Sperm collection and characteristics analysis of the critically endangered Chinese pangolin (Manis pentadactyla).

The Chinese pangolin (Manis pentadactyla) is a critically endangered species. However, there is a paucity of research on the male reproductive gamete biology of this species. The present study was the first to systematically analyse the sperm characterization of the Chinese pangolin, including semen collection, sperm morphometry and ultrastructure. The semen of five male Chinese pangolins was successfully collected using the electroejaculation method. CASA (computer-assisted sperm analysis) was used to assess semen quality and take images for sperm morphometric analysis. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were used for sperm ultrastructure observation. The results showed that the semen of the Chinese pangolin was yellow to pale yellow in colour, viscous, with a fishy odour, and a slightly alkaline pH of between 7.7 and 7.9. The head defects were the main sperm defects; there were 13 kinds of head defects counted in this study. The total sperm length, head length, head width and tail length were 67.62 ± 0.21 µm, 10.47 ± 0.06 µm, 1.33 ± 0.006 µm and 57.16 ± 0.20 µm, respectively. SEM observed that the spermatozoa had a rod-shaped head with a distinct apical ridge, which was different from most mammals and similar to that in avians and reptiles. Interestingly, TEM found that the acrosome membrane of the Chinese pangolin had a double membrane structure rather than a multiple bi-lamellar membrane structure as reported by the previous study. Collectively, this study contributes to the development of artificial breeding efforts and assisted reproductive techniques for the Chinese pangolin, as well as providing technical support for research on germplasm conservation of this species.

Automatic Identification of Pangolin Behavior Using Deep Learning Based on Temporal Relative Attention Mechanism.

With declining populations in the wild, captive rescue and breeding have become one of the most important ways to protect pangolins from extinction. At present, the success rate of artificial breeding is low, due to the insufficient understanding of the breeding behavior characteristics of pangolins. The automatic recognition method based on machine vision not only monitors for 24 h but also reduces the stress response of pangolins. This paper aimed to establish a temporal relation and attention mechanism network (Pangolin breeding attention and transfer network, PBATn) to monitor and recognize pangolin behaviors, including breeding and daily behavior. There were 11,476 videos including breeding behavior and daily behavior that were divided into training, validation, and test sets. For the training set and validation set, the PBATn network model had an accuracy of 98.95% and 96.11%, and a loss function value of 0.1531 and 0.1852. The model is suitable for a 2.40 m × 2.20 m (length × width) pangolin cage area, with a nest box measuring 40 cm × 30 cm × 30 cm (length × width × height) positioned either on the left or right side inside the cage. A spherical night-vision monitoring camera was installed on the cage wall at a height of 2.50 m above the ground. For the test set, the mean Average Precision (mAP), average accuracy, average recall, average specificity, and average F1 score were found to be higher than SlowFast, X3D, TANet, TSN, etc., with values of 97.50%, 99.17%, 97.55%, 99.53%, and 97.48%, respectively. The recognition accuracies of PBATn were 94.00% and 98.50% for the chasing and mounting breeding behaviors, respectively. The results showed that PBATn outperformed the baseline methods in all aspects. This study shows that the deep learning system can accurately observe pangolin breeding behavior and it will be useful for analyzing the behavior of these animals.

Infantile hemangioma in a subadult Chinese pangolin: a case report.

BACKGROUND: Hemangiomas are a relatively common type of tumor in humans and animals. Various subtypes of hemangiomas have been described in the literature. The classification methods for hemangiomas differ between human and veterinary medicine, and the basis for tumor classification can be found in the literature. CASE PRESENTATION: This study describes a tumor in the subcutaneous tissue of the right dorsum of an artificially rescued juvenile Chinese pangolin. Computed tomography (CT) examination yielded the preliminary diagnosis of a vascular malformation, and surgery was performed to resect the tumor. Histopathological examination showed that the tumor mainly was consisted of adipose tissue, capillaries, and spindle cells in the fibrous stroma. Immunohistochemistry showed the positive expression of CD31, CD34, α-SMA, GLUT1 and WT-1 in the tumor tissue, and the tumor was eventually diagnosed as an infantile haemangioma. CONCLUSION: The final diagnosis of infantile hemangioma was depended on the histopathological immunohistochemical and CT examination of the neoplastic tissue. This is the first report of infantile hemangioma in a critically endangered species Chinese pangolin.

Energy budgets of captive Chinese pangolins (Manis pentadactyla).

The Chinese pangolin is an endangered species, and ex situ conservation and captive rescue are important conservation measures. This requires reliable information on nutritional energy requirements and expenditure characteristics. However, we lack sufficient knowledge of their energy physiology to determine their energy requirements for maintenance and growth. An open-flow respirometry system was used to measure the resting metabolic rate (RMR) and the daily energy expenditure (DEE) of Chinese pangolins (Manis pentadactyla), and the dietary digestive energy was measured. The average RMR in Chinese pangolins was 3.23 ml O2 kg-1 min-1 at an ambient temperature (Ta) of 24.5-30°C, which was only 73.0% of the expected value based on body mass (BM). The average DEE values were 744.9 kJ day-1 in animals with BM >3 kg and 597.3 kJ day-1 in those with BM <3 kg, which were only 52.4% and 60.6% of the predicted values, respectively. The RMR and DEE levels of the Chinese pangolin were lower than those of similar-sized eutherian mammals and close to those of anteaters. These characteristics suggest that the Chinese pangolin has a low demand for energy in its diet. Although metabolic level data alone cannot be used to calculate the energy requirements of each Chinese pangolin, we believe they can provide a tangible reference for the relocation of Chinese pangolins. These results provide a scientific basis for future research on the physiology and ecology of endangered wildlife such as the Chinese pangolin.

First case report of pustules associated with Escherichia fergusonii in the chinese pangolin (Manis pentadactyla aurita).

BACKGROUND: Escherichia fergusonii is a common conditionally pathogenic bacterium that infects humans and animals. E. fergusonii has been reported to cause diarrhea, respiratory disease, and septicemia, but it is rarely reported to cause skin infections in animals. E. fergusonii has been isolated from the skin and muscular tissue of Chinese pangolin (Manis pentadactyla aurita). To date, there have been no reports of Chinese pangolins with clinical signs of skin diseases. CASE PRESENTATION: This case report describes the clinical case of a subadult (bodyweight: 1.1 kg) female Chinese pangolin from wild rescue with pustules and subcutaneous suppurative infection due to E. fergusonii in the abdominal skin. Bacterial culture, Biochemical analysis, PCR and histopathology were utilized to identify the bacteria in the pustule puncture fluid and infected tissue. To the best of our knowledge, this is the first report of E. fergusonii-related pustules on a Chinese pangolin. CONCLUSION: This case report presents the first observed skin infection in a Chinese pangolin. E. fergusonii infection should be considered as a possible differential diagnosis of pustules and subcutaneous suppurative skin conditions in Chinese pangolins, and we also provide several recommendations for the diagnosis and treatment of this disease.

Comparison of Venous Blood Gas and Biochemical Parameters in Sunda Pangolin (Manis javanica) and Chinese Pangolin (Manis pentadactyla) before and after Isoflurane Anesthesia.

Venous blood gas analytes are commonly examined in animals, and the results may be important when evaluating the overall health status of an animal. Pangolins are critically endangered mammals, and there is limited information on their physiological reference values in the literature. The aim of this study was to analyze venous blood gas and biochemical parameters before and during isoflurane anesthesia in wild healthy Sunda and Chinese pangolins. The results obtained showed that the blood gas index trends of the two pangolin species before and after isoflurane anesthesia were the same. After anesthesia, the partial pressure of carbon dioxide (pCO2), partial pressure of oxygen (pO2), total carbon dioxide (CO2), mean blood bicarbonate (HCO3-), extracellular fluid compartment (BEecf) base excess and the mean blood glucose (Glu) levels of both pangolin species showed a significant increase compared to the pre-anesthesia period. In contrast, the mean blood potassium (K+), lactate (Lac) and mean blood pH levels were significantly lower. No significant differences in the mean blood sodium (Na+) or blood ionized calcium (iCa) levels were observed during anesthesia. This study is important for future comparisons and understanding the health status of this endangered species.

Multi-step deep neural network for identifying subfascial vessels in a dorsal skinfold window chamber model.

Automatic segmentation of blood vessels in the dorsal skinfold window chamber (DWSC) model is a prerequisite for the evaluation of vascular-targeted photodynamic therapy (V-PDT) biological response. Recently, deep learning methods have been widely applied in blood vessel segmentation, but they have difficulty precisely identifying the subfascial vessels. This study proposed a multi-step deep neural network, named the global attention-Xnet (GA-Xnet) model, to precisely segment subfascial vessels in the DSWC model. We first used Hough transform combined with a U-Net model to extract circular regions of interest for image processing. GA step was then employed to obtain global feature learning followed by coarse segmentation for the entire blood vessel image. Secondly, the coarse segmentation of blood vessel images from the GA step and the same number of retinal images from the DRIVE datasets were combined as the mixing sample, inputted into the Xnet step to learn the multiscale feature predicting fine segmentation maps of blood vessels. The data show that the accuracy, sensitivity, and specificity for the segmentation of multiscale blood vessels in the DSWC model are 96.00%, 86.27%, 96.47%, respectively. As a result, the subfascial vessels could be accurately identified, and the connectedness of the vessel skeleton is well preserved. These findings suggest that the proposed multi-step deep neural network helps evaluate the short-term vascular responses in V-PDT.

Automatic protocol for quantifying the vasoconstriction in blood vessel images.

Vascular targeted photodynamic therapy (V-PDT) has been successfully utilized for various vascular-related diseases. To optimize the PDT dose and treatment protocols for clinical treatments and to elucidate the biological mechanisms for V-PDT, blood vessels in the dorsal skin-fold window chamber (DSWC) of nude mice are often chosen to perform in vivo studies. In this study, a new automatic protocol to quantify the vasoconstriction of blood vessels in the DSWC model is proposed, which focused on tracking the pixels of blood vessels in pre- V-PDT images that disappear after V-PDT. The disappearing pixels indicate that the blood vessels were constricted, and thus, the vasoconstriction image for pixel distribution can be constructed. For this, the image of the circular region of interest was automatically extracted using the Hough transform. In addition, the U-Net model is employed to segment the image, and the Speeded-Up Robust Features algorithm to automatically register the segmented pre- and post- V-PDT images. The vasoconstriction of blood vessels in the DSWC model after V-PDT is directly quantified, which can avoid by the potential of generating new capillaries. The accuracy, sensitivity and specificity of the U-Net model for image segmentation are 90.64%, 80.12% and 92.83%, respectively. A significant difference in vasoconstriction between a control and a V-PDT group was observed. This new automatic protocol is well suitable for quantifying vasoconstriction in blood vessel image, which holds the potential application in V-PDT studies.

[Absorption of aqueous extracts from Salviae Miltiorrhizae Radix et Rhizoma by everted intestinal sac method].

To study the absorptive characteristics of aqueous extracts from Salviae Miltiorrhizae Radix et Rhizoma by in vitro rat everted intestinal sac model. Three representative ingredients in aqueous extracts from Salviae Miltiorrhizae Radix et Rhizome--protocatechuic aldehyde (PAL), posmarinic acid (RA) and salvianolic acid B (SAB), were selected as the study objects. An UPLC method was established to determine and measure their cumulative absorption amount, in order to explain the absorption characteristics of ingredients in different intestinal sections. According to the experimental result, RA and SAB showed the passive absorption in ileum, which conformed to the first-order absorption rate; with low and medium doses, they showed a zero-order absorption rate in jejunum, which was reflected in the coexistence of both positive and passive absorptions; PAL showed a passive absorption manner both in ileum and jejunum. According to the experiment for absorption in different intestinal sections, RA and SAB were mainly absorbed in jejunum, while PAL was absorbed mainly in ileum. All of the three ingredients in aqueous extracts from Salviae Miltiorrhizae Radix et rhizome--PAL, RA and SAB could be absorbed in intestines, but with differences in the absorption rate and mechanism, which indicated that the intestinal absorption of aqueous extracts from Salviae Miltiorrhizae Radix et rhizome was selectivity, instead of a simple semi-permeable membrane penetration process.

[Liquisolid technique for enhancement of dissolution prosperities of tanshinone II(A)].

The technique of liquisolid compress is a new technique developed in 1990s, which was considered to be the most promising technique to improve the dissolution of water-insoluble drugs. In this article, tanshinone II(A) and the extracts of the ester-solubility fractions were chosen as the model drugs to evaluate the effects of the liquisolid technique for enhancement of dissolution properties of tanshinone II(A). Several liquisolid tablets (LS) formulations containing different dosage of drugs and various liquid vehicle were pre-pared and for all the formulations, microcrystalline cellulose and silica were chosen as the carrier and coating materials to evaluate their flow properties, such as angle of repose, Carr's compressibility index and Hausner's ratio. The interaction between drug and excipients in prepared LS compacts were studied by differential scanning calorimetry(DSC) and X-ray powder diffraction (XRPD). The dissolution curves of tanshinone II(A) from liquisolid compacts were investigated to determine the technique's effect in improving the dissolution of tanshinone II(A) and its impacting factors. According to the results, the dissolution increased with the rise in the dissolution of the liquid-phase solvent. The R-value and drug dosage can significantly affect the drug release, but with less impact on active fractions. This indicated that liquisolid technique is a promising alternative for improvement of dissolution property of water-soluble drugs, and can make a synergistic effect with other ester-soluble constituents and bettern improve the release of tanshinone II(A). Therefore, the technique of liquisolid compress will have a better development prospect in traditional Chinese medicines.