (3.4 ± 0.4 mg/kg). Xylazine-ketamine anesthesia was antagonized with tolazoline (3.3 ± 0.8 mg/kg). Dexmedetomidine-ketamine anesthesia was antagonized with atipamezole (272.8 ± 78.2 µg/kg). Both drug combinations showed smooth anesthetic and recovery processes without statistical differences in respiratory rate, heart rate, rectal temperature and hemoglobin oxygen saturation. Dexmedetomidine-ketamine reversed by atipamezole showed a significantly shorter recovery time (1.8 ± 2.3 min) than xylazine-ketamine reversed by tolazoline (4.5 ± 1.7 min) (P < 0.05). Both anesthetic protocols indicated safe and reliable immobilization whereas atipamezole provided better reversal.

Evolution of the DAZ gene and the AZFc region on primate Y chromosomes.

BACKGROUND: The Azoospermia Factor c (AZFc) region of the human Y chromosome is a unique product of segmental duplication. It consists almost entirely of very long amplicons, represented by different colors, and is frequently deleted in subfertile men. Most of the AZFc amplicons have high sequence similarity with autosomal segments, indicating recent duplication and transposition to the Y chromosome. The Deleted in Azoospermia (DAZ) gene within the red-amplicon arose from an ancestral autosomal DAZ-like (DAZL) gene. It varies significantly between different men regarding to its copy number and the numbers of RNA recognition motif and DAZ repeat it encodes. We used Southern analyses to study the evolution of DAZ and AZFc amplicons on the Y chromosomes of primates. RESULTS: The Old World monkey rhesus macaque has only one DAZ gene. In contrast, the great apes have multiple copies of DAZ, ranging from 2 copies in bonobos and gorillas to at least 6 copies in orangutans, and these DAZ genes have polymorphic structures similar to those of their human counterparts. Sequences homologous to the various AZFc amplicons are present on the Y chromosomes of some but not all primates, indicating that they arrived on the Y chromosome at different times during primate evolution. CONCLUSION: The duplication and transposition of AZFc amplicons to the human Y chromosome occurred in three waves, i.e., after the branching of the New World monkey, the gorilla, and the chimpanzee/bonobo lineages, respectively. The red-amplicon, one of the first to arrive on the Y chromosome, amplified by inverted duplication followed by direct duplication after the separation of the Old World monkey and the great ape lineages. Subsequent duplication/deletion in the various lineages gave rise to a spectrum of DAZ gene structure and copy number found in today's great apes.